oral sciences n3 braz j oral sci. 12(2):95-99 original article braz j oral sci. april | june 2013 volume 12, number 2 is bleeding on probing a differential diagnosis between periimplant health and disease? priscila ladeira casado1, ricardo villas-bôas2, luana cristine leão da silva3, cristiana farias de carvalho andrade3, letícia ladeira bonato4, josé mauro granjeiro5 1area of morphology, cell therapy center clinical research unit and biology institute, fluminense federal university – niterói, rj, brazil and orthopedics and traumatology national institute, rio de janeiro, rj, brazil 2area of dentistry, veiga de almeida university, rio de janeiro, rj, brazil 3dentist, veiga de almeida university, rio de janeiro, rj, brazil 4dentist, juiz de fora federal university, juiz de fora, mg, brazil 5area of chemistry, cell therapy center clinical research unit and biology institute fluminense federal university niterói, rj, brazil and national institute of metrology rio de janeiro, rj, brazil correspondence to: priscila ladeira casado núcleo de terapia celular unidade de pesquisa clinica rua marques de paraná 303, 40° andar, cep: 24033-900 centro, niterói, rj, brasil phone / fax: +55 21 26299255 e-mail: pcasado@into.saude.gov.br abstract as far as the periimplant anatomy is considered, the question raised is whether or not healthy periimplant tissues present bleeding on probing (bop). aim: to assess if the criterion bop is strictly related to periimplant disease (pid). methods: 134 patients were included in this study. all periimplant regions were clinically and radiographically evaluated. patients were assigned to 3 groups based on radiographic and clinical aspects in the periimplant region: group a (healthysites) no signs of mucosal inflammation or bone loss; group b (mucositis) red and swollen mucosa, but no radiographic bone loss; group c (periimplantitis) radiographically confirmed pathological bone loss. after this classification, all periimplant sulci were probed at 4 sites (mesial, distal, buccal, lingual/palatal). patients’ mean age was 51.7±12.4 years, 77 women and 57 men, with a total of 486 osseointegrated endosseous implants. results: groups a and c showed significant difference in age and implant region distribution (p=0.009 and p=0.008, respectively). after initial clinical and radiographic diagnosis of periimplant status, 33 (20.1%) regions showed bop in group a. all regions in group b presented bop. in group c, 41 (19.9%) regions showed no bop. all groups differed significantly considering bop as diagnosis parameter (p<0.0001). conclusions: bop was always present in inflamed mucosa, but it was not always absent in healthy mucosa. not all periimplantitis regions showed bop. clinical and radiographic aspects must always be considered together for diagnosis of pid, even if bop is absent. keywords: inflammation, periimplantitis, diagnosis. introduction the soft and hard tissues around endosseous implants share some similarities with the periodontium. however, differences such as the absence of cementum and periodontal ligament in the periimplant region, orientation of the collagen fibers in the periimplant soft tissue, which is parallel to the implant surface and not inserted in the implant surface and periimplant vascularization must be taken into consideration to provide reliable prognosis1. in natural dentition, the junctional epithelium provides sealing on the base received for publication: february 21, 2013 accepted: june 11, 2013 9696969696 braz j oral sci. 12(2):95-99 of the periodontal sulcus against the penetration of chemical pathogens and bacterial substances2. rupture of this sealing or lysis of connective tissue fibers attached to the apical cementum to the junctional epithelium, lead to rapid migration of the sulcular epithelium and consequent pathological pocket formation. since cementum or fiber attachment is not seen around the titanium surface, mucosal seal provides the main barrier against the dissemination of pathological aggressions in the deep periimplant tissues. the sealing around endosseous implants, which has weak adherence to the titanium structure, is provided by the presence of junctional epithelium, sulcular epithelium and connective tissue by hemidesmosomes. the destruction of the mucosal integrity around the titanium leads to the direct extension of the pathological pocket to the bone tissue, which may result in loss of the endosseous implant1-3. several reports emphasize the importance of the presence of healthy gingival tissues around dental implants as being the key factor not only for aesthetics, but also for long-term success2,4-5. however, correct and early clinical diagnosis of periimplant disease status is frequently critical, which makes maintenance of the periimplant tissue difficult6. according to the seventh european workshop on periodontology, the clinical parameters that indicate periimplant disease are bleeding on probing (bop) and increased probing depth7. clinical studies have shown that the key parameter for the diagnosis of periimplant mucositis is bleeding on gentle probing. periimplantitis is characterized by changes in the level of the crestal bone in conjunction with bop with or without concomitant deepening of periimplant pockets. presence of pus, gingival recession, fistula, edema and hyperplasia are other common conditions found in periimplantitis sites6,8. however, the radiographic detection of periimplant bone loss shows only the involvement of the proximal areas to the implant, and thus periimplant probing as a diagnostic procedure is advisable to detect bone loss on all faces9. in addition, probing in periimplant sulci allows evaluating the clinical probing depth, the distance between the marginal soft tissue and a reference point on the implant (for identification of hyperplasia or gingival recession), bop and suppuration from the periimplant pocket10. regarding the clinical probing depth, it is important to consider that in inflamed tissues around the implants the probe penetrates close to the bone level, while in healthy tissues the probe tip tends to stop at the histological level of connective tissue attached to the implant. the inflamed tissue with loss of connective tissue does not seem to inhibit the penetration of the probe beyond the apical extension of the junctional epithelium11-12. quirynem et al.13 (1991) found a relation between the bone level identified by the radiographic exam and the penetration of the probe into the periimplant tissue. in screw-retained implants, the probe tip stops 1.4 mm coronally to the bone level. this way, despite the fact that bop is a diagnosis for periimplant disease, it is important to mention that, according to ericsson and lindhe14 (1993), bleeding, though unusual in healthy periodontium, is frequently found in most healthy periimplant tissues. ferreira et al.15 (2006) stated that it is still not clearly defined if bop of periimplant tissues would be a parameter for identifying the presence of periimplant disease. some studies suggest that periimplant mucosa may be more sensitive to probing forces, causing more bop when compared with teeth16-17. the correct diagnosis of periimplant disease is a critical procedure, which makes it difficult the periimplant tissue maintenance6. actually, a clinical standard to diagnose periimplant disease is based on the presence of bop with probing pocket depth e”4 mm for mucositis diagnosis and additional radiographic bone loss for correct periimplantitis diagnosis8. during the first year after abutment connection, 1 mm of marginal bone loss is allowed, followed by 0.2 mm loss per year18. currently, these criteria are still frequently referred to as the “gold standard” for implant success19. in the present study, we considered previously established clinical characteristics of periimplant tissues that justify the exclusion criterion of bop to diagnose the presence of periimplant disease. based on periimplant anatomy, the tested hypothesis is that healthy periimplant tissues can also present bop. thus, the aim of this study was to assess if bop is directly related to the presence of periimplant disease. material and methods clinical study procedures were conducted according to the veiga de almeida university ethical board’s recommendations (process# 238/10). patient selection one hundred and thirty-four nonsmoking patients without any systemic disease (77 women and 57 men; mean age of 51.7±12.4 years), presenting a total of 486 osseointegrated endosseous implants, 295 in the maxilla and 191 in the mandible, were randomly selected for this study (table 1). patients signed an informed consent form after receiving full information about the study nature and purposes. patients were admitted to the study if they had no medical complications, were not taking medications affecting periodontal status as described by soskolne20 (1997) and had immediate postoperative radiographs showing the vertical bone level around the implant in order to compare bone levels after osseointegration period. patients who had undergone any periodontal or periimplant therapy within the last six months were excluded from the study. all periimplant regions were clinically and radiographically evaluated. clinical examination of the periimplant sites consisted of visual inspection and palpation, analysis of mucosa color, plaque accumulation, edema and implant mobility. conventional periapical radiographs using the paralleling technique measured the presence of vertical bone loss adjacent to the implants. the height of periimplant bone around the implant was recorded according to the exposure of the screw. according to the clinical and radiographic characteristics of the periimplant sites, patients were divided into 3 groups. patients in group a (healthy sites) showed no is bleeding on probing a differential diagnosis between periimplant health and disease? braz j oral sci. 12(2):95-99 visual clinical signs of inflammation in the periimplant mucosa and no signs of bone loss. in group b, periimplant sites characterized as mucositis, presence of mucosae presenting red color and swelling, but no signs of pathologic bone loss. patients in group c (periimplantitis sites) showed implant mobility and suppuration in some cases, and radiographic signs of pathologic bone loss (more than 2 screws exposed). after initial classification, the periimplant sulci from groups a, b and c were gently probed at 4 sites around each implant and the presence of bop was recorded by a previously trained clinician. periimplant measurements were recorded using a millimeter conventional u.n.c. periodontal probe, (hufriedy™, chicago, il, usa). then, if bleeding was detected at any of the sites, a classification of bop was established. statistical analysis the data of each, including clinical and radiographic characteristics, were submitted to descriptive statistical analyses considering age, gender, region and presence of bop using the statistical software spss version 12.0 (spss inc., chicago, il, usa). numerical variables were expressed as frequencies and percentages. the chi-square test was performed to assess the significance of nominal variables between groups. continuous variables as age were expressed as mean and standard deviation. then, after shapiro-wilk test, anova was applied and parametric analysis (student’s t-test) was used to compare means between groups considering that the variable had a normal distribution. the significance level was set at 5%. results taking into consideration baseline characteristics, groups a and c showed statistically significant difference in age and implant region distribution (p=0.009 and p=0.008, respectively). in group a (healthy periimplant tissue), 131 (19.1%) periimplant regions were characterized by the absence of bop while 33 (20.1%) regions showed bop with no clinical or radiographic signs of inflammation. all periimplant regions (100%) in group b (periimplant mucositis) characterized by clinical signs of inflammation (red color of mucosa and swelling) and no radiographic bone loss, presented bop. in group c (periimplantitis), 165 (80.1%) regions around the implants showed bop together with pathologic bone loss and 41 (19.9%) regions presented no signs of bop even with bone loss. periimplant mobility was present in 6 implants in group c (2.9%). group a showed no inflammation in mucosa while group b showed inflammation in all periimplant mucosal tissues, helping differential clinical diagnosis. however, group c showed 118 (57.3%) regions without any sign of mucosal inflammation even when pathological bone loss was present. these results distinguished one group from another by this criterion (p<0.0001). all groups had significant differences considering bop (p<0.0001). periimplant disease groups (b and c) showed higher incidence of bop compared to group a, which showed lower incidence of bop, despite the presence of bleeding. in addition, when comparing disease groups, higher incidence of bop was observed in group b (p<0.0001 in all analyses comparing bop among the 3 groups). table 2 shows the clinical and radiographic findings in each group. discussion this study evaluated the presence of bop in periimplant regions clinically and radiographically characterized as healthy, mucositis and periimplantitis, excluding bop as the initial diagnostic factor. healthy patients presented no signs of visual clinical inflammation (red color or swelling) or radiographic bone loss. regions affected by mucositis were characterized by visible clinical mucosal inflammation without signs of bone loss, and periimplantitis regions (group c) were characterized as all regions with bone loss and more than two exposed screws, considering the radiography obtained to determine alveolar bone levels after physiologic remodeling. the main question was: the presence of bop is really reliable when used as the unique parameter for disease diagnosis? this study showed that after the initial diagnosis 9797979797is bleeding on probing a differential diagnosis between periimplant health and disease? table 1. patients’ baseline findings. *p-values <0.05 are considered significant; ci: confidential interval; ¶chi-square test; **student t-test 9898989898 braz j oral sci. 12(2):95-99 considering other clinical and radiographic parameters of periimplant disease, the presence of bop was secondary for disease identification, taking into consideration that healthy periimplant mucosae (without inflammation or bone loss) showed bop in 20% of cases. according to the seventh workshop of periodontology7 (2011) the presence of bop characterizes periimplant disease. however, despite bop being a diagnosis of periimplant disease, bleeding, unusual in healthy periodontium, is found in most healthy periimplant tissues14 as evident in this work. therefore, according to ferreira et al.15 (2006) it has not been clearly defined whether periimplant bop could represent a reliable parameter for identifying the presence of periimplant disease. some studies suggest that periimplant mucosa may be more sensitive to probing forces, causing more bop when compared with teeth16-17. luterbacher et al.21 affirm that absence of bop represents a stable periimplant condition. however, lack of keratinized tissue, a common finding after implant placement surgery, could also simulate an inflamed tissue, due to gingival manipulation and its red aspect, which can also be associated with non-keratinized mucosa. therefore, swelling, pus and radiographic findings were considered for diagnosis of mucositis. the present study showed that 20% of patients considered clinically and radiographically healthy had bop and all the periimplant regions with a clinical aspect of inflammation (group b mucositis) had bop, which lead to the conclusion that bop is always present in inflamed mucosa, but it will not always be absent in healthy mucosa, obviously due to periimplant anatomical reasons that, even in healthy conditions, do not limit penetration of the probe beyond the barrier in the epithelial junction. however, future studies are required, including in vivo analysis in order to show how the probe penetration can stimulate bleeding in healthy and diseased periimplant mucosa. quirynem et al.13 (1991) found a relation between the bone level identified by the radiographic exam and the penetration of the probe into the periimplant tissue. in screwretained implants, the probe tip appears to stop 1.4 mm coronally from the bone level. in addition, the type of probe used to measure clinically the depth does not seem to influence the result. christensen et al.22 used different types of probe to characterize cpd around endosseous implants and they concluded that the differences between the analyzed probe types during the research were not larger than 0.1 mm. in this study only one type of probe was used, which standardized the obtained results. another important consideration is that previous studies claim that when changes in the clinical parameters indicate disease (bop, increased probing depth); the clinician is encouraged to take a radiograph to evaluate possible bone loss. the results of the performed research showed that 56% of the regions affected by pathological bone loss (periimplantitis – group c) showed healthy mucosa, which in many cases leads the clinician not to perform a radiographic exam and to an erroneous healthy diagnosis. therefore, the radiographic exam must be always considered as a followup measure and not only due to the presence of bop, for if bop is not present and bone loss has been triggered, possible subclinical periimplantitis may be developing. the clinical aspect as well as the radiographic aspect must be used as a diagnostic factor of periimplant disease, even if bop is absent, instead of bop guiding the radiographic analysis. in implantology, the follow-up should be performed by clinical and radiographic examination at least once a year, to identify underlying bone loss in an apparently healthy periimplant gingival tissue and restore bone health before the implant failure. in case of rapid progression of periimplantitis, the following question arises: would rapid progression of periimplantitis be a consequence of a late diagnosis based solely on the clinical aspect of the mucosa? from all patients with more than two exposed threads (pathological bone loss), 20% did not show bop. how can this be explained? the study hypothesis is that in some patients, even with pathological bone loss, the mucosal epithelium remains adhered limiting the penetration of the probe into the tissue due to some of the following reasons: (1) bacterial penetration into the connective tissue is faster and triggers a more aggressive inflammatory response in periimplant bone, which would justify progressive bone loss without prior involvement of the mucosa; (2) at some point, mucosal inflammation might occurr with subsequent periimplant bone loss and spontaneous mucosal healing after routine cleaning procedures performed by the patient, as mucositis is characterized for being a reversible lesion, but the underlying bone shows pathological loss resulting from prior involvement due to the irreversibility of periimplantitis; (3) the thickness of the mucosa may influence the dissemination of the disease to the underlying bone limiting the damage to the thick mucosa, but further studies are needed. is bleeding on probing a differential diagnosis between periimplant health and disease? table 2. clinical aspects in each group *reference for calculation= number of implants; bop= bleeding on probing, measure considering the presence of bleeding in at least one from the 4 analyzed aspects (mesial, distal, buccal, lingual/palatal); p values <0.05 are considered significant; ¶ chi-square test; (—) non measurable value due to values=0. braz j oral sci. 12(2):95-99 9999999999 correct diagnosis of periimplant disease is still difficult to establish. mobility of implants indicates the final stage of the disease, characterized by complete loss of the bone/ implant interface10. therefore, according to heitz-mayfield6 (2008), mobility cannot be a parameter for early diagnosis of periimplant disease, but it may indicate complete lack of osseointegration, which requires the implant to be immediately removed. in order to be able to intervene in the development of periimplantitis before advanced bone loss, it is important to diagnose the disease in its initial stage10. in addition, according to leitão et al.23 (2005) even when significant inflammatory signs are absent in periimplant tissue, the qualitative detection of pathogens may indicate risk of periimplantitis, requiring stricter postoperative control. in summary, several cases of failure found in this research were not directly related to the presence of bop. bop is always present in inflamed mucosa, but it will not always be absent in healthy mucosa. it is also important to consider that not all tissues presenting pathologic bone loss show clinical signs of inflammation. in the present study was considered that bop alone cannot distinguish between the presence and absence of periimplant health and other factors involving a thorough clinical and radiographic characterization of the disease should be considered. the clinical aspect as well as the radiographic aspect must be always used as a diagnostic factor of periimplant disease, even if bop is absent. the authors expect that this research can contribute to a better diagnosis of periimplant disease, thus reducing the rate of failures in implantology. acknowledgements the authors would like to capes/faperj for granting funds for this study (e-26/102.288/2010). references 1. jovanovic sa. diagnosis and treatment of peri-implant diseases. curr opin periodontol. 1994; 194-204. 2. sumi t, braian m, shimada a, shibata n, takeshita k, vandeweghe s et al. characteristics of implant cad/cam abutment connections of two different internal connection systems. j oral rehabil. 2012; 39: 391-8. 3. santos mclg, campos mig, line srp. early dental implant failure: a review of literature. braz j oral sci. 2002; 1: 103-11. 4. kan jy, rungcharassaeng k, lozada jl. bilaminar subepithelial connective tissue grafts for immediate implant placement and provisionalization in the esthetic zone. j can dent assoc. 2005; 33: 865-71. 5. pelegrini aa, costa ces, sendyk wr. connective tissue graft: a clinical alternative perimplant aesthetics. case report. implant news. 2006; 3: 249-54. 6. heitz-mayfield lja. peri-implant diseases: diagnosis and risk indicators. j clin periodontol. 2008; 35: 292-304. 7. lang np, berglundh t. on behalf of working group 4 of the seventh european workshop on periodontology: periimplant diseases: where are we now? – consensus of the seventh european workshop on periodontology. j clin periodontol. 2011; 38(suppl. 11): 178-81 8. zitzmann nu, berglundh t. definition and prevalence of peri-implant diseases. j clin periodontol. 2008; 35: 286-91. 9. albrektsson to, isidor f. consensus report of session iv, 1994 apud salvi ge, persson gr, heitz-mayfield ls, frei m, lang np. adjunctive local antibiotic therapy in the treatment of peri-implantitis ii: clinical and radiographic outcomes. clin oral implants res. 2007; 18: 281-5. 10. mombelli a, muhle t, bragger u, lang np, burgin wb. comparison of periodontal and peri-implant probing by depth-force pattern analysis. clin oral implants res. 1997; 8: 448-54. 11. lang np, wetzel ac, stich h, caffesse rj. histologic probe penetration in healthy and inflamed periimplant tissues. clin oral implants res. 1994; 5: 191-201. 12. khammissa ra, feller l, meyerov r, lemmer j. peri-implant mucositis and peri-implantitis: clinical and histopathological characteristics and treatment. sadj. 2012; 67: 124-6. 13. quirynen m, van steenberghe d, 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long term efficacy of currently used dental implants: a review and proposed criteria of success. int j oral maxillofac implants. 1986; 1: 11-25. 19. levin l. dealing with dental implant failures. j appl oral sci. 2008; 16: 171-5. 20. soskolne wa. subgingival delivery of therapeutic agents in the treatment of peridontal diseases. critic rev oral biol med. 1997; 8: 164-74. 21. luterbacher s, mayfield i, bragger u, lang np. diagnostic characteristics of clinical and microbiological tests for monitoring periodontal and periimplant mucosal tissue conditions. clin oral implan res. 2000; 11: 521-9. 22. christensen mm, joss a, lang np. reproducibility of automated periodontal probing around teeth and osseointegrated oral implants. clin oral implant res. 1997; 8: 455-64. 23. leitão ja, de lorenzo jl, avila-campos mj, sendyk wr. analysis of the presence of pathogens which predict the risk of disease at peri-implant sites through polymerases chain reaction. braz oral res. 2005; 19: 52-7. is bleeding on probing a differential diagnosis between periimplant health and disease? oral sciences n3 original article braz j oral sci. april | june 2015 volume 14, number 2 effect of different endodontic sealers on push-out bond strength of fiber posts kiana ghanadan1, sajjad ashnagar2, ladan ranjbar omrani3, mansooreh mirzaee3 1hamedan university of medical sciences, school of dentistry, department of operative dentistry, hamedan, iran 2university of california los angeles ucla, school of dentistry, department of periodontology, los angeles, california, usa 3tehran university of medical sciences, school of dentistry, department of operative dentistry, tehran, iran correspondence to: ladan ranjbar omrani department of operative dentistry school of dentistry, tehran university of medical sciences north kargar st, amirabad, tehran, iran phone: +98 9122132110 e-mail:ladanonrani@yahoo.com abstract despite the increasing demand for fiber-reinforced composite (frc) posts, their bonding to root canals is still subject to debate. endodontic sealers may affect the bond strength between fiber posts and root canal dentin. aim: to compare the effects of different sealers on fiber post bond strength. methods: sixty teeth were divided into 4 groups according to obturation method: gi, gutta-percha without any sealers; gii, gutta-percha and ah26 resin-based sealer; giii, realseal point and realseal resin-based sealer, giv, guttaflow. fiber posts were cemented into root canals. specimens were sectioned, and the bond strength was measured in the middle area. the failure mode was evaluated. data were analyzed by one-way anova and post hoc test. results: the highest bond strength was observed in the control group (2.95±1.12), and the least was in the guttaflow group (1.15±0.78). there was a significant difference between bond strengths of the control and guttaflow groups and between ah26 and guttaflow groups (p<0.05). the failure mode was mainly adhesive between dentin and resin cement in all groups. conclusions: sealers may have a negative effect on the bond strength of fiber posts to root canal dentin. keywords: root canal filling materials; dentin; dental pulp; tooth; root canal obturation. introduction the selection of a proper type of restoration for natural function and esthetic rehabilitation is a major concern in weakened teeth1. when the crown is clinically almost destroyed, intraradicullar posts are indicated to secure retention between the prosthetic crown and the remaining tooth structure2-3 .this retention plays an important role in the durability of the final restoration4. recent years have seen increased utilization of fiber-reinforced composite (frc) posts5 because of their advantages of desirable esthetics6, a modulus of elasticity similar to dentin, stress distribution along a wider surface area on root walls, and minimal risk of vertical root fracture7-8. relating to the limitations in bonding to root canal dentin, numerous studies have been conducted to improve bonding of posts to tooth structures9. endodontic sealers are among the factors that may have a negative influence on post retention inside the canal2,10 . endodontic sealers should seal the canal laterally and apically and have acceptable adaptation to the root canal dentin11. there is a growing interest in the utilization of resin-based sealers that bond simultaneously to core material and the canal wall, creating a monoblock12. these sealers often have the ability to penetrate lateral canals, accessory canals and dentinal tubules11. consequently, it appears that they cannot be removed completely after canal preparation, which braz j oral sci. 14(2)166-170 received for publication: march 03, 2015 accepted: june 24, 2015 http://dx.doi.org/10.1590/1677-3225v14n2a13 167167167167167 can adversely affect the bond strength between posts and root canal dentin13. guttaflow is a new type of siloxine-based sealer that contains two products in one system: gutta-percha particles (less than 30 µm) and polydimethylsiloxine sealer. this injection system has no gutta-percha shrinkage and exhibits only minor expansion during setting because of the cold filling system and absence of heat. furthermore, removal of this filling material in cases of endodontic re-treatment or preparation of post space would be easier14. this study aimed to assess the effects of two resin-based sealers and guttaflow on the bond strength of fiber posts. we hypothesized that guttaflow would have less effect on the bond strength of fiber posts because there is no chemical bond with radicular dentin. material and methods sixty human anterior maxillary teeth, extracted because of periodontal problems over a period of 3 months were collected. informed consent was obtained from patients (ethical approval number 16897). roots were free of caries, cracks or resorption. teeth were stored in 0.5% chloramine t solution for 1 week and then stored in distilled water. the clinical crowns were sectioned transversely 1 mm up to the cementoenamel junction, with a low-speed diamond disk with air-water cooling (isomet 2000; buehler ltd, lake bluff, il, usa). working length of the remaining roots was measured by the direct method, subtracting 1 mm from the apex by means of a #10 k file. biomechanical preparation was performed with rotary instruments of the mtwo system (vdw gmbh, munich, germany). the apical preparation was extended until #35. the canal was irrigated with 2% chlorhexidine during instrumentation. the teeth were randomly divided into 4 experimental groups (n=15) according to the type of sealer used during the canal filling: gi: control group: teeth were filled with gutta-percha (coltène/whaledent, langenau, germany) without any sealer, by the lateral condensation technique. gii: ah26 group: teeth were filled with ah26 sealer (dentsply detrey gmbh, konstanz, germany) and guttapercha by the lateral condensation technique. root canals were irrigated with 2% chlorhexidin e and dried with absorbent paper points. sealer was introduced into canals by lentulo spirals. giii: realseal group: teeth were filled with realseal sealer (sybronendo, orange, ca, usa) and realseal point. root canals were irrigated with sodium hypochlorite and dried. smear layers were eliminated with 17% edta (smearclear, sybronendo, orange, ca, usa). canals were then irrigated with 2% chlorhexidine and dried with # 30 paperpoints (vdw, munich, germany). two pastes of realseal were mixed and introduced into canals by lentulo spirals. filling was completed with realseal point by the lateral condensation technique. teeth were light-cured with a lava led unit (ultradent products inc., south jordan, ut, usa) (1000 mw/cm2), for 40 s, from the coronal direction. giv: guttaflow group: teeth were filled with the guttaflow system (coltène/whaledent, langenau, germany). root canals were irrigated with chlorhexidine and dried with paperpoints. a guttaflow capsule was then mixed and injected into the canal, and a single gutta-percha cone was inserted into the canal according to the manufacturer’s instructions. after the filling was complete, coronal portions of canals were sealed with temporary cement (cavit g; 3m espe, seefeld, germany). the specimens were kept in 100% humidity for one week at 37 °c. post space was prepared with gates-glidden burs #2 to #4 (dentsply maillefer) at 10mm length. the root canals were washed with distilled water, then dried. fiber glass posts (angelus, londrina, pr, brazil) were cemented by the panavia f2.0 system, and light-cured for 20 sec with a lava led unit (ultradent products inc.) (1000 mw/cm2). samples were then mounted in transparent acrylic resin and sectioned with a cutting device and diamond-covered disc (mecatome, presi, france) in 1-mm slices, by a highspeed sectioning machine. sections were perpendicular to the long axis of the tooth, and the third and fourth sections were selected. the specimens were fixed in a universal testing machine (zwick, ulm, germany), and the push-out test was done at a cross-head speed of 0.5 mm/min, from the apical direction. the plunger was selected according to root canal diameter, which was measured by autocad software 2006; the plunger was between 80 and 90% of canal diameter. it was positioned to touch only the post, without contact with the root canal dentin. the applied force that dislodged the post and cement from the root canal was recorded. the unit of this force was for conversion into megapascal, in which r represents the root canal radius in the coronal portion, r represents the root canal radius in the apical portion, and h represent the height of specimen slices. root canal radius was measured with autocad software 2006. after the push-out test, the failure mode was evaluated by stereomicroscopy (nikon type 102, nikon corp., tokyo, japan) at 40x magnification. failure classification was cohesive if more than 75%, and adhesive if less than 25%, of the luting resin remained on the tooth surface. means and standard deviations were compared by oneway anova and post hoc tests with spss software ver. 16. statistical significance was set at 0.05 for all analyses. results bond strength results of the experimental groups are in table 1. according to these results, the maximum bond strength was observed in the control group (2.95±1.12), with the minimum bond strength in the guttaflow group (1.15±0.78). ah26 group bond strength data (2.68±0.90) are closer to the control group than the realseal group. one-way anova of the data showed significant differences between groups (p< 0.05).the post hoc analysis effect of different endodontic sealers on push-out bond strength of fiber posts braz j oral sci. 14(2)166-170 168168168168168 e x p e r i m e n t a l groups control ah26 realseal guttaflow mean±sd 2.95±1.12 a 2.68±0.90 a 2.02±1.27a 1.15±0.78 b m i n i m u m 1.23 1.38 0.29 0.07 m a x i m u m 5.24 4.64 4.58 2.64 table 1:table 1:table 1:table 1:table 1: statistical specifications of bond strength of fiber post in the experimental groups different letters represent the statistical differences between the groups of data showed that the control group had the highest bond strength and the guttaflow group had the lowest bond strength (figure 1). significant differences were observed between the fiber post bond strengths of the control and guttaflow groups, and between ah26 and guttaflow groups (p<0.05). in both comparisons, the bond strength of guttaflow was less than the other groups. failure mode distribution can be observed in table 2. adhesive failure (between cement and dentin) was the most common failure in all groups. fig. 1. one way anova analysis for comparison of fiber post bond strength among experimental groups. discussion one of the major problems of fiber posts is dislodgement of the post from the root canal. posts are placed in spaces prepared by the removal of filling material without encroaching on the apical portion, then bonded to tooth structures with adhesive cements. the success of fiber posts depends directly upon the quality of bonding among the fiber post/cement/root canal dentin15-16. both non-post-related factors, such as type of cement, irrigation solution during root canal preparation and type of sealer, and post-related factors, such as length, diameter, surface and design of the post may affect retention of the fiber post to the root canal walls17-18. resin-based cements play an important role in the durability of fiber posts. concerning the limitations of the root canal structure, self-etch resin-based luting cements are utilized to bond fiber posts to overcome problems of wetbonding in total-etch systems16,19. the bond would be formed with superficial layers of dentin in self-etch system via smear layer19. concerning root canal wall modification made by endodontic sealers and canal filling materials, it seems that these materials affect the bond between post and canal walls15-16,20. resinous sealers have the ability to penetrate lateral canals, accessory canals, and dentinal tubules7, and their composition and depth of penetration can have specific effects in reducing the post bond strength16. these filled dentinal tubules may be obstructed while post spaces are being prepared, which would interfere with the formation of a hybrid layer of resin cement and dentin. effect of different endodontic sealers on push-out bond strength of fiber posts braz j oral sci. 14(2)166-170 failure mode sealer ah26 control realseal guttaflow adhesive (cement and dentin) 54 58 86.3 91.6 cohesive 22.7 25 9 4.5 adhesive (cement and post) 22.7 17 4.5 4.5 table 2: failure mode distribution in the groups (%) 169169169169169 the present study assessed the effect of three types of sealers on bond strength of fiber posts. the effect of each sealer on the bond strength of fiber posts was evaluated by a thin-slice push-out test, which requires 1-mm-thick root disks21. plunger size diameter was chosen according to root canal diameter. this method results in more reliable, reproducible and clinic-like conditions than other tests22.this study evaluated the bond strength at the middle third using two serial sections, because other studies reported that the portion of the root canal to have a significant effect on the bond strength of fiber posts23-24. results indicated that guttaflow would significantly reduce the fiber post bond strength, in comparison with the control group or the ah26 group. the guttafllow group showed a significant difference from the control group. the highest bond strength was observed in the control group, which lacked any kind of sealer, which agrees with the results of other studies15-16,20. this could be explained by the absence of sealers, whereby cement penetration into root dentin was at the highest level7. according to the results of the present study, it may be observed that while the bond strength is decreasing among the sealer groups, the prevalence of adhesive failure mode between dentin and cement is simultaneously rising (table 2). the lowest bond strength was obtained by the guttaflow group, with the highest occurrence (91.6% of samples) of adhesive failure. the ah26 group showed the highest bond strength among the experimental groups, followed by the realseal group. the lack of significant differences confirms the results of some studies 7, but is in contrast with the results of demiryurek et al.16.this difference may be due to the use of special post drills, which would result in a clean canal surface with high post adaptation, a procedure that would not be used in clinical practice. some studies have stated that the penetration of resinbased sealers into dentinal tubules is higher than that of other sealers11,25. the degree of penetration of resin-based sealers depends on flow, surface tension, viscosity, chemical compounds, working time, setting time, and solubility of the sealers. ah26 is a highly hydrophilic epoxy resin and may set in humid environments26. some studies have reported that the penetration depth of this sealer is acceptable27. the penetration of sealer molecules into dentinal tubules may reduce the bond strength of fiber posts. the present study showed no significant difference between the bond strength of ah26 and the control group, which is in accordance with the findings of aleisa et al.17. realseal se, is a methacrylate resin-based self-adhesive sealer and contains acidic resin monomers that penetrate into the smear layer and bond to canal28. it seems that due to higher penetration of this sealer into dentinal tubules29, less bond strength was observed in comparison with the control group. however, this difference was not statistically significant. despite the fact that this system is based on the theoretical engagement of the smear layer, recent studies have found that these sealers, especially self-etch sealers, cannot establish a proper bond through the smear layer 30. consequently, it is recommended that edta be used prior to the application of sealer in root canals, to eliminate the smear layer, reduce microleakage and enhance sealing ability31. the fiber post bond strength in the guttaflow group was the lowest among the experimental groups. it appears that paramineoil, a composite of guttaflow, could contaminate root canal walls, reducing the bond strength of cemented posts32. previous studies found that oils and lubricants have a significant impact on bond strength of selfetch systems, reducing over half the original bond strength, which is similar to the results of present study32-33. aggarwal et al. found no significant difference between the fiber post bond strengths of the ah26 and guttaflow groups compared with the control group. this may be caused by edta as a chelating agent prior to post cementation, which would clean any remaining sealer oil from root canal surfaces15. notwithstanding the fact that we tried to maximize accuracy of experiment and it‘s comparability to in vivo environment, it is suggested to perform cyclic loading to better simulate oral cavity conditions. it seems that according to their composition, sealers may have negative effects on the bond strength of fiber posts to root canal dentin. it must be highlighted that, because of negligible reductions in the push-out bond strength of samples, ah26 may be used as a safe sealer, with no concerns about negative effects on the bond strength of fiber posts. it is also suggested to consider future restorative treatment plan when selecting guttaflow system while performing a root canal therapy. acknowledgement this research has been supported by the dental school of tehran university of medical sciences (grant number 16897-69-02-91). references 1. teixeira cs, pasternak-junior b, borges ah, paulino sm, sousa-neto md. influence of endodontic sealers on the bond strength of carbon fiber posts. j biomed mater res b appl biomater. 2008; 84: 430-5. 2. alfredo e, de souza es, marchesan ma, paulino sm, gariba-silva r, sousa-neto md. effect of eugenol-based endodontic cement on the adhesion of intraradicular posts. braz dent j. 2006; 17: 130-3. 3. morgano sm. restoration of pulpless teeth: application of traditional principles in presentand future contexts. j prosthet dent. 1996; 75: 375-80. 4. bateman g, ricketts dn, saunders wp. fibre-based post systems: a review. br dent j. 2003; 195: 43-8. 5. stewardson d. non-metal post systems. dent update. 2001; 28: 326-36. 6. morgano sm, rodrigues ah, sabrosa ce. restoration of endodontically treated teeth. dent clin n am. 2004; 48: vi, 397-416. 7. cecchin d, farina ap, souza ma, carlini-junior b, ferraz cc. effect ofroot canal sealers on bond strength of fibreglass posts cemented with self-adhesive resin cements. int endod j. 2011; 44: 314-20. 8. schwartz rs, robbins jw. post placement and restoration of endodontically treated teeth: a literature review. j endod. 2004; 30: 289-301. effect of different endodontic sealers on push-out bond strength of fiber posts braz j oral sci. 14(2)166-170 9. pelegrine ra, de martin as, cunha rs, pelegrine aa, da silveira bueno ce. influence of chemical irrigants on the tensile bond strength of an adhesive system used to cement glass fiber posts to rootdentin. oral surg oral med oral pathol oral radiol endod. 2010; 110: e73-6. 10. menezes ms, queiroz ec, campos re, martins lr, soares cj. influence of endodontic sealer cement on fibreglass post bond strength to root dentine. int endod j. 2008; 41: 476-84. 11. sabadin n, bottcher de, hoppe cb, santos rb, grecca fs. resinbased sealer penetration into dentinal tubules after the use of 2% chlorhexidine gel and 17% edta: in vitro study. braz j oral sci 2014; 13: 308-13. 12. tay fr, pashley dh. monoblocks in root canals: a hypothetical or a tangible goal. j endod. 2007; 33: 391-8. 13. hassanloo a, watson p, finer y, friedman s. retreatment efficacy of the epiphany soft resin obturation system. int endod j. 2007; 40: 633-43. 14. só mvr, saran c, magro ml, vier-pelisser fv, munhoz m. efficacy of protaper retreatment system in root canals filled with gutta-percha and two endodontic sealers. j endod. 2008; 34: 1223-5. 15. aggarwal v, singla m, miglanis, kohli s. effect of different root canal obturating materials on push-out bond strength of a fiber dowel. j prosthodont. 2012; 21: 389-92. 16. demiryurek eo, kulunk s, sarac d, yuksel g, bulucu b. effect of different surface treatments on the push-outbond strength of fiber post to root canal dentin. oral surg oral med oral pathol oral radiol endod. 2009; 108: e74-80. 17. aleisa k, alghabban r, alwazzan k, morgano sm. effect of three endodontic sealers on the bond strength of prefabricated fiber posts luted with three resin cements. j prosthet dent. 2012; 107: 322-6. 18. tjan ah, nemetz h. effect of eugenol-containing endodontic sealer on retention of prefabricated posts luted with adhesive composite resin cement. quintessence int. 1992; 23: 839-44. 19. foxton rm, nakajima m, tagami j, miura h. adhesion to root canal dentine using one and two-step adhesives with dual-cure composite core materials. j oral rehabil. 2005; 32: 97-104. 20. dimitrouli m, gunay h, geurtsen w, luhrs ak. push-out strengthof fiber posts depending on the type of root canal filling and resin cement. clin oral investig. 2011; 15: 273-81. 21. pane es, palamara je, messer hh. critical evaluation of the push-out test for root canal filling materials. j endod. 2013; 39: 669-73. 22. goracci c, grandini s, bossù m, bertelli e, ferrari m. laboratory assessment of the retentive potential of adhesive posts: a review. j dent. 2007; 35: 827-35. 23. soares c, pereira j, valdivia a, novais v, meneses m. influence of resin cement and post configuration on bond strength to root dentine. int end j. 2012; 45: 136-45. 24. calixto l, bandéca m, clavijo v, andrade m, vaz l, campos e. effect of resin cement system and root region on the push-out bond strength of a translucent fiber post. oper dent. 2012; 37: 80-6. 25. gharib sr, tordik pa, imamura gm, baginski ta, goodell gg. a confocal laser scanning microscope investigation of the epiphany obturation system. j endod. 2007; 33: 957-61. 26. ingle ji, bakland lk, baumgartner jc. root canal filling material. in: ingle’s endodontics. 6. ed. hamilton: bc decker; 2008. p. 1034-43. 27. nunes vh, silva rg, alfredo e, sousa-neto md, silva-sousa yt. adhesion of epiphany and ah plus sealers to human root dentin treatedwith different solutions. braz dent j. 2008; 19: 46-50. 28. babb br, loushine rj, bryan te, ames jm, causey ms, kim j, et al. bonding of self-adhesive (self-etching) root canal sealers to radicular dentin. j endod. 2009; 35: 578-82. 29. chadha r, tanejas, kumar m, gupta s. an in vitro comparative evaluation of depth of tubular penetration of three resin-based root canal sealers. j conserv dent. 2012; 15: 18. 30. kim yk, grandini s, ames jm, gu ls, kim sk, pashley dh, et al. critical review on methacrylate resin-based root canal sealers. j endod. 2010; 36: 383-99. 31. kim yk, mai s, haycock jr, kim sk, loushine rj, pashley dh, et al. the self-etching potential of realseal versus realseal se. j endod. 2009; 35: 1264-9. 32. sugawara t, kameyama a, haruyama a, oishi t, kukidome n, takase y, et al. influence of handpiece maintenance sprays on resin bonding to dentin. clin cosmet investig dent. 2010; 2: 13. 33. matos ab, oliveira dc, vieira sn, netto ng, powers jm. influence of oil contamination on in vitro bond strength of bonding agents to dental substrates. am j dent. 2008; 21: 101-4. 170170170170170effect of different endodontic sealers on push-out bond strength of fiber posts braz j oral sci. 14(2)166-170 oral sciences n3 original article braz j oral sci. january | march 2015 volume 14, number 1 is there difference of streptococcus mutans count and adherence on amalgam and resin occlusal restorations? a blind clinical study patricio agustin vildósola grez1, eduardo fernandez godoy1, patricia palma fluxá3, gustavo adolfo moncada cortés4, jose roberto cury saad2, javier martin casielles1 1universidad de chile, dental school, department of restorative dentistry, santiago, chile 2universidade estadual paulista – unesp, araraquara dental school, department of restorative dentistry, araraquara, sp, brazil 3universidad de chile, dental school, department medicine and oral pathology, area of microbiology, santiago, chile 4universidad mayor, dental school, area of cariology, santiago, chile correspondence to: patricio agustin vildósola grez department of restorative dentistry universidad de chile dental school santiago, chile phone: +56 02 29781742 fax +56 02 29781742 e-mail: patovildo@gmail.com abstract aim: to compare the number of colony forming units (cfu) and agar adherence of s. mutans on amalgam (am) and resin composite (rc) occlusal restorations. sixty-five healthy patients older than 18 years with high caries risk who had at least one occlusal am and rc restorations (n=130) were selected. methods: the restorations were evaluated according to the alpha ryge criteria (cohen-kappa 0.8). for each patient, a biofilm sample was taken using an impression tray technique with previously loaded with solid trypticase yeast extract cysteine sucrose with bacitracin agar placed over the am restorations and rc restorations in the same patients. the tray was placed inside an oven at 37 °c for 48 h, and the s. mutans count was then performed. data were analyzed with the test wilcoxon with a 95% confidence level. results: rc restorations had statistically significant higher number of cfu of s. mutans than am restorations (p<0.05). conclusions: in adult patients with high caries risk, rc occlusal surfaces showed greater agar adherence of s. mutans count than am restorations. keywords: streptococcus mutans; composite resins; dental amalgam. introduction dental caries represents one of the most common global diseases. although most industrialized countries have lowered their caries rates, it cannot be completely eradicated. therefore, new treatments are needed, as caries continues to be a significant burden in terms of both morbidity and economics1. among the factors involved in the complex process of caries disease are oral bacteria, which are immersed in a cariogenic biofilm in a balanced oral environment2. this microbiological balance can be modified by a higher intake of carbohydrates, thus increasing the acidogenic bacterial population that is responsible for the demineralization and destruction of tooth surfaces leading to carious lesions3. of the species present in cariogenic biofilm, streptococcus mutans is considered one of the main etiological agents of caries disease, particularly in the case of early disease3. a biofilm is formed immediately on all exposed surfaces in the oral environment4 making the biological interaction between restorative materials and braz j oral sci. 14(1):5-9 received for publication: november 18, 2014 accepted: january 29, 2015 the microorganisms. indeed, different surface restoration properties could directly influence the level of bacterial adhesion and aggregation5. this suggests that restorative materials should be selected according to a patient’s caries risk, which could plays a significant role in the longevity of restorations6-7. for many years, amalgam (am) has been the main restorative material in the posterior zone; however, resin composite (rc) restorations are also a popular filling material because of their good esthetics and adhesion properties8. nevertheless, despite these properties secondary caries is the major reason for the replacement of rc restorations9-10, as they have higher incidence of secondary caries than am restorations11. previous investigations have reported that rc restorations promote bacterial growth on their surface11-12, suggesting that material-specific factors may be involved13. in other way previous studies have reported that am inhibits bacterial adhesion because of its ability to release silver14 however, there is insufficient evidence that the release of these elements has a purely antibacterial effect15. for this reason, it will be interesting to determine the levels of s. mutans colonizing the surfaces of am and rc restorations in the oral environment. the objective of this study was to compare the colonization levels by assessing the colony forming unit (cfu) count and the agar adherence of s. mutans on occlusal surfaces in am and rc restoration s. the alternative hypothesis is that there are differences between the s. mutans cfu count between am and rc restorations. material and methods this research was approved as a dental school project at the universidad de chile, number pri-odo 11-02, by its ethics committee. each patient was informed in detail about the research and signed a written consent. because these clinical characteristic marginal adaptation anatomic form surface roughness secondary caries luster of restoration alpha explorer does not catch or has one way catch when drawn across the restoration/tooth interface the general contour of the restorations follows the contour of the tooth the surface of the restoration does not have any surface defects there is no clinical diagnosis of caries the restoration surface is shiny and has an enamel-like, translucent surface bravo explorer falls into crevice when drawn across the restoration/tooth interface the general contour of the restoration does not follow the contour of the tooth the surface of the restoration has minimal surface defects n/a the restoration surface is dull and somewhat opaque charlie dentin or base is exposed along the margin the restoration has an overhang the surface of the restoration has severe surface defects clinical diagnosis of caries at restoration margin the restoration surface is distinctly dull and opaque and is esthetically displeasing table 1. table 1. table 1. table 1. table 1. ryge criteria patients had a high cariogenic risk, they received preventive measures and a treatment plan according to their risk after study sampling the size of the sample was calculated with analyses a priori using the statistical program g*power©, version 3.1.3 (enrich-heine, universität düsseldorf, düsseldorf, germany), with confidence levels of 95% (α=0.05) and a statistical power of 80%. the size effect was medium (0.5). the analysis indicated that at least 65 patients were required to achieve significant results. the statistical unit was the patient. a total of 584 patients were examined at the operative dentistry clinic of the universidad de chile dental school, and 65 of these and restorations were randomly selected for inclusion in the study (total sample size of 130, with 2 samples from each patient, n=65) using ncss pass 2008, v08.0.15 (ncss statistical software co., kaysville, usa). the following inclusion and exclusion criteria were established: inclusion criteria: patients between 18 and 45 years with homonymous amalgam (original d, wyckle research in., carson city, nv, usa) and composite restorations (filtek supreme, 3m espe, st. paul, mn, usa), restorations must have an alpha value in five parameters, according to the united state public health service (usphs) clinical criteria for the evaluation of restorations16 (table 1). a calibrated clinician evaluated these values (cohen kappa 0.8). . . . . dental restorations placed within a 3-year period in the operative clinic of the universidad de chile dental school, , , , , maxillary and mandibular occlusal posterior restorations were included when they did not exceed one-third of the intercuspal distance, patients with high caries risk according to cariogram 2.01 software (mälmo university, mälmo, sweden). exclusion criteria: patients undergoing treatment with a mouthwash and/or antimicrobial gel, taking antibiotics at the time of the study or in the last 3 months, with fixed or removable prosthetics, fixed or removable orthodontics and any other acrylic device, taking immunosuppressive drugs, is there difference of streptococcus mutans count and adherence on amalgam and resin occlusal restorations? a blind clinical study braz j oral sci. 14(1):5-9 66666 77777 such as corticosteroids, classified as asa iii according to the american society of anesthesiologists, who chew gum at least four days per week, with a physical disability that precludes them from being responsible for their own hygiene. the patient selection procedures were designed by two operators. microbiological sample: prior to sampling the biofilm, trays were prepared to print the biofilm over the restorations17. for this application, we used disposable fluoride gel application trays (deepak products inc., miami, usa), each of which was sterilized in a biosafety hood (esco technologies inc., harboro, usa) under ultraviolet light for 30 minutes. each tray was then charged with 7.5 ml of tycsb agar (casein, yeast extract, l-cysteine, sucrose, bacitracin) (difco laboratories inc., detroit, mi, usa), a selective medium for s. mutans18. subsequently, to individualize the sample collection technique, the trays were cut to fit no more than 3 teeth. immediately after this, the trays were placed in sterile petri plates and stored in sealed plastic bags in the refrigerator for storage. before use, the trays were placed in an incubator/stove (zdp-a2080, labtech co., namyangiu, korea) for 24 h at 37 °c as a quality control measure. the third and fourth operators performed the sampling, which was conducted between 11:00-13:00 to allow for biofilm reorganization following morning brushing. the sampling was performed by gently pressing the tray for 20 s over the rc restoration followed by the homologous am restoration for each patient and then each tray was assigned a number by the third operator into the database. after the sample trays were stored in sterile petri plates, they were transported at 4 °c and then incubated at 37 °c in a microaerophilic (jar candle co2 10%) for 48 h. count of s. mutans: this procedure was performed by the fourth operator who was blinded to which tray was used to print the am or rc restorations. the operator was calibrated for the s. mutans count before this analysis (cohen kappa >0.8). after 48 h of incubation, macroscopic counts of colonies compatible with s. mutans morphology were performed, and the agar adherence of the colonies was observed under an optical microscope with a light source (schott kl 1500, carl zeiss microscopy, thornwood, usa). later, gram staining was performed to determine the micromorphology of the colonies. the s. mutans count was expressed in colony forming units (cfu) of s. mutans from the plates and trays with the tycsb agar. selected colonies that were compatible with s. mutans adhesion and morphological characteristics were suspended in todd-hewitt broth (difco laboratories inc., detroit, mi, usa) and incubated at 37 °c for 48 h. the colonies were then subjected to biochemical tests to identify the species of mutans streptococci and to distinguish s. mutans from s. sobrinus. the biochemical tests included the raffinose fermentation test, the melibiose fermentation and esculin hydrolysis tests. a positive result for all three tests indicated the presence of s. mutans19. after 48 h, each incubated broth sample was centrifuged (bd sero-fuge 2001, clay-adams becton dickinson and co., sparks, md, usa) for 5 min at approximately 1500 rpm to obtain a pellet. the pellet was resuspended in 450 µl of phosphate buffer (ph 7.2) to approximate a mcfarland 5 standard. then, 100 µl of this suspension was inoculated in each biochemical test such as esculin (brain heart infusion, 1% esculin. difco laboratories inc.), raffinose and melibiose (thioglycolate without dextrose and no indicator, 1% of raffinose, 1% of melibiose (difco, laboratories inc.) and incubated for 24 h at 37 °c. subsequently, two drops of ferric ammonium citrate (sigmaaldrich co., st. louis, mi, usa) were added to the melibiose and raffinose broths. analysis of results: statistical analysis was performed by the fifth operator was blinded. the data distribution was assessed using the kolmogorov-smirnov test. for the statistical analysis of the variables it used the wilcoxon test in the statistical package for social sciences (spss) software for windows, version 15.0 (spss inc., chicago, usa), with a significance level of 95%. results sixty-five patients with mean age of 30 years and 130 restorations were evaluated (n=130). the tests gave positive results for s. mutans fermented the raffinose, melibiose (both yellow coloration) and esculin (black coloration). based on the bacterial isolation from plaque samples collected through the tray technique, isolated colonies were obtained with macroscopic properties and adhesion characteristics that were indicative of s. mutans (figure 1 and 2) am restorations had a median of 2.00 cfu. in contrast, rc restorations had a median of 3.00 cfu (graph 1). the median total cfu the both samples in the same patient was 2.5 according to the kolmogorov-smirnov test (p<0.05), the distribution was not normal. the wilcoxon test showed that there were statistically significant differences between the am and rc restorations with p<0.05. fig. 1. s. mutans colonies on tycsb agar exhibit a whitish, rough surface and crystalline appearance. (observed under 4x magnification) is there difference of streptococcus mutans count and adherence on amalgam and resin occlusal restorations? a blind clinical study braz j oral sci. 14(1):5-9 fig. 2. bacterial isolation from surface restoration using tr. (a) colonies of s. mutans adhered to the agar. (b) the red line represents the outline of occlusal surface restoration. discussion when the s. mutans cfu of collected from am and rc restorations using the tray technique were compared, a significantly greater number was found in the rc group (p<0.05). the results of this study thus confirm our original hypothesis; the differences between the sample groups observed here coincide with similar clinical and in vitro studies of s. mutans on restorations, in which higher levels were generally found on rc restorations than on am restorations11,20. the am and rc samples were taken from the same patient to ensure that the biofilm samples deposited on the restorations were from the same oral ecosystem with the same ecological characteristics. furthermore, all samples were taken from restorations that were classified as alpha according to the modified ryge criteria with the same longevity (3 years). this means that the restorations were studied under similar conditions to homogenize the samples, which could indicate that the differences in the cfu of s. mutans on the restorations are mainly due to the characteristics of each material, rather than to ecosystem changes or differences in status, such as a retentive restoration sector that would favor the accumulation of plaque. this is relevant because it has been describe that the characteristics of restorative materials could influence the level of subsequent bacterial growth15. this finding has been associated with the physical and chemical characteristics of the restorative materials20. physical properties, such as the porosity of the restorative material, may be important at the nanometer scale21. indeed, preliminary studies have shown differences in the adhesion of biofilm to resins of different surface roughness22. this could help explaining the increase of bacterial cfu on the rc restorations, as these have a greater roughness at the nanometer scale than do polished am restorations with similar macroscopic characteristics, as assessed by the ryge criteria. furthermore, for these changes to occur on the surfaces of the restorations they must be exposed to the oral environment for a certain amount of time since an in situ study with short-term evaluation period had contradictory results to this study23. chemical surface properties have proven to be a relevant factor in bacterial colonization and adherence. the main influence is the chemical composition of the restoration’s surface, which may have components that benefit the microorganisms20. in this case, am restorations may have certain adhesion inhibition qualities, as they release antibacterial elements such as silver14. however, this alone is not sufficient to clearly show that am releases antibacterial elements15. furthermore, rc is not believed to possess any antibacterial activity and is considered to actually promote bacterial colonization and growth, as its co-monomers can actually stimulate cariogenic bacterial species12,24. there are several studies that describe and explain the stimulation of bacterial growth on resin composites due to monomer degradation, specifically s. mutans. this explains why the action of the esterase enzymes in human saliva causes the degradation of triethylene glycol dimethacrylate (tegdma) in triethylene glycol (teg), which is released in the oral environment and affects the expression of s. mutans glucosyltransferase and thus increases the bacterial synthesis of glucan25. however, there is not clear evidence for this mechanism because the degradation products are found in monomer-dependent concentrations and there is a lack of knowledge about what happens in vivo. in the oral cavity, this process might be directly affected by the biofilm, which is highly complex and heterogeneous, and there might be mechanisms that work to regulate bacterial cell functions in response to the oral environment 26. rc degradation is especially important in this context because it increases the surface roughness of the restoration, thereby promoting adhesion and stimulating the growth of s. mutans 24. additionally, rc restorations may have a greater level of microfiltration than am restorations26, which might also help explain the shorter life of rc restorations. several studies have described this finding in regards to the higher failure rate of rc restorations due to secondary caries, especially in high caries risk patients6,13. opdam6 has reported that the caries risk of a patient plays a significant role in the longevity of a restoration, for example a patient with high caries risk presumably has an oral environment that will negatively affect the restorations. as shown in our study, it might be necessary to assess the is there difference of streptococcus mutans count and adherence on amalgam and resin occlusal restorations? a blind clinical study braz j oral sci. 14(1):5-9 graph 1. distribution of s. mutans cfu on the am and rc restorations. 88888 selection of restorative materials in terms of their longevity in a high caries risk environment. however, in patients with low caries risk, the am and rc restorations have similar longevity, with both being in reportedly good condition 12 years after restoration placement7. within the limitations of the study is that currently caries is known to be a highly complex disease that depends not only on s. mutans, but also on a complex ecosystem of multiple bacteria in association with other factors within the oral environment2. this is the reason why it has been suggested to research others microorganism. it would also be interesting to follow the development of secondary caries among the patients included in this study and to make an accurate determination of the association between s. mutans or other microorganisms and caries risk. within the limitations of this study it can be concluded that in adult patients with high caries risk, rc occlusal surfaces showed greater agar adherence of s. mutans count than am restorations. this result suggests that, for these patients, it would be recommendable to indicate am restorations instead of rc in the posterior teeth, thus helping reducing the potential risk the secondary caries. reference 1. marcenes w, kassebaum nj, bernabé e, flaxman a, naghavi m, lopez a, et al. global burden of oral conditions in 1990-2010: a systematic analysis. j dent res. 2013; 92: 592-7. 2. takahashi n, nyvad b. the role of bacteria in the caries process: ecological perspectives. j dent res. 2011; 90: 294-303. 3. beighton d. the complex oral microflora of high-risk individuals and groups and its role in the caries process. community dent oral epidemiol. 2005; 33: 248-55. 4. moons p, michiels cw, aertsen a. bacterial interactions in biofilms. crit rev microbiol. 2009; 35: 157-68. 5. quirynen m. the clinical meaning of the surface roughness and the surface free energy of intra-oral hard substrata on the microbiology of the supraand subgingival plaque: results of in vitro and in vivo experiments. j dent. 1994; 22(suppl 1): s13-6. 6. opdam nj, bronkhorst em, loomans ba, huysmans mc. 12-year survival of composite vs. amalgam restorations. j dent res. 2010; 89: 1063-7. 7. simecek jw, diefenderfer ke, cohen me. an evaluation of replacement rates for posterior resin-based composite and amalgam restorations in u.s. navy and marine corps recruits. j am dent assoc. 2009; 140: 2009; quiz 49. 8. ferracane jl. resin composite—state of the art. dent mater. 2011; 27: 29-38. 9. opdam nj, van de sande fh, bronkhorst e, cenci ms, bottenberg p, pallesen u, et al. longevity of posterior composite restorations: a systematic review and meta-analysis. j dent res. 2014; 93: 943-9. 10. kopperud se, tveit ab, gaarden t, sandvik l, espelid i. longevity of posterior dental restorations and reasons for failure. eur j oral sci. 2012; 120: 539-48. 11. beyth n, bahir r, matalon s, domb aj, weiss ei. streptococcus mutans biofilm changes surface-topography of resin composites. dent mater. 2008; 24: 732-6. 12. leinfelder kf. do restorations made of amalgam outlast those made of resin-based composite? j am dent assoc. 2000; 131: 1186-7. 13. bernardo m, luis h, martin md, leroux bg, rue t, leitão j, et al. survival and reasons for failure of amalgam versus composite posterior restorations placed in a randomized clinical trial. j am dent assoc. 2007; 138: 775-83. 14. orstavik d. antibacterial properties of and element release from some dental amalgams. acta odontol scand. 1985; 43: 231-9. 15. busscher hj, rinastiti m, siswomihardjo w, van der mei hc. biofilm formation on dental restorative and implant materials. j dent res. 2010; 89: 657-65. 16. ryge g. clinical criteria. int dent j. 1980; 30: 347-58. 17. vildósola grez p, palma fluxa p, zuñiga saavedra p, fernandez godoy e, batista de oliverira jr o, moncada cortes g. recovering streptococcus mutans over restorations by the tray technique: a randomized clinical study. braz j oral sci. 2013; 12: 292-7. 18. van palenstein helderman wh, ijsseldijk m, huis in ‘t veld jh. a selective medium for the two major subgroups of the bacterium streptococcus mutans isolated from human dental plaque and saliva. arch oral biol. 1983; 28: 599-603. 19. coykendall al. classification and identification of the viridans streptococci. clin microbiol rev. 1989; 2: 315-28. 20. auschill tm, arweiler nb, brecx m, reich e, sculean a, netuschil l. the effect of dental restorative materials on dental biofilm. eur j oral sci. 2002; 110: 48-53. 21. mei l, busscher hj, van der mei hc, ren y. influence of surface roughness on streptococcal adhesion forces to composite resins. dent mater. 2011; 27: 770-8. 22. padovani g, fúcio s, ambrosano g, sinhoreti m, puppin-rontani r. in situ surface biodegradation of restorative materials. oper dent. 2014; 39: 349-60. 23. van de sande fh, opdam nj, truin gj, bronkhorst em, de soet jj, cenci ms, et al. the influence of different restorative materials on secondary caries development in situ. j dent. 2014; 42: 1171-7. 24. gregson ks, shih h, gregory rl. the impact of three strains of oral bacteria on the surface and mechanical properties of a dental resin material. clin oral investig. 2012; 16: 1095-103. 25. khalichi p, singh j, cvitkovitch dg, santerre jp. the influence of triethylene glycol derived from dental composite resins on the regulation of streptococcus mutans gene expression. biomaterials. 2009; 30: 452-9. 26. alptekin t, ozer f, unlu n, cobanoglu n, blatz mb. in vivo and in vitro evaluations of microleakage around class i amalgam and composite restorations. oper dent. 2010; 35: 641-8. is there difference of streptococcus mutans count and adherence on amalgam and resin occlusal restorations? a blind clinical study braz j oral sci. 14(1):5-9 99999 oral sciences n3 original article braz j oral sci. october | december 2013 volume 12, number 4 non-white people have a greater risk for maxillofacial trauma: findings from a 24-month retrospective study in brazil luciana domingues conceição1, rafael guerra lund1, gustavo giacomelli nascimento1, ricardo henrique alves da silva2, fábio renato manzolli leite3 1department of restorative dentistry, dental school, federal university of pelotas, pelotas, rs, brazil 2 forensic dentistry, ribeirão preto dental school, university of são paulo, ribeirão preto, sp, brazil 3department of semiology and clinics, dental school, federal university of pelotas, pelotas, rs, brazil correspondence to: fábio renato manzolli leite faculdade de odontologia, universidade federal de pelotas rua gonçalves chaves, 457, cep: 96015-560 centro, pelotas, rs, brasil phone +55 53 32256741 e-mail: leite.fabio@gmail.com abstract aim: to identify the predominant causes and types of maxillofacial trauma in brazil. methods: reports of corporal trauma (7,536) between 2009-2010 in the brazilian institute of forensic medicine were analyzed as to the presence of maxillofacial traumas. victims’ demographic and trauma characteristics were recorded. results: data were submitted to chi-square test and to multivariate poisson regression. 778 reports referred maxillofacial trauma. most victims were men (50.8%) around 27.6 years. main causes were physical aggression (88.1%) and traffic accidents (6.7%). the most affected extraoral area was the middle third (60.7%). risk for trauma in the middle third was significantly higher among patients aged 61-75 (rr 1.32), and non-white patients (black-skinned rr 1.21; brown-skinned rr 1.18); while falls were associated with trauma in the lower third (rr1.79). conclusions: violence was the main cause of maxillofacial trauma. prevention of interpersonal violence may be a key element to prevent maxillofacial trauma. keywords: epidemiology, violence, maxillofacial injuries. introduction the face is usually the first area to be damaged in case of physical aggression, car accidents and falls, which makes the maxillofacial region very susceptible to traumas due to its prominence1. traumas of the maxillofacial complex represent one of the most important health problems worldwide, especially because of the high incidence and the diversity of facial lesions2. moreover, the face represents the center of human attention and sometimes lesions may leave marks or unrepairable sequels that cause physical or psychological damages, burdening the country economy3. within the same country and among different countries the type of maxillofacial trauma is influenced by socio-economic status, cultural and environmental factors, and the period of investigation4. brazil presents the world’s fifth largest geographical area and population. especially after the 2007-2008 crises, the country has strengthened its status as an economic power, developing more employment opportunities, vehicle sales and social mobility, which may influence public policies. however, the economic growth is not being followed by reduction of social inequalities. dark-skinned received for publication: august 30, 2013 a c c e p t e d : november 28, 2013 braz j oral sci. 12(4):313-318 people are still the poorest, consequently, the ones who concentrate more social and health problems5. moreover, as they usually live far from the central urban areas, they are more exposed to violent episodes6. there are many studies worldwide evaluating traumas in the oral and maxillofacial region 1,3,7 and some of them conducted in brazil4,8. literature has identified different causes for traumas in developed and developing countries. there are controversies regarding the association of traffic accident and physical aggression with the country’s economic status3,7,9. authors also observed the high prevalence of falls and sports traumas in both developed and developing countries10. in brazil, the conducted studies presented some limitations: (a) evaluation of a specific population, like children11 or rural population;8 (b) evaluation of fractures and the required treatment only;12 (c) analysis of the dental traumas only13. although there are some minor studies on maxillofacial trauma in some parts of brazil, this is the first report analyzing data of oral and maxillofacial traumas in the last decade. the main purpose of this study is to evaluate the epidemiological characteristics of prevalence, cause and associated factors of maxillofacial traumas in southern brazil in 2009 and 2010. material and methods this retrospective and cross-sectional study was carried out on the records of consecutive patients with maxillofacial traumas who were referred to the brazilian institute of forensic medicine, pelotas, southern brazil, from january 2009 to december 2010 (n=7,536). the institute is a reference for 11 cities with a total of 600,000 inhabitants. from these records a selection was made according to the following inclusion criteria: (1) offense to the integrity and/ or health of the victim and (2) presence of maxillofacial traumas. maxillofacial lesions were grouped in the following extraoral regions: lower third (masseter, mandible and mentum regions), middle third (infraorbital, zygomatic and nasal regions) and oral (intraoral, lips and perioral soft tissues). oral lesions were defined as those involving the following areas: (a) teeth and surrounding supportive tissues (periodontium); (b) oral mucosa including gums, alveolar mucosa in edentulous patient, palate and mucosa; (c) jaw bones (upper and lower); (d) lips (mucosa and skin); (e) tongue; (f) perioral soft tissues (extraoral tissues that surround mouth and cover upper and lower jaw). this study followed the declaration of helsinki on medical protocol and was approved by the institutional review board of the federal university of pelotas, dental school (protocol 88/2009). the selected cases (n=648) were studied for data regarding the victim’s and offender’s demographic characteristics, nature and number of inflicted traumas and their consequences. personal information such as gender, race, age and marital status, and lesion’s characteristics (location, etiology, type) were recorded in an excel spreadsheet. in addition, anatomic location and nature of the trauma were identified to evaluate oral traumas. data were double typed and analyzed by stata 12.0 software (statacorp, college station, tx, usa). descriptive statistics was performed using frequency analysis for categorical variables and descriptive analysis for continuous variables. the statistical significance of the difference in the prevalence of oral and maxillofacial traumas according to gender, age group, skin color and cause of trauma was tested using the chi-square test. multivariate poisson regression analysis was conducted by using traumas in the different parts of face as the dichotomized dependent variable in order to test the association between the outcomes and the independent variables, adjusting it for potential confounders. for variable selection, the stepwise method with backward selection was used. variables with p<0.25 were included in the final model, estimated their risk ratio (rr) and set the interval confidence at 95% . results in this study, out of a total of 7,536 victims only 892 (11.8%) presented maxillofacial traumas. patients with missing data were excluded from the study, totalizing 648 (8.6%) patients presenting 785 traumas. the number of cases was similar in 2009 (n=306; 47.2%) and 2010 (n=342; 52.8%). the majority were men (50.1%), single (75.2%), most of them white (80.6%). the mean age was 27.6 years (sd=7.37), and victims aged between 16 to 30 years were the most affected (46.1%), followed by those from 31 to 45 years (24.0%). the specialized police station for women’s defense (209; 31.9%) referred most of the patients followed by the specialized police station for children and adolescent defense (124; 19.1%) and first assistance police station (120; 18.1%). most of the maxillofacial traumas were due to physical aggressions (563; 86.8%), traffic accidents (47; 7.2%) and falls (32; 4.9%). about the damage caused by lesions, 22 patients (3.3%) presented permanent and irreversible consequences, becoming unable for daily, social and working activities. traumas occurred on all regions of the face in different proportions, with the middle third concentrating most of the traumas (475, 73.3%), followed by the lower third (170, 26.2%) and by the oral region (140, 21.6%). table 1 describes the associations between the traumas and independent variables, which are listed according to the different regions of face. traumas occurred in single thirds or more than one third (figure 1). for dental traumas, tooth fracture was the most prevalent (12, 33.4%) followed by luxation (8, 22.2%) with respect to the intraoral soft tissue lesions, oral and gingival mucosa and tongue were the most affected sites (table 2). when the associations between the occurrence of oral and maxillofacial trauma and explanatory variables were considered simultaneously, in the multivariate regression model, (tables 3 and 4), the risk for trauma in the middle third was significantly higher among patients aged between 61-75 years (rr, 1.32; 95%ci, 1.07-1.62), and non-white patients (brown skinned, rr 1.21, 95% ci, 1.09-1.34; black skinned rr 1.18; 95%ci, 1.01-1.38). the risk for lower third trauma was significantly higher when falls were the main causes of trauma (rr 1.79; 95%ci, 1.18-2.70). non-white people have a greater risk for maxillofacial trauma: findings from a 24-month retrospective study in brazil314 braz j oral sci. 12(4):313-318 middle third (%) lower third (%) oral region (%) p value p value p value gender 0.71 0.54 0.30 male 236 (72.4) 90 (27.3) 77 (23.3) female 239 (74.2) 80 (25.1) 63 (19.8) age 0.44 0.48 0.94 0-15 74 (68.5) 28 (25.9) 24 (22.2) 16-30 225 (74.8) 79 (26.7) 66 (22.1) 31-45 110 (70.8) 47 (30.3) 33 (21.9) 46-60 46 (74.2) 13 (20.1) 14 (22.6) 61-75 16 (88.9) 2 (11.1) 2 (11.1) >75 4 (80.0) 1 (20.0) 1 (20.0) skin color 0.005 0.08 0.48 white 369 (70.5) 147 (28.1) 117 (22.3) brown 29 (82.8) 7 (20.0) 8 (22.9) black 77 (85.6) 16 (17.8) 15 (16.7) cause of trauma 0.85 <0.05 0.31 physical agression 416 (73.9) 139 (24.7) 122 (21.6) trafic accident 33 (70.2) 16 (34.0) 10 (21.3) firearm 4 (66.7) 1 (16.7) 3 (50.0) fall 22 (68.7) 14 (43.8) 5 (15.6) table 1.table 1.table 1.table 1.table 1. lesions distribution in the different regions of the face according to gender, age, skin color and cause of trauma (n=785) fig. 1. isolated and combined maxillofacial lesions distribution and number of victims. discussion information on health is important for planning, monitoring and management of collective and individual health interventions. in the last years, traumas to the maxillofacial region are becoming more common both in the urban and rural areas.1 changes in the global socioeconomic scenery are responsible for switches in the pattern of maxillofacial traumas etiologies. brazil has emerged as an economic power in the last years, resulting in higher employment and immigration rates, but on the other side growth was accompanied by social disparities. in this way, more traumas due to physical aggression are expected. this study shows for the first time that brazilian growth is reflecting in increased reports of trauma due to interpersonal violence. in the present study, the main cause for traumas in all three analyzed regions was physical aggression, followed by car accident and falls, as non-white people have a greater risk for maxillofacial trauma: findings from a 24-month retrospective study in brazil 315 braz j oral sci. 12(4):313-318 independent variables gender female male age (years) 0-15 16-30 31-45 46-60 61-75 >75 skin color white brown black cause of trauma physical agression trafic accident firearm fall rrb (ci 95%) 1.0 0.97 (0.88-1.07) 1.0 1.09 (0.94-1.26) 1.03 (0.88-1.21) 1.08 (0.89-1.31) 1.29 (1.05-1.59) 1.16 (0.73-1.84) 1.0 1.21 (1.09-1.34) 1.17 (1.01-1.37) 1.0 0.95 (0.78-1.15) 0.90 (0.51-1.59) 0.93 (0.73-1.18) p-value 0.61 0.24 <0.001 0.41 rra (ci 95%) 1.0 1.10 (0.95-1.27) 1.03 (0.87-1.21) 1.10 (0.90-1.33) 1.32 (1.07-1.62) 1.15 (0.74-1.78) 1.0 1.21 (1.09-1.34) 1.18 (1.01-1.38) p-value 0.021 <0.001 table 3.table 3.table 3.table 3.table 3. poisson regression crude (b) and adjusted (a) analyzes for occurrence of oral and maxillofacial trauma in the middle third of face. pelotas, brazil (n=75) n % of intraoral traumas % of total traumas soft tissue lesions tongue 3 8.3 0.5 buccal mucosa 2 5.6 0.3 gingival mucosa 4 11.1 0.6 mouthfloor mucosa 0 0 0 palate 0 0 0 dental tissue lesions tooth fracture 12 33.4 1.9 tooth subluxation 0 0 0 tooth luxation 8 22.2 1.2 tooth avulsion 7 19.4 1.1 total 36 100 5.6 table 2.table 2.table 2.table 2.table 2. distribution of intraoral trauma (n = 36) seen in many urban centers in germany14 and the united states15. the explanation for the increase if interpersonal violence is higher alcohol consumption, drug abuse and social disparities due to unequal wealth distribution1,16,17. it was found that skin color, a marker of social inequality, 5,6 represents a risk factor for facial traumas, since black and brown victims tended to have more lesions in the middle third of the face. it is important to emphasize that no previously published paper has reported a social marker as a risk factor for oral and maxillofacial traumas. according to minayo18 (1990), non-white people are the most vulnerable to violence in urban areas with low quality of life, since they live along with violence on a daily basis. as seen in other reports, non-white people at greatest risk of being victims of violence are men, young, single and belonging to low-income families.6 on the other hand, the stiffening of road traffic laws and safety norms such as obligatory use of seat belts, air bags, helmet wearing for motorized two-wheelers and speed surveillance reduced maxillofacial traumas due to traffic accidents16,17,19. according to the age groups, maxillofacial traumas were more frequent in people between 16 and 30 years followed by ages between 31 to 45 years which concurs with previous studies1,17,20-22. people in these age groups have more social interaction than other age groups, with higher alcohol and other drugs consumption1,20. despite of it, an increase of oral and maxillofacial traumas in the elderly is being observed. according to al-khateeb and abdullah9 (2007) this fact is due to an increase in average life expectancy, a more active lifestyle and higher percentage of elderly people in the population. the main cause of traumas in the elderly population was due to falls, which has been related to reduced physical agility, presence of systemic pathologies and use of psychotropic medications. our data corroborate other studies non-white people have a greater risk for maxillofacial trauma: findings from a 24-month retrospective study in brazil316 braz j oral sci. 12(4):313-318 independent variables gender female male age (years) 0-15 16-30 31-45 46-60 61-75 >75 skin color white brown black cause of trauma physical agression trafic accident firearm fall rrb (ci 95%) 1.0 1.08 (0.83-1.40) 1.0 1.02 (0.70-1.48) 1.16 (0.78-1.74) 0.80 (0.45-1.44) 0.42 (0.11-1.64) 0.77 (0.13-4.58) 1.0 0.63 (0.39-1.00) 0.71 (0.36-1.40) 1.0 1.37 (0.90-2.10) 0.67 (0.11-4.06) 1.77 (1.16-2.70) p-value 0.54 0.38 0.07 0.02 rra (ci 95%) 1.0 0.61 (0.38-0.98) 0.72 (0.36-1.42) 1.0 1.39 (0.91-2.12) 0.66 (0.11-3.94) 1.79 (1.18-2.70) p-value 0.16 0.02 table 4.table 4.table 4.table 4.table 4. poisson regression crude (b) and adjusted (a) analyzes for occurrence of oral and maxillofacial trauma in the lower third of face. pelotas, brazil (n=170) where the middle third facial area is more affected in elderly victims, with special regards to the orbital-zygomatic region23,24. another finding was a greater risk for trauma on the lower third associated with falls after adjustment in the final regression model. this fact may be explained by the chin prominence trauma when the victims fall. in addition, iida et al.7,14 (2001, 2003) reported that fall is usually observed as a chin impact leading to condyle fracture, and in less cases, multiple fractures when the impact occurs in the lateral sides of mandible. in contrast to other studies that reported mandible as the most commonly affected site,22,25,26 in the results of this study middle third was more affected (73.3%) than the lower third (26.2%), which agrees with the studies conducted by gandhi et al1 (2011). among the lower third maxillofacial lesions, dentoalveolar traumas presented a low prevalence (3.6%) concurring with previous studies27-30. in this report, crown fracture was the most common (1.9%) followed by luxation (1.2%). as expected, most of these lesions correlate with lowimpact traumas due to interpersonal violence that are usually observed as soft tissue abrasion, hematoma, and dentoalveolar fractures8. it is supposed that the importance, number and severity of the perioral and intraoral lesions would change with the presence of a forensic dentist at the institutes of forensic medicine and their prevalence would increase. an example of specific professional care that has increased the number of notified lesions was the creation of specialized police stations for women’s defense in brazil. these units stimulated the notification of aggression against women and reduced the male-to-female ratio of reported traumas to 1.03:1. some countries have ratios of up to 8:1, but recent studies show a trend towards an equal male-tofemale ratio17,22. the increase in the number of women presenting maxillofacial traumas was attributed to an increase in the women’s working force and many of them working outdoors in more high-risk occupations, thus becoming more exposed to traumas9. in the last decade, changes in global economy reflected in different aspects of the worldwide development. new economies are emerging with consequences to their population. 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[the prevalence of falls and associated factors among the elderly according to ethnicity]. cien saude colet. 2012; 17: 2181-90. non-white people have a greater risk for maxillofacial trauma: findings from a 24-month retrospective study in brazil 317 braz j oral sci. 12(4):313-318 6. soares filho am. homicide victimization according to racial characteristics in brazil. rev saude publica. 2011; 45: 745-455. 7. iida s, kogo m, sugiura t, mima t, matsuya t. retrospective analysis of 1,502 patients with facial fractures. int j oral maxillofac surg. 2001; 30: 286-90. 8. batista am, marques ls, batista ae, falci sg, ramos-jorge ml. urbanrural differences in oral and maxillofacial trauma. braz oral res. 2012; 26: 132-8. 9. al-khateeb t, abdullah fm. craniomaxillofacial injuries in the united arab emirates: a retrospective study. j oral maxillofac surg. 2007; 65: 1094101. 10. van den bergh b, karagozoglu kh, heymans mw, forouzanfar t. aetiology and incidence of maxillofacial trauma in amsterdam: a retrospective analysis of 579 patients. j craniomaxillofac surg. 2012; 40: e165-9. 11. munante-cardenas jl, olate s, asprino l, de albergaria barbosa jr, de moraes m, moreira rw. pattern and treatment of facial trauma in pediatric and adolescent patients. j craniofac surg. 2011; 22: 1251-5. 12. martini mz, takahashi a, de oliveira neto hg, de carvalho junior jp, curcio r, shinohara eh. epidemiology of mandibular fractures treated in a brazilian level i trauma public hospital in the city of são paulo, brazil. braz dent j. 2006; 17: 243-8. 13. gulinelli jl, saito ctmh, garcia-júnior ir, panzarini sr, poi wr, sonoda ck, et al. occurrence of tooth injuries in patients treated in hospital environment in the region of aracatuba, brazil during a 6-year period. dent traumatol. 2008; 24: 640-4. 14. lida s, hassfeld s, reuther t, schweigert hans-gert, haag c, klein j, et al. maxillofacial fractures resulting from falls. j craniomaxillofac surg. 2003; 31: 278-83. 15. laski r, ziccardi vb, broder hl, janal m. facial trauma: a recurrent disease? the potential role of disease prevention. j oral maxillofac surg. 2004; 62: 685-8. 16. bacchieri g, barros aj. traffic accidents in brazil from 1998 to 2010: many changes and few effects. rev saude publica. 2011; 45: 949-63. 17. van beek gj, merkx ca. changes in the pattern of fractures of the maxillofacial skeleton. int j oral maxillofac surg. 1999; 28: 424-8. 18. minayo mcs. violence in adolescence: a public health problem. cad saude publica. 1990; 6: 278-92. 19. de matos fp, arnez mf, sverzut ce, trivellato ae. a retrospective study of mandibular fracture in a 40-month period. int j oral maxillofac surg. 2010; 39: 10-5. 20. lee jh, cho bk, park wj. a 4-year retrospective study of facial fractures on jeju, korea. j craniomaxillofac surg. 2010; 38: 192-6. 21. cheema sa, amin f. incidence and causes of maxillofacial skeletal injuries at the mayo hospital in lahore, pakistan. br j oral maxillofac surg. 2006; 44: 232-4. 22. bakardjiev a, pechalova p. maxillofacial fractures in southern bulgaria a retrospective study of 1,706 cases. j craniomaxillofac surg. 2007; 35: 147-50. 23. gerbino g, roccia f, de gioanni pp, berrone s. maxillofacial trauma in the elderly. j oral maxillofac surg. 1999; 57: 777-82; discussion 82-3. 24. falcone pa, haedicke gj, brooks g, sullivan pk. maxillofacial fractures in the elderly: a comparative study. plast reconstr surg. 1990; 86: 443-8. 25. kieser j, stephenson s, liston pn, tong dc, langley jd. serious facial fractures in new zealand from 1979 to 1998. int j oral maxillofac surg. 2002; 31: 206-9. 26. erol b, tanrikulu r, gorgun b. maxillofacial fractures. analysis of demographic distribution and treatment in 2,901 patients (25-year experience). j craniomaxillofac surg. 2004; 32: 308-13. 27. motamedi mh, sagafinia m, famouri-hosseinizadeh m. oral and maxillofacial injuries in civilians during training at military garrisons: prevalence and causes. oral surg oral med oral pathol oral radiol. 2012; 114: 49-51. 28. castro jc, poi wr, manfrin tm, zina lg. analysis of the crown fractures and crown-root fractures due to dental trauma assisted by the integrated clinic from 1992 to 2002. dent traumatol. 2005; 21: 121-6. 29. santos se, marchiori ec, soares aj, asprino l, de souza filho fj, de moraes m, et al. a 9-year retrospective study of dental trauma in piracicaba and neighboring regions in the state of são paulo, brazil. j oral maxillofac surg. 2010; 68: 1826-32. 30. caldas im, magalhães t, afonso a, matos e. the consequences of orofacial trauma resulting from violence: a study in porto dent traumatol. 2010; 26: 484-9. non-white people have a greater risk for maxillofacial trauma: findings from a 24-month retrospective study in brazil318 braz j oral sci. 12(4):313-318 429 too many requests error 429 too many requests too many requests guru meditation: xid: 38088356 varnish cache server oral sciences n3 original article braz j oral sci. april | june 2015 volume 14, number 2 2d and 3d imaging of the relationship between maxillary sinus and posterior teeth gina delia roque-torres1, laura ricardina ramirez-sotelo1, solange maria de almeida1, gláucia maria bovi ambrosano2, frab norberto bóscolo1 1universidade estadual de campinas unicamp, piracicaba dental school, department of oral diagnosis, piracicaba, sp, brazil 2universidade estadual de campinas unicamp, piracicaba dental school, department of community and preventive dentistry, area of biostatistics, piracicaba, sp, brazil correspondence to: gina delia roque-torres universidade estadual de campinas faculdade de odontologia de piracicaba avenida limeira, 901 bairro areão cep: 13414-903 piracicaba sp brazil phone +55 19 21065327 fax: +55 19 3421 0144 e-mail: ginart87@gmail.com abstract aim: to compare the qualitative and quantitative information yielded by imaging modalities on the evaluation of the relationship between the maxillary sinus and the apices of posterior teeth. methods: three oral radiologists examined 109 panoramic radiographs and cone-beam computed tomography (cbct) images obtained from a digital archive. a total of 1,875 apices were classified according to their topographic relationship in both imaging modalities. agreements between the two imaging techniques were examined statistically. the evaluation was repeated in 25% of the sample to statistically evaluate the intraobserver agreement, with a 30-day interval. results: the values found in the kappa test for qualitative assessment and the intraclass correlation coefficient (icc) for quantitative assessment showed nearly perfect and excellent correlation, respectively. regarding the correlation between the two images, the kappa test in the quality assessment showed a slight correlation between the palatal roots of the right first molars and second molars. moreover, the quantitative evaluation by icc showed poor agreement for the palatal roots of the left first molar and second molar, as well as for the buccomesial of the right second molars and for the buccodistal of the left second molars. conclusions: there is low correlation between panoramic radiography and cbct when roots are in contact or above the maxillary sinus floor. a high level of agreement was observed when roots are below the maxillary sinus floor. keywords: maxillary sinus; tooth apex; cone-beam computed tomography; radiography, panoramic. introduction the inferior sinus wall is a curved structure that is extended between adjacent teeth, or individual roots in about half of the population, creating elevations in the antral surface or protrusions of the root apices into the sinus cavity; its floor is formed by the alveolar process of the maxilla1. in cases where the roots of the posterior maxillary teeth may project into the maxillary sinus, the thickness of the sinus floor is markedly reduced or absent2. information concerning the relationship between the root apices and the inferior wall of the maxillary sinus is crucial in diagnosing and treating sinus pathosis, periodontal or periapical infection of upper premolars and molars, which can spread to the maxillary sinus causing sinusitis 1,3. these anatomical relationships can decide the spread of buccal cellulites4-5. braz j oral sci. 14(2)141-148 received for publication: april 23, 2015 accepted: june 09, 2015 http://dx.doi.org/10.1590/1677-3225v14n2a09 142142142142142 also in the diagnosis and treatment plan for a dental implant6 a positive correlation between the length of the projection roots on the maxillary sinus in panoramic radiographs and the amount of pneumatization after extraction was found. sinus expansion after extraction can greatly decrease bone height available for implant placement2. furthermore, extraction of these same teeth may result in oroantral fistula or root displacement into the sinus cavity, which are usual complications 7. likewise, endodontic procedures and therapies can cause an endo-antral syndrome68, the spread of pulpal disease beyond supporting tissues into the maxillary sinus causing acute or chronic sinusitis1,5,8. finally, it affects orthodontic tooth movements, especially in movements of intrusion or bodily movement of teeth across the sinus floor9-10. panoramic radiograph are frequently used to evaluate teeth and patterns of the craniofacial skeleton, serving as a guide for their diagnosis and planning11. furthermore, some ghost images are formed on the opposite site of the object, mesiodistal and vertical enlargement, adding a limitation to evaluating the anatomic correlation between tooth roots and alveolar bone when using only a panoramic radiography12. in addition, clinical indications involving the roots of teeth are mostly endodontic or related and require single dental x-ray as the best imaging modality with minimal radiation dose. most drawbacks of single dental x-ray are similar to those of panoramic radiography. cone beam computed tomography (cbct) would potentially provide information needed for prosthetic treatment planning, implant selection, and/or surgical placement13. cbct examinations are also reliable for linear measurement 14. furthermore, despite the difference in radiation between cbct and 2d images, it has been demonstrated that decreasing the field of view (fov) helps reducing radiation dose, resulting in 2.7 to 23 msv for a panoramic and 34-89 sv for the cbct13. both modalities have less radiation than computed tomography (ct)15. while some authors16-17 have investigated the relationship between the roots of maxillary teeth and the maxillary sinus floor by ct, others11-12 have compared ct and panoramic radiograph imaging. recent studies 18-21 evaluate this relationship using cbct, and perform classification or measurements without establishing a comparison to the twodimensional technique. hassan (2010) 22 investigated the reliability of both periapical radiographs and panoramic radiography for exact detection of tooth root protrusion in the maxillary sinus by correlating the results with cbct. ok et al. (2014)23 evaluated the relationship between each root of maxillary premolars and molars and the maxillary sinus floor. according to shahbazian et al. (2014)3, cbct provides more information regarding these topographic relationships for maxillary posterior teeth than panoramic radiography. the present study was performed to understand the relationship between the maxillary sinus floor and the apices of maxillary posterior teeth by comparing the qualitative and quantitative information provided by two imaging methods: panoramic radiography and cbct. material and methods one hundred and nine dental records of subjects selected from the digital archive of oral radiology center of piracicaba dental school (unicamp, brazil) were selected after approval of the protocol by the local ethics committee for research on human subjects (protocol #059/2011). the selected subjects comprised 78 women and 31 men, with a mean age of 22 years (range: from 18-30), regardless of ethnic group, social class or other socioeconomic characteristics. from the dental record of each subject, a pair of panoramic radiograph and cbct (dated within 1 month) was selected. each pair depicted the root apices of the maxillary posterior teeth and the sinus floor. the digital panoramic radiographs were all produced by the same orthopantomograph machine dop 100 (instrumentarium corp, imaging division, tuusula, finland), by using settings of 2 mas, 57-60kvp, with time exposure of 17.6 seconds and were stored digitally. all cbct scans were produced by the classic i-cat unit (imaging sciences international, hatfield, pa, usa), using settings of 8 ma, 120 kv, 40 second of acquisition time, voxel size of 0.3 mm and 23 x 17 cm field of view. it was acknowledged that the images had another purpose in the time they were taken. the digital panoramic radiographs were imported to be evaluated in the software radioimp radiomemory (belo horizonte, mg, brazil), and the files of dicoms of tomographic images were imported into ez3d software (korea). images were selected considering a high-level technical standard (i.e. appropriate sharpness, density and contrast), clearly showing the maxillary posterior teeth apices and the sinuses floor. the inclusion criteria were subject to the following: complete permanent dentition, no evidence of supernumerary teeth, no presence of some sort of pathology or radiographic evidence of teeth with marked disruption in the apical third of the root. each root of the first and second premolars and the first and second molars was used in the qualitative and quantitative evaluations. an assessment of the topographic relationship of each root to the maxillary sinus floor was conducted in both panoramic radiograph and in cbct images by three oral radiologists who acted as evaluators of the research for at least 3 years. under dim light conditions, they blindly evaluated the images as described by sharan and madjar (2006)11, using the following scoring system: 0, the apices of the root is not in contact with the cortical borders of the sinus; 1, the apices of the root is in contact with the cortical borders of the sinus; 2, the root is projected laterally to the sinus cavity, and an apices is in contact with the cortical borders sinus; 3, the apices is projecting in the sinus cavity, and; 4, the maxillary sinus has a buckle that goes round the root of the tooth, but its apices is just in contact with the cortical borders sinus. all cases in which the qualitative assessment of the root was scored 0 were given a positive number, those with 1, 2, 4 were numbered as 0 mm, and those with qualitative assessment 3 were given a negative number as a means of quantitatively assessing 2d and 3d imaging of the relationship between maxillary sinus and posterior teeth braz j oral sci. 14(2)141-148 143143143143143 the length of the apical part of the root superior to the sinus inferior wall (figures 1-3). in the panoramic radiograph, this quantitative assessment represented the radiographic projection of the root in the sinus cavity, and in the cbct, it represented the protrusion of the root into the sinus (figures 2-3). the quantitative assessment was made from the root apices to the superior part of the cortical inferior wall of the sinus along the longitudinal axis of the root (figures 1-2) in both images. the oral radiologists were allowed to adjust brightness, contrast and magnification for better observation of anatomic structures in all the views, and choose just one view to make the measurement and scale in the case of cbct. for cases in which the teeth had fused roots, the evaluators gave the same measures and scale for all the roots. thirty days after the first evaluation, 25% of the samples were reevaluated in order to assess intraobserver agreement. through the correlation between panoramic radiography and cbct, the mode for qualitative assessments and the average for quantitative obtained by the three observers in both imaging modalities, for all dental roots was calculated. to calculate statistical differences between the two types of images, based on both qualitative and quantitative assessment, kappa test and intraclass correlation coefficient (icc) were performed, respectively, with a significance level of 5%, using the statistical package sas ® (statistical analysis software institute inc. cary, nc, usa) and spss® version 9.2 (statistical package for the social sciences) version 20.0, respectively. fig. 1. qualitative assessment of the 5 scales of the maxillary posterior teeth roots in relation to the inferior wall of the sinus. a: schematic drawing of the images (sagittal and coronal view). b: illustrations on the panoramic images. c: illustrations on the cbct (sagittal and coronal view). results the values obtained for the intraand inter-observer assessment were good to excellent for kappa and excellent for icc, considering the evaluations for qualitative and quantitative assessments, respectably, for both images. comparison of the qualitative assessment of maxillary tooth roots in relationship to the sinus ûoor in panoramic radiography and cbct images the level of agreement shown for the qualitative assessment according to the kappa test of all roots of the right and left side, with a confidence interval of 95%, was as following: slight agreement (0 0.20) for the palatal roots of the right first molars and second molar; fair (0.21 0.40) for the buccal roots of the second premolars, buccal and palatal the left first premolars, palatal root of the left first molar, buccomesial and buccodistal of the right first molars and second molars; moderate (0.41 0.60) for the buccal root of the right first premolar, buccomesial and bucco distal of the left first molar; substantial (0.60 0.80) for the palatal root of the right first premolars (figure 1, table 1). regarding the quantitative assessment of the relationship between the maxillary tooth roots and the sinus ûoor in panoramic radiography and cbct images, there was poor correlation (icc <0.4) according to the icc for the palatal root of the left first molars, buccomesial and palatal of the right second molars, buccodistal and palatal of the left second molar; satisfactory correlation (0.4 d” icc <0, 75) for the buccal root of the first and second premolars on the right, 2d and 3d imaging of the relationship between maxillary sinus and posterior teeth braz j oral sci. 14(2)141-148 144144144144144 fig. 2. quantitative assessment of the panoramic radiograph images. table 1. table 1. table 1. table 1. table 1. level of agreement between both images modalities by kappa test of all roots, with a confidence interval of 95% (qualitative assessment). k-kappa coefficient, ci-confidence interval, m-molar, pm-premolar, bm-buccomesial, bd-buccodistal, p-palatal, b-buccal. *most of the 2nd pm had only a single root. fig. 3. quantitative assessment of the cbct images. a: sagittal view. b: coronal view. 2d and 3d imaging of the relationship between maxillary sinus and posterior teeth braz j oral sci. 14(2)141-148 table 2. table 2. table 2. table 2. table 2. level of agreement between both images modalities according to the intraclass correlation coefficient (quantitative assessment). icc-intraclass correlation coefficient, ci-confidence interval, m-molar, pm-premolar, bm-buccomesial, bd-buccodistal, p-palatal, b-buccal. table 3.table 3.table 3.table 3.table 3. mean, standard deviation, minimum and maximum values from the quantitative assessment in both images modalities. std dev.-standard deviation, maxmaximum values, minminimum values, m-molar; pm-premolar, cbct-cone beam computed tomography, pan-panoramic radiography, bm-buccomesial, bd-buccodistal, p-palatal, b-buccal. buccal and palatal of the left first premolars, buccodistal for the right second molar, buccomesial for the left second molar, buccomesial and buccodistal for the left first molar and for all the roots of the right first molars; and excellent correlation (icc e” 0.75) for the palatal roots of the first and second premolars on the right and all the roots to the left second premolar (figures 2-3, table 2) as much as 819 (43.7%) out of 1,875 roots seemed to penetrate the maxillary sinus on the panoramic radiographs, but this number dropped to 80 (3.1%) in cbct, which indicates that cbct and panoramic radiographs had a poor correlation when roots were in contact or within the maxillary sinus floor (table 3). based on the average of the quantitative and qualitative assessments of all roots comparing both imaging modalities, it was observed that the buccomesial and buccodistal roots of the second molars were found to be 145145145145145 2d and 3d imaging of the relationship between maxillary sinus and posterior teeth braz j oral sci. 14(2)141-148 146146146146146 table 4. table 4. table 4. table 4. table 4. incidence and classifications of the vertical relationship between the inferior wall of the maxillary sinus and the roots of the maxillary teeth comparing cbct and panoramic radiography images (aualitative assessment). cbct-cone beam computed tomography, m-molar; pm-premolar, bm-buccomesial, bd-buccodistal, p-palatal, b-buccal. closest to the sinus maxillary, whereas the buccal and palatal roots of the first premolars were found to be farthest from the sinus maxillary, by the cbct. however, when comparing the panoramic radiography with cbct, they differed greatly in the palatal roots of the second and first molars, which are teeth closer to the sinus cavity (table 4). the sample comprised 78 women and 31 men. however, considering gender, there was no significant correlation in the comparison of the qualitative and quantitative assessment of the relationship between maxillary tooth roots and the sinus ûoor in panoramic radiography and cbct images. discussion this goal of this study was to compare the qualitative and quantitative information provided by panoramic radiography to cbct in order to assess the relation between maxillary sinus and the apices of the maxillary posterior teeth with or without some kind of pathology. in the literature, other authors used both imaging modalities to evaluate this topographic relationship3, but they reported some difficulty in evaluating this relationship in the presence of apical periodontitis perforating the sinus floor. others22 investigated the reliability of periapical radiographs and orthopantomograms for exact detection of tooth root protrusion in the maxillary sinus by correlating the results with cbct, obtaining a single score for each tooth using each imaging technique. in our study we included sound teeth in order to eliminate drawbacks when evaluating the sinus cortical. also, we assessed each root for each tooth given a single score whether the teeth were single or multirooted as in one recent23. several studies have assessed the relationship between maxillary posterior teeth and the maxillary sinus3,11-12,16-24. in some of those studies, only one observer was used11-12,16,18-21, in others two observers were used7,17,23-24 and four observers were used in one study22. however only two of those studies3,22, however, carried out intra-and inter-observer analysis. according to the majority of studies of roots with projection in the sinus cavities in the panoramic radiographs, only 39-57% showed protrusion into the maxillary sinus on ct scanning. this trend was reported in studies that reported that 2 of 38 subjects (5%) had roots with protrusion into the sinus cavity by ct16. bouquet et al. (2004)12 clearly indicated 2d and 3d imaging of the relationship between maxillary sinus and posterior teeth braz j oral sci. 14(2)141-148 147147147147147 that in certain cases the ct allowed the invalidation of the direct relation between the root to the maxillary sinus. however, 30 panoramic radiographs showed sinus projection on the tooth roots, while the ct showed that only 7 out of 30 cases had this relationship. the other 23 cases had the maxillary sinus far away from the roots of the teeth. one author11 noted that projections of roots into the sinus cavity in panoramic radiograph occurred on 39% of the cases on the average when compared to projections using ct, and the protrusion length is much shorter in ct than in those shown in the panoramic radiographs. in the present study, we also found that 819 (43.7%) out of 1,875 roots seemed to penetrate the maxillary sinus on panoramic radiographs, but only 80 (3.1%) showed penetration of roots in cbct. furthermore, to the best of our knowledge, only one study23 examined each root of all teeth, as we did in the present study23. this closeness between the apices and maxillary sinus floor, which was found to be shorter in molars than in premolars, shows a concordance with the topographic anatomy, in which the anatomical sinus location itself is already a factor to be considered23. other studies showed results for roots assessment just by cbct without comparing with 2d images 19,21, with one only providing assessment for the first and second molar19. studies have shown that the accuracy of the cbct imaging for bone measurement around the apices of the posterior teeth is valid and provide great advantage in obtaining data on a non-invasive form, revealing high predisposition to cbct of 0.4 mm with a standard deviation of 1.1 mm when compared with dissections24. yoshimine et al. (2012)20 recommended the use of cbct because in their study it reliably demonstrates the 3d status of the morphological characteristics of the alveolar bone at the anticipated site of implant insertion. we agree with this conclusion especially in teeth that show root protrusion into the maxillary sinus in 2d images. furthermore, the apices of the first premolar were found beyond the maxillary sinus floor on both sides (mean: 6.54 mm), as reported by some authors16,18,20, average of 7.05 mm, 7.5 mm and 1.13 mm, respectively. moreover, the apices of buccomesial roots of the second molars were found to be below the maxillary sinus floor (mean: -0.21) agreeing with eberhardt et al. (1992)16 and yoshimine et al. (2012)20, who found an average of 1.97 mm and 0.82 mm, respectively. these results are in disagreement with a previous study18 that found that the buccodistal root is the closest to the maxillary sinus floor. ok et al. (2014)23 concluded the maxillary first premolars have no relationship with the maxillary sinus floor, but the maxillary second molars are closer to the sinus floor. even though the panoramic radiograph showed high concordance when the roots were below the maxillary sinus floor, there was a percentage of roots that was on the limit or beyond the maxillary sinus floor, approximately 40%. these results corroborate with those of another study18, according to which when there is a percentage of 36% of teeth that are on the limit or beyond the maxillary sinus floor, cbct should be used as the imaging modality suitable for such evaluations. in addition, jung et al. (2012)19 reported a percentage of 41.2 of first and second molars on the limit or beyond the maxillary sinus floor. panoramic and periapical radiographs have the disadvantage of producing two-dimensional images of threedimensional structures. cbct, on the other hand, is not the chosen method to evaluate individual teeth, but it a highly accurate imaging method in the evaluation of the apex-tooth relationship, especially in cases where the root has protrusion length in the maxillary sinus on radiographs. this was shown in our study, in which the observers found a high percentage of protruded roots in the maxillary sinus on panoramic radiography, and when evaluating the same roots using cbct observed only contact with the maxillary sinus floor. it is also worth mentioning that the study sample was formed recruiting random samples of subjects visiting the oral radiology center of piracicaba dental school, unicamp, brazil, while needing imaging has certain a convenience sample. this can be a problem when we analyzing a significant correlation between genders because of the difference of the number between males and females. however, this accounts for all consecutive patient studies, either clinical or radiological, with the latter unacceptable from an ethical point of view for exposing an external population to ionizing radiation. a limitation in this study is the lack of analysis of histological samples. since the oral radiologists evaluating the cbct images were free to deal with the software and there were no standardized tests in reading the cbct, they simply chose the window with better visualization of the apices in relation to the maxillary sinus floor. it may be concluded that panoramic images enabled a high correlation when compared with cbct in cases when the roots are not in contact with the maxillary sinus floor, and poor correlation when roots were in contact or within the maxillary sinus floor, underestimating values when the roots were projected in the maxillary sinus. for the panoramic radiography and cbct, the same result was obtained for both right and the left sides, considering the dental groups, except for the first molars. the present study also found the first premolar root tip to be the farthest, and the second molar buccomesial root tip to be closest to the sinus floor on both right and left sides. acknowledgements we are grateful to capes for financial support on this research. the authors deny any conflict of interest. references 1. hauman ch, chandler np, tong dc. endodontic implications of the maxillary sinus: a review. int endod j. 2002; 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98: 342-7. 13. tyndall da, price jb, tetradis s, ganz sd, hildebolt c, scarfe wc; american academy of oral and maxillofacial radiology. position statement of the american academy of oral and maxillofacial radiology on selection criteria for the use of radiology in dental implantology with emphasis on cone beam computed tomography. oral surg oral med oral pathol oral radiol. 2012; 113: 817-26. 14. scarfe wc, farman ag, sukovic p. clinical applications of cone-beam computed tomography in dental practice. j can dent assoc. 2006; 72: 75-80. 15. liang x, jacobs r, hassan b, li l, pauwels r, corpas l, et al. a comparative evaluation of cone beam computed tomography (cbct) and multi-slice ct (msct) part i. on subjective image quality. eur j radiol. 2010; 75: 265-9 16. eberhardt ja, torabinejad m, christiansen el. a computed tomographic study of the distances between the maxillary sinus floor and the apices of the maxillary posterior teeth. oral surg oral med oral pathol. 1992; 73: 345-6. 17. ariji y, obayashi n, goto m, izumi m, naitoh m, kurita k, et al. roots of the maxillary first and second molars in horizontal relation to alveolar cortical plates and maxillary sinus: computed tomography assessment for infection spread. clin oral investig. 2006; 10: 35-41. 18. kilic c, kamburoglu k, yuksel sp, ozen t. an assessment of the relationship between the maxillary sinus floor and the maxillary posterior teeth root tips using dental cone-beam computerized tomography. eur j dent. 2010; 4: 462-7. 19. jung yh, cho bh. assessment of the relationship between the maxillary molars and adjacent structures using cone beam computed tomography. imaging sci dent. 2012; 42: 219-24. 20. yoshimine s, nishihara k, nozoe e, yoshimine m, nakamura n. topographic analysis of maxillary premolars and molars and maxillary sinus using cone beam computed tomography. implant dent. 2012; 21: 528-35. 21. pagin o, centurion bs, rubira-bullen ir, alvares capelozza al. maxillary sinus and posterior teeth: accessing close relationship by conebeam computed tomographic scanning in a brazilian population. j endod. 2013; 39: 748-51. 22. hassam ba. reliability of periapical radiographs and orthopantomograms in detection of tooth root protrusion in the maxillary sinus: correlation results with cone beam computed tomography. j oral maxillofac res. 2010; 1: e6. 23. ok e, güngör e, colak m, altunsoy m, nur bg, aðlarci os. evaluation of the relationship between the maxillary posterior teeth and the sinus floor using cone-beam computed tomography. surg radiol anat. 2014; 36: 907-14. 24. howe rb. first molar radicular bone near the maxillary sinus: a comparison of cbct analysis and gross anatomic dissection for small bony measurement. oral surg oral med oral pathol oral radiol endod. 2009; 108: 264-9. 2d and 3d imaging of the relationship between maxillary sinus and posterior teeth braz j oral sci. 14(2)141-148 oral sciences n3 original article braz j oral sci. july | september 2014 volume 13, number 3 impact of light-curing time and aging on dentin bond strength of methacrylateand siloranebased restorative systems anderson catelan1, giulliana panfiglio soares1, ana karina bedran-russo2, débora alves nunes leite lima1, giselle maria marchi1, flávio henrique baggio aguiar1 1universidade estadual de campinas unicamp, piracicaba dental school, department of restorative dentistry, piracicaba, sp, brazil 2university of illinois at chicago, college of dentistry, department of restorative dentistry, chicago, il, usa correspondence to: anderson catelan departamento de odontologia restauradora faculdade de odontologia de piracicaba unicamp av. limeira, 901 areião, caixa postal 52 cep 13414-903 piracicaba, sp, brazil phone: +55 19 2106 5337 fax: +55 19 3421 0144 e-mail: catelan@estadao.com.br received for publication: june 26, 2014 accepted: september 02, 2014 abstract aim: to evaluate the impact of different light-curing times on dentin microtensile bond strength of two restorative systems after 24 h and 6 months of water storage. methods: standardized class ii preparations were performed in 56 freshly-extracted human molars (n = 7), restored with methacrylateor silorane-based restorative systems, and light-cured using a light-emitting diode at 1390 mw/cm2 by the recommended manufacturers’ time or double this time. after storage for 24 h at 37 oc, the teeth were sectioned to yield a series of 0.8-mm thick slices. each slab was trimmed into an hourglass shape of approximately 0.64 mm2 area at the gingival dentin-resin interface. specimens were tested using universal testing machine at crosshead speed of 0.5 mm/min until failure, after 24 h and 6 months of storage. data were statistically analyzed by three-way anova and tukey’s test (α = 0.05). results: the highest bond strength values were recorded for the groups restored with methacrylate system (p<0.001) as well as for extended light-curing time (p = 0.0034). there was no statistically significant difference between 24 h and 6 months storage on bond strength (p>0.05). conclusions: bond strength was influenced by the material and lightcuring time, but the 6-month storage did not affect the bond strength of restorations. keywords: dental bonding; composite resins; methacrylates; silorane resins; polymerization. introduction polymerization of methacrylate-based composite is characterized by volumetric shrinkage1. these photo-activated restorative materials exhibit a significant proportion of methacrylate groups unreacted due to an incomplete conversion of carbon double bonds2. however, the higher the degree of conversion (dc), the higher the shrinkage strain3. polymerization stress may result in cuspal deflection4, de-bonding at composite-dentin interface, post-operative sensitivity5-6, microleakage5, secondary caries formation, marginal staining, restoration and dental fractures6, all reducing the longevity of the restoration. recently a low shrinkage monomer was developed from the reaction of the oxirane and siloxane molecules, termed silorane4,7. silorane presents a cationic ring-opening polymerization mechanism instead of the free radical cure of methacrylate monomers4 and an exended light-curing time to form cations is necessary to initiate the polymerization reaction1,4. it exhibits lower polymerization shrinkage4,8 and mechanical properties comparable to that of methacrylate dental composites7. braz j oral sci. 13(3):213-218 material clearfil se bond(kuraray medical inc. okayama, japan) filtek ls adhesive(3m espe, seefeld, germany) filtek z250(a2 shade; 3m espe, st. paul, mn, usa) filtek ls composite(a2 shade; 3m espe, st. paul, mn, usa) composition* lot. 00955a primermdp, hema, water, cq, hydrophilic dimethacrylate.lot. 01416a bondmdp, bis-gma, hema, cq, hydrophobic dimethacrylate, n,n-diethanol p-toluidine, colloidal silica. lot. 9bn primerbis-gma, hema, water, ethanol, silica treated silica filler, cq, phosphoric acid-methacryloxy-hexylesters mixture, phosphorylated methacrylates, copolymer of acryl and itaconic acid, phosphine oxide.lot. 9bk bondhydrophobic dimethacrylate, phosphorylated methacrylates, tegdma, silane treated silica, cq, stabilizers. lot. n144001brfiller: 60 vol%, aluminum oxide, silica, and zirconium oxide (0.01-3.5 µm).resin: bis-gma, bis-ema, and udma. lot. n183458filler: 55 vol%, silica, and yttrium trifluoride (0.04-1.7 µm).resin: bis-3,4-epoxycyclohexylethyl-phenylmethylsilane and 3,4-epoxycyclohexylcyclopolymethylsiloxane. *as informed by manufacturers. abbreviations – mdp: 10-methacryloyloxydecyl dihydrogen phosphate; hema: 2-hydroxyethylmethacrylate; cq: camphorquinone; bis-gma: bisphenol-a glycidyl dimethacrylate; tegdma: triethylene glycol dimethacrylate; bis-ema: ethoxylated bisphenol-a dimethacrylate; and udma: urethane dimethacrylate. table 1.table 1.table 1.table 1.table 1. materials used in this study. in deep cavity the irradiance that reaches the restorative material surface is decreased by the distance between the guide tip of the light-curing unit and material during the restorative procedure, reducing the degree of conversion, and/or leading to the formation of more polymers with linear structures, presenting inferior physical properties and will result in the weakening of the restoration9. improvement of the physical properties of resin-based materials with increase of the curing time available for the conversion of monomers to polymers has been reported3,10-11. however, few studies have assessed the bond strength of this new restorative system with different light-curing times and after aging. therefore, the objective of this study was to evaluate the influence of different restorative systems and curing times on the microtensile bond strength (microtbs) after 24 h and 6 months. the research hypotheses tested were that: (1) there would be no difference between restorative systems, (2) extended light-curing time would increase bond strength, and (3) aging would decrease microtbs values. material and methods this study was approved by the institutional review board under protocol number 031/2010. fifty-six freshly extracted non-carious, unrestored human third molars were collected and stored in 0.1% thymol solution at 4 oc. the teeth were scaled, cleaned, stored in distilled water at 4 oc, and used within 3 months after extraction. the toot roots were embedded in polystyrene resin (piraglass, piracicaba, sp, brazil) to facilitate the handling, and the occlusal surfaces were ground wet onto 320-grit sic paper in a polishing machine (apl-4, arotec, são paulo, sp, brazil) until the distance between the occlusal surface and cementum-enamel junction was 5 mm. standardized class ii vertical slot preparations were performed on one of the proximal surfaces of human molars with regular-grit cylindrical diamond bur (no. 3100; kg sorensen, barueri, sp, brazil) using a high speed handpiece with water spray coolant. cavity dimensions were 4 mm wide, 6 mm high (1 mm below the cementoenamel junction), and 2 mm of axial depth (from the proximal surface to the axial wall). a custommade preparation device allowed the standardization of the preparations dimensions. the margins were not beveled and burs were replaced after five preparations. table 1 shows the information about the materials used in this study. methacrylate[clearfil se bond (kuraray medical inc. okayama, japan) + filtek z250 (3m espe, st. paul, mn, usa)] and silorane-based [filtek ls system (3m espe)] restorative systems were used in the restorative procedures. the cavities were sequentially randomized in 8 groups (n = 7) (table 2), and the following restorative protocols were accomplished: for the methacrylate groups (1, 2, 5, and 6), clearfil se bond primer (bottle a) was vigorously scrubbed with applicator brushes in the entire cavity during 20 s, a mild air stream was applied for solvent evaporation, the bonding agent (bottle b) was applied, gently air thinned and light-cured for 10 s (g1 and g5) or 20 s (g2 and g6). for silorane groups (3, 4, 7, and 8), filtek ls primer (bottle 1) was actively applied for 15 s, gently air thinned, light cured for 10 s (g3 and g7) or 20 s (g4 and g8), and the bonding agent (bottle 2) was applied, thinned with a gentle air stream and light-cured for 10 or 20 s. after bonding procedures, individual transparent matrices were placed to allow the adequate filling of the proximal preparation. three approximately 2-mm-thick horizontal composite resin increments were inserted, measured with a millimeter periodontal probe with williams’ markings (golgran, são paulo, sp, brazil) positioned parallel to the tooth proximal surface, and light-cured for 20 or 40 s (table 2). the resin materials were light-cured at the occlusal surface using a second-generation light-emitting diode (led) unit (bluephase 16i; ivoclar vivadent, amherst, ny, usa) device at 1390 mw/cm2 of output irradiance (at 0 mm). the optical power (mw) delivered by the device was measured with a power meter (ophir optronics, har hotzvim, jerusalem, israel). the tip diameter was measured with a digital caliper (mitutoyo sul americana, suzano, sp, brazil); recorded 7 mm and tip area was determined in cm2. irradiance (mw/ cm2) was calculated dividing light power by tip area. the 214214214214214impact of light-curing time and aging on dentin bond strength of methacrylateand silorane-based restorative systems braz j oral sci. 13(3):213-218 group restorative system light-curing time* water storage g1 methacrylate recommended by the manufacturers 24 h g2 methacrylate double time 24 h g3 silorane recommended by the manufacturer 24 h g4 silorane double time 24 h g5 methacrylate recommended by the manufacturers 6 months g6 methacrylate double time 6 months g7 silorane recommended by the manufacturer 6 months g8 silorane double time 6 months table 2. table 2. table 2. table 2. table 2. experimental groups. *manufacturer’s recommendation or double the recommended time: adhesive system (10 or 20 s) and composite resin (20 or 40 s). recommended double time methacrylate 24 h *28.01 (4.21) aa *30.68 (4.89) ba 6 months *23.84 (3.91) aa *28.15 (4.81) ba silorane 24 h 17.18 (3.86) aa 20.06 (3.31) ba 6 months 16.93 (3.20) aa 20.37 (3.71) ba light-curing time restorative system distinct letters (capital in the rows and lowercase in the columns) are statistically different (p < 0.05). *differs from the silorane restorative system (p<0.001). the longer light-curing time promoted greater bond strength (p=0.0034). there was no statistically significant difference for aging (p>0.05). table 3.table 3.table 3.table 3.table 3. microtensile bond strength values [mpa (s.d.)] according to restorative system, aging, and curing time. irradiances also were calculated positioning a spacer device (with heights of 4 and 6 mm) between the light guide tip of the curing unit and the surface of the power meter, and beneath resin disks for both composites made using a standardized teflon matrix (with 2 mm of thickness, simulating the first increment) at 4 mm of the top surface of resin disk. the distance between the light guide tip and the bottom of cavity was 6 mm with an irradiance of 610 mw/cm2, when the adhesive systems were cured. the composite increment was approximately 2 mm thick, totalizing 990 mw/cm2 on the top surface of the first composite increment at 4 mm of distance between light guide tip and top surface of the first composite increment. the irradiance on the bottom surface at 6 mm (beneath both 2-mm-thick composite resin disks) was 380 ± 5 mw/cm2. after restorative procedures, specimens were stored in distilled water at 37 oc for 24 h. after this period, the proximal surface was finished and polished with al2o3 abrasive discs (sof-lex pop-on, 3m espe), from coarse to superfine for 30 s with a rotating hand piece at approximately 10,000 rpm. then, the restored teeth were serially sectioned to yield 3 series of 0.8 mm thick vertical slices using a diamond saw (isomet 1000; buehler, lake bluff, il, usa) at 300 rpm. each slab was trimmed into an hourglass shape of approximately 0.64 mm2 area at the gingival resin-dentin interface using a super-fine diamond bur (no. 1090ff; kg sorensen). in the aged groups (g5-g8, table 2), the hourglasses were stored in distilled water at 37 oc for 6 months, changed weekly. all specimens had direct exposure to storage fluid12. this procedure is commonly used and considered a type of accelerated aging13. twenty-four hours or 6 months after water storage at 37 oc, the cross-sectional area of each hourglass was measured with a digital caliper to the nearest 0.01 mm and recorded for the calculation of the dentin bond strength. each bonded slab was individually attached to a flat grip geraldeli device for microtensile testing with cyanoacrylate instant adhesive (super bonder gel; loctite-henkel, são paulo, sp, brazil), and subjected to a tensile force using a universal testing machine (dl 500; emic, são josé dos pinhas, pr, brazil) at crosshead speed of 0.5 mm/min until failure. the number of slabs prematurely de-bonded during specimen preparation was recorded, but no bond strength value was attributed for statistical analysis14. the bond strength values obtained from the 3 slices of each tooth were used to calculate the microtbs of the specimen. means and standard deviations were calculated and expressed in mega pascals (mpa). after microtbs test, the dentin side of the fractured specimens was dried by silica stored in incubator at 37 oc for 48 h, mounted on the aluminum stubs, and gold sputtercoated under high vacuum (scd 050; bal-tec ag, balzers, liechtenstein). a scanning electron microscope (sem; jsm 5600 lv, jeol, tokyo, japan) was used to evaluate the bond failure modes of the fractured specimens on the dentin side with magnifications between 70 and 1000x and classified as follows: (1) cohesive in dentin, (2) adhesive, (3) cohesive in the composite, and (4) mixed. the microtbs data obtained by to assume the normality presuppositions were analyzed by three-way anova and tukey´s test at a 0.05 level of significance. the main factors were restorative system, curing and storage times. results the methacrylate-based restorative system showed higher dentin bond strength than the silorane-based material (p<0.001), the extended light-curing time resulted in higher microtbs values (p = 0.0034) and there was no statistically significant difference between 24 h and 6 months (p>0.05) (table 3). the descriptive analysis of failure modes and the number of pre-testing failures for each experimental group are shown in table 4. discussion the first hypothesis tested was rejected, since the methacrylate materials presented greater dentin bond 215215215215215 impact of light-curing time and aging on dentin bond strength of methacrylateand silorane-based restorative systems braz j oral sci. 13(3):213-218 strength than the low shrinkage restorative system (table 3), in accordance with a previous study that showed higher microtbs for methacrylate than silorane composite regardless of placement technique15. self-etch adhesives are based on absence of rinsing and drying steps, maintaining the ideal dentinal humidity and reducing technique sensitivity13. twostep self-etch adhesive consists in a self-etch primer with acid monomers that demineralize and simultaneously penetrate monomers into dentin subsurface, followed by application of a solvent-free hydrophobic bond agent, which provides better mechanical properties16. all-in-one adhesive contains a mixture of acid, hydrophilic, and hydrophobic monomers, water and organic solvents in a single bottle16. this adhesive is more hydrophilic, allowing deeper penetration with water content increases due to adhesive acidification in water presence, interfering on polymerization, which leads to uncured acid and aggressive monomers that continue etching the dentin, affecting negatively the bonding interface16-17. most one-step self-etch adhesives are severely affected by the hydrolytic degradation18. however, longevity over time was not related to the number of steps of the bonding systems, but to their chemical compositions19. clearfil se bond consists in a hydrophilic self-etch primer and hydrophobic bond agent. this viscous hydrophobic resin-coating layer improves mechanical properties and increases longevity of the bonding interface16. filtek ls low shrinkage composite resin has a dedicated self-etching adhesive. although ls adhesive system is classified by the manufacturer as a two-step self-etch adhesive, the hydrophilic ls primer is applied first and then lightcured forming the hybrid layer1. thus, the bifunctional hydrophobic monomer (phosphorylated methacrylate) of the ls bond applied after the primer cured acts as a low viscosity composite connection liner between methacrylate monomers (by reaction with acrylate group) and silorane monomer (by reaction of the phosphate group with oxirane)8. therefore, ls primer is a one-step self-etch adhesive, which could explain the lower bond strength values1. the mild self-etch primer of clearfil se bond has a ph of 2.016, and is composed by a functional acid monomer mdp, which adheres to the tooth hydroxyapatite most readily and intensely20. this stable chemical bond was left around group g1 g2 g3 g4 g5 g6 g7 g8 pretesting failure 3/21 0/21 3/21 2/21 3/21 2/21 3/21 1/21 cohesive in the dentin adhesive 76.47 42.86 16.67 26.32 88.24 61.11 27.78 30.00 cohesive in the composite mixed 23.53 57.14 83.33 73.68 11.76 38.89 72.22 70.00 table 4. table 4. table 4. table 4. table 4. fracture pattern analysis failure modes (%) the collagen fibrils within the hybrid layer21. the self-etch ls primer has a ph of 2.76 and is classified as ultra-mild1,6,21. transmission electron microscopy (tem) of ls adhesive shows a thin nano-interaction zone, which is probably the combination of the resin-impregnation within smear layer and actual hybridized dentin1,6. smear debris interfere in the interaction between the mild and ultra-mild self-etching adhesives with dentin tissue22. the bonding effectiveness of ultra-mild one-step self-etch adhesive is largely affected by the properties of the produced smear layer because it interacts superficially with the smear layer-covered dentin23. it has been reported that two-step self-etch adhesive systems performed better at bonding ability than one-step self-etch adhesives13,19,21. a longer light-curing time increased the microtbs of the tested restorative systems (table 3); therefore, the second hypothesis was validated. it is now that only 1 mm distance increase between the light guide tip and restorative material decreases the light intensity by approximately 10%24. several studies have related the improvement of the physical properties of resin-based materials with increase of the curing time, due to the higher dc3,10-11. there is a significant correlation between bond strength and total curing time25 with greater dc26. special care should be taken when performing the polymerization of resinous materials with lower light power curing units at deep cavities. the onset of cationic ring-opening polymerization of the silorane is slower due to the required formation of sufficient cations to initiate polymerization, thus a longer light-curing time is required compared with radical cure o f m e t h a c r y l a t e m o n o m e r m o l e c u l e s i n t o p o l y m e r network1,4. the curing device used in this investigation consists in single peak second generation led. this unit presents a high optical power and spectrum between 410 and 530 nm with a peak on at 454 nm that includes the maximum energy absorption peak of the camphorquinone at (468 nm)27, the photo-initiator included in all tested resin-based materials. the light-curing time recommended for silorane composite using quartz-tungsten-halogen (qth) with irradiance between 500-1400 mw/cm2 is 40 s, as well as for leds with output between 500-1000 mw/cm2. for leds with irradiance between 1000-1500 mw/cm2 is advised an exposure light time of 20 s. an irradiation of 10 s is recommended to cure the primer and bond of ls adhesive, without concern about minimum irradiance. in this study it was used a led with irradiance of 1390 mw/cm2, indicating 20 and 10 s of light polymerization for composite and adhesive, respectively. however, the irradiance achieved on the surface of the first composite increment was of 990 mw/ cm2 at 4 mm of tip, and on the adhesive system was of 610 mw/cm2 at 6 mm (applied on cavity bottom) of light guide tip. furthermore, at a 4 mm distance from the light guide tip to the top surface of the composite and curing beneath the restorative material, the irradiance at the bottom surface was 380 mw/cm2. bond strength is influenced by monomer conversion26; thus, extended curing time may have increased 216216216216216impact of light-curing time and aging on dentin bond strength of methacrylateand silorane-based restorative systems braz j oral sci. 13(3):213-218 the dc of adhesives11 and/or composites, and improved the dentin microtbs. the third hypothesis was rejected because the long-term water storage did not affect the bond interface of the restorations (table 3). interface components can be degraded by hydrolysis and water may infiltrate, resulting in the plasticization of the polymeric matrix, by swelling and reduction of the frictional forces between the polymer chains, reducing the mechanical properties, and consequently the bonding interface integrity13. however, the 6 months of water storage did not decrease the microtbs values, similar result as reported elsewhere5. on the other hand, other studies showed a significant decrease in the bond strength after shorter periods (within 3 months)28, and even after longer periods (within 4 years)25. the mdp-contained clearfil se bond adhesive system in contact with the tooth forms the mdp-calcium salt that hardly dissolved in water. therefore the bond between mdp and hydroxyapatite should be stable20. thus, the chemical interaction improves the resistance to hydrolytic breakdown and de-bonding stress, keeping the restoration margins sealed for longer periods21. moreover, since primer application followed by the hydrophobic bonding agent contained mainly cross-linking monomers, clearfil’s bond provides better mechanical properties to clearfil16. this fact, combined with methacrylate filtek z250 composite and high power density, could result in the long-term stability of the bond interface. single-bottle adhesives such as the one-step self-etch ls primer may act as permeable membranes and be more susceptible to aging29. moreover, these adhesives are strongly influenced by light intensity of the photo-curing device16. thus the second viscous hydrophobic coating layer (ls bond) seems to have improved the vulnerability to water sorption resultant of the high hema content in the ls primer, applied and cured previously 6, after long-term water storage. additionally, the active application of one-step self-etch adhesives has been related to improvement in the bonding performance30, along with the increased hydrophobicity of the silorane composite resin due to the presence of siloxane species7. the high irradiance could also contribute to the bond longevity of this new restorative system. the methacrylate restorative system showed more adhesive failures, while silorane exhibited more mixed failures (table 4). most silorane fractures occurred between the bonding agent and the composite, with part of the bonding remaining on dentin surface as well, perhaps due to the lower adhesion compared to adhesion between the methacrylatebased materials. the longer irradiation time increased the occurrence of mixed failures and decreased the adhesive failures for methacrylate restorative system, likely by greater monomeric conversion. water storage increased the adhesive failures percentage for both restorative systems, probably by swelling of the polymer network and reduction of the frictional forces between polymeric chains. the quality and uniformity of the polymerization reaction is an important parameter that affects the conversion of the monomers into structured polymers, and therefore improves the physical properties and clinical performance; however, this process is dependent of various factors, such as design and size of the tip guide, distance of the light guide tip from the material surface, power density, exposure duration, shade and opacity of the composite, increment thickness, materials’ composition, and others9. thus, manufacturers should provide information, such as minimum irradiance and time of light curing required for optimal polymerization of their adhesive systems27, and make clear, in their instructions of use, that the minimum irradiance indicated is the one that reaches the surface of the material and not the optical power of output of the light-curing device. higher irradiance is necessary to adequately cure photoactivated materials in deep cavities, and contribute to the improvement of the longevity of adhesive dental restorations. the longer light-curing time improved the bond strength for both restorative materials and the groups restored with ls restorative system showed the lowest dentin microtbs values; 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15: 317-24. 23. ermis rb, de munck j, cardoso mv, coutinho e, van landuyt kl, poitevin a, et al. bond strength of self-etch adhesives to dentin prepared with three different diamond burs. dent mater. 2008; 24: 978-85. 24. prati c, chersoni s, montebugnoli l, montanari g. effect of air, dentin and resin-based composite thickness on light intensity reduction. am j dent. 1999; 12: 231-4. 25. abdalla ai, feilzer aj. four-year water degradation of a total-etch and two self-etching adhesives bonded to dentin. j dent. 2008; 36: 611-7. 26. oguri m, yoshida y, yoshihara k, miyauchi t, nakamura y, shimoda s, et al. effects of functional monomers and photo-initiators on the degree of conversion of a dental adhesive. acta biomater. 2012; 8: 1928-34. 27. faria-e-silva al, lima af, moraes rr, piva e, martins lr. degree of conversion of etch-and-rinse and self-etch adhesives light-cured using qth or led. oper dent. 2010; 35: 649-54. 28. krajangta n, srisawasdi s. microtensile bond strength of silorane-based resin composite and its corresponding adhesive in class i occlusal restorations. am j dent. 2011; 24: 346-53. 29. tay fr, pashley dh, peters mc. adhesive permeability affects composite coupling to dentin treated with a self-etch adhesive. oper dent. 2003; 28: 610-21. 30. do amaral rc, stanislawczuk r, zander-grande c, michel md, reis a, loguercio ad. active application improves the bonding performance of self-etch adhesives to dentin. j dent. 2009; 37: 82-9. 218218218218218impact of light-curing time and aging on dentin bond strength of methacrylateand silorane-based restorative systems braz j oral sci. 13(3):213-218 oral sciences n3 braz j oral sci. 13(2):133-139 original article braz j oral sci. april | june 2014 volume 13, number 2 dental caries in inland brazilian adolescents and its relationship with social determinants andréa videira assaf1, angela scarparo caldo-teixeira1, flavia maia silveira1, maria isabel bastos valente1, rafael gomes ditterich2, roberta barcelos1 1universidade federal fluminense – uff, nova friburgo dental school, department of specific formation, nova friburgo, rj, brazil 2universidade federal do paraná – ufpr, dental school, department of community health, curitiba, pr, brazil correspondence to: andréa videira assaf rua sílvio henrique braune, n.22, centro cep: 28625-650 nova friburgorj-brasil phone: +55 22 81414848 e-mail: avassaf@gmail.com received for publication: april 05, 2014 accepted: june 10, 2014 abstract aim: to identify the prevalence of dental caries in inland brazilian adolescents, and to analyze the influence of socio-demographic and clinical variables, and access to dental service on caries experience. methods: this study had a non-probabilistic sample comprising 504 adolescents aged 12 years, attending public schools in the city of nova friburgo, mountain region of rio de janeiro, brazil. the world health organization (who, 1997) criterion was used by previously calibrated examiners to report dental caries. variables were obtained by means of a semistructured questionnaire applied to the adolescents’ parents. results: the d3mft mean was 1.90 and the significant caries (sic) index was 4.54, mainly represented by the carious component. multiple logistic regression analysis revealed that parents with over 8 years of schooling (or=0.579), absence of pain (or=0.396) and not visiting the dentist (or=0.270) might suggest protective factors against the disease; the possible risk factors were male gender (or=1.982) and pain, extraction and others were reasons for consultation (or=2.435). conclusions: although the prevalence of caries was slightly below the national mean, polarization of the disease was clearly observed. education and no access to the dentist led to protection against the disease. these results may contribute to planning of oral health actions directed towards this target population for the control of caries. keywords: risk factors; social class; dental caries. introduction many scientific investigations have been published over the last few years, showing what is generally accepted: the pattern of dental caries has decreased in the last decades, especially among younger groups in most developed countries1. this has been mainly associated with factors such as public water supply fluoridation and the dissemination of fluoridated dentifrices1-3. in spite of the trend towards a reduction in dental caries, it is still considered a priority disease, especially in developing countries such as brazil, and this has been demonstrated in the latest national epidemiological surveys4-5. in this context, the polarization of the disease has been observed in groups of younger individuals, with highest incidence (80%) concentrated in a small group of individuals (25 to 30%). this has led to the premise of concentrating efforts especially on certain geographic regions and underprivileged socio-economic groups6. with this in mind, the development of studies focused on investigating the influence of social inequalities on the caries process have become the basic line of approach to a broader understanding of the multifactorial dynamics of the disease, identification of the most vulnerable groups and making the best decisions, based on identifying these groups and directing care towards them6-7. braz j oral sci. 13(2):133-139 in addition to the socio-economic determinants, there has been a welcome emphasis on studies that have pointed out the influence of other factors, both material and symbolic, such as the conditions of the physical, political and cultural environment, aspects related to social behaviors, selfperception and social interaction, on the development of dental caries8. thus, there has been a continuous endeavor to reach broader understanding of the factors involved in the caries process, in order to achieve the desired improvement and technological development focused on care and attention to oral health, based on the planning and adoption of ethical and equitable public policies for the entire society. no epidemiological survey has ever been conducted to obtain data on the oral health of adolescents in the city nova friburgo, rj, brazil. in addition, public water fluoridation, supported by the national oral health policy9, has recently been introduced in that municipality. therefore, studies aimed at identification and longitudinal monitoring are of utmost importance to measure the impact of this factor on the caries and fluorosis indicators on the different population groups. the aims of the present study were: to identify the prevalence of dental caries in 12-year-old adolescents in the municipality of nova friburgo, rj, brazil; to analyze the influence of socio-demographic and clinical variables and access to dental services on caries experience; and to provide “baseline” data, which will serve as a reference for future comparisons of the effects of public water fluoridation on the incidence of dental caries among adolescents. material and methods ethical aspects this study was approved by the research ethics committee of the universidade federal fluminense at nova frigurgo (uff/nf), in accordance with the national health council, ministry of health resolution 196/96, process cep/ cmm/huap n.272/2010 caae n. 0217.0.258.000-10. the participants in the study who needed dental treatment were referred to the school of dentistry of the uff/nf. correspondence was first sent to the municipal and state secretaries for education and later authorization was granted to carry out the project. after this, the directors the public schools in the municipality were contacted and granted their support for conducting the research. parents/guardians of the adolescents also gave authorization by signing an informed consent form. the adolescent was enrolled in the study only after this document was properly filled out and returned to the researchers. study design characterization of the municipality and sample selection the present cross-sectional study consisted in evaluating the residents of the city of nova friburgo, located in the mountain region of the rio de janeiro state, brazil, which has a total area of 938.5 km² and a 0.81 human development index (hdi). the municipality has 8 districts, comprising rural (12.5%) and urban (87.5%) areas, with varying demographic, environmental, socio-economic and cultural characteristics. regarding schooling, these characteristics are worse in comparison with entire rio de janeiro state population, presenting the percentages of 49.5% (nova friburgo) and 39.1% (rio de janeiro state) for the population group with fewer than 8 years of schooling 10, but, the municipality provides differentiated oral health services. the main economic activities in the municipality are agricultural production in the rural zone and underwear manufactures in both rural and urban zones. the sample comprised 12-year-old adolescents from all the 43 state and municipal public schools in nova friburgo, which corresponded to 1081 individuals, representing 76.47% of the individuals in this age group in the municipality. the remaining adolescents were enrolled in private teaching institutions. for sample calculation, a population of 12-year-old adolescents resident in the municipality in the year of 2012 was considered, with a precision of 5% and confidence interval of 95%, for a prevalence of 50% of the disease, since the municipality has no information about dental caries. thus, the largest possible sample was obtained, considering a sample loss of 20% and this resulted in a minimum sample of 461 individuals. individuals were randomly selected from lists of students enrolled in all public schools. the exclusion criteria for participation in the study were: adolescents whose parents/ guardians did not grant permission for participation in the study; or who did not fully respond to the questionnaire, and adolescents with debilitated health. the final sample size was 504 adolescents, which is a larger number than the one calculated for minimum sample. preparatory stage: pilot study and calibration of examiners pilot study the pilot study was conducted before the fieldwork, with adolescents (n=20) and their respective parents/ guardians, from rural and urban regions. the purpose was to verify the parents/guardians’ understanding of the questionnaire and training the researchers as examiners to conduct the study. in addition, some of the parameters with regards to the epidemiological evaluation were reviewed, such as the approach to performing the clinical exams on the adolescents. for this latter requisite, learning was complemented with the stage of examiner training and calibration. examiner training and calibration the entire process of training the five examiners was carried out under the supervision of a researcher experienced in epidemiological surveys, for a 20 h period. in order to provide an initial training phase, theoretical discussions (4 h) and presentation of clinical photographic 134dental caries in inland brazilian adolescents and its relationship with social determinants 135 slides with examples of each criterion were organized. next, clinical training sessions (8 h) were performed, followed by calibration exercises (8 h). for every period of clinical training, each examiner evaluated 10 to 12 adolescents from a public school of nova friburgo, with different levels of dental caries prevalence. in this phase, all examiners discussed about the clinical dental diagnosis to establish a criterion of consensus for the group. after this phase, the examiners undertook two calibration exercises with a 1-week interval between them. a mean kappa interexaminer value of 0.80 was obtained. duplicate exams in 10% of the sample were performed by the examiners during the field research, in order to calculate the intraexaminer kappa, where a mean value of 0.89 was obtained. field stage: questionnaires, exams and epidemiological criteria semi-structured questionnaires in this second stage the semi-structured questionnaires were handed over to the adolescents’ parents/guardians. the semi-structured questionnaire, previously tested in the pilot study, was applied to collect information relative to: 1) sociodemographic characterization of the family; 2) clinical aspects and 3) access to dental services. this data collection instrument was based on the methodology proposed by the last national oral health survey5. clinical evaluation the clinical evaluation of dental caries in the adolescents was performed under natural light, by previously calibrated researchers, with the study subjects seated on ordinary chairs. millimeter periodontal probes with rounded tips (who 621) and plane no.5 dental mirrors were used. the exams were conducted in accordance with the methodology recommended by the who 11. before the exam, the schoolchildren performed toothbrushing with fluoridated dentifrice under supervision of the researchers, to help removing dental biofilm and/or food remains that were left on the tooth surfaces, thereby facilitating visual diagnosis. codes and criteria used for clinical evaluation the indices used for measuring dental caries were the d3mft (total number of decayed, lost and filled teeth) and sci (significant caries index)12, for permanent dentition, according to the criteria and codification of the who oral health surveys basic methods handbook11. the subjects were also evaluated for the presence or absence of fluorosis, using the dean index11 for diagnosis. statistical analysis descriptive analyses of the data were carried out made by calculating the distribution of the independent variables in the sample, the d3mft index and its respective components, percentage of caries-free individuals and sci11-12. for the biand multivariate analyses, d3mft was considered the dependent variable (dichotomized by the median into d3mft<1 and d3mft>1)13. all the independent variables were dichotomized as follows13: zone (rural and urban), gender (female and male), fluorosis (absence and presence), number of residents in the house (<4 and >4), family income (by the median: <3 and >3 brazilian minimum wages), number of assets (by the median: d”7 and >7), parents’ schooling (<8 and >8 years of schooling), need for treatment (yes and no), presence of pain (yes and no), visit to the dentist (yes and no), type of service used (public and private), reason for consultation (routine and pain/extraction/others), problems of an oral nature e.g.: difficulty to eat, to brush the teeth and of a general nature e.g.: irritability, embarrassment to smile, talk, to go parties, difficulties to play sports, problems with sleeping or studying due to the condition of the teeth (yes and no). the chisquare test at 5% level of significance was used to test the association of the independent variables with the dependent variable (d3mft). the variables that presented statistical significance at the level of 20% or lower in the bivariate analysis were selected for multiple logistic regression analysis by the stepwise procedure. the odds ratios (or) and the respective confidence intervals (ci) of 95% were estimated for the variables that remained in the multiple regression model at the level of 5%. all the statistical tests were performed with the sas (sas user’s guide: statistics, version 9.2. cary [ny]: sas institute inc. 2001) software. results the sample comprised a distribution according to gender: male (45.24%) and female (54.76%) subjects. as regards socioeconomic variables, 78.07% of families had an income <3 brazilian minimum wages and most parents/ guardians (61.29 %) had fewer than 8 years of schooling. with respect to dental needs and care, 86.69% of the adolescents needed treatment, and 36.09% had a history of dental pain in the last 6 months. the reasons for consultation of the majority of the subjects were due to pain, extraction, others (72.92%) and most of them alleged to have problems of an oral and general nature due to the condition of their teeth (69.03%). fluorosis was detected in less than 10% of the individuals (table 1). the mean (standard deviation) of the d3mft index was 1.90 (2.25), mainly represented by the decayed component (d=0.98/ds=1.67), followed by the filled (f=0.84/ds=1.46) and missing component (m=0.08/ds=0.42). a total of 201 (39.88%) children were found to be caries-free. the sci index was 4.54 (1.90), with a mean (standard deviation) for the decayed, lost and filled components of 2.23 (2.30), 0.22 (0.71) and 2.09 (1.90), respectively. this specific caries polarization group showed low social indicators, such as income, with 80.75% (n=130) of the individuals with a family income lower than or equal to 3 minimum wages, and schooling, with 65.85% (n=108) of the parents/guardians with eight or fewer years of schooling, and almost all individuals needing dental treatment (95.21%; n=159). the bivariate analysis by the chi-square test showed association between d 3mft and parents’ schooling dental caries in inland brazilian adolescents and its relationship with social determinants braz j oral sci. 13(2):133-139 136 variables n % zone < rural 236 46.83 urban 268 53.17 gender female 276 54.76 male 228 45.24 fluorosis absence 454 90.08 presence 50 09.92 number of residents in house <4 291 59.27 >4 200 40.73 family income <3 bmw* 381 78.07 <3 bmw 107 21.93 number of assets <7 266 58.46 >7 189 41.54 parents’ schooling (years of schooling) <8 285 61.29 >8 180 38.71 treatment need yes 430 86.69 no 66 13.31 presence of pain (in the last 6 months) yes 179 36.09 no 317 63.91 visit to the dentist yes 416 84.21 no 78 15.79 type of service used public 200 48.19 non public 215 51.81 reason for consultation routine 114 27.08 pain, extraction, others 307 72.92 problem of an oral and general nature due to condition of the teeth yes 341 69.03 no 153 30.97 table 1.table 1.table 1.table 1.table 1. sociodemographic and clinical characteristics of the sample and its access to dental services. nova friburgo, rj, brazil, 2012. * bmw brazilian minimum wage. values lower than 504 due to missing data. (or=0.6230), presence of pain (or=0.3914), visit to the dentist (or=0.3758), type of service used (or=0.6095) and reason for consultation (or=3.0382) (table 2). in the multiple logistic regression analysis, it was observed that the suggested protective factors associated with d3mft<1 were: parents’ schooling over 8 years (or=0.579), absence of pain (or=0.396) and not consulting the dentist (or=0.270). therefore, the children whose parents had a level of schooling of over 8 years presented less chance of having d3mft>1. the possible risk factors associated with d3mft>1 were: male gender (or=1.982) and reason for consultation due to pain, extraction and others (or=2.435); that is to say, male children presented 1.982 times more chance of having d3mft>1 than the female children (table 3). discussion the evident decline in caries disease has not been homogeneous, that is, to say, the distribution of the disease is still unequal in the population, and remarkable regional differences are observed in the local and international epidemiological indices1,4-5. this factor represents evident inequalities in oral health, which range from differences in the prevalence of the disease, to access to services, products and protective measures in oral health. a low prevalence of dental caries was found for the 12year-old age group, with a mean of 1.90 for the d3mft index, below the national mean value (d3mft=2.07), but above the mean for the southeastern region (d3mft=1.72) and the goal established by the who for the year 2010 (dmft<1)5,14. it may be observed that the percentage of caries-free individuals was around 39.88%, below both the national (44%) and state-wide (50.6%) means5. moreover, the mean value of the sic index at the value of 4.54 and mainly represented by the carious component, indicates polarization of the disease and is suggestive of correlation with social indicators such as income and schooling. in addition, the adolescents showed a considerable need for attention to oral health, by means of adequate measures of promotion, prevention and rehabilitation. the age group caries indexes in nova friburgo still surpass those of other cities with fluoridated water. this may suggest some relation between the lack of access to this protective measure in the current panorama of dental caries in this city 15-16. this epidemiological scenario in this municipality may be justified by the deficiency of an effective oral health surveillance policy and low coverage by the fluoridated public water supply in the municipality. this is in disagreement with the national oral health policy itself, which prioritizes oral health promotion and preventive actions, including encouragement of healthy dietary habits, fluoridation of public water supply and distribution of dentifrices to the brazilian population9. from this point of view, the development of studies to evaluate the inequalities in oral health are a relevant research topic, in order to identify groups with greater vulnerability, because they directly or indirectly reflect the effects of the relevant determinants, such as precarious housing conditions, low income and access to education and health services, among others. variables such as family income and schooling have been considered good indicators of disease for this age group, since children belonging to families with differentiated income and education present different levels of the disease15,17. in the present study, the schooling of the parents/ guardians was pointed out as a possible indicator for protection from the disease, and had a greater influence than dental caries in inland brazilian adolescents and its relationship with social determinants braz j oral sci. 13(2):133-139 137 variables d3mft o r ci 95% p <1 >1* n % n % zone rural 103 43.64 133 53.36 ref urban 96 35.82 172 64.18 1.3875 0.9695-1.9858 0.0730 gender female 118 42.75 158 57.25 ref male 81 35.53 147 64.47 1.3554 0.9445-1.9449 0.0985 fluorosis absence 175 38.55 279 61.45 ref presence 24 48.00 26 52.00 0.6795 0.3781-1.2211 0.1943 number of residents in house <4 123 42.27 168 57.73 ref >4 73 36.50 127 63.50 1.2737 0.8797-1.8442 0.1997 family income <3 bmw** 156 40.94 225 59.06 ref <3 bmw 39 36.45 68 63.55 1.2089 0.7759-1.8836 0.4015 number of assets <7 103 38.72 163 61.28 ref >7 78 41.27 111 58.73 0.8992 0.6147-1.3155 0.5842 parents’ schooling (years of schooling) <8 102 35.79 183 64.21 ref >8 85 47.22 95 52.78 0.6230 0.4261-0.9108 0.0143 treatment need yes 167 38.84 263 61.16 ref no 31 46.97 35 53.03 0.7169 0.4259-1.2069 0.2091 presence of pain (in the last 6 months) yes 47 26.26 132 73.74 ref no 151 47.63 166 52.37 0.3914 0.2626-0.5835 <0.0001 visit to the dentist yes 151 36.30 265 63.70 ref no 47 60.26 31 39.74 0.3758 0.2290-0.6169 <0.0001 type of service used public 61 30.50 139 69.50 ref non public 90 41.86 125 58.14 0.6095 0.4065-0.9138 0.0162 reason for consultation routine 64 56.14 50 43.86 ref pain, extraction, others 91 29.64 216 70.36 3.0382 1.9496-4.7349 <0.0001 problem of an oral and general nature due to condition of the teeth yes 129 37.83 212 62.17 ref no 69 45.10 84 54.90 0.7408 0.5034-1.0902 0.1275 *level of reference of dependent variable or= odds ratio. ci = confidence interval ** bmw brazilian minimum wage. table 2. table 2. table 2. table 2. table 2. bivariate analysis by chi-square test for association between dependent variable (d3mft) with independent variables. nova friburgo, rj, brazil, 2012. the family income itself (table 3). the results of previous researches have shown that the mother’s schooling seems to be one of the main predictors of child’s health; that is to say, a low level of schooling, especially the mother’s, is related to greater risk for dental caries and to higher treatment needs in children13,18-19. social indicators such as schooling have usually been used to evaluate the advance and reversal in the population’s living conditions. generally, education provides access to better employment conditions, consequently generating a higher income level, which has a direct influence on the assimilation of positive behaviors related to oral health17,20. therefore, inequality in access to education appears to reflect directly on the oral health of the studied population, which leads to the need for of broader discussions and adoption of educational policy measures in a universal and uniform manner. dental caries in inland brazilian adolescents and its relationship with social determinants braz j oral sci. 13(2):133-139 138 variables d3mt>1* o r ci 95% p n % gender female 158 57.25 ref male 147 64.47 1.982 1.246-3.151 0.0108 parents’ schooling (years of schooling) <8 183 64.21 ref >8 95 52.78 0.579 0.370-0.907 0.0115 presence of pain (in the last 6 months) yes 132 73.74 ref no 166 52.37 0.396 0.238-0.660 0.0006 visit to the dentist yes 265 63.70 ref no 31 39.74 0.270 0.071-1.022 0.0432 reason for consultation routine 50 43.86 ref pain, extraction, others 216 70.36 2.435 1.498-3.959 <0.0001 table 3.table 3.table 3.table 3.table 3. multiple logistic regression. nova friburgo, rj, brazil, 2012. *level of reference of dependent variable or = odds ratio. ci = confidence interval conversely, non-use of dental services might also be regarded as a suggestive protection against the development of caries in the studied population, which generates questions about the quality of oral health care for individuals (table 2). this fact may be related to the very practice of dentistry still being based on the surgical-restorative philosophy, in addition to inadequate diagnosis and consequent overtreatment of caries lesions, which generates an increase in the caries indices. this result is in agreement with the studies of nadanovsky et al.21 (1995) and traebert et al.19 (2011), which showed higher caries indices in the groups and populations who go to the dentist more frequently, or those who have never received any oral health care. in this sense, one perceives that there is a greater influence of population strategies in oral health, such as the mere use of fluoridated water and dentifrice, rather than the individual action of the dentist on the reduction of epidemiological dental caries indices7,22. with regard to the other clinical variables, it seems to exist a direct relationship between the reasons for the consultation due to pain or extraction and dmf>1, which tends to demonstrate that individuals with symptoms related to more severe sequelae tend to have greater prevalence of the disease (table 3)23. the variable gender does not appear to be a relevant predictor for dental caries13,23. nevertheless, the results of the present study are controversial, because they show the male gender to be at greater risk for caries than the female gender (table 3), different from other studies which demonstrated a greater risk related to the female gender19,24. from these results, it is suggested that factors of an educational, or even cultural nature, may explain this variable, because there seems to be a consensus that the genders demonstrate different perspectives with regard to their self-care. it should be pointed out that control of the entire research process with the establishment of a pilot stage, examiner calibration process, obtaining results with adequate reproducibility, control at the stage of data collection and analysis, indicate an adequate internal validity of the study. a clear limitation of this study is the fact that private schools adolescents, which represent 23.53% of the total of students in this age group, were not included in the study because most directors of private schools did not grant authorization to carry out the research at their facilities. it was concluded that in spite of the prevalence of dental caries in the 12-year-old adolescents from nova friburgo being slightly below the national mean value, a clear polarization of the disease was observed. education and no access to the dentist led to protection against the disease. these results may contribute to the development of oral health actions directed towards this target population for the control of caries. the present study, which is the first one on oral health conducted in the city, was useful to identify dental caries in the adolescents and the respective factors that limit or favor the onset of caries in this age group. it may provide dentists, especially those from the public health system, with a general framework of the disease that will enable them to identify individuals at higher risk of caries and need for dental care. in view of these findings, it is possible to state that the university has a very important role to play in contributing to the production of epidemiological data which could favor the process of oral health planning by the municipality, with the development of programmed actions by means of adequate individual and population measures required for the control of disease. furthermore, the study is relevant because this survey was concomitant with the beginning of water supply fluoridation in the city, which will form the basis for future epidemiological comparisons for the effect of this public oral health measure on the control of dental caries. dental caries in inland brazilian adolescents and its relationship with social determinants braz j oral sci. 13(2):133-139 acknowledgements the authors wish to thank karine cortellazzi for performing the statistical analysis of data. they also thank faperj for the financial support (grants no. e-26/111.659/ 2010); and faperj and cnpq for the scientific initiation scholarship grants. they also give special thanks to the undergraduate students fernanda balonecker and carla lourenço, to the principals of the schools, teachers and all the children who contributed to the accomplishment of the survey. references 1. marthaler tm. changes in dental caries 1953-2003. caries res. 2004; 38: 173-81. 2. frazão p, peres ma, cury ja. drinking water quality and fluoride concentration. rev saude publica. 2011; 45: 964-73. 3. santos app, nadanovsky p, oliveira bh. a systematic review and meta-analysis of the effects of fluoride toothpastes on the prevention of dental caries in the primary dentition of preschool children. community dent oral epidemiol. 2013; 41: 1-12. 4. brazil. ministry of health. secretary of health care. department of primary care. national coordination of oral health. sb brazil project survey on brazilian population oral health conditions 2002-2003: main results. report. brasília: ministry of health; 2004. 68p. 5. brazil. ministry of health. secretary of health care. department of primary care. national coordination of oral health. sb brazil project 2011: national survey of oral health: main results. report. brasília: ministry of health; 2011. 92p. 6. ditmyer m, dounis g, mobley c, schwarz e. inequalities of caries experience in nevada youth expressed by dmft index vs. significant caries index (sic) over time. bmc oral health. 2011, 11: 12. 7. sheiham a, alexander d, cohen l, marinho v, moyses s, petersen pe, et al. global oral health inequalities: task group—implementation and delivery of oral health strategies. adv dent res. 2011; 23: 259-67. 8. pattussi mp, hardy r, sheiham a. the potential impact of neighborhood empowerment on dental caries among adolescents. community dent oral epidemiol. 2006; 34; 344-50. 9. brazil. ministry of health. secretary of health care. department of primary care. national coordination of oral health. national policy on oral health. guidelines. brasília: ministry of health; 2004. 16p . 10. brazilian institute of geography and statistics – ibge. 2010 census. education and displacement [accessed 2013 apr 2]. available from: http://www.ibge.gov.br/estadosat/ temas.php?sigla=rj&tema=censodemog2010_educ. 11. world health organization. oral health survey: basic methods. 4. ed. geneva: world health organization; 1997. 12. bratthall d. introducing the significant caries index together with a proposal for a new oral health goal for 12-year-olds. int dent j. 2000; 50: 378-84. 13. cortellazzi kl, tagliaferro eps, assaf av, tafner apmf, ambrosano gmb, bittar to, et al. influence of socioeconomic, clinical and demographic variables on caries experience of preschool children in piracicaba, sp. rev bras epidemiol. 2009; 12: 1-11. 14. hobdell mh, myburgh ng, kelman m, hausen h. setting global goals for oral health for the year 2010. int dent j. 2000; 50: 245-9. 15. guerra lm, pereira ac, pereira sm, meneghim mc. assessment of socioeconomic variables in the prevalence of caries and fluorosis in municipalities with and without fluoridated water supplies. rev odontol unesp. 2010; 39: 255-62. 16. benazzi ast, silva rp, meneghim mc, pereira ac, ambrosano gmb. trends in dental caries experience and fluorosis prevalence in 12-year-old brazilian schoolchildren from two different towns. braz j oral sci. 2012; 11: 62-6. 17. freire mcm, leles cr, sardinha lmv, paludetto jr m, malta dc, peres ma. dental pain and associated factors in brazilian adolescents: the national school-based health survey (pense), brazil, 2009. cad saude publica. 2012; 28(suppl): s133-45. 18. lisboa cm, paula js, ambrosano gmb, pereira ac, meneghim mc, cortellazzi kl, et al. socioeconomic and family influences on dental treatment needs among brazilian underprivileged schoolchildren participating in a dental health program. bmc oral health. 2013, 13: 56. 19. traebert j, jinbo y, lacerda jt. association between maternal schooling and caries prevalence: a cross-sectional study in southern brazil. oral health prev dent. 2011; 9: 47-52. 20. machry r v, tuchtenhagen s, agostini b a, teixeira c r s, piovesan c, mendes f m et al. socioeconomic and psychosocial predictors of dental healthcare use among brazilian preschool children. bmc oral health. 2013, 13: 60. 21. nadanovsky p, sheiham a. relative contribution of dental services to the changes in caries levels of 12-year-old children in 18 industrialized countries in the 1970s and early 1980s. community dent oral epidemiol. 1995; 23: 331-9. 22. antunes jl, narvai pc. dental health policies in brazil and their impact on health inequalities. rev saude publica. 2010; 44: 360-5. 23. amaral rc, batista mj, meirelles mpmr, cypriano s, souza mrl. dental caries trends among preschool children in indaiatuba, sp, brazil. braz j oral sci. 2014; 13: 1-5. 24. ferraro m, vieira ar. explaining gender differences in caries: ( a multifactorial approach to a multifactorial disease. int j dent. 2010; 2010:649643. doi: 10.1155/2010/649643. 139 dental caries in inland brazilian adolescents and its relationship with social determinants braz j oral sci. 13(2):133-139 oral sciences n3 braz j oral sci. 13(3):235-241 original article braz j oral sci. july | september 2014 volume 13, number 3 using molecular markers to assess streptococcus mutans variability and the biological risk for caries ivana froede neiva1, mônica moreira1, renata rodrigues gomes2, debora klisiowicz2, ricardo lehtonen rodrigues souza3, vânia aparecida vicente1,2 1universidade federal do paraná – ufpr, department of chemical engineering, areas of engineer of bioprocess and biotechnology curitiba, pr, brazil 2universidade federal do paraná – ufpr, department of basic pathology, areas of microbiology, parasitology and pathology, biological sciences, curitiba, pr, brazil 3universidade federal do paraná – ufpr, department of genetics, curitiba, pr, brazil correspondence to: vânia aparecida vicente universidade federal do paraná ufpr area of biological sciences caixa postal: 19020 cep 81.531-980 curitiba, pr, brasil phone: +55 41 3361 1697 fax: +55 41 3266 2042 e-mail: vicente@ufpr.br received for publication: august 22, 2014 accepted: september 16, 2014 abstract aim: to characterize the genetic variability of streptococcus mutans isolates and to correlate this variability with different colonization profiles observed during dental caries in a sample of children. methods: s. mutans samples were isolated from the saliva of 30 children with varying histories of dental caries, and they were characterized according to morphological and biochemical markers and the sequences of their 16s-23s intergenic spacer region. the genetic variability of the isolates was first assessed using random amplified polymorphic dna (rapd) markers. next, the isolates were differentiated by sequencing a specific region of the gene encoding the enzyme glucosyltransferase b (gtfb). results: characterization using rapd markers uncovered significant genetic variability among the samples and indicated the existence of clusters, which allowed us to reconstruct both the origin and clinical history of the disease. by sequencing the 16s-23s intergenic region, it was found that all of the isolates belonged to the species s. mutans. based on the genetic similarity of the isolates and pattern of amino acid variations identified by partial sequencing of the gtfb gene, base-pair changes were identified and correlated with different virulence patterns among the isolates. conclusions: the partial sequencing of the gtfb gene can be a useful tool for elucidating the colonization patterns of s. mutans. as amino acid variations are likely to be correlated with differences in biological risk, molecular characterization, such as that described in this paper, could be the key for assessing the development of dental caries in children. keywords: streptococcus mutans; dental caries; random amplified polymorphic dna technique; glucosyltransferase. introduction caries is an infectious and transmissible disease associated with bacterial colonization of dental surfaces1. due to its multifactorial nature and microbial origin, the severity and prevalence of caries can be greatly affected by the endogenous conditions within each host individual2. several studies have demonstrated that the colonization and accumulation of streptococcus mutans is associated with dental caries in humans, since they are influenced by various factors in the oral cavity, such as nutrition and hygiene conditions of the host, salivary components, cleaning power and salivary flow braz j oral sci. 13(3):235-241 236236236236236 and characteristics related with microbial virulence factors3. streptococcus mutans is the key contributor to the formation of polysaccharide-based extracellular matrices in dental biofilms4. glucosyltransferases synthesize exopolysaccharides, which are glucans that promote the accumulation of microorganisms at specific sites on dental surfaces, and glucosyltransferases become enzymatically active when exposed to dietary sucrose. streptococcus mutans is widely distributed not only among populations with a moderate-to-high incidence of caries5 but also among populations with little or no incidence of the disease6, which suggests that colonization by this microorganism alone is not sufficient for the development of caries5. therefore, the presence of s. mutans in individuals with low caries experiences could be explained by differences in bacterial virulence factors or endogenous factors within the host populations. this microorganism is only one member of the indigenous oral biota that plays roles in “severe early childhood caries” (s-ecc). as most humans are host to s. mutans but not all carriers manifest dental caries, it has been suggested that certain strains of s. mutans associated with secc could be genetically different from strains found in caries-free individuals7. molecular markers have been used to elucidate the genetic variability among s. mutans isolated from saliva and other sources. rapd (random amplified polymorphic dna) markers have been most widely used for this purpose8-10. in addition, igs sequencing (e.g., sequencing of intergenic regions between the 16s and 23s rrna genes) has been used to taxonomy studies, while partial sequencing of the gene gtfb, which encodes the enzyme glucosyltransferase b, has been used to investigate enzymatic activity and virulence11-12. in the present study, samples of s. mutans isolated from the saliva of children with varying histories of dental caries were characterized using morphological and biochemical markers, 16s-23s igs sequencing, and rapd markers. these isolates were further distinguished by sequencing specific regions of the gtfb gene to characterize the genetic variability at this locus and correlate this information with the colonization profiles of s. mutans observed in the children. material and methods case series and strains for the present study, it was selected 44 strains of streptococcus mutans isolated from the saliva of 30 children aged 6-9 years, attending rural schools affiliated with the university extension program “multidisciplinary strategy for the prevention and control of diseases manifesting clinically in childhood”13. the sample of children exhibiting a range of epidemiological profiles of dental caries was initially characterized according to the dmft (the number of permanent decayed, missing, or filled teeth) and deft (deciduous teeth) metrics. the isolation of the strains and estimation of colony-forming units (cfu) per ml of saliva were performed using mitis salivarius agar. based on the biochemical evidence, as well as factors identified during the clinical interviews and examinations, the 44 s. mutans isolates were divided into three host groups (table 1). the study protocol was approved by the research ethics committee of the federal university of paraná (ufpr), number 085.si 048/04-06. informed consent was provided by the children’s legal representatives. dna extraction and rapd analysis dna extraction was performed according to moreira et al.14 (2010) by applying an ultrasound to a combination of silica and celite (2:1) in ctab (cetyltrimethylammonium bromide). for the rapd reactions, the following six oligonucleotide primers were used: opa 2 (5’ tgccgagctg 3’), opa 3 (5’ agtcagccac 3), opa 5 (5’ aggggtcttg 3’), opa 8 (5’ gtgacgtagg 3’), opa 9 (5’ gggtaacgcc 3’) and opa 13 (5’ cagcacccac 3’). the pcr was performed using the conditions as described previously9. the streptococcus mutans strains atcc 25175 and ua159 were included as reference samples during the amplifications, and the strains s. sobrinus atcc 33478 and s. pyogenes atcc 13540 were included as external control groups. the products resulting from the rapd amplification were analyzed by electrophoresis through 1.6% agarose gels. an analysis of polymorphisms was performed using the non-weighed pair group method with arithmetic mean (upgma)15 (with an estimated jaccard similarity coefficient) using the ntsys 2.1 software program16. the consistency of the clustering was verified by bootstrap analysis with 10,000 re-samplings using the bood 3.03 software program17; clusters exhibiting p values equal to or higher than 75% were considered consistent. amplification and sequencing of the 16s-23s intergenic region and gtfb gene the 16s-23s intergenic region was amplified from the total dna taken from the s. mutans isolates using primers 13bf (5’ gtgaatacgttcccgggcct 3’) and 6r (5’ gggttyccccrttcrgaaat 3’)18. in order to sequence a region of the gtfb gene, the following primers were used: gtfb-f (5’ acttacacacttttcgggtggcttgg 3’) and gtfb-r (5’ cagtataagcgccagtttcatc 3’)19. the sequencing reactions were composed as follows: 50 ng of pcr product purified with peg (polyethylene glycol) according to murphy et al.20 (2005); 125 µm each dntp; 50 nm each primer; 0.5 µl big-dye terminator v 3.1 cycle sequencing kit; 0.5 µl buffer and ultrapure water for a final volume of 10 µl. the amplification conditions were as follows: 1 min at 96 ºc, followed by 35 cycles at 96 ºc for 10 s and 50 ºc for 5 s with a final elongation step at 60 ºc for 4 min. following lyophilization in a speedvac at 60 ºc for 40 min, the samples were sequenced using electrophoresis in an abi prism 377 sequencer (applied biosystems). the sequences were analyzed and corrected using the staden package software program21 and aligned using a version of clustal-w22. in order to obtain the nucleotide and amino acid compositions and calculate the variable nucleotide distances and positions, we used the using molecular markers to assess streptococcus mutans variability and the biological risk for caries 1 7 11 7 11 7 11 7 11 7 1237237237237237 braz j oral sci. 13(3):235-241 clinical group factors isolate c h i l d dmft deft caries activity sm01 a 0 4 yes group i cfu/ml sm02 b 0 5 yes caries sm03 c 0 6 yes sm04 d 0 6 yes sm05 e 4 0 yes sm06 f 1 2 yes sm07 g 0 5 yes sm08 h(1) 4 8 yes sm09 h (2) 4 8 yes sm10 i 0 4 yes sm11 j(1) 0 5 yes sm12 j(2) 0 5 yes sm13 j(3) 0 5 yes sm14 k(1) 4 5 yes sm15 k(2) 4 5 yes sm16 l(1) 1 7 yes sm17 l(2) 1 7 yes sm18 m 1 4 yes sm19 n 2 4 n o group ii cfu/ml sm20 o(1) 4 0 yes caries sm21 o(2) 4 0 yes sm22 p 0 2 yes sm23 q 0 4 yes sm24 r 0 5 yes sm25 s(1) 0 6 yes sm26 s(2) 0 6 yes sm27 t 0 2 yes sm28 u 2 3 yes sm29 v(1) 0 0 n o group iii cfu/ml sm30 v(2) 0 0 n o caries sm31 w(1) 0 0 n o sm32 w(2) 0 0 n o sm33 x(1) 0 0 n o sm34 x(2) 0 0 n o sm35 x(3) 0 0 n o sm36 y(1) 0 0 n o sm37 y(2) 0 0 n o sm38 z 0 0 n o sm39 α 0 0 n o sm40 &(1) 0 1 n o sm41 &(2) 0 1 n o sm42 € 0 1 n o sm43 € 0 1 n o sm44 ¥ 1 0 n o table 1.table 1.table 1.table 1.table 1. the s. mutans isolates used in this study according to the clinical assessment groups 0 to 100 cfu/ml = cfu; above 100,000 cfu/ml = cfu; or caries = higher or lower history of caries, respectively; each child in the studied was assigned a unique letter or symbol (a-z and α, &, €, or ¥). mega 3 software program23. reference strains sequence data were taken from the genbank. results forty-four s. mutans isolates were analyzed in this study. based on the amplification profiles of the rapd markers, polymorphisms were identified among the investigated isolates, and further analysis clustered the isolates into seven groups with bootstrap values above 75% (table 2). thus, isolates from different genetic groups determined by rapd markers, were characterized by intergenic regions (16s-23s) sequencing of rdna and identified as s. mutans (figure 1). based on the analyzed sequences of intergenic using molecular markers to assess streptococcus mutans variability and the biological risk for caries 238238238238238 braz j oral sci. 13(3):235-241 isolates origin/substrate clinical group* bootstrap sm01-sm03 human host**/saliva group i 80% sm08-sm09 human host***/saliva group i 99% sm11-sm12-sm13 human host***/saliva group i 83% sm20-sm21 human host***/saliva group ii 88% sm32sm33 human host**/saliva group iii 80% sm35sm37 human host****/saliva group iii 88% sm39-sm41-sm42 human host**/saliva group iii 75% * clinical group established by epidemiological data of dental caries and s. mutans concentration in saliva (table 1) ** different individuals, ***same individual, ****siblings table 2. table 2. table 2. table 2. table 2. clustering based on rapd markers of the s. mutans isolates derived from saliva samples of children fig. 1. sequence analyses of the 16s-23s igs region of various s. mutans isolates (sm1, sm2, sm6, sm12, sm18, sm20 and sm37) and reference strain s. sobrinus atcc 33478. the tree was constructed using the neighbor-joining method (two-parameter kimura). fig. 2. partial sequencing of the gtfb gene of s. mutans grouped using the neighbor-joining method (two-parameter kimura); s. mutans strain atcc 25175 was used as a reference; sm 1 to sm 37 isolates of s. mutans; d88651 to d89977 represents the nucleotide sequences of the gtfb region of s. mutans deposited in genbank. using molecular markers to assess streptococcus mutans variability and the biological risk for caries 1 7 11 7 11 7 11 7 11 7 1239239239239239 braz j oral sci. 13(3):235-241 region, it was verified the existence of genetic variability among these isolates. the isolates sm01, sm02, sm06, sm12 and sm37 exhibited a high degree of genetic similarity among them (figure 1). the isolates sm01 and sm12 (group i, table 1) showed 100% of similarity. the strain 33478 of s. sobrinus, which was included as an external control group, clustered at a distance from the experimental samples. the characterization of isolates by partial sequencing of the gene encoding the enzyme glucosyltransferase b (gtfb) was also done. as shown in figure 2, isolates sm01, sm02, sm06 and sm12, originated from individuals with similar disease histories (table 1), clustered in the same group; isolates sm01 and sm12 were found to be identical with respect to the analyzed sequences, although they originated from different individuals, but with the same patterns of clinical manifestation, i.e. high caries status (table 1). the analyzed isolates exhibited amino acid sequence variations, which we compared with the sequences of the reference strains as previously described by fujiwara et al.11 (1998). the s. mutans reference strain (mt4239)11 clustered with isolates sm18, sm19, sm20 and sm37 (figure 2), which were similar with respect to amino acid composition in the position 36 of the enzyme (table 3). discussion among the 30 children evaluated in the study, 13 exhibited high concentrations of s. mutans in their saliva (> 105 cfu/ml) and had significant histories of disease (both dmft and deft), whereas 17 children exhibited lower concentrations of s. mutans (<102 cfu/ml) and mostly had only mild histories of disease (table 1). these data indicate a positive correlation between s. mutans concentrations in saliva and the clinical manifestation of caries. however, among the children exhibiting low levels of colonization, one group (group ii) had dental caries, despite having low position* isolates/reference strains of s. mutans 30 36 46 54 57 68 110 120 121 136 141 s. mutans sm01 s a q s s s t n i k s. mutans sm02 . . . . . . . s l . s. mutans sm06 . . . . . . . . l . y s. mutans sm12 . . . . . . . . . . s. mutans sm18 . v . . . n a . l . s. mutans sm19 . v . . . n . . l . s. mutans sm20 . v . . . n a . l . s. mutans sm37 . v . . . n . . l . s. mutans 25175** . . . . . . . . l . s. mutans mt4239 t v . i . . . . l . s. mutans mt4245 . . . . . . . . l . s. mutans mt4251 . . p . f . . . l n s. mutans mt4467 . . . . . . . . l . s. mutans mt8148 . . . . . . . . l . table 3.table 3.table 3.table 3.table 3. amino acid differences in the gtfb gene between s. mutans isolates and reference strains * aminoacid sequence variation : . similar nucleotides, insertion/deletion ** reference strain s. mutans atcc 25175 concentrations of s. mutans in their saliva, which suggests that differences might also exist with respect to the virulence of the isolates. according to the polymorphisms identified by the rapd markers, it was identified a relation between genetic groups of s. mutans and epidemiological data of disease (table 2). it was also verified the clustering of isolates from different individuals, supported by high bootstrap values, suggesting that isolates can be transmitted among individuals. such results correspond to what has been previously reported by domejean et al.8 (2010) who used arbitrary primers and verified the presence of s. mutans corresponding genotypes, proving the horizontal transmission of bacteria among schoolchildren, aging between 5 and 6 years old. furthermore, according to the results presented in table 2, it was verified two isolates grouping proceeding from the same individual (e.g., sm1113 and sm20-21). furthermore, these results (table 2) demonstrate the diversity among isolates, correlated with patterns of variation in the clinical manifestation of the disease, suggesting that each individual may exhibit unique patterns of colonization by s. mutans that could be due host’s endogenous factors, since these individuals had a common pattern of diet, characterized by frequent consumption of refined carbohydrates. according to kreth et al.24 (2005), human mucosal surfaces are colonized by large numbers of bacterial species; these populations exist in a state of homeostasis and play an important role in protecting the host against invasion by exogenous pathogens. however, when this homeostasis is disrupted, indigenous flora can cause pathologies, such as dental caries and periodontal disease. additionally, high colonization levels and diverse range of s. mutans genotypes could be a consequence of frequent consumption of fermentable carbohydrates, and it is possible that the simultaneous action of multiple s. mutans strains with different cariogenic potentials could increase the risk of using molecular markers to assess streptococcus mutans variability and the biological risk for caries 240240240240240 developing dental caries25. in addition, when investigating the children’s oral microbiota, crielaard et al.26 (2011), showed that the salivary microbiota of children aged 3 to 18 years are still maturing and that multiple colonization events can occur during this period. the isolates from different genetic groups determined by rapd markers (table 2), were characterized by intergenic regions (16s-23s) sequencing of rdna, and it was verified the existence of genetic variability among these isolates. the isolates sm01 and sm12 (group i, table 1), which were originated from different children, showed 100% similarity. this result suggested the transmissibility of s. mutans among schoolchildren (figure 1). this way, it can notice that the variability among the isolates, detected by the igs sequencing (figure 1), is not related to the virulence, but may indicate the route of transmissibility. according to the literature, the activity of glucosyltransferase b enzyme seems to be directly related to the adherence of s. mutans to dental enamel4-27. upon partial sequencing of the gtfb gene, five different genotypes were identified among the analyzed isolates. the nucleotide sequences differences were predicted to result in amino acid variations that could influence the patterns of virulence among the isolates. figure 2 shows the grouping of the isolates originating in children with varying disease histories, which were clustered according to their genetic similarity identified by partial sequencing of the gtfb gene. the finding of isolates sm01 and sm12 clustered in the same group, and originated from different individuals with the same patterns of clinical manifestation, i.e. high caries status (table 1), suggests the existence of a similar virulence potential for these two isolates. the genetic similarity of isolates sm20 and sm18 proceeding from individuals with different patterns of colonization, as evidenced by differences in saliva cfu/ml counts (clinical groups i and ii, table 1) and high disease prevalence, also suggested a similar pattern of virulence in these isolates. the isolates sm19 and sm37, proceeding from children with low concentration of s. mutans and with different caries historic (clinical group ii and iii, table 1), was grouped with the latter, indicating that both exogenous and endogenous host factors also might influence disease development. based on the results of amino acid sequence variations described in table 3, amino acid positions ranging from 30 to 141 appeared to be susceptible to variation of s. mutans gtfb gene. according to the literature, the presence of polymorphisms in glucosyltransferase (gfts) genes expressed by different strains of s. mutans is likely to be associated with variations in specific enzymatic activity28. alterations by the site-directed mutagenesis of a single amino acid in the catalytic domain of gtfs are sufficient to considerably change the enzymatic activity of this protein, leading to differences in dental surface adherence patterns for the investigated strains of s. mutans. it is important to emphasize that the isolate sm06 exhibited an insertion of tyrosine at position 141, which differed from the rest of the isolates (table 1). such modification would be likely to influence enzymatic activity, as reports indicate that this is the most highly conserved position in gtfb due to its location within the catalytic domain29. this hypothesis is consistent with the observed pattern of virulence for isolate sm06, which was originated in an individual with a considerable clinical history of disease. in addition, glucosyltransferases produced by s. mutans are also known to play an important role in virulence due to their effects on dental biofilms, according to phylogenetic analysis of glucosyltransferases and implications for the coevolution of mutants streptococci with their mammalian host, as reported by argimón et al.30 (2013). glucosyltransferases can be adsorbed directly onto enamel and catalyze the synthesis of glucans in situ; this promotes sites of avid colonization by microorganisms and helps to create an insoluble matrix of dental bacterial plaque27, which might also favor the development of disease. the occurrence of groups containing children with a high clinical manifestation of disease and low indices of s. mutans in their saliva indicates that in addition to the endogenous and exogenous factors present in each individual, the presence of genetic variations must also be taken into account when attempting to estimate the virulence of wild biotypes of s. mutans. in conclusion, this study demonstrates that partial sequencing of the gene encoding the enzyme glucosyltransferase b can be a useful tool for elucidating the virulence patterns of s. mutans because amino acid variations are likely to justify the differences in colonization pattern of these strains, which could reflect a biological risk of development the dental caries in these children. acknowledgements the authors are grateful for the support of the ufpr, capes (federal agency of support and evaluation of postgraduate education/coordenação de aperfeiçoamento de pessoal de nível superior), cnpq and the 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303-22. 22. thompson jd, higgins dg, gibson tj. clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. nucleic acids res. 1994; 22: 4673-80. 23. kumar s, tamura k, nei m. mega3: integrated software for molecular evolutionary genetics analysis and sequence alignment. brief bioinform. 2004; 5: 150-63. 24. kreth j, merrit j, shi w, qi f. competition and coexistence between streptococcus mutans and streptococcus sanguinis in the dental biofilm. j bacteriol. 2005; 187: 7193-203. 25. alaluusua s, matto j, gronroos, l, innila, s, torkko, h, asikainem, s, et al. oral colonization by more than one clonal type of mutans streptococci in children with nursing-bottle dental caries. arch oral biol. 1996; 41: 167-73. 26. crielaard w, zaura e, schuller aa, huse sm, montijn rc, keijser bj. exploring the oral microbiota of children at various developmental stages of their dentition in the relation to their oral health. bmc med genomics. 2011; 4: 22. 27. bowen wh, koo h. biology of streptococcus mutans-derived glucosyltransferases: role in extracellular matrix formation of cariogenic biofilms. caries res. 2011; 45: 69-86. 28. chia js, hsu ty, teng lj, chen jy, hahn lj, yang cs. glucosyltransferase gene polymorphism among streptococcus mutans strains. infect immun. 1991; 59: 1656-60. 29. fujiwara t, hoshino t, ooshima t, hamada s. differential and quantitative analyses of mrna expression of glucosyltransferases from streptococcus mutans mt8148. j dent res. 2002; 81: 109-13. 30. argimón s, alekseyenko av, desalle r, caufield pw. phylogenetic analysis of glucosyltransferases and implications for the coevolution of mutans streptococci with their mammalian hosts. plos one 2013; 8: e56305. braz j oral sci. 13(3):235-241 using molecular markers to assess streptococcus mutans variability and the biological risk for caries oral sciences n3 original article braz j oral sci. july | september 2013 volume 12, number 3 oral lesions frequency in hiv-positive patients at a tertiary hospital, southern brazil cristiane pedroso peppes1, ana silvia pavani lemos1, renata lins fuente araujo1, magda eline guerrart portugal1, marilene da cruz magalhães buffon1, sonia mara raboni1 correspondence to: sonia mara raboni serviço de infectologia, hospital de clínicas da universidade federal do paraná rua general carneiro 180, 3° andar cep: 800060-900 – curitiba, pr, brasil fax: +55 41 33601811 phone: +55 41 33607974 e-mail: sraboni@ufpr.br raboni.sonia@gmail.com received for publication: may 15, 2013 accepted: september 11, 2013 abstract aim: to report the frequency of oral lesions in hiv-positive patients on highly active antiretroviral therapy (haart), comparing with a non-hiv infected control group, and to correlate the presence of lesions with demographic and clinical features of hiv-seropositive patients. methods: a quantitative case-control study was conducted by a dental professional, using a questionnaire, analysis of medical records of patients and clinical examinations. results: according to the results, oral lesions were found in 23% of hiv-positive patients versus 5% in controls. candidiasis (29%) and periodontal changes (25%) were the most frequent oral lesions found in these patients. gender and viral load values were statistically significant when hiv-positive patients with and without oral lesions were compared. conclusions: the results showed a change in lesion pattern of hiv patients on haart, highlighting a high frequency of these new lesions and reinforcing the need for periodic dental evaluation of hiv-positive patients. keywords: hiv, bacterial infections, candidiasis, opportunist infections. introduction aids has been a serious worldwide public health threat, with a global prevalence in 2010 of about 34 million and an overall number of aids-related deaths of 1.8 million of people. in brazil up to the end of 2011, 608,200 cases were notified, 397,662 (65.4%) in males and 210,538 (34.6%) in females. currently, investments in prevention, scaling up access to hiv testing and to antiretroviral treatment, as well as training of health professionals, keep the aids epidemic under control1. according to the brazilian ministry of health, the prevalence of the disease in 2011 remained stable at around 0.6%, while the incidence was 17.9/100,000 inhabitants. in the state of paraná, southern brazil, 32,273 cases were reported in the same period, with an incidence of 19/100,000 inhabitants. the southern region ranks third in the number of aids cases in brazil, with 21.1% of cases, after the southeast (38.2%) and northeast (21.7%). nevertheless, this is the region where the largest number of new cases were detected in 2009, with a detection rate of 12.6/100,000 inhabitants1. oral manifestations are frequently seen during the course of the disease and can be symptoms of early hiv infection. with their immune system severely compromised by hiv infection, patients are vulnerable to opportunistic infections and cancers, particularly oral lesions such as oral candidiasis, kaposi’s sarcoma, braz j oral sci. 12(3):216-222 1federal university of paraná, curitiba, pr, brazil sores, gingivitis, periodontitis and herpes lesions. the emergence of these lesions is associated with immunodeficiency, and they are often the first symptom of aids or an indication of worsening of disease. hiv patients with associated oral lesions often complain of chewing problems, burning sensation, and difficulty in swallowing and speech among others, which has a negative impact on their quality of life2-4. a wide spectrum of oral manifestations may occur in these patients dependent on factors such as degree of immune impairment, use of antiretroviral therapy, oral hygiene, among others5. since these oral manifestations associated to the progression of hiv infection are common and may be the first clinical signs of disease, the dentist has a very important role in detecting and diagnosing the disease in its earliest stages and in providing appropriate treatment to the patient6. oral manifestations in hiv-positive patients have been the subject of many studies in the pre-haart era. these studies have become less frequent after the introduction of the haart. the aims of this study are to report the frequency of oral lesions in hiv patients on haart, compared with a non-hiv infected control group and correlate the lesion with demographic and clinical features of hiv-seropositive patients. material and methods study design a cross-sectional study was conducted from november 2009 through november 2010 on hiv-positive patients followed at the infectious diseases service at hospital de clínicas, universidade federal do paraná (hc/ufpr). the study was approved by the ethics committee in research on human beings of the hc/ufpr under protocol number 1943.110/2009-05. casuistic the hiv-positive patients and a control group were included in the study after providing written informed consent. a questionnaire was used to collect clinical data based on the analysis of the medical records, obtain demographic and behavioral information provided by the patient and report clinical examinations performed by one dentist using a visual examination method. to select the control group, the authors conducted a search on clinics that offered free dental treatment, such as clinics of trade unions for care workers, which presented socioeconomic level and educational instruction similar to the hiv-positive patients. subjects were randomly invited to participate in the study, and those who accepted and signed the informed consent were examined. from 596 individuals analyzed, 208 that met the criteria for median age similar to the patients of the study groups and previous negative hiv antibody test within 6 months were included as controls. the questionnaire applied to this group was similar to the one used in the study group. sample size calculation sample size calculation was based on the following estimates: α error of 0.05; β error= 0.20 (80% power). for the purposes of these calculations the prevalence of oral lesion in hiv+ patients was arbitrarily set at 15% and in control group at 5%. considering that the number of hiv+ treated at this hospital is around 1,000, the finite population correction factor was used to reduce the standard error. incorporating these estimates into the equation yields a sample size of 164, assuming a 20% loss, at least 197 were required per group. statistical analysis descriptive analysis was performed for all variables. data were compiled using the jmp software version 5.2.1 and analyzed by the graphpad prism software version 5.03. fisher’s exact or÷2 tests were used to assess differences between groups and mann whitney test was used for continuous variables, as appropriate. results of continuous data are expressed as median ± interquartile range (iqr). all p-values were two-tailed and a value of <0.05 was considered significant. results a total of 241 hiv-positive patients and 208 control individuals were enrolled in the study. the control group showed a similar profile to the study group with relation to age and years of education. the median age of the patients was 41 and 36 years for hiv-positive and control group, respectively, and 63% of hiv-positive individuals and 60% of controls had between 4 and 11 years of education. other demographic and epidemiological findings are shown in table 1. hiv-positive patients had been diagnosed with hiv infection for an average of 8.4 (±4.9) years and presented a median of nadir and current cd4+t lymphocyte count of 276.5 cells/mm3 (iqr, 123.3–502.3) and 426 cells/mm3 (iqr, 264–630), respectively. most patients (78.6%) were on haart and in 167 cases (69%) viral load was undetectable. among those with detectable viral load, the median was 3,737 copies/ml (iqr 558 – 26,267), 20.3% had <10,000 copies/ ml, 7% had between 10,000 and 100,000 copies/ml, and only 2.9% of patients had more than 100,000 copies/ml. oral lesions were found in 56/241 (23%) of hiv-positive patients versus 10/208 (5%) in controls (p<0.001). as shown in figure 1, the most frequent lesions in hiv-positive patients were candidiasis and periodontal changes, while herpes lesions were more common in the control group, which was found in 3.5% (2/56) of hiv-positive patients and in 80% (8/10) of control group. idiopathic white patch and ulcerations of oral mucosa were observed in 23% and 5% of hiv-positive patients, respectively. comparison between hiv-positive patients with and without lesion showed that they had similar clinical characteristics, except for gender and viral load values that showed statistically significant differences (table 2). there was a predominance of male patients with lesions (p=0.03), 217217217217217oral lesions frequency in hiv-positive patients at a tertiary hospital, southern brazil braz j oral sci. 12(3):216-222 gender (%) male female age (y) median (iqr) race (%) white brown black yellow ni marital status (%) married single divorced ni sexual behavior (%) m s m heterosexual bisexual ni other risk factors idus blood transfusion or clotting factor occupational exposure ni years of education (%) 1 3 4 – 7 8 – 11 >12 none ni tooth brushing habits (%) 1 x d a y 2 x d a y 3 x d a y ni smoking (%) yes n o ni hiv-positive patients n = 241 102 (42) 139 (58) 41 (32-48) 140 (58) 77 (32) 22 (9) 1 (0.5) 1 (0.5) 82 (34) 158 (65.5) 1 (0.5) 15 (6) 107 (45) 7 (3) 112 (46) 8 (3) 7 (3) 3 (1) 92 (38) 23 (10) 82 (34) 70 (29) 57 (23) 7 (3) 2 (1) 19 (8) 76 (31) 142 (59) 4 (2) 62 (25.5) 178 (74) 01 (0.5) control group n = 208 114 (55) 94 (45) 36 (28-43) 138 (66) 62 (30) 8 (4) 122 (58.5) 67 (32.5) 19 (9) 11 (5) 197 (95) 0 0 0 0 0 not applicable 3 (1)64 (31)81 (39)59 (28.5)1 (0.5) 3 (2) 58 (28) 147 (70) 28 (13) 180 (87) p value 0.008 0.0012 0.07 <0.0001 <0.0001 0.0003 0.001 0.0013 table 1. demographic and epidemiological characteristics of hiv-positive patients and control group individuals ni – not informed. iqr – interquartile range. msm men who have sex with men. idus injecting-drug users the median values of nadir and current cd4+ t lymphocyte detection were 221(67.3 – 451.8) cells/mm3 and 293(147.5 – 502.5) cells/mm3, 64% and 61% had undetectable load viral and median values of viral load were 9,144 (693 – 78,321) copies/ml and 3,018 (553 – 17,293) copies/ml (p=0.02), respectively. discussion the spread of hiv infection in brazil has revealed an epidemic of multiple dimensions, with a significant change in the epidemiological profile over the years, following the global trend. initially restricted to large urban centers and 218218218218218 oral lesions frequency in hiv-positive patients at a tertiary hospital, southern brazil braz j oral sci. 12(3):216-222 markedly masculine, the hiv/aids epidemic is currently characterized by an increase in heterosexual transmission, with a substantial increase in number of cases among women and injecting drug users, spread of disease to small and midsized cities, and pauperization of the infected population7. since 1996 brazil has provided free antiretroviral drugs to all patients with hiv/aids, according to a guideline for the use of antiretroviral agents in hiv-1 infected adults and adolescents. the consolidation of the care policy for people living with hiv in brazil has produced considerably positive results. until 2005, aids-related mortality rate was reduced by almost 40% and the median survival rate of people living with hiv increased from 58 months in 1995-1996 to 108 months in 1998-19998. the availability of antiretroviral drugs on a large-scale has contributed to reduce the frequency and change the pattern of oral manifestations, and, thus, many complaints tended to be overlooked by healthcare professionals. also, the causes of oral diseases in hiv-positive patients were little investigated. this study showed a higher frequency of oral lesions in hiv-positive patients compared with the control group, highlighting the importance of oral clinical examination of these patients, as well as of detection and diagnosis of hiv age (y) median (iqr) gender male/female years of study (%) 1 3 4 – 7 8 – 11 >12 none ni smoking (%) yes n o ni nadir cd4+ t-cells count *median (iqr) current cd4+ t-cells count *median (iqr) viral load** <50 copies/ml (%) median (iqr) haart (%) yes n o hiv-positive patients with oral lesions n = 56 43 (32 – 50) 31/25 9 (16) 21 (38) 14 (25) 8 (14) 3 (5) 1 (2) 17 (30) 39 (70) 221 (67.3 – 451.8) 390 (264 – 540.3) 26 (46) 9.144 (693 – 78.321) 45 (80) 11 (20) hiv-positive patients without oral lesions n = 185 41 (32.2 – 48) 71/114 14 (7.5) 61 (33) 56 (30.5) 49 (26.5) 4 (2) 1(0.5) 45 (24.5) 139 (75) 1 (0.5) 293 (147.5 – 502.5) 447 (310 – 639) 131 (71) 3.018 (553 – 17.293) 144 (78) 41 (22) p value 0.35 0.02 0.08 1.00 0.36 0.57 0.23 0.02 0.84 table 2. clinical, epidemiological and demographic data from hiv-positive patients with and without oral lesions iqr – interquartile range. * cells/mm3. ** copies/ml. ni: not informed. in patients with these symptoms by the dentist. the success or failure of antiretroviral therapy is assessed by viral load and cd4+ t lymphocytes count. undetectable viral load indicates a strong control of viral replication and, therefore, reduced damage to the immune system4. this study showed that 69.3% of patients had undetectable viral load, which shows the success of therapy in most patients. of all the patients with oral lesions, 80% (45/56) were on regular treatment and 20% (11/56) were not on haart. in both groups, periodontal changes were the most common lesions followed by oral candidiasis and idiopathic white patch or leukoplakia, which is an area of keratosis that appears as adherent white spots on the mucous membranes of the oral cavity. it consists predominantly in white lesion of the oral mucosa that cannot be characterized as any other definable lesion. therefore, there was no statistical difference in the prevalence of oral manifestations or in the distribution of lesions between patients on haart and those not on haart. the prevalence of oral lesions in untreated patients was similar to that found previously6, which showed that 20% of patients not on haart had some type of oral lesion. likewise, nittayananta et al. 9 (2002) showed that hyperpigmentation was a very frequent lesion in patients 219219219219219oral lesions frequency in hiv-positive patients at a tertiary hospital, southern brazil braz j oral sci. 12(3):216-222 fig. 1. oral lesions found in hiv-positive patients (n = 56) and control group individuals (n = 10) using haart, which was considered a consequence of zidovudine use, and compared with patients not on haart, patients on haart were at lower risk of caries and periodontal disease. in this study, the frequency of periodontal changes was similar in both groups, regardless of drug use. despite the advances represented by antiretroviral drugs, their serious side-effects may reduce the levels of patient adherence to treatment. these effects may also be associated with oral manifestations, such as sore throat, dry mouth and perioral paresthesia5, such findings were not observed in this study group. hiv-positive patients may have decrease in the salivary flow, which can result from the viral infection or be a side effect of antiretroviral therapy. thus, these patients may show higher caries prevalence, higher rates of periodontal disease, mucositis and opportunistic infections, as well as salivary gland lesions, that signs include swollen glands and a decrease in salivation5. candidiasis was the most prevalent oral manifestation in hiv-positive patients compared with the control group, 29% and 20%, respectively. this infection has been widely associated with hiv infection and was present in all the reviewed articles on the prevalence of oral manifestations in these patients 10. according to shirlaw et al.11 (2002) candidiasis occurs in 50% of hiv-infected individuals and 90% of those with aids. in asymptomatic hiv-infected individuals it is a sign of immunosuppression and often precedes the transition to aids12. periodontal disease (pd) is an inflammatory and chronic bacterial disease, which initially affects the gingival tissues. over time, it can lead to destruction of the tissues that support the teeth. the presence of periodontal involvement (gingivitis, periodontitis, acute necrotizing ulcerative gingivitis anug and acute necrotizing ulcerative periodontitis – anup) is an indication that the individual’s immune system is impaired, and its early recognition may impact hiv-positive patients’ life quality. several studies show that the clinical course of chronic periodontitis is faster in hiv/aids patients compared with hiv-negative individuals. hiv/aids patients have subgingival microorganisms similar to those found in periodontal pockets of hiv-negative individuals. however, in hiv/aids patients there is also the presence of other opportunistic microorganism unrelated to chronic periodontitis, which may be related to the rapid development of periodontal disease. another possible cause of periodontal destruction in hiv patients may be the increased activity of periodontal disease, as a consequence of the high levels of proinflammatory cytokines in the gingival crevicular fluid from sites with active periodontal disease13. in this study, periodontal changes were found in 25% of the cases, while none were found in the control group. of those patients, approximately 40% had current cd4+ t cells counts between 200 and 500 cells/mm 3 and 46% had undetectable viral load. mariano14 did not find significant differences between cases and controls. similarly, other authors did not find a direct relationship between the degree of immunosuppression by hiv and the prevalence of periodontal disease, since most patients with severe periodontal disease were hiv asymptomatic15. these findings were also consistent with the study conducted by scheutz et al.16 (1997) and vastardis, et al.17 (2003), who did not find correlation between cd4+t cells count and severe periodontal disease. laskaris, madjivassilion and stratigos18 (1992) reported the difficulty of correlating periodontal disease with hiv infection, since most patients showed poor oral hygiene, high incidence of dental biofilms, often causing gingivitis and periodontitis. no significant difference was observed regarding tooth cleaning frequency between the groups. therefore, this factor would probably not have influenced these findings. in this study, herpes simplex infection in the lower lip was more common in control group (80%) than in hivpositive patients (3.5%), similarly to chagas, santos and 220220220220220 oral lesions frequency in hiv-positive patients at a tertiary hospital, southern brazil braz j oral sci. 12(3):216-222 ono2 (2009), who reported herpes in 2% of cases. studies conducted in hiv-positive patients in nigeria19 and india20 showed that herpes simplex infection was less prevalent, accounting for 0.9% of patients. only in cambodia the prevalence of herpes simplex lesion was slightly higher, 7.9%21. challacombe, coogan and williams22 (2002) reported that oral herpes are not frequently associated with hiv infection, usually occurring in individuals with hiv infection who develop aids. ulcerations in the oral mucosa that are related to the immune status of the patient are observed in hiv-positive patients23. mouth ulcers are reported in several studies in hiv-infected individuals, with variations in prevalence as seen in india (3.8%)20, china (14.8%)24 and england (23%)25. in the present study, approximately 5% of the patients had such oral lesions, which is similar to the 7% found in the study of chagas, santos and ono2 (2009). the wide availability of haart had a major impact on improving survival of hiv-positive patients. moreover, a change could be seen in the pattern of the clinical disease in this group of patients. a decrease of frequency of some diseases, such as kaposi’s sarcoma was observed, but current findings demonstrate that hiv-positive patients, regardless of their response to therapy, have an increased frequency of other oral lesions, such as periodontal disease, compared with uninfected individuals26. these data reinforce the need for periodic dental evaluation. it should be included in the routine monitoring of these patients, as well as the early referral of patients with these oral lesions to specialist care. in conclusion, after the introduction of haart there was a significant decrease in the frequency of oral lesions on hiv+ patients. however, this study shows a high prevalence of other common oral lesions in hiv/aids patients in comparison with individuals not carrying the virus, such as periodontal diseases and candidiasis. it reinforces the need of a multidisciplinary approach of hiv/aids patients attending specialized centers that include regular dental evaluations aiming at early detection of diseases and prompt treatment, contributing to a better quality of life for these patients. acknowledgements we thank luciane tf silva for participation in data collection, dr. clea elisa ribeiro, infectious disease specialist, and dr. giovana pecharki, dentist and professor of school of dentistry of federal university of paraná for their helpful discussions during the study and comments on the manuscript. references 1. brazil, brazilian ministry of health, division of health surveillance, national program on std/aids. epidemiologic bulletin aids [internet]. [accessed 2012 jun 28] available from: http://aids.gov.br/publicacao/2011/ boletim_epidemiologico_2011. 2. chagas mv, santos lo, ono lm. oral manifestations in hiv patients treated at the tropical medicine foundation of amazonas (fmt-am). rev fac odontol porto alegre. 2009; 50: 10-13. 3. perera m, tsang pc, samaranayake l, lee mp prevalence of oral mucosal lesions in adults undergoing highly active antiretroviral therapy in hong kong. j investig clin dent. 2012; 3: 208-14. 4. watanuki f. oral manifestations associated with hiv after 30 years of the epidemic in brazil [dissertation]. são paulo, sp, br: faculty of dentistry, university of são paulo; 2010. 115 p. 5. tamí-maury im, willig jh, jolly pe, vermund s, aban i, hill jd, et al. prevalence, incidence, and recurrence of oral lesions among hiv-infected patients on haart in alabama: a two-year longitudinal study. south med j 2011; 104: 561–6. 6. khatibi m, moshari aa, jahromi zm, ramezankhani a. prevalence of oral mucosal lesions and related factors in 200 hiv+/aids iranian patients. j oral pathol med. 2011; 40: 659-64. 7. dias cf, nunes cc, freitas io, lamego is, oliveira im, gilli s, et al. high prevalence and association of hiv-1 non-b subtype with specific sexual transmission risk among antiretroviral naïve patients in porto alegre, rs, brazil. rev inst med trop sao paulo. 2009; 51: 191-6. 8. brazil. hiv, aids and viral hepatitis department. 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[accessed 2012 jun 21] available from: http://www.aids.gov.br/aids. 9. nittayananta w, chanowanna n, winn t, silpapojakul k, rodklai a, jaruratanasirikul s, et al. co-existence between oral lesions and opportunistic systemic diseases among hiv-infected subjects in thailand. j oral pathol med. 2002; 31: 163-8. 10. chopra s, arora u. skin and mucocutaneous manifestations: useful clinical predictors of hiv/aids. j clin diagn res. 2012; 6: 1695-8. 11. shirlaw pj, chikte u, macphail l, schmidt-westhausen a, croser d, reichart p. oral and dental care and treatment protocols for the management of hiv-infected patients. oral dis. 2002; 8: 136-43. 12. patton ll, ramirez-amador v, anaya-saavedra g, nittayananta w, carrozzo m, ranganathan k. urban legends series: oral manifestations of hiv infection. oral dis. 2013; 3: 1-18 13. mataftsi m, skoura l, sakellari d. hiv infection and periodontal diseases: an overview of the post-haart era. oral dis. 2011; 17: 13-25. 14. mariano bmf. periodontal disease and oral lesions in the population hiv / aids referral center, control and treatment of the military police of minas gerais [dissertation]. minas gerais, mg, br: federal university of minas gerais; 2008. 169 p. 15. alpagot t, duzgunes n, wolff lf, lee a. risk factors for periodontitis in hiv patients. j periodont res. 2004; 39: 149-57. 16. scheutz f, matee mi, andsager l, holm am, moshi j, kagoma c, et al. is there an association between periodontal conditions and hiv infection? j clin periodontol. 1997; 24: 580-7. 17. vastardis sa, yukna ra, fidel pl jr, leigh je, mercante de. periodontal disease in hiv-positive individuals: association of periodontol indices with stages of hiv disease. j periodontol. 2003; 74: 1336-41. 18. laskaris g, madjivassilion m, stratigos j. oral signs and symptoms in a 60 greek hiv infected patients. j oral pathol med. 1992; 21: 120-3. 19. adurogbangba mi, aderinokun ga, odaibo gn, olaleye od, lawoyin to. oro-facial lesions and cd4 counts associated with hiv/aids in an adult population in oyo state, nigeria. oral dis. 2004; 10: 319-26. 20. sharma g, pai km, suhas s, ramapuram jt, doshi d, anup n. oral manifestations in hiv/aids infected patients from india. oral dis. 2006; 12: 537-42. 21. bendick c, scheifele c, reichart pa 2002. oral manifestations in 101 cambodian patients with hiv infection and aids. j oral pathol med. 2002; 31: 1-4. 22. challacombe sj, coogan nn, williams dn. overview of the fourth international workshop on the oral manifestations of hiv infection. oral dis. 2002; 8: 9-14. 23. owotade fj, shiboski ch, poole l, ramstead ca, malvin k, hecht fm, et al. prevalence of oral disease among adults with primary hiv infection. oral dis. 2008; 14: 497-9. 24. zhang x, reichart pa, song y. oral manifestations of hiv/aids in china: a review. j oral maxillofac surg. 2009; 13: 63-8. 221221221221221oral lesions frequency in hiv-positive patients at a tertiary hospital, southern brazil braz j oral sci. 12(3):216-222 25. eyeson jd, tenant-flowers m, cooper dj, johnson nw, warnakulasuriya ka. oral manifestations of an hiv positive cohort in the era of highly active anti-retroviral therapy (haart) in south london. j oral pathol med. 2002; 31: 169-74. 26. yengopal v, naidoo s. oral lesions associated with hiv affect quality of life? oral surg, oral med, oral pathol, oral radiol endod. 2008; 106: 66-73. 222222222222222 oral lesions frequency in hiv-positive patients at a tertiary hospital, southern brazil braz j oral sci. 12(3):216-222 oral sciences n3 original article braz j oral sci. october | december 2013 volume 12, number 4 evaluation of the use of systemic antimicrobial agents by professionals for the treatment of periodontal diseases aline vicentini monteiro1, fernanda vieira ribeiro1, renato corrêa viana casarin1, fabiano ribeiro cirano1, suzana peres pimentel1, márcio zaffalon casati1,2 1area of periodontics, school of dentistry, paulista university, são paulo, sp, brazil 2department of prosthodontics and periodontics, school of dentistry of piracicaba, university of campinas (unicamp), piracicaba, sp, brazil correspondence to: marcio zaffalon casati departamento de odontologia, universidade paulista (unip) avenida dr. bacelar, 1212, 4o andar, cep: 04026-002 vila clementino, são paulo, sp, brasil phone/fax: +55 11 55864000 e-mail: mzcasati@gmail.com abstract aim: to investigate the indication of systemic antimicrobial agents used by dental professionals for treatment of patients affected by periodontal diseases. methods: interviews by a questionnaire were held with 225 professionals of different dental specialties and who performed periodontal treatment. results: among interviewees, 94% indicated systemic antibiotics as a form of periodontal disease treatment. their main indication was for periodontal abscesses (80%) followed by aggressive periodontitis (62%) and necrotizing diseases (45%). the most frequently used antibiotics were amoxicillin (81%) and metronidazole (57%). the medications were indicated in association with mechanical therapy by 67% of the professionals. as regards the occasion of indication, 60% indicated systemic antibiotic therapy before and after mechanical periodontal scaling and root planing. seventy-eight percent of the professionals indicated antibiotics associated with periodontal surgery for access to scaling, and 76% indicated it before and after the surgical procedure. among the interviewees, 99% took into account systemic involvement for drug administration. conclusions: it was concluded that a considerable portion of professionals indicate systemic antibiotic-therapy in an incoherent manner and in situations in which there is no indication for antibiotic use, or with ineffective protocols. keywords: periodontitis, anti-infective agents, questionnaires. introduction periodontal treatment is based on supra and subgingival dental biofilm control, performed by guidance on oral hygiene, professional supragingival biofilm removal and subgingival periodontal instrumentation. these therapeutic approaches alter the microbiota, allowing the establishment of a larger proportion of bacteria related to periodontal health. however, some studies demonstrated that mechanical therapy used alone, may not combat adequately some types of periodontopathogens, making it difficult to obtain or re-establish periodontal health1. studies demonstrated that the use of systemic antimicrobial agents adjunct to mechanical periodontal treatment may be an important therapeutic strategy in the treatment of chronic2-4and aggressive5-9periodontal diseases. these results were observed with the use of different antibiotics, such as amoxicillin/metronidazole4,8-11, azythromicin12-14, clindamycin15 and clarithromycin16, which showed favorable results as adjunct to therapy for aggressive periodontitis and chronic periodontal received for publication: august 26, 2013 accepted: november 25, 2013 braz j oral sci. 12(4):285-291 disease, when compared with the control group treated with mechanical therapy alone. in addition to the effects of systemic antibiotic therapy based on the isolated use of a certain drug, the use of associations of antibiotics has also shown promising clinical and microbiological results3,5,8,17. another factor of great importance for the correct indication of systemic antimicrobial agent use in therapy for periodontal alterations, in addition to the correct type of drug or association, is the occasion of using systemic antibiotic therapy. studies indicated that due to the characteristics of dental biofilm, it is necessary to disorganize it mechanically before, or soon after the beginning of antimicrobial therapy, to optimize the medication18,19. it is relevant to point out that the conscious use of systemic antibiotics is based on the possibility of adverse reactions to these medications, and particularly on the possibility of selecting resistant microorganisms20-22. adverse reactions may vary from simple nausea and headache, up to the presence of pseudomembranous colitis which, if not correctly treated, may result in the death of the patient affected by this infection. in addition, bacterial resistance is the most important negative factor in the use of indiscriminate and incorrect systemic antibiotic protocols20. in 1996, was made an evaluation of systemic antimicrobial agents use by professionals for periodontal treatment23. in this study, was demonstrated the abusive use of systemic antimicrobial agents. among dentists who performed periodontal treatment, 85% of them mentioned the use of systemic antibiotics in the treatment of chronic periodontitis, without considering the severity of the disease. furthermore, it was observed that the majority of the interviewed professionals prescribed antibiotics for patients with acute processes (periodontal abscesses and necrotizing diseases) without consideration of the patient’s systemic conditions. in view of past evidence of abusive use of antibiotic therapy by professionals who perform therapeutic periodontal procedures, and knowing about the risks of indiscriminate use of this type of medication, the aim of the present study was to make an up-to-date evaluation of systemic antimicrobial agents indication by dental professionals who perform periodontal therapy. material and methods study design this study had a cross-sectional, observational design, using structured questionnaires with multiple-choice questions that allowed completion of responses. sample in the present study, 225 dentists who performed periodontal treatment in private clinics or in public service voluntarily participated in the study. professionals involved in teaching activities in dental schools were excluded from the survey. all the professionals were interviewed by the same person, who followed the structured questionnaire adapted for this study. before the study interviews were held, the questionnaire was tested as regards aspects of understanding the questions, interviewing five dentists not involved in this project. before the interview, the participants were informed about the research and signed a form of free and informed consent. all the procedures performed were approved by the ethics commission of the paulista university (protocol no. 539/10 cep/ics/unip). nine multiple choice questions were asked with regards the use of systemic antibiotic therapy in the treatment of periodontal diseases. during initial questioning, the professionals reported whether they used or not antibiotics in the treatment of periodontal diseases. subsequently, they answered questions about the types of periodontal diseases in which antibiotic therapy was indicated, and could choose one or more of the following clinical situations: gingivitis, chronic periodontitis, aggressive periodontitis, periodontitis modified by systemic factors, periodontal abscesses, and necrotizing or other diseases. questions were also asked as regards the disease severity in which the medications should be indicated (slight, moderate or advanced). after this, the professionals were asked whether the patient’s systemic condition was taken into consideration for prescription of antibiotics. next, the professionals answered questions about whether they indicated the use of antibiotics in association with mechanical therapy, or whether they prescribed medication as monotherapy, and about the occasion of using the medication (prescribing the medication before, before and after, or only after the root scaling and planing procedures). in addition, the professionals were asked whether they prescribed or not antibiotics after performing periodontal surgery for access to root scaling. in cases in which they indicated the use of antibiotics combined with periodontal surgery, the professionals were asked about the occasion of medication use (indicating medication before, before and after, or only after the surgical procedures). the last question asked was about the used medication (amoxicillin, tetracycline, metronidazole, ciprofloxacin, clindamycin, azithromycin, associations of medications and others). results the replies to each question were computed in terms of relative frequency and as a whole, they were analyzed by means of descriptive statistics. interviews were held with 225 dentists from different specialties (figure 1). although the professionals were from different specialties (public health 2.3%, pediatric dentistry 3.8%, restorative dentistry 6.1%, oral surgery 9.9%, endodontics 12.4%, orthodontics 12.4%, periodontics 14.6%, prosthesis15.3% and implantology 23.2%), they all performed periodontal therapy on their patients. as regards the first question about whether they indicated or not antibiotic therapy in the treatment of periodontitis, only 6% of the professionals reported that they did not use systemic antibiotics in the treatment of periodontal diseases. the professionals who used antibiotic therapy reported using evaluation of the use of systemic antimicrobial agents by professionals for the treatment of periodontal diseases286 braz j oral sci. 12(4):285-291 fig. 1 frequency of indication of systemic antimicrobials in the treatment of periodontal diseases by dentists from different specialties. them mainly in cases of periodontal abscesses (80%), aggressive periodontitis (62%) and necrotizing diseases (45%) (figure 2). it should be pointed out that some of the professionals indicated more than one clinical situation for the use of systemic antibiotic therapy. as regards the disease severity for which the medications should be indicated, 87% of the professionals indicated antibiotic therapy for advanced periodontitis, 38% for moderate and 6% for slight periodontitis. asked whether they took the systemic involvement of the patient into consideration when opting to use antibiotic therapy, 99% of the dentists reported that they did take this into consideration when prescribing antibiotics, while 1% did not. when asked about the use of antibiotics as adjunct to mechanical therapy, it was observed that most of the interviewed professionals used antibiotic therapy in association with root instrumentation (67%), although a considerable portion (33%) indicated it without combination fig. 2 – number and percentage of indications for the use of antimicrobials in the different types of periodontal disease. with mechanical therapy. when asked about the occasion of using antibiotics, in case of association with the root scaling and planing, most of the professionals (60%) reported they indicated the use of medication both before and after nonsurgical mechanical instrumentation (i.e. scaling and root planing) (figure 3). asked about the prescription of antibiotics after performing periodontal surgery for access to root scaling, 78% of the professionals replied they indicated the use of medication associated with surgeries, while 22% did not prescribe antibiotics associated with surgeries. as regards the occasion of using antibiotics in cases of periodontal surgeries, most of the professionals (76%) reported that they indicated the use of medication both before and after the surgical procedure (figure 4). with regard to the choice of medications, a predominance of bactericide antibiotic prescriptions (amoxicillin and metronidazole) was observed (figure 5). evaluation of the use of systemic antimicrobial agents by professionals for the treatment of periodontal diseases 287 braz j oral sci. 12(4):285-291 fig. 4 frequency of indication of the use of antimicrobials according to the occasion of administration in the case of association with periodontal surgeries. fig. 3 frequency of indication of the use of antimicrobials according to the occasion of administration, in the case of association with scaling and root planing. fig. 5 – number and percentage of the medications indicated by the interviewed professionals. evaluation of the use of systemic antimicrobial agents by professionals for the treatment of periodontal diseases288 braz j oral sci. 12(4):285-291 discussion in view of previous evidence that demonstrated abusive use of antibiotics by professionals who perform therapeutic periodontal procedures, and bearing in mind the risks associated with the indiscriminate use of this type of medication, the aim of this study was to make an up-to-date evaluation of the indication of systemic antimicrobial agents by dental professionals who perform periodontal therapy. according to the findings of the present study, a large portion of the interviewees (92%) was shown to use antibiotics in the treatment of periodontal diseases, and that in 80% of the cases, this indication was being directed to the treatment of periodontal abscesses. different therapies have been proposed for the treatment of periodontal abscesses, which turned the subject controversial1,24,25. however, the use of antibiotics must be restricted to individuals with systemic involvement such as bad feeling, fever, prostration and lymphoadenopathy 26. indeed, in the present study most of the interviewed professionals (99%) considered this aspect when taking the decision to prescribe the medication. however, as only a small portion of periodontal abscesses develop to a systemic involvement, and 80% of the dentists prescribe antibiotics for the treatment of this acute process, this may indicate excessive use of such medication for this purpose. in the present study, a considerable portion of the interviewees (62%) also reported the use of systemic antibiotic therapy for the treatment of aggressive periodontitis. in this regard, various researchers showed that there was a significant benefit of their use as adjunct to mechanical treatment, when compared with individuals who received mechanical treatment only5,6. haas et al.12 (2008) used an antibiotic regime of 500 mg azythromicin, once a day, for 3 days, associated with root scaling and planing, and after 12 months of follow-up, obtained significant improvement in the evaluated clinical parameters (probing depth and clinical attachment level) in comparison with the group that received mechanical therapy alone. in addition to the clinical benefits, some recent studies also reported the positive effects of the adjunct use of antibiotics on the subgingival microbiota of individuals with aggressive periodontitis. in a randomized, double-blind, placebocontrolled study, 30 individuals with aggressive periodontitis received root scaling and planing alone, or combined with 400 mg of metronidazole plus 500 mg of amoxicillin, 3 times a day for 14 days5. the individuals who received the combination of antibiotics showed better results in the microbial profile, presenting smaller proportions of the red and orange complexes after treatment5. rodrigues et al.7 (2012) also observed a significant reduction in the quantity of subgingival tannerella forsythensis (t.f.) and treponema denticola (t.d.) in patients with aggressive periodontitis treated with amoxicillin and metronidazole, in association with mechanical treatment. although there is scientific evidence supporting the use of systemic antibiotics adjunct to root instrumentation in the treatment of aggressive periodontitis, care must be taken when diagnosing cases of aggressive periodontitis, and differentiate them from advanced chronic periodontitis, for example, since there is a set of specific characteristics for the diagnosis of this type of periodontal disease. this caution is relevant, considering that the use of antibiotics in other types of periodontal disease might not be a suitable option. the results of the present study also demonstrated that a great number of professionals (45%) indicate the use of antibiotics in cases of necrotizing periodontitis. some studies27 affirmed that systemic antibiotics are generally not necessary in the treatment of necrotizing processes, provided that mechanical therapy of the site is sufficient to efficiently contain the disease progression. thus, only in the few cases in which the patient’s response to mechanical therapy is minimal, or in cases in which the patient presents systemic involvement, is indicated the supplementary use of antibiotics. bearing in mind that in the present study, the use of antibiotics was prescribed by almost half of the professionals in the treatment of this periodontal condition, it suggests once again that professionals are making excessive use of this medication. as regards the adjunct use of antibiotics in the treatment of chronic periodontitis, it has been mentioned that generally an efficient clinical response may be obtained by performing mechanical therapy only13. studies emphasized that in order to provide maximum benefit, there is need to reserve the use of these antibiotic agents for individuals with moderate or severe diseases18,28. furthermore, when mechanical therapy alone does not stop the process or prevent recurrence of the disease, a situation more prevalent in smokers, the adjunct use of antibiotics would be justified 2,29. some authors30 demonstrated that the use of azithromycin, in combination with root scaling and planing improved the results of nonsurgical therapy in smokers. according to the authors, greater reduction in probing depth and gains in clinical attachment levels in patients with moderate to advanced attachment loss have been observed when compared with patients who received root scaling and planing only. clinical and microbiological benefits have also been obtained by some authors 2 with the combination of amoxicillin and metronidazole adjunct to mechanical therapy in the treatment of chronic periodontitis in smokers. in the present study, 20% of the professionals reported indicating the use of antibiotics in the treatment of chronic periodontitis, irrespective of the severity of the disease or the patient’s smoking habit, aspects considered relevant when the use of antibiotic therapy is referred to in chronic periodontal disease therapy. in spite of these studies demonstrating positive results with the use of antibiotics as adjunct to the treatment of chronic periodontitis2,3,8,18,28,30, a recent systematic review showed that there is still insufficient and inconclusive evidence to support the use of systemic antibiotics31. although most of the mentioned professionals concern about associating antibiotic with mechanical therapy, 33% of the professionals used antibiotic therapy alone. according to previous evidence, antibiotics must not be used as evaluation of the use of systemic antimicrobial agents by professionals for the treatment of periodontal diseases 289 braz j oral sci. 12(4):285-291 substitutes for mechanical instrumentation of the site18,32 whether it is performed surgically or not, due to the fact that biofilm is relatively resistant to antimicrobial agents19 unless it is mechanically disorganized. the literature is full of studies demonstrating the effectiveness of antibiotics associated with instrumentation, but there is no precise definition of the best occasion to prescribe them. in the present study, most of the interviewed professionals (60%), mentioned using antibiotics before and after non-surgical mechanical instrumentation. it was shown that the administration of antibiotic therapy during the mechanical therapy, that is to say, not before and not after instrumentation, provides greater reduction in probing depth and greater attachment gain in deep pockets32. therefore, it seems that the best form of using systemic antibiotic therapy would be in association with mechanical treatment of all the regions with periodontal involvement, so that the period of antibiotic administration covers the time required to perform full-mouth mechanical therapy, preferably less than one week. as regards the use of antimicrobial agents in the periodontal surgical procedures of root scaling and planing, it was found that the majority of the professionals (76%), used antibiotics before and after the procedures. it is important to point out that although some authors have suggested that the adjunct use of antibiotics may reduce pain and swelling and improve healing, there is no sufficient evidence to support their use associated with periodontal surgery 32. in an extensive review33 of all the records available of periodontal surgeries in a sample of 395 patients and 1.053 periodontal surgery procedures, it was demonstrated that there were no benefits from antibiotics indication in these cases, due to the low rate of post-operative infections. therefore, it is probable that the large number of indications found in the present study were made empirically. one of the few situations in which the use of systemic antibiotics before surgical procedures could be indicated, is for the prevention of bacterial endocarditis34. as discussed in this study, although certain periodontal conditions have been benefitted by the adjunct use of antibiotics, there is no protocol or consensus as regards to medications that must be used in each case1. some studies showed that the association of amoxicillin (a penicillin with broader spectrum) and metronidazole (a nitroimidazole compound) especially in a regime of 400 mg of metronidazole plus 500 mg of amoxicillin, 3 times a day for 7-14 days plays an important role in treating anaeroberelated infection in the oral cavity and may significantly improve the short-term results of non-surgical therapy in patients with aggressive periodontitis5,6,9,10, diminishing the need for surgical therapy. this association has shown good results by presenting a synergic effect between the drugs9,11,35. azythromicin, a member of a new subclass of macrolide, the azalides, has also been shown to be effective as adjunct to the treatment of aggressive periodontitis (regime of 500 mg de azythromicin, once a day, for 3 days)12. therefore, one may affirm that there is scientific support for the use of the antibiotics most frequently indicated by the professionals in the present study (amoxicillin, metronidazole and azythromicin), for the treatment of periodontal diseases, mainly as adjunct to mechanical therapy in the management of aggressive periodontitis. an important subject refeents to the use of antibiotic therapy is the bacterial resistance, which is associated with excessive and/or indiscriminate use of antibiotics21,22. ardila et al.22 (2010) revealed that periodontal microorganisms in patients with chronic periodontitis can be resistant to the antimicrobial agents frequently used in anti-infective periodontal therapy, such as amoxicillin and metronidazole, supporting the position that the indiscriminate use of antimicrobials leads to the appearance of more highly antibiotic-resistant strains of bacteria associated with periodontal diseases. in the survey conducted 15 years ago on the same subject as in of the present study23, only 7% of the professionals reported taking into consideration the systemic manifestation of infection when indicating antibiotics, whereas in the present study 99% reported considering this involvement when indicating this type of medication. this result demonstrates that there has been a considerable change in the knowledge of professionals who indicate antibiotic therapy nowadays. nevertheless, although professionals reported being more prepared for, and conscious of indicating antibiotic therapy in most indications, it could still be observed that there was indiscriminate use, which was scarcely based on scientific evidence. an important aspect to be highlighted is related to the type of questionnaire used in the present study. although a structured multiple-choice questionnaire was used during the interviews, alternative options of answers or other types of questions could have led to different outcomes. in view of the results obtained in the present study, it may be concluded that systemic antibiotics are still used by dentists in an incoherent manner, and scarcely based on the scientific advancements published in the literature, for clinical situations in which antibiotics are not indicated or using protocols that are not effective in periodontal therapy. references 1. herrera d, matesanz p, bascones-martínez a, sanz m. local and systemic antimicrobial therapy in periodontics. j evid based dent pract. 2012; 12: 50-60. 2. matarazzo f, figueiredo lc, cruz se, faveri m, feres m. clinical and microbiological benefits of systemic metronidazole and amoxicillin in the treatment of smokers with chronic periodontitis: a randomized placebo controlled study. j clin periodontol. 2008; 35: 885-96. 3. cionca n, giannopoulou c, ugolotti g, mombelli. amoxicillin and metronidazole as an adjunct to full-mouth scaling and root planing of chronic periodontitis. j periodontol. 2009; 80: 364-71. 4. sgolastra f, gatto r, petrucci a, monaco a. effectiveness of systemic amoxicillin/metronidazole as adjunctive therapy to scaling and root 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beier m, klinger g, pfister w, glockmann e. a 2-step nonsurgical procedure and systemic antibiotics in the treatment of rapidly progressive periodontitis. j periodontol. 2001; 72: 275-83. 16. pradeep ar, kathariya r. clarithromycin, as an adjunct to non-surgical periodontal therapy for chronic periodontitis: a double blinded, placebo controlled, randomized clinical trial. arch oral biol. 2011; 56: 1112-9. 17. cionca n, giannopoulou c, ugolotti g, mombelli a. microbiologic testing and outcomes of full-mouth scaling and root planing with or without amoxicillin/ metronidazole in chronic periodontitis. j periodontol. 2010; 81: 15-23. 18. colombo ap, teles rp, torres mc, rosalem w, mendes mc, souto rm, et al. effects of non-surgical mechanical therapy on the subgingival microbiota of brazilians with untreated chronic periodontitis: 9-month results. j periodontol. 2005; 76: 778-84. 19. marsh pd. dental plaque: biological significance of a biofilm and community lifestyle. j clin periodontol. 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1518-28. 32. herrera d, alonso b, leon r, roldan s, sanz m. antimicrobial therapy in periodontitis: the use of systemic antimicrobials against the subgingival biofilm. j clin periodontol. 2008; 35: 45-66. 33. powell ca, mealey bl, deas d, mcdonnell ht, mortiz aj. post-surgical infections: prevalence associated with various periodontal surgical procedures. j periodontol. 2005; 76: 329-33. 34. wilson w, taubert ka, gewitz m, lockhart pb, baddour lm, levison m, et al. prevention of infective endocarditis: guidelines from the american heart association: a guideline from the american heart association rheumatic fever, endocarditis and kawasaki disease committee, council on cardiovascular disease in the young, and the council on clinical cardiology, council on cardiovascular surgery and anesthesia, and the quality of care and outcomes research interdisciplinary working group. j am dent assoc 2007; 138: 739-45,747-60. 35. slots j. low-cost periodontal therapy. periodontol. 2000.2012; 60: 110-37. evaluation of the use of systemic antimicrobial agents by professionals for the treatment of periodontal diseases 291 braz j oral sci. 12(4):285-291 oral sciences n3 braz j oral sci. 13(2):93-97 original article braz j oral sci. april | june 2014 volume 13, number 2 changes in pharyngeal airway space and soft tissue after maxillary advancement and bimaxillary surgery erika franco freire1, francisco wagner vasconcelos freire filho2, heloisa cristina valdrighi2, viviane veroni degan2, silvia amélia scudeler vedovello2 1centro universitário hermínio ometto uniararas, school of dentistry, area of orthodontics, araras, sp, brazil 2universidade de fortaleza – unifor, school of dentistry, department of oral and maxillofacial surgery, fortaleza, ceará, ce, brazil correspondence to: sílvia amélia scudeler vedovello uniararas av. dr. maximiliano baruto 500 jardim universitário cep: 13607-339 araras, sp, brasil phone: +55 19 35431423 e-mail: silviavedovello@gmail.com abstract aim: to evaluate changes in pharyngeal airway space (nasopharynx and oropharynx), soft palate and lingual vallecula after maxillary advancement surgery and maxillary advancement and mandibular setback surgery (bimaxillary surgery). methods: twenty class iii adult patients were included in the study. ten patients were treated with maxillary advancement and ten with bimaxillary surgery (maxillary advancement and mandibular setback). cephalometric landmark measurements were recorded at 3 different time intervals: pre-surgical, post-surgical and six months after surgery. data collected were subjected to one-way anova (p<0.05). results: nasopharyngeal airway space increased after maxillary advancement and decreased after bimaxillary surgery. there was increase in oropharyngeal dimensions in the region around the uvula and loss of space for lingual vallecula, while in group 2 there was diminished space for both uvula and vallecula. the uvula and vallecula were moved forward in group 1, whereas these structures were moved in the posterior direction in group 2. conclusions: after maxillary advancement surgery there was an increased in space in the nasopharyngeal region. the oropharyngeal region related to the uvula presented an increase in space, whereas there was a reduction in relation to the lingual vallecula. the uvula and lingual vallecula were moved forward. bimaxillary surgery promoted a reduction in the nasopharyngeal and oropharyngeal regions as regards both the uvula and lingual vallecula. the uvula and lingual vallecula were moved in the posterior direction. keywords: orthognathic surgery; pharynx; palate, soft. introduction dentofacial deformities treated by orthognathic surgery may influence functional soft tissue components including the tongue and pharyngeal airway1-6. soft palate, tongue and hyoid bone are directly or indirectly attached to each other, so that maxillary and mandibular movement affects these tissues by changing pharyngeal airway2-3. mandibular and maxillary advancement may offer larger space in the pharyngeal airways7-9, whereas mandibular setback surgery may result in reduction of the respiratory area in this region3,7. a combination of mandibular setback and maxillary advancement are the procedures generally used for correction of severe class iii discrepancies10. however, studies have warned about the risk of orthognathic surgery effects for the correction received for publication: march 04, 2014 accepted: may 29, 2014 braz j oral sci. 13(2):93-97 cephalometric description points and lines n nasion s sella ba basion ptm pterigomaxillary fissure ans anterior nasal spine a most posterior point of the maxillary alveolar bone concavity b most posterior point of the mandibular alveolar bone concavity m e mentum u uvula: uvula, the tip of the uvula v vallecula: the intersection of the epiglottis and the base of the tongue upw linear measurement of the superior pharyngeal wall: intersection between line ptm-ba and the posterior pharyngeal wall mpw linear measurement of the mid-pharyngeal wall: intersection between perpendicular line of ptm-ba at u and the posterior pharyngeal wall chart 1chart 1chart 1chart 1chart 1. cephalometric points and lines used in the study of mandibular prognathism leading to reduction in pharyngeal airway space3,11. reduction in airway space may contribute to the development of obstructive sleep apnea1,3,8,12. the aim of this study was to evaluate changes in pharyngeal airway space (nasopharynx and oropharynx), uvula and lingual vallecula after maxillary advancement surgery, and maxillary advancement and mandibular setback surgery. the main hypothesis is that the pharyngeal space increases with advancement of the maxilla, and diminishes with the combined maxillary advancement and mandibular setback surgery. material and methods this study was approved by the institutional research ethics (process #999/2011) and the research subjects signed an informed consent form prior to their enrollment. a retrospective study was conducted. the sample comprised 20 individuals of both genders (11 men and 9 women), age-range between 25 and 30 years (mean age 27 years and 5 months). inclusion criteria were as follows: adult patients with skeletal and dental class iii deformity, mesiofacial skeletal pattern defined by the frankfortmandibular plane (fma) angle, between 21 o and 29 o, according to tweed; permanent dentition and posterior teeth; who had been submitted to orthognathic surgery for the correction of maxillary retrusion or combined surgery (maxillary advancement and mandibular setback) for the correction of skeletal class iii malocclusion. all patients used internal rigid fixation as a form of osteosynthesis. the following exclusion criteria were applied: previous orthognathic surgery, additional surgical procedures, such as rhinoplasty, infraorbital grafts, craniofacial anomalies, posttraumatic deformity, obstructive sleep apnea and systemic diseases that could affect the stomatognathic system. forty lateral teleradiographs of the head were obtained from the archives comprising files belonging to the team of surgeons who performed the same surgical protocol. for each individual in the sample were analyzed the teleradiographs taken at two time intervals: immediate pre-surgical (t1) and post-surgical after six months (t2). in the initial and final cephalograms (t1 and t2) lines and cephalometric points were marked (fig. 1 and chart 1). fig. 1.cephalometric variables used in the study 94 changes in pharyngeal airway space and soft tissue after maxillary advancement and bimaxillary surgery 95 braz j oral sci. 13(2):93-97 the patients were divided into two groups. group 1 (n=10), composed by individuals subjected to orthodonticsurgical treatment for maxillary advancement (g1) and group 2 (n=10), composed by individuals subjected to orthodonticsurgical treatment for maxillary advancement and mandibular setback (g2). the movements were solely advances and setbacks and therefore they did not cause clockwise or anticlockwise rotations. the 40 radiographs were randomly disposed, and marking and measurements of the points were performed twice by the same investigator. the results were compared to obtain correlation coefficients (r2). to evaluate the results, the pharyngeal airway space was divided into 2 vertical levels (nasopharynx and oropharynx). to delimit these structures the cephalometric variables from chart 2 were used. descriptive statistical analysis of the measurements of groups g1 and g2 was performed, and for comparisons between means of the groups, one-way anova was used, with a 5% significance level. region measurements nasopharynx nasopharyngeal airway upw-u oropharynx oropharyngeal airway mpw-u mpw-v chart 2.chart 2.chart 2.chart 2.chart 2. division of the pharyngeal airway into nasopharynx and oropharynx. group 1 group 2 group 1 group 2 group 1 group 2 upwu nasopharynx mpwu oropharynx mpwv oropharynx 2.72 -1.27 1.25 -1.42 -2.84 -1.32 3.34 3.96 2.97 2.68 2.32 7.60 -3.09 – 7.59 -6.02 – 4.93 -3.40 – 6.31 -5.00 – 2.38 -6.03 – 1.25 -14.44 – 14.21 0.026 0.049 0.553 table 1. table 1. table 1. table 1. table 1. mean value (mm) of changes in the nasopharyngeal and oropharyngeal space. mean of alterations in nasopharynx and oropharynx group region mean s d minimum – maximum p results for the nasopharyngeal region in group 1, a gain of +2.72 mm in anteroposterior space was observed (mpw-u). in group 2, there was a loss of -1.27 mm in anteroposterior space in the nasopharyngeal region (mpw-u) (table 1). statistically significant difference between the groups was found with reference to alterations in airway space in the nasopharynx area, (table 1) in the oropharyngeal region in group 1 occurred a +1.25 mm gain in anteroposterior space (mpw-u) and -2.84 mm loss of space in the mpw-v line. in group 2 a reduction in anteroposterior space occurred in the oropharyngeal region (-1.42 mm in mpw-u and -1.32 mm in mpw-v). statistically significant difference was obtained with reference to alterations in airway space in the oropharynx area between the groups in uvula region. statistically significant difference between the groups was not found with reference to alterations in airway space in the oropharynx area. for both groups occurred a reduction in airway space in the oropharynx area (table 1). uvula and lingual vallecula moved forwards (+0.57 mm and +1.57 mm respectively) in group 1 while in group 2 the structures were moved in a posterior direction (-1.57 mm and -3.12 mm). statistically significant difference between the groups was not obtained with reference to the movement of uvula, while it was observed to the movement of lingual vallecula (table 2). discussion orthognathic surgery has regularly been used to correct dentofacial deformities. surgical procedures that modify the relationship of the bony base may bring about changes both in facial esthetics and in the component structures of the craniofacial complex and its functions. changes in pharyngeal airway space may occur as a result of these procedures2-4,13. studies that evaluated posterior airway space after orthodontic and orthopedic treatment with rapid maxillary expansion and with anterior traction of the maxilla referred to an increase in pharyngeal airway space, both in the nasopharynx and oropharynx14-16. in this study, the patients subjected to maxillary advancement surgery, had the nasopharynx increased. in the oropharyngeal region there was an increase in relation to the uvula and a reduction in relation to the lingual vallecula3,5,13. the interesting point is that as far as orthopedic maxillary changes in pharyngeal airway space and soft tissue after maxillary advancement and bimaxillary surgery group movement of the uvula movement of the lingual vallecula mean s d minimum maximum p mean s d minimum maximum p group 1 0.57 1.86 -2.53 – 3.20 0.077 1.57 3.04 -2.50 – 8.00 0.006 group 2 -1.59 3.12 -5.83 – 2.89 -3.12 3.72 -8.24 – 4.37 table 2.table 2.table 2.table 2.table 2. mean amount of movement of the uvula and lingual vallecula structures in millimeters. 96 advancement is concerned, the nasopharynx gains space but the oropharynx tends to undergo positive changes gain or remains unaltered, whereas in the present study, the result was negative. one could suppose that this factor was caused due to the nature of the procedure, since orthopedics results in gradual alterations, whereas surgery has an immediate result, and may stretch the structures surrounding the operated region. in addition, the orthopedic patient is in the active growth stage and the surgical patient is already full grown. another hypothesis would be the absence of the maxillary disjunction procedure in this study. it was observed in this study that bimaxillary surgery promoted loss of pharyngeal airway space of the nasopharynx and oropharynx, a fact also reported by other authors3,7,10, but with higher values. another study pointed out that the pharyngeal airway space and soft palate were moved to a significant extent in the mandibular setback17. the former was changed in the retropalatine direction and in the retrolingual direction; the latter underwent an increase in length. studies have shown that in bimaxillary surgeries7,13 the mandible underwent a setback and the maxilla an increase. a decrease occurred in the soft palate area and at the base of the tongue. this result demonstrated that, even advancing the maxilla, when combined with mandibular setback, there is a decrease in pharyngeal airway space. the backward movement of the soft palate and the tongue may invade the pharyngeal airway space and is a predisposing factor for obstructive sleep apnea8. this study demonstrated that with mandibular setback surgery, even associated with maxillary advancement, the lingual vallecula and uvula are moved in a backward direction. thus, the oropharynx and nasopharynx airway spaces are diminished. therefore, the professional should be aware of the risk of development of obstructive sleep apnea syndrome after bimaxillary surgeries in class iii patients. in individuals with obstructive sleep apnea, the length of the soft palate may be increased, compared with the individuals that do not present this disturbance and they present reduction in the dimensions of the nasopharynx and oropharynx18. therefore, one should be cautious about surgical movements, even in individuals who do not present signs and symptoms of apnea, in order to avoid elongation by displacement of this structure, and the reduction in oropharyngeal and nasopharyngeal space, thereby hindering the passage of air. the presence of other predisposing factors for the obstructive sleep apnea syndrome must also be observed in candidates for mandibular setback surgery, such as overweight, short neck, tongue volume, wide uvula and excessive amount of soft tissue around the nasopharyngeal region, which may favor the development of the syndrome19-20. in addition, other factors such as the facial type and unsatisfactory neuromuscular adaptation should also be considered15. posterior movement of bony bases may diminish the posterior area of the tongue and retropalatine area, even when the maxilla has been advanced, causing reduction in the nasopharyngeal space21-22. therefore, mandibular setback may potentially diminish this region and generate respiratory disorders, particularly if the individual does not have sufficient neuromuscular adaptation to compensate reduction in the size of space. combined maxillary and mandibular surgeries may be used for the correction of accentuated anteroposterior discrepancies to minimize the side effects on the pharyngeal airway space22. based on the observations in this study, maxillary advancement surgery in skeletal class iii patients must be emphasized because little space is lost in the oropharynx and there is gain in the nasopharynx. caution is strongly recommended for performing bimaxillary surgery in individuals with respiratory diseases, since there is a considerable loss of pharyngeal airway space. it should also be observed that even when advancing the maxilla, mandibular setback seems to influence the amount of functional airway space. it was concluded that after maxillary advancement surgery there was an increase in the space of the nasopharyngeal region. the oropharyngeal region related to the uvula presented an increased space, whereas there was a decrease in relation to the lingual vallecula. the uvula and lingual vallecula were moved forward. bimaxillary surgery promoted a reduction in the nasopharyngeal and oropharyngeal regions as regards both the uvula and lingual vallecula. the uvula and lingual vallecula were moved in the posterior direction. references 1. demetriades n, chang dj, laskarides c, papageorge m. effects of mandibular retropositioning, with or without maxillary advancement, and oro-naso-pharyngeal airway and development of sleep related breathing disorders. j oral maxillofac surg. 2010; 68: 2461-6. 2. hwang s, chung cj, choi yj. huh jk, kim kh. changes of hyoid, tongue and pharyngeal airway after mandibular setback surgery by intraoral vertical ramus osteotomy. angle orthod. 2010; 80: 302-7. 3. aydemir h, memikoðlu u, karasu h. pharyngeal airway space, hyoid bone position and head posture after orthognathic surgery in class iii patients. angle orthod. 2012; 82: 993-1000. 4. becker oe, avelar rl, göelzer jg, dolzan ado n, haas ol jr, de oliveira rb. pharyngeal airway changes in class iii patients treated with double jaw orthognathic surgery—maxillary advancement and mandibular setback. oral maxillofac surg. 2012; 70: 639-47. 5. becker oe, avelar rl, do n dolzan a, haas ol jr, scolari n, de oliveira rb. soft and hard tissue changes in skeletal class iii patients treated with double-jaw orthognathic surgery-maxillary advancement and mandibular setback. int j oral maxillofac surg. 2014; 43: 204-12. 6. ronchi p, cinquini v, ambrosoli a, caprioglio a. maxillomandibular advancement in obstructive sleep apnea syndrome patients: a restrospective study on the sagittal cephalometric variables. j oral maxillofac res. 2013; 4: e5. ecollection 2013. 7. hong js, park yh, kim yj, hong sm, oh km. three-dimensional changes in pharyngeal airway in skeletal class iii patients undergoing orthognathic surgery. oral maxillofac surg. 2011; 69: 401-8. 8. lee y, chun ys, kang n, kim m. volumetric changes in the upper airway after bimaxillary surgery for skeletal class iii malocclusions: a case series study using 3-dimensional cone-beam computed tomography. j oral maxillofac surg. 2012; 70: 2867-75. 9. yuan l, shen g, wu y, jiang l, yang z, liu j, et al. three-dimensional analysis of soft tissue changes in full-face view after surgical correction of skeletal class iii malocclusion. j craniofac surg. 2013; 24: 725-30. braz j oral sci. 13(2):93-97 changes in pharyngeal airway space and soft tissue after maxillary advancement and bimaxillary surgery 97 10. kim ma, kim br, choi jy, youn jk, kim yj, park yh. threedimensional changes of the hyoid bone and airway volumes related to its relationship with horizontal anatomic planes after bimaxillary surgery in skeletal class iii patients. angle orthod. 2013; 83: 623-9. 11. abdelrahman te, takahashi k, tamura k, nakao k, hassanein km, alsuity a, et al. impact of different surgery modalities to correct class iii jaw deformities on the pharyngeal airway space. j craniofac surg. 2011; 22: 1598-601. 12. hasebe d, kobayashi t, hasegawa m, iwamoto t, kato k, izumi n, et al. changes in oropharyngeal airway and respiratory function during sleep after orthognathic surgery in patients with mandibular prognathism. int j oral surgmaxillofac. 2011; 40: 584-92. 13. pereira-filho va, castro-silva lm, de moraes m, gabrielli mf, campos ja, juergens p. cephalometric evaluation of pharyngeal airway space changes in class iii patients undergoing orthognathic surgery. j oral maxillofac surg. 2011; 69: 409-15. 14. oktay h, ulukaya e. maxillary protraction appliance effect on the size of the upper airway passage. angle orthod. 2008; 78: 209-14. 15. kitagawara k, kobayashi t, goto h, yokobayashi t, kitamura n, saito c. effects of mandibular setback surgery on oropharyngeal airway and arterial oxygen saturation. int j oral maxillofac surg. 2008; 37: 328-33. 16. iwasaki t, saitoh i, takemoto y, inada e, kakuno e, kanomi r, et al. tongue posture improvement and pharyngeal airway enlargement as secondary effects of rapid maxillary expansion: a cone-beam computed tomography study. am j orthod dentofacial orthop. 2013; 143: 235-45. 17. muto t, yamazaki a, takeda s, sato y. effect of bilateral sagittal split ramus osteotomy setback on the soft palate and pharyngeal airway space. int j oral maxillofac surg. 2008; 37: 419-23. 18. shigeta y, ogawa t, tomoko i, clark gt, enciso r. soft palate length and upper airway relationship in osa and non-osa subjects. tex dent j. 2013; 130: 203-11. 19. chen f, terada k, hanada k, saito i. predicting the pharyngeal airway space after mandibular setback surgery. j oral maxillofac surg. 2005; 63: 1509-14. 20. xiao y, chen x, shi h, yang y, he l, dong j, et al. evaluation of airway obstruction at soft palate level in male patients with obstructive sleep apnea/hypopnea syndrome: dynamic 3-dimensional ct imaging of upper airway. j huazhong univ sci technolog med sci. 2011; 31: 413-8. 21. bilston le, gandevia sc. biomechanical properties of the human upper airway and their effect on its behavior during breathing and in obstructive sleep apnea. j appl physiol (1985). 2014; 116: 314-24. 22. liukkonen m, vähätalo k, peltomäki t, tiekso j, happonen rp. effect of mandibular setback surgery on the posterior airway size. int j adult orthodon orthognath surg. 2002; 17: 41-6. braz j oral sci. 13(2):93-97 changes in pharyngeal airway space and soft tissue after maxillary advancement and bimaxillary surgery oral sciences n3 original article braz j oral sci. july | september 2013 volume 12, number 3 effect of implant design and bone density in primary stability nathalia ferraz oliscovicz1, antônio carlos shimano2, elcio marcantonio junior3, césar penazzo lepri1, andréa cândido dos reis1 1department of dental materials and prosthodontics, school of dentistry of ribeirão preto, university of são paulo, ribeirão preto, sp, brazil 2department of biomechanics, medicine and rehabilitation of the locomotor, school of medicine of ribeirão preto, university of são paulo, ribeirão preto, sp, brazil 3department of diagnostic and surgery, school of dentistry of araraquara, unesp – univ estadual paulista, araraquara, sp, brazil correspondence to: andréa cândido dos reis avenida do café, s/nº, cep: 14040-904 ribeirão preto, sp, brasil phone: +55 16 36023952 fax: +55 16 36330999 e-mail: andreare73@yahoo.com.br abstract aim: to evaluate the influence of the format and surface treatment of implants, as well as the substrate used in primary stability. methods: thirty-two conexão® implants were used: 8 conical (cc) (11.5 x 3.5 mm) and 24 cylindrical (11.5 x 3.75 mm) – 8 external hexagon implants without surface treatment (ms), 8 external hexagon implants with double porous treatment (mp), 8 internal hexagon implants with porous treatment (ca). they were inserted in nacional® polyurethane in three densities (15, 20 and 40 pcf). the insertion torque (it) (n.cm) was quantified using the digital mackena® torque meter, and the pullout force (pf) (n) by means of axial traction force with a 200 kg load cell, performed in a universal test machine (emic® dl10000) and the tesc 3.13 software. data were analyzed statistically by anova and tukey’s test with a significance level of 5%. results: difference was observed between groups (p<0.05). regarding the it, mp and ms inserted to the substrate 40pcf showed higher values with statistically significant difference with all interactions implants x substrate; the 15 and 20pcf densities was not significant in all groups of implants. mp, ms, cc and ca did not differ significantly, even inserted in a lower density, where cc showed better it compared with other densities. for pf, the best performance was the interaction implant ca x 40pcf substrate, showing a difference from the other implants inserted in all substrates. conclusions: the higher bone density and cylindrical implants with surface treatment provides greater it and pf. keywords: dental implants, biomechanics, bone substitutes, polyurethanes. introduction the oral rehabilitation with implants has high success rates because of the process of osseointegration. for its occurrence, the primary stability must be respected1-5, defined as lack of mobility in the surgical stage. biological and mechanical factors, influenced by surgical technique, bone quality and quantity, and implant’s geometry3,6, are the key factors that define the primary stability and then, the success of osseointegration. bone density is one of the most important parameters for the long-term success of dental implants. to observe the influence of this factor on primary stability in in vitro analysis, composed polyurethane substrates are frequently used3-6, which is used as synthetic bone substitutes of the human bone and is used in studies of received for publication: march 23, 2013 accepted: june 25, 2013 braz j oral sci. 12(3):158-163 159159159159159 implants. the human bones have high variability7 and characteristics that may influence the reliability and validity of measurements, such as fenestrations1, which makes necessary a very large sample to obtain a satisfactory significance in statistical comparisons. this is a hindrance to studies due to problems of availability, handling, preparation and preservation of natural bones7. the knowledge of the bone density is essential for the dentist to come up with the best possible surgery plan, and selects the appropriate implant design to obtain primary stability. the development of new implant designs, surface treatments and a better understanding about bone biology have led to constant changings in implantodonty8. the large number of models available in the market or the absence of manufacturer’s clarification the effectiveness of the different models leave the surgeon with doubts and raise questions about design features. implant design should be widely studied by researchers in order to increase the surface area in contact with the bone, consequently the osseointegration, bone anchorage and load distribution9-10. therefore, the stability is a primary requirement to determine the type of prosthetic treatment that will be started, which should be evaluated prior to application of force11. it is important the determination of a safe and practical method to detect the occurrence the primary stability. the numerical value of the initial stability can be provided for clinical instruments such as periotest and osstell, and can be translated as the insertion torque value measured during the final seating of the implant in the recipient bed, which provides information about the quality of local bone, it can easily be obtained with the aid of a surgical wrench. in addition to the clinical methods used for measuring primary stability, there are mechanical tests that analyze the resistance to movement of the implants and may be related to the physical and chemical properties of the screw. the pullout strength implant, widely studied in the medical orthopedics, is multifactorial and is related to bone mineral density, with the implant design and surgical technique, and it is proportional to thread surface in contact with the bone tissue, to avoid its releasing and classifies it as a property of stability of the screw12. nowadays, there are a few available reports in the literature about the tensile strength of dental implants, which it could certainly contribute to the study of primary stability and its relation to the design of implants, where its proper selection is imperative to decrease the magnitude, quantity, and type of loads imposed on the implant / bone interface13. it is believed that tensile tests associated with the insertion torque may add knowledge regarding to the primary stability of implants, since it can quantify the maximum force required for destabilization of the implant in bone. these studies are important for researchers to engage in search of new materials, devices and designs of screw that can fill the gaps offered by the limitations of the surgical technique and quality and quantity of bone site receiving the implant. the aim of this study was to evaluate the primary stability of dental implants through performance tests to increase the knowledge of designs and bone densities, which allow increasing the primary stability due to a variety of available models with the progress and development in dental industry. material and methods substrates for implant placement for this study was used national® polyurethane (nacional ossos, jaú, são paulo, brazil) in three different densities: 40, 20, 15 pcf or 0.64 g/cm3, 0.32 g/cm3 and 0.24 g/cm3, respectively. the substrates had a rectangular shape with dimensions of 4.2 of height, 17.8 cm wide and 6.5 cm length, so that they make feasible their uses in mechanical testing; and homogeneous density, which allowed a standard analysis for the variable type of bone7 and emphasized the comparison between the screws in relation to the shape on the surface treatment. implants in the studies were used 32 implants conexão® (conexão, jau, sao paulo, brazil) divided in four groups according to the their designs (n=8): cylindrical – master porous (external hexagon with double porous treatment of surface), master screw (external hexagon without surface treatment), master conect ar (internal hexagon with porous surface treatment); and conical master conect conico (external hexagon without surface treatment). the cylindrical implants had 3.75 mm diameter, while the conical implants had 3.5 mm. the length was 11.5 mm for all types of implants. (figure 1) fig. 1 insertion torque of implant inserted in the polyurethane installation of the implants in each sample of polyurethane were installed two groups of implants, in a centralized way with respect to the side of the substrate. the preparation of hole in the polyurethane substrate started with a distance of 7 mm from the marking of the adjacent implant in order to prevent that the tests performed in an implant could interfere in the quality effect of implant design and bone density in primary stability braz j oral sci. 12(3):158-163 160160160160160 of the substrate. the holes were carried out following the sequence of cutters provided by the manufacturer (conexão®). after each drilling and hole preparation, the implant was installed in its place with the supporting of a surgical torque wrench, and then it was measured the maximum insertion torque and then it was subjected to a tensile force until the pullout strength of the polyurethane of respective density. after their analysis, the implants were removed and installed in another density of polyurethane. analysis of insertion torque the primary stability was measured by insertion torque with a support of digital wrench mackenaâ (mackena indústria e comércio ltda., são paulo, são paulo, brazil), model mk-2001; capable of torque measuring up to 120 n.cm. this was coupled to the mount-implants of the respective shapes of the external hexagon implants (cylindrical or conical), and to device developed for the connection with internal hexagon implants; both screwed on implants (figure 1) the value was measured in each turn of the screw, being considered the maximum value obtained, the maximum insertion torque. the torque wrench provided a numerical value which was passed to a formula obtained after calibration of the device, converting the value to n.cm unit: y= 0.0449x 0.7907 , so y= value in n.cm, x= value with reading on the wrench. analysis of pullout strength the analysis of pullout strength is used as a method to compare different shapes of metal screws according to the american society for testing and materials14. to check the maximum pullout strength were used the same devices screwed in internal hexagon implants, and the respective mount-implants of conical and cylindrical external hexagon implants, for connection of the implant to the mobile basis of the testing machine. the substrate made of polyurethane with the implant inserted was located in the lower part of a steel lump with a hole in the center. the accessory developed and the mount implants were connected to the implant by threads present in two structures and after that connected to the mobile basis from machine through a pin, being attached to a piece that served for adaptation in the load cell (200 kg) of the universal testing machine (emic®; emic equipamentos e sistemas de ensaios ltda., são josé dos pinhais, pr, brazil) model dl-10000n. each implant was submitted to the pullout testing by axial force a with a constant velocity of 2 mm/min. (figure 2) through the tesc software 1.13 program was carried out the analysis of results obtained during the mechanics tests. it was evaluated the mechanical properties of tensile ultimate strength. statistical analysis the data were analyzed for their distribution and homogeneities. as the distribution was normal (kolmogorovsmirnov) and homogeneous (test of homogeneity of variances – levene) two-way anova and tukey’s test were applied with a significance level of 5%. fig. 2 pullout test of implant inserted in the polyurethane results analysis of insertion torque regarding the it, mp and ms inserted to the substrate 40pcf showed higher values with statistically significant difference with all interactions implants x substrate; the densities of 15 and 20pcf was not statistically significant in all groups of implants. mp, ms, cc and ca did not differ significantly, even inserted in a lower density. master porous and master screw implants inserted into polyurethane of 40 pcf showed the highest values of insertion torque 31.1 ncm and 24.4 ncm, respectively, without statistically significant difference; but there were differences with the others (p<0.05). however, when placed in substrates with density of 20 pcf and 15 pcf, did not show statistical difference. the cc and ca implants had no statistical difference inserted into polyurethane of any densities. in the polyurethane of 15 pcf, there was no statistically significant difference among all types of implants, which were also similar to the cc implants inserted into the substrate of 20 pcf. at the density of 20 pcf, ca had the greatest results and was different of cc implants (p<0.05). cc implants showed the lowest results in the polyurethane of 20 and 40 pcf, but in a lower density (15 pcf) the results were statistically equals to the other groups (p>0.05) (figure 3). analysis of pullout strength the implants inserted into the polyurethane of 40 pcf showed the highest results of pullout strength and statistical difference (p<0.05) with all implants placed in the substrates of 20 and 15 pcf. among them, the ca implants had the greatest results 1463.21 n with statistically significant difference from all other types of implants (p<0.05), which were similar to each other. in the polyurethane of 20 pcf, the master porous and master screw implants were statistically different from the other types (p<0,05), but were equal to the same models on the polyurethane of 15 pcf. the cc implants did not show statistically significant difference when effect of implant design and bone density in primary stability braz j oral sci. 12(3):158-163 161161161161161 inserted into the polyurethane of 20 and 15 pcf (p<0.05). conect ar implant in polyurethane of 15 pcf did not have statistical difference with the same design, but significant difference was found when the implant was inserted into the substrate of 20 pcf density (figure 4). fig. 4 pullout test of implants master porous, master screw, conect ar and conect conico inserted in the polyurethane of 15, 20 and 40 pcf. discussion the primary stability is essential factor for achievement of osseointegration1,3,6 by preventing the formation of connective tissue in the implant / bone interface and allow the bone formation6 which allows appropriate distribution of masticatory functional loads3,6. important strategies, such as increasing the quantity and quality of bone and appropriate implant designs have been investigated in order to provide the initial stability and achieve a greater predictability of osseointegration, particularly in bones of low density6, where an adequate primary anchorage is hardly achieved. many methods have been proposed to measure the primary stability, such as the periotest, the resonant frequency and insertion torque, non-invasive tools which provide numerical values of the stability of implant through the measurement of stiffness in the bone/implant interface during or after installation15. the insertion torque is the angular moment of force required that the screw advances the screw thread inside the mounting hardware16, and provides fast and objective information about the quality of local bone6-15 and the primary stability at surgery6. according to ottoni et al.17, the risk of loss of implants submitted to immediate load is reduced until 20% for each growth of 9.8 n.cm in the insertion torque, which demonstrates the relation of these values have to do with the primary stability. the insertion torque seems to be one of the most efficient techniques and has fewer contraindications in measuring the primary stability3, so our study has selected this method to quantify this property. regarding the insertion torque the cylindrical implants with external hexagon of double surface treatment and without treatment (master porous and master screw), inserted into the polyurethane of 40 pcf, had higher values of all other types and other densities of substrate, demonstrating that cylindrical implants had a better performance. the comparison among different designs of implants was examined in several studies and noticed that conical implants have a higher insertion torque than cylindrical implants3,9. however, checking the resonant frequency, browers et al.1 found higher values for the cylindrical implants compared with the conical, that confirms our results. statistically, the master porous and master screw implants were equals in the insertion torque of all densities and shows that the presence of surface treatment did not influence the results, despite having selected the use of machined implants (master screw and conect cônico) as negative control, due to better biomechanical characteristics of the treated implants, which its surface roughness, theoretically promotes a larger area of bone-implant contact18-19. so, our results contradict studies that found a higher primary stability at treated implants8,19-20. however, our results can be explained by cunha et al.21, who concluded that the design is more important than the surface for primary stability, when verified that the stability of machined implants was the highest of those treated. on these cylindrical implants with external hexagonal, the insertion in the highest density provided the highest values of insertion torque, but placed at densities of 20 and 15 pcf there was no difference between them, what shows that the density just influenced when was very high. both conical implants (conect cônico) and in the internal hexagon implants (conect ar), at any density results of insertion torque were statistically identical, so there was no influence of density on these types of implants. these screw designs inserted into the polyurethane of 40 pcf showed equal values statistically to the master screw and master porous in polyurethane of pcf 20, pointing out that these cylindrical implants and external hexagon had a better performance. this is repeated in the conical implants at 20 pcf that had equal values of insertion torque to the master fig. 3 insertion torque of implant master porous, master screw, conect ar and conect cônico inserted in the polyurethane of 15, 20 and 40 pcf. effect of implant design and bone density in primary stability braz j oral sci. 12(3):158-163 screw and master porous in the substrate of pcf 15, showing again the best performance of these implants that shows higher values even in a lower density. in the density of 15 pcf there was no statistically significant difference on insertion torque between the shapes of the implants, seeming that there is no influence in the shape in the low density. in the density of 20 pcf, only the internal hexagonal implants, that had the highest values of torque, presented statistically different from the others. the conical implant had better performance of insertion torque when inserted into the lower density in comparison with their results in the other two densities, however it was statistically equal to the cylindrical implants. in addition to the methods already proposed to assess the initial fixation of implants, some mechanical tests have been suggested, such as pullout strength, widely used and researched in studies of orthopedic implants12,22, and dental implants for measuring osseointegration by bonding strength of bone-implant23. the pullout strength is influenced by geometric features of the screw, such as shape, diameter and shape of the threads. in addition to the geometrical characteristics, surgical technique and substrate, especially with respect of density, also have influence about results12. despite the pullout test be static axial force application, these factors are also related to primary stability, so our work has proposed this method to compare different screws, as well its form as surface treatment. when analyzing the maximum pullout strength, there was statistical difference among the groups of implants inserted in 40, 20 and 15 pcf; therefore the density of the substrate influences the stability of the screw. the values of cylindrical internal hexagon implants with treatment placed in the highest density (40 pcf) presented the highest and with statistically difference with all others, but among other implant designs (conical and cylindrical external hexagon, with or without double treatment) in the same material, there was no difference, showing that the shape and treatment did not influence, and probably the type of connection influenced. in the polyurethane of 20 pcf the cylindrical implants and internal hexagon showed once more higher values of pullout strength, and they were statistically equals to those inserted into 15 pcf. they were also equals to the conical in 20 pcf, which they did not differ when inserted into the polyurethane of 15 pcf, showing that the lowest density values do not interfere in the results of the conical and internal hexagon implants. the same happened with cylindrical implants and external hexagon with double treatment and without it, where there wasn’t difference to them as much 20 as 15 pcf, and besides, these types of designs there were not difference, showing that the existence of treatment did not influence the maximum pullout strength. generally, cylindrical implants showed a better performance in pullout strength than the conical ones, in contrast to some studies that pointed out that conical screws had higher values than the cylindrical ones, both in insertion torque and pullout resistance22, due to the progressive increase of the diameter to get promotes the compression of the material around them12. however, the fact of the connection of the implants be internal hexagon, these procedure may have influenced on these superior strength results; and in the densities of 20 and 15 pcf, these conical implants had larger and statistically different results, from that cylindrical implants with external hexagon. moreover, the conical implants had a smaller diameter than the cylindrical ones, due to the availability of the company; and it can have influenced the values of insertion torque and pullout strength as well as the study of lill et al.24, that showed the screws with conical design may present inferior mechanical properties when compared to machine screws in different sizes. according to the methodology and results, considering the limitations of this study, the surface treatment of implants did not influence the analysis of insertion torque and maximum pullout strength; cylindrical implants showed better performance on primary stability compared to the tapered implants in the utilized analysis; and the highest density of the substrate (40 pcf) influenced positively the results of primary stability of dental implants analyzed. the relationship between primary stability and implant design presents different results, because studies are done using implants with different diameters, lengths and designs. additionally, for some authors, the primary stability is more affected by the quantity and quality of the bone than the design of the implant25, as well as our study; and thus, the differences found in the literature may be based in the use of different substrates, synthetic or naturals; or even though with the use of dried bones that have up to 10% of the increase of elasticity modulus in comparison to the fresh bone1 what influences the stiffness of the bone-implant interface. this study examined only the mechanical aspects of the effect of design and surface treatment over the primary stability of dental implants. biological factors, as individual characteristics and local variations of the human bone, as well as modifications in surgical technique to increase the anchoring of implants, are influential in the primary stability in a clinical situation. currently, there are several commercial brands of implants that vary in their shape, size, diameter, surface treatment, spacing of the coils, presence and extension of self-drilling region of implants, and prosthetic connections. the purpose of these modifications is to provide an implant that increases and improves the biological contact of the boneimplant interface, and thus promote a fast osseointegration, a better distribution of stress to the receiver bone bed and greater primary stability10. it should be reminded that the correlation between primary stability and pullout strength is a biomechanical suggestion. thus, the higher stability of the screw in the inner bone suggests that the pullout strength is greater and, according to kim et al.26, this property can be used to test the mechanical stability of implants. acknowledgements the authors would like to thank luiz antônio pitangui, of dentscler indústria de aparelhos odontológicos ltda, for providing us with the motor for implants placement. 162162162162162 effect of implant design and bone density in primary stability braz j oral sci. 12(3):158-163 references 1. brouwers jeig, lobbezoo f, visscher cm, wismeijer d, naeije m. reliability and validity of the instrumental assessment of implant stability in dry human mandibles. j oral rehabil. 2009; 36: 279-83. 2. çehreli mc, kökat am, comert a, akkocaoðlu m, tekdemir i, akça k. implant stability and bone density: assessment of correlation in fresh cadavers using conventional and osteotome implant sockets. clin oral implants res. 2009; 20: 1163-9. 3. chong l, khocht a, suzuki jb, gaughan j. effect of implant design on initial stability of tapered implants. j oral implantol. 2009; 35: 130-5. 4. kahraman s, bal bt, asar nv, turkyilmaz i, tözüm tf. clinical study on the insertion torque and wireless resonance frequency analysis in the assessment of torque capacity and stability of self-tapping dental implants. j oral rehabil. 2009; 36: 755-61. 5. turkyilmaz i & mcglumphy ea. influence of bone density on implant stability parameters and implant success: a retrospective clinical study. bmc oral health. 2008; 8: 1-8. 6. tabassum a, meijer gj, wolke jgc, jansen ja. influence of surgical technique and surface roughness on the primary stability of an implant in artificial bone with different cortical thickness: a laboratory study. clin oral implants res. 2010; 21: 213-20. 7. cristofolini l, viceconti m. mechanical validation of whole composite tibia models. j biomech. 2000; 33: 279-88. 8. martínez-gonzález jm, garcía-sabán f, ferrándiz-bernal j, gonzalolafuente jc, cano-sánchez j, barona-dorado c. removal torque and physico-chemical characteristics of dental implants etched with hydrofluoric and nitric acid. an experimental study in beagle dogs. med oral patol oral cir bucal. 2006; 11: e281-5. 9. o’sullivan d, sennerby l, meredith n. measurements comparing the initial stability of five designs of dental implants: a human cadaver study. clin implant dent relat res. 2000; 2: 85-92. 10. carvalho bm, pellizzer ep, moraes sld, falcón-antenuccil rm, júnior jsf surface treatments in dental implants. rev cir traumatol bucomaxilofac. 2009; 9: 123–30. 11. morton d, jaffin r, weber h-p. immediate restoration and loading of dental implants: clinical considerations and protocols. in: int j oral maxillofac implants. 2004; 19: 103-8. 12. inceoglu s, ferrara l, mclain rf. pedicle screw fixation strength: pullout versus insertional torque. spine j. 2004; 4: 513-8. 13. astrand p, engquist b, dahlgren s, engquist e, feldmann h, gröndahl k. astra tech and branemark system implants: a prospective 5-year comparative studyresults after one year. clin implant dent relat res. 1999; 1: 17-26. 14. american society for testing materials. [cited 2011 jan] available from: http://www.astm.org. 15. turkyilmaz i, mcglumphy ea. influence of bone density on implant stability parameters and implant success: a retrospective clinical study. bmc oral health. 2008; 8: 1-8. 16. daftari tk, horton wc, hutton wc. correlations between screw hole preparation, torque of insertion, and pullout strength for spinal screws. j spinal disord. 1994; 7:139-45. 17. ottoni jmp, oliveira zfl, mansini r, cabral am. correlation between placement torque and survival of single-tooth implants. int j oral maxillofac implants. 2005; 20: 769-76 18. barros rrm, novaes jr. ab, papalexiou v, souza vls, taba jr. m, palioto db et al. effect of biofunctionalized implant surface on osseointegration a histomorphometric study in dogs. braz dent j. 2009; 20: 91-8. 19. klokkevold pr, nishimura rd, adachi ma, caputo a. osseointegration enhanced by chemical etching of the titanium surface. a torque removal study in the rabbit. clin oral implants res. 1997; 8: 442-7 20. sakoh j, wahlmann u, stender e, al-nawas b, wagner w. primary stability of a conical implant and a hybrid,cylindric screw-type implant in vitro. int j oral maxillofac implants. 2006; 21: 560-6. 21. cunha ha, francischone ce, nary filho h, oliveira rcg. a comparisson between cutting torque and resonance frequency in the assessmen of primary stability and final torque capacity of standard tiunite single-tooth implants under immediate loading. int j oral maxillofac implants. 2004; 19: 578-85. 22. zamarioli a, simões pa, shimano ac, defino hla. insertion torque and pullout strength of vertebral screws with cylindrical and conic core. rev bras ortop. 2008; 43: 452-9. 23. ban s, maruno s, arimoto n, harada a, hasegawa j. effect of electrochemically deposited apatite coating on bonding of bone to the ha-gti composite and titanium. j biomed mater res. 1997; 36: 9-15. 24. lill ca, schlegel u, wahl d, schneider e. comparison of the in vitro holding strengths of conical and cylindrical pedicle screws in a fully inserted setting and backed out 180 degrees. j spinal disord. 2000; 13: 259-66. 25. rozé j, babu s, saffarzadeh a, gayet-delacroix m, hoornaert a, layrolle p.correlating implant stability to bone structure.clin oral impl res. 2009; 20: 1140-5. 26. kim jw, baek sh, kim tw, chang yi. comparison of stability between cylindrical and conical type mini-implants. angle orthod. 2008; 78: 692-8. effect of implant design and bone density in primary stability 163163163163163 braz j oral sci. 12(3):158-163 oral sciences n3 original article braz j oral sci. april | june 2013 volume 12, number 2 perception of parents of children with and without disabilities about teething disturbances and practices adopted alessandra maia de castro prado1, fabiana sodré de oliveira1, ludmilla de melo abrão2, myrian stella de paiva novaes1, thaís thereza basso prado2 1pediatric dentistry area, dental school, federal university of uberlândia, uberlândia, mg, brazil 2dental school, federal university of uberlândia, uberlândia, mg, brazil correspondence to: alessandra maia de castro avenida pará 1720, bloco 2g, sala 02, cep: 38405720 campus umuarama, uberlândia, mg, brasil fone/fax: +55 34 32182346 e-mail: odontoinfantil@yahoo.com.br fasoliv@yahoo.com.br received for publication: december 21, 2012 accepted: march 22, 2013 abstract aim: to evaluate and compare the perception of parents of children with and without disabilities about the occurrence of local and systemic manifestations during the eruption of primary teeth and to investigate the parents’ practices used to alleviate teething disturbances. methods: a cross-sectional study was conducted in a sample of parents of children without disabilities (gi) and parents of children with disabilities (gii) treated at a university pediatric dentistry clinic. data from gi and gii were collected using a structured questionnaire applied during an interview with the parents, and were analyzed using descriptive statistics. mann-whitney and wilcoxon tests were employed and the level of significance was set at p<0.05. results: the questionnaires were filled out by 86 parents, being 45 in gi and 41 in gii. for gi, the most frequent local manifestations were edema around the tooth (84.44%) and increased suction (75.56%); and for gii were edema and erythema around the tooth (78.05% and 70.73%, respectively). the most frequent systemic manifestations, according to parents of both groups, were irritability and fever. there was no statistically significant difference between groups (p<0.05). it was observed that 46.66% and 68.3% of parents of gi and gii, respectively, adopted different practices to alleviate teething disturbances. conclusions: during primary teeth eruption, local and systemic manifestations may occur and different practices were adopted by the interviewed parents, mainly those with children with disabilities. keywords: teething, primary teeth, eruption. introduction parents and healthcare professionals consider dental eruption as a significant event in the growth and development of the child1. although it is a physiological process, the relationship between the process of the eruption and the onset of symptoms in child is controversial2-3. some authors believe that there is a clear relationship between general or local disorders and eruption4-5, others consider the eruption a physiological process without any manifestation or correlation with any problem. a third group believes that eruption causes some discomfort in normal physiological process2. a number of local and systemic manifestations have been associated with teething. the local manifestations most frequently cited are: red and sensitive gums, gingival edema, gum rubbing, a drooling rash in chin or face, flushed cheeks, pulling braz j oral sci. 12(2):76-79 7777777777 the ears, heavy drooling, mouth ulcers, cyst of eruption and digital sucking. the systemic manifestations included: fever, gastrointestinal perturbations (diarrhea, constipation, colic and vomiting), irritability, coughing due to excess of saliva, disturbed sleep, nasal discharge, loss of appetite and chewing objects6-8. in a cohort study conducted in the brazilian southern region, mothers of 500 infants reported that 73.0% of their children to suffer teething symptoms. the symptoms most frequently cited were irritability (40.5%), fever (38.9%), diarrhea (36.0%) and itching (33.6%)9. most studies were performed with healthy children, not with children with disabilities9-10. thus, the objectives of this study were to evaluate and compare the perception of parents’ of children with and without disabilities about the occurrence of local and systemic manifestations during the eruption of primary teeth and to investigate the parents’ practices used to alleviate teething disturbances. material and methods ethical approval this study was approved by the ethics committee at the federal university of uberlândia (protocol number 085/ 05). parents and/or guardians were invited to take part in the survey and a signed parental consent was obtained before beginning the study. participants a convenience sample was recruited from parents of children (aged 8 months to 5 years) with primary teeth enrolled at a program of early attention to oral health at dental school of the federal university of uberlandia, brazil. the parents were separated in two groups: gi (parents of children without disabilities) and gii (parents of children with disabilities). parents that did not agree to participate in the study and children aged older 5 years were excluded. data collection the data collection was done through interview using a structured questionnaire with parents, which included the following questions: sociodemographic data about the child (age, gender and with or without disabilities); age that initiated the tooth eruption, a list of local and systemic manifestations that were observed during the tooth eruption, and one question about the practices that the parents adopted to manage these manifestations. all interviews were conducted by a single researcher and the sequence of the questions was not changed. the terms were explained in a clear and understandable manner for parents and no limited number of answers was established for the topics about local and systemic manifestations. the interviews were made in a waiting room of the pediatric dentistry clinic during the appointments of the children. the parents of children with disabilities were interviewed at the department of special needs patients, a unit linked to the dental hospital (school of dentistry – federal university of uberlândia) statistical analysis data was analyzed using descriptive statistics. mann-whitney and wilcoxon tests were conducted to analyze data obtained for gi and gii. the level of significance was set at p<0.05. results in this study, 86 parents and/or guardians of children aged 8 months to 5 years old took part. in gi, 26 children were female and 19 were male (mean age = 2 years and 5 months, with standard deviation = 9 months); and in g ii, 17 children were female and 24 male (mean age = 3 years and 7 months, with standard deviation = 1 year). this group included patients with cerebral palsy (68.28%), down syndrome (12.20), chronic systemic disease (12.20%), neurological development delay (4.88%) and endocrine metabolic disease (2.44%). in tables 1 and 2 are demonstrated the results for the local and systemic manifestations observed by parents of each group. edema around the tooth and irritability were the local and systemic manifestations, respectively, most observed by parents of both groups. tables 3 and 4 demonstrate the different behaviors adopted to manage local and systemic manifestations. it was not found statistically significant difference (p<0.05) between the groups. parents of gi would rather to take child to a pediatric consultation (20.0%), while the use of topical anesthetic was the measure adopted by 17.07% of parents of gii. local manifestations gi (%) gii (%) edema around the tooth 84.44 78.05 increased suction 75.56 41.46 increased salivation 57.78 58.54 erythema around the tooth 46.67 70.73 transient gingival inflammation 40.00 29.27 bulging of mucosa 37.78 7.31 pruritus 8.89 68.29 jugal mucosal erythema 6.67 2.44 eruption hematoma 2.22 eruption cysts 2.22 2.44 mouth ulcers 12.20 table 1. prevalence of local manifestations during dental eruption according to parents of gi and gii systemic manifestations gi (%) gii (%) irritability 84.44 70.73 fever 62.22 53.66 disturbed sleep 53.33 43.90 diarrhea 42.22 53.66 increased intake of liquids 20.00 21.95 vomiting 6.67 7.31 nasal discharge 4.44 12.20 table 2. prevalence of systemic manifestations during dental eruption according to parents of gi and gii perception of parents of children with and without disabilities about teething disturbances and practices adopted braz j oral sci. 12(2):76-79 7878787878 parental conduct % consultation with doctor 20.00 consultation with dentist 15.56 use of a systemic anti-inflammatory 4.44 application of topical anesthetic 4.44 application of topical analgesic + rubber teether 2.22 do nothing/ did not answer 53.34% table 3. practices adopted by parents of gi to manage teething disturbances. parental conduct % application of topical anesthetic 17.07 use of a rubber teether 14.63 application of topical anesthetic + rubber teether 12.20 consultation with a pediatrician 4.88 use of topical anesthetic and systemic analgesic 4.88 consultation of a pediatric dentist and a doctor 4.88 handling objects or solid food to the child (carrots) 4.88 use of systemic analgesics 2.44 use of gauze and water for cleaning 2.44 do nothing/ did not answer 31.7% table 4. practices adopted by parents of gii to manage teething problems. discussion the dental eruption is a normal physiological process, which can be modified by individual and environmental factors11 and has been the subject of studies and interest to pediatric dentists, pediatricians and general practitioners who deal with child patient12. in this study, sample selection was made by convenience and also by age group of children, since eruption of primary teeth extends from the sixth to the thirtieth month child life. although children above thirtieth month were included, it is justified because they still present primary teeth. it should be noted that this was a limitation in this study because mothers could not remember all events that had occurred during dental eruption of their children. however, considering the high percentages obtained, it must not had affected the results. a retrospective study based on the analysis of data collected from 450 medical records of children aged 6-60 months in order to analyze systemic and/or local manifestations related to teething showed that 80.9% of parents noted some alteration4. for children with disabilities, the youngest and the oldest age were higher than the control group. it should be emphasized that the normal evolution of dentition depends on the physiological balance of whole body, being affected by factors of general nature, such as systemic or infectious diseases, genetic syndromes and endocrine alterations13. in children with down syndrome, delayed tooth eruption, in part from delayed tooth formation, may be one of the first features noted and can occur in primary and permanent dentitions14. according to the results, it was evident that all parents interviewed have noted the occurrence of systemic and/or local manifestations during tooth eruption and related it to the process. in a survey of australian parents showed that a high percentage of parents believed that teething causes many systemic symptoms10. another study conducted in israel showed that 76.4%, 83.3% and 55.5% of parents, nurses and physicians, respectively, believed that tooth eruption was associated with infant morbidity15. the results of this study were similar to those obtained by other authors who showed that most parents noticed some changes in child during the eruption of primary teeth5,10,16-24. in addition, more than one local and/or systemic manifestation were observed and related to the process of eruption. according to parents of both groups, edema around the tooth was the most observed local manifestation ( 8 4 . 4 4 % a n d 7 8 . 0 5 % f o r g i a n d g i i , r e s p e c t i v e l y ) . related to systemic manifestations, irritability and fever were the most frequent in both groups (table 2). this result agrees with some studies that showed that the symptoms presented during the eruption of deciduous teeth couldn’t be solely attributed to this process 11,24. m o r e o v e r , t h e y a r e m i l d a n d t r a n s i e n t a n d d o n o t undermine the general state of health of the child in the long term 2 5. other causes should be investigated by health professionals to provide favorable conditions for normal growth and development of children. the perception of pediatric dentists and pediatricians about the relation between general and local manifestations with teething shows many differences. according to these professionals, the local manifestations were more related to the process than systemic manifestations 17,26. they also prescribed various drugs for teething problems20. parents of gii adopted more and different practices to manage teething disturbances than those of gi. for gii, applying a topical anesthetic (17.07%) and use a rubber teether (14.63%) were the most common practices. for gi, parents would rather take their children to visit a doctor (20.0%) or a dentist (15.56%). owais et al.18 observed that a high percentage of parents used systemic analgesics or applied topical analgesics in guns to relieve the symptoms associated with teething. in this study, a low percentage of parents used topical anesthetics, probably because the children have been assisted by an early oral health program i n w h i c h p a r e n t s r e c e i v e i n s t r u c t i o n s a b o u t t h e s e conditions. a cross-sectional study conducted with 1,500 parents treated at maternity and child care centers, showed that 76.1% used systemic analgesics and 65.6% rubbed the gingival tissues with topical analgesics to relieve the symptoms. the authors concluded that parents should be better educated about the teething process and the proper management of teething disturbances by the dental health care providers18. according to results, all parents interviewed agreed that several local or systemic manifestations occurred during primary dental eruption and different practices were adopted to alleviate teething disturbances. perception of parents of children with and without disabilities about teething disturbances and practices adopted braz j oral sci. 12(2):76-79 7979797979 references 1. hulland sa, lucas oj, wake ma, hesketh kd. eruption of the primary dentition in human infants: a prospective descriptive study. pediat dent. 2000; 22: 415-21. 2. mota-costa r, medeiros-júnior a, aciolly-júnior h, araújo-souza gc, clara-costa ic. mothers’ perception of dental eruption syndrome and its clinical manifestation during childhood. rev salud publica. 2010; 12: 82-92. 3. rezende cfm, kuhn, e. perception of mothers and pediatricians from ponta grossa/pr about alterations occurred in infants during the eruption of the primary dentition. pesq bras odontoped clin integr. 2010; 10: 163-7. 4. ferreira fv, machado mvs, ardenghi tm, praetzel, jr. systemic and/ or localized manifestations associated with primary tooth eruption: a retrospective study. pesq bras odontoped clin integr. 2009; 9: 235-9. 5. peretz b, ram d, hermida l, otero mm. systemic manifestations during eruption of primary teeth in infants. j dent child. 2003; 70: 70-3. 6. jones m. teething in children and the alleviation of symptoms. j fam health care. 2002; 12: 12-3. 7. silva fwgp, santos bm, stuani as, mellara ts, queiroz am. dental eruption: symptomatology and treatment. pediatria. 2008; 30: 243-8. 8. noor-mohammed r, basha s. teething disturbances: prevalence of objective manifestations in children under age 4 months to 36 months. med oral patol oral cir bucal. 2012; 17: 491-4. 9. feldens ca, faraco im, otoni ab, feldens eg, vitolo mr. teething symptoms in the first year of life and associated factors: a cohort study. j clin pediatr dent. 2010; 34: 201-6. 10. wake m, heskeyh k, allen ma. parent beliefs about infant teething: a survey of australian parents. j paediatr child health. 1999; 35: 446-9. 11. diniz mb, bolini pda, gaspar amm. local and systemic symptoms related to deciduous tooth eruption. rev paul pediatr. 2006; 24: 71-7. 12. pinheiro ga, casado lem, assunção va. tooth eruption physiological or pathological phenomenon? odontol mod. 1993; 20: 28-33. 13. suri l, gagari e, vastardis h. delayed tooth eruption: pathogenesis, diagnosis, and treatment: a literature review. am j orthod dentofacial orthop. 2004; 126: 432-45. 14. shore s, lightfoot t, ansell p. oral disease in children with down syndrome: causes and prevention. community pract. 2010; 83: 18-21. 15. sarrell em, horev z, cohen z, cohen ha. parents’ and medical personnel’s beliefs about infant teething. patient educ couns. 2005; 57: 122-5. 16. andrade dr, silva c, paiva sm. local and general reactions occurred in children facing the process of eruption of deciduous teeth. rgo. 1999; 47: 219-24. 17. lovato m, pithan sa. perception of pediatrics, pediatric dentistry and parents about the clinical manifestations attributed to the eruption of deciduous teeth. stomatos. 2004; 10: 15-20. 18. owais al, zawaideh f, bataineh o. challenging parents’ myths regarding their children’s teething. int j dent hyg. 2010; 8: 28-34. 19. kiran k, swati t, kamala bk, jaiswal d. prevalence of systemic and local disturbances in infants during primary teeth eruption: a clinical study. eur j paediatr dent. 2011; 12: 249-52. 20. oziegbe eo, esan ta, adekoya-sofowora ca, folayan mo. a survey of teething beliefs and related practices among child healthcare workers in ile-ife, nigeria. oral health prev dent. 2011; 9: 107-13. 21. adimorah gn, ubesie ac, chinawa jm. mothers’ beliefs about infant teething in enugu,south-east nigeria: a cross sectional study. bmc research notes. 2011; 4: 228. 22. kakatkar g, nagarajappa r, bhat n, prasad v, sharda a, asawa k. parental beliefs about children’s teething in udaipur, india: a preliminary study. braz oral res. 2012; 26: 151-7. 23. ramos-jorge j, pordeus ia, jorge mlr, paiva sm. prospective longitudinal study of signs and symptoms associated with primary tooth eruption. pediatrics. 2011; 128: 471-7. 24. 24 vasques efl, vasques efl, carvalho mgf, oliveira pt, granvillegarcia af, costa emmb. clinical manifestations attributed to the eruption of deciduous teeth – perception and attitude of parents. rfo-upf. 2010; 15: 124-8. 25. ginani f, vasconcelos rg, barboza cag. local and systemic symptoms associated to tooth eruption. rev bras sci saude. 2011; 15: 81-6. 26. aragão akr, veloso dj, melo auc. pediatricians and pediatric dentists from joão pessoa opinions’ about deciduous tooth eruption and infantile symptomatology. com cienc saude. 2007; 18: 45-50. perception of parents of children with and without disabilities about teething disturbances and practices adopted braz j oral sci. 12(2):76-79 oral sciences n3 original article braz j oral sci. january | march 2015 volume 14, number 1 water sorption, solubility and surface roughness of resin surface sealants jaqueline biazuz1, patrícia zardo1, sinval adalberto rodrigues-junior1 1universidade comunitária da região de chapecó – unochapecó, school of dentistry, area of restorative dentistry, chapecó – sc – brazil correspondence to: sinval adalberto rodrigues-junior universidade comunitária da região de chapecó área de ciências da saúde caixa postal 1141 av. senador atílio fontana, n. 591-e – efapi cep 89809-000 – chapecó – sc – brasil phone: +55 49 3321-8069 e-mail: rodriguesjunior.sa@unochapeco.edu.br abstract surface sealants have been suggested as final glaze of the surface of composite restorations. however, little is known about bulk and surface properties of these materials aiming the long-term preservation of the surface integrity of these restorations. aim: to evaluate the water sorption, solubility and surface roughness of commercial surface sealants for restorations. methods: five disc-shaped specimens (15 mm diameter x 1 mm high) were made from the surface sealants natural glaze (dfl) and permaseal (ultradent) and were light cured according to the manufacturer’s instructions. the specimens were finished with 1500-grit sic paper. water sorption (ws) and solubility (sl) were assessed as recommended by the iso 4049/2000 and were expressed in µg/mm3. surface roughness was evaluated before and after ws and sl, and was expressed in µm as r1 (before ws and sl) and r2 (after ws and sl). it was obtained from three parallel measurements along a 4mm length. data were analyzed using t-test and paired t-test (α=0.05). results: water sorption and solubility of natural glaze were significantly lower than that of permaseal (p<0.05). degradation of the surface sealants did not affect significantly their surface roughness (p>0.05). conclusions: surface sealants used in composite restorations undergo hydrolytic degradation; however, this degradation seems not to interfere on surface roughness of these materials. keywords: composite resins; solubility; dental restoration, permanent. introduction the quality of the restoration surface is rather important for its longevity, as it affects anatomic form, color stability, surface roughness and luster of the restoration1. it may also influence the speed of water diffusion into the material bulk and its potential degradation. besides, a smooth surface tends to withstand better the wear imposed by tooth brushing and occlusal contact with antagonist teeth or food during mastication2. restorations are submitted to finishing and polishing procedures in an attempt to achieve a smooth surface. the finishing procedures aim at the removal and gross contouring of the restorations and the polishing aims at reducing the roughness of the surface and removing grooves and scratches produced by the finishing instruments. finishing is usually performed with diamond or tungsten carbide finishing burs and polishing is done by abrasive impregnated cups, points and disks3. some manufacturers recommend the use of a felt disk associated to an abrasive paste for final luster, while others advise a final glaze by applying a resin surface sealant. söderholm et al.4 (1984) suggested that micro-fissures and micro-cracks are generated during finishing/polishing procedures, which would accelerate water braz j oral sci. 14(1):27-30 received for publication: january 16, 2015 accepted: march 04, 2015 2828282828 diffusion and surface deterioration, the use of resin sealant being an attempt to fill these defects, as it would preserve the integrity of the surface and improve the longevity of the restoration5-7. however, the use of resin surface sealants to improve the quality of composite restorations is still controversial5,8. dental literature has few studies regarding the surface sealant’s properties and the effect of moisture on them. most surface sealants are constituted by the same dimethacrylatebased substance used in dental composites, and they have no reinforcing fillers9. therefore, water diffusion may affect the sealant in a similar way it affects composite restoratives, which would compromise physical properties important to maintain the integrity of the surface. also, up to the present moment, it is not known whether surface sealants degrade similarly to each other, as they may be composed by different dimethacrylate molecules and proportions. understanding the properties of the sealants may help to identify whether using surface sealants is beneficial for the long-term durability of the restorations, which is of utmost importance, as the application of surface sealants represent additional time and cost for both, clinician and patient5. therefore, this study evaluated some properties of the resin surface sealants and tested the following hypotheses: a) water sorption and solubility of the surface sealants will be different, given the differences in their constitution; b) surface roughness will increase after water sorption and solubility, with no difference between sealants. material and methods five specimens of each surface sealant (table 1) were made using a stainless steel mold with 15 mm diameter x 1 mm thick. the specimens were built according to the iso 404910 for evaluating surface roughness, water sorption and solubility, and they were light cured for the time recommended by the manufacturer (table 1). the structure of the monomer molecules that constitute the surface sealants is depicted in figure 1. eighteen superposed light exposures were performed, nine on the top surface and nine on the bottom surface of the specimen (figure 2). a led light curing unit (ultraled, dabi atlante, ribeirão preto, sp, brazil) was used and had its light irradiance measured (~650mw/cm2) at the beginning and at the end of the session with a led radiometer (ecel, ribeirão preto, sp, brazil). the specimens were removed from the mold and were finished using a 1500 grit sic paper. thickness was averaged from five measurements (four at border of the specimen and one at the center) using a digital caliper (mitutoyo corporation, kanagawa, japan). the diameter was averaged from two measurements10, and was used to calculate the surface sealant manufacturer composition light curing time batch no. natural glaze dfl bis-gma, tegdma 20s 13030488 permaseal ultradent bis-gma 20s b8rpf table 1. table 1. table 1. table 1. table 1. surface sealants assessed in the study fig. 1. monomers constituting the surface sealants volume of the specimens (r=dm/2 and v= .....r².h), where r is the radius of the specimen; dm is the mean diameter, v is the volume, and h is the height of the specimen. the specimens were stored in a desiccator at 37 oc with silica gel and weighed daily until their mass was stabilized (dry mass, m1). the specimens were then stored in distilled water for 7 days to obtain the wet mass (m2). thereafter, the specimens were again stored in the desiccator at 37 oc, and reweighed until a constant dry mass (m3) was attained. weighing was performed with an analytical balance with 0.1 mg accuracy (auw220d; shimadzu corporation, kyoto, japan). water sorption (ws) and solubility (sl) were calculated using the following equations: ws = (m2 – m3)/ v; sl = (m1 – m3)/v, and were expressed in µg/mm3. surface roughness (ra) was measured and averaged from three parallel measurements along a 4 mm length with a cut off setting of 0.8 mm using a profilometer (rp200 roughness meter; instrutherm, são paulo, sp, brazil). surface roughness was measured before and after water sorption and solubility. data of water sorption and solubility between sealants were analyzed using t-test (α=0.05), while data of surface roughness of each sealant before and after water sorption and solubility were analyzed with paired t-test (α=0.05). results the results of water sorption, solubility and surface roughness of the surface sealants evaluated in this study are presented in table 2 (mean and standard deviation). t-test water sorption, solubility and surface roughness of resin surface sealants braz j oral sci. 14(1):27-30 fig. 2. light-curing procedure 2929292929 revealed that permaseal presented higher water sorption (p<0.0001) and solubility (p<0.001) than natural glaze. the paired t-test revealed that water sorption and solubility did not affect the surface roughness of natural glaze (p<0.229) and permaseal (p<0.142). discussion the phenomena of water sorption and solubility are part of the degradation process of restorative materials based on the dimethacrylate chemistry, which ultimately lead to a more porous and fragile restoration11. hydrolytic degradation has been described as the cleavage process of linkage that form polymers, generating oligomers and, finally, monomers12-13. according to gopferich13 (1996), this process is triggered by water infiltration within the polymeric chains. factors such as the hydrophilic nature of polymer, differences in the solubility parameter between the polymer and the solvent, cross-link density and filler are understood as important to determine the extension of the polymer degradation in the aqueous environment11,14. the first hypothesis of this study was accepted, since the water sorption and solubility behavior of the studied sealants presented different results. this may be explained by their constitution, which involves two out of the three most used monomers in dental restorative composites: bisgma (2, 2-bis[4-(20-hydroxy-30-methacryloxypropoxy) phenil]propane) and tegdma (triethyleneglycoldime thacrylate). the bis-gma polymer presents superior mechanical properties when compared to the others. the voluminous aromatic rings in the molecule structure are responsible for the bis-gma’s stiffness15 and for a lower degree of conversion of the resulting polymer. on the other hand, a larger network is formed by tegdma, whose molecules are smaller, therefore more mobile and in higher amount16. this network, in spite of being denser, is also spatially more heterogeneous, presenting parts with higher cross-link density and parts with lower cross-link density, which are particularly more prone to water sorption17-18. both molecules present polar linkages with affinity to water. hydroxyl groups are responsible for the hydrophilic character in bis-gma, while in tegdma the ether linkages are those that attract water11. studies have shown that water sorption by tegdma is higher than by bis-gma11,17. the type of monomer also determines the degree of conversion and the cross-link density of the resulting polymer network and, as a consequence, the degree of deterioration of the material11. for instance, the polymer resulting from a monomer blend based exclusively on bis-gma presents a lower degree of conversion due to the rigidity of the monomer, impairing the union of a polymerizable methacrylate group with another15. the incorporation of tegdma monomers to this blend increases the conversion degree due to its high mobility. also, the higher amount of tegdma monomers proportional to bis-gma increases the extent and the degree of reticulation of the resulting polymer, reducing its susceptibility to water penetration and solubilization11,19. this may explain the lower water sorption and solubility of natural glaze when compared to permaseal, as the former is constituted by bis-gma and tegdma and most likely formed a denser polymer by the presence of this monomer16-17. sideridou, tserki and papanastasiou17 (2003) compared the degree of conversion, water sorption and solubility of a polymer blend formed exclusively by bis-gma and by the combination of bis-gma/tegdma in 50/50 wt% and 70/30 wt%. both combinations of bis-gma/tegdma presented higher degree of conversion than the bis-gma alone. besides, they also presented higher water sorption and lower solubility than bisgma. the amount of water percolating within the material seems to depend on the percentage of tegdma in a bis-gma/ tegdma co-polymer, becoming higher with increase of percentage12. still, tegdma has been shown to be less eluted than bis-gma in a bis-gma/tegdma copolymer17. none of the sealants studied present inorganic filler. the presence of filler may reduce the amount of water sorption and solubility, since it reduces the amount of organic matrix16. besides, the surface of the silanized filler particles is inert and does not absorb fluids9. the initial surface roughness of the sealants was determined by polishing with a 1500-grit sic paper and was inferior to the threshold for bacterial biofilm accumulation of 0,2 µm20 for both sealants. as to the final surface roughness, following water sorption and solubility, one could expect that surface sealants constituted by the same monomers as the composites and with no filler would be increased as consequence of the material deterioration and increase in porosity. nevertheless, surface roughness did not differ significantly from the initial roughness for neither of the sealants, leading to the rejection of the second hypothesis. the increase of surface roughness results from the solubilization process, which in dental composites involves the lixiviation of non-converted monomers, oligomers, catalysts, silane and other by-products20-22. furthermore, this process exposes and dislodges filler particles from the material’s surface, contributing to a rougher surface in composites21. water sorption, solubility and surface roughness of resin surface sealants braz j oral sci. 14(1):27-30 surface sealant water sorption and solubility (µg/mm3) surface roughness (µm) w s s l ra 1 ra 2 natural glaze 4.008x10-5 1.731x10-6 0.1776 0.1328 (0.205x10-5) (0.132x10-6) (0.0861) (0.0591) permaseal 5.183x10-5 2.698x10-6 0.1624 0.1108 (0.234x10-5) (0.299x10-6) (0.0333) (0.0486) table 2. table 2. table 2. table 2. table 2. water sorption, solubility and surface roughness of the surface sealants 3030303030 even so, the final surface roughness of the sealants in absolute numbers was reduced in ~0.05 µm. an explanation may be that this reduction may have been caused by absence of filler load in the material, and roughness resulting from the solubilization process would reflect the regularization of small surface irregularities remaining after abrasion with the sic paper and lixiviation of molecules, whose size did not impact the final roughness. however, this study did not assess the effect of the solubilization process on the surface roughness following additional abrasive challenge, such as the one caused by tooth brushing or mastication20. although surface sealants have been available on the dental market for over 20 years, no previous study had characterized the hydrolytic degradation properties of these materials in view of the maintenance or improvement of the service life of composite restorations. for instance, this rationale should be applied in principle to all materials introduced in the dental market. further research should involve surface sealants containing filler particles and sealants constituted by monomers other than dimethacrylates. dimethacrylate-based surface sealants present different water sorption and solubility due to their different organic composition. surface roughness seemed not to be affected by water sorption and solubilization. references 1. rodrigues-junior sa, chemin p, piaia pp, ferracane jl. surface roughness and gloss of actual composites as polished with different polishing systems. oper dent. 2014 sep 30 [in press]. 2. voltarelli fr, santos daroz cb, alves mc, cavalcanti an, marchi gm. effect of chemical degradation followed by toothbrushing on the surface roughness of restorative composites. j appl oral sci. 2010; 18: 585-90. 3. çelik ç, özgünaltay g. effect of finishing and polishing procedures on the surface roughness of tooth-colored materials. quintessence int. 2009; 40: 783-9. 4. söderholm kj, zigan m, ragan m, fischlschweiger w, bergman m. hydrolytic degradation of dental composites. j dent res. 1984; 63: 1248-54. 5. cilli r, mattos mcr, honorio hm, rios d, araujo pa, prakki a. the role of surface sealants in the roughness of composites after a simulated toothbrushing test. j dent. 2009; 37: 970-7. 6. dickinson gl, leinfelder kf, mazer rb, russell cm. effect of surface penetrating sealant on wear rate of posterior composite resins. j am dent assoc. 1990; 121: 251-5. 7. fonseca as, gerardt lm, pereira gd, sinhoreti ma, schneider lf. do new matrix formulations improve resin composite resistance to degradation processes? braz oral res. 2013; 27: 410-6. 8. lopes mb, saquy pc, moura sk, wang l, graciano fmo, correr sobrinho l, et al. effect of different surface penetrating sealants on the roughness of a nanofiller composite resin. braz dent j. 2012; 23: 692-7. 9. valentini f, oliveira sgd, guimarães gz, barbosa rps, moraes rr. effect of surface sealant on the color stability of composite resin restorations. braz dent j. 2011; 22: 365-8. 10. international standards organization iso 4049. dentistry – polymerbased filling, restorative and luting materials: 2000. 11. ferracane jl. hygroscopic and hydrolytic effects in dental polymer networks. dent mater. 2006; 22: 211-22. 12. bagheri r, tyas mj, burrow mf. subsurface degradation of resin-based composites. dent mater. 2007; 23: 944-51. 13. gopferich a. mechanisms of polymer degradation and erosion. biomaterials.1996; 17: 103-14. 14. sideridou id, karabela mm. sorption of water, ethanol or ethanol/water solutions by light-cured dental dimethacrylate resins. dent mater. 2011; 27: 1003-10. 15. van landuyt kl, snauwaert j, de munck j, peumans m, yoshida y, poitevin a et al. systematic review of the chemical composition of contemporary dental adhesives. biomaterials. 2007; 28: 3757-85. 16. sideridou id, achilias ds. elution study of unreacted bis-gma, tegdma, udma, and bis-ema from light-cured dental resins and resin composites using hplc. j biomed mater res part b: appl biomater. 2005; 74b: 617-26. 17. cramer nb, stansbury jw, bowman cn. recent advances and developments in composite dental restorative materials. j dent res. 2011; 90: 402-16. 18. sideridou i, tserki v, papanastasiou g. study of water sorption, solubility and modulus of elasticity of light-cured dimethacrylate-based dental resins. biomaterials. 2003; 24: 655-65. 19. gajewski ves, pfeifer cs, fróes-salgado nrg, boaro lcc, braga rr. monomers used in resin composites: degree of conversion, mechanical properties and water sorption/solubility. braz dent j. 2012; 23: 508-14. 20. ferracane jl. resin composite – state of the art. dent mater. 2011; 27: 29-38. 21. münchow ea, ferreira aca, machado rmm, ramos ts, rodriguesjunior sa, zanchi ch. effect of acidic solutions on the surface degradation of a micro-hybrid composite resin. braz dent j. 2014; 25: 321-6. 22. van landuyt kl, nawrot t, geebelen b, de munck j, snauwaert j, yoshihara k, et al. how much do resin-based dental materials release? a meta-analytical approach. dent mater. 2011; 27: 723-47. water sorption, solubility and surface roughness of resin surface sealants braz j oral sci. 14(1):27-30 oral sciences n3 original article braz j oral sci. july | september 2013 volume 12, number 3 workers oral health: a cross-sectional study marília jesus batista1, lílian berta rihs2, maria da luz rosário de sousa1 1department of community dentistry, piracicaba dental school, university of campinas, piracicaba, sp, brazil 2municipal secretary of health of piracicaba, piracicaba, sp, brazil correspondence to: maria da luz rosário de sousa avenida limeira, 901, cep 13414-018 piracicaba, sp, brasil phone: +55 19 21065209 fax: +55 19 21065218 e-mail: luzsousa@fop.unicamp.br abstract adults and elderly usually present an expressive tooth loss in household epidemiologic studies. few studies were found to report oral health conditions in economically active adults. aim: to describe the oral health status of adult workers in an extended range age (20-64 years old) of a supermarket chain. methods: this cross-sectional survey was conducted in a company in the state of são paulo. a total of 386 workers aged 20 to 64 years old were examined following the guidelines recommended by the world health organization (1997) with respect to caries, treatment needs for caries, and need and use of dental prostheses. age was stratified into groups for analysis. a descriptive analysis was performed and tooth loss rate was calculated. kruskal walis and tukey’s tests were used for the evaluation of differences in dmtf and chi-square test was used for treatment needs. results: the mean dmft was 14.6 (± 8.3), and differences were found among the 3 groups, mainly due to missing teeth. dmft was 10.8 (±6.95) in the 20-34year-old group, 19.6 (±6.13) in the 35-44-year-old group and 22.1(±7.32) in the 45-64-yearold group. significant differences in tooth loss rate were observed between the age groups (tooth loss rate ranged from 18% to 81%). among the adults, 53.5% had treatment needs for caries. conclusions: the younger adult workers in this study showed better oral conditions and an increase in tooth loss was observed in the older individuals. considering common risk approach, dentistry should work together with health promotion for the studied population of workers in order to meet the oral treatment needs and prevent new tooth losses. keywords: adults, occupational health, epidemiology, oral health. introduction the impact of oral health on daily activities and quality of life is known, as well as the number of working hours lost due to oral diseases1 like untreated caries, severe periodontitis and severe tooth loss, which have been listed among the 100 global burden disease in 20102. although the prevalence of caries has been decreasing in the last decades, this health improvement presents significant differences regarding the occurrences of oral diseases among countries, regions, cities and population groups, like adults1. while studies have demonstrated that reduction of oral disease prevalence in household adult population is still a challenge, workers need to be better investigated. epidemiological studies among adults focusing on dental caries experience, tooth loss and prosthesis need are useful planning tools for public health. brazilian nationwide oral health surveys conducted in 19863, 20034 and 20105 indicate variations of the caries experience index between adolescents, adults and elderly, chiefly as regards the missing teeth component3. while adolescents showed less than one tooth loss, adults from 35 to 44 years of age presented a high number of received for publication: april 28, 2013 accepted: july 31, 2013 braz j oral sci. 12(3):178-183 missing teeth in the national epidemiology survey3-5. this finding may be explained by oral health actions for adults with priority on immediate dental care and restorative dental procedures6. the result of those actions was the predominance of lost teeth (with a 50 % to 90 % variation), as shown by oral health surveys of adults and elderly population in a household sample3. the proportion of economically active adults (older than 20 years) to those who were not working was 2:1, in the são paulo state7. workers are involved with the local productivity and the economically active population, so it is important to know their oral health status focusing dental care and oral health promotion in the working environment. this study is relevant and presents two differentials from the national epidemiological studies carried out at household level: a sample of workers and an extended age range from 20-64 years old. thus, the objective of the present study was to investigate the oral health status among workers aged 20 to 64 years in a discount supermarket chain. material and methods settings and study design this cross-sectional study was carried out in são paulo metropolitan region, which consists of 39 municipalities and has 19,889,559 inhabitants7. data were gathered between june 2008 and august 2009 among employees of a discount supermarket chain. sample the age range of the sample was 20-64 years old, in order extend the who age range8. sample size was determined based on the caries experience variable (dmft) using data of the oral health epidemiological survey of the state of são paulo9. in order to calculate the sample size of adults aged 20-29, it was used the dmft of 19 years old (8.9±5.1); for adults aged 30-64 year-old group, it was used the dmft of 35 to 44 years old (20.3±7.61)9. a 95% confidence interval was adopted with precision of 20% and design effect of 2. it was added 20% more adults to the sample, in order to compensate losses and refuses, resulting in a sample size of 376 individuals, being 224 for adults in the 20-30 years range and 152 for adults in the 31-64 years range. the company management was previously informed about the research objective and methods. twenty-five visits were stipulated to the place and random selection of 16 workers among those present at the day of visit, totalizing 400 examined workers to be enrolled. if the adult was absent the day of the visit, a replace was provided. the universe comprised 2000 employees in 2009. variables oral examinations were performed at the company site using artificial light, cpi probes and plane dental mirror9. the only examiner obtained a 90% concordant diagnosis compared to the reference regarding the clinical conditions adopted10. the intra-examiner agreement was 98.5%11. dmft index, treatment needs for caries, need and use of prosthesis were measured following who guidelines. each volunteer filled in a self-applied questionnaire (61 questions) for verification purposes, like demographic factors and socio-economic factors. the inclusion criteria for the survey were: to belong to one of the established age group categories, to show cognitive abilities to answer a written questionnaire and to agree participating in this research. statistical analysis data were tabulated using the statistical package for the social sciences (spss) 17.0 and the excel. age was stratified into 3 sub-groups: 20-34, 35-44 and 45-64. descriptive analysis was performed. the differences between dmft and between genders were verified by the mann whitney test, those between dmft (and its components: decayed, missing and filled teeth) and the age groups by the kruskal wallis test followed by tukey’s test (post-hoc one-way anova). statistical differences among treatment needs, age groups and gender were analyzed by the chi-square test. when the p value was less than 0.05, it was considered significant. because of the great difference in the number of missing teeth, the sample was stratified by age into 9 groups (with 5year intervals) in order to calculate tooth loss rate for each age class, as proposed by dunning and klein12, using the expression m/dmft, where the missing teeth component (m) is the dividend and dmft index the divisor. in order to soften the cumulative effect of dental loss in time, the number of missing teeth in the older age group was removed from the younger subsequent, so that it would be observed which age range presents addition or reduction of tooth losses. kruskal wallis test, followed by tukey’s test (post-hoc oneway anova) was applied to compare tooth loss rate among age groups. ethical issues considering that human participants were involved in this research, ethical approval was obtained from the research ethics committee of piracicaba dental school state university of campinas (no. 122/2005). results out of the 400 randomly selected workers, 14 refused to participate. thus, 386 employees between the ages of 2064 were examined. the mean age was 32.65 years. after stratifying the age groups into 3 sub-groups, there was a predominance of 62.4% (n=241) of younger adults with ages ranging from 20-34 years, followed by adults from 35 to 44 years old with 24.4% (n=94) and the oldest (13.2%, n=51). regarding the evaluated demographic factors, 211 (54.7%) were women and the majority (61.1%) was born in the city of são paulo. workers’ mean family income was u$715.00. considering the education level, 18.9% (n=73) completed the elementary school, 71% (n=274) started or 179179179179179workers oral health: a cross-sectional study braz j oral sci. 12(3):178-183 completed the second grade. the dmft mean of the general sample was 14.6. by evaluating each component, decayed teeth were 9.5% of the index, missing teeth were 38% and filled teeth 52.5%. no difference was found with respect to the components among the sub-groups, with exception for the m component (p<0.05). regarding the dmft index, a difference was observed in the youngest age group (table 1). the mean of present teeth was 26.6. a variation in the mean values of missing teeth among the nine age groups was observed (table 2). the dental loss rate was higher in the last examined group (81%, between 60-64 years). an increase in tooth loss rate was perceived till the 45 year-old group, and moreover till those aged 5560 years. the biggest difference in missing teeth could be seen between the 30-35-year-old and the 35-39-year-old groups (4.6) (table 2). although the mean values of decayed teeth (1.23 ±1.94) were found not so high in comparison to the other components of the dmft index, 44.4% of the studied population presented active caries. among the examined individuals 53.5% had some treatment needs (table 3). extraction was the only treatment need that showed significant difference in the statistical tests among the 3 sub-groups (p<0.01). regarding the use of prosthesis, it was found a greater use of maxillary rather than mandibular prostheses. divergent values were found comparing the gathered data related to the use of prosthesis among the different age groups (table 4). in the evaluation of prosthesis need it was verified that the most prevalent necessity was for fixed prostheses for the age group(years) n (%) decayed missing filled dmft sound teeth 20-34 241 (62.4) 1.32a (±2.08) 2.35a (±2.79) 6.84a (±5.51) 10.79a (±6.95) 21.21 35-44 94 (24.4) 1.32a (±1.91) 9.36b (±7.18) 8.46a (±6.36) 19.58b (±6.13) 12.42 45-64 51 (13.2) 0.61a (±0.96) 13.45c (±10.12)* 8.38a (±6.40) 22.10b (±7.32)** 9.9 total 386 (100) 1.23(±1.94) 5.38 (±6.88) 7.44 (±5.88) 14.56(±8.31) 17.44 table 1. mean values of decayed, missing and filled teeth components, dmft and sound teeth in workers according to age groups in são paulo, 2009. note: the tukey test was used considering p<0.05 for statistical differences. *p<0.05 **p<0.01 age (years) 60 to 64 55 to 59 50 to 54 45 to 49 40 to 44 35 to 39 30 to 34 25 to 29 20 to 24 n % 4 (1.0) 8 (2.1) 9 (2.3) 30 (7.8) 45 (11.7) 49 (12.7) 58 (15.0) 94 (24.4) 89 (23.1) mean (sd) 24.75 (13.84)c 13.71 (11.15)b 10.78 (9.10)b 12.59 (9.07)b 10.98 (8.44)b 7.89 (5.51)b 3.29 (2.90)a 2.76 (3.19)a 1.30 (1.80)a tooth loss rate m/dmft 0.81 0.65 0.49 0.51 0.54 0.41 0.22 0.24 0.18 differences in tooth loss among the age groups 11.04 2.93 -1.81 1.79 3.09 4.6 0.53 1.46 table 2. mean values of missing teeth, tooth loss rate, difference in the mean values of missing teeth among age groups (5-year intervals) in são paulo, 2009. note: different letters indicate significant differences between the groups according to the tukey´s test (post hoc one-way anova) p<0.01 substitution of one tooth (11.1%). the need for mandibular prostheses was greater compared with maxillary prostheses, and 47.4% did not need mandibular prostheses (table 5). discussion this paper highlights the economically active adult population’s oral health data, aged from 20 to 64, which is an extended age range in relation to the who recommendations8, and requires wider investigation. there are few studies about oral health in the working population and the impact of oral disease is well established on the quality of life and daily activities, like work and study. the dmft index varied from 10.80 among the workers aged 20-34 to 19.58 among those aged 35-44 years, in other words an almost 100 % increase. between the adults in the last group and those in the 45-64-age-group (dmft=22.0) there was no significant difference. however, analyzing only the missing teeth component, differences were observed among the three age groups, with variations from 2.3 to 9.4 and 13.5. in british adults as also in the present study, the youngest presented more sound teeth and less restored teeth, representing healthier teeth than the oldest13. therefore, it is important to investigate the dmft index and its components in different age groups, in order to allow interpretation of the real meaning of this index, which evaluates the caries experience. in the latest brazilian nationwide oral health survey (20115), the adult population (35-44 years) showed dmft of 16.3. that means a better result compared with 2003 data, 180180180180180 workers oral health: a cross-sectional study braz j oral sci. 12(3):178-183 20 to 34 years 35 to 44 years 45 to 64 years total n (%) n (%) n (%) n (%) treatment needs for caries n o 112 (46.5) 43 (45.7) 25 (49) 180 (46.6) yes 129 (53.5)a 51 (54.3)a 26 (51)a 206 (53.4) type of needs restoration of 1 face 60 (24.9)a 25 (26.9)a 12 (23.5)a 97 (25.1) restoration of 2 face 59 (24.5)a 26 (27.7)a 15 (29.4)a 100 (25.9) pulp treatment 44 (18.3)a 16 (17.0)a 8 (15.7)a 68 (17.6) exodontia 14 (5.8)a 13 (13.8)b 3 (5.9)c* 30 (7.8) table 3. absolute and percentage distribution of treatment needs among the examined subgroups in são paulo, 2009. note: chi-square test; p<0.05 was applied for rejection of null hypothesis, *p<0.01 maxillary mandibular use of prothesis 2034 35-44 45-64 2034 35-44 45-64 n (%) n (%) no use 229 (95) 68 (72.3) 29 (56.9) 238 (98.8) 87 (92.6) 41 (80.4) 1 fixed prosthesis 4 (1.7) 3 (3.2) 1 (2.0) 1 (0.4) 1 (1.1) 1 (2.0) 1 removable prosthesis 8 (3.3) 16 (17) 10 (19.7) 2 (0.8) 4 (4.3) 4 (7.8) combination of prostheses 0 0 1 (2.0) 0 0 1 (2.0) total prostheses 0 7 (7.4) 10 (19.6) 0 2 (2.1) 4 (7.8) table 4. use of maxillary and mandibular prostheses among adult workers, são paulo, 2009. maxillary mandibular prosthesis needs 20-34 35-44 45-64 20-34 35-44 45-64 n (%) n (%) no need 187 (77.6) 51 (54.3) 30 (58.8) 143 (59.3) 23 (24.5) 17 (33.3) prosthesis for 1 tooth 28 (11.6) 10 (10.6) 5 (9.8) 38 (15.8) 8 (8.5) 3 (5.9) prosthesis for more than 1 tooth 4 (1.7) 4 (4.3) 4 (7.8) 5 (2.1) 9 (9.6) 3 (5.9) combination of prostheses 22 (9.1) 28 (29.8) 11 (21.6) 55 (22.8) 53 (56.4) 26 (51) total prostheses 0 1 (1.1) 1 (2.0) 0 1 (1.1) 2 (3.9) table 5. maxillary and mandibular prosthesis needs among adult workers, são paulo, 2009. which was 20.14. an improvement on adult oral health across years was found in other countries like united states, canada14 and also among the british adults13. although the percentage of the missing teeth component was reduced from 50%4 to 44.8 %5, it was still the most predominant component of the index. considering the same age range, and the workers of the current study, a higher value of dmft than in the latest national survey was observed, but lower mean values of decayed teeth. the samples of the national surveys of 2003 and 2010 were drawn from individuals at their homes, whilst the present study examined company workers. studies have confirmed the impact of work on the health of an individual. not only is the access of the low-income population restricted and the conventional dental treatment onerous, but dental care is also influenced by individual living conditions1,15. most of the time, the income is the determinant factor in the choice of the individual for a specific kind of treatment and acquisition of an oral health product. extending the view to age groups of adolescents, adults and elderly people, it can be observed in the latest brazilian surveys that there is a considerable difference in the missing teeth component among these groups4. thus, the present study addresses the importance of examining an extended age group that fits in these age intervals. the adolescents examined in the state of são paulo, presented 0.42 missing teeth, adults showed 11.25 and elderly people 26.214. it was observed a greater difference in the mean values of missing teeth between the 60-64 years old and the age ranges of 3034 and 35-39. these data draw the attention to the need of performing more comprehensive epidemiological studies in younger adults in order to make a deeper evaluation of the moment when the teeth losses seem to occur more often. the increase of tooth loss in the older age groups has been reported in several studies16. however, there are no scientific signs that establish a link between the age process and the tooth losses6. in the present study, the rate of missing teeth values showed increasing tendency, which varied from 18 % to 81%, showing differences in the mean values of the nine studied age groups. in brazil, tooth loss is the result of absence of policies that promote the oral health in adults in 181181181181181workers oral health: a cross-sectional study braz j oral sci. 12(3):178-183 the past5 and could be explained in this study by cohort effect17. recently, new policies have been implemented in order to extend the dental services access to other age groups, beyond the students18. lacerda et al.19 examined adult workers in the south of brazil and the missing teeth presented the biggest percentage of dmft (54%). nevertheless, in the present examination filled teeth were the highest dmft component in the total sample. filled teeth could be associated with dental care, but the restorative treatment should take into account the risks and diagnosis methods, to avoid that the patient be driven to a repetitive restoration cycle20. in lebanon, 401 adults in the age group 35 to 44 years had 27% of the dmft (16.3) referred to the decayed teeth component21, higher than in the present study. the importance of interpreting the dmft index is a means of avoiding false conclusions regarding the dental health. the component decayed teeth, depending on the observed need of treatment might be filled or pulled. the verification of the treatment needs is relevant to plan dental services. the sb brasil 20034 demonstrated that the more prevalent necessities were low complexity needs like the oneor two-face restorations observed in the present work. it is important to point out that the non-attendance of these needs can drive the evolution of the disease to the tooth loss, which showed in the present study a significant difference among the age groups, being higher among the younger adults, and implying an increase of tooth losses in the future. untreated caries in permanent dentition affects 35% of the population, being the most prevalent condition at all ages in the global burden disease, which means loss in years of life due to this oral condition2. the effect of tooth loss can be observed by the needs and use of prosthesis. in the present research, 95% of the subjects aged 20 to 34 years did not use prosthesis. the use of prosthesis itself is a factor that leads the adult to be classified as a patient with a potential caries risk22. who and other studies emphasized the importance of controlling caries and periodontal disease, based on risk criteria22. it is recommended to them get involved in a prevention program in order to guarantee prosthesis maintenance and adaptation, and to avoid the occurrence of oral diseases22. this original study focused on the adult population that is the labor force of society, and an extended age group in relation to who criteria, unlike other studies. some difficulties, like sampling limitation, occurred because it was difficult to enroll workers older than 45 years – which is an important factor in reaching the target sample size – as the majority of volunteers were between 20 and 30 years old. this study is not representative of all the working population. however, it presents important data of a sample usually not studied such as workers as age range, which brings new knowledge for public health dentistry. world health organization established as goal for the global oral health the minimization of the impact of oral diseases on health and psychosocial development by the year 2020, in order to reduce the absenteeism from school and work. oral health promotion by early diagnosis and control of diseases is one of the tools to reach this target, which will reflect in the reduction of tooth losses23. this knowledge may help understanding the importance of dentistry and medicine working together with a common risk approach to reduce oral disease and promote a healthy environment. the present study observed that among the examined workers, the youngest presented the best oral health conditions, and showed smaller percentages of need and use of prostheses. the majority of the volunteers needed treatment for caries and the most prevalent necessities were for those of low complexity. the implementation of an efficient program for oral health promotion is needed in order to avoid tooth losses among workers of a supermarket chain. acknowledgments we thank fapesp for financial support [scholarship for master degree number 2007/57547-0, research grant number 2008/53309-0]. special acknowledgement is due to the manager of the supermarket chain roldão, as well as the company workers who participated in this study. references 1. petersen pe. the world oral health report 2003: continuous improvement of oral health in the 21st century – the approach of the who global oral health programme. community dent oral epidemiol. 2003; 31 (suppl.1): 3-24. 2. marcenes w, kasseabaum nj, barnabé e, flaxman a, naghavi m, lopez a, et al. global burden of oral conditions in 1990-2010: a systematic analysis. j dent res. 2013; 92: 592-7. 3. ministry of health of brazil. epidemiological survey of oral health: brazil, urban zone (in portuguese). brasília: centro de documentação do ministério da saúde 1988, 137p. (série c: estudos e projetos, 4). 4. ministry of health of brazil. sb brasil 2003: oral health conditions in the brazilian population 2002/2003: main results. brasília: ministry of health, 2004. 68 p. 5. ministry of health of brazil. sb brasil 2010: oral health conditions in the brazilian population 2010/2011: main results. (in portuguese) brasília: ministério da saúde, secretaria de atenção à saúde, departamento de atenção básica. brasília: ministério da saúde, 2012. 92 p. (série c. projetos, programas e relatórios)._2010.pdf. 6. narvai pc & frazão p. oral health in brazil: more than the roof of the mouth (in portuguese), rio de janeiro: editora fiocruz, 148 p. 7. brazilian institute of geography and statistics (ibge). accessed in january 11, 2009. available from www.ibge.gov.br 8. world health organization, oral heath surveys: basic methods. 4th ed. geneva: world health organization; 1997. 9. são paulo state health department. university of são paulo. oral health conditions in the state of são paulo em 2002 (in portuguese). são paulo: oral health center 22. secretary of health of state of são paulo, 80p. 10. world health organization. basic methods. geneve: world health organization; 1987. 11. frias ac, antunes jlf, narvai pc. precision and validity of epidemiological surveys of oral health: dental caries in the city of são paulo in 2002 (in portuguese). rev bras epidemiol. 2004; 7: 144-54. 12. dunning jm, klein h. saving teeth among home office employees of the metropolitan life insurance company. j am dent assoc. 1944; 31:1632-42. 13. white da, tsakos g, pitts nb, fuller e, douglas gva, murray gg, et al. adult dental health survey 2009: common oral health conditions and their impact on the population. br dent j. 2012; 213: 567-72. 182182182182182 workers oral health: a cross-sectional study braz j oral sci. 12(3):178-183 14. elani hw, harper s, allison pj, bedos c, kaufman js. socio-economic inequalities and oral health in canada and the united states. j dent res. 2012; 91: 865-70. 15. watt rg. emerging theories into the social determinants of health: implications for oral health promotion. community dent oral epidemiol. 2002; 30: 241-7. 16. silva dd, rihs lb, sousa mrl. factors associated to the maintenance of teeth in adults in the state of são paulo, brazil. reports in public health. 2009; 25: 2407-18. 17. batista mj, rihs lb, sousa mlr. risk indicators for tooth loss in adult workers. braz oral res. 2012; 26: 390-6. 18. antunes jlf, narvai pc. dental health policies in brazil and their impact on health inequalities. rev saude publica. 2010, 44: 360-5. 19. lacerda jt, simionato em, peres kg, peres ma, traebert j, marcenes w. dental pain as the reason for visiting a dentist in a brazilian adult population. rev saude publica. 2004; 38:453-8. 20. elderton rj. preventive (evidence-based) approach to quality general dental care. med princ pract. 2003; 12(suppl 1): 12-21. 21. doughan b, kassak k. oral health status and treatment needs of 35-44year olds adults in lebanon. int dent j. 2000; 50: 395-9. 22. featherstone jdb, singh s, curtis da. caries risk assessment and management for the prosthodontic patient. j prosthod. 2011; 20: 2-9. 23. hodbell m, petersen pe, clarkson j, johnson n. global goals for oral health 2020. int dent j, 2003; 53: 285-8. workers oral health: a cross-sectional study 183183183183183 braz j oral sci. 12(3):178-183 oral sciences n3 braz j oral sci. 12(1):16-19 original article braz j oral sci. january | march 2013 volume 12, number 1 detection of bifid mandibular condyle by panoramic radiography and cone beam computed tomography frederico sampaio neves1, laura ricardina ramírez-sotelo1, gina roque-torres1, gabriella lopes resende barbosa1, francisco haiter-neto1, deborah queiroz de freitas1 1department of oral diagnosis, division of oral radiology, piracicaba dental school, university of campinas, piracicaba, sp, brazil correspondence to: frederico sampaio neves departamento de diagnóstico oral, divisão de radiologia oral, fop-unicamp avenida limeira, 901, cep: 13414-903, piracicaba, sp, brasil phone: +55 19 21065327 e-mail: fredsampaio@yahoo.com.br abstract aim: to compare panoramic radiography and cone beam computed tomography (cbct) in the diagnosis of bifid mandibular condyle. methods: the sample consisted of 350 individuals who underwent panoramic radiography and cbct. in the panoramic radiographs and cbct images, the presence or absence of bifid mandibular condyle was determined. results: presence of bifid mandibular condyle was detected in four cases (1.1%). in all cases, the relation of one condylar process to the other was mediolateral and history of trauma was reported. none of the individuals had symptoms. in two cases, panoramic radiography did not reveal the presence of bifid mandibular condyle. conclusions: initial screening for bifid mandibular condyle can be performed by panoramic radiography; however, cbct images can reveal morphological changes and the exact orientation of the condyle heads. keywords: mandibular condyle, computed tomography, temporomandibular joint. introduction the bifid mandibular condyle is a rare disorder, characterized by a division of the head of the mandibular condyle. it was first reported by hrdlièka (1941)1, who found 27 cases of this anomaly while analyzing male and female dried human skulls. after this, only a few clinical cases have been reported. bifid mandibular condyle is usually detected in routine panoramic radiographs. the etiology of bifid mandibular condyle remains uncertain, and developmental anomalies, trauma, nutritional disorders, infection, irradiation, genetic factors, teratogenic embryopathy and surgical condylectomy may all be causal factors2. bifid mandibular condyle usually affects only one condyle, but bilateral cases have also been reported3-13. morphology of the bifid mandibular condyle may vary from a shallow groove to two condyle heads and the orientation may be mediolateral or anteroposterior. currently, three-dimensional (3d) imaging techniques bring information that leads to more accurate and specific diagnosis of mandibular condyle conditions. therefore, the aim of this study was to compare panoramic radiography and cone beam computed tomography (cbct) in the diagnosis of bifid mandibular condyle. received for publication: november 17, 2012 accepted: march 04, 2013 braz j oral sci. 12(1):16-19 material and methods the present retrospective study was carried out following approval of the fop/unicamp ethics committee and informed consent was obtained from all volunteers. the sample consisted of 350 individuals who underwent examination by digital panoramic radiography and cbct. these images were taken as part of routine examination, diagnosis and treatment planning of patients with mandibular condyle conditions. digital panoramic radiographs were obtained using an orthopantomograph op100 d unit (instrumentarium corp., imaging division, tuusula, finland) operating at 66kvp, 2.5ma and exposure time of 17.6 s. cbct images were obtained with an i-cat cbct unit (imaging sciences international, inc, hatfield, pa, usa) operating at 120kvp, 8ma, with 0.25mm voxel size and field of view of 13 cm. the presence or absence of bifid mandibular condyle was determined in the panoramic radiographs and cbct images. the bifid mandibular condyle was considered from the presence of a shallow groove up to two distinct condyle heads. panoramic and cbct images were evaluated by two oral radiologists with at least 2 years experience with oral 1 f 51 r yes mediolateral yes no 2 f 23 r yes mediolateral yes no 3 f 25 l n o mediolateral yes no 4 m 72 r n o mediolateral yes no patient n u m b e r gender age (years) side detected in the panoramic radiography orientation of the bifid mandibular condyle history of trauma clinical symptoms f:female; m:male; r:right; l:left. table 1: cases of bifid mandibular condyle. diagnosis and who jointly evaluated the images on a computer monitor (21-inch lcd monitor with 1280×1024 resolution) under dim lighting conditions. the cbct images were analyzed in all three planes, using xorancat software version 3.0.34 (xoran technologies, ann arbor, mi, usa), using the “zoom” tool and manipulation of brightness and contrast. descriptive analysis of the data was performed. results in the present study, bifid mandibular condyle was detected in only four cases (1.1%). the age, gender and affected side are summarized in the table 1. in all cases of bifid mandibular condyle, the relation of one condylar process to the other was mediolateral and history of trauma was reported. none of the individuals had orofacial pain, inability to open the mouth, infectious history or joint ankylosis (table 1). in two cases, the panoramic radiograph did not show the presence of bifid mandibular condyle (figure 1a), but it was visualized in the cbct images (figure 1b). nevertheless, in these cases, the panoramic radiograph showed altered morphology in the condyle (figure 1a). fig. 1. (a) panoramic radiograph showing hipoplasia of the left condyle, with the presence of a radiopaque image above the condyle. (b) coronal cbct slices of the same patient showing the presence of bifid mandibular condyle. the radiopaque image was not visualized in the cbct images, being the projection of the other condylar head. 1717171717 detection of bifid mandibular condyle by panoramic radiography and cone beam computed tomography 1818181818 braz j oral sci. 12(1):16-19 discussion several etiologies have been suggested for the development of bifid mandibular condyle, but there is no agreement about the main causal factor. the genetic origins of such bone abnormalities have been investigated, but minor trauma or developmental factors in utero or during childhood have shown to be the most significant4. antoniades et al. (1993)14 suggested that the development of the bifid mandibular condyle is caused by insufficient capacity for remodeling. quayle and adams (1986)15 indicated that endocrine disorders, nutritional deficiency, infection, trauma, irradiation and genetic factors may be possible causal factors. two cases of condylar fracture due to a traumatic bicycle accident that resulted in the formation of a bifid condyle have also been reported16. this relationship confirms the findings of poswillo (1972)17 who observed the relationship between the formation of bifid condyle and a history of trauma. in the present study, all individuals reported childhood trauma, which is the probable cause for the formation of bifid mandibular condyle. the prevalence of bifid mandibular condyle is extremely low. miloglu et al. (2010)13 evaluated 10,200 panoramic radiographs of the turkish population and found only 32 cases (0.3%) of bifid mandibular condyle, 24 cases (75%) unilateral and 8 cases (25%) bilateral. menezes et al. (2008)12 examined 50,800 panoramic radiographs of brazilian subjects and found only 9 cases (0.018%) of bifid mandibular condyle, being 7 unilateral (78%) and 2 bilateral (22%). in both studies, all patients denied history of trauma. in another study, the review of 18,798 panoramic radiographs of turkish patients revealed 98 cases (0.52%) of bifid mandibular condyle, being 71 unilateral (72.4%) and 27 bilateral (27.6%)18. history of trauma was not investigated. our results showed a higher prevalence of bifid mandibular condyle (1.1%). both previous studies above used panoramic radiographs for diagnosis, while we used cbct images. therefore, the difference may be attributed to the study method: in the present study the panoramic radiographs failed to detect bifid mandibular condyle in two cases and the diagnosis was based on cbct images. this shows that the panoramic radiography is not a reliable method to visualize bifid mandibular condyle. to the best of our knowledge, no study has yet compared panoramic radiography and cbct in the detection of bifid mandibular condyles. çaglayan and tozoglu (2011)19 evaluated the incidental findings in cbct images of 207 patients and found only 2.9% cases of bifid mandibular condyle. in the present study, the prevalence of bifid mandibular condyle was lower. this difference could be associated with the different populations (brazilian and turkish) in the studies. symptoms associated with the bifid mandibular condyle are variable. however, the overwhelming majority of cases is asymptomatic5,11-13. if present, the most common symptoms are joint sounds4,20, joint pain21-22, ankylosis23-25 and, more rarely, intermittent joint lock26. in the present study, all individuals with bifid mandibular condyle were asymptomatic. morphology of the bifid mandibular condyle can vary from a shallow groove to two distinct condyle heads. the orientation of the two condyle heads can vary between two patterns: mediolateral and anteroposterior. the two patterns of mandibular bifid condyle can be related to its causal factor. according to szentpétery et al. (1990)27, the bifid mandibular condyle in the anteroposterior direction is the result of childhood trauma, while the condition in the mediolateral direction develops due to the persistence of a fibrous septum in the condylar cartilage. however, cowan and fergusson (1997)28 argued that the causal factor does not influence the direction of the bifid condyle, which confirms our case, since the individuals reported history of childhood trauma; however, the bifid mandibular condyle had mediolateral direction in each case. the appropriate treatment for cases of bifid mandibular condyle depends exclusively on the symptoms of the patient. for symptomatic cases, treatment is the same used in patients with temporomandibular disorders, which opts for the administration of analgesics, anti-inflammatory drugs, muscle relaxants and physical therapy. in cases of joint ankylosis, surgical treatment is the first choice7,21. due to absence of symptoms experienced by the individuals, no temporomandibular joint treatment was instituted. cbct is useful in several areas of dentistry because it shows 3d images of dental structures and offers clear structural images with high contrast. the exposure dose is a major advantage of cbct when compared with multislice computed tomography and conventional tomography. examination by cbct produces an adequate image quality of the maxillofacial region using lower patient exposure doses when compared to multislice computed tomography2930. in the present study, 3d images were fundamental in the diagnosis of bifid mandibular condyle. in conclusion, initial screening for the presence of bifid mandibular condyle can be performed by panoramic radiograph, but cbct images can reveal morphological changes and the exact orientation of the condyle heads. references 1. hrdlièka a. lower jaw: double condyles. am j phys anthropol. 1941; 28: 75-89. 2. li z, djae ka, li zb. post-traumatic bifid condyle: the pathogenesis analysis. dent traumatol. 2011; 27: 452-4. 3. shaber ep. bilateral bifid mandibular condyles. cranio. 1987; 5: 191-5. 4. mccormick su, mccormick sa, graves rw, pifer rg, bilateral bifid mandibular condyles. report of three cases. oral surg oral med oral pathol. 1989; 68: 555-7. 5. stefanou ep, fanourakis ig, vlastos k, katerelou j. bilateral bifid mandibular condyles. report of four cases. dentomaxillofac radiol. 1998; 27: 186-8. 6. artvinli lb, kansu o. trifid mandibular condyle: a case report. oral surg oral med oral pathol oral radiol endod. 2003; 95: 251-4. 7. antoniades k, hadjipetrou l, antoniades v, paraskevopoulos k. bilateral bifid mandibular condyle. oral surg oral med oral pathol oral radiol endod. 2004; 97: 535-8. 8. alpaslan s, ozbek m, hersek n, kanl a, avcu n and frat m. bilateral bifid mandibular condyle. dentomaxillofac radiol. 2004; 33: 274-7. detection of bifid mandibular condyle by panoramic radiography and cone beam computed tomography 1919191919 braz j oral sci. 12(1):16-19 9. shriki j, lev r, wong bf, sundine mj, hasso an. bifid mandibular condyle: ct and mr imaging appearance in two patients: case report and review of the literature. ajnr am j neuroradiol. 2005; 26: 1865-8. 10. espinosa-femenia m, sartorres-nieto m, berini-aytes l, gay-escoda c. bilateral bifid mandibular condyle: case report and literature review. cranio. 2006; 24: 137-40. 11. acikgoz a. bilateral bifid mandibular condyle: a case report. j oral rehabil. 2006; 33: 784-7. 12. menezes av, de moraes ramos fm, de vasconcelos-filho jo, kurita lm, de almeida sm, haiter-neto f. the prevalence of bifid mandibular condyle detected in a brazilian population. dentomaxillofac radiol. 2008; 37: 220-3. 13. miloglu o, yalcin e, buyukkurt m, yilmaz a, harorli a. the frequency of bifid mandibular condyle in a turkish patient population. dentomaxillofac radiol. 2010; 39: 42-6. 14. antoniades k, karakasis d, elephtheriades j. bifid mandibular condyle resulting from a sagittal fracture of the condylar head. br j oral maxillofac surg. 1993; 31: 124-6. 15. quayle aa, adams je. supplemental mandibular condyle. br j oral maxillofac surg. 1986; 24: 349-56. 16. thomason jm, yusuf h. traumatically induced bifid mandibular condyle: a report of two cases. br dent j. 1986; 161: 291-3. 17. poswillo de. the late effects of mandibular condylectomy. oral surg oral med oral pathol. 1972; 33: 500-12. 18. sahman h, sekerci ae, ertas et, etoz m, sisman y. prevalence of bifid mandibular condyle in a turkish population. j oral sci. 2011; 53: 433-7. 19. caglayan f, tozoglu u. incidental findings in the maxillofacial region detected by cone beam ct. diagn interv radiol. 2012; 18: 159-63. 20. loh fc, yeo jf. bifid mandibular condyle. oral surg oral med oral pathol. 1990; 69: 24-7. 21. corchero-martín g, gonzalez-terán t, garcía-reija mf, sánchezsantolino s, saiz-bustillo r. bifid condyle: case report. med oral patol oral cir bucal. 2005; 10: 277-9. 22. fernández rf, flores hf, mella hs, lillo tf. bifid condilar process: case report. int j morphol. 2009; 27: 539-41. 23. sales ma, oliveira jx, cavalcanti mg. computed tomography imaging findings of simultaneous bifid mandibular condyle and temporomandibular joint ankylosis: case report. braz dent j. 2007; 18: 74-7. 24. rehman ta, gibikote s, ilango n, thaj j, sarawagi r, gupta a. bifid mandibular condyle with associated temporomandibular joint ankylosis: a computed tomography study of the patterns and morphological variations. dentomaxillofac radiol. 2009; 38: 239-44. 25. balaji sm. bifid mandibular condyle with tempromandibular joint ankylosis a pooled data analysis. dent traumatol. 2010; 26: 332-7. 26. almasam oc, hedesiu m, baciut g, baciut m, bran s, jacobs r. nontraumatic bilateral bifid condyle and intermittent joint lock: a case report and literature review. j oral maxillofac surg. 2011; 69: 297-303. 27. szentpétery a, kocsis g, marcsik a. the problem of the bifid mandibular condyle. j oral maxillofac surg. 1990; 48: 1254-7. 28. cowan df, ferguson mm. bifid mandibular condyle. dentomaxillofac radiol. 1997; 26: 70-3. 29. frederiksen nl, benson bw, sokolowski tw. effective dose and risk assessment from film tomography used for dental implant diagnostics. dentomaxillofac radiol. 1994; 23: 123-7. 30. ludlow jb, davies-ludlow le, brooks sl, howeerton wb. dosimetry of 3 cbct devices for oral and maxillofacial radiology: cbmercuray, newtom 3g and i-cat. dentomaxillofac radiol. 2006; 35: 219-26. detection of bifid mandibular condyle by panoramic radiography and cone beam computed tomography oral sciences n3 original article braz j oral sci. october | december 2013 volume 12, number 4 use of etoricoxib and dexamethasone for postoperative pain prevention and control in mucogingival surgery a randomized parallel double-blind clinical trial ligia nadal zardo1, fábio andré dos santos1, gibson luiz pilatti1 1department of periodontology, dental school, state university of ponta grossa, ponta grossa, pr, brazil correspondence to: ligia nadal zardo universidade estadual de ponta grossa departamento de periodontia avenida carlos cavalcanti 4748, cep: 84030-900 ponta grossa, pr, brasil phone/fax: +55 42 32203741 e-mail: ligizardo@hotmail.com abstract aim: to compare the use of etoricoxib and dexamethasone for postoperative pain prevention and control after mucogingival surgery. methods: fifty-eight patients with indication for mucogingival surgery took part in this randomized parallel double-blind clinical trial. they were divided into three groups (g): g1 – placebo 1 h before surgery; g2 – 8 mg dexamethasone 1 h before surgery; g3 – 90 mg etoricoxib 1 h before surgery. pain intensity was assessed in donor and recipient sites separately using the 101-point numerical rating scale nrs – 101, every hour for the first 8 h after surgery and three times a day on the following 3 days. results: there was a statistically significant difference in the postoperative pain intensity in the donor site between g1 and g3 after 1 h, 2 h, 3 h, 7 h, 8 h and on the second day – in the evening after 32 h; between g1 and g2 after 2 h and 3 h, and between g2 and g3 only after the first hour. pain intensity in the recipient site was statistically significant between g1 and g3 after 1 and 2 h (p<0.05). in addition, there was a lower ingestion of rescue medication in g2 and in g3 than in g1 (p=0.002). conclusions: the use of a pre-emptive single dose of etoricoxib or dexamethasone may be considered an effective protocol for postoperative pain prevention and control after mucogingival surgery. keywords: analgesia; pain, postoperative; surgery, oral. introduction periodontal surgical procedures, such as soft tissue grafts (mucogingival surgeries) or those involving bone resection may have a significant expectation of pain and edema for patients after surgery 1,2. soft tissue grafts are autogenous grafts from masticatory mucosa completely detached from their original site and placed in a prepared recipient bed3. the so called “free gingival graft” has been widely used to increase the width of the keratinised tissue and to treat gingival recessions4,5. the predictability of root coverage procedures was dramatically increased by the use of subepithelial connective tissue graft techniques6. pain following periodontal surgery results from a cascade of events during the inflammatory response triggered by a surgical tissue trauma. to prevent or minimize these effects preemptive analgesia has been used, which consists in the received for publication: august 12, 2013 accepted: december 17, 2013 braz j oral sci. 12(4):345-351 use of a drug regimen prior to nociceptive stimuli in order to prevent hyperalgesia and subsequent pain amplification7,8. the search for drug protocols that provide adequate analgesia and comfort for the patient in the postoperative period has been highlighted in the literature9-14. a few trials15,16 suggested that preoperative administration of different anti-inflammatory drugs reduced postoperative pain intensity and the need for supplementary analgesics. several preand postoperative medication protocols have been used to minimize postoperative pain after third molars extraction9,17-20, endodontic surgery21 and periodontal surgical treatment2,10,12,14,22. etoricoxib is considered a non-steroidal anti-inflammatory drug (nsaid) that selectively inhibits cox-2, predominantly produced during inflammation, thereby inhibiting prostaglandin production and release. it is rapidly absorbed and reaches optimum plasma levels 1 h after administration. it has an elimination half-life of approximately 25 h23. the inhibition of cox-2 by nsaid does not prevent the formation of leukotrienes, which also promote sensitization of nociceptors and result in pain and edema. corticosteroids are steroidal anti-inflammatory drugs (said) which suppress inflammation by several mechanisms. they interact with specific intracellular receptor proteins in the target tissues and alter the expression of genes to corticosteroids. specific steroid receptors in the cell cytoplasm bind to ligands for steroid hormone-receptor complexes that occsionally translocate to the cell nucleus. in the nucleus, these complexes bind to specific dna sequences and alter their expression. the complexes may induce transcription of messenger rna, leading to the synthesis of new proteins. among these proteins, annexin is known to inhibit phospholipase a2 and thus block the production of mediators and arachidonic acid metabolites such as the cox-2 enzyme, leukotrienes and prostaglandins, cytokines, interleukins, adhesion molecules and enzymes such as collagenase24,25. dexamethasone is a said that inhibits phospholipase a2, affects the prostaglandins and leukotrienes synthesis, reducing polymorphonuclear leukocytes chemotaxis. furthermore, saids are able to down-regulate many proinflammatory cytokines such as interleukin 1â, -6, -8, -12 and -18, and alpha tumor necrosis factor, involved in inflammation and immune response. it has a half-life (t 1/2) of approximately 3 h and an apparent distribution volume (vd) of 1.0 l/kg24. there are no published studies comparing the use of dexamethasone and etoricoxib before mucogingival surgery. the hypothesis to be tested is whether there is any difference in the intensity of postoperative pain in patients undergoing mucogingival surgery after different protocols of preemptive analgesia with anti-inflammatory drugs administered in a single preoperative dose. material and methods sixty patients referred for periodontal therapy in the dental school of ponta grossa state university took part in this study, from march 2010 to november 2011. this was a randomized double-blind parallel trial, which included patients of both genders, aged 19 to 67 (mean=36.56 ± sd=9.57) who had at least one area with mucogingival surgery indication of epithelized soft tissue graft or subepithelial connective tissue, such as a narrow width of keratinized tissue, labial frenum with an insertion close to the gingival margin associated with a shallow depth of the vestibule, and class i and ii gingival recessions in an aesthetic area. patients with untreated periodontal disease, poor oral hygiene, history of systemic diseases, pregnant and lactating women, allergy to any of the involved medications, patients making long term use of analgesics and/or anti-inflammatory drugs or at risk of infective endocarditis were excluded from the study. the nature of the study was previously explained to each patient, who signed an informed consent form approved by the university’s institutional ethical committee on human research (protocol 16/2011). the study was conducted in accordance with the helsinki declaration of 1975, as revised in 2000. each patient received a different preoperative medication, one hour before surgery: group 1 received a placebo capsule (n=20); group 2 received two 4-mg tablets of dexamethasone (n=20) and group 3 received one 90-mg tablet of etoricoxib (n=20). both the surgeon and the patient had no knowledge of the drug that was used in the trial. the mucogingival surgeries were performed by an experienced periodontist (lnz) following all preoperative care to avoid cross-infection. patients scheduled to receive an epithelized soft tissue graft received local buccal (1.8 ml) and palatal (0.3-0.4 ml) terminal infiltration anesthesia of 2% lidocaine with 1:100,000 epinephrine (dfl, rio de janeiro, rj, brazil). the recipient site was dissected by an incision with a 15c blade, parallel to the mucogingival junction, removing the epithelium and part of the underlying connective tissue, and exposing the periosteum around the affected teeth. a soft tissue graft consisting of epithelium and connective tissue was gently removed from the palate, adapted and sutured to the recipient site with a 5.0 nylon suture and a non-traumatic needle (figure1). patients who underwent the subepithelial connective tissue graft surgery were anesthetized by the same technique using 2% lidocaine with 1:100,000 epinephrine. the exposed root surface was scaled and planed with 5-6 gracey curettes. two vertical diverging incisions were made laterally from the gingival resection. an intrasulcular incision was made preserving the gingival papillae. a partial thickness flap was raised and the epithelium of the gingival papillae was removed. the graft was taken from the palate using a “trap door” technique and placed on the exposed roots and sutured using resorbable 5.0 vicryl sutures (ethicon, inc., são josé dos campos, sp, brazil). the flap was coronally positioned and sutured with 5.0 nylon interrupted sutures (figure 2). all patients received six 750-mg pills of paracetamol (medley, são paulo, sp, brazil) to be used as a rescue medication every 6 h in case of pain, writing down on a form each time the medication was used. one day after surgery, 346 use of etoricoxib and dexamethasone for postoperative pain prevention and control in mucogingival surgery a randomized parallel double-blind clinical trial braz j oral sci. 12(4):345-351 fig. 1. illustration of the epithelized soft tissue graft technique: a – preoperative recession; b – surgical preparation of the recipient site; c – graft sutured in place; d – clot stabilization in the donor site. fig. 2. illustration of the subepithelial connective tissue graft technique: a – preoperative recession; b – partial thickness flap raised; c – connective tissue being removed from the palate; d – connective tissue graft in place; eflap closure; fsuture of the “trap door” in the palate. 171347use of etoricoxib and dexamethasone for postoperative pain prevention and control in mucogingival surgery a randomized parallel double-blind clinical trial braz j oral sci. 12(4):345-351 the patients were instructed to use 0.12% chlorhexidine gluconate (periogard; colgate-palmolive, são bernardo do campo, sp, brazil) as a 15 ml mouthwash for 1 min, every 12 h for 15 days. the time (in min) for performing the surgery and the size of the graft taken from the palate, measured with a periodontal probe (mm2) were recorded on the patient’s file. postoperative pain intensity was recorded separately for the donor and the recipient site by the patient on a paper form every hour for the first eight hours after surgery and three times a day during the next three days. the 101-points numerical rating scale (nrs-101) was used, which consists of assigning a pain score from 0 (no pain) to 100 (pain as bad as it could be). statistical analysis the power calculation was performed using data previously published by the authors12. when the sample size in each of the three groups was 18, a two-sided test would have 82% power at an effect size of 0.45 and a 0.05 level. these calculations were made using specific software (g*power 3 – http://www.psycho.uni-duesseldorf.de/ abteilungen/aap/gpower3/). duration of the surgery, size of the graft (independent variables), nrs-101 scores for the donor and the recipient sites and rescue medication consumption (dependent variables) did not fit the gauss normal distribution curve nor showed homoscedasticity among variances, even when data were transformed. therefore, non-parametric kruskalwallis test was used to compare the three groups. in case of any differences among the groups, pairs using the mannwhitney post hoc test. pain scores for the donor and the recipient sites were analyzed separately at each time period. for all tests a 0.05 level of significance was used. all tests were performed using spss for windows version 13.0 (spss 13.0 for windows; spss, chicago, il, usa). results fifty-eight from the 60 patients initially enrolled completed the study. two patients did not return the form with the pain scores. no side effects were reported for any used medication. there was no statistically significant difference among the groups concerning the size of the graft (placebo56.9±10.0 mm 2, dexamethasone-53.5±12.6 mm 2 and etoricoxib-58.3±11.6 mm2 p=0.36). the duration of the surgery in the placebo group (50.25±7.1) was shorter than in the other groups (dexamethasone-56.8±6.7; etoricoxib56.0±8.9 p=0.015). the mean and standard error for postoperative pain intensity in the donor site for the placebo, etoricoxib and dexamethasone groups are shown in figure 3. there was a statistically significant difference between the placebo and the etoricoxib groups at 1 h, 2 h, 3 h, 7 h, 8 h and on the second day in the evening (p<0.05). postoperative pain intensity in the dexamethasone group was statistically lower than in the placebo group at 2 h and 3 h periods. finally, the etoricoxib group demonstrated statistically significant lower postoperative pain scores than the dexamethasone group only at the 1 h period. figure 4 shows the mean and standard error for postoperative pain intensity in the recipient site for the placebo, etoricoxib and dexamethasone groups. there was a statistically significant difference between the placebo and etoricoxib groups only at the 1 h and 2 h periods. table 1 shows the mean and standard deviation of rescue medication intake (number of analgesic pills) in each experimental group. patients from the placebo group ingested a statistically significant higher number of analgesic pills (2.85±2.00) than the other 2 groups (dexamethasone: 1.05±1.39 and etoricoxib: 1.54±1.74; kruskal-wallis test – p=0.002). fig. 3. postoperative pain values in the donor site (mean and standard error) for the placebo, dexamethasone and etoricoxib groups after the first eight hours, on the 2nd and 3rd day: ain the morning (24 h), b-in the afternoon (32 h), c-in the evening (after 32 h). * statistically significant difference between placebo and etoricoxib groups; # between dexamethasone and placebo groups; + between etoricoxib and dexamethasone groups. 348 use of etoricoxib and dexamethasone for postoperative pain prevention and control in mucogingival surgery a randomized parallel double-blind clinical trial braz j oral sci. 12(4):345-351 group n mean s d placebo* 20 2.85 2.00 dexamethasone 19 1.05 1.39 etoricoxib 19 1.52 1.74 *statistically significant difference compared to the other groups (kruskal-wallis test – p=0.002). table1.table1.table1.table1.table1. sample size, mean and standard deviation of rescue medication intake (number of analgesic pills). fig. 4. postoperative pain values in the recipient site (mean and standard error) for the placebo, dexamethasone and etoricoxib groups after the first eight hours, on the 2nd and 3rd day: ain the morning (24 h), b-in the afternoon (32 h), c-in the evening (after 32 h). * statistically significant difference between placebo and etoricoxib groups. discussion pain after periodontal surgery is usually associated with tissue damage and has an extremely subjective nature. some factors that influence the perception of pain are gender, type of surgery and duration of the procedure, in addition to psychological aspects such as stress and anxiety 26. in previous studies of our research group, the prevention and control of postoperative pain was assessed using an openflap debridement surgery model12,22. in the first study in 2006, a statistically significant difference in pain scores was found between the placebo group and the 200 mg celecoxib group after 1, 2, 3, 4, 6 and 7 hours post surgery. however, despite the fact that 200 mg of celecoxib was superior to 4 mg of dexamethasone only at the 4-hour period, the dexamethasone group was superior to placebo, only at the 3-h period22. in another study12, three pre-emptive anti-inflammatory protocols were evaluated using the same model of periodontal surgery. etoricoxib (120 mg) was superior to placebo from the third to the eighth postoperative hour and the dexamethasone (8 mg) group was superior to placebo from the fourth to the eighth postoperative hour. however, one of the major drawbacks of these studies was that most of the patients expressed low scores of pain, even in the placebo group. therefore, the choice of a mucogingival surgeries model was adopted, since these surgeries are expected to cause more pain than open-flap surgeries for scaling and root planning1. different scales have been proposed for the clinical evaluation of acute pain intensity, such as the visual analogue scale9,10,14,17,19-22,27, the 101-point numerical rating scale (nrs101)13,16 and the 4-point verbal rating scale (vrs-4)2,12,22, demonstrating a good positive correlation among them. nonetheless, nrs-101 was chosen because it seems to be better understood by patients and therefore easier to be used28. in the present study, pain scores were assessed in the first 8 h and three times a day during the following two days after surgery. the higher postoperative pain scores are usually found in the first 24 h12,14,29. these studies support the assessment of postoperative pain at least in the first 8 h after surgery. since an expressive pain decline was observed 8 h after periodontal surgery, it suggests that a single pre-emptive dose of an anti-inflammatory drug should offer a safe duration of action at least for the first 8 h2,12,14,22. although the sample size did not allow a reliable statistical comparison between donor versus recipient area, and also between surgical procedures (free gingival graft versus subepithelial connective tissue graft), pain intensity was higher in the donor than in recipient area, which agrees with resende et al. (2009)30. patients who underwent free gingival graft also expressed higher pain complaints than those from subepithelial connective tissue grafts group. this may be explained by the fact that the removal of connective tissue grafts in this study used a “trap door” technique which provides a complete wound closure in the palate31. on the other hand, the free gingival graft technique removes both epithelium and connective tissue. therefore, it is not possible to close completely the wound with sutures. these may be considered as limitations of this study. dexamethasone is a synthetic corticosteroid which has 171349use of etoricoxib and dexamethasone for postoperative pain prevention and control in mucogingival surgery a randomized parallel double-blind clinical trial braz j oral sci. 12(4):345-351 a powerful anti-inflammatory action. the period required for dexamethasone to reach the peak plasma concentration ranges from 1 to 2 h8, and there are favorable results for drug administration both in one or two hours prior to a third molar surgery9. an effective pain control has also been reported for open flap periodontal surgeries with the use of dexamethasone, compared to the placebo group12,22. the use of steroids with an analgesic purpose is still controversial. dionne et al. 200332 measured prostaglandin e2 (pge2) and thromboxane b2 (txb2) at molar surgical sites using a microdialysis probe and concluded that the use of 4 mg of dexamethasone suppressed pge2 and txb2 levels in samples collected at pain onset but without any effect on the pain report. therefore, a 4g dexamethasone dose does not suppress pge2 release sufficiently to attenuate peripheral sensitization of nociceptors after tissue injury32,33. in a previous periodontal clinical trial this dexamethasone regimen did not lead also to impressive postoperative pain control compared to placebo after open flap periodontal surgery22. however, steffens et al. (2010)12 reported significant postoperative pain prevention and control with the use of dexamethasone 8 mg in open-flap debridement surgery, compared with placebo. . . . . baxendale et al. (1993)17 reported significant pain prevention with the use of dexamethasone 8 mg after multiple third molar extraction compared to placebo. lin et al. (2006)21 found favorable results in pain control after surgical endodontic treatment. in contrast, laureanofilho et al. (2008)25 had lower levels of swelling and trismus, but no effect on pain control with the administration of 8 mg of the dexamethasone in third molar surgery. additional studies should be conducted to clarify the mechanisms of action and the most adequate dose of dexamethasone for pain control after dental surgeries. favorable results in postoperative pain control were achieved with the preemptive use of 8 mg dexamethasone12,22. in the present study this drug regimen was superior to placebo only at the 2nd and 3rd hour after surgery, considering the donor site. no statistically significant difference could be seen between placebo and dexamethasone at any other period. this may be explained by the pharmacokinetic profile of this drug, and also by the higher pain expectance in mucogingival surgeries. etoricoxib is a second generation of the coxib’s nsaid group. it is highly selective for cox-2, quickly absorbed and reaches optimum plasma levels one hour after administration. it has an elimination half-life of approximately 25 h23. it has a high power of pain relief with an nnt=1.6, according to the oxford league table (2007). these data support the use of this medication in the protocol proposed by this study, promoting analgesia for a sufficient length of time. the use of a single 90 mg dose of etoricoxib prior to mucogingival surgery led to a statistically significant lower pain level in the donor site at 1 h, 2 h, 3 h, 7 h and 8 h period, compared to the placebo group. etoricoxib was also superior to dexamethasone in the first postoperative hour. comparing the three experimental groups regarding postoperative pain intensity in the donor site, the etoricoxib group was superior to the placebo group in the 1 h, 2 h, 3 h, 7 h, 8 h periods, and also after 32 h. on the other hand, the dexamethasone group reported less pain compared to the placebo group in the periods of 2 and 3 h after surgery. similar results were found in the study of pilatti et al. (2006)22, in which the dexamethasone group was superior to the placebo group in the period of 3 h after surgery. the etoricoxib group was superior to the dexamethasone group for controlling postoperative pain only in the period of 1 h. the efficacy of etoricoxib over dexamethasone in the first period may be due to pharmacokinetics, since etoricoxib reaches peak plasma concentration in about 1 h, while dexamethasone may take up to 2 h. in the recipient site, the postoperative pain intensity was lower in the etoricoxib group compared to the placebo group in the 1 h and 2 h periods. no statistically significant difference could be found at any other time periods. therefore, the major concern of the surgeon with postoperative pain in these modalities of mucogingival surgeries seems to be mostly related to the donor site. the use of dexamethasone and etoricoxib as preemptive drugs resulted in a statistically significant lower rescue medication intake (paracetamol 750 mg) compared to the placebo group. these findings also agree with those of previous studies12,14. any comparison between the present study and the literature is quite limited because so far there are no studies comparing the use of anti-inflammatory drugs administered in a single-dose for pain prevention and control in cases of mucogingival surgery. however, based on our findings, it may be suggested that the use of a pre-emptive single dose of 90 mg etoricoxib or 8 mg dexamethasone may be a valuable and effective protocol for postoperative pain prevention and control in patients undergoing mucogingival surgery. acknowledgments the authors would like to thank coordination for the improvement of higher education personnel (capes), brasília, df brazil, for the msc scholarship (lnz) that partially funded the study. reference 1. matthews dc, mcculloch ca. evaluating patient perceptions as short-term outcomes of periodontal treatment: a comparison of surgical and non-surgical therapy. j periodontol. 1993; 64:990-7. 2. vogel ri, desjardins pj, major kvo. comparison of presurgical and immediate postsurgical ibuprofen on postoperative periodontal pain. j periodontol. 1992; 63:914-918. 3. american academy of periodontology (2001) glossary of periodontal terms. 4 ed. [cited 2013 oct 28] available from: http://www.perio.org/sites/default/ files/files/pdfs/publications/glossaryofperiodontalterms2001edition.pdf. 4. bjorn h. coverage of denuded root surfaces with a lateral sliding flap. use of free gingival grafts. odontol revy. 1971; 22:37-44. 5. miller pd jr. root coverage using the free soft tissue autograft following citric acid application.iii. a successful and predictable procedure in areas of deepwide recession. int j periodontics restorative dent. 1985; 5: 14-37. 350 use of etoricoxib and dexamethasone for postoperative pain prevention and control in mucogingival surgery a randomized parallel double-blind clinical trial braz j oral sci. 12(4):345-351 6. chambrone l, sukekava f, araújo mg, pustiglioni fe, chambrone la, lima la. root coverage procedures for the treatment of localized recessiontype defects. cochrane database syst rev. 2009; april15: cd007161. 7. kissin i. pre-emptive analgesia. anesthesiology. 2000; 93: 1138-1143. 8. czock d, keller f, rasche fm, häussler u. pharmacokinetics and pharmacodynamics of systemically administered glucocorticoids. clin pharmacokinet. 2005; 44: 61-98. 9. antunes aa, avelar rl, martins neto ec, frota r, dias e. effect of two routes of administration of dexamethasone on pain, edema, and trismus in impacted lower third molar surgery. oral maxillofac surg. 2011; 15: 217-23. 10. peres mf, ribeiro fv, ruiz kg, nociti-jr fh, sallum ea, casati mz. steroidal and non-steroidal cyclooxygenase-2 inhibitor anti-inflammatory drugs as pre-emptive medication in patients undergoing periodontal surgery. braz dent j. 2012; 23: 621-8. 11. mehlisch dr, aspley s, daniels se, bandy dp. comparison of the analgesic efficacy of concurrent ibuprofen and paracetamol with ibuprofen or paracetamol alone in the management of moderate to severe acute postoperative dental pain in adolescents and adults: a randomized, double-blind, placebo-controlled, parallel-group, single-dose, two-center, modified factorial study. clin ther. 2010; 32: 882-895. 12. steffens jp, santos fa, sartori r, pilatti gl. pre-emptive dexamethasone and etoricoxib for pain and discomfort prevention after periodontal surgery: a double-masked, crossover, controlled clinical trial. j periodontol. 2010; 81: 1153-1160. 13. sotto-maior bs, senna pm, de souza picorelli assis nm. corticosteroids or cyclooxygenase 2-selective inhibitor medication for the management of pain and edema after third-molar surgery. j craniofac surg. 2011; 22: 758-762. 14. steffens jp, santos fa, pilatti gl. the use of etoricoxib and celecoxib for pain prevention after periodontal surgery: a double-masked, parallel-group, placebocontrolled randomized clinical trial. j periodontol. 2011; 82: 1238-44. 15. joshi a, parara e, macfarlane tv. a double-blind randomised controlled clinical trial of the effect of preoperative ibuprofen, diclofenac, paracetamol with codeine and placebo tablets for relief of postoperative pain after removal of impacted third molars. br j oral maxillofac surg. 2004; 42: 299-306. 16. aoki t, yamaguchi h, naito h, shiiki k, izawa k, ota y et al. premedication with cyclooxygenase-2 inhibitor meloxicam reduced postoperative pain in patients after oral surgery. int j oral maxillofac surg. 2006; 35: 613-17. 17. baxendale br, vater m, lavery km. dexamethasone reduces pain and swelling following extraction of third molar teeth. anaesthesia. 1993; 48: 961-4. 18. al-sukhun j, al-sukhun s, penttilä h, ashammakhi n, al-sukhun r. preemptive analgesic effect of low doses of celecoxib is superior to low doses of traditional nonsteroidal anti-inflammatory drugs. j craniofac surg. 2012; 23:526-9. 19. shah r, mahajan a, shah n, dadhania ap. preemptive analgesia in third molar impaction surgery. natl j maxillofac surg. 2012; 3:144-7. 20. aznar-arasa l, harutunian k, figueiredo r, valmaseda-castellón e, gayescoda c. effect of preoperative ibuprofen on pain and swelling after lower third molar removal: a randomized controlled trial. int j oral maxillofac surg. 2012; 41: 1005-9. 21. lin s, levin l, emodi o, abu el-naaj i, peled m. etodolac versus dexamethasone effect in reduction of postoperative symptoms following surgical endodontic treatment: a double-blind study. oral surg oral med oral pathol oral radiol endod. 2006; 101: 814-7. 22. pilatti gl, santos fa, bianchi a, cavassim r, tozetto cw. the use of celecoxib and dexamethasone for the prevention and control of postoperative pain after periodontal surgery. j periodontol. 2006; 77: 1809-1814. 23. shi s, klotz u. clinical use and pharmacological properties of selective cox-2 inhibitors. eur j clin pharmacol. 2008; 64: 233-52. 24. franchimont d. overview of the actions of glucocorticoids on the immune response: a good model to characterize new pathways of immunosuppression for new treatment strategies. ann n y acad sci. 2004; 1024: 124-137. 25. laureano-filho jr, maurette pe, allais m, cotinho m, fernandes c. clinical comparative study of the effectiveness of two dosages of dexamethasone to control postoperative swelling, trismus and pain after the surgical extraction of mandibular impacted third molars. med oral patol oral cir bucal. 2008; 13: 129-132. 171351use of etoricoxib and dexamethasone for postoperative pain prevention and control in mucogingival surgery a randomized parallel double-blind clinical trial braz j oral sci. 12(4):345-351 429 too many requests error 429 too many requests too many requests guru meditation: xid: 31552421 varnish cache server 429 too many requests error 429 too many requests too many requests guru meditation: xid: 38906055 varnish cache server oral sciences n3 braz j oral sci. 13(1):1-5 original article braz j oral sci. january | march 2014 volume 13, number 1 dental caries trends among preschool children in indaiatuba, sp, brazil regiane cristina do amaral1, marília jesus batista1, maria paula maciel rando meirelles1, silvia cypriano2, maria da luz rosário de sousa1 1universidade estadual de campinas unicamp, piracicaba dental school, department of community and preventive dentistry, piracicaba, sp, brasil 2pontíficia universidade católica de campinas puccamp, dental school, department of community and preventive dentistry, campinas, sp, brasil correspondence to: maria da luz rosário de sousa universidade estadual de campinas faculdade de odontologia de piracicaba avenida limeira 901, cep: 13414-903 bairro areão, piracicaba, sp, brasil phone: +55 19 21065364 e-mail: luzsousa@fop.unicamp.br abstract aim: to evaluate caries experience and associated factors in 5-year-old preschool children in the city of indaiatuba, sp, brazil. methods: this was a cross-sectional, representative study, conducted from an epidemiological oral health survey (2010) with 303 children. the sample was established by the systematic probabilistic method, in public and private schools, in accordance with who criteria. the sample was checked for caries experience (dmft) by four trained and calibrated dentists, reaching acceptable levels of agreement for data collection. parents or guardians answered questions related to their education and monthly income, and the children answered questions related to dental care and pain. descriptive and bivariate analyses of independent variables were performed. variables with p<0.20 were included in the model (poisson regression analysis). results: the sample consisted of 151 boys and 152 girls, with a mean dmft of 1.46. the reasons for visiting a dentist due to pain or need for treatment were associated with dmft > 0 (pr=3.76, 95%ci=2.06-6.84) after adjustment of the regression model. conclusions: among the preschool children of this study, pain or need for treatment due to caries disease in the primary dentition and the reason to visit the dentist due pain emphasizing the importance of the professional not only in curative actions, but as a health promoter at the first contact with the child. keywords: oral health; epidemiology; dental caries; preschool. introduction several publications have shown declining trends in caries experience, thus many researchers studied the factors associated with this decline1-4. among the studied factors are family income and mother’s education level1-2. according to the sb brazil 2010 data, there was a 17% reduction in the ‘decayed’ component of dmft index in 5-year-old children, with a decrease in the mean dmft value from 2.8 in 2003 to 2.3 in 20105. nevertheless, brazil did not show a significant decline according to the who (2000) goals for primary dentition (5 years), which established a minimum proportion of 50% of caries-free teeth6, and it is far from fulfilling the who goal for 2010 of 90% of 5and 6-year-old children free of caries7. the city of indaiatuba, sp, brazil, presents a 0.79 human development index (hdi)8, which is above the brazilian national average. this hdi denotes a situation not usually observed in other brazilian cities regarding health indicators. dental caries is a multifactorial disease caused by oral bacteria mediated by dietary carbohydrates, but income and education, which are both considered on hdi determination, might have an influence on caries’ etiological factors. received for publication: november 25, 2013 accepted: march 10, 2014 gisele higa texto digitado http://dx.doi.org/10.1590/1677-3225v13n1a01 braz j oral sci. 13(1):1-5 2 dental caries in the primary dentition is considered a predictor of the disease in adulthood. more than two surfaces with caries experience in primary second molars at 5 years of age may represent a clinically useful predictor for high risk at age 109. thus the knowledge of caries disease in the primary dentition is important to determine whether the oral environment will be favorable or not during eruption of the first permanent molars, which are often more susceptible to caries9. the aim of this study was to evaluate caries experience (dmft) and associated factors in preschool children in the city of indaiatuba, sp, brazil. material and methods study design this research was a cross-sectional study conducted in the city of indaiatuba, sp, brazil, in 2010. the city of indaiatuba is located 112 kilometers from the city of são paulo, in the southeastern region of brazil, has about 201,619 inhabitants and a land area of 311 km2 (ibge)10. to calculate the sample size, it was used the average dmft value and standard deviation obtained in a previous study (ses)11, adopting criteria of the sb brazil project 20105 resulting in 296 pupils with 95% confidence level, 20% precision and design effect (deff) 2, according to the formula used by the sb brazil 2010. twenty percent were added to the total number of volunteers, in order to compensate for losses and refusals, then were selected a sample size of 355 pupils. systematic probabilistic sampling was used, adopting the sampling without replacement rule in order to be representative of the municipality. sample selection occurred in two stages, with the first draw of 20 schools that were organized according to the number of pupils, and then the 355 5 year-old children were randomly selected. ethical aspects this study began after approval was obtained from the ethics committee of piracicaba school of dentistry (no. 105/2010). parents or guardians signed an informed consent form authorizing the enrollment and participation of their children in the study. parents or guardians also participated in the study answering a questionnaire about their socioeconomic conditions. clinical examination for the examinations, a team of 4 examiners (dentists) was calibrated in a 36-h training procedure, divided between theoretical discussions and practical activities, simulating the different conditions and situations that dentists might face during the course of practical work. the percentage of inter-examiner agreement was 95% for caries experience (dmft) and intra-examiner agreement was 98 to 100%. the codes and criteria used for determining the dmft, which measures dental caries experience, were in accordance with the current recommendations of the world health organization (who)12. the children were examined under natural light, using dental mirror and cpi probe for epidemiological surveys (who)12. questionnaire parents or guardians answered questions related to their education, income, number of rooms in the residence and number of children. the parents or guardians were asked whether they had ever been to the dentist; place of the demand for dental service; the reason for seeking care; and whether the children had missed school activities because of toothache. data analysis data were tabulated in the excel program and the spss 17.0 (statistical package for the social sciences; spss inc., chicago, il, usa) for statistical analysis the program was used. the descriptive analysis of the conditions was evaluated considering the weight, i.e. the value of each school relative to the population of 5-year-old children in indaiatuba. later, these variables were grouped and dichotomized for statistical purposes according to the distribution of the sample. the data were further dichotomized relative to the number of rooms in the house; how many people lived in the house; how many children in the family; type of school (private or public), father’s years of education; mother’s years of education; school absence because of pain; if the child has been to the dentist; place of the consultation; reason for consultation, family income, and whether the child had treatment needs with regard to the dmft. the outcome chosen for the study was the dmft=0 and dmft>0. bivariate analyses were performed (chi-square test) and the variables that showed statistical significance (p<0.20) were included in the multivariate analyses and poisson regression with robust variance13. results in 2010, 303 children, 5 years of age, attending public and private schools of indaiatuba, sp were examined. due the loss of the 15% the sample, the final numbers of volunteers was of 303 pupils. the sample consisted of 151 boys and 152 girls. among the 303 children examined, 177 (58.4%) had dmft=0, 25 (8.2%), dmft=1 and 32 (10.5%) dmft=2, with a mean dmft of 1.46. as regards the dmft components, 56.8% of the teeth were decayed and 42.8% were restored (table 1). the variables with p<0.20 in the bivariate analyses were included in the regression model as follows: father’s schooling, missing school due to pain, going to the dentist, reason for consultation and treatment needs. after adjusting the model, only the variables father’s education, income and reason for consultation were maintained in the analysis. the reason for consultation, toothache and treatment need was associated with the presence of dental caries (table 2). dental caries trends among preschool children in indaiatuba, sp, brazil 3 braz j oral sci. 13(1):1-5 p pr ci 95% father’s schooling 9 years or + 0.224 0.780 0.522 1.165 up to 8 years 1 reason for consultation, pain+ need 0.000 3.762 2.069 6.840 routine 1 table 2.table 2.table 2.table 2.table 2. factors associated with caries prevalence in the city of indaiatubasp, brazil, 2010. dental caries trends among preschool children in indaiatuba, sp, brazil characteristics n dmft=0 dmft>0 p gender male 151 87 64 female 152 90 62 0.7 responsible for supporting family father or mother 86 46 40 father and mother 130 81 49 other 11 8 3 0.28 residents in family up to 4 dwellers 157 92 65 5 or more dwellers 70 42 28 0.57 number of children up to 2 children 163 98 65 3 or more 65 37 28 0.61 number of rooms up to 2 rooms 157 90 67 3 or more 65 42 23 0.31 father’s schooling until 8 years 78 43 35 9 years or mores 139 92 47 0.1 mother’s education up to 8 years 76 46 30 9 years or more 152 97 55 0.62 absence from school due totoothache no 218 134 84 y e s 9 1 8 0.04 visit to dentist y e s 153 85 68 no 71 46 25 0.1 place of visit to dentist public health 64 34 30 others 91 52 39 0.6 reason for consultation routine / maintenance 81 62 19 pain / dental caries / bleeding / other 58 16 42 <0.0001 income up to 1500 reais 103 58 45 more than 1500 reais 90 53 37 0.09 school type public 273 155 118 private 30 22 8 0.81 need of treatment does not need treatment 198 175 23 needs treatment 105 2 103 <0.001 table 1.table 1.table 1.table 1.table 1. factors associated with dmft according to bivariate analysis, indaiatuba – sp, brazil, 2010. 4 braz j oral sci. 13(1):1-5 discussion the city of indaiatuba has a history of epidemiological surveys on dental caries dating back to 1992 and there has been a decline in caries experience since then. regarding this decline, differences were observed between the dmft components; for example, in the present study, 56.8% of the teeth were decayed and 42.8% were filled, with a mean dmft of 1.46. in other words, there was higher prevalence of the filled component than was shown in the last survey conducted in 2004 in indaiatuba, which consisted of approximately 12% of cases14. according to the who goals for the year 2000, 57.4% (n=358)13 in 2004 and 58.4% (n=303) in 2010 of the preschool children examined in the city of indaiatuba were free of dental caries. thus, the city of indaiatuba fits in with this goal, and so do other cities in brazil, such as paulínia, sp and salvador, ba, which had a dmft=0 in 54% and 50.4% of the children in 200315 and 200516, respectively. although this fact seems promising, the who established goals for 90% of children aged 5 and 6 years old (who) to be free of dental caries in 20107. however, even in developed countries as belonging to united kingdon this goal does not seem to be feasible since as in the england, wales and scotland the number of children with dmft>0 was 38%, 52.8%, 46% and 39.4%, respectively, which is below the target set for the year 2010. according to the authors, these values characterize the failure to obtain significant improvements in the presented rates, primarily due to vulnerable groups17. on the other hand, there are countries like kosovo, where only 2.1% of children aged 6 years were caries-free, most probably due to dentistry that calls for intervention, with no preventive work being done, and children only seek dental treatment only when they feel pain18. indaiatuba presented low dmft in the study, and has an hdi index considered high (0.79), when compare with the others countries, according to who19. the city has favorable characteristics with regard to education and health programs, and the monthly income of the examined children’s parents was considered high. however, as a factor associated with dental caries, the reason for seeking the dentist was found to be pain or need for treatment. the relationship between the absence and presence of caries had as an associate factor the reason for seeking the dentist. patients with pain or treatment needs showed a 3.7 times higher demand for care than patients who sought the service routinely. similar results were found in the city of recife, pe, where demand of service for treatment was 4.8 times higher than for prevention4, i.e., the population tends to delay seeking dental treatment. socioeconomic status has been considered a determinant factor of the risk for caries. low income may be associated with the level of education, the value attributed to health, lifestyle and access to information on health care. as a result, the family income may be an indirect factor for susceptibility to caries20. thus, in spite of socioeconomic factors considered as predictors of caries distribution in schoolchildren20-21, in similar studies an association was found between socioeconomic factors and dental caries22-23, and such data were not found in the present study. a possible explanation is the fact that, in the present study, there were not many individuals with very low income and the dichotomized income data showed values higher than those of other studies22-23. the results are similar with respect to years of education of the mother (caregiver). some authors define this criterion as an indicator of caries disease4,24 , but in the present study this was only significant for the father’s number of schooling years. other factors, such as number of rooms in the residence, type of school, number of persons residing at home and number of children were not statistically significant in this study. these results were similar to those found by cortellazzi et al., 200825, with regard to the number of people in the same household; however, there was difference in the type of school and dental fluorosis. epidemiological surveys are important tools to diagnose the oral health status of populations and should contribute to the planning of health services. it should be emphasized the importance of the continuity of the oral health programs that have been implemented in the city in order to change the paradigm of seeking the dentist only in case of pain into a model for health maintenance and adherence of parents. for preschool children in the city of indaiatuba, the factor associated with caries disease in the primary dentition was the reason to visit to the dentist due to pain or need for treatment, thus highlighting the importance of the dentist not only in curative actions, but also as health promoters at the first contact with the child. references 1. alcântara tl, batista mj, gibilini c, ferreira np, sousa mlr. factors associated with oral health of preschool children placed in preventive educational program in piracicaba/sp. rpg rev pos-grad. 2011; 18: 102-7. 2. da silva an, mendonça mh, vettore mv. the association between low socioeconomic status mother’s sense of coherence and their child’s utilization of dental care. community dent oral epidemiol. 2011; 39: 115-26. 3. cypriano s, hugo fn, sciamerelli mc, torres lhn, sousa mlr, wada rs. factors associated with the incidence of dental caries among schoolchildren living in a municipality with low prevalence of dental caries. cienc saude colet. 2011; 16: 4095-106. 4. melo mm, souza wv, lima ml, braga c. factors associated with dental caries in preschoolers in recife, pernambuco state, brazil. cad saude publica. 2011; 27: 471-85. 5. brazil. ministry of health. sb brazil. national survey of oral health: 2010. main results. brasília: ministry of health; 2011 [cited 2012 jan 26]. available from: http://189.28.128.100/dab/docs/geral/ projeto_sb2010_relatorio_final.pdf. 6. lucas sd, portela mc, mendonça ll. variations in tooth decay rates among children 5 and 12 years old in minas gerais, brazil. cad saude publica. 2005; 21: 55-63. 7. pan american health brazil [internet]. [cited 2014 mar 10]. available from: http://www.paho.org/bra. dental caries trends among preschool children in indaiatuba, sp, brazil 5 11. secretary of state for health (seh). regional directorate of health of campinas. epidemiological survey on oral health: the state of são paulocampinas dir xii; 1998. [report presented at the conclusion of the research project conducted by the center for studies and research in health systems, school of public health, university of são paulo; 1999]. 12. world health organization. oral health surveys, basic methods. 4th ed. geneva: who; 1997. 13. moraes jfd, souza vba. factors associated with the successful aging of the socially-active elderly in the metropolitan region of porto alegre. rev bras psiquiatr. 2005; 27: 302-8. 14. rihs lb, sousa mlr, cypriano s, abdalla nm. inequalities in distribution of dental caries in teenagers of indaiatuba, são paulo state, 2004. cienc saude colet. 2010; 15: 2173-80. 15. gomes pr, costa sc, cypriano s, sousa mlr. dental caries in paulínia, são paulo state, brazil, and who goals for 2000 and 2010. cad saude publica. 2004; 20: 866-70. 16. almeida tf, cangussu mct, chaves scl, castro e silva di. the dental health of preschool-aged children resident in areas covered by the family health program, in the city of salvador, in the state of bahia, brazil. rev bras saude matern infant. 2009; 9: 247-52. 17. pitts nb, boyles j, nugent zj, thomas n, pine cm. the dental caries experience of 5-year-old children in great britain (2005/6). surveys coordinated by the british association for the study of community dentistry. community dent health. 2007; 24: 59-63. 18. begat a, mega k, siegenthaler d, bearish m, match w. dental health evaluation of children in kosovo. eur j dent. 2011; 5: 32-9. 19. wikipédia. annex: list of countries by human development index [internet]. [cited 2014 jan 24]. available from: http://pt.wikipedia.org/wiki/ anexo:lista_de_países_por_índice_de_desenvolvimento_humano# desenvolvimento_humano_muito_alto 20. solidário dmp, holding jf, moreira d, rodrigues, jao, berilo mfg, rosa ear. dental caries status in deciduous and permanent dentition of brazilian children aged 6-8 years with a socio-economic base. braz j oral sci. 2003; 2: 147-50. 21. piovesan c, mendes fm, antunes jlf, ardenghi tm. inequalities in the distribution of dental caries among 12-year-old brazilian schoolchildren. braz oral res. 2011; 25: 69-75. 22. pereira sm, tagliaferro ep, ambrosano gm, cortellazzi kl, meneghim m de c, pereira ac. dental caries in 12year old schoolchildren and its relationship with socioeconomic and behavioral variables. oral health prev dent. 2007; 5: 299-306. 23. catani db, cypriano s, sousa mlr. clinical and socio-behavioral determining factors of dental caries in deciduous and permanent dentitions in a city with optimal fluoride concentrations in the water supply. arq odontol. 2010; 46: 197-207. 24. tagliaferro eps, ambrosano glb, meneghin mc, pereira ac. risk indicators and risk predictors of dental caries in schoolchildren. j appl oral sci. 2008; 16: 408-13. 25. cortellazzi kl, pereira sm, tagliaferro ep, tengan c, ambrosano gm, meneghim m de c, et al. risk indicators of dental caries in 5 year old brazilian children.community dent health. 2008; 25: 253-6. dental caries trends among preschool children in indaiatuba, sp, brazil braz j oral sci. 13(1):1-5 429 too many requests error 429 too many requests too many requests guru meditation: xid: 36472721 varnish cache server oral sciences n3 braz j oral sci. 13(2):114-117 original article braz j oral sci. april | june 2014 volume 13, number 2 received for publication: april 24, 2014 accepted: june 03, 2014 comparative evaluation of push-out bond strength of a mta-based root canal sealer eduardo diogo gurgel-filho1, felipe martins leite1, jaírton benício de lima1, joão paulo chaves montenegro1, flávia saavedra2, emmanuel joão nogueira leal silva3 1universidade de fortaleza – unifor, school of dentistry, department of endodontics, fotaleza, ce, brazil 2universidade estadual de campinas – unicamp, school of dentistry, department of restorative dentistry, piracicaba, sp, brazil 3unigranrio, school of dentistry, department of endodontics, rio de janeiro, rj, brazil correspondence to: emmanuel joão nogueira leal da silva rua herotides de oliveira 61/902 cep: 24230230 niterói, rio de janeiro. phone: +55 21 83575757 e-mail: nogueiraemmanuel@hotmail.com abstract aim: to evaluate the bond strength to root dentin of three root canal sealers: a mineral trioxide aggregate (mta)-based sealer (mta fillapex®), an epoxy resin-based sealer (ah plus®), and a zinc oxide eugenol-based sealer (endofill®). methods: thirty extracted single-root human teeth of similar sizes and circular canals were prepared using #3 and #2 gates glidden drills in the cervical portion of the canal and k3® rotary instruments to a size #25/0.06 to working length. irrigation with 0.5 ml 2% chlorhexidine gel was used before and 1 ml saline after each instrument. the smear layer was removed with 3 ml 17% edta for 3 min. the samples were sectioned horizontally into eight 1±0.1 mm-thick serial slices and then the push-out test was carried out. two-way analysis of variance (anova) and the post-hoc tukey test were used for the analysis of the data with a significance level of 5%. results: ah plus presented significantly higher bond strengths (p<0.05) than the other sealers, while mta fillapex showed the lowest bond strengths (p<0.05). conclusions: the present study concluded that endofill® sealer and mta fillapex® core combination were not superior to ah plus® sealer and gutta-percha core combination. keywords: endodontics; root canal obturation; root canal filling materials. introduction the aim of endodontic therapy is not only to eliminate microorganisms by cleaning and shaping the root canal, but also to ensure that the root canal system will be fluid free and that a single unit can be created by the filling material (cones and sealer) and root dentin walls. bond strength of endodontic sealers to dentin is an important property of filling materials because it minimizes the risk of filling detachment from dentin during restorative procedures or the masticatory function1, ensuring that sealing is maintained and, consequently, clinical success of endodontic treatment. the push-out bond strength test is a well-known evaluation method used in several other similar studies1-4 with great reliability. thus, its results can be useful for inferring the interfacial strength and dislocation resistance between different root filling materials and the root dentin. mta fillapex® (angelus, londrina, pr, brazil), a sealer based on calcium silicate, was introduced recently on the market. after mixing, its composition is basically mta, salicylate resin, natural resin, bismuth oxide and silica. the manufacturer claims that it has excellent radiopacity, easy handling, a good working time and low solubility, providing sealing of the canal by expansion 115 braz j oral sci. 13(2):114-117 product and manufacturer endofill® (endo fill, dentsply ind. e com. ltda., rio de janeiro, rj, brazil) ah plus® (dentsply, konstanz, germany) mta fillapex® (angelus, curitiba, pr, brazil) composition powder: zinc oxide, staybelite resin, bismuth subcarbonate, barium sulfate, sodium borate anhydrate. liquid: eugenol. paste a bisphenol-a, bisphenol-f calcium tungstate, zirconium oxide, silica iron oxide pigmentspaste b dibenzyldiamineaminoadamantane tricyclodecane-diaminecalcium tungstate, zirconium oxide, silica, silicone oil salicylate resin, diluting resin, natural resin, bismuth trioxide, nanoparticulated silica, mta, pigments. preparation mode the components were combined by mixing the powder into liquid. the components were mixed in equal portions of pastes a and b. the components were combined by using a self-mixing tip attached to a syringe. table 1. table 1. table 1. table 1. table 1. tested sealers and their composition. during setting. recent studies showed suitable radiopacity, ph, flow, working and setting time of mta fillapex5-6. however, controversial results have been presented with respect to its bond strength to root dentin1-2. sagsen et al.1 (2011) concluded that mta fillapex had the lowest pushout bond values to root dentin compared with an epoxybased root canal sealer and different calcium silicate-based root canal sealers. on the other hand, assmann et al.2 (2012) stated that mta fillapex presented acceptable resistance to dislodgement, similar to that observed in samples filled with an epoxy-based root canal sealer. the present study was designed to assess the bond strength of root fillings in canals obturated with mta fillapex. ah plus® (dentsply detrey gmbh, konstanz, germany) and endofill® (endo fill; dentsply ind. e com. ltda., rio de janeiro, rj, brazil) were used as reference materials for comparison, and the push-out bond strength was the outcome variable. the null hypothesis tested is that there was no significant difference in the resistance to dislodgement of the root fillings in canals obturated with the different tested materials. material and methods this study was approved by the local ethics committee (protocol #2011.1.373.58.3). thirty extracted single-root human teeth of similar sizes and circular canals were randomly selected and stored in distilled water at 4 °c. to standardize the working length, a size 15 k-file (dentsply-maillefer, ballaigues, switzerland) was inserted into the root canal until it could be visualized at the apical foramen. the working length was determined by subtracting 1 mm from this length. after measurement, the length of all roots was standardized to 13 mm to prevent the introduction of confounders that could contribute to variations in the preparation procedures7. all teeth were instrumented using #3 and #2 gates glidden drills in the cervical portion of the canal. then, the root canals were instrumented using k3 rotary instruments (sybron endo) to a size #25/0.06 to working length. irrigation with 0.5 ml 2% chlorhexidine gel was used before each instrument and 1 ml 0.9% saline solution after each instrument. the smear layer was removed with 3 ml 17% edta for 3 min. a total of 3 ml saline was used for 3 min as a final rinse. each canal was dried with paper points. obturation procedures were performed using the single gutta-percha cone technique. using a computer algorithm (http://www.random.org), the 30 roots were randomly assigned to 3 groups for obturation with one of the three sealers: ah plus, endofill or mta fillapex. composition of the sealers is shown in table 1. the sealers were prepared according to the manufacturers’ instructions. on completion of these procedures, the specimens were radiographed at different angles to verify the quality of the filling procedure and presence of bubbles. the specimens were placed in 100% humidity for 7 days to ensure complete setting of the sealer. afterwards, each root was sectioned horizontally into eight 1±0.1 mm-thick serial slices by using a low-speed saw with a diamond disk under continuous water irrigation. the root filling of each sample was loaded with a 0.5-mmdiameter stainless steel cylindrical plunger. the plunger tip was sized and positioned to touch only the root filling. the load was always applied in an apical-coronal direction to avoid any constriction interference caused by root canal taper during push-out testing. loading was performed on a universal testing machine (instron corporation, norwood, ma, usa) at a crosshead speed of 0.5 mm/min until debonding occurred. each cross section was coded and measured for the apical and coronal diameters of the obturated area by using an optical stereomicroscope. a load/time curve was plotted during the compression test using real-time software. to express the bond strength in mpa, the load at failure recorded in n was divided by the area of the bonded interface3. the normality test of shapiro-wilkand and levenes variance homogeneity tests were applied to the data showing normal distribution and homogeneity of variance among the groups. two-way analysis of variance (anova) and the posthoc tukey test were used for the data analysis; the independent variables were root canal filling material and root canal third (p<0.05). results all specimens showed measurable adhesive properties to root dentin. in addition, no premature failure occurred. overall, the push-out bond strength was the highest in the coronal third and lowest in the apical third. ah plus specimens comparative evaluation of push-out bond strength of a mta-based root canal sealer group mean (sd)* endo fill® 0.54±0.24a ah-plus® 3.80±1.90b mta fill apex® 0.25±0.10a table 2. table 2. table 2. table 2. table 2. push-out bond strength mean values (mpa) and standard deviation (sd) of the different root canal filling system to root dentin. displayed statistically higher bond strengths (p=0.0012, 0.515.9 mpa). mta fillapex showed the lowest bond strengths. the values of the push-out bond strength data in each experimental group are shown in table 2. discussion gutta-percha does not bond to root dentin and is used in conjunction with a root canal sealer1, so the adhesive properties of endodontic sealers are important. it was suggested that, if a material bonds to the root canal walls, it resists dislodgement of the filling8. it is also believed that chemical bonding to root dentin improves the push out bond strength of sealers to root canal walls9. in this study, the push-out test was used to test the dentin bond strength of different root canal sealers. it has been suggested that this test provides a better evaluation of bond strength than the conventional shear test because in the pushout test, fracture occurs parallel to the dentin-bonding interface, which makes it a true shear test for parallel-sided samples7,10. extrusion testing in dentistry was first described by roydhouse11 (1970). kimura, shimizu and fujii12 (1985) concluded that push-out testing tended to reduce the values for bond strength to dentin. haller and klaiber13 (1991) reintroduced the push-out test and the testing procedure selected for the present investigation used their model. the model has shown to be effective and reproducible. another advantage of this method is that it allows root canal sealers to be evaluated even when bond strengths are low4. during chemo-mechanical preparation, a layer of debris, the smear layer, is formed. current theories of dentine bonding mechanisms involve either chemical modification of the smear layer and bonding directly to it, or removal of the smear layer and bonding to subjacent tooth structures4,14. some studies have shown that removal of the smear layer enhances the adhesion of sealers to the root canal wall15-16. the smear layer can act as a reservoir or substrate for microorganisms17, and can also block the extension of sealer tags into the dentinal tubules, thereby decreasing micromechanical adhesion18. in the current study, 17% edta was used after instrumentation to remove the smear layer. chlorhexidine gluconate (chx) has been suggested as an alternative irrigation solution that could replace naocl. chx is a bactericidal solution because of its ability to precipitate and coagulate bacterial intracellular constituents19. the resin system seems to be sensitive to naocl20, as its use during root canal therapy reduced the bond strength21. the naocl acts to oxidize a component in the dentinal matrix that interferes with free radical propagation at the resin-dentin interface leading to lower bond strength22. the bond strength after use of chx gel and edta differs from that after the use of naocl solely23. the bond strength to pulp chamber dentin decreased when endodontic irrigation was performed with 5.25% naocl either associated or not with 17% edta24-25. thus, the results of these articles support the use of chlorhexidine gluconate associated with edta as root canal irrigation in the present study. mta fillapex is a new salicylate resinand calcium silicate-based sealer. the manufacturer claims that this product provides long-term sealing capacity, high radioopacity and promotes deposition of hard tissue. it contains calcium silicate, salicylate resin, diluting resins, natural resin, nanoparticulated resin and bismuth trioxide. it is anticipated that release of calcium and hydroxyl ions from the set sealer will result in the formation of apatite when the material comes into contact with phosphate-containing fluids25. in the light of the results, the null hypothesis that there was no difference between the groups was rejected. the pushout bond strength of ah plus was statistically superior to that of mta fillapex and endo fill. no statistically significant difference was found between mta fillapex and endo fill. this result corroborates those of pécora et al.16 (2001) and cecchin et al.26 (2012). they also found higher bond strength values for epoxy resin-based cements, like ah plus, compared with zinc oxide-eugenol sealers, like endo fill. several other studies also found that the push-out bond strength of ah plus was superior to that of other root canal sealers27-29. in the present study, the mta-based sealer mta fillapex had the lowest bond strength to root dentin. sarkar et al.25 (2005) suggested that release of calcium and hydroxyl ions from the set sealer will result in the formation of apatite as the material comes into contact with phosphate-containing fluids. reyes-carmona et al.30 (2009), reported that the apatite formed by mta and phosphate buffered saline was deposited within collagen fibrils, promoting controlled mineral nucleation on dentin, seen as the formation of an interface layer with tag-like structures. the reason for the low bond strength of mta fillapex in the present study could be the low adhesion capacity of these tag-like structures, corroborated by the study made by sagsen et al.1 (2011). although previous studies have already shown that mta fillapex had weak bond strength to the dentin wall, this fact is not normally expected, once this sealer has mta as one of its ingredients and some of the well-known components of sealers. mta properties are good adhesion to dentin walls, adequate seal and resistance to dislodgement5. the adhesion of grossman type root canal sealers to dentin is established by electrostatic bonding and not by its penetration into the dentinal tubules16. the low bond strength established with group 1 may be explained by a chelating reaction that occurs while the zinc oxide-eugenol mixture is setting31. this reaction affects both the gutta-percha core material and the root canal dentin. the zinc ion of the zinc oxide may react with the mineral component of the dentin 116 comparative evaluation of push-out bond strength of a mta-based root canal sealer braz j oral sci. 13(2):114-117 117 as well as with the zinc oxide constituent of gutta-percha. also, eugenol may have a softening effect on gutta-percha, thus creating an interlocking meshwork that will increase adhesion between the materials. adhesive strength is only one aspect of the quality of root canal sealing, but it may be considered one of the most important. currently, there are several different types of endodontic sealers available, but as shown by this study, not all of them have the best properties to ensure endodontic success. further investigation of other features of root canal sealers is required. in most cases, the results of laboratory experimental studies cannot be directly applied to clinical situations4. however, they do provide reproducible and reliable means for comparing and testing new and prospective sealers, and for establishing international standards. within the limits of the push-out test method, in the present study, endofill sealer and mta fillapex core combination were not superior to ah plus sealer and guttapercha core combination. on the basis of the findings presented herein it may be concluded that ah plus sealer might provide an advantage over other sealers with respect to bond strength to root dentin. references 1. sagsen b, ustün y, demirbuga s, pala k. push-out bond strength of two new calcium silicate-based endodontic sealers to root canal dentine. int endod j. 2011; 44: 1088-91. 2. assmann e, scarparo rk, böttcher de, grecca fs. dentin bond strength of two mineral trioxide aggregate-based and one epoxy resin-based sealers. j endod. 2012; 38: 219-21. 3. nagas e, cehreli zc, durmaz v, vallittu pk, lassila lv. regional push-out bond strength and coronal microleakage of resilon after different light-curing methods. j endod. 2007; 33: 1464-8. 4. ungor m, onay eo, orucoglu h. push-out bond strengths: the epiphanyresilon endodontic obturation system compared with different pairings of epiphany, resilon, ah plus and gutta-percha. int endod j. 2006; 39: 643-7. 5. vitti rp, prati c, silva ej, sinhoreti ma, zanchi ch, de souza e silva mg, et al. physical properties of mta fillapex sealer. j endod. 2013; 39: 915-8. 6. silva ej, rosa tp, herrera dr, jacinto rc, gomes bp, zaia aa. evaluation of cytotoxicity and physicochemical properties of calcium silicate-based endodontic sealer mtafillapex. j endod. 2013; 39: 274-7. 7. drummond jl, sakaquchi rl, racean dc, wozny j, steinberg ad. testing mode and surface treatment effects on dentin bonding. j biomed mater res. 1996; 32: 533-41. 8. shipper g, ørstavik d, teixeira fb, trope m. an evaluation of microbial leakage in roots filled with a thermoplastic synthetic polymer-based root canal filling material (resilon). j endod. 2004; 30: 342-7. 9. onay eo, ungor m, ari h, belli s, ogus e. push-out bond strength and sem evaluation of new polymeric root canal fillings. oral surg oral med oral pathol oral radiol endod. 2009; 107: 879-85. 10. ureyen kaya b, keceli ad, orhan h, belli s. micropush outbond strengths of gutta-percha versus thermoplastic synthetic polymer-based systems – an ex vivo study. int endod j. 2008; 41: 211-8. 11. roydhouse rh. punch-shear test for dental purposes. j dent res. 1970; 49: 131-6. 12. kimura s, shimizu t, fujii b. influence of dentin on bonding of composite resin. part 1. effect of fresh dentin and storing conditions. dent mater j. 1985; 4: 68-80. 13. haller b, klaiber b. adhesive cementation: prevention of pulp irritation. phillip j. 1991; 8: 373-5. 14. yu xy, joynt rb, davis el, wieczkowski jr g. adhesion to dentine. j calif dent assoc. 1993; 21: 23-9. 15. de gee aj, wu mk, wesselink pr. sealing properties of ketac-endo glass ionomer cement and ah26 root canal sealers. int endod j. 1994; 27: 239-44. 16. pécora jd, cussioli al, gueriºoli dm, marchesan ma, sousa-neto md, brugnera júnior a. evaluation of er: yag laser and edtac on dentin adhesion of six endodontic sealers. braz dent j. 2001; 12: 27-30. 17. pashley dh. smear layer: physiological considerations. oper dent suppl. 1984; 3: 13-29. 18. kouvas v, liolios e, vassiliadis l, parissis-messimeris s, boutsioukis a. influence of smear layer on depth of penetration of three endodontic sealers: an sem study. endod dent traumatol. 1998; 14: 191-5. 19. ferraz cc, gomes bp, zaia aa, teixeira fb, souza-filho fj. in vitro assessment of the antimicrobial action and the mechanical ability of chlorhexidine gel as an endodontic irrigant. j endod. 2001; 27: 452-5. 20. hashem aa, ghoneim ag, lutfy ra, fouda my. the effect of different irrigating solutions on bond strength of two root canal-filling systems. j endod. 2009; 35: 537-40. 21. gonçalves l, silva-sousa yt, raucci neto w, teixeira cs, sousaneto md, alfredo e. effect of different irrigation protocols on the radicular dentin interface and bond strength with a metacrylate-based endodontic sealer. microsc res tech. 2014; 9: 1-7. 22. weston ch, ito s, wadgaonkar b, pashley dh. effects of time and concentration of sodium ascorbate on reversal of naocl-induced reduction in bond strengths. j endod. 2007; 33: 879-81. 23. santos jn, carrilho mr, de goes mf, zaia aa, gomes bp, souzafilho fj, et al. effect of chemical irrigants on the bond strength of a selfetching adhesive to pulp chamber dentin. j endod. 2006; 32: 1088-90. 24. liu c, liu h, zhu s. effect of different irrigating solutions on bonding strength of fiber post to root canal. hua xi kou qiang yi xue za zhi. 2011; 29: 210-3. 25. sarkar nk, caicedo r, ritwik p, moiseyeva r, kawashima i. physicochemical basis of the biologic properties of mineral trioxide aggregate. j endod. 2005; 31: 97-100. 26. cecchin d, souza m, carlini-júnior b, barbizam jv. bond strength of resilon/epiphany compared with gutta-percha and sealers sealer 26 and endo fill. aust endod j. 2012; 38: 21-5. 27. rosa ra, barreto ms, moraes r do a, broch j, bier ca, só mv, et al. influence of endodontic sealer composition and time of fiber post cementation on sealer adhesiveness to bovine root dentin. braz dent j. 2013; 24: 241-6. 28. patil sa, dodwad pk, patil aa. an in vitro comparison of bond strengths of gutta-percha/ah plus, resilon/epiphany self-etch and endorez obturation system to intraradicular dentin using a push-out test design. j conserv dent. 2013; 16: 238-42. 29. al batouty km, hashem aa. push-out bond strength of a newly introduced glass fiber root canal filling material. j adhes dent. 2013; 15: 161-6. 30. reyes-carmona jf, felippe ms, felippe wt. biomineralization ability and interaction of mineral trioxide aggregate and white portland cement with dentin in a phosphate-containing fluid. j endod 2009; 35: 731-6. 31. crisp s, ambersley m, wilson ad. zinc oxide eugenol cements. v. instrumental studies of the catalysis and acceleration of the setting reaction. j dent res. 1980; 59: 44-54. comparative evaluation of push-out bond strength of a mta-based root canal sealer braz j oral sci. 13(2):114-117 429 too many requests error 429 too many requests too many requests guru meditation: xid: 28694560 varnish cache server oral sciences n3 original article braz j oral sci. january | march 2015 volume 14, number 1 does the classification of cerebral palsy influence caries experience in children and adolescents? michele baffi diniz1, renata oliveira guaré1, maria cristina duarte ferreira2, maria teresa botti rodrigues santos3 1universidade cruzeiro do sul – unicsul, school of dentistry, department of pediatric dentistry, são paulo, sp, brazil 2centro universitário senac, department of dentistry, são paulo, sp, brazil 3universidade cruzeiro do sul – unicsul, school of dentistry, department of patients with special needs, são paulo, sp, brazil correspondence to: michele baffi diniz universidade cruzeiro do sul setor de pós graduação rua galvão bueno, 868 liberdade cep: 01506-000 são paulo-sp brazil phone: +55 11 3385 3015 fax: +55 11 3385 3015 e-mail: mibdiniz@hotmail.com abstract aim: to evaluate the influence of the classification of cerebral palsy (cp) on the caries experience in children and adolescents, and determine the types of teeth most affected by dental caries. methods: a total of 181 individuals with cp, aged 8.30±4.10 years were examined for dental caries diagnosis in primary, mixed and permanent dentitions. the sample consisted of 96 quadriplegic individuals, 56 diplegic, 18 hemiplegic and 11 with choreoathetosis. the groups were compared using kruskal-wallis and chi-square tests. univariate logistic regression models were used to identify whether age was a predictor of caries risk (α=5%). results: the sample was homogeneous as regards sex and age, among patients with quadriplegia, diplegia, hemiplegia and choreoathetosis. out of the total sample, 51.4% of the individuals were caries free. the overall mean for the dmft and dmft indices were 1.70±3.30 and 0.70±1.60, respectively. the mean caries experience in primary, mixed and permanent dentitions did not vary significantly according to the cp classification (p>0.05). child´s age was a predictor for dental caries experience (p<0.05). conclusions: the cp classification did not influence on caries experience, and the teeth most affected by dental caries were the primary and first permanent molars. keywords: dental caries; cerebral palsy; oral hygiene; preventive dentistry. introduction cerebral palsy (cp) describes a group of permanent disorders involving movement and posture development that cause activity limitations. these are attributed to nonprogressive disturbances occurring in the developing fetal or infant brain. cp motor disorders are often accompanied by epilepsy, secondary musculoskeletal problems and disturbances of sensation, perception, cognition, communication and behavior. this condition is the most common cause of severe physical disability in childhood1-2. the type of abnormal resting muscle tone or involuntary movement disorder observed or elicited is usually assumed to be related to the underlying pathophysiology of the condition. individuals with spastic cerebral palsy present increased tone, pathological reflexes and hyperreflexia or pyramidal signs, with clinical patterns of involvement including: quadriplegia, more severe motor involvement of all four limbs; diplegia, involvement of corresponding limbs, braz j oral sci. 14(1):46-51 received for publication: january 21, 2015 accepted: march 17, 2015 braz j oral sci. 14(1):46-51 although typically more severe in the legs; and hemiplegia, affecting one side. individuals with dyskinesia (choreoathetosis) present involuntary, uncontrolled, recurring and occasionally stereotyped movements, which may be totally disabling when severe3. dental caries is an oral health burden in individuals with cp, not only in primary but also in permanent dentition, with higher percentages of decayed and missing teeth4-5. children with cp can present a reduced unstimulated salivary flow rate, ph and buffer capacity, which may compromise the protective function of saliva, resulting in increased risk of oral diseases6. maintaining the oral health of individuals with cp requires systematic hygiene practices that demand supervision, or their oral hygiene may eventually have to be performed by family caregivers. a child’s development needs the family’s participation, involvement and support. when this is well-structured, it will positively contribute to the child’s quality of life. however, in individuals with cp, this process of participation, involvement and support is not restricted to the developmental period. the task of taking care of a child with complex disabilities at home may be somewhat daunting for caregivers, and may reflect the way in which these individuals are cared for5,7 the purposes of this study were to assess the influence of the classification of cp on caries experience in children and adolescents, and determine the type of teeth most affected by dental caries. the null hypothesis is that the classification of cp does not influence on caries experience in children and adolescents. material and methods this cross-sectional study was approved by the human research ethics committee of the cruzeiro do sul university, protocol no. 152/2011, and was conducted in accordance with the ethical standards stated in the declaration of helsinki. to calculate the sample size, the used formula considered a confidence interval of 95%, statistical power of 80%, mean caries prevalence of 5.43, standard deviation of 4.17 and a mean minimum difference of 4.56 to be detected8. thus, a minimum of 181 individuals was required. the sample consisted of non-institutionalized children and adolescents (110 male and 71 female) presenting a medical diagnosis of cp, age-range from 1 to 17 years (mean age, 8.30±4.10), and participating in a prevention-based program for oral health. the study was conducted in the state of são paulo, brazil, in a rehabilitation centre between april and december 2011. written informed consent for participation and publication was obtained from the parents or guardians of each individual with cp, who agreed to participate in the study. patient medical records were reviewed for clinical data, including gender, age and cp classification (choreoathetosis, quadriplegia, diplegia or hemiplegia). a single calibrated examiner (weighted kappa=0.89) conducted the oral examination with the help of an assistant to record the data. before clinical examination, professional tooth cleaning was performed in each patient. the patients’ teeth were assessed under a reflector light using a dental mirror and a probe after drying with a 3-in-1 syringe. the world health organization criteria9 were used for recording dental caries experience by the decayed, missing and filled teeth index, using the dmft and dmft indices for primary and permanent dentition, respectively. in children with mixed dentition, dmft and dmft were recorded together. no radiographic examination was performed. statistical analysis descriptive statistical tests were used to assess the demographic data using spss software program (statistics package for social science, version 15.0 for windows). the measures of central tendency (mean) and dispersion (standard deviation) were used for continuous variables. participants were divided into groups according to their clinical type of cp: choreoathetosis (n=11); quadriplegia (n=96); diplegia (n=56); and hemiplegia (n=18). the nonparametric chisquare and kruskal-wallis tests were used to determine significant differences in data. the teeth most affected by dental caries in primary and permanent dentitions were assessed for subsequent regression analysis. univariate logistic regression models were used to identify whether cp child´s age was a predictor of caries risk in each tooth assessed, using the tooth status (presence or absence of caries) as the dependent variable and age as an independent variable. a logistic regression model was also performed considering sex, age in years and cp classification as potential independent variables that could influence on dental caries experience (outcome). the significance level was set at 5%. results table 1 shows the distribution of the 181 participants according to dentition, sex, age (years) and cp classification. the sample was homogeneous as regards dentition (p=0.6454), sex (p=0.6829) and age (0.1932) among the quadriplegia, diplegia, hemiplegia and choreoathetosis groups. the sample consisted of a higher percentage of males (60.8%) than females. according to the cp classification, most of the individuals with cp presented quadriplegia (53.1%), followed by diplegia (30.9%), hemiplegia (9.9%) and choreoathetosis (6.1%). the overall mean for the dmft and dmft indices were 1.70±3.30 and 0.70±1.60, respectively. table 2 shows the distribution of caries experience in primary, mixed and permanent dentition according to the cp classification. in the total sample, 93 participants (51.4%) presented no decay. at least one primary or permanent tooth affected by caries was identified in 88 patients (48.6%). the diplegia group presented more caries experience (60.7%) than the others. the mean caries experience in primary, mixed and permanent dentitions did not vary significantly according to the cp classification (p>0.05). 4747474747does the classification of cerebral palsy influence caries experience in children and adolescents? the mean and standard deviation age of the individuals with cp was 3.6±1.6 years for primary dentition, 8.7±2.1 years for mixed dentition and 13.4±2.0 years for permanent dentition. in primary, mixed and permanent dentitions, 73.2%, 37.5% and 48.9% individuals, respectively, were caries free. although the sample had 60.8% male individuals, there was no statistical difference as regards sex when dental caries was absent (p=0.1384) or present (p=0.8884) according to the primary, mixed and permanent dentitions. figure 1 shows the prevalence of caries in primary and permanent dentitions in individuals with cp. in both maxillary and mandibular arches, primary molars and first permanent molars were the teeth most affected by caries disease. maxillary anterior teeth were more affected by caries than mandibular anterior teeth, particularly in primary dentition. the permanent teeth most affected by dental caries were the mandibular first molars, followed by the maxillary first molars. the primary teeth most affected were second molars, followed by first molars. the univariate logistic regression, considering cp child´s age as a predictor for caries in primary and first permanent molars, confirmed that cp child´s age was not a predictor for caries in primary molars (p>0.05). considering the first permanent molars, cp child´s age was a predictor for caries in tooth #16 and tooth #36 (p<0.05) when they were analyzed separately. however, when the maxillary and mandibular first permanent molars were analyzed together, the results classification of cp caries caries primary mixed permanent (n) experience=0 experiencee>1 dentition dentition dentition n(%) n (%) (n=56) (n=80) (n=45) quadriplegia (96) 53 (55.2) 43 (44.8) 1.5±3.5a 3.4±4.3a 1.3±1.8a diplegia (56) 22 (39.3) 34 (60.7) 1.1±2.5a 3.4±3.1a 2.7±2.8a hemiplegia (18) 11 (61.1) 7 (38.9) 2.6±5.1a 0.9±1.1a 0.0±0.0a choreoathetosis (11) 7 (63.6) 4 (36.4) 7.3±8.4a 1.5±3.0a 2.0±3.5a p value 0.3609 0.1698 0.0664 table 2.table 2.table 2.table 2.table 2. distribution of caries experience (mean±standard deviation) in primary, mixed and permanent dentitions according to the classification of cp *within columns, significant differences are represented by different superscript lowercase letters (kruskall-wallis test; p<0.05). fig. 1. prevalence of caries in primary and permanent dentitions in participants with cp. *significant association between age and presence of caries by logistic regression (p<0.05). confirmed that cp child´s age was a predictor for dental caries (p<0.05). table 3 shows the logistic regression model considering sex, age and cp classification as predictors on dental caries experience. it could be seen that age (years) had a significant effect on dental caries experience (p<0.05) in children with cp. sex and cp classification had no significant influence (p>0.05). 4848484848 does the classification of cerebral palsy influence caries experience in children and adolescents? braz j oral sci. 14(1):46-51 table 1.table 1.table 1.table 1.table 1. distribution (%) of the 181 participants according to dentition, sex, age and the classification of cp *chi-square test (p<0.05) **kruskal-wallis test (p<0.05) dentition classification of cp quadriplegia diplegia hemiplegia choreoathetosis p value (n=96) (n=56) (n=18) (n=11) deciduous 33 (34.4) 12 (21.4) 7 (38.9) 4 (36.4) 0.6454* mixed 39 (40.6) 29 (51.8) 8 (44.4) 4 (36.4) permanent 24 (25.0) 15 (26.8) 3 (16.7) 3 (27.2) s e x male 59 (61.5) 32 (57.1) 13 (72.2) 6 (54.5) 0.6829* female 37 (38.5) 24 (42.9) 5 (27.8) 5 (45.5) age (years) average ± 8.0±4.3 9.3±4.0 7.2±3.9 8.5±3.8 0.1932** standard deviation variable p value odds ratio 95% confidence interval m i n i m u m m a x i m u m cp classification choreoathetosis 0.4190 0.5833 0.1578 2.1563 diplegia 0.2263 1.5210 0.7710 3.0003 hemiplegia 0.3223 0.5820 0.91992 1.7002 age (years) 0.0347* 1.0813 1.0056 1.1627 sex (female) 0.5651 1.1981 0.6472 2.2180 *significant association between by logistic regression (p<0.05). table 3. table 3. table 3. table 3. table 3. logistic regression analysis considering child´s age, sex and cp classification as predictors for the presence of caries discussion clinically, cp varies in severity and highly diverse levels of functional independence can be observed in individuals with the same medical diagnosis, due to the extent of neurodevelopmental disorder1,3. spastic cp (quadriplegia, diplegia and hemiplegia) is by far the most common type of movement disorders in cerebral palsy, occurring in 70% to 80% of all cases1, as observed in this study. this study was conducted in a reference rehabilitation centre for individuals with cp presenting more complex disorders, as observed in the composition of the sample. most of those subjects presented a quadriplegic and diplegic cp classification, as previously described by dos santos and nogueira9 (2005). moreover, 60.8% of children and adolescents with cp were male, which agrees with the results found by bax et al.10 (2006). it was hypothesized that the increased orofacial muscular tonus and involuntary movements in individuals with cp could have an influence on caries experience, since these characteristics are considered factors leading to difficulty with performing effective oral hygiene11. however, it was observed that the cp classification had no influence on caries experience in these individuals. this could be explained by the implementation of an educational program on oral health directed to the caregivers, who acquired knowledge concerning the main diseases that affected the oral cavity of children with cp and forms of prevention and maintenance of oral health. moreover, age was a predictor for dental caries experience. in other words, caries experience increases as the individual´s age increases. it could be observed that cp children with diplegia presented a tendency of higher dmft index in permanent dentition, since these individuals were older than the others and presented more erupted teeth in the oral cavity. in a previous study 9, comparisons between cp classifications determined higher values for dmft and dmft indices among spastic quadriplegic patients. however, the control of caries risk factors is gradually becoming more effective, in a process that involves growing caregiver’s knowledge concerning oral hygiene habits, the use of open mouth devices to facilitate toothbrushing, use of fluoride dentifrice and diet control. the fact that the patients were treated in a rehabilitation centre providing constant preventive care might also be a factor in the low dmft and dmft indices obtained12. with regard to the risk of dental caries, the greatest risk is observed in children from low-income families, most often from minority groups and families that include individuals with special care needs13, particularly cp5,8,10,14. according to the american academy of pediatric dentistry, they show a moderate risk of caries15. dental caries in individuals with cp could be also related to intellectual disability4, oromotor dysfunction8,16, the regular use of sugary anticonvulsant drugs17, shorter mastication endurance time18, biting reflexes9 and worse quality of life and continual burden on the caregivers5,8, which can further influence oral hygiene 8. despite presenting those risk factors, it may be observed that since the individuals with cp are under a continuous preventive follow-up dental care, caries experience can be controlled. moreover, the mean dmft and dmft indices of the individuals with cp were lower than the dental caries indices of normoreactive brazilian children according to the last national survey of dental health19. in this study, there was no difference between age and cp classification. it should be stressed that the mean age was expressed in years and not in number of months and might influence the results. for primary dentition, age was similar to that shown in other studies5,8. in a clinical study involving patients with cp, roberto et al.20 (2012) observed that as the primary teeth erupted, the dmft index increased. for mixed dentition, considering both the dmft and the dmft indices, the caries experience was lower in comparison with previous studies5,8. this difference could be explained by the participation of the caregivers in a systematic prevention program developed at the rehabilitation centre. for permanent dentition, the caries experience was similar in comparison with the study of de carvalho et al.21 (2011), and in disagreement with santos et al.5 (2010), who reported higher dmft indices. these differences may be attributed to the location where the study was conducted and the severity of cp in these two populations. in order to determine the risk of developing oral diseases it is fundamental to identify the types of teeth that are most affected by dental caries in individuals with cp, to determine the types of primary and permanent teeth most affected by caries according to cp child´s age. to the best of the authors’ knowledge, this is the first study to assess the types of primary and permanent teeth most affected by caries according to cp child´s age. the difficulty in maintaining the patient’s mouth open, due to the muscle tonus1, hyper-responsivity to oral hygiene stimulus and oral manipulation1, and non4949494949does the classification of cerebral palsy influence caries experience in children and adolescents? braz j oral sci. 14(1):46-51 cooperative behavior4 could contribute to food residues on the occlusal surfaces of posterior teeth, as observed on the permanent first molars and the primary first and second molars. primary second molars and primary and permanent first molars were the most powerful predictors for assignment to the risk group13. motta et al.22 (2012) found that first permanent molars are the teeth most affected by dental caries. the mandibular anterior teeth were less affected by dental caries than the maxillary anterior teeth, as also occurs in children without any neurological damage. this could be explained by tongue movement and by the fact that a greater salivary film velocity occurs lingual to the mandibular incisors, where biofilm is most readily removed23. with regard to cp child´s age as a predictor for caries in first permanent molars, analysis showed that these teeth presented greater caries risk with increasing age. based on these findings, it seems reasonable to assume that the prevention program requires greater focus on issues specific to this population, including: detailed advice concerning sealant application; fluoride therapy; use of 0.12% chlorhexidine digluconate oral rinse; toothbrushes adapted to facilitate handling by individuals with cp, so they can perform their own oral hygiene wherever possible; the use of dental floss devices; single-tuft toothbrushes for use on occlusal surfaces; and facilitating mouth opening with the use of a dental wooden spatula. all patients with cp should have their dental visits scheduled at shorter time intervals, in order to ensure the best professional care, including constant follow-up of erupting teeth on an individual basis. in this study, caries experience was assessed by the dmft and dmft indices proposed by the who24 (2013), because this population presents limited cooperation for a more detailed clinical examination of dental caries using other indexes, and incipient enamel lesions (i.e., white spot lesions) were not evaluated. the examiner was calibrated for the indices and is a dental professional who frequently examines and treats children with cp. the same examiner has previously conducted other studies using these indexes5,8-9,16,25. moreover, there was no control group of patients without alteration. another research has compared children with cp with normorreactive subjects26, but the mentioned study assessed a small number of patients compared with this investigation. another limitation of the present investigation relates to the different number of subjects in each cp classification, which may have influenced the results. despite the quantitative differences between the assessed groups, they correspond to the percentages found in the population, with a higher prevalence of quadriplegia. in conclusion, the cp classification had no influence on caries experience, and the teeth most affected by dental caries were the primary molars and first permanent molars. references 1. rosenbaum p, paneth n, leviton a, goldstein m, bax m, damiano d, et al. a report: the definition and classification of cerebral palsy april 2006. dev med child neurol suppl. 2007; 109: 8-14. 2. oskoui m, coutinho f, dykeman j, jetté n, pringsheim t. an update on the prevalence of cerebral palsy: a systematic review and meta-analysis. dev med child neurol. 2013; 55: 509-19. 3. bax mc, flodmark o, tydeman c. definition and classification of cerebral palsy. from syndrome toward disease. dev med child neurol suppl. 2007; 109: 39-41. 4. moreira rn, alcântara ce, mota-veloso i, marinho sa, ramos-jorge ml, oliveira-ferreira f. does intellectual disability affect the development of dental caries in patients with cerebral palsy? res dev disabil. 2012; 33: 1503-7. 5. santos mt, biancardi m, guare ro, jardim jr. caries prevalence in patients with cerebral palsy and the burden of caring for them. spec care dentist. 2010; 30: 206-10. 6. santos mt, guaré r, leite m, ferreira mc, nicolau j. does the neuromotor abnormality type affect the salivary parameters in individuals with cerebral palsy? j oral pathol med. 2010; 39: 770-4. 7. abanto j, ortega ao, raggio dp, bönecker m, mendes fm, ciamponi al. impact of oral diseases and disorders on oral-health-related quality of life of children with cerebral palsy. spec care dentist. 2014; 34: 56-63. 8. rodrigues dos santos mt, bianccardi m, celiberti p, de oliveira guaré r. dental caries in cerebral palsied individuals and their caregivers’ quality of life. child care health dev. 2009; 35: 475-81. 9. dos santos mt, nogueira ml. infantile reflexes and their effects on dental caries and oral hygiene in cerebral palsy individuals. j oral rehabil. 2005; 32: 880-5. 10. bax m, tydeman c, flodmark o. clinical and mri correlates of cerebral palsy: the european cerebral palsy study. jama. 2006; 296: 1602-8. 11. dourado m da r, andrade pm, ramos-jorge ml, moreira rn, oliveiraferreira f. association between executive/attentional functions and caries in children with cerebral palsy. res dev disabil. 2013; 34: 2493-9. 12. sehrawat n, marwaha m, bansal k, chopra r. cerebral palsy: a dental update.int j clin pediatr dent. 2014; 7: 109-18. 13. helm s, helm t. correlation between caries experience in primary and permanent dentition in birth-cohorts 1950-70. scand j dent res. 1990; 98: 225-7. 14. cardoso am, gomes ln, silva cr, soares r de s, de abreu mh, padilha ww, et al. dental caries and periodontal disease in brazilian children and adolescents with cerebral palsy. int j environ res public health. 2014; 12: 335-53. 15. american academy of pediatric dentistry. reference manual. guideline on caries-risk assessment and management for infants, children, and adolescents. pediatr dent. 2013; 35: 118-25. 16. santos mtbr, ferreira mcd, guaré ro, nascimento ao, jardim jr. oral hydration in children with cerebral palsy. braz oral sci. 2014; 13: 140-5. 17. siqueira wl, santos mt, elangovan s, simoes a, nicolau j. the influence of valproic acid on salivary ph in children with cerebral palsy. spec care dentist. 2007; 27: 64-6. 18. santos mt, manzano fs, chamlian tr, masiero d, jardim jr. effect of spastic cerebral palsy on jaw-closing muscles during clenching. spec care dentist. 2010; 30: 163-7. 19. ministry of health of brazil. oral health conditions of the brazilian population 2010. main results. brasilia: ministry of health; 2011. 20. roberto ll, machado mg, resende vl, castilho ls, abreu mh. factors associated with dental caries in the primary dentition of children with cerebral palsy. braz oral res. 2012; 26: 471-7. 21. de carvalho rb, mendes rf, prado rr jr, moita neto jm. oral health and oral motor function in children with cerebral palsy. spec care dentist. 2011; 31: 58-62. 22. motta lj, santos jg, alfaya ta, guedes cc, godoy chl, bussadori sk. clinical status of permanent first molars in children aged seven to ten years in a brazilian rural community. braz j oral sci. 2012; 11: 475-80. 23. dawes c. why does supragingival calculus form preferentially on the lingual surface of the 6 lower anterior teeth? j can dent assoc. 2006; 72: 923-6. 5050505050 braz j oral sci. 14(1):46-51 does the classification of cerebral palsy influence caries experience in children and adolescents? braz j oral sci. 14(1):46-51 24. world health organization. oral health surveys: basic methods. 5th ed. geneva: world health organization; 2013. 25. santos mt, ferreira mc, mendes fm, de oliveira guaré r. assessing salivary osmolality as a caries risk indicator in cerebral palsy children. int j paediatr dent. 2014; 24: 84-9. 26. guaré rde o, ciamponi al. dental caries prevalence in the primary dentition of cerebral-palsied children. j clin pediatr dent. 2003; 27: 287-92. 5151515151does the classification of cerebral palsy influence caries experience in children and adolescents? braz j oral sci. 14(1):46-51 429 too many requests error 429 too many requests too many requests guru meditation: xid: 36472718 varnish cache server oral sciences n3 original article braz j oral sci. october | december 2014 volume 13, number 4 comparison of 2 types of treatment of skeletal class ii malocclusions: a 5-year post-retention analysis ana de lourdes sá de lira1, margareth maria gomes souza2, ana maria bolognese2, matilde nojima2 1universidade estadual do piauí – uespi, faculty of dentistry, department of orthodontics, teresina, pi, brazil 2universidade federal do rio de janeiro – ufrj, faculty of dentistry, department of orthodontics, rio de janeiro, rj, brazil correspondence to: ana de lourdes sá de lira departamento de ortodontia, faculdade de odontologia universidade federal do rio de janeiro av. professor rodolpho paulo rocco, 325 ilha do fundão cep: 21941-617 rio de janeiro rj brasil e-mail: anadelourdessl@hotmail.com abstract aim: to compare 2 types of treatment for class ii malocclusion assessing mandibular behavior in subjects submitted to full orthodontic treatment with standard edgewise appliance and cervical headgear (kloehn appliance) and those who used cervical headgear in the first period and with full orthodontic appliance in the second period. methods: the sample consisted of 80 children treated with either cervical headgear combined with full fixed appliances (n=40, group 1), or with cervical headgear at first (n=40, group 2). in both groups, lateral cephalometric radiographs were compared with those made at the beginning of treatment, at its end and at 5-year post-retention phase, in order to quantify the cephalometric measures (8 angular and 3 linear), presenting the mandibular behavior in the antero-posterior and vertical directions. all patients were treated with no extraction and no use of class ii intermaxillary elastics during the full orthodontic treatment. results: in both groups, the effective treatment of skeletal class ii malocclusion did not interfere in the direction and amount of growth of mandibular condyles and remodeling at the lower border, with no influence on the anti-clockwise rotation of the mandible. the mandibular growth also was observed after the orthodontic treatment, suggesting that it is influenced by genetic factors. conclusions: these observations may lead to the speculation that growing patients with skeletal class ii malocclusion and low mandibular plane are conducive to a good treatment and long-term stability with one or two periods of treatment. keywords: malocclusion; orthodontics; control. introduction the growth potential of individuals with class ii malocclusion is of interest to practicing orthodontists because this type of malocclusion comprises a significant percentage of the cases they treat1. using angle’s classification as their criterion, several authors have attempted to describe the cephalometric characteristics of the class ii, division 1 malocclusion. the resulting convex profile involves maxillary protrusion, mandibular retrusion or combination of both2. class ii malocclusion may be accompanied or not by a skeletal discrepancy. the mandible may be normal or retruded relative to the maxilla or the maxilla could be protruded or normal relative to the mandible3-7. a successful treatment of class ii malocclusion in young people depends on the proper orthodontic mechanics, patient cooperation and how satisfactorily the growth spurt occurs, in ages from 10 to 13 for girls and 11 to 14 years for boys4. ricketts8 reported that condylar growth towards antero-superior direction would received for publication: july 21, 2014 accepted: november 25, 2014 braz j oral sci. 13(4):251-256 increase the facial depth and the brachiocephalic pattern. however, a condylar growth towards a posterior-superior direction will result in an increase in the face height with dolicocephalic trends. kloehn9 has suggested that class ii malocclusions should be treated with cervical traction during mixed dentition followed by fixed orthodontic appliance without tooth extractions, because of the mandibular alveolar processes and forwards shift of teeth in normal growth. most corrections result from a combination of a normal jaw growth pattern accompanied by changes in the maxillary alveolar process and dentition10-11. however, these data are not corroborated by hubbard et al.12, who reported that there are several variables involved in it, such as angulation of mandibular plane, techniques for using and adjusting the kloehn appliance, along with the patient’s age. patients with normal vertical face proportions undergoing orthodontic treatment in the growth spurt phase are more likely to have favorable results and long-term stability10-13. the objective of the present study was to assess the changes in mandibular behavior of patients submitted to full orthodontic treatment with standard edgewise appliance and cervical traction headgear with those who used cervical headgear first and full orthodontic treatment later. material and methods the ufrj’s ethics committee approved the development of this study under the protocol number (caae 54/20090050.0.339.000/09). this clinical research was based on one group of 40 individuals (group 1), 21 girls and 19 boys, who received conventional edgewise fixed appliance and kloehn cervical headgear treatment during a 25-month period with 12 h/day wearing time of the cervical headgear in just one period of the treatment. in the other group, 40 individuals (group 2), 23 girls and 17 boys, were treated using a cervical headgear for 12 months (first period) and conventional edgewise fixed appliance and kloehn cervical headgear for 25 months (second period). the force of cervical headgear applied for the 80 patients averaged 400 g. the onset of treatment was either at the late mixed dentition or at the beginning of the permanent dentition. all patients were evaluated three times by lateral cephalometric radiographs: at the beginning of the treatment (t0), at the end of the active orthodontic treatment (t1), and at least 5 years out of retention (t2). all subjects were in the pubertal growth spurt period at the beginning of treatment of the skeletal class ii malocclusions, (anb angle > 5o) and exhibited dental relationship of class ii, division 1 malocclusion according to angle’s classification. all the individuals also exhibited sngogn angle <35o. all patients were treated nonextraction and no use of class ii intermaxillary elastics, in the postgraduate orthodontic program of the federal university of rio de janeiro. in group 1, the mean age for female patients at t0 was 11.4 years (±1.5 years); at t1 was 13.6 years (±1.6 years) and at t2 was 26 years (±1.2 years). for male patients at t0 was 12.2 years (±1.7 years), at t1 was 14.4 years (±1.5 years) and at t2 was 28 years (±1.4 years). in group 2, the mean age of female patients was 9.8 years (± 1.2 years) in phase t0; 12.9 years (± 1.7 years) in phase t1 and 26 years (± 1.3 years) in phase t2. the mean age of male patients was 10.8 years (± 1.6 years) in phase t0; 14.5 years (± 1.3 years) in phase t1 and 28 years (± 1.7 years) in phase t2. the cephalograms were obtained by delimiting skeletal, dental, and tegumentary structures. the measurements from cephalometric tracings regarding t0, t1 and t2 were tabulated for statistical analysis, with angle measurements rounded up whenever decimal fraction existed. changes in mandibular displacement were measured in relation to skull base by using the following angles: snb, snd, sngogn, sngome, cdgogn, y-axis, facial angle and fma (figure 1). the linear measurements were used to describe separately the mandibular components: cdgo (height of mandibular ramus); cdpog (total mandibular length) and gopog (mandibular body length) (figure 2). means and standard deviations were calculated for each cephalometric measurement at t0, t1 and t2. the statistical treatment of the data between t0 x t1 as well as between t1 x t2 was performed using the paired student’s t test with 5% significance level. unpaired t tests were used to evaluate the differences in therapeutic effects and the length of active treatments between the groups. pearson’s r correlation coefficient was applied to determine whether any skeletal or dental characteristics and age were related to the length of active treatment. fig. 1. cephalogram illustrating angular measurements used in the study: snb, snd, sngogn, sngome, cdgogn, y-axis, face angle and fma. 252252252252252comparison of 2 types of treatment of skeletal class ii malocclusions: a 5-year post-retention analysis braz j oral sci. 13(4):251-256 fig. 2. cephalogram showing linear measurements (mm) used in the study: cdgo, cdpog, and gopog. error of the method to evaluate the error of the method, 30 radiographs chosen at random were traced and digitized by the same investigator on 2 separate occasions at least 2 months apart. the dahlberg14 formula was used: me =\/σ d2/2n, where n is the number of duplicate measurements. random errors varied between 0.26 and 0.92mm for linear measurements and between 0.280 and 0.370 for angular measurements. results both groups had comparable mean cephalometric values for angular measurements: snb, snd, y-axis, facial angle snb (o) 76.25 ± 2.67 6.75 ± 2.09 0.50 -0.65 .51 n s snd (o) 73.25 ±2.67 74.05 ± 1.84 0.80 -1.1 .027 n s sngogn (o) 31.85± 2.08 29.75 ± 1.61 2.10 3.55 .001 * * sngome (o) 32.90± 2.10 31.15 ± 1.56 1.75 2.98 .005 * cdgogn (o) 124.25±5.48 120.95± 3.95 3.30 2.18 .03 * y-axis (o) 58.25± 4.94 56.70 ± 4.48 1.55 1.03 .30 n s face (°) 83.75± 3.91 84.75 ± 3.53 1.0 -0.84 .40 n s fma (o) 26.05± 5.36 23.60 ± 3.95 2.45 1.64 .10 n s cdgo (mm) 5.0 ± 0.41 4.70 ± 0.44 0.30 2.20 .03 * cdpog(mm) 10.68± 0.56 10.29 ± 0.56 0.39 2.18 .03 * gopog mm) 7.23 ± 0.47 7.08 ± 0.40 0.14 1.04 .30 n s group 1 n=40 mean sd mean sd difference t0 p significance group 2 n=30 table 1.table 1.table 1.table 1.table 1. comparison between the group 1 and group 2 at t0 sd = standard deviation **= 1% significance level *= 5% significance level ns= non significant and fma, and for linear measurements, gopog. however, the mandibular plane angles: sngogn and sngome were 2.1 0 and 1.8 0 greater in group 1 than in the group 2, respectively (table 1). the angular measurement cdgogn was 3.30° bigger in the group 1 than in group 2. the linear measurements cdgo and cdpog were slightly larger in group1 (0.3 cm and 0.39 cm respectively) than in group 2 (table 2). during the treatment, the snb angle increased an average 1.70 (± 0.650) in both groups. in group i the snd angle increased an average 1.650 (± 0.870) and 1.350 (± 0.670) in group 2. the angles sngogn, sngome and cdgogn decreased respectively 1.200, 1.550 and 2.80° in group 1 and 0.850, 0.800 and 1.950 in the group 2. in both groups the yaxis angle decreased 0.40 whereas the facial angle increased 1.750 (± 1.610) in group 1 and 2.30 in group 2. in both groups the linear measurements cdgo, cdpog and gopog increased with approximate values. the mean differences between the 2 groups for all analyzed measurements were not statistically significant. at the post-retention phase the snb angle increased an average 1.050 (± 0.220) in both groups, whereas the snd angle increased 1.10 in group 1 and 0.950 in group 2. the angles sngogn, sngome and cdgogn also decreased in average respectively 1.050, 1.050 and 2.10 in group1 and 0.30, 0.30 and 20 in group 2. the y-axis increased slightly 0.150 (± 0.480) in group 1 and 0.10 (± 0.640) in group 2. the facial angle increased 1.10 (± 0.710) in group 1 and 1.050 (± 0.510) in group 2, whereas the fma angle decreased slightly, in average 10 in group1 and 0.450 in group 2. in both groups the linear measurements also increased, but with approximate values. the mean differences between the 2 groups for all analyzed measurements were not statistically significant. treatment was moderately inversely related to age at t0 in both groups (r=-0.33 for group1 and r=0.34 for group 2); the younger patients had longer treatments. in the same way the y-axial, face and fma angles were inversely related to age at t0 in group 1. in group 2, the angular measurements snb, snd, sngogn, sngome were also inversely related to comparison of 2 types of treatment of skeletal class ii malocclusions: a 5-year post-retention analysis braz j oral sci. 13(4):251-256 253253253253253 age at t0. there was no correlation with the other initial tested variables in both groups. discussion the skeletal changes resulting from face growth, which occurs during the transition from deciduous to permanent dentition, do not correct the class ii malocclusion established at an earlier age. it probably happens due to the morphological characteristics of the class ii malocclusion, justifying the therapeutic intervention during the growth spurt15-16 as was observed in this study. for dental class ii and moderate skeletal discrepancy in growing children, two methods of treatment were compared to assess their cephalometric effects at the end of comprehensive treatment and at post retention. while assessing the mandibular behavior of the study snb (o) 1.7 ± 0.65 1.7 ± 0.65 ns snd (o) 1.65 ± 0.87 1.35 ± 0.67 0.3 ns sngogn (o) 1.20 ± 0.41 0.85 ± 0.36 0.35 ns sngome (o) 1.55± 1.39 0.80 ± 0.52 0.75 ns cdgogn (o) 2.80 ± 2.50 1.95 ± 0.94 0.85 ns y-axis (o) 0.45 ± 1.53 0.40 ± 1.63 0.05 ns face (o) 1.75 ± 1.61 2.3 ± 1.72 0.55 ns fma (o) 1.85 ± 2.03 1.50 ± 1.82 0.35 ns cdgo (mm) 0.67 ± 0.27 0.72 ± 0.24 0.05 ns cdpog(mm) 0.98 ± 0.15 1.06 ± 0.25 0.08 ns gopog mm) 0.66 ± 0.32 0.70 ± 0.14 0. 04 ns sd = standard deviation ns= non significant table 2.table 2.table 2.table 2.table 2. treatment effects on cephalometric values and differences between both groups at t1 mean difference sd mean difference sd mean difference significance group 1 t1-t0 n=40 group 2 t1-t0 n=30 group 1 vs group 2 mean difference sd mean difference sd mean difference significance group 1 t1-t0 n=40 group 2 t1-t0 n=30 group 1 vs group 2 snb (o) snd (o) sngogn (o) sngome (o) cdgogn (o) y-axis (o) face (o) fma (o) cdgo (mm) cdpog(mm) gopog mm) 1.05 ± 0.22 1.1 ± 0.30 1.05 ± 0.39 1.05 ± 0.75 2.1 ± 1.48 0.15 ± 0.48 1.10 ± 0.71 1.0 ± 0.85 0.34 ± 0.30 0.68 ± 0.05 0.52 ± 0.16 1.05 ± 0.22 0.95 ± 0.39 0.30 ± 0.57 0.30 ± 0.57 2.0 ± 1.48 0.1 ± 0.64 1.05 ± 0.51 0.45 ± 0.51 0.28 ± 0.17 0.71 ± 0.02 0.58 ± 0.15 0.15 0.75 0.75 0.1 0.05 0.05 0.55 0.06 0.03 0.06 n s n s n s n s n s n s n s n s n s n s n s table 3.table 3.table 3.table 3.table 3. cephalometric values and differences between both groups (t2 – t1) sd = standard deviation. ns= non significant. group, it was observed that the mechanics of a conventional edgewise fixed appliance and kloehn cervical headgear used for orthodontic treatment did not interfere with the mandibular growth and displacement, since the mean values for snb angle had a statistically significant increase in the t0 t1 interval. this was observed in group 1 that received conventional edgewise fixed appliance and kloehn cervical headgear treatment for 25 months, as well as in group 2 that was treated using a cervical headgear for 12 months (first period) and conventional edgewise fixed appliance and kloehn cervical headgear for 25 months (second period). this demonstrated a favorable mandibular growth in relation to the skull base during the phase of active orthodontic treatment, which was confirmed by an expressive increase in the snd angle. therefore there are no outcome differences between the two types of treatment because the selected sample showed horizontal facial growth, , , , , with favorable mandibular and maxillary growth, in down and forward comparison of 2 types of treatment of skeletal class ii malocclusions: a 5-year post-retention analysis braz j oral sci. 13(4):251-256 254254254254254 duration (years) age (years) snb (o) snd (o) sngogn (o) sngome (o) cdgogn (o) y-axis (o) face (o) fma (o) cdgo (mm) cdpog(mm) gopog mm) group 1 n=40 mean sd 2.1 ± 0.38 11.8 ± 0.8 76.25 ± 2.67 73.25 ± 2.67 31.85 ± 2.08 32.90 ± 2.10 124.25 ±5.48 58.25 ± 4.94 83.75 ± 3.91 26.05 ± 5.36 5.0 ± 0.41 10.68 ± 0.56 7.23 ± 0.47 0.33 0.08 0.06 0.04 0.018 0.16 0.38 0.45 0.40 0.10 0.03 0.17 * n s n s n s n s n s * * * n s n s n s group 2 n=30 mean sd 3.1 ± 0.17 10.2 ± 0.6 6.75 ± 2.09 74.05 ± 1.84 29.75 ± 1.61 31.15 ± 1.56 120.95 ± 3.95 56.70 ± 4.48 84.75 ± 3.53 23.60 ± 3.95 4.70 ± 0.44 10.29 ± 0.56 7.08 ± 0.40 1 0.34 0.41 0.36 0.28 0.29 0.04 0.07 0.19 0.09 0.12 0.009 0.10 difference difference * * * * * n s n s n s n s n s n s n s table 4: table 4: table 4: table 4: table 4: correlations with duration of active treatment in both groups sd = standard deviation. *= 5% significant level. ns= non significant directions. similar conditions were observed in the t1 t2 interval regarding the mean snb and snd angles, which could be the result of residual mandibular growth after the active orthodontic treatment period (tables 2 and 3)15. interestingly, the 12 months difference in treatment length corresponded approximately to the time that only the kloehn cervical headgear was worn. an explanation of this variation in treatment duration is the age at t0; kloehn cervical headgear was used relatively early in patients of group 2, who were far from full permanent dentition or far from their maximum growth potential during puberty. this was confirmed in younger patients, who showed slower increase in the snb,snd, sngogn and sngome measurements and total treatment time longer than group 1 (table 4). with regard to the profile, a mean reduction of the face convexity was found in the time intervals, which was confirmed by a significant increase of the face angle. this fact may be supported by the anterior positioning of the mandible during facial growth (tables 2 and 3) as well as bone apposition in the pogonion region in both groups5,17. the cephalometric evaluation showed a decreasing trend of the angles related to the mandibular plane during growth due to the intrinsic morphogenetic characteristic of the studied cases18-19. all patients submitted to orthodontic treatment presented low mandibular plane, which is a crucial factor for using cervical traction as mentioned in other studies2,5,17. the mean values for sngogn, sngome, cdgogn and fma angles showed a significant reduction in the time interval, suggesting that rotation of the mandible is ruled by the direction and amount of condylar growth and remodeling at the lower border of the mandible in both groups (table 2)15,19-20. according to the structural analysis established by björk3, the mandibular rotation depends on the morphogenetic pattern, which is determined by the mandible morphology. the vertical growth of mandibular condyles should be greater than that of posterior alveolar processes and is an important factor in the anti-clockwise rotation of the mandible 21. nevertheless, the changes observed in the y-axis angle revealed the harmonic pattern of face growth in both groups during orthodontic treatment and post-retention phases (tables 2 and 3)11,22-23. analysis of the linear measurements cdgo, cdpog and gopog (tables 2 and 3) showed a significant increase in t0 t1 and t 1 t 2 intervals. these data also suggest that mandibular growth occurs during the active orthodontic treatment as well as post-retention period, including an increase in both mandibular ramus and body due to the condylar growth and bone apposition in the pogonion region. according to the literature, the mandibular growth is more prominent than the maxillary growth, continuing for an additional period of time11,22,24. amount and direction of mandibular growth are genetically determined. the lower border of the mandible influences the mandibular plane angle because of bone remodelling (tables 2 and 3)10. the mean values for sngogn, sngome, cdgogn, and fma angles were reduced in both groups of patients between t0 t1, demonstrating favorable condylar growth and remodeling at the lower border of the mandible. in this way, anti-clockwise rotation of the mandible was observed in the patients, which was confirmed by the significant reduction in cdgogn and fma angles2,4. between t1 t2, all the angular measurements cited above were found to be significantly decreased for all patients, suggesting that both growth and displacement of the mandible are determined by genetic factors (tables 2 and 3)3,10. analysing the mean values regarding the linear measurements cdgo, cdpog, and gopog (tables 2 and 3), a significant increase in both time intervals for both groups was found. this emphasized the mandibular growth observed during and after the active orthodontic treatment phase. similar results were also found by other authors, who reported a residual mandibular growth11,22,24. the apposition in the region of pogonion occurs continuously even after the active treatment is finished17-19. full corrective orthodontic treatment, using concurrently comparison of 2 types of treatment of skeletal class ii malocclusions: a 5-year post-retention analysis braz j oral sci. 13(4):251-256 255255255255255 standard edgewise technique and cervical headgear (kloehn appliance), was considered effective in patients with skeletal class ii malocclusions and low mandibular plane as well as just cervical headgear was used at first period, followed by full corrective orthodontic treatment and cervical headgear. the treatments did not interfere in mandibular growth, which happened during the active treatment as well as after it finished. these observations may lead to the speculation that growing patients with skeletal class ii malocclusion and low mandibular plane are conducive to a good treatment and long-term stability with one or two periods of treatment. references 1. angle e. classification of malocclusion. dent cosmos. 1899; 41: 248-64. 2. jacob hb, buschang ph. mandibular growth comparisons of class i and class ii division skeletofacial patterns. angle orthod. 2014; 84: 755-61. 3. janson g, goizueta oefm, garib dg, janson m. relationship between maxillary and mandibular base lengths and dental crowing in patients with complete class ii malocclusions. angle orthod. 2011; 81: 217-21. 4. boccetti t, stahl f, mcnamara ja jr. dentofacial growth changes in subjects with untreated class ii malocclusion from late puberty through young adulthood. am j orthod dentofacial orthop. 2009; 135: 148-54. 5. 5.hassan ah. cephalometric characteristics of class ii division 1 malocclusion in a saudi population living in the western region. saudi dent j. 2011; 23: 23-7. 6. 6.al-khatecb ea, al-khatecb sn. anteroposterior and vertical components of class ii division 1 and division 2 malocclusion. angle orthod. 2009; 79: 859-66. 7. saltazi h, floresmir c, mazor pw, yowsef m. the relationship between vertical facial morphology and overjet in untreated class ii subjects. angle orthod. 2012; 82: 432-40. 8. ricketts rm. planning treatment on the basis of the facial pattern and an estimate of its growth. angle orthod. 1957; 27: 14-37. 9. kloehn s. evaluation of cervical anchorage force in treatment. angle orthod. 1961; 31: 91-104. 10. bjork a. variations in the growth pattern of the human mandible: longitudinal radiographic study by the implant method. j dent res. 1963; 42: 400-11. 11. fidler bc, artun j, joondeph dr, little rm. long-term stability of angle class ii, division 1 malocclusions with successful occlusal results at end of active treatment. am j orthod dentofacial orthop. 1995; 107: 276-85. 12. hubbard gw nrs, currier g f. a cephalometric evaluation of non extraction cervical headgear treatment in class ii malocclusion. angle orthod. 1994; 64: 359-70. 13. franchi l, pavoni c, faltin k jr, mcnamara ja jr, cozza p. long term skeletal and dental effects and treatment timing for functional appliances in class ii malocclusion. angle orthod. 2013; 83: 334-40. 14. dahlberg g. statistical methods for medical and biological students. london: allen & unwin; 1940. 15. flores-mir c, mcgrath l, heo g, major p. efficiency of molar distalization associated with molar eruption stage. a systematic review. angle orthod. 2013; 83: 735-40. 16. baccetti t, franchi l, kim lh. effect of timing on the outcomes of 1-phase nonextraction therapy of class ii malocclusion. am j orthod dentofacial orthop. 2009; 136: 501-9. 17. garbui iu, nouer pr, nouer df. cephalometric assessment of vertical control in treatment of class ii malocclusion with a combined maxillary splint. braz oral res. 2010; 24: 34-9. 18. lira als, souza mmg, bolognese am. long-term maxillary behavior in treated skeletal class ii malocclusion. braz j oral sci. 2012; 11: 120-4. 19. carter ne. dentofacial changes in untreated class ii division 1 subjects. br j orthod. 1987; 14: 225-34. 20. kim j, nielsen la. a longitudinal study of condilar growth and mandibular rotation in untreated subjects with class ii malocclusions. angle orthod. 2002; 72: 105-11. 21. klocke a, nanda rs, kahl-nieke b. skeletal class ii paterns in the primary dentition. am j orthod. 2002; 121: 596-601. 22. lira als, izquierdo a, prado s, nojima li, nojima m. mandibular behavior in the treatment of skeletal class ii malocclusion: 5 years post-retention analysis. braz j oral sci. 2009; 8: 166-70. 23. kirjavainen m, hurmerinta k, kirjavainen t. facial profile changes in early class ii correction with cervical headgear. angle orthod. 2007; 77: 960-7. 24. lira als, izquierdo a, prado s, nojima m, maia l. anteroposterior dentoalveolar effects with cervical headgear and pendulum. comparison of 2 types of treatment of skeletal class ii malocclusions: a 5-year post-retention analysis braz j oral sci. 13(4):251-256 256256256256256 oral sciences n3 braz j oral sci. 12(2):119-124 original article braz j oral sci. april | june 2013 volume 12, number 2 a comparative leakage study on er,cr:ysgg laserand burprepared class v cavities restored with a low-shrinkage composite using different filling techniques fernanda strohmayer sarabia1, andréa dias neves lago2, sérgio brossi botta3, cynthia soares de azevedo1, narciso garone-netto1, adriana bona matos1 1department of operative dentistry, school of dentistry, university of são paulo (usp), são paulo, sp, brazil 2department of operative dentistry, school of dentistry, university of minas gerais (ufmg), belo horizonte, mg, brazil 3department of operative dentistry, school of dentistry, nove de julho university (uninove), são paulo, sp, brazil correspondence to: adriana bona matos av. prof. lineu prestes, 2227, cep: 05508-900 são paulo, sp, brasil phone: +55 11 30917839 ext 224 fax: +55 11 30917839 e-mail: bona@usp.br received for publication: march 23, 2013 accepted: june 25, 2013 abstract aim: to evaluate the leakage on er,cr:ysgg laserand burprepared class v cavities restored with a silorane-based composite resin using different insertion techniques methods: 40 cavities were outlined according to: the type of instrument [er,cr:ysgg laser (3.0 w power, energy per pulse of 150 mj, fluence of 53.57j/cm2, pulse duration of 140-200 µs, 20 hz repetition rate and 55/65% air/water spray) or diamond bur]; and the type of filling technique (bulk increment or incremental). four experimental groups were obtained (n=10): g1diamond bur (db) and incremental (i); g2db and bulk increment (bi); g3er,cr:ysgg and i; and g4er,cr:ysgg and bi. specimens were restored with a silorane-based composite resin (filtek p90, 3m/espe), subjected to 500 thermal cycles, sealed, infiltrated with 2% (w/v) methylene blue and sectioned in halves. specimen analysis was scored based on a scale. statistical analyses were done using the kruskal-wallis and student newman-keuls tests (α=0.05). results: statistically significant differences were observed between g2 and g4 (p=0.003) and between g1 and g2 (p=0.028). the filling technique did not influence the pattern of dye leakage in the cavity walls (p=0.151). conclusions: less leakage was observed when er,cr:ysgg cavities were restored with silorane-based composite resin, using the bulk increment technique. nevertheless, cavities done using diamond bur have less leakage only when incrementally restored. keywords: dental leakage, lasers, polymerization, composite resins, tooth preparation. introduction dentistry has become more conservative, using minimally invasive techniques allied with new technologies1. among several alternatives for preparation of conservative cavities, the use of er,cr:ysgg (erbium, chromium:yttrium-scandiumgallium-garnet) laser has been proposed due to its high absorption of water and hydroxyapatite2. er,cr:ysgg laser, when safely used in conjunction with air/water spray3 and 120120120120120 braz j oral sci. 12(2):119-124 appropriate irradiation parameters, have some advantages over conventional cavity preparation techniques, namely production of less noise and vibration, preservation of more tooth structure and less injury to pulp tissue, antibacterial properties, and no need of anesthesia during preparation4-5. together, these advantages have led to an increased use of er,cr:ysgg laser in clinical practice, especially for patients who are anxious about pain and discomfort6. er,cr:ysgg laser irradiation does not generate a smear layer7-9. thus, the tooth surface presents exposed tubule apertures8-9 with characteristics that suggest greater permeability of the irradiated surface. reports are still controversial regarding the microleakage and bonding of resin monomers to irradiated dentin. while some authors10-13 suggest improved adhesion and less microleakage on irradiated surfaces, when compared with diamond burs, others show that microleakage is lower in irradiated dentin14. there is yet a third group of authors who claim no difference in adhesion to surfaces prepared with either laser irradiation or rotary instruments15. more information is needed to resolve the debate regarding irradiated dentin and resin monomer interaction and its ability to bond to tooth structure. this discussion is even more important when new materials are introduced in the dental market. silorane-based composite resins associated with its specific adhesive system are widely available for clinical application. these restorative systems, whose matrix is composed of organic silorane, claim to have lower polymerization shrinkage. this is a result of the silorane chemical reaction, which occurs through a cationic benzene ring-opening, promoting reduced resin shrinkage, when compared with methacrylate-based resins16. by reducing resin shrinkage, the clinical durability of restorations is increased as a result of good marginal sealing. according to the manufacturer instructions, this composite allows use of larger increments (2 mm), if inserted horizontally into the cavity. thus, the chair-side time is reduced, which is highly desirable for shortening the treatment time. however, some authors still claim that the layering technique remains recommended even for low-shrinking materials17. it is fundamental to point out that these studies have been performed over conventionally prepared dentin, using diamond or carbide burs. to our knowledge, there are currently no studies on the use of silorane-based composites and er,cr:ysgg laser-irradiated dentin. therefore it might be expected that the different superficial dentin morphology originated by burs and lasers would influence leakage when silorane-based composite is used. the aim of this study was to compare the effects of the er,cr:ysgg laser cavity preparation technique with conventional preparation instruments, such as, diamond bur18-20 on the marginal leakage of silorane-based composite restorations. moreover, the effects on leakage of the restorative technique used to insert the composite resin into the cavity was also evaluated. material and methods the factors under study were cavity preparation method (er,cr:ysgg laser and diamond bur) and restorative technique (bulk increment and incremental). the experimental units consisted of 40 cavities on erupted human third molars, randomly divided in 4 groups (n=10). leakage was assessed qualitatively. tooth selection after the approval of the research project by the institutional ethics committee (protocol 108/10), 20 freshly extracted, erupted human third molars obtained by donation of the patients were collected and stored in distilled water at 4°c. teeth were analyzed using a stereoscopic magnifying glass (olympus, hongo, tokyo, japan) at 25× magnification to selected only those without cracks or restorations. cavity preparation procedures the dimensions of the standardized cavities on both buccal and lingual/palatal surfaces were 2.0 mm occlusogingival, 4.0 mm mesiodistal and 2.0 mm depth. cavity outline was standardized using a template and its depth was guided by a periodontal probe. cavities were made in the buccal and lingual/palatal surface of each tooth18-19, resulting in 40 cavities that divided into 4 groups (n=10), according to method of cavity preparation and restorative technique. all cavities were prepared at the cervical third of the teeth, but all margins were located in enamel to produce a better marginal sealing. in groups g1 and g2 cavities were prepared with a #1090 cylindrical diamond bur (kg sorensen, barueri, sp, brazil) at high speed under constant water spray coolant2021. in groups g3 and g4, cavities were prepared with a er,cr:ysgg laser (millennium, biolase, san clemente, ca, usa) belonging to the biophotonics laboratory of the nuclear and energy research institute; são paulo, sp, brazil; fapesp-98/14270-8 cepid project). this laser has a 2.78 µm wavelength with pulse duration of 140-200 µs. a sapphire fiber, model “g,” with 600 nm in diameter and 4 mm in length, was attached to a #2415 handpiece (millennium, biolase), which virtually touches the tooth. irradiation parameters used for these groups were 3.0 w power, energy per pulse of 150 mj, fluence of 53,57 j/cm2, 150 mj energy per pulse and 20 hz repetition rate7,21cooled by a 55/65% air/water spray. for a precise tissue irradiation control, an automatic pitch shifter xyz micrometer (model esp 300 newport corporation, ca, usa) was used in such a way that the specimens were displaced during irradiation with standardized speed (6.0 mm/s) and distances (200 µm) between the pulses, avoiding any gaps between the laser pulses22. restorative technique cavities were restored using a silorane-based composite resin (filtek p90, 3m/espe, st. paul, mn, usa) associated with filtek p90 self-etch bonding agent. following the manufacturer’s instructions, primer solution was actively applied for 15 s and light-cured for 10 s. the adhesive resin was then applied to all cavity walls and light-cured for 10 s. after applying the adhesive system, composite resin was inserted using different restorative techniques: incremental (g1 and g3) or bulk increment (g2 and g4). groups filled a comparative leakage study on er,cr:ysgg laserand burprepared class v cavities restored with a low-shrinkage composite using different filling techniques braz j oral sci. 12(2):119-124 with the incremental technique had composites inserted in approximately two horizontally oriented increments, each one light-cured for 40 s23-24, using a jetlite 4000 halogen lamp curing unit (j. morita, irvine, ca, usa), at a 1000 mw/cm2 intensity. for groups treated with bulk increment, the composite resin was inserted at once, covering the cavity along the entire length and depth and polymerized for 40 s25-26. finishing and polishing of restorations after the restorative procedure, specimens were stored in distilled water at 37oc for 24 h. at the end of this period, finishing and polishing procedures were performed with silicone tips27-28 (kit enhance; caulk dentsply, milford, de, usa) at low speed (kavo brazil, joinville, sc, brazil). restorations were polished until regular and smooth surfaces were obtained with well-defined borders and no excess material. marginal adaptation was evaluated using a stereomicroscope at 40× magnification. the specimens were then stored in distilled water at 37oc for additional 24 h. thermal aging test specimens were subjected to 500 thermal cycles23-24, alternating baths between 5oc and 55°c for 1 min at each temperature, and 3 s of transfer time between baths. leakage test after thermal cycling, the specimens were sealed with two coats of nail polish (risqué, são paulo, sp, brazil) up to 1 mm from the edge of the restoration. for the leakage test, specimens were then immersed in a 2% (w/v) methylene blue solution (merck, darmstadt, germany)29 for 4 h at 37°c, followed by rinsing under running water for 15 min to eliminate the dye from the specimen surface. leakage evaluation specimens were vertically sectioned in a buccolingual direction, through the center of the restoration. all halves were observed at 40× magnification (miview usb digital microscope video camera, cosview technologies co., ltd, longgang district, shenzhen, china) and the half with a larger amount of dye was chosen to represent the specimen. the specimens were scored according to the following criteria: 0 = no dye penetration (figure 1); 1 = partial dye penetration along the occlusal or gingival wall (figure 2); 2 = dye penetration along the occlusal or gingival wall but not including the axial wall (figure 3); 3 = dye penetration to and along the axial wall (figure 4)30. the dye penetration was assessed by three independent calibrated examiners using a light microscope under 40× magnification. cohen’s kappa for inter-and intraexaminer reproducibility was 0.90 and 0.95, respectively. data were analyzed with the program bioestat 5.0 (institute for sustainable development mamirauá, belém, am, brazil) and submitted to the nonparametric kruskal-wallis test followed by group comparison with student-newmankeuls test (α=0.05). fig. 1 image representative of score 0 (no dye penetration). fig. 2 image representative of score 1 (partial dye penetration along the occlusal or gingival wall). fig. 3 image representative of score 2 (dye penetration along the occlusal or gingival wall but not including the axial wall). 121121121121121 a comparative leakage study on er,cr:ysgg laserand burprepared class v cavities restored with a low-shrinkage composite using different filling techniques braz j oral sci. 12(2):119-124 122122122122122 fig. 4 image representative of score 3 (dye penetration to and the axial wall). results the frequency distribution of dye penetration scores for the groups are presented in table 1. the kruskal wallis test indicated that there is a significant difference between the tested groups (p=0.013). in groups restored with a bulk increment, er,cr:ysgg laser cavity preparation (g4) showed significantly less leakage than conventional preparation with diamond bur (g2) (p=0.003). as for the incremental technique, there was no statistically significant difference between g1 (diamond bur) and g3 (er,cr:ysgg laser) (p=0.515). when cavities were prepared with diamond bur (g1 x g2), there was less leakage when the restoration was done using the incremental technique (p=0.028). however, when cavities were prepared with er,cr:ysgg laser, the filling technique did not influence the pattern of dye leakage in the cavity walls (p=0.151). the mean leakage score of experimental groups can be increasingly arranged (table 2). groups (n=10) g1 g2 g3 g4 median 1.78 2.93 2.12 1.37 table 2. median microleakage score in each group group score 0 score 1 score 2 score 3 g1 3 3 1 3 g2 1 0 0 9 g3 2 2 2 4 g4 3 6 0 1 table 1. frequency distribution of dye penetration scores for each group discussion this study evaluates leakage of silorane-based composite inserted using the bulk increment and incremental technique in cavities prepared with either er,cr:ysgg laser or diamond burs. the obtained results show that restoration of cavities made with er,cr:ysgg was beneficial with respect to the marginal sealing when the bulk increment technique was used. it could be attributed to the presence of silorane in the composite resin matrix. this restorative system consists of a silorane radiopaque low shrinkage base and a hydrophobic matrix, yielding lower polymerization shrinkage and reduced water absorption23,31. during the silorane benzene ring break, polymer volume is only slightly lower than the non-cured resin16,23. this polymer also presents a low tension of polymerization, which is determined by the following factors: polymerization shrinkage, internal flow of material, polymerization kinetics, and polymerization rate16. because the laser produces cavity walls with invaginations, the final surface area is larger, if compared with conventional methods of preparation. this reduces the tension associated with polymerization and the formation of marginal gaps, which in turn leads to less leakage than that observed when conventional methods are applied17. moreover, the adhesive system used in conjunction with the silorane-based composite resin was specially developed for this type of resin, which optimizes filling due to its structural viscosity. an important advantage of this silorane-based resin is the possibility of using larger increments, thereby reducing errors during the restorative procedure. according to the results of this study, er,cr:ysgg laser (g3 and g4) showed similar results of leakage irrespective of the restorative technique used. it is fundamental to point out that the best leakage performance was observed in er,cr:ysgg laser preparations restored with a bulk increment. paradoxically, when cavities were prepared with diamond bur (g1 and g2), the use of the incremental filling technique showed less leakage when compared with bulk resin insertion, keeping the use of incremental technique for resin insertion fundamental, when cavities are conventionally prepared16,23. it should be highlighted that due to the different composition and low polymerization shrinkage of the silorane-based composite resin, the manufacturer claims that this type of material can be used in layers up to 2 mm, which means that the incremental composite insertion technique would be unnecessary for shallow cavities. this study confirmed this manufacturer’s recommendation only when cavities were prepared with er,cr:ysgg laser, indicating that incremental insertion is still necessary for bur-prepared cavities, even though using a silorane-based resin32-33. several reports have evaluated microleakage of cavities made with erbium lasers11-13,15 and in some cases, the laser appears to be a detrimental factor, since it does not always form an appropriate hybrid layer, which leads to adhesive failure4,9,11. authors claim that irradiation keep calcium attached to peritubular dentin, impairing the penetration of the adhesive systems and its proper hybrid layer formation3,511. this is the first experiment that tested the association of silorane-based resins in laser prepared cavities. although other studies using additional methodologies are still needed to confirm the optimal performance of this a comparative leakage study on er,cr:ysgg laserand burprepared class v cavities restored with a low-shrinkage composite using different filling techniques 123123123123123 braz j oral sci. 12(2):119-124 resin in cavities made with lasers, our leakage results for silorane-based composite resin inserted in cavities prepared with er,cr:ysgg laser are very promising2. the little leakage observed in er,cr:ysgg laser preparation indicates that filtek p90 self-etch bonding agent adequately interacts with irradiated dentin and thus may be the adhesive that authors have been looking for since this laser was introduced in restorative dentistry for cavity preparation. it may be concluded that silorane-based composite resin bulk increment restorations presented less leakage when cavities were made with er,cr:ysgg laser. conversely, for 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and staining susceptibility of composite resins after finishing and polishing. j esthet restor dent. 2011; 23: 34-43. 29. antonson sa, yazici ar, okte z, villalta p, antonson de, hardigan pc. effect of resealing on microleakage of resin composite restorations in relationship to margin design and composite type. eur j dent. 2012; 6: 389-95. 30. arslan s, yazici ar, gorucu j, pala k, antonson de, antonson sa, et al. comparison of the effects of er,cr: ysgg laser and different cavity disinfection agents on microleakage of current adhesives. lasers med sci. 2012; 27: 805-11. 31. baracco b, perdigao j, cabrera e, giraldez i, ceballos l. clinical evaluation of a low-shrinkage composite in posterior restorations: one-year results. oper dent. 2012; 37: 117-29. a comparative leakage study on er,cr:ysgg laserand burprepared class v cavities restored with a low-shrinkage composite using different filling techniques 124124124124124 braz j oral sci. 12(2):119-124 32. poureslami hr, sajadi f, sharifi m, farzin ebrahimi s. marginal microleakage of low-shrinkage composite silorane in primary teeth: an in vitro study. j dent res dent clin dent prospects. 2012; 6: 94-7. 33. schmidt m, kirkevang ll, horsted-bindslev p, poulsen s. marginal adaptation of a low-shrinkage silorane-based composite: 1-year randomized clinical trial. clin oral investig. 2011; 15: 291-5. a comparative leakage study on er,cr:ysgg laserand burprepared class v cavities restored with a low-shrinkage composite using different filling techniques 429 too many requests error 429 too many requests too many requests guru meditation: xid: 39003552 varnish cache server oral sciences n3 braz j oral sci. 14(2):100-105 original article braz j oral sci. april | june 2015 volume 14, number 2 influence of chemical degradation and abrasion on surface properties of nanorestorative materials andréia bolzan de paula1, roberta caroline bruschi alonso2, giovana albamonte spagnolo de araújo1, julia puppin rontani1, lourenço correr-sobrinho1, regina maria puppin-rontani3 1universidade estadual de campinas – unicamp, piracicaba dental school, area of dental materials, piracicaba, sp, brazil 2faculdade anhanguera, dental school, area of biomaterials, são paulo, sp, brazil 3universidade estadual de campinas – unicamp, piracicaba dental school, area of pediatric dentistry, piracicaba, sp, brazil correspondence to: andreia bolzan de paula universidade estadual de campinas faculdade de odontologia de piracicaba departamento de odontologia restauradora av. limeira, 901 cep: 13414-903 piracicaba, sp, brasil phone: +55 19 2106 5286 / fax: +55 19 2106 5218 e-mail: andbol_63@hotmail.com abstract aim: the aim of this in vitro study was to investigate the synergistic effect of chemical degradation (erosion) and three-body abrasion (mechanical degradation) on the surface roughness (ra) and hardness (khn) of two nanorestorative materials and two conventional materials. methods: discshaped specimens (5 mm in diameter, 2 mm thick) of filtek z350tm and tph spectrumtm composites and ketac nanotm and vitremertm light-curing glass ionomer cements, nanomaterials and conventional materials were prepared according to the manufacturer’s instructions. after 24 h, polishing procedures were performed and initial measurements of ra and khn were taken in all specimens. the specimens were divided into 12 groups (n = 10) according to material and storage media: artificial saliva, orange juice, and coca-cola®. after 30 days of storage, the specimens were submitted to mechanical degradation and re-evaluated for ra and khn. data were tested for significant differences by repeated-measure three-way anova and tukey’s tests (p<0.05). results: erosion and abrasion wear significantly decreased hardness of all materials. only filtek z350 roughness, however, was not affected by erosion and abrasion. all materials showed a significant increase in surface roughness after erosion and abrasion, except for filtek z350. after chemical and mechanical degradation, the khn of all samples had decreased significantly. after mechanical degradation, the acidic drinks (coca-cola® and orange juice) were more aggressive than artificial saliva to all materials. conclusions: a synergistic effect was observed by the increase in roughness for all materials, except for filtek z350; hardness values decrease for all materials, regardless of whether they were nanofilled or not. the rmgics were more susceptible to degradation than the composites, considering both hardness and roughness surface parameters. keywords: nanotechnology; tooth erosion; tooth abrasion. introduction the application of nanotechnology to dental materials was introduced in past few decades. in addition to improved optical properties, nanomaterials present better mechanical behavior 1, since the nanometric size of particle allows incorporating greater amount of filler load in the restorative materials2. nanofillers and nanofiller “clusters” are combined to improve mechanical properties, as threebody wear resistance. the nanofiller components also provide superior aesthetics and excellent polishing, with higher gloss and smoother surfaces than other resinreceived for publication: march 04, 2015 accepted: may 22, 2015 http://dx.doi.org/10.1590/1677-3225v14n2a01 101101101101101 modified glass ionomers (rmgics), while offering fluoride release similar to that of a conventional rmgic. a new rmgic has been introduced for operative dentistry recently: ketac nano. this material contains nanofillers and clusters of nano-sized zirconia/silica that result in a highly packed filler composition. it is important to compare this material to a traditional rmgic and a nanocomposite in order to establish whether the nano-ionomer shows a behavior similar to that of ionomeric and composite materials, thus predicting its mechanical and chemical properties. although it is possible to improve the material physical properties by incorporating nanofillers into restorative materials, it should be considered that the restorative materials are constantly subject to thermal, mechanical, and chemical challenges on the oral environment. de paula et al.3 (2011) found that nanotechnology incorporated in restorative materials, is important for the superior resistance to biomechanical degradation. those challenges can negatively influence the material properties by causing degradation of the matrix in resin influences the degradation of resin composites and glassionomer restorative materials4. soft drinks may contain several different types of acid that contribute to their low ph value5. a study reported by jensdottir et al.6 (2004) have found carbonated drinks, especially carbonated cola drinks, to be associated with erosion. an in vitro study has shown, however, that fruit juices may also be erosive, due their high titrability7. the erosive attack can induce matrix and filler degradation of restorative materials, and also potentially jeopardize the clinical performance of these materials8. not only erosive attack can jeopardize the restorative materials surface, but also the abrasion process produced by oral hygiene methods can adversely affect the surface characteristics of restorative materials1. this process may interfere with both health and materials ketactm nano (3m espe) vitremer (3m espe) filtek z350 (3m espe) tph spectrum (dentsply) composition paste a: silane-treated glass, silane-treated zirconia oxide silica, polyethylene glycol dimethacrylate (5–15%), silane-treated silica, hema, bis-gma (< 5%), tegdma (< 5%), hema (1–10%)paste b: silane-treated ceramic, silane-treated silica, copolymer of acrylic and itaconic acids, hema (1–10%) powder: fluoroaluminosilicate glass; redox systemliquid: aqueous solution of a modified polyalkenoic acid, hema (15–20%) 58–60 vol. % (78.5 wt. %) combination of aggregated zirconia/silica cluster filler with primary particles size of 5–20 nm, and non-agglomerated 20 nm silica filler, bis-ema, bis-gma; udma; tegdma polymer matrix: bis-gma, bis-ema and tegdma; filler: 57 vol% of ba-al-borosilicate glass and colloidal silica with mean particle size of 0.8 µm mean filler size (µm) 5–25 nm 3.0 µm 5–20 nm 0.6–1.4 µm (clusters) 0.8 µm manufacturer/batch # 3m-espe, st. paul, mn, usa m3m3 3m-espe, st. paul, mn, usa p: 6lp l: 6fh 3m-espe, st. paul, mn, usa 8 n u dentsply ind. e com. ltd., petropolis, rj, brazill797977 table 1.table 1.table 1.table 1.table 1. materials tested in this study. bis-gma = bisphenol glycidyl methacrylate; tegdma = triethylene glycol dimethacrylate; hema = 2hydroxyethyl methacrylate; bis-ema = ethoxylated bisphenol-a dimethacrylate; udma = urethane dimethacrylate. aesthetics, since rough surfaces may predispose teeth to biofilm accumulation. de paula et al. 3 (2011) have found that nanomaterials, when exposed to a cumulative effect of biofilm/ abrasion, shows superior resistance to biomechanical degradation in comparison with conventional restorative materials. it may therefore, be hypothesized that toothbrush abrasion and erosion caused by an acidic diet have a synergic effect on the substance loss of dental materials. in this way, restorative materials are in a constant process of degradation in the oral cavity, and nanotechnology has been investigated for its possible application to the materials as a way to minimize the cumulative deleterious effects of this process. the aim of this in vitro study was to investigate the synergistic effect of chemical degradation (erosion) and three-body abrasion (mechanical degradation) on the surface roughness (ra) and hardness (khn) of two nanomaterials and two conventional materials. material and methods specimen preparation and initial analysis four different types of tooth-colored restorative materials were tested in this study (table 1): two rmgics (vitremer and ketac nano, 3m espe, st. paul, mn, usa) and two composites: filtek z350 (3m espe), and tph spectrum (dentsply, caulk, usa). thirty specimens of each material were manipulated according to the manufacturer’s instructions. materials were inserted into plastic molds with internal dimensions of 5 mm diameter and 2 mm thickness. the top surface of the fulfilled mold was covered by a polyester strip and pressed flat by a glass slab. the top surface of all materials was cured according to the manufacturer’s cure times using an elipar trilight curing light unit (3m espe), with a mean light intensity of about 800 mw/cm2 influence of chemical degradation and abrasion on surface properties of nanorestorative materials braz j oral sci. 14(2):100-105 102102102102102 food/drink coca-cola® orange juice (minute maid®) artificial saliva main ingredients carbonated water, sugar, caramel color, phosphoric acid, natural flavors, caffeine water, orange juice, sugar, citric acid, natural flavor, antioxidant ascorbic acid calcium (0.1169 g of calcium hydroxide/l of deionized water); 0.9 mm of phosphorus and potassium (0.1225 g potassium phosphate monobasic/l of deionized water); 20 mm tris buffer (2.4280 g tris buffer/l of deionized water) p h 2.49 3.23 7.0 table 2. table 2. table 2. table 2. table 2. main characteristics of the storage solutions studied checked with a curing light meter (hilux dental curing light meter, benliglu dental inc., turkey). the surface of vitremer was protected with finishing gloss (3m espe). all specimens were maintained at 100% relative humidity and 37 °c for 24 h. then, the surfaces were wetpolished with on a sequence of waterproofed silicon carbide paper (600-, 1200-, and 2000-grit) and ultrasonically cleaned (ultrasonic cleaner, model usc1400, unique co, são paulo, sp, brazil) in distilled water for 10 minutes to remove polishing debris. the specimens were randomly distributed into 12 groups (n=10), according to material and storage medium: artificial saliva (control), orange juice (minute maid, coca-cola), and coca-cola® (table 2). before erosion testing, specimens were analyzed for surface roughness and knoop hardness. for surface roughness testing, the specimens were analyzed using a surfcorder se1700 instrument (kosaka corp, tokyo, japan), with cutoff length of 0.25 mm, at a tracing speed of 0.1 mm/s. the mean surface roughness values (ra, mm) of each specimen were obtained from three successive measurements of the center of each disk in different directions (total length analyzed of 3.750 mm)99999. then, hardness tests were carried out by a knoop indenter (shimatzu, tokyo, japan) and a 50 g load, 15 s dwell time. three readings were taken for each specimen, and the mean khn was calculated. erosion storage in acidic drinks all specimens were immersed individually in 4 ml of storage solutions: coca-cola® (ph 2.49), orange juice (ph 3.23) and artificial saliva (ph 7.00), for 30 days. the solutions were weekly changed and ph-tested by a portable ph meter (orion model 420a, analyzer, são paulo, sp, brazil). in all cases, the ph electrodes were calibrated immediately before use, by standard buffer solutions at ph 4.0 and 7.0. at the end of the storage period, the specimens were ultrasonically washed for 10 min. three-body abrasion test after erosion, the tooth-brushing test was performed in all specimens at 250 cycles/min, for 30,000 cycles with a 200 g load. colgate total dentifrice (colgate palmolive co., são bernardo do campo, são paulo, brazil) diluted in distilled water (1:2) was used as an abrasive third body. the specimens were ultrasonically washed for 10 min, then dried and evaluated for roughness and hardness. surface roughness readings were made on each specimen perpendicular to the brushing movement10. statistical analysis data were evaluated by the proc lab from sas in order to check the equality of variances and confirm a normal distribution. hardness and roughness data were submitted to repeated-measure three-way anova and tukey’s test with a significance level of 5%. results regarding roughness, there was significant interaction between the factors “materials” and “erosion/abrasion effect” (p<0.0001), and also between “storage solution” and “erosion effect” (p<0.0001). there was a significant difference among the three factors (p<0.0001). it was not observed any significant interaction between “materials” and “storage solution” (p=0.2372). the means and standard deviations of surface roughness of each material after erosive/ abrasive challenge are presented in table 3. regardless of the storage solution, both composites (filtek z350 and tph spectrum) presented similar roughness values (p>0.05) and significantly lower roughness values than glass ionomer cements, both before and after erosive challenge/abrasion. there was no significant difference in roughness values between ketac nano and vitremer, in all storage conditions (p>0.05). in addition, when different storage solutions were compared concerning each material after erosive challenge and abrasion, it was observed that there was no statistically significant difference in surface roughness for tph composite. however, the orange juice was more aggressive than the artificial saliva for filtek z350, ketac nano, and vitremer, increasing the surface roughness. in all cases, however, the cumulative effect of erosive challenge plus abrasion roughened the specimens of all materials, except the filtek z350 surface. table 4 shows the means and standard deviations of the knoop hardness of each material after erosive/abrasive challenge. there was significant interaction among the three factors (p=0.0062). there was no significant interaction between the factors “materials” and “storage solution” (p=0.6294), or between “materials” and “erosion/abrasion influence of chemical degradation and abrasion on surface properties of nanorestorative materials braz j oral sci. 14(2):100-105 103103103103103 effect” (p<0.0665). between “storage solution” and “erosion and abrasion effect” (p<0.0001), however, there was significant interaction. in addition, there was significant difference among materials studied (p<0.0001), among storage solutions (saliva/juice/coca-cola®; p<0.0177), and between erosion/abrasion effects (p<0.0001). before erosion/abrasion challenge, it was observed that both composites (filtek z350 and tph spectrum) presented similar or significantly higher values than the rmgics, which also presented similar values between them. regarding erosion/abrasion effects on each material’ surface, exposure to any storage solutions produced significantly lower hardness values for all materials tested. it was also observed that the storage solution influenced the materials: the acidic drinks (coca-cola® and orange juice) were more aggressive than artificial saliva to all materials. in addition, composites presented significantly higher hardness values than ionomeric materials after chemical/abrasion degradation. discussion wear of a dental material involves various processes, such as abrasion and erosion. on exposure to dental biofilm materials storage solutions saliva coca-cola juice initial tph *0.17 (0.04) ab *0.19 (0.03) ab *0.19 (0.04) ab filtek z350 0.12 (0.05) ab 0.13 (0.03) ab 0.12 (0.03) ab ketac nano *0.32 (0.11) aa *0.34 (0.10) aa *0.33 (0.10) aa vitremer *0.45 (0.15) aa *0.43 (0.13) aa *0.41 (0.09) aa after erosion tph 0.30 (0.04) ab 0.30 (0.14) ab 0.37 (0.06) ab and abrasion filtek z350 0.13 (0.02) bb 0.16 (0.02) abb 0.23 (0.08) ab ketac nano 0.71 (0.18) ba 0.59 (0.19) ba 1.14 (0.67) aa vitremer 0.63 (0.20) ba 0.74 (0.13) ba 1.58 (0.82) aa table 3. table 3. table 3. table 3. table 3. surface roughness mean (standard deviation in parentheses) (µm) of restorative materials submitted to erosion/abrasion challenge. capital letters indicate comparison among storage solutions (horizontal). lowercase letters demonstrate comparison among materials (vertical) within each storage solution and each erosion condition (before or after). asterisks represent a significant statistically difference between erosion effects (before and after). groups denoted by the same letter/symbol represent no significant difference (p > 0.05). materials storage solutions saliva coca-cola juice initial tph *82.24 (11.15) aa *79.9 (9.11) aa *80.43(10.97)aa filtek z350 *80.10 (8.2) aa *84.0(9.79)aa *83.5 (10.31) aa ketac nano *41.8 (5.24) ab *40.5 (5.56) ab *39.8 (6.73) ab vitremer *39.8 (5.53) ab *40.51 (6.82) ab *40.31 (8.51) ab after erosion tph 67.54 (10.42) aa 58.33 (4.88) ba 66.75 (4.38) ba and abrasion filtek z350 73.60 (9.80) aa 66.19 (7.99) ba 67.53 (6.60) ba ketac nano 38.78 (6.47) ab 29.20 (4.98) bb 29.20 (4.34) bb vitremer 31.55 (8.07) ab 25.95 (4.79) bb 25.74 (4.73) bb table 4. table 4. table 4. table 4. table 4. knoop hardness mean (standard deviation in parentheses) (khn) of restorative materials submitted to erosion/abrasion challenge. capital letters indicate comparison among storage solutions (horizontal). lowercase letters demonstrate comparison among materials (vertical) within each storage solution and each erosion condition (before or after). asterisks represent a significant statistically difference between erosion effects (before and after). groups denoted by the same letter/symbol represent no significant difference (p > 0.05). acids, food-simulating constituents and enzymes, resin-based restorative materials can be softened. consumption of certain beverages, such as coffee, tea, soft drinks, fruit juices, and alcoholic beverages, may affect the aesthetics and physical properties of composite resins11. usually after consuming beverages and foods, people brush their teeth to prevent caries development, exerting mechanical forces on enamel/restorative material surface12. the wear resistance of composites and rmgic is greatly influenced by the size and shape of the filler particles. according to de paula et al.3 (2011) and de fúcio et al.13 (2012), the greater the size of filler particles, the greater the amount of material lost. this study evaluated the cumulative effect of erosion and abrasion in composites and rmgic. higher roughness values were observed for rmgic than for composite resins before the erosion/abrasion challenge. the differences observed at baseline among materials regarding their means of surface roughness are mainly related to differences in their filler particle size, shape, volume, and distribution, and to their interaction with the organic matrix, allowing better polishing characteristics for the composites14. also, those results may be occurred through the handling of rmgics, since they are in a powder: liquid or paste: paste formulation influence of chemical degradation and abrasion on surface properties of nanorestorative materials braz j oral sci. 14(2):100-105 and air can be trapped in the material structure, resulting in surface bubbles and exposure of porosities after finishing/ polishing procedures. similar roughness values between the nanofilled and conventional materials were observed before erosion/abrasion challenge, for both the composite and rmgic groups. cavalcante et al.15 (2009) have demonstrated, however, that nanofilled composites present lower roughness values and better polishing characteristics than do hybrid composites, thanks to the presence of nanofillers. most likely, the resinous matrix of the materials used in this study was not totally removed by initial finishing/polishing procedures, leaving a matrix layer over the fillers. the erosive/abrasive challenge affected surface roughness of tph spectrum, but it was observed that there was no statistically significant difference in surface roughness for tph composite, concerning storage solutions. the ethoxylated version of the bis-gma (bis-ema) existing in the composition of tph spectrum matrixes probably contributed to their hydrolytic and biochemical stability, by the hydrophobicity of this monomer. yap et al.16 (2000) have also showed that the surface roughness of a bis-emabased composite is not affected by acidic beverages. bisema shows a decreased flexibility and increased hydrophobicity due to the elimination of the hydroxyl groups, when compared with composites formulated with bis-gma17. hence, the reduction in water uptake may be partially responsible for the chemical stability of composites that contain bis-ema. for the other materials (filtek z350, ketac nano, and vitremer), orange juice resulted in higher surface roughness values than did saliva and coca-cola®, indicating that solutions produced different effects in materials. there are two ways to quantify the acid content of a beverage include ph and total or titrable acidity. barbour and shellis18 (2007) have shown that fruit juices may also be potentially erosive, because of their high content of titrable acid. it was shown that, the higher the value of titrable acidity, the greater were the erosion effects. coca-cola® contains phosphoric acid that has low titratiability, and has been shown to contain almost no carboxylic acid. only filtek z350 specimens retained similar roughness values before and after erosion and abrasion challenge. the biomechanical degradation resistance of nanocomposite filtek z350 is basically related to its chemical composition. with regard to filler particles, this material is formulated by a combination of nanosized particles with the nanocluster formulations18. the higher filler loading with smaller particle size provides a reduction in the interstitial spacing, which effectively protects the softer matrix, reduces the incidence of filler exfoliation, and enhances the material’s overall resistance to abrasion19. when the nanocomposite undergoes toothbrush abrasion, only nanosized particles are plucked away, leaving the surfaces with defects smaller than light wavelength1. another parameter used in this study to measure the surface changes caused by erosion/abrasion was knoop hardness. according to the present results, both composites (filtek z350 and tph spectrum) presented higher hardness values than the rmgics before and after the erosion/abrasion challenge. the different constitution of organic matrices and higher filler loading, could explain the behavior of these materials. in addition, the initial characteristics of hardness are not affected by the presence of nanofillers in the different materials studied. after erosion/abrasion, all materials showed a significant reduction of hardness for all storage solutions. this reduction appears to have originated from hydrolysis20. according to sarkar21 (2000), corrosive wear begins with water absorption that diffuses internally through the resin matrix, filler interfaces, pores, and other defects, accelerated by the solution’s low ph. moreover, the rmgics showed a greater loss of hardness than the resin composites after erosion/ abrasion. thus, the chemical degradation rates of different materials depend on their hydrolytic stabilities, which are mainly related to the resin matrix. as the resin matrix of composites is known to absorb a small percentage of water22, composites were more degradation-resistant than hydrophilic materials, such as rmgics23. in addition, the storage solutions may promote dissolution near the glass particles, which could be the result of dissolution of the siliceous hydrogel layer of rmgics24. on the other hand, the acid could also attack the resin (to a lesser extent), softening the methacrylate-based polymers, possibly by leaching the comonomers, such as triethylene glycol dimethacrylate (tegdma), and thus decreasing the surface hardness of these materials4. this process is also emphasized by abrasion challenge 15. abrasion commonly takes place through a gradual removal of the softened organic material. this removal eventually leaves the fillers unsupported and susceptible to exfoliation25, which may have had a part in reducing the hardness of all the materials. it can be concluded that, according to the chemical composition of the material and storage medium, a synergistic effect can be observed by the increase in roughness for all materials used, except for filtek z350; hardness values decreased for all materials, regardless of whether they were nanofilled. rmgic is more susceptible to degradation than are composites, in both hardness and roughness surface parameters. this study showed that restorative materials might undergo degradation when exposed to acidic solutions and abrasive wear. however, an in vitro study presents some limitations, and thus in vivo studies should be performed to confirm these results in the oral environment. references 1. schmidt c, ilie n. the effect of aging on the mechanical properties of nanohybrid composites based on new monomer formulations. clin oral investig. 2013; 17: 251-7. 2. senawongse p, pongprueksa p. surface roughness of nanofill and nanohybrid resin composites after polishing and brushing. j esthet restor dent. 2007; 19: 265-73. 3. de paula ab, fucio sb, ambrosano gm, alonso rc, sardi jc, puppinrontani rm. biodegradation and abrasive wear of nano restorative materials. oper dent. 2011; 36: 670-7. influence of chemical degradation and abrasion on surface properties of nanorestorative materials 104104104104104 braz j oral sci. 14(2):100-105 105105105105105 4. asmussen e. softening of bis-gma-based polymers by ethanol and by organic acids of plaque. scand j dent res. 1984; 92: 257-61. 5. edwards m, creanor sl, foye rh, gilmour wh. buffering capacities of soft drinks: the potential influence on dental erosion. j oral rehabil. 1999; 26: 923-7. 6. jensdottir t, arnadottir ib, thorsdottir i, bardow a, gudmundsson k, theodors a, et al. relationship between dental erosion, soft drink consumption, and gastroesophageal reflux among icelanders. clin oral investig. 2004; 8: 91-6. 7. jensdottir t, bardow a, holbrook p. properties and modification of soft drinks in relation to their erosive potential in vitro. j dent. 2005; 33: 569-75. 8. francisconi lf, honório hm, rios d, magalhães ac, machado ma, buzalaf ma. effect of erosive ph cycling on different restorative materials and on enamel restored with these materials. oper dent. 2008; 33: 203-8. 9. lopes mb, saquy pc, moura sk, wang l, graciano fm, correr sobrinho l, et al. effect of different surface penetrating sealants on the roughness of a nanofiller composite resin. braz dent j. 2012; 23: 692-7. 10. carvalho fg, sampaio cs, fucio sb, carlo hl, correr-sobrinho l, puppin-rontani rm. effect of chemical and mechanical degradation on surface roughness of three glass ionomers and a nanofilled resin composite. oper dent. 2012; 37: 509-17. 11. seow ll, chong sy, lau mn, tiong sg, yew cc. effect of beverages and food source on wear resistance of composite resins. malaysian dent j. 2008; 29: 34-9. 12. addy m, hunter ml. can tooth brushing damage your health? effects on oral and dental tissues. int dent j. 2003; 53: 177-86. 13. de fúcio sb, de paula ab, de carvalho fg, feitosa vp, ambrosano gm, puppin-rontani rm. biomechanical degradation of the nano-filled resin-modified glass-ionomer surface. am j dent. 2012; 25: 315-20. 14. gladys s, van meerbeek b, braem m, lambrechts p, vanherle g. comparative physico-mechanical characterization of new hybrid restorative materials with conventional glass-ionomer and resin composite restorative materials. j dent res. 1997; 76: 883-94. 15. cavalcante lm, masouras k, watts dc, pimenta la, silikas n. effect of nanofillers’ size on surface properties after toothbrush abrasion. am j dent. 2009; 22: 60-4. 16. yap au, low js, ong lf. effect of food-simulating liquids on surface characteristics of composite and polyacid-modified composite restoratives. oper dent. 2000; 25: 170-6. 17. wu w, mckinney je. influence of chemicals on wear of dental composites. j dent res. 1982; 61: 1180-3. 18. barbour me, shellis rp. an investigation using atomic force microscopy nanoindentation of dental enamel demineralization as a function of undissociated acid concentration and differential buffer capacity. phys med biol. 2007; 52: 899-910. 19. turssi cp, ferracane jl, vogel k. filler features and their effects on wear and degree of conversion of particulate dental resin composites. biomaterials. 2005; 26: 4932-7. 20. cilli r, pereira jc, prakki a. properties of dental resins submitted to ph catalysed hydrolysis. j dent. 2012; 40: 1144-50. 21. sarkar nk. internal corrosion in dental composite wear. j biomed mater res. 2000; 53: 371-80. 22. ferracane jl. hygroscopic and hydrolytic effects in dental polymer networks. dent mater. 2006; 22: 211-22. 23. mohan m, shey z, vaidyanathan i, vaidyanathan tk, munisamy s, janal m. color changes of restorative materials exposed in vitro to cola beverage. pediatr dent. 2008; 30: 309-316. 24. turssi cp, hara at, serra mc, rodrigues al jr. effect of storage media upon the surface micromorphology of resin-based restorative materials. j oral rehabil. 2002; 29: 864-71. 25. condon jr, ferracane jl. in vitro wear of composite whit varied cure, filler level, and filler treatment. j dent res. 1997; 76: 1405-11. influence of chemical degradation and abrasion on surface properties of nanorestorative materials braz j oral sci. 14(2):100-105 429 too many requests error 429 too many requests too many requests guru meditation: xid: 37279077 varnish cache server 1http://dx.doi.org/10.20396/bjos.v18i0.8657264 volume 18 2019 e191636 original article 1 graduate program in clinical dentistry. universidade positivo. curitiba, paraná, brazil. 2 department of stomatology. universidade federal do paraná. curitiba, paraná, brazil. corresponding author: melissa rodrigues de araujo address: rua francisco juglair 749. mossungue. curitiba – paraná. brazil. zip code: 81200-230. phone number: +55 41 3360 4050 email: melissararaujo@ufpr.br https://orcid.org/0000-0002-2180-8223 conflict of interest statement: all authors declare that there are no conflicts of interest. received: april 24, 2019 accepted: august 23 2019 impact of oral medicine training on oral cancer-related knowledge among undergraduate dental students gisele reisdoerfer1, gilmar josé begnini1, flares baratto-filho1, juliana feltrin de souza2, carla castiglia gonzaga1, melissa rodrigues de araujo1,* aim: the aim of this study was to evaluate knowledge and attitudes regarding potentially malignant oral lesions and oral cancer among undergraduate dental students with or without training in the oral medicine. methods: a cross-sectional study was undertaken to assess all undergraduate dental student (1st to 5th year) in two private universities in the state of paraná, brazil. a structured questionnaire about knowledge regarding potentially malignant oral lesions, continuing education, and different approaches to diagnosis was administered to dental students with or without training in the oral medicine discipline between may and july 2015. descriptive statistics were obtained, and the data were analyzed using a chi-square test to compare knowledge in dental students. results: a total of 662 undergraduate dental students were invited to participate, and the response rate was 97.6% (n = 646). of the responders, 472 were female (73.1%), 168 were male (26.0%), and 6 did not declare what sex they were (0.9%). undergraduate dental students who had previously studied oral medicine tended to report that they always perform complete intraoral examination (76.3%) and identified alcohol (87%) and tobacco consumption (97%) and sun exposure (80%) as major risk factors for developing oral cancer. while students who had not yet studied oral medicine poorly identified these factors. students who had previously studied the discipline did not identify cheilitis actinica (26%) and erythroplakia (32%) as potentially malignant lesions. only, 32.6% of dental students participate in continuing education during the past year. conclusions: dental students trained in the oral medicine discipline exhibited satisfactory knowledge necessary for the prevention and early identification of potentially malignant oral lesions and oral cancer. continuing education during undergraduate and after academic training is very important. keywords: students, dental. knowledge. mouth neoplasms. oral medicine. https://orcid.org/0000-0002-2180-8223 2 reisdoerfer et al. introduction head and neck cancers are currently the seventh most commonly diagnosed types of cancers in the world, and two-thirds of cases occur in men. they account for 2.1% of cancers worldwide and 1.8% of deaths, 77% of which occur in less-developed regions1. tobacco and alcohol consumption are independently associated with increased risks of head and neck cancer. when combined these factors have synergistic effects, increasing the risk of developing a malignant oral neoplasm2. the likelihood of developing squamous cell carcinoma (scc), which accounts for 90% of all oral cancers3, can be reduced by 30% via smoking cessation2. other recognised risk factors include human papillomavirus (hpv), which is associated with oropharyngeal cancer onset, older age, immunosuppression, and low fruit and vegetable intake4. notably however, community knowledge pertaining to the risk factors and early signs and symptoms of oral cancer is unclear and evidently insufficient5. most cases of oral cancer are diagnosed in the advanced stages6. community awareness regarding the onset of the first signs and symptoms of oral cancer, as well as the education of health professionals, are directly associated with early diagnosis, reduction of morbidities related to treatment, and increased cure rates3. dentists play a fundamental role in the primary prevention of oral cancer by informing their patients about the importance of avoiding the major risk factors5, detecting early potentially malignant lesions7 (pmls), and performing routine systematic examination of the oral mucosa2. the average level of oral cancer knowledge possessed by dentists is insufficient, and the subject is reportedly not well understood by undergraduate students8. this suggests a need for more structured academic programs, which would result in improved professional skills and abilities9. the aim of the current study was to evaluate knowledge and attitudes about pmls and oral cancer in undergraduate dental students with and without training in oral medicine. materials and methods this cross-sectional study was based on a survey administered to undergraduate dental students in two dental schools. the research ethics committee of the university of positivo, brazil (protocol number 45245615.0.0000.0093), approved the study. the sample comprised 662 undergraduate dental students ranging from 1st year to 5th year derived the schools of dentistry of two institutions in paraná, brazil, the university of positivo (4th and 5th-year undergraduates) and união de ensino do sudoeste do paraná (unisep) (5th-year undergraduates) from may to july in 2015. the sample was divided into students who had not received information about oral cancer or completed an oral medicine course (i.e. 1st and 2nd-year students) and those who had completed an oral medicine course (i.e. 3rd, 4th and 5th-year students). as inclusion criteria students were required to be regularly enrolled in the dentistry course, and present at the time the survey was administered. participation was voluntary, and participants were informed that their responses would remain anonymous and confidential. all participants provided written informed consent. all data obtained 3 reisdoerfer et al. in the study were self-reported based-paper questionnaire. two researchers were involved, and a pilot study and calibration were conducted with 10 students in order to obtain the average time needed to answer the questionnaire and sign the informed consent term. the research tool was a questionnaire comprising 19 closed questions adapted from those developed by andrade et al.10 and angheben et al.11. the questions pertained to sociodemographic variables including sex, age and graduation year, and knowledge about pmls and the initial stages of malignancy, as well as knowledge about other aspects of oral cancer including clinical signs, common histological types, risk factors, age effects, commonly involved anatomical sites, and relevant diagnostic tools. risk factors for oral cancer were determined based on the literature12,13. knowledge about oral precursor lesions was evaluated with reference to classifications proposed by warnakulasuriya et al.14 in 2007 and the world health organization15 in 2017. statistical analysis was performed using the statistical package for social sciences® program (spss version 20.0; spss inc., chicago, il, usa). descriptive statistics were obtained and described, when appropriate, as absolute (n) and relative frequencies (%), and means and standard deviations (sd). chi-square tests were used to determine if there is a significant relationship between two nominal (categorical) variables. in the present study, the two variables were the questions included in the questionnaire and whether the undergraduate student had completed an oral medicine course. the frequency of each category for one nominal variable was compared across the categories of the second nominal variable. the data was presented in contingency tables (tables 2, 3 and 4) where the rows represent a category for one variable and columns represent a category for the other variable. chi-square tests were performed with a significance level of 5% (p < 0.05). results a total of 662 undergraduate dental students were invited to participate, and the response rate was 97.6% (n = 646). of the responders, 472 were female (73.1%), 168 were male (26.0%), and 6 did not declare what sex they were (0.9%). the mean age was 22.1 years (sd 4.9 years). a total of 290 students (44.9%) had studied oral medicine, whereas 356 (55.1%) had not (table 1). table 1. demographics of the study population (n= 646; paraná, brazil). variables n (%) sex female 472 (73.1) male 168 (26.0) no response 6 (0.9) age (mean sd) 22.1 ± 4.9 studied oral medicine yes 290 (44.9) no 356 (55.1) abbreviations: sd, standard deviation. 4 reisdoerfer et al. levels of knowledge pertaining to complete clinical examinations and palpation of the ganglionic chains in the head and neck region were used to evaluate perceptions and capacity for the detection of oral cancer signs and symptoms, and how the students would proceed if the diagnoses were confirmed (table 2). levels of knowledge about risk factors for oral cancer were evaluated, and the data are summarised in figure 1. there was a difference for the risk factors: alcohol, tobacco, sun exposure table 2. undergraduate dental students’ attitudes regarding oral cancer prevention (n = 646; paraná, brazil). questions oral medicine course p-valueyes n (%) no n (%) do you submit the patient to a complete intraoral examination during the first consultation? n=592 n 287 305 <0.001 always 219 (76.3) 137 (44.9) usually 40 (13.9) 44 (14.4) sometimes 22 (7.6) 27 (8.8) rarely 6 (2.09) 13 (4.2) never 0 (0.0) 84 (27.5) do you performs lymph node palpation during the first appointment? n= 587 n 286 301 <0.001 always 160 (55.9) 72 (23.9) usually 52 (18.1) 51 (16.9) sometimes 55 (19.2) 44 (14.6) rarely 13 (4.5) 29 (9.6) never 6 (2.1) 105 (34.8) what is your reason for not completing a complete physical examination? n=151  n 19 132   does not know how to perform a clinical examination and palpation. 5 (26.3) 64 (48.4) <0.001 does not know how to perform a clinical examination. 0 (0.0) 19 (14.3) <0.001 does not know how to palpate. 12 (63.1) 35 (26.5) <0.001 does not have enough time. 1 (5.2) 8 (6.1) <0.001 does not find it important. 1 (5.2) 6 (4.5) <0.001 have you previously detected a malignant lesion?  n=572 n 279 293 0.009yes 20 (7.1) 7 (2.3) no 259 (92.8) 286 (97.6) if you find oral lesions suspected to be malignant, how would you proceed?  n=582 n 281 301   perform diagnostic procedures. 18 (6.4) 7 (2.3) 0.023 refer to an oral medicine specialist. 125 (44.9) 90 (29.9) <0.001 refer to a physician. 8 (2.8) 25 (8.3) 0.006 refer to the faculty of dentistry. 23 (8.9) 12 (4.0) 0.037 refer to a specialized hospital. 15 (5.3) 24 (7.8) 0.246 wait for manifestation of the patient. 0 (0.0) 2 (0.7) 0.5 note: chi square test. significance level of 0.05. in each question, missing values correspond to non-response. 5 reisdoerfer et al. and hpv infection (p<0.001). levels of knowledge pertaining to pmls are represented in figure 2. leucoplakia was identified as a pml by 73% students who had previously studied oral medicine, while only 46% of students who had not yet studied oral medicine identified it. other pmls such as erythroplakia, lichen planus, and actinic cheilitis were only correctly identified by approximately 30% of students who had studied oral medicine and 10% of students who had not. figure 1. risk factors and non-risk factors for oral cancer identified by students who had completed training in oral medicine and those who were yet to undergo training in this discipline. hpv: human papillomavirus al co ho l to ba cc o su n e xp os ur e hp v fr uit s a nd ve ge ta ble s fa m ily hi sto ry of ca nc er po or fit tin g d en tu re s po or or al hy gie ne ba d t ee th ho t b ev er ag es an d f oo d risk factors oral medicine course non-risk factors no oral medicine course 120% 100% 80% 60% 40% 20% 0% 87% 55% 97% 86% 80% 31% 48% 32% 4% 3% 79% 71% 51% 31% 21% 34% 7% 15% 31% 18% figure 2. percentage of students who correctly identified potentially malignant lesions, according to whether they had completed the oral medicine course. leukoplakia erythroplakia candidiasis pemphigus lichen planus stomatitis actinic cheilitis melanocytic nevus do not know no oral medicine course oral medicine course 0% 10% 20% 30% 40% 50% 60% 70% 80% 46% 73% 11% 32% 10% 2% 14% 26% 13% 33% 9% 4% 4% 26% 16% 46% 33% 2% 6 reisdoerfer et al. the students’ knowledge about oral cancer is summarised in table 3. the majority of students were evidently aware that the prevalence of oral cancer is higher in people aged 40 years and over, and awareness of this was higher in students who had studied oral medicine. the majority of respondents correctly identified scc as the most common histological type of oral cancer, and again awareness of this was higher in students who had studied oral medicine. table 3. undergraduate dental students’ knowledge regarding oral cancer (n= 646; paraná, brazil). questions oral medicine course p-valueyes n (%) no n (%) what is the most common age group in which oral cancer is found? n=625 n 283 342 <0.001 <18 years 0 (0.0) 2 (0.6) 18–39 years 46 (16.3) 86 (25.1) >40 years 230 (81.3) 185 (54.1) do not know. 7 (2.5) 69 (20.2) what are the characteristics of suspected malignant lesions? n=633 n 287 346 painful and ulcerated. 69 (24.0) 144 (41.6) <0.001 painless with rigid edges. 215 (74.9) 179 (51.7) <0.001 fetid and purulent. 39 (13.6) 37 (10.7) 0.272 is scc the most frequently encountered type? n=605 n 279 326 <0.001 yes 207 (74.9) 193 (59.2) is the tongue the site that has the highest occurrence of scc? n=609 n 280 329 0.032 yes 109 (38.9) 100 (30.4) what are the initial clinical signs of oral cancer? n=633 n 287 346 abundant salivation. 7 (2.4) 18 (5.2) 0.023 painless ulcers. 151 (52.6) 105 (30.4) 0.001 hard nodules. 125 (43.5) 135 (39.0) 0.140 severe pain. 18 (6.3) 19 (5.5) 0.736 do not know. 21 (7.3) 89 (25.7) <0.001 what are the characteristics of lymph nodes of cervical metastases linked to oral cancer felt during palpation? n=628 n 284 344 hard, sore and mobile. 46 (16.2) 46 (13.4) 0.418 hard, pain-free, and with or without mobility. 195 (68.7) 148 (43.0) 0.064 soft, sore, and mobile. 8 (2.8) 20 (5.8) 0.006 soft, pain-free, and with or without mobility. 7 (2.5) 21 (6.1) 0.002 do not know. 30 (10.6) 116 (33.7) 0.037 note: abbreviations: scc, squamous cell carcinoma; chi square test. significance level of 0.05. in each question, missing values correspond to non-response. 7 reisdoerfer et al. the tongue was not correctly identified as the intraoral site with the highest incidence of scc by the majority in either group, indicating a lack of knowledge about the most frequent site of scc occurrence. with regard to the most common initial clinical observation in cases of oral cancer, most students who had studied oral medicine correctly reported that it was a painless ulcer. moreover, most students who had studied oral medicine correctly reported that pmls appeared as painless lesions with rigid margins. with regard to the clinical features of metastatic lymph nodes in cases of oral cancer, in both groups most students correctly reported that such lymph nodes tended to be hard, not associated with pain, and either mobile or fixed. the importance of dentists in the prevention and early diagnosis of oral cancer, and perceptions about levels of knowledge and continuing education are summarised in table 4. discussion the early detection of oral cancer is positively associated with a better prognosis, therefore it is imperative that dental students be sufficiently trained and competent in the diagnosis and multidisciplinary treatment of the disease4. a dentist’s ability to identify pmls and initial malignant lesions depends largely on the knowledge and skills they acquire during their undergraduate course16,17. in the present study undergraduate dental students’ levels of knowledge about oral cancer were considered reasonable, which is consistent with previous reports18,19. the comparative appeal of table 4. perception of knowledge, dentists’ importance, and continuing education (n= 646; paraná, brazil). questions oral medicine course p-valueyes n (%) no n (%) what is the importance of dentists in the prevention and early diagnosis of oral cancer? n=641 n 287 354 0.007 high 283 (98.6) 331 (93.5) average 3 (1.0) 14 (4.0) low 1 (0.3) 1 (0.3) do not know. 0 (0.0) 8 (2.3) what do you perceive your level of oral cancer knowledge to be? n=620 n 275 345 <0.001 great 3 (1.1) 3 (0.9) good 84 (30.5) 38 (11.0) regular 153 (55.6) 163 (47.2) insufficient 35 (12.7) 141 (40.9) when did you last participate in continuing education? n=282 n 282 n/a <0.001 during the past year 92 (32.6) n/a from 2–5 years 64 (22.7) n/a up to 5 years 1 (0.4) n/a never 75 (26.6) n/a do not know 50 (17.7) n/a abbreviations: n/a, not applicable. chi square test. significance level of 0.05. in each question, missing values correspond to non-response. 8 reisdoerfer et al. areas of dentistry related to aesthetics, implant dentistry and prosthetic rehabilitation, which may be more profitable for clinicians, may lessen students’ interest in the diagnosis of oral lesions. if this is the case, a new mentality with regard to the value of preventive oral health in the context of disease needs to be fostered. tobacco use and alcohol abuse have been identified as prominent aetiologies for oral cancer4,17-19,20. both groups in the present study were aware that these two parameters are positive risk factors, but students who had studied oral medicine were more certain of these factors, which is concordant with the results of previous studies21,22. hpv infection, sun exposure, and a previous history of oral cancer are evidently prominent risk factors for oral carcinogenesis18. notably however, awareness of hpv infection4,17 and low fruit and vegetable intake4,17-20 as risk factors remains poor, even among professionals23. the two groups surveyed in the present study did not correctly identify hpv infection or low fruit and vegetable intake as risk factors. sun exposure was also generally poorly identified, but it was identified more often by students who had studied oral medicine, which is concordant with previous studies17,19. the consumption of hot food and beverages and spicy foods, obesity, poor oral hygiene, poor-fitting dentures, a family history of cancer, heredity, and chronic infections were not considered risk factors by the majority of students in both groups in the present study, although they have been implicated as aetiological factors for scc4,18,19. thus, a substantial lack of knowledge of scientifically proven risk factors and non-risk factors was observed, as it has been in previous studies17,18. the correct identification of pmls is fundamental with regard to malignant oral neoplasms22. in the present study the vast majority of students who had studied oral medicine identified leucoplakia as a pml. in contrast, other equally important pmls such as erythroplakia, lichen planus, and actinic cheilitis tended not to be identified in either group, which is concordant with a study reported by alami et al.12 in 2013 and another reported by jayasinghe et al.22 in 2016. in the current study students who had studied oral medicine tended to consider painless and rigid-edged oral lesions ‘suspicious lesions’, and this has also been observed in similar previous studies12. conversely, students who had not studied oral medicine often wrongly described suspicious oral lesions as painful and ulcerated, which are generally only characteristic of oral lesions when they reach an advanced stage. inaccurate knowledge about clinical aspects of oral lesions among practitioners was also reported by razavi et al.23 in 2013. in the present study just over half the undergraduates who had not studied oral medicine and most of those who had correctly identified scc as the most common histological type of oral cancer, and those observations are concordant with other studies that have evaluated dental students22. caucasians, men and individuals aged > 40 years have the highest incidence of scc24,25. in the present study students who had studied oral medicine tended to correctly identify the aged > 40 years age group as being associated with a higher prevalence of scc, but it was less frequently identified in the other group. the tongue and floor of the mouth are the most common sites of oral cancer lesions4. in the present study the tongue tended not to be cited as a common site of scc in 9 reisdoerfer et al. either of the two groups surveyed. notably, when professionals were evaluated in two previous studies this common site was identified by the majority in one23, but only a small percentage in another20. this suggests that there may be considerable variation in awareness of this parameter, even among fully qualified professionals. in the current study students who had studied oral medicine tended to correctly report that oral cancer is initially asymptomatic. however, neither group satisfactorily identified initial signs. the signs and symptoms considered initial warnings for established oral cancer include white and red spots, localised hardening, bleeding, ulceration, necrosis, and painless fixed or mobile lymph nodes4. visual inspection is the most effective method for identifying oral lesions4. the majority of students in the current study reported that they performed a complete intraoral examination during the first appointment, and similar findings have been reported in previous studies18,20,26. lymph node palpation was reportedly not performed frequently however, with students asserting that they did not know how to perform such a procedure. in the present study most students reported that they would prefer to refer a patient to a specialist for detailed diagnostic procedures, when needed. it has previously been suggested that there is a lack of adequate knowledge pertaining to oral cancer among dental professionals8, and that a protocol for the early detection of oral cancer is necessary27. prior to the administration of the questionnaire the respondents were specifically instructed not to guess at the correct answer to a question if they did not know it, simply for the purposes of returning a ‘complete’ questionnaire. accordingly, in some cases questions were left blank by the respondents. these absent responses did not detract from the validity of the study results at all, and on the contrary they added to it by way of eliminating any potentially confounding effects resulting from ‘lucky guesses’. it is also notable with regard to validity that only 0.93% of potential respondents abstained from the questionnaire. the study was limited by the fact that only students from two institutions were evaluated. a study conducted at a national level will assess students’ knowledge about oral cancer more representatively. the present study demonstrates a need to update undergraduate curricula in dentistry using methods that engage students’ interest in preventive measures pertaining to oral lesions. it is likely that the appeal of components of modern dentistry such as aesthetic procedures, implantology and prosthetic treatments—which are associated with comparatively high financial yields—divert the interest of dentistry students away from preventive dentistry. in conclusion, the early detection of risk factors for oral cancer via the use of effective diagnostic tools by dentists has become imperative. in the present study a higher rate of correct responses pertaining to oral cancer-related knowledge was observed in students who had studied oral medicine. there was a general lack of satisfactory identification of certain risk factors for head and neck cancer, including hpv, chronic sun exposure and nutritional deficiency. thus, it is of fundamental importance that knowledge about oral cancer be well developed in undergraduate dental students, and that continuing education be part of the ongoing dental curricula, even after the conclusion of undergraduate training. 10 reisdoerfer et al. references 1. ferlay j, soerjomataram i, dikshit r, eser s, mathers c, rebelo m, et al. cancer incidence and mortality worldwide: sources, methods and major patterns in globocan 2012. int j cancer. 2015 mar 1;136(5):e359-86. doi: 10.1002/ijc.29210. 2. perdomo 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[oral cancer: assessment of knowledge and conduct of dentists in primary health care]. rev bras odontol. 2014;71:42-47. portuguese. 11. angheben pf, salum fg, cherubini k, figueiredo maz. [oral cancer knowledge background of dental school students of the pontifical catholic university of rio grande do sul]. rev odontol bras central. 2013;21(60):33-40. portuguese. 12. alami ay, el sabbagh rf, hamdan a. knowledge of oral cancer among recently graduated medical and dental professionals in amman, jordan. j dent educ. 2013 oct;77(10):1356-64. 13. american cancer society. oral cancer; 2007. available from: www.cancer.org. 14. warnakulasuriya s, johnson nw, van der waal i. nomenclature and classification of potentially malignant disorders of the oral mucosa. j oral pathol med. 2007 nov;36(10):575-80. 15. el-naggar ak, chan jkc, grandis jr, takata t, slootweg pj. who classification of head and neck tumours. in: who/iarc classification of tumours. 4th ed. lyon, france: iarc; 2017. p. 347. 16. cerero-lapiedra r, esparza-gómez gc, casado-de la cruz l, domínguez-gordillo aa, corral-linaza c, seoane-romero jm. ability of dental students in spain to identify potentially malignant disorders and oral cancer. j dent educ. 2015 aug;79(8):959-64. 17. frola mi, barrios r. knowledge and attitudes about oral cancer among dental students after bologna plan implementation. j cancer educ. 2017 sep;32(3):634-9. doi: 10.1007/s13187-016-0990-9. 11 reisdoerfer et al. 18. kujan o, alzoghaibi i, azzeghaiby s, altamimi ma, tarakji b, hanouneh s, et al. knowledge and attitudes of saudi dental undergraduates on oral cancer. j cancer educ. 2014 dec;29(4):735-8. doi: 10.1007/s13187-014-0647-5. 19. rahman b, hawas n, rahman mm, rabah af, al kawas s. assessing dental students’ knowledge of oral cancer in the united arab emirates. int dent j. 2013 apr;63(2):80-4. doi: 10.1111/idj.12017. 20. kebabcioğlu ö, pekiner fn. assessing oral cancer awareness among dentists. j cancer educ. 2018 oct;33(5):1020-1026. doi: 10.1007/s13187-017-1199-2. 21. silva sr, juliano y, novo nf, weinfeld i. comparative study of knowledge about oral cancer. einstein (sao paulo). 2016 jul-sep;14(3):338-345. doi: 10.1590/s1679-45082016ao3729. 22. jayasinghe rd, sherminie lpg, amarasinghe h, sitheeque mam. level of awareness of oral cancer and oral potentially malignant disorders among medical and dental undergraduates. ceylon med j. 2016 jun;61(2):77-9. doi: 10.4038/cmj.v61i2.8289. 23. razavi sm, zolfaghari b, foroohandeh m, doost me, tahani b. dentists, knowledge, attitude, and practice regarding oral cancer in iran. j cancer educ. 2013 jun;28(2):335-41. doi: 10.1007/s13187-013-0460-6. 24. garrote lf, herrero r, reyes rm, vaccarella s, anta jl, ferbeye l, et al. risk factors for cancer of the oral cavity and oro-pharynx in cuba. br j cancer. 2001 jul 6;85(1):46-54. 25. udeabor se, rana m, wegener g, gellrich nc, eckardt am. squamous cell carcinoma of the oral cavity and the oropharynx in patients less than 40 years of age: a 20-year analysis. head neck oncol. 2012 may 30;4:28. doi: 10.1186/1758-3284-4-28. 26. soares trc, carvalho mea, pinto lss, falcão ca, matos ftc, santos tc. oral cancer knowledge and awareness among dental students. braz j oral sci. 2014 mar;13(1)28-33. doi: 10.1590/1677-3225v13n1a06. 27. lehew ch, epstein jb, kaste lm, choi yk. assessing oral cancer early detection: clarifying dentists’ practices. j public health dent. 2010 spring;70(2):93-100. doi: 10.1111/j.1752-7325.2009.00148.x. 429 too many requests error 429 too many requests too many requests guru meditation: xid: 63618219 varnish cache server oral sciences n3 original article braz j oral sci. october | december 2014 volume 13, number 4 efficiency of occlusal splints on police officers with tmd paulo henrique ferreira caria1, reinaldo josé a. faria2, claudia regina sgobbi de faria3, carla scanavini croci1, rubens negrão filho3 1universidade estadual de campinas unicamp, piracicaba dental school, department of morphology, piracicaba, sp, brazil 2universidade do oeste paulista unoeste , dental school, department of health sciences, presidente prudente, sp, brazil 3universidade estadual paulista unesp, school of science and technology, department of physiotherapy, presidente prudente, sp, brazil correspondence to: paulo henrique ferreira caria faculdade de odontologia de piracicaba, unicamp caixa postal 52 cep: 13414-903, piracicaba, sp, brasil phone: +55 19 2106520 e-mail: phcaria@fop.unicamp.br abstract aim: to evaluate of the effect of two different occlusal splints on police officers with tmd. methods: thirty police officers were selected based on research diagnostic criteria for tmd and on clinical exams. volunteers (ten per group) were distributed according to occlusal splints: group a – control, group b michigan occlusal splint (mos), and group c – planas appliance (pa). experimental groups were analyzed using a visual analog pain scale (vaps), subject to a clinical evaluation of temporomandibular joint. bilateral surface electromyographic activities of anterior and posterior temporal, masseter and suprahyoid muscles were analyzed at rest and during clenching, before and after four weeks using the occlusal splints. results: the left and right temporal and masseter muscles sensitivity decreased after using both splints. pain symptoms increased for group a (control) and decreased for group c. conclusions: planas appliance was more efficient on pain reduction than the michigan occlusal splint. keywords: temporomandibular joint disorders; stress, physiological; electromyography, masticatory muscles, occlusal splints. introduction temporomandibular joint disorder, commonly referred to as tmd, is a compound of disorders characterized by orofacial pain, chewing dysfunction, or a combination of both. common symptoms of facial pain include actual pain, headache, joint discomfort or dysfunction, earaches, tinnitus, dizziness, pain in the upper and lower back, or neck pain. stress is one of the most important factors causing temporomandibular disorder (tmd) and professional activity is a significant source of stress1. police officers are exposed to high levels of stress and therefore susceptible to chronic diseases and disorders like tmd2. the incidence of signs and symptoms associated with tmd includes 6% to 93% of the population, but only 3.6% to 7% requires treatment3. some studies report high incidence of signs and symptoms associated with tmd in war veterans4-5, but few reports evaluated incidence of tmd in police officers6. stressful situations may intensify tmd symptoms, which are frequently treated using occlusal splints. in most cases, this therapy balances the masticatory muscles’ activity and reduces bruxism and tmd symptoms7. surface electromyography is a non-invasive exam that enables the evaluation of muscle activity in people with tmd as well as the analysis of the occlusal splints’ effect on masticatory muscles8-11. braz j oral sci. 13(4):292-296 received for publication: september 01, 2014 accepted: december 05, 2014 293293293293293 based on the connection between stressful activity and tmd, this study suggests that the effect of two different occlusal splints on the temporal, masseter and suprahyoid muscles of police officers with tmd be evaluated based on clinical symptoms and electromyographic activity. material and methods research diagnostic criteria for temporomandibular disorders (rdc/tmd). rdc/tmd is a dual-axis system developed by dworkin and leresche in order to define the subtypes of tmd and to standardize their diagnosis. axis i is a physical measure that outlines the clinical characteristics of tmds, separating them into three categories: myofascial pain disorder (mpd); disc displacements (dd); and degenerative joint conditions (djd). axis ii assesses psychosocial factors commonly seen in patients with tmd. volunteers a total of 905 police officers in the state of são paulo completed the rdc/tmd axis ii12 after being approved by the human research ethics committee of unicamp in brazil. 256 volunteers were selected and subject to rdc axis i, including the ones who showed myogenic or mixed tmd, tmj pain for at least three months and joint tenderness on palpation on at least one side. thirty volunteers (15 women and 15 men) with mean age of 29 years and an indicative diagnosis of myogenic or mixed tmd were selected. each volunteer filled out a visual analog pain scale (vaps) questionnaire to evaluate muscle pain sensibility. volunteers were randomly divided into three different groups according to occlusal splints as follows: group a – control group b – michigan occlusal splint (mos); group c – planas appliance (pa). after four weeks of treatment, volunteers completed again the rdc axis i and axis ii12 in the morning, supervised by the same examiner. occlusal splints occlusal splits were used during four weeks while the volunteers were sleeping. 1. mos – a 4.5mm thick rigid acrylic resin plate which was checked every 7 days. all necessary adjustments were performed on it. 2. pa –two (maxillary and mandibular) bilateral rigid acrylic resin plates parallel to campers’ plane. volunteers were instructed to avoid contact with each other during treatment to keep differences between occlusal splints and control groups confidential. instrumentation electromyography (emg) records were performed on temporal, masseter and suprahyoid muscles before and after using occlusal splints for four weeks. volunteers remained seated, facing forward, feet on the floor, legs at a 90º angle, hands resting on thighs and frankfort plane parallel to the floor. occlusal splints were removed during emg documentation. all emg signals were obtained by lynx data acquisition system (mcs1000-v2) with 16 channels, 12-bit resolution of dynamic range, butterworth filter, 500 hz band pass, 20 hz high-pass filter and unit gain of 2000 times. software aqdados 5, lynx was used with a sampling frequency of 1000 hz and active single differential surface electrodes with entrance impedance of 10 g, cmrr (common mode rejection ratio) of 80 db, impedance of 1012 w / % pf, and unit gain of 20 times. a reference electrode was positioned on the sternum bone of the volunteers. surface electrodes were bilaterally placed on the masseter, temporal and suprahyoid muscles, according to muscle palpation and their function. electrodes were attached to the skin (previously cleaned with alcohol) using double-sided adhesive tape over the center of the muscle and parallel to the muscle fibers, placing the silver bars perpendicular to their direction to maximize signal capture and minimize noise interference. emg analysis was performed in three different moments: firstfirstfirstfirstfirst: 5 seconds of muscle resting. secondsecondsecondsecondsecond : maximum voluntary contraction (mvc) bilaterally biting two pieces of elastic cord (lemgruber® no. 201), each 2.5cm long. thirdthirdthirdthirdthird: maximum voluntary contraction of the suprahyoid muscle at maximal mouth opening (mmo). signals were stabilized using rms (root mean square) of three mvc measurements. each emg exam was performed after 3 minutes of rest to physiologically recover and to avoid muscle fatigue. the statistical analyses were: tukey’s test (5% level of significance) and student’s t-test for emg data; shapiro-wilk tests (5% level of significance) for normality; mann-whitney and student t-test for data from the clinical evaluation and rdc. results palpation in group c (pa) showed statistically significant reduction (p<0.05) to sensitivity on the left side of the posterior fibers of the temporal muscle and on both sides of the medial fibers, as well as on both sides of the superficial fibers of the masseter muscle (p<0.10) (table 1). control group did not show statistically significant differences. vaps data showed a statistically significant difference in the means of groups a and c compared to group b, indicating an increase of pain symptoms in group a (control group) and symptom alleviation in group c (pa) (figure 3). rms values of emg exam (figures 2 and 3) showed association with the clinical aspects. emg signal analysis showed a statistically significant difference (p<0.05) only for the right masseter muscle during maximal clenching effort in groups b and c. discussion most epidemiological studies about the association efficiency of occlusal splints on police officers with tmd braz j oral sci. 13(4):292-296 294294294294294 right temporal posterior fibers initial 12.6 17.6 16.3 final 15.95 16.05 14.5 medial fibers initial 12.85 13.8 19.85* final 18.9 14.5 13.1* anterior fibers initial 13.95 15 17.55 final 18.1 12.8 15.6 left temporal posterior fibers initial 11.55 14 20.95* final 17.2 14 15.3* medial fibers initial 11.55 14 20.95* final 17.2 14 15.3* anterior fibers initial 14.95 15.1 16.45 final 18.65 14.05 13.8 right masseter initial 13.7 14.9 17.9** final 17.5 14.9 14.1** left masseter initial 15.4 13.9 17.2** final 18.5 14.7 13.3** right suprahyoid initial 16.5 16.5 13.5 final 16.5 15 15 left suprahyoid initial 15.45 15.45 15.6 final 16 16 14.5 group a group b group c * 5% level of significance **10% level of significance table 1. table 1. table 1. table 1. table 1. means of palpation value for right and left temporal, masseter and suprahyoid muscles recorded before and after splint appliance. control group (group a); michigan occlusal splint (group b); planas appliance (group c). fig. 1. means of rms (mv) value for right and left temporal, masseter and suprahyoid muscles recorded at maximal mouth opening. control group (group a); michigan occlusal splint (group b); planas appliance (group c). fig. 2. means of rms (mv) value for right and left temporal, masseter and suprahyoid muscles recorded at maximal clenching effort. control group (group a); michigan occlusal splint (group b); planas appliance (group c). efficiency of occlusal splints on police officers with tmd braz j oral sci. 13(4):292-296 295295295295295 fig. 3. means of palpation value for right and left masseter and suprahyoid muscles recorded before and after the use of splint appliance. control group (group a); michigan occlusal splint (group b); planas appliance (group c). between occlusal factors and tmd have used samples representing general populations13 in addition, studies about tmd often evaluate heterogeneous samples in patients with different kinds and degrees of tmd that compromise repeatability and data comparison and limit clinical application14. police work is highly stressful, since it is one of the few occupations where employees are constantly asked to face physical dangers and put their lives on the line any time15 .hence, we decided to use this specific population, divided in homogenous subgroups, matched by age and gender, and evaluated by standardized criteria and blind experimental designs to reduce observer bias. occlusal splints have been the preferred modalities in the management of myofascial temporomandibular disorders (tmds)16. some authors advocate the use of these splints as a first step for therapeutic treatment in order to minimize the neuromuscular unbalance since it is the prevalent factor in this kind of dysfunction16-17. the use of occlusal splints seems to relieve tmd symptoms18-19. a significant number of clinical works evaluated the therapeutic effect of occlusal splints on muscular hyperactivity20-22. emg is a valuable resource that evaluates muscle activity able to recognize signs related to tmd, thus helping establish differential diagnosis23-24. differences in the clinical protocols used to establish tmd diagnoses may be responsible for the high result variability between studies. the introduction of research diagnostic criteria for temporomandibular disorders (rdc/ tmd) in 1992 12 was expected to increase the level of consistency between studies by the use of standardized diagnostic criteria. rdc/tmd provides criteria for a dualaxis diagnosis, i.e. the patient receives physical diagnosis (axis i) along with a psychosocial assessment (axis ii). clinical exam showed statistically significant reduction to bilateral palpation sensitivity in group c’s temporal and masseter muscles, which means a reduction of muscle hyperactivity, since occlusal mechanoreceptor splints cancel neuromuscular activity, favor blood flow and remove unnecessary substances resulting from excessive muscle contraction 25. in group a any statistically significant difference was noticed because volunteers did not receive any kind of tmd treatment and so the symptoms remained. the results obtained by vaps showed significant muscle pain reduction in group c. this confirms the clinical results, in which the use of occlusal splints relieves tmd symptoms19 and reinforces the theory about blood flow increase and unnecessary substance removal inside the muscle 25. in addition, occlusal splints change the maxillomandibular relation repositioning articular structures while balancing the muscular action3. mos had already been tested as a tmd treatment26, whereas pa had no scientific report as such. in our research, pa also caused muscle stretching by increasing vertical dimension of occlusion and eliminating occlusal interferences, which reduced periodontal proprioception causing muscle relaxation. emg activity of the masseter and temporal muscles in patients with tmd using occlusal appliances is a controversial subject. some authors report reduction of emg activity in these muscles 27 while others stated that there are no statistically significant differences in the muscle activity11,28. even after following isek recommendations, personal dissimilarities can develop different outcomes and interfere on emg results, as well as different muscle structures29. in this study, emg values for the right masseter muscle in groups b and c showed statistically significant differences during maximal clenching effort. the contraction of the masseter muscle during mouth opening occurs as a response to a protective reflex in patients who have muscle pain while opening their mouths25. the temporal muscle is a mandibular positioner, and since occlusal splints interrupt the proprioceptive information of the mechanoreceptors located in the periodontal ligament, which balances muscular activity, they provide a better mandible positioning and therefore loosen that muscle30. emg activity of muscles studied in these circumstances did not show significant changes. however, there was a clinically significant reduction in the palpation sensitivity of temporal and masseter muscles in group c, indicating relief for tmd symptoms, as stated by other authors11. emg activity and pain sensation are probably distinct due to longer latency of emg responses, when compared to pain reduction, so a prolonged time in the use of oral efficiency of occlusal splints on police officers with tmd braz j oral sci. 13(4):292-296 appliances could improve emg results, relieving muscle pain. on the other hand, pa keeps teeth without occlusal contact and its prolonged use can lead to occlusal alterations17. in conclusion, planas appliance was more efficient in pain reduction than the michigan occlusal splint; the type of occlusal splints chosen did influence on pain reduction; occlusal splints showed advanced clinical outcomes. acknowledgements this research was supported by capes. the authors wish to thank the force unit commander of the 18th district presidente prudente police department. references 1. slade gd, diatchenko l, bhalang k, sigurdsson a, fillingim rb, belfer i et al. influence of psychological factors on risk of temporomandibular disorders. j dent res. 2007; 86: 1120-5. 2. collins pa, gibbs ac. stress in police officers: a study of the origins, prevalence and severity of stress-related symptoms within a county police force. occup med (lond). 2003; 53: 256-64. 3. leresche l, mancl l, sherman jj, gandara b, dworkin sf. changes in temporomandibular pain and other symptoms across the menstrual cycle. pain. 2003; 106: 253-61. 4. uhac i, kovac z, muhvic-urek m, kovacevic d, franciskovic t, 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(part 4): effects of occlusal splints and other treatment modalities. int j comput dent. 2013; 16: 225-39. 10. hugger s, schindler h, kordass b, hugger a. clinical relevance of surface emg of the masticatory muscles. (part 1): resting activity, maximal and submaximal voluntary contraction, symmetry of emg activity. int j comput dent. 2012; 15: 297-314. 11. landulpho ab, e silva wa, e silva fa, vitti m. electromyographic evaluation of masseter and anterior temporalis muscles in patients with temporomandibular disorders following interocclusal appliance treatment. j oral rehabil. 2004; 31: 95-8. 12. dworkin sf, leresche l. research diagnostic criteria for temporomandibular disorders: review, criteria, examinations and specifications, critique. j craniomandib disord. 1992; 6: 301-55. 13. alanen p. occlusion and temporomandibular disorders (tmd): still unsolved question? j dent res. 2002; 81: 518-9. 14. suvinen ti, kemppainen p. review of clinical emg studies related to muscle and occlusal factors in healthy and tmd subjects. j oral rehabil. 2007; 34: 631-44. 15. territo l, vetter hj. stress and police personnel. j police sci admin. 1981; 9: 195-208. 16. zhang f, wang x, dong j, zhang j, lü y. effect of occlusal splints for the management of patients with myofascial pain: a randomized, controlled, double-blind study. chin med j. 2013; 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20: 473-82. 22. dimitroulis g, gremillion h, dolwick m, walter j. temporomandibular disorders. 2. non-surgical treatment. aust dent j. 1995; 40: 372-6. 23. visser a, naeije m, hansson tl. the temporal/masseter co-contraction: an electromyographic and clinical evaluation of short-term stabilization splint therapy in myogenous cmd patients. j oral rehabil. 1995; 22: 387-9. 24. kawazoe y, kotani h, hamada t, yamada s. effect of occlusal splints on the electromyographic activies of masseter muscles during maximum clenching in patients with myofascial pain-dysfunction syndrome j prosthet dent. 1980; 43: 578-80. 25. lund jp, donga r, widmer cg, stohler cs. the pain-adaptation model: a discussion of the relationship between chronic musculoskeletal pain and motor activity. can j physiol pharmacol. 1991; 69: 683-94. 26. proff p, richter ej, blens t, fanghanel j, hutzen d, kordass b et al. a michigan-type occlusal splint with spring-loaded mandibular protrusion functionality for treatment of anterior disk dislocation with reduction. ann anat. 2007; 189: 362-6. 27. ciancaglini r, gherlone ef, radaelli g. unilateral temporomandibular disorder and asymmetry of occlusal contacts. j prosthet dent. 2003; 89: 180-5. 28. sato s, nasu f, motegi k. analysis of post-treatment electromyographs in patients with non-reducing disc displacement of the temporomandibular joint. j oral rehabil. 2002; 29: 1126-30. 29. vianna-lara ms, caria ph, tosello dde o, lara f, amorim mm. electromyographic activity of masseter and temporal muscles with different facial types. angle orthod. 2009; 79: 515-20. 30. vitti m. electromyographic analysis of the musculus temporal in basic movements of the jaw. electromyography. 1971; 3(suppl 4): 389. 296296296296296efficiency of occlusal splints on police officers with tmd braz j oral sci. 13(4):292-296 oral sciences n3 original article braz j oral sci. october | december 2014 volume 13, number 4 effect of different enamel conditionings on the bond strength of glass ionomer cement and ceramic brackets dauro douglas oliveira1, thomas gerard bradley2, matheus melo pithon3, mariele cristina garcia pantuzo1, emílio akaki1 , virendra dhuru4 1pontifícia universidade católica de minas gerais puc minas, department of dentistry, area of orthodontics, belo horizonte, mg, brazil 2marquette university, school of dentistry, department of developmental sciences, area of orthodontics, milwaukee, wi, usa 3universidade estadual do sudoeste da bahia uesb, school of dentistry, department of healthy, area of orthodontics, jequié, ba, brazil 4marquette university, school of dentistry, department of general dental sciences, area of dental materials, milwaukee, wi, usa correspondence to: dauro douglas oliveira pontifícia universidade católica de minas gerais puc minas avenida. dom josé gaspar, 500 prédio 46, sala 101. belo horizonte, mg,brasil e-mail: dauro.bhe@gmail.com abstract aim: to evaluate the effect of different enamel conditionings on the shear bond strength (sbs) and bond failure patterns of a resin modified glass ionomer cement (rmgic) used to bond ceramic brackets. methods: 105 human premolars extracted for orthodontic reasons were divided into 7 groups according to the enamel surface treatments: conditioners (35% phosphoric acid and 10% polyacrylic acid), type of adhesive (transbond® and fuji ortho lc capsule®), washing time and drying of the surface. results: a significant p<0.05 was observed among the shear bond strength values obtained with seven different types of enamel preparation prior to bonding. the group that had the tooth surface conditioned with polyacrylic acid and enamel surface slightly dried before bracket placement showed no significant difference (p>0.05) to the group that had brackets bonded with composite resin. the two groups that had enamel prepared with polyacrylic acid and brackets bonded with gic on a wet surface showed significantly lower shear bond strength than the control group (p<0.05). conclusions: moisture contamination decreased sbs, but not enough to preclude the use of rmgic as an alternative to composite resin (cr) for direct bonding of ceramic brackets. keywords: glass ionomer cements; shear strength; dental bonding; orthodontic brackets. introduction the introduction of direct bonding was a major development in orthodontics and bonding brackets with composite resin (cr) has been considered the standard of care for many years1-2. however, bonding on an acid-etched tooth surface presents some disadvantages, such as enamel loss during conditioning and adhesive removal3. furthermore, contamination with oral fluids increases bond failure and the risk of developing caries during orthodontic therapy may be higher because the fixed appliances make an adequate oral hygiene more difficult4. thus, biofilm levels surrounding the brackets are increased and demineralization often occurs in these areas5. an adhesive system that provides sufficient retention to orthodontic brackets and tubes, decreasing the failure rate due to moisture contamination and preventing decalcification would be ideal. the fluoride releasing properties of glass ionomer cement (gic) made it an interesting material for orthodontic use. since gic presented similar retention received for publication: september 21, 2014 accepted: november 25, 2014 braz j oral sci. 13(4):270-275 properties as zinc phosphate and zinc polycarboxylate cements6 and also release/uptake fluoride7, it became the material of choice for cementation of orthodontic bands8. gic was also tested as a direct bonding agent in orthodontics. in the early studies, conventional gic has not shown the same effectiveness as cr to bond orthodontic brackets, but resin modified glass ionomer cements (rmgic) have shown good potential to bond metallic brackets9. there are few studies reporting the use of rmgic for bonding ceramic brackets, especially when it is used after different enamel conditionings. this investigation evaluated the effect of different enamel conditioning procedures on the shear bond strength (sbs) and bond failure pattern (bfp) of a rmgic used for bonding ceramic brackets. material and methods the bonding agents used in this study were a cr (transbond xt, 3m/unitek, monrovia, ca, usa) and a rmgic (fuji ortho lc capsule, gc america, alsip, il, usa). two types of enamel conditioners were evaluated in this study, 35% phosphoric acid (pha) (transbond xt etching gel, 3m/unitek,) and 10% polyacrylic acid (paa) (fuji ortho conditioner, gc america). the ceramic premolar brackets used in this experiment were standard edgewise 0.022 x 0.028-in slot with mechanical retention on their bases (clarity, 3m/unitek). fig. 1. mounting fixture used to standardize sample preparation. a. brackets bonded to the acrylic cup rim; b. premolar suspended in the center of the copper end-cap; c. premolar with occluso-labial surface above end-cap rim; d. mounted premolar with labial surface exposed for bracket placement. preparation of specimens one-hundred and five human premolars, extracted for orthodontic purposes, with intact labial surface and caries or restorations-free were collected and randomly assigned to 7 groups containing 15 teeth per group. the roots were sectioned and removed about 2 mm apically from the cementoenamel junction. a mounting fixture was fabricated to guarantee parallelism of the teeth labial surfaces and the base of the specimen holders. two 0.022x0.028-in slot, standard edgewise stainless steel brackets (3m/unitek,) were bonded to the rim of the acrylic cup (figure 1a). a copper end cap that had half of its volume filled with self-curing acrylic was placed on the acrylic holder. a piece of 0.021x0.025-in stainless steel wire was bent to create a 2.5 mm step up in the wire. a ceramic bracket identical to those used for the experiment was tied to the wire by an elastic ligature, so that it was positioned at the center of the crown, both occluso-gingivally and mesio-distally (figure 1b) and fixed to the tooth with sticky wax (figure 1c). labial surfaces of the mounted premolars were cleaned and dried (figure 1d). the following groups of enamel surface treatments were performed: (1) ct (control):(1) ct (control):(1) ct (control):(1) ct (control):(1) ct (control): enamel was etched for 30 s with 35% pha, rinsed for 10 s with tap water and dried with oilfree compressed air for 10 s. a thin coat of primer was placed and light-cured for 10 s. (2) pa-w:(2) pa-w:(2) pa-w:(2) pa-w:(2) pa-w: 10% paa for 20 s and rinsed with tap water effect of different enamel conditionings on the bond strength of glass ionomer cement and ceramic brackets271271271271271 braz j oral sci. 13(4):270-275 fig. 2. sbs apparatus. a: specimen secured in a vise clamp onto the load cell; b: frontal close up of fixture for shear loading. c. shear loading blade perpendicular to tooth-bracket interface. for 10 s. the bonding surfaces of the teeth were wiped with a water-moistened cotton roll. (3) pa-d-w: (3) pa-d-w: (3) pa-d-w: (3) pa-d-w: (3) pa-d-w: 10% paa for 20 s, rinsed for 10 s with tap water and dried with oil-free compressed air for 10 s. the surfaces were wiped with a water-moistened cotton roll. (4) pa-ld:(4) pa-ld:(4) pa-ld:(4) pa-ld:(4) pa-ld: 10% paa for 20 s and rinsed with tap water for 10 s. the surfaces were lightly dried with oil-free compressed air for 1 s. (5) ph-w:(5) ph-w:(5) ph-w:(5) ph-w:(5) ph-w: 35% pha for 15 s and rinsed with tap water for 10 s. the surfaces were wiped with a water-moistened cotton roll. (6) ph-d-w:(6) ph-d-w:(6) ph-d-w:(6) ph-d-w:(6) ph-d-w: 35% pha for 15 s, rinsed for 10 s with tap water and dried with oil-free compressed air for 10 s. the surfaces were wiped with a water-moistened cotton roll. (7) ph-ld:(7) ph-ld:(7) ph-ld:(7) ph-ld:(7) ph-ld: 35% pha for 15 s and rinsed for 10 s with tap water. the surfaces were lightly dried with oil-free compressed air for 1 s. the brackets in group ct were bonded with cr while rmgic was used in all other groups. enamel conditioning in the pa-w group followed strictly the manufacturers’ recommendations. the tested rmgic was pre-proportioned in capsules and mixed by a mixing machine (gc america) to avoid possible inaccuracy of the powder/liquid ratio and to assure thorough mixing of the components. the bracket was placed at the center of the labial surface of the prepared tooth with sufficient pressure to obtain the thinnest layer of cement between the bracket and the tooth. after the removal of any excess of cement surrounding the bracket base, the cement was cured with a light unit (demetron/kerr, danbury, ct, usa) for 10 s on each margin of the bracket at a maximum distance of 1 mm. the light intensity was tested using the built-in radiometer to assure that the correct wavelength (above 450 nm) was used. all samples were stored in distilled water at room temperature for 24 h until the test was carried out. sbs test a universal testing machine (instron corp., canton, ma, usa) was equipped with a 50 kg load cell and used for all sbs tests. the test specimens were secured in a vise clamped onto the load cell platter and the load was applied to the bracket base (figure 2) at a crosshead speed of 0.5 mm/min. bond failure analysis bracket bases and corresponding enamel surfaces were evaluated with an optical stereomicroscope at 10x magnification. the percentage of adhesive that remained on both surfaces was quantified according to the adhesive remnant index (ari)10: (0) no adhesive on the enamel; (1) less than 50% of the adhesive remained on the tooth; (2) more than 50% of the adhesive was left on the enamel; (3) 100% of the adhesive remained on the tooth. results one-way anova revealed a significant difference (p<0.05) among the sbs obtained with the different enamel conditionings. the mean sbs values and standard deviations (sd) for the different treatment groups were compared with tukey multiple comparison test (table 1). it showed no statistically significant differences (p>0.05) between ct and all groups bonded with rmgic that had enamel etched with pha and the pa-ld group. however, the two groups that had enamel prepared with paa and brackets bonded with rmgic on a wet surface showed significantly lower sbs than ct (p<0.05). the group that followed the manufacturers’ recommendations (pa-w) showed a significantly lower sbs (p<0.05) than 4 out of the other 5 experimental groups (pald, ph-w, ph-d-w, and ph-ld). the only exception was the group that used a different moisturizing technique and that also had the tooth surface conditioned with paa (pa-dw). even though the results of pa-w and pa-d-w were not significantly different (p>0.05), pa-d-w showed higher sbs mean values. in consequence, pa-d-w was not significantly different (p>0.05) from pa-ld and ph-d-w. the results also indicated that there was a statistically significant difference (p<0.05) in the bfp among the groups. effect of different enamel conditionings on the bond strength of glass ionomer cement and ceramic brackets 272272272272272 braz j oral sci. 13(4):270-275 ct group presented bond failures basically in the bracket base-cement interface, while most of the failures with rmgic were within the cement resulting in some adhesive being left both on the enamel and bracket base. discussion comparison among the paa treated groups moisture contamination resulted in a decrease in sbs of rmgic, which is in agreement with rix et al.11 and cheng et al.12. additionally, cacciafesta et al.13 reported higher sbs when enamel was kept moist prior to bonding mechanically retained ceramic brackets with rmgic. they suggested that moisture contamination appeared to increase the retention of these brackets. even though the design of the studies was similar, there were some differences that could explain why their conclusions could not be supported by the findings of the present study. while in this experiment human pre-molars were used, cacciafesta and his coworkers13 utilized bovine incisors. they ground the labial surfaces with carbide paper. therefore, the surface was less irregular, which may have contributed for the higher sbs in their study. jobalia et al.14 reported that moisture was necessary for an ideal performance of fuji ortho lc for bonding of orthodontic brackets. however, they used metal brackets and performed a tensile test instead of a shear test. in this study, two different methods to keep the tooth surface moist were use. the first followed strictly the manufacturer’s instructions and consisted in using a moist cotton roll right after rinsing the enamel conditioner. the second method had an additional step that consisted of drying the enamel for 10 s after rinsing the acid and then applying moisture with a cotton roll. the inclusion of the later group intended to mimic an accidental moisture contamination that is commonly observed in an orthodontic clinic. although there was no statistically significant difference (p>0.05) between the results of the groups that used these two methods (pa-w, pa-d-w), the first method (pa-w) resulted in lower and more variable sbs values. the variation coefficient of the group that followed the manufacturer’s recommendations was 36%, which is considered relatively high. this may be due to the fact that moisture contamination is a difficult variable to control and could become even more unpredictable if tooth surfaces were not dried before the final moisturizing step. the third group treated with paa had the enamel slightly dried before bracket placement (pa-ld). it produced sbs values that were higher than both groups bonded with moist enamel (pa-w, and pa-d-w). however, statistically significant difference (p<0.05) was only from the group that never had the enamel dried after surface conditioning (paw). lippitz et al.15 reported similar results, suggesting that with a slightly dried surface, rmgic had sbs equivalent to those observed with cr. however, they used a different storage medium (synthetic saliva), a different rmgic and metal brackets. comparison within the pha treated groups once again, moisture contamination reduced the sbs of rmgic, but not to a statistically significant level (p>0.05). there were no statistically significant differences (p>0.05) among the three groups with enamel treated with pha for 15 s. valente et al.16 reported similar results. however, wiltshire17 and shanilec et al.18 reported a significant increase in the retention force of metal brackets bonded with rmgic after enamel was etched with 35% pha for 30 s. comparison between paa and pha groups the group treated with paa that had enamel surface slightly dried presented results that were not statistically different (p>0.05) from any of the groups treated with pha. the pa-dw group did not have statistically significant different (p>0.05) results from its counterpart treated with pha. in addition, the group that followed the manufacturers’ instructions (pa-w) had statistically different (p<0.05) results than any of the pha groups. overall, there was a tendency to get higher and less variable bond failure loads when pha was used. as reported earlier, pha creates treatment* mean standard variation m i n i m u m m a x i m u m deviation coefficient **(mpa) (mpa) (%) (mpa) (mpa) c p 17.9 c 3.4 19 12.2 23.4 pa-w 10.8a 3.9 36 4.6 15.6 pa-d-w 12.6ab 3.0 24 7.5 16.7 pa-ld 16.2bc 3.0 20 12.4 24.2 ph –w 16.4c 3.4 24 9.0 24.3 ph-d-w 15.8bc 2.9 19 11.7 20.6 ph-ld 18.0c 3.4 19 11.6 24.3 table 1.table 1.table 1.table 1.table 1. mean shear bond strength of ceramic brackets bonded with two different adhesives to enamel surface subjected to different treatments. * cp: phosphoric acid, rinse, dry, primer, composite; pa-w: polyacrylic acid, rinse, wet cotton roll, gic; pa-d-w: polyacrylic acid, rinse, dry, wet cotton roll, gic; pa-ld: polyacrylic acid, rinse, lightly dry, gic; ph-w: phosphoric acid, rinse, wet cotton roll, gic; ph-d-w: phosphoric acid, rinse, dry, wet cotton roll, gic; ph-ld: phosphoric acid, rinse, lightly dry, gic. ** sbs with the same superscript letters are not significantly different. effect of different enamel conditionings on the bond strength of glass ionomer cement and ceramic brackets273273273273273 braz j oral sci. 13(4):270-275 micropores on the enamel surface that are filled by resin tags increasing the retention of adhesives used in dentistry. if the resin component of the rmgic is able to penetrate the porosity created by the acid, mechanical interlock between enamel and the adhesive may happen. therefore, in addition to the well-reported chemical adhesion of gics to tooth structure, mechanical adhesion would be obtained resulting in increased sbs. this phenomenon is probably not observed when paa is applied, because it removes debris and smear layer without creating so many and so deep pores as with pha. comparison of all experimental groups to ct when rmgic was used with pha and the enamel was slightly dried (ph-ld), sbs results were very similar to the ct. the paa groups bonded with moist enamel (pa-w, and pa-d-w) showed statistically significant lower (p<0.05) sbs values than ct. many other studies using not only ceramic, but also metal brackets, have observed similar characteristics1920. in contrast, cacciafesta et al.13 demonstrated that rmgic had higher sbs than cr when mechanically retained ceramic brackets were used, but not at a statistically significant level. the other experimental groups (pa-ld, ph-w, and ph-dw) presented lower, but not a statistically different (p>0.05) sbs than cp. bond failure site comparisons an evaluation of the fracture site revealed a statistically significant difference (p<0.05) among the 7 groups. ct had all the adhesive remaining on the tooth surface on 100% of the specimens. rmgic groups with enamel conditioned with paa showed a tendency to have cohesive failure resulting in most of the cement remaining on the bracket base. about 67% of the samples in this subgroup had less than 50% of the adhesive remaining on the enamel. other 24% of the specimens had more than 50% of the adhesive remaining on the teeth, and less than 1% had all the adhesive remaining either on the tooth or on the bracket base, which is characteristic of an adhesive failure. rmgic groups with tooth prepared with pha presented a performance closer to the one observed with cr, which is in agreement with other studies21. this could reinforce the assumption that the percentage of resin material added to the rmgic could be able to mechanically adhere to tooth structure roughened by the acid conditioning. about 45% of the failures occurred on the bracket-adhesive interface resulting that all adhesive was left on the enamel. other 37% of the specimens of these three groups had more than 50% of adhesive remaining on tooth surface, and finally only 18% of the specimens that used pha had less than 50% of the adhesive remaining on the enamel. these results were similar to those reported by jou et al.22, haydar et al.23 and summers et al.19 who showed ari (adhesive remnant index) scores similar to those in the present study for ceramic brackets bonded with rmgic and completely different results for cr failure. while the present study found that 100% of the specimens had all the adhesive remaining on the enamel, they found that 80% of the specimens had the entire adhesive attached to the enamel and the other 20% had less than 50% of the adhesive on the tooth. the difference may be explained by the fact that the studies used ceramic brackets with a different retention mechanism. it may be concluded that: enamel conditioning with 35% pha resulted in higher and more consistent sbs values than when paa was used; moisture contamination significantly decreased sbs of rmgic on enamel conditioned with paa and all brackets bonded with composite resin presented adhesive failure at the bracket-adhesive interface, while the rmgic groups showed a more variable pattern with mostly cohesive failures. references 1. da rocha jm, gravina ma, da silva campos mj, quintao cc, elias cn, vitral rw. shear bond resistance and enamel surface comparison after the bonding and debonding of ceramic and metallic brackets. dent press j orthod. 2014; 19: 77-85. 2. costa ar, vedovello-filho m, correr ab, vedovello sa, puppin-rontani rm, ogliari fa, et al. bonding orthodontics brackets to enamel using experimental composites with an iodonium salt. eur j orthod. 2014; 36: 297-302. 3. ryf s, flury s, palaniappan s, lussi a, van meerbeek b, zimmerli b. enamel loss and adhesive remnants following bracket removal and various clean-up procedures in vitro. eur j orthod. 2012; 34: 25-32. 4. pithon mm, batista vo, d’el rey nc. effect of different methods for decontaminating tooth enamel after contact with blood before bonding orthodontic buttons. j oral maxillofac surg. 2012; 70: 2035-40. 5. da silva fidalgo tk, pithon mm, do santos rl, de alencar na, abrahao ac, maia lc. influence of topical fluoride application on mechanical properties of orthodontic bonding materials under ph cycling. angle orthod. 2012; 82: 1071-7. 6. kocadereli i, ciger s. retention of orthodontic bands with three different cements. j clin pediatr dent. 1995; 19: 127-30. 7. santos rl, pithon mm, fernandes ab, carvalho fg, cavalcanti al, vaitsman ds. fluoride release/uptake from different orthodontic adhesives: a 30-month longitudinal study. braz dent j. 2013; 24: 410-4. 8. pithon mm, dos santos rl, de oliveira mv, ruellas ac, romano fl. metallic brackets bonded with resin-reinforced glass ionomer cements under different enamel conditions. angle orthod. 2006; 76: 700-4. 9. komori a, ishikawa h. evaluation of a resin-reinforced glass ionomer cement for use as an orthodontic bonding agent. angle orthod. 1997; 67: 189-95. 10. artun j, bergland s. clinical trials with crystal growth conditioning as an alternative to acid-etch enamel pretreatment. am j orthod. 1984; 85: 333-40. 11. rix d, foley tf, mamandras a. comparison of bond strength of three adhesives: composite resin, hybrid gic, and glass-filled gic. am j orthod dentofacial orthop. 2001; 119: 36-42. 12. cheng hy, chen ch, li cl, tsai hh, chou th, wang wn. bond strength of orthodontic light-cured resin-modified glass ionomer cement. eur j orthod. 2011; 33: 180-4. 13. cacciafesta v, jost-brinkmann pg, sussenberger u, miethke rr. effects of saliva and water contamination on the enamel shear bond strength of a light-cured glass ionomer cement. am j orthod dentofacial orthop. 1998; 113: 402-7. 14. jobalia sb, valente rm, de rijk wg, begole ea, evans ca. bond strength of visible light-cured glass ionomer orthodontic cement. am j orthod dentofacial orthop. 1997; 112: 205-8. 15. lippitz sj, staley rn, jakobsen jr. in vitro study of 24-hour and 30-day shear bond strengths of three resin-glass ionomer cements used to bond orthodontic brackets. am j orthod dentofacial orthop. 1998; 113: 620-4. effect of different enamel conditionings on the bond strength of glass ionomer cement and ceramic brackets 274274274274274 braz j oral sci. 13(4):270-275 16. valente rm, de rijk wg, drummond jl, evans ca. etching conditions for resin-modified glass ionomer cement for orthodontic brackets. am j orthod dentofacial orthop. 2002; 121: 516-20. 17. wiltshire wa. shear bond strengths of a glass ionomer for direct bonding in orthodontics. am j orthod dentofacial orthop. 1994; 106: 127-30. 18. shanilec cf, staley rn, jakobsen jr. orthodontic bracket bond strengths with hybrid-glass ionomer cement. j dent res. 1998; 77: 298. 19. summers a, kao e, gilmore j, gunel e, ngan p. comparison of bond strength between a conventional resin adhesive and a resin-modified glass ionomer adhesive: an in vitro and in vivo study. am j orthod dentofacial orthop. 2004; 126: 200-6; quiz 254-5. 20. godoy-bezerra j, vieira s, oliveira jh, lara f. shear bond strength of resin-modified glass ionomer cement with saliva present and different enamel pretreatments. angle orthod. 2006; 76: 470-4. 21. baysal a, uysal t. resin-modified glass ionomer cements for bonding orthodontic retainers. eur j orthod. 2010; 32: 254-8. 22. jou gl, leung rl, white sn, zernik jh. bonding ceramic brackets with light-cured glass ionomer cements. j clin orthod. 1995; 29: 184-7. 23. haydar b, sarikaya s, cehreli zc. comparison of shear bond strength of three bonding agents with metal and ceramic brackets. angle orthod. 1999; 69: 457-62. effect of different enamel conditionings on the bond strength of glass ionomer cement and ceramic brackets275275275275275 braz j oral sci. 13(4):270-275 oral sciences n3 original article braz j oral sci. january | march 2014 volume 13, number 1 how are children and adolescents cleaning their orthodontic appliances? a cross-sectional study in private schools mabel miluska suca salas1, rita regina souza lamas1, tatiana pereira cenci1, rafael guerra lund1 1 universidade federal de pelotas ufpel, school of dentistry, department of restorative dentistry, pelotas, rs, brasil correspondence to: rafael guerra lund faculdade de odontologia da universidade federal de pelotas rua gonçalves chaves 457 cep: 96015568 pelotas, rs, brazil phone: +55 53 3222 6690 e-mail: rafael.lund@gmail.com received for publication: february 11, 2014 accepted: march 12, 2014 abstract aim: to determine the prevalence and the hygiene methods used by 6-16-year-old private school children and adolescents to clean removable orthodontic appliances (roa) in the city of pelotas, rs, brazil. methods: a cross-sectional study was conducted in private schools located in the urban area after the school tutors signed an informed consent form. questionnaires were applied to children using removable orthodontic appliances. data regarding hygiene methods of orthodontic appliances were collected. descriptive analysis was performed. pearson’s chi-square test and linear trend with a confidence level of 95% were used for analytical analysis. results: children using roa in private schools were 7.6%. the most frequent hygiene method used was mechanical cleansing with toothbrush and toothpaste (85.6%). daily cleansing was the frequency most reported by children (51.6%). conclusions: use of roa in students from private schools was low and the most used hygiene method was brushing with toothpaste. keywords: removable orthodontic appliances; hygiene; disinfection; child; adolescent. introduction the demand1 and use2 of orthodontic appliances have increased in recent years. studies have demonstrated that 16-28% children use removable orthodontic appliances (roa)2-4. the main reported reasons are aesthetics5 and occlusal dysfunctions6. roa are indicated in occlusal correction and for dental retention after fixed treatment. studies have reported the increase of microorganisms such as streptoccoccus mutans7, candida albicans8, actinomyces, among others after the placement of roa in children9-10. the use of roa increases the risk of developing dental caries7, gingivitis11 and halitosis8. studies have demonstrated higher incidence of initial caries lesions white spots associated with orthodontic appliances in children12. the presence of caries or gingival problems could compromise oral health of children and interfere indirectly in the social interaction of children13. proper hygiene can control the presence of bacterial biofilms, preventing oral problems14. studies have investigated several hygiene methods for acrylic appliances15-20, with contradictory results. some investigations showed that the combination of mechanical and chemical methods may reduce significantly the amount of bacteria compared to other methods16-17. other studies found a significant reduction of oral microbiota with mechanical cleaning alone16,21, chlorhexidine solution22 or effervescent tablets23-24. however, it is not clear whether children are braz j oral sci. 13(1):34-36 gisele higa texto digitado http://dx.doi.org/10.1590/1677-3225v13n1a07 35 actually using the most efficient methods for cleaning of orthodontic appliances and reduction of bacterial load. the aim of the study was to determine the most used hygiene methods to clean roa by 6-16-years-old children and adolescents from private schools in the city of pelotas, rs, brazil. material and methods the present investigation was an observational crosssectional study. the population was children and adolescents from primary and secondary private schools in the urban area of pelotas, a city in southern brazil. the number of private schools in the city was obtained by a list provided by the education department of pelotas. day care centers and special schools were excluded. prior to the beginning of the research, the study was approved by the institutional ethics committee (registration number 216/2011) of the federal university of pelotas. the tutors responsible for each school signed an informed consent form for participation of children in the study. children formally enrolled in private schools of the city that were users of roa and had ability to answer the questionnaires were included. the teachers applied questionnaires in the classrooms. the collected data included the total number of children per class, age of each child, number of students using removable orthodontic appliances, cleaning methods and frequency of cleaning of the removable appliances. the statistical analysis was descriptive. pearson’s chi-square with linear trend was used between children with removable appliances and age. the confidence level was 95%. results all private schools of the city (n=21) participated in this study. a total of 6,706 students were invited and enrolled as participants. the response rate was 100%. at the time of the survey, out of the 6,706 students, 507 were using removable orthodontic appliances, which represent a prevalence of 7.6%. a significant trend toward older children using roa compared with younger children was observed (table 1). most children (98.0%) with roa cleaned their own appliances. the most commonly used method was mechanical cleaning with toothbrush and regular toothpaste (90.0%), followed by corega tabs® effervescent tablets (5.0%). children cleaned their appliances at least once a day (52.0%). twenty one percent of children cleaned twice a day, 15.0% 3 times a day and 12.0% rarely cleaned the orthodontic appliances. discussion children from private schools using roa were 7.6%. this result revealed a lower prevalence of roa users than in a recent study performed in germany4 (2012), in which 16% of children aged 11-14 years old used roa. chesnutt et al.2 (2006) reported a frequency of 28% and 18% in 12 and 15 years old children, respectively, in england. this difference can be explained by the fact that germany4 and england2 include orthodontic treatment as part of their health insurances. this makes the percentage of children with orthodontic treatment higher than in other countries. for instance, germany is the country with the highest frequency of roa treatments in europe4. in brazil, some private and public insurances include dental orthodontic treatments in dental health polices, but in a limited way. our sample was composed of students from private schools. other studies observed that children with higher socioeconomic level were more frequently users of orthodontic appliances than those with lower socioeconomic level2-3. in our study, 7.6% of children used roa. on the other hand, there is a significant trend toward older children using removable orthodontic appliances. a trend could be observed regarding fixed and removable appliances. studies usually found that younger children used roa more frequently than older children2,25 and an opposite situation could be observed when children used fixed appliances2-3,25. younger children usually used interceptive orthodontics as retainers and space maintainers in high frequency (93%), older children often used roa as dental space retainers and braces3. this could explain our findings. most of the older children used fixed appliances and at the time of evaluation were wearing a dental retainer after the fixed orthodontic treatment. this study showed that most of the children clean their roa using a common toothbrush and toothpaste. the findings of the present study regarding the cleaning method used by children are consistent with those reported by eichenauer et al.26 (2011), who stated that dentists reported to frequently indicate brushing with toothpaste the removable orthodontic appliances, followed by effervescent tablets (corega tabs®). studies have investigated the hygiene methods used by users of roa or acrylic removable prosthetic appliances. these how are children and adolescents cleaning their orthodontic appliances? a cross-sectional study in private schools braz j oral sci. 13(1):34-36 orthodontic treatment n o yes total n (%) 6,199 507 6 703(11.3) 7(1.4) 7 624(10.1) 11 2.1) 8 573 (9.2) 17 (3.4) 9 605(9.8) 19 (3.8) 10 565 (9.1) 53 (10.5) 11 630(10.2) 71 (14.0) 12 522 (8.4) 27(5.3) 13 482(7.8) 46 (9.1) 14 486 (7.8) 80 (15.8) 15 494 (8.0) 74 (14.6) 16 515(8.3) 102(20.1) p value* <0.001 age (years) table 1.table 1.table 1.table 1.table 1. frequency of orthodontic treatments with removable appliances in 6-16 years old children and adolescents from private schools in the city of pelotas, brazil, 2011. *linear trend 36 studies found that acrylic appliance users also frequently clean their appliances with toothbrush and toothpaste17, toothbrush and water21 or with effervescent tablets24. the cleaning method using only mechanical removal, without any chemical disinfectant agent, was reported as insufficient to remove the biofilm from the acrylic and does not adequately decrease the bacterial load16,18,20. an adjuvant chemical cleaning is required16,18-20. proper hygiene can control the presence of bacterial biofilm on tooth surfaces7. data on the most appropriate method for cleaning orthodontic appliances are scarce, and there is not a standardized indication regarding the most efficient and effective technique or material to be used19. oral hygiene instructions for oral care and cleaning of the roa acrylic baseplate by orthodontic patients are important keys to the promotion of health. healthy behaviors such as control of dental biofilm can prevent gingival inflammation27 and tooth caries7. some limitations must to be pointed out. this study had a crossover design, which is limited to the time when data were collected. furthermore, a specific group of the population was investigated, private school children and adolescents, which probably showed a different prevalence of roa in child population. despite the limitations, our findings may contribute to the knowledge of the methods used by children to clean their roa and may add to future strategies for preventing biofilm control in orthodontic patients. in conclusion, the prevalence of roa used by students of primary and secondary private schools in the city pelotas was low. most of the children brush their appliances with regular toothpaste. acknowledgments the authors are grateful to the state secretary of education, the municipal secretary of education and the direction of private schools, which allowed the study to be performed. references 1. ucuncu n, ertugay e. the use of the index of orthodontic treatment need (iotn) in a school population and referred population. j orthod. 2001; 28: 45-52. 2. chestnutt ig, burden dj, steele jg, pitts nb, nuttall nm, morris aj. the orthodontic condition of children in the united kingdom, 2003. br dent j. 2006; 200: 609-12. 3. pietila t, pietila i, widstrom e, varrela j, alanen p. extent and provision of orthodontic 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amsel r, et al. comparison of two popular methods for removal and killing of bacteria from dentures. j can dent assoc. 1991; 57: 937-9. 25. kavaliauskiene a, sidlauskas a, zaborskis a. demographic and social inequalities in need for orthodontic treatment among schoolchildren in lithuania. medicina (kaunas). 2010; 46: 767-73. 26. eichenauer j, serbesis c, ruf s. cleaning removable orthodontic appliances: a survey. j orofac orthop. 2011; 72: 389-95. 27. dersot jm. [plaque control, a key element of successful orthodontics]. orthod fr. 2010; 81: 33-9. how are children and adolescents cleaning their orthodontic appliances? a cross-sectional study in private schools braz j oral sci. 13(1):34-36 oral sciences n3 original article braz j oral sci. january | march 2014 volume 13, number 1 in vitro antimicrobial photoinactivation with methylene blue in different microorganisms bruna paloma de oliveira1, carla cabral dos santos accioly lins2, fátima alves diniz3, liliane lima melo3,célia maria machado barbosa de castro4 1universidade federal de pernambuco ufpe, school of dentistry, department of prosthodontics and oral facial surgery, recife, pe, brasil 2universidade federal de pernambuco ufpe, department of anatomy, recife, pe, brasil 3universidade federal de pernambuco ufpe, laboratory of immunopathology keiso asami lika, recife, pe, brasil 4universidade federal de pernambuco ufpe, department of tropical medicine, recife, pe, brasil corrrespondence to: bruna paloma de oliveira rua mamanguape, 518, apto 2701 boa viagem cep: 51020250 recife, pe, brasil phone: +55 81 92853170 e-mail: bruna_paloma@msn.com abstract aim: to evaluate the in vitro antimicrobial effects of photodynamic therapy (pdt). methods: the microorganism indicators were: candida albicans, pseudomonas aeruginosa,enterococcus faecalis and staphylococcus aureus. a microbial pool was prepared (108 cells/ml), from which aliquots were transferred to culture plates for carrying out the pdt using methylene blue (50 µm) and low-power laser (660 nm, 100 mw and 9 j).the effect of methylene blue alone, low power laser and the absence of treatments were evaluated. then, aliquots of 1 µl were plated in a media culture, the number of colony forming units (cfu/ml) was obtained and the data submitted to the f test (anova) with tamhane’s comparisons. results:the laser radiation in the presence of methylene blue was able to eliminate 74.90% of c. albicans, 72.41% of p. aeruginosa, 96.44% of e. faecalis and 95.42% of s. aureus, thus statistically significant differences were found among the groups (p<0.001). conclusions: pdt was effective in reducing the number of viable cells in the studiedmicroorganisms, especially e. faecalis and s. aureus. keywords: endodontics; enterococcus faecalis; methylene blue; microbiology; photodynamic therapy. introduction microorganisms play an essential role in the development and maintenance of pathologies that affect the pulp and the periapical region1, and their removal during the biomechanical preparation is crucial to the success of endodontic treatment2. pseudomonas aeruginosa and staphylococcus aureus have been commonly associated with persistent infections 3-5.special attention has been given to enterococcus faecalis, a tough gram-positive bacterium, which has a much higher incidence in cases of endodontic treatment failure6-7. this microorganism has the property of survival in extremely alkaline ph environments, with scarce nutrients, invading and growing within dentinal tubules, colonizing the root canal and reinfecting the root-filled teeth8-9. fungi are occasionally found in the primary infection of root canals, but occur more frequently in teeth obturated with lesions refractory to treatment. candida albicans is the most prevalent fungal species, a microorganism that has affinity for dentin and is resistant to some intracanal medications, for example, those based on calcium hydroxide10. braz j oral sci. 13(1):53-57 received for publication: january 28, 2014 accepted: march 20, 2014 gisele higa texto digitado http://dx.doi.org/10.1590/1677-3225v13n1a11 54 braz j oral sci. 13(1):53-57 the antibacterial activity of low power lasers associated with a photosensitizer has been studied as adjuvant treatment together with conventional endodontic therapy 11. photodynamic therapy (pdt) assumes that the interaction of light with an appropriate wavelength, when associated with a nontoxic photosensitizing dye in the presence of oxygen, results in free radicals of high cytotoxicity, such as superoxides and singlet oxygen. these highly reactive species can cause serious damage to microorganisms via irreversible oxidation of cellular components12. however, this treatment presents other challenges regarding its susceptibility to different microorganisms, according to their physiology13-14.therefore, it is still necessary to set specific parameters so that pdt can be used for maximum effectiveness in removing microorganisms that cause endodontic infections. the aim of this study was to contribute to other studies that seek to clarify the effects of antimicrobial pdt, evaluating the effects of in vitro photosensitization of methylene blue by laser irradiation in suspensions containing various species of microorganisms. materials and methods microorganisms and preparation of microbial suspensions the microorganisms used in the study were obtained from the department of microbiology and antibiotics of the federal university of pernambuco, one yeast and three bacterial strains: candida albicans (atcc 10231), staphylococcus aureus (atcc 29213), pseudomonas aeruginosa (atcc 27853) and enterococcus faecalis (atcc 6057) previously cultured in agar nutrient (difco, detroit, mi, usa). four microbial suspensions of 3 ml each were formed in test tubes, in which microorganism indicators were diluted using sterile saline (0.9% nacl). the suspensions of the microorganisms had the optical density adjusted spectrophotometrically to approximately 1.0 x 10 8 colonyforming units (cfu) ml-1(equivalent to 1.0 mcfarland scale)5,15. from each microbial suspension, 2 ml was removed and a mixture with the four microorganisms was prepared (microbial pool). description of experimental groups aliquots of 200µlwere removed from the microbial pool and transferred to culture plates with 24 wells each. experimental groups were formed as follows (n=10): group l-p-: positive control (microbial pool); group l+p-: formed by the microbial pool that received the isolated action of the laser; group l-p+: microbial pool that received 20µl of the photosensitizer for two minutes, and group l+p+: microbial pool that received 20µl of the photosensitizer for two minutes and then laser irradiation. laser and photosensitizer the photosensitizer used in the study was a solution of methylene blue 50µm (chimiolux ®; hypofarma, belo horizonte, mg, brazil). the light source came from a low power laser (equipment whitening lase ii, dmc equipment ltd) with a wavelength of 660 nm, 100 mw, at an irradiation time of 3 min. this resulted in an energy dose of 9 j for each sample. photosensitization in vitro irradiation of samples was performed under aseptic conditions in a laminar flow hood (a/b3 cass ii; air tech, tokyo, japan). throughout the experiment, all the samples were handled in the dark. a bulkhead was made using an opaque black paper sheet with a central hole with a diameter similar to the wells, to prevent the same well from being irradiated more than once. a burette clamp was used in order to standardize the distance of 3 cm between the tip of the laser and the bottom of each well on the plate. to evaluate the antimicrobial treatment, aliquots of 1µlwere obtained from each well and plated in agar sabouraud (difco) growth medium and in blood agar (difco). after incubation for 48 h at 37°c in a bacteriological incubator, the cfu/ml was counted through observation of the morphology of the colonies. all experiments were conducted in triplicate. statistical analysis for data analysis, statistical measures were obtained using the average and standard deviation of the colonies count (in cfu/ml). calculation of percentage (descriptive statistics) was made using the f test (anova), with comparisons using tamhane’s inferential statistics technique. the hypothesis verification of equal variances was performed using levene’s f test with p<0.001 considered as statistically significant. the statistical program used was spss (statistical package for social sciences) version 15 (spss inc., chicago, il, usa). results the microbial effectiveness of the group treated with laser in the presence of the photosensitizer (l+p+) in all the microorganisms tested showed the lowest average value of cfu/mlwith significant difference between the groups (p<0.001) (table 1). table 2 shows the percentage of reduction in cfu/ml observed for the l+p+ group compared to the l-p-. among the evaluated microorganisms, p. aeruginosa was the most resistant to pdt, followed by c. albicans, s. aureus and e. faecalis. figure 1 shows the average and the standard deviation of cfu/ml obtained for the various microorganisms studied in each experimental group. in the group l+p-, c. albicans and p. aeruginosa showed a reduction in the number of cfu/ ml, similar to l+p+; whereas in group l+p+, e. faecalis and s. aureus showed a significant reduction compared to l+p-.the cfu/ml number in l-p+ was similar to the group l-p-. when the groups l+pandl-p+ were compared, a significant decrease of the microbial growth in all the studied microorganisms was observed. in vitro antimicrobial photoinactivation with methylene blue in different microorganisms 55 braz j oral sci. 13(1):53-57 species log (10) cfu/ml l+p l-p+ l+p+ l-p p-value c. albicans 9.66 ± 0.21 (a) 10.02 ± 0.42 (b) 9.51 ± 0.49 (a) 10.21 ± 0.32 (b) p(1)< 0.001* p. aeruginosa 10.10 ± 0.19 (a) 10.70 ± 0.02 (b) 10.09 ± 0.24 (a) 10.69 ± 0.02 (b) p(1)< 0.001* e. faecalis 9.39 ± 0.17 (a) 10.70 ± 0.02 (b) 7.30 ± 3.73 (c) 10.69 ± 0.02 (b) p(1)< 0.001* s. aureus 10.18 ± 0.18 (a) 10.69 ± 0.02 (b) 9.14 ± 0.46 (c) 10.69 ± 0.02 (b) p(1)< 0.001* table 1. table 1. table 1. table 1. table 1. average and standard deviation of the logarithm of colony-forming units per milliliter (cfu/ ml) for the following groups (n=10): l+p= group treated only with laser, lp+ = group treated only with photosensitizer, l+p+ = group irradiated with laser in the presence of photosensitizer, l-p= positive control group which has not been treated with laser or photosensitizer. (*): significant difference at level of 5.0%. (1) by f test (anova). note: if all the letters in parentheses are different, it shows a significant difference between the corresponding groups, using tamhane’scomparison method. species l+p+ l-preduction in cfu/ml (%) c. albicans 50.06 ± 37.04 199.50 ± 133.89 74.90% p. aeruginosa 131.85 ± 44.56 492.10 ± 24.03 72.41% e. faecalis 17.43 ± 24.96 489.20 ± 23.51 96.44% s. aureus 22.43 ± 24.22 490.20 ± 22.57 95.42% table 2. table 2. table 2. table 2. table 2. percentage of reduction, expressed in the average and standard deviation of the values (cfu/ml) in the cell viability of the microorganisms exposed to the laser in the presence of a photosensitizer (l+p+) compared to positive control (l-p-). fig. 1. average and standard deviation obtained for c. albicans, p. aeruginosa, e. faecalis and s. aureus in all experimental groups (n=10): l+p= group treated only with laser, lp+ = group treated only with photosensitizer, l+p+ = group irradiated with laser in the presence of photosensitizer, l-p= positive control. a, b and c: statistically significant difference (test f (anova): p<0.001) discussion the application of pdt, as an adjuvant treatment, has been indicated in endodontics, seeking to help the conventional therapy in eradicating the resistant pathogens of the root canal16-19. microbial agents are considered the main etiological factors to the progression and perpetuation of pulp and periradicular inflammatory diseases20.the pathogens used in the present study were selected because of their clinical importance and association with endodontic infection21. various dyes have been used to perform pdt, such as toluidine blue and methylene blue14. the latter had its chemical properties tested in several studies that proved its antimicrobial efficacy, which motivated the choice for using this product in the present study22-24. the results obtained in this study demonstrated that when methylene blue was used alone, there was no significant reduction in the number of cfu/ml for all studied species. this result indicates that the concentration and the amount used in the present study showed no cytotoxic effect on the test microorganisms, corroborating the findings of pupo et al.25 (2011) and miyabe et al. 26 (2011) who used only methylene blue at 100 mg/ml in c. albicans and at 3 mm in in vitro antimicrobial photoinactivation with methylene blue in different microorganisms braz j oral sci. 13(1):53-57 s. aureus respectively. these results, however, are different from those of foschi et al.27 (2007), who reported a 19.5% reduction in viability of e. faecalis when 6.25 mg/ml of methylene blue was used without photosensitization in extracted single-rooted teeth. regarding the laser effects in the absence of a photosensitizer, p. aeruginosa and c. albicans showed a reduction in the number of cfu/ml similar to the group treated with pdt, differing from the findings of queiroga et al.28 (2011),who found no reduction in cell viability of c. albicans after their exposure to the laser in the parameters of 60 j/cm2, 120 j/cm2 and 180 j/cm2. thus, in comparison with other groups, pdt behaved better in microbial reduction using methylene blue with a concentration of 50 µm at 660 nm, 100 mw and 9j, corroborating other studies that showed that the use of the laser associated with a photosensitizer is effective against various microorganisms16,29-31. microbial reduction by photodynamic effect faces various challenges when used against gram-positive bacteria, gram-negative and fungi. e. faecalis was the microorganism with the highest reduced percentage of cfu/ml (96.44%), followed by s. aureus (95.42%), c. albicans (74.90%) and p. aeruginosa (72.41%). in general, the literature shows that gram-positive bacteria are more susceptible to the action of pdt compared to gramnegative bacteria. this is due to differences in the physiology of these microorganisms, since gram-positive bacteria have a relatively porous outer membrane formed by a thicker layer of peptidoglycan and lipoteichoic acid14. this feature allowsa greater diffusion of the photosensitizer within the microbial cells,sincethey can be eliminated by various types of dye and lower doses of radiation, which explains the greater susceptibility of e. faecalis and s. aureusto pdt in this study. on the other hand, the outer membrane of gram-negative bacteria (pseudomonas aeruginosa) is thinner and complex, being formed by a heterogeneous composition of proteins with porin function, lipopolysaccharides and lipoproteins that act as an effective barrier to limit the penetration of various substances14. regarding fungi, besides their nuclear membrane and increased cellular volume, they possess a cell wall composed of a thick layer of beta glucan and chitin, which promotes an intermediate permeability barrier between the grampositive and gram-negative bacteria32. variables such as exposure time and laser energy density, type and dye concentration influence the number of microorganisms affected by pdt12. in this study, a reduction in the number of cfu/ml c. albicans to 74.90% was achieved. on the other hand, de souza et al.33 (2006) obtained a reduction of cfu/ml in a suspension of c. albicans to 88.6% when 0.1 mg/ml of methylene blue, 685 nm of laser light and an energy dose of 28 j/cm2 was used. the differences in results between these studies may be attributed to the dye concentration or to parameters used for laser irradiation. in summary, despite the pdt not reducing the microorganisms completely, the results obtained lead to the conclusion that the treatment was able to promote the reduction of microbial cell viability using the selected parameters. acknowledgements this study was supported by grants from pernambuco state foundation for science and technologyfacepe (bic0874-4.02/10) and cnpq brazil. the english version of this study has been revised by sidney pratt, canadian, ba, mat (the johns hopkins university), rsadip (tefl). references 1. siqueira jf jr, 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hydroxide against intracanal enterococcus faecalis. int endod j. 2013; 46: 499-505. 32. pereira ca, romeiro rl, costa ac, machado ak, junqueira jc, jorge ao. susceptibility of candida albicans, staphylococcus aureus, and streptococcus mutans biofilms to photodynamic inactivation: an in vitro study. lasers med sci. 2011; 26: 341-8. 33. de souza sc, junqueira jc, balducci i, koga-ito cy, munin e, jorge ao. photosensitization of different candida species by low power laser light. j photochem photobiol b. 2006; 83: 34-8. in vitro antimicrobial photoinactivation with methylene blue in different microorganisms oral sciences n3 original article braz j oral sci. october | december 2014 volume 13, number 4 dental development of children and adolescents with cleft lip and palate correspondence to: francielle topolski departamento de odontologia infantil e social disciplina de ortodontia preventiva – faculdade de odontologia de araçatuba – unesp rua josé bonifácio, 1193. vila mendonça cep: 16015-050 araçatuba, sp, brazil phone/fax: +55 18 3636-3236 +55 41 9168-8717, +55 18 99600-8717 e-mail: frantopolski@gmail.com abstract aim: to evaluate the dental development of brazilian children and adolescents with cleft lip and palate. methods: the sample consisted of 107 panoramic radiographs of children and adolescents with cleft lip and/or palate (cleft group) and 107 panoramic radiographs of children and adolescents without cleft lip and/or palate (control group), with chronological ages ranging from 6 to 15 years, matched in gender and chronological age within 60 days. radiographs were digitized and masked and dental age was assessed using the method described by demirjian et al. (1973). three trained examiners conducted the assessments. each examiner evaluated the radiographs three times. data were statistically analyzed using non-parametric tests and univariate linear regression (p<0.05). results: the dental age was overestimated in relation to the chronological age in both groups (p<0.0001). compared to the control group, there was a delay in the dental age in the cleft group of 0.17 years (2.1 months). however, no statistically significant difference in the dental age between the cleft and the control group was found even when considering the different cleft types (p=0.152). conclusions: there was no statistically significant difference in the dental age between the cleft and the control groups. the evaluation of dental development in individuals with cleft lip and palate should be approached in the same way as in individuals without clefts, with a focus on the individualization of diagnosis and treatment planning. keywords: cleft lip; cleft palate; dentition, permanent. introduction cleft lip and palate is the most common congenital malformation of the face. this morphologic deformity affects 1 in every 1000 births1 and impacts on quality of life. specifically, cleft lip and palate compromise dentomaxillofacial aesthetics and function culminating in psychosocial disorders1-2. in this context, treatment is challenging and requires a continuous multidisciplinary approach2-5. mostly, orthodontic treatment is essential for rehabilitation of aesthetics and function of cleft lip and palate children. however, this treatment is often prolonged due to the complexity of the situation. in order to simplify the treatment approach, accurately knowing the time for orthodontic intervention, assessment of dental maturity is of great clinical relevance2. additionally, information concerning the ideal time for alveolar bone grafting is obtained in forehand, enabling a better surgical planning4,6. in accordance to the clinical relevance of assessing dental maturity in cleft braz j oral sci. 13(4):319-324 francielle topolski1, rafael boscheti de souza2, ademir franco3, osmar aparecido cuoghi1, luciana reichert da silva assunção2, ângela fernandes2 1universidade estadual paulista – unesp, araçatuba dental school, department of pediatric and community dentistry, araçatuba, sp, brazil 2universidade federal do paraná – ufpr, school of dentistry, department of stomatology, curitiba, pr, brazil 3katholieke universiteit leuven, oral health sciences, area of forensic odontology, leuven, belgium received for publication: october 07, 2014 accepted: december 16, 2014 1 7 11 7 11 7 11 7 11 7 1320320320320320 lip and palate patients, several authors reported outcomes of investigations in specific populations, such as north americans7-10 and dutch11-13. howerer, the accessment of dental maturity in cleft lip and palate patients was not performed in a brazilian population up to the present date. based on that, the present study aims to compare the dental development of brazilian cleft lip and palate patients with a control group paired by chronological age and gender, contributing for further clinical diagnosis and surgical planning. materials and methods sample selection the study was approved by the ethics committee of the division of health sciences, federal university of paraná (curitiba, pr, brazil). the sample consisted of 107 panoramic radiographs of children and adolescents with cleft lip and/or palate (cleft group) and 107 panoramic radiographs of children and adolescents without cleft lip and/or palate (control group), with chronological ages ranging from 6 to 15 years, matched in gender and chronological age within 60 days. the cleft group sample was obtained from the analysis of medical records and panoramic radiographs of 160 patients with cleft lip and/or palate in a specialized treatment center. patients presenting incomplete medical records, syndromes, systemic diseases, dental agenesis or missing permanent teeth in the mandibular left hemiarch, low-quality panoramic radiographs, and age below 6 years old or over 15 years old at the radiographic exam date were excluded from the study. the control group consisted of the data of medical records and panoramic radiographs of children and adolescents without cleft lip and/or palate. the exclusion criteria applied for the case group were repeated for control group sampling. no racial or ethnic data were recorded for both case and control groups. the subjects included in the sample are mainly from the state of paraná, located in southern brazil. the population of this region is mainly of european descent, but also has individuals of african and indigenous origin. therefore, the sample is characterized by large racial and ethnic heterogeneity, like most of the brazilian population. scanning and blinding in order to avoid potential bias during the analysis, radiogr aphs were digitized masking the identification information, including gender and date of birth. the radiographs were digitized using a scanner (scanjet g4050) and the software digitalização hp (hewlett-packard company, palo alto, ca, usa). the levels of brightness and contrast indicated by the software were respected. the used standard resolution was 150 ppi (pixels per inch), on a 100% scale. images were saved in tiff (tagged image file format), which allows generation of high quality scanned images. to identify the digitized image files, a legend of random numbers was created. only after completing all the analyses, the examiners had access to the original identification information. in addition, the region corresponding to the maxilla was previously cut, preventing visualization of cleft areas. evaluation of dental age dental age was determined using the method described by demirjian et al.14 (1973). this method is based on the evaluation of the seven left mandibular permanent teeth, excluding the third molar. the formation of each tooth is divided into eight stages, from a to h, from appearance of the first points of calcification until apex closure. each stage of tooth development corresponds to a score, which is different for males and females. the sum of the scores of the seven evaluated teeth on a panoramic radiograph corresponds to the maturity score, represented on a scale of 0 to 100. the score of maturity, in turn, must be converted into the dental age, using two tables one for males and other for females. radiographs were evaluated in a 20-inch computer monitor in low light environment. three trained examiners evaluated the radiographs: an oral radiologist, an orthodontist and a dental practitioner. after studying the method and training with 30 radiographs, wich were not included in the sample, each examiner evaluated the radiographs three times with a three-day interval between assessments. an average of the maturity scores obtained in the three assessments of each examiner was calculated and then a final average from the three examiners was established. the final average of maturity scores of the three examiners was converted into the dental age of each patient. statistical methods data were analyzed using the statistical package for the social sciences (version 15.0, spss inc., chicago, il, usa). the outcome variable considered for statistical analysis was the dental age. as there was no normal distribution of this variable (kolgomorov-smirnov test, p<0.001), nonparametric tests for bivariate analyses were used (chi-square and mann-whitney u). to compare the dental age between the cleft and control groups, wilcoxon test was used due the dependence between observations. this test was also used for the comparison between dental age and chronological age. the relationship between dental age and chronological age in both groups was also analysed by linear regression analysis. a significance level of 5% was adopted. in order to test accuracy and reability of the method, the intraclass correlation coefficient was used to assess intraand inter-examiner agreement. the test was applied to the maturity scores obtained in the assessments of the first 30 radiographs. for intra-examiner analysis, the scores of maturity achieved in each of the three assessments of each examiner were compared. for inter-examiner analysis, the averages of the scores of maturity of the three examiners were compared. results error of method intra-examiner analysis showed intraclass correlation braz j oral sci. 13(4):319-324 dental development of children and adolescents with cleft lip and palate 321321321321321 coefficients of 0.999, 0.998 and 0.994. the result of the interexaminer analysis was 0.995. these results indicate very good intraand inter-examiner agreement. sample characteristics of the 107 pairs of children and adolescents evaluated in the study, 68 (63.6%) were males and 39 (36.4%) were females. the average chronological age was 10.3 years for boys and 9.8 years for girls. among the individuals in the case group, 72 (68.2%) had cleft lip and palate (clp), 27 (25.2%) had cleft lip with or without cleft alveolus (cl±a) and 2 (1.9%) had cleft palate (cp). in 6 patients (4.7%), there was an association between cl±a and cp (cl±a + cp). for the clp and the cl±a, unilateral clefts were more frequent (77%) and the left side was more often affected (63.6%). the distribution of cleft types by gender is shown in table 1. clp was the most frequent type of cleft followed by cl±a for both genders. the relative frequency of clp was higher for boys (72.1%) than for girls (61.5%). oppositely, the relative frequency of cl±a was higher for girls (28.2%) than for boys (23.5%). however, considering the clp and cl±a groups, no statistically significant difference was found between cleft type and gender (p=0.464). in the cp and cl±a + cp group, the small number of individuals prevented the application of statistical tests to the variables gender and cleft type. dental development table 2 shows the descriptive statistics for the variables cleft type boys girls boys + girls n % n % n % cl±a* 16 23.5 11 28.2 27 25.2 clp** 49 72.1 24 61.5 73 68.2 cp*** 1 1.5 1 2.6 2 1.9 cl±a + cp**** 2 2.9 3 7.7 5 4.7 all cleft types 68 100.0 39 100.0 107 100.0 table 1. table 1. table 1. table 1. table 1. distribution of cleft types by gender *cleft lip with or without cleft alveolus **cleft lip and palate ***cleft palate ****association between cleft lip with or without cleft alveolus and cleft palate chronological dental age dental age age (cleft group) (control group) n 107 107 107 mean 10.12 10.93 11.10 median 9.90 10.70 11.00 mode 6.70 16.00 16.00 standard deviation (2.42) (2.66) (2.74) percentiles 25 8.00 8.50 8.60 50 9.90 10.70 11.00 75 12.10 13.00 13.50 m i n i m u m 6.00 6.80 6.40 m a x i m u m 14.90 16.00 16.00 table 2. table 2. table 2. table 2. table 2. descriptive statistics for the variables chronological age and dental age (y) chronological age and dental age in cleft and control groups. the average dental age in both the cleft and the control groups was higher than the average chronological age. dental age, therefore, was advanced in relation to chronological age in both groups, which was statistically significant for both groups (p<0.0001). specifically, the advance corresponded to 0.81 years (9.7 months) for the cleft group and 0.98 years (11.8 months) for the control group. this result shows a delay of 0.17 years (2.1 months) in dental age in the cleft group compared to the control group; however, this difference was not statistically significant (p=0.152). the dental ages of the cleft and control groups were also compared considering the different genders, cleft types and chronological age groups (6-9; 9.1 to 12; 12.1 to 15 years). once again, no statistically significant difference (p>0.05) was found as shown in table 3. table 4 shows the comparison of dental development between different genders and groups (cleft and control), considering the mean of differences between the dental and chronological age. as it can be seen, there was no statistically significant difference (p>0.05). a strong and positive correlation between dental age and chronological age in the control and cleft groups was observed (r=0.9348, p<0.001, r=0.9198, p<0.001, respectively). linear regression was used to show how much of the variation in dental age was explained by chronological age. estimated regression showed that chronological age alone explained 87.38% of the dental age variation in the control group (figure 1) and 84.61% in the cleft group (figure 2). braz j oral sci. 13(4):319-324 dental development of children and adolescents with cleft lip and palate 1 7 11 7 11 7 11 7 11 7 1322322322322322 dental age cleft vs control cleft group control group m d * q(25)** q(75)*** m d * q(25)** q(75)*** age group 6-9 8.3 7.8 8.6 8.3 7.7 8.9 p=0.690**** 9.1-12 11.1 10.0 12.3 11.4 10.5 12.0 p=0.101**** 12.1-15 14.3 13.2 15.8 14.7 13.5 16.0 p=0.899**** gender boys 10.9 8.5 12.8 11.1 8.7 14.1 p=0.162**** girls 10.6 8.3 13.1 10.9 8.4 13.2 p=0.557**** cleft type cl±a 11.2 8.4 13.2 10.7 8.6 14.1 p=0.333**** c l p 10.7 8.5 12.8 11.2 8.6 13.4 p=0.401**** c p 11.9 7.8 16.0 12.2 8.4 16.0 p=0.180**** table 3. table 3. table 3. table 3. table 3. dental age in cleft vs control group by chronological age group, gender and cleft type *median **1st quartile ***3rd quartile ****wilcoxon test fig. 2. diagram showing the relationship between chronological age in years and dental age in the cleft group. estimated regression equation, correlation coefficient (r), coefficient of determination (r2) and significance level (p) are also shown. fig. 1. diagram showing the relationship between chronological age in years and dental age in the control group. estimated regression equation, correlation coefficient (r), coefficient of determination (r2) and significance level (p) are also shown. braz j oral sci. 13(4):319-324 dental development of children and adolescents with cleft lip and palate 323323323323323 discussion in the medical literature, several authors reported population-specific results for the assessment of dental maturity in cleft lip and palate patients by comparison between cleft and control groups. in some of them, however, the cleft and the control groups were not paired by gender and/or chronological age7-8,11-13. in the present study, a control group was designed pairing patients by gender and chronological age, making the present outcomes more suitable to a population-specific survey. moreover, in the present investigation a larger sample is observed if compared to previous reports7,9-13,15-19 highlighting a strong reliability. in addition, contrarily to previous studies79,11-13,15,17-20 a blinding methodology was applied to avoid potential bias during the image analysis. the blinding methodology plays an important part in the study reliability, since the method of demirjian et al.14 (1973) presents a certain degree of subjectivity. some teeth are in intermediate stages of development (e.g. between stages d and e) potentially hampering the process of age estimation. if the examiner knows that the radiography belongs to an individual with cleft, he may tend to choose a premature stage, since the literature reports that the dental development in these individuals is delayed. therefore the importance of blinding is justified. furthermore, in relation to the image analysis, it is relevant to note that three calibrated examiners performed the assessment of dental maturity. only two previous studies12,17 report these standards to achieve greater reliability. the method of demirjian et al.14 (1973) was applied in the present sample, revealing a slight developmental delay of the dental age in the cleft group, therefore not statistically significant. similarly, eerens et al.16 (2001) reported a slight delay in the dental development of belgian children. despite the concordance, both studies contradict most of the previous investigations 7,9-13,15,17-20, which reported statistically significant delays in the dental development of individuals with cleft lip and palate. moreover, some studies lack information concerning the performed methodology 7,15, hampering adequate interpretations. yet other authors performed different methods for dental age assessment, not allowing for accurate comparisons 11,17. in the study of bindayel et al.19 (2014) no control group was used, which may compromise the reliability of the results. thus, the differences observed in the current study when compared with other studies that evaluated the dental development of individuals with cleft lip and palate may be explained by the applied methodological design, considering aspects such as sample size, pairing of the sample, blinding, number of examiners and number of radiograph evaluations per examiner. moreover, ethnic and racial differences among the surveyed populations may have contributed to these differences. furthermore, in the present study individuals with agenesis in mandibular left hemiarch were excluded. the etiologic factors of delayed dental development of individuals with clefts seem to be the same factors responsible for the occurrence of dental anomalies in these individuals, as well as for the manifestation of the cleft itself7,16,21. moreover, the delay tends to be more pronounced in individuals with agenesis21. this may explain the absence of delayed dental development in the individuals with cleft lip and palate observed in this study. we excluded individuals who had agenesis and a possible greater probability of changes in dental development. on the other hand, ruiz-mealin et al.22 (2012) observed delays in the dental development of subjects with agenesis and without any syndrome or medical condition (meaning without clefts). this may suggest that delayed dental development is more associated with the presence of agenesis than with cleft lip and palate. there was a strong correlation between dental age and chronological age in the control and cleft groups. however, the dental age was advanced in relation to chronological age in the cleft and control groups with a statistically significant difference for both groups. this finding was also reported by other studies23-25 that aimed to estimate the chronological age by assessing the dental age using the method described by demirjian et al.14 (1973). these results demonstrate that this method tends to overestimate the dental age when applied to different populations. for the present study, however, this fact was not relevant, since the goal was not to estimate the chronological age from the dental age. the demirjian et al.14 (1973) method was used with the purpose of comparing the dental age of individuals from cleft and control groups with the same chronological age. no statistically significant difference in dental age between the cleft and control groups was found even when considering the different genders, cleft types and chronological age groups separately. therefore, one can conclude that the analysis of dental development in individuals with cleft lip and palate should have the same approach used for individuals without clefts, with a focus on the individualization of diagnosis and treatment planning. acknowledgement this work was supported by a brazilian research funding agency (capes). references 1. world health organization. global strategies to reduce the health-care burden of craniofacial anomalies. geneva: the organization; 2002. boys girls p** mean (y) sd mean (y) sd cleft group 1.06 (0.80) 1.02 (0.82) 0.702 control group 1.16 (0.89) 1.03 (0.76) 0.555 p * 0.415 0.971 table 4. table 4. table 4. table 4. table 4. means of the differences between dental and chronological ages *wilcoxon test **mann-whitney test braz j oral sci. 13(4):319-324 dental development of children and adolescents with cleft lip and palate 1 7 11 7 11 7 11 7 11 7 1324324324324324 2. freitas jas, garib dg, oliveira tm, lauris rcmc, almeida alpf, neves lt, et al. rehabilitative treatment of cleft lip and palate: experience of the hospital for rehabilitation of craniofacial anomalies – usp (hrac/ usp) – part 2: pediatric dentistry and orthodontics. j appl oral sci. 2012; 20: 268-81. 3. david dj, smith i, nugent m, richards c, anderson pj. from birth to maturity: a group of patients who have completed their protocol management. part iii. bilateral cleft lip-cleft palate. plast reconstr surg. 2011; 128: 475-84. 4. toscano d, baciliero u, gracco a, siciliani g. long-term stability of alveolar bone grafts in cleft palate patients. am j orthod dentofacial orthop. 2012; 142: 289-99. 5. farronato g, kairyte l, giannini l, galbiati g, maspero c. how various surgical protocols of the unilateral cleft lip and palate influence the facial growth and possible orthodontic problems? which is the best timing of lip, palate and alveolus repair? literature review. stomatologija. 2014; 16: 53-60. 6. osawa t, omura s, fukuyama e, matsui y, torikai k, fujita k. factors influencing secondary alveolar bone grafting in cleft lip and palate patients: prospective analysis using ct image analyzer. cleft palate craniofac j. 2007; 44: 286-91. 7. bailit hl, doykos jd, swanson lt. dental development in children with cleft palates. j dent res. 1968; 47: 664. 8. loevy ht, aduss h. tooth maturation in cleft lip, cleft palate, or both. cleft palate j. 1988; 25: 343-7. 9. pham and, seow wk, shusterman s. developmental dental changes in isolated cleft lip and palate. pediatr dent. 1997; 19: 109-13. 10. borodkin af, feigal rj, beiraghi s, moller kt, hodges js. permanent tooth development in children with cleft lip and palate. pediatr dent. 2008; 30: 408-13. 11. brouwers hjm, kuijpers-jagtman am. development of permanent tooth length in patients with unilateral cleft lip and palate. am j orthod dentofacial orthop. 1991; 99: 543-9. 12. heidbüchel klwm, kuijpers-jagtman am, ophof r, van hooft rjm. dental maturity in children with a complete bilateral cleft lip and palate. cleft palate craniofac j. 2002; 39: 509-12. 13. huyskens rwf, katsaros c, van’t hof ma, kuijpers-jagtman am. dental age in children with a complete unilateral cleft lip and palate. cleft palate craniofac j. 2006; 43: 612-5. 14. demirjian a, goldstein h, tanner jm. a new system of dental age assessment. hum biol. 1973; 45: 211-27. 15. harris ef, hullings jg. delayed dental development in children with isolated cleft lip and palate. arch oral biol. 1990; 35: 469-73. 16. eerens k, vlietinck r, heidbüchel k, olmen av, derom c, willems g, et al. hypodontia and tooth formation in groups of children with cleft, siblings without cleft, and nonrelated controls. cleft palate craniofac j. 2001; 38: 374-8. 17. mitsea ag, spyropoulos mn. premolar development in greek children with cleft lip and palate. quintessence int. 2001; 32: 639-46. 18. tan ely, yow m, kuek mc, wong hc. dental maturation of unilateral cleft lip and palate. ann maxillofac surg. 2012; 2: 158-62. 19. bindayel na, alsultan ma, elhayek so. timing of dental development in saudi cleft lip and palate patients. saudi med j. 2014; 35: 304-8. 20. lai mc, king nm, wong hm. dental development of chinese children with cleft lip and palate. cleft palate craniofac j. 2008; 45: 289-96. 21. ranta r. a review of tooth formation in cleft lip/palate. am j orthod dentofacial orthop. 1986; 90: 11-8. 22. ruiz-mealin ev, parekh s, jones sp, moles dr, gill ds. radiographic study of delayed tooth development in patients with dental agenesis. am j orthod dentofacial orthop. 2012; 141: 307-14. 23. koshy s, tandon s. dental age assessment: the applicability of demirjian’s method in south indian children. forensic sci int. 1998; 94: 73-85. 24. willems g, olmen av, spiessens b, carels c. dental age estimation in belgian children: demirjian’s technique revisited. j forensic sci. 2001; 46: 893-5. 25. kýrzýoðlu z, ceyhan d. accuracy of different dental age estimation methods on turkish children. forensic sci int. 2012; 216: 61-7. braz j oral sci. 13(4):319-324 dental development of children and adolescents with cleft lip and palate oral sciences n3 original article braz j oral sci. july | september 2014 volume 13, number 3 influence of surface moisture condition on the bond strength to dentin of etch-and-rinse adhesive systems eliseu aldrighi münchow, lisia lorea valente, mayara bossardi, tanize cezar priebe, cesar henrique zanchi, evandro piva universidade federal de pelotas ufpel, school of dentistry, department of operative dentistry, pelotas, rs, brazil correspondence to: eliseu aldrighi münchow rua gonçalves chaves, 457 cdc-bio cep: 96015-560 pelotas, rs, brasil phone: +55 53 32226690 / 135 e-mail: eliseumunchow@hotmail.com received for publication: may 15, 2014 accepted: august 08, 2014 abstract aim: to evaluate the immediate microtensile bond strength (µtbs) of three two-step etch-andrinse adhesive systems applied under different dentin surface moisture conditions. methods: class v cavities were prepared in seventy-two bovine incisors. each tooth was randomly allocated into three groups, according to the adhesive system used: single bond 2 (sb), prime & bond 2.1 (pb) and xp bond (xpb). each group was divided in three subgroups, according to the dentin moisture condition: over-wet, moist and dry (n=8). for the moist subgroups the adhesive was applied as to the manufacturer’s instructions; for the over-wet ones, without drying the cavity after the rinsing procedure; and for the dry subgroups, drying the surface for 20 s. the teeth were restored with filtek z-250 and stored in distilled water (24 h); next, each restoration was sectioned in beam-shaped specimens which were stored for 24 h at 37 °c. each specimen was submitted to µtbs test (emic), and data were analyzed using two-way anova and tukey test (p<0.05). results: there was a statistically significant interaction between the adhesive system type and the dentin surface moisture condition (p=0.003). sb and xpb presented higher bond strength in the moist dentin condition, and pb showed high µtbs values in the dry substrate. conclusions: the moisture condition influenced the bond strength between the adhesives and dentin. sb performed better in the moist condition, whereas pb and xpb showed satisfactory bond strength in the moist and in the dry substrates. the over-wet dentin condition only impaired bond strength to sb and xpb. keywords: tensile strength; solvents; humidity; in vitro. introduction differently from the enamel substrate, the adhesive process in dentin is more technique-sensitive, depending on the operator’s experience1, on the surface moisture conditions of the tooth cavity2-4 and also on the substrate morphology5. therefore, to promote dentin adhesion, contemporary dental adhesives are categorized into two classes of materials according to the strategy of etching the tooth substrate: the etch-and-rinse or the self-etch techniques. the former uses separate application of an acid solution (commonly phosphoric acid) to etch the surface, whereas the latter uses the adhesive itself6. finished the etching step, a primer and a resin adhesive material should also be applied to complete the adhesive system application. with regard to the use of etch-and-rinse adhesives in dentin, the acid solution braz j oral sci. 13(3):182-186 1 8 31 8 31 8 31 8 31 8 3 is applied for 15 s followed by rinsing and drying the surface. after that, an ideally moist surface must be achieved to maintain the collagen fibrils in expansion, allowing them to entangle with the resin monomers and to enhance the adhesion process (the so called wet bonding technique)7. however, these procedures of rinsing and drying are considered the most critical factors when using etch-andrinse materials; in addition, extensive or complex tooth cavities may present different degrees of wetness, where overwet and dry regions may exist on the same tooth surface8-9. as a consequence, that ideally moist surface is difficult to achieve. besides the possibility of different moisture degrees remaining in the same tooth cavity after the etching/rinsing/ drying procedures, the adhesion process is also influenced by the adhesive system’s composition, mainly the type of solvent used9. among the most common solvents present in dental adhesives there are water, ethanol, acetone and tetrahydrofuran10-11. differently from this pattern, xp bond® adhesive system (dentsply caulk, milford, ma, usa)12, which contains tert-butanol as solvent, is a two-step etch-and-rinse adhesive which is marketed as being a good adhesive agent even in over-wet or dry dentin conditions. hence, considering that the solvent plays important roles in the collagen fibrils expansion, in their affinity for the entanglement with the resin monomers, and in the residual water removal of the tooth cavity13, the aim of this study was to evaluate the influence of surface moisture condition on the immediate bond strength to dentin of different etchand-rinse adhesive systems. the tested hypothesis was that the adhesives would present different bond strength results at different dentin surface moisture conditions after 24 h of water storage. material and methods tooth preparation and bonding procedures seventy-two bovine incisors were obtained, cleaned and stored in an aqueous 0.5% solution of chloramine t for one week. class v cavities (6 mm length, 4 mm width, and 2 mm thickness) were then prepared using diamond bur (fg #1045, kg sorensen, cotia, sp, brazil) in high-speed handpiece; the bur was replaced by a new one after every 8 teeth prepared. the cavity walls were parallel with each other, as a parallelometer was used. after being prepared, each tooth was randomly allocated in three groups, according to the adhesive system applied: adper™ single bond 2 (sb); prime & bond 2.1 (pb), and xp bond (xpb). the brand name, manufacturer, lot number, composition information and application protocol (following manufacturer’s instructions) of each adhesive are shown in table 1. each group was further divided in three subgroups, according to the dentin surface moisture condition maintained in the cavity: (1) an over-wet condition; (2) a moist condition; or (3) a dry condition (n=8). a 35% phosphoric acid gel solution (condac 37; fgm, joinville, sc, brazil) was applied on the dentin substrate for 15 s and rinsed by water-spray for 15 s. the adhesives were then applied (manufacturer’s instructions) for all groups (table 1), except in the over-wet groups where the dentin substrate was maintained plenty of water without surface drying, and in the dried groups where the dentin was thoroughly dried with an air-spray for 20 s. the moist groups where obtained by gently drying the substrate with paper points until a glossy dentin substrate was achieved, according to the wet bonding technique7. next, the adhesives were applied and light-activated for 20 s using a light-emitting diode (led) light-curing unit (radii; sdi, bayswater, vic, australia). the radiance was measured with a digital power meter (ophir optronics, danvers, ma, usa) and it was 900 mw/cm2. after light-activation, the composite resin (filtek z-250, 3m espe, st. paul, mn, usa) was placed in three 2mm-thick increments, which were built up separately using opposite tooth surfaces in an attempt to diminish the c-factor; each increment was then light-activated for 20 s. the restored teeth were stored in distilled water at 37 °c for 24 hs. microtensile bond strength (µtbs) test the teeth were sectioned longitudinally and transversally using a refrigerated diamond saw at low speed (isomet 1000; buehler ltd, lake bluff, il, usa) to obtain specimens with approximately 0.8 mm² of transverse-sectional area. the specimens were stored in distilled water at 37 °c for 24 h and were fixed with cyanoacrylate adhesive (super bonder gel, loctite ltda., são paulo, sp, brazil) to a metallic device for the microtensile test14. the µtbs test was performed in a universal testing machine (dl-500; emic, são josé dos pinhais, pr, brazil) at a crosshead speed of 1 mm/min. bond strengths were calculated and expressed in mpa, and the premature failures were registered but were not used for the statistical analysis. fracture analysis the fractured specimens were examined by stereomicroscopy at 40x magnification. the failure patterns were classified as: adhesive at the interface (when the failure occurred at the hybrid layer), cohesive in resin (at the adhesive resin and/or composite), cohesive in dentin, or mixed (when the failure involved different failure patterns). statistical analysis the statistical program sigmastat version 3.5 (systat software inc., chicago, il, usa) was used for data analysis. the immediate µtbs results were submitted to two-way analysis of variance (anova) (adhesive system and dentin moisture condition as factors) and post hoc tukey’s test for multiple comparisons. statistical significance was established at 5%. results there was a statistically significant interaction between the adhesive system type and the dentin surface moisture condition (p=0.003). means (standard deviations) of µtbs influence of surface moisture condition on the bond strength to dentin of etch-and-rinse adhesive systems braz j oral sci. 13(3):182-186 adhesive system adper™ single bond 2 (sb) prime & bond 2.1 (pb) xp bond (xpb) manufacturer 3m espe, st. paul, mn, usa dentsply caulk, milford, ma, usa dentsply caulk, milford, ma, usa lot number 9xb 4301070 1105001715 composition ethanol, water, bis-gma, mdp, hema, dimethacrylates, silanated colloidal, cq, silica, polyalkenoic acid copolymer acetone, bis-gma, penta, udma, edab, photoinitiators butylated hydroxytoluene, cetylamine hydrofluoride t-butanol, penta, udma, tegdma, hema, stabilizers, edab, cq, functionalized amorphous silica application protocol* a (15 s); b; c; d (2 coats); e; f (20 s) a (15 s); b; c; d (3 coats); e; f (20 s) a (15 s); b; c; d (1 coat); e (5 s); f (20 s) table 1.table 1.table 1.table 1.table 1. brand names, manufacturers, lot number, composition, and application directions of adhesives. abbreviations: bis-gma: bisphenol a diglycidyl methacrylate; mdp: 10-10-methacryloyloxydecyl dihydrogen phosphate; hema: 2-hydroxyethyl methacrylate; cq: camphorquinone; penta: dipentaerythritol pentacrylate monophosphate; udma: urethane dimethacrylate; tegdma: tryethyleneglicol dimethacrylate; edab: 4-ethyl dimethyl aminobenzoate. *procedures according to manufacturer’s directions: (a) acid etching; (b) rinse; (c) dry with absorbent paper; (d) apply adhesive; (e) gently air-dry; (f) light curing. adhesive systems dentin surface moisture condition over-wet moist dry single bond (sb) a 25.8 (9.1) b a 41.1 (11.9) a ab 25.3 (10.3) b prime & bond (pb) ab 18.6 (10.0) b b 27.6 (10.4) ab a 30.1 (7.5) a xp bond (xpb) b 12.7 (7.4) b b 22.1 (10.1) a b 19.9 (7.6) ab table 2.table 2.table 2.table 2.table 2. immediate microtensile bond strength to dentin (mpa) and standard deviation (sd) presented by the adhesive systems at different dentin surface moisture conditions. superscripts capital letters in a same column represent statistically significant differences among the adhesive systems (p<0.05) and superscripts small letters in the same row represent statistically significant differences among dentin conditions (p<0.05). are displayed in table 2. in the over-wet condition, sb demonstrated similar µtbs mean to pb (p=0.308), but higher than xpb (p<0.001), which presented the lowest immediate bond strength mean of this study, but similar to pb (p=0.441). within the moist dentin condition, sb showed higher bond strength results than the other adhesive systems (p<0.001), which had not differed between each other (p=0.228). in the dry condition, pb demonstrated similar µtbs mean to sb (p=0.310), but higher than xpb (p=0.008), which showed similar bond strength mean to sb (p=0.289). figure 1 shows the fracture pattern analysis of the µtbs results, where a predominance of adhesive failures has occurred in the over-wet condition; equilibrium of mixed and adhesive failures in the moist dentin surface; and presence of some dentin cohesive failures in the dry condition. discussion the current investigation selected different types of twostep, etch-and-rinse adhesive systems containing different organic solvents in an attempt to evaluate their influence on the bond strength to dentin in over-wet, moist or dry surface moisture conditions. while single bond (sb) is a waterethanol-based adhesive, prime & bond (pb) and xp bond fig. 1. fracture pattern (in percentage) of the immediate microtensile bond strength results of the adhesive systems under different dentin surface moisture conditions. (xpb) are composed by acetone and tert-butanol, respectively. it was hypothesized that the adhesive systems would produce different bond strength results in the different dentin moisture conditions. according to the results displayed in table 2, this hypothesis can be partially accepted, as sb performed better on the moist dentin substrate, pb on the dry when compared to over-wet circumstance, and xpb showed higher bond strength in the moist substrate than in the over-wet condition. the highest bond strength results of the present study were obtained by the application of sb on the moist dentin (table 2). this material presents a mixture of water and ethanol, which are considered strong hydrogen bonding solvents, although the latter has a higher vapor pressure than the former (43.9 versus 17.5 mm hg at 20 °c, respectively)15, leading to its faster evaporation. several studies have stated that ethanol-based adhesive systems seem less sensitive to the amount of moisture in dentin16-18; nevertheless, this might be true only when a moist dentin is achieved, as the excess influence of surface moisture condition on the bond strength to dentin of etch-and-rinse adhesive systems1 8 41 8 41 8 41 8 41 8 4 braz j oral sci. 13(3):182-186 of water (over-wet groups) or its absence (dry groups) resulted in a significant reduction of the bond strength values. even though moisture is necessary for a good bonding to dentin, its excess may hamper the water removal, preventing complete monomer infiltration into the demineralized dentin, or even compromising the adhesive polymerization19-20. the over-wet moisture condition prepared in the present study, which was obtained without drying the surface after rinsing the tooth cavity, may have probably diluted the adhesive material, turning the hybrid layer porous and more permeable. in the dry surface condition, the over-drying procedure (20 s of airstream application after rinsing) might have collapsed the collagen fibrils, enabling the monomers to properly infiltrate through the collagen matrix11. differently from a waterethanol-based material, pb is formulated with acetone. according to reis et al.18 (2003) acetone-based adhesive systems require a wetter dentin surface to achieve high bond strengths. likewise, cardoso et al.21 obtained higher bond strength results in wet dentin when compared to dry dentin. notwithstanding, da silva et al. 22 showed similar bond strengths in both wet and dry substrates when using an acetone-based adhesive. these results corroborate those of the present study, in which higher mean µtbs was found in the dry subgroup but without statistically significant difference in comparison with the moist subgroup. a reasonable explanation is that acetone presents high vapor pressure (184 mm hg at 20 °c)15, which makes it extremely volatile, with fast evaporation from dentin; as a consequence, residual solvent into the adhesive layer is less likely to remain, guaranteeing optimal monomer infiltration/polymerization23. also, complete residual solvent removal increases the quality of adhesives by forming a structure, which is less permeable, more cohesive and densely-packed24. on the other hand, pb applied on the over-wet dentin condition resulted in significantly lower µtbs mean than the value obtained in the dry condition. acetone, which presents lower polarity than water and ethanol, does not form hydrogen bonds, thus it would not be able to interact easily with over-wet dentin. consequently, residual water may have been entrapped within the fibrils, enabling the resin diffusion and subsequent polymerization11. despite the water-ethanol and acetone-based adhesive systems being more common in dentistry, xpb contains tertbutanol as the organic solvent. it has been claimed to be less technique-sensitive, due to its improved ability of diffusing through collagen fibrils, even in a collapsed state12. thus, it was supposed to be a good bonding agent option to be applied in some adverse conditions such as over-wet or over-dry dentins. according to an in vivo clinical evaluation, xpb met the criteria for a provisional acceptance (american dental association guidelines), presenting less than 5% failure rate after 6 months of clinical performance 25. additionally, other studies have demonstrated satisfactory bond strength results when using this adhesive 26-27. nevertheless, the present study results showed that xpb was associated with the lowest µtbs values in all the three surface conditions evaluated (table 2), although similar bond strength means for pb (in the over-wet and moist conditions) and for sb (in the dry condition) could be observed. a possible reason for this result is the lower vapor pressure of tert-butanol (26 mm hg at 20 °c)28 when compared to ethanol and acetone (43.9 and 184 mm hg at 20 °c, respectively), hampering its ability to remove the residual water molecules and its own evaporation from the dentin surface. another possible explanation is that monomers such as hema can decrease the vapor pressure of water, interfering with the removal of any residual water29, and considering that xpb contains hema (table 1), the tert-butanol ability of controlling dentin wetness may have been even more reduced. all the afore-mentioned different bond strength results are in accordance with the failure patterns obtained in this study (figure 1). the highest µtbs means were obtained in the moist dentin condition, which has shown a balance of adhesive and mixed failures for all the adhesive systems applied. on the other hand, in the dry condition, which may have reduced the cohesive strength of the dentin substrate, a predominance of adhesive failures has occurred plus the appearance of some dentin cohesive failures. differently, the over-wet dentin condition was marked by a predominance of adhesive failures, suggesting that the hybrid layer was at least in some extent defective and poorly produced. within the limitations of the present study, it may be concluded that the moisture condition influenced the microtensile bond strength to dentin among the evaluated adhesive systems. single bond 2 performed better in the moist condition, whereas prime & bond and xp bond showed adequate bond strength in the moist and dry substrates. notwithstanding, the over-wet dentin condition only significantly impaired the bond strength of the single bond 2 and xp bond adhesive systems. acknowledgements the authors acknowledge the brazilian national council for scientific and technological development (cnpq) for supporting the present study (grant 156232/2010-3) and capes/mec (brazilian government) for scholarship. references 1. unlu n, gunal s, ulker m, ozer f, blatz mb. influence of operator experience on in vitro bond strength of dentin adhesives. j adhes dent. 2012; 14: 223-7. 2. jacquot b, durand jc, farge p, valcarcel j, deville de periere d, cuisinier f. influence of temperature and relative humidity on dentin and enamel bonding: a critical review of the literature. part 1. laboratory studies. j adhes dent. 2012; 14: 433-46. 3. plasmans pj, creugers nh, hermsen rj, vrijhoef mm. the influence of absolute humidity on shear bond adhesion. j dent. 1996; 24: 425-8. 4. pupo ym, michel md, gomes om, lepienski cm, gomes jc. effect of the regional variability of dentinal substrate and modes of application of adhesive systems on the mechanical properties of the adhesive layer. j conserv dent. 2012; 15: 132-6. 5. cavalcanti an, de souza es, lopes gs, de freitas ap, de araújo rpc, mathias p. effect of a desensitizing dentifrice on the bond strength of different adhesive systems. braz j oral sci. 2013; 12: 148-52. influence of surface moisture condition on the bond strength to dentin of etch-and-rinse adhesive systems 1 8 51 8 51 8 51 8 51 8 5 braz j oral sci. 13(3):182-186 6. pashley dh, tay fr, breschi l, tjaderhane l, carvalho rm, carrilho m, et al. state of the art etch-and-rinse adhesives. dent mater. 2011; 27: 1-16. 7. kanca j, 3rd. improving bond strength through acid etching of dentin and bonding to wet dentin surfaces. j am dent assoc. 1992; 123: 35-43. 8. miyazaki m, onose h, moore bk. effect of operator variability on dentin bond strength of two-step bonding systems. am j dent. 2000; 13: 101-4. 9. tay fr, gwinnett aj, wei sh. the overwet phenomenon: a transmission electron microscopic study of surface moisture in the acid-conditioned, resin-dentin interface. am j dent. 1996; 9: 161-6. 10. fontes st, ogliari fa, lima gs, bueno m, schneider lf, piva e. tetrahydrofuran as alternative solvent in dental adhesive systems. dent mater. 2009; 25: 1503-8. 11. manso ap, marquezini l jr, silva sm, pashley dh, tay fr, carvalho rm. stability of wet versus dry bonding with different solvent-based adhesives. dent mater. 2008; 24: 476-82. 12. dentsply xp bond for extra performance. scientific compendium. konstanz: dentsply detrey; 2006. 13. koliniotou-koumpia e, kouros p, koumpia e, helvatzoglou-antoniades m. shear bond strength of a “solvent-free” adhesive versus contemporary adhesive systems. braz j oral sci. 2014; 13: 64-9. 14. münchow ea, bossardi m, priebe tc, valente ll, zanchi ch, ogliari fa, et al. microtensile versus microshear bond strength between dental adhesives and the dentin substrate. int j adhes adhes. 2013; 46: 95-9. 15. gallo jr, burgess jo, xu x. effect of delayed application on shear bond strength of four fifth-generation bonding systems. oper dent. 2001; 26: 48-51. 16. finger wj, balkenhol m. practitioner variability effects on dentin bonding with an acetone-based one-bottle adhesive. j adhes dent. 1999; 1: 311-4. 17. jacobsen t, soderholm kj. effect of primer solvent, primer agitation, and dentin dryness on shear bond strength to dentin. am j dent. 1998; 11: 225-8. 18. reis a, loguercio ad, azevedo cl, de carvalho rm, da julio singer m, grande rh. moisture spectrum of demineralized dentin for adhesive systems with different solvent bases. j adhes dent. 2003; 5: 183-92. 19. spencer p, wang y. adhesive phase separation at the dentin interface under wet bonding conditions. j biomed mater res. 2002; 62: 447-56. 20. tay fr, gwinnett ja, wei sh. micromorphological spectrum from overdrying to overwetting acid-conditioned dentin in water-free acetonebased, single-bottle primer/adhesives. dent mater. 1996; 12: 236-44. 21. cardoso p de c, lopes gc, vieira lc, baratieri ln. effect of solvent type on microtensile bond strength of a total-etch one-bottle adhesive system to moist or dry dentin. oper dent. 2005; 30: 376-81. 22. da silva ma, rangel pm, barcellos dc, pagani c, rocha gomes torres c. bond strength of adhesive systems with different solvents to dry and wet dentin. j contemp dent pract. 2013; 14: 9-13. 23. bail m, malacarne-zanon j, silva sm, anauate-netto a, nascimento fd, amore r, et al. effect of air-drying on the solvent evaporation, degree of conversion and water sorption/solubility of dental adhesive models. j mater sci mater med. 2012; 23: 629-38. 24. ye q, spencer p, wang y, misra a. relationship of solvent to the photopolymerization process, properties, and structure in model dentin adhesives. j biomed mater res a. 2007; 80: 342-50. 25. blunck u, knitter k, jahn kr. six-month clinical evaluation of xp bond in noncarious cervical lesions. j adhes dent .2007; 9(suppl 2): 265-8. 26. hegde m, manjunath j. bond strength of newer dentin bonding agents in different clinical situations. oper dent. 2011. jun 24. [epub ahead of print]. 27. margvelashvili m, goracci c, beloica m, papacchini f, ferrari m. in vitro evaluation of bonding effectiveness to dentin of all-in-one adhesives. j dent. 2010; 38: 106-12. 28. teagarden dl, baker ds. practical aspects of lyophilization using nonaqueous co-solvent systems. eur j pharm sci. 2002; 15: 115-33. 29. takahashi m, nakajima m, hosaka k, ikeda m, foxton rm, tagami j. long-term evaluation of water sorption and ultimate tensile strength of hema-containing/-free one-step self-etch adhesives. j dent. 2011; 39: 506-12. influence of surface moisture condition on the bond strength to dentin of etch-and-rinse adhesive systems1 8 61 8 61 8 61 8 61 8 6 braz j oral sci. 13(3):182-186 oral sciences n3 braz j oral sci. 13(2):98-103 original article braz j oral sci. april | june 2014 volume 13, number 2 maxillofacial infection. a retrospective evaluation of eight years bruno veronez1, fernando pando de matos1, marcelo silva monnazzi1, alexander tadeu sverzut1, cássio edvard sverzut1, alexandre elias trivellato1 1universidade de são paulo usp, dental school of ribeirão preto, department of oral and maxillofacial surgery and periodontology, ribeirão preto, sp, brazil abstract aim: to evaluate medical records from patients who had maxillofacial infections between august 2002 and may 2010. methods: analysis of 157 patients’ data looking for epidemiology, causes of the infection, type of treatment established and complications. results: 113 patients had odontogenic infections and 44 had non-odontogenic infections. the white ethnicity was the most affected (64.33%), prevailing the men (53.5%). the odontogenic infections occurred mostly at the mandible (55.74%), involving the posterior teeth (82%). dental decay was the main etiology (90.90%). the most affected facial anatomic region was the submandibular (42.85%). surgical treatment was required in 76% of the cases. conclusions: maxillofacial infections should be treated as soon as possible. even without culture and antibiogram results, it was possible to treat the infection and to reestablish tissue function. keywords: focal infection, dental; epidemiology; retrospective studies. introduction maxillofacial infections are not rare and could be considered as a public health problem due to their great potential of spreading to important and vital anatomical structures, such as the respiratory system and mediastinum, increasing the risk of septicemia and death for the affected patients1-2. usually those infections are limited and easily treated3. however, there are cases of fatal outcomes due to airway obstruction, since they can progress rapidly if not adequately treated 3. maxillofacial infections (mi) are characterized as polymicrobial, endogenous, opportunistic, dynamical and mixed (aerobic and anaerobic bacteria)4. the literature shows that mi affects mainly male patients either in adult or child populations. the odontogenic infection (oi) has multiple possible triggering factors. it could be related to dental decay, periapical and/or periodontal abscess, pericoronitis, pulpitis, osteitis, apart from others5-7. peterson et al. (2002)3 point out two major causes for the oi, the periapical (due to pulp necrosis and subsequent bacterial invasion) and the periodontal (as a result of periodontal disease) that allows inoculation of bacteria into deep tissues. studies concerning the facial infections are needed to evaluate the treated population and to upgrade whenever required the initial evaluation and treatment in order to improve the results. these studies are also an important contribution to the expertise education8-9. the aim of this study was to evaluate retrospectively the epidemiology, treatment and complications of the mi in patients who presented odontogenic or correspondence to: marcelo silva monnazzi, rua voluntários da pátria, 2777, cep: 14801-320 ap 1001, araraquara, sp, brasil phone: + 55 16 33845822 e-mail: monnazzi@ig.com.br received for publication: march 14, 2014 accepted: april 16, 2014 braz j oral sci. 13(2):98-103 99 non-odontogenic infections in hospitals treated by our team of surgeons in ribeirão preto, sp, brazil, between august 2002 and may 2010. material and methods the present epidemiologic survey was carried out by collecting data from patient records in the department of oral and maxillofacial surgery of ribeirão preto dental school of the são paulo university – usp. the collected data ranged from august 2002 to may 2010. this research was approved by the ribeirão preto dental school (usp) ethics committee under the registration number: 2011.1.177.58.2. the medical records of all patients who presented mi of odontogenic or nonodontogenic cause during the aforementioned period were revised and included in this study, without any kind of distinction. the only exclusion criteria were incorrect or incomplete records. medical records of 157 patients were evaluated. the following data were collected: gender, age, race, first evaluation date, intervention date, medical history, addictions, dentition, oral hygiene, etiology, signs and symptoms, affected facial regions, adopted therapeutics, discharge date and complications, such as dehiscence, need for reintervention or medication change and residual scars. for patients whose treatment required surgical intervention, it was performed by the following procedures: removal of the etiologic agent, puncture and aspiration of the affected area, incision and drainage of the cellulitis and/ or abscess, necrotic tissues debridement and abundant irrigation with saline. penrose and irrigation drain insertion was done and the drains were kept for at least 48 h. the harvested secretion was sent for microbiological analysis for all patients who required surgical intervention, but all culture and antibiogram results were negative. empiric antibiotic therapy was prescribed for all patients. the first choice for antibiotic prescription was amoxicillin associated to clavulanate with the dosage of 1 g every 6 h, by intravenous administration during the hospital stay. however, in the initial years of this study were found records describing the use of cephalothin associated to metronidazole, by intravenous administration every 6 and 8 h respectively, because the amoxicillin/clavulanate was not a standard drug at the hospitals back then preoperative image exams were obtained providing radiographic diagnosis for the infection cause; the main exams were the postero-anterior mandible radiographs and in more severe cases computed tomography scans were provided. surgically treated patients received saline irrigation through irrigation drains previously inserted at the infection site, twice a day until secretion was reduced. these patients were discharged after the involution of the infectious signs and symptoms and improvement of the systemic medical conditions. at discharge time the drains were removed and the patient maintained the use of antibiotic medications by oral administration until completing seven days after surgery the follow-up protocol was weekly until completing one month postoperatively, and after that it was scheduled once every month, for at least 6 months. however, this protocol was not followed in all cases, because some patients did not return. the data were collected by a single examiner and tabulated for analysis in microsoft access® 2007 software, then the data were shown in tables and a descriptive analysis of the results was done. the authors also state having read the helsinki declaration and following its guidelines in this investigation, and that none has any kind of conflict of interest. results the medical record data collection revealed that, out of the 157 patients that were affected by facial infection, 113 cases had odontogenic cause (72.15%) and 44 patients presented nonodontogenic infection (27.85%). men were the most affected (53.5%), while women were less affected (46.50%). caucasians were more affected by the facial infection (64.33%), followed by the brown (23.41%), black (7.64%) and asian (0.63%) populations. the records revealed 3.99% files without ethnicity data. when the odontogenic infections were evaluated regarding the affected dental arch, it was verified that the mandibular arch was more affected (55.73%) in comparison to the maxillary arch (44.26%). the permanent dentition was more affected by the infectious process (87.34%), followed by the primary (4.43%) and mixed dentitions (3.79%). posterior teeth were the most related to odontogenic infections (82%) compared with anterior teeth (18%). the involved teeth were recorded in all cases and the left mandibular third molar was most often related to the odontogenic infection (13.63%), followed by the left mandibular first molar (11.3%), 48 (7.14%) and left mandibular second molar (6.49%). regarding the side of the facial involvement it was verified that the left side was most frequently affected (56.75%), followed by the right side (32.97%) and both sides at the same time (10.27%). evaluating the facial spaces affected and the respective tooth, it was found that the submandibular space was the most affected due to the left mandibular third and first molar (table 1). the canine space (10 cases) and the oral vestibulum space (8 cases) were the most affected when the infections were from the anterior teeth (table 2). the oral space (5 cases) was most commonly involved when the infection originated from the premolars, followed by the canine and submandibular spaces with 3 cases each (table 3). usually the deciduous teeth were related to oral vestibulum region infections (table 4). about half of the evaluated patients presented very bad oral hygiene during the initial clinical examination (50.63%), 18.98% of the patients presented regular oral hygiene and only 22.78% presented good oral hygiene. dental decay was the main etiologic factor for the odontogenic infections (90.90%), followed by unsatisfactory root canal treatments maxillofacial infection. a retrospective evaluation of eight years 100 (4.54%), pericoronitis (3.24%) and post-extraction complications (1.29%) (figure 1). surgical treatment was used in 76% of the cases and nonsurgical in 24%. from all the surgically treated patients, 63% had the procedures done under local anesthesia and 37% required general anesthesia. the patients managed with general anesthesia were hospitalized for 3.52 days in average, with 2.90 days from the surgery to the hospital dismissal. the follow-up of these patients was in average 30 days. relative to the drain, penrose drain number 1 was used in 38% of the cases. in 33% of the surgically managed patients no drains were used, and the association of penrose braz j oral sci. 13(2):98-103 maxillofacial infection. a retrospective evaluation of eight years facial spaces teeth * total 16 17 18 26 27 28 36 37 38 46 47 48 submandibular 0 0 0 0 0 0 8 3 9 4 6 5 35 buccal 2 0 0 3 1 2 2 3 0 3 3 1 20 sublingual 0 0 0 0 0 0 1 1 0 0 0 0 2 sublingual + submandibular 0 0 0 0 0 0 1 1 1 0 0 1 4 parapharyngeal + submandibular 0 0 0 0 0 0 1 0 1 0 0 1 3 submandibular+buccal+submental+sublingual 0 0 0 0 0 0 1 0 0 0 0 0 1 paranasal sinuses+orbital 0 0 0 1 1 0 0 0 0 0 0 0 2 submandibular+submental+sublingual 0 0 0 0 0 0 0 0 1 0 0 0 1 retropharyngeal 0 0 0 0 0 0 0 0 1 0 0 0 1 subperiosteal 0 0 0 0 0 0 0 0 1 0 0 0 1 retropharyngeal + submandibular 0 0 0 0 0 0 0 0 1 0 0 0 1 parapharyngeal + submandibular+ buccal 0 0 0 0 0 0 0 1 0 0 0 1 2 oral vestibule 1 0 0 0 0 0 1 0 1 0 0 0 3 total 3 0 0 4 2 2 15 9 16 7 9 9 76 table 1.table 1.table 1.table 1.table 1. facial spaces affected distribution and respective posterior teeth that were the cause of the infection. * 16 – right maxillary first molar; 17 – right maxillary second molar; 18 – right maxillary third molar; 26 – left maxillary first molar; 27 – left maxillary second molar; 28 – left maxillary third molar; 36 – left mandibular first molar; 37 – left mandibular second molar; 38 – left mandibular third molar; 46 – right mandibular first molar; 47 – right mandibular second molar; 48 – right mandibular third molar. facial spaces teeth * total 11 12 13 21 22 23 31 32 33 41 42 43 canine 0 1 5 0 0 4 0 0 0 0 0 0 10 submandibular 0 0 0 0 0 0 0 0 0 0 0 1 1 sublingual 0 0 0 0 0 0 0 2 0 0 0 0 2 submandibular+submental+sublingual 0 0 0 0 0 0 0 0 1 0 0 1 2 canine + buccal 1 0 1 0 0 0 0 0 0 0 0 0 2 parapharyngeal + submandibular+ buccal 0 0 0 0 0 0 0 0 0 1 0 0 1 oral vestibule 1 1 0 2 1 1 0 0 1 0 0 1 8 total 2 2 6 2 1 5 0 2 2 1 0 3 26 table 2.table 2.table 2.table 2.table 2. facial spaces affected distribution and the respective anterior teeth that was the cause of the infection. * 11 – right maxillary central incisor. 12 – right maxillary lateral incisor; 13 – right maxillary canine; 21 – left maxillary central incisor; 22 – left maxillary lateral incisor; 23 – left maxillary canine; 31 – left mandibular central incisor; 32 – left mandibular lateral incisor; 33 – left mandibular canine; 41 – right mandibular central incisor; 42 – right mandibular lateral incisor; 43 – left maxillary canine. facial spaces teeth * total 14 15 24 25 34 35 44 45 submental 0 0 0 0 1 0 0 0 1 canine 0 0 3 0 0 0 0 0 3 submandibular 0 0 0 0 1 0 1 1 3 buccal 0 1 0 2 0 2 0 0 5 sublingual 0 0 0 0 1 0 1 0 2 canine + buccal 1 0 0 0 0 0 0 0 1 oral vestibule 1 0 0 0 0 1 0 0 2 total 2 1 3 2 3 3 2 1 17 table 3.table 3.table 3.table 3.table 3. facial spaces affected distribution and the respective premolars that was the cause of the infection. * 14 – right maxillary first premolar; 15 – right maxillary second premolar; 24 – left maxillary first premolar; 25 – left maxillary second premolar; 34 – left mandibular first premolar; 35 – left mandibular second premolar; 44 – right mandibular first premolar; 45 – right mandibular second premolar. 101 fig. 1 – study etiology distribution. drain and irrigation tubes (number 12) was used in 29% of the cases. that association was used in the most severe cases where saline irrigation through the drains was required and it was removed as soon as the secretion diminished. some patients reported paresthesia, and out of them 60% complained about the mentual nerve area and 40% about the infraorbital area; none of the patients complained of long lasting paresthesia. the association of amoxicillin with clavulanate was the more often applied antibiotic drug therapy (29.74%), followed by the association of cephalexin and metronidazole (25.94%) and amoxicillin and metronidazole (15.82%); other associations or isolated antibiotics were applied in some cases (15.82%). in this sample there was only one case in which reintervention was necessary for a new surgical incision and drainage of the facial abscess. complications like dehiscence, need for drug change and hypertrophic scar or even keloid at the incision site, were not observed in this study. discussion in this study, men were more affected (53.33%), which is in agreement with the findings in the scientific literature. although the reason is unclear, dodson et al. (1989) 10 reported that the male gender has greater tendency to suffer facial and neck traumas, which could lead to an exacerbation of chronic processes. they usually have a worse hygiene condition than women, beyond the fact that men neglect more often light infections in the mouth and face. indresano et al. (1992)11 has estimated that 80% of the population have at least one third molar and they are commonly associated to complications such as pain, swelling and decay, being one of the probable causes of deep facial space infections. in this study it was verified that the posterior teeth were the main responsible for odontogenic infections (82%), especially the posterior mandibular teeth, among which the left mandibular third molar was the most affected (13.6%). this is probably due to the higher difficulty in brushing and cleaning the posterior teeth. decay was the cause for 90.9% of the odontogenic infections, as previously described by flynn et al. (2006)12-13 who reported it as the main etiologic factor (65%). another reason for the number of decayed teeth could be a social problem found in this country. amaral et al. (2014)14 reported presence of 58.6% decayed teeth in a study that evaluated 303 children in a school of a são paulo state city; xavier et al. (2012)15 also state the need of planning educational activities and adoption of preventive policy measures to change this reality. rega et al. (2006)16 reported that the submandibular space was the most often affected in cases of facial infection. in the present study this same facial space was the most affected (30.3%). according to dodson et al. (1991)5 and schuknecht et al. (2008)17 the origin and localization of the infection are strongly related because the abscess origin defines its localization as well as the localization defines its origin. thirty seven percent of the patients were submitted to general anesthesia for the surgical treatment in this sample. the authors agree with the hospitalization criteria for patients who present facial infections described by flynn et al. (2006)12-13. according to this criterion the patients selected for hospital admission are those who presented big swelling in one or more deep facial or neck spaces that could be lifethreatening due to the proximity of vital structures and/or airway obstruction; body temperature over 38.5 °c; need of previous concomitant systemic disease control and need of surgical treatment under general anesthesia. the hospitalization period was in average of 3.52 days (time between the admission and the discharge), a period quite similar to the one found by krishnan et al. (1993)1 which was 4 days; and not similar to the results described by indresano et al. (1992)11 who presented an average of 6.2 days of hospitalization and flynn et al. (2006) 12-13 who reported an average of 5.1 days with a standard deviation of ± 3. indresano et al. (1992)11 and wang et al. (2005)2 state that long-term hospitalization could become a negative factor for the patient and the society. braz j oral sci. 13(2):98-103 maxillofacial infection. a retrospective evaluation of eight years facial spaces teeth * total 54 84 51 64 74 buccal 1 1 0 0 0 2 oral vestibule 0 0 1 3 1 5 total 1 1 1 3 1 7 table 4.table 4.table 4.table 4.table 4. facial space distribution and the respective deciduous teeth that was the cause of the infection. * 54 – right maxillary first decidous molar; 84 – right mandibular first deciduous molar; 51 – right maxillary deciduous central incisor; 64 – left maxillary first deciduous molar; 74 – left mandibular deciduous first molar. 102 according to peterson et al. (2002)3 and miloro et al. (2011)18 the main objectives of the facial infection treatment are: pain relief, function recovery, vital structure preservation, to prevent flare or relapses and to limit the incapacity period. in the present study the surgical treatment was performed in 76% of the cases and was made in order to extinguish the etiologic agent, puncture and aspiration of the affected area (for secretion examination by culture and antibiogram), cellulitis and facial abscess incision and drainage, necrotic tissues removal, abundant saline irrigation, drains and irrigation tubes insertion and daily dressing. nonsurgical treatment was adopted in 24% of the total cases, usually where the infection was chronic and limited. all patients included in this study had complete resolution of the infectious disease and perfect reestablishment of the affected tissues. this agrees with wang et al. (2005)2 who stated that microbiologic culture and antibiogram do not seem to be clinically useful; probably because the surgical treatment performed adequately and the previous knowledge of the microbiology involved in facial and oral infection sites lead to an empiric choice of antibiotic medication that could be employed with a considerable confidence level18. according to peterson (2002)3 and miloro et al. (2011)18 the antibiotic medication choice must follow four criteria. first, the antibiotic must be effective against all the microorganisms that are usually responsible for facial infections. second, the medication should have a limited spectrum, when possible, in order not to interfere on the normal microbiota of the patient. third, the antibiotic must be as less toxic as possible and fourth, the drug must be bactericidal, because the patient affected by facial infection could be defenseless and the bacteriostatic antibiotics could lead to a slow recovery. rega et al. (2006)16 state that the penicillins still are the empiric first choice antibiotic for odontogenic infections, due to their effectiveness, minimal side effects, low cost, good patient tolerance and easy accessibility. other authors state that basic beta lactum antibiotics are key antibiotics to start treating odontogenic infections, as these infections are predominantly of gram-positive aerobes19-20. on the other hand, some authors21 described the organisms causing infection as aerobes (68.2%), mixed (13.6%) and anaerobes (9.1%), and they state that according to their results the microflora-causing maxillofacial infection did not change, and penicillin remains the drug of choice in treating these infections21. only in one case the reintervention was needed in this sample, and it was the single complication found by the authors in this study. it was the case of a woman (53 years old) who used chronic steroids, and probably had the organic defense capacity decreased due to the immunosuppressive properties of the drug. additionally, for infections with special difficulty to treat, fungal contamination should be investigated22. based on the results, the authors concluded that the facial infections should be promptly treated by proper antibiotic therapy and surgical management; and even without the results for culture and antibiogram the authors achieved success in all facial infections treated. acknowledgements fapesp for the financial support to this study (n°2010/ 09430-0). references 1. krishnan v, johnson jv, helfrick jf. management of maxillofacial infections: a review of 50 cases. j oral maxillofac surg. 1993; 51: 868-73. 2. wang j, ahani a, pogrel ma. a five-year retrospective study of odontogenic maxillofacial infections in a large urban public hospital. int j oral maxillofac surg. 2005; 34: 646-9. 3. peterson lj, ellis iii e, hupp jr, tucker mr. contemporary oral and maxillofacial surgery. saint louis: mosby; 2002. p.343-79. 4. maestre vjr. treatment options in odontogenic infection. med oral patol oral cir bucal. 2004; 9(suppl): s19-31. 5. dodson tb, barton ja, kaban lb. predictors of outcome in children hospitalized with maxillofacial infections: a linear logistic model. j oral maxillofac surg. 1991; 49: 838-42. 6. sánchez r, mirada e, arias j, paño jr, burqueño m. severe odontogenic infections: epidemiological, microbiological and therapeutic factors. med oral patol oral cir bucal. 2011; 16: e670-6. 7. mckellop ja, bou-assaly w, mukherji sk. emergency head & neck imaging: infections and inflammatory processes. neuroimag clin n am. 2010; 20: 651-61. 8. mathew gc, ranganathan lk, gandhi s, jacob me, singh i, solanki m, et al. odontogenic maxillofacial space infections at a tertiary care center in north india: a five-year retrospective study. int j infect dis. 2012; 16: e296-302. 9. sato frl, hajala fac, freire filho fwv, moreira rwf, moraes m. eightyear retrospective study of odontogenic origin infections in a postgraduation program in oral and maxillofacial surgery. j oral maxillofac surg. 2009; 67: 1092-7. 10. dodson tb, perrott dh, kaban lb. pediatric maxillofacial infections: a retrospective study of 113 patients. j oral maxillofac surg. 1989; 47: 327-30. 11. indresano at, haug rh, hoffman mj. the third molar as a cause of deep space infections. j oral maxillofac surg. 1992; 50: 33-5. 12. flynn tr, shanti rm, levi mh, adamo ak, kraut ra, trieger n. severe odontogenic infections, part 1: prospective report. j oral maxillofac surg. 2006; 64: 1093-103. 13. flynn tr, shanti rm, hayes c. severe odontogenic infections, part 2: prospective outcomes study. j oral maxillofac surg. 2006; 64: 1104-13. 14. amaral rc, batista mj, meirelles mpmr, cypriano s, sousa mlr. dental caries trends among preschool children in indaiatuba, sp. brazil. braz j oral sci. 2014; 13: 1-5. 15. xavier a, carvalho fs, bastos rs, caldana ml, bastos jrm. dental caries-related quality of life and socioeconomic status of preschool children, bauru, sp. braz j oral sci. 2012; 11: 463-8. 16. rega aj, aziz sr, ziccardi vb. microbiology and antibiotic sensitivities of head and neck space infections of odontogenic origin. j oral maxillofac surg. 2006; 64: 1377-80. 17. schuknecht b, stergiou g, graetz k. masticator space abscess derived from odontogenic infection: imaging manifestation and pathways of extension depicted by ct and mr in 30 patients. eur radiol. 2008;18: 1972-9. 18. miloro m, ghali ge, larsen p, waite p. peterson´s principles of oral and maxillofacial surgery. shelton: pmph-usa; 2011. p.841-61. 19. kuriyama t, nakagawa k, karasawa t, saiki y, yamamoto e, nakamura s. past administration of beta-lactam antibiotics and increase in the emergence of beta-lactamase-producing bacteria in patients with orofacial odontogenic infection. oral surg oral med oral pathol oral radiol endod. 2000; 89: 186-92. braz j oral sci. 13(2):98-103 maxillofacial infection. a retrospective evaluation of eight years 103 20. walia is, borle rm, mehendiratta d, yadav ao. microbiology and antibiotic sensitivity of head and neck space infections of odontogenic origin. j maxillofac oral surg. 2014; 13: 16-21. 21. yuvaraj v, alexander m, pasupathy s. microflora in maxillofacial infection a changing scenario? j oral maxillofac surg. 2012; 70: 119-25. 22. almeida op, scully c. fungal infections of the mouth. braz j oral sci. 2002; 1: 19-26. braz j oral sci. 13(2):98-103 maxillofacial infection. a retrospective evaluation of eight years oral sciences n3 braz j oral sci. 12(1):20-22 original article braz j oral sci. january | march 2013 volume 12, number 1 is depression associated with periodontal status in elderly? leonel ramonnd ferreira viana1, consuelo penha castro1, hellen-bry wanderley pereira2, adriana de fátima vasconcelos pereira1, fernanda ferreira lopes1 1department of dentistry, dental school, federal university of maranhão, são luís, ma, brazil 2federal university of maranhão, são luís, ma, brazil correspondence to: fernanda ferreira lopes rua das jaqueiras, no.5, qd.55 bairro renascença i, cep: 65075-220 são luís, ma, brasil phone: +55 98 33018575 / 33018577 e-mail: fernanda.f.lopes@gmail.com abstract aim: to examine whether the depression is associated with periodontitis in elderly and to evaluate oral hygiene of these patients. methods: one hundred and ninety one individuals aged 60 years or more were randomly selected. periodontal examination was performed in full mouth by probing depth and clinical attachment loss in 6 sites for each tooth. the simplified oral hygiene index (ohi-s) per individual was also obtained. depression was assessed by the geriatric depression scale (gds-15) to show how the elderly have been feeling during the last week. results: only sohi was statistically significant (t = 4.7169, p<0.001), which better explains the variance in periodontal status. the variable gds-15 revealed no significant values (t=0.3901, p=0.6971). conclusions: there was no association between periodontitis and depression in elderly, but there was association between periodontitis and oral hygiene. keywords: depression, periodontitis, oral hygiene, elderly. introduction aging is a constant and complex process that occurs in all types of body cells, although it presents special features in certain organs and systems1. according to the world health organization (who), the chronological level of 60 years of age is used to define an elderly population in developing countries2. oral conditions and depressive symptoms in elderly have been little studied. there are few studies about the association between periodontal disease and depression in elderly3; however, several studies have addressed the association between periodontal disease and stress4-6. the depressed activity of neutrophils and macrophages linked to human physical and psychological states, such as family problems, aging and unemployment, can clarify the role of depression on periodontal condition7. considering that depression and periodontal diseases are accompanied by an activation of inflammatory responses, the aim of this study was to examine whether depression in elderly may be a systemic factor associated with periodontitis and to evaluate oral hygiene of these patients. material and methods this cross-sectional study was conducted in accordance with the resolution 196/96 of the brazilian national health council and complementary, and was received for publication: november 24, 2012 accepted: march 04, 2013 2121212121 braz j oral sci. 12(1):20-22 approved by the ethics committee of the federal university of maranhão (protocol number 2315-012694/2008-90). population and sample sample size calculation was performed using the bioestat 3.0 (ayres m., mct-cnpq, belém, pa, brazil), which indicated a minimum sample of 188 elders with a power test of 80% and alpha level of 5%8. the sample was formed by random number table and was composed by 191 subjects aged 60 years or more from those attending the university for thirdage persons of federal university of maranhão. smokers, edentulous individuals and those who underwent periodontal treatment less than 6 months before were excluded. data collection a questionnaire was used to assess personal health history and physical factors. the geriatric depression scale (gds15) was applied to detect depressive symptoms in elderly by 15 negative/affirmative questions. one point was assigned for each positive answer, where 0-4 points was considered normal, 5-10 points were diagnosed with a probable light depression and 11-15 points with a probable severe depression9. a full-mouth periodontal examination was performed. the extent measure of attachment loss was performed by measurements of probing depth (pd) and clinical attachment loss (cal) using a williams periodontal probe (hu friedy®, chicago, il, usa) parallel to the tooth long axis10. the simplified oral hygiene index (ohi-s) for every individual11 was also obtained, and classified as adequate and inadequate (regular and poor). severe periodontitis was diagnosed based on pd ³ 4mm and cale” 5mm12. statistical analysis logistic regression model was used to verify whether at least one of the independent variables (the ohi-s and gds-15) had influenced the dependent variable (severe periodontitis). the chi-square test was used to analyze the distribution of severe periodontitis, oral hygiene and geriatric depression. for all the tests was used the significance level of 5%. results among the 191 participants, 156 (81.7%) were female and 35 (18.3%) were male aged 60 years or more, and the table 2ordinal logistic regression analysis measuring the outcome variable (periodontal status) and exposure variables (depression, oral hygiene). variable or confidence intervals (95%) p depression 1.1102 0.541 to 2.228 0.7755 oral hygiene 3.4060 1.930 to 6.012 <0.001 variable severe periodontitis absent present oral hygiene adequate inadequate n o depression 63 75 27 111 probable mild/severe depression 24 29 4 49 total 87 104 31 160 p * 0.9634 0.0437 table 1sample distribution according to severe periodontitis, oral hygiene and geriatric depression scale (gds-15). *chi-square test (α=0.05) mean age was 68.8 years. the educational status of the elderly was an average 5.6 years of study. most participants were married corresponding to 71 (37.17%), followed by widowed (64/33.51%), single (40/20.94%) and divorced (16/8.38%). severe periodontitis was present in 104 (54.4%) elders, while 160 (83.7%) revealed inadequate oral hygiene and 138 (72.3%) did not have depression. table 1 illustrates the sample distribution according to depression, oral hygiene and severe periodontitis. ordinal logistic regression model measured the outcome variable (periodontal status) and the exposure variables (depression, oral hygiene) and showed an association between periodontal status and oral hygiene, which was used as adjustment for confounding bias, and lack of association between periodontal status and depression in elderly (table 2). the periodontal dependent variable and the independent variables, ohi-s and depression, were also submitted to the multiple regression test, which showed the value of f (regression) as significant (f=11.4549, p<0.001), accepting that at least one of the variables had influence on periodontal status. among the partial regression coefficients, only the ohi-s was statistically significant (t=4.7169, p<0.001) which may better explain the variance on periodontal status. the gds-15 revealed no significant values (t=0.3901, p=0.6971). discussion depression is the result of several interdependent neurobehavioral symptoms. the variation may be explained by stress factors that have been associated with a decrease in immune function and increase of the susceptibility to infections. moreover, if the relation stress/periodontal disease is true, greater prevalence of this disease can be found among patients with severe depression5. this study evaluated depression as a perceived risk factor associated with periodontal disease in elderly, but no is depression associated with periodontal status in elderly? braz j oral sci. 12(1):20-22 significant association was found between these variables. similar data were observed by peruzzo et al.5, who found no association between depression and established periodontitis in subjects with an age range of 19 to 67 years. tobacco smoking has been associated with an increased prevalence and severity of periodontal diseases, and there is evidence that systemic and local malondialdehyde (mda) levels are increased by smoking in addition to having an impact on periodontitis13. in this study, smokers were excluded to eliminate this potential confounder, based on the fact that smoking may be considered a risk factor for further periodontal disease progression among healthy elderly people aged 70 years and over8. periodontal disease in elderly is a common process of aging and there were no signiûcant correlations between mean serum levels of disease markers and additional attachment loss8, so the risk factors for systemic diseases such as diabetes and coronary heart disease were included in our study. the world health organization2 classifies the aging process in four stages: the middle-elderly from 45 to 59 years old, the elderly from 60 to 74 years old, the ancient from 75 to 90 years old and extreme old age from 90 years onwards. thus, the age group chosen for evaluation in this study was composed by elders aged 60 years or more, since the epidemiological studies indicated greater severity6,14 and prevalence15 of periodontal disease in people over 50 years6,14-15. a case-control study has been conducted to determine whether severe periodontitis and its treatment are associated with oxidative stress16. the authors found that patients with severe periodontitis exhibited higher diacron-reactive oxygen metabolites (d-rom) levels, so they suggest a positive association between severe periodontitis and oxidative stress. croucher et al.17 showed that periodontitis may be related to psychological stress in adults. however, these studies have non-homogenous samples, so it is possible that depression among young and old subjects is different and the elderly who reported depression may only have developed this condition recently, instead of having a psychiatric disorder earlier in life. periodontal examination was conducted in all teeth and not only in the index teeth. this choice provides the best way to assess accurately the prevalence and severity of periodontal disease in a population18. the partial examination underestimates the extension, severity and prevalence of periodontitis when compared with the full mouth method10. within the limitations of this study, it was concluded that there was no difference in frequency distribution of periodontitis between elderly with depression or not, but the elderly with depression showed significantly more inadequate oral hygiene. so, there was no association between periodontitis and depression in elderly, but there was association between periodontitis and inadequate oral hygiene, which it was used as adjustment for confounding bias. due to the high frequency of inadequate oral hygiene in elderly, great need for dental care to this age group is required, so this study has provided data to emphasize the need of dentists in multidisciplinary elderly care teams. acknowledgements the authors thank the state of maranhão research foundation (fapema) for the scholarship grant to the graduate program. references 1. oliveira mc, schoffen jpf. oxidative stress action in cellular aging. braz arch biol technol. 2010; 53: 1333-42. 2. who library cataloguing in publication data. integrating poverty and gender into health programmes: a sourcebook for health professionals: module on ageing. geneva: who; 2006. 3. persson gr, persson re, macentee ci, wyatt cc, hollender lg, kiyak ha. periodontitis and perceived risk for periodontitis in elders with evidence of depression. j clin periodontol. 2003; 30: 691-6. 4. goyal s, jajoo s, nagappa g, rao g. estimation of relationship between psychosocial stress and periodontal status using serum cortisol level: a clinico-biochemical study. indian j dent res. 2011; 22: 6-9. 5. peruzzo dc, benatti bb, ambrosano gm, nogueira-filho gr, sallum ea, casati mz et al. a systematic review of stress and psychological factors as possible risk factors for periodontal disease. j periodontol. 2007; 78: 1491-1504. 6. wimmer g, janda m, wieselmann-penkner k, jakse n, polansky r, pertl c. coping with stress: its influence on periodontal disease. j periodontol. 2002; 73: 1343-51. 7. bartolomucci a. social stress, immune functions and disease in rodents. front neuroendocrinol. 2007; 28: 28-49. 8. ogawa h, yoshihara a, amarasena n, hirotomi t, miyazaki h. risk factors for periodontal disease progression among elderly people. j clin periodontol. 2002; 29: 592-7. 9. sousa rl, medeiros jgm, moura acl, souza clm, moreira if. validity and reliability of the geriatric depression scale for the identification of depressed patients in a general hospital. j bras psiquiatr. 2007; 56: 1027. 10. borges-yáñez as, irigoyen-camacho me, maupomé g. risk factors and prevalence of periodontitis in community-dwelling elders in mexico. j clin periodontol. 2006; 33: 184-94. 11. albandar, jm, rams te. global epidemiology of periodontal diseases: an overview. periodontol 2000. 2002; 29: 7-10. 12. armitage gc. development of a classification system for periodontal diseases and conditions. ann periodontol. 1999; 4: 1-6. 13. tonguç mö, öztürk o,sütçü r, ceyhan bm, kýlýnç g, sönmez y, yetkin ay z et al. the impact of smoking status on antioxidant enzyme activity and malondialdehyde levels in chronic periodontitis. j periodontol. 2011 82: 1320-8. 14. hilgert jb, hugo fn, bandeira dr, bozzetti mc. stress, cortisol, and periodontitis in a population elderly 50 years and over. j dent res. 2006; 85: 324-8. 15. heitz-mayfield lj, schätzle m, löe h, bürgin w, anerud a, boysen h, lang np. clinical course of chronic periodontitis. ii. incidence, characteristics and time of occurrence of the initial periodontal lesion. j clin periodontol. 2003; 30: 902-8. 16. d’aiuto f, nibali l, parkar m, patel k, suvan j, donos n. oxidative stress, systemic inflammation, and severe periodontitis.j dent res. 2010; 89: 1241-6. 17. croucher r, marcenes ws, torres mc, hughes f, sheiham a. the relationship between life events and periodontitis. a case control study. j clin periodontol. 1997; 24: 39-43. 18. ramachandra ss, mehta ds, sandesh n, baliga v, amarnath j. periodontal probing systems: a review of available equipment. compend contin educ dent. 2011; 32: 71-77. 2222222222is depression associated with periodontal status in elderly? oral sciences n3 braz j oral sci. 10(3):217-220 original article braz j oral sci. july | september 2011 volume 10, number 3 clinical assessment of masticatory efficiency in the rehabilitation of edentulous patients maria elioneide de oliveira apolinário1, wilson mestriner junior2, fábio roberto dametto3, cícero romão gadê-neto3, samira albuquerque de sousa3 1graduate student, master degree program in dentistry, department of dentistry, area of prosthetic dentistry, dental school, potiguar university (laureate international universities), natal, rn, brazil 2department of pediatric clinics , preventive and social dentistry, faculty of dentistry of ribeirão preto, university of são paulo, ribeirão preto, sp, brazil 3dds, msc, phd, professor, graduate program in dentistry, department of dentistry, area of integrated clinical, potiguar university (laureate international universities), natal, rn, brazil correspondence to: maria elioneide de oliveira apolinário faculdade de odontologia da universidade potiguar – unp av. senador salgado filho, 1610, lagoa nova natal-rn, brazil cep 59.056-000 phone: +55 84 215 1272 fax: +55 84 215 1230 e-mail: eli.one.ide@hotmail.com abstract rehabilitation of edentulous patients has been a constant concern in dentistry. several studies have reported a reduction in the masticatory function in these patients. osseointegrated implants have been used in order to obtain better masticatory efficiency, but more studies are needed to confirm these results. aim: to evaluate the masticatory function of patients with conventional dentures and implant-supported dentures. methods: a double-blinded controlled clinical study was conducted. the sample was composed of 60 patients divided into three groups: g1 with 20 patients with conventional upper (maxillary) and lower (mandibular) complete dentures, g2 with 20 patients with mandibular overdentures and upper (maxillary) complete dentures, and g3 with 20 patients with lower fixed implant-supported complete dentures (protocol). objective data were collected through the masticatory efficiency test performed by the colorimetric method with the beads, in which capsules of a synthetic material enclosing fuchsine-containing granules were used. results: a statistically significant difference was found for masticatory efficiency between groups g1 and g2 (pd”0.05) and between g1 and g3 (pd”0.05), and there was no statistically significant difference between g2 and g3 (pe”0.05). conclusions: the results suggest that placement of osseointegrated implants in complete denture wearers improves their masticatory efficiency. keywords: complete dentures, complete dentures over implant, masticatory efficiency, edentulousness. introduction reestablishing the masticatory function is fundamental to preserve the stomatognathic system’s health. the purpose of rehabilitating completely edentulous patients by replacing lost teeth is to provide adequate physical, functional and psychological rehabilitation. good performance of mastication is related to the dental conditions. individuals with complete natural dentition show high masticatory performance rates, while edentulous individuals show minimal performance. however, according to the type of dentition and rehabilitation procedure performed, there are intermediate rates between the extreme ones1. mastication is therefore considered one of the most important functions of the stomatognathic system for it is the initial stage of the digestive procedure. on received for publication: july 12, 2011 accepted: september 09, 2011 braz j oral sci. 10(3):217-220 218 the other hand, ingested food bolus properties may be affected by the oral conditions2. before the advent of osseointegrated implants, there were no rehabilitation options for completely edentulous patients other than the mucosa-supported complete dentures. several patients did not feel safe with this treatment due to the poor retention of the mandibular denture in comparison with the maxillary one. a previous study has shown that 22% of the examined patients were unsatisfied with their maxillary complete dentures and 55% with their mandibular dentures3. at present, completely edentulous patients may be rehabilitated by conventional complete dentures (cd) or implant-supported complete dentures (overdentures or protocol). the use of implants to support dentures significantly improves the masticatory performance of these individuals. the assessment of the masticatory function is undoubtedly a method to determine the effectiveness of rehabilitation procedures mentioned for edentulous patients by using objective tests that measure masticatory efficiency through artificial test materials4-8. there are several studies that show a reduction in the masticatory efficiency of edentulous patients rehabilitated with bimaxillary complete dentures9-10. however, few studies have compared the different types of rehabilitations for this group of patients by assessing their masticatory efficiency. the aim of this study was to compare clinically the masticatory efficiency of edentulous patients rehabilitated with conventional complete dentures and implant-supported complete dentures, seeking scientific evidences of the benefits for the masticatory function of these types of prosthetic rehabilitations. material and methods the clinical study protocol was submitted to the ethics committee of the potiguar university (laureate international universities) and approved in accordance with the report no nº156/2009. sixty volunteers of both sexes were selected from the patients treated at the study center of osseointegrated implants of the department of dentistry at the federal university of rio grande do norte (ufrn) and the dental research and study center of paraiba (coesp), brazil. the volunteers were included in the study if they had bimaxillary conventional complete dentures or maxillary conventional complete dentures, but not implant-supported complete mandibular dentures. those who did not have good general health, intraoral conditions, and dentures in satisfactory conditions and minimum time of use from 3 to 6 months were excluded. an informed consent form was signed by all the participants before the beginning of the study. the volunteers were divided into 3 groups. the first group was composed of 20 completely edentulous volunteers, 17 women and 3 men (mean age of 65.55 ± 10 years), rehabilitated with conventional bimaxillary complete dentures. the second group was composed of 20 volunteers, 18 women and 2 men (mean age of 62.60 ± 8.88 years), rehabilitated with mucosa-implant-supported complete mandibular overdentures (miscod) and maxillary conventional complete dentures. the third group was composed of 20 volunteers, 14 women and 6 men (mean age of 64.07 ± 10.30 years), rehabilitated with mandibular fixed implant-supported dentures and maxillary conventional complete dentures. assessment of the masticatory function the masticatory function of the groups studied was assessed by the colorimetric method. the beads were the artificial test-food used to measure masticatory efficiency8. they are composed of a pvc capsule with an internal diameter of 7.6mm and external diameter of 8.95mm. inside each capsule, there is approximately 250mg of standardized beads which contain violet fuchsine as the pigment of choice mixed and crushed with a small amount of crystalline cellulose, lactose, starch and other substances. each pigmented bead was covered with a coat of the substance eudragit e100® (rohm pharma gmhh, germany), in a standardized size of approximately 1 mm in diameter. the participants were instructed to chew the beads for 20 s, without adding any other additional instruction on how to chew it with the purpose of reproducing habitual mastication. for this reason, they were seated in a chair with a backrest and both feet supported on the ground. with regard to mastication, the grains contained inside the capsule were ground and the pigment released in proportion to the energy used, and then collected in an identified receptacle. in the laboratory, the capsule was opened and its content dissolved in 5 ml of distilled water and mechanically agitated (mechanical agitator model q.222.2 – quimis aparelhos cientificos ltda, campinas, sp, brazil) for 30 s. afterwards the solution was filtered using a 0.5% grey paper filter (qualitative paper filter; nalgon equipamentos científicos ltda., iyupeva, sp. brazil) to remove the grains which were not ground. thus, the masticatory efficiency was calculated by measuring the absorbance concentration of the color intensity of the fuchsine solution with the aid of a spectrophotometer (sp-22 uv, biospectro inc., curitiba, pr, brazil). therefore, the higher the concentration of fuchsine in the solution, the higher was absorbance and masticatory efficiency. the analysis of the beads was carried out at the analytical chemistry laboratory of the department of pharmacy of the potiguar university, rn, brazil. the absorbance values were compared with the different types of rehabilitation using the one-way analysis of variance (anova) and descriptive statistics. the variables with significant differences (p<0.05) were compared with bonferroni’s post test. results there was a statistically significant difference between groups 1 and 2 (p<0.05) and groups 1 and 3 (p<0.05). however, there was no statistically significant difference between groups 2 and 3 (p>0.05) (table1). clinical assessment of masticatory efficiency in the rehabilitation of edentulous patients 219 braz j oral sci. 10(3):217-220 groups (g) n mean(abs = absorbance) standard deviation* bimaxillary complete dentures (g1) 20 0.244 ± 0.065 a overdentures (g2) 20 0.327 ± 0.139 b protocol (g3) 20 0.301 ± 0.073 b table 1 data for masticatory efficiency. *different letters indicate statistically significant difference in accordance with the bonferroni test (pd”0.05). discussion the aim of this study was to assess if patients with implant-supported complete dentures had better masticatory efficiency than those with conventional complete dentures since there are no studies on this subject using the three methods of rehabilitation. the term masticatory efficiency was defined in this study as a degree of fragmentation of a certain food-test after mastication for a pre-determined time11-21. the predetermined mastication time in this study was 20 s, which is the time needed to breakdown the food 5-12. differently from other investigations that assessed masticatory efficiency using natural test food1-11, this clinical study used artificial test food. this method was used for being more reliable for testing masticatory efficiency, enabling greater standardization of the tests, while the physical properties of the natural food are variable and difficult to standardize, which may alter the final results5-13-9. the obtained results showed a significant difference for the masticatory efficiency between g1 and g2 and g1 and g3, but no significant difference was found between g2 and g3. the significant lower masticatory efficiency in patients rehabilitated with conventional bimaxillary complete dentures (g1) has been reported in other studies9-10, and a significant improvement was observed in the masticatory function after treatment with overdentures, irrespective of the retention modality (magnet, sphere and bar/clip)14. this was also found in other studies in which the masticatory efficiency was better for patients with overdentures in comparison with conventional complete dentures15-17. however, some studies have shown that patients with mean mandibular ridge height rehabilitated with bimaxillary complete dentures present masticatory performance similar to the ones rehabilitated with mandibular overdentures and maxillary complete dentures18. this suggests that the residual ridge height is a critical factor to assess masticatory efficiency. group g2 showed a higher masticatory efficiency mean than group g3. this result is extremely important since overdentures are a simpler and cheaper treatment option when compared with implant-supported fixed dentures. furthermore, they provide a significant improvement in terms of stability and retention for patients with severe adaptation problems to conventional mandibular dentures16. however, this unexpected result is probably due to the following factors: increase in retention and stability of overdentures using additional retentions fixed to implants, more favorable condition for mounting the teeth with a centralized occlusal platform in the crest of the mandibular ridge and the presence of a conventional complete denture as an antagonist. no studies were found in the literature that compared masticatory efficiency with the above-mentioned groups. it is also important to emphasize the diagnostic factor and previous planning before placing the implants. the human factor in planning and technical performance are decisive for the success of rehabilitations. mandibular complete overdentures and protocol dentures with conventional complete dentures as antagonists would bring more benefits for edentulous patients with great resorption of the ridges or difficulties in adapting to bimaxillary conventional complete dentures19 in addition to offering the possibility of overcoming some limitations of these dentures with regard to masticatory efficiency20. therefore, more scientific evidence must be found through clinical trials comparing these types of rehabilitation. based on the results, it may be concluded that the use of osseointegrated implants improves masticatory efficiency of patients with complete dentures. references 1. manly rs, braley lc. masticatory performance and efficiency. j dent res. 1950; 29: 448-62. 2. mioche l, bourdiol p, peyron m. influence of age on mastication: effects on eating behavior. nutr res rev. 2004; 17: 43-54. 3. brodeur jm, laurin d, vallee r, lachapelle d. nutrient intake and gastrointestinal disorders related to masticatory performance in the edentulous elderly. j prosthet dent. 1993; 70: 468-73. 4. kayser af, van der hoeven. colorimetric determination of the masticatory performance. j oral rehabil. 1977; 4: 145-8. 5. edlund j, lamm c.j. masticatory efficiency. j oral rehabil. 1980; 7: 123-30. 6. nakasima a, higashi k, ichinose m. a new, simple and accurate method for evaluating masticatory ability. j oral rehabil. 1989; 16: 373-80. 7. mestriner-jr w, mazzeto mo, felício cm, freitas o, spararo acc. comparação da eficiência mastigatória avaliada pelo uso de um método colorimétrico nas dentições decídua e permanente. j bras ortodon ortop facial. 2005; 57: 242-8. 8. escudeiro santos c, freitas o, spadaro ac, mestriner-júnior w. development of a colorimetric system for evaluation of the masticatory efficiency. braz dent j. 2006; 17: 95-9. 9. slagter ap, olthoff lw, steen wha, bosman f. comminution of food by complete-denture wearers. j dent res. 1992; 71: 380-6. 10. prado mms, borges tf, prado cj,gomes vl, neves fd. função mastigatória de indivíduos reabilitados com próteses totais mucoso suportadas. pesq bras odontoped clin integr 2006; 6: 259-66. 11. 11.helkimo e, carlsson ge, helkimo m. chewing efficiency and state of dentition, a methodologic study. acta odontol scand. 1978; 36: 33. clinical assessment of masticatory efficiency in the rehabilitation of edentulous patients braz j oral sci. 10(3):217-220 220 12. ohara a, tsukiyama y, ogawa t, koyano k. a simplified sieve method for determining masticatory performance using hydrocolloid material. j oral rehabil. 2003; 30: 927-35. 13. olthoff lw, van der bilt a, bosman f,kleizen h h. distribuition of particle sizes in food comminuted by human mastication. arch oral biol. 1984; 29: 899-903. 14. van kampen fmc, van der bilt a, cune ms, fontijn-tekamp fa, bosman f. masticatory function with implant-supportes overdentures. j dent res. 2004; 83: 708-11. 15. geertman me, slagter ap, van waas m.a, kalk w. comminution of food with mandibular implant-retained overdentures. j dent res. 1994; 73: 1858-64. 16. 16.awad ma, lund jp, shapiro sh, locker d, klemetti e, chehade a, et al. oral health status and treatment satisfaction with mandibular implant overdentures and conventional dentures: a randomized clinical trial in a senior population. int j prosthod. 2003; 16: 390-6. 17. 17.carlsson ge, lindquist lw. ten-year longitudinal study of masticatory function in edentulous patients treated with fixed complete dentures on osseointegrated implants. int j prosthodont. 1993; 7: 448-53. 18. 18.garret nr, kapur kk, hamada mo, roumanas ed, freymiller e, han t, et al. a randomized clinical trial comparing the efficacy of mandibular implant-supported overdenture in diabetic patients. part ii. comparisons of masticatory performance. j prosthet dent. 1998; 79: 632-40. 19. fueki k, kimoto k, ogawa t,garret nr. effect of implant-supported or retained dentures on masticatory performance: a systematic review. j prosthet dent. 2007; 98: 470-7. 20. allen f, mcmillan a. food selection and perceptions of chewing ability following provision or implant and conventional prostheses in complete denture weares. clin oral implant res. 2002; 13: 320-6. 21. 21.van der bilt a, mojet j, tekamp fa, abbink jh. comparing masticatory performance and mixing ability. j oral rehabil. 2010; 37: 79-84. clinical assessment of masticatory efficiency in the rehabilitation of edentulous patients isotretinoin 121 isotretinoin: action on mice tooth germs and palate eleny balducci roslindo1 vanessa camila da silva2 alessandra rezende peris3 karina gonzales silvério4 1assistant professor department of morphology – faculty of dentistry at araraquara – state university of são paulo-unesp 2graduate student, msc in periodontics program faculty of dentistry at araraquara – state university of são paulo-unesp 3graduate student – faculty of dentistry at araraquara – state university of são paulounesp 4graduate student, msc, phd in periodontics program – faculty of dentistry at araraquara – state university of são paulounesp received for publication: april 16, 2002 accepted: november 27, 2002 correspondence to: profª. drª. eleny balducci-roslindo rua humaitá, 1680, caixa postal 331 centro cep 14801-903 araraquara – sp brasil e-mail: eleny@foar.unesp.br fone/fax: 55 11 5575-1337 abstract vitamin a and its derivates, acid retinoic, tretinoin and isotretinoin, are currently used in dermatological treatments. the administration of high doses of this vitamin provokes malformation in the following systems: central nervous, cardiovascular, skeletal, brain and face. this study compares the tooth germs of the first maxillary and mandibular molars and the palate of fetal mice submitted to isotretinoin during organogenesis. twelve 60-day-old female mus musculus were divided into two groups on the 7th day of pregnancy: treated group – 2 mg isotretinoin per kg body wight, dissolved in vegetable oil, was administered orally from the 7th to the 13th day of pregnancy; control group – vegetable oil in an equivalent volume was administered orally for the same period. on the 16th day of pregnancy, the females were sacrificed, the fetuses were removed and their heads were amputted. after standard laboratory procedures, 6-mm-thick serial sections were stained with hematoxylin and eosin for light microscopy examination. the results showed that the control group had closed palates with no traces of epithelial cells and two fetuses of treated group had insufficient development in the lateral palate processes, having lack of fusion in the midline. the first molar germs of the isotretinoin-treated animals showed delayed development compared to the control animals. in conclusion, isotretinoin was shown to be toxic causing retardation of tooth germs and palate development. key words: isotretinoin, tooth germ, development, palate. braz j oral sci. october/december 2002 vol. 1 number 3 122 braz j oral sci. 1(3): 121-125 isotretinoin: action on mice tooth germs and palate introduction vitamin a acts on vertebrate organisms during ontogenesis and preand post-birth cellular differentiation process, reproduction, normal growth, maintenance of visual function, regulation and proliferation of tissue epithelial structures1. besides this, derivates of vitamin a as retinoids were observed at the endogenous form in embryonic mouse mandible being important in the formation of the dental lamina at the beginning of odontogenesis and in defining the morphology of incisors and molars6. among the synthetic derivates of vitamin a, we may find isotretinoin (13-cis-retinoic acid), currently indicated in the treatment of ichthyosis, illness of darrier, psoriasis, acne, carcinoma of basal cells and in the prevention of carcinogenesis. in spite of its clinical effectiveness, it may cause some undesirable side effects such as: depression, psychotic behaviors, hydrocephaly and facial malformation. teratologic effects of vitamin a include malformation of the central nervous, cardiovascular, skeletal, and brain systems and face deformation2. the use of isotretinoin by women during the first quarter of pregnancy caused defects in the members as well as gastric and hepatic systems of children3. it was also noticed that the critical period was between the 2nd and 5th weeks after conception. in the rats, mice and monkeys, the placental transference of 13-cis-retinoic acid during the pregnancy period was observed when the clorio-allantoic placenta was well developed7. when another derivate of vitamin a, isotretinoin, was systemically administered at the concentration of 1 mg per kg body weight to pregnant mice, the studies8,9 verified that this drug produced structural alterations in the fetus as alterations at the development of the tooth germ and in the palate closure. besides these alterations, studies4,5 observed total and partial fusion of the maxilary incisors and the mandibular molars, agenesis of the incisors, formation of diastema in the incisors region, supernumerary teeth and absence of temporomandibular articulation in mice fetuses. however, as metabolism in rodents is more active than in humans, it is necessary to correlate the exposition period of the embryo to the administered drug doses and its concentration in order to obtain the results that would confirm the described alterations in the literature. considering the lack of information on isotretinoin effect in human organism, the aim of the present work was to study the palate closure and tooth germ development of the maxillary and mandibular first molars in mice fetuses submitted to isotretinoin during the organogenesis in the concentration of 2 mg per kg. material and methods twelve 60-day-old female mus musculus (albino swiss variation) primiparous mice were used. the animals were fed with granular ration and water ad libitum. the gestation period was determined by identifying the vaginal plug as day “zero” of pregnancy after the mice mated during the night, in the proportion of two females for each male of the same species. on the 7th day of pregnancy, the females were divided into two groups: treated group – 2 mg of isotretinoin per kg body weight, dissolved in vegetable oil, was administered orally, once a day, from the 7th to the 13th day of pregnancy; control group – vegetable oil in an equivalent volume was administered orally for the same period. after 16 days of pregnancy, the females of both groups were sacrificed by ip injection of 10% chloral hydrate (4 ml/100g body weight). following an abdominal incision, the uterus was removed and placed on a petri dish containing saline solution. fetuses were removed, weigh and measured in the brain tail direction and examined macroscopically for the identification of possible morphologic alteration. after macroscopic analyses, fetuses had their heads removed and fixed in bouin solution, decalcified by morse10 solution for histological analyses of palate and first mandibular and maxillary molar germ development. the heads were embedded in paraffin, and serial 6-mm-thick slices were stained with hematoxylin and eosin. the slides were analyzed by light microscopy. results macroscopic findings all specimens in number of 6 fetuses for each female mouse were histologically analyzed using serial 6um frontal incisions of similar depth and thickness (figures 1 and 6). the macroscopic evaluation of the sacrificed fetuses on the 16th day of fetal life did not show external malformation, reabsorption or dead born mice. however, it was verified that the isotretinoin-treated fetuses presented average body size and weigth (1,13 g) inferior to the control fetuses (1,42 g). microscopic findings control group the palate was totally fused without epithelial cells remaining in the fusion line of the palate process with the nasal septum (figure 1). bone trabeculae with variable size and thickness growing toward the midline, containing in its surface osteoblasts was also observed (figure 2). tooth germs in cap stage were connected to oral epithelium through the dental lamina. figures 3 and 4, demonstrate that the enamel organ was constituted by outer epithelium formed with a layer of cubic cells and the stellate reticulum formed by polygonal cells. the intermediary stratum was constitued by elongated cells. adjacently, the internal ephitelium in the cusp area was formed by highly cylindrical cells arranged perpendicularly to the dental papilla forming the preameloblasts, and in the other areas the cells were short and cylindrical. in the peripheral cells of the dental papilla, 123 especially in cusp regions, short cylindrical cells were identified, the future odontoblasts arranged in parallel exhibiting central rounded nuclei. in the central region, it was possible to observe a loose connective tissue, highly cellularized, containing ectomesenchymal cells, fibroblasts, and blood vessels of small diameter. the dental follicles involving the whole embryo were constituted by loose connective tissue rich in cells arranged in parallel rows with dental organ surface. in the lateral areas of the dental organs, neoformed thin immature bone trabeculae was noticed, with large medullar space, showing osteoblasts in their periphery and osteocytes identified in the interior of mature trabeculae. treated group the microscopic analyses revealed that the majority of the specimens had fused palates however, two fetuses p r e s e n t e d i n s u f f i c i e n t f u s i o n i n t h e l a t e r a l p a l a t e processes, having lack of fusion in the midline (figure 5). the fetuses with fused palates showed an absence of b o n e t r a b e c u l a e w i t h c e l l u l a r d i f f e r e n t i a t i o n a n d m u l t i p l i c a t i o n m a i n l y a r o u n d t h e b l o o d v e s s e l s characteristics of the beginning and end of the ossification process (figure 7). the tooth germs of the first molars were found in different development stages including the cap or initial bellshaped phase for some inferior embryos (figures 8, 9, and 10). in the cap phase (figure 9), three different layers of cells were observed. the external layer was constituted by cuboid cells forming the external epithelium. the inner l a y e r, o r i n n e r e p i t h e l i u m , w a s c o n s t i t u t e d b y s h o r t cylindrical cells in the cusp regions, and in the others r e g i o n s b y c u b o i d c e l l s . o n t h e c e n t r a l l a y e r, a n agglomeration of rounded and indifferentiated cells that would form the stellate reticulum and the intermediary stratum with discrete deposition of intracellular substance between stellate reticulum cells beginning a differentiation of enamel organ cell layers was noticed. figures 8, 9 and 10, demonstrate a higher concentration of ectomesenchymal cells and small diameter vessels in the concavity of the enamel organ. beyond the dental f o l l i c l e a r o u n d t h e d e n t a l g e r m w a s c o n s t i t u t e d b y connective loose tissue highly cellularized with fibroblasts arranged in parallel rows, except in the region of dental lamina. adjacent to the dental organ, in the majority of specimens, thin irregular immature bone trabeculae with wide medullar spaces, showing osteoblasts with spherical nuclei located on its surface was noticed. osteocytes in small number were seen in the interior of mature bone trabeculae. figure 1 – control group – frontal slice of the head of embryos. h&e. magnification: 32x. figure 2 – control group – close palatal processes in the midline and formation of bone trabeculae. h&e. magnification: 125x. figure 3 – control group – first maxillary molar germs. cap phase. h&e. magnification: 125x. braz j oral sci. 1(3): 121-125 isotretinoin: action on mice tooth germs and palate figure 4 – control group – first mandibular molar germs. cap phase. h&e. magnification: 125x. 124 discussion odontogenesis in mice begins at the 9th day of fetal life and the formation of dental bud occurs at the beginning of the 12th day11,12. in this period, drugs that have teratogenic potential, such as vitamin a and derivates, may cause the formation of the supernumerary bud and molar substitution by incisors in the molar region, as verified by kronmiller et al.5 when they administered all-trans-retinoic acid in the exogenous form in the period previous to the formation of the dental lamina. studies13,14 that administered isotretinoin and its metabolie 4-oxi-isotretinoin to pregnant female mice, showed that these drugs may exert inhibitory effect on the migration of the neural-crested cells, besides promoting reduction of members and palate opening. ritchie & webster15 carried out some studies with the purpose of determining the teratogenicity of isotretinoin and showed in vitro that use of this drug in the concentration of 500 ng/ml for a period of a least 6 hours of exposure before the migration of the cells in the neural crest, was enough to induce severe defects on the second visceral arch in most of the exposed embryos. these results are likely to explain the two fetuses with palate braz j oral sci. 1(3): 121-125 isotretinoin: action on mice tooth germs and palate figure 7 – treated group – closed palatal processes in the midline and no formation of bone trabeculae. h&e. magnification: 125x. figure 6 – treated group frontal slice of the head of embryos. h&e. magnification: 32x figure 8 – treated group – first maxillary molar germs. bellshaped phase. h&e. magnification: 125x. figure 9 – treated group – first mandibular molar germ. bellshaped phase. h&e. magnification: 125x. figure10 – treated group – first mandibular molar germ. cap phase. h&e. magnification: 125x. figure 5 – treated group – palatal processes having lack of fusion in the midline. h&e. magnification: 125x. 125 opening, caused by the insufficient development of lateral palatine processes, when 2 mg of isotretinoin per kg of body weight was administered to pregnant female mice, during the period between the 7th and the 13th day of gestation. concerning odontogenesis, we know that tooth development is related to a series of complex inductive interations between two embryonic tissues, the epithelium in the first branchial arch and the ectomesenchyme derived from neural crest cells. in the gastrula phase of embryonic development, the ectoderm and the mesoderm respond more to the excess of vitamin a, and the endoderm responds less 16,17. during organogenesis, vitamin a produces an interruption in the cephalic ectomesenchyme or alters significantly cell properties18. subsequently, the action of vitamin a has more negative effects on bones, cartilages, and dental organs than positive effects on growth in general. it is knwon that vitamin a is mobilized in liver stocks and it is taken to the peripheral tissues by means of highly regulated transportation. takahashi & smith19 verified that in rat embryos, during the pregnancy period, the presence of protein connects to retinol (rbp) and that the transportation system works in the intra-uterine life of mammals. the presence of those proteins in fetal organs was also studied by lorente & miller20, who verified that a direct action of vitamin a on tissues and organs is more probable than placental alterations. in the literature we did not find histological works on placentas showing the systemic action of isotretinoin. however, we observed in this study some placental alterations in the treated group (not shown). these alterations indicated an unfavourable development for their gestational age. since the development of teeth can be influenced by nutritional status21,22 the placental alterations would no be responsible for the embryological alterations caused by isotretinoin. the data obtained from the fetuses from the treated group suggest that isotretinoin, even when used in low dose, 2 mg per kg of body weight, but administered from the 7th to the 13th day of pregnancy may cause insufficient development and growth of the lateral palatine processes, delay in the development of the first mandibular and maxillary tooth germs, and lower size and weight, when compared to the fetuses from the controlled group. when balducci-roslindo9 et al. administered 1 mg per kg of isotretinoin in female mice during the period from the 7th to the 13th day of pregnancy, they noted the same alterations in fetal development and growth and in their bone formation. we can conclude that isotretinoin had an adversed effect on the size and weigth of the fetuses examed. additionaly, this drug may affect the formation of bone tissue in the palatine process and a delay in the development of the first mandibular and maxillary molars. acknowledgements the authors would like to thank the financial support provided by pibic/cnpq (1998-1999). references 1 . di giovanna jj. retinoids for the future: oncology. j acad dermatol 1992; 27: 534-7. 2 . lammer ej, chen dt, hoer rn. retinoic acid embryopathy. n engl j med 1985; 313: 837-41. 3 . rosa fw, wilk al, kelsey fo. teratogen update: vitamin a congeners. teratology 1986; 33: 355-64. 4 . knudsen pa. fusion of upper incisors at bud or cap stage in mouse embryos with exencephaly induced by hipervitaminose a. acta odont sacnd 1965; 23: 549-65. 5 . kronmiller je, beeman cs, nguyent, t, berndt, w. blockade of the initration of murine odontogenesis in vitro by citral, an inhibitor of endoge nous retinoic. acid synthesis. arch oral biol 1993; 40: 646-52. 6 . beeman cs, kronmiller je. temporal distribution of endogenous retenoids in the embryonio mouse mandible. arch oral biol 1994; 39: 733-9. 7 . tzimas g, collins md, nau h. developmental stage-associated differences in the transplacental distribution of 13-cis-and alltrans-retinoic acid as well as their glucuronides in rats and mice. toxicol appl pharmacol 1995; 133: 91-101. 8 . barboza mcjp, jorge m a, balducci-roslindo e, gonzaga hfs. estudo da ação da isotretinoína sistêmica na organogênese e na estrutura placentária de fetos de camundongos. ix congresso de iniciação científica,campus de jaboticabal-unesp, out. 1997. p.145. resumos. 9 . balducci-roslindo e, silvério kg, jorge ma, gonzaga hfs. effect of isotretinoin on tooth germ and palate development in mouse embryos. braz dent j 2001; 12: 115-9. 1 0 . morse a .formic acid-sodium citrate descalcification and butyl alcohol dehydration of teeth and bone for sectioning in parafin. j dent res 1945; 24: 143-53. 1 1 . cohn sa. development of the molar teeeth in the albino mouse. am j anat 1957; 101: 295-319. 1 2 . hay mf. the development in vivo and in vitro of the lower incisor and molars of the mouse. arch.oral biol 1961; 3: 86109. 1 3 . webster ws, johnston mc, lammer ej, sulik kk. isotretinoin embriopathy and cranial neural crest. an in vivo and in vitro study. j craniofac genet dev biol 1986; 6: 211-22. 1 4 . kochhar dm, penner jd. developmental effects of isotretinoin and 4-oxo-isotretinoin: the role of metabolism in teratogenicityy. teratology 1987; 36: 67-75. 1 5 . ritchie h, webster ws. parameters determining isotretinoin teratogenicity in rats embryo culture. teratology 1991; 43: 718 1 . 1 6 . morris gm. morphogenisis of the malformations induced in rat embryos by maternal hypervitaminosis a. j ant 1972; 113: 2 4 1 5 0 . 1 7 . morris gm. vitamin a and congenital malformations. int j vitamin nutr res 1976; 46: 220-2. 1 8 . morris gm. the ultrastructural effects of excess maternal vitamin a on the primitive streak stage rat embryo. j embryol exp morphol 1973; 30: 219-42. 1 9 . 19.takahashi yi, smith je. metabolism of retinol binding protein (rbp) in the fetal rat. federation proc 1974; 33: 688. 2 0 . lorente ca, miller sa. fetal and maternal vitamin a levels in tissues of hypervitaminotic a rats and rabbbits. j nutr 1977; 107: 1816-21. 2 1 . deol ms, truslove gm. genetical studies on the skeleton of the mouse. xx. maternal physiology and variation in the skeleton of c57bl mice. j genet 1957; 55: 288-312. 2 2 . searle a g. genetical studies on the sckeletal skeleton of the mouse. xi. the influence of diet of variation within pure lines. j genet 1954; 52: 413-24. braz j oral sci. 1(3): 121-125 isotretinoin: action on mice tooth germs and palate oral sciences n3 braz j oral sci. 14(1):31-35 original article braz j oral sci. january | march 2015 volume 14, number 1 shear bond strength of ceramic brackets after different pre-treatments in porcelain surface naudy brodbeck may1, élito araújo2, luiz clovis cardoso vieira2 1universidade do sul de santa catarina unisul, school of dentistry, department of orthodontics, palhoça, sc, brazil 2universidade federal de santa catarina ufsc, school of dentistry, department of orthodontics, florianópolis, sc, brazil correspondence to: naudy brodbeck may rua vitor konder, 66 ap 900 cep: 88.015-400 – florianópolis, sc, brasil phone: +55 48 96405776 e-mail: naudy.may@unisul.br abstract aim: to evaluate the bond strength of brackets bonded after different surface treatments on two dental ceramics. methods: one hundred and twenty discs (5 mm thick and 7 mm diameter) of two ceramic types were made and randomly divided in 8 groups. groups 1, 3, 5, and 7 used eris ceramic and groups 2, 4, 6, and 8 used d.sign ceramic. the ceramic surfaces were treated with 10% hydrofluoric acid (g1 and g2), 10% hydrofluoric acid + silane (g3 and g4), aluminum oxide blasting + 35% phosphoric acid + silane (g5 and g6), cojet blasting + 35% phosphoric acid + silane (g7 and g8). metallic brackets were cemented with concise cement. mechanical test was performed in a universal testing machine until failure. results: the average values (mpa) obtained (g1 7.30; g2 6.12; g3 17.49; g4 19.54; g5 18.80; g6 21.93; g7 6.81 e g8 9.77) were submitted to anova and tukey test (p<0.05). the fracture patterns were analyzed in stereoscopic microscope (25´x) and representative samples of each group were analyzed in sem. conclusions: it was possible to conclude that use of silane after hydrofluoric acid increased the bond strength values. keywords: adhesives; dental bonding; dental porcelain; orthodontic brackets; shear strength. introduction the advances in cosmetic dentistry and the increased aesthetic requirements have led to a growing demand for orthodontic treatments1-4 and orthodontists have sometimes to deal with patients that ceramic laminates or crowns from previous dental interventions3-5. it is known that the orthodontic treatment will be successful only if there is an adequate bond strength between the substrate (tooth, ceramic, or ceromer) and the orthodontic appliance (bracket); notwithstanding, the bond strength between ceramic/brackets has proven to be unsatisfactory1,3-6. buonocore7 (1955) reported that it was possible to bond the resin to the tooth based on a study about enamel. ever since, several researches have been performed aiming to establish a clinical protocol for the bonding procedure using other substrates like dentin, metal, composite and ceramic8-9. as regards ceramics, due to their different compositions, diverse methods have been developed for the treatment of their surfaces10-11. different pretreatment methods have been proposed for substrates that will receive orthodontic brackets, as polishing with sandpapers 2,12, creation of irregularities with diamond burs2,6, laser13, hydrofluoric acid etching3,5,12, aluminum oxide blasting2-3,6, phosphoric acid etching5,13, maleic acid etching13, acidulated phosphate fluoride application2,12, silane agent application2,4-5,12, and lastly, the combination of one of the above-mentioned treatments with or without silane received for publication: january 22, 2015 accepted: march 04, 2015 3232323232 surface treatment eris d.sign ceramic ceramic 10% hydrofluoric acid g1 g2 10% hydrofluoric acid g3 g4 + silane agent aluminum oxide blasting g5 g6 + 35% phosphoric acid + silane agent cojet blasting g7 g8 + 35% phosphoric acid + silane agent table 1.table 1.table 1.table 1.table 1. groups according to the pre-treatment and the type of ceramic. application4-6. however, failures have been normaly found when a bracket is cemented to a ceramic restoration1,3-6. considering the lack of conclusive studies regarding the most effective pretreatment technique for bonding orthodontic appliances to teeth with ceramic prosthesis, the aim of this study was to evaluate the bond strength of brackets bonded after different surface treatments on two dental ceramics. the null hypothesis is that there are no significant differences among pre-treatment with 10% hydrofluoric acid, 10% hydrofluoric acid with silane agent, aluminum oxide blasting with 35% phosphoric acid and silane and cojet blasting with 35% phosphoric acid and silane. material and methods for this study were made 60 discs for each ceramic (eris (ivoclar vivadent, schaan, liechtenstein) and d.sign (ivoclar vivadent)) using a plastic matrix (5 mm thick and 7 mm diameter). the specimens were embedded in pvc tubes (amanco, são paulo, sp, brazil), with 20 mm external diameter by 20 mm height. the pvc tubes were filled with polystyrene resin (central fiberglass, florianópolis, sc, brazil), prepared according to manufacturer’s recommendations. for easier identification of the groups, different pigmentations (clássico, são paulo, sp, brazil) of polystyrene resin were used for each group. then they were divided into 8 groups (n=15) according to the surface treatment and type of ceramic (table 1). for g1 and g2, the surface treatment was 10% hydrofluoric acid for 20 s, rinse for 15 s and 10 s drying. for g3 and g4 the same procedures were performed plus the application of 3 layers of silane agent, dryed for 15s and photo-activated for 10 s. in g5 and g6, the aluminum oxide blasting was performed during 15 s, with pressure at 80 psi (using a needle gauge to measure pressure) at a 5 mm distance; 35% phosphoric acid etching for 30 s, rinsing during 15 s, drying for 10 s, application of 3 layers of silane agent, drying during 15 s and photo-activation during 10 s. at last, the specimens of g7 and g8, after the same previous procedures as in g5 and g6, were blasted by the cojet system followed by application of 35% phosphoric acid for 30 s and 3 layers of silane agent. concise cement (3m espe, st paul, mn, usa) was prepared according to the manufacturer’s instructions and inserted on the specimen’s surface. next, using tweezers (dental morelli ltda, sorocaba, sp, brazil), standard metallic brackets (dental morelli) for central incisors, with area of 6.08 mm2, were positioned at the center of ceramic blocks with manual pressure. the excess resin was removed using explorer catheter (duflex ss white group, gloucester, uk). the ceramic/bracket specimens were stored in an oven (quimis, model q317b, diadema, sp, brazil) at 37 °c immersed in distilled water for 24 h. after that, the specimens were subjected to 800 thermal cycles (ética equip. cient. s.a., series 96, no 0364, model 521-e, são paulo, sp, brazil), for 30 s at 5 °c and 30 s at 55 c, with 10 s dell-time between baths. the shear strength test was performed in a universal testing machine (instron model 4444, canton, ma, usa), at a cross-speed of 0.5 mm/min. the specimens were positioned in a metallic glove (20.5 mm diameter x 20 mm high). the test was performed using a chisel as load on the ceramic/ bracket interface, trying to simulate the oral cavity environment, as well as the masticatory loads. the fracture patterns produced after the shear test were observed with a stereomicroscope (xlt30, nova optical systems, piracicaba, sp, brazil) at 25× magnification, and classified according to vaz et al.14 (2011) as: (1) adhesive fracture: cement/bracket; (2) adhesive fracture: ceramic/ cement; (3) mixed fracture: cement/bracket; (4) mixed fracture: ceramic/bracket/cement. representative specimens of each group were randomly selected to determine the fracture pattern in a scanning electron microscope (philips xl-30, mahwah, nj, usa) set at 20 kv. sem micrographs were obtained at 15× and 60× magnifocations. the shear strength data were subjected to two-way anova and tukey’s test for multiple comparisons (p=0.05). fracture pattern data were analyzed statistically by kruskalwallis test (p=0.05). paiwise comparisons of groups were performed with mann-whitney u test (p=0.05). the correlation between shear strength and fracture pattern was performed by spearman’s rank correlation. results anova showed that there were statistically significant differences among the ceramics (p=0.01) and surface treatments (p=0.0001), but it did not show interaction among them (p=0.14). means compared by the tukey’s test (p<0.05) are described in table 2. faiure mode analysis is described in table 3. groups mean (mpa) standard deviation g1 7.30a 3.5043 g2 6.12a 3.1490 g3 17.49b 3.9677 g4 19.54b 4.3325 g5 18.80b 4.9050 g6 21.93b 4.6884 g7 6.81a 2.4816 g8 9.77a 4.5717 table 2.table 2.table 2.table 2.table 2. bond strength means and standard deviations. same letter indicate statistically significant difference between them (tukey test, p<0.05) shear bond strength of ceramic brackets after different pre-treatments in porcelain surface braz j oral sci. 14(1):31-35 3333333333 groups fracture standard (%) adhesive adhesive mixed mixed (cement/bracket) (ceramic/cement) (cement/bracket) (cement/bracket/ ceramic) g1 0 100 0 0 g2 0 100 0 0 g3 46.66 0 53.33 0 g4 0 60 40 0 g5 33.33 0 6.66 60 g6 26.66 0 6.66 66.66 g7 93.33 0 6.66 0 g8 100 0 0 0 table 3. table 3. table 3. table 3. table 3. distribution in percentiles of fracture standards after shear test. initially, the groups were compared relative to the fracture pattern scores by the kruskal-wallis test (h=52.49; p<0.0001). in the mann-whitney u test, the groups were compared in a pairwise fashion (table 4). the table 4 indicates significance values between the groups that did not present statistical difference related to ceramics. the spearman’s rank correlation showed that the higher the bond strength value, the more severe the fracture pattern, including fracture of ceramic or bracket (r=0.372; p<0.0001). sem micrographs (figures 1, 2, 3 and 4) illustrate representative fracture patterns found in each group. fig. 1. fracture pattern in g1 and g2 after shear test (adhesive ceramic/cement). comparisons among u p * the groups g1xg2 112.500 1 g3xg4 96.000 0.460419 g5xg6 104.500 0.695646 g7xg8 105.000 0.317310 table 4. table 4. table 4. table 4. table 4. comparisons of fracture patterns by mann-whitney u test. *statistically significant difference (p<0.05). fig. 2. fracture pattern in g3 and g4 after shear test (mixed cement/bracket). discussion based on the results of this study, the null hypothesis that there are no significant differences among the different ceramics treatments was rejected. the use of 10% hydrofluoric acid alone produced lower bond strength values (g1 7.30 fig. 3. fracture pattern in g5 and g6 after shear test (mixed cement/bracket/ ceramic). shear bond strength of ceramic brackets after different pre-treatments in porcelain surface braz j oral sci. 14(1):31-35 3434343434 mpa and g2 6.12 mpa). however, when it was combined with silane agent, the bond strength increased significantly (g3 – 17.49 mpa and g419.54 mpa), corroborating previous findings12,15. the increase in bond strength could be due to silane capacity of increasing the energy on the substrate surface, optimizing resin penetration in the created microregions5,12. the groups treated with aluminum oxide blasting (50 ìm) followed by phosphoric acid etching (35%) + silane agent, obtained values of 18.80 mpa (g5) and 21.93 mpa (g6), and did not differ significantly from groups treated with hydrofluoric acid + silane agent (g3 and g4). these findings agree with those of recent studies2-3, 6,16-17. a possible explanation could be the fact the impact produced by this treatment raises temperature locally, causing an incorporation of particles up to 15 ìm from ceramic structure18, which increases surface roughness and makes it more retentive18-19 and receptive to the chemical bond with silane agent20. g7 (6.81 mpa) and g8 (9.77 mpa) bond strength means were significantly lower than those of g3-g6, and similar to those of g1 and g2. it is possible that the micro-retentions generated by cojet system, whose particles are 30 ìm in size, could be lower than those created by the aluminum oxide blasting (50 ìm particles). these findings agree with girish et al.21 (2012), who reported that blasting with larger particles (110 ìm) produced higher bond strength values than those found with cojet. ozcan22 (2014), who evaluated the cojet in ceramic, metal-ceramics, metal-mechanics and metallic substrates, achieved the best results with metallic substrates. according to wady et al.20 (2014), the efficiency of cojet depends on the mechanical properties of the ceramic, working better in leucite-based ceramics than is feldspathic ones. on the other hand, passos et al.23 (2013) reported bond strength of 13.2 mpa after 37% phosphoric acid etching of enamel for 60 s. zhang et al.4 (2013) disagree with this values theretofore considered adequate clinically by silveira et al.24 (2014). based on the results of bracket bond to ceramic, there is a common sense to use vijayakumar25 (2014) findings as a reference to indicate the best pretreatment to be performed fig. 4. fracture pattern in g7 and g8 after shear test (adhesive cement/bracket) or at least the one that produces the closest to values obtained in enamel. this author suggested that the appropriate bond strength values to enamel bracket be 6-8 mpa. therefore, in the present study, all treatments produced adequate values (g1 – 7.30; g2 – 6.12; g3 – 17.49; g4 – 19.54; g5 – 18.80; g6 – 21.93; g7 – 6.81 and g8 – 9.77 mpa). according to the classification used in this study for analysis of fracture patterns, it is possible to observe that g1 and g2 presented exclusively (100%) adhesive failures on ceramic/cement interface. this suggests that the bond strength between cement and ceramic was weak (g1 – 7.30 mpa and g2 – 6.12 mpa), according to findings by okuda3 (2014). statistically similar, g7 (6.81 mpa) and g8 (9.77mpa), also presented prevalence of adhesive failures; however, they occurred in the cement/bracket interface (93.33% and 100%, respectively), which denotes more effectiveness in the treatment of the surface. in g3 and g5, the treatment was hydrofluoric acid application and silane agent. it was observed that the bond performance was different between the ceramics: it was predominantly adhesive in ceramic/ cement (60%) interface in d.sign and mixed in cement/ bracket (53.33%) interface in eris. g5 and g6 were also statistically similar (18.80 and 21.93 mpa, respectively), with prevalence of mixed fails (cement/bracket/ceramic), g7 (60%) and g8 (66.66%). comparing the bond strength values and fracture patterns obtained in this study, it may be observed that despite the significant correlation (r=0.372; p<0.0001), a tendency could be identified that the higher bond strength, the higher the fracture scores; in other words, higher quantity of fractures on the ceramic. this led the authors to believe that the higher retention created by the pre-treatment, more severe can be the structural damage, according to the findings by grewal bach2 (2014), gavake et al.5 (2013), lung et al.6 (2015), kumar et al.26 (2014) and okuda3 (2014). further research should be done including thermal and mechanical cycling treatment as well the ideal strength to avoid the bracket displacement damage to the ceramic structure. based on data obtained in this study, it is possible to conclude that: (1) despite the different types of surface pretreatments, the tested ceramics performed similarly in therms of bond strength; (2) the use of silane after hydrofluoric acid etching was responsible for the increase of bond strength values; (3) pretreatment of ceramic substrate by 10% hydrofluoric acid etching during 20 s followed by silane application, as well as aluminum oxide (50 ìm) blasting for 15 s, followed by 35% phosphoric acid etching and silane application provided significantly higher bond strength values to metallic brackets; (4) the cojet system did not result in significantly higher values t h a n t h o s e o b s e r v e d f o r a l u m i n u m o x i d e b l a s t i n g , becoming similar to the groups treated with hydrofluoric acid without silane application; (5) aluminum oxide blasting followed by phosphoric acid etching and silane presented results similar to the treatment with hydrofluoric acid and silane. shear bond strength of ceramic brackets after different pre-treatments in porcelain surface braz j oral sci. 14(1):31-35 3535353535 references 1. komori a, takemoto k, shimoda t, miyashita w, scuzzo g. precise direct lingual bonding with the kommonbase. j clin orthod. 2013; 47: 42-9. 2. grewal bach gk, torrealba y, lagravère mo. orthodontic bonding to porcelain: a systematic review. angle orthod. 2014; 84: 555-60. 3. okuda w. predictable replacement of failing porcelain restorations. gen dent. 2014; 62: 21-3. 4. zhang zc, giordano r, shen g, chou ll, qian yf. shear bond strength of an experimental composite bracket. j orofac orthop. 2013; 74: 31931. 5. gayake pv, chitko ss, sutrave n, gaikwad pm. the direct way of indirect bonding—the combined effect. int j orthod milwaukee. 2013; 24: 15-7. 6. lung cy, liu d, matinlinna jp. silica coating of zirconia by silicon nitride hydrolysis on adhesion promotion of resin to zirconia. mater sci eng c mater biol appl. 2015; 46: 103-10. 7. buonocore mg. a simple method of increasing the adhesion of acrylic filling materials to enamel surfaces. j dent res. 1955; 34: 849-53. 8. trakyali g, malkondu o, kazazoðlu e, arun t. effects of different silanes and acid concentrations on bond strength of brackets to porcelain surfaces. eur j orthod. 2009; 31: 402-6. 9. rathke a, tymina y, haller b. effect of different surface treatments on the composite-composite repair bond strength. clin oral investig. 2009; 13: 317-23. 10. rao s, chowdhary r. comparison of fracture toughness of all-ceramic and metal-ceramic cement retained implant crowns: an in vitro study. j indian prosthodont soc. 2014; 14: 408-14. 11. bieniaœ j, surowska b, stoch a, matraszek h, walczak m. the influence of sio2 and sio2-tio2 intermediate coatings on bond strength of titanium and ti6al4v alloy to dental porcelain. dent mater. 2009; 25: 1128-35. 12. valentini f, moraes rr, pereira-cenci t, boscato n. influence of glass particle size of resin cements on bonding to glass ceramic: sem and bond strength evaluation. microsc res tech. 2014; 77: 363-7. 13. geraldo-martins vr, lepri cp, faraoni-romano jj, palma-dibb rg. the combined use of er,cr: ysgg laser and fluoride to prevent root dentin demineralization. j appl oral sci. 2014; 22: 459-64. 14. vaz rr, hipolito vd, d’alpino ph. bond strength and interfacial micromorphology of etch-and-rinse and self-adhesive resin cements to dentin. j prosthodont. 2012; 21: 101-11. 15. kim jh, chae s, lee y, han gj, cho bh. comparison of shear test methods for evaluating the bond strength of resin cement to zirconia ceramic. acta odontol scand. 2014; 72: 745-52. 16. brunharo ih, fernandes dj, de miranda ms, artese f. influence of surface treatment on shear bond strength of orthodontic brackets. dental press j orthod. 2013; 18: 54-62. 17. denry i, kelly jr. emerging ceramic-based materials for dentistry. j dent res. 2014; 93: 1235-42. 18. kosyfaki p, swain mv. adhesion determination of dental porcelain to zirconia using the schwickerath test: strength vs. fracture energy approach. acta biomater. 2014; 10: 4861-9. 19. bunek ss, swift ej jr. contemporary ceramics and cements. j esthet restor dent. 2014; 26: 297-301. 20. wady af, paleari ag, queiroz tp, margonar r. repair technique for fractured implant-supported metal-ceramic restorations: a clinical report. j oral implantol. 2014; 40: 589-92. 21. girish pv, dinesh u, bhat cs, shetty pc. comparison of shear bond strength of metal brackets bonded to porcelain surface using different surface conditioning methods: an in vitro study. j contemp dent pract. 2012; 13: 487-93. 22. ozcan m. surface conditioning protocol for multiple substrates in repair of cervical recessions adjacent to ceramic. j adhes dent. 2014; 16: 394. 23. passos sp, kimpara et, bottino ma, júnior gc, rizkalla as. bond strength of different resin cement and ceramic shades bonded to dentin. j adhes dent. 2013; 15: 461-6. 24. silveira gs, bittencourt lp, mucha jn. scoring of ceramic bracket bases for easier debonding. j clin orthod. 2014; 48: 441-2. 25. vijayakumar rk, jagadeep r, ahamed f, kanna a, suresh k. how and why of orthodontic bond failures: an in vivo study. j pharm bioallied sci. 2014; 6: 85-9. 26. kumar m, maheshwari a, lall r, navit p, singh r, navit s. comparative evaluation of shear bond strength of recycled brackets using different methods: an in vitro study. j int oral health. 2014; 6: 5-11. shear bond strength of ceramic brackets after different pre-treatments in porcelain surface braz j oral sci. 14(1):31-35 oral sciences n3 original article braz j oral sci. april | june 2013 volume 12, number 2 biomechanical analysis on different fixation techniques for treatment of mandibular body fractures marcela ribeiro1, andrezza lauria1, fábio ricardo loureiro sato1, roger william fernandes moreira1 1area of oral and maxillofacial surgery, piracicaba dental school, university of campinas, piracicaba, sp, brazil correspondence to: andrezza lauria departamento de diagnóstico oral faculdade de odontologia de piracicaba – unicamp avenida limeira, 901, cep:13414900 caixa postal: 52, piracicaba, sp, brasil phone: +55 19 21065708 e-mail: andrezzalauria@gmail.com received for publication: december 26, 2012 accepted: april 01, 2013 abstract aim: to biomechanically analyze two fixation techniques in polyurethane hemi-mandibular body fractures, using a universal testing machine. methods: the study employed 10 polyurethane hemi-mandible replicas, which simulated simple fractures of the mandibular body, divided into two groups: one group comprised 5 hemi-mandibles with two 2.0 mm system plates in the tension and compression zones, while the other group contained 5 hemi-mandibles with an erich bar and a 2.0 mm system plate in the tension and neutral zones, respectively. data were analyzed statistically by the student’s t-test (α=0.05) results: the test results indicated that the fixation using 2.0 mm system plates offered significantly more resistance to the loads and presented significantly larger displacement compared to the fixation using just one 2.0 mm system plate and the erich bar. conclusions: it may be concluded that the use of two plates in the 2.0 mm system had greater mechanical strength than a single 2.0 mm plate combined with an erich bar. clinically, it is known that both techniques can provide good results, but patients receiving the combination of erich bar and one plate are required to be more cooperative during the postoperative period, especially with respect to the prescribed diet in order to avoid failures in this system. keywords: mandibular fractures, bone plates, internal fixation of fractures. introduction mandibular body fractures are considered common but there are discrepancies among the studies, such as epidemiological surveys, regarding their incidence. martini et al.1 (2006), noticed a higher incidence of mandibular body fracture (30.9%). atilgan et al.2 (2006) observed that mandibular body fractures were more frequent in adults than in children, but occurred in only 12% of the 532 turkish patients included in the study. in the surgical treatment of facial fractures, there are principles that should be followed for a successful outcome: fracture reduction (replacement of bone segments in their correct anatomical positions), fixation and containment of bone segments and immobilization at the fracture site3. specifically to the mandible, the treatment can follow a more conservative approach such as the maxillomandibular fixation (mmf) with stainless steel wires or the procedures under general anesthesia with surgical exploration, reduction and fixation of the fracture4. the use of mini plates has been changing the treatment of fractures in the last two decades5, in addition to various methods of mandible fixation, such as braz j oral sci. 12(2):80-83 8181818181 compressive and non-compressive plates, reconstructive plates, isolated screws, either by intra or extra oral access, have been used with varying degrees of success6. regarding to mandibular body fixation, milloro et al.7 (2009) reported that for simple linear fractures, the application of a single mini plate of the 2.0 mm system along the lower edge, combined with a bar, is usually an adequate fixation. other possibilities are the use of lag screws for oblique fractures of mandibular body, a hybrid technique with lag screw and miniplate8-9, 2.4 mm reconstruction plate placed along the inferior border of the mandible or two in the mandibular tension and compression zone10. few studies have been published on the analysis of fixation techniques for mandibular body fractures5,7-8,10-11, and further research is needed relating to the biomechanical study of the different techniques for the internal fixation of mandibular body fractures. when evaluating the functional stability of the fixations by simulating the vectors and load intensity that they will be subjected to, the results may be associated with the clinical practice, which implicates the need for a model simulating the human mandible anatomy. for the present study, a polyurethane hemi-mandible replica, already reported in the literature, was employed as a substitute for the human mandible during the biomechanical tests12-13. the literature offers no clear evidence that one rigid internal fixation technique for the treatment of simple (linear) fractures of the mandibular body is superior to another11. therefore, the objective of this study was to evaluate the mechanical strength of plates and screws from the 2.0 mm system in two types of fixation techniques, using human hemi-mandible polyurethane resin replicas, subjected to mechanical linear load testing. material and methods a rigid polyurethane hemi-mandible with teeth and standardized density of 200g/l was used for the tests, manufactured by nacional™ (franceschi & costa and silva ltda., jaú, sp, brazil). the samples were submitted to sectioning simulating mandibular body fracture (in the premolar and first molar regions), and fixed with plates and screws in accordance with their respective group by making a colorless chemically activated acrylic resin guide (dental vipi ltda., pirassununga, sp, brazil) for standardizing the fixation the samples were grouped as follows: group 1: 5 polyurethane hemi-mandibles with rigid internal fixation system, using a tóride ® 2.0 mm system with 2 plates containing 4 titanium extension holes and screws linearly inserted with a 90º angle to the hemi-mandible. the first plate was fixed to the tension zone using 2.0 mm x 5 mm screws; the second was secured to the compression zone with 2.0 mm x 11 mm screws (ure 1). group 2: 5 polyurethane hemi-mandibles with an erich arch-bar were secured to the tension zone, and one 2.00 mm system titanium plate with 4 fixed extension holes to the neutral zone with 2.0 mm x 5 mm screws, linearly arranged and inserted at 90º angle o the hemi-mandible (figure 2). all samples were embedded in a block of pink bonding acrylic resin (artigos odontológicos clássico ltda., campo fig. 2. group 2: an erich arch bar in the compression zone and one 2.0 mm miniplate in the neutral zone. fig. 1. group 1: one 2.0 mm mini-plate in the compression zone and another one in the tension zone. limpo paulista, sp, brazil) to the full extent of the mandibular branch to allow its positioning and fixation to the holder during the test. the load test was undertaken to evaluate the fastening systems resistance to the forces exerted on them. the load testing was performed in an instron universal testing machine, (model 4411; instron corp., norwood, ma, usa) at a crosshead speed of 1 mm/min for applying progressive load (in kgf) on the system. when the load resistance (peak and ultimate loads) was reached, the displacement (mm) imposed by test was recorded in these two moments. the load was always applied on the distal segment at a fixed point, which received the device load, previously fabricated at the lingual canine region. data were analyzed statistically using the student’s ttest at 5% significance level. biomechanical analysis on different fixation techniques for treatment of mandibular body fractures braz j oral sci. 12(2):80-83 8282828282 sample peak load displacement peak 1 28.47 9.48 2 11.91 6.18 3 16.07 10.27 4 23.67 6.64 5 19.17 9.54 table 1. values of peak load and peak displacement in group1 sample peak load displacement peak 1 26.38 9.01 2 6.71 2.37 3 11.34 3.07 4 11.7 6.00 5 9.101 2.89 table 2. values of peak load and peak displacement in group 2. results the peak load (kgf) and peak displacement (mm) means and standard deviations of the two test groups are presented in tables 1 and 2. the peak load values indicated significantly greater resistance for the fixation using two plates of 2.0 mm system compared with fixation with one plate of the 2.0 mm system and one erich bar (p<0.05) (figure 3). the peak displacement values indicated significantly greater displacement from the fixation using two plates of 2.0 mm system compared with the fixation with one plate of the 2.0 mm system and one erich bar (p<0.05) (figure 4). discussion isolated fractures of the mandibular body tend to displace the superior surface compressed towards the inferior surface fig. 3. comparison os treatment depending on the peak load when subjected to masticatory forces. the superior surface is called tension zone (separation) while the lower edge is called compression zone. the fixation on the upper edge (dentate) is more effective in preventing the separation when the mandible is subjected to efforts7,14-15. thus, when applying fixation materials to the mandible during fracture treatments, typically a plate is installed in the tension zone, where the bone surfaces are separated. this study is based on the principle that some kind of material is required in the tension zone, either an erich bar or a fixation plate, during the force testing. various combinations of rigid internal fixation are available for mandibular body fractures: two fixation plates are required, one in the tension zone with monocortical screws because of the presence of teeth, and one titanium plate in the compression zone with bicortical screws. lag screws are reserved for oblique fractures and the maxillo-mandibular fixation for a more conservative approach. the rigid internal fixation technique can be performed with a 2.0 mm system plate, a combination of the 2.0 mm and the 2.4 mm systems, or a 2.4 mm system with an erich bar6,8-10. this way, evaluation of the different fixation methods is important to provide a direction for the stability and predictability of each option. this biomechanical study evaluated two forms of fixation for the surgical treatment of mandibular body fractures because there is no consensus on the routinely used methods11 and no studies were found comparing the combined use of the 2.0 mm mini-plate system in the neutral area and an erich bar in the tension zone. the biomechanical stability is an important factor in the choice of material to be used, although other factors influence the outcome of the treatment, since each technique presents clinical advantages and disadvantages that should be taken into consideration during the surgical planning. the use of two fixation plates increases the treatment cost due to the larger amount of material required, the risk of biomechanical analysis on different fixation techniques for treatment of mandibular body fractures braz j oral sci. 12(2):80-83 8383838383 fig. 4. comparison of treatment depending on the peak displacement iatrogenic injury to the tooth roots is increased when using the plate in the tension zone, and a greater chance of a wound dehiscence and exposure of the upper plate11. despite its potential drawbacks, biomechanically, this technique had the best behavior in this study, requiring larger force for displacement and consequent failure. the main advantage of using the erich bar in combination with a fixation plate is associated with its low cost and versatility. conversely, the patient must be dentated for the stabilization of the erich bar (which can damage the periodontium due to the steel wires), and requires greater patient compliance during the postoperative period, especially in relation to hygiene and diet7. it may be then concluded that the use of 2 plates in the 2.0 mm system is capable to withstand a greater load before failure. from a clinical perspective, it is known that both techniques can produce good results, but patients receiving the combination of erich bar in the tension zone and a plate in the compression zone during treatment of a fractured mandible, should be more collaborative during the postoperative period, especially with respect to the prescribed diet in order to avoid failures in this system. acknowledgments the authors would like to acknowledge toride® (tóride indústria e comércio ltda. – mogi mirim, sp, brasil) for their generous assistance in providing the fixation plates and screws for this study. references 1. martini mz, takahashi a, oliveira neto hg, carvalho júnior jp, curcio r, shinohara eh. epidemiology of mandibular fractures treated in a brazilian level i trauma public hospital in the city of são paulo, brazil. braz dent j. 2006; 17: 243-8. 2. atilgan s, erol b, yaman f, yilmaz n, ucan mc. mandibular fractures: a comparative analysis between young and adult patients in the southeast region of turkey. j appl oral sci. 2010; 18: 17-22. 3. peterson jl, ellis iii e, hupp jr, tucker mr. contemporary oral and maxillofacial surgery. 4th ed. saint louis: mosby; 2002. 4. fonseca rj, marciani rd, turvey ta. oral and maxillofacial surgery. 2nd ed. saint louis: saunder elsevier; 2009. v.2. 5. sauerbier s, schön r, otten je, schmelzeisen r, gutwald rj. the development of plate osteosynthesis for the treatment of fractures of the mandibular body and a literature review. j craniomaxillofac surg. 2008; 36: 251-9. 6. ellis iii e. treatment methods for fractures of the mandibular angle. int j oral maxillofac surg. 1999; 28: 243-52. 7. miloro m, ghali eg, larsen ep, waite dp. peterson’s principles of oral and maxillofacial surgery. hamilton: bc decker; 2004. 8. ellis iii e. use of lag screws for fractures of the mandibular body. j oral maxillofac surg. 1996; 54: 1314-6. 9. ramalho ra, araújo fac, santos fsm, caubi af, sobreira t. management of the mandible fracture: miniplates and screws vs. lag screws – case report. rev cir traumatol buco-maxilo-facial. 2011; 11: 9-12. 10. assael al, klotch wd, manson np, prein j, rahn ab, schilli w. manual of internal fixation in the cranio-facial skeleton. berlin: springer-verlag; 1998. 11. ellis iii e. a study of 2 bone plating methods for fractures of the mandibular symphysis/body. j oral maxillofac surg. 2011; 69: 1978-87. 12. haug rh, peterson gp, goltz m. a biomechanical evaluation of mandibular condyle fracture plating techniques. j oral maxillofac surg. 2002; 60: 73-80. 13. ziccardi vb, schneider re, kummer fj, wurzburg lag screw plate versus four hole miniplate for the treatment of condylar process fractures. j oral maxillofac surg. 1997; 55: 602-7. 14. ellis iii e. management of fractures through the angle of the mandible. oral maxillofacial surg clin n am. 2009; 21: 163-74. 15. kimsal j, baack b, candelaria l, khraishi t, lovald s. biomechanical analysis of mandibular angle fractures. j oral maxillofac surg. 2011; 69: 3010-4. biomechanical analysis on different fixation techniques for treatment of mandibular body fractures braz j oral sci. 12(2):80-83 oral sciences n3 braz j oral sci. 13(1):6-11 original article braz j oral sci. january | march 2014 volume 13, number 1 intracanal dressing paste composed by calcium hydroxide, chlorhexidine and zinc oxide for the treatment of immature and mature traumatized teeth adriana de jesus soares, thiago farias rocha lima, juliana yuri nagata, brenda paula figueiredo de almeida gomes, alexandre augusto zaia, francisco josé de souza-filho universidade estadual de campinas unicamp, piracicaba dental school, department of restorative dentistry, area of endodontics, piracicaba, sp, brasil correspondence to: adriana de jesus soares área de endodontia, faculdade de odontologia de piracicaba, unicamp avenida limeira, 901, cep: 13414-018 piracicaba, sp, brasil phone: +55 19 3251-7138 e-mail: ajsoares.endo@uol.com.br abstract aim: to evaluate clinical and radiographic aspects before and after endodontic treatment with an intracanal dressing paste composed of calcium hydroxide, chlorhexidine and zinc oxide in traumatized teeth followed-up for 1 year. methods: patients (n=105) treated at the dental trauma service of piracicaba school of dentistry, brazil were enrolled in the study. two groups of teeth were formed: immature (g1) (n=28) and completely developed teeth (g2) (n=174). all teeth were endodontically treated and received an intracanal dressing with a paste composed by calcium hydroxide, 2% chlorhexidine gel and zinc oxide at a 2:1:2 rate. clinical and radiographic aspects were evaluated initially, monthly and after 1-year. results: most of the immature teeth suffered extrusive luxation (39.3%), whereas intrusive luxation (40.8%) was more common in completely developed teeth. there was a significant reduction in pain on percussion and mobility (p=0.0001) for immature teeth. mature teeth showed reduction of spontaneous pain, fistula, mobility and pain on percussion (p<0.0001). radiographic examination showed decrease in all evaluated parameters for both groups, in addition to stabilization of root resorption. conclusions: this new intracanal dressing paste for traumatized teeth showed promising results in both immature and completely developed teeth, and may prevent posttraumatic complications. keywords: calcium hydroxide; dental trauma; treatment. introduction dental trauma may cause damage to the pulp and periodontal tissues1. depending on the intensity and type of injury, damage may be reversible or present an unfavorable prognosis for these tissues2. among the dental trauma complications, the most common are pulp necrosis and microbial infection, which may accelerate the root resorption process 2-3. in order to minimize these complications, endodontic treatment should be performed at an appropriate time to reduce infective and irritant agents. in dental trauma situations, endodontic treatment involves the use of intracanal dressing between sessions as a complementary factor to eliminate and reduce microorganisms, prevent or stabilize root resorption, induce dentin formation received for publication: january 12, 2014 accepted: march 10, 2014 gisele higa texto digitado http://dx.doi.org/10.1590/1677-3225v13n1a02 7 braz j oral sci. 13(1):6-11 and periapical tissue repair4. another factor that may influence endodontic therapy is the stage of root development of the traumatized tooth. immature teeth present a wide open apex that may favor revascularization and repair of the vascular bundle after trauma. in these teeth the frequency of pulp necrosis is lower (13.6%) than in teeth completely closed roots (63.1%)3. despite the more favorable prognosis for immature teeth, the need for endodontic therapy may be a challenge to conventional endodontic treatment. in these situations, some types of treatment have been proposed, including apexification with periodic changes of intracanal dressing, apexification with an apical plug, and more recently pulp revascularization56. apexification is the classic treatment and involves periodic changes of intracanal dressing during a period of 6 to 24 months until a calcified mineralized barrier is formed, allowing apical sealing of the tooth7. more recently, pulp revascularization has demonstrated promising results, with root end development in immature teeth with indication for endodontic therapy8-10. nevertheless, there are situations in which pulp revascularization may not be the first choice, considering the need for rehabilitation with intracanal retainers, root resorption and possibility of complications of this little known therapy. contrasting with the uncertainty about this new treatment, apexification is a well-known and successful procedure with long-term followup studies 1 1. although it is a well-known procedure, apexification also presents some disadvantages, due to the lack of patient cooperation for intracanal dressing changes, long-term weakening of the root considering the hygroscopic and proteolytic properties of calcium hydroxide, increase of fracture risk and pulp space contamination12. considering the disadvantage of intracanal dressing changes in traumatized teeth, an intracanal dressing paste composed by calcium hydroxide, chlorhexidine and zinc oxide, which does not require periodic changes, has been proposed13-14. this paste may be used regardless of the level of root development, and its qualities include low cost, ease-to-use, high radiopacity, no need for periodic replacement and capacity of inducing mineralization13-15. in addition, it has been reported that avulsed teeth with unfavorable prognosis may be treated with this paste for long-term periods without dissolution, resulting in periapical lesion repair16. the aim of this study was to evaluate the clinical and radiographic aspects before and after endodontic treatment with an intracanal dressing paste composed by calcium hydroxide, chlorhexidine and zinc oxide in traumatized teeth followed up for 1 year. material and methods sample one hundred and five patients presenting 202 traumatized teeth were divided in two groups. the first group (g1) comprised patients with immature teeth (n=28), and the second one (g2) comprised those having teeth with completely developed roots (n=174) (table 1). all these patients were treated at the dental trauma service of piracicaba school of dentistry – unicamp, endodontics area. the research was approved by institutional ethics committee and the identities of the patients and their legal representatives were kept confidential. patients received explanations and were informed that both pictures and periapical radiographs would be used for scientific purposes only. the sample was selected by the clinical charts of patients who were attended in regional hospitals and health centers of the neighboring cities, and from the files of the oral diagnosis department, oral and maxillofacial area of piracicaba school of dentistry, unicamp. after the emergency visit, patients were referred to the local dental trauma service of piracicaba school of dentistry, unicamp, endodontics area, and treated by 3 trained endodontic specialists. information was collected about age, gender, number of traumatized teeth, trauma date, etiology of trauma and first attendance date. all patients diagnosed with pulp necrosis, periodontal tissue involvement and radiographic signs suggesting root resorption were included in the research. teeth with inflammatory exudate or severe alveolar bone ridge destruction were excluded from the study. pulp sensitivity was evaluated by the thermal test, using a mixture of butane at -50ºc (endo-frost, roeko, langenau, germany), in association with radiographic findings. in teeth with immature root development (group 1), the pulp sensitivity test was confirmed by the cavity test. endodontic treatment in groups 1 and 2 in both immature (g1) and completely developed teeth (g2) cavity access was performed in a similar manner, using #1014 diamond burs (k.g. sorensen, barueri, sp, brazil) in a high-speed hand piece with copious sterile saline irrigation. outline shape was completed with #3082 blunt-end cylindrical diamond burs (k.g. sorensen). afterwards, the teeth were isolated with a rubber dam and stabilized with super bonder adhesive (loctite brasil ltda., itapevi, sp, brazil). chemomechanical preparation in immature teeth was group patients gender age teeth male female <7 7-14 15-20 21-26 >26 1 23(21.9%) 16 7 5 18 0 0 0 28(13.9%) 2 82(78.1%) 57 25 0 42 20 10 10 174(86.1%) total 105(100%) 73(69.5%) 32(30.5%) 5(4.8%) 60(57.1%) 20(19%) 10(9.6%) 10(9.6%) 202(100%) table 1.table 1.table 1.table 1.table 1. characterization of groups (g1 and g2). intracanal dressing paste composed by calcium hydroxide, chlorhexidine and zinc oxide for the treatment of immature and mature traumatized teeth 8 braz j oral sci. 13(1):6-11 modified due to the thickness of dentin walls and open apex. decontamination in the cervical and middle thirds was performed with gates-glidden burs #5, 4, 3 and 2 (dentsply/ maillefer, petrópolis, rj, brazil). length determination was performed by radiographic analyses using a k-file (dentsply/ maillefer) inserted up to the apical constriction. next, the apical third was instrumented with k-files (dentsply/ maillefer,), #50 to 80, inserted up to the apical limit. in some cases, it was necessary to use hedstroem files (dentsply/ maillefer), #50 to 60, to the same length, for removing pulp debris. all root canals were filled with 2% chlorhexidine gel during the entire mechanical preparation procedure and were irrigated with sterile saline solution at each file change. at the conclusion of chemomechanical preparation, 17% edta solution was used for 3 min, and a final irrigation by sterile saline solution after this. after aspiration, the root canals were dried with fitted paper points (konne indústria e comércio de materiais odontológicos ltda., belo horizonte, mg, brazil). teeth with closed apex (g2) were subjected to the same decontamination protocol, with the difference that root canal length determination was performed using an electronic apex locator (novapex; forum technologies, rishon-le-zion, israel), and instrumentation with smaller diameter files. manipulation and insertion of intracanal dressing the intracanal dressing paste was prepared using calcium hydroxide (biodin âmica™, ibiporã, pr, brazil), 2% chlorhexidine gel (farmácia de manipulação ltda., itapetininga, sp, brazil) and zinc oxide (s.s. white artigos dentários ltda., rio de janeiro, rj, brazil), ata2:1:2 rate. this paste was manipulated to a firm consistency similar to coltosol (vigodent s/a indústria e comércio, rio de janeiro, rj, brazil), and was inserted in increments, using mediumand fine-medium-sized vertical condensors (konne) (figure 1) in the entire extent of the root canal up to the apical constriction. a control radiograph was performed to verify the apical limit of the intracanal dressing paste. after this, the teeth were sealed with coltosol (vigodent s/a indústria e comércio), restored with composite resin (filtek z350, 3m dental products, saint paul, mn, usa), and then radiographed (figures 2 and 3). the patients were followedup for at least one year. definitive root canal filling with fig. 2. immature tooth (group 1) treated with the root filling paste. (a) initial radiograph. (b) treatment with the root filling paste. (c) 12-month follow-up. fig. 1. preparation of the root filling paste sealer and gutta-percha was performed only in cases where it was observed dissolution of the paste and need for an esthetic restoration with intracanal posts. clinical and radiographic evaluation evaluations were made before and after insertion of the intracanal paste. the clinical examination showed presence or absence of spontaneous pain, abscess, fistula, mobility and sensitivity to percussion. the radiographic examination revealed presence or absence of cortical alveolar bone, periodontal ligament thickness, root resorptions (inflammatory and replacement types) and periapical radiolucency. for radiographic analyses, rapid speed periapical films (kodak, são josé dos campos, sp, brazil) and radiographic exposure of 55 kvp and 15 ma for 0.7 s were used. an intraoral film holder (indusbello indústria de instrumentos odontológicos ltda., londrina, pr, brazil) was used to standardize and improve the quality of radiographs. the radiographs of groups 1and 2were evaluated using a intracanal dressing paste composed by calcium hydroxide, chlorhexidine and zinc oxide for the treatment of immature and mature traumatized teeth fig. 3. mature tooth (group 2) treated with the root filling paste. (a) initial radiograph. (b) treatment with the root filling paste. (c) 12-month follow-up. 9 braz j oral sci. 13(1):6-11 dental trauma group 1 group 2 subluxation 5 (17.9%) 8(4.7%) extrusive luxation 11 (39.3%) 46(26.4%) lateral luxation 5(17.9%) 15(8.6%) intrusive luxation 1(3.5%) 71(40.8%) avulsion 6(21.4%) 34(19.5%) total 28(100%) 174(100%) table 2.table 2.table 2.table 2.table 2.types of dental trauma affecting immature (group 1) and completely developed teeth (group 2). radiographic aspects group 1 (n=28) group 2 (n=174) before after p* before after p* periodontal ligament thickness 16(57.1%) 2(7.1%) 0.0001 95(54.6%) 41(23.6%) <0.0001 cortical alveolar bone 7(25%) 21(75%) 0.0001 58 94 <0.0001 inflammatory resorption 3(10.7%) 2(7.1%) 1 42(24.1%) 10(5.7%) <0.0001 replacement resorption 3(10.7%) 6(21.4%) 0.25 11(6.3%) 49(28.1%) <0.0001 periapical radiolucence 5(17.9%) 2(7.1%) 0.25 70(40.2%) 31(17.8%) <0.0001 * mcnemar test, p<0,05 table 4.table 4.table 4.table 4.table 4. radiographic evaluation observed before and after intracanal dressing with filling paste in immature and completely developed teeth. clinical aspects group 1 (n=28) group 2 (n=174) before after p* before after p* spontaneous pain 1(3.7%) 0(0%) 1 15(8.6%) 0(0%) 0.0001 fistula 3(10.7%) 0(0%) 0.25 10(5.7%) 0(0%) 0.002 abscess 4(14.3%) 0(0%) 0.125 5(2.9%) 0(0%) 0.0625 mobility 12(42.9%) 1(3.7%) 0.001 97(55.7%) 15(8.6%) <0.0001 pain on percussion 15(53.6%) 1(3.7%) 0.0001 95(54.6%) 7(4%) <0.0001 table 3.table 3.table 3.table 3.table 3.clinical evaluation before and after intracanal dressing with filling paste in groups 1 and 2. * mcnemar test, p<0.05 intracanal dressing paste composed by calcium hydroxide, chlorhexidine and zinc oxide for the treatment of immature and mature traumatized teeth light box (lumatron; encor indústria fotográfica ltda, rio claro, sp, brazil), coupled to a lens providing 4 times enlargement. statistical analysis the results were evaluated in the bioestat 5.0 program at a level of significance level of 5%. the mcnemar test was used to analyze clinical and radiographic parameters before and after insertion of the intracanal paste. results groups 1 and 2 were affected by subluxation, extrusive luxation, lateral luxation, intrusive luxation and avulsion. most of the immature teeth suffered extrusive luxation (39.3%), whereas in completely developed teeth, intrusive luxation (40.8%) was more common (table 2). in the clinical evaluation it was observed that in both immature and completely developed teeth there was complete reduction of spontaneous pain, fistula and abscess after the use of the studied intracanal dressing paste(table 3). this decrease was significant for pain on percussion (p=0.001) and mobility (p=0.0001) in immature teeth. for the completely developed teeth, this decrease was statistically significant for spontaneous pain (p=0.0001), fistula (p=0.002), mobility (p<0.0001) and pain on percussion (p<0.0001). the radiographic evaluation before and after use of the intracanal dressing paste in immature and completely developed teeth is shown in table 4. discussion dental trauma, depending on its severity, may lead to complications in the pulp and periodontal tissues. literature reports have shown that intrusive luxation and tooth avulsion are most frequently related to pulp necrosis and root resorptions (76.2%), requiring endodontic treatment3. in the present study this relationship was also observed for completely developed teeth, since most of the sample presented intrusive luxation (40.8%). root development must be considered for the treatment to be adopted, since immature teeth present thin dentin walls and open apices, which could make it difficult to achieve appropriate apical sealing and may weaken the root structure. literature reports have shown promising treatments for these conditions with pulp revascularization and apexification17. revascularization has been studied as an alternative treatment in some cases of incomplete root formation, because it stimulates the thickening and apical closure of immature teeth18-19. however, revascularization may present potential clinical and biological complications, such as crown discoloration20, development of resistant bacterial strains and allergic reaction to the intracanal medication21. moreover, the mechanism of pulp revascularization, the type of tissue that develops on the root canal walls and the clinical outcome of a long follow-up period are still unclear. considering these aspects, a more predictable treatment (apexification) was the 10 braz j oral sci. 13(1):6-11 intracanal dressing paste composed by calcium hydroxide, chlorhexidine and zinc oxide for the treatment of immature and mature traumatized teeth treatment of choice in the present study. apexification usually involves refreshing the calcium hydroxide paste every three months, requiring multiple visits with heavy demands on patients and operators, inevitable clinical costs, and the increased risk of tooth fracture, since the use of calcium hydroxide requires many dressing changes until a calcified barrier isformed12,22. this study demonstrated an alternative to periodic changes of intracanal medication in apexification, using a paste composed by calcium hydroxide, chlorhexidine and zinc oxide. this dressing reduced clinical and radiographic pathological signs, in addition to promoting apical closure even without periodic changes. in previous reports on immature teeth, this medicament was also successfully used in apexification13-15. concurring with the present study, a recent clinical research showed that most of the immature teeth (74%) needed only a single application of calcium hydroxide for complete apexification and replacements were required only for teeth presenting displacement and/or sinus tracts23. endodontic treatment in traumatized teeth with completely developed roots follows the same protocol as for teeth with pulp necrosis due to dental caries. some authors suggested the need for calcium hydroxide as intracanal medication to prevent complications such as root resorptions24. the present study also treated these completely formed teeth with intracanal dressing composed by calcium hydroxide, chlorhexidine and zinc oxide. no dissolution of the dressing was observed, which may have acted as a physical barrier preventing contamination of the root canal and periapical region. in addition, it produced a reduction of the clinical and radiographic symptoms and prevented root resorption. the intracanal dressing paste used in the present study may be used both for immature and completely developed teeth after dental trauma. technically, it is easy to manipulate, simple to insert with condensers and presents radiopacity, allowing observation of complete filling of the root canal. in addition, it acts as a temporary material and may be more effective than a paste dressing for long periods of follow-up. considering severe trauma such as tooth avulsion and intrusive luxation that may require long-term follow-up due to high probability of progressive root resorptions, this intracanal dressing paste may be an alternative to gutta-percha obturation. endodontic filling with gutta-percha and sealer does not dissolve, differently from what occurs with this intracanal dressing paste, since it may dissolve at the same time as it comes into contact with the periapical tissues. this paste also demonstrated antimicrobial properties, and maintained alkaline ph for one week25. it is likely that after some weeks, only zinc oxide was present, since ca(oh)2 should have undergone complete dissolution14. the presence of zinc oxide may have worked as an inert sealing material, preventing contamination, and allowing apical repair and barrier formation in cases of open apices. the absence of a good sealing and the presence of radiographic and clinical symptoms may indicate the need to replace the medication. further studies should be conducted on the composition, mechanism of action and long term follow-up of cases treated with this intracanal dressing. more important than the intracanal dressing, is the cleaning of root canal system. several studies demonstrated that reduction of microorganisms within the root canal allows periapical repair and formation of a calcified tissue barrier in open apex teeth11,17. in this study, 2% chlorhexidine gel was used as auxiliary chemical substance. it was demonstrated that 2% chlorhexidine gel has excellent antimicrobial activity and is more biocompatible than sodium hypochlorite25. the new intracanal dressing paste for traumatized teeth evaluated in this study showed promising results for both immature and completely developed teeth. all patients presented remission of clinical signs and radiographic reduction or repair of radiolucent lesions and root resorption. the paste, which has calcium hydroxide, chlorhexidine and zinc oxide as main components, may be an alternative for dental trauma cases diagnosed with pulp necrosis, and may prevent post-traumatic complications. references 1. andreasen jo. pulp and periodontal tissue repair regeneration or tissue metaplasia after dental trauma. a review. dent traumatol. 2012; 28: 19-24. 2. al-jundi sh. type of treatment, prognosis, and estimation of time spent to manage dental trauma in late presentation cases at a dental teaching hospital: a longitudinal and retrospective study. dent traumatol. 2004; 20: 1-5. 3. hecova h, tzigkounakis v, merglova v, netolicky j. a retrospective study of 889 injured permanent teeth. dent traumatol. 2010; 26:466-75. 4. mohammadi z, dummer pm. properties and applications of calcium hydroxide in endodontics and dental traumatology. int endod j. 2011; 44: 697-730. 5. al ansary ma, day pf, duggal ms, brunton pa. interventions for treating traumatized necrotic immature permanent anterior teeth: inducing a calcific barrier & root strengthening. dent traumatol. 2009; 25: 367-79. 6. turkistani j, hanno a. recent trends in the management of dento alveolar traumatic injuries to primary and young permanent teeth. dent traumatol. 2011; 27:46-54. 7. shah n, logani a, bhaskar u, aggarwal v. efficacy of revascularization to induce apexification/apexogensis in infected, nonvital, immature teeth: a pilot clinical study. j endod. 2008; 34: 919-25. 8. cotti e, mereu m, lusso d. regenerative treatment of an immature, traumatized tooth with apical periodontitis: report of a case. j endod. 2008; 34: 611-6. 9. petrino ja, boda kk, shambarger s, bowles wr, mcclanahan sb. challenges in regenerative endodontics: a case series. j endod. 2010; 36: 536-41. 10. chen my, chen kl, chen ca, tayebaty f, rosenberg pa, lin lm. responses of immature permanent teeth with infected necrotic pulp tissue and apical periodontitis/abscess to revascularization procedures. int endod j. 2012; 45: 294-305. 11. chala s, abouqal r, rida s. apexification of immature teeth with calcium hydroxide or mineral trioxide aggregate: systematic review and metaanalysis. oral surg oral med oral pathol oral radiol endod. 2011; 112: 36-42. 12. andreasen jo, farik b, munksgaard ec. long-term calcium hydroxide as a root canal dressing may increase risk of root fracture. dent traumatol. 2002; 18: 134-7. 13. soares aj, lima tfr, lins ff, herrera dr, gomes bpfa, de souzafilho fj. um nuevo protocolo de medicación intraconducto para dientes com necrosis pulpar y rizogénesis incompleta. rev estomatol herediana. 2011; 21: 145-9. braz j oral sci. 13(1):6-11 11intracanal dressing paste composed by calcium hydroxide, chlorhexidine and zinc oxide for the treatment of immature and mature traumatized teeth 14. soares aj, nagata jy, casarin rcv, almeida jfa, gomes bpfa, zaia aa, et al. apexification by using a new intra-canal medicament: a multidisciplinary case report. iran endod j. 2012; 7: 165-70. 15. soares aj, prado m, lima tfr, zaia aa, souza-filho fj. the multidisciplinary management of avulsed teeth: a case report. iran endod j. 2012; 7: 203-6. 16. buck clbp, soares aj, nagata jy, buck a, zaia a, souza-filho fj. evaluation of reimplanted teeth submitted to a new therapeutic protocol. rev assoc paul cir dent. 2013; 67: 22-6. 17. aggarwal v, miglani s, singla m. conventional apexification and revascularization induced maturogenesis of two non-vital, immature teeth in same patient: 24 months follow up of a case. j conserv dent. 2012; 15: 68-72. 18. thibodeau b, teixeira f, yamauchi m, caplan dj, trope m. pulp revascularization of immature dog teeth with apical periodontitis. j endod. 2007; 33: 680-9. 19. friedlander lt, cullinan mp, love rm. dental stem cells and their potential role in apexogenesis and apexification. int endod j. 2009; 42: 955-62. 20. kim jh, kim y, shin sj, park jw, jung iy. tooth discoloration of immature permanent incisor associated with triple antibiotic therapy: a case report. j endod. 2010; 36: 1086-91. 21. reynolds k, johnson jd, cohenca n. pulp revascularization of necrotic bilateral bicuspids using a modiûed novel technique to eliminate potential coronal discoloration: a case report. int endod j. 2009; 42: 84-92. 22. rafter m. apexification: a review. dent traumatol. 2005; 21: 1-8. 23. yassen gh, chin j, mohammedsharif ag, alsoufy ss, othman ss, eckert g. the effect of frequency of calcium hydroxide dressing change and various preand inter-operative factors on the endodontic treatment of traumatized immature permanent incisors. dent traumatol. 2012; 28: 296301. 24. diangelis aj, andreasen jo, ebeleseder ka, kenny dj, trope m, sigurdsson a, et al. international association of dental traumatology guidelines for the management of traumatic dental injuries: 1. fractures and luxations of permanent teeth. dent traumatol. 2012; 28: 2-12. 25. de souza-filho fj, soares ade j, vianna me, zaia aa, ferraz cc, gomes bp. antimicrobial effect and ph of chlorhexidine gel and calcium hydroxide alone and associated with other materials. braz dent j. 2008; 19: 28-33. oral sciences n3 original article braz j oral sci. january | march 2015 volume 14, number 1 civil liability related to imaging exams in brazil mathias pante fontana1, gabriela salatino liedke1, helena da silveira fontoura2, heraldo luis dias da silveira1, heloísa emilia dias da silveira1 1universidade federal do rio grande do sul ufrgs, dental school, department of surgery and orthopedics, porto alegre, rs, brazil 2universidade federal do rio grande do sul ufrgs, school of law, porto alegre, rs, brazil correspondence to: heloisa emilia dias da silveira faculdade de odontologia universidade federal do rio grande do sul rua ramiro barcelos 2492. cep: 90035-003 porto alegre, rs, brasil phone/fax: +55 51 3308 5199 e-mail: heloisa.silveira@ufrgs.br abstract aim: to analyze all court lawsuits in brazil in relation to civil liability involving radiographic and tomographic images up to february 2014. methods: all brazilian courts were surveyed for “civil liability,” “error,” “radiology,” “radiography,” and “tomography,” returning 3923 second-instance lawsuits. out of them were excluded labor legislation, health insurance coverage of radiological examinations, and criminal liability cases and 359 were selected. compliance with expert reports, involvement of imaging exams, the defendant professional, the reasons of claims and convictions, and indemnity were evaluated. results: of the 359 selected lawsuits, physicians were defendants in 71%, radiologist physicians in 10.6% and dentists in 18.4%. the prevalence of physicians found liable was related to the lack or delay in requesting the imaging exams (49.6%), and among radiologist physicians, misdiagnosis (47.1%). considering the dentists, imaging exams had mostly an indirect involvement, and failure of the proposed treatment (73.8%) was the most prevalent cause of dentists found liable; no radiologist was sued. regarding indemnity, 50% of lawsuits resulted in compensation up to r$ 20,000 (us$ 8,583). conclusions: misdiagnosis was the main cause of claims and radiologists were found liable. the medical field showed the largest absolute number of claims and physicians were found liable, but the highest proportion was directed to dentists. keywords: damage liability; jurisprudence; malpractice; tomography; radiography. introduction diagnostic radiology focuses on the detection of abnormalities in an imaging examination and their accurate diagnoses. discrepancies and misinterpretations were shown when physicians’ and radiologists’ interpretation were compared1; suggesting that image acquisition and interpretation should be the competency of a radiologist. this emphasizes the importance of expert knowledge for adequate image interpretation, particularly with respect to more advanced techniques. the development of radiology services along with the increase in litigation, especially in medicine, has increased the radiologist’s responsibilities 2-4. allegations of missed radiologic diagnosis accounted for an average of 42% of all cases over a 20-year period, according to a study conducted in the state of illinois, usa4. however, the failure to detect abnormalities is not necessarily malpractice: negligence occurs when the error violates the basic principles of interpretation or is a substantial cause of injury to patient’s health5-6. according to the american college of radiology, an official report must be made and filed following any examination. where findings require immediate intervention, the report should be submitted to the referring physician7. failure to communicate the results of the exam directly to the patient in case of selfreferral or if the physician cannot be reached, has been cause for lawsuits against radiologists2. braz j oral sci. 14(1):10-15 received for publication: december 16, 2014 accepted: march 04, 2015 region state available lawsuits (%) online since southeast são paulo 1980 134 (37.3) minas gerais 1983 101 (28.1) rio de janeiro 1975 58 (16.2) 293 (81.6) south rio grande do sul 1960 20 (5.6) paraná 1983 17 (4.7) santa catarina 1982 8 (2.2) 45 (12.5) midwest mato grosso do sul 2000 13 (3.6) mato grosso 1998 4 (1.1) 17 (4.7) northeast paraíba 2000 1 (0.3) pernambuco 1979 1 (0.3) 2 (0.6) north roraima 2006 2 (0.6) 2 (0.6) total 359 (100.0) table 1table 1table 1table 1table 1. lawsuits selected after on-line search at brazilian courts web-sites (grouped by geographical region) lawsuits have also grown against dentists around the world8-16. a study in germany found that dentistry was the second most frequent discipline confronted with claims of medical malpractice (16.4%), following surgery accidents and orthopedics (30.2%)17. furthermore, surveys conducted in denmark and the united kingdom showed a condemnation ratio of 43% and 54%, respectively, in cases involving dentists18-19. in brazil, complaints to the regional councils of dentistry showed orthodontics, prosthodontics, oral and maxillofacial surgery and implants as the specialties with the largest number of claims against dentists20-21. this trend was also observed in brazilian courts, and has shown a growth tendency in the last years22. even though no studies dealing with lawsuits involving dental radiologists have been found, dentists are intimately linked to imaging exams, and their acquisition, interpretation and storage are the dentists’ responsibility23-24. brazil represents the largest population, economy, and land area of south america, but its unique reality seldom stands out in global policy. bearing in mind the importance of the topic, as well as the growth of reports in the literature, the aim of this study was to analyze court decisions in brazil relative to liability involving radiographic and tomographic images. this study focuses on the mentioned imaging exams because of the higher frequency of use in both areas of interest, allowing comparisons between the medical and dental fields. material and methods the official web pages of all brazilian courts, available online up to february 2014, were surveyed for all second instance decisions involving civil liability and radiographic or tomographic images. the researched period varied according to the digitalization of the lawsuits in each brazilian state (table 1). the search strategy, comprising the terms “civil liability,” “error,” “radiology,” “radiography,” and “tomography,” yielded 3923 lawsuits (figure 1). the wide variety of terms used here aimed to identify the highest number of claims, since lawsuit language often lacks specific keywords or expressions regarding scientific vocabulary. fig. 1 study design flow chart two oral radiologists and a senior year law student examined the summary of each lawsuit, containing the claim and the verdict report. claims dealing with labor legislation, coverage of radiological exams by health insurance and criminal liability were excluded. the remaining 429 lawsuits were reviewed in full, and those in which imaging exams did not influence the decision, those without decision on their merits and repetitions of previously selected cases were excluded. the final selection comprised 359 lawsuits that contemplated professional or entity civil liability related to radiographic or tomographic examination. each one was thoroughly studied regarding the claim and its decision, as well as its legislative content, to allow grouping of data. data analysis: six topics of interest were observed in each lawsuit: compliance with expert reports, direct or indirect involvement of the imaging exam, the defendant (professional), the claim’s reasons, conviction causes and indemnity. direct involvement was indicated when the image was the reason to instigate the lawsuit. where the imaging exam was a probative element and not the merit of the claim, were considered as indirect involvement cases. the defendants were divided into four groups: physicians of any specialty other than radiology, hospitals, and medical clinics (group 1); radiologist physicians, radiology services linked to hospitals, and private radiology clinics (group 2); dentists of any specialty other than radiology and dental clinics (group 3); dentists and specialists in radiology and dental radiology clinics (group 4). the settlement in both material and moral harm, if present, was computed, and the amount paid was calculated in brazilian reais (r$) and u.s. dollars (us$), updated to the completion of data collection. to allow pooling of data, the paid indemnity was categorized into six ranges of values. civil liability related to imaging exams in brazil 1111111111 braz j oral sci. 14(1):10-15 when the conviction also involved the payment of a monthly pension to the plaintiff, the amount to be paid and the duration of payments were also registered. the institutional research committee approved the study (n. 20753). data retrieved from the webpages of the courts are public and therefore ethics committee submission was waived. data analysis was performed with the spss version 13.0 software package (spss, chicago, il, usa). results among the 27 brazilian states, 11 featured claims involving imaging exams (table 1). the south and southeast regions accounted for 94.1% of total occurrences. out of the studied 359 lawsuits, the expert report, whenever present was upheld by the judge in 96% of cases. image involvement was considered direct in 231 (64.3%) cases and indirect in 128 (35.7%) cases. claims that gave rise to the lawsuits classified as direct involvement included misdiagnosis, lack of or delay in requesting the imaging exam, exchange of exams, failure in performance or in quality of the exam, complications in the use of contrast, and complications with the device. treatment failure, on the other hand, was considered an indirect image involvement claim. group 1 had defendants in 255 cases (71%), group 2 in 38 cases (10.6%) and group 3 in 66 cases (18.4%). no cases fig. 3 prevalence of acquittals and convictions within each group against group 4 were found. figure 2 shows the distribution of the type of image involvement among the groups. figure 3 shows the prevalence of lawsuits in each group, and the proportion of acquittals and convictions within each case. in 198 cases (55.2%), the professionals or health entities involved were convicted, including 139 cases (54.5% of the group total) in group 1, 17 cases (44.7% of the group total) in group 2, and 42 cases (63.6% of the group total) in group 3. figure 4 shows the reasons that gave rise to convictions in relation to the professionals involved in each group. the highest frequency of convictions in group 1 related to the lack of an imaging exam or delay in requesting it (49.6%), while in group 2 the main reason was misdiagnosis (47.1%). on the other hand, most convictions in group 3 were due to the failure of a proposed treatment (73.8%); here, imaging exams were used as evidence in the lawsuit. only two dentists were sued for misdiagnosis, but none was found liable. regarding the quantum of indemnity, 49.5% convictions resulted in compensation for damages amounting up to r$ 20,000 (us$ 8,583), with compensation in 9.4% of cases exceeding r$ 160,000 (us$ 68,670). group 1 had the highest amount of compensations, reaching r$ 715,116.30 (us$ 306,916.90). figure 5 shows the distribution of compensation for material and moral harm per group. twenty-seven convicted professionals from group 1 (19.6%) and two from group 2 (12.5%) were sentenced to pay a monthly pension. fig. 2 distribution of lawsuits among the groups and image involvement within each one (%) fig. 4 reasons for the convictions in each group (%).tf = treatment failure, m = misdiagnosis, ldre = lack of or delay in requesting the exam, ee = exchange of exams, fpqe = failure in performance or quality of the exam, cc = complications with the use of contrast, o = others 1212121212 civil liability related to imaging exams in brazil braz j oral sci. 14(1):10-15 fig. 5 distribution of compensation for material and moral harm per group (%). range 1: up to r$ 10,000 (us$ 5,102), range 2: > r$ 10,000 r$ 20,000 (us$ 5,102 us$ 10,204), range 3: > r$ 20,000 r$ 40,000 (us$ 10,204 us$ 20,408), range 4: > r$ 40,000 r$ 80,000 (us$ 20,408 us$ 40,816), range 5: > r$ 80,000 r$ 160,000 (us$ 40,816 us$ 81,633), range 6: more than r$ 160,000 (us$ 81,633) discussion currently, physicians and dentists have increased responsibilities regarding the request for, performance and interpretation of imaging exams. considering the increase of claims against these professionals, studies conducted in the united states, united kingdom, australia and italy warn about the legal implications of errors in radiology, and suggest solutions to minimize the number of faults and their consequences2,4-6,24-26. lacking information about this reality in brazil, the aim of this study was to analyze court decisions covering imaging exams in medicine and dentistry. the study specifically addressed radiographic and tomographic images to allow comparison between the two areas, excluding other exams, such as ultrasonography, and mammography, which are seldom or not performed by dentists. the data search strategy selected 359 lawsuits, 94.1% of which were cases from southern and southeastern brazil, following the country’s size and development in those regions. the main reason for litigation against non-radiologist physicians and hospitals were the lack or delay in requesting the imaging exam (38.8%). the absence of a thorough investigation of a patient’s complaint that could be assessed by specific exam, or the excessive time spend to request it, causing sequelae or even death, were responsible for the conviction of 49.6% professionals from group 1. data from the united states showed that diagnostic error was responsible for 42% of lawsuits involving radiologists4. in the present study, 26.7% of the claims against group 1 and 52.6% against group 2 were due to misdiagnosis. among radiologists, 47.1% of them resulted in conviction. there were two false-negative and three false-positive cases of cancer. in three other cases, the diagnoses were a false negative for fracture, bleeding, and presence of a foreign body. driscoll, halpenny and guiney25 (2012) also observed that most errors were due to false-negative diagnoses, and occurred more frequently in radiographs (46.85%), followed by computed tomography (41.44%). radiological error is multifactorial, and could arise from poor technique, failures of perception, lack of knowledge and misjudgment27. an objective classification suggests dividing cases into procedural or diagnostic errors; errors can also be classified in observer errors, interpretation errors, failure to suggest the next appropriate procedure, and failure to communicate in a timely and clinically appropriate manner6,26,28. perception error is the failure to identify an abnormal radiological finding, while interpretation error is the failure to correctly interpret a radiological abnormality5. perception errors may be due to human fallibility, and can be explained by external factors (excessive time load and inappropriate workplace), technical mistakes, presence of abnormalities that were not under investigation, and by different lesions found in the same exam. on the other hand, interpretation errors may be influenced by inexperience, insufficient knowledge, or underestimation of radiographic signs that could lead to the correct diagnosis5,29. failures in detection are generally attributed to the subtlety of an abnormality, low incidence, or its poor definition: when the incidence of a particular disease is very low, most cases are correctly diagnosed (true negatives), but very rarely the lesion is present and not detected (false positives) 28,30. however, the phenomenon of nonidentification of “obvious” and easily recognized abnormalities in a second analysis was not yet satisfactorily explained. misinterpretations may result in harm to the patient and legal consequences to the professional. for this reason, errors are often kept secret. even though disclosure is important to the patient, reducing suffering, increasing quality of care and limiting the consequences of damage, there seems to be no evidence that it modifies the likelihood that the professional will be sued26. in the present study, the second largest cause of convictions in group 2 was due to complications arising from the use of contrast. the convictions were mainly due to the failure in obtaining informed consent, and failing to investigate the medical history of patients, which could avoid 1313131313civil liability related to imaging exams in brazil braz j oral sci. 14(1):10-15 1414141414 the risks involved with the procedure. in one case, a minor with microencephaly underwent a computed tomography scan and had complications triggered by the use of contrast, which placed the minor in a vegetative state. the radiologic clinic was fined in r$ 89,672.85 (us$ 38,486.20) as moral harm, and a monthly pension was paid to the victim until age 60. there is no consensus in the literature regarding the requirement of informed consent to the use of contrast in imaging exams, and the decision to obtain it or not from the patient depends on local legislation and policy of the hospital31. in the authors’ view, this case suggests that informed consent should be adopted by health professionals to avoid the risk of legal complications, corroborating mavroforou, et al.32 (2003). imaging exams, when properly requested after a careful clinical investigation, help establishing the diagnosis, treatment plan and follow-up routine. in this study, 27.8% of claims against group 1 and 75.8% against group 3 were due to treatment failure, generating 22.3% and 73.8% of convictions, respectively. the most litigated areas in dentistry were orthodontics, surgery, endodontics and implantology, in which imaging exams play an important role regarding the planning and monitoring of cases, corroborating previous findings in the literature9-12,14-16,18,20-22. moreover, 26.2% of convictions in group 3 were due to the lack of a request for an imaging exam, mainly in cases of tooth extractions without radiograph analysis, dental implant surgeries made unsuccessful by the lack of tomographic planning, and orthodontic movement resulting in root resorption that was not diagnosed and monitored. these results emphasize the importance of imaging exams requested by the professional, when appropriate, to start and follow-up treatment. this behavior allows proving that the professional acted with all expected carefulness and used the best techniques available to the case. the knowledge regarding ethical and legal implications of exercising a profession is fundamental33. giffoni filho et al.21 (2013) suggest that the study of ethics and bioethics should be part of the dental curriculum, to teach the student legal aspects of the career and to reduce future infractions. most lawsuits against health professionals included an expert report performed by a physician or dentist; when available, they were upheld by the judge in 96% of cases. however, complying with the report is the judge’s choice, according to his own conviction. additionally, the use of imaging exams as evidence in lawsuits involving dentists is common and its non-presentation when otherwise indicated for the correct diagnosis and treatment may suggest condemnation of the professional. in this study, most of the claims, in absolute numbers, were against those in the medical field. however, dentists showed a higher frequency of convictions. comparing indemnity ranges, higher amounts were paid by groups 1 and 2, and group 3 had 48.8% of convicted professional within the first range. the present study has some limitations. the search was limited to digitized decisions of lawsuits, which vary according to the region (table 1). since it is a time consuming and ongoing process, not all brazilian states retrieved results. in addition, only second instance decisions were included, since they generally are the final judgment of the court, except in case of extraordinary appeals. however, while only one country was studied, brazil is a continental country, representing the largest population, economy and land area of south america. other studies have also assessed the legal situation in a single country4,17-19,24-25, each one with its specific legal structure. law and liability depends on each country’s construction doctrine and jurisprudence. most of brazilian courts understand the duty assumed by health professionals as being of means and not of results, except in cases of aesthetic treatments. the duty is not to cure the patient, but to use the most appropriate treatment according to the state of science, carefully and consciously. nevertheless, in some cases the judges considered guilty those professionals who have not achieved the results expected by the patients. in other lawsuits, healthcare entities and radiology clinics were sentenced due to strict liability, based on article 14 of the brazilian consumer defense code34, which states that the service provider must respond, regardless the presence of guilt, to compensate for damages caused to the patient by faults in provided services, as well as those secondary to inadequate or insufficient information pertaining to the risks inherent in the provided care. strict liability, however, does not apply to liberal professionals. reasons for conviction in cases directly or indirectly involving imaging exams varied according to the studied occupational group. misdiagnosis was the highest cause of claims and convictions against radiologists and radiologic clinics. despite the largest number of physicians accused and convicted, the highest proportion of convictions occurred among dentists. references 1. alfaro d, levitt ma, english dk, williams v, eisenberg r. accuracy of interpretation of cranial computed tomography scans in an emergency medicine residency program. ann emerg med. 1995; 25: 169-74. 2. berlin l. are radiologists contracted by third parties to interpret radiographs liable for not communicating results directly to patients? ajr am j roentgenol. 2002; 178: 27-33. 3. smith jj, berlin l. the infected or substance abuse-impaired radiologist. ajr am j roentgenol. 2002; 178: 567-71. 4. berlin l, berlin jw. malpractice and radiologists in cook county, il: trends in 20 years of litigation. ajr am j roentgenol. 1995; 165: 781-8. 5. olivetti l, fileni a, de stefano f, cazzulani a, battaglia g, pescarini l. the legal implications of error in radiology. radiol med. 2008; 113: 599608. 6. pinto a, brunese l, pinto f, reali r, daniele s, romano l. the concept of error and malpractice in radiology. semin ultrasound ct mr. 2012; 33: 275-9. 7. american college of radiology. practice guideline for communication of diagnostic imaging findings resolution 11. reston: acr, 2010. 7p. 8. baker p, 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[updated 2013 jun 17; cited 2014 feb 11]. available from: http://www.planalto.gov.br/ccivil_03/leis/l8078.htm. 1515151515civil liability related to imaging exams in brazil braz j oral sci. 14(1):10-15 oral sciences n3 original article braz j oral sci. january | march 2015 volume 14, number 1 a finite element study on the mechanical behavior of reciprocating endodontic files mohamed i. el-anwar1, ayman o. mandorah2, salah a. yousief3, tarek a. soliman4, tarek m. abd el-wahab5 1national research centre, department of mechanical engineering, giza, egypt 2 king faisal hospital, dental department, makkah, ksa and umm-alqura university, faculty of dentistry, department of endodontics, makkah, ksa 3 al-farabi dental college, department of restorative dentistry, jeddah, ksa, and university of al azhar, faculty of oral and dental medicine, department of crown & bridge, assiut, egypt 4university of mansoura, faculty of dentistry, department of dental biomaterials, mansoura, egypt 5university of mansoura, faculty of dentistry, department of conservative dentistry, mansoura, egypt correspondence to: mohamed i. el-anwar national research centre department of mechanical engineering 33 el bohouth st., dokki, giza egypt. p.o. 12622 phone: +2 0122 2431297 fax: +2 02 33370931 e-mail: anwar_eg@yahoo.com abstract aim: to evaluate the mechanical behavior of reciprocating endodontic files, comparing nickeltitanium (niti) and stainless steel 316l (st.st. 316l) as manufacturing material for such instruments. methods: a three-dimensional finite element model was designed for this study. the simplified instrument model geometry was created on commercial cad/cam software. real strain stress curves of st.st. 316l and niti were used in the analysis. non-linear static analysis was performed to simulate the instrument inside root canal at an angle of 45° in the apical portion, and subjected to torsion of 0.3 n.cm. results: non-linear niti material showed super elasticity and high functionality in such applications. very high levels of stress appeared in the file at 3 mm from the tip close to yield point. conclusions: st. st. 316l is not suitable for manufacturing reciprocating instruments. modeling of the instrument with equivalent circular cross-sectional area did not affect results quality. reciprocating instruments have short lifespan, thus manufacturers recommend using one file per tooth. reciprocating instruments are recommended for less experienced dentist. keywords: endodontics; stainless steel; nickel; titanium. introduction the goals of endodontic instrumentation are to shape without deviating from the original canal position, to enlarge until the walls are smooth and free of soft tissues, to completely remove microorganisms and debris, and to create a canal form that converges toward the foramen 1. stainless steel endodontic instruments, whose characteristics include stiffness that increases with size, may set limitations to successful shaping. during enlargement of the apical third, this characteristic may be responsible for curvature defects such as apical transportation, ledging or zipping, which might compromise the outcome of treatment2. in the early 1960s, a nickel-titanium (niti) alloy was developed by buehler, during the investigation of nonmagnetic, salt resisting, and waterproof alloys for the space program at the naval ordnance laboratory in silver springs, maryland, usa3. nitinol is the name given to a family of inter-metallic alloys of nickel and titanium, which has unique properties of shape memory and super-elasticity. nickel titanium instruments are more flexible than stainless steel instruments and have the ability to revert to their original shape after flexure. it has been reported that niti instruments are 2 to 3 times more flexible than stainless steel instruments braz j oral sci. 14(1):52-59 received for publication: november 11, 2014 accepted: march 17, 2015 braz j oral sci. 14(1):52-59 5353535353 and more resistant to fracture4. however, despite these advantages, the main problem with rotary niti instruments is a probable failure of the instruments. instrument fracture is a serious problem and can jeopardize the outcome of the root canal treatment. separation of rotary niti instruments can occur due to two reasons: torsional fracture or cyclic fatigue5. torsional failure occurs when the tip of the instrument is locked in the canal while the shaft continues to rotate. if the elastic limit of the metal is exceeded, the instrument undergoes plastic deformation, which can be followed by fracture if the load is high enough. failure by torsional overload was reported as the most common cause of separation of rotary niti instruments6. endodontic files are subjected to stress when they are bent around a curve. every bent segment of the file experiences a cycle of both compressive and tensile stresses; this may lead to cyclic fatigue. due to initiation, propagation, coalescence of micro-fractures it may ultimately cause an overt fracture of the file. because the micro-fractures cannot be seen even with the aid of a surgical operating microscope, there is no warning preceding fracture7. possible strategies to increase efficiency and safety of niti rotary instrument include improving the manufacturing process or using new alloys that provide superior mechanical properties. the stiffness and flexibility of endodontic files are greatly dependent on their geometric design, including taper, helix angle, cross section shape, tip size and length8. in an attempt to increase the resistance to cyclic fatigue of the rotary instruments, m-wire was introduced by applying a series of heat treatments to niti wire blanks9. before the grinding process, the alloy was thermally treated to improve its properties. the final goal was to produce instruments with superelastic behavior (reduced generation and accumulation of lattice defects during each load-unload cycle) and increased resistance to cyclic fatigue, compared to those constructed from traditional niti alloy 10. the first commercially available endodontic rotary system using the new m-wire niti material was gtx (dentsply tulsa dental specialties, tulsa, ok, usa). recently, costly experimental studies 11-13 were implemented on different brands and types of instruments like reciproc, waveone, hyflex, mtwo, protaper, endowave, etc., according to iso 3630-1. tens of instruments were tested in order to statistically prove that heat treated instruments have better cutting efficiency and higher fatigue resistance than conventional niti files. in 2008 yared14 proposed a new preparation motion (reciprocating), using only f2 protaper rotary file to prepare the root canal. it has been claimed that rotary niti endodontic files show more resistance to cyclic fatigue, when used in reciprocating motion. various cyclic fatigue tests have been conducted to compare file systems which allow the files to rotate till fracture. flexural-fatigue failure occurs when the instrument rotates inside a curved canal while subjected to an excessive number of tensile-compressive strain cycles in the region of maximum canal curvature. the stresses generated during flexural loading are directly associated to the fatigue life of the material15. the stress conditions within instruments cannot be revealed by inspection of broken segments but require a mathematical/ numerical simulation. finite element analysis (fea) has been applied as alternative to study the mechanical behavior of endodontic instruments for detailed assessment of stress distributions in instruments15-16. arruda santos et al.17 confirmed the potential of fea as a numerical method to assess the mechanical behavior of endodontic instruments comparing the behavior of three different types of instruments: mtwo (vdw, munich, germany), race (fkg dentaire, la-chaux-de-fonds, switzerland) size 25, .06 taper (0.25 mm tip diameter, 0.06% conicity) and ptu f1 (dentsply maillefer, ballaigues, switzerland) experimentally and numerically by fea17. the aim of this study was to evaluate the mechanical behavior of reciprocating endodontic files, comparing nickeltitanium (niti) and stainless steel 316l (st.st. 316l) as manufacturing material for such instruments. material and methods in this study the waveone (dentsply maillefer, ballaigues, switzerland) primary file (equivalent to protaper f2 file) was modeled in 3d (figure 1). the file tip size was iso-25 with an apical taper of 8% that reduces towards the coronal end. the 3d geometric model was prepared on a commercial general purpose cad/cam software (autodesk inventor version 8.0; autodesk inc., san rafael, ca, usa). although the cross section of the wave one primary file is a convex triangle, it was simplified in this study to be circular with equivalent cross-sectional area, while the change in cross section in the apical part was neglected. the niti instruments and wires with different cross sections showed similar behavior in previous studies17-18 which inspired the simplification of the modeled file cross section. the geometric model was transferred as iges file to the meshing and finite element analysis package (ansys version 14.5; ansys inc., canonsburg, pa, usa). the meshing element was 20 nodes “solid 186” which has three degrees of freedom (translations in the global directions). mesh density is a parameter that improves the result accuracy and reduces artificial peak stresses by improving the representation of the actual geometry. the mesh density effect was evaluated before extracting results with 20,935 nodes and 13,057 elements. multi-linear material was defined as presented in figure 2. niti alloy and aisi 316l stainless steel, widely used in biomedical applications and described by an elasto-plastic constitutive model with kinematic hardening 19. in both material models, the values of the characteristic parameters were derived from the literature. considering the niti alloy, the transformation starting stress σsssss and the transformation finishing stress σfffff, are the stress values when, at the working temperature, the transformation between austenite and singlevariant martensite starts and finishes, respectively. the limit a finite element study on the mechanical behavior of reciprocating endodontic files 5454545454 transformation strain εlllll is the amplitude of the transformation strain interval. the capacity to recover all the deformation (i.e. pseudo-elastic behavior) ends when the martensitic yielding stress ‘σyyyyy’’’’’ and the martensitic yielding strain ‘εyyyyy’’’’’ (indicated as pseudo-elastic limits) are assessed19. the boundary conditions were imposed to simulate the behavior of the files under bending and torsional conditions in compliance with the iso 3630-1 specification20. to test the bending resistance, the bending moment was calculated while the file was clamped 3 mm from the tip and the shaft was deflected until 45° inclination21. to evaluate the torsional resistance, the file was held at 3 mm from the tip, and a clockwise torsional moment of 0.3 n.cm was applied. the boundary conditions used in the bending and torsional simulations are presented in figure 3. the model used in this study was confronted with fig. 1: modeled file geometry on inventor screen fig. 2: niti and st.st. 316l material properties (uniaxial stress-strain curves)19 previously published researches18-19, and showed close and comparable results. in this study was planned to perform non-linear static analysis under the worst loading conditions. this analysis was followed by fatigue failure check on st.st. 316l and niti, stress vs number-of-cycles curves of both materials to estimate the instrument lifespan, as evaluation procedure steps in previous study by cheung et al.18. as presented in figure 4(a) cheung et al.18 used bannantine et al.22 equations and tabulated parameter values (table 1) to estimate the instrument lifespan. in figure 4(b) norwich et al.23 showed the endurance strain amplitude % limit of 0.6 for 0.23 mm diameter niti wire. …. (1) 18 …. (2) 18 a finite element study on the mechanical behavior of reciprocating endodontic files braz j oral sci. 14(1):52-59 5555555555 symbol stainless steel nickel-titanium (niti) (304 annealed) alloy (super elastic) cyclic strain hardening exponent n′ 0.334 0.1 cyclic strength coefficient k′ 2275 mpa 733 mpa fatigue strength exponent b -0.139 -0.06 fatigue strength coefficient σ′f 1267 mpa 705 mpa fatigue ductility exponent c -0.415 -0.6 fatigue ductility coefficient ε′f 0.174 0.68 table 1.table 1.table 1.table 1.table 1. parameters used in evaluating fatigue behavior of st.st. and niti wires18 fig. 3: (a) file outlines and regions, (b) boundary conditions for the bending and torsion simulations21 (a) (b) fig. 4: (a) comparison between triangle and square cross sections of stst and niti wires life-time vs. tip displacement18 and (b) niti (ti-55.8/55.9 wt%ni) 0.23 & 0.61 mm heat treated wire diameters; strain % vs. life-time curves23. lcf is low cycle fatigue, hcf is low cycle fatigue, sq is square cross section, tr is triangular cross section. (a) (b) a finite element study on the mechanical behavior of reciprocating endodontic files 5656565656 where e is young’s modulus, εapapapapap is plastic strain amplitude, εa a a a a and σaaaaa are total strain and total stress amplitudes respectively. the solid modeling and finite element non-linear static analysis were performed on a server hp prolaint ml150, with intel xeon 3.2 ghz processors (with 12 mb l2 cache), 10 gb ram. results the linear static analysis showed unrealistic stress level, thus a multi-linear stress strain curve is essential in nonlinear analysis. therefore, two runs on the constructed model were performed, simulating the use of the files under bending and torsional conditions in compliance with the iso 3630-1 specification20. first simulation was performed for st.st. 316l as the file material and its stress strain curve was imported to ansys as multi-linear material. as presented in figures 2, 4, and 5, the generated stress and strain levels on the instrument exceeded the fracture point of st.st. 316l, which indicated fig. 5: st.st. 316l file behavior (a) von mises stress distribution and (b) plastic von mises strain distribution, with deformed/un-deformed shape instrument failure. thus st.st. 316l as a material is not suitable for manufacturing rotary/reciprocating files. second simulation was performed for niti as the file material, and its stress strain curve was imported to ansys as multi-linear material, as illustrated in figure 2. the meshed model, deformed shape and total deformation of the studied model are presented in figure 6. figures 7(a) and 8(a) illustrated the distributions of von mises strain (ca 0.61) and stress (ca 480 mpa) respectively, showing the critical points at file tip and outer layer in the bending region. the total plastic strain in figure 7(b) represents the majority of total strain that indicated fatigue failure expectation. maximum tensile stress in figure 8(b) dominated the total stress and showed the critical point (maximum total stress) at the outer layer at the bending region (3 mm from tip). discussion using basics of mechanics from literature5-6 to compare between the two systems (rotary and reciprocating) with the fig. 6: niti file (a) mesh & deformed shape and (b) total deformation distribution a finite element study on the mechanical behavior of reciprocating endodontic files braz j oral sci. 14(1):52-59 (a) (b) (a) (b) 5757575757 same file will give good indications. the reciprocating system will have three advantages and one drawback. the first advantage is reducing the torsional stress, since during reciprocation movement the instrument engages dentin at its tip during the counterclockwise movement, whereas the clockwise movement disengages the instrument immediately afterwards24. second, extended fatigue life of the niti file when used in reciprocation movement compared to continuous rotation, which may be explained by smaller angular movement that induces less stress on the file in case of locking25. finally, reciprocating action working as hammer at the file’s cutting edge, increased the applied load on the root canal to double its value. the drawback is an expected shorter lifespan in comparison to rotating one, due to increased load applied at the blade tip. finite element analysis has been applied for detailed assessment of stress distributions in instruments. few researches have studied the influence of reciprocating motion on cyclic fatigue resistance of rotary niti files using finite element analysis16. in this research, st.st. 316l file analysis showed that the generated stress levels on the instrument exceed the fracture stress (ca 568 mpa)26, which indicated instrument failure. this may be attributed to the rigidity of stainless steel alloy, which is not suitable for instrumentation of curved canals under high torsional load. on the other hand, the niti file analysis showed maximum values of total von mises stress (ca 480 mpa) and strain (ca 0.61) as indicated in figures 7 and 8 which are matching similar studies 22,27, although with less instruments’ cross sectional details in current study. plastic stress and strain components are dominating the total von mises stress and strain that indicates fatigue failure expectation after certain number of such type of load(45° bend without locking). the expected number of cycles under such level of stress may be obtained from figure 4, is approximately 500 cycles (file lifespan before failure). pessoa et al.28 reported a comparable average number of 310 cycles to failure for race rotary niti files (fkg dentaire, la-chaux-de-fonds, switzerland), size #5, taper 0.04 inside buccal canals with an angle of curvature of 40°, a finite element study on the mechanical behavior of reciprocating endodontic files fig. 8: niti file (a) von mises stress and (b) maximum tensile stress distributions fig. 7: niti file (a) von mises strain and (b) total plastic strain distributions (a) (b) (a) (b) curvature of 5 mm radius and 21 mm long, and the beginning of the curve was positioned 14 mm from the canal orifice. three roots per tooth require about 2 min to be treated by reciprocating instrument, if the average rotating speed of the instruments is 250 rpm. that was experimentally proven by you et al.29 who estimated the time for one root preparation by reciprocating instrument at 21.15 ± 6.70 second29 i.e. less than 2 min. in addition, applying equations 1 and 2 on the obtained results with niti instrument with locking at 3 mm from tip and 45° bend with 3 n.cm torque (as the worst possible loading condition), it will resist up to three locking cases during its life, which is too difficult to occur during root preparation of one tooth. in addition, the improvement in cyclic fatigue resistance shown by the reciprocating movement is related to two main factors. first, the rotation cycles are slightly reduced and consequently the overall number of rotations is reduced. second, during reciprocation there is a different distribution of the same tensile stress values in time and this may reduce the overall accumulation of fatigue 2 9. reciprocating preparation with only one file was much faster than root canal instrumentation with continuous rotation. however, one file can be safely used a limited number of times in reciprocating motion29. inexperienced operators achieved better canal preparations with reciprocating and/or rotary ni-ti instruments than with manual stainless steel files. however, rotary preparation was associated with significantly more fractures30. within the limitations of this study, the following conclusions can be drawn: finite element analysis indicated that modeling the instrument with fewer details (using equivalent circular cross sectional area) did not affect results quality. st.st. 316l, the traditional material used in manufacturing manual files, is not suitable for rotary or reciprocating instruments. reciprocating instruments have short lifespan, and it is usually recommended to use one file per tooth. reciprocating system has great advantages over other root therapy instruments. therefore, it is recommended to be used by less experienced dentist. references 1. del bello p, wang n, roane jb. crown-down tip design and shaping. j endod. 2003; 29: 513-8. 2. schäfer e, tepel j, hoppe w. properties of endodontic hand instruments used in rotary motion, part 2, instrumentation of curved canals. j endod. 1995; 21: 493-7. 3. saunders em. hand instrumentation in root canal preparation. endod top. 2005; 10: 163-7. 4. walia h, brantley wa, gerstein h. an initial investigation of the bending and torsional properties of nitinol root canal files. j endod. 1988; 14: 346-51. 5. fishelberg g, pawluk jw. nickel titanium rotary file canal preparation and intracanal file separation. compend contin educ dent. 2004; 25: 17-24. 6. sattapan b, nervo gj, palamara je, messer hh. defects in rotary nickel titanium files after clinical use. j endod. 2000; 26: 161-5. 7. pruett jp, clement dj, carnes dl. cyclic fatigue testing of nickel titanium endodontic instruments. j endod. 1997; 23: 77-85. 8. he r, ni j. design improvement and failure reduction of endodontic files through finite element analysis: application to v-taper file designs. j endod. 2010; 36: 1552–7. 9. shen y, zhou hm, zheng yf, peng b, haapasalo m. current challenges and concepts of the thermomechanical treatment of nickel-titanium instruments. j endod. 2013; 39: 163-72. 10. pereira es, peixoto if, viana ac, oliveira ii, gonzalez bm, buono vt, et al. physical and mechanical properties of a thermomechanically treated niti wire used in the manufacture of rotary endodontic instruments. int endod j. 2012; 45: 469-74. 11. plotino g, rubini ag, grande nm, testarelli l, gambarini g. cutting efficiency of reciproc and waveone reciprocating instruments. j endod. 2014; 40: 1228-30. 12. braga lc, faria silva ac, buono vt, de azevedo bahia mg. impact of heat treatments on the fatigue resistance of different rotary nickel-titanium instruments. j endod. 2014; 40: 1494-7. 13. pedullà e, lo savio f, boninelli s, plotino g, grande nm, rapisarda e, et al. influence of cyclic torsional preloading on cyclic fatigue resistance of nickel titanium instruments. int endod j. 2014 oct 29. doi: 10.1111/ iej.12400. 14. yared g. canal preparation using only one ni-ti rotary instrument: preliminary observations. int endod j. 2008; 41: 339-44. 15. câmara as, de castro martins r, viana ac, de toledo leonardo r, buono vt, de azevedo bahia mg. flexibility and torsional strength of protaper and protaper universal rotary instruments assessed by mechanical tests. j endod. 2009; 35: 113-6. 16. min-h o lee, anth eunis versluis, byung-min kim, chan-joo lee, bock hur, hyeon-cheol kim. correlation between experimental cyclic fatigue resistance and numerical stress analysis for nickel-titanium rotary files. j endod. 2011; 37: 1152-7. 17. de arruda santos l, lópez jb, de las casas eb, de azevedo bahia mg, buono vt. mechanical behavior of three nickel-titanium rotary files: a comparison of numerical simulation with bending and torsion tests. mater sci eng c. 2014; 37: 258-63. 18. cheung gs, zhang ew, zheng yf. a numerical method for predicting the bending fatigue life of niti and stainless steel root canal instruments. int endod j. 2011; 44: 357-61. 19. necchi s, petrini l, taschieri s, migliavacca f. a comparative computational analysis of the mechanical behavior of two nickel-titanium rotary endodontic instruments. j endod. 2010; 36: 1380-4. 20. international organization for standardization. 3630-1: dentistry. rootcanal instruments part 1: general requirements and test methods. 2nd ed. geneva: iso; 2008. 21. santos la, bahia mg, casas eb, buono vt. comparison of the mechanical behavior between controlled memory and superelastic nickeltitanium files via finite element analysis. j endod. 2013; 39: 1444-7. 22. bannantine ja, comer jj, handrock jl. fundamentals of metal fatigue analysis. upper saddle river: prentice hall; 1989. p.38. 23. norwich dw, fasching a. a study of the effect of diameter on the fatigue properties of niti wire. jmepeg. 2009; 18: 558-62. 24. pirani c, ruggeri o, cirulli pp, pelliccioni ga, gandolfi mg, prati c. metallurgical analysis and fatigue resistance of waveone and protaper nickel-titanium instruments. odontology. 2014; 102: 211-6. 25. lopes hp, vieira mvb, elias cn, siqueira jr jf, mangelli m, lopes wsp, et al. fatigue life of waveone and protaper instruments operated in reciprocating or continuous rotation movements and subjected to dynamic and static tests. endod pract today. 2013; 7: 217–22. 26. neeharika vb, narayana ks, krishna v, kumar mp. tensile and creep data of 316l (n) stainless steel analysis. j eng appl sci. 2014; 9: 699-705. 27. arbab-chirani r, chevalier v, arbab-chirani s, calloch s. comparative analysis of torsional and bending behavior through finite-element models of 5 ni–ti endodontic instruments. oral surg oral med oral pathol oral radiol endod. 2011; 111: 115-21. 5858585858 a finite element study on the mechanical behavior of reciprocating endodontic files braz j oral sci. 14(1):52-59 5959595959 28. pessoa of, da silva jm, gavini g. cyclic fatigue resistance of rotary niti instruments after simulated clinical use in curved root canals. braz dent j. 2013; 24: 117-20. 29. you sy, bae ks, baek sh, kum ky, shon wj, lee w. lifespan of one nickel-titanium rotary file with reciprocating motion in a curved root canals. j endod. 2010; 36: 1991-4. 30. sonntag d, guntermann a, kim sk, stachniss v. root canal shaping with manual stainless steel files and rotary ni-ti files performed by students. int endod j. 2003; 36: 246-55. a finite element study on the mechanical behavior of reciprocating endodontic files oral sciences n3 braz j oral sci. 12(2):100-104 original article braz j oral sci. april | june 2013 volume 12, number 2 microleakage in combined amalgam/composite resin restorations in mod cavities kosmas tolidis1, christina boutsiouki1, paris gerasimou1 correspondence to: christina boutsiouki 12 kallidopoulou str. 54642, thessaloniki, greece phone: +30 6945822764 fax: +30 2310863631 e-mail: christinaboutsiouki@gmail.com abstract aim: to compare marginal seal at tooth-material and material-material interfaces in the proximal box in combined amalgam/composite resin restorations. methods: mesio-occlusal-distal (mod) cavities were prepared in 35 premolars and permanent molars with carbide bur. the distal proximal box was restored with amalgam (permite, sdi) until reaching the height of pulpal floor. dental tissues were etched with 37% acid and a bonding agent (bond 1-sf, pentron) was applied and cured. composite resin (filtek z250, 3m-espe) was placed in layers in the mesial proximal box and occlusally, and light cured. marginal adaptation was evaluated at the following interfaces: amalgam-tooth (a), amalgam-composite resin (ac) and composite resin-tooth (c). microleakage was evaluated by means of methylene blue infiltration after 7-day water storage and thermocycling regimen (1500 cycles). microleakage was assessed as percentage depth of horizontal dye penetration. results: anova showed statistically significant difference between a-ac and a-c (p<0.01). no statistically significant difference was found between ac-c interfaces (p>0.05). mean microleakage values were a (73.529/28.71), ac (34.118/34.6) and c (40.435/ 34.965), according to tukey’s test. conclusions: although the bonding mechanism between amalgam and composite has not yet been completely explained, amalgam/composite resin interface exhibited the lowest microleakage scores. since amalgam/composite resin restorations exhibited lower microleakage scores than composite resin on the cervical surface, combined restorations can be considered as a biological and aesthetic alternative to conventional class ii composite or amalgam restorations. keywords: combined restorations, amalgam-composite, microleakage, class ii cavity, mod cavity. 1department of operative dentistry, school of dentistry, aristotle university of thessaloniki, greece introduction secondary caries is still cause for composite resin restoration failure1. teeth restored using composite resins are especially prone to this phenomenon due to stress generated within the tooth-restoration interface following resin contraction during polymerization, known as polymerization shrinkage 2. should the accumulated polymerization contraction stress result in tooth-composite adhesive failure3-4, bacterial aggregation at the disrupted tooth-restoration margin may occur resulting in microleakage and later secondary caries. furthermore, bonding on the cervical surface of class ii composite restorations is complex. despite the favorable presence of cervical enamel, composite resin bonding on cervical surface of proximal box usually takes place on aprismatic enamel or dentine. bonding onto aprismatic enamel is compromised by altered etching pattern5 and dentin bond is degraded, especially in permanent teeth 5-6. lower level of dentin received for publication: february 09, 2013 accepted: may 22, 2013 braz j oral sci. 12(2):100-104 101101101101101 mineralization, challenging moisture control for application of adhesive system, presence of tubular fluid and bonddegrading matrix-metalloproteinases hinder bonding to dentin5, making composite placement a technique-sensitive procedure7. thereby, both polymerization shrinkage and quality of the bond seem to be responsible for the degradation of marginal adaptation. microleakage is strongly controlled by marginal adaptation and is thought to be one of the major disadvantages of resin composite restorations8. although composite resins exhibit better initial marginal adaptation than amalgam9, amalgam restorations rarely fail due to secondary caries10. amalgam surface corrosion and deposition of oxides improve marginal auto-sealing over time11. in contrast to composite resins, amalgam is dimensionally stable. the aim of the present study was to compare marginal seal in tooth-material and material-material interfaces in the proximal box in combined amalgam/composite resin restorations via microleakage. the null hypothesis was that there is no difference in microleakage values between any of the tested interfaces. material and methods a group of 35 freshly extracted maxillary and mandibular premolars and permanent molars was collected from a private dental office. authorization was obtained from the owner of the dental office and only teeth extracted for orthodontic and periodontal reasons or impacted third molars were included in the study, for ethical reasons. teeth free of caries and fractures were stored in saline and not allowed to dry throughout the whole experiment. standard mesio-occlusal-distal (mod) cavities were prepared with carbide burs at high speed under air-water spray (depth of 1 mm beneath dentinoenamel junction, buccolingual width of 2.5 mm, mesiodistal width of cervical surface of proximal box of 2 mm). all margins were placed on enamel and proximal boxes were extended until 1 mm above cervix (figure 1). bur was discarded after each preparation. teeth were solely mounted with no contact points. a 7 mm-wide metallic matrix was used. distal proximal box was restored with amalgam (permite; sdi limited, bayswater, australia) until reaching the height of pulpal floor. each increment was condensed with maximum hand pressure, using the appropriate condenser size. surface mercury-rich amalgam layer was removed after condensation. after 5 minutes, dental tissues and amalgam surface were etched with 37% phosphoric acid (ultra-etch; ultradent products inc., south jordan, ut, usa) and bonding agent (bond 1-sf; pentron clinical technologies, los angeles ca, usa) was applied on both and cured for 10 s according to the manufacturer’s instructions. no additional preparation was performed for the amalgam surface. composite resin (filtek z250; 3m-espe gmbh, neuss, germany) was inserted in layers in mesial proximal box and was cured for 20 s each layer. subsequently, the occlusal surface was restored with the same composite, covering both proximal boxes. marginal adaptation was evaluated at the following interfaces: amalgam-tooth (a), amalgam-composite resin (ac), composite resin-tooth (c). teeth were kept in saline at room temperature (20oc) for 1 week. all teeth were thermocycled for 1,500 cycles at 5o c – 36o c – 55o c – 36o c with a dwell time of 15 s. these temperatures were chosen in an effort to reproduce thermal changes in the intraoral environment. teeth were then covered with nail varnish except for the restoration area and 1 mm around it, in order to avoid false positive results via dye penetration from another point rather than the restoration margins. following that, the teeth were immersed in 5% aqueous solution of methylene blue for 24 h. after that they were washed with saline and cut longitudinally by a microtome (figure 2). in order to assess the degree of microleakage at the occlusal margin, photographs were taken for each cut, under a stereomicroscope at 100x magnification. two specimens were excluded from the survey due to fig. 2. microtome cut. the degree of dye penetration is evident at the interfaces: amalgam-tooth (a), amalgam-composite resin (ac) and composite resin-tooth (c). microleakage in combined amalgam/composite resin restorations in mod cavities fig. 1. preparation of mod cavity and its dimensions 102102102102102 braz j oral sci. 12(2):100-104 additional dye penetration through minor fractures. microleakage was assessed as percentage depth of horizontal penetration (infiltration extent/cavity extension) (table 1). descriptive statistics including means and standard deviations were calculated for the microleakage analysis. the obtained data were subjected to one-way analysis of variance (anova) and tukey-kramer multiple-comparison test to determine significant differences among the three interfaces. the level of significance was set at p =0.05. all statistical analyses were performed using spss 12.0 (spss inc., chicago, il, usa). results mean microleakage values, from higher to lower, were as follows: a (73.529/28.71), c (40.435/34.965) and ac microleakage in combined amalgam/composite resin restorations in mod cavities n o . amalgam – tooth amalgam – composite composite – tooth (a) (ac) (c) 1 100 80 100 2 70 50 0 3 70 30 40 4 100 0 100 5 100 10 20 6 70 40 40 7 50 40 0 8 30 10 100 9 70 30 40 10 100 100 50 11 70 30 40 12 100 0 20 13 80 70 0 14 50 0 0 15 20 0 50 16 excluded excluded excluded 17 70 30 40 18 50 20 0 19 100 30 80 20 70 30 30 21 50 20 60 22 70 30 40 23 excluded excluded excluded 24 70 30 40 25 100 100 30 26 70 30 40 27 60 30 30 28 50 10 0 29 60 30 40 30 100 40 20 31 70 30 40 32 70 30 40 33 70 30 40 34 50 20 40 35 70 30 50 table 1 microleakage exhibited as percentage % depth of horizontal penetration (infiltration extent / cavity extension) in the interfaces examined. (34.118/34.6). statistically significant difference was observed between a-ac and a-c (p<0.01), but no statistically significant difference could be found between ac-c interfaces (p>0.05). discussion the first combined amalgam/composite case report was published in 1982 and presented a mandibular premolar, which was restored occlusally with composite resin in order to mask the unaesthetic amalgam12. combined amalgam/ composite restorations have been investigated in the recent literature in terms of bonding strength13 or are suggested as an alternative for amalgam repair without sacrificing healthy tissues14 or as a means for increasing cusp fracture resistance14. there are few studies on marginal seal14-18 and fewer still do 103103103103103 braz j oral sci. 12(2):100-104 microleakage in combined amalgam/composite resin restorations in mod cavities investigate microleakage at the amalgam/composite resin interface14,17-18. franchi et al. (1994)17 and franchi et al. (1999)15 demonstrated that microleakage at the amalgam/ composite resin interface was in between amalgam/enamel (higher) and composite resin/enamel (lower), results which partially match our findings. the present study showed that microleakage at the materials’ interface was lower than microleakage around amalgam or composite. however, contrary to the used methodology, specimens in those studies were not thermocycled, assuming that thermal loading may have an effect on microleakage at amalgam/composite interface and may explain the opposite findings. in contrast, cehreli et al. (2010)14 demonstrated that microleakage at the amalgam/composite resin interface was higher than at the tooth interface, but dealt with old amalgam as a substrate. it seems that use of fresh amalgam is favorable in terms of sealing ability at the interface of an amalgam/ composite combined restoration. a similar study has been performed with primary molars, reaching the same results for amalgam/composite interface as in the conducted study19. kournetas et al. (2010)18 used a qualitative scale and concluded that marginal adaptation in amalgam/composite resin interface is comparable to composite/tooth with self-etch adhesive. type of materials tested, cavity preparation, type of dental tissue, tooth age, amalgam condensation, amalgam surface conditioning, amalgam and composite bonding procedures, composite placement and polymeri-zation, use of oxidation solution for amalgam, thermocycling and type of used dye, all affect microleakage values and impose limitations for direct comparisons between the published papers. moreover, in vitro microleakage does not necessarily predict in vivo restoration failure due to secondary caries. even though a threshold marginal gap size for clinical failure of the restorations has not been established20, restorations with marginal defects fail more frequently 21. in spite of these limiting aspects, microleakage was chosen in this study because of its longterm report in the literature. concerning the present study, since teeth had no contact points, a matrix system was used in order to condense amalgam into the proximal box. despite the fact that fresh amalgam has high surface tension22, which affects negatively the wettability of the surface, composite resin was inserted after only 5 min in order to mimic clinical conditions. this approach seems to be time saving and the use of a temporary restoration over the placed amalgam is avoided. composite was applied in layers in order to control polymerization shrinkage. since high-copper amalgam was placed, no oxidation solution was used, in order to comply with previous studies. higher microleakage values at the amalgam/tooth interface, which are not confirmed by clinical experience10, could be attributed to condensation technique and lack of oxidation solution. in contrast, ideal presence of cervical enamel for bonding and placement of the material in layers, seem to be the reasons for lower microleakage at the composite resin/tooth interface. the use of amalgam in the cervical surface of proximal boxes has been related to a good marginal seal23. adding the advantageous time-dependent auto-sealing due to gradual oxide deposition11, amalgam is regarded as the material of choice regarding optimum marginal behavior. this study showed that the amalgam/composite resin interface performs even better, concluding that despite the use of two completely different materials, there is an excellent marginal seal. since the bonding mechanism is not fully understood24, questions arise regarding the irregularities of the amalgam surface, the higher surface tension of fresh amalgam, the entrapment of air, the presence of a “hybrid” surface or the proliferation of the setting reaction, which could possibly affect marginal integrity. however, even mechanical loading does not affect marginal adaptation of the interface18, eliminating the issue of a possible failure of the interface due to occlusal forces. a recent in vivo study on one-hundred posterior teeth, demonstrated that combined restorations performed better for contact, contour and retention than conventional composite resin or amalgam restorations25. combined amalgam/composite restorations are not thoroughly researched, as shown by the aforementioned drawbacks, but in vitro microleakage results are convincing. lacking alternative conservative aesthetic restorative options, which would improve the quality of the cervical area of class ii restorations, and considering the research limitations, combined amalgam/composite resin restorations can be suggested. combined restorations should be employed in challenging clinical situations, particularly in cases of proximal boxes with cervical margins located near the gingiva or beneath the cementoenamel junction. controlled clinical trials involving the implementation of this technique should be performed to determine its usefulness, durability and longevity. the conclusion is that despite the fact that amalgam and composite 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a. composite resin-amalgam compound restorations. quintessence int. 1994; 25: 577-82. 18. kournetas n, kakaboura a, giftopoulos d, chakmachi m, rahiotis c, geis-gerstofer j. marginal behaviour of self-etch adhesive/composite and combined amalgam-composite restorations. europ j prosthodont restor dent. 2010; 18: 70-7. 19. hovav s, holan g, lewinstein i, fuks ab. microleakage of class 2 superbond-lined composite restorations with and without a cervical amalgam base. oper dent. 1995; 20: 63-7. 20. jokstad a, bayne s, blunck u, tyas m, wilson n. quality of dental restorations. fdi commission project 2-95. int dent j. 2001; 51: 117-58. 21. hayashi m, wilson nh. marginal deterioration as a predictor of failure of a posterior composite. eur j oral sci. 2003; 111: 155-62. 22. morge s, adamczak e, linden la. variation in human salivary pellicle formation on biomaterials during the day. arch oral biol. 1989; 34: 669-74. 23. demarco ff, ramos ol, mota cs, formolo e, justino lm. influence of different restorative techniques on microleakage in class ii cavities with gingival wall in cementum. oper dent. 2001; 26: 253-9. 24. ozcan m, vallittu pk, huysmans mc, kalk w, vahlberg t. bond strength of resin composite to differently conditioned amalgam. j mater sci mater med. 2006; 17: 7-13. 25. kaur g, singh m, bal c, singh u. comparative evaluation of combined amalgam and composite resin restorations in extensively carious vital posterior teeth: an in vivo study. j conserv dent. 2011; 14: 46-51. 104104104104104 microleakage in combined amalgam/composite resin restorations in mod cavities braz j oral sci. 12(2):100-104 oral sciences n3 original article braz j oral sci. april | june 2015 volume 14, number 2 systemic and oral alterations associated with stress in nurses of public referral hospital danilo rodrigues1, dagmar de paula queluz1 1 universidade de campinas – unicamp, piracicaba dental school, department of community and preventive dentistry, piracicaba, sp, brazil correspondence to: dagmar de paula queluz departamento de odontologia social faculdade de odontologia de piracicaba unicamp avenida limeira, 901, cep: 13414 900 piracicaba, sp, brasil phone: +55 19 21065277 e-mail: dagmar@fop.unicamp.br abstract jobs that require great responsibility can cause serious damage to the body such as stress and its consequences. stress can be one of the triggers of disease systemic and oral diseases in different professionals. aim: to associate emotional stress with the systemic and oral alterations in the nurses of public referral hospital. methods: all 60 nurses of both genders, with higher education level, different ethnicities and ages were invited to join this study. nurses filled out the inventory of stress symptoms for adults (issl) and questionnaire of diseases/psychosomatic symptoms. next, they were subjected to a stomatological examination of the oral cavity, according to the boraks (1996) criteria. stress was associated with most outstanding psychosomatic and oral variables by the fisher’s exact test for calculating the p value (0.05). results: the sample was comprised of 37 nurses, most of them female (91.9%), young (83.7% are less than 36-years old), full range 2250 years old, white ethnicity (86.5%). the level of stress in level ii (resistance) and iii (exhaustion) was observed in 51.3% of the nurses. symptoms of headache and gain of body weight lately were present in 48.6% of the nurses. with regard to oral alterations, 32.4% reported cold sore sometimes and 59.5% nibbled mucosa always. there was no significant association between the stress and psychosomatic and oral variables. conclusions: based on the results of this study it may be concluded that the nursing profession can lead to emotional stress, although no significant association between stress and disease/psychosomatic and oral symptoms was found. future studies should be performed to evaluate this association. keywords: stress; occupational health; psychosomatic medicine; oral abnormalities. introduction in analyzing an individual’s needs as a whole, we should take into account his/her cultural universe and living conditions, i.e., his/her interaction with the environment and the reflection of this in everyday life. according to moreira (1985)1 and arantes and vieira (2002)2, the original concept of stress was introduced in 1936 by hans selye. from the 1980s, the number of jobs involving emotional stress increased significantly, possibly because of the very conditions of life and livelihood in big cities, but with its effects also being extended to smaller population groups, sometimes even specific as seen among students in test periods3-5. the mental or psychological stress occurs daily in our lives. the ability to react physiologically and emotionally is a natural and necessary response. however, the heightened reactivity to mental stress identifies individuals at higher risk of developing hypertension and may cause cardiovascular events and sudden death6. nurses, bank employees and police officers as well as entrepreneurs are more braz j oral sci. 14(2)171-175 received for publication: may 04, 2015 accepted: june 26, 2015 http://dx.doi.org/10.1590/1677-3225v14n2a14 172172172172172 exposed to factors that generate this kind of stress, which usually leads to a great intake of coffee, alcohol and tobacco. this fact usually favors the occurrence of circulatory, metabolic, immune or even emotional diseases contributing to an organic overload that produces emotional stress at different levels, according to the number of working hours, sector, and deal of responsibility7-8. considering the population as a whole, the frequency of systemic diseases directly or indirectly associated with emotional factors, may cause long bouts of tonsillitis at the start to more severe and difficult to control problems such as diabetes mellitus, hypertension and depression9-10. emotional stress in nurses due to the strain of everyday life can lead to risk of death for patients at their care, affecting this group of professionals not only psychologically but also generating serious health problems4,11-12. allergy problems, asthma and cancer are linked to emotional factors. traces of bitterness, sadness, depression and anger may be observed in people suffering from these diseases before organic manifestations become evident13. with regard to oral lesions caused by stress, godoy et al.9 (2005) reported canker sores, herpes simplex and nibbled mucosa. however, gingivitis is stands out with its multifactorial etiology. despite being within a specific context, diseases and conditions related to general oral hygiene cannot be discarded because its etiology is associated with the immune system. the association between high levels of stress and changes in the oral mucosa has been reported in patients with immunosuppression resulting from a poor emotional status13. the nursing professionals that are part of hospital medical staff in different functions subjected to stressful conditions of varying degrees by either superior demands or the operating area14-17. the body always tries to adapt to stress, using large amounts of adaptive energy for this18. several studies have investigated the impact of stress and quality of life at work (qvt)12,19-27. the objective of the present study was to assess the association between emotional stress and systemic and oral alterations in nurses working at a public referral hospital in a brazilian mid-sized city. material and methods this cross-sectional, descriptive and quantitative study was approved by the ethics committee of the piracicaba dental school – unicamp (protocol number 052/2013). the study was conducted in a public referral hospital with an installed capacity of 266 beds, located in piracicaba sp brazil, , , , , a mid-sized city of the southern region of brazil. the study population was the entire clinical nursing staff, consisting of 60 nurses of both genders, with higher education level, different ethnicities and ages, who signed an informed consent form, and responded to questionnaires. the data was collected between august and december 2013. the identity of the individuals was preserved. four instruments were used for data collection. the first was a questionnaire addressing the profile/demographic information: age, gender, and ethnicity. the second was to obtain data of psychological character, with a selfadministered questionnaire on symptoms perceived by the respondent himself and applied for checking the level of stress (level i (alert), ii (resistance), iii (exhaustion)), according to the lipp’s stress symptoms inventory for adults (issl)28. the third was a self-administered questionnaire also referring to psychosomatic diseases or systemic symptoms, installed as a result of emotional stress. this instrument assessed organic and functional changes of neuro-endocrine-metabolic nature, caused by emotional stress, including diabetes mellitus, heart disease, depression, headache, weight gain, high blood pressure15,29-30. the fourth instrument consisted of an intraoral stomatological clinical examination performed by the researcher at the hospital31. data were statistically analyzed by descriptive and estimate analyses. the analyses were performed using sas software version 9.1, using a significance level of 5% (p<0.05). the variable “stress” was classified as present or absent based on the existence of three levels/phases i (alert), ii (resistance) and iii (exhaustion), indicating presence of stress. the variable “psychosomatic events” (headache and weight gain) was classified as yes and no. the presence of cold sore, herpes and nibbled mucosa in the questionnaire was classified as always, never and sometimes, and then grouped into present (always or sometimes) or absent (never). these variables were combined to obtain the p-value by fisher’s exact test. results from the total number of nurses, the final sample consisted of 37 (61.7%). tables 1-3 show the sample profile/stress level, the psychosomatic diseases or systemic symptoms, stomatological evaluation of the oral cavity (oral abnormalities), respectively. the most important data are: age group 31-35 (n=13, 35.1%), average 30.6 ± 5.59 years old, young (n=31, 83.7% are less than 36-years old), full range 22-50 years old, female predominance (n=34, 91.9%), white ethnicity (n=32, 86.5%), no stress (n=18, 48.7%), no diabetes mellitus (n=35, 94.5%), no heart disease (n=35, 94.5%), no depression (n=33, 89.1%). considering headache, yes and no answers showed the same frequency (n=18, 48.6%). yes and no answers showed the same frequency (n=18, 48.6%) for weight gain. no was the prevalent answer for high blood pressure (n=34, 91.85), never for cold sore (n=21, 56.8%), never for herpes (n=22, 59.5%), always for nibbled mucosa (n=22, 59.5%). in tabulating the data and calculated the absolute and relative frequencies obtained: level ii resistance (n=10, 27.0%), level iii exhaustion (n=9, 24.3) (table 1). however, 48.6% (n=18) reported having frequent headache as well; 48.6% (n=18) reported weight gain (table 2). sporadic presence of cold sore (sometimes) was observed in one group (n=12, 32.4%). herpes was also another finding reported sometimes (n=9, 24.3%). others (n=22, 59.5%) reported nibbled mucosa (table 3). systemic and oral alterations associated with stress in nurses of public referral hospital braz j oral sci. 14(2)171-175 173173173173173 variables categories n % age group* 22-25 6 16.2 26-30 12 32.4 31-35 13 35.1 >36 4 10.8 no answer 2 5.5 total 37 100 gender male 3 8.1 female 34 91.9 total 37 100 ethnicity white 32 86.5 others 5 13.5 total 37 100 level of stress level i (alert) 0 0.0 level ii (resistance) 10 27.0 level ill (exhaustion) 9 24.3 no stress 18 48.7 total 37 100 table 1-table 1-table 1-table 1-table 1distribution of absolute frequency (n), relative frequency (%) characteristics variables of public referral hospital (2013). variables categories n % diabetes mellitus yes 1 2.7 n o 35 94.5 no answer 1 2.8 total 37 100 heart disease yes 1 2.7 n o 35 94.5 no answer 1 2.8 total 37 100 depression yes 3 8.1 n o 33 89.1 no answer 1 2.8 total 37 100 headache yes 18 48.6 n o 18 48.6 no answer 1 2,8 total 37 100 weight gain yes 18 48.6 n o 18 48.6 no answer 1 2.8 total 37 100 high blood pressure yes 2 5.4 n o 34 91.8 no answer 1 2.8 total 37 100 table 2 table 2 table 2 table 2 table 2 distribution of absolute frequency (n), relative frequency (%) of psychosomatic disease or systemic symptoms variables of public referral hospital (2013). variables categories n % cold sore a l w a y s 2 5.4 never 21 56.8 sometimes 12 32.4 no answer 2 5.4 total 37 100 herpes a l w a y s 4 10.8 never 22 59.5 sometimes 9 24.3 no answer 2 5..4 total 37 100 nibbled mucosa a l w a y s 22 59.5 never 8 21.6 sometimes 4 10.8 no answer 3 8.1 total 37 100 table 3 -table 3 -table 3 -table 3 -table 3 distribution of absolute frequency (n), relative frequency (%) of oral abnormalities variables of public referral hospital (2013) regarding the association of stress levels with headache, weight gain, cold sore, herpes and nibbled mucosa, no statistically significant association was found (table 4). discussion in the beginning, all 60 nurses were expected to participate. however, respecting the term of consent, only 37 nurses participated characterizing the sample. considering the literature used for this study, several studies19,26-27,32 analyzed data from samples of similar size. in this study, 91.9% of the participants were females with a mean age of 30 years. the female has a historical prevalence in nursing due to greater physical proximity to the patient and caregiver personality, as confirmed by several studies26-27,33. similar work was carried out by montes-berges and augusto-landa27 (2014) reporting similar age and predominant proportion of females. variables with stress no stress p n(%) n(%) headache yes 8(22.2) 10(27.7) 1 n o 8(22.2) 10(27.7) gained weight yes 7(19.4) 10(27.7) 0.74 n o 9(25) 10(27.7) with cold sore yes 5(14.2) 9(25.7) 0.49 n o 11(31.4) 10(28.5) with herpes yes 4(11.4) 9(25.7) 0.29 n o 12(34.3) 10(28.5) with nibbled mucosa yes 13(37.1) 13(37.1) 0.46 n o 3(8.6) 6(17.1) table 4 -table 4 -table 4 -table 4 -table 4 association of psychosomatic illnesses/systemic symptoms and oral abnormalities with stress. * two-tailed p values obtained by fisher’s exact test (p < 0.05) from the analysis of 2x2 contingency tables for association of variables. systemic and oral alterations associated with stress in nurses of public referral hospital braz j oral sci. 14(2)171-175 174174174174174 considering the sample, emotional stress with 27.0% in level ii (resistance) and 24.3% in level iii (exhaustion) is a high stress level (total 51.3%). the hospital working environment involves many health professionals, with their different personalities, a fact that predisposes the emergence of emotional stress. hooper et al.33 (2010) conducted a survey of emergency nurses and three other specialty units self selected to participate in a cross-sectional study. participants completed a demographic profile and the professional quality of life, satisfaction, compassion and fatigue. about 82% of emergency nurses had moderate to high levels of burnout, and nearly 86% had moderate to high levels of fatigue. differences between emergency nurses and those working in three other specialties: oncology, nephrology and intensive care, had the subscales: fatigue, satisfaction and compassion, but did not achieve a level of statistical significance. however, dozens of emergency nurses showed a risk to less compassion and satisfaction, while nurses in intensive care and oncology showed higher risk for burnout. during the analysis of stress symptom18 in the present study, we observed the occurrence of symptoms of stress similar to those found by other authors when checking the items indicated in the level ii questionnaire26,32. the class of general nurses ends up being responsible for activities from the good reception of the patient and activities related to the handling, following his recovery or death to the end. this generates emotional and even professional instability. there are also the pressures due to the hierarchy of labor relations and the strong sense of charge both internal and external. wright17 (2014) described that prolonged stress or work in situations where nurses feel trapped can cause stress levels that can have a lasting effect on health. the stressful condition caused by an imbalance between the demands placed on an individual and the resources he/she has available to deal with these demands. the requirements and the resources available to deal with are affected by skills, the individual thoughts and beliefs about himself/herself and work, available support structures and his/her personal life. the author concluded that managers have a duty to reduce stress in the workplace and provide structures and advice to ease and provide opportunities for nurses to regularly relax and relieve stress. in the present study, it was observed that eating at work incorrectly or even due to stress, can lead to gained weight, where 48.6% said they had weight gain, 5.4% said they had high blood pressure, but taking proper medication. han et al.15 (2011) conducted a survey of 2,103 female nurses in the united states and examined the relationship between working hours and stress associated with obesity. stress at work and shift work are known risk factors for obesity. this finding was measured using the body mass index, and 55% of the sample reported being overweight. factors such as nurse position, mental or emotional distress, behaviors and covariates related to the family were considered. regarding the oral alterations, 37.9% of the participants had some frequency of cold sore. also, 35.1% said they had herpes at some point, and these two findings are commonly associated with immunosuppression and emotional stress. similar data were also found by lorette et al.34 (2006). an abnormality usually associated with stress is the nibbled mucosa, which was present in this study in 70.3% of the cases. with respect to orofacial pain, 48.6% said they had headaches. lacerda et al.35 (2008) investigated the prevalence of orofacial pain and its relation to absenteeism in workers of metallurgical and mechanical sector of the city of xanxerê, sc, in the southern region of brazil. workers with severe pain reported higher absenteeism percentage (p<0.001), and the prevalence of orofacial pain was high in this population. after general analysis of the results, table 4 shows the most prominent associations between variables. emotional stress was associated with headache and obesity as observed in other studies15,36. the same process was done with the variables cold sore, herpes and nibbled mucosa, aiming to calculate the p value10,37, but considering the sample size, the association was not considered statistically significant. in view of the obtained and discussed data, there is need for further studies with larger samples in order to investigate possible association between emotional stress and systemic and oral diseases in nurses. based on the results of this study, it may be concluded that the nursing profession can lead to emotional stress, although no significant association was found between stress and disease/ psychosomatic and oral symptoms. future studies should be performed to evaluate this association. references 1. moreira ms. the stress syndrome. j bras med. 1985; 48; 19-32 portuguese. 2. arantes mac, vieira mjf. stress: psychoanalytic clinic. são paulo: casa do psicólogo; 2002 portuguese. 3. douglas cr. general pathophysiology. mechanism disease. são paulo: robe; 2000. p. 827-85 portuguese. 4. silva mcm, gomes, ars. occupational stress in health 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psychosomatics. a consensus statement of the european association of consultation-liaison psychiatry and psychosomatics (eaclpp) and the academy of psychosomatic medicine (apm). j psychosom res. 2011; 70: 486-91. systemic and oral alterations associated with stress in nurses of public referral hospital braz j oral sci. 14(2)171-175 oral sciences n3 original article braz j oral sci. july | september 2013 volume 12, number 3 legal analysis of the information contained in dental plaster packs mário marques fernandes1,2, ricardo mattana2, carolina mattana2, ana paula reckziegel2, rachel lima ribeiro tinoco3, rogério nogueira oliveira1 1department of community dentistry, school of dentistry, university of são paulo, são paulo, sp, brazil 2department of forensic dentistry, brazilian dental association, porto alegre, rs, brazil 3department of community dentistry, school of dentistry, university of campinas, piracicaba, sp, brazil correspondence to: mário marques fernandes laboratório de antropologia e odontologia forense (oflab – oflab), universidade de são paulo avenida prof. lineu prestes 2227, cep: 05508-000 cidade universitária, são paulo, sp, brasil phone: +55 11 30917891 fax: +55 11 30917874 e-mail: mario-mf@live.com received for publication: may 15, 2013 accepted: september 11, 2013 abstract aim: the aims of this study were: a) to verify if the instructions displayed on the packages and labels of dental plaster available for purchase in the city of porto alegre are in accordance with the brazilian laws, and b) to analyze how information is available on packages and labels of dental plaster acquired by dentists. methods: for this study, it was used the method proposed by silva et al. (2010) modified, according to which the information related to instructions for product use displayed on packages and labels, if present, provided by manufacturers and/or importers from each brand of plaster was reviewed: how to use, composition, price, expiration date, health risks and disposal instructions. the collected information was subsequently confronted with the requirements laid out in national legislation on trade of dental plaster. results: no product showed the price or the instructions for disposal. one brand showed no information of the package and another one showed only the composition of the product; this requirement was present in most products. the mode of use and the expiration date were present in almost all packages, except for two brands. only two brands presented information on health risks, and yet unsatisfactory. conclusions: the different brands and types of dental plaster analyzed presented some kind of violation of the provisions of the brazilian legislation, most with missing or unsatisfactory information. keywords: calcium sulfate, forensic dentistry, consumer advocacy, product packaging, dental materials. introduction dental plaster is generally used to make casts for study, prosthetic procedures, and to integrate the patient records. however, although yet little noticed, dental casts play an important role as pieces of evidence for forensic expert exams, either for human identification by comparing information contained in previous examinations, either for its use in civil litigations. for this reason, dental casts should be well archived and kept by dentists1. currently in brazil, the consumer is supported by the consumer defense code (cdc), approved by law 8.078/1990, which aims at protecting the public order as well as the social interest in consumption relations in order to preserve the more vulnerable part of this relationship: the consumer. in opposition, there are the suppliers,that develop activities, such as production, assembly, creation, construction, transformation, import, export, distribution or trading of products braz j oral sci. 12(3):223-227 and services. these companies must respect the consumer rights, under penalty of responding to civil and/or criminal lawsuits, for any damage caused. by purchasing products, dental instruments or equipment, dentists begin to act as a consumer, being supported by the cdc. in this sense, the cdc states in its article 6, section iii, as a basic right of consumers the access to adequate and clear information about the various products and services, with correct specification of quantity, characteristics, composition, quality and price, as well as potential risks2-3. this way, the brazilian national agency of sanitary surveillance (anvisa), on standardizing labels of dental plaster, considers dental plaster as exempt from registration at the regulating organ because they are classified as risk level 1, as stated in the user manual contained in resolution no. 185/ 2001, which provides guidance on registration, modification, revalidation and cancellation of registration of medical products. however, in annex v (guide for risk assessment), there rule i states this this applies to: ‘product that has no contact with the patient or touches only intact skin4-5. it is worth noting that the plaster is found in many countries, particularly in germany and scotland. chemically, the mineral used for dental purposes is almost a pure dihydrate calcium sulfate. the plaster used in dentistry is obtained by the removal of part of the water of crystallization of the dihydrate calcium sulfate by the process of plaster calcination. the criterion for use of a certain type of plaster depends on its use, and consequently, on the physical properties needed for that particular application. the american dental association (ada) specification no. 25 classifies the dental plaster into 5 types: a) type i – molding plaster, b) type ii ordinary plaster, c) type iii – plaster stone, d) type iv high strength plaster stone, and type v extra-hard plaster stone. the ordinary plaster is generally used in prosthetic dentistry to make study and anatomical models, fixing models in articulators, including complete and removable dentures in furnaces, among others. plaster stone is most commonly used in dentistry for producing various types of prosthetic models, orthodontic models, study models, and others. it is thus up to the dentist choose which type of plaster will be used6. the aims of this study were: a) to verify if the instructions displayed on the packages and labels of dental plaster available for purchase in the city of porto alegre are in accordance with the brazilian law, and b) to analyze how information is available on packages and labels of dental plaster acquired by dentists. material and methods in this research, all commercial registered establishments that sale dental products in the district center of porto alegre were visited, in the search of different brands and types of dental plaster available. the products were requested to employees, who provided the product for analysis. the sample consisted of nine products (n = 9) from six brands: yamay® (atibaia, sp, brazil) y, asfer® (são caetano do sul, sp, brazil) a, vigodent® (rio de janeiro, rj, brazil) v, dam proben® (cachoeirinha, rs, brazil) dp, dentsply® (petrópolis, rj, brazil) – dy, and polidental® (cotia, sp, brazil) p. all brands are registered belonging to brazilian companies, and two of them produce more than one type of plaster – asfer® (three types) and vigodent® (two types). for this study, it was used the method proposed by silva et al. (2010)7 modified, according to which the information related to instructions for product use displayed on packages and labels, if present, provided by manufacturers and/or importers from each brand of plaster was reviewed: how to use, composition, price, expiration date, health risks and disposal instructions. the collected information was subsequently confronted with the requirements laid out in national legislation on trade of dental plaster. for each data assessed it was associated the letters “s” (satisfactory information), “u” (unsatisfactory information) or “a” (absence of information). for this analysis, photographs of the package of each brand were taken with a digital camera, with the aid of a dark background and a millimeter scale. the images were made of both the front and the back of the label and/or package. results with respect to how information about the products was available, all of them had information displayed on the package or on the label, in the form adhesives or printed externally. no product showed the price or the instructions for disposal. the brand damproben® did not show information of the package and the brand polidental® exhibited only the composition of the product. composition was present in the majority of products, but it was unsatisfactory in the brands of plaster type iv. the mode of use and the expiration date were shown in almost all packages, except for the brands of plaster type iv. regarding health risks, only the brands yamay® and asfer® presented information, and yet unsatisfactory (table 1). discussion all commercial establishments that sell dental products in the city of porto alegre were contacted. most dental products stores are located in the district center, according to the list provided by anvisa. therefore, for sample collection, were accessed five establishments of a total of nine. still considering the sample analyzed it must be understood the features of the product, according to the supervisory body. the dental plaster is a product that, although considered not medical, is handled by health professionals, such as dentists, in various fields and specialties – including forensic experts and forensic dentistry specialists –, as well as prosthetic technicians2,4-5,7-10. according to the anvisa’s table coding of medical products, the dental plaster is encoded in dental consumer item, number 2101190 (plaster for dentistry). anvisa classifies dental plaster as exempt from registration at the regulating organ. however, it needs to be enrolled according 224224224224224 legal analysis of the information contained in dental plaster packs braz j oral sci. 12(3):223-227 information type ii plaster type iii plaster type iv plaster type v plaster dam proben® vigodent® asfer ® yamay ® asfer ® asfer ® vigodent® dentsply® polidental® how to use a s s s s s s s a composition a s s s s u u u s price a a a a a a a a a expiration date a s s s s s s s a health risks a a u u u a a a a disposal instructions a a a a a a a a a table 1 classification of information included on the labels and/or packages of the dental plaster brands analyzed in the study (s = satisfactory information; u = unsatisfactory information; a = absence of information). asfer® has three different types of plaster and vigodent® has two different types of plaster to the resolution 260/02, art. 2, which states the health products subject to registration. in annex i of that resolution, obeying certain requirements and conditions, all medical products framed in any risk class, including parts and accessories, must be registered at anvisa. this way, figure 1 was elaborated to show clearly, which justifies the no need of registration of dental plaster: the analysis of variables established in this study showed that instructions for use and expiration date were the most respected requirements and displayed in 78% (n = 7) of the packages, which goes head-on against the cdc guidelines. none of the brands presented price on the label, limiting the access of the consumers to clear information and reducing their ability to make a more consistent choice within their personal budget. the cdc, in article 6, subsection iii, determines as consumer law: “iii adequate and clear information about the various products and services, with correct specification of quantity, characteristics, composition, quality and price, as well as the risks that present”2-3,7,11-12. expiration date, requirement still respected, being mentioned in 78% (n = 7) of the products. however, this important information was missing in two brands, which may cause serious damage during the clinical work or forensic exam on not taking prey when handling the product, for being out of date or having its hardening time excessively increased. in this sense the consumer defense code also protects consumers, pointing in article 31, in full: “the supply and presentation of products or services should ensure correct, clear, precise, and in portuguese on their characteristics, qualities, quantity, composition, price, warranty, expiration date and origin, among other data, as well as the risks posed to the health and safety of consumers”23,10-12. another information that missing in 56% (n=5) of brands and unsatisfactory in 34% (n=3) of them was health risks. it is important to highlight that plaster is handled by dental professionals and mainly by dental technicians. so, those professionals are exposed to plaster dust produced mostly during preparation of work or study plaster models. it is known that excessive exposure to plaster powder (silica powder) can cause respiratory diseases to workers not wearing without personal protective equipment. as an example, it can be cited pneumoconiosis, which virtually block the pulmonary alveoli and prevent breathing. the disease starts with silicosis caused by plaster, aluminum oxide and ceramic powders, pumice and spain white. still, the major causative agent of this severe disease is silica, which is found on most products like plaster, coating, and ceramic used in dental laboratories9-11,13-14. in this regard, the cdc describes on its article 63 about what is considered violation of the norm: “omit ostensive signs or sayings about the harmful or hazardous product, on the packaging, wrappers, receptacles or advertisements”, and predicts a sentence from six months to two years and fine. it fig. 1. flowchart of framework of health products according to registration (anvisa, 20028). 225225225225225legal analysis of the information contained in dental plaster packs braz j oral sci. 12(3):223-227 also clearly states as violation (article 66): “making false or misleading statements, or omit relevant information about the nature, characteristics, quality, quantity, safety, performance, durability, price or warranties of products or services,” which punishable by detention from three months to one year and a fine3,11,14. this information of extreme importance that should be included on labels and packages was simply ignored by a significant number of manufacturers. finally, it was analyzed the presence of instructions on product disposal. it must be said that when purchased, the use of the product becomes a responsibility of the consumer, which means, it is not yet considered a residue at this point; it will become later, when the generator of waste will be responsible for managing this waste, as stated in the health legislation15-16. following the resolution no. 306, which establishes technical standards for waste management for health services, special attention must be given to chapter v, where it is stated that any generator of waste from health service should prepare the plan of waste management from health services, which consists of a document that governs the actions relating to solid waste management, observing the characteristics of the facilities and covering aspects related to the generation, separation, packaging, collection, storage, transportation, treatment and disposal, as well as public health protection. in chapter iv, there is the classification of waste of health services, which aims at highlighting the composition of the waste according to their biological, physical, chemical, state of matter and origin for their safe handling15. in group “d”, called ordinary waste, are all wastes generated in the services covered by this resolution which do not require different processes related to packaging, identification and treatment, and should be considered urban solid waste. fall into this group: plaster, gloves, adhesive tape, cotton, gauze, bandages, serum catheter and the like, that have not had contact with blood, tissues or body fluids (in case of contamination, they should be classified in group “a”)15-16. none of the evaluated packages contained guidance on the disposal of the product. although it is a manufacturer’s obligation, since the product purchased by the dentist is not yet a residue, social responsibility and education on waste disposal belong to everybody, including those who produce the product. therefore, dental schools should address more deeply themes of biosecurity directed to waste disposal in such a way that future dentists can be aware of their clinical, ethical and legal obligations in order to promote safe and healthy conditions to the patients, workers and environment17. finally, it is noteworthy that most plaster brands did not have some basic information related to the products, probably because the regulating bodies seem to act discreetly in relation to control and supervision. therefore, the manufacturers do not have a major commitment in guiding the consumer and clearly disobey what the cdc dictates regarding a product that is widely used not only in dental clinics. it is also largely employed in forensic exams, for activities like facial reconstruction and analysis of bite marks, being an important adjunct to unravel crimes, clarify investigations and support dilemmas of processes in civil, criminal, labor, social security, ethical or administrative fields1-2,7,9-10,18-20. given the described survey, it is fair to conclude that the different brands and types of dental plaster analyzed presented some kind of violation of the provisions of the brazilian legislation, most of them with missing or unsatisfactory information. references 1. silva rf, ramos dia, pereira sdr, daruge jr e, daruge e. plaster models: forensic importance and guidance for archiving. rev assoc paul cir dent. 2007; 61: 381-4. 2. silva rf, prado mm, freitas gc, oliveira hcm, portilho cdm, daruge jr e. legal analysis of packs of limes kerr part 1: information about manufacturers, importers and product features. rev odontol unesp. 2008; 37: 337-43. 3. brazil. common law number 8,078 of september 11, 1990. treats of consumer protection and other measures. federal official gazette of brazil, legislative power 1990; 1990 sept 12; p.1 (col. 1). 4. national agency of sanitary surveillance. resolution rdc. 56, of april 6th, 2001. establishes the essential requirements for safety and efficacy applicable to health products, referred to in the technical regulation annexed to this resolution. federal official gazette of brazil 2001, 2001 apr 6. 5. national agency of sanitary surveillance. user manual of resolution rdc 185/2001guidance on registration, enrollment, modification, maintenance and cancellation of registration of medical products. brasília, df, version updated in january 7th, 2005. 6. fleming ps, marinho v, johal a. orthodontic measurements on digital study models compared with plaster models: a systematic review. orthod craniofac res. 2011; 14: 1-16. 7. silva rf, prado mm, oliveira hcm, portilho cdm, daruge jr e. legal analysis of information about usage instructions present in packs of limes kfile. rsbo. 2010; 7: 200-7. 8. national agency of sanitary surveillance. resolution rdc n. 260, of september 23, 2002. treats of the health products subjected to enrollment provided in article 3 of resolution rdc n. 185/01. federal official gazette of brazil, 2002, 2002 oct 3. 9. brazil. common law number 5,081 of august 24, 1966 regulates the practice of dentistry. federal official gazette of brazil, legislative power 1966; p. 9843 (col. 1). 10. federal council of dentistry. repeals dental code of ethics approved by resolution cfo-42/2003 and approves another instead. resolution 118/ 2012, of may 11, 2012. federal official gazette of brazil 2012; 2012 may 11. 11. brazil. common law n. 10,406 of january 11, 2002. establishing the civil code. federal official gazette of brazil, legislative power, 2002 jan 11; p. 1 (col. 1). 12. pereira t, santos jr gc, rubo jh, ferreira pm, valle al. plaster type iv: influences of manipulation techniques. rev fac odontol. 2002; 10: 150-5. 13. algranti e, chibante ams. pneumoconiosis in workers from dental laboratory. rev assoc paul cir dent. 1993; 47: 969-72. 14. brazil. law number 2,848 of 07 december 1940. criminal code. federal official gazette of brazil, executive power, 1940; 1940 dec 31; p. 23911 (col. 2). 15. national agency of sanitary surveillance. resolution rdc no. 306 of december 7, 2004 treats of technical standards for waste management of health services federal official gazette of brazil, brasília, df, 2004 dec 10. 16. national council of the environment. resolution no. 358 of 29 april 2005. treats of the treatment and disposal of waste from health services and takes other measures federal official gazette of brazil, brasília, df, 2005 may 4. 226226226226226 legal analysis of the information contained in dental plaster packs braz j oral sci. 12(3):223-227 17. fernandes mm, francesquini jr l, daruge jr e, daruge e, maduell ta, pereira s. knowledge of graduating students in dentistry about plan of waste management of health services legal and ethical issues. rco. 2009; 11: 13-6. 18. mutalik vs, menon a, jayalakshmi n, kamath a, raghu ar. utility of cheiloscopy, rugoscopy, and dactyloscopy for human identification in a defined cohort. j forensic dent sci. 2013; 5: 2-6. 19. santos kc, fernandes cms, serra mc. evaluation of a digital methodology for human identification using palatal rugoscopy. braz j oral sci. 2011; 10: 199-203. 20. verma ak, kumar s, bhattacharya s. identification of a person with the help of bite brand analysis. j oral biol craniofac res. 2013; 3: 88-91. 227227227227227legal analysis of the information contained in dental plaster packs braz j oral sci. 12(3):223-227 oral sciences n3 original article braz j oral sci. october | december 2013 volume 12, number 4 radiographic evaluation of the dental condition of elderly people treated at a brazilian public university francisco ivison rodrigues limeira1, daniela pita de melo1, daliana queiroga de castro gomes2, sérgio d’ávila lins bezerra cavalcanti3, patrícia meira bento4 1area of dental radiology, department of dentistry, state university of paraiba, campina grande, pb, brazil 2area of oral diagnosis, department of dentistry, state university of paraiba, campina grande, pb, brazil 3area of public health, department of dentistry, state university of paraiba, campina grande, pb, brazil 4area of oral pathology, department of dentistry, state university of paraiba, campina grande, pb, brazil correspondence to: francisco ivison rodrigues limeira avenida baraunas, 351, cep: 58109-753 universidade estadual da paraíba campus universitário bodocongó, campina grande, paraíba, brasil phone: +55 83 88579262 / +55 83 96113409 e-mail: ivisonodontoce@hotmail.com received for publication: september 29, 2013 accepted: december 17, 2013 abstract aim: to assess the dental status of elderly patients examined in the clinical dentistry course of a brazilian public university. methods: an observational and descriptive study based on the analysis of panoramic radiographs. the sample consisted of 60 elderly patients who met the inclusion criteria. two observers who had been trained in appropriate conditions performed the radiographic analysis. data were stored in a specific form, recorded in a database and analyzed using descriptive statistics (measures of central tendency and variability) and inferential statistics (mann-whitney u test, kruskal-wallis test, chi-square analysis and calculation of the cramer’s v coefficient). results: most patients had at least one tooth in the oral cavity (71.7%), while 28.3% were totally edentulous, an average of 10.5 teeth per individual. the average number of teeth was 11.36 in males and 9.89 in females. the number of healthy teeth was 328 (5.47 per patient). in this study, 88.3% of the subjects had periodontal bone loss, with prevalence of moderate (35.0%) and severe (28.3%) bone loss. conclusions: given the high incidence of edentulous individuals, the high number of restored teeth and poor periodontal conditions, it is concluded that the overall oral health status of the evaluated elderly subjects is poor. keywords: oral health, panoramic radiography, epidemiology. introduction according to the world health organization (who), the chronological level of 60 years of age and over is used to define the elderly population in developing countries1. it is estimated that there are approximately 17.6 million elderly brazilians and that by the year 2025, the country will have the sixth largest elderly population in the world, over 30 million people2,3. among the main challenges faced by the healthcare system is the generation of new demands4. regarding oral health, such changes have predictable consequences and should be instrumental in defining effective measures to prevent disease and loss of teeth along an individual’s lifetime5. preliminary results of the most recently released survey showed that more than 3 million seniors need complete dentures and other 4 million need partial dentures6. braz j oral sci. 12(4):352-356 aging is an ongoing and complex process that occurs in all types of body cells, although it presents special features in certain organs and systems7. according to matos et al.8 and meloto et al.9, the aging process is associated with many changes in the mouth, some of them related to systemic problems (cardiovascular and cerebrovascular diseases, diabetes mellitus, oral cancer, osteoporosis, alzheimer’s and parkinson’s diseases) and their treatments, where the loss of teeth is not a consequence of aging but rather the result of complex interaction between dental diseases and the lack of preventive measures implemented either by the dentist or the individual. thus, dental professionals must understand the complexities inherent to older people, their special needs and their ability to undergo and respond to care. many of these senior citizens still have their natural teeth, requiring significant levels of maintenance, which is beneficial to their overall health, considering the advantages of better nutrition and life quality10. however, these benefits can only be maintained if the individual’s dental health is preserved, increasing the emphasis on preventive care11. given the importance of understanding the oral health status of this population, the aim of this study was to survey the dental status of elderly patients examined in the clinical dentistry course of a brazilian public university. material and methods this is an observational and descriptive study based on the analysis of panoramic radiographs of elderly patients (people with 60 years or more) examined in the clinical dentistry course of a public university in brazil from august 2009 to june 2010. during this period, 78 senior citizens were examined, but some panoramic radiographs without conditions for a good interpretation were excluded. the sample consisted of 60 senior citizens of both genders, enrolled by a non-probabilistic sampling, who had panoramic radiography as prerequisite for a treatment plan. the research project was approved by uepb ethics committee with caae (presentation of certificate of appreciation for ethics) protocol number 0539.0.133.00009, according to resolution 466/12 of the cns (national health council). two double-blinded investigators who had received previous theoretical and practical training to ensure a consistent interpretation of the established criteria performed the radiographic evaluation. a pilot study was conducted with 20 elders to test the methodology and instruments to be used. three weeks later this group was re-evaluated. the calibration for the radiographic analysis was carried out according to the recommendations of a dental radiologist, and the kappa agreement index was 0.91. the radiographic interpretation was performed in the same light box, with appropriate light intensity, using a 4x magnifying lens and a sequence by quadrants. all information obtained in the radiographic examinations was recorded on a structured form containing each individual’s socioeconomic information, including age, gender, and ethnicity, as well as the radiographic findings, describing the condition of the patient’s teeth and oral bone structures. in the radiographic analysis were considered as healthy teeth without evidence of caries (radiolucent image) or radiopaque restoration. the teeth restored with amalgam, metal crowns, ceramic metals, or other radiopaque materials were considered as radiopaque restoration. teeth with healthy roots were included in this study, and residual roots were not included in the number of present teeth. the following scores were adopted for the classification of periodontal bone loss in the periodontal assessment: 0 no bone loss; 1 cervical bone loss; 2 bone loss in the middle third; 3 bone loss in the apical third. the data were recorded using the statistical package for social sciences (spss) for windows®, version 15.0, and analyzed using descriptive statistics (measures of central tendency and variability) and inferential statistics (mannwhitney u test, kruskal-wallis test, chi-square analysis and calculation of the cramer’s v coefficient). finally, a 95% confidence interval at a 5% significance level (p<0.05) was set for data analysis. results in the examined sample, most individuals (58.3%) were female and aged 60"64 years (43.3%). the percentages corresponding to the other age groups were: 65"69 years, 30.0%; 70"74 years, 13.3%; and 75 or older, 13.3%. among the participants, 56.7% had at least one tooth in both arches, 28.3% were totally edentulous, 13.3% were edentulous in the maxillary arch, and 1.7% in the mandibular arch. regarding the teeth present, it was verified that out of a total of 630, there were 293 teeth in the maxillary arch and 337 teeth in the mandibular arch, resulting in a mean of 10.5 (sd = 8.74) teeth per individual. among the participants, 20% had 1"10 teeth, 46.7% had 11"20 teeth, and 5.0% had over 20 teeth. the mean number of teeth in males was 11.36 and 9.89 in females. despite this variation, there were no statistically significant differences by gender (u=393.5, p=0504) (table 1). regarding dental characteristics, the total sum of healthy teeth was 328 (5.47 per patient). residual root tissue was present in 26.7% of participants (0.33 per patient), and radiopaque fillings were present in 70.0% of patients (2.87 per patient) (table 2). there was a progressive decrease in the number of teeth corresponding to higher age. the average number of teeth present in those aged 60"64 years was 15.23 teeth, while the average decreased to 11.50 in those aged 65"69 years, and, more drastically, to 1.75 or 1.63 in the group aged 70 and above (table 2). this difference was statistically significant (kw=24.09, p<0.001) (table 3). bone loss occurred in 88.3% of the participant individuals at the following levels: cervical/mild, 25.0%; medium/ moderate, 35.0%; and apical/severe, 28.3% (table 4). tooth loss was statistically correlated with the age of participants (÷²=10.363, p=0.016). the results suggested 171353radiographic evaluation of the dental condition of elderly people treated at a brazilian public university braz j oral sci. 12(4):352-356 source: clinical dentistry of the state university of paraíba. table 1: table 1: table 1: table 1: table 1: distribution of the total number of teeth depending on age and gender. campina grande, pb, brazil. 2013 table 3: table 3: table 3: table 3: table 3: distribution of teeth according to age and tooth characteristics. campina grande, pb, brazil. 2013 source: clinical dentistry of the state university of paraíba. **statistically significant difference at 1%. tttttable 4:able 4:able 4:able 4:able 4: bone loss according to the age. campina grande, pb, brazil. 2013 source: clinical dentistry of the state university of paraíba. **statistically significant difference at 1%. characteristics f (total) % (total) mean/patient healthy 328 63.08 5.47 radiopaque 172 33.07 2.87 restorations residual root 20 3.85 0.33 total 520 100.00 table 2:table 2:table 2:table 2:table 2: evaluation of teeth for health, presence of radiopaque restorations and residual roots. campina grande, pb, brazil. 2013 source: clinical dentistry of the state university of paraíba. 171354 radiographic evaluation of the dental condition of elderly people treated at a brazilian public university braz j oral sci. 12(4):352-356 a moderate association (v=0.486) and statistical significance (÷²=42.453, p<0.001), mainly regarding the apical loss in those aged 70 years and older. discussion in brazil, there are few studies assessing the current oral health status of the elderly. this may be due to the low demand for dental treatment by the elderly, who consider poor oral health an inevitable consequence of aging. the extant studies show edentulism, coronal and root carious lesions, periodontopathy and soft-tissue lesions to be the most frequent changes in this population12-13. regarding the gender of the participants, most were female (58.3%), corroborating a strong trend in the recent epidemiological studies in elderly populations13-16. the greater female presence may be associated with the phenomenon of the “feminization of old age”, as women are majority in the world’s elderly population, and there is a global difference in life expectancy between the genders. mastroeni et al.18 and costa et al.19, affirm that the current predominance of women over 60 years, in proportions that increase with age, is an important aspect to consider in the planning of local programs to assist senior citizens. the age group with the largest number of participants was the 60-64year-old age group, which represented 43.3% of the total. the behavior was similar in both genders. however, the subgroup that grew most rapidly in the last 10 years is the group aged 75 years or more, according to data from the american geriatrics society20. in the analysis of oral conditions, it was observed that 28.3% showed total edentulism, similar to the findings of talwar, malik and sharma21. other studies show an even worse situation, such as the one reported by costa et al.19 (31,1%), simunkovic et al.22 (45,3%) and mack et al.23: 16% in the age group from 60-65 years and 30% in the age range from 75 to 79 years..... other studies that evaluated the oral health status of the elderly population by radiographs also found a high incidence of total edentulism, such as the study conducted by soikkonen et al.24, who evaluated the oral radiographs of 293 senior citizens between 76 and 86 years old and found, that 124 were completely edentulous. additionally, the study conducted by karhunen et al.25 with radiographic evaluation of the dental health of men aged 33"69 years by panoramic radiographs taken in autopsies, found that 17.4% of men were completely edentulous. according to alcantara et al.17, the suboptimal status of the elderly brazilian population’s oral health is due to the lack of preventive attitudes by the dentists, who, for some years performed unnecessary extractions that were possibly iatrogenic, and this increased the rate of edentulism in the country. the partial or total loss of teeth is a large part of an irreversible and cumulative process. according to rihs et al.26 this scenario may reflect the difficulty of access to dental services for a large portion of the brazilian population or even the lack of importance assigned by these individuals to oral health along their lives. based on the situations observed in the current study, it is believed that the poor oral health conditions arise from the fact that there has been no policy for oral health education for this population in their lives. this implies that this population does not actively seek dental care because they are not aware that poor dental health is not a necessary part of aging. regarding the teeth found present in the study, there was a total of 630 teeth, with an average of 10.5 teeth per individual. the average number of teeth in males was 11.36 and 9.89 in females. the healthy teeth amounted to a total of 328, an average of 5.47 per patient, and 172, or an average of 2.87 per participant showing radiopaque restoration. these results are similar to those found in other studies that show poor oral conditions, such as mack et al.23. in their study, 26% of the participants aged 60"69 years and 17% of those in the aged 70-79 years had the examined teeth restored, and dental caries were found in 2% of the teeth in both age groups. simunkovic et al.22 reported that the average number of decayed teeth was 1.03 per participant and 0.74 restored teeth per participant, and approximately 9 teeth per person needed treatment. periodontal disease, expressed as gingival inflammation and retraction, tooth root exposure, and loss of alveolar bone structure supporting the teeth, was found as a major cause of tooth loss in adults5. in their study, carneiro et al.12 selected 293 subjects, stratified according to age and gender. more than 94% of the subjects showed calculus as the ultimate sign of periodontal neglect; only 1.8% had healthy periodontal conditions, and 33.3% had insertion losses of 6"8 mm. in this study, 88.3% of the elders had periodontal bone loss, which varied from moderate (35.0%) to severe (28.3%), similar to the study of mack et al.23 where the prevalence of periodontal disease was higher in men and among the younger subjects (men aged 60-69 years: 85% vs. 71% in 70-79-year-old men; women aged 60-69 years: 71% vs. 62% in 70-79-years), which is possibly associated with oral hygiene, according to viana et al.16. aging with dignity is a desire in every society, but this will only be achieved when it is translated into a better quality of life. the national oral health policy of the ministry of health emphasizes its goal to increase the number of brazilian adults and senior citizens who possess a satisfactory quality of life level relative to their oral health. however, according to saintrain et al.27 and matheus et al.28, the dentistry area as a whole should incorporate a new mind frame in training professionals, based on interdisciplinary and comprehensive health care for the elderly individuals, to meet the demands of patients in this age group. this would require training professionals to specialize in geriatric dentistry, specific to all levels of senior citizens’ care, involving not only the professional team, but also the senior citizens and their families, the authorities and the community through more objective and effective integrated actions. given the high incidence of edentulism, the large number of restored teeth and poor periodontal conditions found in elderly test subjects, it is clear that there is a need to take steps in the community towards providing full and comprehensive oral health care to senior citizens. 171355radiographic evaluation of the dental condition of elderly people treated at a brazilian public university braz j oral sci. 12(4):352-356 acknowledgements the authors are grateful to the national council for scientific and technological development (cnpq) for financial support. references 1. who library cataloguing in publication data. integrating poverty and gender into health programmes: a sourcebook for health professionals: module on ageing. geneva: who; 2006. 2. carvalho jam, garcia ra. the aging process in the brazilian population: a demographic approach. reports in public health. 2003; 19: 725-33. 3. lima-costa mf, veras r. aging and public health. reports in public health. 2003; 19: 700-1. 4. pan american health organization. managerial network of informations for health. report of situations and trends: demographic and health. brasília: 2009. 5. cardoso em, parente rcp, vettore mv, rebelo mab. oral health conditions of elderly residents in the city of manaus, amazonas: estimates by sex. brazilian journal of epidemiology. 2011; 14: 131-40. 6. brazil. health of ministry. department of health care. department of primary care. sb brasil 2010. national survey of oral health: preliminary results. brasília: health of ministry; 2011. 7. oliveira mc, schoffen jpf. oxidative stress action in cellular aging. braz arch biol technol. 2010; 53: 1333-42. 8. matos fd, freitas ev, py l et al. diseases of high digestive tract. in: freitas ev, py l, cancado fax et al. treaty of geriatrics and gerontology. brasil: guanabara koogan, 2006: 641-642. 9. meloto cb, rizzatti-barbosa cm, gomes sgf, custodio w. dental practice implications of systemic diseases affecting the elderly: a literature review. braz j oral sci. 2012; 27: 1691-1699. 10. haikal ds, paula amb, martins amebl, moreira an, ferreira ef. selfperception of oral health and impact on quality of life among the elderly: a quantitative-qualitative approach. ciência & saúde coletiva. 2011; 16: 3317-3329. 11. newton jp. oral health for older people. gerodontology. 2006; 23: 1-2. 12. carneiro rmv, silva dd, sousa mls, wada rs. oral health of institutionalized elderly in the eastern zone of são paulo, brazil, 1999. reports in public health. 2005; 21: 1709-1716. 13. andrade fb, lebrão ml, santos jlf, duarte yao. relationship between oral health and frailty in community-dwelling elderly individuals in brazil. j am geriatr soc. 2013; 61: 809–814. 14. rodrigues sm, oliveira ac, vargas amd, moreira an, ferreira ef. implications of edentulism on quality of life among elderly. int j environ res public health. 2012; 9: 100-109. 15. lemos mmc, zanin l, jorge mlr, flório fm. oral health conditions and self-perception among edentulous individuals with different prosthetic status. braz j oral sci. 2013; 12: 5-10. 16. viana lrf, castro cp, pereira hbw, pereira, afv, lopes ff. is depression associated with periodontal status in elderly? braz j oral sci. 2013; 12: 20-22. 17. alcântara cm, dias ca, rodrigues sm, reis fa. comparative study of the oral health status of non-institutionalized elderly from governador valadares-mg, with the goal proposed by the world health organization, 2010. physis. 2011; 21: 1023-1044. 18. mastroeni mf, erzinger gs, mastroeni ssbs, silva nn, marucci mfn. demographic profile of the elderly in the city of joinville, santa catarina: a household survey. brazilian journal of epidemiology. 2007; 10:190-201. 19. costa am, guimarães mcm, pedrosa sp, nóbrega ot, bezerra acb. oral health status of elderly women from the brazilian federal district. ciência & saúde coletiva. 2010; 15: 2207-2213. 20. american geriatrics society. geriatrics in brazil: a big country with big opportunities. j am geriatr soc. 2005; 53: 2018-2022. 21. talwar m, malik g, sharma s. prevalence of dental caries and periodontal disease in the elderly of chandigarh a hospital based study. j indian dent assoc. 2012; 6: 78-82. 22. simunkoviæ sk, boras vv, panduriæ j, ziliæ ia. oral health among institutionalized elderly in zagreb, croatia. gerodontology. 2005; 22: 238-241. 23. mack f, mojon p, budtz-jorgensen e, kocher t, splieth c, schwahn c, et al. caries and periodontal disease of the elderly in pomerania, germany: results of the study of health in pomerania. gerodontology. 2004; 21: 27-36. 24. soikkonen k, wolf j, salo t, tilvis r. radiographic periodontal attachment loss as an indicator of death risk in the elderly. j clin periodontol. 2000; 27: 87-92. 25. karhunen v, forss h, goebeler s, huhtala h, ilveskoski e, kajander o, et al. radiographic assessment of dental health in middle-aged men following sudden cardiac death. j dent res. 2006; 1: 89-93. 26. rihs lb, silva dd, sousa mlr. dental caries in an elderly population in brasil. j appl oral sci. 2009; 17: 8-12. 27. saintrain mvl, souza eha, caldas júnior af. geriatric dentistry in brazilian universities. gerodontology. 2006; 23: 231-236. 28. matthews dc, clovis jb, brillant mgs, filiaggi mj, mcnally me, kotzer rd et al. oral health status of long-term care residents a vulnerable population. j can dent assoc. 2012; 78: 1-10. 171356 radiographic evaluation of the dental condition of elderly people treated at a brazilian public university braz j oral sci. 12(4):352-356 oral sciences n3 original article braz j oral sci. october | december 2013 volume 12, number 4 recovering streptococcus mutans over restorations by the tray technique a randomized clinical study patricio vildósola grez1, patricia palma fluxa2, paula zuñiga saavedra1, eduardo fernandez godoy1, osmir batista de oliveira junior3, gustavo moncada cortés1 1department of restorative dentistry, operative dentistry, dental school, university of chile, santiago, chile 2department of medicine and oral pathology, microbiology dental school, university of chile, santiago, chile 3department of restorative dentistry, araraquara dental school, unesp – univ estadual paulista, araraquara, sp, brazil correspondence to: patricio vildósola grez sergio livingstone 943, office 304, independencia, santiago, chile phone: +56 02 29781742 / fax: +56 02 29781742 e-mail: patovildo@gmail.com abstract aim: to correlate the tray technique for isolation and counting of streptococcus mutans over dental restoration with the toothpick technique. methods: forty 18-50-year-old patients of both genders with high cariogenic risk were selected. inclusion criteria were: occlusal restoration evaluated as alpha, according to the ryge’s criteria (examiner´ cohen-kappa 0.8) and 2 years old maximum age. each patient provided a saliva sample (control group s) and two samples of dental biofilm from amalgam or resin-based composite restorations in the same patient, one obtained with the toothpick technique (group tt), and the other from homologous tooth restoration with the tray technique (group tr). this method involves only a direct impression on the occlusal surfaces by a section of a fluoride tray with trypticase yeast extract cysteine sucrose with bacitracin (tycsb) agar previously prepared and solidified. the samples were incubated at 37 ºc for 48 h and the colony-forming units (cfu) were counted. the results were statistically analyzed with spss software using the linear regression method at 95% confidence level. results: tr was able to isolate s. mutans, and there was a significant positive correlation (r=0.95) with tt. there was no significant correlation between tr and control group s (r=0.47). conclusions: tr was able to isolate and count s. mutans from the dental biofilm of dental restorations with a significant correlation with the tt group. keywords: streptococcus mutans, dental caries, dental restoration. introduction caries is one of the most common diseases in the whole world and although the industrialized countries have lowered their rates, it cannot be completely eradicated; therefore a treatment is required, consuming large human and economic resources1,2. among the factors in the complex process of caries disease are the bacteria, which are immersed in cariogenic biofilm in balance with the oral environment3-5. this balance can be altered by microbial carbohydrate intake, increasing the population of acidogenic bacteria responsible for demineralization and destruction of hard tooth tissue, thereby causing the caries lesion6. such ecological disruptions of the microbial community are prone to occur because of microbial acid-induced adaptation and selection processes5. received for publication: august 26, 2013 accepted: november 28, 2013 braz j oral sci. 12(4):292-297 in this cariogenic biofilm are found mutans streptococci, that are the main etiologic agents of dental caries in humans7,8 and are classified into seven species. out of these, streptococcus mutans (s. mutans), is the most frequently isolated pathogen from biofilm9. the levels of s. mutans in dental plaque samples have been shown to be associated with the development of caries and their detection has been employed to predict caries risk, to monitor caries activity10 and also play an important role in the secondary caries11, because dental caries is a localized disease of the teeth12. in order to facilitate the isolation of this microorganism selective gold culture media have been used 13, and different methods have been proposed in an attempt to simplify s. mutans culture in laboratory for its detection and quantification14. the s. mutans samples are processed and inoculated in selective culture media, such as mitis salivarius agar with bacitracin (msb) and trypticase yeast extract cysteine sucrose with bacitracin (tycsb)15. several sampling methods for s. mutans have been proposed, such as counting s. mutans in saliva or taking direct samples of biofilm from teeth or restorations. in the case of counting s. mutans from saliva, it can be done stimulating or not the saliva16. this method is easy and inexpensive, but it presents some limitations, e. g. a person with low counts of s. mutans in saliva could have high concentrations of s. mutans on a restoration17. therefore, it has been suggested that the direct identification of the s. mutans colonies on the restoration margins allow a better assessment of the presence of the bacteria and a more accurate estimation of the failure risk of dental restorations16. for this reason, a few years ago, wallman and krasse18 proposed a more precise method for counting s. mutans on restorations. they suggested that plaque samples should be collected from restorations using the tip of a sterile wooden instrument (a toothpick). it is a simple method of collection to perform in the dental office. however, the authors pointed to a great disadvantage: this technique often results in underestimating the presence of microorganisms, which may not be accessed by the toothpick, due to its shape and size. the toothpick technique was one of the most used methods. the present paper describes a new method that allows the identification and quantification of s. mutans directly from restored teeth with a tray technique by a previously solidified selective medium. the objective of this study was to evaluate the recovery of the tray technique (tr) for the isolation and counting of s. mutans populations on occlusal surfaces of restored teeth and correlate it with the toothpick method. the hypothesis tested is that the tr is able to the recover s. mutans from occlusal surfaces of restorations and has correlation with the toothpick method. material and methods experimental design this experimental, randomized and blind study was part of project pri-odo 11-02 and was approved by the ethics committee of the dental school at universidad de chile protocol 2011/07. the objectives of this study were explained to all participants, who provided written consent. the study was conducted in full accordance with ethical principles, including the world medical association’s declaration of helsinki (2002) and local regulations. a sample size equal to or greater than 40 restored teeth was determined with a confidence interval of 95% (á=0.05) and a statistical power of 0.9 (g*power 3.1 software)19. a total of 584 patients of the operative dentistry clinic of the dental school, universidad de chile were examined and 40 patients were randomly selected to participate using the ncss pass 2008, v08.0.15 software, and their data were stored in a microsoft office excel (version 2007) spreadsheet. inclusion criteria • age between 18 and 50 years • high caries risk patients according to the cariogram software • occlusal restoration of the same material in the contralateral arch • full dentition (>28 teeth) • amalgam and resin-based composite restorations • restorations must have alpha value in five parameters according to the united states public health service (usphs/ ryge) criteria20 (table 1), assessed by a calibrated clinician (cohen kappa=0.8) • dental restorations placed within a maximum period of 2 years in the operative dentistry clinic of the dental school, universidad de chile • occlusal restorations in premolars and molars of no more than 1/3 of the intercuspal distance exclusion criteria • bravo and charlie restorations according to the usphs/ ryge criteria (table 1) • patients taking drugs that are proven to reduce salivary flow, such as antidepressants, narcotics, antihistamines, antihypertensive, antiemetic and diuretics • patients undergoing treatment with mouth rinses and/or other oral antiseptics and/or toothpaste with fluoride concentrations greater than or equal to 2500 ppm fluoride ions at the time of the study or in the previous last 3 months • patients taking antibiotics at the time of the study or in the last 3 months • patients taking immunosuppressive drugs (corticosteroids) • patients classified as asa iii according to the american society of anesthesiologists, • patients who chew gum at least four days per week21 • patients with a physical disability that precludes them being responsible for their own hygiene sampling and isolation of s. mutans selected patients were instructed on bass technique, and their performance was evaluated one week later, prior to sample taking, using the plaque index (modified by loe). all samples were collected in the morning between 10:00recovering streptococcus mutans over restorations by the tray technique a randomized clinical study 293 braz j oral sci. 12(4):292-297 13:00 am. on one side of the mouth, a sample of plaque was taken from the restoration using the tray technique (tr), while on the opposite side the sample was obtained using the toothpick technique (tt). while biofilm samples from restorations were obtained by the tr and tt methods from each patient, non-stimulated whole saliva samples were collected into sterile glass beaker for 5 min22. in the tr technique, biofilm on the surface of restorations was collected using a disposable fluoride gel application tray (deepak products inc., miami, fl, usa). each tray was sterilized in a type ii biosafety hood (esco technologies, inc., harboro, pa, usa) under ultraviolet light for 20 min and filled with tycsb agar. the composition of the media per litre was: casein (15.0 g), yeast extract (5.0 g), l-cysteine (200 ml) (difco laboratories inc., detroit, mi, usa), sodium sulfite (0.1 g), sodium chloride (1.0 g), sodium phosphate 12 hydrate (2.0 g), sodium bicarbonate (2.0 g), sodium acetate trihydrate (20.0 g), sucrose (50.0 g), agar (15.0 g), distilled water (qsp), 0.2 u/ml bacitracine23. the biofilm collected with tt was removed from the surfaces with the tip of a sterile, triangular, wooden toothpick. patient was asked to rinse the mouth with water, and the teeth were isolated using cotton rolls and gently air dried for 5 s to avoid contamination with saliva. the tt samples were transferred to 500 µl of rtf (reduced transport fluid) and stored at 4 °c until laboratory processing. all samples were transported to the laboratory and manipulated on the same day. saliva was homogenized in a vortex homogenizer (maxi mix ii type 37600, mixer barnstead thermolyne, dubuque, ia, usa). in addition to the trays used for sample collection, trays loaded with tycsb were incubated at 37 °c (zdp-a2080; labtech co., namyangju-city, korea) for 24 h as a quality control. all of these processes were performed at oral microbiology laboratory, department medicine of oral pathology, dental school, universidad de chile. bacteriological procedures the plaque samples obtained by the toothpick technique were homogenized in a vortex mixer (maxi mix ii type 37600 clinical characteristic marginal adaptation anatomic form surface roughness secondary caries luster of restoration alpha explorer does not catch or has one way catch when drawn across the restoration/tooth interface the general contour of the restorations follows the contour of the tooth the surface of the restoration does not have any surface defects there is no clinical diagnosis of caries the restoration surface is shiny and has an enamel-like, translucent surface bravo explorer falls into crevice when drawn across the restoration/tooth interface the general contour of the restoration does not follow the contour of the tooth the surface of the restoration has minimal surface defects n/a the restoration surface is dull and somewhat opaque charlie dentin or base is exposed along the margin the restoration has an overhang the surface of the restoration has severe surface defects clinical diagnosis of caries at restoration margin the restoration surface is distinctly dull and opaque and is esthetically unpleasing table 1.table 1.table 1.table 1.table 1. ryge criteria mixer, barnstead thermolyne) for 45 s. then, 100 µl of the homogenate of plaque samples was added to a tube with 900 µl of phosphate (ph 7.2) and shaken for 15 s in a vortex. a 100 µl aliquot was obtained after the first dilution and added to a second tube with 900 µl of phosphate buffer. thus, dental plaque dilutions of 1:10 and 1:100 were obtained. the same procedure was performed with the saliva samples to obtain dilutions of 1:1000 and 1:10 000. 100 µl of each of the diluted plaque and saliva samples was plated in tycsb agar plates using a micropipette. the entire plating process was performed under sterile conditions in a hood or type ii biosafety cabinet. the plates were placed in a candle jar system and incubated at 37 ºc for 48 h. isolation and identification of s. mutans from the different samples (tr, tt and saliva), s. mutans colonies were identified based on colony morphology (macroscopic) and adherence to the agar colonies observed under a stereomicroscope (stemi 2000; carl zeiss microscopy, thornwood, ny, usa) with a light source (schott kl 1500, carl zeiss microscopy). the s. mutans count was expressed as colony forming units (cfu) of s. mutans from the plates and trays with tycsb agar. then, the selected colonies that were compatible with s. mutans adhesion and morphology characteristics were suspended in todd-hetwitt broth (difco laboratories inc.) and incubated at 37 ºc for 48 h. the colonies were then subjected to biochemical tests to identify the species of mutans streptococci and distinguish s. mutans from streptococcus sobrinus. biochemical tests were performed, including raffinose fermentation, melibiose and esculin hydrolysis. when all three are positive they indicate presence of s. mutans24. after 48 h, each incubated broth sample was centrifuged (bd sero-fuge 2001, clay-adams becton, dickinson and co., sparks, md, usa) for 5 min at approximately 1500 rpm to obtain a pellet. the pellet was resuspended in 450 µl of phosphate buffer (ph 7.2) to obtain a near mcfarland 5: 100 µl of this suspension was inoculated in esculin (brain heart infusion, 1% esculin. difco laboratories inc.) in raffinose recovering streptococcus mutans over restorations by the tray technique a randomized clinical study294 braz j oral sci. 12(4):292-297 (thioglycolate without dextrose and no indicator, 1% of raffinose. difco laboratories) and melibiose (thioglycolate without dextrose and no indicator, 1% of melibiose. difco, laboratories) and incubated for 24 h at 37 ºc. subsequently, two drops of ferric ammonium citrate were added to melibiose and raffinose broths, respectively. the hydrolysis of esculin is considered positive if the broth quickly exhibits black color, and the fermentation of raffinose and melibiose was identified by yellow color, which indicated a positive test. both situations confirm the diagnosis of s. mutans. counting of s. mutans cfu from the samples was performed by a single calibrated examiner (cohen-kappa > 0.8). statistical analysis a single blind statistician analyzed the results using the statistical package for social sciences (spss) software for windows, version 15.0 (spss inc., chicago, il, usa). analyses were used to verify the normality of the data distribution and the homogeneity of variance with simple linear regression correlation. the significance level was set at 5% results identification and isolation of s. mutans based on the bacterial culture and isolation from dental plaque samples by the tr, tt and s methods in tycsb agar, isolated colonies were obtained with macroscopic properties and adhesion characteristics that were indicative of s. mutans. (figs. 1 and 2) the smears of the colonies showed gram staining of selected cocci forms: they were gram positive and arranged in chains, which is typical of s. mutans bacteria. tests for esculin hydrolysis and fermentation of raffinose and melibiose were positive for all isolates; therefore, the presence of s. sobrinus was not detected. quantification of s. mutans approximately 87.5% (35 out of 40) of the tr, tt and s samples showed the presence of s. mutans. cfu average was tr 7.98 (±8.70) cfu/cm2, tt 3.13 (±4.77)x103 cfu/ ml, s 5.05 (±6.40)x105 cfu/ml. tr and tt correlation a simple linear regression was calculated to determine the correlation between the s. mutans counts obtained by tr and tt. the obtained result was r=0.95 (fig. 3). a positive value of r greater than or equal to 0.65 was considered a significant correlation. the correlation between the counts of s. mutans from the plaque samples of the restorations obtained by tr and s was positive but not significant (r=0.47) (fig. 4).fig. 1. colonies of s.mutans on tycsb agar exhibited a whitish, rough surfaceand adhered to agar (observed under 4x magnification) fig. 2. bacterial isolation from dental plaque samples using tr (a) colonies of s. mutans had a round, smooth, crystalline appearance and adhered to the imprint of the occlusal surface of the restored tooth. (b) the red line represents the outline of the occlusal surface of the tooth. recovering streptococcus mutans over restorations by the tray technique a randomized clinical study a b 295 braz j oral sci. 12(4):292-297 fig. 4. correlation between the counts of s. mutans by s and tr from dental plaque samples on restorations. fig. 3. correlation between the counts of s. mutans by tt and tr from dental plaque samples from restorations. discussion this study demonstrated a significant correlation between tr and tt methods of collecting plaque samples of s. mutans in occlusal restorations in a selective medium for s. mutans (tybsc). additionally, a positive but nonsignificant correlation was observed between the results of tr and saliva, and similar results were observed when correlating the results of tt and saliva. this result may be explained mainly by the difficulty of removing the biofilm from different areas of the teeth by chewing on paraffin wax17. in this research the patients were standardized by sampling on restorations with alpha rating, mainly to homogenize the samples. however it would be interesting to investigate what happens with patients who have bravo rating restorations, because they have a greater biofilm accumulation, therefore a higher caries risk. it is known that s. mutans levels can vary among restorations, even in the same subject11. for this reason, wallman and krasse proposed the toothpick method that collects s. mutans from the restoration’s margins18. this method has been used extensively and it is considered to be the simplest available method. it is a non-invasive test for monitoring s. mutans in dental restorations. nevertheless, it has been reported that tt could not collect the complete range of the oral microbiota on restorations25. the tr is able to recover the bacteria deposited in all the surface occlusal restorations. in addition, tr is the most suitable recovering streptococcus mutans over restorations by the tray technique a randomized clinical study296 braz j oral sci. 12(4):292-297 technique for the monitoring of s. mutans in dental restorations due to the several advantages of this method relative to tt. for example tr involves a simpler method of microbiological processing due to the elimination of dilution and seeding steps, so it requires fewer logistical resources. the tr technique involves taking a direct impression of the restoration, and it was very well tolerated by the patients. the possibility of gag reflex was avoided because there was little contact with culture medium and no patient complained of bad taste. furthermore, it is a non-invasive technique for detecting microorganisms on surfaces, which could be used for monitoring early caries lesions9,26 and assessing local risk factors for the development of caries lesions. in the present study, only tycsb agar was used due to its high specificity for s. mutans, but it is recommended to use other culture media, such as sb or sb-20m to determine more specifically strains of s. sobrinus27 with the s. mutans, because they are an important factor in the development of dental caries5,28,29. this technique may be used for clinical research with new antibacterial materials in a very simple way. current studies on antibacterial materials are focused on s. mutans, but it is necessary to investigate other bacteria that are involved in the caries process. the technique is limited to free dental surfaces and is not indicated for proximal surfaces due to the low resistance of the culture medium to tearing and its deformation, which impedes the access to the other area of interest. the following conclusions can be drawn based on the obtained results: 1. the tray technique may recover and count s. mutans on occlusal surfaces of dental restorations, having a positive correlation with tt; 2. the tray technique may be considered as less time consuming and simpler than the toothpick technique for being a direct and non-invasive method of detecting s. mutans. acknowledgements the authors are indebted to dra. marta estela saravia of department of preventive dentistry, school of dentistry national of tucumán, argentina for her helpful assistance in the microbiological procedures. references 1. marcenes w, kassebaum nj, bernabé e, flaxman a, naghavi m, lopez a, et al. global burden of oral conditions in 1990-2010: a systematic analysis j dent res. 2013; 92: 592-7. 2. richards d. oral diseases affect some 3.9 billion people evid based dent. 2013; 14: 35. 3. nyvad b, crielaard w, mira a, takahashi n, beighton d. dental caries from a molecular microbiological perspective. caries res. 2013; 47: 89-102. 4. takahashi n, nyvad b. caries ecology revisited: microbial dynamics and the caries process. caries res. 2008; 42: 409-18. 5. takahashi n. nyvad b. the role of bacteria in the caries process: ecological perspectives. j dent res. 2011; 90: 294-303. 6. beighton d. the complex oral microflora of high-risk individuals and groups and its role in the caries process. community dent oral epidemiol. 2005; 33: 248-55. 7. aas ja, griffen al, dardis sr, lee am, olsen i, dewhirst fe, et al. bacteria of dental caries in primary and permanent teeth in children and young adults j clin microbiol. 2008; 46(4): 1407-17. 8. preza d, olsen i, aas ja, willumsen t, grinde b, paster bj. bacterial profiles of root caries in elderly patients. j clin microbiol. 2008; 46: 2015-21. 9. tanzer jm, livingston j, thompson am. the microbiology of primary dental caries in humans. j dent educ. 2001; 65: 1028-37. 10. fontana m, zero dt. assessing patients’ caries risk j am dent assoc. 2006; 137: 1231-9. 11. bentley cd, broderius ca, drake cw, crawford jj. relationship between salivary levels of mutans streptococci and restoration longevity. caries res. 1990; 24: 298-300. 12. fejerskov o, nyvad b, kidd eam. pathology of dental caries. in: fejerskov o, kidd eam, editors. dental caries. the disease and its clinical management. 2nd ed. oxford: blackwell munksgaard; 2008. p. 19-48. 13. gold og, jordan hv, van houte j. a selective medium for streptococcus mutans. arch oral biol. 1973; 18: 1357-64. 14. westergren g, krasse b. evaluation of a micromethod for determination of streptococcus mutans and lactobacillus infection. j clin microbiol. 1978: 82-3. 15. wan ak, seow wk, walsh lj, bird ps. comparison of five selective media for the growth and enumeration of streptococcus mutans. aust dent j. 2002; 47: 21-6. 16. park jh, tanabe y, tinanoff n, turng bf, lilli h. minah ge. evaluation of microbiological screening systems using dental plaque specimens from young children aged 6-36 months. caries res. 2006; 40: 277-80. 17. wallman c, krasse b. mutans streptococci in margins of fillings and crowns j dent. 1992; 20: 163-6. 18. wallman c, krasse b. a simple method for monitoring mutans streptococci in margins of restorations. j dent. 1993; 21: 216-9. 19. faul f, erdfelder e, buchner a, lang ag. statistical power analyses using g*power 3.1: tests for correlation and regression analyses. behav res methods. 2009; 41: 1149-60. 20. ryge g. clinical criteria. int dent j. 1980; 30: 347-58. 21. zibell s, madansky e. impact of gum chewing on stress levels: online self-perception research study. curr med res opin. 2009; 25: 1491-500. 22. silva tc, valarelli tm, sakal vt, tessarolli v, machado maam. oral antibacterial effect of chlorhexidine treatments and professional prophylaxis in children. braz j oral sci. 2013; 12: 132-7. 23. van palenstein hwh, ijsseldijk m, huis in ‘t veld jh. a selective medium for the two major subgroups of the bacterium streptococcus mutans isolated from human dental plaque and saliva. arch oral biol. 1983; 28: 599-603. 24. coykendall al. classification and identification of the viridans streptococci clin microbiol rev. 1989; 2: 315-28. 25. wennerholm k, lindquist b, emilson cg. the toothpick method in relation to other plaque sampling techniques for evaluating mutans streptococci. eur j oral sci. 1995; 103: 36-41. 26. coogan mm, mackeown jm, galpin js, fatti lp. microbiological impressions of teeth, saliva and dietary fibre can predict caries activity. j dent. 2008; 36: 892-9. 27. saravia me, nelson-filho p, silva ra, de rossi a, faria g, silva la. emilson cg. recovery of mutans streptococci on msb, sb-20 and sb20m agar media. arch oral biol. 2013; 58: 311-6. 28. saravia me, nelson-filho p, ito iy, da silva la, da silva ra, emilson cg. morphological differentiation between s. mutans and s. sobrinus on modified sb-20 culture medium. microbiol res. 2011; 166: 63-7. 29. peterson sn, snesrud e, schork nj, bretz wa. dental caries pathogenicity: a genomic and metagenomic perspective. int dent j. 2011; 61(suppl 1): 11-22. recovering streptococcus mutans over restorations by the tray technique a randomized clinical study 297 braz j oral sci. 12(4):292-297 oral sciences n3 braz j oral sci. 13(3):242-245 original article braz j oral sci. july | september 2014 volume 13, number 3 antifungal efficacy of azadirachta indica (neem) an in vitro study srinidhi surya raghavendra1, ketaki dattatray balsaraf1 1sinhgad dental college & hospital, school of dentistry, department of conservative dentistry & endodontics, pune, maharashtra, india. received for publication: june 06, 2014 accepted: september 17, 2014 abstract aim: to evaluate antimicrobial ability of neem leaf extract, 3% sodium hypochlorite (naocl) and 2% chlorhexidine (chx) against candida albicans. methods: neem leaf extract was prepared by using absolute ethanol with fresh neem leaves, filtering the extract through muslin cloth, coarse residue and filter paper. cultures of c. albicans were maintained on brain heart infusion broth and agar. the antimicrobial efficacy was checked using the agar diffusion and the zones of inhibition were measured. the results were statistically analysed using anova test. inter-group comparison was checked using kruskal wallis anova and mann whitney tests (a=0.05). results: there was statistically significant difference between the zones of inhibition seen with 3% naocl and neem extract with 2% chx (p<0.05). there was no significant difference observed between neem extract and 3% naocl. conclusions: efficacy of neem extract is comparable to 3% naocl against c. albicans and it is significantly better than 2% chx. keywords: candida albicans; chlorhexidine; sodium hypochlorite. introduction microorganisms and their by products are considered to be the primary etiologic agents in endodontic diseases1. it is evident that an infected root canal system is a unique niche for selective species of microorganisms. endodontic therapy aims at removal of bacteria and fungi from the root canal space and to prevent re infection. the basis of success of endodontic treatment and retreatment depends on identifying and eliminating the causative factors in the development of apical periodontitis, so that healing can be achieved. the role of bacteria, fungi and their by products in the pathogenesis of apical periodontitis has been clearly established. the reduction and elimination of bacteria, fungi and their by products should be given the utmost importance towards achieving a successful endodontic therapy1. the most effective way to achieve this aim is by means of instrumentation and irrigation. failure during and after endodontic treatment is associated with the presence of bacteria in the root canal1. enterococcus faecalis and candida albicans are known to be important resistant species in infected root canals, and they may cause treatment failures1. chemical treatment of the root canal system can be arbitrarily divided into irrigants, rinses and inter appointment medicaments2. sodium hypochlorite (naocl) has been widely used as an irrigant since its introduction in endodontics. naocl has been the irrigant of choice for non-surgical endodontic procedures because of its tissue dissolving ability and anti bacterial property3. however, it has many deleterious effects if pushed beyond the apex. unpleasant taste and odor4, toxicity5, correspondence to: srinidhi s r department of conservative dentistry & endodontics sinhgad dental college & hospital, pune, maharashtra, india phone: +91 9372342232 e-mail: srinidhi73@gmail.com 1 7 11 7 11 7 11 7 11 7 1243243243243243 braz j oral sci. 13(3):242-245 serial n u m b e r 1 2 3 4 5 groups 3 % naocl neem leaf extract 2 % chx ethanol (control) saline (control) mean ± sd(in mm) 19.66 16.33 8.66 8.33 p value of kw anova 0.011* p value of m.w. test 2&1= 0.043* 2&3= 0.046* 2&4 =0.046* 2&5=0.034* sd = standard deviation. *p<0.05 = statistical significance. table 1.table 1.table 1.table 1.table 1. resorption6, inability to remove smear layer and fully eradicate microbes from the infected canals5 are the main disadvantages of this popular irrigant. it can be used in various concentrations like 1.5%, 2.5%, 3%, 5.25% and 6%. chlorhexidine gluconate (chx), a synthetic cationic bisbiguanide is a broad-spectrum antimicrobial agent effective against gram positive and gram negative bacteria. it has substantivity and long lasting antimicrobial effect due to its binding with hydroxyapatite. a 0.5% chx solution can kill all yeast cells by 5 min6. however, its activity is ph dependent and it is toxic to human periodontal ligament (pdl) cells4. the use of plants and plant products as medicines could be traced as far back as the beginning of human civilization. the earliest mention of medicinal use of plants in hindu culture is found in “rigveda”. herbs like neem and green tea might have a potential use as irrigants as they have been found to possess antimicrobial and antifungal properties5,7. neem has been extensively used in ayurveda, unani and homoeopathic medicine. the literature has shown that neem (azadirachta indica) has antimicrobial and therapeutic effects, suggesting its potential to be used as an endodontic irrigant7, but there is lack of extensive documentation or data regarding use of neem extract as an irrigant in endodontics. the need for this study arose keeping in mind the disadvantages of conventional endodontic irrigants like naocl and chx. even though they have been proven effective against c. albicans, we are in constant search of alternatives that has the same efficacy but without the side effects. the purposes of this in vitro study was to compare the antimicrobial activity of 3% naocl , 2% chx and neem leaf extract against c. albicans and to assess the antimicrobial property of neem leaf extract against c. albicans using the agar diffusion method. material and methods the composition of the neem leaf extract is as follows: 3% naocl (hyposept, ups hygienes pvt ltd., mumbai, india), 2% chx (asep-rc, anabond stedman pharma research, chennai, india), absolute ethanol (department of oral pathology), normal saline (0.9% w/v, claris life sciences ltd., ahmedabad, india), c. albicans (atcc 10231) culture (department of microbiology and pathology). in preparation of the herbal extracts, 25 g of fresh neem leaves were added to 50 ml of absolute ethanol. mixture was macerated for 1-2 min and then the extract was filtered through muslin cloth for coarse residue. extraction process was repeated again using coarse residue and 25ml ethanol. both the extracts were pooled together and filtered through fast filter paper. alcohol part was removed from the extract on water bath till the volume was about 25 ml. extract was prepared and stored in airtight amber colored container. for the agar diffusion test, c. albicans cultures were maintained on brain heart infusion (bhi) broth and agar. cultures grown overnight at 37oc in brain heart infusion (bhi) broth on a rotary shaker (150 rpm) and bacterial growth was checked by changes in turbidity after 24 h. to check the antimicrobial efficacy of neem leaf extract, 3% naocl and 2% chx, agar well diffusion method was performed. bhi agar plates were prepared and cultures (200 ml) were spread on agar plates. wells of 6mm diameter were made in the agar surfaces. neem leaf extract, naocl, chx, positive control (absolute ethanol) and negative control (normal saline) were added to the respective wells and the plates were incubated for 24 h at 37oc in an incubator. after the incubation period, plates were removed and zones of inhibition were recorded. the test was performed two times and the average values were recorded. results the results were tabulated and analyzed statistically by anova. data was compiled on excel, statistical analysis was done using spss software (v.17.0) (spss inc., chicago, il, usa). for intergroup comparisons, kruskal-wallis anova test was used, followed by mann whitney test for pair wise comparison (table 1). no statistically significant difference was observed between zone of inhibition for neem leaf extract and 3% naocl. there was statistically significant difference between 3% naocl and neem extract with 2% chx (p<0.05). discussion fungi are not common members of the microbiota associated with primary endodontic infections. fungi have occasionally been found in primary root canal infections, but they seem to be more common in root-filled teeth with failed endodontic treatment. sundqvist et al. 2 found c. albicans in 2 out of 24 teeth with endodontic treatment failure. pinheiro et al.6 studied the flora in 60 root-filled teeth with persisting periapical lesion. microorganisms were isolated from 51 teeth, and candida species from 2 teeth. peciuliene et al.8 studied the occurrence of yeasts, enteric gram-negative rods, and e faecalis, especially in root-filled teeth with chronic apical periodontitis. forty teeth were included in the study and growth was detected in 33 teeth using conventional culturing methods including selective media for yeasts (tsbv and sabouraud plates). yeasts were isolated from 6 teeth (18% of the culture-positive teeth). all isolates belonged to the species c. albicans. antifungal efficacy of azadirachta indica (neem) an in vitro study 244244244244244 braz j oral sci. 13(3):242-245 egan et al.9 also showed that the probability to have yeasts in the root canal was 13.8 times greater, when the patient had cultivable yeasts in saliva, the difference being statistically significant. previous root canal treatment, coronal leakage, or previous antibiotic therapy did not seem to have an association with the occurrence of yeasts. in another study, dutta and kundabala7 analyzed the antimicrobial efficacy of 5 irrigants formulated from different parts of the azadirachta indica and compared them with 2.5% naocl and 0.2% chx through an agar diffusion test using c. albicans cultures. the authors found that naocl inhibited c. albicans completely and the neem extract had better efficacy than chx. an in vitro evaluation of 5 different herbal extracts as endodontic irrigants against e. faecalis and c. albicans using quantitative polymerase chain reaction revealed that neem was highly efficient to 5.25% naocl in reducing the counts of these microorganisms within the root canals when compared with other extracts10. bohora et al.11 compared the antibacterial efficacy of neem leaf extract and 2% naocl against e. faecalis, c. albicans and mixed culture, and found that neem leaf extract is a viable medicament against both microorganisms and even against mixed culture. additionally, it is a biocompatible irrigant when compared to 2% naocl. a strong correlation has been observed between apical periodontitis and the presence of bacteria and fungi in canals; if they persist in the root canal system at the time of obturation, there is a higher risk of failure1,10. c. albicans is the second most common cause of recalcitrant infections after e. faecalis. fungi have been demonstrated to possess virulence attributes that may play a role in disease causation. the mechanisms believed to be involved in pathogenesis are (1) adaptability to a variety of environmental conditions, (2) adhesion to a variety of surfaces, (3) production of hydrolytic enzymes like proteinase, (4) morphologic transition like phenotypal switching, (5) biofilm formation12. in this study, selective yeast media were not used. naocl and chx at various concentrations have been proven effective against c. albicans but they have certain disadvantages as already discussed. the need is for an endodontic irrigant that can be effective against these resistant organisms while also being biocompatible. in dentistry, neem has been investigated due to its antimicrobial potential against oral microorganisms especially those associated with gingivitis and periodontitis10. the use of neem as an endodontic irrigant might be advantageous because it is a biocompatible antioxidant and thus not likely to cause the severe injuries to patients that might occur due to naocl accidents. neem acts on oral microflora because of its anti-adherence activity by altering bacterial and fungal adhesion. nimbidin and nimbolide, which are constituents of neem, have been found to possess antibacterial and antifungal properties. these components cause lysis of the bacterial and fungal cell wall 3. one study concluded that azadirachta indica is highly efficacious in the treatment of periodontal disease thus exhibiting its biocompatibility with human pdl fibroblasts. neem is bitter in taste and can be altered by different formulations due to addition of sweeteners and flavors to increase patient compliance and acceptability13. the results obtained in the present in vitro study showed that neem leaf extract is a viable medicament against c. albicans. in conclusion, as the global scenario is now changing towards the use of non-toxic plant products that have traditional medicinal use, extensive research and developmental work therefore can be undertaken on neem and its products for their better efficacy, biocompatibility and economics. under the limitations of this study, it was concluded that the efficacy of neem leaf extract against c. albicans is comparable to 3% naocl. further, the action of neem extract was significantly better than that of 2% chx. the microbial inhibition potential of neem leaf extract observed in this study opens avenues for its use as an intracanal medication. preclinical and clinical trials are needed to evaluate biocompatibility and safety before neem can conclusively be recommended as an intracanal irrigating solution, but in vitro observation of neem effectiveness seems promising. acknowledgements study conducted as part of indian council of medical research sponsored sts (short term studentship) project id no: 2013-03543. the authors are grateful for their support. references 1. tirali re, turan y, akal n, karahan zc. in vitro antimicrobial activity of several concentrations of naocl and octenisept in elimination of endodontic pathogens. oral surg oral med oral pathol oral radiol endod. 2009; 108: 117-20. 2. sundqvist g, figdor d, persson s, sjogren u. microbiologic analysis of teeth with failed endodontic treatment and the outcome of conservative retreatment. oral surg oral med oral pathol. 1998; 85: 86-92. 3. prabhakar j, senthilkumar m, priya ms, mahalakshmi k, sehgal pk, sukumaran vg. evaluation of antimicrobial efficacy of herbal alternatives (triphala and green tea polyphenols), mtad and 5% sodium hypochlorite against e faecalis biofilm formed on tooth substrate: an in vitro study. j endod. 2010; 36: 83-6. 4. malkhassian g, manzur aj, legner m, fillery ed, manek s, basrani br, et al. antibacterial efficacy of mtad final rinse and 2% chlorhexidine gel medication in teeth with apical periodontitis: a randomized doubleblinded clinical trial. j endod. 2009; 35: 1483-90. 5. chang yc, huang fm, tai kw, chou my. the effect of sodium hypochlorite and chlorhexidine on cultured human periodontal ligament cells. oral surg oral med oral pathol oral radiol endod. 2001; 92: 446-50. 6. pinheiro et, gomes bp, ferraz cc, teixeira fb, zaia aa, souza filho fj. evaluation of root canal microorganisms isolated from teeth with endodontic failure and their antimicrobial susceptibility. oral microbiol immunol. 2003; 18: 100-3. 7. dutta a, kundabala m. antimicrobial efficacy of endodontic irrigants from azadirachta indica: an in vitro study. acta odontol scand. 2013; 71: 1594-8. 8. peciuliene v, reynaud ah, balciuniene i, haapasalo m. isolation of yeasts and enteric bacteria in root filled teeth with chronic apical periodontitis. int endod j. 2001; 34: 429-34. 9. egan mw, spratt da, ng yl, lam jm, moles dr, gulabivala k. prevalence of yeasts in saliva and root canals of teeth associated with apical periodontitis. int endod j. 2002; 35: 321-9. antifungal efficacy of azadirachta indica (neem) an in vitro study 1 7 11 7 11 7 11 7 11 7 1245245245245245 braz j oral sci. 13(3):242-245 10. vinothkumar ts, rubin mi, balaji l, kandaswamy d. in vitro evaluation of 5 different herbal extracts as an antimicrobial endodontic irrigant using real time quantitative polymerase chain reaction. j conserv dent. 2013; 16: 167-70. 11. bohora a, hegde v, kokate s. comparison of the antibacterial efficiency of neem leaf extract and 2% sodium hypochlorite against e. faecalis, c. albicans and mixed culture an in vitro study. endodontology. 2010; 22: 8-12. 12. obando-pereda ga, anibal pc, furlatti vf, hofling jf. evidence of heterokaryon compatibility on candida albicans biofilm. braz j oral sci. 2008; 27: 1678-81. 13. dutta a, kundabala m. comparative anti microbial efficacy of azadirachta indica irrigant with standard endodontic irrigants: a preliminary study. j conserv dent. 2014; 17: 133-7. antifungal efficacy of azadirachta indica (neem) an in vitro study oral sciences n3 original article braz j oral sci. april | june 2015 volume 14, number 2 agreement between rapd, api20c aux, chromagar candida and microculture on oral candida identification emanuene galdino pires1, edimilson martins de freitas2, paulo rogério ferreti bonan1, sérgio avelino mota nobre3 1universidade federal da paraíba ufpb, dental school, department of clinical and social dentistry, joão pessoa, pb, brazil 2universidade estadual de montes claros – unimontes, dental school, department of social dentistry, montes claros, mg, brazil 3universidade estadual de montes claros – unimontes, center of biological and health sciences, montes claros, mg, brazil correspondence to: emanuene galdino pires ccs odontologia cidade universitária, castelo branco cep: 58051-970 joão pessoa, pb, brasil phone: +55 83 93142930 e-mail: emanuene@hotmail.com abstract aim: to measure the agreement of methods for identification of candida species in oral cavity samples, comparing the chromagar candida, microculture, api 20c aux and rapd techniques. methods: ninety-one colonies of candida were isolated and presumptively identified in chromagar candida, submitted to microculture, api 20c aux and rapd techniques. after this, agreement among methods using kappa test was performed. results: agreement rates between rapd and chromagar candida, showed significant accuracy for c. albicans, c. tropicalis, c. dubliniensis and c. krusei (kappa: 0.760, 0.640, 0.416 and 0.360, respectively, p<0.05). comparing rapd results with microculture, the highest agreement was for c. albicans (kappa: 0.851 p<0.05) but no significant agreement for c. lusitaniae, c. krusei and c. guilliermondii was obtained (p>0.05). the agreement was significant for all identified species when rapd (ope-18) and api 20c aux (p<0.05) were used. critical levels of agreement between rapd and microculture were observed when c. lusitaniae, c. krusei and c. guilliermondii were identified. conclusions: api 20c aux presented the best agreement with molecular random identification and chromagar showed good agreement for c. albicans, c. tropicalis, c. dubliniensis and c. krusei identification. keywords: candida; mouth; methods. introduction candida species are commensal microorganisms of the oral cavity. they have several virulence factors, which in the presence of local and systemic host failures may result in their transition from commensal to pathogenic organisms1, causing oral and systemic infections that pose significant public health problems. their isolation is used in investigations related to salivary disfunction, oral candidiasis, orofacial pathologies, and immune suppressant status2-4. there is a variety of methods for identifying candida species from clinical samples in the oral cavity5. the chromagar candida differential medium is commonly used to isolate and identify presumptive c. albicans, c. dubliniensis, c. tropicalis and c. krusei. their sensitivity and specificity are considered satisfactory for these species2,6-7. the microculture analysis has considerable accuracy and presents low cost2, braz j oral sci. 14(2)149-153 received for publication: march 10, 2015 accepted: june 19, 2015 http://dx.doi.org/10.1590/1677-3225v14n2a10 150150150150150 but it requires visual experience, sometimes limited by the resolution of optical microscopy and confused by similarities among species’ expressions. the biochemical characterization could be performed using the api® 20c aux (biomerieux, france) which relies on variations in the assimilation of carbohydrates7-9. however, it presents limitations related to cost and to distinguish between some species2. a study of 159 clinical isolates of candida species identified by the very similar kit api® candida aux (biomerieux, marcy l’etoile, france) reported that 12 isolates (7.5%) were incorrectly identified10-11. in recent decades, traditional methods of microorganism phenotyping have been replaced or added by the procedures associated to recombinant dna12-14. methods based on molecular markers are useful not only for phenotyping, but also for differentiation of candida species15-16. the rapd (random amplification polymorphic dna) allows the amplification of dna sequences and is a simple and quick technique that does not require prior knowledge on the genomes to characterize organisms, using one randomly determined (usually a decamer) primer17. it is used for genetic characterization of a range of organisms, plants, animals or microorganisms, including candida species, for different purposes18-21. the sensitivity, specificity and resolution of the ope-18 primer for identification of candida species has been reported and could be used for epidemiological candida identification11,22. due to scarce information about presumptive, biochemical and molecular agreement on candida identification, this study aimed to measure the assertive correlation between the presumptive identification of candida species from oral cavity using chromagar candida , microculture, api® 20c aux and ope-18 genotyping. material and methods ethical procedures ethical considerations in accordance with helsinki declaration have been observed. this research was conducted according to the ethical principles of research involving human participants, as stipulated by resolution 196/96 of the national health council of the ministry of health of brazil. the collection and analysis of data in this study were certified by the research ethics committee of the state university of montes claros, mg, brazil, protocol cep nº. 1111/08. origin of samples the candida isolates resulted from salivary collections of oral cavity of patients irradiated on head and neck due to malignant neoplasms (n=29) and elderly volunteers (n=63). the collection comprised 91 isolates of candida species. isolation and presumptive identification of candida species the isolation of yeasts was made in salivary samples collected from the buccal mucosa and tongue with a swab and sterile saline solution (nacl, 0.85%) as diluent. the isolation and presumptive identification was made by drawing aliquots (100 µl) from each sample and placing them on plates containing chromagar candida and incubated at 37 °c for 24 to 48 h, in duplicate. yeast identification was made by considering the morphology and color of the colonies2,23. each colony of candida was cataloged and then stored at -20 °c in sabouraud dextrose broth (dsb, oxoid ltd., london, england) amended with glycerol (40% v/v). atcc 10231 of c. albicans was used as quality control (qc). microculture characterization of isolates microcultures with cornmeal agar-tween 80 (rheum, lenexa, ks, usa)23 were made to highlight blastospores, chlamydospores, pseudohyphae and true hyphae of the isolates. to differentiate c. albicans and c. dubliniensis from other candida species, germ tube production was viewed on bovine serum 24-25. to distinguish c. albicans from c. dubliniensis, cultivation on sabouraud dextrose agar (oxoid, hampshire, england) for 48 h was made at 42 °c, using atcc 10231 as qc. identification by api 20c aux the inoculum used to this procedure was obtained from cultured yeast on sabouraud agar. the procedures for inoculation and interpretation were performed according to the instructions provided by the manufacturer (biomerieux, france). identification list on these indexes was considered as excellent (%id>99.9, t>0.75), very good (>99.0% id and t>0.5) or acceptable (%id>90.0 and t >0.5)7. identification of isolates by rapd (random amplification polymorphic dna) the extraction and purification of dna from isolates of candida spp was made with the purelink genomic dna® kit (invitrogen k1820-02, brazil). the used dna was obtained from cells grown in ypd broth (1% malt extract powder, 2% bacteriological peptone and 2% dextrose d-glucose) at 37 °c and shaking (150 rpm for 24 h)11. a total of 50 µl of concentrated suspension of each isolate was obtained by centrifugation (3,500 rpm for 30 min). the purification of dna was made by adding 200 µl of digestion buffer, 20 µl proteinase k and 20 µl rnase. we added to 200 µl of binding buffer and then the tubes were heated for 10 min at 80 °c in a water bath. to neutralize the detergent and to allow the connection with the silica column, 200 µl of absolute ethanol was added (merck, darmstadt, germany). the tubes were centrifuged at 13,000 rpm for 1 min and the pellet was discarded. subsequently were added 500 µl of the first washing buffer and centrifuged again at 13,000 rpm for 1 min and the precipitate discarded. the column with the silica was passed to the second tube and added 500 µl of the second washing buffer and centrifuged to 13,000 rpm for 1 min and for 3 additional minutes to enhance drying. for the first extraction, 200 µl of sterile water were added to milli-q heated to 60 °c in the column in a second agreement between rapd, api20c aux, chromagar candida and microculture on oral candida identification braz j oral sci. 14(2)149-153 151151151151151 method method of identification sensibility r a p d random aleatory polymorphic dna. molecular identification. high microculture culture and morphological evaluation. intermediary(some species) chromagar culture and evaluation by color. intermediary (some species) api20c aux assimilation of carbohidrates. high table 1.table 1.table 1.table 1.table 1. comparation of methods used in this study. tube. then it was centrifuged at 13,000 rpm for 1 min. for the second extraction, 200 µl of elution buffer of the same column were placed in a third tube and centrifuged it at 13,000 rpm for 1 min. the products of rapd-pcr were obtained with ope18 primer (5'-ggactgcaga-3') (gibco brl, grand island, ny, usa). the preparation of reactions for each isolate was done by adding 1 µl of primer, 5 µl of dntp mix (datp, dctp, and dttp dgpt invitrogen, brazil), 2.5 µl 10x pcr buffer rxn, 1 µl mgcl2 (50 mm), 0.5 µl taq dna polymerase (2.5 u invitrogen platinum®, brazil) and 5.5 µl milli-q. the final volume was 25 µl, 15 µl of mix and 10 ìl of extracted dna. the amplification consisted of 39 one-minute cycles at 94 °c, 1 min at 36 °c, 2 min at 72 °c followed by a 10 min cycle at 72 °c11. pcr products were separated by agarose gel eletroforesis (1.4% / v 5µl ethidium bromide – 10 mg/ml), 80 v for 5 h. we used atcc 10231 as qc and two molecular weights were incorporated (100 bp and 250 bp invitrogen, são paulo, sp, brazil). the dna bands were observed and photographed in transillumination and the images were analyzed considering the literature reports11,22. table 1 shows comparison of the methods used in this study. results among the 91 isolates, c. albicans was the most prevalent, identified presumptively in 35 (38.5%) of colonies by chromagar candida. on the confirmatory identification, 31 (34.1%) of these isolates were confirmed on microculture as c. albicans, while 30 (32.9%) were confirmed by the api 20c aux® and 29 (31.9%) by rapd. rapd identified 29 (31.9%) c. albicans, 4 (4.4%) c. dubliniensis, 10 (10.9%) c. tropicalis, 9 (9.9%) c. krusei, 12 (13.2%) c. glabrata, 9 (9.9%) c. parapsilosis, 6 (6.6%) c. guilliermondii, 6 (6.6%) c. lusitaniae and 5 (5.5%) c. kefyr. figure 1 shows rapd with different species of candida. the chromagar candida® presumptively identified 28 (30.8%) of isolates as other candida species (c. dubliniensis, c. tropicalis, and c. krusei). the agreement between genetic typing and chromagar candida® was higher for c. albicans and lower for c. krusei. table 2 shows the agreement coefficient (kappa) between rapd (ope-18) and chromagar candida. in the rapd technique only one isolate (1.1%) presented an undefined pattern, followed by five (5.5%) in api 20c aux® and 8 (8.8%) in microculture. considering the comparative analysis between the rapd characterization and microculture evaluation, we can observe that the identifications of c. albicans, c. dubliniensis, c. tropicalis, c. glabrata, c. kefyr and c. parapsilosis were significantly concordant in decreasing levels among the methods, in that order. when rapd and api 20c aux® were compared, the species c. tropicalis, c. albicans, c. glabrata, c. kefyr, c. dubliniensis, c. lusitaniae, c. krusei and c. guilliermondii showed significant decreasing agreement, in that order. table 3 shows the kappa coefficient among rapd, api 20c aux and microculture. discussion the presumptive identification of yeasts may be crucial in the diagnosis and treatment of fungal infections. it is a fundamental recognition and validation of methods that should be fast, accurate and inexpensive. bernal et al.25 (1996) using the chromagar candida for presumed identification fig. 1. rapd showing different species of candida. (1) ladder (250 pb), (2) c. albicans atcc,(3) c. albicans atcc, (4) c. tropicalis; (5) c. krusei, (6) c. albicans atcc, (7) c. guilliermondii, (8) c. albicans. agreement between rapd, api20c aux, chromagar candida and microculture on oral candida identification braz j oral sci. 14(2)149-153 152152152152152 of 593 colonies, revealed 341 (57.5%) c. albicans, 339 (57.2%) of them featuring green characteristic color. all 35 (5.9%) c. krusei and 73 (12.3%) of c. tropicalis presented specific characteristics identified on chromagar candida. in the present study, among the 91 isolates, 35 (38.46%) were pale green, 18 (30.7%) pale pink with white halo and 5 (5.5%) were blue on chromagar candida. using rapd, 29 (31.8%) were identified as c. albicans, 8(8.8%) as c. krusei and 9 (9.9%) as c. tropicalis (kappa coefficient 0.760, 0.360 and 0.640 respectively p<0.05), showing a good accuracy of chromagar candida identification of these species. studies with ope-18 primer11,22 showed different monomorphic bands for the species c. glabrata, c. guilliermondii and c. lusitaniae. baires-varguez et al.11(2007) using ope 18 by rapd-pcr with 92 clinical isolates revealed 20 (21.7%) c. albicans, 14 (15.2%) c. glabrata, 10 (10.9%) c. guilliermondii, 11 (11.95%) c. lusitaniae and 15 (16.3%) c. tropicalis with a 91% sensitivity for the total isolates, being very specific and sensitive for the c. glabrata, c. guilliermondii,c. tropicalis, c. pelliculosa, c. albicans, c. krusei and c. lusitaniae species. among the 91 isolates in the analysis using the same technique and the same primer, were obtained 29 (31.9%) c. albicans, 12 (13.2%) c. glabrata, 6 (6.6%) c. guilliermondii and 6 (6.6%) c. lusitaniae. the sensitivity and specificity in the present study was respectively 96% and 97% for c. albicans, 80% and 100% for c. glabrata, 89% and 95% for c. parapsilosis and 100% and 98% for c. tropicalis. several studies used the api® 20c aux as identification and confirmation of candida species26-28. silva and candido26 (2005) using the api® 20c aux identified 92% (46) of yeasts used in their study, 76% (38) did not require additional tests chromagar candida candida species c. albicans c. dubliniensis c. tropicalis c. krusei kappa(p*) 0.760 (0.000) 0.416 (0.000) 0.640 (0.000) 0.360 (0.000) * kappa coefficient probability. table 2.table 2.table 2.table 2.table 2. kappa coefficient between the presumptive identification of candida species by chromagar candida and rapd (ope18) candida spp microculture api 20c aux kappa (p*) kappa (p* ) c. albicans 0.851 (0.000) 0.925 (0.000) c. dubliniensis 0.852 (0.000) 0.657 (0.000) c. tropicalis 0.806 (0.000) 0.946 (0.000) c. krusei 0.059 (0.565) 0.474 (0.000) c. glabrata 0.712 (0.000) 0.897 (0.000) c. parapsilosis 0.588 (0.000) 0.732 (0.000) c. guilliermondii 0.056 (0.587) 0.323 (0.000) c. lusitaniae 0.004 (0.908) 0.578 (0.000) c. kefyr 0.739 (0.000) 0.883 (0.000) undefined species 0.020 (0.724) 0.019 (0.808) table 3.table 3.table 3.table 3.table 3. kappa coefficient applied to comparative identification between api® 20c aux and microculture analysis with reference to the products of rapd (ope 18) * kappa coefficient probability. and 16% (8) required some additional analysis. the results are closer to sand and rennie27 (1999), who found 96.5% accuracy after 72 h. good results were also obtained by smith et al.9 (1999), who found 95.6% of identification without extra tests. in this analysis, among the 91 isolates, rapd identified 12 (13.2%) c. glabrata, 9 (9.9%) c. parapsilosis, 6 (6%) c. guilliermondii, 6 (6%) c. lusitaniae and 5 (5.5%) c. kefyr. when the same species were submitted to the api® 20c aux, the agreement was statistically significant (p<0.05). the agreement between rapd (ope-18) and api® 20c aux is evidently higher. critical levels of agreement between rapd and microcultive were observed when c. lusitaniae, c. krusei and c. guilliermondii were identified. for presumptive identification, chromagar candida is adequate for c. albicans, c. dubliniensis, c. tropicalis and c. krusei identification. acknowledgements we would like to thank the foundation for research support of minas gerais fapemig minas gerais and cnpq, brazil, for the financial support provided to our research and we also wish to thank marise silveira for the biostatistical services. references 1. rossoni rd, barbosa jo, vilela sfg, santos jdd, jorge aoc, junqueira jc. correlation of phospholipase and proteinase production of candida with in vivo pathogenicity in galleria mellonella. braz j oral sci. 2013;12, 199-204. 2. beighton d, ludford r, clark dt, brailsford sr, pankhurst cl, tinsley gf, et al. use of chromagar candida medium for isolation of yeasts from dental samples. j clin microbiol. 1995; 33: 3025-7. 3. reichart pa, samaranayake lp, samaranayake yh, grote m, pow e, cheung b. high oral prevalence of candida krusei in leprosy patients in northern thailand. j clin microbiol. 2002; 40: 4479-85. 4. alnuaimi ad, wiesenfeld d, o’brien-simpson nm, reynolds ec, peng b, mccullough mj. the development and validation of a rapid genetic method for species identification and genotyping of medically important fungal pathogens using high-resolution melting curve analysis. molecular oral microbiology.2014; 29: 117-130. 5. neppelenbroek kh, seó rs, urban vm, silva s, dovigo ln, jorge jh, et al. identification of candida species in the clinical laboratory: a review of conventional, commercial, and molecular techniques. oral diseases. 2014; 20: 329-44. 6. da costa k, ferreira j, komesu m, candido r. candida albicans and candida tropicalis in oral candidosis: quantitative analysis, exoenzyme activity, and antifungal drug sensitivity. mycopathologia. 2009; 167: 73-9. 7. yücesoy m, marol s. performance of chromagar candida and biggy agar for identification of yeast species. ann clin microbiol antimicrob. 2003; 2: 1-7. agreement between rapd, api20c aux, chromagar candida and microculture on oral candida identification braz j oral sci. 14(2)149-153 153153153153153 8. hata dj, hall l, fothergill aw, larone dh, wengenack nl. multicenter evaluation of the new vitek 2 advanced colorimetric yeast identiûcation card. j clin microbiol. 2007; 45: 1087-92. 9. smith m, dunklee d, hangna v, woods g. comparative performance of the rapid yeast plus system and the api 20c aux clinical yeast system. j clin microbiol. 1999; 37: 2697-8. 10. ramani r, gromadzki s, pincus dh, salkin if, chaturvedi v. efficacy of api 20c and id 32c systems for identification of common and rare clinical yeast isolates. j clin microbiol. 1998; 36 : 3396-8. 11. baires-varguez l, cruz-garcía a, villa-tanaka l, sánchez-garcía s, gaitán-cepeda la, sánchez-vargas lo, et al. comparison of a randomly amplified polymorphic dna (rapd) analysis and atb id 32c system for identification of clinical isolates of different candida species. rev iberoam micol. 2007; 24: 148-51. 12. sullivan dj, henman mc, moran gp, o’neill lc, bennett de, shanley db, et al. molecular genetic approaches to identification, epidemiology and taxonomy of non-albicans candida species. j med microbiol. 1996; 44: 399-408. 13. elie cm, lott tj, reiss e, morrison cj. rapid identification of candida species with species-specific dna probes. j clin microbiol. 1998; 36: 3260-5. 14. joly s, pujol c, rysz m, vargas k, soll dr. development and characterization of complex dna fingerprinting probes for the infectious yeast candida dubliniensis. j clin microbiol. 1999; 37: 1035-44. 15. neppelenbroek kh, campanha nh, spolidorio dm, spolidorio lc, seó rs, pavarina ac. molecular fingerprinting methods for the discrimination between c. albicans and c. dubliniensis. oral dis. 2006; 12: 242-53. 16. ahmad s, khan z, asadzadeh m, theyyathel a, chandy r. performance comparison of phenotypic and molecular methods for detection and differentiation of candida albicans and candida dubliniensis. bmc infectious diseases. 2012; 12: 230. 17. babu kn, rajesh mk, samsudeen k, minoo d, suraby ej, anupama k, et al. randomly amplified polymorphic dna (rapd) and derived techniques. methods mol biol. 2014; 1115: 191-209. 18. mucciarelli m, ferrazzini d, belletti p. genetic variability and population divergence in the rare fritillaria tubiformis subsp. moggridgei rix (liliaceae) as revealed by rapd analysis. plos one. 2014; 9 :e101967. 19. yuan g, sun j, li h, fu g, xu g, li m, et al. identification of velvet antler by random amplified polymorphism dna combined with non-gel sieving capillary electrophoresis. mitochondrial dna. 2014; 8: 1-7. 20. paluchowska p, tokarczyk m, bogusz b, skiba i, budak a. molecular epidemiology of candida albicans and candida glabrata strains isolated from intensive care unit patients in poland. mem inst oswaldo cruz. 2014; 109: 436-41. 21. nielsen kl, godfrey pa, stegger m, andersen ps, feldgarden m, frimodt møller n. selection of unique escherichia coli clones by random amplified polymorphic dna (rapd): evaluation by whole genome sequencing. j microbiol methods. 2014; 103: 101-3. 22. bautista-muñoz c, boldo x, villa-tanaca l, hernández-rodríguez c. identification of candida spp. by randomly amplified polymorphic dna analysis and differentiation between candida albicans and candida dubliniensis by direct pcr methods. j clin microbiol. 2003; 41: 414-20. 23. koehler ap, chu k-c, houang ets, cheng afb. simple, reliable and cost-effective yeast identification scheme for the clinical laboratory. j clin microbiol. 1999; 37: 422-6. 24. odds fc. quantitative microculture system with standardized inocula for strain typing, susceptibility testing, and other physiologic measurements with candida albicans and other yeasts. j clin microbiol. 1991; 29: 2735-40. 25. bernal s, martín mazuelos e, garcía m, aller ai, martínez ma, gutiérrez m. evaluation of chromagar candida medium for the isolation and presumptive identification of species of candida of clinical importance. diagn microbiol infect dis. 1996. 24: 201-4. 26. silva j, candido r. evaluation of the api20c aux system for the identification of clinically important yeasts. rev soc bras med trop. 2005; 38: 261-3. 27. sand c, rennie r. comparison of three commercial systems for the identification of germ-tube negative yeast species isolated from clinical specimens. diagn microbiol infect dis. 1999; 33: 223-9. 28. kirkpatrick wr, revankar sg, mcatee rk, lopez-ribot jl, fothergill aw, mccarthy di, et al. detection of candida dubliniensis in oropharyngeal samples from human immunodeficiency virus-infected patients in north america by primary chromagar candida screening and susceptibility testing of isolates. j clin microbiol. 1998; 36: 3007-12. agreement between rapd, api20c aux, chromagar candida and microculture on oral candida identification braz j oral sci. 14(2)149-153 oral sciences n3 358358358358358 original article braz j oral sci. july | september 2013 volume 12, number 3 accuracy of face castings employing thermoplastic custom trays for facial molding kaue campos pavanello1, priscila galzo marafon1, beatriz silva câmara mattos1 1department of maxillofacial surgery, prosthesis and traumatology, school of dentistry, university of são paulo, são paulo, brazil correspondence to: beatriz silva câmara mattos faculdade de odontologia da universidade de são paulo avenida lineu prestes 2227, cep: 05508-000 cidade universitária, são paulo, sp, brasil phone/fax: +55 11 30917879 e-mail: bscmatto@usp.br abstract aim: to evaluate the dimensional stability of a new facial molding technique using thermoplastic custom molding tray. methods: the designs established demarcation of facial anthropometrics landmarks, making linear measurements with a digital caliper. facial molding was carried out using thermoplastic custom trays, constructing a facial plaster cast with the anthropometric landmarks already transferred by measuring the linear dimensions on the plaster cast and statistical analysis. results: all linear measurements in the palpebral and labial regions presented statistically significant distortions. only one of the linear measurements from the orbital region did not demonstrate any significant distortion. the nasal region presented the least amount of distortion. conclusions: although statistically significant, distortions were due to the method of breathing and were considered to be clinically irrelevant. the reduction in the working time, the comfort provided to the patient and the dimensional accuracy of the plaster cast obtained by the facial molding technique using custom perforated molding tray suggest that this technique should be employed for making facial prostheses. keywords: facial prosthesis, molding material, anthropometrics. introduction in most instances, facial prosthesis represents the only possibility for rehabilitation of facial mutilation resulting from trauma or cancer surgery, with immediate esthetic and functional results that will enhance the patient’s quality of life. it is important to obtain a mold casting of the defective area, the morphology of the surrounding tissues, as well as the corresponding unaffected side, in order to sculpt the prostheses or, as more recently used, to adapt a wax prototype before ascertain its adjustment on the patient’s face. ariani et al.1 (2013), reviewing the techniques and materials used to rehabilitate maxillofacial defects, observed that it has not yet been described in the literature a fully digital workflow for scanning, designing, and fabricating maxillofacial prostheses to be placed directly onto the patient’s defect, without plaster nor wax casts. low viscosity and good elastic recovery are important for the accurate facial molding and consequently of the final plaster cast. additional amount of cold water provides a smooth fluid mix with a delayed set2. during the conventional facial impression technique an initial layer of irreversible hydrocolloid is spread over the face and when it is set, a plaster layer is applied to act as reinforcement to the initial impression3. this technique is time consuming and uncomfortable for the patient. it also requires too much material, resulting in excessive bulk and weight, which may distort the soft facial tissues and the dimensional stability of the irreversible hydrocolloid may be jeopardized by exothermic reaction during the plaster setting. received for publication: april 05, 2013 accepted: july 31, 2013 braz j oral sci. 12(3):164-168 165165165165165 the addition of two to eight drops of sodium phosphate increases the setting time, providing the professional an extended working time with no change in the elastic recovery of the irreversible hydrocoloid4. however, the significant amount of plaster required to provide rigidity for backing the impression material may deform the soft facial tissues and the final weight of the mold must be controlled by the professional in order to avoid distortion of the cast. positioning the face in a vertical plane reduces the gravitational effect and ensures correct muscle toning, but complicates the contention of material in the area to be molded5. the requirements for an ideal face impression material include the ability to record the defect, the adjacent structures and undercuts with minimal distortion, and to prevent soft tissue deformation by weight of the material. the supine position allows greater gravitational interference on the facial tissues, leading to deformation as a result of the excessive weight of the molding material6. care should be taken when using conventional impression techniques, since the final weight of the plaster material may eventually deform the impression of a large facial area7. the impression material must have low viscosity and good elasticity in order to accomplish these aspects3. specific clinical situations, such as tissue movement during function, may require a preliminary conventional facial impression with irreversible hydrocolloid and then a customized acrylic tray with tissue-conditioning material may be used to produce a functional impression of the moveable areas. lowand high-viscosity polyvinyl siloxane reduces the impression material’s weight on the soft tissue and presents dimensional stability and high tear strength when compared to irreversible hydrocolloid. this material has been used to duplicate the defect site, where severe undercuts were blocked out using modeling wax of a previous facial plaster cast obtained from an irreversible hydrocolloid moulage8 twopiece impression procedure is necessary when there are impression posts placed onto the implant abutments. the impression of the basal tissues of the defect area may be performed using medium-body polyether material and then the impression of the entire face is made with polyvinyl siloxane impression material9. however, extra-oral impressions require excessive material and its high cost is a disadvantage. facial impressions made with a thermoplastic custom tray and irreversible hydrocolloid proved to be effective in conventional situations. the tray is lightweight, which reduces the possibility of soft tissue displacement during the impression and can be disinfected, which allows storage for future use. it was observed that the irreversible hydrocolloid moldings and the perforated trays produced the same level of dimensional accuracy along the length and breadth of the dental arch10. modern computer technology for capturing threedimensional digital facial images and for constructing prototypes using the cad/cam system introduces a new direction for obtaining facial casts. plaster casts and molds created by capturing three-dimensional digital images proved to be statistically equivalent11. the distortion from the use of irreversible hydrocolloid was assessed by comparing data gathered from digitalization by scanning the patient’s face and results obtained from the respective plaster mold produced by using the molding technique. the main deviations were found in the regions of the lips, nose, cheeks and the whole region covered by the lower third of face12. facial casts made of plaster, obtained from conventional impression with irreversible hydrocolloid proved to have good reproducibility, enabling laboratorial adaptation of a prototype before the clinical first adjustment13. nevertheless, conventional facial molding techniques demand excessive time and discomfort for the patient, obliging the technician to maintain a control over the possibility of facial tissue distortion. this study proposes and assesses the dimensional accuracy of the facial molding technique utilizing irreversible hydrocolloid and a thermoplastic custom tray. material and methods the study group comprised 15 adult volunteers, over 18 years of age and from both genders, following approval by the ethics committee, school of dentistry, university of são paulo 204/2008. the volunteers were placed in dental chairs and the anthropometric landmarks were marked using a black dermatographic pencil koh-i-noor, no. 3260-5, according to farkas14. the anthropometric landmarks g, sn and ls, located on the facial median line, were established in the first moment. the other landmarks were bilateral; being marked on both right and left hemi-faces. the anthropometric landmarks were grouped in regions as follows: 1. orbital region: g – en; g – ex; g – os; g – sci; sci – os 2. palpebral region: en – ex; en – ps; ex – os 3. nasal region: or – prn; or – al; or – sn; ac – ac; ac – al 4. labial region: cph – cph; sn – cph; ls – sn all the linear measurements were recorded for both right and left hemi-faces as well as the ls – sn measures, which are central to the face and the ac – ac and cph – cph measures, which consisted of isolated landmarks. data were collected with a digital caliper (cd – 6´´cx’b; mitutoyo sul americana ltda., suzano, sp, brazil) by a single examiner and the data were registered in millimeters. for assessing the intra-examiner variability, two separate data collections were made from the faces of 10 individuals, with a 30 min interval between the first and second collection. the data were submitted to statistical analysis cronbach’s alpha (p<0.05). the custom molding was made with perforated aquaplast® (patterson medical holdings, inc., bolingbrook, il, usa) 1.6-mm-thick thermoplastic strips with moderate resistance to stretching, good adaptability and 100% elastic memory15. plasticization occurred at 70º c inside a plastifying tray, then it was slowly cooled and applied to the individual’s face, according to the following limits: lower – upper lip; lateral – in front of the tragus; upper – a horizontal line through the glabella (figure 1). after making the thermoplastic tray, the anthropometric points were marked and the linear measurements made. accuracy of face castings employing thermoplastic custom trays for facial molding braz j oral sci. 12(3):164-168 166166166166166 the volunteers were seated in a dental chair, with normal axial relaxation and reclined 30° to the horizontal plane. the nasal openings were occluded with humid sterile cotton wool, soaked in physiological saline solution, to prevent penetration of the molding material into the nose. ventilation was maintained with oral breathing, by introducing a spacer, 1.0 cm thick, previously fixed to a sheet of rose wax no. 7 (epoxiglass ind. e com. de produtos químicos ltda., diadema, sp, brazil), between the anterior teeth, in a way to ensure oral breathing and restrain any run-down of the irreversible hydrocolloid (jeltrate type ii; dentisply ind. e comércio ltda., petrópolis, rj, brazil). the material was prepared in the water/powder proportion of 1.5/1, a consistency more fluid than the one recommended by the manufacturer, to allow greater fluidity and a better flow of the material16 (figure 2). fig. 1 thermoplastic material being cooled on the face. fig. 2 facial molding with thermoplastic custom tray; mouth breathing. the landmarks marked on the face were automatically transferred to the molding material in direct contact with the skin, by black pigment absorption and could be identified on the inner surface of the mold. these points were highlighted with a dermatographic pen before immediately proceeding to make the plaster cast using plaster rock type iii (asfer tipo iiitm, asfer ind. química ltda., são caetano do sul, sp, brazil) to avoid any deformation17. in the same way, these points were transferred to the plaster in contact with the ink, becoming evident on the outer surface of the plaster cast. the same linear measurements, which were registered on the faces of the volunteers, were now taken from the facial casts. the statistical analysis wilcoxon signed-rank test (p<0.05) was set for evaluating the dimensional acuity of the casts resulting from the facial molding technique employing thermoplastic custom trays. results the reliability of the values collected by the same technician from the faces of the 10 volunteers, maintaining an interval of 30 min between the two data collections, was carried out by statistical analysis conbrach’s alpha (p<0.05), demonstrating internal consistency of the sample, with a high level of reliability. the statistical analysis of the data, using wilcoxon signed-rank test (p<0.05), for assessing the accuracy of the facial cast obtained by using thermoplastic custom trays, is presented according to the facial regions, considering first the right and then the left side. all the linear measurements obtained from the orbital region of the face and from the plaster cast demonstrated significant statistical difference, except for the sci-ps measurement, on the left side (table 1). the measurements obtained from palpebral region showed statistically significant difference on both left and right sides (table 2). the nasal region showed no significant difference for the or-prn and measurement g-en g-ex g-os g-sci sci-ps g-en g-ex g-os g-sci sci-ps variable odf odm odf odm odf odm odf odm odf odm oef oem oef oem oef oem oef oem oef oem n * 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 mean 28.75 29.67 57.82 59.11 35.69 36.49 44.53 45.36 20.46 21.30 28.71 29.33 57.96 59.45 37.62 38.36 45.09 44.22 20.95 20.87 s d 1.69 1.89 2.70 3.29 3.61 4.01 4.53 5.14 3.05 2.98 1.72 1.73 3.04 3.52 4.16 4.41 2.87 5.48 3.26 4.08 sig.(p) 0.001 0.001 0.006 0.008 0.012 0.002 0.001 0.001 0.015 0.112 table 1 orbital region – wilcoxon signed-rank test. p<0.05. *number of cases. accuracy of face castings employing thermoplastic custom trays for facial molding braz j oral sci. 12(3):164-168 167167167167167 measurement en-ex en-ps ex-ps en-ex en-ps ex-ps variable pfd pmd pfd pmd pfd pmd pfe pme pfe pme pfe pme n * 15 15 15 15 15 15 15 15 15 15 15 15 mean 35.02 35.87 22.68 23.15 23.33 23.60 34.82 35.31 22.02 24.43 22.51 23.03 s d 2.18 2.51 1.74 1.58 2.27 2.49 2.75 2.95 3.15 5.09 1.73 1.97 sig. (p) 0.002 0.027 0.041 0.005 0.001 0.012 table 2 – palpebral region – wilcoxon signed-rank test. p<0.05. *number of cases. measurement or-prn or-al or-sn ac-ac ac-al or-prn or-al or-sn ac-al variable nfd nmd nfd nmd nfd nmd n f n m nfd nmd n f e n m e n f e n m e n f e n m e n f e n m e n * 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 mean 54.03 54.34 32.46 33.50 51.05 51.69 29.34 29.95 10.84 10.99 55.63 56.02 33.07 33.52 51.72 52.13 10.16 10.34 s d 3.82 4.14 3.78 2.37 3.43 3.48 4.19 4.32 1.49 1.39 4.12 3.99 2.51 2.67 3.38 3.22 1.63 1.55 sig. (p) 0.256 0.041 0.041 0.003 0.268 0.061 0.009 0.069 0.017 table 3 – nasal region – wilcoxon signed-rank test. p< 0.05. *number of cases. ac-al measurements on the right side, as well as for the orprn and or-sn measurements on the left side (table 3). the analysis of measurements obtained from the labial region of the face and from the facial cast demonstrated significant difference for all evaluated measurements (table 4). discussion strategies in handling irreversible hydrocolloid are used in order to increase the working time and the viscosity of the material during the performance of facial molding. therefore, the deliberate increase of the water to powder ratio to 1.5/1, corresponds to 27 ml of water for every 7 g of irreversible hydrocolloid powder16, resulting in an increase in the working time and the viscosity of the material without compromising its elasticity3. a more fluid molding material will present greater viscosity and diminish compression during the act of molding. this reduction in compression will minimize the amount of displacement suffered by the soft facial tissues, providing greater precision to the mold. the correct positioning of the patient is also a significant factor for achieving proper impression fidelity, as a large area leads to greater volume of material and, consequently, a greater weight and possibility of deforming the soft facial tissues6. the supine position allows greater gravitational interference on the facial tissues, leading to deformation as a result of the excessive weight of the molding material6. the position of the face at 30o in relation to the horizontal plane was employed in this study in order to compensate the distorting effects mentioned above and allows greater control of the run-off of the molding material. the use of a perforated molding and a uniform layer of irreversible hydrocolloid with a more fluid consistency minimizes the displacement of facial tissues and enables the perforations to mechanically retain the molding material3. these observations confirmed the advantages of using a perforated molding tray for performing facial moldings. when marking the area to be molded in this study, the use of nasal tubes was discarded as ventilation was maintained by placing a strip of rose wax between the anterior teeth, which was attached to a rose wax laminate adapted to the contours of the lip in order to contain any possible runoff of the molding material. claustrophobia and other sensations created by the contact of molding material on the face make it imperative that the individual is well oriented in order not to contract the mimic muscles. however, the fact that all linear measurements from the orbital region presented statistical differences, except for the left side scips measurement, suggests that peri-orbital muscular contraction did occur during the molding. similarly, the palpebral region presented significant differences between the linear measurements obtained from the face and the facial cast, indicating that possible muscular contractions did occur. the nasal region presented the greatest dimensional stability, with four measurements demonstrating statistical similarities. the use of nasal tubes is liable to create deformations around the nasal wings, where the nasal apex and the sub-nasal regions tend to present the greatest distortions12. in this study, the use of oral respiration may have avoided tissue deformation, preserving the linear measures and providing greater accuracy in the nasal region despite the fact that this region is not supported by underlying bone tissue. measurement cph-cph sn-cph ls-sn sn-cph variable lfd lmd lfd lmd lfd lmd lfe lme n * 15 15 15 15 15 15 15 15 mean 12.52 13.06 13.65 14.30 14.09 14.65 13.45 14.25 s d 2.64 2.60 1.89 1.93 2.01 2.20 2.15 2.52 sig.(p) 0.001 0.001 0.003 0.002 table 4 – labial region wilcoxon signed-rank test. p<0.05. *number of cases. accuracy of face castings employing thermoplastic custom trays for facial molding braz j oral sci. 12(3):164-168 all linear measurements obtained from the labial region demonstrated significant difference. this fact may have resulted from deformities provoked by placing a strip of wax in the anterior maxillary region and confirms previous observations that in the case of oral respiration the upper lip is subjected to the greatest distortions12. it should be reminded that the ventilation system interferes in the accuracy of the final cast and, in a clinical situation, the professional should choose for nasal or oral breathing, depending on the area of the facial injury. the perforated thermoplastic custom trays and irreversible hydrocolloid material technique for facial molding evaluated in this study confirmed the importance of minimizing the factors that interfere in the accuracy of the final cast: the position of the patient’s face and the great weight of the mold, as a result of the plaster layer used in the conventional molding technique. it also allowed for proper ventilation and restrained any rundown of the irreversible hydrocolloid material proportioned to present greater fluidity and a better flow. although statistically significant, the majority of the differences observed were restricted to tenths of millimeters. minor values of this size are irrelevant in clinical situations, as the final sculpturing and adaptation of the facial prosthesis should necessarily be made directly on the patient’s face during a clinical session. the shorter working time and the greater comfort created by using the perforated thermoplastic custom mold evaluated in this study suggests that this technique should be employed for making facial prostheses. the slight dimensional alterations observed in the plaster casts are within the dimensional accuracy criteria required for making facial prostheses. references 1. ariani n, visser a, van oort rp, kusdhany l, rahardjo tbw, krom bp et al. current state of craniofacial prosthetic rehabilitation. int j prosthodont. 2013; 26:57-67. 2. veerareddy c, nair c, reddy r. simplified technique for orbital prosthesis fabrication: a clinical report. j prosthodont. 2012; 21: 561-8. 3. rommerdale eh. maxillofacial technology. 1. , part one. introduction to facial impressions. trends tech contemp dent lab. 1990; 7: 36-9. 4. lemon jc, okay dj, powers jm, martin jw, chambers s. facial moulage: the effect of a retarder on compressive strength and working and setting times of irreversible hydrocolloid impression material. j prosthet dent. 2003; 90: 276-81. 5. alsiyabi as, minsley ge. facial moulage fabrication using a two-stage poly(vinyl siloxane) impression. j prosthodont. 2006; 15: 195-7. 6. holberg c, schwenzer k, mahaini l, rudzki-janson i. accuracy of facial plaster casts. angle orthod.ont. 2006; 76: 605-11. 7. siadat h, mirfazaelian a. a handle for facial casts. j prosthet dent. 2003; 89: 323. 8. shaikh sr, patil pg, puri s. a modified technique for retention of orbital prosthesis. ind j dent res. 2011; 22: 863-5. 9. ozcelick tb, yilmaz b. two-piece impression procedure for implantretained orbital prostheses. int j oral maxillofac surg. 2012; 27: 93-5. 10. al-athel. the effect of selected variables on the retention of irreversible hydrocolloid impression material. j contemp dent pract. 2008; 9: 57-64. 11. littlefield tr, cherney jc, luisi jn, beals sp, kelly km, pomatto jk. comparison of plaster casting with three-dimensional cranial imaging. cleft palate craniofac j. 2005; 42: 157-64. 12. holberg c, schwenzer k, mahaini l, rudzki-janson i. accuracy of facial plaster casts. angle orthodont. 2006; 76: 605-11. 13. marafon pg, mattos bs, sabóia ac, noritomi py. dimensional accuracy of computer-aided design/computer-assisted manufactured orbital prostheses. int j prosthodont. 2010; 23: 271-6. 14. farkas lg. anthropometry of the head and face. 2nd ed. new york: raven press; 1994. 15. politec importação e comércio ltda.. produto – placa termoplástica aquaplast – sammons preston rolyan [cited 2013 apr] available from:. accessible from http://www.politecsaude.com.br [april 10, 2013]. 16. sykes lm. custom made ocular prostheses. a clinical report. j prosthet dent. 1996; 75: 1-3. 17. rodrigues sb, augusto cr, leitune vc, samuel sm, collares fm. influence of delayed pouring on irreversible hydrocolloid properties. braz oral res. 2012; 26: 404-9. accuracy of face castings employing thermoplastic custom trays for facial molding168168168168168 braz j oral sci. 12(3):164-168 429 too many requests error 429 too many requests too many requests guru meditation: xid: 97459278 varnish cache server oral sciences n3 original article braz j oral sci. april | june 2014 volume 13, number 2 oral hydration in children with cerebral palsy maria teresa botti rodrigues santos1, maria cristina duarte ferreira2, renata oliveira guaré1, oliver a. nascimento3, jose r. jardim3 1 universidade cruzeiro do sul unicsul, school of dentistry, area of pediatric, são paulo, sp, brazil 2 serviço nacional de aprendizagem comercial senac, area of pediatric, são paulo, sp, brazil 3 universidade federal de são paulo – unifesp, paulista school of medicine, respiratory division, são paulo, sp, brazil correspondence to: maria teresa botti rodrigues santos universidade cruzeiro do sul rua constantino de souza, 454, apto 141 cep: 04605-001 são paulo sp brasil phone: +55 11 999722301 fax: +55 11 50930865 e-mail: drsantosmt@yahoo.com.br received for publication: april 07, 2014 accepted: june 10, 2014 abstract salivary osmolality reflects the hydration status of individuals with cerebral palsy necessary for adequate unstimulated salivary flow rate. aim: to investigate whether oral motor performance is determinant for the hydration status and the effect of a supplemental oral fluid supply on salivary osmolality. methods: the sample consisted of 99 children with cerebral palsy aged 6 to 13 years old. in this study, children participated in 2-day evaluations: 1st day baseline: saliva collection, caries experience and oral motor performance evaluations; and 2nd day: saliva collection after supplemental fluid supply. prior to each evaluation, the participants were trained for saliva collection. unstimulated whole saliva was collected using cotton roll at baseline, with the amount of fluid usually offered by caregivers, and 48 h after baseline, with as much as twice the normal daily fluid intake previously offered. salivary osmolality was measured using a freezing point depression osmometer. caries experience index for decayed, missed and filled teeth (dmft) was evaluated. according to the oral motor assessment scale, the children were classified into subfunctional or functional groups. chi-square, student’s t test and pearson’s correlation coefficient were used. results: the subfunctional group presented a higher percentage of quadriplegic children (p<0.001), with significantly higher values for caries experience (p<0.001) and salivary osmolality (p<0.001), which did not diminish when supplemental fluid supply was offered, compared with the functional group (p=0.001). conclusions: the effectiveness of oral motor performance plays an important role in the hydration status of children with cerebral palsy and those with worse oral motor performance may be at higher risk of oral diseases. keywords: cerebral palsy; motor skills disorders; muscle spasticity; osmolar concentration; fluid therapy. introduction cerebral palsy describes a group of chronic disorders that involve movement and posture development, often accompanied by epilepsy, secondary musculoskeletal problems and disturbances of sensation, perception, cognition, communication and behavior. it is the most common cause of severe physical disability in childhood1, with an estimated prevalence of 2.4 per 1000 children2. alterations in mastication and swallowing in children with cerebral palsy result in feeding dysfunctions that may lead to reduced dietary intake, prolonged feeding times and poor nutritional status accompanied by compromised physical performance3-7. children with cerebral palsy present low unstimulated salivary flow rate, ph and buffer capacity8, and variations in the activity of enzymes and sialic acid concentrations9. it has also been reported that children with cerebral palsy present increased salivary osmolality and total protein concentration10, together with increased salivary, urine and plasma osmolalities, characterizing an impaired braz j oral sci. 13(2):140-145 hydration status11. decreased levels of hydration (dehydration) may cause diminished salivary output 12, which could compromise the protective function exerted by saliva and increase the risk of oral diseases11. considering that an adequate fluid intake and homeostasis of total body water is essential for human health and survival, the aims of this study were to investigate whether (i) oral motor performance is determinant for hydration status and (ii) the effect of a supplemental oral fluid supply on salivary osmolality in children with cerebral palsy. the tested hypothesis was that compromised oral motor performance interferes in fluid intake, resulting in a diminished health status for these children. material and methods participants ninety-nine non-institutionalized male and female children (aged 6 to 13 years-old) with a medical diagnosis of spastic cerebral palsy13, who were referred to a specialized rehabilitation center in são paulo, sp, brazil, were consecutively included in this study. the adults responsible for each child provided written informed consent to participate in the study, which was approved by the cruzeiro do sul university review board for human studies (#0152/2011). children using any drug that could interfere with saliva secretion (anticholinergic, neuroleptic and benzodiazepine drugs) for at least 72 h prior to examination or with a history of head and neck radiation or surgical procedures to reduce drooling were excluded from the study. demographic and clinical data, including sex, age and gross motor function classification system14, were obtained for all children. children participated in 2-day evaluations: 1st day baseline saliva collection, caries experience and oral motor performance evaluations; and 2nd day saliva collection after supplemental fluid supply. prior to each evaluation, the participants were trained for saliva collection. saliva collection and assessment the caregivers were carefully instructed to record the total amount of fluid (water, milk, juice, soda, soup) offered to their children in the 48 h prior to the first saliva collection (baseline). after the first saliva collection, caregivers were instructed to maintain the same intake for the children and to offer a supplemental volume of fluid (as much as twice the normal daily fluid intake) for the following 48 h. then, a new saliva sample was collected (supplemental volume supply). at least 2 h after the previous meal, unstimulated whole saliva was collected by a single calibrated examiner between 8:00 and 9:00 a.m., using the absorbent method15. after the mouth was dried with sterile gauze, one absorbent cotton roll (salivette®; sarstedt, nümbrecht, germany) was placed in the mouth under the tongue for exactly 5 min, with the children comfortably seated in a ventilated and illuminated room. immediately after, the saliva collection sample was frozen in dry ice, transported to the laboratory and stored at -80 °c until analysis. salivary osmolality was measured using a freezing point depression osmometer (model vapro vapor pressure osmometer 5600; new instrument, washington, dc, usa). caries experience dental clinical evaluation was performed by a single calibrated examiner under standardized conditions, using optimal artificial lighting, compressed air to dry the teeth, a plane buccal mirror and a who probe. caries experience was recorded according to the world health organization criteria16, and decayed, missed and filled teeth were evaluated using the dmf-t / dmf-t index for primary and permanent dentition, respectively. in children with mixed dentition, dmft and dmf-t were recorded together. oral motor performance the same trained oral health care professional evaluated the performance of the masticatory muscles of all participants using the oral motor assessment scale 17. the kappa coefficient was used to evaluate the intra-rater consistency of the test-retest measurements. the significance level was set at p<0.05 and the kappa value for the final score was 1.00. the oral motor assessment scale was applied according to the following description. three types of food were offered to the child: paste (danonin® brand “petit suisse” cheese), solid (“waffle” style cracker), and water in a cup with a straw. the child was asked to eat normally. the researcher observed the child’s oral motor ability and indicated a score for each of the following items: mandibular closing, labial sealing with the utensil, anterior lingual sealing when swallowing, control of solid food when swallowing, suction with a straw, control of liquid when swallowing, and chewing. the researcher did not guide or modify the way in which the caregiver helped or how the child ate. in cases in which the child exhibited more than one type of oral motor function for the same item, the most frequent behavior during the evaluation was determined. the final classification score for motor ability was determined based on the most frequent score. a score of 0 indicated the greatest degree of impairment (passive), 1 indicated subfunctional impairment, 2 indicated semi-functional impairment, and 3 indicated normal function, meeting efficiency standards. children defined as having a final predominance type of passive and subfunctional (severely and moderately compromised oral motor performance) were classified into a single group that was named the subfunctional group, while the semi-functional and functional individuals (slightly and very slightly compromised oral motor performance) were named the functional group. statistical methods statistical analyses were performed using the statistical package for sciences (version 19.0; spss inc., chicago, il, usa). to evaluate proportional differences between the studied groups, the chi square test was used. the student t test was used to verify the hypothesis of equality between 171141oral hydration in children with cerebral palsy braz j oral sci. 13(2):140-145 the two groups. pearson´s correlation coefficient was used to associate the behavior among the studied variables. the power of this sample was calculated using descriptive statistics of mean and standard deviation of saliva osmolarity collected at baseline, using a confidence interval of 95%. a p value of 0.05 was the criterion for significance. values are presented as means ± sd. results the power of the sample of 99 children who participated in this study was 0.837. the subfunctional (n=49) and functional (n=50) groups did not differ regarding sex (p=0.366) and age (p=0.621). however, the subfunctional group presented a higher percentage of quadriplegic children (p<0.001), with level v of the gross motor function classification system (p<0.001). regarding caries experience, the subfunctional group presented significantly higher dmf-t/dmf-t values (p<0.001) (table 1). in relation to salivary osmolality, the subfunctional group presented significantly higher values (p<0.001) not only at baseline, but also when the supplemental fluid supply was offered compared with the functional group. concerning the supplemental fluid supply, both groups showed an increased volume (subfunctional: 46.7 ± 121.4 ml; functional: 112.1 ± 138.1 ml) in the follow-up period. despite this fact, only the functional group showed reduction in salivary osmolality. for the subfunctional group, no effects on salivary osmolality were observed following supplemental fluid supply. variables sex, n (%) female male age years (mean± sd) clinical pattern, n (%) quadriplegic diplegic hemiplegic gmfcsx, n (%) i ii iii iv v caries experience total (n=99) 39 (39.4) 60 (60.6) 9.6±2.4 50 (50.5) 40 (40.4) 9 (9.1) 9 (9.1) 9 (9.1) 7 (7.1) 52 (52.5) 22 (22.2) 2.1±1.8 subfunctional (n=49) 22 (44.9) 27 (55.1) 9.8±2.6 38 (77.5) 11 (22.5) 0 (0) 0 ( 0) 0 ( 0) 1 (2.1) 28 (57.1) 20 (40.8) 3.5±1.4 functional (n=50) 17 (34.0) 33 (66.0) 9.5±2.2 12 (24.0) 29 (58.0) 9 (18.0) 9 (18.0) 9 (18.0) 6 (12.0) 24 (48.0) 2 (4.0) 0.7±0.3 significance 0.366a 0.621b <0.001*a <0.001*a <0.001*b groups xgmfcs (gross motor function classification system) levels13: i indicates walking without restrictions with limitations in more advanced gross motor skills; ii indicates walking without assistive devices with limitations walking outdoors and in the community; iii indicates walking with assistive mobility devices with limitations walking outdoors and in the community; iv indicates self-mobility with limitations, the patient is transported or uses powered mobility outdoors and in the community; v indicates that self-mobility is severely limited even with assistive technology. the data were compared with the following: a. chi square test, b. student t test, *p<0.05 table 1.table 1.table 1.table 1.table 1. descriptive characteristics of children with cerebral palsy classified into subfunctional and functional groups. figure 1 shows the values of the pearson correlation c o e f f i c i e n t f o r s a l i v a r y o s m o l a l i t y a n d f l u i d s u p p l y values. salivary osmolality was negatively correlated with fluid supply at baseline (p<0.001; a) and after 48 h (p<0.001; b). discussion understanding the impact of oral motor dysfunction in children with cerebral palsy may be helpful in identifying individuals at high risk of developing oral diseases. to our knowledge, this is the first study to evaluate the effect of a supplemental fluid supply on salivary osmolality in children with spastic cerebral palsy. analysis of these results confirm the hypothesis of this study, that is, compromised oral motor performance interferes in fluid intake, resulting in increased salivary osmolality and suggesting that greater neurological impairment causes the diminished hydration status. clinically, cerebral palsy varies in severity and extremely diverse levels of functional independence can be observed in individuals with the same medical diagnosis, due to the extent of neurodevelopmental disorder1,18. spastic cerebral palsy is by far the most common type of movement disorder in cerebral palsy, occurring in 70% to 80% of all cases1. regarding oral motor performance, spastic cerebral palsy children from the subfunctional group were more severely compromised, represented by the quadriplegic clinical pattern and gross motor function classification system level v. salivary osmolality has been described as a reliable parameter of hydration status and reflects changes in plasma 142 oral hydration in children with cerebral palsy braz j oral sci. 13(2):140-145 fig.1. illustration of pearson’s correlation between salivary osmolality and fluid supply values for baseline (a) and after 48 h (b) (r = pearson’s correlation coefficient, p<0.001). variables baseline osmolality (mosml) supplemental fluid supply osmolality (mosml) subfunctional (n=49) 106.3±37.4 101.1±32.7 functional (n=50) 74.3±11.7 67.5±12.1 significance <0.001* <0.001* table 2.table 2.table 2.table 2.table 2. salivary osmolality (mosm/kg h2o) before and after supplemental fluid supply in children with spastic cerebral palsy classified into subfunctional and functional groups. the data were compared by the student t test. *p<0.05 and urine osmolality in cerebral palsy individuals11. the higher salivary osmolality observed in the spastic cerebral palsy children with subfunctional oral motor performance is very similar to the osmolality presented by individuals in dehydration conditions19. in this study, analysis of the initial observations revealed that there is a causal relationship between the events of hydration and oral dysfunction. the reduction in salivary osmolality in the subfunctional and functional groups following supplemental fluid supply was 4.3% and 9.6%, 171143oral hydration in children with cerebral palsy braz j oral sci. 13(2):140-145 respectively. patients in the most compromised group (subfunctional) drank less fluid (6.7%) than those in the functional group (12.3%). the issue of hydration status and fluid diet intake in individuals with cerebral palsy has been rarely described in the literature10-11. solid and semisolid food consistencies provide greater proprioceptive mouth stimulus, which facilitates the control of swallowing. in individuals with cerebral palsy who cannot sustain lip closure, the liquid diet intake usually flows out of the mouth, resulting in a lack of negative pressure for swallowing, which may cause choking. in individuals with cerebral palsy, water intake is generally below the required standards20 and the management of solid alimentary bolus is easier than liquid ingestion; on the other hand, small liquid boluses are more easily swallowed than large liquid boluses. moreover, these individuals tend to consume small volume of liquids21. the total daily fluid supply recorded by caregivers from the functional group, was similar to those described in children with severe cerebral palsy20. one limitation of this study was the fact that the volume supply was not fixed, since cerebral palsy children present difficulties in swallowing, although a higher fluid intake was observed during the observational period. it is also relevant to this discussion to emphasize that, in this study, the severely oromotor compromised cerebral palsy individuals presented higher values of the caries experience index for both dentitions 22-24, with higher prevalence of untreated caries25, which is in agreement with the literature26-27. in addition, the presence of pathological oral reflex of tonic bite4 makes tooth brushing, biofilm control and dental floss use more difficult, which directly impacts on oral hygiene27. further studies should assess the effectiveness of supplying a high liquid content with different consistencies to cerebral palsy children wit h severe oral motor compromise, since this may eventually help reducing salivary osmolality, increase salivary flow rate, improve oral hydration and reduce oral diseases. the findings of the present study suggest that oral motor performance is determinant for hydration status in cerebral palsy children and the most compromised individuals present a higher salivary osmolality, which is associated with an inadequate fluid intake. acknowledgements this study was supported by the fundação de amparo a pesquisa do estado de são paulo, fapesp process no.11/ 12475-8). references 1. richards cl, malouin f. cerebral palsy: definition, assessment and rehabilitation. handb clin neurol. 2013; 111: 183-95. 2. hirtz d, thurman dj, gwinn-hardy k, mohamed m, chaudhuri ar, zalutsky r. how common are the “common” neurologic disorders? neurology. 2007; 68: 326-37. 3. calis ea, veugelers r, rieken r, tibboel d, evenhuis hm, penning c. energy intake does not correlate with nutritional state in children with severe generalized cerebral palsy and intellectual disability. clin nut. 2010; 29: 617-21. 4. dos santos mt, nogueira ml. infantile reflexes and their effects on dental caries and oral hygiene in cerebral palsy individuals. j oral rehabil. 2005; 32: 880-5. 5. fung eb, samson-fang l, stallings va, conaway m, liptak g, henderson rc, et al. feeding dysfunction is associated with poor growth and health status in children with cerebral palsy. j am diet assoc. 2002; 102: 361-73. 6. sullivan pb. gastrointestinal disorders in children with neurodevelopmental disabilities. dev disabil res rev. 2008; 14: 128-36. 7. troughton ke, hill ae. relation between objectively measured feeding competence and nutrition in children with cerebral palsy. dev med child neurol. 2001; 43: 187-90. 8. santos mt, guaré r, leite m, ferreira mc, nicolau j. does the neuromotor abnormality type affect the salivary parameters in individuals with cerebral palsy? j oral pathol med. 2010; 39: 770-4. 9. rodrigues santos mt, siqueira wl, nicolau j. amylase and peroxidise activities and sialic acid concentration in saliva of adolescents with cerebral palsy. quintessence int. 2007; 38: 467-72. 10. santos mt, guaré ro, leite mf, ferreira mc, durão ms, jardim jr. salivary osmolality in individuals with cerebral palsy. arch oral biol. 2010; 55: 855-60. 11. santos mt, batista r, guaré ro, leite mf, ferreira mc, durão ms, et al. salivary osmolality and hydration status in children with cerebral palsy. j oral pathol med. 2011; 40: 582-6. 12. amstrong le. assessing hydration status: the elusive gold standard. j am coll nutr. 2007; 6: 575s-84s. 13. world health organization. 10th international classification of diseases and related health problems, icd-10, 2010. [cited 2014 apr 10]. available from: http://www.who.int/classifications/icd/en . 14. palisano rj, rosenbaum p, bartlett d, livingston mh. content validity of the expanded and revised gross motor function classification system. dev med child neurol. 2008; 50: 744-50. 15. rotteveel lj, jongerius ph, van limbeek j, van den hoogen fj. salivation in healthy schoolchildren. int j pediatr otorhinolaryngol. 2004; 68: 767-74. 16. world health organization. oral health surveys: basic methods. 5th ed. geneva: who; 2013. 17. ortega ade o, ciamponi al, mendes fm, santos mt. assessment scale of the oral motor performance of children and adolescents with neurological damages. j oral rehabil. 2009; 36: 653-9. 18. bax mc, flodmark o, tydeman c. definition and classification of cerebral palsy. from syndrome toward disease. dev med child neurol suppl. 2007; 109: 39-41. 19. walsh np, laing sj, oliver sj, montague jc, walters r, bilzon jl. saliva parameters as potential indices of hydration status during acute dehydration. med sci sports exerc. 2004; 36: 1535-42. 20. veugelers r, benninga ma, calis ea, willemsen sp, evenhuis h, tibboel d, et al. prevalence and clinical presentation of constipation in children with severe generalized cerebral palsy. dev med child neurol. 2010; 52: e216-21. 21. andrew mj, parr jr, sullivan pb. feeding difficulties in children with cerebral palsy. arch dis child educ pract ed. 2012; 97: 222-9. 22. ferreira de camargo ma, frias ac, antunes jl. the incidence of dental caries in children and adolescents who have cerebral palsy and are participating in a dental program in brazil. spec care dentist. 2011; 31: 210-5. 23. moreira rn, alcântara ce, mota-veloso i, marinho sa, ramos-jorge ml, oliveira-ferreira f. does intellectual disability affect the development of dental caries in patients with cerebral palsy? res dev disabil. 2012; 33: 1503-7. 24. santos mt, biancardi m, guare ro, jardim jr. caries prevalence in patients with cerebral palsy and the burden of caring for them. spec care dentist. 2010; 30: 206-10. 144 oral hydration in children with cerebral palsy braz j oral sci. 13(2):140-145 25. de camargo ma, antunes jl. untreated dental caries in children with cerebral palsy in the brazilian context. int j paediatr dent. 2008; 18: 131-8. 26. rodrigues dos santos mt, bianccardi m, celiberti p, de oliveira guaré r. dental caries in cerebral palsied individuals and their caregivers’ quality of life. child care health dev. 2009; 35: 475-81. 27. santos mt, guare ro, celiberti p, siqueira wl. caries experience in individuals with cerebral palsy in relation to oromotor dysfunction and dietary consistency. spec care dentist. 2009; 29: 198-203. 171145oral hydration in children with cerebral palsy braz j oral sci. 13(2):140-145 oral sciences n3 braz j oral sci. 12(2):125-131 original article braz j oral sci. april | june 2013 volume 12, number 2 influence of dialysis duration and parathyroid hormone on the clinical and radiographic oral conditions of pre-transplant patients with chronic kidney disease stênio medeiros queiroz1, adriana gomes amorim2, ana luiza dias leite de andrade2, manuel antonio gordón-núñez2, roseana de almeida freitas2, hébel cavalcanti galvão1 1center of health sciences, rio grande do norte federal university, natal, rn, brazil 2area of oral pathology, department of dentistry, rio grande do norte federal university, natal, rn, brazil correspondence to: hébel cavalcanti galvão avenida general gustavo cordeiro de farias, s/n, cep: 59010-180 – petrópolis, natal, rn, brasil phone/fax: +55 84 33429776 / 32154108 e-mail: hebel.galvao@yahoo.com.br received for publication: march 11, 2013 accepted: june 25, 2013 abstract aim: to evaluate the oral conditions of patients with chronic kidney disease undergoing dialysis and to determine the influence of dialysis duration and bone metabolism on the prevalence and severity of the alterations found. methods: the simplified oral hygiene index (ohi-s), prevalence of dental caries (decayed, missing filled teeth index, dmft), and the periodontal screening and recording (psr) index were evaluated in 154 patients. parathyroid hormone (pth), calcium, phosphorus and urea measurements, as well as panoramic radiographs, were obtained from all patients. to evaluate the effect of duration of dialysis treatment on oral health, the patients were divided into two groups: (1) <5 years and (2) > 5 years. regarding blood levels of pth, patients were divided into three groups: (1) 0-149 pg/ml, (2) 150-584 pg/ml, and (3) >585 pg/ml. the ohi-s identified the accumulation of biofilm and calculus around the teeth. results: gingival inflammation was found in 100% of dentate patients, with 2 being the predominant psr score (72.3%). the dmft index was high (17.52). dental calculus was the most common radiographic finding (70.8%). no significant correlation was observed between dialysis duration, biochemical alterations, and oral health. conclusions: most patients undergoing dialysis presented precarious oral hygiene, periodontal inflammation, and bone alterations. however, these manifestations were not influenced by the duration of dialysis or bone metabolism. keywords: chronic kidney disease, parathyroid hormone, oral health, hemodialysis. introduction chronic kidney disease (ckd) is a global public health problem. the disease is characterized by a slow, progressive and irreversible decline in the number of functional nephrons, which results in a decrease of glomerular filtration rate and in the accumulation of various, often toxic, substances that are normally excreted by the kidneys, causing uremic syndrome1-5, as well as disturbances in normal homeostatic mechanisms that control the water-electrolyte balance in the braz j oral sci. 12(2):125-131 126126126126126 organism1. diabetes mellitus, hypertension, glomerulonephritis, and renal cystic disease are the most common causes of kidney failure, whose incidence and prevalence have increased over the last few years2. ckd is defined when kidney function decreases by 5 to 10% of its original capacity. treatment includes hemodialysis (hd) or continuous ambulatory peritoneal dialysis (capd) as replacement of renal filtration, and a kidney transplant as definitive therapy6. patients with ckd show a broad spectrum of oral manifestations that affect the soft or hard tissues7, including xerostomia, uremic breath, uremic stomatitis, radiographic changes of the jaw bones, accumulation of calculus on the teeth, periodontitis, and other less frequent abnormalities23,8. some of these conditions are more severe in patients undergoing dialysis than in healthy individuals9-12. the duration of dialysis can influence the prevalence of oral abnormalities observed may be related to worsening of oral health11,13. with respect to bone alterations commonly observed in these individuals, bone metabolism disorders caused by altered blood levels of calcium (ca) and phosphorus (p) in response to lack of activation of vitamin d by the kidneys promote the development of secondary hyperparathyroidism, contributing to alveolar bone loss by changing the normal skeletal remodeling, in addition to favoring the development of intra-osseous lesions such as brown tumor14. these oral conditions can become a source of infection since patients with ckd are highly susceptible due to the immunodeficiency caused by uremia3. after transplantation, the persistence of oral infections may be a cause of morbidity in patients on immunosuppressive therapy10. it is therefore important that dentists are aware of the main conditions affecting the oral cavity of these patients in order to prevent or treat them before transplantation4. the objective of the present cross-sectional cohort study was to evaluate cross through clinical, biochemical and radiographic oral conditions in a brazilian population with ckd that was preparing for a kidney transplant, investigating the influence of dialysis duration and status on bone metabolism the prevalence and severity of the changes found. material and methods the sample consisted of 154 patients of both genders with ckd undergoing hd or capd for at least 3 months. the patients were recruited from the central preparation center for kidney transplantation, onofre lopes university hospital (rio grande do norte, brazil), were older than 18 years, and had not received dental treatment during the previous 6 months. the study was conducted in accordance with the declaration of helsinki’s guidelines and was approved by the ethics committee of federal university of rio grande do norte (ufrn) (protocol 082/09). all patients gave written informed consent to participate in the study. a clinical form containing the following data was completed for each patient: demographic data; data about the cause of ckd; type, duration and place of dialysis; medications used; results of routine biochemical tests; frequency of toothbrushing; use of dental floss; presence of bitter taste in the mouth; xerostomia; use of dentures, and date of the last visit to the dentist. the patients underwent intraoral clinical examination at the dental office of the family and community health unit of onofre lopes university hospital, ufrn, using a dental mirror, exploratory probe, basic fuchsine (eviplac®) for biofilm detection, and a periodontal probe (oms-621). this examination was performed to evaluate the presence of alterations in oral soft tissues (lip, palate, tongue, floor of the mouth, cheek mucosa) such as candidiasis and petechiae. next, the teeth were examined regarding the presence of attrition, erosion, abrasion, mobility, stains and hypoplasia. the prevalence of caries was analyzed using the decayed, missing, filled teeth (dmft) index recommended by the world health organization. oral hygiene was evaluated using the simplified oral hygiene index (ohi-s) proposed by greene and vermilion15 (1964), which combines two components: biofilm index and calculus index. six sites of selected teeth were examined after application of a biofilm stain: buccal surface of teeth 16, 26, 11 and 31, and lingual surface of teeth 36 and 46. a score of 0 to 3 was attributed to each surface according to the amount of biofilm or calculus detected. for the biofilm index, scores were attributed as follows: 0 (no staining), 1 (biofilm covering no more than 1/3 of the tooth surface), 2 (biofilm covering more than 1/3 and less than 2/3 of the tooth surface), and 3 (biofilm covering more than 2/ 3 of the tooth surface). the following scores were attributed for determination of the calculus index: 0 (no calculus), 1 (supragingival calculus covering no more than 1/3 of the tooth surface), 2 (supragingival calculus covering more than 1/3 and less than 2/3 of the tooth surface), and 3 (calculus covering more than 2/3 of the tooth surface, or a band of subgingival calculus in the cervical region of the tooth). the ohi-s is calculated as the sum of scores of the two components. the periodontal screening and recording (psr) system was used to analyze the need for periodontal treatment. for this system, the mouth is divided into sextants and the tip of the periodontal probe is inserted into the gingival sulcus of the teeth present. all tooth surfaces are examined and the highest score is recorded. the following scores are attributed: 0: colored part of the probe completely visible. absence of calculus and defective restorations. healthy periodontal tissues. 1: gingivitis. colored part of the probe completely visible but bleeding on probing. absence of calculus and defective restorations. 2: gingivitis or mild periodontitis. colored part of the probe completely visible but bleeding on probing. presence of supraor subgingival calculus. 3: moderate periodontitis. colored part of the probe partially visible, with a pocket depth of 4 to 5 mm. 4: advanced periodontitis. colored part of the probe disappearing completely inside the gingival pocket. clinical evaluation was performed by a single calibrated examiner. kappa statistics was used to evaluate the level of intraexaminer agreement for ohi-s, dmft, and psr. influence of dialysis duration and parathyroid hormone on the clinical and radiographic oral conditions of pre-transplant patients with chronic kidney disease 127127127127127 braz j oral sci. 12(2):125-131 calibration was performed on 10 patients at an interval of one hour between analyses. all kappa values were close to 0.70, indicating almost perfect agreement. panoramic radiographs were taken from all patients to identify sources of infection, the presence of calculus, and bone alterations. all participants received detailed instructions and were encouraged to practice oral hygiene. patients who required dental treatment were referred to the department of dentistry of ufrn where they underwent the adequate procedures. routine biochemical parameters obtained at the dialysis centers, including parathyroid hormone (pth), ca, p, and pre and post-dialysis urea, were used to evaluate the correlation between bone and mineral metabolism, which is commonly altered in this group of patients, and the clinical and radiographic alterations observed in this study. since pth concentration indicates the presence of bone disease, the patients undergoing dialysis were divided into three groups according to pth levels: group 1: 0 to 149 pg/ml (low turnover bone disease); group 2: 150 to 584 pg/ml (absence of bone disease); group 3: > 585 pg/ml (high turnover bone disease). the ca x p product was calculated for each patient and the sample was divided into two groups according to the risk of extraskeletal metastatic calcifications: group 1 (ca x p < 55 mg/dl) and group 2 (ca x p > 55 mg/ dl). in addition, the urea reduction ratio (urr) was obtained for each participant to evaluate the efficiency of dialysis. a urr > 65% was considered to indicate adequate dialysis. for determination of the effect of dialysis duration on dmft, ohi-s, psr, radiographic bone alterations, dental calculus, ca x p product, and pth, the patients undergoing dialysis were divided into two groups: group 1 consisting of patients undergoing dialysis for <5 years, and group 2 consisting of patients undergoing dialysis for >5 years. the data were entered into microsoft excel spreadsheets and analyzed using the spss 17.0 software (spss inc., chicago, il, usa). variables are expressed as absolute (n) and relative (%) frequencies and as means. the kolmogorovsmirnov test was used to determine whether numerical variables showed a normal distribution. differences between normally distributed quantitative variables were evaluated by the student t-test. associations between qualitative variables were tested by the chi-squared test or fisher’s exact test. mean and median biochemical parameters were compared between groups using the nonparametric kruskal-wallis test for pth and the mann-whitney test for urr and ca x p product. a level of significance of 5% (0.05) was adopted. results the mean age was 41.8 years. patients aged 36 to 45 years were the predominant age group, corresponding to 22.7%. most patients were males (55.8%) and 46.1% were caucasian. hypertension (43.50%) was the most common causes of ckd, followed by unspecified chronic glomerulonephritis (9.74%). hemodialysis was the most frequent type of dialysis (92.2%) and 94 (61%) patients with ckd had started treatment at least 5 years earlier. sixteen (10.4%) of the 154 variables location of oral soft tissue alterations oral soft tissue alterations tooth alterations radiographic alterations jugal mucosa hard palate lip petechiae candidiasis nicotine stomatitis actinic cheilitis hemangioma fibroma fibrous hyperplasia attrition abrasion mobility erosion stains hypoplasia calculus tooth decay horizontal bone loss vertical bone loss residual roots impacted teeth diffuse bone rarefaction circumscribed bone rarefaction n (%) 16 (10.4) 6 (3.9) 6 (3.9) 13 (8.5) 4 (2.6) 2 (1.3) 1 (0.6) 2 (1.3) 2 (1.3) 2 (1.3) 48 (48.5) 23 (23.2) 15 (15.2) 6 (6.1) 5 (5.1) 2 (1.3) 109 (70.8) 96 (62.3) 96 (62.3) 61 (39.6) 46 (29.9) 30 (19.5) 28 (18.2) 9 (5.8) table 1. soft and hard tissue alterations in the oral cavity and radiographic findings in patients undergoing dialysis. patients were completely edentulous and 59 (38.3%) used some type of denture. with respect to oral hygiene habits, 53.9% of the patients brushed their teeth three times per day; however, only 24.7% used dental floss regularly. at the time of the present study, 44.8% of the participants reported to have visited the dentist in the previous year. with respect to the use of vitamins and medications for the control of plasma calcium and potassium levels, the patients also reported the use of other drugs, including antihypertensives (55.8%), hypoglycemics (5.8%), antiulcer drugs (14.3%), and anxiolytics (5.2%). the most frequent oral clinical finding was coated tongue (31.2%), which is generally associated with inadequate hygiene. other changes observed in soft tissues were petechiae (8.5%), candidiasis (2.6%), nicotine stomatitis (1.3%), hemangioma (1.3%), fibroma (1.3%), fibrous hyperplasia (1.3%), and actinic cheilitis (0.6%). xerostomia was reported by 42.9% of the patients and altered taste by 37%. attrition was the most common dental problem (48.5%), followed by abrasion (23.2%) and tooth mobility (15.2%). bruxism was present in 21 (13.6%) patients. the most frequent radiographic alteration was dental calculus, observed in 109 (70.8%) patients. other findings were dental caries (62.3%), horizontal bone loss (62.3%), vertical bone loss (39.6%), residual roots (29.9%), lost teeth (85.1%), and impacted teeth (19.5%). signs of pulp necrosis, such as diffuse bone rarefaction were observed in 28 (18.2%) patients and circumscribed bone rarefaction in 9 (5.8%) (table 1). one patient had a brown tumor. vertical and horizontal bone loss was significantly correlated with age (p = 0.004 influence of dialysis duration and parathyroid hormone on the clinical and radiographic oral conditions of pre-transplant patients with chronic kidney disease braz j oral sci. 12(2):125-131 128128128128128 and p < 0.001, respectively). analysis of oral hygiene conditions in dentate patients using the ohi-s showed deficient hygiene in 36.8% of these patients. evaluation of the need for periodontal treatment revealed a psr score of 2 in 72.3% of the patients. these results indicate the presence of supraor subgingival calculus and bleeding on probing. psr was not performed in one patient and the ohi-s was not obtained in five because they did not attend the scheduled exam. the chi-squared and fisher’s exact tests revealed no significant association of dialysis duration, ohi-s, psr, pth or ca x p product with radiographic bone alterations (vertical and horizontal bone resorption, dental calculus, diffuse and circumscribed bone rarefaction, bone sclerosis, and bone repair) (table 2). the mean dmft was 17.52 (d = 2.29, m = 11.87, and f = 3.36). analysis of dmft according to the duration of dialysis showed a mean (± standard deviation) index of 16.8 (± 9.8) in the group < 5 years of dialysis and of 18.5 (± 8.1) in the group > 5 years of dialysis, but the difference was not significant (p = 0.268, student t-test). there was no significant association between the three pth ranges and radiographic bone alterations (calculus, circumscribed and diffuse bone rarefaction, sclerosis, bone repair, horizontal and vertical bone loss, and periapical lesion). in addition, no association was observed between this biochemical parameter and the calculus component of the ohi-s (table 3). the latter was also not significantly associated with the ca x p product (p=0.404), or with any of the bone alterations apparent on panoramic radiographs. the frequency of calculus demonstrated by radiography was higher in patients with a urr <65%, but this difference was not statistically significant (p=0.579). none of the biochemical parameters studied was correlated with periodontal disease based on psr score. discussion the therapeutic modalities for patients with ckd such as dialysis increase the life expectancy of this population. however, patients undergoing dialysis are more susceptible to infections due to their state of immunodeficiency. these patients, particularly transplant candidates, should therefore be submitted to careful oral examination to eliminate any source of infection. in the present study, 154 pre-transplant patients with ckd undergoing dialysis were examined to identify oral conditions associated with the disease. in addition, the influence of dialysis duration and bone and mineral metabolism on the oral manifestations of these patients was evaluated. descriptive analysis showed that coated tongue was the most common oral clinical finding in soft tissues, a condition generally associated with inadequate hygiene. in addition, there was a high prevalence of dry mouth, a finding that might be related to the restricted intake of fluids by patients undergoing dialysis or to the use of medications. similar results have been reported by dirschnabel et al.7. petechiae were observed in 16.4% of the sample and might be related table 2. comparison of ohi-s, psr, dmft, biochemical parameters (pth, ca x p product, pre-dialysis urea), and radiographic findings (diffuse and circumscribed bone rarefaction, sclerosis, bone repair, dental calculus, vertical and horizontal bone loss) between patients undergoing dialysis for < 5 years and >5 years. ohi-s satisfactory 10 (7.5%) 7 (5.3%) 0.9101 regular 24 (18%) 15 (11.3%) deficient 30 (22.6%) 19 (14.3%) very poor 15 (11.3%) 13 (9.8%) psr grade 1 5 (3.6%) 2 (1.5%) 0.7801 grade 2 61 (44.5%) 39 (28.5%) grade 3 13 (9.5%) 12 (8.8%) grade 4 3 (2.2%) 2 (1.5%) dmft total 16.8 ± 9.8 18.5 ± 8.1 0.2682 pth 0 a 149 27 (17.8%) 20 (13.2%) 0.7331 150 a 584 42 (27.6%) 23 (15.1%) 585 + 24 (15.8%) 16 (10.5%) ca x p < 55 54 (35.1%) 36 (23.4%) 0.7541 > 55 40 (26%) 24 (15.6%) u r r < 65 % 21 (13.8%) 11 (7.2%) 0,6201 > 65 % 73 (48%) 47 (30.9%) pre-dialysis urea 15 a 100 3 (2%) 0 (0%) 0.2291 101 a 200 82 (53.9%) 49 (32.2%) > 200 9 (5.9%) 9 (5.9%) diffuse bone rarefaction yes 15 (9.7%) 13 (8.4%) 0.3701 no 79 (51.3%) 47 (30.5%) circumscribed bone rarefaction yes 6 (3.9%) 3 (1.9%) 0.5083 no 88 (57.1%) 57 (37.0%) bone sclerosis yes 1 (0.6%) 1 (0.6%) 0.6293 no 93 (60.4%) 59 (38.3%) bone repair yes 11 (7.1%) 12 (7.8%) 0.1591 no 83 (53.9%) 48 (31.2%) vertical bone loss yes 34 (22.1%) 27 (17.5%) 0.2751 no 60 (39.0%) 33 (21.4%) horizontal bone loss yes 55 (35.7%) 41 (26.6%) 0.2201 no 39 (25.3%) 19 (12.3%) dental calculus yes 68 (44.2%) 41 (26.6%) 0.5941 no 26 (16.9%) 19 (12.3%) < 5 years > 5 years time of dialysis p value 1. p value of chi-square test. 2. p value of the student’s t test. 3. p value of fischer’s exact test. influence of dialysis duration and parathyroid hormone on the clinical and radiographic oral conditions of pre-transplant patients with chronic kidney disease braz j oral sci. 12(2):125-131 129129129129129 pth1 p 0 a 149 150 a 584 >585 diffuse bone rarefaction yes 13 (8.6%) 8 (5.3%) 6 (3.9%) 0.0962 no 34 (22.4%) 57 (37.5%) 34 (22.4%) circumscribed bone rarefaction yes 3 (2.0%) 5 (3.3%) 1 (0.7%) 0.5423 no 44 (28.9%) 60 (39.5%) 39 (25.7%) bone sclerosis yes 1 (0.7%) 0 (0%) 1 (0.7%) 0.4643 no 46 (30.3%) 65 (42.8%) 39 (25.7%) bone repair yes 6 (3.9%) 12 (7.9%) 4 (2.6%) 0.4513 no 41 (27.0%) 53 (34.9%) 36 (23.7%) vertical bone loss yes 19 (12.5%) 27 (17.8%) 14 (9.2%) 0.7912 no 28 (18.4%) 38 (25.0%) 26 (17.1%) horizontal bone loss yes 32 (21.1%) 35 (23.0%) 27 (17.8%) 0.2142 no 15 (9.9%) 30 (19.7%) 13 (8.6%) calculus yes 36 (23.7%) 41 (27.0%) 30 (19.7%) 0.2292 no 11 (7.2%) 24 (15.8%) 10 (6.6%) psr grade 1 2 (1.5%) 4 (3.0%) 1 (0.7%) 0.6462 grade 2 31 (21.0%) 44 (32.6%) 24 (17.8%) grade 3 7 (5.2%) 8 (5.9%) 10 (7.4%) grade 4 2 (1.5%) 1 (0.7%) 1 (0.7%) ohi-s calculus 0.82 ± 0.6 0.83 ± 0.65 0.82 ± 0.6 0.9664 group 1: 0 to 149 pg/ml (low turnover bone disease); group 2: 150 to 584 pg/ml (absence of bone disease); group 3: > 585 pg/ ml (high turnover bone disease). 1. p value of chi-square. 2. p value of fischer’s exact test. 3. p value of the kruskal-wallis. table 3. absolute and relative frequencies of the main radiographic findings, psr score and calculus component of the ohi-s according to parathyroid hormone levels. to the use of heparin during the hd sessions. halitosis and altered taste can be the result of xerostomia and the presence of oral microorganisms that metabolize urea (found at high levels in the saliva of these patients) and produce ammonia. about one-third of patients undergoing dialysis complain of a bad taste in their mouth2,6 in agreement with the present study in which 37% of the sample reported this symptom. attrition was the most common dental problem. according to klassen and krasco16 this condition is secondary to aging or xerostomia. most patients studied here presented poor oral hygiene conditions. souza3 and gürkan et al.4 suggested that some oral problems may occur due to the negligence of preventive health measures since the patients are more concerned with the treatment of ckd. however, since the present patients reported a good frequency of daily tooth brushing, the precarious oral conditions might have been due to the lack of knowledge about adequate brushing techniques. in addition, most patients did not use dental floss, a fact favoring biofilm accumulation on the interproximal surfaces of teeth. the ohi-s also indicated the abundant presence of calculus in these patients, a fact contributing to the high scores observed. a high concentration of salivary urea can influence the formation of calculus3,6. other factors that favor the development of calculus in these patients are the intake of calcium supplements, hyperparathyroidism, and inadequate hygiene17. although almost half the sample (44.8%) had seen a dentist during the year prior to the study, this fact did not improve oral hygiene since this care focused on isolated treatment and not on prevention. these results highlight the need to invest in oral health promotion programs for this population. a high dmft index (17.52) was found in the sample studied. similar results have been reported by other investigators6,17 who observed no significant differences in dmft indices between patients with ckd and control groups. the high dmft index observed in the present study was mainly due to the large number of missing teeth (11.87) when compared to decayed (2.29) and filled teeth (3.36). according to dias et al.18 these findings are not directly related to kidney disease, but rather to the socioeconomic conditions of the patients and limited access to preventive/ curative treatment. as a consequence, the patient only sought influence of dialysis duration and parathyroid hormone on the clinical and radiographic oral conditions of pre-transplant patients with chronic kidney disease 130130130130130 braz j oral sci. 12(2):125-131 treatment when experiencing pain and tooth extraction was frequently required in these cases. with respect to periodontal disease, chamani et al.12 observed some degree of gingival inflammation in all patients studied, in agreement with the present investigation in which 100% of dentate patients had gingivitis, with a predominance of psr score 2 (72.3%). periodontal disease is highly prevalent and severe in patients with kidney failure. prophylaxis and early dental treatment should be reinforced and may have a positive impact on the general health status of the patient19. the investigation of sources of infection was complemented by panoramic radiography, which permitted the visualization of the teeth and their supporting bone structures. studies using radiographic analysis for ckd patients undergoing dialysis are scarce in the literature. in the present study, calculus was the most common radiographic alteration, followed by dental caries. thorman et al. 20 compared the panoramic radiographs of 93 patients with ckd (pre-dialysis, hd, and capd) with a control group and found a higher incidence of periapical infections in the first three groups. these results highlight the importance of radiographic investigation in all pre-transplant patients undergoing dialysis since they may present inflammatory lesions of the teeth that cannot be detected by clinical examination alone. pth concentration can be used as a biochemical marker of bone remodeling. an increase of this hormone occurs in response to altered blood levels of ca and p in the organism caused by the loss of renal function. this condition, in turn, leads to secondary hyperparathyroidism, which promotes the development of high turnover bone disease. on the other hand, renal osteodystrophy can manifest as a low turnover bone disease associated with low pth levels, particularly in diabetic patients21. in the present study, no correlation was observed between blood pth levels and bone alterations observed on panoramic radiographs in any of the three groups studied. frankenthal et al.14 also found no influence of secondary hyperparathyroidism on alveolar bone loss or periodontal indices in 35 patients with ckd undergoing dialysis. in addition, urr or ca x p product was not associated with calculus accumulation, periodontal disease or bone loss. these data indicate that bone and mineral metabolism, which is frequently altered in patients undergoing dialysis, did not influence the oral alterations observed in this study. analysis of the effect of dialysis duration on the oral conditions of patients with ckd showed no significant difference in psr, ohi-s or dmft between the two groups studied (< 5 years and > 5 years). these results agree with other investigators who suggested that the duration of dialysis exerts no additional effect on dental or periodontal conditions in patients with ckd12. in contrast, some studies reported a higher accumulation of biofilm and calculus and a high degree of gingival bleeding in long-term patients undergoing dialysis 9,11,13. bone and mineral metabolism evaluated based on blood pth concentrations and ca x p product was also not influenced by the duration of dialysis. no significant difference in radiographic bone alterations was observed between the two groups (<5 years and >5 years). we found no studies in the literature investigating the influence of dialysis duration on oral conditions detected by panoramic radiography. most patients with ckd undergoing dialysis presented poor oral hygiene, calculus accumulation and gingival inflammation, which represent sources of infection. however, the duration of dialysis was not associated with poor oral conditions. the state of bone or mineral metabolism also did not influence oral health manifestations in the patients studied. taken together, the results indicate the need for oral health care in these patients as an important part of the kidney transplant preparation protocol. acknowledgements the authors thank the foundation for research support of the state of rio grande do norte, brazil, for financial support. references 1. marakoglu i, gursoy uk, demirer s, sezer h. periodontal status of chronic renal failure patients receiving hemodialysis. yonsei med j. 2003; 44: 648-52. 2. proctor r, kumar n, stein a, moles d, porter s. oral and dental aspects of chronic renal failure. j dent res. 2005; 84: 199-208. 3. souza crd, libério sa, guerra rnm, monteiro s, silveira ejd, pereira ala. evaluation of periodontal condition of kidney patients on dialysis. rev assoc med bras. 2005; 51: 285-9. 4. gürkan a, köse t, atilla g. oral health status and oral hygiene habits of an adult turkish population on dialysis. oral health prev dent. 2008; 6: 37-43. 5. carvalho aa, farsura pp, bastos mg, vilela em. influence of nonsurgical periodontal treatment on hematological and biochemical parameters of patients with chronic renal failure in pre-dialysis. periodontia. 2011; 21: 27-33. 6. bots cp, poorterman jhg, brand hs, kalsbeek h, van amerongen bm, veerman ec et al. the oral health status of dentate patients with chronic renal failure undergoing dialysis therapy. oral dis. 2006; 12: 176-80. 7. dirschnabel aj, martins as, dantas sag, ribas mde o, grégio am, alanis lr et al. clinical oral findings in dialysis and kidney-transplant patients. quintessence int. 2011; 42: 127-33. 8. kho h, lee s, sung-chang chung s, kim y. oral manifestations and salivary flow rate, ph, and buffer capacity in patients with end-stage renal disease undergoing hemodialysis. oral surg oral med oral pathol oral radiol endod. 1999; 88: 316-9. 9. bayraktar g, kurtulus i, duraduryan a, cintan s, kazancioglu r, yildiz a et al. dental and periodontal findings in hemodialysis patients. oral dis. 2007; 13: 393-7. 10. bayraktar g, kurtulus i, kazancioglu r, bayramgurler i, cintan s, bural c et al. evaluation of periodontal parameters in patients undergoing peritoneal dialysis or hemodialysis. oral dis. 2008; 14: 185-9. 11. cengiz mi, sümer p, cengiz s, yavuz u. the effect of the duration of the dialysis in hemodialysis patients on dental and periodontal findings. oral dis. 2009; 15: 336-41. 12. chamani g, zarei mr, radvar m, rashidfarrokhi f, razazpour f. oral health status of dialysis patients based on their renal dialysis history in kerman, iran. oral health prev dent. 2009; 7: 269-75. 13. sekiguchi rt, pannuti cm, silva jr ht, medina-pestana jo, romito ga. decrease in oral health may be associated with length of time since beginning dialysis. spec care dent. 2012; 32: 6-10. influence of dialysis duration and parathyroid hormone on the clinical and radiographic oral conditions of pre-transplant patients with chronic kidney disease braz j oral sci. 12(2):125-131 14. frankenthal s, nakhoul f, machtei ee, green j, ardekian l, laufer d et al. the effect of secondary hyperparathyroidism and hemodialysis therapy on alveolar bone and periodontium. j clin periodontol. 2002; 29: 479-83. 15. greene jc, vermillion jr. the simplified oral hygiene index. j am dent assoc. 1964; 68: 7-13. 16. klassen jt, krasko bm. the dental health status of dialysis patients. j can dent assoc. 2002; 68: 34-8. 17. souza cm, braosi apr, luczyszyn sm, casagrande rw, pecoitsfilho r, riella mc et al. oral health in brazilian patients with chronic renal disease. rev med chil. 2008; 136: 741-6. 18. dias crs, sá tcv, pereira ala, alves cmc. evaluation of oral condition in chronic renal patients undergoing hemodialysis. rev assoc med bras. 2007; 53: 510-4. 19. borawski j, wilczyñska-borawska m, stokowska w, myœliwiec m. the periodontal status of pre-dialysis chronic kidney disease and maintenance dialysis patients. nephrol dial transplant. 2007; 22: 457-64. 20. thorman r, neovius m, hylander b. clinical findings in oral health during progression of chronic kidney disease to end-stage renal disease in a swedish population. scand j urol nephrol. 2009; 43: 154-9. 21. hamdy nat. calcium and bone metabolism preand post-kidney transplantation. endocrinol metab clin n am. 2007; 36: 923-35. 131131131131131 influence of dialysis duration and parathyroid hormone on the clinical and radiographic oral conditions of pre-transplant patients with chronic kidney disease oral sciences n3 braz j oral sci. 13(1):76-82 original article braz j oral sci. january | march 2014 volume 13, number 1 evaluation of health parameters, use of drugs, and alcohol intake among an elderly population in são josé dos campos, sp, brazil simone bernardes de paula1, patricia fernanda braga mendonça1, mateus bertolini fernandes dos santos2, jarbas francisco fernades dos santos1,3, leonardo marchini4 1universidade do vale do paraíba univap, school of dentistry, area of prosthodontics, são josé dos campos, sp, brasil 2universidade estadual de campinas unicamp, piracicaba dental school, department of prosthodontics and periodontics, piracicaba, sp, brasil 3universidade de taubaté unitau, department of dentistry, area of prosthodontics, taubaté, sp, brasil 4university of iowa, college of dentistry, department of preventive and community dentistry, iowa city, ia, usa correspondence to: mateus bertolini fernandes dos santos departamento de prótese e periodontia faculdade de odontologia de piracicaba unicamp av. limeira, 901 cep 13414-903 piracicaba, sp, brazil phone: +55 19 2106-5297 e-mail: mateusbertolini@yahoo.com.br received for publication: february 04, 2014 accepted: march 24, 2014 abstract aim: to evaluate health parameters (blood pressure, heart rate and blood glucose), the use of medicines and alcohol consumption in elderly residents in the city of são josé dos campos, sp, brazil. methods: a sample of 500 elderly individuals (98 men and 402 women, with mean age of 69.5 years) was examined. personal data and medicines used by the patients were recorded, general health aspects were assessed and the alcohol use disorders identification test (audit) was applied to survey alcohol consumption. results: two hundred and four patients (40.8%) presented high blood pressure and 93 patients (18.6%) had hyperglycemia. the most used pharmacological groups were antihypertensive drugs, antilipidemics, drugs to control hypothyroidism and hypoglycemic agents. possible pharmacological interactions with alcohol were present in 60.9% of the used medicines. the audit results showed that 91% of the sample presented a low-level intake of alcohol and only 1% presented characteristics of alcohol addiction. associations were found among audit scores with age (p=0.037), since a larger number of alcohol addicts were found among younger patients, and use of drugs (p=0.046), since patients who consumed more daily medicines made less use of alcohol (low-level). conclusions: the studied sample presented a high prevalence of hypertension and relatively low incidence of diabetes. a low use of drugs was verified and women made more use of daily medications than men. low alcohol consumption was also observed, and women consumed less alcohol than men. keywords: medicines; drugs; alcohol; elderly; general health. introduction the whole world is experiencing a growth in the elderly population. a demographic study in brazil showed that in 10 years (1998-2008) the elderly increased from 8.8% to 11.1% of the entire population1, totalizing approximately 21 million people, which is more than the number of elderly in the united kingdom, france, and italy. alcohol abuse among the elderly has been called an “invisible epidemic” because it often does not follow stereotypes and therefore goes unnoticed. as the elderly population continues to increase, there is a growing need to re-evaluate the problem of alcoholism in this group of the population2. abusive intake of gisele higa texto digitado http://dx.doi.org/10.1590/1677-3225v13n1a15 17177 braz j oral sci. 13(1):76-82 alcohol by the elderly can have serious health consequences, undermining the length and quality of their lives3. alcohol addiction prevalence is currently lower in people over 65 years compared to other age groups, but there is some evidence of a growing proportion of elderly people who use alcohol at inappropriate levels. because older people are more vulnerable to alcohol’s effects, the current definitions of abuse and alcohol dependence may also be more stringent for this population4. although alcohol-related problems are common among the elderly, physicians almost never recognize them early because of the difficulty in making early diagnoses, since the problems related to alcohol are not usually addressed in elderly patients5, especially in women6. however, the elderly are particularly prone to the adverse consequences of alcohol use, such as depression, sleep disorders, gastritis, hypertension, problems with heart rate, diabetes, falls, bradycardia and arrhythmias. this is due to functional decreases caused by age-related physiological changes in the distribution of alcohol, the effects of alcohol on the central nervous system and the increased use of medications associated with age7-8. furthermore, the concomitant use of drugs and alcohol in the elderly may decrease the effectiveness of medications, increase the incidence of undesirable drug side effects7,9, and also affect negatively the elderly’s general health. in view of this, the aim of this study was to evaluate some health parameters (blood pressure, heart rate and blood glucose), the use of medicines, and alcohol use in an elderly sample of residents of the city of são josé dos campos, sp, brazil. the tested hypothesis was that the use of drugs among this population is low10, but an important impact on general health parameters might be found among alcohol abusers. material and methods participants considering a total elderly population of 40,524 individuals in são josé dos campos, a sample of 500 patients was included in the study, which has 86.64% power to represent the target population (minitab 15 software package). the individuals were recruited among the community centers for the independent elderly registered in the municipal committee for the elderly, until the desired sample size was achieved. the participants signed an informed consent form approved by the university of vale do paraíba ethics committee (protocol #h105/cep/2010). evaluation of personal data, general health, and drugs administration personal data of the patients, such as name, age, sex, and education level (divided into illiterate, primary school, high school, college and post-graduation, complete or incomplete) were registered during initial interviews. for general health evaluation, blood pressure and glucose were checked; 140 mg/dl was considered the normal value of postprandial blood glucose, according to the american diabetes association11, and 120/80 mmhg the normal value for blood pressure. a single professional made all examinations following a standard procedure. the examinations and interviews take place at the community center where the participants were recruited. drugs used by the patients were also recorded, and the names, frequency of intake and possible pharmacological interactions with alcohol were evaluated. alcohol intake evaluation for such evaluation, the alcohol use disorders identification test (audit) was applied to the patients. this test was designed by the world health organization to identify disorders caused by alcohol abuse in facilities for primary health care4, and it has been shown to be valid in the detection of dangerous alcohol use in large-scale studies with active adults and elderly patients12-13. a brazilianportuguese version of the test had been previously validated10,14 and was used in this study. all interviews were conducted by a single operator, who had been previously calibrated for interviewing and completing the questionnaire forms. data analysis the collected data were tabulated and descriptive analysis was made. parametric tests were used due to normal distribution of the data (kolmogorov-smirnov test). one-way anova was used for intergroup comparisons and the chisquared test was used for verification of possible associations. the significance level was set at 5% pharmacological groups possible interactions with alcohol antibiotics – metronidazol vomit, palpitations, low blood pressure, breathing difficulty antibiotics – eritromicine liver damage, vomit, palpitations, low blood pressure, breathing difficulty, inhibits the effect of the drug anticonvulsants breathing difficulty, inhibits the effect of the drug, dizziness, intoxication antidepressives inhibits the effect of the drug, increases the sedative effect antifungals liver damage antihypertensives dizziness, fainting, cardiac arrhythmia antiarrhythmics further depresses normal heart function anxiolytics i nhibits the effect of the drug, increases the sedative effect, risk of coma cholesterol control drugs liver damage, stomach bleeding insulin vomit, palpitations, headache, hypoglycemia chart 1 chart 1 chart 1 chart 1 chart 1 – possible interactions of different pharmacological groups with alcohol. evaluation of health parameters, use of drugs, and alcohol intake among an elderly population in são josé dos campos, sp, brazil pharmacological group n % antihypertensives 416 37.48% antilipidemics 106 9.54% hypothyroidism control drugs 86 7.75% hypoglycemiant drugs 67 6.04% osteoporosis control drugs 57 5.14% anticoagulants 50 4.50% anxiolytics 49 4.41% antiulcerous 40 3.60% antiarrhythmic 19 1.71% hypoglycemia control drugs 18 1.62% table 1table 1table 1table 1table 1 – distribution of the most used pharmacological groups. audit average standard n confidence p-value deviation interval age low risk 69.6 6.6 455 0.6 0.037* addiction 62.6 2.4 5 2.1 harmful abuse 68.5 6.3 39 2.0 blood glucose low risk 120.5 35.7 455 3.3 0.534 addiction 133.2 46.4 5 40.7 harmful abuse 116.1 22.1 39 6.9 heart rate low risk 71.3 8.2 455 0.8 0.143 addiction 65.8 4.6 5 4.0 harmful abuse 73.0 7.3 39 2.3 use of drugs low risk 2.3 1.9 455 0.2 0.046* addiction 1.6 1.7 5 1.5 harmful abuse 1.5 1.4 39 0.5 table 2 –table 2 –table 2 –table 2 –table 2 – comparison of age, blood glucose, heart rate and use of drugs among audit groups. the asterisk (*) indicates a statistically significant difference (one-way anova). results the sample comprised 98 men and 402 women and the mean age was 69.5 years old, with 101 and 60 being the highest and lowest ages, respectively. as much as 54.2% of the participants had only primary education, 13.6% completed college, 1.2% had post-graduation education and 2.4% were illiterate. two hundred and four individuals (40.8%) presented high blood pressure and 39 (7.8%) hypotension. the mean rate of the patients was 71.4, with 100 and 43 being the highest and lowest values, respectively. with regard to blood glucose, 93 patients (18.6%) presented hyperglycemia and 6 (1.2%) presented hypoglycemia. regarding the use of drugs, the most used pharmacological groups were antihypertensives, antilipidemics, hypothyroidism control drugs, and hypoglycemic agents (table 1). the mean daily drug use was 2.2 and the highest value was 14 drugs taken on a daily basis. the most used drug in this sample was simvastatin (antilipidemic), followed by captopril (antihypertensive). among all the drugs used by the patients, 60.9% presented possible pharmacological interactions with alcohol. when sex was compared regarding the use of drugs, it was found that women consumed more drugs than men (p=0.003). table 1 presents the possible interactions of different pharmacological groups used by the patients in this study and the possible interactions with alcohol. when different blood pressure groups were compared by age, blood glucose levels, heart rate levels and number of drugs used daily, it was observed that patients presenting hypertension also presented higher heart rate (p=0.015). no significant differences were found among different blood pressure groups to the other variables. the audit results showed that 91% of the interviewed elders present a low-level intake of alcohol and only 1% presented characteristics of alcohol addiction. table 2 shows the comparisons of different audit groups regarding age, blood glucose levels, heart rate levels and number of daily used drugs. significant differences among different audit groups regarding the age (p=0.037) were found. a large number of alcohol addicts were found among younger patients and use of drugs (p=0.046), the patients who consumed more medicines daily were those who consumed less alcohol. table 3 presents the associations among sexes and blood pressure, blood glucose, educational level, and audit classifications. an association between sex and education level (p=0.017) was verified, where women predominated among those who had elementary school education and men, among those who with high school education. another association was found between sex and audit classification (p<0.001), where women presented lower scores of alcohol intake than men. no correlation was found between blood pressure and glucose and the audit scores. discussion in this study, a very marked predominance of women (80.4%) was observed. the elderly subjects had a mean age of 69.5 years, with a minimum of 60 and a maximum of 101 years. these results were similar to those described by de deco et al.9, who reported a mean age of 74.3 years and 17178 evaluation of health parameters, use of drugs, and alcohol intake among an elderly population in são josé dos campos, sp, brazil braz j oral sci. 13(1):76-82 17179 women men total p-value n % n % n % blood pressure hypotension 35 9% 4 4% 39 8% 0.293 hypertension 161 40% 43 44% 204 41% normal 206 51% 51 52% 257 51% blood glucose high 75 19% 18 18% 93 19% 0.694 l o w 4 1% 2 2% 6 1% normal 323 80% 78 80% 401 80% education level illiterate 11 3% 1 1% 12 2% 0.017* primary school 231 57% 40 41% 271 54% high school 107 27% 36 37% 143 29% college 48 12% 20 20% 68 14% post-graduation 5 1% 1 1% 6 1% audit low risk 374 93% 81 83% 455 91% <0.001* addiction 1 0% 4 4% 5 1% harmful abuse 26 6% 13 13% 39 8% total 402 100% 98 100% 500 100% table 3 table 3 table 3 table 3 table 3 – association of sexes and blood pressure, blood glucose, educational level and audit groups. the asterisk (*) indicates a statistically significant association (chi-square test). women also accounted for the vast majority, 70.3% of the sample. the american heart association (aha) considers as normal blood pressure when systolic/diastolic pressure is 120/ 80 mmhg and persons presenting blood pressure of 140/90 mmhg are considered to be hypertensive, although one may argue about the fact that elderly persons may have different parameters in blood pressure than the aha recommends. however, the blood pressure uk, formerly known as blood pressure association, suggests that the level for high blood pressure does not change with age 15. based on these parameters, in the studied sample, 51.4% of the elders presented normal blood pressure values, 40.8% were hypertensive, and 7.8% presented hypotension. in campinas, sp, brazil, zeitune et al.16 found a hypertension prevalence of 51.8% among 426 adults aged 60 years or older, with higher incidence in women (55.9%). in japan, blood pressure was measured in 499 elderly community dwellers, with a mean age of 80 years, and mean systolic and diastolic blood pressures were 149.6 mmhg and 78.5 mmhg respectively17. de deco et al.9 showed a slightly higher systolic pressure in the group of institutionalized elders compared with community dwellers, and diastolic blood pressure was higher in the community dwellers. hypertension is considered an aggravating factor in health and may lead to higher morbidity and mortality, mainly linked to cardio-vascular disorders (such as heart attacks and strokes). thus, control of blood pressure should be a goal to be pursued by all health professionals. it is important to mention that hormones play an important role in hypertension. the renin-angiotensinaldosterone axis controls the sodium and potassium balance and arterial blood pressure. the atrial hormone is also reported to counter-influence the renin-angiotensin-aldosterone axis in situations involving high blood pressure or sodium surfeit18. however, more studies are required to further elucidate the hypertension etiopathogenesis. heart rate is expressed as the number of heart beats per minute. problems with heart rhythm and rate are quite common in the elderly. low heart rate (bradycardia) and arrhythmias such as atrial fibrillation are also common. the results of this study showed an average of 71.4 bpm, with maximum and lowest values of 100 and 43, respectively. the results of this study are in agreement with those of previous studies, ranging from 60 to 100 bpm at rest9 and an average of 69.6 bpm17. no correlation was observed between blood pressure and audit scores, which differs from a classic study19 that evaluated the influence of alcohol consumption and blood pressure on 83,947 adult patients and found that regular use of three or more drinks of alcohol per day should be considered a risk factor for hypertension due to its increase in systolic and diastolic pressures as well as higher prevalence of pressures higher than 160/95 mmhg. this discrepancy between the results presented here and those from klatsky et al.19 is probably due to the smaller sample size in the present study, as the number of participants showing characteristics of alcohol addiction was very small, weakening the statistical power to detect a possible correlation between blood pressure and audit scores. the concepts proposed by the american diabetes association were used to classify the obtained levels of blood glucose. two hours after meals, normal blood glucose should be less than 140 mg/dl11, and higher values are indicative of diabetes. using these parameters, it was found that 80.2% of the subjects had levels within the normal range of blood glucose and 18.6% had levels indicative of diabetes. de deco et al.9 found 32% of diabetes among institutionalized elderly and 30% among community-dwelling elderly. the lower incidence of diabetes found in this study can be attributed to health programs targeted at the elderly, using both controlled diet and specific medication. furthermore, no correlation was observed between blood glucose with evaluation of health parameters, use of drugs, and alcohol intake among an elderly population in são josé dos campos, sp, brazil braz j oral sci. 13(1):76-82 17180 audit scores. as mentioned above, the number of participants showing characteristics of alcohol addiction in the present study was very small, weakening the statistical power to detect a possible correlation between blood glucose and audit scores. further studies should also test the possible influence of eating habits and even oral health, which interferes on the chewing pattern, on diabetes prevalence. the high prevalence of hypertension in this study, despite the high use of antihypertensive drugs is probably due to the low adhesion of patients to the anti-hypertensive treatment (patients do not take their medications as they should and/ or do not honor recall appointments as they should). thus the observed hypertension levels were high, despite the patients’ claim to use the anti-hypertensive medicines. it should signal red to the dentist when the patient tells that he/she has controlled hypertension. the dentists do better double-checking though. regarding the diabetes, prevalence and use of medicines were low in this group, which indicates a greater participation of this group of elderly individuals in the treatment of diabetes. it may be due to obesity control and personal appearance, which is increasingly prized by aged people, especially in this group, which leads a fairly active social life. the most used drugs by the elderly were the ones to control hypertension (37.48%), cholesterol (9.54%), hormone replacement therapy or hormone supplementation in patients with hypothyroidism (7.75%), and hypoglycemic drugs (6.04%), with an average of 2.2 medications used daily. similar results were found by de deco et al.9, who found an average of 1.78 medicines for non-institutionalized elderly. in other countries, such as finland, it was verified that elders use more drugs on a daily basis (7.9 drugs)20 than they do in brazil. the relatively low use of drugs among the elderly population should be seen as a positive fact, since a good control of health parameters is achieved, because it reduces the risk of iatrogenic effects of drugs and drug interactions. however, it may also be caused by the low income of the elderly in brazil, because cost plays an important role in the choice of drugs8. some of the most used drugs in the present study, such as simvastatin, captopril and insulin, have government subsidies and are purchased by low-income populations at almost 10% of the normal over the counter cost, since they are considered as essential drugs by the brazilian ministry of health8. the relatively low use of drugs among this elderly population may also be related with the absence of regular recall examinations, which may lead to a lower rate of diagnoses, leading to a lower use of drugs. the absence of regular recall examinations is a chronic problem in the brazilian health system, as access to physicians is not universal. it is important to consider that a low medication intake may affect the control of several health parameters, which may negatively influence the general health of the patients. however, a limitation of this study is related to the absence of drug dosage verification, and without dosage records it is not possible to check for problems related to this variable. it is also important to consider that the low medication intake may affect the control of several health parameters, which may negatively influence the general health of the patients. women presented an average use of 2.3 medicines on a daily basis, while men used 1.7. these results were expected, because women visit medical clinics and seek to prevent diseases more often than men, showing more concern about health, while men mostly seek medical advice only when they are already experiencing a health problem. the audit allowed evaluating the use of alcohol in this elderly sample. it can be argued that specific tests such as audit have a better performance in the elderly compared to young people21, and audit seems to be a reliable method to identify alcohol-related problems and assist in planning appropriate services for the treatment and prevention of alcoholism4. to calculate the frequency of alcohol intake, scores were assigned to each question, and after that the final score was obtained. questions 1 to 8 covered aspects such as quantity, frequency and the way the person usually ingest alcohol. the answer scores ranged from 0 to 4 points and the answers to questions 9 (“have you ever got hurt or hurt someone because you have drunk?”) and 10 (“anytime a relative, friend, doctor, or health care professional expressed concern for your alcohol intake or suggested that you should quit drinking?”) are answered with 0, 2, or 4 points. the results are expressed in values between 0 and 40. scores ranging from 1 to 7 are considered low-level drinkers; from 8 to 19 are considered harmful abuse and scores ranging from 20-40 are considered alcohol addiction. the present results showed a higher prevalence of lowlevel intake of alcohol (91.0%), 7.8% for harmful use / abuse, and only 1.0% of dependency. these results are similar to those of jeong et al.22, who conducted a study involving 997 elderly koreans and observed that only 23% of the sample consumed alcohol. among those who consumed alcohol, 150 met the criteria for social drinking, 46 for atrisk drinking, 17 for alcohol abuse, and 23 for alcohol dependence at the baseline assessment22. in a recent study23, it was verified that only 23.6% in a sample of 169 elderly men from the metropolitan region of são paulo were lifetime abstainers and 38% could be considered alcohol addicts in the past. however, the results of such a study cannot be directly compared to the results of the present study because these authors evaluated alcohol consumption in the past, and the present study used a specific tool to measure the current alcohol intake (audit). the predominance of women in the present sample may also have influenced the results regarding alcohol intake. in this sample, comparing alcohol consumption among sexes women showed lower intake of alcohol compared to men. these results are in agreement with kim et al.24, in which alcohol was reported as a common problem especially among men. a previous study by denneson et al.12 evaluated, by a cross-sectional study, a sample of 1,105 subjects aged 90 years in which 96.19% were men that took part in world war ii. the alcohol intake measurement was also made by evaluation of health parameters, use of drugs, and alcohol intake among an elderly population in são josé dos campos, sp, brazil braz j oral sci. 13(1):76-82 17181 audit and their results showed that 60.3% of the men were abstainers, 36.9% were low-level drinkers, and only 2.8% were mediumor high-level drinkers, while 46.9% of the women were abstainers, 43.6% were low-level drinkers, and the remaining 9.5% were medium-level drinkers. the comparison of these results with the findings of the present study should take into consideration that the sample in this study was mostly composed by women and the average age was 69.5 years old, which differs from the above-mentioned study12. another study22 also affirmed that no women reported high-level alcohol consumption. when the use of medicines and alcohol intake were evaluated, it was possible to verify that patients who took a higher number of drugs made lower use of alcohol, decreasing the risk of alcohol-drug interactions. patients older than 65 years have a significantly increased risk for drug-related problems not only because aging affects how their body handles medications but also because they take more drugs when compared with younger patients8. adverse side effects in older patients are a common cause for hospitalization and an important cause of morbidity and death25. side effects are associated with polypharmacy. carbonin et al.26 suggested that the risk of adverse side effects is rather exponentially than linearly related to the number of medicines taken by the patient. also, the adverse side effects lead to increased expenses for the public health system. the audit commission27 found that each year the nhs (uk) spends approximately £0.5 billion for longer stays in hospitals due to errors in medicines and medication. as raised by the present study hypothesis, the concomitant use of drugs and alcohol by the elderly may decrease the efficacy of the drugs and increase the incidence of undesirable side effects7,9. in this study, it was possible to verify high prevalence of hypertension, a relatively low incidence of diabetes, and low use of drugs, where antihypertensives and antilipidemics were the most used, since they are available for free under governmental assistance8. women use more daily medications than men. low alcohol consumption was also observed, and women consume less alcohol than men. the generalization of the results presented here should be made with caution, since a specific group with some homogeneous variables, such as social status and access to full health services, may not be the reality of the entire population of this city. acknowledgments this study was presented as an oral presentation in the 36th annual conference of the european prosthodontic association, in rotterdam. this study received financial support from a research agency (fapesp 2010/16121-3). references 1. brazilian institute of geography and statistics. national survey by household sample. brasília: ibge; 2008. 2. sorocco kh, ferrell sw. alcohol use among older adults. j gen psychol. 2006; 133: 453-67. 3. culberson jw. alcohol use in the elderly: beyond the cage. part 1 of 2: prevalence and patterns of problem drinking. geriatrics. 2006; 61: 23-7. 4. philpot m, pearson n, petratou v, dayanandan r, silverman m, marshall j. screening for problem drinking in older people referred to a mental health service: a comparison of cage and audit. aging ment health. 2003; 7: 171-5. 5. suwala m, gerstenkorn a. detection of alcohol problems among elderly people. psychiatr pol. 2007; 41: 703-13. 6. sedlak ca, doheny mo, estok pj, zeller ra. alcohol use in women 65 years of age and older. health care women int. 2000; 21: 567-81. 7. moore aa, beck jc, babor tf, hays rd, reuben db. beyond alcoholism: identifying older, at-risk drinkers in primary care. j stud alcohol. 2002; 63: 316-24. 8. marchini amps, de deco cp, silva mrv, lodi kb, rocha rf, marchini l. use of medicines among a brazilian elderly sample: a cross-sectional study. int j gerontol. 2011; 5: 94-7. 9. de deco cp, do santos jf, da cunha v de p, marchini l. general health of elderly institutionalised and community-dwelling brazilians. gerodontology. 2007; 24: 136-42. 10. santos g, barreto e, santos jf, marchini l. alcohol and quality of life among social groups for the elderly in são josé dos campos, brazil. appl res qual life 2013: 1-9. doi: 10.1007/s11482-013-9225-y. 11. american diabetes association. ada live from american diabetes association [internet]. 2011 [accessed 2011 may 2]. available from: http://www.diabetes.org. 12. denneson lm, lasarev mr, dickinson kc, dobscha sk. alcohol consumption and health status in very old veterans. j geriatr psychiatry neurol. 2011; 24: 39-43. 13. saunders jb, aasland og, babor tf, de la fuente jr, grant m. development of the alcohol use disorders identification test (audit): who collaborative project on early detection of persons with harmful alcohol consumption—ii. addiction. 1993; 88: 791-804. 14. méndez eb. a brazilian version of audit alcohol use disorders identification test [mastering thesis]. pelotas: federal university of pelotas; 1999. 15. blood pressure uk. blood pressure chart [internet]. 2008 [accessed 2014 jan 4]. available from: http://www.bloodpressureuk.org/ bloodpressureandyou/thebasics/bloodpressurechart. 16. zeitune mp, barros mb, cesar cl, carandina l, goldbaum m. arterial hypertension in the elderly: prevalence, associated factors and control practices in campinas, são paulo, brazil. cad saude publica. 2006; 22: 285-94. 17. matsumura k, ansai t, awano s, takehara t, abe i, iida m, et al. association of dental status with blood pressure and heart rate in 80-yearold japanese subjects. jpn heart j. 2003; 44: 943-51. 18. laragh jh. atrial natriuretic hormone, the renin-aldosterone axis, and blood pressure-electrolyte homeostasis. n engl j med. 1985; 313: 133040. 19. klatsky al, friedman gd, siegelaub ab, gerard mj. alcohol consumption and blood pressure kaiser-permanente multiphasic health examination data. n engl j med. 1977; 296: 1194-200. 20. hosia-randell hm, muurinen sm, pitkala kh. exposure to potentially inappropriate drugs and drug-drug interactions in elderly nursing home residents in helsinki, finland: a cross-sectional study. drugs aging. 2008; 25: 683-92. 21. berks j, mccormick r. screening for alcohol misuse in elderly primary care patients: a systematic literature review. int psychogeriatr. 2008; 20: 1090-103. 22. jeong hg, kim th, lee jj, lee sb, park jh, huh y, et al. impact of alcohol use on mortality in the elderly: results from the korean longitudinal study on health and aging. drug alcohol depend. 2012; 121: 133-9. 23. de oliveira jb, santos jl, kerr-correa f, simao mo, lima mc. alcohol screening instruments in elderly male: a population-based survey in metropolitan sao paulo, brazil. rev bras psiquiatr. 2011; 33: 347-52. evaluation of health parameters, use of drugs, and alcohol intake among an elderly population in são josé dos campos, sp, brazil braz j oral sci. 13(1):76-82 17182 24. kim kw, choi ea, lee sb, park jh, lee jj, huh y, et al. prevalence and neuropsychiatric comorbidities of alcohol use disorders in an elderly korean population. int j geriatr psychiatry. 2009; 24: 1420-8. 25. routledge pa, o’mahony ms, woodhouse kw. adverse drug reactions in elderly patients. br j clin pharmacol. 2004; 57: 121-6. 26. carbonin p, pahor m, bernabei r, sgadari a. is age an independent risk factor of adverse drug reactions in hospitalized medical patients? j am geriatr soc. 1991; 39: 1093-9. 27. anon a. a spoonful of sugar; medicines management in nhs hospitals. london: audit commission; 2001. evaluation of health parameters, use of drugs, and alcohol intake among an elderly population in são josé dos campos, sp, brazil braz j oral sci. 13(1):76-82 oral sciences n3 original article braz j oral sci. january | march 2013 volume 12, number 1 fracture resistance of endodontically treated teeth restored with different intraradicular posts with different lengths jefferson ricardo pereira1, elias manoel ribeiro neto2, saulo pamato2, accácio lins do valle3, vitor guarçoni de paula4, hugo alberto vidotti4 1department of prosthodontics, university of southern santa catarina (unisul), tubarão, sc, brazil 2department of prosthodontics, university of southern santa catarina (unisul), tubarão, sc, brazil 3department of prosthodontics, university of são paulo (usp), bauru, sp, brazil 4department of prosthodontics, university of são paulo (usp), bauru, sp, brazil correspondence to: jefferson ricardo pereira rua recife, 200 apto 601, cep: 88701-420 bairro recife, tubarão, sc, brasil phone: +55 48 36222894 fax: +55 48 36264088 e-mail: jeffripe@rocketmail.com abstract aim: this study compared the resistance to fracture of endodontically treated teeth restored with different intraradicular posts with different lengths and full coverage metallic crowns. methods: sixty extracted human canine teeth were randomly divided into 6 groups. groups cp5, cp75 and cp10 were restored using custom cast post and core (cp) and groups pf5, pf75 and pf10 were restored with provisional pre-fabricated tin post (pf) and composite resin core at 5 mm, 7.5 mm and 10 mm of intraradicular length, respectively. the specimens were submitted to dynamic cyclic loading and those that resisted to this load were submitted to load compression using a universal testing machine. compressive load was applied at a 45-degree angle to the long axis of the tooth until failure. results: kruskal-wallis one-way analysis of variance by ranks showed statistically significant differences among the groups (p<0.0001). however, when the means were compared using the tukey’s test, significant differences were noted between groups cp5 and cp10 and between groups cp10 and pf5. all groups presented root fractures and post displacements during mechanical cycling. all teeth in groups cp5 and pf5 failed the dynamic cycling test. conclusions: this study showed that increasing intraradicular post length also increases resistance to fracture of endodontically treated teeth. on the other hand, most endodontically treated teeth restored with pre-fabricated tin posts (provisional posts) failed in the dynamic cycling test. keywords: post and core technique, prosthesis failure, mechanical stress. introduction numerous techniques for restoring endodontically treated teeth succeed depending on post length1-2, its surface shape and configuration1-7, amount of remaining dentinal structure3-5, and techniques and materials used for build-ups6-7. restoring endodontically treated teeth is a frequent task for clinicians. devitalized teeth are known to present higher risks of biological (inflammation) and mechanical (root fracture) failure than vital teeth1,8. the generally accepted explanation for this fact is the substantial loss of tooth structure for endodontic access, root canal and post preparation6. posts are necessary to allow for retention received for publication: august 25, 2012 accepted: january 17, 2013 braz j oral sci. 12(1):1-4 22222 for the subsequent coronal restoration9. the price for such an increase in retention, however, may become a risk of further damage to the tooth structure. cast posts have been accepted as the restorative component of choice for endodontically treated teeth when coronal structure is absent. nevertheless, the use of prefabricated posts is growing, since most stages can be concluded at chairside and good prognosis is expected3. some authors3,10 argue that roots restored with cast posts show significantly higher internal tension than the ones with prefabricated posts. post length in relation to the root length is a controversial issue11. with the recent improvements in dentin bonding, good adhesion may positively influence the success of treatment1214. in vitro studies have demonstrated that increasing post length results in better stress distribution along the post2,15-16 and higher resistance to fracture1. furthermore, a clinical study reported a higher survival rate relative to the increase in post length7. nonetheless, other studies have shown minimal difference in stress distribution17 and resistance to fracture18 with the increase in post length. it is important to note that it is not always possible to use a long post, especially when the root is short or curved. several studies have suggested that it is important to preserve 3 to 5 mm of gutta-percha to maintain the quality of the apical seal19. the aim of this study was to evaluate the effect of length and type of prefabricated posts on the resistance to fracture of endodontically treated teeth. the research hypotheses are: 1) there is no difference in tooth fracture resistance between different prefabricated post lengths and 2) a significant difference exists between the types of posts. material and methods sixty freshly extracted human canine teeth were obtained from the university of southern santa catarina (unisul) tooth bank for this study. the inclusion criteria were that all teeth should present similar anatomy and lengths varying between 15 to 18 mm. the teeth were kept in saline at room temperature during the experiments, following the guidelines of unisul dental school’s ethics committee (protocol #10.585.4.02.iii). the teeth were endodontically treated using the crowndown technique and filled by cold lateral condensation. kfiles #20 to #35 (dentsply maillefer, ballaigues, switzerland) were used 1 mm short of the apex. canals were irrigated with 2.5% sodium hypochlorite (asfer industrial química, são paulo, sp, brazil) dried with paper points (tamari; tamariman industrial ltda., macaçaruru, am, brazil). guttapercha points (tamari, tamariman industrial ltda.) and sealer 26 (dentsply ind. e com. ltda., petrópolis, rj, brazil) were used for root canal filling. following the endodontic treatment, the tooth crowns were sectioned perpendicular to their long axes using doublesided diamond disks (kg sorensen, barueri, sp, brazil), leaving a standardized 15 mm root length. the teeth were then mounted in cylinders (30 mm high x 22 mm diameter), leaving 1.5 mm of the root exposed, and were randomly divided into 6 groups (n=10). groups cp5, cp75 and cp10 were restored with custom cast post and core and groups pf5, pf75 and pf10 with prefabricated tin posts (metalpost, angelus, londrina, pr, brazil). in groups cp5 and pf5, the posts were positioned 5 mm into the canal, in groups cp75 and pf75 7.5 mm and in groups cp10 and pf10 10 mm. the length of the post was standardized using a digital caliper. different post preparations were standardized using a #5 reamer (largo; dentsply ind. e com., ltda.). five millimeters of gutta-percha (apical to the cementoenamel junction cej) were removed from each filled canal in groups cp1 and pf1, 7.5 mm in groups cp2 and pf2, and 10 mm in groups cp3 and pf3. for the custom cast post and cores, impressions of the root canal were made using acrylic resin (duralay; reliance dental mfg. co. chicago, il usa). the cores were standardized using preformed acetate matrices (tdv dental, pomerode, sc, brazil), mounted (cristobalite; whip-mix corporation, louisville, ky, usa) and cast in a cu-al alloy (npg aalbadent, cordelio, ca, usa). occasional minor casting imperfections were removed. the post/cores were fitted in their respective teeth. all posts were cemented with glass-ionomer cement (vidrion c; s.s. white artigos dentários, rio de janeiro, rj, brazil) according to the manufacturer’s instructions. the cement was spun into the canals using a lentulo spiral (lentulo; dentsply maillefer, ballaigues, switzerland). the post was coated with cement before insertion into the canal and maintained under 5 kg pressure for 5 min. pressure was removed and the cement was left to set. cement excess was removed and the specimens were placed back in saline. the prefabricated posts were cemented in the same manner as the custom cast post and core. the coronal aspect of the root was etched with 37% phosphoric acid for 30 s, washed for 30 s and gently air thinned. two layers of the adhesive system adapter single bond 2 (3m espe, sumaré, sp, brazil) were applied and light-cured for 20 s each using a curing light system with a 750 mw light intensity (ultradent; dabi atlante, ribeirão preto, sp, brazil). the core was built using the same acetate matrices as per the custom cast post and cores. five increments of composite resin (charisma heraeus kulzer, hanau, germany) were used for core build-up. each increment was light-cured for 40 s (ultraled; dabi atlante) with the light source at a 10 mm distance from the core. all teeth were restored with a full-coverage cast metal crown. silicone impressions were taken from the teeth before preparation (aquasil, dentsply, konstanz, germany) to facilitate the crown wax-up (kerr corporation, ca, usa). a ni-cr alloy (durabond, são paulo, sp, brazil) was used to cast the crowns, which were cemented with glass-ionomer cement (vidrion c; s.s. white artigos dentários, rio de janeiro, rj, brazil). the post-core-crowned teeth were submitted to dynamic load with impact simulation. the tests were carried out with 5 specimens at a time and a frequency of 2 cycles per second, braz j oral sci. 12(1):1-4 fracture resistance of endodontically treated teeth restored with different intraradicular posts with different lengths 33333 groups with same superscripts do not differ significantly (p<0.05) (tukey’s test). cp: custom cast post and core (5, 7.5 and 10 mm); pf: pre-fabricated tin post (5, 7.5 and 10). group median 25% 75% cp5 0.000 ac 0.000 0.000 cp75 8.800 abc 0.000 19.500 cp10 32.100 b 25.400 41.900 pf5 0.000 ac 0.000 0.000 pf75 0.000 abc 0.000 10.800 pf10 0.000 abc 0.000 14.000 table 1. median, 25 percentile and 75 percentile of fracture resistance of the endodontically treated teeth. kruskal-wallis one-way analysis of variance by ranks and tukey’s test for multiple comparisons. depth (mm) event post type custom cast post and core pre-fabricated tin post 5.0 tooth fracture 70% 10% post release 30% 30% post fracture 0% 60% 7.5 tooth fracture 100% 10% post release 0% 40% post fracture 0% 50% 10 tooth fracture 100% 20% post release 0% 10% post fracture 0% 70% table 2. percentage distribution of events according to the type of post placement and depth comparison among groups after dynamic and static load. making up 250,000 thousand cycles with a peak load of 250 n at 37ºc (± 1º c). the load was applied to the palatal surface of the crowns at a 45º angle to the long axis of the tooth. load values were noted at failure, i.e. root or post fracture or crown/post displacement. specimens that did not fail at the dynamic load test were then submitted to progressive static compression testing (kratos, são paulo, sp, brazil) at 0.5 mm/min and 100 kg load cell. compression was applied at a 45º angle to the long axis of the tooth. statistical analysis of the results was performed using kruskal–wallis one-way analysis of variance by ranks and multi-comparison tukey’s test at 5% significance level. results there were statistically significant differences in fracture resistance among the groups (p=0.02) (table 1). significant differences in fracture resistance were found comparing 10mm-long custom cast post and core with 5-mm-long custom cast post and core and 5-mm-long pre-fabricated posts (p<0.05). the fracture patterns for all groups are found in table 2. all teeth that were treated with 5 mm long posts failed during the fatigue test. considering the 7.5 mm long devices, 5 of the custom cast post and core and 6 of the pre-fabricated posts failed during the fatigue test. among the 10 mm long posts, 2 from the custom cast post and core and 7 from the pre-fabricated posts failed before compressive load. twenty-seven failures in the custom cast post and core groups occurred due to root fractures. however, in the provisional prefabricated post groups most failures occurred as a result of post fracture (18) or post displacement8. discussion this study confirms the hypothesis that there is a significant difference in the effect of post length on fracture resistance. similarly the types of post also show significant differences. it was observed that roots restored with a 10 mm custom cast post and core showed a significantly higher resistance to fracture (p<0.05), when compared with shorter posts (5 and 7.5 mm). such findings were also observed by pereira et al.1, standlee et al.2. and holmes et al.16, who reported that an increase in post length resulted in higher resistance to fracture of endodontically treated teeth. this could be explained by the higher resistance of the ni-cr alloy and its higher module of elasticity10 as well as a reduction of the wedge effect generated by shorter posts2,15-16. furthermore, 50% of the 7.5 mm and 100% of the 5 mm custom cast post and cores failed during the mechanical cycling. in vitro studies have demonstrated that longer posts allow for an even stress distribution along their length, whereas shorter posts generate stresses that overload the tooth/post junction resulting in failure2,15-16, which in the case of custom cast post and core means root fracture12. on the other hand, the present study demonstrated that an increase in length of prefabricated tin posts and composite resin cores did not increase the fracture strength in endodontically treated teeth. this could be explained because tin posts are rather malleable and present low hardness, which performs well as a provisional restoration and, because of this, the major failure when they were used was post fracture. the results of this study are in line with the findings of a previous one14 in which the necessary stress that lead to failure of the resin/post combination was lower than the one to cause root fracture. the results of the present study show that most prefabricated posts failed the dynamic loading test due to post fracture or displacement. braz j oral sci. 12(1):1-4 fracture resistance of endodontically treated teeth restored with different intraradicular posts with different lengths 44444 restorative work using prefabricated posts and composite resin is a viable technique for endodontically treated teeth3-6. failures of such restorations during occlusal loads may be considered a positive event because it preserves the remaining root12. however, such failures may occur under greater loads than those found intraorally. in the present study, the use of prefabricated tin posts suggests that their chances of achieving treatment success are limited, because the vast majority failed the mechanical cycling stage and those that did passed the test showed significantly lower results than those expected for the maximum physiological occlusal load19. the material the posts are made from could explain this result. the limitations of this study include its in vitro background, which does not necessarily reflect the oral environment. for more significant results, future studies should incorporate thermocycling that can cause an alternating increase and decrease of deformation between material and tooth structure, thus changing the results. references 1. pereira jr, valle al, juvêncio tm, fernandes tmf, ghizoni js, só mvr. effect of post length on endodontically-treated teeth: fracture resistance. braz j oral sci. 2010; 9: 176-80. 2. pereira jr, valle al, shiratori fk, ghizoni js. effect of post length on endodontically-treated teeth: analysis of tensile strength. braz j oral sci. 2011; 10: 277-81. 3. pereira jr, de ornelas f, conti pc, do valle al. effect of a crown ferrule on the resistance of endodontically treated teeth restored with prefabricated posts. j prosthet dent. 2006; 95: 50-4. 4. pereira jr, mendonça neto t, porto v de c, pegoraro lf, valle al. influence of the remaining coronal structure on the resistance of teeth with intraradicular retainer. braz dent j. 2005; 16: 197-201. 5. pereira jr, valle al, shiratori fk, ghizoni js, melo mp. influence of intraradicular post and crown ferrule on the fracture strength of endodontically treated teeth. braz dent j. 2009; 20: 297-302. 6. zogheib lv, pereira jr, valle al, oliveira ja, pegoraro lf. fracture resistance of weakened roots restored with composite resin and glass fiber post. braz dent j. 2008; 19: 329-33. 7. oliveira ja, pereira jr, valle al, zogheib lv. fracture resistance of endodontically treated teeth with different heights of crown ferrule restored with prefabricated carbon fiber post and composite resin core by intermittent loading. oral surg oral med oral path oral radiol endod. 2008; 106: 52-7. 8. faria ac, rodrigues rc, de almeida antunes rp, de mattos mda g, ribeiro rf. endodontically treated teeth: characteristics and considerations to restore them. j prosthodont res. 2011; 55: 69-74. 9. zarow m, devoto w, saracinelli m. reconstruction of endodontically treated posterior teeth—with or without post? guidelines for the dental practitioner. eur j esthet dent. 2009; 4: 312-27. 10. fraga rc, chaves gsb, mello jf, siqueira jr. fracture resistance of endodontically treated roots after restoration. j oral rehabil. 1998; 25: 809-13. 11. fernandes as, shetty s, coutinho i. factors determining post selection: a literature review. j prosthet dent. 2003; 90: 556-62. 12. abdalla al, alhadainy ha. 2-years clinical evaluation of class i posterior composites. am j dent. 1996; 9: 150-2. 13. goracci c, ferrari m. current perspectives on post systems: a literature review. aust dent j. 2011; 56: 77-83. 14. bowen rl, cobb en. a method for bonding to dentin and enamel. j am dent assoc. 1983; 107: 1070-6. 15. leary jm, aquilino, sa, svare cw. an evaluation of post length within the elastic limits of dentin. j prosthet dent. 1987; 57: 277-81. 16. holmes dc, arnold am, leary jm. influence of post dimensions on stress distribution in dentin. j prosthet dent. 1996; 75: 140-7. 17. burns da, krause wr, douglas hb, burns dr. stress distribution surrounding endodontic posts. j prosthet dent. 1990; 64: 412-8. 18. isidor f, brondum k, ravnholt g. the influence of post length and crown ferrule on the resistance to cyclic loading of bovine teeth prefabricated titanium post. int j prosthodont. 1999; 12: 79-82. 19. kvist t, rydin e, reit c, the relative frequency of periapical lesions in teeth with root canal –retained posts. j endodont. 1989; 15: 578-80. braz j oral sci. 12(1):1-4 fracture resistance of endodontically treated teeth restored with different intraradicular posts with different lengths oral sciences n3 braz j oral sci. 13(2):118-123 original article braz j oral sci. april | june 2014 volume 13, number 2 dimensional stability of a novel polyvinyl siloxane impression technique moira pedroso leão, camila paloma pinto, ana paula sponchiado, bárbara pick ornaghi1 universidade positivo up, school of dentistry, area of prosthodontics, curitiba, pr, brazil correspondence to: bárbara pick ornaghi rua prof. pedro viriato parigot de souza, 5300 cep: 81280-330, curitiba, pr, brasil phone: +55 41 33173000 , +55 41 99796310 e-mail: bpo@up.com.br, barbara@pick.com.br received for publication: april 08, 2014 accepted: june 05, 2014 abstract aim: to introduce a modification of the reline impression technique (mrit), and compare the dimensional changes of impressions obtained by mrit and by conventional reline impression technique (crit). methods: an acrylic resin tablet was milled by a cad-cam system to simulate three abutments (a, b and c) with different distances among them. the abutments were molded using both impression techniques. for mrit, before completing the putty silicone polymerization, the relieve procedure was made by compression and it was immediately repositioned to complete the polymerization. impressions were stored dry at room temperature for different periods (immediately, 1 h, 2 days and 7 days). the distances were obtained by scanning. the differences between the impressions and their respective matrix reference measurements were calculated to determine the dimensional changes. data were subjected to anova and tukey’s test (p<0.05). results: for ab and bc distances, there was no statistically significant difference between crit and mrit (p=0.0597 and p=0.2167, respectively). for ac, there was statistically significant difference between the techniques for the immediate storage time (p=0.006). in general, for crit the material showed expansion, while for mrit it showed contraction. conclusions: it was verified that the addition silicon impressions obtained by both impression techniques showed dimensional stability, except for the immediate time-point. keywords: dimensional measurement accuracy; dental impression materials; dental impression technique; laboratory research. introduction in the process of dental prosthesis production, impression making plays a key role, because it transfers the clinical situation to a cast, which must reproduce accurately the oral structures and simulate the occlusion with its antagonist1-3. a precise reproduction of dental structures provides prostheses with good marginal quality. the different characteristics of commercially available elastomeric impression materials influence the accuracy and the dimensional stability of the impressions1-4. according to the american dental association (ada) specification #19, elastomers are polymeric impression materials, classified into four categories by their chemical composition and polymerization reaction: condensation-cured silicones, addition-cured silicones, polysulfides (mercaptans) and polyethers2-5. ideally, the dimensional stability of an impression material reflects its ability to maintain the accuracy of the impression over time which, according to specification #19 of ada, the elastomeric impression materials must be able to reproduce in fine details of 25 ìm or less3-6. the dimensional changes in elastomeric materials may occur due to many factors such as hydrophilicity, polymerization shrinkage, byproduct evaporation from polymerization reaction, shrinkage from braz j oral sci. 13(2):118-123 119 thermal alteration, incomplete elastic recovery, time elapsed for impression pouring, mishandling, thickness and adhesion of the material to the tray2,3,7-9. polyvinyl siloxanes do not present byproduct formation after the polymerization reaction, which is expected to guarantee dimensional stability. it was shown that it remains dimensionally stable for up to 7 days and, also after the preparation of a first cast, it is possible to make a subsequent cast from the same mold 4,8,10. however, the additional polymerization reaction involves linking a vinyl siloxane in the base material with a hydrogen siloxane via a platinum catalyst, where it is expected to occur some material shrinkage. moreover, the reaction produces hydrogen, which is scavenged by platinum or palladium2,9,11. therefore, mold distortion is inevitable and should be minimized by improving the impression techniques. polyvinyl siloxanes are found in different viscosities (from very low to very high viscosity materials), making it possible to use different impression techniques: regular onestep (single phase), putty wash one-step (simultaneous impression technique) and putty wash two-step (reline impression technique)2,4,8,10,12-19. the single phase impression technique employs only a regular body monophase material, which is inserted into an individual tray. both the simultaneous and the reline impression techniques use silicone materials of two different viscosities, but in the last technique handling of both materials is performed at different times. in this case, a first impression is made with heavybody material (putty viscosity). after its polymerization, the impression is removed from the mouth and a uniform relief of approximately 1 to 3 mm is made in order to obtain a space for the fluid-body material (wash viscosity). after that, the fluid polyvinyl siloxane material is handled, inserted into the relieved mold and over the prepared teeth, and the tray is repositioned in the mouth until the polymerization reaction is complete8,10,13-14,18-19. the main disadvantages of the reline impression technique are the time spent to relieve the putty material, the greater amount of debris generated by the relieving procedure in the clinical environment and lack of adhesion between the heavybody putty and light-body wash materials 7,20. in order to eliminate this step, without losing the quality of the final work, this study aimed to present a modification of the reline impression technique, which we will call as modified reline impression technique (mrit), as well as to assess the dimensional changes of polyvinyl siloxane impressions obtained by the mrit and compare it with the conventional reline impression technique (crit) at different storage times (immediately, 1 h, 2 days and 7 days). material and methods matrix fabrication an acrylic resin matrix was made by a cad-cam system (zirkonzahn, gais, italy) for obtaining the polyvinyl siloxane specimens. for this purpose, an acrylic resin tablet (temp premium, zirkonzahn) with 95 mm diameter and 16 mm thick was milled. the zirkonzahn archiv software (zirkonzahn) was used to design three abutments (a, b and c) similar to a canine tooth randomly arranged from each other. after acrylic resin tablet milling (zirkonzahn frasen), the cusp tips of the teeth were cut using a tungsten cutter (edenta, são paulo, sp, brazil) to generate a flat surface. next, a cross was carved in this region on each tooth with a thin grained cutting disc (kg sorensen, são paulo, sp, brazil). moreover, three acrylic resin spacers (duralay, polidental, cotia, brazil) were made in the resin matrix to allow polyvinyl siloxane material flow during the impressions and thereby avoid the presence of bubbles (figure 1). fig. 1. acrylic resin matrix used to obtain the addition silicone specimens, where it is possible to observe the three abutments (a, b, c) similar to canine teeth that had their cusp tips sectioned. polyvinyl siloxane specimen preparation in order to obtain the polyvinyl siloxane specimens (ad futura, dfl, rio de janeiro, rj, brazil), the acrylic resin matrix was molded using both impression techniques (n=5): the conventional reline impression technique (crit) and the modified reline impression technique (mrit). round metallic devices (43 mm diameter and 5 mm high) were fabricated and used as trays. conventional reline impression technique (crit) initially, the high viscosity polyvinyl siloxane (ad futura) was handled according to manufacturer’s instructions, inserted into a metallic tray and positioned over the acrylic resin matrix. the set (tray/polyvinyl siloxane/acrylic resin matrix) was kept under pressure (250 kg) for 3 min by a hydraulic press (vh, araraquara, sp,brazil). then, the impression was removed from the matrix and the relieving procedure was made using a tungsten cutter (edenta, são paulo, sp, brazil), maintaining a uniform reduction of approximately 1.5 mm21. next, the low viscosity polyvinyl dimensional stability of a novel polyvinyl siloxane impression technique 120 siloxane (futura ad) was inserted over the relieved impression with a self-mixing dispenser, repositioned over the matrix, and the set was taken to the hydraulic press (250 kg, vh) for 3 min (figure 2 a and b). modified reline impression technique (mrit) the high viscosity polyvinyl siloxane (futura) was handled according to the manufacturer’s instructions, inserted into a metallic tray and positioned over the acrylic resin matrix. the set (tray/polyvinyl siloxane/acrylic resin matrix) was kept under pressure of 250 kg (vh). however, before completing the polyvinyl siloxane polymerization, the impression was separated from the matrix. immediately, the impression’s relieving procedure was made by compression using the handle of a #36 dental cement spatula (duflex ss white, rio de janeiro, rj, brazil). even before the end of the polyvinyl siloxane polymerization, the impression was repositioned over the matrix and the end of its polymerization was awaited under a pressure of 250 kg for 3 min (vh). after that, the impression was removed from the matrix. then, the low viscosity polyvinyl siloxane (futura) was inserted over the relieved impression with a self-mixing dispenser, repositioned over the matrix, and the set was taken again to the hydraulic press (250 kg, vh) for 3 min (figure 2 c and d). fig. 2. a and b refer to the crit, where a shows the impression relieving procedure with a tungsten cutter and b shows the final impression. c and d refer to the mrit, where c shows the impression relieving procedure by compression with a spatula handle and d shows the final impression. dimensional changes measurement of the polyvinyl siloxane impressions all impressions were stored dry at room temperature and the dimensional measurement changes were done at different storage times: immediately (i), 1 h (1h), 2 days (2d) and 7 days (7d). initially, the distances among the a, b and c abutments of the acrylic resin matrix were assessed using the zirkonzahn modellier software (zirkonzahn). in order to decrease the superficial brightness of the matrix and allow the measurement of the distances, it was necessary to coat it with a white suspension solution (spot check magnaflux skd-s2, são paulo, sp, brazil). thus, three measurements were taken as reference for evaluation of the impressions’ dimensional changes: ab = 12.896 mm, bc = 17.183 mm and ac = 18.846 mm. after that, the same distances (ab, bc and ac) in all polyvinyl siloxane impressions were obtained by the zirkonzahn modellier software at each storage time (figure 3). the differences between the impressions and their respective matrix reference measurements were calculated to determine the dimensional changes. data for each distance (ab, bc and ac) were subjected to one-way anova. tukey’s test was used for multiple comparisons. tests were performed with a pre-set significance level of 5%. dimensional stability of a novel polyvinyl siloxane impression technique braz j oral sci. 13(2):118-123 121 fig. 3. zirkonzhan modellier software image of an addition silicone impression showing the measurement of the distances among the three abutments (ab, bc and ac). results there was no statistically significant difference among the mean dimensional changes of the impressions made by both techniques, irrespective of the storage times, for ab impression technique storage time mean standard deviation confidence interval m a x i m u m m i n i m u m crit i 116.8 119.6 236.4 -2.8 1h 38.8 151.1 189.9 -112.3 2d 118.4 143.4 261.8 -25.0 7d 76.8 73.8 150.6 3.0 mrit i -42.2 142.8 100.6 -185.0 1h -83.0 25.4 -57.6 -108.4 2d 71.0 69.2 140.2 1.8 7d -8.8 103.5 94.7 -112.3 table 1.table 1.table 1.table 1.table 1. means, standard deviations and confidence intervals (in µm) of dimensional changes for the ab distance of both impression techniques (crit and mrit) according to the storage times: immediate (i), 1 h (1h) 2 days (2d) and 7 days (7d). * * there was no statistically significant difference between the groups (p=0.0597). impression technique storage time mean standard deviation confidence interval maximum minimum crit i 98.8 156.1 254.9 -57.3 1h 65.2 111.9 177.1 -46.7 2d 95.8 118.6 214.4 -22.8 7d 50.8 169.2 220.0 -118.4 mrit i -62.8 192.2 129.4 -255.0 1h -49.2 68.9 19.7 -118.1 2d -22.4 78.8 56.4 -101.2 7d -55.6 73.2 17.6 -128.8 table 2. table 2. table 2. table 2. table 2. means, standard deviations and confidence intervals (in µm) of dimensional changes for the bc distance of both impression techniques (crit and mrit) according to the storage times: immediate (i), 1 h (1h) 2 days (2d) and 7 days (7d).* * there was no statistically significant difference between the experimental groups (p=0. 2167). (p=0.0597) and bc (p=0.2167) distances. tables 1 and 2 show the mean dimensional changes, standard deviations and confidence intervals (in µm) of the experimental groups for ab and bc distances, respectively. on the other hand, there was a statistically significant difference for dimensional change means of the experimental groups for the ac distance (p=0.006). table 3 and figure 4 show the means, standard deviations and confidence intervals (in µm) of dimensional changes of the tested experimental groups. for the immediate storage time, the means for both impression techniques were statistically different, in which crit impressions showed expansion, and mrit impressions showed contraction in relation to the resin matrix dimensions. however, for other storage times, no statistically significant difference among the means of both impression techniques was found, but the same tendency of the crit impressions presenting expansion and mrit impressions presenting contraction was observed. discussion factors such as non-simultaneous polymerization shrinkage of polyvinyl siloxane materials with different viscosities, the deficiency of detailed reproduction of the high viscosity impression material and high flow under pressure of the low viscosity material during the impression, dimensional stability of a novel polyvinyl siloxane impression technique braz j oral sci. 13(2):118-123 122 impression technique storage time mean standard deviation confidence interval maximum minimum crit i 195.2a 161.5 236.4 -2.8 1h 172.6ab 200.7 189.9 -112.3 2d 83.6ab 126.1 261.8 -25.0 7d 135.4ab 143.8 150.6 3.0 mrit i -100.8b 141.1 100.6 -185.0 1h -75.2ab 139.1 -57.6 -108.4 2d -52.8ab 85.0 140.2 1.8 7d -33.2ab 96.5 94.7 -112.3 table 3. table 3. table 3. table 3. table 3. means, standard deviations and confidence intervals (in µm) of dimensional changes for ac distance of both impression techniques (crit and mrit) according to the storage times: immediate (i), 1 h (1h) 2 days (2d) and 7 days (7d).* * same letters indicate no statistically significant difference (p>0.05). seem to explain the superiority of the reline impression technique to the simultaneous technique8,17-18. however, some disadvantages of the reline impression technique were reported, as the difficulty in repositioning the relieved impression in the mouth, the increase of clinical time and the amount of debris due to the impression’s relieve procedure, which are commonly performed with a razor blade, drill or milling cutter7,10. the modification of the conventional reline impression technique proposed in the present study eliminates such problems because the impression’s relieving is performed by compression using the handle of a dental cement spatula and the repositioning is done before completing the polymerization of the material. several studies comparing different impression techniques found superior dimensional stability obtained by the conventional reline technique, which indicates that the impression’s relieving procedure result in less distortion8,1718. furthermore, it was demonstrated that this result was obtained both for 30 s after impression and 30 days of the impression’s storage 7. in this study, the impressions’ dimensional changes obtained by the two techniques, in which both used some impression relieve procedure, was assessed immediately, 1 h, 2 days and 7 days after the impression, which were the storage times previously reported as being able to identify possible dimensional changes of impression materials4,7-8,14. in both techniques and for most fig. 4. dimensional changes (in µm) of the impression techniques (crit and mrit) according to the storage times (immediately, 1 h, 2 days and 7 days) for ac distance. of storage times, except for the immediate time, the impressions maintained their dimensional stability, which is probably due to the fact that they were made with polyvinyl siloxane, were under constant pressure during the polymerization, presented a uniform thickness for the low viscosity polyvinyl siloxane, were free of bubbles and presented correct proportion between the base and catalyst pastes, due to the material handling with a self-mixing dispenser21. in a previous study, the dimensional change of additional curing silicone and polyether was evaluated by plaster casts. casts were made by multiple pouring at 30 min, 6 h, 24 h and 30 days after impression making. all deviations in casts made from silicone impression material were considered within a clinically acceptable range. on the other hand, for polyether the distortions were clinically unacceptable7. this result could be attributed to the good elastic recovery of the polyvinyl siloxanes, which corroborates the results of the present research (except for the immediate time for the ac distance) and other studies7,9. besides the dimensional stability of polyvinyl siloxane, the elastic recovery is an essential property these materials must have to guarantee the impression’s accuracy4. elastic recovery of an impression is defined as the ability of a material to return to its original dimensions without significant distortion upon removal from the mouth3,9. the significant difference between the evaluated techniques for the immediate storage time for the ac distance may be the result of an incomplete elastic recovery of the impression material. the measurements of the present study did not consider any tooth movement. if it were to consider the periodontal ligament space, which was previously reported to range from 90 to 240 µm22, the dimensional changes found in the impressions of the present study, which ranged from 9 to 195 µm, could be compensated by periodontal movement and this material distortion could be considered clinically acceptable7. another important result observed in this study is that generally the crit promoted the polyvinyl siloxane expansion (positive values of dimensional changes), while the mrit allowed the material shrinkage (negative values of dimensional changes). considering that the polyvinyl dimensional stability of a novel polyvinyl siloxane impression technique braz j oral sci. 13(2):118-123 123 siloxanes are polymers that naturally undergo polymerization shrinkage, a more coherent response occurred by mrit. several studies evaluated the dimensional changes of impression materials by means of plaster casts and the measurements were carried out by different devices such as microscope, digital caliper, three-dimensional zeiss meter and stereomicroscope4,7,13-14,23. however, the measurements in the present study were performed directly in the impressions using the zirkonzahn system, which ensured accuracy and practicality in impression evaluation, and allowed the visualization of three-dimensional images. thus, this methodology excluded the possibility of changes in plaster casts and wrong positioning of the specimens during measurement. based on that and according to ada’s specification #19 and previous studies, five specimens of each experimental group are appropriate to perform linear dimensional change tests5,7,16,24. moreover, the use of a cadcam system to fabricate the acrylic resin matrix resulted in a master model with good mechanical resistance and surface finishing. moreover, considering that the impression is a timedependent procedure14, it is also recommended to wait at least 1 h to pour when it is made with a polyvinyl siloxane material. however, more experimental studies to verify detail reproduction and clinical studies are required for this new impression technique. based on the results of this study it was possible to conclude that: 1. for all distances and both impression techniques, there was no significant difference among the dimensional change means for 1 h, 2 days and 7 days of storage time; 2. for the ac distance, there was a significant difference between the dimensional change means obtained by the crit and mrit for immediate storage time; 3. the mrit may be recommended due to its simplicity, with a waiting time for pouring of at least 1 h. within the limitations of this research, it is feasible to suggest that the modification of the reline impression technique proposed in the present study is recommended because it is easy to carry out, reduces the time spent for putty material relieving and decreases the amount of debris generated by the relieving procedure. acknowledgements the authors would like to thank dr. marcelo filietaz of the masterdent prótese odontológica ltda, brazil for the cad-cam system availability. references 1. giordano r. issues in handling impression materials. gen dent. 2000; 48: 646-8. 2. giordano r. impression materials: basic properties. gen dent. 2000; 48: 510-2, 514, 516. 3. hamalian ta, nasr e, chidiac jj. impression materials in fixed prosthodontics: influence of choice on clinical procedure. j prosthodont. 2011; 20: 153-60. 4. chen sy, liang wm, chen fn. factors affecting the accuracy of elastomeric impression materials. j dent. 2004; 32: 603-9. 5. ansi/ada: specification n.19 – dental elastomeric impression materials. chicago: american dental association; 2004. 6. tjan ah, li t, logan gi, baum l. marginal accuracy of complete crowns made from alternative casting alloys. j prosthet dent. 1991; 66: 157-64. 7. thongthammachat s, moore bk, barco mt, hovijitra s, brown dt, andres cj. dimensional accuracy of dental casts: influence of tray material, impression material, and time. j prosthodont. 2002; 11: 98-108. 8. al-bakri ia, hussey d, al-omari wm. the dimensional accuracy of four impression techniques with the use of addition silicone impression materials. j clin dent. 2007; 18: 29-33. 9. papadogiannis d, lakes r, palaghias g, papadogiannis y. effect of storage time on the viscoelastic properties of elastomeric impression materials. j prosthodont res, 2012; 56: 11-8. 10. shiozawa m, takahashi h, iwasaki n. effects of the space for wash materials on sulcus depth reproduction with addition-curing silicone using two-step putty-wash technique. dent mater j. 2013; 32: 150-5. 11. derrien g, le menn g. evaluation of detail reproduction for three die materials by using scanning electron microscopy and two-dimensional profilometry. j prosthet dent. 1995; 74: 1-7. 12. christensen gj. what category of impression material is best for your practice? j am dent assoc. 1997; 128: 1026-8. 13. franco eb, da cunha lf, herrera fs, benetti ar. accuracy of singlestep versus 2-step double-mix impression technique. isrn dent. 2011; 2011: 341-546. 14. franco eb, da cunha lf, benetti ar. effect of storage period on the accuracy of elastomeric impressions. j appl oral sci. 2007; 15: 195-8. 15. idris b, houston f, claffey n. comparison of the dimensional accuracy of oneand two-step techniques with the use of putty/wash addition silicone impression materials. j prosthet dent. 1995; 74: 535-41. 16. hung sh, purk jh, tira de, eick jd. accuracy of one-step versus twostep putty wash addition silicone impression technique. j prosthet dent. 1992; 67: 583-9. 17. caputi s, varvara g. dimensional accuracy of resultant casts made by a monophase, one-step and two-step, and a novel two-step putty/light-body impression technique: an in vitro study. j prosthet dent. 2008; 99: 274-81. 18. nissan j, rosner o, bukhari ma, ghelfan o, pilo r. effect of various putty-wash impression techniques on marginal fit of cast crowns. int j periodontics restorative dent. 2013; 33: e37-42. 19. nissan j, gross m, shifman a, assif d. effect of wash bulk on the accuracy of polyvinyl siloxane putty-wash impressions. j oral rehabil. 2002; 29: 357-61. 20. purk jh, hung sh, chappell rp, casper rl, eick jd. the effect of time on the adhesion of light-body to heavy-body express in the two-step reline polyvinylsiloxane impression technique. am j dent. 1990; 3: 249-52. 21. levartovsky s, levy g, brosh t, harel n, ganor y, pilo r. dimensional stability of polyvinyl siloxane impression material reproducing the sulcular area. dent mater j. 32: 25-31. 22. coolidge ed. the thickness of the human periodontal membrane. j am dent assoc. 1937; 24: 1260-70. 23. thielke s, serrano jg, lepe x. a method for true coordinate threedimensional measurement of casts using a measuring microscope. j prosthet dent. 1998; 80: 506-10. 24. klooster j, logan gi, tjan ah. effects of strain rate on the behavior of elastomeric impression. j prosthet dent. 1991; 66: 292-8. dimensional stability of a novel polyvinyl siloxane impression technique braz j oral sci. 13(2):118-123 oral sciences n3 braz j oral sci. 14(2):106-111 original article braz j oral sci. april | june 2015 volume 14, number 2 th2 cells and the ifn-γ r1 subunit in early and advanced experimental periodontitis in rats; an immunohistochemical study janaína cavalcante lemos1, bruno césar de vasconcelos gurgel2, roseana de almeida freitas3 1universidade potiguar – unp, school of dentistry, department/area of periodontics, natal, rn, brazil 2universidade federal do rio grande do norte – ufrn, school of dentistry, department/area of periodontics, natal, rn, brazil 3universidade federal do rio grande do norte – ufrn, school of dentistry, department/area: oral pathology, natal, rn, brazil correspondence to: janaína cavalcante lemos av. amintas barros 3735 bl. c ap. 603 bairro lagoa nova cep: 59075-250 natal rn brasil phone: +55 (084) 3201 5017 (084) 9985 5455 e-mail: nainalemos@gmail.com abstract aim: to evaluate the involvement of th2 cells in different periods of the active phase of experimental periodontal disease and expression of the r1 subunit of the receptor for ifn-γ during the early and advanced progression of the disease. methods: experimental periodontitis was induced in 30 male wistar rats by placing cotton ligatures around the mandibular first molars. the rats were then randomly assigned into two groups: g1=15 and g2=15, in group g1, ligatures were maintained for 2 days, a period that corresponds to the initial stage of periodontal disease in rats, in g2 ligatures were left for 15 days, a period that corresponds to the advanced stage of periodontal disease. the contra-lateral teeth served as controls (unligated). an immunohistochemical investigation of the gingival tissue was performed to detect the presence of the th2 specific transcription factor (gata3). results: light microscopy analysis revealed a decreased expression of gata-3-positive cells when bone loss progressed. ifn-γ r1 was detected at an early stage during the active phase of disease, but the expression of positive cells remained unaltered during the remaining period of the study. conclusions: these results indicate that the th2 response may have a protective role during the pathogenesis of experimental periodontal disease, and that the ifn-γ r1 subunit may not be associated with periodontal disease progression. keywords: rats; periodontal diseases; th1 cells; th2 cells; cytokines; immunohistochemistry. introduction periodontal disease is the result of microbial aggression as well as metabolic and environmental factors and life-style habits influence the pathogenesis of this disease1. bacteria are capable of damaging directly the host tissue, but it can also be destroyed by inflammatory processes resulting from this aggression as consequence of the exaggerated immunoinflammatory and proteolytic processes2 that activate osteoclasts3. cd4 and cd8 t lymphocytes play an import role in the inflammatory response, as these cells may manage the profile of cytokines produced against an infectious agent4. cytokines are mediators that, among other functions, can cause bone resorption and dissolution of the extracellular matrix. progression of periodontal lesions is caused by dysregulation of molecules (cytokines) released by specific cell populations5. cd4 t cells may assume different phenotypes under different stimulations, changing to th1 and th2 cells. they can be distinguished based on the profile of received for publication: january 21, 2015 accepted: may 28, 2015 http://dx.doi.org/10.1590/1677-3225v14n2a02 107107107107107 cytokines produced: th1 cells produce characteristic cytokines such as il-2, ifn-γ, tnf-β, il-12, while active th2 cells secrete il-4, il-5, il-6, il-10 and il-136. in addition to th1 and th2 cells, th17, treg and t fh cells have also been described7, treg cells are associated with reduction of clinical scores of disease in soft and hard tissues8. usually, protective and destructive roles are assigned to the th1 and th173,9-11 cells, while th2 and treg cells are more involved in processes that reduce the destruction of the periodontium10. in order to differentiate into th1 or th2 cells, cd4 t cells must become antigen-activated. differentiation into th1 depends on the presence of ifn-γ and il-12, which bind to receptors on the surface of cd4 t cells5. this binding initiates a cascade of events that culminates in differentiation into th1 cells, by increasing the transcription factor t-bet12. th2 cells have a differentiation process which is dependent on il-4, which causes the activation of the transcription factor stat6. this signal together with the t cell receptor (tcr) induces the expression of gata-3. gata-3 acts as a master regulator of th2 differentiation12-13. gata-3 and ifn-γ r1 subunit expressions were evaluated during different stages of induced periodontal disease (early and advanced) and these data were correlated with the severity of periodontal destruction, which was determined clinically and histologically. material and methods study design thirty male adult wistar rats (rattus norvegicus albinus), weighing between 300 and 400 g at the beginning of the experiment and aged approximately 10 weeks, were included in this study. they were kept in polypropylene cages with 5 animals per cage during the whole experimental period and received standard laboratory chow and water ad libitum. the protocol was approved by institutional animal care and use committee of the potiguar university, brazil (protocol number: 079/07). after an adaptation period of 7 days for the animals, experimental periodontitis was induced in animals. previously, general anesthesia was obtained by intramuscular administration of xylazine hydrochlorate (virbaxyl® 2% – virbac do brasil ind e com. ltda – 0.3 mg/kg) and ketamine (francotar® – virbac do brasil ind e com. ltda 1ml/kg). a cotton ligature (no 10; coats corrente, sp, brazil) was randomly assigned to one mandibular first molar, in a submarginal position, as previously described14. the control side remained unligated and was used as control. subsequently, the animals were randomly assigned into two groups of 15 animals each: • group 1 (initial stage) ligatures were maintained for 2 days to enable the development of established gingival lesions (g1t – test group 1). this period should not induce bone loss on any surface around the tooth. contralateral teeth were used as controls (g1c control group 1). • group 2 (advanced stage) ligatures were maintained for 15 days (g2t – test group 2). this period can cause bone loss (advanced lesions) on any surface around the tooth14. contralateral teeth were also used as controls (g2c control group 2). following these protocols, the animals were sacrificed by deep anesthesia and the jaws were removed, dissected and fixed in 10% neutral formalin for 48 h. blocks containing the tooth, the inter-radicular bone and soft tissue were obtained. the soft tissues that represent the region of the periodontal pocket were carefully removed with a 15c scalpel blade. the fragments were processed routinely and stained with hematoxylin and eosin (he) and for immunohistochemistry. the remaining block containing hard tissue after soft tissue removal was demineralized in a 10% formic acid solution. this solution was renewed every two days for 12 days, to confirm the presence and absence of bone loss. morphological analysis the fragments from soft tissue were embedded in paraffin and histological serial sections of 5 mm were obtained, followed by he staining. likewise, after the demineralization process, serial sections of 5 mm from hard tissue were also obtained from the buccolingual position (starting at the buccal bone plate) and stained with he. after the staining process, the intensity of the inflammatory infiltrate (mild, moderate and intense), the prevailing cell types (neutrophils, lymphocytes, plasma cells, macrophages) and vascularization (scarce, moderate and intense) were analyzed in the soft tissue under an optical microscope (magnification of 400x). presence or absence of bone loss was also analyzed in hard tissue, by visual examination under an optical microscope. immunohistochemical methods for the immunohistochemical study, 3-µm-thick sections were obtained from paraffin-embedded gingival tissue blocks. the tissue sections were de-paraffinized and immersed in 3% hydrogen peroxide to block endogenous peroxidase activity. antigen retrieval was performed in a pascal pressure cooker with citrate buffer, ph 6.0, and tris/edta, for g r1 and gata-3, respectively. after treatment with normal serum, the sections were incubated with the primary anti-ifn-g r1 antibody diluted 1:100 (santa cruz biotechnology, santa cruz, ca, usa) for 18 h and anti-gata-3 diluted 1:250 (santa cruz biotechnology, santa cruz, ca, usa) for 60 min. the tissue sections were then washed twice in tris ph 7.4 and treated with an immunoperoxidase-based kit (envision + dual link system peroxidase; dako, carpinteria, ca, usa) at room temperature to bind the primary antibody. peroxidase activity was visualized by immersing tissue sections in diaminobenzidine (liquid dab + substrate; dako, capinteria, ca, usa) for 3 min, resulting in a brown reaction product. finally, the tissue sections were counterstained with mayer haematoxylin and cover slipped. as a negative control, sections of gingival tissues from animals (test and control sides) were treated as earlier, except for the primary antibody, which was replaced by a solution of bovine serum albumin in pbs (phosphate-buffered saline). th2 cells and the ifn-g r1 subunit in early and advanced experimental periodontitis in rats; an immunohistochemical study braz j oral sci. 14(2):106-111 108108108108108 immunostaining and statistical analysis for quantitative analysis, immunostained cells were evaluated for each histological tissue in five fields on each slide; the 5 histological fields were photographed using an olympus cx41 camera. images were downloaded and the immunostained cells were counted using an image analysis system (image tool for windows, version 3.0). the total number of immunostained cells in each field was recorded and the averages corresponding to each slide calculated. averages were registered for each animal and each group. data interpretation was performed by a trained examiner, without knowledge of the different groups (blinded study). a statistical software was used for statistical analysis (spss statistical package for social sciences, v. 13). immunohistochemical expression of ifn-γ r1 and gata-3 was compared in the two independent groups, group 1 (2 days) and group 2 (15 days). the shapiro-wilk test was performed to confirm the normal distribution of the groups. levene’s f test was applied for verification of equal variances. to compare inter and intra-group differences, student’s t test was used and the mann-whitney and wilcoxon tests were used for groups without normal distribution. the level of significance was set at 5%. results morphological data most of the samples from the g1c (control group 1) and g2c (control group 2) groups exhibited a mild inflammatory infiltrate that was compatible with clinically healthy gingiva. lymphocytes were identified as the predominant cells and bone loss was not observed in the furcation or interproximal region. for the g1t (test group 1), 7 cases exhibited moderate inflammatory infiltration and 8 cases demonstrated intense infiltration. in this group, the infiltrate was predominantly lymphocytic, and foci of osteoclastic cells and bone resorption were observed in the furcation and interproximal regions in 4 cases. the g2t (test group 2) presented mild (3 cases), moderate (7 cases) and intense infiltration (5 cases). in this group, lymphocytes predominated in seven cases and lymphoplasmacytic infiltrate predominated in 6 cases, bone resorption was observed in all cases. neutrophils were observed in 100% cases in the test groups. immunohistochemical data all samples showed staining for each antibody. ifn-g r1 was detected in the cytoplasm of all inflammatory cells (figure 1a) and gata-3 positivity was found in the lymphocytes (figure 1b). for the expression of gata-3, intragroup analysis (g1c/g1t, g2c/g2t) was statistically significant (p<0.05), indicating a higher expression of gata-3 in ligated teeth (test side). positive cells for gata3 expression were found in groups 1 (means: control side = 45.83/test side = 149.27) and 2 (means: control side = 30.37/test side = 89.84). these results were compatible with the greater fig. 1. test group: 1a inflammatory infiltrate showing immunostaining to ifn-γ r1 subunit of the receptor for ifn-γ (200x). 1b lymphocytic infiltrate showing intracytoplasmic immunostaining to the transcription factor gata-3(400x). inflammatory response seen in these groups. with regards to gata-3 expression, no statistically significant differences were observed comparing the control groups (g1c/g2c). between the test groups (g1t/g2t), a higher expression was observed in g1t (p<0.05). these results indicate that duration of the cotton ligature could have a significant effect on the expression of gata-3. intragroup analysis (g1c/g1t, g2c/g2t) for ifn-γ r1 showed statistically significant differences between the test and control sides (p<0.05), demonstrating an increased expression of this receptor in ligated teeth (test side). positive cells for ifn-γ r1 expression in groups 1 (means: control side=66.25/test side=138.98) and 2 (means: control side= 61.44/test side=102.11). however, no statistical differences were observed between test groups (g1t/g2t), i.e., the ligation time was not decisive for ifn-γ r1 expression. in addition, differences were not observed between the control groups (g1c/g2c). discussion the interaction of bacterial challenge and host response induces the expression of several molecules, including th2 cells and the ifn-g r1 subunit in early and advanced experimental periodontitis in rats; an immunohistochemical study braz j oral sci. 14(2):106-111 metalloproteinases, involved in the process of destructive periodontal disease15 and cytokines5,16-17. the cytokines exhibit a strong regulatory role in immune response, regulating the differentiation of native t cells into th1 and th2 cells. numerous cytokines are produced during the immune response, where il-12 and ifn-γ are involved in the development of the th1 and th2 cytokines, and il-4 induces the differentiation of th2 cells17. other cytokines are also directly involved in the pathogenesis of periodontal disease, such as interleukin 6 (il-6), interleukin 8 (il-8) and tumor necrosis factor α (tnf-α)16. the pathogenesis of periodontitis is classically described as stable and destructive. based on the paradigm of th1/th2 cells, periodontal lesions with a predominantly th1 profile may be termed as a stable lesion (gingivitis). in the lesions with a predominance of th2 cells, a destructive profile (periodontitis) may be present18. however, some authors have shown a protective role of the th2 cells10,19-20. since the exact knowledge of this response remains unclear, the object of this study was to analyze by immunohistochemistry the expression of th2 cells in the destructive phase of periodontal disease as well as to evaluate possible differences in the expression of surface ifn-γ r1. the expression of this receptor is crucial for an effective th1 response during the evolution of this model of experimentally induced periodontal disease in rats, which has been widely used in several studies21-26. in the control groups, presence of a mild lymphocytic cellular infiltrate was typically observed. in absence of ligatures these cells are a possible indicator of a constant response to bacterial challenge in the gingival sulcus, as described in other studies 23. in the experimental groups, an acute cellular inflammatory response was observed; during the first 2 days of ligature, the cellular infiltrate became moderate to intense and remained even after 15 days. similar results were observed in human specimens27, where the authors analyzed the immunohistochemical phenotype of cells participating in the immune response in gingivitis and periodontitis. no statistically significant differences were observed in relation to t and b lymphocyte densities or in the number of dendritic cells in the described inflammatory conditions. regarding the type of cellular infiltrate in the test groups, lymphocytes and plasma cells predominated, whereas in controls groups the lymphocytes were the predominant cells. however, the presence of neutrophils was frequent in all test groups (g1t, g2t). this indicates that the ligatures were able to induce an inflammatory response and that the total number of cells remained during disease progression. as described in the literature, cell density (t and b cells) can remain unchanged between different inflammatory conditions, such as gingivitis and periodontitis 27. other studies attributed a pathological role for b cells in periodontal disease5,28. these findings imply a greater involvement of th2 cells at this stage of disease, since th2 cytokines such as il-4, il-5, il-6 stimulate the proliferation and differentiation of b cells29. the results of this study demonstrate the involvement of th2 cells in the test groups, i.e., at the stage of the increased inflammatory response in experimental periodontitis. bone loss was predominantly observed at 15 days in the g2t group as well as initial foci of bone resorption in 4 cases of the g1t group. this finding indicates that in this experimental model of disease the inflammatory process installs quickly, with consequent destruction of periodontal tissue. it probably makes this model ideal for the study of acute and destructive phases of periodontal disease. some authors23,30 reported that the presence of the ligature around teeth induces an acute inflammatory response, resulting in bone loss and loss of connective tissue attachment. the active phase of experimental periodontal disease was consistent with the increased expression of gata-3, compared to the control groups, suggesting a greater involvement of th2 cells in the higher inflammatory response phase. several studies have demonstrated that the periodontitis lesion involves predominantly b cells; this implies a greater involvement of th2 cells in the destructive phase of the disease18,31. however, when experimental groups were compared, i.e. when the course of the disease was evaluated, the expression of gata-3 was found to decrease significantly (p<0.05), together with a reduction in the amount of th2 cells (g1t = 149.27 / g2t = 89.84). with this reduction, the anti-inflammatory th2 responses are probably smaller. some authors have shown a protective role of the th2 cells10,19-20,32-34. there is an association between the absence of il-10 (lower th2 response) with the presence of severe bone loss induced by p. gingivalis19 and patients with chronic periodontitis have suppressed anti-inflammatory cytokine production that can be partly restored by neutralizing pro-inflammatory cytokines, showing a cross-talk between the production of these cytokines33. this protective role was described in a clinical study that measured the levels of several cytokines, including il-4, before and after nonsurgical periodontal therapy in humans. the levels of il-4 significantly increased following treatment, contributing to reduce periodontal inflammation32. however, a destructive role is also assigned to these cells, since they can reduce the th1 response, resulting in failure to control the infection35. this contradictory role of th cells can be explained by the conversion of th1 to th2 cells, influenced by epigenetic modifications in the environment36. it is clear that a group of t cells with destructive role, named th17, play an important role in osteoclast formation. the involvement of these cells in periodontal disease has been reported5,34. the results show that in periodontal lesions, il-17 levels are significantly greater than il-4, which plays a protective role in the pathogenesis of periodontal disease34. based on the results of the present study and the reviewed literature, it may be suggested that the decreased expression of gata-3 (lower number of th2 cells) led to a reduction in the anti-inflammatory potential in the microenvironment, which culminated with the greatest bone loss seen in the g2t group. as this model seems to stabilize over time23, it may be suggested that studies involving a longer period of observation may result in the return of this marker expression. evaluation of the expression of the ifn-γ r1 subunit of 109109109109109 th2 cells and the ifn-g r1 subunit in early and advanced experimental periodontitis in rats; an immunohistochemical study braz j oral sci. 14(2):106-111 110110110110110 the ifn-γ receptor was carried out to observe whether the microenvironment is prepared for the th1 response. the presence of functional receptors for ifn-γ on cells could be associated with the presence of the cytokine in the extracellular environment11. comparisons between the test and control teeth for the ifn-γ r1 subunit have shown an increased expression of this subunit during the active phase of periodontal disease. however, inter-group analysis demonstrated that the expression of this receptor did not change. these findings show that there was a direct relationship between increased inflammatory response and ifn-γ r1 expression. this result partially agrees with another author35, who showed that cells carrying ifn-γ increased significantly with the expansion of inflammation due to the increase in inflammatory response observed during periodontal tissue destruction. however, in the present study, the expression of the ifn-γ r1 subunit did not change with increased bone loss. these results demonstrate that with the progression of periodontal disease the expression of this receptor remained unchanged. this can also be explained based on the cell types that express this receptor. the ifn-γ r1 subunit of the ifn-γ receptor is not exclusive for th cells37 and it is moderately expressed in almost all inflammatory cells or non-inflammatory cells, where gene expression of the r1 chain appears to be constitutive in these cell types. the analysis of the promoter region of this gene revealed a structure that resembles that of the “housekeeping” genes (genes involved in basic functions of the cells and that are constitutively expressed). these authors also stated further that the expression of this subunit of the ifnreceptor is not regulated by external stimuli, unlike the r2 subunit, which can be regulated positively or negatively depending on the stimulus. analyzing this information, the r1 subunit of the receptor for ifn-γ did not work as an indicator of the presence of th1 cytokines in the microenvironment. when these results are compared with those of the literature37-38, it is accepted that this subunit is not regulated by the presence of the cytokine in the environment, since several other inflammatory cell types constitutively express this subunit. therefore, it may be suggested that the development of the inflammatory response was associated with the increase in ifn-γ expression, due to the increased number of inflammatory cells that carry this subunit. in addition, the expression of this subunit did not change between the test groups, as the amount of the inflammatory infiltrate also remained the same. regulatory mechanisms of the r1 subunit have been described37 after antigen recognition that there is a temporary down-regulation in native t cells. without this subunit, the cross-talk with the cytokine does not occur, and the antiproliferative effects associated with ifn-γ are nullified. this may explain the slight reduction in the expression of this subunit in the g2t in the present study. as described, the participation of th2 cells in periodontitis was observed herein; however, it is still unclear whether these cells are more protective or destructive in the context of periodontal pathogenesis, since they exhibit conflicting roles, i.e., they may be either protective or destructive according to their expression profile. some studies have reported the participation of both th1 and th2 cytokines in periodontal disease 5,39. as previously described, these two groups of cytokines play an important role in maintaining the homeostasis of the alveolar bone39; however, differences in the balance of cytokines in the environment may result in the progression of periodontal disease7. according to the results of the present study, it may be suggested that the decrease in positive th2 cells during the phase of higher tissue destruction, associated with experimentally induced periodontitis, may have contributed to the evolution of the destructive process. additionally, the significant expression of ifnγ r1 subunit in the test group and the lack of alteration in expression observed in the ligated teeth throughout the experiment (between g1t and g2t) suggests that this receptor is expressed at the beginning of inflammatory response development. this alteration makes these microenvironments more prepared for the th1 response, but there was no association between the presence of this receptor and increased bone loss. further experiments are necessary in order to better understand the host response against the biofilm in the periodontal tissues. understanding the regulatory mechanisms of ifn-γ and gata-3 during the development of t cell lines may also provide a better understanding of the th1/th2 response in periodontal disease as well as allow the development of methods of treatment modulation that may be able to balance this response. in conclusion, the current study demonstrates, in different periods of the active phase of experimental periodontal disease, that the decrease in the expression of the gata-3 transcription factor was associated with increased bone loss, suggesting that a controlled th2 response may be associated with protective mechanisms during the active phase of periodontal destruction induced by ligatures in rats and the ifn-γ r1 subunit may not be associated with the progression of experimental periodontal disease. acknowledgements this study was supported by the post-graduate program in oral pathology of the federal university of rio grande do norte and part of this research was conducted at the university potiguar – department of dentistry – rn, brazil. references 1. ianni m, bruzzesi g, pugliese d, porcellini e, carbone i, schiavone a, et al. variations in inflammatory genes are associated with periodontitis. immun ageing. 2013; 10:1-8. 2. yucel-lindberg t, båge t. inflammatory mediators in the pathogenesis of periodontitis. expert rev mol med. 2013; 15: e7. 3. kayal ra. the role of osteoimmunology in periodontal disease. biomed res int. 2013; 2013: 1-12. 4. yamamoto m, fujihshi k, hiroi t, mcghee jr, van dyke te, kiyono h. th2 cells and the ifn-g r1 subunit in early and advanced experimental periodontitis in rats; an immunohistochemical study braz j oral sci. 14(2):106-111 111111111111111 molecular and cellular mechanisms for periodontal diseases: role of th1 and th2 type cytokines induction of mucosal inflammation. j 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ifn-ã, il-10, il-17 levels. asian pac j trop med. 2013; 6: 489-92. 11. baker pj, boutaugh nr, tiffany m, roopenian dc. b cell igd deletion prevents alveolar bone loss following murine oral infection. interdiscip perspect infect dis. 2009; 2009: 1-6. 12. chakir h, wang h, lefebvre de, webb j, scott fw. t-bet/gata-3 ratio as a measure of the th1/th2 cytokine profile in mixed cell populations: predominant role of gata-3. j immunol methods. 2003; 278: 157-69. 13. teng yta. mixed periodontal th1-th2 cytokine profile in actinobacillus actinomycetencomitans – specific osteoprotegerin ligand (or rank-l) – mediated alveolar bone destruction in vivo. infect immun. 2002; 70: 5269-73. 14. kuhr a, popa wagner a, schmoll h, schwahn c, kocher t. observations on experimental marginal periodontitis in rats. j periodontal res. 2004; 39: 101-6. 15. nagasupriya a, rao db, ravikanth m, kumar ng, ramachandran cr, saraswathi tr. immunohistochemical expression of matrix metalloproteinase 13 in chronic periodontitis. int j periodontics restorative dent. 2014; 34: 79-84. 16. noh mk, jung m, kim sh, lee sr, park kh, kim dh, et al. assessment of il-6, il-8 and tnf-á levels in the gingival tissue of patients with periodontitis. exp ther med. 2013; 6: 847-51. 17. o’garra a. cytokines induce the development of functionally heterogeneous t helper cell subsets. immunity. 1998; 8: 275-83. 18. gemmell e, seymour gj. immunoregulatory control of th1/th2 cytokine profiles in peridontal disease. periodontology 2000. 2004; 35: 21-41. 19. sasaki h, okamatsu y, kawai t, taubman m, stashenko p. the interleukin10 knockout mouse is highly susceptible to porphyromonas gingivalisinduced alveolar bone loss. j periodontal res. 2004; 39: 432-41. 20. zhao l, zhou y, xu y, sun y, li l, chen w. effect of non-surgical periodontal therapy on the levels of th17/th1/th2 cytokines and their transcription factors in chinese chronic periodontitis patients. j clin periodontol. 2011; 38: 509-16. 21. graves dt, fine d, teng yt, van dyke te, hajishengallis g. the use of rodent models to investigate host-bacteria interactions related to periodontal diseases. j clin periodontol. 2008; 35: 89-105. 22. cavagni j, soletti ac, gaio ej, rösing ck. the effect of dexamethasone in the pathogenesis of ligature-induced periodontal disease in wistar rats. braz oral res. 2005; 19: 290-4. 23. gurgel bc, duarte pm, nociti fh jr, sallum ea, casat mz, sallum aw, et al. impact of an anti-inflammatory therapy and its withdrawal on the progression of experimental periodontitis in rats. j periodontol. 2004; 75: 1613-8. 24. galvão mp, chapper a, rösing ck, ferreira mb, de souza ma. methodological considerations on descriptive studies of induced periodontal disease in rats. pesq odontol bras. 2003; 17: 56-62. 25. souza dm; ricardo lh; rocha rf. effects of alcohol intake in periodontitis progression in female rats: a histometric study. braz j oral sci. 2013; 13: 229-34. 26. oz hs, puleo da. animal models for periodontal diseases. j biomed biotechnol. 2011; 18. 27. lins rd, figueiredo cr, queiroz lm, da silveira ej, freitas ra. immunohistoquemical evaluation of the inflammatory response in periodontal disease. braz dent j. 2008; 19: 9-14. 28. oliver-bell j, butcher jp, malcolm j, macleod mk, adrados planell a, campbell l, et al. periodontitis in the absence of b cells and specific antibacterial antibody. mol oral microbiol. 2015; 30: 160-9. 29. yamazaki k, nakajima t, hara k. immunohistological analysis of t cell functional subsets in chronic inflammatory periodontal disease. clin exp immunol. 1995; 99: 384-91. 30. nyman s, schroeder he, lindhe j. supression of inflammation and bone resorption by indomethacin durin experimental periodontitis in dogs. j periodontol. 1979; 50: 450-61. 31. arun kv, talwar a, kumar ts. t-helper cells in the etiopathogenesis of periodontal disease: a mini review. j indian soc periodontol. 2011; 15: 4-10. 32. pradeep ar, roopa y, swati pp. interleukin-4, a t-helper 2 cell cytokine, is associated with the remission of periodontal disease. j periodontal res. 2008; 43: 712-6. 33. berker e, kantarci a, hasturk h, van dyke te. blocking proinflammatory cytokine release modulates peripheral blood mononuclear cell response to porphyromonas gingivalis. j periodontol. 2013; 84: 1337-45. 34. behfarnia p, birang r, pishva ss, hakemi mg, khorasani mm. expression levels of th-2 and th-17 characteristic genes in healthy tissue versus periodontitis. j dent (tehran). 2013; 10: 23-31. 35. ukai t, mori y, onoyama m, hara y. immunohistological study of interferon-gammaand interleukin-4-bearing cells in human periodontitis gingiva. arch oral biol. 2001; 46: 901-8. 36. talwar a, arun kv, kumar tss, clements j. plasticity of t helper cell subsets: implications in periodontal disease. j indian soc periodontol. 2013; 17: 288-91. 37. bach ea, aguet m, schreiber rd. the ifn gamma receptor: a paradigm for cytokine receptor signaling. ann rev immunol. 1997; 15: 563-91. 38. skarenta h, yang y, pestka s, fathman cg. ligand-independent downregulation of ifn-? receptor 1 following tcr engagement. j immunol. 2000; 164: 3506-11. 39. alayan j, ivanovski s, farah cs. alveolar bone loss in t helper 1/t helper 2 cytokine-deficient mice. j periodontal res. 2007; 42: 97-103. th2 cells and the ifn-g r1 subunit in early and advanced experimental periodontitis in rats; an immunohistochemical study braz j oral sci. 14(2):106-111 1http://dx.doi.org/10.20396/bjos.v18i0.8657271 volume 18 2019 e191670 original article 1 graduate program in gerontology, university of campinas, brazil; department of oral rehabilitation, university of talca, chile 2 department of stomatology, federal university of santa maria, brazil 3 paulista university, brazil 4 department of preventive and social dentistry, federal university of rio grande do sul, brazil 5 graduate program in gerontology, university of campinas, brazil 6 department of social dentistry, piracicaba dental school, university of campinas, brazil corresponding author: maria da luz rosario de sousa department of social dentistry, piracicaba dental school, p.o. box 52 university of campinas unicamp 13414-903, piracicaba, sp, brazil email: luzsousa@fop.unicamp.br received: may 26, 2019 accepted: september 09, 2019 validity of self-report of oral conditions in older people maría jesús arenas-márquez1, luísa helena do nascimento tôrres2, debora dias da silva3, juliana balbinot hilgert4, fernando neves hugo4, anita liberalesso neri5, maria da luz rosario de sousa6,* aim: to verify if self-report is a valid instrument to study the clinical oral condition in older people without cognitive deficit. methods: a cross-sectional study was conducted with 647 older people from the community, without cognitive deficit, living in campinas, brazil. a self-report questionnaire assessing the presence or absence of teeth (edentulism) and use of complete denture was applied, identifying the location of the denture, whether in the upper and/or lower arch. in the same session oral clinical exams were performed, considered the gold standard. the self-report validation was performed by calculating sensitivity, specificity, predictive values, odds ratios and kappa agreement. results: there were high percentages of sensitivity (95–99%), specificity (84–97%), positive (81–97%) and negative (95–98%) predictive values, obtaining an elevated level of confidence and intrinsic quality of the self-report. agreement with the clinical examination was excellent for all variables (greater than 0.80). the likelihood ratios showed compelling evidence that with self-report an edentulous individual (+lr 32), non-edentulous (-lr 0.06) and absence of complete denture (-lr 0.01) could be correctly identified, with moderate evidence to identify the presence and location of complete denture use (+lr 6.5 to 6.9). conclusion: self-report is a valid instrument to study the clinical oral condition in the older people of the community. keywords: reproducibility of results. self-report. oral health. aged. 2 arenas-márquez et al. introduction epidemiological studies are a fundamental source of information to know the state of a population’s oral health. they allow us to understand the patterns of diseases, causes, risk factors and their vigilance over time1. considering that the world population continues to age rapidly, epidemiological studies are critical to planning public policies and effective interventions that address the specific needs of this age group2. the oral health needs of older people are complex. the final marker of oral diseases burden is edentulism3. its impact on nutrition, quality of life, and its association with disability and mortality4,5, place it as one of the main public health issues6. despite the decline of edentulism in the last decades, it remains being a highly prevalent reality in old age7, and the problems derived from this condition are further accentuated when functionality is not restored with dental prostheses8. therefore, it is essential to monitor these conditions in older people population. the gold standard in oral health research is clinical examination. however, its achievement demands many resources in terms of qualified personnel, training, facilities, time and economic cost9. as an alternative, self-report have been frequently used10, since it offers among its advantages, the obtaining of reliable, quicker and cheaper data collection1. also, it allows to reach more distant populations or with mobility limitations, since its application can be done both in person, by telephone or by mail11. large scale multidisciplinary longitudinal studies have been using self-report to investigate the health status of the population12, including questionnaires for evaluation of oral conditions. the national health interview survey (nhis) is recognized as the leading source of us health information, known for obtaining data from household interviews over 50 years13. in the same way, in brazil there are the national health survey (pesquisa nacional de saúde pns)14, with focus on older people populations, the health, well-being and aging study (saúde, bem-estar e envelhecimento sabe)15, the brazilian longitudinal study of aging (estudo longitudinal da saúde dos idosos brasileiros elsi)16, and the frailty in brazilian elderly study (fragilidade do idoso brasileiro fibra)17, which are also using this instrument. every instrument used to replace another must ensure that the measuring condition is accurate in reference to the gold standard18, as well as the self-report. a literature review verified the diagnostic validity of self-reported oral diseases in population surveys, revealing that the largest volume of studies were conducted in developed countries12. the review found acceptable results for the evaluation of the number of teeth, use and need for a denture, but recognize the need for research that certifies its validity in brazil12. additionally, with a growing number of studies about aging, such as those already mentioned15-17, it is necessary to evaluate its validity for brazilian older people. thus, the purpose of this study is to verify if self-report is a valid instrument to study the clinical oral condition in older people without cognitive deficit. 3 arenas-márquez et al. materials and methods study design and participants this cross-sectional study was performed with secondary data from the frailty in brazilian elderly study (fragilidade do idoso brasileiro fibra), developed in 2008 and 200917. the ethics committee of the school of medical sciences of the state university of campinas (nº 208/2007) approved all the procedures performed. a representative sample was collected, consisting of 900 older people from campinas, brazil. a probabilistic, cluster sample was used, taking into consideration urban census sectors (90 of the 835 in the city) randomly selected. on average, 10 older individuals randomly selected too in each census sector, were invited to take part in the study from their homes. the number of elderly individuals in campinas was calculated as 82 560 (≥65 y old), corresponding to 7.8% of the city’s population. based on this number, the sample was calculated through the formula of finite population, taking into account the achievement of statistical representativeness to describe the prevalence of frailty, use and need of dental prosthesis, presence of teeth, and oral soft tissue injuries. a detailed description of the methodology has been previously published17. in this study were included all participants aged 65 to 97 years, who had complete data for the variables of interest related to their clinical and self-reported oral status. older people with cognitive deficit, determined by the mini mental state examination (mmse), were excluded, using cut-off points established for the brazilian population according to schooling years19. oral clinical condition (gold standard) oral clinical examinations were carried out following the world health organization (who) criteria for epidemiological studies on oral health20. the oral clinical examination was performed by three trained dentists. examiners were provided with a manual describing the study, the clinical examination protocol and criteria. they were instructed to review the material independently. afterwards, they had a meeting with a trainer who revised the information, described and explained the criteria, and answered their doubts. no calibration was performed. the presence and absence of four oral conditions was verified: edentulism, use of complete denture (cd) and its location, if in the upper and/or lower arch. the edentulism was evaluated by the number of teeth present, being considered edentulism the absence of teeth. regarding the variables related to cd use, the prosthetic condition of each dental arch was examined individually, as established by who20. the cd as use was considered in its presence at the time of the clinical examination, and the non-use, the absence of cd, or the use of another type of denture. this criterion was also used to evaluate the location of cd. self-reported oral condition in the same session, a self-report questionnaire with structured answers was applied, which evaluated the same four variables measured in the clinical examination. for 4 arenas-márquez et al. edentulism it was asked: “do you have any natural teeth?” for the variables related to the use and location of cd(s), the following was asked: “do you wear dentures?” and “in which arch do you wear dentures?” the answers to this last question (upper, lower, both and not used) were subdivided to create the two variables that specified the location of the cd: use in the upper arch (yes: upper use/both; no: only lower/not use) and use in the lower arch (yes: lower use/both; no: only upper/not use). finally, it was registered whether older people had used dental services during the past year, and how they evaluate their oral health. this last question was dichotomized as positive (great/good evaluation) and negative (bad/regular evaluation). sociodemographic information age, gender, race/color, schooling and household income were registered. the race/ color was dichotomized as whites and not whites (category that included blacks, biracial, oriental and indigenous). schooling was dichotomized, according to the years of study, as up to four years and five years or more. household income was classified according to the minimum wage (mw), valued at r$415.00 / us$ 231.00 in 2008, being dichotomized in up to three mw, and four or more mw. statistical analysis the study population was characterized using descriptive statistics. for the validation, the self-reported and clinical variables were dichotomized as yes or no, whose equivalences are presented in table 1. subsequently, a contingency table was created with the distribution of self-report responses according to the clinical oral condition, to calculate the percentages and confidence intervals of: sensitivity, specificity, positive predictive values (ppv) and negative predictive values (npv). values greater than 80% were considered valid, and the sum of sensitivity plus specificity is equal to or greater than 160%12. additional information on the quality of the self-report were obtained by calculating the positive likelihood ratio (+lr) and negative likelihood ratio (-lr), where values ≥ 10 and ≤ 0.10 were respectively considered as strong evidence that self-report is a good indicator of the clinical oral condition21. finally, the agreement level between the table 1. equivalences between clinical examination and self-report issues to assess oral health condition. condition clinical protocol (gold standard) self-report issues edentulism number of teeth • n = 0 = edentulous • n ≥ 1 = not edentulous do you have any natural teeth? • no = edentulous • yes = not edentulous use of cd prosthetic condition • uses upper and/or lower cd • does not use, uses fdp and/or rpd do you wear dentures? • yes • no use of upper cd condition of upper prosthetic • uses maxillary cd • does not use, uses fdp and/or rpd in which arch do you wear dentures? • uses upper/both • do not use/uses lower use of lower cd condition of lower prothesis • uses mandibular cd • does not use, uses fdp and/or rpd in which arch do you wear dentures? • use lower/both • do not use/uses upper cd, complete denture; fdp, fixed dental prosthesis; rpd, removable partial denture. 5 arenas-márquez et al. self-reported and clinical variables was evaluated using the kappa coefficient, considering values above 0.80 as excellent22. all analyses were performed with spss version 23 (ibm spss®, armonk, ny, usa). results characteristics of participants from the 900 participants in the fibra survey, 647 older people without cognitive deficit had complete data for validation (table 2). the mean age was 72.2 (± 5.3) years, women were predominant (69%), as well as older individuals with up to four years of schooling (72.1%), and approximately half used dental services in the last year (51%). table 2. characteristics of participants (n = 647) variables n (%) age (mean ± sd*) 72.2 (± 5.3) gender male 200 (31) female 447 (69) color white 477 (74) not white 168 (26) education level up to 4 years of study 466 (72.1) 5 years of study or more 181 (27.9) household income* up to 3 mw 262 (46.1) 4 mw or more 306 (53.9) use of odontological services in the last year yes 327 (51) no 314 (49) self-assessment of oral health positive 460 (71.9) negative 180 (28.1) edentulism** yes 309 (47.8) no 338 (52.2) use of complete denture** yes 423 (65.4) no 224 (34.6) use of complete upper denture** yes 418 (64.6) no 229 (35.4) use of complete lower denture** yes 248 (38.3) no 399 (61.7) *mw, minimum wage in 2008: 3 mw = r$1245.00/on average us$693.00. ** oral conditions evaluated clinically. sd, standard deviation. 6 arenas-márquez et al. regarding the oral condition of the participants (figure 1), the clinical prevalence of edentulism was 47%, and the use of cd 65% (in the upper arch 64% and in the lower arch 38%). estimates made by self-report were equivalent in edentulism and overestimated between 5 and 7% in the variables related to denture use. as to the distribution of self-report responses according to the clinical oral condition (supplementary table), a high number of true positives and false negatives and a small number of true negatives and false positives are observed in all variables. validation of oral condition self-report. table 3 revealed that the self-report of older people is valid when compared with the clinical examination. sensitivity and specificity analyzes showed that clinical oral condition was reflected by self-report. from older people who reported having any of the oral conditions evaluated, there was a high percentage that truly had it (sensitivity 95–99%). thus, among older people who reported not having the conditions, a high percentage did not have them (specificity 84–97%). predictive values revealed high odds that the self-report agrees with clinical reality. the four measured variables showed that the probability of truly having a condition when reported was between 81–97% (ppv), and the probability of not having a condition when it was informed so was between 95–98% (npv). figure 1. prevalence of oral conditions. estimates made by clinical examination and self-report are similar. cd, complete denture. clinical prevalence self-reported prevalence edentulism cd upper cd lower cd 100 50 0 table 3. validation of self-reported oral health condition according to clinical oral condition in older people without cognitive deficit. edentulism use of cd use of upper cd use of lower cd sensitivity* (ci) 94.5 (91.3 96.8) 99.3 (97.9 99.8) 99.3 (97.9 99.8) 97.2 (94.3-98.9) specificity* (ci) 97 (94.6-98.6) 84.4 (79.7 89.3) 84.7 (79.4 89.1) 86 (82.2 89.2) ppv* (ci) 96.7 (94.1 98.2) 92.3 (89.8 94.2) 92.2 (89.7 94.1) 81.1 (77.1 84.6) npv* (ci) 95.1 (92.4 96.8) 98.5 (95.5 99.5) 98.5 (95.4 99.5) 98 (95.9 99) +lr value (ci) 31.9 (17.33 58.85) 6.61 (4.87 8.97) 6.50 (4.79 8.81) 6.92 (5.43 8.84) -lr value (ci) 0.06 (0.04 0.09) 0.01 (0.00 0.03) 0.01 (0.00 0.03) 0.03 (0.02 0.07) kappa coefficient** 0.92 0.87 0.87 0.80 ppv, positive predictive value; npv, negative predictive value; +lr, positive likelihood ratio; -lr, negative likelihood ratio; ci, confidence interval; cd, complete denture. * values expressed as percentages. ** p < 0.0001 in chi-square or fisher’s exact tests for all variables. 7 arenas-márquez et al. the likelihood ratio expressed the practicality of self-report as a measure of the true clinical oral condition. the values showed compelling evidence that an edentulous individual (+lr = 32), not edentulous (-lr = 0.06) and that does not use cd (-lr = 0.01) can be properly identified with the self-report. however, it is moderate to identify the presence and location of cd (+lr 6.5 to 6.9). finally, self-report agreement with clinical reality was higher than 0.80 in all variables (kappa coefficient). discussion this research confirms the reliability of self-report for brazilian older people, correctly identifying edentulism and cd use, essential indicators in oral health studies in old age. the high level of confidence and intrinsic quality of the self-report is evidenced by the excellent agreement with the clinical examination, which together with high percentages of sensitivity, specificity and predictive values justify the validity of self-report in individuals without cognitive deficit. the likelihood ratios also confirm this finding. there is strong evidence that self-report can correctly identify presence or absence in most of the evaluated oral conditions. however, the evidence is moderate to identify the presence and location of cd. it is suggested as a hypothesis, maybe due to the clinical criterion used to consider the use or not of cd (presence or absence of cd at the time of oral examination), because even with a denture, some older people only use it occasionally, when feeding, for example. thus, it could be that self-report is measuring reality and clinical judgment, underestimating its use. research on validation of self-report for number of teeth and use of cd has great heterogeneity in the literature. while the majority encompass adult populations23,24, or adults and older people together9,25-29, only one study was conducted with older people population exclusively30. in this study, the agreement between the number of teeth obtained by clinical examination and the one estimated by self-report was verified through telephone interviews with older people in the united states. the researchers did not find significant differences when comparing averages, concluding that self-report is a valid instrument30. literature is also heterogeneous regarding the tests used to validate self-report. only five studies were found using universal measures for validation25-29, and three of them obtained good or excellent values27-29. note that there are no studies conducted in brazil evaluating these oral conditions or exclusively with older people population. this research contributes to this knowledge, involving a representative sample of older people living in the community17 and verifying through the mmse, that the participants had the cognitive capacity to answer the questions. the self-report use may provide additional benefits, allowing the exploration of interrelationships between health self-assessment, behavior and awareness about it, health service use, and sociodemographic variables11. participants’ responses may be influenced by factors, such as the recent use of dental services31 and educational level32. higher schooling is associated with greater ease in recognizing a health need and seeking care33. however, it is considered that these factors were not relevant to the 8 arenas-márquez et al. results obtained in this research, since only a third of them completed the first phase of elementary education and half went to the dentist in the last year. as a limitation of this study is the exclusion of older people who scored below the cutoff in the mmse. this fact may have contributed to the achievement of high values in the validation tests, probably limiting its use to those who do not have cognitive deficit but giving greater fidelity to the measured data. note that the results of this study refer to a population with a high prevalence of the conditions studied, which in brazil have been stable over the years34. however, the evaluation of other oral health conditions, such as the use of other types of denture, periodontal condition and presence of root caries, are frequent in older people and important to consider in future research. additionally it is suggested to review the language used in the formulation of the questions, since maybe not all older people understand the term “natural tooth” (which when restored may not be considered as a natural tooth by all older people) or the term “denture” (which may confuse a patient with removable partial dentures, leading to classifying this prosthetics as denture). considering these observations in future research could make it easier for the answers to be even more representative of reality. finally, note that who recommends countries to establish an oral health information system for follow-up and ongoing evaluation of the national programs35. this organization recognizes the importance of self-report for the identification of appropriate approaches in the promotion and prevention of oral health1. thus, the results obtained in this research contribute to the valorization of this instrument in brazil. this research verified that the self-reported oral condition reflects the clinical oral condition, since older people without cognitive deficit have accurately identified conditions such as edentulism and denture use. it is confirmed that the self-report is a valid instrument to be used in the brazilian context, in epidemiological studies that evaluate these oral conditions in this age group. acknowledgments the authors thank espaço da escrita – pró-reitoria de pesquisa unicamp for the language services provided. arenas-márquez mj was supported by capes (1644168). this research was funded by cnpq (555082-2006/7). arenas-márquez mj was supported by capes (1644168). to national council for scientific and technological development (cnpq) from which j.b.h. and f. n.h. hold a research productivity (pq-2) fellowship. supplementary table. oral health condition self-reported according to the oral clinical condition. oral clinical condition/gold standard edentulism use of cd use of upper cd use of lower cd self-report yes no total yes no total yes no total yes no total yes 292 10 302 420 35 455 415 35 450 241 56 297 no 17 328 345 3 189 192 3 194 197 7 343 350 total 309 338 647 423 224 647 418 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[national health survey in brazil: design and methodology of application]. cien saude colet. 2014 feb;19(2):333-42. portuguese. 15. lebrão ml, de oliveira duarte ya. o projeto sabe no município de são paulo: uma abordagem inicial. brasília: organização pan-americana de saúde, opas/oms; 2003. 255 p. 16. lima-costa mf, de andrade fb, de souza prb, neri al, de oliveira duarte ya, castro-costa e, et al. the brazilian longitudinal study of aging (elsi-brazil): objectives and design. am j epidemiol. 2018 jul 1;187(7):1345-1353. doi: 10.1093/aje/kwx387. 17. neri al, yassuda ms, araujo lf, eulalio mdo c, cabral be, siqueira me, et al. [methodology and social, demographic, cognitive, and frailty profiles of community-dwelling elderly from seven brazilian cities: the fibra study]. cad saude publica. 2013 apr;29(4):778-92. portuguese. 18. cohen jf, korevaar da, altman dg, bruns de, gatsonis ca, hooft l, et al. stard 2015 guidelines for reporting diagnostic accuracy studies: explanation and elaboration. bmj open. 2016 nov 14;6(11):e012799. doi: 10.1136/bmjopen-2016-012799. 10 arenas-márquez et al. 19. brucki sm, nitrini r, caramelli p, bertolucci ph, okamoto ih. [suggestions for utilization of the minimental state examination in brazil suggestions for utilization of the mini-mental state examination in brazil]. arq neuropsiquiatr. 2003;61(3b):777-81. doi: 10.1590/s0004-282x2003000500014. portuguese. 20. world health organization. oral health surveys-basic methods. geneva: who; 1997. 21. farmer j, ramraj c, azarpazhooh a, dempster l, ravaghi v, quinonez c. comparing self-reported and clinically diagnosed unmet dental treatment needs using a nationally representative survey. j public health dent. 2017 sep;77(4):295-301. doi: 10.1111/jphd.12205. 22. sopelete mc. métodos de análise em estudos sobre diagnóstico. in: mineo j, silva d, sopelete m, leal g, vidigal l, tápia l, et al., editors. [biomedical research: from planning to publishing]. uberlândia: edufu; 2009. p. 203-23. portuguese. 23. blizniuk a, ueno m, zaitsu t, kawaguchi y. association between self-reported and clinical oral health status in belarusian adults. j investig clin dent. 2017 may;8(2). doi: 10.1111/jicd.12206. 24. ueno m, zaitsu t, shinada k, ohara s, kawaguchi y. validity of the self-reported number of natural teeth in japanese adults. j investig clin dent. 2010 nov;1(2):79-84. doi: 10.1111/j.2041-1626.2010.00016.x. 25. axelsson g, helgadottir s. comparison of oral health data from self-administered questionnaire and clinical examination. community dent oral epidemiol. 1995 dec;23(6):365-8. 26. palmqvist s, soderfeldt b, arnbjerg d. self-assessment of dental conditions: validity of a questionnaire. community dent oral epidemiol. 1991 oct;19(5):249-51. 27. gilbert gh, chavers ls, shelton bj. comparison of two methods of estimating 48-month tooth loss incidence. j public health dent. 2002 summer;62(3):163-9. 28. allen f, burke f, jepson n. development and evaluation of a self-report measure for identifying type and use of removable partial dentures. int dent j. 2005 feb;55(1):13-6. 29. pitiphat w, garcia ri, douglass cw, joshipura kj. validation of self-reported oral health measures. j public health dent. 2002 spring;62(2):122-8. 30. douglass cw, berlin j, tennstedt s. the validity of self-reported oral health status in the elderly. j public health dent. 1991 fall;51(4):220-2. 31. blicher b, joshipura k, eke p. validation of self-reported periodontal disease: a systematic review. j dent res. 2005 oct;84(10):881-90. 32. liu h, maida ca, spolsky vw, shen j, li h, zhou x, et al. calibration of self-reported oral health to clinically determined standards. community dent oral epidemiol. 2010 dec;38(6):527-39. doi: 10.1111/j.1600-0528.2010.00562.x. 33. almeida a, nunes bp, duro sms, facchini la. socioeconomic determinants of access to health services among older adults: a systematic review. rev saude publica. 2017 may 15;51:50. doi: 10.1590/s1518-8787.2017051006661. 34. ministry of health of brazil. [sb brazil 2010: national research on oral health: main results]. brasília: ministry of health; 2012. 116p. portuguese. 35. petersen pe. improvement of global oral health--the leadership role of the world health organization. community dent health. 2010 dec;27(4):194-8. oral sciences n3 original article braz j oral sci. october | december 2013 volume 12, number 4 effects of zirconia nanoparticles addition to experimental adhesives on radiopacity and microhardness gislaine cristine martins1, marcia margarete meier2, alessandro dourado loguercio3, fabielle cecchin3, osnara maria mongruel gomes3, alessandra reis3 1department of restorative dentistry, dental school, pontifical catholic university of paraná, curitiba, paraná, pr, brazil 2department of chemist, state university of santa catarina, joinville, sc, brazil 3department of restorative dentistry, dental school, state university of ponta grossa, ponta grossa, pr, brazil correspondence to: alessandra reis departamento de odontologia, universidade estadual de ponta grossa avenida carlos cavalcanti, 4748, cep: 84030-900 uvaranas, ponta grossa, pr, brasil e-mail: reis_ale@hotmail.com abstract aim: to evaluate the radiopacity and microhardness (khn) of experimental dental adhesives (ex). the experimental adhesive resins of the present study were formulated based on the simplified adhesive system ambar (fgm). methods: five ex with different concentrations of zirconia nanoparticles [0(ex0), 15(ex15), 25(ex25), 30(ex30) e 50%(ex50)] were incorporated in a udma/hema adhesive (control). adper single bondtm 2 (sb, 3m espe) was used as a commercial reference. for the radiopacity (n=5), khn (n=5), adhesive specimens were fabricated using a stainless steel mold. data were submitted to one-way anova and tukey´s test (α=0.05). results: the filler addition on the ex showed radiopacity similar to enamel and higher than sb. the ex25, ex35 and ex50 showed higher khn values when compared to the commercial sb. ex25, ex35 and ex50 showed higher khn values when compared to the commercial sb. conclusions: the results of the present investigation suggest that the addition of zirconia nanoparticles seems to be a good alternative to produce radiopaque adhesives with increased microhardness. keywords: radiology, contrast media, nanoparticles. introduction the radiopacity of adhesive materials is clinically relevant, mainly in technique-sensitive restorations, where evaluation of the tooth/restorative interface is critical1,2. unfortunately, the great majority of the commercial adhesives available are radiolucent3 and they cannot be clearly detected in radiographs4 when applied in layers thicker than 40 µm. this is a pertinent clinical concern since adhesive layers should be distinguished from marginal gaps with potential for secondary caries. the radiopacity of esthetic restorative materials has been established as an important requirement, improving the radiographic diagnosis. to the extent of our knowledge, the flowable composites5,6, composite resins7 or resin cements6,8 available are radiopaque and only bonding agents are still radiolucent9. the development of radiopaque adhesive systems can avoid inappropriate replacements10,11 due to misinterpretations in the diagnosis of secondary caries2,10,12 and detection of gaps near the restoration4,12. thus, the studies should focus on received for publication: september 09, 2013 accepted: december 03, 2013 braz j oral sci. 12(4):319-322 table 1. table 1. table 1. table 1. table 1. composition of adhesive systems and application mode. adhesive systems adper single bond™ 2(3m espe, st. paul, mn, usa) experimental adhesives* (fgm dental products, joinville, sc, brazil) composition bis-gma; polyalkenoic acid copolymer; dimethacrylates; hema; photoinitiators; ethanol; water; nanofiller particles udma (5-40), hema (5-40), methacrylate acidic monomers (1– 20), methacrylate hydrophilic monomers (5-40), silanized silicon dioxide (<1), camphorquinone (<1), 4-edamb (<1), ethanol (<20) application mode 1. apply generous amounts of adhesive. actively scrub for 15s; 2. application of a second coat of adhesive, as above; 3. removal of excess solvent by gently drying with an airstream for 15 s at 15 cm away from the surface, gradually bringing it to within 10 mm of distance; 4. light cure for 10 s batch number 8rf 2011-05 e x 0 : 0606231 ex15: 080410 ex25: 080410 ex35: 080410 ex50: 080410 bis-gma: bisphenol-glycydil methacrylate; hema: 2-hydroxyethyl methacrylate; udma: urethane dimethacrylate; 4-edamb: ethyl-4-dimethyl. *to the original composition of the adhesive ambar, varied concentrations of barium-borosilicate glass particles were added to produce the ex0, ex15, ex25, ex35 and ex50 formulations, described in the materials and methods section. the investigation of bonding features and mechanical properties of radiopaque adhesive systems. recently, there has been a great interest in the application of nanotechnology in resin-based materials13,14 to produce dental composite materials with increased hardness7,13,15. these promising findings led investigators to evaluate the effects of filler addition on the mechanical properties16-20 as well as bonding features of adhesives18,22. however few of them incorporated nano-sized filler particles into the adhesive formulations13,15 and evaluated the role of nanofillers on the material’s radiopacity18,20. therefore, the aim of the present investigation was to evaluate the incorporation of varied concentrations of zirconia nanofiller into a two-step etch-and-rinse adhesive on the material´s radiopacity and microhardness. material and methods this research project was approved by the institutional review board from the local dental school under protocol # 28/2010. the experimental adhesive resins from the present study were formulated using the simplified adhesive system ambar (fgm dental products, joinville, sc, brazil) as base. this material was specifically formulated for this study without any filler content. the detailed composition of this adhesive system, as provided by the manufacturer, can be seen in table 1. the simplified etch-and-rinse commercial adhesive system (adper single bondtm 2; 3m espe, st. paul, mn, usa) was used as reference (table 1). zirconia oxide nanoparticles (20-30 nm average particle size) (transparent materials, rochester, nova york, usa) were silanized by gamma-methacryloxypropyltrimethoxysilane (aldrich chemical co., milwaukee, wi, usa) as reported before23. after the silanization process, the nanoparticles were dried for 24 h at 37°c and then disaggregated in a pistil. five experimental adhesive systems were formulated according to the filler weight percentage (wt%): 0 (ex0), 15 (ex15), 25 (ex25), 35 (ex35) and 50% (ex50). using a circular stainless steel mold, five specimens measuring 5.0 mm in diameter and 1.0 mm thick were prepared for each material. the adhesive was dispensed in the mold until complete filling. all visible air bubbles trapped in the adhesive solution were carefully removed. the solvent was evaporated by gentle air blowing from a dental syringe for 40 s. each specimen was polymerized for 80 s with a visiblelight curing unit (vip; bisco inc., schaumburg, il, usa) with a power density 450 mw/cm2. enamel and dentin specimens were obtained from 1.0-mm thick longitudinal sections of human third molars previously stored in 0.5% thymol and used within 6 months after extraction. slices were prepared using a low-speed diamond blade (isomet 1000; buehler, lake bluff, il, usa) mounted in a cutting machine under water cooling. a total of five radiographs were made. each radiograph was taken with one specimen of each experimental condition and the enamel-dentin slice positioned on the digital sensor. an digital radiography was then taken with an exposure time of 0.2 s. the radiographic position was standardized: the radiographic central beam focusing in a 90° angle with the surface of the image receptor, at a 30 cm focus-object distance and parallelism between the sensor and the specimens with the heliodent vario machine (sirona, bensheim, germany). the digital radiopacity (% white) was measured by pixels counting using the uthscsa imagetool 3.0 software (department of dental diagnostic science, university of texas health science center, san antonio, tx, usa). data for each property was subjected to one-way anova. effects of zirconia nanoparticles addition to experimental adhesives on radiopacity and microhardness320 braz j oral sci. 12(4):319-322 post-hoc multiple comparisons were performed using tukey’s test at a significance level of 5%. results one-way anova detected statistically significant difference among groups (table 2, p<0.001). all experimental bonding adhesives showed radiopacity similar to enamel, except for ex0 and sb. these two adhesives showed radiopacity similar to the dentin substrate. for khn oneway anova detected significant differences between groups (table 2, p<0.0001). in regard to khn, the addition of filler loading equal or higher than 25% produced materials with increased microhardness when compared with the commercial sb. the unfilled ex0 and the lightly filled ex15 showed intermediate microhardness between these extremes. groups pixel intensity microhardness (khn) enamel 68.8±15.3 a — dentin 33.0±8.1 b — sb 26.9±1.2 b 3.3±1.2 c ex0 27.1±1.0 b 5.8±0.7 bc ex15 60.5±7.9 a 5.7±0.3 bc ex25 71.2±6.7 a 7.5±1.5 ab ex35 61.0.9±9.2 a 8.8±1.8 a ex50 73.3.0±7.4 a 8.8±1.8 a table 2. table 2. table 2. table 2. table 2. means and standard deviations the enamel, dentin and adhesive systems radiopacity by pixel intensity as well as knoop microhardness of the adhesive solutions. comparisons are valid within columns. averages identified with the same letters indicate statistically similar means (p>0.05). discussion it is noteworthy to mention that the monomeric composition of the commercially available two-step etchand-rinse adhesive ambar (fgm dental products, brazil) was employed. this simplified adhesive contains nanofillers, which were especially removed by the manufacturer preparing the evaluated experimental adhesives. therefore, the experimental adhesives contained only the filler loading added by the authors. the radiopacity of esthetic restorative materials has been established as an important requirement, improving radiographic diagnosis 1,2,24. radiolucent areas around restorations may result from either a halo effect or the radiographic density of the adhesives. therefore, the use of an adhesive with radiopaque fillers can avoid inappropriate replacements10,11 due to misinterpretations in the diagnosis of secondary caries2,10,12. for adequate assessment of the restoration quality both at baseline and in recall examinations, radiographic evaluations are very useful and for such, dental materials should be sufficiently radiopaque to be detected against a background of sound and caries-affected enamel and dentin substrate, and thus allow correct evaluation of the presence of secondary caries, marginal defects, contour of restoration, and contact with adjacent teeth, cement overhangs and interfacial gaps1,2,9. to the extent of the authors’ knowledge only one study investigated the impact of filler addition on the adhesive radiopacity22 and this is therefore a novel study that attempts to investigate this issue. the results of the present study showed that the addition of zirconia nanoparticles yielded enamel radiopacity to the experimental adhesives evaluated. even the smallest percentage of zirconia nanoparticles evaluated in this study (15%) produced a radiopaque experimental adhesive similar to the radiopacity of the enamel substrate. similarly schulz et al. also observed increased adhesive radiopacity after inclusion of agglomerated ta2o5/sio2 nanoparticles. dental adhesives are intricate mixtures of components and they are designed to bond composite resins to enamel and dentin25,26. irrespective of the number of bottles, an adhesive system typically contains resin monomers, curing initiators, inhibitors, stabilizers, solvents and inorganic filler. each one of these components has a specific function25. although the addition of filler to adhesives has shown to be beneficial20 this finding is not consensual in the literature20,27. the addition of filler particles to composite resins is made in an attempt to improve the mechanical properties of dental composites13,25,28,29. similarly, the present investigation showed that the addition of filler loading higher equal to or higher than 25% allowed the increase in the microhardness of the experimental adhesives. a recent study also demonstrated that the addition of hydroxyapatite nanoparticles30 and niobium pentoxide31 also produced an adhesive layer with increased microhardness and other superior properties. the commercial sb adhesive showed the lowest khn. this means that the attainment of higher microhardness is not dependent only on the filler loading. an adhesive system with adequate and balanced monomer composition blend may also lead to the production of a polymeric material with increased properties. compared to the commercial sb adhesive, the unfilled experimental adhesive (ex0) showed higher knh and such difference may be attributed to composition differences in monomer blends. the addition of filler loading in moderate concentrations (ex25 and ex35) produced radiopaque materials. compared to the commercial sb adhesive, the unfilled experimental adhesive (ex0) with higher knh that were either improved or remained unchanged compared to the unfilled version. the results of the present investigation suggest that the addition of zirconia nanoparticles seems to be a good alternative to produce radiopaque adhesives with increased microhardness. further studies should focus on the enamel and dentin bonding strength of adhesive systems with zirconia nanoparticles. acknowledgments the authors would like to thanks fgm dental products for the generous donation and manipulation of the products employed in this study. this study was partially supported effects of zirconia nanoparticles addition to experimental adhesives on radiopacity and microhardness 321 braz j oral sci. 12(4):319-322 by the national council for scientific and technological development (cnpq) under grants 301937/2009-5 and 301891/2010-9. the authors of this study are very grateful to iverson e. woyceichoski for the help in the radiographs and luiz lima junior for the help in microhardness test. references 1. tveit ab, espelid i. radiographic diagnosis of caries and marginal defects in connection with radiopaque composite fillings. dent mater. 1986; 2: 159-162. 2. espelid i, tveit ab, erickson rl, keck sc, glasspoole ea. radiopacity of restorations and detection of secondary caries. dent mater. 1991; 7: 114-117. 3. hotta m, yamamoto k. comparative radiopacity of bonding agents. j adhes dent. 2009; 11: 207-212. 4. 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m, et al. microtensile bond strength of a filled vs unfilled adhesive to dentin using self-etch and total-etch technique. j dent. 2006; 34: 283-291. 21. lee yk, pinzon lm, o’keefe kl, powers jm. effect of filler addition on the bonding parameters of dentin bonding adhesives bonded to human dentin. am j dent. 2006; 19: 23-27. 22. schulz h, schimmoeller b, pratsinis se, salz u, bock t. radiopaque dental adhesives: dispersion of flame-made ta2o5/sio2 nanoparticles in methacrylic matrices. j dent. 2008; 36: 579-587. 23. martins gc, meier mm, loguercio ad, reis a, gomes jc, gomes om. effects of adding barium-borosilicate glass to a simplified etch-and-rinse adhesive o radiopacity and selected properties. j adhes dent. 2013 oct 2. [epub ahead of print]. 24. matteson sr, phillips c, kantor ml, leinedecker t. the effect of lesion size, restorative material, and film speed on the detection of recurrent caries. oral surg oral med oral pathol. 1989; 68: 232-237. 25. van landuyt kl, snauwaert j, de munck j, peumans m, yoshida y, poitevin a, et al. systematic review of the chemical composition of contemporary dental adhesives. biomaterials. 2007; 28: 3757-3785. 26. finger wj,fritz ub. resin bonding to enamel and dentin with one-component udma/hema adhesives. eur j oral sci. 1997; 105: 183-186. 27. lee jh, um cm, lee ib. rheological properties of resin composites according to variations in monomer and filler composition. dent mater. 2006; 22: 515-526. 28. conde mc, zanchi ch, rodrigues-junior sa, carreno nl, ogliari fa, piva e. nanofiller loading level: influence on selected properties of an adhesive resin. j dent. 2009; 37: 331-335. 29. kim kh, ong jl, okuno o. the effect of filler loading and morphology on the mechanical properties of contemporary composites. j prosthet dent. 2002; 87: 642-649. 30. leitune vc, collares fm, trommer rm, andrioli dg, bergmann cp, samuel sm. the addition of nanostructured hydroxyapatite to an experimental adhesive resin. j dent. 2013; 41: 321-327. 31. leitune vc, collares fm, takimi a, de lima gb, petzhold cl, bergmann cp, et al. niobium pentoxide as a novel filler for dental adhesive resin. j dent. 2013; 41: 106-113. effects of zirconia nanoparticles addition to experimental adhesives on radiopacity and microhardness322 braz j oral sci. 12(4):319-322 oral sciences n3 original article braz j oral sci. october | december 2014 volume 13, number 4 taste alteration, mouth dryness and teeth staining as side effects of medications taken by elderly camila porto de deco1, michelle roberta vieira silva reis2, adriana mathias pereira da silva marchini1, rosilene fernandes da rocha1, mateus bertolini fernandes dos santos3, leonardo marchini4 1universidade estadual paulista – unesp, são josé dos campos dental school, department of biosciences and oral diagnosis, são josé dos campos, sp, brazil 2private practice – são josé dos campos, sp, brazil 3universidade federal de pelotas – ufpel, school of dentistry, department of restorative dentistry, pelotas, rs, brazil 4university of iowa, college of dentistry, department of preventive and community dentistry, iowa city, ia, usa correspondence to: mateus bertolini fernandes dos santos universidade federal de pelotas faculdade de odontologia rua gonçalves chaves, 457/502 cep: 96015-560 pelotas, rs, brasil phone: +55 53 9934 9134 e-mail:mateusbertolini@yahoo.com.br abstract elderly patients generally use several types of medication, some of which may cause oral side effects. aim: to investigate the oral side effects caused by medication in an elderly sample. methods: three hundred patients were interviewed about their use of medication and were divided in two groups: institutionalized (n=150) and community-dwelling (n=150) elderly. results: the most used drugs were antihypertensives (53%) for community-dwelling elders and antiulceratives (76%) for the institutionalized ones. the more prevalent side effects were taste alterations that occurred in 19%, dry mouth in 17% and teeth staining in 2%. conclusions: a high prevalence of oral side effects from medications used by the elderly was found in this study. the health professionals should be aware of the possible side effects caused by prescribed medications. keywords: aged; drug interactions; xerostomia. introduction aging is characterized by accentuated changes in many organs of the human body1. the association of these changes and age-related pathologies usually result in the need for essential medication to maintain the elderly’s quality of life. generally, the elderly use several types of medication2. however, the use of multiple medications must be undertaken with caution, as aging also promotes important changes concerning their absorption. many medications could be dangerous if used together and a great number of medications used by the elderly could also result in an increase of undesirable side effects3. many undesirable oral side effects may be caused by commonly used medications by the elderly. the most common side effect is mouth dryness (xerostomia)4-5. xerostomia can affect eating, drinking, swallowing and the retention of removable dentures4, reducing the elderly’s quality of life6. another undesirable oral side effect is taste alteration7. abnormal taste perceptions in the elderly may impair nutritional status and also negatively affect quality of life8. the aim of this study was to investigate which oral side effects could be the result of medication taken by an elderly population sample. considering the received for publication: july 27, 2014 accepted: november 25, 2014 braz j oral sci. 13(4):257-260 institutionalized community-dwelling total antihypertensives 47% (n=82) 53% (n=92) 58% (n=174) diuretics 61% (n=42) 39% (n=27) 23% (n=69) nsaids* 50% (n=34) 50% (n=34) 23% (n=68) vitamin supplements 80% (n=52) 20% (n=13) 22% (n=65) antidiabetics 55% (n=27) 45%(n=22) 16% (n=49) antiulceratives 76% (n=32) 24% (n=10) 14% (n=42) table 1 table 1 table 1 table 1 table 1 pharmacological classification of medication used by an elderly sample of são josé dos campos, sp, brazil in 2012. *nsaids= non-steroidal anti-inflammatory drugs pharmacological class antihypertensives diuretics nsaids* vitamin supplements antidiabetics diuretics <0.001 nsaids* <0.001 0.923 vitamin supplements <0.001 0.695 0.768 antidiabetics <0.001 0.040 0.050 0.096 antiulceratives <0.001 0.005 0.006 0.014 0.426 table 2 -table 2 -table 2 -table 2 -table 2 p-values chart comparing the use of the pharmacological classes in the sample (two ratios equality test). *nsaids= non-steroidal anti-inflammatory drugs results of previous studies 4-5, it was hypothesized that xerostomia could be the most common oral side effect. material and methods sample size the sample size for this study was calculated using the minitab (minitab inc., state college, pa, usa) power and sample size tool. three hundred elderly subjects aged 60 years or over from the elderly residents of são josé dos campos, sp, brazil, were randomly included and evaluated. this number represented about 1.27% of the city’s elderly population, as described previously in another study2. the study sample received a sample power score of 0.94. the included patients were divided in two groups: institutionalized (n=150) and community-dwelling (n=150). the use of any medication was not considered as inclusion criterion. this survey was approved by the research ethics committee of the university of vale do paraíba (protocol l102/2004/cep)2. one single professional visited and interviewed all the patients and the obtained data were recorded on a custommade form, medications were then classified and their possible oral side effects were evaluated by a literature review before being documented. the whole data set was then analyzed statistically. statistical analysis the data were evaluated using two ratios equality test, kruskal-wallis test, anova, mann-whitney test and spearman’s correlation test. all variables (gender, institutionalized or community-dwelling, use and number of medications, pharmacological class and side effects) were compared to check for possible significant differences and/ or correlations. the level of significance adopted in all tests was set at 95%. results sample description in the sample, 70% (n=211) were women. the institutionalized group was older than the communitydwelling group (anova, p<0.001). the average age of the institutionalized group was 76.69 years, and among the community-dwelling group it was 71.89 years. use of medication in this sample, only 52 (17.3%) did not use any medications, which was significantly different from the number of patients that took at least one medication (two ratios equality test; p<0.001), the institutionalized elderly used more (kruskalwallis, p<0.001) medication (3.26) than the communitydwelling group (1.97), as seen previously in another study2. there was no correlation between age and number of medications used (spearman’s correlation, p<0.173). the most common type of medication used by the elderly were antihypertensive drugs (table 1), which presented a statistically significant difference to the other medications (58%, two ratios equality test, p<0.001), as observed in table 2. oral side effects taste alteration was the most frequent oral side effect, occurring in 19% of the total sample, followed by dry mouth (17.3%) and teeth staining (2.3%). table 3 illustrates the oral side effects, which could be 258258258258258taste alteration, mouth dryness and teeth staining as side effects of medications taken by elderly braz j oral sci. 13(4):257-260 taste alterations xerostomia teeth staining antihypertensives inst 57% (n=47) 17% (n=14) c-d 80% (n=74) 9% (n=8) diuretics inst 12% (n=5) c-d nsaids* inst 3% (n=1) c-d 5% (n=2) supplements inst 4% (n=2) 29% (n=15) c-d 23% (n=3) 23% (n=15) antidiabetics inst 18% (n=5) c-d 4% (n=1) antiulcerants inst 12% (n=4) 3% (n=1) c-d 10% (n=1) 40% (n=4) table 3 -table 3 -table 3 -table 3 -table 3 oral side effects possibly caused by each pharmacological classification group that were reported by the elderly patients. *nsaids = non-steroidal anti-inflammatory drugs, inst. = institutionalized elderly, c-d= communitydwelling elderly. caused by each medication group and the percentage of elderly who presented the effects. antihypertensives were the most common type of medication to cause oral side effects. discussion as previously stated, xerostomia is a common undesirable side effect of polypharmacy among the elderly45, which negatively affects the quality of life6. in the studied sample, xerostomia was the second most common undesirable oral side effect, after taste alteration. thomson et al.9, in a longitudinal study with australian elderly, observed that the prevalence and incidence of xerostomia was strongly associated with medication. they observed that diuretics caused xerostomia more frequently. also, in this longitudinal study, other medications such as aspirin (on a daily basis), non-steroidal anti-inflammatory drugs (nsaids) and antidepressants caused side effects9. in the present sample, it was possible to notice that patients using antihypertensive drugs, diuretics, nsaids, antiulcerants and even vitamin supplements reported xerostomia. it is important to state that since the vast majority of the patients did not use only one single medication, it is difficult to conclude what pharmacological class of the medications caused xerostomia as a side effect or if it was a result of polypharmacy. pajukoski et al.10 compared 175 hospitalized patients with 252 patients from outpatient clinics, in helsinki. they concluded that the greater the number of medications, the greater the probability of xerostomia. considering that xerostomia was the second most common oral side effect found and that there was considerable polypharmacy in the sample of this study, the here results presented corroborate them. furthermore, some authors have observed that physiological age-related changes in salivary glands could have a synergistic effect with polypharmacy for causing xerostomia4-5. the management of the xerostomia condition should include patient education, where the patient receives information about the potential sequelae of hyposalivation, including dental caries, candidiasis and mucosal complications11-12. it was also found that the use of palliative treatments for dry mouth, such as rinses, sprays, gels and chewing gums could alleviate the symptoms of xerostomia12. however, according to a cochrane review made by furness et al.13, there is no strong evidence that any topical therapy is effective for relieving the symptoms of dry mouth. the most common undesirable oral side effect found in this study was abnormal taste perception. this may have negative effects on the elderly’s general health as it could impair nutritional status. indeed, taste alteration can affect physical and mental health, and even survival rates8. the results presented here suggest that commonly used antidiabetic and antihypertensive medication may cause taste alterations. most taste alterations induced by medication may reduce after medicine withdrawal, but this could take up to 4 months8. strategies to reduce the risk of adverse drug events include discontinuing medications, prescribing new medications sparingly, reducing the number of prescriptions and frequent medications reconciliation14. however, in some cases, discontinuing treatment or changing the medication is not an option, as some medications are required to be taken for the patient’s entire life. health professionals should be aware observing these changes and to dialogue with the medical staff about possible changes in the medication of patients in order to promote the elderly’s well-being and quality of life. in elderly patients, stains are caused by extrinsic pigmentation of teeth and commonly caused by tea, coffee, red wine, tobacco smoking, food with dyes, and numerous medications, such as chlorhexidine, oral iron salts in liquid form, essential oils and co-amoxiclav15. tooth staining is not considered to be a public health problem. however, it has been reported that drug-induced tooth staining is a common cause of decline in use or withdrawal of certain medications16. on the other hand, it is important to state that most of extrinsic stains in teeth may be removed by external cleaning15. 259259259259259 taste alteration, mouth dryness and teeth staining as side effects of medications taken by elderly braz j oral sci. 13(4):257-260 the results of this study suggest that medication used by the elderly may cause a high prevalence of oral side effects, such as taste alterations, dry mouth and also teeth staining. these side effects could compromise feeding, talking, esthetics and even nutritional status. the interpretation of the present results should consider some limitations inherent to this study. it is very important that future studies assess what medications have higher potential to cause oral side effects and/or what combination of medications may cause such problems. also, the city where the study was conducted is considered to be a well-developed city in brazil, both economically and socially, which may explain the large number of patients that made use of at least one medication. references 1. tamine k, ono t, hori k, kondoh j, hamanaka s, maeda y. age-related changes in tongue pressure during swallowing. j dent res. 2010; 89: 1097-101. 2. de deco cp, dos santos jf, da cunha v de p, marchini l. general health of elderly institutionalised and community-dwelling brazilians. gerodontology. 2007; 24: 136-42. 3. fick dm, cooper jw, wade we, waller jl, maclean jr, beers mh. updating the beers criteria for potentially inappropriate medication use in older adults: results of a us consensus panel of experts. arch intern med. 2003; 163: 2716-24. 4. gueiros la, soares ms, leao jc. impact of ageing and drug consumption on oral health. gerodontology. 2009; 26: 297-301. 5. leal sc, bittar j, portugal a, falcao dp, faber j, zanotta p. medication in elderly people: its influence on salivary pattern, signs and symptoms of dry mouth. gerodontology. 2010; 27: 129-33. 6. gerdin ew, einarson s, jonsson m, aronsson k, johansson i. impact of dry mouth conditions on oral health-related quality of life in older people. gerodontology. 2005; 22: 219-26. 7. souza lm, riera r, saconato h, demathe a, atallah an. oral drugs for hypertensive urgencies: systematic review and meta-analysis. sao paulo med j. 2009; 127: 366-72. 8. schiffman ss. effects of aging on the human taste system. ann n y acad sci. 2009; 1170: 725-9. 9. thomson wm, chalmers jm, john spencer a, slade gd, carter kd. a longitudinal study of medication exposure and xerostomia among older people. gerodontology. 2006; 23: 205-13. 10. pajukoski h, meurman jh, halonen p, sulkava r. prevalence of subjective dry mouth and burning mouth in hospitalized elderly patients and outpatients in relation to saliva, medication and systemic diseases. oral surg oral med oral pathol oral radiol endod. 2001; 92: 641-9. 11. ismail ai, tellez m, pitts nb, ekstrand kr, ricketts d, longbottom c, et al. caries management pathways preserve dental tissues and promote oral health. community dent oral epidemiol. 2013; 41: e12-40. 12. plemons jm, al-hashimi i, marek cl. managing xerostomia and salivary gland hypofunction: executive summary of a report from the american dental association council on scientific affairs. j am dent assoc. 2014; 145: 867-73. 13. furness s, worthington hv, bryan g, birchenough s, mcmillan r. interventions for the management of dry mouth: topical therapies. cochrane database syst rev. 2011; (12): cd008934. 14. pretorius rw, gataric g, swedlund sk, miller jr. reducing the risk of adverse drug events in older adults. am fam physician. 2013; 87: 331-6. 15. abdollahi m, rahimi r, radfar m. current opinion on drug-induced oral reactions: a comprehensive review. j contemp dent pract. 2008; 9: 1-15. 16. kumar a, kumar v, singh j, hooda a, dutta s. drug-induced discoloration of teeth: an updated review. clin pediatr (phila). 2012; 51: 181-5. 260260260260260taste alteration, mouth dryness and teeth staining as side effects of medications taken by elderly braz j oral sci. 13(4):257-260 oral sciences n3 original article braz j oral sci. october | december 2014 volume 13, number 4 reliability of measurements on virtual models obtained from scanning of impressions and conventional plaster models débora duarte moreira1, bruno frazão gribel1, gina delia roque torres1, karla de faria vasconcelos1, deborah queiroz de freitas1, gláucia maria bovi ambrosano2 1universidade estadual de campinas unicamp, piracicaba dental school, departament of oral diagnosis, area of oral radiology, piracicaba, sp, brazil 2universidade estadual de campinas unicamp, piracicaba dental school, departament of community and preventive dentistry, area of biostatistics, piracicaba, sp, brazil correspondence to: débora duarte moreira faculdade de odontologia de piracicaba unicamp departamento de diagnóstico oral avenida limeira, 901 caixa postal 52 cep: 13414-903 piracicaba, sp, brasil phone: +55 19 21065327 e-mail: dededm@hotmail.com abstract aim: to evaluate the reliability of linear measurements in virtual models by comparing measurements performed on virtual models obtained from alginate impression scans, plaster model and measurements performed on conventional plaster model. methods: the sample comprised 26 randomly selected patients to have impressions of their upper and lower jaws taken using alginate and their bite registration using a wax bite. the virtual models were obtained by scanning the alginate impression and the plaster model in a laser surface scanner (r700; 3shape, copenhagen, denmark), and the measurements were performed using the ortho analyser (3shape) proprietary software. the linear measurements of the size of the teeth mesial to distal, arch perimeter, intercanine distance and intermolar distance in the upper and lower arches were performed on plaster models, digital impressions and digital models, by three observers and repeated after 15 days on 8 models for intra-observer evaluations. data were tabulated and analyzed statistically. intra-class correlation to check the agreement of intra and inter-observers and anova test were used to analyze the differences between measurements of digital models from impression and digital models from plaster. results: the results showed a statistically significant difference (pd”0.05) for the posterior teeth, anterior teeth, upper arch perimeter and lower inter-canine distance, comparing the digital models with plaster models, but these differences are considered clinically non-significant. conclusions: digital models were proven be reliable and clinically acceptable for measuring tooth width, perimeter arches, intercanine and intermolar distances. keywords: diagnosis; orthodontics; dental models; digitalis. introduction the study of plaster models is an essential prerequisite for successful orthodontic treatment planning1-2. traditionally, information is gathered from plaster orthodontic models, direct measurement or 2d photographs and radiographs3-5. however, analyzing plaster models can be a time-consuming procedure3. since 1990, digital technology is becoming part of the orthodontic records in many orthodontic practices. in order to improve the quality and efficiency of the record-taking consultation, digital photos, x-rays and more recently 3d study models are becoming the standard orthodontic record in many practices across the world6-9. more recently companies have developed scanning technologies to produce received for publication: september 02, 2014 accepted: december 05, 2014 braz j oral sci. 13(4):297-302 298298298298298 digital models not only by direct plaster model scans but also direct alginate impression scans. these digital models can be used for visualization as well as for taking digital measurements using proprietary software4. moreover, no physical space is necessary to store the records and they facilitate retrieving and sharing information with dental labs and colleagues in multidisciplinary treatments, contributing also for better practice management6-8. on the other hand, digital models require investment in hardware acquisition and training for hardware/software correct manipulation4,10,12. few studies have looked at the accuracy, reproducibility and reliability of measurements taken from 3d digital models in order to validate its use in the routine orthodontic practices1,3,5,8,10. the accuracy and reproducibility of such measurements may be influenced by a number of factors including tooth position (rotation, inclination and angulations), anatomical variations, position of the interproximal contact points, as well as inter-examiner variability due to lack of familiarity with the software4. the reliability of measurements on virtual models may also be influenced by some factors such as scanners and softwares. the use of digital models is of great interest to all orthodontic practitioners, particularly if the impression can be scanned and the plaster models are not required to be poured, trimmed and polished. some studies evaluated the reliability of measurements obtained of digital models from plaster models3,4,6-7,10-11,13-14. however, there are no studies that evaluated the reliability of digital models scanned by this kind of scanner and from alginate impression. the aim of this study was to evaluate the reliability of linear measurements in virtual models by comparing measurements taken from plaster model with those taken from digital models obtained by two different scanning methods: 1) direct plaster model scan and 2) direct impression model scan. material and methods this study was approved by the ethics committee in research of the state university of campinas (protocol # 120/2011). a sample size of 26 individuals was statistically determined to be adequate. subjects were on average 34.7 years of age (18 to 58 years-old) and reported to a radiology clinic for regular orthodontic record taking. a written consent was obtained from each patient. acquisition of models impressions of the upper and lower arches were taken using ortho print (zhermack, rovigo, italy) and their bites registered using conventional utility wax 1 mm. the inclusion criterion was permanent dentition without braces bonded to the teeth. patients with removable dentures were requested to remove it so that the impression and bite registration could be taken. all scans (impressions and models) were made using a surface laser scanner r700 (3shape, copenhagen, denmark). the scanner was calibrated using the recommended calibration kit twice daily to ensure that the optimal accuracy claimed by the manufacturer (20 µm) could be reached. the scans were made according to the manufacturer’s instructions in the handbook. first the upper impression was placed inside the scanner using the impression holding fixture. next, after the automated scanning of the upper impression, the lower impression was placed inside the scanner. at last, the bite record was placed inside the scanner using the bite registration fixture and scanned. the total scanning time including the creation of a virtual base was approximately 40 min. after the impressions were scanned, the plaster model was poured (using conventional white plaster) and an upper and lower model was also scanned separately. after the upper and lower models were placed in occlusion using the bite registration, the set was scanned using the 2-cast fixture, provided by the scanner manufacturer. the total scanning time including the creation of a virtual base was approximately 30 min. model analysis three different observers that had a short 5-case training for calibration purposes performed the measurements. each observer was trained independently and the 5 cases used for this purpose were not included in this study. all measurements were taken in the following order: plaster models, alginate impression scans and plaster model scans. each observer measured 3 sets (upper and lower) of models per day. the plaster models were measured using a digital caliper (sc-6 digital caliper, mitutoyo corporation, tokyo, japan) and all digital measurements were made using ortho analyser software (3shape). the measurements used in this study were: tooth width (greatest mesial-distal distance, measured from the buccal view); intercanine distance icd (distance between the right and left canine incisor tip). intermolar width imd (distance between the right and left first molar lingual/palatal sulcus). arch perimeter ap (sum of the distance between the mesial of the first molar to the distal of the canines and the distal of the canines to the mesial of the central incisors, measured at the papilla level). a total of 731 measurements were taken from the 26 model sets. eight model sets were randomly chosen to be reassessed by the observers within a period of 15 days to verify the reproducibility of the measurements. statistical analysis data were analyzed using sas software 9.1 (sas institute, cary, nc, usa). the intraclass correlation coefficient (icc) was calculated for the intraand interobserver agreement. all measurements were analyzed statistically using anova with tukey correction. the significance level was 5%. braz j oral sci. 13(4):297-302 reliability of measurements on virtual models obtained from scanning of impressions and conventional plaster models 299299299299299 results intraand inter-observer agreement the values of icc were good or excellent, according to szklo and nieto15.the intra-observer agreement of the observer 1 varied from 0.78 to 1.00; observer 2 varied from 0.74 to 1.00 and observer 3 from 0.75 to 1.00. the observer agreement varied from 0.53 to 1.00 for the plaster models measurements; from 0.51 to 1.00 for the impression models scans and from 0.68 to 0.99 for the plaster model scans. digital models accuracy table 1 shows the means and standard deviations (sd) of all measurements. there was a statistically significant difference between the tooth width measurements, upper arch perimeter and lower intercanine distance between the digital and plaster models. table 1. table 1. table 1. table 1. table 1. descriptive statistics and results of the statistical analysis of the measurements performed on the plaster model, virtual molding and virtual model # confidence interval of 95%. means followed by different letters horizontally differ (p < 0.05). ci, confidence interval; t, tooth; uap, upper arch perimeter; uicd, maxillary intercanine distance; uimd, maxillary intermolar distance; lap, mandibular arch perimeter; licd, mandibular intercanine distance; limd, mandibular intermolar distance. tooth plaster model impression scan model scan mean standard ic95%# mean standard ic95%# mean standard ic95%# deviation deviation deviation t16 9.78c 0.58 9.54-10.02 10.21a 0.66 9.93-10.48 9.99b 0.60 9.74-10.23 t15 6.56b 0.53 6.35-6.78 6.86a 0.38 6.71-7.01 6.80a 0.43 6.63-6.97 t14 6.91c 0.50 6.69-7.12 7.18a 0.54 6.94-7.41 7.06b 0.53 6.83-7.28 t13 7.77a 0.39 7.62-7.93 7.81a 0.43 7.64-7.98 7.77a 0.48 7.57-7.97 t12 6.50a 0.87 6.14-6.86 6.52a 0.79 6.20-6.85 6.50a 0.80 6.17-6.83 t11 8.43a 0.54 8.21-8.64 8.43a 0.51 8.22-8.63 8.44a 0.56 8.22-8.67 t21 8.48a 0.62 8.23-8.73 8.47a 0.54 8.25-8.69 8.44a 0.60 8.20-8.69 t22 6.45a 0.79 6.13-6.77 6.49a 0.76 6.18-6.80 6.53a 0.82 6.20-6.87 t23 7.71a 0.42 7.54-7.88 7.63a 0.47 7.44-7.82 7.69a 0.52 7.48-7.90 t24 6.84b 0.48 6.64-7.05 7.04a 0.49 6.84-7.25 7.07a 0.48 6.86-7.27 t25 6.41b 0.57 6.17-6.65 6.65a 0.52 6.43-6.87 6.66a 0.61 6.40-6.92 t26 9.72b 0.64 9.45-9.99 9.93a 0.61 9.67-10.19 9.92a 0.68 9.64-10.21 t36 11.02b 0.68 10.70-11.33 11.35a 0.62 11.06-11.64 11.34a 0.61 11.06-11.62 t35 7.06b 0.78 6.73-7.39 7.39a 0.73 7.08-7.70 7.28a 0.78 6.96-7.61 t34 6.79b 0.55 6.55-7.03 7.02a 0.51 6.79-7.24 7.07a 0.55 6.83-7.31 t33 6.77a 0.35 6.63-6.91 6.83a 0.41 6.66-6.99 6.81a 0.39 6.65-6.97 t32 5.81a 0.51 5.60-6.01 5.75a 0.46 5.57-5.94 5.87a 0.40 5.71-6.03 t31 5.28a 0.50 5.07-5.48 5.29a 0.52 5.08-5.50 5.40a 0.46 5.21-5.58 t41 5.28a 0.35 5.14-5.43 5.34a 0.40 5.18-5.50 5.35a 0.37 5.20-5.51 t42 5.85b 0.51 5.64-6.06 5.86b 0.46 5.67-6.05 6.01a 0.55 5.78-6.23 t43 6.66b 0.41 6.49-6.82 6.75a 0.42 6.58-6.92 6.77a 0.41 6.60-6.94 t44 6.76b 0.51 6.55-6.98 7.03a 0.63 6.77-7.30 6.91ab 0.54 6.69-7.14 t45 6.87b 0.60 6.61-7.13 7.13a 0.64 6.85-7.41 7.18a 0.58 6.93-7.43 t46 11.03b 0.58 10.76-11.30 11.31a 0.61 11.02-11.60 11.37a 0.66 11.06-11.68 uap 71.99b 4.43 70.20-73.79 73.12a 4.34 71.36-74.87 73.06a 4.54 71.23-74.89 uicd 33.16a 2.29 32.23-34.08 33.06a 2.17 32.19-33.94 33.22a 2.18 32.34-34.10 uimd 40.01a 3.88 38.34-41.69 39.77a 3.17 38.40-41.14 39.98a 3.24 38.58-41.38 lap 63.33a 5.71 60.86-65.80 65.15a 4.46 63.22-67.07 64.64a 5.80 62.13-67.15 licd 25.50b 2.03 24.68-26.32 26.32a 1.98 25.52-27.12 26.01a 2.10 25.16-26.86 limd 36.08b 3.33 34.37-37.80 36.21b 3.40 34.45-37.96 36.92a 3.40 35.18-38.67 there was no statistically significant difference between the measurements taken from the maxillary and mandibular anterior tooth width (p>0.05) except from tooth 42 and 43. there were also no statistically significant differences between the maxillary intercanine and intermolar width and mandibular dental arch perimeter (p>0.05) the greatest differences were found in the maxillary and mandibular posterior tooth width. the differences on the maxillary arch length, mandibular intercanine and intermolar distances were also statistically significant. all differences are listed in the descriptive statistics in table 2. discussion analyzing study models is a key factor for a good orthodontic diagnosis and successful orthodontic treatment plan, because it enables not only the analysis of the present and the required space for the correct leveling and aligning braz j oral sci. 13(4):297-302 reliability of measurements on virtual models obtained from scanning of impressions and conventional plaster models 300300300300300 measurement plaster x impression scan (mm) plaster x model scan (mm) model scan x impression scan (mm) d16 0.43 0.21 0.22 d15 0.30 0.24 — * d14 0.27 0.15 0.12 d24 0.20 0.23 — * d25 0.24 0.25 — * d26 0.21 0.20 — * d36 0.33 0.32 — * d35 0.33 0.22 — * d34 0.28 0.23 — * d42 — * 0.16 0.15 d43 0.09 0.11 — * d44 0.27 — * — * d45 0.26 0.31 — * d46 0.28 0.34 — * pas 1.13 1.07 — * dici 0.82 0.51 — * dimi — * 0.84 0.71 table 2.table 2.table 2.table 2.table 2. differences between the digital models and the plaster models —* no statistically significant difference of the teeth, but also the individual tooth sizes and discrepancies that can ultimately lead to a poor indentation of the occlusion. furthermore, the anatomical variations observed on a plaster model can help for a better position of brackets either using direct or indirect bonding, determine the need for tooth extraction or maxillary expansions. traditionally, most of the information required for an orthodontist to develop a diagnosis and treatment plan comes from photographs, radiographic images and measurements obtained directly from the patient’s mouth or from plaster models3-5. recent technological advancements allowed these 3d measurements to be made digitally using virtual study models scanned from plaster models or alginate impressions5,16. some studies evaluated the accuracy and validity of measurements obtained of digital models from plaster models3-4,6-7,10-11,13-14,16. however, we emphasize that there are no studies that evaluated the reliability of digital models scanned from alginate impression. the total scanning time was of particular interest in this study. although the instruction handbook says it takes from 2 to 3 min to scan a plaster model and 7-10 min to scan an impression, the total scanning time was greater than initially expected because it involves virtual reconstructions, virtual trimming and virtual base creation. these processes are very dependent on the used hardware and the experience of the operator. in the present study the measurements of both different virtual model sets were compared to those of the physical plaster model, with was judged to be the gold standard, since it would be ethically and clinically impossible to obtain the exact tooth measurements to be used as the gold standard12,8-9,11,16. the statistically significant differences found in some measurements can be partially explained by the fact that even though the alginate used in this study has a high stability, in the first 100 h, some contraction may have occurred from the time the impression was scanned to the time the models were poured and expansion caused by the plaster once it hardens. another influence could be the disinfection process of the alginate impressions4,9-11. additionally, in this study the observers were not trained orthodontists but radiologists and possibly had some difficulties placing the landmarks on more crowded dentitions due to increased overlapping, rotation and inclination of the teeth4,11,13-14. this makes it more difficult to exactly identify the same landmarks at the contact points in the interproximal areas and duplicate them exactly on 3 different model sets.4-5,11,13-14 although the intra-class correlation was high for all groups indicating a high reproducibility, the differences between the digital and physical group was greater than the differences between the two digital groups. this could be due to the fact that the two digital model sets, differently from the physical model set, allow the observer to enhance visualization, zoom and maintain each landmark at the exact same position while performing a measurement.10-11,14 in this study the most of significant differences were found in the posterior region with overestimation of the measurements. this may have occurred due to the scanning precision, since the positioning of cameras and laser beam can be less accurate in this region causing image distortion. stevens et al.11 have advocated that the measurements in digital models could be more accurate than those made on plaster models, because they are not limited by the physical limitations of the caliper at the landmarks. in spite of having found a number of statistically significant differences between the plaster model tooth width measurements and those made on the digital models (0.09 mm to 0.43 mm), according to santoro et al.10 and leifert et al.4 differences smaller than 0.5 mm, can be considered clinically insignificant. braz j oral sci. 13(4):297-302 reliability of measurements on virtual models obtained from scanning of impressions and conventional plaster models 301301301301301 in the present study the greatest differences (0,51 mm to 1,13 mm) were found on the linear measurements: maxillary arch perimeter (uap), maxillary intercanine distance (uicd), maxillary intermolar distance (uimd), mandibular arch perimeter (lap), mandibular intercanine distance (licd), mandibular intermolar distance (limd). these measures have greater difference because they are measurements at greater distances. m o r e o v e r , a c c o r d i n g t o t o m a s s e t t i e t a l . 1 7 a n d wiranto et al.9, only differences greater than 1.5 mm are considered clinically significant. although this may be true in some cases, but if one wishes to use these digital models further for appliance production this threshold may be too high, one could want to keep these differences to a minimum. the use of digital models can lead to numerous advantages in the daily orthodontic practice. the files can be easily stored, retrieved and shared, improving patient communication and data sharing with orthodontic labs and other players in multidisciplinary cases. the measurements taken from the digital files are accurate and reproducible making this type of record likely to become rapidly the standard of treatment planning in orthodontics. in conclusion, although some measurements of digital models were different from those obtained from plaster models in the studied scanner, virtual models from either plaster model scans or alginate impression scans are reliable and sufficiently accurate for orthodontic diagnosis and treatment planning, since the differences agree with the literature. acknowledgements we would like to acknowledge capes for the financial support. fig. 1. landmarks and caliper measurements used to determine the arch perimeter on the 3 different model sets: plaster model (a), impression model scan (b) and model scan (c). a b c references 1. luu ns, nikolcheva lg, retrouvey jm, flores-mir c, el-bialy t, carey jp, major pw. linear measurements using virtual study models. a systematic review. angle orthod. 2012; 82: 1098-1106. 2. luu ns, mandich ma, flores-mir c, el-bialy t, heo g, carey jp, major pw. the validity, reliability, and time requirement of study model analysis using cone-beam computed tomography-generated virtual study models. orthod craniofac res. 2014; 17: 14-26. 3. zilberman o, huggare jav, parikakis ka. evaluation of the validity of tooth size and arch width measurements using conventional and threedimensional virtual orthodontic models. angle orthod. 2003; 73: 301-6. 4. leifert mf, leifert mm, efstratiadis ss, cangialosi tj. comparasion of space analysis evaluations with digital models and plaster dental casts. am j orthod dentofacial orthop. 2009; 136: 16.e1-4. 5. el-zanaty hm, el-beialy ar, el-ezz, ama, attia, kh, el-bialy ar, mostafa ya. three-dimensional dental measurements: an alternative to plaster models. am j orthod dentofacial orthop. 2010; 137: 259-65. 6. whetten jl, williamson pc, heo g, varnhagen c, major pw. variations in orthodontic treatment planning decisions of class ii patients between virtual 3-dimensional models and traditional plaster study models. am j orthod dentofacial orthop. 2006; 130: 485-91. 7. okunami tr, kusnoto b, begole e, evans ca, sadowsky c, fadavi s. assessing the american board of orthodontics objective grading system: digital vs plaster dental casts. am j orthod dentofacial orthop. 2007; 131: 51-6. 8. akyalcin s, dyer dj, english jd, sar c. comparison of 3-dimensional dental models from different sources: diagnostic accuracy and surface registration analysis. am j orthod dentofacial orthop. 2013; 144: 831-7. 9. wiranto mg, engelbrecht wp, nolthenius het, meer wj, ren y. validity, reliability, and reproducibility of linear measurements on digital models obtained from intraoral and cone-beam computed tomography scans of alginate impressions. am j orthod dentofacial orthop. 2013; 143: 140-7. 10. santoro m, galkin s, teredesai m, nicolay of, cangialosi tj. comparison of measurements made on digital and plaster models. am j orthod dentofacial orthop. 2003; 124: 101-5. 11. stevens dr, mir cf, nebbe b, raboud dw, heo g, major pw. validity, realiability, and reproducibility of plaster vs digital study models: comparison of peer assessment rating and bolton analysis and their constituent measurements. am j orthod dentofacial orthop. 2006; 129: 794-803. braz j oral sci. 13(4):297-302 reliability of measurements on virtual models obtained from scanning of impressions and conventional plaster models 302302302302302 12. brusco n, andreetto m, lucchese l, carmignato s, cortelazzo gm. metrological validation for 3d modeling of dental plaster casts. med eng phys. 2007; 29: 954-66. 13. costalos pa, sarraf k, cangialosi tj, efdtratiadis s. evaluation of the accuracy of digital model analysis for the american board of orthodontics objective grading system for dental casts. am j orthod dentofacial orthop. 2005; 128: 624-9. 14. sousa mv, vasconcelos ec, janson g, garib d, pinzan a. accuracy and reproducibility of 3-dimensional digital model measurements. am j orthod dentofacial orthop. 2012; 142: 269-73. 15. szklo r, nieto fj. epidemiology beyond the basis. aspen publications; 2000. p. 343-404. 16. lione r, buongiorno m, franchi l, cozza p. evaluation of maxillary arch dimensions and palatal morphology in mouth-breathing children by using digital dental casts. int j pediatr otorhinolaryngol. 2014; 78: 91-5. 17. tomassetti jj, taloumis lj, denny jm, fischer jr jr. a comparison of 3 computerized bolton tooth-size analyses with a commonly used method. angle orthod. 2001; 71: 351-7. braz j oral sci. 13(4):297-302 reliability of measurements on virtual models obtained from scanning of impressions and conventional plaster models oral sciences n3 original article braz j oral sci. october | december 2014 volume 13, number 4 intraoperative accidents associated with surgical removal of third molars aline monise sebastiani1, sara regina barancelli todero1, giovana gabardo1, delson joão da costa1, nelson luis barbosa rebelatto1, rafaela scariot2 1universidade federal do paraná – ufpr, school of dentistry, department of stomatology, area of oral and maxillofacial surgery-maxillo-facial, curitiba, pr, brazil 2universidade positivo up, school of dentistry, area of oral and maxillofacial surgery-maxillo-facial, curitiba, pr, brazil correspondence to: aline monise sebastiani rua maurício nunes garcia, 250 apto 603 jardim botânico cep: 80210-150 curitiba, pr, brasil phone: +55 41 9693 6973 email: sebastiani.aline@gmail.com abstract aim: to evaluate the prevalence of intraoperative accidents associated with extraction of third molars and identify possible risk factors. methods: prospective study with patients undergoing third molar surgery by residents of the oral and maxillofacial surgery service at the federal university of parana. epidemiological data were collected from preoperative evaluation forms. during the surgical procedure were evaluated the radiographic position classifications of all third molars removed, using methods such as osteotomy and/or tooth section, time for the procedure and occurrence of any complication. results: the students extracted a total of 323 teeth. the mean surgical time was 45 min. conclusions: the prevalence of intraoperative accidents during extraction of third molars was 6.19%. the most prevalent accident was maxillary tuberosity fracture, followed by hemorrhage. age, positioning of the teeth and use of the techniques of osteotomy and tooth section are possible risk factors. keywords: molar third; surgery oral; radiography; intraoperative complications; tooth extraction. introduction extraction of third molars is routinely performed by non-specialist dentists. however, it is a procedure associated with some difficulties, like the molar’s close anatomical relationship with the noble anatomical structures, the angle of teeth crowns and impactions. besides, there are surgical complications like bleeding, nerve damage, injuries to adjacent teeth, fracture of maxillary tuberosity, displacement of the tooth to other anatomical structures and fracture of the dental apex1. bleeding occurs in 0.2% to 5.8% of third molar extractions, transoperatively or postoperatively, locally or systemically. it’s four times more common in mandibular third molars than maxillary third molars. the highest incidence is in the deeply impacted distoangular teeth and in older patients2. injury to the inferior alveolar nerve after removal of third molars occurs in 0.4 to 8.4% of cases, less than 1% permanent. lingual nerve damage ranges from 0 to 23%3. damage to the second molars is reported in 0.3% to 0.4% of these surgeries. the positions of third molars of greatest risk for this complication in maxilla are mesial associated with class b (pell and gregory4), already in the mandible, the position is vertical2. the position of the maxillary third molar at the end of the dentoalveolar arch is such that the posterior portion cannot be supported, and the internal composition of the bone may be of the maxillary sinus, or porous bone. this facilitates fracture of maxillary tuberosity, which is more associated with erupted third molars and excessive use of force5. moreover, these teeth have close received for publication: august 14, 2014 accepted: november 26, 2014 braz j oral sci. 13(4):276-280 anatomical relationship with the maxillary sinus. thus, a tooth extraction can lead to an accidental opening of the sinus or displacement of the tooth in the sinus, especially due to inadequate use of extractors6. displacement of the tooth into other adjacent structures may occur occasionally, such as the infratemporal fossa 7. another common complication, the fractured tooth root apex, may occur mainly in root lacerations such as hypercementosis and ankylosis, conditions that increase resistance to avulsion8. according to araujo, 20119, some variants are related to accidents and complications occurring during surgery of these teeth: patient age, tooth position, surgeon’s experience and time of surgery. attention to surgical details, including patient preparation, asepsis, careful handling of tissues, control of instrument force, control of hemostasis and adequate postoperative instructions, reduce the rate of complications. the aim of this study was to evaluate the prevalence of intraoperative accidents in patients undergoing extraction of third molars and identify possible risk factors. material and methods data for the development of this research were collected by a prospective study. it was approved by ethical research committee on human beings at the human health department under number cep/sd:1021.146.10.10 and caae:0086.0.091.000-10. all patients gave signed an informed consent. sample selectionsample selectionsample selectionsample selectionsample selection: all patients undergoing third molar surgery by residents of the oral and maxillofacial surgery service at the federal university of parana were invited to participate in the study (september 2010 to september 2011). for this purpose, the patients were informed about the research and invited to sign the term of consent, granting permission for the collection and use of information arising from the surgery, knowing that it would not influence their procedure. surgeons prescribed preoperative (injectable steroids – betamethasone one hour before the procedure) and postoperative (nsaids, analgesics and antibiotics if needed) medications. inclusion criteria comprised patients who had their third molars removed during the period covered by the study under local anesthesia, 18 years of age or older, presenting records with panoramic radiographs, and signing the informed consent form. patients with some systemic changes (hypertension, anemia, and diabetes) were excluded. data collection:data collection:data collection:data collection:data collection: epidemiological data (age, gender and ethnics) were collected in preoperative evaluation files. the researcher in charge of data collection followed the entire surgical procedure, analyzing panoramic radiographs exposed in the operating room, giving the radiographic position of all removed third molars, following the classifications by winter10 and pell and gregory4. winter classified third molars as vertical, horizontal, mesioangular, and distoangular, according to the angulation of the long axis of the third molar in relation to the long axis of the second molar9. pell and gregory4 related the tooth to the ramus of the mandible. in their class i, the crown is completely in front of the anterior border of the ascending branch; in their class ii, the tooth is partially within the mandibular branch, and in class iii, the tooth is located completely in the ascending branch of the mandible. they also classify teeth in relation to the occlusal plane as follows: in a, the occlusal surface of the third molar is the occlusal plane of the second molar; in b, the occlusal surface of the tooth is not erupted between the occlusal plane and cervical line of the second molar; and in c, the occlusal surface of the unerupted tooth is below the cervical line of the second molar9. all radiographs were performed by the equipment at the department of dental radiology, orthophos model 90 kv/12 ma (siemens corp.,erlangen, germany) at preoperative evaluation of the patient. the researcher recorded methods such as osteotomy and/ or tooth section, time of the procedure in minutes and any complications, such as hemorrhage, displacement of teeth to the adjacent spaces, damage to adjacent teeth, soft tissue injuries, maintenance apex/root, tuberosity fracture, and dislocation of the mandible. any active bleeding observed during osteotomy, tooth section and avulsion which had not ceased after five minutes of compression with gauze was considered hemorrhage. statistics:statistics:statistics:statistics:statistics: data of patients were entered in a microsoft excel for windows® spreadsheet specifically developed for the study. statistical evaluation was performed using frequency analysis and specific statistical tests (statistical package for social sciences-spss, version 15.0, spss inc. chicago, il, usa), with a 95% confidence interval. results the sample consisted of 150 patients who underwent surgery for removal of third molars. the mean age was 24 (18-62). there were more females than males (n=95/63.3%). a total of 323 teeth were extracted: 164 maxillary third molars and 159 mandibular third molars. osteotomy was performed in 26 maxillary third molars and in 113 mandibular third molars. tooth section was not done in maxillary third molars, but it was in 84 mandibular third molars. the minimum surgical time was 15 min and the maximum was 1 h and 40 min, with a mean time of 45 min. there was no statistical association between intraoperative accidents and gender (chi-square test p=0.526) or ethnics (chi-square test p=0.361). however, there was a significant correlation between accidents and age (mannwhitney test p=0.041). the most prevalent accident was fracture of maxillary tuberosity; the mean age of patients who had this complication was 24.3. the second most prevalent accident was hemorrhage; the mean age for these patients was 29. there was no relationship between intraoperative accidents and surgical time. intraoperative accidents occurred during the removal of 20 third molars (6.19%). table 1 shows these accidents. table 2 shows the relationship of tuberosity fracture with radiographic position of the teeth and use of osteotomy and tooth section. table 3 relates hemorrhage to the same variables. intraoperative accidents associated with surgical removal of third molars braz j oral sci. 13(4):276-280 277277277277277 complication n % fracture of maxillary tuberosity 8 2.48 hemorrhage 3 0.92% maintenance apex/root 2 0.62% soft tissue injuries 2 0.62% anesthesia of the facial nerve 1 0.31% damage of the second molar 1 0.31% oral sinus communication 1 0.31% others 2 0.62% table 1 table 1 table 1 table 1 table 1 prevalence of intraoperative accidents in 323 third molars removed at ufpr between september 2010 and september 2011 48 mesioangular class a-ii yes yes 48 distoangular class b-ii yes yes 48 mesioangular class b-iii yes yes tooth winter pell e gregory osteotomy tooth section classification classification table 3 table 3 table 3 table 3 table 3 radiographic position of teeth associated with hemorrhage and surgery techniques performed. tooth winter pell e gregory osteotomy tooth section classification classification 18 vertical class a yes n o 18 vertical class a n o n o 18 vertical class a yes n o 18 vertical class a n o n o 28 vertical class a n o n o 28 vertical class a n o n o 28 vertical class a n o n o 28 vertical class a n o n o table 2 table 2 table 2 table 2 table 2 radiographic position of teeth associated with tuberosity fracture and surgery techniques performed. discussion impacted third molars are developmental pathological medical deformities characteristic of modern civilization, accounting for 98% of all impacted teeth11. transoperative accidents during third molar surgery are not uncommon and the professional who performs this procedure must be able to solve these possible operative complications. in the literature complications have been associated with the experience of the surgeon. berge and gilhuurs compared complications following surgical removal of third molar in two groups of patients. surgery was performed in the first group by four general dental practioners and in the second group by consulting oral surgeons, who had fewer complications and shorter surgeries12. in a retrospective study, bui et al. (2003)13 found an operative complication rate of 2.2%. chiapasco (2006)14, found an incidence of intraoperative accidents of 1.1% and side effects of mandibular third molar surgery of 4% for maxillary third molar surgery. in the present study, the prevalence of transoperative accidents was 6.19%, the difference due to varying definitions of complications, study design, and different methods of evaluating study variables. these two studies were retrospective, which reduces the authenticity of these prevalences: many trans-operative complications may have occurred and not registered in the records. in the chuang (2007)15 prospective cohort study, the intraoperative complication rate was 3.9%, however, it does not mention the accidental maxillary tuberosity fracture, which was the most prevalent transoperative accident in this study, neither do the above cited studies13-14. kato et al. (2010)16, while investigating accident rates and complications in third molar surgery, found a higher rate of accidents and complications in female patients (73.91%) than in male patients (27.28%). however, in this study the vast majority of patients (70.45%) were females, thereby increasing the chances of an accident or complication in this group of patients. this study found no statistical relationship between gender or ethnics and the complications. a statistically significant difference with age was found. the older the patients, the more complications associated with the procedure. a prospective cohort study of a sample of subjects having extracted at least one third molar found that those over 25 years old had an increased risk of complications14. in a study of benediktsdóttir et al. (2004)17, older groups had more than twice the risk of a prolonged operating time than the youngest group. according to hupp5, in cases of extreme age, surgical removal of the third molars is contraindicated because as the patient ages, the bone becomes increasingly more calcified and thus less flexible. as a result, more bone should be removed during the procedure. postoperative reaction is less favorable in these patients and post-operative sequel more likely. therefore, in older patients (usually over 40) with asymptomatic impacted teeth, removal is not indicated. the osborn et al. (1985)18 prospective study evaluated surgical and postsurgical problems of patients of a wide range of ages who had third molars removed and concluded that removal during teenage years decreased operative and postoperative morbidity. oliveira (2006)1 correlated the incidence of accidents and complications with time of surgery. dental extractions performed in less than 60 min, had an accident rate of 9.6% but those over 120 min had one of 83.33%. those authors also observed more accidents and complications relative to the complexity of surgical procedures. other studies show the post-operative morbidity increase with longer procedures19. this study found no relationship between complications and surgical time. bui et al. (2003)13 found in their study that the most prevalent complication was inferior alveolar damage (1%), followed by bleeding (0.7%), oroantral communication (0.3%), and incomplete root removal (0.2%). in the present study, no cases of inferior alveolar damage occurred. however, bleeding was the second most prevalent complication (0.92%), ahead of communication (0.31%) and incomplete root removal (0.62%). this study showed a higher prevalence of bleeding than nerve damage because only intraoperative intraoperative accidents associated with surgical removal of third molars braz j oral sci. 13(4):276-280 278278278278278 complications were observed, unlike other studies that evaluated postoperative complications such as paresthesia. the highest prevalent complication in this study can be seen in table 2. all teeth associated with tuberosity fracture presented vertical position according to the classification of winter and “class a” according to pell and gregory4, which means that the occlusal surface of the third molar was the same of the second molar, corresponding to completely erupted teeth. this occurs when the tooth is completely erupted, it gets occlusal load, reducing the periodontal ligament, therefore favoring fracture of the tuberosity. bui et al.13 found a statistical relationship between position of the teeth according to the classification of winter and complications. they never conducted tooth section, and only twice performed osteotomy, maneuvers that could have presented tuberosity fracture. ngeow (1998)20 stated that if a pre-extraction radiograph revealed a large antrum, an appropriate technique to avoid fracture of the tuberosity would be sectioning of the tooth and removal of one root at a time, or removing an adequate amount of bone from the sides of the tooth to facilitate a gentle levering out of its socket without disturbing the fractured bone. if a maxillary tuberosity is fractured, the surgeon must determine the extent of the fracture by palpating the mobile fragment. if the fracture is small, the maxillary tuberosity is removed with the tooth. if the bony fragment is large, a sharp instrument should be inserted into the distobuccal cervical area of the tooth and used to separate the alveolar bone from its roots, keeping the remaining bone attached to the periosteum so that it will be perfused continuously, and it must be compressed against the communication. the gingiva should then be sutured. the sutures should be removed only after 2 weeks. in some cases the tuberosity fracture involves the maxillary sinus. in such cases, antibiotics, nasal decongestant, and antiinflammatory analgesics should be prescribed to help preventing maxillary sinusitis. the patient should be advised against blowing his or her nose20. in this study, only one patient had oral sinus communication. according to boulox et al. (2007)2, hemorrhage is more prevalent in older patients with deeply impacted teeth and four times more associated with mandibular third molars than maxillary third molars. in this study the mean age in patients who suffered hemorrhage was 29, higher than the general mean (24). all teeth associated with hemorrhage were mandibular third molars and bent, with some portion localized in the mandibular branch (table 3), therefore harder to extract. in all cases, there was osteotomy and tooth section, which can lead directly to bleeding, because when the drill breaks the bone marrow, it may cause a medullar bleeding. in some cases the inferior alveolar vascular-nervous plexus is reached, causing a vascular hemorrhage. in both hemorrhage cases in this study, hemostasis was done with gauze and fibrin sponge. agrawal et al. (2014) 21 found in their study that extractions associated with both osteotomy and odontotomy are associated with higher risk of complications21. for delamare (2012)22, the panoramic radiograph should be routinely used as an auxiliary examination for treatment planning of mandibular third molar removal, due to its wide availability, low cost, and relatively low exposure dose. other traditionally accepted options for estimation of difficulty and risk during lower third molar removal are the classifications of pell & gregory and winter. the association of intraoperative accidents with the radiographic position of the third molars in this study shows the usefulness of both methods. fractured roots are the most common problem associated with tooth extraction, according to hupp (2009)5. this accident is usually associates with long, curves and diverge roots’, in a dense bone. in some situations, the risks of removing a small fragment of the fractured apex may outweigh the benefits, like a risk of moving it to any anatomical structure. the two teeth that had their apexes buried were mandibular third molars and were near to alveolar nerves. if the root meets the following four conditions, it should be left in the alveolar process: the fragment has to be less than 5 mm long, deeply inserted into the bone, free of infection and showing no radiolucent areas around the root apex. this study also had two cases of injury to the soft tissues, which may occur when instruments such as a lever straight line or periosteal peeler slip from the surgical field and puncture or lacerate adjacent tissues. this can be avoided with controlled force, with special attention to the support of a finger or hand stand against the anticipation of sliding. abrasions or burns of the lips can still occur due to oral commissure or patchwork resulting from rotational friction rod. the surgeon and his assistant should be vigilant against this5. malamed (2012)23 mentioned transient facial paralysis as a possible complication of the inferior alveolar nerve block anesthetic technique. this was commonly caused by the introduction of local anesthetic into the capsule of the parotid gland, which is on the posterior border of the mandibular branch, where the terminal branches of the facial nerve extend. symptoms include the inability to close the eyelid and lower slope of the upper lip on the affected side. the reflex protective cover of the eye is abolished, but it retains its corneal reflex and lubricates with tears when irritated. the loss of motor function of the muscles will not last more than a few hours. the problems are cosmetic. when the needle tip is in contact with the bone (on the medial branch) prior to deposition of the solution, it keeps the local anesthetic solution off the parotid gland. the patient was informed of what was happening and was monitored with a cap holding the eye closed until the anesthesia effect passed. damage to adjacent teeth can be minimized if care is taken to visualize the entire operating field rather than the tooth being extracted. susarla (2003)24 noted that a surgeon aware of the periphery of the operating field is often able to anticipate possible damage and take action to prevent its occurrence24. teeth with large restorations or carious lesions are always at risk of fracture or damage upon elevation. correct use of surgical elevators and bone removal can help to prevent this. preoperative discussion should take place with highrisk patients2. if an adjacent tooth is luxated or avulsed inadvertently, the most common course of action is intraoperative accidents associated with surgical removal of third molars braz j oral sci. 13(4):276-280 279279279279279 repositioning the tooth followed by fixation. the conclusion of this study is that the possible risk factors of intraoperative accidents identified in this study were age, positioning of teeth, and use of osteotomy and tooth section. the surgeon who performs the surgical removal of third molars should be aware of the risk factors and able to solve possible complications during the procedure. references 1. oliveira lb, schmidt db, assis ap, gabrielli mac, vieira eh, pereira filho va. review of accidents and complications associated with extraction of third molars. rev cir traumatol buco-maxilo-fac. 2006; 6: 51-6. 2. bouloux gf, steed mb, perciaccante vj. complications of third molar surgery. j oral maxillofacial surg clin n am. 2007; 19:117-28. 3. ziccardi vb, zuninga jr. nerve injuries after third molar removal. oral maxillofacial surg clin n am. 2007; 19: 105-15. 4. pell gj, gregory gt. report a ten-year study of a tooth division technique for the removal of impacted teeth. am j orthod. 1942; 28: 660. 5. hupp jr, ellis e, tucker mr. oral maxillofacial surgery and contemporary. 5. ed. rio de janeiro: elsevier; 2009. 6. graziani, m. oral and maxillofacial surgery. 8. ed. rio de janeiro: guanabara koogan; 1995. 7. primo bt, stringhini dj, klüppel le, 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extractions. j oral maxillofac surg. 2003; 61: 1379-89. 14. chiapasco m, cicco l, marrone g. side effects and complications associates with third molar surgery. oral surg oral med oral pathol. 2006; 76: 412-20. 15. chuang sk, perrott dh, susarla sm, ba, dodson tb. age as a risk factor for third molar surgery complications. j oral maxillofac surg. 2007; 65:1685-92. 16. kato rb; bueno rbl, oliveira pj neto, ribeiro mc; azenha mr. accidents and complications associated with third molar surgery performed by dental students. rev cir traumatol buco-maxilo-fac. 2010; 10: 45-54. 17. benediktsdóttir is, wenzel a, petersen jk, hintze h. mandibular third molar removal: risk indicators for extended operation time, postoperative pain, and complications. oral surg oral med oral pathol oral radiol endod. 2004; 97: 438-46. 18. osborn tp, frederickson g jr, small ia, torgerson ts. a prospective study of complications related to mandibular third molar surgery. j oral maxillofac surg. 1985; 43: 767-9. 19. pathak s, vashisth s, mishra s, singh sp, sharma s. grading of extraction and its relationship with post-operative pain and trismus, along with proposed grading for trismus. j clin diagn res. 2014; 8: zc09-11. 20. ngeow wc. management of the fracture maxillary tuberosity: an alteramtive method. quintessence int. 1998, 29: 189-90. 21. agrawal a, yadav a, chandel s, singh n, singhal a. wisdom toothcomplications in extraction. j contemp dent pract. 2014; 15: 34-6. 22. delamare el, liedke gs, vizzotto mb, silveira hld, azambuja twf, silveira hed. topographic relationship of impacted third molars and mandibular canal: correlation of panoramic radiograph signs and cbct images. braz j oral sci. 2012, 11: 411-5. 23. malamed sf. handbook of local anesthesia. 6. ed. saint louis: mosby elsevier; 2012. 24. susarla sm, blaeser bf, maganalnick d. third molar surgery and associated complications. oral maxillofacial surg clin n am. 2003; 15: 177-86. intraoperative accidents associated with surgical removal of third molars braz j oral sci. 13(4):276-280 280280280280280 oral sciences n3 original article braz j oral sci. january | march 2014 volume 13, number 1 reasons related to tooth loss among adolescents in são paulo, brazil luísa helena nascimento tôrres1, juliana zanatta1, raquel aparecida pizolato1, cássia maria grillo1, antônio carlos frias, maria da luz rosário de sousa1 1universidade estadual de campinas unicamp, piracicaba dental school, department of community dental health, piracicaba, sp, brasil 2universidade de são paulo usp, school of dentistry, department of community dentistry, são paulo, sp, brasil correspondence to: maria da luz rosário de sousa universidade estadual de campinas faculdade de odontologia de piracicaba avenida limeira 901, bairro areião cep: 13414-903 piracicaba, sp, brasil phone: +55 19 21065364 e-mail: luzsousa@fop.unicamp.br abstract aim: to estimate tooth loss prevalence among adolescents in são paulo, considering socioeconomic and demographic factors, dental service use and pain. methods: data were obtained from the são paulo oral health survey 2008. the sample comprised 2858 adolescents from 15 to 19 years old from public and private schools, who underwent through an oral examination. in addition, a questionnaire was applied regarding the characterization of factors related to socioeconomics, demographics, use of dental services and pain in the last six months. at least one tooth lost was the outcome. the independent variables included gender, ethnicity, parents’ schooling, type of school, number of rooms, people and cars per home, family income, dental service use, decayed teeth, toothache. a multivariate logistic regression model was used. results: the prevalence of tooth loss was 7%. the independent variables decayed tooth (rp=1.71), toothache (rp=2.04), father’s schooling elementary (rp=1.40) and per capita family income less than 1/ 2 a minimum salary (rp=1.45) were associated with the outcome. conclusions: the results suggest that socioeconomic factors may contribute to the increase of early tooth loss among adolescents. keywords: tooth loss; adolescent; income. introduction adolescence is the period of life comprised between the ages of 10 and 19 years, according to the world health organization1 (1995), and is divided into two stages: the first from 10 to 14 years, and the second from 15 to 19 years of age. according to data from the brazilian institute of geography and statistics (ibge)2 (2007), the young population from 15 to 19 years of age in the são paulo state comprised 1,174,920 adolescents. at this stage the youngsters are in a state of constant biopsychosocial development, and therefore, it is common for them to present behaviors that place them at risk for their health3. caries is a disease that affects a large sector of the population, in spite of the advancements of dentistry in scientific and structural terms. nevertheless, the majority of researches have focused on children at the schooling age, and there are insufficient data in the literature about the prevalence of dental caries among adolescents4. the rights of adolescents are preserved by the brazilian statute of the child and adolescent (“estatuto da criança e do adolescente eca”)5, issued in 1990. in addition, there is the health program for the adolescent (“programa saúde do adolescente prosad”)6 (1996), created by the ministry of health in 1989, which addresses std/aids, drug addiction, traffic accidents and premature braz j oral sci. 13(1):37-42 received for publication: december 01, 2013 accepted: march 17, 2014 gisele higa texto digitado http://dx.doi.org/10.1590/1677-3225v13n1a08 38 pregnancy, but as far as we know, there is no specific program that includes oral health directed towards this age group. on the other hand, the federal government developed a national oral health policy within the national health system (“sistema único de saúde – sus”), known as “brasil sorridente”. by means of a set of actions like reorganization of basic care and creation of dental specialty centers (“centros de especialidades odontológicas ceos”), this strategy widened the access to dental treatment and specialized treatment by the population in the public network. among youngsters aged between 15 and 19 years, only 55% have all their teeth7, according to statistical data. in the project “sb brasil 2003”, the age group from 15 to 19 years was evaluated in a nation-wide epidemiologic survey for the first time8. in this survey, the component “lost” was approximately 15% of the dmft index in the 15-19year-old group. the study of barbato and peres9 (2009) observed a prevalence of tooth loss of 38.9% in adolescents, based on secondary data from the project “sb brasil 2003”. in the são paulo state in 2002, it was observed that 6.51% of the dmft corresponded to the component m (number of missing teeth) in this age group10. in order to study the oral health of the adolescent, it is fundamental to reflect on the particular history of each family, considering the social context in which it lives. the study of cimões et al.11 (2007) showed that social class influenced the ratio of tooth losses among adults, with the number of losses due to caries being higher in the groups of lower social class. tooth loss is considered a relevant theme to be evaluated by public health authorities, since it is considered a result of the most prevalent oral diseases12, among them dental caries (costa et al, 2013)13. in the qualitative study of silva, magalhães and ferreira (2010)14, in the analysis of content, the main causes pointed out for tooth loss were found to be the lack of knowledge, methods for maintenance, difficult access to dental services, lack of financial resources for treatment, iatrogenic experiences and fear of pain. nevertheless, according to costa et al.13, it seems that there is no lack of resources nor access to dental service for tooth extraction in comparison with other dental treatments it does not seem to lack neither resources nor access to dental service. the aim of this study was to estimate the tooth loss prevalence of the population in the age-range between 15 and 19 years, considering socioeconomics, demographics, use of services and pain factors in the municipality of são paulo, sp, brazil, in 2008, and thereby contribute to the process of planning and actions on oral health directed towards this group. material and methods this study was performed using secondary data collected in the epidemiological survey on oral health in the city of são paulo in the year 2008. the sample comprised 2858 adolescents aged from 15 to 19 years. the data were collected by clinical oral examination; the index used for dental caries was the dmft (in accordance with who criteria)15, and a questionnaire, all applied in previously drawn public and private schools. the questionnaire contained questions to enable the characterization of factors related to socioeconomics, demographics, use of dental services and pain in the last six months. the study was conducted in two stages with probability proportional to size (pps) in the conglomerates (schools) of the adolescent population. in the second stage the draw was systematic, pondered by the number of pupils. the goal was to guarantee the ponderability of the draw in both stages, draw of the school and draw of the adolescent in the school16. in this survey, 75 oral health teams (ohts) participated, being a dentist, an oral health assistant and a municipal health service officer16. in the calibration process, agreement on the results between examiners was evaluated, using the kappa coefficient as instrument of agreement (dental caries kappa 0.954 ic 0.95-0,96) and general percentage of agreement (gpa) in addition to the parameters of observation consistency proposed previously17-18. in this cross-sectional study, the outcome was loss of at least one tooth and its analyzed independent variables were the socioeconomic factors (family income, schooling of the father and mother, number of rooms, number of persons living in the same house, car, type of school), demographics (age, gender and ethnicity), use of services (visit to the dentist in the last six months), toothache in the last six months and tooth with caries. the independent variables were grouped into categories and re-coded. the variable school was dichotomized into public (public and contracted schools), and ethnicity into non-white (yellow, indian, mulatto and black) and white. schooling was dichotomized into primary schooling and middle, aggregated to higher learning. family income was dichotomized into less than four minimum wages and four or more minimum wages, whereas family income per house inhabitant into less than half a minimum wage per person and higher than or equal to half a minimum wage per inhabitant. the variable visit to the dentist, into never and at least one consultation in the last six months. for the variable age, the ages from 15 to 16 years and 17 to 19 years were grouped. the variables number of rooms and persons in the house, from 1 to 4 and more than 4. the variable car was categorized as none, one and two or more cars. as regards caries experience the mean dmft in the sample was 2.41 in this age group, and the component “missing” accounted for 7% in this index. to evaluate whether there was association between the variable outcome (tooth loss) and the other independent variables, bivariate analysis was performed. all the independent variables that showed association with p<0.25 were subjected to multivariate analysis. the variables that did not contribute with the model were discarded and a new model was constructed. the data were entered in the epi-info6 software and stata 10 was used to analyze the data for the bivariate and multivariate analyses. reasons related to tooth loss among adolescents in são paulo, brazil braz j oral sci. 13(1):37-42 parents or legal guardians of the adolescents were asked to sign an informed consent form, according to the report approved by the research ethics committee of the municipal secretary of health of são paulo (048/08) on march 18, 2008. results tooth loss prevalence with at least 1 lost tooth was 7% (5.93 7.82) among the adolescents. between the ages of 15 and 16 years it was 6.2% and between 17 and 19 years it rose to 9.1%, representing the accumulation of need in the course of time. the sample was almost equally distributed according to the gender (females 50.7%). table 1 contains the data of the association between lost teeth and the independent variables (untreated tooth with caries, toothache, type of school, father and mother educational level, family income, crowding in the home and family income per person in the house) in adolescents from 15 to 19 years of age in the municipality of são paulo in 2008. it was observed that adolescents with an untreated carious tooth had a 1.71 times higher prevalence of tooth loss than those without a tooth with untreated caries. all these variables in table 1 showed strong association with variables n yes n o pr* ic 95% tooth with untreated caries 2858 yes 1174 131 1043 1.71 1.53 — 1.91 no 1684 64 1620 toothache 2858 yes 799 104 695 2.04 1.76 — 2.36 no 2059 91 1968 school 2858 public 2271 189 2082 1.23 1.20 — 1.28 private 587 06 581 father’s schoolingk 2312 basic schooling 1328 119 1209 1.40 1.28 — 1.54 middle higher education 984 32 952 mother’s schoolingk 2376 basic schooling 1314 107 1207 1.30 1.17 — 1.45 middle higher education 1062 44 1018 family income (minimum wage mw = r$ 450,00)k 2010 less than 4 mw 1559 119 1440 1.19 1.12—1.26 4 or more mw 451 11 440 crowding in the homek 2279 fewer than 1 person/room 1481 114 1367 1.23 1.13—1.35 1 or more person/room 798 30 768 family income per person in the house (mw / person house)k 1909 less than ½ mw/person 1110 101 1009 1.45 1.32—1.59 > ½ mw/person 799 22 777 table 1table 1table 1table 1table 1 – association of the presence of lost teeth with demographic, socioeconomic variable and oral health conditions in adolescents (15 to 19 years), municipality of são paulo, são paulo, 2008. *pr – prevalence ratio adjusted by the cluster sampling model and ponderation by population weight. kdata with missing cases. tooth loss (p<0.01). table 2 presents the multivariate data analysis of the factors associated with lost teeth in adolescents in the municipality of são paulo in 2008. tooth with caries, toothache, father’s schooling (basic schooling) and family income lower than half a minimum wage per inhabitant in the house were statistically significant with regards to the outcome tooth loss, different from age (p>0.05). age was placed in the model for the adjustment of variables in the multivariate model. discussion even in a young age group the social and economic disparities might already reflect in the oral condition of the adolescents in the city of são paulo. this emphasizes the need for oral health promotion and preventive measures in the early stages to prevent the continuous tooth losses that lead to edentulism, so common in adults and the elderly. the increased prevalence of tooth loss between the ages of 15 and 16 (6.2%) and 17 to 19 (9.1%) may be justified mainly by the fact that preventive programs focus on younger schoolchildren, and when they conclude middle school terms, 39reasons related to tooth loss among adolescents in são paulo, brazil braz j oral sci. 13(1):37-42 40 variables pr* std. error ic 95% p tooth with caries 2.63 0.71 1.54 — 4.49 0.000 toothache 1.64 0.18 1.30 — 2.05 0.000 father’s schooling (basic schooling) 2.20 0.75 1.12 — 4.33 0.022 family income lower than ½ a mw per person resident in house 2.81 0.83 1.56 — 5.05 0.001 age 1.17 0.14 0.91 — 1.49 0.204 table 2table 2table 2table 2table 2 – multivariate analysis of the factors associated with the prevalence of lost teeth in adolescents (15 to 19 years), municipality of são paulo, são paulo, 2008. *pr – prevalence ratio adjusted by the cluster sampling model and ponderation by population weight, adjusted for age. they lose the follow-up by oral health teams who may be working in the schools on preventive and health promotion activities. this accumulation of need with age has previously been reported in a study that showed an increase in dental caries in each year from 15 to 19 years9. this demonstrates the need for intervention and inclusion and/or continuity of the health promotion measures in this age group. in a study conducted by jovino-silveira, et al.19 (2005), caries and its sequelae were shown to be the main reason for tooth losses, particularly when comparing the individuals in the 18-39year-old age range to those over 40 years of age. there is a social gradient on tooth loss, the lower the income and schooling the higher the loss of teeth20. in the survey in the são paulo state in 2002, the adolescents from 15 to 19 years of age with a family income between 1 and 3 minimum wages presented 0.48 as the mean of the m component of the dmft, with the mean value of lost teeth diminishing as the family income increased. furthermore, it was observed in this study that youngsters whose family income exceeded 10 minimum wages had approximately one tooth less affected by dental caries, thus diminishing the possibilities of tooth loss due to caries21. these findings resemble those of the present study since lower family income was associated with tooth loss. in addition, higher income inequality may result in more tooth loss possibly due to disinvestment in public resources, erosion of social cohesion, stress-induced oral-health-related behaviors and physiological effects22. gushi, et al. 8 (2005) worked with data from epidemiological surveys in the são paulo state from 1998 and 2002 involving a population at the ages of 12 and 18 years. with regard to the treatment needs for those aged 18 years, the percentage of need for extractions increased from 5.9% in 1998 to 7.1% in 2002, however without statistical significance. the authors found a similar result when they evaluated a group of high and low dental caries experience in 1998 and 2002, presenting high values for the age, thus confirming the increase in early tooth loss. in 2010 another data collection took place in são paulo state and within an 8 years period in adolescents aged 15-19 years, tooth extraction need maintained the same values over the years23. susin, et al.24 (2006), evaluated tooth loss among individuals from 14 to 29 years of age in the metropolitan region of porto alegre, southern brazil, and found a 26% prevalence of tooth loss in the group from 14 to 19 years of age. they also observed that the group with the worse socioeconomic status presented the highest prevalence of tooth loss. furthermore, according to the authors, individuals with a higher educational and economic level are more conscious for maintaining the teeth in the mouth and financing more conservative treatments. the present study also found a significant association between tooth loss and family income but among adolescents. this demonstrates the importance of socioeconomic factors in determining tooth loss as well as the need of adopting equitable measures of access to oral health services, qualification of the service provided (both curative and preventive) and health promotion, which are the demands and needs of this group. moreover, the importance of the health care of adolescents is linked to the fact that they may go into the adult stage with healthier dentition, preserving their quality of life, since tooth loss causes psychological discomfort, functional limitation, shame25, malposition of the teeth, facial asymmetry, alterations in the tmj, esthetic problems, alteration in phonation10 and in the selection of foods26. according to lisboa and abegg27 (2006), the adolescents were shown to be one of the most assiduous groups as regards visits to the dentist and did so in a time interval that ranged from six months to every 2 years. this fact may be explained by the importance youngsters place on esthetics. in the present study, it was observed that adolescents’ visits to the dentist were linked to toothache, rather than esthetics. in this study toothache was associated with tooth loss. however, there are other consequences of dental pain that affect adolescents and their families. goes et al.28 (2007) evaluated the impact of toothache on the daily activities of 1052 brazilian adolescents from 14 to 15 years of age, and observed that the main impacts affected their concentration at school, in sports and home activities. in addition, the adolescents that had a lower income were those most frequently affected, with impacts of longer duration that extended also to the family. peres, et al.29 (2010) evaluated adolescents of 12 and 15 years of age by the data of the epidemiological survey of são paulo (2008) and also observed greater prevalence of pain of dental origin among the adolescents that inhabited regions with a lower human development index, affecting approximately 25% of the sample. in this study, the father’s low schooling was associated with tooth loss. in a qualitative research conducted in reasons related to tooth loss among adolescents in são paulo, brazil braz j oral sci. 13(1):37-42 41 sweden, 17 adolescents from 15 to 19 years of age, with high risk for caries and patients of a public dental clinic identified the parents and not the oral health professionals as the main source of information30. although the study did not explain the degree of the parents’ education, this demonstrates the importance of the parents’ role in the construction of healthy habits. family crowding was a variable that drew attention, since the present results presented a higher number of individuals with tooth loss in homes with less than one person per room. however, the question could have been more specific by asking how many rooms were used as bedrooms, so that the variable might have been shown to be more sensitive. in the study of lisboa and abegg 27 (2006), 2,627 individuals from 14 to 49 years of age were evaluated, and it was found that almost half of this population visited the dentist exclusively in a situation of pain. in the study of pitanga fernandes et al.31 (2010), adolescents from 15 to 18 years of age with a higher concentration of caries (caries index – sic) in a city in southeastern brazil, presented a relation with toothache reported in the six months before the study, which could be explained by the authors as being due to the progression of untreated caries disease itself; or due to the difficult access to dental services in this age group. for the adolescents there are differences in seeking public medical and dental services, as seeking dental assistance is directed towards the private sector. this may be explained by the fact that part of this age group is inserted in the work market, so they are at work during the hours when attendance is offered, which hampers the access to the public service. however, the authors pointed out that the solution to this is not the availability of dental treatment, but that the individual’s knowledge of the factors associated with the experience of dental caries that contribute to the preparation of health actions that favor improvements in the oral health conditions in this age group. this age group has not yet been widely studied, particularly on account of the history of providing oral health services in brazil, where the actions planned and developed in the majority of the municipalities, were directed to the age group from 6 to 12 years; that is to say, schoolchildren, with emphasis on curative measures32. preventive and health promotion actions, as well as extending access to health services, when adopted in population strategies, contribute to the maintenance of health and to the development of healthy habits. nevertheless, according to silva et al.33 (2006), it must be considered that the individual’s perception of his/her oral health is the factor that determines whether or not he/she seeks attendance. in a study with children and young persons, youngsters with less than optimal oral health status may be those who do not seek care and might avoid dental visits, and to whom the lack of preventive care may contribute to their poor oral health status34. in this way, socioeconomic and cultural conditions make the perception and appreciation of oral health difficult, which is particularly true in persons with low income, due to the lack of knowledge about the maintenance of oral health16. these results suggest that socioeconomic factors may contribute to the increase of early tooth loss among adolescents. the formulation of public policies directed towards this age group, especially oral health policies, must consider that this population is in the process of development, which requires full care, capable of stimulating awareness about the importance of oral health and self-care, developed in conjunction with preventive and curative actions. references 1. world health organization (who). the health of youth: a challenge and a hope. geneva: who; 1995. 2. brazilian institute of geography and statistics (ibge). population count 2007 [internet]. 2007 sep [accessed 2010 sep 23]: [about 2p.]. available from: http://www.ibge.gov.br/home/estatistica/populacao/contagem2007/ contagem_final/tabela1_2_20.pdf. 3. farias júnior jc, nahas mv, barros mvg, loch mr, oliveira esa, de bem mfl et al. health risk behaviors among adolescents in the south of brazil: prevalence and associated factors. rev panam salud publica. 2009; 25: 344-52. 4. truin gj, koning kg, kalsbeek h. trends in dental caries in the netherlands. adv dent res. 1993; 7: 15-8. 5. federative republic of brazil. law no. 8069, 1990 jul 13. statute of child and the adolescent and other measures rectification. brasília: diário offcial da união. 1990 sep 13. 6. brazilian health ministry. adolescent health program (prosad). programmatic basis. brasília, brazil: health ministry; 1996. 7. pucca jr ga. the national politics of buccal health as social demand. cienc saude colet. 2006; 11: 243-6. 8. gushi ll, rihs lb, soares mc, forni tib, vieira v, wada rs, et al. dental caries in 15-to-19-year-old adolescents in são paulo state, brazil, 2002. cad saude publica. 2005; 21: 1383-91. 9. barbato pr, peres ma. tooth loss and associated factors in adolescents: a brazilian population-based oral health survey. rev saude publica. 2009; 43: 13-25. 10. wolf smr. the psychological meaning of losing one’s teeth in adult subjects. rev assoc paul cir dent. 1998; 52: 307-15. 11. cimões r, caldas jr. af, souza eha, gusmão es. influence of social class on clinical reasons for tooth loss. cienc saude colet. 2007; 12: 1691-6. 12. mendonça tc. dental mutilation: rural workers’ concepts of responsibility for tooth loss. cad saude publica 2001; 17: 1545-7. 13. costa sm, abreu mhng, vasconcelos m, lima rcgs, verdi m, ferreira ef. inequalities in the distribution of dental caries in brazil: a bioethical approach. cienc saude colet. 2013; 18: 461-70. 14. silva mes, magalhães cs, ferreira ef. dental loss and prosthetic replacement expectation: qualitative study. cienc saude colet. 2010; 15: 813-20. 15. world health organization (who). oral health surveys, basic methods. geneva: world health organization; 1997. 16. são paulo. municipal health secretariat of são paulo. epidemiological survey on oral health. city of são paulo, 2008-2009. summary of the first phase: children and adolescents. são paulo: coordination of primary care; 2009. 17. landis jr, kock gg. the measurement of observer agreement for categorical data. biometrics. 1977; 33: 159-74. 18. frias ac, antunes jlf, narvai pc. reliability and validity of oral health surveys: dental caries in the city of são paulo, 2002. rev bras epidemiol. reasons related to tooth loss among adolescents in são paulo, brazil braz j oral sci. 13(1):37-42 42 2004; 7: 144-54. 19. jovino-silveira rc, caldas jr. af, souza eh, gusmão es. primary reason for tooth extraction in a brazilian adult population. oral health prev dent. 2005; 3: 151-7. 20. peres ma, barbato pr, reis scgb, freitas chsm, antunes jlf. tooth loss in brazil: analysis of the 2010 brazilian oral health survey. rev saude publica. 2013; 47 (supl 3): 78-89. 21. brazilian state department of health. school of public health, university of são paulo. oral health conditions in the state of são paulo in 2002 final report. são paulo: fsp-usp; 2002. 22. bernabé e, marcenes w. income inequality and tooth loss in the united states. j dent res. 2011; 90: 724-9. 23. sousa mlr, rando-meirelles mpm, tôrres lhn, frias ac. dental caries and treatment needs in adolescents from the state of são paulo, brazil. rev saude publica. 2013; 47(supl 3): 50-8. 24. susin c, haas an, opermann rv, albandar jm. tooth loss in a young population from south brazil. j public health dent. 2006; 66: 110-5. 25. silva mês, vilhaça el, magalhães cs, ferreira ef. impact of tooth loss in quality of life. cienc saude colet. 2010; 15: 841-50. 26. de marchi, r. j, hugo fn, hilgert jb, padilha dmp. association between oral health status and nutritional status in south brazilian independent-living older people. nutrition. 2008; 24: 546-53. 27. lisboa ic, abegg c. oral hygiene habits and use of dental services by adolescents and adults in the municipality of canoas, rio grande do sul state, brazil. epidemiol serv saude. 2006; 15: 29-39. 28. goes psa, watt r, hardy r, sheiham a. impacts of dental pain on daily activities of adolescents aged 14-15 years and their families. acta odontol scand. 2007; 66: 7-12. 29. peres ma, peres kg, frias ac, antunes jlf. contextual and individual assessment of dental pain individual assessment of dental pain period prevalence in adolescents: a multilevel approach. bmc oral health. 2010; 10: 20. 30. hattne k, folke s, twetman s. attitudes to oral health among adolescents with high caries risk. acta odontol scand. 2007; 65: 206-13. 31. pitanga fernandes et, duarte vargas am, oliveira ac, camargo da rosa ma, dutra lucas s, ferreira e. factors related to dental caries in adolescents in southeastern brazil. eur j paediatr dent. 2010; 11: 165-70. 32. gibilini c, esmeriz cec, volpato lf, meneghim zmap, silva dd, sousa mlr. access to dental services and self-perception of oral health in adolescents, adults, and the elderly. arq odontol. 2010; 46: 213-23. 33. silva cjp, ferreira ef, magnaco fm, alves rg. the perception of oral health of the population of coimbra, minas gerais. rev fac odontol porto alegre. 2006; 47: 23-8. 34. bell jf, huebner ce, reed sc. oral health need and access to dental services: evidence from the national survey of children’s health. matern child health. j 2012; 16 (suppl 1): s27-34. reasons related to tooth loss among adolescents in são paulo, brazil braz j oral sci. 13(1):37-42 oral sciences n3 original article braz j oral sci. july | september 2013 volume 12, number 3 histometric analysis of alveolar bone regeneration with expanded polytetrafluoroethylene (e-ptfe) and latex membranes gustavo otoboni molina1, marcelo tomás de oliveira2, leonardo buss1, joão davi faraco peruchi1, jefferson ricardo pereira4, janaina salomon ghizoni3 1department of periodontology, dental school, university of southern santa catarina (unisul), tubarão, sc, brazil 2department of dental materials, dental school, university of southern santa catarina (unisul), tubarão, sc, brazil 3department of oral pathology, dental school, university of southern santa catarina (unisul), tubarão, sc, brazil 4department of prosthodontics, dental school, university of southern santa catarina (unisul), tubarão, sc, brazil correspondence to: jefferson ricardo pereira rua recife 200, apto 601, cep: 88701-420 bairro recife, tubarão, sc, brasil phone: +55 48 36471571 fax: +55 14 36264088 e-mail: jeffripe@rocketmail.com abstract aim: to investigate the amount of connective tissue migrated into the extraction socket using eptfe and latex membranes. methods: seventeen rats were selected and randomly divided into 3 groups: e-ptfe membrane (n = 6), latex membrane (n = 6) and control (no membrane, n=5). after extraction of the maxillary right incisor, the animals of the test groups were subjected to alveolar guided bone regeneration (gbr) surgery and received an expanded polytetrafluoroethylene (e-ptfe) and a latex membrane, respectively. thirty days after surgery, the animals were killed and histometric analysis was done to evaluate the migration of connective tissue. data were analyzed statistically by one-way anova and multiple-comparison tukey’s test at 5% significance level. results: there was statistically significant difference between groups eptfe and latex (p=0.001), and between groups e-ptfe and control (p=0.012), but no significant difference was found between groups latex and control (p=0.416). conclusions: the eptfe membrane showed better results and appeared more adequate for gbr therapy, forming a barrier to prevent the migration of connective tissue into the extraction socket. the latex membrane, on the other hand, did not show benefits over the control group. keywords: bone regeneration, membranes, oral surgery, tooth extraction. introduction when all attempts maintaining the tooth have failed and extraction of teeth is inevitable, the dentist is faced with the concern of maintaining the height and thickness of the alveolar ridge, which are critical for rehabilitation with implantsupported dentures. alveolar bone resorption after tooth extraction results in a significant reduction in bone height. the connective tissue can have great influence on osteogenesis during alveolar healing and results in narrowing of the alveolus after approximately one month of extraction, due to local bone resorption. this received for publication: may 20, 2013 accepted: august 16, 2013 braz j oral sci. 12(3):184-188 leads to aesthetic and restorative complications, such as decrease of bone volume for future installation of osseointegrated implants1. periodontal regeneration and bone ridge restoration using physical barriers are well-established procedures in reconstructive surgery. it is possible to find different techniques using physical barriers2-7 and the characteristics of the biomaterial and the design of the membrane used in guided tissue regeneration play an important role in obtaining good results8. expanded polytetrafluoroethylene (e-ptfe) membranes have been the standard materials for clinical treatment with guided bone regeneration (gbr), achieving good results when used as mechanical barriers covering sites of extraction eptfe is a polymer with high stability in biological systems, which provides better tissue organization, infection resistance and no induction of inflammatory reactions 1. e-ptfe membranes are used as mechanical barriers to protect the blood clot and allow bone cells to be selected to repopulate the bone defect, preventing the epithelial tissue to migrate into the defect1. however, care should be taken during placement because exposure of the membrane during the healing of the bone defect can lead to significant a decrease in bone tissue regeneration9. in dentistry, gbr is commonly understood as a surgical technique to improve bone defect in a particular region through new bone formation. this technique is based on melcher’s10 (1970) observation that the type of tissue formed in a given area depends on the type of cells populating that area. therefore, the aim of gbr to exclude soft tissue with the use of barriers in such a way that only bone cells populate the region to be regenerated. dahlin et al.11 (1988) was the first to demonstrate that bone defects created on mandibles of rats can be healed successfully using gbr procedures. gbr has been accepted as an excellent option for periodontal treatment and, after several decades of use, the advantages and disadvantages of this technique are already well known. non-absorbable membranes show good results when used in large bone defects12. murray13 (1957) placed plastic domes in the iliac and femoral regions of dogs, noting that the entire areas were filled with blood clot and consequently bone tissue. philips14 (1990) reported that the new bone is formed only where there is biomechanical stability of the membrane, that is, the membrane should be well fixed and remain stable as micro-movements may influence the type of tissue to be formed. bartee15 (2001) have stated that one of the benefits of the use of occlusive membranes is that there is less bone resorption in the early stages of healing, but the mechanism responsible for this result is not very clear. latex membrane is the result of biotechnological development and is available as a thin, translucent elastic, easy-to-use biomembrane originated from natural material (latex polymer extracted from plants). its structure is composed of polyisoprene chains and proteins, so like the cell membranes. the latex membrane has a microarchitecture that allows protein and cellular adhesion as well as stimulation of the various cell types adhered, in particular the macrophages involved in the healing process. the analysis of the micro-geometry of the latex membrane surface at 50and 500-fold increases reveals a “lunar surface” appearance, and examination by scanning electron microscopy at 1500-fold increase confirms the existence a rough exterior with recesses and protrusions. these structures have an important role in the processes of cell adhesion and maintenance of vascularization of tissue-interface membrane of the fibrous capsule, which begins when the latex membrane is applied on the implanted tissue. it has great plasticity and can be used as a dressing on areas of different sizes, with the advantage of being easily removable. frade et al.16 (2004) investigated the effect of latex biomembrane to treat leg ulcers compared with the traditional treatment (antibiotic ointments and proteolytic enzymes). biopsies of the lesion were collected before and 30 days after treatment and subjected to histopathological and immunohistochemical analyses. the result showed that the biomembrane facilitated lesion healing, offering the advantage of a low cost and ease of use. it was also observed that the biomembrane led to a clinical and histopathologic differentiation of tissue healing, with increase in the detection of growth factors, such as vascular endothelial growth factor (vegf) and transforming growth factor â1 (tgfâ1), and reduction of expression of the enzyme inducible nitric oxide synthase (inos), compared with controls. mrue et al.17 (2000) used latex membranes in the treatment of chronic ulcers of different etiologies with 2 to 18 years of evolution by covering the lesions with the membranes every 24/48 h. the results showed that from the 3rd day of treatment, the granulation tissue was clear, becoming lush and full on the 12th day. after granulation, the process of reepithelization occurred at around 75% of patients quickly and spontaneously. closure of the lesions with the use of latex membrane ranged from 4 days to about 6 months. in the remaining 25%, despite the formation of granulation tissue, reepithelization was unsatisfactory, and a split-thickness skin graft was used with 100% success rate. an important aspect of concern that could influence the success and predictable result in the healing of bone defects is bacterial infection. it has been suggested that immediate postoperative infection in periodontal defects and colonization of membrane surface could be the reason for poor results in some cases. for example, periodontal pathogens can colonize a membrane within 3 min of intraoral handling. the presence of bacteria on the surface of a membrane in contact with the gingiva 6 weeks after surgery has been shown to affect significantly the gain of clinical insertion. controlling bacterial colonization in the first stage of healing and reducing the spread of infections can increase the predictability of results18. occlusive membranes and e-ptfe space promoters have been developed to assist bone regeneration in supraalveolar periodontal defects. polimeni et al.19 (2006) estimated the effect of cell occlusion and space provision by use of membranes on periodontal regeneration. space-providing occlusive and porous e-ptfe membranes were implanted to histometric analysis of alveolar bone regeneration with expanded polytetrafluoroethylene (e-ptfe) and latex membranes 185185185185185 braz j oral sci. 12(3):184-188 table 1. mean depths of connective tissue migration into the extraction sockets (in mm) of the three groups group mean s.d. e-ptfe membrane 0.3338 a 0.1267 latex membrane 1.866 bc 0.1841 control (no membrane) 1.516 c 0.3921 same letters indicate no statistically significant difference (p<0.05). provide for guided-tissue regeneration in supraalveolar periodontal defects. the gingival ûaps were advanced for primary intention healing that was allowed to progress for 8 weeks. a histometric analysis assessed alveolar bone regeneration relative to space provision by the e-ptfe membranes. the bivariate analysis showed that space provision and membrane occlusivity enhanced signiûcantly bone regeneration. sites that received the occlusive membrane and those with enhanced space provision presented signiûcantly greater bone regeneration than sites receiving the porous membrane (p=0.03) or exhibiting more limited space provision (p=0.0002). however, a signiûcant association was found between bone regeneration and space provision at sites receiving occlusive (b = 0.194, p<0.02) and porous (b = 0.229 p<0.0004) membranes, regardless of the treatment, which means that the relationship between space provision and regeneration was signiûcant for both types of membranes. regeneration followed similar patterns in both groups. the authors assumed that the healing assisted by these membranes is similar, or at least similarly inûuenced by space provision. however, the magnitude of regeneration was signiûcantly enhanced at sites receiving the occlusive membranes compared with that at sites receiving the porous membrane, when adjusted for wound area. thus, while space provision appears to be critical for regeneration, membrane occlusivity seems to have adjunctive effects. although it is not possible to affirm that cell occlusion is an absolute prerequisite for periodontal regeneration, it seems clear that the use of cell occlusive membranes may optimize the magnitude of periodontal regeneration. the purpose of this study was to compare the use of eptfe membrane, latex membrane and no biomembrane in the maintenance of the extraction socket, assessing the healing processes and tissue responses caused by membranes. material and methods the study was approved by the ethics committee of the university of southern santa catarina (protocol number 07.303.4.04iii). seventeen 50-70-day-old male wistar rats (rattus norvegicus albinus), weighing between 180 and 200 g were kept in individual cages under controlled conditions of lighting (12 h of light/12 h of darkness) and temperature (21 to 25 °c), fed a balanced solid diet. the animals were randomly divided into 3 groups: two tests groups using eptfe membrane (membrana de teflon; bionnovations, bauru, sp, brazil) or latex membrane (biocure; pele nova biotecnologia, ribeirão preto, sp, brazil), and a control group, which did not receive a membrane. the test groups were subjected to gbt surgeries. the animals were sedated by inhalation of sulfuric ether (rioquímica, são josé do rio preto, sp, brazil) and anesthetized with an intraperitoneal injection of sodium thiopental (thiopentax; cristal pharma ltda, contagem, mg, brazil; 0.2 ml/100 g body weight). it was used the infiltrating local anesthetic administered mepivacaine 2% with epinephrine 1:100000 (dfl, rio de janeiro, rj, brazil). the maxillary right incisor of each animal was extracted using instruments specially adapted for this purpose. the extraction socket was curetted, gently rinsed with saline and either covered with e-ptfe or latex membranes or left with the blood clot only. wen placed, the membranes were carefully adapted to the bone margins to avoid migration of the connective tissue into the extraction socket. the periosteum was sutured with 5.0 nylon thread using a 1.5 cm a needle with a triangular cross section (techsuture; techsuture industria de comercio de produtos cirurgicos ltda, bauru, sp, brazil). after 30 days of experimental surgical procedures, all animals were sedated by inhalation of sulfuric ether (rioquímica) and were killed by decapitation. the maxilla was separated from the head and the left maxilla was separated from the right maxilla with the aid of a chisel, by making an incision at the median sagittal plane along the intermaxillary suture. a straight cut was done with a pair of surgical scissors (hu-friedy, chicago, il, usa) tangential to the distal side of the molars. samples of the surgical areas were removed in blocks containing alveolar bone and surrounding soft and hard tissues. the blocks were embedded in paraffin and 6ìm-thick longitudinal sections were obtained and stained with hematoxylin and eosin. the sections were viewed with video camera (sony, dcr-sr42, 40x optical zoom, carl zeiss lens, oberkochen, germany) and the image j (rasband, w.s., imagej, u. s. national institutes of health, bethesda, md, usa) software was used for analyzing histomorphometrically the depth of connective tissue invagination into the extraction socket in millimeters. a first line was traced on the image displayed on the computer screen connecting the buccal and the palatal alveolar crests and, from this line, a second line was traced towards the deepest portion of the connective tissue invagination into the extraction socket. the results were analyzed statistically by analysis of variance (p<0.05) and tukey’s test for multiple comparison with a significance level of 5%. results there was statistically significant difference between groups e-ptfe and latex (p=0.001) and between groups eptfe and control (p=0.012), but no statistically significant difference could be found between groups latex and control (p=0.416). the mean depths of connective tissue migration into the extraction sockets (in mm) of the three groups are presented in table 1. 186186186186186 histometric analysis of alveolar bone regeneration with expanded polytetrafluoroethylene (e-ptfe) and latex membranes braz j oral sci. 12(3):184-188 discussion in the present study, the e-ptfe membrane showed better results allowing less migration of connective tissue into the extraction socket, while the protection offered by the latex membrane against connective tissue invagination did not differ significantly from that of the control group (without membrane). this can be explained because the membrane bioabsorbable (latex), in general, increases bone regeneration more than e-ptfe membranes. however, if the dehiscence of the soft tissues does not exist, the e-ptfe membrane allows bone regeneration slightly better than the membrane bioabsorbable. from a clinical point of view, the use of membranes simplifies the management and stabilization of bone-graft substitute materials, but from a biological point of view, the use of barriers promotes a recruitment of defense cells20. frade et al.16 (2004) reported that treatment with latex membrane leads the organization of scar tissue consequent to increased production of steam cells. ereno et al.21 (2010) showed that latex membrane accelerated healing in critical bone defects. in another study19, porous membranes implanted in supraalveolar periodontal defects were compared with occlusive membranes. it was observed that the occlusion tissue is not an absolute requirement for periodontal regeneration, as the sites that received the membranes showed significant regeneration of cementum, periodontal ligament and alveolar bone, similar to the sites treated with occlusive membranes, which is in accordance with the present study. in the present study, the e-ptfe membrane showed significantly better results than the control group. in a previous study using a tetracycline-coated e-ptfe membrane, it was suggested that the antimicrobial properties during the initial healing period could result in a gain of clinical integration22. the results of the present study are in accordance with those of schenk et al.23 (1994), who assessed the pattern of bone regeneration in canine mandibles using standard and prototype reinforced e-ptfe membranes. after a healing period of 2 and 4 months, the control sites (without membranes) exhibited incomplete bone healing, with a persisting defect, while the test sites (with membranes) presented significantly enhanced bone formation, although bone regeneration was not complete after 4 months. the histological analysis showed that, once activated, bone regeneration progressed in a programmed sequence of maturation steps, which closely resembles bone development and growth pattern. a limitation of this study is that experimental animal models do not precisely replicate the “in vivo” human conditions, and so further research using different membranes in human patients and with different healing periods is required. the results of this study demonstrate that treatment with gbr is certainly a successful procedure, when an adequate technique is used. comparing the two types of membranes, the e-ptfe membrane achieved better results, preventing the migration of tissue into the site of extraction. the latex membrane did not achieve good results in this type of periodontal therapy. few studies have investigated the use of latex membranes in the treatment of periodontal defects through gbr procedures, needing more studies on this subject. references 1. diès f, etienne d, abboud nb, ouhayoun jp. bone regeneration in extraction sites after immediate placement of an e-ptfe membrane with or without a biomaterial. a report of 12 consecutive cases. clin oral impl res. 1996; 7: 277-85. 2. montanari m, callea m, yavuz i, maglione m. a new biological approach to guided bone and tissue regeneration. bmj case rep. 2013; apr 9, 2013. doi:pii: bcr2012008240. 3. shue l, yufeng z, mony u. biomaterials for periodontal regeneration: a review of ceramics and polymers. biomatter. 2012; 2: 271-7. 4. horowitz r, holtzclaw d, rosen ps. a review on alveolar ridge preservation following tooth extraction. j evid based dent pract. 2012; 12(3 suppl): 149-60 5. al salamah l, babay n, anil s, al rasheed a, bukhary m. guided bone regeneration using resorbable and non-resorbable membranes: a histological study in dogs. odontostomatol trop. 2012; 35: 43-50. 6. matsumoto g, hoshino j, kinoshita y, sugita y, kubo k, maeda h, et al. evaluation of guided bone regeneration with poly(lactic acid-co-glycolic acid-co-å-caprolactone) porous membrane in lateral bone defects of the canine mandible. int j oral maxillofac implants. 2012; 27: 587-94. 7. scheyer et, schupbach p, mcguire mk. a histologic and clinical evaluation of ridge preservation following grafting with demineralized bonematrix, cancellous bone chips, and resorbable extracellular matrix membrane. int j periodontics restorative dent. 2012; 32: 543-52. 8. macedo nl, de macedo lg, matuda fde s, ouchi sm, monteiro as, carvalho yr. guided bone regeneration with subperiosteal implants of ptfe and hydroxyapatite physical barriers in rats. braz dent j. 2003; 14: 199-24. 9. lekovic v, kenney eb, weinlaender m, han t, klokkevold p, nedic m, et al. a bone regenerative approach to alveolar ridge maintenance following tooth extraction. report of 10 cases. j periodontol. 1997;68: 563-70. 10. melcher ah. repair of wounds in the periodontium of the rat. influence of periodontal ligament on osteogenesis. arch oral biol. 1970; 15: 1183-204. 11. dahlin c, linde a, gottlow j, nyman s. healing of bone defects by guided tissue regeneration. plast reconstructive surg. 1988; 81: 672-6. 12. walters sp, greenwell h, hill m, drisko c, pickman k, scheetz jp. comparison of porous and non-porous teflon membranes plus a xenograft in the treatment of vertical osseous defects: a clinical reentry study. j periodontol. 2003; 74: 1161-8. 13. murray c, holden d, roachlau w. experimental and clinical study of new growth of bone in a cavity. am j surg. 1957; 95: 385-7. 14. phillips rw, jendresen md, klooster j, mcneil c, preston jd, schallhorn rg. reports of the committee on scientific investigation of the american academy of restorative dentistry. j prosthetic dent. 1990; 64: 74-110. 15. bartee kb. extraction site reconstruction for alveolar ridge preservation. part 2: membrane-assisted surgical technique. j oral implantol. 2001; 27: 194-7. 16. frade mac, cursi, ib, andrade ff, netto, jc, barbetta fmb, foss nt. management of diabetic skin wounds with a natural latex biomembrane. med cutan iber lat am. 2004; 32: 157-62. 17. mrue, f. tissue neoformation induced by natural latex biomembrane with polylysine. application in esophageal and abdominal wall neoformation. experimental study in dogs [thesis]. ribeirao preto: university of sao paulo; 2000. 18. sipos pm, loos bg, abbas f, timmerman mf, van der velden u. the combined use of enamel matrix proteins and tetracycline-coated expanded polytetrafluoroethylene barrier membrane in treatment of intra-osseous defects. j clin periodontol. 2004; 32: 765-72. 187187187187187histometric analysis of alveolar bone regeneration with expanded polytetrafluoroethylene (e-ptfe) and latex membranes braz j oral sci. 12(3):184-188 19. polimeni g, koo k-t, qahash m, xiropaidis av, albandar jm, wikesjo¨ ume. prognostic factors for alveolar regeneration: effect of tissue occlusion on alveolar bone regeneration with guided tissue regeneration. j clin periodontol. 2004; 31: 730-5. 20. hämmerle chf, jung re. bone augmentation by means of barriers membranes. periodontol 2000. 2003; 33: 36-53. 21. ereno c, guimarães sa, pasetto s, herculano rd, silva cp, graeff cf, et al. latex use as an occlusive membrane for guided bone regeneration. j biomed mater res a. 2010; 95: 932-9. 22. zarkesh n, nowzari h, morrison jl, slots j. tetracycline-coated polytetrafluorethylene barrier membranes in treatment of intraosseous periodontal lesions. j periodontol. 1999; 70: 1008-16. 23. schenk rk, buser d, hardwick wr, dahlin c. healing pattern of bone regeneration in membrane-protected defects: a histologic study in the canine mandible. int j oral maxillofac implants. 1994; 9: 13-29. 188188188188188 histometric analysis of alveolar bone regeneration with expanded polytetrafluoroethylene (e-ptfe) and latex membranes braz j oral sci. 12(3):184-188 oral sciences n3 braz j oral sci. 13(1):58-63 original article braz j oral sci. january | march 2014 volume 13, number 1 effect of astaxanthin and fish oil on enzymatic antioxidant system and α-amylase activity of salivary glands from rats mariana ferreira leite1, amanda martins de lima1, simone jee sun kang1, maria teresa botti rodrigues dos santos1, rosemari otton2 1universidade cruzeiro do sul unicsul, biological and health sciences, department of pediatric dentistry, são paulo, sp, brasil 2universidade cruzeiro do sul unicsul, biological and health sciences, department of health sciences, são paulo, sp, brasil correspondence to: mariana ferreira leite universidade cruzeiro do sul avenida ussiel cirilo 225 cep: 08060-070 são miguel paulista são paulo sp – brasil phone: +55 11 2037-5744 e-mail: mariana.leite@cruzeirodosul.edu.br received for publication: january 29, 2014 accepted: march 20, 2014 abstract salivary glands contribute to oral health. it is therefore of interest to study therapies that may favor their function and protection. aim: to evaluate the effect of astaxanthin, fish oil and association of them on enzymatic antioxidant system and functional parameters of salivary glands. methods: healthy rats (n=32) were divided into 4 groups: untreated-control, astaxanthin-treated (1 mg/kg body weightbw), fish oil-treated (10 mg epa/kg bw and 7 mg dha/kg bw), and fish oil plus astaxanthin-treated. a prophylactic dose was administered in each group daily by gavage, for 45 days. superoxide dismutase (sod), catalase, glutathione peroxidase, reductase, and α-amylase activities were determined in salivary glands and compared by anova and tukey post-test (p<0.05). results: parotid gland presented increased catalase and glutathione system and unaffected sod activity after astaxanthin and astaxanthin plus fish oil treatment (p<0.05). fish oil stimulated only glutathione peroxidase activity of parotid gland (p<0.05). submandibular gland presented stimulated sod and catalase, and reduced glutathione reductase activities after fish oil and fish oil plus astaxanthin treatment (p<0.05). sod and glutathione reductase activities were reduced by astaxanthin treatment in submandibular gland (p<0.05). parotid gland presented increased α-amylase activity in all groups supplemented and submandibular glands presented no changes (p<0.05). conclusions: astaxanthin, fish oil and combination of them stimulated the antioxidant system and functional parameter of salivary glands, which could be beneficial to oral health. keywords: astaxanthin; fish oil; antioxidant system; α-amylase; salivary glands. introduction the salivary glands produce saliva, which contributes to maintaining oral health. the parotid gland has serous cells in abundance, which produce a salivary secretion with abundant water and electrolytes responsible for the buffering capacity and protection of dental surface1. the proteins synthesized by parotid acinar cells are stored in large secretory granules whose composition includes α-amylase, leucine-rich parotid secretory protein (psp), and proline-rich proteins (prps), in addition to multiple minor components2-3 related to digestive and protective functions. the major regulated secretory pathway involves large granules that are exocytosed in response to autonomic stimulation4. in some diseases, for example sjögren’s syndrome (ss), there is a hyposalivation related to organic disorders of salivary glandular tissue. increased oxidative stress due to high production of gisele higa texto digitado http://dx.doi.org/10.1590/1677-3225v13n1a12 59 braz j oral sci. 13(1):58-63 reactive oxygen species (ros) is proposed to be involved in pathogenesis of ss5. the submandibular gland is composed of a predominance of cells characterized by mucus secretion. salivary seromucous glands are regulated predominantly by parasympathetic activation of muscarinic receptors, resulting in exocrine secretion of mucins and macroglobulin responsible by lubrication and protection of oral mucosa3-4. in addition to the autonomic innervation, neuropeptides and hormones can influence the secretion and vascularization of submandibular6. some pathological conditions related to symptoms of xerostomia, such as radiotherapy, diabetes and ss, can alter the secretion of both parotid and submandibular glands5,7-8. a number of endogenous systems, such as the aerobic metabolism and electron transport chains, generate highly reactive molecules with important biological functions known as reactive oxygen species (ros), including superoxide and hydrogen peroxide (h2o2). in order to prevent oxidative damage, the antioxidant system presents a group of cellular enzymes (sod, catalase and glutathione system) responsible for the control of free radicals. while sod catalyzes the dismutation of superoxide anion (o2") to h2o2, catalase and glutathione (peroxidase, reductase) system reduce cellular toxicity degrading peroxides into oxygen and water9. nonenzymatic antioxidant system also maintains the balance of reactive oxygen species (ros), including vitamin c, carotenoids and fish oil. antioxidants are expected to serve as potentially therapeutic agents for oxidative stress-related diseases. astaxanthin (ast) is a xanthophyll carotenoid and current human dietary intake is almost exclusively from seafood. ast has been used with efficacy and safety due to its biological properties, such as antioxidant, antiinflammatory and immunemodulatory properties and cardioprotective effect in therapies for aging, diabetes, cardiovascular disease and other systemic disease10-11. the antioxidant action of ast is mainly due to the presence of oxygenated groups contained in each additional ring structure of the molecule, which reduces the effect of peroxyl, superoxide radicals and singlet oxygen10-11. fish oil is a compound rich in polyunsaturated fatty acids (pufas) mainly represented by eicosapentaenoic acid (epa) and docosahexaenoic acid (dha) that regulates a wide range of functions in the body including blood pressure, blood clotting, modulation of inflammatory response, and correct development and functioning of brain and nervous systems 12. epidemiological studies suggest that among populations ingesting large amounts of pufas, mainly present in fish oil, there are reduced risk of neurodegenerative disorders such as alzheimer’s disease, lower incidence of acute myocardial infarction and chronic inflammatory diseases such as rheumatoid arthritis, ulcerative colitis, psoriasis, among other inflammatory diseases13. recently, our research group published a study evaluating the effect of ast administration on antioxidant parameters of salivary glands in diabetic rats, showing a positive effect after supplementation 14. however, ast presented a modest antioxidant effect on salivary glands from healthy rats. for this purpose we measured the enzymatic antioxidant system of parotid and submandibular gland of healthy rats to evaluate whether the combination of ast with fish oil could be more effective than ast and fish oil alone. moreover, it was also evaluated the α-amylase activity as a functional parameter of salivary gland. material and methods chemicals and natural products all purified chemicals were purchased from sigmaaldrich chemical company (st. louis, mo, usa), except for common laboratory solutions and buffers, which were obtained from labsynth (diadema, são paulo, sp, brazil). fish oil (fo) capsules were purchased from pharmanostra (são paulo, sp, brazil). each fo capsule of 500 ml contains 9 kcal (38 kj), 2.0 mg of mixed tocopherols, and 1.0 g of total fat, out of which 30% are from saturated fats, 20% from monounsaturated fats (mostly palmitoleic and oleic acids), and 50% of polyunsaturated fatty acids (300 mg epa and 200 mg dha). natural asta supplements (astareal a1010) were obtained as a donation bioreal ab (gustavsberg, sweden). astareal a1010 is an astaxanthinrich natural microalgae haematococcus pluvialis product that contains 5.2-5.8% of total carotenoids, whereas 5.0-5.6% are purely astaxanthin (3.9% as monoesters, 0.9% diesters, and 0.1% in free form). based on that composition, we calculated the astareal a1010 biomass per gavage volume (of 10% tween-80 aqueous solution, v/v) and animal body weight (bw) to reach the aforementioned mg asta/ kg bw. animals adult wistar male rats (225.6±17.1g) were housed in plexiglas cages (4 rats/cage) under standard laboratory conditions: 12 h light/dark cycle; lights on at 7:00 a.m.; 22±2°c and ad libitum access to water and purina rat chow. the animals used were handled in accordance with guidelines of the committee on care and use of laboratory animals resources. the research ethics committee of the federal university of são paulo approved the experimental protocol (protocol number 1938/09). supplementation protocols four experimental groups of 8 animals each were formed: control (fed with 400 µl of 10% tween-80 aqueous solution (v/v)); asta (fed with 1 mg asta/kg body weight (bw)); fish oil (fed with 10 mg epa/kg bw and 7 mg dha/ kg bw) and fo+asta (fed with 1 mg asta/kg bw, 10 mg epa/kg bw and 7 mg dha/kg bw). the animals were treated orally by gavage in a constant volume of 1 ml/kg, 5 days a week, for 45 days. a maximum volume of 400 µl was established in order to prevent regurgitation or stomach discomfort of the animals. fish oil content of capsules was diluted in 10% tweeneffect of astaxanthin and fish oil on enzymatic antioxidant system and α-amylase activity of salivary glands from rats 60 80 aqueous solution (v/v) to reach final n-3 pufas concentrations of 10 mg epa/kg bw and 7 mg dha/kg bw. an identical procedure was conducted for animal supplementation with 1 mg asta/kg bw. for combined fo and asta treatments (fo+asta), both components were diluted in the same stock 10% tween-80 aqueous solution (v/v) to reach previously described concentrations. experimental procedure and preparation of homogenates after forty-five days of treatment, fed rats were killed by decapitation. the salivary glands were immediately removed, weighed (50mg), homogenized on ice-cold condition at 10%, with 0.5 ml of 50 mm sodium phosphate buffer, ph 7.4, vortexed briefly and broken down by ultrasonication in a vibra-cell ultrasonic liquid processing equipment (sonics & materials, inc. newtown, ct usa). a refrigerated centrifugation step was included (10000 x g for 10 min at 4oc) and supernatant was then used for further analysis. measurement of antioxidant enzymes • assay of superoxide dismutase activity (sod) the activity of superoxide dismutase (sod) was measured according to ewing and janero15. the complete reaction buffer included 50 mm sodium phosphate buffer, ph 7.4, 0.1 mm edta, 50 µm nitrobluetetrazolium (nbt), 78 µm nadh, and 3.3 µm phenazine methosulphate (pms) used as an o2 generator. the kinetic absorbance variation at 560 nm was monitored for 2 min to evaluate o2 dependent reduction of nbt. a control system lacking pms revealed negligible change in absorbance at 560 nm with an ultrospec 3000 spectrophotometer (pharmacia biotech, little chalfont, uk). • assay of catalase activity the decomposition of h2o2 can be followed directly by the decrease in absorbance at 240 nm (ε240 = 0.0394 ± 0.0002 l.mm-1cm-1). one catalase unit is defined as the enzyme concentration required for the decomposition of 1 ìmol of h2o2 per min at 25oc, as described by aebi16. the complete reaction system for catalase consisted of 0.1 mm phosphate buffer, ph 7.4 and 10 mm h2o2. the reaction was initiated by the addition of 10 mm h2o2 and absorbance was monitored for 2 min at 240 nm with the ultrospec 3000 spectrophotometer (pharmacia biotech, little chalfont, uk). • assay of glutathione peroxidase (gpx) and reductase activities (gr) gpx activity was measured according to the method described by mannervik17. enzyme activity was determined using 2.5 u/ml of glutathione reductase (gr), 10 mm reduced glutathione (gsh), 250 µm sodium azide (as a catalase inhibitor) and 1.2 mm nadph in the presence of 4.8 mm tert-buthyl hydroperoxide used as substrate. the oxidation of nadph was monitored at 340 nm for 2 min in 0.2 m phosphate buffer (ph 7.4) in the ultrospec 3000 spectrophotometer (pharmacia biotech). glutathione reductase (gr) activity was measured using the same methodology described by mannervik17. alternatively, gr activity was determined using 3.6 mmnadph and 10 mm oxidized glutathione (gssg). again, the nadph oxidation was monitored in 0.2 m phosphate buffer, ph 7.4, at 340 nm for 2 min with the ultrospec 3000 spectrophotometer (pharmacia biotech). • assay of α-amylase activity and total protein measurement α-amylase activity was determined by the method described by fisher and stein18, using maltose as standard. the samples were incubated with 1% starch solution in 20 mm phosphate buffer ph 7.0 for 5 min at 30oc. the reaction was interrupted by the addition of an alkaline solution of dinitrosalicylic acid and the mixture was maintained in boiling water for 5 min. the absorbance was determined at 530 nm with the ultrospec 3000 spectrophotometer (pharmacia biotech). specific enzyme activities were all related to protein concentrations, which were estimated by bradford19 using bovine serum albumin as a standard. statistical analysis to increase data reliability, each sample was evaluated in duplicate. the data are presented as mean ± standard deviation (sd). the anderson-darling test was applied for the evaluation of the frequency distribution of the data. after confirming the normality and homogeneity of data distribution, the biochemical parameters of the groups studied were compared by analysis of variance and tukey’s multiplecomparison test, using the graphpad instat software (graphpad software, inc., san diego, ca, usa). the level of significance adopted was 5%. results tables 1 and 2 show the enzymatic activities of antioxidant system and α-amylase of parotid and submandibular, respectively. the results of superoxide dismutase (u/mg protein), catalase (µmol/mg protein), glutathione peroxidase (u/mg protein), glutathione reductase (u/mg protein), and α-amylase activities (mg malt/mg protein) are presented as mean and standard deviation. in the parotid gland (table 1), fish oil treatment (fo) stimulated the glutathione peroxidase activity (65%) compared to the control group. astaxanthin treatment (asta) and its association with fish oil (fo+asta) also increased the catalase (43 and 61 %, respectively), glutathione peroxidase (43 and 86%, respectively), and glutathione reductase activities (72 and 73%, respectively) as compared to the control group (p<0.05). there was no significant effect on the sod activity in all groups. in the submandibular gland (table 2), fish oil treatment effect of astaxanthin and fish oil on enzymatic antioxidant system and α-amylase activity of salivary glands from rats braz j oral sci. 13(1):58-63 sod cat gp gr amylase activity c 73.85 ± 7.03a 42.01 ± 11.53 a 0.38 ± 0.04a 0.42 ± 0.03a 0.30 ± 0.09 a f o 85.57 ± 6.21b 71.28 ± 15.18 b 0.38 ± 0.10a 0.17 ±0.02 b 0.26 ±0.10 a asta 47.33 ± 6.23c 54.17 ± 10.41ab 0.21 ± 0.03 b 0.32 ± 0.04 c 0.33 ± 0.10 a fo+asta 89.43 ± 6.64b 72.27 ± 16.73 b 0.27 ± 0.07 b 0.36 ± 0.07 c 0.28 ± 0.05 a table 2table 2table 2table 2table 2 – superoxide dismutase (sod) (u/mg prot), catalase (cat) (µmol/mg prot), glutathione peroxidase (gp) (u/mg prot), glutathione reductase (gr) (u/mg prot), and α-amylase activity (mg malt/ mg prot) of submandibular gland from rats of untreated control (c) (n=8), fish oil-treated (fo) (n=8), astaxanthin-treated (asta) (n=8), and fish oil/astaxanthin-treated groups (fo+asta) (n=8). mean± sd. statistically significant differences compared among groups for the same parameter are represented by distinct letters (p<0.05). table 1table 1table 1table 1table 1 – superoxide dismutase (sod) (u/mg prot), catalase (cat) (µmol/mg prot), glutathione peroxidase (gp) (u/mg prot), glutathione reductase (gr) (u/mg prot), and α-amylase activity (mg malt/ mg prot) of parotid gland from rats of untreated control (c) (n=8), fish oil-treated (fo) (n=8), astaxanthin (asta)-treated (n=8), and fish oil/astaxanthin-treated groups (fo+asta) (n=8). mean± sd. statistically significant differences compared among groups for the same parameter are represented by distinct letters (p<0.05). sod cat gp gr amylase activity c 32.38 ± 12.46a 24.18 ± 4.38 a 0.23 ± 0.08a 0.18 ± 0.04a 0.45 ± 0.16a f o 37.34 ± 16.26 a 25.90 ± 5.86 a 0.38 ± 0.06b 0.19 ±0.05a 1.00 ± 0.39b asta 35.08 ± 10.68 a 34.79 ± 6.99 b 0.33 ± 0.09 b 0.31 ± 0.07 b 0.70 ± 0.29 b fo+asta 31.80 ± 6.35 a 38.93 ± 4.24 b 0.43 ± 0.14 b 0.32 ± 0.08 b 0.93 ± 0.32 b (fo) stimulated the sod (16%) and catalase activities (70%), whereas it reduced glutathione reductase activity (60%) as compared to the control group (p<0.05). submandibular gland from rats treated with asta presented a reduction of sod (36%), glutathione peroxidase (45%), and glutathione reductase (24%) activities and an increase in the catalase activity (29%) compared to the control group (p<0.05). association of fish oil and astaxanthin (fo+asta) promoted a stimulating effect on the sod and catalase activities in the submandibular and an inhibitory effect on glutathione system (peroxidase and reductase, 29 and 15%, respectively) (p<0.05). fish oil-treated, asta-treated and fish oil plus astatreated groups presented increased α-amylase activity in the parotid gland (120, 55, and 106%, respectively) compared to the control group (p<0.05) (table 1). no changes were observed in the submandibular gland (table 2). discussion our research group has developed scientific studies evaluating the actions of different elements present in the diet on salivary glands and dental pulp14,20-21,with positive effects on oral healthy. in the present study, we evaluated the effect of astaxanthin, fish oil and association of them on antioxidant system and amylase activity of salivary glands from healthy rats. we observed a stimulating effect of parameters studied after supplementation. the impact of nutritional changes on the oral cavity of human individuals is a topic of interest in dentistry22-23. dental caries is considered a public health problem that has the diet as one of the main etiological factors, with increased risk by association between inadequate intake of fruits and vegetables and excessive consumption of sugar sweetened beverages and foods22.the effect of diet has also been investigated on periodontal disease and individuals who had a poor diet presented higher number of missing teeth, higher average clinical attachment loss, which were significantly associated with increased odds of periodontitis23. considering the contribution of salivary glands in maintaining oral health, it seems of interest to study elements that can act therapeutically in salivary glands, such as vitamins, carotenoids, and minerals. some studies have shown that administration of vitamins are very beneficial to the salivary glands for modulating the quality and quantity of saliva production and promoting tissue protection in cases of systemic diseases24-25. asta, fish oil and the combination of them presented a different pattern of stimulation on antioxidant system according to the type of salivary gland. studies have shown that the parotid and submandibular glands have different responses to oxidative challenge such as diabetes and exposure to fluorides, particularly with reduced activity of some antioxidant enzymes and increased lipid peroxidation and oxidative damage 14, 26-27 in the submandibular gland, and greater stability and regenerative ability of parotid gland7,14.when submitted to antioxidant therapies, the salivary glands also exhibit different behavior from each other14, which agrees with our results. it is known that the parotid can be more prepared to oxidative damage by reactive oxygen species by presenting a predominantly aerobic metabolism28, however there are no reports in the literature to clarify the specific mechanism of action of parotid 17161effect of astaxanthin and fish oil on enzymatic antioxidant system and α-amylase activity of salivary glands from rats braz j oral sci. 13(1):58-63 62 and submandibular when exposed to oxidative or antioxidants conditions. further studies are required to evaluate the signaling pathways and expression of antioxidants proteins in salivary glands. the asta action on the antioxidant system of salivary glands from healthy rats has been previously evaluated by our research group, presenting mild antioxidant effects14. for this reason, we decide to combine another element, fish oil, which also has a potential antioxidant role. the antioxidant effects observed after combination with fish oil were markedly improved and could be beneficial to salivary glands by enhancing the protective action of enzymatic antioxidant system of parotid and submandibular glands. in addition, we demonstrated that astaxanthin administered in smaller doses (1mg/kg bw) for a longer time (45 days) stimulates enzymatic antioxidant system of parotid gland compared to the protocol used before (20mg/kg bw for 30 days).these results show that a diverse diet may protect salivary glands14. some agents have oxidative property, but at low concentrations stimulate cellular enzymes of antioxidant system, acting as pro-oxidant29..... among all biomolecules, lipids are the most sensitive to free radicals. double bonds in fatty acids form peroxide products by reacting with free radicals and lipid radicals can be formed subsequently upon removal of electrons. excessive consumption of lipids, including polyunsaturated fatty acid (pufa), increases lipid peroxidation significantly and may raise the susceptibility of tissues to free radical oxidative damage30. a previous published study showed that submandibular gland presents increased resistance against oxidative damage depends on the source of dietary pufa31. in the present study, the fish oil administered in low concentration stimulated the enzymatic antioxidant system of submandibular gland, probably acting as pro-oxidant agent. on the other hand, parotid gland also responded positively to treatment with antioxidants showing increased amylase activity. the response of salivary cells in the secretion process depends on the subtype of autonomic receptors that appears to be different on each gland 32, submandibular and parotid glands have a predominance of muscarinic and adrenergic receptors, respectively. some study showed that the beta-adrenergic agonist induced an increase of camp in both salivary glands, but while in the parotid it triggered amylase release, in the submandibular it was unable to increase α-amylase secretion32. parotid α-amylase release was dependent on adenylate cyclase activation32.dietary omega 3 fatty acids change the fatty-acid composition of the membrane phospholipids of submandibular salivary glands, accompanied by higher adenylate-cyclase activity33. the increased α-amylase activity in parotid gland could be related to adenylate-cyclase activity stimulation, particularly in the groups that received the fish oil supplementation that showed a more expressive increase of amylase activity. moreover, further studies are required in order to explain the stimulatory effect of astaxanthin in the α-amylase activity of parotid gland. α-amylase is highly abundant salivary protein responsible for the initial digestion of starch, favoring the formation of the food bolus. its main function is to split the α-1,4-glicosidic bindings of several glycans, such as starch (amylopectin), producing oligosaccharides (dextrin) disaccharides (maltose, isomaltose) and monosaccharide glucose. its action is inactivated in the acid portions of the gastrointestinal tract and is consequently limited to the mouth34.α-amylasehas been studied as a biomarker for sympathetic nervous system and functional capacity of salivary glands7,35. if by one side α-amylase provides substrate for bacteria, favoring the formation of dental biofilm36, in contrast, it presents specific binding sites with affinity for microorganisms (cariogenic and periodontopathogenic), forming bacterial agglomerates diluted in saliva that are easily eliminated by swallowing and consequently suffer acid digestion by the stomach37. the stimulation of α-amylase activity in parotid gland of rats supplemented with astaxanthin, fish oil and the combination of them was an interesting result, which could express an improved functional capacity of salivary gland and defense properties of the enzyme. further studies are needed to assess the impact of supplementation with antioxidant agents on oral cavity of human healthy individuals, using salivary and clinical parameters that represent the oral immunity. moreover, there is a lack of studies that assess the expression of antioxidant proteins or key enzymes of pathway signaling of salivary glands from rats subjected to treatment with antioxidants such as asta and fish oil, which justifies further studies in this research line. in conclusion, our results showed that antioxidant therapy could stimulate parotid gland, by increasing the α-amylase activity. fo and asta as well as the combination had some antioxidant effect, especially on parotid glands (increase of glutathione and catalase activity) and partially on submandibular gland (increase of catalase activity for all treatments). acknowledgements this research was supported by research foundation of the state of são paulo (fapesp 2009/12342-8). the authors are grateful to dra. rita mattei from são paulo university and dr. marcelo paes de barros, for providing supplemented rats. references 1. lee mg, ohana e, park hw, yang d, muallem s. molecular mechanism of pancreatic and salivary gland fluid and hco3 secretion. physiol rev. 2012; 92: 39-74. 2. gorr 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in the regulatory mechanism of amylase release by rat parotid and submandibular glands. arch oral biol. 2002; 47: 717-22. 33. alam sq, alam bs. in-vivo incorporation of omega 3 fatty acids into membrane lipids of rat salivary glands and changes in adenylate-cyclase activity. arch oral biol. 1988; 33: 295-9. 34. de almeida pv, grégio am, machado ma, de lima aa, azevedo lr. saliva composition and functions: a comprehensive review. j contemp dent pract. 2008; 9: 72-80. 35. nater um, rohleder n. salivary alpha-α-amylase as a non-invasive biomarker for the sympathetic nervous system: current state of research. psychoneuroendocrinol. 2009; 34: 486-96. 36. scannapieco fa, torres g, levine mj. salivary α-amylase: role in dental plaque and caries formation. crit rev oral biol med. 1993; 4: 301-7. 37. choi s, baik je, jeon jh, cho k, seo dg, kum ky, et at. identification of porphyromonas gingivalis lipopolysaccharide-binding proteins in human saliva. mol immunol. 2011; 48: 2207-13. effect of astaxanthin and fish oil on enzymatic antioxidant system and α-amylase activity of salivary glands from rats braz j oral sci. 13(1):58-63 oral sciences n3 braz j oral sci. 13(1):12-16 original article braz j oral sci. january | march 2014 volume 13, number 1 tooth erosion and dental caries in schoolchildren: is there a relationship between them? mabel miluska suca salas¹, raquel venâncio fernandes dantas¹, hugo ramalho sarmento¹, fabiana vargas-ferreira², dione torriani¹, flávio fernando demarco1,2 ¹universidade federal de pelotas ufpel, dental school, departament of restorative dentistry, pelotas, rs, brasil ²universidade federal de pelotas ufpel, dental school, area of epidemiology, pelotas, rs, brasil correspondence to: flávio fernando demarco faculdade de odontologia universidade federal de pelotas rua gonçalves chaves 457 cep: 96015568 pelotas, rs, brasil phone: +55 53 3222 6690 e-mail: flavio.demarco@pq.cnpq.br abstract aim: to identify a possible association between the occurrence of dental caries and tooth erosion and to correlate the dietary factor with an increased risk of dental caries. methods: a crosssectional study in a multistage random sample of 1,211, 8 to 12-years-old brazilian schoolchildren in private and public schools was conducted in pelotas, brazil. the prevalence of dental caries and tooth erosion was assessed. dietary habits data were collected by a structured questionnaire. data were analyzed using poisson regression model taking into account the cluster sample (prevalence ratio pr; 95% confidence interval ci). results: the prevalence of dental caries and tooth erosion was 32.4% (95% ci: 30.0; 35.2) and 25.0% (95% ci: 23.4; 29.0), respectively. most children had high consumption of sweets (65%). consumption of fruits, such as oranges (31%), strawberries (23.6%), pineapple (12%), and natural fruit juices (oranges, lemon) was low. soft drinks consumption, 3 or more times a week was observed in 40% of the children. conclusions: dental caries was not statistically associated with tooth erosion. the consumption of acidic fruits/drinks was not associated with dental caries. dental caries and dental erosion were not associated with each other and with acidic fruits or drinks consumption. keywords: dental caries; tooth erosion; epidemiology; child; diet. introduction dental caries is the main oral disease affecting children and adolescents, particularly among underprivileged groups in the developed and developing countries1. data from brazil in 2010 show that 43.5% of 12-year-old children had dental caries, with a mean dmft (decayed, missed, filled teeth) of 2.072. despite the high prevalence of caries in children, there is a worldwide trend of caries reduction3, raising interest for other oral health problems, such as the increase of tooth erosion, mainly in young individuals4. tooth erosion has been defined as a progressive loss of hard dental tissue due to a chemical process that does not involve bacteria5. it has the potential to substantially impact on dentition, due to its cumulative and progressive nature6. the prevalence of tooth erosion in brazilian children (age 6 to 16 years old) ranged from 7.2 to 26 percent7-10. it is well established that dietary habits play an important role in promoting and maintaining oral health among individuals. dietary habits are important risk factor for dental caries11 and also main source of acids, contributing to the development of tooth erosion10. it has been suggested that children with dental caries may also be predisposed to tooth erosion12. many types of food and soft drinks are not only acidic but also contain high percentage of sugar13 and, therefore, received for publication: january 13, 2014 accepted: march 10, 2014 gisele higa texto digitado http://dx.doi.org/10.1590/1677-3225v13n1a03 13 braz j oral sci. 13(1):12-16 dental erosion and caries may occur simultaneously in the same individual, but on different tooth surfaces14-15. however, this potential association remains unclear and contradictory, while some studies found an association between the two diseases, others did not find any association12,14,16. variability between investigations regarding localization and the studied population may influence the results 14-15. we hypothesized that presence of caries is associated with tooth erosion and diet. the aim of this study was to test if dental caries occurrence may be associated with tooth erosion in brazilian schoolchildren. additionally, it was tested whether dietary habit patterns may increase the risk for dental caries. material and methods study population and design in 2010, a cross-sectional study was performed in the city of pelotas, rs, brazil, using a multi-stage random representative sample of 8-12-year-old schoolchildren of the city. pelotas is a middle-sized city of the southern region of brazil, which has 328,275 inhabitants, nearly 85% residing in the urban area. more detailed information can be found elsewhere17. for the sample calculation to assess the prevalence and association between dental caries and independent variables, the following parameters were used: caries prevalence of 39.3 percent18, a standard error of 4 %, a confidence interval (ci) of 95% and power equal to 80%. a design effect of 1.4 9 was considered and the sample size was increased by 20% for possible refusals or losses. the ratio of unexposed to exposed tooth erosion was 3:1 and the prevalence ratio to be detected was at least 1.6, and the minimum sample size to satisfy these requirements was 616 children. a two-stage random sampling procedure was adopted to select the sample. the first stage units were public and private schools of the city. twenty schools were randomly selected. the second stage units were the classrooms. in each school, five classes, from 2nd to 6th grade, were randomly selected and all children enrolled in these classes were initially eligible for the study. subjects intellectually and physically able to answer the questionnaire were included in the study. from the 1,744 children invited to participate, 1,211 children were enrolled in the study with a 70% response rate. reasons for non-participation were mainly due to the lack of parental consent and absence on the examination day. the actual sample size was larger than the minimum size to satisfy the sample size requirements because this study was part of a major project including other outcomes that required a larger sample. data were collected with structured interviews and clinical oral examinations, and six calibrated dentists participated. the calibration included theoretical activities for the diagnostic criteria for dental caries (dmft)19 and tooth erosion20. to assess inter-examiner consistency for caries, a calibration exercise was performed on 10 children that were not included in the final sample, which were re-examined by the same examiners 2 weeks after the first examination. for tooth erosion, a range of different levels of erosion – based on the diagnosis of photographic images – was used in the calibration exercise (in lux), in agreement with the literature. for dental caries and tooth erosion, inter-examiner kappa values ranged from 0.68 to 0.90 and from 0.60 to 0.65, respectively. examinations were conducted in the classrooms, with children in a sitting position, under artificial light, using periodontal probes (cpi; “ball point”) and dental mirrors. sterile gauze pads were used to clean and dry the tooth surfaces. dental caries was assessed in all teeth, using the dmft – decayed, missed, filled teeth index according to the who recommendations 19 which measure the lifetime experience of tooth caries in permanent dentition. tooth erosion was assessed by the modified version 8 of the o´sullivan index 20 on maxillary incisors, by visual examination of facial, lingual, and incisal surfaces20. for this study, tooth erosion was dichotomized as present or not. the presence of one erosive lesion was considered “with tooth erosion”. site of erosion, severity and affected tooth surface area were not analyzed. a previously used questionnaire based on the literature7,9, was employed to investigate dietary habits (type and frequency of consumption of acidic drinks, including non-diet/non-light and diet/light soft drinks, fruit and sweets). data were completed in schoolroom before the clinical examination. dietary foods and drinks consumption was initially recorded by three frequencies: never, 1 or 2 times and 3 times or more (during a week) and later dichotomized in two categories: low (never or 1-2 times a week consumption) and high (3 or more times a week consumption)9. this cut off was chosen because in brazilian children, the acidic uptake more than three times a week was observed to increase twice the chances for tooth erosion7. to assess the applicability of the questionnaire, a pre-test was performed with 10 % of the study population before the survey, showing satisfactory levels of acceptability and ease of completion. data analyses were performed using stata software 12.0 (stata corp., college station, tx, usa). descriptive and bivariate analyses were conducted to provide summary statistics and preliminary assessment of the association of predictor variables and the outcome (dental caries) was performed using the chi-square pearson and mann-whitney test. poisson regression model taking into account the cluster sample (prevalence ratio and 95 percent confidence interval) was performed to assess the association between the predictor variables and the outcome. a backward stepwise procedure was used to include or exclude explanatory variables in the fitting of models. explanatory variables presenting a p-value < 0.20 in the assessment of correlation with each outcome (bivariate analyses) were included in the fitting of the model. explanatory variables were selected for the final models only if they had a p-value < 0.05 after adjustment. the study was approved by the human research ethics committee of the federal university of pelotas (approval tooth erosion and dental caries in schoolchildren: is there a relationship between them? 14 protocol n°160/2010) and informed consent was obtained prior of starting data collection. results demographic, socioeconomic, behavioral and clinical characteristics of the sample are presented in table 1. there were 574 (47.4%) boys and 637 (52.6%) girls. prevalence of dental caries and tooth erosion were 32.4% (95%ci 30.0; 35.2) and 25.0% (95%ci 23.4; 29.0) respectively. in relation to sweets consumption, a prevalence of 65% was found. for the 1,211 schoolchildren the dmft index result was 0.64 (standard deviation ± 1.18). the mean dmft index was not statistically different for children with tooth erosion 0.75 (± sd 1.35) compared with those without erosion 0.59 (±sd 1.11). in the preliminary assessment, the outcome (dental caries) was not associated with any dietary or clinical factors (tooth erosion) (table 2). in the multivariate analysis, dental caries was not associated with dietary habits (pr:1.15; 95% ci: 0.97;1.35) (table 3). similarly, dental caries was not associated with tooth erosion (pr: 1.15; 95% ci: 0.96;1.37) (table 3). discussion in this study a prevalence of 32.4% of dental caries was found among 8to 12-year-old children (95% ci: 30.0;35.0). this prevalence was lower than observed in other studies21,22. a significant decrease in caries prevalence in brazilian children was observed in the last decades 10,23. some longitudinal investigations indicated an increase of erosion incidence in children24-25. the prevalence found in this study (25.0%) was higher than those observed in other brazilian studies investigating mixed and/or permanent dentitions8-9. different indexes used or dietary habits between countries and regions may account for this variability. younger children (aged 8 to 12 years) were included in the present study, turning the acidic contact with tooth structure as the main potential etiologic factor7. in this study, 8.9% of children had caries and tooth erosion. however, the mean dmft scores were not statistically different for children with tooth erosion (0.75) compared to those without erosion (0.59). similar findings were observed in children aged 13-14 years in brazil, england and libya10,15,26. in a brazilian study with adolescents, while 35% of them had dental caries and tooth erosion, 32% without caries had also tooth erosion, and no association was detected between the two outcomes10. in australia, children who had caries were more likely to have severe tooth erosion in both dentitions12. in an english cohort, tooth erosion was associated with dental caries and with high consumption of carbonated drinks at age 1216. in saudi arabia, direct relationship between tooth erosion and dental caries was observed, suggesting that the carious process could be a potential risk factor for tooth erosion, with possible common risk factors14. children may fail to maintain a noncariogenic diet, which could also be a potentially erosive tooth erosion and dental caries in schoolchildren: is there a relationship between them? braz j oral sci. 13(1):12-16 variables/category n * % gender 1,211 male 574 47.0 female 637 53.0 skin color 1,173 white 856 73.0 dark-skinned black 189 16.0 light-skinned black 128 11.0 age (years) 1,211 8 182 15.0 9 312 26.0 10 295 24.5 11 259 21.0 12 163 13.5 family income (quartiles) 1,030 1st (poorest) 246 24.0 2nd 271 26.2 3rd 241 23.0 4th (richest) 279 27. 0 mother’s schooling (years) 1,176 < 12 502 43.0 9-11 121 10.0 5-8> 4 127426 11.036.0 consumption of sweets 1,208 l o w 423 35.0 high 785 65.0 consumption of natural orange juice 1,208 l o w 779 64.5 high 429 35.5 consumption of natural lemon juice 1,207 l o w 976 81.0 high 231 19.0 consumption of flavored juice 1,206 l o w 792 66.0 high 414 34.0 consumption of soft drink 1,208 l o w 727 60.0 high 481 40.0 consumption of strawberry 1,207 l o w 922 76.4 high 285 23.6 consumption of pineapple 1,207 l o w 1,067 88.0 high 140 12.0 consumption of orange 1,206 l o w 832 69.0 high 374 31.0 dental caries 1,210 present 392 32.4 absent 818 67.6 tooth erosion 1,202 present 302 25.0 absent 900 75.0 table 1table 1table 1table 1table 1clinical, demographic, behavioral and socioeconomic characteristics of the sample. pelotas, southern brazil, 2010. (n=1,211) *values lower than 1,211 due to incomplete information 15 dental caries p value* tooth erosion 0.117 without 622 278 with 194 108 natural orange juice 0.439 l o w 532 246 high 284 145 natural lemon juice 0.627 l o w 662 313 high 153 78 flavored fruit juices 0.342 l o w 527 264 high 287 127 soft drinks 0.116 l o w 504 223 high 312 168 strawberry 0.904 l o w 624 297 high 192 93 pineapple 0.889 l o w 722 344 high 94 46 orange 0.541 l o w 568 264 high 248 125 sweets 0.922 l o w 82 40 high 734 351 * chi-squared (÷²) test table 2 –table 2 –table 2 –table 2 –table 2 – prevalence of dental caries and associated factors, pelotas, southern brazil, 2010. (n=1,211) diet15. higher levels of mutans streptococci were found in children at risk of tooth erosion27. the loss of protective factors, such as saliva, may place the children at risk for both diseases12. tooth erosion and caries have a multifactorial etiology5. the acids responsible for erosion are not produced by the oral biofilm, but derived from dietary intrinsic acids or occupational sources5. on the opposite, caries development relies on the acids produced by the oral biofilm. studies have shown an association between tooth erosion, dental caries and dietary habits14,16. in the present study it was assessed the type and frequency of consumption of acidic food and drinks. these variables were not statistically associated with caries, in agreement with another previously reported investigation10. this result could be attributed to the frequency of consumption of acidic diet. frequent consumptions, more than 3 times a day, increased the risk for both oral conditions 1.5 times16. the increased consumption of beverages and foods, containing high percentage of sugar and acidic products rises concerns for their impact on general health, especially in children and adolescents, being associated with cardiopathy, diabetes and obesity12. it should be highlighted that the process and site specificity are different for both conditions. in general, the surfaces more prone to erosion are not those where caries occurs26 and tooth erosion is often located in plaque-free areas, in opposition to dental caries, which is located in plaque accumulation sites5. dental caries and tooth erosion can occur independently. the association between them may not always be found, and in some cases, the rapid and destructive nature of caries may force removal of the clinical evidence of erosion5. the lack of association between dental caries, tooth erosion and dietary habits may be due to the influence of other factors, such as quantity and composition of saliva, tooth composition and structure, oral hygiene practices and medical conditions4,7,10. moreover, tooth erosion has been mostly studied in populations where the prevalence of dental caries is low and the association may be more difficult to demonstrate14. studies have focused on dietary habits because there is a possible connection between the two conditions by one common item, sugary and acidic “soft drinks”14. to have a better understanding of dietary consumption, for both oral conditions, one possibility is to evaluate the diet for 3 days at least6,14. this could not be done in the present study together other oral and systemic health outcomes because it was carried out in schools making it difficult to include extensive questionnaires or successive visits were investigated. the reliability of self-reporting dietary habits by the schoolchildren may be an other limitation, as there is possibility of underor over-reporting10. unfortunately, there is no standardized questionnaire for tooth erosion in epidemiological surveys and this may be associated to those findings9. the obtained data must be considered with caution. the findings are limited by the cross-sectional nature of data collection. this study design cannot show the temporal effect27 of the diet on the development of caries and/or tooth erosion. dental caries and tooth erosion may not occur together in some point of the time. the severity of tooth caries and tooth erosion could make the presence of both conditions in the same individual more or less obvious. in permanent teeth, superimposition of a greater and more rapid destruction of caries could mask or remove any evidence of erosion14. further investigations with longitudinal designs using the life course epidemiology approach 28 are required to confirm these findings and to clarify the association between dental caries and tooth erosion. tooth erosion and dental caries in schoolchildren: is there a relationship between them? braz j oral sci. 13(1):12-16 with dental caries variables prc (95%ci) p value* pr adj (95%ci) p value* tooth erosion 0.111 0.130 without 1.00 1.00 with 1.16 (0.97;1.39) 1.15 (0.96;1.37) soft drinks 0.114 0.101 l o w 1.00 1.00 high 1.14 (0.97;1.34) 1.15 (0.97;1.35) table 3 –table 3 –table 3 –table 3 –table 3 – prevalence of dental caries and associated factors (prevalence ratio: 95% ci), pelotas, southern brazil, 2010. (n=1,211) *wald test; prc = crude prevalence ratio; pr adj. = adjusted prevalence ratio; ci = confidence interval 16 it has been shown that dental caries is suffering a phenomenon called polarization, with most of the disease concentrated in a small part of the population, especially those more deprived. therefore, the investigation of factors that could be associated with caries occurrence should be investigated in epidemiological surveys in order to establish preventive measures29. in the present study, there was no statistically significant association between dental caries and tooth erosion in brazilian schoolchildren. dietary habits were not associated with all dental caries. acknowledgments the authors are grateful to the state secretary of education, the municipal secretary of education and the direction of private schools, which allowed the study to be performed. also, the authors would like to thank brazilian national council for scientific and technological development (cnpq) for the research grant (process #402350/ 2008-1 and 579996/2008-5) provided to the principal investigator (ffd). also, the authors would like to thank the twas (the world academy of sciences for the advancement of science in developing countries process # 190268/2010-7) for the scholarship provided to the first author (mmss). references 1. traebert j, guimaraes l do a, durante ez, serratine ac. low maternal schooling and severity of dental caries in brazilian preschool children. oral health prev dent. 2009; 7: 39-45. 2. freire mcm, reis scgb, figueiredo n, peres kg, moreira rs, antunes jlf. individual and contextual determinants of dental caries in brazilian 12-year-olds in 2010. rev. saude publica 2013; 47:40-9. 3. whelton h. overview of the impact of changing global patterns of dental caries experience on caries clinical trials. j dent res. 2004; 83 spec no c: c29-34. 4. truin gj, van rijkom hm, mulder j, van’t hof ma. caries trends 19962002 among 6and 12-year-old children and erosive wear prevalence among 12-year-old children in the hague. caries res. 2005; 39: 2-8. 5. taji s, seow wk. a literature review of dental erosion in children. aust dent j. 2010; 55: 358-67. 6. okunseri c, okunseri e, gonzalez c, visotcky a, szabo a. erosive tooth wear and consumption of beverages among children in the united states. caries res. 2011; 45: 130-5. 7. correr gm, alonso rc, correa ma, campos ea, baratto-filho f, puppin-rontani rm. influence of diet and salivary characteristics on the prevalence of dental erosion among 12-year-old schoolchildren. j dent child. (chic) 2009; 76: 181-7. 8. peres kg, armenio mf, peres ma, traebert j, de lacerda jt. dental erosion in 12-year-old schoolchildren: a cross-sectional study in southern brazil. int j paediatr dent. 2005; 15: 249-55. 9. vargas-ferreira f, praetzel jr, ardenghi tm. prevalence of tooth erosion and associated factors in 11-14-year-old brazilian schoolchildren. j public health dent. 2011; 71: 6-12. 10. auad sm, waterhouse pj, nunn jh, moynihan pj. dental caries and its association with sociodemographics, erosion, and diet in schoolchildren from southeast brazil. pediatr dent. 2009; 31: 229-35. 11. perera i, ekanayake l. relationship between dietary patterns and dental caries in sri lankan adolescents. oral health prev dent. 2007; 8: 165-72. 12. kazoullis s, seow wk, holcombe t, newman b, ford d. common dental conditions associated with dental erosion in schoolchildren in australia. pediatr dent. 2007; 29: 33-9. 13. yip hh, wong rw, hagg u. complications of orthodontic treatment: are soft drinks a risk factor? world j orthod. 2009; 10: 33-40. 14. al-malik mi, holt rd, bedi r. the relationship between erosion, caries and rampant caries and dietary habits in preschool children in saudi arabia. int j paediatr dent. 2001; 11: 430-9. 15. huew r, waterhouse p, moynihan p, kometa s, maguire a. dental caries and its association with diet and dental erosion in libyan schoolchildren. int j paediatr dent. 2012; 22: 68-76. 16. dugmore cr, rock wp. a multifactorial analysis of factors associated with dental erosion. br dent j. 2004; 196: 283-6. 17. goettems ml, correa mb, vargas-ferreira f, torriani dd, marques m, domingues mr,et al. methods and logistics of a multidisciplinary survey of schoolchildren from pelotas, in the southern region of brazil. cad saude publica. 2013; 29: 867-78. 18. piovesan c, antunes jl, guedes rs, ardenghi tm. impact of socioeconomic and clinical factors on child oral health-related quality of life (cohrqol). qual life res. 2010; 19: 1359-66. 19. world health organization. oral health survey: basic methods. genebra: who; 1997. 20. o’sullivan ea. a new index for the measurement of erosion in children. eur j paediat dent. 2000; 2: 69-74. 21. eslamipour f, borzabadi-farahani a, asgari i. the relationship between aging and oral health inequalities assessed by the dmft index. eur j paediatr dent. 2011; 11: 193-9. 22. peres ma, barros aj, peres kg, araujo cl, menezes am. life course dental caries determinants and predictors in children aged 12 years: a population-based birth cohort. community dent oral epidemiol 2009; 37: 123-33. 23. pereira sm, tagliaferro ep, ambrosano gm, cortelazzi kl, meneghim m de c, pereira ac. dental caries in 12-year-old schoolchildren and its relationship with socioeconomic and behavioural variables. oral health prev dent. 2007; 5: 299-306. 24. el aidi h, bronkhorst em, huysmans mc, truin gj. multifactorial analysis of factors associated with the incidence and progression of erosive tooth wear. caries res. 2011; 45: 303-12. 25. nunn jh, gordon ph, morris aj, pine cm, walker a. dental erosion — changing prevalence? a review of british national childrens’ surveys. int j paediatr dent. 2003; 13: 98-105. 26. bardolia p, burnside g, ashcroft a, milosevic a, goodfellow sa, rolfe ea, et al. prevalence and risk indicators of erosion in thirteento fourteenyear-olds on the isle of man. caries res. 2010; 44: 165-8. 27. linnett v, seow wk. dental erosion in children: a literature review. pediatr dent. 2001; 23 : 37-43. 28. demarco ff, peres kg, peres ma. life course epidemiology and its implication for oral health. braz oral res. 2014. 24:0. [epub ahead of print] 29. oliveira ljc , correa mb, nascimento gc , goettems ml, tarquínio sbc , torriani dd, demarco ff. inequalities in oral health: are schoolchildren receiving the bolsa família more vulnerable? rev saude publica 2013;47:1-9. tooth erosion and dental caries in schoolchildren: is there a relationship between them? braz j oral sci. 13(1):12-16 oral sciences n3 original article braz j oral sci. july | september 2014 volume 13, number 3 evaluation of two methods for mixed dentition analysis using the method error bruna santos da cruz, eduardo kant colunga rothier, beatriz de souza vilella, oswaldo de vasconcellos vilella, rizomar ramos do nascimento universidade federal fluminense uff, school of dentistry, department of orthodontics, niterói, rj, brazil correspondence to: rizomar ramos do nascimento departmento de ortodontia uff rua mário santos braga, 30, 2° andar, centro cep: 22050-001 niterói rj brasil phone: + 55 21 2622 1621/+55 21 9944 5095 fax: +55 21 2629 9812 e-mail: rizonascimento@gmail.com received for publication: march 20, 2014 accepted: august 08, 2014 abstract the most commonly used tests to assess the mesiodistal width of the unerupted permanent canines and premolars are divided in two groups: those performed directly on plaster models, using mathematical equations that can generate tables, and those using radiographs. aim: in order to determine the reliability between two of these methodologically different method, this study evaluated the systematic and random errors of the method proposed by tanaka and johnston, which is based on the sum of mandibular permanent incisors, and the huckaba method, which uses radiographs. methods: in a random sample of 28 plaster models of mandibular dental arches belonging to individuals of both genders, aged six to eleven years old, a single investigator performed the measurement of required space, according to the two methods evaluated. after 15 days, the measurements were repeated, and each of them was performed twice in sequence to calculate the repeatability and reproducibility conditions, and the systematic and random errors for each method. results: the random error of the method proposed by huckaba was larger in terms of reproducibility (1.53 mm) and repeatability (0.57 mm) compared with the analysis proposed by tanaka and johnston (0.20 mm and 0.12 mm, respectively). conclusions: the method proposed by huckaba was proved to be inadequate in relation to reproducibility, with respect to the random error, and should be used with caution to measure the required space in the mandibular arch. keywords: dental arch; dentition, mixed; orthodontics. introduction the mixed dentition analysis is a valuable tool in preventive and interceptive orthodontic treatments. it makes it possible to predict the mesiodistal (md) width of unerupted permanent canines and premolars, and assess whether there is enough space to align these teeth on the dental arch, reducing the occurrence of crowding and deviations of the eruption pattern1-4. usually performed in the mandibular arch, one of the main references for orthodontic treatment planning5,6, the mixed dentition analysis is the basis for diagnosis and orthodontic treatment planning, and takes into account that the deciduous canines and molars are replaced by teeth with smaller md width1,7. this difference in tooth size can be used to prevent or intercept future malocclusions8. the most commonly used tests are divided in two groups: those using mathematical equations that can generate tables and those using radiographs to assess the size of the unerupted teeth1,2,9-11. the decision on the appropriate method to be used for each patient depends on certain circumstances. braz j oral sci. 13(3):163-167 tanaka and johnston10 (1974) developed an analysis with 75% of probability, where half the sum of mandibular incisor md widths (x), plus a predetermined value (10.5 mm for the mandibular hemiarch), provides the likely md width of unerupted canines and premolars (y), being y = x/2 +10.5 mm. the values are then duplicated to match both sides of the arch. in an initial appointment, the method proposed by tanaka and johnston10 (1974) has some advantages, since radiographs are not required. it employs prediction equations based on md width of erupted permanent teeth during the mixed dentition, and it´s easy to memorize. however, this analysis tends to overestimate the size of unerupted teeth1. on the other hand, the methods that use radiographs have the advantage of measuring unerupted teeth, and provide more accurate results, but the patient is exposed to ionizing radiation, and it requires additional radiographs, which implicates financial costs12. huckaba9 (1964) developed a method for overcoming the effect of radiographic distortions while measuring the permanent canines and premolars md width, using an equation relating the measures of erupted teeth to their radiographic images in order to obtain the proportional dimensions of unerupted teeth, as follows: the tanaka and johnston analysis is a practical way to predict the md width of the unerupted permanent canines and premolars, as it does not require a table and can be performed in an initial appointment or using plaster models. on the other hand, methods using radiographs have been considered more accurate, but also less practical, more expensive, more time-consuming and require specific equipment11. comparing these two methods for estimating the md width of unerupted permanent teeth in the mixed dentition analysis, this study aimed to determine which one is more reliable, by assessing the method error13. material and methods the present study was approved and monitored by the local ethics committee (protocol 231/10). a total of 650 plaster casts of dental arches from the archives of the fluminense federal university orthodontic clinic (niterói, rj, brazil) were examined and selected for the study, according to the following criteria: 1) mixed dentition, in which the four permanent mandibular incisors, and deciduous canines, first and second molars were present (one of the deciduous canines could be missing, since the successor was unerupted); 2) no previous orthodontic treatment; 3) complete documentation (record query, periapical radiographs); and 4) the mandibular incisors fully erupted and presenting well-defined contact areas, no cavities, malformations or restorations on the proximal surfaces. using these criteria, 28 plaster casts were selected, belonging to patients of both genders, aged 6-11 years. damaged models and incomplete or poor-quality radiographs were excluded. the md width of the mandibular permanent incisors was assessed8 using a digital caliper (lee tools, brazil), with +0.02 mm accuracy, and a reproducibility of +0.01 mm on the plaster models. the caliper was positioned as perpendicular as possible to the clinical crown, with its active probes touching the proximal surfaces (mesial and distal), thus covering the largest md width portion. when the tooth position did not allow the caliper adjustment in this way, the measurement was made with the instrument parallel to the incisal border. the sum of the widths recorded was divided by 2, and 10.5 mm were added, to estimate the size of permanent canines and premolars in one hemiarch. a 75% level of prediction was used. this digital caliper was also used to measure the md width of deciduous teeth in periapical radiographs9 and the permanent teeth in the plaster models. subsequently, the above-mentioned equation was used9. to improve the reliability of the measurements, this investigation adopted some recommended procedures, such as (1) use of high-quality dental casts made of dental stone, (2) use of calipers with digital displays to greatly reduce eye fatigue and the possibility of reading error, and (3) assessing intra-examiner variability using dahlberg´s formula14. measurements were carried out by an examiner calibrated by one of the supervisors of the fluminense federal university orthodontic clinic, on 10 sets of plaster models, and the intraobserver variability was assessed by measuring 10 sets of randomly selected casts twice at one-week interval. measurements were performed following calibration and were repeated after 15 days. each measurement was performed twice in sequence. therefore, data were obtained in two different conditions: with an interval of 15 days between them, and in successive measurements. subsequently, the systematic and random errors of each method were calculated for both situations. statistical treatment the sample size was calculated using the formula described by pocock and recommended by pandis15, with 90% power levels, considering 0.05 the desired statistical significance and 0.5 mm the minimum difference to be detected. this indicates that the required sample size for the present research was 22.3. thus, the sample used (28) is within the recommendation to carry out this study. bland-altman analysis was used to compare the measurements at three different times in each method. the paired student’s t test was used to obtain the systematic error, as recommended by houston 13, where represents the average of the differences in each of the pairs formed by the two measurements, sd corresponds to the standard deviation of the differences and n is the number of pairs of the sample, as follows: sd nx t = evaluation of two methods for mixed dentition analysis using the method error1 6 41 6 41 6 41 6 41 6 4 braz j oral sci. 13(3):163-167 to estimate the magnitude of the random or casual error, the following formula was proposed by dahlberg14, where d is the difference between the two measurements and n is the number of duplicate determinations: a significance level of 5% (p<0.05) was set for all tests. results table 1 shows the mean values of the required space, obtained in the first measurement (tj1 and h1) and in the two subsequent measurements, performed 15 days later (tj2a and tj2b, and h2a and h2b), using the huckaba method8 (h) and the tanaka and johnston analysis10 (tj). n d se 2 2∑= table 2 displays the random and systematic errors in both evaluated methods. for systematic error, the student’s t test was applied to compare values obtained in the first moment and the mean values obtained at the second time, and also between the values obtained in the second measurement. table 3 demonstrates the mean values and standard deviations (sd) from the three assessments, and table 4 shows bland-altman mean differences, standard deviations (sd) and 95% limits of agreement comparing intra examiner measurements in huckaba method (h) and tanaka and johnston analysis (tj). in figure 1, bland-altman plots show correlations between first and second measurements (a; d), first and third measurements (b; e) and second and third measurements (c; f), performed for both assessed methods. a good correlation can be noted between measurements, especially in the tanaka and johnston analysis10. discussion the random error of the method proposed by huckaba was larger in the comparisons between the first measurements and the mean values of the second ones, as well as between the second measurements (1.53 mm and 0.57 mm, respectively), in relation to the analysis proposed by tanaka and johnston (0.20 mm and 0.12 mm, respectively). an important factor to be considered in studies involving variable measurements is an adequate review of the method error. some factors that may contribute to the test procedure variability are: the operator, the used equipment and the interval between the measurements. the present study found that the correlation between both measurements was excellent. therefore, the intraand interobserver error of method was considered of minor importance. this result validated the methodology employed to data collection. previous studies, comparing methods in which x-rays are not used, showed variable results when assessing different methods of mixed dentition analysis for different populations11,16-21. the differences in the ethnic origin of the samples and the methods of measurement may explain these findings16. analysis based on 45o cephalometric radiographs and computed tomography scans are considered the most precise method to predict the md widths of unerupted permanent canines and premolars16,18. however, these methods are less practical, because they are more expensive and require more time and specific equipment. these facts may explain why most researches compared methods based on tables to perform the mixed dentition analysis11. in contrast, the present study aimed to compare the reliability of two methodologically different methods, one that uses periapical x-rays and another that uses mathematical equations to predict the md width of unerupted permanent canines and premolars, in order to identify which one has higher reliability, by assessing the method error. an important aspect when using any measurement method is assessing its ability of repeatability and tanaka and johnston method (75%) huckaba method plaster tj1 tj2a tj2b h1 h2a h2b model 1 42.71 42.57 42.89 45.00 43.85 44.13 2 41.81 41.58 41.26 40.65 40.38 40.62 3 44.08 44.16 44.47 43.07 42.80 42.71 4 44.52 44.40 44.42 35.51 41.76 43.67 5 43.73 43.67 43.86 52.57 51.43 49.50 6 42.26 42.14 42.18 43.33 43.87 44.71 7 44.15 44.15 44.88 49.49 50.79 50.06 8 43.60 43.18 43.17 43.31 46.43 46.84 9 43.19 43.58 43.55 43.40 42.90 43.51 10 42.02 42.25 42.17 39.59 40.36 41.21 11 44.61 44.63 45.06 48.69 48.92 49.55 12 43.42 44.28 44.24 51.79 53.01 52.27 13 43.82 44.10 43.99 46.50 44.63 44.75 14 42.60 42.12 42.06 43.90 42.07 41.52 15 43.17 43.46 43.33 51.10 51.25 51.00 16 42.63 42.89 42.85 44.33 43.12 42.97 17 43.60 43.66 43.66 42.78 40.87 41.78 18 42.34 42.28 42.28 44.18 46.00 45.22 19 43.10 43.16 43.37 45.03 46.05 46.12 20 42.25 42.45 42.24 41.27 39.59 38.77 21 42.26 42.49 42.57 47.19 44.00 44.37 22 43.35 43.22 43.29 45.51 45.53 45.40 23 42.50 42.46 42.41 45.44 45.42 45.74 24 43.63 43.14 43.08 42.22 41.91 42.00 25 43.48 43.54 43.58 51.32 45.41 47.37 26 43.03 43.16 43.21 46.90 47.17 46.60 27 43.24 43.24 43.23 48.61 48.96 48.91 28 43.68 43.57 43.55 44.69 46.74 47.27 table 1. table 1. table 1. table 1. table 1. values of the mesiodistal widths of permanent canines and premolars (mm), in the two quadrants of the 28 models, obtained in the first measurement (tj1 and h1) and in two consecutive measurements taken 15 days later (tj2a and tj2b and h2a and h2b) by using digital caliper according to the method used. evaluation of two methods for mixed dentition analysis using the method error 1 6 51 6 51 6 51 6 51 6 5 braz j oral sci. 13(3):163-167 tanaka and johnston (75%) comparison between the mean values obtained in the first (tj1) and the second moment (tj2) comparison between the mean values obtained in the second stage (tj2a and tj2b) comparison between the mean values obtained in the first (h1) and in the second moment (h2) comparison between the mean values obtained in the second stage (h2a and h2b) huckaba 0.20 0.30 0.77ns* 0.12 0.25 0.80ns* 1.53 0.05 0.96ns* 0.57 0.21 0.83ns* methods of mixed dentition analysis for predicting the required space re t p table 2. verification of the random error (re) (mm) and the systematic error of the method using paired t test (t) and its significance level (p), between values obtained in the first moment and the mean values obtained in the second time, and also between the values obtained at the second time of measurement. *ns = not significant measurements mean ± sd minimum maximum standard error h 45.2 ± 3.9 35.51 52.57 0.741 h2a 45.2 ± 3.6 39.59 53.01 0.685 h2b 45.3 ± 3.4 38.77 52.27 0.635 tj1 43.2 ± 0.8 41.81 44.61 0.141 tj2a 43.2 ± 0.8 41.58 44.63 0.149 tj2b 43.24 ± 0.9 41.26 43.55 0.171 table 3. table 3. table 3. table 3. table 3. mean values and standard deviations (sd) from three assessments for huckaba´s method9 (h) and tanaka and johnston analysis10 (tj). mean difference 95% limit of agreement (sd) tj1 x tj2a -0.02 (0.27) -0.56; 0.51 tj1 x tj2b -0.07 (0.33) -0.73; 0.58 tj2a x tj2b -0.04 (0.20) -0.45; 0.35 h1 x h2a 0.07 (2.14) -4.13; 4.28 h1 x h2b -0.04 (2.32) -4.59; 4.51 h2a x h2b -0.11 (0.81) -1.71; 1.47 table 4.table 4.table 4.table 4.table 4. bland-altman mean difference, standard deviation (sd) and 95% limits of agreement comparing intra examiner measurements in huckaba method (h) and tanaka and johnston analysis (tj) reproducibility. repeatability occurs when there is no variation of factors that can affect the variability of the method’s results, and reproducibility when at least one of these factors is varied. according to albuquerque jr. et al.21, reproducibility can be assessed when at least one of the factors that may contribute to the variability of results from a test method is varied21,22. the systematic error tends to overestimate or underestimate the true value of the magnitude. the random error results from unpredictable factors, such as the limitations of the equipment, the measurement procedure, or a variety of other factors, often making it impossible to accurately identify its source21,23. evaluation of two methods for mixed dentition analysis using the method error fig. 1. bland-altman plots comparing intra examiner measurements in huckaba method9 (a, b and c) and in tanaka and johnston analysis10 (d, e and f). 1 6 61 6 61 6 61 6 61 6 6 braz j oral sci. 13(3):163-167 however, the greater the number of measurements, the algebraic sum of the differences more clearly tends to annul itself, and all these differences are subject to a probability distribution law known as the normal distribution or gausslaplace curve22. it was observed that some measurements showed a significant discrepancy between the two investigated analyses (table 1, plaster models 5, 7, 12, 15 and 25). two aspects can be considered in this finding. first, the discrepancy occurs in measurements between the two methods, however presents proportionality in consecutive measurements within each of them. moreover, differently from the tanaka and johnston analysis, the huckaba method uses radiographs, which can be distorted at the time of their acquisition, and this sort of error might explain the disharmony observed in this study. it was demonstrated in this study that the systematic error showed no statistically significant differences. on the other hand, the random error of the method proposed by huckaba9 was larger in terms of reproducibility (1.53 mm) and repeatability (0.57 mm), when compared with the analysis proposed by tanaka and johnston10 (0.20 and 0.12 mm, respectively). this finding could be relevant to routine clinical practice. the orthodontic planning can be modified depending on the outcome presented by the mixed dentition analysis. an error greater than one millimeter in relation to the true discrepancy, for example, may be sufficient to modify it. in conclusion, as far as the systematic error is concerned, no significant differences were found between the methods of mixed dentition analysis. however, with respect to the random error, the method proposed by huckaba was proven to be inadequate in terms of reproducibility, and should be used with caution to measure the required space in the mandibular arch. this suggests that care must be taken in borderline cases where the decision to extract or not teeth depends on meticulous analysis of the diagnostic elements. acknowledgements this study was performed by b. s. c. as fulfillment of her pibic research, which was supported by grant from cnpq, brazil. references 1. bherwani ak, fida m. development of a prediction equation for the mixed dentition in a pakistani sample. am j orthod dentofacial orthop. 2011; 140: 626-32. 2. bernabé e, biostat c, flores-mir c, orth c. are the lower incisors the best predictors for unerupted canine and premolars sums? an analysis of a peruvian sample. angle orthod. 2005; 75: 202-7. 3. oliveira mv, pithon mm, ruellas aco. comparative evaluation of three methods for the calculation of request space in the mixed dentition analysis. j dent sci. 2007; 22: 148-53. 4. rubin rl, baccetti t, mcnamara jr. a. mandibular second molar eruption difficulties related to the maintenance of arch perimeter in the mixed dentition. am j orthod dentofacial orthop. 2012; 141: 146-52. 5. melgaço ca, araújo mts, ruellas aco. mandibular permanent first molar and incisor width as predictor of mandibular canine and premolar width. am j orthod dentofacial orthop. 2007; 132: 340-5. 6. paranhos lr, andrews wa, jóias rp, bérzin f, daruge júnior e, triviño t. dental arch morphology in normal occlusions. braz j oral sci. 2011; 10: 65-8 7. zilberman o, huggare jav, parikakis ka. evaluation of the validity of tooth size and arch width measurements using conventional and three dimensional virtual orthodontic models. angle orthod. 2003, 73: 301-6. 8. tome w, ohyama y, yagi m, takada k. demonstration of a sex difference in the predictability of widths of unerupted permanent canines and premolars in a japanese population. angle orthod. 2011; 81: 938-44. 9. huckaba gw. arch size analysis and tooth size prediction. dent clin north am. 1964: 11: 431-40. 10. tanaka mm, johnston le. the prediction of the size of unerupted canines and premolars in a contemporary orthodontic population. j am dent assoc. 1974; 88: 798-801. 11. burhan as, nawaya fr. prediction of unerupted canines and premolars in a syrian sample. prog in orthod. 2014; 15: 4. 12. ritshel r, bechtold te, berneburg m. effect of cephalograms on decisions for early orthodontic treatment. angle orthod. 2013; 83: 1059-65. 13. houston wjb. the analysis of errors in orthodontic measurements. am j orthod. 1983; 83: 382-90. 14. dahlberg g. statistical methods for medical and biological students. new york: interscience; 1940. 15. pandis n, polychronopoulou a, eliades t. sample size estimation: an overview with applications to orthodontic clinical trial designs. am j orthod dentofacial orthop. 2011; 140: e141-6. 16. brito fc, nacif vc, melgaço ca. mandibular permanent first molars and incisors as predictors of mandibular permanent canine and premolar widths: applicability and consistency of the method. am j orthod dentofacial orthop. 2014; 145: 393-8. 17. dasgupta b, zahir s. comparison of two non-radiographic techniques of mixed dentition space analysis and their evaluation of their reliability for bengali population. contemp clin dent. 2012; 3: 146-50. 18. legovic m, novosel a, scrinjaric t, legovic a, mady b, ivancic n. a comparison of methods for predicting the size of unerupted permanent canines and premolars. eur j orthod. 2006; 28: 485-90. 19. miyamura zy, tubel cam, ciruffo pad, paludo ah. prediction of mesiodistal diameter of permanent canines and premolars unerupted. evaluation of methods of moyers and tanaka & johnston. rev gaucha orthodontol. 2006; 54: 52-7. 20. philip ni, prabhakar m, arora d, chopra s. applicability of the moyers mixed dentition probability tables and new prediction aids for a contemporary population in india. am j orthod dentofacial orthop. 2010; 138: 339-45. 21. albuquerque hr jr, santos-pinto a, santos-pinto lam. precisão de medidas cefalométricas: validade de métodos de ensaio. dental press j orthod. 2002; 7: 5762. 22. machado la, vilella ov, agostinho lmag. evaluation of two techniques to measure the available space in the mandibular dental arch using the method error. rev odonto cienc. 2012; 27: 228-32. 23. mendes ajd, santos-pinto a. algumas diretrizes estatísticas para avaliação do erro do método na mensuração de variável quantitativa. rev dental press ortod ortop facial. 2007; 12: 78-83. evaluation of two methods for mixed dentition analysis using the method error 1 6 71 6 71 6 71 6 71 6 7 braz j oral sci. 13(3):163-167 oral sciences n3 analgesic effect of hypericum perforatum, valeriana officinalis and piper methysticum for orofacial pain luciana cristina nowacki1, paulo roberto worfel1, paulo francisco arant martins2, rosane sampaio dos santos2, josé stechman-neto2, wesley mauricio de souza2,3 1universidade tuiuti do paraná – utp, dental school, department of pharmacology, curitiba, pr, brazil 2universidade tuiuti do paraná – utp, dental school, graduate program in communication disorders, curitiba, pr, brazil 3universidade federal do paraná – ufpr, pharmacy school, department of clinical analysis, curitiba, pr, brazil correspondence to: wesley mauricio de souza campus prof. sydnei lima santos (barigui) rua sydnei a. rangel santos, 238 cep: 82010-330 – curitiba, pr, brazil phone: +55 41 96868656 e-mail: wesley.souza@utp.br abstract aim: to evaluate in vivo the association of hypericum (hypericum perforatum), valerian (valeriana officinalis) and kava (piper methysticum) with analgesia by assessing their effects in reducing orofacial pain as well as the possible hepatic, hematologic and biochemical alterations induced by regular administration of these extracts. methods: orofacial pain was induced in mice with the administration of 2.5% formalin in the upper lip. after 60 min, the animals were treated with saline, carbamazepine and hydroalcoholic plant extracts. the nociceptive intensity was determined by the timing at which the animal remained rubbing the injected area. to assess the hepatotoxic effect, mice were chronically treated for 25 days with saline, carbamazepine and hydroalcoholic extract. the animals were euthanized and the liver weighed, followed by a differential count of leukocytes and measurement of alanine transaminase and alkaline phosphatase. results: the evaluation of analgesic activity in phase 1 reduced the time of rubbing compared to the control by 86% (0.05 ml/10 g) and 76% (0.10 ml/10 g). in phase 2, the extracts reduced rubbing time by 94% and 85%, respectively. in the evaluation of alkaline phosphatase, the groups treated with extracts at doses of 0.05 ml/10 g and 0.1 ml/10 g increased by 16.1% and 9.5% compared to the control group and a reduction of 8.5% and 9.1% in the evaluation of alanine transaminase respectively. it was demonstrated that in the differential counts showed an increase in eosinophils in the treated group with 0.05 ml/10 g. conclusions: the use of hydroalcoholic extract of the associated plants reduced the orofacial formalin-induced pain with better results than carbamazepine, at both the neural conductor level of pain (phase 1) and in inflammatory or later pain (phase 2) without presenting hepatotoxicity. the observed eosinophilia is suggestive of a phenomenon called hormesis. keywords: temporomandibular joint disorders; facial pain; hypericum; valerian; kava. introduction orofacial pain is the field of dentistry devoted to the diagnosis and management of chronic and complex facial pain, and oromotor disorders1-2. it may be defined as pain localized in the region above the neck, in front of the ears and below the orbitomeatal line, as well as pain in the oral cavity3, and is widely prevalent in the community. orofacial pain, like pain elsewhere in the body, is usually the result of tissue damage and the activation of nociceptors, which transmit a noxious stimulus to the brain. however, due to the rich innervations of the head, original article braz j oral sci. january | march 2015 volume 14, number 1 braz j oral sci. 14(1):60-65 received for publication: january 12, 2015 accepted: march 17, 2015 6161616161 face and oral structures, orofacial pain entities are often very complex and can be difficult to diagnose4. patients with orofacial pain seek a variety of clinicians, including headache physicians, dentists, maxillofacial surgeons, otolaryngologists, neurologists, chronic pain clinics, psychiatrists and related health professionals such as physiotherapists and psychologists5-6. orofacial pain is associated with significant morbidity and high levels of health care use6. because of the wide variety of conditions, many different synthetic classes of drugs are used7. currently, professionals and official programs worldwide have advised the use of medicinal plants for the treatment of several common diseases8. brazil has a very large number of native plant species that are considered medicinal, but many do not have any scientific assessment of their medicinal potential, which is essential to be used safely by the population9. the search for new drugs has consistently been an effective and safe alternative in reducing neuropathic pain and chronic inflammation. kava (pyper methysticum), hypericum (hypericum perforatum) and valerian (valeriana officinalis) mainly composed of kavalactones, hypericin and valeric acids, respectively, are described in the literature as plants that present several therapeutic properties such as antidepressants and antiinflamatories10. however, they can significantly harm the body when consumed chronically and in associated use with other plants11. health professionals prescribe these plant extracts at low doses to patients with temporomandibular disorder with significant improvements in chronic pain. there are many studies showing the therapeutic effects of these three plants separately10, but there are few studies concerning the association of them in treatments related to pain and inflammation. the purpose of this study was to evaluate in vivo the association of hypericum (hypericum perforatum), valerian (valeriana officinalis) and kava (piper methysticum) with analgesia by assessing their effects in reducing orofacial pain as well as the possible hepatic, hematologic and biochemical alterations induced by regular administration of these extracts. material and methods extract preparation alcoholic extracts of valeriana officinalis 10% (equivalent to 0.8% of valeric acid), hypericum perforatum 10% (equivalent to 0.27-0.33 mg of hypericin), and piper methysticum 10% (equivalent to 22.5 mg of kavalactones) were used. the dyes were commercially available in the city of colombo, pr, brazil, along with the product label and technical certification. the alcohol extracts were mixed at a ratio of 3:1:0.5, respectively, and evaporated in an air circulating oven until complete dryness and re-suspended in sterile distilled water at a concentration of 100 mg/ml. after sterilization by a 0.22 µm pore-size filter, aliquots were stored at -20°c. animals experiments were performed on male swiss albino mice (30-40 g), obtained from the production and research center of immunobiology (cppi) in piraquara, pr, brazil, where they were kept in a temperature-controlled room (21±2 °c), with light/dark cycles of 12 hours each, and were allowed free access to food and water. the experiments were performed with the approval of the committee for the use of animals in experiments at tuiuti university of paraná under protocol number 008/10p/ceua/utp and in accordance with cns resolution 196/96 and guidelines of the committee for the control and supervision in animal experiments (nih publication no. 86-23, 1985). orofacial pain test induced by formalin the orofacial pain test consisted of the treatment of all groups with 2.5% formalin (20 µl) in the upper lip using a hamilton’s hypodermic syringe. this volume and percentage concentration of formalin were obtained from pilot studies that showed a pain-related biphasic behavioral response (facerubbing) of greater intensity at periods of 0-5 min (first phase) and at 15-30 min (second phase). the nociceptive intensity was determined for each period by counting the time (in s) that the animal rubbed the injected area with the rear and/or front paws, indicative of pain12. to evaluate the analgesic effect of the plant mixture, groups of mice (n=10) were treated orally 60 min prior to formalin administration with: i) saline (0.1 ml/10 g); ii) carbamazepine (medley pharmaceuticals; 4 mg/kg), and iii) the hydroalcoholic extract mix of hypericum perforatum, valeriana officinalis and pyper methysticum at doses of 0.05 0.1 ml/10 g. doses were chosen by previous studies reported in the literature for general plants13-15. hepatotoxicity study to assess hepatotoxic effect of the plant combination, the groups of mice (n=10) were treated orally for 25 consecutive days using respectively: i) saline (0.1 ml/10 g), ii) carbamazepine (4 mg/kg) and iii) the hydroalcoholic extract of the plant combination of hypericum perforatum, valeriana officinalis, and pyper methysticum at doses of 0.05 0.1 ml/10 g. on the 26th day the animals were euthanized with ether inhalation, supervised by a veterinarian, their blood collected with edta, and their livers removed and weighed. whole blood smears were performed on slides and stained by the may grunwald-giemsa method for differential leukocyte count. subsequently, the whole blood was centrifuged for 10 min at 2.500 rpm to separate the plasma and stored in eppendorf tubes at 2-8 °c until use. the serum levels of the liver enzymes alanine transaminase (alt) and alkaline phosphatase (alp) were assessed in a semi-automated spectrophotometer (tp analyzer plus®, thermoplate, china) using labtest kits and validation with control samples (qualitrol company llc, fairport, ny, usa). statistical analysis statistical analyses were performed using the statistical packages jmp (version 8.0; sas institute inc., cary, n.c., usa) and sigmastat (version 3.5; systat software inc., erkrath, analgesic effect of hypericum perforatum, valeriana officinalis and piper methysticum for orofacial pain braz j oral sci. 14(1):60-65 6262626262 germany). results were expressed as mean±standard error of the mean (sem) and used anova followed by bonferroni test, and the statistical difference was considered to be p<0.05, p<0.01 or p<0.001. results in the formalin lip test to determine the analgesic activity of the extract, the obtained data show that the control group showed rubbing time in phase 1 was 44±7 s and in phase 2 it was 57±17 s. in phase 1, the carbamazepine cut the rubbing time compared to the control group by 55% and the extracts in doses 0.05 ml/10 g and 0.1 ml/10 g cut rubbing time by 86% and 76%, respectively, when administered 1 h before the formalin. in phase 2 of the formalin lip test, the carbamazepine reduced the rubbing time by 60% and the extracts in doses of 0.05 ml/10 g or 0.1 ml/10 g by 94% and 85%, respectively (figure 1). the liver evaluation results showed that the chronic use of the combination of plants when showed no hepatotoxicity. in assessing the alkaline phosphatase (alp) treated with the saline control group, amounts were 62.3±19.5 u/l. these values decreased by 3% in the group treated with carbamazepine and the groups treated with extracts in volumes of 0.05 ml/10 g and 0.1 ml/10 g increased the values by 16.1% and 9.5%,, respectively (figure 2a). in the evaluation of the control group, alt values were 60.31±10.55 u/l, the group treated with carbamazepine decreased by 12.25%, and the groups treated with extracts decreased by 8.5% and 9.1% (figure 2b). in assessing the weights of the livers, the control group had values of 1.83±0.16 g, the group treated with carbamazepine showed a 12.7% increase of these values, and the groups treated with the plant combination decreased by 13.1% and 5.3% with no significant differences (figure 2c). these variations in the figures for liver weight without statistical differences fig. 1. effect of hydroalcoholic extract of plant on formalin-induced orofacial pain related face rubbing response in mice. saline (sal – ml/10 g), carbamazepine (carba – g/kg) or hydroalcoholic extract was administered orally 1 h before formalin 2.5% injection (20 µl) into the vibrissa pad. (i) phase 1 (0-5 min) and (ii) phase 2 (15-30 min) after formalin injection. each value represents the means±sem of 10 animals. *p<0.01, **p<0.001 compared to control (anova followed by bonferroni’s test). can be justified by the difference in weight between the groups of animals, ranging from 38.1±2.76 for the group treated with carbamazepine to 32.1±2.64 for the group treated with a dose of 0.05 ml/10g (figure 2d). the neutrophil count for the control group treated with saline showed values of 17.92±0.99%, the group treated with carbamazepine showed an increase of 1.67%, and values in the groups treated with extracts at doses of 0.05 ml/10 g and 0.1 ml/10 g had decrease of 18.90% and 9.49%, respectively. in the eosinophil count, the control group had values of 1.1±0.49%, the group treated with carbamazepine had values 9.09% lower, and the group treated with the plant extract at a dose of 0.05 ml/10 g showed increased values by 172,7%, while the group treated with 0.1 ml/10 g dosage decreased by 27.27%. in the monocyte count, the control group had values of 1.7±0.7%, the group treated with carbamazepine showed an increase of 58.82%, and values in the groups treated with 0.05 ml/10 g and 0.1 ml/10 g presented reductions of 23.52% and 17.64%, respectively. in the lymphocyte count, the control group showed values of 79.20±1.22%, the group treated with carbamazepine decreased those values by 1.38%, and the groups treated with the extracts showed 2.52% and 2.27% higher values, respectively, for the dosages of 0.05 ml/10 g and 0.1 ml/10 g. the results of this study showed that neutrophils, monocytes and lymphocytes remained at normal levels in different cell groups with no significant difference, but there was an increase in eosinophils in the group treated with a dose of 0.05 ml/10 g of extract presenting a statistically significant difference (p<0.05) (table 1). discussion the mice orofacial formalin test is a useful pre-clinical model of inflammatory trigeminal pain for evaluating antinociceptive activity of analgesics and their combinations. injection of formalin in the mice lip induces stereotyped response (rubbing), consisting of two distinct phases: a first ‘‘phasic’’ phase and a second ‘‘tonic’’ phase16. the orofacial region is the body area most densely innervated by the trigeminal nerve and focuses some of the most common acute pains17. the test is a widely accepted model of tonic pain in the corresponding region, which allows for the study of both behavioral and neurophysiological aspects of this pain condition18. the present study demonstrated that the used associated plants reduced formalin-induced orofacial pain with better results than carbamazepine, a drug used to treat neuropathic pain. carbamazepine is an anticonvulsant that inhibits synaptic depolarization in the pain conduction pathway and limits the repetitive firing of action potentials evoked by persistent depolarization by slowing down the recovery of sodium channels19-20 and has been used for its effectiveness in about 60% to 80% of patients21. moreover, the results for the evaluation of hepatic markers (alt and alp) did not differ significantly between the treatments and the hematological evaluation showed eosinophilia in the group treated with the lower dose (0.05 ml/10 g). braz j oral sci. 14(1):60-65 analgesic effect of hypericum perforatum, valeriana officinalis and piper methysticum for orofacial pain 6363636363 neutrophils (%) eosinophils (%) lymphocytes (%) monocytes (%) saline 17.9±0.99 1.1±0.49 79.2±1.22 1.7±0.70 carbamazepine 18.2±3.25 1.0±0.36 78.1±3.19 2.7±0.53 0.05 ml/ 10g 14.5±1.72 3.0±0.77* 81.2±2.04 1.3±0.42 0.10 ml/10g 16.2±2.59 0.8±0.37 81.0±2.84 1.4±0.45 table 1.table 1.table 1.table 1.table 1. values of neutrophils, eosinophils, lymphocytes, and monocytes in groups treated with saline, carbamazepine, extracts in doses of 0.05 ml/10g and 0.1 ml/ 10g expressed in mean±sem (n=10). anova test followed by bonferroni’s test (*p<0.05). fig. 2. absolute values of alkaline phosphatase (a), alanine transaminase (b), liver weight (c), and animal weight (d) in mice in the control groups (c – saline ml/10g), carbamazepine (carb – mg/kg), and extracts in doses of 0.05 ml/10 g and 0.1 ml/10 g after chronic treatment for 25 days (p.o.). the results are expressed in mean ± sem (n=10). *p<0.05 compared to control (anova followed by bonferroni’s test). fig. 3. dose-response curve showing the quantitative features of hormesis braz j oral sci. 14(1):60-65 analgesic effect of hypericum perforatum, valeriana officinalis and piper methysticum for orofacial pain 6464646464 there are few studies involving the three plants that were chosen to use a ratio for the plants of 3:1:0.5, respectively, in order to reduce individual toxicities and seek a synergistic analgesic effect in mice. the proportions of the drugs used account for about 1/3 of the therapeutic dose. in this sense, for the analgesia induced by the formalin test in the trigeminal nerve, were obtained significant results because the plant combination reduced direct formalin pain in the pain conducting neuron (trigeminal), called phase 1, and reduced inflammatory or later pain, called phase 2, which is the release of pro-inflammatory products such as bradykinin, tnf and prostagladins22. studies have shown that piper methysticum, valeriana officinalis and hypericum perforatum used separately exhibit antidepressant, muscle relaxant, anti-inflammatory and sedative properties 10. alcoholic kava extract inhibits cyclooxygenase 1 and 2. in addition to having a variety of effects on the central nervous system, including anxiolytic, sedative, anticonvulsant, local anesthetic, spasmolytic and analgesic properties, probably for its inhibitory effects of nf-kb, it is an important factor in the anti-inflammatory activity of the cascade of prostaglandins 23. yao et al.24 demonstrated the analgesic effect of kava on abdominal contortions and formalin tests on rats’ paws. recent studies suggest that valerian reduces dopamine transport for chronic pain in rats. valeriana officinalis has anxiolytic, antidepressant and myorelaxing properties, important factors in cases of mild insomnia, orofacial pain and temporomandibular disorders25. the alcoholic and methanolic extracts of hypericum have analgesic and anti-inflammatory effects in formalin and abdominal contortions tests26. hepatic changes can occur due to reactions involving cytochrome p45027-28. liver damage may be hepatocellular, causing increased serum of the alanine aminotransferase (alt) and aspartate aminotransferase (ast) enzymes, or cholestatic, leading to increased bilirubin, alkaline phosphatase (alp) and gamma-gt29. studies have shown that chronic use of valerian causes hepatic toxicity in addition to side effects such as dyspepsia, allergic skin reactions, headache, and agitation30. kava may exacerbate the status of patients with liver failure and hepatitis in those with a history of recurrent liver disease. there are reports of acute hepatitis with severe hepatocellular necrosis, requiring liver transplantation in patients after ingestion of kava extract doses considered to be therapeutic. in susceptible patients, some symptoms may appear after a short time. evaluations of liver function tests indicate changes in the results after 1-2 months of use, with symptoms of hepatomegaly and early encefalopatia10. hypericum also has reports of liver damage in rat fetuses whose mothers were exposed to hypericin11. despite reports of hepatic disorders related to the use of hypericum, kava and valerian in therapeutic doses of 900 mg/kg, 300 mg/kg and 150 mg/kg respectively29-30, the obtained results showed that the use of combined smaller doses (1/3 of the therapeutic doses) are not detrimental considering the biochemical parameters. eosinophilia in the group treated with the lower dose (0.05 ml/10 g) can be explained by a phenomenon called hormesis. this is a term for generally favorable biological responses to low exposures to stressors. chemical substances like, for example, kavalactones, hypericin and valeric acid, or toxins showing hormesis thus have the opposite effect in small doses as in large doses resulting in either a j-shaped or an inverted u-shaped dose response (figure 3). a related concept is mithridatism, which refers to the willful exposure to a substance in an attempt to develop immunity against it31. evidence is presented which supports the conclusion that the hormetic dose-response model is the most common and fundamental in the biological and biomedical sciences, being highly generalized across biological model, measured endpoint and chemical class and physical agent. the hormesis model of dose response is vigorously debated 32. the biochemical mechanisms by which hormesis works are not well understood. the conjecture is that low doses of stressors may activate the repair mechanisms of the body. the repair process fixes not only the damage caused by the chemical substance, but also other low-level damage that may have accumulated before, without triggering the repair mechanism. kava (piper methysticum), hypericum (hypericum perforatum) and valerian (valeriana officinalis) are described in the literature as plants with several biological properties, but that may present severe hepatic impairment related to chronic consumption and in combination with other plants. in low doses, they are prescribed by health professionals to patients with orofacial pain of nociceptive origin, such as pain of temporomandibular origin and bucofacial neuropathic pain with significant improvements in levels of chronic pain. in conclusion, this study is the first demonstrated that p.o pretreatment of an association of hypericum (hypericum perforatum), valerian (valeriana officinalis) and kava (piper methysticum) reduced the nociceptive response in both the first and the second phases in orofacial formalin test. in addition, there were no changes in liver markers and white blood cells count, but one eosinophilic at the lowest dose of extract. the authors believe that this research contributes to the health knowledge in the area of orofacial pain treatment by providing data for possible use of these plants as adjuvants or substitutes of the currently used drugs because of their proven analgesic and anti-inflammatory powers. it is important to mention that the present study has some limitations. studies comprising other analgesic activity tests, histopathological studies and pro-inflammatory markers are required to understand the involved mechanisms. taken together, these observations will provide an insight for similar future studies. references 1. american academy of orofacial pain. the scope of tmd/orofacial pain (head and neck pain management) in contemporary dental practice. dental practice act committee of the american academy of orofacial pain. j orofac pain. 1997; 11: 78-83. 2. coderre tj, katz j, vaccarino al, 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extracts of valeriana officinalis l. s.l. show anxiolytic and antidepressant effects but neither sedative nor myorelaxant properties. phytomedicine. 2008; 15: 2-15. 14. perazzo ff, lima ml, padilha mm, rocha lm, sousa pjc, carvalho jc. anti-inflammatory and analgesic activities of hypericum brasiliense (willd) standardized extract. braz j pharmacog. 2008; 18: 320-5. 15. tasleem f, azhar i, ali sn,perveen s, mahmood za. analgesic and antiinflammatory activities of piper nigrum l. asian pac j trop med. 2014: 7s1: s461-8. doi: 10.1016/s1995-7645(14)60275-3. 16. raboisson p, dallel r. the orofacial formalin test. neurosci biobehav rev. 2004; 28: 219-26. 17. bonjardim lr, silva am, oliveira mg, guimarães ag, antoniolli ar, serafini mr, et al. sida cordifolia leaf extract reduces the orofacial nociceptive response in mice. phytother res. 2011; 25: 1236-41. 18. clavelou p, pajot j, dallel r, raboisson p. application of the formalin test to the study of orofacial pain in the rat. neurosci lett. 1989; 103: 349-53. 19. koppenol wh. concurrent cooperativity and substrate inhibition in the epoxidation of carbamazepine by cytochrome p450 3a4 active site mutants inspired by molecular dynamics simulations. biochemistry. 2014; doi: 10.1021/bi5011656. 20. brunton ll. goodman & gilman: the pharmacological basis of therapeutics. rio de janeiro: mcgraw-hill; 2012. 21. góes tmpl, fernandes rsm. trigeminal neuralgia: diagnosis and treatment. int j dent. 2008; 7: 104-11. 22. carvalho wa; lemônica l. specific cyclooxygenase-2 inhibitor analgesics: therapeutic advances. rev bras anestesiol. 1998; 48: 137-58. 23. shaik aa, hermanson dl, xing c. identification of methysticin as a potent and non-toxic nf-jb inhibitor from kava, potentially responsible for kava’s chemopreventive activity. bioorg med chem lett. 2009; 19: 5732-36. 24. yao cy, wang j, dong d, qian fg, xie j, pan sl. laetispicine, an amide alkaloid from piper laetispicum, presents antidepressant and antinociceptive effects in mice. phytomedicine. 2009; 16: 823-9. 25. hattesohl m, feistel b, sievers h, lehnfeld r, hegger m, winterhoff h. extracts of valeriana officinalis l. show anxiolytic and antidepressant effects but neither sedative nor myorelaxant properties. phytomedicine. 2008; 15: 2-15. 26. sanchez-mateo cc, bonkanka cx, hernandez-perez m, rabanal rm. evaluation of the analgesic and topical anti-inflammatory effects of hypericum reflexum l. fil. j ethnopharmacol. 2006; 107: 1-6. 27. bertolami mc. mechanisms of hepatotoxicity. arq bras cardiol. 2005; 85: 25-7. 28. rodrigues mg, mendonça mm, de paula jam. analysis of rational use of hypericum perforatum from the profile of the prescriptions dispensed in pharmacies anapolis-go.rev eletron de farm. 2006; 3: 45-52. 29. pearl pl, drillings im, conry ja. herbs in epilepsy: evidence for efficacy, toxicity, and interactions. semin pediatr neurol. 2011; 18: 203-8. 30. prasad r, naime m, routray i, mahmood a, khan f, ali s. valeriana jatamansi partially reverses liver cirrhosis and tissue hyperproliferative response in rat. methods find exp clin pharmacol. 2010; 32: 713-9. 31. calabrese ej. hormesis and medicine. br j clin pharmacol. 2008; 66: 594-617. 32. kaiser j. sipping from a poisoned chalice. science. 2003; 302: 376-9. braz j oral sci. 14(1):60-65 analgesic effect of hypericum perforatum, valeriana officinalis and piper methysticum for orofacial pain oral sciences n3 original article braz j oral sci. july | september 2014 volume 13, number 3 influence of water/powder ratio in the mineral and synthetic casts rudys rodolfo de jesus tavarez1, rufino josé klug1, mayana soares vieira1, gisele lima bezerra1, matheus coelho bandeca1, leily macedo firoozmand2 1universidade ceuma uniceuma, school of dentistry, department of prosthodontics, são luis, ma, brazil 2universidade federal do maranhão ufma, school of dentistry, department of dental materials, são luis, ma, brazil correspondence to: rudys rodolfo de jesus tavarez universidade ceuma pós-graduação em odontologia rua josué montello, nº1, renascença ii cep 65.075-120 são luís ma brasil phone: +55 98 3214 4127 e-mail: rudysd@uol.com.br received for publication: july 17, 2014 accepted: september 02, 2014 abstract aim: to evaluate the influence of varying the water/powder ratio on the compressive strength of type iv mineral and synthetic casts. methods: four commercial brands of type iv mineral and synthetic casts were evaluated: durone, herostone, fuji rock, and elite rock. ninety-six test samples were prepared from a silicone matrix, according to ada’s standard no. 25. the samples were prepared according to the manufacturer’s recommendations with a normal water/powder ratio (n = 12) and with 20% extra water (n = 12), forming the control (a) and experimental (b) subgroups, respectively. compressive strength tests were performed using a universal testing machine emic (dl 2000) with a load cell of 2,000 kgf/cm2. the obtained data were analyzed statistically using two-way anova and tukey’s test (α=5%). results: the synthetic elite rock cast was statistically different from the one obtained when the portion of water indicated by the manufacturer was used; no significant differences were found in the remaining casts when the proportion of water was increased by 20%. conclusions: the groups of synthetic and mineral casts differed and the water increase (20%) did not cause significant difference on the compressive strength of the materials. keywords: calcium sulfate; compressive strength; dental materials introduction models for fabricating dies and prosthetic pieces require the use of casts with high resistance, surface hardness and resistance to abrasion. moreover, high accuracy is required to reproduce details and for dimensional stability1-3. type iv and v casts are the materials of choice in these cases, as they have favorable mechanical properties such as high resistance, minimal setting expansion, and high surface hardness4-7. depending on the fabrication process, casts may be classified as mineral or synthetic. to obtain their inherent characteristics, mineral casts are fabricated by heating calcium sulfate dihydrate (caso4·2h2o), which is converted into calcium sulfate hemihydrate (caso4·1/2h2o). depending on the calcination method, different forms of hemihydrate may be obtained, i.e., the β-hemihydrate form is known as a plaster cast, which consists of particles of large rhomboid crystals of irregular shape with capillary pores; while α-hemihydrate, which contains smooth and dense particles, the powder being mainly indicated for die fabrication8. however, it is possible to produce αand β-hemihydrate from the by-products of phosphoric acid production, resulting in the so-called synthetic casts. they are generally more expensive than mineral casts and their properties are identical or superior to those of conventional casts. using type iv mineral and synthetic casts, it is possible to fabricate models braz j oral sci. 13(3):225-228 with smooth and hard surfaces, important characteristics that allow the waxing and sealing of edges with minimal abrasion during the production of fixed prostheses. these are some of the most frequently used casts, easy to produce and compatible with molding materials3-4. however, these casts expand in the first 24 h and shrink for up to two weeks after setting9. despite the high resistance and hardness of type iv casts, care must be taken during their fabrication. the water/powder ratio is considered a determining factor of the physical and chemical properties of a cast, and change in this ratio may alter these properties. an increase in the water/powder ratio increases the setting time and the possibility of producing a cast with fewer crystals per volume, lower resistance, and lower setting expansion8. on the other hand, less water will decrease the fluidity of the cast, preventing the exact replication of mold details10. this ratio varies among different commercial brands; between 0.22 and 0.24 ml of water per 100 g are required for type iv casts8. according to the american dental association (ada) specification no. 25, type iv casts, 1 h after mixing, must have a compressive strength of 5000 psi, equivalent to 351.53 kgf/cm2 (ada 25)11. the compressive strength of the casts is directly associated with their surface hardness and resistance to abrasion during handling in a clinic or laboratory, or during die casting, articulation, cutting, and duplication of the model12-13. the highest resistance to diametral traction of these casts occurs 120 min after setting14. the null hypotheses tested in this study were: (1) there is no change in compressive strength between different brands of type iv mineral and synthetic casts; and (2) a 20% increase in the water/powder ratio does not affect the compressive strength of the tested casts. materials and methods ninety-six test samples were prepared, with dimensions of 20 x 40 mm, from four different commercial brands of mineral and synthetic type iv casts (table 1). the samples were prepared using a standardized silicone matrix according to standard no. 25 of the ada (figure 1). test samples in each group of casts were prepared according to the manufacturers’ instructions, with normal water/powder ratio (n = 12) and an extra 20% of water group mineral synthetic 1 2 3 4 description durone iv cast herostone iv cast elite rock iv cast fuji rock iv ep cast, peal white brand dentsply indústria e comércio, petrópolis, rj, brazil vigodent s/a indústria e comércio, rio de janeiro, rj, brazil zhermacktechnical, badiapolesine (rovigo), italy gc europe n v, leuven, belgium batch expiration date 649874e mar/15 00712 jul/2015 130508 sep/2014 1201042 jan/2015 normal(a) 19 ml 20 ml 20 ml 20 ml 20% extra(b) 22.8 24 ml 24 ml 24 ml table 1.table 1.table 1.table 1.table 1. specifications of the type iv casts used in the study. fig. 1. silicone mold and test sample. (n=12), resulting in the control (a) and experimental (b) subgroups, respectively. the cast was prepared at a controlled temperature of 25 ± 2 °c and a relative humidity of approximately 50 ± 10% (table 1). the casts were weighed on a precision scale (bioprecisa fa2104n, curitiba, pr, brazil) and mixed mechanically under vacuum (polidental, são josé dos campos, sp, brazil) for 30 s. after mixing, the casts were poured into silicon matrices with the help of a gypsum vibrator (vh goldline, araraquara, sp, brazil) for 1 min. a sheet of glass was placed on the matrices, maintaining a constant weight of 1 kg as the casts set. after 1 h, the test samples were removed and assessed for elements that may interfere with the compression test, such as bubbles, cracks and faults. the test samples with visible imperfections were discarded. one hour after mixing, the test samples were subjected to compressive strength tests in a universal testing machine (emic dl2000, são josé dos pinhais, pr, brazil) with a load cell of 2,000 kgf/cm2 and a displacement velocity of 0.5 mm/min. kolmogorov-smirnov and shapiro-wilk (0.05%) tests were used to confirm whether the analyzed data followed a normal distribution. as this was the case, the data were then analyzed statistically by two-way anova and tukey’s tests (α5%). the same operator analyzed the fractures in the test samples, but he was blinded as regards the materials. the 226226226226226 braz j oral sci. 13(3):225-228 influence of water/powder ratio in the mineral and synthetic casts mineral g1: durone 1127.0 (177.7)* 1006.2 (167.3)* 1067.6 (179.1) g2: herostone 1102.3 (315.1)* 955.9 (107.9)* 1029.1 (241.2) synthetic g3: elite rock 1314.6 (196.7)* 958.4 (161.9)* 1136.5 (253.2) g4: fuji rock 1119.7 (65.6)* 952.5 (93.6)* 1036.1 (116.4) total 1165.9 (218.5)* 968.3 (133.1)* 1067.1 (205.4) indicated h2o proportion (control) a increased h2o proportion b totalgroups compressive strength (kgf) *n = 12 table 2.table 2.table 2.table 2.table 2. mean and standard deviation of compressive strength for each studied group. effect sq g l qm f p cast 144389.680 3 48129.893 1.549 0.209 h2o × powder ratio 781255.109 1 781255.109 25.146 0.000* interaction effect 172884.954 3 57628.318 1.855 0.145 residual 2236923.998 72 31068.389 total 9.444e7 80 table 3.table 3.table 3.table 3.table 3. two-way anova for compressive strength data for different experimental groups. *p < 0.05 groups fracture (score) 1 2 3 mineral g1: durone a 2 (20%) 1 (10%) 7 (70%) b 3 (30%) 3 (30%) 4 (40%) g2: herostone a 3 (30%) 1 (10%) 6 (60%) b 4 (40%) 1 (10%) 5 (50%) synthetic g3: elite rock a 1 (10%) 0 9 (90%) b 3 (30%) 3 (30%) 4 (40%) g4: fuji rock a 3 (0%) 1 (10%) 6 (60%) b 2 (20%) 2 (20%) 6 (60%) table 4.table 4.table 4.table 4.table 4. distribution of fracture types in studied samples. final assessment was made on the basis on the following scores: 1, medial longitudinal fracture; 2, lateral longitudinal fracture; and 3, composite fracture (both longitudinal and/or transversal associated with oblique fractures and multiple fragments). results the mean and standard deviation of the studied groups and subgroups are shown in table 2. two-way anova showed that both the cast type and the interaction effect did not significantly differ between the studied groups. however, when the effect of changing the water/powder ratio of the studied casts was analyzed, it was found to be statistically significant (table 3). the highest mean of compressive strength between the subgroups with the water/powder ratio recommended by the manufacturer was found for the elite rock synthetic cast g3 (table 2, figure 2). with regard to the different fracture types in the test fig. 2. minimum and maximum values, mean and standard deviation (kgf) values for groups and tukey’s test (α = 5%) results after compressive strength test. 0 500 1000 1500 2000 g2-a a -b a -b a g3-a b -b a g4-a a g1-a a g1 g2 g3 g4 ______ ______ ______ _____ a b a b a b a b -b a k g f samples, table 4 shows that the predominant type was type 3 (multiple fractures) in all groups, mainly when the water/ powder ratio recommended by the manufacturer was used. discussion currently, mineral casts are commonly used for preparing study models, and it is noticed that the use of synthetic casts has become widely accepted in dentistry. thus, when the compressive strength of type iv synthetic and mineral casts was analyzed, the first null hypothesis was not rejected but the second one was rejected, since the synthetic elite rock cast differed significantly from the other groups. the compressive strength test is especially important, as it relates to the surface hardness of a model12. cast resistance is directly affected by two factors, water/powder ratio and mixing time. in this study, the mixing time was standardized so that the results were not negatively influenced3,15. the results obtained in this study showed that only the comparison of casts and the interaction effect did not significantly differ between the studied groups, confirming the results found in other studies1,16. however, when the water/ powder ratio was varied, significant differences were found 227227227227227 braz j oral sci. 13(3):225-228 influence of water/powder ratio in the mineral and synthetic casts among the test samples. the compressive strength of casts appears to be materialdependent, as the post hoc statistical test demonstrates that only the elite rock synthetic cast had a higher mean resistance when the water/powder ratio indicated by the manufacturer was analyzed. the mineral casts and the other studied synthetic cast (fuji rock) differed from the elite rock cast but did not show significant differences from each other. after the extra 20% water was added, there was difference in compressive strength for the synthetic elite rock cast compared with the normal ratio. moreover, the decrease in resistance to compression of this synthetic cast was found to be significant. when the amount of water was increased, small dihydrate crystals, which acted as anchor points for larger crystals, precipitated and the bonds between them were destroyed, increasing the porosity and hence lowering the resistance of the cast17-18. therefore, an increase in the water/ powder ratio produces a more porous and less resistant cast, since the water evaporates and leaves empty spaces between the particles. with regard to the types of fracture in the test samples, the obtained results showed that type 3 was the most frequent; few studies reported in the literature have assessed the type of fracture in test samples12. it is assumed that this predominance occurs because of the mechanical behavior of gypsum products during setting, since the greater the hardness of the material, the more friable it becomes. this explains the greater incidence of multiple fractures, especially when the normal water/powder ratio was maintained15,18-19. according to the results of this study, it was concluded that there is no significant difference between compressive strength of synthetic and mineral casts and there is a significant decrease in compressive strength with additional 20% water in relation to powder, particularly in the synthetic elite rock cast. further studies are required to prove the benefits and behavior of synthetic casts introduced in the market. references 1. duke p; moore bk; haug sp; andres cj. study of the physical properties of type iv gypsum, resin-containing, and epoxy die materials. j prosthet dent. 2000; 83: 466-73. 2. lindquist tj; stanford cm; knox e. influence of surface hardener on gypsum abrasion resistance and water sorption. j prosthet dent. 2003; 90: 441-6. 3. he lh; van vuuren lj; planitz n, swain mv. a micro-mechanical evaluation of the effects of die hardener on die stone. dent mater j. 2010; 29: 433-7. 4. ragain jc; grosko ml; raj m; ryan tn; johnston wm. detail reproduction, contact angles, and die hardness of elastomeric impression and gypsum die material combinations. int j prosthodont. 2000; 13: 214-20. 5. sharma a, shetty m, hegde c, shetty ns, prasad dk. comparative evaluation of dimensional accuracy and tensile strength of a type iv gypsum using microwave and air drying methods. j indian prosthodont soc. 2013; 13: 525-30. 6. gujjarlapudi mc, reddy sv, madineni pk, ealla kk, nunna vn, manne sd. comparative evaluation of few physical properties of epoxy resin, resin-modified gypsum and conventional type iv gypsum die materials: an in vitro study. j contemp dent pract. 2012; 13: 48-54. 7. valente vs, zanetti al, feltrin pp, inoue rt, de moura cd, pádua le. dimensional accuracy of stone casts obtained with multiple pours into the same mold. isrn dent. 2012; 2012:730674. doi: 10.5402/2012/730674. 8. anusavice kj. phillips’ science of dental materials. 11th ed. saint louis: elsevier; 2003. 9. michalakis kx, asar nv, kapsampeli v, magkavali-trikka p, pissiotis al, hirayama h. delayed linear dimensional changes of five high strength gypsum products used for the fabrication of definitive casts. j prosthet dent. 2012; 108: 189-95. 10. alsadi s, combe e, cheng ys. properties of gypsum with the addition of gum arabic and calcium hydroxide. j prosthet dent. 1996; 76: 530-4. 11. american national standards/american dental association specification no. 25. dental gypsum products. new york: american national standards institute; 2000. 12. schneider rl, taylor td. compressive strength and surface hardness of type iv die stone when mixed with water substitutes. j prosthet dent. 1984; 52: 510-4. 13. hiraguchi h, nakagawa h, wakashima m, miyanaga k, saigo m,nishiyama m. effects of disinfecting alginate impressions on the scratch hardness of stone models. dent mater j. 2006. 25: 172-6. 14. hersek n, canay s, akça k, ciftçi y. tensile strength of type iv dental stones dried in a microwave oven. j prosthet dent. 2002; 87: 499-502. 15. craig gc, powers jm. restorative dental materials. 10th ed. missouri: mosby year book; 1997. p. 63-73. 16. silva ma, vitti rp, consani s, sinhoreti ma, mesquita mf, consani rl. linear dimensional change, compressive strength and detail reproduction in type iv dental stone dried at room temperature and in a microwave oven. j appl oral sci. 2012; 20: 588-93. 17. khan z, morris jc, von fraunhofer ja. effect of irreversible hydrocolloid impressions on surface hardness of dental stone. j prosthet dent. 1984; 52: 435-7. 18. van noort r. introduction to dental materials. 4. ed. london: mosby; 2013. 19. schwedhelm er, lepe x. fracture strength of type iv and type v die stone as a function of time. j prosthet dent. 1997; 78: 554-9. 228228228228228 braz j oral sci. 13(3):225-228 influence of water/powder ratio in the mineral and synthetic casts oral sciences n3 braz j oral sci. 12(1):23-29 original article braz j oral sci. january | march 2013 volume 12, number 1 keratocystic odontogenic tumor related to nevoid basal cell carcinoma syndrome: clinicopathological study luana eschholz bomfin1, ana paula m. vivas1, andre caroli rocha2, maria isabel w. achatz3, clovis antonio l. pinto4, fabio abreu alves5 1 department of stomatology, são paulo university, são paulo, sp, brazil 2 department of stomatology, a. c. camargo hospital, são paulo, sp, brazil 3 department of oncogenetics, a. c. camargo hospital, são paulo, sp, brazil 4 department of anatomical pathology, a. c. camargo hospital, são paulo, sp, brazil 5 department of stomatology, university of são paulo, department of stomatology, a. c. camargo hospital, são paulo, sp, brazil correspondence to: fábio abreu alves rua prof. antônio prudente 211, liberdade, cep: 01509-900 são paulo, sp, brasil phone: +55 11 21895129 fax: +55 11 21895133 e-mail: falves@accamargo.org.br abstract aim: to assess clinicopathological features of patients with keratocystic odontogenic tumor (kcot) associated with nevoid basal cell carcinoma syndrome (nbccs) in a single brazilian institution. methods: after histopathological analyses of kcot related to nbccs, the medical charts of 14 patients were assessed. these patients presented a total of 31 primary and 8 recurrent kcot. results: out of 14 patients, 8 presented a single kcot, 4 showed synchronous tumors, 1 had 3 metachronous lesions and another patient had 2 synchronous lesions at initial evaluation and then developed other 3 metachronous lesions. besides the 31 primary kcots, 18 lesions were located in mandible and 13 in maxilla. most tumors presented unilocular pattern and association with a tooth. conclusions: kcot is a frequent manifestation of nbccs and can be its first sign, mainly in young patients. in contrast to a previously published series, most patients presented a single lesion. keywords: gorlin syndrome,keratocyst, keratocystic odontogenic tumor, nevoid basal cell carcinoma syndrome. introduction nevoid basal cell carcinoma syndrome (nbccs) was first described in 1960 by gorlin and goltz and was characterized by multiple basal cell carcinomas (bcc), odontogenic keratocysts (okc) and bifid ribs1. a multidisciplinary colloquium was recently organized and its aims were to better define the physical findings associated with nbccs. the participants reviewed the diagnostic criteria of the syndrome and there was no consensus for a formal recommendation. consequently, a suspected diagnosis of nbccs should be considered based on the findings of less stringent criteria of: (1) one major criterion and molecular confirmation; (2) two major criteria; or (3) one major and received for publication: january 14, 2013 accepted: march 05, 2013 braz j oral sci. 12(1):23-29 2424242424 two minor criteria. in addition, medulloblastoma (mb) should be considered a major criterion as it may lead to increased early detection of the syndrome. both major and minor criteria according to this colloquium are shown in table 12. basal cell carcinoma (bcc) and okc are the most common incident manifestations of the syndrome3. in 2004, agaram et al. showed significant loss of heterozygosity of tumor suppressor genes in sporadic lesions4. consequently, these authors hypothesized the neoplastic nature of the lesion and the world health organization later recommended changing the name of the lesion from okc to keratocystic odontogenic tumor (kcot). there are few reported cases of kcot associated with nbccs from brazil and most data feature biological behavior. in addition, we have found only 13 welldocumented patients have been found in english language literature with such association in the brazilian population in the last 20 years5-10. the purpose of this paper is to describe clinical and histopathological aspects of kcots associated with nbccs and report other manifestations associated with the syndrome. furthermore, this study shows the largest clinical data about kcot associated with nbccs ever compiled in the brazilian population. material and methods characterization of the study this study consisted of a retrospective analysis of patients with kcot diagnosed between 1970 and 2009. a total of 74 patients presented koct, 14 being related to nbccs. clinical criteria for nbccs diagnosis were based on bree et al.2 the present study was approved by the ethics committee of ac camargo hospital (number 1322/2009). clinical data the medical charts of 14 patients presenting kcot related to nbccs were evaluated. these patients presented a total of 31 primary and 8 recurrent kcot. data including major criteria 1. basal cell carcinomas (bcc) prior to 20years old or excessive numbers of bcc out of proportion to prior sun exposure and skin type 2. odontogenic keratocystic of the jaw prior to 20 years of age 3. palmar or plantar pitting 4. lamellar calcification of the falx cerebri 5. medulloblastoma, typically desmoplastic 6. first degree relative with nbccs minor criteria 1. rib anomalies 2. macrocephaly determined after adjustment for height 3. other specific skeletal malformations and radiologic changes (i.e., vertebral anomalies, kyphoscoliosis, short fourth metacarpals, postaxial polydactyly). 4. cleft/lip palate 5. ovarian/cardiac fibroma 6. lymphomesenteric cysts 7. ocular abnormalities (i.e., strabismus, hipertelorisms, congenital cataracts, glaucoma, coloboma) table 1. major and minor criteria stated by first international colloquium on nbccs bree et al2. age, gender, race, signs, symptoms, radiographic features, treatment and recurrence were analyzed. histopathological analysis the hematoxylin/eosin (he)-stained slides of kcots were retrieved and submitted to histopathological exam. all slides were reviewed by an oral pathologist and findings were confirmed by a second general pathologist (pinto cal). statistical analysis frequency distribution tables were built in order to record patient clinical data, tumors and histopathological features. the recurrence rate was calculated by kaplan-meier estimator. all data analysis was performed using r version 2.13.0 software (http://www.r-project.org) results clinical features of 14 patients six (42.86%) patients with kcot were referred by the cutaneous oncology department due to previous diagnosis of nbccs and the remaining 8 (57.14%) had kcot as first manifestation of the syndrome. the patient’s age at kcot diagnosis ranged from 8 to 66 years (mean age of 31 years), and most of the lesions occurred in the second decade of life. nine out of 14 patients (64.29%) were female and 12 patients (85.71%) were caucasian (table 2). among the 14 patients, 8 patients presented a single kcot, 4 presented kcot synchronous tumors (total of 15 lesions), 1 had 3 kcot metachronous lesions and 1 patient had 2 synchronous lesions at the initial evaluation and developed other 3 metachronous lesions during the followup period (table 2). in addition to kcots, the most common clinical manifestations were basal cell carcinoma (cbc), palmar pits, abnormal ribs and vertebrae, and calcification of the falx cerebri (figure 1). the nbccs clinical manifestations are listed in table 3. keratocystic odontogenic tumor related to nevoid basal cell carcinoma syndrome: clinicopathological study 2525252525 variable category n % age 0-9 17.14 10-19 5 35.71 20-29 2 14.29 30-39 4 28.57 40-69 2 14.28 gender female 9 64.29 male 5 35.71 race caucasian 12 85.71 not caucasian 2 14.29 main complaint swelling 5 35.71 none 9 64.29 extra-oral examination none 10 71.43 facial asymmetry 3 21.43 fistula 1 7.14 intra-oral examination lump 6 42.86 fistula 1 7.14 none 7 50.00 number of kcot 1 8 57.14 3 3 21.43 4 1 7.14 5 2 14.29 patient’s status alive without disease 12 85.71 alive with disease 1 7.14 out of follow-up 1 7.14 total 14 100 table 2. clinical features of 14 patients with nbccs. braz j oral sci. 12(1):23-29 fig. 1: some of clinical manifestations of nbccs. a. multiple palmar pits (patient 13). b. basal cell carcinoma located in fluff region (patient 13). c. one kcot located in ramus and posterior mandible and with multilocular pattern (patient 4). d. presence of four synchronous kcot with unilocular pattern involving 3rd impacted molar (patient 11). e. chest radiograph demonstrating bifid ribs (patient 12). f. antero-posterior skull radiograph showing calcification of the falx cerebri (patient 2). clinicopathological features of 31 primary kcot in 14 patients at initial evaluation, 5 (35.71%) patients complained of swelling and 9 (64.29%) had no symptoms. the time of complaint ranged between 15 days and 6 months (mean=2 months). on extra-oral examination, 3 patients (21.43%) presented facial asymmetry, 1 patient extra-oral fistula (7.14%) and no alterations were observed in 10 patients (71.43%). on intra-oral examination, 6 patients (42.86%) presented lumps, 1 patient (7.14%) a fistula and 7 patients (42.86%) presented no alterations. radiographic analysis of 31 primary kcots demonstrated that 18 (58.06%) lesions were located in the mandible and 13 (41.94%) in the maxilla. most of the tumors presented a unilocular pattern and had tooth association (figure 1). the size of the lesions ranged from 2.5 to 10 cm (mean = 6.14 cm). according to kcot treatment, enucleation associated with curettage was performed in 30 cases (96.78%) and the other case was treated by marsupialization and curettage (table 4). out of the 31 kcots, 6 presented a single recurrence and one tumor recurred twice. the time between the treatment and the recurrence ranged between 9 to 149 months (median=66.25 months) (table 4). histopathological data of 31 primary and 8 recurrent kcots typical kcot epithelium was found in 26 (66.66%) keratocystic odontogenic tumor related to nevoid basal cell carcinoma syndrome: clinicopathological study braz j oral sci. 12(1):23-29 2626262626 patient pkcot rkcot majo r criteria mino r criteria other clin ical manifestations kcot cf pp bc mb fd mc hy pc cl fb ar 1 l mand r mand* ant maxilla r maxilla* l maxilla 2 1 1 + + + + epidermoid cyst, endometrial duplications, mitral valve prolapsed, nevi, scoliosis 2 r mand + + + + + + + + broad nasal base, epidermoid cyst, hypocromic lesions in the conjunctiva, multiple nevi, nail clubbing 3 l+r mand* r maxilla* l maxilla* + + 4 r mand 1 + + bilateral kidney stones, breasts cysts, epidermoid cyst, hypercalciuria, lipoma, nevi, ovarian myoma, scoliosis 5 l mand + + + 6 r maxilla + + ductal carcinoma of breast, multiple nevi 7 r mand 1 + + marginal osteophytes, nevi, ovarian myoma, scoliosis 8 r mand l mand r maxilla 1 + + + + + breast cysts, cholecystitis, epidermoid cyst, ovarian myoma and pelvis cysts. 9 r maxilla 1 + + + + multiple nevi, ovarian myoma 10 r mand + + + + + broad nasal base, nevi and prognathism. 11 r mand* l mand* r maxilla* l maxilla* + + + mitral valve prolapsed, multiple nevi. 12 r mand + + + + + facial milia, scoliosis 13 r maxilla* l maxilla* l mand* r mand* ant mand* + + + + + + broad nasal base, high-arched palate, prognathism and sebaceous cyst. 14 r mand* l mand* r maxilla* + + + + hypothyroidism, liver adenoma, multiple nevi, renal cyst and thyroid goiter. total 31 8 14 6 5 9 3 2 4 2 1 2 5 1 46 table 3. clinical features of the 14 patients with nbccs distributed according to main major and minor nbccs criteria2. pkcot=primary keratocystic odontogenic tumor, rkcot=recurrent keratocystic odontogenic tumor, cf=calcification of the falx cerebri, pp=palmar pits, bc=basal cell carcinoma, mb=meduloblastoma, fd=first degree relative with nbccs, mc=macrocephaly, sc=scoliosis, hy=hypertelorism, pc=pectus cavitatum, cl=clinodactilia, fb=frontal bossing, ar=abnormal ribs, l=left, r=right, mand=mandible, * synchronous kcot, +presence, absence. tumors and 13 lesions had typical areas of kcot and areas of epithelial hyperplasia. furthermore, detachment of the epithelium was seen in 27 (69.23%) cases. interestingly, only one case presented evident epithelium dysplasia. variable category n % location mandible posterior 7 22.58 posterior + ramus 6 19.35 ramus 3 9.68 anterior 2 6.45 total 18 58.06 maxilla posterior 12 38.71 anterior 1 3.23 total 13 41.94 radiographic pattern multilocular 6 19.35 unilocular 15 48.39 not informed 10 32.26 tooth association yes 13 54.17 n o 11 45.83 not informed 7 22.58 treatment enucleation 30 96.78 and curettage marsupialization and 1 3.23 curettage recurrence n o 24 77.42 yes 7 22.58 table 4. clinical and radiographic features of the 31 primary kcot. in relation to the connective tissue, 21 cases (53.84%) demonstrated mild inflammation in 11 tumors (28.20%), moderate in 5 (12.82%), and severe in 5 cases (12.82%). satellite cysts, remnants of odontogenic epithelium, budding and dystrophic calcification were found in 11 (28.20%), 14 (35.89%), 9 (23.07%), 6 cases (15.38%), respectively (figure 2). discussion bccs and kcot are the main features observed in patients with nbccs3. similarly, kimonis et al. evaluated clinical and radiological data of 105 persons with nbccs. pits, bccs, jaw cysts and falx calcification were the most common anomalies, and according to their results the authors suggested some major and minor criteria for nbccs diagnosis 11. comparing these criteria with the first international colloquium on nbccs criteria2, there were two important alterations. the latter suggested changing rib anomalies to minor criterion and mb to major criterion. interestingly, amlashi et al. evaluated 76 patients with mbs and three of them had syndromic mbs. additionally, the authors reviewed the literature and found other 33 patients with syndromic mbs. the mean age of syndromic mbs was 4 years (earlier than sporadic mbs) and most syndromic patients were younger than 2 years12. only one of these patients developed mb at 3 years. at 17 years he presented 3 synchronous kcots, and at 18 years calcification of the falx cerebri. it is worthy of note that calcification of the falx cerebri had been previously investigated in this patient. keratocystic odontogenic tumor related to nevoid basal cell carcinoma syndrome: clinicopathological study 2727272727 braz j oral sci. 12(1):23-29 fig. 2: a. typical epithelium lining of a kcot (h.e., 10.3x). b. daughter cyst observed in cystic capsule (h.e., 4.8x). c. budding of the basal cell layer of the lining epithelium (h.e., 9.8x). d. remnants of odontogenic epithelium in the cystic wall (h.e., 9.3x). kimonis et al. evaluated the falx cerebri calcification in 82 individuals with nbccs11. this calcification was present in 23 out of 29 (79%) individuals over the age of 40, 20 out of 26 (77%) individuals between the ages of 20 and 40 and 10 out of 27 (37%) individuals under the age of 20. in the stomatological system, besides kcot, other benign and malignant tumors have been described in nbccs patients such as ameloblastoma, myxoma, fibrosarcoma, squamous cell carcinoma, adenoid cystic carcinoma and lymphoma. furthermore, development defects such as cleft lip/palate, dental ectopy/heterotopy, impacted teeth, dental agenesis, malocclusion, mandibular prognathism, high-arched palate, skeletal open bite and hyperplasia of mandibular coronoid process have also been reported10,13-17. interestingly, ponti et al.18 evaluated 41 ameloblastomas and two of them were related to nbccs. in addition, ptch 1 germline mutations were also detected in both cases and negative in the others. the authors suggested including ameloblastoma as a criterion for syndrome identification. the present series reveals that only one patient presented high-arched palate associated with prognatism. in the present study, 74 patients with kcot were reviewed and 14 (19.17%) of them also presented nbccs. kcot was the first sign of the syndrome in 8 (57.14%) patients. in a similar study, lo muzio et al.19 evaluated 37 individuals with nbccs, and 34 of them had kcot (92%). in approximately 70% of these patients, the first manifestation of the syndrome was kcot. in general, most of the patients with nbccs are females and kcot occurs in the second decade of life with a mean age ranging from 17 to 26 years1921. in the present series, 9 out of 14 patients were females and there were two peaks of age, in the second and fourth decades. as a consequence, the mean age was 31 years, differing from the above-mentioned studies. in the largest series in english literature, woolgar et al. evaluated 164 kcots in syndromic patients and 379 kcots not associated with nbccs20. it was observed that the posterior area of the mandible was the main affected site, followed by the maxillary molar region in both groups. since the syndromic patients almost always have more than one tumor, it is to be expected that more maxillary tumors are present in these patients. such data were also demonstrated in the present study, in which multiple lesions were found in 6 patients and accounted for 23 tumors (12 in the mandible and 11 in the maxilla). interestingly, 8 patients presented a single lesion, 6 affecting the mandible and 2 the maxilla. in general, the mandible was the main location of the lesions (18 cases 58.06%), 13 being in the body/ramus and 5 in the anterior area. in addition, there were 12 cases (38.71%) in the posterior and 1 case in the anterior region of the maxilla. other studies have also shown multiple lesions affecting syndromic patients. kimonis et al. reported that 78 (74%) nbccs patients presented kcot with number of tumors ranging from 1 to 2811. however, the authors did not clarify which tumors were primary or recurrent. furthermore, it was also shown that 5 individuals had more than 10 kcots in their lifetime. ahn et al. reviewed 33 well-documented case keratocystic odontogenic tumor related to nevoid basal cell carcinoma syndrome: clinicopathological study 2828282828 braz j oral sci. 12(1):23-29 reports of nbccs published between 1981 and 2002. out of the total, 30 patients (90.9%) had kcot and the number of lesions per patient ranged from 1 to 6 (mean 2.7 lesions)21. in a recent nbccs case series reported in indian patients, all 6 patients developed multiple kcot (range 3 to 6) 22. differently, most of the patients in this study (8 out of 14) had a single kcot, and the other 6 patients presented 23 tumors (range 3 to 5). the above-mentioned research involving brazilian patients with kcot related to nbccs reports only 13 patients, and there was no information on 3 patients, 3 had a single lesion, 4 had 2 lesions, 2 had 3 lesions and one patient had 5 lesions5-10. regarding the treatment of kcot, zecha et al. demonstrated that 58 patients who did not have an nbccs diagnosis and were treated with enucleation alone had recurrence in 20.7% of the cases23. a lower rate was described by boffano et al. accounting for 11.9% of 261 tumors treated by enucleation and curettage24. in this study, the association of enucleation and curettage was performed in 30 (96.78%) cases (13 patients). recurrence was observed in 7 tumors (22.58%), which corresponded to 5 patients. one other patient was at first treated by marsupialization and after 10 months by curettage. this patient has been asymptomatic for 5 years. recurrences usually manifest within the first 5 to 7 years. however, zhao et al. demonstrated recurrence after 13 years of follow-up25. similarly, our study demonstrated 2 patients who had recurrence after 10 years. in summary, kcot related to nbccs can affect patients at a younger age than sporadic kcot and multiple tumors are commonly found. interestingly, in this series, 8 out of 14 patients (57%) had a single lesion. early diagnosis of the syndrome and a long follow-up period is important due to the severity of clinical manifestations. moreover, a multidisciplinary team is required, including dentists, dermatologists, geneticists and neurologists to improve the diagnosis and quality of life. acknowledgments we wish to thank ana laura miziara for text revision. funding this study was supported by são paulo state research foundation (fapesp). references 1. gorlin rj, goltz rw. multiple nevoid basal-cell epithelioma, jaw cysts and bifid rib. a syndrome. n engl j med. 1960; 62: 908-12. 2. 2bree af, shah mr; bcns colloquium group. consensus statement from the first international colloquium on basal cell nevus syndrome (bcns). am j med genet a. 2011; 155a: 2091-7. 3. evans dg, ladusans ej, rimmer s, burnell ld, thakker n, farndon pa. complications of the naevoid basal cell carcinoma syndrome: results of a population based study. j med genet. 1993; 30: 460-4. 4. agaram np, collins bm, barnes l, lomago d, aldeeb d, swalsky p et al. molecular analysis to demonstrate that odontogenic keratocysts are neoplastic. arch pathol lab med. 2004; 128: 313-7. 5. tincani aj, martins as, andrade rg, franco jr efm, camargo mab, martins as. nevoid basal-cell carcinoma syndrome: literature review and case report in a family. sao paulo med j. 1995; 113: 917-21. 6. melo es, kawamura jy, alves ca, nunes fd, jorge wa, cavalcanti mg. imaging modality correlations of an odontogenic keratocyst in the nevoid basal cell carcinoma syndrome: a family case report. oral surg oral med oral pathol oral radiol endod. 2004; 98: 232-6. 7. lopes nn, caran em, lee ml, silva ns, rocha ac, macedo cr. gorlin-goltz syndrome and neoplasms: a case study. j clin pediatr dent. 2010; 35: 203-6. 8. visioli f, martins ca, heitz c, rados pv, sant’ana filho m. is nevoid basal cell carcinoma syndrome really so rare? proposal for an investigative protocol based on a case series. j oral maxillofac surg. 2010; 68: 903-8. 9. casaroto ar, loures dc, moreschi e, veltrini vc, trento cl, gottardo vd et al. early diagnosis of gorlin-goltz syndrome: case report. head face med. 2011; 2: 1-5. 10. pereira cm, lopes ap, meneghini aj, silva af, botelho tl. oral diffuse b-cell non-hodgkin’s lymphoma associated to gorlin-goltz syndrome: a case report with one year follow-up. indian j pathol microbiol. 2011; 54: 388-90. 11. kimonis ve, goldstein am, pastakia b, yang ml, kase r, digiovanna jj et al. clinical manifestations in 105 persons with nevoid basal cell carcinoma syndrome. am j med genet. 1997; 69: 299-308. 12. amlashi sf, riffaud l, brassier g, morandi x. nevoid basal cell carcinoma syndrome: relation with desmoplastic medulloblastoma in infancy. a populationbased study and review of the literature. cancer. 2003; 98: 618-24. 13. gorlin rj. nevoid basal-cell carcinoma syndrome. medicine (baltimore). 1987; 66: 98-113. 14. hasegawa k, amagasa t, shioda s, kayano t. basal cell nevus syndrome with squamous cell carcinoma of the maxilla: report of a case. j oral maxillofac surg. 1989; 47: 629-33. 15. yilmaz b, goldberg lh, schechter nr, kemp bl, ruiz h. basal cell nevus syndrome concurrent with adenoid cystic carcinoma of salivary gland. j am acad dermatol. 2003; 48: s64-6. 16. eslami b, lorente c, kieff d, caruso pa, faquin wc. ameloblastoma associated with the nevoid basal cell carcinoma (gorlin) syndrome. oral surg oral med oral pathol oral radiol endod. 2008; 105: e10-3. 17. lo muzio l. nevoid basal cell carcinoma syndrome (gorlin syndrome). orphanet j rare dis. 2008; 25: 32. 18. ponti g, pastorino l, pollio a, nasti s, pellacani g, mignogna md et al. ameloblastoma: a neglected criterion for nevoid basal cell carcinoma (gorlin) syndrome. fam cancer. 2012; 11: 411-8. 19. lo muzio l, nocini pf, savoia a, consolo u, procaccini m, zelante l et al. nevoid basal cell carcinoma syndrome. clinical findings in 37 italian affected individuals. clin genet. 1999; 55: 34-40. 20. woolgar ja, rippin jw, browne rm. the odontogenic keratocyst and its occurrence in the nevoid basal cell carcinoma syndrome. oral surg oral med oral pathol. 1987; 64: 727-30. 21. ahn sg, lim ys, kim dk, kim sg, lee sh, yoon jh. nevoid basal cell carcinoma syndrome: a retrospective analysis of 33 affected korean individuals. int j oral maxillofac surg. 2004; 33: 458-62. 22. gupta sr, jaetli v, mohanty s, sharma r, gupta a. nevoid basal cell carcinoma syndrome in indian patients: a clinical and radiological study of 6 cases and review of literature. oral surg oral med oral pathol oral radiol. 2012; 113: 99-110. 23. zecha ja, mendes ra, lindeboom vb, van der waal i. recurrence rate of keratocystic odontogenic tumor after conservative surgical treatment without adjunctive therapies a 35-year single institution experience. oral oncol. 2010; 46: 740-2. keratocystic odontogenic tumor related to nevoid basal cell carcinoma syndrome: clinicopathological study 2929292929 braz j oral sci. 12(1):23-29 24. boffano p, ruga e, gallesio c. keratocystic odontogenic tumor (keratocyst): preliminary retrospective review of epidemiologic, clinical, and radiologic features of 261 lesions from university of turin. j oral maxillofac surg. 2010; 68: 2994-9. 25. zhao yf, wei jx, wang sp. treatment of odontogenic keratocysts: a follow-up of 255 chinese patients. oral surg oral med oral pathol oral radiol endod. 2002; 94: 151-6. keratocystic odontogenic tumor related to nevoid basal cell carcinoma syndrome: clinicopathological study oral sciences n3 braz j oral sci. 14(2):112-116 original article braz j oral sci. april | june 2015 volume 14, number 2 pharyngeal airspace in patients undergoing orthognathic surgery for mandibular advancement guilherme dos santos trento1, fernando anunziato ogg de salles santos1, leandro eduardo klüppel1, delson joão da costa1, nelson luis barbosa rebellato1, rafaela scariot1,2 1universidade federal do paraná – ufpr, school of dentistry, department of stomatology, area of oral and maxillofacial surgery, curitiba, pr, brazil 2universidade positivo up, school of dentistry, area of oral and maxillofacial surgery, curitiba, pr, brazil correspondence to: rafaela scariot rua professor pedro viriato parigot de souza, 5300 curitiba pr brazil cep 81280-330 phone: +55 41 3317-3000 e-mail: rafaela_scariot@yahoo.com.br abstract aim: to evaluate the increase of pharyngeal airway space (pas) in patients undergoing mandibular advancement. methods: a retrospective cross-sectional study was performed in thirteen patients who underwent mandibular advancement and were evaluated by cephalometric tracing in pre and postoperative lateral radiographs. in cephalometric tracing, the pas was assessed by measuring the distance from the lower portion of the soft palate to the posterior pharyngeal wall (up-phw) and from the tongue base to the posterior pharyngeal wall (tb-phw). results: patients’ age ranged from 22 to 42 years with an average of 28.54 ± 2.23 years. a preoperative mean of 9.20 ± 4.56 mm in the up-phw measure and 10.53 ± 5.84 mm in the tb-phw measure were obtained. the mean values found for those measurements in the postoperative period were 11.61 mm and 13.95 mm, respectively. there was an average increase of 2.4 mm in the up-phw and of 2.95 mm in the tb-phw. the mean mandibular advancement in evaluated patients was 5 mm. there was no statistical correlation between pas increase and the amount of mandibular advancement for up-phw (p=0.058) and tb-phw (p=0.53), as there was no such correlation either between pas increase and the age of patients for up-phw (p=0.16) and tb-phw (p=0.26). a greater effect of the retrolingual dimension in mandibular advancement was observed, with an average increase of 24.52% while in the retropalatal dimension an average increase of 20.75% was obtained. conclusions: surgical advancement of the mandible increases the size of the pharyngeal airway space. keywords: mandible; pharynx; tongue; orthognathic surgery. introduction orthognathic surgery is one of the most used procedures for treatment and correction of dentofacial deformities. changes in facial skeleton produced by the surgery affect the bones of the facial skeleton and their relationship with soft tissues. an aspect to be considered is the impact of the skeleton movement on the pharyngeal airspace (pas) of patients undergoing orthognathic surgery1-2. in this context, it is observed that the movement of the facial skeleton produces changes in the tension of the soft tissues and muscles inserted in the jaw. soft palate, tongue, hyoid bone and associated musculature are directly or indirectly inserted in the jaw, therefore movements in those structures induce alterations in the oral and nasal cavity and on the pas1-5. studies1,4-5 have been conducted to quantify the regular measurements of pas, as well as the measurements in patients diagnosed with obstructive sleep received for publication: february 10, 2015 accepted: may 28, 2015 http://dx.doi.org/10.1590/1677-3225v14n2a03 113113113113113 apnea (osa). those studies are based on lateral cephalometric analysis1,6 and indicate that the most significant anatomic variation is the lower position of the hyoid bone and consequently the lower position of the tongue base in osa patients4-5,7. surgeries for mandibular advancement, bimaxillary advancement and counterclockwise rotation of the occlusal plane of the maxillomandibular complex promote the increase in pas bringing improvements in the respiratory status of patients with osa. arikasa et al.8, in 2014, argue that orthognathic surgery increases airway space and improves osa. faria et al. 1, in 2013, stated that mandibular advancement significantly provokes an advancement of the lingual and suprahyoid increasing the inter-maxillary space. mehra et al.9, in 2001, showed a 76% increase in the retrolingual and oropharyngeal air dimensions in relation to a certain amount of mandibular advancement. on the other hand, some studies 10-12 have reported that surgery for maxillomandibular advancement (7.5 mm mandibular advancement) with counterclockwise rotation (4.2º out of decrease in angulation of occlusal plane) increases 3.5 mm on the retropalatal region and 5.7 mm on the retrolingual region of the pas. given the potential for soft tissue adaptation and post-surgical recurrence of skeletal parts, it is important to know whether the changes produced by surgery in pas will remain stable over time. yu et al.13 conducted a retrospective study with data from 26 patients of orthognathic surgery in which there were 16 mandibular advancements and 10 maxillomandibular advancements. preoperative radiographs, immediate postoperative and late postoperative (15 months) lateral radiographs were evaluated. the patients were divided into two groups: group 1 comprised patients with preoperative pas measure greater than 11 mm, and group 2 those with preoperative pas measure under 11 mm. patients from group 1, who underwent mandible advancement surgery, showed a bigger change in the dimension of their airways with an average of 3.5 ± 3.2 mm aspect ratio of increased airway, and the mandibular advancement reached a 0.62:1 ratio in late postoperative period13. such results are similar to those found by other authors.10-12 patients from group 2 reported minor changes, with a mean 0.9 ± 3.6 mm increase in pas, measured in the late postoperative period, with a ratio between mandibular advancement and gain of airway dimension of approximately 0.19:113. within the aforementioned context, this study aims to quantify and correlate the changes of the pas in patients undergoing orthognathic surgery for mandibular advancement in the department of oral and maxillofacial surgery of the federal university of paraná. material and methods sample selection records of 13 patients from the department undergoing orthognathic surgery of mandibular advancement were selected in descending chronological order. in order to be part of the sample, the patient had to present mandibular retrognathia and to have undergone orthognathic surgery to advance the mandible with bilateral sagittal split osteotomy and stable internal fixation with plates and/or screws. the records should contain pre and postoperative lateral radiographs of the patients in the study. this study was approved by the ethics and research committee of the department of health sciences, federal university of paraná, brazil, with registration cep/sd: 934.059.10.05 and caae: 0034.0.91.000-10. inclusion criteria patients with mandibular retrognathia who underwent orthognathic surgery for mandibular advancement with bilateral sagittal split osteotomy and stable internal fixation with plates and/or screws. the records should contain pre and postoperative lateral radiographs of the patients included in this study. exclusion criteria patients who underwent orthognathic surgery for mandibular advancement with vertical osteotomy. records that do not contain pre and postoperative lateral radiographs or contain low-quality radiographs leading to an erroneous diagnosis. data collection a single researcher collected the data. in all the charts, data such as gender and age of the patient included in the study were evaluated. data were tabulated in a sheet for frequency analysis. in addition, lateral radiographs from the medical records of the patients were evaluated (one week before surgery and three months after surgery). the lateral radiographs were obtained by an orthophos® radiological equipment model (siemens ag, berlin, germany) in the dental radiology department of the federal university of paraná. all the radiographs were taken with the patient in constant head position with the frankfurt plane parallel to the ground. the radiographic interpretation was performed in a dark room with an appropriate light box. the radiographs were evaluated by the performance of cephalometric tracing while outlining craniofacial structures and the cephalometric points of interest for the assessment of changes introduced by orthognathic surgery in pas. the pre and postoperative radiographs were drawn three times with a one-week interval between measurements in order to reduce the memory bias. in order to evaluate the reliability of the obtained data, the intraclass correlation coefficient was calculated (cci=0.977) demonstrating that the measurements were reliable for the research. the cephalometric tracing was performed by outlining all the important craniofacial structures for the study. then, we assessed the amount of mandibular advancement produced by measuring the distance between the pogonion point and the nasion perpendicular line in pre and postoperative radiographs. pharyngeal airspace in patients undergoing orthognathic surgery for mandibular advancement braz j oral sci. 14(2):112-116 114114114114114 the dimension of the pas was evaluated by two lines parallel to the frankfurt plane, one passing by the gonion (go) while intersecting the base of the tongue and the posterior pharyngeal wall (phw-tb), and another drawn from the lower portion of the soft palate to the posterior pharyngeal wall (up-phw) (figure 1). afterwards, the anteroposterior dimension of the pas from those lines on lateral radiographs was measured with a digital caliper (vonder®, curitiba, pr, brazil). statistical analysis data were recorded on individual sheets and tabulated using microsoft excel 2007® software for windows xp. furthermore, correlations between variables were performed with statistical tests (spss for windows 15.0 – spss inc., chicago, il, usa) with a confidence interval of 95%. fig 1. points and reference lines used in the study. phw = pharyngeal wall; up = lower portion of the soft palate; tb = tongue base. results thirteen patients, twelve female and one male were included in the survey. patients’ age ranged from 22 to 42 years with an average of 28.54 ± 2.23 years. no statistically significant correlation was observed between the increase of pas and the age of patients undergoing surgery for mandible advancement in up-phw (p = 0.16) and tb-phw (p=0.26) measurements by the pearson’s correlation coefficient. the values found for pas to measure up-phw and tbphw, both pre and postoperatively, and the amount of mandibular advancement is shown in table 1. the pas average for up-phw was 9.20 ± 4.56 mm and 11.61 ± 5.14 mm pre and postoperatively, respectively. in the preoperative period, the pas average for tb-phw was 10.53 ± 5.84 mm. the postoperative tb-phw mean was 13.95 mm (11.97 – 15.01). a greater effect in the retrolingual dimension was observed by mandibular advancement with an average of 24.52%, while an average increase of 20.75% was obtained in the retropalatal measure. the mean mandibular advancement in patients undergoing surgery was 5 mm (4 8). the average increase of the upphw dimension was 2.4 ± 0.4 mm and for the tb-phw measurement it was 2.95 ± 0.5 mm. there was no statistically significant correlation between mandibular advancement and pas increase in both the up-phw (p=0.058) and tb-phw (p=0.53) measurements by the spearman correlation coefficient. the impact of surgical advancement of the mandible on the pas is very variable and unpredictable, because patients with larger mandibular advancement do not always have the greatest impact on pas. gender, age, amount of advancement of the mandible and the difference in pas for both measurements are shown in table 2. discussion dentofacial deformities are congenital or growth malformations during the development which may cause functional and aesthetic problems2-3. changes in the airways after orthognathic surgery have been the subject of great interest in recent years1,4,8,13. some studies have shown that repositioning of both the maxilla and mandible generate changes in pharyngeal airspace 10,13. moreover, cases of respiratory disorders after mandibular setback have been reported related to narrowing of the pharyngeal airspace1. the pas may become reduced under various conditions including obesity, hypertrophy of the tonsils, adenoids, macroglossia and decrease in the size of the jaws. a narrow pas causes increased resistance to airflow accounting for possible respiratory disorders including obstructive sleep apnea and snoring14. the final study sample included twelve females and one male patient, which made it impossible to conduct any statistical test to compare the effects of mandible advancement surgery in different genders. the average age of patients participating in the survey was 28.54 years; however, there was no statistically significant correlation between changes in pas and the age at which patients underwent the procedure, probably due to the small amplitude of the age range of patients included in the study. rilley et al.10, in 1993, reported an increase of the pas dimension with an average of 5 to 6 mm after maxillomandibular advancement surgery in a group of patients with obstructive sleep apnea. mehra et al.9, in 2001, reported 76% increase in the retrolingual and oropharyngeal dimensions related to the amount of mandibular advancement, compared to other studies10-12 which related an average increase of 42% to 51% in the same dimensions and to the current study that obtained an average increase of 20.75% in the retropalatal dimension (up-phw) and of 24.52% in the retrolingual dimension (tb-phw). pharyngeal airspace in patients undergoing orthognathic surgery for mandibular advancement braz j oral sci. 14(2):112-116 table 1. table 1. table 1. table 1. table 1. pre and postoperative up-phw and tb-phw measurements with the respective amount of mandibular advancement. spearmen correlation coefficient between pas increase and the amount of mandibular advancement: up-phw (p=0.058) and tb-phw (p=0.53) patient preoperative postoperative amount of mandibular up-phw tb-phw up-phw tb-phw advancement ( m m ) ( m m ) ( m m ) ( m m ) ( m m ) 1 12.2 14.34 11.9 14.9 4 2 8.99 10.44 8.16 9.4 4 3 9.98 10.85 14.69 15.77 7 4 9.35 11.72 13.93 14.66 8 5 7.4 7.99 10.75 13.23 5 6 10.91 12.98 13.96 17.16 5 7 8.89 9.79 11.76 14.56 7 8 8.45 10.66 9.91 13.43 6 9 6.97 6.02 9.77 7.48 7 10 11.4 11.52 12.37 13.75 4 11 6.9 9.3 10.58 13.28 4 12 9.78 11.16 11.91 13.95 5 13 8.43 11.59 11.25 14 7 patient gender age (years) amount of mandibular up-phw tb-phw advancement mean difference mean difference ( m m ) ( m m ) ( m m ) 1 f 22 4 -0.12 1.24 2 f 34 4 -0.98 -1.61 3 f 42 7 4.86 5.1 4 f 35 8 4.24 2.54 5 f 42 5 3.3 5.06 6 f 28 5 2.83 4.94 7 f 23 7 2.58 4.78 8 f 18 6 1.13 3.3 9 f 18 7 3.1 1.3 10 f 27 4 1.28 0 11 f 30 4 2.92 4 12 m 22 5 1.45 2.74 13 f 30 7 2.44 2.43 table 2. table 2. table 2. table 2. table 2. pre and post operative mean difference of the up-phw/tb-phw measurements and the amount of mandibular advancement according to gender. pearson correlation coefficient between pas increase and patients’ age: up-phw (p=0.16) and tb-phw (p=0.26) the mean mandibular advancement in patients in the survey was 5 mm, resulting in a 2.40 mm average increase of the up-phw and of 2.95 mm in the tb-phw. these results contrast with the findings of yu et al.13, in 1994, which had an average increase in the pas size of 3.5±3.2 mm in patients with a pas preoperative measurement under 11 mm, and of 0.9±3.6 mm in patients with a pas preoperative measure greater than 11 mm. changes in pharyngeal airway space after mandibular setback surgery are controversial since some studies show changes in the airways space dimension, but do not affect their capacity15-16. in most studies1,6 pas was measured by radiograph in lateral view in which the position of the head (craniofacial angulation) was not always constant. additionally, the lateral radiographs show a two-dimensional aspect of the patient. mandibular advancement surgery has a greater impact on lower airways, whereas patients showed more changes in airways space17. this probably occurs because the tension of the suprahyoid muscles and other muscles inserted in the jaw causes a greater shift in the lower pharyngeal airway space. furthermore, the inter-maxillary dimension increases and the tongue proportion decreases leading to an increase of the tongue space for its function, resulting in a more anterior position while extending the retroglossal airway. according to the data, age presented no influence on the relationship between the amount of mandibular advancement and pas changes. moreover, skeletal alterations produced by mandibular advancement surgery have repercussions on pas dimensions, particularly on the retrolingual dimension. however, it was not found significant association between the amount of mandibular advancement and the changes produced in the pas, both for the retropalatal and for retrolingual measures. 115115115115115 pharyngeal airspace in patients undergoing orthognathic surgery for mandibular advancement braz j oral sci. 14(2):112-116 116116116116116 references 1. faria ac, xavier sp, silva jr sn, voi trawitzcki lv, de mello-filho fv. cephalometric analysis of modification of pharynx due to maxillamandibular advancement surgery in patients with obstructive apnea. int j oral maxillofac surg. 2013; 42; 579-84. 2. freire ef, freire filho fwv, valdrighi hc, degan vv, vedovello sas. changes in pharyngeal airway space and soft tissue after maxillary advancement and bimaxillary surgery. braz j oral sci. 2014; 13: 93-7. 3. soh cl, narayanan v. quality of life assessment in patients with dentofacial deformity undergoing orthognathic surgery—a systematic review. int j oral maxillofac surg. 2013; 42: 974–80 4. zinser mj, zachow s, sailer hf. bimaxillary ‘rotation advancement’ procedures in patients with obstructive sleep apnea: a 3-dimensional airway analysis of morphological changes. int j oral maxillofac surg. 2013; 42: 569-78. 5. candido msc, monazzi ms, gabrielli mac, spin-neto r, gabrielli mfr, pereira-filho va. pharyngeal airway space cephalometric evaluation in transverse maxillary deficient patient after sarme. braz j oral sci. 2014; 13: 288-91. 6. kim s, kim k, park j, kim s. cephalometric predictors of therapeutic responde to multilevel surgery in patients with obstructive sleep apnea. j oral maxillofac surg. 2012; 70: 1404-12. 7. mickelson sa. hyoid advancement to the mandible (hyo-mandibular advancement). oper tech otolaryngol. 2012; 23: 56-9. 8. arikasa t, ito c, sato k, tonogi m, yamane g, nakajima t. examination of changes in the pharyngeal airway space under anterior traction of the mandible: influence of detachment of the periosteum during orthognathic surgery. j oral maxillofacial surg med pathol. 2014; 26: 540-4. 9. mehra p, downie m, pita mc, wolford lm. pharyngeal airway space changes after counterclockwise rotation of the maxillomandibular complex. am j orthod dentofacial orthop. 2001; 120: 154-9. 10. riley rw, powell nb, guilleminault c. maxillary, mandibular, and hyoid advancement for treatment of obstructive sleep apnea: a review of 40 patients. j oral maxillofac surg. 1990; 48: 20-6. 11. li kk, guilleminault c, riley rw, powell nb. obstructive sleep apnea and maxillomandibular advancement: an assessment of airway changes using radiographic and nasopharyngoscopic examinations. j oral maxillofac surg. 2002; 60: 526-31. 12. farole a, mundenar mj, braitman le. posterior airway changes associated with mandibular advancement surgery: implications for patients with obstructive sleep apnea. int j adult orthodont orthognath surg. 1990; 5: 255-8. 13. yu lf, pogrel am, ajayi m. pharyngeal airway changes associated with mandibular advancement. j oral maxillofac surg. 1994; 52: 40-4. 14. mannarino m, di filippo f, pirro m. obstructive sleep apnea syndrome. eur j intern med. 2012; 23: 586-93. 15. park j, kim n, kim j, kim m, chang y. volumetric, planar, and linear analyses of pharyngeal airway change on computed tomography and cephalometry after mandibular setback surgery. am j orthod dentofacial orthop. 2010; 138: 292-9. 16. fernandez-ferrer l, montiel-company j, pinho t, almerich-silla a, bellotarcís c. effects of mandibular setback surgery on upper airway dimensions and their influence on obstructive sleep apnoea a systematic review. j craniomaxillofacl surg. 2015; 43: 248-53. 17. becker o, avelar r, göelzer j, dolzan a, haas júnior o, oliveira r. pharyngeal airway changes in class iii patients treated with double jaw orthognathic surgery – maxillary advancement and mandibular setback. j oral maxillofac surg. 2012; 70: 639-47. pharyngeal airspace in patients undergoing orthognathic surgery for mandibular advancement braz j oral sci. 14(2):112-116 oral sciences n3 original article braz j oral sci. april | june 2013 volume 12, number 2 influence of plaster drying on the amount of residual monomer in heat-cured acrylic resins tarcisio josé de arruda paes-junior1, rodrigo furtado de carvalho1, sâmia carolina mota cavalcanti1, guilherme de siqueira ferreira anzaloni saavedra1, alexandre luiz souto borges1 1department of dental materials and prosthodontics, school of dentistry, institute of science and technology, unesp – univ estadual paulista, são josé dos campos, sp, brazil correspondence to: tarcisio josé de arruda paes-junior avenida josé longo, 777, são dimas cep: 12245-000 são josé dos campos, sp, brasil phone: +55 12 39479371 e-mail: tarcisio@fosjc.unesp.br received for publication: february 04, 2013 accepted: march 21, 2013 abstract aim: to evaluate the influence of plaster condition, dry or not, on the amount of residual monomer in heat-cured acrylic resin. methods: thirty acrylic resin specimens (65x10x3 mm) were fabricated and randomly assigned to 5 groups (n=6). the evaluated resins were heat-cured acrylic resins by conventional or microwave polymerization techniques and the plaster was previously dried in microwave oven in two groups. each specimen was individually immersed in a test tube containing methanol (7 days) for surface analysis. in the groups for which internal monomer was evaluated, the specimens were fragmented and the small fragments were weighed prior to immersion in methanol. the analysis was made by high performance liquid chromatography (hplc). data were analyzed by anova and tukey test (p<5%) results: showed statistical differences among the groups. conclusions: the previous plaster drying influenced the residual monomer amount showing a decrease of these levels. keywords: acrylic resins, polymers, chromatography, laboratory research. introduction several materials have been consolidated due to their broad use in several areas of dentistry1. acrylic resin is one of the most typical examples, which since the 1940’s has been established as the material of choice in the preparation of prosthetic works, such as complete and partial removable dentures2. advantages such as biocompatibility, no taste and odor, adequate thermal properties, dimensional stability, and simple technique are prevalent in these indications3-4. although its extensive use has established defined criteria concerning its polymerization, especially in the case of hot water bath, some questions persist regarding the procedure for triggering the polymerization by microwave energy and the effects on the properties of the final product. ilbay et al.5 (1994), focusing on the mechanism of action of microwave energy, described that it causes the vibration of water molecules in a substance two to three billion times per second and that it produces friction that results in the heating of substance. those authors demonstrated that microwaves cause the molecules of the acrylic resin (monomer) to vibrate with higher frequency, creating friction and the heat that triggers the polymerization5. studies on the heating of the monomer molecules that determine the formation of polymer chains by microwaves do not explain whether this process is incremented or not by heating the water contained in the plaster3,5. de clerck6 (1987) found braz j oral sci. 12(2):84-89 8585858585 group acrylic resin plaster drying polymerization 1 a lucitone 550 n o microwave 2 a vipi-wave n o microwave 1 b lucitone 550 yes microwave 2 b vipi-wave yes microwave 3 lucitone 550 n o water bath table 1groups set based on the type of resin, plaster and condition of polymerization cycle that when the monomer’s boiling temperature (100.8°c) is reached, there is formation of pores in the resin, which is more easily achieved when the heat generated by the resin cannot be released. verifying the factor related to the condition of plaster, studies have proposed to see how different types of plaster behave when submitted to drying in a microwave oven4,7-8. luebke and chan8 (1985) reported an increase in surface hardness in some of the tested dental plasters when microwave drying was performed. berg et al.9 (2007) assessed the degree of disinfection of dental casts by means of microwave, and noticed that performing a cycle of 5 min at 900w resulted in an effective disinfecting of these models without dimensional changes or macroscopic defects. one of the points of major importance in the study of acrylic resins lies on analyzing the amount of monomer present in the polymerized material, which is not converted during the resin processing and can determine tissue damage, such as irritations or allergic reactions in oral tissues, and also cause undesirable changes in the mechanical properties of the material10. according to international standards, the content of monomer should not exceed 2.2% for heat-cured resin11. urban et al.12 (2007) compared the effect of the amount of residual monomer when acrylic resin was subjected to post-polymerization by immersion in hot water bath and found similar reduced values of residual monomer for conventional or microwaving techniques. it is questionable if the water contained in the plaster would influence in the resin process because in the conventional process the heat reaches the mass of resin indirectly, requiring first to heat the flask and the cast. using microwave a dry plaster provides a more effective degree of polymerization. then, the objective of this research was to determine whether previous plaster drying when polymerized by microwaves affects the superficial and internal amount of monomer. material and methods preparation of specimens two types of acrylic resins, one heat-cured in water bath (lucitone 550; dentsply ind. com ltda, petrópolis, rj brazil) and the other cured by microwave (vipi-wave; dental-vipi ltda., pirassununga, sp, brazil) were used in this study. rectangular stainless steel bars (12.6x67.0x3.0 mm) with sharp edges was included in fiberglass reinforced microwave flask (vipi-stg; dental-vipi ltda.), which was also used in the hot water bath in the conventional technique of acrylic resin polymerization. the process was initiated by embedding the base of the flask in freshly hand-mixed type ii plaster (plaster-rio; bussioli me, rio claro, sp, brazil) and then a condensation silicone (vipi-sil; dental-vipi ltda.) was applied around the metallic bars and proceeded to fill the flask with a new quantity of plaster. five groups (table 1) were formed according to type of acrylic resin, plaster drying and polymerization and, after 1h, the metallic bars were removed and the acrylic resin was prepared according to the manufacturer’s recommendations and placed in the flask when reached the plastic phase. after that, the flask was kept closed for 30 min under pressure, for all groups. for the control group the polymerization process was undertaken using a long cycle in water bath when the flasks were positioned in cool water, then taken to 72ºc for 9 h and next the temperature was increased to 100ºc for 1 hour. groups 1a, 2a, 1b, 2b, in which the proposal was the previous drying of plaster, the methodology described by hersek et al.13 (2002) was used proceeding the drying in a microwave oven for 10 min at 600 w, where parts of the flask were kept separate during the cycle. after this cycle, flasks were left at room temperature for 1 h until complete cooling was reached. the flask parts were kept open and were stored in an oven at 37oc for 2 hours for drying and then the polymerization method was established. for the microwave polymerization a continental aw30 microwave oven (bs continental amazonia ind. e com., manaus, am, brazil) was used with a turntable plate, frequency of 2450 mhz and maximum power of 900 w, and the cycle was to maintain 20% of the device power for 20 min, followed by an additional cycle at 60% of the power for another 5 min. completing the polymerization cycles proposed for each group, the flask was kept for about 2 h at room temperature until complete cooling. analysis of residual monomer for the analysis of the surface residual monomer, glass test tubes with lids were filled with 20 ml of methyl alcohol pa methanol. one specimen was immersed in each of the tubes and was kept in this condition at approximately 2°c for 7 days. after this period, the specimens were removed from test tubes and the residual monomer in the solvent was quantified. high performance liquid chromatography (hplc) was used with a chromatographer ls 10 ad (dionex corporation, sunnyvale, ca, usa), column rp-18 250 mm long by 2.5 mm diameter, attached to a detector uv-vis (dionex corporation), at a wavelength of 225 nm. calibration curve for the pure monomer (liquid) was determined from each of the tested resins. regarding the analysis of groups, just as for the preparation of the calibration curve, from each test tube it was withdrawn an aliquot of 20 µl. the area was identified numerically and for each monomer concentration three samples were injected and the average value of these areas was calculated. with the average value of the areas for all checked influence of plaster drying on the amount of residual monomer in heat-cured acrylic resins braz j oral sci. 12(2):84-89 dilutions (chromeleon version 6.70 (chromeleon inc., sunnyvale, ca, usa) program did the mathematical calculation and rendered the area number for the unit of measurement in µg/ml. calculation of values for each sample was divided by this constant measure of surface area, which allowed obtaining results in µg/cm2 (figure 1). for the analysis of the internal monomer the polymerized material was reduced to small particles, to enable greater contact of the substance and solvent (methanol). specimens previously weighed as described above (n=3) were targeted without warming the material during this process. the mass of the particulate material was recorded on a digital precision scale and the results varied from 0.540 mg to 0.650 mg. this was put into test tubes with lids, containing methanol, similar to that described for the analysis of residual monomer surface. after 7 days the particulate material was removed from the solvent and the monomer quantified. the figures were compiled, distributed and assessed comparatively by one-way anova and tukey’s test at 5% significance level (statistic for windows program, statsoft south america, são caetano do sul, sp, brazil). results there were statistically significant differences among groups (p<0.05), with group 3 (control) showing the lowest levels of residual surface monomer (table 2), but no significant was found among the other groups. among the studied groups, one that showed higher values of surface monomer was group 2a (vipi-wave) in the fig. 1. chromatogram for the sample of vipi-wave of methyl methacrylate for both groups. groups*(µµµµµg/cm 2) 1 a 2a 1b 2b 3 11.91+0.89 b 39.54+ 7.18 c 7.75+0.83 ab 10.46+3.16 ab 3.46+0.51 a table 2means, standard deviations and tukey’s test results for superficial residual monomer numbers in µg/cm2 for the analyzed groups different lowercase letters in the row reveal differences among the means. microwave cycle set by the manufacturer. the chart in figure 2 illustrates average values for the surface monomer. the results for the internal monomer showed statistically significant difference (p<0.05) only between group 2a and the other groups, with 2a showing the highest values. the values and averages of groups, including tukey test results, are shown in tables 3 and 4 and figure 3. total values in ìg of the percentages in each internal monomer of all groups are shown in table 5. discussion several studies have been focusing on this analysis by considering the influence of aspects such as methods and polymerization cycles and also storage time of the polymerized material14-15. another point discussed in literature involves tissue behavior in excessive amounts of free monomer and in the oral environment, which explain irritation of smaller proportions and the existence of severe hypersensitivity reactions to this product16. based on the idea of de clerck6 (1987) who claimed that it is relevant for effective polymerization that during a microwave polymerization cycle the plaster contained in the flask is rather dry, it was considered important to analyze the possible effects of it in the final outcome of work, focusing on assessing the amount of residual monomer. it is known that the microwave enables the vibration of molecules of liquids, mainly water17. according to nishii18 (1968), the monomer molecules present in the mass of the resin are agitated by the electromagnetic wave generated by 8686868686 influence of plaster drying on the amount of residual monomer in heat-cured acrylic resins braz j oral sci. 12(2):84-89 8787878787 fig. 2. means and standard deviations for the amount of surface residual monomer in µg/cm2. fig. 3. means and standard deviations for the amount of internal residual monomer in µg/ml. the device, and the friction of these molecules would promote the release of heat required to trigger the conversion of monomer and forming polymer, hence, new polymer chains5-6. there was a decrease in the quantity of surface monomer, when the plaster was dried prior to resin pressing. both resins, one formulated for microwave use and the other for influence of plaster drying on the amount of residual monomer in heat-cured acrylic resins braz j oral sci. 12(2):84-89 monomer µg/cm2 monomer µg/cm2 8888888888 sample µµµµµ g / m l groups 1 a 2 a 1 b 2 b 3 i 14.52 71.75 17.00 8.86 7.67 ii 25.44 57.60 13.90 13.92 6.77 iii 19.03 35.46 11.83 10.79 12.46 means 19.66 54.93 14.24 11.19 8.96 sd 5.48 18.29 2.60 2.55 3.05 table 3means and standard deviations (s.d.) for the amounts of internal residual monomer in µg/ml samples groups 1 a 2 a 1 b 2 b 3 total mass (mg) 630 540 607 629 627 i(%) 0.04 0.26 0.05 0.02 0.02 total mass(mg) 613 603 643 649 631 ii(%) 0.08 0.18 0.04 0.04 0.01 total mass(mg) 640 563 644 655 647 iii(%) 0.06 0.12 0.03 0.03 0.03 table 5total values in µg of the percentages in each internal monomer samples conventional water bath, were used in the microwave oven. in this sense, it may be hypothesized that for microwave processing the amount of water plaster does not aid in the polymerization process. on the contrary, it interferes partially with the polymerization, probably “absorbing” a part of the electromagnetic energy, which act on the molecules of the monomer, confirming the indicative data of de clerck6 (1987). the importance of quantifying the monomer by hplc chromatography lies in the possibility of accurately verifying differences in these quantities, even though, from the clinical point of view, for the type of used materials, the values obtained are within acceptable standards for use of acrylic resin. several authors used this approach and found similar results19. other aspects are relevant in this analysis, which proved the possibility of microwave polymerization of a conventional heatcured resin, presenting similar monomer values to those found for the resin formulated for microwave when the plaster was dry. these data corroborate with the studies of lombardo et al.20 (2012) which confirmed the possibility of polymerizing in microwave a conventional heat-cured resin, such as lucitone 550. for the analysis of internal monomer, the variation of individual values of the samples was higher than the quantification of the surface monomer. this may possibly be explained by the larger number of different segments of the resin which were immersed in methanol and resulting in a greater heterogeneity of surface areas in contact with the solvent. the control group had lower levels of residual monomer than the groups polymerized by microwave. other works presented a similarity of concentrations for water bath cycles and microwave; although when considering the time factor, the microwave cycle proposed by the manufacturer of vipi wave resin and applied to the other groups, it was a total of 25 min and did not present greater values of residual monomer than the control group, which used the long cycle in warm water for a total of 13 h. these data agree with the results obtained by urban et al.12(2007), jorge et al.21 (2003), zissis et al.22 (2008), who found low levels of residual monomer for heat-cured resins regardless of the polymerization method. another important and justified concern is about the relationship between the setting time and power for better polymerization. same researchers agree on the fact that starting a microwave cycle with lower power and longer time and ending it with increased power and decreased time, the polymerization becomes more effective17. data obtained in this study showed variations that may be an indication of possible changes in the characteristics of the material when considering increases in the thickness of acrylic resin, because it is known that there is a correlation between degree of polymerization and amount of free monomer on the mechanical properties, such as hardness, transverse resistance and roughness17. therefore, other studies are required involving plaster drying, in order to verify whether there is variation in the behavior of the resin when considering other properties, such as flexural strength, porosity, dimensional stability, toughness and other mechanical characteristics. it is possible to conclude that: plaster drying prior to the resin polymerization resulted in a decreased amount of monomer, regardless of acrylic resin brand tested; regarding the amount of internal residual monomer, plaster drying caused a significant decrease of these values for the groups of vipi-wave resin; the conventional polymerization of acrylic resin in hot water bath (control group) determined the lowest levels of surface residual monomer; the polymerization of the resin lucitone in microwave was feasible, presenting low levels of surface and internal residual monomer, a fact that corroborates the use of microwave cycle for this resin as a reliable alternative to polymerization. acknowledgments foundation for development of unesp-fundunesp for financial support. vera lucia tedeschi savoy, dds são paulo biological institute references 1. baydas s, bayindir f, akyil ms. effect of processing variables (different compression packing processes and investment material types) and time on the dimensional accuracy of polymethyl methacrylate denture bases. dent mater j. 2003; 22: 206-13. influence of plaster drying on the amount of residual monomer in heat-cured acrylic resins braz j oral sci. 12(2):84-89 table 4results of tukey test for the values of surface residual monomer µg/cm2 for the analyzed groups * different lowercase letters in the rows reveal differences among the averages. 1 a 2 a 1 b 2 b 3 19.66 a 54.93 b 14.24 a 11.19 a 8.96 a 8989898989 2. kalsi hj, wang yj, bavisha k, bartlett d. an audit to assess the quality and efficiency of complete and partial dentures delivered by junior hospital staff. eur j prosthodont restor dent. 2010; 18: 8-12. 3. yannikakis s, polychronakis n, zissis a. temperature rise during intraoral polymerization of self-cured hard denture base liners. eur j prosthodont restor dent. 2010; 18: 84-8. 4. lopes mc, consani rl, mesquita mf, sinhoreti ma, consani s. effect of monomer content in the monomer-polymer ratio on complete denture teeth displacement. braz dent j. 2011; 22: 238-44. 5. ilbay sg, guvener s, alkumru hn. processing dentures using a microwave technique. j oral rehabil. 1994; 21: 103-9. 6. de clerck jp. microwave polymerization of acrylic resins used in dental prostheses. j prosthet dent. 1987; 57: 650-8. 7. ghani f, kikuchi m, lynch cd, watanabe m. effect of some curing methods on acrylic maxillary denture base fit. eur j prosthodont restor dent. 2010; 18: 132-8. 8. luebke rj, chan kc. effect of microwave oven drying on surface hardness of dental gypsum products. j prosthet dent. 1985; 54: 431-5. 9. berg e, nielsen o, skaug n. efficacy of high-level microwave disinfection of dental gypsum casts: the effects of number and weight of casts. int j prosthodont. 2007; 20: 463-4. 10. melilli d, currò g, perna am, cassaro a. cytotoxicity of four types of resins used for removable denture bases: in vitro comparative analysis. minerva stomatol. 2009; 58: 425-34. 11. yilmaz h, aydin c, caglar a, yasar a. the effect of glass fiber reinforcement on the residual monomer content of two denture base resins. quintessence int. 2003; 34: 148-53. 12. urban vm, machado al, oliveira rv, vergani ce, pavarina ac, cass qb. residual monomer of reline acrylic resins. effect of water-bath and microwave post-polymerization treatments. dent mater. 2007; 23: 363-8. 13. hersek n, canay s, akça k, ciftçi y. tensile strength of type iv dental stones dried in a microwave oven. j prosthet dent. 2002; 87: 499-502. 14. consani rl, monteiro vl, mesquita mf, consani s. the influence of storage on dimensional changes in maxillary acrylic denture bases and the effect on tooth displacement. eur j prosthodont restor dent. 2011; 19: 105-10. 15. rizzatti-barbosa cm, ribeiro-dasilva mc. influence of double flask investing and microwave heating on the superficial porosity, surface roughness, and knoop hardness of acrylic resin. j prosthodont. 2009; 18: 503-6. 16. de andrade lima chaves c, machado al, vergani ce, de souza rf, giampaolo et. cytotoxicity of denture base and hard chairside reline materials: a systematic review. j prosthet dent. 2012; 107: 114-27. 17. lai cp, tsai mh, chen m, chang hs, tay hh. morphology and properties of denture acrylic resins cured by microwave energy and conventional water bath. dent mater. 2004; 20: 133-41. 18. nishii m. curing of denture base resins with microwave irradiation: with particular reference to heat-curing resins. j osaka dent univ. 1968; 2: 23-40. 19. urban vm, machado al, vergani ce, giampaolo et, pavarina ac, de almeida fg, et al. effect of water-bath post-polymerization on the mechanical properties, degree of conversion, and leaching of residual compounds of hard chairside reline resins. dent mater. 2009; 25: 662-71. 20. lombardo ce, canevarolo sv, reis jm, machado al, pavarina ac, giampaolo et, et al. effect of microwave irradiation and water storage on the viscoelastic properties of denture base and reline acrylic resins. j mech behav biomed mater. 2012; 5: 53-61. 21. jorge jh, giampaolo et, machado al, vergani ce. cytotoxicity of denture base acrylic resins: a literature review. j prosthet dent. 2003; 90: 190-3. 22. zissis a, yannikakis s, polyzois g, harrison a. a long term study on residual monomer release from denture materials. eur j prosthodont restor dent. 2008; 16: 81-4 influence of plaster drying on the amount of residual monomer in heat-cured acrylic resins braz j oral sci. 12(2):84-89 oral sciences n3 original article braz j oral sci. january | march 2015 volume 14, number 1 streptococcus mutans adhesion and releasing of metallic ions in dental alloys adriana cristina zavanelli1, ricardo alexandre zavanelli2, josé vitor quinelli mazaro1, rosse mary falcón-antenucci1 1universidade estadual paulista – unesp, araçatuba dental school, department of dental materials and prosthodontics, araçatuba, sp, brazil 2universidade federal de goiás – ufg, dental school, department of oral rehabilitation, goiania, go, brazil correspondence to: adriana cristina zavanelli. rua josé bonifácio, 1193 – vila mendonça cep 16015-050 araçatuba, são paulo, brasil phone/fax: + 55 18 3636-3246/3636-3245 e-mail: zavanelliac@foa.unesp.br abstract aim: to evaluate the adherence of streptococcus mutans to the surface of the amalgam and copper/aluminum alloy samples and also evaluate the release of metallic ions. methods: the prepared medium was changed every 72 h and analyzed by atomic absorption spectrophotometer. samples were removed from the prepared medium at 15, 30, 48 and 60 days. results: the result shows that ions released were statistically different among all groups, and so were both biofilm and pits formation and the corrosion induced by the s. mutans in both types of samples. sem observation of the samples immersed in the prepared medium with s. mutans showed adherence of microorganisms on the whole surface, in all groups. conclusions: the s. mutans adhere to both amalgam and copper/aluminum alloy causing corrosion of those restorations. s. mutans produced a greater ions release in cu/al alloy; in amalgam, the ions release was not influenced by exposure to s. mutans. keywords: dental alloys; corrosion; ions. introduction biocorrosion in dentistry is the classic electrochemical corrosion induced by the biofilm1, which is a complex aggregation of microorganisms growing on a solid substrate2. dental biofilm also known as dental plaque is usually disastrous. it colonizes and also contaminates not only dental surfaces but also restorations, metallic surfaces of prostheses and implants3-5, causing corrosion in pits, in a similar way as demineralized areas and decalcified cavities on tooth enamel. although metal-free restorations are more popular nowadays, metal restorations such as amalgam and copper/aluminum alloy are still being used widely at universities, national health services and some practices all over the world6-8. copper/aluminum alloy and amalgam restorations still face the problem of corrosion resulting in dissatisfied patients regarding the aesthetics and also in the longevity of those restorations. doubts still remain regarding the deleterious effects on the properties of the metallic surfaces of these alloys and their resistance to corrosion9-10. it is known that restoring materials should be resistant to corrosion to avoid biological effects caused by it and also to avoid jeopardy to esthetics3,10-12. however, in oral environment, these restorations are exposed to certain conditions directly related to ions release, such as ph reduction13 caused by streptococcus mutans (s. mutans) after liquids and food intake14. the exposure of alloys to ph reduction intensifies metallic ions bleaching to tissues of surrounding oral mucosa10,14 and more pronounced in nickel alloys10,13-14. according to wataha et al.15 the metallic ions released from alloys can be braz j oral sci. 14(1):36-40 received for publication: december 12, 2014 accepted: march 06, 2015 3737373737 toxic, cause inflammatory, allergenic and mutagenic reactions and can also irritate adjacent tissues. however, the toxicity of metal-ceramic alloys depends on quantity and quality of the metallic ions released, possible synergistic or antagonist effects, and the time they remain in contact with organic tissues9,16-17. the challenge now is to focus on this association in trying to prevent the colonization by the microorganisms and consequent corrosion. hence, the aim of this in vitro study was to evaluate the adherence of s. mutans to the surface of the amalgam and copper/aluminum alloy samples and also the release of metallic ions such as copper, nickel, iron, zinc, silver, manganese, tin, aluminum and mercury on those samples when colonized by s. mutans. material and methods sample preparation amalgam velvalloy samples (s. s. white s.a., rio de janeiro, rj, brazil) were prepared in accordance to the manufacturer’s recommendations, triturated in the silamat amalgamator (silamat s6, ivoclar vivadent inc., amherst, ny, usa) and hand-condensed into a circular stainless steel matrix (12 mm x 3 mm). excesses were removed and burnished 5 min after condensation. after 25 min, samples were removed and stored in oven at 37 oc with 100% relative humidity for 24h. four samples were produced for each studied material. two samples were prepared for the observation of biofilm formation and the other two for the observation of pit formation. they were then divided into the following groups: group i burnished, sterilized in ethylene oxide gas camera (sercon mp 3000 hg, são paulo, sp, brazil) and immersed in the prepared medium with s. mutans; group ii burnished, sterilized and immersed in the prepared medium without s. mutans; group iii – metallographic polishing, sterilized and immersed in the prepared medium with s. mutans; and group iv metallographic polishing, sterilized and immersed in the prepared medium without s. mutans. copper/aluminum alloy samples (duracast ms, são paulo, sp, brazil) were obtained by the lost wax technique using the same matrix mentioned above. they were divided into the following groups: group v polished with abrasive roads, sterilized and immersed in the prepared medium with s. mutans; group vi polished with abrasive roads, sterilized and immersed in the prepared medium without s. mutans; group vii – metallographic polishing, sterilized and immersed in the prepared medium with s. mutans; and group viii – metallographic polishing, sterilized and immersed in the prepared medium without s. mutans. induction of biocorrosion samples were aseptically immersed in polystyrene conical tubes (falcon 50.0 ml) containing 15.0 ml of prepared medium and mili-q water (millipore, billerica, ma, usa), in accordance to the manufacturer’s specifications. the prepared medium used was the mueller-hinton broth (difco laboratories inc, detroit, mi, usa lot 27006) with 5.0% sucrose (reagen lot 961038) and 200 ìl (106 microorganisms/ ml) s. mutans. the control samples were immersed in the same prepared medium without s. mutans. samples were then autoclaved for 15 min at 121 oc. tubes were incubated at 37 oc, in the orbital agitator (marconi, piracicaba, sp, brazil) with constant agitation of 100 rpm for 60 days. the prepared medium was changed every 72 h, for 60 days and reserved for analysis in the atomic absorption spectrophotometer (aas) (shimadzu corporation, kyoto, japan) to detect the metallic ions release. all the samples were removed from prepared medium on the 15th, 30th, 48th and 60th day, and observed by scanning electron microscopy (sem) (jsm 5410; jeol, tokyo, japan). preparation of specimens to sem two samples were immersed in edta 10% (merck, darmstadt, germany) during 24 h to confirm the absence of biofilm. the other two samples were immersed in α glutaraldehyde 3.0% to confirm the presence of biofilm. samples were washed in sterilized distilled water and fixed in á glutaraldehyde 3.0% sodium cacodylate 0.1 m, 5oc, ph 7,4 for 12 h. they were then postfixed in osmium tetroxide 2.0%, 5 oc for 4 h, dehydrated for 15 min in increasing percentages of alcohol 15, 30, 50, 75, 95 and 100% and dried in the critical point dryer using co2 (denton vacuum – desk ii, japan) before sem analysis. qualitative and quantitative analysis of metals a solution containing 1.0 ml of prepared medium and 9.0 ml mili-q water was used for the aas analysis. the reading of the diluted prepared medium was analyzed using standard titrisol (merck). the experimental model chosen in this study was described by pizzolitto et al.18. mili-q water was used to avoid metallic ions in the prepared medium or even during cleaning of the material. results the result shows both biofilm and pits formation and the corrosion induced by the s. mutans in both amalgam and copper/aluminum samples. metallic ion concentrations released from metal alloys are shown in figures 1a to 1f. the highest levels of metal concentration released at 60 days: copper (approx. 80 µg/ml) from group v, zinc (approx. 4.5 µg /ml) from groups ii and i and nickel (approx. 4.5 µg / ml) from group vii. tables 1 and 2 show the statistical analysis. aluminum, tin and mercury were not detected in the study due to the sensibility of the method. the results for cu/al alloy (table 1) showed statistically significant difference (p>0.05) between control and experimental groups. the highest values were observed in experimental group, especially in cu and ni ions, respectively. the results of amalgam (table 2) ions release showed no statistically significant difference for silver streptococcus mutans adhesion and releasing of metallic ions in dental alloys braz j oral sci. 14(1):36-40 3838383838 fig. 1. a: copper released from copper/aluminum alloy. figure b: iron released from copper/aluminum alloy. figure c: manganese released from copper/aluminum alloy. figure d: zinc released from both amalgam and copper/aluminum alloy. figure e: nickel released from both amalgam and copper/aluminum alloy. figure f: silver released from amalgam and nickel ions. the experimental group with metallographic polishing exhibited statistically significant difference (p>0.05) in comparison with the burnished experimental group. figures 2a to 2f show the sem analysis of the surface of the samples immersed in prepared medium either with or without microorganism (control group). sem observation of the samples immersed in the prepared medium with s. mutans streptococcus mutans adhesion and releasing of metallic ions in dental alloys braz j oral sci. 14(1):36-40 metals group silver zinc nickel control – burnished 0.002 a* 0.217 a 0.001 a control – metallographic polishing 0,002 a 0.186 ab 0.006 a experimental – burnished 0.003 a 0.215 a 0.004 a experimental – metallographic polishing 0.004 a 0.139 b 0.007 a table 2. table 2. table 2. table 2. table 2. results of metal ions released from culture medium with or without microorganisms s. mutans to amalgam. * values following by same letter in column to each metal were not different by tukey’s test at 5% significance level (p>0.05). 3939393939 metals groups copper i r o n manganese zinc nickel control – abrasive roads 0.591 b* 0.022 b 0.005 b 0.018 b 0.034 c control – . metallographic polishing 0.463 b 0.025 b 0.004 b 0.020 b 0.033 c experimental – abrasive roads 3.517 a 0.043 a 0.026 a 0.032 a 0.169 b experimental – metallographic polishing 3.308 a 0.054 a 0.032 a 0.030 a 0.204 a table 1.table 1.table 1.table 1.table 1. results of metal ions released from culture medium with or without microorganisms s. mutans to copper/aluminum alloy. * values followed by same letter in column to each metal were not different by tukey’s test at 5% significance level (p>0.05). fig. 2. a: sem of amalgam burnished (a), evidencing pits (b) and s. mutans 60th day – edta 10%. b: sem of amalgam metallographic polishing surface evidencing s. mutans 30th day – α glutaraldehyde 3%. c: sem of amalgam metallographic polishing surface, evidencing pits 60th day – edta 10%. d: sem of cooper/aluminum alloy surface – abrasive roads 15th day – edta 10%. e: sem of copper/aluminum alloy surface – abrasive roads 15th day – α glutaraldehyde 3%. f: sem of copper/aluminum alloy surface metallographic polishing 60th day – edta 10%. streptococcus mutans adhesion and releasing of metallic ions in dental alloys braz j oral sci. 14(1):36-40 4040404040 showed adherence of microorganisms on the whole surface of both amalgam and copper/aluminum alloy in all groups. the release of metallic ions did not inhibit the growth of microorganisms. discussion the alloy surfaces presented microorganisms’ adherence and ions release was influenced by s. mutans, but only for cu/al alloy. despite the many studies reporting adherence of streptococcus mutans on the surface of dental alloys5,910,13,15,17, this study has proved that even release of metal ions with the samples incubated in constant agitation, there was adherence and development of the colonies. in other studies that did not use those conditions, the samples remained in the resting tube. sem results showed that the exposure to s. mutans caused microorganisms adherence on the surface of both specimens. this fact, along with the presence of gaps on the surface, makes alloys more susceptible to corrosion, and the low corrosion resistance may lead to greater ions release19. mcginley et al.14 verified s. mutans corrosive effect on metallic alloy disks. corrosion is always a concern not only regarding the esthetic, but also the longevity of restorations and the possibility of causing severe allergic reaction20-21. the atomic absorption spectrophotometer indicated that the values of ions release for cu/al alloy were significantly affected by exposure to s. mutans. this was also observed by the study of mcginley et al.14 who assessed the influence of s. mutans on dental alloys’ toxicity and observed that ions release significantly increased in the presence of these bacteria, as it leads to ph reduction. on the other hand, mutlusagesen et al.22 stated that ph reduction is directly related to the great ions release by dental alloys. the cu/al alloy released copper, iron, manganese, zinc and nickel ions, but copper presented the highest ions release. benatti et al.23 showed that cu and ni alloys exhibit high in vitro corrosion as well as in oral cavity, mainly in areas of difficult hygiene. the ions released by amalgam were silver, zinc and nickel. however, there was no statistically significant difference between control and experimental group. zinc ion (with s. mutans) presented lower release in group with metallographic polishing, perhaps due to the formation of a stable passivating layer in more polished surfaces (smoother). ions release can potentially alter the oral tissues’ biological response in contact with dental alloys. special attention should be given regarding the release of metallic ions in patients that presents metal allergies. the challenge now is to focus on the nature of this intimate association and try to prevent the colonization by the microorganisms and consequent corrosion. within the limitations of this study, the following conclusions were drawn: s. mutans adhere to both amalgam and copper/aluminum alloy causing corrosion of those restorations; s. mutans produced higher ions release in cu/ al alloy. the amalgam ions release was not influenced by exposure to s. mutans. acknowledgements the authors thank heitor panzeri and izabel yoko ito (in memoriam) for their contributions to this study. references 1. beech ib, sunner j. biocorrosion: towards understanding interactions between biofilms and metals. curr opin biotechnol. 2004; 15: 181-6. 2. beech ib, sunner ja, hiraoka k. microbe-surface interactions in biofouling and biocorrosion processes. int microbiol. 2005; 8: 157-68. 3. garhammer p, schmalz g, hiller ka, reitinger t. metal content of biopsies adjacent to dental cast alloys. clin oral invest. 2003; 7: 92-7. 4. laurent f, grosgogeat b, reclaru l, dalard f, lissac m. comparison of corrosion behaviour in presence of oral bacteria. biomaterials. 2001; 22: 2273-82. 5. elshahawy w, watanabe i, koike m. elemental ion release from four different fixed prosthodontic materials. dent mat. 2009; 25: 976-81. 6. leinfelder kf. an evaluation of casting alloys used for restorative procedures. j am dent assoc. 1997; 128: 37-45. 7. wataha jc, messer rl. casting alloys. dent clin north am. 2004; 48: 499-512. 8. darwell bw. effect of corrosion on the strength of dental silver amalgam. dent mat. 2012; 28: 160-7. 9. elshahawy w, ajlouni r, james w, abdellatif h, watanabe i. elemental ion release from fixed restorative materials into patient saliva. j oral rehabil. 2013; 40: 381-8. 10. can g, akpinar g, aydin a. the release of elements from dental casting alloy into cell-culture medium and artificial saliva. eur j dent. 2007; 2: 86-90. 11. galo r, ribeiro rf, rodrigues rc, rocha la, de mattos mda g. efects of chemical composition on the corrosion of dental alloys. braz dent j. 2012; 23: 141-8. 12. lu y, chen w, ke w, wu s. nickel-based (ni-cr and ni-cr-be) alloys used in dental restorations may be a potential cause for immune-mediated hypersensitivity. med hypotheses. 2009; 73: 716-7. 13. wataha jc, lockwood pe, khajotia ss, turner r. effect of ph on element release from dental casting alloys. j prosthet dent. 1998; 80: 691-8. 14. mcginley el, dowling ah, moran gp, fleming gjp. influence of s. mutans on base-metal dental casting alloy toxicity. j dent res. 2013; 92: 92-7. 15. wataha jc, malcolm ct, hanks ct. correlation between cytotoxicity and the elements released by dental casting alloys. int j prosthod. 1995; 8: 9-14. 16. wataha jc. biocompatibility of dental casting alloys: a review. j prosthet dent. 2000; 83: 223-34. 17. oyar p, can g, atakol o. effects of environment on the release of ni, cr, fe, and co from new and recast ni-cr alloy. j prosthet dent. 2013; 112: 64-9. 18. pizzolitto el, lochagin n, bernardi aca, ito iys, guastaldi ac. microbial corrosion of biomaterials. j dent res. 1998; 35: 348. 19. wylie cm, shelton rm, fleming gjp, davenport aj. corrosion of nickelbased dental casting alloys. dent mat. 2007; 23: 714-23. 20. hansen pa, west la. allergic reaction following insertion of a pd-cu-au fixed partial denture: a clinical report. j prosthodont. 1997; 6: 144-8. 21. gokcen-rohlig b, saruhanoglu a, cifter ed, evlioglu g. applicability of zirconia dental prostheses for metal allergy patients. int j prosthodont. 2010; 23: 562-5. 22. mutlu-sagesen l, ergun g, karabulut e. ion release from metal-ceramic alloys in three different media. dent mat j. 2011; 30: 598-610. 23. benatti ofm, miranda wg, muench a. in vitro and in vivo corrosion evaluation of nickel-chromium-and copper-aluminum-based alloys. j prosthet dent. 2000; 84: 360-3. streptococcus mutans adhesion and releasing of metallic ions in dental alloys braz j oral sci. 14(1):36-40 oral sciences n3 braz j oral sci. 12(2):105-108 original article braz j oral sci. april | june 2013 volume 12, number 2 could idiopathic osteosclerosis have correlations with palatally impacted maxillary canines? cesar augusto rodenbusch poletto1, claudinéia itiberê2, sérgio aparecido ignácio3, lucilia kuriki4, orlando motohiro tanaka4, elisa camargo4 1area of orthodontics and radiology, university of planalto catarinense (uniplac), lages, sc, brazil and pontifical catholic university of paraná (pucpr), curitiba, pr, brazil 2 dentist, curitiba, pr, brazil 3area of biostatistics, dental school, pontifical catholic university of paraná (pucpr), curitiba, pr, brazil 4area of orthodontics, dental school, pontifical catholic university of paraná (pucpr), curitiba, pr, brazil correspondence to: cesar augusto rodenbusch poletto rua rubens de almeida, 75, cep: 88523-180 lages, sc, brasil phone / fax: +55 49 32232275 e-mail: cesarpoletto2@gmail.com abstract aim: to investigate the association between palatally impacted maxillary canines (pic) and idiopathic osteosclerosis. methods: a sample of 54 subjects (28 females and 26 males, mean age of 12.98±1.59 years) with pic was selected from the records of 1,650 orthodontic patients treated at the discipline of orthodontics clinics at the dental school of the pontifical catholic university of paraná (pucpr), in curitiba, pr, brazil. a control group of 54 subjects with normally erupted canines was also selected from the same files (mean age of 12.93±1.58 years). panoramic, lateral skull, postero-anterior skull, periapical and occlusal radiographs, as well as stone casts of the patients were examined. the kolmogorov-smirnov test revealed a normal distribution of gender and age in the groups. the results were analyzed with the chi-square test (α=0.05). results: there were no statistically significant differences (p>0.05) between the groups. four patients from each group had idiopathic osteosclerosis (7.41%), a rate that falls in the prevalence range reported in the literature. conclusions: no correlation was observed between palatally impacted maxillary canines and idiopathic osteosclerosis. keywords: diagnosis, tooth, unerupted, osteosclerosis. introduction idiopathic osteosclerosis is an asymptomatic, non-expansive and localized increase of bone radiopacity caused by an increase in the width of the bone trabeculae at the expense of medullary space. it is usually located in the mandibular premolar region1-3. the lesion has benign characteristics and seems not to increase over time4. peck5 listed several dental anomalies that seemed to indicate a pattern. among them were missing teeth, conoid teeth, teeth with reduced size (generalized or localized), delayed tooth formation and eruption (generalized or localized), infraocclusion (more frequently in primary teeth), palatally impacted canines, maxillary braz j oral sci. 12(2):105-108 106106106106106 canine/premolar transposition, mandibular canine/lateral incisor transposition, and distal angulation of a non-erupted second mandibular premolar. according to this author, other associations will be discovered as researchers focus on the genetic pattern of these anomalies. after the third molars, the permanent maxillary canine is the most frequently involved tooth in cases of impaction6-7. palatally impacted maxillary canines seem connected to a genetic condition, and are often associated with other dental and skeletal disturbances7. the aim of the present study was to investigate whether an increased prevalence of idiopathic osteosclerosis exists in patients with palatally impacted maxillary canines. material and methods a total of 1,650 patient records from the files of the discipline of orthodontics of the dental school of the pontifical catholic university of paraná (pucpr) were reviewed for this study. the experimental group (eg) consisted of 54 (3.27%) patients who presented 62 palatally impacted maxillary canines; these patients had ages starting at 12 years and 5 months to ensure a definite diagnosis of palatal impaction. according to ericson and kurol8, the best age to establish a clinical diagnosis of maxillary canine impaction using digital palpation and checking for the presence of a buccal bulge would be starting at 9 years because the clinical signs are not evident before this age. as for gender, 28 cases were observed among females (51.85%) and 26 among males (48.15%). the control group (cg) consisted of 54 patients, 28 females and 26 males, with approximately the same age (12.93±1.58 years) as those of the patients in the eg (12.98±1.59 years), with normally erupted canines. the kolmogorov-smirnov test indicated normal distribution of gender and age in the groups. the patients’ orthodontic documentation, which included a set of radiographs (panoramic, lateral skull, postero-anterior skull, periapical and occlusal), photographs and stone casts, was used for diagnosing the cases of palatally impacted maxillary canines. the methods were the three-dimensional localization using orthogonal radiographs and the clark method. the canine was considered palatally impacted whenever it was unerupted and the patient was already in the c5 cervical maturation stage, as proposed by baccetti9. the diagnosis of idiopathic osteosclerosis was also established based on the patient’s radiographic examinations. idiopathic osteosclerosis was observed as a bone radiopacity increase located inside the bone, measuring 3 mm or more, with no radiolucent halo and no apparent cause (figures 1 and 2), as proposed by langlais et al.1, and white and pharoah3. radiopaque lesions associated with dental caries, deep restorations or tooth extraction regions were not diagnosed as idiopathic osteosclerosis, since they could be the result of condensing osteitis or residual condensing osteitis, when located in a tooth extraction region. lesions fig. 1. palatally impacted right maxillary canine and idiopathic osteosclerosis in the right mandibular first premolar region. located in tooth extraction regions could also be the result of alveolar sclerosis. exostoses of the torus type were excluded by evaluating the intraoral photographs and the gypsum casts of the orthodontic documentation. the projected image of a soft tissue calcification was discarded by analyzing the radiograph with a projection perpendicular to the panoramic radiograph or to the lateral skull radiograph, namely the posteroanterior skull radiograph. initially, canine impaction was assessed and idiopathic osteosclerosis was identified in 30 patient records (15 from the cg and 15 from the eg), at two time points (day zero and 30 days later), by a single radiology specialist, and by applying the kappa agreement test. the test indicated agreement between the assessments (p<0.05), obtaining the lowest kappa value of 0.7830 (a value greater than 0.60 is recommended). all radiographs of both groups were then analyzed and the data were tabulated for application of the chi-square statistical test (α=0.05). fig. 2. periapical view of an idiopathic osteosclerosis. could idiopathic osteosclerosis have correlations with palatally impacted maxillary canines? braz j oral sci. 12(2):105-108 107107107107107 results four cases of osteosclerosis were observed in eg and 4 cases cg (7.41%). no significant correlation was found between osteosclerosis and either of the groups (p>0.05). discussion one of the most important requisites for treating and preventing a disease is to know its etiologic factor. the term idiopathic has been used whenever science has failed to define the cause of a certain change. nevertheless, pursuit for the origin goes on and nowadays several pathological conditions have been shown to have a genetic condition as a causal factor. some changes overlap and often share the same origin. these associations may help in the early diagnosis of some serious disease as the gardner syndrome, for example. one of its first manifestations in the oral cavity is the presence of osteomas in the maxillary bones. later on, the patient develops malignant intestinal tumors that are hard to treat and have a more doubtful prognosis. a dentist on the alert may suspect a new case of gardner syndrome by observing atypical osteoma-like lesions in a young patient and referring him/her to medical evaluation10. palatally impacted maxillary canines seem to have a genetic etiology6,11, and are frequently associated with other changes, such as microdontia of maxillary lateral incisors11-12, delayed tooth eruption13 and fusion of the clinoid processes of the sella turcica14. idiopathic osteosclerosis is a bone tissue structural change whose etiologic factor remains unknown. it is also known as enostosis or dense bone island, terms used more frequently by medical literature to describe radiopaque lesions found occasionally, mostly in the long bones. its most common location in the jaws is the mandible, mainly in the premolar and molar regions. although idiopathic osteosclerosis is not a rare entity, little importance has been given by orthodontists to its occurrence, as demonstrated by the small number of studies published on this topic in orthodontic journals. in a study on the incidental findings made on panoramic radiographs of orthodontic patients, bondemark et al.15 observed that idiopathic osteosclerosis was the most frequent finding, outnumbering even periapical inflammatory lesions and marginal bone resorptions. diagnosing idiopathic osteosclerosis accurately is a difficult task, since this lesion may be radiographically mistaken for exostosis, including torus mandibularis and palatinus, residual condensing osteitis, alveolar calcification after exodontia (whether complicated or not), bone architectural change in response to occlusal trauma on an inclined tooth, particularly on mandibular second molars, when the first molars are missing. the lesion may even be mistaken for a radiographic projection over bone of a soft tissue calcification. the mere presence of teeth, their carious lesions, occlusal traumas and infectious processes, as well as the primary teeth shedding process, may also cause bone tissue architectural changes, adding to the abovementioned enostoses and dense bone islands. the dentist’s approach towards idiopathic osteosclerosis is also not unanimous in the literature. williams and brooks4 followed adult patients with idiopathic osteosclerosis for a decade and concluded that it remained stable. mcdonaldjankowski2 studied idiopathic osteosclerosis lesions in radiographic examinations after a 9-year interval and found that lesion size may decrease and that that there may be complete remission in some cases. these findings reinforce the recommendation for no intervention. there are, however, reports of complications arising from the presence of idiopathic osteosclerosis in the maxillary bones. bennett and mclaughlin16 warned that bone sclerosis might delay or even obstruct space closure after tooth extractions during orthodontic treatment. nakano et al.17 reported a case of tooth inclination caused by idiopathic osteosclerosis growth. marques-silva et al.18 described a case where idiopathic osteosclerosis caused deviation of a tooth eruption route. this, in turn, led to the resorption of the adjacent tooth. mah et al.19 suggested that caution should be taken in biomechanical planning and orthodontic movement in sclerotic bone areas, and also that computerized tomography examination may aid in this planning. alveolar sclerosis is a condition histologically very similar to idiopathic osteosclerosis. baumgaertel20 reported a very interesting case where the tooth movement process through an alveolus repaired with sclerotic bone was not possible using the conventional retraction technique, requiring extra anchorage obtained by means of mini-implants. the patient of that case also displayed agenesis of a maxillary lateral incisor and palatal impaction of a maxillary canine, both conditions that seem to share the same genetic origin. considering the possible difficulty in performing tooth movement when faced with a case of osteosclerosis, the orthodontist should register its presence on the patient’s records and follow the patient radiographically. if moving a tooth through the zone of osteosclerosis is required, caution must be taken during the procedure since the lesion may render tooth movement difficult. to the best of our knowledge, no previous study has investigated the association between idiopathic osteosclerosis and palatally impacted permanent maxillary canines. a single study21 was found comparing the prevalence of this lesion in certain malocclusion types, although the criterion adopted by the authors for diagnosing malocclusion was based solely on the gypsum model records. the authors did not distinguished the skeletal from the dental aspects of the sagittal malocclusions, a distinction seemingly relevant when studying possible correlations between this malocclusion type and a bone lesion. a possible association between a greater prevalence of idiopathic osteosclerosis among patients with palatally impacted maxillary canines could suggest that idiopathic osteosclerosis is a genetic condition and possibly also the manifestation of the complex condition of palatally impacted maxillary canines. the prevalence of palatally impacted maxillary canines found in the present study (3.27%) and the prevalence of could idiopathic osteosclerosis have correlations with palatally impacted maxillary canines? 108108108108108 braz j oral sci. 12(2):105-108 idiopathic osteosclerosis (7.41%) were similar to those reported elsewhere22. finding an identical number of idiopathic osteosclerosis cases in both groups suggests that there is no association of this condition with palatally impacted maxillary canines. also, the small number of idiopathic osteosclerosis cases observed in this study may have interfered with establishing a possible association. further investigation is required with a greater sample of patients to effectively discard an association between the two pathologies or possibly demonstrate a common genetic origin. the results showed no correlation between palatally impacted maxillary canines and idiopathic osteosclerosis. references 1. anic-milosevic s, varga s, mestrovic s lapter-varga m, slaj m. dental and occlusal features in patients with palatally displaced maxillary canines. eur j orthod. 2009; 31: 367-73. 2. baccetti t. risk indicators and interceptive treatment alternatives for palatally displaced canines. semin orthod. 2010; 16: 186-92. 3. bennett j c, mclaughlin r p. controlled space closure with preadjusted appliance system. j clin orthod. 1990; 4: 251-60. 4. baumgaertel s. socket sclerosisan obstacle for orthodontic space closure? angle orthod. 2009; 79: 800-3. 5. bishara s e. impacted maxillary canines: a review. am j orthod dentofac orthop. 1992; 101: 159-71. 6. bondemark l, jeppsson m, lindh-ingildsen l, rangne k. incidental findings of pathology and abnormality in pretreatment orthodontic panoramic radiographs. angle orthod. 2006; 76: 76-98. 7. cankaya ab, erdem ma, isler sc, cifter m, olgac v, kasapoglu c, et al. oral and maxillofacial considerations in gardner’s syndrome. int j med sci. 2012; 9: 137-41. 8. chaushu s, sharabi s, becker a. dental morphologic characteristics of normal versus delayed developing dentitions with palatally displaced canines. am j orthod dentofacial orthop. 2012; 121: 339-46. 9. ericson s, kurol j. longitudinal study and analysis of clinical supervision of maxillary canine eruption. community dent oral epidemiol. 1986; 8: 133-40. 10. garib dg, alencar bm, lauris jr, baccetti t. agenesis of maxillary lateral incisors and associated dental anomalies. am j orthod dentofacial orthop. 2010; 137: 732-6. 11. jacoby h. the etiology of maxillary canine impaction. am j orthod dentofacial orthop. 1983; 84: 125-32. 12. langlais rp, langland oe, nortjé cj. generalized radiopacities. in: cooke d, editor. diagnostic imaging of the jaws. baltimore: williams & wilkins; 1995. p.565-615. 13. leonardi r, barbato e, vichi m, caltabiano m. skeletal anomalies and normal variants in patients with palatally displaced canines. angle orthod. 2009; 79: 727-32. 14. marques-silva l, guimarães als, dilascio mlc, castro wh, gomez rs. a rare complication of idiopathic osteosclerosis. med oral patol oral cir bucal 2007, 12: e233-4. 15. mah jk, yi l, huang rc, choo hr. advanced applications of cone beam computed tomography in orthodontics. semin orthod. 2011; 17: 57-71. 16. mcdonald-jankowski d s. idiopathic osteosclerosis in the jaws of britons and of the hong kong chinese: radiology and systematic review. dentomaxillofac radiol. 1999; 28: 357-63. 17. nakano k, ogawa t, sobue s, ooshima t. dense bone island: clinical features and possible complications. int j paediatr dent. 2001; 12: 433-7. 18. peck s. dental anomaly patterns (dap): a new way to look at malocclusion. angle orthod. 2009; 19: 1015-6. 19. white sc, pharoah mj. benign tumours of the jaws. in: white sc, pharoah mj. oral radiology: principles and interpretation. saint louis: mosby; 2000. p.378-419. 20. williams tp, brooks sl. a longitudinal study of idiopathic osteosclerosis and condensing osteitis. dentomaxillofac radiol. 1998; 27: 275-8. 21. lee s,park i, jang i, choi d, cha b. a study on the prevalence of the idiopathic osteosclerosis in korean malocclusion patients. korean j oral maxillofac radiol. 2010; 40: 159-63. 22. sisman y, ertas et, ertas h, sekerci ae. the frequency and distribution of idiopathic osteosclerosis of the jaws. eur j dent. 2001; 5: 409-15. could idiopathic osteosclerosis have correlations with palatally impacted maxillary canines? oral sciences n3 braz j oral sci. 13(2):104-108 original article braz j oral sci. april | june 2014 volume 13, number 2 oblique or orthoradial cbct slices for preoperative implant planning: which one is more accurate? frederico sampaio neves1, taruska ventorini vasconcelos2, anne caroline costa oenning2, sergio lins de-azevedo-vaz3, solange maria de almeida2, deborah queiroz freitas2 1união metropolitana de educação e cultura – unime, school of dentistry, departament of clinical and surgical propaedeutics, salvador, ba, brazil 2universidade estadual de campinas – unicamp, school of dentistry, department of oral diagnosis, piracicaba, sp, brazil 3universidade federal do espírito santo – ufes, school of dentistry, department of clinical dentistry, vitoria, es, brazil correspondence to: frederico sampaio neves departamento de propedêutica clínica e cirúrgica, união metropolitana de educação e cultura – unime rua professor fernando rocha, 326 cep: 41194-020 saboeiro, salvador, ba, brasil phone: +55 71 92363724 fax: +55 71 3358005 e-mail: fredsampaio@yahoo.com.br abstract aim: to assess which slice inclination would be more accurate in measuring sites for implant placement: the oblique or the orthoradial slice. methods: five regions of eight edentulous mandibles were selected (incisor, canine, premolar, first molar and second molar). the mandibles were scanned with a next generation i-cat cbct unit. two previously calibrated oral radiologists performed vertical measurements in all the selected regions using both the oblique and orthoradial slices. the mandibles were sectioned in all the evaluated regions in order to obtain the gold standard. the wilcoxon signed rank test compared the measurements obtained in the oblique and orthoradial slices with the gold standard. results: the bone height measurements for the first and second molar regions using the orthoradial slices were statistically different from the gold standard. conclusions: using the orthoradial slices to obtain cross-sectional images may offer insufficient accuracy for implant placement in the posterior region. keywords: cone-beam computed tomography; dental implants; mandible; software. introduction cone beam computed tomography (cbct) is a radiographic method that has been used in several areas of dentistry, because it shows three-dimensional images of the dental structures with high contrast1. this image modality became commercially viable and suitable in the dental practice given its small dimension, easy handling, inherently fast image acquisition, relatively low cost and radiation doses1-3. dental implant placement depends remarkably on the estimation of the distance between anatomical landmarks and bone dimensions, in which the surgeon uses linear cbct measurements. inaccurate bone measurements can lead to a risky relationship between the dental implant and important anatomical structures (e.g. mandibular canal and mental foramen). it may result in injury to the neurovascular bundle and cause some postoperative complaints such as neuroma, paresthesia, anesthesia and hemorrhage4-5. it has been stated that cbct anatomical linear measurements are accurate and reliable6-13. however, the inclination of the patient’s head during cbct image acquisition can cause distortion in measurements for preoperative implant received for publication: april 30, 2014 accepted: june 03, 2014 105 planning14-17. on the other hand, there are several types of software designed to assess cbct images; some of them allow the operator obtaining oblique slices while others do not. the hypothesis of this study is weather the inclination of the slices would be relevant for preoperative implant planning. therefore, this study aimed to assess which slice would be more accurate in measuring sites for implant placement: the oblique or the orthoradial slice. material and methods this study was conducted after approval from the ethic research committee of unicamp’s school of dentistry, brazil. eight dry mandibles (totaling 16 hemi-mandibles) with completely resorbed alveolar processes were selected to the study. the regions to be assessed were determined according the criteria proposed by neves et al. (2012)13: incisive (1 cm distal from the median sagittal plane), canine (1 cm distal from the incisive region), premolar (at the mental foramen level), first molar (1 cm distal from the mental foramen) and second molar (2 cm distal from the mental foramen) regions. one of the investigators (tvv) draw perpendicular lines to the base of the mandibles with a permanent marker pen (bic, cajamar, sp, brazil) and fixed a 0.7-mm sphere with a radiopaque marker (gutta-percha point) in all extension of the buccal cortical plate above each line (fig. 1). these procedures aimed at establishing controlled sites, i.e. standard regions for measurements. fig. 1. mandible with the radiopaque markers in the buccal cortical. in order to simulate soft-tissue attenuation, each mandible was placed in a polystyrene box filled with water prior to the cbct examination. the mandibles were scanned with a next generation i-cat cbct unit (imaging sciences international, inc, hatfield, pa, usa), under specific exposure parameters: 8x8 cm field of view (fov), full-scan mode (360°), 37.07 ma, 120 kvp, 0.20 mm voxel size and 26.9 s exposure time. all measurements were performed in cross-sectional images using the ondemand3d®™ software (version 1.0, cybermed inc., seoul, south korea) with both oblique and orthoradial slices (fig. 2). the investigator adjusted the oblique slices by inclination of the cutting plane in the multiplanar reconstruction (mpr) view, until the slice showed all the gutta-percha point in the extension of the buccal cortical plate. the orthoradial slices were obtained by using the curved planar reformat tool of the software to create crosssectional images, without any adjustment to the gutta-percha points. the investigator positioned the cutting lines at the center of the radiopaque spheres for both the oblique and orthoradial slices and saved the images as projects, so the observers only had to open the projects to assess the images without moving or adjusting any line. this procedure guaranteed that all observers assessed the same regions. fig. 2. schematic drawing showing the oblique and orthoradial cbct images. under dim light conditions, two previously calibrated oral radiologists with 5-years of cbct experience assessed independently the saved projects to obtain the measurements. the observers obtained the bone height by measuring the distance from the upper to the lowest point in the cortical boundaries of the mandibular body in all the predefined regions of the mandible. additionally, they measured the distance from the superior cortical ridge of the mental foramen (premolar region) or the mandibular canal (molar regions) to the highest point of the alveolar ridge (fig. 2). the mandibular canal could not be precisely located in six hemi-mandibles so these measurements were not obtained. after the observers measured all images, the mandibles were sectioned using an electric saw in all the assessed regions. the slices were done immediately before the drawn lines, so the bone tissue in the site related to the tomographic image was not lost. the investigator obtained the vertical measurements for each site using a digital caliper (sc-6 digital caliper; mitutoyo corporation, tokyo, japan), repeating three times the direct measurements. the means of these measurements were considered the gold standard for the study (fig. 3). data were analyzed using the spss version 18.0 software (statistical package for the social sciences, chicago, il, usa). the variables and factors in study were the measurements and post-processing view (oblique or orthoradial), braz j oral sci. 13(2):104-108 oblique or orthoradial cbct slices for preoperative implant planning: which one is more accurate? 106 fig. 3. direct measurement in the mandible section. gold standard oblique images p value orthoradial images p value incisive bone height 26.28 (3.76) 26.17 (3.87) 0.38 26.57 (3.90) 0.19 canine bone height 24.85 (3.49) 25.00 (3.44) 0.91 25.17 (3.56) 0.15 premolar bone height 23.47 (3.18) 23.43 (3.30) 0.55 23.78 (3.46) 0.07 mental foramen height 10.55 (2.18) 10.63 (2.75) 0.55 10.37 (2.53) 0.61 first molar bone height 21.23 (2.51) 21.25 (2.64) 0.91 20.90 (2.63) 0.005* mandibular canal height 9.21 (0.97) 9.04 (1.31) 0.59 8.79 (1.11) 0.31 second molar bone height 20.06 (1.93) 19.78 (2.13) 0.07 19.66 (2.00) 0.03* mandibular canal height 8.97 (2.28) 8.78 (1.44) 0.64 8.67 (1.54) 0.91 table 1. table 1. table 1. table 1. table 1. mean (s.d.) of the gold standard, oblique and orthoradial images in each region. *statistically significant difference according wilcoxon test. respectively. the intraclass correlation coefficient (icc) quantified the interobserver agreement. descriptive statistics were calculated as mean and standard deviation. the wilcoxon signed rank test compared the measurements obtained in the oblique and orthoradial slices to the gold standard. the null hypothesis considered that the orthoradial and the oblique slices would not result in statistically significant differences from the gold standard. the significance level was set at 5%. results the interobserver agreement found in the study was excellent (icc = 0.99; 95% confidence interval [ci] = 0.99 0.99). table 1 shows the mean and standard deviation values of the measurements obtained using the digital caliper (gold standard), the oblique and the orthoradial slices. the results from the wilcoxon test are also shown for the comparisons of oblique and orthoradial slices vs gold standard. the null hypothesis was only rejected after measuring bone height for the first and second molar regions using the orthoradial slice (p<0.05). these measurements were underestimated in comparison with the gold standard. discussion in this study, the oblique slices were obtained by inclination of the cutting plane in the mpr view while the orthoradial slices were obtained using the curved planar reformat tool to create cross-sectional images. the mpr and especially the cross-sectional images are the views that clinicians mostly use when assessing cbct images. in some cbct software – like the one that was used in the study – while the mpr images allow tilting the cut plane along an object of interest (represented in this study by the gutta-percha point), the orthoradial images mostly do not. therefore, it was aimed at assessing which of the two slices was the most accurate in determining measurements for implant placement. the literature advises that implants should be placed with their axis parallel to the oclusal forces. therefore, implant axial angulation should be planned to distribute the occlusal forces vertically along the implant axis18. considering the spee curve, where the maxillary posterior teeth are slightly tilted distally, implants in the posterior region of the mandible may require a mesio-inclined position. also, the adoption of tilted implants has been proposed to avoid injury to the alveolar inferior nerve in edentulous maxilla/mandible cases19. braz j oral sci. 13(2):104-108 oblique or orthoradial cbct slices for preoperative implant planning: which one is more accurate? 107 therefore, it was speculated if the use of orthoradial images which are very much used by clinicians – would be accurate enough for preoperative planning of tilted implants. the hypothesis was that the clinician should consider the orientation of the implant axis in order to guide accordingly the tomographic cutting plane. the results demonstrated statistically significant differences between the orthoradial slices and the gold standard for bone height in the first and second molar regions. the other regions and the oblique slice did not provide any statistically significant differences from the gold standard, which indicated that most of the measurements performed in both slices were accurate. this is also demonstrated by the mean values of the measurements performed with the digital caliper (gold standard), the oblique and the orthoradial slices, which were all close to each other. the gutta-percha points fixed along the buccal cortical plate of the mandibles aimed to set radiopaque marks in the regions to be assessed by the observers. they represented the inclination of hypothetically planned implants. as only the oblique slices could be adjusted along the gutta-percha points, they were more accurate in measuring both the bone and mandibular canal height in the first and second molar regions. it is important to consider that inferior alveolar nerve injury can result in some postoperative complaints, and it is more prone to occur when the length of the implant to be placed is greater than the implant site4,13. this can lead to higher morbidity and additional treatment costs for the patient. several studies have shown the influence of maxillomandibular inclination in the bone measurements14-17. variations in the patient position during cbct image acquisition can lead to inaccurate bone height and width measurements in different regions of the maxilla and mandible. when the planning is incorrect, important anatomical structures may be harmed, such as the nasal cavity floor and the incisor branch of the inferior alveolar nerve. dantas et al. (2008)14 and visconti et al. (2013) 17 evaluated the influence of mandible positioning in bone height measurements taken in ct and cbct images, respectively. the authors reported that for some slices in the orthoradial image, principally those in the premolar region, at the site of what appeared to be the anatomical repair corresponding to the studied area, the radiopaque marker was not present. this confirmed that the considered differences did not correspond to distortions in the reformatted images, but to the performance of measurements at different locations. in this format, the slices obtained would not be perpendicular to the mandible base, but they rather would be oblique in relation to the reference system. this principle may be used in the present study, due to the fact that the oblique and orthoradial slices are in different positions, the radiopaque marker not being completely observed in the orthoradial images. in the present study, no statistically significant differences were found in the anterior region of the mandible comparing both the orthoradial and oblique slices with the gold standard. this corroborates other authors14,15,17, because they also did not find differences in the anterior region comparing different positions of the skull with normal position. this could probably be justified by the fact that the anterior region of the mandible is straighter, remaining more perpendicular to the horizontal plane when compared with the posterior regions. the observers could not locate the mandibular canal in six hemi-mandibles, which is justified by the reduction of mineral bone density caused by old age and teeth loss. lindh et al. (1995)20 found that the compact bone surrounding the neurovascular bundle was missing in some histological sections of edentulous mandibles, with the result that the canal could not be identified in radiographs. in conclusion, the preoperative planning for implant placement in mandibles using cbct orthoradial slices to obtain cross-sectional images should be made with special caution. these images may offer insufficient accuracy for implant placement in the posterior region, especially when planning tilted implants. references 1. scarfe wc, farman ag. what is cone-beam ct and how does it work? dent clin north am. 2008; 52: 707-30. 2. mozzo p, procacci c, tacconi a, martini pt, andreis ia. a new volumetric ct machine for dental imaging based on the cone-beam technique: preliminary results. eur radiol. 1998; 8: 1558-64. 3. arai y, tammisalo e, iwai k, hashimoto k, shinoda k. development of a compact computed tomographic apparatus for dental use. dentomaxillofac radiol. 1999; 28: 245-8. 4. neves fs, de almeida sm, bóscolo fn, haiter-neto f, alves mc, crusoé-rebello i. et al. risk assessment of inferior alveolar neurovascular bundle by multidetector computed tomography in extractions of third molars. surg radiol anat. 2012; 34: 619-24. 5. neves fs, nascimento mc, oliveira ml, almeida sm, bóscolo fn. comparative analysis of mandibular anatomical variations between panoramic radiography and cone beam computed tomography. oral maxillofac surg. 2013 aug 24. (in press). 6. lascala ca, panella j, marques mm. analysis of the accuracy of linear measurements obtained by cone beam computed tomography (cbctnewtom). dentomaxillofac radiol. 2004; 33: 291-4. 7. kamburoðlu k, kiliç c, ozen t, yüksel sp. measurements of mandibular canal region obtained by cone-beam computed tomography: a cadaveric study. oral surg oral med oral pathol oral radiol endod. 2009; 107: e34-42. 8. kim ts, caruso jm, christensen h, torabinejad m. a comparison of cone-beam computed tomography and direct measurement in the examination of the mandibular canal and adjacent structures. j endod. 2010; 36: 1191-4. 9. ganguly r, ruprecht a, vincent s, hellstein j, timmons s, qian f. accuracy of linear measurement in the galileos cone beam computed tomography under simulated clinical conditions. dentomaxillofac radiol. 2011; 40: 299-305. 10. panmekiate s, apinhasmit w, petersson a. effect of electric potential and current on mandibular linear measurements in cone beam ct. dentomaxillofac radiol. 2012; 41: 578-82. 11. torres mg, campos ps, segundo np, navarro m, crusoé-rebello i. accuracy of linear measurements in cone beam computed tomography with different voxel sizes. implant dent. 2012; 21: 150-5. 12. waltrick kb, nunes de abreu junior mj, corrêa m, zastrow md, dutra vd. accuracy of linear measurements and visibility of the mandibular canal of cone-beam computed tomography images with different voxel sizes: an in vitro study. j periodontol. 2013; 84: 68-77. braz j oral sci. 13(2):104-108 oblique or orthoradial cbct slices for preoperative implant planning: which one is more accurate? 108 13. neves fs, vasconcelos tv, campos ps, haiter-neto f, freitas dq. influence of scan mode (180°/360°) of the cone beam computed tomography for preoperative dental implant measurements. clin oral implants res. 2014; 25: 155-8. 14. dantas ja, montebello filho a, campos ps. computed tomography for dental implants: the influence of the gantry angle and mandibular positioning on the bone height and width. dentomaxillofac radiol. 2005; 34: 9-15. 15. tomasi c, bressan e, corazza b, mazzoleni s, stellini e, lith a. reliability and reproducibility of linear mandible measurements with the use of a cone-beam computed tomography and two object inclinations. dentomaxillofac radiol 2011; 40: 244-50. 16. sheikhi m, ghorbanizadeh s, abdinian m, goroohi h, badrian h. accuracy of linear measurements of galileos cone beam computed tomography in normal and different head positions. int j dent. 2012; 214954. doi: 10.1155/2012/214954. 17. visconti ma, verner fs, assis nm, devito kl. influence of maxillomandibular positioning in cone beam computed tomography for implant planning. int j oral maxillofac surg. 2013; 42: 880-6. 18. machtei ee, oettinger-barak o, horwitz j. axial relationship between dental implants and teeth: a radiographic study. j oral implantol. 2012 sep 10. (in press). 19. del fabbro m, bellini cm, romeo d, francetti l. tilted implants for the rehabilitation of edentulous jaws: a systematic review. clin implant dent relat res. 2012; 14: 612-21. 20. lindh c, petersson a, klinge b. measurements of distances related to the mandibular canal in radiographs. clin oral implants res. 1995; 6: 96-103. braz j oral sci. 13(2):104-108 oblique or orthoradial cbct slices for preoperative implant planning: which one is more accurate? oral sciences n3 braz j oral sci. 13(1):17-21 original article braz j oral sci. january | march 2014 volume 13, number 1 age estimation by teeth periodontosis and transparency: accuracy of lamendin’s method on a brazilian sample juliana ribeiro lopes1, simone borges braga dos santos queiroz1, mário marques fernandes1,3, luiz airton saavedra de paiva2, rogério nogueira de oliveira1 1universidade de são paulo usp, school of dentistry, department of community dentistry, area of forensic dentistry, são paulo, sp, brasil 2instituto de medicina legal de guarulhos iml, center for forensic sciences studies and research, guarulhos, sp, brasil 3ministério público do rio grande do sul, biomedical service, porto alegre, rs, brasil correspondence to: mário marques fernandes av. prof. lineu prestes, 2227 cidade universitária cep 05508-900 são paulo-sp, brasil phone: +55 11 30917891 fax: +55 11 30917874 e-mail:mario-mf@live.com abstract aim: to apply the lamendin et al. (1992) technique on a brazilian sample to assess its accuracy on this specific population. methods: the authors present two measurements in single-rooted teeth: the peridontosis and transparency of the root. then, these variables are inserted in an equation to estimate the individual’s age. the sample comprised 49 teeth obtained from the collection of the forensic medicine institute of guarulhos, brazil. statistical analysis was performed with t and paired t tests, comparing chronological and estimated ages. results: it was possible adjust the original formula for brazilians by linear regression analysis: a = (p x 0.18) + (t x 0.47) + 31.77. where, a = age; p = (periodontosis height x 100)/root height; and t = (transparency height x 100)/root height. conclusions: the method is accurate only for young adults but it could be used with caution in individuals between 45 and 60 years of age to assist in estimating an age range. however, the technique loses its efficacy in older individuals. keywords: forensic anthropology; age determination by teeth; forensic dentistry. introduction age determination by teeth is an important part of forensic dentistry and anthropology, since it valuably assists in cases of identification. for many years and still now, the scientific community have produced a large number of researches on this topic. several of them are based on developmental stages of teeth, which makes them suitable for estimating age of children and adolescents1-4. for adults, the methods are often based on degenerative modifications, such as attrition, periodontosis, transparency of the root, secondary dentin, cementum apposition and root resorption. since gustafson5 (1950) first published his work on age determination by degenerative changes in teeth, several authors6-8 developed other methods based on the characteristics he described, including lamendin et al.9 (1992). the technique was developed to be plain and with the purpose to estimate adults’ age at death. it consists in the analysis of two dental variables (periodontosis and transparency) and three height measurements (periodontosis, transparency and root),applying the numbers to the following formula: a (age) = (p x 0.18) + (t x 0.42) + 25.53. where, a = age; p = (periodontosis height x 100)/root height; received for publication: january 28, 2014 accepted: march 10, 2014 gisele higa texto digitado http://dx.doi.org/10.1590/1677-3225v13n1a04 18 braz j oral sci. 13(1):17-21 and t = (transparency height x 100)/root height. the method is a good option to be used in forensic cases. therefore, the aim of this study was to apply lamendin’s technique to a brazilian sample in order to assess its efficacy on this population. material and methods the technique, as described by lamendin et al.9 (1992), was applied to 49 single-rooted teeth from 26 skeletonized individuals. the sample consisted of a collection of corpses formerly buried in a cemetery in guarulhos, sp, brazil, and then donated by their families to the forensic sciences study and research center at the forensic medicine institute of guarulhos. donations were made 3 years after death and burial. age and gender were known with certainty. the region of guarulhos has a humid subtropical climate with annual averages of 19°c temperature, 81.1 air relative humidity and1470 mm rainfall. the rain concentrates in the summer months, contrasting with the dry period, which corresponds to the winter months (july and august). the soil from where the skeletons were taken has a ph around 5.5, which means that the concentration of hydrogen ions is high, making it an acid soil. the original method advocates the measurements to be made on the labial surface of the root, which was honored in this study. however, in some cases this surface was exposed due to lack of bone structure in this area and was damaged during cleaning of the skulls, impairing visualization on this surface. in such cases, measurements were made on the most labial part of the mesial surface. as defined by the authors, the periodontosis is a yellowish area darker than the enamel, but lighter than the rest of the root. it corresponds to the maximum distance between the cement enamel junction and insertion line of soft tissues, as shown in figure 1. the root transparency results from hydroxyapatite crystals deposition inside the dentinal tubules, and the maximum length of transparency is measured from the apex. to avoid erroneous measurements, in the present study all measurements were taken at a starting point on an imaginary line at the height of the root apex, as illustrated in figure 2. a digital sliding caliper was used to make all measurements and a light box was used to aid visualization of root transparency. the observer was unaware of the chronological age during this process. to evaluate intraand inter-observer variation, blandaltman analysis with pitman’s test of difference in variance was used. to verify efficacy of the method, the estimated ages obtained by applying measurements to lamendin’s formula were compared to chronological ages and statistically analyzed with t and paired t tests. all analyses were performed with stata® 12 (stata corp., college station, tx, usa), and a 5% level of significance was set. the study was approved by the ethics research committee of university of são paulo school of dentistry under protocol number 76/11 and caae 0086.0.017.000-11. results at the time the investigation was conducted, there were 66 individuals in the collection; 27 of them had viable teeth for the research. however, after the measurements, it was revealed that one skull belonged to a 17 year-old boy, which led to its exclusion from the sample, due to the lamendin’s formula constant (25.53), which does not allow for estimating the age of individuals less than 25 years old. therefore, the final sample comprised 49 teeth from 26 skulls. whenever possible, two teeth were extracted from the same individual, thus 14 teeth from 7 females and 35 teeth from 19 males were obtained. two weeks after measurements, 10 teeth were randomly selected to test intraand inter-observer variation. the second fig. 1: periodontosis measure: the maximum distance between the soft tissue insertion line (point a) and the cement enamel junction (point b). always made on the labial surface of root, and facing the observer. fig. 2: root transparency measurement: the maximum length of transparency from the apex on labial surface. the tooth is positioned with the proximal face in contact with light box to aid visualizing root transparency on the labial surface. two parallel lines are drawn, one on the upper region (point a) and other on the lower region (point b) of labial transparency area. age estimation by teeth periodontosis and transparency: accuracy of lamendin’s method on a brazilian sample 19 braz j oral sci. 13(1):17-21 observer was calibrated and also blinded. variations were never higher than 1mm and bland-altman analysis with pitman’s test of difference in variance showed that the measurements were statistically equal between observers and the main observer (p>0.05). ages at death varied from 30 to 81 years, with a mean of 49.08 years. the sample was divided into age groups, as displayed in table 1. this table also shows the comparative analysis between estimated and chronological ages for each group performed with paired t test. the sample was also divided according to gender (table 2). table 3 shows compares the differences in age estimation using lamendin’s formula with and without adjustment to brazilian population. discussion although lamendin et al.9 (1992) states that the method is not suitable to estimate the age of young adults, the formula produced better results in that group of the brazilian sample with a mean error (me) of 1.22 in the <39-year-old group. it is worth noting that, as individuals between 40 and 45 years were included in the group, accuracy and variation improved, suggesting that this technique is very efficient in this age group. several authors had similar results10-14, but others also presented better outcomes in groups over 50 years of age15-16. the other age groups did not present statistically significant results, but the 40-59-year-old group had a much lower me than group >60. this shows that the method is not suitable for individuals over 60 years and it may be used to estimate only the age range for subjects between 40 and 60 years. most studies show similar results, in which me for adults over 60 years of age presents a high variation (me>15)10-14,16. it is also agreed among the researchers that in older groups, age is underestimated while in younger adults, age is overestimated10-13,15-16. in the total sample, age was best estimated in males, which could be explained by the difference in sample size. although no significant differences were observed between genders, it may be noticed that in the 40-59-year-oldgroup females showed a much lower me than males. a study on a larger sample is required to clarify if this difference has statistical significance. the literature shows diverging results as to the correlation of the technique with gender; some authors found best results for males10,14, as opposed to others that observed better estimates forfemales16, while other study reported no difference regarding gender15. in general, authors9,11,13-14,16 do not specify several sample conditions, such as inhumation, exposure to substances or state of teeth. in the present study such aspects are reported because they may influence or even determine the conservation of anatomical structures (teeth), which directly affects the method application. it was also observed that some studies do not make clear whether they were blinded or not, but performed intraand inter-examiner10,16 correlations, one of theminaccurate11. in the present study both criteria were respected to assure greater reliability and reproducibility of results. ubelaker and prince15 (2002) observed differences between blacks and whites, but did not go deep into the subject, not specifying whether a group would be more accurate than the other, unlike martrille et al.16 (2007) who observed better results in whites. foti et al.10 (2001) found higher accuracy in the mandible than in the maxilla. in the present study these classifications were not considered, mainly to have a closer approach to the forensic reality, where the age estimation by teeth periodontosis and transparency: accuracy of lamendin’s method on a brazilian sample year n mean mean mean error standard p-value range estimated age chronological age (me) deviation (sd) <39 16 36.00 34.37 1.62 1.22 0.204 40-59 18 38.88 46.44 -7.55 2.52 0.008 >60 15 46.86 67.93 -21.06 2.43 0.000 <45 26 38.00 37.38 0.61 0.97 0.532 >45 23 43.08 62.30 -19.21 2.06 0.000 table 1table 1table 1table 1table 1estimated age compared with chronological age gender and age male (total) female (total) <39 male female 40 – 59 male female >60 male female n 35 14 12 4 14 4 9 6 mean estimated age 39.91 41.57 35.33 38.00 38.78 39.25 47.77 45.50 mean chronological age 47.57 52.85 34.00 35.50 47.28 43.50 66.11 70.66 m e -7.65 -11.28 1.33 2.50 -8.50 -4.25 -18.33 -25.16 s d 11.77 14.48 5.46 3.00 12.00 2.50 7.95 10.66 p 0.000 0.012 0.416 0.194 0.020 0.042 0.000 0.002 table 2table 2table 2table 2table 2estimated age compared with chronological age according to gender. male x female p=0.413 20 braz j oral sci. 13(1):17-21 expert does not know initially the age of a skull or a single tooth. most of the collected teeth were canines (n=20) and lateral incisors (n=17); there were only 3 premolars and 6 central incisors. therefore, the number of different types of teeth was insufficient to perform a statistical analysis. it has been suggested that there are significant differences between populations 11-15,17. for this reason, a specific formula for brazilians was developed by linear regression analysis. the new formula is as follows: a = (p x 0.18) + (t x 0.47) + 31.77 where, a = age; p = (periodontosis height x 100)/ root height; and t = (transparency height x 100)/root height. age estimation by teeth periodontosis and transparency: accuracy of lamendin’s method on a brazilian sample table 3table 3table 3table 3table 3 – sample data, chronological age and estimates by the lamendin original method and adjusted to the brazilian population sample chronological ages (ca) lamendin original (lo) ages difference 1 (lo-ca) lamendin adjusted(la) ages difference 2 (la-ca) 1 32 37.75 5.75 45.26 13.26 2 43 53.43 10.43 62.99 19.99 3 76 49.38 -26.62 58.32 -17.68 4 57 36.59 -20.41 43.76 -13.24 5 43 39.21 -3.79 46.61 3.61 6 61 53.75 -7.25 63.35 2.35 7 46 38.96 -7.05 46.35 0.35 8 34 32.72 -1.28 39.81 5.81 9 37 42.61 5.61 50.15 13.15 10 34 39.89 5.89 47.59 13.59 11 37 36.80 -0.20 44.18 7.18 12 36 35.30 -0.70 42.40 6.40 13 65 53.20 -11.80 62.74 -2.26 14 74 48.01 -25.99 56.42 -17.58 15 30 36.11 6.11 43.60 13.60 16 58 33.12 -24.88 40.03 -17.97 17 77 42.48 -34.52 50.47 -26.53 18 81 52.22 -28.78 61.15 -19.85 19 35 37.13 2.13 44.75 9.75 20 61 41.30 -19.70 49.09 -11.91 21 41 39.26 -1.74 46.94 5.94 22 44 39.04 -4.96 46.89 2.89 23 64 44.65 -19.35 53.09 -10.91 24 61 51.01 -9.99 60.28 -0.72 25 46 39.24 -6.76 47.12 1.12 26 40 39.60 -0.40 47.00 7.00 27 34 33.88 -0.12 41.12 7.12 28 30 32.82 2.82 39.93 9.93 29 77 41.89 -35.11 49.74 -27.26 30 36 29.14 -6.86 35.81 -0.19 31 58 27.27 -30.73 33.51 -24.49 32 74 46.01 -27.99 54.69 -19.31 33 65 49.22 -15.78 58.11 -6.89 34 35 36.66 1.66 44.15 9.15 35 44 37.49 -6.51 44.93 0.93 36 61 55.05 -5.95 64.81 3.81 37 46 37.01 -8.99 44.62 -1.38 38 61 36.69 -24.31 44.26 -16.74 39 40 44.20 4.20 52.06 12.06 40 41 38.28 -2.72 45.81 4.81 41 43 42.95 -0.05 50.98 7.98 42 32 27.96 -4.04 34.20 2.20 43 37 39.06 2.06 46.45 9.45 44 61 43.96 -17.04 52.40 -8.60 45 46 44.91 -1.09 53.46 7.46 46 34 48.52 14.52 57.25 23.25 47 57 34.24 -22.76 41.51 -15.49 48 37 38.35 1.35 45.95 8.95 49 43 42.16 -0.84 49.72 6.72 mean error (me) -8.25518 -0.39132 21 braz j oral sci. 13(1):17-21 in order to test its accuracy in a brazilian population, a different sample would be required, which was not available for this research. in addition, the sample used to develop this formula was relatively small, and the youngest individuals were 30 years old, which made the constant to be relatively high and impaired age estimation of adults younger than 31 years of age. lamendin’s method is a suitable option for estimating ages of brazilians between 30 and 45 years old. the technique loses its efficacy in other age groups, but with proper care, it could be used to help establishing age ranges for individuals between 45 and 60 years. further research is required to test the suggested formula or even develop a new one with better results in the brazilian population. references 1. moorrees cf, fanning ea, hunt ee jr. age variation of formation stages for ten permanent teeth. j dent res. 1963; 42: 1490-502. 2. demirjian a, goldstein h, tanner jm. a new system of dental age assessment. hum biol. 1973; 45: 211-27. 3. fernandes mm, tinoco rlr, braganca dpp, lima shr, francesquini junior l, daruge junior e. age estimation by measurements of developing teeth: accuracy of cameriere’s method on a brazilian sample. j forensic sci. 2011; 56: 1616-9. 4. cameriere r, ferrante l, cingolani m. age estimation in children by measurement of open apices in teeth. int j legal med. 2006; 120: 49-53. 5. gustafson g. age determination on teeth. j am dent assoc. 1950; 41: 45-54. 6. bang g, ramm e. determination of age in humans from root dentin transparency. acta odontol scand. 1970; 28: 3-35. 7. kvaal si, kolltveit km, thomsen io, solheim t. age estimation of adults from dental radiographs. forensic sci int. 1995; 74: 175-85. 8. cameriere r, ferrante l, cingolani m. variations in pulp/tooth area ratio as an indicator of age: a preliminary study. j forensic sci. 2004; 49: 317-9. 9. lamendin h, baccino e, humbert jf, tavernier jc, nossintchouk rm, zerilli a. a simple technique for age estimation in adult corpses: the two criteria dental method. j forensic sci. 1992; 37: 1373-9. 10. foti b, adalian p, signoli m, ardagna y, dutour o, leonetti g. limits of the lamendin method in age determination. forensic sci int. 2001; 122: 101-6. 11. sarajliæ n, cihlarz z, klonowski ee, selak i, brkiæ h, topiæ b. twocriteria dental aging method applied to a bosnian population: comparison of formulae for each tooth group versus one formula for all teeth. bosn j basic med sci. 2006; 6: 78-83. 12. meinl a, huber cd, tangl s, gruber gm, teschler-nicola m, watzek g. comparison of the validity of three dental methods for the estimation of age at death. forensic sci int. 2008; 178: 96-105. 13. prince da, konigsberg lw. new formulae for estimating age-at-death in the balkans utilizing lamendin’s dental technique and bayesian analysis. j forensic sci. 2008; 53: 578-87. 14. ubelaker dh, parra rc. application of three dental methods of adult age estimation from intact single rooted teeth to a peruvian sample. j forensic sci. 2008; 53: 608-11. 15. prince da, ubelaker dh. application of lamendin’s adult dental aging technique to a diverse skeletal sample. j forensic sci. 2002; 47: 107-16. 16. martrille l, ubelaker dh, cattaneo c, seguret f, tremblay m, baccino e. comparison of four skeletal methods for the estimation of age at death on white and black adults. j forensic sci. 2007; 52: 302-7. 17. gonzález-colmenares g, botella-lópez mc, moreno-rueda g, fernández-cardenete jr. age estimation by a dental method: a comparison of lamendin’s and prince & ubelaker’s technique. j forensic sci. 2007; 52: 1156-60. age estimation by teeth periodontosis and transparency: accuracy of lamendin’s method on a brazilian sample art 1 blood clot braz j oral sci. 13(2):83-88 original article braz j oral sci. april | june 2014 volume 13, number 2 blood clot stabilization on root dentin conditioned by the combination of tetracycline and edta gustavo giacomelli nascimento1, amauri antiquera leite2, elza regina manzolli leite2, josé eduardo cezar sampaio3, fábio renato manzolli leite1 1 universidade federal de pelotas – ufpel, school of dentistry, department of semiology and clinics, pelotas, rs, brazil 2 universidade estadual paulista unesp, school of pharmaceutical sciences, department of clinical analysis, araraquara, sp, brazil 3 universidade estadual paulista unesp, araraquara dental school, department of diagnosis and surgery, araraquara, sp, brazil correspondence to: fábio renato manzolli leite universidade federal de pelotas faculdade de odontologia departamento de semiologia e clínica rua gonçalves chaves, cep 96015-560 centro, pelotas, rs, brasil phone: +55 53 3225 6741 e-mail: leite.fabio@odontoufpel.com.br abstract aim: to assess the combined use of tetracycline (ttc) and ethylenediaminetetraacetic acid (edta) on clot formation, considering that edta may neutralize ttc acidity. methods: planed human tooth roots were treated with saline solution, edta, ttc and their combination (edta followed by ttc and ttc before edta). fresh human blood was applied on the conditioned surfaces to check clot adhesion and stabilization. a previously calibrated (kappa = 0.93) and blinded examiner scored scanning electron micrographs of the samples. statistical analyses were performed using one-way anova and tukey’s test. results: application of ttc before edta presented the best results with the highest number of cells adhered to the root surface (p=0.046). use of edta alone and edta before ttc disturbed clot stabilization when compared to control group (p<0.01). conclusions: the use of ttc before edta seems to be able to keep blood cells viable to establish an organized clot and could be used by clinicians together with the conventional mechanical root scaling and planing. keywords: ethylenediaminetetraacetic acid; periodontal regeneration; scanning electron microscopy; smear layer. introduction one of the main goals of periodontal therapy is to obtain new connective tissue attachment or reattachment to roots already exposed to oral environment1. histological and ultrastructural studies have demonstrated that dental roots exposed to the oral cavity or to periodontal pockets present reduced collagen fiber insertion2, change their mineral density and contaminate them by bacteria and their products. scaling and root planing are the best techniques for mechanical decontamination. however, these methods per se are not able to fully eliminate the etiological contaminants, and they produce a compact smear layer that covers the instrumented surface inhibiting periodontal reattachment 3. for these reasons, the use of demineralizing agents has been studied as an adjunct to periodontal therapy, since these agents present a great potential not only for removing smear layer4, but also to expose the underlying radicular collagen fibrils and to restore the biocompatibility of the roots5. these properties are necessary to increase fibrin received for publication: january 24, 2014 accepted: march 25, 2014 84 network linkage to root collagen fibers, resulting in a better periodontal regenerative procedure4,6. some reports have demonstrated that demineralization of the root surface could not only exert neutralizing effects on endotoxins from periodontal pathogens, but also induce fibroblast proliferation, synthesis and attachment 7. additionally, studies have shown that in comparison to nonconditioned teeth, acid conditioned tooth roots presented a greater tendency to maintain fibrin clot, exposing collagen fibrils and increasing the levels of proteoglycans8. an improvement of biological response on root surface after conditioning with demineralizing agents has been described9. in vivo human9 histological studies have shown improved biological response when decalcifying/chelating agents are used to condition the root surface. citric acid, phosphoric acid, ethylenediaminetetraacetic acid (edta) and tetracycline hydrochloride (ttc)10 are considered the most used agents for this purpose. despite the well-described uses of these agents, there is lack of standardization of the procedures employed by clinicians and researchers. thus, the great variability of protocols has avoided reliable comparisons among them. moreover, there is only one systematic review about this subject, published by mariotti in 200311. the author concluded that use of citric acid, ttc or edta to modify the root surface provided no clinically significant benefit for regeneration in patients with chronic periodontitis11. nevertheless, several factors such as lack of controls, noncalibrated examiners, masked reference standards and small sample sizes among others, reduced the quality of relevant studies, so the conclusions of mariotti’s review must be carefully considered. many studies showed that edta might have controversial results on tissue repair, since its use could disturb clot formation and ttc may kill connective tissue cells due to its acid ph. thus, the aim of this study was to compare the influence of root conditioning with ttc, edta and their combination on blood elements adsorption and adhesion to root surfaces. the main goal was to check the possible combined use of ttc with edta on clot stabilization, since edta may neutralize ttc acidity. material and methods this study was approved by the research ethics committee of the school of dentistry at araraquara, unesp – univ estadual paulista (protocol #10/04). all patients signed an informed consent. freshly extracted human teeth with periodontal disease characterized by at least 6 mm attachment loss and bleeding on gentle probing, absence of caries or restorations below the cementoenamel junction4 were used. teeth were individually placed in capped tubes containing saline solution. sample preparation: the cervical third of roots was chosen for sample preparation. specimens were prepared by making two parallel 0.5-mm-deep grooves on the buccal and lingual root surfaces using a high-speed cylindrical bur (kg sorensen, barueri, sp, brazil) under copious irrigation. one groove was made at the cementoenamel junction and the other approximately 3 mm distant from this point, in the apical direction. the same bur was used to remove the surface layer of the root between the two grooves. the area between grooves was then scaled with 50 apical-cervical strokes using a sharp #56 gracey curette (hu-friedy, chicago, il, usa) in order to create a smear layer. samples were stored in containers with saline solution (n=50). specimens were divided into five groups (n=10): 1) irrigation with 10 ml of saline solution (control gi); 2) fresh tetracycline hydrochloride 25 mg/ml (valde química, são paulo, sp, brazil) (ttc gii)12; 3) application of 24% edta prefgel (biora ab, malmö, sweden) (giii); 4) ttc used before edta (giv); 5) edta used before ttc (gv). substances were applied with a soft brush for 3 min, changing solutions every 30 s. immediately after, teeth were washed with 10 ml of sterile saline solution. fresh human blood from a healthy male donor was placed on dentin surface and allowed for 20 min to form clot in a humidified chamber at 37 °c (figure 1). specimens were then rinsed three times for 5 min in pbs. washes and rinses of the root specimens were performed in small petri dishes with a gentle swirling motion using a rotating tabletop shaker at low speed. after rinsing, the specimens were fixed in 1% formaldehyde diluted in pbs for 15 min. after three 5-min pbs rinses, the samples were incubated for 10 min in 0.02 m glycine diluted in pbs and rinsed again. specimens were post-fixed in 2.5% glutaraldehyde in pbs for 30 min and rinsed again. dehydration was performed by immersion in an increasing ethanol series: 30%, 50%, 75%, 90%, 95% and three exchanges of 100%. the specimens were dried in a carbon dioxide critical point drier (baltec cpd 030; baltec union ltd., liechtenstein) and then dried at room temperature. samples were mounted on aluminum stubs with colloidal graphite, sputter coated with gold palladium in a specified device (baltec scd 050, bal-tec union ltd.,), and stored and desiccated at room temperature for 3 days10. sem examination: sem micrographs (500× and 1000×) from the central area of each specimen were obtained in a scanning electron microscope (jeol jsm-t330a; miaka, tokyo, japan) adjusted to 20 kv and evaluated by a trained and calibrated (kappa = 0.93) examiner blinded to the experimental groups10,12. sem micrographs of the specimens that received blood application were evaluated by the blood elements adhesion index (beai)10 shown in figure 2: score 0: absence of fibrin network and blood cells. score 1: scarcely distributed fibrin network and/or blood cells. score 2: moderate number of blood cells and thin fibrin network with poor interlacing. score 3: dense fibrin network with rich interlacing and presence of blood cells. blood clot stabilization on root dentin conditioned by the combination of tetracycline and edta braz j oral sci. 13(2):83-88 85 braz j oral sci. 13(2):83-88 fig. 2 – blood elements adhesion index (beai) score. a: absence of fibrin network and blood cells (score 0); b: scarcely distributed fibrin network and/or blood cells (score 1); c: moderate number of blood cells and thin fibrin network with poor interlacing (score 2); d: dense fibrin network with rich interlacing and presence of blood cells (score 3). fig. 1 – sample preparation. a: parallel grooves; b: root scaling; c: dentin samples; d: solution application with soft brush; e: blood drop allowed to clot. blood clot stabilization on root dentin conditioned by the combination of tetracycline and edta 86 braz j oral sci. 13(2):83-88 each sem micrograph was evaluated three times in intervals of at least 7 days. the score attributed to each sample was the predominant score in the three evaluations. statistical analysis beai results for each group were compared by one-way anova and tukey’s test at a significance level of p<0.05. analyses were performed using the graphpad instat software 5.05 (graphpad software, inc., san diego, ca ). results giv presented the best results among the groups, with 80% of the specimens presenting scores 2 and 3. sixty percent of the specimens in the control group (gi) received score 2. frequency distribution according to groups and scores is exhibited in table 1. a great variation in morphological aspects of fibrin network and blood cells amount was observed. analyzing separately the scores of each group, the samples of giii and gv had a similar distribution of scores, showing that these treatments are able to provide homogeneous final results. in gi and gii the scores ranged from 0 to 2. anova test rejected the hypothesis that all groups have the same performance in clot stabilization (p<0.001). variation among groups’ means was significantly greater than expected by chance. edta presented the worst results regarding clot organization compared to all groups. the use of ttc before edta increased the clot stabilization potential (p=0.046) and ttc before edta improved clot organization when compared to the use of ttc after edta (p<0.05). edta alone and edta before ttc conditioning disturbed clot stabilization compared to control group (p<0.01). figure 3 summarizes anova and tukey test results. discussion to the best of our knowledge, this is the first study to evaluate the effects of edta and ttc used in combination. both agents are the most used for conditioning root surface adjunct to conventional periodontal treatment. as previously described, scaling and root planing are unable to decontaminate completely the root surface and produce a smear layer13 formed by remnants of calculus, plaque and contaminated dental hard tissues (cement and dentin). smear control (gi) ttc (gii) edta (giii) ttc+edta (giv) edta+ttc (gv) n(%) n(%) n(%) n(%) n(%) score 0 2 (20) 5 (50) 10 (100) 0 (0) 9 (90) score 1 2 (20) 3 (30) 0 2 (20) 1 (10) score 2 8 (60) 2 (20) 0 3 (30) 0 score 3 0 (0) 0 0 5 (50) 0 total 10 10 10 10 10 table 1: table 1: table 1: table 1: table 1: frequency distribution of scores according to blood elements adhesion index (beai) score 0: absence of fibrin network and blood cells; score 1: scarce fibrin and cells; score 2: moderate cells and thin fibrin network; score 3: dense fibrin with blood cells; ttc: tetracycline; edta: ethylenediaminetetraacetic acid layer is composed by very small particles of organic and inorganic material varying in size from less than 1 µm to over 15 µm. this layer is in intimate contact with the tooth surface and it is only removed by applying a demineralizing solution14. smear layer removal by conditioning agents promotes collagen union between the exposed root surface fibrils and soft tissue collagen fibrils15. the binding of the two sources of collagen by fibronectin in the early stages of wound repair inhibits the downgrowth of oral epithelium and enhances fibroblast chemotaxis, migration and attachment15. in this way, clinical outcome tends to improve with more consistent results. ttc became one of the most widely used and studied demineralizing agent since in vitro studies16 suggested its potential use in regenerative procedures. ttc is an antibiotic with affinity for mineralized tissues; it is adsorbed and then slowly released to surrounding tissues for up to 14 days. many concentrations and application times were tested ranging from 0.5% to 200% and from 0.5 to 10 min17. the low ph of the saturated solutions of ttc has been suggested as one of the reasons for the reduced cellular insertion and for the unpredictable results, as it could denature the organic matrix of dentin16. it was also suggested that acid etching would interfere on periodontal healing by its necrotizing effect on the surrounding progenitor cells5. fig. 3 blood clot stabilization on root dentin conditioned by tetracycline and/or edta. results of blood elements adhesion index (beai) comparison between groups by anova and tukey’s tests (mean and standard deviation). groups with different letters had statistically significant differences (p<0.05). blood clot stabilization on root dentin conditioned by the combination of tetracycline and edta 87 braz j oral sci. 13(2):83-88 edta was used in this study to investigate the effect of non-acidic substances to stabilize clot. our findings demonstrated that 24% edta failed to produce a compatible root surface for clot stabilization, corroborating previous studies18-19. all samples in this group had no attached cells or fibrin. edta at 12%-24% concentrations with neutral ph was introduced aiming to remove the smear layer without damaging biological structures20. notwithstanding, some authors 8 presented reliable findings using monoclonal antibodies and field emission in-lens scanning electron microscopy (feisem) showing that edta treatment is able to etch and expose collagen fibrils and proteoglycans without any degradation of dentin collagen matrix. it is speculated whether edta residues interfere on clot stabilization. a meta-analysis study suggested that guided tissue regeneration-based root coverage with root conditioning could be used successfully to repair gingival recession defects21. according to those results, the use of absorbable membranes and root conditioning, resulted in significantly improved percentages of sites with complete root coverage. clot stabilization seen in the present results may be important as a scaffold for the penetration and proliferation of the surrounding tissues. in this study, edta improved ttc (giv) results increasing the amount of samples with score 3 (dense fibrin network with presence of blood cells). a possible explanation is that ttc action by low ph denatures the organic matrix of dentin and using edta as a neutralizing agent, probably avoids an intense dentin demineralization, preserving a collagen scaffold for clot stabilization and proliferation of connective tissue cells22. clot formation and adhesion are the initial steps of wound healing after periodontal therapy. a rapid adherence of the clot to root surface acts as a physical barrier reducing epithelial apical migration and avoiding a long junctional epithelium formation4. acid conditioning agents such as citric acid and ttc cause the acidification of extracellular medium, which induces cell death, and some authors suggest that ph elevation can prevent or reduce tissue damage5. the use of edta after ttc as a neutralizing agent showed promising results in clot organization. in vivo studies should be carried out to verify whether this combination maintains the connective tissue cells more viable to adhere on root surface and if the slow release of ttc after therapy is maintained. a previous study already tested the combination of ttc with citric acid as subgingival irrigants after root scaling and planing 23. the authors found a synergistic effect of the two agents and a more powerful demineralizing potential than ttc-containing gel. the only systematic review about this subject indicated no significant clinical effect of root conditioning on tissue repair11. however, the author highlighted that several factors such as lack of controls, non-calibrated and non-blinded examiners, and small sample sizes reduced the quality of most studies. thus, the author stated that the conclusions of his review must be critically considered11. it was concluded that compared with ttc alone, the use of edta after ttc in an attempt to neutralize its acid ph, improved clot formation and organization in vitro, indicating that this may be a promising therapy. more clinical studies are required to verify the effects of root conditioning as an adjunct to conventional periodontal treatment. acknowledgements this study was supported by the são paulo state foundation fapesp (grants 03/04252-2 and 03/04754-8). references 1. theodoro lh, zezell dm, garcia vg, haypek p, nagata mj, de almeida jm, et al. comparative analysis of root surface smear layer removal by different etching modalities or erbium:yttrium-aluminum-garnet laser irradiation. a scanning electron microscopy study. lasers med sci. 2010; 25: 485-91. 2. adriaens pa, adriaens lm. effects of nonsurgical periodontal therapy on hard and soft tissues. periodontol 2000. 2004; 36: 121-45. 3. blomlof j, lindskog s. root surface texture and early cell and tissue colonization after different etching modalities. eur j oral sci. 1995; 103: 17-24. 4. leite fr, moreira cs, theodoro lh, sampaio je. blood cell attachment to root surfaces treated with edta gel. braz oral res. 2005; 19: 88-92. 5. zandim dl, leite fr, da silva vc, lopes bm, spolidorio lc, sampaio je. wound healing of dehiscence defects following different root conditioning modalities: an experimental study in dogs. clin oral investig. 2013; 17: 1585-93. 6. leite fr, nascimento gg, leite er, leite aa, sampaio je. effect of the association between citric acid and edta on root surface etching. j contemp dent pract. 2013; 14: 5. 7. fardal o, lowenberg bf. a quantitative analysis of the migration, attachment, and orientation of human gingival fibroblasts to human dental root surfaces in vitro. j periodontol. 1990; 61: 529-35. 8. ruggeri a jr, prati c, mazzoni a, nucci c, di lenarda r, mazzotti g, et al. effects of citric acid and edta conditioning on exposed root dentin: an immunohistochemical analysis of collagen and proteoglycans. arch oral biol. 2007; 52: 1-8. 9. gamal ay, mailhot jm. the effects of edta gel conditioning exposure time on periodontitis-affected human root surfaces: surface topography and pdl cell adhesion. j int acad periodontol. 2003; 5: 11-22. 10. dantas aa, fontanari la, ishi ede p, leite fr, zandim dl, rached rs, et al. blood cells attachment after root conditioning and prp application: an in vitro study. j contemp dent pract. 2012; 13: 332-8. 11. mariotti a. efficacy of chemical root surface modifiers in the treatment of periodontal disease. a systematic review. ann periodontol. 2003; 8: 205-26. 12. leite fr, sampaio je, zandim dl, dantas aa, leite er, leite aa. influence of root-surface conditioning with acid and chelating agents on clot stabilization. quintessence int. 2010; 41: 341-9. 13. baker dl, stanley pavlow sa, wikesjo um. fibrin clot adhesion to dentin conditioned with protein constructs: an in vitro proof-of-principle study. j clin periodontol. 2005; 32: 561-6. 14. cavassim r, leite fr, zandim dl, dantas aa, sampaio je. smear layer removal for collagen fiber exposure after citric acid conditionings. j contemp dent pract. 2010; 11: e001-8. 15. heritier m. effects of phosphoric acid on root dentin surface. a scanning and transmission electron microscopic study. j periodontal res. 1984; 19: 168-76. 16. amaral ng, rezende ml, hirata f, rodrigues mg, sant’ana ac, greghi sl, et al. comparison among four commonly used demineralizing agents for root conditioning: a scanning electron microscopy. j appl oral sci. 2011; 19: 469-75. blood clot stabilization on root dentin conditioned by the combination of tetracycline and edta 17. isik ag, tarim b, hafez aa, yalcin fs, onan u, cox cf. a comparative scanning electron microscopic study on the characteristics of demineralized dentin root surface using different tetracycline hcl concentrations and application times. j periodontol. 2000; 71: 219-25. 18. minocha t, rahul a. comparison of fibrin clot adhesion to dentine conditioned with citric acid, tetracycline, and ethylene diamine tetra acetic acid: an in vitro scanning electron microscopic study. j indian soc periodontol. 2012; 16: 333-41. 19. preeja c, janam p, nayar br. fibrin clot adhesion to root surface treated with tetracycline hydrochloride and ethylenediaminetetraacetic acid: a scanning electron microscopic study. dent res j (isfahan). 2013; 10: 382-8. 20. bogle g, garrett s, crigger m, egelberg j. new connective tissue attachment in beagles with advanced natural periodontitis. j periodontal res. 1983; 18: 220-8. 21. al-hamdan k, eber r, sarment d, kowalski c, wang hl. guided tissue regeneration-based root coverage: meta-analysis. j periodontol. 2003; 74: 1520-33. 22. fontanari la, pinto sc, cavassim r, spin-neto r, ishi ede p, sampaio je. influence of dental exposure to oral environment on smear layer removal and collagen exhibition after using different conditioning agents. braz dent j. 2011; 22: 479-85. 23. george rp, kumar s, ramakrishna t, emmadi p, ambalavanan n. effects of tetracycline-containing gel and a mixture of tetracycline and citric acid-containing gel on non-surgical periodontal therapy. indian j dent res. 2013; 24: 52-9. 88 blood clot stabilization on root dentin conditioned by the combination of tetracycline and edta braz j oral sci. 13(2):83-88 oral sciences n3 original article braz j oral sci. july | september 2013 volume 12, number 3 screw loosening of different ucla-type abutments after mechanical cycling marcela c. junqueira, ricardo f. ribeiro, adriana claudia l. faria, ana paula macedo, rossana p. almeida department of dental materials and prosthodontics, dental school of ribeirão preto, university of são paulo, ribeirão preto, sp, brazil correspondence to: rossana pereira de almeida faculdade de odontologia de ribeirão preto universidade de são paulo av. do café s/n, cep: 14040-904 ribeirão preto, sp, brasil phone: +55 16 36024411 fax: +55 16 36024780 e-mail: rpaa@forp.usp.br received for publication: may 28, 2013 accepted: september 20, 2013 abstract aim: to evaluate the loss of applied torque (detorque) values in cast and pre-machined abutments for external hex abutment/implant interface of single implant-supported prostheses subjected to mechanical cycling. methods: ten metal crowns were fabricated using two types of ucla abutments: cast and pre-machined with metal base in nicrti alloy and tightened to regular external hex implants with a titanium alloy screw, with an insertion torque of 32 n.cm, measured with a digital torque gauge. samples were embedded with autopolymerizing acrylic resin in a stainless steel cylindrical matrix, and positioned in an electromechanical machine. dynamic oblique loading of 120 n was applied during 5 x 105 cycles. then, each sample was removed from the resin and detorque values were measured using the same digital torque gauge. the difference of the initial (torque) and final (detorque) measurement was registered and the results were expressed as percentage of initial torque. the results of torque loss were expressed as percentage of the initial torque and subjected to statistical analysis by the student’s t-test (p<0.05) for comparisons between the test groups. results: statistical analysis demonstrated that mechanical cycling reduced the torque of abutments without significant difference between cast or pre-machined ucla abutments (p=0.908). conclusions: within the limitations of this in vitro study, it may be concluded that the mechanical cycling, corresponding to one-year use, reduced the torque of the samples regardless if cast or pre-machined ucla abutments were used. keywords: dental implant, single tooth implant, external hexagon. introduction the mechanical stability of implant-supported fixed restorations may be considered to improve long-term stability and minimize complications1. the stability of the connection between different implant parts is important for the success of the rehabilitation, especially for single tooth restorations. loosening of abutment screws, mainly with the external hex implants, has been a technical problem that occurs in the first two years of use2. the stability of the external implant-abutment connection has been improved by altering the screw alloys and their surfaces and applying proper torque values to establish higher initial preloads3-5. a systematic review compared the complications of screw-retained prosthesis showing that the most frequent complication was related to abutment screw loosening (10-55.5%). the incidence of abutment screw loosening was 4.3% in short-term studies and 10% in long-term studies1. mechanical factors, such as the implant-abutment fit and the abutment screw preload are involved in the success of implant rehabilitation6. the preload loss during the occlusal load favors the misfit of the implant-abutment connection and may cause screw loosening and fracture. implant biological factors may be braz j oral sci. 12(3):228-232 affected due to microgap formation, which can cause periimplantitis7. in vitro and clinical studies have demonstrated the correlation between rotation of the abutment and prosthetic screw loosening and showed the importance of reducing to a minimum the implant-abutment misfit in order to avoid mechanical complications8-9. the hexagonal configuration prevents abutment rotation on the implant surface and provides a stable screw joint assembly. the amount of freedom between the implant hexagonal extension and its abutment counterpart has also been implicated as a factor in screw joint instability. the applied torque and the masticatory load could generate micromovements, deforming the implant hexagon. studies have indicated a direct correlation between implant-abutment rotational misfit and screw loosening9-12. preload is the tension on a screw generated when a torqueing force is applied to the screw head. occlusal forces play an important role in screw loosening of hexagonal connection implants; preload is the only force that resists to functional occlusal forces in order to maintain the abutment stability, preventing its separation from the implant. when the preload is exceeded by the occlusal force, the screw will loosen13-14. several mechanisms can cause screw loosening; one is the embedment relaxation of mating thread surfaces15. normally, when a screw is tightened, most of the screw responds elastically (plastic deformation occurs only at spots of machining microroughness and asperities at thread flanks). thus, preload produces a clamping force between the screw head and its seat. the behavior and life of a screw joint depends mainly on the magnitude and stability of that clamping force. in general, the greater the clamped force (preload), the tighter the clamped joint. however, preload values should not be too high and should be within the elastic limit, because retaining screws may yield or break under repeated functional bite forces. on the other hand, the preload values should not be too low in order to retain loose screws under repeated functional forces16. eccentric and compressive forces are generated during chewing movements and influence the screw retention3,8,17-20. the optimal preload values for the implant/abutment screw joint have not been fully identified and in single tooth implants this value is critical for screw joint stability21. the purpose of this study was to evaluate the loss of applied torque (detorque) values in cast and pre-machined (ucla) type abutments for external hex abutment/implant interface of single implant-supported prostheses subjected to mechanical cycling. the null hypothesis was that torque loss of cast and pre-machined ucla abutments submitted to the mechanical cycling is similar. material and methods ten implants with external hexagon (titamax ti cortical; neodent, curitiba, pr, brazil) measuring 3.75 mm in diameter and 13 mm long, five castable ucla abutments (neodent) and five tilite pre-machined ucla abutments (neodent), both with 4.1 mm platform size, were used in this study. ten metal crowns were fabricated using the two types of abutments: cast and pre-machined ucla abutments. all crowns were fabricated according to a silicone matrix (silicone master; talmax, curitiba, pr, brazil) to present similar dimensions. the patterns were invested in a rapid cycle, carbonfree, phosphate-bonded investment (castorit super c; dentaurum, ispringen, germany) and cast using a nickelchromium alloy (ni-cr, verabond ii; aalba dent inc., cordelia, ca, usa) to the castable ucla abutments and nickelchromium-titanium alloy (ni-cr-ti, tilite omega; talladium, valencia, ca, usa) to the pre-machined ucla abutments. castings were allowed to bench cool and after divesting were lightly abraded by airborne particles of 100-µm aluminum oxide (polidental, são paulo, sp, brazil) at 90 psi pressure, followed by water washing and air drying. no further polishing or finishing was performed. the abutments were fixed to each implant using titanium screws (ti6al4v) (neodent). the implants were fixed in a metallic matrix with a lateral screw in order to prevent implant rotation. the set implant/metallic matrix was placed at the base of a torque application device developed in the department of dental materials and prosthodontics of the dental school of ribeirão preto, university of são paulo. a digital torque meter was attached to the top of the device (tq-680; instrutherm, são paulo, sp, brazil). initially, the crowns were slightly screwed to the implants by hand22. then the set was placed in a socket at the device base. this socket allows only rotational movement. the crowns were torqued to the implants (32 n.cm), according to manufacturer’s recommendation, using a hexagonal wrench. after 3 min, the screw was retightened to the same torque to minimize embedment relaxation23. the placement torque was measured by the digital torquemeter with a 0.1 precision. the sequence is shown on figure 1 (a-d). the implant and metal crown were embedded in autopolymerizing acrylic resin (jet; clássico produtos odontológicos ltda., são paulo, sp, brazil) in a stainless steel cylindrical matrix to standardize the positioning with a 30° inclination relative to the vertical axis24. an autopolymerizing acrylic resin was used due to its appropriate elastic modulus (3gpa) for a bone analog material25. the replicas were positioned in an electromechanical machine (msfm; elquip, são carlos, sp, brazil) and immersed in distilled water at 37±2 °c. dynamic oblique loading of 120 n was applied to each replica during 5x105 cycles. the load was applied with a metal cylinder with 4 mm in diameter. the machine was set to work at a frequency of 101 cycles per minute, simulating to the human chewing frequency26 (fig. 2). after mechanical loading, each sample was removed from the resin and returned to the torque application equipment in the same initial position, and detorque values were measured with the digital torquemeter. to remove the screw, torque was applied in a counterclockwise direction, using a hexagonal wrench attached to the digital torquemeter. the digital torque was recorded immediately after releasing the screw. the results of torque loss were expressed as percentage of the initial torque and subjected to statistical analysis by kolmogorov-smirnov normality test. the student’s t-test 229229229229229screw loosening of different ucla-type abutments after mechanical cycling braz j oral sci. 12(3):228-232 230230230230230 a b c d fig. 1. (a) digital torque meter and torque application device. (b) hexagonal wrench positioned. (c) crown and wrench before torque application. (d) application of torque using hexagonal wrench attached to a digital torque meter. fig. 2. (a) electromechanical machine. (b) the load was applied through a metal cylinder with 4 mm in diameter. samples were immersed in distilled water at 37°±2° c. screw loosening of different ucla-type abutments after mechanical cycling braz j oral sci. 12(3):228-232 a b (p<0.05) was used for comparisons between the groups, using the statistical program spss 17.0 (statistic package for the social science, version 17; spss inc., chicago, il, usa). results table 1 shows the results obtained for the different samples after mechanical cycling. no statistically significant difference was observed (p=0.908) when the two types of ucla abutments (cast or pre-machined) were compared (table 2). discussion since the branemark system was introduced in the market with an external hexagon to facilitate the implant insertion rather than to provide an antirotational device, several competing systems have used well this design over the years27. even though there were some failures and other design connections were introduced to overcome these failures, many patients received this design connection. currently, there is lack of conclusive evidence regarding abutment screw loosening to external hexagon implants, mainly those related to single restorations25. several factors may cause reduction or loss of preload in single tooth restorations such as casting procedures, superstructure inaccuracy, occlusal morphology and insertion torque, occlusal overload and physical properties of the screw materials28-29. kano et al. 21 studied the casting effect on torque maintenance by detorque measurements of ucla-type abutments and observed a detorque mean of 92.3% for the machined titanium abutments, 81.6% for the pre-machined palladium abutments cast with palladium, 86.4% for plastic abutments cast with nickel-chromium, and 84.0% for plastic abutments cast with cobalt-chromium alloy. in this study, there was no statistically significant difference between cast and pre-machined abutments (p=0.908). the detorque measurement after mechanical cycling revealed a reduction to 13.26±4.32 n.cm (41.4%) for the cast abutment and 13.72±7.36 n.cm (42.9%) for the pre-machined. despite the use of different abutments, torque reduction was observed for both groups, suggesting changes in the mating surfaces. any irregularities in the mating surfaces will likely result in preload reduction28. the casting procedures may have contributed to these results since the table 1. detorque data, expressed as percentage of initial torque. groups n mean standard deviation m i n i m u m median m a x i m u m cast 5 41.4 13.5 21.6 41.9 59.4 pre-machined 5 42.9 23.0 20.3 31.5 75.9 comparison mean difference p-value confidence interval lower limit upper limit cast x pre-machined -1.420 0.908 -28.914 26.074 table 2. comparison between cast and pre-machined ucla abutments 231231231231231 integrity of screw joint23 and material properties of metal components may be altered during casting21. therefore, it is important to point out that any irregularity in the mating surfaces should be detected, as changes occur between contacting parts when the screw is tightened because all the metallic contacting surfaces flatten slightly and the microscopic distance between contacting surfaces decreases21. other studies also compared plastic, pre-machined and machined abutments and concluded that lower preload has developed for all components subjected to casting15,21,30. this study confirms such results and shows that casting procedures can decrease detorque values even in pre-machined cast abutments, like those used. the reason seems to be the irregularities and roughness of contacting surfaces that may result from the casting process, which causes greater embedment relaxation and consequent preload loss15,30. the applied torque is distributed to the friction between the screw and the abutment, and between the threads of the screw and the implant, causing loosening. thus, screw loosening is only avoided if the applied preload remains constant. preload is a tension created in screw when a torque force is applied to the screw head and is affected by the screw material’s properties. preload produces a clamping force between the screw head and its seat. the behavior and durability of a screw joint depends mainly on the magnitude and stability of that clamping force. in this study, titanium screws were used, which have a higher friction coefficient than other materials, like gold. although the torque values have decreased after mechanical cycling, no movements of the replicas were observed macroscopically, which may indicate the maintenance of screw stability. then, this torque loss may not immediately reflect in an evident loosening of the joint, but if the process is allowed to continue, it may result in joint instability and separation of abutment from the implant15,31, fracture, patient discomfort and biological complications, such as periimplantitis, because of the microgap created at the interface7. further studies are required to verify the effects of a larger number of cycles on the longterm retention and stability of different abutments with external connection. it is difficult to predict clinical results by in vitro studies because there are many factors affecting the oral environment, but the results of the present study allow suggesting that the use of cast or pre-machined ucla abutments can present similar values of preload and torque loss after simulated use. it is also important to evaluate the mechanisms of the screw loosening of different ucla-type abutments after mechanical cycling braz j oral sci. 12(3):228-232 232232232232232 abutment/implant retention screw joint with the study of stress distribution. additional studies would be helpful to establish the clinical relevance of the present findings. within the limitations of this in vitro study, it may be concluded that the mechanical cycling, corresponding to one year of use, reduced the torque of the samples regardless if cast or pre-machined ucla abutments were used. references 1. chaar ms, att jr, strub jr. prosthetic outcome of cement-retained implant-supported fixed dental restorations: a systematic review. j oral rehabil. 2011; 38: 697-711. 2. simon rl. single implant-supported molar and premolar crowns: a tenyear retrospective clinical report. j prosthet dent. 2003; 90: 517-21. 3. siamos g, winkler s, boberick kg. the relationship between implant preload and screw loosening on implant-supported prostheses. j oral implantol. 2002; 28: 67-73. 4. ricomini filho ap, fernandes f, staioto fg, silva wj, del bel cury aa. preload loss and bacterial penetration on different implant-abutment connection systems. braz dent j. 2010; 21: 123-9. 5. guzaitis kl, knoernschild kl, viana mag. effect of repeated screw joint closing and opening cycles on implant prosthetic screw reverse torque and implant and screw thread morphology. j prosthet dent. 2011; 106: 159-69. 6. weng d, nagata mj, bell m, bosco af, melo lg, richter ej. influence of microgap location and configuration on the periimplant bone morphology in submerged implants. an experimental study in dogs. clin oral implants res. 2008; 19:1141-7. 7. jung re, pjetursson be, glauser r, zembic a, zwahlen m, lang np. a systematic review of the 5-year survival and complication rates of implantsupported single crowns. clin oral implants res. 2008; 19: 119-30. 8. binon pp. the effect of implant/abutment hexagonal misfit on screw joint stability. int j prosthodont. 1996; 9: 149-60. 9. lang la, wang rf, may kb. the influence of abutment screw tightening on the screw joint configuration. j prosthet dent. 2002; 87: 74-9. 10. vigolo p, majzoub z, cordioli g. measurement of the dimensions and abutment rotational freedom of gold-machined 3i ucla-type abutments in the as-received condition, after casting with a noble metal alloy and porcelain firing. j prosthet dent. 2000; 84: 548-53. 11. theoharidou a, petridis hp, tzannas k, garefis p. abutment screw loosening in single-implant restorations: a systematic review. int j oral maxillofac implants. 2008; 23: 681-90. 12. malaguti g, denti l, bassoli e, franchi i, bortolini s. dimensional tolerances and assembly accuracy of dental implants and machined versus cast-on abutments. clin implant dent relat res. 2011; 13: 134-40. 13. schwarz ms. mechanical complications of dental implants. clin oral implants res 2000; 11 suppl: 156-8. 14. coppedê ar, mattos mgc, rodrigues rcs, ribeiro rf. effect of repeated torque/mechanical loading cycles on two different abutment types in implants with internal tapered connections: an in vitro study. clin oral implants res. 2009; 20: 624-32. 15. binon pp. the external hexagonal interface and screw-joint stability: a primer on threaded fasteners in implant dentistry. quint dent technol. 2000; 23: 91-105. 16. jabbari ysa, fournelle r, ziebert g, toth j, iacopino am. mechanical behavior and failure analysis of prosthetic retaining screws after long-term use in vivo. part 3: preload and tensile fracture load testing. j prosthodont. 2009; 17:192-200. 17. sotto-maior bs, senna pm, silva wj, rocha ep, delbel cury aa. influence of crown-to-implant ratio, retention system, restorative material, and occlusal loading in stress concentration in single short implants. int j maxillofac implants. 2012; 27:.e13-8. 18. akour sn, fayyad ma, nagy ww, fournelle ra, dhuru vb, tzenakis gk, et al. finite element analyses of two antirotational designs of implant fixtures. implant dent. 2005; 14: 77-81. 19. kitagawa t, tanimoto y, odaki m, nemoto k, aida m. influence of implant/ abutment joint designs on abutment screw loosening in a dental implant system. j biomed mater res part b appli biomater. 2005; 75b: 457-63. 20. yousef h, luke a, ricci j, weiner s. analysis of changes in implant screws subject to occlusal loading: a preliminary analysis. implant dent. 2005; 14:378-85. 21. kano sc, binon p, bonfante g, curtis da. effect of casting procedures in ucla-type abutments. j prosthodont. 2006; 15: 77-81. 22. cardoso m, torres mf, lourenço ejv, telles dm, rodrigues rcs, ribeiro rf. torque removal evaluation of prosthetic screws after tightening and loosening cycles: an in vitro study. clin oral implants res. 2012; 23: 475-80. 23. jorge jr, barão var, delben ja, assunção wg. the role of implant abutment system on torque maintenance of retention screws and vertical misfit implant-supported crowns before and after mechanical cycling. int j oral maxillofac implants. 2013; 28: 415-22. 24. international organization for standardization. iso 1480. (2003) dentistry fatigue test for endosseous dental implants. 2007: 1-9. 25. tsuge t, hagiwara y. influence of lateral-oblique cyclic loading on abutment screw loosening of internal and external hexagon implants. dent mater j. 2009; 28: 373-81. 26. piermatti j, yousef h, luke a, mahevich r, weiner s. an in vitro analysis of implant screw torque loss with external hex and internal connection implant systems. implant dent. 2006; 15: 427-35. 27. gracis s, michalakis k, vigolo p, von steyern pv, zwahlen m, sailer i. internal vs. external connections for abutments/reconstructions: a systematic review. clin oral implants res. 2012; 23: 202-16. 28. hanses g, smedberg ji, nilner k. analysis of a device for assessment of abutment and prosthesis screw loosening in oral implants. clin oral implants res. 2002; 13: 666-70. 29. tavarez rrj, bonachela wc. effect of cyclic load on vertical misfit of prefabricated and cast implant single abutment. j appl oral sci. 2011; 19: 16-21. 30. dixon dl, breeding lc, sadler jp, mckay ml. comparison of screw loosening, rotation and deflection among three implant-designs. j prosthet dent. 1995; 74: 270-8. 31. gracis s, michalakis k, vigolo p, von steyern pv, zwahlen m, sailer i. internal vs. external connections for abutments/reconstructions: a systematic review. clin oral implants res. 2012; 23: 202-16. screw loosening of different ucla-type abutments after mechanical cycling braz j oral sci. 12(3):228-232 oral sciences n3 braz j oral sci. 12(2):132-137 original article braz j oral sci. april | june 2013 volume 12, number 2 oral antibacterial effect of chlorhexidine treatments and professional prophylaxis in children thiago cruvinel silva1, thaís marchini oliveira valarelli1, vivien thiemy sakai2, vanessa tessarolli1, maria aparecida de andrade moreira machado1 1department of pediatric dentistry, orthodontics, and public health, bauru school of dentistry, university of são paulo, bauru, sp, brazil 2department of clinic and surgery, alfenas, school of dentistry, alfenas federal university, mg, brazil correspondence to: thiago cruvinel silva alameda octávio pinheiro brisolla, 9-75, vila universitária, cep: 17012-901 bauru, sp, brasil phone: +55 14 32358224 fax: +55 65 32234679 e-mail: thiagocruvinel@yahoo.com.br received for publication: march 20, 2013 accepted: june 25, 2013 abstract aim: the primary aim of this longitudinal study was to evaluate additional effects of 4-week chlorhexidine digluconate (chx) gel treatments to control aggregatibacter actinomycetemcomitans counts in children after professional dental prophylaxis. porphyromonas gingivalis and streptococcus mutans counts were also determined to evaluate the secondary effects of anti-plaque treatments on microbial shifts. methods: twenty-six children with a. actinomycetemcomitans counts >4 log10/ ml of saliva and/or quigley-hein plaque index >3.0 were enrolled in this study. patients were randomly assigned to groups gi (placebo gel), gii (0.5% chx gel), giii (1% chx gel), and giv (2% chx gel). four sessions of treatment were performed during 4 weeks after a session of professional dental prophylaxis. real-time polymerase chain reaction (pcr) was used to determine viable microorganism counts in non-stimulated whole saliva samples collected at baseline, one week, one month and three months after interruption of treatments. results: a reduction of all bacterial counts was detected after the 3-month follow-up in all groups. lower counts of p. gingivalis were achieved from 1 week on after treatments. the 2% chx concentration seemed to contribute to lower a. actinomycetemcomitans levels and increase s. mutans levels. conclusions: professional dental prophylaxis was effective to control salivary levels of a. actinomycetemcomitans, p. gingivalis and s. mutans. additional antimicrobial effects, however, were not observed by the combination of professional dental prophylaxis and 4-week chlorhexidine gel treatments. keywords: aggressive periodontitis, professional prophylaxis, chlorhexidine, aggregatibacter actinomycetemcomitans, porphyromonas gingivalis, streptococcus mutans. introduction aggressive periodontitis (agp) affects younger individuals and is characterized by a rapid loss of the supporting connective tissue and alveolar bone around teeth in the presence of oral biofilm1. the prevalence of agp is higher among black individuals than among caucasian individuals, ranging from 0.1% to 7.6%, with a tendency for familiar clustering of cases2. increased detection levels of aggregatibacter actinomycetemcomitans in the oral cavity is an indicator for the risk of development of agp in children, adolescents and young adults3–6. a. actinomycetemcomitans is a gram-negative facultative anaerobic microorganism acquired via maternal transmission7-8. it plays an important role in the onset of localized agp because of its virulence factors, such as rtx leukotoxin and cytolethal distending toxin, which trigger braz j oral sci. 12(2):132-137 133133133133133 gel formulations 0%, 0.5%, 1% or 2% chlorhexidine concentrations 2% hydroxyethylcellulose 0.1% benzalkonium chloride 95 v/v 0.18% methylparaben 0.05% propylparaben 0.05% menthol crystal 7% ethyl alcohol 96 v/v 0.5% polysorbate 20 15% sorbitol 100 ml of distilled water chemical properties ph 5.8-6.5 viscosity ∼ 300.000 cps specific density 1.0546 g/ml table 1 – formulations and chemical properties of different gels. immunological host responses activating osteoclastogenesis and leading to tissue breakdown9. antimicrobial chemotherapeutic methods can be used to prevent agp. chlorhexidine digluconate (chx) is effective against a large spectrum of gram-positive and gram-negative oral microorganisms10. short-term adjunctive use of chx gel and/or mouthrinses decreases periodontopathogen counts1112 and limits supra/subgingival biofilm re-growth after mechanical removal of dental plaque13–16. microbial recolonization of tooth surfaces, however, begins soon after antimicrobial treatments17, which might induce shifts in the composition of biofilms. simultaneous identification of certain bacteria may also change the clinical severity of periodontal diseases. ramsey and whiteley18 (2009) demonstrated an enhanced resistance and virulence of a. actinomycetemcomitans in the presence of oral streptococci. de soete et al.19 (2005) observed higher streptococcus mutans counts in saliva of periodontal patients after scaling root planning (srp). mayorga-fayad et al.20 (2007) reported more prevalent detection of porphyromonas gingivalis in agp patients. to the best of our knowledge, salivary microbial shifts after the association between mechanical removal of dental plaque and chx gel treatments have not yet been investigated. the primary aim of this longitudinal study was to evaluate additional effects of 4-week chx gel treatments to control a. actinomycetemcomitans counts in children after professional dental prophylaxis. p. gingivalis and s. mutans counts were also determined to evaluate the secondary effects of anti-plaque treatments on microbial shifts. material and methods participants the present study was conducted after approval by the ethics committee of bauru school of dentistry, university of são paulo (process #47/2006). parents of patients attending the clinic of pediatric dentistry were invited to enroll their children in the study. parental agreement was documented by signing a written informed consent. in a preliminary screening, forty-eight 7-12-year-old children donated saliva and were examined by a calibrated dentist (intraobserver concordance 8>0.9) to determine löe and silness21 (1963) gingival index (g-index) and quigley & hein modified by turesky dental plaque index22 (dpindex). patients with a. actinomycetemcomitans counts >4 log10/ml of saliva and/or dp-index >3.0 could be included in the study. consumption of antibiotics and non-steroidal anti-inflammatory drugs up to 30 days before the study, presence of supragingival calculus, severe malocclusion, oral lesions in soft tissues and use of orthodontic appliances were considered as exclusion criteria. twenty-six children were enrolled in the study and randomly assigned to groups gi (control, placebo gel), gii (0.5% chlorhexidine gel), giii (1% chlorhexidine gel), and giv (2% chlorhexidine gel) by microsoft excel 2003® software. clinical procedures non-stimulated whole saliva was collected into sterile glass beaker for 5 min. saliva samples were stored at -20oc until dna extraction of specific microorganisms23. professional dental prophylaxis with sodium bicarbonate jet was performed before the first session of treatment. for that purpose, dental plaque was previously disclosed by a solution of 0.2% fuchsine to ensure the quality of procedures. then, chx gels were dispensed into disposable trays and applied on teeth for 1 min. formulations and chemical properties of chx gels are depicted in table 1. excesses of gel were removed and children were instructed to restrict liquid and food intake for next 30 min. treatments were conducted once a week for 28 days. clinical appointments were scheduled 1 week, 1 month and 3 months after the interruption of gel treatments to collect saliva samples and determine gingival and dental plaque indexes. real time pcr tests real time pcr tests were performed to allow the detection and quantification of a. actinomycetemcomitans, p. gingivalis, and s. mutans. after defrosting and homogenizing the saliva samples, two aliquots of 500 µl saliva each were diluted in 1 ml of sterile demineralized water (1:2) and centrifuged (10,000 xg) at 4oc for 5 min. supernatants were discarded and pellets were washed three times with sterile demineralized water (10000 xg, 4oc, 5 min)23. one bacterial pellet was incubated with 100 µl of a solution for cellular lyses (20 u/ml mutanolysin plus 0.2 mg/ml lysozyme) at 37oc for 2 h. this mixture was boiled (100oc, 10 min) to extract bacterial dna from s. mutans cells. other bacterial pellet was used to extract dna from a. actinomycetemcomitans and p. gingivalis cells. the pellet was suspended in a mixture of 100 µl dnase-free water and 100 µl instagene matrix (bio-rad laboratories inc., hercules, ca, usa), and incubated at 56oc for 30 min. then, the samples were vortexed and boiled for 10 min. unbroken cells and large debris were removed by subsequent centrifugation oral antibacterial effect of chlorhexidine treatments and professional prophylaxis in children braz j oral sci. 12(2):132-137 134134134134134 sequence (5’ – 3’) a.actinomycetemcomitans forward cagcatctgcgatccctgta reverse gccctttgtctttcctaggt p. gingivalis forward tacccatcgtcgccttggt reverse cggactaaaaccgcatacacttg s. mutans forward gcctacagctcagagatgctattct reverse gccatacaccactcatgaattga table 2 – sequences of oligonucleotide primers. baseline 1 week 1 month 3 months g-index 1.12±0.31 1.05±0.37 0.97±0.32 1.09±0.24 gi dp-index 3.07±0.50 3.30±0.50 3.51±0.43 2.75±0.95 g-index 0.84±0.54 0.84±0.42 1.00±0.34 1.12±0.12 gii dp-index 2.90±0.59 3.25±0.56 3.16±0.60 3.41±0.83 g-index 1.15±0.30 1.04±0.25 1.07±0.36 1.02±0.19 giii dp-index 3.03±0.44 3.39±0.27 3.41±0.64 3.05±0.94 g-index 0.88±0.53 1.06±0.30 1.25±0.29 1.05±0.46 giv dp-index 3.29±0.78 3.52±0.54 3.33±0.72 3.45±1.09 table 3 – mean (±sd) of gingival index (g-index) and dental plaque index (dp-index) of groups gi (placebo gel), gii (0.5% chx gel), giii (1% chx gel), and giv (2% chx gel) after different follow-up times. (10,000 xg, 3 min). supernatants were used for real-time pcr dna analysis. real-time pcr dna analysis was performed in an abi prism 7000 system (applied biosystems, warrington, uk), 1x universal pcr master-mix (abi), 200 nm of specific primers, 1µl of the supernatant, and 250 nm of each quencher dye (6-carboxyfluorescein and 6-carboxytetramethylrhodamine). the specificities of the primers were initially confirmed by blast with the national center for biotechnology information server (http://www.ncbi.nlm.nih.gov). oligonucleotide primer sequences are shown in table 2. bacterial dna levels were determined using the ct method and normalized by the volume of the supernatants. statistical analysis data were analyzed by ncss/pass® 2000 statistical software (kaysville, ut, usa). microorganism counts were log-transformed before statistical tests. normality and homogeneity of data were respectively evaluated by kolmogorov-smirnov and levene tests. statistical differences were assessed using parametric one-way anova and bonferroni post-hoc test. correlations between gingival or dental plaque indexes and microorganism counts were observed by pearson’s test. the p values <0.05 were considered significant. results gingival and dental plaque indexes fourteen male and 12 female children were enrolled in the study (8.9±1.2 years). correlations between gingival and dental plaque indexes and a. actinomycetemcomitans (gindex r=-0.24, dp-index r=-0.10), p. gingivalis (g-index r=-0.03, dp-index r=-0.10), and s. mutans (g-index r= -0.14, dp-index r=0.01) counts were not observed at baseline. both clinical indexes were statistically similar in different treatment groups and times (table 3). microorganism counts p. gingivalis and s. mutans were identified in 61.5% and 96.2% of children, respectively, at baseline. bacterial counts did not differ significantly between groups throughout the study (p>0.05). the effectiveness of treatments was demonstrated by statistical differences observed among bacterial counts in different follow-up times. considering all groups (table 4), the mean of a. actinomycetemcomitans counts at 3 months (4.20±3.90) was significantly lower than at baseline (5.98±1.96) (p<0.05). similar differences were verified in s. mutans counts between 3-month follow-up (3.14±2.67) and baseline (4.29±2.31) (p<0.05). p. gingivalis counts were statistically reduced from 1 week (1.73±2.74) after interruption of treatments compared with baseline (5.18±4.72) (p<0.05). although p. gingivalis counts gradually increased to 3.21±3.42 at 1-month and 3.37±3.12 at 3-month follow-ups, the levels still remained significantly lower than at baseline (p<0.05). the maintenance of lower p. gingivalis and s. mutans counts were noticed in 100% and 84.6% children, respectively, with initial p. gingivalis and s. mutans counts >4 log10/ml of saliva. on the other hand, respectively 54.5% and 69.2% children with initial p. gingivalis and s. mutans counts <4 log10/ml of saliva showed higher microorganism counts after 3 months of follow-up (figures 1a, b and c). a reduction in the percentage of children with salivary a. actinomycetemcomitans counts >4 log10/ml of saliva was observed in all groups since chx treatments. the highest chx concentration (2%) seemed to contribute to obtain lower levels of a. actinomycetemcomitans in most patients at the end of the study (figure 1a). in comparison with baseline, the percentage of patients with p. gingivalis counts >4 log10/ml of saliva decreased after 1 week and 1 month of placebo and 0.5% chx gel treatments, whereas the percentage of patients did not vary after 1% and 2% chx gel treatments. after 3 months, different oral antibacterial effect of chlorhexidine treatments and professional prophylaxis in children 135135135135135 braz j oral sci. 12(2):132-137 chx concentrations differed in controlling p. gingivalis levels. higher percentage of children with p. gingivalis counts >4 log10 was noticed in the group giii as compared to groups gii and giv (figure 1b). the results supported a trend of simultaneous increment of chx gel concentrations and s. mutans counts along time. treatments with 2% chx gel increased s. mutans levels in most patients 1 week and 3 months after chx treatments (figure 1c). discussion a positive impact on the prevention of agp could be achieved by a short-term reduction of salivary periodontopathogens. this preliminary study demonstrated that one session of professional dental prophylaxis can be effective to reduce salivary counts of a. actinomycetemcomitans, p. gingivalis and s. mutans in children. additional antimicrobial effects of chx gels could not be demonstrated. after a 3month follow-up, most children showed lower salivary counts of at least one microorganism, especially in patients with specific bacterial counts >4 log10 at baseline. the amounts of microbial deposits are inconsistent with the severity of periodontal tissue destruction in agp patients23. high proportions of a. actinomycetemcomitans and p. gingivalis can be associated with the onset of this disease. the rapid identification and quantification of periodontal microorganisms, therefore, permits early treatments to prevent the colonization of tooth surfaces for the maintenance of homeostasis of the periodontium. c u r r e n t l y , i t i s p o s s i b l e t o m o n i t o r o r a l m i c r o b i a l compositions using diagnostic tools as real-time pcr2425. umeda et al.23 (1998) demonstrated that pcr is a specific and highly sensitive method for detection of periodontopathogens in whole saliva. our study focused on the potential application of chx gels to reduce salivary pathogens, which would be able to colonize tooth and radicular surfaces, and increase the risk of developing periodontal diseases. in the present study, disposable trays were used to deliver chx gels in the oral cavity of children. dental plaque and gingival indexes of all groups did not differ significantly after treatments. francis et al.14 (1987) demonstrated efficient inhibition of dental plaque and control of gingival bleeding oral antibacterial effect of chlorhexidine treatments and professional prophylaxis in children treatment patient a. actinomycetemcomitans p. gingivalis s. mutans group baseline 1 week 1 m 3 m baseline 1 week 1 m 3 m baseline 1 week 1 m 3 m 1 8.94 10.44 7.36 5.23 10.12 8.97 3.79 4.49 9.45 8.07 7.30 7.15 2 8.81 9.27 5.29 9.86 8.07 2.28 nd 4.60 9.10 nd 6.11 10.03 3 5.13 2.06 2.29 nd 2.96 nd nd 4.45 3.29 2.56 3.21 nd gi 4 8.47 6.72 9.25 10.57 9.30 nd 8.55 2.89 8.10 6.44 1.56 nd 5 4.88 3.78 3.19 2.43 nd nd nd nd 2.51 4.08 0.54 nd 6 3.02 3.47 nd nd nd nd nd nd 5.23 5.09 5.28 nd 7 3.34 2.87 nd nd nd nd nd nd 5.06 7.21 5.19 5.22 mean±sd 6.1±2.6 5.5±3.3 3.9±3.6 4.0±4.6 4.4±4.7 1.6±3.4 1.8±3.3 2.3±2.3 6.1±2.8 4.8±2.8 4.2±2.5 3.2±4.2 8 4.22 nd 3.23 2.44 6.91 nd nd nd 5.17 6.56 3.96 nd 9 6.47 6.54 8.00 5.51 8.14 2.51 5.87 5.37 5.02 5.62 4.05 nd 10 5.90 nd 7.85 5.98 9.60 2.30 3.21 5.47 5.50 nd 4.42 3.80 gii 11 5.55 5.84 6.81 2.87 7.25 nd 4.04 4.56 6.02 5.86 3.03 4.00 12 8.15 6.46 5.91 2.89 6.64 nd 2.95 3.82 1.53 7.78 6.28 4.71 13 4.58 6.74 nd nd 5.89 1.71 nd nd 1.91 nd 3.18 3.64 mean±sd 5.8±1.4 4.3±3.3 5.3±3.1 3.3±2.2 7.4±1.3 1.1±1.2 2.7±2.3 3.2±2.6 4.2±1.9 4.3±3.4 4.2±1.2 2.7±2.1 14 8.21 6.64 9.52 10.07 13.94 nd 10.11 11.11 5.42 nd 5.12 4.24 15 5.43 6.65 6.87 11.78 11.74 3.99 3.33 nd 4.27 4.45 4.39 nd 16 6.91 6.08 5.89 5.36 6.02 nd nd 4.09 nd 5.54 nd 3.53 giii 17 6.45 3.76 nd nd 13.14 2.70 5.64 6.09 5.61 4.40 3.48 4.18 18 4.33 10.68 6.09 5.72 nd 7.30 4.86 5.83 3.07 2.36 6.76 5.79 19 6.33 9.01 5.50 5.04 nd 6.30 nd 4.98 2.58 nd 2.72 3.84 20 5.35 4.30 8.82 8.87 nd nd 7.76 7.03 2.87 nd 2.12 nd mean±sd 6.1±1.3 6.7±2.4 6.1±3.1 6.7±3.9 6.4±6.5 2.9±3.1 4.5±3.8 5.6±3.3 3.4±1.9 2.4±2.4 3.5±2.4 3.1±2.2 21 4.39 2.39 2.77 nd nd nd 3.00 nd 2.18 4.00 3.76 nd 22 6.58 5.20 5.50 2.59 nd nd 2.73 4.52 2.61 6.09 3.18 4.66 23 6.77 9.28 8.94 nd 8.42 7.00 7.94 nd 3.50 4.65 2.94 5.13 giv 24 3.21 3.95 6.93 nd nd nd nd nd 2.33 2.83 4.96 3.40 25 8.31 4.94 2.93 2.59 6.44 nd nd nd 6.16 2.44 0.78 4.15 26 5.87 6.78 9.27 9.40 nd nd 9.75 8.31 3.06 nd 3.11 4.10 mean±sd 5.9±1.8 5.4±2.4 6.1±2.8 2.4±3.6 2.5±3.9 1.2±2.9 3.9±4.1 2.1±3.5 3.3±1.5 3.3±2.1 3.1±1.4 3.6±1.8 table 4 – mean (±sd) of salivary counts of a. actinomycetemcomitans, p. gingivalis, and s. mutans (log10) at baseline, 1 week and 3 months after treatments with placebo gel (gi), 0.5% chx gel (gii), 1% chx gel (giii), and 2% chx gel (giv). nd means microorganism not detected. braz j oral sci. 12(2):132-137 fig. 1 – percentage of children with a. actinomycetemcomitans (a), p. gingivalis (b) and s. mutans (c) counts >4 log10/ml of saliva in different groups and followup times. in patients treated with 4-week tray application of 1% chx gel. slot et al.16 (2010) reported an improvement of dental plaque control after tray application of 1% chx gel compared to 0.12% chx dentifrice gel. our contrasting results could be explained by the inclusion of healthy periodontal patients with low scores of dental plaque and gingival indexes at baseline. according to berchier et al.26 (2010), the increase of chx concentration seems to enhance the dental plaque control, although this could not be considered clinically relevant. since the diversity of microorganisms from saliva and dental plaque has been estimated in about 19,000 specieslevel phylotypes27, multiple microbial clusters are possible among individuals. in our study, the host-specific microbial load can be illustrated by the detection of p. gingivalis and s. mutans in respectively 61.5% and 73% children at baseline. similar treatments resulted in diverse antimicrobial responses in different patients: 4-week 1% chx gel treatment was effective to reduce all bacterial counts in patient #17, whereas the same regimen increased all microorganism counts in patient #18. lower s. mutans and p. gingivalis counts and higher a. actinomycetemcomitans were detected in patients #14 and #15. filoche et al.28 (2008) showed a host specificity of antimicrobial agents against ex vivo microorganisms. after a 3-month follow-up, professional dental prophylaxis was effective to reduce at least one specific bacterial count in all patients of group gi. periodic dental visits may also have contributed to the enhancement of dental plaque control in children. the application of 0.5% chx gel decreased simultaneously a. actinomycetemcomitans, p. gingivalis and s. mutans counts in most patients. higher counts of bacteria were detected in four out of seven patients treated with 1% chx gel. two percent chx gel treatments were responsible for lowering a. actinomycetemcomitans counts in 83.3% patients and raising s. mutans counts in 66.7% patients at the end of the study. although speculative, our findings could indicate a possible trend of microbial shifts between periodontopathogens and cariogenic microorganisms with increasing concentration of chx gels. de soete et al.19 (2005) reported a significant increase of detection of s. mutans after srp. clinical interventions could induce local shifts in composition of biofilms from predominant anaerobic and proteolytic microorganisms to aerobic and acidogenic microorganisms. other microbial relationships can be expressed by association among specific substrate, microbiological composition and metabolism. for example, lactate a metabolite of cariogenic bacteria can be used as a carbon source for veilonella sp. these bacteria are able to produce considerable amount of menaquidone, an organic source of vitamin k, essential to growth of p. gingivalis and t. denticola29. thresholds of minimal p. gingivalis and s. mutans counts were not established in our inclusion criteria. independently of treatment group, most patients with initial s. mutans and p. gingivalis counts >4 log10/ml of saliva presented a trend to reduction of bacterial counts after 3-month follow-up, whereas respectively 69.2% and 54.5% patients with s. mutans and p. gingivalis counts <4 log10/ml of saliva presented a trend to increase bacterial counts along time. moreover, faster reductions of p. gingivalis counts seemed to indicate more sensitivity to mechanical and chemotherapeutic treatments than a. actinomycetemcomitans and s. mutans. these findings suggest shifts in microbial compositions after treatments for dental plaque control. sekino et al.12 (2004) demonstrated that the combination of a 0.2% chx mouthrinse and a 1% chx gel decreased salivary microorganisms and blocked tooth recolonization by a. actinomycetemcomitans and p. gingivalis. a significant reduction of periodontopathogen counts after srp and fullmouth disinfection with chx was also described by quirynen 136136136136136oral antibacterial effect of chlorhexidine treatments and professional prophylaxis in children braz j oral sci. 12(2):132-137 137137137137137 et al.17 (1999). perinetti et al.11 (2004) achieved a reduction of a. actinomycetemcomitans and s. mutans counts in periodontal pockets after irrigation with 1% chx gel. some issues should be discussed regarding the results. it is possible that significant differences were not observed among the different groups of this study due to some limitations: (1) small sample size, (2) allocation of participants into four distinct groups, and (3) diversity of antimicrobial responses from each patient. also, inclusion criteria limited the enrollment of participants in this study, especially a. actinomycetemcomitans counts >4 log10/ml of saliva. additionally, the follow-up could be extended to 6 months to adequately assess the effect of chlorhexidine substantivity on clinical and microbiological parameters. in conclusion, this study demonstrated that mechanical dental plaque control can be used as an effective preventive strategy to control salivary counts of a. actinomycetemcomitans, p. gingivalis and s. mutans, contributing to the maintenance of periodontal health. further randomized clinical trials are needed to establish safe protocols for demonstrating additional benefits of chlorhexidine treatments against pathogenic microorganisms in saliva, with minimum modifications of oral homeostasis. acknowledgements the authors thank thiago josé dionísio for his valuable technical support. this study was financially supported by the são paulo state research foundation – fapesp, brazil (process # 2007/00962-6). references 1. albandar jm, buischi yap, barbosa mfz. disease in adolescents. a 3year longitudinal study. j periodontol. 1991; 62: 370-6. 2. ickenstein gw, klotz jm, langohr hd. kopfschmerz bei polycythaemia vera. schmerz. 1999; 13: 279-82. 3. armitage gc. periodontal diagnoses and classification of periodontal diseases. periodontol 2000. 2004; 34: 9-21. 4. armitage gc. comparison of the microbiological features of chronic and aggressive periodontitis. periodontol 2000. 2010; 53: 70-88. 5. jardim jr eg, bosco jmd, lopes am, landucci lf, jardim ecg, carneiro srs. occurrence of actinobacillus actinomycetemcomitans in patients with chronic periodontitis, aggressive periodontitis, healthy subjects and children with gingivitis in two cities of the state of são paulo, brazil. j appl oral sci. 2006; 14: 153-6. 6. shaddox lm, huang h, lin t, hou w, harrison pl, aukhil i, et al. microbiological characterization in children with aggressive periodontitis. j dent res. 2012; 91: 927-33. 7. lamell cw, griffen al, mcclellan dl, leys ej. acquisition and colonization stability of actinobacillus actinomycetemcomitans and porphyromonas gingivalis in children. j clin microbiol. 2000; 38: 1196-9. 8. fine dh, markowitz k, furgang d, velliyagounder k. aggregatibacter actinomycetemcomitans as an early colonizer of oral tissues: epithelium as a reservoir? j clin microbiol. 2010; 48: 4464-73. 9. henderson b, ward jm, ready d. aggregatibacter (actinobacillus) actinomycetemcomitans: a triple a* periodontopathogen? periodontol 2000. 2010; 54: 78-105. 10. emilson cg. susceptibility of various microorganisms to chlorhexidine. scand j dent res. 1977; 85: 255-65. 11. perinetti g, paolantonio m, cordella c, ercole ds, serra e, clinical pr. clinical and microbiological effects of subgingival administration of two active gels on persistent pockets of chronic periodontitis patients. j clin periodontol. 2004; 31: 273-81. 12. sekino s, ramberg p, uzel ng, socransky s, lindhe j. the effect of a chlorhexidine regimen on de novo plaque formation. j clin periodontol. 2004; 31: 609-14. 13. löe h, schiott cr. the effect of mouthrinses and topical application of chlorhexidine on the development of dental plaque and gingivitis in man. j periodontal res. 1970; 5: 79-83. 14. francis jr, hunter b, addy m. a comparison of three delivery methods of chlorhexidine in handicapped children. i. effects on plaque, gingivitis, and toothstaining. j periodontol. 1987; 58: 451-5. 15. vinholis ah, figueiredo lc, marcantonio júnior e, marcantonio ra, salvador sl, goissis g. subgingival utilization of a 1% chlorhexidine collagen gel for the treatment of periodontal pockets. a clinical and microbiological study. braz dent j. 2001; 12: 209-13. 16. slot de, rosema nam, hennequin-hoenderdos nl, versteeg pa, van der velden u, van der weijden ga. the effect of 1% chlorhexidine gel and 0.12% dentifrice gel on plaque accumulation: a 3-day non-brushing model. int j dent hyg. 2010; 8: 294-300. 17. quirynen m, mongardini c, pauwels m, bollen cml, van eldere j, van steenbergh d. one stage fullversus partial-mouth disinfection in the treatment of chronic adult or generalized early-onset periodontitis. ii. longterm impact on microbial load. j periodontol. 1999; 70: 646-56. 18. ramsey mm, whiteley m. polymicrobial interactions stimulate resistance to host innate immunity through metabolite perception. proc natl acad sci usa. 2009; 106: 1578-83. 19. de soete m, dekeyser c, pauwels m, teughels w, van steenberghe d, quirynen m. increase in cariogenic bacteria after initial periodontal therapy. j dent res. 2005; 84: 48-53. 20. mayorga-fayad i, lafaurie gi, contreras a, castillo dm, barón a, aya m del r. microflora subgingival en periodontitis crónica y agresiva en bogotá, colombia: un acercamiento epidemiológico. biomedica. 2007; 27: 21-33. 21. löe h, silness j. periodontal disease in pregnancy. i. prevalence and severity. acta odontol scand. 1963; 21: 533-51. 22. turesky s, gilmore nd, glickman i. reduced plaque formation by the chloromethyl analogue of victamine c. j periodontol. 1970; 41: 41-3. 23. umeda m, contreras a, chen c, bakker i, slots j. the utility of whole saliva to detect the oral presence of periodontopathic bacteria. j periodontol. 1998; 69: 828-33. 24. price rr, viscount hb, stanley mc, leung kp. targeted profiling of oral bacteria in human saliva and in vitro biofilms with quantitative real-time pcr. biofouling. 2007; 23: 203-13. 25. atieh ma. accuracy of real-time polymerase chain reaction versus anaerobic culture in detection of 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2019 e191668 original article 1 department of infant and social dentistry, araçatuba school of dentistry, univ. estadual paulista – unesp, araçatuba, brazil. corresponding author: cléa adas saliba garbin/ julia arruda batista department of infant and social dentistry, araçatuba school of dentistry, unesp, josé bonifácio street, 1193. neighborhood: vila mendonça, araçatuba, sp –brazil. e-mail: cgarbin@foa.unesp.br / jarrudabaptista@gmail.com https://orcid.org/0000-0001-5069-8812 received: may 23, 2019 accepted: august 23, 2019 prevalence of musculoskeletal pain in dental students and associated factors renata reis dos santos1,*, cléa adas saliba garbin1,*, julia arruda batista1, tânia adas saliba1, artênio josé isper garbin1 aim: the objective of this research was to evaluate the prevalence of painful symptoms among dental academics, as well as their associated factors. methods: a cross-sectional study was carried out at a public higher education institution, and the study population (n = 303) included undergraduate students who studied at least one subject that included clinical activities. for data collection, two self-administered questionnaires were delivered to the participants, the first of which consisted of sociodemographic variables, academic life, harmful habits, physical activity practice, and general health. the validated nordic musculoskeletal questionnaire was used to identify musculoskeletal symptoms, the need to seek health resources, and to assess whether the disorders interfered with the work activities of academics. results: the presence of pain in the past 12 months was observed in 199 participants (82.6%). several variables, including gender, number of courses performed, physical activity, and general health status, had an association with painful symptoms in at least one area of the body (ga) over the past 12 months. in addition, variable general health was associated with pain symptoms in any area of the body (dg) over the past seven days. conclusion: there was a high prevalence of musculoskeletal symptoms, especially in the upper limbs, and there were associations between muscular pain and the number of disciplines studied and between muscular pains and the general health of the students. keywords: dentistry. musculoskeletal disorders. occupational health. students. pain. https://orcid.org/0000-0001-5069-8812 2 santos et al. introduction musculoskeletal disorders are common morbidities in dental practice and are characterized by persistent pain and/or discomfort in the musculoskeletal system, including joints, ligaments, tendons, nerves, and structures that support the limbs; these disorders may present as inflammatory or degenerative conditions that occur either alone or by cumulative trauma, causing painful symptomatology in different parts of the body1-5. in addition to pain, they include complaints such as tingling, numbness, weight, and early fatigue; thus, these diseases contribute to the inability of the professional from his job, and even their withdrawal from the position6. among occupational diseases in health workers, musculoskeletal disorders affect approximately 63% to 93% of dentists, and their early onset is a very common problem for these professionals1. a career in dentistry requires clinical care to be carried out in a restricted area (the mouth); thus, the professional remains in the same position for a long time, requiring extreme precision and strength of the movements of the hands and wrists, often repetitively1,7. the necessity of this positioning during the work activity can increase the risk of developing work-related osteomuscular disorders (dort)7. these disorders arise from the interplay of many factors, including a lack of knowledge surrounding the need for correct ergonomic posture, work posture associated with repetitive movements, long working hours, and a lack of physical activity and muscle strengthening7,8. although the prevalence of musculoskeletal problems among dental surgeons is high (63% to 93%), there are few studies focused on this profession9. in a study carried out in academics in brazil, it was found that an incorrect posture was assumed during the execution of work7. in another study, there was an increased risk of the development of musculoskeletal disorders and painful or chronic conditions due to the professional attitudes during the execution of the work8. therefore, this study aimed to evaluate the prevalence of pain symptoms among dental students, as well as their association with other factors. materials and methods this was a cross-sectional study involving dentistry students enrolled at the university, who attended at least one course that included clinical activities in its teaching plan. during the collection period (september 2013 to november 2013), 303 students were enrolled who met this criterion. students who had any congenital physical impairment involving the upper or lower limbs were excluded, as were pregnant women, infants, and those who did not consent to participate in the study. an explanation of free and informed consent was attached to the questionnaire in order to clarify any questions regarding the voluntary participation in the research. this study was approved by the araçatuba school of dentistry committee for ethics research unesp, and followed all national and international regulations (caae: 18569513.2.0000.5420) each participant responded to two self-administered questionnaires, which were delivered at the end of the theoretical lectures so as not to disrupt the teaching activities. 3 santos et al. the questionnaires were distributed to the participants by previously trained graduate students, who explained and clarified doubts, thus avoiding errors in the completion and understanding of the questionnaire. the first questionnaire consisted of a series of questions about socio-demographic variables (gender, age, and marital status); academic life (number of subjects taken); harmful habits (use of tobacco, alcohol, and other drugs); the performance of physical activities (what, how long, frequency, and monitoring by a specialist); and general health. further information on weight and height were collected in order to calculate the body mass index (bmi). an individual was considered obese when their bmi was ≥ 30 kg/m2, overweight when the bmi was between 25 and 29.9 kg/m2, and normal when the bmi was between 20 and 24.9 kg/m2. in the second questionnaire, a portuguese adaptation of the nordic musculoskeletal questionnaire, assessed musculoskeletal disorders10. this questionnaire has been validated, and has a reliability ranging from 0.88 to 1, according to the kappa coefficient11. the questionnaire is used internationally and is accepted for the assessment of musculoskeletal disorders. it consists of questions on nine areas of the body divided into three areas of the upper extremities, three of the lower extremities, and three of the spine. the questionnaire assesses symptoms of pain in the neck, shoulder, elbow, forearm, wrist/hand/finger, dorsal, lumbar, hip/thigh, knee, and ankle/foot. the participants report if they have had any experience of pain or discomfort in any of the nine areas of the body during the past 12 months or 7 days, and whether they sought professional help for these pains. positive pain symptoms were identified when pain or discomfort was reported in at least one of the body areas. the students who reported pain or discomfort were also asked whether they were using drugs to relieve the pain and/or discomfort, and if so, were asked to indicate which drug was used. statistical analysis was performed using the statistical package for the social sciences (spss), version 21.0. descriptive statistics were used to characterize the sample (gender, age, marital status, bmi, courses taken, harmful habits, physical activity, general health problems, use of medications, and symptoms of pain). in order to verify the association between pain symptoms and the variables studied, a response of “yes” was given when at least one positive was reported in any area of the body for each question of the nordic musculoskeletal questionnaire. thus new variables were obtained as follows: symptoms in the last 12 months in any area of the body (ga), impairment of normal activities as a result of this problem in any area of the body in the past 12 months (gb), consultation with a health professional because of this condition in the past 12 months (gc), and symptoms in past 7 days concerning any area of the body (gd). the fisher’s exact chi-square test and the likelihood ratio were used to determine the association between variables. results following completion of the questionnaire, 241 students were obtained for participation in this study. the average age of the subjects was 22:31 years (sd ± 1.97), and the majority of the respondents (62.7%) were women. regarding marital status, 97.9% of the respondents were single. regarding the number of clinical disciplines 4 santos et al. studied, the average was 12 (sd ± 4.25). the bmi analysis of the students showed that 62.7% were in the normal range, 14.5% were considered overweight, 5.4% were classified with obesity, and no less than a disorder 17.4% were considered underweight. in terms of unhealthy habits, the majority of students did not use alcohol, tobacco, or other drugs (table 1). physical activity was reported as routine for 59.8% of the undergraduate students, with most of the practiced activities being classified as resistance (weight training or some type of contact sport) (54.2%); these activities were practiced on average 4 times per week or more, and were mostly supervised by professionals. although only a few students reported having been diagnosed with a general health issue in the past 12 months (n = 24), 33.3% of these were diagnosed with problems related to musculoskeletal disorders. regarding the use of medication to control pain, 44.4% reported using painkillers, anti-inflammatories, or a combination of these drugs to eliminate symptoms of pain (table 2). table 1. sociodemographic variables of undergraduate students of araçatuba dental schoolunesp. araçatuba, 2013. variables n % genre female 151 62.7 male 90 37.3 age     19 years 4 1.7 20 a 24 years 209 86.7 25 a 29 years 27 11.1 32 years 1 0.4 marital status married 4 1.7 single 236 97.9 no information 1 0.4 bmi underweight 42 17.4 normal weight 151 62.7 overweight 35 14.5 obesity 13 5.4 subjects less than 12 124 51.5 12 or more 117 48.5 harmful habits alcohol 81 33.6 tobacco 2 0.8 no habit 158 65.6 5 santos et al. table 2. variables related to physical activity, general health and medication use by undergraduate students of araçatuba dental schoolunesp. araçatuba, 2013. variables n % do you do any type of physical activity? yes 144 59.8 no 97 40.2 what activity? resistive 78 54.2 aerobic 33 22.9 both 32 22.2 no information 1 0.7 how long?     less than 6 months 38 26.4 from 6 a 12 months 42 29.2 from 12 a 24 months 13 9 more than 24 months 48 33.3 no information 3 2.1 how many times a week? once 7 4.9 twice 19 13.2 three time 47 32.6 four time or more 70 48.6 no information 1 0.7 do you have guidance of a professional to do physical activity? yes 91 63.2 no 53 36.8 has a problem of general health diagnosed in the last 12 months? yes 24 10 no 217 90 what general health problem? wmsd 8 33.3 other 16 66.7 medicine use? yes 107 44.4 no 134 55.6 what medicine? analgesic 36 33.6 anti inflammatory 11 10.3 combination 15 14 other 43 40.2 no information 2 1.9 6 santos et al. the presence of pain in the past 12 months was observed in 199 participants (82.6%). more than half of the respondents reported neck pain (51.5%), while 48.1% had upper back pain, 38.6% had pain in the wrist/hand, and 49.8% reported pain in the lumbar region. musculoskeletal pain in the lower extremities (hips/thighs, knees, calves/legs, ankles/feet) was reported by less than 30% of the respondents (table 3). regarding the impairment of activities, 25% of the students reported that pain interfered with task performance in the past 12 months. in table 3, the frequency and percentage of students who had some impairment due to pain, and the respective areas, can be observed in detail. only a small number of students sought medical advice or treatment for pain or symptoms that they reported in the past 12 months (28.2%). regarding the painful symptoms in the past 7 days, nearly half of the students (48.5%) reported pain in at least one area of the body (table 3). the analysis of the association between the socio-demographic variables and the general variables of each of the nordic questions demonstrated an association between pain symptoms in the past 12 months in at least one area of the body (ga) and sex; ga and the number of courses taken; ga and the practice of physical activity and general health; and ga and between pain symptoms in the last 7 days in any area of the body (gd) and general health (table 4). discussion work associated musculoskeletal disorders have a high prevalence among dental surgeons; given the impairment on work and normal activities, research in this field has increased worldwide9. table 3. associations between body regions and musculoskeletal symptoms according to the dimensions of the nordic musculoskeletal questionnaire. body area symptoms in the last 12 months impediment to the normal activities because of perform this problem over the past 12 months consultation with a professional in the area of health because of this condition in the past 12 months symptoms in the last 7 days yes no yes no yes no yes no n % n % n % n % n % n % n % n % neck 124 51.5 117 48.5 18 7.5 223 92.5 15 6.2 226 93.8 51 21.2 190 78.8 shoulder 102 42.3 139 57.7 14 5.8 227 94.2 14 5.8 227 94.2 42 17.4 199 82.6 upper back 116 48.1 125 51.9 11 4.6 230 95.4 23 9.5 218 90.5 48 19.9 193 80.1 elbow 18 7.5 223 92.5 1 0,4 240 99.6 3 1,2 238 98.8 4 1.7 237 98,3 wrist/hand 93 38.6 148 61.4 17 7.1 224 92.1 12 5.0 238 95.0 24 10.0 217 90.0 lower back 120 49.8 121 50.3 20 8.3 221 91.7 28 11.6 213 88.4 52 21.6 189 78.4 hip/haunch 41 17.0 200 83.6 5 2.1 236 97.9 5 2.1 236 97.9 13 5.4 228 94.6 knee 66 27.4 175 72.6 17 7.1 224 92.9 17 7.1 224 92.9 26 10.8 215 89.2 ankle/foot 55 22.8 186 77.2 8 3.3 238 96.7 6 2.5 235 97.5 8 3.3 233 96.7 7 santos et al. in the united states, since 1995, posture training activities undertaken during working hours are presented in the curricula of the courses of dentistry10. since occupational problems can affect students of dentistry, it is necessary to include these activities in the curriculum. in this study, a high prevalence of painful symptomatology was observed in the study population; indeed, high pain rates have been previously reported by dentists of both sexes, with early onset5. these indexes were also high when only the students who already perform activities clinical practices were analyzed11,12. table 4. associations between sociodemographic variables and the variables of the nordic musculoskeletal questionnaire. araçatuba, 2013 . variable ga p gb p gc p gd pyes no yes no yes no yes no n % n % n % n % n % n % n % n % gender         female 131 66 20 48 0,027 37 63 114 63 0.992 43 63 108 62 0.907 79 68 72 58 0.129 male 68 34 22 52   22 37 68 37   25 37 65 38   38 33 52 42   marital status         married 3 1.5 1 2 0.539* 4 2.2 0.575* 2 3 2 1 0.318* 2 2 2 2 1.000* singles 195 99 41 98   59 100 177 98   66 97 170 99   115 98 121 98   bmi         normal 126 63 25 60 0.308** 39 66 112 62 0.934** 47 70 104 60 0.292** 73 62 78 63 0.252*** overweight 29 15 6 14 8 14 27 15 10 15 25 15 19 16 16 13 obesity 8 4 5 12 3 5,1 10 5.5 4 5 9 5 3 2,6 10 8,1 underweight 36 18 6 14   9 15 33 18   7 10 35 20   22 19 20 16   subjects         less than 12 109 55 15 36 0.025 30 51 94 52 0.915 37 54 87 50 0.564 67 57 57 46 0.079 12 or more 90 45 27 64   29 49 88 48   31 46 86 50   50 43 67 54   physical activies         yes 126 63 18 43 0.014 38 64 106 58 0.401 39 57 105 61 0.634 68 58 76 61 0.616 no 73 37 24 57   21 36 76 42   29 43 68 39   49 42 48 39   harmful habits         alcohol 71 36 10 24 0.158 21 36 60 33 0.75 20 29 61 36 0.356 39 34 42 34 0.932 no habit 127 64 31 76   38 64 120 67   48 71 110 64   77 66 81 66   health         yes 23 12 1 2 0.089* 9 15 15 8 0.118 16 24 8 4,6 0 19 16 5 4 0.002 no 176 88 41 98   50 85 167 92   52 77 165 95   98 84 119 96   medicine         yes 86 43 21 50 0.421 30 51 77 42 0.251 34 50 73 42 0.273 56 48 51 41 0.293 no 113 57 21 50   29 49 105 58   34 50 100 58   61 52 73 59   x2 test * fischer’s test ** likelihood ratios 8 santos et al. in a study using the rula methodology, we found a high risk for the development of musculoskeletal disorders and, especially, in the upper limbs, of the students who were performing preclinical activity11. in the present study, when the individual body areas were analyzed, the upper body (neck, shoulder, and upper back) was found to be the most affected by pain. these are commonly the most affected areas in both dentistry professionals and students13. among the possible pathologies are the degeneration of the intervertebral discs of the cervical region, such as the scapulohumeral periarteritis or bursitis, and physiological muscular contracture4. these lesions on the upper limbs often result in a temporary or permanent incapacity to work2. another region with a high prevalence of pain among students in this study was the lumbar area of the spine, however, the prevalence was lower than other studies. lower back pain is among one of the main chronic health complaints, leading to the need for medical care and also to absenteeism14. in a study of students from the first to the last year of graduation in dentistry (n = 154), 95 (62.5%) of the students complained of lower back pain15. however, when analyzed, the lower body was found to be affected to a lesser extent, and the majority of the discomfort or pain in these areas is due to the fact that students do not work with their feet supported on the floor11. a good sitting position, with a support base of the expanded body, avoids potential changes in the circulatory system, such as varicose veins, edema, pain, and inflammation, which may occur as a result of muscle compression of the lower extremities which impedes venous return7. despite the high prevalence of pain among students, there were few reports of impairments of normal activities because of pain and/or discomfort. consequently, the search for a health professional to treat or minimize the problems was also low. an association was found between pain symptoms (ga) and the number of disciplines studied, with a smaller number of disciplines being associated with a greater complaint of pain. this is likely due to the fact that the discipline of vocational guidance is given clinical disciplines concomitantly; this complicates the application of ergonomic knowledge in the practices of pre-clinical activities, which is reflected in their performance in clinical practice11,13. it is important to highlight the fact that students who are starting their clinical and preclinical activities focus on developing the proposed activities and are not aware of their posture during the execution of the work. this reinforces the need for ever-present ergonomic guidance during pre-clinical and clinical training so that the student receives constant information about the mistakes that they make and to ensure that they are promptly corrected11. in a study conducted by botta et al.16, it was possible to verify that the students understanding was limited in relation to the risk factors that contribute to the musculoskeletal disorders; this can be explained by a lack of knowledge of ergonomics. thus, the integration of preventive-educational programs and ergonomic training in the university dentistry curriculum is essential not only for the expansion of knowledge, but also to allow for the early diagnosis of musculoskeletal disorders. in addition, monitoring 9 santos et al. and/or advising of the ergonomic habits of future professionals, particularly in the initial phase of their clinical activities, is pertinent to the adoption of an adequate postural awareness, considering that these students are developing their manual skills, and are best placed to avoid future musculoskeletal damages16,17. just over half of the students interviewed stated that they performed physical activities, and the habit of exercising, essentially aerobics and stretching, is considered to be an ergonomic preventive means. the high prevalence of musculoskeletal pain is associated with the long working hours and the sedentary lifestyle of these professionals. studies have shown that because of the ability to increase oxygen flow, improve cardiovascular/musculoskeletal function and help reduce muscle tension due to incorrect posture, these physical activities may be relevant in reducing pain symptoms in work-associated musculoskeletal disorders related to work18-22. thus regular physical activity is suggested and is included in the curricula of undergraduate programs for the prevention of muscular disorders, especially back pain15. the present study demonstrated an association between the pain symptoms in the past year (ga) and the past 7 days (gd), and general health. indeed, in some previous studies, dentists have presented with poor general health7,23,24. it is important to emphasize that musculoskeletal pain can affect other areas of life, not just the performance and limitations of work activities. indeed, in a study conducted in brazil with teaching professionals who had musculoskeletal symptoms, it was shown that these pains correlated with the quality of life of the professionals23,25. the majority of the existing epidemiological studies involving dentistry students are cross-sectional and use different methods of identification and classification of painful symptomatology, which makes it difficult to compare existing studies. in addition, they present with the characteristic limitations of cross-sectional studies including an increase in the possibility of bias and not allowing for causal inference; however, they do provides essential evidence for future research in the field of ergonomics. another possible limitation of this study is associated with the use of self-administered instruments that only show the students’ occupational health at a specific moment in time7,24,25. in the current study, the subjects had a high prevalence of musculoskeletal symptoms (82.6%), especially in the upper limbs. we demonstrated an association between muscular pain and the number of disciplines studied, as well as between muscular pains and the general health of the students. thus, it is necessary to begin measures to reduce and prevent musculoskeletal disorders early, while students are still in training, since the correction of bad posture habits is easier at this early stage. references 1. aminian o, banafsheh alemohammad z, sadeghniiat-haghighi k. musculoskeletal disorders in female dentists and pharmacists: a cross-sectional study. acta med iran. 2012;50(9):635-40. 2. hayes m, cockrell d, smith dr. a systematic review of musculoskeletal disorders among dental professionals. int j dent hyg. 2009 aug;7(3):159-65. doi: 10.1111/j.1601-5037.2009.00395.x. 10 santos et al. 3. presoto cd, corrocher pa, campos jadb, garcia ppns. [risk factors for musculoskeletal disorders at the workplaces of undergraduate dental students]. pesq bras odontoped clin integr. 2012;12(4):549-54. doi: 10.4034/pboci.2012.124.16. portuguese. 4. régis filho gi, michels g, sell i. [work related musculoskeletal disorders in dentists]. rev bras epidemiol. 2006 sep;9(3): 346-9. doi: 10.1590/s1415-790x2006000300009. portuguese. 5. rising dw, bennett bc, hursh k, plesh o. reports of body pain in a dental student population. j am dent assoc. 2005 jan;136(1):81-6. 6. ribeiro nf, fernandes rde c, solla dj, santos junior ac, de sena junior as. [prevalence of musculoskeletal disorders in nursing professionals]. rev bras epidemiol. 2012 jun;15(2):429-38. portuguese. 7. garbin aj, garbin ca, diniz dg, yarid sd. dental students’ knowledge of ergonomic postural requirements and their application during clinical care. eur j dent educ. 2011 feb;15(1):31-5. doi: 10.1111/j.1600-0579.2010.00629.x. 8. de carvalho mv, soriano ep, de frança caldas a jr, campello ri, de miranda hf, cavalcanti fi. work-related musculoskeletal disorders among brazilian dental students. j dent educ. 2009 may;73(5):624-30. 9. freire a, soares g, rovida t, garbin c, garbin a. musculoskeletal disorders among dentists in northwest area of the state of são paulo, brazil. br j oral sci; 2017 jul/sep;15(3):199-204. doi: 10.20396/bjos.v15i3.8649979. 10. moodley r, naidoo s, wyk jv. the prevalence of occupational health-related problems in dentistry: a review of the literature. j occup health. 2018 mar 27;60(2):111-125. doi: 10.1539/joh.17-0188-ra. 11. peros k, vodanovic m, mestrovic s, rosin-grget k, valic m. physical fitness course in the dental curriculum and prevention of low back pain. j dent educ. 2011 jun;75(6):761-7. 12. biswas r, sachdev v, jindal v, ralhan s. musculoskeletal disorders and ergonomic risk factors in dental practice. indian j dent sci 2012 mar;4(1):70-4. 13. corrocher pa, presoto cd, campos ja, garcia pp. the association between restorative preclinical activities and musculoskeletal disorders. eur j dent educ. 2014 aug;18(3):142-6. doi: 10.1111/eje.12070. 14. de barros en, alexandre nm. cross-cultural adaptation of the nordic musculoskeletal questionnaire. int nurs rev. 2003 jun; 50(2):101-8. 15. pinheiro fa, troccoli bt, carvalho cv. [validity of the nordic musculoskeletal questionnaire as morbidity measurement tool]. rev saude publica. 2002 jun;36(3):307-12. portuguese. 16. khan sa, chew ky. effect of working characteristics and taught ergonomics on the prevalence of musculoskeletal disorders amongst dental students. bmc musculoskelet disord. 2013 apr 2;14:118. doi: 10.1186/1471-2474-14-118. 17. botta ac, presoto cd,wajngarten d,campos j, garcia p. perception of dental students on risk factors of musculoskeletal disorders. eur j dent educ. 2018 nov;22(4):209-214. doi: 10.1111/eje.12328. 18. garcia p, wajngarten d, campos j. development of a method to assess compliance with ergonomic posture in dental students. j educ health promot. 2018 apr 3;7:44. doi: 10.4103/jehp.jehp_66_17. 19. vakili l , halabchi f, mansournia ma , khami mr , irandoost s , alizadeh z. prevalence of common postural disorders among academic dental staff. asian j sports med. 2016 jan 16;7(2):e29631. doi: 10.5812/asjsm.29631. 20. carvalho fm, vidal cl, reis ac. [signs and symptoms of work-related musculoskeletal disorders in dentistry: evaluation of professors of the dental clinic of universidade vale do rio verde (unincor)]. clin lab res dent. 2019:1-9. doi: 10.11606/issn.2357-8041.clrd.2019.151007. portuguese 11 santos et al. 21. gupta d, bhaskar dj, gupta kr, karim b, kanwar a, jain a. et al. use of complementary and alternative medicine for work related musculoskeletal disorders associated with job contentament in dental professionals: indian outlook. ethiop j health sci. 2014 apr;24(2):117-24. 22. thakar s, shivlingesh kk, jayaprakash k, gupta b, gupta n, anand r, et al. high levels of physical inactivity amongst dental professionals: a questionnaire based cross sectional study. j clin diagn res. 2015 jan;9(1):zc43-6. doi: 10.7860/jcdr/2015/10459.5466. 23. kumar dk, rathan n, mohan s, begum m, prasad b, prasad erv. exercise prescriptions to prevent musculoskeletal disorders in dentists. j clin diagn res. 2014 jul;8(7):ze13-6. doi: 10.7860/jcdr/2014/7549.4620. 24. lindfors p, von thiele u, lundberg u. work characteristics and upper extremity disorders in female dental health workers. j occup health. 2006 may;48(3):192-7. 25. fernandes mh, da rocha vm, fagundes aa. [impact of osteomuscular symptoms on the quality of life of teachers]. rev bras epidemiol. 2011 jun;14(2):276-84. portuguese. 26. freire acgf, soares gb, rovida tas, garbin cas, garbin, ají. [musculoskeletal disorders and disability in brazilian dentists in são paulo.] rev dor. 2017;18(2):97-102. doi: 10.5935/1806-0013.20170020. portuguese. 27. garcia ppns, pinelli c, derceli j dos r, campos j álvares db. musculoskeletal disorders in upper limbs in dental students: exposure level to risk factors. braz j oral sci. 2012 apr/jun;11(2):148-53. doi: 10.20396/bjos.v11i2.8641448. oral sciences n3 original article braz j oral sci. october | december 2013 volume 12, number 4 bolton ratio in subjects with normal occlusion and malocclusion ivan delgado ricci1, marco antonio scanavini1, armando koichiro kaieda1, henrique damian rosário2, luiz renato paranhos3 1department of orthodontics, methodist university of são paulo, são bernardo do campo, sp, brazil 2department of oral biology, sacred heart university, bauru, sp, brazil 3department of orthodontics, school of dentistry, federal university of sergipe, lagarto, se, brazil correspondence to: luiz renato paranhos departmento de ortodontia universidade federal de sergipe rua pe alvares pirangueira, 248, cep: 49400-000, centro, lagarto, se, brasil phone: +55 19 983367619 fax: +55 19 38044002 e-mail: paranhos@ortodontista.com.br abstract aim: to verify the presence of bolton anterior and total discrepancy in brazilian individuals with natural normal occlusion and angle’s class i and class ii, division 1 malocclusions. methods: the sample was divided in three groups (n=35 each): natural normal occlusion; class i malocclusion; class ii, division 1 malocclusion. of the 105 caucasian brazilian individuals, 24 were boys and 81 were girls aged from 13 to 17 years and 4 months. the mesiodistal width of the maxillary and mandibular teeth, from the left first molar to the right first molar, was measured on each pre-treatment dental plaster cast using a digital caliper accurate to 0.01 mm resolution. values were tabulated and the bolton ratio was applied. the kolmogorov-smirnov test was used to verify if data were normally distributed (p>0.2). for comparison between the values obtained and those from the bolton standard, student’s t test was used and one-way anova was used for comparisons among the 3 groups, with a significance level of 5% (p<0.05). results: for groups 1, 2 and 3, respectively, the total ratio found was 90.36% (sd 1.70), 91.17% (sd±2.58) and 90.76% (sd±2.45); and the anterior ratio was 77.73% (sd 2.39), 78.01% (sd 2.66) and 77.30% (sd 2.65). conclusions: there was no significant difference among the groups regarding the values indicated in the bolton ratio. keywords: malocclusion, angle class i, malocclusion, angle class ii, tooth abnormalities. introduction a detailed planning phase is critical to the success of orthodontic treatment, by which the professional may identify and prevent occlusal disorders that impede treatment completion1,2. in addition, in this stage tooth discrepancies have greater influence. anatomical changes in dental proportions preclude obtaining an balanced occlusion with good intercuspation, as well as appropriate overjet and overbite3. according to proffit4, although natural teeth have a good ratio in most individuals, part of the population (5%) has some degree disproportion in tooth size. freeman et al.5 found that the excess of mass is greater in the mandible (19.7%) than in the maxilla (10.8%). in the beginning of the last century, black became interested in the study of dental morphology and size6. among the numerous analyses used by orthodontists, the study of casts allows to verify the existence and severity of interarch dental discrepancy7,8. the bolton analysis2 came up with the proposal to locate and determine tooth size discrepancies – intra and intermaxillary – thus avoiding the need to perform set up for such purpose. the bolton method assists in the decision to perform interdental stripping, dental extractions, or even increase of dental crown with restorative material. braz j oral sci. 12(4):357-361 received for publication: october 03, 2013 accepted: december 17, 2013 although several studies3,9-17 investigated the difference in the incidence of the bolton discrepancy between genders, there are still conflicting results in the literature. another relevant question is the relationship between the bolton ratio and the various types of malocclusion1,7,12,14-18. again, although there is evidence in the literature11,12,15-17,19,20 on the prevalence of the bolton discrepancy in diverse populations, there is no consensus about its relationship with the different types of malocclusions classified by angle. given the importance of these topics, this study aimed to evaluate the presence of the bolton anterior and total discrepancy in individuals with natural normal occlusion, class i and class ii, division 1 malocclusions. additionatly, it was investigated whether there was influence of sexual dimorphism on the obtained values. material and methods sample this study was undertaken after approval by the ethics committee of the methodist university of são paulo (umesp), brazil, under registration number #296120-09. the sample consisted of 105 pairs of plaster casts as part of the orthodontic records of umesp, corresponding to caucasian brazilian patients, with a minimum age of 13 years and maximum age of 17 years and 4 months (mean age: 15 years and 2 months), who presented teeth with intact proximal surfaces and centric relation near the habitual position. the sample exclusion criteria were: previous orthodontic treatment, agenesis and dental extractions. the sample was divided into three groups: group 1 (n=35) with natural normal occlusion; group 2 (n=35) with angle’s class i malocclusion; and group 3 (n=35) with angle’s class ii, division 1 malocclusion. measurement of the plaster casts the greatest mesiodistal distance was measured in each tooth in the maxillary and mandibular arches (except for 2nd and 3rd molars) using a mitutoyo digital caliper (model 500144, suzano, sp, brazil), with capacity of 150 mm and accurate to 0.01 mm. a previously trained single operator performed the procedures maintaining carefully the models and the caliper parallel to the ground. when the measurement of an arch was finished, the caliper was closed and zeroed again. its continued use could implicate errors due to the total closure of the instrument, and sometimes the display did not indicate zero. such procedure provided the method a greater accuracy. in order to evaluate the intraexaminer’s method error (table 1), measurements were taken twice at an interval of * statistically significant difference (p<0.05). ns=not statistically significant difference tooth 1st measurement 2nd measurement t p error mean s d mean s d 16 10.45 0.50 10.36 0.52 2.069 0.052ns 0.15 15 6.88 0.49 6.80 0.44 2.882 0.009* 0.10 14 7.23 0.47 7.08 0.49 4.595 0.000* 0.15 13 7.92 0.48 7.90 0.46 0.410 0.686ns 0.10 12 7.09 0.60 7.02 0.59 3.147 0.005* 0.09 11 8.85 0.52 8.83 0.51 0.561 0.581ns 0.09 21 8.86 0.53 8.80 0.54 2.026 0.056ns 0.11 22 7.01 0.54 6.96 0.57 1.285 0.214ns 0.12 23 7.91 0.43 7.73 0.53 3.462 0.002* 0.22 24 7.32 0.49 7.22 0.46 2.371 0.028* 0.14 25 6.91 0.53 6.83 0.52 3.003 0.007* 0.10 26 10.46 0.50 10.38 0.51 2.990 0.007* 0.09 36 10.98 0.62 10.93 0.60 2.263 0.035* 0.09 35 7.22 0.47 7.17 0.46 1.518 0.145ns 0.13 34 7.20 0.41 7.12 0.40 3.297 0.004* 0.09 33 6.96 0.41 6.85 0.42 4.875 0.000* 0.10 32 6.04 0.41 6.04 0.40 0.096 0.925ns 0.14 31 5.49 0.34 5.50 0.33 0.313 0.758ns 0.13 41 5.54 0.36 5.54 0.36 0.051 0.960ns 0.09 42 6.00 0.38 5.98 0.45 0.425 0.675ns 0.14 43 6.79 0.42 6.72 0.42 1.564 0.133ns 0.15 44 7.22 0.42 7.17 0.45 2.153 0.044* 0.10 45 7.31 0.50 7.23 0.53 3.249 0.004* 0.10 46 10.95 0.53 10.92 0.56 0.988 0.335ns 0.10 16-26 96.88 4.70 95.92 4.77 5.603 0.000* 0.87 13-23 47.64 2.63 47.24 2.73 3.965 0.001* 0.42 36-46 87.70 3.92 87.16 4.00 3.483 0.002* 0.62 33-43 36.81 1.91 36.64 1.97 1.306 0.207ns 0.45 bolton’s total 90.56 1.60 90.90 1.86 2.433 0.024* 0.51 bolton’s anterior 77.32 2.24 77.59 2.18 1.401 0.177ns 0.65 table 1.table 1.table 1.table 1.table 1. mean and standard deviation of two measurements, paired t-test and dahlberg’s error used to assess the systematic and random error. 171358 bolton ratio in subjects with normal occlusion and malocclusion braz j oral sci. 12(4):357-361 group variable female male t p mean s d mean s d normal boltontotal 90.36 1.70 90.44 1.20 -0.132 0.896ns bolton anterior 77.73 2.39 76.68 1.19 1.195 0.241ns class i boltontotal 91.17 2.58 91.25 3.24 -0.068 0.946ns bolton anterior 78.01 2.66 78.66 3.64 -0.561 0.579ns class ii boltontotal 90.76 2.45 90.37 2.35 0.405 0.688ns bolton anterior 77.30 2.65 77.27 2.08 0.029 0.977ns ns=not statistically significant difference table 2.table 2.table 2.table 2.table 2. anterior and total ratio analyzed according to gender and malocclusion (student’s t test). table 3.table 3.table 3.table 3.table 3. comparison between the bolton anterior and total ratio among the three groups (n=35 each). ns=not statistically significant difference group bolton total bolton anterior mean s d mean s d normal 90.38 1.58 77.49 2.20 class i 91.19 2.70 78.16 2.87 class ii 90.67 2.40 77.29 2.51 anova (p value) 0.324ns 0.331ns bolton group obtained standard t p mean s d mean s d total normal 90.38 1.58 91.30 1.91 2.386 0.019* class i 91.19 2.70 91.30 1.91 0.232 0.817ns class ii 90.67 2.40 91.30 1.91 1.377 0.172ns anterior normal 77.49 2.20 77.20 1.65 0.714 0.477ns class i 78.16 2.87 77.20 1.65 2.009 0.048* class ii 77.29 2.51 77.20 1.65 0.204 0.839ns table 4.table 4.table 4.table 4.table 4. anterior and total ratio (n=35) compared with the bolton ratio (n=55) (student’s t test). * statistically significant difference (p<0.05). ns=not statistically significant difference 15 days by randomly selecting 20% of the 105 pairs of plaster casts (n=21), resulting in 7 pairs per group. for suc purpose, the paired t test was used with a significance level of 5%. for determining the random error, the calculation error proposed by dahlberg21 was applied. to verify if data were normally distributed, was used the kolmogorov-smirnov test (p>0.2). for comparison between genders and obtained values, and those from the bolton standard, was used the student’s t test. one-way anova was used for comparisons among the 3 groups, with a significance level of 5% (p<0.05). results there was no statistically significant difference between genders for both anterior and total ratio in each group (table 2). since no occurrence of gender dimorphism was found, the sample was grouped so that it could be possible to compare a potential relationship between natural normal occlusion and the different types of malocclusion (class i and class ii, division 1). the presence or not of tooth size discrepancy was also verified. table 3 shows that there was no statistically significant difference for anterior and total ratio among the three studied groups. when the groups were compared in relation to the bolton standard (table 4), no statistically significant difference could be found for the anterior and total ratio, except for the total ratio of the normal occlusion group, and the anterior ratio of the class i malocclusion group. discussion the diagnosis of dental discrepancies has been proven to be of great importance in planning orthodontic treatments. according to bolton2, a good occlusion depends on a correct ratio between the dental masses in the maxillary and mandibular arches. by measuring the greatest mesiodistal width of each permanent tooth, including all the teeth since the 1st left to the 1st right permanent molar, this author2 found a ratio of 91.3% (sd 1.91). when only the six anterior teeth of the arch were evaluated, the ratio was 77.2% (sd 1.65). for bolton2, patients with means of anterior and total tooth size ratio above or below 2% of the values established in his research, should be classified as having tooth size discrepancy. other authors 5,8,12,19,22,23 agree that such dental relationship is mandatory for the orthodontic planning and finishing, in addition to influencing on occlusal factors. the significant values that were applied in this research for tooth size discrepancy followed the bolton criterion. nonetheless, the bolton’s sample consisted only of class i individuals, without gender differentiation. similar methodology was used in other studies1,3,5,9,10,14-18,20,24,25. it is worth noting that the plaster casts in the sample came from brazilians, which reflects multiethnicity and consequent genetic variety. individuals carry peculiarities and genetic features from their parents, such as teeth size and shape1,26,27. in a study with homoand heterozygous twins, 171359bolton ratio in subjects with normal occlusion and malocclusion braz j oral sci. 12(4):357-361 it was concluded that the mesiodistal width of the teeth and the anterior tooth ratio are genetically influenced27. another study3 reported a marked difference between the studied brazilian population and the bolton’s sample, probably owing to the extensive miscegenation of the population in brazil. in the present study, however, the studied population showed values for total and anterior ratio very close to those proposed by bolton, thus allowing his table to be used, even though his values were based on a north american caucasian population18. the literature 5,12,26,28,29 reported significant differences between races, both for dental size and proportions. there are reports in the literature 5,12,26,28,29 indicating a trend of men presenting teeth greater than those of women concerning the mediodistal width. these findings stimulated one of the aims of this research, which was to determine if there is sexual dimorphism in the different types of malocclusion in relation to tooth size discrepancy. since there was none, the groups had to be categorized by gender. in the group with natural normal occlusion, the observed values were similar regarding the means obtained for the total ratio in females (90.36%) and males (90.44%). in addition, close values were also found for the anterior ratio – 77.73% and 76.68% respectively. similar results were obtained by several authors1,9,12,14,30. the only measure that was shown to be increased in females was the amount of crowding in the lower anterior region (observed during measurement), although with no statistical significance. uysal et al.18 found sexual dimorphism in patients with normal occlusion only for the total ratio (91.73% in women and 89.83% in men). the authors18 point out that this difference may be related to features of the studied population, and that different racial groups should be treated according to their own characteristics. smith et al.28 corroborate the presence of sexual dimorphism among genders and races with respect to the parameters proposed by bolton. sexual dimorphism was not found in class i and ii malocclusions, with similar means obtained for the total ratio in males (91.17%) and females (91.25%). similarly, close values were also found between the means for the anterior ratio – 78.01% and 78.66% respectively for class i. with regards to the class ii, division 1 group, the results were 90.76% and 90.37%, for males and females respectively. an approximate value between means was also found for the anterior ratio – 77.30% and 77.27% respectively. these results are similar to those of several authors5,17,18,24. it is likely that racial variation between samples explains the differences between studies1,3,18. regarding the bolton ratio, there was no significant difference between the groups, i.e., the means obtained for this measure in individuals with natural normal occlusion, class i and class ii, division 1 were very close, indicating that the relationship between all the maxillary and mandibular teeth does not influence the occurrence of malocclusions. however, it is important to point out that the selection criterion for the type of malocclusion used here was proposed by angle, which takes into account only the mesiodistal relationship between the maxillary and mandibular first molars, excluding facial sagittal and vertical discrepancies that may be masked by dental compensations. other studies have found similar results1,12,14,15,17. yet, oktay and ulukaya30 identified differences only for the posterior region. in the samples of fattahi et al.24 and alkofide and hashim14 there was a higher incidence of the bolton discrepancy in individuals with class iii malocclusion. however, when only the anterior region was analyzed, there was a higher incidence of discrepancy for class iii, but only when compared to class ii14. motta et al.11 obtained opposite results, reporting a higher incidence in individuals with class i and ii malocclusions. on the other hand, based on a sample of 300 brazilians, araujo and souki3 reported an increased incidence in class i and iii patients. only when the anterior region was analyzed, class iii subjects had a higher incidence of discrepancy. the comparison of the bolton tooth ratio between normal occlusion and class i and class ii, division 1 malocclusions, showed no significant differences. there was no sexual dimorphism among tooth size discrepancies when compared with the natural normal occlusion and the different types of malocclusions investigated. references 1. akyalçin s, dogan s, dinçer b, erdinc ame, öncag g. bolton tooth size discrepancies in skeletal class i individuals presenting with different dental angle classifications. angle orthod. 2006; 76: 637-43. 2. bolton wa. disharmony in tooth size and its relations to the analysis and treatment of malocclusion. angle orthod. 1958; 28: 113-30. 3. araujo e, souki m. bolton anterior tooth size discrepancies among different malocclusion groups. angle orthod. 2003; 73: 307-13. 4. proffit wr. contemporary orthodontics, 4th edn. st louis: elsevier; 2007. 5. freeman je, maskeroni aj, lorton, l. frequency of bolton tooth size discrepancies among orthodontic patients. am j orthod dentofacial orthop. 1996; 11: 24-7. 6. harris fe, burris gb. contemporary permanent tooth dimensions, with comparisons to g.v. blacks data. j tenn dent assoc. 2003; 83: 25-9. 7. al-khateeb sn, alhaija esja. tooth size discrepancies and arch parameters among different malocclusions in a jordanian sample. angle orthod. 2006; 76: 459-65. 8. al-omari ik, al-bitar zb, hamdan am. tooth size discrepancies among jordanian schoolchildren. eur j orthod. 2008; 30: 527-31. 9. johe rs, steinhart t, sado n, greenberg b, jing s. intermaxillary toothsize discrepancies in different sexes, malocclusion groups, and ethnicities. am j orthod dentofacial orthop. 2010; 138: 599-607. 10. bernabé e, villanueva km, flores-mir, c. tooth width ratios in crowded and noncrowded dentitions. angle orthod. 2004; 74: 765-8. 11. motta ats, rodrigues s, quintão cca, capelli jr. j. analysis of tooth size discrepancy in patients of rio de janeiro state university orthodontic clinic. dental press j orthod. 2004; 9: 83-90. 12. o’mahony g, millett dt, barry mk, mcintyre gt, cronin ms. tooth size discrepancies in irish orthodontic patients among different malocclusion groups. angle orthod. 2011; 81: 130-4. 13. alkofide e, hashim h. intermaxillary tooth size discrepancies among different malocclusion classes: a comparative study. j clin pediatr dent. 2002; 26: 383-7. 14. basaran g, selek m, hamamci o, akkus z. intermaxillary bolton tooth size discrepancies among different malocclusion groups. angle orthod. 2006; 76: 26-30. 171360 bolton ratio in subjects with normal occlusion and malocclusion braz j oral sci. 12(4):357-361 15. kumar p, singh v, kumar p, sharma p, sharma r. effects of premolar extractions on bolton overall ratios and tooth-size discrepancies in a north indian population. j orthodont sci. 2013; 2: 23-7. 16. naseh r, padisar p, zarenemati p, moradi m, shojaeefard b. comparison of tooth size discrepancy in cl ii malocclusion patients with normal occlusions. j dent (shiraz). 2012; 13: 151-155. 17. endo t, abe r, kuroki h, oka k, shimooka s. tooth size discrepancies among different malocclusions in a japanese orthodontic population. angle orthod. 2008; 78: 994-9. 18. uysal t, sair z, basciftci fa, memili b. intermaxilary tooth size discrepancy and maloclusion: is there a relation? angle orthod. 2005; 75: 208-13. 19. santoro m, ayoub me, pardi va, cangialosi tj. mesiodistal crown dimensions and tooth size discrepancy of the permanent dentition of dominican americans. angle orthod. 2000; 70: 303-7. 20. manopatanakul s, watanawirun n. comprehensive intermaxillary tooth width proportion of bangkok residents. braz oral res. 2011; 11: 67-70. 21. houston, wjb. the analysis of errors in orthodontic measurements. am j orthod. 1983: 83: 382-90. 22. bolton wa. the clinical application of a tooth-size analysis. am j orthod. 1962; 48: 504-29. 23. heusdens m, dermaut l, verbeeck r. the effect of tooth size discrepancy on occlusion: an experimental study. am j orthod dentofacial orthop. 2000; 117: 184-91. 24. fattahi hr, pakshir hr, hedayati z. comparison of tooth size discrepancies among different malocclusion groups. eur j orthod. 2006; 28: 491-5. 25. endo t, uchikura k, ishida k, shundo i, sakaeda k, shimooka s. thresholds for clinically significant tooth-size discrepancy. angle orthod. 2009; 79: 740-6; 26. paredes v, gandia jl, cibrian r. determination of bolton toothsize ratios by digitization and comparison with the traditional method. eur j orthod. 2006; 28: 120-5. 27. garn sm, lewis ab, kerewsky rs. sex difference in tooth size. j dent res. 1964; 43: 306. 28. smith ss, buschang ph, watanabe e. interarch tooth size relationships of 3 populations: does bolton’s analysis apply? am j orthod dentofacial orthop. 2000; 117: 169-74. 29. bishara se, jakobsen jr, treder je, stasi mj. changes in the maxillary and mandibular tooth size-arch length relationship from early adolescent to early adulthood. am j orthod dentofacial orthop. 1989; 95: 46-59. 30. oktay h, ulukaya e. intermaxillary tooth size discrepancies among different malocclusion groups. eur j orthod. 2010; 32: 307-12. 171361bolton ratio in subjects with normal occlusion and malocclusion braz j oral sci. 12(4):357-361 oral sciences n3 original article braz j oral sci. january | march 2013 volume 12, number 1 oral health conditions and self-perception among edentulous individuals with different prosthetic status maria madalena canuto lemos1,2, luciane zanin3, maria letícia ramos jorge1, flávia martão flório2 1department of dentistry, faculty of basic and health sciences, federal university of valle do jequitinhonha e mucuri, diamantina, mg, brazil 2preventive dentistry, são leopoldo mandic school of dentistry and research center, campinas, sp, brazil 3preventive dentistry, hermínio ometto university center, araras, sp, brazil correspondence to: flávia martão flório rua josé rocha junqueira, 13, cep:13045-755 ponte preta, campinas, sp, brasil e-mail: flavia.florio@yahoo.com abstract aim: to evaluate the objective oral health conditions and self-perception of edentulous individuals wearing functional complete dentures (fcd) and non-functional complete dentures (nfcd) and completely edentulous non-denture wearers, and identify the factors that influence self-evaluation and the impact on quality of life. methods: the convenience sample was selected at the integrated clinic of a dentistry course (n=193) and was divided into 3 groups: fcd wearers (n= 54); nfcd wearers (n= 65); 3edentulous non-denture wearers (n= 74). data collection was performed by means of interviews, application of the geriatric oral health assessment index (gohai) and clinical exams. data were submitted to univariate analysis and logistic regression, considering dichotomization of the sample by the median value of gohai (=30). results: individuals with nfcd are 4.5 times more likely to show a low gohai score than individuals with fcd; edentulous individuals are 7.5 times more likely to show a low gohai score than individuals with fcd; individuals that consider their oral health as regular, poor or bad are 3.1 times more likely to show a low gohai score than individuals who consider their oral health as good or excellent. conclusions: being completely edentulous or wearing nfcd, and having oral health classified as regular, poor or extremely bad were important features for a negative self-perception of oral health, with impact on quality of life. keywords: oral health survey, quality of life, complete dentures,edentulism. introduction life expectancy of the brazilian population has increased significantly over the last few years, showing the importance of quality of life to the healthy aging and consequent need for specific health policies for this population1-4. edentulism is a prevalent condition in elderly individuals worldwide5. in brazil, recent studies have pointed out the presence of edentulism in 7 million individuals aged 65 to 74 years6, and it could be affirmed that complete dentures (cd) continue to be significant instruments of oral rehabilitation for this population7. various studies have pointed out the strong association between oral health and quality of life3-15. compromised oral health may affect the nutritional level as well as physical and mental well being, in addition to diminishing the pleasure of an active social life16-17. edentulism is a preponderantly negative factor for the oral and general health of individuals, with strong impact on quality of life. received for publication: october 04, 2012 accepted: january 10, 2013 braz j oral sci. 12(1):5-10 absence of teeth reduces masticatory function, resulting in alterations to dietary habits9,14. adequate cd are capable of restoring function and promoting social and psychological well being3,15. nevertheless, it is important to observe that the majority of these appliances are in an unsatisfactory condition for use1819, which compromises oral health, with impact on quality of life. oral lesions, such as stomatitis, inflammatory hyperplasia, traumatic ulcers and angular cheilitis are directly associated with the use of inadequate cd2. little research has been done about the self-perception of oral health and its impact on the quality of life of individuals7-8,10,15. different instruments have been developed to obtain data about self-perception of the oral condition and the impact on quality of life. one of these instruments is the geriatric oral health assessment index (gohai)20, which groups oral health self-perception in three basic functions: physical, psychosocial and pain/discomfort21. in this way, the perceived oral health becomes an important tool for diagnosing the priority requirements of this population and implementing actions that result in an improvement in quality of life11, through the development of educational, preventive and social measures and policies specifically for this population. the aims of this study were to evaluate the objective oral health conditions and self-perception of edentulous individuals wearing functional complete dentures (fcd) and non-functional complete dentures (nfcd) and completely edentulous nondenture wearers, and to identify the factors that influence self-evaluation and the impact on quality of life. material and methods the research consisted of a study involving questions about objective and subjective oral health in a convenience sample composed of 193 participants, age-range between 29 and 100 years (59.7±13.6), who were treated after screening at the integrated clinic of the dentistry course of the federal university of vale do jequitinhonha e mucuri – ufvjm, diamantina, mg, brazil, between april and december, 2009. the study was approved by the institutional ethics committee (protocol # 026/09). a pilot study was conducted with 30 participants to test the methodology and instruments to be used. two weeks later this group was re-evaluated. calibration for the clinical exam was carried out in accordance with the recommendations of the examiner calibration manual of sb brazil22, and the kappa agreement index was 0.91. the data collection instruments were composed of an interview form, application of the gohai index and a clinical exam form. by means of the interview form, sociodemographic data were obtained, and two questions about self-perception were applied: 1do you consider that you need treatment at present? (yes/no); 2how would you classify your oral health? (excellent/good/regular/poor/extremely bad). interviews were conducted individually by a calibrated member of the integrated clinic staff. for self-evaluation of oral health the gohai20 questionnaire was used, composed of 12 items that deal with oral health conditions, by self-perception in three dimensions: the physical, psychosocial and that of pain or discomfort. the physical dimension included diet, speech and swallowing. the psychosocial included concern about and interest in oral health, as well as dissatisfaction with appearance. the dimension of pain and discomfort included the use of medications to alleviate pain and discomfort provided that they appeared in the mouth. for each question, the questionnaire offers the alternatives “always”, “sometimes” and “never”, which were given scores 1, 2 and 3 respectively. the index is the result of the sum of scores to the questions on a scale from 12 to 36, so that the higher the score the better the oral health self-evaluation. the researcher performed the clinical exams to determine the objective conditions of the individual’s oral health. for each exam, the dental chair, reflector illumination, #5 dental mirror and a wooden spatula were used. the following features were observed: oral lesions, use and need for cd, time of denture wearing and its condition. the clinical exam criteria were based on the who manual23, as described in the examiner manual22. the volunteers were divided into three groups: group 1fcd wearers (n=54), group 2nfcd wearers (n= 65) and group 3completely edentulous individuals who did not wear dentures (n= 74). fcd were considered to be those that met the clinical requisites of stability, fit, occlusion, esthetics, retention and integrity19,24. nfcd were considered to be those that had compromised at least one of the requisites required for fcd. edentulous individuals were considered to be completely edentulous volunteers, at least in one arch, without the presence of cd at the time of the exam. for statistical treatment of the data the statistical package for social science (spss), version 15.0 software was used. the impact of the oral condition on the quality of life evaluated by means of dichotomization of gohai was chosen as a dependent variable. univariate analysis was performed to verify the association between each of the independent variables with gohai. for dichotomization of the sample, individuals with a positive perception of oral health were considered those whose scores presented values equal to or greater than the median (>30) and negative perception of oral health was considered with the scores presented values below the median. association of this variable with the independent variables was initially evaluated by means of the chi-square test. the level of significance as set at 5%. multiple logistic regression was performed to verify whether the impact of the oral condition on the quality of life was independently associated with the variables. results the volunteers’ ages ranged from 29 to 100 years (59.7±13.6), the majority were women (73.6%), 66.3% came from the urban area, 74% had 4 or more years of schooling 35935935935935966666 braz j oral sci. 12(1):5-10 oral health conditions and self-perception among edentulous individuals with different prosthetic status and 79.2% had a monthly income of approximately us$170 per month or less. as regards the subjective need for treatment, 66.3% affirmed that they needed treatment, but the majority of the volunteers (68%) classified their oral health as good group functional denture wearers non-functional denture wearers edentulous age 29 to 60 years 61 to 100 years gender female male place of origin rural urban schooling > 4 years < 4 years personal monthly income > 1 minimum wage < 1 minimum wage need for treatment (subjective) n o yes classification of oral health (subjective) excellent/good regular/poor/extremely bad need for denture (normative) n o yes time of use of mandibular denture <10 years >10 years time of use of maxillary denture <7 years >7 years oral lesions absent present > median (%) 43 (79.6) 21 (32.3) 23 (31.1) 38 (39.2) 49 (51.0) 62 (43.7) 25 (49.0) 27 (41.5) 60 (46.9) 61 (42.7) 26 (52.0) 23 (69.7) 55 (43.7) 48 (73.8) 39 (30.5) 73 (55.7) 14 (22.6) 44 (80.0) 43 (31.2) 27 (61.4) 19 (40.4) 47 (65.3) 25 (37.3) 81 (48.5) 6 (23.1) < median (%) 11 (20.4) 44 (67.7) 51 (68.9) 59 (60.8) 47 (49.0) 80 (56.3) 26 (51.0) 38 (58.5) 68 (53.1) 82 (57.3) 24 (48.0) 10 (30.3) 71 (56.3) 17 (26.2) 89 (69.5) 58 (44.3) 48 (77.4) 11 (20.0) 95 (68.8) 17 (38.6) 28 (59.6) 25 (34.7) 42 (62.7) 86 (51.5) 20 (76.9) p * <0.001 0.098 0.510 0.481 0.253 0.008 <0.001 <0.001 <0.001 0.046 0.001 0.015 gohai table 1 – relationship between the independent variables and the dependent variable (gohai) chi-square tests 77777 braz j oral sci. 12(1):5-10 oral health conditions and self-perception among edentulous individuals with different prosthetic status 88888 group functional denture wearers non-functional denture wearers edentulous personal income > 1 minimum wage < 1 minimum wage need for treatment (subjective) n o yes classification of oral health (subjective) excellent/good regular/poor/extremely bad need for denture (normative) n o yes time of use of maxillary denture < 7 years > 7 years time of use of mandibular denture < 10 years > 10 years presence of oral lesions absent present or not adjusted* (ic 95%)* 1.0 8.2 (3.5-19.0) 8.7 (3.8-19.8) 1.0 2.9 (1.3-6.7) 1.0 6.4 (3.3-12.6) 1.0 4.3 (2.2-8.6) 1.0 8.8 (4.2-18.8) 1.0 3.2 (1.6-6.3) 1.0 2.3 (1.1-5.4) 1.0 3.1 (1.2-8.2) p <0.001 <0.001 0.009 <0.001 <0.001 <0.001 0.001 0.048 0.020 or adjusted (ic 95%) 1.0 4.5 (1.6-12.5) 7.5 (2.7-20.8) ns* ns 1.00 3.1 (1.3-7.3) ns ns ns ns p 0,003 <0,001 0,008 gohai or= odds ratio; ci=confidence interval; ns= not significant table 2 – multivariate analysis, logistic regression and evaluation of presence of impact of oral condition and gohai. or excellent. the normative need for dentures was verified in 71.5% of the participants. the time of use of the mandibular cd ranged from 1 to 45 years and that of the maxillary cd between 1 and 55 years. oral lesions were present in 13.5% of the participants. table 1 shows the association of the dependent variable gohai, dichotomized as a function of the median value (=30), with the independent variables, using the chi-square statistical test. significant association was verified between the following variables and the impact on quality of life (gohai): study groups, treatment need (subjective), classification of oral health (subjective), need for complete denture use, personal income, time of use of the mandibular denture, time of use of the maxillary denture and presence of oral lesions. the impact was greater among the edentulous patients (68.9%) and nfcd wearers (67.7%), in comparison with the fcd wearers (20.4%). the impact was also greater among the participants who considered their oral health regular, poor or extremely bad (77,4%), in comparison with those who considered it good or excellent (44.3%). table 2 shows that individuals who were nfcd wearers presented 4.5 times more chance of a low gohai score than those with fcd. edentulous individuals presented 7.5 times more chance of presenting a low gohai score than the fcd wearers. individuals who classified their oral health as regular, poor or extremely bad presented 3.1 times more chance of presenting a low gohai score than those who classified it as good or excellent. discussion diagnosis of the conditions of health/disease in population groups has traditionally been made by means of approaches based on the biomedical model, in which health braz j oral sci. 12(1):5-10 oral health conditions and self-perception among edentulous individuals with different prosthetic status 99999 is understood simply as the absence of disease, and determination of health needs is obtained only from the normative point of view4,11,25. this point of view ignores important socio-behavioral aspects that must be considered in the evaluation of oral health conditions26. self-evaluation or oral health has been one of the indicators of quality of life widely used in dentistry as it reflects the subjective experience of individuals about their functional, social and psychological well being12,27. in this study, clinical-objective and subjective approaches to oral health were used, based on the selfperception of individuals. to evaluate the subjective conditions of oral health, a transculturally translated version21 of the gohai index20 was used. it is a multidimensional evaluation instrument, and has been widely used10,25,27-29. although findings in this study were limited by the use of a convenience sample and the perceptions of oral health status in a select sample at the integrated clinic of a dentistry course, in the present investigation, a strong association was verified between the variable ‘group’ (fcd, nfcd and edentulous) and ‘gohai’, with impact on the quality of life. oral rehabilitation by means of cd continues to be significant in the dental clinic, in spite of the great technical-scientific advancement of the profession7,30. however, the success and satisfaction with these appliances are directly associated with the physical, functional and psychosocial self-perfection of oral health by individuals31-33. the majority of the participants in the fcd group presented positive evaluation of oral health, corroborating the findings of other studies7-8,15, demonstrating that adequate cd are efficient instruments for recovering the masticatory function and promoting physical, social and psychological health of edentulous individuals, promoting satisfaction, increasing self-esteem and significantly enhancing their quality of life. this was the group with the highest scores for gohai, similar to the values obtained by veyruneet al19. the nfcd group presented 4.5 times more chance of having a low gohai score in comparison with the fcd group and presented a negative evaluation of oral health, demonstrating that the deficient condition of the cd exerts a strong influence on the individual’s oral health, resulting in a negative impact on the quality of life, because of the discomfort, difficulty with chewing and expression, reflected in his/her functional, social and psychological well being19,34. most dentures are found to be in unsatisfactory conditions18-19 and one of the reasons is their long time in use25 because functional qualities such as stability, retention, occlusion and vertical dimension become progressively unfavorable over time, and a specialized follow up is necessary for the maintenance of these dentures16,32. in this investigation it was verified that the majority of individuals in the edentulous group presented a negative evaluation of oral health, and had 7.5 times more chance of having a low gohai score than those in the fcd group, demonstrating that this was the worst condition. these results corroborate those of other investigations that verified that tooth loss has a strong impact on people’s life, and involves negative consequences, affecting significantly their quality of life5,7,9,13-14,17. as regards the subjective classification of oral health, the majority of the volunteers considered their oral health good or excellent, and presented a positive self-perception of oral health, corroborating the results of other investigations4,21. it is worth mentioning that most people see their oral condition in a favorable manner, even under unsatisfactory clinical conditions. this was probably because the clinical criteria of health used by the professional do not coincide with the individuals’ perception of oral health21,27, as occurred in this study, demonstrating that objective health measures are insufficient for evaluating the individual’s oral health4,35. participants who classified their oral health as regular, poor or extremely bad presented 3.1 times more chance of presenting a low gohai score than those who classified it as good or excellent, showing a negative evaluation of oral health. these results corroborate those of martins et al.4, who verified that subjective conditions, related to the impact on quality of life, were more strongly associated with negative selfevaluation of oral health, than objective conditions of health. the edentulous group presented the worst self-perception of oral health with the greatest impact on quality of life, followed by the nfcd wearers. the need to use a cd or even the use of a nfcd and the classification of oral health as regular, poor or extremely bad, were risk factors for the negative self-evaluation of oral health, demonstrating that the oral condition exerts a strong impact on people’s life. the findings of this study suggest that being completely edentulous, or wearing nfcd, and oral health classification into regular, poor or extremely bad were important features for negative self-perception of oral health, with impact on the volunteers quality of life. references 1. souza rf, patrocínio l, pero ac, marra j, compagnoni ma. reliability and validation of a brazilian version of the oral health impact profile for assessing edentulous subjects. j oral rehabil. 2007; 34: 821-6. 2. freitas jb, gomez rs, de abreu mhng, ferreira ef. relationship between the use of full dentures and mucosal alterations among elderly brazilians. j oral rehabil. 2008; 35: 370-6. 3. silva mes, magalhães cl, ferreira ef. complete removable prostheses: from expectation to (dis)satisfaction. gerodontology. 2009; 26: 143-9. 4. martins amebl, barreto sm, pordeus ia. objective and subjective factors related to self-rated oral health among the elderly. cad saúde pública.2009; 25: 421-35. 5. petersen pe, yamamoto t. improving the oral health of older people: the approach of the who global oral health. community dent oral epidemiol. 2005; 33: 81-92. 6. brazil. ministry of health care/health surveillance. departament of primmary care. coordination of oral health. project sb brazil 2010 – main results. brasilia; 2011. 7. carlsson ge, omar r. the future of complete dentures in oral rehabilitation: a critical review. j oral rehabil. 2009; 37: 143-56. 8. koshino h, hirai t, ishijima t, tsukagoshi h, ishigami t, tanaka y. quality of life and masticatory function in denture wearers. j oral rehabil. 2006; 33: 323-9. 9. tramini p, montal s, valcarcel j. tooth loss and associated factors in longterm institutionalised elderly patients. gerodontology. 2007; 24: 196-203 10. ellis js, pelekis nd, thomason jm.conventional rehabilitation of edentulous patients: the impact on oral health-related quality of life and patient satisfaction. j. prosthod. 2007; 16: 37-42. braz j oral sci. 12(1):5-10 oral health conditions and self-perception among edentulous individuals with different prosthetic status 1010101010 11. mesas ae, andrade sm, cabrera mas. factors associated with negative self-perception of oral health among elderly people in brazilian community. gerodontology. 2008; 25: 49-56. 12. baker sr, pearson nk, robinson pg. testing the applicability of a conceptual model of oral health in housebound edentulous older people. community dent oral epidemiol. 2008; 36: 237-48. 13. gagliardi di, slade gd, sanders ae. impact of dental care on oral healthrelated quality of life and treatment goals among elderly adults. aust dent j. 2008; 53: 26-33. 14. brennan ds, spencer aj, thomson-roberts kf. tooth loss, chewing ability and quality of life. qual life res. 2008; 17: 227-35. 15. kuo hc, yang yh, lai sk, yap sf, ho ps. the association between health-related quality of life and prosthetic status and prosthetic needs in taiwanese adults. j oral rehabil. 2009; 36: 217-25. 16. carlsson ge. clinical morbidity and sequelae of treatment with complete dentures. j prosthet dent.1998; 79: 17-23. 17. pan s, awad m, thomason mj, dufresne e, kobayashi t, kimoto s et al. sex differences in denture satisfaction. j dent. 2008; 36: 301-8. 18. mcnaugher ga, benington ic, freeman r. assessing expressed need and satisfaction in complete denture wearers. gerodontology. 2001; 18: 51-7. 19. veyrune jl, tubert-jeannin, dutheil c, riordan, pj. impact of new prostheses on the oral health related quality of life of edentulous patients. gerodontology. 2005; 22: 3-9. 20. atchison ka, dolan ta. development of the geriatric oral health assessment index. j dent educ 1990; 54: 680-7. 21. silva src, fernandes rac. self-perception of oral health status by the elderly. rev saude publica. 2001; 35: 349-55. 22. brazil. ministry of health. public policy department of health, department of primary care, technical department of oral health. project sb 2000. oral health status of the population in 2000: examiner’s manual. brasilia: ministry of health; 2001. 49p. 23. world health organization. oral health surveys: basic methods. 4th ed. geneva.: who; 1997. 24. de baat c, van aken aam, mulder j, kalk w. “prosthetic condition” and patients’ judgment of complete dentures.j prosthet dent. 1997; 78: 472-8. 25. colussi cf, freitas sft, calvo mcm. the prosthetic need who index: a comparison between self-perception and professional assessment in na elderly population. gerodontology. 2009; 26: 187-92. 26. reis scgb, higino masp, melo hmd, freire mcm. oral health status of institutionalized elderly in goiânia-go, brazil, 2003. rev bras epidemiol. 2005; 8: 67-73. 27. locker d, gibson b. discrepancies between self-ratings of and satisfaction with oral health in two older adult populations. community dent oral epidemiol. 2005; 33: 280-8. 28. locker d, matear d, stephens m, lawrence h, payne b. comparison of the gohai and ohip-14 as measures of the oral health-quality of life of the elderly. community dent oral epidemiol. 2001; 29: 373-81. 29. abud mc, dos santos jff, da cunha vpp, marchini l. tmd and gohai índices of brazilian institutionalised and community-dwelling elderly. gerodontology. 2009; 26: 34-9. 30. silva mes, villaça el, magalhães cl, ferreira ef. impact of tooth loss in quality of life. cienc saude col. 2010; 15: 841-50. 31. bae kh, kim c, paik di, kim jb.a comparison of oral health related quality of life between complete and partial removable denture-wearing older adults in korea.j oral rehabil. 2006; 33: 317-22. 32. fenlon mr, sherriff m. an investigation of factors influencing patients’ satisfaction with new complete dentures using structural equation modelling. j dent. 2008; 36: 427-34. 33. emami e, alisson pj, de grandmont p, rompré ph, feine js. better oral health related quality of life: type of prosthesis or psychological robustness? j dent. 2010; 38: 232-36. 34. john mt, koepsell td, hujoel p, miglioretti dl, leresche l, micheelis w. demographic factors, denture status and oral health-related quality of life.community dent oral epidemiol. 2004; 32: 125-32. 35. lacerda jt, castilho ea, calvo mcm, freitas sft. oral health and daily performance in adults in chapecó, santa catarina state, brazil. cad. saude publica. 2008; 24: 1846-58. braz j oral sci. 12(1):5-10 oral health conditions and self-perception among edentulous individuals with different prosthetic status oral sciences n3 original article braz j oral sci. july | september 2013 volume 12, number 3 profile of salivary gland flow dysfunctions in patients with differentiated thyroid carcinoma submitted to radioiodine therapy anna clara fontes vieira1, aline sampaio lima rodrigues1, maria carmen fontoura nogueira da cruz1,2, fernanda ferreira lopes1,2 1department of dentistry, dental school, federal university of maranhão, são luís, ma, brazil 2oral pathology department, dental school, federal university of maranhão, são luís, ma, brazil correspondence to: fernanda ferreira lopes avenida dos portugueses, campus do bacanga s/n, cep: 65085-580 prédio de odontologia, programa de pós-graduação, são luís, ma, brasil phone: +55 98 99717343 e-mail: fernanda.f.lopes@bol.com.br abstract aim: to evaluate the effects of radioiodine therapy on salivary flow in patients with differentiated thyroid cancer. methods: a sample comprising 88 patients submitted to ablation with iodine 131 was included in the study. the patients were submitted to sialometry and evaluation of the presence of xerostomia before, 10 days and 3 months after radioiodine therapy. results: xerostomia was observed in 36.4% of the patients before radioiodine therapy, 59.15% at 10 days after therapy, and 25% at 3 months after therapy. significant differences were observed in non-stimulated salivary flow rates between the second and third evaluations (p<0.020) and in stimulated salivary flow between the first and second evaluations (p<0.010). conclusions: the results suggest that changes in salivary flow resulting from radioiodine therapy are more pronounced during the first weeks after treatment and seem to regress after 3 months. keywords: thyroid gland tumors, iodine radioisotopes, salivary glands, saliva, xerostomia, hyposalivation. introduction differentiated thyroid carcinoma is a curable tumor, especially when diagnosed early. the most adequate treatment istotal thyroidectomy followed by actinic ablation with radioactive iodine (iodine 131) and has a very good prognosis (in more than 80% of cases) with excellent long-term survival, similar to that of the population that never had cancer1. iodine 131 is taken up by the thyroid through the sodium/iodide symporter (nis), a protein that is also expressed in other tissues such as the salivary glands, stomach and breast. as a consequence, iodine 131 can cause damage to these structures since it is absorbed at these sites after administration of therapeutic doses2-4. the beta radiation of iodine 131 exerts cytotoxic effects on the salivary glands because these organs are highly radiosensitive5. all salivary glands are involved in the transport of radioactive iodine into the saliva6. saliva is essential to maintain adequate oral functions such as lubrication, chewing and swallowing, speech, oral ph balance, taste perception, and cleanliness. quantitative and qualitative changes in salivary flow can compromise these functions. consequently, subjects with salivary gland dysfunction are more susceptible to periodontal disease, rampant caries, and fungal and bacterial oral received for publication: april 30, 2013 accepted: july 31, 2013 braz j oral sci. 12(3):169-172 170170170170170 infections7. longitudinal studies investigating the progression and intensity of salivary gland dysfunction in patients submitted to ablation with iodine 131 are scarce in the literature2,4,8-9. the objective of the present study was to evaluate the effects of iodine 131 on the salivary glands by subjective symptom of dryness in the mouth (xerostomia) and reduced salivary flow (hyposalivation) of patients with differentiated thyroid carcinoma submitted to radioiodine therapy. xerostomia and salivary flow were compared before and after therapy to establish the progression and frequency of salivary changes considering the high survival rate of these patients. material and methods a clinical longitudinal study with of 88 patients with differentiated thyroid carcinoma (papillary and follicular) who underwent complementary treatment with high single doses of iodine 131 (100 to 150 mci). the patients were treated at the radioiodine therapy service of instituto maranhense de oncologia aldenora bello and had not used drugs to treat other comorbidities. the collected data were: personal identification (name, age, gender, educational level and marital status), anatomopathological definition of the tumor, data of radioiodine therapy (dose), presence or absence of xerostomia and salivary flow. xerostomia and salivary flow were evaluated at three time points: before radioiodine therapy, 10 days after treatment (when a complete body scanning for the detection of metastases was done), and after 3 months. xerostomia was evaluated by subjective symptoms of dryness in the mouth during anamnesis, and classified as present or absent. the patients did not receive any recommendation for home salivary gland stimulation, during the period of this study. stimulated and non-stimulated whole saliva samples were collected for analysis of salivary flow10. the patients did not drink or eat 90 min before sialometry. all tests were performed at the same time (13:00 to 15:00 h) to minimize variations in salivary secretion11. descriptive statistics was used for analysis and the results were compared by the paired student’s t-test, adopting a 5% significance level. the study was approved by the ethics committee of the federal university of maranhão (protocol 23115005754/ 2009-00) and all patients signed an informed consent form. results in a convenience sample of 88 patients submitted to ablation with iodine 131, differentiated thyroid carcinoma was more prevalent among women (n=80) than men (n=8). patient age ranged from 18 to 72 years (mean: 43.4 years). seventy-seven patients had papillary carcinoma and 11 had follicular carcinoma. most participants (52.3%) were married, 5.7% were illiterate, and 39.8% completed high school. saliva was collected initially from 88 patients, but only 25 continued to participate in the study for sialometry 3 months after the administration of iodine 131. a decrease in the mean non-stimulated and stimulated salivary flow rates was observed between the first (before radioiodine therapy) and second evaluation (10 days after therapy). in contrast, mean non-stimulated and stimulated salivary flow increased between the second and third evaluation (3 months after therapy). these differences were statistically significant for non-stimulated salivary flow between the second and third evaluations (p<0.020) and for stimulated salivary flow between the first and second evaluations (p<0.010) (tables 1 and 2). xerostomia before radioiodine was reported by 36.4% of the patients. after 10 days of radioiodine therapy, 71 (100%) participants were reassessed and 42 (59.15%) reported xerostomia. twenty patients did not report xerostomia prior to therapy. after 3 months, only 5 (25%) among the 25 (100%) patients had xerostomia, and three of these five reported symptoms prior to therapy (table 3). discussion it is important to provide best-quality care to patient with differentiated thyroid carcinoma, so knowledge of non-stimulated salivary flow 1st evaluation 2nd evaluation mean sd mean sd t p 0.4384 0.3039 0.3600 0.2540 1.383 0.179 1st evaluation 3rd evaluation mean sd mean sd t p 0.4384 0.3039 0.5428 0.4564 -1.074 0.293 2nd evaluation 3rd evaluation mean sd mean sd t p 0.3600 0.2540 0.5428 0.4564 -2.491 0.020 table 1. comparison of non-stimulated salivary flow before radioiodine therapy (1st evaluation), 10 days (2nd evaluation) and 3 months (3rd evaluation) after therapy in 25 patients submitted to thyroidectomy*. results are reported as mean and standard deviation (sd). p<0.05 (paired t-test). *only 25 participants were submitted to sialometry 3 months after the administration of iodine 131. profile of salivary gland flow dysfunctions in patients with differentiated thyroid carcinoma submitted to radioiodine therapy braz j oral sci. 12(3):169-172 171171171171171 xerostomia before* after 10 days* after 3 months* n =88 (100%) n=71 (100%) n=25 (100%) present 32(36.4%) 42(59.15%) 5 (25%) absent 56(63.6%) 29(40.85%) 20(75%) table 3. distribution of patients with xerostomia before, after 10 days and after 3 months of radioiodine 131 therapy. *radioiodine 131 therapy complications and associated factors to high doses of radioactive iodine therapy (>100 mci) must be investigated12. there are few brazilian studies about repercussion of iodine ablation on the salivary glands12-13. although xerostomia was evaluated in these studies, no measurement of salivary flow to diagnose hyposalivation was done in these investigations. the present study showed an increase of patients with symptoms of xerostomia from 36.4% (before radioiodine therapy) to 59.15% (10 days after therapy). after 3 months, only 5 (25%) of the 25 reevaluated patients reported this symptom. similar results were described in a longitudinal study in which the population was submitted to more than one evaluation and a decrease in the percentage of patients with xerostomia was observed several months after treatment12. that study had a follow-up period of 3 months while in the preent investigation the follow up period was 8 months12. caglar et al.14 evaluated subjective symptoms of salivary gland dysfunction in 39 patients submitted to radioiodine therapy. twenty-one (54%) patients developed xerostomia after ablation with iodine 131. in contrast, grewel et al.9 evaluated retrospectively 262 patients in the first year of follow-up after treatment with high doses of iodine 131 and reported 17% of xerostomia. this incidence of xerostomia is similar to the 25% observed in the present study after 3 months of radioiodine therapy. however, in both cited studies salivary flow was not measured as in the present investigation. both non-stimulated and stimulated mean salivary flow rates decreased between the first (before radioiodine therapy) and second (10 days after therapy) evaluation. in contrast, an increase of non-stimulated and stimulated mean salivary flow rates was observed between the second and third (3 months after therapy) evaluations, i.e., salivary flow rates tended to return to baseline after 3 months. these findings suggest transient alterations in salivary flow as demonstrated by the observation of significant differences in non-stimulated salivary flow between the second and third evaluations (p<0.020) and in stimulated salivary flow between the first and second evaluations (p<0.010). although there are few data about salivary gland dysfunction after radioiodine therapy, apparently there is higher prevalence of transient alterations9. there are various longitudinal studies about postablation salivary gland dysfunction, but they are based on subjective prospective analyses (interviews and questionnaires) of glandular function8-9,12. generally, objective analysis of glandular function has been done by scintigraphy, but most of these studies applied a cross-sectional method to demonstrate the occurrence of salivary disorders within months after treatment13-14. prospectively, solans et al.3 evaluated glandular dysfunction by scintigraphy over a period of 3 years. however, the exam was performed annually, i.e., the first evaluation of salivary gland dysfunction was made only one year after treatment. contrastingly, in the present study the glandular function was evaluated objectively in 25 patients at three different time points (before, 10 days and 3 months after radioiodine therapy). analysis by sialometry showed reduced salivary flow as early as 10 days after treatment. this finding indicates the need for complete dental follow-up of these patients since the time of admission for iodine 131 treatment. there is no consensus on how to prevent adverse effects of ablation with high doses of radioactive iodine12. several protocols for the prevention of salivary gland injury are used. the most common recommendation is sucking lemon candies or ingestion of lemon juice during the period of iodine 131 treatment15. considering the definition of non-stimulated and stimulated hyposalivation (salivary flow rate<0.1 ml/min and < 0.7 ml/min, respectively)14, 7 of the studied patients had non-stimulated hyposalivation and 18 patients had stimulated hyposalivation at baseline. the prevention of salivary gland injuries after radioiodine ablation is of utmost importance because salivary gland dysfunction as an adverse effect of this therapy may turn the patients more susceptible to carious lesions and periodontal problems16. the results of this study suggest that changes in salivary flow resulting from radioiodine therapy are more pronounced during the first weeks after treatment and it seems to retreat after 3 months. however, further clinical studies are needed for evaluating qualitative changes of salivary composition in patients undergoing radioiodine therapy. after radioiodine therapy, atients require efficient measures to prevent or reduce salivary gland dysfunction in order to improve their quality of life. acknowledgements we thank capes/ education and culture government for the master’s fellowship granted to anna clara fontes vieira. we also thank fapema for publishing support and stimulated salivary flow 1st evaluation 2nd evaluation mean sd mean sd t p 1.5808 0.877 1.4144 0.9452 2.780 0.010 1st evaluation 3rd evaluation mean sd mean sd t p 1.5808 0.877 1.6864 1.0774 -0.815 0.423 2nd evaluation 3rd evaluation mean sd mean sd t p 1.4144 0.9452 1.6864 1.0774 -1.992 0.058 table 2. comparison of stimulated salivary flow before radioiodine therapy (1st evaluation), 10 days (2nd evaluation) and 3 months (3rd evaluation) after therapy in 25 patients submitted to thyroidectomy*. results are reported as mean and standard deviation (sd). p<0.05 (paired t-test). *only 25 participants were submitted to sialometry 3 months after the administration of iodine 131. profile of salivary gland flow dysfunctions in patients with differentiated thyroid carcinoma submitted to radioiodine therapy braz j oral sci. 12(3):169-172 172172172172172 dr. ibrahim assub and the nuclear medicine clinic of maranhão for providing the physical space for sample collection and for the interview with the volunteers of the study. references 1. ward ls, assumpção lv. thyroid cancer: prognostic factors and treatment. arq bras endocrinol metabol. 2004; 48: 126-36. 2. van nostrand d. sialoadenitis secondary to 131i therapy for well-differentiated thyroid cancer. oral dis. 2011; 17: 154-61. 3. solans r, bosch ja, galofré p, porta f, roselló j, selva-o’callagan a, et al. salivary and lacrimal gland dysfunction (sicca syndrome) after radioiodine therapy. j nucl med. 2001; 42: 738-43. 4. almeida jp, sanabria ae, lima en, kowalski lp. late side effects of radioactive iodine on salivary gland function in patients with thyroid cancer. head neck. 2011; 33: 686-90. 5. liu b, kuang a, huang r, zhao z, zeng y, wang j, tian r. influence of vitamin c on salivary absorbed dose of 131i in thyroid cancer patients: a prospective, randomized, single-blind, controlled trial. j nucl med. 2010; 51: 618-23. 6. jentzen w, balschuweit d, schmitz j, freudenberg l, eising e, hilbel t, et al. the influence of saliva flow stimulation on the absorbed radiation dose to the salivary glands during radioiodine therapy of thyroid cancer using (124)i pet(/ct) imaging. eur j nucl med mol imaging. 2010; 37: 2298-306. 7. aframian dj, helcer m, livni d, markitziu a. pilocarpine for the treatment of salivary glands’ impairment caused by radioiodine therapy for thyroid cancer. oral dis. 2006; 12: 297-300. 8. silberstein eb. reducing the incidence of 131i-induced sialadenitis: the role of pilocarpine. j nucl med. 2008; 49: 546-9. 9. grewal rk, larson sm, pentlow ce, pentlow ks, gonen m, qualey r, et al. salivary gland side effects commonly develop several weeks after initial radioactive iodine ablation. j nucl med. 2009; 50: 1605-10. 10. amenábar j m, pawlowski j, hilgert j b, hugo fn, bandeira d, lhüller f, et al. anxiety and salivary cortisol levels in patients with burning mouth syndrome: case-control study. oral surg oral med oral pathol oral radiol endod. 2008; 105: 460-5. 11. van den berg i, pijpe j, vissink a. salivary gland parameters and clinical data related to the underlying disorder in patients with persisting xerostomia. eur j oral sci. 2007; 115: 97-102. 12. rosário pws, maia ffr, barroso a, padrão el, rezende l, purish s. sialoadenitis following ablative therapy with high doses of radioiodine for treatment of differentiated thyroid cancer. arq bras endocrinol metabol. 2004; 48: 310-4. 13. rosário pws, cardoso ld, barroso al, padrão el, rezende ll, purish s. safety of radioiodine therapy in patients with thyroid carcinoma younger than 21 years. arq bras endocrinol metabol. 2005; 49: 241-5. 14. caglar m, tungel m, alpar r. scintigraphic evaluation of salivary gland dysfunction in patients with thyroid cancer after radioiodine treatment. clin nucl med. 2002; 27: 767-71. 15. van nostrand d, atkins f, bandaru vv, chennupati sp, moreau s, burman k, et al. salivary gland protection with sialogogues: a case study. thyroid. 2009; 19: 1005-8. 16. bomeli sr, desai sc, johnson jt, walvekar rr. management of salivary flow in head and neck cancer patients a systematic review. oral oncol. 2008; 44: 1000-8. profile of salivary gland flow dysfunctions in patients with differentiated thyroid carcinoma submitted to radioiodine therapy braz j oral sci. 12(3):169-172 oral sciences n3 original article braz j oral sci. april | june 2015 volume 14, number 2 braz j oral sci. 14(2)176-181 electromyography and asymmetry index of masticatory muscles in undergraduate students with temporomandibular disorders gisele harumi hotta1, ana izabela sobral de oliveira1, anamaria siriani de oliveira1, cristiane rodrigues pedroni2 1universidade de são paulo usp, ribeirão preto medical school, department of biomechanics, medicine and rehabilitation of the locomotor apparatus, ribeirão preto, sp, brazil 2universidade estadual paulista unesp, school of philosophy and sciences, department of physical terapy and occupational therapy, marilia, sp, brazil correspondence to: gisele harumi hotta prédio da fisioterapia e terapia ocupacional da faculdade de medicina de ribeirão preto da universidade de são paulo avenida bandeirantes, 3900, bairro monte alegre cep: 14049-900 ribeirão preto, sp, brasil phone: +55 16 33150737 e-mail: harumi.hotta@usp.br abstract aim: to compare the electromyographic activity and the asymmetry index among degrees of severity of temporomandibular disorders (tmd). methods: surface electromyography (emg) of the right and left masseter and temporalis muscles was performed in 126 undergraduate students at rest and at maximal voluntary contraction. three measurements were performed for five seconds of maximal contraction and mandibular rest. the degree of temporomandibular dysfunction was identified according to the fonseca anamnestic index. the analysis of the asymmetry index for two pairs of muscles during maximal voluntary contraction was based on the asymmetry index proposed by naeije, mccarrol and weijs (1989). results: 48.41% of the sample had mild tmd, followed by volunteers without tmd (26.98%), moderate tmd (19.05%) and severe (5.56%). the survey results show absence of correlation between the fonseca anamnestic index and electromyographic activity at rest and at maximal voluntary contraction in undergraduate students in both muscles (p>0.05) and the asymmetry index did not differ between the analyzed groups. conclusions: for the population of undergraduate students, there is no evidence that the presence and severity of tmd influence the emg activity of masseter and temporalis muscles and the muscle asymmetry index at rest and maximal voluntary contraction. keywords: electromyography; evaluation; stomatognathic system. introduction temporomandibular dysfunction (tmd) is a disorder of the stomatognathic system characterized by the presence of pain in the preauricular region, fatigue of masticatory muscles (mm), limitation or deviation during movement of the temporomandibular joint (tmj) and could be associated to noise during opening and closing the mouth1-2. the difficulty of identifying the tmd is related to its multifactorial etiology and the complex relationship of tmj with other structures of the head, neck and scapular waist3, apart from an important relationship with emotional factors, especially in adolescents2,4. the main muscles affected in this dysfunction are the temporal (anterior and posterior), masseter (superficial and deep), pterygoid (medial and lateral) and digastric muscles5. the prevalence of tmd symptoms has been shown to vary from 16 to 59% in general populations 1 while other studies showed that nearly 70% of received for publication: may 05, 2015 accepted: june 26, 2015 http://dx.doi.org/10.1590/1677-3225v14n2a15 undergraduate students present some symptoms of tmd3,6. despite such a high prevalence, only 7% of subjects classified as moderate and severe tmd patients reported to had ever sought medical care6. undergraduate students are vulnerable to signs and symptoms of tmd6, but the future impact of these symptoms on this population is unknown. several questionnaires were prepared for the assessment of tmd signs and symptoms, and currently the most widely used method for classification is the research diagnostic criteria for temporomandibular disorders (rdc/tmd)4. nevertheless, the index proposed by helkimo7 (1994) has been clinically used to classify the severity levels of tmd, especially in epidemiological survey studies1-4,6. fonseca et al.8 (1994) based on the helkimo index7 translated and validated the questionnaire into portuguese with 95% reliability and good correlation with the helkimo index. the fonseca et al.8 (1994) questionnaire has the advantage of being self-manageable, short application time, low cost and may be used for the screening of patients1-2,6. surface electromyography is widely used for evaluation and observation of the electrophysiological behavior of the muscles under different physiological conditions 4-5. this method is a non-invasive evaluation of the bioelectric phenomena of muscle activity at rest and during muscle contraction and is useful for assessing the electrical activity of mm9-10. research of electromyographic characteristics among undergraduate students with tmd is essential for an early detection of bioelectric characteristics of muscles that may predispose individuals to pain and muscle dysfunction and/or joint in the adult age4,9. electromyography studies in mm have shown that the mean values for electrical activity of the masseter and temporal muscles at rest range 2.2 4.0 v in individuals with tmd and 2.9 3.8 v in healthy subjects, without a significant difference among the groups10-11. however, glaros et al.12 (1997) state that the electrical activity is slightly higher in the tmd patients,, averaging 2.9 to 3.3 v for the masseter and 4.5 to 5.7 v for temporal. in healthy volunteers, the average was 2.2 to 2.3 for the masseter and 3.6 to 3.7 for the temporal. in literature, the consensus is that the temporal muscle has a higher electrical activity at rest compared with the masseter10-12. at maximal voluntary contraction (mvc), adult tmd patients have a lower electrical activity, ranging from 64.1 to 79.2 v, compared to healthy subjects, ranging from 140.2 to 182.8 v10-11, but such values have not been found for the college age group. in this context, the aim of this study was to compare the electromyographic activity and the asymmetry index with the severity degrees of tmd. material and methods participants the convenience sample was recruited from the são paulo state university unesp, marilia, brazil. individuals using orthodontic appliances or orthopedic functional, fixed or removable dentures were excluded from the study and also abusers of painkiller-type drugs, anti-inflammatory drugs, muscle relaxants and antidepressants, as well as those with some type of neurological disorder. this study was approved by the ethics in research committee of the são paulo state university in accordance with resolution of the national health council 196/96 (reference number: 0457/2012) and the rights of the subjects were protected. all participants were informed about the study and signed an informed consent form before participating. instruments and assessment procedures fonseca anamnestic index (iaf) the iaf was initially applied to each individual by a physical therapist blinded to the electromyographic evaluation, determining the presence and severity of tmd. the questionnaire consists of 10 questions, where the yes answer scores 10, sometimes scores 5 and no scores 0. the sum of the answer values corresponds to the following classification: 0-15 (no tmd), 20-40 (mild tmd), 45-65 (moderate tmd) and 70-100 (severe tmd). surface electromyography simultaneous sampling of signals was used to record the electromyographic signals of the mm (bilateral masseter and anterior temporal muscles) by the lynx technology ltd (lynx® são paulo, sp, brazil) a/d board, composed of six active channels with analog band pass filters with cut off frequency at 20-1000 hz and digitized with 16-bit resolution. the equipment was connected to a battery with a capacity of 10 ah, 12 v, to reduce the noise from the power supply. the guidelines of surface electromyography for non-invasive assessment of muscles (seniam) were followed to prepare the skin13. during signal acquisition, the subjects remained comfortably seated in a chair with back support and no support for the head and upper limbs, maintaining the hands rested on the femur, the soles of the feet flat on the floor with the knees in a 90° flexion, head erect and looking to the horizon. the volunteer got no visual feedback from the computer screen. before each data collection, the volunteer underwent familiarization with the equipment and the movements to be performed, getting all the necessary instructions and information. the electrodes were placed bilaterally along the anterior fibers of the temporal and masseter muscles14. the recordings were made in the resting mandibular position and maximal voluntary contraction (mvc) with the use of parafilm m® (wertheim, baden-württemberg, germany) folded similarly to a trident gum (3.5x1.5)15 and placed bilaterally between the premolars, first and second mandibular and maxillary molars. there were three measurements during mvc. all recordings were performed for 5 s with a two-minute interval between them. the data acquisition and storage of digitized signals were performed by aqdados software (lynx®), version 7.2 177177177177177 electromyography and asymmetry index of masticatory muscles in undergraduate students with temporomandibular disorders braz j oral sci. 14(2)176-181 1 7 11 7 11 7 11 7 11 7 1178178178178178 groups sample size age iaf without tmd 34 (26.98 %) 21.59 ± 5.21 9.56 ± 4.33 mild tmd 61 (48.41 %) 21.15 ± 2.85 27.79 ± 6.31 moderate tmd 24 (19.05 %) 21.69 ± 3.40 55 ± 6.35 severe tmd 7 (5.56 %) 22 ± 2.55 80.63 ± 9.43 tmd: temporomandibular dysfunction; iaf: fonseca anamnestic index. table 1:table 1:table 1:table 1:table 1: sample characterization according to the score obtained from the fonseca anamnestic index8 for windows. the used sensors were passive surface double halves of silver/silver chloride circles, adhesive, disposable, 42 mm long, 20 mm wide, 20 mm from center to center (miotec® porto alegre, rs, brazil), coupled to a lynx technology ltd preamp with a 10 gω input impedance, 130db common rate of rejection and gain of 20 times. data analysis the electromyographic signal was processed in the time domain by root mean square (rms). all registration procedures and analyses of the electromyographic signal followed the standards for reporting emg data16. data from the mvc and at rest were standardized on the average obtained by three repetitions of the mvc. the statistical program used was spss statistics version 17.0 for windows (spss inc, chicago, il). the kolmogorovsmirnov test was used to verify the normality of data. the kruskal-wallis test was used for comparison between groups. the spearman correlation coefficient was used to calculate the association between variables. for all data processing the significance level was set at 5%. the asymmetry values of muscle activity of the two muscle pairs during isometric contraction were based on the asymmetry index (ai), proposed by naeije, mccarrol and weijs 17 (1989). if the muscle activation level is fully symmetrical ia is 0%, while the full asymmetry corresponds to 100%. the formula is: ia = [(rms right left rms)/ (rms right + left rms) * 100]. results the clinical and demographic characteristics of the sample are shown in table 1. the participants were 126 undergraduate students with mean age of 21.59 (±5.21) years and 74.24% of the sample were females. among the students, 34 (26.98%) presented no tmd, 61 (48.41%) mild tmd, 24 (19.05%) moderate tmd and 7 (5.56%) severe tmd. comparing the groups according to the iaf severity and disregarding the right and left sides, the rms values of electromyography activity showed no difference (p<0.05) for the masseter and temporalis at rest (figures 1 and 3) and in mvc (figures 2 and 4). the analysis of the electromyographic activity and the iaf carried out by spearman correlation coefficient showed that there is no association between the variables. correlation was not found between the rms value of the masseter and iaf (r=-0.066), and the temporal and iaf with mvc (r=-0.081). fig. 1: comparison between groups in relation to masseter electromyographic at rest. tmd: temporomandibular dysfunction there is no evidence that the temporal (r=0.063) and the masseter (r=-0.001) correlate with the iaf at rest (table 2). the groups showed a degree of asymmetry index with greater evidence at rest, however, statistical differences were not found between groups. descriptive data are presented in table 3. fig. 3: comparison between groups in relation to temporal electromyographic activity at rest. tmd: temporomandibular dysfunction electromyography and asymmetry index of masticatory muscles in undergraduate students with temporomandibular disorders braz j oral sci. 14(2)176-181 fig. 2: comparison between groups in relation to masseter electromyographic activity at maximal voluntary clench. tmd: temporomandibular dysfunction 179179179179179 correlation r s p valor iaf (score) x mear (rms) -0.001 0.99 iaf (score) x tear (rms) 0.063 0.47 iaf (score) x meamvc (rms) -0.066 0.44 iaf (score) x meamvc (rms) -0.081 0.35 iaf: fonseca anamnestic index; mear: masseter’s electromyographic activity at rest; tear: temporal electromyographic activity at rest; meamvc: masseter electromyographic activity at maximal voluntary contraction; teamvc: temporal electromyographic activity at maximal voluntary contraction. rs: spearman’s correlation coefficient table 2: table 2: table 2: table 2: table 2: correlation between the fonseca anamnestic index and electromyographic activity at rest and at maximal voluntary contraction. masseter temporalis resting (%) mvc (%) resting (%) mvc (%) without tmd 17.29 ± 18.10 3.47 ± 7.32 22.61 ± 16.83 5.85 ± 9.15 mild tmd 14.65 ± 15.81 5.28 ± 9.75 16.93 ± 16.61 5.16 ± 8.97 moderate tmd 19.24 ± 25.05 8.53 ± 16.23 27.82 ± 19.42 5.00 ± 8.46 severe tmd 20.51 ± 23.71 12.83 ± 13.61 29.36 ± 26.96 7.27 ± 11,93 table 3:table 3:table 3:table 3:table 3: descriptive data of the asymmetry index, according naije et al17 (1989). data expressed as mean ± standard deviation. mvc: maximal voluntary contraction; tmd: temporomandibular dysfunction. fig. 4: comparison between groups in relation to temporal electromyographic activity at maximal voluntary clench. tmd: temporomandibular dysfunction discussion the study results showed that among the undergraduate students population, there are many individuals with some type of tmd signs or symptoms. the emg activity of the masseter and temporal does not differ between the presence or absence of symptoms and between different degrees of tmd, both at rest and the maximal voluntary contraction (mvc). there is also no evidence of changes in muscle asymmetry index at rest and mvc in individuals with signs and symptoms of tmd. there is no correlation between the score obtained by iaf and the emg activity of masseter and temporalis muscles. the high incidence of mild tdm in the study population corroborates those of studies that classify mild tmd as the most prevalent type among brazilian male and female undergraduate students1-2. this large number of undergraduate students with tmd signs or symptoms emphasizes the importance of studying tmd in this age group, considering possible progression of the disorder and future need of specialized medical care2,6. the results of this study are consistent with those found of hugger et al.10 (2012) and weggen et al.18 (2012), who analyzed emg activity in mvc and did not identify differences between groups of healthy participants and those with tmd10,18. this finding is justified by the fact that to create accurate jaw movements, entries of various sensory receptors must be received by the central nervous system by afferent nerve fibers. the brain assimilates and organizes this information and generates appropriate motor activities by the efferent nerve fibers. these motor activities involve the contraction of some muscle groups and the inhibition of others. chewing is a subconscious activity, but it may be controlled consciously at any moment 5 declaring the individual’s ability to control the use of the mm in unnecessary activities, avoiding increase in electrical activity of the muscle in this phase. healthy subjects when compared with individuals with tmd exhibit reduction of electrical potentials of the masseter and temporal muscles at rest19-21. however, these differences are not consistent in the literature, and the increase of electrical potentials at rest is considered in most cases as the result of chronic pain10. although some authors11,19-21 confirm the increased emg activity at rest and decrease in activation capacity during mvc and it is known that during mastication the relationship between muscle actions is generally similar among individuals of the same gender. however, there are significant differences in bite between men and women and between young and adults,5 justifying the difference between the results of this study with the above mentioned, since the populations consisted of different age groups. the chewing movements of patients with myofascial pain had the same pattern of healthy individuals. thus, people with pain in mm or joint noises may have normal mandibular range of motion5. in both groups at the start of the closing phase of mastication, the ipsilateral side of the temporal muscle to the bite is first activated and then the contralateral temporal muscle and masseter muscles act simultaneously. the emg activity in these muscles is very low, but it increases gradually, electromyography and asymmetry index of masticatory muscles in undergraduate students with temporomandibular disorders braz j oral sci. 14(2)176-181 1 7 11 7 11 7 11 7 11 7 1180180180180180 reaching a peak at the end of the closing movement5. the muscle activity changes during mvc and at rest are relatively small, but these small changes in muscle activity at rest were reported as clinical indicators of tmd, but considering these small changes in emg activity as a factor in the pathogenesis of muscle pain in these patients is still controversial22. the muscle tension is characterized as causal factor for tmd, therefore the electrical activity is not necessarily increased in the mm on these patients. however, it should be stressed that a possible increase in muscle activity may precede and produce pain, and that once a group of motor units are tired or painful they are centrally inhibited from being recruited22. the electromyographic data from this study, along with the data from carlson et al.22 (1998) do not support the use of emg on an integrated surface as a means to differentiate patients with tmd from controls, even when they are exposed to standard stresses with a significant level of physiological and emotional activation23. in most studies the emg activity is increased at rest for tmd patients, corroborating the above information. nevertheless, chandu et al.23 (2004) and hugger et al.10 (2012) concluded that the isolated assessment of emg activity of the masseter and temporal muscles is not able to evaluate and distinguish the tmd patients from orofacial pain-free individuals. the muscle asymmetry during chewing activity is seen predominantly in patients with tmd24. however, rodriguesbigaton et al.25 (2010) found no significant difference between tmd or healthy patients when assessing muscle asymmetry. it is observed that all individuals including the healthy ones have some degree of mm asymmetry, and that the temporal muscle is more active compared to the masseter in all subjects10,24-25. tartaglia et al.21 (2011) found that the emg activity of the masseter, temporalis and sternocleidomastoid muscles is less symmetrical in tmd patients, however this difference is not significant, while hugger et al.10 (2012) report that there is no difference between the groups as regards symmetry of the masseter muscle activity, but analyzing the temporal muscle this difference is verified. there is no difference between tmd patients and healthy individuals when observing asymmetry at rest and it can be seen that healthy individuals exhibit a significant dominance of temporalis in relation to the masseter muscles. however, this prevalence does not exist while analyzing asymmetry during cvm, additionally, dominance between the sides in the same patient was found in both groups25. in conclusion, for this population of undergraduate students, there is no evidence that the presence and severity of tmd influence the emg activity of masseter and temporalis muscles and the muscle asymmetry index at rest and maximal voluntary contraction. acknowledgements the authors are grateful to são paulo state university and pró-reitoria de pesquisa (prope) for financial support. no conflicts of interest declared. references 1. bevilaqua-grossi d, chaves tc, de oliveira as, monteiro-pedro v. anamnestic index severity and signs and symptoms of tmd. cranio j craniomandib pract. 2006; 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kinesiol. 2012; 22: 266-72. 15. ap biasotto-gonzalez d, berzin f, da costa jm, de gonzalez to. electromyographic study of stomatognathic system muscles during chewing of different materials. electromyogr clin neurophysiol. 2010; 50: 121-7. 16. merletti r. standards for reporting emg data. j electromyogr kinesiol. 1999 [cited 2015 may 4]; 24: iii-iv. available from: http: // www.jelectromyographykinesiology.com/article/s1050641114000066/ abstract 17. naeije m, mccarroll rs, weijs wa. electromyographic activity of the human masticatory muscles during submaximal clenching in the intercuspal position. j oral rehabil. 1989; 16: 63-70. electromyography and asymmetry index of masticatory muscles in undergraduate students with temporomandibular disorders braz j oral sci. 14(2)176-181 181181181181181 18. weggen t, schindler hj, kordass b, hugger a. clinical and electromyographic follow-up of myofascial pain patients treated with two types of oral splint: a randomized controlled pilot study. int 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electromyography in asymptomatic subjects and patients with temporomandibular pain and dysfunction. j oral rehabil. 2004; 31: 530-7. 24. scopel v, alves da costa gs, urias d. an electromyographic study of masseter and anterior temporalis muscles in extra-articular myogenous tmj pain patients compared to an asymptomatic and normal population. cranio j craniomandib pract. 2005; 23: 194-203. 25. rodrigues-bigaton d, berni kcs, almeida afn, silva mt. activity and asymmetry index of masticatory muscles in women with and without dysfunction temporomandibular. electromyogr clin neurophysiol. 2010; 50: 333-8. electromyography and asymmetry index of masticatory muscles in undergraduate students with temporomandibular disorders braz j oral sci. 14(2)176-181 oral sciences n3 braz j oral sci. 13(2):146-151 original article braz j oral sci. april | june 2014 volume 13, number 2 indicators of the risk mechanics for class-i and class-ii amalgam and composite resin restorations eduardo fernández1, erik dreyer arroyo2, claudia letelier pardo1, osmir batista oliveira junior3, gustavo moncada cortés1, javier martín casielles1 1university of chile, department of restorative dentistry, independencia, santiago, chile 2university of chile, department of conservative dentistry, independencia, santiago, chile 3universidade estadual paulista unesp, araraquara dental school, department of restorative dentistry, araraquara, sp, brazil correspondence to: eduardo fernández university of chile, director of department restorative dentistry, olivos 943 – independencia santiago – chile phone: +56 998854770 e-mail: edofdez@yahoo.com received for publication: april 24, 2014 accepted: june 10, 2014 abstract aim: to determine indicators of prognosis for mechanical risks of amalgam and composite resin restorations in permanent teeth. methods: thirty-nine adult patients with direct clinical, photographic, radiographic and model examinations. a total of 256 restorations were classified as “not satisfactory,” with bravo or charlie values according to the modified ryge /usphs criteria. the total “n” was divided into bravo and charlie groups according to the value obtained in the “marginal adaptation” parameter. each of the groups was sub-divided by the type of material (amalgam and composite resins) and the class: occlusal (o) and proximal (mod). results: comparing the bravo and charlie groups, the statistically significant indicators were: the mesiodistal dimension (p=0.037), the distal isthmus (p<0.05), the average of the isthmuses (p<0.05), the distal (p<0.05) cavity depth, and the average depth of the mod (p<0.05) cavities. it was concluded that the type and the class of the restoration are not indicators for sampling. conclusions: with regard to the design of the cavity preparation, the valid mechanical risk indicators include the mesiodistal dimension, the distal isthmus, the average of the isthmuses, the depth of the distal cavity and the average depth of the mod cavities. a simple clinical assessment does not provide sufficient information to establish the indicators for mechanical failure risk of restorations. keywords: risk management; composite resins; dental amalgam. introduction restorations exist in a septic environment and are functionally tested during chewing, a series of mechanical loads and flexural compressions that are increased in magnitude if the patient presents bruxism or has a reduced number of teeth1. due to multiple reasons, these restorations may fail and require replacement2-10. while treatment variables are often studied, it is necessary to study the causes of failure and within those causes, to search for indicators that can be easily recognized, assessed and compared over time to establish the predictors of mechanical failure risk for the restored teeth. among the causes of most important mechanical failures are marginal defects in composite resins restorations. it is important for the assessment of defective restorations to understand how the failure occurs11. despite the development of new materials that compensate phenomena such as the c factor in operative cavities, 171147 it is important to understand what would be the main risk indicators to improve proper decisions12. the present work aimed to identify the indicators for the prognosis of mechanical risks for unsatisfactory resin restorations in a follow-up. the tested hypothesis was that it is possible to identify mechanical risk indicators of failed defective amalgam and composite resin restorations. material and methods this study was approved by the ethics committee of the research office of the dental school of university of chile ascribed to pri-odo 12-005. all patients signed informed consent forms and completed a registration form. thirty-nine adult patients underwent a clinical examination (direct examination), along with photographic, radiographic and model examinations (indirect tests) as proposed by the literature. the clinical examinations were performed using the ryge/usphs modified criteria13-14 by a previously calibrated operator (cohen’s kappa 85% concordance), in which all class i and ii composite resin and amalgam restorations were classified either as “satisfactory” (alpha) or “not satisfactory” (bravo and charlie). the photographic study consisted of digital photographs (nikon d100, tokyo, japan) of both arches and the individual restored teeth in all patients. the radiological examination consisted of standardized bilateral bitewing radiographs in all patients. the analysis of models consisted of standardized impressions of the arches, obtained at the time of surgery with polyether impression material impregum penta soft (3m espe, st paul, mn, usa), which were later poured with extrahard plaster (sintec ltda, santiago, chile). all patients of the operative dental clinic of university of chile of a period (1 year) (n=156) were randomly selected and enrolled in a representative sample of 39 patients. the total number of failed restorations (n=256) was divided into two groups, bravo and charlie, according to the value obtained in the “marginal adaptation” evaluation by the modified ryge/usphs criteria. the following indicators of the prognosis of mechanical risk were evaluated for each restoration in a standardized fashion using previously obtained x-rays and models, as described in the literature1,15. indicators of the prognosis of mechanical risk 1. class of restoration: occlusal (o), proximal (p), mesioocclusal-distal (mod) restorations. 2.type of restorative material: amalgam or resin composite. 3. design of the cavity preparation: the size of the restoration as a class was measured on the radiographs and models using a millimeter ruler (staedtler, nüremberg, germany) with 0.5-mm increments. in the models, the mesiodistal dimension, the buccolingual dimension, the mesial isthmus and the distal width were measured. in the radiographs were measured the mesiodistal dimension and the depths of the mesial, occlusal, and distal (mod) cavities. all of the collected data were entered into an excel spreadsheet to be statistically analyzed. the confidence level was set at p=0.05. the sspsv21.0 statistical program (ibm, new york, ny, usa) was used. statistical analysis the total number of “unsatisfactory” restorations was divided into two groups, bravo and charlie, which constituted independent variables, each with different n values. at an early stage, they were compared generally with student’s t-test for the indicators. then, one-way anova was used to compare the indicators of design in both groups by the type of restoration (amalgam and composite resin), without considering the class. later, the classes were compared among themselves according to group and class with a scheffé test. results i. general comparison of the bravo and charlie groups: the statistically significant indicators were: the mesiodistal dimension in both model restorations (p=0.037) and radiographs (p=0.014), the distal isthmus (p<0.05), the average of the isthmuses (p<0.05), the distal cavity depth (p<0.05), and the average of the depths of the mod cavities (p<0.05). the data are presented in table 1. ii. comparison of each class (occlusal, proximal, mod) by group and type (amalgam and resin) the distribution of the sample is presented in table 2. using one-way anova, the amalgam and resin restorations were compared by groups (bravo and charlie), without comparing their types. the results of this analysis are presented in tables 3 and 4. table 3 presents that there were significantly different indicators between the composite resin and the amalgam restorations in the bravo group. table 4 presents that there were indicators that had statistically significant differences between the composite resin and the amalgam restorations in the charlie group. the results of the comparison of classes (occlusal, proximal and mod) by group and type of restoration are presented in tables 5 and 6. table 5 shows that there were significant differences within the amalgam restorations of the bravo group (anova and scheffé test, p<0.05) for the following indicators: mesiodistal dimension (p<0.05), the average of the isthmuses (p<0.05) and the average of the depths of the mod cavities (p<0.05). these three indicators were significant in the comparison of types: mod, proximal occlusal vs. mod, and proximal occlusal. as shown in the table, the p values were not significant statistically for the labio-linguo-palatal dimension among the three classes. this demonstrates that, within the resin composite restorations of the bravo group, indicators of the risk mechanics for class-i and class-ii amalgam and composite resin restorations braz j oral sci. 13(2):146-151 148 indicator mesiodistal dimension buccolingual/palatal dimension mesial isthmus distal isthmus average of isthmuses mesiodistal dimension (bitewing radiograph) occlusal cavity depth (bitewing radiograph) mesial cavity (bitewing radiograph) distal cavity depth (bitewing radiograph) average depth of mod cavities (bitewing radiograph) group bravo charlie bravo charlie bravo charlie bravo charlie bravo charlie bravo charlie bravo charlie bravo charlie bravo charlie bravo charlie n 178 78 178 78 178 78 178 78 178 78 178 78 178 78 178 78 178 78 178 78 average 6.355 7.018 4.008 4.369 0.992 1.305 0.671 1.541 0.831 1.463 6.537 7.335 3.201 3.358 1.140 1.346 0.986 2.333 0.831 1.463 d s 2.337 2.308 1.652 1.845 1.689 1.862 1.464 1.903 1.219 1.230 2.502 2.295 1.016 1.140 2.095 2.129 2.035 2.461 1.219 1.230 means difference 0.663 0.361 0.393 0.870 0.631 0.798 0.157 0.206 1.347 0.631 p value 0.037 0.121 0.098 0.000 0.000 0.014 0.275 0.476 0.000 0.000 table 1.table 1.table 1.table 1.table 1. comparative values (in mm) for group indicators regardless of type and class of the restoration. restoration occlusal proximal mod total n (%) n (%) n (%) n (%) amalgam 101 (48) 86 (41) 23 (11) 210 (100) resin 31 (67) 13 (28) 2 (5) 46 (100) table 2.table 2.table 2.table 2.table 2. distribution of the sample according to type and class of the restoration. indicator p-values amalgam (n=141) p-values composite (n=37) mesiodistal dimension 0.000 0.098 buccolingual/palatal dimension 0.442 0.388 average isthmuses 0.000 0.000 average depth of mod cavities 0.000 0.000 table 3.table 3.table 3.table 3.table 3. comparison of the results of amalgam and composite restorations in bravo group, according to the indicators. indicator p-values amalgam (n=69) p-values composite (n=9) mesiodistal dimension 0.021 0.160 buccolingual/palatal dimension 0.006 0.510 average isthmuses 0.000 0.057 average depth of mod cavities 0.000 0.013 table 4.table 4.table 4.table 4.table 4. comparison of the results of amalgam and composite restorations in charlie group, according to the indicators. there was a significant difference in the average of the isthmuses and the depth averages of the mod cavities. the first indicator was significant for the comparison of types: mod, proximal occlusal vs. mod, and proximal occlusal. the second indicator was significant for the comparison of types: occlusal vs. mod and proximal occlusal, as shown in the table. table 6 shows that the four indicators were significant in the comparison of the different types of amalgam restorations in the charlie group. the mesiodistal dimension was significantly different between the proximal and mod restorations. the bucco-linguo-palatal dimension is significant in the comparison of proximal vs. occlusal classes. the average of the isthmuses and the average of the depths of the mod cavities are significant when comparing mod, proximal occlusal vs. mod, and proximal-occlusal restorations, as shown in the table. it shows that the significant indicators relative to different types of resin composite indicators of the risk mechanics for class-i and class-ii amalgam and composite resin restorations braz j oral sci. 13(2):146-151 171149 indicator type of restoration mod amalgam occlusal amalgam mod composite occlusal composite n=141 n=141 n=37 n=37 p p p p mesiodistal dimension mod occlusal 0.000 0.523 proximal 0.000 0.035 0.142 buccolingual/palatal dimension mod occlusal 0.483 0.471 proximal 0.474 0.991 0.772 0.741 average isthmuses mod occlusal 0.000 0.000 proximal 0.000 0.000 0.000 0.000 average radiographic depth mod of mod cavities occlusal 0.000 0.000 proximal 0.000 0.000 0.883 0.000 table 5.table 5.table 5.table 5.table 5.values of p with scheffé test. comparison of classes among themselves, according to type of restoration (mod, proximal and occlusal) and indicators (mesiodistal dimension, buccolingual/palatal dimension, average isthmuses and average depth of mod cavities) in bitewing radiographs for bravo group amalgam and composite restorations. indicator type of restoration mod amalgam occlusal amalgam occlusal composite n=69 n=69 n=9 p p p mesiodistal dimension mod occlusal 0.427 proximal 0.034 0.271 0.160 buccolingual/palatal dimension mod occlusal 0.105 proximal 0.997 0.007 0.510 average isthmuses mod occlusal 0.000 proximal 0.000 0.000 0.057 average depth of mod cavities mod occlusal 0.000 proximal 0.000 0.000 0.013 table 6.table 6.table 6.table 6.table 6. values of p with scheffé test. comparison of classes among themselves, according to type of restoration (mod, proximal and occlusal) and indicators (mesiodistal dimension, buccolingual/palatal dimension, average isthmuses and average depth of mod cavities) in bitewing radiographs for charlie group amalgam and composite restorations. restorations (proximal vs. occlusal) in the charlie group were: the average of the isthmuses and the average of the depths of the mod cavities. notice that the comparison of the mod restorations could not be performed between the groups with low n values (n=2). discussion the pre-determined indicators of the mechanical risk prognosis were chosen from a number of other indicators that are vaguely described in the literature, as these were objectively analyzed within a pool of restorations previously classified as “unsatisfactory” or “failed,” meaning that within a medium (bravo) or short (charlie) period of time, these restorations required repair or replacement16-21. in addition, this study design corresponds to descriptions at a specific time in the long-term life of the restorations (cross-sectional) and not to a follow-up assessment from the origin of the restoration22. in the general comparison of the bravo and charlie groups, without considering type and class, the following factors were found to be influential indicators: the mesiodistal dimension, the distal isthmus, the average of the isthmuses, the depth of the distal cavity, and the average depths of the mod cavities. this finding is consistent with the literature, which states that greater restorations showed increased risk of mechanical failure due to the loss of tissue resistance23-24. it was also demonstrated that the behavior of both groups was similar, with no differences between the bravo and charlie restorations. this means that the magnitude of the failure determinant of restoration initial or late damage does not matter because they exhibited similar behavior in terms of prognosis failure by mechanical risk 1. therefore, a restoration that presents small mechanical failure, such as indicators of the risk mechanics for class-i and class-ii amalgam and composite resin restorations braz j oral sci. 13(2):146-151 150 bravo restorations, or extensive failure, such as charlie restorations, should be replaced or repaired promptly. any mechanical failure, regardless of its magnitude, is a determining factor to carry out the repair or replacement of restoration. with respect to the cavity dimensions, the effects of small restoration on the distribution of stress in a tooth are not significant. however, when the width of the cavity increases, the stress in the distal third of the cervical portion increases rapidly, which is harmful to tooth structure and can easily cause the fracture of the cavity wall. the influence of the size indicators of the distal isthmus, the average of the isthmuses, the distal cavity depth and the average of the depth of the mod cavities and their relationship with the prognosis of a restoration could be due to the increase in the degree of difficulty for the operator to properly perform a posterior class ii restoration in a distal tooth compared to the one located in a mesial tooth, likely involving variable access, visibility or technique. table 1 indicates that a restoration with isthmuses greater than 2 mm and distal cavities greater than 4.5 mm increased the likelihood of mechanical failure compared to a restoration with smaller dimensions. the results also suggest that increasing the depth of one cavity in a mod restoration is sufficient to influence the prognosis of the complete restoration. for the amalgam restorations of the bravo group, the significant indicators were the mesiodistal dimension, the average of the isthmuses, and the mod cavities depth. in contrast, for the composite resins from the same group, the average of the isthmuses and the depths of the mod cavities were significant. in the charlie group, the four relevant indicators were influential in amalgams, but for resins only the average of the mod cavities depth was influential. this discrepancy could be due to the heterogeneity of the sample and, therefore, to the low number of posterior composite resin restorations25-26. a comparison of the types of restorations (occlusal, proximal and mod) demonstrated that the only indicator that was significant for all classes, regardless of group and type, was the average of the mod cavities depth, whose value for 256 restorations was 1.949 mm with a sd of 1.181 mm. this finding agrees with conclusions described in the literature: changes in the cavity depth also have large effects on the stress distribution and the loads required to fracture the tooth (failure of a restoration)27-28. after the deep restoration of a decayed tooth, the maximum stress increases about five times and focuses on the cavity angles. the values of the loads needed to fracture the tooth decrease nearly one-fifth in relation to non-decayed teeth. in addition, deep cavity preparations have been linked to the high cuspal deflection, which predisposes the tooth to fractures10. the evaluation of indicators in relation to the prognosis of restorations suggests that for deep mod carious lesions, a pedagogical protocol must be established, different from the occlusal approach; an alternative might be indirect restoration6,15,25. furthermore, class ii mod restorations presented significant indicators more frequently than occlusal and proximal restorations. the indicators were as follows: the average of the isthmuses and the average of the mod cavities depth for both amalgam and composite resins in both groups. this finding is consistent with the literature, which states that the larger the quantity of restored surfaces and/or amplitude of the isthmus, the higher the possibility of cusp fracture1,25. based on the obtained results, it may be concluded, in contrast to the literature, that the size of the restoration, generally described as “bandwidth,” introduces specific indicators in performing a critical evaluation of a restoration. these indicators are the mesiodistal dimension, the size of the distal isthmus, the average of the isthmuses, and the average depth of mod cavities, which must be evaluated in both models and bitewing radiographs. the depth of the mod cavity should be assessed with a radiograph and complemented by other indicators obtained from the model (mesiodistal dimension, the size of the distal isthmus and the average of the isthmuses). the dimensions of these indicators influence the prognosis of a restoration. these indicators should be considered a tool to help decide whether to perform replacement therapy or to repair the affected tooth, with the same material or a combination29-30. in conclusion, the type of restoration (proximal and mod) is an indicator for the prognosis of mechanical risk for the sample. the restorative material (amalgam or composite resin) for the sample type is not an indicator of the prognosis for mechanical risk. in relation to the design of the cavity preparation, the indicators of the prognosis for mechanical risk assessing a restoration are the mesiodistal dimension, the distal isthmus, the average of the isthmuses, the distal cavity depth, and the average of the mod cavities depths. the most significant indicator of the risk of mechanical failure is the average of the mod cavities depth for composite resins. for amalgam, the most significant predictors of the risk of mechanical failure are the mesiodistal dimension, the distal isthmus, the average of the isthmuses, the depth of the distal cavity, and the average of the mod cavities depth. the depth of the mod cavity is a significant indicator of the risk of failure for both groups (bravo and charlie) and both types (amalgam and composite resin) of restorations. therefore, indirect restorations techniques should be considered in treating clinical situations that present this indicator. a single clinical assessment does not provide sufficient information to establish the indicators for the risk of mechanical failure of restorations. references 1. dennison jb ,sarrett dc. prediction and diagnosis of clinical outcomes affecting restoration margins. j oral rehabil. 2012; 39: 301-18. 2. blum ir, jagger dc ,wilson nh. defective dental restorations: to repair or not to repair? part 1: direct composite restorations. dent update. 2011; 38: 78-84. 3. burke fj, wilson nh, cheung sw , mjör ia. influence of patient factors on age of restorations at failure and reasons for their placement and replacement. j dent. 2001; 29: 317-24. 4. chrysanthakopoulos na. placement, replacement and longevity of composite resin-based restorations in permanent teeth in greece. int dent j. 2012; 62: 161-6. indicators of the risk mechanics for class-i and class-ii amalgam and composite resin restorations braz j oral sci. 13(2):146-151 171151 5. demarco ff, correa mb, cenci ms, moraes rr , opdam nj. longevity of posterior composite restorations: not only a matter of materials. dent mater. 2012; 28: 87-101. 6. forss h , widström e. reasons for restorative therapy and the longevity of restorations in adults. acta odontol scand. 2004; 62: 82-6. 7. goldstein gr. the longevity of direct and indirect posterior restorations is uncertain and may be affected by a number of dentist-, patient-, and materialrelated factors. j evid based dent pract. 2010; 10: 30-1. 8. heintze sd, rousson v. clinical effectiveness of direct class ii restorations a meta-analysis. j adhes dent. 2012; 14: 407-31. 9. mjör ia, dahl je, moorhead je. age of restorations at replacement in permanent teeth in general dental practice. acta odontol scand. 2000; 58: 97-101. 10. campos ea, andrade mf, porto-neto st, campos la, saad jr, deliberador tm, et al. cuspal movement related to different bonding techniques using etch-and-rinse and self-etch adhesive systems. eur j dent. 2009; 3: 213-8. 11. palotie u ,vehkalahti mm. reasons for replacement of restorations: dentists’ perceptions. acta odontol scand. 2012; 70: 485-90. 12. ishikiriama sk, valeretto tm, franco eb, mondelli rf. the influence of “c-factor” and light activation technique on polymerization contraction forces of resin composite. j appl oral sci. 2012; 20: 603-6. 13. ryge g, snyder m. evaluating the clinical quality of restorations. j am dent assoc. 1973; 87: 369-77. 14. mjör ia ,ryge g. comparison of techniques for the evaluation of marginal adaptation of amalgam restorations. int dent j. 1981; 31: 1-5. 15. hickel r, peschke a, tyas m, mjor i, bayne s, peters m, et al. fdi world dental federation clinical criteria for the evaluation of direct and indirect restorations. update and clinical examples. j adhes dent. 2010; 12: 259-72. 16. blum ir, lynch cd, schriever a, heidemann d, wilson nh. repair versus replacement of defective composite restorations in dental schools in germany. eur j prosthodont restor dent. 2011; 19: 56-61. 17. fernández em, martin ja, angel pa, mjör ia, gordan vv, moncada ga. survival rate of sealed, refurbished and repaired defective restorations: 4-year follow-up. braz dent j. 2011; 22: 134-9. 18. gordan vv, riley jl, 3rd, blaser pk, mondragon e, garvan cw, mjor ia. alternative treatments to replacement of defective amalgam restorations: results of a seven-year clinical study. j am dent assoc. 2011; 142: 842-9. 19. moncada g, martin j, fernández e, hempel mc, mjör ia, gordan vv. sealing, refurbishment and repair of class i and class ii defective restorations: a three-year clinical trial. j am dent assoc. 2009; 140: 425-32. 20. moncada g, fernández e, martín j, arancibia c, mjör ia ,gordan vv. increasing the longevity of restorations by minimal intervention: a twoyear clinical trial. oper dent. 2008; 33: 258-64. 21. moncada gc, martin j, fernandez e, vildosola pg, caamano c, caro mj, et al. alternative treatments for resin-based composite and amalgam restorations with marginal defects: a 12-month clinical trial. gen dent. 2006; 54: 314-8. 22. knibbs pj. methods of clinical evaluation of dental restorative materials. j oral rehabil. 1997; 24: 109-23. 23. mjör ia, moorhead je, dahl je. reasons for replacement of restorations in permanent teeth in general dental practice. int dent j. 2000; 50: 361-6. 24. asmussen e ,peutzfeldt a. class i and class ii restorations of resin composite: an fe analysis of the influence of modulus of elasticity on stresses generated by occlusal loading. dent mater. 2008; 24: 600-5. 25. deliperi s. functional and aesthetic guidelines for stress-reduced direct posterior composite restorations. oper dent. 2012; 37: 425-31. 26. forss h ,widström e. from amalgam to composite: selection of restorative materials and restoration longevity in finland. acta odontol scand. 2001; 59: 57-62. 27. mjör ia, um cm. survey of amalgam and composite restorations in korea. int dent j. 1993; 43: 311-6. 28. mjör ia, gordan vv. failure, repair, refurbishing and longevity of restorations. oper dent. 2002; 27: 528-34. 29. tolidis k, boutsiouki c, gerasimou p. microleakage in combined amalgam/ composite resin restorations in mod cavities. braz j oral sci. 2013; 12: 100-4. 30. hickel r, brushaver k ,ilie n. repair of restorations—criteria for decision making and clinical recommendations. dent mater. 2013; 29: 28-50. indicators of the risk mechanics for class-i and class-ii amalgam and composite resin restorations braz j oral sci. 13(2):146-151 oral sciences n3 original article braz j oral sci. april | june 2015 volume 14, number 2 dental bleaching agents with calcium and their effects on enamel microhardness and morphology andréia mara andrade pizani1, beatriz tholt2, sidnei paciornik3, katia regina hostilio cervantes dias4, pedro paulo albuquerque cavacanti de albuquerque5, celso silva queiroz2 1universidade paulista – unip, school of dentistry, department of restorative dentistry, são paulo, sp, brazil 2universidade veiga de almeida – uva, school of dentistry, department of restorative dentistry, rio de janeiro, rj, brazil 3pontifícia universidade católica do rio de janeiro puc-rio, school of materials engineering, department of materials engineering dema, rio de janeiro, rj, brazil 4universidade federal do rio de janeiro – ufrj, school of dentistry, department of restorative dentistry, rio de janeiro, rj, brazil 5universidade de são paulo – usp, school of dentistry, departament of biomaterials and oral biology, são paulo, sp, brazil correspondence to: celso silva queiroz universidade veiga de almeida uva rua ibituruna, 108, tijuca, cep: 20271-021 rio de janeiro, rj phone: +55 21 25748871 e-mail: celsoq@yahoo.com received for publication: december 18, 2014 accepted: june 20, 2015 abstract aim: to evaluate enamel microhardness and morphology after bleaching with hydrogen peroxide containing calcium in different concentrations. methods: one hundred specimens of human teeth were ground and polished and had the initial microhardness evaluated. the specimens were randomly assigned into five groups (n=20): group 1 control group (no treatment); group 2 home peroxide 6% (without calcium); group 3 home peroxide 7.5% (without calcium); group 4 white class 6% (with calcium); group 5 white class 7.5% (with calcium). for each group, the bleaching was performed according to the manufacturer’s specifications. the specimens were bleached once a day for 5 days and subjected to ph cycling. microhardness and scanning electron microscopy (sem) analysis were performed before and after bleaching. results: the results showed that groups submitted to bleaching treatment presented hardness loss compared to the control group. the group of 7.5% hydrogen peroxide with calcium showed a lower percentage of hardness loss in relation to other groups. conclusion: calcium in association with a higher hydrogen peroxide concentration may decrease microhardness changes on enamel. keywords: bleaching agents; calcium; hydrogen peroxide; dental enamel. introduction several techniques have been used to promote teeth whitening, including home bleaching technique and the one performed at the office. there are commercially available gels in different chemical formulations and concentrations, including carbamide peroxide (cp) from 10% to 37% and hydrogen peroxide (hp) from 6% to 38%. dental enamel is composed of a huge number of highly mineralized prisms, and its hardness is due to a high percentage of inorganic matrix (95%) made of hydroxyapatite crystals (calcium phosphate) and low percentage of organic protein nature matrix (0.36 to 2%) together with polysaccharides1. the enamel permeability is low, but it acts as a semipermeable membrane allowing the diffusion of water and some ions. teeth whitening usually generates concern about the possibility of enamel braz j oral sci. 14(2)154-158 http://dx.doi.org/10.1590/1677-3225v14n2a11 155155155155155 groups 1 control* 2 home peroxide 6% dmc® 3 home peroxide 7.5% dmc® 4 white class 6% fgm® 5 white class 7.5% fgm® calcium n o n o yes yes composition hydrogen peroxide, sodium fluoride, potassium nitrate, water, carbomer, amine, preservative,flavorizer. hydrogen peroxide, sodium fluoride, potassium nitrate, water, carbomer, amina, preservative,flavorizer. hydrogen peroxide, sodium fluoride, potassium nitrate, water , aloe vera, calcium. hydrogen peroxide, sodium fluoride, potassium nitrate, water , aloe vera, calcium. * control group no bleaching treatment and subjected to ph cycling. the manufacturers do not provide all components of the formulations. table 1.table 1.table 1.table 1.table 1. basic composition and manufacturer of each bleaching agent. chemical changes, especially when peroxides with great diffusivity are applied in high concentrations 2-3. some bleaching gels are presented in low ph solutions to ensure the stability of hydrogen peroxide, so these bleaching solutions with acidic ph, may promote microstructure and chemical changes in the enamel 3. changes in chemical composition of enamel may reflect on modified values of mechanical properties, resulting in undesirable effects such as reduction in hardness and/or flexural strength4. on the other hand, different authors stated that bleaching is effective and unable to cause deleterious effects on the enamel surface57. thus, regardless of the aesthetic results achieved by different whitening techniques, the effects of bleaching agents on enamel are yet not completely understood. assuming that enamel is mainly composed by calcium, phosphorus, oxygen and carbon ca10(po4)6(oh)2, in vitro assays designed to identify these components are desirable to reveal possible alterations of enamel’s chemical structure. some studies showed that dental bleaching alters the enamel chemically and have shown decreased calcium values in enamel5-6,8. in order to minimize such demineralization after contact with bleaching agents, some biomaterials such as fluoride, hydroxyapatite, calcium ions and amorphous calcium have been added to the composition of those whitening agents9. beneficial effects of combining these substances with bleaching agents have been verified in previous studies3,9-10, which concluded that such association may protect the enamel from demineralization and also may avoid hypersensitivity during the procedure. however, fluoride and calcium addition to the bleaching agents were not sufficient to increase the microhardness of enamel11. thus, the aim of this study was to evaluate the microhardness of enamel after bleaching with hydrogen peroxide 6% and 7.5%, with or without calcium. additionally, scanning electron microscopy (sem) was employed to analyze the structure of the enamel surface before and after application of peroxides. material and methods one hundred human permanent molars, obtained from the local tooth bank, were used after approval by the ethics committee in research (protocol number 705/09). the crowns were separated from roots and an enamel block (3x3x2 mm) was removed from the buccal surface of the crowns. the specimens (enamel blocks) were ground wet in a mechanical grinding machine with aluminum oxide discs of sequentially decreasing granulation and polished with diamond pastes and felt discs under mineral oil cooling. the specimens were identified and remained stored in humidified atmosphere at 37 °c during the experiment. surface microhardness was assessed before the bleaching and ph cycling procedures. the measurements were performed in 3,500 blocks using a microhardness tester (future tech fm 300; shimadzu, tokyo, japan) with a knoop indenter (25 g/ 5 s). three lines with five indentations each were made on the enamel surface at a distance of 100 µm, resulting in fifteen indentations. after obtaining the initial microhardness, one hundred blocks were selected (153 ± 10 khn – baseline) and randomly divided into five groups (n=20) in according to the bleaching treatment. the control group was subjected to ph cycling, but not to bleaching (table 1). the basic composition of bleaching agents is shown in table 1. the specimens were submitted to ph cycling for 5 days at 37 °c, in order to simulate the effects of demineralization and re-mineralization that occur in the oral cavity. the specimens were immersed in a demineralizing solution (2.0 mm ca and p in 0.075 m acetate buffer, ph 4.3 6.25 ml/ mm2) for 6 h and immersed in remineralizing solution (1.5 mm and 0.9 mm p in 0.1 m tris buffer, ph 7.0) for 17 h12. the specimens had the bleaching agent applied for 1 h, between the demineralizing and remineralizing solutions, once daily for five days. bleaching technique was carried out according to the manufacturer’s specifications after prophylaxis with pumice stone and water. a calibrated syringe was used to place 0.02 ml of each bleaching agent on the specimen. the bleaching agents were removed from the dental specimens with distilled deionized water, causing no changes on the enamel surface. after ph cycling, surface microhardness was again assessed on each specimen as previously mentioned. five specimens from each group were randomly chosen and analyzed by sem (ssx – 550, shimadzu) and just one unbleached specimen served as control group. the specimens were dried, fixed on aluminum stubs and sputter-coated with gold. the surface morphology of enamel was qualitatively examined at 5000 x magnification. statistical analysis was carried out by anova in a split plot randomized design, considering the bleaching product (gel) factor. the tukey’s test was used for comparison with a 5% dental bleaching agents with calcium and their effects on enamel microhardness and morphology braz j oral sci. 14(2)154-158 156156156156156 groups 1-control 2-home peroxide 6.0% (without calcium) 3home peroxide 7.5% (without calcium) 4-white class 6.0% (with calcium) 5white class 7.5% (with calcium) initial 337.45 ± 17.37 281.43 ± 21.57 309.89 ± 13.70 307.96 ± 25.79 272.31 ± 18.20 final* 221.90 ± 16.34a 87.54 ± 3.07b 85.09 ± 2.78b 95.97 ± 3.63b 110.95 ± 7.43c %sml -34.72 -59.61 -71.23 -64.05 -53.43 table 2.table 2.table 2.table 2.table 2. initial and final microhardness means and standard deviations and percentage loss of surface microhardness (%sml) in experimental groups * mean values with different letters differ statistically from each other (p<0.05). confidence level (α=0.05). the percentage of hardness reduction (mean) was assessed by the microhardness values. the statistical program was sas 7.0 (sas institute, cary, nc, usa). results groups treated with bleaching gels showed a statistically significant hardness loss compared to the control group (table 2). comparison of groups treated with home peroxide without calcium at 6.0% and 7.5%, showed no statistically significant differences (table 2). groups treated with home peroxide with calcium at 6.0 and 7.5%, showed different values (p<0.05). however, only the 7.5% group with calcium was statistically different (p<0.05) from the others (table 2). the control group lost 35% of the enamel hardness; the groups without calcium at 6.0% and 7.5% lost 60% and 71% respectively. groups with calcium at 6.0% and 7.5% lost 64% and 53% respectively (table 2). the sem images revealed changes in the enamel structure as result of bleaching with home peroxide and white class with or without calcium (figures 1 – 5). discussion bleaching agents are primarily made of hydrogen peroxide and are classified by the u.s. food and drug administration – fda as class i agents, i.e., products generally recognized as safe and effective. however, their fig. 1. control group no bleaching treatment. fig. 2. home peroxide 6.0% without calcium. fig. 3. home peroxide 7.5% without calcium. indiscriminate use, especially at higher concentrations, led the scientific council of the american dental association to establish criteria for bleaching agents’ approval, since it should have neutral ph, its formula must be specified because of each ingredient function, the action mechanism of active agents and user’s instructions. oxidation-reduction reaction of bleaching agents produce hydrogen ions (h +) that can create an acidic environment and lead to dissolution of the organic and inorganic enamel13-14. the action of acid on the enamel dental bleaching agents with calcium and their effects on enamel microhardness and morphology braz j oral sci. 14(2)154-158 157157157157157 fig. 4. white class 6.0% with calcium. fig. 5. white class 7.5% with calcium. structure may result in erosion that reduces its translucency, promoting opacity, which is a common effect in bleaching treatments5. however, whether this erosion is caused directly by the bleaching agent’s ph, is still controversial. enamel re-mineralization is expected due to the action of saliva, especially in the case of agents used in this study, with concentrations advised for home technique, which usually lasts between 14 and 30 days. in this technique, the tooth enamel in contact with the whitening gel is subject to the action of the demineralization and re-mineralization processes in the oral environment, factors that can greatly affect the results of erosion on dental enamel3. the ph changes that occur in the mouth may interfere with the action of bleaching agents, thus the ph cycling was performed in this study in an attempt to simulate the clinical condition15. several studies have shown enamel microhardness loss after the use of bleaching agents16. thus, a microhardness test is often used for in vitro studies to evaluate possible deleterious effects of tooth whitening17.the results of this study showed that treatment with hydrogen peroxide gels may decrease the microhardness values, which was observed in previous findings17-20. the control group showed a decrease of 34.7% between the initial and final microhardness values. this change was also demonstrated in another study 20. although the demineralizing (de) and remineralizing (re) solutions were buffered to regular ph, it is believed that washing with distilled water and the six hours’ time of the de solution, exceeds the mineral replacement action of the mineral re solution. in order to minimize the enamel mineral loss during tooth whitening, introduction of calcium in its composition was proposed21-22. according to our results, 7.5% hydrogen peroxide-based whitening gel with calcium showed the smallest microhardness decrease (p<0.05), while the whitening gel at 6,0% with calcium showed the same behavior as the one without calcium. the concentration of hydrogen peroxide (7.5%) may lead to greater enamel demineralization and promote ionic imbalance, so the calcium contained in the composition of the bleaching agent may be more effective incorporating into the enamel surface. some studies pointed out that different bleaching agents have reduced the enamel microhardness values, although these values were recovered after treatment by contact with saliva19. an ideal system of remineralization should provide not only calcium ions, but also phosphorus and fluoride ions, since they are also lost by enamel11. the amorphous calcium phosphate (acp) is a remineralizing substance and when stabilized by casein phosphopeptide (cpp), its remineralizing potential is higher (calcium, phosphorus and fluoride ions) compared with the acp and sodium fluoride23. the same authors also related the positive effects of acp incorporation as more important to low concentrations of hydrogen peroxide in combination with saliva. for this reason, the authors believe that incorporating one or more biomaterials such as calcium, hydroxyapatite or fluoride in the bleaching agents composition is quite promising. the present study showed that a higher concentration of hydrogen peroxide (7.5%) with calcium was more effective; however, comparison must be viewed with caution, because the authors used amorphous calcium in saliva and not calcium ions in artificial saliva. the sem analysis revealed structural changes on the enamel surface after bleaching agent application. the images corresponding to the experimental groups show erosions and discontinuities on enamel surface compared to the control group. others authors identified craters, depression areas, erosion, aprismatic layer removal and exposure of the enamel rods24. this may be explained by the radicals stemming from oxidizing agents, i.e., diffusion of them at about 100 ìm on the enamel surface23. thus, according to our results it may be suggested that the enamel’s external surface is more susceptible to bleaching. according to the results and inherent limitations of this research, further studies should be carried out to minimize the change in enamel mechanical and structural properties when subjected to bleaching agents, either by incorporating remineralizing components, or using them in association with the whitening techniques. all bleaching gels cause loss of hardness on human dental enamel. however, the inhibition dental bleaching agents with calcium and their effects on enamel microhardness and morphology braz j oral sci. 14(2)154-158 158158158158158 of enamel demineralization may be related not only to the presence of calcium, but to the concentration of bleaching gel. acknowledgements the authors wish to thank the postgraduate laboratory of paulista university (unip) and the laboratory technician mr. felipe barros for his assistance. thanks also to mr. vitor brandão and professor marcos henrique pinho maurício (dema-puc) for helping with the laboratory procedures and sharing their expertise. references 1. özcan m, dündar m, çömlekoðlu e. adhesion concepts in dentistry: tooth and material aspects. j adhes sci technol. 2012; 26: 2661-81. 2. cakir fy, korkmaz y, oztas efss, gurgan s. chemical analysis of enamel and dentin following the application of three different at-home bleaching systems. oper dent. 2011; 36: 529-36. 3. xu b, li q, wang y. effects of ph values of hydrogen peroxide bleaching agents on enamel surface properties. oper dent. 2011; 36: 554-62. 4. khoroushi m, mazaheri h, manoochehri a. effect of ccp-acp application on flexural strength of bleached enamel and dentin complex. oper dent. 2011; 36: 372-9. 5. 5.. soares dg, basso fg, pontes ecv, garcia lfr, hebling j, de souza costa ca. effective tooth-bleaching protocols capable of reducing h2o2 diffusion through enamel and dentine. j dent. 2014; 42: 351-8. 6. tamam e, aydin k, bilgiç s. electrochemical corrosion and surface analyses of a ni-cr alloy in bleaching agents. j prosthodont. 2014; 23: 549-58. 7. sasaki rt, arcanjo aj, flório fm, basting rt. micromorphology and microhardness of enamel after treatment with home-use bleaching agents containing 10% carbamide peroxide and 7.5% hydrogen peroxide. j appl oral sci. 2009; 17: 611-6. 8. pedreira de freitas ac, botta sb, teixeira f de s, salvadori mc, garone-netto n. effects of fluoride or nanohydroxiapatite on roughness and gloss of bleached teeth. microsc res tech. 2011; 74: 1069-75. 9. jin j, xu x, lai g, kunzelmann kh. efficacy of tooth whitening with different calcium phosphate-based formulations. eur j oral sci 2013; 121: 382-8. 10. de abreu dr, sasaki rt, amaral flb, flório fm, basting rt. effect of home-use and in-office bleaching agents containing hydrogen peroxide associated with amorphous calcium phosphate on enamel microhardness and surface roughness. j esthetrestor dent. 2011; 23: 158-68. 11. kim ys, kwon hk, kim bi. effect of nano-carbonate apatite to prevent restain after dental bleaching in vitro. j dent. 2011; 39: 636-42. 12. queiroz, cs, hara at, paes leme af, cury ja. ph-cycling models to evaluate the effect of low fluoride dentifrice on enamel deand remineralization. braz dent j. 2008; 19: 21-7. 13. oskoee pa, navimipour ej, oskoee ss, moosavi n. effect of 10% sodium ascorbate on bleached bovine enamel surface morphology and microhardness. open dent j. 2010 21: 207-10. 14. sulieman m, addy m, macdonald e, rees js. a safety study in vitro for the effects of an in-office bleaching system on the integrity of enamel and dentine. j dent. 2004; 32: 581-90. 15. mahmoud s, elembaby a, zaher a, grawish m, elsabaa h, el-negoly s, et al. effect of 16% carbamide peroxide bleaching gel on enamel and dentin surface micromorphology and roughness of uremic patients: an atomic force microscopic study. eur j dent. 2010; 4: 175-82. 16. featherstone jdb, ten cate jm, shariati m, arends j. comparison of artificial caries-like lesions by quantitative microradiography and microhardness profiles. caries res. 1983; 17: 385-91. 17. smidt a, weller d, roma i, gedalia i. effect of bleaching agents on microhardness and surface morphology of tooth enamel. am j dent. 1998; 11: 83-5. 18. attin t, schmidlin pr, wegehaupt f, wiegand a. influence of study design on the impact of bleaching agents on dental enamel microhardness: a review. dent mater. 2009; 25: 143-57. 19. mondelli rfl, gabriel trcg, rizzante fap, magalhães ac, bombonatti jfs, ishikiriama sk. do different bleaching protocols affect the enamel microhardness? eur j dent 2015; 9: 25-30. 20. tezel h, ertas os, ozata f, dalgar h, korkut zo. effect of bleaching agents on calcium loss from the enamel surface. quintessence int. 2007; 38: 339-47. 21. cavalli v, arrais ca, giannini m, ambrosano gm. high-concentrated carbamide peroxide bleaching agents effects on enamel surface. j oral rehabil. 2004; 31: 155-9. 22. cochrane nj, cai f, huq nl, burrow mf, reynolds ec. new approaches to enhanced remineralization of tooth enamel. j dent res. 2010; 89: 1187-97. 23. titley k, torneck cd, smith d. the effect of concentrated hydrogen peroxide solutions on the surface morphology of human tooth enamel. j endod. 1988; 14: 69-74. 24. ferreira s da s, araújo jl, morhy on, tapety cm, youssef mn, sobral ma. the effect of fluoride therapies on the morphology of bleached human dental enamel. microsc res tech. 2011; 74: 512-6. dental bleaching agents with calcium and their effects on enamel microhardness and morphology braz j oral sci. 14(2)154-158 oral sciences n3 original article braz j oral sci. october | december 2013 volume 12, number 4 cost analysis of materials used in class iii, iv and v composite resin restorations eduardo hebling1, rodolfo luís gonçalves2, dagmar de paula queluz1 1department of community dentistry, piracicaba dental school, university of campinas, piracicaba, sp, brazil 2dentist of the brazilian federal court, rio de janeiro, rj, brazil correspondence to: eduardo hebling faculdade de odontologia de piracicaba universidade estadual de campinas avenida limeira, 901, cep: 13414-903 areião, piracicaba, sp, brasil phone: +55 19 21065280 fax: +55 19 21065218 e-mail: hebling@fop.unicamp.br abstract aim: to assess the total cost of direct and indirect materials used in class iii, iv and v composite resin direct restorations. methods: the calculation of costs was based on the method of variable costing system. a list of the materials was obtained by a panel of experts and based on the excellence standards established in the literature for dental team treatment. the cost considered for each material was obtained from an average of the costs found in the regional supplier market (us$1.0=r$2.12). the repetitions were obtained from class iii, iv and v cavities in artificial premanufactured teeth. the cavities were classified as shallow, medium and deep. the materials were quantified for each type of preparation. seven brands of composite resins were used and weighed on a precision scale after their insertion in each cavity. the data were analyzed by descriptive statistics and non-parametric friedman’s test (α=0.05). results: the mean costs were us$7.96 (r$16.88) for class iii restoration, us$8.13 (r$17.24) for class iv, and us$7.84 (r$16.62) for class v. there was statistically significant difference in cost between the types of cavities and depth classification. the small cost difference among the different resin brands resulted in no statistically significant differences in the total cost of the restorations. conclusions: the costs obtained in this survey may be used in the calculation of the final cost of restorative procedures, helping in the management of public or private dental care services. keywords: dentistry, costs and cost analysis, dental materials, composite resins. introduction composite resins are commonly used materials in direct dental restorations. the similarity of the original color of the teeth, afforded by the use of composite resins, allows the maintenance and restoration of the aesthetics of the smile, increasing the preference for this restorative material in both anterior and posterior teeth by professionals and patients1. the materials used in dental procedures can be classified into direct and indirect materials2. drills, matrix strips, wedges and composite resin are examples of direct materials used in a restorative procedure. disinfecting solutions, gloves, cap and glasses are examples of used indirect materials. dental care services in both private and public sectors should be managed with the same principles applied to a commercial enterprise. although fixed costs can differ from one place to another, some direct variable costs related to the implementation of the service have their values established by some criteria that do not depend exclusively on the operator or manager of the service, such as the cost of dental materials. the price determined by the manufacturers and suppliers received for publication: august 02, 2013 accepted: november 25, 2013 braz j oral sci. 12(4):298-306 of dental products, the amount of material required by cavity size, and the type of these materials established by clinical and scientific evidence, are examples of these criteria3. a costing system consists in determining a criterion by which the costs are apportioned for the production. according the system employed, certain costs may or may not be part of the production costs4,5. in the variable costing system is appropriate to services only the variable costs of production, both direct and indirect5. the cost of materials is part of the calculation of the value of the fees for direct dental restorations. knowing the cost of the materials used can help planning of actions to manage the dental care services and determination of dental restoration fees3,5-7. despite the continuous need for less costly dental treatments8 and standardization of dental procedures based on scientific evidence9, there is remarkably scarce information on the costs of different restorative materials and their use10. the few studies available on this subject are mostly based on estimates of longevity of the restoration, in performance of retreatments in the medium and long term, and on their relative cost and benefits and effectiveness11. as up to date there are no publications that calculate the costs for direct and indirect materials used for direct dental restorations. thus, this study was designed to determine the total cost of direct and indirect materials used in class iii, iv and v cavities. an specific aim was to compare the mean cost of direct and indirect materials used in those types of cavity with different sizes. the null hypothesis was that there is no difference in mean cost values among these variables. material and methods this experimental study was developed at the piracicaba dental school, university of campinas, piracicaba, brazil in 2012. the calculation of costs was based on the method of variable costing system5. the list of materials was obtained through consultation by a panel of experts and based on the excellence standards established in the literature for dental team treatment, applying restorative techniques with efficiency and productivity2, and respecting the biosecurity of the patient and the dental team12,15, the use of materials16,17 and the ethical principles18. this panel of experts was formed by 10 dentists with over 20 years of experience (4 restorative dentistry clinical specialists, 2 general dentists, 3 restorative dentistry professors and 1 dental materials professor) and 2 dental hygiene technicians with 10 years of experience. a list of materials to be used was prepared by the authors. the referees reviewed each item according to the likert scale19 and attributed the following classifications: 1) strongly disagree; 2) disagree; 3) neither agree nor disagree; 4) agree; 5) strongly agree. the referees were also given the possibility of including a suggestion. the materials and quantities classified with scores 4 and 5 by the referees were maintained in the final list of materials. the costs of the materials were obtained from an average of the cost found in the supplier market of the administrative region of campinas-sp, brazil, in three different resellers. the obtained costs in local currency (real) were converted to american dollars (us$1.00 = r$2.12). this cost was adjusted according to the amount of material to be used. for the non-disposable materials, the cost was adjusted considering their mean use life. the amount of material was stipulated by simulating a clinical restorative procedure. this quantity was measured with the use of a graduated measuring cylinder, for liquid materials, and precision digital scale (model ab-s; mettler toledo®,barueri, sp, brazil,) with reading range of 0.01 mg to 0.1 mg, weighting capacity from 51 g to 320 g, for solid materials. the repetitions were obtained from classes iii, iv, and v cavities preparations in artificial permanent pre-manufactured teeth. each type of cavity was classified in relation to the preestablished depth as shallow, medium and deep, totalizing 9 anterior artificial teeth. the depth classification was established by the authors in the artificial teeth, which are supplied by the manufacturer with cavities prepared according to the currently recommended conservative techniques16,17. the list of materials was set up considering the dental care performed by the dental team (operator and auxiliary), following the biosecurity principles and the currently recommended conservative restorative techniques based on scientific evidence 15-17. the operative and restorative procedures were standardized as follows: specific diamond drills20 were sterilized before use and changed after being used in 10 patients21,22; no liner was used; absolute isolation was provided; use of condensation composite resin; an incremental placement technique was employed for the restorative materials; occlusal adjustment, finishing and polishing were performed with carbon paper and specific tips and sandpaper discs and strips16,17. the materials were classified into 7 different groups and for each one were established the following standardization criteria23: group 1: group 1: group 1: group 1: group 1: materials used for the maintenance and cleaning of equipment. • dental operating room equipment containing: 1) two ultra-speed air handpieces, a set of slow-speed air handpiece micro-motor with contra angle and three-way syringe; 2) an auxiliary unit containing one spittoon, vacuum system with saliva and blood suction hoses, one three-way syringe and one light-curing device; 3) a chair with the seat and back without seams or buttons, drive command by feet and arm support on both sides; 4) a monofocal spotlight with single cable; 5) stools, one for the operator and one for the auxiliary; 6) mobile auxiliary table, size 60 x 50 cm; • lubrication of high and slow-speed handpieces: two sprays of lubricating oil into the drive air line; lubricant oil recommended by the manufacturer for each four hours of use; • disinfection: use of two gauzes and disinfectant solution. sterilization: use of sterilization packaging following the manufacturer’s guidelines12,13. group 2:group 2:group 2:group 2:group 2: materials used as disposable personal protective equipment (ppe) and for the biosecurity of the team and the patient. • use of ppe and washing of hands by helping to perform cost analysis of materials used in class iii, iv and v composite resin restorations 299 braz j oral sci. 12(4):298-306 the disinfection of dental equipment; use of ppe and washing of hands by the operator and the auxiliary to the clinical care; use of protective barriers for the equipment; protective barriers and eyeglass for the patient; use of preoperative mouthwash with antiseptic solution by the patient. group 3: group 3: group 3: group 3: group 3: materials used for the sterilization of instruments. • sterilization in autoclave with polypropylene thermoplastic seal packaging. group 4:group 4:group 4:group 4:group 4: materials for anesthesia • local anesthesia; topical anesthetic gel, applied with cotton ball; solution of lidocaine 2% with epinephrine 1:100,000 (two tubes per patient); long needle. group 5:group 5:group 5:group 5:group 5: materials for absolute isolation and cavity preparation. • use of sterilized and reused drills for up to 10 patients21, 22; • number of drills: two types of high-speed diamond drills; low-speed carbide drills20; • use of restorative instruments (packaged in drilled holster), metal tray, rubber dam, template, punch, clamp forceps, isolation arc, scissors, ivory clamps (packaged in drilled holster with separations), drills (wrapped in small drilled holster for eight drills). group 6:group 6:group 6:group 6:group 6: materials for tooth restoration • materials were sub-classified in common use materials for classes iii, iv, and v cavities, and specific materials for classes iii and iv cavities (such as anatomic wooden wedges); • adper single bond 2 (3m espe, sumaré, sp, brazil) adhesive system for all types of resin; • composite resins brands: llis (fgm, joinvile, sc, brazil); fill magic (vigodent, rio de janeiro, rj, brazil); charisma (heraeus-kulzer, são paulo, sp, brazil); prisma aph (dentsply, petrópolis, rj, brazil); z350 (3m espe,); herculite xrv (kerr, são paulo, sp brazil); tetric n-ceram (ivoclar vivodent, são paulo, sp, brazil). group 7:group 7:group 7:group 7:group 7: materials for finishing and polishing of the tooth restoration: • assembled tips: use of materials in up to 10 patients21,22; • carbon paper and finishing and polishing discs and trips: single use, disposable. the main goal of the present study was to determine the cost of the materials in group 6group 6group 6group 6group 6. in this group, the materials were quantified for each type of preparation and depth classification. the amount of each material determined the final cost individually. the simulation of material use was performed by calibrated professionals (kappa>0.85). the calibration process consisted by one hour of theory discussion, four hours of practical training, including both repetitions of insertion and weighing and intra-examiner differences. the amount of adhesive was measured in drops, predetermined by the panel of experts. the amount of resin used was determined after its insertion in the artificial tooth of each type of preparation and cavity depths, following the realization of a pre-sculpture of the tooth anatomy, removing the excesses and the weighing the artificial tooth with the material, discounting the tooth weight. the final value of the cost of the direct and indirect materials was calculated by the sum of the individual costs of each used material. the independent variables studied in this study were: composite resin trade mark (seven types); cavity preparation (classes iii, iv and v) and depth of the cavity preparation (shallow, medium and deep). the data of mean cost of direct and indirect materials used in classes iii, iv and v composite resin dental restorations were used to analyze comparatively all the independent studied variables. the dependent variable was the total cost of the used direct and indirect materials. descriptive analysis of the mean cost of direct and indirect materials used in classes iii, iv and v composite resin restorations were performed. mean values found for the seven groups of materials were considered. for each evaluated material it was determined the amount of material required, the amount of material per package, the average price of materials per package and the adjusted price for the quantity required for the achievement of the restorative procedure. data were assessed by the non-parametric friedman’s test for dependent variable. this test was applied to two factors, the first factor was a fixed independent variable and the second factor were the blocks of each combination of the other two independent variables. when the friedman’s test indicated significant differences between the groups, the siegel-castellan test of multiple comparisons was applied. a 5% significance level was used to check which groups were statistically different. results the values of the cost of the direct and indirect materials included in groups 1, 2, 3, 4, 5 and 7 relating to materials for biosecurity, for local anesthesia, for absolute isolation and for cavity preparation were presented in a previous study23. the mean total cost found for the biosecurity materials (groups 1 to 3) was of us$4.18 (r$8.86). out of this amount, the materials of group 2, which correspond to ppe disposable materials and to the biosecurity for the dental team and the patient, represented 56.72 % and, in groups 1 and 3 corresponded to 6.89% and 36.39%, respectively. the mean total cost found for the materials used for local anesthesia (group 4) was of us$1.01 (r$2.14), the anesthetic represented 90.23% of that value, and the topical anesthetic and the needle corresponded to 1.4% and 8.37%, respectively. materials used for absolute isolation and cavity preparation (group 5) showed mean a total value of us$1.0 (r$2.12), the materials used to cavity preparation represented 67.30% and those for absolute isolation corresponded to 32.7% of this value. the distribution of costs adjusted for these materials showed that the cost of the drills of low and high-speed represented the highest found value, corresponding to 64% of the total cost for this group. the cost of the rubber dam, used in absolute isolation was responsible for 26% of the mean total value of this group, which corresponds to the mean value of us$0.26 (r$0.55)23. materials used for dental finishing and polishing (group 7) showed mean total value of us$1.23 (r$2.61). the distribution of costs adjusted for materials showed that the cost analysis of materials used in class iii, iv and v composite resin restorations300 braz j oral sci. 12(4):298-306 costs of carbon paper and assembled part of silicone for finishing corresponded to 74.23% of the mean total value of this group23. the calculation of the weights (in grams) of the evaluated composite resins is shown in table 1. the mean weight found for class iii was 0.027 (±0.015) g, for class iv was 0.052 (±0.040) g and for class v was 0.021 (±0.007) g. the calculation of the adjusted value (in us$) by gram from evaluated composite resins was shown in table 2. the mean adjusted value found was us$6.77 (r$14.35). the calculation of the cost (in us$) of the evaluated composite resins according the studied cavity preparation is shown in table 3. mean values found for class iii were us$0.18±0.10 (r$0.38), for class iv was us$0.35±0.26 (r$0.74), and for class v was us$0.14±0.05 (r$0.30). the calculation of the cost (in us$) of the materials used for tooth restoration according the type of cavity preparation (group 6) was shown in table 4. adjusted cost found for class iii was us$0.55 (r$1.17), for class iv was us$0.72 (r$1.53), and for class v was us$0.43 (r$0.91). the differences found are due to the greater amount of resin used for class iv than for classes iii and v, and to the use of polyester matrix and wedges for classes iii and iv. table 1. table 1. table 1. table 1. table 1. calculation of weight (in grams) of the composite resins according the type of cavity. costs (in us$) of the direct and indirect materials used in class iii, iv and v composite resin dental restoration were shown in table 5. the mean total cost found for class iii was us$7.96 (16.88), for class iv was us$8.13 (r$17.24), and for class v was us$7.84 (16.62). there were no significant differences in total cost of restorative materials between the types of cavity. figure 1 shows the non-parametric statistical data. the slot a shows the difference between the total costs of the materials to the types of studied composite resin. charisma, prisma aph, z350, and tetric n-ceram composite resin trade marks showed higher total mean cost than llis, fill magic, and herculite xrv composite resin brands. there were statistically significant differences between llis and fill magic to z350 and charisma composite resins; between magic fill, charisma, prisma aph, z350 to tetric n-ceram composite resins; and between herculite xrv and z350 to charisma composite resins. the slot b shows the difference between the total costs of the materials to the types of cavity preparation. there were statistically significant differences of class iii and iv to class v. the slot c shows the difference between the total costs of the materials to the types of depth classification. there was statistically significant difference between the types of depth classification of the cavity preparation. cost analysis of materials used in class iii, iv and v composite resin restorations 301 braz j oral sci. 12(4):298-306 table 2. table 2. table 2. table 2. table 2. calculation of the adjusted value (in us$) per gram of the evaluated composite resins. table 3.table 3.table 3.table 3.table 3. calculation of the cost (in us$) of the composite resins according the type of cavity. cost analysis of materials used in class iii, iv and v composite resin restorations302 braz j oral sci. 12(4):298-306 1 microbrushtm (fgm, kg sorensen, brazil). 2 composite resin: mean weight of composite resin used in class iii, iv and v (table 1). mean of the values of the studied composite resins (table 3). table 4:table 4:table 4:table 4:table 4: calculation of the cost (in us$) of the materials used for dental restoration according the type of cavity (group 6). table 5.table 5.table 5.table 5.table 5. mean cost of the direct and indirect materials used in class iii, iv and v composite resin dental restoration in us$ and percent value (%). 1 – secondary data from hebling & trentin, 2013 (23). cost analysis of materials used in class iii, iv and v composite resin restorations 303 braz j oral sci. 12(4):298-306 fig. 1 slot-plots of the non-parametric statistical data: adifference between the total costs of the materials to types of studied composite resin; bdifference between the total costs of the materials to types of cavity; cdifference between the total costs of the materials to types of depth classification. discussion this is the first study in which the cost analysis of materials used in classes iii, iv, and v composite resin dental restorations has been analyzed in brazil. to the best of our knowledge, no cost analysis on any aspect has been published in brazil or worldwide, which makes it difficult to establish comparisons of our results. the list drawn up by researchers and evaluated by the panel of referees represents an ideal pattern of care, including the requirements for excellence in productivity and quality of restorative procedures. all the biosecurity measures were included, while respecting the ethical and legal precepts12-15. this standard of excellence of care should be observed in all types of dental care services, be they public or private, when performing direct composite resin dental restorations. cost analysis of materials used in class iii, iv and v composite resin restorations304 braz j oral sci. 12(4):298-306 reduction of excellence of care may result in reduction of the cost of biosecurity. however, the risks to the health of patients and dental team hardly compensates for this reduction. direct dental restorations are low-risk contamination procedures. even so, the principles of biosecurity must be respected12-15. the use of protection barriers reduces the risk of cross-contamination for both the treated patients and the dental team15. the low total cost of biosecurity in this type of procedure ($4.18/r$8.86) is still much lower than the values to be spent to minimize the possible effects of a contamination of the team or the patient by any infectious disease. the risks of inability to work and death of the dental team should also be considered, which may occur even in low-risk procedures, as is in case of tooth restorations. the lack of other brazilian or international articles on the same subject made it difficult to compare these results. the economic stability of brazil, with control of inflation and reduction in the rate of increase in materials’ prices, reflects in the present study. there were no statistically significant differences in the total costs of the studied materials among the three evaluated suppliers. the present study showed that the mean cost of materials for class iii restoration was us$7.96 (r$16.88), for class iv it was us$8.13 (r$17.24) and for class v it was us$7.84 (r$16.62). there was a statistically significant difference in cost among classes iii, class iv and v restoration. the cost of materials for class iv is higher than for classes iii and v due to the high involvement of the tooth surfaces in the cavity preparation, requiring more restorative material than for the other two types. although it had a lower cost, the polyester matrix and wedge made the material costs for classes iii and iv to be higher than for class v. this value does not represent the value of dental care fees to be charged to the patient. the cost of the material is part of the variable costs to be considered in the calculation of the value of dental care fees. for this calculation it must be considered both the fixed and the variable costs associated with the dental care service3-5. differences in the properties of composite resins have been demonstrated in other studies16,17. however, the ultimate goal of achieving a direct restorative procedure is to obtain esthetic and functional results of the tooth, with long-lasting acceptable procedure, and it can be achieved with any type of the commercial brands of resin available on the market. thus, the small difference in the cost of the different evaluated resins results that there is no significant difference in the final total cost of the used materials. this fact allows that composite resins with better physical and aesthetic characteristics be elected as primary choice. in 2012, the national commission of covenants and accreditations24 established the reference values for dental procedures and stated minimum values of dental fees to be complied with in the private sector. the values suggested for class iii restoration in composite resin type was us$43.20 (us$1.0=r$2.12), for class iv it was us$61.31 and for class v it was us$40.98. the cost of the materials presented in this study, which represent only one of the items to be considered in the calculation of the fees5, corresponded to 18.45% of the fees for class iii, 13.27% for class iv, and 19.15% for class v. this fact reinforces the need for constant review of these fees. in the public sphere, the deployment of oral health funding policy for the municipalities with full management modified the lending forms to health financing. the financial resources are no longer intended for production according to the type of procedure to be performed, but according to the type of oral health program to which the municipality joins, according to the operations per inhabitant and program25. in this way, the results of this study cannot be compared with the resources transferred to the municipality. however, managers of dental services, both public and private may use the data of this study as parameters in strategic decision-making from choosing the type of composite resin up to the operability of using absolute isolation. the use or further development of this methodology for other groups of researchers in different scenarios and countries must be stimulated, thus allowing comparisons of the cost estimations presented in this study. based on the obtained results, it may be concluded that there was statistically significant difference in cost of materials between the types of cavities and teeth. the observation of a small difference in the values of the different resins evaluated revealed no significant differences in the final costs. this present study showed values of direct and indirect materials to be used. these values can be used as parameters for the calculation of the dental care fees. again, the lack of other studies on the same subject makes it difficult to compare these results. this fact can be considered as one of the limitations of this type of study. in addition, other factors to be considered are the possible regional differences in the values of the presented materials. however, the methodology described in this present study, as well as the weight of composite resin increments can be easily reproduced in other studies, allowing the comparison of the present results. future studies should be conducted to evaluate the costbenefit ratio from the studied resins, as well as to compare the costs found in this present study with other costs in different regions and countries. references 1. mackenzie l, parmar d, shortall ac, burke fj. direct anterior composites: a practical guide. dent update. 2013; 40: 297-9. 2. wilson nh, dunne sm, gainsford id. current materials and techniques for direct restorations in posterior teeth. part 2: resin composite systems. int dent j. 1997; 47: 185-93. 3. drummond f. methods for the economic evaluation of health care programmes. new york: oxford; 2005. 379 p. 4. shim jk, siegel jg. modern cost management and analysis. 3rd ed. new york: barron’s education series; 2009. 376 p. 5. creese al, packer d. cost analysis in primary health care: a training manual for programme managers. geneva: world health organization; 1994. 147 p. 6. gapenski lc. healthcare finance: an introduction to accounting and financial management. 5th ed. portland: health administration press; 2011. 724 p. cost analysis of materials used in class iii, iv and v composite resin restorations 305 braz j oral sci. 12(4):298-306 7. logan em. dentistry’s business secrets: proven growth strategies for your new or existing practice. bloomington: authorhouse; 2011. 439 p. 8. donaldson c. economic evaluation in dentistry: an ethical imperative? dent update. 1998; 25: 260-4. 9. bader j, ismali a, clarkson j. evidence-based dentistry and the dental research community. j dent res. 1999; 78: 1480-3. 10. smales rj, hawthorne ws. long-term survival and cost-effectiveness of five dental restorative materials used in various classes of cavity preparations. int dent j. 1996; 46: 126-30. 11. mjor ia, burke fj et al. the relative cost of different restorations in the uk. br dent j. 1997; 182: 286-9. 12. miller ch, palenik cj. infection control and management of hazardous materials for the dental team. saint louis: mosby; 2010. 365 p. 13. bonehill ja. managing health and safety in the dental practice: a practical guide. ames, iowa: wiley-blackwell; 2010. 264 p. 14. lemay ca, cashman sb, mcdonald a, graves jr. a new approach to ensuring oral health care for people living with hiv/aids: the dental case manager. prev chronic dis. 2012; 9: e158. 15. thomas mv, jarboe g, frazer rq. infection control in the dental office. dent clin north am. 2008; 52: 609-28. 16. dalli m, çolak h, mustafa hamidi m. minimal intervention concept: a new paradigm for operative dentistry. j investig clin dent. 2012; 3: 167-75. 17. robinson s, nixon pj, gahan mj, chan mf. techniques for restoring worn anterior teeth with direct composite resin. dent update. 2008; 35: 551-2. 18. marinozzi s, corbellini g, ottolenghi l, ripari f, pizzuti a, pezzetti m, et al. from nuremberg to bioethics: an educational project for students of dentistry and dental prosthesis. ann stomatol. 2013; 4: 138-41. 19. likert r. a technique for the measurement of attitudes. arch psychol. 1932; 140: 1-55. 20. siegel sc, von fraunhofer ja. dental burs: what bur for which application? a survey of dental schools. j prosthodont. 1999; 8: 258-63. 21. siegel sc, von fraunhofer ja. assessing the cutting efficiency of dental diamond burs. j am dent assoc. 1996; 127: 763-72. 22. gureckis km, burgess jo, schwartz rs. cutting effectiveness of diamond instruments subjected to cyclic sterilization methods. j prosthet dent. 1991; 66: 721-6. 23. hebling e, trentin ef. cost analysis of materials used in composite resin direct posterior dental restorations. rev odontol unesp. 2013; 42: 144-51. 24. brazil. brazilian dentistry council. reference values for dental procedures. brasília: brazilian dentistry council; 2012. 25. brazil. health ministry. [operational guidelines: pacts by life, in defense of the sus and management]. 2nd ed. brasilia: health ministry; 2006. portuguese. cost analysis of materials used in class iii, iv and v composite resin restorations306 braz j oral sci. 12(4):298-306 oral sciences n3 original article braz j oral sci. july | september 2014 volume 13, number 3 in situ assessment of the saliva effect on enamel morphology after microabrasion technique núbia inocencya pavesi pini1, débora alves nunes leite lima1, renato herman sundfeld2 , gláucia maria bovi ambrosano3, flávio henrique baggio aguiar1, josé roberto lovadino1 1universidade estadual de campinas unicamp, piracicaba dental school, department of restorative dentistry, piracicaba, sp, brazil 2universidade estadual paulista unesp, araçatuba dental school, department of restorative dentistry, araçatuba, sp, brazil 3universidade estadual de campinas unicamp, piracicaba dental school, department of social dentistry, area of statistics, piracicaba, sp, brazil correspondence to: débora alves nunes leite lima departamento de odontologia restauradora universidade estadual de campinas – unicamp faculdade de odontologia de piracicaba avenida limeira 901 – caixa postal 52 cep: 13414-903, piracicaba, sp, brasil phone: +55 19 2101 5340 e-mail: deboralima@fop.unicamp.br abstract aim: this study evaluated saliva effects on enamel morphology surface after microabrasion technique. methods: enamel blocks (16 mm2) obtained from bovine incisors were divided into 9 groups as follows: one control group (no treatment), four groups with microabrasion treatment using 35% phosphoric acid and pumice (h3po4+pum) and other four groups treated with 6.6% hydrochloric acid and silica (hcl+sil). one group of each treatment was submitted to 4 frames of saliva exposure: without exposure, 1-h exposure, 24-h exposure, and 7-days exposure on in situ regimen. nineteen volunteers (n=19), considered as statistical blocks, used an intraoral appliance containing the specimens, for 7 days. enamel roughness (ra) was tested before and after treatment, and after saliva exposure. confocal laser scanning microscopy (clsm) was used to evaluate qualitatively the enamel morphology. results: all groups exhibited increased ra after microabrasion. with regards to saliva exposure, the treatment with hcl+sil presented more susceptibility to the saliva action, but no period of time was efficient in re-establishing this characteristic compared with the control group. clsm analysis showed reduction of the micro-abrasive damages during the experimental times. conclusions: seven days of saliva exposure were not sufficient for the treated enamel to reach its normal characteristics compared with the control group. keywords: dental enamel; enamel microabrasion; saliva. introduction the microabrasion technique is a treatment to achieve dental esthetics in cases of superficial tooth stains and enamel decalcification and defects, improving color and surface texture of the tooth1-2. it is considered a safe and conservative technique, since it causes a non-significant loss of enamel, and it provides lasting and satisfactory results in a short clinical time1,3-4. currently, the microabrasive agents used are 6.6% hydrochloric acid with silica, in a commercial presentation, or pumice added to 35% phosphoric acid, with mechanical application under low-speed rotation5. the application of the microabrasive system is commonly followed by enamel polishing with diamond paste6-7 or fluoridated paste2,4. the clinical success of this technique has been documented1,2,5. however, little is known about the effects of the microabrasive systems used on enamel surface, such as roughness and microhardness6-7 and its behavior in the oral braz j oral sci. 13(3):187-192 received for publication: may 21, 2014 accepted: august 07, 2014 environment in contact with saliva. since microabrasion provides both erosive and abrasive challenges by an acid, and abrasive and mechanical application, it is expected that the enamel surface will undergo mineral loss due to demineralization, in which saliva and its components have a role in maintaining the integrity of the tissue8-10. surface roughness is important for enamel, since it is related to color, smooth and brittle appearance of the tooth3,11. compared with in vitro models, in situ and in vivo studies can simulate more closely the clinical conditions8,11 and may really reveal influences of microabrasion on enamel. the in situ models are more accessible than the in vivo studies and are considered reliable as they allow the control of some clinical variables 8, e.g. in relation to the standardization of the enamel surface to be treated with microabrasion and exposed to the oral environment. the literature lacks in situ studies evaluating the treatment of microabrasion and in those cases where in vitro studies are available3,7,12-13 only few of them analyzed the influence of artificial saliva on the enamel after microabrasion6-7 and none of them used human saliva. the use of artificial saliva in studies about demineralization creates an optimum environment for the mineral recovery of enamel surface, since it contains only mineral ions in its composition14. on the other hand, the treatment of enamel surface with human saliva presents differences in this process, as it contains proteins such as mucin, which is deposited on the surface and may hamper the remineralization process15. so, the use of human saliva, in an in situ model, allows the analysis of microabraded enamel, reproducing more reliably the in vivo conditions. since there is no study evaluating the behavior of enamel after microabrasion and relating it to human saliva exposure, the present work used an in situ model to investigate the effect of saliva exposure on enamel morphology after different microabrasion techniques. material and methods the study design is presented in figure 1. volunteers and ethical issues the study protocol was approved by the local ethics committee in research (piracicaba dental school protocol no. 037/2011). nineteen adult volunteers (20-30 years old) met the inclusion criteria exhibiting no fixed or removable orthodontic appliances, good general and oral health, no antibiotic use during 2 months prior to the study, and normal salivary flow. for standardization purposes, the volunteers were instructed to use fluoridated toothpaste (colgate total 12, colgate-palmolive, são paulo, sp, brazil) and manual toothbrushes (colgate professional clean colgatepalmolive, são paulo, sp, brazil) for 7 days prior to and during the course of the experiment. preparation of specimens enamel slabs (4 x 4 x 2 mm) were obtained from bovine incisors using a precision saw and a high-concentration diamond disc. to obtain flat and standardized enamel surfaces, the blocks were leveled using water-cooled carborundum discs with increasing grit size in a circular watercooled polishing machine. the resulting surface was then polished with felts associated with a diamond paste of 1, 1/2 fig. 1. experimental design of study (a) bovine incisor and separation of coronal portion; (b) sectioning of crowns to obtain specimens; (c) enamel/dentin blocks with 16 mm2; (d) polishing of specimens; (e) roughness analysis; (f) enamel microabrasion; (g) roughness analysis after treatment; (h) division of groups according to salivary exposition; (i) groups without salivary exposition stored in distilled water; (j-k) groups with salivary exposition distributed for the volunteers; (l) roughness analysis after salivary exposition; (m) sectioning of the specimens; (n) confocal analysis. in situ assessment of the saliva effect on enamel morphology after microabrasion technique188 braz j oral sci. 13(3):187-192 and 1/4µm grit sizes and greased with specific oil. between the polishing steps and after the final polishing, all the slabs were sonicated with distilled water for 15 min. the specimens were sterilized with ethylene oxide. groups nineteen volunteers took part in the study and were considered as statistical blocks (n=19), since they used a palatal appliance containing one specimen of each group designed to receive saliva exposure. the samples were divided into nine groups (n=19) in randomized order and according to the microabrasion technique and storage time in human saliva (in situ phase). the experimental groups were the following: one control group, four groups treated with h3po4+pum and four groups treated with hcl+sil. the groups with treatment were subdivided according to the in situ regimen: without saliva exposure, 1 h, 24 h and 7 days of saliva exposure. surface treatment the enamel microabrasion was performed by 35% phosphoric acid and pumice (h 3po 4+pum) or 6.6% hydrochloric acid and silica (hcl+sil). in the first case, equal parts of 35% phosphoric acid (h3po4) and pumice were measured with a metering spoon and mixed to compound the microabrasive mixture. both products were placed on the enamel surface with a syringe until covering the specimen. the microabrasion was achieved with specific rubber cups coupled to an electric micro-motor operating at low speed (12,000 rpm). the treatment regimen was 10 applications of 10 s each. palatal appliances the intra-oral palatal appliances containing seven cavities measuring 4 x 4 x 3 mm (three at each side and one at the center of the appliance) were made for each volunteer in acrylic resin to set the slabs (figure 1). the enamel slabs were fixed inside the cavities with wax in a manner that the enamel surface was positioned at the same level of the cavity, allowing their contact with the salivary fluid. each volunteer used its appliance containing seven bovine dental enamel specimens, representing different experimental groups, during an experimental phase of 7 days. when the specimen of the group with 1 hour or 24 hours of salivary exposure was removed from the appliance, the cavity was full of wax. preferably, the appliances were worn for 24 h, except during meals and periods of oral hygiene. with the appliances in situ, the participants were instructed not to eat, being only allowed to drink water. after meals, a period of at least 15 min elapsed prior to reinsertion of the appliance. in the evening, the appliances, but not the specimens, were cleaned with a toothbrush without toothpaste. afterwards, the appliances were immersed for 1 min in chlorhexidine digluconate solution (periogard; colgate-palmolive, são paulo, sp, brazil) to avoid plaque formation. analysis of the enamel roughness the enamel roughness (ra) was analyzed at three time frames: before and after microabrasion, and after saliva exposure using a profilometer roughness tester (mitutoyo surfitest 211, são paulo, sp, brazil). three readings were made in three different directions on the enamel surface, and the measurements were averaged. confocal laser scanning microscopy (clsm) to visualize enamel surface structures with the confocal laser scanning microscope (leica tcs sp2-se microscopy, mannheim, germany), representative specimens of each group were sectioned to obtain slices. each slice was polished by carbide papers and felts associated with a diamond paste of 1 µm grit size, according to the same protocol described for specimen preparation, resulting in slices about 0.3 µm thick. subsequently, the samples were immersed into a freshly prepared 0.1 mm rhodamine b solution and left for 1 h, without further rinsing16. cslm analysis was performed in fluorescence mode using a 40x magnification objective and oil immersion. statistical analysis after an exploratory analysis, the data were analyzed by mixed models for repeated measures (proc mixed), followed by tukey-kramer and dunnett tests. analysis of variance was applied since different treatments were performed in the same sample, at different moments. the significance level was set at 5%. results all the experimental groups presented increased enamel roughness (table 1) after microabrasion, with statistically significant differences in comparison with both initial analysis and control group, but without significant differences between them. after saliva exposure, only the group treated with h3po4+pum and 1-h exposure showed no decrease in enamel roughness, with statistically significant difference compared with the ra analysis after microabrasion. in the other groups with this treatment, the groups with 24 h and 7 days of exposure to saliva showed statistical significance in reducing enamel roughness. with regard to the groups treated with hcl+sil, all exposure periods to saliva (1 h, 24 h and 7 days) resulted in decreased enamel roughness compared with the group without saliva exposure. the groups with this treatment and saliva exposure for 24 h and 7 days to the intraoral environment showed greater reduction of roughness, with statistically significant difference compared with the group without saliva exposure and the group with 1-h saliva exposure. the effect of saliva on enamel roughness for both microabrasive systems showed that groups treated with h3po4+pum and exposed to saliva for 1 and 24 h presented the greatest enamel roughness, with statistically significant difference compared with the groups treated with hcl+sil and the group with the same exposure time to the intraoral environment. in situ assessment of the saliva effect on enamel morphology after microabrasion technique 189 braz j oral sci. 13(3):187-192 the clsm analysis showed detectable lesions depending on the group, characterized by sites of microwear on enamel surface, for both treatments. during the in situ regimen, there was a tendency towards reduction of this wear by filling of the micro-sites in order to recover the continuous and uniform line of the enamel surface, as found in the control. for both treatments, after 7 days of saliva exposure, almost no microwear sites could be observed and an almost unchanged line characterized the enamel surface (figure 2). discussion this study evaluated the action of saliva on the effects of microabrasion techniques, combining an acid, an abrasive agent and mechanical application3,12-13, on enamel surface. the clinical protocol proposed by some authors1-2,4-5 advises to perform a final polishing of enamel surface after microabrasion. however, this procedure is probably against the purpose of this study, as the polishing reestablishes the enamel roughness6-7. as a consequence, it could be stated that if saliva has a positive effect on enamel, it would not be necessary to perform polishing after microabrasion, avoiding additional enamel wear on a demineralized surface. the results showed changes in enamel roughness, which are considered a function of variations in both surface porosity15-16 and mineral loss15,17 resulting from the erosiveabrasive challenge of the microabrasion. in accordance with the results, both microabrasive treatments using h3po4+pum or hcl+sil increased the enamel roughness, with statistically significant differences compared with initial analysis and without differences between them. these results are corroborated by previous findings that reported mineral loss and increased enamel porosity after treatment with erosive17 and/or abrasive9-10,12,18-19 treatment and low ph14 compounds. enamel microabrasion may be considered an erosiveabrasive model, since the erosive and abrasive challenges cause dissolution of the hydroxyapatite crystals20. although proteins comprise a minor part of the enamel, they have the function of attaching hydroxyapatite crystals together to maintain the hierarchical structure of enamel21. therefore, the effects of microabrasion techniques on the mineral and protein components may include the mechanical behavior of enamel. both acids showed similar alterations of enamel roughness. this may be related to the concentration of hydrochloric acid at 6.6%, different from those initially proposed for the technique, e.g., 36% and 18%1,4,22, which produce a similar conditioning pattern of 35% phosphoric acid 23. with regard to the abrasive, pumice presented excessive abrasiveness and deep erosion compared with the other 19. as h 3po 4+pum is a microabrasive mixture customized by the clinician, the peculiarities of these components should be considered, as their sensitivity to variations may have clinical outcomes. as the treated surface became less mineralized, it was possible to observe the saliva buffering capacity during the in situ regimen9,17. fluoride action may be considered even with 1 h of saliva exposure, since this component is found in oral environment at high concentration up to 10 h after the last use24. in this study, the volunteers were instructed to use fluoridated toothpaste because its role should be taken into account. this analysis showed a decrease in roughness, which varied depending on the saliva exposure time in situ regimen and the used microabrasive system. the treatment with hcl+sil could be considered more prone to mineral reposition of saliva than that with h3po4+pum because, after 1 h of saliva exposure, it was possible to observe decreased roughness, with statistically significant difference compared with the analysis after microabrasion for this group. for both treatments, 24-h exposure to saliva was efficient in reducing roughness, with statistical differences compared with the analysis after microabrasion. the 7-day exposure time resulted in less roughness than the other exposure times, for both microabrasion treatments. although the reestablishment of roughness was not observed in the total storage period in saliva applied in this study (7 days), analyzing the progressive results of increase in enamel roughness over time and comparing it with studies that used artificial saliva for 15 days, the authors hypothesized that normal enamel roughness probably would be reached even in a longer evaluation time. this result raises the necessity of performing further studies on this subject. salivary microabrasion exposition time before treatment after treatment after saliva h3po4 + pumice no exposure 0.14 (0.03) ca #0.36 (0.03) aa #0.36 (0.02) aa 1 h 0.14 (0.03) ca #0.41 (0.05) aa *#0.38 (0.02) aa 24 h 0.13 (0.02) ca #0.41 (0.03) aa *#0.35 (0.04) ba 7 days 0.14 (0.04) ca #0.38 (0.03) aa #0.21 (0.05) bb hcl + silica no exposure 0.14 (0.03) ca #0.39 (0.05) aa #0.36 (0.03) aa 1 h 0.15 (0.03) ca #0.41 (0.05) aa #0.28 (0.03) bb 24 h 0.17 (0.04) ca #0.38 (0.04) aa #0.25 (0.04) bbc 7 days 0.16 (0.02) ca #0.38 (0.03) aa #0.22 (0.02) bc control 0.15 (0.03) 0.17 (0.03) 0.16 (0.02) table 1.table 1.table 1.table 1.table 1. roughness analysis (ra) means followed by pattern error of all groups. means followed by different letters (uppercase in horizontal and lowercase in vertical compare time within each category of microabrasion) differ (p<0.05). *differ from the group treated with hcl + silica with the same time of salivary exposition. #differ from the control in the same time of analysis. in situ assessment of the saliva effect on enamel morphology after microabrasion technique190 braz j oral sci. 13(3):187-192 comparing both treatments, the groups treated with hcl+sil presented more evidence of superficial mineral reposition as their results after 1 h and 24 h of saliva exposure exhibited the lowest mean values of roughness when compared with h3po4+pum. the best effects of saliva with the treatment with hcl+sil may be related to specific action of the compounds hcl and silica. the ion chloride in the buccal environment has been associated with the suppression of hydroxyapatite dissolution 25 beside the fact that the hydrochloric acid presents lower ph than the phosphoric acid 26, which favors surface rehardening and fluoride uptake17,20. in relation to silica, it has been used in a bioactive material (ca3sio5) as an important agent to induce the remineralization of an acid-etched enamel27, since it can provide the link between the calcium from hydroxyapatite and from saliva, leading to the formation of a new apatite layer18,27. therefore, regarding the better results achieved with this treatment, it may be assumed that this compound is formed in the enamel after microabrasion using hcl+sil. none of the treatments was able to re-establish the enamel roughness, as found in control group or in the initial condition, in none of the periods of saliva exposure. in previous in vitro findings, the enamel roughness treated with microabrasion was restored after 15 days of immersion in artificial saliva6-7. the differences between these studies6-7 and the present one are related to both time of saliva exposure and type of saliva. according to another investigation15, the deposition of proteins from saliva (e.g. mucins) on the demineralized substrate may be a factor related to the reduction of re-mineralization. also, the absence of organic content in artificial saliva may have created an optimized system, thus enhancing the mineral reposition on the eroded surface. to the best of the authors’ knowledge, this is the first study that evaluated the effect of the human saliva on the enamel after microabrasion, under in situ conditions. the delay in the reestablishment of the enamel roughness, which did not occur within 7 days, probably is due to the irregular deposition of ions between the enamel prisms, that happen first in the 10 and 25 µm layers, possibly due to the topographic characteristics of this region, where retzius lines have a regular periodicity28-29. despite of the above information, the in vitro model does not reflect the dynamic conditions of the mouth, such as continuous flow and clearance of saliva, even using artificial saliva29. apart from the similar calcium, phosphate and fluoride ions in artificial saliva, human saliva has some advantages that may account for its superior protective ability, such as formation of an acquired enamel pellicle11. this pellicle forms within seconds of exposure to saliva11,14 and it has anti-erosive potential because it acts as a diffusion barrier as well as a semi-permeable membrane11,23, being important for the enamel re-mineralization. moreover, saliva action may be enhanced by the presence of re-mineralizing agents containing fluoride8,24. with regards to the roughness means obtained with the salivary exposure, it was shown that this property could be possibly restored in a short time. therefore, it should not be stated that the recovery of mineral content in the enamel after microabrasion is more feasible with artificial saliva, such as in vitro models, since they do not mimic the real circumstances of the oral environment. the clsm results showed that the enamel surface had remaining erosive effects from the combined action of acid, abrasive agent and mechanical pressure. the images showed an irregular surface with discontinuity points, characterized by microwear points. the recurrence of these points in the groups with only enamel microabrasion reflects the results on enamel roughness. the clsm images (figure 2) for the groups in early stages of saliva exposure (1 and 24 h) showed gradual reduction of these points, possibly completing the enamel surface with minerals and ions from saliva. the images also showed that this deposition occurs possibly in a random manner, which could be observed with the roughness results too. after 7 days of saliva exposure in the oral environment, the images demonstrated that the enamel surface tended to be uniform and continuous, as found in the control group, showing the re-mineralizing action of saliva. a limitation of the present study design should be considered: in clinical situations, enamel microabrasion is performed on the buccal surface of the teeth, whereas in the current study, the enamel blocks were placed on the palate, mainly for the comfort of the volunteers and esthetic implications. moreover, previous in situ studies8-9,11,24 chose the palatal model to simulate the condition of the human fig. 2: representative images of the groups obtained from confocal laser scanning microscopy (clsm). arrows show surface microwear sites on enamel surface resulting from the microabrasion technique and its behavior in the oral environment. (a) control group; (b) h3po4+pumice; (c) hcl+sil; (d), (e) and (f) h3po4+pumice with 1 h, 24 h and 7 days of salivary exposure respectively; (g), (h) and (i) hcl+sil with 1 h, 24 h and 7 days of salivary exposure respectively. in situ assessment of the saliva effect on enamel morphology after microabrasion technique 191 braz j oral sci. 13(3):187-192 oral cavity and successfully proved its superiority over the in vitro strategy. this is the first in situ study investigating the action of saliva on the enamel morphology subjected to microabrasion treatment. the results showed that the technique causes a significant alteration in enamel roughness, without differences between the tested microabrasive systems. in addition, saliva plays an important role in re-establishing the characteristics of the enamel, since a 7-day exposure to saliva reduced the roughness mean values and the alterations on enamel surface, according to the clsm analysis. acknowledgements the authors would like to gratefully acknowledge all the volunteers who participated in this study. this study was supported by fapesp (proc. 2011/004067-7). references 1. croll tp. enamel microabrasion: observations after 10 years. j am dent assoc. 1997; 128: 45s-50s. 2. sundfeld rh, rahal v, croll tp, de alexandre rs, briso al. enamel microabrasion followed by dental bleaching for patients after orthodontic treatment-case reports. j esthet and restor dent. 2007; 19: 71-7. 3. meireles ss, andre d de a, leida fl, bocangel js, demarco ff. surface roughness and enamel loss with two microabrasion techniques. j contemp dent pract. 2009; 10: 58-65. 4. sundfeld rh, croll tp, briso al, de alexandre rs, sundfeld-neto d. considerations about enamel microabrasion after 18 years. am j dent. 2007; 20: 67-72. 5. wang y, sa y, liang s, jiang t. minimally invasive treatment for esthetic management of severe dental fluorosis: a case report. oper dent. 2012; 38: 358-62. 6. bertoldo ces, pini nip, miranda da, catelan a, ambrosano gmb, lima danl, et al. physicochemical properties of enamel after microabrasion technique. j res dent. 2014; 2: 176-88. 7. fragoso ls, lima da, de alexandre rs, bertoldo ce, aguiar fh, lovadino jr. evaluation of physical properties of enamel after microabrasion, polishing, and storage in artificial saliva. biomed mater. 2011; 6: 035001. 8. calvo af, tabchy cp, del bel cury aa, tenuta lm, da silva wj, cury ja. effect of acidulated phosphate fluoride gel application time on enamel demineralization of deciduous and permanent teeth. caries res. 2012; 46: 31-7. 9. grazziotin gb, rios d, honorio hm, silva sm, lima je. in situ investigation of the remineralizing effect of saliva and fluoride on enamel following prophylaxis using sodium bicarbonate. eur j dent. 2011; 5: 40-6. 10. ribeiro hz, lima je, vono bg, machado ma, da silva sm. airpolishing effect on bovine enamel and the posterior remineralizing effect of saliva. an in vitro study. j appl oral sci. 2006; 14: 193-7. 11. sa y, sun l, wang z, ma x, liang s, xing w, et al. effects of two in-office bleaching agents with different ph on the structure of human enamel: an in situ and in vitro study. oper dent. 2013; 38: 100-10. 12. paic m, sener b, schug j, schmidlin pr. effects of microabrasion on substance loss, surface roughness, and colorimetric changes on enamel in vitro. quint int. 2008; 39: 517-22. 13. schmidlin pr, gohring tn, schug j, lutz f. histological, morphological, profilometric and optical changes of human tooth enamel after microabrasion. am j dent. 2003; 16: 4a-8a. 14. siqueira wl, custodio w, mcdonald ee. new insights into the composition and functions of the acquired enamel pellicle. j dent res. 2012; 91: 1110-8. 15. hara at, gonzalez-cabezas c, creeth j, zero dt. the effect of human saliva substitutes in an erosion-abrasion cycling model. eur j oral sci. 2008; 116: 552-6. 16. de abreu da, sasaki rt, amaral fl, florio fm, basting rt. effect of home-use and in-office bleaching agents containing hydrogen peroxide associated with amorphous calcium phosphate on enamel microhardness and surface roughness. j esthet restor dent. 2011; 23: 158-68. 17. gonzalez-cabezas c, jiang h, fontana m, eckert g. effect of low ph on surface rehardening efficacy of high concentration fluoride treatments on non-cavitated lesions. j dent. 2012; 40: 522-6. 18. dong zc j, deng y, joiner a. in vitro remineralization of acid-etched human enamel with ca3sio5. app surf sci. 2010; 256: 3. 19. honorio hm, rios d, abdo rc, machado ma. effect of different prophylaxis methods on sound and demineralized enamel. j app oral sci. 2006; 14: 117-23. 20. buzalaf ma, hannas ar, magalhaes ac, rios d, honorio hm, delbem ac. ph-cycling models for in vitro evaluation of the efficacy of fluoridated dentifrices for caries control: strengths and limitations. j app oral sci. 2010; 18: 316-34. 21. dominguez ja, bittencourt b, michel m, sabino n, gomes jc, gomes om. ultrastructural evaluation of enamel after dental bleaching associated with fluoride. microsc res tec. 2012; 75: 1093-8. 22. dalzell dp, howes ri, hubler pm. microabrasion: effect of time, number of applications, and pressure on enamel loss. ped dent. 1995; 17: 207-11. 23. wiegand a, bliggenstorfer s, magalhaes ac, sener b, attin t. impact of the in situ formed salivary pellicle on enamel and dentine erosion induced by different acids. acta odontol scand. 2008; 66: 225-30. 24. paes leme af, dalcico r, tabchy cp, del bel cury aa, rosalen pl, cury ja. in situ effect of frequent sucrose exposure on enamel demineralization and on plaque composition after apf application and f dentifrice use. j dent res. 2004; 83: 71-5. 25. jager dh, vieira am, ligtenberg aj, bronkhorst e, huysmans mc, vissink a. effect of salivary factors on the susceptibility of hydroxyapatite to early erosion. caries res. 2011; 45: 532-7. 26. meyer-lueckel h, paris s, kielbassa am. surface layer erosion of natural caries lesions with phosphoric and hydrochloric acid gels in preparation for resin infiltration. caries res. 2007; 41: 223-30. 27. wang y, li x, chang j, wu c, deng y. effect of tricalcium silicate (ca3sio5) bioactive material on reducing enamel demineralization: an in vitro ph-cycling study. j dent. 2012; 40: 1119-26. 28. torres-rodriguez c, gonzalez-lopez s, bolanos-carmona v, sanchezsanchez p, rodriguez-navarro a, attin t. demineralization effects of phosphoric acid on surface and subsurface bovine enamel bleached with in-office hydrogen peroxide. j adhes dent. 2011; 13: 315-21. 29. li c, risnes s. sem observations of retzius lines and prism crossstriations in human dental enamel after different acid etching regimes. arch oral biol. 2004; 49: 45-52. in situ assessment of the saliva effect on enamel morphology after microabrasion technique192 braz j oral sci. 13(3):187-192 oral sciences n3 braz j oral sci. 13(2):124-128 original article braz j oral sci. april | june 2014 volume 13, number 2 strength of 3y-tzp and feldspathic porcelain subjected to different cooling methods antonio alves de almeida-júnior1, diogo longhini2, beatriz regalado galvão3, claudinei dos santos4, gelson luis adabo2 1universidade tiradentes – unit, school of dentistry, department of oral rehabilitation, aracaju, se, brazil 2universidade estadual paulista – unesp, school of dentistry, department of dental materials and prosthodontics, araraquara, sp, brazil 3universidade potiguar unp, school of dentistry, department of oral rehabilitation, natal, rn, brazil 4universidade do estado do rio de janeiro – uerj, resende school of technology, department of engineering materials, resende, rj, brazil correspondence to: gelson luis adabo rua humaitá, 1680. sala 415. centro cep 14801-903 araraquara, sp, brasil phone: +55 16 3301-6415 fax: +55 16 3301-6406 e-mail: adabo@foar.unesp.br abstract aim: to investigate the effect of the cooling rate on flexural strength of monolayer and bilayer porcelain/zirconia (y-tzp) bars. methods: forty-five specimens were made for each design group: (pm) monolithic specimens of veneer porcelain vita vm9 (vita, germany); (zm) monolithic specimens of zirconia (zihp; protmat, brazil); (pb) bilayer specimens zirconia/porcelain with porcelain on lower surface; and (zb) bilayer specimens porcelain/zirconia with zirconia on lower surface. each group was cooled by three different methods after porcelain sintering: slow – specimens were cooled inside the turned-off furnace; normal – specimens were removed from the furnace and cooled in air at room temperature; and fast – specimens were removed from the furnace at 910°c and cooled by compressed air for 10 s. specimens were polished and flexural strength was measured in water at 37 °c (n=15). maximum load at fracture was recorded, and equations for simple (monolayer) and composite (bilayer) structures were used to calculate the flexural strength. results were analyzed using one-way anova (p<0.05) and tukey test separately for each design. results: the results of one-way anova were statistically significant only for the pb group. the post-hoc tukey test showed the highest flexural strength for fast cooling and the lowest for slow cooling; the normal cooling was statistically similar to both. conclusions: cooling methods affected only the flexural strength of bilayer specimens with porcelain on low surface (under tension) when the slow cooling method was used. keywords: dental porcelain; zirconium; material resistance. introduction medical applications of ceramic system based on yttria-stabilized zirconia (3y-tzp) have increased due to its biological and mechanical properties and improvement in computer-aided design/computer-aided manufacturing (cad/ cam) technology. zirconia was introduced into the dental market as framework for ceramic fixed partial dentures due to its sufficient strength and toughness, and it is believed to resist the masticatory forces in posterior teeth, comparable to several metal-ceramic alloys1. however, high crystalline zirconia is an opaque material, and, for esthetical reasons, 3y-tzp frameworks have to be veneered with specific feldspathic dental porcelains2. nevertheless, the veneer porcelain is the weakest part of this system due to its low content of crystalline phase. moreover, this system seems to behave received for publication: april 06, 2014 accepted: june 09, 2014 braz j oral sci. 13(2):124-128 125 differently from conventional metal-ceramic prostheses. after three years of service time, chipping of zirconia porcelains is described to have a failure rate of 15%, while this rate for metal-ceramic prostheses is less than 0.5% for crowns and 3% for fixed partial denture in five years3-6. among the reasons for failure, residual tension is the most discussed issue in the literature7-11. incompatible thermal expansion coefficient7, non-uniform porcelain thickness12, inadequate substructure design13, low thermal conductivity of zirconia, fast cooling rates11, and intrinsic strength of these ceramic materials14-15 may develop tension stress in the system and induce the occurrence of failures in 3y-tzp/porcelain prostheses10,15. moreover, zirconia and metal frameworks behave differently after porcelain sintering. during the cooling, an excessive compressive stress may be compensated by plastic flow or thermal creep of the metal, while similar compensation is not possible in 3y-tzp frameworks because of its high rigidity. therefore, veneering materials for allceramic system should have high mechanical strength16. different cooling rates after porcelain firing schedule have been proposed for reducing those residual stresses10,17-18. the study of flexural strength of veneering porcelains and zirconia using faster or slower cooling is an important parameter to improve the clinical behavior and performance of veneered zirconia prostheses. the aim of this study was to investigate if cooling methods change the flexural strength on monolayer and bilayer specimens of zirconia and feldspathic porcelain subjected to different cooling methods. the null hypothesis was that the cooling method did not modify the flexural strength of monolayer and bilayer zirconia/porcelain specimens. material and methods a three-point flexural strength test was performed in four different designs (figure 1) of monolayer or bilayer bar specimens of 3y-tzp (zihp; protmat materiais avançados, são paulo, sp, brazil) and feldspathic porcelain (vita vm9; vitazahnfabrik, bad säckingen, germany) subjected to three different cooling methods. forty-five specimens of each design were prepared according to the dimensions recommended by iso 6872:2008 for a three-point flexural strength test (4±0.25 mm in width, 1.2±0.2 mm in thickness and 22 mm in length). the fig. 1.design groups: (pm) monolithic specimens of porcelain; (pb) bilayer specimens 3ytzp/vm9 with porcelain in lower surface; (zm) monolithic specimens of 3ytzp; (zb) bilayer specimens vm9/3y-tzp with zirconia in lower surface thickness of bilayer specimens was in a ratio of 1:1. zirconia specimens were cut approximately 25% larger than the final dimensions with a diamond disk (15lc; buehler ltd, lake bluff, il, usa) in a precision saw (isomet 2000; buehler ltd.) at low speed. after cutting, the specimens were ground using 600-grit silicon carbide (sic) abrasive papers (norton abrasivos, são paulo, sp, brazil) on a mechanical polisher (buehler metaserv 2000; buehler uk ltd., coventry, england) under running water. the specimens were sintered in a mosi2 oven (inti fe 1800; maitec, são carlos, sp, brazil) at 1.530°c for 2 h, heating rate of 8 °c/min, and cooling rate of 5 °c/min, according to the manufacturer’s recommendation. the firing schedules for zirconia and porcelain are shown in table1. porcelain specimens were made by mixing vm9 powder and vita modeling liquid (vitazahnfabrik). slurry was prepared and condensed into polyether mold (impregum f; 3m espe, seefeld, germany) 20% larger than the final dimensions, to compensate for porcelain contraction. after the excess liquid had been soaked up with an absorbent tissue, the specimens were fired in an oven for ceramics (aluminipress; edg, são carlos, sp, brazil) according to the recommendations of the porcelain manufacturer (table 1). step zirconia base dentine base washbake vm9 dentine vm9 pre-drying time (min) 2 6 pre-drying temperature (°c) 500 500 heating rate (°c/min.) 8 55 55 firing temperature (°c) 1530 950 910 holding time (min) 120 1 1 table 1.table 1.table 1.table 1.table 1. firing schedules of the materials for bilayer specimens, the porcelain manufacturer recommends the base dentine washbake firing previous to dentine porcelain. washbake porcelain was applied on one side of the zirconia bilayer samples and fired according to the manufacturer’s instructions (table 1). after the washbake layer cooling, dentine porcelain was applied as previously described. after porcelain firing (groups pm, pb, and zb) or porcelain firing simulation for the zm group, three cooling methods were performed (n=15): slow – samples were left inside the closed turned-off furnace until it reached the room temperature; normal – the elevator of the furnace was down, strength of 3y-tzp and feldspathic porcelain subjected to different cooling methods 126 braz j oral sci. 13(2):124-128 and when the temperature inside the furnace reached 500°c, the samples were removed and cooled in air at room temperature; fast – samples were blasted directly by compressed air immediately after removal from the furnace. when cooling was finished, the porcelain in the specimens were grounded and polished using 120-, 220-, 320-, 400-, 600-, and 1200-grit sic abrasive papers (norton abrasivos brasil, são paulo, sp, brazil) on a mechanical polisher (buehler metaserv 2000; buehler uk ltd.) under running water. bilayer specimens were randomly allocated to the design group. the three-point flexural strength test was performed in a universal testing machine (dl 2000; emic, são josé dos pinhais, pr, brazil) with a 5.0 kn load cell and at crosshead speed of 1.0 mm/min until failure. the specimens were placed in the sample holder, which had a span of 15 mm between the two 0.8 mm radius rounded bearers and loaded by a 1.6 mm radius rounded steel knife edges. testing was carried out in distilled water at 37 °c with the load applied at the midpoint of the samples. the flexural strength was calculated for the monolayer specimens according to equation 1. where σ is the maximum center tensile stress (mpa), f is the load at fracture (n), l is the distance of the two supports (mm), w is the width of the specimen (mm), and h is the height of the specimen (mm). for bilayer specimens, flexural strength was calculated using equation 2, where σf is the maximum center tensile stress (mpa), l is the distance of the two supports (mm), p is the load at fracture (n), et is the young modulus (according to the manufacturer) of material under tensile stress (gpa), tt is the height of the material under tensile stress (mm), ec is the young modulus (according to the manufacturer) of material under compression (gpa), tc is the height of the material under tensile stress (mm), and w is the width of the specimen (mm). the flexural strength data for each design group were analyzed using one-way anova (α=0.05), and tukey posthoc test (α=0.05) was used to identify differences among the cooling methods. results the mean values (mpa), standard deviations, and coefficients of variance for three-point flexural strength are presented in table 2. the results of one-way anova for bilayer specimens 3y-tzp/vm9 with porcelain on lower surface (pb) were statistically significant (table 3). tukey post-hoc test showed the highest flexural strength for fast cooling and the lowest for slow cooling; the normal cooling was not different from fast and slow cooling. the results of the one-way anova for pm (table 4), zm (table 5), and zb (table 6) were not statistically significant (α>0.05). group cooling average s d covar p m slow 74.4 9.0 12.1% normal 75.4 7.7 10.2% fast 77.9 11.1 14.2% z m slow 835.5 129.9 15.5% normal 873.0 140.1 16.1% fast 817.1 95.2 11.6% pb slow 49.3 10.1 20.5% normal 56.1 11.0 19.6% fast 67.5 16.7 24.8% z b slow 572.5 136.5 23.8% normal 590.6 117.4 19.9% fast 691.3 177.8 25.7% table 2.table 2.table 2.table 2.table 2. mean (mpa) of flexural strength, standard deviation, and coefficient of variance. discussion the null hypothesis was partially rejected. in monolayer specimens, the cooling method did not change the flexural strength. the lack of effect of the cooling rate for zirconia monolayer specimens was possibly because the temperature of simulating the feldspathic porcelain sintering was below the temperature required to induce phase transformation in the used y-tzp. however, for porcelain monolayer specimens, could be expected thermal material tempering on fast cooling method due to very high heat transfer between the material and the environment7,19-20 or microstructural changes on vitreous matrix on slow cooling21-22. moreover, the pm group, as monolayer specimens, did not have any effect of thermal expansion coefficient mismatch on the framework material and consequent residual tension. for bilayer specimens, in zb groups, the cooling method was not able to affect the flexural strength because zirconia was on the lower surface and it was more directly under tension and might be responsible for the whole sample strength23-24. in pb groups, the cooling method affected the flexural strength. this could be attributed to residual tension. at temperatures above the glass transition temperature (tg – around 600 °c) stresses are relieved by plastic deformation since the porcelain behaves as a viscoelastic liquid and allows the rearrangement of the atoms within the structure. when the temperature declines to the glass transition region, atomic displacement is more difficult to occur. thus, the viscous liquid porcelain gets denser with the atoms in closer packing. at temperatures below the tg, porcelain is solid and structural rearrangements are impossible. at this moment, residual stress develops from the potential discrepancy in volume, density and viscosity between layers of porcelain that are below (external) and above (internal) the glass transition phase. this process could be affected by the cooling rate, thickness, thermal conductivity, and the mismatch in coefficient of thermal expansion of both the porcelain and zirconia core7-11. in the fast cooling rate, the external regions cool faster strength of 3y-tzp and feldspathic porcelain subjected to different cooling methods eq 1 eq 2 127 braz j oral sci. 13(2):124-128 sum of squares df mean square f-ratio significance inter-group 24,400.0 2 12,200.0 0.8025 0.541 intra-group 638,000.0 42 15,200.0 total 662,400.0 44 table 4.table 4.table 4.table 4.table 4. one-way anova results of flexural strength values of zirconia monolayer design sum of squares df mean square f-ratio significance inter-group 123,000.0 2 61,500.0 2.8802 0.066 intra-group 897,000.0 42 21,300.0 total 1020,000.0 44 table 5.table 5.table 5.table 5.table 5. one-way anova results of flexural strength values of zirconia bilayer design sum of squares df mean square f-ratio significance inter-group 2,530.0 2 1,270.0 7.5813 0.002 intra-group 7,010.0 42 167.0 total 9,540.0 44 table 6.table 6.table 6.table 6.table 6. one-way anova results of flexural strength values of porcelain bilayer design sum of squares df mean square f-ratio significance inter-group 97.58 2 48.792 0.5574 0.582 intra-group 3680.00 42 87.527 total 3777.58 44 table 3.table 3.table 3.table 3.table 3. one-way anova results of flexural strength values of porcelain monolayer design and the temperature gradients through the porcelain increase, concentrating stresses near the surface. thus, stress development increases with higher porcelain thickness, faster cooling rate, and lower thermal conductivity10. moreover, the mismatch between thermal expansion coefficient and thermal gradients inevitably makes the layered structures subject to a high residual stress when cooled from a furnace temperature11. the highest flexural strength observed in fast cooling in the pb group might be associated to compressive forces on the surface. however, when those structures are subjected to cyclic loads such as chewing, chipping or delamination may occur. thus, the dental laboratory technician must be careful with the fast cooling method. the slow cooling method has been proposed to decrease the mismatch of coefficients of thermal expansion and thermal diffusion. it was expected that this method would allow cooling of both materials at a more uniform rate. however, annealing of the porcelain occurs when the restoration is cooling slowly. this reduces substantially the possibility of surface compressive force formation, which is believed to strengthen the restoration. however, this speculation was not confirmed in this study. in fact, the slow cooling method could be harmful since it adds more heat to the restoration and, thus, increases the potential for induction of thermal strains and possible zirconia phase transformation 25. nevertheless, the interaction of zirconia and porcelain during veneering requires more investigation, at several principles of thermodynamics. it is important to observe that flexural strength with bar samples is a simplified method for predicting clinical performance of these materials. however, fixed partial dentures may show a different behavior than bars due to their complex geometry. additionally, in the oral environment these materials are susceptible to different chemical and physical fatigues that were not reproduced in this study. then, different cooling methods affected only the flexural strength of bilayer specimens with porcelain in lower surface. the complex residual thermal stresses generated in bilayer specimens could 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strength of 3y-tzp and feldspathic porcelain subjected to different cooling methods oral sciences n3 original article braz j oral sci. october | december 2014 volume 13, number 4 long-term bond strength, degree of conversion and resistance to degradation of a hema-free model adhesive fabrício mezzomo collares1, vicente castelo branco leitune1, fernando freitas portella1, fabrício aulo ogliari2, susana maria werner samuel1 1universidade federal do rio grande do sul – ufrs, school of dentistry, dental materials laboratory, porto alegre, rs, brazil 2universidade federal de pelotas – ufpel, materials engineering school, pelotas, rs, brazil correspondence to: fabrício mezzomo collares faculdade de odontologia laboratório de materiais dentários universidade federal do rio grande do sul rua ramiro barcelos 2492 cep: 90035-003 porto alegre, rs, brasil phone: +55 51 33085198 e-mail: fabricio.collare@ufrgs.br abstract aim: to evaluate the long-term bond strength, degree of conversion and resistance to degradation in ethanol of hema-containing and hema-free model adhesive resins of a three-step etch-andrinse adhesive system. methods: the superficial dentin of 16 bovine incisor teeth was exposed, and the teeth were divided in two groups according to the hema concentration in the experimental adhesive (0% and 15%). in each tooth were made 6 cylindrical composite restorations. half of the tooth restorations were submitted to microshear bond strength test after 24 h and the other half after 6 months. degree of conversion of experimental resins was determined by fourier transform infrared spectroscopy. crosslink density was indirectly determined by the knoop hardness of five specimens per group before and after immersion in ethanol for 6 h. results: the group with 0% hema showed no difference in bond strength as compared to the group with 15% hema after 24 h or 6 months. there was no difference in degree of conversion and crosslink density between groups. conclusions: hema content of the adhesive resin did not influence the bond strength to dentin, degree of conversion or resistance to degradation in ethanol. keywords: dental bonding; dentin-bonding agents; light-curing of dental adhesives. introduction the longevity of dental restorations is an important clinical concern1-2. efficient adhesive resin infiltration and polymerization at the tooth/resin interface are related to the preservation of the results of clinical procedures3. improvement of the adhesive systems has been associated with the development of different system formulations, as the incorporation of resin monomers with hydrophilic groups increases the bond strength4. almost all commercial etch-and-rinse adhesive systems include 2hydroxyethyl methacrylate (hema) or other hydrophilic monomer in their composition5. this hydrophilic monomer is required to enhance infiltration of hydrophobic components into demineralized dentin to promote micromechanical retention of curable monomers. however, the presence of hydrophilic components in the hybrid layer could promote water penetration and degradation of the polymer over time6-8 whereas hema increases permeability of the adhesive layer, taking up water and decreasing the mechanical properties of hybrid layer. the influence of hema on mechanical properties of polymer structure may be attributed to the received for publication: august 14, 2014 accepted: november 25, 2014 braz j oral sci. 13(4):261-265 group composition %wt 0% hema bisphenol a glycol dimethacrylate 42 ethoxylated bisphenol a glycol dimethacrylate 6 42 triethylene glycol dimethacrylate 16 2-hydroxyethyl methacrylate 0 15% hema bisphenol a glycol dimethacrylate 36.5 ethoxylated bisphenol a glycol dimethacrylate 6 36.5 triethylene glycol dimethacrylate 12 2-hydroxyethyl methacrylate 15 table 1.table 1.table 1.table 1.table 1. composition of the adhesive resins. low degree of conversion exhibited by polymers containing increased concentration of hema9. it is known that a low degree of conversion is related to a low crosslink density10 and decreased mechanical properties of the formed polymer. the bond strength to tooth substrate is directly related to the mechanical properties of the adhesive layer11. the effect of hema on adhesive resin properties has already been examined in a previous study and showed that an increased ratio of hema decreases the degree of conversion and ultimate tensile strength, and increases the water sorption and solubility of polymer9. however, more studies are needed to evaluate other properties and the longevity of the adhesive/dentin bond of adhesive resins with or without hema, since the presence of a hydrophilic monomer in the adhesive layer could influence the bond strength over time. hence, the present study tested the null hypothesis that the addition of 15% hema in a model adhesive resin will not influence the microshear bond strength, degree of conversion and resistance to chemical degradation. material and methods materials the monomers used were bisphenol a glycol dimethacrylate (bisgma), ethoxylated bisphenol a glycol dimethacrylate 6 (bisema), triethylene glycol dimethacrylate (tegdma) and 2-hydroxyethyl methacrylate (hema). two blends with different ratios of hema were prepared, one with 0% and another with 15% in weight (table 1). for each group, 1% mol of camphoroquinone (cq, esstech, essington, pa, usa), used as photosensitizer and 1% mol of n,n-dimethyl-para-toluidine (dmpt, fluka, everett, wa, usa) used as a reducing agent were added to transform the mixtures into light polymerizing blends. the photoactivation for all tests was initiated by a light-emitting diode light source (radii, sdi, bayswater, victoria, australia), and the irradiance value was confirmed with a digital power meter (ophir optronics, north andover, ma, usa) with 1200mw/cm2. microshear bond strength sixteen bovine maxillary incisor teeth, stored in 4 °c distilled water for no more than 3 months, were used in this study. the teeth were embedded in acrylic resin and the labial enamel was ground down to expose the superficial dentin. the dentin was ground with 600-grit sic paper for 30 s in running water12. the teeth were divided in two groups according to the hema presence in the model adhesive resins (0% and 15%). the dentin was conditioned for 15 s with 37% phosphoric acid gel and washed for the same time. the water was gently removed with an absorbent paper. a commercial primer composed by water (40-50%), hema (3545%) and polyalkenoic acid (10-20%) (primer scotch bond multi purpose, 3m espe, st. paul, mn, usa) was agitated using disposable applicators on the dentin surface for 10 s and dried for 10 s with an air stream at a distance of 10 cm. then the model adhesive resins was applied for 5 s using disposable microbrush tips and polymerized for 20 s. in each tooth, 6 cylindrical composite restorations (z250, 3m espe) were made using metallic cylindrical moulds 2 mm high, resulting restorations with 0.88 (± 0.03) mm2 of adhesive area13. restorations were polymerized for 40 s, and the teeth were stored in 37 °c distilled water. three of these restorations in each tooth were randomly submitted to a microshear bond strength test after 24 h and the other three after 6 months of storage. the specimens were mounted in a universal testing machine (dl-2000, emic, são josé dos campos, sp, brazil), and shear force was applied at a 1 mm/ min cross-head speed using a steel wire (± 0.4 mm). the wire was positioned in the bond line, and the cylinder was pulled. the bond strengths were expressed in mpa. the failure mode of each specimen was determined under a stereomicroscope at 60x magnification and designated as adhesive, mixed or cohesive failure in either adhesive or resin composite. the means and standard deviations of the groups were analyzed for statistically significant differences by two-way anova for microshear bond strength evaluation. to compare the pattern of failure between groups, the kruskal-wallis test was used. statistical significance was defined as p<0.05. degree of conversion degree of conversion of the experimental adhesives was evaluated using fourier transform infrared spectroscopy (ftir) with a shimadzu prestige 21 (shimadzu, kyoto, japan) spectrometer equipped with an attenuated total reflectance device with a horizontal znse crystal and a 45° mirror angle (pike technologies, madison, wi, usa). a support was coupled to the spectrometer to fix the light curing unit and standardize the distance between the fiber tip and sample at 5 mm. irsolution software in monitoring scan mode was used, with happ-genzel appodization in a range of 1750 to 1550 cm-1 and resolution of 8 cm-1. analysis was performed at a controlled room temperature of 23±1 °c and 60±1% relative humidity after sample (3 µl) polymerization, which was directly dispensed onto the znse crystal and lightactivated for 20 s., the test was repeated three times (n=3). the degree of conversion was calculated as described in a previous study14, considering the intensity of carbon-carbon double bond stretching vibration (peak height) at 1635 cm1 and using the symmetric ring stretching at 1610 cm-1 from the polymerized and unpolymerized samples as an internal standard. the means of the degree of conversion of the groups 262262262262262long-term bond strength, degree of conversion and resistance to degradation of a hema-free model adhesive braz j oral sci. 13(4):261-265 were compared using the t-test, with p<0.05 indicating statistical significance. softening in ethanol to determine the resistance to degradation, the experimental adhesives were placed in circular elastomeric molds with 4 mm diameter and 2 mm deep, covered with polyester strips and photoactivated for 20 s. five specimens (n=5) were prepared for each experimental adhesive and then embedded in a acrylic resin with the top in contact with a glass plate and polished in a polisher (model 3v; arotec, cotia, sp, brazil) with a felt disc embedded with alumina suspension (alumina 1.0 µm, arotec) after the specimens were stored at 37 °c for 24 h. the specimens were subjected to a microhardness test in which 9 indentations (15 g/10 s), 100 µm apart from each other and were assessed using a digital microhardness tester (hmv 2, shimadzu, tokyo, japan). the initial knoop hardness number (khn1) was registered, and then the specimens were subjected to softening in absolute alcohol for 6 h at 37 °c, when the hardness test was repeated, and the post-conditioning hardness value was measured (khn2)15. the hardness values between groups were compared by t-test, and the values before and after ethanol immersion were compared by paired t-test, being statistically different if p<0.05. results microshear bond strength values of experimental adhesive with 0 and 15% of hema (0% hema and 15% hema, respectively) to bovine dentin showed no difference between groups (table 2). the 0% hema group showed no statistical difference to the 15% hema group at 24 h or 6 months (p>0.05). the fracture mode of almost all specimens was classified as mixed for all groups (figure 1) and presented no significant difference in failure pattern when correlated with presence of hema and storage times. the data for degree of conversion and softening in ethanol are shown in table 3. for the degree of conversion in 20 s, no significant difference between groups was detected (p=0.262). initial microhardness evaluation showed no statistical difference among 0% hema and 15% hema adhesive resins (p=0.211). after 6 h in absolute ethanol, the 15% hema resin showed no statistical difference in knoop microhardness to 0% hema adhesive resin (p=0.346). however, 15% hema and 0% hema adhesive resins showed groups 24 h 6 months 0% hema 13.77 (± 3.90) aa 12.83 (± 5.02)aa 15% hema 14.02 (± 4.13) aa 13.82 (± 3.55)aa table 2.table 2.table 2.table 2.table 2. mean and standard deviation, in mpa, of microshear bond strength of hema and hema-free adhesive resins in 24 h and 6 months. same capital letter indicates no statistically significant difference in same column (p>0.05). same small letter indicates no statistically significant difference in same row (p>0.05). group dc (%) khn1 khn2 0% hema 49.78 (± 0.73)* 16.02 (± 1.15) aa 6.98 (± 0.71)bb 15% hema 47.16 (± 3.40)* 17.18 (± 1.54) aa 7.56 (± 1.09)bb table 3.table 3.table 3.table 3.table 3. mean and standard deviation of degree of conversion (%), initial and final knoop microhardness value (khn1 and khn2, respectively) of hema and hema-free adhesive resins same symbol (*) indicates no statistical difference in the same column (p=0.262). same capital letter indicates no statistically significant difference in the same column (p>0.05). different small letter indicates no statistically significant difference in the same row (p<0.05). fig. 1. failure pattern, in percentage, of debonded specimens of hema and hema-free groups at 24 h and 6 months. a statistical reduction in knoop microhardness values after 6 h in absolute ethanol immersion (p<0.001). discussion immediate and long-term bond strength at the adhesive/ resin interface influences the efficiency of the resin bond to dentin. almost all commercial dental adhesive systems contain hema in their composition in order to improve wetting of the dentin substrate, promote hydrophobic monomer infiltration and enhance bond strength. this study evaluated the long-term microshear bond strength of experimental adhesive resins with different ratios of hema to bovine dentin and showed no statistical difference between groups despite the presence of hema or the storage time. despite the non-significant difference shown for degree of conversion, the knoop microhardness of both adhesive resins decreased after immersion in ethanol. there is morphological evidence that hydrophilic adhesive systems behave as semi-permeable membranes16-17. porous regions in the bonded interface with water-rich and hydrophilic monomer zones could lead to channels for water sorption and leaching of unpolymerized monomers, thus promoting hydrolytic degradation of the polymer. long-term long-term bond strength, degree of conversion and resistance to degradation of a hema-free model adhesive braz j oral sci. 13(4):261-265 263263263263263 hybrid layer degradation is explained by the degradation of polymer matrix8 and/or collagen fibrils18 by hydrolysis due to water penetration from the dentin and oral environment through porosities and intermolecular spaces of the polymer network interface with dentin substrate, decreasing the mechanical properties of the polymer formed. despite the increase in the percentage of hydrophilic and low molecular weight monomer of the 15% hema group, the same adhesive resin compositions showed no difference in ultimate tensile strength between each other, 85.4 and 81.1 mpa for 0% hema and 15% hema respectively, in a previous study9. however, the water sorption and solubility of 15% hema adhesive resin presented significantly higher values than 0% hema9. nevertheless, in the present study, the bond strength showed no difference between the two groups even in a long-term bond strength test. this could be explained by the low viscosity of hema, which increases the penetration of adhesive resin into the demineralized dentin of the 15% hema group, thus increasing the proportion of hydrophobic monomers in the hybrid layer. a previous study shows that the microshear bond strength of adhesive resins to bovine dentin did not differ from human dentin19. the same pattern was confirmed in an evaluation using microtensile bond strength test20. moreover, the scanning electronic microscopy images reveal that bovine and human dentin present similar dentinal morphology after phosphoric acid etching20. hema monomer hydrophilicity contributes to promote bonding to tooth substrate4. due to its low molecular weight and size, hema may easily penetrate demineralized dentin tissue4,21, thus promoting hybrid layer formation. however, increased hybrid layer hydrophilicity could lead to bond interface that is more prone to degradation. in a previous study21, a transmission electronic microscopy evaluation showed the same pattern of spot and cluster-like nanoleakage for a hemafree and a hema-containing adhesive systems, whereas the hema-free adhesives present lower immediate dentin bond strength than the hema-containing adhesives. despite this difference in initial bond strength, a long-term evaluation is still required to confirm the effects of hema-containing adhesives’ hydrophilicity on the preservation of bonded interface. in this study, no difference between bond strength of 0 and 15% hema adhesive resins were found neither immediately nor after 6 months of water storage. the failure pattern of specimens was almost all mixed for both groups. the microshear bond strength test revealed a nonhomogeneous stress concentration at the dentin substrate22 which could explain these results. however, no significant difference was observed in the failure pattern when correlated with presence of hema and storage times. an explanation for these results may be the presence of bisema in the composition of the adhesive resins. the bisema molecule is similar to bisgma, with a phenyl central core without the two hydroxyl groups in the backbone, which decreases the viscosity of the comonomer blend23. a decreased viscosity could lead to a higher interpenetration of monomer into the demineralized dentin, proxying the hema function in the adhesive resin. both adhesive resins evaluated in this study presented a similar degree of conversion. the 0% hema showed no difference on softening in ethanol when compared with 15% hema. resistance to degradation after immersion in ethanol is affected by the crosslink density of polymers24. networks with high crosslink density have reduced solvent uptake due to reduced free space between the polymer chains. therefore, it is expected that organic solvents would cause less softening in these polymers8. in polymers with a low crosslink density, alcohol can form strong secondary bonds with the polymer chains, penetrate and replace the interchain secondary bonds, and dissolve the material, causing the softening25. the polymerization behaviors could be affected by increased hema content, reducing the degree of conversion, due perhaps to lower monomer reactivity. the 0% hema adhesive resin’s low polymerization ratio in the initial polymerization seconds9 may result from the high bisgma content (42% wt). bisgma has a stiff central core with a hydroxyl group in the backbone that hinders monomer diffusion through the solidifying adhesive and reduces the mobility of unreacted pendant double bonds9. it is known that a high ratio of monofunctional/bifunctional monomers may result in a polymer with low crosslink density, due to its less reactive double bonds26, but this fact was not observed in this study. the crosslink density, indirectly assessed by the softening in ethanol, of the 15% hema adhesive resin and of the adhesive resin without monofunctional components showed no significant difference. nonetheless, the addition of a iodonium salt (e.g. diphenyliodonium hexafluorphosphate) as an alternative photoinitiator could improve the reactivity of methacrylate monomers 27, enhancing the degree of conversion27 and improving dentinbond strength28, and thus offsetting the drawbacks of a high viscosity blend, as the 0% hema. an in vitro study29 showed similar results for bond strength of hema-free experimental adhesive systems compared to a commercial three-step etchand-rinse adhesive system. additionally, a clinical study showed a high retention rate of non-carious class v restorations after 5 years in function that did not differ from a three-step etch-and-rinse adhesive system containing hema30. the commercial primer (adper scotch bond multi purpose, 3m espe) used in restorative procedures presents 35-45 wt% of hema in its composition, which provide an adequate diffusion of monomers on etched dentin and help to explain the lack of difference in longitudinal microshear bond strength between 0 and 15% hema adhesive resins verified in this study. the polymerized hema that remained entrapped on the adhesive layer of both tested groups could also make less sensible the detection of long-term bond strength changes promoted by hema addition to bonding resin. even with the significantly higher water sorption rate and solubility of the 15% hema adhesive resin used9, a significant degradation of bond strength was not observed. however, the storage period used could be not long enough for a noticeable degradation to occur and differences in bond strength of the tested adhesive resins to be perceived. within the limitations of this study, the content of long-term bond strength, degree of conversion and resistance to degradation of a hema-free model adhesive braz j oral sci. 13(4):261-265 264264264264264 hema in the adhesive resin showed no influence on the degradation of bond strength to dentin in the three-step etchand-rinse adhesive system use din this study. it neither influenced the degree of conversion and resistance to degradation of adhesive resin. acknowledgements the author f.f.p. gratefully acknowledges capes for the scholarship. references 1. demarco ff, corrêa mb, cenci ms, moraes rr, opdam nj. longevity of posterior composite restorations: not only a matter of materials. dent mater. 2012; 28: 87-101. 2. pallesen u, van dijken jw, halken j, hallonsten al, höigaard r. longevity of posterior resin composite restorations in permanent teeth in public dental health service: a prospective 8 years follow up. j dent. 2013; 41: 297-306. 3. peumans m, kanumilli p, de munck j, van landuyt k, lambrechts p, van meerbeek b. clinical effectiveness of contemporary adhesives: a systematic review of current clinical trials. dent mater. 2005; 21: 864-81. 4. pashley dh, zhang y, agee ka, rouse cj, carvalho rm, russell cm. permeability of demineralized dentin to hema. dent mater. 2000; 16: 7-14. 5. van landuyt kl, snauwaert j, de munck j, peumans m, yoshida y, poitevin a et al. systematic review of the chemical composition of contemporary dental adhesives. biomaterials. 2007; 28: 3757-85. 6. de munck j, van landuyt k, peumans m, poitevin a, lambrechts p, braem m, et al. a critical review of the durability of adhesion to tooth tissue: methods and results. j dent res. 2005; 84: 118-32. 7. de munck j, van meerbeek b, yoshida y, inoue s, vargas m, suzuki k, et al. four-year water degradation of total-etch adhesives bonded to dentin. j dent res. 2003; 82: 136-40. 8. ferracane jl. hygroscopic and hydrolytic effects in dental polymer networks. dent mater. 2006; 22: 211-22. 9. collares collares fm, ogliari fa, zanchi ch, petzhold cl, piva e, samuel sm. influence of 2-hydroxyethyl methacrylate concentration on polymer network of adhesive resin. j adhes dent. 2011; 13: 125-9. 10. barszczewska-rybarek i, gibas m, kurkok m. evaluation of the network parameter in aliphatic poly (urethane dimethacrylate)s by dynamic thermal analysis. polymer. 2000; 41: 3129-35. 11. bae jh, cho bh, kim js, kim ms, lee ib, son hh, et al. adhesive layer properties as a determinant of dentin bond strength. j biomed mater res b: appl biomater. 2005; 74: 822-8. 12. koliniotou-koumpia e, kouros p, koumpia e, helvatzoglou-antoniades m. shear bond strength of a “solvent-free” adhesive versus contemporary adhesive systems. braz j oral sci. 2014; 13: 64-9. 13. leitune vc, portella ff, bohn pv, collares fm, samuel sm. influence of chlorhexidine application on longitudinal adhesive bond strength in deciduous teeth. braz oral res. 2011; 25: 388-92. 14. collares fm, portella ff, leitune vc, samuel sm. discrepancies in degree of conversion measurements by ftir. braz oral res. 2014; 28: 9-15. 15. leitune vc, collares fm, trommer rm, andrioli dg, bergmann cp, samuel sm. the addition of nanostructured hydroxyapatite to an experimental adhesive resin. j dent. 2013; 41: 321-7. 16. tay fr, frankenberger r, krejci i, bouillaguet s, pashley dh, carvalho rm, et al. single-bottle adhesives behave as permeable membranes after polymerization. in vivo evidence. j dent. 2004; 32: 611-21. 17. tay fr, pashley dh, yoshiyama m. two modes of nanoleakage expression in single-step adhesives. j dent res. 2002; 81: 472-6. 18. hebling j, pashley dh, tjaderhane l, tay fr. chlorhexidine arrests subclinical degradation of dentin hybrid layers in vivo. j dent res. 2005; 84: 741-6. 19. schilke r, bauss o, lisson ja, schuckar m, geurtsen w. bovine dentin as a substitute for human dentin in shear bond strength measurements. am j dent. 1999; 12: 92-6. 20. reis af, giannini m, kavaguchi a, soares cj, line sr. comparison of microtensile bond strength to enamel and dentin of human, bovine, and porcine teeth. j adhes dent. 2004; 6: 117-21. 21. mine a, de munck j, van landuyt kl, poitevin a, kuboki t, yoshida y, et al. bonding effectiveness and interfacial characterization of a hema/ tegdma-free three-step etch&rinse adhesive. j dent. 2008; 36: 767-73. 22. placido e, meira jb, lima rg, muench a, de souza rm, ballester ry. shear versus micro-shear bond strength test: a finite element stress analysis. dent mater. 2007; 23: 1086-92. 23. ogliari fa, ely c, zanchi ch, fortes cb, samuel sm, demarco ff, et al. influence of chain extender length of aromatic dimethacrylates on polymer network development. dent mater. 2008; 24: 165-71. 24. schneider lf, moraes rr, cavalcante lm, sinhoreti ma, correr-sobrinho l,consani s. cross-link density evaluation through softening tests: effect of ethanol concentration. dent mater. 2008; 24: 199-203. 25. soh ms, yap au. influence of curing modes on crosslink density in polymer structures. j dent. 2004; 32: 321-6. 26. rueggeberg f, tamareselvy k. resin cure determination by polymerization shrinkage. dent mater. 1995; 11: 265-8. 27. gonçalves ls, moraes rr, ogliari fa, boaro l, braga rr, consani s. improved polymerization efficiency of methacrylate-based cements containing an iodonium salt. dent mater. 2013; 29: 1251-5. 28. leal fb, lima gs, collares fm, samuel sm, petzhold cl, piva e, et al.iodonium salt improves the dentin bonding performance in an experimental dental adhesive resin. int j adhesion and adhesives. 2012; 38: 1-4. 29. zanchi ch, münchow ea, ogliari fa, chersoni s, prati c, demarco ff et al. development of experimental hema-free three-step adhesive system. j dent. 2010; 38: 503-8. 30. van landuyt kl, de munck j, ermis rb, peumans m, van meerbeek b. five-year clinical performance of a hema-free one-step self-etch adhesive in noncarious cervical lesions. clin oral investig. 2014; 18: 1045-52. 265265265265265 long-term bond strength, degree of conversion and resistance to degradation of a hema-free model adhesive braz j oral sci. 13(4):261-265 oral sciences n3 original article braz j oral sci. october | december 2013 volume 12, number 4 bulk and surface properties related to composite filler size kátia gerhardt1, andrea soares quirino da silva1,2, guilherme rego3, mário alexandre coelho sinhoreti4, vinicius esteves salgado5, luis felipe j. schneider1,3 1department of oral rehabilitation, dental materials research lab, dental school, veiga de almeida university, rio de janeiro, rj, brazil 2department of restorative dentistry, dental school, rio de janeiro federal university, rio de janeiro, rj, brazil 3department of restorative dentistry, dental school, federal fluminense university, niterói, rj, brazil 4area of dental materials, piracicaba dental school, university of campinas, piracicaba, sp, brazil 5area of dental materials, dental school, pelotas federal university, pelotas, rs, brazil correspondence to: luis felipe jochims schneider universidade veiga de almeida rua ibituruna 108, casa 3, cep: 20271-020 rio de janeiro, rj, brasil phone: +55 21 81020039 e-mail: felipefop@gmail.com abstract there are few studies that fully characterize the effect of the filler size on the surface, optical and mechanical properties of resin composites. aim: to determine the influence of the filler size content on surface, optical and mechanical properties before and after accelerated aging. methods: seven resin composites were investigated: filtek supreme® (3m/espe), estelite σ quick® (tokuyama), evolu-x® (dentsply), esthet-x hd® (dentsply), opallis® (fgm), herculite xrv ultra® (kerr) and filtek z250® (3m/espe). elastic modulus (em) and flexural strength (fs) were determined by three-point bending of specimens immersed or not in absolute ethanol for seven days. roughness (ra) and gloss (g.u.) analyses were determined before and after ethanol storage for seven days and cielab color change (∆e*) was obtained with a spectrophotometer. results were analyzed by two-way anova (general linear model) for em and fs, with repeated measures for roughness and gloss, and one-way anova for ∆e*. all comparisons were performed by tukey’s test (5%). pearsons’ correlation test was performed to determine the correlation between em, fs, roughness, gloss, and ∆e* with the mean filler sizes. results: mechanical properties decreased for all materials when submitted to immersion in ethanol. increasing filler size gradually increased em, fs and roughness values; gloss values decreased and didn’t significantly affects ∆e*. nanofiller composites presented higher gloss values than the others. conclusions: it was possible to determine that the average filler size is a good predictor just for fs and gloss retention. keywords: nanocomposites, resin cements, surface properties. introduction for years the increasing demand for restorative materials to mimic the dental structures has lead to the search for materials with improved physical properties. the resin composites combine adequate esthetics, acceptable lifetime and clinical performance with reasonable cost. these materials consist basically of an organic matrix and inorganic fillers, surrounded by a coupling agent. since their introduction, almost fifty years ago, studies have been conducted in order to characterize the best composition of filler content1,2. the shape, amount and size of the fillers employed in commercial formulations led to the establishment of different classifications of resin composite materials since 19833,4. reported data and technological improvements addressed the broad received for publication: august 25, 2013 accepted: december 03, 2013 braz j oral sci. 12(4):323-329 material filtek supreme estelite σ quick sigma herculite xrv ultra opallis filtek z250 esthet x hd evolu-x composition bis-gma, udma, tegdma, pegdma, bis-ema. zro2/sio2 bis-gma, tegdma, zro2-sio2 bis-gma tegdma, al-b-si glass,sio2 bis-gma, bis-ema,udma, tegdma. ba-al-sio2 bis-gma, udma, bis-ema. zro2-sio2 cluster bis-gma, bis-ema, tegdma, ba-f-al-b-si glass, sio2 bis-gma, bis-ema,tegdma, ba-al si glass, ba-al-f-b-si glass nanofiller filler average size cluster 0.6 to 1.4 ¼m particules of 5 to 20 nm) – zr si 0.2 ¼m 0.47 ¼m 0.5 ¼m 0.6 ¼m 0.6-0.8 ¼m 0.6 – 0.,8 ¼m10 – 20 nm filler fraction (% vol) 55.6 71 59 58 60 60 58 manufacturer 3m espe, usa tokuyama dental co, japan kerr, usa fgm, brazil 3m espe, usa dentsply, brazil dentsply, brazil table 1:table 1:table 1:table 1:table 1: restorative materials used in the current study. all the technical information was obtained from the manufacturers. range of formulation, properties, characteristics and esthetic possibilities for each type of resin composite. initially, it was proposed a classification according to the average filler size as: macrofill composites (range 10-50 mm), that were difficult to polish and retain surface smoothness and microfill composites (range 40-50 nm), that exhibit inferior mechanical properties when compared to conventional composites, which are described by ferracane4 as those composites with filler sizes that exceeded 1 µm. associating the benefits of macrofill and microfill composites, hybrid resins composites are used universally for most anterior and posterior applications. these composites are classified as traditional hybrid composites (ranging from 10-50 mm to 40 nm), small particle hybrid minifill (0.6-1mm to 40 nm), small particle hybrid midfill (1-10 mm to 40 nm) 4-6. more recently nanofill-based composites (ranging from 5 to 100 nm) were launched on the market5. the hybrid composites, due to the incorporation of different filler sizes, provide excellent mechanical properties 4. the introduction of nano-scale fillers in composites might produce adequate esthetic, required for cosmetic applications, and adequate mechanical properties for posterior applications7. they combine the good mechanical strength of the hybrid composites and the polish of the microfills composites. the reduced size of the particles may improve the optical properties of composites, such as color stability, retention of the gloss and diminished wear rate5,8. although filler particles play an important role in the final physical properties, there are few studies that systematically characterize the effect of the fillers on the mechanical, surface and optical properties of resin composites. therefore, the aim of this study was to assess the influence of filler size content of seven commercial resin composites on the mechanical, surface and optical properties, before and after aging. material and methods seven commercial light-cured restorative resin composites (a2 shade), with different filler sizes were analyzed: filtek supreme® (3m espes, st paul, mn, usa), estelite σ quick® (tokuyama dental, tokyo, japan), evolux® (dentsply, catanduva, são paulo, brazil), esthet-x hd® (dentsply, catanduva, são paulo, brazil), opallis® (fgm, joinville, santa catarina, brazil), herculite xrv ultra® (keer corporation, oragende, ca, usa) and filtek z250® (3m espes, st paul, mn, usa). information provided by the manufacturers is listed in table 1. for photoactivation was employed a light-emitting-diode based unit (ultrablue plus, dmc, são carlos, sp, brazil) with light radiance of 600 mw/ cm². the light radiance, calculated by dmc, was confirmed with a hand-held radiometer (demetron led radiometer, kerr corporation, orange, ca, usa). mechanical properties analyses twenty bar-shaped specimens of each composite were made using a stainless steel mold (10 mm long x 2 mm wide x 1 mm thick, adapted from iso 4049). the composite was uniquely positioned in the mold, filling the space, with cellulose acetate strips on both upper and lower sides in order to give the specimens a smooth surface. the curing time was 40 s on both sides. ten specimens of each composite were dry stored and the other ten in absolute ethanol (biocloro indústria e comércio, campinas, sp, brazil) immersion for seven days. before mechanical analysis, all specimens immersed in absolute ethanol were washed and dried. three-point flexural test was conducted with a universal testing machine (instron 4411, canton, ma usa) at a crosshead speed of 0.5 mm/min, until its total collapse or failure. the software was supplied with individual data of each specimen that were previously measured with digital caliper (digimess instrumentos de precisão, são paulo, sp, brasil). elastic modulus “e” (gpa) and flexural strength “ó” (mpa) were measured according to iso 4049 and were calculated according to the following formulas: where fmax was the fracture force (n), … the support distance (mm), b the specimen thickness (mm), h height and f (mm) the extension during loading. surface properties analyses five disk-shaped specimens of each composite were made in a stainless steel mold (8 mm diameter x 2 mm thickness). after the materials were inserted in the mold, mylar bulk and surface properties related to composite filler size324 braz j oral sci. 12(4):323-329 strips were placed below and above the orifice of the mold to obtain a smooth surface on both sides. the material was light cured for 40 s in both sides. the initial measures were obtained 24 h after curing. then, the specimens were stored in absolute ethanol for 7 days, washed, dried and submitted to analyses of surface properties again. a surface profiler (surfcorder se 1700; kosakalab, tokyo, japan) to evaluate the roughness was used. the diamond stylus travelled at a constant speed of 1 mm/s across the surface with a force of 6 mn. six line scans were performed on the top surface of each specimen, three in horizontal and three in perpendicular directions9. the cut-off length was 0.25 mm and the measuring length 2 mm. the average of all six readings per specimen was used. the amplitude parameter ra, which relates the arithmetic mean between the peaks and valleys of the surface, was used. the gloss was measured with a glossmeter (zgm 1110; zehntner gmbh testing instruments, gewerbestrasse, switzerland), which was calibrated against a black glass standard provided by the manufacturer. measurements were performed at 60º light incidence and reflection angles relative to the vertical axis10. an average of four readings (in the center of top surface) for each sample was calculated. the measuring window employed was 4.7 mm x 2 mm. color measurement color was measured according to the cie l*a*b* parameters in the reflectance mode, with the sci mode11 over zero calibrating box (cie l* = 0.0, cie a* = 0.0, and cie b* = 0.0) and white background (cie l* = 93.2, cie a* = -0.3, and cie b* = 1.6), using a spectrophotometer with illuminating/measuring geometry d/8° (cm-2600d: konica minolta, tokyo, japan). the used measuring aperture size was 6 mm of diameter. illuminating and viewing configurations complied with cie 10º observer geometry and d65 illuminant. an average of three readings (in the center of top surface) for each sample was calculated. cie l*, a*, and b* values were automatically calculated by the machine. the cie l* axis is the brightness, where 100 is white and 0 is black. the axes cie a* and cie b* are the red-green and yellow-blue chromatic coordinates. a positive cie a* or cie b* value represents a red or a yellow shade, respectively. the initial measures were obtained 24 h after curing. then, the specimens were stored in absolute ethanol for seven days, washed, dried and submitted again to color measurement. the cielab color difference (∆e*) was calculated from the average of l*a*b* values of each specimen by using the formula: ∆e = [(∆l) 2 + (∆a) 2 + (∆b) 2] 1/2 where ∆l*, ∆a* and ∆b* are the mathematical differences between cie l*, cie a* and cie b* obtained after 48 h in absolute ethanol with the initial measures. statistical analyses all data was subjected to analyses of variance, general linear model for elastic modulus (em) and flexural strength (fs), analyses of variance with repeated measures for roughness and gloss, and one-way analysis of variance for ∆e*, followed by tukey’s post-hoc test performed at a preset α of 0.0512. pearson’s correlation tests were performed to analyze the relationship between average filler size and the studied properties. it is important to mention that for supreme was considered a value of 0.001 mm for the average filler size in order to consider the nanometric scale. results figure 1 shows the dry and ethanol storage values of elastic modulus (em) for all resin composites evaluated in this study. concerning dry storage, there were no statistical differences among the tested groups. in comparison with dry storage, all materials tended to present em loss in ethanol storage, resulting from the softened structure. however, tukey’s pairwise comparisons did not verify specific differences among the tested materials. the lowest loss percentage was observed in filtek supreme (p<0.05), as shown in figure 2. flexural strength (fs) values are presented in figure 3. evolu-x presented the highest value of fs in dry storage specimens (p<0.05) while estelite σ quick presented the lowest (p<0.05). with the exception of z250, all resin composites presented lower fs values after ethanol storage and presented different fs percentage loss. as can be seen in figure 4, xrv-ultra exhibited the highest fs loss (p<0.05) followed by opallis and estelite σ quick, and filtek z250 the lowest (p<0.05). figure 5 presents the roughness of all resin composites before and after immersion in absolute ethanol for 7 days. different behaviors were observed between restorative materials. the only resin composite that presented significant difference after ethanol storage was estelite σ quick (p<0.05), which exhibited a potential roughness increase. gloss values are displayed in figure 6. before ethanol storage, esthet-x presented the highest gloss (p<0.05) followed by evolux, while estelite σ quick and filtek z250 presented the lowest values (p<0.05). after ethanol storage, different behaviors of the groups were observed. estelite σ quick composites presented an increase of gloss values after ethanol immersion, while opallis, filtek z250, esthet-x and evolux presented significant decrease of their values. supreme and xrv-ultra maintained constant values. figure 7 shows the cielab color difference (∆e*) values. no significant difference was observed among the groups, despite the xrv-ultra exhibiting the highest numerical value, while esthet-x and evolux, the lowest ones. the pearson’s correlation tests demonstrated that there was a direct and significant relationship between average filler size and flexural strength (p=0.049). according to the average filler size, the elastic modulus, the flexural strength and the roughness analyses exhibited increased values as the filler size increased. in contrast, reduced filler size may lead to higher maintenance of gloss and better color stability, as shown in figure 8, but without a significant relationship. bulk and surface properties related to composite filler size 325 braz j oral sci. 12(4):323-329 fig. 1. mean values and standard deviation of elastic modulus. different capital letters indicate statistically significant differences considering readings performed after ethanol storage. no statistically significant differences were observed when considering readings taken before ethanol. fig. 2. percentage of elastic modulus decrease of materials exposed to ethanol storage. fig. 3: mean values and standard deviation of flexural strength. different lowercase letters indicate statistically significant differences considering readings performed before ethanol storage. different capital letters indicate statistically significant differences considering readings performed after ethanol storage. horizontal line indicates statistically significant difference when considering readings performed before and after ethanol storage for that material. fig. 4: percentage of flexural strength decrease of materials exposed to ethanol storage. fig. 5: mean values and standard deviation of roughness before and after ethanol storage.different lowercase letters indicate significant differences considering readings performed before ethanol storage. horizontal line indicates statistically significant difference when considering readings performed before and after ethanol storage for that material. no statistical differences were observed when considering readings taken after ethanol. fig. 6: mean values and standard deviation of gloss before and after ethanol storage. different lowercase letters indicate significant differences considering readings performed before ethanol storage. different capital letters indicate significant differences considering readings performed after ethanol storage. horizontal line indicates statistically significant difference when considering readings performed before and after ethanol storage for that material. bulk and surface properties related to composite filler size326 braz j oral sci. 12(4):323-329 fig. 7: mean values and standard deviation of cielab color differences. fig. 8: correlations between elastic modulus, flexural strength, roughness, gloss and color stability with average filler size. elastic modulus, flexural strength, roughness and gloss were considered before ethanol storage. discussion the success of a composite restoration involves many factors and requirements such as color, roughness, polishing and optical properties. many of these factors are associated with the morphological and physical characteristics of the inorganic particles, but there is lack of studies that systematically evaluate such properties before and after the degradation process. the resin composite’s degradation is a process that involves several factors such wear and staining13, absorption of liquids14, polymerization15, finishing and polishing16. all resin composites used in this study are based on bis-gma and, according to previous studies17 it is known that ethanol is a suitable choice of solvent to simulate the aging of dental composites. when a composite is placed in a great amount of ethanol, the monomer is released in less time than if it were placed in water. the ethanol molecules are retained in the monomeric matrix, increasing the distance between the polymer chains, resulting in a soft matrix18 and for that reason it was employed in this study. due to the higher amount of [-oh] present in ethanol, there is a higher absorption by the polar portion of the matrix, causing swelling of the material. this dimensional change in the matrix causes stress at the matrix-silane-filler particle interfaces, resulting in degradation of this bond. in consequence, inorganic particles detach from the surface, causing an increase in roughness and consequently reduction in the gloss of the specimens. the present investigation demonstrated that there was no correlation between the filler size and the elastic modulus (em). on the other hand, flexural strength (fs) was shown to be directly dependent on the average filler size. it is important to add that there is no valid information about the amount of small, medium or large particles given by manufacturers. regarding the information provided in figure 1, there is a trend of em values to increase according to the average bulk and surface properties related to composite filler size 327 braz j oral sci. 12(4):323-329 filler size, which is likely no to occur with filtek supreme composite. the lowest em percentage loss is highlighting the composite with reduced polymerization shrinkage19, due to its high content of small particles, and is well known as a true nano-based material, since its particles are made from agglomerates of nanometric fillers. therefore, for the correlation tests was considered a value of 0.001 mm for the average filler size in order to consider the nanometric scale. if it were considered the value of 0.6 mm (stated by the manufacturer as the average filler size), a positive relationship would be established. the similar performance in em between filtek z250 and opallis composites may be related to the average filler size (0.6 µm and 0.5 µm respectively) and volume weight (60% and 58% respectively). on the basis of the above argumentation, the characteristics of nanohybrid composites, which involve nanoparticles and high filler fractions (e.g. estelite quick 71% vol) did not improve the elastic modulus neither the flexural strength. the standard specimens according to iso 4049 for flexural strength and elastic modulus would be 10 mm long x 2 mm wide x 2 mm thick. the different specimen dimensions used in this study had been previously used20, where no influence was observed in the final result. in the present study, the herculite xrv ultra and opallis microhybrid composites were the most affected after immersion in ethanol considering fs, while the filtek z250 microhybrid composite was the least affected. it may be considered that the shape and composition of the microhybrid composites with large and irregular particles are the main reason for the lower resistance21. differences in gloss between a restoration and the surrounding enamel are clinically relevant, as the human eye can easily detect differences in gloss even if their color is matched22. higher gloss that indicates smooth and highluster surface23 reduces the effect of a color difference, since the color of reflected light is predominant rather than the color of underlying composite material 24. the angle of illumination affects the amount of reflected light. according to silikas et al.10, 60º light incidence is considered more reliable from a clinical perspective, since it is closer to the perception of tooth gloss by an observer. considering the gloss values after ethanol storage, it is clear that the material’s gloss was inversely related to the filler particles’ average sizes. after ethanol storage, the resin matrix of the surface is removed, exposing the surface fillers and showing that the smaller the filler size, the higher the gloss. previous studies used commercially available composites to study the influence of the filler size on physical properties. janus et al.25 studied the surface roughness and morphology of a hybrid composite (tetric ceram, ivoclar) and three nanocomposites (one “pure” nanofilled composite, filtek supreme/ 3m espe; and two “nano-hybrid” composites, grandio/voco; synergy d6/coltène) and verified a positive correlation between the average filler size and the surface roughness. on the opposite, berger et al.26 investigated the influence of filler size and finishing systems on the surface roughness and staining of three commercial dental composites (“nanofilled” filtek supreme/3mespe; “minifill” esthet-x dentsply, and “microfill” renamel microfill/cosmedent) and concluded that a smaller filler size does not necessarily result in a low surface roughness and staining susceptibility. in the same way, gönülol and yilmaz27 evaluated the effects of different finishing and polishing techniques on the surface roughness and color stability of commercial composites (two “nanohybrid” composites, grandio/voco and aelite aesthetic/bisco, two “nanofillied” composites, filtek supreme xt/3mespe dentin and translucent, and a “microhybrid” composite, filtek z250/3m/espe) and concluded that composites with smaller filler size did not necessarily show lower roughness and discoloration than the others and, furthermore, the authors stated that staining of composite resins was dependent on monomer structure and surface irregularities. as it can be seen, studies that use commercially available composites have found conflicting results and this may be expected since such materials do not differ only in the filler content. it is suggested that color characteristics can be assigned to the size and number of particles in a composite and its relationship with the resinous matrix. it might be considered that color change could be directly related to the load particle size. composites with larger particles would be more susceptible to water sorption and color alteration28. on the other hand due to its nanometric particles, the nanofiller composite would demonstrate the slightest color change29 even when submitted to the effects of artificial discoloration by saliva, juice, coffee, cola soft drinks and wine 30. in addition to the type of filler (glass, silica, etc.), the resin matrix can also influence the stability of color, because the more hydrophilic the material, the more opaque the color; in a hydrophobic matrix, water is less absorbed, so there is less color change12. dental composites with lower concentration of load particles present higher values of color change 31. the cielab color difference represents the change in color after a designated period, and was defined as the distance between two points in space, relative to the initial and final color. with respect to the results of the current study, microhybrid composite herculite xrv showed the highest value of color change and the highest filler average size (0.47 µm); on the contrary, nanohybrid composites esthet-x (filler average: 0.7 µm) and evolu-x (filler average: 0.7 µm with nanofillers) showed the highest color stability. considering that both composites have the same manufacturer, their compositions are similar and so is the behavior. the lack of statistically significant differences might be related to the fact that only intrinsic discoloration was considered. further studies using extrinsic pigments are currently being developed. in conclusion, the present investigation demonstrated that it was not possible to establish a viable correlation between bulk and surface properties with the filler classification as provided by manufacturers. indeed, it was possible to determine that the filler average size is a good bulk and surface properties related to composite filler size328 braz j oral sci. 12(4):323-329 predictor for fs and gloss retention ability, as demonstrated by the correlation tests. although it was not possible to differentiate the microhybrid, nanohybrid and “pure-nano” behaviors with respect to the studied properties in the current research, it could be observed that fs and gloss are determined by the inorganic filler average size. references 1. chen mh. update on dental nanocomposites. j dent res. 2010; 89: 549-60. 2. kim kh, ong jl, okuno o. the effect of filler loading and morphology on the mechanical properties of contemporary composites. j prosthet dent. 2002; 87: 642–9. 3. lutz f, philips rw. a classification and evaluation of composite resin systems. j prosthet dent. 1983; 50: 480–8. 4. ferracane jl. resin composite state of the art. dent mater. 2011; 27: 29-38. 5. mitra sb, wu d, holmes bn. an application of nanotechnology in advanced dental materials. j am dent assoc. 2003; 134: 1382-90. 6. rastelli an, jacomassi dp, faloni ap, queiroz tp, rojas ss, bernardi mi et al. the filler content of the dental composite resins and their 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2013; 41: 385–392. 13. bagheri r, burrow mf, tyas m. influence of food-simulating solutions and surface finish on susceptibility to staining of aesthetic restorative materials. j dent. 2005; 33: 389-98. 14. buchalla w, attin t, hilgers r, hellwig e. the effect of water storage and light exposure on the color and translucency of a hybrid and a microfilled composite. j prosthet dent. 2002; 87: 264-70. 15. sakaguchi rl. a review of the curing mechanics of composites and their significance in dental applications. compend contin educ dent. 1999; 20: 16-23. 16. sirin karaarslan e, bulbul m, yildiz e, secilmis a, sari f, usumez a. effects of different polishing methods on color stability of resin composites after accelerated aging. dent mater j. 2013; 32: 58-67. 17. schneider lf, moraes rr, cavalcante lm, sinhoreti ma, correr-sobrinho l, consani s. cross-link density evaluation through softening tests: effect of etanol concentration. dent mater. 2008; 24: 199-203. 18. asmussen e. softening of bisgma-based polymers by ethanol and by organic acids of plaque. scandinavian j dent res. 1984; 92: 257-61. 19. gonçalves f, kawano y, braga rr. contraction stress related to composite inorganic content. dent mater. 2010; 26: 704-9. 20. muench a, correa ic, grande rhm, joão m. the effect of specimen dimensions on the flexural strength of a composite resin. j appl oral sci. 2005; 13: 265-8. 21. ilie n, hichel r. investigations on mechanical behavior of dental composites. clin oral investig. 2009; 13: 427-38. 22. ardu s, braut v, uhac i, benbachir n, feilzer aj, krejci i. influence of mechanical and chemical degradation on surface gloss of resin composite materials. am j dent. 2009; 22: 264-8. 23. kakaboura a, fragouli m, rahiotis c, silikas n. evaluation of surface characteristics of dental composites using profilometry, scanning electron, atomic force microscopy and gloss-meter j mater sci. 2007; 18: 155-163. 24. o’brien wj, johnston wm, fanian f. the surface roughness and gloss of composites. j dent res. 1984; 63: 685-8. 25. janus j, fauxpoint g, arntz y, pelletier h, etienne o. surface roughness and morphology of three nanocomposites after two different polishing treatments by a multitechnique approach. dent mater. 2010; 26: 416-25. 26. berger sb, palialol ar, cavalli v, giannini m. surface roughness and staining susceptibility of composite resins after finishing and polishing. j esthet restor dent. 2011; 23: 34-43. 27. gönülol n, yilmaz f. the effects of finishing and polishing techniques on surface roughness and color stability of nanocomposites. j dent. 2012; 40: e64-70. 28. pinto gd, dias kc, cruvinel dr, garcia lr, consani s, pires-desouza fp. influence of finishing/polishing on color stability and surface roughness of composites submitted to accelerated artificial aging. indian j dent res. 2013; 24: 363-8. 29. lee yk. influence of scattering/absorption characteristics on the color of resin composites. dent mater. 2007; 23: 124-31. 30. topcu ft, sahinkesen g, yamanel k, erdemir u, oktay ea, ersahan s. influence of different drinks on the colour stability of dental resin composites. eur j dent. 2009; 3: 50-6. 31. schulze ka, marshall sj, gansky sa, marshall gw. color stability and hardness in dental composites after accelerated aging. dental mater. 2003; 19: 612-9. bulk and surface properties related to composite filler size 329 braz j oral sci. 12(4):323-329 oral sciences n3 braz j oral sci. 12(2):138-142 original article braz j oral sci. april | june 2013 volume 12, number 2 effectiveness of different obturation techniques in surpassing the ledge formed in simulated curved canals marilisa carneiro leão gabardo1, wander josé da silva2, letícia machado gonçalves2, marili doro andrade deonízio3 1area of collective health, school of health and biosciences, pontifical catholic university of paraná, curitiba, pr, brazil 2department of prosthodontics and periodontology, piracicaba dental school, university of campinas, piracicaba, sp, brazil 3area of endodontics, department of dentistry, federal university of paraná, curitiba, pr, brazil correspondence to: marilisa carneiro leão gabardo rua professor joão argemiro loyola, 452, cep: 80240-530, seminário, curitiba, pr, brasil phone: +55 41 32422304 e-mail: marilisagabardo@e-odonto.com received for publication: april 10, 2013 accepted: june 25, 2013 abstract aim: to compare the effectiveness of different obturation techniques in surpassing the ledge formed in simulated curved root canals. methods: eighty acrylic-resin blocks with curved canals were instrumented with gates-glidden drills to simulate a ledge formation. then, a k-file #10 was used for trying to surpass the deviation, and the blocks that permitted surpassing were rejected. the remaining blocks were divided into 4 groups according to the obturation technique: lateral condensation, tagger’s hybrid technique, thermafil and system b. the blocks had their images digitalized using a scanner before and after the obturation procedures. the images were analyzed with image tool 3.0 software. statistical analysis was performed by one-way anova at a significant level of 5%. results: the system b resulted in the highest obturated area (p<0.001). there were no significant differences between the tagger’s hybrid technique and thermafil system regarding the effectiveness in surpassing ledge (p>0.05). the lateral condensation resulted in the worst ability in filling the ledge space (p<0.001). conclusions: within the limitations of this study, it was possible to conclude that techniques using heat condensation or gutta-percha thermoplastification were more effective in surpassing the ledge formed in curved canals than the lateral condensation. keywords: endodontics, root canal therapy, root canal obturation, root canal filling materials. introduction the major objective during an endodontic therapy is the cleaning, shaping and also filling the root canal properly1. however, the root canal system has a very complex anatomy that could difficult all steps of the treatment2. during the root canal instrumentation, especially in curved canals, the maintenance of the original trajectory without causing deformations is a challenge3. among the possible complications, an error characterized by a deviation that not reaches the periodontal ligament, is termed ledge formation or ledging. it is possible to detect when the file no longer achieves de curvature and brakes on a “deadlock”1,4-6. actually, there is a wide range of iatrogenic errors associated with ledge formation, but the inappropriate application of the technique prevails4. of these, 139139139139139 braz j oral sci. 12(2):138-142 there are two major causes for the creation of ledged canals: inadequate extension of the access opening to allow straight access to the apical part of the root canal5, and using a noncurved stainless steel instrument that is too large for a curved canal7. in addition to the presence of root canal curvature, other contributory factors involved in ledge formation include tooth type, canal location, working length (wl), master apical file size, and clinician’s level of experience in endodontics6. once a root canal is ledged, the endodontic treatment becomes difficult to complete. considering that the wl was not reached, the formation of a deviation implies an incomplete shaping and disinfection of root canal and, consequently, the obturation will also be compromised, which might lead to treatment failure4,8-10. there are several techniques available to assist the negotiation and bypassing of a ledged canal, usually involving prebending the tip of files and/or using ultrasonic tips4,6. thus, an alternative procedure in cases that the ledge was not surpassed and the wl was not recovered is the apical extrusion of the filling material with thermoplasticized techniques11. considering the high incidence of ledge formation in endodontic clinical practice5,12-14, and the limited information about apical extrusion of filling material as an alternative treatment in these cases, this study aimed to compare the effectiveness of different obturation techniques in surpassing the ledge formed in simulated curved root canals. material and methods preparation of simulated canals with ledge a single operator performed all technical procedures for standardization purposes. eighty transparent acrylic-resin blocks simulating root canals (p-oclusal, são paulo, brazil) with 20º curvature degree, 17 mm length and diameter and taper equivalent to a flexofile #15 (dentsply maillefer, ballaigues, switzerland) were used. the ledge in the simulated canals was prepared with gates-glidden drills (dentsply maillefer) as follows: number 1 and 2 at 15 mm, and number 3 at 13 mm (figure 1a). the procedures were performed under irrigation with 1% naocl. then, a k-file #10 (dentsply maillefer) was used for trying to surpass the deviation by slightly prebending the file, with a slight rotation motion combined with a picking motion to help advance the instrument4. the blocks that allowed this passage were rejected (figure 1b). the remaining blocks (n=64) were dried with paper points #60 (tanariman indústria ltda., manacapuru, am, brazil) and covered with adhesive tape during the new trial to surpass the accident produced. this procedure was done to avoid seeing the file manipulation. then, the blocks were divided into 4 groups containing 16 blocks each, according to the obturation technique: lateral condensation, tagger’s hybrid technique, thermafil (tulsa dental, tulsa, ok, usa) and system b (sybronendo, west collins, ca, usa). ah-plus sealer (dentsply maillefer) was used in all groups for obturation. fig. 1. (a) simulated root canal with ledge in an acrylic-resin block. (b) k-file #10 surpassing the ledge formed in the canal. the sealer was prepared according with the manufacturer’s instructions. lateral condensation in this technique, a well-fitting master gutta-percha cone #60 (dentsply maillefer) coated with sealer was taken up to the wl of 15 mm. the master cone was left seated and the lateral condensation was performed using a finger spreader b (dentsply maillefer). this spreader was inserted with the sealer towards the canal walls and then counter-clock wisely removed to create room for the insertion of the accessory gutta-percha cones (dentsply maillefer) medium-fine(mf) (dentsply maillefer). this procedure was repeated until the insertion of new accessory cones was not possible. after the obturation completion, the excess of filling material was removed and cold vertical condensation was performed (figure 2a). tagger’s hybrid technique in this technique, the master cone was seated as described above. an initial lateral condensation was performed with the finger spreader b, and one accessory gutta-percha cone was inserted. next, a mcspadden compactor #70 (dentsply maillefer) was coupled to a low-speed contra-angle handpiece and introduced passively into the root canal. the penetration of the compactor inside root canal was obtained effectiveness of different obturation techniques in surpassing the ledge formed in simulated curved canals braz j oral sci. 12(2):138-142 140140140140140 fig. 2. acrylic-resin blocks obturated with (a) lateral condensation, (b) tagger’s hybrid technique, (c) thermafil and (d) system b. with the aid of a rubber marker 1 mm short of the wl. with the mcspadden compactor inside the root canal, next to the gutta-percha cones, it was driven by forward-backward movements until reaching wl, and then left at that point for about 1 s. the compactor was removed from the root canal with the motor still operating maintaining gentle pressure on one side of the canal wall. next, vertical condensation of the plasticized gutta-percha was performed with heat paiva’s condensers to obtain a better adaptation of the filling material (figure 2b). thermafil initially, the appropriated size of gutta-percha plastic obturator was selected considering the diameter in the acrylicresin blocks. the size selected was #45. then, the prepared sealer was coated all over the root canal walls with the aid of a paper point #60. the plastic obturator was heated in the thermaprep plus oven (tulsa dental, tulsa, oklahoma, usa) and inserted into the canal at the wl of 15 mm. the time required for obturation was selected according to the obturator size. then, the external portion of the plastic obturator was removed with a round bur at high speed (figure 2c). system b in this technique, the plugger f was selected (sybronendo, west collins, california, usa). a gutta-percha cone #60 (dentsply maillefer) was coated in sealer and then inserted into the canal. the plugger f was heated to approximately 200ºc and introduced up to the site of deviation, being maintained there for approximately 10 s. next, the equipment was turned off and the plugger was kept in position with apical pressure. the equipment was turned on for just 1 s to remove the plugger. the backfilling was done placing a fine-medium accessory gutta-percha cone (dentsply maillefer) coated with sealer into the empty backfill space. the system-b heat source, with the same plugger used before, was heated to 100°c and activated during about 0.5 s. the plugger was firmly pushed and then held for 5 s with apical pressure maintained. the system-b plugger was rotated to break it loose. next, with light back pressure, the plugger was rotated again to separate it from the condensed backfill cone. a second backfill cone was placed with sealer into the remaining backfill space left by the system-b plugger. so, the temperature was back up to 200°c and seared the cone off at the orifice level. finally, the material in excess was also removed and condensed (figure 2d). digitalization and analysis of the images the acrylic-resin blocks had their images digitalized using a scanner (genius hr7x, china) before and after the obturation procedure. the images analysis was performed using the image tool 3.0 software (uthscsa, san antonio, tx, usa). the area observed before the obturation was measured in mm2 and denominated as initial area (ia). after performing the obturation technique the blocks were digitalized again and a new measurement was done. in this case, the material that was able to surpass the deviation was denominated as the final area (fa). the obturated area (oa) was calculated using the following equation: oa = ia – fa (figure 3). data analysis the data were analyzed using the sas/lab package (sas software, version 9.0; sas institute inc., cary, nc, usa) with the significant level set at 5%. differences between the obturation techniques were evaluated by one-way anova. fig. 3. illustration of the initial area (ia) and final area (fa). effectiveness of different obturation techniques in surpassing the ledge formed in simulated curved canals braz j oral sci. 12(2):138-142 results the mean of the obturated areas obtained by the different techniques is shown in figure 4. according to the results, the obturation using system b resulted in the highest obturated area (p<0.001). there were no significant differences between the tagger’s hybrid and thermafil techniques regarding the efficacy in surpassing ledge (p>0.05). the lateral condensation resulted in the worst ability in filling the ledge space (p<0.001). it was not possible to identify the difference between gutta-percha and sealer when extrusion of the filling material occurred through the original trajectory. fig. 4. obturated areas according to the obturation techniques evaluated in the study. different letters indicate statistically significant differences among the techniques. discussion the maintenance of the original trajectory during the instrumentation of curved root canals remains as a challenge in endodontic clinical practice 3,6. actually, during instrumentation, accidents such as the ledge formation, have been described in the literature 5,12, especially when the curvature of the root canal is accentuated13. prevention of these iatrogenic procedures is improved by training and experience, mainly with the application of techniques of cervical preflaring6,15-16. however, in case of its occurrence, handling must be done with an initial negotiation and bypassing the ledge and a combination of file’s movements. if such movements fail, an area will not be reached and disinfected, representing a potential cause of poor prognosis4. thus, the present study aimed to compare the effectiveness of different obturation techniques in surpassing ledge formed in simulated curved root canals, as an alternative to minimize unfavorable endodontic treatment outcomes. despite the limitation of in vitro studies, acrylic-resin blocks have been widely used in studies involving canal shaping and filling17-19, being confirmed as a reproducible model20. in the present study, acrylic-resin blocks were used and having a single operator ensured standardization of all procedures. also, a pilot-study confirmed that a deviation located at 15 mm was the length that permitted less surpassing the ledge. several techniques have been developed to achieve an adequate three-dimensional obturation of the instrumented root canal1 and the gutta-percha-filled area tends to decrease at the apical level21. the most common technique is the lateral condensation of gutta-percha in combination with a sealer2224. however, in the present study, the lateral condensation showed the worst ability in surpassing the ledge formed in simulated curved canals. it is possible that the cold condensation of gutta-percha difficult the penetration of the filling material, especially nearby apical region25-26. also, as the finger spreader penetrates until de ledge, it was not possible to introduce accessory cones in the ledge space23. in addition to the classical cold lateral condensation, techniques have been introduced utilizing heat condensation of gutta-percha or thermoplasticized material in the canal. according to our results, the tagger’s hybrid technique, a heat condensation technique, and the thermafil, a thermoplasticized one, resulted is similar obturated area. the tagger’s hybrid technique is known to be a technique able to produce a great overextension of filling material in curved and straight canals27. however, it seems that this technique is limited in surpassing ledges. actually, techniques in which thermoplastification is used, have a superior potential to extrude the material beyond the accident24,27-28. also, a systematic review and meta-analysis confirmed that the root canal obturation by warm gutta-percha demonstrated a higher rate of overextension29. however, in our study, the thermafil was less effective than the system b. the thermafil system involves a heated alpha-phase guttapercha using a plastic carrier30. this system is claimed to be fast, easier and applicable in curved and narrow canals, the same characteristics proposed by the continuous wave of obturation technique, the system b31. despite the advantages, under the tested conditions, it is possible that the plastic carrier used in thermafil stopped at the level of the deviation, which did not allowed great material extrusion. on the other hand, system b resulted in the highest obturated area, which could be explained by the depth capability of plugger penetration32. furthermore, our results corroborate with those of previous investigations, indicating a direct correlation with the apical extrusion of the filling material33-34. thus, this technique should be considered as a useful alternative in cases in which the return to the canal trajectory is not possible. overall, the heat condensation and thermoplasticized obturation were superior to the lateral condensation in surpassing the ledge formed in simulated curved canals. in these cases, the thermoplasticized obturation technique should be indicated for it higher ability of gutta-percha apical penetration. however, it is important to highlight that the tagger’s hybrid technique has advantages such as being fast and not requiring expensive devices, which could encourages 141141141141141effectiveness of different obturation techniques in surpassing the ledge formed in simulated curved canals braz j oral sci. 12(2):138-142 142142142142142 its use by general dentists and endodontic specialists. within the limitations of this study, it was possible to conclude that techniques using thermoplasticized gutta-percha were more effective in surpassing ledge formed in curved canals than the lateral condensation. references 1. schafer e, nelius b, burklein s. a comparative evaluation of gutta-percha filled areas in curved root canals obturated with different techniques. clin oral investig. 2012; 16: 225-30. 2. goldberg f, artaza lp, de silvio a. effectiveness of different obturation techniques in the filling of simulated lateral canals. j endod. 2001; 27: 362-4. 3. burklein s, benten s, schafer e. shaping ability of different single-file systems in severely curved root canals of extracted teeth. int endod j. 2013; 46: 590-7. 4. jafarzadeh h, abbott pv. ledge formation: review of a great challenge in endodontics. j endod. 2007; 33: 1155-62. 5. kapalas a, lambrianidis t. factors associated with root canal ledging during instrumentation. endod dent traumatol. 2000; 16: 229-31. 6. terauchi y. correction of ledged canals with ultrasonic tips. dent today. 2008; 27: 140: 2-5. 7. wilcox lr, roskelley c, sutton t. the relationship of root canal enlargement to finger-spreader induced vertical root fracture. j endod. 1997; 23: 533-4. 8. vire de. failure of endodontically treated teeth: classification and evaluation. j endod. 1991; 17: 338-42. 9. peters lb, wesselink pr, moorer wr. the fate and the role of bacteria left in root dentinal tubules. int endod j. 1995; 28: 95-9. 10. de deus g, murad cf, reis cm, gurgel-filho e, coutinho filho t. analysis of the sealing ability of different obturation techniques in ovalshaped canals: a study using a bacterial leakage model. braz oral res. 2006; 20: 64-9. 11. bernardineli n. accidents and complicantions in instrumentation. in: bramante cm, berbert a, bernardineli n, moraes ig, editors. accidents and complications in endodontic treatment clinical solutions. são paulo: santos publishing; 2003: p.50-106. 12. eleftheriadis gi, lambrianidis tp. technical quality of root canal treatment and detection of iatrogenic errors in an undergraduate dental clinic. int endod j. 2005; 38: 725-34. 13. greene kj, krell kv. clinical factors associated with ledged canals in maxillary and mandibular molars. oral surg oral med oral pathol. 1990; 70: 490-7. 14. santos smc, soares ja, césar cas, brito-júnior m, moreira an, magalhães cs. radiographic quality of root canal fillings performed in a postgraduate program in endodontics. braz dent j. 2010; 21: 315-21. 15. goerig ac, michelich rj, schultz hh. instrumentation of root canals in molar using the step-down technique. j endod. 1982; 8: 550-4. 16. torabinejad m. passive step-back technique. oral surg oral med oral pathol. 1994; 77: 398-401. 17. burroughs jr, bergeron be, roberts md, hagan jl, himel vt. shaping ability of three nickel-titanium endodontic file systems in simulated sshaped root canals. j endod. 2012; 38: 1618-21. 18. goldberg m, dahan s, machtou p. centering ability and influence of experience when using waveone single-file technique in simulated canals. int j dent. 2012; 2012: 206-321. 19. hamze f, honardar k, nazarimoghadam k. comparison of two canal preparation techniques using mtwo rotary instruments. iran endod j. 2011; 6: 150-4. 20. lam tv, lewis dj, atkins dr, macfarlane rh, clarkson rm, whitehead mg, et al. changes in root canal morphology in simulated curved canals over-instrumented with a variety of stainless steel and nickel titanium files. aust dent j. 1999; 44: 12-9. 21. marciano ma, bramante cm, duarte ma, delgado rj, ordinola-zapata r, garcia rb. evaluation of single root canals filled using the lateral compaction, tagger’s hybrid, microseal and guttaflow techniques. braz dent j. 2010; 21: 411-5. 22. dummer pm. comparison of undergraduate endodontic teaching programmes in the united kingdom and in some dental schools in europe and the united states. int endod j. 1991; 24: 169-77. 23. dummer pm, kelly t, meghji a, sheikh i, vanitchai jt. an in vitro study of the quality of root fillings in teeth obturated by lateral condensation of gutta-percha or thermafil obturators. int endod j. 1993; 26: 99-105. 24. gulabivala k, holt r, long b. an in vitro comparison of thermoplasticised gutta-percha obturation techniques with cold lateral condensation. endod dent traumatol. 1998; 14: 262-9. 25. luccy ct, weller rn, kulild jc. an evaluation of the apical seal produced by lateral and warm lateral condensation techniques. j endod. 1990; 16: 170-2. 26. brosco vh, bernardineli n, moraes ig. “in vitro” evaluation of the apical sealing of root canals obturated with different techniques. j appl oral sci. 2003; 11: 181-5. 27. mann sr, mcwalter gm. evaluation of apical seal and placement control in straight and curved canals obturated by laterally condensed and thermoplasticized gutta-percha. j endod. 1987; 13: 10-7. 28. clinton k, van himel t. comparison of a warm gutta-percha obturation technique and lateral condensation. j endod. 2001; 27: 692-5. 29. peng l, ye l, tan h, zhou x. outcome of root canal obturation by warm gutta-percha versus cold lateral condensation: a meta-analysis. j endod. 2007; 33: 106-9. 30. beasley rt, williamson ae, justman bc, qian f. time required to remove guttacore, thermafil plus, and thermoplasticized gutta-percha from moderately curved root canals with protaper files. j endod. 2013; 39: 125-8. 31. buchanan ls. the continuous wave of obturation technique: ‘centered’ condensation of warm gutta-percha in 12 seconds. dent today. 1996; 15: 60-2, 64-7. 32. zhang w, suguro h, kobayashi y, tsurumachi t, ogiso b. effect of canal taper and plugger size on warm gutta-percha obturation of lateral depressions. j oral sci. 2011; 53: 219-24. 33. guess gm, edwards kr, yang ml, iqbal mk, kim s. analysis of continuous-wave obturation using a single-cone and hybrid technique. j endod. 2003; 29: 509-12. 34. jung iy, lee sb, kim es, lee cy, lee sj. effect of different temperatures and penetration depths of a system b plugger in the filling of artificially created oval canals. oral surg oral med oral pathol oral radiol endod. 2003; 96: 453-7. effectiveness of different obturation techniques in surpassing the ledge formed in simulated curved canals oral sciences n3 original article braz j oral sci. october | december 2013 volume 12, number 4 the wits appraisal among a nigerian sub-population: an assessment of dental base geometric factors ifesanya joy ucheonye1, adeyemi abigail tokunbo1, otuyemi olayinka donald2 1department of child oral health, college of medicine, university of ibadan, nigeria 2department of child dental health, college of health sciences, obafemi awolowo university, ile-ife, nigeria correspondence to: ifesanya joy u orthodontic unit, department of child oral health, college of medicine university of ibadan, nigeria, phone: +2348055623129 e-mail: joyifesanya@yahoo.co.uk abstract the wits appraisal is a common linear cephalometric analytic tool established by jacobson. it has been reported to be superior to angular cephalometric measures as it is not dependent on pericranial structures and their variations both during growth and orthodontic treatment. aim: to obtain reference values for the wits appraisal among nigerians and assess the effect of the dental base geometry on it. methods: lateral cephalometric radiographs of 100 nigerian subjects with normal occlusion were analyzed to obtain the wits appraisal, and the effect of the vertical distance from subspinale to supramentale, length of the palatal and mandibular planes, the mandibular plane angle as well as the palatal plane angle were assessed using the anova and linear regression analysis. results: the mean age was 20.69 ± 4.9 years. thirty-four (34%) were males and 66(66%) were females. mean wits value of -3.43 ± 3.24 mm was obtained based on the functional occlusal plane (fop). linear regression analysis showed that the vertical height from subspinale (a point) to supramentale (b point) caused a significant change in the wits appraisal reading in participants still undergoing active growth process (less than 16 years of age) (p=0.02). conclusions: it is advocated that the wits appraisal be used as a moderator to the anb angle among nigerians. however, as a result of dental height variations its use in growing nigerian children requires caution. keywords: wits appraisal, nigeria, dental base, cephalometry. introduction radiographic analysis is useful in diagnosis, treatment monitoring and assessment of achieved goals at the end of orthodontic treatment. it also influences the course of treatment for malocclusion especially when decisions for or against extractions are involved1. the anb angle used in steiner’s analysis2-3 has been the most commonly used and possibly the simplest radiographic parameter in sagittal skeletal base position assessments4. however, the reliability of the anb angle has been reported to be dependent on the antero-posterior position of the nasion and the subspinale5, the length of the cranial base, the inclination of the mandible in reference to the anterior cranial base, inclination of the occlusal plane, and the vertical dental height from a to b points5-8. thus, the anb may not always offer an accurate data for the antero-posterior relationships of the jaws9. numerous cephalometric parameters have been proposed as adjuncts to the anb10-14. received for publication: june 10, 2013 accepted: september 27, 2013 braz j oral sci. 12(4):275-279 the wits appraisal among a nigerian sub-population: an assessment of dental base geometric factors the wits appraisal, which is based on the occlusal plane, was established by jacobson9,15-16 in 1975. it has become one of the most popular linear cephalometric modalities used in assessing orthodontic patients. its primary importance is to serve as a source of supplementary information used in conjunction with the anb angle for assessment of sagittal skeletal base discrepancies. this is expedient in patients whose angular cephalometric (anb) value show great divergence from the clinical assessment. since the wits appraisal does not use cranial or pericranial landmarks, it should, theoretically, give a more accurate picture of any anteroposterior skeletal disharmonies existing between the maxilla and the mandible17. the wits appraisal has been reported to show no significant age changes in people with normal occlusion18 though contrary report exists19. the assessment of sagittal jaw positions using the wits appraisal is however reported to be affected by the inclination of the occlusal plane20 and the vertical distance from the a to the b point11. this study presents reference values for the wits appraisal among a group of nigerians and investigates the effect of the dental base geometry on it. material and methods ethical approval was obtained from the university of ibadan/ university college hospital ethical review board. participants with normal occlusion based on the following criteria were recruited from secondary and tertiary institutions in ibadan, nigeria (figures 1a , 1b). 1. age between 11-30years of age 2. angle’s class i molar relationship 3. overjet between 2-4 mm and overbite not exceeding half of the labial surface of the lower incisors 4. arch crowding or spacing not exceeding 2mm 5. no buccal or lingual crossbites 6. no rotations of teeth 7. a coincident midline 8. no supernumeraries or congenitally missing teeth with the exception of the third molars 9. no previous exodontia of permanent teeth non-nigerians or those of mixed origin as well as individuals with special needs and other craniofacial anomalies were excluded from the study. all participants had never had orthodontic treatment. intraoral examination was performed by the principal investigator (iju) under natural light using a sterile wooden spatula and a dental mirror. lateral cephalometric radiographs of all participants were taken(blue-x imaging s/no 2402kk0164 assago, italy) with participants’ head held in a cephalostat, looking forward with the frankfort horizontal plane parallel to the floor and the teeth in maximum intercuspation. the distance from mid-sagittal plane of each participant to the source of radiation and the film was maintained at 150 cm and 15 cm respectively. soft and hard tissue tracings were obtained manually on a 0.003 inch matte finish acetate tracing paper and a sharpened 2h pencil over a light viewing box in a dark room. the a point (subspinale: located on the deepest point on the anterior outline of the bony maxilla), b point (supramentale: located on the deepest point on the anterior outline of the bony mandible) as well as the functional occlusal plane (defined by a straight line passing through the intercuspation of the first premolar and the first molar) were determined. the wits appraisal was obtained by projecting straight lines from the a and b points respectively unto the functional occlusal plane at 90° and measuring the horizontal distance form point ao to bo (figure 2). the vertical distance from subspinale to supramentale, the length of the palatal and mandibular planes, the mandibular plane angle and the palatal plane angle were also ascertained. intraexaminer variability was assessed by retracing 20 randomly selected radiographs at a two-week interval. the correlation coefficient was used for assessment of variability of measurements. correlation value of 0.88 (p<0.01) was obtained, showing an acceptable level of agreement between both readings. data was analyzed using the statistical package for social sciences (spss), version 19 (spss, inc., chicago, il, usa). the t-test was used to assess any gender-based difference in the wits appraisal. the anova was used to assess the relationship between the wits appraisal values and the other dental base factors, while linear regression analysis was used for assessing the effect of growth phase on the wits appraisal. the level of significance was set at p<0.05. fig. 1a facial intra-oral view of a participant with class i normal occlusion fig. 1b profile intra-oral view of a participant with class i normal occlusion 276 braz j oral sci. 12(4):275-279 277the wits appraisal among a nigerian sub-population: an assessment of dental base geometric factors gender n mean± std. desviation (mm) f-test score p value male 34 -4.15 ± 3.65 2.744 0.10 female 66 -3.06 ± 2.96 table 2.table 2.table 2.table 2.table 2. gender distribution of wits appraisal. fig. 2 the wits appraisal results one hundred participants were evaluated, with mean age of 20.69±4.9 years (age range of 11-30 years), and the majority (66%) being females. though all participants had class i molar relationship and clinical type i facial profile, 48% of them were radiographically determined to be class i skeletal pattern, 35% were class ii, and 17% were class iii based on the anb. however, using the wits appraisal, 68% were class i skeletal pattern, 17% were class ii and 15% were class iii. this difference in diagnostic accuracy was statistically significant (p<0.01) as shown in table 1. linear cephalometric values for the wits appraisal using the functional occlusal plane (fop) showed a range of -12mm to 3.5mm (mean -3.43 ± 3.24 mm). gender distribution of the wits values did not show any significant variation (table 2). one-way anova showed no significant variation in the wits appraisal values with respect to the vertical distance from the subspinale to the supramentale, length of the palatal or mandibular plane as well as the mandibular or palatal plane angles (table 3). a linear regression model constructed based on age of the participants showed that with exception of the vertical dental base height (p<0.05), the skeletal base variables caused no significant change (p>0.05) in the wits appraisal values among participants still actively growing (age of 16 years and less). however, among those in whom active growth had ceased none of the dental base factors was associated with a significant change (p>0.05) in the wits appraisal value (table 4). discussion the wits appraisal obtained from this study using the functional occlusal plane as originally described reveals a more negative value than that reported by jacobson9. results similar to jacobson’s have been reported among the saudi, cappadocian, turkish and american populations 21-23. a southern chinese study had reported a similar finding to that obtained in this study24. nigerians exhibit bi-maxillary protrusion25 and this may be responsible for the difference in wits appraisal values. it has been established that angular measurements do not compensate for facial divergence hence linear measurements such as the wits appraisal offer a more reliable assessment of sagittal skeletal base discrepancy irrespective of the degree of malocclusion26. in congruence with this, the stability of the wits appraisal values in the studied population, especially among those who have passed their active growth phase, may mean that the wits appraisal in itself is a sufficient diagnostic tool among this group of people. however in actively growing patients, caution must be exercised because of the effect of the vertical dental base height: a finding corroborating a previous report19. a recent study reported no change in the wits appraisal in the 8-14year-old age group in class i subjects with clinically acceptable occlusion and subjects with class iii malocclusion 27. similarly, the anb and wits appraisal obtained from cbct images has been reported not to show any significant correlation with age. the latter study however assessed a sample with a wide age disparity28 and this wide age gap might have masked the true influence of age in the sample. this study found no significant influence of age on the wits appraisal only among participants older than 16 years. on the other hand, it has been shown that the anb and wits appraisal decreased among cleft lip, alveolus and palate variable class i class ii class iii total malocclusion malocclusion malocclusion n(%) n(%) n(%) n(%) anb angle 48 (48) 35 (35) 17 (17) 100 (100) mm° bisector plane wits appraisal 70 (70) 14 (14) 16 (16) 100 (100) bop wits appraisal 68 (68) 17 (17) 15 (15) 100 (100) fop wits appraisal 68 (68) 17 (17) 15 (15) 100 (100) table 1.table 1.table 1.table 1.table 1. classification of participants’ skeletal pattern as obtained using anb angle and wits appraisal among participants with normal occlusion. braz j oral sci. 12(4):275-279 278 the wits appraisal among a nigerian sub-population: an assessment of dental base geometric factors dental base geometric variable mean ± std. desviation (mm) f test score p-value vertical distance from a to b point 44.3 ± 0.4 1.09 0.38 n.s length of anterior cranial base 71.2 ± 0.6 0.86 0.66 n.s length of palatal plane 55.7 ± 0.4 1.35 0.16 n.s length of mandibular plane 83.6 ± 0.6 0.86 0.66 n.s mandibular plane angle 32.6 ± 0.6 1.08 0.38 n.s palatal plane angle 6.3 ± 0.3 0.74 0.80 n.s maxillomandibular plane angle 27. 2 ± 0.5 1.15 0.31 n.s table 3.table 3.table 3.table 3.table 3. relationship between wits appraisal values and dental base geometry n.s-not significant patients from serial cephalograms taken at ages 10, 15 and 18years29. a similar finding was repeated among 10-14year-old chinese children30 and our study corroborates this finding in participants aged 16 years and younger. roth’s proposal of projecting the wits measurement from a standardized point 50mm along the vertical distance ab to the occlusal plane (the wits50) as a means of eliminating the effect of the vertical dental height among growing patients19 may be applicable in growing nigerian children. an added advantage of using wits 50 is that it describes the molar relationship relative to the occlusal plane and presents a better separation among cases in the three classes of malocclusion19. the difference in diagnostic accuracy between the anb angle and the wits appraisal shows the wits appraisal as being more reliable than the anb in assessment of sagittal dental base discrepancy. this is similar to findings from other studies9,31-32 and, based on this, it is wise to use the wits appraisal as a moderator of the anb angle in the management of orthodontic patients. in conclusion, despite the obvious advantages of the wits appraisal, using it as a moderator of the anb angle among nigerians must be done with caution. this is pertinent especially in actively growing children where the effect of dental height variations may confound wits value. references 1. kannan s, goyaliya a, gupta r. comparative assessment of sagittal maxillo-mandibular jaw relationship a cephalometric study. j oral health comm dent. 2012; 6: 14-7. 2. steiner cc. cephalometrics for you and me. am j orthod. 1962; 39: 720-55. 3. oktay ha. a comparison of anb, wits , af-bf and apdi measurement. am j orthod. 1975; 99: 122-8. 4. italia s, bhatia af. palatal plane and thier comparison with “angle anb” and “wits appraisal”: a cephalometric study. j ahmedabad dental college hosp. 2011; 2: 22-6. 5. rotberg s, fried n, kane j, shapiro e. predicting the “wits” appraisal from the anb angle. am j orthod. 1980; 77: 636-42. 6. bishara se, fahl ja, peterson lc. longitudinal changes in anb angle and wits appraisal: clinical implications. am j orthod 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jacobson a. application of wits appraisal. am j orthod. 1976; 70: 179-89. 16. jacobson a. update on the wits appraisal. angle orthod. 1988; 58: 205-19. 17. jabbar a, mahmood a. correlation of overjet , anb and wits appraisal for assessment of sagittal skeletal relationship. pakistan orthod j. 2012; 4: 17-23. 18. lux cj, burden d, conradt c, komposch g. age-related changes in sagittal relationship between the maxilla and mandible. eur j orthod. 2005; 27: 568-78. 19. roth r. the “wits” appraisal its skeletal and dento-alveolar background. eur j orthod . 1982; 4: 21-8. 20. sachdeva k, singla a, mahajan v, jaj hs seth v, nanda m. comparison of different angular measurements to assess sagittal skeletal discrepancya cephalometric study. indian j dent sci. 2012; 4: 27-9. 21. al-barakati sf. the wits appraisal in a saudi population sample. saudi dent j. 2002; 14: 89-92. 22. ramoðlu sý, yaðci a, uysal t. wits appraisal in cappadocian turkish population. j health sci. 2009; 18: 111-7. 23. davis gs, cannon jl, messersmith ml. determining the sagittal relationship between the maxilla and the mandible/ : a cephalometric analysis to clear up the confusion. j tennessee dent assoc. 2013; 22-30. 24. so lly, dvis jp, king nm. wits appraisal in southern chinese children. angle orthod. 1990; 60: 43-8. age <16 years age >16 years variable p value confidence interval p value confidence interval mandibular plane angle 0.69 -2.49 1.71 0.188 -0.143 0.715 palatal plane angle 0.51 -1.41 2.71 0.640 -0.601 0.372 length palatal plane 0.47 -0.31 0.62 0.319 -0.122 0.368 length mandibular plane 0.20 -0.56 0.13 0.677 -0.120 0.183 vertical distance a to b point 0.02* -1.27 -0.14 0.314 -0.328 0.107 maxillomandibular plane angle 0.63 -1.68 2.69 0.711 -0.479 0.320 table 4.table 4.table 4.table 4.table 4. influence of growth on the relation of wits appraisal and dental base geometry * -significant braz j oral sci. 12(4):275-279 the wits appraisal among a nigerian sub-population: an assessment of dental base geometric factors 25. isiekwe mc, sowemimo goa. cephalometric findings in a normal nigerian population sample and adults with unrepaired clefts. cleft palate journal. 1984; 21: 323-8. 26. kapoor dn, chandna a, shalini t. linear assessment of anteroposterior jaw relationships. j indian soc pedo prev dent. 2004; 22: 187-92. 27. chen f, terada k, wu l, saito i. longitudinal evaluation of the intermaxillary relationship in class iii malocclusions. angle orthod. 2006; 76: 955-61. 28. zamora n, cibrián r, gandia j, paredes v. study between anb angle and wits appraisal in cone beam computed tomography(cbct ). med oral patol oral cir bucal. 2013; 18: e725-32. 29. lisson ja, heib n, von moeller s spitzer wj, ludwig b. treatment result at 10, 15 and 18 years of age in patients with complete bilateral cleft lip and palate: an intercentre comparison. cleft palate craniofac j. 2013; 50: 19-24. 30. wu jyc, hägg u, wong rwk, mcgrath c. comprehensive cephalometric analyses of 10 to 14 year old southern chinese. open anthropol j. 2010; 3: 85-95. 31. zhou l, mok c-w, hägg u, mcgrath c, bendeus m, wu j. anteroposterior dental arch and jaw-base relationships in a population sample. angle orthod. 2008; 78: 1023-9. 32. shendre s, karan gk, ravinarayana rpr, mamtha t. correlation of the anteroposterior relationships of the dental arch and jaw-base in subjects with class i, class ii and class iii malocclusions. int j contemporary dent. 2011; 2: 68-73. 279 braz j oral sci. 12(4):275-279 oral sciences n3 original article braz j oral sci. october | december 2014 volume 13, number 4 analysis of anatomical landmarks of the mandibular interforaminal region using cbct in a brazilian population paloma rodrigues genú1, ricardo josé de holanda vasconcellos2, bruna paloma de oliveira3, bruna caroline gonçalves de vasconcelos3, nádia cristina da cruz delgado3 1universidade federal de pernambuco – ufpe, faculty of dentistry, department of social medicine, recife, pe, brazil 2universidade de pernambuco upe, faculty of dentistry of pernambuco, department of oral medicine, camaragibe, pe, brazil 3universidade federal de pernambuco – ufpe, faculty of dentistry, department of prosthetics and oral and facial surgery, recife, pe, brazil corrrespondence to: bruna paloma de oliveira universidade federal de pernambuco departamento de prótese e cirurgia buco-facial pós-graduação em odontologia av. prof. moraes rego, 1235 cidade universitária cep: 50670901, recife, pe, brasil phone: +55 81 92853170 e-mail: bruna_paloma@msn.com abstract aim: to evaluate the position, presence, appearance and extent of various anatomical landmarks in the mandibular interforaminal region of brazilian patients using cone-beam computed tomography (cbct). methods: a total of 142 cbct examinations were analyzed to determine the most common location of the mental foramen (mf), the presence and extent of the anterior loop (al) of the inferior alveolar nerve, and the appearance and length of the incisive canal (ic). the presence of sexual dimorphism and differences with relation to the left and right sides were also evaluated. results: most of the mf (45.5%) was located below the second premolar. the al and the ic were observed in 18.9 and 96.5% of the images respectively. the average length of al and ic was 3.14±1.25 mm and 13.68±5.94 mm respectively. no significant differences (p>0.05) between genders or left and right sides were observed for all evaluated parameters. conclusions: the most common location of the mf, the high rate of visualization of the ic and the occasional presence of al in the studied brazilian population demonstrate the importance of using threedimensional images of the mandibular anterior region, allowing proper surgical planning and preventing injury to the neurovascular bundle. keywords: mandible; cone-beam computed tomography; surgery, oral; mandibular nerve. introduction one of the most frequent accidental complications that may occur during surgical procedures in the mandibular interforaminal region is a neurosensory disturbance in the chin and lower lip. this complication occurs when important structures such as the mental foramen (mf), the anterior loop (al) of the inferior alveolar nerve and the incisive canal (ic) are not properly identified and protected1. during its path, the inferior alveolar nerve runs through the mandibular canal and it is divided into two segments near the mf: the mental, which emerges in this foramen innervating the mental region and the lower lip; and the incisive, which continues intraosseous and runs through the ic and innervates the anterior teeth. however, in some individuals, the terminal portion of the inferior alveolar nerve may extend, passing below the inferior border of the mf and after giving off the incisive nerve branches, the main branch curves back to the mf, which emerges as the mental nerve. this section of the nerve in front of the mf can be braz j oral sci. 13(4):303-307 received for publication: september 03, 2014 accepted: december 16, 2014 304304304304304 described as the al of the inferior alveolar nerve 2. additionally, the mf is presented as a structure that may have different anatomical variations in terms of size, shape and location3. the use of appropriate imaging techniques is therefore essential to enable the accurate identification and location of these vital structures, avoiding potential injuries when surgical procedures are performed in the mandibular interforaminal region, including insertion of dental implants, mentoplasty and rehabilitation after trauma4. cone-beam computed tomography (cbct) is a relatively new imaging modality that provides a detailed evaluation of important bony structures5. this technique has high resolution, a relatively low radiation dose and produces images that demonstrate on different planes the real size of anatomical structures of the interforaminal region6-7. in recent years, several studies have analyzed the characteristics of anatomical landmarks in the mandibular anterior region in various populations around the world2,8-10. nevertheless, to date, few studies have evaluated by cbct the characteristics of the mandibular interforaminal anatomy in populations from the northeastern brazil. the objective of this study was to examine by means of cbct the most common location of mf, the presence and extent of al, and the appearance and length of ic in brazilian patients, investigating the presence of sexual dimorphism and differences with respect to the left and right sides. material and methods after local institutional research ethics committee approval (no. 67222), this retrospective study included a total of 143 cbct scans of brazilian patients, obtained between september and december 2012 from a radiology clinic located in recife, pe, brazil. the study population consisted of 43.4% male and 56.6% female patients with a mean age of 49.84 years (range 21-79 years). the examinations were performed as part of the planning procedure for rehabilitation with dental implant placement. the selection criterion of the scans used in the research was: (i) bilateral presence of the first and second premolars, (ii) absence of pathology that could affect the position of mf, al or ic. the radiographic examinations were performed with a cone-beam volumetric tomography device, i-cat (imaging sciences international, hartsfield, pa, usa), adjusted at 120 kvp, 5 ma, voxel size of 0.25 mm, and field of view of 6 cm. the acquired images were reconstructed into multipleplane views (axial, panoramic and cross-sectional) for evaluation of the following parameters: 1 .1 .1 .1 .1 . mf.mf.mf.mf.mf. location: (i) between canine and first premolar; (ii) below the first premolar; (iii) between first and second premolars; (iv) below the second premolar; (v) between second premolar and first molar; (vi) below the first molar. 2 .2 .2 .2 .2 . al.al.al.al.al. presence and extent. extent was measured by the distance between the anterior border of the mf and the anterior border of al7 (figure 1). 3 .3 .3 .3 .3 . ic.ic.ic.ic.ic. appearance and length. length was determined fig. 1. schematic presentation of measurement of anterior loop of the inferior alveolar nerve on a panoramic reconstruction image. (a) line tangent to inferior border of mandible; (b) line perpendicular to a tangent to anterior border of mental foramen; (c) line perpendicular to a tangent to anterior border of the anterior loop; (d) anterior loop = shortest distance between b and c by the distance between the anterior border of the mf and the last mesial slice where the canal was definitely visible in cross-sectional images (figure 2). in addition, differences with respect to the left and right sides and the presence of sexual dimorphism were also investigated. all measurements were performed by one of the authors with experience in the interpretation of cbct and in oral and maxillofacial surgery. this researcher was blind to the gender of the patients. the results were expressed in percentages and statistical measures: mean and standard deviation. data were statistically analyzed using fisher’s exact test and mcnemmar test in the categorical variables, and t-student test with equal variances for numeric variables. hypothesis verification of equal variances was performed using levene’s f test. a level of significance of 0.05 was adopted. the spss software (statistical package for the social sciences, version 17, chicago, usa) was used. results no significant differences (p>0.05) between genders or right and left sides were observed for all parameters. mental foramen one mf was found on each side in all patients. the most common location of the mf was below the second premolar (position iv), followed by a location between the first and second premolars (position iii) (table 1). presence of mf in the region located between the canine and the first premolar (position i) was not observed. braz j oral sci. 13(4):303-307 analysis of anatomical landmarks of the mandibular interforaminal region using cbct in a brazilian population 305305305305305 anterior loop of the inferior alveolar nerve al was visualized in 18.9% of the images (13 males and 14 females), most of them found unilaterally: 5.6% on the right side, 6.3% on the left side and 7% on both sides. the mean length of al was 3.14±1.25 mm. table 2 describes the mean length of the al according to gender and side. incisive canal in 96.5% of the images (61 males and 77 females) it was possible to identify the anterior extension of the ic, 91.6% bilaterally, 2.8% on the right side and 2.1% on the left side. the mean length of the ic was 13.68±5.94 mm. mental foramen right left position total female n male n total n female n male n p valuea total n n(%) n(%) (%) n(%) (%) (%) (%) (%) i 0 0 0 0 0 p > 0.05 0 ii 10 (7) 7 (8.6) 3 (4.8) 11 (7.7) 7 (8.6) 4 (6.5) p > 0.05 21 (7.3) iii 47 (32.9) 26 (32.1) 21 (33.9) 49 (34.3) 26 (32.1) 23 (37.1) p > 0.05 96 (33.6) iv 67 (46.9) 37 (45.7) 30 (48.4) 63 (44.1) 33 (40.7) 30 (48.4) p > 0.05 130 (45.5) v 16 (11.2) 9 (11.1) 7 (11.3) 18 (12.6) 13 (16) 5 (8.1) p > 0.05 34 (11.9) vi 3 (2.1) 2 (2.5) 1 (1.6) 2 (1.4) 2 (2.5) 0 p > 0.05 5 (1.7) total 143 (100) 81(100) 62(100) 143 (100) 81 (100) 62(100) p > 0.05 286 (100) table 1 table 1 table 1 table 1 table 1 gender distribution of the mental foramen position on the right and left sides aindicates statistical significance at level of p less than 0.05. gender side male female p valuea mean ± sd mean ± sd length of the right 3.31 ± 0.88 2.99 ± 1.50 p=0.608 anterior loop left 3.25 ± 1.18 3.00 ± 1.51 p=0.699 length of the right 14.30 ± 6.11 13.28 ± 6.25 p=0.346 incisive canal left 14.20 ± 5.76 13.15 ± 5.64 p=0.291 table 2 -table 2 -table 2 -table 2 -table 2 mean and standard deviation of the length (mm) of the anterior loop and the incisive canal by side according to gender. aindicates statistical significance at level of p less than 0.05. analysis of anatomical landmarks of the mandibular interforaminal region using cbct in a brazilian population braz j oral sci. 13(4):303-307 the mean length of the ic according to the gender and side is described in table 2. discussion to avoid potential injury to the neurovascular bundle during surgical procedures in the mandibular anterior region, it is essential to define the exact location of the mf, as well as to determine the extent of the ic and consider the possibility of al located mesially from the mf11. fig. 2. in this example on cross-sectional reconstructions, the incisive canal could be seen on the images 86-93 (arrows). since the step was 1 mm, the total visible length of the incisive canal was 8 mm 306306306306306analysis of anatomical landmarks of the mandibular interforaminal region using cbct in a brazilian population braz j oral sci. 13(4):303-307 this radiographic study aimed to evaluate the most common location of the mf, as well as to analyze the presence and extent of al, and the appearance and length of ic in a population from the northeastern brazil. also investigated were dimorphism and differences with respect to the right and left sides. panoramic radiographs have been used to study anatomical landmarks of the mandibular interforaminal region1213. however, cbct is a modern imaging technique, providing a more precise three-dimensional evaluation, enabling the identification of anatomical variations5. in order to get a better visualization of anatomic structures and clinical applicability, the present study opted to use images of this type for examination. according to the results of this study, the most frequent position of mf was below the apex of the second premolar (45.5%), followed by the position between the apex of the premolars (33.6%). this result is in agreement with the one reported by previous studies conducted in dry mandibles or panoramic radiographs of populations from the southeastern and northeastern brazil14-17, and in malaysian18, turkish19 and indian20 populations. however, manhães jr et al.21 (2008), amorim et al.22 (2009), almeida filho et al.23 (2011), and guedes et al.24 (2011) have shown by panoramic radiographs the position between the apex of the premolars as the most common for the mf in brazilian populations from the southeastern and center-west regions. the same was observed in nigerian25, jordanian26 and iranian13 populations and on dry mandibles of the late byzantine period27. when analyzing the position of mf in three different populations, santini and alayan28 (2012) verified that among chinese, the most common location was below the second premolar, whereas between europeans and indians, the mf was more frequently located between the first and second premolars. the differences among populations from various countries or in the same country can be attributed to variations in the dietary habits, which may subsequently affect the development of the mandible19. in addition, it must be noted that in the majority of these studies both locations, between the apices of the premolars or along with the second premolar, are the first two main positions for the mf. in the present study, no statistically significant differences were found between males and females, neither between right and left sides with respect to the position of the mf. this is in agreement with the results of previous studies9,15,29. regarding the prevalence and extent of the al of the inferior alveolar nerve, there is considerable disagreement among studies, because the radiographic visualization of this structure, especially in edentulous patients, may be adversely affected by poor bone qualities30. this information is of great importance to clinicians during the preoperative evaluation of the region. additionally, patients with significant anterior extensions of the inferior alveolar nerve (larger than 2 mm) are more likely to suffer sensory disturbances or hemorrhagic complications when dental implants are installed in the most distal area of the interforaminal region7. in the present investigation, al was verified in 18.9% of cases, a result lower than the one observed in the filo et al.2 (2014) study, which evaluated cbct of a swiss population and observed the occurrence of al in 69.7% of patients; as well as that of li et al.31 (2013), which verified a prevalence of 83.1% of al using spiral computed tomography scans of a chinese population. however, jacobs et al.32 (2002) reported the presence of the al in only 7% of the ct in belgian patients. these differences in incidence may be related to geographic/ethnic differences, as well as to methodological discrepancies. according to de oliveira-santos et al.7 (2012), different radiographic techniques, different methods of measurements and the inexistence of a specific definition of al are recurrent in the literature. anterior loops ranging from 0 to 9.0 mm have been reported7-8,30,33. in our study, the mean value of the al was 3.14 mm, coinciding with the values found by other authors also using cbct11. however, in a study performed in southeastern brazil34 was found a mean 2.41 mm length of al in the analyzed cbct scans. as in other studies2,35, the results of the present study showed no statistically significant differences related to gender or to the right and left sides. however, other investigations8,10,31,3334 have demonstrated that males have longer al extensions. ic was first described by olivier36 (1928), who defined it as a continuation of the inferior alveolar nerve, traveling through a canal or through vacuoles in spongy bone mesially from the mf. a relatively precise knowledge of the location of this anatomical structure is of extreme importance prior to any surgical procedure in the region in order to avoid potential neurovascular complications4. the results of our study demonstrated that visualization of the ic was possible in 96.5% of cases. these findings are in agreement with the results of makris et al.4 (2010) and rosa et al.34 (2013) that detected ic in 91% and 98.5% of cases respectively, using cbct. according to pires et al.37 (2012), the ics could not be identified in all of the cbct images due to the small diameter of this structure. the mean length of ic observed in this study was 13.68 mm, longer than the one found in the study by rosa et al.34 (2013) which found a mean length of 9.11 mm in patients from southeastern brazil. however, it was shorter than the one found by makris et al.4 (2010), who verified, also by using cbct, an ic average length of 15.1 mm in greeks. none of the previously cited studies observed significant differences in appearance or length of the ic with respect to gender or sides, confirming the results of the present research. in conclusion, the variation in the location of mf, the high rate of ic and the occasional presence of al in the brazilian population discussed here, demonstrate the importance of using cbct for assessment of bone morphology and anatomical dimensions of the mandibular anterior region, allowing proper surgical planning and preventing injury to the neurovascular bundle. acknowledgements the authors are grateful to marco frazão for providing the scans that were used in this study. the english 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for the mandibular canal and diameter of the mandibular incisive canal to avoid nerve damage when installing endosseous implants in the interforaminal region. j oral maxillofac surg. 2007; 65: 1772-9. 34. rosa mb, sotto-maior bs, machado v de c, francischone ce. retrospective study of the anterior loop of the inferior alveolar nerve and the incisive canal using cone beam computed tomography. int j oral maxillofac implants. 2013; 28: 388-92. 35. apostolakis d, brown je. the anterior loop of the inferior alveolar nerve: prevalence, measurement of its length and a recommendation for interforaminal implant installation based on cone beam ct imaging. clin oral implants res. 2012; 23: 1022-30. 36. oliver e. the inferior dental nerve and its nerve in the adult. br dent j. 1928; 49: 356-8. 37. pires ca, bissada nf, becker jj, kanawati a, landers ma. mandibular incisive canal: cone beam computed tomography. clin implant dent relat res. 2012; 14: 67-73. oral sciences n3 original article braz j oral sci. july | september 2013 volume 12, number 3 impact of oral health conditions on school performance and lost school days by children and adolescents: what are the actual pieces of evidence? janice simpson de paula1, fábio luiz mialhe1 1department of public health dentistry, piracicaba dental school, university of campinas, piracicaba, sp, brazil correspondence to: janice simpson de paula faculdade de odontologia de piracicaba universidade estadual de campinas avenida limeira 901 cep: 13414-903 – bairro areão, piracicaba, sp, brasil phone: +55 19 21065279 e-mail: janicesimpsondp@yahoo.com.br received for publication: may 31, 2013 accepted: september 11, 2013 abstract aim: to investigate evidence of associations between oral health status of children and adolescents and their school performance and lost school days due to dental problems. methods: pubmed electronic database was searched for scientific papers published between 1990 and 2013. twenty-one papers that attempted to investigate the impact of oral health on school performance and lost school days were retrieved. brief descriptions of each study’s methodology and outcomes were presented and discussed. results: although the papers reported statistically significant associations between school performance or lost school days and oral health conditions of schoolchildren, all of them were cross-sectional and ecological studies with an observational design, which may not provide full information about causes and effects. in addition, the lack of standardized criteria did not allow comparisons among the studies retrieved in the search. conclusions: oral diseases appear to impact on lost school days and school performance of children and policy-makers should address this issue when planning health promotion interventions in school settings. however, standardized materials and methodologies as well as longitudinal studies using valid and reliable criteria are needed to confirm the causes or risks of oral health factors in school performance, generating hypotheses for future research and providing important data for determining effective actions in school health programs. keywords: school performance, absenteeism, oral health. introduction in 1948, the world health organization defined health as being “a state of complete physical, mental and social well-being, and not merely the absence of disease or infirmity”1. oral health is considered an integral element of general health and well being because it enables individuals to eat, communicate and socialize with others2. moreover, oral health is considered a mirror of general health and the mouth is a portal for infectious organisms to enter the whole organism2. therefore, according to sheiham3 “the compartmentalization involved in viewing the mouth separately from the rest of the body must cease because oral health affects general health”. in spite of a range of oral diseases affecting the world’s population, dental caries continues to be the most prevalent oral disease in children and adolescents worldwide, leading to pain, poor nutrition and time out of school, interfering in their quality of life4-6. several studies have investigated the effect of chronic diseases7, such as asthma8, allergic rhinitis9, inflammatory demyelination of the central nervous system10 and diabetes11 on school performance. braz j oral sci. 12(3):189-198 in relation to oral health, gift, et al.12 (1992) showed that in 1989, over 51 million school hours were missed annually by north-american school-aged children as result of visits to dentists or oral problems. moreover, according to the us general accounting offices13 children with poor oral health are 12 times more likely to have restricted-activity days than those children without oral disease. although studies evaluating the impact of oral health conditions on school performance and lost school days are of growing concern to educational and health researchers, there remains an astounding lack of strict scientific inquiry that reviews the studies developed on this topic in order to ascertain the validity of the conclusions. considering the importance of this issue in planning health promotion activities in schools, the objectives of this study were to investigate the pieces of evidence and discuss the methods and results of studies that assessed the existence of associations between oral health status of children and adolescents, their school performance and lost school days due to dental problems. this evaluation aimed at providing researchers and decision makers with a more solid background needed to enlighten recommendations and interventions in the school settings. material and methods the questions addresses by this review were “what evidence is there of an association between oral health and school performance?” and “what are the materials and methods used for studies about oral health and school performance?” for this purpose, an extensive literature search was carried out using medline, isi, lilacs and scielo databases. the intent was to review all full-text papers published in biomedical journals between 1990 and 2013. the search strategy included the key words “school performance”, “oral health” and “absenteeism”. the studies were screened according to the inclusion criteria: (1) research papers, (2) publications related to oral health, school performance and/or lost school days due to dental problems, (3) english-language papers. studies conducted and published in other languages were excluded. two reviewers selected and reviewed the papers. first, each reviewer independently selected the papers after reading their abstracts and checking their contents. to validate the selection procedure, the reviewers examined the potentially relevant arguments against the inclusion criteria and all discrepancies were discussed until agreement was reached. results the initial search retrieved 512 studies. one paper was excluded because it was duplicated. the titles and abstracts of the remaining 511 papers were reviewed for a more detailed evaluation. after review and discussion in situations of disagreement, only 17 papers met the inclusion criteria. the reasons for exclusion of the other papers are shown in figure 1. in a second round, a secondary search was conducted by investigating the reference list of the gathered literature and four more papers were included in the review due to the importance of the studies and strong relationship with the aim of this review5,14-16. table 1 shows the results of the studies. all papers included in this review were classified as having an observational clinical study design (cross-sectional studies and ecological studies). 190190190190190 data sources medline, pubmed central (jan/1970– apr/2013) key words: performance school, oral health, absenteeism inclusion criteria: research papers, publications related to oral health, school performance and/or lost school days due to dental problems, and english-language papers. titles and abstracts reviewed: first round (n = 512) excluded: papers repeated papers related to clinical dentistry techniques and materials papers related to systemic conditions and human plasma papers related to orofacial disorders papers studying substances, foods and medicines papers related the dental care, health services and health education papers presenting studies with bacteria papers related to training of human resources in health care/procedures papers related to quality of life papers on literature reviews that associate school performance and oral health titles and abstract reviewed: second round (n = 17) included: references listed in the gathered literature (n=4) papers included in the review (n = 21) fig. 1. diagram of literature search. ����� ����� impact of oral health conditions on school performance and lost school days by children and adolescents: what are the actual pieces of evidence? braz j oral sci. 12(3):189-198 191191191191191 t a b le 1 . r es u lt s o f re fe re n ce s ap p ra is ed impact of oral health conditions on school performance and lost school days by children and adolescents: what are the actual pieces of evidence? braz j oral sci. 12(3):189-198 192192192192192 t a b le 1 . r es u lt s o f re fe re n ce s ap p ra is ed ( co n t. ) impact of oral health conditions on school performance and lost school days by children and adolescents: what are the actual pieces of evidence? braz j oral sci. 12(3):189-198 193193193193193 t a b le 1 . r es u lt s o f re fe re n ce s ap p ra is ed ( co n t. ) impact of oral health conditions on school performance and lost school days by children and adolescents: what are the actual pieces of evidence? braz j oral sci. 12(3):189-198 194194194194194 t a b le 1 . r es u lt s o f re fe re n ce s ap p ra is ed ( co n t. ) impact of oral health conditions on school performance and lost school days by children and adolescents: what are the actual pieces of evidence? braz j oral sci. 12(3):189-198 195195195195195 oral health status of students as regards the evaluation of the students’ oral health status, the researchers used mainly objective criteria by direct visual inspection of the oral cavity14-15,17-22 and data from government researches5,23-24. dental caries was the most common oral condition evaluated in the studies, generally following the who diagnostic criteria 14-15,17-19,23-24. furthermore, other oral conditions were also evaluated, namely oral hygiene, obtained by the simplified oral hygiene index (ohi-s)18,20; dental trauma, obtained by the number of fractured anterior teeth and untreated severe traumatic dental injuries, according to the who criteria15,18,24; and periodontal disease, obtained by the community periodontal index (cpi)15. evaluation of school performance with regard to data related to school performance in t h e r e v i e w s t u d i e s , t h e y w e r e g e n e r a l l y o b t a i n e d subjectively by questionnaires applied to parents4,25. for example, questions such as “in the last 12 months, how would you describe your child’s performance in school” (excellent, above average, average, below average, or poor) were used in the study of blumenshine et al.4 (2008). other studies used self-reports of children and adolescents as regards their school performance, ranked as low, moderate or high21,26-27 or even questions such as “in your opinion, what does your class teacher think about your school performance compared with that of your classmates? (good or poor)”’18. other studies, such as freire et al.19 (2008) used the question: ‘have you ever failed an examination at school?’ and the responses were “no” and “yes” (failed once or more than once). instruments developed to assess the quality of life related to oral health (ohrqol) such as the oral impact on daily performance (oidp) and child oral impacts on daily performances (child-oidp) were also applied to students in some studies as an indirect means of obtaining their school performance17,20,28-30. in only three studies there were objective data about school performance obtained from standardized achievement tests. in the study by muirhead and locker23 (2006), data were obtained from broad governmental educational research databases that used the criteria of the education quality and accountability office (eqao) standard tests. other indexes of academic performance were obtained from the linguistic awareness of reading readiness test of the emergent literacy test (larr)24 and human development report5. the criteria used in eqao were the percentages of grade three and grade six children scoring below the provincial average in reading, writing and mathematics23. in the larr, school performance data included the results of baseline english, mathematics and literacy tests22. the study by seirawan et al.22 (2012) used the los angeles unified school district (lausd) office data and accountability, which provide information about students’ number of absent days, california standards test scores, proficiency levels. evaluation of lost school days another indirect way used in some of the reviewed studies to evaluate the school performance of schoolchildren was by quantifying the students’ lost school days. generally the studies used questionnaires applied to parents and schoolchildren to obtain these data4,14-16, 31-32. results of studies all the evaluated studies showed associations among clinically detected (normative needs) and/or self-perceived oral health status (subjective needs) with school performance and school days lost by children and adolescents. discussion to the best of our knowledge, this is the first study to review the quality of evidence related to the impact of oral health conditions on school performance and school days lost by children and adolescents. it was observed that all reviewed studies found statically significant associations between school performance or lost school days and the oral health conditions of schoolchildren. however, due to several limiting methodological factors observed in the studies, the associations observed could be inaccurate, generating weak evidence. designs of the studies as regard the research design characteristics of the reviewed studies, it was observed that all of them were crosssectional and ecological observational studies. observational studies are fast and have lower cost than longitudinal ones, but these studies are unable to show the causality or risk factors related to outcomes33. in spite of this, they can indicate associations that may exist and are therefore useful in generating hypotheses for future research, providing important data to determine effective actions in public health34. thus, it is recommended that longitudinal studies be developed in the future to evaluate the relative risk of the oral conditions impacting on school performance and absence from school due to dental problems, generating stronger evidence. evaluation of oral health conditions the researchers in the reviewed studies used diverse ways of obtaining clinical data on oral health, by means of objective clinical data or subjective reports. most of the studies evaluated the oral needs of participants by normative clinical evaluation, such as caries and periodontal indexes14-15,17-20,22,24. direct inspection of the oral cavity through well-established indexes and criteria, usually collected in a standardized manner by trained personnel, could be considered a reliable and valid method for evaluating oral health conditions. on the other hand, the analysis of oral health conditions from the perceptions of parents, teachers or students’ selfreports, as observed in some studies, might have subjective interpretations that could generate a systematic error arising impact of oral health conditions on school performance and lost school days by children and adolescents: what are the actual pieces of evidence? braz j oral sci. 12(3):189-198 196196196196196 from inaccurate measurement of the outcomes of the studied variables26-29,32. thus, it is recommended that oral health indexes be used by calibrated teams of researchers in future studies for more reliable associations. evaluation of school performance with regard to school performance, no standardized manner of collecting and measuring was observed in the reviewed studies, hindering the replication of studies and comparison of the data. only three ecological studies used standardized data obtained from a government source for evaluating children’s school performance5,22-24. although measures of school performance based on standardized tests have received criticisms due to the largely uncontrolled bias that could interfere in the measurement of intelligence, leading to failure in accurately predicting academic performance, it is likely that a strict standardization of the criteria used in these studies could permit comparisons among them 35. with this assumption, the national standard achievement tests have commonly been used in studies evaluating associations between chronic diseases and school performance6,36-37. other studies obtained data about missed school days or school performance from the reports of the schoolchildren’s parents or directly from the schoolchildren4,25. the acquisition of data related to children’s school performance obtained from parents or from schoolchildren must be scrutinized for the probability of information bias, since their perception could be mediated by subjective variables and cannot match the reality, producing data that are not reliable for research purposes. in addition, instruments such as oidp and child-oidp were used in some studies as an indirect means of evaluating student school performance. these instruments were originally developed to assess the quality of life related to oral health and measure the impact of oral problems on performing the daily life activities of individuals. considering that school performance is a part of daily activities, this could be considered an indirect criterion used in studies for evaluating this objective. however, they are not specific instruments or criteria for evaluating school performance, which could generate inaccurate measurements or measurement biases. mikaeloff et al.10 (2010) used grade retention as another criterion for evaluating children´s school performance. however, the use of grade retention data for this purpose depends on the educational system and criteria adopted in each region or country, and do not allow comparison among studies. associations between oral health, school performance and lost school days the reviewed studies showed associations between dental problems of children and their school performance. according to pourat and nicholson38 (2012) missed school days due to dental problems may have implications for the school performance of children, since absences from school mean missed opportunities for learning and academic advancement, and have significant negative social and economic consequences. it was also observed that the majority of studies investigating the school days lost due to dental problems did not investigate the specific oral problem related to absenteeism, such as pain, dental caries, orofacial trauma or other oral problems. lack of this information is a limiting study factor because it does not allow determining the specific oral causes associated with poor school performance and prevents comparisons among the studies. the influence of other variables on school performance analyzing the factors that may interfere with school performance, it is important to control the confounding variables, such as general or systemic health of schoolchildren. blumenshine et al.4 (2008) stated that children with poor oral and general health are more likely to have poor school performance. thus, general health could function as a confounding variable for the relationship between oral health and school performance4,39. other confounding variables were related to socio-environmental conditions of the children, such as low socioeconomic status and low education level of the family, which exert great influence on disturbed schooling, together with the burden of disease10,40. this represents a challenge to public health interventions, especially in some populations, such as immigrants and lowincome individuals in whom oral problems are more frequent and who cannot afford private treatment 32,38,41. furthermore, other factors must be considered in the association with school performance, as studies have related this variable to perceptions of children’s oral health-related quality of life42-43. these recent findings provide evidence of the relationship between social determinants, such as quality of life and environment, and school performance and school absenteeism. from the results of the present review, it was observed a need for developing longitudinal studies to evaluate whether oral health is a risk factor for school absenteeism and school performance, in order to understand the actual influence of oral health conditions on an individual’s school performance, using more accurate and reliable data. in addition, the use of multivariate statistics can be an important way to control the confounding factors in these analyses, such as general health, quality of life and socio-environmental conditions. all the reviewed studies encountered associations between oral conditions and school performance of children and adolescents. however, the lack of longitudinal studies, standardized methodologies for comparisons of results and objective and standardized criteria for data collection on oral conditions, school performance and absenteeism, could make the current evidence on the association of oral conditions with low school performance and absence from school seem inconclusive or weak. the need for developing studies with a longitudinal design using more reliable and valid criteria is emphasized in order to assess the causality or risk of oral health factors impacting on school performance, generating hypotheses for future research and providing impact of oral health conditions on school performance and lost school days by children and adolescents: what are the actual pieces of evidence? braz j oral sci. 12(3):189-198 important data for determining effective actions in school health programs. the outcomes of this study should be interpreted in the context of some limitations. the main objective was to collect important publications about school performance and oral health, though it was not included an appraisal of the quality of the papers selected in the systematic review. acknowledgements this study was supported by fapesp (process nº 2009/ 06081-7), são paulo, brazil. references 1. who. constitution of the world health organization. basic documents. 45 ed. supplement, october 2006 [access 2013, sept 17]. available from: http://www.who.int/governance/eb/who_constitution_en.pdf. 2. watt rg. strategies and approaches in oral disease prevention and health promotion. bull world health organ. 2005; 83: 711-8. 3. sheiham a. oral health, general health and quality of life. bull world health organ. 2005; 83: 644-5. 4. blumenshine sl, vann wf jr, gizlice z, lee jy. children’s school performance: impact of general and oral health. j public health dent. 2008; 68: 82-7. 5. egri m, gunay o. association between some educational indicators and dental caries experience of 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[access 2012, march 13]. available from: http:// healthpolicy.ucla.edu/publications/search/pages/detail.aspx?pubid=92. 39. sheiham a. oral health, general health and quality of life. bull world health organ. 2005; 83: 644-5. 40. xavier a, carvalho fs, bastos rs, caldana ml, bastos jrm. dental caries-related quality of life and socioeconomic status of preschool children, bauru, sp braz j oral sci. 2012; 11: 463-8. 41. moyses sj. inequalities in oral health and oral health promotion braz oral res.2012; 26: 86-93. 197197197197197impact of oral health conditions on school performance and lost school days by children and adolescents: what are the actual pieces of evidence? braz j oral sci. 12(3):189-198 42. paula js, ambrosano gmb, mialhe fl. oral disorders, socioenvironmental factors and subjective perception impact on children’s school performance. oral health prev dent. 2013 [in print]. 43. piovesan c, antunes jl, mendes fm, guedes rs, ardenghi tm. influence of children’s oral health-related quality of life on school performance and school absenteeism. j public health dent. 2012; 72: 156-63. impact of oral health conditions on school performance and lost school days by children and adolescents: what are the actual pieces of evidence?198198198198198 braz j oral sci. 12(3):189-198 oral sciences n3 original article braz j oral sci. january | march 2015 volume 14, number 1 assessment of torque angle of brackets from different brands anelisa dos anjos1, daniela daufenback pompeo2, gilson josé enricone dos anjos1, gustavo mussi stefan oliveira3, henrique damian rosário1 1faculdades integradas do norte de minas – funorte, dental school, department of orthodontics, florianópolis, sc, brazil 2universidade sagrado coração – usc, dental school, department of oral biology, bauru, sp, brazil 3university of louisville school of dentistry, department of general dentistry & oral medicine, louisville, ky, usa correspondence to: henrique damian rosário rua adhemar da silva, 235, sala 3, kobrasol cep: 88101-090 são josé, sc, brasil phone: +55 48 30351211 fax: +55 48 30351211 e-mail: drhenriquerosario@icloud.com abstract aim: to measure torque angle values of brackets designed for canines, comparing it to their prescription values. methods: one hundred and sixty maxillary (-2o) and mandibular (-11o) canine brackets of roth prescription from the following brands were selected: abzil, eurodonto, morelli and ormco. the brackets were set in wax and images were obtained by scanning. reference points were determined over these images, lines drawn and the torque angle was measured. the student’s t-test and the wilcoxon signed-rank test were used at a significance level of 5%. results: for maxillary canine brackets, the medium torque angle values were: abzil 0.93o (± 0.88o); eurodonto 0.13o (± 0.34o); morelli -2.56o (± 0.50o), and ormco -1.16o (± 1.27o). for mandibular canine brackets, the values were: abzil -11.76o (± 0.40o); eurodonto -10.40o (± 0.25o); morelli -11.18o (± 0.56o), and ormco -11.36o (± 0.30o). for maxillary canine brackets, the brands abzil, morelli, and ormco presented statistically diferente values from those indicated for prescription. for mandibular canine brackets, the brands abzil, eurodonto, and ormco presented statistically different values from the prescribed ones. conclusions: some marketed brands present differences between the torque angle found in the brackets and those recommended in the prescription. however, these differences are clinically acceptable. keywords: orthodontic brackets; torque; orthodontics. introduction during the 70’s, andrews introduced the straight-wire appliance and the 11 andrews’ prescriptions. the concept described a completely programmed appliance, created by a tridimensional system of brackets. these brackets were designed with ideal angular characteristics of each tooth, for a regular occlusion, adjustments embedded in brackets and ideal tooth positioning1-3. another characteristic of these brackets is torque, which may be defined from clinical and mechanical standpoints4. regarding mechanics, it indicates torsion of a structure in its longitudinal axis resulting in a torsion angle. clinically, it represents a buccolingual inclination of the crown/root of a tooth. the terms moment, moment of torsion, biomechanics torque and third-order torque can also be found in the literature. roth5 modified some of the bracket prescription values from the original straight-wire system, producing a universal prescription that is used in every case, and named it the second generation of pre-adjusted appliances. for maxillary canines, a decrease in lingual crown torque from -7o to -2o was recommended and braz j oral sci. 14(1):66-70 received for publication: december 15, 2014 accepted: march 20, 2015 for mandibular canines, the -11o lingual crown torque was maintained. a mesial rotation of 4o in maxillary canines and 2o in mandibular canines was also added. straight-wire appliances correct dental positions if brackets are well placed by the orthodontist, and dental crowns present a typical morphology6. the expression of torque over teeth is influenced by factors such as: anatomy of the buccal aspect of the tooth, tooth size, bracket position, bracket size7, gap between the wire and the bracket slot8-9, differences in direct bonding10, unusual crown shape and angulation11, application of strength outside the tooth center of resistance5, dental arch shape and dental eruption position12. due to the number of possible variations in the torque expression of a bracket, it is important that their angulation values meet the values recommended by the author of the technique, so that the orthodontic treatment is successful, resulting in favorable occlusion, function and esthetic conditions. the aim of this study was to assess the agreement among the torque angle values of metallic brackets in the roth prescription from four different commercial brands, with what was recommended in the prescriptions. material and methods this laboratory study used a total of 160 metallic orthodontic brackets of four commercial brands with 0.022" x 0.028" slot of the roth prescription. the sample was divided in 4 groups of brackets (n=40, with 20 maxillary canines and 20 mandibular canines), according to the respective commercial brands: group 1 – 3m abzil brackets (são josé do rio preto, sp, brazil); model kirium line; group 2 – eurodonto brackets (curitiba, pr, brazil); model 280; group 3 – morelli brackets (sorocaba, sp, brazil); model roth max; group 4 ormco brackets (glendora, ca, usa); model mini-damon. for image capturing and more accurate torque measurement, the bracket was placed over a glass plate, with the mesial aspect facing down (in contact with the glass plate surface). a plastic set square (professional drawing set square, acrimet, são paulo, sp, brazil) was used to place the bracket along with the utility wax (wilson polidental, cotia, sp, brazil), measuring 1.5 cm x 1.0 cm x 0.5 cm (figure 1). the wax stabilized the bracket so it could be scanned (scanner epson stylus tx 220®, taiwan, china) for image capturing, providing a total of 160 images. the identification of brackets was performed during the scanning process, considering the first letter as the commercial brand, the two following numbers indicated the tooth, and the two subsequent ones indicated the order of image capturing. for example, a bracket with the specification “morelli, tooth 13, number 1” would receive the following nomination: m130. numeration was omitted during measurements so that the evaluator was blind to which group was being assessed. the images were transferred to a notebook computer (acer ink aspire® 4740, i3, 3mb, 250gb, taiwan, china). the resolution used was 9600 pixels for an area of 1.016 cm2. some points and lines of reference were drawn to measure torque according to streva13, and are shown in figures 2 and 3. point b1: point b1: point b1: point b1: point b1: point of cervical edge of the bracket body base. point b2:point b2:point b2:point b2:point b2: point of occlusal edge of the bracket body base. point c1: point c1: point c1: point c1: point c1: angle vertex between the base of the bracket slot and the gingival flap. point c2: point c2: point c2: point c2: point c2: angle vertex between the base of bracket slot and the incisal slot edge. line b:line b:line b:line b:line b: resulting from the junction of b1 and b2 points, it defined the base of the bracket body. line c:line c:line c:line c:line c: resulting from the junction of c1 and c2 points. this line defined the slot base, and therefore its torque. the torque angle was defined by the angle formed between the bracket body base (line b), and the slot floor (line c), as illustrated in figure 3. a single evaluator measured these angles during three consecutive days. for this purpose, the images were viewed on the windows 7® display and software mb-ruler® (figure 4) was used. then, the data were recorded in a spreadsheet. fig. 1 – bracket positioning over glass plate, stabilized in the wax block for later image capturing. fig. 2 – points b1 and b2 in the base of the bracket body. points c1 and c2 limiting the floor of the slot of the bracket. 6767676767assessment of torque angle of brackets from different brands braz j oral sci. 14(1):66-70 fig. 3 – reference lines used to measure torque angle. line b, and line c. fig. 4 – application of software mb ruler® to measure torque angle. assessment of measurement error two images of maxillary brackets and two images of mandibular brackets of each assessed brand were randomly selected for analysis of method error; the evaluator measured each image at least twice with a 7-day interval between measurements. the paired t-test was used to verify the intra-examiner systematic error for both repetitions. dahlberg error calculation was used to determine the casual error13: error \/σd2/2n considering d=difference between 1 st and 2nd measurements, and n=number of measurements replications. the results of the systematic error assessment and the casual error are shown in table 1. the method error estimate was between 0.18º and 0.25º. comparison measurement 1 measurement 2 t p error mean sd mean s d 1st x 2nd -6.26 5.38 -6.29 5.48 0.432 0.672ns 0.18 1st x 3rd -6.26 5.38 -6.30 5.44 0.433 0.671ns 0.25 2nd x 3rd -6.29 5.48 -6.30 5.44 0.110 0.914 ns 0.25 table 1 – table 1 – table 1 – table 1 – table 1 – mean, standard deviation of both measurements, paired t-test, and dahlberg error to assess the systematic error and casual error ns – non-significant statistical difference. data analysis data were described by mean, standard deviation, minimum value, and maximum value parameters. the shapiro-wilk test was used to verify whether the groups were regularly distributed. for groups 1, 2 and 4, the maxillary canine brackets were not regularly distributed. the mandibular brackets of groups 1, 2 and 4 were regularly distributed. in group 3, all brackets (maxillary and lower mandibular) were regularly distributed. the student’s t-test was used for groups with regular distribution to compare the values obtained with the norm, and the wilcoxon signed-rank test was used in groups with irregular distribution. one-criterion variance analysis and the tukey’s post hoc test were used to compare brands, the difference among the obtained values and the norm in groups of mandibular canine brackets. the kruskal-wallis nonparametric test and the miller’s post hoc test were used in groups of maxillary canine brackets. a significance level of 5% (p<0.05) was set for all statistical tests. results the values of medium, minimum and maximum torque angle, and standard deviations of maxillary canine brackets are described in table 2. the torque values of eurodonto brackets were not statistically different when compared with the recommended norm. table 3 presents the results obtained for mandibular canine brackets. mandibular brackets of the morelli brand presented torque values that were not statistically different from the norm. the remaining brands presented statistically significant differences. the comparison among all four brands’ values and the norm, in maxillary and mandibular canine brackets, is described in tables 4 and 5, respectively. discussion streva et al.14 and bóbbo15 studied the values of bracket torque angles, and compared them with the values recommended by certain techniques. they set the brackets in a jig, capturing images by light microscopy. the present study used a methodology that allowed the correct placement of the bracket, since the use of a set square perpendicular to the bracket would not allow its rotation, which could interfere in the image recording and torque angle measuring processes. image was obtained by scanning. 6868686868 assessment of torque angle of brackets from different brands braz j oral sci. 14(1):66-70 brand mean s d m i n . max. n o r m d i f f e r . p abzil -0.93 0.88 -2.16 0.00 -2 1.07 0.001* eurodonto -0.13 0.34 -1.39 0.14 0 -0.13 0.060ns morelli -2.56 0.50 -3.40 -1.72 -2 -0.56 < 0.001* ormco -1.16 1.27 -2.76 1.84 0 -1.16 0.008* table 2 – table 2 – table 2 – table 2 – table 2 – mean, standard deviation, minimum, maximum, and comparison to the norm of maxillary canine brackets. * – significant statistical difference (p<0.05). ns – non-significant statistical difference. brand mean s d m i n . max. n o r m differ. p abzil -11.76 0.40 -12.53 -11.09 -11 -0.76 <0.001* eurodonto -10.40 0.25 -10.88 -10.04 -11 0.60 <0.001* morelli -11.18 0.56 -12.29 -10.28 -11 -0.18 0.173ns ormco -11.36 0.30 -11.90 -11.00 -11 -0.36 <0.001* table 3 – table 3 – table 3 – table 3 – table 3 – mean, standard deviation, minimum, maximum, and comparison to the norm of mandibular canine brackets. * – significant statistical difference (p<0.05). ns – non-significant statistical difference. brand differ. s d m i n . max. p abzil 1.07 0.88 -0.16 2.00 <0.001* a eurodonto -0.13 0.34 -1.39 0.14 b morelli -0.56 0.50 -1.40 0.28 b c ormco -1.16 1.27 -2.76 1.84 c table 4 table 4 table 4 table 4 table 4 comparison among all four brands of value differences obtained, and the norm from each manufacturer in maxillary canine brackets. * – significant statistical difference (p<0.05). brands with the same letter do not present significant statistical difference between themselves. table 5 table 5 table 5 table 5 table 5 comparison among all four brands of value differences obtained, and the norm from each manufacturer in mandibular canine brackets brand differ. s d m i n . max. p abzil -0.76 0.40 -1.53 -0.09 <0.001* a eurodonto 0.60 0.25 0.12 0.96 b morelli -0.18 0.56 -1.29 0.72 ab ormco -0.36 0.30 -0.90 0.00 ab * – significant statistical difference (p<0.05). brands with the same letter do not present significant statistical difference among themselves. the reference points used for measurement were the same established in the works by streva et al.14 and bóbbo15, which made torque angles easy to identify. using these points also allowed the results to offer a standard of comparison to those studies. bóbbo15 analyzed the torque of maxillary and mandibular incisor brackets, and streva et al.14 assessed maxillary and mandibular canine brackets. both assessed the brackets of the m.b.t. prescription from different commercial brands. generally, the researchers observed that some brands obtained statistically different torque angle values from what was recommended by the m.b.t. prescription. the results observed in this study, which are in agrément with the findings of those authors, showed that variations generally occur in the 6969696969assessment of torque angle of brackets from different brands braz j oral sci. 14(1):66-70 accuracy of torque values of brackets when compared with the values recommended by the technique and different manufacturers. this finding represents another aspect that interferes with the final buccolingual position of teeth. torque is related to the dental position desired at the end of treatment, and it must be individualized for the achievement of correct intercuspation, esthetics, and adequate function14,16. a change in the torque of brackets may have significant clinical consequences. this variability may cause either an end-to-end ratio or, more severely, an anterior crossbite when the inclination of the maxillary canine decreases or the mandibular one increases. clinically, this result would be undesirable especially in patients with class iii malocclusion. on the other hand, a torque increase in the maxillary canine bracket, or a reduction in the mandibular one, causes an increase in the overjet, hindering the treatment of patients with class ii malocclusion. torque is influenced by factors such as: anatomy of the buccal aspect of teeth, teeth size, bracket positioning, bracket size7, gap between the wire and the bracket slot4,8-9, differences in direct bonding10, unusual crown shape and angulation11, application of strength outside the tooth center of resistance6, teeth with unusual morphology, and position of dental eruption14. the clinical responses for torque angle variations of brackets presented in this study should be interpreted carefully. considering the nature of a laboratory study, it is very difficult to define precisely which changes in dental positions would occur due to the small variations in the accuracy of the torque of brackets, in light of the high number of variables associated with the final tooth position. professionals should exercise attention to identify variables and controlling torques individually, especially in the final stages of the treatment. archambault et al.4 reported the existence of a gap between the wire and the slot of the bracket, which allows a torque variation. the authors observed that a gap in the wire with greatest diameter 0.018" x 0.025" in a 0.018" slot, and a 0.021" x 0.025" wire in a 0.22" slot-created gaps of 2° 7070707070 assessment of torque angle of brackets from different brands braz j oral sci. 14(1):66-70 and 6°, respectively. the difference found between the torque angle values of brackets used in this study and the roth prescription is lower than the gap of the last wire of the technique. furthermore, the orthodontists often use a 0.019" x 0.025" wire in a 0.022" slot. this leads to an even larger gap of around 10°. it is not possible to affirm that the statistical differences found between the brackets from some brands and the recommended norms could lead to clinically relevant outcomes. further clinical studies are required to investigate the clinical outcomes of the discrepancies found between torque angles of brackets and their norms. considering the limitations of this study, it may be concluded that the medium torque angle values were significantly different for maxillary canine brackets from brands abzil, morelli and ormco; for mandibular canines from brands abzil, eurodonto and ormco regarding. comparison s among the brands revealed significant differences for all brands, except for morelli and eurodonto, and morelli and ormco for maxillary canines. for mandibular canines, only the brands abzil and eurodonto differed significantly between each other. despite the differences found among the torque angles of the evaluated brackets , compared with those recommended in the prescription, these variations are clinically acceptable. references 1. andrews lf. straight wire – the concept and the appliance. san diego: wells; 1989. 407p. 2. moesi b, dyer f, benson pe. roth versus mbt: does bracket prescription have an effect on the subjective outcome of pre-adjusted edgewise treatment? eur j orthod. 2011; 36: 1-8. 3. jain m, varghese j, mascarenhas r, mogra s, shetty s, dhakar n. assessment of clinical outcomes of roth and mbt bracket prescription using the american board of orthodontics objective grading system. contemp clin dent. 2013; 4: 307-12. 4. archambault a, lacoursiere r, badawi h, major pw, carey j, flores mir c. torque expression in stainless steel orthodontic brackets. angle orthod. 2010; 80: 201-10. 5. roth rh. the straight-wire appliance 17 years later. j clin orthod. 1987; 21: 632-42. 6. tominaga j, chiang pc, ozaki h, tanaka m, koga y, bourauel c, et al. effect of play between bracket and archwire on anterior tooth movement in sliding mechanics: a three-dimensional finite element study. j dent biomech. 2012; 3: 1-7. 7. dolci gs, spohr an, zimmer er, marchioro ee. assessment of the dimensions and surface characteristics of orthodontic wires and bracket slots. dental press j orthod. 2013; 18: 69-75. 8. meireles jks, ursi w. optimization of orthodontic treatment using the centrex system to retract anterior teeth. dental press j orthod. 2012; 17: 29-44. 9. arreghini a, lombardo l, mollica f, siciliani g. torque expression capacity of 0.0018 and 0.022 bracket slots by changing archwire material and crosssection. prog orthod. 2014; 15: 61-79. 10. ousehal l, lazrak l. the accuracy of brackets placement in direct bonding technique: a comparison between the pole-like bracket positioning gauge and the star-like bracket positioning gauge. open journal of stomatology. 2011; 1: 121-5. 11. polak pt, moro a, bié mdd, lopes sk, spada pp, moresca r, et al. influence of variation in the bracket positioning on the lingual surface of upper canines on the torque expression. ortho sci orthod sci pract. 2010; 3: 315-9. 12. sardarian a, danaei sm, shahidi s, boushehri sg, geramy a. the effect of vertical bracket positioning on torque and the resultant stress in the periodontal ligament – a finite element study. prog orthod. 2014; 15: 50-60. 13. houston wjb. the analysis of errors in orthodontic measurements. am j orthod. 1983; 83: 382-90. 14. streva am, cotrim-ferreira fa, garib dg, carvalho peg. are torque values of preadjusted brackets precise? j appl oral sci. 2011; 19: 313-7. 15. bóbbo mf. torque evaluation to incisors brackets of the mbt technique. [master‘s thesis]. são paulo: universidade cidade de são paulo; 2006. 108p. 16. capistrano a, cordeiro a, siqueira df, capelozza filho l, cardoso ma, almeida-petrin rr. from conventional to self-ligant bracket systems: is it possible to aggregate the experience with the former to the use of the latter? dental press j orthod. 2014; 19: 139-57. oral sciences n3 original article braz j oral sci. october | december 2014 volume 13, number 4 correlation between temporomandibular disorders, occlusal factors and oral parafunction in undergraduate students fabiane maria ferreira1, paulo cézar simamoto-júnior1, veridiana resende novais2, marcelo tavares3, alfredo julio fernandes-neto1 1universidade federal de uberlândia – ufu, school of dentistry, department of occlusion, fixed prosthesis and dental materials, uberlândia, mg, brazil 2universidade federal de uberlândia – ufu, school of dentistry, department of operative dentistry and dental materials, uberlândia, mg, brazil 3universidade federal de uberlândia – ufu, school of mathematics, uberlândia, mg, brazil correspondence to: fabiane maria ferreira faculdade de odontologia foufu av. pará, 1720 campus umuarama bloco 4l anexo b sala 34 uberlândia mg cep 38400-902 phone: +55 34 3218 2222 fax.: +55 34 3218 2279 e-mail: fabianemariaferreira@yahoo.com.br received for publication: september 03, 2014 accepted: december 02, 2014 abstract aim: to investigate the prevalence of temporomandibular disorders (tmd) in undergraduate students and to correlate its prevalence with occlusal factors and parafunctional habits. methods: 201 undergraduate students were evaluated. the research diagnostic criteria for temporomandibular disorders (rdc/tmd) was filled out at the beginning of the study, followed by occlusal analysis based on morphological and functional alterations. the identification of tooth grinding and clenching was carried out by self-reports. statistical analysis was based on chisquare and multivariate logistic regression analyses. p-value for all statistical analysis was set at 5%. results: according to rdc/tmd, 18.4% of subjects experienced myofascial pain (gmpd), and 12.4% had joint disorder with disc displacement (g-dd). tooth clenching was statistically associated with tmd (p=0.000). in the occlusal factors, overjet showed statistically significant correlation only with myofascial pain. no association between functional alteration and tmd was found. no statistically significant correlation was found between g-dd and occlusal alterations or parafunctional habits. conclusions: overjet and tooth clenching were correlated with g-mpd. occlusal alterations or parafunctional habits did not show correlation with g-dd. keywords: myofascial pain syndromes; temporomandibular joint disorders; bruxism; dental occlusion. introduction epidemiological studies are performed to determine the prevalence of temporomandibular disorder (tmd) in various populations. higher prevalence rates are observed in patients who have sought some sort of treatment, compared to non-patient populations1. however, diseases and disorders cannot be understood only by the study of people seeking treatment, but also by the expression of the disease in the population as a whole2. currently, tmd can be considered the most frequent cause of chronic orofacial pain, and its most common symptoms are pain and/or tenderness in the preauricular region, cervical and masticatory muscles; restricted or deflection mandibular movements; and temporomandibular joint (tmj) sounds3-4. this dysfunction has multifactorial etiology5-6, and biomechanical, neuromuscular, biopsychosocial and neurobiological factors may contribute to the disorder7. these factors are classified as predisposing (structural, metabolic and/or psychological conditions), initiating braz j oral sci. 13(4):281-287 282282282282282 (trauma or repetitive adverse loading of the masticatory system) and aggravating (parafunctional, hormonal or psychosocial factors) to emphasize their roles in the progression of tmd8-9. studies have evaluated the important role of occlusal alteration in the etiology of tmj disorders. some results suggested that tmd was associated with posterior crossbite, anterior open bite, angle class iii malocclusion, extreme maxillary overjet, discrepancy between centric relation and maximal intercuspation position, interference on the nonworking side, absence of effective canine guidance and occlusal instability5-6,10-12. although occlusion is commonly considered to be a major risk factor for tmd, there is limited un derstanding of the causal relationship between the occurrence of tmd symptoms and occlusion, and of the possible role of different aspects of occlusion in the etiology of tmd13. parafunctional habits such as bruxism and tooth clenching might increase the risk of developing tmd14-15; when the adaptive capacity of the joint is exceeded16. bruxism and clenching reportedly leads to joint space reduction, followed by disc compression and resulting pain in masticatory muscles17. considering the multiplicity of symptoms of tmd, a standardized diagnostic system with proper intraoral and extraoral exams is required to assess risk factors and to identify conditions requiring prevention and treatment. for this purpose, classification systems have been proposed and used by many epidemiological studies. thus, the introduction of an index called the research diagnostic criteria for temporomandibular disorders (rdc/tmd) aimed to standardize the diagnosis and classification of different clinical settings of tmd18-19. in this context, the aim of this study was to determine the prevalence of tmd in undergraduate students considered a non-patient population, to investigate a potential correlation among some occlusal factors and parafunctional habits, using the rdc/tmd as a diagnostic measure, based on a clinical exam. the tested hypothesis is that the etiology of dysfunction is related to occlusal variables and parafunctional habits. material and methods the research protocol was approved with number 373/ 08 prior to study initiation. two hundred and one undergraduate students between 17 and 34 years of age, from the same university and with a mean age of 20.5 years were enrolled. participants’ selection and tmd diagnosis all participants answered a questionnaire developed for this study, which contained the inclusion and exclusion criteria11, and questions related to parafunctional habits. subjects who had undergone previous occlusal adjustment, extracted teeth, except third molars and premolars for orthodontic reasons, or who wore occlusal splints in the last six months were excluded from this study. in addition, subjects with any history of severe facial trauma, relevant head and neck pathologies, systemic diseases, or drugs that may reflect muscle activity were not included in order to obtain a homogeneous sample with similar characteristics. all other students continued their participation in the research. the identification of parafunctional habits was carried out by self-reports. the undergraduate students answered the following questions: 1are you aware of grinding and/or clenching your teeth? and 2have ever your parents, siblings or bed partners already heard grinding sounds? later, medical history was reviewed and clinical examination was performed according to the rdc/tmd guidelines18 using the validated brazilian version of the rdc/tmd instrument available since 2010 on the rdc/tmd consortium web site20. all steps were undertaken by a single calibrated examiner. in this study, only axis i was considered, and this axis can be divided into three subgroups. for each person, this axis provides the score and obtains the single or combined rdc/ tmd group diagnosis. the first subgroup (g-mpd) is related to myofascial pain disorder with or without limited opening; the second subgroup (g-dd) comprises disc displacement with reduction, disc displacement without reduction with limited opening, and disc displacement without reduction and without limited opening. finally, the third subgroup comprises degenerative joint diseases (g-djd) namely arthralgia, osteoarthritis and osteoarthrosis. only the subgroups g-mpd and g-dd were considered in this study due to lack of complementary exams to validate the g-djd diagnostic group. occlusal examination occlusal analysis was carried out without knowledge of the rdc/tmd results from each patient. evaluation of occlusal alterations was carried out into two steps: the first a functional or dynamic analysis, verifying the discrepancy in position between the centric relation (cr) and maximal intercuspal position (mip), occlusal interferences in lateral movements and protrusion. the second step was morphological or static analysis, based on measurements and observations of the morpho-skeletal characteristics, such as overjet (normal value <4 mm), overbite (normal value > 0 < 5 mm), anterior/ posterior open bite and anterior/posterior crossbite11. discrepancy between jaw positioning in cr and mip was observed using lucia jig for muscle relaxation. after approximately five minutes of use, this device eliminates the reflex arc responsible for the acquired mandible closure trajectory in mip, determined by memory traces and the teeth 21. subsequently, mandible manipulation (frontal technique) was used to identify the centric relation position until first contact between opposing teeth (premature contact) occurred. at this time, the teeth in contact were marked with a pencil registering the cr position. the maxillary tooth was used as a fixed reference point; subsequently, the subject was instructed to open and close the mouth in mip. thus, the mark present in the mandibular tooth enabled other demarcation in the maxillary arch in this new position. the correlation between temporomandibular disorders, occlusal factors and oral parafunction in undergraduate students braz j oral sci. 13(4):281-287 283283283283283 distances between the maxillary markers were measured and reported as discrepancy between cr and mip in the anteriorposterior direction. group function, mesiotrusion and laterotrusion interferences during lateral movements, and posterior interferences during protrusion were identified using the double-sided articulator film (accufilm ii ® parkell, farmingdale, ny, usa). discrepancy between cr and mip >2 mm, mesiotrusion and laterotrusion interferences, posterior interference during protrusive movement, and mouth opening less than 40 mm were also considered functional alterations. statistical analysis the statistical analysis was performed using the statistical package for social sciences 15.0 (spss, inc., chicago, il, usa). the chi-square (x2) test and multivariate logistic regression analysis were used. in x2 test, each variable was tested individually to find some kind of dependence on the diagnostic groups. self-reported parafunctional habits by participants were also included in the statistics to identify any correlation. two multivariate logistic regression models were created (g-mpd and g-dd) to identify significant associations with occlusal factors and parafunctional habits. in this analysis, all variables were tested simultaneously for each group, promoting an inter-relationship between them and simulating the standard multifactorial etiology of tmd. the tested hypothesis was accepted when p<0.05. results among the 201 subjects, 146 (72.6%) were women and 55 (27.4%) were men. according to the diagnoses obtained by the rdc/tmd, 18.4% participants (30 women and 7 men) were in g-mpd, and 12.4% (22 women and 3 men) in g-dd. all disc displacement cases were with reduction (table 1). analysis of the results by the x2 test revealed no statistically significant difference between gender and prevalence of both disorders (p=0.141). tooth clenching was reported by 85 participants (42.3%), and 26 of them were classified in g-mpd and 13 in g-dd. tooth grinding was reported by only 26 subjects (12.9%), 7 classified in g-mpd and 6 in g-dd. according to the chisquare test, only tooth clenching showed statistically significant correlation with g-mpd (p=0.000) (table 1). the x2 test did not show any statistically significant association between occlusal alterations and dysfunction groups (tables 2 and 3). in the logistic regression model related to g-mpd, only tooth clenching and overjet showed some degree of correlation with myofascial pain (table 4). the model presented r2=0.108. the logistic regression model related to g-dd did not show any statistically significant association with occlusal alterat ions or parafunctional habits. discussion the hypothesis that the etiology of dysfunction is related to occlusal factors and parafunctional habits was partially confirmed. overjet and tooth clenching showed statistically significant correlation with myofascial pain. the basic premise of the population’s perspective is that diseases and disorders cannot be understood exclusively by the study of persons seeking treatment. rather, to understand a disease, one must understand the expression of the disease in the population as a whole. clinical samples reflect not only the manifestations of the disease itself, but also all the biological, psychological and social factors associated with an individual’s motivation to seek care and with access to correlation between temporomandibular disorders, occlusal factors and oral parafunction in undergraduate students braz j oral sci. 13(4):281-287 table 1table 1table 1table 1table 1. contingency table between variables: parafunctional habits (grinding and clenching teeth) and tmd diagnosis (g-mpd and g-dd) (n total = 201, % total = 100) 20 156 6 1 9 284284284284284 table 3table 3table 3table 3table 3. contingency table between occlusal functional alteration and tmd diagnosis (g-mpd and g-dd) (n total = 201, % total = 100) correlation between temporomandibular disorders, occlusal factors and oral parafunction in undergraduate students braz j oral sci. 13(4):281-287 table 2. table 2. table 2. table 2. table 2. contingency table between occlusal morphological alteration and tmd diagnosis (g-mpd and g-dd) (n total = 201, % total = 100) 142 22 68 9 6 285285285285285 care2. thus, to determine the prevalence of tmd in this study, a population of students as a non-patient population was assessed. among the studied subjects, 18.4% were diagnosed with myofascial pain and 12.4% with disc displacement with reduction. muscle disorders group was the most frequent diagnosis and this value is moderately higher than those found in a prior study performed in non-patient populations1. it is possible that these small prevalence variations occur due to socioeconomic and cultural differences inherent to each population. however, it is puzzling that such a high number of undergraduate students did not seek treatment since it is an informed population with access to the health system. although a general population perspective is that half of the cases of temporomandibular disorder pain have never sought treatment, and only one-quarter sought treatment in the past six months22, it is worrisome that other brazilian non-patient populations may present prevalence rates equal to or higher than these. more studies should be performed in brazil so that new health policies can be introduced in the country. in general, tmd occurs in populations over 18 years of age; it is primarily a condition of young and middle-aged adults, rather than of children or elderly, and it is approximately twice as common in women as in men2. this study in an adult population between 17 and 34 years did not observe statistically significant correlation between gender and tmd, but a female predominance was observed in g-mpd and g-dd. the higher prevalence rates for adult women than for adult men may indicate that biologic, behavioral, psychological, and/or social factors associated with female gender increase the risk of experiencing pain in the temporomandibular region2. a significant association between tooth clenching and g-mpd was observed. this probably can be explained by the association of muscle tension in the jaw, face, head or a combination of them. this is caused by parafunctional behavior, which is strongly related to levels of jaw and facial pain23. repetitive strain injury to the muscle, resulting from parafunctional activities such as teeth clenching or grinding may cause pain in the masticatory muscles by the induction of localized tissue ischaemia and/or release of algogenic substances such as serotonin or glutamate to excite and sensitize muscle nociceptors16. it is possible that the longer the clenching habit the more likely the development of tmd signs and symptoms. no statistically significant correlation between tooth table 4. table 4. table 4. table 4. table 4. variables remaining at the end of the multivariate logistic regression equation: g-mpd diagnosis positive versus negative grinding and tmd was achieved, but this might be attributed to the low prevalence of this disorder in the studied population. complete absence of correlation may have been neglected, because tooth grinding episodes act as microtraumas in the stomatognathic system, which can precipitate pain and system changes4. further studies need to be carried out with methodologies specific to bruxism diagnosis24. the complete clinical examination to assess the impact of bruxism on oral structures is important because self-reporting participants may eventually overor underestimate their tooth grinding and clenching habits24. as described in tables 2 and 4, although overjet was not considered statistically significant in the x2 test, the logistic regression analysis revealed that this was the only morphological variable associated with g-mpd, confirming previous results5-6,10,12. excessive overjet predisposes to large mandibular movements, most probably for functional reasons, speech articulation and bite, which may stress the masticatory muscles6. it is likely that the longer the clenching habit the more likely to develop signs and symptoms of tmd. furthermore, recent study showed that large overjet or anterior open bite associated with clenching had a significantly higher prevalence of combined diagnoses, namely, disorders involving both the jaw muscles and the temporomandibular joints25. functional occlusal alterations were more frequent than morphological alterations (tables 2 and 3), but showed no statistically significant correlation with muscle or joint disorders. high frequency of these occlusal variables was also found among undergraduate students without tmd. previous study asserts that it is difficult to determine associations between tmd and occlusion due to the high prevalence of occlusal interferences in the general population, so a standardized control group without occlusal disorders is not possible26. sometimes the control group is also compromised by the inclusion of patients with mild symptoms or adaptation27. to minimize this difficulty, these authors suggest conducting studies among populations based on a control group with minimal symptoms and on an experimental group with maximum degree of the disease27. multivariate logistic regression models were created to cluster the possible risk factors and presence of the disorder, considering its multifactorial character. analyses of g-mpd and g-dd showed considerably low r2 values, meaning that the model explains little about the results (tables 4 and 5). this low value was due to the low-frequency manifestations of the disorder among the study subjects. thus, even if the correlation between temporomandibular disorders, occlusal factors and oral parafunction in undergraduate students braz j oral sci. 13(4):281-287 associations found are not considered strong, it is known that tmd is multifactorial in origin and the association of several factors determines their etiology. these factors are classified as predisposition (structural, metabolic and/or psychological conditions), initiation (trauma or repetitive adverse loading of the masticatory system), and aggravation (parafunctional, hormonal, and psychosocial factors)8. thus, both stress and occlusal factors are required for development of this disorder, and occlusion is one of the causal factors10. other biopsychosocial factors such as depression and somatization disorders should not be underestimated28-30. due to its epidemiological nature, the study had to be conducted in a population composed by non-patients. however, one limitation of the study was its omission of a group of symptomatic patients to better fulfill the objective of determining correlations between occlusion variables, parafunctional habits and tmd. therefore, further studies should be performed with representative samples of patients with bruxism and occlusal morphological changes, in comparison with standardized control groups. in addition, prospective longitudinal studies should be conducted to observe fluctuation in the lifelong signs and symptoms of tmd, and the incidence and remission of cases of parafunctional habits. the follow-up of patients with occlusal changes that are currently asymptomatic should also be the focus of longitudinal studies, in order to determine the effects of these variations in the long term. in conclusion, only overjet and tooth clenching were correlated with myofascial pain. no occlusal alterations or parafunctional habits showed correlation with disc displacement with reduction. acknowledgements the authors are grateful to capes for financial support. no conflicts of interest declared. references 1. manfredini d, guarda-nardini l, winocur e, piccotti f, ahlberg j, lobbezoo f. research diagnostic criteria for temporomandibular disorders: a systematic review of axis i epidemiologic findings. oral surg oral med oral pathol oral radiol endod. 2011; 112: 453-62. 2. leresche l. epidemiology of temporomandibular disorders: implications for the investigation of etiologic factors. crit rev oral biol med. 1997; 8: 291-305. 3. manfredini d, chiappe g, bosco m. research diagnostic criteria for temporomandibular disorders (rdc/tmd) axis i diagnoses in an italian patient population. j oral rehabil. 2006; 33: 551-8. 4. mcneill c. management of temporomandibular disorders: concepts and controversies. j prosthet dent. 1997; 77: 510-22. 5. celic r, jerolimov v, panduric j. a study of the influence of occlusal factors and parafunctional habits on the prevalence of signs and symptoms of tmd. int j prosthodont. 2002; 15: 43-8. 6. pahkala r, qvarnström m. can temporomandibular dysfunction signs be predicted by early morphological or functional variables? eur j orthod. 2004; 26: 367-73. 7. suvinen ti, reade pc, kemppainen p, könönen m, dworkin sf. review of aetiological concepts of temporomandibular pain disorders: towards a biopsychosocial model for integration of physical disorder factors with psychological and psychosocial illness impact factors. eur j pain. 2005; 9: 613-33. 8. oral k, bal küçük b, ebeoðlu b, dinçer s. etiology of temporomandibular disorder pain. agri. 2009; 21: 89-94. 9. xie q, li x, xu x. the difficult relationship between occlusal interferences and temporomandibular disorder insights from animal and human experimental studies. j oral rehabil. 2013; 40: 279-95. 10. pahkala rh, laine-alava mt. do early signs of orofacial dysfunctions and occlusal variables predict development of tmd in adolescence? j oral rehabil. 2002; 29: 737-43. 11. landi n, manfredini d, tognini f, romagnoli m, bosco m. quantification of the relative risk of multiple occlusal variables for muscle disorders of the stomatognathic system. j prosthet dent. 2004; 92: 190-5. 12. thilander b, rubio g, pena l, mayorga c. prevalence of temporomandibular dysfunction and its association with malocclusion in children and adolescents: an epidemiologic study related to specified stages of dental development. angle orthod. 2002; 72: 146-54. 13. wang c, yin x. occlusal risk factors associated with temporomandibular disorders in young adults with normal occlusions. oral surg oral med oral pathol oral radiol. 2012; 114: 419-23. 14. magnusson t, egermarki i, carlsson ge. a prospective investigation over two decades on signs and symptoms of temporomandibular disorders and associated variables. a final summary. acta odontol scand. 2005; 63: 99-109. 15. carlsson ge, egermark i, magnusson t. predictors of signs and symptoms of temporomandibular disorders: a 20-year follow-up study from childhood to adulthood. acta odontol scand. 2002; 60: 180-5. 16. cairns be. pathophysiology of tmd pain — basic mechanisms and their implications for pharmacotherapy. j oral rehabil. 2010; 37: 391-410. 17. tanaka e, detamore ms, mercuri lg. degenerative disorders of the temporomandibular joint: etiology, diagnosis, and treatment. j dent res. 2008; 87: 296-307. 18. dworkin sf, leresche l. research diagnostic criteria for temporomandibular disorders: review, criteria, examinations and specifications, critique. j craniomandib disord. 1992; 6: 301-55. 19. dworkin sf. research diagnostic criteria for temporomandibular disorders: current status & future relevance. j oral rehabil. 2010; 37: 734-43. 20. international rdc-tmd consortium. tmd assessment/diagnosis. [cited 2014 jan 28]. available from: http://www.rdc-tmdinternational.org/ tmdassessmentdiagnosis/rdc-tmd/translations/portuguese (brazil).aspx. 21. nassar ms, palinkas m, regalo sc, sousa lg, siéssere s, semprini m, et al. the effect of a lucia jig for 30 minutes on neuromuscular reprogramming, in normal subjects. braz oral res. 2012; 26: 530-5. 22. von korff m, dworkin sf, le resche l, kruger a. an epidemiologic comparison of pain complaints. pain. 1988; 32: 173-83. 23. glaros ag, williams k, lausten l. the role of parafunctions, emotions and stress in predicting facial pain. j am dent assoc. 2005; 136: 451-8. 24. lobbezoo f, ahlberg j, glaros ag, kato t, koyano k, lavigne gj, de leeuw r, manfredini d, svensson p, winocur e. bruxism defined and graded: an international consensus. j oral rehabil. 2013; 40: 2-4. 25. manfredini d, vano m, peretta r, guarda-nardini l. jaw clenching effects in relation to two extreme occlusal features: patterns of diagnoses in a tmd patient population. cranio. 2014; 32: 45-50. 26. lipp mj. temporomandibular symptoms and occlusion: a review of the literature and the concept. ny state dent j. 1990; 56: 58-66. 27. pullinger ag, seligman da, gornbein ja. a multiple logistic regression analysis of the risk and relative odds of temporomandibular disorders as a function of common occlusal features. j dent res. 1993; 72: 968-79. 286286286286286correlation between temporomandibular disorders, occlusal factors and oral parafunction in undergraduate students braz j oral sci. 13(4):281-287 287287287287287 28. manfredini d, ahlberg j, winocur e, guarda-nardini l, lobbezoo f. correlation of rdc/tmd axis i diagnoses and axis ii pain-related disability. a multicenter study. clin oral investig. 2011; 15: 749-56. 29. dougall al, jimenez ca, haggard ra, stowell aw, riggs rr, gatchel rj. biopsychosocial factors associated with the subcategories of acute temporomandibular joint disorders. j orofac pain. 2012; 26: 7-16. 30. resende cm, alves ac, coelho lt, alchieri jc, roncalli ag, barbosa ga. quality of life and general health in patients with temporomandibular disorders. braz oral res. 2013; 27: 116-21. correlation between temporomandibular disorders, occlusal factors and oral parafunction in undergraduate students braz j oral sci. 13(4):281-287 oral sciences n3 original article braz j oral sci. april | june 2015 volume 14, number 2 periodontal status and treatment need among adolescents in ibadan, southwestern nigeria bamidele olubukola popoola1, elizabeth bosede dosumu2, joy ucheonye ifesanya1 1university of ibadan, college of medicine, faculty of dentistry, department of child oral health, ibadan, oyo, nigeria 2 university of ibadan, college of medicine, faculty of dentistry, department of periodontology and community dentistry, ibadan, oyo, nigeria correspondence to: bamidele o. popoola department of child oral health faculty of dentistry university college hospital and university of ibadan zip code: 200009, ibadan, oyo, nigeria. phone: +23 48028999960 e-mail: olubukolap@gmail.com abstract a previous nigerian study had reported high incidence of periodontal disease in young nigerians though global studies show the contrary. aim: to determine the periodontal status and treatment needs of adolescents attending private and public secondary schools in ibadan, south-western nigeria. methods: one thousand, five hundred and twenty two secondary school children in ibadan were assessed. sociodemographic data was obtained using a data extraction form. intraoral examination was performed on all participants. oral cleanliness was assessed using the plaque index of silness and loe, while periodontal health status and treatment needs were assessed using the community periodontal index of treatment needs (cpitn). data were analyzed with the statistical package for social sciences (spss) version 22. statistical significance was set at p<0.05. results: only eight participants (0.5%) had healthy component of periodontal status. the mean plaque index was 1.12±0.41. the cpitn scores varied significantly with the gender of participants (p<0.001). number of sextants affected by periodontal diseases varied significantly with the socioeconomic status (p=0.02). conclusions: gingival ill-health is prevalent among adolescent nigerians with a great need for oral hygiene instructions and professional cleaning. there is need for oral health education among this sub-population of nigerian children and provision of mobile dental clinics to help meet their treatment needs. keywords: periodontium; adolescent health; treatment outcome; student. introduction periodontal disease is a spectrum of health anomalies affecting the surrounding structures of the teeth including the gums, periodontal ligament and alveolar bone. these anomalies may be restricted to the gingiva (gingivitis) or may extend to the periodontal ligament space and alveolar bone (periodontitis). periodontitis may lead to tooth mobility, pathologic migration and eventual loss of teeth. the clinical signs of periodontal disease may appear at any age1 and epidemiological studies indicate that gingivitis, which varies in severity, is almost a universal finding in children and adolescents, while destructive periodontitis is of lower prevalence in younger individuals when compared with adults2. a nigerian study has reported a relatively high occurrence of deep pockets in young nigerians3. however, whether these pockets are true (pockets greater than 6 mm deep) or false pockets (less than or equal to 6 mm) was not specified. the first step in proper planning for oral health among young children is to establish the prevalent oral health problem, which in this case is the periodontal health condition. this will help to promote evidence-based intervention programs4. the community periodontal index for treatment needs is thus a valuable tool in assessment for health policy making and strategic planning for oral health implementation programs5 and this is true even among children. braz j oral sci. 14(2):117-121 received for publication: march 02, 2015 accepted: may 28, 2015 http://dx.doi.org/10.1590/1677-3225v14n2a04 118118118118118 epidemiological trends have shown waxing and waning in both oral cleanliness and occurrence of gingivitis in children and adolescents. deterioration appears to be more evident when the age range of subjects studied is wide6. an earlier nigerian study in ile-ife reported a prevalence of periodontal diseases of 84.2% among 3-20-year olds3, while a more recent study in benin city revealed a prevalence rate of 99.2% among 5-19-year old7. both studies reported a high occurrence of gingivitis and heavy deposits of calculus but little evidence of damage to the periodontal tissues. however, these studies used the gingival index of loe and silness, plaque index of silness and loe and the oral hygiene index of greene and vermilion in their assessment. in a study among institutionalized mentally handicapped nigerian children using the cpitn8, a high level of gingivitis and periodontal diseases was reported, with 92.9% of assessed sextants involved in periodontal tissue changes. a similar finding was reported in a similar population of iranian children9. a study comparing public and private school children found that presence of calculus and gingivitis was less in private school children10. this suggests that oral hygiene was better when socio-economic status was high, as it has been previously reported11. another study has reported that only 17.8% of 11-14 years old nigerian children had good oral hygiene, while about 50.4% had fair oral hygiene12. this study corroborated the gender difference in oral cleanliness reported by previous studies7,10, with female children exhibiting better hygiene status than males. it has also been reported that severity of gingivitis lessens as age increases13. this study aimed to describe the pattern of periodontal health status of secondary school adolescents in private and public institutions in ibadan as well as their treatment needs. material and methods in this study, children aged 11 to 16 years old from secondary schools in ibadan, nigeria were examined for periodontal diseases. sample size was determined using the formula for determining the sample for cross-sectional studies assessing qualitative variables as shown below: n= z α 2 p(1-p)/d2 where: n= minimum sample size z α = standard normal variety at p<0.05 =1.96 p= prevalence of periodontal diseases among children in this environment from a previous study =71% =0.71 1-p= 29% =0.29 d= minimum acceptable error=5%. 1.962x0.71x0.29/0.052 n=316. the participants for the study were selected from public and private secondary schools in five local government areas in ibadan metropolis. the list of all public and private secondary schools in these local government areas was obtained from oyo state ministry of education and 20 schools were selected using proportionate sampling method. ethical clearance for this study was obtained from oyo state ministry of health and permission to visit the schools was obtained from the principals of the various schools. these schools have on average 3 branches per class with approximately 30 students in each class. at each school, total population samples of students who gave assent and whose parents consented to their participation in the study were collated. random selection from the classes by simple random technique using ballot process was used to obtain the study participants. demographic data for each participant were obtained and socioeconomic status assessed according to the criteria of oyedeji14 (1985). oral examinations were conducted by the field team that included two dentists (pbo & iju). intra oral examinations were done under natural light outdoors with sterile mouth mirrors and who cpitn probe. examination for periodontal disease was carried out using the community periodontal index for treatment needs as instituted by ainamo et al.15 (1982). the index teeth in each sextant were examined by running the cpitn probe around the entire sulcus of each tooth and the highest score recorded. the participants were categorized as: score 0= healthy periodontium score 1= gingival bleeding after gentle probing score 2=supra or sub-gingival calculus score 3=pathologic pockets 4-5.99 mm deep score 4= pathologic pocket 6 mm and above. treatment needs were classified according to the highest score recorded in all assessed sextants. tn 0=no need for treatment tn 1=need for oral hygiene instructions tn 2=need for oral hygiene instructions and professional cleaning tn 3=need for complex treatment. oral cleanliness was assessed using the plaque index of sillness and loe16. calibration of the two examiners was done by repeat examination of 20 children on two occasions. the results were subjected to cohen’s kappa statistics and intra class correlation coefficient for inter and intra-examiner reliability; values were 0.8 and 0.8 respectively. data obtained were analyzed using the statistical package for social sciences (spss) version 22. descriptive statistics was used to summarize the variables in the data set. qualitative variables were assessed using the chi square test, while the independent sample student t-test and anova were employed to test association involving descriptive data in two or more groups. level of significance was set at p<0.05. results a total of 1522 secondary school children were seen. mean age was 13.5±1.1years. other socio-demographic variables are as shown in table 1. periodontal status and treatment need among adolescents in ibadan, southwestern nigeria braz j oral sci. 14(2):117-121 119119119119119 variables n (%) gender male 629(41.3) female 893(58.7) total 1522(100.0) socioeconomic status high 213(14.0) middle 878(57.8) l o w 430(28.2) total 1522 (100.0) school private 605(36.8) public 917(60.2) total 1522(100.0) table 1:table 1:table 1:table 1:table 1: socio-demography of study participants variables cpitn scores and status 0. no sign of disease 1. gingival bleeding after gentle probing 2. supra or sub-gingival calculus 3. pathologic pockets 4-5 mm deep 4. pathologic pockets >6 mm cpitn treatment needs 0. no needs for treatment 1. need for oral hygiene instructions 2. need for professional cleaning and oral hygiene instructions 3. need for complex periodontal treatment mean number of sextant affected per cpitn score per child 0. healthy 1.83 1. bleeding 3.09 2. calculus 3.89 4. shallow pockets 1.23 n (%) 8 (0.5) 158 (10.4) 1313 (86.3) 43 (2.8) 0 (0.0) 8 (0.5) 158 (10.4) 1356 (89.1) 0 (0.0) proportion of children exhibiting the cpitn score. 499 (32.8) 824 (54.3) 1443 (94.8) 53 (3.5) table 2:table 2:table 2:table 2:table 2: cpitn scores, treatment needs and sextant affectation of the study participants none of the children had a missing or edentulous sextant. of the 9132 sextants assessed, 8219 (90.0%) were involved in periodontal tissue changes. only eight children (0.5%) had no sign of periodontal disease as assessed with the cpitn. periodontal health status and treatment needs as well as the mean number of sextants affected by periodontal diseases in the children assessed are as shown in table 2. the mean plaque index was 1.12±0.41. the males had higher plaque scores than females (p<0.001). the cpitn scores varied significantly with oral cleanliness as assessed by the plaque index (p<0.001). though younger adolescents (age 11 to 13) had worse plaque scores than older adolescents (14 to 17 years old), there was no significant difference in the plaque scores on the basis of age (p=0.06). the cpitn scores did not vary significantly with the age group (p=0.12), socioeconomic status (p=0.12), nor with the type of school attended by the children (p=0.37). in contrast, cpitn scores varied significantly with the gender p<0.001. while more males had cpitn scores 0 and 1, more females had scores 2 and 3 (table 3). the number of sextants affected by periodontal diseases increased as the socioeconomic status of the children worsened (p=0.02). though females had more sextants affected by periodontal diseases than males, this relationship was not statistically significant (p=0.05). discussion this study has observed a high prevalence of adverse periodontal tissue changes among nigerian adolescents with about 90% of the sextants assessed involved in various stages of periodontal ill health. this figure slightly improves on but is similar to that reported by denloye8 among mentally handicapped children. the present finding is an improvement because our participants have better musculoskeletal capability than the handicapped children. according to the who stipulated goal for periodontal health among children aged 15 years as at the year 2010, 100% of 15-year-olds should have at least five healthy sextants5. however, findings from this study demonstrated that less than one third of the studied population had less than two healthy sextants. this finding falls severely short of the global goal for periodontal health and implies that there is still a great periodontal health need among these children. the study also found that the plaque score was higher among children with cpitn scores 1, 2 and 3. this is similar to the observation in an iranian study among 15-19-year olds 5, and further asserts the relationship between plaque and gingival health17. contrary to a previous study18, this study observed that there was no significant association between periodontal tissues status and the type of school attended or to the socioeconomic status of the children. however, we did find periodontal status and treatment need among adolescents in ibadan, southwestern nigeria braz j oral sci. 14(2):117-121 120120120120120 table 3:table 3:table 3:table 3:table 3: relationship between cpitn scores and socio-demographic factors variables cpitn score 0 1 2 3 total p-value socioeconomic status high 1(0.5) 12(5.6) 194(91.1) 6(2.8) 213(100.0) p=0.10 middle 5(0.6) 90(10.3) 762(86.8) 21(2.4) 878(100.0) chi sq. 10.52 l o w 2(0.5) 56(13.0) 357(82.8) 16(3.7) 429(100.0) total 8(0.5) 158(10.4) 1313(86.3) 43(2.8) 1522(100.0) gender male 5(0.8) 101(16.1) 506(80.4) 17(2.7) 629(100.0) p<0.001 female 3(0.3) 57(6.4) 807(90.4) 26(2.9) 893(100.0) chi sq. total 8(0.5) 158(10.4) 1313(86.3) 43(2.8) 1522(100.0) 39. 02 school private 4(0.7) 53(8.8) 531(87.8) 17(2.8) 605(100.0) p=0.37 public 4(0.4) 105(11.4) 782(85.3) 26(2.8) 917(100.0) chi sq. total 8(0.5) 158(10.4) 1313(86.3) 43(2.8) 1(100.0) 3.16 that children of lower socioeconomic class had more diseased sextants than those from higher social class. this suggests a need for a firmer surveillance system that will enable proper measurement of both the extent of spread and control of oral health conditions as well as impact of oral health promotion strategies19. the prevalence of periodontal disease in the studied group is high and is accounted for largely by gingival bleeding, presence of calculus and shallow pockets. this is similar to previous findings in this environment based on other assessment criteria3,7,12 and has been attributed to the combined effect of dental cleaning ability as reflected by the association between gingival health scores and plaque scores5. change in bacterial composition of the dental plaque, inflammatory cell response and the hormones of puberty are other factors which may be responsible for this poor gingival health20. this study also found no difference between periodontal health and socio-economic status of all the children assessed. this is contrary to a previous report that has documented worsened periodontal status in children of lower socioeconomic strata10,21. however, since the present study also found that children in the lower socioeconomic class had more sextants affected by periodontal pathologic changes, the absence of an association between the previous variables may not be the true status of association. this occurred because the methods of classifying socioeconomic strata in the previous studies were not specified. on the other hand, the observed lack of association may be a pointer to the emergence of an alarming trend where high social class parents are beginning to ignore their children’s oral health care. in congruence with previous studies12,21, the females had better oral cleanliness than males. we also found a significant relationship between gender and gingival health in the present study as in previous documentations7,12,10,21. however in this study, more boys had healthy gingival status and cpitn score 1 than the girls, who had more calculus accumulations and shallow pockets. considering that the girls cleaned better that their male counterparts, the effect of the puberty hormones especially oestrogen and progesterone which are responsible for most of the modulator actions seen in hormone based gingivitis22-23 may be responsible for this finding. especially since these hormonesare found in higher concentration in females. in conclusion, gingival ill-health is prevalent among adolescent nigerians resident in ibadan oyo state with a great need for oral hygiene instructions and professional cleaning. there is need for oral health education among this sub-population of children and provision of mobile dental clinics to help meet their treatment needs. references 1. mârþu s, solomon s, potârnichie o, ãrin lpãs, mârþu a, nicolaiciuc o, et al. evaluation of the prevalence of the periodontal disease versus systemic and local risk factors. int j med dent. 2013; 3: 212-8. 2. chauhan vs, chauhan rs, devkar n, vibhute a, more s. gingival and periodontal diseases in children and adolescents. j dent allied sci. 2012; 1: 26-9. 3. akpata es. oral health in nigeria. int dent j. 2004; 54: 361-6. 4. folayan mo, adeniyi aa, chukwumah nm, onyejaka n, esan ao, sofola oo, et al. programme guidelines for promoting good oral health for children in nigeria: a position paper. bmc oral health [internet]. 2014 jan [cited 2015 feb 26]; 14: 128. 5. sane a-s, nikhbakht-nasrabadi a. periodontal health status and treatment needs in iranian adolescent population. arch iran med. 2005; 8: 290-4. 6. hatem ae. epidemiology and risk factors of periodontal disease. in: manakil dj, editor. periodontal diseasea clinician’s guide. intech; 2012. p.213-30. 7. odai cd, azodo cc, braimoh om, obuekwe on. oral health profile of primary and post primary school children at a health facility in uselu, benin city. benin j postgrad med. 2009; 11: 34-9. 8. denloye oo. periodontal status and treatment needs of 12-15 year old institutionalized mentally handicapped school children in ibadan , nigeria. odontostomatol trop trop. 1999; 86: 8-10. periodontal status and treatment need among adolescents in ibadan, southwestern nigeria braz j oral sci. 14(2):117-121 121121121121121 9. nematollahi h, makareem a, noghani ar. periodontal treatment needs amongst 9-14 year old institutionalized mentally retarded children in mashhad, iran. shiraz univ dent j. 2010; 10: 15-20. 10. adenubi jo. the gingival health of eight year old nigerian children. j public health dent. 1984; 44: 67-72. 11. khan ma, khan d, qureshi zur. dental ailments among low and high socioeconomic status school children aged 11-12 years. pakistan oral dent j. 2011; 31: 388-91. 12. kolawole ka, oziegbe eo, bamise ct. oral hygiene measures and the periodontal status of school children. int j dent hyg. 2011; 9: 143-8. 13. pauraite j, milciuviene s, sakalauskiene j. the prevalence of gingivitis among 4-16 year old schoolchildren in kaunas. stomatol balt dent maxillofac j. 2003; 5: 97-100. 14. oyedeji ga. socioeconomic and cultural background of hospitalised children in ilesha. nig paed j. 1985; 12: 111-7. 15. ainamo j, barnes d, beaagrie g, cutress t martin j, sardo-infirri j. development of the world health organization (who) community periodontal index of treatment needs. int dent j. 1982; 32: 281-91. 16. silness j, loe h. periodontal disease in pregnancy ii: corelation between oral hygiene and periodontal condition. acta odontol scand. 1964; 22: 121-35. 17. fukuda h, ogada cn, kihara e, wagaiyu eg, hayashi y. oral health status among 12-year-old children in a rural kenyan community. j dent oral heal. 2014; 2: 1-5. 18. awad f, idris i. periodontal disease prevalence and some related factors among 15 years old school children in khartoum state, sudan. sudan j public health. 2010; 5: 187-92. 19. petersen pe, ogawa h. strengthening the prevention of periodontal disease: the who approach. j periodontol. 2005; 76: 2187-93. 20. borrell ln, papapanou pn. analytical epidemiology of periodontitis. j clin periodontol. 2005; 32: 132-58. 21. agbelusi ga, jeboda so. oral health status of 12 year old nigerian children. west afr j med. 2006; 25: 195-8. 22. güncü gn, tözüm tf, ça”layan f. effects of endogenous sex hormones on the periodontium – review of literature. aust dent j. 2005; 50: 138-45. 23. bhardwaj a, bhardwaj sv. effect of androgens , estrogens and progesterone on periodontal tissues. j orofac res. 2012; 3: 165-70. periodontal status and treatment need among adolescents in ibadan, southwestern nigeria braz j oral sci. 14(2):117-121 oral sciences n3 original article braz j oral sci. january | march 2014 volume 13, number 1 dental caries in mother-child pairs from xingu lucila brandão hirooka1, wilson mestriner-junior2, soraya fernandes mestriner2, selma aparecida chaves nunes2, pablo natanael lemos3, laércio joel franco1 1universidade de são paulo usp, medical school of ribeirão preto, department of community medicine, ribeirão preto, sp, brasil 2universidade de são paulo usp, school of dentistry of ribeirão preto, department of pediatric dentistry, ribeirão preto, sp, brasil 3universidade de são paulo usp, project xingu, são paulo, sp, brasil correspondence to: laércio joel franco departamento de medicina social, faculdade de medicina de ribeirão preto universidade de são paulo, avenida bandeirantes, 3900 cep: 14049-900 ribeirão preto, sp, brasil phone: +55 16 6022536 fax: +55 16 6331386 e-mail: lfranco@fmrp.usp.br received for publication: december 05, 2013 accepted: march 17, 2014 abstract aim: to describe cases of dental caries in indian mother-child pairs of the middle and lower xingu river – xingu indigenous park. methods: a total of 246 children aged 3-5 years old and their respective mothers took part in this study. caries indexes dmft and dmft were analyzed for deciduous and permanent dentitions, respectively, according to criteria proposed by the world health organization. results: analysis of the dmft index showed a mean value of 14.3 for mothers. in mothers aged between 35 and 44 years, tooth loss accounted for more than 80% of the total index score. with regard to the children, dmft index was 4.7, on average, and only 13.4% were caries-free. no significant correlation was found between mother and child caries experience (p = 0.16). conclusions: these results suggest that it is important to consider the community as a whole, and not only the mother, regarding the oral health of indigenous children. community should be involved in the planning of strategies for caries prevention and health promotion, taking into account the socio-cultural complexity and specificities of indigenous population and adapt them to the local reality for these strategies to become actually effective. keywords: oral health; dental caries; mother-child relations; indians; south american. introduction oral health is a determining factor for quality of life and personal development, and according to the world health organization (who), political, social, economic, cultural, environmental, behavioral and biological factors may improve or impair oral health1. with regard to mother-child relationships, microbiological studies have associated the mother’s salivary levels of s. mutans to the degree of colonization and/or caries experience in her children2. other studies show a positive relationship between mother and child caries experience, suggesting that this is a good predictor of caries risk in children3. in the etiological context of caries disease, bacterial infection is necessary but it is not the sole factor contributing to its development, since occurrence and severity of caries in a population result from an interaction with the causing elements4. dental caries in childhood is strongly related to the mother, who not only is a transmitter of bacteria but also the model of habits, values and attitudes for her child5. according to arantes6 (2010), it is possible to notice a common course of the oral health in indigenous people when they have permanent contact with settler societies. socioeconomic, cultural and political changes resulting from such a process interfere with the subsistence food supply and introduce new braz j oral sci. 13(1):43-46 gisele higa texto digitado http://dx.doi.org/10.1590/1677-3225v13n1a09 44 types of foods, thus altering oral health patterns. however, socioeconomic, environmental and cultural determinants, which are highly complex and diversified, assume a particular role in relation to indigenous ethnic groups. the xingu indigenous park (xip) was established in 1961, according to the act 51.084 signed by the then president of brazil on 31 july, 1961, mainly aiming to ensure medical, social and educational support for indigenous people so that they could survive physically and have their culture preserved7. partnerships with national health foundation (funasa), federal university of são paulo (unifesp) and the school of dentistry of ribeirão preto (forp-usp) have been established in order to promote an improvement in the oral health conditions of the middle and lower xingu river population. this work is intended to describe dental caries experience in mother-child pairs living in the xip (middle and lower xingu region) in 2007. material and methods a descriptive study was performed from data collected by the epidemiological survey on oral health in middle and lower xingu conducted in 2007. the entire xip’s indigenous population covering the area of the middle and lower xingu were invited to participate in the study, provided that they were present at the moment of examination. a total of 1,911 individuals were examined, 83.12% of the population. the clinical examinations were performed by 5 calibrated examiners (kappa = 0.91) assisted by oral health indigenous agents (aisb), with natural light, using a dental mirror and who probe (hu-friedy, chicago, il, usa), using the methodology recommended by the who8. data were processed using the brazilian ministry of health’s sb data software9. local of research the xip covers an area of 2.8 million ha in the northern region of mato grosso state, brazil. in the middle and lower xingu region, there are approximately 2,299 indigenous individuals from several ethnical groups, including kisêdje, tapayuna, kaiabi, ikpeng, yudjá, mehinaku, waurá, panará, kamaiurá and trumai. study population children aged between 3 and 5 years who participated in the epidemiological survey with their respective mothers were selected for study. six mother-child pairs were excluded because their mothers were older than 45 years. therefore, 246 mothers and 246 children were examined. mother and child experience of dental caries was analyzed using dmft and dmft indexes, respectively. the research project was approved by the research ethics committee of the school of dentistry of ribeirão preto, university of são paulo (process 2010.1.33.58.0). for data analysis, tables and graphs were used as well as distribution of absolute and relative frequencies, in addition to mean values and standard deviations at significance level of 5%. spearman’s correlation coefficient was employed to verify the correlation between dmft and dmft indexes. results the mean age of the mothers was 27.7 years old, with minimum of 15 and maximum of 44 years. analysis of the dmft index showed a mean value of 14.3 for mothers. in the age group of 35-44 years, tooth loss was the component responsible for more than 80% of the total index scores (table 1). analysis of the dmft index showed a mean value of 4.7 for children, reaching maximum value (6.4) in the age group of 60-71 months old. caries was the component most contributing to dmft index in all age groups. the prevalence of dental caries in indigenous children was 86.6%. in the age group of 48-59 months, it was observed that 95.2% of the children had already experienced dental caries in their deciduous dentition (table 2). no significant correlation was found between mother and child dental caries experience (p = 0.16). table 1. table 1. table 1. table 1. table 1. mean (standard deviation) and percentage of dmft index components according to mother age groups. table 2. table 2. table 2. table 2. table 2. mean (standard deviation) and percentage of dmft index components according to child age groups. dental caries in mother-child pairs from xingu braz j oral sci. 13(1):43-46 45 discussion the oral health of mother-child pairs was characterized by high prevalence of dental caries in children and significant tooth loss in mothers. also, the mother and child experience of dental caries tended to increase with age, a common phenomenon if one considers the cumulative character of dmtf and dmft indexes10. comparing the data on the children’s caries experience to those obtained from the brazilian ministry of health national epidemiological survey on oral health conditions9, it may be observed that, at 5 years of age, the mean dmft values were greater than those reported in this study (6.4 vs. 2.8, respectively). with regard to the prevalence of dental caries, the national rate is almost 60%, whereas the great majority of the indigenous children experienced caries in at least one deciduous tooth (94.5%), which is far from the who proposed goal for the year 2000 (50% of caries-free children). the severity of children’s dental caries, expressed by dmft index values found in the present study, is close to the one reported by carneiro et al.11 (2008) for baniwa 5-yearold children (6.4 vs 6.3, respectively). for the author, besides the factors related to high levels of caries observed in that community, it should be pointed out that the distribution of oral hygiene materials by the alto rio negro indian special sanitary district to a high-risk population was interrupted. in the xip, the problem regarding distribution of oral hygiene material is alleviated by donations from dental manufacturers. however, not only distribution is currently unavailable, but also there is neither enough material nor logistics for the delivery to the villages. an increase in the proportion of tooth loss with age was observed in indigenous mothers. detogni12 (1994) states that xingu native population accept “naturally” the dental loss as if the absence of a substantial number of teeth was something to be expected in life, a fact possibly associated to the aggravating process of oral health conditions. the high contribution of tooth loss among mothers may reflect a greater access to dental services, but it may also mean difficult access to secondary healthcare services. in the xip, high demand, difficult transportation from village to town, and low supply of secondary services in the neighboring towns, among other factors, compromise the establishment of reference and counter-reference networks. despite the importance of these networks, we agree with palmier et al.13 (2012), who state that socio-economic changes play a more prominent role in the reduction of dental caries indexes among children than the contribution of healthcare services. several authors have studied the association between oral health conditions of mothers and children. noce et al.14 (2008) reported that the presence or absence of caries in children was not associated with the level of dental caries in their parents. in their study, no correlation between mother and child experiences of dental caries was observed. according to laitala et al.3 (2012), s. mutans colonization avoided at early ages may lead to favorable long-term effects on caries experience in children. however, caries is a multifactorial disease and no isolated variable, biological, social, economic or cultural, seems to be able to predict its development5. indigenous children are nurtured in an environment of great freedom, and the elderly pass their knowledge on through oral tradition to the young people. although the parents are the main caregivers, the wider socialization process of indigenous children is also conducted by close relatives as well as by the community as a whole15. according to mattila et al.16 (2000), attention to the children’s oral health alone is not enough. it is necessary to address the factors related to the family and community habits and lifestyle in order to promote health maintenance rather than treatment and control of the disease. therefore, it is important to consider the entire community and not only the mothers in the oral health of indigenous children. pacagnella17 (2007) observed an improvement in the caries levels during the 2001-2006 period in the xip for most of the age groups. such a decline may be related to various factors. the establishment of indigenous special sanitary districts in 1999 allowed political stability and increased the federal investment in the healthcare of indigenous people. as a result of the partnerships with forpusp and dental manufacturers, indigenous people started having wider access not only to dental services, but also to educative and preventive programs18. the partnerships also allowed the conduction of the “epidemiological survey of oral health conditions of middle and lower xingu” in 2007 to collect data on the oral health conditions and support the planning/evaluation of actions at different management levels in this area. this survey enabled the studies that clarify oral health conditions of the middle and lower xingu as well as mother-child pairs in this region. nevertheless, the high caries indexes found may be related to an increase in the contact between xingu people and settler’s society. the xingu indigenous park has a geographically limited area, which is subject to the impact of the advance of the agricultural activity of logging and mining fields. the emergence of new cities and highways in the neighborhood intensify the contact relationships, resulting in changes that made the people of the xingu indigenous park more vulnerable to certain diseases. a more sedentary lifestyle has been observed, a result of changes in the way of life due to the introduction of technologies and wages of indians who participate in various activities organized by institutions working in the xingu6. the increased mobility of the indigenous population and the visits to nearby cities have also contributed to the change in eating habits 19. industrialized food has been increasingly introduced and replacing the traditional foods, which impairs child nutrition and raises the levels of dental caries6,19. the model of health care that has was been consolidated seeks to provide answers to key problems arising from social and historical process of contact between indians and settlers, prioritizing intersectoral actions and the growing involvement of xingu population 20. furthermore, the importance of promotion and prevention of both the collective dental caries in mother-child pairs from xingu braz j oral sci. 13(1):43-46 46 and individual, decentralized and multiplicative, with the purpose of promoting the interaction of oral health teams, indigenous individuals and indigenous health agents, who are the pillars of the education process continued in communities. the current epidemiological scenario of the oral health of xingu indigenous mothers and children unravels the importance of evaluating periodically the oral healthcare services and re-structuring the already developed work by identifying new needs. the findings of this study show that the whole community, and not only the mothers, should be considered in the oral health of the indigenous children. the community should be involved in the planning of strategies for caries prevention and health promotion, adapted to local reality, so that they can be really effective. references 1. world health organization. the world oral health report, 2003. continuous improvement of oral health in the 21 st century – the approach of the who global oral health programe. geneve: world health organization; 2003. 2. zhan l, tan s, den besten p, featherstone jd, hoover ci. factors related to maternal transmission of mutans streptococci in high-risk childrenpilot study. pediatr dent. 2012; 34: 86-91. 3. laitala m, alanen p, isokangas p, söderling e, pienihäkkinen k. a cohort study on the association of early mutans streptococci colonisation and dental decay. caries res. 2012; 46: 228-33. 4. gomes d, daros ma. the etiology of caries: the construction of a thoughtstyle. ciênc. saúde coletiva 2008; 13: 1081-090. 5. reisine s, tellez m, willem j, sohn w, ismail a. relationship between caregiver’s and child’s caries prevalence among disadvantaged african americans. community dent oral epidemiol. 2008; 36: 191-200. 6. arantes r, santos rv, frazão p. oral health in transition: the case of indigenous peoples from brazil. int dent j. 2010; 60: 235-40. 7. brazil. decree-law n.º 51.084. 1961july 31. 8. world health organization. oral health surveys: basic methods. 4.ed. geneva: orh/epid; 1997. 9. brazil. health ministry of brazil. sb brazil 2003 project – oral health conditions of the brazilian population 2002-2003. brasília, df; 2004. 68p. 10. cogulu d, ersin nk, uzela a, eronat n, aksit s. a long-term effect of caries-related factors in initially caries-free children. int j paed dent. 2008; 18: 361-7. 11. carneiro mcg, santos rv, garnelo l, rebelo mab, coimbra jr cea. dental caries and need for dental care among the baniwa indians, rio negro, amazonas. ciec saude colet. 2008; 13: 1985-92. 12. detogni am. back to basics. rev abo nac. 1994; 2: 138-48. 13. palmier ac, andrade da, campos acv, abreu mhng, ferreira ef. socioeconomic indicators and oral health services in an underprivileged area of brazil. rev panam salud publica. 2012; 32: 22-9. 14. noce e, rubira cmf, rosa ops, silva smb, bretz wa. streptococcus mutans acquisition and dental caries development in first-born children. pesq bras odontoped clin integr. 2008; 8: 239-44. 15. silva al, macedo avls, nunes a. indigenous children anthropological essays. são paulo: global editora; 2002. 16. mattila ml, rautava p, sillanpaa m, paunio p. caries in five year-old children and associations with family-related factors. j dent res. 2000; 79: 875-81. 17. pacagnella rc. epidemiological profile of oral health of the population of the xingu indigenous park, between the years 2001 and 2006 [doctoral thesis]. ribeirão preto: faculty of medicine of ribeirão preto, são paulo university; 2007. 101p. 18. mestriner s f, lemos pn, hirooka lb, nunes sac, arantes r, mestriner jr w. the oral health care model in middle and low xingu: partnerships, processes, and perspectives. cienc saude colet. 2010; 15: 1449-56. 19. rodrigues d. the federal university of são paulo, the xingu project and policy attention to health of indigenous peoples. in: baruzzi r, junqueira c, editors. xingu indigenous park: health, culture and history. são paulo: federal university of são paulo /terra virgem; 2005. p. 259-73. 20. mesquita lp, lemos pn, hirooka lb, nunes sac, mestriner sf, taba jr m, et al. periodontal status of an indigenous population at the xingu reserve. braz j oral sci. 2010; 9: 43-7. dental caries in mother-child pairs from xingu braz j oral sci. 13(1):43-46 oral sciences n3 braz j oral sci. 13(3):229-234 original article braz j oral sci. july | september 2014 volume 13, number 3 effects of alcohol intake in periodontitis progression in female rats: a histometric study daniela martins de souza1, lucilene hernandes ricardo2, rosilene fernandes da rocha3 1fundação universitária vida cristã faculdade de pindamonhangaba (funvic/fapi) – school of dentistry, department of periodontology, pindamonhangaba, são paulo, brazil 2private clinic, pindamonhangaba , são paulo, brazil 3universidade estadual paulista – unesp, são josé dos campos dental school, são josé dos campos, são paulo, brazil correspondence to: daniela martins de souza centro clínico da faculdade de pindamonhangaba rua marechal deodoro da fonseca, 316, centro cep: 12 401-010, pindamonhangaba/brasil phone: +55 12 3648-8323 e-mail: danimart.voy@terra.com.br; danimart.voy@gmail.com received for publication: july 25, 2014 accepted: september 16, 2014 abstract aim: to evaluate histometrically the effect of low and high caloric value of ethanol on ligatureinduced bone loss in female rats. methods: sixty female rats were divided into five groups of 12 animals each: normal control (water), test a (low ethanol), control a (low iso), test b (high ethanol) and control b (high iso). control groups a and b received diets with the same amount of calories consumed by test groups a and b, respectively, with ethanol replaced by isocaloric amounts of carbohydrate. four weeks prior to the end of the experimental period, half of the rats in each group were randomly assigned to receive a ligature on mandibular molar, whereas the other half of group was left unligated. at 8 weeks, the animals were sacrificed, and the specimens were processed to obtain decalcified sections. the area of periodontal ligament and/or bone loss in the furcation region of the first molars was histometrically measured in five sections per specimen (mm²). results: ethanol intake did not have effect on the alveolar bone loss in unligated teeth (p>0.05). however, in ligature-induced periodontitis, high value of calories associated with ethanol feeding enhanced the area of bone loss (p<0.05). conclusions: the results of this study demonstrated that a low-ethanol diet did not affect the periodontium, while a high-ethanol diet may aggravate the progression of periodontitis, as demonstrated by the increased furcation region bone destruction in periodontal disease. keywords: ethanol; alveolar bone loss; periodontitis; rats. introduction periodontitis involves the destruction of supporting structures of the teeth, including the periodontal ligament, alveolar bone and gingival tissues1. however, some individuals are more susceptible than others to progressive periodontitis2. a large variety of factors exist that influence disease progression, including social and behavioral factors, systemic factors, genetic factors, tooth factors and microbial composition of the dental biofilm. excessive alcohol consumption is considered a behavioral factor that is associated with periodontal disease3. investigations have suggested that alcohol consumption is associated with increased severity of periodontitis and may be a risk indicator for periodontal disease4-5. however, more recent studies on humans have shown no relation between periodontal disease and alcohol consumption6-7. the use of rat models has been applied in the evaluation of periodontal pathogenesis8 and regarding the influence of risk indicator/factors, such as estrogen deficiency9-10, alcohol consumption11-16, nicotine17 and simultaneous nicotine and alcohol use18 on disease progression. braz j oral sci. 13(3):229-234 230230230230230 in the presence of experimental periodontitis, a high dose of ethanol (36% of total calories in the diet) was associated with higher density of polymorphonuclear leukocytes, increasing the severity of periodontal inflammation in the ligature model of male rats15. another study demonstrated that in male rats, ethanol increases ligature-induced bone loss when representing a low value (22%) in total caloric value13. considering that more studies are necessary to test the hypothesis that alcohol consumption can aggravate destruction of the periodontium, the aim of this study was to evaluate histometrically the effect of low and high caloric values of ethanol consumption on alveolar bone loss associated with ligature-induced bone loss in female rats. material and methods animals the institutional animal research committee at the university of são josé dos campos (são paulo, brazil) approved the protocol for all experimental (protocol nº 026/ 2003-pa/cep). sixty adult female wistar rats (4 months-old) weighing 270 g on average were randomized into five groups (12 rats/ group): normal control (water); test a (low ethanol), composed of rats fed an ethanol-containing liquid diet with ethanol representing low caloric value; control a (low iso), rats fed a pair-fed control diet (ethanol replaced by isocaloric amounts of carbohydrate); test b (high ethanol), rats fed an ethanol-containing liquid diet with ethanol representing high caloric value; and control b (high iso), rats fed a pair-fed control diet (ethanol replaced by isocaloric amounts of carbohydrate). experimental procedures prior to the experimental period, the rats comprising test a and test b groups were subjected to an adaptation period in which the ethanol concentration was increased until it reached experimental concentrations. a solution containing 5% ethanol was administered to ethanol test a group for 7 days and ethanol test b group received 5% and 10% ethanol for 7 days each. after this period, the rats received a diet containing the experimental concentrations for 8 weeks. in test a group, the rats received an ethanol solution (ecibra, cetus, santo amaro, sp, brazil) at a concentration of 10% ethanol v/v (by volume) and in test b group, at a concentration of 20% ethanol v/v, in addition to standard rat chow (guabi nutrilador, mogiana alimentos, campinas, sp, brazil), both provided ad libitum. the mean quantities of rat chow and ethanol solution consumed were calculated daily and one day after ethanol administration, the control a and b rats were fed an equal amount of rat chow as that consumed by the associated alcohol group and an equal volume of liquid diet, with the ethanol replaced by isocaloric amounts of carbohydrate. four weeks after beginning of experiment, general anesthesia was induced by intramuscular administration with solution of 13 mg/kg of 2% xylazine hydrochloride and 33mg/ kg of ketamine hydrochloride. in order to induce periodontitis, cotton ligatures were placed around the cervix of the mandibular left first molar in six rats, leaving other six rats unligated to serve as a control. the rats were sacrificed four weeks after ligature placement and their mandibles were removed and fixed in 10% neutral formalin for 48 h. the left side of each mandible was submitted for histometric evaluation of area of periodontal ligament and/or bone loss in the furcation region of the first molars. histometric analysis the left mandibles were resected from each rat and immediately demineralized in 20% plank-rychlo solution (aluminum chloride, hydrochloric acid and formic acid). these specimens were dehydrated, embedded in paraffin, sectioned into 5-µm-thick slices along the molars in a mesiodistal direction and stained with hematoxylin and eosin. using the image analysis system image tool v.3.0 (uthscsa, san antonio, tx, usa), the area of periodontal ligament and/or bone loss in the furcation region of the first molars was measured in 5 semi serial sections with an interval of four sections per specimen (mm²). the histometric analysis showed the area limited by the bone crest and cementum surface of unligated (figure 1) and ligated (figure 2) teeth, as described by nociti jr et al.17. measurements from the five sections were averaged to permit statistical analysis. fig. 1. photomicrograph of the periodontal ligament area ( ) in the furcation region of an unligated tooth in high ethanol group (x25 h&e). effects of alcohol intake in periodontitis progression in female rats: a histometric study 231231231231231 braz j oral sci. 13(3):229-234 diet test b (high ethanol) control b (isocaloric) total calories consumed 51.72 ± 7.45 50.27 ± 8.10 calories derived from protein 31.13 ± 5.79 30.69 ± 5.86 calories derived from alcohol 20.59 ± 2.90 19.58 ± 3.51 or carbohydrate % calories of liquid diet 40.10 ± 4.43 39.02 ± 4.77 % calories of solid diet 59.90 ± 4.43 60.98 ± 4.77 table 2. table 2. table 2. table 2. table 2. mean and standard deviation of analysis of diet (kcal/day) for test and control groups associated with high caloric values. statistical analysis the independent sample t-test (p<0.05) was used for comparisons between differences in weigh gain and between the diets consumed in the test and control groups. for histometric analyses the data were expressed as mean and standard deviation (mm²). the independent samples ttest was used for comparisons between alcohol groups and control groups in presence or absence of periodontitis induced. one-way analysis of variance and tukey’s test for subsequent multiple comparisons were used to separately determine significant differences in area of periodontal ligament and/or bone loss among the treatment groups for ligated or unligated rats. differences were considered significant at p<0.05. results diet and weight analysis diet analysis showed no significant differences in diet consumption (p>0.05) between the test and control groups for low (table 1) or high (table 2) caloric values, considering the total number of calories consumed (kcal/day), calories derived from protein, calories derived from alcohol or carbohydrate and the percentage of calories of the liquid and solid diets. the systemic effect of alcohol consumption observed by analysis rat’s body weight showed that the ethanol feed 24% kcal per day (test a) and pared-fed to low (control a) ethanol consumption gained weight (19.33 g; p=0.0000) during the experiment. however, no statistically significant difference was observed between baseline and final weight in the rats receiving 40% kcal per day of ethanol (test b). paired-fed to high (control b) ethanol consumption lost weight. histologic evaluation eight weeks after the beginning of the experiment, greater bone loss in the furcation region with most of the area filled for connective tissue was noted in rats that received ligature (figure 2) compared with those without ligature (figure 1). in the group of high caloric value of ethanol with ligature, the extent of bone loss in the furcation region was greater than in the other groups with ligature. there was a less dense periodontal ligament, characterized by the presence of smaller collagen fibers oriented horizontally to the root surface (figure 2) and reabsorbed bone with some osteoclasts (figure 3). fig. 2. photomicrograph of the bone loss area in the furcation region of a ligated tooth in high ethanol group (x25 h&e). effects of alcohol intake in periodontitis progression in female rats: a histometric study diet test a (low ethanol) control a (isocaloric) total calories consumed 45.32 ± 5.80 43.64 ± 5.72 calories derived from protein 34.28 ± 4.65 33.06 ± 4.59 calories derived from alcohol or carbohydrate % calories of liquid diet 24.41 ± 2.42 24.35 ± 2.61 % calories of solid diet 75.59 ± 2.42 75.65 ± 2.61 table 1.table 1.table 1.table 1.table 1. mean and standard deviation of analysis of diet (kcal/day) for test and control groups associated with low caloric values. 11.04 ± 1.61 10.58 ± 1.67 232232232232232 braz j oral sci. 13(3):229-234 fig. 4. mean and standard derivation of the periodontal ligament area around unligated teeth and periodontal bone loss area around ligated teeth (mm²) *statistically significant (anova and tukey’s test; p<0.05). fig. 3. photomicrograph of the osteoclasts ( ) in region of a ligated tooth in high ethanol group (x400 h&e). histometric assessment histometric analysis showed that cotton ligatures placed around the teeth induced alveolar bone loss, which was confirmed by the greater bone loss (p<0.05) observed in ligated teeth compared to unligated teeth. in unligated groups, no statistically significant differences (p>0.05) were observed in the periodontal ligament area among the groups normal control, test a (low ethanol), control a (low control), test b (high ethanol) and control b (high control). however, when ligature was placed, analysis revealed a significantly greater area of bone loss (p<0.05) in the furcation region of ligated teeth in rats that received high caloric value of ethanol (test b) than in rats receiving high caloric value of carbohydrate (control b), low caloric value of ethanol (test a), low caloric value of carbohydrate (control a) and water (normal control) (figure 4). ethanol affected ligature-induced bone loss when it represented a high value in the total of calories. discussion research suggests that alcohol consumption is associated with the increase of severity of periodontitis in humans4-5,19, periodontal inflammation14-15 and alveolar bone loss in periodontal disease in rats11-13. however, others studies on humans have shown no relation between periodontal disease and alcohol intake9-10. the effects of alcohol on periodontal disease exist and this relation has been explained by biological plausibility through different mechanisms. chronic alcoholic patients show an increased risk of developing severe infection, which may be due to altered immune response20. alcohol has a toxic effect on the liver causing a negative effect on the clotting mechanism and frequently in alcoholic present nutritional disorders resulting from protein and vitamin deficiency21. additionally, ethanol alters bone metabolism, as demonstrated by the dramatic effects on rat bone22. as a consequence of the toxic effects on the liver, bone, immune system and nutrition, alcohol may interfere in the mechanisms of inflammatory response in periodontal disease. alcohol consumption can affect the bone metabolism both directly and indirectly 23. directly, it causes the suppression of tissue formation due to toxic effect on osteoblastic activity and proliferation24, while indirectly, ethanol can aggravate bone loss as a consequence of the effects of malnutrition23. a population study that used a self-reported questionnaire regarding alcohol consumption observed a significant relationship between alcohol, gingival inflammation and clinical attachment loss, after controls to account for major confounding variables 4. another cross-sectional study 19 showed that alcohol dependence might be associated with periodontal disease. this study evaluated 49 alcoholic and 49 non-alcoholic men in brazil and observed increased severity of clinical attachment level and probing depth associated with alcohol dependence. an important cross-sectional study involving 13,198 individuals found a moderate but consistent dose-dependent relation between alcohol consumption and increased severity of clinical attachment loss in periodontal disease 5. the present study demonstrated that the effect of alcohol consumption might accentuate periodontal bone loss in ligature-induced periodontitis in female rats in a dosedependent manner. similar findings were observed in other studies in rats12-13. a previous study7 related alcohol consumption with periodontitis and obtained conflicting results, showing that higher alcohol consumption appears to have an inverse association with attachment loss in men, but not in women. the authors emphasized that further investigation is necessary to observe the association between alcohol and periodontitis, establishing prospective cohort studies with participants free of periodontitis at baseline. effects of alcohol intake in periodontitis progression in female rats: a histometric study 233233233233233 braz j oral sci. 13(3):229-234 a longitudinal study6 that associated alcohol consumption with dental health used questions on alcohol consumption, clinical and radiographic examinations. the marginal bone level and longitudinal bone loss was determined by assessments on the proximal surfaces of all measurable teeth on radiographs from 1970 and 1990. only teeth that were measurable in both examinations were included in the study and the results did not support an association between alcohol consumption and periodontal disease. the reduction in human body weight during chronic alcohol consumption is associated with the fact that part of the daily diet calorie intake is provided by the alcoholic beverage25. however, this impact on weight is rarely observed in rats26. in the present study, the systemic effect of alcohol consumption observed by analysis of rat’s body weight verified that groups that consumed low ethanol or carbohydrate showed gain of weight. however, in the rats receiving high caloric values of ethanol the weight did not change and those with high carbonhydrate consumption showed weight lost. in contrast, irie et al.15 showed a weight gain in ethanol and isocaloric groups during the experimental period associated with a high caloric value of ethanol (36%). analyzing rat’s body weight, souza and rocha13 also observed that groups that consumed ethanol or carbohydrate in low or high caloric values gained weight. irie et al.15 and dantas et al.14 demonstrated that ethanol consumption increased the severity of periodontal inflammation in the ligature model in rats. irie et al.15 suggest that chronic alcohol consumption increased periodontal inflammation, oxidative damage, tnf-alpha production and had an additive effect on polymorphonuclear leukocyte infiltration and gingival oxidative damage. dantas et al.14 suggested that ethanol consumption could represent a risk indicator for periodontal disease since augments the expression of inflammatory markers in healthy rats, and increases them significantly, at short term, in the presence of disease. in this study, analysis of the results verified that chronic ethanol feeding may not be capable of causing bone loss in unligated rats associate with low (24%) and high (40%) caloric values. souza and rocha13 found similar findings in unligated rats associated with low (22%) or high (36%) caloric values of ethanol. souza et al. 12 also observed that alcohol consumption at concentrations of 10% and 20% by volume may not be capable of causing alveolar bone loss without ligature-induced periodontitis in rats. in contrast, irie, et al.15 showed in unligated rats, that the distance between the cementoenamel junction and alveolar bone crest was greater in the ethanol group (36% calories derived from ethanol) than in the respective control group. the present study demonstrated that high caloric values (40%) of ethanol increased ligature-induced bone loss in rats. similarly, souza and rocha13 showed that ethanol intake representing low caloric values (22% of total caloric value) aggravated bone loss in the presence of experimental periodontitis. and souza et al.12 also evaluated rats receiving a concentration of 20% by volume showed significantly greater alveolar bone loss in ligature-induced periodontitis in female rats12. dantas et al.14 demonstrated that short-time ethanol administration increased the length of the periodontal ligament in rats submitted to ligature induced periodontitis and diminished the alveolar bone volume, which suggest that alcohol consumption promotes the progression of this disease in the short term. differently, irie et al.15 reported that a high dose of ethanol itself (36% of total calories in the diet) was not capable of affecting ligature-induced bone resorption. in conclusion, the present study demonstrated that a low-ethanol diet did not affect the periodontium, but that a high-ethanol diet may aggravate the progression of periodontitis, as demonstrated by the increased furcation region bone destruction in periodontal disease. acknowledgements the present study was partially funded by the coordination for the improvement of higher education (capes), brazil. references 1. kinane df. causation and pathogenesis of periodontal disease. periodontol 2000. 2001; 25: 8-20. 2. loe h, anerud a, boysen h, morrison e. natural history of periodontal disease in man. rapid, moderate and no loss of attachment in sri lankan laborers 14 to 46 years of age. j clin periodontol. 1986; 13: 431-40. 3. genco rj. current view of risk factors for periodontal diseases. j periodontol. 1996; 64: 1041-9. 4. tezal m, grossi sg, ho aw, genco rj. the effect of alcohol consumption on periodontal disease. j periodontol. 2001; 72: 183-9. 5. tezal m, grossi sg, ho aw, genco rj. alcohol consumption and periodontal disease. the third national health and nutrition examination survey. j clin periodontol. 2004; 31: 484-8. 6. jansson l. association between alcohol consumption and dental health. j clin periodontol. 2008; 35: 379-84. 7. kongstad j, hvidtfeldt ua, gronbaek m, jontell m, stoltze k, holmstrup p. amount and type of alcohol and periodontitis in the copenhagen city heart study. j clin periodontol 2008; 35: 1032-9. 8. weinberg ma, bral m. laboratory animal models in periodontology. j clin periodontol. 1999; 26: 335-40. 9. amadei su, souza dm, brandão aah, rocha rf. influence of different durations of estrogen deficiency on alveolar bone loss in rats. braz oral res. 2011; 25: 538-43. 10. milhan nvm, forte lfbp, vasconcelos lmr, balducci i, carvalho yr. influence of ovariectomy combined with lack of masticatory force in the evolution of periodontal disease. braz j oral sci. 2014; 13: 47-52. 11. de souza dm, ricardo lh, prado mde a, prado fde a, rocha rf. the effect of alcohol consumption on periodontal bone support in experimental periodontitis in rats. j appl oral sci. 2006; 14: 443-7. 12. souza dm, ricardo lh, kantoski kz, rocha rf. influence of alcohol consumption on the alveolar bone level associated with ligature-induced periodontitis in rats. braz oral res. 2009; 23: 326-32. 13. souza dm, rocha rf. low caloric value of ethanol itself increases alveolar bone loss in ligature-induced periodontitis in male rats. braz oral res. 2009; 23: 460-6. 14. dantas am, mohn ce, burdet b, zorrilla zubilete m, mandalunis pm, elverdin jc, et al. ethanol consumption enhances periodontal inflammatory markers in rats. arch oral biol. 2012; 57: 1211-7. effects of alcohol intake in periodontitis progression in female rats: a histometric study 234234234234234 braz j oral sci. 13(3):229-234 15. irie k, tomofuji t, tamaki n, sanbe t, eruni d, azuma t, et al. effects of ethanol consumption on periodontal inflammation in rats. j dent res. 2008; 87: 456-60. 16. bastos mf, gaag gld, romero jr, gabrili jjm, marques mr, duarte pm. effects of cachaça, a typical brazilian alcoholic beverage, on alveolar bone loss and density: a study in peripubertal rats. arch oral biol. 2014; 59: 82-91. 17. nociti fh jr, nogueira-filho gr, primo mt, machado ma, tramontina va, barros sp, et al. the influence of nicotine on the bone loss rate in ligature-induced periodontitis. a histometric study in rats. j periodontol. 2000; 71: 1460-4. 18. vasconcelos dfp, da silva mad, marques mr, gibilini c, vasconcelos accg, barros sp. effects of simultaneous nicotine and alcohol use in periodontitis progression in rats: a histomorphometric study. j clin exp dent. 2013; 5: e95–e99. 19. amaral cd, luiz rr, leão a. the relationship between alcohol dependence and periodontal disease. j periodontol. 2008; 79: 993-8. 20. messingham kan, faunce de, kovacs ej. alcohol, injury, and cellular immunity. alcohol. 2002; 28: 137-49. 21. lieber cs. relationships between nutrition, alcohol use, and liver disease. alcohol res health. 2003; 27: 220-31. 22. hogan ha, argueta f, moe l, nguyen lp, sampson hw. adult-onset alcohol consumption induces osteopenia in female rats. alcohol clin exp res. 2001; 25: 746-54. 23. klein r.f. alcohol-induced bone disease: impact of ethanol on osteoblast proliferation. alcohol clin exp res. 1997; 21: 392-9. 24. gonzalez-calvin jl, garcia-sanches a, bellot v, muñoz-torres m, raiaalvares e, salvatierra-rios d. mineral metabolism, osteoblastic function and bone mass in chronic alcoholism. alcohol alcohol. 1993; 28: 571-9. 25. lieber cs. relationships between nutrition, alcohol use, and liver disease. alcohol res health. 2003; 27: 220-31. 26. hefferan te, kennedy am, evans gl, turner rt. disuse exaggerates the detrimental effects of alcohol on cortical bone. alcohol clin exp res. 2003; 27: 111-7. effects of alcohol intake in periodontitis progression in female rats: a histometric study oral sciences n3 original article braz j oral sci. july | september 2014 volume 13, number 3 radiant exposure effects on physical properties of methacrylate and silorane-composites anderson catelan1, yoshio kawano2, paulo henrique dos santos3, gláucia maria bovi ambrosano4, ana karina bedran-russo5, flávio henrique baggio aguiar1 1universidade estadual de campinas unicamp, piracicaba dental school, department of restorative dentistry, piracicaba, sp, brazil 2universidade de são paulo usp, institute of chemistry, department of fundamental chemistry, são paulo, sp, brazil 3universidade estadual paulista unesp, araçatuba dental school, department of dental materials and prosthodontics, araçatuba, sp, brazil 4universidade estadual de campinas unicamp, piracicaba dental school, department of social dentistry, area of statistics, piracicaba, sp, brazil 5university of illinois at chicago, college of dentistry, department of restorative dentistry, chicago, il, usa correspondence to: anderson catelan department of restorative dentistry, piracicaba dental school, unicamp av. limeira, 901, areião, caixa postal 52, cep 13414-903, piracicaba, sp, brasil phone: +55 19 2106-5337 e-mail: catelan@estadao.com.br abstract aim: to evaluate the effect of different radiant exposures on the degree of conversion (dc), knoop hardness number (khn), plasticization (p), water sorption (ws), and solubility (s) of different monomer resin-based composites. methods: circular specimens (5 x 2 mm) were manufactured from methacrylate and silorane composite resins, and light-cured at 19.8, 27.8, 39.6, and 55.6 j/cm2, using second-generation led at 1,390 mw/cm2. after 24 h, dc was obtained using a ft-raman spectrometer equipped with a nd:yag laser, khn was measured with 50-g load for 15 s, and p was evaluated on the top and bottom surfaces by the percentage of hardness reduction after 24 h immersed in absolute alcohol. ws and s were determined according to iso 4049. data were subjected to statistical analysis (α=0.05). results: methacrylate material presented higher dc, khn, p, and ws than silorane (p<0.05). there was no difference in the s values (p>0.05). the increased radiant exposures improved only the khn (p<0.05). in general, top surfaces showed higher dc and khn than bottom, for both materials (p<0.05). the increase of the radiant exposure did not improve most physical properties of the composites and were monomerbase dependent. conclusions: chemical composition of the composite resins resulted in different physical properties behavior and could affect the clinical longevity of dental restorations, but overall these properties were not influenced by the different radiant exposures evaluated in the study. keywords: composite resins; polymerization; physical properties. introduction since the development of the dental composite resin in the 1960s, numerous improvements in its composition and characteristics were made and with the increased demand by patients for esthetic restorations and simplification of the bonding procedures, light-cured composites have been widely used in the dental practice1. currently dental composites are classified in nanofilled, microfilled, or micro/nano hybrid composite resins2. these materials are composed basically by polymeric matrix based on methacrylate monomers, inorganic filler particles, silane and photo-initiator system3. bis-gma (bisphenol-a glycidyl dimethacrylate) is received for publication: may 25, 2014 accepted: august 08, 2014 braz j oral sci. 13(3):168-174 material methacrylate (filtek z250, a2 shade; 3m espe, st. paul, mn, usa) silorane (filtek ls, a2 shade; 3m espe, st. paul, mn, usa) composition* lot. n144001br filler: 60 vol%, aluminum oxide, silica, and zirconium oxide (0.01-3.5 µm). resin: bis-gma, bis-ema (ethoxylated bisphenol a dimethacrylate), and udma. lot. n183458 filler: 55 vol%, silica, and yttrium trifluoride (0.04-1.7 µm). resin: bis-3,4-epoxycyclohexylethyl-phenyl-methylsilane and 3,4epoxycyclohexylcyclopolymethylsiloxane. table 1. table 1. table 1. table 1. table 1. tested materials *according to manufacturer’s information. the most used monomer in dental composites, however due to its high molecular weight, high viscosity and low mobility, other monomers with lower viscosity and/or higher mobility, as tegdma (triethylene glycol dimethacrylate) and udma (urethane dimethacrylate), are used to increase the degree of conversion (dc) and crosslinking of the resulting polymer3,4. methacrylate restorative materials exhibit volumetric polymerization shrinkage5, ranging from 1.9 to 3.5 vol%6, and a significant proportion of unreacted monomer due to incomplete c=c bond conversion7. however, a higher dc increases the shrinkage strain and the resulting polymerization stress may result in cuspal deflection8, de-bonding at adhesive interface, post-operative sensitivity 9,10, microleakage 9, marginal staining, secondary caries formation, restoration and dental fractures10, affecting the restoration durability. in the attempt to reduce these problems some techniques were proposed to decrease the shrinkage stress effects, such as different incremental composite placement10, light-curing protocols and intermediate layer with hybrid glass ionomer or flowable composite11. a low shrinkage monomer was developed from the reaction of the oxirane and siloxane molecules, termed silorane8. silorane network is generated by cationic ringopening polymerization mechanism instead of free radical curing of methacrylate monomers8, and more light-curing time to form cations is required to initiate the polymerization reaction5. it exhibits lower polymerization shrinkage6,8, less than 1 vol%6, and mechanical properties comparable to conventional bis-gma composites6,11. one factor that cannot be controlled by the dentist during the restorative procedure in deep cavities is the reduction of the light intensity reach into the material due to the distance between guide tip and resinous material surface. thus, restoration weakening may occur by lower dc and/or formation of more linear polymers, presenting inferior physical properties, such as reduced hardness, increased wear, solubility, and discoloration12. the increase of the curing time, and consequently the radiant exposure available for the monomer conversion can improve the physical properties of resin-based materials13,14, and thus increase the long-term durability of adhesive restorations2. the dc is an important physical property that may have some impact on the restoration longevity, but this property alone is not enough to characterize the 3-dimensional dental composite structure, as different c=c bond concentrations coexist in the same polymer15. the same dc value may result in different linear polymer content, which is more susceptible to softening than a more cross-linked polymer16. thus, the study of other physical properties together with dc measurement are better for assessing the performance of dental materials. the objectives of this study were to evaluate the effect of the radiant exposure on the dc, knoop hardness number (khn), plasticization (p), water sorption (ws) and solubility (s) of methacrylateand silorane-based composite resins. the research hypotheses tested were that: (1) there is no difference between the materials and (2) the highest radiant exposure improves the tested physical properties. material and methods table 1 presents the materials’ composition. circular specimens (5 mm diameter and 2 mm thick) were made for khn and p (n=10), and for dc, ws, and s (n=5). a circular polytetrafluoroethylene (teflon) mold (figure 1a) was filled with the composite resin held between two glass slabs separated by mylar strips and pressed with a 500-g load, to prevent bubble formation and to remove excess material. cavities were filled with only one increment of composite, which was randomly light-cured set at 0 or 4 mm from the top surface of the mold using a second-generation lightemitting diode (led) bluephase 16i (vivadent, bürs, austria) device at 1,390 mw/cm2 of irradiance according to table 2. a holder coupled to the light source was used to standardize the distance between the light guide tip and material (figure 1b and 1c), controlled by digital caliper (mitutoyo sul americana, suzano, sp, brazil) (figure 1d). the optical power (mw) delivered by the device was measured with a power meter (ophir optronics, har hotzvim, jerusalem, israel). the tip diameter was measured with a digital caliper (mitutoyo sul americana, suzano, sp, brazil) to determine tip area (cm2). irradiance (mw/cm2) was calculated dividing optical power by tip area; and radiant exposure (j/ cm2) is the irradiance multiplied by curing time and divided by 1000. simulating clinical restorative procedure in a cavity 6 mm deep, on top surface of first composite increment resulted in 19.8 and 39.6 j/cm2 radiant exposure, when light polymerized for 20 and 40 s respectively, at 990 mw/cm2. radiant exposure effects on physical properties of methacrylate and silorane-composites 169 braz j oral sci. 13(3):168-174 group and material curing time, light-curing radiant exposure distance and irradiance g1 methacrylate / g5 silorane 20 s, 4 mm, 990 mw/cm2 19.8 j/cm2 g2 methacrylate / g6 silorane 20 s, 0 mm, 1,390 mw/cm2 27.8 j/cm2 g3 methacrylate / g7 silorane 40 s, 4 mm, 990 mw/cm2 39.6 j/cm2 g4 methacrylate / g8 silorane 40 s, 0 mm, 1,390 mw/cm2 55.6 j/cm2 table 2.table 2.table 2.table 2.table 2. experimental groups. fig. 1. (a) teflon ring mold and specimen (5 mm and 2 mm thick), (b) holder used to standardize the distance between the light guide tip and material, (c) composite polymerization (d) at 0 and 4 mm distance controlled by a digital caliper. the irradiance was calculated at 4 mm of distance between tip of curing light device and top surface of first composite increment, due its had 2 mm of thickness. control groups were light-cured for 20 and 40 s set at 0 mm from restorative material surface at 1,390 mw/cm2, resulting in 27.8 and 55.6 j/cm2, respectively (table 2). after polymerization, the specimens were removed from the molds, dry stored in lightproof containers at 37 oc for 24 h, and polished with 1200-grit silicon carbide (sic) grinding paper (carbimet 2 abrasive discs; buehler, lake bluff, il, usa). the degree of c=c conversion assessment was recorded in scattering mode using a fourier transform raman (ft-raman) spectrometer (rfs 100/s; bruker optics inc., billerica, ma, usa), equipped with a nd:yag laser. absorption spectra of the cured and uncured composites were obtained on the top and bottom surfaces with 64 scans at 4 cm-1 resolution in the region between 1000-2000 and 6002000 cm-1 for methacrylate-and silorane-based and siloranebased resins, respectively (figures 2a-2d). to calculate the dc, the ratios (r) between the peak heights of the c=c aliphatic (1638 cm -1) and aromatic (1608 cm -1) for methacrylate, and oxirane (1263 cm-1) and siloxane (1000 cm-1) for silorane band absorptions for cured and uncured composite were used. according to the formula: dc (%) = [1 – (r cured/r uncured)] x 100. initial microhardness (mhi) reading was measured on the top and bottom surfaces of each specimen using a microhardness tester (hmv-2t; shimadzu, tokyo, japan) with a knoop diamond indenter under 50-g load for 15 s (figures 3a and 3b). five indentations were made on each surface of the specimen, one at the center and other four at a 100 µm distance from the central location. the average of the five khn values was calculated for each specimen. plasticization analysis was evaluated by percentage of the microhardness reduction (%mhred) after absolute alcohol storage16. after mhi assessments, all specimens were immersed in 100% ethanol for 24 h. following this period, a second microhardness measurement (mhf) was made as previously described. the same operator did the khn test, before and after alcohol storage. the results were tabulated, and the p was calculated using the following equation: %mhred=100– [(mhf x 100)/mhi]. the water sorption and solubility were performed in compliance with iso 4049:2009 standard specifications, except for the specimen dimensions and curing protocol. the specimens were stored in a desiccator at 37 ºc (figure 3c) containing silica gel and weighted daily on an analytical scale (tel marke; bel quimis, são paulo, sp, brazil) accurate to 0.001 mg (figure 3d), constituting a weighing cycle every 24 h. the complete cycle was repeated to obtain a constant radiant exposure effects on physical properties of methacrylate and silorane-composites170 braz j oral sci. 13(3):168-174 fig. 2. absorption spectrum of methacrylate (a-b) and silorane-based composite resin (c-d) uncured and cured, respectively. asterisk represents the reference peak and arrow the reaction peak. fig. 3. hardness tester used for khn measures (a-b). desiccator (c) and analytical balance (d) used in the water sorption and solubility tests. mass (m1): until the mass loss of each specimen was no more than 0.1 mg per 24 h cycle. thickness (4 measurements at four equally located points on the circumference) and diameter (2 measurements at right angles) of each specimen were made using a digital electronic caliper. mean values were used to calculate the volume (v) of each specimen (in mm3). thereafter, the specimens were stored in water at 37 ºc for 7 days, the volume for immersion being at least 6 ml per specimen. specimens were weighed again after being carefully wiped with an absorbent paper; this value was recorded as m2. after this weighting, the specimens were returned to the first desiccators, the mass reconditioning cycle was completely repeated and the constant mass was recorded as m3. the values for ws and s, in micrograms per cubic millimeters, were calculated using the following equations: ws = (m2–m3)/v and s = (m1–m3)/v. this study had a two-factor experimental design: radiant exposure effects on physical properties of methacrylate and silorane-composites 171 braz j oral sci. 13(3):168-174 material radiant degree of conversion† microhardness‡ plasticization§ exposure top* bottom top bottom top bottom methacrylate** 19.8 62.68 (1.67) 61.09 (0.72) 63.23 (1.91) ab 60.83 (1.73) bb 47.52 (1.90) 45.93 (2.08) 27.8 63.23 (1.39) 63.27 (1.95) 64.41 (1.17) aab 62.85 (1.09) aab 47.27 (1.09) 46.19 (1.65) 39.6 63.90 (2.73) 62.25 (2.45) 66.17 (3.58) aa 64.86 (1.55) aa 45.93 (2.57) 44.06 (1.72) 55.6 63.93 (1.83) 62.97 (2.29) 66.65 (0.95) aa 64.35 (1.80) ba 46.31 (1.36) 44.94 (1.57) silorane 19.8 45.61 (2.91) 44.82 (2.09) 52.77 (0.90) aa 46,42 (0.89) bb 15.32 (3.52) 17.09 (3.27) 27.8 45.63 (1.78) 44.60 (0.78) 53.18 (1.53) aa 48.75 (1.41) ba 14.93 (3.25) 16.43 (4.42) 39.6 45.42 (2.36) 44.85 (2.08) 53.56 (0.96) aa 47.78 (1.15) bab 13.91 (2.53) 16.76 (2.73) 55.6 46.43 (3.30) 45.04 (1.92) 52.72 (1.48) aa 49.23 (0.98) ba 13.43 (2.66) 14.98 (3.67) table 3.table 3.table 3.table 3.table 3. degree of conversion (%), knoop microhardness number (kg/mm2), and plasticization (%) means (standard deviation) of the composite resins according to material, radiant exposure (j/cm2) and test surface. †there was no statistical difference for radiant exposure (p>0.05). *it differs from the bottom surface (p<0.001). **it differs from the silorane (p=0.015). ‡distinct letters (uppercase letters in the rows and lowercase letters in the columns within each composite) are statistically different (pd”0.05). **it differs from the silorane (p<0.001). §there was no statistically significant difference for radiant exposure (p=0.0586) and test surface (p=0.5504). **it differs from the silorane (p<0.001). radiant sorption† solubility‡ exposure methacrylate* silorane methacrylate silorane 19.8 16.41 (2.47) 8.97 (2.48) 0.00 (-4.92; 0.00) 0.00 (-4.55; 0.00) 27.8 16.61 (2.89) 8.48 (2.16) 0.00 (-4.58; 0.00) 0.00 (-4.29; 0.00) 39.6 15.53 (2.26) 8.44 (2.25) 0.00 (-4.70; 0.00) 0.00 (0.00; 0.00) 55.6 15.46 (2.18) 8.33 (2.09) 0.00 (0.00; 0.00) 0.00 (0.00; 0.00) table 4.table 4.table 4.table 4.table 4. water sorption (µg/mm3) and solubility (µg/mm3) values of the composite resins according to material and radiant exposure (j/cm2). †means (standard deviation). *it differs from the silorane (p<0.001). there was no statistical difference for radiant exposure (p>0.8368). ‡median (minimum value; maximum value). there was no statistically significant difference among the radiant exposures (p=0.4565 and p=0.2544 for methacrylate-and silorane, respectively) and between the composite resins (p=0.9089, p=0.4604, p=0.2126, and p=1 for 19.8, 27.8, 39.6, and 55.6 j/cm2, respectively). material in 2 levels: methacrylateand silorane-based microhybrid composite resins and radiant exposure in 4 levels: 19.8, 27.8, 39.6 and 55.6 j/cm2. for dc, khn and p one subfactor in 2 levels was added: top and bottom surfaces. dc, khn, and p data were subjected to 2-way split-plot anova and tukey’s test at a pre-set level of 0.05. the factors material and radiant exposure were considered in the parcels and the sub-factor surface (top and bottom) was considered in the sub-parcel. ws was analyzed by two-way anova and tukey test (α=0.05). s data did not present homoscedasticity and were submitted to non-parametric kruskal wallis, dunn, and mann whitney tests at 5% significance level. results table 3 illustrates the dc of the composite resins. the methacrylate material presented higher curing degree than silorane (p<0.001), the dc of the top surface also was higher than bottom (p=0.015), and no difference was observed for radiant exposure (p>0.05). silorane material showed lower khn than methacrylate (p<0.001) as well as bottom compared to top surface (p<0.001). the factor radiant exposure (p<0.001) and the interaction of the factor (composite resin) and sub-factor (test surface) showed statistical differences (p<0.001). in general, the highest radiant exposure presented higher khn, except for the top surface of the silorane composite, which showed no difference among the tested radiant exposures (table 3). the p test exhibited lower softening after ethanol storage for silorane than methacrylate resin-based composite (p<0.001). no difference for radiant exposure (p=0.0586) and test surface (p=0.5504) was found (table 3). in table 4, silorane also presented lower water sorption than methacrylate (p<0.001), but no statistical difference for radiant exposure (p=0.8368). there is no difference between the materials and radiant exposures for s (p>0.05) (table 4). discussion composite resins are widely used as restorative materials in the dental practice and several clinical studies have reported an adequate durability of resin-based restorations even after an extended period of time2,17. the first hypothesis that both materials showed similar performance was partially rejected. methacrylate composite presented higher dc, khn, p, and ws than silorane (tables 3 and 4); only in the s test no difference was found between the materials (table 4). according to results of this radiant exposure effects on physical properties of methacrylate and silorane-composites172 braz j oral sci. 13(3):168-174 investigation, higher dc 15,18 and khn15 were found for methacrylate compared to silorane composite. the udma monomer contained in the filtek z250 composite provides more mobility and has been related to the increase of dc3; and differences in the mechanism of the polymerization reaction can explain these results. methacrylate is cured by radical intermediates and cycloaliphatic oxirane polymerizes via cationic intermediates6. moreover, the onset of cationic ring-opening polymerization of the silorane is slower due to the required formation of sufficient cations to initiate the polymerization, thus a longer light-curing time is required compared with radical cure of methacrylate monomer molecules into polymer network5. improvements of the mechanical properties have been associated with the increase of monomer conversion into polymer13,14 and also related to the filler fraction of composite resins4. the increase of the filler content has been associated with lower volumetric shrinkage due to the reduced volume of organic matrix, but it can affect negatively the dc by mobility restrictions imposed on the reactive species and light scattering19. low hardness usually results in poor wear resistance and has been related to the filler fraction4,20. silorane exhibits 55 vol% and methacrylate 60 vol% of inorganic filler. a positive relation between the volume fraction of filler and hardness was found4. thus, the higher dc and filler content of methacrylate could have influenced the higher khn values than silorane. aqueous environment may cause softening of the resinbased materials by swelling of the polymer network and reduction of the frictional forces between polymer chains, resulting in lower hardness15. moreover, the insufficiently cross-linked polymer is more susceptible to plasticization effect by the chemical substances that get in contact during eating and drinking21. silorane is a merger of siloxane and oxirane. siloxane presents good biocompatibility even in uncured condition and high hydrophobicity; oxirane has low shrinkage and high reactivity6. unchanged knoop hardness was reported for silorane after storage in water due to the presence of siloxane moiety, while the methacrylate composites were susceptible to softening15, and lower ethanol degradation18. thus, the more hydrophobic nature of silorane22 may contribute for lower ws and p compared with a conventional methacrylate-based resin. the plasticization test has been used to evaluate indirectly the polymer cross-linked structure16. the absorption of ethanol molecules by the polar portion of the matrix causes swelling of the resinous material23. however, with the different hydrophobicity of the tested monomers, this property is difficult to be correlated, as a material less susceptible to the deleterious effects and absorption of alcohol and with lower crosslink density could present a smaller hardness decrease after ethanol exposure. the solubility of the composite is strongly influenced by monomer conversion24 and the high hydrophobicity of siloxane species may decrease the s of the silorane15. despite of the greater dc of methacrylate, the higher hydrophobicity of silorane composite could have compensated its lower monomer conversion and resulted in the same s values. the light-curing time recommended for a silorane composite resin using a quartz-tungsten-halogen (qth) unit with irradiance between 500-1,400 mw/cm2 is 40 s, as well as for leds with output between 500-1,000 mw/cm2. for leds with irradiance between 1,000-1,500 mw/cm 2 is indicated an exposure light time of 20 s. a second generation led was used in this study; this device shows a single peak and high irradiance (1,390 mw/cm 2), indicating 20 s polymerization. this curing unit emits a narrow spectrum (between 410 and 530 nm, with a peak at 454 nm) that includes the maximum energy absorption peak of the camphorquinone at 468 nm, which absorbs a wide spectrum of wavelengths from 360 to 510 nm 25, a photo-initiator included in both tested composites. the light radiant exposure that reaches the material is an important factor that provides better physical properties, a higher distance between the tip of light source and irradiated surface decreases the irradiance and can affect the polymerization effectiveness 12,26,27. several studies have reported to improve the physical properties of resin-based materials with the increase of radiant exposure, due to the higher dc13,14. in this investigation, the greater radiant exposures increased only the hardness (table 3), and the second hypothesis also was partially rejected. during the restorative procedure it is usual that the distance between the light guide tip and first composite increment surface placed at the bottom of cavity be 4 mm (cavity depth 6 mm and composite increment 2 mm). in this clinical simulation the achieved irradiance was 990 mw/ cm2 at 4 mm, very near to the limit for light-curing during 20 s. maybe the high light power was sufficient to form more cross-linked polymers7, which in spite of improving the mechanical behavior and polymer resistance to degradation, results in the deceleration of the polymerization reaction and limits the conversion rate26. thus, with the used high power led, the physical properties were little affected. however, special care should be taken when the polymerization of restorative materials using light-curing units with low irradiance power in deep cavities. the top surface of composites showed higher dc and khn than bottom, except for methacrylate at 27.8 and 39.6 j/cm2 (table 3). light curing beneath the restorative material at a 4 mm distance decreased the irradiance to approximately 380 mw/cm2 at bottom surface. the increased distance between the light guide tip and material, light scattering by filler particles and the thickness of the composite decreased the light intensity that reached the bottom surface of the restorative material12,19, resulting in lower dc and for most hardness values of the bottom compared to the top surface of the material. a previous retrospective longitudinal study showed good performance of two posterior composite resins over 22 years; yet a lower annual failure rate for the higher filler loaded composite was observed, suggesting that physical properties of the material may have some influence on the restoration longevity2. so, the small differences in the physical properties radiant exposure effects on physical properties of methacrylate and silorane-composites 173 braz j oral sci. 13(3):168-174 174 could result in identical short-term clinical performance, but not over an extended period. the clinical performance of dental composite restorations could be influenced by the physical properties. overall, the different radiant exposures evaluated did not affect the tested physical properties, but these properties were influenced by the chemical composition of the composite resin. acknowledgements this study was supported by fapesp (#2010/05666-9 and #2010/15076-4). references 1. catelan a, briso al, sundfeld rh, dos santos ph. effect of artificial aging on the roughness and microhardness of sealed composites. j esthet restor dent. 2010; 22: 324-30. 2. da rosa rodolpho pa, donassollo ta, cenci ms, loguércio ad, moraes rr, bronkhorst em, et al. 22-year clinical evaluation of the performance of two posterior composites with different filler characteristics. dent mater. 2011; 27: 955-63. 3. filho jd, poskus lt, guimarães jg, barcellos aa, silva em. degree of conversion and plasticization of dimethacrylate-based polymeric matrices: influence of light-curing mode. j oral sci. 2008; 50: 315-21. 4. baek dm, park jk, son sa, ko cc, garcia-godoy f, kim hi, et al. mechanical properties of composite resins light-cured using blue dpss laser. lasers med sci. 2013; 28: 597-604. 5. van ende a, de munck j, mine a, lambrechts p, van meerbeek b. does a low-shrinking composite induce less stress at the adhesive interface? dent mater. 2010; 26: 215-22. 6. weinmann w, thalacker c, guggenberger r. siloranes in dental composites. dent mater. 2005; 21: 68-74. 7. asmussen e, peutzfeldt a. influence of selected components on crosslink density in polymer structures. eur j oral sci. 2001; 109: 282-5. 8. palin wm, fleming gj, nathwani h, burke fj, randall rc. in vitro cuspal deflection and microleakage of maxillary premolars restored with novel low-shrink dental composites. dent mater. 2005; 21: 324-35. 9. duarte s jr, phark jh, varjão fm, sadan a. nanoleakage, ultramorphological characteristics, and microtensile bond strengths of a new low-shrinkage composite to dentin after artificial aging. dent mater. 2009; 25: 589-600. 10. mine a, de munck j, van ende a, cardoso mv, kuboki t, yoshida y, et al. tem characterization of a silorane composite bonded to enamel/ dentin. dent mater. 2010; 26: 524-32. 11. leprince j, palin wm, mullier t, devaux j, vreven j, leloup g. investigating filler morphology and mechanical properties of new lowshrinkage resin composite types. j oral rehabil. 2010; 37: 364-76. 12. aguiar fh, lazzari cr, lima da, ambrosano gm, lovadino jr. effect of light curing tip distance and resin shade on microhardness of a hybrid resin composite. braz oral res. 2005; 19: 302-6. 13. borges bc, souza-junior ej, catelan a, ambrosano gm, paulillo la, aguiar fh. impact of extended radiant exposure time on polymerization depth of fluoride-containing fissure sealer materials. acta odontol latinoam. 2011; 24: 47-51. 14. heo yj, lee gh, park jk, ro jh, garcía-godoy f, kim hi, et al. effect of energy density on low-shrinkage composite resins: diode-pumped solid state laser versus quartz-tungsten-halogen light-curing unit. photomed laser surg. 2013; 31: 28-35. 15. kusgoz a, ülker m, yesilyurt c, yoldas oh, ozil m, tanriver m. silorane-based composite: depth of cure, surface hardness, degree of conversion, and cervical microleakage in class ii cavities. j esthet restor dent. 2011; 23: 324-35. 16. schneider lf, moraes rr, cavalcante lm, sinhoretti ma, corrersobrinho l, consani s. cross-link density evaluation through softening tests: effect of ethanol concentration. dent mater. 2008; 24: 199-203 17. opdam nj, bronkhorst em, loomans ba, huysmans mc. 12-year survival of composite vs. amalgam restorations. j dent res. 2010; 89: 1063-7. 18. boaro lc, gonçalves f, guimarães tc, ferracane jl, pfeifer cs, braga rr. sorption, solubility, shrinkage and mechanical properties of “low-shrinkage” commercial resin composites. dent mater. 2013; 29: 398-404. 19. gonçalves f, kawano y, braga rr. contraction stress related to composite inorganic content. dent mater. 2010; 26: 704-9. 20. say ec, civelek a, nobecourt a, ersoy m, guleryus c. wear and microhardness of different resin composite materials. oper dent. 2003; 28: 628-34. 21. ferracane jl. hygroscopic and hydrolytic effects in dental polymer networks. dent mater. 2006; 22: 211-22. 22. giannini m, di francescantonio m, pacheco rr, cidreira boaro lc, braga rr. characterization of water sorption, solubility, and roughness of siloraneand methacrylate-based composite resins. oper dent. 2014; 39: 264-72. 23. gerhardt k, silva as, rego g, sinhoreti ma, salgado ve, schneider lf. bulk and surface properties related to composite filler size. braz j oral sci. 2013; 12: 323-9 24. da silva em, almeida gs, poskus lt, guimarães jg. relationship between the degree of conversion, solubility and salivary sorption of a hybrid and a nanofill resin composite. j applied oral sci. 2008; 16: 161-6. 25. faria-e-silva al, lima af, moraes rr, piva e, martins lr. degree of conversion of etch-and-rinse and self-etch adhesives light-cured using qth or led. oper dent. 2010; 35: 649-54. 26. catelan a, ambrosano gm, lima da, marchi gm, aguiar fh. influence of radiant exposure on degree of conversion, water sorption and solubility of self-etch adhesives. int j adhes adhes. 2013; 46: 40-3. 27. torres sa, silva gc, maria da, campos wr, magalhães cs, moreira an. degree of conversion and hardness of a silorane-based composite resin: effect of light-curing unit and depth. oper dent. 2014; 39: e137-46. radiant exposure effects on physical properties of methacrylate and silorane-composites braz j oral sci. 13(3):168-174 1http://dx.doi.org/10.20396/bjos.v18i0.8657269 volume 18 2019 e191651 original article 1 department of restorative dentistry, endodontics and dental materials, bauru school of dentistry, university of são paulo (usp), bauru, sp, brazil. 2 department of operative dentistry, western paraná state university (unioeste), cascavel, pr, brazil. corresponding author: fabiana scarparo naufel 2069 universitária street, cascavel, pr, 85814-110, brazil phone +55 45 32203168, e-mail: biberes@terra.com.br received: may 05, 2019 accepted: august 20 2019 effect of sports drinks on the surface properties of composite resins after prolonged exposure in vitro study daniella cristo santin1, fabiana scarparo naufel2,*, rafael francisco lia mondelli1, adriano piccolotto2, vera lúcia schmitt2 aim: using dietary supplements may affect the properties of composite resins due to their acidic ph. the present in vitro study aimed to assess the surface roughness and color stability of two composite resins nanohybrid (empress direct) and nanoparticulate (filtek z350) after prolonged exposure to dietary supplements. methods: we produced 30 specimens from each composite (8x2-mm discs) and divided them into six groups (n=10). after the initial measurement of the surface properties (roughness and color), we exposed the specimens to a degradation process in maltodextrin and whey protein for 22.5 and 7.5 days, respectively, using deionized water as the control solution. at the end of 22.5 days, we reassessed the specimens. after verifying data normality with the kolmogorov-smirnov test, we performed anova followed by tukey’s test at 5%. results: we found significant differences for materials immersed in the whey protein solution (p<0.05). the roughness of empress direct was higher (0.45+0.07) than filtek z350 (0.22+0.05). the composites tested also showed color change (δe>3.3) after the immersion period (p<0.001). in maltodextrin, the empress direct group presented (4.52+1.23) and filtek z350 (4.04+0.66), while after immersion in whey protein, they showed (5.34+1.68) and (4.26+1.02), respectively. conclusion: sports drinks changed the surface roughness and color stability of the composite resins studied. the filtek z350 group showed lower color variation than the empress direct composite in both solutions evaluated. keywords: composite resins. beverages. dietary supplements. surface properties. 2 santin et al. introduction despite the improvements in the physical properties of composite resins, this restorative material is still subject to deterioration in the oral cavity. the unfavorable interaction between the oral environment and external factors such as dietary habits may compromise the longevity of restorations1-3. some studies claim that ingesting acidic beverages may cause dental erosion and affect the properties of restorative materials1,4,5. in this context, the effect of regularly consuming dietary supplements requires further investigation. consuming sports drinks serves for nourishing/hydration, aiming to improve athletic performance during physical exercise4,5. nevertheless, they contain preservatives, acidulants, and large amounts of carbohydrates (glucose, fructose, sucrose, and maltodextrin), which create an acidic oral environment after consumption1 and may change the structure of composite resins6,7. the chemical challenges provided by an acidic diet cause the degradation of the resin-based restorative materials, resulting in a softer organic matrix and increased surface roughness6,8,9. in this case, daily toothbrushing would mechanically remove superficial layers of the restoration and modify the surface roughness7,10,11. this would lead to dental biofilm deposition and reduce restoration brightness7,10,12. as a result, the restorations may suffer an extrinsic color change due to the penetration of pigments from sports drinks into the porosities, compromising the color stability and the longevity of restorations6. thus, this in vitro study aimed to assess the effects of the daily exposure to dietary supplements on the surface properties (surface roughness and color stability) of composite resins. the null hypotheses tested in this study were: i) dietary supplements do not affect the surface roughness and color stability of composite resins; ii) the composition of composite resin materials does not affect the surface roughness and color stability after the exposure to dietary supplements. materials and methods experimental design this study analyzed two factors: composite resin (in two levels) and solution of immersion (in three levels), whereas the surface roughness and color stability were the response variables. following the manufacturer’s instructions for treatment, the consumption of maltodextrin (peter food, são paulo, brazil) should be fractioned during training, while the consumption of whey protein (new millen, são paulo, brazil) should occur immediately after training. thus, for the daily use of the supplements tested in the present study, we established a daily 30-min exposure to maltodextrin and 10-min exposure to whey protein. we calculated the exposure to sports drinks for three years of consumption, resulting in a total exposure time of 540 hours for maltodextrin and 180 hours for whey protein. specimen preparation in the present study, we tested the following two composite resins of color a2 for enamel: nanoparticulate (filtek z350, 3m espe, saint paul, usa) and nanohybrid 3 santin et al. (empress direct, ivoclar vivadent, schaan, liechtenstein). table 1 presents details of the materials tested. we prepared 60 disc-shaped specimens (30 from each resin) using an addition silicone mold with 8 mm in diameter and 2 mm in height. we covered the set with a transparent polyester strip and pressed it with a glass plate. then, we photoactivated the increment through the polyester strip with a led device (valo ultradent, south jordan, usa) for 20 seconds and measured the irradiance (1000 mw/cm2) of the curing light with a power meter (hilux dental curing light meter, benlioglu dental inc., ankara, turkey). then, we stored the specimens in deionized water for 24 hours at 37˚c. we polished the surfaces of the specimens sequentially using #600, #1200, and #2000 grit silicon carbide abrasive papers under water cooling for 10, 20, and 30 seconds, respectively4. at each swap, we cleaned the specimens ultrasonically in deionized water for 5 minutes (u.s. thornton electronic ltda., são paulo, brazil). after polishing, we identified the specimens and immersed them in deionized water for another 24 hours at 37˚c. surface roughness measurements (ra) we measured surface roughness (ra, µm) with a surfcorder se1700 surface roughness measuring instrument (kosaka corp, tokyo, japan). we took the measurements before and after the immersion period. for standardizing the readings, we divided the disc-shaped specimens virtually into two parts, marking the side of the specimen at the 12-hour position. then, on the left side of the disk, we took three readings from each specimen in three different directions (45°, 90°, and 135°) (figure 1), always starting from the most central region of the disc towards the periphery, and traveling 1.25 mm with a cut of 0.25 mm; minimum t = 0.01 μm and maximum t = 8.00 μm. we determined the ra value of each specimen by calculating the means of the three roughness readings for each time assessed (before and after immersion). assessment of color stability (δe) we used a cm-700d spectrophotometer (konica minolta, tokyo, japan) to obtain the initial and final colors of the specimens according to the ciel*a*b*. we transported the discs to a sample carrier and performed the readings in a light cabinet. we analyzed the color change by the difference between the initial and final δl, δa, and δb measurements13, using the following formula: δe = (δl*2 + δa*2 + δb*2]1/2. figure 1. description of the directions considered for measuring the surface roughness. 12h 45° 90° 135 4 santin et al. immersion of specimens in the solutions after measuring the initial roughness and color, we stored the specimens again in deionized water for 24 hours at 37˚c. degradation started for the three solutions studied (n=10): deionized water (control), maltodextrin, and whey protein (table 1). figure 2 shows the immersion protocol performed according to the groups evaluated. final roughness and color measurements after 22.5 days of immersion, we performed the final measurements of surface roughness and color stability, as described previously (figure 3). statistical analysis for surface roughness, we tabulated the data considering the factors of resin, time, and treatment, while for color stability (δe), the factors were resin and treatment. primarily, we analyzed the results using a kolmogorov-smirnov test to verify normal distribution. we continued with three-way anova with repeated measures for roughness and two-way anova for color stability (5%). tukey’s test (5%) determined statistically significant differences. table 1. composition of composite resins and dietary supplements. material manufacturer and batch composition color/taste ph filtek z350 3m espe (saint paul, mn, usa) 1507000832hb004134407 bis-gma, udma, tegdma, pegdma, bis-ema. non-aggregated silica and zirconia particles, silica/zirconia. ea2 empress direct ivoclar vivadent (schaan, liechtenstein) u02602 bis-gma, udma, tegma, barium glass particles, ytterbium trifluoroethane, mixed oxides, silica dioxide, copolymers. ea2 maltodextrin peter food (são paulo, brazil) 10686 maltodextrin, citric acid, tangerine flavor, ascorbic acid, sodium saccharin, sodium hydrochloride, titanium dioxide, dye yellow dye. tangerine 2.67 whey protein new millen (são paulo, brazil) 36807 whey protein isolated and concentrated, hydrolyzed whey protein, modified waxy maize starch, cocoa powder, magnesium pyruvate, zinc l-aspartate, xanthan gum, flavoring and sucralose sweetener. black forest 5.99 *abbreviations: bis-gma, bisphenol glycidyl methacrylate; udma, urethane dimethacrylate; tegdma, triethylene glycol dimethacrylate; pegma, polyethylene glycol dimetacrylate; bis-ema, ethoxylated bisphenol-a dimethacrylate. figure 2. description of the groups and immersion protocols evaluated. groups empress direct filtek z350 geco gzco control empress direct filtek z350 gemd gzmd maltodextrin 5ml of deionized water in each compartment renewed 1x week 22g of maltodextrin + 110ml of deionized water renewed 2x daily for 22.5 days 16g of whey protein + 110ml of deionized water renewed 2x daily for 7.5 days + 15 days in deionized water (renew 1x week) empress direct filtek z350 gewp gzwp whey protein immersion protocols 5 santin et al. results table 2 presents the mean and standard deviation of roughness values before and after immersing the composite resins in the solutions tested. the anova results showed statistically significant differences in material roughness for resin, immersion solution, and time (p<0.001). we observed a significant double interaction for the variables of immersion solution with time and resin and a triple interaction for all factors (p<0.001). tukey’s test (5%) revealed that, throughout the immersion protocol, the surface roughness of both resins decreased in all treatment solutions, except for the empress direct resin immersed in whey protein (p<0.05). for color variation (δe), anova indicated significant statistical differences for the factors of isolated resin and immersion solution and for the double interaction between the two variables (p<0.001). the data described in table 3 (tukey’s test at 5%) show that both resins presented a visible color change in all solutions, considering they showed δe greater than or equal to 3.3. the empress direct resin showed a higher δe in the control solution than in both sports drinks, and the z350 resin presented equivalent δe in all three solutions. for the variation of luminosity (δl) (table 3), anova showed statistically significant differences (p<0.001) for the isolated solution factor, figure 3. flowchart representing the research steps: a insertion of composite resin in the matrix; b – photoactivation; c – storage of the specimens in deionized water for 24 hours in a 37˚c stove; d – polishing with #600, #1200, and #2000 granulation files, followed by ultrasonic cleaning for 5 minutes; e – storage of the specimens for additional 24 hours at 37˚c; f, g – initial roughness and color measurements, respectively; h – immersion protocol in solutions of deionized water, maltodextrin, and whey protein; i – specimens immersed in individual plastic compartments containing the solutions assessed; j – storage in a 37˚c stove for 22.5 days for the control group and maltodextrin, and 7.5 days for whey protein (to complete 22.5 days, the specimens were immersed in deionized water for 15 days); k – cleaning of specimens in ultrasonic bath; l – final roughness and color measurements. a i h b j g c k f d l e 6 santin et al. and there was a lower luminosity reduction for both resins evaluated when immersed in maltodextrin, compared to the control group and whey protein (p<0.05). for the redgreen axis (δa), anova detected a significant difference for the interaction between the factors of resin and immersion solution (p<0.001). positive values (table 3) indicate a decrease in the green color and an increase in the red one. the interaction showed that for the a* ordinate, the empress direct composite resin showed greater variation in the control solution, while filtek z350 showed greater variation in the maltodextrin solution (p<0.05). the δa for whey protein was similar in both resins. for the δb, anova found a statistical significance for the variables of resin (p<0.001), immersion solution (p<0.05), and for the double interaction (p<0.001). the negative values of the b* ordinate (table 3) indicate a decrease of blue coloration, which is mainly evident in the specimens of the empress direct resin immersed in the control solution. the empress direct resin showed higher δb than z350, with statistical significance in the control solution and whey protein (p<0.05). discussion aesthetic restorative materials are subject to the gradual degradation process in the oral environment due to changes in ph, temperature, chewing, brushing, and composition of the restorative material2,3. because of the increased consumption of dietary supplements, this in vitro study aimed to assess the effects of sports drinks on the surface properties of composite resins. table 2. mean (µm) and standard deviation (sd) of surface roughness of the composite resins for the three factors studied. deionized water maltodextrin whey protein initial final initial final initial final empress direct 0.66 (0.15) aa 0.26 (0.03) ba 0.36 (0.12) aa 0.27 (0.04) ba 0.32 (0.02) bb 0.45 (0.07) aa filtek z350 0.37 (0.08) abb 0.28 (0.04) ba 0.39 (0.08) aa 0.28 (0.08) ba 0.44 (0.09) aa 0.22 (0.05) bb *different lower-case letters in the columns and upper-case letters in the rows indicate statistically significant differences (p<0.05). table 3. mean and standard deviation (sd) of δe, δl, δa* and δb* of resins after immersion in solutions. deionized water maltodextrin whey protein δe empress direct 7.54 (1.38) aa 4.52 (1.23) ab 5.34 (1.68) ab filtek z350 4.25 (2.14) ba 4.04 (0.66) aa 4.26 (1.02) aa δl* empress direct -2.79 (0.65) ba -1.57 (1.83) aa -2.33 (1.17) ba filtek z350 -2.32 (2.62) ba -0.71 (1.00) aa -3.04 (0.71) ba δa* empress direct 3.70 (1.08) aa 2.23 (0.89) bb 2.50 (2.00) aab filtek z350 1.50 (1.01) bb 3.39 (0.61) aa 2.54 (0.72) aab δb* empress direct -5.87 (1.12) bb -2.88 (1.60) aa -3.53 (1.54) ba filtek z350 -1.03 (2.66) aa -1.50 (1.16) aa -1.26 (1.02) aa *for each variation, different lower-case letters in the columns and upper-case letters in the rows indicate statistically significant differences (p<0.05) for each factor. 7 santin et al. in this study, we selected maltodextrin and whey protein because they are often consumed for increasing the physical performance and for muscle gain, respectively4. the results showed that the composites resins exposed to whey protein were mostly affected, potentially for being protein-based. the literature reports that the composite resin exposed to the oral environment allows adsorbing proteins on its surface14, which may have contributed to the increase in surface roughness and consequently greater retention of pigments. the tendency to staining is one of the disadvantages of composite resins1,8,9, therefore this study assessed whether resins immersed in dietary supplements would present color changes. hence, the study results rejected the first null hypothesis, considering that the analysis of δe values showed that both maltodextrin and whey protein solutions changed the color of the resins assessed. this resulted in δe values equal to or greater than 3.3, which is clinically perceptible to the human eye13. we may justify increased δe values after immersion in all solutions, including deionized water, because of the resin matrix potential of absorbing liquids8,15. the water absorbed carries the pigments of sports drinks and may stain the composites8. the results of the present study agree with the studies by erdemir et al.6 (2016), which state that the acidity of beverages may dissolute the organic matrix and increase the absorption of dyes that stain composite restorations, impairing their longevity. low-ph beverages (maltodextrin: ph=2.67; whey protein: ph=5.99) can solubilize restorative materials1,16. increased surface wear may occur due to organic matrix softening, resulting in the loss of structural ions and decreased resistance1,17,18. however, we did not assess the ph effect of the experimental solutions on wear resistance, which requires further investigation to evaluate such an effect. when comparing the two composite resins, the empress direct showed higher δe values than filtek z350 in all treatment solutions, rejecting the second null hypothesis, which we may attribute to the chemical composition19. potentially, against chemical and/or frictional wear, the inorganic particles of larger size (0.04–3 μm) in the empress direct release from the organic matrix, leaving craters on the surface and increasing roughness. this may have contributed to the increase of the extrinsic pigmentation and loss of brightness of the specimens6,8,19. in addition, its organic matrix based on bis-gma and udma appears to be more susceptible to dissolution1,17,18. it is worth noting that, in the oral cavity, the salivary cleaning action, toothbrushing, and polishing of restorations may reduce the staining susceptibility of composite restorations, which would contribute to greater clinical longevity. at the end of 22.5 days, the immersion in the solutions assessed reduced the surface roughness in all groups, except for the nanohybrid resin immersed in whey protein, which presented increased surface roughness values. according to previous studies1,2,19, the effect of treatment solutions varied depending on the type of material, rejecting again the second null hypothesis. for the empress direct composite resin, we verified the highest roughness value in whey protein, whereas for filtek z350, it occurred in deionized water and maltodextrin. this may occur because roughness is a property affected by the water sorption capacity of composite resins. composites with increased loads tend to absorb less liquid20. 8 santin et al. the simulation of the frequent consumption of sugary and/or acidic solutions (whey protein and maltodextrin) did not affect surface roughness. the organic phase of the composite resins, which is subject to water sorption, potentially determined the hygroscopic expansion, which relieves the stresses generated during polymerization shrinkage and may consequently reduce cracks and irregularities, as well as the roughness of composites1,21,22. the wear resistance of the filtek z350 nanoparticulate composite may relate to its chemical composition. the formulation based on nanoparticles and nanoclusters provides less interstitial space between the inorganic particles, making it more difficult to displace them against wear7,11. the smaller particles released leave minor surface defects, affecting the surface roughness and color stability of the restorations to a lesser extent11,19. the literature states that resins formulated with bis-ema tend to be less susceptible to dissolution when compared with those formulated with bis-gma and udma1,17,18. the resin-based composites made of inorganic particles of silica, zirconia, or barium glasses tend to suffer greater degradation than quartz particles1,16. analyzing the composition of the empress direct resin allows inferring that its exclusive composition of bis-gma and udma, in addition to the larger and soft spherical particles (silica and barium glass), determined an increased softening and leaching of inorganic fillers1,17. in situ and in vivo studies are required to complement the findings of the present study regarding the degradation of composite resins exposed to dietary supplements. under the experimental conditions described, sports drinks changed the surface properties of the resins assessed, considering there were color changes after the immersion in the beverages tested and increased surface roughness for the empress direct resin immersed in whey protein. the nanoparticulate composite resin presented increased resistance to surface degradation, showing the best results for both properties evaluated. references 1. erdemir u, yildiz e, eren mm, ozel s. surface hardness evaluation of different composite resin materials: influence of sports and energy drinks immersion after a short-term period. j appl oral sci. 2013 mar-apr;21(2):124-31. doi: 10.1590/1678-7757201302185. 2. münchow ea, ferreira aca, machado rmm, ramos ts, rodrigues-jr sa, zanchi ch. effect of acidic solutions on the surface degradation of a micro-hybrid composite resin. baz dent j. 2014;25(4):321-6. 3. fatima n, hussain m. effect of two different commonly available energy drinks on surface micro hardness of tooth color restorative materials. j research dent. 2014 may/jun;2(3):269-76. doi: 10.19177/jrd.v2e32014269-276. 4. vidal mg, de oliveira ph, lima-arsati ybo, rodrigues ja. the effect of dilution on the erosive potential of maltodextrin-containing sports drinks. rev odontol unesp. 2017 jan-feb;46(1):28-32. doi: 10.1590/1807-2577.12316. 5. cochrane nj, yuan y, walker gd, shen p, chang ch, reynolds c, et al. erosive potential of sports beverages. aust dent j. 2012 sep;57(3):359-64; quiz 398. doi: 10.1111/j.1834-7819.2012.01708.x. 9 santin et al. 6. erdemir u, yildiz e, saygi g, altay ni, eren mm, yucel t. effects of energy and sports drinks on tooth structures and restorative materials. world j stomatol. 2016 feb;5(1):1-7. doi: 10.5321/wjs.v5.i1.1. 7. paula ab, fúcio sbp, ambrosano gmb, alonso rcb, sardi jco, puppin-rontani rm. biodegradation and abrasive wear of nano restorative materials. oper dent. 2011 nov-dec;36(6):670-7. doi: 10.2341/10-221-l. 8. erdemir u, yildiz e, eren mm. effects of sports drinks on color stability of nanofilled and microhybrid composites after long-term immersion. j dent. 2012 dec;40 suppl 2:e55-63. doi: 10.1016/j. jdent.2012.06.002. 9. lepri cp, palma-dibb rg. surface roughness and color change of a composite: influence of beverages and brushing. dent mater j. 2012 apr;31(4):689-96. 10. barbosa rp, pereira-cenci t, da silva wm, coelho-de-souza fh, demarco ff, cenci ms. effect of cariogenic biofilm challenge on the surface hardness of direct restorative materials in situ. j dent. 2012 may;40(5):359-63. doi: 10.1016/j.jdent.2012.01.012. 11. de oliveira gu, mondelli rfl, rodrigues mc, franco eb, ishikiriama sk, wang l. impact of filler size and distribution on roughness and wear of composite resin after simulated toothbrushing. j appl oral sci. 2012 sep-oct;20(5):510-6. 12. quirynem m, bollen cml. the influence of surface roughness and surface-free energy on supra and subgingival plaque formation in man: a review of the literature. j clin periodontol. 1995 jan;22(1):1-14. 13. ruyter ie, nilner k, moller b. color stability of dental composite resin materials for crown and bridge veneers. dent mater. 1987 oct;3(5):246-51. doi: 10.1007/s10266-018-0350-9. 14. zhang n, zhang k, melo ma, weir md, xu dj, bai y, et al. effects of long-term water-aging on novel anti-biofilm and protein-repellent dental composite. int j mol sci. 2017 jan 18;18(1). pii: e186. doi: 10.3390/ijms18010186. 15. ardu s, duc o, di bella e, krekci i, daher r. correction to: color stability of different composite resins after polishing. odontology. 2018 jul;106(3):350. doi: 16. valinoti ac, neves bg, silva em, maia lc. surface degradation of composite resins by acidic medicines and ph-cycling. j appl oral sci. 2008 jul-aug;16(4):257-65. 17. erdemir u, yildiz e, eren mm, ozel s. surface hardness of different restorative materials after long-term immersion in sports and energy drinks. dent mater j. 2012;31(5):729-36. 18. tanthanuch s, kukiattrakoon b, siriporananon c, ornprasert n, mettasitthikorn w, likhitpreeda s, et al. the effect of different beverages on surface hardness of nanohybrid resin composite and giomer. j conserv dent. 2014 may;17(3):261-5. doi: 10.4103/0972-0707.131791. 19. al-samadani k. effect of energy drinks on the surface texture of nanofilled composite resin. j contemp dent pract. 2013 sep;14(5):830-5. doi: 10.5005/jp-journals-10024-1411. 20. tavangar m, bagheri r, kwon ty, mese a, manton dj. influence of beverages and surface roughness on the color change of resin composites. j investig clin dent. 2018 aug;9(3):e12333. doi: 10.1111/jicd.12333. 21. bowen rl, rapson je, dickson g. hardening shrinkage and hygroscopic expansion of composite resins. j dent res. 1982 may;61(5):654-8. 22. hanssen ek. visible light-cured composite resins: polymerization contraction, contraction pattern and hygroscopic expansion. scand j dent res. 1982 aug;90(4):329-35. oral sciences n3 braz j oral sci. 13(1):64-69 original article braz j oral sci. january | march 2014 volume 13, number 1 shear bond strength of a “solvent-free” adhesive versus contemporary adhesive systems eugenia koliniotou-koumpia1, pantelis kouros1, effimia koumpia2, maria helvatzoglou-antoniades1 1aristotle university of thessaloniki, dental school, department of operative dentistry, thessaloniki, greece 2aristotle university of thessaloniki, dental school, department of orthodontics, thessaloniki, greece correspondence to: eugenia koliniotou-koumpia department of operative dentistry dental school, aristotle university of thessaloniki 54124 thessaloniki greece phone: (0030) +2310342957 e-mail: jeny@dent.auth.gr received for publication: february 02, 2014 accepted: march 20, 2014 abstract aim: to compare the shear bond strength (sbs) of a solvent free self-etch adhesive with solvent containing adhesives. methods: forty-five human teeth were sectioned longitudinally to expose superficial dentin and substrates polished with 600-grit sic paper. the adhesive area was isolated with a cylindrical teflon mold 3x4 mm. fifteen specimens were prepared for each material. were evaluated a solvent free self-etch adhesive (bond 1 sf), an ethanol self-etch adhesive (futurabond m), and a water-acetone-ethanol self-etch adhesive (optibond all-in-one). all specimens were subjected to an aging procedure by thermo-cycling (5000 cycles). thirty-six specimens were stressed in shear at a rate of 0.5mm/min. mean data values were analyzed statistically using the welch robust analysis of variance and the games-howell statistic. failure patterns were analyzed using stereomicroscope and scanning electron microscopy (sem). additional more dentin specimens were prepared for sem. results: the bond 1 sf showed the statistically significant lowest sbs to dentin (welch statistic p<0.001). failures for bond 1 sf were mainly adhesive failures with partial cohesive failures in the adhesive resin, while for futurabond m and optibond all-in-one were mainly mixed. sem findings confirm the results. conclusions: eliminating solvents from self-etch adhesive systems may decrease the bonding strength to dentin. keywords: adhesives; bonding systems; shear bond strength; solvents. introduction clinically, the most attractive current adhesive systems are the one-step selfetch systems, since an additional rinse and drying step is no longer needed1.selfetch adhesive systems are reported to exhibit low technique sensitivity regarding the conditions of the interface, which may be due to the fact that penetration of the acid components and the co-monomers occurs to the same depth2. self-etch dental adhesive systems are a complex mixture of components, including reactive monomers, an association of dissolved hydrophilic and hydrophobic monomers, cross linkers, initiators and solvents (such as water, acetone and ethanol)3. hema(2-hydroxyethyl methacrylate) is a widely used, water-soluble low-molecular-weight methacrylate monomer that enhances the penetration efficacy of an adhesive into demineralized dentin4,and has been reported to positively influence the bond strength to dentin5. hema also acts as a solvent and helps prevent hydrophilic and hydrophobic phase separations in one-step systems2,4,6-7. furthermore, adhesive systems also comprise organic molecules of lower gisele higa texto digitado http://dx.doi.org/10.1590/1677-3225v13n1a13 braz j oral sci. 13(1):64-69 17165 polarity, which may form a homogeneous phase when proper co-solvents are used, such as acetone, ethanol or butanol8. several researches have been carried out to determine the role of each ingredient found in adhesive systems 2-4. severalaspects have been studied, such as resin dentin bond strengths as a function of the nature, amount, and evaporation rate of incorporated solvents3,9. authors have reported lower bonding effectiveness for simplified adhesive systems1,10. recently, the presence and evaporation of solvents was determined to have an effect on monomer infiltration and polymerization9.some authors have dealt with the effect of the solvent in the dentin primer of three-step adhesive systems on the bond strength and the marginal adaptation11. however, there is no current study that compares the influence of a solvent free self-etch adhesive system versus an ethanol self-etch adhesive system and a water-acetoneethanol self-etch adhesive system. therefore, the purpose of this laboratory study was to investigate the shear bond strength of a new solvent-free self-etch adhesive system versus two contemporary self-etch adhesive systems containing different solvents used on superficial dentin. the null hypothesis tested was that self-etch adhesive systems containing different solvents or no solvent at all are equally effective in terms of shear bond strength when bonded to superficial dentin substrates. material and methods forty-five freshly extracted non-carious human third molars were stored in 0.5% chloramine solution at 4oc until use in the current study. all procedures detailed in the present investigation were performed in accordance with the protocol outlined by the ethics committee of the school of dentistry, aristotle university of thessaloniki, greece (protocol 280/ 25.01.2012), regarding the recommended standard practices for biological investigations in extracted human teeth. the roots of the teeth were removed 2 mm below the cemento-enamel junction using a water-cooled diamond saw. the crowns were sectioned perpendicularly to the tooth long axis, exposing the superficial dentin using low speed sectioning machine (isomet 1000; buehler ltd, lake bluff, il, usa). the exposed dentin substrates were abraded using 600-grit sic paper, rinsed with water, and air dried for 3 s with dental air syringe at a distance of 15cm and air pressure of 3.8 kg/cm2. the specimens were randomly assigned to three groups in order to test shear bond strength of each material to dentin substrates. the dentin samples were embedded in a self-cure resin (concise, 3m dental products, st paul, mn, usa) in the middle of a metal ring mold. the surfaces to be tested were delimited using 3 mm diameter holes punched in teflon tape (ptfe teflon tape, cs hyde company, inc. illinois, usa) using a modified rubber-dam punch. this step was necessary to restrict the bonding of the adhesive system and the composite resin restorative material to the test area. the adhesive systems tested were: a solvent free selfetch adhesive system bond 1 sf/artiste (pentron clinical corporation, wallingford ct, usa), an ethanol self-etch adhesive system futurabond m /grandio so (voco gmbh, cuxhaven, germany), and a water-acetone-ethanol self-etch adhesive system optibond all-in-one /herculite ultra xrv(kerrcorporation, orange, ca, usa). the materials and their chemical compositions are listed in chart 1. twelve dentin samples were treated with each adhesive bonding system. the adhesive systems were applied on the surfaces following the manufacturers’ instructions. bond-1 sf was applied in one coat, rubbing with the specific dip applicator for 20 s and then light cured for 10 sec. futurabond m was applied with a brushing motion for 20 s in a single coat, thoroughly air-dried for at least 5 s to evaporate the solvent and light cured for 10 sec. optibond all-in-one was applied in two coats by a brush with scrubbing motion for 20 s each, thoroughly air-dried for at least 5 s to evaporate the solvent and light cured for 10 s. after curing the adhesive systems, a polytetrafluoroethylene mold (3 mm in diameter and 4 mm in height) was placed over the specimen and filled with the respective manufacturer’s composite resin in two increments. each increment was light-cured for 40 s using a bluephase (ivoclarvivadent ag, schaan, liechtenstein) device. this procedure resulted in cylindrical specimens of composite resin measuring 3 mm in diameter and 4 mm in height being bonded to the dentin. all specimens were subjected to an aging procedure by thermo-cycling (5000 cycles between 5 and 55oc, dwell time 30s). the shear bond strength (sbs) was determined after storage in water at room temperature (25oc) for 24 h. shear testing was conducted using a universal testing machine (testometric ax, m 350-10kn, rochdale, england) at a crosshead speed of 0.5 mm/min with the load of a metal chisel until fracture. the load at fracture, expressed in mpa, was calculated by dividing the peak load by the bonding area. the data were subjected to statistical analyses. the assumption of normality was tested with the shapiro-wilk test; which demonstrated normality with the data. levene’s test for equality of variances was used to test the homogeneity of variances, which was rejected. accordingly, the hypothesis concerning the equality between mean data values was tested using the welch robust analysis of variance. pair wise comparisons between mean data values were conducted using the games-howell statistic. the analysis was performed with the spss 16.0 software and the statistical significance was set for p<0.05. failure patterns at the dentin/restorative system interface were analyzed under a stereomicroscope at 40 x magnification (olympus co, tokyo, japan) to determine the failure modes. failure was considered to be: a) adhesive, if it occurred at the dentin/adhesive interface; b) cohesive, if it occurred in the material or in the substrate and c) mixed, when involving both the interface and the material. the bond failure patterns were not statistically analyzed. three specimens of each fracture failure mode from the three groups tested for shear bond strength were randomly selected for evaluation using scanning electron microscopy (sem). in order to investigate the effect of solvents on the adhesive interface, three more shear bond strength of a “solvent-free” adhesive versus contemporary adhesive systems 66 futurabond m optibond all-in-one bond 1 sf 20.9 38 10.34 23.02 13.9 11.51 29.24 19.71 9.87 9.57 22.47 8.68 32.14 31.43 7.1 20.82 41.19 13.99 17.86 30.26 10.05 15.37 20.47 9.55 3.95 26.9 3.67 18.23 40.62 8.89 13.31 40.62 12.21 24.04 24.5 10.6 mean (sd) 19.04 (7.91) 28.36 (8.67) 9.71 (2.6) shear b o n d strength (mpa) table 1.table 1.table 1.table 1.table 1.shear bond test results expressed in mpa for each test group dentin specimens of each adhesive system were prepared for sem examination. in these additional specimens, the processing followed the same procedures as described above, but the specimens were sectioned longitudinally to expose the dentin-adhesive junction. the sem examinations were done on the longitudinally sectioned hybridized areas prepared in the proceeding procedures12.the sections were polished with no. 1200-grit paper discs and immersed in 6mol/l hydrochloric acid (hcl) for 30s. this was followed by immersion in a 1wt% sodium hypochlorite (naocl) for 10 min. specimens were rinsed with distilled water and stored in desiccators overnight to dry. the specimens were then mounted on stubs, sputter-coated with carbon and examined by one evaluator under sem (jeol, j.s.m.-840 tokyo, japan) at 19 kv. results sbs test results are shown at table 1. statistically significant differences were found between the three materials (welch statistic: f (2, 15.904)=30.863, p<0.001). the solvent free adhesive system (bond 1 sf) exhibited the statistically significant lowest bond strength (11.3±1.54 mpa) when compared with the other two adhesive systems, braz j oral sci. 13(1):64-69 shear bond strength of a “solvent-free” adhesive versus contemporary adhesive systems chart 1.chart 1.chart 1.chart 1.chart 1. materials studied and their chemical compositions as provided by manufactures. 17167 fig. 2. a) sem micrograph of a mixed failure for futurabond m at x500. “r” indicates resinous material while “d” indicates dentin. b) representative sem micrograph at x500 of dentin-adhesive-resin interface formed by futurabond m; hybrid layer of thickness 2 to 4 µm is visible. (rresin, hhybrid layer, ddentin). fig. 3. a) sem micrograph of a mixed failure for optibond all-in-one at x500. “r” indicates resinous material while “d” indicates dentin. b) representative sem micrograph at x500 of dentin-adhesive-resin interface formed by optibond all-inone; hybrid layer of thickness 1 to 1,5 µm is visible. (rresin, hhybrid layer, ddentin). fig. 4. a) sem micrograph of an adhesive failure for bond-1 sf at x500. dentinal tubules are visible on the dentin surface (d), while a small amount of resin fillers (r) are detectable on the surface. b) representative sem micrograph at x500 illustrating dentin-adhesive interface formed by self-etch adhesive bond-1 sf. a gap is visible between the resinous material (r) and the dentin surface (d). futurabond m (20.2±6.11 mpa) and optibond all-in-one (29.2±9.35 mpa) (games-howell statistic, p=0.001 and p<0.001, respectively). additionally, the bond strength of futurabond m, was statistically significant lower than optibond all-in-one (games-howell statistic, p=0.031). optibond all-in-one showed the highest bond strength (figure 1). when analyzing failure patterns under a stereomicroscope and by sem, it was possible to observe a variety of different zones of failure on the same surface. a mixed-failure (8/12) mode was predominantly observed in fractured specimens that were treated with futurabond m (figure 2a), the remaining fractures were adhesive (4/12). failure modes in dentin/restorative material interface in fractured specimens that were treated with optibond allin-one were cohesive in dentin (4/12) or mixed (6/12) (figure 3a), although two failures were adhesive (2/12). on the other hand, the groups treated with the solvent free adhesive (bond-1 sf) exhibited a higher percentage of adhesive fractures (8/12) (figure 4a) and some mixed failure modes (4/12). sem imaging of the dentin-adhesive interfaces formed by futurabond m showed that the fiber network was entirely integrated into the adhesive layer (figure 2b). the interface morphology can be described as similar to optibond allin-one, although it was not as uniform and the resin tags were more scattered and thinner. the hybrid layer thickness was approximately 1µm. for the optibond all-in-one adhesive system, the sem images of the dentin–adhesive interfaces illustrated a uniform hybrid layer between 1-1.5µm thick. optibond all-in-one delivers excellent and extensive penetration into dentinal tubules forming prominent tags. the junction between the adhesive and dentin appeared tight and continuous (figure 3b). regarding the hybrid layer which forms bond 1 sf, sem analysis reveals an inconsistency. it appears that only parts of the material achieving contact with the dentin surface and there is a formation of arcs between the contact areas, which are probably due to the polymerization shrinkage of the material (figure 4b). fig. 1. shear bond strength of three one step self-etching adhesives to dentin. discussion according to this study’s results, the null hypotheses claiming that that self-etch adhesive systems containing different solvents or no solvent at all are equally effective in terms of shear bond strength when bonded to superficial dentin substrates has to be rejected. many factors can influence the bonding performance of adhesive systems to dentin, among these are: dentin substrate13, dentin treatment14 and chemical composition of the adhesive systems, including the solvents. bond 1 sf is a light-cured, one-coat self-etch adhesive system in which the manufacturer has removed the solvent that is contained in virtually all other adhesive systems. all-in-one adhesive systems are currently composed of hydrophilic and hydrophobic monomers and solubilized in water and/or braz j oral sci. 13(1):64-69 shear bond strength of a “solvent-free” adhesive versus contemporary adhesive systems 68 organic solvents, such as acetone or ethanol. such monomers can penetrate the smear layer and perform a mild demineralization of the underlying dentin, forming covalent bonds with the dentin collagen and ionic bonds with hydroxyapatite15-16. a high solvent content, in particular water, is necessary for adequate ionization of the acidic monomer17.increasing the water concentration reduces the monomer concentration and may reduce bond strengths, possibly by reducing the degree of monomer polymerization17. while remembering that bond 1 sf contains the 4-met monomer, which was proven to form ionic bonds with hydroxyapatite15,it was reasonable to expect a certain degree of chemical interaction and to observe a shallow resin-dentin interface under sem. sem samples showed only partial formation of hybrid layer in this group. a gap was present between the material and the dentin substrate. the lack of a solvent in this adhesive system could be correlated with all facts described above and may explain the relatively lower bond strength of bond 1 sf adhesive system. additionally, the ph value of bond 1 sf (ph=3-4) is comparatively higher than optibond all-in-one (ph=2.5) and futurabond m (ph1.4) and this may decrease the dentin demineralization depth and among other factors as the composition of the material and the infiltration capacity may lead to lack formation and lower bond strength. however, further studies are needed to support those hypotheses. a previous study suggested that the presence of moisture on the dentin surface is essential when the bond 1 solvent free adhesive system is applied18. their results showed that the duration of air drying of the dentin surface affected microtensile bond strength and that prolonged air-drying of the dentin surface decreased the bond strengths. although in this study the dentin surface was air dried prior adhesion for 3 sec, the solvent free adhesive system failed to achieve satisfactory bond strength values. optibond all-in-one is a single component, light cure, one step self-etch adhesive system. the results of this current study showed that optibond all-in-one revealed the highest shear bond strength values. optibond all-in-one values were statistically significantly higher than futurabond m. the differences between these two one-step self-etch adhesive systems are attributed to the actual functional monomer and solvent system provided, consisting of a mixture of water, ethanol and acetone. the bonding performance obtained by self-etch adhesive systems varies considerably, depending on the composition and, more specifically, on the functional monomer included in the adhesive formulation 19.the chemical composition of optibond all-in-one adhesive system differs from the other adhesive systems examined in this present study due to its dimethacrylate monomers and gpdm (glycerol phosphate dimethacrylate). glycerol phosphate dimethacrylate is a monomer that has a low viscosity and improved water solubility. the concentrations of individual monomers in the adhesive system and their interactions determine the extent of infiltration, ionization, and cross-linking obtained during polymerization and subsequent mechanical properties of the adhesive system16,19.it was determined that the ultra-structure of dentin-adhesive interfaces formed by self-etch adhesive systems depends on the interaction of functional monomers with dentin and on the acidity (ph=2.5) of the self-etch solution16,19. as mentioned in the results section, sem images of the dentin–adhesive interfaces formed by optibond all-in-one adhesive system illustrated a uniform hybrid layer and excellent penetration into dentinal tubules, forming prominent tags. the junction between the adhesive and dentin appeared tight and continuous. in addition, several possible mechanisms may be responsible for the improved durability of resin-dentin bonds made with water-acetone-ethanol saturated dentin. it is possible that the ternary solvent system provides enhanced self-life stability and effective bond strength. incomplete adhesive solvent removal following air-drying for all-in-one adhesive systems20-21, may impair polymerization22,and result in reduced bond strength23.the presence of acetone in optibond all-in-one seems to help residual solvent and excess water removal following air-drying. solvents and excess water need to be removed prior to or during monomer polymerization to obtain a pore free adhesive layer. despite the presence of hema or hydrophilic groups in the final copolymer, reversible water uptake is inhibited by adequate crosslinking of the adhesive3. solvents are used to dissolve polar and non-polar components3. solvents containing the products examined in this current study showed large variations on shear bond strengths from one product to another. evaporation of solvents may also contribute to the elimination of water from the hybrid layer precursor before polymerization3. furthermore, solvents modify pre-polymer viscosities and help adhesive spreading and substrate wetting3.futurabond m is also an all in one self-etch adhesive system reinforced with nanoparticles. manufacturers suggest that nano-scaled silicon dioxide particles with a diameter of ca. 20 nm (0.00002 mm) provided for cross-linking of the bond’s resin components and improve its film building properties. the adhesive can thus optimally wet the released collagen fibers and micro-retentive etching pattern. as a result, the sensitive collagen fiber network cannot collapse and is entirely integrated into the adhesive layer. the far-reaching resin tags in the dentine tubules harden during polymerization and strengthen the retentive bond of the collagen fiber-bonding hybrid layer. the solvent system of this adhesive is ethanol, which is a polar solvent that forms hydrogen bonds. however, due to its much lower dielectric constant, ethanol is also a more appropriate solvent for less polar solutes7. in futurabond m, ethanol helps the adhesive to optimally wet the released collagen fibers in order to strongly bond to dentin24. this was confirmed by the results of this present study. the interface morphology can be described as similar to optibond all-in-one, although it was not as uniform and resin tags were more scattered and thinner. this adhesive system also differs from optibond all-in-one in that the hybrid layer was thinner. the long-term stability of adhesive restorations depends not only on the thickness of braz j oral sci. 13(1):64-69 shear bond strength of a “solvent-free” adhesive versus contemporary adhesive systems 17169 the hybrid layer, but also on the quality of the hybrid layer25. nonetheless, acetone is preferred as a solvent medium, due to its better hydrolytical stability of the functional monomers in acetone when compared to ethanol10.the reduced bonding effectiveness of futurabond m can be assumed to be due to incorporating only ethanol, while optibond allin-one contains both ethanol and acetone. however the etching ability of the acidic monomers, which is included in each self-etch adhesive system and the contained polymerization initiators may be improving the bond strength to dentin16. the current results are in accordance with one in vitro study11 that examined the influence of acetone /water versus ethanol/water solvent mixture in a dentin primer of a three-step adhesive system. those authors found that the marginal integrity of non-retentive composite fillings might be affected11. those results also indicated that it is not only the water content of the primer that was a crucial factor, but also the type of organic solvent used (acetone or ethanol)11. also the current results are in accordance with the results of a new study that evaluated the bonding effectiveness of this new solvent free adhesive system26. within the limitations of this study, it may be concluded that elimination of the solvent from a self-etch adhesive system may lead to decrease of infiltration of the adhesive components into the dental tissue’s microstructures, debility of hybrid zone formation and eventually to a decrease of the bond strength to the dentin. acknowledgements the kind help from the physics department, scanning electron microscopy, aristotle university of thessaloniki, greece and from dr. oikonomidis stavros in performing sem procedures for this present work is very much appreciated. references 1. sarr m, kane aw, vreven jj, mine a, van landuyt kl, peumans m, et al.microtensile bond strength and interfacial characterization of 11 contemporary adhesives bonded to bur-cut dentin. oper dent. 2010; 35: 94-104. 2. moszner n, salz u, zimmermann j. chemical aspects of self-etching enamel-dentin adhesives: a systematic review. dent mater. 2005; 21: 895-910. 3. grégoire g, dabsie f, dieng-sarr f, akon b, sharrock p. solvent composition of one-step self-etch adhesives and dentine wettability. j dent. 2011; 39: 30-9. 4. nakabayashi n, takarada k. effect of hema on bonding to dentin. dent mater. 1992; 8: 125-30. 5. nakaoki y, nikaido t, pereira pn, inokoshi s,tagami j. dimensional changes of demineralized dentin treated with hema primers. dent mater. 2000; 16: 441–6. 6. van landuyt kl, de munck j, snauwaert j, coutinho e, poitevin a, yoshida y, et al. monomer-solvent phase separation in one-step self-etch adhesives. j dent res. 2005; 84: 183-8. 7. van landuyt kl, snauwaert j, peumans m, de munck j, lambrechts p, van meerbeek b. the role of hema in one-step self-etch adhesives. dent mater. 2008; 24: 1412-9. 8. van landuyt kl, snauwaert j, de munck j, peumans m, yoshida y, poitevin a, et al. systematic review of the chemical composition of contemporary dental adhesives. biomaterials 2007; 28: 3757-85. 9. klein-júnior ca, zander-grande c, amaral r, stanislawczuk r, garcia ej, baumhardt-neto r et al. evaporating solvents with a warm airstream: effects on adhesive layer properties and resin-dentin bond strengths. j dent. 2008; 36: 618-25. 10. van landuyt k, peumans j, de munck j, lamprechts p, van meerbeek b. extension of one-step self-etch adhesive into a multi-step adhesive. dent mater. 2006; 22: 533-44. 11. balkenhol m., huang j, wöstmann b, hannig m. influence of solvent type in experimental dentin primer on the marginal adaptation of class v restorations. j dent. 2007; 35: 836-44. 12. miyasaka k,& nakabayashi n. effect of phenyl-p/hema acetone primer on wet bonding to edta-conditioned dentin. dent mater. 2001; 17: 499503. 13. cavalcanti an, de souza es, lopes gds, de freitas ap, de araújo rpc, mathias p. effect of a desensitizing dentifrice on the bond strength of different adhesive systems. braz j oral sci. 2013; 12: 2. 14. menezes fch, borges ga, valentino ta, oliveira mahm, turssi cp, correr-sobrinho l. effect of surface treatment and storage on the bond strength of different ceramic systems. braz j oral sci. 2009; 8: 119-23. 15. yoshida y, nagakane k, fukuda r, nakayama y, okazaki m, shintani h et al. comparative study on adhesive performance of functional monomers. j dent res. 2004; 83: 454-8. 16. ikemura k, kadoma y, endo t. a review of the developments of selfetching primers and adhesives. effects of acidic adhesive monomers and polymerization initiators on bonding to ground smear layer-covered teeth. dent mater. j 2012; 30: 769-89. 17. hiraishi n, nishiyama n, ikemura k, yau jy, king nm, tagami j et al. water concentration in self-etching primers affects their aggressiveness and bonding efficacy to dentin. j dent res. 2005; 84: 653-8. 1. 18.takai t, hosaka k, kambara k, thitthaweerat s, matsui n, takahashi m et al. effect of air-drying dentin surfaces on dentin bond strength of a solvent-freeone-step adhesive. dent mater. j 2012; 31: 558-63 18. van meerbeek b, yoshihara k, yoshida y, mine a, de munck j, van landuyt kl. state of the art of self-etch adhesives. dent mater. 2011; 27: 17-28. 19. sidhu sk, omata y, tanaka t, koshiro k, spreafico d, semeraro s, et al. bonding characteristics of newly developed all-in-one adhesives. j biomed mater res b. 2007; 80: 297-303. 20. van landuyt kl, snauwaert j, de munck j, coutinho e, poitevin a, yoshida y, et al. origin of interfacial dropletswith one-step adhesives. j dent res. 2007;86: 739-44. 21. nunes tg, garcia fcp, osorio r, carvalho r, toledano m. polymerization efficacy of simplified adhesive systems studied by nmr and mri techniques. dent mater. 2006; 22: 963-72. 22. sadr a, shimada y, tagami j. effects of solvent drying time on micro shear bond strength and mechanical properties of two self-etching adhesive systems. dent mater. 2007; 23:1114-9. 23. li f, liu xy, zhang l, kang jj, chen jh. ethanol-wet bonding technique may enhance the bonding performance of contemporary etch-and-rinse dental adhesives. j adhes dent. 2012; 14:113-20. 24. eliguzeloglu e, genc ö, özcopur b, belli s, eskitascioglu g, özcan m. influence of powdered dentin on the shear bond strength of dentin bondingsystems. dent mater j. 2012; 31: 758-64. 25. shirban f, khoroushi m, shirban m. a new solvent-free one-step selfetch adhesive: bond strength to tooth structures. j contemp dent pract. 2013; 14: 269-74. braz j oral sci. 13(1):64-69 shear bond strength of a “solvent-free” adhesive versus contemporary adhesive systems oral sciences n3 braz j oral sci. 14(1):41-45 original article braz j oral sci. january | march 2015 volume 14, number 1 relations between oral health and work ability among administrative workers rafael aiello bomfim1, edgard crosato2, luiz eugênio nigro mazzilli2 1 universidade federal do mato grosso do sul – ufms, school of dentistry, departament of community dentistry, campo grande, ms, brazil 2 universidade de são paulo – usp, school of dentistry, departament of community dentistry, são paulo, sp, brazil correspondence to: rafael aiello bomfim cidade universitária faodo/ufms av. senador fillinto muller, s/n, vila ipiranga cep 79080-190 campo grande ms brasil phone: +55 11 99991 0203, +55 67 3345 7379 e-mail: rafael.aiello@ufms.br abstract aim: to analyze the relationships between perceived oral health quality of life and work ability index. methods: the data regarding administrative workers of a private textile company in são paulo brazil, included socio-demographic, occupational characteristics, self-perceived oral health (ohip 14) and self-perceived work ability index (wai). results: the response rate of the questionnaires was 75.20% and the reliability of the instruments (conbrach’s alpha) was 0.89 for ohip 14 and 0.64 for wai. linear multiple regression analyses showed that ohip 14 was associated with educational level (p=0.009) and work ability index (p=0.001) of workers, regardless of other variables. conclusions: these results showed the importance of adopting oral health programs in private companies to improve oral health and work ability. keywords: occupational dentistry; oral health; work capacity evaluation. introduction oral health status is a matter of great importance not only for individuals in terms of their quality of life1 but also considering the demands and requirements related to occupational practice. in fact, anyone who experienced an oral disease knows how these situations affect the work ability leading to a dilemma: to work in unsatisfactory conditions (presenteism) or sick leave. since health related absenteeism rate is commonly used as an indicator of the health status of employees, some authors2-5 performed studies regarding oral diseases impacts on absenteeism and concluded that pulp pathologies (icd-10, k04) and tmj dysfunction (icd-10, k07.6) are usually its major causes. despite its relevance, this information fails to evaluate the perception of social, physical and psychological disabilities and functional limitations in the work environment6, which suggests the use of other approaches and instruments to assess these impacts7. among the classical quality of life instruments, were adopted the work ability index (wai), developed by the finnish institute of occupational health (fioh) and the oral health impact profile-14 (ohip-14). the wai index is based on the assumption of a combination of human resources in relation to social, mental and physical demands, as well as its relationship to management, organizational culture, community and the workplace8. its concept is expressed by the question “how well is, or will be, a worker in the present or near future, and how he will be able to perform his work according to the requirements of his status of health, physical and mental habilities?”9 this instrument has been validated for use in a brazilian population10. the ohip is a well-known instrument that also had been validated to brazilian portuguese and is used to record and to analyze the oral health received for publication: january 20, 2015 accepted: march 16, 2015 4242424242 impact profile. the 14-question version assesses seven dimensions: functional limitation; pain; physiologic discomfort, physical disability; physiologic disability; social disability and inability7. the aims of this research were to assess and analyze the relationship between perceived oral health quality of life and the work ability among administrative workers of a private textile company in são paulo, brazil. material and methods this study was conducted in the administrative department of a private company that had 134 employees at the time of the study. the research project was presented to the management and leadership to clarify the objectives and the ethical aspects involved. the workers were informed about the research objectives and invited to participate. all the participants signed a free and informed consent form prior to participation. the data collection (wai and ohip-14) was performed without any identification of the participant. instructions for calculation of both are available in tuomi et al.11 (2005) and in oliveira and nadanovsky7 (2005) respectively. the dependent variable was the measurement of ohip 14 as it represents the premise that oral health influences the work ability (better conditions of self-perceived oral health improve work ability index). the independent variables related to sociodemographics characteristics were sex, age, marital status, educational level and income. independent variables related to occupational characteristics were job title, work shift, company affiliation time and job role. the work ability index (wai) was also used to analyze relations between the variables. the statistical analysis was conducted in stata v.13 (stata corp., college st.,tx, usa). kolmogorov-smirnov test was performed to assess normality of ohip 14 measurements. to analyze the correlation between ohip 14 and other variables, the spearman correlation coefficient was used. for comparison of the mean ohip 14 with dichotomous qualitative variables without constant variance, the mannwhitney test was used. to compare means between qualitative variables with three or more categories, the kruskal-wallis test was used. a multiple linear regression including variables with p<0.05 was performed followed by a residual analysis. the level of significance was 5%. the research project was approved by the ethics research committee of the university of são paulo school of dentistry (approval protocol 181/2008), and the study followed all the ethical guidelines for human research. results response rate among all the employees only one did not participate as he was on sick leave; six were in external projects and six others were excluded since they participated in the “pretest” (applied for preliminary verification and prior analysis). variables n % s e x male 50 54.95 female 41 45.05 marital status single 33 36.26 married/partner 52 57.14 divorced 6 6.6 educational level elementary school 1 1.1 middle school 1 1.1 high school 18 19.78 university 71 78.02 age mean (sd) l o w high 36.31(9.6) 18 67 table 1 table 1 table 1 table 1 table 1 demographic characteristics wai (points) n % lower (7-27) 0 0 moderate (28-36) 11 12.09 good (37-43) 30 32.96 excellent (44-49) 50 54.95 table 2 table 2 table 2 table 2 table 2 work ability index (wai) of the remaining 121, 92 workers consented to participate. only one participant failed to fill completely the questionnaires and was excluded. the response rate was 75.20%. questionnaires’ reliability internal reliability of the questionnaires was analyzed under cronbach’s alpha, with a confidence interval of 95%. ohip 14 score was 0.89 and wai score 0.64, which showed good internal reliability. demographic characteristics of all participants 54.95% were male and 57.14% married or living with a partner and had complete university level (78.02%). mean age was 36.31 years (sd = 9.6) ranging from 18 to 67 years old, shown in table 1. functional characteristics all participants were full-time workers and their employment average time was 95.95 months (sd = 77.5). the monthly salary ranged between us$ 409 to 816. experts in textile area were 42.85% and 34.07% belonged to the administrative or engineering sectors. characteristics of work ability table 2 summarizes work ability characteristics. the average wai score was 43.19 points (sd 4.44), ranging from 32 to 49 points. “excellent” score met 54.95% of the participants. descriptive analysis of oral health score the oral health score was 7.70 (sd 7.26) varying from 0 to 28 points on a scale that ranges from 0 to 56 points. as relations between oral health and work ability among administrative workers braz j oral sci. 14(1):41-45 variables n mean (sd) p demographic s e x male 50 5.72 (7.34) 0.01 female 41 10.12 (7.26) marital status not married 39 8.25 ( 6.5 ) 0.56 married/partner 52 7.28 ( 6.0 ) educational level not university 20 10.05 (9.5) 0.004 university 71 6.53( 4) functional job title administrative 28 6.6 (2.76) 0.03 specialists 39 5.79(1.05) attendants 19 10.92(1.55) director 5 7.15(1.41) salary range (us$) < 408 2 6.5(3.5) 0.67 409 to 816 23 9.69(1.75) 817 to 1224 20 8.05(1.78) 1225 to 1632 16 7(1.7) 1633 to 2040 13 8.69(1.96) 2041 to 2449 4 5.25(1.88) 2450 to 2857 2 1.5(0.5) 2858 or more 11 5(1.65) job role administrative 31 7.64(1.14) 0.96 credit 12 8.75(2.99) human resources 4 5(2.12) accounts 19 8.94(1.90) sales 16 6.93(1.67) marketing 9 6.44(1.95) table 3 -table 3 -table 3 -table 3 -table 3 association score ohip 14 with functional and demographic characteristics this variable was not normally distributed (p<0.05, kolmogorov-smirnov test) nonparametric tests were used to perform the statistical analysis. for ohip 14 dimensions regarding problems in mouth or teeth in the past six months, were found the following results: functional limitation represented by speaking difficulty or deteriorated food taste was reported by 29.67% participants; pain in the mouth or teeth reported by 60.43%; psychological discomfort represented by worry or stress was reported by 62.64%; physical handicap like chewing limitation or having to interrupt meals was reported by 40.65%; psychological disabilities like being ashamed or having trouble to relax reported by 48.35%; social disabilities like being angry to others or difficulty to perform daily activities, reported by 36.26%; limitations or disabilities like being unable to perform daily activities or felling that life in some way got worse because of oral or dental problems were reported by 25.27% of the participants. analysis of factors associated with oral health analysis didn’t show significant differences between mean scores of ohip 14 and age (p=0.48) or marital status (p=0.56). significant differences regarding sex (p=0.01) and educational level (p=0.004) were observed. the results are shown in table 3. although no significant associations were seen between ohip-14 scores, work sector and salary range, associations were observed between ohip-14 scores and job title variable, where specialists and attendants presented significant differences (p<0.05). a significant correlation (p=0.0014) was observed between ohip-14 scores and wai. a multiple linear regression analysis including all variables was performed, age being considered as an adjustment variable. in this model the following variables showed significant associations for ohip-14 score: wai (p=0.0014), educational level (p=0.009), sex (p=0.133), job title (p=0.863) and age (p=0.611) (table 3). after analysis it was realized that wai and educational level had significant associations with oral health status, as shown in table 4. the squared adjusted coefficient (r2a) of 0.24 indicates that 24% of the variability of ohip-14 could be explained by the variables that remained in the multivariate model. analysis of the residues between the ohip 14 (dependent variable) and the wai score (independent variable) performed by d’agostino-pearson normality test, and the p value (p=0.44) showed that the residues belong to a normal curve, with the presence of some outliers (outliers). the conclusion is that the model is unbiased. 4343434343relations between oral health and work ability among administrative workers braz j oral sci. 14(1):41-45 4444444444 variables b ci95% b r r 2a p-valor work ability index -0.546 (-0.86; -0.22) 0.001 educational level -3.695 (-6.43; -0.95) 0.009 job title 0.148 (-1.55; 1.85) 0.29 0.24 0.863 s e x 2.171 (-0.67; 5.02) 0.133 age 0.037 (-0.10; 0.18) 0.611 table 4 table 4 table 4 table 4 table 4 linear multiple regression analysis: ohip 14 dependent variable p model=0.000; standard error= 11.83. discussion the theoretical framework adopted in this research considers oral health, described by locker6 (1988), as any limitation or loss of ability to perform daily activities. a good example is a person who does not pronounce the words correctly and, as a result, have problems in engaging in a conversation. the result is a limitation, characterized by adverse situations like experiencing problems at work due to its inability to communicate clearly6. these concepts emphasize qualitative differences in the experience of “social impact” as well as their characteristics. individuals are more likely to have any social, physical and psychological disability if they experienced both pain and functional limitation. in addition, this pattern is part of the theoretical framework used to assess the work ability. this would result in job demands and resources of the individual to cope with them. if an individual’s ability to meet these demands is reduced, it may be a stress trigger causing wear and consequently impairs working ability12. this study evaluated the factors associated with oral health among administrative workers of a private textile company aiming to investigate its relationship to their quality of life1 and work ability. the analyzed questionnaires showed good reliability by cronbach’s alpha analysis13. ohip 14 score showed internal reliability of 0.89, a value above 0.7, which is recommended13. the work ability index, as well as other studies14 showed a lower but acceptable reliability. the explanation is that there are issues with different weights in wai, and when an issue is absent, the chance of changing the value of the overall score increases, generating a lower reliability. in ohip-14 questionnaire, all questions have the same value, ranging from 1 to 4 points, so that an absent question does not change the overall value of the questionnaire, keeping its internal reliability. while studies of absenteeism due to dental reasons are limited to icd-10 information (diseases, causes), subjective tests with ohip-14 instrument can identify physical, social and psychological disabilities, impairments or limitations. it could contribute to better elucidate the “social impact” of oral disorders 15. additionally, this approach allows identifying presenteeism2-5, in which despite of not being estranged from its labor activity, the employee’s performance, safety and well being is reduced. in this study, oral health was associated with sex (p=0.01) and educational level (p=0.004), and was not associated with age (p=0.48) or economic status (p=0.44), differently from oliveira and nadanovsky 7 (2005) and robinson et al.16 (2001), who reported that ohip-14 was inversely proportional related to age. a possible explanation may be the low age profile of the participants in the present study (mean 36.31 years old, sd 9.6 years). in close agreement with sanders and spencer 17 (2004) and oliveira and nadanovsky7 (2005) studies, sex and educational level relationships with oral health status were significant. while sex was dependent on work ability, educational level (with and without university grade) was independent of other variables, which is consistent with the findings of sanders and spencer17 (2004). job title was significantly associated with oral health status (p=0.03) and was related to work ability and educational level, as seen on the analysis between the expert professionals and the attendant ones (p<0.05). the explanation may be the different educational level between the groups, as expert workers were considered those who had university grade. income (salary range) and job role variables did not show significant associations with oral health status, although the best oral health conditions were seen in those with higher educational level. these findings agree with those of sanders and spencer17 (2004). work shift differences were not assessed as all participants worked full-time. oral health status may be affected not only by age but also as a consequence of work requirements and circumstances (i.e. chemical, or physical hazards). as a matter of fact both work requirements and oral health status may diminish the work ability, meaning the longer the worker is exposed to some circumstances, the more his work ability will be affected10,14. in the present research, multiple regression analysis confirmed oral health association with work ability. the squared adjusted coefficient (r2a) of 0.24 indicates that 24% of the variability of ohip-14 could be explained by the variables that remained in the multivariate model, meaning that work ability and educational level were independently associated to other variables. this results agree with guerra et al.18 (2014), who showed that 39% of ohip-14 scores was explained by educational level (p=0.03), age (p=0.03), reason for visiting a dentist (p=0.01), oral health perception (p<0.01) and satisfaction with teeth and mouth (p<0.01). this result indicates that ohip-14 can be useful for planning actions for oral health workers. the prevalence of orofacial pain in the study of lacerda et al.19 (2011) was 32.2%, where the toothache was most frequent (25.5%) and was the only one to have a high impact on workers in southern santa catarina state (brazil), and this was associated with level education (p=0.010), corroborating relations between oral health and work ability among administrative workers braz j oral sci. 14(1):41-45 4545454545 the present findings. given the results and limitations of this research and considering oral health a result of several interactions between people and working environment, some suggestions are presented: promotion, protection and recovery of the oral health of workers work ability was proven to be an independent factor statistically associated with oral health, which justifies actions related to the promotion, protection and recovery of workers’ oral health. this evidence not only justifies, but also reinforces the importance of oral health practices as a regular component of occupational health programs in order to integrate surveillance actions. oral health informative and educational procedures at workplace, as well as periodic exams are also of utmost importance not only to improve oral and general health and well being, but to integrate occupational activities and health care. new researches the cross-sectional design of this study does not support causal analysis between oral health and any of the tested independent variables. given this limitation as well as the scarcity of studies assessing oral health at workplace, new studies are suggested (designed in a longitudinal way) to assess causal relations between oral health and those variables. the findings will be of great importance to evaluate the influence of oral health on general health status and to investigate work ability of different groups of workers in different occupational scenarios. cohort studies, for example, can lead to a better understanding of the causal link between oral disease and occupational activity and how this affects the work ability. references 1. macedo cg, queluz dp. quality of life and self-perceived oral health among workers from a furniture industry. braz j oral sci. 2011; 10: 226-32. 2. santos ej, queluz dp. factors involved in dentistry absenteeism since the foundation of the labour court from 1986 to 2008. braz j oral sci. 2012; 11: 492-504. 3. mazzili len, crosato e. analysis of sick leave due to dental reasons in municipal civil servants of são paulo undergoing occupational expertise from 1996 to 2000. rpg rev pós grad. 2005; 12: 444-53. 4. bomfim ra, mazzilli len, michel-crosato e, camanho edl, crosato e. absenteeism in guarulhos municipal workforce due to oral and maxillofacial affections. j manag prim health care. 2013; 4: 169-75. 5. togna grd, melani rhr, crosato e, michel-crosato e, biazevic, mgh. use of the international classification of diseases in analysis of dental absenteeism. rev saúde pública. 2011; 45: 512-8. 6. locker d. measuring oral health: a conceptual framework. community dent health. 1988; 2: 3-18. 7. oliveira bh, nadanovsky p. psychometric properties of the brazilian version of the oral health impact profileshort form. community dent oral epidemiol. 2005; 33: 307-14. 8. ilmarinen j. aging and work. occup environ med. 2001; 58: 546-51. 9. martinez mc, latorre mrd, fisher fm. work ability: literature review. ciênc saúde col. 2010; 15(suppl1): 1553-61. 10. martinez mc, latorre mrdo, fisher fm. validity and reliability of the brazilian version of the work ability index. rev saúde pública. 2009; 43: 525-32. 11. tuomi k. ilmarinen j, jahkola a, katajarinne l, tulkki a, organizators. work ability index. são carlos: edufscar; 2005. 12. ilmarinen j, tuomi k, eskelinen l, nygard ch, huuhtanen p, klockars m. background and objectives of the finnish research project on aging workers in municipal occupations. scand j work environ health. 1991; 17: 7-11. 13. bland jmb; altmann dg. cronbach´s alpha. bmj. 1997; 314: 572. 14. martinez mc, latorre mrdo. health and work ability index for electricians in the state of são paulo. ciênc saúde col. 2008; 13: 1061-73. 15. reisine s. the impact of dental conditions on social functioning and the quality of life. ann rev public health. 1988; 9: 1-19. 16. robinson pg, gibson b, khan fa, birnbaum w. a comparision of ohip14 and oidp as interviews and questionnaires. community dent health. 2001; 18: 144-9. 17. sanders ae; spencer aj. job characteristics and the subjective oral health of australian workers. aust n j public health. 2004; 28: 259-66. 18. guerra mjc, greco rm, leite icg, ferreira ef, paula mvq. impact of oral health conditions in worker´s quality of life. ciência & saúde coletiva 2014; 19: 4777-86. 19. lacerda jt, ribeiro jd, ribeiro dm, traebert j. prevalence of orofacial pain and its impact on the oral health-related quality of life of textile industries workers of laguna, sc, brazil. ciênc saúde col. 2011; 16: 4275-82. relations between oral health and work ability among administrative workers braz j oral sci. 14(1):41-45 oral sciences n3 original article braz j oral sci. january | march 2015 volume 14, number 1 effects of estrogen deficiency combined with chronic alcohol consumption on rat mandibular condyle miriane carneiro machado salgado1, adriana mathias pereira da silva marchini1, tábata de mello tera1, rosilene fernandes da rocha1, leonardo marchini2 1universidade estadual paulista – unesp, instituto de ciência e tecnologia, department of dentistry, são josé dos campos, sp, brazil 2 university of iowa, college of dentistry, department of preventive and community dentistry, iowa city, iowa, united states of america correspondence to: leonardo marchini 337-1 dental science n, iowa city, iowa usa, 52772 phone: +1 319 3358285 fax : +1 319 3357187 e-mail : leonardo-marchini@uiowa.edu abstract estrogen deficiency and chronic alcohol consumption may have a synergistic and deleterious effect on bone tissue. aim: to investigate the effects of estrogen deficiency associated with chronic alcohol consumption on the mandibular condyle in rats. methods: fifty-four female rats were first divided equally into two groups: ovariectomized (ovx) and simulated ovariectomy (sham). one month after the surgeries, these groups were equally sub-divided according to their dietary treatment: g1: sham/ad-libitum diet; g2: sham/alcohol; g3: sham/isocaloric; g4: ovx/ad-libitum diet; g5: ovx/alcohol, g6: ovx/isocaloric. eight weeks after starting the diets, all animals were anesthetized and sacrificed. the condyles were analyzed histologically, histomorphometrically, and immunohistochemically using the antibodies for bone sialoprotein (bsp), osteocalcin (occ) and receptor activator of nuclear factor kappa-b ligand (rankl). results: histological analysis of the mandibular condyles showed that ovx and sham groups presented almost the same characteristics. the histomorphometric analysis showed that there was a statistically significant difference only between ovx/isocaloric and ovx/ad-libitum groups (p=0.049). no difference was observed in the intensity of bsp, occ, and rankl antibody staining between the ovx/alcohol and the other groups. conclusions: it may be concluded that there was no histomorphometric, histological, or rankl, bsp, and occ staining differences between the ovx/alcohol group and other experimental groups. keywords: mandible; mandibular condyle; ethanol; ovariectomy. introduction estrogen is a steroid hormone that is important for the growth and maintenance of the female skeleton and inhibits bone resorption1. estrogen deficiency is associated with increased bone resorption relative to bone formation, causing excessive loss of bone mineral density. bone resorption increases due to increased osteoclastogenesis and decreased apoptosis of osteoclasts2. these events culminate in the imbalance of bone remodeling, which can contribute to the occurrence of osteoporosis3. osteoporosis is a chronic and progressive skeletal disorder related to bone metabolism in which the bones become less resistant to fractures. the amount of bone tissue is reduced, the bone presents larger resorption areas and bone surface becomes irregular. thus, there is skeletal fragility and, consequently, an increased braz j oral sci. 14(1):16-22 received for publication: january 14, 2015 accepted: march 04, 2015 1717171717 risk of fractures4. with the growing elderly population, more people may be likely to develop osteoporosis and consequently suffer from fractures. considering that fractures lead to high-cost treatment, osteoporosis is considered a public health problem3. the effects of osteoporosis are greater in long bones, such as the femur and radius, and also in vertebrae5. however, there is evidence that osteoporosis also occurs in the jaw4. the relationship between systemic bone loss and bone loss in the jaw for patients with osteoporosis has been reported in the literature6. bone metabolism in the mandibular condyle is heavily influenced by sex hormones, especially estrogen, suggesting that the absence of estrogen may influence condyle bone remodeling and potentially lead to degenerative changes in the temporomandibular joint 7. studies have shown that ovariectomy induces bone loss in the whole mandible, including its condyle7-11. structural defects in mandibular condyles were found in patients diagnosed with severe osteoporosis after menopause, indicating that osteoporosis can change mandibular condyle structure12. alcohol consumption inhibits bone formation by decreasing osteoblast count and proliferation. however, the decrease in bone formation and increase in resorption may indicate that alcohol not only inhibits osteoblastic activity, but also stimulates osteoclastic activity. excessive alcohol consumption results in progressive bone loss and increased risk for osteoporosis development and, consequently, increased risk of osteoporosis-related fractures13,14. in the orofacial region, the bone most commonly affected by fractures is the mandible15. two-thirds of the fractures of the maxillofacial region occur in the mandible, and the condyle region is the most commonly fractured mandibular site, followed by the symphysis, body, alveolar region, angle, and ramus16. thus, considering 1) estrogen deficiency and its relationship with osteoporosis; 2) osteoporosis and its implications for bone fragility; 3) alcohol consumption and its consequences for bone tissue; 4) the high incidence of condyle fractures among orofacial fractures; and 5) the absence of studies that investigate the influence of osteoporosis in conjunction with alcohol consumption in the mandibular condyle region, this study aims to investigate the effects of estrogen deficiency combined with chronic alcohol consumption on the mandibular condyle of rats. in this way, the hypothesis to be tested is that estrogen deficiency combined with chronic alcohol consumption might have deleterious effect on the bone structure of the mandibular condyle in rats. material and methods animals fifty-four female rats (rattus norvegicus albinus, variation wistar) were used. the animals were three months of age in the beginning of the experiment, and the study was carried out according to the ethical principles established by the brazilian college of experiments involving animals (cobea in portuguese) and approved by the ethics committee for research involving animals of the state university of são paulo-unesp, under the protocol #0122011-pacep. the animals were first equally divided into two groups: ovariectomized (ovx) and simulated ovariectomy (sham). one month after surgeries, these groups were equally subdivided according to the dietary treatment: g1: sham/adlibitum diet; g2: sham/alcohol; g3: sham/isocaloric; g4: ovx/ad-libitum diet; g5: ovx/alcohol, g6: ovx/isocaloric. therefore, the animals were divided into six experimental groups with nine (n=9) animals in each group (totalizing 54 animals in the whole experiment). the solid food offered to all groups was a commercial rat food (labina, purina of brazil, paulínia-brazil). for g2 and g5, that received alcohol, 50 grams of food were provided daily to each animal. the next day, the remaining food was weighed and averaged, and the average quantity of food was provided to each animal in g3 and g6, the isocaloric groups. g1 and g4 received ad-libitum solid food. animals from groups g2 and g5 received 20% alcohol solution obtained by diluting absolute ethanol (abs.acs99.5°, ecibra, são paulo, sp, brazil) in purified water. initially, these animals were submitted to a nine-day adaptation period: three days receiving a 5% alcohol solution followed by three days receiving 10% alcohol solution, and then three days receiving 15% alcohol solution. subsequently, they received the prepared 20% alcoholic solution daily for eight weeks. g2 and g5 groups (the alcohol groups) began one day before g3 and g6 (the isocaloric groups), and they were provided 50 ml of alcohol solution per animal. the next day, the remaining solution was measured and the intake averaged. the isocaloric solution was then prepared by dissolving 266 g of sucrose into 1 l of water. the calculations for the quantity of isocaloric solution to be offered to g3 and g6 were made by taking into consideration the alcohol concentration used in this study (20%), the density of absolute alcohol (0.787 g/ml), the caloric values of sucrose (4.1 kcal/ g) and alcohol (7.1 kcal/g). in addition to the isocaloric solution, groups g3 and g6 also received water ad-libitum. the average values for solid and liquid consumption per cage were recorded daily. from these data, the average consumption per animal was calculated for each experimental group as food intake (g and kcal), liquid solution (ml and kcal), total calories (kcal), and percentage of calories from liquid and solid food. anesthesia the rats were anesthetized by intramuscular injection of 0.1 ml/kg of 1.25:1 mixture of xylazine chloride (anasedan; vetbrands, jacareí, são paulo, sp, brazil) and ketamine chloride (dopalen; vetbrands) whenever necessary during the procedures described below. ovariectomy and sham surgery the rats belonging to ovx groups (g4, g5, and g6) were ovariectomized at three months of age. animals from effects of estrogen deficiency combined with chronic alcohol consumption on rat mandibular condyle braz j oral sci. 14(1):16-22 1818181818 sham groups (g1, g2, and g3) had their ovaries exposed and immediately reseated in the abdominal cavity at the same procedure to simulate the surgical stress of an ovariectomy. after the surgery, all rats were given a single dose of intramuscular antibiotics (benzathine benzylpenicillin, procaine benzylpenicillin, benzylpenicillin potassium, and dihydrostreptomycin sulphate [pentabiótico; fort dodge saúde animal, campinas, sp, brazil, 1.35 ml/kg]) and an anti-inflammatory (diclofenac sodium 75 mg [voltaren injetável; ciba-geigy, rio de janeiro, rj, brazil, 1 mg/kg]). euthanasia eight weeks after starting diets, all animals were anesthetized and sacrificed. all animals were weighed at the beginning of the experiment and immediately prior to sacrifice. histological and immunohistochemical analysis the mandibles were cleaned and placed in 10% formaldehyde solution for 48 h. then, the samples were decalcified in edta solution and subsequently embedded in paraffin. histological sections were performed in the frontal plane of the condyle. 3-µm sections were mounted on silanized slides for immunohistochemical procedures, and 4-µm sections were used for hematoxylin-eosin staining. the primary antibodies used in this study were those for bone sialoprotein (bsp-lf-87), osteocalcin (occ fl110: sc-30044), and receptor activator of nuclear factor kappab ligand (rankl n-19: sc-7628). their titration, time and temperature of incubation, and antigen recovery are listed in table 1. samples were incubated with a biotinylated secondary antibody for 30 min and exposed to a streptavidin”peroxidase tertiary complex, also for 30 min (universal dako lsab® kit, peroxidase, carpinteria, ca, usa). they were then incubated with diaminobenzidine solution for 2 min (dako liquid dab). sections were counterstained with mayer’s hematoxylin. negative controls were achieved using an equivalent solution for primary antibody dilution, but without the antibody itself. positive controls were made according to the instructions of primary antibody manufacturers. the images of histological and immunohistochemical slides were photographed by a high-resolution digital camera (axiocam mrc5; carl zeiss, oberköchen, germany) coupled to a microscope (axiophot 2; carl zeiss). the camera is connected to a computer containing image acquisition and analysis software (axiovision release 4.7.2). in the immunohistochemical analysis, the following cells were analyzed: osteoblasts, osteoclasts, chondrocytes and fig.1 was considered staining positive for osteocyte when the osteocyte lacunae was stained (arrows) antibody – rankl. osteocytes. staining was considered positive for osteocyte when the osteocyte lacunae were stained (figure 1). for all groups, cell types and antibodies, the staining intensity was classified either as weak, moderate or intense17. the staining analysis took into account both degree of color (lighter colors were considered weaker) and degree of heterogeneity (more heterogeneous patterns were considered weaker). for the cases were analysis was challenging, the cells were visualized in higher magnification. histomorphometric analysis the image-j software was used for histomorphometric analysis of trabecular bone by counting points in a grid. only the points that fell on trabecular bone were counted, excluding points on osteocytes, medullary spaces, osteoblasts, osteoclasts, and bone marrow cells. data analysis weight changes and variations in food and liquid intake were subjected to kruskal-wallis and mann-whitney tests. the data obtained from the histomorphometric analysis were subjected to anova and tukey variance tests. all tests used a significance level of 5%. histological and immunohistochemical data were analyzed qualitatively. results there was an average weight gain in all groups. the group that gained the most weight was the ovx/ad-libitum group (average gain of 40.83% ± 8.59%), which gained antibody titration incubation antigenic recovery bone sialoprotein (bsp) 1:100 room temperature, 1h citrate osteocalcin (occ) 1:100 4 ºc, overnight citrate receptor activator of nuclear 1:75 4 ºc, overnight pepsin factor kappa-b ligand (rankl) table 1table 1table 1table 1table 1 – antibody, titration, time and temperature of incubation, and antigenic recovery. effects of estrogen deficiency combined with chronic alcohol consumption on rat mandibular condyle braz j oral sci. 14(1):16-22 1919191919 significantly more weight than the other groups according to the kruskal wallis and mann whitney tests. the group that gained the least weight was the sham/isocaloric group (average gain of 4.10% ± 7.25%), also statistically different from the others except the sham/alcohol and ovx/alcohol groups. the group that consumed the most food was the ovx/ ad-libitum group (19.26 g ± 4.05), statistically different from the other groups with the exception of the sham/ad-libitum group (kruskal-wallis and mann-whitney tests). average food intake by the isocaloric groups was similar to the average from the alcoholic groups, indicating that isocaloric groups consumed all the food offered to them. regarding liquid intake, animals from isocaloric groups did not consume all of the solution offered to them. the animals that were given alcohol ingested the equivalent of 8.43 g of absolute alcohol per kg per day; an average of 44.5% of their caloric intake came from alcohol. histological analysis of the mandibular condyles showed that ovx and sham groups presented almost the same characteristics. osteoclasts were present in all condyles from the sham/alcohol and sham/ad-libitum groups. only three condyles from sham/isocaloric group presented osteoclasts, ovx/isocaloric group presented osteoclasts in two condyles, ovx/ad-libitum in four, and ovx/alcohol in three. osteoblasts were present in all groups. ovx/alcohol ovx/ad-libitum ovx/isocaloric sham/alcohol sham/ ad-libitum sham/isocaloric average 57.83 55.42 65.10 59.72 56.09 63.29 median 57.81 57.29 66.14 59.89 54.68 63.54 sd 12.79 12.81 11.18 12.76 14.00 9.53 min 36.45 30.72 40.10 32.29 32.39 37.50 m a x 80.20 76.04 86.45 88.54 86.45 80.72 n 27 29 25 27 29 29 p-value 0.019* table 2table 2table 2table 2table 2 – comparison among experimental groups regarding trabecular bone area (in percentage [%] of the total area). the asterisk (*) indicates a statistically significant difference. n = number of slides used in the histological analysis in each experimental group. fig. 2 rankl staining intensity. intense osteoclast staining ( ), intense osteoblast staining ( ) and moderate osteoblast staining ( ). the histomorphometric analysis showed that there was a statistically significant difference (anova) among the groups, as can be observed in table 2. using the tukey’s multiple comparison test, there was a statistically significant difference only between ovx/isocaloric and ovx/ad-libitum groups (p=0.049). immunohistochemical analysis showed that rankl is expressed by osteoclasts, osteoblasts, osteocytes and chondrocytes. all experimental groups showed variations of staining intensities between weak, moderate and intense (figure 2). the pattern of these variations can be observed in table 3. the bone sialoprotein (bsp) staining presented variations in intensity, which varied according to the group and cell type, as shown in table 4. bsp reacted positively on osteoblasts (figure 3), osteocytes and chondrocytes. osteoclast staining occurred only in sham groups. the sham/ alcohol group presented all three staining intensities, the sham/ad libitum group presented weak and moderate osteoclast staining and the sham/isocaloric group presented only moderate osteoclast staining. osteocalcin (occ) was expressed by osteoblasts (figure 4), osteocytes and chondrocytes. occ staining also presented variations of intensity, which varied according to the group and cell type, as shown in table 5. fig. 3 bsp staining intensity. weak ( ) and moderate ( ) osteoblast staining. effects of estrogen deficiency combined with chronic alcohol consumption on rat mandibular condyle braz j oral sci. 14(1):16-22 2020202020 osteoblasts osteoclasts osteocytes condrocytes sham/ alcohol moderate weak intense weak sham/ isocaloric intense weak moderate weak sham/ ad-libitum intense moderate weak moderate ovx/alcohol moderate intense weak moderate ovx/ isocaloric moderate weak weak weak ovx/ad-libitum moderate intense moderate weak table 3 –table 3 –table 3 –table 3 –table 3 – receptor activator of nuclear factor kappa-b ligand (rankl) staining intensity. fig. 4 occ staining intensity. weak ( ) and moderate ( ) osteoblast staining osteoblasts osteoclasts osteocytes condrocytes sham/ alcohol moderate weak weak moderate sham/ isocaloric moderate moderate weak weak sham/ ad-libitum weak weak weak intense ovx/alcohol moderate weak weak ovx/ isocaloric intense weak ovx/ad-libitum intense weak moderate table 4 –table 4 –table 4 –table 4 –table 4 – bone sialoprotein (bsp) staining intensity. osteoblasts osteoclasts osteocytes condrocytes sham/ alcohol weak weak weak sham/ isocaloric weak weak weak sham/ ad-libitum weak weak weak ovx/alcohol weak weak weak ovx/ isocaloric weak weak weak ovx/ad-libitum moderate weak weak table 5 – table 5 – table 5 – table 5 – table 5 – osteocalcin (occ) staining intensity. discussion in the present study, the average alcohol consumption of 8.43 g alcohol per kg per day suggests that the animals exhibited chronic and excessive alcohol consumption. the harmful effects of alcohol on the bone are noted in alcohol abuse, but not in moderate alcohol consumption 18,19. a previous study20 observed animals consuming 7.6 g of 95% alcohol per kg per day, which would be equivalent to the daily consumption of 2.5 l beer or 1 l wine for an adult man. another study21 reported that 8.76 g of alcohol per kg per day were consumed by their animals, close to the values found in this study. in this study, an average of 44.5% of caloric intake was derived from alcohol. however, these calories from alcohol are empty, i.e. are not related to protein, vitamins or minerals, leading to nutritional deficiencies due to low nutrient intake22. it was decided to use isocaloric groups as nutritional controls to simulate the nutrient deficiency associated with alcohol consumption21,23. however, one of the limitations of this study was that the animals of the isocaloric groups were not pushed to consume the same amount of calories from liquid, as did those of the alcohol groups. therefore, the isocaloric groups ingested less sucrose volume compared to the alcohol groups, and so they had an even more calorierestricted diet compared to the alcohol groups. in regard to solid diet, the animals of the isocaloric groups consumed the entire amount of provided food (average of 9.39 g per day per animal). animals from all groups gained weight, which was reported previously24,25. in the present study, the ovx/adlibitum group gained significantly more weight from all the other groups, as reported earlier21, which contradicts another study25 that found no statistically significant differences. the ovx/ad-libitum group also consumed more food, as reported earlier21. ovariectomy can increase food intake and weight gain26,27, and a study27 verified that ovx animals showed a greater increase in body weight compared to sham-operated effects of estrogen deficiency combined with chronic alcohol consumption on rat mandibular condyle braz j oral sci. 14(1):16-22 animals. in addition, the alcohol groups ingested a much smaller amount of food than the groups that were on adlibitum diets. this was expected, since alcohol is a substance with low nutritional value and high energy content, causing the animals to feel satiety. histological results showed no significant difference between the ovx/alcohol group and the other groups. additionally, ovx groups showed a smaller number of osteoclasts relative to sham groups. bone metabolism in the mandibular condyle is heavily influenced by estrogen, suggesting that the absence of estrogen influences condylar bone remodeling and may lead to degenerative changes in the temporomandibular joint7. in addition to ovariectomy increasing the number of osteoclasts7, decreased numbers of osteoclasts and osteoblasts in both sham and ovx groups were observed sixty days after surgeries28. however, contrary to the present results, another study found that the number of osteoclasts in ovx animals was higher than in sham animals11. histomorphometric results also revealed that the ovx/ alcohol group was not different from other groups. only between the ovx/ad-libitum and ovx/isocaloric groups there was a statistically significant difference regarding the percentage of trabecular bone. this result shows that diet seems to have an influence on the amount of mandibular condyle trabecular bone in ovx rats, since the ovx-ad-libitum group consumed more food. however, in sham groups there was no difference between the ad-libitum, isocaloric or alcohol groups. in the present study, antibodies for receptor activator of nuclear factor kappa-β ligand (rankl), bone sialoprotein (bsp), and osteocalcin (occ) were used for immunohistochemical analysis. they are some of the noncollagenous proteins in the bone matrix. non-collagenous proteins play an important role in the organization of the collagen matrix and in regulating the formation and growth of hydroxyapatite crystals29. rankl is a cytokine that belongs to the family of tumor necrosis factors (tnf)30. it increases the activity and prolongs the lifetime of osteoclasts by decreasing apoptosis. rankl is a mediator of the formation, function and survival of osteoclasts, and it is considered a main mediator of bone resorption31,32. bsp is produced by osteoblasts, osteoclasts, osteocytes and hypertrophic chondrocytes33. bsp is also a powerful regulator of the differentiation and activity of osteoblasts, and it has fundamental importance in the early stages of osteogenesis34. however, bsp is also recognized to induce cell adhesion, increasing the synthesis of osteoclasts and bone resorption. occ is a small protein found exclusively in mineralized tissues31. it is considered a marker of osteoblast activity and bone formation13. occ is expressed in the final stages of osteoblast differentiation in the bone maturation and mineralization processes35. a previous study 36 reported intense and moderate rankl staining on osteoblasts and osteoclasts. positive bsp staining was also previously reported12 during the first week after ovariectomy and occ expression was clearly changed by ovariectomy37. in this study, slides were analyzed qualitatively according to the intensity of immunohistochemistry reaction, as previously conducted by other authors17,38,39. qualitative analysis has some limitations, since it does not provide numerical estimates and rely on subjective human observation. furthermore, the antigen antibody reactions could be not be stoichiometric, so a strong intensity reaction (darkness of stain) does not necessarily mean greater amount of reaction products, which can be considered another limitation of this study. considering these effects of ovariectomy and reported deleterious effects of alcohol consumption on bone tissues14,21,25, important modifications of rankl, bsp, and occ staining intensities were anticipated in the ovx/alcohol group compared to other groups. however, similar to the histological and histomorphometric results, no differences were found between the ovx/alcohol group and other groups, thus rejecting the study hypothesis. acknowledgements the authors would like to thank fapesp for its support (são paulo state research support foundation, grant #2011/ 03447-0). the authors also thank ms. anna okulist for her native english proofreading of this manuscript. references 1. manolagas sc, kousteni s, jilka rl. sex steroids and bone. recent prog horm res. 2002; 57: 385-409. 2. manolagas sc. birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. endocr rev. 2000; 21: 115-37. 3. faloni aps, cerri ps. cellular and molecular mechanisms of the estrogen in the bone 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sciences n3 original article braz j oral sci. july | september 2014 volume 13, number 3 quality of life in temporomandibular disorder patients with localized and widespread pain maísa soares gui1, marcele jardim pimentel2, marta cristina da silva gama1, glaucia maria bovi ambrosano3, célia marisa rizzatti barbosa2 1universidade estadual de campinas unicamp, piracicaba dental school, department of anatomy, piracicaba, sp, brazil 2universidade estadual de campinas unicamp, piracicaba dental school, department of prosthesis and periodontology, piracicaba, sp, brazil 3universidade estadual de campinas unicamp, piracicaba dental school, department of community dentistry, area of statistics, piracicaba, sp, brazil correspondence to: maísa soares gui faculdade de odontologia de piracicaba unicamp av. limeira, 901 cp 52 cep 13414-903 piracicaba, sp, brasil. phone: +55 19 2106-5200 e-mail: maisa_gui@yahoo.com.br abstract aim: to compare temporomandibular (tmd) subgroups classified according to the presence of localized pain (lp) or widespread pain (wp) in order to assess the quality of life domains and verify which components affect most the functional capacity of facial pain patients. methods: a cross-sectional study was conducted and the short form-36 health survey was applied in order to assess quality of life. thirty-nine tmd/wp patients, 37 tmd/lp patients and 40 subjects free of tmd complaints were evaluated. results: tmd/wp patients differed significantly from healthy controls in all sf-36 components and tmd/lp patients ranked between them. it was also observed that patients with bodily pain and tmd with wp are respectively, 4.16 and 49.42 times more likely to have low functional capacity. conclusions: functional capacity in tmd subgroups was only affected by the presence of bodily pain and wp. these patients feature high chance of low functional capacity. furthermore, tmd patients with localized and widespread pain share roleemotional impairments. keywords: facial pain; temporomandibular joint dysfunction syndrome; quality of life. introduction temporomandibular disorders (tmd) are defined as a set of conditions affecting the masticatory muscles or joints and exhibiting pain as their primary characteristic1-2. it has been described that individuals with tmd could display diffuse hyperalgesia and allodynia3-4 and it was suggested that they have a fundamental problem with pain or sensory processing rather than an abnormality confined to a specific region of the body where pain is perceived to originate5-6. recently, two tmd clinical subgroups were proposed based on findings showing a group of tmd patients that was split with respect to patients’ tender point score (one of the diagnosis criteria for fibromyalgia) into an insensitive subgroup resembling healthy control subjects and into a sensitive subgroup resembling patients with fibromialgia1. the distinction between localized and generalized pain in tmd patients was recognized as important for both, patient diagnosis and for proper understanding of the etiology and pathophysiology of chronic pain4,7. numerous psychological and behavioral factors are well-established influences on a wide range of pain conditions including tmd pain2. an orofacial pain prospective study identified that psychological and behavioral factors have become significant influences on tmd pain 8. another study also supported the received for publication: may 18, 2014 accepted: august 12, 2014 braz j oral sci. 13(3):193-197 interpretation that psychosocial parameters may be independent predictors for the development of chronic pain conditions and their generalization1. the short-form-36 is a global health-related quality of life measurement9 that could help identify the similarities and differences in those tmd patients. indeed, the knowledge of how physical and mental components influence the quality of life and how people realize these events in their life has increased regarding the etiology of chronic pain. previous studies clearly demonstrated the psychological process, i.e., emotion could modulate pain and vice-versa10-11. the aim of this study was to compare tmd subgroups that were classified according to the presence of localized or widespread pain in order to assess the quality of life domains and to verify which components most affect the functional capacity of facial pain patients. material and methods study design a cross-sectional study was conducted in pain-free healthy subjects and two subgroups of tmd patients recruited from the clinic of the piracicaba dental school/unicamp and the communities surrounding the school between january 2010 and november 2012. ethical procedures this study was approved by the ethics committee on research involving human subjects under protocol number 103/ 2009. after a verbal presentation of the project, the volunteers signed an informed consent form to participate in the study. participants for tmd case, patients with myogenic facial pain diagnosed using the research diagnostic criteria for tmd (rdc/tmd) 12 were invited to participate. calibrated examiners performed the rdc/tmd clinical examination on all subjects. the inclusion criteria were gender (female), due to the higher prevalence of tmd and longer duration of the condition in women13-14 and presence of symptomatic tmd. exclusion criteria were the presence of systemic diseases, polyarthritis, exposure to macro facial trauma, dislocated joints, use of orthodontic braces, dental pain, and the presence of sinusitis, ear infections, cancer and hormonal disorders. after that, subgroups of tmd patients were defined according to the presence or absence of widespread pain, palpation tenderness. the subgroup with widespread pain (tmd/wp) was identified on the basis of their tender point count, which is an easy practicable screening tool for those patients1. briefly, widespread pain was present when the palpation of 18 body sites elicited pain at diagonally opposite quadrants of the body (i.e., above and below the waist, on both the left and right sides)1,7. three pounds of digital palpation pressure were applied bilaterally for 2 s to each site by calibrated examiners. at each location, a response of pain to palpation was recorded as tenderness. the tmd patients without widespread pain were classified as having “localized pain” (tmd/lp subgroup). control subjects had neither tmd nor widespread pain classification. then, a control group of female individuals without complaint, which were free of any bodily or facial pain condition, was also recruited and invited to participate. variables and data sources quality of life was assessed by a generic multidimensional instrument: the short form-36v2® health surveys (sf-36)9. briefly, this questionnaire measures eight health domains: physical functioning, role-physical, bodily pain, general health, vitality, social functioning, role-emotional and mental health9. the score for each scale varies from 0 to 100, and the higher score corresponds to better life and provides psychometricallybased physical component summary (pcs) and mental component summary (mcs) scores. the mental health measure has been shown to be useful in screening for psychiatric disorders, as has the mcs measures 15. the mcs had a sensitivity of 74% and a specificity of 81% in detecting patients diagnosed with depressive disorder15. the sf-36 has been widely used in research with excellent metric properties (sensitivity, validity and reliability)16 and it was translated and validated for the portuguese language17. statistical methods data were analyzed by kruskal wallis and dunn considering a significance level of 5%. the values of life quality items and age were dichotomized by the median of the sample. bivariate analysis was performed by associating each variable with functional capacity. variables with p<0.20 in bivariate analysis were tested in a multiple logistic regression model, remaining in the model those with p<0.05. results from the initial sample size of 120 subjects (40 per group), one tmd/wp patient was lost to follow up and three tmd/lp patients withdrew from the study. therefore, 40 tmd-free healthy controls (mean age 50.93±12.34), 37 tmd/lp patients (mean age 24.92±5.0) and 39 tmd/wp patients (mean age 53.21±9.34) were evaluated. there was statistically significant difference (p<0.001) in the mean age of tmd/lp patients when compared with tmd/wp patients and controls, possibly because localized facial pain appears earlier than widespread facial pain. the main result of this study is that tmd/wp patients significantly differ from healthy controls in all components, while tmd/lp patients ranked in between (table 1). however, the emotional factors did not differ between tmd subgroups and general health, mental health, physical function and role-physical domains were not different between tmd/lp patients and controls. regardless of the other variables it could also be observed that patients with more bodily pain and widespread quality of life in temporomandibular disorder patients with localized and widespread pain1 9 41 9 41 9 41 9 41 9 4 braz j oral sci. 13(3):193-197 groups controls (n=40) tmd/lp patients (n=37) tmd/wp patients (n=39) median m i n i m u m m a x i m u m median m i n i m u m m a x i m u m median m i n i m u m m a x i m u m physical functioning 97.5 a 65.0 100.0 90.0 a 60.0 100.0 35.0 b 10.0 95.0 role-physical 100. a 0.0 100.0 100.0 a 0.0 100.0 0.0 b 0.0 100.0 bodily pain 84.0 a 31.0 100.0 61.0 b 0.0 84.0 22.0 c 0.0 82.0 general health 79.5 a 40.0 100.0 72.0 a 5.0 100.0 47.0 b 5.0 92.0 vitality 80.0 a 45.0 100.0 45.0 ab 0.0 80.0 20.0 b 0.0 90.0 social functioning 100.0 a 37.5 112.5 75.0 b 0.0 100.0 25.0 c 0.0 100.0 role-emotional 100.0 a 0.0 100.0 66.7 b 0.0 100.0 0.0 b 0.0 100.0 mental health 76.0 a 32.0 96.0 60.0 a 1.0 92.0 44.0 b 4.0 100.0 sf-36 scale (0-100) table 1. table 1. table 1. table 1. table 1. median, minimum and maximum values obtained for the eight components of the sf-36. zero is the worst score and a hundred is the best score. medians followed by different letters horizontally differ (p<0.05). pain are, respectively, 4.16 and 49.42 times more likely to have lower functional capacity than healthy controls (table 2). discussion the quality of life components of tmd/lp patients ranked in between tmd/wp patients and healthy controls. furthermore, only the presence of widespread pain and bodily pain affected the functional capacity of the individual. the present results also show that role-emotional (problems with work or other daily activities as a result of emotional problems) is not significantly different between tmd subgroups and could represent a common point that differentiates them from the control group. however, there quality of life in temporomandibular disorder patients with localized and widespread pain variable categories low physical functioning gross analysis adjusted analysis (logistic regression) n n % odds ic p odds ic p 95% 95% group controls 40 4 10.0% ref ref tmd/lp 37 11 29.7% 3.81 1.090.0580 2.67 0.680.1243 13.30 10.40 tmd/wp 39 37 94.9% 37.00 10.45<0.0001 49.42 7.49<0.0001 130.97 325.92 role-physical l o w 51 41 80.4% 20.13 7.80<0.0001 51.92 h i g h 65 11 16.9% ref bodily pain l o w 57 42 73.7% 13.72 5.58<0.0001 4.16 1.220.0224 33.75 14.14 h i g h 59 10 17% ref ref general health l o w 58 38 65.6% 5.97 2.66<0.0001 13.41 h i g h 58 14 24.1% ref vitality l o w 58 42 72.4% 12.60 5.16<0.0001 30.74 h i g h 58 10 17.2% ref social functioning l o w 52 39 75% 11.77 4.91<0.0001 28.22 h i g h 64 13 20.3% ref role-emotional l o w 49 32 65.3% 4.42 2.01<0.0001 9.72 h i g h 67 20 29.8% ref mental health l o w 52 35 67.3% 5.69 2.55<0.0001 12.70 h i g h 64 17 26.5% ref age l o w 58 18 31% ref h i g h 58 34 58.6% 3.15 1.460.0028 6.75 table 2.table 2.table 2.table 2.table 2. influence of the sf-36 components age and group in the component functional capacity of the research subjects. the values of the quality of life items and age were dichotomized by the median of the sample. 1 9 51 9 51 9 51 9 51 9 5 braz j oral sci. 13(3):193-197 was a great difference between patients with localized and widespread pain (respectively, 66.7 and 0). one of the limitations of the study, is the fact that this was cross-sectional research and temporal conclusions cannot be drawn (e.g., it is unknown if the emotional problems occurred before or after pain). in addition, there is a lack of control group matching with the tmd/lp subgroup with respect to age. potential psychosocial risk factors for chronic tmd were identified, revealing components constructs as stress and negative affectivity, global psychosocial symptoms, passive pain coping, and active pain coping8 that provide evidences of associations between psychosocial factors and tmd. furthermore, strong support was provided for the hypothesis that chronic widespread pain is one manifestation of the somatization process, which was described as the expression of personal and social distress through physical symptoms18, and high nocturnal masseter muscle activity was related to higher intensity of headache and higher somatization in tmd patients19. however, it was previously described that tmd subgroups (“sensitive” with generalized increase of evoked pain, and “insensitive” with localized pain complaint) did not differ with respect to psychological parameters and sensitive tmd had shorter pain duration than fibromyalgia patients1. in general, painful stimuli elicit considerable cognitive and emotional activity in the brain 20. the notion that widespread pain syndromes, as fibromyalgia, might represent generalized neurobiological amplification of sensory stimuli has some support from functional imaging studies suggesting that the insula is the most consistently hyperactive neurocortical region of the pain matrix. this region has been noted to play a critical role in sensory integration, with the posterior insula having a more exclusive sensory role, and the anterior insula being associated with the emotional processing of sensations 5. another recent study demonstrated that rejection and physical pain are similar not only in that they are both distressing, but also they share the same common somatosensory representation 21. emotional modulation of muscle pain was associated with polymorphisms in the serotonin-transporting gene and indicated that polymorphisms that lead to a high expression of the serotonin transporter gene are highly associated with the ability to modulate deep types of pain in relation to the emotional state. further, only the studied participants with a high expression of serotonin transporter experienced a signiûcantly changed perception of jaw muscle pain depending on their emotional state22. taken together, all these factors seem to indicate that emotional characteristics could be predisposing factors of these chronic facial pain conditions. despite the age difference, physical function and rolephysical domains did not differ between tmd/lp patients and healthy controls. low physical functioning was considerably more related to tmd with widespread pain, which means very limited in performing all physical activities, including bathing or dressing 15. it could be related to pain but, particularly, with helplessness and small practice of pain coping. this refers to a belief that nothing can be done to solve a problem, characterized by emotional, motivational, and cognitive deficits23. while positive emotions lead to pain reduction10, pain catastrophizing may lead to hyperalgesia via independent processes of spinal nociception, perhaps related to the subjective evaluation of pain (e.g., memory, attention) 24. the present findings also show that the distinction between localized and generalized pain in tmd is important both for patient diagnosis and for treatment target. since psychosocial factors play a role in the pathogenesis of musculoskeletal pain25, the knowledge of tmd subgroups characteristics and their functional impairments could help to target treatment approach. it may be concluded that functional capacity in the tmd subgroups was only affected by the presence of bodily pain and widespread pain. these patients feature high chance of having low functional capacity. furthermore, tmd/lp and tmd/wp patients shared role-emotional impairments. acknowledgements coordination of improvement of higher education personnel – capes, for a two-years post-graduation sponsorship (#2009-2010). references 1. pfau db, rolke r, nickel r, treede rd, daublaender m. somatosensory profiles in subgroups of patients with myogenic temporomandibular disorders and fibromyalgia syndrome. pain. 2009; 147: 72-83. 2. ohrbach r, fillingim rb, mulkey f, gonzalez y, gordon s, gremillion h, et al. clinical findings and pain symptoms as potential risk factors for chronic tmd: descriptive data and empirically identified domains from the 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large population based study. arthritis rheum. 2001; 44: 940-6. 19. shedden mora m, weber d, borkowski s, rief w. nocturnal masseter muscle activity is related to symptoms and somatization in temporomandibular disorders. j psychosom res. 2012; 73: 307-12. 20. bradley la. recent approaches to understanding osteoarthritis pain. j rheumatol. 2004; 70: 54-60. 21. kross e, berman mg, mischel w, smith ee, wager td. social rejection shares somatosensory representations with physical pain. proc natl acad sci u s a. 2011; 108: 6270-5. 22. horjales-araujo e, demontis d, lund ek, vase l, finnerup nb, børglum ad, et al. emotional modulation of muscle pain is associated with polymorphisms in the serotonin transporter gene. pain. 2013; 154: 1469-76. 23. venkataramanan v, gignac m, dunbar m, garbuz d, gollish j, gross a, et al. the importance of perceived helplessness and emotional health in understanding the relationship among pain, function, and satisfaction following revision knee replacement surgery. osteoarthr cartil. 2013; 21: 911-7. 24. rhudy jl, williams ae, mccabe km, russell jl, maynard lj. emotional control of nociceptive reactions (econ): do affective valence and arousal play a role? pain. 2008; 136: 250-61. 25. barbosa gas, morais mhst. differential diagnosis between post-polio syndrome symptoms and temporomandibular disorder: clinical case. braz j oral sci. 2013; 12: 57-60. quality of life in temporomandibular disorder patients with localized and widespread pain 1 9 71 9 71 9 71 9 71 9 7 braz j oral sci. 13(3):193-197 oral sciences n3 original article braz j oral sci. april | june 2014 volume 13, number 2 clinical setting influences physiological responses in dental implant patients milene cardoso candido1, roberto andreatini2, joão cesar zielak1, juliana feltrin souza1, estela maris losso1 1universidade positivo up, school of dentistry, area of implantology, curitiba, pr, brazil 2 universidade federal do paraná – ufpr, department of pharmacology, area of biological sciences, curitiba, pr, brazil correspondence to: estela maris losso universidade positivo – curso de odontologia rua pedro viriato parigot de souza, 5300 campo comprido cep: 81280-330, curitiba, pr, brasil phone: +55 41 33173456 fax: +55 41 33365962 email: lossoem@gmail.com abstract anxiety is often observed in dental procedures and may cause promote alteration in the physiological responses during implant surgeries. aim: to evaluate changes in blood pressure and heart rate in patients undergoing dental implant procedures, considering the dental setting as the main variable. methods: fifty-five patients who underwent dental implant surgery were evaluated. thirty-seven were treated at a university clinic and 18 were treated at a private office. blood pressure and heart rate were measured at the following time-points: at the appointment prior to surgery (t0), immediately before the surgical procedure (t1), during anesthesia (t2), during implant installation (t3), immediately after the surgical procedure (t4) and at the first follow-up appointment after surgery (t5). the data were analyzed by two-way analysis of variance and student’s t-test. results: the university clinic patients had an increase in heart rate at t5 (t53=2.62, p<0.05) compared with private office patients. systolic blood pressure in university clinic patients was higher at t2 (t53=2.86, p<0.01), t3 (t53=2.64, p<0.05), and t4 (t53=3.15, p<0.01). diastolic blood pressure at t2 (t53=3.15, p<0.01) and t3 (t53=3.86, p<0.01) were also higher in university clinic patients. conclusions: these results suggest that the dental setting is a relevant factor when planning dental implant surgery. keywords: dental implants; blood pressure; heart rate. introduction anxiety is a phenomenon characterized by subjective feelings of tension, apprehension, nervousness and preoccupation that may be experienced when an individual contacts an aggressor, such as dental surgery1-4. anxiety is a consciously perceived emotional reaction that intensifies the activity of the autonomic nervous system. the sympathetic nervous system acts directly on the adrenal gland to promote epinephrine and norepinephrine release. activation of this system produces responses that include changes in heart rate (hr), breathing, and blood pressure (bp) patterns, and restlessness, tremors and increased sweating1,5-7. blood pressure and hr can be altered in dental procedures8. beck and weaver9 (1981) studied bp and hr in nonstressful and stressful (dental surgery) procedures. they reported that systolic and diastolic bp did not vary on the day of surgery. however, hr varied in all phases of the treatment, with a higher variation on the day of surgery. the authors also suggested that the pretreatment period appears not to influence significantly the bp in healthy adults, with no gender differences. liau et al.10 (2008) analyzed dental anxiety (corah’s dental anxiety) in 180 patients who underwent tooth extraction under local anesthesia in the mandible received for publication: april 23, 2014 accepted: june 03, 2014 braz j oral sci. 13(2):109-113 braz j oral sci. 13(2):109-113 and reported anxiety with cardiovascular alterations. the treatment occurred in a university hospital. the anxiety scale was applied before anesthesia, and cardiovascular parameters (i.e., bp, hr, oxygen saturation and electrocardiography) were measured every 5 min up to 15 min after anesthesia. the highest alterations in hr occurred during the induction of anesthesia in individuals who were more anxious, in patients who reported a traumatic history with dental treatment and in patients who reported pain during the induction of anesthesia. patients with pain had increased systolic bp, but no differences were found in diastolic bp. bispo et al.8 (2011) also studied bp and hr in adult patients in different phases of root canal filling in molar teeth (class i) using different anesthetic drugs. they reported that the cardiovascular alterations were similar among the different anesthetic drugs, but the cardiovascular parameters were influenced by the phase of the dental treatment. the cardiovascular parameters were higher before the beginning of treatment and before local anesthesia, during the test of pulp vitality after anesthesia, during the procedure itself and at the end of treatment. it may be assumed that anxiety had an effect on these cardiovascular parameters during the root filling treatment. to the best of our knowledge, there are no studies assessing the influence of the clinical environment on cardiovascular responses to dental procedures. considering the importance of cardiovascular function in surgical trauma11, the objective of the present study was to evaluate bp and hr in two different dental settings in patients who underwent dental implant surgery or a related procedure. material and methods this was a prospective study (implant surgeries) in which physiological alterations in bp and hr were evaluated in patients treated at the positivo university surgical center and at a private clinic, both in city of curitiba, pr, brazil. approval was obtained from the ethics committee of positivo university (075/10). the participants were 55 patients of both genders (45 women, 75%) aged 30 to 69 years who sought dental implant treatment. thirty-seven were treated at positivo university surgical center (university clinic [uc] group) and 18 at a private dental office (private office [po] group). the inclusion criteria were: literate patient with good general health, not using any drug that could produce cardiovascular alterations. the criteria for exclusion were the following: patients with syndromes, systemic disease, cardiac problems and use of drugs that produce cardiovascular alterations. the patients were evaluated during three different phases of treatment, according to the following schedule: • consultation before surgery (t0). the consultation before surgery occurred between 7 and 21 days before the procedure. at this time, the patients were identified by recording their date of birth, gender and medical history (overall health and use of medications). blood pressure and hr were recorded. • day of the procedure (t1, t2, t3, and t4). during the consultation on the day of dental implant, the systolic and diastolic bp and hr were measured immediately before the surgical procedure in the waiting room (t1). in the procedure room, bp and hr were measured at the moment of anesthesia induction (t2), during the procedure when the dental implant motor was used (t3) and immediately after surgery (t4). • postsurgery consultation (t5). during the first followup visit for suture removal, between 7 and 10 days after surgery systolic and diastolic bp and hr were measured again. surgery and cardiovascular parameters in order to obtain the cardiovascular parameters, a properly calibrated noninvasive semiautomatic digital bp monitor (model mf-621 se; more fitness, são paulo, sp, brazil) was used to measure systolic and diastolic bp and hr simultaneously. the patients received the following instructions: to empty the bladder, not to exercise, not to ingest coffee or alcoholic beverages and not to eat in excess or smoke cigarettes within the 30 min prior to the evaluation. the patients received an explanation about the measurement procedure that was performed while sitting and were asked to rest for 5-10 min. the brachial artery was located by palpation, and the cuff was firmly positioned, with the rubber bag centered over the brachial artery. the patient’s arm was positioned at heart level, and the patient was asked not to speak during the measurement procedure. the data were recorded on the patient’s chart. in case of equipment error, new measurements were performed after 1-2 min. the cardiovascular parameter measurements and surgeries were performed in both locations by the same properly trained team. for the surgeries, the procedures were performed using the same technique in all patients. in the procedures that involved surgery anesthesia was performed used with 4% articaine chloride and epinephrine (1:100,000), or 2% mepivacaine chloride with epinephrine (1:100,000). the amount varied according to the need for surgery and did not exceed the maximal recommended dose. during the postoperative period, ibuprofen was used for analgesia. amoxicillin (500 mg) was administered every 8 h, or clindamycin (600 mg) was administered every 6 h for 7 days. statistical analysis the data were analyzed statistically using oneor twoway analysis of variance (anova), depending on the conditions. individual comparisons were performed using student’s t-test or duncan’s test for multiple comparisons. for all tests, the significance level was set to 5%. results figure 1 presents the bp results for groups uc and po. the anova revealed a significant effect of group on systolic bp (f5.270=5.81, p<0.001). systolic bp at t4 (i.e., the end of surgery) was significantly higher compared with the other time-points (p<0.01), except for t1 (p>0.05). systolic bp 110 clinical setting influences physiological responses in dental implant patients 111 braz j oral sci. 13(2):109-113 fig. 1. systolic and diastolic blood pressure (bp) in the uc and po groups. the data are expressed as mean + standard error (n=55). #p<0.05, compared with t4; +p<0.05, compared with t1. at t5 was lower than at t4 (p<0.01). diastolic bp was significantly higher at t4 compared with the other evaluations (f5,270=9.58, p<0.001), which did not differ from each other. analyzing the systolic bp (figure 2) as a function of treatment setting, anova revealed significant effects of setting (f1,53=7.53, p<0.01) and time (f5,265=3.97, p<0.01) but no interaction between these factors (f 5,265=1.69, p>0.05). statistically significant differences were found between the uc and po groups at t2 (t53=2.86, p<0.01), t3 (t53=2.64, p<0.05), and t4 (t53=3.15, p<0.01; fig. 2). the intra-group analysis revealed statistically significant differences in systolic bp in the uc group (f5,180=5.53, p< 0.01), with an increase at t4 compared with the other timepoints (p<0.05). however, systolic bp at t5 was lower than at t2 and t4 (p<0.05). in the po group, no statistically significant differences were found in systolic bp (f5.180=1.67, p>0.05) at any of the time-points. the analyses of diastolic bp as a function of treatment setting (uc and po groups) revealed significant effects of setting (f1.53=8.33, p<0.01) and time (f5.265=9.48, p<0.01) but no interaction between these factors (f 5.265=0.87, p>0.05). statistically significant differences were found between the uc and po groups at t2 (t53=3.15, p<0.01) and t3 (t53=3.86, p<0.01; fig. 2). the intra-group analyses revealed a significant effect of group on diastolic bp (f5.180=4.70, p<0.01), with an increase at t4 compared with the other time-points (p<0.05). statistically significant differences in diastolic bp were also found in the po group (f5.180=7.08, p<0.01), with an increase at t4 compared with the other time-points (p<0.05), except for t1. additionally, a reduction of diastolic bp was observed at t3 compared with the other time-points (p<0.05), except for t3. the comparison of hr between the uc and po groups (fig. 3) revealed a statistically significant effect of time (f5.265=2.99, p<0.01) and a statistically significant time ´ setting interaction (f5.265=3.01, p<0.05), but no effect of setting was found (f1,53=0.86, p>0.05). the only difference observed between the uc and po groups was a reduction of hr at t5 in the po group (t53=2.62, p<0.05). no statistically fig. 2. systolic and diastolic blood pressure (bp) in dental implant patients in the uc group (n=37) and po group (n=18). the data are expressed as mean + standard error. *p<0.05, compared with po group at the same time-point; #p<0.05, compared with all other time-points in the same group; +p<0.05, t2 vs. t4 in the same group; $p<0.05, compared with t0, t2, t3 and t5 in the same group; @p<0.05, compared with t0, t1, t4 and t5 in the same group. fig. 3. heart rate (hr) in dental implant patients of the uc and po groups. *p<0.05, compared with po group; #p<0.05, compared with t0, t2, t4 and t5 in the same group; +p<0.05, compared with t1, t2, t3 and t4 in the same group. significant differences were found for the other comparisons (t<1.88, p>0.05). the intra-group analysis showed that hr did not differ in the uc group (f 5.180=1.24, p>0.05). statistically significant differences in hr were found in the po group (f5.85=5.41, p<0.01), with an increase at t3 compared with the other time-points (all p < 0.05), except for t1. a reduction of hr was also observed at t5 compared with the other time-points (p<0.05), except for t0. discussion the main result of the present study was that the treatment setting was sufficient to produce changes in the bp of patients who underwent dental implant procedures. the patients who were treated in the private clinic had lower bp during all phases of the study, but mainly during anesthesia, use of the dental implant motor and at the end of surgery. the clinical environment might have caused such changes because several surgical procedures are performed simultaneously at the university surgical center, with various clinical setting influences physiological responses in dental implant patients 112 noise stimuli, such as high-speed rotary instruments and saliva suction devices. the use of a dental implant motor is a stressful factor that can alter bp. on the other hand, treatment in the private clinic occurs individually with fewer noise stimuli. the average systolic and diastolic bp had higher peaks at the end of the surgery, regardless of the treatment setting. this effect may be attributed to the fact that the patients were beginning to feel pain and were tired, and this could be related to an increase in anxiety and hemodynamic changes2,6,12-13. these findings are partially consistent with the results of beck and weaver9 (1981), who did not report variations in bp during some phases of dental treatment, but reported an increase in bp at more stressful moments (i.e., during the surgical procedure and use of the dental implant motor) in the same way as in the present study. the location where the surgical procedures were performed (i.e., university surgical center or private clinic) was not mentioned in their paper. liau et al.10 (2008) reported greater alterations in hr during anesthesia induction in more anxious individuals and increased systolic bp in patients who reported pain, with no differences in diastolic bp. in that study, the treatment occurred in a university setting. the noise of the dental implant motor is a stressful agent that can explain the maximal peak hr during its use in the treatment both at the university and private clinic settings14-16. beck and weaver9 (1981), liau et al.10 (2008) and bispo et al.8 (2011) reported that changes in hr occurred in more anxious individuals, suggesting a relationship between stress during dental treatment and hr. eli et al.17 (2008) observed that increased anxiety and the expectation of feeling pain had profound effects on patients capacity to recognize information during dental procedures, causing difficulties in processing information that was provided before and during the dental procedure. although the surgeries were performed at both locations by the same properly trained team, the university environment could be considered a more stressful agent. although no scientific evidence is presented herein, it can be ascertained that this occurrence may be associated with a possible collective sense existing among patients subjected to procedures performed by professionals undergoing training programs, who could be have less experience and skills than what is normally expected at private clinics. as a health professional, the dentist should not limit its role to the dental treatment itself. dentists should also be prepared to identify physiological alterations in their patients and investigate their possible sources. hence, the evocation of fear could be avoided, improving cardiovascular alterations. unfortunately, the observation and identification of behavioral changes are not easy tasks for the dental clinician, whose training seldom includes behavioral observations or knowledge about behavior management. technical acumen is a product of a teaching system in which manual skills and technological procedures are taught and enhanced, many times contrasting with the need for a more humane approach, which often is required when considering the physical proximity between the patient and dentist, and the emotional vulnerability of many people6. further studies are required to identify the factors in the treatment environment that can increase physiological changes or reduce anxiety (e.g., the use of lavender or orange essence in the waiting room)18-20. it may be concluded that the clinical setting is an important factor when planning dental surgeries. moreover, identifying the characteristics of the environment that negatively influence physiological responses in dental implant patients is necessary to reduce risk, especially at places with high attendance rates, such as university clinics. references 1. spielberg c, gorgush r, lushene r. manual for the strait-trait anxiety inventory. palo alto, ca: consulting psycologists press; 1970. 2. muglali m, komerik n. factors related to patients’ anxiety before and after oral surgery. j oral maxillofac sur. 2008; 66: 870-7. 3. kim s, lee yj, lee s, moon hs, chung mk. assessment of pain and anxiety following surgical placement of dental implants. int j oral maxillofacial implants. 2013; 28: 531-5. 4. humphris g, crawford jr, hill k, gilbert a, freeman r. uk population norms for the modified dental anxiety scale with percentile calculator: adult dental health survey 2009 results. bmc oral health. 2013; 13: 29. 5. hall je. guyton and hall textbook of medical physiology: enhanced ebook: elsevier health sciences; 2010. 6. sanikop s, agrawal p, patil s. relationship between dental anxiety and pain perception during scaling. j oral sci. 2011; 53: 341-8. 7. romano mm, soares ms, pastore ca, tornelli mj, de oliveira guare r, adde ca. a study of effectiveness of midazolam sedation for prevention of myocardial arrhythmias in endosseous implant placement. clin oral implants res. 2012; 23: 489-95. 8. bispo cg, tortamano ip, rocha rg, francischone ce, borsatti ma, da silva jc jr, et al. cardiovascular responses to different stages of restorative dental treatment unaffected by local anaesthetic type. aust dent j. 2011; 56: 312-6. 9. beck fm, weaver jm. blood pressure and heart rate responses to anticipated high-stress dental treatment. j dent res. 1981; 60: 26-9. 10. liau fl, kok sh, lee jj, kuo rc, hwang cr, yang pj, et al. cardiovascular influence of dental anxiety during local anesthesia for tooth extraction. oral sur oral med oral pathol oral radiol endond. 2008; 105: 16-26. 11. desborough jp. the stress response to trauma and surgery. br j anaesth. 2000; 85: 109-17. 12. eli i, schwartz-arad d, baht r, ben-tuvim h. effect of anxiety on the experience of pain in implant insertion. clin oral implants res. 2003; 14: 115-8. 13. vassend o, roysamb e, nielsen cs. dental anxiety in relation to neuroticism and pain sensitivity. a twin study. j anxiety disord. 2011; 25: 302-8. 14. corah nl. development of a dental anxiety scale. j dent res. 1969; 48: 596. 15. oosterink fm, de jongh a, aartman ih. what are people afraid of during dental treatment? anxiety-provoking capacity of 67 stimuli characteristic of the dental setting. eur j oral sci. 2008; 116: 44-51. 16. 16malamed sf. knowing your patients. j am dent assoc. 2010; 141(suppl 1): 3s-7s 17. eli i, schwartz-arad d, bartal y. anxiety and ability to recognize clinical information in dentistry. j dent res. 2008; 87: 65-8. 18. lehrner j, marwinski g, lehr s, johren p, deecke l. ambient odors of orange and lavender reduce anxiety and improve mood in a dental office. physiol behav. 2005; 86: 92-5. braz j oral sci. 13(2):109-113 clinical setting influences physiological responses in dental implant patients 113 19. lehrner j, eckersberger c, walla p, potsch g, deecke l. ambient odor of orange in a dental office reduces anxiety and improves mood in female patients. physiol behav. 2000; 71: 83-6. 20. kritsidima m, newton t, asimakopoulou k. the effects of lavender scent on dental patient anxiety levels: a cluster randomised-controlled trial. community dent and oral epidemiol. 2010; 38: 83-7. braz j oral sci. 13(2):109-113 clinical setting influences physiological responses in dental implant patients oral sciences n3 original article braz j oral sci. january | march 2013 volume 12, number 1 morphological aspects of dentin-pulp complex development in the offspring of rats treated with fluoxetine during pregnancy isabela maria de albuquerque santiago1, luciana silva regueira1, priscylla gonçalves correia1, robério josé barbosa de alcântara2, joaquim evêncio neto3, liriane baratella-evêncio4 1department of dentistry, federal university of pernambuco, recife, pe, brazil 2dental surgeon, recife, pe, brazil 3department of morphology and animal physiology, federal rural university of pernambuco, recife, pe, brazil 4department of histology and embryology, federal university of pernambuco, recife, pe, brazil correspondence to: isabela maria de albuquerque santiago avenida prof. moraes rego, 1235, cep: 50670-901, recife, pe brasil phone/fax: + 55 81 21268515 e-mail: isabelasantiago1@yahoo.com.br abstract aim: to evaluate the morphological aspects of coronal dentinogenesis in the first molars of 1and 5-day-old rats whose mothers were treated with fluoxetine hydrochloride during pregnancy. methods: twelve pregnant wistar rats were divided randomly into three groups: group c (control), group fl (fluoxetine administered at 10 mg/kg bodyweight), and group fx (fluoxetine administered at 20 mg/kg bodyweight). saline (0.9%) solution or fluoxetine hydrochloride was administered subcutaneously for the first 21 days of pregnancy. subsequently, the offspring of these animals was subdivided into subgroups according to age of tooth germ development to be studied: 1 and 5 days of life. c1 and c5 (control group 1 and 5 days of age); fl1 and fl5 (groups treated with 10 mg/kg fluoxetine at 1 and 5 days of age); fx5 and fx1 (groups treated with 20 mg/ kg fluoxetine at 1 and 5 days of age). results: no structural changes in the dentin-pulp complex of rats whose mothers were treated with fluoxetine hydrochloride were observed at either dose. conclusions: fluoxetine, at the doses administered during pregnancy in this study, did not alter the morphological development of the coronal dentin-pulp complex in their offspring. keywords: fluoxetine, serotonin, dental germ, dentin-pulp complex. introduction clinical depression is a recognized public health problem, interfering decisively in the personal, professional, social and economic standing of patients. in recent years, a greater number of women during pregnancy have been diagnosed with depression. the pharmacological treatment of this disease has reduced morbidity and improved the clinical outcomes of thousands of patients with depression globally1-2. several drugs cross the placental barrier and some have teratogenic effects on the fetus, which may cause dysfunction and disorders during embryonic development, particularly during the first three months of intrauterine life2. in 1987, the us food and drug administration approved the first antidepressant (fluoxetine) in a group of selective serotonin reuptake inhibitors (ssris)3. fluoxetine hydrochloride is the most widely prescribed antidepressant in the world, which received for publication: november 25, 2012 accepted: march 15, 2013 braz j oral sci. 12(1):30-36 3131313131 acts by inhibiting serotonin (5-ht) reuptake and enhancing serotonergic neurotransmission4-5. the 5-ht neurotransmitter regulates important pathways of mammalian metabolism and is synthesized from the phenylalanine, tyrosine and tryptophan amino acids5. in the human brain, the first 5-ht-releasing neurons are present from the fifth week and their numbers increase markedly by the tenth week of gestation6. 5-ht interacts with its receptors, which alters cell metabolism and influences several stages of organogenesis7. serotonergic neurotransmission modulates cell proliferation in several tissues, but is involved mainly in the morphogenesis of the craniofacial region8. the metabolism of mineralized tissues can be influenced by the central nervous system (cns)9. neuroendocrine mechanisms, particularly those related to 5-ht, are associated with the differentiation and activation of osteoblasts and osteoclasts. reports have demonstrated that fluoxetine hydrochloride can induce bone resorption in rats by blocking 5-ht reuptake10. 5-ht also has a stimulatory role in tooth germ development by inducing the formation of enamel and dental papilla to form the bell and crown stages. these phenomena are cytoand histo-differentiation steps that occur during odontogenesis and give rise to teeth11-12. extensive renovation of the epithelium, cell proliferation, apoptosis and changes in the shape and positioning of cell groups are determined by morphogenetic gradients that play critical roles during the morphogenesis of teeth13. the tooth germ develops in five stages: the bud, cap, bell, crown and root. after bell formation is complete, the tooth germ has the required structure to form the tooth and its supportive and protective tissues14. the beginning of the crown phase is characterized by odontoblast maturation and deposition of dentin, followed by enamel secretion by ameloblasts to form the mineralized tissues of the future crown15. therefore, it is possible that chronic exposure to therapeutic doses of ssris alters the development of the serotonergic system. however, such evidence is yet unclear and more studies are required. the objective of this study was to evaluate the morphological aspects of coronal dentinogenesis in the first molars of 1and 5-day-old rats whose mothers were treated with fluoxetine hydrochloride during pregnancy. material and methods this study was conducted at the animal facility of the center for experimental surgery at the department of histology and embryology and at the laboratory of histotechnical graduate programs in pathology, federal university of pernambuco. the experiment protocol was approved by the committee for ethics in animal research of the center for biological sciences, federal university of pernambuco, recife, brazil, number 23076.006899/2008 – 51. twelve female albino wistar rats received a standard animal care facility diet and water ad libitum. they were kept in a room maintained at 23 ± 2°c and with a 12:12 h light/dark cycle. after mating and pregnancy confirmation by vaginal smear, the rats were divided into three groups, including four in a control group (c group), four in a group treated with fluoxetine at 10 mg/kg bodyweight (fl group) and four in a group treated with fluoxetine at 20 mg/kg bodyweight (fx group). each group was subdivided into two subgroups according to the age of the offspring (1 or 5 days of age), totaling six subgroups, according to the figure 1. two pregnant rats were used for each subgroup and each litter was used for three pups of both genders. six animals in each subgroup were obtained from pregnant rats treated fig. 1: twelve pregnant rats were used, 4 rats for each group (c, fl and fg). for each subgroup, 2 pregnant rats were used and 3 pups of each pregnant rats were analyzed, totaling 6 animals per subgroup. morphological aspects of dentin-pulp complex development in the offspring of rats treated with fluoxetine during pregnancy braz j oral sci. 12(1):30-36 3232323232 according to the study groups from which they belonged for the first 21 days of pregnancy. in group c, mothers received 0.9% saline at 10 ml/g injected subcutaneously daily between 7 and 8 am. mothers in the fl and fx groups were treated daily with fluoxetine injected subcutaneously using the same protocol established for the control group. animals at 1 day of age were cryo-anesthetized and those at 5 days of age were anesthetized by subcutaneous injection of ketamine (25 mg/kg) and xylazine (10 mg/kg). the animals were decapitated, their jaws removed and the maxillary first molar with the left and right tooth germs were sectioned tangentially to the mesial of the first molar. specimens were fixed in 10% buffered formalin for 24 h at room temperature. then, the 5-day-old specimens were decalcified in an aqueous 10% nitric acid solution followed by conventional histology and embedding in paraffin. serial 4-µm thick sections were prepared, stained with hematoxylin-eosin, mounted in entellan® mounting medium (merck kgaa, darmstadt, germany), were and observed and photographed under an eclypse 51® optical microscope (nikon, tokyo, japan). the crown of each tooth germ was divided into cusp, middle and cervical thirds. the histological characteristics of the dentin, pre-dentin and odontoblastic layers were observed and described in these regions. the eclypse 51® optical microscope (nikon, tokyo, japan). with a miniature camera connected to a computer containing an image capture board (ati) and the image j® software (national institute of mental health, bethesda, md, usa) was used for the histometric analysis. the crowns were analyzed, and each tissue was assigned 10 fields in which the thicknesses of the pre-dentin and dentin as well as the height of odontoblasts were measured. the means and standard deviations of these data were calculated for statistical analysis. the statistical analysis was performed using a oneway anova and tukey’s test. the level of significance was set at p<0.05. results one-day-old animals it was observed that the tooth germ was in the initial of crown stage. initial deposition of a very thin dentin matrix with light pink coloration was noted in the third cuspidate. the palisade odontoblastic layer with highly prismatic aspects and a nucleus polarized toward the dental papilla was observed beneath the dentin matrix. the odontoblast cell layer resembled pseudo-stratified epithelium in the region of the cusp curvature due to the proximity of the cells. the preameloblast cell layer, which has highly prismatic cells, was observed above the dentin matrix, but the synthesis of enamel matrix had not yet begun (figure 2). dentin matrix was observed in the middle third down the slope of the crown, as thick as the third cuspidate, which tapered toward the cervical third. the odontoblasts underlying this layer were less prismatic, but the nuclei were biased toward the dental papilla, indicating cytodifferentiation activity. in the same way, the preameloblasts were prismatic, fig. 2. photomicrographs of parts the dental germ of 1-day-old rat maxillary first molar. it is observed in a (subgroup c1), group of odontoblasts (o) in the equivalent region of the future dental cusp and early production of dentin matrix (dm). b (subgroup fl1) and c (subgroup fx1) show an area of dentinogenesis (*) in the region of the middle third of the crown. in d (subgroup c1), it can be noted that the loop region is not yet occurring cervical deposition of dentin matrix (dm). sl, sub-odontoblastic layer. dp, dental papilla. eo, enamel organ. pa, pre-ameloblasts. v, blood vessels. ec, ectomesenchymal cells on differentiation. ie, inner epithelium enamel organ. ee, external epithelium enamel organ. sr, stellate reticulum. he staining. morphological aspects of dentin-pulp complex development in the offspring of rats treated with fluoxetine during pregnancy braz j oral sci. 12(1):30-36 3333333333 but less so than those in the anterior third (figure 2). no deposition of dentin matrix was observed in the cervical third, and dental papilla cells were more condensed and in the cellular differentiation phase. such cells had a star appearance, were basophilic and had rounded nuclei. odontoblasts and preameloblasts were observed in this region, and the inner epithelium of the enamel organ cells had a low prismatic or cuboid aspect with a central nucleus (figure 2). no structural differences in the tooth germ were observed among the groups of animals of this age. no significant changes in the thickness of the deposited dentin matrix were observed when compared to the c1, fl1, and fx1 subgroups. no significant changes in the length of the odontoblast cells in the region of the future cusp on the middle third of the crown were observed in any of the subgroups (figure 3). five-day-old animals it was observed that the tooth germ was at an advanced crown stage. no structural differences in tooth germ were observed in the animals of this age in the three study groups. the dentin layer was thicker in the third cuspidate than in the younger group, with a dark pink color, and the predentin layer was light pink. the enamel matrix was stained purple-blue above the dentin layer. highly prismatic ameloblasts above the enamel matrix were observed. below the pre-dentin layer, odontoblasts were observed arranged in a palisade pattern with high prismatic aspects and polarized nuclei toward the dental papilla. the odontoblastic cell layer resembled a pseudo-stratified epithelium in the cusp curvature region, due to proximity and rearrangement of the cells (figure 4). in the middle third, down the slope of the crown, were observed the predentin and dentin layers, which were thinner than the cuspidate, tapered toward the cervical third. the odontoblasts underlying the predentin layer were prismatic with polarized nuclei toward the dental papilla, but lower than those in the third cusp. above the dentin layer was the enamel matrix, thinner in the anterior third. prismatic ameloblasts above the enamel matrix were observed, but lower than those in the anterior third (figure 4). dentin and pre-dentin layers in the cervical third were found, but they were thinner than those in the cusp and middle third. above the dentin layers, preameloblasts with prismatic features were observed but no enamel matrix in this region. below the predentin layer, the odontoblast layer with a low prismatic aspect and nuclei toward the dental papilla was found, indicating synthesis activity (figure 4). no structural differences in tooth germ were observed in the animals of this age in the three study groups. no significant changes in the thickness of the predentin tissue deposited in the region of the future cusp, in the middle third, nor the cervical third of the crown were observed in the three subgroups. the mineralized dentin had the same thickness in all three subgroups and in the crowns of the analyzed tooth germs. the length of the odontoblastic layer was not different between the fluoxetine-treated groups and the control group (figure 5). discussion the development of rat molars is a classical model to study odontogenesis. in a series of histological studies, the molars of rats have been analyzed for their reactions to various drugs and experimental conditions16. mammalian teeth represent a combination product during the development of the ectoderm and ectomesenchyme14. the influence of the oral epithelium on the mesoderm plays an important role in tooth germ development17. no differences were found in the histological characteristics of the 1-day-old animals in any of the groups, i.e., the odontogenesis process began at the dental cusps that presented in a more advanced stage of dental crown morphological aspects of dentin-pulp complex development in the offspring of rats treated with fluoxetine during pregnancy braz j oral sci. 12(1):30-36 fig. 3. in 3a shows the evaluation of the effect of fluoxetine on the thickness (in µm) of dentin matrix deposited in the region of future dental cusp and in the middle third of the crown. in 3b shows the evaluation of the effect of fluoxetine on the length (in µm) of odontoblasts layer in the region of future dental cusp and in the middle third of the crown. animals with 1 day of life, comparing subgroups c1, fl1 and fx1. anova single factor test was used followed by tukey test, p<0.05. 3434343434 fig. 5. in 5a and 5b shows the evaluating the effect of fluoxetine on the thickness (in µm) of pre-dentin and dentin deposited in the region of future dental cusp, middle third and the cervical third of the crown. in 5c shows the evaluating the effect of fluoxetine on the length (in µm) of odontoblasts layer in the region of future dental cusp, middle third and the cervical third of the crown. animals aged 5 days old, comparing subgroups c5, fl5 and fx5. one-way anova was used, followed by tukey test, p<0.05. fig. 4. photomicrographs of parts the tooth germ of 5-day-old rat maxillary first molar. in a (subgroup fx5), it is observed in third cuspidate a thicker layer of dentin (d). in b (subgroup c5) and c (subgroup fl5), it can be noted that odontoblasts (o) show an aspect prismatic lowest in the region of middle third. in d (subgroup fl5), it can noted that the cervical loop region already has the pre-dentin (pd) and dentin (d). eo, enamel organ. a, ameloblasts. pa, pre-ameloblasts e, enamel. p, dental pulp. d, dentin. pd, pre-dentin. o, odontoblasts. sl, sub-odontoblastic layer. dp, dental papilla. v, blood vessels. he staining. morphological aspects of dentin-pulp complex development in the offspring of rats treated with fluoxetine during pregnancy braz j oral sci. 12(1):30-36 3535353535 development. an undifferentiated stage was observed down the slopes of the crowns to the cervical loop, as the ectomesenchymal dental papilla prepared for dentin formation. this process evolved gradually, and the dentin approached the cervical third of the crown in 5-days old animals. these data agree with the description of beverlander and hiroshi (1966)18 who reported that dentin begins to be formed in the advanced bell stage as a function of the dental papilla. the cells that differentiate in the periphery of the dental papilla are responsible for the formation and mineralization of the organic dentine matrix. the odontoblasts are juxtaposed with the predentin layer and are present in greater numbers in the portion closer to the cusp19. several sections showed pseudostratification of the odontoblast layer; however, this variation in tissue morphology can be explained by the difference in the angle of the thick and sloped region of the cusp in the tooth germ section. moreover, the crown of a rat molar has a specific anatomy, with more than one cusp and deep grooves that contribute to formation of a larger number of bends15. these characteristics may be related to differences in thickness of the dentin matrix and the length of the odontoblast layer, depending on the selected region. the odontoblasts synthesized the organic dentin matrix as dentinogenesis advanced, then retreated and left a cell extension that made contact with the basement membrane near the dentin-enamel junction. a pink band, w h i c h s t a i n e d w i t h l e s s i n t e n s i t y , l a y b e t w e e n t h e odontoblasts and dentin and stained more intensely in the 5-day-old animals, which characterized the deposition of predentin. these findings are consistent with results describing the presence of a matrix rich in sulfur and less acidophilic than the organic dentin matrix (predentin) in adjacent areas of the odontoblast layer20. the dentin matrix stains acidophilic, acquiring a light pink hue when stained with hematoxylin and eosin, which indicates the presence of type-i collagen fibers and proteoglycans. these findings were reported by reith (1968)21 who found that odontoblasts differentiate by location and size and are flanked by an extracellular compartment with collagen fibers and an amorphous substance. in 1-day-old animals, the histometric data showed no significant differences in the thickness of the dentin matrix or the length of odontoblasts in any part of the tooth germ crown. just as in the 5-day-old animals, all the parameters measured in histometric analysis were similar. as the mothers were treated only during pregnancy, fluoxetine administration did promote significant morphological and quantitative changes in those tissues. even 5-day-old animals that had been breastfed and would receive fluoxetine through breast milk, showed no significant changes in tooth germ. the choice of fluoxetine hydrochloride was justified for this study, because it is the most widely prescribed antidepressant worldwide. it is capable of inhibiting 5-ht reuptake4 and has little affinity for other neuroreceptors, which increases its tolerability by the body and diminishes side effects5. few studies have investigated use of ssris (fluoxetine hydrochloride) and the development of mineralized tissues that form the tooth, as most related studies investigated bone tissue. bliziotes et al. (2006)9 found that 5-ht 1a and 5-ht 2a receptors in bone may be related to possible interactions with the serotonergic system. a previous study found that 5-ht is present during morphogenesis of the craniofacial region; however, they did not discuss tooth development22. other experimental evidence has also revealed that 5-ht may influence embryogenesis and growth23. battaglino et al. (2007)24 reported reduced cortical bone and trabecular bone mass in rats treated with fluoxetine, and concluded that fluoxetine inhibits normal bone growth in rats. those authors claimed that 5-ht plays an important role in the differentiation of osteoblasts and osteocytes, and that interference of 5-ht by fluoxetine could reduce bone mass. bonnet et al. (2007)25 reported deleterious effects on the architecture, microarchitecture and biomechanics of bones in animals treated with fluoxetine hydrochloride. similarly, battaglino et al. (2004)10 concluded that fluoxetine inhibits the differentiation of osteoclasts derived from bone marrow cells. lauder et al. (2000)26 revealed that 5-ht 2a , 5-ht 2b , and 5-ht 2c receptors in the developing tooth germ play a stimulating role during odontogenesis; however, they did not investigate development of the dentin-pulp complex. corroborating these findings, moiseiwitsch, lauder (1996)11 and moiseiwitsch et al. (1998)12 analyzed the inhibitory effects that ssris have during organogenesis by culturing rat mandibles with different 5-ht concentrations. those authors concluded that 5-ht could influence the dental papilla to induce the ameloblasts to differentiate from the external epithelium, inner epithelium, stellate reticulum, and stratum intermedium, which are essential tissues for growth and development of the tooth germ. however, they made no statements regarding dentinogenesis. cavalcanti et al. (2009)27 evaluated the morphological and embryonic developmental changes occurring in skeletal bones of animals whose mothers were treated with 10 mg/kg body weight fluoxetine hydrochloride. in the present study the temporomandibular joint was chosen because it is related to craniofacial development, which is influenced by the serotonergic system. the findings of the present study were similar to those of silva et al. (2010)28, who analyzed the direct effect of fluoxetine on the development of tooth enamel. in this study were evaluated the morphological and structural changes that this drug could produce in the enamel organ of the maxillary first molars of rats. the results showed no structural changes in the development of tissues that form the tooth enamel. thus, from these results, it may be suggested that fluoxetine hydrochloride administration during pregnancy, up to 20mg/kg, does not interfere on the dentin-pulp complex structural development. further research is required with a different design and using a longer evaluation period to confirm the present data. morphological aspects of dentin-pulp complex development in the offspring of rats treated with fluoxetine during pregnancy braz j oral sci. 12(1):30-36 acknowledgements the financial support given by the national council for scientific and technological development (cnpq). references 1. morrison jl, riggs kw, rurak dw. fluoxetine during pregnancy: impact on fetal development. reprod fertil dev. 2005; 17: 641-50. 2. patel sr, wisner kl. decision making for depression treatment during pregnancy and the postpartum period. depress anxiety. 2011; 28: 589-95. 3. patil as, kuller ja, rhee eh. antidepressants in pregnancy: a review of commonly prescribed medications. gynecol surv. 2011; 66: 777-87. 4. lanza di scalea t, wisner kl. antidepressant medication use during breastfeeding. clin obstet gynecol. 2009; 52: 483-97. 5. feijó fm, bertoluci mc, reis c. serotonina e controle hipotalâmico da fome: uma revisão. rev assoc med bras. 2011; 57: 74-7. 6. canto-de-souza a, souza rln, pelá ir, graeff fg. involvement of the midbrain periaqueductal gray 5-ht1a receptors in social conflict induced analgesia in mice. eur j pharmacol. 1998; 345: 253-6. 7. buznikov ga, lambert hw, lauder jm. serotonin and serotonin-like substances as regulators of early embryogenesis and morphogenesis. cell tissue res. 2001; 305: 177-86. 8. lauder jm, luo x, persico am. serotonergic regulation of somatosensory cortical development: lessons from genetic mouse models. devel neurosci. 2003; 25: 173-83. 9. bliziotes m, eshleman a, burt-pichat b, zhang xw, hashimoto j, wiren k, et al. serotonin transporter and receptor expression in osteocytic mloy4 cells. bone. 2006; 39: 1313-21. 10. battaglino r, fu j, späte u, ersoy u, joe m, sedaghat l, et al. serotonin regulates osteoclast differentiation through its transporter. j bone miner res. 2004; 19: 1420-31. 11. moiseiwitsch jrd, lauder jm. simulation of murine tooth development in organotypic culture by the neurotransmitter serotonin. arch oral biol. 1996; 41:161-5. 12. moiseiwitsch jrd, raymond jr, tamir h, lauder jm. regulation by serotonin of tooth-germ morphogenesis and gene expression in mouse mandibular explant cultures. arch oral biol. 1998; 43: 789-800. 13. lesot h, brook ah. epithelial histogenesis during tooth development. arch oral biol. 2008; 19: 1-9. 14. bei m. molecular genetics of tooth development. curr opin genet dev. 2009; 19: 504-10. 15. goldberg m, kulkarni ab, young m, boskey a. dentin: structure, composition and mineralization. front biosci. 2011; 1: 711-35. 16. pinzon rd, kozlov m, burch w. histology of rat molar pulp at different ages. j dent res. 1967; 46: 202-8. 17. thesleff i, tummers m. tooth organogenesis and regeneration. stembook [internet]. cambridge (ma): harvard stem cell institute; 2009. 18. bevelander g, hiroshi n. the formation and mineralization of dentine. anat rec. 1966; 156: 303-23. 19. sasaki t, garant pr. structure and organization of odontoblasts. anat rec. 1996; 245: 235-49. 20. goracci g, mori g, baldi m. terminal end of the human odontoblasts process: a study using sem and confocal microscopy. clin oral investig. 1999; 3: 126-32. 21. reith ej. collagen formation in developing molar teeth of rats. j ultrastruct res. 1968; 21: 383-414. 22. reisoli e, de lucchini s, nardi i, ori m. serotonin 2b receptor signaling is required for craniofacial morphogenesis and jaw joint formation in xenopus. development. 2010; 137: 2927-37. 23. bliziotes mm, eshleman aj, zhang xw, wiren km. neurotransmitter action in osteoblasts: expression of a functional system for serotonin receptor activation and reuptake. bone. 2001; 29: 477-86. 24. battaglino r, vokes m, schulze-späte u, sharma a, graves d, kohler t et al. fluoxetine treatment increases trabecular bone formation in mice (fluoxetine affects bone mass). j cell biochem. 2007; 100: 1387-94. 25. bonnet n, bernard p, beaupied h, bizot jc, trovero f, courteix d et al. various effects of antidepressant drugs on bone microarchitectecture, mechanical properties and bone remodeling. toxicol appl pharmacol. 2007; 221: 111-8. 26. lauder jm, wilkie mb, wu c, singh s. expression of 5-ht2a, 5-ht2b and 5-ht2c receptors in the mouse embryo. int j dev neurosci. 2000; 8: 653-62. 27. cavalcanti udnt, baratella-evêncio l, neto je, castro rm, cardona as, melo mlm et al. morphological aspects of the embryonic development of the tmj in rats (rattus norvegicus albinus) treated with fluoxetine. int j morphol. 2009; 27: 899-903. 28. silva ihm, leão jc, evêncio lb, porter sr, de castro rm. morphological analysis of the enamel organ in rats treated with fluoxetine. clinics. 2010; 65: 61-6. 3636363636morphological aspects of dentin-pulp complex development in the offspring of rats treated with fluoxetine during pregnancy braz j oral sci. 12(1):30-36 oral sciences n3 original article braz j oral sci. october | december 2013 volume 12, number 4 orofacial and dental condition in leprosy raony môlim de sousa pereira1, thalisson saymo de oliveira silva1, luciana saraiva e silva2, tanit clementino santos3, carlos alberto monteiro falcão1,3, lucielma salmito soares pinto2,3 1department of diagnosis and clinical dentistry, dental school, state university of piauí, parnaíba, pi, brazil 2departament of basic sciences, medical school, state university of piauí, teresina, pi, brazil 3department of diagnosis and clinical dentistry, dental school, uninovafapi, teresina, pi, brazil correspondence to: lucielma salmito soares pinto centro universitário de saúde ciências humanas e tecnológicas do piauí uninovafapi, faculdade de odontologia rua vitorino orthigues fernandes, 6123 cep: 64073-505 bairro uruguai, teresina, pi, brasil phone: +55 86 21060712 e-mail: lucielma@yahoo.com.br abstract aim: to verify the orofacial and dental complex in individuals affected by leprosy concerning lesions, prevalence of dental caries and biofilm storage. methods: this study was performed on 56 patients with leprosy: 28 under treatment and 28 treated for the disease. a clinical form was prepared to include demographic and leprosy data. the prevalence of caries was analyzed using the decayed, missing, filled teeth (dmft) index and oral hygiene was evaluated using the plaque control record. the statistical significance was measured by chi-square and student’s t-test. results: the mean age was 56.4 years, and there was predominance of males. the treatment group had more women (pearson ÷2=12.47, p=0.0004) and younger patients (pearson ÷2=9.688, p=0.0079) than the healed group. as much as 30.4% of patients had orofacial complaints related to the disease. the most often observed variations of normality as racial melanin pigmentation (67.8%) and lingual varices (32.1%), and lesions such as atrophy of the anterior nasal spine (25%), inflammatory papillary hyperplasia (17.8%), and collapse of the nasal bridge (14.3%). there were no specific oral lesions among leprosy patients. the mean dmft was 20.8 and the plaque control record was 70.2%. the treated group had higher dmft than the treatment group (student’s t p=0.003). conclusions: the oral health status of individuals with leprosy is poor, with high levels of plaque and dmft. no oral lesions associated with the disease were seen. keywords: leprosy, mycobacterium leprae, oral health. introduction leprosy is a chronic infectious disease with interpersonal transmission caused by mycobacterium leprae, a microorganism that has an affinity for the skin and peripheral nerves and, in more advanced stages affects internal organs and mucous membranes, with a high potential for causing physical disability1-4. it is present in less developed countries and most often affects the population with low socioeconomic status. with variable prevalence, india and brazil are respectively the first and second countries with the highest number of leprosy cases1,5. the pan american health organization (paho) includes leprosy in the group of neglected diseases and other poverty-related infections, and considers it a public health problem6. the prevalence rate of leprosy in brazil, an indicator that monitors the progress towards elimination of this disease as a public health problem, had a gradual reduction in recent years. this reduction results from the decrease in the number of new cases of the disease, the shorter treatment time and the improvement of braz j oral sci. 12(4):330-334 received for publication: september 25, 2013 accepted: december 03, 2013 information systems. the prevalence rate of leprosy in brazil in 2012 was 1.51 cases per 10,000 inhabitants, which represents a decrease of 12% compared to the value of this coefficient in 2004. despite these advances, the spatial distribution of leprosy remains the same in the country, with coefficients still high in the cities of northern and northeastern brazil6. despite the oral examination being part of the comprehensive examination of a patient with leprosy, there are few studies describing the oral health and dental care of these patients. unlike the cutaneous manifestations of the disease, which are well documented in the literature, descriptions of oral findings are scarcely detailed, making comparisons difficult, and old if the treatment of the disease was not effective2-4,7,8. it is important that the dentist be aware of the reactions caused by leprosy in the orofacial complex. thus, the present study aimed to evaluate the oral health status of patients with leprosy in relation to the presence of associated lesions, prevalence of caries and plaque accumulation. material and methods the sample consisted of 56 patients of both genders with a history of leprosy: 28 under treatment and 28 were already treated for the disease. the patients were recruited from the hospital colônia do carpina and basic health units in the city of parnaíba, pi, brazil in 2011 and 2012. the study was conducted in accordance with the declaration of helsinki guidelines and was approved by the ethics committee of uninovafapi (0269.0.043.000-10 protocol). all patients gave written informed consent to participate in the study. a clinical form containing the data was filled for each patient, as follows: demographic data, type and duration of leprosy, treatment adopted for leprosy, and complaints about the disease. facial and oral examinations were performed. the prevalence of caries was analyzed using the decayed, missing, filled teeth (dmft) index as recommended by the world health organization (who). oral hygiene was evaluated using the plaque control record by o’leary, which was obtained by the examination of four dental surfaces of all teeth and included the percentage of tooth surfaces with dental plaque9. leprosy diagnosis was carried out according to who criteria1. biopsies, histopathological examinations, smears, and photographic registration were undertaken as required. the spss software version 18.0 (spss inc., chicago, il, usa) was used and the statistical significance was analyzed by pearson’s chi-square test and student’s t-test. results fifty-six patients were evaluated, 28 under treatment and 28 treated patients. the mean and median age was 56.4 years and 58.5, respectively, ranging from 23 to 83 years. the treatment group had a mean age of 51.4 and the treated group had a mean age of 61.2 years. the treated group had patients older than the treatment group (pearson ÷2 =9.688; p=0.0079). out of all analyzed patients, 33 (58.9%) were male; however, the treatment group showed greater prevalence of women (pearson ÷2 =12.47; p=0.0004) (table 1). among patients with ongoing disease, 16 (57.14%) had lepromatous or multibacillary type leprosy and 12 (42.86%) had tuberculoid or paucibacillary. orofacial complaints were reported by 17 (30.4%) patients: nodules, swelling, ulcers, spots on the face and ears, loss of the nasal bridge and spine; ulcers and nodules in the tongue and gingiva, as well as tooth weakness. most patients (n=42, 75%) reported following proper hygiene measures regarding toothbrushing and doing it with minimum frequency of three times a day; however, dental floss was not used by any individual. fifty-three subjects (94.6%) denied having received specific dental guidance after diagnosis. alcoholism and smoking were reported by 29 (51.7%) respondents. twenty-two subjects (39.3%) reported at least one family member with a history of the disease. physical examination revealed orofacial variations of normality, with 38 cases of melanin pigmentation (67.8%) and 18 cases of lingual varices (32.1%), whereas among the lesions were seen most frequently: atrophy of the nasal spine (25%), inflammatory papillary hyperplasia (17.8%) and collapse of the nasal bridge (14.3%). prevalence of normality variations and non-specific lesions, candidiasis and inflammatory papillary hyperplasia, showed no significant difference between groups. however, the lesions associated with leprosy were more prevalent in the healed group. there were no specific oral lesions of leprosy among the evaluated patients (table 2). the overall mean dmft was 20.8; in the group of treated patients it was 23.8 and among patients under treatment, it 17.8 (student’s t; p=0.003). the mean plaque control record was 70.2%; among the healed patients it was 75.3% and 66.6% in under treatment subjects (student’s t; p=0.297) (table 3). table 1 -table 1 -table 1 -table 1 -table 1 distribution of gender and age among disease groups *pearson chi-square 331orofacial and dental condition in leprosy braz j oral sci. 12(4):330-334 table 2 table 2 table 2 table 2 table 2 distribution of orofacial findings among the groups. table 3 -table 3 -table 3 -table 3 -table 3 distribution of the dmft and plaque control record index among disease groups. *student’s t test discussion this study evaluated 56 patients with leprosy, 28 treated and 28 in treatment. the treatment group had a mean age of 51.4 years, agreeing with the literature that describes average ages 35.9 to 53 years for patients at diagnosis or during multidrug therapy3,10–14. the group of healed patients had a mean age of 61.2 years. the significantly larger number of treated seniors in the group can be understood considering that the treatment of leprosy is long-lasting and there are frequent dropouts and interruptions in its course, resulting in a longer healing time14. the disease affected a larger number of men, except in the group under treatment, in which 64.3% of patients were women, with a statistically significant difference. the literature shows a higher prevalence in men3,8,11,12,15, though some authors describe more cases in women10,16. due to a higher risk of exposure, men are usually more affected, but the gender ratio 332 orofacial and dental condition in leprosy braz j oral sci. 12(4):330-334 has declined in the past few years 17. still, 39.3% of respondents had at least one family member with the disease living in the same household, which shows the importance of family contact as a source of the disease spreading, evidenced by the presence of anti-pgl-1 in household contacts18,19. most patients with active disease had multibacillary leprosy (57.14%), usually seen in older individuals or with decreased immune resistance, reflecting a greater spread of the bacillus in the body. leprosy is a chronic infection with diverse clinical presentations that vary according to the individual’s immune response. the two ends of this spectrum correspond to the forms tuberculoid (paucibacillary) and lepromatous (multibacillary). the first is characterized by a more intense immune response, fewer skin lesions and few bacilli in tissue specimens. the lepromatous form displays a relatively anergic state, and often occurs in patients with reduced cell-mediated immune response. there is a group called borderline, with less common forms and intermediate lesions. the main clinical features of the disease are hypopigmented or reddish spots on the skin with loss of sensitivity, involvement of peripheral nerves, and muscle weakness. they involve most often the skin, nerves, and mucosa of the upper respiratory tract3,20. orofacial complaints during the course of the disease were common, being reported by 30.4% of the patients. according to the perception of individuals, were reported lesions such as nodules and stains on the face, as well as sequelae of the disease, particularly affecting the nasal structure. the perception is the personal interpretation concerning his/her health status, involving objective and subjective factors. due to the great potential of developing visible deformities that affect areas of the face, it is important to highlight that leprosy’s impacts are not only physical, but also psychological21. the extraoral examination revealed changes associated with leprosy such as collapse of the nasal bridge (32.6%), atrophy of the nasal spine (18.6%), spots on the face (14%), and loss of eyebrows (11.6%). the typical face of leprosy includes atrophy of the anterior nasal spine, atrophy and recession of the alveolar process of the premaxilla, and endonasal inflammatory reactions12. other manifestations such as facial nerve involvement, eye injuries, and facial deformity have also been described3. significantly, higher prevalence of changes observed in the healed group was mainly the disease sequelae. patients treated for a longer time, as in the present study, particularly until the 1980s, underwent monotherapy with dapsone or were confined in colonies. many had the disease for a long period without the correct diagnosis, resulting in greater exposure to the bacillus and consequently numerous sequels8. variations of normality were observed by the intraoral examination: melanin pigmentation (46.9%), lingual varices (22.2%), fissured tongue (13.6%), fordyce granules (12.3%) and smoker’s melanosis (4.9%). common injuries were also noticed, such as inflammatory papillary hyperplasia associated with the use of prosthesis (71.4%) and candidiasis (28.6%). no specific leprosy lesion was perceived within the oral cavity. agreeing with these findings, other authors in similar studies observed fissured tongue, candidiasis, inflammatory papillary hyperplasia, fibroma, traumatic ulceration. however, there were no lesions caused by m. leprae in the oral cavity, as reported in the literature7,8,10,15. the absence of specific leprosy lesions may be attributed to the natural scarcity of m. leprae in the oral mucosa, when compared to the colder places of the skin7, as well as the scarcity provided by the action of multidrug therapy, since the patients were under treatment 7,8,10,15. however, the m. leprae genome was identified by polymerase chain reaction in oral mucosa of patients even during treatment22. oral lesions are characteristic of advanced disease. they are rare, especially in the tuberculoid form; however, they may be recognized in about 20–60% of cases in the lepromatous type2,3,12,20. they are usually asymptomatic and may vary between yellowish spots or reddish papules or nodules that may develop sessile tissue necrosis and ulceration 4,12. hard and soft palate, uvula, tongue belly, lips, and gingiva are prevailing affections, in addition to the destruction of the anterior maxilla and tooth loss12. it is noteworthy that clinical abnormalities of the oral cavity in patients with leprosy do not imply in a specific lesion of the disease, but there is need for histopathological confirmation. moreover, even in patients with normal clinical appearance, it is possible to see specific histopathological disease signs2. the mean dmft value found among all study subjects was 20.8, considered very high. a higher mean dmft was observed with increasing age. the range of 41 to 60 years had mean dmft of about 20 and from 61 to 83 years, around 25 in both groups, so there was no statistically significant difference when considering each age group. the age range of 23 to 40 years had this analysis compromised since the group had only one healed patient. the mean dmft in the group of cured patients was 23.8 and 18 in the treatment group, with a statistically significant difference between them. this may be attributed to the difference between the age groups, due to the higher number of elderly individuals in the healed group, which eventually raised the mean dmft of this group. the results of the national oral health project (sb brazil 2010) indicate mean dmft of 17.83 and 28.47 for the age groups 35–44 years and 65–74 years, respectively, considering the values for northeastern brazil 23. thus, although the results are bad, they do not differ from the brazilian reality for the general population in that region, agreeing with souza8. during the interview, 75% of patients reported brushing their teeth at least three times a day, despite not using dental floss. still, average rates of plaque control record of 70.2% were observed. indexes above 20% are associated with poor oral hygiene (9). the lack of specific dental guidance reported by 94.6% of patients, may be responsible for the accumulation of dental plaque evidenced in this study. despite the high frequency of reported toothbrushing, people do not always use the correct technique, which may reflect in a poor dental condition24. 333orofacial and dental condition in leprosy braz j oral sci. 12(4):330-334 it may be concluded that the oral health status of individuals treated and healed from leprosy is poor, with high levels of plaque and dmft. additionally, variations of normality and common oral lesions were seen, such as candidiasis and inflammatory papillary hyperplasia. the patients did not present leprosy-specific lesions. nevertheless, the situation shown in the present study cannot be considered worse than the one of the general population in northeastern brazil. better methods of oral hygiene and regular dental monitoring should be adopted in order to increase the quality of life of these patients. references 1. who. expert committee on leprosy, 8th report. who, technical report series 968, 2012. 2. abreu mamm, michalany ns, weckx llm, pimentel drn, hirata chw, alchorne mma. the oral mucosa in leprosy: a clinical and histopathological study. rev bras otorrinolaringol. 2006; 72: 312-6. 3. taheri jb, mortazavi h, moshfeghi m, bakhshi m, bakhtiari s, azarimarhabi s et al. oro-facial manifestations of 100 leprosy patients. med oral patol oral cir bucal. 2012; 17: 728-32. 4. pallagatti s, sheikh s, kaur a, aggarwal a, singh r. oral cavity and leprosy. indian dermatol online j. 2012; 3: 101-4. 5. alencar ch, ramos an jr, dos santos es, richter j, heukelbach j. clusters of leprosy transmission and of late diagnosis in a highly endemic area in brazil: focus on different spatial analysis approaches. trop med int health. 2012; 17: 518-25. 6. brazil. ministry of health. epidemiological bulletin. epidemiological situation of leprosy in brazil – analysis of the selected last decade indicators and challenges for disposal. 2013; 44. 7. santos gg, marcucci g, marchese lm. oral aspects of specific and unspecific lesions in hansen’s disease patients. pesqui odontol bras. 2000; 14: 268-272. 8. souza va, emmerich a, coutinho em, freitas m, silva eh, merçon fg et al. dental and oral condition in leprosy patients from serra, brazil. lepr rev. 2009; 80: 156-163. 9. buczkowska-radliñska j, pol j, szmidt m, biñczak-kuleta a. the influence of polymorphism of the muc7 gene on the teeth and dental hygiene of students at a faculty of dentistry in poland. postepy hig med dosw. 2012; 19: 204-9. 10. de araújo navas ea, inocêncio ac, almeida jd, back-brito gn, mota aj, jorge ao et al. oral distribution of candida species and presence of oral lesions in brazilian leprosy patients under multidrug therapy. j oral pathol med. 2009; 38: 764-7. 11. dave b, bedi r. leprosy and its dental management guidelines. int dent j. 2013; 63: 65-71. 12. rawlani sm, rawlani s, degwekar s, bhowte rr, motwani m. oral health status and alveolar bone loss in treated leprosy patients of central india. indian j lepr. 2011; 83: 215-24. 13. motta acf, furini rb, simão jcl, vieira mb, ferreira man, komesu mc et al. could leprosy reactions episodes be exacerbated by oral infections? rev soc bras med trop. 2011; 44: 633-5. 14. heukelbach j, andré chichava o, de oliveira ar, häfner k, walther f, de alencar ch et al. interruption and defaulting of multidrug therapy against leprosy: population-based study in brazil’s savannah region. plos negl trop dis. 2011; 5: 1031-9. 15. martins md, russo mp, lemos jb, fernandes kp, bussadori sk, corrêa ct et al. orofacial lesions in treated southeast brazilian leprosy patients: a cross-sectional study. oral dis. 2007; 13: 270-3. 16. gomes ccd, gonçalves hs, pontes maa, penna go. clinical and epidemiological profile of patients diagnosed with leprosy in a reference center in the northeast of brazil. an bras dermatol. 2005; 80: 283-8. 17. monteiro ld, alencar chm, barbosa jc, braga kp, castro md, heukelbach j. physical disabilities in leprosy patients after discharge from multidrug therapy in northern brazil. cad saude publica. 2013; 29: 909-20. 18. cardona-castro n, beltrán-alzate jc, manrique-hernández r. survey to identify mycobacterium leprae-infected household contacts of patients from prevalent regions of leprosy in colombia. mem inst oswaldo cruz. 2008; 103: 332-6. 19. bazan-furini r, motta ac, simão jc, tarquínio dc, marques w, barbosa mh et al. early detection of leprosy by examination of household contacts, determination of serum anti-pgl-1 antibodies and consanguinity. mem inst oswaldo cruz. 2011; 106: 536-540. 20. ghosh s, gadda rb, vengal m, pai km, balachandran c, rao r et al. oro-facial aspects of leprosy: report of two cases with literature review. med oral patol oral cir bucal. 2010; 15: 459-62. 21. almeida jrs, alencar ch, barbosa jc, dias aa, almeida mel. selfperception of people afflicted with leprosy regarding their oral health and the need for treatment. cien saude coletiva. 2013; 18: 817-26. 22. santos gg, marcucci g, guimarães júnior j, margarido lc, lopes lhc. molecular detection of mycobacterium leprae by polymerase chain reaction in oral mucosa biopsy specimens. an bras dermatol. 2007; 82: 245-9. 23. brazil. [department of primary care. department of health care project sb brazil 2010: national oral health survey main results. brasilia: ministry of health], 2011.portuguese. 24. belmonte pcr, virmond mcl, tonello as, belmonte gc, monti jfc. characteristics of periodontal disease in leprosy. bol epidemiol paulista. 2007; 4: 4-9. 334 orofacial and dental condition in leprosy braz j oral sci. 12(4):330-334 oral sciences n3 original article braz j oral sci. january | march 2014 volume 13, number 1 effects of various hydrogen peroxide bleaching concentrations and number of applications on enamel sandrine bittencourt berger1, luis eduardo silva soares2, airton abrahão martin2, gláucia maria bovi ambrosano3, cínthia pereira machado tabchoury4, marcelo giannini5 1universidade do norte do paraná unopar, school of dentistry, department of restorative dentistry, londrina, pr, brasil 2universidade do vale do paraíba univap, research and development institute (ip&d), laboratory of biomedical vibrational spectroscopy, são josé dos campos, sp, brasil 3universidade estadual de campinas unicamp, piracicaba dental school, department of community dentistry and biostatistics, piracicaba, sp, brasil 4universidade estadual de campinas unicamp, piracicaba dental school, department of physiological science, piracicaba, sp, brasil 5universidade estadual de campinas unicamp, piracicaba dental school, department of restorative dentistry, piracicaba, sp, brasil correspondence to: marcelo giannini departamento de odontologia restauradora faculdade de odontologia de piracicaba unicamp avenida limeira, 901, cep: 13414-900 piracicaba, sp, brasil phone: + 55 19 21065340. fax: 55 19 21065218 e-mail: giannini@fop.unicamp.br received for publication: january 19, 2014 accepted: march 11, 2014 abstract aim: to evaluate the effects of three hydrogen peroxide (hp) concentrations on enamel mineral content (mc), following three peroxide applications and using three methods to measure the mc. methods: forty samples were obtained from bovine incisors and randomly divided into 4 groups (n=10). the control group remained untreated, while the experimental groups were tested for three hp concentrations (10%, 35% and 50%). the hp gel was applied 3 times on the enamel surface for 30 min per application and the samples were analyzed after each application. the mc of the enamel was determined before and after bleaching using fourier transform (ft-raman) spectroscopy and micro energy-dispersive x-ray fluorescence spectrometry (µedxrf). the calcium (ca) lost from the bleached enamel was quantified with an atomic absorption spectrometer (aas). the data were analyzed statistically by anova, tukey and dunnett´s tests (p<0.05). results: the ft-raman showed a decrease in mc for all bleaching treatments, without influence of the different hp concentrations or the number of applications. µedxrf did not detect any changes in mc. conclusions: ca loss was observed by the aas, with no difference among the three hp concentrations. the ft-raman and aas analyses detected mc reduction and ca loss after hp bleaching. keywords: dental enamel; bleaching agents; hydrogen peroxide. introduction tooth bleaching is a well-accepted method of treating stained and discolored teeth. the techniques comprise the application of a strong oxidizing agent as an active ingredient on a tooth surface to achieve a whitening effect1. the bleaching mechanism is based on the decomposition of peroxides into free radicals, which react with the organic pigment molecules, transforming them into smaller and less pigmented compounds2. bleaching agents designed for professional use only contain a high concentration of peroxides (30% to 40%), while the active ingredient of patient-applied (at-home) tooth bleachers, present at lower concentrations, are carbamide peroxide (10% to 20%) and hydrogen peroxide (3% to 7.5%)3. since the introduction of at-home bleaching in 1989 by haywood and braz j oral sci. 13(1):22-27 gisele higa texto digitado http://dx.doi.org/10.1590/1677-3225v13n1a05 23 heymann 4, many studies have evaluated the effects of hydrogen and carbamide peroxides on mineralized and dental pulp tissues. a number of studies have reported some important side effects, such as increased roughness5, decreased microhardness5-7, reduction of enamel and dentin strength8, color changes9 and significant decrease in the calcium (ca), phosphate or carbonate concentration in enamel10-11. on the other hand, some authors have found no significant adverse effects following the bleaching12-13. such differences in the adverse effects on enamel and dentin caused by bleaching agents are due to the large number of bleaching protocols, peroxide types and concentrations, and evaluation methods. regarding the methods, microhardness 2,5, scanning electron and polarized light microscopy7,14, micro energy-dispersive x-ray fluorescence spectrometry (µedxrf)11, fourier transform-raman (ftraman) spectroscopy10 and atomic absorption spectrometer (aas)10 have been used to determine the adverse effects resulting from bleaching techniques. the objective of this study was to analyze the effect of different hydrogen peroxide (hp) concentrations (10%, 35% and 50%) after 1, 2 and 3 applications on an enamel surface, comparing three methodologies commonly used to evaluate the changes in the mineral contents of teeth. the research hypothesis was that hp promotes alterations in the mineral content of enamel regardless of the peroxide concentration and the number of peroxide applications. material and methods tooth preparation and experimental groups forty bovine incisors having the same age were used in this study. after extraction, the teeth were cleaned and stored in 0.1% thymol solution at 4 ºc for 30 days. they were then submitted to manual debridement with a periodontal curette to remove organic debris. the root portion was removed with a diamond saw 2 mm below the dentin-enamel junction. the enamel surface of all samples was flattened using 600-grit al2o3 abrasive paper and polished with 1000and 1200-grit aluminum oxide abrasive papers to remove irregularities from the bovine enamel surface6,10,156,10,156,10,156,10,156,10,15. the enamel surfaces of all teeth were coated with 2 layers of nail varnish (revlon, new york, ny, usa), except for a standardized, central buccal area (1 cm2) for application of hp. the teeth were randomly divided into 4 groups (n=10), according to the hp concentration (10%, 35% or 50%) and an unbleached control group. the teeth from the control group were kept in distilled water at 37 °c. ft-raman spectroscopy spectra of the teeth before (baseline) and after enamel treatments (bleaching or control) were obtained using a ftraman spectrometer (rfs 100/s; bruker, karlsruhe, germany). all spectra were processed by adjusting five raman vibrational stretching modes: 430 cm-1 (p1), 449 cm-1 (p2), 586 cm-1 (p3), 1043 cm-1 (p4) and 1073 cm-1 (p5). the p1–p3 represent phosphate modes and p4 and p5 are carbonate vibrations (type b carbonate)15-16. for the qualitative and semiquantitative spectral analysis, the spectra were corrected at baseline and then normalized to the 960 cm-1 peak17-18. the area of each band was obtained by gaussian shapes. microcal origin software (microcal software, northampton, ma, usa) was used for calculating the integrated areas of the peaks for the unbleached and bleached samples19. the averages of integrated areas of the evaluated raman peaks (p1 to p5) were calculated for the baseline and treated data. µedxrf spectrometry analysis the semi-quantitative elemental analyses of calcium (ca) (ca wt.%) and phosphorus (p) (p wt.%) were conducted by an energy-dispersive micro x-ray fluorescence spectrometer, model µedx 1300 (shimadzu, kyoto, japan), equipped with a rhodium x-ray tube and a si (li) detector cooled by liquid nitrogen (n2) and coupled to a computer system for data processing. the voltage in the tube was set at 15 kv, with an automatic adjustment of the current and a beam diameter of 50 µm. three spectra from each specimen were collected before and after bleaching treatments. the measurements were performed with a count rate of 100 s per point (live time) and a dead time of 25%. the energy range of the scans was 0.0 to 40.0 ev. the equipment was adjusted using a certified commercial reagent of stoichiometric hydroxyapatite [aldrich, synthetic ca10(po4)6(oh)2, grade 99.999%, lot 10818ha] as reference. the measurements were collected under fundamental parameters of characteristic x-ray emissions of ca and p, and the elements o and h were used as a chemical balance17,20. the energy calibration was performed using the internal standards of the equipment, as previously reported17,20. bleaching procedures the bleaching agents were prepared using a rate of 10 parts of hp (dinâmica reagentes analíticos, são paulo, sp, brazil) to 1 part of thickener (carbopol ultrez 10; proderma, piracicaba, sp, brazil), and ph adjusted to 6.0, according to a pilot study. a 2-mm thick layer of hp gel (0.2 g) was applied 3 times on the enamel surface for 30 min per application. between applications, the samples were rinsed with 5 ml distilled and deionized water; this rinse was kept for aas analysis. atomic absorption spectrometer the rinse (hp and water) was evaluated using aas. after each hp application, a standardized amount of water (5 ml) was used for rinsing and removing the peroxide gel from the enamel surface. the ca concentration in the solution was measured using aas (varian spectra aa50). before the sample analysis, the equipment was calibrated with ca standards ranging from 0.2 to 2.0 µg/ml. the concentrations of ca in the peroxide gels were previously analyzed and were not found in significant amounts. each analysis used lanthanum solution to reduce the interference of phosphate in the calcium analysis. effects of various hydrogen peroxide bleaching concentrations and number of applications on enamel braz j oral sci. 13(1):22-27 24 data analysis the sas software system (sas institute, cary, nc, usa) was used for data analysis, with the significance set at 5%. data from the ft-rs analysis were analyzed by two-way anova and tukey’s test. edxrf data were analyzed by two-way anova and dunnett’s test to compare the treated groups with the control group. aas data were analyzed by two-way anova, tukey’s test and dunnett’s test. results the selected range of raman spectra from the phosphate and carbonate main vibrational modes of enamel after 1, 2 and 3 applications of hp are shown in figures 1 to 3, respectively. the raman spectra of the inorganic content did not show obvious reduction in intensity after treatments, compared with the non-treated and control group (figures 1 3). the peaks at 430 and 449 cm-1 were associated to the í2po43modes. the peak at 586 cm ”1 was attributed to phosphate í4po43vibrations and the peak 960 cm”1 was related to í1po43vibration. the peaks at 1043 and 1073 cm”1 were attributed to bending and stretching modes of carbonate (co32-), respectively (figures 1-3)21. table 1 presents the means and standard deviations (sd) of the integrated area of the raman peaks for the enamel before and after treatments. two-way anova revealed statistically significant differences for evaluation time (p = 0.0386). however, there was no significant difference between the hp gel concentrations (p = 0.2144) or the number of applications (p = 0.8982). table 2 exhibits the mean ca, p and ca/p ratio of enamel at baseline and after treatment, obtained by µedxrf fig. 1 raman spectra of untreated enamel (normal, i.e., control group) and bleached enamel after 1 application of hydrogen peroxide (hp): 10% hp, 35% hp and 50% hp. raman spectra are shown in the 1250 to 650 cm-1 range, with inorganic peaks as follows: 431 and 449 cm-1 (v2po4 3vibrations), 589 cm -1 (phosphate v4po4 3vibrations), 960 cm-1 (phosphate v1po4 3vibrations), 1044 and 1070 cm-1 (bending and stretching modes of carbonate co3 2-). fig. 2 raman spectra of untreated enamel (normal, i.e., control group) and bleached enamel after 2 applications of hydrogen peroxide (hp): 10% hp, 35% hp and 50% hp. raman spectra are shown in the 350 to 1250 cm-1 range, with inorganic peaks as follows: 431 and 449 cm-1 (v2po4 3vibrations), 589 cm-1 (phosphate v4po4 3vibrations), 960 cm-1 (phosphate v1po4 3vibrations), 1044 and 1070 cm-1 (bending and stretching modes of carbonate co3 2-). fig. 3 raman spectra of untreated enamel (normal, i.e., control group) and bleached enamel after 3 applications of hydrogen peroxide (hp): 10% hp, 35% hp and 50% hp. raman spectra are shown in the 350 to 1250 cm”1 range, with inorganic peaks as follows: 431 and 449 cm-1 (í2po 4 3vibrations), 589 cm-1 (phosphate v4po4 3vibrations), 960 cm-1 (phosphate v1po4 3vibrations), 1044 and 1070 cm-1 (bending and stretching modes of carbonate co3 2-). analysis. there was no statistically significant difference among the peroxide concentrations (p = 0.1344), number of applications (p = 0.1951), evaluation times (p = 0.4420) or the control group compared with the bleached experimental groups (p = 0.6259). the results of aas are depicted in table 3. all experimental bleached groups differed from the unbleached control group, according to dunnett’s test (p < 0.05). the effects of various hydrogen peroxide bleaching concentrations and number of applications on enamel braz j oral sci. 13(1):22-27 25 groups before bleaching (baseline) after bleaching control group 67.8 (4.1) a a 65.4 (3.6) a a 10% hp + 1 application 68.0 (7.5) a a 56.0 (5.8) b a 10% hp + 2 applications 67.0 (8.2) a a 56.4 (5.7) b a 10% hp + 3 applications 63.8 (2.4) a a 58.7 (2.4) b a 35% hp + 1 application 70.3 (8.4) a a 61.5 (5.6) b a 35% hp + 2 applications 67.9 (8.6) a a 58.4 (8.8) b a 35% hp + 3 applications 71.2 (4.1) a a 60.5 (8.3) b a 50% hp + 1 application 69.4 (9.8) a a 56.7 (3.6) b a 50% hp + 2 applications 69.3 (4.1) a a 59.8 (7.5) b a 50% hp + 3 applications 69.2 (6.6) a a 60.6 (4.8) b a table 1 –table 1 –table 1 –table 1 –table 1 – mean (standard deviation) of the integrated area of raman peaks (p1 to p5) at baseline and after bleaching. means followed by different letters, capital letters in rows and lowercase letters in columns, are statistically different by tukey’ test (p<0.05). hp: hydrogen peroxide hp concentration had no influence on ca loss in any of the applications (p = 0.0884). the first and second applications of hp released significantly higher values of ca in the rinse compared with the third (p < 0.0001). discussion µedxrf, ft-raman and aas are versatile and nondestructive techniques that allow use of the same samples for the various types of dental enamel analysis. these methods provide precise concentrations of ca, phosphate and carbonate and the ca/p ratio, as shown in the results section. the hypothesis that hp promotes alterations in the mineral content of enamel regardless of peroxide concentration or number of peroxide applications was accepted only after using the ft-raman and aas methods. ft-raman analysis showed that hp decreased the integrated area of raman peaks of enamel samples (table 1 and figures 1 3). on the other hand, the control group, in which the specimens were not bleached, no enamel chemical changes were observed, as expected. alterations in mineral content of enamel promoted by peroxides have also been detected by other studies using ft-raman10,15,22. in this study, effects of various hydrogen peroxide bleaching concentrations and number of applications on enamel braz j oral sci. 13(1):22-27 there was no statistically significant difference among the experimental groups. hp: hydrogen peroxide groups element content (wt%) ca (mean ± sd) p (mean ± sd) ca/p (mean ± sd) before after before after before after treatments treatments treatments treatments treatments treatments control group 30.7 (7.8) 32.3 (7.7) 15.6 (3.9) 16.1 (4.4) 2.0 (0.1) 2.0 (0.1) 10% hp + 1 application 34.0 (5.8) 32.8 (3.9) 17.3 (2.5) 16.8 (2.3) 2.0 (0.1) 2.0 (0.1) 10% hp + 2 applications 30.1 (3.9) 28.9 (6.1) 16.5 (2.8) 15.0 (3.7) 1.8 (0.1) 1.9 (0.2) 10% hp + 3 applications 33.9 (2.4) 31.7 (2.6) 17.7 (1.2) 16.1 (1.6) 1.9 (0.1) 2.0 (0.1) 35% hp + 1 application 34.0 (7.8) 30.4 (2.8) 17.5 (5.9) 15.1 (1.7) 2.0 (0.3) 1.9 (0.1) 35% hp + 2 applications 37.2 (3.7) 31.6 (7.1) 19.1 (1.9) 16.4 (3.7) 1.9 (0.1) 1.9 (0.1) 35% hp + 3 applications 34.7 (5.1) 33.2 (4.4) 17.9 (1.7) 16.9 (2.0) 1.9 (0.1) 2.0 (0.1) 50% hp + 1 application 32.7 (1.0) 35.3 (1.9) 17.4 (0.7) 18.2 (0.7) 1.9 (0.1) 1.9 (0.1) 50% hp + 2 applications 35.6 (4.0) 33.9 (3.2) 19.1 (1.9) 19.4 (2.2) 1.9 (0.1) 1.9 (0.1) 50% hp + 3 applications 30.4 (3.9) 33.0 (2.2) 17.5 (6.3) 16.7 (1.5) 1.8 (0.3) 2.0 (0.1) table 2 –table 2 –table 2 –table 2 –table 2 – means of element content in wt% (standard deviation) in enamel surface. means followed by different letters, capital letters in rows and lowercase letters, in columns are statistically different by tukey’s test (p<0.05). means followed by asterisk (*) differ from control group by dunnett’s test (p<0.05). table 3 –table 3 –table 3 –table 3 –table 3 – concentration of calcium (µg/ml) in hydrogen peroxide (hp) gels and rinsing water from the bleached enamel surfaces. hp concentration number of applications 1 application 2 applications 3 applications 10% 142.6 (27.5) aa* 128.7 (44.8) aa* 42.3 (15.9) ba* 35% 131.8 (17.7) aa* 149.5 (25.4) aa* 29.8 (6.3) ba* 50% 120.1 (28.0) aa* 102.5 (20.8) aa* 33.6 (8.4) ba* control group 2.8 (1.6) 26 the number of applications did not decrease the integrated area of raman peaks, indicating that, up to 3 hp applications, the effects of peroxides were similar. however, bistey et al.22 reported that changes in the enamel surface were directly proportional to the hp concentration. in addition, these changes in the area of the peaks were more obvious when the duration of bleaching was 30 and 60 min. the 30-min time used in this study may not have been able to promote more alterations in enamel surface after first, second or third applications of bleaching agent regardless of the hp concentration. a previous10 has shown similar results on human enamel after using 35% hp, testing three commercially available, bleaching agents applied for 30 min. the µedxrf analysis provides information about tooth mineral content and could be used as an analytical technique to study chemical changes on enamel20. however, in the conducted study it was not possible to identify any changes in mineral phase using this method, even after three 50% hp applications. smidt et al.13 also found no significant changes in the enamel ca/p ratio after bleaching. conversely, some authors reported a reduction in the ca/p ratio12,23. rotstein et al.12 found a significant reduction in the ca/p ratio following treatment with 35% hp for 7 days. also, oltu and gurgan24 observed a significant decrease in the ca/p ratio after bleaching with 35% carbamide peroxide compared with low carbamide peroxide concentrations. in that study, the bleaching agents were applied for 8 h/day for 6 weeks. poorni et al.25 found decrease of ca/p ratio in human enamel after 30-min exposure to 35% hp. some reasons to the for variations of results among studies are due to different bleaching protocols, peroxide concentrations, type of bleaching agent and morphological differences between human and bovine enamel. the current study used flat and polished bovine enamel in order to remove the irregularities present in the buccal surface, which could interfere in the µedxrf and ft-raman analysis5,10,16. previous studies have detected that peroxide bleaching induces ca loss from dental enamel10,26. these investigations used aas in an attempt to quantify the concentration of ca lost after bleaching. this study compared three hp concentrations and found that, with the same number of applications, there were no significant differences among the hp bleaching agents regardless of using high and low concentrations. conversely, tezel et al.27 found more ca ions lost from the enamel surface after 35% and 38% hp bleaching than with 10% carbamide peroxide treatment. the authors suggested that the significant enamel ca loss was due to the high potential of concentrated hp to cause dental demineralization. the low stability of highly concentrated peroxides and the fast peroxide degradation reaction may lead to less interaction of the peroxide with the enamel structure28, causing the same alteration when using low or high concentrations, as observed in this study. al-salelhi et al.6 investigated the effect of 24 h non-stop 3%, 10% or 30% hp concentration on mineral loss and found increased ion release with increasing hp concentration. it may be speculated that the higher values of ca loss may be due to the longer exposure time (24 h versus 30 min used in the present work). the ca bonded weakly to the hydroxyapatite and it was lost mostly after the first and second peroxide applications, according to the aas method. thus, after the third application of peroxide, the ca concentration decreased significantly in the rinsing solution (hp and water). according to wang et al.29 the release of ca from the enamel apatite may occur via atomic diffusion through the apatite channels along the crystallographic c-axis and the inter-crystallites and inter-rod special voids with openings on the surface. another reason for bleaching having enhanced ca leaching could be the degradation of the enamel organic component, which removes related mineral elements. calcium removed by the hp application is the one present in the hydroxyapatite crystal, the building block of dental hard tissues. probably polishing of the enamel surface, made in the present study in order to remove irregularities from the bovine enamel surface, produced a more reactive surface and in the first and second applications of hp, independent of its concentration, a greater amount of ca was removed from the crystal. another hypothesis that could help explaining the present findings is that the mineral content of enamel changes from the surface to inside and ca concentrations were shown to decrease from the surface to the inside of the enamel30. however, further studies are required to elucidate this mineral loss due to hp application. bovine teeth were used in this study because their chemical properties and the prismatic morphology are very similar to human enamel as reported by yassen et al.31 and reis et al.32, respectively. studied have used bovine teeth as substitute for human teeth also in cosmetic dentistry and dental bleaching 6,21. however, the comparison of these findings with studies using human teeth must be careful because of the variations in bovine tooth age and the different methodologies used to prepare the samples. this study did not test commercial whitening products; the used bleaching gels were a mixture of hp and a thickener. the commercially available bleaching agents have in their composition some additives like fluoride ions, potassium nitrate, carpobol, propylene glycol, sodium fluoride, calcium and others. these additives may minimize the mineral loss and produce different results from those obtained in this study. more studies are required to investigate the role of each component in the enamel mineral loss. an important factor that must be considered is that in this study the amount of bleaching agent used was standardized (2 mm thick on the enamel surface), since many studies do not mention the amount of bleaching agent used. there are no studies in the literature correlating the amount of bleaching agent and its effects on tooth structure. different methods were used in the present study to assess the mineral content of enamel after the bleaching treatment. while µedxrf did not identify any changes in mineral content after bleaching, ft-raman and aas detected alterations in mineral components and ca loss, respectively. the analysis of mineral content of enamel after bleaching effects of various hydrogen peroxide bleaching concentrations and number of applications on enamel braz j oral sci. 13(1):22-27 27 requires the knowledge of the accuracy and limitations of each method used. the effect of dental bleaching on enamel mineral content has been frequently evaluated using edxrf, ft-raman and aas, and the findings of the present study showed that these methods are accurate for this purpose. when used separately, different results were found for each method and this should be considered when comparing studies with different methodologies. according to the ft-raman and aas methods of detection, hp bleaching causes a reduction in the mineral content of enamel. increasing the hp concentration did not increase the mineral loss. acknowledgments this study was supported by grants #01/14384-8, #05/ 50811-9, #07/54784-1 and #05/60696-2 from the são paulo state research foundation (fapesp). 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applications on enamel oral sciences n3 braz j oral sci. 12(2):90-94 original article braz j oral sci. april | june 2013 volume 12, number 2 a multiparametric assay to compare the cytotoxicity of different storage media for avulsed teeth emmanuel joão nogueira leal silva1, carolina bluzarca rollemberg2, tauby de souza coutinho-filho3, renato liess krebs3, alexandre augusto zaia1 1department of restorative dentistry, area of endodontic, piracicaba dental school, university of campinas, piracicaba, sp, brazil 2dentist, rio de janeiro, rj, brazil 3department of endodontic, state university of rio de janeiro (uerj), rio de janeiro, rj, brazil correspondence to: emmanuel joão nogueira leal silva rua herotides de oliveira 61/902 cep: 24230-230 – niterói, rj, brasil phone: +55 21 83575757 e-mail: nogueiraemmanuel@hotmail.com abstract aim: to evaluate the cytotoxicity of several storage media (coconut water, whole milk, isotonic solution, hank´s balanced salt solution and tap water) using a multiparametric cytotoxicity analysis employing 3t3 cells. methods: plates containing confluent 3t3 fibroblasts were exposed to the various media for 24h, at 37°c with 5% co 2 , and cell viability was evaluated by a multiparametric assay assessing sequentially, on the same cells, mitochondrial activity (xtt), membrane integrity (neutral red test), and total cell density (crystal violet dye exclusion test). results from each test were compared by one-way analysis of variance (anova). results: statistical analysis showed that whole milk and hbss were the most effective media in maintaining cell viability at all tested times (p<0.05). isotonic and tap water showed the highest cytotoxicity effects. conclusions: this study shows that whole milk and hbss are more efficient in maintaining the viability of 3t3 fibroblasts as demonstrated by three different cell viability tests. keywords: tooth avulsion, fibroblasts, cell viability. introduction tooth avulsion is one of the main issues in dental traumatology because it is a severe dental injury. due to the complexity of this injury, the neurovascular supply is severely compromised in most cases, causing loss of pulp vitality1. the main etiological factors are trauma after fighting and sports, as well as falls and bumps against hard objects and/or the floor2. the incidence of tooth avulsion accounts approximately 1-16% of all traumatic injuries in the permanent dentition and 7-21% in the primary dentition3-4. when dental avulsion occurs, immediate replantation at the trauma site is the ideal procedure for maintaining the viability of periodontal ligament (pdl) cells. however, immediate replantation is rarely achieved5, principally because of lack of dental knowledge from parents/tutors at the moment of the accident. the viability of pdl cells relies on three critical factors: the duration of extra-alveolar time, preservation of the root and storage media of the tooth, all of which determine the prognosis for dental replantation2-5. inflammatory resorption and replacement received for publication: january 21, 2013 accepted: may 08, 2013 9191919191 braz j oral sci. 12(2):90-94 resorption along with dental alveolar ankylosis are the most signiûcant and common complications after replantation of avulsed teeth2-3. thus, to avoid further damage do the pdl cells, it is important to select an appropriate storage medium to keep their viability. different transport media for avulsed teeth have been investigated to evaluate their capacity to preserve the vitality of pdl components and dental pulp tissue until replantation1,6-9. the ideal storage medium should preserve cell vitality, adherence and clonogenic capacity10 and must be easily accessible and available at the site of the accident11. examples are: saliva, milk, hank’s balanced salt solution (hbss), savea-tooth system (phoenix-lazerus, shartlesville, pa, usa), and viaspan (dupont phamaceuticals, wilmington, de, usa). other storage media include egg white, powdered milk, gatorade (the gatorade co., chicago, il, usa), and propolis, all of which were recently studied and tested1,5-11. the simultaneous evaluation of different cell viability parameters, in a multiparametric assay, may identify more accurately any possible cytotoxic effects of storage media with immortalized cells intimately related to the in-vivo tissue. therefore, the aim of this study was to compare the cytocompatibility of different storage media. cytocompatibility was assessed by a multiparametric assay employing 3t3 fibroblasts. material and methods cell viability in the following storage media was evaluated: long-shelf-life coconut water (taeq, fortaleza, ce, brazil), isotonic beverage (gatorade®, são paulo, sp, brazil), hank´s balanced salt solution (gibco brl, grand island, ny, usa), long-shelf-life whole milk (parmalat, são paulo, sp, brazil) and tap water. cells cultured in dulbecco’s modified eagle medium (dmem) served as positive control. the ph of all solutions was measured with a digital ph meter (hanna instruments, ann arbor, mi, usa) at room temperature. the osmolality was tested with an automatic cryoscopic osmometer (osmomat 030, gonotec, berlin, germany). fibroblast cells (lineage 3t3) were obtained from the american type culture collection and were cultured in dmem supplemented with 10% fetal bovine serum (fbs) (gibco, grand island, ny, usa), 100 µg/ml streptomycin, and 100 mg/ml penicillin at 37°c in a humidified incubator at room atmosphere containing 5% co 2 . confluent cells were detached with 0.25% trypsin and 0.05% ethylenediaminetetraacetic acid (gibco, grand island, ny, usa) for 5 min, and aliquots were sub-cultured. for the experimental set, 5 x103 cells were cultured in 96-well culture plates and allowed to achieve 80% confluence. after 24h, the medium was removed from each well and replaced by 200 µl of one of the tested storage media for 24 h. cytotoxicity was evaluated with a commercial kit (cytotox; xenometrix, germany) that measures three different cell viability parameters sequentially in the same cell culture: xtt, neutral red test (nr), and crystal violet dye exclusion test (cvde). the xtt test is based on the ability of mitochondrial enzymes from metabolically active cells to reduce 2,3-bis(2-methoxy-4–nitro-5-sulphophenyl)-2htetrazolium-5-carboxanilide (xtt) molecules to a soluble salt of formazan, detectable by its absorbance at 480 nm, as measured by a spectrophotometer (urit 660; urit, china). the same cells submitted to the xtt test were washed and assayed with the neutral red uptake test (nr), which determines the levels of viable cells by their membrane integrity. the vital dye nr is incorporated by endocytosis and accumulates preferentially on the lysosomes of intact viable cells membrane. after 3 h of exposure to the dye, cells were fixed and the nr was extracted and measured by the optical density (od) of the supernatant at 540 nm, which directly relates to the proportion of viable cells. after the nr test, fixed cells were washed and evaluated for the total density of adhered cells, as estimated by the crystal violet dye exclusion test (cvde). cvde is a simple assay that evaluates cell density by staining dna; after elimination of excess dye, the absorbance at 540 nm is proportional to the amount of cells in the well. data from the assays are presented as means ± standard deviation (sd). the results were subjected to the kolmogorov-smirnov test to evaluate the normal distribution, and data were analyzed using one-way analysis of variance (anova). statistical differences among the groups were analyzed using the tukey test at a significance level of 5%. data were analyzed using the statistical software spss® (spss, inc., chicago, il, usa). results figure 1 shows cell viability as evaluated by three different assays, after exposure to 24-h of the different storage media, expressed as a percentage of the control (cells exposed to dmem). as seen in panels a, b, and c, tap water and isotonic have higher cytotoxic effects, as measured by all three employed methods. coconut water had better results than tap water and isotonic (p < 0.05). no significant difference was found between whole milk and hbss (p < 0.05) that have the lowest cytotoxic effects. the results of ph and osmolality are expressed in table 1. discussion dental trauma is described in the literature as a common problem among children and adolescents throughout the world5,12-14. among the different types of dental trauma, material osmolality (mosmol/kg) p h coconut water 375 4.6 tap water 2 8.2 long shelf-life milk 283 6.8 gatorate 404 3.0 dmen 311 8.0 hbss 280 7.6 table 1 – ph and osmolality of different storage media a multiparametric assay to compare the cytotoxicity of different storage media for avulsed teeth braz j oral sci. 12(2):90-94 fig. 1. cytotoxic effects of storage media on 3t3 cells by xtt (a), nr (b), and crystal violet dye exclusion test (c), expressed as percentage of control (cells exposed to culture medium). bars indicate mean ± sd. (*) mean statistically significant differences between tested groups (p<0.05). avulsion leads to the greatest functional and esthetic impairment due to its worse prognosis2-5. the ideal outcome after replantation of an avulsed tooth is regeneration of the periodontal ligament, which is possible when the vitality of pdl cells is maintained. the cells that remain on the root after exarticulation are deprived of their blood supply and immediately begin to deplete their stored cell metabolites. hammer15, demonstrated that the length of survival of a replanted tooth is directly related to the amount of viable periodontal ligament and the major factors that may influence the viability of the periodontal ligament fibroblasts are the extra-alveolar time and the storage medium2-5,8,16. therefore, many studies aimed to preserve avulsed tooth in a proper medium 1,6-11,16. the storage medium should be able of preserving cell vitality and adherence capacity and also be readily available at the moment of avulsion to allow rapid access to it17-18. in previous studies, various techniques were used to quantify the number of viable pdl cells including the trypan blue assay1,7-8,10,16, xtt assay6 and mtt assay9. in the present study, storage media cytotoxicity was tested employing a methodological strategy that differs from most previous works on these materials by employing a multiparametric assay with three different cell viability tests. in this manner, three different parameters were evaluated on the same sample: (1) mitochondrial metabolism and respiratory toxicity, (2) liposomal integrity and membrane permeability and (3) presence of dna and cell proliferation. this method increases the chance of detection of cytotoxic effects, allows correlation of different parameters, and sometimes provides hints about the mechanisms of toxicity19-20. a 3t3 fibroblast cell line was used in this study for its easy preparation and handling. 9292929292 a multiparametric assay to compare the cytotoxicity of different storage media for avulsed teeth 9393939393 braz j oral sci. 12(2):90-94 studies with established cell lines are used because of the reproducibility of the results and besides they multiply rapidly with an unlimited life span21-22. fibroblasts are major constituents of connective tissue and the predominant cell type of periodontal ligament. many materials have been evaluated for use as storage media. hbss is a widely used standard solution recommended by the international association of dental traumatology as a storage medium for avulsed teeth5. in this study, hbss, tap water, milk, coconut water and isotonic solution were tested. hbss and long shelf-life milk showed the best results when compared with the other tested solutions (p<0.05). hbss is a standard saline solution, which is widely used in biomedical research to support the growth of many cell types23-24. a commercially available tooth preserving system utilizing hbss as a storage medium, save-a-tooth, has become available for storage of avulsed teeth until replantation. an added advantage of this system is an inner suspension net and a removable basket which permits general washing and removal of the tooth, without crushing the periodontal ligament by the dentist25. in the present study, hbss was used simulating the save-a-tooth system. although hbss showed excellent results in the multiparametric assay, it is not easily available in pharmacies or drug stores at the site of an accident. therefore, milk is known as the appropriate storage medium for avulsed tooth for its physiological properties, including ph and osmolality compatible to those of the cells from the pdl; the easy availability, the advantage of not requiring refrigeration and for being bacteria-free6,8,10-11,18. the favorable results of milk probably occur due to the presence of nutritional substances, such as amino acids, carbohydrates and vitamins6,10-11,17-18,25-26. coconut water showed better results when compared with gatorade® and tap water (p<0.05). coconut water is biologically pure and sterile, with a rich presence of amino acids, proteins, vitamins and minerals8,25. the acidic ph (4.6) could be deleterious to cell metabolism, explaining the intermediate results. among the studied storage media, tap water and gatorade® showed the worst results. although gatorade® could be a potential storage media because it is commonly found at sports events, it did not turn out to be an adequate storage media for avulsed teeth due its acid ph (3.0). when the cells are exposed to gatorade®, it is possible that their membrane gets damaged because of this low ph, which turns impossible cell growth18. the hypertonic osmolality (404.0) also can make the cells lose water. different explanation could be given to tap water, as it causes rapid cellular lyses of pdl due to its hypotonic osmolality (2.0). according to the results of this study, it could be concluded that milk and hbss have the best results using the multiparametric assay, corroborating their use in cases of tooth avulsion. references 1. martin mp, pileggi r. a quantitative analysis of propolis: a promising new storage media following avulsion. dent traumatol. 2004; 20: 85-9. 2. trope m. avulsion of permanent teeth: theory to practice. dent traumatol. 2011; 27: 281-94. 3. soares aj, gomes bp, zaia aa, ferraz cc, de souza-filho fj. relationship between clinical-radiographic evaluation and outcome of teeth replantation. dent traumatol. 2008; 24: 183-8. 4. andreasen jo, borum mk, jacobsen hl, andreasen fm. replantation of 400 avulsed permanent incisors 1. diagnosis of healing complications. endod dent traumatol. 1995; 11: 51-8. 5. flores mt, andersson l, andreasen jo, bakland lk, malmgren b, barnett f et al. international association of dental traumatology. guidelines for the management of traumatic dental injuries. ii. avulsion of permanent teeth. dent traumatol. 2007; 23: 130-6. 6. gjertsen aw, stothz ka, neiva kg, pileggi r. effect of propolis on proliferation and apoptosis of periodontal ligament fibroblasts. oral surg oral med oral pathol oral radiol endod. 2011; 112: 843-8. 7. moazami f, mirhadi h, geramizadeh b, sahebi s. comparison of soymilk, powdered milk, hank´s balanced salt solution and tap water on periodontal ligament cell survival. dent traumatol. 2012; 28: 132-5. 8. gopikrishna v, baweja ps, venkateshbabu n, thomas t, kandaswamy d. comparison of coconut water, propolis, hbss, and milk on pdl cell survival. j endod. 2008; 34: 587–9. 9. hwang jy, choi sc, park jh, kang sw. the use of green tea extract as a storage medium for the avulsed tooth. j endod. 2011; 37: 962-7. 10. ashkenazi m, marouni m, sarnat h. in vitro viability, mitogenicity and clonogenic capacity of periodontal ligament cells after storage in four media at room temperature. endod dent traumatol. 2000;16: 63-70. 11. huang sc, remeikis na, daniel jc. effects of long-term exposure of human periodontal ligament cells to milk and other solutions. j endod. 1996; 22: 30-3. 12. bastone eb, freer tj, mcnamara jr. epidemiology of dental trauma: a review of the literature. aust dent j. 2000 ;45: 2-9. 13. piovesan c, guedes rs, casagrande l, ardenghi tm. socioeconomic and clinical factors associated with traumatic dental injuries in brazilian preschool children. braz oral res. 2012; 26: 464-70. 14. correa mb, schuch hs, collares k, torriani dd, hallal pc, demarco ff. survey on the occurrence of dental trauma and preventive strategies among brazilian professional soccer players. j appl oral sci. 2010; 18: 572-6. 15. hammer h. replantation and implantation of teeth. int dent j. 1955; 5: 439-57. 16. khademi aa, saei s, mohajeri mr, et al. a new storage medium for an avulsed tooth. j contemp dent pract. 2008; 9: 25-32. 17. santos cl, sonoda ck, poi wr, panzarini sr, sundefeld ml, negri mr. delayed replantation of rat teeth after use of reconstituted powdered milk as a storage medium. dent traumatol. 2009; 25: 51-7. 18. chamorro mm, regan jd, opperman la, kramer pr. effect of storage media on human periodontal ligament cell apoptosis. dent traumatol. 2008; 24: 11-6. 19. scelza mz, linhares ab, da silva le, granjeiro jm, alves gg. a multiparametric assay to compare the cytotoxicity of endodontic sealers with primary human osteoblasts. int endod j. 2012; 45: 12-18. 20. de-deus g, canabarro a, alves g, linhares a, senne mi, granjeiro jm. optimal citocompatibility of a bioceramic nanoparticulate cement in primary human mesenchymal cells. j endod. 2009; 35: 1387-90. 21. loushine ba, bryan te, looney sw, gillen bm, loushine rj, weller rn et al. setting properties and cytotoxicity evaluation of a premixed bioceramic root canal sealer. j endod. 2011; 37: 673-7. 22. bouillaguet s, wataha jc, tay fr, brackett mg, lockwood pe. initial in vitro biological response to contemporary endodontic sealers. j endod. 2006; 32: 989-92. 23. casaroto ar, hidalgo mm, sell am, franco sl, cuman rk, moreschi e et al. study of the effectiveness of propolis extract as a storage medium for avulsed teeth. dent traumatol. 2010; 26: 323-31. 24. krasner p, person p. preserving avulsed teeth for replantation. j am dent assoc. 1992; 23: 80-8. a multiparametric assay to compare the cytotoxicity of different storage media for avulsed teeth 9494949494 braz j oral sci. 12(2):90-94 25. thomas t, gopikrishna v, kandaswamy d. comparative evaluation of maintenance of cell viability of an experimental transport media “coconut water” with hank’s balanced salt solution and milk for transportation of an avulsed tooth: an in vitro cell culture study. j conserv dent. 2008; 11: 22-9. 26. marino tg, west la, liewehr fr, mailhot jm, buxton tb, runner rr, et al. determination of periodontal ligament cell viability in long shelf-life milk. j endod. 2000; 26: 699-702. a multiparametric assay to compare the cytotoxicity of different storage media for avulsed teeth oral sciences n3 braz j oral sci. 13(2):89-92 original article braz j oral sci. april | june 2014 volume 13, number 2 bacterial microleakage at the implant-abutment interface in morse taper implants joão paulo da silva-neto1, marina de freitas fratari majadas2, marcel santana prudente2, thiago de almeida prado naves carneiro2, mario paulo amante penatti3, flávio domingues das neves2 1universidade federal do rio grande do norte ufrn, school of dentistry, department of prosthodontics, natal, rn, brazil 2universidade federal de uberlândia ufu, school of dentistry, department of occlusion, fixed prostheses, and dental materials, uberlândia, mg, brazil 3universidade federal de uberlândia ufu, technical school of health, uberlândia, mg, brazil correspondence to: joão paulo da silva neto departamento de prótese faculdade de odontologia universidade federal do rio grande do norte avenida sen. salgado filho 1787 cep: 59056-000 lagoa nova, natal, rn, brasil phone: + 55 84 32153883 e-mail: joaopaulosneto@gmail.com abstract aim: to evaluate the microleakage at the implant-abutment (i-a) interface of morse tapered implants inoculated with different volumes of bacterial suspension. methods: morse tapered ia sets were selected and divided in two groups depending on the type of abutment: passing screw (ps) and solid (s), and then subdivided into four subgroups (n=6) according to the suspension volume: ps1: 0.1 µl; ps3: 0.3 µl; ps5: 0.5 µl; ps7: 0.7 µl; s1: 0.1 µl; s3: 0.3 µl; s5: 0.5 µl and s7: 0.7 µl. a control test was performed to verify the presence of external contamination during the inoculation and the implants were incubated for microbiological analysis. the microleakage was evaluated every 24 h for 7 days by the clarity of solution. after this period, the implants were disassembled for confirmation of bacterial viability. results: all the specimens with 0.7 µl and one sample of s5 presented turbidity in the control test indicating external contamination, and were excluded from the study. after 7 days of observation, none of the specimens presented positive results for microleakage and the bacterial viability was confirmed in all specimens. the 0.1 µl and 0.3 µl volumes did not present bacterial microleakage, meaning that these volumes may be inadequate for analysis. conclusions: none of the sets evaluated showed bacterial microleakage at the i-a interface and the volume of 0.7 µl exceeded the internal capacity of the implants. keywords: dental implants; dental abutments; microbiology. introduction failures in implant therapy have been associated with lack of stability or misfit at the implant-abutment (i-a) interface1. two-piece implants have a microgap depending on the interface type or system, but presence of fluid flow at this interface and its relationships are very variable2-18. this has been correlated to the presence of bacterial infiltration and inflammatory cells that may lead to bone loss around this area19. implant manufacturers try to reduce bacterial infiltration by increasing the accuracy and stability of the jointed pieces by fabricating very high precision mechanical parts 7. internal conical joints have greater mechanical stability of the i-a interface and have shown no crest bone loss20-21. this has been explained by the stress distribution of the implant’s long axis during function. the greater stability of the soft tissues provided by the tapered abutment design and its reduced diameter relative to the platform and absence of abutment micromobility by the self-locking feature reduces bacterial leakage at the interface or even prevents it3-7. received for publication: february 20, 2014 accepted: march 28, 2014 90 braz j oral sci. 13(2):89-92 in vitro bacterial studies at the i-a interface of morse taper implants have attempted to assess the actual advantage of mechanical locking of these joints, but variable results were found 2-8,18. these variations may be related to methodological bias in the tests such as: holding implants with forceps; hand inoculation of the bacterial suspension into the implants; using one single torquemeter for all samples; failure to measure the inner volume of the implant; using a higher amount of bacterial suspension than should be used; type of bacteria and its viability within the study conditions, and the sterile technique used when studying the inward flow from the outer part of the implant15. different volumes of bacterial suspension for implants inoculation could minimize the variations found in the literature. therefore, the objective of this study was to assess the microleakage at the i-a interface of morse tapered implants with different abutments and to verify the influence of different volumes of bacterial suspension. material and methods forty-eight morse taper implants (3.75 x 11.5 mm neodent®, curitiba, pr, brazil) and 24 passing screw abutment (ps) and solid abutments (s) (4.5x6x1.5 mm; 11.5º neodent®, curitiba, pr, brazil) were selected and assigned to two groups according to the type of abutment (ps and s). the assemblies of both groups were further subdivided into four subgroups according to the amount of bacterial suspension inoculated into the inner part of the implants (n=6). ps1: 0.1 µl; ps3: 0.3 µl; ps5: 0.5 µl; ps7: 0.7 µl; s1: 0.1 µl; s3: 0.3 µl; s5: 0.5 µl and s7: 0.7 µl (table 1). escherichia coli strain atcc 35218 was selected for bacterial microleakage evaluation because it is a facultative anaerobic bacterium, able to survive in adverse situations and has a good motility, and has been used in other in vitro tests2,5,7,13-14. this bacterium was cultivated in brain heart infusion broth – bhi (biolife, milano, italy) for 24 h in an incubator at 37 °c (biomatic, porto alegre, rs, brazil). all the used instruments were autoclaved at 121 oc and 15 psi for 15 min. all the procedures and necessary materials were conducted under the clean conditions of a laminar flow hood (veco, campinas, sp, brazil). a standard bacterial suspension dilution of 0.5 mcfarland (108 colony forming unit/ml – cfu/ml) was used for minimizing the environmental adversity inside of the implants. the implants were set on a holder and an automatic pipette (0.1-20 µl, labmate+ ht; pz htl s.a, warsaw, poland) was fixed to the vertical shaft of a prosthetic surveyor (bioart, são carlos, sp, brazil.)14. the pipette tip was inserted and loaded with the amount that was required for each experimental group. after the inner part of the implant had been inoculated, the abutment was opened and carefully installed using sterile pliers. it was then tightened according to the manufacturer’s recommendations by a manual torquemeter individually calibrated for each sample. control test.control test.control test.control test.control test. all assemblies were immersed for 30 s in tubes containing sterile bhi broth until the i-a interface was completely coated. this test determined if there was any external contamination during the abutment installation and screw tightening process. the tubes of the control test were maintained in an incubator at 37 °c for up to 7 days in order to evaluate every 24 h the changes in the broth turbidity. a turbid broth indicated bacterial leakage during inoculation and/or abutment installation and that sample was excluded. for negative control, the abutment was not connected to the implant and was not subjected to bacterial suspension and showed no turbidity. for positive control, the abutment was not connected to a fixture, but was subjected to the same bacterial culture as the groups and presented turbidity. after the implants immersion in the control tubes, all the i-a samples were kept in microcentrifuge tubes (eppendorf, hamburg, germany) with another sterile bhi broth until reaching the i-a interface coating. the i-a samples were also evaluated every 24 h by the same method of the control test. after 7 days, if no bacterial growth was observed, the i-a samples were opened in the laminar flow hood and the disassembled components were placed in a new tube with sterile bhi to assess the viability of the bacteria by turbidity. if the bacteria were not viable after this period, the sample would be excluded to prevent a false negative result. results to validate the colonization and detection techniques, abutments similar to those in ps and s were kept disassembled and either exposed to bacterial culture or maintained sterile. zero cfus of e. coli were detected from sampling of abutments that were maintained sterile (negative control) (figure 1a), whereas 10 mcfarland (30 x 108 cfu/ml) of e. coli were detected in samples from abutments exposed to bacterial culture (positive control) (figure 1b). after the first 24 h of follow up one specimen of the control groups s5 and all the ps7 and s7 samples presented turbidity of the solution, which indicates bacterial contamination and they were excluded from the study. during the period of 7-day follow up, none of the other specimens presented positive results for bacterial microleakage (table 2) (figure 1c). the viability test using the disassembled components of i-a samples confirmed that e. coli survived inside the implants. groups n volume (µµµµµl) abutment recommended tightening torque mtps 6 for 0.1 0.3 0.5 0.7 passive 15 ncm each s c r e w mts volume 0.1 0.3 0.5 0.7 solid 32 ncm table 1. table 1. table 1. table 1. table 1. features of the groups tested. bacterial microleakage at the implant-abutment interface in morse taper implants 91 braz j oral sci. 13(2):89-92 groups 1 3 5 ps s table 2. table 2. table 2. table 2. table 2. results fig. 1. a. negative control; b. positive control; c. negative results for bacterial microleakage during the 7 days period. discussion all the specimens inoculated with 0.7 µl presented turbidity of the solution in the control test after the first 24 h, which indicated bacterial contamination. this showed that during the installation of the abutments, the volume of inoculation exceeded the internal capacity of the implants. only one specimen of the s5 group presented positive results in the control test, but it may be due to problems in the experimental step rather than an inability to accommodate the volume in the inner parts of the implants. these results showed that for the studied system, the maximum volume is around 0.5 µl. other studies evaluating morse tapered systems used 0.1 µl to 0.5 µl of volume inoculation2,4-5,7. however, only two studies reported the evaluation of the best inner volume for the studied system and used the same volume for different conical systems4-5. the other studies did not find any difference among the internal volumes of the systems2,7. the groups inoculated with 0.7 µl were excluded due to the positive results of the control test. for the other groups, after the period of 7-day follow up, none of the evaluated specimens showed positive results for bacterial microleakage at the i-a interface. these results corroborate studies under static conditions where morse tapered implants showed reduced levels of bacterial flow in the i-a interface 4,6-7, although there was no consensus5-6. alterations in the shape of the abutments may also have harmed the mechanical imbrication of the conical interfaces allowing greater bacterial flow in the interface3,6, which was not observed in this study. the use of reduced volumes of bacterial suspension for inoculation, with high concentrations (superior to 1.5 x 108 cfu/ml) and longer periods of follow up, over 7 days, may create an extremely adverse environment for bacterial survival inside the implants, reducing significantly the possibility of microleakage, because of the bacterial competition. these volumes may also lead to low levels of bacterial suspension in relation to the i-a interface, hindering the microleakage15 and showing negative results for microleakage as in previous studies7,16. these results reinforce the theory that high concentrations of bacteria and reduced volumes may mask the microleakage14-15. moreover, it may lead to false negative results caused by bacterial death inside the implants. in order to avoid this, beyond the precautions on the concentration of bacterial suspension, bacterial viability was confirmed after the follow up period by disassembling the specimens that presented negative results for microleakage. precaution involving the concentration of bacterial suspensions is already a reality;4,5,14 but confirmation of bacterial viability was made only in two studies5,14. the chosen post-inoculation control test was immersion of the implants up to the i-a interface coating for 30 s. the overflow control by immersion was used in a previous study and showed good results 9. currently, in vitro tests for microleakage with corpuscular bacteria showed no flow at the i-a interface, even in hexagonal joints, contradicting what was previously thought9,14. even though these findings may be different in situations of mechanical load, inner joints are more stable and could be more beneficial, but few studies compared this situation3,13. moreover, tests using dyes and bacterial toxins of molecular sizes, which are the real cause of marginal bone crest resorption, showed that microleakage occurs even in morse tapered joints regarded as hermetically stable, and even in absence of load, its flow increases with time8,11. therefore, more studies are required using bacteria, dyes and bacterial toxins under load conditions. these studies will guide the understanding about the flow that occurs at this interface and its relation to the microgap. this may solve clinical problems related to this process, mainly during the first years in function. the present study verified that despite the fact that tests for bacterial viability showed that the volumes of 0.1 and 0.3 µl allowed the survival of bacteria during the period of 7 days, it may be insufficient to evaluate the microleakage at the i-a interface, since it did not occupy the maximum capacity of the inner parts of the implant. the 0.5 µl volume showed that it could be used for in vitro tests using morse tapered implants with different abutments (ps and s), without overflow of the volume after abutment installation. the need of determining and using the closest volume of the inner parts of the implants and controlling the bias related to the methodology should be underlined in order to avoid false positive or negative results. within the limitations of this study, it may be concluded that the volume of 0.7 exceeds the inner capacity and should not be used. none of the other evaluated specimens showed bacterial microleakage at the i-a interface. however, the volumes of 0.1 and 0.3 may be insufficient for future tests. bacterial microleakage at the implant-abutment interface in morse taper implants 92 braz j oral sci. 13(2):89-92 this suggests that the 0.5 µl volume seems to be closer to ideal for the studied groups. acknowledgements this research was supported by capes: coordination for the improvement of higher education personnel, brazil. the authors would like to thank neodent implante osteointegrável for the donation of implants and abutments and the microbiology laboratory of the technical school of health – federal university of uberlândia. references 1. goodacre cj, bernal g, rungcharassaeng k, kan jy. clinical complications with implants and implant prostheses. j prosthet dent. 2003; 90: 121-32. 2. jansen vk, conrads g, richter ej. microbial leakage and marginal fit of the implant-abutment interface. int j oral maxillofac implants. 1997; 12: 52740. 3. koutouzis t, wallet s, calderon n, lundgren t. bacterial colonization of the implant-abutment interface using an in vitro dynamic loading model. j periodontol. 2011; 82: 613-8. 4. aloise jp, curcio r, laporta mz, rossi l, da silva am, rapoport a. microbial leakage through the implant-abutment interface of morse taper implants in vitro. clin oral implants res. 2010; 21: 328-35. 5. deconto ma, salvoni ad, wassall t. in vitro microbiological bacterial seal analysis of the implant/abutment connection in morse taper implants: a comparative study between 2 abutments. implant dent. 2010; 19: 158-66. 6. tesmer m, wallet s, koutouzis t, lundgren t. bacterial colonization of the dental implant fixture-abutment interface: an in vitro study. j periodontol. 2009; 80: 1991-7. 7. dibart s, warbington m, su mf, skobe z. in vitro evaluation of the implantabutment bacterial seal: the locking taper system. int j oral maxillofac implants. 2005; 20: 732-7. 8. harder s, dimaczek b, acil y, terheyden h, freitag-wolf s, kern m. molecular leakage at implant-abutment connection—in vitro investigation of tightness of internal conical implant-abutment connections against endotoxin penetration. clin oral investig. 2010; 14: 427-32. 9. do nascimento c, pedrazzi v, miani pk, moreira ld, de albuquerque rf, jr. influence of repeated screw tightening on bacterial leakage along the implant-abutment interface. clin oral implants res. 2009; 20: 1394-7. 10. do nascimento c, miani pk, pedrazzi v, gonçalves rb, ribeiro rf, faria ac, et al. leakage of saliva through the implant-abutment interface: in vitro evaluation of three different implant connections under unloaded and loaded conditions. int j oral maxillofac implants. 2012; 27: 551-60. 11. coelho pg, sudack p, suzuki m, kurtz ks, romanos ge, silva nr. in vitro evaluation of the implant abutment connection sealing capability of different implant systems. j oral rehabil. 2008; 35: 917-24. 12. jaworski me, melo ac, picheth cm, sartori ia. analysis of the bacterial seal at the implant-abutment interface in external-hexagon and morse taperconnection implants: an in vitro study using a new methodology. int j oral maxillofac implants. 2012; 27: 1091-5. 13. steinebrunner l, wolfart s, bossmann k, kern m. in vitro evaluation of bacterial leakage along the implant-abutment interface of different implant systems. int j oral maxillofac implants. 2005; 20: 875-81. 14. silva-neto jp, prudente ms, carneiro t de a, nobilo ma, penatti mp, neves fd. micro-leakage at the implant-abutment interface with different tightening torques in vitro. j appl oral sci. 2012; 20: 581-7. 15. silva-neto jp, nobilo ma, penatti mp, simamoto pc, jr., neves fd. influence of methodologic aspects on the results of implant-abutment interface microleakage tests: a critical review of in vitro studies. int j oral maxillofac implants. 2012; 27: 793-800. 16. besimo ce, guindy js, lewetag d, meyer j. prevention of bacterial leakage into and from prefabricated screw-retained crowns on implants in vitro. int j oral maxillofac implants. 1999; 14: 654-60. 17. do nascimento c, miani pk, pedrazzi v, muller k, de albuquerque rf jr. bacterial leakage along the implant-abutment interface: culture and dna checkerboard hybridization analyses. clin oral implants res.. 2012; 23: 1168-72. 18. gross m, abramovich i, weiss ei. microleakage at the abutment-implant interface of osseointegrated implants: a comparative study. int j oral maxillofac implants. 1999; 14: 94-100. 19. broggini n, mcmanus lm, hermann js, medina r, schenk rk, buser d, et al. peri-implant inflammation defined by the implant-abutment interface. j dent res. 2006; 85: 473-8. 20. bozkaya d, muftu s. mechanics of the taper integrated screwed-in (tis) abutments used in dental implants. j biomech. 2005; 38: 87-97. 21. donovan r, fetner a, koutouzis t, lundgren t. crestal bone changes around implants with reduced abutment diameter placed non-submerged and at subcrestal positions: a 1-year radiographic evaluation. j periodontol. 2010; 81: 428-34. bacterial microleakage at the implant-abutment interface in morse taper implants oral sciences n3 case report braz j oral sci. july | september 2013 volume 12, number 3 unusual yet isolated oral manifestations of persistent thrombocytopenia – a rare case report amit byatnal1, neha mahajan2, shrinivas koppal1, a ravikiran3, thriveni r1, parvathi devi m k1 1department of oral medicine & radiology, ames dental college and hospital, raichur, karanataka 2department of oral medicine & radiology, private consultant shimla, himachal pradesh 3department of oral medicine and radiology, sibar institute of dental sciences, guntur, andhra pradesh correspondence to: amit byatnal department of oral medicine & radiology, ames dental college and hospital, raichur, karnataka 584103 mobile phone: +91 80 88086642 e-mail: amitbyatnal@gmail.com abstract patients with platelet-mediated disorders often present clinical manifestations of bruising and bleeding. although these changes are detected most frequently on the skin, the oral cavity also may exhibit signs of bleeding. in this report, we describe a patient who presented isolated oral features of hemorrhagic bullae with bleeding, indicative of a bleeding disorder. results of laboratory tests revealed severe thrombocytopenia and a careful history disclosed dengue fever as the cause. the importance of recognizing the oral manifestations of thrombocytopenia is highlighted here, since the oral cavity is a frequent site of hemorrhage and could be the only manifestation of the disease. keywords: thrombocytopenia, platelet disorder, dengue fever. introduction platelets or thrombocytes are a critical component of vascular ‘plugs’ that form during hemostasis to limit blood loss secondary to vascular damage. consequently, disorders accompanied by insufficiencies of platelet number or platelet dysfunction may contribute to pathologic bleeding states1. a platelet count of 150-450x103/ mm3 is considered normal2. thrombocytopenia is a hematologic disorder that is characterized by a markedly decreased number of circulating blood platelets. a reduction in platelet number or function can occur through a variety of mechanisms, including autoimmune destruction, spleen sequestration, bone marrow infiltration by tumor cells, infection (e.g. dengue fever), and adverse drug reaction. regardless of the cause, platelet disorders typically manifest with petechiae, purpura, and bleeding of the mucous membranes3. although these changes are detected most frequently on the skin; the oral cavity, the nasal and genital mucosa, as well as the renal and gastrointestinal systems, also may exhibit signs of bleeding4. spontaneous clinical hemorrhages are usually not observed until platelet counts fall below 30x103/mm3. gingival bleeding, either spontaneous or in response to minor trauma (i.e. tooth brushing, flossing), is often the first sign of thrombocytopenia. the oral mucosa, most notably the soft palate and buccal mucosa, may demonstrate petechiae and ecchymoses. deep red to black hemorrhagic bullae may occur with very low platelet counts3. in case of dengue viral fever, mucocutaneous manifestations play significant received for publication: december 10, 2013 accepted: april 12, 2013 braz j oral sci. 12(3):233-236 role in its diagnosis. though most of these manifestations are noted on ocular mucosa like conjunctiva, sclera, there have been reports of dengue fever presenting as small vesicles on soft palate, erythema and crusting of lips and tongue5. here we report a case of thrombocytopenia associated with dengue fever, which showed only oral manifestations, thus highlighting the significance of recognizing the oral signs. case report a 50-year-old female patient reported to the department of oral medicine and radiology with a history of spontaneous and profuse bleeding of tongue for the last six months. patient had suffered from dengue fever six months before, for which she had undergone blood transfusion, as per her previous medical reports. patient was married for the past 20 years and had 2 children. her family history and review of systems also did not reveal any significant findings. intraoral examination revealed the presence of multiple hemorrhagic bullae on left sublingual mucous membrane as well as left lateral surface of the tongue and floor of the mouth (figure 1). overlying surface was blue-black in color, and profuse bleeding occurred on slight palpation. right side of palate and right posterior buccal mucosa revealed the presence of brown color plaques with rough surface. these lesions also showed bleeding on touch (figures 2 and 3). based on the clinical examination, hematological disorder due to dengue fever was chiefly considered, while differential diagnoses included oral squamous cell carcinoma of tongue and buccal mucosa and other infections like influenza, measles, rubella and bacterial sepsis. hematological examinations revealed platelet level less than 40,000/mm3, along with decrease in level of hemoglobin, red blood cells count, packed cell volume and erythrocyte sedimentation rate. based on the history of dengue fever with the presence of oral manifestations of hemorrhagic bullae and a laboratory report confirming decreased platelet count, a final diagnosis of persistent thrombocytopenia after dengue fever was given. the patient was referred to general medicine department for further systemic treatment. but we lost the patient to follow up as she succumbed to her illness. fig. 1 multiple hemorrhagic bullae on left sublingual mucous membrane, left lateral border of tongue and floor of the mouth discussion dengue is probably the most imperative mosquito-borne viral disease in the world, in terms of morbidity and mortality. it is caused by dengue virus that belongs to flaviviridae group, while the vector is aedes aegypti mosquito that once infected remains infected for life and is able to transmit to susceptible individuals. dengue affects tropical and subtropical regions very commonly6. co-circulation of the four different types of dengue viruses and expansion of dengue epidemic has given rise to infection enhancement and a big expansion of clinical aspects of the disease7. dengue virus infections cause a spectrum of illness ranging from asymptomatic, mild undifferentiated fever to classical dengue fever (df), and dengue fever with fig. 2: brown plaques on the right palatal surface fig. 3: brown plaques on the right posterior buccal mucosa unusual yet isolated oral manifestations of persistent thrombocytopenia – a rare case report234234234234234 braz j oral sci. 12(3):233-236 hemorrhagic manifestations, or dengue hemorrhagic fever (dhf) and the dengue shock syndrome (dss). each year an estimated 100 million cases of df occur and between 250,000 and 500,000 cases of dhf are reported to who6. the southeast asian countries such as india, indonesia, myanmar and thailand are at the highest risk of df/dhf, accounting for nearly half the global risk. criteria for diagnosis of dengue hemorrhagic fever include a probable or confirmed case of dengue infection while hemorrhagic tendencies are evidenced by one or more of the following: a positive result from tourniquet test; petechiae, ecchymoses or purpura; bleeding from mucosa, gastrointestinal tract, injection sites; hematemesis or melena; and thrombocytopenia (<100,000/mm3) with an evidence of plasma leakage due to increased vascular permeability8. in the present case, history of previously confirmed diagnosis of dengue infection along with oral mucosal lesions exhibiting bleeding tendency, in association with platelet count <40000/mm3 pointed toward dengue hemorrhagic fever. as there were no other findings to fulfill the criteria of dhf, it is significant to consider dengue fever and thrombocytopenia as one of the prime diagnosis based on oral manifestations. thrombocytopenia and elevated transaminases have been observed in patients with classic dengue fever9. cases of hemorrhagic bullae and other oral manifestations of thrombocytopenia have been reported10-12. bleeding in dengue is one of the dreaded complications and is associated with higher mortality in dengue dhf/dss. bleeding manifestations are highly variable and do not always correlate with the laboratory abnormalities in the coagulation profile. factors like mild degree of disseminated intravascular coagulation (dic), hepatic derangement and thrombocytopenia act synergistically to cause bleeding in dengue patient 13. severe bleeding is related to severe thrombocytopenia14. hemorrhagic manifestations include skin hemorrhages in the form of petechiae, purpurae, ecchymoses; gingival bleeding, nasal bleeding, gastrointestinal bleeding in form of hematemesis, melena, hematochezia; hematuria and increased menstrual flow. up to half of patients with dengue fever develop a characteristic rash, which is variable and may be maculopapular or macular, and, petechiae and purpura develop as hemorrhagic manifestations, commonly at venepuncture sites. an aberrant immune over activation after dengue virus infection not only impairs the immune response to clear the virus, but also results in overproduction of cytokines that affect monocytes, endothelial cells and hepatocytes, as well as the abnormal production of autoantibodies to platelets and endothelial cells. a molecular mimicry occurs between platelets or endothelial cells and dengue virus antigens. dengue virus-induced vasculopathy and coagulopathy are involved in the pathogenesis of hemorrhage15. vaughn et al.16 demonstrated the correlation of virus serotype with disease severity in pediatric patients, but there are no available data regarding this correlation in the adult population. differential diagnosis of acquired thrombocytopenia includes disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, drug induced post-transfusion purpura and idiopathic or immune mediated thrombocytopenic purpura (itp)12. dengue fever is usually a self-limited illness with no specific currently available antiviral treatment. the world health organization (who) has provided guidelines for treatment of dengue fever/dengue hemorrhagic fever, which included supportive care with analgesics, fluid replacement and bed rest. however, in the present case the patient unfortunately succumbed to her illness before the treatment could be initiated. the two clinical tests used to evaluate primary hemostasis are the platelet count and bleeding time (bt). as platelet count reduces in patients with thrombocytopenia, bleeding time increases. bt is usually considered to be normal between 1 and 6 minutes (by modified ivy’s test) and is prolonged when greater than 15 minutes. prothrombin time (pt) or international normalized ratio (inr) and activated partial thromboplastin time (aptt) remains normal17. patients with platelet counts greater than 30x103/mm3 require no treatment unless they are about to undergo a surgical procedure. first-line therapy consists of oral corticosteroids 1 mg/kg body weight. second-line therapy involves splenectomy, to which the majority of patients respond. other treatments include high-dose intravenous (i.v.) igg, i.v. anti-d, vinca alkaloids, danazol and immunosuppressive agents such as azathioprine, cyclosporine and dapsone. there is also interest in the use of specific monoclonal antibodies such as rituximab, as well as recombinant thrombopoietin. platelet transfusions are reserved for intracranial or other extreme hemorrhage, where emergency splenectomy may be justified. transfusion of 6 to 10 units of platelets will increase patient platelet counts by 17 to 31x103/mm3, respectively. with advances in apheresis technology, a single donor in one apheresis sitting can produce 6 to 10 units of leukocytereduced platelets (3 to 8x1011 platelets). as a consequence, single-donor infusions can be used in place of platelets pooled from multiple donors. the advantages of this practice are reduced donor exposure, lower incidence of alloimmunization and lower refractoriness to platelet transfusions18. dengue should hence be suspected in patients with hematological disorders and autoimmune features in endemic regions7. it is also important to recognize the oral manifestations of thrombocytopenia since the oral cavity is a frequent site of hemorrhage and may be the only manifestation of the disease. proper identification permits the prompt institution of treatment and prevention of serious complications. references 1. cleveland db, rinaggio j. oral and maxillofacial manifestations of systemic and generalized disease. endodontic topics. 2003; 4: 69-90. 2. nicki rc, brian rw, stuart hr, editors. davidson’s principles and practice of medicine. 21th ed. churchill living stone elsevier; 2010. 3. schlosser bj, pirigyi m, mirowski gw. oral manifestations of hematologic and nutritional diseases. otolaryngol clin n am. 2011;44: 183-203. unusual yet isolated oral manifestations of persistent thrombocytopenia – a rare case report 235235235235235 braz j oral sci. 12(3):233-236 4. eisen d, essell j. drug-induced thrombocytopenia presenting with isolated oral lesions: report of two cases. cutis. 1998; 62: 193-5. 5. thomas ea, john m, kanish b. mucocutaneous manifestations of dengue fever. indian j dermatol. 2010; 55: 79-85. 6. world health organization: dengue and dengue haemorrhagic fever. factsheet n.117. genebra: who; 2002. 7. jardim dl, tsukumo dm, angerami rn, carvalho filho ma, saad mj. autoimmune features caused by dengue fever: a case report. braz j infect dis. 2012; 16: 92-5. 8. chuansumrit a, tangnararatchakit k. pathophysiology and management of dengue haemorrhagic fever. transfus altern transfus med. 2006; 8: 3-11. 9. dengue: profile of haematological and biochemical dynamics. rev bras hematol hemoter. 2012; 34: 36-41. 10. eisen d, essell j. drug-induced thrombocytopenia presenting with isolated oral lesions: report of two cases. cutis. 1998; 62: 193-5. 11. james wd, guiry cc, grote wr. acute idiopathic thrombocytopenic purpura. oral surg oral med oral pathol. 1984; 57: 149-51. 12. helms ae, schaffer ri. idiopathic thrombocytopenic purpura with black oral mucosal lesions. cutis. 2007; 79: 456-8. 13. shivbalan s, anandnathan k, balasubramanian s, datta m, amalraj e. predictors of spontaneous bleeding in dengue. indian j pediatr. 2004; 71: 33-6. 14. chairulfatah a, setiabudi d, agoes r, colebunder r. thrombocytopenia and platelet transfusions in dengue haemorrhagic fever and dengue shock syndrome. who dengue bulletin. 2003; 27: 141-3. 15. lei hy, huang kj, lin ys. immunopathogenesis of dengue haemorrhagic fever. am j infect dis. 2008; 4: 1-9. 16. vaughn dw, green s, kalayanarooj s, innis bl, nimmannitya s, suntayakorn s et al. dengue viremia titer, antibody response pattern and virus serotype correlate with disease severity. j infect dis. 2000; 181: 2-9. 17. greenberg ms, glick m, editors. burket’s oral medicine: diagnosis and treatment. 11th ed. lewiston, ny: b.c. decker; 2008. 18. drews re. critical issues in haematology: anaemia, thrombocytopenia, coagulopathy, and blood product transfusions in critically ill patients. clin chest med. 2003; 24: 607-22. unusual yet isolated oral manifestations of persistent thrombocytopenia – a rare case report236236236236236 braz j oral sci. 12(3):233-236 oral sciences n3 literature review braz j oral sci. july | september 2013 volume 12, number 3 prevalence of orofacial alterations in sickle cell disease: a review of literature poliana ramos braga santos1, paula dioné casais e silva machado2, cristina pinho passos3, marcio cajazeira aguiar4, roberto josé meyer nascimento5, maria isabela guimarães campos4 1dental school, federal university of bahia, salvador, ba, brazil 2medical school, federal university of bahia, salvador, ba, brazil 3institute of health science, federal university of bahia, salvador, ba, brazil 4department of biomorphology, institute of health science, federal university of bahia, salvador, ba, brazil 5department of biointeraction, institute of health science, federal university of bahia, salvador, ba, brazil correspondence to: marcio cajazeira aguiar instituto de ciências da saúde departamento de biomorfologia av. reitor miguel calmon, s/n° vale do canela, cep: 40.110-902, salvador, ba, brasil phone: +55 71 32838888 fax: +55 71 32838884 e-mail: mcaguiar@ufba.br received for publication: may 07, 2013 accepted: september 17, 2013 abstract aim: to evaluate the manifestations of sickle cell disease on the orofacial complex through a review of current literature concerning prevalence of dental caries, periodontal disease, temporomandibular joint disorders and radiographic alterations of maxillofacial bones. methods: full-text papers retrieved from medline and lilacs electronic databases were critically reviewed. results: alterations of maxillofacial bones are well documented in the literature, but studies reporting caries, periodontal condition and temporomandibular joint alterations in are scarce and inconclusive. conclusion: further well-designed epidemiological studies are needed to indicate the real impact of this disease on the stomatognathic health, collaborating to improve public health policies. keywords: sickle cell anemia, hemoglobin c disease, dental caries, periodontal diseases, temporomandibular joint, diagnostic x-ray. introduction sickle cell disease (scd) includes genetic blood disorders in which morphologic alterations of erythrocytes are caused by presence of the sickle hemoglobin (hbs). hbs is characterized by a mutation in the sixth position of â globulin chain, replacing the glutamic acid by valine1-3. sickle cell anemia (sca) is the most common and severe form of scd, resulting from genetic inheritance of hbs genes from both progenitors (ss genotype)1. the sc disease is the most second common manifestation of scd, presenting less severe symptoms4, and is characterized by heterozygosis of two mutant hemoglobins, hbs and hbc (sc genotype). in sickle cell trace, the heterozygosis for genes of normal (hba) and mutant (hbs) hemoglobins (as genotype) does not exhibit clinical symptoms of the disease under physiological conditions3,5. scd can be found in several countries affecting people all over the world, but primarily affects african descendents5 . in brazil, it’s estimated the existence of more than 2 million carriers of the gene for hbs, with 700 to 1000 new cases of scd every year, turning such disorder into a public health problem6. braz j oral sci. 12(3):153-157 the sickle erythrocyte presents reduced flexibility and becomes more adherent to vascular endothelium, developing vaso-occlusion of microvasculature and subsequent local hypoxia1,7. premature destruction of sickle red blood cells leads to hemolytic anemia. these mechanisms are the mean hallmarks of scd and are responsible for its clinical manifestations1,8. algic crises is the most common and important clinical symptom of this disease9. several other complications have been described, including: stroke, pulmonary infarct with decreased lung function, priapism, chronic renal failure, splenic and hepatic dysfunctions, jaundice, retinal ischemia causing transient or permanent blindness, chronic leg ulceration, apathy, cardiac alterations, convulsion and osseous alterations like osteonecrosis, osteomyelitis, osteopenia and osteoporosis 4,10,11. it is noted a wide interindividual variety of disease severity that has been suggested as result of polymorphisms in several genes12, mainly of fetal hemoglobin which has been pointed as a target for promising treatments13. little research has been done on the possible impact of this disease in the stomatognathic health. the aim of this paper was to review the literature regarding the actual knowledge concerning orofacial conditions in patients with scd. dental caries there are few studies in the literature concerning prevalence of caries lesions in scd patients and even those presented controversial results. in 1986, okafor et al.14 found a reduced caries prevalence (35.13%) among 37 scd adult patients (14-33 years old) when compared to 20 control subjects (54%) paired by age and gender. this finding was attributed to a reduced ingestion of sweets but its methodology was not specified in details, neither statistic comparisons were presented. in a retrospective study, no statistically significant difference was observed in the prevalence of dental caries determined by dmfs index between 35 hbss subjects and 140 control volunteers, both under treatment at a dental school and frequency matched on enrollment period and age. however, the great age variability among volunteers (5-92 years old) in this study may have influenced its results15. the controversial results can be related to socioeconomic factors that may influence caries risk rather than the hematologic disorder itself 16,17. comparing dmfs index of 102 sickle subjects (82 hbss, 15 hbsc and 5 hbs betathalassemic) and 103 control volunteers between 18 and 70 years old, the statistical analysis, adjusted for scd severity, age, gender, risk factors for caries and socioeconomic data, demonstrated that for low-income african americans, those with scd presented significantly more decayed and fewer filled surfaces18. since control volunteers were selected at dental colleges, these findings should be considered with regards. reduced prevalence of decayed teeth was also reported among 60 hbss children when compared to a control group paired by age and race. prolonged use of penicillin by falcemic children prevents acquisition of streptococcus mutans, reflecting the significant reduction in dental caries in these individuals19. in the scd patients, risk factors for caries seem to be similar to that observed in healthy subjects. passos et al.20 (2012) investigated the prevalence of dental caries and periodontal condition in 99 subjects with scd and a control group comprising 91 without disease, analyzing some associations with disease severity. their findings suggested that the sickle condition or the disease‘s clinical severity were not the main risk factors for the development of caries and periodontal disease. in this study, older age, female gender and daily smoking proved to be more important risk factors for higher decayed, missing and filled teeth index (dmft). their data suggested that risk factors known to affect the occurrence of dental caries were more important than scd on the dental condition of subjects with scd. periodontal conditions it has been reported that hygiene and oral care are important factors that influence the severity of periodontal disease and can prevent complications and infections in patients with sca17,21 a case report of a 14-year-old boy, during his ninth episode of sickle cell crisis, described an unusual complication, characterized by swelling on the right side of the face without any infection related. gingival enlargement, firm to palpation, was found in the lower arch on both sides. biopsies suggested that gingival edema was a result from repeated hemorrhage episodes followed by fibrous tissue repair formation22. crawford23 (1988), evaluating clinical and radiographically 78 african patients with sca, sc disease or thalassemia, did not observe significant association between scd and gingivitis or periodontitis when compared to control group. the authors believe that in a larger population, in which more severe cases of scd could be reached, periodontal disease might be affected by scd. arowojolu et al. 24 (1997), through a prospective comparative study over 6 months, analyzed gingival and plaque indexes and probing depths in 50 sca patients and 50 normal ones (11-19 years). no significant differences were found when groups were compared, suggesting that sca did not lead to increased periodontal disease. additionally, statistical significant difference was observed in probing depths only when female patients were considered (2.71 and 2.06 mm, for sca and controls, respectively). even though probing depths until 3 mm are accepted as normal, it was suggested that these findings might be of clinical importance with advancing age21. the same results were observed by guzeldemir et al.25 (2011). they evaluated 55 scd patients and observed that there was no difference regarding the periodontal disease between scd patients and healthy subjects. the authors were unable to assert that the disease could be a risk factor for periodontal disease. according to most of literature available, scd does not appear to predispose to periodontal complications. passos et al.20 (2012) did not observe association between sca and periodontal disease in scd patients. their study showed that 154154154154154 prevalence of orofacial alterations in sickle cell disease: a review of literature braz j oral sci. 12(3):153-157 higher risk for periodontal pockets was associated with older age and the absence of daily use of dental floss, which are recognized as risk factors for periodontal disease. nevertheless, new investigations should be performed in order to assess the role of periodontal infection in the worsening of scd picture, initiating or exacerbating vasoocclusive episodes. temporomandibular joint (tmj) disorders scd patients are commonly affected by bone and joint complications. the most common area of bone destruction is the femoral head, but other regions have been described: humeral head, thoracic and lumbar spine and temporomandibular joint (tmj)26. there are few papers in the literature about tmj disorders in scd, all reporting clinical cases. it was reported a clinical case of a 23 years old saudi female with sca that early demonstrated signs of bony infarction of humeral head and avascular necrosis of tmj and head femur, all in the left side, diagnosed by clinical examination and computed tomography. the authors suggested that despite the protective mechanisms of tmj, it was not absolutely immune against vaso-occlusive episodes of sca nature27. another case of tmj disorder was reported in a 23– year-old woman with sca, who presented no trauma history or orthognathic surgery. during clinical examination, it was observed limited mouth opening, central line deviation to the right side and a slight facial asymmetry because of the short right ramus height. a flattened condyle and avascular necrosis of glenoid fossa were demonstrated on the right joint through radiological examination and computed tomography. authors suggested that subtle onset of tmd symptoms should been taken seriously, especially in sca adolescents (11 to 15 years), when bone changes become more obviously. prevention of overloading and conservative approaches can prevent the development of irreversible deformities of tmj28. caracas et al.29 (2013) reported a rare case of aseptic arthritis in the tmj of a 22-year-old woman with sca. the patient experienced pain in the left preauricular region, hyperalgesia of the left masseter muscle, mouth opening limitation and mild edema. aseptic arthritis in the left condylar head secondary to a sickle cell crisis was diagnosed after the evaluation of the laboratory tests and images. the treatment consisted parenteral opioid analgesia and nonsteroidal anti-inflammatory drugs for manage of intense pain and blood transfusion because of severe anemia. health professionals should be aware that aseptic arthritis may also be provoked by scd. laboratory tests and diagnostic imaging are important for differential diagnosis. radiographic alterations scd patients usually present common radiographic features due to lesions in the cortical bone and bone marrow, often affecting the maxillofacial area30. maxillary bone alterations in scd are classified as: (1) lesions with osteoporotic appearance due to bone marrow hyperplasia, (2) radiopaque images associated to vaso-occlusive phenomena and (3) osteomyelitis lesions due to infections. all these maxillofacial features are similar to the ones found in the rest of the skeleton31. hemolytic anemia in scd patients promotes a compensatory marrow hyperplasia resulting in trabecular bone changes associated with expansion of the jaws 28. enlarged bone marrow spaces usually appear as radiolucent areas between the root apices of posterior teeth and in the inferior border of the mandible, creating in some cases a horizontal trabecular pattern described as “step-ladder”9. demirbas et al.32 (2004) reported decreased trabecular bone density and enlarged bone marrow spaces in 67% of 36 sca patients. however, “stepladder trabecular” pattern was observed in only 28% of these patients. faber et al.33 (2002) evaluated intertrabecular spacing in periapical digital radiographs from 18 sca patients and control volunteers (mean age 20.8 years). significant increase of intertrabecular spacing in both jaws and reduction of trabecular complexity were noted in sca individuals. demirbas et al.34 (2008) investigated trabecular bone complexity of posterior mandible in 35 sca patients (age ranged 11 to 40 years) and 26 control individuals using fractal dimension analysis from panoramic radiographs. sca patients under age 20 showed significant lower fractal dimension values, pointing to scarcity of trabecular bone, when compared to control individuals. neves et al.35 (2011) used panoramic radiograph to evaluate the radiographic features in the oral and maxillofacial region in brazilian patients with scd and healthy subjects. the healthy group showed a significantly smaller number of radiographic alterations when compared to the scd patients. increased intertrabecular distance, decreased trabecular complexity and the absence of mandibular canal corticalization were observed in subjects with hbss. these findings were statistically significant when compared to the control group. the alterations of the trabecular pattern may be related to presence of hemolytic anemia in subjects with sc. theses alterations may be found in healthy patients, but the occurrence of increased spacing of bone trabecular is more common between patients with scd. these data provide important information for identifying the disease. bone marrow expansion promotes maxilla overgrowth, which can increase the distance between upper and lower incisors teeth, resulting in altered interlabial distance with an untoward physical appearance and occlusion problems36. brown et al.37 (1986), analyzing lateral skull radiographs of 50 sca patients and 25 control volunteers matched by age and gender, observed in the first group a significant difference in maxillary protrusion due to increase in palate-alveolarridge angle. licciardello et al. 36 (2007) evaluated cephalometric radiographs from 36 patients with and 36 control volunteers (18.5-51 years). patients with scd presented a significantly maxillary incisor proclination and protrusion of the lower and upper lip. however the most severe craniofacial changes occurred in ss patients when compared to thalassemic ones. a less frequently radiographic finding is the expansion 155155155155155prevalence of orofacial alterations in sickle cell disease: a review of literature braz j oral sci. 12(3):153-157 of bone marrow in the calvarium, which is more often observed in very young patients. the widening of the diploe narrows the outer table, while perpendicular orientation of trabeculae to inner table gives a “hair-on-end” or “bristlelike” appearance30,31. radiopaque lesions presented by scd patients are commonly associated to bone infarction and osteonecrosis3031. in the first situation, long bones are affected more often, yet osteonecrosis usually takes place in articular surfaces38. it is suggested that the mandibular posterior region is the most affected by these lesions, coinciding with severe facial pain during sickle cell crisis. the affected area is ischemic and aseptic. initially the decalcification is surrounded by reactive sclerosis and later it is separated from cortex by a thinning radiolucent area, characterizing a “bone-withinbone” appearance31. kavadia-tsatala et al.31 (2004), evaluating panoramic and cephalometric radiographs, found radiopaque lesions associated to bone infarct in 6 of 42 scd patients (20-65 years old). lesions were related to vaso-occlusive phenomena, since it was followed by a painful crises episode and without any dental pathology associated. podlesh et al.39 (1995) reported a case of an 21 years-old man, hospitalized during a sickle cell crisis, who presented a profound anesthesia of the right mental nerve, fever, acute pain and tenderness to palpation on the right mandible. laboratory data showed no evidence of infection, however scans revealed cortical bone and bone marrow infarction of the right mandible. hamdoun et al.40 (2012) reported a case of an 15-year-old boy who showed no sensation to pain or touch of the entire lower lip and chin. the finding by magnetic resonance imaging were consistent with bone infartion. this case is considered unique because the patient was a child and had bilateral mental neuropathy. moreover, patients with scd are more likely to report pain than a lack of sensitivity. it is possible that frequency of complications such mental nerve neuropathy in patients with sca be high. mendes et al.41 (2011), comparing the prevalence of oral manifestations in 330 patients comprising subjects with sca and clinically normal patients, observed that the prevalence of previous mental nerve neuropathy in patients with sca was 2.2 times greater than observed in individuals without the disease, particularly in the female. osteomyelitis occurs up to 200 times more frequently in scd subjects than in the rest of population38,42. it is suggested that bone infarcts are the initial incident. the infarcted area is a propitious environment for bacteria growth, especially salmonella and staphylococcus aureus that migrate from several sources38,43. mandibular osteomyelitis seems to develop from direct extension of periapical abscess38 or pericoronitis43 and displays the same signs of bone infarcts like pain, soft tissue swelling, fever and leukocytosis 39. differential diagnosis between infarction and osteomyelitis can be very difficult, however this distinction is important as these two bone lesions are treated differently44,. magnetic resonance imaging was suggested to be useful to investigate these lesions. however, only techniques able to identify the infectious organism are reliable to establish a correct verdict42. final considerations considering the currently available literature, radiographic alterations of orofacial bones are better documented, while there is no precise knowledge concerning the prevalence and impact of caries, periodontal condition and tmd in the scd. it seems like orofacial complications depends not only on the presence of sickle disease, but also on factors related to the subject such as oral hygiene, diet habits and social conditions. although there is no direct association between scd and orofacial problems, limitations from systemic complications of this disease can reduce the patient’s availability for dental care. a poor buccal condition can act as an infectious focus for vasoocclusive crisis precipitation, compromising even more health and social life of these individuals. further well-designed epidemiological studies are needed to understand and recognize oral manifestations related with scd, indicating the real impact of this disease on stomatognathic health and collaborating to improve public health policies. references 1. steinberg mh. pathophysiology of sickle cell disease. baillieres clin haematol. 1998;11:163-184. 2. da fonseca m, oueis hs, casamassimo ps. sickle cell anemia: a review for the pediatric dentist. pediatr dent. 2007;29:159-69. 3. pauling l, itano ha, singer sj, wells, ic. sickle cell anemia a molecular disease. science. 1949;110:543-8. 4. nagel rl, fabry me, steinberg mh. the paradox of hemoglobin sc disease. blood rev. 2003;17:167–178. 5. creary m, williamson d, kulkarni r. sickle cell disease: current activities, public health implications, and future directions. j womens health (larchmt). 2007;16:575-82. 6. ministry of health. secretary of attention to health. manual of oral health in sickle cell disease. brasilia: ministry of health, 2005. 52 p. 7. stuart mj, nagel rl. sickle-cell disease. lancet. 2004;364:1343-60. 8. kelleher m, bishop k, briggs p. oral complications associated with sickle cell anemia: a review and case report. oral surg oral med oral pathol oral radiol endod. 1996;82:225-8. 9. ramakrishna y. dental considerations in the management of children suffering from sickle cell disease: a case report. j indian soc pedod prev dent. 2007;25:140-3. 10. serjeant gr. sickle-cell disease. lancet. 1997;350:725-30. 11. kato gj, gladwin mt, steinberg mh. deconstructing sickle cell disease: reappraisalof the role of hemolysis in the development of clinical subphenotypes. blood rev. 2007;21:37-47. 12. steinberg mh, adewoye ah. modifier genes and sickle cell anemia. curr opin hematol. 2006;13:131-136. 13. orkin sh, higgs dr. sickle cell disease at 100 years. science. 2010;329:291-292. 14. okafor la, nonnoo dc, ojehanon pi, aikhionbare o. oral and dental complications of sickle cell disease in nigerians. angiology. 1986;37:672-5. 15. laurence b, reid bc, katz rv. sickle cell anemia and dental caries: a literature review and pilot study. spec care dentist. 2002;22:70-4. 16. luna ac, rodrigues mj, menezes va, marques km, santos fa. caries prevalence and socioeconomic factors in children with sickle cell anemia. braz oral res. 2012; 26:43-9. 17. javed f, correa fo, nooh n, almas k, romanos ge, al-hezaimi k. orofacial manifestations in patients with sickle cell disease. am j med sci. 2013;345:234-7. 156156156156156 prevalence of orofacial alterations in sickle cell disease: a review of literature braz j oral sci. 12(3):153-157 18. laurence b, george d, woods d, et al. the association between sickle cell disease and dental caries in african americans. spec care dentist. 2006;26:95-100. 19. fukuda jt, sonis al, platt os, kurth s. acquisition of mutans streptococci and caries prevalence in pediatric sickle cell anemia patients receiving long-term antibiotic therapy. pediatr dent. 2005;27:186-90. 20. passos cp, santos, prb, aguiar mc, cangussu mct, toralles mbp, da silva m c bo, nascimento rjm, campos mi g. sickle cell disease does not predispose to caries or periodontal disease. special care in dentistry. 2012;32:55–60. 21. arowojolu mo. periodontal probing depths of adolescent sickle cell anaemic (sca) nigerians. j periodontal. res 1999;34:62-4.22. 22. scipio je, al-bayaty hf, murti pr, matthews r. facial swelling and gingival enlargement in a patient with sickle cell disease. oral dis. 2001;7:306-9. 23. crawford jm. periodontal disease in sickle cell disease subjects. j periodontol. 1988;59:164-9. 24. arowojolu mo, savage ko. alveolar bone patterns in sickle cell anemia and non-sickle cell anemia adolescent nigerians: a comparative study. j periodontol. 1997;68:225-8. 25. guzeldemir e, toygar hu, boga c, cilasun u. dental and periodontal health status of subjects with sickle cell disease. journal of dental sciences. 2011;6:227-234 26. aguilar c, vichinsky e, neumayr l. bone and joint disease in sickle cell disease. hematol oncol clin north am. 2005;19:929-41. 27. el-sabbagh am, kamel m. avascular necrosis of temporomandibular joint in sickle cell disease. clin rheumatol. 1989;8:393-7. 28. baykul t, aydin ma, nasir s. avascular necrosis of the mandibular condyle causing fibrous ankylosis of the temporomandibular joint in sickle cell anemia. j craniofac surg. 2004;15:1052-6. 29. caracas mda s, jales sp, jales neto lh, da silva castro jc, suganuma lm, fonseca gh, gualandro sf, de siqueira jt. temporomandibular joint arthritis in sickle cell disease: a case report. oral surg oral med oral pathol oral radiol. 2013 feb;115(2):e31-5.30. 30. yanaguizawa m, taberner gs, cardoso fnc, natour j, fernandes arc. diagnóstico por imagem na avaliação da anemia falciforme. rev bras reumatol. 2008;48:102-5. 31. kavadia-tsatala s, kolokytha o, kaklamanos eg, antoniades k, chasapopoulou e. mandibular lesions of vasoocclusive origin in sickle cell hemoglobinopathy. odontology. 2004;92:68-72. 32. demirbas ak, aktener bo, unsal c. pulpal necrosis with sickle cell anaemia. int endod j. 2004;37:602-6. 33. faber td, yoon dc, white sc. fourier analysis reveals increased trabecular spacing in sickle cell anemia. j dent res. 2002;81:214-8. 34. demirbas ak, ergun s, guneri p, aktener bo, boyacioglu h. mandibular bone changes in sickle cell anemia: fractal analysis. oral surg oral med oral pathol oral radiol endod. 2008;106:e41-8. 35. neves fs, de almeida da, oliveira-santos c, dos santos jn, toralles mb, da silva mc, campos mi, crusoé-rebello i. radiographic changes of the jaws in hbss and hbsc genotypes of sickle cell disease. spec care dentist. 2011 jul-aug;31(4):129-33 36. licciardello v, bertuna g, samperi p. craniofacial morphology in patients with sickle cell disease: a cephalometric analysis. eur j orthod. 2007;29:238-42. 37. brown dl, sebes ji. sickle cell gnathopathy: radiologic assessment. oral surg oral med oral pathol. 1986;61:653-6. 38. lawrenz dr. sickle cell disease: a review and update of current therapy. j oral maxillofac surg. 1999;57:171-8. 39. podlesh sw, boyden dk. diagnosis of acute bone/bone marrow infarction of the mandible in sickle hemoglobinopathy. report of a case. oral surg oral med oral pathol oral radiol endod. 1996;81:547-9. 40. hamdoun e, davis l, mccrary sj, eklund np, evans ob. bilateral mental nerve neuropathy in an adolescent during sickle cell crises. j child neurol. 2012;27:1038-41. 41. mendes ph, fonseca ng, martelli dr, bonan pr, de almeida lk, de melo la, martelli h jr. orofacial manifestations in patients with sickle cell anemia. quintessence int. 2011;42:701-9. 42. anand aj, glatt ae. salmonella osteomyelitis and arthritis in sickle cell disease. semin arthritis rheum. 1994;24:211-21. 43. olaitan aa, amuda jt, adekeye eo. osteomyelitis of the mandible in sickle cell disease. br j oral maxillofac surg. 1997;35:190-2. 44. royal je, harris vj, sansi pk. facial bone infarcts in sickle cell syndromes. radiology. 1988;169:529-31. 157157157157157prevalence of orofacial alterations in sickle cell disease: a review of literature braz j oral sci. 12(3):153-157 oral sciences n3 original article braz j oral sci. april | june 2015 volume 14, number 2 effect of different torques in cyclic fatigue resistance of k3 rotary instruments dirce akemi sacaguti kawakami1, george táccio de miranda candeiro2, eduardo akisue1, celso luiz caldeira2, giulio gavini2 1universidade santa cecília – unisanta, dental school, department of endodontics, santos, sp, brazil 2universidade de são paulo usp, faculty of dentistry, department of restorative dentistry, são paulo, sp, brazil correspondence to: giulio gavini departamento de dentística faculdade de odontologia universidade de são paulo av. prof. lineu prestes, 2227 cidade universitária cep: 05508-000 são paulo, sp, brasil phone: +55 11 30917839 e-mail: ggavini@usp.br abstract aim: to assess the effect of different torque values on cyclic fatigue resistance of k3 rotary nickeltitanium (niti) files. methods: eighty k3 files, size 25 mm, taper 0.04 were divided in 4 groups according to different torques (0.5, 1, 2 and 6 ncm) and were submitted to a cyclic fatigue test. this test was performed with a device that allowed the file to rotate inside a stainless steel artificial curved canal, simulating the pecking motion. files rotated until fracture occurred and time to failure was recorded in seconds with a stopwatch. data were analyzed by anova and tukey tests (p<0.05). results: all groups were compared and only the group of 6 n.cm showed statistically significant difference (p=0.0002). conclusions: for #25.04 k3 files, the evaluated torques up to 2 n.cm had no influence on cyclic fatigue resistance. using 6 n.cm torque value resulted in lower resistance to cyclic fatigue. keywords: dental instruments; endodontics; fatigue; root canal preparation; torque. introduction endodontic file fracture is a catastrophic accident that may occur during root canal therapy. despite greater flexibility and torsion resistance of niti files, fracture is the major concern in these files, especially after prolonged use1. unfortunately, most of these fractures occur unexpectedly, with no sign of permanent deformation. cyclic alloy fatigue, with successive tension and compression loads on the curved areas of the root canal can be the most destructive form of cyclic load. therefore, most cases of mechanical failure of niti rotary files during clinical use have been associated with cyclic fatigue2. many factors can affect the cyclic fatigue behavior of niti files, such as radius3,4 and root canal curvature5, number of uses of the files, motor torque3-,4, dentist’s expertise6-8, motor speed, sterilization method9 and surface treatment of files10. the frequency of use of rotary niti files and the motor torque setting are parameters that might affect the cyclic fatigue resistance of these files. studies have investigated the dynamic cyclic fatigue using an apparatus3,10-14 that simulates the pecking motion, and use of this movement during instrumentation by niti rotary files appears to significantly extend the life span of the file. as there is close relationship between torque and file separation by cyclic fatigue, the aim of this study was to assess the cyclic fatigue behavior of k3 files (sybronendo, orange, usa) submitted to different torques using an experimental cyclic fatigue testing apparatus that simulates the pecking motion in curved canals. the null hypothesis was that different torque values have no influence on cyclic fatigue resistance during instrumentation in simulated curved canals. braz j oral sci. 14(2):122-125 received for publication: march 07, 2015 accepted: may 28, 2015 http://dx.doi.org/10.1590/1677-3225v14n2a05 123123123123123 torque n time to failure (s) standard deviation mean cycles to fracture standard deviation 0.5 n.cm 20 143.6a 6.87 718.0a 34.37 1.0 n.cm 20 146.3a 5.87 731.5a 29.37 2.0 n.cm 20 156.0a 6.44 780.0a 32.24 6.0 n.cm 20 112.9b 5.17 564.5b 25.86 table 1.table 1.table 1.table 1.table 1. means of cycles to fracture, standard deviations and time to failure, expressed in seconds, for the different groups different letters indicate statistically significant difference (p<0.05). material and methods a total of 80 k3 files #25.04 were divided into four groups of 20 files each, based on torques of 0.5, 1.0, 2.0 and 6.0 n.cm to which they would be subjected. the files were used with an electric motor (x-smart; dentsply maillefer, ballaigues, switzerland) at 300 rpm. cyclic fatigue test was performed with a custom-made apparatus specifically designed to allow dynamic testing by simulating the pecking motion, made essentially of aluminum (figure 1), according to previous studies10-13. platforms were moved using a ring with internal sulcus until reaching a position that allowed the file to remain curved and free to rotate between the cylinder and the steel jig, thus simulating rotary instrumentation of a canal with a 40-degree, 5-mm radius curvature. care was taken to ensure that the file was well positioned in the cylinder groove to avoid file displacement. the file tip remained visible throughout the experiment, touching the sensor when the maximum displacement of the pneumatic system was achieved. thus, the whole micromotor/contra-angle/file set was powered by the pneumatic system, reproducing the pecking motion, with a 2 mm forward and backward movement, where the file slides in the groove created on the ring made of tempered steel. this movement was repeated at the speed of one cycle per second. a sensor detected the instrument fracture at the moment the counter and timer were stopped. testing time was registered with a digital stopwatch (casio, tokyo, japan), which started at the moment the motor was turned on and stopped by fracture detection. this procedure was sequentially repeated for all groups. after completion of all tests, the mean time to failure observed in each group was recorded in seconds. due to the fact that this study included an independent set of samples with normal distribution and equal variances, anova and tukey tests were used to check statistically significant differences (p<0.05). statistical analysis was performed with spss 17.0 statistical software (spss, chicago, il, usa). results fatigue resistance data were assessed with regard to central tendency (mean) and dispersion (standard deviation). anova revealed that the torque affected significantly the cyclic fatigue resistance (p=0.0002). table 1 shows fracture data obtained from each group and tukey test results for the used torques, respectively. it was observed that torques of 0.5, 1.0 e 2.0 had no influence on the cyclic fatigue resistance (p>0.05). however, the 6.0 n.cm torque influenced significantly the cyclic fatigue resistance (p<0.05). discussion in the present study, the null hypothesis was rejected, since the torque values affected the cyclic fatigue resistance in niti files. the present study assessed the cyclic fatigue resistance of k3 files submitted to different torques, using an experimental cyclic fatigue testing apparatus that simulates the pecking motion. file size (#25) and taper (0.04) were chosen since they are compatible with instrumentation of apical thirds in curved canals, mainly due their flexibility for cleaning and shaping these root canals. the option for k3 files is based on the idea that the file cross-sectional area is strongly related to cyclic fatigue resistance which occurs along the 16 mm of the working portion of these files15. mechanical stress of niti files is strongly related to the root canal curvature and dentin hardness5, but it is also proportional to the motor torque3-4 and the cyclic fatigue resistance decreases with prolonged clinical use3,16. cyclic fatigue occurs at the instrument’s maximal flexure rotating freely inside curved canals, without prior indication of failure17. continuous traction and compression cycles in fig 1. cyclic fatigue testing apparatus. letters a, b and c – rectangular platforms; d – grading rings; e – mechanical arm with locking ring to support micromotor/ contra-angle/file; f – pneumatic cylinder to produce the pecking motion. effect of different torques in cyclic fatigue resistance of k3 rotary instruments braz j oral sci. 14(2):122-125 124124124124124 curved canals are the most destructive form of cyclic fatigue and fracture for endodontic files2,4,8,14,18. although many studies have assessed cyclic fatigue and the dynamics of niti rotary files3-4,19, the relationship between force exerted during preparation of the root canal and clinical risk of distortion and fracture of files has not yet been properly studied. this study aimed to investigate the pecking motion mechanisms associated with cyclic fatigue test in the fracture of k3 niti rotary files. the methodology allowed the files to rotate freely at a standardized curvature. other studies34,16,18 have also indicated that these methodological characteristics are the most suitable for cyclic fatigue assessment in rotary niti files, since static tests do not reproduce the real conditions faced in clinical practice: automated instrumentation systems have been designed to enter the root canal in motion, with previously determined torque and speed values, whereas load distribution over a large area prolonged the life span of file. recently, gambarrasoares et al.20 (2013) reported that despite the fact that the static test complies with the no. 28 ansi/ada specifications, the dynamic cyclic fatigue test provides more reliable data regarding the lifespan determination of endodontic files and a better representation of clinical practice. occurrence of maximum flexion in the same location, in the same point, will decrease the lifespan of the file. continuous tension and compression in the curved area of the root canal promote a destructive load on niti rotary files3,14. during the pecking motion, the files were always stressed in the curved canal, but the pecking distance provides the files a time interval before it is once again subjected to the area of highest stress. according to li et al.11 (2002) the pecking motion may be a crucial factor in preventing the fracture of niti rotary files. the pecking motion minimizes the stress on files in curves, decreasing the chance of occurring fracture. to avoid rupture of the niti rotary file, li et al.11 (2002) advise the use of appropriate rotational speed and continuous pecking motion in root canals. in the present work, it was also observed that the effect of high torque (6 n.cm) on cyclic fatigue resistance was statistically significant (p=0.0002). a possible explanation for this result is that the mechanical stress on the niti files is also proportional to motor torque. if a high torque motor is used, the maximum torque limit of file is often exceeded, increasing mechanical stress and risk of plastic deformation or file fracture. even though some good results were obtained with high torques12,18, instrumentation technique and the dentist’s expertise play an important role in file failure19. gambarini13 (2001) also observed that the endodontic motor with lower torque values reduced cyclic fatigue of nickeltitanium rotary instruments. the results of this in vitro study must be critically interpreted, and comparisons with clinical practice must be made with caution, because only two of the many variations of root canal preparation were assessed. during this procedure, there are different types of stress from different mechanisms, which are correlated and can affect the lifespan of niti rotary files. although there is still no consensus regarding the maximum torque allowed for each file system, according to results of this study, it may be concluded that 25.04 k3 files can be used with a maximum torque of 2 n.cm without affecting their cyclic fatigue behavior. further studies should be carried out in dynamic pecking motion, with torques ranging from 2 to 6 n.cm in order to prevent accidents that may occur during preparation of root canals. new designs and alloys, and different motions have been introduced to increase the cyclic fatigue resistance of nickeltitanium (niti) files10,21. a recent research showed that the reciprocating motion improved significantly the resistance of k3 files to cyclic fatigue21. therefore, it is important to develop strategies to improve the resistance to fracture of endodontic files in order to avoid a catastrophic file separation that may contribute negatively to success of treatment. further works should be performed to better understand the behavior of files inside the root canal. the analysis of the results obtained in the present study allowed concluding that for k3 files, torques up to 2 n.cm had no influence on cyclic fatigue resistance. the use of 6 n.cm torque value resulted in lower resistance to cyclic fatigue. references 1. spanaki-voreadi ap, kerezoudis np, zinelis s. failure mechanism of protaper ni-ti rotary instruments during clinical use: fractographic analysis. int endod j. 2006; 39: 171-8. 2. haïkel y, serfaty r, bateman g, senger b, allemann c. dynamic and cyclic fatigue of engine-driven rotary nickel-titanium endodontic instruments. j endod. 1999; 25: 434-40. 3. pruett jp, clement dj, carnes-jr dl. cyclic fatigue testing of nickeltitanium endodontic instruments. j endod. 1997; 23: 77-85. 4. zelada g, varela p, martín b, bahillo jg, magán f, ahn s. the effect of rotational speed and the curvature of root canals on the breakage of rotary endodontic instruments. j endod. 2002; 28: 540-2. 5. mesgouez c, rilliard f, matossian l, nassiri k, mandel e. influence of operator experience on canal preparation time when using the rotary ni-ti profile system in simulated curved canals. int endod j. 2003; 36: 161-5. 6. yared g, bou dagher fe, kulkarni k. influence of torque control motors and the operator’s proficiency on protaper failures. oral surg oral med oral pathol oral radiol endod. 2003; 96: 229-33. 7. yared gm, bou dagher fe, machtou p. influence of rotational speed, torque and operator’s proficiency on profile failures. int endod j. 2001; 34: 47-53. 8. melo mcc, bahia mga, buono vtl. fatigue resistance of enginedriven rotary nickel-titanium endodontic instruments. j endod. 2002; 28: 765-9. 9. gavini g, pessoa of, barletta fb, vasconcellos maz, caldeira cl. cyclic fatigue resistance of rotary nickel-titanium instruments submitted to nitrogen ion implantation. j endod. 2010; 36: 1183-6. 10. gavini g, caldeira cl, akisue e, candeiro gtm, kawakami das. resistance to flexural fatigue of reciproc r25 files under continuous rotation and reciprocating movement. j endod. 2012; 38: 684-7. 11. li um, lee bs, shih ct, lan wh, lin cp. cyclic fatigue of endodontic nickel-titanium rotary instruments: static and dynamic tests. j endod. 2002; 28: 448-51. 12. yao jh, schwartz sa, beeson tj. cyclic fatigue of three types of rotary nickel-titanium files in a dynamic model. j endod. 2006; 32: 55-7. effect of different torques in cyclic fatigue resistance of k3 rotary instruments braz j oral sci. 14(2):122-125 125125125125125 13. gambarini g. cyclic fatigue of nickel-titanium rotary instruments after clinical use with low and high-torque endodontic motors. j endod. 2001; 27: 772-4. 14. eggert c, peters o, barbakow f. wear of nickel-titanium lightspeed instruments evaluated by scanning electron microscopy. j endod. 1999; 25: 494-7. 15. sattapan b, nervo gj, palamara jea, messer hh. defects in rotary nickel-titanium files after clinical use. j endod. 2000; 26: 161-5. 16. yared gm, bou dagher fe, machtou p. cyclic fatigue of profile rotary instruments after clinical use. int endod j. 2000; 33: 204-7. 17. sattapan b, nervo gj, palamara jea, messer hh. torque during canal instrumentation using rotary nickel-titanium files. j endod. 2000; 26: 156-60. 18. pessoa of, silva jm, gavini g. cyclic fatigue resistance of rotary niti instruments after simulated clinical use in curved root canals. braz dent j. 2013; 24: 117-20. 19. cho oi, versluis a, cheung gs, ha jh, hur b, kim hc. cyclic fatigue resistance tests of nickel-titanium rotary files using simulated canal and weight loading conditions. restor dent endod. 2013; 38: 31-5. 20. gambarra-soares t, lopes hp, oliveira jcm, souza lc, vieira vtl, elias, cn. dynamic or static cyclic fatigue tests: which best determines the lifespan of endodontic files? endod pract today. 2013; 7: 101-4. 21. pérez-higueras jj, arias a, de la macorra jc. cyclic fatigue resistance of k3, k3xf, and twisted file nickel-titanium files under continuous rotation or reciprocating motion. j endod. 2013; 39: 1585-8. effect of different torques in cyclic fatigue resistance of k3 rotary instruments braz j oral sci. 14(2):122-125 oral sciences n3 original article braz j oral sci. july | september 2013 volume 12, number 3 bismuth subsalicylate as filler particle for an experimental epoxy-based root canal sealer eduardo schwartzer1, bruna genari1, fabrício mezzomo collares1, vicente castelo branco leitune1, fabrício aulo ogliari2, susana maria werner samuel1 1dental materials laboratory, school of dentistry, federal university of rio grande do sul, porto alegre, rs, brazil 2school of engineering, federal university of pelotas, pelotas, rs, brazil correspondence to: fabrício mezzomocollares rua ramiro barcelos, 2492, cep: 90035-003 rio branco, porto alegre, rs, brasil phone: +55 51 33085198 e-mail: fabricio.collares@ufrgs.br abstract aim: to evaluate the influence of bismuth subsalicylate addition in different concentrations on theproperties ofan experimental epoxy-based root canal sealer. methods: bismuth subsalicylate in 20%, 40%, 60%, 80%, 100% and 120 wt% was added tothe sealer. flow, film thickness, working time, setting time, dimensional change, sorption, solubility and cytotoxicity were evaluated according to iso standard. data were statistically analyzed by one-way anova and tukey’stest with a significance level of 5% for all tests. results: the flow, working and setting times significantly decreased withincreasing particle concentration. the film thickness, dimensional change, water sorption and solubility values significantly increased with higher particle amount. the results for cytotoxicity showed no statistically significant differences among the particle proportions. conclusions: the results suggest that the addition up to 80% wt of bismuth subsalicylate appears to be a promising filler particle to root canal sealer development. keywords: root canal, cement, endodontics, bismuth subsalicylate. introduction root canal filling is an important step in endodontic therapy after appropriate shaping and cleaning of the canals to seal off the root canal system of any irritants that remain after enlargement1. new root canal sealers have been developed to improve properties like sealing, solubility and dimensional stability2-4. these materials are composed, in general, by a main organic component5 and inorganic elements such as radiopacifiersand filler particles, such as bismuth compounds6. bismuth is a chemical element with different applications7. in medicine, bismuth compounds such as bismuth subsalicylate can be used for the treatment of various gastrointestinal illnesses8-10. in dentistry, bismuth compounds (e.g. bismuth oxide)provide an acceptable radiopacity to root canal sealers6,11-12. considering that resin-based endodontic sealers present good physical properties and ensure adequate biological performance2-3, bismuth subsalicylate should be studied. the aim of this study was to evaluate the influence of bismuth subsalicylate addition in different concentrations into an experimental epoxy-based root canal sealer regarding the flow, film thickness, working time, setting time, dimensional change, sorption and solubility and cytotoxicity of an experimental epoxy-based root canal sealer. received for publication: may 28, 2013 accepted: september 13, 2013 braz j oral sci. 12(3):173-177 material and methods experimental sealer formulation an experimental epoxy-based root canal sealer containing bisphenol-a and epichlohydrin, (fiberglass, porto alegre, rs, brazil) at 2:1 (base: catalyst) was used in this study. to this sealer was added bismuth subsalicylate in several concentrations: 20%, 40%, 60%, 80%, 100% and 120%, in weight. the particle size distribution was assessed using a laser diffraction particle size analyzer (cilas 1180, orleans, france). the mean diameter particle was 9.61 µm and particle size distribution is shown in figure 1. colloidal silica (particle diameter of 7nm) was added at 0.05wt% to adjust the viscosity of the sealers. flow the flow test was conducted in accordance with iso 687613. a total of 0.05ml of each experimental sealer was placed on a glass plate (40x40x5mm) with a graduated syringe (1.5 ml). another plate with a mass of 20±2g and a load of 100g was applied on top of the material. ten minutes after the start of mixing, the load was removed, and the major and minor diameters of the compressed material were measured using a digital caliper (digimess, são paulo, fig. 1.the particle size distribution of bismuth subsalicylate particle. sp,brazil). for each experimental group, the test was conducted three times and the mean value was recorded. film thickness this evaluation was made according to iso 687613. two glass plates (5x10mm) were placed together and their combined thickness was measured. an amount of 0.5ml of experimental sealer was placed at the center of one of the plates, and a second plate was placed on top of the material. at 180±5s after the start of mixing, a load of 150±3n was applied vertically onto the top glass plate. ten minutes after the start of mixing, the thickness of the two glass plates and the interposed sealer film was measured using a digital caliper. the film thickness was recorded by the difference between the thickness of the two glass plates with and without sealer. the mean value of three measurements was recorded as the film thickness of the material. working time the test to measure the time to mix the components to obtain the cement with appropriate properties was based in iso 687613. this test had the same sequence of the flow test, but it was repeated at longer time intervals between manipulation and setting time. the working time was bismuth subsalicylate as filler particle for an experimental epoxy-based root canal sealer174174174174174 braz j oral sci. 12(3):173-177 recorded when the diameter of the specimen were 10% lowerthan the diameter of the immediate manipulated cement. the test was repeated three times and the mean values were recorded. setting time the setting time was recorded according to iso 687613. rings measuring 10mm in diameter and 1mm in height were filled with the material. these specimens were maintained under controlled temperature and humidity conditions, 37±1oc and 95% respectively. measurements were conducted using gilmore needles, weighing 100±0.5g and a flat end of 2.0±0.1mm diameter. the needle was lowered vertically onto the horizontal surface of each sample in such a way that it touched the surface every 5 min. the setting time was recorded when the needle did not produce any visible indentation on the sealer surface. dimensional change following setting the dimensional change was measured based in iso 687613. the cylindrical matrixes were filled with the sealer. these specimens were positioned between two glass plates (25x70x1mm). five minutes after the start of mixing, the specimens were placed in desiccators at 37oc and 95% relative humidity and held for a period three times the setting time. the specimens were removed from the matrixes and the thickness was measured. then, they were immersed in distilled water at 37oc during 30 days and a new measurement was made. a micrometer (aus-jena, jena, germany) with 0.001mm accuracy was used for measuring purposes. the difference between before and after storage was calculated. measurements were made three times and the mean value of these measurements was recorded as the dimensional change of the material. water sorption and solubility water sorption and solubility were determined based on the iso 404914. root canal sealer disks were produced in a silicone matrix (10.0mm diameter, 1.0mm thick). specimens were placed in a desiccator at 37°c for22 hand in a desiccator at 23°cfor2 h. the disks were repeatedly weighed in an analytical balance (shimadzu corp., tokyo, japan) until a constant mass (m 1 ) was obtained (i.e., until the mass variance of each specimen was no more than 0.1mg in any 24 h period). flow (mm) film thickness working time setting time (µm) (min) (h) 20% 21.72 (0.36)a 113 (20.8)e 53.33 (01.15)a 06:35 (00:27)a 40% 20.9 (0.84)a 173 (15.3)d 48.33 (01.15)b 06:31 (00:15)a 60% 18.69 (0.54)b 223 (20.8)d 48.67 (01.15)b 06:15 (00:18)a,b 80% 17.16 (0.43)c 333 (11.5)c 40.33 (01.15)c 05:25 (00:20)b 100% 16.30 (0.55)c 400 (20)b 32.00 (01.73)d 04:17 (00:17)c 120% 15.13 (0.37)c 483 (20.8)a 25.67 (01.15)e 03:51 (00:12)c table 1. flow, film thickness, working time and setting time of the sealers with bismuth subsalicylate in different proportions. different letters in same column represent statistically significant differences (p<0.05). diameter and thickness of each specimen were measured with a digital caliper to calculate the volume (v) of each disk (in mm3). thereafter, the specimens were stored in sealed glass vials with 10 ml of distilled water at 37°c for seven days. after 7days, the disks were weighed after being washed under running tap water and gently wiped with an absorbent paper to obtain themass (m 2 ) and then returned to the desiccator. next, the specimens were weighed until a constant mass (m 3 ) was obtained (as described for m 1 ). water sorption (ws) and solubility (sl), in micrograms per cubic millimeter, were calculated using the following formulae: ws = m 2 – m 3 / v (1) sl = m 1 – m 3 / v (2) cytotoxicity according to iso 10993-515, the cell viability was analyzed using mononuclear cells obtained from human peripheral blood. these cells were routinely maintained in dulbecco’s modified eagle’s medium (dmem) with hepes hdmem, with 10% fetal calf serum. the cells were maintained with endodontic sealers incubated for 72 h at 37oc and 5% co 2 . the controls consisted of cells incubated without endodontic cement. the rate of viable cells was quantified by testing(3-4,5dimethylthiazol-2-yl)-2,5-diphenol tetrazoliumbromide) mtt assay after 24hin contact with the endodontic cement. statistical analysis data normality distribution was analyzed by kolmogorovsmirnov and the tests used in this study were one-way anova and tukey’s multiple-comparison test with a significance level of 5% for all tests. results flow the flow significantly decreased with increasing filler particles concentration comparing 20% and 40% with the other percentages(p<0.05), ranging from 15.13 to 21.72mm. the results of flow measurements are set out in table 1. film thickness the film thickness values significantly increased with increasing filler particle concentration (p<0.05), ranging from bismuth subsalicylate as filler particle for an experimental epoxy-based root canal sealer 175175175175175 braz j oral sci. 12(3):173-177 dimensional change (%) sorption (µg/mm3) solubility (µg/mm 3) 20% -0.14 (0.02)a 33.85 (4.81)e 13.19 (6.33)c 40% -0.31 (0.14)b 44.02 (6.42)d,e 18.4 (5.05)b,c 60% -0.57 (0.06)b 59.12 (9.67)c,d 22.96 (1.15)b 80% -0.75 (0.04)c 69.55 (5.36)c 23.62 (6.01)b 100% * 123.17 (7.36)b 21.47 (2.83)b,c 120% * 177.9 (15.22)a 39.45 (3.23)a * it was not possible to evaluate because of disintegrationof the specimens. different letters in the same column represent statistically significant differences (p<0.05). table 2. dimensional change, sorption, solubility and radiopacity of the sealers with bismuth subsalicylate in different proportions. 113 to 483 µm. the film thickness measurements are shown in table 1. working time the means and standard deviations of working time are shown in table 1. the working time significantly decreased with increasing filler particle concentration (p<0.05), ranging from 53.3 to25.67 min. setting time the setting time significantly decreased with increasing filler particle concentration (p<0.05). these values varied from 03:51 to 06:35h. the setting time measurements are shown in table 1. dimensional change following setting the means and standard deviations of the dimensional change are shown in table 2. the significantly highest dimensional change occurred with 80% filler particle concentration and the lowest was with 20%. the values ranged from -0.14 to -0.75%. the specimens of the groups with 100 and 120 wt% of filler particles were solubilized and the dimensional change measurement could not be performed. water sorption and solubility water sorption and solubility means and standard deviations are shown in table 2. water sorption significantly increased with higher addition of filler particle concentration (p<0.05), ranging from 33.85 to 177.9 µg/mm3. solubility of the specimens with 120wt% filler particle concentration was statistically higher than groups with other filler particle concentration (p<0.05). the values of solubility ranged from 13.19 to 39.45µg/mm3. cytotoxicity there were no statistically significant differences (p>0.05) amongthe filler particle concentrations regarding cytotoxicity. the results are shown in figure 2. discussion in the present study, the addition of bismuth sub salicylate influenced the tested properties. the groups higher than 60% did not achieve the standards13. previous study fig. 2.cytotoxicity of the sealer with bismuth subsalicylate in different proportions. that evaluated flow presented values of 8.9 mm for gutta flow, 10.9 mm for ah plus and 12.2 mm for epiphany16. a high flow value could lead to an increased risk of sealer extrusion, but low flow values could difficult the penetration of materials in dentin root canal walls17-18.the sealers with bismuth subsalicylate showed higher values than the standard (50 µm)13. increased concentration of particles led to an increased volume of sealer particles, decreasing the flow and film thickness of sealer. however, flow and film thickness values of the experimental sealer of the present study are consistent to the values presented by commercial resin-based sealers19.the working and setting times must be long enough to manipulate the material to fill adequately the root canal20. iso 687613 requires that a sealer should present working timeless than 30 min and setting time ranged from 30 min to 72 h. in the present study, the working time of group with 120 wt% fulfilled the requirement. for the setting time, all groups fulfilled the requirements. water sorption could cause hydrolysis and plasticizing of the resin-based materials. these processes could result in the separation of the polymer chains, changing the dimension of the material, and consequently leading to gap formation and fluid infiltration21. according to iso687613, the endodontic sealer should not shrink more than 1% and swell more than 0.1% to avoid gaps between sealer and dentin. in the present study, the experimental endodontic sealers presented swelling higher than 0.1%13 accordingly to commercial available sealers22-24.any uptake of water is bismuth subsalicylate as filler particle for an experimental epoxy-based root canal sealer176176176176176 braz j oral sci. 12(3):173-177 determined by the intrinsic hydrophilicity of the copolymer, the type and amount of filler particles25and it could result in lower mechanical properties of root canal sealers21,26.the water sorption could also lead to degradation of the sealer, due to the unreacted monomers leach through porosities21.according to iso 404914, the water sorption of the resin-based material cannot be more than 40 µg/mm3 and the water solubility must be up to 7.5 µg/mm3. in the present study, the water sorption of the sealers with 20 wt% of bismuth subsalicylate presented water sorption in accordance to the requirement. the other concentrations did not fulfill the standard. solubility also presented higher values than required. however, this standard is for restorative materials; there is no specific standard for resin-based root canal sealers. the values of water sorption and solubility are consistent with commercial resin-based root canal sealers27. solubility of unreacted components could lead to cytotoxicity of periapical region tissues21,28. low cytotoxicity is a desirable characteristic for new root canal sealers. in the present study, addition of bismuth subsalicylate did not increase the cytotoxicity of the experimental sealer. it was concluded that addition up to 80% wt of bismuth subsalicylate appears to be a promising filler particle for root canal sealer development. references 1. schilder h. filling root canals in three dimensions. j endod. 2006; 32: 281-90. 2. schwartz rs. adhesive dentistry and endodontics. part 2: bonding in the root canal system -the promise and the problems: a review. j endod. 2006;32: 1125-34. 3. salz u, poppe d, sbicego s, roulet jf. sealing properties of a new root canal sealer. int endod j. 2009; 42: 1084-9. 4. rosa pcf, mancini mng, camargo sea, garrido adb, camargo chr; rode sm. dimensional alterations and solubility of new endodontic sealers. braz dent j. 2010; 21: 301-4. 5. al-hiyasat as, tayyar m, darmani h. cytotoxicity evaluation of various resin based root canal sealers. int endod j. 2010; 43: 148-53. 6. collares fm, ogliari fa, lima gs, fontanella vr, piva e, samuel sm. ytterbium trifluoride as a radiopaque agent for dental cements. int endod j. 2010; 43: 792-7. 7. li h, sun h. recent advances in bioinorganic chemistry of bismuth (review). curr opin chem biol. 2012; 15: 74-83. 8. andrews pc, deacon gb, forsyth cm, junk pc, kumar i, maguire m. towards a structural understanding of the antiulcer and anti-gastritis drug bismuth subsalicylate. angew chem int ed engl. 2006; 45: 5638-42. 9. chande n, macdonald jk, mcdonald jw. interventions for treating microscopic colitis: a cochrane inflammatory bowel disease and functional bowel disorders review group systematic review of randomized trials. am j gastroenterol. 2009; 104: 235-41. 10. temmerman f, baert f. collagenous and lymphocytic colitis: systematic review and update of the literature. dig dis. 2009; 27(suppl 1): 137-45. 11. kim ec, lee bc, chang hs, lee w, hong cu, min ks. evaluation of the radiopacity and cytotoxicity of portland cements containing bismuth oxide. oral surg oral med oral pathol oral radiol endod. 2008; 105: e54-7. 12. húngaro-duarte ma, kadre gdoe, vivan rr, tanomaru jmg, tanomaru filho m, de moraes ig. radiopacity of portland cement associated with different radiopacifying agents. j endod. 2009; 34: 737-40. 13. international organization for standardization. dental root canal sealing materials. n° 6876:2001(e); second edition. 14. international organization for standardization. dentistry polymer-based restorative materials. n° 4049:2009(e); fourth edition. 15. international organization for standardization. biological evaluation of medical devices part 5: tests for in vitro cytotoxicity. n° iso10993-5:2001(e); third edition. 16. nawal rr, parande m, sehgal r, naik a, rao nr.a comparative evaluation of antimicrobial efficacy and flow properties for epiphany, gutta flow and ah-plus sealer. int endod j. 2011; 44: 307-13. 17. gambarini g, testarelli l, pongione g, gerosa r, gagliani m. radiographic and rheological properties of a new endodontic sealer. aust endod j. 2006; 32: 31-4. 18. bernardes ra, de amorim campelo a, junior ds, pereira lo, duarte ma, moraes ig, et al. evaluation of the flow rate of 3 endodontic sealers: sealer 26, ah plus, and mta obtura. oral surg oral med oral pathol oral radiol endod. 2010; 109: e47-9. 19. hosoya m, kurayama h, iino f, arai t. effects of calcium hydroxide on physical and sealing properties of canal sealers. int endod j. 2004; 37: 178-84. 20. mcmichen fr, pearson g, rahbaran s, gulabivala k. a comparative study of selected physical properties of five rootcanal sealers. int endod j. 2003; 36: 629-35. 21. ferracane jl. hygroscopic and hydrolytic effects in dental polymer networks. dent mater. 2006; 22: 211–22. 22. versiani ma, carvalho-junior jr, padilha miaf, lacey s, pascon ea, sousa-neto md. a comparative study of physicochemical properties of ah plustm and epiphany tm root canal sealants. int endod j. 2006; 39: 464-71. 23. carvalho-junior jr, correr-sobrinho l, correr ab, sinhoreti ma, consani s, sousa-neto md. solubility and dimensional change after setting of root canal sealers: a proposal for smaller dimensions of test samples. j endod. 2007; 33: 1110–6. 24. resende lm, rached-junior fja, versiani ma, souza-gabriel ae, miranda ce, silva-sousa yt et al. a comparative study of physicochemical properties of ah plus, epiphany, and epiphany se root canal sealers. int endod j. 2009; 42: 785-93. 25. johns ji, o’donnell jn, skrtic d. selected physicochemical properties of the experimental endodontic sealer. j mater sci mater med. 2010; 21: 797-805. 26. francisconi lf, de freitas ap, scaffa pm, mondelli rf, francisconi pa. water sorption and solubility of different calcium hydroxide cements. j appl oral sci. 2009; 17: 427-31. 27. baldi jv, bernardes ra, duarte mah, ordinola-zapata r, cavenago bc, moraes jcs, et al. variability of physicochemical properties of an epoxy resin sealer taken from different parts of the same tube. int endod j. 2012; 19: 1-6. 28. loushine ba, bryan te, looney sw, gillen bm, loushine rj, weller rn, et al. setting properties and cytotoxicity evaluation of a premixed bioceramic root canal sealer. j endod. 2011; 37: 673-7. bismuth subsalicylate as filler particle for an experimental epoxy-based root canal sealer 177177177177177 braz j oral sci. 12(3):173-177 oral sciences n3 braz j oral sci. 13(2):129-132 original article braz j oral sci. april | june 2014 volume 13, number 2 received for publication: april 23, 2014 accepted: june 09, 2014 staining of esthetic brackets by plaque disclosing solutions luiza novelino acatauassú ismael¹, mauro de amorim acatauassú nunes2, ana maria novelino acatauassú nunes3, rogério heládio lopes motta4, ana paula dias demasi5, flávia martão flório6 1 universidade federal do pará – ufpa, school of dentistry, department of orthodontic, belém, pa, brazil 2 universidade federal do pará – ufpa, school of dentistry, department of traumatology, belém, pa, brazil 3 associação brasileira de odontologia abo, area of pediatric dentistry, belém, pa, brazil 4 faculdade são leopoldo mandic slmandic, dental school and research center, department of pharmacology, anesthesiology and therapeutics, campinas, sp, brazil 5 faculdade são leopoldo mandic – slmandic, dental school and research center, department of pathology, campinas, sp, brazil 6 faculdade são leopoldo mandic – slmandic, dental school and research center, department of dental public health, campinas, sp, brazil correspondence to: flávia martão flório rua josé rocha junqueira, 13 bairro swift cep: 13041-445 campinas, sp, brazil phone: + 55 19 32113650 e-mail: flaviaflorio@yahoo.com abstract aim: to evaluate the staining of esthetic orthodontic brackets by plaque disclosing solutions. methods: two types of brackets manufactured by gac/dentsply® were evaluated: ceramic (n=30) and polycarbonate (n=30). the brackets were divided into 6 groups. two control groups (n=6) were immersed in absolute ethanol: gi ceramic brackets and gii polycarbonate brackets. four experimental groups (n=12) were immersed in different plaque disclosing solutions: giii (ceramic brackets) and giv (polycarbonate brackets) were immersed in replak®; gv (ceramic brackets) and gvi (polycarbonate brackets) were immersed in replasul “s”®. relative quantitative analysis of the influence of plaque disclosing tablets on bracket staining was performed using reflectance spectrophotometry of stain deposition. exploratory analysis of the data was performed using analysis of variance (anova) in a 2x2 factorial setup (bracket x immersion) with additional treatments (controls). results: the results demonstrated that the ceramic brackets presented the highest amount of staining when replasul “s”® was used (pd”0.05). however, when replak® was used, no statistically significant difference was found in comparison with the control group (p>0.05). for polycarbonate brackets, staining was detected for both disclosing solutions (p>0.05). conclusions: the disclosing solutions caused stain formation on polycarbonate brackets and, under the tested conditions, use of replak® on ceramic brackets did not cause staining. keywords: orthodontic brackets; spectrophotometry; biofilms. introduction the strategy of applying a tailored preventive program for each patient, considering both the risk to plaque-related disease and the level of adherence to the necessary measures, is mandatory for a satisfactory correction of occlusion without compromising the oral tissue health1-3. the dental materials industry has been investing in manufacturing custom brackets that combine both adequate esthetics and technical performance4. the first esthetic brackets developed in the 1960s were made of polycarbonate5, which presented disadvantages in terms of clinical performance such as deformation, structural fragility, low adhesion and superficial staining6-8. consequently, the use of such appliances was recommended with caution and was limited to short braz j oral sci. 13(2):129-132 130 times of treatment. these materials present unique features that must be fully understood by orthodontists so they can prescribe them safely, taking the adequate clinical care to select the cases that would benefit most from the treatment with esthetic brackets9. ceramic orthodontic brackets were first made available in the late 1980s with the aim of eliminating the esthetic issue imposed by metallic brackets and the disadvantages of polycarbonate brackets. the early appliances represented significant clinical and esthetic advances due to their shade stability and resistance to oral fluids10-15. the advantages and disadvantages of polycarbonate and ceramic brackets have been investigated in vitro, especially in terms of changes in optical properties due to discoloration and staining by pigmented substances from foods and drinks 9,16-20. the latter aspect is of great importance to orthodontists as patients become increasingly demanding of appliances that do not show and remain as such, especially in terms of shade,16,21-25. the optical properties of esthetic brackets have a direct influence on visual perception26. once bonded, brackets make oral hygiene more difficult and may serve as a retention sites for stagnation of foods and bacterial biofilm accumulation, increasing the risk of enamel demineralization and periodontal problems27. mechanical plaque removal using a toothbrush is the bestknown and readily available method for prevention and control of such problems, which may be effective if performed appropriately. plaque disclosing substances should also be part of the educational process, since they play a fundamental role of guiding and motivating orthodontic patients to tooth brushing, and among the available plaque disclosing solutions, basic fuchsine and replak® are largely used in clinical practice28. the purpose of tooth brushing should not be limited to the automatic and mechanical fulfillment of a mere cosmetic ritual, but it must achieve an adequate disorganization of the bacterial biofilm. passing this message across clearly to the patients, so that they are motivated throughout the course of orthodontic treatment, is no easy task. effective communication between patients, parents, orthodontists and general dental practitioners must be in place in order to reduce the incidence of white lesions in patients wearing fixed orthodontic appliances29. studies on the possible shade changes caused by known pigmented solutions such as coca-cola®, tea, coffee, red wine, etc. to polycarbonate and ceramic brackets have been performed17,21. there is, however, a lack of reports on the degree of staining of such appliances by plaque disclosing solutions. this could partially explain why most orthodontists avoid procedures of plaque disclosure prior to prophylaxis and patients prior to brushing, in other words, there is no scientific evidence that such dyes will not cause staining of polycarbonate and ceramic brackets. the purpose of this study was to evaluate whether esthetic brackets are stained by different plaque disclosing solutions. material and methods the study sample was composed by 60 esthetic brackets, 30 ceramic and 30 polycarbonate, from gac/dentsply (islandia, ny, usa) for the maxillary right central incisor (11). the brackets were divided into 2 control groups (n=6) and 4 experimental groups (n=12). two disclosing solutions replak® (dentisply, petrópolis/rj, brazil), based on red and blue food dyes and replasul “s”® (iodontosul, porto alegre/ rs, brazil), based on 0.04% basic fuchsine] and two types of esthetic brackets [ceramic (mystique mb®, dentsply/gac, bohemia, ny)] or polycarbonate (elation mb®, dentsply/gac, bohemia, ny) were used. the groups were named gi (control), esthetic ceramic brackets immersed in absolute ethanol; gii (control), esthetic polycarbonate brackets immersed in absolute ethanol; giii, esthetic ceramic brackets immersed in replak®, giv; esthetic polycarbonate brackets immersed in replak®; gv, esthetic ceramic brackets immersed in replasul “s”®; and gvi, esthetic polycarbonate brackets immersed in the plaque disclosing solution replasul “s”®. the relative quantitative analysis of the influence of plaque disclosing solutions on shade changes of brackets by stain deposition was performed using reflectance spectrophotometry17,21,30-31. an amount of 250 ìl of each dye and ethanol was pipetted into the wells of a 96-well plate. in order to avoid cross-contamination of dyes through spillage, the pipetting sequence was: replak®, replasul “s”® and ethanol. each well received one type of bracket using dental tweezers. all brackets remained immersed in ethanol (control group) and in their respective plaque disclosing solution for 1 h. the plates were kept at room temperature in the dark, preventing the interference of light in shade change of the brackets17,21,. the test specimens were removed using a pair of tweezers and rinsed in distilled water by immersion for 15 s in the appropriate well of a 24-well plate17,21,30. the brackets were subsequently dried in absorbent paper for 1 min, always following a random sequence30-31. the brackets were then immersed in 250 ìl absolute ethanol in the wells of a 96-well plate30-31 which was placed in an orbital shaker (biomixer, chatswood, nsw, australia) for 24 h to elute the dye that was deposited onto the specimen. the brackets were removed from the wells and the plate was placed in a reflectance spectrophotometer (epoch biotek™ instruments, inc., winooski, vt, usa) to quantify and observe the degree of absorbance of the experimental solutions, which was registered and printed using the gen 5tm getting started guide (biotek™ instruments, inc. – dec. 2009) software. readings were taken from the absorption spectrum peaks, since each dye showed better results at different wavelengths. the readings for each solution were compared against the control group within the same absorbance spectrum30-31. in order to establish the relationship between the absorbance of the red and blue solutions (replak®) and 0.04% basic fuchsine (replasul “s”®), the spectrophotometer was calibrated at the of 600 and 550 nm wavelengths, respectively (maximum absorbance spectrum of each solution). the exploratory analysis of the data required a square root adjustment so that the values fulfilled the assumptions staining of esthetic brackets by plaque disclosing solutions 131 of a parametric analysis. analysis of variance (anova) was then applied using a 2x2 factorial setup (bracket x immersion) and additional treatments (control groups). for replak®, ethanol measured at a wavelength of 600 nm was considered, whilst for replasul “s”®, ethanol was measured at 550 nm, always with the respective bracket type. the comparisons with the controls were performed using the dunnett’s test with p=0.05. all analyses were performed using the statistics sas software (sas inc., cary, nc, usa). results table 1 shows the amount of staining of the studied esthetic brackets. for the ceramic brackets, the highest degree of staining was observed with replasul “s”® (p<0.05), whereas for the polycarbonate brackets no significant difference was found between the tested disclosing solutions (p>0.05), but staining was observed for both products. regarding the plaque disclosing solutions, replak® on polycarbonate brackets caused the highest average stain readings (p<0.05). the use of replak® on ceramic brackets did not show statistically significant difference against the control group (p<0.05). immersion bracket type ceramic polycarbonate ethanol (550 nm control replasul) 0.08 (0.06) 0.06 (0.02) ethanol (600 nm control replak) 0.06 (0.03) 0.05 (0.01) replak 0.11 (0.05) bb *0.17 (0.07) aa replasul “s” *0.27 (0.06) aa *0.19 (0.09) ba table 1.table 1.table 1.table 1.table 1. mean staining (standard deviation) according to type of bracket and immersion solution means followed by different letters (uppercase horizontally and lowercase vertically) indicate significant difference (p<0.05). * different from the ethanol group for the same bracket type (p<0.05). discussion the present study assessed the shade changes caused by plaque disclosing solutions on polycarbonate and ceramic brackets using reflectance spectrophotometry. this method aims to determine the concentration of a substance generally present in a solution, using a known concentration of the same compound as a reference31. it is a method previously used to quantify a dye that impregnates specimens17,21,30-31 and it is in an analytical technique to quantitatively determine chemical species. the results demonstrated that the use of a fuchsine-based plaque disclosing solution (replasul “s”®) caused the ceramic bracket to present the highest degree of staining, however, when replak® was used, no difference was observed when compared with the control group, suggesting that this disclosing solution was not adsorbed onto the bracket surface in the time adopted for this study, thus causing no shade change to the bracket. therefore, the use of replak® on ceramic brackets did not cause staining, suggesting it as the plaque disclosing solution of choice for patients undergoing orthodontic treatment. previous studies have reported that ceramic brackets are stain and discoloration resistant to any chemical substance encountered in the oral environment, which contradicts the findings from this study 10-13,23. esthetic material can undergo shade or color change due to both extrinsic and intrinsic factors. according to some authors10,14,18-19,27 the intrinsic factors include the material discoloration itself, as a result of matrix changes (ageing, physicochemical conditions such as visible light radiation, ultraviolet rays and moisture) and the extrinsic factors include absorption and adsorption of substances. consequently, it is possible to infer that the fuchsine-based disclosing solution bonded to the ceramic both intrinsically and extrinsically via incorporation of the dye into the bracket. the food dyes present in replak®, however, did not show intrinsic or extrinsic affinity to the ceramic when compared with the control group, thus making it the plaque disclosing product of choice for patients wearing esthetic ceramic brackets. this corroborates other reports on the esthetic advantages of ceramic over polycarbonate brackets9,14,16,18-22,24-25,27; however, ceramic brackets can also be stained30. according to a study that evaluated the chromatic changes of some types of ceramic brackets, the results demonstrated that the discolorations were significant when time was taken into account for all tested solutions17. this study demonstrated the staining of polycarbonate brackets by both disclosing solutions replasul “s”® and replak®, showing that shade or color change by extrinsic physicochemical factors were observed in this type of material. another study reported clinical problems with discoloration when using polycarbonate brackets even in the absence of a dye18, despite the excellent hardness of such resin material, thus corroborating the results from this study. as reported in the literature, polycarbonate and ceramic brackets have different patterns of color or shade change for each solution used 6-9,14,16,18,22,24-25,27. the present study corroborates those findings in terms of changes to optical properties of such brackets in the oral environment, as a result of staining by coloring substances present in foods and beverages9,16-17,21-22,24-25. the planning of orthodontic treatment either due to esthetics or for oral health purpose3,13 must include strategies of plaque control and, the use of plaque disclosing solutions must not be dismissed from the motivational arsenal and home care kit. therefore, further studies are needed on the factors that interfere with color and shade stability of such devices, since the demand for more discrete appliances increases rapidly, just as the optical properties of esthetic brackets are the least investigated directly9. this study demonstrated that plaque disclosing agents might interfere with bracket shade. the choice of the most appropriate product for each bracket type should be cautious. the findings from this study suggest that patients wearing ceramic brackets ought to opt for replak®. the tested plaque disclosing products caused color changes on polycarbonate brackets, which should be further investigated clinically to determine its relevance for patients wearing such appliances. staining of esthetic brackets by plaque disclosing solutions braz j oral sci. 13(2):129-132 132 in conclusion, the plaque disclosing solutions tested in this study should not be used on polycarbonate brackets and, under the conditions described in this study, the use of replak® on ceramic brackets did not cause staining. references 1. derks a, kuijpers-jagtman am, frencken je, van’t hof ma. caries preventive measures used in orthodontic practices: an evidence-based decision? am j orthod dentofacial orthop. 2007; 132: 165-70. 2. behnan sm, arruda ao, gonzález-cabezas c, sohn w, peters m. invitro evaluation of various treatements to prevent demineralization next to orthodontic brackets. am j orthod dentofacial orthop. 2010; 138: 712.e1-7; discussion 712-3. 3. mckiernan exf, mckiernan f, jones ml. psychological profiles and motives of adults seeking orthodontic treatment. int j adult orthodon orthognath surg. 1992; 7: 187-98. 4. jena ak, duggal r, mehrotra ak. physical properties and clinical characteristics of ceramic brackets: a comprehensive review. trends biomater artif organs. 2007; 20: 101-5. 5. newman gv. adhesive and orthodontic plastic attachments. am j orthod dentofacial orthop. 1969; 56: 573-88. 6. dobrin rj, kamel il, musich dr. load-deformation characteristics of polycarbonate orthodontic brackets. am j orthod. 1975; 67: 24-33. 7. eliades t, lekka m, eliades g, brantley wa. surface characterization of ceramic brackets: a multi-technique approach. am j orthod dentofacial orthop. 1994; 105: 10-8. 8. feldner jc, sarkar nk, sheridan jj, lancaster dm. in vitro torquedeformation characteristics of orthodontic polycarbonate brackets. am j orthod dentofacial orthop. 1994; 106: 265-72. 9. maltagliati la, feres r, figueiredo maf, siqueira df. aesthetic brackets clinical aspects. rev clín ortodon dental press. 2006; 5: 89-95. 10. swartz ml. ceramic brackets. j clin orthod boulder. 1988; 22: 82-8. 11. aknin pc, nanda rs, duncanson mg, currier f, sinha pk. fracture strength of ceramic brackets during arch wire torsion. am j orthod dentofacial orthop. 1996; 109: 22-7. 12. sinha pk, nanda rs. esthetic orthodontic appliances and bonding concerns for adults. dent clin north am. 1997; 41: 89-109. 13. olsen me, bishara se, jakobsen jr. evaluation of the shear bond strength of different ceramic bracket base designs. angle orthod. 1997; 67: 179-82. 14. winchester lj. aesthetic brackets: to perfect or to reject? dent update. 1992; 19: 107-10, 112-4. 15. phillips rw, anusavice kj. physical properties of dental materials. in: phillips rw. dental materials. 10. ed. rio de janeiro: guanabara koogan; 1998. p.18-27. 16. ghafari j. problems associated with ceramic brackets suggest limiting use to selected teeth. angle orthod. 1992; 62: 145-52. 17. guignone bc. color instability of ceramic brackets when immersed in potentially staining substances. in vitro study [dissertation]. belo horizonte: pontifícia universidade católica de minas gerais; 2008. portuguese. 18. dietschi d, campanile g, holz j, meyer jm. comparison of the color stability of ten new-generation composites an in vitro study. dent mater. 1994; 10: 353-62. 19. sham ask, chu fcs, chai j, chow tw. color stability of provisional prosthodontic materials. j prosthet dent. 2004; 91: 447-52. 20. lee yk. changes in the reflected and transmitted color of esthetic brackets after thermal cycling. am j orthod dentofacial orthop. 2008; 133: 641.e1-641.e6. 21. silva lk. color instability of polycarbonate brackets when immersed in potentially staining substances: in vitro study [dissertation]. belo horizonte: pontifícia universidade católica de minas gerais; 2008. portuguese. 22. bishara se, fehr de. ceramic brackets: something old, something new, a review. semin orthod. 1997; 3: 178-88. 23. karamouzos a, athanasiou ae, papadopoulos ma. clinical characteristics and properties of ceramic brackets: a comprehensive review. am j orthod dentofacial orthop. 1997; 112: 34-40. 24. fernandez l, canut ja. in vitro comparison of the retention capacity of new aesthetic brackets. eur j orthod. 1999; 21: 71-7. 25. bishara se. ceramic brackets: a clinical perspective. world j orthod. 2003; 4: 61-6. 26. filho hl, maia leg, araújo mva, ruellas aco. influence of optical properties of esthetic brackets (color, translucence and fluorescence) on visual perception. am j orthod dentofacial orthop. 2012; 141: 460-7. 27. faltamier a, bürgers r, rosentritt m. bacterial adhesion of streptococcus mutans to esthetic bracket materials. am j orthod dentofacial orthop. 2008; 133(4 suppl): s99-103. 28. brito rl, silva sc, freitas jm. brushing techniques adopted by periodontology disciplines of 13 dental schools in the northeast of brazil. rev periodont. 1999 [cited 2014 jun 2]; 8: 14-20. 1999 available from: http://www.revistasobrape.com.br/arquivos/edicao_anterior/ ed_maidez_99/file____d__1999_mai_dez_tec_esco_tec_esco.pdf] 29. maxfield bj, hamdan am, tufekçi e, shroff b, best alm, lindauer sj. development of white spot lesions during orthodontic treatment: perceptions of patients, parents, orthodontists, and general dentists. am j orthod dentofacial orthop. 2012; 141: 337-44. 30. lima fap. evaluation of the plaque disclosing in the staining of two glassionomer/composite resin hybrid materiais [dissertation]. piracicaba: universidade estadual de campinas. piracicaba dental school; 1997. portuguese. 31. dorini al. the influence of different finishing and polishing systems on two composites surface roughness and pigmentation [dissertation]. piracicaba: universidade estadual de campinas. piracicaba dental school; 1999. portuguese. staining of esthetic brackets by plaque disclosing solutions braz j oral sci. 13(2):1129-132 oral sciences n3 original article braz j oral sci. april | june 2015 volume 14, number 2 chemical salivary composition and its relationship with periodontal disease and dental calculus anne alejandra hernández-castañeda1, gloria cristina aranzazu-moya1, gerardo mantilla mora2, dagmar de paula queluz3 1universidad santo tomás, dentistry school, department of oral medicine, semiology and pathology, bucaramanga, santander, colombia 2universidad industrial de santander, school of medicine, department of biochemistry, bucaramanga, santander, colombia 3universidade estadual de campinas – unicamp, piracicaba dental school, department of community and preventive dentistry, piracicaba, sp, brazil correspondence to: anne alejandra hernández castañeda universidad santo tomás carrera 18 n 9-27 colombia phone: 6800801 ext. 2502 e-mail: anne.usta@hotmail.com abstract aim: to determine the relationship between the chemical composition of saliva, periodontal disease and dental calculus. methods: an observational analytical cross-sectional study was conducted with patients over 55 years of age. ethical principles of autonomy and risk protection were applied according to the international standards. sociodemographic and diagnosis variables (presence of dental calculus and periodontal status) were considered to measure salivary concentrations of glucose (by the glucose oxidase/peroxidase method, amylase (by the colorimetric test), urea (by the amount of indophenol), total protein (by the bradford method) and albumin (by the nephelometric method). patients chewed a sterile rubber band and 3 ml of stimulated saliva were collected. the samples were stored at -5 °c, centrifuged at 2,800 rpm for 10 min, and the supernatant was removed and stored at -20 °c. data were presented as frequencies and proportions for qualitative variables and measures of central tendency and dispersion for quantitative variables. data were analyzed by either analysis of variance or kruskal wallis test . a p value <0.05 was considered statistically significant. results: significant relationships were observed between the concentration of salivary urea and periodontal status (p = 0.03) and the presence of dental calculus and urea (p = 0.04) was demonstrated. conclusions: a relationship between the salivary urea concentration and the presence of periodontal disease and dental calculus is suggested. keywords: dental calculus; urea; periodontal diseases; saliva. introduction saliva is an important biological fluid in oral physiology1. the reduction of salivary secretion or changes in the properties of the saliva are responsible for a lot of dental and oral problems, such as cavities or periodontal disease, as they remain major diseases, with a direct impact on the quality of life of patients affected by them2-4. among the important components of saliva, there are several enzymes released by stromal, epithelial cells and by bacteria. salivary amylase involved in digestion of starches also acts as a buffer to protect oral pharyngeal and esophageal mucosae from ingested acids. saliva protects the teeth against acid by its bicarbonate as buffer and urea 5. moreover, drugs like cyclic antidepressants and others could affect the levels of salivary amylase, total proteins and urea6-7. braz j oral sci. 14(2)159-165 received for publication: april 07, 2015 accepted: june 17, 2015 http://dx.doi.org/10.1590/1677-3225v14n2a12 160160160160160 analysis of the enzymes in salivary secretion and in the crevicular fluid may help to clarify the pathogenesis and improve the early diagnosis and prognosis of periodontal disease8-10. among salivary composition studies are those concerning lactoferrin, urea, glucose, total proteins, which provide advantages such as elasticity, moisture, buffering effect and repair, thus promoting oral health11-15. with respect to periodontal disease, investigations evaluate the salivary chemical compounds, which contribute in the destruction and/or protection of the periodontal tissues. concentration of some salivary compounds increases or decreases in patients with periodontal disease because it contains enzymes from periodontal damaged cells, which can be identified in saliva and gingival crevicular fluid16-17. thus, these biomarkers could be used routinely in clinical practice to assess disease progression. the aim of this study was to determine the chemical components in saliva and their relationship with periodontal status and dental calculus in the elderly. material and methods population and sample an observational analytical cross-sectional study was conducted. the sample consisted of senior patients of the school of dentistry at universidad santo tomás colombia. the sample size was calculated based on a population of 120 individuals, treated at the elderly clinic of universidad santo tomás, between january and december 2013 with 95% confidence level, 5% margin of error and a 30% rate of affected patients. the sample size established for obtaining significant differences was 88 individuals, with an approximate loss of 10%, for a total sample of 98 subjects. people under 55 years old and those who did not wish to participate in the study were not included. in addition, privacy and autonomy was respected, by signing the informed consent and ethical support of the universidad santo tomás, according to national and international standards of human research, # protocol 18/04072013. evaluated variables sociodemographic and socioeconomic data such as age, gender (male and female), origin (rural and urban) and educational level (illiterate, elementary, high school, postsecondary) were collected and the medical condition was registered in a clinical record form. after standardization, clinical assessment was performed to evaluate the periodontal status with a who probe according to the criteria established by bassani et al. (2006)18 and tan (2003)19. for the measurement of dental calculus, the greene and vermillion modified method was used, according to criteria established by aguilar agulló et al. (2003)20. the concentration of amylase, glucose, urea, total proteins and albumin in saliva was evaluated. procedures saliva collection: the patients were instructed not to drink or eat 120 min before saliva sample collection and then asked to chew a previously sterilized rubber band. samples of 3 ml of saliva were collected, stored in 6 ml sterile falcon tubes and kept refrigerated at 5 ºc for 1 h21. the samples were centrifuged at 2,800 rpm for 10 min and the supernatant was separated from the substrate and frozen at -20 ºc. next, the sample was defrosted at room temperature, in groups of twenty samples, and centrifuged at 3,000 rpm. the supernatant was separated again and processed to determine the concentrations of glucose, amylase activity, urea, total protein and albumin. chemical analysis glucose: the concentration of glucose was determined by the glucose oxidase / peroxidase22 method. the technique is based on the following reaction: glucose+h2o+o2 gluconic acid+h2o2 h2o2+4 aminophenazone–fenol quinonaimine+h2o considering the following detection limits: detection limit: 0.23 mg/dl=0.0126 mm/l linearity limit: 500 mg/dl=27.5 mm/l measured by spectrophotometer at 505 nm. total proteins: the concentration of proteins was determined following the bradford method (qian et al., 20149) based on the binding of a dye, coomassie blue g-250 to proteins and considering the following detection limits: detection limit: 250 µg/ml linearity limit: 5 mg/ml albumin it was determined by the nephelometry method23. using spheres that increase the sensitivity, loaded with antibodies against albumin, it was quantified by colorimetry24 in an acid medium with bromocresol green, using a spectrophotometer at 600 nm. the following detection limits were considered: detection limit: 25/µg/ml linearity limit: 2.5 mg/ml urea the urea was quantified by determining the amount of green colored indophenol 25. it was quantified by spectrophotometer at 600 nm. the technique is based on the following reaction: urease urea+h2o nh4+co2 nitroprusside nh4 salicylate+naclo indophenol considering the following detection limits: detection limit: 1.3 mg/dl linearity limit: 300 mg/dl amylase the amylase was quantified by the colorimetric test using chemical salivary composition and its relationship with periodontal disease and dental calculus braz j oral sci. 14(2)159-165 161161161161161 a 2chloro – 4 nitrophenil – malatotriosido (cnpg3) substrate. by longline action of amylase the cnpg3 degrades releasing 2 –chlor– 4 nitrophenol by increasing the absorbance measured on a spectrophotometer at 405 nm26. the technique is based on the following reaction: α amylase 5cnpg3 3cnp+2cnpg2+3g3+2g all the techniques were verified for accuracy and precision processing a sample of known concentration 10 times for each one, always following the same protocol. with these results, standard deviation, mean, coefficient of variation, absolute error and relative error were calculated. normal values of chemicals in saliva: as normal parameters, the following control values reported by carda et al. (2006)27 were considered: glucose: <2 mg/dl; amylase: 11.9-304.7 u/ml; urea: 17-41 mg/dl; albumin: 246-344mg/l; total protein: 1.1-1.8 g/l. parameters were considered high when they were above the reference value and low when they were not equal to or lower than reference values. processing and analysis of data the results of the quantification of quality controls as well as their calculations were entered into excel and the information was processed in spss 21. description of the data, frequencies and proportions were calculated for qualitative variables and measures of central tendency and dispersion for quantitative variables. either an analysis of variance (anova) or a kruskal wallis test was conducted, according to the nature and distribution of the variables. a p<0.05 was considered statistically significant. results the sample included 98 subjects, 55.1% (n=54) of them were women. the mean age of the subjects was 66.92 years with a standard deviation (sd) of 9.23. the mean age of periodontal status in advanced periodontitis was 70.8±14.4 years (figure 1.a) and according to their procedence; urban individuals had a higher participation with 60% in gingivitis and 40% of incipient periodontitis (figure 1.b). the registered medical condition showed that 4 (4.1%) of the fig. 1. sociodemographyc and periodontal status. ¨ chemical salivary composition and their relationship with periodontal disease and dental calculus¨ part a: periodontal status and age; part b: periodontal status and procedence, part c: periodontal status and educational level, part d: periodontal status and sex. spc=supragingival calculus/ sbc= subgingival calculus/pd=probing depth. chemical salivary composition and its relationship with periodontal disease and dental calculus braz j oral sci. 14(2)159-165 162162162162162 patients had acute kidney disorders and chronic kidney conditions were not present. regarding the association between educational level and periodontal status, showed participation of elementary educational level with similar distribution in all cases (figure 1.c). regarding the association between sex and periodontal status, 30 females had periodontal disease, 21 (48.8%) of them presented gingivitis; while 39 males had periodontal disease, 20 (48.8%) of them with incipient periodontitis. there was statistically significant (p=0.017) between sex and periodontal status (figure 1.d). regarding dental calculus, the age had similar distribution (figure 2.a). according to the precedence, the rural group patients without dental calculus had a higher distribution of cases (n=32; 43.2%) (figure 2.b). regarding the educational level, elementary education had the highest participation and 15 (19.2%) patients had no dental calculus (figure 2.c). in the analyses of dental calculus versus sex, higher percentages of dental calculus were found in males (figure 2.d). the salivary components were evaluated for total proteins, glucose, albumin, urea and amylase, which were measured quantitatively (by mean and standard deviation) and qualitatively (high, low and normal concentration of chemicals). the average values found globally and according to reference data are shown in table 1. regarding to confidence intervals, glucose and total protein were found in high concentrations in nearly all patients, except for a subject with an average glucose of 2.0 mg/dl. low albumin concentrations were found in a large part of the population and the patients presented normal concentrations of urea and amylase (table 1). regarding periodontal status and its relationship with the chemical concentrations of the various salivary components, increased amylase and albumin concentrations was observed in patients with advanced periodontitis, but with no statistically significant differences. on the other hand, a significant association (p = 0.03) was found for urea concentration and periodontal disease (table 2), especially in patients with high concentrations of urea and diagnosed with advanced periodontitis. regarding the presence of dental calculus, a higher concentration of amylase was observed in the presence of calculus in more than 2/3 of the tooth surface with no significant difference. regarding urea, as well as periodontal fig. 2. sociodemographyc and presence of calculus.¨salivary chemical characteristics and its relationship with periodontal disease & dental calculus¨ part a: presence of calculus and age; part b: presence of calculus and procedence, part c: presence of calculus and educational level, part d: presence of calculus and sex. chemical salivary composition and its relationship with periodontal disease and dental calculus braz j oral sci. 14(2)159-165 163163163163163 total protein (g/l) 2.1-2.5 2.3±1.0 2.6-3.1 2.8±0.9 0.4-1.4 0.9±0.5 1.35-1.65 1.5±0.4 glucose (mg/dl) 2.6-2.7 2.7±0.2 2.6-2.7 2.7±0.2 ——— ——— ——— 2.0 albumin (mg/l) 1.9-215.1 206.2±44.6 ——— ——— 185.2-201.9 193.5±37.2 243-274.6 258.8±32.8 urea (mg/dl) 30.3-44.1 37.2±34.2 62.8-95.0 78.9±40.7 11.4-13.6 12.5±2.7 24.9-29.3 27.1±7.0 amylase (u/ml) 240.4-395.3 317.9±386.2 447.8-959.6 703.7±619.9 ——— ——— 171.9-199.6 185.7±59.3 i c x i c x ±sd i c x±sd i c x±sd global h i g h low normal salivary chemistry confidence interval 95%. x: mean sd: standard deviation. high: those above the reference value. low: those not equaled and lower than the reference value. normal: glucose: <2 mg / dl; amylase: 11.9-304.7u / ml; urea: 17-41mg / dl; albumin: 246-344mg / l; total protein: 1.1-1.8g / l table 1:table 1:table 1:table 1:table 1: concentration of chemicals in saliva. dental calculus salivary composition total proteins (g/l) albumin (mg/l) urea (mg/dl) amylase (u/ml) glucose (mg/dl) global i c 2.18-2.66 198.9-218.7 29.02-42.08 247.2-439.9 2.64-2.67 no calculus n=32 x±sd 2.3±1.1 206.6±28.0 27.2±22.4 326.4±229 2.7±0.2 calculus <1/3 of the tooth n=29 x±sd 2.4±1 213.9±47.2 38±32.7 316.2±231.3 2.6±0.2 calculus >1/3 <2/3 of the tooth n=13 x±sd 2.6±1 208.4±67 41.8±27.1 247±192.3 2.7±0.31 calculus >2/3 of the tooth n=34 x±sd 2.2±0.8 190.7±40.1 63±38 993±1698.6 2.8±0.3 p 0.38* 0.66* 0.04** 0.80** 0.61* table 3:table 3:table 3:table 3:table 3: dental calculus and salivary composition *anova **kruskal wallis x: mean n:subjects sd: standard deviation disease, a statistically significant difference (p=0.04) was observed when relating urea and the presence of dental calculus (table 3), especially when it is observed in more than 2/3 of the tooth structure. generally, it could be established that the higher the concentration of urea, the greater the presence of calculus and the severity of periodontal disease. in order to assess the concentration of salivary urea and kidney disease, the urea average was 28.8 mg/dl in patients with acute kidney conditions. compared with patients without it, in whom the urea mean was 37.6 mg/dl there was no statistically significant difference (p=0.46). discussion protein concentration is considered a biomarker for periodontal disease, since plasma protein leakage occurs as a result of the inflammatory process, which raises the concentration of total proteins in saliva. in this study, higher concentrations of total proteins in the whole population were observed. shaila et al. (2013)28 also found an increase in the concentration of proteins in the presence of gingivitis and periodontitis; however, no differences were found in the group of edentulous patients. it is known that salivary protein content averages 3,000 mg/l. due to these proportions, salivary proteins are involved in numerous biological processes, including cellular support, tissue strain and flexibility, immune response and participation in enzymatic reactions. the functions of these proteins vary according to the type of molecule, so there are some that regulate the maintenance of dental and mucosal integrity, soft tissue repair, regulators of ph and antimicrobial activity. the salivary concentration of this component may be influenced by factors such as the circadian rhythm, the presence of hormones, psychological disorders, tooth brushing and/or exercise29-30. with respect to albumin, which is considered a marker chemical salivary composition and its relationship with periodontal disease and dental calculus braz j oral sci. 14(2)159-165 periodontal status salivary composition total proteins (g/l) albumin (mg/l) urea (mg/dl) amylase (u/ml) glucose (mg/dl) 2.1-2.6 199.1-217.9 29.1-42.1 245.2-424.7 2.64-2.76 gingivitis (bleeding on probing) n=30x±sd 2.2±0.8 201.2±37.4 38.2±38.4 323.3±232.3 2.6±0.2 incipient periodontitis (supragingival calculus) n=39x±sd 2.4±1.2 209.9±47.3 30.1±19.6 293.4±216.1 2.7±0.2 moderate periodontitis (ps=4-5mm) n=10x±sd 2.3±1.0 215.2±46.9 27.7±14.3 257.1±203.2 2.6±0.3 advanced periodontitis (ps=>6mm) n=5x±sd 2.7±0.4 228.2±39.7 79.1±30.5 884.4±1486.4 2.8±0.2 p 0.62* 0.46* 0.03** 0.89** 0.63* confidence interval 95%. ps= probing depth. *anova **kruskal wallis x: mean n:subjects sd: standard deviation table 2:table 2:table 2:table 2:table 2: periodontal status and salivary composition. global i c 164164164164164 of disease, as it originates at a sulcus level. shaila et al. (2013)28 and cheaib and lussi (2013)31 have suggested that the albumin present in saliva is a consequence of contamination by traces of blood or gingival fluid, considering albumin as an infiltration of serum into the mouth. its presence has also been reported in patients with periodontitis32. terrapon et al. (1996)32 in their study, observed an average of over 218.5 mg/l of albumin. these values are close to the average of albumin in our study, with 206.23 mg/l. the same authors found a positive correlation between albumin concentration and the severity of periodontitis, increasing the concentration of salivary albumin. however, those authors did not find a significant relationship between pocket depth and the concentration of albumin. in this study, no significant differences in the concentration increase of albumin were observed in the presence of periodontal disease. nevertheless, an increment in the concentration of albumin was observed as the severity of the disease increased, possibly due to the release of gingival exudate, which increases with the presence of this pathology. shaila et al. (2013)28 found a significant difference in the concentration increase of albumin and periodontal destruction, concurring with terrapon et al. (1996)32, who mentioned that the saliva in edentulous patients contained five times less albumin than that of dentate patients. they suggested the gingival sulcus as origin of the albumin. likewise, this study found lower concentrations of albumin in edentulous patients with no statistically significant differences, confirming the data reported by other authors. meurman et al. (2002) 33 evaluated the salivary albumin, observing higher concentrations of albumin in systemically compromised or weak older adults; what the authors suggest can be used as a reference for comprehensive care of the elderly. regarding the presence of dental calculus, banderastarabay et al. (1997)34 found that the protein concentration decreases in the presence of dental calculus, contrary to this study, which generally found no protein concentration decrease in the presence of calculus. however, a lower total protein concentration in those with more calculus than others or more than 2/3 of the dental surface without statistically significant differences was observed specifically. this may occur due to the presence of subgingival calculus, preventing the release of plasma proteins as a result of the inflammatory process, decreasing the concentration of total proteins. leite et al. (2014)35 suggested that there are reduced risks of inflammatory disease in patients who have a polyunsaturated fatty acid (pufas) rich diet. pufas could be related with an increment of salivary amylase. on the other hand, in this study amylase concentration showed no differences between groups of periodontal disease. it is important to mention urea as a salivary component since it plays a primary role in the formation of dental calculus. this is why significant values were observed in this study with the presence of dental calculus and urea concentration, possibly due to the alkalization of saliva as a result of high concentrations of urea. in this regard, tomás et al. (2008)36 found alkaline saliva and high urea concentration, which shows a relationship with the presence of dental calculus. indirect evidence showed that the metabolism of urea might enhance caries resistance which was seen in patients with chronic renal failure. they are able to produce 10 to 50-fold greater salivary urea levels. patients with low levels of urease have low capacity to neutralize glucolite acidification even though high levels of urea are available. this urease could come from bacteria and be a potential caries control strategy37. other studies have shown the relationship between dental calculus, phosphate and urea concentration in renal patients3839. in this study, the salivary urea concentration was not related to a renal condition because only 4 (4.1%) patients had acute kidney conditions, without significant relationship. queiroz et al. (2013)40 found urea reduction ratio of <65%, in patients with higher frequencies of calculus in pre-transplant patients with chronic kidney disease. based on the results, the information regarding salivary composition and its relationship with the different oral alterations has increased, as demonstrated by periodontal disease and the presence of dental calculus. these findings suggest a relationship between dental calculus and variation in the concentration of salivary urea, which makes the study of saliva meaningful as diagnostic and prognostic aids for clinical assessment in dental practice. acknowledgements the research project had financial support from the departamento administrativo de ciencia, tecnología e innovación colciencias (colombia) and the dentistry school of universidad santo tomás colombia. references 1. llena-puy c. the role of saliva in maintaining oral health and as an aid to diagnosis. med oral patol oral cir bucal. 2006; 11: e449-55. 2. muñoz sl, narvaez cg. salivary ph, buffer capacity, total proteins and salivary flow in controlled hypertensive patients diuretic users. int j odontostomat. 2012; 6: 11-7. 3. hernández castañeda aa, aranzazu moya gc. characteristics and physical-chemical properties of saliva: a review. ustasalud. 2012; 11: 101-11 [spanish]. 4. bretas i, rocha m, vieira m, rodríguez a. flow rate and buffering capacity of the saliva as indicators of the susceptibility to caries disease. pesq bras odontoped clin integr. 2008; 8: 289-93 [portuguese]. 5. dawes c, pedersen am, villa a, ekström j, proctor gb, vissink a, et al. the functions of human saliva: a review sponsored by the world workshop on oral medicine vi. arch oral biol. 2015; 60: 863-74. 6. milton ba, bhambal a, nair p. sialochemical analysis: windfall to the oral physician (a hospital-based clinical cross-sectional study in depressive disorders). j int oral health. 2014; 6: 82-9. 7. milton ba, bhambal a. sialochemical analysis: a portal for the oral diagnostician. j clin diagn res. 2014; 8: zc43-8. 8. cosío arévalo dj, ortega cambrains a, vaillard jiménez e. determination salivary ph before, during and after consumption of sweets in children aged 3, 4 and 5 years old. oral. 2010; 11: 642-5 [spanish]. chemical salivary composition and its relationship with periodontal disease and dental calculus braz j oral sci. 14(2)159-165 9. qian x, dong h, hu x, tian h, guo l, shen q, et al. analysis of the interferences in quantitation of a site-specifically pegylated exendin-4 analog by the bradford method. anal biochem. 2014; 465: 50-2. 10. hegde mn, malhotra a, hegde nd. salivary ph and buffering capacity in early and late human immunodeficiency virus infection. dent res j (isfahan). 2013; 10: 772-6. 11. martins c, buczynski ak, maia lc, siqueira wl, castro gf. salivary proteins as a biomarker for dental caries-a systematic review. j dent. 2013; 41: 2-8. 12. al kawas s, rahim zh, ferguson db. potential uses of human salivary protein and peptide analysis in the diagnosis of disease. arch oral biol. 2012; 57: 1-9 13. acevedo am, laguna f. salivary mucins and their implications for the rheology of human saliva and saliva substitutes. acta odontol venez. 2009; 47. [spanish]. [cited 2014 apr 10] available from: http:// www.scielo.org.ve/pdf/aov/v47n2/art24.pdf. 14. felizardo kr, gonçalves rb, schwarcz wd, poli-frederico rc, maciel sm, andrade fbd. an evaluation of the expression profiles of salivary proteins lactoferrin and lysozyme and their association with caries experience and activity. rev odonto cienc. 2010; 25: 344-9. 15. sajewicz e. effect of saliva viscosity on tribological behavior of tooth enamel. tribology international. 2009; 42: 327-32. 16. wolf dl, lamster ib. contemporary concepts in the diagnosis of periodontal disease. dent clin n am. 2011; 55: 47-61. 17. castrillón rle, ramos ap, cabrera sm. innovative study on lactoferrin in periodontal disease. rev odont mex. 2011; 15: 231-8. 18. bassani dg, da silva cm, oppermann rv. validity of the community periodontal index of treatment needs´(cpitn) for population periodontitis screening. cad saude publica. 2006; 22: 277-83. 19. tan b. basic periodontal examination a simple screening tool in general practice. singapore dent j. 2003; 25: 55-7. 20. aguilar agulló mj, cañamas sanchis mv, ibáñez cabanelli p, gil loscos f. importance of the use of index in the periodontal practice by dental hygienist periodoncia (barc.) 2003; 13: 233-44. [spanish]. 21. vuletic l, peros k, spalj s, rogic d, alajbeg l. time-related changes in ph, buffering capacity and phosphate and urea concentration of stimulated saliva. oral health prev dent. 2014; 12: 45-53. 22. abikshyeet p, ramesh v, oza n. glucose estimation in the salivary secretion of diabetes mellitus patients. diabetes metab syndr obes. 2012; 5: 149-54. 23. florvall g, basu s, helmersson j, larsson a. microalbuminuria measured by three different methods, blood pressure and cardiovascular risk factors in elderly swedish males. anal chem insights. 2008; 3: 69-74. 24. chen ww, guo ym, zheng ws, xianyu yl, wnag z, jiang xy. recent progress of colorimetric assays based on gold nanoparticles for biomolecules. chin j anal chem. 2014; 42: 307-14. 25. shirzaiy m, heidari f, dalirsani z, dehghan j. estimation of salivary sodium, potassium, calcium, phosphorus and urea in type ii diabetic patients. diabetes metab syndr. 2013 mar 14. doi: 10.1016/ j.dsx.2013.02.025. [cited 2014 apr 10]. available from: http://dx.doi.org/ 10.1016/j.dsx.2013.02.025. 26. rohleder n, nater um. determinants of salivary á-amilase in humans and methodological considerations. psychoneuroendocrinology. 2009; 34: 469-85. 27. carda c, mosquera-lloreda n, salom l, gomez de ferraris me, peydro a. structural and functional salivary disorders in type 2 diabetic patients. med oral patol oral cir bucal. 2006; 11: e309-14. 28. shaila m, pai p, shetty p. salivary protein concentration, flow rate, buffer capacity and ph estimation: a comparative study among young and elderly subjects, both normal and with gingivitis and periodontitis. j indian soc periodont. 2013; 17: 42-6. 29. gutiérrez nova p, olivares navarrete r, leyva huerta er. epidermal growth factor and total protein in saliva of smokers and non smokers. av odontoestomatol. 2008; 24: 377-83 [spanish]. 30. zárate daza an, leyva huerta er, franco martínez f. determination of ph and total proteins in saliva in patients with and without fixed orthodontic appliances (pilot study). rev odontol mex. 2004; 8: 59-63 [spanish]. 31. cheaib z, lussi a. role of amilase, mucin, iga and albumin on salivary protein buffering capacity: a pilot study. j biosci. 2013; 38: 259-65. 32. terrapon b, mojon ph, mensi n, cimasoni g. salivary albumin of edentulous patients. arch oral biol.1996; 41: 1183-5. 33. meurman j, rantonen p, pajukoski h, sulkava r. salivary albumin and other constituents and their relation to oral and general health in the elderly. oral surg oral med oral pathol oral radiol endod. 2002; 94: 432-8. 34. banderas-tarabay ja, gonzález-begné m, sánchez-garduño m, milláncortéz e, lópez-rodriguez a, vilchis-velázquez a. flow and protein concentration in human whole saliva. salud publica mex. 1997; 9433-43 [spanish]. 35. leite mf, lima hm, vang sjs, santos mt, ohon r. effect of astaxanthin and fish oil on enzymatic antioxidant. system and a-amylase activity of salivary glands from rats. braz j oral sci. 2014; 13: 58-63. 36. tomás i, marinho js, limeres j, santos mj, araújo l, diz p. changes in salivary composition in patients with renal failure. arch oral biol. 2008; 53: 528-32. 37. reyes e, martin j, moncada g, neira m, palma p, gordan w, et al. caries-free subjects have high levels of urease and arginine deiminase activity. j appl oral sci. 2014; 22: 235-40. 38. martins c, siqueria wl, oliveira e, nicolau j, primo lg. dental calculus formation in children and adolescents undergoing hemodialysis. pediatr nephrol. 2012; 27: 1961-6. 39. andrade mr, salazar sl, de sá lf, portela m, ferreira-pereira a, soares rm, et al. role of saliva in the caries experience and calculus formation of young patients undergoing hemodialysis. clin oral investig. 2015 mar 20 [in press]. doi: 10.1007/s00784-015-1441-4. 40. queiroz sm, amorim ag, andrade ald, gordón-núñez ma, freitas ra, galvao hc. influence of dialysis duration and parathyroid hormone on the clinical and radiographic oral conditions pre-transplant patients with chronic kidney disease. braz j oral sci. 2013; 12: 125-31. 165165165165165 chemical salivary composition and its relationship with periodontal disease and dental calculus braz j oral sci. 14(2)159-165 429 too many requests error 429 too many requests too many requests guru meditation: xid: 37279030 varnish cache server oral sciences n3 original article braz j oral sci. january | march 2015 volume 14, number 1 comparison of two types of biomecanics for deep overbite correction ana de lourdes sá de lira, yarasmin nolêto de sousa alexandrino universidade federal do piauí – ufpi, dental school, department of orthodontics, teresina, pi, brazil correspondence to: ana de lourdes sá de lira universidade federal do piauí, faculdade de odontologia, departamento de ortodontia. rua senador joaquim pires, 2076 -iningacep: 64049-590 teresinapibrasil e-mail: anadelourdessl@hotmail.com abstract aim: to compare two types of treatment for class ii deep overbite malocclusion assessing maxillary and mandibular arches behavior in subjects submitted to full orthodontic treatment with standard edgewise appliance and those who used straight wire appliance. methods: the sample consisted of 50 patients treated with full fixed appliances either with edgewise appliance (n=25, group 1), or with straight wire appliance (n=25, group 2). in both groups lateral cephalometric radiographs were compared with those done at the beginning of treatment and at its end, in order to quantify the cephalometric measures (8 linear and 6 angular) presenting the maxillary and mandibular arches behavior in the anteroposterior and vertical directions. all patients were treated without extraction or use of class ii intermaxillary elastics during the full orthodontic treatment. results: in both groups the treatment of malocclusion contributed for mandibular forward displacement, reduction of deep overbite and overjet, reduction of mandibular plane with anti-clockwise rotation and labial projection of maxillary incisors. conclusions: in both groups the sample showed favorable mandibular displacement, reduction of facial convexity, and profile improvement with anti-clockwise rotation. the correction of deep overbite was due to labial projection and intrusion of maxillary incisors. keywords: malocclusion; overbite; orthodontics. introduction deep overbite is a malocclusion with skeletal dental and neuromuscular implications characterized by the excessive vertical trespass of incisors1. excessive overlapping of the maxillary incisors over mandibular incisors is noted when values above of 40% by vertical trespass of upper incisors are observed2. it can be found in class i and class ii malocclusions, particularly in class ii division 2 malocclusion3, and be associated with incisors’ wear, palatal lesions and damaged esthetics4, periodontal disease, functional deviations, inadequate mastication, occlusal trauma, teeth grinding and temporomandibular joint dysfunction5. several etiologic factors have been associated with the occurrence of deep overbite6-7 and they may be of genetic or dentofacial development source origin2, involving change of maxillomandibular growth, modification of labial and lingual functions and dentoalveolar alterations8. among these factors are overstated incisors eruption, excessive overjet, incisor mesiodistal width, incisor inclination, canine position, molars infraocclusion, molar cusp height, mandibular rami height and vertical facial type6. deep bite has potentially harmful effects to mandibular development and may be associated to functional disturbances9. the treatment allows favorable mandibular growth9-10. if not treated, it may be observed mandibular displacement difficulty, faulty mandibular functional movement, masticatory cycle alteration, braz j oral sci. 14(1):71-77 received for publication: january 23, 2015 accepted: march 23, 2015 7272727272 pronounced anterior guide, interference on protrusion and lateral movements, loss of vertical dimension, facial alterations and periodontal disease. it is considered the most harmful malocclusion to dental and alveolar tissues, causing modifications of masticatory function and in temporomandibular articulation, with implications in facial esthetics. maxillary incisor intrusion may be adequate to individuals who exhibit maxillary vertical excess, gum smile, lack of labial seal, short upper lip, increased inferior facial height and inclined occlusal plane10-12. it is indicated to persons with good facial harmony, normal occlusal plane or a little inclined and with excessive spee curve in the mandibular arch12. posterior tooth extrusion is ideal for patients with hypodivergent facial pattern (short anterior facial height) redundant lips, excessive spee curve, incisor exposition from moderate to minimal in the growth spurt phase. this procedure will provide inferior facial height increase, mandibular plane opening, facial convexity increase and reduction of inferior lip projection4,10-12. the evaluation of post-treatment results may reveal whether the treatment goals were achieved6. for nanda4, the stability of deep bite treatment depends on specific dental correction (intrusion, extrusion or inclination), growth spurt and neuromuscular adaptation. the few studies that dealt with this subject indicate that active treatment is able to induce a moderate improvement of the overbite, but unfortunately the few studies assessing the effects of orthodontic treatment in deep bite patients either did not include untreated control subjects13-18 or used controls with normal overbite values19. due to the scarcity of studies investigating skeletal and dental modifications caused by deep overbite treatment, two types of biomechanics with fixed appliances were searched. the objective of the present study was to compare two types of treatment for class ii deep overbite malocclusion assessing maxillary and mandibular arch behavior in individuals submitted to full orthodontic treatment with standard edgewise appliance or those who used straight wire appliance. material and methods the ufpi’s ethics committee approved the development of this study under the protocol number (caae 35309414800005209). this clinical research was based on 50 caucasian brazilian individuals, 13 girls and 12 boys, who underwent full edgewise appliance (group 1); and 15 girls and 10 boys that were treated using straight wire appliance (group 2) during a 26-month period in the post-graduation orthodontic course of the federal university of piauí. all patients were treated in a single phase without extractions and no association of intermaxillary elastics. each patient was evaluated two times by lateral cephalometric radiographs: at the beginning of the treatment (t0) and at the end of the active orthodontic treatment (t1). all the subjects were in the pubertal growth spurt period at the beginning of the orthodontic treatment, with skeletal pattern of class ii evidenced by anb angle>5o and wits>0 mm, overbite in average between 5 5.5 mm (80% 90%) and overjet between 3-3,5 mm. the skeletal maturity stage of all individuals was analyzed by hand and wrist radiographs. the dental relationship was class ii, according to angle classification. the individuals also exhibited in average sn.gogn angled 33o. in group 1 the mean age for female patients at t0 was 11.8 (±0.61) years; at t1 it was 13.10 (±1.43) years. for male patients at t0 was 12.9 (±0.62) years; at t1 it was 14.11 (±1.42) years. in group 2 the mean age for female patients at t0 it was 12.2 (±0.54) years; at t1 it was 14.4 (1.22) years. for male patients at t0 it was 12.9 (±0.56) years; at t1 it was 14.11 (±1.35) years. the cephalograms were obtained by delimitating skeletal, dental and tegumentary structures. the measurements from cephalometric tracings regarding t 0 and t 1 were tabulated for statistical analysis, with angular measurements rounded up whenever decimal fraction existed. eight linear and six angular measurements were obtained in the cephalometric tracing regarding t0 and t1 (figures 1 and 2). to measure skeletal linear modifications a perpendicular line was traced from sn plane (skull base), passing by s point, and called s’ line. a parallel line to sn plane was traced between s’ and ena point (s’ena) to describe the anterior maxillary displacement and between s’ and pog point (s’pog) to measure the horizontal mandibular displacement. the lengths of palatal plane linear and of mandibular plane in t0 and t1 were measured to examine fig. 1cephalogram showing linear measurements (mm) s’-ena, s’-pog, palatal plane, go-gn, 1-na, 1-nb, overjet and overbite. comparison of two types of biomecanics for deep overbite correction braz j oral sci. 14(1):71-77 fig. 2cephalogram illustrating angular measurements: sn.pp, sn.gogn, sna, snb, 1.na, 1.nb. probable maxillary and mandibular horizontal growth. to describe skeletal vertical changes the sn-palatal plane and sn-gogn angles were evaluated. sna and snb angles were measured to analyze skeletal horizontal changes. initial and final overbite were obtained from the distance between incisal border of maxillary central incisor to mandibular central incisor. overjet was measured from least distance of vestibular face of mandibular central incisor to palatal face of maxillary central incisor (figure 1). the incisor anteroposterior modifications were observated from linear measurements (1-na, 1-nb and overjet) (figure 1) and angular measurements (1.na and 1.nb) (figure 2). vertical changes also were evaluated from linear measurements (figures 1, 3 and 4). in the molar tracings, vertical linear measurements were made to quantify the extrusion or intrusion on maxillary and mandibular partial superimpositions at t0 and t1 (figures 3 and 4). a line was traced on the long axis of these teeth, at a 90º to another line tangent to the incisal border and molar oclusal border. the intersection between these lines was used as a reference point. the difference in vertical direction between initial (black tracing) and final (red tracing) positions of teeth on their tracings (figures 3 and 4) was measured. total superimpositions of initial and final tracing from sn plane (sella-nasion) with register for s were made to analyze the growth direction, the orthodontic treatment and dental movements. means and standard deviations were calculated for each fig. 3 partial superimposition for ena with linear measurements showing vertical changes of maxillary teeth. cephalometric measurement at t0 and t1. the statistical treatment of the data between t0 versus t1 was analyzed by using the paired student’s t test with 5% significance level. unpaired t tests were used to evaluate the differences in therapeutic effects and the lengths of active treatment between both groups. the pearson’s r correlation coefficient was applied to determine whether any skeletal or dental characteristics and age were related to the appliance of active treatment. error of the methods the error of the method was evaluated by 30 radiographs chosen at random, traced and digitized by the same investigator on two separate occasions at least two months apart. the dahlberg20 formula was used: me =\/σ d2/2n, where n is the number of duplicate measurements. random errors varied between 0.26 and 0.92 mm for linear measurements and between 0.28 º and 1.1º for angular measurements. fig. 4 partial superimposition for symphysis internal contour with linear measurements showing vertical changes of mandibular teeth. 7373737373comparison of two types of biomecanics for deep overbite correction braz j oral sci. 14(1):71-77 7474747474 results both groups had comparable mean cephalometric values for linear measurements: s’ -ena, s’-pog, palatal plane, 1na, 1-nb, overjet and overbite and angular measurements: snpp, sngogn, sna, snb, 1-na and 1-nb. comparison between group 1 and group 2 at t0 is shown in table 1. treatment effects on cephalometric values and differences between the two groups may be seen in table 2. dental intrusion and extrusion (mm) at the end of treatment is shown in table 3. the correlation with type of appliance during orthodontic treatment in both groups is presented in table 4. 6± 0.53 28.5± 0.58 87± 0.52 80± 0.47 20± 0.41 41± 0.44 12± 0.51 33± 0.54 86± 0.51 78± 0.55 15 ± 0.44 30± 0.52 6 4.5 1 1 5 11 <0.001 <0.001 0.003 0.001 <0.001 <0.001 * * * * * * * * * * * * measurements group 1 group 2 n=20 n=20 mean sd mean sd difference p significance s´ena(mm) 79 ± 0.56 67 ± 0.56 12 <0.001 ** s´pog(mm) 62 ± 0.47 49± 0.40 13 <0.001 ** palatal plane(mm) 58 ± 0.57 51 ± 0.49 7 <0.001 ** go-gn(mm) 76.5± 0.41 77± 0.52 0.5 0.001 ** 1-na(mm) 4.5± 0.43 3.5± 0.53 1 <0.001 ** 1-nb(mm) 10.5± 0.52 6.5± 0.46 4 <0.001 ** overjet(mm) 3± 0.52 3.5± 0.48 0.5 0.001 ** overbite(mm) 5± 0.37 5.5± 0.49 0.5 0.001 ** sn.pp(0) sn.gogn(0) sna(0) snb(0) 1.na(0) 1.nb(0) table 1. table 1. table 1. table 1. table 1. comparison between group 1 and group 2 at t0 sd= standard deviation; **=1% significance level. s´ena(mm) s´pog(mm) palatal plane(mm) go-gn(mm) 1-na(mm) 1-nb(mm) overjet(mm) overbite(mm) sn.pp(0) sn.gogn(0) sna(0) snb(0) 1.na(0) 1.nb(0) mean sd difference 1 ± 0.25 0.8 ± 0.22 1 ± 0.17 1.5± 0.11 0.5± 0.23 0.5± 0.12 -1.5± 0.21 -2± 0.17 1± 0.23 -0.5± 0.31 -1± 0.62 0.3± 0.72 5± 0.41 -2± 0.64 mean sd difference 1 ± 0.22 1± 0.25 1 ± 0.19 1.3± 0.12 1.5± 0.53 0.5± 0.16 -1.5± 0.28 -3± 0.21 0.3± 0.21 -0.4± 0.34 -1± 0.59 1± 0.65 9 ± 0.44 3± 0.52 mean difference -0.2 0.2 -1 -1 1 0.7 -0.1 -0.7 -4 -5 significance n s n s n s n s * * * * n s * * * n s n s * * * * * measurements group 1 t1-t0 group 2 t1-t0 group 1 n=20 n=20 group 2 sd= standard deviation ** =1% significance level * = 5% significance level ns= non-significant. table 2.table 2.table 2.table 2.table 2. treatment effects on cephalometric values and differences between the two groups in both groups was observed anterior mandibular displacement, reduction of deep overbite and overjet, reduction of mandibular plane with anti-clockwise rotation and labial projection of maxillary incisors. in group 1, significant vertical displacement contributed to reduce the overbite. discussion in this study the cephalometric evaluation demonstrated that in both groups there was skeletal class ii with mandibular retraction, deep overbite and favorable vector of facial growth (table 1). the group tracings were compared in relation to the comparison of two types of biomecanics for deep overbite correction braz j oral sci. 14(1):71-77 7575757575 group 1 group 2 incisors maxillary intrusion (1.0) intrusion (2.0) mandibular intrusion (1.0) intrusion (2.0) molars maxillary extrusion (0.5) intrusion (0.5) mandibular extrusion (1.0) intrusion (0.5) table 3. table 3. table 3. table 3. table 3. dental intrusion and extrusion (mm) at the end of the treatment s´ena(mm) s´pog(mm) palatal plane(mm) go-gn(mm) 1-na(mm) 1-nb(mm) overjet(mm) overbite(mm) sn.pp(0) sn.gogn(0) sna(0) snb(0) 1.na(0) 1.nb(0) mean sd 79 ± 0.56 62 ± 0.47 58 ± 0.57 76.5± 0.41 4.5± 0.43 10.5± 0.52 3± 0.52 5± 0.57 6± 0.53 28.5± 0.58 87± 0.52 80± 0.47 20± 0.41 41± 0.44 difference -0.002 -0.001 -0.003 -0.08 -0.06 -0.03 -0.017 -0.06 -0.02 0.03 -0.05 -0.08 -005 -0.04 n s n s n s n s n s n s n s n s n s n s n s n s n s n s mean sd 67 ± 0.56 49± 0.40 51 ± 0.49 77± 0.52 3.5± 0.53 6.5± 0.46 3.5± 0.48 5.5± 0.42 12± 0.51 33± 0.54 86± 0.51 78± 0.55 15 ± 0.44 30± 0.52 difference -0.03 0.04 0.03 -0.05 -0.01 -0.03 -0.05 -0.04 -0.06 0.04 -0.02 -0.03 -0.05 0.02 n s n s n s n s n s n s n s n s n s n s n s n s n s n s group 1 n=20 group 2 n=20 sd= standard deviation ns= non–significant. table 4.table 4.table 4.table 4.table 4. correlation with type of appliance during orthodontic treatment skull base (sn) and it was diagnosed that the group 1 showed brachiocephalic pattern, lower mandibular plane (sn.gogn) and both maxillary and mandibular arches were more advanced (s’-ena e s’-pog) than group 2. in this group the mesocephalic pattern prevailed (sn.pp, sn.gogn), with more mandibular retrognathism (s’-pog, snb and overjet) than group 1. overbite also was more pronounced, probably due to retroinclination of maxillary and mandibular incisors (table 1). the main strategy of deep bite treatment for both groups consisted of combined extrusion of the posterior teeth, intrusion and vestibular inclination of the anterior teeth (maxillary and mandibular incisors). this is confirmed in the literature because the decision of extrusion or intrusion should be based on skeletal pattern, vertical pattern, facial esthetic, growth spurt, occlusal plane, severity of final malocclusion, occlusion stability or the combination of these factors1,4-7,10,13-19. at first in group 1 segmented arches (rickets’ technique) 0.018´x 0.025´ were used for intrusion and projection of maxillary and mandibular incisors, enabling the decrease of deep overbite, with anterior brackets glued in the most possible incisal level. in group 2 continuous arches of nickel and titanium 0.014´ e 0.016´ with pronounced spee curve in the maxillary arch and reverse curve in the mandibular arch were utilized for little molar intrusion, labial projection and incisor intrusion in deep overbite correction. since they are flexible, their sockets are complete in all teeth from the beginning of the treatment, as observed by burstone7 and chen et al.10. after this, in groups 1 and 2 continuous arches of leveling were sequentially used to maintain spee curve alterations and obtain the best possible mandibular displacement in downward and forward directions during growth spurt. this approach is corroborated by martinelli et al.19, who reported that the favorable mandibular displacement enables achieving the best harmonious profile, and by woods18 who affirmed that the extrusive movement of only 1 mm of maxillary and mandibular molars reduces effectively the overbite between 1.5 to 2.5 mm. in group 1, based on total cephalometric superimposition, skeletal pattern and facial profile maintained the same proportions with maxillary and mandibular residual growth vector to the down and forward directions, associated with the orthodontic mechanics of extrusion to maxillary and mandibular molars during deep bite correction (tables 1-3), (figure 5). the reduction of sna angle with retraction of a point probably resulted from maxillary incisors projection. in spite of residual growth with anterior displacement and anticlockwise rotation of the mandible, there was little reduction of snb angle, probably due the forward nasion displacement (table 2), (figure 5). the deep overbite correction occurred because there were intrusion and labial inclination of maxillary incisors and extrusion of maxillary and mandibular molars. the overjet comparison of two types of biomecanics for deep overbite correction braz j oral sci. 14(1):71-77 7676767676 fig. 6total superimposition of tracing for sn at t0 and t1 to group 2. fig. 5total superimposition of tracing for sn at t0 and t1 to group 1. decreased demonstrating late growth of the mandible, in spite of the labial inclination of maxillary incisors and the uprighting of mandibular incisors (tables 2 and 3). the correction of overbite may be supported because the molar extrusions and the mandibular displacement forward and downward occurred harmoniously, although the mean value of sn.gogn reduction was 0.5 degree (tables 2 and 3). the anti-clockwise rotation during puberal growth spurt, representative of brachiocephalic pattern, was not observed probably due the molar extrusions, corroborating with the studies of some authors13-19 . the total cephalometric superimposition between t0 and t1 of group 2 showed forward displacement and anticlockwise rotation of the mandible, together with profile improvement. this was confirmed by increased snb angle and linear measurements s’–pog and go-gn, as well as decreased sn.gogn angle and overjet. the reduction of sna angle probably happened due to a point backward displacement with labial inclination of maxillary incisors (table 2 and fiure 6). the deep overbite correction occurred basically with labial projection and intrusion of maxillary and mandibular incisors. the intrusion of maxillary molars was necessary to correct the reverse spee curve. nevertheless, in the mandibular arch molar intrusion was observed to decrease the spee curve, contributing for an average 0.4 º decrease of sn.gogn and anti-clockwise rotation of the mandible (tables 2 and 3) (figure 6). these data were corroborated by baccetti et al.15 and martinelli et al.19, who reported that the overbite correction resulted of the intrusion of maxillary incisors as well as mandibular incisors accompanied by mandibular growth. similar results were found by quintao et al.17 and woods18 who investigated the dental movement during deep overbite correction of 87 subjects with class ii division 1 malocclusion and they concluded that the correction basically occurred by intrusion and labial projection of mandibular incisors. there were no outcome differences between the two groups during the treatment of class ii deep overbite malocclusion in relation to the orthodontic technique applied (table 4). similar results were found by ghafari et al.2, who compared intrusive mechanics by segmented arches with the treatment using continuous arches of nickel and titanium and observed real intrusion of maxillary and mandibular incisors. woods18 evaluated initial and final cephalograms of patients class ii division 1 and division 2 at the end of mixed dentition to determine if skeletal and dentoalveolar alterations with deep overbite were influenced by direction of mandibular growth during the treatment. he concluded that in patients with brachiocephalic and mesiocephalic patterns the point b moved significantly more forward than in dolicocephalic patients. similar outcomes also were found in this study. in conclusion, deep overbite correction in group 1 was supported especially by incisors intrusion and molars comparison of two types of biomecanics for deep overbite correction braz j oral sci. 14(1):71-77 7777777777 extrusion, associated with mandibular displacement, due to growth spurt with anti-clockwise rotation. in group 2 the incisors’ intrusion and labial projection of maxillary incisors provided the deep overbite correction. meanwhile the molars intrusion to correct spee curve promoted mandibular forward displacement with anti-clockwise rotation. in both groups the treatment showed reduction of facial convexity and improvement in profile. references 1. baccetti t, franchi l, mcnamara ja jr. longitudinal growth changes in subjects with deepbite. am j orthod dentofacial orthop. 2011; 140: 202-9. 2. ghafari jg, macari at, haddad rv. deep bite: treatment options and challenges. seminars orthodontics. 2013; 19: 253-66. 3. lindauer sj, lewis sm, shroff b. overbite correction and smile aesthetics. semin.orthod. 2005; 11: 62-6. 4. nanda sk. growth patterns in subjects with long and short faces. am j orthod dentofacial orthop. 1990; 98: 247-58. 5. schutz-fransson u, bjerklin k, lindsten r. long-term follow-up of orthodontically treated deep bite patients. eur j orthod. 2006; 28: 503-12. 6. parker cd, nanda rs, currier gf. skeletal and dental changes associated with the treatment of deep bite malocclusion. am j orthod dentofacial orthop. 1995; 107: 382-93. 7. burstone cj. biomechanics of deep overbite correction. seminars orthodontics. 2001; 7: 26-33. 8. franchi l, baccetti t, giuntini v, masucci c, vangelist a, defraia e. outcomes of two-phase orthodontic treatment of deep bite malocclusions. angle orthod. 2011; 81: 941-52. 9. mcdowell eh, baker im. the skeletodental adaptations in deep bite correction. am j orthod dentofacial orthop. 1991; 100: 370-5. 10. 10 chen yj, yao ccj, chang hf. nonsurgical correction of skeletal deep overbite and class ii division 2 malocclusion in an adult patient. am j orthod dentofacial orthop. 2004; 126: 371-8. 11. lira als, souza mmg, bolognese am. long-term maxillary behavior in treated skeletal class ii malocclusion. braz j oral sci. 2012; 11: 120-4. 12. lira als, souza mmg, bolognese am, nojima m. comparison of 2 types of treatment of skeletal class ii malocclusion: a 5-years postretention analysis. braz j oral sci. 2014; 13: 251-6. 13. martinelli fl, ruellas ac, lima em, bolognese am. natural changes of maxillary first molars in adolescent with esqueletal class ii malocclusion. am j orthod dentofacial orthop. 2010; 137: 775-81. 14. engel g, cornforth g, damerell jm, gordon j, levy p, mcalpine j et al. treatment of deep bite cases. am j orthod. 1980; 77: 1-13. 15. baccetti t, franchi l, giuntini v, masucci c, vangelisti a, defraia e. early vs late orthodontic treatment of deep bite: a prospective clinical trial in growing subjects. am j orthod dentofacial orthop. 2012; 142: 75-82. 16. millett dt, cunningham sj, o’brien kd, benson pe, oliveira cm. treatment and stability of class ii division 2 malocclusion in children and adolescents: a systematic review. am j orthod dentofacial orthop. 2012; 142: 159-69. 17. quintao cca, miguel jam, brunharo ip, zanardi g, feu d. is traditional treatment a good option for na adult with a class ii deepbite malocclusion. am j orthod dentofacial orthop. 2012; 141: 105-12. 18. woods mg. sagittal mandibular changes with overbite correction in subjects with different mandibular growth directions: late mixed-dentition treatment effects. am j orthod dentofacial orthop. 2008; 133: 388-94. 19. martinelli fl, reale cs, bolognese am. class ii malocclusion with deep overbite: a sequential approach. dental press j orthod. 2012; 17:76-82. 20. dahlberg g. statistical methods for medical and biological students. london: allen & unwin; 1940. comparison of two types of biomecanics for deep overbite correction braz j oral sci. 14(1):71-77 oral sciences n3 original article braz j oral sci. july | september 2013 volume 12, number 3 correlation of phospholipase and proteinase production of candida with in vivo pathogenicity in galleria mellonella rodnei dennis rossoni1, júnia oliveira barbosa1, simone furgeri godinho vilela1, jéssica diane dos santos1, antonio olavo cardoso jorge1, juliana campos junqueira1 1 department of biosciences and oral diagnosis, institute of science and technology, unesp univ estadual paulista, são josé dos campos, sp, brazil correspondence to: rodnei dennis rossoni department of biosciences and oral diagnosis universidade estadual paulista – unesp avenida eng. francisco josé longo 777 cep: 12245-000 são josé dos campos, sp, brasil e-mail: dennisrossoni@hotmail.com abstract an essential factor to the virulence of the genus candida is the ability to produce enzymes and this may be crucial in the establishment of fungal infections. aim: this study investigated in vitro enzymatic activities of candida species and their virulence in an in vivo galleria mellonella experimental model. methods: twenty-four clinical strains of candida spp. isolated from the human oral cavity were evaluated, including the following species: c. albicans, c. dubliniensis, c. glabrata, c. tropicalis, c. krusei, c. parapsilosis, c. norvegensis, c. lusitaniae and c. guilliermondii. all candida strains were tested in vitro for production of proteinase and phospholipase. the candida strains were also injected into galleria mellonella larvae to induce experimental candidiasis, and after 24 hours, the survival rate was assessed. results: phospholipase and proteinase activity were observed in 100% of the c. albicans strains. in the non-albicans species, proteinase and phospholipase activity were observed in 25 and 43% of the studied strains, respectively. the most pathogenic candida species in g. mellonella were c. albicans, c. dubliniensis and c. lusitaniae, whereas c. glabrata was the least virulent species. furthermore, a positive significant correlation was found between both enzymatic activities with virulence in g. mellonella. conclusions: the virulence of candida strains in g. mellonella is related to the quantity of proteinases and phospholipases production of each strain. keywords: candida, virulence factors, invertebrates. introduction as the most common yeasts in humans, candida spp. are responsible for most fungal diseases. therefore, understanding the mechanisms by which these microorganisms colonize and cause disease in humans is a great challenge for planning and establishing treatments1. candida species have many virulence factors, which in the presence of local and systemic host failures may result in their transition from commensal to pathogenic organisms. the virulence factors vary between different candida species. c. albicans is the most virulent species, and it has virulence factors that allow it to adhere to oral tissues, invade tissue, escape host defenses, form germ tubes and hyphae, and produce histolytic enzymes such as proteinases and phospholipases2-3. there are two major families of histolytic enzymes produced by candida species: the secretory aspartyl proteinases (sap) and phospholipases (pl). proteinases are central virulence factors that facilitate the colonization and invasion of host received for publication: june 12, 2013 accepted: september 13, 2013 braz j oral sci. 12(3):199-204 tissues via degradation of physiological substrates, such as albumin, immunoglobulins and skin proteins, and inhibition of phagocytosis-inducing inflammatory reactions 4-6. phospholipases also play a role in candida pathogenesis, as they may damage the host cell membranes. various candida species produce proteinases and phospholipases, although in lower amounts than c. albicans6. the pathogenic mechanisms of candida yeasts and fungus-host interactions have been studied in rat and mouse experimental models of candidiasis7-11. recent in vivo studies in invertebrate models of candidiasis investigated the humoral and cellular immune responses of the host. invertebrates have several advantages over the conventional mammalian models, including lower costs, faster results and fewer ethical issues12-13. galleria mellonella, the greater wax moth larva, is an interesting model for in vivo studies of pathogenic yeasts, such as c. albicans, c. parapsilosis, c. glabrata, c. tropicalis, and c. krusei, and bacteria, including pseudomonas aeruginosa, proteus mirabilis, escherichia coli and bacillus cereus14. aside from its low costs, g. mellonella allows systemic inoculation of these pathogens. moreover, g. mellonella is the insect most widely used to study human immune responses15-16 . brennan et al.17 demonstrated that virulence deviations of hyphal-deficient mutants detected in galleria mellonella are similar to those observed in mice. the response of insects to infection shows strong similarities to that found in mammals and opens the possibility of reducing the need to use mammals for testing the virulence of mutants by employing galleria larvae as a primary screening mechanism. the ability to screen a large number of mutants in a short time using g. mellonella would have a number of cost and labor advantages compared to the use of conventional vertebrate models. since enzymatic activity may be crucial in the establishment of fungal infections18-19, the aim of this study was to correlate the production of proteinases and phospholipases by candida species with their virulence in g. mellonella. materials and methods candida isolates a total of 24 clinical candida strains recovered from the oral cavity of hiv-positive patients seen at the emílio ribas institute of infectious diseases (são paulo, brazil) were studied. the isolates included the following species: c. albicans (n=8), c. glabrata (n=4), c. dubliniensis (n=3), c. tropicalis (n=2), c. krusei (n=2), c. parapsilosis (n=2), c. guilliermondii (n=1), c. lusitaniae (n=1) and c. norvegensis (n=1). the identification of candida species was done by growth on hicrome candida (himedia, mumbai, maharashtra, india), germ tube test, clamydospore formation on corn meal agar, and api20c for sugar assimilation (biomerieux, marcy l’etoile, france). the identity of c. dubliniensis was determined by a multiplex polymerase chain reaction (pcr) procedure. the study was approved by the ethics committee of the school of dentistry of são josé dos campos/unesp (protocol 21/2010/cepa), according to the declaration of helsinki, and followed the guidelines established by “ethical principles for animal experimentation of the brazilian college of animal experimentation” (cobea). all isolates were revived from stock cultures maintained in ypd broth (himedia) with 20% glycerol (amresco, solon, oh, usa) at -80ºc. the cultures in maintenance medium were transferred onto fresh sabouraud’s dextrose agar (sda) plates (himedia) and incubated at 37 ºc for 24-48 h. the fresh cultures were used in enzyme assays and in the g. mellonella infection model. phospholipase activity the candida isolates were evaluated for phospholipase production using the egg yolk agar plate method described by price et al.20 and pereira et al.21. the sda plates contained 57.3 g nacl, 0.55 g cacl 2 and 8% sterile egg yolk emulsion. the test strains were spot inoculated (~6 mm), and the plates were incubated at 37 ºc for up to 5 days. each isolate was tested in quadruplicate. the diameter of the colony and the surrounding precipitation zone (pz) were measured, and phospholipase activity was scored as per the described method20. the pz value represented the ratio of the colony diameter to the diameter of the colony plus the precipitation zone. the results were classified as follows: no activity (pz=1), moderate activity (0.64>pz<1) and strong activity (pz<0.64). by this classification, a high pz value indicates low enzymatic activity. proteinase activity all candida isolates were tested for proteinase secretion in bovine serum albumin (bsa) agar that contained yeast carbon base (1.17%), yeast extract (0.01%) and bsa (0.2%) using the method of ruchell et al.22.the medium was adjusted to ph 5.0, sterilized by filtration and added to autoclaved 2% agar. the test strains were spot inoculated (~6 mm) and the plates were incubated at 37 ºc for up to 5 days. each isolate was tested in quadruplicate. the post-incubation clearance zone around the colony was recorded, and the pz value for proteinase activity was calculated as described above. galleria mellonella infection model g. mellonella were infected with candida as previously described by fuchs et al.23 and junqueira et al. 24. in brief, g. mellonella caterpillars in the final instar larval stage (entomology and plant pathology laboratory, state university of north fluminense, rj, brazil) were stored in the dark and used within 7 days from the date of shipment. sixteen randomly chosen caterpillars (330±25 mg) were infected for each candida isolate for a total of 384 assays. candida inocula were prepared by growing 5 ml ynb broth (1% yeast extract, 1% dextrose and 2% peptone) cultures overnight at 37 ºc. the cells were pelleted at 2000×g for 10 min and washed three times in pbs (laborclin, pinhais, 200200200200200 correlation of phospholipase and proteinase production of candida with in vivo pathogenicity in galleria mellonella braz j oral sci. 12(3):199-204 candida species(number of strains) proteinase activity phospholipase activity ++ + ++ + c. albicans (n=8) 3 5 0 5 3 0 c. glabrata (n=4) 0 0 4 0 0 4 c. dubliniensis (n=3) 0 2 1 0 2 1 c. parapsilosis (n=2) 2 0 0 0 0 2 c. tropicalis (n=2) 0 0 2 1 1 0 c. krusei (n=2) 0 0 2 0 2 0 c. norvegensis (n=1) 0 0 1 0 1 0 c. lusitaniae (n=1) 0 0 1 0 0 1 c. guilliermondii (n=1) 0 0 1 0 0 1 table 1. activity of proteinase and phospholipase for the candida species studied according to the classification of price et al.20: strong activity (+ +), moderate activity (+) and no activity (-) candida species number of deaths mortality rate (number of g. mellonella) (24 h post-infection) (24 h post-infection) c. albicans (n = 128) 125 97.65% c. glabrata (n = 64) 6 9.37% c. dubliniensis (n=48) 47 97.91% c. parapsilosis (n=32) 18 56.25% c. tropicalis (n=32) 6 18.75% c. krusei (n=32) 13 40.62% c. norvegensis (n=16) 2 12.5% c. lusitaniae (n=16) 15 93.75% c. guilliermondii (n=16) 5 31.25% total (n=384) 237 61.71% table 2. g. mellonella larvae killed 24 h after infection by candida species pr, brazil). the cell densities were determined by hemacytometer counting. the candida inocula were confirmed by determining the colony-forming units per milliliter (cfu/ml) on sda. a hamilton syringe (hamilton, reno, nv, usa) was used to deliver candida inocula (105 cells in 10 µl) into the hemocoel of each larva via the last left proleg. the larvae were incubated in plastic containers (37 ºc) and the number of dead g. mellonella was counted after 24 h of infection. statistical analysis percent survival and killing curves of g. mellonella were plotted and statistical analysis was performed by the logrank (mantel-cox) test using graphpad prism statistical software (graphpad software, inc., california, ca, usa) the pearson test was used to correlate phospholipase and proteinase activity with virulence in g. mellonella. this test was performed in the minitab program with a 5% significance level. results all c. albicans isolates (100%) were positive for proteinase and phospholipase activity. only 25% and 43.75% of the nonalbicans species showed proteinase and phospholipase activity, respectively (table 1). the most pathogenic candida species in vivo were c. albicans, c. dubliniensis and c. lusitaniae, which caused over 90% mortality at 24 h post-infection. the least virulent species was c. glabrata with 9.37% mortality (log-rank test: p = 0.0001) (table 2). the results obtained for each candida strain, including the pz values for enzymatic activity and the g. mellonella mortality rates, are shown in table 3. using a pearson test, a significant correlation between the proteinase pz values and g. mellonella mortality was observed (pearson correlation=-0.571, p=0.004). there was a similar correlation between phospholipase pz values and in vivo mortality (pearson correlation=-0.447, p=0.029). according to pereira et al.21, low pz values indicate high enzymatic activity. this study demonstrated that greater production of proteinases and phospholipases by candida strains led to increased virulence in g. mellonella (fig. 1). discussion the identification of virulence factors in candida strains can elucidate their adhesion, invasion and infection process, thus leading to the development of more efficient antifungal therapies25. in the present study was evaluated the production of two types of extracellular enzymes, proteinases and phospholipases, which are virulence factors that are crucial to candida pathogenesis. furthermore, the enzymatic activity 201201201201201correlation of phospholipase and proteinase production of candida with in vivo pathogenicity in galleria mellonella braz j oral sci. 12(3):199-204 fig. 1. scatter plot and respective regression line of mortality rate versus enzymatic activity. a) proteinase: y = -109.0*x + 150.8; r2= 0.326. b) phospholipase: y = 88.84*x + 131.8; r2 = 0.200. there was significant difference between mortality rate and enzymatic activity (p<0.05). of various candida strains was correlated with in vivo virulence in g. mellonella. all c. albicans isolates (100%) were positive for proteinase and phospholipase production. this result is in agreement with pereira et al.21, who studied the enzymatic activity of 51 strains of candida in oral isolates and found that all c. albicans strains were positive for proteinase and phospholipase activity. martins et al.26 also observed presence of these enzymes in all tested isolates of c. albicans. in nonalbicans species, the enzymatic activity was lower. only 25% of the strains produced proteinases and 43% of the strains candida strain(identity code) proteinase(pz) phospholipase (pz) g. mellonella(n=16) c. albicans (15-s) 0.64 0.54 16 c. albicans (7) 0.64 0.54 13 c. albicans (21) 0.80 0.73 16 c. albicans (60) 0.48 0.72 16 c. albicans (10-s) 0.56 0.46 16 c. albicans (24-s) 0,70 0.55 16 c. albicans (31-s) 0.86 0.75 16 c. albicans (39-s) 0.50 0.41 16 c. glabrata (55) 1.00 1.00 0 c. glabrata (217-s) 1.00 1.00 4 c. glabrata (89-s) 1.00 1.00 2 c. glabrata (12-s) 1.00 1.00 0 c. dubliniensis (222-s) 0.75 0.67 16 c. dubliniensis (183-s) 1.00 1.00 16 c. dubliniensis (155-s) 0.75 0.87 15 c. parapsilosis (127-s) 0.50 1.00 13 c. parapsilosis (156-s) 0.43 1.00 5 c. tropicalis (140-s) 1.00 0.70 3 c. tropicalis (212s) 1.00 0.59 3 c. krusei (54-s) 1.00 0.84 10 c. krusei (64-s) 1.00 0.90 3 c. norvegensis (52-s) 1.00 0.67 2 c. lusitaniae (114-s) 1.00 1.00 15 c. guilliermondii (166-s) 1.00 1.00 5 table 3. pz values for enzymatic activity and g. mellonella larvae killed 24 h after infection with different candida strains produced phospholipases. these data agree with a study by junqueira et al.19 that evaluated the enzymatic activity of 64 candida isolates. among the 31 non-albicans species, 21% had proteinase activity and 39% had phospholipase activity. previous studies also reported that proteinases and phospholipases are produced at high levels in c. albicans, whereas non-albicans species generally have lower production of these enzymes. in a work by mane et al. 27, enzymatic activity was evaluated in 65 strains of candida (39 c. albicans and 26 non-albicans) isolated from hiv-positive, oral candidiasis patients. the 202202202202202 correlation of phospholipase and proteinase production of candida with in vivo pathogenicity in galleria mellonella braz j oral sci. 12(3):199-204 authors verified that most c. albicans strains had active proteinases (89% of strains) and phospholipases (59%), while only 13% or 6% of non-albicans strains were positive for proteinase or phospholipases, respectively. in the present study, was used an in vivo g. mellonella infection model to evaluate the pathogenicity of oral candida isolates. there are some benefits to using g. mellonella larvae as a model host. for example, the larvae can be maintained at a temperature range from 25 °c to 37 °c, thus facilitating a number of temperature conditions under which fungi exist in either natural environmental niches or mammalian hosts12,25. recently, mesa-arango et al.28 investigated the efficacy of antifungal drugs in this infection model and verified that amphotericin b, caspofungin, fluconazole and voriconazole had a protective effect at concentrations equivalent to therapeutic doses used in human, concluding that g. mellonella offers a simple and feasible model to study drug efficacy. c. albicans, c. dubliniensis and c. lusitaniae were more pathogenic in the g. mellonella experimental model. c. albicans is the most pathogenic candida species due to its virulence factors, which include hwp1, a hyphal cell wall protein responsible for adherence and biofilm formation29. cotter et al.14 tested the pathogenicity of candida species in g. mellonella experimental models. microbial suspensions were inoculated into hemolymphs of larvae, which were then incubated at 30 °c for 72 h. the resulting mortality rates were similar to those in the present study. c. albicans killed 90% of the larvae, while the mortality rates for other species were 70%, 45%, 45%, 20% and 0% for c tropicalis, c. parapsilosis, c. pseudotropicalis, c. krusei and c. glabrata, respectively. the virulence of candida strains in g. mellonella was dependent on the intensity of enzyme production. this study was the first to correlate in vitro enzyme production with in vivo pathogenicity. in candida strains with higher proteinase and phospholipase activity, g. mellonella virulence was greater. this correlation may be due to the high protein and lipid content in g. mellonella bodies30. fuchs et al.23 correlated c. albicans filamentation with pathogenicity in g. mellonella. five mutant strains lacking the genes bcr1, flo8, kem1, tec1 and suv3, which are required for filamentation, were studied. only the mutant strain flo8 did not form filaments in g. mellonella, and this strain also showed reduced virulence in the larvae. in contrast, the tec1 mutant strain exhibited reduced pathogenicity and hyphae formation in larvae. the authors concluded that filamentation was not sufficient for a lethal infection in g. mellonella and suggested that other candida virulence factors were associated with in vivo pathogenicity. the number of studies using g. mellonella as a model host has increased significantly in the last few years. in addition, there has been an improvement in the techniques used with this model, which allows further studies of candida virulence factors and pathogenicity in this experimental model of infection that will elucidate 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fidel pl jr, odds fc. animal models of mucosal candida infection. fems microbiol lett. 2008; 283: 129-39. 17. brennan m, thomas dy, whiteway m, kavanagh k. correlation between virulence of candida albicans mutants in mice and galleria mellonella larvae. fems immunol med microbiol. 2002; 34: 153-7. 18. ghannoum ma. potential role of phospholipases in virulence and fungal pathogenesis. clin microbial rev. 2000; 13: 122-43. 19. junqueira jc, vilela sf, rossoni rd, barbosa jo, costa ac, rasteiro vm, et al. oral colonization by yeasts in hiv-positive patients in brazil. rev inst med trop são paulo. 2012; 54: 17-24. 203203203203203correlation of phospholipase and proteinase production of candida with in vivo pathogenicity in galleria mellonella braz j oral sci. 12(3):199-204 20. price m.f, wilkinson id, gentry lo. plate method for detection of phospholipase activity in candida albicans. sabouraudia. 1982; 20: 7-14. 21. pereira ca, costa ac, machado ak, beltrame júnior m, zöllner ms, junqueira jc, et al. enzymatic activity, sensitivity to antifungal drugs and baccharis dracunculifolia essential oil by candida strains isolated from the oral cavities of breastfeeding infants and in their mothers’ mouths and nipples. mycopathologia. 2011; 171: 103-9. 22. ruchell r, tegeller r, trost ma. a comparison of secretory proteinases from different strains of candida albicans. sabouraudia. 1982; 20: 233-44. 23. fuchs bb, eby j, nobile cj, el khoury jb, mitchell ap, mylonakis e. role of filamentation in galleria mellonella killing by candida albicans. microbes infect. 2010; 12: 488-96. 24. junqueira jc, fuchs bb, muhammed m, coleman jj, suleiman jm, vilela sf, et al. oral candida albicans isolates from hiv-positive individuals have similar in vitro biofilm-forming ability and pathogenicity as invasive candida isolates. bmc microbiol. 2011; 11: 247. 25. abaci o. investigation of extracellular phospholipase and proteinase activities of candida species isolated from individual dentures wearers and genotypic distribution of candida albicans strains. curr microbiol. 2011; 62: 1308-14. 26. martins js, junqueira, jc, faria rl, santiago nf, rossoni rd, colombo ce, et al. antimicrobial photodynamic therapy in rat experimental candidiasis: evaluation of pathogenicity factors of candida albicans. oral surg oral med oral pathol oral radiol endod. 2011; 111: 71-7. 27. mane a, pawale c, gaikwad s, bembalkar s, risbud a. adherence to buccal epithelial cells, enzymatic and hemolytic activities of candida isolates from hiv-infected individuals. med mycol. 2011; 49: 548-51. 28. mesa-arango ac, forastiero a, bernal-martínez l, cuenca-estrella m, mellado e, zaragoza o. the non-mammalian host galleria mellonella can be used to study the virulence of the fungal pathogen candida tropicalis and the efficacy of antifungal drugs during infection by this pathogenic yeast. med mycol. 2013; 51: 461-72. 29. naglik jr, moyes dl, wachtler b, hube b. candida albicans interactions with epithelial cells and mucosal immunity. microbes infect. 2011; 13: 963-76. 30. bulushova nv, elpidina en, zhuzhikov dp, lyutikova li, ortego f, kirillova ne, et al. complex of digestive proteinases of galleria mellonella caterpillars: composition, properties, and limited proteolysis of bacillus thuringiensis endotoxins. biochemistry (mosc). 2011; 76: 581-9. 204204204204204 correlation of phospholipase and proteinase production of candida with in vivo pathogenicity in galleria mellonella braz j oral sci. 12(3):199-204 oral sciences n3 braz j oral sci. 12(1):11-15 original article braz j oral sci. january | march 2013 volume 12, number 1 influence of acid etching on shear strength of different glass ionomer cements carolina carvalho bortoletto1, walter gomes miranda junior2, lara jansiski motta3, sandra kalil bussadori4 1master’s degree student in rehabilitation sciences, university nove de julho, são paulo, sp, brazil 2department of dental material, school of dentistry, university of são paulo, são paulo, sp, brazil 3department of pediatric dentistry, university nove de julho, são paulo, sp, brazil 4department of rehabilitation sciences, university nove de julho, são paulo, sp, brazil correspondence to: carolina carvalho bortoletto rua rui barbosa, 133 – 134 bairro santo antônio cep: 09530-240 são caetano do sul, sp brasil phone: +55 11 42251500 e-mail: carolbortoletto@ig.com.br abstract aim: to assess the influence of dental etching on the shear strength of different glass ionomer cements. methods: the crown of 60 bovine incisors was prepared to obtain a flat, smooth surface, were attached to a pvc tube and randomly divided into six groups: group 1 – riva self cure without etching; group ii – riva self cure with pre-etching; group iii – maxxion r without etching; group iv – maxxion r with pre-etching; group v – ketac™molar easymix without etching; and group vi – ketac™molar easymix with pre-etching. etching was performed with 10% polyacrylic acid for 30 s. a standardizing device was used for preparation of the test specimens (sbs test method). following preparation, the specimens were stored in distilled water at 37º c for 24 h. the shear test was performed on a kratos universal testing machine at a crosshead speed of 0.5 mm/min. the specimens were then viewed under a stereomicroscope at a magnification of x90 for analysis of fractures. results: the results revealed that pre-etching increased the shear strength of riva glass ionomer cement (sdi) alone, whereas no statistically significant differences were found with regard to the other materials tested. conclusions: pre-etching with 10% polyacrylic acid for 30 s increased the shear strength of riva glass ionomer cement. keywords: glass ionomer cement, shear strength, dentin. introduction glass ionomer cements have been commonly used in different dental procedures in recent decades1-4. these materials offer advantages such as physiochemical adhesion to the dental structure, biocompatibility, the release of fluoride for a long period of time, a thermal expansion coefficient similar to that of teeth and ease of use5-6. despite these properties, glass ionomer cements have limitations, such as low diametral tensile and compressive strength and consequent low border strength as well as their considerable sensitivity to contraction and infiltration, especially in the first moments following placement in the oral cavity6-7. strong, durable adhesion between the restorative material and tooth is essential, as the adequate adaptation of the material reduces microleakage, pulp irritation and risk of caries recurrence8. shear bond strength tests are the method of choice in the assessment of restorative materials, especially traction and shear strength tests9-10. the difficulty of performing a strength test in the oral cavity has led most researchers to the use of extracted teeth9. bovine teeth are often employed for this purpose due to the similarities to human teeth in the results achieved in comparison to the use received for publication: november 09, 2012 accepted: february 04, 2013 1212121212 braz j oral sci. 12(1):11-15 of teeth from dogs or goats11-13. despite divergent opinions regarding the reliability of laboratory tests for the assessment of the clinical performance of restorative materials, such tests indeed achieve a very good correlation with clinical performance, thereby justifying their use14. a number of studies have demonstrated that the treatment of the material in the oral cavity increases the bond strength of the cement to dentin. however, other studies suggest that there is no need for prior treatment for a glass ionomer cement to adhere to the tooth. others however report that bond strength can be improved by treating the tooth surface with different solutions15-17. glass ionomer cements have the capacity to form a chemical bond with the tooth, especially the enamel, which is a more mineralized structure. the adhesion process occurs through the chemical bond between carboxyl groups of polyacids (chelation agents of the restorative materials) and calcium ions in the tooth. despite this ability, the bond strength of glass ionomer cements is considered low. in order to enhance this strength, the application of a 10% polyacrylic acid solution is indicated, which removes unwanted residue, alters the wetting capacity and improves the adaptation of the material18-19. polyacrylic acid removes the smear layer and surface contaminants, while altering the surface energy and exposing the mineralized dental structure for the diffusion of the acid and ionic exchanges20. however, there are divergent opinions regarding the need or non-need for etching prior to restoration with glass ionomer cement as well as the substances used for this purpose21. considering the increase in the number of restorations carried out with this material, especially following the introduction of the concept of atraumatic restorative treatment, further studies on the use of glass ionomer cement are needed. with the aim of optimizing working time and increase restoration longevity, the purpose of the present study was to assess the influence of dentin etching on the shear strength of different glass ionomer cements. material and methods sixty bovine incisors with a healthy crown free of cracks and enamel fractures were used. the roots were sectioned and the crowns were prepared in such a way as to obtain a surface of exposed dentin approximately 5 x 5 mm. the specimens were then attached to pvc tubes using chemically activated acrylic resin, maintaining the dentin surface table 1: experimental groups group (n=10) surface treatment restorative material i no treatment riva self cure – sdi – bayswater, vic, au ii 10% polyacrylic acid for 30 s riva self cure – sdi – bayswater, vic, au iii no treatment maxxion r – fgm – joinville, sc, brazil iv 10% polyacrylic acid for 30 s maxxion r – fgm – joinville, sc, brazil v no treatment ketac™ molar easymix; – 3m espe ag, seefeld, ge vi 10% polyacrylic acid for 30 s ketac™ molar easymix; – 3m espe ag, seefeld, ge exposed. this surface was ground wet onto sandpaper of different grits (220, 320, 400 and 600) until obtaining a flat, smooth surface. the specimens were then randomly divided into six groups, as displayed in table 1. a standardizing device was used for the preparation of the test specimens (sbs test method – sdi – bayswater, vic, au), which allowed similar volume, shape and pressure. all glass ionomer cements were blended following the manufacturer’s instructions. pre-etching with 10% polyacrylic acid for 30 s was performed in groups ii, iv and vi, followed by water-jet cleaning and air-jet drying. the materials were placed in the cylinder of the standardizing device and submitted a constant force of 0.4 kg for 10 s. with the pressure maintained, the materials were stored at 37º c for 10 min, after which the pressure was removed and the specimens were stored in distilled water at 37º c for 24 h. the specimens were submitted to the shear test on a kratos universal testing machine at a crosshead speed of 0.5 mm/min. after the shear test, the specimens were viewed under a stereomicroscope at x90 for analysis of failure mode. the failure modes were classified as follows: adhesive – when the dental surface was visible in more than 75% of the area tested (figure 1); cohesive – when the cement covered more than 75% of the area tested (figure 2); and mixed – when the area tested exhibited 25 to 75% adhesive and cohesive fractures (figure 3)22. the non-parametric kruskal-wallis test was used for the statistical analysis, with the level of significance set at 5% (p < 0.05). fig. 1: adhesive fracture influence of acid etching on shear strength of different glass ionomer cements 1313131313 braz j oral sci. 12(1):11-15 failure mode group i group ii group iii group iv group v group vi adhesive 100 40 60 60 90 100 cohesive 0 30 30 30 10 0 mixed 0 30 10 10 0 0 table 3: failure modes (%) group i group ii group iii group iv group v group vi mean 23.21 35.43 14.21 18.77 14.78 12.59 standard deviation 9.45 15.26 15.95 14.16 13.83 7.98 table 2: arithmetic bond strength means and standard deviation for each group no statistical difference was found. results mean and standard deviation of shear strength obtained in each group are presented in table 2. pre-etching with polyacrylic acid only increased the bond strength in group ii (riva self cure). a number of premature fractures occurred during the tests6. in such cases, the shear strength value was considered zero. the results of the microscopic analysis are displayed in table 3. fig. 3: mixed fracture fig. 2: cohesive fracture discussion adhesiveness is an important characteristic of glass ionomer cements, as these materials dispense of the need for an intermediate adhesive. the ionomer forms a chemical union with the tooth due to the action of carboxyl groups of polyacids in the calcium found in the apatite of the enamel and dentin. the bond strength is stronger with the enamel due to its greater mineral component6-7,23-24. the adhesion mechanism of ionomer materials is the formation of an ionic bond between the liquid component of the cement and the calcium of the hydroxyapatite of the tooth. moreover, micromechanical adhesion to the collagen fibers exposed on the surface of the dentin may occur following the action of a weak de-mineralizing agent used to remove the smear layer. this occurs more with resin-modified ionomer cements than conventional ionomer cements25. a number of studies have been carried out in the search for improvements to the mechanical properties of glass ionomer cements. however, there is no consensus to date regarding the treatment of the tooth surface in order to ensure better adhesion of the cement. the bond strength of restorative materials is influenced by a number of factors related to the material itself as well as the substrate and even the technique employed. such factors include the nature of the substrate (healthy or carious dentin), proximity of the pulp, surface roughness, age of the dentin, whether the dental structure is a primary or permanent tooth, dentin permeability, method of carious tissue removal, presence of wrinkles and bubbles in the interior of the material, alterations in the powder/liquid proportions, alterations in the blending and the different treatments the substrate may received, such as acid etching or the use of antimicrobial agents7,13. an important factor to consider in the study of glass ionomer cements is the type of fracture displayed. it is very common to find a high rate of cohesive and mixed fractures, which illustrate the cohesion strength rather than the adhesion strength of the material. a standardizing device (sbs test method) was used in the present study. this device has a metallic cylinder that envelops the glass ionomer cement until the moment of fracture, the aim of which is to provide greater cohesive strength and thereby allow the assessment of adhesive strength. the high rate of adhesive fractures in the influence of acid etching on shear strength of different glass ionomer cements 1414141414 braz j oral sci. 12(1):11-15 present study demonstrates the validity of the use of this device. the statistical analysis of the results demonstrated that pre-etching only improved the bond strength of the riva self cure, whereas no differences were found between etching and non-etching with the other materials. this finding is also made clear by the analysis of fractures with this same material. in group i, riva self cure was used without pre-etching and 100% of the failures were adhesive fractures, demonstrating that the material detached from the dentin due to the fact that adhesive strength was less than cohesive strength. in group ii, riva self cure was used with pre-etching and 40% of the fractures were adhesive, 30% were cohesive and 30% were mixed fractures, demonstrating a greater adhesive force, with a consequent greater number of cohesive and mixed fractures. the same did not occur with the maxxion r or ketac™ molar easymix restorative materials. in this study it was also possible to observe that the pre-etching with 10% polyacrylic acid increased adhesiveness of riva self cure and maxxion r. for the ketac™ molar easymix this treatment decreased the adhesiveness of the material, which may occurred due to the presence of polycarbonic acid and tartaric acid , that may have promoted a reaction with polyacrylic acid. as self cure and maxxion r present polyacrylic acid in their composition, this reaction does not occur, increasing the material’s adhesion. it’s important to emphasize that the variation of the adhesion was not statistically significant. riva self cure had higher adhesion values under both conditions in comparison to the other materials, which may be explained by the fact that this product is a encapsulated cement and its mechanical blen ding achieves a more homogeneous mixture with less porosity. this creates a larger contact surface between the restorative material and tooth, thereby enhancing bond strength. there is also an increase in cohesive strength due to the reduction in porosity7. it is difficult to compare the results of bond strength obtained in different studies due to the differences in the methodologies employed, such as the substrate used, means of storage and crosshead speed applied during the test. a number of studies report that pre-etching provides no significant difference in the bond strength of glass ionomer cements21,26-28. however, others have found an improvement in bond strength when pre-etching is performed15,28-29. further studies are needed to establish the adhesion mechanisms of glass ionomer cements to carious dentin tissue and determine the best manner for enhancing this adhesion, especially in the context of the more widespread employment of atraumatic restorative treatment. the results of the present study revealed that preetching with 10% polyacrylic acid for 30 s only increased the shear strength of the riva glass ionomer cement, whereas no statistically significant differences were found between the previously etched and non-etched groups with regard to maxxion r or ketac™ molar easymix. references 1. blatt ja, goes fm. microleakage in cavities prepared on primary and sclerotic dentin restored with resin modified glass ionomer. rev fac odontol sao jose campos. 2001; 4: 61-6. 2. gateau p, sabek m, dayley b. in vitro fatigue resistance of glass ionomer cements used in post-and-core applications. j prosthet dent. 2001; 86: 149-55. 3. frança trt, sedycias m, silva rj, beatrice lcs, silva chv. use of glass ionomer cement : a systematic review. pesq bras odontoped clin integr. 2010; 10: 301-7. 4. ngo h. glass-ionomer cements as restorative and preventive materials. dent clin north am. 2010; 54: 551-63. 5. costa cas, ribeiro apd, giro ema, randall rc, hebling j. pulp response after application of two resin modified glass ionomer cements (rmgics) in deep cavities of prepared human teeth. dent mater. 2011; 27: e158-e170. 6. choi k, oshida y, platt ja, cochran ma, matis ba, yi k. microtensile bond strenght of glass ionomer cements to artificially created carious dentin. oper dent. 2006; 31: 590-7. 7. fagundes tc. influence of ultrasound on the bond strength of glass ionomer cements to dentin. bauru: universidade de são paulo; 2005. 104p. 8. çehrli cz, akca t, altay n. bond strengths of polyacid-modified resin composites and a resin-modified glass-ionomer cement to primary dentin. am j dent. 2003; 16: 47a-50a. 9. mussolino zm, borsatto mc, turbino ml. shear bond strength of a sealant using components of na adhesive system. rev odontol univ sao paulo. 1998; 12: 389-94. 10. cardoso pec, braga rr, carrilho mro. evaluation of micro-tensile, shear and tensile tests determining the bond strength of three adhesive systems. dent mater. 1998; 14: 394-8. 11. tao l, pashley dh, mcguckin r. in vivo bond strength : effect of depth and tooth type. j dent res. 1990; 69: 285 [abstract 1411]. 12. gray se, burgess jo. an in vivo an in vitro comparison of two dentin bonding agents. dent mater. 1991; 7: 161-5. 13. pashley el, tao l, matthews wg, pashley dh. bond strengths to superficial, intermediate and deep dentin in vivo with four dentin bonding systems. dent mater. 1993; 9: 19-22. 14. chain mc, chain jb, leinfelder kf. hybrid glass-ionomer cements. dentin bond strength and bondin mechanism. using electron microscopy. rgo. 2000; 48: 42-9. 15. powis dr, folleras t, merson sa, wilson ad. improved adhesion of a glass ionomer cement to dentin and enamel. j dent res. 1982; 61: 1416-22. 16. raggio dp, sonego fg, camargo lb, marquezan m, imparato jc. efficiency of different polyacrilic acid concentrations on the smear layer, after art technique, by scanning electron microscopy (sem). eur arch paediatr dent. 2010; 11: 232-5. 17. castro mfs, costa jf, costa el, padilha ln, lopes ff. effect of chlorhexidine on the adhesion of glass ionomer cement used in atraumatic restorations, using microleakage test. rgo. 2010; 58: 167-71. 18. van meerbeek b, al e. buonocore memorial lecture. adhesion to enamel and dentin : current status and future challenges. oper dent. 2003; 28: 215-35. 19. de munck j, al e. a critical review of the durability of adhesion to tooth tissue : method and results. j dent res. 2005; 84: 118-32. 20. cunnigham mp, meires jc. the effect of dentin desinfectants on shear bond strength of resin-modified glass-ionomer materials. quintessence int. 1997; 28: 545-51. 21. inoue s, abe y, yoshida y, de munck j, sano h, suzuki k, lambrechts p, van meerbeek b. effect of conditioner on bond strength of glassionomer adhesive to dentin/enamel with and without smear layer interposition. oper dent. 2004; 29: 685-92. influence of acid etching on shear strength of different glass ionomer cements 1515151515 braz j oral sci. 12(1):11-15 22. hibino y, kuramochi k, harashima a, honda m, yamazaki a, nagasawa y, et al. correlation between the strength of glass ionomer cements and their bond strength to bovine teeth. dent mater j. 2004; 23: 656-60. 23. tanumiharja m, burrow mf, tyas mj. microtensile bond strengths of glass ionomer (polyalkenoate) cements to dentin using four conditioners. j dent. 2000; 28: 361-6. 24. rekha cv, varma b, jayanthi. comparative evaluation of tensile bond strength and microleakage of conventional glass ionomer cement, resin modified glass ionomer and comomer: an in vitro study. contemp clin dent. 2012; 3: 282-7. 25. tanumiharja m, burrow mf, cimmino a, tyas mj. the evaluation of four conditioners for glass ionomer cements using field-emission scanning electron microscopy. j dent. 2001; 29: 131-8. 26. hinoura ko, moore bk, phillips rw. influence of dentin surface treatments on the bond strength of dentin lining cements. oper dent. 1986; 11: 14754. 27. banomyong d, palamara jea, burrow mf, messer hh. effect of dentin conditioning on dentin permeability and micro-shear bond strength. eur j oral sci. 2007; 115: 502-9. 28. el-askary fs, nassif ms, fawzy as. shear bond strength of glassionomer adhesive to dentin : effect of smear layer tickness and different dentin conditioners. j adhes dent. 2006; 10: 471-9. 29. glasspoole ea, erickson rl, davidson cl. effect of surface treatments on the bond strength of glass ionomers to enamel. dent mater. 2002; 18: 454-62. influence of acid etching on shear strength of different glass ionomer cements oral sciences n3 original article braz j oral sci. october | december 2013 volume 12, number 4 prevalence of dental fractures and associated factors in students of valinhos, sp, brazil andressa reisen1, rubia raquel santos do nascimento1, cristiana carina de barros lima dantas bittencourt1, roberta tagliari da rosa1, luciane zanin2, flavia martão flório1 1department of social sciences, são leopoldo mandic school of dentistry and research center, campinas, sp, brazil 2department of social sciences, hermínio ometto university center, araras, sp, brazil correspondence to: andressa reisen avenida vitória, 1729, cep: 29040-780 jucutuquara, vitória, es, brasil phone: +55 27 33312124 e-mail: areisen@hotmail.com abstract aim: to investigate the prevalence of dental fractures and their association with risk factors in the permanent dentition of adolescents in valinhos, sp, brazil. methods: the study population was obtained using the probability sampling method and comprised 379 students between 13 and 19 years old enrolled in the eight state schools of the city, who were examined by nine pairs of calibrated dentists (kappa>0.80). the presence of dental fractures in permanent anterior incisors, as well as the presence of considerable overjet (>>>>> 5mm) and lip seal was evaluated in a clinicalepidemiological examination. results: the prevalence of dental trauma was 27.1%. the most frequent lesions were enamel fractures (72.6%) of which falls were the main cause (45.7%). no association was found between the presence of considerable overjet and deficient lip seal (chisquare test; p>0.05). conclusions: the prevalence of dental trauma in the studied population was expressive when compared with the literature, but the analyzed oral aspects did not contribute to its occurrence. keywords: dental fractures, risk factors, students, oral health, epidemiology. introduction the reduction in dental caries worldwide1-2 in recent years has drawn attention to other aspects of oral health3, such as dental trauma4, which is a public health issue on the increase5-6, making it the second most common cause (after dental caries7) of dental treatment. epidemiological studies in oral health that include the diagnosis of dental trauma are quite rare compared with data on caries and periodontal disease6. few population-based studies on this subject have been carried out in latin america, and the majority of the reports come from emergency services, which is not representative of the population8. information could be lost, especially when carious lesions coexist, since the importance of caries prevails over trauma9. the main causes of dental trauma are falls6,10-11, activities related to infancy12, sports13, car accidents5 and violence13. amongst the biological factors related to an increased risk of dental trauma is an accentuated overjet8 and an inadequate lip seal4,14. the aim of this study was to investigate the prevalence of dental fractures and their association with risk factors in the permanent dentition of adolescents in a city of the the state of são paulo, brazil. received for publication: june 18, 2013 accepted: september 20, 2013 braz j oral sci. 12(4):280-284 material and methods a cross-sectional epidemiological survey was carried out in the city of valinhos, in the state of são paulo, involving students of both genders, between 13 and 19 years of age, regularly enrolled in the eight schools from the state’s public school system (n=2,962) in june 2012. the study followed the regulations established in resolution 196, 10/10/1996, of the national health council of the ministry of health, and was carried out with the approval of the ethics committee in research of the são leopoldo mandic dental research center (registration number 2012/0127) and with the written informed consent of the parents or guardians. a probability sampling method was used. sample size was calculated considering a trauma prevalence of 50%3, with its respective 95% confidence interval, 10% accuracy and a 10% non-response rate, with adjustment for finite populations, considering 2,962 individuals in the specified age group in 2011, which resulted in a minimum sample size of 374 students. the allocation of the sample elements followed the recommendations from the latest national oral health survey, sb, brazil15. the inclusion criteria were: enrolment in the public state school network of valinhos; age between 13 and 19 years at the time of examination; presence of maxillary and mandibular anterior teeth; and no extensive carious lesions6. the exclusion criteria were: student absence on the day of the survey; limited mouth opening; lesions that prevented examination; orthodontic appliances; and one or more maxillary and/or mandibular incisors with a crossbite or rotation6. calibration of the examiners for dental fractures was performed using photographs of patients with dental fractures with different degrees of severity. presence or absence of treatment, overjet measurement and lip seal were calibrated by examining patients. intra-examiner error was assessed by re-examining 10% of the sample6. inter-examiner agreement was verified using the kappa test16, obtaining a value of 0.80, which was considered adequate for this study. data collection was performed by 18 dental surgeons, properly trained and calibrated, divided into 9 groups, each of them with one examiner and one annotator, according to the method used in the national oral health survey, sb, brazil 201015. only the criteria for signs of coronal fracture and dental absence of the national oral health survey, sb, brazil, 201015 were used to evaluate the presence of dental trauma. the permanent maxillary and mandibular incisors were considered for the study15. the clinical examination was carried out at the schools, during lesson times, under sufficient natural light, with the examiner and patient facing one another. the permanent maxillary and mandibular incisors were examined without drying, using a wooden tongue depressor. all health and safety procedures were strictly followed15. in addition to fractures, the presence of any restorative treatment to the traumatized tooth was also assessed. patients with teeth needing treatment were referred to the local healthcare units of the unified health system (sus). the presence of anterior overjet was assessed as the distance in millimeters between the labial aspect of the most prominent maxillary incisor and the corresponding mandibular incisor15. wooden tongue depressors were used to measure overjet, previously marked at their flat end at 5 mm, with the aid of a millimeter ruler and a pencil. the tongue depressors were then inspected by an independent examiner and then autoclaved. the student had his/her occlusion positioned in centric relation and the flat end of the wooden tongue depressor was put in contact with the labial aspect of the most lingually positioned incisor. the tongue depressor was placed parallel to the incisal surface at a right angle to the normal arch line. those who showed coincidental overjet or beyond the mark were classified with “presence of an acentuated overjet”. in case of avulsion, the cause of the tooth absence was inquired and included in the trauma prevalence analysis, if it had been the cause. lip seal was assessed using the method proposed by o’mullane17, which is defined as the upper lip covering the maxillary incisors at rest. the seal was otherwise considered as inadequate. in order to assess this, the student was instructed to silently read a document, without knowing that he/she was being observed18. in cases of doubt, this step was repeated until the examiner was satisfied with the lip position. all data were collected on a specific form where clinical data, age, gender, etiology and trauma location were recorded. the data were transferred to excel spreadsheets (microsoft, inc, redmond, wash) and analyzed using the bioestat 5.0 software (http://mamiraua.org/cms/content/public/documents/ bioestat-5.3-portugues.zip, free for academic use). results four hundred and thirteen (413) volunteers participated in this study, 91.8% fitted in the inclusion criteria (n=379), 59.4% females and 40.6% males. the prevalence of fractures was 27.1%, predominantly in females (59.4%). in terms of marked overjet, the prevalence was 8.3% and inadequate lip seal 13.7%, as described in table 1. table 2 illustrates that 58.4% of the volunteers reminded the etiology and the location where the fractures occurred. fall was the main cause of fractures, 45.7%, and 50.8% happened during leisure time, whereas 6.8% occurred at school. from the 101 volunteers presenting dental fractures, 128 teeth were affected, 38.2% involving the permanent maxillary right central incisor, which was the most representative when compared with the other 7 incisors. enamel fractures (72.6%) had the highest prevalence, with no evidence of pulp exposure or absence of the tooth due to trauma. only 17.9% of the fractured teeth had been treated (figure 1). the permanent maxillary right central incisor was the most frequently treated tooth, representing 56.5% of the prevalence of dental fractures and associated factors in students of valinhos, sp, brazil 281 braz j oral sci. 12(4):280-284 gender/lip trauma p-value(*) seal/overjet absence of trauma presence of trauma total female 162 60 222 adequate lip seal 139 50 189 0.95 without overjet 131 47 178 with overjet 8 3 11 inadequate lip seal 23 10 33 0.12 without overjet 21 7 28 with overjet 2 3 5 male 109 41 150 adequate lip seal 96 36 132 0.50 without overjet 91 33 124 with overjet 5 3 8 inadequate lip seal 13 5 18 0.95 without overjet 8 3 11 with overjet 5 2 7 total 271 101 372 table 1.table 1.table 1.table 1.table 1. absolute frequency distribution (n) of dental trauma in terms of gender, lip seal and overjet. (*): chi-squared test. treated fractures. restorations involving both dentin and enamel were the most common when compared to enamel restorations only, representing 34.7% and 21.7%, respectively. there was no association between presence of trauma and gender (p=0.95), or between trauma and accentuated overjet (p=0.27), or insufficient lip seal (p=0.69). however, the association between marked overjet and lip seal was significant (p<0.0001). discussion there are very few reports in the literature regarding dental fractures and the age group evaluated in this study. from the available studies, the fracture prevalence varies from 17.1%19 to 50.8%10, which places the 27.1% encountered in this study within the range. it is likely that such variation in prevalence may be associated to analysis of specific populations, to the sample selection or to the place where the study was conducted. in a recent national oral health study, dental trauma was included and evaluated as a specific measurement in 12-year-old children, which represents an important advancement in establishing a baseline that can be used as an action-planning tool and to determine future targets2. in this study, a predominance of females was encountered. however, the association did not reach significance between genders (p=0.95), which is in accordance with the findings of paiva6 and traebert et al.18 and contrasts with the results of most other studies, which reported a male predominance 5,8,14,2021. the gender equivalence may be related to a greater concern of female volunteers in terms of health issues22. the dental fractures reported in this study affected only the coronal portion of the teeth23. most traumas occurred to etiology/location gender p-value(*) f m total home 15 10 25 0.85 eating 3 2 5 collision 2 2 4 others 2 2 fall 8 6 14 school 2 2 4 0.83 collision 1 1 2 fall 1 1 2 leisure 17 13 30 0.67 eating 1 1 collision 5 3 8 sports practice 4 6 10 fall 7 4 11 total 34 25 59 (*): fisher’s exact test. table 2.table 2.table 2.table 2.table 2. absolute frequency distribution (n) of the etiology and location of dental trauma, according to the gender prevalence of dental fractures and associated factors in students of valinhos, sp, brazil fig. 1. percentage of traumatized teeth with and without treatment. 282 braz j oral sci. 12(4):280-284 the anterior region, mainly the permanent maxillary central incisors, corroborating the findings of a survey carried out with students from belo horizonte, mg, brazil14. the most frequent traumatic injury was enamel fracture, confirming the findings from other studies6,8,10. although trauma prevalence of 10.5%, 15.29% and 19.9% in adolescents has been identified in previous studies45,11 in valinhos (sp), a high prevalence among the volunteers was demonstrated in this study (27.1%). among the moment of occurrence of the fractures, leisure time accounted to 50.8% of the cases. no avulsion was encountered, which agrees with the findings of paiva6. this suggests an association with the location of data collection and may also suggest that avulsed teeth have been replanted, since valinhos offers adequate access to dental treatment. the results demonstrated that the main causes of dental fractures were falls (45.7%), followed by collisions (23.7%) and sports (16.9%), which is in line with other studies6,10-11. the relationship between marked overjet and dental trauma was evaluated. this association has been investigated by several authors who reported that individuals with an increased overjet, greater than 5 mm, are significantly more prone to suffer traumatic injuries4,14. however, in this study, no significant association between those variables was detected, thus further increasing the disagreement among the results observed in brazilian studies. likewise, inadequate lip seal also showed no association with trauma prevalence. however, some authors reported that individuals with inadequate lip protection were more susceptible to dental trauma4,14. a possible explanation may be that the lips may absorb part of the impact to the teeth, therefore in cases of adequate lip seal, dental trauma could be less frequent24. conversely, the likelihood of trauma would be higher when inadequate lip seal was detected, due to the lack of lip protection. this, combined with a marked overjet, would further increase the probability of trauma, clarifying the significant association found in this study between overjet and lip seal (p<0.0001). the lack of information may be responsible for the scarcity of care by health professionals. appropriate training is needed to deal with affected individuals, in order to develop health promotion strategies that include both treatment and a psychosocial approach to dental trauma25. it is also very important that parents and/or guardians have access to information on the best conduct when faced with dental trauma, so that they may be prepared to act appropriately. the present results indicate that, regarding the prevalence of dental fracture in adolescents in valinhos, sp, brazil, a high number of cases with no associated risk factors was observed. acknowledgements the authors acknowledge the principals of the public state schools of valinhos, sp, brazil and the master’s degree students in public health at the são leopoldo mandic dental school and dental research center (class 519/11), for their help in carrying out this research. references 1. pedroni lbg, barcellos la, miotto mhmb. treatment of traumatized permanent teeth. pesq bras odontopediat clín integr. 2009; 9: 107-12. 2. brazil. ministry of health. national oral health survey 2010. main results. brasília: ministry of health; 2011. 3. patel mc, sujan sg. the prevalence of traumatic dental injuries to permanent anterior teeth and its relation with predisposing risk factors among 8-13 years school children of vadodara city: an epidemiological study. j indian soc pedod prev dent. 2012; 30: 151-7. 4. soriano ep, caldas af jr, carvalho mvd, amorim haf. prevalence and risk factor related to traumatic dental injuries in brazilian schoolchildren. dent traumatol. 2007; 23: 23-40. 5. vasconcellos rjh, oliveira dm, porto gg, silvestre h, silva e. ocurrence of traumatic dental injury in students of a recife´s public school. rev cir traumatol buco-maxilo-fac [internet]. 2003 oct-dec [accessed 2012 mar 30]; 3: 9-12. available from: www.revistacirurgiabmf.com/2003/ v3n4/v3n4.htm. 6. paiva pcp. prevalence and risk factors associated with dental trauma among schoolchildren in montes claros [dissertation]. belo horizonte: school of dentistry, pontifical catholic university of minas gerais; 2005. 7. brito as, carvalho b, heimer m, vieira s, colares v. prevalence of dental trauma in adolescents aged 15-19. rev saude-ung [internet]. 2010; [accessed 2013 mar 30] 4(3). available from: http://revistas.ung.br/ index.php/saude/rt/printerfriendly/496/826. 8. traebert j, almeida ics, garghetti c, marcenes w. prevalence, treatment needs, and predisposing factors for traumatic injuries to permanent dentition in 11-13-year-old schoolchildren. cad saude publica. 2004; 20: 403-10. 9. brazil. ministry of health. national oral health survey 2010. manual of field team. brasília: ministry of health; 2009. 10. caldas jr af, burgos mea. a retrospective study of traumatic dental injuries in a brazilian dental trauma clinic. dent traumatol. 2001; 17: 250-3 11. silva ac, passeri la, mazonetto r, moraes m, moreira wf. incidence of dental trauma associated with facial trauma in brazil: a 1-year evoluation. dent traumatol. 2004; 20: 6-11. 12. prata thc, duarte msr, miquilito jl, valera mc, araújo mam. etiology and frequency of the dental trauma injuries in patients from dental trauma center in the são josé dos campos school of dentistry, são paulo state university – unesp. rev odontol unesp. 2000; 29: 43-53. 13. damé-teixeira n, alves ls, susin c, maltz m. traumatic dental injury among 12-year-old south brazilian schoolchildren: prevalence, severity, and risk indicators. dent traumatol. 2013; 29: 52-8. 14. côrtes mi, marcenes w, sheiham a. prevalence and correlates of traumatic injuries to the permanent teeth of school-children aged 9-14 years in belo horizonte, brazil. dent traumatol. 2001; 17: 22-6. 15. brazil. ministry of health national survey of oral health 2010. technical project. brasília: ministry of health; 2009. 16. landis jr, koch gg. the measurement of observer agreement for categorical data. biometrics. 1977; 33: 159-74. 17. o’mullane dm. injured permanent incisor teeth: an epidemiological study. j ir dent assoc. 1972; 18: 160-73. 18. traebert j, facenda f, lacerda jt. prevalence and treatment needs due to traumatic dental injuries in schoolchildren of joaçaba, sc. rev fac odontol porto alegre. 2008; 12: 11-4. 19. sakai vt, magalhães ac, pessan jp, silva smb, machado maam. urgency treatment profile of 0 to 15 year-old children assisted at urgency dental service from bauru dental school, university of são paulo. j appl oral sci. 2005; 13: 340-4. 20. traebert j, marcon kb, lacerda jt. prevalence of traumatic dental injuries and associated factors in schoolchildren of palhoça, santa catarina state. cienc saude colet. 2010; 15 suppl 1: 1849-55. 21. carvalho b, franca c, heimer m, vieira s, colares v. prevalence of dental trauma among 6-7-year-old children in the city of recife, pe, brazil. braz j oral sci. 2012; 11: 72-5. prevalence of dental fractures and associated factors in students of valinhos, sp, brazil 283 braz j oral sci. 12(4):280-284 22. gomes r, nascimento ef, araújo fc. why do men use health services less than women? explanations by men with low versus higher education. cad saude publica. 2007; 23: 565-74. 23. carrascoz a, ferrari ch, simi j, medeiros jmf. epidemiology and etiology of dental trauma in permanent teeth in the region of bragança paulista. dentária.com [internet]. 2004 [accessed 2012 mar 12]; [about 9 p.]. available from: http://www.dentaria.com/artigos/ver/?art=69. 24. bonini gc, bönecker m, braga mm, mendes fm. combined effect of anterior malocclusion and inadequate lip coverage on dental trauma in primary teeth. dent. traumatol. 2012; 28: 437-40. 25. antunes laa, leão at, maia lc. the impact of dental trauma on quality of life of children and adolescents: a critical review and measurement instruments. cienc saude colet. 2012; 17: 3417-24. prevalence of dental fractures and associated factors in students of valinhos, sp, brazil284 braz j oral sci. 12(4):280-284 oral sciences n3 braz j oral sci. 12(2):143-147 original article braz j oral sci. april | june 2013 volume 12, number 2 proliferation of human periodontal ligament mesenchymal cells on polished and plasma nitriding titanium surfaces rodrigo alves ribeiro1, rodrigo gadelha vasconcelos1, fernanda ginani2, josé sandro pereira da silva1, clodomiro alves-júnior3, carlos augusto galvão barboza2 1area of oral and maxillofacial surgery, department of dentistry, federal university of rio grande do norte, natal, rn, brazil 2department of morphology, federal university of rio grande do norte, natal, rn, brazil 3department of mechanical engeneering, federal university of rio grande do norte, natal, rn, brazil correspondence to: carlos augusto galvão barboza avenida senador salgado filho, 3000, cep: 59072-970 – lagoa nova, natal, rn, brasil phone: +55 84 94015955 fax: +55 84 32119207 e-mail: cbarboza@cb.ufrn.br received for publication: february 28, 2013 accepted: june 25, 2013 abstract aim: to evaluate the proliferative capacity of mesenchymal cells derived from human periodontal ligament on polished and plasma-treated titanium surfaces. methods: eighteen titanium disks were polished and half of them (n=9) were submitted to plasma nitriding using the cathodic cage technique. mesenchymal cells were isolated from periodontal ligament of impacted third molars (n=2) and cultured on titanium disks (polished and nitrided) and on a plastic surface as a positive control of cell proliferation. cell proliferation was analyzed and growth curves were constructed for the different groups by determining the number of cells adhered to the different surfaces at 24, 48 and 72 h after plating. results: higher cell number was observed for the nitrided surface at 24 and 48 h. however, no statistically significant difference in cell proliferation was observed between the two different surface treatments (p>0.05). conclusions: we concluded that plasma nitriding produced surfaces that permitted the proliferation of human periodontal ligament mesenchymal cells. associated to other physical and chemical properties, it is possible to assume the feasibility of plasma nitriding method and its positive effect on the early cellular events of osseointegration. keywords: biocompatible materials, titanium, cell proliferation, periodontal ligament. introduction cell cultures have been extensively used in implantology to evaluate the effect of the substrate on the behavior of osteoblasts during osseointegration. various biological events associated with bone healing on implant surfaces can be investigated separately using appropriate cell cultures, such as cell adhesion, proliferation and differentiation, as well as the production and mineralization of extracellular matrix. the use of these cultures provides cellular and molecular data that favor a nanostructural engineering approach in implant design and permits the testing of different hypotheses. within this context, cell cultures offer unique insights into the process and phenomenon of osseointegration1. in vitro models have been proposed in an attempt to better understand the early stages of osseointegration and how cells interact with surfaces modified by different methods. the functional and structural adhesion of bone tissue to the surface of load-bearing orthopedic implants seems to be a determinant factor for in vivo success2. 144144144144144 braz j oral sci. 12(2):143-147 there is consensus in the literature that the physicochemical properties of implant surfaces play a fundamental role in the success of osseointegration. several surface treatments were developed to improve the molecular and cellular events that guide the early stages of this process3. however, the adequate parameters required to obtain an ideal surface are still not defined. in this respect, new surface modification techniques were studied4. surface treatment permits the modification of the dental implants characteristics, such as chemical composition, morphology, topography and rugosity5. plasma nitriding, which consists of generating an electrical discharge in gas mixture containing low-pressure nitrogen, has been applied to modify biomaterials6. the basic concept in using ionic nitriding to improve titanium (ti) surface properties is based on the possibility of forming nitrides or carbides below the alloy surface. ti nitrides and carbides are brittle materials that improve tribologic surface properties, which means that they increase resistance to corrosion and surface roughness7. the search for a titanium surface favoring cellular events that would permit faster and more effective osseointegration encouraged the present study. in this respect, this study evaluated the proliferation of mesenchymal cells derived from the periodontal ligament of human third molars on polished and plasma-nitrided titanium surfaces. material and methods the study was approved by the ethics committee of the federal university of rio grande do norte – ufrn (process #080/2010). periodontal ligament mesenchymal cells were obtained from two healthy human teeth (impacted third molars). the teeth were extracted from patients with a surgical indication due to orthodontic reasons, who had no oral or systemic diseases. grade ii astm f86 titanium disks, 1.5 mm thick and measuring 15 mm in diameter, were used. preparation of the titanium disks eighteen titanium disks were embedded in polyester resin and gradually polished with 220-, 360-, 400-, 600-, 1,200and 2,000-grit silicon carbide sandpaper in running tap water, followed by polishing with an apl-2 polishing machine (series 212560, arotec, cotia, sp, brazil) and cleaning with an opchem polishing cloth, colloidal silica (sio 2 ) suspension and hydrogen peroxide (h 2 o 2 ) 20 v until a final finishing resulted in a surface with a 0.04 µm roughness. next, the samples were embedded and cleaned to remove contaminants that could interfere with the ion nitriding process. for this purpose, the same samples were washed in an ultrasound bath for 30 min, divided into three steps of 10 min each. enzyme detergent (endozime aw plus, planitrade, porto alegre, rs, brazil) was used in the first wash, absolute alcohol in the second, and distilled water in the last wash. the samples were then dried at room temperature and stored in an appropriate container until the time for use. nine titanium disks were submitted to nitriding treatment using a previously described protocol8. the plasma atmospheres were set up using a gas flow of 15 sccm (table 1). next, all samples were transferred to 24-well culture plates (2 cm2) and sterilized by gamma irradiation. the total irradiation dose per sample was 25 kgy, with a mean dose rate of 8.993 kgy/h (2 h and 46 min at a distance of 50 mm), and a gammacell 220 excel irradiator (mds nordion, ottawa, on, canada) was used. isolation of periodontal ligament cells mesenchymal cells were isolated from periodontal ligament of impacted third molars, as previously described by vasconcelos et al.9 (2012). each tooth was immediately stored in a 50-ml falcon tube containing alpha-mem culture medium (cultilab, campinas, sp, brazil), and then washed three times for 15 min each with alpha-mem medium (cultilab) supplemented with 10,000 iu/ml penicillin, 10,000 µg/ml streptomycin, 100 mg/ml gentamicin, and 250 µg/ ml amphotericin b (all antibiotics were purchased from gibco, grand island, ny, usa). the periodontal ligament was removed by gently scraping the root surface with a scalpel, followed by enzymatic digestion with 3 mg/ml collagenase i (gibco) and 4 mg/ml dispase (gibco) for 1 h at 37°c. the solution was aspirated, filtered through a 70-µm filter (bd falcon, bredford, ma, usa), and centrifuged at 1,200 rpm for 5 min. the supernatant was discarded and the precipitated cells were resuspended in culture medium. the periodontal ligament mesenchymal cells were cultured in 25-cm2 bottles (ttp®, usa) in basic alpha-mem medium (cultilab) supplemented with 15% fetal bovine serum (cultilab). the cultures were incubated at 37ºc in a 5% co 2 atmosphere and the medium was changed at intervals of 3 days until 70 to 90% confluence was reached. cell characterization was performed by expression of cd29/integrin β-1 (bd bioscience, usa), a mesenchymal stem cell marker, by flow cytometry. additionally, the multilineage differentiation potential of periodontal ligament cells was confirmed by culturing the cells in osteogenic and adipogenic differentiation media (stempro® differentiation kits, invitrogen, carlsbad, ca, usa) for up to 21 days. cell culture on titanium disks after the third passage, the periodontal ligament cells were transferred to 24-well plates (ttp®, trasadingen, table 1. parameters of the plasma nitriding treatment. group plasma atmosphere treatment time plasma pressure plasma temperature total gas flow polished nitrided 20% n + 80% h 1 h 2.5 mbar 450°c/17.5 ma 15 sccm proliferation of human periodontal ligament mesenchymal cells on polished and plasma nitriding titanium surfaces 145145145145145 braz j oral sci. 12(2):143-147 switzerland) at a density of 2 x 104 cells per well. eighteen titanium disks were used, nine for each group (polished p and nitride n). the same cell density was cultivated in wells without discs, as a positive control of cell proliferation. the disks are the same size of the well, so the growth area of disks and controls were the same. analysis of cell proliferation cell proliferation was analyzed and growth curves were constructed for the different groups by determining the number of cells adhered to the titanium surfaces (polished and nitrided groups) at 24, 48 and 72 h after plating. the number of cells per well was obtained by counting viable cells in a hemocytometer using the trypan blue exclusion method. cell counts are reported as the mean of three samples per group for each time interval (24, 42 and 72 h). differences were compared between groups by the mann whitney tests. a p value <0.05 was considered to indicate statistical significance. results by flow cytometry, 97.2% of the periodontal ligament cells expressed cd29/integrin â-1. after 21 days under osteogenic and adipogenic induction, the cells produced typical mineralized nodules and showed adipocyte morphology by light microscopy (figure 1). the growth curve of periodontal ligament-derived undifferentiated mesenchymal cells cultured on different surfaces is illustrated in figure 2. cell growth increased linearly on the control surface (plastic) over the period studied, since it is the gold standard surface for cell adhesion and proliferation. comparing the studied titanium surfaces, higher number of cells was observed for the nitrided surface at 24 h. however, no statistically significant difference in cell proliferation was observed between the two different surface treatments (table 2). nitrided surfaces favored cell proliferation at 48 h when compared with the polished surfaces. on the other hand, cell proliferation was increased at 72 h in the polished group, although no statistically significant difference was observed between polished and nitrided surfaces in both time intervals (table 2). polished nitrided mean sd mean sd p value* 24 h 3.33 0.58 4.00 1.00 0.48 48 h 4.67 0.58 5.33 0.58 0.30 72 h 7.33 1.53 4.67 1.53 0.12 p*: mann whitney test. table 2. analysis of cell proliferation for each time interval (24, 48 and 72 h) after plating in the different studied groups. fig. 1. photomicrograph of periodontal ligament mesenchymal cells in the undifferentiated stage (a) and subjected to osteogenic (b) and adipogenic differentiation (c). (light microscopy, a: 40x; b: von kossa stain, 40x; c: oil red stain, x100). discussion cell adhesion to titanium surfaces is known to be the key factor for osseointegration. biocompatibility assays testing new titanium surfaces employ different types of cells (primary cells, clones, immortalized cells) that retain specific properties in terms of the degree of interaction with the microenvironment of the material. immortalized cell lines that are able to express phenotypic characteristics of osteoblasts are generally used to study cell/material interactions, which are essential for the development of new materials10. according to seo et al.11 (2004), the human periodontal ligament contains cells that, like bone marrow stem cells, possess the potential to differentiate into osteoblasts, chondrocytes and adipocytes. in addition, these periodontal ligament stem cells can be used for regeneration of the fig. 2. growth curve of periodontal ligament mesenchymal cells at different time intervals. the plastic surface was used as a positive control of cell proliferation. proliferation of human periodontal ligament mesenchymal cells on polished and plasma nitriding titanium surfaces 146146146146146 braz j oral sci. 12(2):143-147 ligament itself and of cementum. this cell type was chosen in the present study because undifferentiated mesenchymal cells of the periodontal ligament have been reported to be responsible for both the regeneration of alveolar bone and the osseointegration of titanium implants placed immediately after tooth extraction from the alveolus12. studies have focused on the superficial properties of titanium as the main factor responsible for the adhesion and proliferation of cells with different phenotypes and the consequent formation of mineralized tissue at the implant interface13-14, although cell adhesion and proliferation have been shown to be similar on surfaces with different rugosities15-16. the present study evaluated the effect of surface modification of pure grade ii titanium disks by plasma nitriding. this process is applied to modify materials used as dental implants and consists of depositing nitride onto metallic surfaces under nitrogen plasma6. the cathodic cage technique was used for plasma nitriding, in which the samples were surrounded by a titanium cage in order to enhance the effect of the plasma8. plasma nitriding of metals is a wellestablished method that has numerous industrial applications since it results in peculiar physical and chemical characteristics of the materials, such as increased hardness and wear and oxidation resistance, in addition to proven biocompatibility17. characterization of polished and nitrided surfaces used in this study was previously published by da silva et al.18 (2011). in the present study, periodontal ligament mesenchymal cells reacted differently to the tested surfaces. cell proliferation was more pronounced on the plastic surface as expected, since plastic differs chemically from titanium and is the standard environment for cell culture. this behavior is frequently observed in surface biocompatibility studies8,19. rough surfaces seem to favor cell adhesion when compared with the polished ones20. moreover, integrins are known to play a role in human periodontal ligament cells attachment to substrates and integrin receptors are necessary for attachment of fibroblasts to titanium substrates. the number or density of integrin receptors adhering the cell to the titanium surface varies depending on the type of titanium surface21. among the different titanium surfaces tested in the present study, the number of cells was higher on the nitrided surface at 24 h, indicating that the surface roughness obtained with this treatment favors cell adhesion, in agreement with the findings of previous studies18,22-23. these results suggest a positive effect of the cathodic cage nitriding on the capacity to generate homogenous textured surfaces due to the concentration of energized ions close to the samples. in addition, the surface of nitrided samples in the cathodic cage may contain a higher concentration of titanium nitride (tin) residues due to the high density of ions on the samples, providing the topographic and chemical features necessary for the interaction of cells with a surface18. the proliferation of undifferentiated mesenchymal cells derived from adult human periodontal ligament increased linearly in the polished group over the studied period. in the nitrided group, cell proliferation increased until 48 h, followed by a decline at 72 h. similar results have been reported by da silva et al.18 (2011), who studied the proliferation of preosteoblastic mc3t3 cells on titanium surfaces subjected to plasma nitriding using the cathodic cage technique, demonstrating that different cell types behave similarly on the nitrided surface. the decline in the number of cells probably corresponds to the onset of cell differentiation characterized by a reduction in proliferative activity and modification in the extracellular matrix components produced in response to the surface treatment by plasma nitriding and the resulting increase of the surface roughness. in fact, a previous study with mg-63 cells showed that increasing ti roughness decreases cell proliferation and increases cell differentiation24. further studies are needed to prove this hypothesis. the results confirmed the feasibility of the plasma nitriding method and its positive effect on the early cellular events of osseointegration. this method permitted to modify the titanium surface while maintaining the biocompatibility characteristics of the material22,25. the study of surface treatment techniques has shown that the nanoscale topographical features are particularly important for the development of strategies aiming to functionalize biomaterial surfaces with molecules that are known to promote cell adhesion26. in conclusion, both polished and plasma nitriding surfaces 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2004; 21: 93-7. 14. nebe b, lüthen f, lange r, becker p, beck p, beck u, et al. topographyinduced alterations in adhesion structures affect mineralization in human osteoblasts on titanium. mat sci eng c. 2004; 24: 619-24. 15. rosa al, beloti mm. rat bone marrow cell response to titanium and titanium alloy with different surface roughness. clin oral implants res. 2003; 14: 43-8. 16. anselme k, bigerelle m. statistical demonstration of the relative effect of surface chemistry and roughness on human osteoblast short-term adhesion. j mater sci mater med. 2006; 17: 471-9. 17. figueroa ca, alvarez f. on the hydrogen etching mechanism in plasma nitriding of metals. app surf sci. 2006; 253: 1806-9. 18. da silva js, amico sc, rodrigues ao, barboza ca, alves-júnior c, croci at. osteoblastlike cell adhesion on titanium surfaces modified by plasma nitriding. int j oral maxillofac implants. 2011; 26: 237-44. 19. linez-bataillon p, monchau f, bigerelle m, hildebrand hf. in vitro mc3t3 osteoblast adhesion with respect to surface roughness of ti6a14v substrates. biomol eng. 2002; 19: 133-41. 20. bächle m, kohal rj. a systematic review of the influence of different titanium surfaces on proliferation, differentiation and protein synthesis of osteoblast-like mg63 cells. clin oral implants res. 2004; 15: 683-92. 21. kramer pr, janikkeith a, cai z, ma s, watanabe i. integrin mediated attachment of periodontal ligament to titanium surfaces. dent mater. 2009; 25: 877-83. 22. alves lb, ginani f, da silva jsp, alves-júnior c, barboza cag. bone marrow mesenchymal cell adhesion to polished and nitrided titanium surfaces. braz j oral sci. 2011; 10: 258-61. 23. sá jc, brito ra, de moura ce, silva nb, alves mbm, alves-júnior c. influence of argon-ion bombardment of titanium surfaces on the cell behavior. surf coat technol. 2009; 203: 1765-70. 24. wirth c, grosgogeat b, lagneau c, jaffrezic-renault n, ponsonnet l. biomaterial surface properties modulate in vitro rat calvaria osteoblast response: roughness and or chemistry? mater sci eng c. 2008; 28: 9901001. 25. guerra-neto clb, silva mam, alves-júnior c. osseointegration evaluation of plasma nitrided titanium implants. surf eng. 2009; 25: 434-9. 26. bueno rbl, adachi p, castro-raucci lms, rosa al, nanci a, oliveira pt. oxidative nanopatterning of titanium surfaces promotes production and extracellular accumulation of osteopontin. braz dent j. 2011; 22: 179-84. 147147147147147proliferation of human periodontal ligament mesenchymal cells on polished and plasma nitriding titanium surfaces oral sciences n3 braz j oral sci. 14(1):1-4 original article braz j oral sci. january | march 2015 volume 14, number 1 chemical analysis and vickers hardness of orthodontic mini-implants christiane cavalcante feitoza1, guilherme josé pimentel lopes de oliveira2, rafael leonardo xediek consani3, eloisa marcantonio boeck1, karina eiras dela coleta pizzol1, nadia lunardi1 1centro universitário de araraquara – uniara, department of dentistry, area of orthodontics, araraquara, sp, brazil 2 universidade estadual paulista – unesp, araraquara dental school, department of diagnosis and surgery, area of periodontology, araraquara, sp, brazil 3universidade estadual de campinas – unicamp, piracicaba dental school, department of oral rehabilitation, piracicaba, sp, brazil correspondence to: nádia lunardi av. maria antonia camargo de oliveira,170 cep: 14707-120, araraquara, sp, brasil phone: +55 19 991648770 e-mail: nadialunardi@yahoo.com.br abstract orthodontic mini-implants are used in clinical practice to provide efficient and aesthetically-pleasing anchorage. aim: to evaluate the hardness (vickers hardness) and chemical composition of miniimplant titanium alloys from five commercial brands. methods: thirty self-drilling mini-implants, six each from the following commercial brands, were used: neodent (neo), morelli (mor), sin (sin), conexão (con), and rocky mountain (rmo). the hardness and chemical composition of the titanium alloys were performed by the vickers hardness test and energy dispersive x-ray spectroscopy, respectively. results: vickers hardness was significantly higher in sin implants than in neo, mor, and con implants. similarly, vh was significantly higher in rmo implants than in mor and neo ones. in addition, vh was higher in con implants than in neo ones. there were no significant differences in the proportions of titanium and aluminum in the mini-implant alloy of the five commercial brands. conversely, the proportion of vanadium differed significantly between con and mor/neo implants. conclusions: mini-implants of different brands presented distinct properties of hardness and composition of the alloy. keywords: orthodontics; dental materials; hardness. introduction in recent decades, there has been a growing demand for orthodontic treatment in dental offices by adult patients, which required the development of an efficient and aesthetically-pleasing anchorage system to enable and expedite treatment. the orthodontic mini-implant is a temporary skeletal anchorage device that allows the orthodontist to work safely because it eliminates the ensuing side effects on teeth used as anchorage in conventional treatment and does not depend on patient compliance1. the ease of installation and removal, possibility of insertion in different intraoral regions, low cost, small healing time, and good patient acceptance all contribute to the diffusion of the technique2-4. several studies have demonstrated the clinical efficacy of this anchorage technique5-7, but clinical practice exposes some disadvantages such as screw breakage during installation, the possibility of osseointegration making removal difficult, and the lack of stability with subsequent mini-implant loss. thus, further studies on the efficacy of mini-implants are needed to solve these issues. the resistance of mini-implants is determined by the interaction between mini-implant design and chemical composition of the alloy. the alloy used for mini-implant production should be nontoxic, biocompatible, have good mechanical received for publication: november 05, 2014 accepted: january 29, 2015 22222 group sin neo c o n r m o m o r manufacturer sin sistema de implantes nacional neodent conexão rocky mountain orthodontics morelli origin são paulo/sp, brazil curitiba/pr, brazil arujá/sp, brazil seoul, south korea sorocaba/sp, brazil *nd (mm) 1.60 1.60 1.50 1.60 1.60 †nl (mm) 6.00 7.00 6.00 6.00 6.00 profile (mm) 0.00 1.00 1.00 0.00 2.00 shape cylindrical cylindrical conical cylindrical cylindrical lead thread single single double single single table 1.table 1.table 1.table 1.table 1. mini-implant features according to manufacturer’s specifications. *nd = nominal diameter; †nl = nominal length properties, and be stress, tension, and corrosion resistant8-9. the use of orthodontic mini-implants in different bone regions, including roots, requires that screws have reduced size and thickness and resist higher torsional loads than those required for insertion, in addition to orthodontic and orthopedic forces. to meet these requirements, the titanium alloy (ti-6al-4v) chosen for manufacture of mini-implants incorporates aluminum (al) and vanadium (v) into its composition along with commercially pure titanium (cpti)10. ti-6al-4v alloy has higher fatigue resistance than (cpti), while having the same corrosion resistance and low toxicity11. the amount of each component in the alloy, in addition to the manufacturing quality, can alter its mechanical properties such as hardness and resiliency, which are responsible for the fracture resistance of the mini-implant. given the wide diversity of mini-implant types available in the market, this study aimed to evaluate the chemical composition and vickers hardness (vh) of five orthodontic mini-implant commercial brands to determine whether their properties are suitable for clinical use. material and methods orthodontic mini-implants thirty self-drilling mini-implants were used, six each from five commercial brands (sin – sin, neodent – neo, conexão – con, rocky mountain – rmo, and morelli – mor) with the largest number of similar characteristics to enable comparisons (table 1). the macrostructural aspects of each mini-implant design are shown in figure 1. three mini-implants of each brand were used for the vickers hardness analysis while the other three were used for the eds analysis. vickers hardness vickers hardness was determined by penetration length of the pyramidal diamond tip. a single operator previously calibrated by the repetition process performed hardness tests using a shimadzu hmv-2micro hardness tester at a load of 300 gf and 4.904 n for 5 sec. the measurements were performed over the mini-implant head, which is the region with the highest stability for diamond penetration. each of the three mini-implants from the five commercial brands was measured three times, totaling nine measurements per brand. energy dispersive x-ray spectroscopy (eds) the remaining mini-implants were used for semi quantitative analysis of alloy components. each mini-implant was removed from its packaging only at the time of analysis so that the surface was not manipulated or contaminated by external agents, thus preserving the original characteristics. six surface micrographs of each mini-implant (two of the mini-implant head, two of the body, and two closer to the mini-implant tip) were taken in panoramic view using a scanning electron microscope coupled with noran instruments eds detector with a vantage digital acquisition engine. the values for the chemical composition of the alloy were then obtained from these measurements. statistical analysis due to the small number of samples, non-parametric tests were used for the statistical analysis using bioestat 5.0 software (belém, pa, brazil). the kruskall-wallis complemented by the post-hoc test of dunn was used to evaluate the statistical differences regarding the hardness fig. 1. micrographic aspects of mini-implant design from five commercial brands (18x magnification). a) sin-sin, b) neodent – neo, c) conexão – con, d) rocky mountain – rmo, and e) morelli – mor chemical analysis and vickers hardness of orthodontic mini-implants braz j oral sci. 14(1):1-4 33333 and the chemical composition of the alloy between the brands. the bioestat 5.0 software (belém, pa, brazil) was used for the analysis and the significance level was set at 5% (p<0.05) for all the tests. results vickers hardness there were significant differences in vh values between commercial brands. vickers hardness was significantly higher in sin implants than in neo, mor, and con implants. similarly, vh was significantly higher in rmo implants than in mor and neo ones. in addition, vh was higher in con implants than in neo ones (table 2). brands vickers hardness 1-sin 400.66 ±35,142,3,5 2-neodent 337.11±4,66 3-conexão 366.11±19,572 4-rockmo 393.00±4,932,5 5-moreli 342,44±14,52 table 2.table 2.table 2.table 2.table 2. vickers hardness values of three mini-implant samples each from five commercial brands. 2mini-implants with higher hardness than the neodent mini-implants (kruskal wallis with dunn); 3mini-implants with higher hardness than the conexão mini-implants (kruskal wallis with dunn); 5 mini-implants with higher hardness than the morelli mini-implants (kruskal wallis with dunn). groups ti a l v 1-sin 87.10 8.51 4.06 2-neo 86.89 8.39 4.293 3-con 86.94 8.99 3.91 4-rmo 86.04 8.31 4.11 5-mor 86.85 8.86 4.323 table 3.table 3.table 3.table 3.table 3. proportion of metal components in orthodontic mini-implant alloys from five commercial brands determined using energy dispersive x-ray spectroscopy (eds). ³mini-implants with higher proportion of vanadium than the conexão mini-implants (kruskall wallis with dunn). energy dispersive x-ray spectroscopy (eds) there were no significant differences in the proportions of titanium and aluminum in the mini-implant alloy of the five commercial brands. conversely, the proportion of vanadium differed significantly between con and mor/ neo implants. even though the differences in mean values between brands were small, the low proportion of vanadium in the alloy causes small variations to be readily detectable (table 3). discussion vickers hardness values showed little variation within each commercial brand except for con implants. conversely, vh values differed significantly between brands, and sin and rmo implants exhibited the highest vh values. our findings are consistent with the study by eliades et al. (2009)12, who evaluated mini-implants from a single commercial brand and found vh values of 342 ± 14 hv for the body and 354 ± 16 hv for the surface. these vh values are slightly higher than the ones found in our study, which ranged from 337 ± 4.66 hv (neo) to 440 ± 35.14 hv (sin). moreover, the values of both studies are higher than the value (325.0 ± 10.1 hv) found by lima et al. (2011)13, who evaluated implants made with the same alloy. energy dispersive x-ray spectroscopy (eds) is a semi quantitative analysis that determines the chemical elements in the specimen and quantifies their approximate proportions. the results of our study confirmed the presence of ti, al, and v in mini-implants alloys, albeit at different proportions than those specified by manufacturers (ti6al4v). nevertheless, the fact that we found a higher proportion of aluminum (approximately 8%) may be an artifact of the technique. the difference in vh among brands may be due to differences in the proportion of titanium and vanadium in mini-implant alloys. even though the proportion of titanium was similar in all commercial brands, titanium alloys can have different amounts of alpha-phase and beta-phase titanium, possibly due to differences in the manufacturing process of the alloy. for instance, cotrim-ferreira et al. (2010)11 showed that there were quantitative differences in alpha-phase and beta-phase titanium in mini-implant alloys from three commercial brands (sin, dewimed, and con), even though they were within the guidelines of the “technical committee of european titanium producers” described in publication ettc-2. the crystalline microstructure of the alloy, i.e., the amount of alpha and beta titanium in the alloy, is responsible for differences in its mechanical properties. an alloy with a higher amount of beta-phase titanium has higher tensile strength than an alloy with a higher amount of alpha-phase titanium, whereas alpha titanium has higher corrosion resistance than beta titanium10,14. the addition of vanadium (a beta stabilizer) and an increase in temperature can both result in increased betaphase titanium in the alloy15. this study showed that there were differences in the proportion of vanadium among commercial brands. even though these differences were not high, because the proportion of vanadium in the alloy is low, any small deviations are readily detectable and become statistically significant. thus, the differences in vh among mini-implant brands may have been due to the proportion of vanadium in the alloy or to differences in heating and cooling temperatures during the manufacturing process of miniimplants14. the difficulty in the casting process of titanium miniimplants may also have been responsible for the differences in vh observed in our study. the low density of titanium, its high melting temperature, and high chemical reactivity with surface elements and atmospheric gases make casting of this alloy extremely costly and laborious due to the need chemical analysis and vickers hardness of orthodontic mini-implants braz j oral sci. 14(1):1-4 for special equipment to keep the titanium in a vacuum oxygen-free environment or in the presence of inert gases1618. additional tests such as x-ray diffraction are needed to test the hypothesis that titanium phases affect alloy hardness. the variation in alpha and beta phases of mini-implants may occur during manufacturing of the alloy or even during the manufacturing process of the mini-implant. the knowledge of mini-implant hardness and composition is vital to assist in choosing the ideal commercial brand and model for each installation site, thus minimizing the risk of fracture. however, tensile strength of mini-implants is determined not only by their chemical composition and hardness, but mini-implant design is also fundamental for mini-implant choice since these parameters influence the possibility of the mini-implants fracture. based on the results of this research it can be concluded: 1. there were significant differences in vickers hardness between sin and neodent/morelli/conexão; rmo and neodent/morelli; and neodent and conexão brands; 2. the proportion of vanadium in the alloy differed significantly between commercial brands; 3. the effect of these differences in the clinical practice needs to be test in clinical trials. references 1. kanomi r. mini-implant for orthodontic anchorage. j clin orthod. 1997; 31: 763-7. 2. nienkemper m, pauls a, ludwig b, wilmes b, drescher d. multifunctional use of palatal mini-implants.j clin orthod. 2012; 46: 679-86. 3. lehnen s, mcdonald f, bourauel c, jäger a, baxmann m. expectations, acceptance and preferences of patients in treatment with orthodontic miniimplants: part ii: implant removal. j orofac orthop. 2011; 72: 214-22. 4. lehnen s, mcdonald f, bourauel c, baxmann m. patient expectations, acceptance and preferences in treatment with orthodontic mini-implants. a randomly controlled study. part i: insertion techniques. j orofac orthop. 2011; 72: 93-102. 5. rodriguez jc, suarez f, chan hl, padial-molina m, wang hl. implants for orthodontic anchorage: success rates and reasons of failures. implant dent. 2014; 23: 155-61. 6. nienkemper m, wilmes b, pauls a, drescher d. multipurpose use of orthodontic mini-implants to achieve different treatment goals. j orofac orthop. 2012; 73: 467-76. 7. motoyoshi m. clinical indices for orthodontic mini-implants. j oral sci. 2011; 53: 407-12. 8. pan cy, chou st, tseng yc, yang yh, wu cy, lan th, et al. influence of different implant materials on the primary stability of orthodontic miniimplants. kaohsiung j med sci. 2012; 28: 673-8. 9. serra g, morais l, elias cn, semenova ip, valiev r, salimgareeva g, et al. nanostructured severe plastic deformation processed titanium for orthodontic mini-implants. mater sci eng c mater biol appl. 2013; 33: 4197-202. 10. morais ls, serra gg, muller ca, andrade lr, palermo ef, elias cn, et al. titanium alloy mini-implants for orthodontic anchorage: immediate loading and metal ion release. acta biomater. 2007; 3: 331-9. 11. cotrim-ferreira fa, quaglio cl, peralta rpv, carvalho peg, siqueira df. metallographic analysis of the internal microstructure of orthodontic mini-implants. braz oral res. 2010; 24: 438-42. 12. eliades t, zinelis s, papadopoulos ma, eliades g. characterization of retrieved orthodontic miniscrew implants. am j orthod dentofacial orthop. 2009; 135: 10-7. 13. lima gma, soares ms, penha ss, romano mm. comparison of the fracture torque of different brazilian mini-implants. braz oral res. 2011; 25: 116-21. 14. alsamak s, bitsanis e, makou m, eliades g.morphological and structural characteristics of orthodontic mini-implants. j orofac orthop. 2012; 73: 58-71. 15. kent d, wang g, dargusch m. effects of phase stability and processing on the mechanical properties of ti-nb based â ti alloys. j mech behav biomed mater. 2013; 28: 15-25. 16. donachie jr mj. titanium. a technical guide. ohio: asm international; 2000. 17. taira m, moser jb, greener eh. studies of ti alloys for dental castings. dent mater. 1989; 5: 45-50. 18. voitik aj. titanium dental castings, cold worked titanium restorations-yes or no? trends tech contemp dent lab. 1991; 8: 23-34. chemical analysis and vickers hardness of orthodontic mini-implants braz j oral sci. 14(1):1-4 44444 oral sciences n3 braz j oral sci. 12(2):109-113 original article braz j oral sci. april | june 2013 volume 12, number 2 a survey on dental undergraduates’ knowledge of oral radiology sergio lins de-azevedo-vaz1, karla de faria vasconcelos2, karla rovaris2, naiara de paula ferreira3, francisco haiter neto2 1department of clinical dentistry, dental school, federal university of espirito santo, vitória, es, brazil 2department of oral diagnosis, division of oral radiology, piracicaba dental school, university of campinas, piracicaba, sp, brazil 3 department of community dentistry, piracicaba dental school, university of campinas, piracicaba, sp, brazil correspondence to: sergio lins de-azevedo-vaz federal university of espirito santo, school of dentistry, department of clinical dentistry avenida marechal campos 1468, cep: 29043-900 – maruípe, vitória, es, brasil phone: +55 27 33357242 abstract dentists’ incorrect behavior with regards to oral radiology, as reported in the literature, has been related to inadequate training of undergraduates. aim: this study assessed dental undergraduates’ knowledge of oral radiology. methods: a questionnaire containing 30 questions pertaining to three domains general principles, radiobiology/radioprotection and technique/interpretation was used as data collection instrument. a total of 195 students answered the questionnaires. results: no statistically significant differences were found between second-, thirdand fourthyear students (p>0.05) when the whole questionnaire and the general principles domain (p>0.05) were considered. the technique/interpretation domain presented a borderline statistical significance level (p=0.051), with more correct answers attributed to second-year students. a statistically significant difference (p<0.05) was seen for the radiobiology/radioprotection domain, in which the fourth-year students performed better. conclusions: dental undergraduates’ knowledge of oral radiology did not increase or decrease significantly comparing the undergraduate years. however, with the exception of the technique/interpretation domain, students of more advanced undergraduate years answered more correctly the questions. nevertheless, the technique/ interpretation domain should be reinforced throughout the undergraduate course. keywords: dental education, radiology, dental radiography, radiation protection, questionnaires. introduction literature shows that many dentists have neglected not only the basic principles of radiology, but also certain national laws on radiograph application1-6. inappropriate procedures mostly involve inadequate development of films, use of cone indicators, incorrect radiographic techniques, excessive exposure time, failure to protect patients during radiographic exposure, and improper disposal of processing solutions and lead foil3. these procedures result in radiographs of inadequate quality for diagnosis, higher radiation doses for patients and damage to the environment3. some authors associate such failings with an inadequate training of undergraduates3,7-8. currently, education of dental students is being discussed all over the world with a view to changes9-12. in brazil, the ministry of education has proposed new curriculum guidelines for dental courses10. these guidelines propose an interdisciplinary general graduation in dentistry. however, 10 years after the guidelines were first proposed, little discussion has taken place in terms of education received for publication: february 25, 2013 accepted: june 11, 2013 110110110110110 braz j oral sci. 12(2):109-113 on oral radiology. as an example, this subject is taught during a 1-year period in brazilian undergraduate dental courses, although dental radiographs are taken throughout the whole undergraduate course. piracicaba dental school and other brazilian universities are implementing new curricula based on the abovementioned guidelines. learning deficiencies must be assessed in order to propose methodological strategies and improve the course. with such a background, this study set out to investigate the development of oral radiology knowledge during an undergraduate dental course. material and methods this cross-sectional study was conducted at the piracicaba dental school, university of campinas, piracicaba, sp, brazil. its design received full approval from the local research ethics committee (protocol #095/2011) and the participants were second-, thirdand fourth-year undergraduate students. first-year students did not participate because oral radiology is not a subject in their curriculum. in order to evaluate the students’ knowledge, a selfadministered questionnaire containing 30 questions on oral radiology was applied at the end of the academic year. the chart 1 shows the questionnaire with the answers considered correct. the questions pertained to three domains: general principles, radiobiology/radioprotection and technique/ interpretation. the questionnaire had been validated earlier according to the protocol described by ferreira et al.13 (2012). the students received the questionnaire after agreeing with their participation and giving their written informed consent. the questionnaire was not used for graduation purposes and the students were not obliged to fill it out. questions were answered with “true”, “false”, or “i don’t know” statements. the aim of the “i don’t know” statement was to prevent random responses by guessing the question response* general principles 1. x-rays are electromagnetic radiation. true 2. during examination, the x-ray operator must protect himself from the reflected rays. false 3. the x-ray machine must be turned off when not in use in order to avoid inadequate x-ray emission. false 4. it takes 5 seconds after an exposure to scatter radiation be dissipated. false 5. it is not possible to generate x-rays without power supply. true 6. the oil in the tube head is heated when the x-ray machine is turned on, even if no exposure is performed. false 7. the room must be immediately isolated if a x-ray tube is broken. false 8. a radiograph fixed within 15 seconds is adequate for diagnosis. false 9. covering the processing solutions can extend their usage time. true 10. rinse stops the action of the developer in the manual processing. false radiobiology/radioprotection 11. routine radiographic examination with a six-month interval cannot cause stochastic biological effects. false 12. radiographic examination in pregnant women must be performed only in the second trimester of pregnancy in order to reduce the chance of deleterious effects. true 13. protecting gonads from radiation is not necessary, because dental radiographs are taken in the head and neck region. true 14. all human tissues have the same radiosensitivity. false 15. whole body low-intensity-fractionated irradiation is more dangerous than high-intensity-localized irradiation. true 16. x-ray operators have minimal chance of somatic effects if they correctly adopt the radioprotection rules. true 17. barriers like lead walls are mandatory to ensure adequate protection for the operator. false 18. an adequate maintenance of the x-ray machine results in better productivity, and protection for both operator and patient. true 19. parents should hold films in children’s mouth if they do not cooperate during examination. true 20. periapical radiographs are strictly indicated for children only in cases of emergency. false technique/interpretation 21. bite-wing radiographs are indicated to investigate dental decay. true 22. oclusal radiographs are indicated to investigate buccolingual bone expansion. true 23. panoramic radiographs are indicated to investigate incipient caries lesions. false 24. a full-mouth series (fmx) is indicated if many teeth are absent during physical examination. false 25. an unerupted superior left-canine had dislocated coincidently with the x-ray tube in the clark method. therefore, it is localized in a palatal position. true 26. an elliptical radioluscence in the apex of vital lower pre-molars with intact lamina dura probably refers to the mentual foramen. true 27. a diffuse radiolucency in the mandibular body, apically to lower molars, may indicate an aggressive lesion named “stafne bone defect”. false 28. since it onset, dental decay is radiographically detected. false 29. multilocular ameloblastoma has a ground-glass appearance. false 30. tooth displacement and bone expansion are typical of malignant lesions. false chart 1: questions of the questionnaire applied and the respective correct answers. the students were asked to answer each question as “true”, “false” or “i don’t know”. a survey on dental undergraduates’ knowledge of oral radiology 111111111111111 braz j oral sci. 12(2):109-113 variables gender age (years) household income female male < 20 20-23 24-27 > 27 up to 1 mw from 1 to 2 mw from 2 to 4 mw from 5 to 10 mw above 10 mw n (%) 145 (74.4) 47 (24.1) 16 (8.2) 131 (67.1) 19 (9.7) 2 (1.0) 1 (0.6) 3 (1.8) 38 (22.8) 63 (37.7) 62 (37.1) mw = minimum wage. note: several students left unanswered some questions of the sociodemographic survey. table 1 – demographics of students in study. table 2 – correct and incorrect answers per year group. 2nd year 3rd year 4th year total n (%) n (%) n (%) n (%) incorrect 743 (36.4) 808 (35.9) 517 (33.1) 2,068 (35.3) correct 1,297 (63.6) 1,442 (64.1) 1,043 (66.9) 3,782 (64.7) total 2,040 2,250 1,560 5,850 χ2=4.559 p=0.102 answers. the spss software version 18.0 (spss inc., chicago, il, usa) was used for statistical analysis and the data were dichotomized into correct and incorrect answers (including the “i don’t know” answers). the correct and incorrect frequencies were tested in relation to the students’ year group, using the chi-square test. these frequencies were also tested in relation to each of the three main domains of the questionnaire. significance level was set at 5%. each undergraduate year group invited to participate in this study had approximately 80 students regularly enrolled in the undergraduate course, so the sample comprised a population of 240 potential respondents. results the overall response rate was 81.2% (n=195) and the final sample comprised 68 second-year (34.9%), 75 thirdyear (38.5%) and 52 fourth-year students (26.7%). the sociodemographic survey (table 1) showed predominance of female students (74.4%) aged 20 to 23 years (67.1%), with a household income above five minimum wages (74.8%). table 2 shows the percentage of correct and incorrect answers to the questionnaire. the chi-squared test did not show statistically significant difference between these values (p>0.05). analyzing the questions separately, question 18 (“an adequate maintenance of the x-ray machine results in better productivity, and protection for both operator and patient”) yielded the largest number of correct answers. on the other hand, question 17 (“barriers like lead walls are mandatory to ensure adequate protection for the operator”) presented the largest number of incorrect answers. question 11 (“routine radiographic examination with a six-month interval cannot cause stochastic biological effects”) was also answered incorrectly by most students. analyzing each questionnaire domain (table 3), no statistically significant difference was found either for the general principles or for the technique/interpretation domains (p>0.05). in the general principles domain, fourthyear students answered more questions correctly, while the second-year students performed better in the technique/ interpretation domain. a statistically significant difference (p<0.05) was seen for the radiobiology/radioprotection domain with more correct answers attributed to the fourthyear students. discussion this study set out to investigate to what extent dental undergraduates’ knowledge of oral radiology developed throughout their course. brazil has the largest number of oral radiology courses and oral radiologists14. the incorrect behavior of dentists in terms of oral radiology is reported in the literature and it is correlated to a deficiency in the undergraduate education3,7-8. various parameters are used to determine the effectiveness of education, namely student’s performance, satisfaction, attitudes and skills, accomplishment of course goals and objectives, teachers’ perceptions and evaluation of the course15-16. in this study, students’ knowledge was evaluated by means of a self-applied questionnaire, a methodology also used in similar studies17-19. the questionnaire used in this research had been previously validated according to a protocol referred to in the literature13. the aim of this validation process was to obtain a trustworthy instrument for evaluation of the students. it included specialist’s opinion in the field, pilot tests to verify students’ understanding of the questions, consistency of data, and reproducibility of the questionnaire. undergraduates at advanced levels of a dentistry course have more study content than students at initial levels. therefore, it is expected that the more advanced the student level, the better their knowledge of course content. in this study, fourth-year students answered more questions correctly than did either secondor third-year students. since the questionnaire was not used to grade the students in levels, it is believed that the obtained results are close to the real situation and thereby provide a reliable evaluation of the students’ knowledge. some deficiencies were, however, detected for fourth-year students in relation to the specific domains evaluated in the study. the general principles domain presented uniform behavior throughout the course, with no statistically significant difference between the students (p>0.05). the evaluated students answered correctly about 61 to 65% of this domain, showing satisfactory knowledge of the fundamentals of oral radiology. a survey on dental undergraduates’ knowledge of oral radiology braz j oral sci. 12(2):109-113 undergraduates’ knowledge of the radiobiology/ radioprotection domain increased to a significant extent (p<0.05). since the second-year students had not studied this subject when the research was conducted, they produced more incorrect answers than other students. on the other hand, fourth-year students registered more correct answers than did third-year students. such results strongly indicated that dental students showed a significant increase in their knowledge of radioprotection procedures and the consequences of exposure to radiation along the course. it is important to emphasize that only the radiobiology/radioprotection domain had not been taught to the second-year students when the questionnaire was applied. the technique/interpretation domain showed a borderline statistical significance level (p=0.051). secondyear students performed better than fourth-year students, which in turn, answered more questions correctly than did the third-year. possibly, the second-year students answered more questions correctly because they had studied this subject just before the questionnaire was applied. despite the decrease in the knowledge levels, as observed in the technique/interpretation domain, it presented the highest percentage of correct answers. dental radiographs are a valuable diagnostic tool for patient assessment and treatment planning in most clinical specialties of dentistry20. the obtained results demonstrated that thirdand fourth-year students showed less knowledge of the technique/interpretation domain. these students are closest to entering the professional field and this lower knowledge should concern the clinical practice because a thorough knowledge of the various available radiographic modalities, their application, and accurate interpretation of the images and obtained data is necessary for the ethical and efficient practice of dentistry20. question 29 (“multilocular ameloblastoma has a ground-glass appearance”) presented the highest percentage of incorrect answers in the technique/ interpretation domain. education systems worldwide are undergoing remarkable changes, as courses and programs are being designed in new ways19, moving away from the passive teacher-centered to a more active learner-centered learning21. the brazilian guidelines for dental education10 encourage a more active learner-centered learning. the literature has shown that this methodology presents better results in oral radiology20. therefore, it can be suggest that an active learner-centered methodology be introduced in the evaluated school in order to improve students’ knowledge of the technique/ interpretation domain. it is important to emphasize that this was a cross-sectional study, which evaluated only one dental school, which means that the obtained results refer to a group of students who took part in the survey and, undoubtedly, there are differences between courses. hence, further longitudinal investigations involving other dental schools are recommended to provide more data for discussion on education in oral radiology. additionally, the present study evaluated only the students’ theoretical knowledge and not their practical skills. in conclusion, dental undergraduates’ knowledge of oral radiology did not increase or decrease significantly comparing the first-, secondand fourth-year groups. however, except for the technique/interpretation domain, the more advanced the undergraduate year, the more correctly the students answered the questions. nevertheless, we believe that the technique/interpretation domain needs to be reinforced throughout the course. references 1. aps jk. flemish general dental practitioners’ knowledge of dental radiology. dentomaxillofac radiol. 2010; 39: 113-8. 2. jacobs r, vanderstappen m, bogaerts r, gijbels f. attitude of the belgian dentist population towards radiation protection. dentomaxillofac radiol. 2004; 33: 334-9. 3. shahab s, kavosi a, nazarinia h, mehralizadeh s, mohammadpour m, emami m. compliance of iranian dentists with safety standards of oral radiology. dentomaxillofac radiol. 2011; 41: 159-64. 112112112112112a survey on dental undergraduates’ knowledge of oral radiology d o m a i n 2nd year 3rd year 4th year total n (%) n (%) n (%) n (%) general principles incorrect 265 (39.0) 290 (38.6) 179 (34.4) 735 (37.7) correct 415 (61.0) 460 (61.4) 341 (65.6) 1,215 (62.3) total 680 750 520 1,950 χ2=3.166 p=0.205 radiobiology/ radioprotection incorrect 316 (46.5) 299 (39.8) 196 (37.6) 811 (41.6) correct 364 (53.5) 451 (60.2) 324 (62.4) 1,139 (58.4) total 680 750 520 1,950 χ2=10.965 p=0.004 technique/ interpretation incorrect 160 (23.5) 219 (29.2) 142 (27.3) 522 (26.7) correct 520 (76.5) 531 (70.8) 378 (72.7) 1,428 (73.3) total 680 750 520 1,950 χ2=5.969 p=0.051 table 3 – correct and incorrect answers per year group in relation to the questionnaire domains. braz j oral sci. 12(2):109-113 113113113113113 4. svenson b, söderfeldt b, gröndahl hg. attitudes of swedish dentists to the choice of dental x-ray film and collimator for oral radiology. dentomaxillofac radiol. 1996; 25: 157-61. 5. svenson b, söderfeldt b, gröndahl hg. analysis of dentists’ attitudes towards risks in oral radiology. dentomaxillofac radiol. 1996; 25: 151-6. 6. tosoni gm, campos dm, silva mr. frequency and quality management of intraoral radiographic examinations in private dental practices. rev odontol unesp. 2003; 32: 25-9. 7. melo mf, melo sl. radioprotection in dentistry offices. cienc saude colet. 2008; 13(suppl 2): 2163-70. 8. neves fs, vasconcelos tv, bastos lc, góes la, freitas dq. attitudes of dentists in relation to radiological protection, according to brazilian law. rev odontol bras central. 2010; 19: 301-5. 9. hendricson wd, andrieu sc, chadwick dg, chmar je, cole jr, george mc et al. educational strategies associated with development of problem-solving, critical thinking, and self-directed learning. j dent educ. 2006; 70: 925-36. 10. brazil. ministry of education. national council of education. resolution cne/ces. board of higher education. national curriculum guidelines for undergraduate dentistry. official federal gazette; 2002 mar 4. 11. haden nk, andrieu sc, chadwick dg, chmar je, cole jr, george mc et al. the dental education environment. j dent educ. 2006; 70: 1265-70. 12. linjawi ai, walmsley ad, hill kb. online discussion boards in dental education: potential and challenges. eur j dent educ. 2012; 16: e3–9. 13. ferreira np, batista mj, sousa mlr, cury ja. validation of the questionnaire about knowledge cariology [in portuguese]; 2012 [in press]. 14. ruprecht a. the status of oral and maxillofacial radiology worldwide in 2007. dentomaxillofac radiol. 2009; 38: 98-103. 15. olapiriyakul k, scher jm. a guide to establishing hybrid learning courses: employing information technology to create a new learning experience, and a case study. internet higher educ. 2006; 9: 287-301. 16. handal b, groenlund c, gerzina t. dentistry students’ perceptions of learning management systems. eur j dent educ. 2010; 14: 50-4. 17. neuhaus kw, schegg r, krastl g, amato m, weiger r, walter c. integrate learning in dentistry: baseline data and first evaluation at the dental school of basel. eur j dent educ. 2008; 12: 163-9. 18. woltering v, herrler a, spitzer k, spreckelsen c. blended learning positively affects students’ satisfaction and the role of the tutor in the problem-based learning process: results of a mixed-method evaluation. adv health sci educ. 2009; 14: 725-38. 19. kavadella a, tsiklakis k, vougiouklakis g, lionarakis a. evaluation of a blended learning course for teaching oral radiology to undergraduate dental students. eur j dent educ. 2012; 16: e88-95. 20. kumar v, gadbury-amyot cc. a case-based and team-based learning model in oral and maxillofacial radiology. j dent educ. 2012; 76: 330-7. 21. pahinis k, stokes cw, walsh tf, tsitrou e, cannavina g. a blended learning course taught to different groups of learners in a dental school: follow-up evaluation. j dent educ. 2008; 72: 1048-57. a survey on dental undergraduates’ knowledge of oral radiology oral sciences n3 original article braz j oral sci. october | december 2013 volume 12, number 4 changes in root canal anatomy using three nickel-titanium rotary system a cone beam computed tomography analysis carlos menezes aguiar1, fernanda araujo donida1, andréa cruz câmara1, marco frazão2 1department of prosthetics and oral and facial surgery, faculty of dentistry, federal university of pernambuco, recife, pe, brazil 2private practice, recife, pe, brazil correspondence to : carlos menezes aguiar rua aristides muniz, 70/501, cep: 51020-150 boa viagem, recife, pe, brasil phone: +55 81 34676821 e-mail: cmaguiar.ufpe@yahoo.com.br abstract aim: cone beam computed tomography (cbct) was used to evaluate the ability of three niti rotary systems to maintain the original root canal anatomy. methods: sixty mesiobuccal canals of human mandibular first molars were divided into three groups with 20 root canals each. all teeth were scanned by cbct before instrumentation. the images were captured digitally for further analysis using the image tools software. the images were sectioned in three points, located at 9 mm, 6 mm and 3mm from the apex. in group 1, the root canals were instrumented with protaper universal™ rotary system; in group 2, with twisted file™ rotary system; and in group 3, with mtwo™ rotary system. instrumented teeth were scanned again using cbct and the images of the uninstrumented canals were compared with images of the instrumented canals. the results were statistically analyzed using the one-way anova test. a level of significance of 0.05 was adopted. results: the means of d1 at distances of 9 mm, 6 mm, and 3 mm from the apex were, respectively: group 1: 0.88±0.257, 1.00±0.000, and 1.00±0.000; group 2: 0.79±0.745, 0.65±0.669, and 0.25±0; group 3: 0.50±0.745, 0.33±0.472, and 0.03±0.104. the means of d2 at distances of 9 mm, 6mm, and 3mm from the apex were respectively: group 1: 1.00±0.00, 1.00±0.00, and 1.00±0.00; group 2: 0.41±0.299, 0.30±0.428, and 0.50±0.707; group 3: 0.58±0.910, 0.85±1.857, and 0.31±0.643. conclusions: the cbct analysis revealed that the protaper universal™ produced centered preparations and while the twisted file™ and mtwo™ rotary systems produced canal deviation. keywords: root canal preparation, tomography, emission-computed. introduction root canal preparation is an important part of endodontic treatment. one of the main objectives in root canal preparation is to develop a shape that tapers from apical to coronal, maintaining the original canal shape1. instruments should remain centered in the root canal throughout the preparation2. nickel-titanium (niti) rotary instruments, due to their superelastic behavior, are able to maintain the original canal shape without significant deviation or create irregularities such as zipping, ledges, perforations or danger zones in curved canals. many types of rotary root canal instruments have been introduced, varying in cross-section, blade, pitch design and taper3. the protaper™ niti rotary system has been upgraded to the protaper universal™ rotary system, which includes shaping, finishing and retreating instruments. it received for publication: august 26, 2013 a c c e p t e d : november 25, 2013 braz j oral sci. 12(4):307-312 incorporates a shallow, u-shaped groove at each of its convex triangular sides in cross-section, supposedly to improve flexibility in the larger instruments. the modified design has also been suggested to reduce the subjective feeling of the instrument being “pulled” into the canal or so-called screw-in effect4-6. a completely different manufacturing process has been developed by sybronendo to create a new rotary file for root canal preparation called the twisted file™ (sybronendo, orange, ca, usa). these files have a triangular cross section with constant tapers of .04, .06, .08, .10, and .12. they are available in five tip sizes from 25 to 50. the manufacturer claims that the three new manufacturing processes of these files, namely r-phase heat treatment, twisting of the metal, and special surface conditioning, significantly increase the instrument’s resistance to cyclic fatigue and flexibility, even with .06-, .08-, .10-, and .12-tapered instruments, maintaining the original canal center and minimizing canal transportation even in severely curved root canals1,7,8. mtwo™ (vdw; antaeus, munich, germany) is a newly developed niti rotary system. mtwo instruments have an sshaped cross-sectional design, a positive rake angle with 2 cutting edges, no radial lands, progressive blade camber (pitch) in the apical–coronal direction and a noncutting tip9-11. in order to investigate the efficiency of instruments and techniques developed for root canal preparation, a number of methods have been used to compare canal shape before and after preparation. cone beam computed tomography (cbct) imaging techniques have been evaluated as noninvasive methods for the analysis of canal geometry and efficiency of shaping techniques. using this technique it is possible to compare the anatomic structure of root canal before and after instrumentation1,12. t h e p r e s e n t s t u d y w a s s e t o u t t o d e t e r m i n e t h e c h a n g e s i n r o o t c a n a l a n a t o m y u s i n g t h e p r o t a p e r universal™, twisted file™ and m two™ rotary systems analyzed by cbct. material and methods selection and preparation of samples sixty mesiobuccal canals of extracted human mandibular first molars (length 20-21 mm) obtained from the tooth bank of the department of prosthetics and oral and facial surgery of the federal university of pernambuco, brazil were selected with the approval of the ethics in research committee of the center of health sciences of the university. the mesiobuccal roots had completely formed apices and curved root canals whose curvature ranged from 25° to 30° according to the canal access angle (caa) technique13. the access cavities were prepared, and to determine the working length (wl) a #10 senseusflexofile (dentsply/maillefer, ballaigues, switzerland) was inserted into the mesiobuccal canal until it was visible at the apical foramen. the wl was calculated to be 1 mm less than the length obtained with this initial file. preoperative images the roots were stored in alginate hydrogel (jeltrade; dentsply, petrópolis, rj, brazil) poured in plastic containers to ensure a very close approximation of the preoperative and postoperative images according to a previously described method14. after the alginate solidified, all teeth were scanned by cbct (i-cat®; imaging sciences international, hatfield, pa, usa) to determine the root canal shape before instrumentation. the exposure time was 26.9 s, operating at 120 kv and 7 ma. the images were sectioned in three points, located respectively at 9 mm (coronal level), 6 mm (middle level) and 3mm (apical level) from the apex. the images were stored in a computer for later comparison. root canal preparation the specimens were randomly divided into three groups with 20 root canals each. all instrumentation was performed according to each manufacturer’s instructions. random distribution of the groups considered the degree of canal curvature, allowing the average curvature, as well as the more severe cases, to be evenly allocated to each group: group 1: protaper universal™ rotary system. the canals were instrumented at a rotational speed of 300 rpm as follows: (a) the sx file was used up to one half of the wl, (b) the s1 file was used up to 4 mm short of the apex, (c) the s1 and s2 files were used to the full wl, and (d) the f1 and f2 files were used to the full wl. group 2: twisted file™ rotary system. the canals were instrumented at a rotational speed of 300 rpm as follows: (a) the #25.08 file was used up to the coronal third of the root canal, (b) the #25.06 file was used up to 4 mm short of the wl, and (c) the #25.04 and #25.06 files were used up to the full wl. group 3: m two™ rotary system. the canals were instrumented at a rotational speed of 300 rpm as follows: the #10.04, #15.05, #20.06, and #25.06 files were used up to the full wl. the specimens were fastened to a morse (neboluz, são paulo, sp, brazil) to keep them fixed during preparation. after use of each file, the root canals were irrigated with 3 ml of a freshly prepared 1% sodium hypochlorite solution (roval, recife, pe, brazil). glyde™ (dentsply maillefer) was used as a lubricant during instrumentation. a single operator experienced in rotary systems prepared all root canals. each instrument was changed after five canals. instruments were examined after every use to record and reject deformed or fractured instruments. an electric motor (driller endo-pro torque, são paulo, sp, brazil) was used. postoperative images after instrumentation, the specimens were scanned under the same conditions as the initial scans and the postoperative images were captured in the same way as mentioned before. evaluation of centering ability using the image tool software (university of texas health science center, san antonio, tx, usa) the changes in root canal anatomy using three nickel-titanium rotary system a cone beam computed tomography analysis308 braz j oral sci. 12(4):307-312 preoperative and postoperative images were compared (figure 1, a and b). gambill et al.15 (1996) defined centering ratio as the measurement of the ability of the instrument to stay centered in the canal. this ratio was calculated for each section using the following ratio (figure 2): d1: (x1 x’1)/(x2 x’2) d2: (y1 y’1)/(y2 y’2) where d1= the buccolingual measurement and d2= the mesiodistal measurement. x1= shortest distance from the buccal aspect of the root to the periphery of the uninstrumented canal. x’1= shortest distance from the buccal aspect of the root to the periphery of the prepared canal. x2= shortest distance from the lingual aspect of the root to the periphery of the uninstrumented canal. x’2= shortest distance from the lingual aspect of the root to the periphery of the prepared canal. y1= shortest distance from the mesial aspect of the root to the periphery of the uninstrumented canal. y’1= shortest distance from the mesial aspect of the root to the periphery of the prepared canal. y2= shortest distance from the distal aspect of the root to the periphery of the uninstrumented canal. y’2= shortest distance from the distal aspect of the root to the periphery of the prepared canal. according to this formula, a result of 1 indicates a perfect centering ability; the closer the result is to zero, the worse is the ability of the instrument to remain centered. fig. 1. cbct scan images showing the centering ability of the protaper universal™ before (a) and after (b) preparation at apical level. fig. 2. schematic presentation of the image used in the evaluation. statistical analysis of the data the categorical data were summarized by means of absolute frequency and relative percentage, and the numerical data by means of the usual descriptive statistics of location and dispersion. the results were statistically analyzed using one-way anova test. a level of significance of 0.05 was adopted, using the statistical package for the social sciences, version 13 (spss, chicago, il, usa). results table 1 and figure 3 present the main descriptive statistics of the buccolingual measurement (d1) and mesiodistal measurement (d2) according to the rotary system used and the instrumented root segment at distances of 9 mm (coronal), 6 mm (middle) and 3 mm (apical) from the apex. the means of d1 ranged from 0.03 to 1.00. in group 1, the mean deviation was 0.88±0.257, 1.00±0.000, and 1.00±0.000 at distances of 9 mm (coronal), 6mm (middle) and 3mm (apical) from the apex, respectively. in group 2, the mean deviation was 0.79±0.745, 0.65±0.669, and 0.25±0.425 at distances of 9 mm (coronal), 6 mm (middle) and 3 mm (apical) from the apex, respectively. in group 3, the mean deviation was 0.50±0.745, 0.33±0.472, and 0.03±0.104 at distances of 9 mm (coronal), 6mm (middle) changes in root canal anatomy using three nickel-titanium rotary system a cone beam computed tomography analysis 309 braz j oral sci. 12(4):307-312 instrument third ptu tf mtwo mean±sd mean±sd mean±sd p value coronal d1 0.88±0.257 0.79±0.745 0.50±0.745 0.171 coronal d2 1.00±0.000 0.41±0.299 0.58±0.910 0.001 middle d1 1.00±0.000 0.65±0.669 0.33±0.472 0.012 middle d2 1.00±0.000 0.30±0.428 0.85±1.857 0.003 apical d1 1.00±0.000 0.25±0.425 0.03±0.104 <0.001 apical d2 1.00±0.000 0.50±0.707 0.31 0.643 0.005 table 1.table 1.table 1.table 1.table 1. mean and standard deviation of the buccolingual and mesiodistal measurements according to the instrument used and the root segment instrumented. ptu: protaper universal; tf: twisted file; sd: standard deviation; d1: buccolingual measurement; d2: mesiodistal measurement fig. 3. mean of the d1 (buccolingual) and d2 (mesiodistal) measurements according to the used instrument and the instrumented root segment. and 3 mm (apical) from the apex, respectively. the means of d2 ranged from 0.30 to 1.00. in group 1, the mean deviation was 1.00±0.00, 1.00±0.00, and 1.00±0.00 at distances of 9 mm (coronal), 6 mm (middle), and 3 mm (apical) from the apex, respectively. in group 2, the mean deviation was 0.41±0.299, 0.30±0.428, and 0.50±0.707 at distances of 9 mm (coronal), 6 mm (middle) and 3 mm (apical) from the apex, respectively. in group 3, the mean deviation was 0.58±0.910, 0.85±1.857, and 0.31±0.643 at distances of 9 mm (coronal), 6 mm (middle) and 3 mm (apical) from the apex, respectively. there were statistically significant differences (p<0.05) among the three groups, except for coronal d1. discussion the introduction of niti alloy allowed the manufacture of instruments that were able to prepare curved root canals with safety, less deviations and in less working time, in comparison with stainless steel instruments3. new niti rotary systems are continuously being marketed, each having subtly different design features, which are claimed to improve flexibility, cutting efficiency, safety and ultimately canal shaping16,17. the present study evaluated the ability of three niti rotary systems to maintain the original root canal anatomy by using the cbct. several methodologies have been used to evaluate the final shape of root canal preparations such as the serial sectioning technique and optical microscopy. however, when using these methods, part of the specimen structure is lost, because there is need to cut the tooth before the postoperative evaluation. the use of simulated root canals in resin blocks, in spite of allowing for a high degree of reproducibility and standardization, does not reflect the clinical behavior of the instruments, because of the different hardness of resin and dentine. radiographic evaluation 18, however, is not destructive, but allows only a two-dimensional evaluation of the root canal. cbct has been adapted for dentistry and changes in root canal anatomy using three nickel-titanium rotary system a cone beam computed tomography analysis310 braz j oral sci. 12(4):307-312 compared with medical tomography that leads to increased precision and resolution, as well as reducing the image acquisition time and, as ain consequence, the exposure time to radiation . another advantage of this method is that there is no destruction of the sample19. as reported by flores et al.20 (2012), cbct and specialized software (i-cat cone beam) were successfully used in the present research for measurements before and after instrumentation of root canals and for the calculations of the centering ability of three niti rotary systems during cleaning and shaping of the root canals. the present study evaluated the effects of protaper universal™, mtwo™, and twistedfile™ on root canal anatomy. cbct analysis showed that the protaper universal™ showed the ability for producing centered preparations and the twisted file™ and mtwo™ rotary systems produced canal deviation. the use of this methodology was based on the study of sanfelice et al.14 (2010) which showed it to be reliable, without destructive sectioning of the specimens or loss of the root material during sectioning. previous studies showed that cbct used to evaluate root canals prepared with niti rotary instruments provided a nondestructive and reproducible method12,14,21,22. data obtained with this technique enable the identification of morphologic changes associated with different biomechanical preparations including canal deviation, dentin removal and final canal preparation. a major advantage of cbct is the possibility to obtain highly accurate evaluation of root canal shape by the superimposition and measurement of 3d renderings23. in the present research, in the group where the mtwo™ rotary system was used, a larger number of deviations was observed. hin et al.11 (2013) observed that instrumentation with mtwo could cause damage to root canal dentin. these results are in disagreement with previous studies24,25. schäfer et al.26 (2006) also demonstrated the efficiency of cleaning and shaping of this new instrument. yang et al.9 (2011) showed that the mtwo™ rotary system maintained the original curvature of the root canal as well as provided a good centering ability. the differences found in this research compared with previous studies9,24,25,26 may be due to the fact that in this study mesiobuccal roots and curved root canals were used. the current study showed that the twisted file™ rotary systems produced morphological changes, such as apical deviation. these results are in disagreement with a previous study27, which stated that the twisted file™ rotary system can be used in any clinical case, regardless of the anatomical aspect of the tooth. according to duran-sindreu et al.28 (2012), the twisted file™ instruments were designed to have improved properties in relation to root canal preparation as compared with other instruments. stern et al16 (2012) reported that the twisted file™ was able of producing centered preparations. other authors1,29,30 also demonstrated the ability of the twisted file™ rotary system to maintain the original root anatomy in curved canals. the results of the present research showed that the protaper universal ™ rotary system produced centered preparations maintaining the original root canal anatomy even in extremely curved canals, as demonstrated in previous studies31-34. these results may be related to the modified cross section design of the protaper universal™ rotary system. the manufacturer reported a decrease of the area in contact with the dentin wall, and that u-shaped grooves had been added at each of the instrument’s convex triangular sides to improve flexibility and reduce transportation21. decreasing tapers of the finishing files and increased flexibility of s1 and f1 may have had favorable effects on the performance of the protaper universal™ rotary system. the protaper universal™ instruments showed better performance than the conventional protaper™ files evaluated previously, probably because the file tip has been changed from the “modified guiding tip” to the “rounded safe tip”. these changes in the instruments may give rise to favorable clinical behavior35. on the other hand, hashem et al.36 (2012) observed that the protaper universal™ system recorded a significantly lesser centering ratio and higher canal transportation than the twisted file, profile gt series x, and revo-s systems. similar results were reported and were attributed to the sharp cutting edges and the multiple tapers along the cutting surface of the files1. the use of cbct revealed that the protaper universal™ had the capacity of producing centered preparations while twisted file™ and mtwo rotary systems produced canal deviation. acknowledgments this study was supported by grants from national council for scientific and technological development (cnpq) brazil. references 1. gergi r, rjeily ja, sader j, naaman a. comparison of canal transportation and centering ability of twisted files, pathfile-protaper system, and stainless steel hand k-files by using computed tomography. j endod. 2010; 36: 904-7. 2. el ayouti a, dima e, judenhofer ms, löst c, pichler bj. increased apical enlargement contributes to excessive dentin removal in curved root canals: a stepwise microcomputed tomography study. j endod. 2011; 37: 1580-4. 3. câmara ac, aguiar cm, figueiredo jap de. assessment of the deviation after biomechanical preparation of the coronal, middle, and apical thirds of root canals instrumented with three hero rotary systems. j endod. 2007; 33: 1460-3. 4. aguiar cm, mendes d de a, câmara ac, figueiredo jap de. assessment of canal walls after biomechanical preparation of root canals instrumented with protaper universal™ rotary system. j appl oral sci. 2009; 17: 590-5. 5. mendes d de a, aguiar cm, câmara ac. comparison of the centering ability of the protaper universal, profile and twisted file rotary systems. braz j oral sci. 2011; 10: 282-7. 6. ünal gç, maden m, savgat a, onur orhan e. comparative investigation of 2 rotary nickel-titanium instruments: protaper universal versus protaper. oral surg oral med oral pathol oral radiol endod. 2009; 107: 886-92. 7. kim hc, yum j, hur b, cheung gsp. cyclic fatigue and fracture characteristics of ground and twisted nickel-titanium rotary files. j endod. 2010; 36: 147-52. 8. larsen cm, watanabe i, glickman gn, he j. cyclic fatigue analysis of a new generation of nickel titanium rotary instruments. j endod. 2009; 35: 401-3. changes in root canal anatomy using three nickel-titanium rotary system a cone beam computed tomography analysis 311 braz j oral sci. 12(4):307-312 9. yang g, yuan g, yun x, zhou x, liu b, wu h. effects of mtwo versus protaper universal, on root canal geometry assessed by micro–computed tomography. j endod. 2011; 37: 1412–6. 10. foschi f, nucci c, montebugnoli l, marchionni s, breschi l, malagnino va, prati c. sem evaluation of canal wall dentine following use of mtwo and protaper niti rotary instruments. int endod j. 2004; 37: 832–9. 11. hin es, wu mk, wesswlink pr, shemesh h. effects of self-adjusting file, mtwo, and protaper. j endod. 2013; 39: 262-4. 12. bernardes ra, rocha ea, duarte mah, vivan rr, moraes ig de, bramante as, azevedo jr de. root canal area increase promoted by the endosequence and protaper systems: comparison by computed tomography. j endod. 2010; 36: 1179-82. 13. günday m, sazak h, garip y. a comparative study of three different root canal curvature measurement techniques and measuring the canal access angle in curved canals. j endod. 2005; 31: 796–8. 14. sanfelice cm, costa fb da, só mvr, vier-pelisser f, bier cas, grecca fs. effects of four instruments on coronal pre-enlargement by using cone beam computed tomography. j endod. 2010; 36: 858-61. 15. gambill jm, alder m, del rio ce. comparison of nickel-titanium and stainless steel hand-file instrumentation using computed tomography. j endod. 1996; 22: 369–75. 16. stern s, patel s, foschi f, sherriff m, mannocci f. changes in centring and shaping ability using three nickel-titanium instrumentation techniques analysed by micro-computed tomography (µct). int endod j. 2012; 45: 514-23. 17. venkateshbabu n, emmanuel s, santosh gk, kandaswamy d. comparison of the canal centring ability of k3, liberator and ez fill safesiders by using spiral computed tomography. aust endod j. 2012; 38: 55-9. 18. sydney gb, batista a, de mello ll. the radiographic platform: a new method to evaluate root canal preparation in vitro. j endod. 1991; 17: 570–2. 19. pasternak-júnior b, sousa-neto md, siva rg. canal transportation and centring ability of race rotary instruments. int endod j. 2009; 42: 499-506. 20. flores cb, machado p, montagner f, gomes bpf de a, dotto gn, schmitz m da s. a methodology to standardize the evaluation of root canal instrumentation using cone beam computed tomography. braz j oral sci. 2012; 11: 84-7. 21. özer sy. comparison of root canal transportation induced by three rotary systems with noncutting tips using computed tomography. oral surg oral med oral pathol oral radiol endod. 2011; 111: 244-50. 22. michetti j, maret d, mallet jp, diemer f. validation of cone beam computed tomography as a tool to explore root canal anatomy. j endod. 2010; 36: 1187-90. 23. pasqualini d, bianchi cc, paolino ds, mancini l, cemenasco a, cantatore, castelluci a, berutti e g. computed micro-tomographic evaluation of glide path with nickel-titanium rotary pathfile in maxillary first molars curved canals. j endod. 2012; 38: 389-93. 24. schäfer e, oitzinger m. cutting efficiency of five different types of rotary nickel-titanium instruments. j endod. 2008; 34: 198 –200. 25. machado me de l, sapia lab, cai s, martins ghr, nabeshima ck. comparison of two rotary systems in root canal preparation regarding disinfection. j endod. 2010; 36: 1238-40. 26. schäfer e, erler m, dammaschke t. comparative study on the shaping ability and cleaning efficiency of rotary mtwo instruments. part 2. cleaning effectiveness and shaping ability in severely curved root canals of extracted teeth. int endod j. 2006; 39: 203–12. 27. mounce re. making endo fun again: get twisted. dent econ. 2008; 98:23. 28. duran-sindreu f, garcía m, olivieri jg, mercadé m, morelló s, roig m. a comparison of apical transportation between flexmaster and twisted files rotary instruments. j endod. 2012; 38: 993-5. 29. marzouk am, ghoneim ag. computed tomographic evaluation of canal shape instrumented by different kinematic rotary nickel-titanium systems. j endod. 2013; 39: 906-9. 30. siqueira júnior jf, alves frf, versiani ma, rôças in, almeida bm, neves mas, sousa-neto md. correlative bacteriologic and microcomputed tomographic analysis of mandibular molar mesial canals prepared by self-adjusting file, reciproc, and twisted file systems. j endod. 2013; 39: 1044-50. 31. aguiar cm, sobrinho pb, teles f, câmara ac, figueredo jap. comparison of the centering ability of the protapertm and protaperuniversaltm rotatory systems for preparing curved root canals. aust endod j. 2013; 39: 25-30. 32. hartmann msm, barletta fb, fontanella vrc, vanni jr. canal transportation after root canal instrumentation: a comparative study with computed tomography. j endod. 2007; 33: 962–5. 33. huang x, ling j, gu l. quantitative evaluation of debris extruded apically by using protaper universal tulsa rotary system in endodontic retreatment. j endod. 2007; 33: 1102–10. 34. versiani ma, leoni gb, steier l, de-deus g, tassani s, pécora jd, sousa-neto md. micro–computed tomography study of oval-shaped canals prepared with the self-adjusting file, reciproc, waveone, and protaper universal systems. j endod. 2013; 33: 1060–6. 35. câmara as, martins r de c, viana acd, leonardo r de t, buono vtl, bahia mg de a. flexibility and torsional strength of protaper and protaper universal rotary instruments assessed by mechanical tests. j endod. 2009; 35: 113-6. 36. hashem aar, ghoneim ag, lutfy ra, foda my, omar gaf. geometric analysis of root canals prepared by four rotary niti shaping systems. j endod. 2012; 38: 996-1000. changes in root canal anatomy using three nickel-titanium rotary system a cone beam computed tomography analysis312 braz j oral sci. 12(4):307-312 429 too many requests error 429 too many requests too many requests guru meditation: xid: 35937707 varnish cache server 429 too many requests error 429 too many requests too many requests guru meditation: xid: 87468700 varnish cache server oral sciences n3 original article braz j oral sci. january | march 2015 volume 14, number 1 different methods of dental caries diagnosis in an epidemiological setting renato pereira da silva1, andréa videira assaf2, gláucia maria bovi ambrosano3, fábio luiz mialhe4, marcelo de castro meneghim5, antonio carlos pereira5 1 universidade federal de viçosa – ufv, department of nutrition and health, area of public health, viçosa, mg, brazil. 2 universidade federal fluminense – uff, dental school, department of specific formation, nova friburgo, rj, brazil 3 universidade estadual de campinas – unicamp, piracicaba dental school, department of community and preventive dentistry, area of biostatistics, piracicaba, sp, brazil. 4 universidade estadual de campinas – unicamp, piracicaba dental school, department of community and preventive dentistry, area of health education, piracicaba, sp, brazil. 5 universidade estadual de campinas – unicamp, piracicaba dental school, department of community and preventive dentistry, area of preventive dentistry and public health, piracicaba, sp, brazil. correspondence to: renato pereira da silva universidade federal de viçosa departamento de nutrição e saúde av. peter henry rolfs, s/n 36570-900, viçosa, mg, brazil phone: +55 31 3899 2545 fax: +55 31 38992108 e-mail: renatop.silva@ufv.br abstract aim: to evaluate the performance of dental caries detection when adjunct methods are associated and their applicability in epidemiological survey of dental caries, at d3 (cavitated carious lesions at dentin layer) and d1+d3 (non cavitated and cavitated carious lesions at enamel/dentin layer) diagnostic thresholds. methods: a total of 2189 posterior teeth from 165 12-year-old schoolchildren underwent visual examination without (cl1) and with artificial lighting (cl2), radiographic bitewing (bw), fiber-optic transillumination/foti (ft), diagnodenttm (dd) and associations of these methods. reproducibility was calculated by kappa statistics and validity was calculated by sensitivity, specificity and accuracy tests. anova (scott-knott test) was performed in order to compare the average values of dmf-s obtained by the diagnostic methods. results: the cl2ftddbw (d3) and cl2bw (d1+d3) exams presented the highest values for accuracy at epidemiological setting. the dmf-s index obtained for those exams was statistically different at d3 and d1+d3 thresholds. conclusions: the association of adjunct methods increased the validity of dental caries examination in an epidemiological setting. however, the potential of cl2bw (traditional caries detection methods) or visual exam performed under a more refined diagnostic criteria must be considered in dental caries epidemiological surveys. keywords: dental caries; diagnosis, oral; epidemiology. introduction with the purpose of improving the quality of dental caries diagnosis, the association of adjunct diagnostic methods with the conventional visual examination has been proposed, and it has been successful to some extent in studies conducted under epidemiological conditions and adopting more sensitive diagnostic thresholds of dental caries1. there has been underestimation of caries prevalence in surveys verified in groups of 12-year-old or in older children, when examined by the visual method2. in addition, the visual method of examination and who criteria have not been sufficient to properly identify the whole spectrum of the dental caries and received for publication: january 23, 2015 accepted: march 23, 2015 braz j oral sci. 14(1):78-83 1 7 11 7 11 7 11 7 11 7 17979797979 subsequent treatment needs in posterior teeth3. according to the systematic review of bader et al.4, the strength of scientific evidence for adjunct methods of caries detection is poor. however, some studies have presented satisfactory results with the use of auxiliary resources (often low-tech and low-cost actions that improve visual examination as previous tooth brushing and drying or artificial lighting, for example) and adjunct methods (often high-tech devices as fiber-optic transillumination/foti, diagnodent™, electronic caries monitor/ecm or digital radiography, for example) for the detection of caries5-6. apart from adding new diagnostic adjuncts with the aim of designing a more reliable nosological chart, more sensitive diagnostic thresholds recording the presence of initial carious lesions and/or activity have been proposed with some success in the literature1,3,7. nowadays, epidemiological studies have shown that initial caries lesions in posterior teeth are very common. the detection of the whole spectrum of dental caries is an important tool for oral health services planning and evaluation. the aim of this study was to evaluate the performance of dental caries detection when adjunct methods are associated and their feasibility in epidemiological survey of dental caries, at d3 (cavitated carious lesions at dentin layer) and d1+d3 (non cavitated and cavitated carious lesions at enamel/dentin layer) diagnostic thresholds. materials and methods the study was conducted in accordance with resolutions 196/96 of national health council of brazilian ministry of health, and resolution cfo 179/93 of the dental professional code of ethics, started after its approval by research ethics committee protocol no. 082/2006. dental examinations the epidemiological data were gathered by clinical visual examination without (cl1) or with artificial lighting (cl2), radiographic bitewing examination (bw), foti (ft), laser fluorescence examination (diagnodenttm/ dd) and their respective associations: cl2ft, cl2dd, cl2bw, cl2ftdd, cl2ftbw, cl2ddbw and cl2ftddbw. the codes and criteria1,7-10 are shown in table 1. * adapted from assaf et al.1, fyffe et al.7 and who8 ** adapted from hintze et al.9 ***criteria and codes from zanin et al.10 table 1.table 1.table 1.table 1.table 1. diagnostic criteria and codes for dental examinations different methods of dental caries diagnosis in an epidemiological setting braz j oral sci. 14(1):78-83 8080808080 the cl1 and cl2 exams were carried out by using dental mirror, who probe, previous tooth brushing and air-drying for 5 s per each dental surface. the d3 diagnostic criterion, which considers cavitated carious lesions in dentine, and the d1+d3 diagnostic criterion, which considers cavitated and non-cavitated carious lesions in enamel and/or dentine were adopted. bitewings were taken of the schoolchildren’s posterior teeth in a separate session, by an examiner experienced in this type of dental examination. the radiographical film agfa dentus m2 comfort m2-58, e/f-speed, and a spectro 70x x-ray device (dabi-atlante, ribeirão preto, sp, brazil) of 70 kvp, 8 ma and 0.4 s exposure time were used. radiograph processing was standardized and performed in an automatic processor. the radiographs were analyzed in a viewing box (vh, araraquara, sp, brazil) without magnification by the benchmark examiner and the participant examiner. for the examination of proximal dental surfaces by foti, a fiberlite® pl-800 series device (dolan-jenner elaborate, lawrence, ma, usa), with a 0.5 mm diameter tip (positioned below the proximal point of contact) was used. the laser fluorescence examination by a diagnodent™ 2095 device (kavo, biberach, germany) was performed according to the manufacturer’s recommendations for the occlusal dental surfaces, but considering the classification scale and interpretation of the reading values proposed by zanin et al.10, which was more adequate for the study proposals. sample one hundred and sixty five 12-year-old schoolchildren from public schools in piracicaba, sp, brazil, with low to high prevalence of dental caries, were randomly selected. the sample size was determined considering an agreement proportion of 90%, disagreement proportion of 5%, test power of 92% and an alpha of 5% under a bilateral test. schoolchildren who presented fixed orthodontic devices, severe fluorosis and enamel hypoplasia or a serious systemic disease were replaced by other children without these conditions. only the mesial, distal and occlusal dental surfaces of permanent posterior teeth were considered in this study, totaling 6565 surfaces of 2189 teeth. two dental surfaces were excluded because they could not be examined by all the diagnostic methods. calibration of examiner the calibration between the benchmark examiner and the participant examiner consisted of 9 sessions lasting 4 h, contemplating lectures, clinical training and calibration exercises for cl1, cl2, bw, foti and diagnodent™. a time interval of 10 days, needed to measure the intra-examiner agreement (only for the participant examiner), was adopted. thirteen schoolchildren, with low to high caries prevalence, exclusively examined for this purpose, were involved in the final calibration exercise. the cohen’s kappa statistics was used for both intra and inter-examiner agreement. statistical analysis sensitivity (sn), specificity (sp) and accuracy (a) tests were used to evaluate the validity of the dental caries examinations. the standard for the validation of exams was the cl2bw exam performed by the benchmark examiner. each diagnostic method was compared with the cl2bw (performed by the benchmark examiner) by the mcnemar test (á=0.05). anova was performed in order to compare the average values of dmf-s obtained by the diagnostic methods. the scott-knott test, which is used when there are too many treatment modalities, and there is interest to separate their results without ambiguity, was used for this purpose. results the reproducibility was calculated by cohen’s kappa statistics. at the d3 diagnostic criterion, the kappa values for the inter-examiner agreement were 0.87 for cl1 and cl2, 0.94 for bw, 0.92 for foti, and 0.34 for dd exam, while the intra-examiner agreement was 0.99 for cl1 and cl2, 1.00 for bw and foti, and 0.42 for dd exam. at the d1+d3 diagnostic criterion, the kappa values for inter-examiner agreement were 0.86 for cl1 and cl2, 0.88 for bw, 0.89 for foti and 0.48 for dd exam, while for the intra-examiner agreement the values were 0.98 for cl1, 0.97 for cl2, 1.00 for bw, 0.95 for foti and 0.42 for dd exam. the validity of the dental caries examinations at the d3 and d1+d3 diagnostic thresholds are shown in the table 2. at both diagnostic thresholds the cl2ftddbw exam presented the best values of sensitivity (sn). however, the cl2bw exam produced a satisfactory clinical performance with good values of sensitivity (sn) and accuracy (a) (table 2). the mean dmf-s index values as well as their differences among the dental caries examinations, measured by anova (scott-knott test), are presented in table 3. at both diagnostic thresholds, the association of bw with the cl2 exam (cl2bw) was responsible for a considerable improvement in determining the dmf-s index. however, the simultaneous association of the bw and dd exams with the cl2 exam was statistically similar to the performance of cl2ftddbw and differed statistically from the cl2bw performance for all diagnostic thresholds (table 3). discussion diagnosis of dental caries has been considered a complex process, under both clinical and epidemiological conditions, due to the current pattern of disease, characterized by a smaller number and size of lesions and a concentration of them mainly in the posterior teeth2,11. a great number of studies deal with the development and the use of new technologies to improve the diagnosis of dental caries, especially at the early stages5,12-19. generally, such studies are performed under controlled conditions, in vitro (laboratory settings) or in vivo (clinical settings) methodology. none was performed in epidemiological settings. the first limitation of the present study was its “gold standard” to validate the diagnostic methods. despite the different methods of dental caries diagnosis in an epidemiological setting braz j oral sci. 14(1):78-83 1 7 11 7 11 7 11 7 11 7 18181818181 occlusal + approximal surfaces d3 criterion d1+d3 criterion examinations s n s p a s n s p a cl1 4.56 99.88 89.69* 56.61 98.61 91.21* cl2 4.99 99.88 89.73* 57.99 98.43 91.30* bw 77.08 99.26 96.85* 59.74 99.18 92.16* cl2ft 5.13 99.86 89.74* 59.46 97.73 90.98* cl2dd 33.48 97.99 91.09* 68.19 95.97 91.08* cl2bw** 77.49 99.23 96.91* 86.78 97.69 95.76 cl2ftdd 33.62 97.97 91.08* 69.66 95.27 90.76* cl2ftbw 77.49 99.22 96.90* 87.12 96.99 95.24 cl2ddbw 82.62 97.37 95.79 90.15 95.25 94.35* cl2ftddbw 82.62 97.36 97.78 90.49 94.55 93.83* table 2.table 2.table 2.table 2.table 2. validity of the dental caries examinations * significant difference between diagnostic method and validation method (pd”0.05). ** performed by participant examiner. all examinations were tested against the cl2bw exam performed by the benchmark examiner. examinations d3 criterion d1+d3 criterion dmf-s s d dmf-s s d cl1 1.32a 2.45 6.58a 4.97 cl2 1.37a 2.50 6.92a 5.14 bw 4.55c 2.17 6.88a 3.74 cl2ft 1.39a 2.51 7.67a 8.02 cl2dd 3.29b 2.96 8.38b 4.98 cl2bw** 5.32d 2.58 9.06b 4.95 cl2ftdd 3.32b 2.99 8.68b 5.11 cl2ftbw 5.59d 2.56 9.00b 4.75 cl2ddbw 5.97e 2.58 9.72c 4.84 cl2ftddbw 6,23e 2.61 10.08c 4.78 table 3.table 3.table 3.table 3.table 3. dmf-s index values obtained by the examinations at the d3 and d1+d3 diagnostic thresholds. mean values followed by distinct letters on column-wise are statistically different (p<0.05). ** perfomed by participant examiner. controversy around the association between visual (cl2) and bitewing radiography (bw) exams performed by an experienced examiner as “gold standard”, such association was adopted. ideally, the histological validation and the opening lesions are the most indicated methods to such purposes. however, those validation methods could not be adopted because the design of the present study simulates a real epidemiological survey of dental caries. satisfactory results from the cl2 or cl2bw exams have been related in literature 13,20, especially when immediate operative intervention is not implemented12,15. the exclusive use of bitewing radiographs as baseline in permanent dentition may be considered for populations with an overall low caries prevalence. that recommendation, along with the correctness of technique, the use of faster films, intraoral bitewing holders and leaded protective aprons with a thyroid collar, minimize the hazards of ionizing radiation21. the purposes of an epidemiological survey of dental caries include registering the trend of the disease and the general treatment needs of a particular population8 aiming to organize the demand and the oral health services. for this reason, dental examinations at epidemiological survey are not suitable for caries diagnosis and treatment. to detect carious lesions is different from to diagnosing the dental caries disease. for caries diagnosis and subsequent decision treatment, a set of other factors must be considered. such factors include the initial caries signs, the rest of the dentition, the patient’s case history and population factors, fluoride availability and sugar intake. diagnostic decision making is a balancing act5. although there is low strength to the evidence as regards the performance of diagnostic methods4, there are positive results described in the literature2,6-7,22 about the association of auxiliary resources and adjunct diagnostic methods for identifying initial carious lesions. this trend was evident and was in agreement with the results of this study. although the poor reproducibility of the dd exam in epidemiological setting, it was maintained exclusively as an adjunct technique22. such reproducibility may be explained by the tooth cleaning method used in the study. the diagnodent® manufacturer’s advice is to perform professional prophylaxis previous to the examination. however, it is time-consuming and not required for visual examination. a previous tooth brushing (and drying procedure) is sufficient for that purpose. nevertheless, toothpaste and biofilm remaining over occlusal dental surfaces may have influenced the dd readings23. in the present study, the association of the clinical method with different adjunct technologies improved the sensitivity to detect caries, corroborating the results of pereira et al.5. it is especially important to use methods or their association with others, which generate high sensitivity results in detecting caries in populations that present moderate to high prevalence of non-cavitated or hidden dentin lesions. therefore, the sensitivity values of cl2ddbw and cl2ftddbw at both diagnostic thresholds could be better indicated for these populations. however, since the cl2ddbw and cl2ftddbw dmf-s means did not differ statistically, the use of cl2ddbw could be suggested first, assuming that such examination is simpler to perform than the cl2ftddbw. different methods of dental caries diagnosis in an epidemiological setting braz j oral sci. 14(1):78-83 8282828282 on the other hand, cl1, cl2 and cl2ft at the d3 diagnostic threshold, and cl1, cl2 and bw at the d1+d3 diagnostic threshold, would be better indicated in populations with low caries prevalence due to the high specificity of their results. since the dmf-s means did not differ statistically, cl1 and cl2 at both diagnostic thresholds could be recommended in surveys, although the most accurate methods were cl2ftddbw at the d3 diagnostic threshold and cl2bw at the d1+d3 diagnostic threshold. although the dmf-s obtained by cl2bw is statistically different from the values from cl2ddbw and cl2ftddbw (table 3), its results (tables 2 and 3) cannot be neglected due its availability and familiarity among dentists. the purposes of a dental caries surveys support such differences. furthermore, cl1 and cl2 accuracy results at the d1+d3 diagnostic threshold, indicated that a well-trained and competent professional is able to detect caries lesions. therefore, training strategies should be focused not only on private but also on public services, because professional experience may influence the diagnosis of caries 5 and treatment of the disease, which is often underestimated by professionals24. low sensitivity values for cl1 and cl2 methods, at the d3 threshold, could be explained by the fact that dentin lesions under intact enamel are rarely detected without the use of an adjunct method, mainly the bitewing radiographic method. in addition, results are sometimes compromised by the examiners’ subjectivity due to their previous clinical experience, verified even among calibrated examiners, such as those who conducted this survey. an improvement in the sensitivity of cl1, cl2 and bw, with the association of diagnodent™ was found in this survey. that improvement in sensitivity is followed by a decrease in specificity. this is critical in populations whose caries prevalence is low, increasing the probability of false positive diagnoses5,12,21. in spite of its performance in non-cavitated caries lesions on occlusal surfaces 13,19, use of diagnodent™ in epidemiological surveys might be not recommended mainly due to its high cost. it is important to mention that improvement in detecting caries with the diagnodent™ could be also explained by its trend to produce false-positive diagnoses4. according to pereira et al.5, to have data available from multiple methods may influence on the number of surfaces indicated for operative treatment. so, the adoption of diagnostic adjuncts in epidemiological surveys must be only used to supplement the decision to plan preventive strategies for initial dental caries16. nowadays, professionals should be more prepared to diagnose properly a higher number of intact teeth or teeth with initial caries, than to detect cavitated lesions in dentin, due to the current less progressive pattern of disease. for this reason, it is recommended to “wait and watch” and not to immediately “drill and fill” teeth25. according to novaes et al.17, visual inspection alone seems to be sufficient to detect dental caries satisfactorily. the results from cl1 and cl2 exams corroborate that statement in some degree. the training for dental caries diagnosis under a more refined threshold is also needed nowadays12. however, studying alternatives to the traditional diagnostic methods and criteria does not mean to substitute them in all circumstances. the availability to acquire such diagnostic adjuncts and subsequent opportunity to apply them must always be considered. the who criteria8 still have their important role in dentistry. it is also relevant to know the advantages and disadvantages of diagnostic technologies and their associations, because there is no modality of examination that can be successfully used alone for diagnosing caries and able to produce representative data of the disease in a population5,14. in conclusion, the association of adjunct diagnostic methods with the clinical method generated higher validity results than the clinical method alone, at both d3 and d1+d3 diagnostic thresholds, under epidemiological examination conditions. diagnosing is a balancing act. therefore, considering the familiarity of the professionals with clinical and radiological examinations, the present results show the potential of the cl2bw in epidemiological surveys of dental caries. the visual exam (cl1 and cl2), under more refined diagnostic criteria, also showed its potential at epidemiological surveys. however, studies with similar methodologies are needed to endorse the association of adjunct diagnostic methods in epidemiological settings. acknowledgement the authors would like to thank the volunteers and those responsible for the schoolchildren of the public schools in piracicaba, sp, brazil. the first author received a scholarship from proex/capes-unicamp during the doctorate course in dentistry. the authors acknowledge the financial support of the fapesp (grants #06/58881-9). references 1. assaf av, meneghim mc, zanin l, mialhe fl, pereira ac, ambrosano gmb. assessment of different methods for diagnosing dental caries in epidemiological surveys. community 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traditional and novel methods for occlusal caries detection: performance on primary teeth. laser med sci. 2013; 28: 287-95. doi: 10.1007/s10103012-1154-8. 20. wenzel a. bitewing and digital bitewing radiography for detection of caries lesions. j dent res. 2004; 83 spec no c: c72-5. 21. espelid i, mejàre i, weerheijm k. eapd guidelines for use of radiographs in children. eur j paediatr dent. 2003; 4: 40-8. 22. bader jd, shugars da. a systematic review of the performance of a laser fluorescence device for detecting caries. j am dent assoc. 2004; 135: 1413-26. 23. lussi a, reich e. the influence of toothpastes and prophylaxis pastes on fluorescence measurements for caries detection in vitro. eur j oral sci. 2005; 113: 141-4. 24. traebert j, marcenes w, kreutz jv, oliveira r, piazza ch, peres ma. brazilian dentist’s restorative treatment decisions. oral health prev dent. 2005; 3: 53-60. 25. bader jd, shugars da. the evidence supporting alternative management strategies for early occlusal caries and suspected occlusal dentinal caries. j evid based dent pract. 2006; 6: 91-100. different methods of dental caries diagnosis in an epidemiological setting braz j oral sci. 14(1):78-83 oral sciences n3 original article braz j oral sci. january | march 2013 volume 12, number 1 comparison of methods to evaluate implant-abutment interface karina oliveira de faria1, clébio domingues da silveira-júnior1, joão paulo da silva-neto2, maria da glória chiarello de mattos3, marlete ribeiro da silva2, flávio domingues das neves1 1department of occlusion, fixed prosthesis and dental materials, federal university of uberlandia, school of dentistry, uberlândia, mg, brazil 2department of prosthodontics and periodontology, piracicaba dental school, university of campinas, piracicaba, sp, brazil 3department of prosthodontics and dental materials, university of são paulo, school of dentistry of ribeirão preto, ribeirão preto, sp, brazil correspondence to: flávio domingues das neves department of occlusion, fixed prostheses, and dental materials federal university of uberlândia, school of dentistry avenida pará, bloco 2b, sala 01, umuarama, cep: 38400-970, uberlândia, mg, brasil e-mail: neves@triang.com.br received for publication: november 27, 2012 accepted: march 15, 2013 abstract aim: to compare two main methods of two-dimensional measurement of fit at the implant prosthodontic interface, testing the hypothesis that optical microscopy (om) can reliably and efficiently scanning electronic microscopy (sem). methods: four frameworks with four titanium abutments joined with titanium bars were used. the implant-abutment interfaces were examined by three different methods, forming 3 groups: analysis by om (40x), and analysis by sem at 300x and 500x. readings were taken at the mesial and distal proximal surfaces on the horizontal and vertical axes of each implant (n=32). one-way anova with a significance level of 5% was used for statistical analysis. results: neither the horizontal fit nor vertical fit values of the 3 groups presented statistically significant differences (p=0.410 and p=0.543). conclusions: om was found to be an accurate two-dimensional method for abutment-framework or implant-abutment interface measurements, with lower costs than sem. sem micrographs at 500x presented technical difficulties for the readings that might produce different results. keywords: dental implant, scanning electron microscopy, methodology. introduction framework passive fit is one of the most important factors in the longevity of implant treatment1-2. the laboratory and clinical steps for denture fabrication may contribute to implant-prosthesis misfit3-4. the load on the non-passive dental prosthetic system could result in mechanical complications, such as loosening or fracture of screws, fractures of components and of the implant itself5-6, and biological complications, such as mucositis, periimplantitis7, and loss of osseointegration8. according to the literature, a perfect adaptation does not exist5, but values for vertical fit less than 10 µm are considered acceptable3,9. despite several implantology studies investigating the problems arising from lack of passive fit, reliable methods for the clinical and laboratory evaluation of these interfaces have not yet been defined1,3,10. the main two-dimensional evaluation methods use optical microscopy (om)5,11-13 and scanning electron microscopy (sem)3-4,9-10,14-16 to measure fit at the implant-prosthesis or implantabutment interface. om uses a series of glass lenses to bend light waves and create a limited magnification and resolution image (spatial resolution of 50 to 100 nm), while sem uses electrons and creates higher magnification and resolution images. magnification refers to the degree to which a part has been enlarged. braz j oral sci. 12(1):37-40 3838383838 higher magnification does not necessarily mean better image definition, which is a factor of resolution. resolution is the ability to distinguish between two discrete objects in an image. people in science and industry have used optical measuring and inspection techniques to get enhanced views of their products. these techniques enable users to observe, measure and analyze parts for quality control, quality assurance and other purposes. sem is routinely used to generate high-resolution images of shapes of objects and to show spatial variations in chemical compositions. the relative accuracy of abutment-framework or implantabutment interface measurements generated by these two microscope types, each associated with difficulties and limitations, has not yet been determined. the lack of such comparative studies has resulted in confusion among clinicians and researchers. this paper thus discusses these two main methods of two-dimensional measurement of fit at the implant-framework interface. the study is based on the hypothesis that sem can be efficiently and reliably replaced by om, simplifying and reducing the costs of studies that evaluate implantabutment/framework interface. material and methods an aluminum master cast was made in which four implants (3.75 mm diameter x 10 mm height; master screw, conexão sistemas de próteses, são paulo, sp, brazil) were placed. these implants were placed parallel to each other, but at variable distances, as occurs in the majority of clinical situations. a working cast with implant analogues (013020, conexão sistemas de próteses,) was fabricated from a transfer impression of the master cast. the implants were numbered from 1 to 4, from right to left. four metallic frameworks were fabricated under this cast, using laser titanium abutments connected by titanium bars (055024 laser abutment, 400204 laser bars; conexão sistemas de próteses), as shown in figure 1. fig. 1. metallic frameworks the interfaces between the abutments and the implants were examined in three different ways, making up three study groups: om group was analyzed with an om at 40x magnification; the sem300 group was analyzed by sem at 300x magnification; and the sem500 group was analyzed by sem at 500x magnification. all the frameworks presented real misfits, just as in clinical practice. the values obtained for the fits based on the obtained measurements were statistically compared, showing similarity or not, and thus we determined that it was not required to create a control group with previously determined measurements for fits. for the interface analyses, the frameworks were screwed with a tightening torque of 20 ncm over the implants of the master cast, in the following order: implant 2, 4, 1, 3. readings were taken at the mesial and distal proximal surfaces (n=32) on the horizontal and vertical axes of each implant. all readings were repeated three times by the same examiner to obtain the mean value. optical microscopy analysis: om group the frameworks (a, b, c and d) were screwed onto the master cast and evaluated with an optical comparator microscope (mitutoyo tm-500, tokyo, japan). the mesial and distal aspects of the abutment and implant interfaces were measured. this is a monocular microscope with two digital micrometers, 0.001 mm resolution, with an objective lens of 2x magnification and an ocular lens of 20x magnification, resulting in a 40x magnification. the microscope has a table on which the samples are positioned and moves on the x and y axes, allowing measurement of horizontal and vertical misfits. scanning electron microscopy analysis: sem300 and sem500 groups for sem analysis (leo vp-435, carl zeiss, oberkochen, germany), the samples were processed in an ultrasonic cleaner with acetone, dried and stored in a container with silica. the same procedure was done with the master cast. this microscope operates in varied pressure conditions (vp), and in the conventional way, high pressure was controlled by a computer using the windows operating system. loquif software (leo user interface, carl zeiss, oberkochen, germany) allows the entire process of sample examination to be done as if the user were operating a common desktop computer, with “mouse” and/or “keyboard” controls. movement of the samples is made with a “joystick”. the resulting images can be stored in a computer’s hard drive or in diskettes (standard or zip drive), and printed on a standard or thermal printer. magnifications of 300x and 500x were used for groups sem300 and sem500, respectively. the images generated during examination were stored in the computer’s hard drive and then transferred to zip drives. the measurements were taken on the printed images, referred to the micrometer scale (ìm) printed at the base of each image. the horizontal fit measurement was made by drawing a tangent to the implant profile and one to the abutment profile, which passed through the highest number of points. a horizontal line referring to the implant radius was also drawn. misfits were quantified by the distance between the two profile tangent intersections with this horizontal line (figure comparison of methods to evaluate implant-abutment interface braz j oral sci. 12(1):37-40 3939393939 2). the vertical fit measurement was made by drawing a line on the implant platform and another that passes through the abutment base. misfits were defined as the distance between these two lines, represented by a vertical line drawn perpendicular to the two horizontal lines (figure 3). fig. 2. horizontal fit measurement (300x). horizontal fit vertical fit group mean s.d. 7.03 7.24 o m 34.02 23.15 8.53 6.22 sem300 41.82 33.97 8.81 7.26 sem500 44.04 35.48 7.03 7.24 table 1: means and standard deviation (s.d.) of the horizontal and vertical fit values (µm). fig. 3. vertical fit measurement (300x). statistical analysis one-way anova test was used to compare data among the three groups, considering interface situations and vertical and horizontal interface values. all data analyses were performed using spss 12.0 for windows (spss inc., chicago, il, usa) and p values of less than 0.05 were considered significant. results the horizontal and vertical fit values for the 3 groups are presented in table 1. no statistically significant difference was found among the groups for either horizontal (p=0.410) or vertical (p=0.543) fit values. discussion the hypothesis was confirmed as om was proven to be an accurate two-dimensional method for interface measurements in implant systems compared with sem. the magnitude of the implant-abutment gap has received significant attention,1-4,8-10,12-14,16 and different methodologies have been used for such investigation3-4,914,16. however there is still no consensus on the most appropriate methodology1 and comparisons between methods were not performed. the present results showed that no statistically significant differences were found among the three methods used to evaluate horizontal and vertical misfits. it was noticed, however, that the interface values for both vertical and horizontal misfits were consistently larger at the highest magnification. these results might be due the technical difficulty of examining the sem micrographs. it was more difficult to define the implant and abutment profiles at higher magnifications. these profiles are necessary to guarantee correct fit measurements. for example, figures 2 and 3 show sem micrographs at 300x magnification. at this scale, it is easier to draw the implant and abutment profiles than with a sem micrograph at 500x magnification. figure 4 illustrates several profile possibilities, which can influence the outcome of the analysis. thus, the same researcher may find different results for the same sem micrograph. larger misfits are also more difficult to evaluate with the sem, as the abutment and implant profile visualization is hampered by extension of the samples beyond the visual field of the sem micrographs. the lower (40x) magnification used with om in this study facilitates the visualization of the abutment and implant profiles and thus reduces the possibility of errors. this microscope shows the x and y coordinate axes in the eye lens, which facilitates measurement. although there is still no consensus on the acceptable fit values between implant-abutment interface3,9-10,16, the horizontal and vertical fit values found in this study showed similarity with previous studies for all analyzed methods3-4,913,16. the difference between the means of the horizontal misfit values evaluated by om (34.0 ± 23.15 µm) and by sem at 500x magnification (44.0 ± 35.4 µm) was approximately 10µm. for vertical misfit values the difference between means was even smaller, about 1.8 µm. these differences are comparison of methods to evaluate implant-abutment interface braz j oral sci. 12(1):37-40 4040404040 insignificant considering these magnifications and possible errors due the difficulty of measurement in the sem500. within the limitations of this study, it is possible to conclude that om is an accurate two-dimensional method for abutment-framework or implant-abutment interface measurements, with lower costs compared with sem. it was also observed that sem micrographs at 500x magnification presented technical difficulties to make the readings that might produce different results, depending on the reference point adopted by each researcher. acknowledgments the authors would like to thank elliot watanabe kitajima (nap/mepa – esalq – usp) for his assistance and permission to use sem. references 1. abduo j, bennani v, waddell n, lyons k, swain m. assessing the fit of implant fixed prostheses: a critical review. int j oral maxillofac implants. 2010; 25: 506-15. 2. goodacre cj, bernal g, rungcharassaeng k, kan jy. clinical complications with implants and implant prostheses. j prosthet dent. 2003; 90: 121-32. 3. neves fd, elias ga, dantas lc, silva-neto jp, mota as, fernandesneto aj. comparison of implant-abutment interface misfits after casting and soldering procedures. j oral implantol. 2012; jan 15. [epub ahead of print]. 4. da silveira-junior cd, neves fd, fernandes-neto aj, prado cj, simamoto-júnior pc. influence of different tightening forces before laser welding to the implant/framework fit. j prosthodont. 2009; 18: 337-41. 5. hecker dm, eckert se. cyclic loading of implant-supported prostheses: changes in component fit over time. j prosthet dent. 2003; 89: 346-51. 6. mendonça g, mendonça db, fernandes-neto aj, neves fd. management of fractured dental implants: a case report. implant dent. 2009; 18: 10-6. fig. 4. implant and abutment profile possibilities that can influence analysis outcome (500x) 7. hultin m, komiyama a, klinge b. supportive therapy and the longevity of dental implants: a systematic review of the literature. clin oral implants res. 2007; 18(suppl 3): 50-62. 8. jemt t, book k. prosthesis misfit and marginal bone loss in edentulous implant patients. int j oral maxillofac implants. 1996; 11: 620-5. 9. barbosa ga, bernardes sr, das neves fd, fernandes neto aj, de mattos mda g, ribeiro rf. relation between implant/abutment vertical misfit and torque loss of abutment screws. braz dent j. 2008; 19: 358-63. 10. kano sc, binon pp, curtis da. a classification system to measure the implant-abutment microgap. int j oral maxillofac implants. 2007; 22: 879-85. 11. costa hm, rodrigues rc, mattos mda g, ribeiro rf. evaluation of the adaptation interface of one-piece implant-supported superstructures obtained in ni-cr-ti and pd-ag alloys. braz dent j. 2003; 14: 197-202. 12. koke u, wolf a, lenz p, gilde h. in vitro investigation of marginal accuracy of implant-supported screw-retained partial dentures. j oral rehabil. 2004; 31: 477-82. 13. sartori ia, ribeiro rf, francischone ce, de mattos mda g. in vitro comparative analysis of the fit of gold alloy or commercially pure titanium implant-supported prostheses before and after electroerosion. j prosthet dent. 2004; 92: 132-8. 14. eisenmann e, mokabberi a, walter mh, freesmeyer wb. improving the fit of implant-supported superstructures using the spark erosion technique. int j oral maxillofac implants. 2004; 19: 810-8. 15. o’mahony a, macneill sr, cobb cm. design features that may influence bacterial plaque retention: a retrospective analysis of failed implants. quintessence int. 2000; 31: 249-56. 16. coelho al, suzuki m, dibart s, da silva n, coelho pg. cross-sectional analysis of the implant-abutment interface. j oral rehabil. 2007; 34: 508-16. comparison of methods to evaluate implant-abutment interface braz j oral sci. 12(1):37-40 oral sciences n3 original article braz j oral sci. july | september 2014 volume 13, number 3 bonding durability of dental sealants to deciduous and permanent teeth sandra kiss moura1, letícia vargas freire martins lemos2, suellen myszkovisk1, maria gisette arias provenzano1, ivan balducci3, silvio issao myaki2 1universidade norte do paraná – unopar, school of dentistry, department of restorative dentistry, londrina, pr, brazil 2universidade estadual paulista – unesp, school of dentistry, department of paediatric dentistry, são josé dos campos, sp, brazil 3universidade estadual paulista unesp, school of dentistry, department of mathematics and statistics, são josé dos campos, sp, brazil correspondence to: sandra kiss moura universidade norte do paraná (unopar) curso de odontologia rua marselha, 183 jardim piza cep 86041-140 londrina, paraná, brasil phone: +55 43 3371 7820 fax: +55 43 3341 8122 e-mail: kissmoura@gmail.com received for publication: may 18, 2014 accepted: august 25, 2014 abstract aim: to evaluate the bonding durability of materials used as sealants on different dentitions. methods: deciduous (d) and permanent (p) molars were divided into four groups (n=5) and sealed with fluroshield (f) and optibond fl (fl). blocks of composite resin were built, stored in distilled water (24 h/37 °c), sectioned into 0.8 mm2 and tested at tensile (0.5 mm/min) after 24 h and 6 months. the fractures were observed and classified into adhesive, cohesive or mixed types. the data were analyzed using repeated measures anova and tukey test (α=5%), with tooth as the experimental unit. results: there were differences for dentition (p=0.0097), dental sealant (p=0.0019) and time (p=0.0001). at 24h the highest bond strength was observed for optibond fl on deciduous and permanent teeth, similar to fluroshield at both dentitions. after 6 months the bond strength of optibond fl did not decrease in the permanent teeth, but decreased similar to fluroshield in deciduous teeth, the bond strength of fluroshield decreased similarly in both dentitions. conclusions: the bond strength of dental sealants varied with the experimental conditions. keywords: dental enamel; longevity; pit and fissure sealants. introduction the enamel of deciduous and permanent teeth differs in terms of chemical composition, morphology and physiology1-4. deciduous teeth have thinner enamel2 and thicker aprismatic layer2-3. in addition, they are more susceptible to dental caries and erosion, and the bonding of dental materials may vary from that of permanent teeth5-6. it is also known that 81.5% of dental caries found in children 6-36 months of age occur in the posterior teeth7. a recent study8 has shown that pain and dental caries in the primary dentition were the main reasons to visit dentists and the relationship between dental caries in deciduous teeth is a predictor for the disease in permanent dentition. morphology of the occlusal surface favors the accumulation of biofilm and increases the risk for development of dental caries, as shown in a study with children between 2 and 3 years old, in which 74.5% of them had dental caries on the occlusal surface9. dental sealants act as a mechanical barrier to the accumulation of biofilm, minimizing the risk for development of dental caries10. however, application of some types of sealants may be complicated in children, due to the risk of saliva contamination during the procedure. sealants are traditionally hydrophobic and hydrophilic materials investigated as an alternative to overcome the challenges braz j oral sci. 13(3):198-202 199199199199199 in sealant application11-14. difference in formulations of dental materials may have an impact on the bonding ability of dental sealants to deciduous and permanent teeth. this would be relevant to understand the importance of testing different materials for pit and fissure sealants15-16. a previous study has shown superior adhesion of optibond fl compared with a traditional hydrophobic sealant used in pediatric dentistry, 24 h after being applied to the occlusal surface of molars17. other studies have confirmed the feasibility of using certain sealant adhesive systems on occlusal surfaces in pediatric dentistry12-13. however, there are few studies that assess the durability of this procedure, especially comparing the deciduous and permanent dentitions. the efficacy of sealants is related to their retention to enamel18. since deciduous and permanent teeth have different properties, it is important to test different sealant materials in both dentitions. therefore, the aim of this study was to evaluate the microtensile bond strength of sealant materials to the enamel of primary and permanent teeth, after 24 h and 6 months. the tested hypothesis is that the microtensile bond strength of different dental sealants to deciduous and permanent tooth does not vary along the time. material and methods ten deciduous molars obtained no longer than 6 months after exfoliation13 were disinfected in 0.5% chloramine solution at 4 °c and cleaned with pumice and water slurry. the same procedure was used with 10 extracted, caries-free third permanent molars. all teeth were used for bond strength measurement. the research protocol was approved by the local ethics committee (protocol pp0096/11). material (batch number) optibond fl (primer 3124126; adhesive 3101101) fluroshield (047065a) filtek z250 (6br) adper single bond 2 (8rl) composition primer: 37.5% phosphoric acid; ethyl alcohol (20-25%), alkyl dimethacrylate resins, water; adhesive: uncured methacrylate ester (50-60%), tegdma (5-10%), ytterbium trifluoride (12-17%), inert mineral fillers, photoinitiators, stabilizers dental gel etchant: phosphoric acid, water, colloidal silica, inorganic colorfluroshield (50% inorganic fillers): bis-gma modified urethane, tegdma, aluminum and barium borosilicate, phosphoric acid tetracyclic ester, sodium fluoride, n-methyldiethanolamine, camphoroquinone. bis-gma, tegdma, udma, bis-ema, silica/zirconium (0,6 um), camphoroquinone bis-gma, hema, urethane dimethacrylate, polyalkenoic acid copolymer, camphorquinone, water, ethanol, 1.3 glycerol dimethacrylate, 10% silica (5 nm) application mode 1acid etching (15 s); 2wash (15 s);3dry (5 s); 4apply one coat of primer (15 s); 5dry (5 s at 10 cm); 6apply 1 coat of adhesive (15 s);7light cure (20 s at 600 mw/ cm²)8-apply one coat of adhesive (15 s);9light cure (20 s at 600 mw/cm²) 1acid etching (15 s); 2wash (15 s); 3dry (5 s at 10 cm); 4apply one coat of sealant; 5light cure (20 s at 600 mw/cm²) apply composite in 2 mm-thick increments and light cure (30 s) 37% h3po4 (15 s); wash (15 s); air stream (10 s); adhesive (2 coats); air stream (10 s); light cure (10 s at 600mw/ cm²); composite restoration table 1 table 1 table 1 table 1 table 1 used materials, composition and application modes hema (2-hydroxyethylmethacrylate); udma (urethane dimethacrylate); tegdma (triethyleneglycol dimethacrylate); bis-gma (bisphenol a-glycidyl methacrylate); bis-ema (bisphenol ethyl methacrylate). the deciduous and permanent teeth were randomly assigned to four groups (n=5), according to the type of dentition and dental sealant: permanent optibond fl, permanent fluroshield, deciduous optibond fl and deciduous fluroshield. the occlusal surfaces of all teeth were cleaned with pumice and water prior to sealing. the sealants were a hydrophobic pit-and-fissure sealant (fluroshield; dentsply, petrópolis, rj, brazil) and a conventional adhesive system (optibond fl; kerr co, orange, ca, usa). a trained operator performed all sealing procedures in accordance with the manufacturers’ recommendations (table 1). the pulp chamber of each primary tooth was restored to prevent e namel cracking during preparation of the specimens for the microtensile bond strength test13. this procedure is required because after exfoliation only a thin layer of dentin remains in the crown. the pulp chambers of 10 primary molars were acid-etched with 37.5% phosphoric acid for 15 s, washed for 15 s, dried, and restored with adper single bond 2 adhesive system and filtek z250 composite (shade a3,5; 3m espe dental products; st paul, mn, usa), according to the manufacturer’s directions (table 1). after application, the restoring materials were photoactivated with light intensity of 600 mw/cm2 (vip; bisco, schaumburg, il, usa). the sealants were placed on the occlusal surfaces in a single increment and according to the manufacturer’s instructions. in sequence, filtek z250 resin blocks (3m espe dental products, st paul, mn, usa) were built up in three 2mm-increments on sealant surface. after storage in distilled water for 24 h at 37 °c, the restored teeth were serially sectioned12-13 with a diamond disk (extec 12205 high concentration; enfield, ct, usa) in a bonding durability of dental sealants to deciduous and permanent teeth braz j oral sci. 13(3):198-202 200200200200200 high-precision cutting machine (isomet 1000; buehler ltd, lake bluff, il, usa). perpendicular sections to the bonding interface in the mesiodistal direction were obtained, resulting in 0.9-mm-thick slices. each slice was individually positioned and the flattest area of the occlusal interface was delimited for a second section, in the buccolingual direction, producing stick-shaped samples of 0.8 mm2. the stick samples of each tooth were allocated into two groups (24-hour and six-month) and subjected to tension at 0.5 mm/min in a universal testing machine (emic dl2000; são josé dos pinhais, pr, brazil). bond strength analysis was performed 24 h after application of the sealants12-13. after 6 months of water storage in distilled water at 37 ºc, the procedure was repeated and fragments were observed in an optical microscope (bel microimage analyser; bel photonics do brasil ltda, osasco, são paulo, brazil) at 40x magnification. fractures were classified as cohesive (enamel or composite), adhesive (interface), or mixed (presence of composite and/or enamel in the same fragment). percentage of fracture modes and specimens fractured before testing, were recorded for all groups13. for statistical purposes, the tooth was considered as the experimental unit. average values of bond strength (mpa) of each group were analyzed by repeated measures anova and tukey’s test (α= 5%). results table 2 presents the microtensile bond strength after 6 months for all experimental groups. there were differences for dentition (p=0.0097), dental sealant (p=0.0019) and time (p=0.0001). at 24 h, the highest bond strength was observed for optibond fl in deciduous and permanent dentition, similar to fluroshield in deciduous and permanent dentitions. after 6 months of water storage, the bond strength of optibond fl decreased in deciduous teeth similar to fluroshield in permanent teeth. the lowest bond strength type of dentition/ time material deciduous permanent 24 h 6 months 24 h 6 months optibond fl 31.35(0.40)a 16.91(1.20)b,c 29.89(2.50)a 24.42(5.19)ab fluroshield 24.62(2.77)ab 14.73(1.84)c 27.97(3.61)a 18.42(5.60)bc table 2 -table 2 -table 2 -table 2 -table 2 mean (standard deviation) of bond strength in mpa in the groups different letters indicate statistically significant difference (p<0.05). fracture pattern material deciduous permanent 24 h 6 months 24 h 6 months (m/op) (m/op) (m/op) (m/op) optibond fl (25/0) (100/0%) (15/10) (40/0) (27/13) (60/40%) (100/0%) (67,5/32.5%) fluroshield (20/0) (100/0%) (13/7) (22/0) (17/5) (65/35%) (100/0%) (77/23%) table 3 -table 3 -table 3 -table 3 -table 3 distribution of fractures in the sticks of the experimental groups according to the type of dentition and time. m-fracture mixed; op-other patterns (sum of cohesive resin, of cohesive enamel and, premature failures). after this time was observed for fluroshield in deciduous teeth. the bond strength of optibond fl did not decrease in permanent dentition after 6 months. the repeated measures anova did not indicate statistically significant differences for the three-way interaction effect (statistics fdf(1:12) = 2.74; p-value = 0.1240 > 0.05), and both for two-way effects: tooth and time (statistic f df (1:12) = 3.20; p-value = 0.0990>0.05) and for tooth and sealant (statistic f df (1:12) = 0.05; p-value = 0.8197>0.05). table 3 presents the observed fracture types. in all groups mixed fractures were prevalent over “other fractures” (where cohesive fractures in enamel, cohesive fractures in resin and premature failures were grouped because their percentages were lower than those of mixed fractures and were not used for statistical purposes). adhesive fractures were not observed. discussion the hypothesis of this study was rejected since the bonding durability of resin materials to enamel varied among the experimental conditions. the american academy of pediatric dentistry19 recognizes that the application and continued maintenance of sealants can prevent pit and fissure caries in children’s teeth. however, enamel adherence to the occlusal surface of primary teeth via adhesive systems has not yet been widely investigated12-13,17. a few studies have tested the effectiveness and durability of hydrophilic adhesive sealants in caries prevention11,20. in 2006, feigal and donly 21 reported a modified technique using a bonding primer and adhesive layer between the etched enamel and the sealant. the technique overcame successfully the negative effects of salivary contamination that often occurs in pediatric patients. it has been shown22 that the inclusion of the primer and adhesive layer improves the bonding to the enamel and minimizes microleakage under conditions of saliva contamination. bonding durability of dental sealants to deciduous and permanent teeth braz j oral sci. 13(3):198-202 201201201201201 this study aimed at analyzing the bonding durability of a hydrophobic sealant and an hydrophilic adhesive system on deciduous and permanent teeth along 6 months of water storage. results at 24 h indicated high bond strength for optibond fl in deciduous and permanent teeth, which was similar to fluroshield in both dentitions. these similarities may be partially explained by the filler content in both pit and fissure sealants, which is around 50% in fluroshield and 48% in the hydrophobic adhesive optibond fl13,17. both dental sealants were applied to deciduous and permanent teeth following acid etching with phosphoric acid, so the micro-retentive surfaces to be filled with dental sealants may have been produced similarly in both dentitions and materials. according to pashley et al. 201123, adhesive systems that present acid, primer and hydrophobic adhesive in separate bottles and no solvent in adhesive are superior in terms of adhesion durability, since there is no influence of hydrophilic compounds on the degree of conversion of the polymer, and optibond fl is an example of this. this may explain why the bond strength of optibond fl did not decrease from 24 h to 6 months in the permanent dentition. other researches have described the good performance for optibond fl13,17 in deciduous teeth. the advantage of using a hydrophilic adhesive system as sealant was emphasized13,17 by minimizing the risk of saliva contamination in pediatric dentistry12-13. this was emphasized by romito et al.13,17, who obtained the worst results for hydrophobic sealant under saliva contamination in their in vitro results. fluroshield is a hydrophobic sealant that may have undergone the same consequences under saliva contamination as tested in the present in vitro study. regarding the type of dentition, differences may be considered in terms of the aprismatic enamel layer, which is thicker in deciduous than in permanent teeth2-3 and more resistant to acid etching24. this could explain the similar decrease of bond strengths of optibond fl and fluroshield in deciduous teeth after 6 months, considering the pit-andfissure sealant itself in the present study. on the other hand, the type of dentition did not influence the bond strength of fluroshield over time, because it decreased similarly in deciduous and permanent teeth. after 6 months of water storage, decreased bonding was also observed for optibond fl on deciduous teeth, similar to fluroshield on permanent teeth. the bond strength of optibond fl in permanent teeth was also similar to fluroshield in deciduous teeth after 24 h. apart from the differences related to the type of dentition, due to the aprismatic enamel layer and composition of materials, these findings may also be explained by the method used to evaluate the bond strength in enamel, the microtensile bond strength. as enamel is a friable substrate, the diamond disc to obtain the specimens produces flaws that should be minimized by using diamond wires as previously described25. this may be considered a limitation of the present study, but the choice for using microtensile to test the bond to enamel was supported by a systematic review that revealed that this methodology is advantageous over micro-shear and other bond strength tests for allowing comparison of the adhesion of in vitro results to that of class v in clinical circumstances26. in addition to the differences observed among the experimental groups, the fracture analysis of dental specimens showed prevalence of mixed fractures in all groups, which means that the enamel/interface was tested in tensile as expected. they were considered for statistical purposes, and the percentage of other fracture types in all groups was low. lemos et al.27 reported that the assiduity factor positively influences the dental caries prevalence. it is therefore essential, in every dental visit, not only the choice of the dental sealant, but to assess the risk of the child to develop dental caries. a recent literature review28 showed that the application of sealants to the occlusal surfaces of permanent molars in children and adolescents reduced caries onset up to 48 months when compared with no sealant application. it is important to consider this modality of treatment for children, regarding the maintenance of the masticatory function29 by means of a caries-free dentition. other studies should be encouraged to evaluate clinically the use of different types of materials as pit-and-fissure sealants. references 1. gwinnett aj, garcia-godoy f. effect of etching time and acid concentration on resin shear bond strength to primary tooth enamel. am j dent. 1992; 5: 237-9. 2. mortimer kv. the relationship of deciduous enamel structure of dental disease. caries res. 1970; 4: 206-23. 3. fava m, watanabe i, moraes ff, costa lrrs. prismless enamel in human non erupted deciduous molar teeth: a scanning electron microscopic study. rev odontol univ são paulo. 1997; 11: 239-43. 4. sonju clasen ab, ruyter ie. quantitative determination of type a and type b carbonate in human deciduous and permanent enamel by means of fourier transform infrared spectrometry. adv dent res. 1997; 11: 523-7. 5. marquezan m, da silveira bl, burnett lh jr, rodrigues cr, kramer pf. microtensile bond strength of contemporary adhesives to primary enamel and dentin. j clin pediatr dent. 2008; 32: 127-32. 6. wang l, sakai vt, kawai es, buzalaf ma, atta mt. effect of adhesive systems associated with resin-modified glass ionomer cements. j oral rehabil. 2006; 33: 110-6. 7. mattos-graner ro, rontani rm, gavião mb, bocatto ha. caries prevalence in 6-36-month-old brazilian children. community dent health. 1996; 13: 96-8. 8. amaral rc, batista mj, meirelles mpmr, cypriano s, sousa mlr. dental caries trends among preschool children in indaiatuba, sp, brazil. braz j oral sci. 2014; 13: 1-5. 9. hicks mj, flaitz cm. caries formation in vitro around a fluoride-releasing pit and fissure sealant in primary teeth. asdc j dent child. 1998; 65: 161-8. 10. feldens eg, feldens ca, araujo fb, souza ma. invasive technique of pit and fissure sealants in primary molars: a sem study. j clin pediatr dent. 1994; 18: 187-90. 11. grande rhm, lima acp, rodrigues filho le, witzel mf. clinical evaluation of an adhesive used as a fissure sealant. am j dent. 2000; 13: 167-70. 12. ramires-romito ac, reis a, loguercio ad, goes mf, grande rh. micro-tensile bond strength of adhesive systems applied on occlusal primary enamel. j clin pediatr dent. 2004; 28: 333-8. 13. ramires-romito ac, reis a, loguercio ad, hipólito vd, de goes mf, singer jm, et al. microtensile bond strength of sealant and adhesive systems applied to occlusal primary enamel. am j dent. 2007; 20: 114–20. 14. vargas cm, casper js, altema-johnson d, kolasny cr. oral health bonding durability of dental sealants to deciduous and permanent teeth braz j oral sci. 13(3):198-202 202202202202202 trends in maryland. j public health dent. 2012; 72: s18-22. 15. kanehira m, finger wj, ishihata h, hoffmann m, manabe a, shimauchi a et al. rationale behind the design and comparative evaluation of an all-inone self-etch model adhesive. j dent. 2009; 37: 432-9. 16. gomes gb, almeida lh, oliveira as, moraes rr. influence of water concentration on the etching aggressiveness of self-etch primers to ground primary enamel. pediatr dent. 2012; 34: 226-30. 17. lemos lvfm, felizardo kr, myaki si, lopes mb, moura sk. bond strength and morphology of resin materials applied to the occlusal surface of primary molars. int j paediatr dent. 2012; 22: 435-41. 18. simonsen rj. pit and fissure sealant: review of the literature. pediatr dent. 2002; 24: 393-414. 19. american academy of pediatric dentistry. policy on third-party reimbursement of fees related to dental sealants. pediatr dent. 2012; 33: 85-6. 20. grande rh, ballester r, singer jm, santos jf. microleakage of a universal adhesive used as a fissure sealant. am j dent. 1998; 11: 109-13. 21. feigal rj, donly kj. the use of pit and fissure sealants. pediatr dent. 2006; 28: 143-50; discussion 192-8. 22. hebling j, feigal rj. use of one-bottle adhesive as an intermediate bonding layer to reduce sealant microleakage on saliva-contaminated enamel. am j dent. 2000; 13: 187-91. 23. pashley dh, tay f, breschi l, tjasderhane l, carvalho rm, carrilho m et al. state of the art of etch and rinse adhesives. dent mater. 2011; 27: 1-16. 24. daronch m, de goes mf, grande rh, chan dc. antibacterial and conventional self-etcing primer system: morphological evaluation of intact primary enamel. j clin pediatr dent. 2007; 27: 251-6. 25. sadek ft, monticelli f, muench a, ferrari m, cardoso pe. a novel method to obtain microtensile specimens minimizing the cutting flaws. j biomed mater res b appl biomater. 2006; 78: 7-14. 26. heintze s. systematic reviews i. the correlation between laboratory tests on marginal quality and bond strength. ii. the correlation between marginal quality and clinical outcomes. j adhes dent. 2007; 9: 77-106. 27. lemos lvfm, barata tje, myaki si, walter lrf. dentistry for babies: caries experience vs. assiduity in clinical care. braz j oral sci. 2012; 11: 486-91. 28. ahovuo-saloranta a, forss h, walsh t, hiiri a, nordblad a, makela m et al. sealants for preventing dental decay in the permanent teeth. cochrane database syst rev. 2013; 3: cd001830. 29. motta lj, santos jg, alfaya ta, guedes cc, godoy chl, busadori sk. clinical status of permanent first molars in children aged seven to ten years in a brazilian rural community. braz j oral sci. 2012; 11: 475-80. bonding durability of dental sealants to deciduous and permanent teeth braz j oral sci. 13(3):198-202 oral sciences n3 original article braz j oral sci. april | june 2014 volume 13, number 2 received for publication: april 17, 2014 accepted: june 11, 2014 electromyographic evaluation of the effect of ultrasound with muscle stretching in temporomandibular disorder: a clinical trial jalusa boufleur1, eliane castilhos rodrigues corrêa1, lais chiodelli1, ana maria toniolo da silva2, lilian gerdi kittel ries3 ¹universidade federal de santa maria – ufsm, school of physical therapy, department of physical therapy and rehabilitation, santa maria, rs, brazil 2universidade federal de santa maria ufsm, school of speech therapy, department of speech therapy, santa maria, rs, brazil 3 universidade do estado de santa catarina udesc, school of physical therapy, department of physical therapy, florianópolis, sc, brazil correspondence to: jalusa boufleur rua machado de assis, 221 apto. 402 cep: 97050-450 bairro nossa senhora das dores santa maria-rs phone: + 55 55 33476364; + 55 55 91292052 e-mail: jalusaboufleur@yahoo.com.br abstract aim: to investigate the electromyographic activity of masticatory muscles in women with myogenic or mixed tmd treated with ultrasound and ultrasound associated with stretching. methods: sixteen women with myogenic or mixed tmd, according to the research diagnostic criteria for temporomandibular disorder (rdc/tmd), participated in the study. the patients were evaluated using surface electromyography (semg) of the masseter and anterior temporalis muscles, during maximum intercuspation, before and immediately after application of therapeutic resources. all patients were treated by ultrasound (us), ultrasound associated with stretching and placebo ultrasound, by turns, once a week with a one-week minimum interval between them. results: there was greater symmetry of the masseter muscle electrical activity after ultrasound associated with stretching (p=0.03). the electromyographic values for the maximum intercuspation as well as the symmetry of anterior temporal muscle (p=0.47, p=0.84, p=0.84) and anteroposterior coefficient (p=0.07, p=0.84, p=0.57) showed no statistically significant difference after the intervention, placebo, ultrasound and ultrasound with stretching. conclusions: these results indicate that a single application of ultrasound and ultrasound associated with stretching were not able to modify the activity pattern of the masticatory muscles during maximum intercuspation, except the symmetry of the masseter muscle that increased with the last, in women with myogenic or mixed temporomandibular disorder. keywords: temporomandibular joint disorder; electromyography; ultrasonics; muscle stretching exercise. introduction temporomandibular disorder (tmd) is a multifactorial condition characterized by symptoms, most of them painful, which affect the stomatognathic system, including the ligaments, muscles and the temporomandibular joint1-2. in the tmd treatment, non-invasive therapies such as physical therapy are recommend, which aims at relief the of musculoskeletal pain, improvement of inflammation and restoration of oral function3-4. ultrasound is one the physical therapeutic modalities used in the tmd treatment. it promotes pain relief and muscle relaxation, since muscle hyperactivity, braz j oral sci. 13(2):152-157 considered one of the tmd etiologic factors, may lead to the pain-muscle spasm cycle 5-6. increase in the musculotendinous length and decrease of the muscle stiffness may be achieved by stretching7. thus, the restoration of the musculotendinous length, associated to pain relief, may reflect on the masticatory muscle electromyographic (emg) potentials during a certain activity, with the improvement of the muscular balance. the muscle electrical activity at rest is higher in tmd patients, while the emg potentials during the voluntary maximal contraction are reduced, compared to healthy subjects. asymmetries also are observed in the masticatory muscles in these patients. the evaluation of the functional symmetry of the orofacial complex, involves the pattern of the mandibular movement and the masticatory muscles activity. the contraction pattern of a pair of muscles can be investigated by the surface emg8-9. some authors10-11 have investigated the effect of specific physiotherapeutic protocols on the pain and on the functional limitation of tmd. frequently, it is not known the isolated effect of a determined therapeutic modality on the signs and symptoms of the disorder and which of them was responsible for the patient‘s clinical improvement. the pain relief and mandibular range of movement increase obtained with the physical therapy, allows for a more effective and faster effect of the speech therapy and dental treatments on the functional restoration of the stomatognathic system12-13. thereby, the aim of the present study was to evaluate the immediate effect of ultrasound, ultrasound combined with stretching of the mandibular elevator muscles and placebo on the activation pattern and symmetry of these muscles in women with myogenic and mixed tmd. material and methods subjects the clinical trial registration (nct02139735) and the research ethics committee of the federal university of santa maria (protocol number 0281.0.243.000-08) approved the study and informed consent was obtained from each subject. twenty-nine prospective female subjects recruited from the ufsm occlusal clinical and from direct advertising for research subjects on electronic media were initially selected. out of these, 16 women with mean age of 25.69±5.13 years, with myogenic and mixed tmd took part in the experimental procedures of the research. all participants were treated by three therapeutic modalities (ultrasound, ultrasound with stretching and placebo). the modalities were applied in a random sequence (crossover design), with a one-week interval (washout period) among them, in order to dissipate the residual effect of the previous intervention14. selection criteria the inclusion criteria were: (1) age from 20 to 35 years old; (2) diagnosis of myogenic or mixed tmd, according the research diagnostic criteria for temporomandibular disorders (rdc/tmd). the exclusion criteria were: (1) neuropsicomotor disease; (2) history of trauma on the temporomandibular region, orofacial surgery or malformation; (3) consumption of anti-inflammatory, analgesic, antidepressant or myorelaxant drugs; (4) presence of acute pain in tmj and (5) previous speech therapy or physical therapy (in the last six months). additionally, to avoid the influence of the estrogenic hormone reduction15, the final period of the menstrual cycle (seven days before the menstruation) and the beginning of the menstruation (two first days) were skipped for the evaluation of women who did not use oral contraceptive and the ones who paused their use. surface electromyographic signal acquisition the electromyographic evaluation of the masticatory muscles (masseter and temporalis anterior, bilaterally) was conducted with the equipment miotool 400 (miotec®; porto alegre, rs, brasil), with four channels, 14-byte resolution, 2,000 hz sampling frequency, 110 db common mode rejection and a band pass filter of 20-500 hz. the emg data were collected by miograph (miotec®) software and stored in a portable computer acer, model aspire 5250-0866. during signal acquisition, the subjects were sat on a wooden chair, with their feet parallel on a rubber carpet, hands over the thighs and looking at a target placed at eye level in front of them. bipolar surface electrodes ag-agcl, 20 mm center to center interelectrode distance (hal indústria comércio de metais ltda,. são paulo, sp, brazil) connected to active sensors with differential input (miotec®) were positioned on the region of greater volume of the muscle belly during maximum intercuspation position. previous to the electrode placement, the skin was cleaned with gauze soaked with 70% alcohol (isek international society of electromyography and kinesiology). a common grounding to all channels was placed on the subject‘s sternum bone to avoid the interferences and noises in the emg signal16. the emg recordings were acquired during maximum intercuspation (mi) and the patients were oriented to clench their teeth as much as possible and keep this contraction for 5 s. for the voluntary maximal contraction emg recording, the patients were oriented in the same manner, but they clenched their teeth with parafilm material, folded with similar width and thickness of a trident chewing gum (3.5 x 1.5 cm)17, placed between the first and second molar, bilaterally. each recording was repeated twice with a 2-min interval. electromyographic data processing the emg signal was processed in the time domain by the root mean square (rms). the best quality emg signals were visually selected for posterior cutout and qualitative analysis. in all the recorded tests one second with the highest signal amplitude was selected from the total collection period. the emg signal normalization was performed using the maximal voluntary contraction as a reference value. thus, the amplitude of the muscular activation of each muscle in 171153electromyographic evaluation of the effect of ultrasound with muscle stretching in temporomandibular disorder: a clinical trial braz j oral sci. 13(2):152-157 the different situations was expressed as a percentage of the reference value, calculated using the following formula: where x is the measurements data, i is the measurements index and n is the measurements length. the percentage overlapping coefficient (poc%) represents the pair of muscles (right and left side) symmetry, obtained by the overlapping of the normalized values of the amplitude emg signal from the right and left sides. the anteroposterior coefficient (apc), which evaluates the balance between masseter and temporal muscles, was also calculated. this coefficient is the relation between the overlapping and non-overlapping areas obtained from the normalized values of the signal amplitude of the masseter and temporalis muscles from both sides. if only one pair of muscles contracts (for example: right and left masseter), there is an unbalanced contraction between these muscles, with an anterior or posterior displacement of the occlusal center, according the prevalence of the activity of the temporal or masseter muscles, respectively9,16,18-20. it ranges from 0 (without balance) to 100% (complete balance). for the poc and apc calculation, the emg potentials were filtered with a band-pass of 20-500 hz. the two coefficients were obtained with the mean potential calculated in rms on the 25 ms moving windows (50 data). the data were processed with the matlab (the mathworks®, r2006b version) software. physical therapy procedures the subjects underwent three procedures: ultrasound, ultrasound with stretching and placebo. all of them were carried out with the patient lying supine with a towel roll under their cervical column. ultrasound: the sonopulse iii 1 and 3 mhz model equipment (ibramed ltda., amparo, sp, brazil), set at 3 mhz, continuous mode and 1.0 w/cm2 intensity, was applied during 6 min (3 min on each side) at the tmj and masseter muscle regions21. ultrasound simultaneous with masseter muscle stretching: the patient should open the mouth in a confortable position, keeping the lips closed, while the physical therapist applied a slight digital support on her mentum to keep the stretching during 30 s (adapted from pertes and gross21(2005). the stretching was repeated twice with a 10-s rest between the repetitions (figure 1). ultrasound placebo: same procedures of the ultrasound group except that equipment was turned off. statistical analysis the effect of the three therapeutic interventions was evaluated in sixteen patients, comparing the results among them, before and after each intervention. the statistica 9.1 software was used for the statistical analysis. data normality was verified by the shapiro wilk fig. 1. ultrasound with masseter muscle stretching test. the comparison between the mean values of emg signal amplitude before and after the intervention was carried out by the student’s t test for dependent samples or wilcoxon test, according to the characteristic of the variables, parametric or non-parametric. two-way anova was used to compare the values of emg amplitude before and after intervention, among the three modalities. a significance level of 5% was used. results the comparative analysis of the emg amplitude showed, for the three modalities, that there was no statistically significant difference between values obtained before and after intervention, during the maximum intercuspation, in all evaluated muscles (table 1). 154 electromyographic evaluation of the effect of ultrasound with muscle stretching in temporomandibular disorder: a clinical trial ultrasound muscle pre post p value r m 73.42±25.13 74.38±29.30 0.90 l m 72.88±31.13 69.30±29.08 0.52 rat 90.59±29.12 84.09±20.00 0.20 lat 85.70±36.46 79.88±21.81 0.39 ultrasound with stretching muscle pre post p r m 74.21±28.23 71.39±24.47 0.41 l m 73.60±23.58 72.11±25.86 0.75 rta 87.63±22.65 84.53±20.99 0.44 lta 78.47±20.89 83.21±24.79 0.46 placebo muscle pre post p md 68.16±21.56 73.43±22.14 0.19 m e 70.13±24.02 74.17±22.03 0.44 rat 84.42±28.89 87.54±23.19 0.45 lat 83.00±26.70 83.72±23.54 0.91 table 1.table 1.table 1.table 1.table 1.normalized values of emg amplitude obtained during maximum intercuspation position, on the three modalities (ultrasound, ultrasound with stretching and placebo (n=16) right masseter (rm), left masseter (le), right anterior temporalis (rat), left anterior temporalis (lta). student’s t test for dependent samples. p<0.05 braz j oral sci. 13(2):152-157 muscle right masseter left masseter right anterior temporalis left anterior temporalis treatment ultrasound ultrasound+ stretching placebo ultrasound ultrasound+ stretching placebo ultrasound ultrasound+ stretching placebo ultrasound ultrasound+ stretching placebo difference pre and post treatment 0.95 -2.82 5.32 -3.58 -1.48 4.04 -6.49 -3.10 3.12 -5.82 4.74 0.71 p value 0.60 0.51 0.26 0.54 table 2 -table 2 -table 2 -table 2 -table 2 differences of the normalized emg potentials, during maximum intercuspation, before and after each intervention (n=16) anova (two way). p<0.05 fig. 3. mean value of the symmetry (%) of the masseter muscle (poc m) and anterior temporalis muscle (poc t) and cap, pre and post intervention (ultrasoundn=16) fig. 4. mean value of the symmetry (%) of the masseter muscle (poc m) and anterior temporalis muscle (poc t) and cap, pre and post intervention (ultrasound with stretching n=16). 171155electromyographic evaluation of the effect of ultrasound with muscle stretching in temporomandibular disorder: a clinical trial fig. 2. mean value of the symmetry (%) of the masseter muscle (poc m) and anterior temporalis muscle (poc t) and cap, pre and post intervention (placebo – n=16) table 2 shows the differences preand post-intervention on the emg potentials obtained among the modalities during the maximum intercuspation position for all the evaluated muscles. regarding the symmetry evaluation (poc) of the emg amplitude, it was verified a statistically significant increase in the right and left masseter muscles with ultrasound and stretching. apc showed no difference after intervention in all of the three modalities. these results are presented in the figures 2-4. discussion studies regarding the specific and immediate effect of the ultrasound on the masticatory muscles activity were not found in the literature. nevertheless, laser 22 and tens23(transcutaneous electrical nerve stimulation) application as single interventions have been investigated, in addition to the immediate effect of the stabilization splint13,24. a study evaluating the immediate effect of tens on the emg activity at rest in women with artrogenic tmd23 verified decrease in the sternocleidomastoid, digastric, masseter and anterior temporalis muscles activity after this intervention. diversely, the present study evaluated the emg activity during the maximum intercuspation after another modality of electrotherapy (ultrasound with stretching), by normalized emg data, which hinders the comparison between the studies. in the present study, the temporal anterior muscle presented higher values of the normalized emg potentials than the masseter muscles during the maximum intercuspation, before and after the intervention. this result was also found by ferrario et al.25 (2000), but in healthy cap 7 braz j oral sci. 13(2):152-157 156 electromyographic evaluation of the effect of ultrasound with muscle stretching in temporomandibular disorder: a clinical trial individuals with normal occlusion. the values of the emg activity found in patients with tmd in the present study, during maximum intercuspation, were lower than the ones found in healthy individuals by ferrario et al. 25 (2000), suggesting a light level of dysfunction in these patients. mean poc values of 87.01% and 88.60% were observed by tartaglia et al.16 (2011) in a emg evaluation of masseter and anterior temporalis muscles, respectively, in women without tmd. women with tmd presented lower values of symmetry, 85.85% and 84% for the masseter and anterior temporalis muscles, respectively. the symmetry values pre intervention of the present study resemble the control group results of the tartaglia et al. study16. the normalization of the emg signal is recommended when comparing different subjects, days, muscles or studies19 and it consists in converting the data acquired in a given activity in percent values of a reference activity. however, not always the differences observed in rms are confirmed after normalization26. botelho et al.13 (2010) found a significant increase in the masseter muscle symmetry during maximum intercuspation in 15 subjects with tmd after the use of a resilient splint. in this study, the poc values may be considered within normality compared to the values of healthy participants in the control group of the abovementioned study. even so, a significant increase may be observed in the symmetry values of the masseter muscle after intervention with ultrasound combined to stretching. another study24 also verified, immediately after the use of stabilization splint, a significant increase in the symmetry of the masseter muscle, different from the temporalis muscle, which did not change after treatment. the high apc values, possibly within the normality, previously to the intervention may explain the absence of statistically significant difference between pre and post intervention observed in this study, in all three modalities. similar values were observed by ferrario et al.18 (2006) in healthy subjects regarding the symmetry in the apc of masseter and anterior temporalis muscles. based on this, it is believed that the participants of this study, apart from being diagnosed as tmd patients by rdc/tdm instruments, presented a slight degree of the dysfunction. the mentioned instruments do not allow determination of tmd severity27. before the interventions, the participants presented some emg potential values within the normality patterns, probably due to adaptations. acording to türp et al.28 (2007), 75 – 95% of acute tmd patients present significant improvements due to adaptive processes at the biological and psychological levels. this result also agrees with manfredini et al.29 findings who observed spontaneous remission in most of tmd cases, regardless of the tmd diagnostic group, between the 2nd to 3rd year after the diagnosis. therefore, as the patients of this study presented tmd for more than three months and did not carry out any physical therapeutic intervention in this period, it is possible that spontaneous improvements may have occurred. besides, the myofascial tmd is categorized by episodes of symptoms exacerbation and remission30. these periods with symptoms remission may explain the emg results found in this study, similar to the healthy subjects. the present study presented some limitations, such as the sample size and the absence of tmd severity classification of the research subjects. it is believed that studies with more severely affected patients may demonstrate some evidence of immediate response after the intervention. occlusal evaluation must also be included, since it may influence the emg activity. in further studies, it is advisable to overcome these limitations. the evaluation of craniocervical posture and the treatment of cervical pathology have been suggested for tmd patients20. this study’s findings, elicit the need of researches that investigate the duration, frequency and combination of therapeutic modalities, including the cervical vertebrae and muscles, in order to obtain positive and more definitive outcomes in the tmd treatment. the results of this study showed that the ultrasound and ultrasound with masticatory muscle stretching did not modify the emg potentials of the evaluated muscles with a single application, in women with myogenic and mixed tmd. however, the symmetry between the left and right masseter muscles increased immediately after the ultrasound with stretching. acknowledgements we would acknowledge the capes for the support of this study. references 1. amaral ap, politti f, hage ye, arruda eec, amorin cf, biasotto-gonzalez da. immediate effect of nonspecific mandibular mobilization on postural control in subjects with temporomandibular disorder: a single-blind randomized controlled clinical trial. braz j phys ther. 2013; 17: 121-7. 2. la touche r, fernández-de-las-peñas c, fernández carnero j, escalante k, angulo-díaz-parreño s, paris-alemany a, et al. the effects of manual therapy and exercise directed at the cervical spine on pain and pressure pain sensitivity in patients with myofascial temporomandibular disorders. j oral rehabil. 2009; 36: 644-52. 3. carrara sv, conti pcr, barbosa js. statement of the 1st consensus on temporomandibular disorders and orofacial pain. dental press j orthod. 2010; 15: 114-20. 4. cuccia am, caradonna c, annunziata v, caradonna d. osteopathic manual therapy versus conventional conservative therapy in the treatment of temporomandibular disorders: a randomized controlled trial. j bodyw mov ther. 2010; 14: 179-84. 5. tosato j, biasotto-gonzalez da, caria phf. effect of massage therapy and of transcutaneous eletrical nerve stimulation on pain and electrotromyographic activity in patients with temporomandibular dysfunction. fisioter pesq. 2007; 14: 21-6. 6. watson t. ultrasound in contemporary physiotherapy practice. ultrasonics. 2008; 48: 321-9. 7. behm dg, peach a, madiggan m, aboodarda sj, disanto mc, button dc. et al. massage and streching reduce spinal reflex excitability without affecting twitch contractile properties. j electromyogr kinesiol. 2013; 23: 1215-21. 8. pinho jc, caldas fm, mora mj, santana-penín u. electromyographic activity in patients with temporomandibular disorders. j oral rehabil. 2000; 27: 985-90. braz j oral sci. 13(2):152-157 171157electromyographic evaluation of the effect of ultrasound with muscle stretching in temporomandibular disorder: a clinical trial 9. botelho al, brochini apz, martins mm, melchior mo, silva ambr, silva mamr. an electromyographic assessment of masticatory muscles asymmetry in normal occlusion subjects. rev odontol univ são paulo. 2008; 13: 7-12. 10. nikolakis p, erdogmus b, kopf a, djaber-ansari a, piehslinger e, fialkamoser v. exercise therapy for craniomandibular disorders. arch phys med rehabil. 2000; 81: 1137-42. 11. furto es, cleland ja, whitman jm, olson ka. manual physical therapy interventions and exercise for patients with temporomandibular disorders. cranio. 2006; 24: 283-91. 12. felício cm, melchior mo, ferreira clp, rodrigues dsmam. otologic symptoms of temporomandibular disorder and effect of orofacial myofunctional therapy. cranio. 2008; 26: 118-25. 13. botelho al, silva bc, gentil fhu, sforza c, silva mamr. immediate effect of the resilient splint evaluated using surface electromyography in patients with tmd. cranio. 2010; 28: 265-73. 14. petrie a, sabin c. medical statistics. são paulo: roca; 2007. 15. turner ja, mancl l, huggins kh, sherman jj, lentz g, leresche l. targeting temporomandibular disorder pain treatment to hormonal fluctuations: a randomized clinical trial. pain. 2011; 152: 2074-84. 16. tartaglia gm, lodetti g, paiva g, de felício cm, sforza c. surface electromyographic assessment of patients with long lasting temporomandibular joint disorder pain. j electromyogr kinesiol. 2011; 21: 659-64. 17. biasotto-gonzalez da, bérzin f, costa jm, gonzalez to. electromyographic study of stomatognathic system muscles during chewing of different materials. electromyogr clin neurophysiol. 2010; 50: 121-7. 18. ferrario vf, tartaglia gm, galletta a, grassi gp. the influence of occlusion on jaw and neck muscle activity: a surface emg study in healthy young adults. j oral rehabil. 2006; 33: 341-8. 19. ferrario vf, tartaglia gm, luraghi fe, sforza c. the use of surface electromyography as a tool in differentiating temporomandibular disorders from neck disorders. man ther. 2007; 12: 372-9. 20. ries lgk, alves mc, bérzin f. asymmetric activation of temporalis, masseter and sternocleidomastoid muscles in temporomandibular disorder patients. cranio. 2008; 26: 59-64. 21. pertes ap, gross. sg. clinical treatment of temporomandibular disorders and orofacial pain. são paulo: quintessense; 2005. 22. sattayud s, bralley p. a study of the influence of low intensity laser therapy on painful temporomandibular disorders patients. laser therapy. 2012; 21: 183-92. 23. monaco a, sgolastra f, pietropaoli d, giannoni m, cataneo r. comparison between sensory and motor transcutaneous electrical nervous stimulation on electromyographic and kinesiographic activity of patients with temporomandibular disorder: a controlled clinical trial. bmc musculoskelet disord. 2013; 14: 2-8. 24. ferrario vf, sforza c, tartaglia gm, dellavia c. immediate effect of a stabilization splint on masticatory muscle activity in temporomandibular disorder patients. j oral rehabil. 2002; 29: 810-5. 25. ferrario v, sforza c, colombo a, ciusa v. an electromyographic investigation of masticatory muscles symmetry in normo-occlusion subjects. j oral rehabil. 2000; 27: 33-40. 26. kroll cd, bérzin f, alves m. clinical evaluation of activity of masticatory muscles during usual mastication a study on the normalization of emg data. rev odontol unesp. 2010; 39: 157-62. 27. milanesi j, weber p, pasinato f, corrêa ecr. severity of the temporomandibular disorder and its relationship with craniocervical cephalometric measures. fisioter mov. 2013; 26: 79-86. 28. türp jc, jokstad a, motschall e, schindler hj, windecker-gétaz i, ettlin da. is there a superiority of multimodal as opposed to simple therapy in patients with temporomandibular disorders? a qualitative systematic review of literature. clin oral implants res. 2007; 18: 138-50. 29. manfredini d, favero l. gregorini g, cocilovo f, guarda-nardini l. natural course of temporomandibular disorders with low pain-related impairment: a 2-to-3 year follow up study. j oral rehabil. 2013; 40: 436-42. 30. la touche r, parís-alemany a, von piekartz h, manheimmer js, fernández-carnero j, rocabado m. the influence of cranio-cervical posture on maximal mouth opening and pressure pain threshold in patients with myofascial temporomandibular pain disorders. clin j pain. 2011; 27: 48-55. braz j oral sci. 13(2):152-157 oral sciences n3 original article braz j oral sci. october | december 2012 volume 11, number 4 factors associated with the use of herbal medicines for oral problems by patients attending the clinics of the school of dentistry, federal university of juiz de fora, brazil janice simpson de paula1, alexandre marques de resende2, fábio luiz mialhe3 1dds, ms, phd student of dentistry, department of community dentistry, piracicaba dental school, university of campinas (unicamp) piracicaba, sp, brazil 2phd, associate professor, dental school, federal university of juiz de fora, juiz de fora, mg, brazil 3phd, associate professor, department of community dentistry, piracicaba dental school, university of campinas (unicamp), piracicaba, sp, brazil correspondence to: janice simpson de paula departmento de odontologia social faculdade de odontologia de piracicaba caixa postal 52 cep: 13414-903 piracicaba, sp,brasil e-mail: janicesimpsondp@yahoo.com.br received for publication: july 16, 2012 accepted: october 18, 2012 abstract aim: to evaluate differences in sociodemographic characteristics (gender, age, educational level and income) between users and nonusers of phytotherapy for dental purposes; the degree of population’s knowledge of herbal medicines for dental needs; and whether patients inform the health professional about their use of phytotherapy. methods: a questionnaire was applied to 100 patients in waiting rooms of the school of dentistry of the federal university of juiz de fora, in 2008, to inquire about sociodemographic variables and other factors associated with the use of phytotherapy. statistical analyses were carried out using the chi square statistical test to calculate association between the variables, with 0.05 as level of significance. results: it was observed that 64.8% of participants were women with a mean age of 43.9±15.1 years. phytotheraphy use for treatment of oral problems was reported by 37% of patients interviewed in this study. significant differences were found between users and non-users of herbal medicines for oral problems, associated with the patient’s age (p<0.05) and previous experience with use of phytotherapy to treat general health problems (p<0.001). conclusions: the results emphasize the need for more scientific evidence of the efficacy of herbal medicinal products already incorporated in the popular knowledge to treat oral problems in order to make it an accessible and alternative method for prevention and therapy in dentistry. keywords: medicinal plants, dentistry, public health. introduction herbal products have been used since antiquity by humans as a way to reach or recover health. over the decades, many plants with biological and antimicrobial properties have been studied by pharmaceutical companies as sources for new phytotherapeutic agents1. since the declaration of the alma-ata in 1978, the world health organization (who) has expressed the need to appreciate the use of medicinal plants in public health systems, as some studies have indicated that almost 80% of world population uses these plants in primary care2-3. in the first us national survey, eisenberg, et al.4 reported an increase in the braz j oral sci. 11(4):445-450 prevalence of the use of complementary and alternative medicine (cam). in 1990, this prevalence in the american adult population was 33.8% and increased to 42.1% seven years later. as regards people using herbal therapies, the same author detected 10% increase from 1990 to 1997. according to druss, et al.5, the majority of people use cam therapies in conjunction with the conventional medical treatment. in brazil, the population and municipal health systems have shown growing interest in the use of cam since the 1980s. in 2006, the national policy for complementary and integrative practices within the brazilian national health system6 was established. among the practices, traditional chinese medicine, acupuncture, homeopathy, phytotherapy (herbal medicines), anthroposophical medicine and thermalism-crenotherapy have become prominent6. the use of these practices has also been incorporated into dentistry. numerous studies have been conducted to evaluate vegetable species in dentistry, natural agents that are economically feasible and provide effective alternatives for treating oral diseases1,7-8. over the years, the brazilian population has used different forms of plant extracts to treat diseases of the oral cavity9. singh, et al.10 showed that the prevalence of oral diseases is high and medicinal plants are increasingly gaining attention because of their antimicrobial properties. abdulwahab and al-kholani 11 confirmed the increasing interest in natural products by presenting a study about a herbal dentifrice tested in a randomized trial, and demonstrating that there was no difference between using this dentifrice in comparison with the traditional one. although various studies have evaluated the reasons for and individual factors associated with the use of herbal remedies by primary care patients, the characteristics of the population that use herbal medicines for oral health problems are relatively unknown. however, by means of studies along these lines, health managers and researchers are able to find strategies for future studies on herbal medicines and health professional training. thus, studies such as those of tomazzoni et al.12 are encouraged in dentistry because they support the inclusion of phytotherapies in the brazilian public health care system. the aims of this exploratory study were to evaluate: (1) differences in sociodemographic characteristics (gender, age, educational level and income) between users and nonusers of phytotherapy for dental purposes; (2) the degree of population’s knowledge of herbal medicines for dental needs; and (3) whether patients inform the health professional about their use of phytotherapy. material and methods the research project was submitted to and approved by the research ethics committee (nº 336/2008) of the federal university of juiz de fora, brazil. the participants’ consent was obtained for inclusion in the study. the study had a cross-sectional research design and the sample was determined by convenience, selected from the patients present in the waiting room of all clinics of the school of dentistry, federal university of juiz de fora, from august 2008 to november 2008. juiz de fora is one of the pioneering cities in the industrial state of minas gerais, brazil, and its predominating economic sectors are industry and services. the city has about 570,000 inhabitants, spread over a wide range of socioeconomic backgrounds, of whom 98.91% have access to fluoridated water13. a self-administered questionnaire was applied by a researcher before the scheduled visit. the survey instrument included questions about the patient’s social and demographic characteristics, including age, sex, education, household income and use of phytotherapy for treatment oral and general health conditions (figure 1). for participation in the study, patients had to be at least 18 years of age and be under treatment at one of the dental school clinics. patients who agreed to participate were given the survey questionnaire to complete on their own. the researcher was available onsite to answer any questions the patients had in order to help to improve their understanding of the questionnaire used in the survey. descriptive and statistical analyses were performed using spss statistical software version 17 (spss inc., chicago, il, usa) for data analysis at a 0.05 level of significance. the chisquare test was used to test the association of phytotherapy use for oral health problems and the sociodemographic characteristics of the sample, such as gender, age and use of phytotherapy for other health problems. 1. do you use phytotherapy for general health problems or treating oral problems? 2. name of herbal medicine used for dental problems. 3. the main reason for using phytotherapy for oral health treatment. 4. form of consumption of the plant: tea, mouthwash or tampons. 5. duration of use 6. satisfaction with this type of treatment 7. adverse effects 8. do you inform the health professional (physician or dentist) during the clinical interview that you are using herbal medicines? if not, explain the reasons. 9. do you desire to receive more information on the therapies with herbal fig. 1. questions about use of herbal medicines for oral problems by patients present in the clinics of the school of dentistry, federal university of juiz de fora, brazil. results of the 100 eligible study participants, 64.8% were women with a mean age of 43.9 years with standard deviation of ± 15.1 years (age range: 18-78 years). the characteristics of the study sample, the use of phytotherapy for general health problems and its association with use of phytotherapy for oral health problems are summarized in table 1. of all patients interviewed in this study, 71 affirmed that they used phytotherapy for general health problems and 37 were using some form of phytotherapy for treating oral problems. in the association of phytotherapy with the sociodemographic characteristics of the sample, it was 446446446446446 braz j oral sci. 11(4):445-450 factors associated with the use of herbal medicines for oral problems by patients attending the clinics of the school of dentistry, federal university of juiz de fora, brazil use of phytotherapy for oral health problems n no yes p-value * n % n % gender female 75 45 71.4 30 81.1 0.282 male 25 18 28.6 7 18.9 age groups < 30 24 20 31.7 4 10.8 0.007 31 a 64 71 38 60.3 33 89.2 > 65 5 5 7.9 0 0 educational level < 8 years 52 29 46.0 23 62.2 0.119 > 8 years 48 34 54.0 14 37.8 household income per month** < 2 minimum wages 60 35 55.6 25 67.6 0.236 > 2 minimum wages 40 28 44.4 12 32.4 use of phytotherapy for general health problems n o 29 28 44.4 1 2.7 0.000 yes 71 35 55.6 36 97.3 table 1. sociodemographic characteristics of the sample, use of phytotherapy for general health problems and its associations with the use of phytotherapy for oral health problems (n=100) * chi-square test ** brazilian minimum wage in effect at time of data collection = us$ 290 popular name (in portuguese) folha de batata doce transagem terramicina vermelha arnica folha de algodão hortelã malva arruda boldo camomila cana de macaco cinco folhas erva cidreira folha de cana folha de tomate jaborandi maria-dormideira necroton rebenta pedra urucum total scientific name ipomoea batatas plantago lanceolata alternanthera brasiliana arnica montana gossypium hirsutum l. mentha piperita l. malva sylvestris ruta graveolens l. peumus boldus chamomilla recutita dichorisandra thyrsiflora panax ginseng melissa officinalis saccharum officinarum l. lycopersicum esculentum pilocarpus jaborandi mimosa pudica allium cepa phyllanthus niruri l. bixa orellana no. cited (%) 19 (36.5%) 10 (19.2%) 2 (3.8%) 2 (3.8%) 2 (3.8%) 2 (3.8%) 2 (3.8%) 1 (1.9%) 1 (1.9%) 1 (1.9%) 1 (1.9%) 1 (1.9%) 1 (1.9%) 1 (1.9%) 1 (1.9%) 1 (1.9%) 1 (1.9%) 1 (1.9%) 1 (1.9%) 1 (1.9%) 52 (100%) oral problem indications pain, inflammation, infection and swelling pain, inflammation, swelling pain pain, infection pain , infection pain inflammation pain , infection infection pain pain pain pain pain pain inflammation pain and inflammation pain pain inflammation table 2. list of phytotherapy most commonly used in dentistry by respondents of the study survey observed that the age group (p<0.05) and use of phytotherapy for general health problems (p<0.001) were found to be significantly associated with use of phytotherapy for oral health problems. the 37 patients using herbal medicines for dental problems mentioned 52 plants, listed in table 2. the most common phytotherapy products reported as being used at present were ipomoea batatas and plantago lanceolata, for pain, inflammation, infection or swelling. table 3 presents the information about use the phytotherapy for oral health treatment. the main reason for using phytotherapy for oral health treatment was that it was recommended by family tradition (78.4%), and in most cases the plants were obtained the plants were grown by the patients 447447447447447 braz j oral sci. 11(4):445-450 factors associated with the use of herbal medicines for oral problems by patients attending the clinics of the school of dentistry, federal university of juiz de fora, brazil question main reason for using phytotherapy for oral health treatment form of consumption of the plant duration of use satisfaction with this type of treatment adverse effects informed the health professional (physician or dentist) during clinical the interview of being using herbal medicines response family tradition indication from another person tea mouthwash and tampons < 10 days > 10 days yes n o yes n o yes n o frequency 78.4% 21.6% 43.2% 56.8% 94.5% 5.4% 100% 0% 0% 100% 54.1% 45.9% table 3. information about use the phytotherapy for oral health treatment. themselves (56.8%). the users of phytotherapy for oral problems reported consumption of the plant in the form of tea (43.2%), mouthwash and tampons (56.8%). in almost all cases, the duration of use was up to 10 days. all patients who used medicinal plants for oral problems reported satisfaction with this type of treatment and absence of adverse effects. among them, 54.1% affirmed that they generally informed the health professional (doctor or dentist) in the clinical interview that they were using herbal medicines. the justifications for not reporting their use to the health professional were, in a descending order: the patient did not think it was important (6 participants); the professional did not ask (4 participants); the plant is not harmful to health (2 participants); tea is simple to take and it does not interfere with treatment (1 participant); some doctors do not like the patient to use medicinal plants (1 participant). three participants did not justify the reason for not reporting the use of medicinal plants to health professionals. among all participants of the research, 94% wanted more information on the therapies with herbal medicines for treating health problems. discussion in the present study it was found that the majority of users of herbal medicines for oral problems were women between the ages of 31 and 64 years, with fewer than 8 years of schooling, and family income less of than two minimum wages. however, gender, educational level and household income were not signiûcantly associated with the use of medical plants for these problems. this is similar to ûndings by tam, et al.14 studying patients treated at dental clinics and dental practices in the u.k., and the study of elder et al.15, which found no significant differences in the use of cam therapies associated with the above-mentioned variables. on the other hand, hasan, et al.16 found signiûcant associations between phytotherapy use for oral problems and age, educational level and income in a study on cam users. in comparison with descriptive data in another brazilian study, santos et al.17, observed that the prevalence results were similar: most of the sample were women with family income of fewer than two minimum wages. one of the most important findings of this study was the strong association (p<0.001) between use of herbal medicines for other general health problems and use for oral problems. as affirmed by hasan, et al.16, the popularity of unconventional therapies indicated an increase in patients’ preference for a holistic approach to health care. the research results showed that 71% of participants reported using herbal medicines for general health problems, proving the high popularity of this type of therapy. this fact makes it important for health professionals to address the use of phytotherapy and unconventional therapies by their patients, by explaining the benefits and limitations of this type of treatment to them, and more importantly to establish whether there are any potential drug interactions or adverse drug reactions. hasan el al.16 also proposed that conventional health practitioners should acquire and develop essential skills in the area of cam practice and its education should be incorporated into the medical and health science curriculum. the proportion of participants that related the use of phytotherapy for oral diseases was 37%, but it was difficult to make a comparison with other results because there are few similar studies in the literature. in studies evaluating phytotherapy users, the authors found that 53%9, 55%12, 26%5, 50%10 of the studied samples used herbal medicines for general health problems. only the study of santos, et al.17 presents this data, stating that 80% of participants used herbs for dental problems. a significant number of patients who reported using herbal medicines for oral health problems omitted this information from their physicians, pharmacists or dentists (45.9%). similar to the data found by hasan, et al.16 , clay, et al.18 and santos, et al.17, as these authors verified that 54.6%, 20.6% and 77,5%, respectively, of the patients did not inform their health care professionals about cam use. in the study 448448448448448 braz j oral sci. 11(4):445-450 factors associated with the use of herbal medicines for oral problems by patients attending the clinics of the school of dentistry, federal university of juiz de fora, brazil of druss, et al.5 with patients who used cam, 91.2% of patients reported that they did not inform the physician of their use of unconventional therapies. when relating these findings to those of the study of viegas jr.19, one can assume that the reason why many patients do not report the use of herbal health professionals may be due to the culture of selfmedication, very common among brazilians. the relevant number of patients who affirmed that they did not inform health professionals of their use of medicinal plants demonstrates their insecurity in discussing their other options of health care treatments in addition to those of traditional medicine. this information confirms the professionals’ need to be aware of the profile of the population served and to include questions related to this topic in the patient’s medical history in order to attribute the appropriate value to and popularity of the use of herbal medications. according to the literature, elder et al.15 confirmed that it is important for physicians to know about their patients’ use of alternative medicine. eisenberg et al.4, affirmed that it is a very common belief worldwide that ‘herbal products’ are safe. spector et al.20, little21, clay et al.18, tam et al.14 and santos et al.17 warned that dentists must be better informed of the adverse effects and contraindications of cam, based on scientific evidences. in a study conducted in a health center, genovés et al.22 found that one in every five patients being treated with medicines was also taking herbal medicines by self-medication, confirming that the health authorities and physicians should know and advise the population about the possible risks to health and the contraindications of these products. among all participants, 94% wanted to be better informed about these therapies with herbal medicines for health problems, suggesting the need for more scientific evidence to be developed and transmitted to the population about the use of cam. little21 affirmed that clinical trials demonstrated that certain cam treatments are effective and safe and they can be incorporated into conventional medicine and dentistry. based on their use and popular knowledge, the important growth of phytotherapy within preventive and curative programs has stimulated the evaluation of the action of different plant extracts23. studies should be conducted and information disseminated to the population. in the present study, 79% of participants used medicinal herbs by family tradition, ignoring scientific data confirming their safety. begnami and mialhe 24 found that even with the advancement of pharmaceutical discoveries, the tradition of using medicinal plants was not abandoned. therefore, this research presents the people’s interest in this type of therapy and public health policies, such as the policy for complementary and integrative practices within the ministry of health in brazil6, are valid as regards encouraging the use of herbal medicines. for palombo25, given the incidence of oral disease, increased resistance of bacteria to antibiotics, adverse affects of some antibacterial agents at present used in dentistry and financial considerations in developing countries, there is a need for alternative prevention and treatment options that are safe, effective and economical. the search for alternative products continues and natural phytochemicals isolated from plants used as traditional medicines are considered good alternatives1,25. however, the data of this research should be interpreted within the context of some limitations. the study was exploratory; the sample does not represent the entire population, since a convenience sample was used instead of identifying patients by a randomized sampling. this study did not include measures of quality of life or questions about patient satisfaction with the use of herbal medication exclusively in order to determine how the treatment with phytotherapy interfered with patients’ quality of life. from the results of this investigation, it would appear that phytotherapy is a matter of great importance to the population, and is of interest to the different areas of health. since the first step taken by the who in 1978, there have been a growing number of studies on medicinal plants throughout the world. the present study characterized the profile of the patients in the sample using and enjoying herbal remedies for oral problems, allowing new questions to be raised for other researches in this area, collaborating with the planning of public health services in dentistry. the present study verified that a large number of patients use phytotherapy for the treatment of general health problems, and among them, 37% use it for the treatment of oral health problems. previous history of use for general problems and the patient’s gender were variables associated with use of herbal medicines for oral problems. the development of further studies is necessary to promote scientific evidence of the efficacy and safety of products already enshrined in popular knowledge in order to enable them to become accessible alternatives in public health services for the prevention and treatment of oral health problems. however, dentists need to be aware of their patients’ use of herbal remedies, know their potential risks, side effects and possible drug interactions, in order to provide the population with the best care. references 1. groppo fc, bergamaschi cc, kogo k, franz-montan m, motta rhl, andrade ed. use of phytotherapy in dentistry. phytother res. 2008; 22: 993-8. 2. rosa c, câmara sg, béria ju. representations and use intention of phytotherapy in primary health care. cienc saude col. 2011; 16: 311-8. 3. ministry of health (brasil). decreto n.º 5.813, de 22 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mode of use by the population. braz j pharmacogn; 2008; 18: 308-13. 20. spector ml, fischer m, dawson dv, holmes dc, kummet c, nisly nl et al. complementary and alternative medicine usage by patients of a dental school clinic. spec care dentist. 2012; 32: 177-83. 21. little jw. complementary and alternative medicine: impact on dentistry. oral surg oral med oral pathol oral radiol endod. 2001; 98: 137-45. 22. genovés js. larrea vp, gomis er, martínez-mir i. consumo de hierbas medicinales y medicamentos. atenc prim. 2001; 28: 311-4. 23. santos rl, guimarães gp, nobre msc, portela as. analysis about phytotherapy as an integrating practice in the brazilian unified health system (uhs). rev bras plantas med. 2011; 13: 486-91. 24. begnami af, mialhe fl. survey of medicinal plants and their application in the municipal district of piracicaba-sp. rev uning. 2008; 18: 49-62. 25. palombo ea. traditional medicinal plant extracts and natural products with activity against oral bacteria: potential application in the prevention and treatment of oral diseases. evid based complement alternat med. 2009; 10: 1-15. 450450450450450 braz j oral sci. 11(4):445-450 factors associated with the use of herbal medicines for oral problems by patients attending the clinics of the school of dentistry, federal university of juiz de fora, brazil oral sciences n3 original article braz j oral sci. april | june 2015 volume 14, number 2 expression analysis of notch signaling pathway molecules in shed cultured in keratinocyte growth medium siti aisyah mohd taha1, joanne koh su ling1, nur izyan binti azmi1, thirumulu ponnuraj kannan1,2, ahmad azlina1, khairani idah mokhtar3 1universiti sains malaysia, school of dental sciences, kota bharu, kelantan, malaysia 2universiti sains malaysia, school of medical sciences, human genome centre, kota bharu, kelantan, malaysia 3international islamic universiti malaysia, kulliyah of dentistry, kuantan, pahang, malaysia correspondence to: thirumulu ponnuraj kannan school of dental sciences, universiti sains malaysia health campus 16150 kota bharu, kelantan, malaysia phone: +609 767 5847 fax: +609 767 5505 e-mail: kannan@usm.my abstract aim: to detect the expression of molecules associated with notch signaling pathway in stem cells from human exfoliated deciduous teeth (shed) cultured in specific differentiation medium, namely, keratinocyte growth medium (kgm). methods: rna was extracted from shed harvested on day 1, 3 and 7. rna was reverse-transcribed to obtain the cdna and then proceeded with pcr using specific primers for the notch signaling pathway molecules (notch1, jagged-1, jagged-2 and, hes1) as well as stem cell marker (nanog). pcr products were electrophoresed on a 2% agarose gel and stained with sybr green. results: notch-1 was highly expressed in shed cultured in kgm and showed increase in density as the days progressed, while jagged-1 showed a decrease. jagged-2 on the other hand, showed a slight increase on day 3 followed by a decrease on day 7. however, hes-1 was not expressed in shed cultured in kgm. nanog showed expression only on day 3 and gradually increased in expression on day 7. conclusions: notch signaling pathway associated molecules; notch-1, jagged-1, jagged-2, and stem cell marker nanog are expressed in shed cultured in kgm which may be involved in the differentiation into epithelial-like cells in human dental pulp tissues. keywords: receptors, notch; gene expression; stem cells; tooth, deciduous; culture media. introduction stem cells from human exfoliated deciduous teeth (shed) are multipotent stem cells derived from the pulp tissues of extracted deciduous teeth1. shed has the ability to be differentiated to specific cell lineages such as odontoblasts and osteoblasts as well as epithelial like cells. shed was able to differentiate into epithelial like cells when cultured in keratinocyte growth medium (kgm)2. since the notch signaling pathway molecules play an important role in differentiation of epithelial cells, it is important to identify the presence of notch signaling molecules in shed during the process of cell differentiation. the notch signaling pathway provides important intercellular signaling mechanisms essential for cell fate specification and it regulates differentiation and proliferation of stem or progenitor cells by para-inducing effects3-4. the core components of a notch signaling pathway involves three different molecules; the dsl-type ligand, a notch-type receptor and a transcription factor of the csl family braz j oral sci. 14(2)135-140 received for publication: april 09, 2015 accepted: june 09, 2015 http://dx.doi.org/10.1590/1677-3225v14n2a08 136136136136136 (cbf1; c promoter binding factor/ suppressor of hairless/ lag1). notch signaling pathway is also involved in the regulation of epithelial cell differentiation in various tissues5-6. the aim of this study was to detect the expression of molecules associated with notch signallng pathway in shed cultured in specific differentiation medium, namely, kgm. knowledge on the expression analysis of notch signaling pathway molecules in shed cultured in kgm could highlight its involvement in controlling the biological activity of these stem cells, particularly during odontogenesis and other developmental process. material and methods shed culture stem cells from human exfoliated deciduous teeth (shed) (allcells, alameda, ca, usa) were employed in the current study. shed was cultured and maintained in t25 cm2 culture flask using minimum medium alpha (α-mem) from gibco, usa. the medium was changed 3 days after culture and were sub-cultured once they reached 70% confluence. at each passage, the cells were counted, photographed using an inverted phase-contrast microscope (carl zeiss microscopy, llc, thornwood, ny, usa) and cyro-preserved in cyrovial tube for further culture and later for rna analysis. the cryopreserved shed was subcultured for 2 passages in α-mem. after washing, 1x10ˆ6 cells were seeded and cultured in kgm (lonza group ltd., basel, switzerland). total rna was extracted from the cells cultured in kgm after 1, 3 and 7 days. rna extraction the total rna was extracted from the cells using rna extraction kit, rneasy mini kit (qiagen inc., valencia, ca usa) following the protocol provided by the manufacturer. cdna synthesis reverse-transcriptase enzyme was used to convert extracted rna into cdna using reverse transcriptase cdna gene β-actin7 notch-18 jagged-19 jagged-28 hes-110 nanog11 primer sequences f: 5’-tggcaccacaccttctacaatgagc-3’ r: 5’-gcacagcttctccttaatgtcacgc-3’ f: 5’-ccgcctttgtgcttctgtt-3’ r: 5’-tcctcctcttcctcgctgtt-3’ f: 5’-gatcctgtccatgcagaacg-3’ r: 5’-ggatctgatactcaaagtgg-3’ f: 5’-ttccagtgcgatgcctaca-3’ r: 5’-gtgctcggtggctcttct-3’ f: 5’-gacagcatctgagcacagaaatg-3’ r: 5’-gtcatggcattgatctgggtcat-3’ f: 5’-cccaaaggcaaacaacccacttct-3’ r: 5’-aactgtgttctcttccacccagct-3’ size of pcr product (bp) 437 490 436 731 374 107 table 1.table 1.table 1.table 1.table 1. primer sequences of the different genes with their respective product sizes. synthesis kit (mmlv rt 1st-strand cdna synthesis kit, k o r e a ) f o l l o w i n g t h e p r o t o c o l p r o v i d e d b y t h e manufacturer. 8.23 microlit (200 ng) of total rna was used for cdna synthesis using oligo(dt)21 primer. the synthesized cdna was stored at -20 oc and was used as a template for pcr technique using specific primer pairs. the quality of cdna was observed by running the sample on a 2% agarose gel. polymerase chain reaction (pcr) the synthesized cdna was used for pcr amplification using specific pair of primers for the respective genes, notch1, jagged-1, jagged-2 ligands and hes-1 transcription factor genes and nanog, stem cell marker gene. the sequence of the primers with their references and respective product sizes are presented in table 1. the house-keeping gene used in this study was β-actin. the pcr master mix was made in a total volume of 50 µl of reaction mixture containing 1 µl (200 ng) of cdna template, 1 µl (10 µm) of each forward and reverse primers, 25 µl of pcr ready-to-use mixture (mytaq hs mix 2x, bioline, london, uk) and 22 µl of distilled water. the pcr conditions used were similar for all genes but with different annealing temperatures using pcr machine (c1000 thermal cycler, biorad, hercules, ca, usa) as shown in table 2. two µl of the pcr products were electrophoresed on 2% agarose gel in lb buffer at 100v (power pac hc, biorad) and visualized under uv after sybr green staining. the gel was photographed under uv light using digital image analyzer (gel doc xr, biorad). appropriate product size of the specific genes analyzed was indicated by the production of a discrete single band on the 2% agarose gel. the experiments were run in triplicates and the average density value (adv) of the pcr products for each gene was calculated. statistical analysis the data obtained were analyzed using the kruskalwallis rank test. expression analysis of notch signaling pathway molecules in shed cultured in keratinocyte growth medium braz j oral sci. 14(2)135-140 137137137137137 gene β-actin notch-1 jagged-1 jagged-2 hes-1 nanog initial denaturing (oc) for 5 min 94 94 94 94 94 94 denaturing (oc) for 30 s 94 94 94 94 94 94 annealing temperature (oc) for 30 s 59.0 58.3 58 58.3 59 60.6 elongation (oc) for 1 min 72 72 72 72 72 72 final elongation (oc) for 7 min 72 72 72 72 72 72 table 2. table 2. table 2. table 2. table 2. pcr conditions for the different genes. the cycle was repeated 35 times. results the house keeping gene β-actin expressed with a band size of 437 bp in all the samples. the product size for βactin of shed cultured in kgm for day 1, 3 and 7 are shown in figure 1a and the adv is presented in figure 2. the samples analyzed for the expression of notch-1 receptor in this study cultured in kgm showed expression of notch-1, which increased in expression as the days progressed from day 1 to, 3 and 7. this is shown in figure 1b with the adv in figure 2. the samples were tested for the expression of jagged-1. all the samples showed expression of jagged-1, which decreased with increase in days. the expression for jagged1 of shed cultured in kgm for day 1, 3 and 7 is shown in figure 1c and the adv is presented in figure 2. fig. 1. gel electrophoresis of pcr products. lane m = 100 bp dna ladder; lane 1 = negative control; lanes 2, 3 and 4 = day 1, 3 and 7; a: β-actin gene b: notch1 gene c: jagged-1 gene d: jagged-2 gene e: hes-1 gene f: nanog gene the samples tested for the expression of jagged-2 in this study showed expression of jagged-2 on all the days but showed a different pattern of expression. the expression increased from day 1 to day 3 and then followed by a decrease on day 7. the expression of jagged-2 of shed cultured in kgm for day 1, 3 and 7 are shown in figure 1d and the adv is presented in figure 2. the samples were also analyzed for the expression of hes-1 transcription factor. in this study, all samples did not show any expression of hes-1 in shed cultured in kgm for day 1, 3 and 7 as shown in figure 1e. the expression analysis for stem cell marker, nanog was also performed. nanog was not detected on day 1 but was expressed at day 3 and 7 as shown in figure 1f. the average density value (adv) for all analyzed pcr products is presented in figure 2. expression analysis of notch signaling pathway molecules in shed cultured in keratinocyte growth medium braz j oral sci. 14(2)135-140 138138138138138 fig. 2. the average density value (adv) of the different genes expressed in shed cultured in kgm discussion to evolve specific developmental programs, notch signals control cells that respond to intrinsic or extrinsic developmental cues. development of morphology of organs and their evolution are affected by differentiation, proliferation, and apoptotic programs via the notch activity4. the dsl-type ligand, a notch-type receptor and a transcription factor of the csl family are the three different molecules which are the main components of notch signaling pathway3,12,. when the ligands on neighboring cells bind to notch receptor on one cell, it will generate an active notch intracellular fragment (nic) or notch intracellular domain (nicd). in order to trigger the transcription of notch target genes, the active nicd will be released into the cytoplasm before it translocates into the nucleus12. the main functions of notch signaling are differentiation of odontoblasts and osteoblasts, cusp pattern formation, tooth roots generation and calcification of tooth hard tissue. the notch signaling can also be triggered in dental stem cells of the pulp to differentiate it into odontoblast, and forming fresh dentin tissue after tooth eruption3. additionally, notch signaling has been shown to be required for epithelial stem cell survival and amelogenesis13. shed is a mesenchymal stem cell derived from the pulp tissues of extracted deciduous teeth and is found to be a population of highly proliferative, clonogenic cells capable of differentiating into a variety of cell types including neural cells, adipocytes, and odontoblasts1. as it is deemed that the notch signaling pathway molecules have a role to play in the differentiation of epithelial cells, it would be appropriate to investigate the notch signaling molecules in shed during the process of cell differentiation. the notch receptor family is mainly expressed in tooth germ during the early development stage, throughout the dental epithelium and during the differentiation stage in the stratum intermedium, gradually extending to the pulp mesenchyme14. in addition to mediating tissue-tissue interactions, growth factor signaling also participates in mediating cellcell interactions within epithelial and mesenchymal tissues during organogenesis15. this short-range signaling between cells is usually accomplished by one cell possessing a transmembrane receptor and the neighboring cell possessing a membrane-bound ligand. notch signaling is one such system implicated in tooth morphogenesis14-15. notch-1 gene encodes a member of the notch family. members of this type 1 transmembrane protein family share structural characteristics including an extracellular domain consisting of multiple epidermal growth factor-like (egf) repeats, and an intracellular domain consisting of multiple, different domain types16. in this study, notch-1 gene was detected in shed cultured in kgm, with marked increase from day 1 to day 3 and day 7 but it was not statistically significant (p>0.05). this shows that notch-1 is being expressed, suggesting the presence of notch signaling pathway receptor. this is parallel to the study shown by mitsiadis and his co-workers17 where the expression of notch-1, -2, and -3 was detected in the dental epithelium from the initiation stage to the later differentiation stage. jagged transmembrane proteins are ligands to the notch receptors. intimate cell-cell contacts aid in binding of the membrane-bound ligands to the notch receptor and triggers the release of the cytoplasmic domain of notch that functions as a transcription factor in the nucleus. the expression of several notch ligands, one of them jagged-1, has been detected in developing teeth18. this study showed presence expression analysis of notch signaling pathway molecules in shed cultured in keratinocyte growth medium braz j oral sci. 14(2)135-140 139139139139139 of jagged-1 gene in the shed cultured in kgm. this showed that there is a membrane-bound ligand to notch receptors, indicating the function played by notch signaling pathway during the differentiation process. according to zhang et al. the upregulation of jagged-1 indicated that shed might differentiate into epithelial-like cells and inhibit to differentiate into odontoblast-like cells19. in the current study, though the expression of jagged-1 showed a declining trend, yet, it was not statistically significant (p>0.05). jagged-2 is another ligand to notch receptors. jagged-2 gene provides instructions for making a protein, which is part of jak/stat pathway that promotes the growth and proliferation of cells. this pathway transmits chemical signals from extracellular to the cell’s nucleus20. in our study, it was found that jagged-2 was expressed at day 1, increased at day 3 but eventually, the level of expression decreased by day 7, where, day 7 had the lowest level of expression of this gene. though there was fluctuation in the expression of jagged-2, yet, there was no statistical significance as the p>0.05. it was down regulated as compared to β-actin. this showed that jagged-2 is also involved in the differentiation process of shed into epithelial-like cells through notch signaling pathway. interestingly, the current study also showed that both ligands; jagged-1 and jagged-2 were down regulated at day 7 while notch-1 expression was up regulated at this point. this may suggest that as the process of differentiation continues, the expression of ligands start to subside. in our study, the hes-1 gene showed no expression at all throughout the process. hes-1 is activated transcriptionally by the notch signaling pathway during notch-mediated lateral inhibition 21. hes-1 is one of the downstream transcription factors involved in notch signaling pathway and has been shown to be important in regulating the maintenance of the progenitor or stem cells. there are seven members in the hes family (hes1-7), thus suggesting that other members of hes family might be involved in this process of controlling the stem cell differentiation22-23. though hes-1 is a downstream target of notch signaling, may be it is not expressed in shed cultured in kgm. nanog is the protein coding gene that acts as a transcription regulator involving in inner cell mass and embryonic stem (es) cell proliferation and self-renewal10-24. in our study, nanog started showing the expression at day 3 and subsequently increased in density at day 7, which shows the stemness of this shed. however, there was no statistical significance seen (p>0.05). this study showed that the notch signaling pathway associated molecules, notch-1, jagged-1, jagged-2, and stem cell marker nanog are expressed in shed cultured in kgm. this indicates that these molecules may be involved in the differentiation into epithelial-like cells in human dental pulp tissues. acknowledgements the authors would like to acknowledge the staff of craniofacial science laboratory, school of dental sciences, universiti sains malaysia for their technical support. this study was supported by universiti sains malaysia research university grant (1001/ppsg/813077). references 1. miura m, gronthos s, zhao m, lu b, fisher lw, robey pg, et al. shed: stem cells from human exfoliated deciduous teeth. proc natl acad sci usa. 2003; 100: 5807-12. 2. nam h, lee g. identification of novel epithelial stem cell-like in human deciduous dental pulp. biochem biophys res commun. 2009; 386: 135-9. 3. cai x, gong p, huang y, lin y. notch signalling pathway in tooth development in adult dental cells. cell prolif. 2011; 44: 495-507. 4. schwanbeck r, martini s, bernoth k, just u. the notch signaling pathway: molecular basis of cell context dependency. eur j cell biol. 2011; 90: 572-81. 5. leong kg, karsan a. recent insights into the role of notch signalling in tumorigenesis. blood. 2006; 107: 2223-33. 6. vandussen kl, carulli aj, keeley tm, patel sr, puthoff bj, magness st, et al. notch signaling modulates proliferation and differentiation of intestinal crypt base columnar stem cells. development. 2012; 139: 488-97. 7. karaoz e, dogan bn, aksoy a, gacar g, akyuz s, ayhan s, et al. isolation and in vitro characterisation of dental pulp stem cells from natal teeth. histochem cell biol. 2010; 133: 195-12. 8. zhang xp, zheng g, zou l, liu hl, lou lh, zhou p, et al. notch activation promotes cell proliferation and the formation of neural stem cell-like colonies in human glioma cells. mol cell biochem. 2008; 307: 101-8. 9. tachikawa y, matsushima t, abe y, sakano s, yamamoto m, nishimura j, et al. pivotal role of notch signalling in regulation of erythroid maturation and proliferation. eur j haematol. 2006; 77: 273-81. 10. chambers i, colby d, robertson m, nichols j, lee s, tweedie s, et al. functional expression cloning of nanog, a pluripotency sustaining factor in embryonic stem cells. cell. 2003; 113: 643-55. 11. park sw, lim hy, do hj, sung b, huh sh, uhm sj, et al. regulation of human growth and differentiation factor 3 genes expression by nanog in human embryonic carcinoma nccit cells. febs letter. 2012; 586: 3529-35. 12. radtke f, fasnacht n, macdonald hr. notch signaling in the immune system. immunity. 2010; 32: 14-27. 13. felszeghy s, suomalainen m, thesleff i. notch signalling is required for the survival of epithelial stem cells in the continuously growing mouse incisor. differentiation. 2010; 80: 241-8 14. mitsiadis ta, graf d, luder h, gridley t, bluteau g. bmps and fgfs target notch signalling via jagged 2 to regulate tooth morphogenesis and cytodifferentiation. development. 2010; 137: 3025-35. 15. thesleff i, mikkola m. the role of growth factors in tooth development. int rev cytol. 2002; 217: 93-135. 16. notch1 [homo sapiens (human)] [internet]. hugo gene nomenclature committee – hgnc. bethesda: national center for biotechnology information, u.s. national library of medicine [cited 2014 oct 15]. available from: http://www.ncbi.nlm.nih.gov/gene/4851. 17. mitsiadis ta, feki a, papaccio g, catón j. dental pulp stem cells, niches, and notch signaling in tooth injury. adv dent res. 2011; 23: 275-9. 18. mitsiadis t, henrique d, thesleff i, lendahl u. mouse serrate-1 (jagged-1) expression in the developing tooth is regulated by epithelial-mesenchymal interactions and fibroblast growth factor-4. development. 1997; 124: 1473-83. 19. zhang c, chang j, sonoyama w, shi s, wang cy. inhibition of human dental pulp stem cell differentiation by notch signaling. j dent res. 2008; 87: 250-5. 20. genetics home reference. your guide to understanding genetic conditions [internet]. bethesda: national center for biotechnology information, u.s. national library of medicine [cited 2014 oct 15]. available from: http://ghr.nlm.nih.gov/gene/jak2. expression analysis of notch signaling pathway molecules in shed cultured in keratinocyte growth medium braz j oral sci. 14(2)135-140 21. jarriault s, brou c, logeat f, schroeter eh, kopan r, israel a. signalling downstream of activated mammalian notch. nature. 1995; 377: 355-8. 22. kageyama r, ohtsuka t, kobayashi t. the hes gene family: repressors and oscillators that orchestrate embryogenesis. development. 2007; 134: 1243-51. 23. kobayashi t, kageyama r. hes1 regulates embryonic stem cell differentiation by suppressing notch signaling. genes cells. 2010; 15: 689-98. 24. mitsui k, tokuzawa y, itoh h, segawa k, murakami m, takahashi k, et al. the homeoprotein nanog is required for maintenance of pluripotency in mouse epiblast and es cells. cell. 2003; 113: 631-42. 140140140140140expression analysis of notch signaling pathway molecules in shed cultured in keratinocyte growth medium braz j oral sci. 14(2)135-140 1http://dx.doi.org/10.20396/bjos.v18i0.8657268 volume 18 2019 e191649 original article 1 department of oral diagnosis, division of oral radiology, piracicaba dental school, university of campinas (unicamp), piracicaba, são paulo, brazil. 2 pediatric dentistry department, piracicaba dental school, university of campinas (unicamp), av. limeira, 901, 13414-903, piracicaba, são paulo, brazil. corresponding author: deborah queiroz freitas university of campinas. piracicaba dental school, department of oral diagnosis. av. limeira, 901, zip code 13414-903, piracicaba, são paulo, brazil. phone/fax: +55 19 2106-5327. e-mail: deborahq@unicamp.br https://orcid.org/0000-0002-1425-5966 received: may 03, 2019 accepted: august 30 2019 efficacy of digital radiographic systems in the quality assessment of intracanal materials used for primary teeth guilherme fantini ferreira1, larissa pereira lagos de melo1, mariana rocha nadaes1, fernanda maria mazoni reis2, fernanda miori pascon2, deborah queiroz freitas1,* aim: to evaluate the performance of three digital radiographic systems in the analysis of root canal filling quality using different intracanal materials for primary teeth. methods: twenty-five bovine teeth were divided into 5 groups: calen® combined with iodoform; calen® combined with zinc oxide; zinc oxide and eugenol; ultracal®xs, and 2% chlorhexidine combined with ca(oh)2 + zinc oxide. periapical radiographs were obtained with the vistascan, express, and snapshot systems. the quality of the images was evaluated objectively (radiopacity) and subjectively (apical sealing and filling homogeneity). as the reference standard, the teeth were scanned with a micro-ct device. results: radiopacity differed among the radiographic systems and materials tested. in general, the greatest difference was observed between the express and vistascan systems; calen® combined with iodoform resulted in the highest radiopacity. the radiographic systems did not differ in terms of homogeneity. however, calen® combined with iodoform differed from the other materials and exhibited the best results. regarding apical sealing, the snapshot system and calen® combined with zinc oxide provided the best results. conclusion: direct digital systems show better performance in evaluating the quality of endodontic treatment in primary teeth and should be preferred for this purpose. keywords: radiography, dental, digital. tooth, deciduous. root canal filling materials. https://orcid.org/0000-0002-1425-5966 2 ferreira et al. introduction conservative management is currently the focus of dentistry, notably in pediatric dentistry, which consists of preserving the primary dentition in the dental arch until the period of physiological exfoliation1. endodontic treatment can prevent the extraction of those teeth if it is correctly indicated. periapical radiography is first indicated to evaluate the quality of these treatments, as well as the periapical status of endodontically treated teeth, since it enables visualization of the tooth and its relationships with adjacent tissues2. nowadays, digital radiographic systems using either direct (solid complementary metal-oxide-semiconductor sensors cmos) or semidirect acquisition methods (photostimulable phosphor plates psp)3 are applied in clinical practice. these systems mainly differ in their acquisition process and spatial and contrast resolution. the influence of different characteristics of digital receptors has been studied for some diagnostic tasks, such the evaluation of caries lesions4,5, periapical lesions6,7, root fractures8,9, and fractured endodontic instruments10. however, we found no studies investigating the effect of these systems on the quality assessment of root canal filling. it is therefore important to establish whether different radiographic systems show diverse performance in evaluating endodontic treatment. this would allow dental professionals to use more adequate systems for this type of procedure, resulting in an accurate and reliable diagnosis, treatment and follow-up. an adequate analysis of radiographic images is necessary for diagnostic purposes and to ensure the success of endodontic treatment in primary teeth and of their follow-up. adequate radiographic images are important for assessing endodontic treatment quality. since periapical radiography is the first choice for this purpose and considering the wide range of digital systems available for clinical use and the lack of studies about the topic, our aim was to investigate the influence of three digital radiographic systems using cmos and psp technologies on the evaluation of root canal filling quality when different materials designed for the treatment of primary teeth were used. the null hypotheses stated that there would be no differences among the different digital systems or materials. material and methods selection and preparation of the samples twenty-five bovine primary teeth were selected. their crowns were removed and a 22-mm length of the root was kept in order to standardize the size of the roots. the length was determined with the aid of an endodontic ruler and confirmed with a digital caliper. the roots were randomly divided into 5 different groups according to the filling material: • calen® (s.s. white, batch #0080713) combined with iodoform (biodinâmica, batch #59813) (ca + io); • calen® combined with zinc oxide (biodinâmica, batch #38513) (ca + zno); • zinc oxide and eugenol (biodinâmica, batch #48013) (zoe); • ultracal®xs (ultradent, batch #b7w8p) (ultracal); 3 ferreira et al. • chlorhexidine digluconate 2% (essencial pharma, batch #1009364) combined with calcium hydroxide [ca(oh)2] (biodinâmica, batch #114113) + zno (chlorhexidine). the chemical-mechanical preparation was carried out by the same operator using the step-back technique with 1st series files k (#15 to #40) (maillefer, tulsa, ok, usa) and 0.5% sodium hypochlorite (naocl) (biodinâmica, ibiporã, pr, brazil) + endo-ptc (urea peroxide, polysorbate 80 and polyethylene glycol) (biodinâmica, ibiporã, pr, brazil) after every file size exchange. the combination of naocl and endo-ptc is frequently used in the endodontic treatment of primary teeth. the peroxides react chemically, releasing large amounts of nascent oxygen that explains their bactericidal activity11,12. circular movements were performed and 2.5 ml of 0.5% naocl was used for irrigation. the opening of the apical foramen was standardized with a # 40 k-file so the canal had a cylindrical shape. finally, the canals were irrigated with 5 ml of 0.89% saline solution and dried with an absorbent paper cone. the filling materials were then introduced into the root canals with a # 20 endodontic file (maillefer, tulsa, ok, usa), except for the ultracal®xs group, in which a syringe provided by the manufacturer was used. vertical condensation technique was employed by vertical compression was carried out with cotton balls and the root canal was sealed in the cervical region with a resin composite restoration (z250 xt, 3m espe, saint paul, mn, usa). radiographic images after root canal filling, digital radiographs (figure 1) were obtained with the following devices: • phosphor plate, 2 sizes, of the express digital system (instrumentarium imaging, tuusula, finland), scanned immediately after acquisition, 17 lp mm-1 theoretical spatial resolution according to the manufacturer, and cliniview software (instrumentarium imaging); • phosphor plate, 2 sizes, of the vistascan digital system (dürr dental, bietigheim-bissingen, germany), scanned immediately after acquisition, 25.6 lp mm-1 theoretical spatial resolution according to the manufacturer, and dbswin software (dürr dental); ca+io ca+zno zoe ultracal chlorexhidine express vistascan snapshot figure 1. cropped periapical radiographs of teeth with different intracanal materials obtained with the radiographic systems studied. 4 ferreira et al. • solid cmos sensor, 1 size, of the snapshot digital system (instrumentarium imaging, tuusula, finland), 26.3 lp mm-1 theoretical spatial resolution according to the manufacturer, and cliniview software (instrumentarium imaging). the radiographic exposures were obtained using a focus x-ray device (instrumentarium dental, tuusula, finland) operating at 70 kv and 7 ma, with an exposure time of 0.1 s for the vistascan and express systems and of 0.063 s for the snapshot system. the exposure times were established in a pilot study. for image acquisition, each tooth was placed parallel to the radiographic receptor and the radiation was set perpendicular to both, with a focus-receiver distance of 30 cm. in addition, an aluminum step wedge with uniform incremental steps from 1 to 10 mm was placed on the receptor adjacent to the tooth. evaluation of the radiographic images the images were evaluated objectively by determining the mean gray values of the intracanal materials and aluminum wedge, as well as subjectively through the scores assigned by the examiners to the filling quality. in both analyses, the examiners were blinded to the digital system and material used. the examiners were previously calibrated and performed the evaluation on a 24.1’’ lcd monitor with a resolution of 1920x1200 pixels (mdcr-2124, barco, kortrijk, belgium) in a dimly lit and quiet environment. objective analysis was carried out by an experienced examiner. for this purpose, the images were exported in tiff format for analysis with the imagej 1.49 software (national institutes of health, bethesda, maryland, usa). regions of interest (roi) of 0.4 x 0.4 mm were determined in coronal, middle and apical root thirds of the filling material, as well as in the second, third and fourth step of the aluminum wedge (figure 2). after determination of the rois, the gray levels were obtained. in the imagej program, value 0 assigns the color black and value 255 the color white. the final radiopacity of each material was calculated by averaging the values of the three thirds. additionally, the values obtained between the third and second steps and between the fourth and third steps were subtracted and the mean was taken for expressing the average contrast that each radiographic system exhibited for the same aluminum wedge. figure 2. regions of interest (roi) in the coronal, middle and apical thirds of roots filled with the intracanal material, and in the second, third and fourth step of the aluminum wedge. 5 ferreira et al. for subjective evaluation, two oral and maxillofacial radiologists with over 5 years of experience in the evaluation of digital radiographic images assessed the images together and reached a consensus. the examiners were allowed to adjust brightness and contrast and to use the zoom tool freely. for each image, the examiners classified the homogeneity of the filling on a two-point scale: 1 = adequate, or 2 = inadequate. score 1 was attributed if the root canal filling showed no empty spaces, no failure in internal adaptation, and no bubbles. if these occurred, score 2 was attributed. the scores were recorded separately for each third of the root. regarding apical sealing, score 1 (adequate) was attributed when the root canal was sealed and the material was 0 to 2 mm from the radiographic apex. if the apical limit of the filling was more than 2 mm from the radiographic apex and/or the material did not seal the canal, score 2 (inadequate) was attributed (figure 3). after 30 days, the subjective evaluation was repeated in 40% of the sample to obtain the intra-examiner agreement. reference standard as reference standard for subjective analysis, the teeth were scanned with a micro-computed tomography (ct) device (skyscan 1178; bruker, kontich, belgium). scanning was performed at a voltage of 65 kv, current of 615 µa, and voxel size of 8.46 µm. the images were reconstructed with the nrecon software (v1.6.4.8, bruker, kontich, belgium), and the dataviewer software (v1.5.1.2, bruker, kontich, belgium) was used for the analysis. in this analysis, one observer determined if the endodontic filling material was homogeneously distributed inside the root canal or if it contained bubbles, as well as if the apex was sealed. data analysis the spss 22.0 program (spss, inc., chicago, illinois, usa) was used for statistical analysis, adopting a significance level of 5%. normality of the data was tested using the shapiro-wilk test. a b figure 3. examples of (a) inadequate score (2% chlorhexidine digluconate combined with ca(oh)2 + zno) and (b) adequate score (calen® combined with zinc oxide) for the two parameters evaluated (sealing and homogeneity). 6 ferreira et al. the gray values of the material’s radiopacity and those corresponding to the contrast of the aluminum wedge obtained in the objective analysis were compared by analysis of variance (two-way anova: digital systems and filling materials). the tukey test was used for post hoc analysis. the null hypothesis considered the absence of significant differences among the different digital systems or materials. the scores attributed in the subjective evaluation were compared using the friedman test. the null hypothesis considered the absence of influence of the different digital systems, different materials or different thirds of the root on the evaluation. additionally, the kappa test was used to assess intra-examiner agreement. results the mean radiopacity values of the materials are presented in table 1. the systems differed from each other (p<0.005) and the greatest difference was observed between the express and vistascan systems. a difference was also observed between some materials being ca + io different from the other materials, with ca + io exhibiting the highest radiopacity. table 2 shows the mean contrast of each radiographic system for the aluminum wedge. all systems differed from each other (p<0.005) and the express system exhibited the highest contrast. table 1. mean and standard deviation radioapacity values of the filling materials in the radiographic systems studied material express vistascan snapshot ca+io 254.05 (2.13) aa 207.94 (5.35) ba 241.25 (10.71) aa ca+zno 207.17 (10.32) abc 146.20 (6 .44) bb 143.75 (6.97) bbc zoe 209.84 (13.23) ab 156.42 (7.69) bb 162.48 (7.96) bb ultracal 182.21 (25.64) ac 130.98 (12.28) bbc 128.02 (15.84) bc chlorhexidine 143.66 (17.32) ad 116.65 (6.43) bc 133.29 (2.19) ac ca + io calen® + iodoform ca + zno calen® + zinc oxide zoe zinc oxide and eugenol ultracal ultracal®xs chlorhexidine chlorhexidine digluconate 2% + calcium hydroxide + zinc oxide different uppercase letters indicate differences between the systems (columns) and lowercase letters indicate differences between the materials (rows). table 2. mean and standard deviation (sd) contrast of aluminum scale in the radiographic systems studied system mean (sd) express 48.77 (2.53) a vistascan 21.34 (1.83) b snapshot 16.87 (1.41) c different letters indicate statistical differences. 7 ferreira et al. the results of subjective evaluation and of the reference standard are illustrated in tables 3 and 4. regarding homogeneity (table 3), no differences were observed among the radiographic systems (p = 0.573). however, the root third and the material influenced homogeneity (p=0.0001 and 0.0007, respectively). in general, the apical third received more inadequate scores. moreover, ca + io differed from the other materials and showed the best results, receiving more adequate scores, whereas chlorhexidine and ultracal provided the worst results. table 3. adequate homogeneity scores according to the filling materials, radiographic systems and reference standard (%) material* ca + io (a) calen + zno (b) zoe (b) ultracal (c) chlorexhidine (c) third** n (%) n (%) n (%) n (%) n (%) system*** cervical middle apical cervical middle apical cervical middle apical cervical middle apical cervical middle apical express 25 (100) 25 (100) 25 (100) 25 (100) 0 (0) 0 (0) 20 (80) 15 (60) 0 (0) 0 (0) 4 (20) 0 (0) 10 (40) 0 (0) 0 (0) vistascan 25 (100) 25 (100) 15 (60) 15 (60) 4 (20) 4 (20) 20 (80) 10 (40) 0 (0) 4 (20) 10 (40) 0 (0) 4 (20) 0 (0) 0 (0) snapshot 20 (80) 25 (100) 15 (60) 20 (80) 4 (20) 0 (0) 15 (60) 4 (20) 0 (0) 0 (0) 4 (20) 0 (0) 10 (40) 0 (0) 4 (20) microct 20 (80) 20 (80) 15 (60) 25 (100) 4 (20) 0 (0) 20 (80) 10 (40) 4 (20) 0 (0) 0 (0) 0 (0) 4 (20) 4 (20) 0 (0) ca + io calen® + iodoform ca + zno calen® + zinc oxide zoe zinc oxide and eugenol ultracal ultracal®xs chlorhexidine chlorhexidine digluconate 2% + calcium hydroxide + zinc oxide * some materials differed in the homogeneity evaluation (p=0.0007) – difference indicated by the different letters between the parentheses. ** the thirds differed between themselves, regarding the homogeneity (p=0.0001). *** the radiographic systems did not differ between themselves in the homogeneity evaluation (p=0.573). table 4. adequate apical sealing scores according to the filling materials, radiographic systems and reference standard (%) material* ca + io (ab) calen + zno (a) zoe (ab) ultracal (b) chlorexhidine (b) system** n (%) n (%) n (%) n (%) n (%) express (b) 20 (80) 25 (100) 20 (80) 10 (40) 15 (60) vistascan (b) 20 (80) 25 (100) 20 (80) 4 (20) 10 (40) snapshot (ab) 15 (60) 20 (80) 20 (80) 10 (40) 10 (40) microct (a) 15 (60) 20 (80) 15 (60) 0 (0) 4 (20) ca + io calen® + iodoform ca + zno calen® + zinc oxide zoe zinc oxide and eugenol ultracal ultracal®xs chlorhexidine chlorhexidine digluconate 2% + calcium hydroxide + zinc oxide * some materials differed in the apical sealing evaluation (p=0.0133) – difference indicated by the different uppercase letters between the parentheses in horizontal. ** some systems differed in the apical sealing evaluation (p=0.0127) – difference indicated by the different lowercase letters between the parentheses in vertical. 8 ferreira et al. regarding apical sealing (table 4), some differences were observed among the radiographic systems (p = 0.0127). snapshot was the only system that did not differ from the micro-ct scans (reference standard). in addition, some differences were found among the materials (p = 0.0133), with ca + zoe providing better results than ultracal and chlorhexidine. the intra-examiner agreement was excellent for both homogeneity and apical sealing (kappa = 0.954 and 1.0, respectively). discussion current x-ray detectors usually show satisfactory performance in terms of spatial resolution, contrast, and exposure latitude in quantitative analyses13. however, in view of the variety of digital systems and their diverse performance in different diagnostic tasks, it is important to test the technologies (cmos and psp) for not yet studied tasks, such as root filling quality. in this respect, we found that one of the digital systems studied provided some improvement in the evaluation of apical sealing. in radiographic images, one factor that influences on detail discernment is the spatial resolution of the x-ray detector, which refers to the size of the smallest possible feature that can be detected. in this respect, a higher spatial resolution seems to have some benefit in the evaluation of apical sealing since snapshot permitted better analysis. the same radiographic system provided some improvement in the evaluation of fractured endodontic files and vertical root fractures in teeth with glass fiber posts9,10. these previous studies also attributed the better performance to the higher spatial resolution of the system. it is important to note that the systems studied use different technologies, a fact that could also influence the results. however, they were controlled during the experiment. regarding image acquisition, psps require scanning for imaging assessment. therefore, a decrease in image quality may occur over time or secondary to exposure to ambient light through the electron decay of psp’ traps in higher energy states14. the vistascan and express systems (psps) are capable of producing high-quality images, even if they are manipulated in bright rooms. however, if these images are evaluated objectively, the result of incorrect handling can be quantified14. nevertheless, psps spontaneously release captured electrons over time and attention must therefore be paid to the scanning process14. to avoid these biases, we exposed the plates to x-rays and scanned them immediately in a room with little light. in addition to higher spatial resolution, the solid sensors (cmos) produce a digital image immediately after radiation exposure. therefore, the quality of the final image is not influenced by the external light. moreover, in cmos, the dexels of the receptors are separate from each other, so that each one is connected directly to a converter. this ensures the individualization of the voltage of the pixel transfer and, thus, individual evaluation. contrast resolution is the ability to distinguish between differences in intensity in an image and depends on the bit depth. images with 8-bit depth can provide 256 shades of gray per pixel. a high contrast resolution increases the availability of shades of gray in an image. theoretically, this could improve diagnosis with more 9 ferreira et al. shades of gray in image until a limit considering the ability that the human eye can see and the contemporary computer monitors can support15. however, our results showed that spatial resolution seems to be more important than contrast resolution, as snapshot exhibited better performance but lower contrast resolution when the aluminum wedge was evaluated. in addition to the influence of radiographic systems, the different intracanal materials used for the endodontic treatment of primary teeth could interfere with the evaluation of root canal filling quality. however, the filling materials must meet some criteria for use in the root filling of primary teeth, such as being antibacterial, being resorbable at the same proportion as the root of the tooth, being biocompatible, easily filling the channels, adhering to the walls, not causing discoloration of the tooth, and being radiopaque16. although some of these characteristics were not tested in the present study, radiopacity is an important feature to verify the full extent of the material and the presence of faults and voids and was therefore evaluated in the study. nowadays, several types of filling materials for primary teeth are available, with materials containing zinc oxide, ca(oh)2 and iodoform being commonly used 17,18. zinc oxide and eugenol has been recommended by the american academy of pediatric dentistry (aapd) as the material of choice for endodontic treatment of primary teeth until 2008. however, this material is not considered biocompatible because it causes an inflammatory reaction in periapical tissues and promotes slow root resorption, which can damage the permanent successor18. therefore, the aapd started to recommend materials based on ca(oh)2 and/or iodoform as an alternative to zinc oxide and eugenol. materials based on ca(oh)2 are poorly soluble in water, requiring a longer time to be dissolved, and cause no damage when in contact with apical tissues. in addition, they are easy to apply and are resorbed faster than the roots of primary teeth. on the other hand, these materials have some undesirable properties such as low radiopacity and viscosity that require their combination with other compounds such as iodoform and/or zinc oxide19,20. in addition to the known good biomechanical qualities of the combination with iodoform or zinc oxide, those materials also showed good results in terms of radiopacity, homogeneity and apical sealing. in addition, the combination of iodoform with other materials such as ca(oh)2 increases radiopacity21. thus, calen® combined with iodoform provided better results in objective and subjective analyses. the combination of ca(oh)2, 2% chlorhexidine and zinc oxide (2: 1: 2 ratio) has been proposed in cases of dental trauma. this medication exhibits excellent antimicrobial activity, high remineralizing capacity and relative radiopacity, is inexpensive, and does not require periodic replacement22. thus, this intracanal material may become a viable alternative for obturation of primary teeth and was therefore tested in this study. however, this material did not perform well in terms of the parameters studied. further studies are necessary to test other properties. the flow of filling material can influence homogeneity, particularly in the apical third of the root. a good material should allow easy filling of the entire root canal, reduc10 ferreira et al. ing the formation of bubbles and the possibility of unsealed parts. we believe that some of the materials tested do not have adequate flow as they received inadequate evaluations (score 2) in the apical third. another explanation could be related to anatomical variations in the root canal, since bovine primary teeth were used. however, considering the similar root dentin morphology of bovine and human teeth, permitting similar penetration of the endodontic obturation material into the dentinal tubules23,24, we believe that the use of these teeth did not influence our results. finally, the large number of inadequate scores was a surprising finding, as we expected more adequate homogeneity and apical sealing because this was an in vitro study of anterior teeth. thus, the working condition is better than in the oral cavity, facilitating endodontic treatment. we do not believe that the evaluations were flawed because the intra-examiner agreement was high and the micro-ct images confirmed the inadequate scores. our hypothesis is that the images were acquired without superimposition of other structures as occurs in clinical studies in which maxillary bones could mask bubbles or other failures. therefore, the evaluations would favor to identify the irregularities in the homogeneity and apical sealing. however, it is also important to remember that the success rates of endodontic treatment in primary teeth do not only depend on a complete seal, but also on a set of clinical factors such as correct diagnosis, adequate chemical-mechanical root canal preparation, use of materials with desirable and biological properties, and an adequate coronal seal. the results of the present study suggest that direct digital systems with higher resolution should be preferred for quality assessment of endodontic treatment in primary teeth since they improve the analysis of apical sealing. acknowledgments this work was partially supported by pibic-cnpq. the authors declare no conflict of interest. references 1. seale ns. indirect pulp therapy: an alternative to pulpotomy in primary teeth. tex dent j. 2010 nov;127(11):1175-83. 2. akbar i. radiographic study of the problems and failures of endodontic treatment. int j health sci. 2015 apr;9(2):111-8. 3. udupa h, mah p, dove sb, mcdavid wd. evaluation of image quality parameters of representative intraoral digital radiographic systems. oral surg oral med oral pathol oral radiol. 2013 dec;116(6):774-83. doi: 10.1016/j.oooo.2013.08.019. 4. pontual aa, de melo dp, de almeida sm, boscolo fn, haiter neto f. comparison of digital systems and conventional dental film for the detection of approximal enamel caries. dentomaxillofac radiol. 2010 oct;39(7):431-6. doi: 10.1259/dmfr/94985823. 5. abesi f, mirshekar a, moudi e, seyedmajidi m, haghanifar s, haghighat n, et al. diagnostic accuracy of digital and conventional radiography in the detection of non-cavitated approximal dental caries. iran j radiol. 2012 mar;9(1):17-21. doi: 10.5812/iranjradiol.6747. epub 2012 mar 25. 11 ferreira et al. 6. özen t, kamburoglu k, cebeci ar, yuksel sp, paksoy cs. 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systems in detecting vertical root fractures. int endod j. 2015 sep;48(9):864-71. doi: 10.1111/iej.12382. 10. ramos brito ac, verner fs, junqueira rb, yamasaki mc, queiroz pm, freitas dq, et al. detection of fractured endodontic instruments in root canals: comparison between different digital radiography systems and cone-beam computed tomography. j endod. 2017 apr;43(4):544-9. doi: 10.1016/j.joen.2016.11.017. 11. pascon fm, kantovitz kr, puppin-rontani rm. influence of cleansers and irrigation methods on primary and permanent root. dentin permeability: a literature review. braz j oral sci. 2006;5(18):1063-9. doi: 10.20396/bjos.v5i18.8641897. 12. pinheiro sl, araujo g, bincelli i, cunha r, bueno c. evaluation of cleaning capacity and instrumentation time of manual, hybrid and rotary instrumentation techniques in primary molars. int endod j. 2012;45(4):379-85. doi: 10.1111/j.1365-2591.2011.01987.x. 13. farman ag, farman tt. a comparison of 18 different x-ray detectors currently used in dentistry. oral surg oral med oral pathol oral radiol endod. 2005 apr;99(4):485-9. 14. nascimento ha, visconti ma, ferreira lm, suarez ma, haiter neto f, freitas dq. effect of delayed scanning on imaging and on the diagnostic accuracy of vertical root fractures in two photostimulable phosphor plates digital systems. int endod j. 2016 oct;49(10):973-9. doi: 10.1111/iej.12547. 15. vasconcelos tv, santaella gm, nascimento ha, rovaris k, ambrosano gm, freitas dq. digital radiographs displayed on different devices: effect on the detection of vertical root fractures. int endod j. 2016;49(4):386-92. doi: 10.1111/iej.12466. 16. gupta s, das g. clinical and radiographic evaluation of zinc oxide eugenol and metapex in root canal treatment of primary teeth. j indian soc pedod prev dent. 2011 jul-sep;29(3):222-8. doi: 10.4103/0970-4388.85829. 17. navit s, jaiswal n, khan sa et al. antimicrobial efficacy of contemporary obturating materials used in primary teethan in-vitro study. j clin diagn res. 2016;10(9):zc09-zc12. 18. american academy on pediatric dentistry clinical affairs committee. guideline on pulp therapy for primary and immature permanent teeth. pediatr dent. 2016 oct;38(6):280-8. 19. silva la, leonardo mr, oliveira ds, silva ra, queiroz am, hernández pg, et al. histopathological evaluation of root canal filling materials for primary teeth. braz dent j. 2010 jan;21(1):38-45. 20. pramila r, muthu ms, deepa g, farzan jm, rodrigues sj. pulpectomies in primary mandibular molars: a comparison of outcomes using three root filling materials. int endod j. 2016 may;49(5):413-21. doi: 10.1111/iej.12478. 21. ordinola-zapata r, bramante cm, garcía-godoy f, moldauer bi, gagliardi minotti p, tercília grizzo l, et al. the effect of radiopacifiers agents on ph, calcium release, radiopacity, and antimicrobial properties of different calcium hydroxide dressings. microsc res tech. 2015 jul;78(7):620-5. doi: 10.1002/jemt.22521. 12 ferreira et al. 22. de jesus soares a, yuri nagata j, casarin rc, almeida jfa, gomes bp, augusto zaia a, et al. apexification with a new intra-canal medicament: a multidisciplinary case report. iran j radiol. 2012;7(3):165-70. 23. costa bm, iwamoto as, puppin-rontani rm, pascon fm. comparative analysis of root dentin morphology and structure of human versus bovine primary teeth. microsc microanal. 2015 jun;21(3):689-94. doi: 10.1017/s1431927615000434. 24. xavier sr, pilownic kj, gastmann ah, echeverria ms, romano ar, geraldo pappen f. bovine tooth discoloration induced by endodontic filling materials for primary teeth. int j dent. 2017;2017:7401962. doi: 10.1155/2017/7401962. oral sciences n3 braz j oral sci. 12(2):114-118 original article braz j oral sci. april | june 2013 volume 12, number 2 evaluation of human enamel permeability exposed to bleaching agents diego horning1, giovana mongruel gomes1, bruna fortes bittencourt1, lucas manuel ruiz1, alessandra reis1, osnara maria mongruel gomes1 1department of restorative dentistry, dental school, state university of ponta grossa, ponta grossa, pr, brazil correspondence to: giovana mongruel gomes rua engenheiro schamber 452, apto 21, cep: 84010-340 ponta grossa, pr, brasil phone: +55 42 32226560 / fax: +55 42 32247351 e-mail: giomongruel@gmail.com received for publication: march 21, 2013 accepted: june 25, 2013 abstract aim: to evaluate the human enamel permeability after different external bleaching techniques. methods: the coronal portions of 20 maxillary human incisors were covered with an acid resistant varnish, except the labial surface, to prevent dye penetration of silver nitrate. the specimens were divided randomly into four groups (n=5) according to the bleaching treatments: group 1 (g1) without bleaching agent (control group), group 2 (g2) 35% hydrogen peroxide (hp) – one session of three 10-min applications, group 3 (g3) 35% hp – 2 sessions of three 10-min applications, and group 4 (g4) 6% hp – 14 days (1h/day). g1 was exposed to artificial saliva only and in the other groups the bleaching agents were applied following the manufacturers’ recommendations. after bleaching, teeth were immersed in a 50% silver nitrate solution for 2 h and photodeveloped for 16 h. then, three longitudinal slices of each tooth were photographed by an optical microscope (100x). the degree of dye penetration was analyzed in the middle third of the dental crowns using a 0 to 4 score system (0= no dye penetration, 1= less than half the enamel thickness, 2= half of the enamel thickness, 3= full extent of enamel without reaching the dentin, and 4= tracer agent reaching dentin). the data were analyzed statistically by the kruskalwallis and dunn tests (α=0.05). results: the median (1st/3rd interquartiles) ranges were: g1: 0(0/0), g2: 2(2/3), g3: 3(3/3) and g4: 3(3/3). g2, g3 and g4 showed significantly higher dye penetration compared with g1 (p=0.0017). conclusions: the use of bleaching agents increased dental enamel permeability, regardless of the bleaching technique. keywords: dental enamel, bleaching agents, permeability. introduction the current search for beauty and esthetic standards has increased the number of patients in dental offices drawn by tooth bleaching procedures1. this popularity increased the products availability to treat discolored teeth. the literature has several reports confirming that bleaching gels, on their various concentrations, are effective for tooth whitening2-3. treatment modalities are basically two: athome and in-office techniques. at-home technique utilizes trays and lowconcentration bleaching gels, while in-office bleaching requires higher concentration gels, under professional supervision4. it is well known that bleaching agents release oxygen in dental structures, which, due to its low molecular weight and associated to dental permeability, may diffuse through enamel and dentin substrates, acting on the pigments. these molecules, by means of redox reactions, can break macromolecules (pigments) into smaller molecular chains, which are totally or partially removed from dental structure by diffusion5. 115115115115115 braz j oral sci. 12(2):114-118 while some authors state that no significant chemical or morphological alterations may occur in the dental structure after bleaching procedures using different bleaching agents and concentrations5, other studies report that bleaching products may cause enamel mineral loss at different levels6 and alterations in surface morphology7-8. theses alterations may vary depending on the product concentration, time of application9-11 and ph of the product10-11. although several studies were conducted to evaluate the effects of dental bleaching on the tooth structure, divergent results are found in the literature, which requires further studies in order to investigate the safety of tooth bleaching in relation to teeth, as well as the effectiveness of the treatments12. a model study was designed to assess penetration of bleaching agents through dental hard tissues using silver nitrate13. this methodology can track the diffusion channels promoted by bleaching agents that may penetrate into enamel structure14 and may be an indirect indicator of adverse effects of this treatment on the surface morphology of tooth structures12. so far, the studies that evaluated the permeability of dental enamel to bleaching procedures were mainly conducted in bovine enamel14. although bovine teeth have been considered a good substitute for human teeth15, there are clear morphological differences between then, as human teeth are more susceptible to penetration of bleaching agent than bovine teeth16. therefore the aim of this study was to compare the human enamel permeability after exposure to at-home and in-office bleaching agents. material and methods the research project was approved by the research ethics committee of the dental school of the state university of ponta grossa, report number 50/2008 and protocol number 13033/08. twenty extracted human central incisors were stored in distilled water at 4oc and used within 6 months after extraction (iso/ts 11405, 200317). the teeth were scaled for the removal of periodontal tissue remnants and other debris, and then ultrasonically cleaned in distilled water (dabi atlante, ribeirão preto, sp, brazil) in order to remove all organic and inorganic materials adhered to the coronal and root surfaces. all teeth were examined under 20x magnification (zeiss, estec, são paulo, sp, brazil) to detect enamel cracks, fractures, caries or other defects. in case such features were observed, the teeth were excluded from the sample. the crowns of the selected teeth were separated from their roots at the cementoenamel junction (cej) using a lowspeed diamond saw (isomet 1000, buehler, lake bluff, il, usa) under water cooling at 300 rpm. then, the specimens were randomly divided into four groups (n=5) according to the bleaching treatments (table 1). the labial surface of all specimens was subjected to the bleaching procedures, according to the experimental groups, following the manufacturers’ instructions. the control group (g1) was exposed only to artificial saliva at 37°c in a dark environment and was not subjected to any bleaching treatment. for g2 and g3, an in-office bleaching technique was used with 35% hydrogen peroxide agent (ph35, whiteness hp maxx, fgm). three drops of peroxide for 1 drop of thickener were mixed in a plate included in the bleaching kit. with the aid of a spatula, the labial surfaces were covered with a gel layer 1 mm thick. then, the bleaching agent was light activated for 3 min (whitening laser ii, dmc, são carlos, sp, brazil) and 7 min without light exposure. this procedure was repeated 3 times, with no waiting time between the applications. the gel was removed with a cotton pellet. for g3, another session was performed, repeating the same procedures, totalizing 2 sessions of three 10-min applications, with a 7-day interval. during this period the specimens were stored in artificial saliva at 37° c in a dark environment. for g4, an at-home bleaching protocol was used with 6% hydrogen peroxide agent (white class calcium, fgm). the gel was applied for 14 consecutive days, 1 h per day. in the remaining period, the specimens were also stored in artificial saliva at 37° c in a dark environment during the treatment period. evaluation of human enamel permeability exposed to bleaching agents groups (n=5) g1 g2 g3 g4 bleaching agent/ composition/ ph whiteness hp maxx (fgm dental products, joinville, sc, brazil)35% hydrogen peroxide (ph35), thickening agent, colorant, glycol, inorganic filler and deionized water ph ~ 5.5 whiteness hp maxx (fgm dental products, joinville, sc, brazil)35% hydrogen peroxide (ph35), thickening agent, colorant, glycol, inorganic filler and deionized water ph ~ 5.5 white class calcium 6% (fgm dental products, joinville, sc, brazil)6% hydrogen peroxide (ph6), neutralized carbopol, potassium nitrate, sodium fluoride, aloe vera, calcium gluconate, stabilizer, deionized water and surfactant. ph ~ 6.2 application procedure without bleaching agent (control group) one session of three 10-min applications two sessions of three 10-min applications 1 h daily exposure for 14 days table 1 – experimental groups, bleaching agents, composition and application procedure used in this study. 116116116116116 braz j oral sci. 12(2):114-118 after the end of the bleaching procedures, all specimens were stored in artificial saliva for 48 h, in a dark environment at 37ºc. after this period, the specimens were ultrasonically cleaned once again for 5 min, in order to remove any other debris from the enamel external surfaces. after these procedures, the specimens were dried at room temperature for 12 h and the lingual, proximal surfaces and the cej were sealed with cyanoacrylate resin (super bonder loctite, henkel ltda, são paulo, sp, brazil) and a nail varnish (rebu, risqué niasi, taboão da serra, sp, brazil) to prevent dye penetration of silver nitrate (tracer agent) through this area. the nail varnish was also used to delimitate an area of 4 x 4 mm in the middle third of the labial surface. specimens were then immersed in 50% aqueous silver nitrate solution (vetec química fina, xerém, rj, brazil) for 2 h, in a dark and closed environment. subsequently, the specimens were photodeveloped for 16 h (developing solution, kodak, eastman kodak company, rochester, ny, usa). after this period, the specimens were cleaned with tap water and the nail varnish layer was removed with manual cutting instruments. the specimens were then embedded in a polyvinyl chloride (pvc) tube with acrylic resin (duralay, reliance, dental mfg. co., worth, il, usa), and three longitudinal slices of each tooth with approximately 0.4 mm thick were obtained in a buccolingual direction, with a low-speed diamond saw (isomet 1000, buehler, lake bluff, il, usa), under water cooling. the slices were then photographed under an optical microscope (leica®, olympus bx41-u-ca, tokyo, japan) with 100x magnification. the images were taken with a digital camera, resolution of 5.1 megapixels. the dye penetration degree was analyzed by three previously calibrated evaluators, using a 0 to 4 score system: score 0 = no dye penetration; score 1 = less than half the enamel thickness; score 2 = half of the enamel thickness; score 3 = full extent of enamel without reaching the dentin and score 4 = tracer agent reaching dentin. a flow-chart of the different scores of the dye penetration degree may be seen in figure 1. this evaluation was determined by visualizing the middle third of the dental crowns. if there was any disagreement between the evaluators, the sample under discussion was analyzed jointly until a consensus was reached. the median scores of the images from the same tooth were considered for statistical analysis using the kruskal wallis and dunn tests (α= 0.05). results the medians and interquartile ranges for the permeability degree obtained from each group are shown in table 2. the statistical analysis revealed a significant effect for the groups medians(1st/3rd interquartiles) significance * g1 0 (0/0) a g2 2 (2/3) b g3 3 (3/3) b g4 3 (3/3) b table 2 – medians (1st/3rd interquartiles) for permeability degree and significance of all experimental groups. * different letters indicate statistically significant differences (p< 0.05). fig. 1 – flowchart of the different scores for the dye penetration degree. e – enamel; d – dentin. evaluated factor (p=0.0017). no significant differences were observed between the bleached groups (g2, g3 and g4) but all these groups showed significantly higher permeability compared to control group (g1). in figure 2, one can observe that there was no dye penetration in g1, and in g2, g3 and g4 the tracer agent penetrated just on enamel, without reach dentin. fig. 2 – stereomicroscopic image (100 x magnification) of the permeability degree obtained from each group. it may be visualized that there was no dye penetration in g1, and in g2, g3 and g4 the tracer agent (arrow) penetrated only on enamel, without reaching dentin. e – enamel; d – dentin. discussion there are currently three techniques for bleaching purposes: in-office bleaching, at-home bleaching and overthe-counter products3. this study verified the influence of the in-office and at-home approaches on enamel permeability by silver nitrate penetration. the results showed no differences between the bleaching protocols. a ft-ir spectroscopy study found that enamel alterations after in-office and at-home bleaching are dependent on the concentration and treatment time, since treatments for longer periods and at higher concentrations resulted in higher structural alterations in enamel9. however, in the present study, there were no significant differences between the bleached groups, regardless the used bleaching agent and the bleaching protocol. lower enamel permeability could be expected with the at-home product. however, an earlier study found no significant differences between 1, 7 or 14 applications (days) of a 10% carbamide peroxide agent18. additionally, although the at-home bleaching agent has a lower hydrogen peroxide concentration, the product was used for 14 days and the effects on enamel permeability were only evaluated at the end of this 14-day period. thus, the low concentration gel evaluation of human enamel permeability exposed to bleaching agents braz j oral sci. 12(2):114-118 was applied for a longer period than the in-office gel, which may explain the similar results obtained. furthermore, there are studies reporting that the ph values of the bleaching agents have a direct influence on the chemical and morphological structure of bleached enamel. neutral bleaching agents (30% hydrogen peroxide) caused minor deleterious effects on enamel compared to an acidic bleaching agent (also hydrogen peroxide) at the same concentration of 30%10. usually, at-home bleaching agents have lower ph than in-office systems19 and this may also interfere with the enamel permeability. in the present investigation, care was taken to select at-home and bleaching system with similar ph (whiteness hp maxx ph ~ 5.5; white class 6% ph ~ 6.2) in order to minimize the effects of ph on enamel permeability19. in the present research, all bleached groups showed a higher permeability compared to the control group. likewise, mendonça et al.14 reported a higher degree of silver nitrate penetration in bleached enamel specimens, explaining that bleaching procedure opens diffusion channels in the tooth structure through which the oxygen molecules are carried into the dentin substrate. additionally, the superficial alterations occurred in enamel structure, observed by sem14, and it may be speculated that subsurface damages also occurred in that tissue. in contrast, another study revealed, also by sem observations, that despite some morphological alterations that may occur, bleaching may be considered safe for enamel, as a regional variation of the dental substrate may amplify the effects of 35% hydrogen peroxide and 10% carbamide peroxide20. the methodology employed in this study was first reported by iwamoto et al.13 who observed silver nitrate penetration through dental structures subjected to bleaching therapy. however, different from the findings of this study, those authors found no penetration of the agent through the tooth structure. perhaps this fact may be attributed to a shorter exposure period of the specimens to silver nitrate, which was only 1 h. in the present investigation a 2-hour immersion period was used, which agrees with earlier studies14. the adverse effects caused by bleaching procedures on dental structures have been extensively studied. researches demonstrated several alterations on dental hard structures, as increased surface roughness7-8, reduction in microhardness values6 and pulpal responses21, as showed in a previous study that found direct damage to odontoblasts and decreased metabolic activity of these cells22. this effect may be due to the fact that low molecular weight reactive oxygen is more likely to penetrate dental structure than the silver nitrate used in this study. these findings suggest the need to supervise the use of bleaching agents, since oxygen molecules released by decomposition of these agents may reach the pulp chamber16 and cause toxic effects to pulp cells23-24. some in vitro studies use remineralizing solutions to store the specimens, such as hbss (hank’s saline solution), basically formed by salts25. another alternative is the use of artificial saliva, like in the present study, because it simulates accurately intraoral conditions26, compared to storage in distilled water. also, due to its high mineral content, artificial saliva27 or natural human saliva11 may increase remineralization of bleached enamel, different from distilled water, which has no remineralizing effect. since this is an in vitro study, clinical extrapolations are limited. vongsavan and matthews28 reported that the results obtained from in vitro studies do not correspond to in vivo reality. the authors state that in the oral cavity, there is a fluid movement within the dentinal tubules and enamel porosities. this may render the penetration of bleaching agents under in vivo conditions but not under in vitro conditions29. considering all the above-mentioned physiological factors that may affect the results, more clinical studies should be performed, in order to determine the actual effect of bleaching agents on enamel. references 1. tin-oo mm, saddki n, hassan n. factors influencing patient satisfaction with dental appearance and treatments they desire to improve aesthetics. bmc oral health. 2011; 11: 6 2. haywood vb, heymann ho. nightguard vital bleaching. quintessence int. 1989; 20: 173-6. 3. heymann ho. tooth whitening: facts and fallacies. brit dent j. 2005; 198: 514. 4. li y. safety controversies in tooth bleaching. dent clin n am. 2011; 55: 255-63. 5. haywood vb, hook v, heymann h. nightguard vital bleaching effects of various solutions on enamel surface texture and color. quintessence int. 1991; 22: 775-82. 6. al-salehi sk, wood dj, hatton pv. the effect of 24h non-stop hydrogen peroxide concentration on bovine enamel and dentin mineral content and microhardness. j dent. 2007; 35: 845-50. 7. cavalli v, arrais cag, giannini m. influence of low-concentrated bleaching agents on the human enamel roughness and morphology. clipeodonto – unitau. 2009; 1: 14-9. 8. martin jm, de almeida jb, rosa ea, soares p, torno v, rached rn, et al. effects of fluoride therapies on the surface roughness of human enamel exposed to bleaching agents. quintessence int. 2010; 41: 71-8. 9. bistey t, nagy ip, simó a, hegedus c. in vitro ft-ir study of the effects of hydrogen peroxide on superficial tooth enamel. j dent. 2007; 35: 325-30. 10. sun l, liang s, sa y, wang z, ma x, jiang t, et al. surface alteration of human tooth enamel subjected to acidic and neutral 30% hydrogen peroxide. j dent. 2011; 39: 686-92. 11. sa y, chen d, liu y, wen w, xu m, jiang t, et al. effects of two in-office bleaching agents with different ph values on enamel surface structure and colour: an in situ vs. in vitro study. j dent. 2012; 40: e26-34. 12. minoux m, serfaty r. vital tooth bleaching: biologic adverse effects – a review. quintessence int. 2008; 39: 645-59. 13. iwamoto n, shimada y, tagami j. penetration of silver nitrate into bleached enamel, dentin, and cementum. quintessence int. 2007; 38: e183-8. 14. mendonça lc, naves lz, garcia lfr, correr-sobrinho l, soares cj, quagliatto ps. permeability, roughness and topography of enamel after bleaching: tracking channels of penetration with silver nitrate. braz j oral sci. 2011; 10: 1-6. 15. krifka s, börzsönyi a, koch a, hiller ka, schmalz g, friedl kh. bond strength of adhesive systems to dentin and enamel—human vs. bovine primary teeth in vitro. dent mater. 2008; 24: 888-94. 16. camargo sea, valera mc, camargo chr, mancini mng, menezes mm. penetration of 38% hydrogen peroxide into the pulp chamber in bovine and human teeth submitted to office bleach technique. j endod. 2007; 33: 1074-7. 117117117117117 evaluation of human enamel permeability exposed to bleaching agents 118118118118118 braz j oral sci. 12(2):114-118 17. international standardization for organization. guidance on testing of adhesion to tooth structure. geneve, switzerland; 2003. cd tr 11405. 18. soares dgs, ribeiro apd, sacono nt, coldebella cr, hebling j, souza costa ca. transenamel and transdentinal cytotoxicity of carbamide peroxide bleaching gels on odontoblast-like mdpc-23 cells. int endod j. 2011; 44: 116-25. 19. freire a, archegas lr, de souza em, vieira s. effect of storage temperature on ph of in-office and at-home dental bleaching agents. acta odontol latinoam. 2009; 22: 27-31. 20. spalding m, taveira la, de assis gf. scanning electron microscopy study of dental enamel surface exposed to 35% hydrogen peroxide: alone, with saliva, and with 10% carbamide peroxide. j esthet restor dent. 2003; 15: 154-64. 21. de oliveira mavc, quagliatto ps, magalhães d, biffi jcg. effects of bleaching agents and adhesive systems in dental pulp: a literature review. braz j oral sci. 2012; 11: 428-32. 22. dias ribeiro ap, sacono nt, lessa fcr, nogueira i, coldebella cr, hebling j, et al. cytotoxic effect of a 35% hydrogen peroxide bleaching gel on odontoblast-like mdpc-23 cells. oral surg oral med oral pathol oral radiol endod. 2009; 108: 458-64. 23. coldebella cr, ribeiro apd, sacono nt, trindade fz, hebling j, costa cas. indirect cytotoxicity of a 35% hydrogen peroxide bleaching gel on cultured odontoblast-like cells. braz dent j. 2009; 20: 267-74. 24. dantas cmg, vivan cl, ferreira ls, freitas pm, marques mm. in vitro effect of low intensity laser on the cytotoxicity produced by substances released by bleaching gel. braz oral res. 2010; 24: 460-6. 25. chuang sf, chen hp, chang ch, liu jk. effect of fluoridated carbamide peroxide gels on enamel microtensile bond strength. eur j oral sci. 2009; 117: 435-41. 26. uysal t, basciftci fa, usumez s, sari z, buyukerkmen a. can previously bleached teeth be bonded safely? am j orthod dentofacial orthop. 2003; 123: 628-32. 27. cavalli v, reis af, giannini m, ambrosano gm. the effect of elapsed times following bleaching on enamel bond strength of resin composite. oper dent. 2001; 26: 597-602. 28. vongsavan n, matthews b. the permeability of cat dentin in vivo and in vitro. arch oral biol. 1991; 36: 641-6. 29. berger sb, pavan s, dos santos ph, giannini m, bedran-russo ak. effect of bleaching on sound enamel and with early artificial caries lesions using confocal laser microscopy. braz dent j. 2012; 23: 110-5. evaluation of human enamel permeability exposed to bleaching agents oral sciences n3 original article braz j oral sci. july | september 2013 volume 12, number 3 effects of masticatory hypofunction on mandibular morphology, mineral density and basal bone area fernanda da silva guerreiro1, péricles diniz2, paulo eduardo guedes carvalho1, eduardo cargnin ferreira3, sandra regina paulon avancini4, rívea inês ferreira-santos1 1 department of pediatric dentistry and orthodontics, university of são paulo city (unicid), são paulo, sp, brazil 2 department of morphology, federal university of santa catarina (ufsc), florianópolis, sc, brazil 3 department of genetics, federal university of santa catarina (ufsc), florianópolis, sc, brazil 4 department of nutrition, federal university of santa catarina (ufsc), florianópolis, sc, brazil correspondence to: rívea inês ferreira-santos universidade cidade de são paulo (unicid) pós-graduação (mestrado em ortodontia) rua cesário galeno, 448/bloco a, cep: 03071-000 tatuapé, sp, brasil phone: +55 11 21781310 fax: +55 11 21781355 e-mail: riveaines@gmail.com abstract aim: this experimental study investigated the association between masticatory hypofunction and mandibular morphological dimensions and internal bone characteristics. methods: twentyfour 21-day-old male wistar rats were randomly divided into two groups, according to the diet consistency. the control group (cg) was fed a solid diet (pellets) and the experimental group (eg) received a powdered diet during 50 days. all animals were euthanized and their mandibles removed and processed for histomorphometric analysis. a calibrated examiner performed linear and angular measurements (mandibular body length and height, mandibular lengths, ramus depth and height, mandibular base depth, mandibular head and gonial angle) on photographs, estimated bone density in the mandibular ramus region on digital radiographs and assessed the area of cortical and trabecular bone tissue in the second molar region, in 5-µm-thick serial cuts stained with cason’s trichrome. measurements for the study groups were compared using mann-whitney test (α=0.05). results: some of the macroscopic dimensions evaluated on photographs were significantly smaller in eg compared to cg, specifically mandibular ramus height (10.77 mm vs. 11.11 mm, p=0.0375), mandibular body length (21.67 mm vs. 22.36 mm, p=0.0165) and height (4.24 mm vs. 4.54 mm, p=0.0016), as well as mandibular base depth (1.24 mm vs. 1.47 mm, p=0.0325). the relative mineral bone density was significantly decreased in eg (1.04) compared to cg (1.25), p<0.001. rats in the eg also presented smaller trabecular and cortical bone area (2.36 mm²) than those in cg (3.16 mm²), p<0.001. conclusions: based on the above-mentioned measurements, it may be concluded that masticatory hypofunction induced by a powdered diet affected mandibular morphology and was associated with significantly reduced bone content. keywords: mastication, diet, mandible, bone development. introduction the adoption of a soft diet is related to histological, morphological and biochemical alterations in muscle fibers, which in turn impair the normal development of masticatory muscles1-2 and mandibular growth1,3-4. similarly, experimental studies using botulinum neurotoxin type a to induce masticatory received for publication: july 03, 2013 accepted: september 11, 2013 braz j oral sci. 12(3):205-211 muscle atrophy reported decreased craniofacial growth and development5-6. when bone is subjected to mechanical load, areas of tension and compression are generated in different regions, resulting in bone deposition and resorption as part of the remodeling process7-8. the mandibular ramus exhibits a posterior concave and anterior straight border, which favors the development of compression and tension loads during mastication. therefore, it was hypothesized that mechanical load arising in the mandibular ramus during mastication would generate the physiologic stimulus for bone remodeling and mandibular growth. some evidence indicates that bone natural curvatures may restrict tissue deformation during mechanical load, so the bone tissue becomes more adapted and resistant to a normal pattern of strain distribution 8. furthermore, bone curvatures tend to amplify the functional stimulus produced by mechanical loads9-10. considering that the mandible receives intense mechanical load during mastication, it may also be hypothesized that myofunctional alterations due to soft diet consistency would affect mandibular overall remodeling process with consequences on bone curvatures. thus, to avoid biased assumptions, this study assessed the relationship between masticatory muscles hypofunction, induced by a prolonged powdered diet, and mandibular morphological measurements (i.e. assessment of bone curvatures, sagittal and vertical dimensions), estimation of mineral bone density and quantification of basal area. the null hypothesis stated that there are no differences between animals fed powdered or solid diet. material and methods the experimental protocol was approved by an institutional review board on the use of animals (#20070138). experimental design the sample comprised 24 male wistar rats (albinus norvegicus), 21 days old, randomly and equally divided into two groups, according to the diet consistency. the control group received solid diet, i.e. laboratory chow for rats in a hard commercial pellet form without modifications (nuvilab cr-1®; nuvital, colombo, pr, brazil), while the experimental group received a powdered diet, which consisted of ground and sieved commercial pellets. similar amounts of food and water were offered ad libitum. apart from the diet, there was no other material or object that could induce masticatory stimulus inside the cages. body weight was measured on a calibrated precision scale once a week to assure normal development as related to growth and health for the animals (figure 1). despite the minor differences between body weight measurements of control and experimental animals, both groups had normal development. even in the experimental group, there fig. 1. mean (±se: bars) body weight in control and experimental groups, from the first to the last (seventh) week of experiment. 206206206206206 effects of masticatory hypofunction on mandibular morphology, mineral density and basal bone area braz j oral sci. 12(3):205-211 was progressive increase in body weight (figure 1). all animals were euthanatized by anoxia in co 2 chamber on the 50th day of the experiment. the mandibles were removed, separated from the soft tissue, split in two halves and placed in identified recipients containing 4% buffered paraformaldehyde solution (phosphate buffered saline-pbs, ph 7.2; sigma-aldrich® co., saint louis, mo, usa) for 48 h. macroscopic measurements standardized photographs of the hemi-mandibles were taken with a digital camera (e10; olympus corp., kobe, japan) at a resolution of 300 dpi and stored in tiff (tagged image file format), 8 bits. a millimeter scale was included in the photographic field. a tripod was used for steadying the camera at right angle to the hemi-mandible. macroscopic measurements were carried out on digital photographs using the image analysis software scion image® (nih image; scion corp., frederick, md, usa). some reference landmarks and measurements used in this study were adapted from cephalometric measurements in a previous investigation4. biometric points (figure 2) m (menton): the lowest point on the mental symphysis contour iia (alveolar point of the mandibular incisor): the lowest point of the buccal alveolar bone contour of the mandibular incisor ma (mandibular alveolar point): the deepest point of the upper part of the alveolar crest between the mandibular incisors and first molar co (condylion): the most posterior and superior point of the mandibular head go (gonium): the most posterior point of the mandibular angle contour got (gonial tangent): the lowest point of the mandibular fig. 2. biometric points and measurements. mandibular length i (co-iia); mandibular body length (go-iia); mandibular length ii (cr-iia); mandibular ramus curvature depth (rd at right angle to co-go); mandibular head (co at right angle to rd-ic); mandibular base curvature depth (mb at right angle to got-m); ramus height (co-got); mandibular body height (m-ma); gonial angle (intersection between co-go and got-m). angle contour cr (coronoid): the most superior and posterior point of the coronoid process rd (ramus depth): the deepest point in the concavity of the mandibular ramus ic: point located in the notch between the coronoid process and the mandibular head mb (mandibular base): the deepest point of the mandibular base concavity sagittal measurements (figure 2) mandibular length i: distance between points co and iia mandibular body length: distance between points go and iia mandibular length ii: distance between points cr and iia mandibular ramus curvature depth: distance from point rd at right angle to the co-go line mandibular head: distance from point co at right angle to the rd-ic line vertical measurements (figure 2) mandibular base depth: distance from point mb at right angle to the got-m line ramus height: distance between points co and got. ramus height could also be measured as the distance between points cr and got. however, the anatomical region where point cr would be located consists of a very thin bone structure, which is often fractured during dissection. therefore, measurements were carried out using the distance co-got. mandibular body height: distance between points m and ma angular measurement (figure 2) gonial angle: intersection of lines passing through points co-go and got-m. 207207207207207effects of masticatory hypofunction on mandibular morphology, mineral density and basal bone area braz j oral sci. 12(3):205-211 mineral bone density assessment standardized digital radiographs of the hemi-mandibles were obtained using the denoptix ® system (dentisply international/gendex® dental x-ray division, des plaines, il, usa), with an occlusal size (57 mm x 76 mm) storage phosphor plate. the x-ray unit ge 1000® (general electric co., milwaukee, wi, usa) operated in a constant regime at 70 kvp and 10 ma, 2.5 mm total aluminum filtration, and a 50 cm focus–receptor distance. an aluminum step wedge was positioned on the storage phosphor plate during the exposures. an acrylic device was manufactured to hold the storage phosphor plate, hemi-mandibles, aluminum step wedge and the x-ray beam indicator device in a reproducible relationship. the images were acquired using the standard commands of the digital radiographic system. the denoptix® storage phosphor plate scanning was performed at a resolution of 300 dpi. by this procedure, the pixel size is estimated at 85 µm2 and image resolution is equivalent to 6 line pairs per millimeter. the digital radiographs were stored in compact disc-recordable media as 8-bit tiff images. subsequently, mineral bone density was assessed using the image analysis software scion image®, which compared the gray levels of the region of interest in the hemi-mandibles with those of the aluminum step wedge. the mean mineral bone density was calculated in the mandibular ramus area, except for the mandibular incisors’ roots (figure 3). the results were expressed in values equivalent to those of the aluminum step wedge. histomorphometry the hemi-mandibles were then immersed in 5% nitric acid solution for removing all mineral content. thereafter, the specimens were embedded in paraffin according to the histological protocols. longitudinal sections (5 µm-thick) were obtained along the second molar region. a total of 5 fig. 3. radiographic image of the mandibular ramus, selected to perform measurements of mineral bone density. sections stained with cason’s trichrome were randomly selected for analysis. photomicrographs of the cut sections were acquired using a 3.3 megapixels cooled color digital camera (qcolor 3c; qimaging, surrey, bc, canada) connected to an optic microscope (olympus bx41, center valley, pa, usa) by the qcapture pro 5.1 software (qimaging). a 2 mmlong micrometric scale was positioned in the image field. the images were captured at a resolution of 640 x 512 pixels through a 4x magnification objective and stored as 8-bit tiff. all photomicrographs corresponding to the multiple parts of one cut section were assembled into one image using the ptgui pro 7.1 software (new house internet services b.v., rotterdam, the netherlands). the final image was rotated until the most prominent upper and lower points in the lingual surface were vertically aligned. other image processing software (photoshop® cs, adobe®, san jose, ca, usa) was used to erase structures other than bone from the image. subsequently, the image corresponding to the second molar and its alveolar process was erased by drawing a horizontal line passing on the roof of the mandibular canal. the remaining images of the mandibular basal bone were converted to gray scale (figure 4), stored as tiff and analyzed using the scion image® software, which provided the area measurements in mm2. method error to estimate reproducibility, macroscopic, bone density and histomorphometric measurements were performed twice. the images were renamed by a second operator in order to prevent the examiner to identify the groups during measurements. a 15-day interval was allowed to elapse between the first and second assessments. wilcoxon test and dahlberg’s formula were used for analyses of systematic and casual errors, respectively. systematic errors were not statistically significant (p>0.05). the casual errors for macroscopic (0.01 – 0.25 mm; 0.01º), bone density (0.0065) 208208208208208 effects of masticatory hypofunction on mandibular morphology, mineral density and basal bone area braz j oral sci. 12(3):205-211 fig. 4. cut section vertically aligned. soft tissue, alveolar process and tooth were removed from the image to measure the area of mandibular basal bone. and histomorphometric (0.0414 mm2) measurements were considered acceptable. statistical analysis all measurements obtained for the study groups were compared using mann-whitney test, which is an alternative to the student’s t-test and can be useful for small samples. given two independent samples, it tests whether one feature tends to have values higher than the other. 11 spearman coefficients were calculated to test any possible correlation between body weight after 50 days of experiment and morphological measurements, bone density and basal area. this kind of analysis can provide evidences that insufficient food intake is actually associated with decreased mandibular development, and not hypofunctional load. the significance level was set at 0.05. statistical analyses were carried out using the stata 8.0 package (statacorp. lp, college station, tx, usa). results the spearman coefficients obtained for all measurements were lower than 0.70, evidencing weak correlations between body weight and mandibular development features. in general, the experimental group presented smaller morphological measurements compared to the control group. concerning the sagittal measurements, mandibular body was significantly shorter in experimental animals compared to the controls. all vertical measurements were decreased in the experimental group (table 1). no significant difference was found between groups for the gonial angle. both radiographic bone density and mandibular basal bone area in the second molar region were significantly reduced in the experimental group compared to the control group (table 2). not only the amount of bone structure, but also the mineral composition was decreased in animals fed a powdered diet. discussion masticatory hypofunction may affect facial bone physiology12 and morphology4-6,13-14. the effects of soft diet on the mandibular bone development have already been studied by x-ray-based imaging4,13,15 or histological 1 6 methods. however, the present study used different methodologies, which not only complement each other, but also give distinct results to prove the rationale of this investigation. as shown in tables 1 and 2, the mandibular body length and height, the ramus height, as well as the mandibular basal bone area and the radiographic density in the mandibular ramus were significantly decreased in the experimental group, compared to the control group. this study was able to demonstrate that low masticatory demands were associated with remarkable changes in the mandibular morphology and alveolar process. these results corroborate those registered in other experimental studies4,13,15-16. powdered diet affected all three vertical linear dimensions of the mandible (table 1). reduced ramus and mandibular body heights in the experimental group might be resulting from inhibition or delay in basal bone and alveolar process growth. in agreement with these findings, a previous study reported that masticatory hypofunction was associated with reduced cortical bone deposition, particularly 209209209209209effects of masticatory hypofunction on mandibular morphology, mineral density and basal bone area braz j oral sci. 12(3):205-211 measurement control group experimental group p value mean (standard deviation) mean (standard deviation) mandibular length i (mm) 23.42 (0.44) 23.04 (0.51) 0.0567 mandibular body length (mm) 22.36 (0.63) 21.67 (0.83) 0.0165* mandibular length ii (mm) 20.29 (0.78) 20.03 (1.14) 0.4288 mandibular ramus curvature depth (mm) 2.67 (0.22) 2.66 (0.20) 0.6232 mandibular head (mm) 4.80 (0.21) 4.83 (0.21) 1.0000 mandibular base depth (mm) 1.47 (0.20) 1.24 (0.19) 0.0325* ramus height (mm) 11.11 (0.28) 10.77 (0.41) 0.0375* mandibular body height (mm) 4.54 (0.16) 4.24 (0.21) 0.0016** gonial angle (º) 81.06 (2.83) 82.91 (2.18) 0.1333 table 1. comparative analysis of the mean values for sagittal, vertical and angular measurements, obtained on photographs, between control and experimental groups. *p < 0.05, ** p < 0.01 in the mandibular basal region17 and inhibition of the alveolar process development2-3. several parameters of mechanical load influence bone shape, but bone adapts its structure more readily in response to the peak of strain induced by mechanical load than to the load frequency or duration of the stimulus18. therefore, the lack of vigorous masticatory stimulus caused by a powdered diet might have reduced the intensity of mechanical load applied to the mandible and compromised the ramus cortical sliding, leading to a decrease in mandibular size as observed in the experimental animals. according to the mechanostat theory18, bone tissue tends to increase in volume whenever it is exposed to mechanical loads over 1,500 microstrains. nevertheless, bone has a trend to maintain or decrease its volume when exposed to mechanical loads below 1,500 microstrains 18. strain is a measure of deformation. cortical bone deposition on the periosteal surface, stimulated by mechanical load, increases bone resistance to reduce the peak strain to a level that does not compromise tissue integrity9-10. differently from other parts of the human body subjected to high intensity mechanical load resulting from impact activities, the mandibular bone has a distinct feature associated with a noticeably great resistance to quasi-static loads imposed by the masticatory function. the mandibular body resistance to flexural deformation is proportional to the cross-sectional area where a perpendicular load is applied, divided by the depth of curvature caused by tissue deformation10. the higher the intensity of the applied load, the greater must be the cross-sectional area of bone tissue to resist the deformation. although the level of strain in the mandible was not measured in the present study, it is possible that animals fed a powdered measurement control group experimental group p value mean (standard deviation) mean (standard deviation) radiographic bone density 1.25 (0.07) 1.04 (0.04) < 0.0001 (aluminum equivalent) basal bone area (mm2) 3.16 (0.21) 2.36 (0.16) < 0.0001 table 2. comparative analysis of the mean values for radiographic bone density of the mandibular ramus and mandibular basal bone area in the second molar region measurements, between control and experimental groups. diet experienced lower level of strains in the mandibular basal bone due to the lack of vigorous masticatory stimulus, while mastication of hard pieces of pellets by the control rats might have induced sufficient strains to stimulate cortical bone deposition on the mandibular periosteal surface. therefore, it may be assumed that masticatory hypofunction due to a powdered diet could be associated with reduction in the cross-sectional area of the mandibular basal bone by inhibiting or postponing cortical bone deposition. this supposition is in agreement with previous findings17, in which inhibition of periosteal bone deposition on the mandible was observed after posterior teeth disocclusion. however, after reestablishment of normal occlusion or masticatory function, bone qualitative and quantitative characteristics can be improved, denoting the so-called catchup effect of functional adaptation2,17. the mandibular base depth, measured in an area of mandibular body curvature, was significantly reduced in the experimental group in comparison to the control group (table 1). it has been suggested that the shape of the cross-sectional area and the bone curvatures could act jointly to react to high levels of strain during deformation of the tissue subjected to mechanical load. however, bone architecture would restrict and coordinate the direction of load to avoid potentially dangerous overloads on the unusual axes10. moreover, an axis for mechanical load application provided by bone architecture would enable less tissue accumulation without jeopardizing resistance, since it would not be necessary to increase bone mass to resist the strain generated by mechanical loads in several directions. accordingly, it is reasonable to assume that the mandibular bone mass adapted its structure 210210210210210 effects of masticatory hypofunction on mandibular morphology, mineral density and basal bone area braz j oral sci. 12(3):205-211 in the experimental group, not only by reducing its crosssectional area, but also by attenuating the mandibular base curvature in response to a decreased mechanical load. although the mandibular base curvature has been attenuated due to the reduced muscular stimulus, the mandibular ramus curvature depth was not significantly affected (table 1). this feature might not have been affected because the strain on the posterior ramus border was not high enough to promote changes in the curvature depth or presumably because ramus structure in the sagittal plane was sufficiently thicker to avoid flexure when masticatory load is applied18. it may be suggested that the lack of vigorous masticatory stimulus may affect ramus thickness to a greater extent in relation to the mandibular ramus curvature depth. the significant decrease in radiographic bone density of the mandibular ramus in the experimental group, as shown in table 2, corroborates this assumption. even though significantly decreased cortical bone deposition has been observed in the mandibular basal region of interest, it is important to take into account that this decrease in radiographic bone density in the experimental group may also have resulted from bone hypomineralization. other authors mentioned the negative influence of muscular hypofunction induced by the alteration of diet consistency on the mandibular cortical bone deposition17 as well as on bone density in different regions of the mandible, such as the alveolar process4,16, coronoid process and mandibular angle4. the present study registered a decrease in radiographic bone density encompassing a large area of the mandibular ramus, instead of isolated spot measurements. unlike previous reports13, in this study hypofunctional masticatory load was not related to a significant decrease in the mandibular head (table 1). in another study19, altered functional temporomandibular joint (tmj) load in mice for 2-6 weeks was associated with loss of condylar cartilage and transient decrease in density of the mandibular subchondral bone. however, masticatory hypofunction did not change the mandibular head length. in fact, long-term alteration in masticatory function due to liquid diet feeding may impair jaw-opening reflex maturation12, but it appears that genetic and adaptive remodeling changes have greater influence on the mandibular head growth. despite the valid contribution of this study to understand the interplay between diet consistency and mandibular development, like most related scientific investigations, it was carried out under experimental conditions. nevertheless, the ontogenetic changes in mandibular morphology are supported by the role of the functional mechanical load due to diet consistency during growth20. on the basis of these study findings, it may be concluded that masticatory hypofunction induced by a powdered diet promoted significant changes in mandibular development in rats. the affected mandibles showed significant decrease in ramus and mandibular body heights, mandibular body length, and mandibular base curvature depth. in addition, radiographic bone density in the ramus and mandibular basal bone area in the second molar region were also significantly reduced. references 1. shimizu y, ishida t, hosomichi j, kaneko s, hatano k, ono t. soft diet causes greater alveolar osteopenia in the mandible than in the maxilla. arch oral biol. 2013; 58: 907-11. 2. mavropoulos a, odman a, ammann p, kiliaridis s. rehabilitation of masticatory function improves the alveolar bone architecture of the mandible in adult rats. bone. 2010; 47: 687-92. 3. kiliaridis s. masticatory muscle function and craniofacial morphology. an experimental study in the growing rat fed a soft diet. swed dent j suppl. 1986; 36: 1-55. 4. maki k, nishioka t, shioiri e, takahashi t, kimura m. effects of dietary consistency on the mandible of rats at the growth stage: computed x-ray densitometric and cephalometric analysis. angle orthod. 2002; 72: 468-75. 5. tsai cy, yang ly, chen kt, chiu wc. the influence of masticatory hypofunction on developing rat craniofacial structure. int j oral maxillofac surg. 2010; 39: 593-8. 6. tsai cy, shyr ym, chiu wc, lee cm. bone changes in the mandible following botulinum neurotoxin injections. eur j orthod. 2011; 33: 132-8. 7. lanyon le. functional strain as a determinant for bone remodeling. calcif tissue int. 1984; 36(suppl 1): s56-61. 8. turner ch, pavalko fm. mechanotransduction and functional response of the skeleton to physical stress: the mechanisms and mechanics of bone adaptation. j orthop sci. 1998; 3: 346-55. 9. currey jd. effects of differences in mineralization on the mechanical properties of bone. philos trans r soc lond b biol sci. 1984; 304: 509-18. 10. rubin ct. skeletal strain and the functional significance of bone architecture. calcif tissue int. 1984; 36(suppl 1): s11-8. 11. hart a. mann-whitney test is not just a test of medians: differences in spread can be important. bmj. 2001; 323: 391-3. 12. changsiripun c, yabushita t, soma k. masticatory function and maturation of the jaw-opening reflex. angle orthod. 2009; 79: 299-305. 13. enomoto a, watahiki j, yamaguchi t, irie t, tachikawa t, maki k. effects of mastication on mandibular growth evaluated by microcomputed tomography. eur j orthod. 2010; 32: 66-70. 14. patullo im, takayama l, patullo rf, jorgetti v, pereira rm. influence of ovariectomy and masticatory hypofunction on mandibular bone remodeling. oral dis. 2009; 15: 580-6. 15. mavropoulos a, kiliaridis s, bresin a, ammann p. effect of different masticatory functional and mechanical demands on the structural adaptation of the mandibular alveolar bone in young growing rats. bone. 2004; 35: 191-7. 16. kingsmill vj, boyde a, davis gr, howell pg, rawlinson sc. changes in bone mineral and matrix in response to a soft diet. j dent res. 2010; 89: 510-4. 17. shimomoto y, chung cj, iwasaki-hayashi y, muramoto t, soma k. effects of occlusal stimuli on alveolar/jaw bone formation. j dent res. 2007; 86: 47-51. 18. frost hm. wolff’s law and bone’s structural adaptation to mechanical usage: an overview for clinicians. angle orthod. 1994; 64: 175-88. 19. chen j, sorensen kp, gupta t, kilts t, young m, wadhwa s. altered functional loading causes differential effects in the subchondral bone and condylar cartilage in the temporomandibular joint from young mice. osteoarthritis cartilage. 2009; 17: 354-61. 20. holmes ma, ruff cb. dietary effects on development of the human mandibular corpus. am j phys anthropol. 2011; 145: 615-28. 211211211211211effects of masticatory hypofunction on mandibular morphology, mineral density and basal bone area braz j oral sci. 12(3):205-211 oral sciences n3 case report braz j oral sci. january | march 2013 volume 12, number 1 combined therapy with mineral trioxide aggregate, and guided tissue regeneration for a large radicular cyst: a 13-year follow-up pedro felício estrada bernabé1, joão eduardo gomes-filho1, eloi dezan-júnior1, annelise katrine carrara prieto1, renata oliveira samuel1, luciano tavares angelo cintra1 1department of endodontics, araçatuba school of dentistry, unesp – univ estadual paulista, araçatuba, sp, brazil correspondence to: luciano tavares angelo cintra araçatuba school of dentistry, são paulo state university rua josé bonifácio, 1193, araçatuba, sp, brasil phone: +55 18 36363252 fax: +55 18 36363279 e-mail:lucianocintra@foa.unesp.br abstract biomaterials such as membrane barriers and/or bone grafts are often used to enhance periapical new bone formation. a combination of apical surgery and these biomaterials is one of the latest treatment options for avoiding tooth extraction. in case of periapical lesions, guided tissue regeneration (gtr) is attempted to improve the self-regenerative healing process by excluding undesired proliferation of the gingival connective tissue or migration of the oral epithelial cells into osseous defects. in many cases, gtr is necessary for achieving periodontal tissue healing. this report describes the healing process after surgery in a challenging case with a long-term followup. in this case report, endodontic surgery was followed by retrograde sealing with mineral trioxide aggregate (mta) in the maxillary right central incisor and left lateral incisor. apicectomy was performed in the maxillary left central incisor and a 1-mm filling was removed. the bone defect was filled with an anorganic bone graft and covered with a decalcified cortical osseous membrane. no intraoperative or postoperative complications were observed. after 13 years of follow-up, the patient showed no clinical signs or symptoms associated with the lesion and radiographic examination showed progressive resolution of radiolucency. in conclusion, the combination of apical surgery and regenerative techniques can successfully help the treatment of periapical lesions of endodontic origin and is suitable for the management of challenging cases. keywords: guided tissue regeneration, apical surgery, mta. introduction radicular cysts are common inflammatory cystic lesions that develop in the apical tissues as consequence of an infected and necrotic pulp1. although in most cases small cystic lesions heal after endodontic therapy, in case of larger lesions, additional treatment may be needed2. apical surgery for radicular cysts generally involves apical root resection and sealing with endodontic material3. currently, the preferred root-end filling material is mineral trioxide aggregate (mta) because it has some biological properties, such as induction of calcification that enables biological sealing4-6. the physiochemical and biological properties of mta have been reported in numerous papers4-7. however, the ideal scenario would be to improve the benefits offered by the mta with the aid of other techniques that promote tissue regeneration. received for publication: october 25, 2012 accepted: december 11, 2012 braz j oral sci. 12(1):66-70 retrospective studies have shown that the success rate of apical surgery is not as high as expected8-9. apical surgery was considered unsuccessful in about 1 out of every 4 cases in 1960-197910. from the 1980s, the success rates increased to 50%, and the sizes of the lesions reduced in more than 25% of the cases11. however, the success rate of surgery remains low in cases of endoperiodontal lesions8. the high failure rate of apical surgery is directly related to the variety of factors that can influence the healing process in the periapical region7,9. adequate results were obtained in studies related to the healing process and the regeneration of tissues, especially in the ones pertaining to the support and protection of the periapex12-13. these results were obtained by developing regenerative and reparative techniques that helped to reestablish the periodontal structures and to preserve the biological width of the involved tissues14. use of guided tissue regeneration (gtr) in apical surgery can increase the success rate of this procedure 15. the technique helps creating ideal conditions for the restoration of original structures and normal functioning of the tissues that were lost because of infectious and inflammatory processes16. the basic principle of gtr is cellular selectivity. the technique aims at enhancing the quality and quantity of new bone and accelerating bone growth around the bone cavity17. the barrier is put on the bone defect and may frequently be associated with osseous grafting materials. this avoids the penetration of cells from both the epithelial tissue and gingival connective tissue. the use of the barrier membrane affords the time needed for the differentiation, proliferation and migration of the cells from the ligament, and from periodontal and alveolar bones to the bone cavity, favoring the healing process. furthermore, the space created by the membrane enables undifferentiated mesenchymal cells to migrate to this area and differentiate, thus promoting osteogenesis without the interference of other types of competitor cells12-13. the aim of the present case report was to describe a clinical situation in which combined therapy with mta and gtr was performed to treat a large radicular cyst. case report a 45-year-old woman who had previously undergone endodontic treatment for apical lesions associated with the maxillary left central incisor sought treatment at the apical surgery center at the araçatuba school of dentistry at the unesp. the patient presented poorly adapted prostheses in the maxillary right central incisor and left lateral incisor, as well as a large resin restoration in the maxillary left central incisor (figure 1a). radiographic examination revealed extensive apical lesions associated with the maxillary right and left central incisors and the left lateral incisor, in which root canal treatment had failed (figure 1b). the first suggested treatment option was the removal of fig. 1. (a) clinical aspects of poorly adapted prostheses in the maxillary right central incisor and left lateral incisor; large resin restoration in maxillary left central incisor. (b) preoperative radiographs of the central maxillary right, left central and left lateral incisors. radiography revealed extensive periapical lesions for which root canal treatments had failed. (c) exposed surgical area with root-end surgery in the maxillary right central, left central and left lateral incisors. (d) excision for periapical lesions. (e) in the maxillary right central and left lateral incisors, retrograde endodontic treatment was performed and teeth were sealed with mineral trioxide aggregate (mta). in the maxillary left central incisor, apicectomy and removal of the filling (1 mm) was performed. (f) anorganic bovine-bone particles (gen-ox; genius). (g) the bone cavity was filled with anorganic bone graft particles and had a blood clot. (h) radiographic aspect after apicetomy, root-end filling and bone filling with anorganic bovine bone. (i) the bone cavity and the bone graft were covered by a decalcified cortical osseous membrane (gen-derm; genius). (j) the flap was repositioned and secured by interrupted 4.0 sutures. combined therapy with mineral trioxide aggregate, and guided tissue regeneration for a large radicular cyst: a 13-year follow-up 6767676767 braz j oral sci. 12(1):66-70 fig. 2. (a) histological specimens revealed a periapical cyst lined by non-keratinized stratified squamous epithelium. note the moderate inflammatory response (h.e. staining, 40× magnification). (b, c) a follow-up radiograph obtained 32 months after surgical root canal therapy shows healing of periapical lesions. (d-f) radiograph obtained at the 13-year follow-up visit. the crowns/posts and retreatment of the maxillary right central incisor and left lateral incisor. however, the patient refused the proposed treatment due to the risks involved and opted for apical surgery. complementary laboratory exams showed that the patient had no systemic alteration. prophylactic antibiotics were prescribed 1 h prior to the surgery. the surgical area was disinfected with iodine solution and 0.12% chlorhexidine gluconate. prilocaine hydrochloride (3%) with octapressin (dentsply, petrópolis, rj, brazil) was used for local anesthesia. the flap design consisted of 2 releasing incisions connected by a sulcular incision (figure 1c). the apical lesion was removed with size 85 lucas surgical curettes (hu-friedy, chicago, il, usa) and size 35 and 36 curettes (dentsply maillefer, tulsa, ok, usa) (figure 1d). apical roots were sectioned (3-mm sections) perpendicular to the long axis of the root with a zekrya bur (dentsply maillefer) with a high-speed handpiece (figure 1e). different treatment approaches were performed for each tooth. for the maxillary central right incisor, retrograde endodontic treatment was performed using pre-bent files with a 4 mm length and filled with pro-root mta® (dentsply maillefer). apicectomy and removal of the apical filling (1 mm) was performed in the maxillary central left incisor because it was properly treated. for the maxillary lateral left incisor, retrograde endodontic treatment was performed using 6 mm pre-bent files and pro-root mta® as the root-end filing material. pro-root mta® was prepared according to the manufacturer’s instructions and inserted using the map system device (produits dentaires, vevey, switzerland). after the surgical procedures, radiographs were taken to verify the quality of the root-end treatments. the bone defect was filled with an anorganic bone graft (gen-ox; genius, são josé dos campos, sp, brazil) (figure 1f-h) and covered with a decalcified cortical osseous membrane (gen-derm; genius, são josé dos campos, sp, brazil) (figure 1i). finally, the flap was sutured with a simple interrupted suture using 4.0 silk (ethicon, são josé dos campos, sp, brazil) (figure 1j). after the surgery, the patient received antibiotics and medication to control pain. a full histological study of the cystic capsule was performed to confirm the previous diagnostic hypothesis (figure 2a). after 7 days, the sutures were removed and the patient was examined. the patient experienced no pain and showed no swelling. no intraoperative or postoperative complications were observed. at the 32-month follow-up, the tooth had no clinical signs or symptoms and radiographic examination showed progressive resolution of radiolucency (figure 2b, c). a follow-up evaluation performed after 13 years confirmed clinical silence and normal apical radiographic aspects (figure 2d-f). it was also verified that the patient had not changed the prosthetic crowns, preserving the posts, following the recommendations after surgery. discussion radicular cysts are also known as periapical cysts, dental cysts or apical periodontal cysts. a radicular cyst is generally asymptomatic, grows slowly and rarely grows large enough to erode extensively the adjacent bone structures. enucleating combined therapy with mineral trioxide aggregate, and guided tissue regeneration for a large radicular cyst: a 13-year follow-up6868686868 braz j oral sci. 12(1):66-70 the cyst is one of the recommended treatments1, but surgical procedures alone are not sufficient for a successful treatment of radicular cysts. one of the main goals of conventional endodontic treatment is to prevent the invasion of bacteria and their byproducts from the root canal system into the periradicular tissues of teeth in cases of apical periodontitis18. the use of gtr techniques has been proposed as an adjunct to endodontic surgery to favor bone healing19. gtr has been accepted as a viable treatment for gingival recession20, intrabone defects21-22, vertical ridge augmentation23, furcation defects24, circumferential periodontal and dental implantassociated defects25-26, and in apical microsurgery12,18. membrane barriers and/or bone graft materials have also been used in periapical surgery to enhance new bone formation3. however, there are significant differences in the application of gtr between periodontal regenerative therapy and apical surgery. regeneration represents replacement of damaged tissue by the cells of the same tissue. repair occurs when the healing process results in the formation of new tissue with cells and structures that have the ability to behave differently from the original ones18. in apical surgery, the resected root end cannot be regenerated. complete periapical wound healing after periapical surgery includes regeneration of the alveolar bone, periodontal ligament and cementum27. the application of a membrane barrier and/or bone graft during periapical surgery may not result in a complete regeneration of the apical tissues3. the adequate apical healing would be deposition of the cementum on the resected surface and root-end filling material4-6 and re-establishment of the biological width and periodontal ligament12-13. in this case report, a large bone defect involving 3 teeth was detected and the use of gtr was justified. in a previous study it was observed that the use of biomaterials such as genox® combined with the genderm® membrane provided better results than blood clot for treating critical-size defects28. th e distance between the margins of the bone cavity determined the type of cells that would first migrate to the defect site and consequently the tissue that would be formed8. fibrous scar formation may occur in cases in which gtr is not used. the membranes used in apical surgeries, which may or may not be in contact with bone graft materials, have the special function of guiding the formation of the new bone in the apical defect and may enhance the healing process. the bone graft material used in this case was an anorganic bovine bone that participated in the development of the new bone tissue and can act as an osteoconductive material. in the present case, a retrograde, not direct, endodontic retreatment was performed for the maxillary central right incisor and left lateral incisor because of the presence of the prostheses. on the other hand, for the maxillary left central incisor, which had been subjected to endodontic treatment recently, a curettage was performed followed by apicoectomy and remodeling of the filling 1 mm from the apex. in this case, the lesions had a positive response to the combination of surgical treatment and biomaterials. the use of membrane barriers and other agents, such as bone graft materials or tissue growth factors, has been reported as a viable treatment option 1,3,27-28. no intraoperative or postoperative complications were observed. at the 13-year follow-up, the patient showed no clinical signs or symptoms associated with the lesion and radiography showed progressive resolution of the radiolucency. compared to the traditional methods of endodontic surgery, gtr techniques have significantly improved the outcomes for periapical lesions29. a review of literature suggests that there is a lot of optimism about regenerative procedures. however, despite the success achieved with these procedures, as seen in this case report, they should be applied with caution. biological studies in experimental models should be conducted to evaluate the need for gtr use with apical surgery. references 1. sagit m, guler s, tasdemir a, akf somdas m. large radicular cyst in the maxillary sinus. j craniofac surg. 2011; 22: e64-5. 2. martin sa. conventional endodontic therapy of upper central incisor combined with cyst decompression: a case report. j endod. 2007; 33: 753-7. 3. lin l, chen my, ricucci d, rosenberg pa. guided tissue regeneration in periapical surgery. j endod. 2010; 36: 618-25. 4. cintra lt, de moraes ig, estrada bp, gomes-filho je, bramante cm, garcia rb et al. evaluation of the tissue response to mta and mbpc: microscopic analysis of implants in alveolar bone of rats. j endod. 2006; 32: 556-9. 5. gomes-filho je, de moraes costa mt, cintra lt, lodi cs, duarte pc, okamoto r et al. evaluation of alveolar socket response to angelus mta and experimental light-cure mta. oral surg oral med oral pathol oral radiol endod. 2010; 110: e93-7. 6. gomes-filho je, de moraes costa mm, cintra lt, duarte pc, takamiya as, lodi cs et al. evaluation of rat alveolar bone response to angelus mta or experimental light-cured mineral trioxide aggregate using fluorochromes. j endod 2011; 37: 250-4. 7. bernabé pf, gomes-filho je, rocha wc, nery mj, otoboni-filho ja, dezan-júnior e. histological evaluation of mta as a root-end filling material. int endod j. 2007; 40: 758-65. 8. douthitt jc, gutmann jl, witherspoon de. histologic assessment of healing after the use of a bioresorbable membrane in the management of buccal bone loss concomitant with periradicular surgery. j endod. 2001; 27: 404-10. 9. song m, jung iy, lee sj, lee cy, kim e. prognostic factors for clinical outcomes in endodontic microsurgery: a retrospective study. j endod. 2011; 37: 927-33. 10. rud j, andreasen jo, jensen je. a follow-up study of 1,000 cases treated by endodontic surgery. int j oral surg. 1972; 1: 215-28. 11. gutman j, harrison j. surgical endodontics. cambridge: blackwell scientific; 1991. p.355-7. 12. britain sk, arx t, schenk rk, buser d, nummikoski p, cochran dl. the use of guided tissue regeneration principles in endodontic surgery for induced chronic periodontic-endodontic lesions: a clinical, radiographic, and histologic evaluation. j periodontol. 2005; 76: 450-60. 13. von arx t, britain s, cochran dl, schenk rk, nummikoski p, buser d. healing of periapical lesions with complete loss of the buccal bone plate: a histologic study in the canine mandible. int j periodontics restorative dent. 2003; 23: 157-67. 14. nandlal b, daneswari v. restoring biological width in crown-root fracture: a periodontal concern. j indian soc pedod prev dent. 2007; 25 (suppl): s20-4. 15. taschieri s, corbella s, tsesis i, bortolin m, del fabbro m. effect of guided tissue regeneration on the outcome of surgical endodontic treatment of through-and-through lesions: a retrospective study at 4-year follow-up. oral maxillofac surg. 2011; 15: 153-9. combined therapy with mineral trioxide aggregate, and guided tissue regeneration for a large radicular cyst: a 13-year follow-up 6969696969 braz j oral sci. 12(1):66-70 16. gagnon k, morand ma. [guided tissue regeneration in endodontics. [part 1]. j can dent assoc. 1999; 65: 394-8. 17. pecora g, baek sh, rethnam s, kim s. barrier membrane techniques in endodontic microsurgery. dent clin north am. 1997; 41: 585-602. 18. artzi z, wasersprung n, weinreb m, steigmann m, prasad hs, tsesis i. effect of guided tissue regeneration on newly formed bone and cementum in periapical tissue healing after endodontic surgery: an in vivo study in the cat. j endod. 2012; 38: 163-9. 19. taschieri s, del fabbro m, testori t, saita m, weinstein r. efficacy of guided tissue regeneration in the management of through-and-through lesions following surgical endodontics: a preliminary study. int j periodontics restorative dent 2008; 28: 265-71. 20. rosetti ep, marcantonio ra, cirelli ja, zuza ep, marcantonio e jr. treatment of gingival recession with collagen membrane and dfdba: a histometric study in dogs. braz oral res. 2009; 23: 307-12. 21. arikan f, becerik s, sonmez s, gurhan i. effect of platelet-rich plasma on gingival and periodontal ligament fibroblasts: new in-vitro growth assay. braz j oral sci. 2007; 6: 1432-7. 22. kaur m, ramakrishnan t, amblavanan n, emmadi p. effect of plateletrich plasma and bioactive glass in the treatment of intrabony defects a split-mouth study in humans. braz j oral sci. 2010; 9: 108-14. 23. rothamel d, schwarz f, herten m, ferrari d, mischkowski ra, sager m, et al. vertical ridge augmentation using xenogenous bone blocks: a histomorphometric study in dogs. int j oral maxillofac implants. 2009; 24: 243-50. 24. sallum ea, pereira lss, caffesse rg, nociti fh, casatti mz, sallum, aw. gtr in class iii furcation defects with resorbable polylactic acid membranes. a histomorphometric study in dogs. braz j oral sci. 2002; 1: 76-83. 25. markou n, pepelassi e, kotsovilis s, vrotsos i, vavouraki h, stamatakis hc. the use of platelet-rich plasma combined with demineralized freezedried bone allograft in the treatment of periodontal endosseous defects: a report of two clinical cases. j am dent assoc. 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12(1):66-70 oral sciences n3 braz j oral sci. 14(1):23-26 original article braz j oral sci. january | march 2015 volume 14, number 1 gender evaluation in human beings by occlusal radiographs letícia ferreira dos santos1, rodrigo galo2, ricardo henrique alves da silva1 1universidade de são paulo, school of dentistry of ribeirão preto, department of stomatology, public health, and forensic dentistry, ribeirão preto, sp, brazil 2universidade de são paulo – usp, school of dentistry of ribeirão preto, department of dental materials and prosthodontics, ribeirão preto, sp, brazil correspondence to: ricardo henrique alves da silva faculdade de odontologia de ribeirão preto – usp departamento de estomatologia, saúde coletiva e odontologia legal avenida do café, s/n, bairro monte alegre cep: 14040-904 ribeirão preto, sp. phone:+ 55 16 3315 3969 fax:+ 55 16 3633 0999 e-mail: ricardohenrique@usp.br abstract aim: to analyze the dental arches by occlusal radiographs for possible gender differentiation. methods: one hundred occlusal radiographic images were obtained. first, a median line and a line touching the vestibular portions of the projections of the maxillary central incisors were drawn. the intersection of these lines was called point i and from this point a line to the canine most vestibular portion was drawn, from the second premolar and second molar, from the left and right sides, in the maxillary and mandibular arches, thus obtaining the angle measurements. line measurements relative to the width of the arches in three segments, canines, premolars and second molars were made. results: the analyses performed by t-test indicated that all linear values obtained were statistically different (p<0.05), as may be seen in the values obtained from canine to canine, in which the female average was 3.48 cm (0.16) while the male average was 3.74 cm (0.21) and in respect to the angle measurements, only two angles have shown applicability in the definition of gender. conclusions: gender can be determined by analysis of occlusal radiographs with greater reliability when linear measurements are performed. keywords: forensic anthropology; forensic dentistry; radiography; gender identify. introduction the need of identifying people not only reflects a social demand; but also follows mainly legal requirements1. to give someone an identity is to recognize this person’s fulfillment of rights and duties. therefore, proving someone’s identity is not restricted to the legal sphere but also involves civil interests. human identification can be done by different methods like fingerprint analysis, the most used when soft tissues are preserved. although situations in which the corpse is charred or skeletonized, an anthropological and dental analysis may be necessary in order to determine the person’s identity2. it is known that in cases where first the corpses were considered unrecognizable, medical and dental records (radiographs) can assist in a relevant way the human identification processes3. however, when human bones are found and the gender needs to be identified, first is taken into consideration the pelvic girdle which gives a great number of qualitative and quantitative data for gender determination, although, in some cases, the coroner has only the skull or just a part of it, which provides some data to assume genders. therefore, the aim of tis study was to analyze dental arches to verify measures related to gender differentiation, taking into consideration anterior, central and posterior portions by linear and angular measurements. received for publication: january 14, 2015 accepted: march 04, 2015 2424242424 braz j oral sci. 14(1):23-26 material and methods the project was approved by the institutional research ethics committee, under the protocol (#0022.0.138.000-10). a total of 50 subjects were selected from a population of 360 dental students, by simple random sampling method. the study group included 25 females (22.12±2.50 years old) and 25 males (21.92±2.58 years old) with an age range of 19-27 years. the subjects were previously informed of all the procedures related to the research and the inclusion criteria were acceptance to participate in the projects’ activities, signing of an informed consent form, as well as fitting to the description above. all 50 subjects enrolled in the study had no morphological abnormalities in their teeth and had fully erupted dentition without attrition, caries and were never submitted to dental extractions (except for the third molar in any dental arch), avoiding possible changes in mandibular and maxillary dental arches anatomy. one hundred occlusal radiographs were taken (spectro 70x; dabi atlante®, ribeirão preto, sp, brazil) following the technique developed by saliba4 in order to obtain centralized and standardized radiographic images of anterior teeth up to the second maxillary and mandibular molar. all occlusal radiographs of the maxillary arch were obtained using occlusal type films (eastman kodak co, rochester, ny, usa), with 1.5 s exposure time and 70 kpv radiation, 40 cm distance between the x-ray source and the x-ray film, with 1.5 s exposure time. in all radiographs, protection rules for the patient were respected, with the use of lead rubber aprons. for the radiographs of maxillary and mandibular arches, the x-ray film was placed in the machine in a way that it was perfectly perpendicular to the main x-ray beams. the patient was told to keep maxillary and mandibular teeth closed, biting the plates of the machine. after getting the radiographic images 4 the analysis started attributing capital letters to the maxillary arch and small letters to the mandibular arch and the letters l indicating the left side and r indicating the right side, as fig. 1 – schematic illustration. angular measurements. seen in figure 1. a median line and a line touching the vestibular portions of the projections of the maxillary central incisors were drawn. the intersection of these points was called point “i”. from this point, another line was drawn towards the most vestibular portion of the canine tooth (“c” point), and this line was named linear measure “ic”. the angle formed by these two points and “a” point, locating in the line that coincides with the union of the palatal processes of maxillary bone, i.e. medial line, was named “cia” (figure 1). following the same methodology, a line from “i” point to the most vestibular portion of the second pre-molar (“p” point) was drawn, named linear measure “ip”. the angle obtained by these two points and “b” point, locating in the line that coincides with the union of the palatal processes of maxillary bone, i.e. medial line, was called “pib” (figure 1). at last, the line starting from “i”, following until the second molar vestibular side most prominent radiograph projection (“m” point) was named “im”. the angle obtained by these two points and “c” point, locating in the line that coincides with the union of the palatal processes of maxillary bone, i.e. medial line, was named “mic” (figure 1). then the same drawings were made in the mandibular arch, following the same sequence but naming the abbreviations in low case letters, so the angle measures became “cia”, “pib”, “mic” and linear measures became “ic”, “ip” and “im”. measures related to the maxillary and mandibular dental arches width were made in three portions (figure 2): (i) canine portion, linear measure “cc” (maxillary arch) and “cc” (mandibular arch), corresponding to a line drawn from the most vestibular portions of the left and right canines’ radiograph projections; (ii) premolars portion, linear measure “pp” (maxillary arch) and “pp” (mandibular arch), corresponding to a line drawn from the most vestibular portions of left and right second premolars radiographic projections; (iii) second molars portion, linear measure “mm” (maxillary arch) and “mm” (mandibular arch), corresponding to a line drawn from the most vestibular fig. 2 – schematic illustration. linear measurements. gender evaluation in human beings by occlusal radiographs “ c i a ” r “pib”r “ m i c ” r “ c i a ” l “pib”l “ m i c ” l “cia”r “ p i b ” r “ m i c ” r “cia”l “ p i b ” l “ m i c ” l male mean 32.68 59.16 77.72 34.44 59.44 78.52 17.92 49.80 74.28 22.12 52.48 75.76 s d 5.07 4.29 2.76 5.25 3.83 2.98 6.42 5.68 3.54 6.59 4.49 3.45 female mean 32.20 59.48 77.50 31.90 59.18 76.40 20.56 49.24 73.72 27.64 54.40 76.88 s d 5.38 4.79 4.04 4.75 5.08 3.32 7.00 7.54 3.91 6.15 5.33 3.30 table 1 – table 1 – table 1 – table 1 – table 1 – means and standard deviations of angular measurements, male and female, ribeirão preto, brazil. c c pp m m cc p p m m male mean 3.7400 5.1840 6.3920 3.0480 4.6880 6.2000 s d 0.21409 0.28089 0.23615 0.19391 0.23695 0.20817 female mean 3.4880 4.8840 5.9320 2.8400 4.4280 5.8080 s d 0.16912 0.27031 0.39021 0.13540 0.24752 0.31744 table 2 table 2 table 2 table 2 table 2 – means and standard deviation of linear measurements, male and female, ribeirão preto, brazil. portions of left and right second molar radiographic projections. experimental data were statistically analyzed by anova, since data distribution was normal, to check gender influence in human identification. t test (p<0.05) was used as comparison basis. statistical analysis was made with the software spss for windows, version 12.0 (spss inc., chicago, il, usa). results means and standard deviations obtained for male and female research subjects can be seen in tables 1 and 2. linear variables analyzed by t-test were statistically significant (p<0.05) for gender differentiation, while angular values have shown statistical differences (p<0.05) between male and female only in “mic”l and “cia”l angle measures. discussion a human identification process must attend biological demands like oneness and immutability of analyzed characteristics and technical requirements such as classification and practicability of these findings5. based in these requirements, fingerprinting is the most used human identification technique around the world, although in situations where the corpses have significant changes or soft tissues destruction, anthropology and forensic dentistry5 can be used together, giving more reliable results. dna is also is commonly used as well, with excellent results, but depends specifically on structure and human resources. in an anthropological analysis the coroner uses statistical parameters, associating qualitative and quantitative characteristics to a determined populational group, mainly in the attempt of estimating data like: species, gender, ethnical group, age, height and weight6, searching the establishment of general characteristics for a general identification, since in these cases there are no data before the person’s death7. gender estimation must be the first item to be stabilished in an identification and the skull is the focus regarding gender differentiation by morphological aspects observation or qualitative and metric or quantitative8. most studies on gender differentiation in human skulls refer to qualitative variables as glabella characteristics, bone surface and muscle inserts9, superciliary arches10 and frontonasal joint2. however, not always skulls from male subjects show standardized characteristics, having some similarities with female skulls, which provide unreliable results8 and population differences11. as for the quantitative variables, different measures between pre-set points were studied by many authors, specially through skull radiographs in order to identify gender differentiation1,12-13, knowing that the skull isolated from the skeleton offers major difficulties in the identification process. it is valuable to highlight that differences between genders are not always perceptible, since there are features that overlap both genders. additionally, gender dimorphism does not express itself equally between individuals, it goes through morphological influences from the ethnic group, cultural habits and age14. recenetly, gender dimorphism has been studied with metric parameters and has the advantage of distinguishing gender dimorphism among different populations, with reliable results14. this is the goal of this research, that by linear and angular measures obtained from occlusal radiographs, was noticed a statistical difference between males and females, especially related to linear measures, demonstrating to be possible non-identified corpse gender identification with this analysis. gender dimorphism is the system to identify differences in shapes of men and women. in addition, gender identification allows the elimination of half the population, leading to a more precise search of the dead person’s identity14. some authors have used linear measures to determine gender, especially through craniometric measurements15, but there are few researches relating the size and shape of dental arches. merz16 studying the size of dental arches did not find significant differences between genders, which disagrees with this research. therefore the synergism of teeth size effect together with the arch shape may contribute to the differences between the studied groups, since caucasian women usually have smaller teeth and african descent males have the biggest teeth in the dental arches17. based in these data, relationship 2525252525gender evaluation in human beings by occlusal radiographs braz j oral sci. 14(1):23-26 2626262626 between arch size can vary among gender and ethnical group, indicating bigger sizes in males, as seen in the present study of linear measures. therefore, as demonstrated by dalidjan et al.18, discordance and a slight relationship between teeth size and arches shape can be observed. there are many things that influence the size of teeth and the shape of arches. dentofacial complex relationship, the tissue that involves them, occlusion, just as dental arches variability in shape and genetic component are related to the differences found in the maxilla and mandible18. coronal morphology and permanent teeth sizes are unchanged during growth and development process, except for specific conditions of nutritional abnormality or disorders inherited in other pathological conditions. thus, odontometry can be used in gender determination. finally, this is a simple, quick and accurate technique for gender determination, which is always population-specific, allowing gender determination through occlusal radiographs, highlighting the obtained linear values. acknowledgements to fapesp for financial support of this research. to mr. tarcísio josé rodrigues júnior (radiology technician) for the support in radiograph taking. references 1. borges sr. sexual dimorphic characteristics. quantitative variations in facial areas and distances, by means of lateral cephalometric radiographs in white adult. rev bras odontol. 1977; 31: 118-27. 2. gupta a, kumar k, shetty dc, wadhwan v, jain a, khanna ks. stature and gender determination and their correlation using odontometry and skull anthropometry. j forensic dent sci. 2014; 6: 101-6. 3. kirk nj, wood re, goldstein m. skeletal identification using the frontal sinus region: a retrospective study of 39 cases. j forensic sci. 2002; 47: 318-23. 4. daruge e, nouer df, saliba ta. the study of linear and angular measures of is dental superior and inferior arches and pericial important. braz dent j. 2000; 79: 1136-9. 5. robinson ms, bidmos ma. the skull and humerus in the determination of sex: reliability of discriminant function equations. forensic sci int. 2009; 186: 86-95. 6. panchbhai as. dental radiographic indicators, a key to age estimation. dentomaxillofac radiol. 2011; 40: 199-212. 7. passalacqua nv. forensic age-at-death estimation from the human sacrum. j forensic sci. 2009; 54: 255-62. 8. jaja bn, ajua co, didia bc. mastoid triangle for sex determination in adult nigerian population: a validation study. j forensic sci. 2013; 58: 1575-8. 9. celbis o1, iscan my, soysal z, cagdir s. sexual diagnosis of the glabellar region. leg med. 2001; 3: 162-70. 10. garvin hm, sholts sb, mosca la. sexual dimorphism in human cranial trait scores: effects of population, age, and body size. am j phys anthropol. 2014; 154: 259-69. 11. lopez mc, galdames ics, matamala daz, smith rl. sexual dimorphism determination by piriform aperture morphometric analysis in brazilian human skulls. int j morphol. 2009; 27: 327-31. 12. naikmasur vg, shrivastava r, mutalik s. determination of sex in south indians and immigrant tibetans from cephalometric analysis and discriminant functions. forensic sci int. 2010; 197: 122.e1-6. 13. ghoubril jv, abou obeid fm. three-dimensional facial architecture in normodivergent class i caucasian subjects. odontostomatol trop. 2013; 36: 5-14. 14. camps fe. gradwohl’s legal medicine. 3rd ed. wright and son: bristol john; 1976. 15. nascimento correia lima n, fortes de oliveira o, sassi c, picapedra a, francesquini l jr, daruge e jr. sex determination by linear measurements of palatal bones and skull base. j forensic odontostomatol. 2012; 30: 38-44. 16. merz m. tooth diameters and arch perimeters in a black and white population. am j orthod dentofac orthop. 1991; 100: 53-8. 17. altherr er, koroluk ld, phillips c. influence of sex and ethnic tooth-size differences on mixed-dentition space analysis. am j orthod dentofacial orthop. 2007; 132: 332-9. 18. dalidjan m, sampson w, townsend g. prediction of dental arch development: an assessment of pont’s index in three human populations. am j orthod dentofacial orthop. 1995; 107: 465-75. gender evaluation in human beings by occlusal radiographs braz j oral sci. 14(1):23-26 oral sciences n3 braz j oral sci. 11(2):116-119 received for publication: february 27, 2012 accepted: may 03, 2012 evaluation of linear tomography and cone beam computed tomography accuracy in measuring ridge bone width for planning implant placement arthur rodriguez gonzalez cortes1, andrea ferraz de arruda monteiro gomes2, maria josé albuquerque pereira de sousa e tucunduva1, emiko saito arita3 1dds, ms, graduate student, department of oral radiology, school of dentistry, university of são paulo, brazil 2dds, oral radiologist, radi institute, brazil 3dds, ms, phd, associate professor, department of oral radiology, school of dentistry, university of são paulo, brazil correspondence to: arthur rodriguez gonzalez cortes departamento de radiologia,faculdade de odontologia,universidade de são paulo av. prof. lineu prestes, 2227 cep: 05508-000 são paulo, sp brazil phone/fax: +55 11 30917831 e-mail: arthuro@usp.br abstract the development of oral implantology has led to the establishment of various image-acquisition methods as important surgical diagnosis tools, such as linear (lt) and cone beam computed tomography (cbct), indicated for planning implant placement surgeries. however, there still is little information in the literature regarding details on the difference between the accuracy of these methods. aim: the aim of the present study was to assess the difference between the accuracy of lt and cbct in measuring ridge bone width. methods: a sample of ten human skulls was used, totaling 40 edentulous sites, marked with 2-mm gutta-percha balls in the buccal and lingual plates. buccal-lingual measurements of ridge width were performed on the images of both tomography types. direct caliper measurements were used as control values, to which all lt and cbct measurements were compared. results: cbct images showed significantly more accurate results in comparison with the direct caliper measurements (p<0.05). conclusions: cbct proved more reliable than lt regarding ridge bone measurements for dental implant planning. keywords: linear tomography, cone beam computed tomography, dental implant, surgical planning. introduction with the development of implant-related treatments, presurgical assessment has became critical to evaluating the dimensions of the available alveolar bone, and to locating important anatomical structures, such as the mandibular canal, especially in cases with atrophic alveolar ridges and great bone loss1. standard panoramic and periapical radiographs do not provide cross-sectional information, and are therefore insufficient for implant site evaluation2-3. tomographic images are useful for assessing information on ridge measurements three-dimensionally, considered essential for the surgical planning of implant placement4-5. linear tomography (lt) is an accessible radiographic technique used for the cross-sectional imaging of edentulous jaws and is relatively inexpensive compared original article braz j oral sci. april | june 2012 volume 11, number 2 braz j oral sci. 11(2):116-119 117117117117117 with computed tomography (ct). it has the advantage of exposing the patient to less radiation than ct when used for a single site6. in the last decade, the development of volumetric tomography devices – considered important techniques for using a three-dimensional image navigation system – allowed the professional to perform image-guided surgical planning and to fabricate a stereolithographic surgical guide7-8. cone beam computed tomography (cbct) units have been widely used in maxillofacial studies. compared with other ct methods, cbct offers advantages such as reduced effective radiation doses, shorter acquisition scan times, easier imaging, and lower costs9-10. the buccal-lingual ridge width has been described as an important measurement for implant treatment planning, since it can be used as a parameter to evaluate and compare the accuracy of different image-acquisition methods 11-12. furthermore, although radiographic methods have been compared and evaluated for the surgical planning of implant placement cases13-14, there still is little information in the literature regarding details on the difference between the accuracy of commonly used tomographic methods, lt and cbct, in measuring ridge width. thus, the objective of the present study was to compare the accuracy of lt and cbct in measuring the ridge bone width of edentulous sites in human skulls. material and methods ten human dry skulls were used as a sample, in which two edentulous sites in the maxilla and two in the mandible were selected, totaling 40 sites. measurements were conducted according to a previously described methodology 15. the skulls were marked with 2-mm gutta-percha balls, one in the buccal plate and other in the lingual plate. the balls were placed at two points located 3 to 8 mm perpendicularly away from the crest of the ridge. the buccal-lingual ridge width was calculated by measuring the distance between the two gutta-percha balls, performed for each of the edentulous sites evaluated. the skulls were supported on the lt and cbct devices by a chin holder and a head strap. the orientation of the skull for scan was with the frankfort horizontal parallel to the floor and the midsagittal plane perpendicular to the floor. direct caliper measurements were performed and considered the control values of this study, to which all lt and cbct measurements were compared. lt images were obtained with a panoramic machine that has a linear tomographic function (vera view scope x600, morita, tokyo, japan), with exposure conditions of 60 kv and 4 ma and with a 0.5-mm copper filter. the tomographic projection angle was set at 60º with nominally 1.0-mm thick slices at 1.0-mm intervals. the cross-sectional images obtained were processed and used to perform buccallingual measurements (figure 1), which were traced on acetate paper using a clear plastic ruler and a mechanical #2 lead pencil. the measurements obtained were adjusted for magnification error. a cbct unit (classic i-cat, image sciences international, hatfield, pa, usa) was used and configured with a diagnostic protocol used for dental implants (0.25mm voxel, 120kvp, 3 to 8ma), in order to obtain digital cross-sectional images (figure 2), in which buccal-lingual measurements were performed by using ct imaging software (implantviewer 2.709, anne solutions, são paulo, sp, brazil) on a personal computer (figure 3). fig.1. a. lt cross-sectional image of a marked maxillary edentulous site used to perform measurements. b. indication of measurement (asterisk) position between the buccal and lingual markers in the same image. fig. 2. cbct cross-sectional image of a marked mandibular edentulous site, which was digitally measured by using the imaging software. fig. 3. screen view of the ct imaging software used to perform cbct measurements. all caliper, lt and cbct measurements (in millimeters) were recorded separately in a random order by two trained independent observers. measurement reproducibility was evaluation of linear tomography and cone beam computed tomography accuracy in measuring ridge bone width for planning implant placement braz j oral sci. 11(2):116-119 118118118118118 assessed by having each observer repeat both caliper and cbct measurements separately after a 2-week interval to eliminate memory bias. measurement reliability assessment of replicate measurements was made using the concordance correlation coefficient (ccc). one observer served as the main observer and intra-observer reliability was estimated between measurements performed 2 weeks apart. for the purpose of data analysis, deviations in lt and cbct measurements compared to direct caliper measurements were recorded and grouped into four different deviation degrees: up to 0.5 mm, from 0.51 to 1 mm, from 1.01 to 2 mm, and over 2 mm. the statistical analysis was performed using the wilcoxon t test, a hypothesis test indicated to detect possible significant differences between two related samples or repeated measurements on a single sample, and commonly mentioned in articles on accuracy comparisons between different imageacquisition methods13,16. a p value under 0.05 was considered a statistically significant difference. results comparison between data for image-acquisition methods and direct caliper control measurements are shown in table 1. intra-observer reproducibility was confirmed, insofar as the ccc ranged between 0.83 and 0.91 for the measurements. in assessing the buccal-lingual measurements, cbct presented more accurate results than lt, in comparison with the control measurements. this fact was confirmed by statistical analysis, insofar as a p value under 0.05 was found for the comparison between lt and direct caliper measurements. additionally, most edentulous sites analyzed (62.5%) showed differences from 0 to 0.2 mm between cbct and direct caliper measurements. positive deviations (larger values for tomographic images than for direct caliper measurements) were more frequent than negative deviations in both image-acquisition methods (67% of the analyzed cases). however, negative deviations were observed more often in cbct images than in lt images. discussion the lower consistency of lt measurements in comparison with cbct measurements, observed in the present study, was in agreement with the findings of a previous investigation, which compared measurement results among tomographic techniques for dental implant planning, not including cbct, as the analysis showed that lt results were less accurate in comparison with all ct methods analyzed13. the importance of evaluating ridge bone dimensions in cases of dental implant surgeries has been described in the literature7-9. as observed in the present study, cbct has been regarded as a high-quality reliable image-acquisition method for the dentomaxillofacial area, in comparison with other tomographic methods 16-17. however, a study comparing different ct methods found that a protocol of multidetector ct yielded more accurate linear measurements in comparison with those performed by cbct images18. for the purpose of evaluating preoperative assessment accuracy with tomographic images, lt and cbct buccallingual measurements have also been compared with ridgemapping preoperative buccal-lingual measurements, performed after penetration of patients’ soft tissues with calipers11-12. a study on lt found no significant differences between lt and ridge-mapping measurements, although both methods underestimated ridge dimensions11. on the other hand, a similar study on cbct found that ridge-mapping preoperative measurements were more consistent than cbct measurements 12. the findings of the above-mentioned studies, taken together, contrast with those of the present study, insofar as cbct images provided reliable and more accurate measurements in comparison with lt, supporting the fact that there is still controversy in the literature on image-acquisition methods for measuring ridge width. the findings of the present study are in agreement with those of a recent similar study on cbct measurements in human dry skulls, which indicated the effectiveness of both 2-d and 3d cbct reconstructions in measuring specific distances19. the authors also stated that skull orientation during cbct scanning has not been found to affect accuracy or reliability of measurements, as also observed in the present study. in conclusion, cbct was presented as a more accurate image-acquisition method in comparison with lt in providing information on ridge width measurements, which important to perform a precise surgical planning for implant placement. references 1. simon bi, von hagen s, deasy mj, faldu m, resnansky d. changes in alveolar bone height and width following ridge augmentation using bone graft and membranes. j periodontol. 2000; 71: 1774-91. 2. lecomber ar, downes sl, mokhtari m, faulkner k. optimisation of patient doses in programmable dental panoramic radiography. dentomaxillofac radiol. 2000; 29: 107-12. image-acquisition methods lt cbct number of sites showing deviation degrees in comparison with direct caliper measurements up to 0.5 mm 21 (52.5%) 36 (90%) from 0.51 to 1mm 15 (37.5%) 4 (10%) from 1.01 to 2 mm 3 (7.5%) over 2 mm 1 (2.5%) p=0.004 p=0.244 p* table 1 comparison between data for image-acquisition methods and direct caliper control measurements. lt = linear tomography; cbct= cone beam computed tomography. *significant level according to the wilcoxon t test. evaluation of linear tomography and cone beam computed tomography accuracy in measuring ridge bone width for planning implant placement 119119119119119 braz j oral sci. 11(2):116-119 3. danforth ra, dus i, mah j. 3-d volume imaging for dentistry: a new dimension. j calif dent assoc. 2003; 31: 817-23. 4. jeffcoat m, jeffcoat rl, reddy ms, berland l. planning interactive implant treatment with 3-d computed tomography. j am dent assoc. 1991; 122: 40-4. 5. cavalcanti mg, ruprecht a, vannier mw. 3d volume rendering using multislice ct for dental implants. dentomaxillofac radiol. 2002; 31: 21823. 6. dula k, mini r, van der stelt pf, buser d. the radiographic assessment of implant patients: decision-making criteria. int j oral maxillofac implants. 2001; 16: 80-9. 7. wanschitz f, birkfellner w, figl m, patruta s, wagner a, watzinger f et al. computer-enhanced stereoscopic vision in a head-mounted display for oral implant surgery. clin oral implant res. 2002; 13: 610-6. 8. sarment dp, sukovic p, clinthorne n. accuracy of implant placement with a stereolithographic surgical guide. int j oral maxillofac implants. 2003; 18: 571-7. 9. scarfe wc, farman ag, sukovic p. clinical applications of cone beam computed tomography in dental practice. j can dent assoc. 2006; 72: 75-80. 10. hirsch e, wolf u, heinicke f, silva ma. dosimetry of the cone beam computed tomography veraviewepocs 3d compared with the 3d accuitomo in different field of views. dentomaxillofac radiol. 2008; 37: 268-73. 11. perez la, brooks sl, wang hl, eber rm. comparison of linear tomography and direct ridge mapping for the determination of edentulous ridge dimensions in human cadavers. oral surg oral med oral pathol oral radiol endod. 2005; 99: 748-54. 12. chen lc, lundgren t, hallström h, cherel f. comparison of different methods of assessing alveolar ridge dimensions prior to dental implant placement. j periodontol. 2008; 79: 401-5. 13. naitoh m, katsumata a, kubota y, ariji e. assessment of three-dimensional x-ray images: reconstruction from conventional tomograms, compact computerized tomography images, and multislice helical computerized tomography images. j oral implantol. 2005; 31: 234-41. 14. aguiar mf, marques ap, carvalho ac, cavalcanti mg. accuracy of magnetic resonance imaging compared with computed tomography for implant planning. clin oral implants res. 2008; 19: 362-5. 15. berco m, rigali ph, miner m, deluca s, anderson nk, will la. accuracy and reliability of linear cephalometric measurements from conebeam computed tomography scans of a dry human skull. am j orthod dentofacial orthop. 2009; 136: 171-9. 16. al-ekrish aa, ekram m. a comparative study of the accuracy and reliability of multidetector computed tomography and cone beam computed tomography in the assessment of dental implant site dimensions. dentomaxillofac radiol. 2011; 40: 67-75. 17. hashimoto k, kawashima s, kameoka s, akiyama y, honjoya t, ejima k et al. comparison of image validity between cone beam computed tomography for dental use and multidetector row helical computed tomography. dentomaxillofac radiol. 2007; 36: 465-71. 18. loubele m, van assche n, carpentier k, maes f, jacobs r, van steenberghe d et al. comparative localized linear accuracy of small-field cone beam ct and multislice ct for alveolar bone measurements. oral surg oral med oral pathol oral radiol endod. 2008; 105: 512-8. 19. kamburoðlu k, kolsuz e, kurt h, kiliç c, özen t, paksoy cs. accuracy of cbct measurements of a human skull. j digit imaging. 2011; 24: 787-93. evaluation of linear tomography and cone beam computed tomography accuracy in measuring ridge bone width for planning implant placement oral sciences n3 original article braz j oral sci. july | september 2015 volume 14, number 3 masticatory performance with different types of rehabilitation of the edentulous mandible flávio domingues neves1, francielle alves mendes2, tânia de freitas borges2, daniela baccelli silveira mendonça3, marisa martins da silva prado4, karla zancopé1 1universidade federal de uberlândia ufu, school of dentistry, department of occlusion, fixed prosthodontics and dental materials, uberlândia, mg, brazil 2faculdade de patos de minas fpm, school of dentistry, department of prosthodontics, patos de minas, mg, brazil 3university of north carolina at chapel hill, laboratory of bone biology and implant therapy, department of prosthodontics, chapel hill, nc, usa 4universidade federal de uberlândia ufu, technical school of dentistry, uberlândia, mg, brazil correspondence to: flávio domingues das neves av. pará, 1720, bloco 4la sala 4la-42, campus umuarama, cep: 38405-320, uberlândia, minas gerais, brasil phone: +55 34 3218 2222 fax: +55 34 3218 2626 e-mail: neves@triang.com.br abstract aim: to compare the masticatory performance associated with different rehabilitation strategies for patients with edentulous mandibles. methods: one portion of the test food “optocal” was provided to groups: natural dentition (n = 15), mandibular fixed implant-supported prosthesis with maxillary fixed prosthesis (n = 8), mandibular fixed implant-supported prosthesis with maxillary removable prosthesis (n = 14), mandibular implant-retained overdenture with maxillary removable prosthesis (n = 16), and complete dentures (n = 16). the portion was collected after 40 chewing strokes, and then passed through a stack of eight sieves with decreasing apertures. masticatory performance was determined by weighing the portion of food on each sieve. results: the masticatory performance was: 71.00% for natural dentition, 41.57% for mandibular fixed implant-supported prosthesis with maxillary fixed prosthesis, 31.44% for mandibular fixed implant-supported prosthesis with maxillary removable prosthesis, 27.70% for mandibular implant-retained overdenture, and 14.33% for complete dentures. the data were statistically compared using student’s t-test (p < 0.05). natural dentition and complete denture groups were statistically different from all other groups, with the natural dentition and complete denture groups exhibiting the highest and lowest masticatory performance values, respectively. conclusions: osseointegrated implants improved the masticatory performance of all implant-supported groups compared to the complete dentures group. keywords: mastication; prostheses and implants; dental implants; dental prosthesis retention. introduction digestive enzymes and proper mastication must transform complex molecules into simple structures1. deficient masticatory function often forces an individual to choose foods that are easier to chew rather than foods that fulfill daily nutritional needs. complete denture wearers commonly avoid hard foods2, which typically leads to a poor-quality diet3. even when these patients try to eat hard foods, they swallow larger portions, which decreases nutrient absorption and may not provide enough nutrients to maintain good general health4. swallowing larger portions of food also increases the risk for gastrointestinal problems and diseases related to malnourishment3,5. however, gender-specific interventions and counseling aimed at slowing the rate of ingestion could be promising behavioral treatments for obese persons6. received for publication: may 27, 2015 accepted: august 05, 2015 braz j oral sci. 14(3):186-189 http://dx.doi.org/10.1590/1677-3225v14n3a02 187187187187187 for an edentulous patient, good masticatory performance is the most difficult function to reestablish. prior to the development of dental implants, the only option was complete dentures. oral rehabilitation using dental implants provides better retention and stability to prostheses. previous studies have evaluated the effect of missing teeth on masticatory function7-9, but comparisons among these studies are difficult because the methods were not standardized. the sieves method, use of natural test food, the optocal10 and optical scanning11 are the most usually employed test foods to evaluate masticatory efficiency. the aim of this study was to compare masticatory performance between different rehabilitations for patients with edentulous mandibles. the hypothesis of the present study was that masticatory performance in patients with complete dentures would be improved by dental implants to retain and/or support the prosthesis. material and methods the federal university of uberlândia research ethics committee approved this research (141/07). a written, informed consent was obtained from each subject before the tests. five groups of subjects were enrolled in this study: (nd) natural dentition or control group (n = 15, 20–28 years old), (isp-f) mandibular fixed implant-supported prosthesis with maxillary fixed prosthesis (n = 8, 55-–80 years old), (isp-r) mandibular fixed implant-supported prosthesis with maxillary removable prosthesis (n = 14, 55–80 years old), (od) mandibular implant-retained overdenture and maxillary complete denture (n = 16, 30–76 years old), and (cd) mandibular and maxillary complete dentures (n = 16, 30– 76 years old). all prostheses were made at the dental school of federal university of uberlândia 3 to 48 months before the tests were conducted. prior to implant treatment, rehabilitation strategies included partial or complete removable prostheses that were used for approximately 2 years. twelve patients in both isp groups received immediate loading of the implant prostheses. the control group comprised 15 subjects with natural dentition. subjects had all their second molars, no prior orthodontic treatment, and no signs or symptoms of traumatic occlusion, periodontal disease or temporomandibular dysfunction. exclusion criteria for the cd and isp groups were any signs or symptoms of temporomandibular dysfunction. methods used in this study have been previously published9. masticatory performance of all subjects was determined by chewing cubes of optocal test food for 40 sequential strokes. preparation of the optocal test food has been also already described9. after mixing all components, the homogenized paste was placed in an aluminum mold 12.5 ´ 12.5 cm and 0.56 cm deep. samples were placed in an electrical stove at 65 °c for 16 h to ensure complete polymerization. cubes were obtained using a device developed by the school of mechanical engineering of the federal university of uberlândia. shore a durometer (mitutoyo, são paulo, sp, brazil) was used to confirm the hardness of the optocal food, which ranged from 30 to 35 shore-a units. each portion consisted of seventeen cubes. during the experiment, subjects were comfortably seated in dental chair, and instructed to chew normally. the test food was provided in one portion of 17 cubes (~3 cm3). after 40 masticatory strokes the chewed particles were spit into a 300-ml plastic cup. the subject was then asked to rinse his/ her mouth carefully with water, which was spit into the same cup. the examiner confirmed clinically that no pieces of test food remained in the oral cavity. the triturated particles were disinfected with 2% chlorhexidine solution (pharmus, uberlândia, mg, brazil), according to a previous study12. the chewed particles were passed through a stack of eight sieves (bertel, são paulo, sp, brazil) with apertures that ranged from 5.6 to 0.5 mm. food particles were placed on the upper sieve and washed with 1000 ml of water for 30s. the sieves were then placed on a dental laboratory vibrator (vibramold, são paulo, sp, brazil) set at 1/2 speed for 2 min. chewed particles from each sieve were then collected and placed in individual rigid plastic recipients (2 cm diameter and 1 cm high). the particles were dried in an electrical stove at 60 °c for 3 h. after drying, the particles from each recipient were weighed on an analytic scale (sauter kg, ebingen, germany) with 0.0001 g accuracy. based on the weight of optocal retained on each sieve, the geometric mean diameter (gmd) of the chewed particles was calculated using excel (microsoft, redmond, wa, usa). superior masticatory performance was considered as breaking up the test food into smaller particles. to quantify particle reduction during chewing, it was necessary to define the maximal gmd value. it was therefore simulated a situation in which all particles remained intact after chewing and were retained on the first sieve (5.6 mm). the maximal particle gmd was defined as 6,660 µm. for each patient, the mean particle-reduction value was the difference between gmd and 6,660 µm (maximum possible gmd). to compare masticatory performances of isp-f, ispr, od, and cd groups to the control (nd), the particle reduction of the nd group was considered as 100%. since the data followed a normal distribution, a parametric test was used for the objective analysis. the differences between groups were analyzed using the student’s t-test (α≤ .05), performed with spss 15.0 (spss, chicago, il, usa). results the masticatory performance test revealed a statistically significant difference between nd and all other groups. cd group also exhibited a statistically significant difference in relation to all other groups. no significant difference was shown between isp-f group and isp-r or od. nd group demonstrated the highest value for masticatory performance and the cd group was the least effective (table 1). figure 1 shows the corrected masticatory performance values after the nd value was set as 100%. masticatory performance with different types of rehabilitation of the edentulous mandible braz j oral sci. 14(3):186-189 group natural dentition (nd) fixed implant-supported mandible prosthesis with fixed maxilla prosthesis (isp-f) fixed implant-supported mandible prosthesis with removable maxilla prosthesis (isp-r) implant-retained mandible overdenture (od) complete dentures (cd) mean ± sd (statistical categories)* 71.00 3.36 (a) 41.57 14.94 (b) 31.44 17.72 (b) 27.70 17.46 (b) 14.33 14.42 (c) *different letters indicate statistically significant differences (p < 0.05). table 1table 1table 1table 1table 1 – masticatory performance (%) associated with each group. fig. 1 – relative masticatory performance when the nd (control group) value was set to 100%. discussion the hypothesis of the present study was accepted, since the dental condition influenced the masticatory performance. the number of teeth rather than the extension of occlusal restoration13, the number of posterior teeth and the type of antagonist14 influence the chewing performance. subjects with natural dentition showed the highest level of masticatory performance2. to set the masticatory performance score of the nd group to 100%, a group of patients with natural dentition was selected for the present study, with the highest level of masticatory performance. it is important to emphasize that even the nd group did not achieve 100% masticatory performance (table 1), explaining the importance of higher numbers of chewing cycles before swallowing, decreasing the risk for gastrointestinal problems and diseases related to malnourishment3,5. for populations with fewer resources, complete dentures are often the only choice for edentulous mandible rehabilitation. therefore it is necessary to improve the conventional management of edentulous patients. although a large number of edentulous patients are satisfied using complete dentures, a small number of patients are unable to adapt15. the best option for these patients is the dental implant. for other patients, the knowledge of their level of masticatory performance could help them choose between treatment strategies, and a period of adaptation, before choosing a treatment with dental implants, should be considered16. when possible, the implant-retained overdenture should be offered to edentulous patient to provide a better masticatory condition rather than a complete denture. patients with complete dentures often complain about the lack of retention and stability. these events are associated with aging, loss of muscle strength, reduced salivary flow and other factors, often forcing individuals to change their dietary pattern by choosing soft foods. this could lead to patient malnutrition8. in this study, patients with complete dentures exhibited only 20% of the masticatory performance of individuals with natural dentition. this finding raises questions about the efficiency of complete dentures in providing satisfactory oral rehabilitation. oral rehabilitation with dental implants leads to better masticatory function in edentulous patients17. previous study18 revealed a decrease in electromyographic amplitude for the masseter muscles during swallowing in elderly individuals with mandibular fixed implant-supported prostheses. this may indicate adaptation to new conditions of stability provided by fixation of the complete denture in the mandibular arch. studies have demonstrated that the retention of complete dentures also influences the masticatory performance level of patients19, and treating complete denture wearers with implants to support the denture improves the chewing efficiency, increases maximum bite force and improves satisfaction20. a multidisciplinary approach is necessary to evaluate the general health of patients with “poor oral condition” and to determine how chewing performance has affected their systemic health. the digestive system depends on the stomatognathic system, and the dentist must inform patients 188188188188188masticatory performance with different types of rehabilitation of the edentulous mandible braz j oral sci. 14(3):186-189 189189189189189 about the importance and potential benefits of improving the retention and stability of complete dentures by installing two dental implants, at least. this is a simple treatment that could improve a patient’s masticatory and psychological condition19. the present study indicated that subjects with natural dentition exhibited better masticatory performance than completely edentulous patients, but the use of osseointegrated implants improved the masticatory performance of all implantsupported groups when compared to the cd group. acknowledgements the authors would like to thank adérito soares da mota and carlos josé soares for their assistance and critical reading of the manuscript. they also would like to thank gustavo mendonça for reviews and comments while preparing this manuscript, fapemig, capes, cnpq and nepro research group for their support. references 1. kay rf. the functional adaptations of primate molar teeth. am j phys anthropol. 1975; 43: 195-216. 2. manly rs, braley lc. masticatory performance and efficiency. j dent res. 1950; 29: 448-62. 3. hutton b, feine j, morais j. is there an association between edentulism and nutritional state? j can dent assoc. 2002; 68: 182-7. 4. n’gom p i, woda a. influence of impaired mastication on nutrition. j prosthet dent. 2002; 87: 667-73. 5. papas as, palmer ca, rounds mc, russell rm. the effects of denture status on nutrition. spec care dentist. 1998; 18: 17-25. 6. park s, shin ws. differences in eating behaviors and masticatory performances by gender and obesity status. physiol behav. 2015; 138: 69-74. 7. borges t de f, mendes fa, de oliveira tr, gomes vl, do prado cj, das neves fd. mandibular overdentures with immediate loading: satisfaction and quality of life. int j prosthodont. 2011; 24: 534-9. 8. borges t de f, mendes fa, de oliveira tr, do prado cj, das neves fd. overdenture with immediate load: mastication and nutrition. br j nutr. 2011; 105: 990-4. 9. mendonca db, prado mm, mendes fa, do prado cj, das neves fd. comparison of masticatory function between subjects with three types of dentition. int j prosthodont. 2009; 22: 399-404. 10. oliveira nm, shaddox lm, toda c, paleari ag, pero ac, compagnoni ma. methods for evaluation of masticatory efficiency in conventional complete denture wearers: a systematized review. oral health dent manag. 2014; 13: 757-62. 11. eberhard l, schneider s, eiffler c, kappel s, giannakopoulos nn. particle size distributions determined by optical scanning and by sieving in the assessment of masticatory performance of complete denture wearers. clin oral investig. 2015; 19: 429-36. 12. campos ss, pereira vp, zangerônimo mg, marques ls, pereira lj. influence of disinfectant solutions on test materials used for the determination of masticatory performance. braz oral res. 2013; 27: 238-44. 13. akeel r, nilner m, nilner k. masticatory efficiency in individuals with natural dentition. swed dent j. 1992; 16: 191-8. 14. julien kc, buschang ph, throckmorton gs, et al. normal masticatory performance in young adults and children. arch oral biol. 1996; 41: 69-75. 15. carlsson ge, omar r. the future of complete dentures in oral rehabilitation. a critical review. j oral rehabil. 2010; 37: 143-56. 16. farias-neto a, carreiro ada f. changes in patient satisfaction and masticatory 17. efficiency during adaptation to new dentures. compend contin educ dent. 2015; 36: 174-7; quiz 178,190. 18. vieira ra, melo ac, budel la, gama jc, de mattias sartori ia, thomé g. benefits of rehabilitation with implants in masticatory function: is patient perception of change in accordance with the real improvement? j oral implantol. 2014; 40: 263-9. 19. berretin-felix g, nary filho h, padovani cr, trindade junior as, machado wm. electromyographic evaluation of mastication and swallowing in elderly individuals with mandibular fixed implant-supported prostheses. j appl oral sci. 2008; 16: 116-21 20. thomason jm, feine j, exley c, moynihan p, müller f, naert i, et al. mandibular to implant-supported overdentures as the first choice standard of care for edentulous patients—the york consensus statement. br dent j. 2009; 207: 185-6. 21. boven gc, raghoebar gm, vissink a, meijer hj. improving masticatory performance, bite force, nutritional state and patient’s satisfaction with implant overdentures: a systematic review of the literature. j oral rehabil. 2015; 42: 220-33. masticatory performance with different types of rehabilitation of the edentulous mandible braz j oral sci. 14(3):186-189 oral sciences n3 braz j oral sci. 11(3):377-380 original article braz j oral sci. july | september 2012 volume 11, number 3 gender as risk factor for mouth breathing and other harmful oral habits in preschoolers lara jansiski motta1, thays almeida alfaya2, analúcia ferreira marangoni3, raquel agnelli mesquita-ferrari4, kristianne porta santos fernandes4, sandra kalil bussadori4 1phd, professor of pediatric dentistry of nove de julho university (uninove), são paulo, sp, brazil 2dds, graduate student of dental clinic, graduate program of federal fluminense university (uff), niterói, rj, brazil 3msc, private practice, são paulo, sp, brazil 4phd, professor of rehabilitation sciences, graduate program, nove de julho university (uninove), são paulo, sp, brazil correspondence to: thays almeida alfaya rua doutor calandrine, 235 a cep: 24755-160 são gonçalo, rj, brasil phone: +55 21 99515428 e-mail: thalfaya@gmail.com abstract aim: to analyze associations between the mouth-breathing pattern and other harmful oral habits among preschoolers. methods: an observational, cross-sectional study was carried out involving 198 children from 3 to five 5 of age. a questionnaire, clinical evaluation and specific tests (mirror and water tests) were used for confirmation of the mouth-breathing pattern. results: mean age of the participants was 4.13 ± 0.8 years and 57.1% were male. a total of 87.4% exhibited one or more harmful oral habits. harmful habits were more common in the male gender (61.8%); this association was statistically significant (p<0.001). mouth breathing was the most prevalent habit (49%), followed by biting/sucking on objects (33.3%). regarding gender, statistically significant associations were found for bottle feeding (p=0.02) and nail biting (p=0.02). mouth breathing was associated with biting on object (p=0.00), pacifier use (p=0.02) and thumb sucking (p=0.00). conclusions: the results of the present study suggest that mouth breathing is significantly associated with biting/sucking on objects, pacifier use and thumb sucking in preschoolers and that the occurrence of harmful oral habits is more prevalent among the male gender. early diagnosis and intervention should be established in order to avoid future consequences involving the orofacial region. keywords: mouth breathing, habits, child. introduction habit is defined as “a custom or practice acquired by the frequent repetition of a single act that begins in a conscious manner and subsequently becomes unconscious.” oral habits are classified as physiological and non-physiological. physiological habits include nasal breathing, chewing, speaking and swallowing. non-physiological habits are often called harmful or parafunctional and include thumb sucking, pacifier sucking, bottle feeding, the tongue placing pressure on the teeth and the mouth-breathing pattern1. particular habits may involve emotional factors2-3, such as anxiety4 and sensitivity to stress5. with regard to age, harmful oral habits are common among children and there is a lesser frequency among adolescents6. such habits have a direct influence over quality of life7 and can affect the stomatognathic system, leading to an imbalance between external and internal muscle forces1. this occurs received for publication: may 16, 2012 accepted: august 02, 2012 378378378378378 braz j oral sci. 11(3):377-380 when physiological tolerance is surpassed (response to the action performed), leading to alterations in the dentition, musculature and temporomandibular joint8. temporomandibular disorder may be caused by the intensity of the action of a particular harmful habit9-11. bone malformations may also result from the duration and frequency of harmful habits1. as such habits require a multidisciplinary approach to provide integral care to pediatric patients, the aim of the present study was to analyze associations between the mouth-breathing pattern and other harmful habits in children aged 3 to 5 years. material and methods an observational, cross-sectional study was carried out involving students enrolled in public preschools in the city of são roque, state of são paulo, brazil. convenience sampling was employed, with the evaluation of all children enrolled in the schools between three and five years of age in 2008. all parents/guardians received information regarding the objectives of the study and signed a statement of informed consent in compliance with resolution 196/96 of the brazilian national health council. this study received approval from the local human research ethics committee under process #82622/08. a questionnaire containing objective, closed-ended questions was used to gather information on age, gender and the presence of harmful oral habits. this questionnaire was filled out by the parents/guardians with no time constraint. the aim of the questionnaire was to investigate the presence/absence of harmful habits and associations between the mouth-breathing pattern and nail biting, biting/sucking on objects, thumb sucking, pacifier sucking, bottle feeding and bruxism (teeth clenching/grinding). children using systemic medications for treatment of the airway problems (colds and influenza) and those with orthodontic or orthopedic appliances of the maxillae were excluded from the study. besides the questionnaire, a clinical evaluation and specific tests (mirror and water tests) were performed by a single examiner who had undergone a training process and was supervised by an otolaryngologist for the confirmation of the mouth-breathing pattern. the clinical evaluation involved the determination of the presence/absence of the following signs: long face, drooping eyes, dark circles under the eyes, thin upper lip, dry lips, hypotonic lips, inverted lower lip, narrow nostrils, high-arched palate, inadequate lip seal and anterior open bite. the mirror test consisted of placing a two-sided mirror below the child’s nostrils and observing the formation of vapor condensation stemming from respiration. fogging on the upper part of the mirror indicates nasal breathing and fogging on the lower part or both parts indicates mouth breathing9. the water test was performed after the mirror test. for such, the child held a small amount of water in his/her mouth and maintained the lips in contact without swallowing for three minutes, during which time the effort of the lip commissure was observed. children who were unable to maintain the lips in contact for three minutes were considered mouth breathers12. data analysis was performed using the spss 17 program (ibm corp., chicago, il, usa) and involved the chi-squared (÷2) test and fisher’s exact test, with the level of significance set to 5% (p<0.05). results the sample was made up of 198 children between three and five years of age. mean age was 4.13 ± 0.8 years and 57.1% were male. a total of 87.4% (n = 173) exhibited one or more harmful oral habits: 29.3% (n = 58) had one habit, 30.8% (n = 61) had two habits, 18.2% (n = 36) had three habits and 9.1% (n = 18) had four habits. harmful habits were more common in the male gender (61.8%; n = 107) than the female gender (38.2%; n = 66). the association between the male gender and harmful habits was statistically significant (x 2 = 12.773, p<0.001). regarding the type of habit, the mouth-breathing pattern gender as risk factor for mouth breathing and other harmful oral habits in preschoolers mouth breathing biting/sucking on objects bottle feeding pacifier sucking nail biting thumb sucking bruxism (clenching and/or grinding) gender male female male female male female male female male female male female male female present n (%) 61 (54.0%) 36 (42.2%) 42 (37.2%) 24 (28.2%) 41 (35.3%) 18 (21.2%) 30 (26.5%) 20 (23.5) 18 (15.9%) 24 (28.2) 12 (10.6%) 11 (12.9%) 11 (9.7) 6 (7.1) absent n (%) 52 (46.0%) 49 (57.6%) 71 (62.8%) 61 (71.8%) 72 (63.7%) 67 (78.8%) 83 (73.5%) 65 (76.5%) 95 (84.1%) 61 (71.8%) 101 (89.4%) 74 (87.1%) 102 (90.3) 79 (92.2) total n (%) 113 (100%) 85 (100%) 113 (100%) 85 (100%) 113 (100%) 85 (100%) 113 (100%) 85 (100%) 113 (100%) 85 (100%) 113 (100%) 85 (100%) 113 (100) 85 (100) p-value 0.70 0.18 0.02* 0.62 0.02* 0.61 0.50 table 1: occurrence of harmful oral habits according to gender * statistically significant (p < 0.05) 379379379379379 braz j oral sci. 11(3):377-380 present absent total p-value n (%) n (%) n (%) biting/sucking on objects 24 (36.4%) 42 (63.6%) 66 (100%) 0.00* bottle feeding 30 (50.8%) 29 (49.2%) 59 (100%) 0.42 pacifier sucking 31 (62.0%) 19 (38.0%) 50 (100%) 0.02* nail biting 18 (42.9%) 24 (57.1%) 42 (100%) 0.23 thumb sucking 17 (73.9%) 6 (26.1%) 23 (100%) 0.00* bruxism (clenching and/or grinding) 12 (70.6%) 5 (29.4%) 17 (100%) 0.05 table 2: associations between mouth breathing and other harmful oral habits * statistically significant (p < 0.05) was the most prevalent (49%; n = 97), following by biting/ sucking on objects (33.3%; n = 66). gender was significantly associated with bottle feeding and nail biting (table 1). the mouth-breathing pattern was significantly associated with biting/sucking on objects, bottle feeding and thumb sucking (table 2). discussion the results of the present study suggest that the mouthbreathing pattern is significantly associated with biting/ sucking on objects, bottle feeding and thumb sucking and that the occurrence of harmful oral habits is more prevalent among the male gender. mouth breathing is a potential etiological factor for the alterations to the occlusion and normal facial growth. when combined with other parafunctional habits, the harm to the stomatognathic system is even greater. according to cattoni et al. (2007)13, children with this habit exhibit pathologic adaptations regarding postural and morphological characteristics of the stomatognathic system. thus, the early diagnosis of mouth breathing and proper intervention are suggested to avoid the emergence of orofacial abnormalities. a number of acute and chronic conditions can lead to the mouth breathing pattern. acute conditions included infectious processes and the introduction of foreign bodies. chronic conditions include choanal atresia, adenoid hypertrophy, chronic tonsil hypertrophy, nasal septum deformity, nose fracture, allergic rhinitis (and respective medication), polyps, tumors and narrow nasal cavities14. in order to avoid bias in the results of the present investigation, children in use of systemic medication for the treatment of the airways and those with orthodontic or orthopedic appliances on the maxillae were excluded from the study. however, non-reported chronic conditions could have led to the mouth-breathing pattern in the children evaluated. considering the aim of the study, the researchers did not seek to establish the reason for the habit, but rather its presence or absence. the present study found a high percentage of harmful habits among the children evaluated. similar findings are reported in the literature6,15-16. emodi-perlman et al. (2012)6 evaluated parafunctional habits in children in the primary and mixed dentition phases, reporting high prevalence rates for biting/sucking on objects and nail biting. the authors point out that stressful events in life are related to an increase in the number of harmful oral habits in children. bruxism was the least prevalent habit in the present study and was not associated with mouth breathing. while few studies have been carried out on this subject, serra-negra et al. (2010)17 report a high prevalence rate of this habit (33.0%). carra et al. (2011)15 assessed the prevalence and risk factors of sleep-related bruxism and wake-time tooth clenching in a population ranging from seven to 17 years of age who sought orthodontic treatment. the results indicate that sleep-time and wake-time parafunctions are often associated with signs and symptoms of temporomandibular disorder, sleep problems and behavioral problems and therefore merit attention during dental evaluations. one limitation of the present study was the failure to evaluate the presence of snoring, which is reported to be common in children. the repercussions of snoring are mainly related to cognitive development 18-19 and high blood pressure20. moreover, snoring may be common in individuals with the habit of bruxism21. in general, the presence of such habits can compromise the stomatognathic system1,8-11. a study involving both children and adults sought to determine associations between parafunctional habits and the emergence of temporomandibular disorder through the evaluation of the frequency of diurnal bruxism and nail biting, the results of which demonstrated that the female gender was at a significant risk for myofascial pain10. another study with the same line of reasoning found that parafunctional habits were associated to important symptoms of orofacial pain, indicating that such habits are risk factors for temporomandibular disorder. a study with a 20-year follow up found that parafunctional habits can be persistent, as angle class ii malocclusion and tooth wear in childhood were predictors of increased tooth wear in adulthood22. these findings underscore the importance of the early diagnosis of harmful oral habits and proper intervention in order to avoid future consequences involving the orofacial region. references 1. agurto pv, diaz rm, cadiz od, bobenrieth fk. oral bad habits frequency and its association with dentomaxilar abnormal development, in children three to six year old in santiago oriente. rev chil pediatr. 1999; 70: 47082. gender as risk factor for mouth breathing and other harmful oral habits in preschoolers 380380380380380 braz j oral sci. 11(3):377-380 2. castelo pm, barbosa ts, gaviao mb. quality of life evaluation of children with sleep bruxism. bmc oral health. 2010; 10: 16. 3. manfredini d, lobbezoo f. role of psychosocial factors in the etiology of bruxism. j orofac pain. 2009; 23: 153-66. 4. restrepo cc, medina i, patino i. effect of occlusal splints on the temporomandibular disorders, dental wear and anxiety of bruxist children. eur j dent. 2011; 5: 441-50. 5. serra-negra jm, paiva sm, flores-mendoza ce, ramos-jorge ml, pordeus ia. association among stress, personality traits, and sleep bruxism in children. pediatr dent. 2012; 34: 30-4. 6. emodi-perlman a, eli i, friedman-rubin p, goldsmith c, reiter s, winocur e. bruxism, oral parafunctions, anamnestic and clinical findings of temporomandibular disorders in children. j oral rehabil. 2012; 39: 126-35. 7. simoes-zenari m, bitar ml. factors associated to bruxism in children from 4-6 years. pro fono. 2010; 22: 465-72. 8. okeson jp. management of temporomandibular disorders and occlusion. 6. ed. saint louis: mosby; 2008. 9. cortese sg, biondi am. relationship between dysfunctions and parafunctional oral habits, and temporomandibular disorders in children and teenagers. arch argent pediatr. 2009; 107: 134-8. 10. michelotti a, cioffi i, festa p, scala g, farella m. oral parafunctions as risk factors for diagnostic tmd subgroups. j oral rehabil. 2010; 37: 157-62. 11. sari s, sonmez h. investigation of the relationship between oral parafunctions and temporomandibular joint dysfunction in turkish children with mixed and permanent dentition. j oral rehabil. 2002; 29: 108-12. 12. jorge ep, gandini júnior lg, santos-pinto a, guariza filho o, castro abbat. evaluation of the effect of rapid maxillary expansion on the respiratory pattern using active anterior rhinomanometry: case report and description of the technique. dental press j orthod. 2010; 15: 71-9. 13. cattoni dm, fernandes fd, di francesco rc, latorre mdo r. characteristics of the stomatognathic system of mouth breathing children: anthroposcopic approach. pro fono. 2007; 19: 347-51. 14. motonaga sm, berte lc, anselmo-lima wt. mouth breathing: causes and changes of the stomatolognathic system. braz j otorhinolaryngol. 2000; 66: 373-9. 15. carra mc, huynh n, morton p, rompre ph, papadakis a, remise c, et al. prevalence and risk factors of sleep bruxism and wake-time tooth clenching in a 7to 17-yr-old population. eur j oral sci. 2011; 119: 386-94. 16. medeiros pkm, cavalcanti al, bezerra pm, moura c. malocclusions, breast feeding and deleterious buccal habits preschool children: an association study. pes bras odontoped clin integr. 2005; 5: 267-74. 17. serra-negra jm, paiva sm, seabra ap, dorella c, lemos bf, pordeus ia. prevalence of sleep bruxism in a group of brazilian schoolchildren. eur arch paediatr dent. 2010; 11: 192-5. 18. 18piteo am, kennedy jd, roberts rm, martin aj, nettelbeck t, kohler mj, et al. snoring and cognitive development in infancy. sleep med. 2011; 12: 981-7. 19. 19piteo am, lushington k, roberts rm, van den heuvel cj, nettelbeck t, kohler mj, et al. prevalence of snoring and associated factors in infancy. sleep med. 2011; 12: 787-92. 20. li am, au ct, ho c, fok tf, wing yk. blood pressure is elevated in children with primary snoring. j pediatr. 2009; 155: 362-8. 21. ng dk, kwok kl, poon g, chau kw, 2002. habitual snoring and sleep bruxism in a paediatric outpatient population in hong kong. singapore med j. 2001; 43: 554-6. 22. carlsson ge, egermark i, magnusson t. predictors of bruxism, other oral parafunctions, and tooth wear over a 20-year follow-up period. j orofac pain. 2003; 17: 50-7. gender as risk factor for mouth breathing and other harmful oral habits in preschoolers 429 too many requests error 429 too many requests too many requests guru meditation: xid: 32663444 varnish cache server oral sciences n3 braz j oral sci. 13(3):203-208 original article braz j oral sci. july | september 2014 volume 13, number 3 influence of mouthwashes on the physical properties of orthodontic acrylic resin fabrício mezzomo collares, flavia veronezi rostirolla, érika de oliveira dias de macêdo, vicente castelo branco leitune, susana maria werner samuel universidade federal do rio grande do sul – ufrs, school of dentistry, area of dental materials, porto alegre, rs, brazil correspondence to: fabrício mezzomo collares área de materiais dentários universidade federal do rio grande do sul rua ramiro barcelos, 2492 rio branco cep 90035-003 porto alegre, rs, brasil phone: +55 51 3308 5198 e-mail: fabricio.collares@ufrgs.br received for publication: june 22, 2014 accepted: september 02, 2014 abstract aim: the aim of this study was to evaluate the influence of acrylic resin immersion in different mouthwashes on hardness, roughness and color. methods: specimens of an orthodontic selfcured acrylic resin (orto clas®) were produced and immersed in five mouthwashes: plax® classic (colgate®); plax® alcohol-free (colgate®); listerine® (johnson & johnson®); periogard® (colgate®) and periogard® alcohol-free (colgate®). nine different immersion times were studied: 1, 2, 4, 6, 8, 10, 12, 24 h and 7 days, totalizing 45 groups. the specimens were evaluated before and after immersion by knoop microhardness (n=5), roughness in ra parameter (n=5), and colorimetric analysis, cielab (n=3). results: all mouthwashes softened the acrylic resins after 7 days of immersion. plaxò alcohol-free showed no statistically significant difference of softening between the immersion times. listerine® showed softening after immersion at all times. plax® alcohol-free and listerine® showed significantly increased values of roughness after 12 h of immersion (p<0.05). listerine® presented a significant increase in color variation after 12 h of immersion. conclusions: immersion in mouthwashes could influence acrylic resin hardness, roughness and color. keywords: acrylic resins; mouthwashes; hardness. introduction orthodontic treatment for myofunctional therapies and small movements increase the surface microbial contamination of orthodontic appliances in the oral cavity1. biofilm formation occurs on orthodontic wires2, clasps and springs3 and acrylic baseplates4. acrylic removable devices are more prone to colonization due to subsurface porosities5. to avoid the biological proliferation tooth brushing seems to be an adequate way to perform the removal of the biofilm attached on the acrylic surface . however, factors such as age and manual dexterity of patients may interfere in the quality of its results6. the use of antimicrobial agents, such as mouthwashes, can help maintaining the oral health of removable orthodontic appliance users bycontrolling the growth of biofilm7. the most commonly used mouthwashes for biofilm control due to their efficiency, are chlorhexidine8, triclosan9, cetylpyridinium chloride10 and essential oil-based11 solutions. on the other hand, a disinfectant for removable acrylic appliances should have no negative influence on the materials’ properties after immersion. immersion in solutions may cause plasticization of polymer chains, leading to material degradation by increased water sorption and solubility12. acrylic resin used for removable orthodontic appliances is a material that must fulfill certain requirements for use, such as dimensional and color stability13, and ability to be polished and disinfected14. chemical disinfectants such as braz j oral sci. 13(3):203-208 chlorhexidine, peroxides, sodium perborate, sodium hypochlorite and glutaraldehyde have shown surface and property alterations, such as transverse strength6,15, color stability16, roughness16-17 and hardness17 of acrylic resins subjected to chemical disinfection. an alternative for disinfection of acrylic baseplates is the home-care chemical solutions4. however, to the best of our knowledge there is no study evaluating long-term immersion of orthodontic acrylic resin devices in mouthwashes. therefore, the aim of this study was to evaluate the influence of mouthwashes on the properties of orthodontic acrylic resin. the null hypothesis tested is that the different solutions and immersion times have no influence on the microhardness and roughness of acrylic resin. material and methods test specimen production a self-cured acrylic resin (orto clas®; são paulo, sp, brazil) was manipulated according to the manufacturer’s instructions and 360 square-shaped specimens (10x10x5 mm) were obtained. the sample consisted of 225 specimens for the microhardness and roughness tests (n=5) and 135 specimens for the color test (n=3). after polymerization, all specimens were visually inspected, and checked for a smooth surface without voids or porosity. samples with voids or porosity were discarded. the specimens for the microhardness and roughness tests were embedded in a self-cured acrylic resin (jet; campo limpo paulista, sp, brazil), in order to prevent any problems with the alignment that could interfere in the tests. the samples for knoop hardness and roughness tests had the acrylic resin excess removed during polishing using progressively 600to 1200–grit silicon carbide paper (3m do brasil; são paulo, sp, brazil). to obtain a smooth and flat surface, the specimens were finished with 220, 400 and 600-grit sandpaper (norton abrasives; saint-gobain, vinhedo, sp, brazil) impregnated with a diamond suspension in a polishing machine (arotec; cotia, sp, brazil) under water cooling. the specimens for color analysis were not polished. five commercial mouthwashes, plax® classic (colgate®), material plax® classic plax® alcohol-free listerine® cool mint periogard® periogard® alcohol-free composition aqua, sorbitol, alcohol, glycerin, sodium lauryl sulfate, sodium methyl cocoyl taurate, pvm/ma copolymer, aroma, dosodium phosphate, sodium hydroxide, triclosan, sodium saccharin, cl 16035 aqua, glycerin, propylene glycol, sorbitol, peg-40 hydrogenated castor oil, aroma, phosphoric acid, sodium benzoate, cetylpiridinium chloride, sodium fluoride 0.05%, sodium saccharin thymol, eucalyptol, methyl salicylate, menthol, aqua, sorbitol, alcohol, poloxamer 407, benzoic acid, aroma, sodium saccharin, sodium benzoate, ci 42053 0.12% chlorhexidine digluconate, 11.6% alcohol, glycerin, peg-40 sorbitan diisostearate, flavor, sodium saccharin, and fd&c blue no. 1. aqua, glycerin, sorbitol, peg-40, hydrogenated castor oil, chlorhexidine digluconate, aroma, citric acid, cl 42090 batch numbers br122a br123b 3140b13 12br121a br123a table 1. table 1. table 1. table 1. table 1. mouthwashes, composition and batch numbers of used solutions. plax® alcohol-free (colgate®), listerine® cool mint (johnson & johnson®), periogard® (colgate®) and periogard® alcoholfree (colgate®) were used. the used mouthwashes, as well as their composition and batch numbers, are shown in table 1. softening after immersion the initial and final surface knoop microhardness of each acrylic resin specimen was determined by using a hardness tester (hmv-2, shimadzu, tokyo, japan). the longest diagonal of the indentation was measured and its value was used in the following formula to calculate khn: khn = [(14228 c)/(d2)] where: c = applied load in g d = length of the longest indentation diagonal in mm 14228 = constant number at first, 3 indentations were made on the surface of each specimen using a 15 g load for 10 s and the mean value from the 3 indentations was considered the specimen’s khn value. after the first measurement, the specimens were randomly divided into 45 groups (n=5) in accordance with the immersion time and the tested mouthwashes. the groups were immersed in 30 ml of plax® classic, plax® alcoholfree, listerine®, periogard® and periogard® alcohol-free mouthwashes for 1, 2, 4, 6, 8, 10, 12, 24 h and 7 days. after mouthwash immersion the specimens were washed with distilled water for 10 s and dried with compressed air for 1 minute. then, the second microhardness measurements were made as described for the initial measurements. the softening of acrylic resin (∆khn) was recorded as the percent difference between final and initial knoop microhardness (khnf khni). roughness mean surface roughness from each specimen was obtained from 3 measurements in different areas using a roughness surface analyzer (sj-201 mitutoyo, tokyo japan). the surface roughness ra is the value assigned to the area peaks and valleys divided by the distance traveled by the sensor in a straight line, providing roughness ra in µm. the roughness surface analyzer provides the average of three 0.25 204204204204204 influence of mouthwashes on the physical properties of orthodontic acrylic resin braz j oral sci. 13(3):203-208 205205205205205 µm lines. the specimens for the roughness test were the same as those used in the microhardness test (figure 1). after the initial measurements were made, the specimens were randomly divided into 45 groups (n=5) in accordance with the time of immersion and the tested chemical solutions. the specimens from each group were immersed in 30 ml of each mouthwash for 1, 2, 4, 6, 8, 10, 12, 24 h and 7 days. after the mouthwash immersion each specimen was washed with distilled water for 10 s and dried with compressed air for 1 min. then, the second roughness measurements were made in the same way as described for the initial measurement. the difference was calculated. colorimetric analysis the 135 specimens were randomly divided in 5 groups (n=45), each one subdivided into 9 (n=3), as for the roughness and hardness evaluation. color measurements (cm2600, konica minolta, osaka, japan) were made before and after the immersion times. color was assessed by measurements of l*a*b* coordinates with d65 cie illuminant (average daylight) and a 10 degree viewing angle geometry. l* represents the lightness or darkness of the object. a* represents the red-green chromaticity of the object (+a* towards red and -a* towards green). b* represents the yellow-blue chromaticity of the object (+b* towards region and -b* towards blue). three measurements were made in each sample and the mean value was recorded. color variation of specimens (∆e) was calculated between the initial measurement and after each immersion interval using the following equation: ∆e = ([∆l*]) 2 + ([∆a*]) 2 + ([∆b*]) 2) 1/2 after three measurements for each group, the mean values of l*, a* and b* taken before and after immersion, the ∆e fig. 1. flowchart of the study methodology. value was calculated for each mouthwash and immersion time. mouthwash ph the ph values from the chemical solutions were measured at room temperature (20°c) using a digital ph meter (ph 21, hanna instruments inc., woonsocket, ri, usa). the equipment was calibrated with deionized water before the samples were measured. samples of 40 ml of each solution were analyzed before specimen immersion. after 5 measurements, the mean value for each mouthwash was recorded. statistical analysis the normality of data was evaluated using the kolmogorof-smirnov test. statistical analysis was performed using two-way anova (mouthwash and immersion time), one-way anova (ph) and tukey’s post-hoc test at a 0.05 level of significance. results softening of acrylic resin by microhardness all mouthwashes decreased the surface microhardness values after 7 days of immersion. plax® alcohol-free showed no statistically significant differences among immersion times between 1 h to 7 days. at 7 days, plax® alcohol-free s h o w e d l o w e r v a l u e o f s o f t e n i n g t h a n t h e o t h e r mouthwashes (p<0.05). at 7 days, periogard® showed its h i g h e s t m i c r o h a r d n e s s v a l u e o v e r t i m e ( p < 0 . 0 5 ) . listerine® showed decrease in microhardness values in all tested times (figure 2). influence of mouthwashes on the physical properties of orthodontic acrylic resin 206206206206206 braz j oral sci. 13(3):203-208 roughness the mouthwashes increased the surface roughness in all tested groups. however, only plax® alcohol-free at 12 h and listerine® at 12 h showed statistically significant increased values, within the same mouthwash and when compared with others (p<0.05). ∆ra values are shown in figure 3. fig. 2. variation of knoop hardness values after and before immersion in mouthwashes. fig. 3. variation of roughness value, in µm, after and before specimen immersion in mouthwashes. colorimetric analysis the colorimetric analysis using ∆e revealed differences between times within each mouthwash (figure 4). however, no significant difference was shown in plax® alcohol-free, plax® classic and periogard® groups independent of time. when solutions were compared with each other, listerine® was the only one presenting a significant increase in color variation after 12 h immersion. mouthwash ph the ph solutions values ranged from 4.93 (±0.02) to 7.00 (±0.04), showing statistically significant difference among groups. plax® classic ph value was 7.00 (±0.04). listerine® showed the lowest ph (table 2). mouthwash ph mean (sd) periogard® 5.05 (±0.03)b periogard® alcohol-free 4.99 (±0.02)c listerine® cool mint 4.11 (±0.07)e plax® classic 7.0 (±0.04) a plax® alcohol-free 4.93 (±0.02)d table 2 table 2 table 2 table 2 table 2 ph values of commercial mouthwashes before sample immersion. *different letters indicate statistically significant difference among the groups (p<0.05). discussion the use of removable orthodontic appliances (roa) results in greater biofilm accumulation on dental surface and retentive sites of the appliance components1. reducing the levels of microorganisms may prevent caries18, halitosis19 and candidiasis 20. therefore, antimicrobial agents, such as mouthwashes, have been advised for orthodontic patients to aid in the control of bacterial biofilm formation7. according to a systematic review, the mean wearing time of orthodontic removable appliances is 13.4 (±10.3) months21. based on this data, nine periods of immersion in mouthwashes were defined to simulate, and even extrapolate, situations at home disinfection practice by orthodontic patients, as a possible attempt to offer research-based data for a home disinfection protocol to be prescribed by orthodontists. disinfection by immersion in chemical solutions, such as mouthwashes may predispose the acrylic resin to structural impairment due to sorption of water molecules and increase of solubility12. this study evaluated the effect of five of the most commonly used commercial mouthwashes, on the properties of an orthodontic self-cured acrylic resin. the results of this study showed that the acrylic resin properties were influenced by fig. 4. variation of e value of colorimetric analyses for all immersion times in the mouthwashes. influence of mouthwashes on the physical properties of orthodontic acrylic resin 207207207207207 braz j oral sci. 13(3):203-208 immersion in mouthwash solutions. therefore, the null hypothesis was rejected. roughness of acrylic surface is of paramount importance to microorganism colonization on the acrylic surface. the five tested mouthwashes increased the acrylic surface roughness. however, for microbial adhesion to occur, it is necessary a minimal roughness of 0.2 mm, which is the acceptable threshold value22. in this study, only two groups showed values higher than 0.2 mm. listerine® after 12 h immersion, presented values ranging from 0.6 to 1.6 mm. plax alcohol-free showed an increase on roughness values after 12 h immersion, from 0.12 to 1.05 mm. the ph of this mouthwash was 4.93. listerine® had the lowest ph value compared with other mouthwashes, showing a ph of 4.11. it may be assumed that the higher acidity may have caused the degradation of the superficial layer of the acrylic resin, resulting in an increase of the roughness values. the continuous exposure of the material to these mouthwashes promoted a removal of this superficial layer, exposing a subsurface region of the acrylic resin, which could explain the decreased roughness values from 24 h to 7 days immersion in plax® alcohol-free. this roughening effect, shown in listerine® 12h and plax® alcohol-free 12h groups, could cause patient discomfort and became more susceptible to microorganism colonization and biofilm formation. the softening of acrylic resin evaluated by microhardness is closely related to surface characteristics of roa, which are subjected to continuous abrasion during cleaning methods and by storage environment. acrylic resin immersion in all mouthwashes resulted in an increase of softening after 7 days of immersion. the plax® alcohol-free mouthwash showed a pattern similar to mouthwashes with alcohol, probably due to superficial alterations produced by phosphoric acid. at 7 days, periogard® showed the highest softening values over time (p<0.05). asad et al.23 showed similar results when immersed acrylic resin specimens in a 0.5% chlorhexidine gluconate solution and showed alterations of microhardness values after 7 days of storage. in the present study, test specimens were disinfected with 0.12% chlorhexidine digluconate. periogard ® is an alcohol-based mouthwash (11.6%) and the alcohol solvent action could also explain the changes on the acrylic surface. the resin may slowly absorb the disinfectant, altering the structure of polymer24. listerine® showed an increased softening process in all immersion times. the antibacterial activity of listerine® is claimed to be due to the combination of four essential oils: eucalyptol, menthol, methyl salicylate, and thymol. the essential oils act on the resin surface and are a potential source of harm considering the solvent action for acrylic and other thermoplastic resins25. this may explain the increased softening showed by the acrylic resin after immersion in listerine®. at 7 days of immersion in listerine®, the specimens showed a slight but significant increase in final hardness values, and decrease of softening, which is in accordance with the softening effect of ethanol on acrylic resins6,24. it seems that ethanol, as well as water, helps moving the polymer chains apart and allows them to slide (deform plastically) more easily6. this decrease in final hardness values for pmma is also caused by the plasticization effect enhanced by ethanol, which penetrates the matrix and expands the space between the chains26. colorimetric analysis using the ∆e as a parameter, showed that at the 12-h immersion period, listerine® presented a significantly greater variation compared to other mouthwashes, probably due to changes in surface topography of the acrylic resin that influence the parameters of colorimetric measurement27-28. moreover, there was visible interaction of the green dye (ci 42053) in listerine® with the acrylic resin, especially after a 12 h immersion, confirmed by the colorimetric assessment. this colorant influences the values of -a* and +b* parameters, changing the final ∆e by an increase in ∆a and ∆b of the samples. this could be explained by what is referred to as a crowding effect29, when higher pigment concentrations result in an interaction among the colorant particles and lead to deviation from the linear behavior. regarding the plax® alcohol-free, plax® classic and periogard® groups, the lack of significant difference in the values of ∆e among times within the groups can be related to changes on the surface, since in such groups the difference in roughness also showed no significance27-28. the limitations of this study include the fact that the immersion procedures were conducted without time intervals and the specimens were subjected to the mouthwash action continuously. the patients do not expose continuously their removable appliances to mouthwash solutions. the immersion procedures are intermittent, and use of an acrylic device in oral environment between immersion periods could elute chemical components like ethanol and acids during the use. then, the acrylic resin will adsorb water molecules, which act as a plasticizer to a lesser extent than ethanol. in other words, the harmful effects on acrylic resin surface and color might be less significant. however the influence of immersion in mouthwashes could represent several months of acrylic device use, considering daily immersions. it was concluded that immersion in mouthwashes could influence acrylic resin hardness, roughness and color. it is therefore recommenced that the orthodontist indicates mouthwashes to their patients with caution when establishing an at-home disinfection protocol. acknowledgements the authors would like to express their gratitude to the brazilian government through capes (coordination of improvement of higher education personnel) for the financial support provided and the professor carlos otávio petter from the laprom laboratory (mineral processing laboratory) of the 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natural products against oral candida isolates from denture wearers. bmc complement altern med. 2011; 26: 11: 119. 21. mavreas d, athanasiou ae. factors affecting the duration of orthodontic treatment: a systematic review. eur j orthod. 2008; 30: 386-95. 22. bollen cm, lambrechts p, quirynen m. comparison of surface roughness of oral hard materials to the threshold surface roughness for bacterial plaque retention: a review of the literature. dent mater. 1997; 13: 258-69. 23. asad t, watkinson ac, huggett r. the effects of various disinfectant solutions on the surface hardness of acrylic resin denture base material. int j prosthodont. 1993; 6: 9-12. 24. vlissidis d, prombonas a. effect of alcoholic drinks on surface quality and mechanical strength of denture base materials. j biomed mater res. 1997; 38: 257-61. 25. smith dc. the cleansing of dentures. dent pract dent rec. 1966; 17: 39-43. 26. polydorou o, trittler r, hellwiga e. elution of monomers from two conventional dental composite materials. dent mater. 2007; 23: 1535-41. 27. kingery wd. introduction to ceramics. new york: wiley; 1960. 28. ghinea rg, ugarte-alvan l, yebra a, pecho oe, paravina rd, perez mdm. influence of surface roughness on the color of dental-resin composites. j zhejiang univ sci b. 2011; 12: 552-62. 29. no ia, kleist ulf, rigdahl m. color of pigmented plastics measurements and predictions. polym plast technol eng. 2004; 44: 141-52. influence of mouthwashes on the physical properties of orthodontic acrylic resin 429 too many requests error 429 too many requests too many requests guru meditation: xid: 86750099 varnish cache server oral sciences n3 original article braz j oral sci. july | september 2015 volume 14, number 3 soft denture liners and sodium perborate: sorption, solubility and color change marina xavier pisani1, vanessa maria fagundes leite1, maurício malheiros badaró1, antônio de luna malheiros-segundo1, helena de freitas de oliveira paranhos1, cláudia helena lovato da silva1 1 universidade de são paulo – usp, dental school of ribeirão preto, department of dental materials and prosthodontics, ribeirão preto, sp, brazil correspondence to: cláudia helena lovato da silva faculdade de odontologia de ribeirão preto usp departamento de materiais dentários e prótese av. do café s/n cep:14040-904 ribeirão preto, sp, brasil phone: +55 16 33154006 e-mail: chl@forp.usp.br abstract aim: to evaluate the sorption, solubility and color change in two lining materials after 120 days of immersion in either sodium perborate or artificial saliva. methods: thirty disk-shaped specimens (15×3 mm) of each material, mucopren® soft (ms) and elite® soft (es) were manufactured and divided into two groups. the specimens in the control group (cg, n=15) were stored in artificial saliva at 37 °c. the specimens in the experimental group (eg, n=15) were stored in artificial saliva at 37 °c and immersed in sodium perborate daily for 5 min. the analysis of sorption and solubility was based on the initial dry weight and on the wet and dry weights after immersion. the color was assessed with a portable spectrophotometer and the nbs system. anova and tukey test (p<0.05) were used to analyze color and sorption. the solubility was analyzed by kruskalwallis test (p<0.05). results: sorption was higher in the eg group (0.31±0.08) than in the control group (0.26±0.05), and higher in elite® soft relining (0.34±0.07) than in mucopren® soft (0.23±0.06). there was no interaction between the factors. elite® soft presented a higher solubility when immersed in artificial saliva (cg: 0.16±0.07 and eg: 0.13±0.06; p=0.00). mucopren® soft showed no solubility in either treatment. regarding the color changes, there was a significant difference between the groups (cg: 9.2±1.2 and eg: 9.9±1.2; p=0.025) but not between the materials (mucopren® soft: 9.4±1.3 and elite® soft: 9.7±1.0; p=0.34). using the nbs system, we verified that both materials presented a high color change. conclusions: the daily use of sodium perborate promoted changes in the liners’ sorption and color. elite® soft relining was more prone to changes than mucopren® soft. keywords: denture, complete; denture liners; hygiene; physical properties. introduction heat-polymerized acrylic resin is commonly used as denture base material. however, as the supporting tissues (mucosa and alveolar ridge) are sensitive to the pressure caused by this rigid denture base, soft denture liners can be used as a cushion to overcome this disadvantage1. soft liners are able to distribute the functional load on the denture support area, and improve the denture’s retention and adaptation2. these characteristics can improve the patient’s comfort and quality of life during denture use2-3. the resilient denture liners are divided into two groups based on their chemical composition: acrylic polymers and silicone polymers3-4. both have short and longterm use and can be polymerized at room temperature or at high temperatures5-6. the structure of the soft lining materials and their surface roughness may promote biofilm accumulation7, which in turn can create favorable conditions to colonization by microorganisms (candida spp.), which may cause denture http://dx.doi.org/10.1590/1677-3225v14n3a09 received for publication: july 04, 2015 accepted: september 24, 2015 braz j oral sci. 14(3):219-223 220220220220220 stomatitis7-8, unpleasant odor and stains. the risk of infections, such as aspiration pneumonia in immunocompromised patients, is caused by the ingestion of microorganisms from the denture biofilm. these problems may be reduced by an adequate denture biofilm control 7. thus, it is extremely necessary to avoid material degradation during the daily use and hygiene procedures5,9. although brushing is the most commonly used method of biofilm control, it may promote adverse effects such as the increase in surface roughness8 and therefore, it is not the most adequate method for soft liner hygiene. chemical disinfectants such as sodium perborate, which are available as solutions and as effervescent tablets, have detergent and antiseptic action that prevent microorganism proliferation on dentures5-6,10. however, due to immersion in these disinfectants, the soft lining materials may become stiff and porous. in addition, liquid sorption or the loss of components 11-14 to the environment may occur, and discoloration15-18 and surface roughness may increase1. in this way, it is important to evaluate whether chemical disinfectants may have a negative influence on the physical properties of soft liners used in association with acrylic resin bases11,19-21. in the present study, we evaluated the influence of sodium perborate, a alkaline peroxide, on three clinically relevant properties of two different soft liner materials. the specific aims of this research were: 1) to investigate the differences between two soft liner materials and whether sorption takes place after immersion in sodium perborate and 2) to investigate whether there is an increase in their solubility and/or color changes after immersion. the hypothesis to be tested was that sodium perborate would not cause negative effects to the sorption, solubility and color change on the relining denture bases and there would be no difference between the materials. material and methods experimental design we evaluated two soft lining materials, mucopren® soft and elite® soft relining after immersion in sodium perborate commercial name mucopren soft elite soft relining corega tabs artificial saliva *material soft relining (polyvinylsiloxane) andethyl-acetate adhesive soft relining (polyvinylsiloxane)and dichloromethane adhesive effervescent tablet sodium perborate, ph 6,8 potassium phosphate diacid, potassium phosphate dibasic, potassium chloride, sodium chloride, magnesium chloride (6 h2o), calcium chloride (2 h2o), sodium fluoride, sorbitol 70%, flavoring and coloring, preservatives (nipagin/nipasol), thickener, water q.s.p, ph 6,8 manufacturer kettenbach gmbh & co. kg, eschenburg, germany. zhermack s.p.a., badia polesine, italy. glaxosmithkline brazil ltd., rio de janeiro, brazil. faculty of pharmaceutical sciences of ribeirao preto university of sao paulo, ribeirao preto, brazil. table 1 – table 1 – table 1 – table 1 – table 1 – commercial name, materials and manufacturer used in the study. * information according manufacturers. for 120 days. the analyzed quantitative variables were sorption, solubility and color change. thirty specimens of each material were randomly assigned to one of two groups: the experimental group (eg, n=15) in which specimens were immersed in sodium perborate and a control group (cg, n=15) where specimens were immersed in artificial saliva. the soft liner materials, hygiene solution and the used artificial saliva are in table 1. specimen preparation a stainless-steel matrix with five open disc-shaped molds (15×3 mm) was placed on a glass plate and fixed with adhesive strip. to obtain the soft lining specimens, the materials were injected into each mold and the matrix was pressed against another plate until the final polymerization of the materials. following the manufacturer’s instructions, the polymerization time was 10 min at room temperature. after polymerization, the specimens were detached from the molds and the excess material was removed with a stainless steel scalpel (#15 stainless steel scalpel blade; cirurgica passos, curitiba, pr, brazil). thirty disc-shaped specimens with a 15 mm diameter and 3 mm thick were obtained from each soft liner material. immersion procedures the specimens in the control group (15 of each material) were stored in a container with 300 ml of artificial saliva, which was changed daily (table 1). this container was kept in an incubator at 37±2 °c. in the experimental groups, the 15 specimens of each material were immersed in the hygiene solution, which was prepared daily for each immersion, according to the manufacturer’s instructions, using 1400 ml water at 50 °c and 7 sodium perborate tablets (200 ml water per tablet). the number of tablets was defined according to the specimens’ weight, similar to the weight of a medium-sized denture. the specimens were immersed in the fresh hygiene solution for 5 min per day for 120 days. prior and after each immersion, the specimens were rinsed in tap water for 10 s and stored in fresh artificial saliva for the next 24 h at 37 °c. soft denture liners and sodium perborate: sorption, solubility and color change braz j oral sci. 14(3):219-223 221221221221221 sorption and solubility the specimens’ sorption and solubility were evaluated according to the methodology described by ada #12 specification for denture base polymer22. all specimens were weighted immediately after being obtained and then placed in a desiccator with silica gel (dpv chemical products, sao paulo, sp, brazil) at room temperature. the specimens were weighted daily in an analytical balance until obtaining constant weight (±0.001 g), which was considered as the specimens’ initial weight (w1). after the total immersion period (120 days), the specimens were washed in tap water, dried with filter paper and weighted in an analytical balance. this value was used in the sorption calculation (w2), i. e., %sorption=(w2–w3)/w1 × 10011, where w1 is the initial weight, w2 the weight after sorption and w3 the final weight after desiccation. then, the specimens were placed in the desiccator, weighted daily until stable readings of the dry weight (w3). solubility (in %) was given by the formula: %solubility=(w1–w3)/w1 × 100 color change test the amount of color change was obtained using a portable colorimeter (color guide 45/0; byk-gardner gmbh geretsried, germany), calibrated according to the manufacturer’s instructions and using the standard commission internationale de l’eclairage (cie lab) color system. a white background was used for the measurements, and for each specimen the following readings were obtained: brightness (l*), red-green proportion (a*) and the yellowblue proportion (b*). the color change (∆e) was determined as the variation between the values obtained before and after immersion using the formula: ∆e=[(∆l)2+(∆a)2+(∆b)2]½, where ∆l, ∆a, and ∆b are the differences between the initial and final values of l*, a*, and b*, respectively. in addition to this analysis, the critical observation of color change was also quantified by the national bureau of standards (nbs), which is given by nbs=∆e×0.92 (table 2). data analysis all variables were tested for normal distribution (shapirowilk test) and homogeneity (levene test). once the normality and homogeneity of sorption and color change were confirmed, these variables were analyzed using anova (twoway) and the tukey hst test. due to the non-normal distribution of solubility data, the kruskal-wallis test was employed. regardless of the test, significance level was set critical observation of color change units nbs very light 0.0 – 0.5 light 0.5 – 1.5 remarkable 1.5 – 3.0 appreciable 3.0 – 6.0 high 6.0 – 12.0 very high >12.0 table 2 -table 2 -table 2 -table 2 -table 2 classification unit by nbs. at p<0.05. all analyses were made using spss statistics base 18 (spss inc., chicago, il, usa). results sorption and solubility test the average values showed that specimens in sodium perborate (0.31±0.08) had a higher average sorption than the ones in saliva (0.26±0.05), with a significant difference (p=0.006). the materials also differed in sorption (p=0.0), where the elite® soft relining (0.34±0.07) had a higher average sorption than mucopren® soft (0.23±0.06). there was no interaction between the factors (p=0.06). elite® soft relining had a higher average solubility when immersed in artificial saliva whereas mucopren® soft showed no solubility in either group. the comparison of the individual averages and standard deviations (sd) in each group/material are shown in table 3. color change there was a significant difference in color change (p=0.025) between the control and experimental groups, and eg showed the highest average color change. there was no difference (p=0.34) between the materials and no interaction between the factors (p=0.18). the averages and sd are shown in table 4. the critical observation of color change by the nbs, revealed that both materials presented color changes independent of the immersion group (table 4). discussion one of the main disadvantages and even the reason behind the failure of the resilient denture liners is the difficulty in maintaining them clean. in addition, there is lack of information regarding the ideal hygiene products and methods that would not affect negatively the physical properties of soft denture liners1,5-6. sorption, solubility and color stability are important properties for maintaining quality of the materials23 and these properties can be affected by the denture cleanser and periods of immersion24-25. increase of sorption and solubility may result in dimensional change, discoloration, unpleasant odor, separation from the complete denture base and loss of resilience11,23,26. these changes result in a lower sorption of the occlusal impact force and in patient’s dissatisfaction11. ideally, a soft liner should have low sorption and solubility15 but such material is still lacking26. alkaline peroxide (sodium perborate) was used in this study because it has specific indication for denture hygiene and has been largely used in different studies5-6,10,15,17,19 due to its antiseptic and disinfecting properties, capacity to eliminate stains on the denture surface, and a more favored flavor. in this study, the hypothesis tested for sorption was rejected, since there was change in this property after use of perborate and there was difference between the materials. for solubility, the hypothesis was partially accepted, since soft denture liners and sodium perborate: sorption, solubility and color change braz j oral sci. 14(3):219-223 222222222222222 sorption solubility c g eg material average c g eg mucoprem soft 0.22 (0.04)aa 0.24 (0.08)ba 0.23 (0.06)a 0.00aa 0 .00aa elite soft 0.30 (0.06)ab 0.38 (0.08)bb 0.34 (0.07)b 0.16 (0.07)ab 0.13 (0.06)bb groups average 0.26 (0.05)a 0.31 (0.08)b table 3 table 3 table 3 table 3 table 3 comparison of averages (%) and sd of the sorption and solubility of the materials and groups after immersion. same letters indicate statistical equality; capital letter: comparison between columns; lower case: comparison between lines. cie lab nbs cg eg c g eg mucopren soft 9.07 (1.4)a 9.7 (1.2)b 8.34° 8.92° elite soft relining 9.3 (0.96)a 10.04 (1.09)b 8.54° 9.23° table 4 -table 4 -table 4 -table 4 -table 4 evaluation of the color change in the materials after immersion according the cie lab system (average and sd) and classification of nbs. for cie lab: same letters indicate statistical equality. for nbs: # remarkable; * appreciable; high. there was difference between materials. the sorption was higher after immersion of the materials in the hygiene solution, and the same results were found by mansoor24 (2014). moreover, the solubility was higher when elite® soft relining was immersed in saliva, whereas for mucopren®, there was no change in solubility between the immersion groups. it is known that high ionic concentration (potassium and sodium) of denture cleansers, as compared to the water, caused higher dissolution of soluble components 12. as regards solubility, goll et al.21 (1983) and rodrigues-garcia et al.14 (2003) also found loss in weight of resilient materials after 30 days of immersion in water and sanitizer. the results for the control group were as expected, because the saliva contains ions and is based on a polar solvent (water), which may encourage diffusion processes of ionic and polar species from the lining material into the solution5,11. although both analyzed materials in this study were silicone based, differences between them were observed. water sorption depends on the hydrophobicity and porosity13 of the material, as well as on the presence of cross-linking agents11. further studies should be conducted to evaluate the hydrophobicity and the cross-linking agents of the materials used here. micro-structural analysis may identify the differences between the used materials. according to hekimoglu and anil12 (1999) and kazanji and watkinson11 (1988), in daily use dentures are in contact with saliva and can be kept in a sanitizing solution as a hygiene method. such situations can increase the denture’s solubility, and consequently, water sorption. soft liners can release chemical compounds such as monomers (methyl methacrylate, ethyl methacrylate, dodecyl methacrylate), plasticizers (dibutyl phthalate, diethyl phthalate, tributyl acetylcitrate), and others (e.g., benzophenone) after immersion in a chemical denture cleanser and in saliva19. color stability is an important physical property for soft denture liners, which besides interfering in the esthetics of the material, also indicates the degree of aging16. an ideal soft denture lining material should therefore not easily change in color and/or become stained after long use23. in this study, the tested hypothesis was rejected, since there was change color due to hygiene solution and materials. the immersion in sodium perborate caused higher color change than immersion in artificial saliva. similar results were found by tan et al.17 (2000) after immersion for 90 days in a hygiene solution that was also based on sodium perborate. goll et al.21 (1983) found different results in color change caused by cleansers with similar composition (sodium perborate). they concluded that quantitative differences in their formulation, ph and in water temperature used with the cleanser solution, apparently influenced the liner color. saraç et al.20 (2007) agreed that the oxidant is the cause for color change. salloum et al.25 (2014) affirmed that the chemical type influenced soft denture liners’ color stability and that the color changes depend on the period of immersion. in this study, both the saliva and sanitizer solution had a 6.8 ph. therefore, in this case the oxidant action hypothesis would be better accepted. according to jin et al.15 (2003), many factors may contribute to the discoloration of the lining materials; for example, the accumulation of stains, dehydration, hydrolysis and the decomposition chain reaction. the chain reaction is the oxidation of carbon double bonds reacting with peroxide (present in some hygiene solutions) and produce colored compounds. these compounds in turn continue to degrade the colored products. matsumura et al.27 (2001) report that color stability is related with the direct polymerization of the material in the oral cavity due to the presence of a low polymerization surface layer, compared to indirect polymerization. no comparison was made with the indirect technique of polymerization, but this fact may have contributed to the color changes observed in this study. further studies should be performed with this purpose. although the literature indicates that the color change is related with liquid sorption18, the present study did not find a relationship between these properties. ma et al.18 (1997) in a laboratory investigation, observed the color changes of four relining materials after immersion in sanitizing solutions and found that, although statistically significant, the results were not clinically detectable. however, in critical analysis of the color changes based on the nbs classification, the present study found that both materials and both groups had high color variation. maintaining the material’s color is important for patients, and they are more satisfied when the soft liner color is stable and indistinguishable from the color of the denture base soft denture liners and sodium perborate: sorption, solubility and color change braz j oral sci. 14(3):219-223 223223223223223 material16. in our study, both materials showed nbs values higher than 2, demonstrating a clinically noticeable color change, which indicates that the combination of the lining materials with the evaluated hygiene method was not favorable. in conclusion, daily use of sodium perborate as a chemical denture hygiene method may promote sorption and color alterations in the soft denture liners studied here and elite® soft relining was more prone to changes compared to mucopren® soft. acknowledgements to fapesp (04/09878-0) for providing funds for this work. references 1. pisani mx, da silva ch, paranhos hf, souza rf, macedo ap. evaluation of experimental cleanser solution of ricinus communis: effect on soft denture liner properties. gerodontology. 2012; 29: e179-85. 2. pisani mx, malheiros-segundo al, balbino kl, souza rf, paranhos hf, lovato da silva ch. oral health related quality of life of edentulous patients after denture relining with a silicone-based soft liner. gerodontology. 2012; 29: 474-80. 3. santawisuk w, kanchanavasita w, sirisinha c, harnirattisai c. mechanical properties of experimental silicone soft lining materials. dent mater j. 2013; 32: 970-5. 4. gupta s. effect of surface treatment on the flexural strength of denture base resin and tensile strength of autopolymerizing silicone based denture liner bonded to denture base resin: an in vitro study. j indian prosthodont soc. 2010; 10: 208-12. 5. bro¿ek r, koczorowski r, rogalewicz r, voelkel a, czarnecka b, nicholson jw. effect of denture cleansers on chemical and mechanical behavior of selected soft lining materials. dent mater. 2011; 27: 281-90. 6. murata h, chimori h, hong g, hamada t, nikawa h. compatibility of tissue conditioners and denture cleansers: influence on surface conditions. dent mater j. 2010; 29: 446-53. 7. segundo al, pisani mx, do nascimento c, souza rf, paranhos hf, silva-lovato ch. clinical trial of an experimental cleaning solution: antibiofilm effect and integrity of a silicone-based denture liner. j contemp dent pract. 2014; 15: 534-42. 8. huh jb, lim y, youn hi, chang bm, lee jy, shin sw. effect of denture cleansers on candida albicans biofilm formation over resilient liners. j adv prosthodont. 2014; 6: 109-14. 9. liao wc, pearson gj, braden m, wright ps. the interaction of various liquids with long-term denture soft lining materials. dent mater. 2012; 28: e199-206. 10. malheiros-segundo al, pisani mx, paranhos hfo, souza rf, silvalovato ch. effect of a denture cleanser on hardness, roughness and tensile bond strength of denture liners. braz j oral sci. 2008; 7: 1596-601. 11. kazanji mnm, watkinson ac. soft lining materials; their sorption of, and solubility in, artificial saliva. br dent j. 1988; 165: 91-4. 12. hekimoglu c, anil n. sorption and solubility of soft denture liners after accelerated aging. am j dent. 1999; 12: 44-6. 13. hayakawa i, akiba n, keh e, kasuga y. physical properties of a new denture lining material containing a fluoroalkyl methacrylate polymer. j prosthet dent. 2006; 96: 53-8. 14. rodrigues-garcia rcm, leon bl, oliveira vmb, del bel cury aa. effect of a denture cleanser on weight, surface roughness, and tensile bond strength of two resilient denture liners. j prosthet dent. 2003; 89: 489-94. 15. jin c, nikawa h, makihira s, hamada t, furukawa m, murata h. changes in surface rougness and colour stability of soft denture lining materials caused by denture cleansers. j oral rehabil. 2003; 30: 125-30. 16. villar a, pesun ij, brosky me, fines c, hodges js, delong r. clinical evaluation of a new resilient denture liner. part 1: compliance and color evaluation. j prosthodont. 2003; 12: 82-9. 17. tan h, woo a, kim s, lamoureux m, grace m. effect of denture cleansers, surface finishing, and temperature on molloplast b resilient liner color, hardness, and texture. j prosthodont. 2000; 9: 148-55. 18. ma s, johnson gh, gordon ge. effects of chemical disinfectants on the surface characteristics and color of denture resins. j prosthet dent. 1997; 77: 197-204. 19. brozek r, rogalewicz r, koczorowski r, voelkel a. the influence of denture cleansers on the release of organic compounds from soft lining materials. j environ monit. 2008; 10: 770-4. 20. saraç d, saraç s, kurt m, yüzbasioglu e. the effectiveness of denture cleansers on soft denture liners colored by food colorant solutions. j prosthodont. 2007; 16: 185-91. 21. goll g, smith de, plein jb. the effect of denture cleansers on temporary soft liners. j prosthet dent. 1983; 50: 466-72. 22. council of dental materials and devices: revised american dental association specification no. 12 for denture base polymers. j am dent assoc. 1975; 90: 451-8. 23. kasuga y, akiba n, minakuchi s, uchida t, matsushita n, hishimoto m, hayakawa i. development of soft denture lining materials containing fluorinated monomers. nihon hotetsu shika gakkai zasshi. 2008; 52: 183-8. 24. mansoor ns. effect of denture cleansers on sorption and solubility of soft denture lining materials immersed in different time interval. med j babylon. 2014; 11(2): 465-75. 25. salloum am. effect of 5.25 % sodium hypochlorite on color stability of acrylic and silicone based soft liners and a denture base acrylic resin. j indian prosthodont soc. 2014; 14: 179-86. 26. wiêckiewicz w, kasperski j, wiêckiewicz m, miernik m, król w. the adhesion of modern soft relining materials to acrylic dentures. adv clin exp med. 2014; 23: 621-5. 27. matsumura h, tanoue n, kawasaki k, atsuta m. clinical evaluation of a chemically cured hard denture relining material. j oral rehabil. 2001; 28: 640-4. soft denture liners and sodium perborate: sorption, solubility and color change braz j oral sci. 14(3):219-223 429 too many requests error 429 too many requests too many requests guru meditation: xid: 38315897 varnish cache server oral sciences n3 original article braz j oral sci. april | june 2014 volume 13, number 2 effect of finishing and polishing on surface roughness of composite resins after bleaching bruna fortes bittencourt, giovana mongruel gomes, felipe auer trentini, mônica regina de azevedo, joão carlos gomes, osnara maria mongruel gomes universidade estadual de ponta grossa uepg, school of dentistry, area of dental materials, ponta grossa, pr, brazil correspondence to: bruna fortes bittencourt paula xavier st. 1399 ap. 142 84010-270 ponta grossa, pr, brazil. phone: +55 42 32245164 fax: +55 42 32241166 e-mail: brubita@hotmail.com abstract aim: to evaluate the influence of finishing and polishing techniques on the surface roughness of two composite resins (crs) subjected to bleaching procedure. methods: forty-eight cr specimens were divided into six groups (n=8). for g1 to g3, a microhybrid cr (opallis; fgm) was used, and g4 to g6, received a nanohybrid cr (brilliant newline; coltène/whaledent). all specimens were subjected to bleaching procedure with 35% hydrogen peroxide (two 45-min applications, with a 5-day interval). the surface roughness of all specimens was evaluated before and after the bleaching and/or finishing/polishing (ra parameter) by a roughness meter. after bleaching, the groups were subjected to finishing and polishing procedures: g2 and g5 felt discs + diamond pastes; and g3 and g6 silicon rubber tips. the control groups (g1 and g4) had no finishing or polishing treatment after bleaching. data were analyzed by anova and tukey’s posttest, and t test for paired samples (α=0.05). results: bleaching treatment increased ra values for the nanohybrid cr specimens, but both finishing/polishing techniques were able to reduce these values; for the microhybrid specimens, only finishing/polishing with silicon rubber tips decreased the roughness values. conclusions: for both microhybrid and nanohybrid crs, the silicon rubber tips were effective to reduce the surface roughness after bleaching procedure. keywords: composite resins; dental polishing; tooth bleaching. introduction the beauty of the smile is extremely important in daily life. with aesthetics being increasingly valued, professionals use composite resins (crs) as the first choice materials in dental offices, since these materials combine suitable mechanical properties with excellent aesthetics1. in a parallel development, tooth bleaching modalities also became widely known and considered a safe and effective technique to treat discolored teeth2-3. thus, in dental practice, it is common to have patients whose restorations will be subjected to bleaching procedures4. therefore, a larger number of studies have been performed to confirm whether the bleaching agents may cause undesirable effects on dental tissues5-6 and restorations4,7-11. therefore, it is important that dentists understand the consequences of these procedures on restorations’ longevity, and to substitute8 or to maintain4 the crs after bleaching, whenever required. however, the decision to maintain or not the restoration depends on several factors, including marginal integrity, color and surface characteristics. several studies have reported material-dependent surface roughness alterations of restorative materials after bleaching8,10-12. for the bleaching agent factors such as application time, ph and concentration may influence the topography8; for the composite, the organic matrix and particle size are the main factors related to surface alterations10. braz j oral sci. 13(2):158-162 received for publication: may 08, 2014 accepted: june 11, 2014 surface smoothness of the restorations is one of the main factors of aesthetic success, since rough surfaces contribute to staining and bacteria deposition, and decreased brightness13. thus, the surface roughness is an important property to evaluate the surface integrity of restorations, determining the polishing ability and wear rate of these materials14. final finishing and polishing are extremely important to increase the quality of restorations in oral function since they ensures surface smoothness and consequently, aesthetics and longevity15-16. after bleaching treatment, in particular, resin-based restorations should be carefully finished and polished in order to remove the outermost layer damaged by the bleaching agent. based on this, the present study evaluated the influence of finishing and polishing techniques on the surface roughness of two crs after bleaching. the null hypothesis was that finishing and polishing techniques do not influence the surface roughness of the bleached crs. material and methods two brands of crs were used: a microhybrid (mh) opallis (fgm, joinvile, sc, brazil) and a nanohybrid (nh) brilliant newline (coltène/whaledent, altstatten, switzerland). the specimens were made using a metallic matrix 2 mm thick and 5 mm diameter. the surface smoothness of the specimens was standardized: the matrices were placed over a mylar strip (mylar; dupont, wilmington, de, usa) and a glass slide. the matrix was filled with cr, and a new mylar strip was positioned over the composite/matrix set. then, another glass slide was used, and a 1 kg device was put on top of the glass slide, in order to obtain a flat and smooth composite surface. the weighing device and the upper glass slide were removed, and the specimens were light-cured for 20 s (ledemetron; kerr corp., orange, ca, usa) with a power intensity of 800 mw/cm2. after 48 h, the initial roughness (ir) of all specimens was obtained in a digital roughness meter (mitutoyo surftest301; mitutoyo-kawasaki, kanagawa, japan). five measures were performed on each specimen (one at the center and four in each quadrant, in a clockwise direction), and the arithmetic mean was obtained from these values. this mean was considered as the ir value. surface roughness reading was performed using the ra parameter (µm) and the measuring profile iso2001, a 0.25 mm cut-off, 1.25 mm in length and 0.1 mm/s speed. after the ir analysis, bleaching treatment was performed with 35% hydrogen peroxide (hp) (whiteness hp blue calcium; fgm, joinville, sc, brazil), according to the manufacturer’s instructions: two 45-min applications, with 5 days interval between each application. after the last bleaching agent application, the specimens were subjected to the finishing and polishing procedures, according to the cr and finishing/polishing treatment (n=8): for g2 and g5: felt discs + diamond pastes (felt discs diamond flex and diamond aci, acii and diamond r; fgm, joinville, sc, brazil); g3 and g6: silicon rubber tips (astropol; ivoclarvivadent, schaan,liechtenstein). the control groups g1 and g4 did not receive any finishing or polishing treatment (figure 1). for g2 and g5, the specimen’s surfaces were finished with a medium granulation paste (aci) with the felt fig. 1. flowchart of the experimental design of the study. 171159effect of finishing and polishing on surface roughness of composite resins after bleaching braz j oral sci. 13(2):158-162 material composite resin opallis composite resin brilliant newline whiteness hpblue diamond ci and cii (medium and fine granulation) diamond r (extra-fine granulation) astropol manufacturer fgm, joinville, sc, brazil coltène/whaledent, altstatten, switzerland fgm, joinville, sc, brazil fgm, joinville, sc, brazil fgm, joinville, sc, brazil ivoclar-vivadent, schaan,liechtenstein composition bis-gma, bis-ema, tegdma, silanized barium-aluminum glass and nanoparticles – particle size: 40 nm to 3 µm (78.5 – 79.8 wt%) methacrylates, dental glass -barium glass and amorphous silica – particle size: 0.04 to 2.8 µm (77 – 78 wt%). 35% hydrogen peroxide, thickening agents, color mixtures, calcium gluconate, glycol and deionized water aluminum oxide (aci: 80 µm; acii: 30 µm), carbowax, thickeners, mint essence, water aluminum oxide (6 8 µm), carbowax, thickeners, mint essence, water caoutchouc, silicon carbide, aluminum oxide, titanium oxide, iron oxide (coarse gray [45 mm], fine green [1 mm]);caoutchouc, silicon carbide, aluminum oxide, titanium oxide, iron oxide, diamond dust (extra-fine-pink [0.3 mm]) table 1.table 1.table 1.table 1.table 1. materials used in the study disc rotating at low-speed. the initial polishing of the specimens was carried out with small amount of the acii paste (fine granulation) on the felt disc. final polishing was achieved with the diamond r paste with another felt disc. for g3 and g6, the procedures were performed in three steps: step 1 – finishing with astropol f (gray) – with this tip, excess of material was removed and the rough surface of the restoration was smoothened; step 2 – polishing with astropol p (green), in order to produce a complete final smooth surface; step 3 – high-gloss polishing with astropol hp (pink) – to produce a high-gloss surface. all tips were used at low speed. all materials, manufacturers and respective compositions used in this study are shown in table 1. for all groups that were subjected to finishing and polishing procedures, the materials were changed after use in every 2 specimens. all procedures were performed by a single calibrated operator, in an analytical balance, with a mean pressure of 0.2 kgf. all procedures were carried out under cold tap water to ensure removal of residues. intermittent movements were applied and each instrument was used for 20 s to avoid cracks and grooves. the specimens were stored in artificial saliva at 37 °c (benzoate 1 g; cmc 10 g; magnesium chloride 0.05 g; potassium chloride 0.62 g, sodium chloride 0.025 g; sorbithol 42.74 g, distilled water 944.53 ml; dibasic potassium phosphate 0.8035 g; monobasic potassium phosphate 0.326 g), which was changed every day. after 48 h of the final polishing, new surface roughness readings were performed in the same way as described for ir to obtain the final roughness values (fr). data were analyzed statistically by anova and tukey’s post-test for independent samples; and t test for paired samples (ir and fr of each group), confidence interval of 95% and α=0.05. results mean values and standard deviation of ir and fr roughness (in µm) for mhcr and nhcr are shown in tables 2 and 3, respectively. no statistically significant differences were found between ir of all groups, for both mhcr (p=0.95) and nhcr (p=0.93). comparing the ir and fr values from g1 (mh cr), roughness values increased, but this difference was not statistically significant (p=0.0769). there were no statistically significant differences between g2 and g1 (p>0.05), and g3 showed statistically significant lower final values compared with g1 (p<0.05). comparing the different finishing and polishing procedures for mh cr, g3 presented lower roughness values than to g2 (p<0.05). comparing ir and fr from g4, it was observed that the bleaching procedure significantly increased the roughness values for nhcr (p=0.0036). analyzing the fr values for the finishing and polishing techniques, it may be observed that both g5 and g6 had lower values than the control group g4 (p<0.05). g6 also had significantly lower final roughness values than g5 (p<0.05). discussion after dental bleaching, it is necessary to evaluate some characteristics of cr restorations, such as color change, marginal integrity and surface roughness, in order to indicate the restoration maintenance or replacement. surface roughness is an important parameter to be observed, since rough surfaces may appear after hp bleaching10. roughness also influences aesthetic restoration, since it may lead to discoloration and wear, as well as biologic consequences to the periodontal health, especially the occurrence of secondary caries and gingivitis13. during the specimen preparation in this study, a mylar strip was used to produce specimens with standardized top surfaces15. after composite polymerization, the specimens covered with the mylar strips which did not receive any finishing/polishing before and after bleaching, were used as control group, as reported in other studies15,17-19. in the present study, both crs demonstrated increased surface roughness after bleaching procedures, as reported in previous studies7,20, but this alteration was significant only for the nhcr composite. despite the commercial 160 effect of finishing and polishing on surface roughness of composite resins after bleaching braz j oral sci. 13(2):158-162 groups initial roughness (ir) final roughness (fr) g1 0.282 ± 0.295 aa 0.427 ± 0.419 aa g2 0.290 ± 0.173 aa 0.263 ± 0.114 aa g3 0.274 ± 0.174 aa 0.135 ± 0.040 bb table 2.table 2.table 2.table 2.table 2. mean values and standard deviation (±) of initial (ir) and final (fr) surface roughness (µm) for all experimental groups of the microhybrid composite resin (*) (*) lowercase letters indicate the comparisons within each column. uppercase letters indicate the comparison within each row. identical letters indicate statistically similar means (p > 0.05). groups initial roughness (ir) final roughness (fr) g4 0.463 ± 0.234 aa 0.654 ± 0.326 bb g5 0.457 ± 0.184 aa 0.242 ± 0.072 bc g6 0.476 ± 0.264 aa 0.174 ± 0.082 bd table 3.table 3.table 3.table 3.table 3. mean values and standard deviation (±) of initial (ir) and final (fr) surface roughness (µm) for all experimental groups of the nanohybrid composite resin (*) (*) lowercase letters indicate the comparisons within each column. uppercase letters indicate the comparison within each row. identical letters indicate statistically similar means (p > 0.05). nomenclature, both crs used in this study have the same medium particle size: 0.5 µm for the mhcr (opallis) and 0.6 µm for the nhcr (brilliant newline). it is known that bleaching agents may act mainly on the resinous matrix of crs whereas the inorganic particles are inert, even in an acidic environment7. this suggests that matrix erosion occurs, with the consequent displacement of inorganic particles. in this study, both crs also have the same mass fraction, approximately 77 wt%. a previous study reported that the presence of lower filler amount and greater organic matrix content makes the crs more susceptible to erosive action by the bleaching agents, leading to particle exposure and crack formation20. for the nhcr brilliant newline, both finishing/ polishing procedures were able to decrease the surface roughness of the bleached specimens. however, in this study, better results were found when silicon rubber tips were used, as demonstrated elsewhere21. these results are probably due to the increased abrasiveness of silicon rubber tips compared to diamond pastes, removing the roughness caused by bleaching treatment on the top layer of the crs. another hypothesis to explain the results may be the composition of the astropol tips. hence, the abrasiveness of the used finishing/polishing system may directly influence the restoration’s final roughness21. on the other hand, researchers have found that astropol system caused the highest roughness values on crs compared to several finishing/polishing procedures22, due to its abrasive potential. in this case, a bleaching treatment was not performed before the finishing and polishing procedure; in other words, there was no previous rougher surface. particle type and size of crs may influence the surface roughness 23, determining the final polishing of the restorations24-25. however, some material’s intrinsic factors, like the resin monomer composition and filler content also play a critical role in the clinical behavior of these restorative materials18. berger et al.18, reported that the most appropriate technique would be the use of a cr and finishing/polishing agent developed by the same manufacturer, since the use of these agents depend on the filler hardness, size and content of each resin; and those features the manufacturer must know. this study encourages further investigations to evaluate crs with different inorganic particle size and weight. it is important to note that this is an in vitro study, and it has limitations. only the surface roughness of cr was analyzed, without concerning with aesthetic factors. bleaching agents may affect the color of existing composite restorations26. so, when dealing with anterior restorations, not only the topography, but also the aesthetics of the restoration should be evaluated, in order to indicate the correct restorative procedure. as recently reported, bleaching may cause increased surface roughness on composite materials, and the clinicians should be aware whether the cr restoration will be exposed to a bleaching procedure27. however, if there is an optimal finishing and/or polishing procedure, this seems to have no clinical significance27-28. this study demonstrated that despite the alterations in the surface roughness of crs by the bleaching procedure, maintenance of these restorations might be indicated if there is no aesthetic involvement. finishing and polishing techniques are effective to reduce bleaching adverse effects cr surface roughness. specifically, the silicon rubber tips showed satisfactory results for both mhcr and nhcr after bleaching procedure. according to the results of this study, it may be concluded that for the mhcr, only the finishing and polishing procedure with silicon rubber tips reduced the roughness values of these restorations after bleaching. for the nhcr, both finishing and polishing techniques were able to reduce roughness values of restorations after bleaching, and the silicon rubber tip technique was more effective than felt discs and diamond pastes. references 1. mitra sb, wu d, holmes bn. an application of nanotechnology in advanced dental materials. j am dent assoc. 2003; 134: 1382-90. 2. kwon sr. whitening the single discolored tooth. dent clin north am. 2011; 55: 229-39. 3. li y. safety controversies in tooth bleaching. dent clin north am. 2011; 55: 255-63. 4. polydorou o, mönting js, hellwig e, auschill tm. effect of in-office tooth bleaching on the microhardness of six dental esthetic restorative materials. dent mater. 2007; 23: 153-8. 5. zantner c, beheim-schwarzbach n, neumann k, kielbassa am. surface microhardness of enamel after different home bleaching procedures. dent mater. 2007; 23: 243-50. 6. cavalli v, rodrigues lk, paes-leme af, soares le, martin aa, berger sb, et al. effects of the addition of fluoride and calcium to low-concentrated carbamide peroxide agents on the enamel surface and subsurface. photomed laser surg. 2011; 29: 319-25. 7. moraes rr, marimon jl, schneider lf, correr-sobrinho l, camacho gb, bueno m. carbamide peroxide bleaching agents: effects on surface roughness of enamel, composite and porcelain. clin oral invest. 2006; 10: 23-8. 171161effect of finishing and polishing on surface roughness of composite resins after bleaching braz j oral sci. 13(2):158-162 8. gurgan s, yalcin f. the effect of 2 different bleaching regimens on the surface roughness and hardness of tooth-colored restorative materials. quintessence int. 2007; 38: 83-7. 9. mujdeci a, gokay o. effect of bleaching agents on the microhardness of tooth-colored restorative materials. j prosthet dent. 2008; 95: 286-9. 10. dutra ra, branco jr, alvim hh, poletto lt, albuquerque rc. effect of hydrogen peroxide topical application on the enamel and composite resin surfaces and interface. indian j dent res. 2009; 20: 65-70. 11. yu h, li q, cheng h, wang y. the effects of temperature and bleaching gels on the properties of tooth-colored restorative materials. j prosthet dent. 2011; 105: 100-7. 12. lima da, de alexandre rs, martins ac, aguiar fh, ambrosano gm, lovadino jr. effect of curing lights and bleaching agents on physical properties of a hybrid composite resin. j esthet restor dent. 2008; 20: 266-75. 13. heintze sd, forjanic m, ohmiti k, rousson v. surface deterioration of dental materials after simulated tooth brushing in relation to brushing time and load. dent mater. 2010; 26: 306-19. 14. tanoue n, matsumura h, atsuta m. wear and surface roughness of current prosthetic composites after toothbrush/dentifrice abrasion. j prosthet dent. 2000; 84: 93-7. 15. rai r, gupta r. in vitro evaluation of the effect of two finishing and polishing systems on four esthetic restorative materials. j conserv dent. 2013; 16: 564-7. 16. barakah hm, taher nm. effect of polishing systems on stain susceptibility and surface roughness of nanocomposite resin material. j prosthet dent. 2014 apr 7. pii: s0022-3913(14)00045-6. doi: 10.1016/ j.prosdent.2013.12.007. (in press). 17. korkmaz y, ozel e, attar n, aksoy g. the influence of one-step polishing systems on the surface roughness and microhardness of nanocomposites. oper dent. 2008; 33: 44-50. 18. berger sb, palialol ar, cavalli v, giannini m. surface roughness and staining susceptibility of composite resins after finishing and polishing. j esthet restor dent. 2011; 23: 34-43. 19. schmitt vl, puppin-rontani rm, naufel fs, ludwig d, ueda jk, corrersobrinho l. effect of finishing and polishing techniques on the surface roughness of a nanoparticle composite resin. braz j oral sci. 2011; 10: 105-8. 20. turker sb, biskin t. effect of three bleaching agents on the surface properties of three different esthetic restorative materials. j prosthet dent. 2003; 89: 466-73. 21. marghalani hy. effect of finishing/polishing systems on the surface roughness of novel posterior composites. j esthet restor dent. 2010; 22: 127-38. 22. paravina rd, roeder l, lu h, vogel k, powers jm. effect of finishing and polishing procedures on surface roughness, gloss and color of resinbased composites. am j dent. 2004; 17: 262–6. 23. marghalani hy. effect of filler particles on surface roughness of experimental composite series. j appl oral sci. 2010; 18: 59-67. 24. reis af, giannini m, lovadino jr, ambrosano gm. effects of various finishing systems on the surface roughness and staining susceptibility of packable composite resins. dent mater. 2003; 19: 12-8. 25. takanashi e, kishikawa r, ikeda m, inai n, otsuki m, foxton rm, et al. influence of abrasive particle size on surface properties of flowable composites. dent mater j. 2008; 27: 780-6. 26. canay s, cehreli mc. the effect of current bleaching agents on the color of light-polymerized composites in vitro. j prosthet dent. 2003; 89: 474-8. 27. yu h, li q, wang yn, cheng h. effects of temperature and in-office bleaching agents on surface and subsurface properties of aesthetic restorative materials. j dent. 2013; 41: 1290-6. 28. markovic l, jordan ra, glasser mc, arnold wh, nebel j, tillmann w, et al. effects of bleaching agents on surface roughness of filling materials. dent mater j. 2014; 33: 59-63. 162 effect of finishing and polishing on surface roughness of composite resins after bleaching braz j oral sci. 13(2):158-162 oral sciences n3 braz j oral sci. 10(4):233-235 original article braz j oral sci. october | december 2011 volume 10, number 4 received for publication: march 14, 2011 accepted: october 11, 2011 association between dentin thickness and presence of accessory foramina in human permanent mandibular molars luana de nazaré silva santana¹, luciana brandão freitas², tamea lacerda monteiro², thais de mendonça petta2, ana cássia reis-costa³, rafael rodrigues lima³ 1dental surgeon and master’s candidate in animal science, federal university of pará, brazil 2undergraduate dental students, federal university of pará, brazil 3institute of biological sciences, federal university of pará, brazil correspondence to: rafael rodrigues lima laboratório de neuroproteção e neurorregeneração experimental, instituto de ciências biológicas, universidade federal do pará rua augusto corrêa n. 1. campus do guamá cep: 66075-900. belém – pará, brasil phone/fax: 00 5591 3201 7891 e-mail: rafalima@ufpa.br rafaelrodrigueslima@hotmail.com abstract the roots and periodontal system in human dentition are closely correlated from the early stages of dental formation, maintaining this connection after teeth are established in the oral cavity through the apical foramen or other communications. aim: therefore, the aim of this study was to evaluate the correlation between the presence of foramina on the pulp chamber floor surface and in the furcation region and the thickness of dentin in this area. methods: forty sound permanent mandibular molars were submitted to scanning electron microscopy (sem) to determine the presence of foramina on the pulp chamber floor and in the furcation region, and to correlate the presence of foramina with the variation in wall thickness in these regions. results: the results showed a mean thickness of 2.16 mm for the teeth analyzed and a 25% frequency of foramina on the pulp chamber floor and 92.5% in furcation area, with only 22.5% showing foramina on both sides. the foramina found on both surfaces showed varied diameters and shapes and locations dispersed throughout the area. conclusions: there was no significant difference between the mean thicknesses of teeth featuring foramina and those without foramina. keywords: pulp chamber floor, furcation region, foramina. introduction the anatomic relationship between dental pulp and periodontium in the furcation region trough foramina and accessory canals has prompted several research studies searching for possible pathologic consequences of this close communication1-10. this aberrant contact between tissues in this region is the result of failed differentiation of odontoblasts due to defect formation in hertwig’s sheath4. thus, these foramina can also contribute to the communication between periodontium and dental pulp. studies with primary and permanent teeth using scanning electron microscopy (sem)6 and topographic analyses2,9-10 found a significant incidence of foramina in the furcation area. moreover, some studies using a simple technique (light microscopy) observed the presence of accessory canals1,3-4,7-8. one study compared 234234234234234 braz j oral sci. 10(4):233-235 the incidence of foramina in permanent and primary teeth, and found a greater incidence in primary teeth10. in the literature, there are few data on the influence of pulp chamber floor surface thickness and furcation area on the presence or absence of foramina. therefore, the purpose of this research was to determine the presence of foramina in the pulp chamber floor and furcation area of human permanent mandibular molars and correlate the presence of foramina with the variation of wall thickness in these regions. material and methods forty sound permanent mandibular molars belonging to the tooth bank of the oral histology discipline (opinion 08/2009, bioethics committee of the college of dentistry, ufpa) were examined. the crown and roots were sectioned transversely along the tooth axis with a double-faced steel disc 2 mm from the cementoenamel in the direction of the crown and 3 mm in the direction of the root apex to obtain a reduction in the longitudinal axis in order to facilitate the later stages of evaluation. the samples were cleaned by immersion in 1% sodium hypochlorite (asfer indústria química ltda) for 5 min, and then in 17% edta (farmáciaescola, federal university of pará, brazil) for 30 s. samples were then subjected to a final rinse with distilled water in an ultrasonic bath (ms 200 thornton, impec eletrônica, são paulo, sp, brazil) for 30 s and dried at room temperature overnight. following sample preparation, wall thickness between the pulp chamber floor and the furcation area was measured using a thickness caliper (jon comércio de produtos odontológicos ltda. são paulo, sp, brazil) accurate to the nearest 0.1 mm). later, the samples were metalized and subjected to scanning electron microscopy (model leo1430/laboratory of scanning electron microscopy – labmev do institute of geosciences of ufpa), at 90 ma electron beam current, constant acceleration voltage of 15 kv and working distance of 10 mm. the sem micrographs were analyzed under different magnifications, and the presence/absence of foramina on the pulp chamber floor and furcation area was recorded, divided the sample in two groups. the difference in mean thickness between the group of teeth with foramina on both surfaces and teeth without foramina on both surfaces was evaluated by student’s t-test. results the foramina found on both surfaces showed varied diameters and shapes and locations dispersed throughout the area (figures 1 and 2). the results showed a mean thickness of 2.15 ± 0.41 mm (sd) among the teeth analyzed. the data for the sample was evaluated by the kolmogorov-smirnov test, and a non-significant value of p=0.804 was obtained, confirming the normality of the sample. the study recorded a frequency of 25% of foramina on the pulp chamber floor and 92.5% in the furcation region, with only 22.5% showing foramina on both sides. fig. 1. sem micrograph of foramina in the pulp chamber floor (original magnification x30). fig. 2. sem micrograph of foramina in the furcation region (original magnification x600). the difference in mean thickness between the group of teeth with foramina on both surfaces and teeth without foramina on both surfaces was evaluated by student’s t-test. there was no statistically significant difference for the pulp chamber floor (t = -0.7587 and p = 0.4527) or for the furcation area (t = 0.5712 and p = 0.5712). hence, there was no difference in mean thickness between the teeth with and without foramina. discussion these anatomic communications are of great clinical importance with regard to the periodontal-endodontic interrelation due to its role in the etiopathogenicity of endoperiodontal lesions 11-12. different investigations have examined the presence of pulpoperiodontal canals between the pulp chamber floor and the furcation area and evaluated the possible pathological consequences of this relation5. in the literature, studies already exist showing the incidence of foramina in the furcation area and the pulp association between dentin thickness and presence of accessory foramina in human permanent mandibular molars braz j oral sci. 10(4):233-235 chamber floor1-3,5-9. in the present study, there were a larger number of foramina, both in the furcation area and on the pulp chamber floor, compared with previous investigations. kramer (2003)9 found a prevalence of 53% in the external furcation area and 25% in the internal furcation area using sem analysis9. burch (1974)2 found 76% of foramina in the furcation area using a dissection microscope2. vertucci (1974)3 found 46% lateral canals in the furcation area and 13% on the pulp chamber floor using a dissection microscope3. haznedaroglu (2003)8 found a 21% incidence of patent furcal accessory canals using a stereomicroscope8. however, in the current study, we found 25% of foramina on the pulp chamber floor and 92.5% in the furcation area, with only 22.5% showing foramina on both sides. this result is probably due to the method used, in which organic content and inorganic residue were removed from the teeth by the combination of sodium hypochlorite, edta and ultrasonic bath. this resulted in satisfactory cleanness, clearing the foramina and providing better visualization. thickness was not significantly influenced by the presence of foramina on the pulp chamber floor or in the furcation area. furthermore, the higher frequency of foramina in the furcation area compared to the pulp chamber floor suggests the presence of blind foramina (accessory canals originating from the pulp floor and/or periodontium and ending in dentin without going on to another surface) or loop foramina (originating from the pulp floor and/or periodontium, going through dentin, and returning to pulp chamber or periodontium), which has already been described in another investigation13. hence, in this study, even with 22.5% of the sample showing foramina on both surfaces, it is still impossible to confirm whether there is a real communication between these regions even in these teeth. the dentin-pulp complex and periodontium are closely related since odontogenesis, maintaining this interconnection through the apical foramen. however, there are other accessory pathways of communication, such as lateral apical foramina or even foramina between chamber floor and furcation areas 8,10. these communications have great relevance in endodontic therapy, because their unsealing can result in the maintenance of accessory ways of communication, which, in the presence of an infection process, can facilitate its spread between the periodontium and root canal system, in both directions3. from the obtained results, this study did not show a correlation between thickness and the presence/absence of foramina. although a higher frequency of foramina was observed in the furcation area compared with the pulp chamber floor, it is not possible to infer that the frequency of foramina is associated with the frequency of communication between the surfaces, which suggests the formation of blind or loop foramina. references 1. lowman jv. patent accessory canals: incidence in molar furcation region. oral surg oral med oral pathol. 1973; 36: 580-4. 2. burch jg. a study of the presence of accessory foramina and the topography of molar furcations. oral surg oral med oral pathol. 1974; 38: 451-5. 3. vertucci fj. furcation canals in the human mandibular first molar. oral surg oral med oral pathol. 1974; 38: 308-14. 4. gutman jl. prevalence, location, and patency of accessory canals in the furcation region of permanent molars. j periodontol. 1978; 49: 21-6. 5. goldberg f. accessory orifices: anatomical relationship between the pulp chamber floor and the furcation. j endod. 1987; 13: 176-81. 6. paras lg. an investigation of accessory foramina in furcation areas of primary molars: part 1 – sem observations of frequency, size and location of accessory foramina in the internal and external furcation areas. j clin pediatr dent. 1993; 17: 65-9. 7. wrbas kt. microscopic studies of accessory canals in primary molar furcations. j dent child. 1997; 64: 118-22. 8. haznedaroglu f. incidence of patent furcation accessory canals in permanent molars of a turkish population. int endod j. 2003; 36: 515-9. 9. kramer pf. a sem investigation of accessory foramina in the furcation areas of primary molars. j clin pediatr dent. 2003; 27: 157-61. 10. dammaschke t. scanning electron microscopic investigation of incidence, location and size of accessory foramina in permanent molars. quintessence int. 2004; 35: 699-705. 11. chen sy, wang hl, glickman gn. the influence of endodontic treatment upon periodontal wound healing. j clin periodontol. 1997; 24: 449-56. 12. pilatti gl, toledo bec. as comunicações anatômicas entre polpa e periodonto e suas conseqüências na etiopatogenia das lesões endoperiodontais. rev paul odontol. 2000; 5: 38-42. 13. zuza e. prevalence of different types of accessory canals in the furcation area of third molars. j periodontol. 2006; 77: 1755-61. 235235235235235association between dentin thickness and presence of accessory foramina in human permanent mandibular molars oral sciences n3 original article braz j oral sci. january | march 2013 volume 12, number 1 effect of elastomeric ligatures on frictional forces between the archwire and orthodontic bracket flávia ramos venâncio1, sílvia amélia scudeler vedovello1, carlos alberto malanconi tubel1, viviane veroni degan1, adriana simone lucato1, letícia nery lealdim1 1program in orthodontics, uniararas, araras, sp, brazil correspondence to: sílvia amélia scudeler vedovello avenida maximiliano baruto, 500, araras, sp, brasil. phone: +55 19 35431423 e-mail: silviavedovello@gmail.com abstract aim: to evaluate the frictional force between the archwire and orthodontic bracket generated by elastomeric ligatures with polymer coating (super slick, tp orthodontics) and conventional ligatures (morelli) using two types of insertion techniques. methods: forty elastomeric ligatures, 20 with polymer coating and 20 conventional, were evaluated. each type of ligature was separated into two groups (n=10), according to the insertion mode: conventional or crossed (from mesial to distal region crossed in front). to analyze friction, 40 5-cm-long segments of stainless steel orthodontic archwire 0.019" x 0.025" (morelli) and edgewise brackets (slot 0.022" x 0.028"; morelli) were used. each set (bracket, wire and elastic) was submitted to frictional testing in a universal test machine (instron 4411) at a crosshead speed of 5 mm/min. each bracket was moved 5 mm on the wire, with maximum friction and mean friction being recorded by software. three readouts were taken for each bracket. data were submitted to two-way anova and tukey’s test (p<0.05). results: it was shown that for maximum and mean friction, the polymer-coated ligature did not differ statistically from the conventional type in a dry environment condition. ligatures placed in crossed mode promoted significantly greater friction than those placed in conventional mode, irrespective of the type of elastomeric ligature. conclusions: friction depended on the insertion mode, but not on the type of elastomeric ligature. keywords: friction, elastomer, orthodontics. introduction sliding mechanics may be used in cases of tooth extraction, severe crowding, or for problems of discrepancy between the dental arches1, involving movement of brackets along archwires2. however, the disadvantage of using this type of mechanics is the friction generated between the bracket and wire during orthodontic movement3, in which the friction produced at the bracket-wire interface tends to prevent the desired movement. according to burrow and charlotte (2009)4, friction is defined as the force of resistance exerted by the surfaces opposed to the movement. the area of contact is influenced by the roughness and force with which the surfaces are pressed against each other. there are two types of friction, static and kinetic. static friction is opposed to any application of force, and its magnitude is exactly what it should be to prevent movement between two surfaces, up to the point when it is overcome and movement begins, that is, the force applied is not sufficient to move the object. on the other hand, kinetic friction is opposed to the direction of movement of the object and occurs when the bodies are in motion, that is, kinetic friction is irrelevant during the orthodontic movement of teeth, as continuous movement of braz j oral sci. 12(1):41-45 received for publication: november 07, 2012 accepted: february 25, 2013 the tooth along an arch is rare4. clinically, dental sliding movement is always preceded by inclination and rotation of the teeth5. during orthodontic movement, at the stage of alignment, leveling and in sliding mechanics during space closure, the desirable situation is that there should be little or no friction at the bracket-wire interface. various factors interfere in friction, but alterations to the elastomeric ligatures deserve attention as they play an important role with regard to friction between the bracket and wire during orthodontic mechanics. a reduction in friction between the bracket and orthodontic wire may be obtained with the use of lubricated elastomeric ligatures, or with alterations in their composition6. recently, a ligature with polymer coating was launched on the market: super slick (tp orthodontics, la porte, in, usa), with the purpose of reducing friction, in comparison with conventional ligatures. according to the manufacturer, this ligature is covered with a polymer coating, allowing greater sliding of the wire over this material. in orthodontic treatment, the most frequently used modes of insertion of elastomeric ligatures are the conventional and crossed types. ligatures inserted in crossed mode are responsible for a significant increase in frictional force in comparison with other insertion techniques, in a dry and moist medium7. however, little is known about studies of friction generated by elastomeric ligatures with surface alterations using different insertion techniques for retention of the wire to the orthodontic bracket. it is thus important to study the friction generated by elastomeric ligatures available on the market with the purpose of making orthodontic treatment efficient, fast and inexpensive. according to leander and kumar (2011)8, ligature is the most used method for uniting the wire to the bracket, as ligatures are comfortable for the patient, offer fewer risks of causing damage to the mucosa, have better acceptance due to the possibility of choosing colors and increase motivation as regards treatment. therefore, the aim of this study was to evaluate the frictional force between the archwire and orthodontic bracket generated by elastomeric ligatures coated with polymer (super slick, tp orthodontics) and conventional ligatures (morelli) using two types of insertion techniques. material and methods fabrication of the support for the friction test a rectangular acrylic plate (14 cm long by 4 cm wide by 0.5 cm thick), with a groove (1.5 cm deep by 1.2 cm wide) at a distance of 2 cm from one of the extremities was used for the friction test. initially, the bracket bonding area was abraded with 120 grit abrasive papers (3m, sumaré, sp, brazil), then edgewise brackets, slot 0.022"x 0.028" (morelli ltda ref. 10.30.208), were bonded using chemically activated orthodontic concise (3m unitek corporation, monrovia, ca, usa) at a distance of 0.8 cm between them and at 1.6 cm in the region of the groove. the distance from the top edges of the brackets to the top extremity of the plate was 0.4 cm (figure 1). before resin polymerization occurred, a stainless steel wire (0.021’’x 0.025’’) was used to align the brackets and removed after polymerization. after this, a 5-cm-long stainless steel wire segment (0.019"x 0.025"; morelli; ref. 55.03.014) was inserted into the channel of the brackets, with its two extremities bent to prevent the wire from being displaced from the channels during the friction test. fig.1. a) stainless steel tie wire holding the bracket-universal test machine; b) bracket test; c) acrylic plate; d) weight simulating the premolar. test specimen fabrication the extremity of an orthodontic wire 1.00 mm in diameter by 14 mm long (morelli; 55.06.100) was fixed to the back of the edgewise orthodontic bracket slot 0.022" x 0.028" (morelli ref. 10.30.208) with resin z250 (3m espe, st. paul, mn, usa), light activated with an xl 2500 lightcuring unit (3m espe), for 20 s, with the bracket slot at 90o in relation to the long axis of the wire. a groove was made 10 mm from the center of the bracket, using a carborundum disc (kg sorensen, são paulo, sp, brazil) in order to place a 100 g weight, simulating the weight of a tooth, in the universal test machine (figure 2). for each test specimen, a bracket was used with a stainless steel wire segment 0.019"x 0.025" (morelli ltda ref. 55.03.014) and an elastomeric ligature with polymer covering 4242424242effect of elastomeric ligatures on frictional forces between the archwire and orthodontic bracket braz j oral sci. 12(1):41-45 frictional means (n) elastic conventional b crossed a mean (sd) m i n i m u m m a x i m u m mean (sd) m i n i m u m m a x i m u m tp a 1.89 (0.24) 1.52 2.39 3.26 (0.28) 2.87 3.70 morellia 1.99 (0.32) 1.51 2.41 3.09 (0.29) 2.72 3.59 table 1. friction means (n) of the elastomeric ligatures inserted using two modes. different lowercase letters in column and capital letters in row differ significantly between them (tukey’s test; p<0.05). (super slick, tp orthodontics, usa) or conventional type (morelli; ref. 60.03.317). the elastomeric ligatures were inserted in the conventional mode with the elastic tie applicator (morelli) embracing the mesial and distal winglets of the brackets. in crossed mode they were first placed in the applicator in the same way as before, followed by removal and insertion in the bracket with a mathieu needle holder (mocar, são paulo, sp, brasil) from the mesial to distal direction, crossing in the center of the bracket. a total of 40 test specimens were made, 20 for the conventional (10 tp ligatures and 10 morelli ligatures) and 20 for the crossed technique (10 tp ligatures and10 morelli ligatures). fig. 2. segment of an orthodontic wire (1.00 mm diameter x 14 mm long) fixed to the back of the edgewise orthodontic test bracket. resistance to friction test to analyze friction, the acrylic plate was placed in the universal test machine. for bracket movement, a stainless steel tie wire 0.025mm (morelli) was used, fixed to one of the extremities in the test machine by means of a vise, and the other extremity fixed on the bracket. friction analysis was performed at a crosshead speed of 5 mm/min, the peak and mean values recorded with bluehill 2.0 software coupled to the test machine. the bracket was moved 5 mm on the wire and the friction evaluated during the run. for each set, 3 repetitions were performed and the mean obtained. the mean and maximum friction analysis data were submitted to two-way anova (insertion mode and elastomeric ligature) and tukey’s test (p<0.05). results anova showed that for friction there was significant influence of the elastomeric ligature placement mode (p<0.001), but not for elastomeric ligature (p=0.92) and the interaction between them (p=0.203). tukey’s test (table 1) showed that friction of the elastomeric ligatures inserted in the crossed mode was higher in comparison with the conventional mode. there was no statistically significant difference between ligatures for conventional and crossed insertion modes. discussion the technological advancement of elastic materials has increased their applicability in contemporary orthodontic treatments. in order to reduce the friction caused in the bracket/orthodontic archwire/ligature set, ligatures with polymer coating have been introduced on the market. in the present study, they were compared with conventional ligatures, using two insertion modes. some factors have been attributed to the increase in friction in orthodontic systems, including the alloy of which the wire is made, wire deflection and thickness, the material of the bracket channel, bracket width, lubrication and the method of ligation9-11. it is known that the frictional force tends to increase with rectangular cross-section wires in comparison with round wires. cacciafesta et al. (2003)12 reported higher frictional force with an increase in orthodontic archwire thickness. in ceramic brackets, there is significantly higher friction in comparison with metal or plastic brackets13-15. according to 4343434343 effect of elastomeric ligatures on frictional forces between the archwire and orthodontic bracket braz j oral sci. 12(1):41-45 gandini et al. (2008)16 the metal ligature produces less frictional force in comparison with elastomeric ligatures; however, friction depends on the tying force between the metal ligature and orthodontic archwire, differently from elastomeric ligatures17. moreover, longer clinical chairtime is required when metal ligatures are used in comparison with elastomeric ligatures. in order to reduce the friction caused in the bracket/ orthodontic archwire/ ligature set, self-ligating bracket systems have been introduced on the market. in addition to reducing chair time, these devices have shown significantly lower levels of friction in comparison with the system of brackets with conventional ligatures12-14. nevertheless, these brackets present a higher increase in frictional force when used with wires with rectangular cross sections12. in this in vitro study, it was verified that the elastomeric ligature with a polymer covering super slick (tp orthodontics) promoted similar friction to that of the conventional ligature (morelli). according to the manufacturer, the products with this technology are made with a polymer coating, insoluble in water, modifying the elastomeric surface, making it extremely smooth, slippery and lubricated. it is likely that the super slick ligature produced similar friction to that generated by the conventional ligature because the tests in this study were performed in a dry environment condition. on the other hand, some studies have reported that the use of artificial saliva acts as an adhesive17-18, and others that saliva reduces friction19-20, while a third point of view indicated that saliva makes no difference on the effect of friction21. according to magno et al. (2008)22, the polymer coating could be altered during stretching of the ligature with the elastic tie applicators for placement in the bracket, thus modifying the quality of the surface and responsible for the increase in friction. it is known that alterations in the structure of the elastomeric ligature surfaces may vary according to the time and place of storage23, and may affect the polymer covering. although this is not a variable that is under the orthodontist’s control it should be duly considered, because the form of storage, transportation conditions and time may vary from one region to the other, considering the geographic dimension of brazil and the place of manufacture (usa). the final quality of the elastomeric ligature depends on the raw material, the used technology and quality control. although the ligatures used in this study were from the same lot, according to wong (1976)24 ligatures from the same lot may present different properties. the elastic ligatures from morelli and tp orthodontics are synthetic, produced from petroleum-derived polyurethane materials; therefore, they are obtained by means of chemical transformations of coal, petroleum and some vegetable alcohols, which exact composition is not disclosed by the manufacturers. thus, in the present study, it was verified that the ligatures placed in the crossed mode were responsible for a significant increase in frictional force between the orthodontic wire and bracket when compared with the conventional insertion technique, irrespective of the used brand. this probably occurred due to the increase of contact surface between the elastomeric ligature and the orthodontic archwire. in this mode of inserting, the elastomeric ligature is crossed over the wire at the points of contact (mesial, distal and center) with the orthodontic archwire surface, whereas in the conventional mode, there are only two points where the ligature touches the wire. another cause of the increase in friction in this mode of insertion may be the stress generated by stretching, which is transmitted to the orthodontic wire. in this case, when the ligature is stretched, it is tenser and does not return to the original form, and is therefore inserted into the bracket in an extended form so that it will be crossed over it. thus, the friction increases by the increase in force with which the surfaces are pressed against each other. it would be recommendable therefore to use elastomeric ligatures or forms of insertion that promote a lower degree of friction in the initial stages of alignment and leveling, as well as in the stages of space closure, so that efficient tooth movement occurs. on the other hand, greater friction would be required during the correction of teeth rotation in the initial stages, when greater contact of the orthodontic wire inside the bracket slot is necessary, in the final stages when torque is necessary25 and in teeth that form part of anchorage units in some orthodontic mechanics. in summary, the present study demonstrated that no significant difference could be identified between the friction of conventional ligatures and ligatures with polymer coating in a dry environment condition. the crossed mode of inserting the elastomeric ligatures promoted greater friction than the conventional insertion mode, irrespective of the type of ligature. references 1. souza rs, pinto as, shimizu rh, sakima mt, gandini junior lg. avaliação do sistema de forças gerado pela alça t de retração préativada segundo o padrão unesp-araraquara. rev dent press ortod ortop facial. 2003; 8: 113-22. 2. ogata rh, nanda rs, duncanson mg, sinha pk, currier gf. frictional resistances in stainless steel bracket-wire combinations with effects of vertical deflections. am j orthod dentofacial orthop. 1996; 109: 535-42. 3. chimenti c, franchi l, giuseppe mg, lucci m. friction of orthodontic elastomeric ligatures with different dimensions. angle orthod. 2005; 75: 421-25. 4. burrow sj, charlotte nc. frictional and resistance to sliding in orthodontics: a critical review. am j orthod dentofacial orthop. 2009; 135: 442-7. 5. loftus bp, artun j. a model for evaluating friction during orthodontic tooth movement. eur j orthod. 2001; 23: 253-61. 6. baccetti t, franchi l. friction produced by types of elastomeric ligatures in treatment mechanics with the preajusted appliance. angle orthod. 2006; 76: 211-6. 7. edwards gd, davies eh, jones sp. the ex vivo effect of ligation technique on the static frictional resistance of stainless steel brackets and archwires. br j orthod. 1995; 22: 145-53. 8. leander d, kuramar jk. comparative evaluation of frictional characteristics of coated low friction ligatures super slick ties with conventional uncoated ligatures. indian j dent res. 2011; 22: 90-4. 9. griffiths hs, sherriff m, ireland aj. resistance to sliding with 3 types of elastomeric modules. am j orthod dentofacial orthop. 2005; 127: 670-5. 10. hain m, dhopatkar a, rock p. the effect of ligation method on friction in sliding mechanics. am j orthod dentofacial orthop. 2003; 123: 416-22. 4444444444effect of elastomeric ligatures on frictional forces between the archwire and orthodontic bracket braz j oral sci. 12(1):41-45 11. frank ca, nikolai rj. a comparative study of frictional resistances between orthodontic bracket and arch wire. am j orthod dentofacial orthop. 1980; 78: 593-609. 12. cacciafesta v, sfondrini mf, ricciardi a, scribante a, klersy c, auricchio f. evaluation of friction of stainless steel and esthetic self-ligating brackets in various bracket-archwire combinations. am j orthod dentofacial orthop. 2003; 124: 395-402. 13. mendes k, rossouw pe. friction: validation of manufacturer’s claim. seminars in orthod. 2003; 9: 236-50. 14. pithon mm, dos santos rl, nascimento le, ayres ao, alviano d, bolognese am. do self-ligating brackets favor greater bacterial aggregation? braz dent j. 2011; 10: 3,. 208-12. 15. keith o, orth m, jones sp, davies eh. the influence o bracket material, ligation force and wear on frictional resistance of orthodontic brackets. br j orthod. 1993; 20: 109-15. 16. gandini p, orsi l, bertoncini c, massironi s, franchi l. in vitro frictional forces generated by three different ligation methods. angle orthod. 2008; 78: 917-21. 17. khambay b, millet d, mchugh s. archwire seating forces produced by different ligation methods and their effect on frictional resistance. eur j orthod. 2005; 27: 302-8. 18. downing a, mc cabe jf, gordon ph. the effect of artificial saliva on the frictional forces between orthodontic brackets and archwire. br j orthod. 1995; 22: 41-6. 19. thorstenson ga, kusy rp. effects of ligation type and method on the resistance to sliding of novel orthodontic brackets with second-order angulation in the dry and wet states. angle orthod. 2003; 73: 418-30. 20. baker kl, nieberg lg, weimer ad, hanna m. frictional changes in force values caused by saliva substitution. am j orthod dentofacial orthop. 1987; 91: 316-20. 21. kusy rp, whitley jq, prewitt mj. comparison of the frictional coefficients for selected archwire-bracket slot combinations in the dry and wet states. angle orthod. 1991; 61: 293-302. 22. magno af, enoki c, ito iy, matsumoto, faria g, filho pn. in vivo evaluation of the contamination of super slick elastomeric rings by streptococcus mutans in orthodontic patients. am j orthod dentofacial orthop. 2008; 133: 4-9. 23. edwards ir, spary dj, rock wp. the effect upon friction of the degradation of orthodontic elastomeric modules. eur j orthod. 2012; 34: 618-24. 24. wong ak. orthodontic elastic materials. angle orthod. 1976, 46: 196205. 25. ioi h, yanase y, uehara m, hara a, nakasima n, nakasima a. frictional resistance in plastic preadjusted brackets ligated with low-friction and conventional elastomeric ligatures. journal of orthodontics 2009; 36: 17-22. 4545454545 effect of elastomeric ligatures on frictional forces between the archwire and orthodontic bracket braz j oral sci. 12(1):41-45 oral sciences n3 braz j oral sci. 10(4):268-271 original article braz j oral sci. october | december 2011 volume 10, number 4 prevalence and microscopic features of enamel pearls from permanent human molars estela kaminagakura1, cristiane ribeiro salmon2, douglas campideli fonseca3, maria cândida almeida lopes4, rubens nisie tango5 1professor of stomatology, são josé dos campos dental school, unesp-university estadual paulista, brazil 2 phd, bucco-dental biology area, piracicaba dental school, university of campinas, brazil 3professor of clinical periodontology, dental school, university center of lavras, brazil 4 professor of oral surgery, federal university of piauí, brazil 5professor of dental materials, são josé dos campos dental school, unesp-university estadual paulista, brazil correspondence to: rubens nisie tango rua engenheiro francisco josé longo, 777 são josé dos campos, sp cep 12245-000 brasil phone/fax: +55 12 3947 9369 e-mail: tango@fosjc.unesp.br abstract enamel pearls are ectopic structures observed mainly on the roots of permanent teeth and could be related to periodontal diseases. aim: to evaluate the prevalence of enamel pearls in extracted human molars and characterize their structures using light and scanning electron microscopy. methods: the study comprised 2,201 extracted human permanent molars. the teeth were analyzed and classified according to morphological features. the presence, location and shape of enamel pearls were investigated. fifteen human molars with enamel pearls were embedded in acrylic resin and observed by light microscopy. results: seventy-one enamel pearls were identified on third molar root. microscopically, most pearls were composed of prismatic irregular enamel and normal dentin. the dentinoenamel junction presented an irregular course. the number of dentinal tubules was normal and they presented curvature to continue within the root dentin of the carrier tooth. dentinal tubules below the enamel pearls were closer to each other. conclusions: scanning electron microscopic analysis revealed that the enamel pearls were similar to coronal enamel. keywords: microscopy, electron, scanning, enamel, tooth abnormalities. introduction enamel is normally restricted to the anatomical crowns of human teeth1. however, ectopic structures, called enamel pearls, that are firmly adherent to the roots of deciduous and permanent teeth may occur1-2. their prevalence ranges from 1.1 to 9.7% among molar teeth and the majority are detected on maxillary third molars1,3. one pearl per root is the most common occurrence, but two or more pearls have also been identified1,3-4. microscopically, they can consist entirely of enamel, but large pearls may also contain dentin and pulp tissue. the pearls are characterized by a similar structure to that found on dental crown; however, the shape and direction of enamel prisms and dentinal tubules might be irregular5. striae of retzius, huntershreger bands and aprismatic enamel have been observed6 and, occasionally, enamel pearls are covered by a thin cementum layer5-6. received for publication: august 25, 2011 accepted: december 06, 2011 269 braz j oral sci. 10(4):268-271 enamel pearls have a distinct predilection for the furcation area of molar teeth and for concavities or furrows within the root structure1,3-4. previous studies have reported that the composition, size and topographic relation to furcation may contribute to periodontal disease1,7. the aims of this study were to evaluate the prevalence of enamel pearls in extracted molars from the unilavras human tooth bank and to characterize their enamel and dentin by light microscopy (lm) and scanning electron microscopy (sem). material and methods the material comprised 2,201 clean and sterile extracted human permanent molars stored in the human tooth bank of the university center of lavras, (centro universitário de lavras, unilavras), lavras, mg, brazil. the dates of and reasons for the extractions were unknown and unavailable. the teeth were analyzed and classified according to morphological features. the presence, location and shape of the enamel pearls were investigated under a loupe at ×20 magnification. all teeth were analyzed by a single examiner. this study was approved by the local ethics committee (caae 0002.0.189.000-05). light microscopy (lm) briefly, 15 molars with enamel pearls were dehydrated in an increasing series of ethanol concentrations, dried and embedded in acrylic resin (vipi crill®, vipi, são paulo, sp, brazil) for 2 h under vacuum. infiltration was achieved with metacrylate with 0.25% benzoyl peroxide overnight at 16oc and methacrylate with 0.5% benzoyl peroxide for 2 h under vacuum. the teeth were placed in glass test tubes and maintained at 25oc until the resin cured. next, the tubes were broken, the resin was trimmed and the teeth were sectioned longitudinally under water coolant through enamel pearls with a diamond saw (extec 12205; extec corp, enfield, ct, usa) mounted on an automatic cutter (model 650; south bay technology inc., san clemente, ca, usa). sections measuring 20 µm in thickness were cut, ground and polished. the samples were ultrasonicated, dried and mounted on glass slides. the enamel pearls were analyzed by light microscope regarding composition, enamel prisms and dentin tubule organization. scanning electron microscopy (sem) five enamel pearls were removed from their respective carrier teeth with a diamond saw, washed with distilled water and acid etched with 35% phosphoric acid (dental gel, dentsply ind. e com. ltda., petrópolis, rj, brazil) for 30 s. after washing, the samples were fixed on metal stubs, dehydrated in silica for 2 h, gold sputtered and examined with scanning electron microscope (jeol v, tokyo, japan). images from the sem were obtained using working distances (wds) of 15 mm and 40 mm, 15kv. results seventy-one enamel pearls were detected on 63 permanent molars of a total of 2,201 teeth analyzed (2.86%); in 8 of these teeth (12.69%) 2 enamel pearls were observed. all enamel pearls were observed in the furcation or furrow on the root of the third molar (figure 1). hemispherical or spherical were the most common forms observed. the macroscopic features were the same as coronal enamel in relation to color and texture. microscopically, two pearls were composed entirely by enamel and 13 by enamel and dentin (figure 2). irregularities were detected on the external surface. in most cases, the enamel observed was prismatic with an irregular course. close to the dentinoenamel junction, the enamel appeared disorganized. in some cases, the dentinoenamel junction presented an irregular course. the number of dentinal tubules was normal and they presented curvature to continue within the root dentin of the carrier tooth. dentinal tubules below the enamel pearls were closer to each other than normal root dentinal tubules. in the sem analysis, the pearls showed both prismatic and aprismatic enamel similar to regular coronal enamel (figure 3a and b) and irregularities could be observed on the pearl surface. fig. 1. clinical appearance of spherical enamel pearl located in the furcation region of the root. fig. 2. sem micrograph showing an oval enamel pearl (x50, ground section). prevalence and microscopic features of enamel pearls from permanent human molars 270 braz j oral sci. 10(4):268-271 discussion in this study, the prevalence of enamel pearls on extracted teeth was 2.8%, similar to previous reports8, and on about 87% of the teeth, only one pearl was observed. more than two pearls on a single tooth are rare5. all the enamel pearls were detected on third molars, in disagreement with a radiographic study that reported they are detected more frequently on the mandibular first molar, followed by the maxillary first molar9. analyses of samples from human tooth banks can lead to incomplete demographic data and clinical information, because in many cases the donor cannot be identified, as reported previously by chrcanovic1 (2010). additionally, third molars are the most frequent teeth available in human tooth banks. the enamel pearls were easily differentiated from the cementum surface by their color and texture and the fact that they appear as discrete, glass-like globular bodies attached to the root by a sessile base3. some authors identified these structures using the naked eye under direct light 1; however, a loupe was used in the present investigation. fig. 3. sem micrographs showing a) spherical enamel pearl located in the furrow of the root and b) prismatic and aprismatic enamel (conditioned enamel). enamel pearls could contribute to periodontal disease onset and progression because of their composition, morphology and location1. the enamel covering the pearl can interfere in connective tissue attachment, permitting only a hemidesmosomal junction less resistant to periodontal breakdown; moreover, their morphology enhances plaque retention and protects organisms from the action of salivary enzymes and oral hygiene measures7,10. in addition, their topographic relation to the furcation can hinder treatment10, as the presence of superficial concavities filled with organic material on the enamel pearls have been described11. in the present study, irregularities on the enamel pearl surface were observed by sem. the majority of the enamel pearls analyzed were formed by enamel and dentin 3, particularly the large pearls. microscopically, enamel pearl structure is similar to that of a dental crown, but it shows localized defects with irregularities in the shape and course of the prisms6, which are likely related to the aberrant timing of enamel pearl formation4. the enamel identified on the roots resembles immature enamel3. it shows irregular areas of hypomineralization and the prisms do not always end on the free surface, often presenting thin layers without structure 5. occasionally, hunter-schreger bands and striae of retzius are present5. when dentin is present, it generally appears normal. the number and course of dentinal tubules is also normal and they continue into the root dentin of the tooth without interruption6. cavanha5 (1965), however, reported that the direction of the dentinal tubules is irregular and less mineralized. in the present study, disorganized dentin was only observed close to the dentinoenamel junction and the latter presented an irregular course, as observed by gaspersic12 (1992). interglobular dentin and tome’s granular layer in large pearls and a conical-shaped core of dentin that resembles a cusp have been reported3. enamel pearls represent the localized activity of portions of hertwig’s epithelial root sheath, which retain a latent potential to become functional ameloblasts and enamel organ that produces enamel deposits on the root3,12. the mechanism that permits this latent capacity remains unknown12. genetic factors do not play a decisive role in enamel pearl formation for some authors4,6,8, but others disagree4,11. our research group agrees with moskow and canut3 (1990), who suggested that enamel pearls are aberrations. the mineral contents of composite enamel and dentin from pearls are similar. the mineral-content gradient in the enamel pearl is the same as that of premolar enamel, as both have higher mineral contents at the surface. however, for pearl dentin, the opposite is true; i.e., compared with coronal dentin, it presents a reverse mineral-content gradient. this suggests that the process of mineralization in pearl dentin differs from that of permanent coronal dentin12. another study indicated that the enamel pattern of large pearls is structurally and biochemically identical to that of the carrier tooth6. the findings of the present study do not fully support this assumption, since the dentinal tubules that continue into the root dentin of the carrier teeth were closer to each other and, because of such organization, this area seemed to be prevalence and microscopic features of enamel pearls from permanent human molars braz j oral sci. 10(4):268-271 271 more mineralized. in conclusion, the prevalence of enamel pearls in teeth collected from the unilavras human tooth bank was 2.8%; only one pearl was detected in approximately 87% of the teeth examined; the third molar was most affected tooth; and the macro and microscopic features of enamel pearl were similar to those of coronal enamel. references 1. chrcanovic br, abreu mh, custódio al. prevalence of enamel pearls in teeth from a human teeth bank. j oral sci. 2010; 52: 257-60. 2. anderson p, elliott jc, bose u, jones sj. a comparison of the mineral content of enamel and dentine in human premolars and enamel pearls measured by x-ray microtomography. archs oral biol. 1996; 41: 281-90. 3. moskow bs, canut pm. studies on root enamel (2). enamel pearls. a review of their morphology, localization, nomenclature, occurrence, classification, histogenesis and incidence. j clin periodontol. 1990; 17: 275-81. 4. risnes s. enamel pearls and cervical enamel projections on 2 maxillary molars with localized periodontal disease: case report and histologic study. oral surg oral med oral pathol oral radiol endod. 2000; 89: 493-7 5. cavanha ao. enamel pearls. oral surg oral med oral pathol. 1965; 19: 373-82. 6. gaspersic d. enamel microhardness and histological features of composite enamel pearls of different size. j oral pathol med. 1995; 24: 153-8. 7. matthews dc, tabesh m. detection of localized tooth-related factors that predispose to periodontal infections. periodontol 2000. 2004; 34: 136-50. 8. risnes s. the prevalence, location, and size of enamel pearls on human molars. scand j dent res. 1974; 82: 403-12. 9. darwazeh a, hamasha aa. radiographic evidence of enamel pearls in jordanian dental patients. oral surg oral med oral pathol oral radiol endod. 2000; 89: 255-8. 10. romeo u, palaia g, botti r, del vecchio a, tenore g, polimeni a. enamel pearls as a predisposing factor to localized periodontitis. quintessence int. 2011; 42: 69-71. 11. saini t, ogunleye a, levering n, norton ns, edwards p. multiple enamel pearls in two siblings detected by volumetric computed tomography. dentomaxillofac radiol. 2008; 37: 240-4. 12. gaspersic d. histogenetic aspects of the composition and structure of human ectopic enamel, studied by scanning electron microscopy. arch oral biol. 1992; 37: 603-11. prevalence and microscopic features of enamel pearls from permanent human molars oral sciences n3 braz j oral sci. 10(3):167-170 original article braz j oral sci. july | september 2011 volume 10, number 3 received for publication: march 18, 2011 accepted: july 06, 2011 force degradation of different elastomeric chains and nickel titanium closed springs bruno ubirajara pires1, rafael evangelista de souza2, mario vedovello filho3, viviane veroni degan3, julio cesar bento dos santos3, carlos alberto malanconi tubel3 1dds, graduate student in orthodontics, fundação hermínio ometto-uniararas, araras, sp, brazil 2undergraduate student dentistry, fundação hermínio ometto-uniararas, araras, sp, brazil 3phd, professor of the graduate program in orthodontics, fundação hermínio ometto-uniararas, araras, sp, brazil correspondence to: mario vedovello filho av. dos trabalhadores , 2991 jd jacira mogi guaçu sp brazil phone/fax: 19 – 38610472 // 19 -3861 0178 e-mail: vedovelloorto@terra.com.br abstract aim: the purpose in this study was to evaluate the degradation force of conventional synthetic orthodontic elastics and synthetic orthodontic elastics with memory properties. methods: specimens of each material (plastic chain, memory chain and closed spring niti) were stretched and adapted to the test specimens composed of resin plates and orthodontic wires, simulating retraction units. degradation force was verified in an instron universal test machine at the following intervals: 1, 2, 18, 24, 48 h; 7, 14, 21, and 28 days. data (gf) were analyzed statistically using friedman and kruskal-wallis tests at 5% significance level. results: it was observed a significant force reduction of plastic chain and memory chain after 2 h (p<0.05). for niti spring significantly force reduction was observed after 18 h, but no significant change was showed up to 21 days. niti spring showed force significantly higher than synthetic elastomeric materials (p<0.05). there was no significant difference between memory chain and plastic chain up to 24 h. however, from 48 h to 21 days memory chain showed force significantly higher than plastic chain. conclusions: there was higher force degradation in the synthetic elastomeric materials in comparison with niti springs, which allows the preferential indication of these space closure jigs for clinical use. keywords: orthodontics, biomechanics, tensile strength, elastomeric chains, closed nickel titanium. introduction interdental space closure is a common procedure in daily orthodontic clinical practice. in this context, different space closing systems have been proposed1-5. although a great deal is known about these systems, several studies are still being conducted in search of the ideal interdental space closure system. there is literary consensus in the sense that the ideal space closure mechanism should have mechanical properties that provide a light and continuous force that closes the orthodontic space in the shortest possible time3,6-8. firstly, this type of force would have the advantage of causing the least possible periodontal and root trauma, in addition to reducing the time of orthodontic treatment. in this context, many space closure mechanisms have been suggested, among these elastomeric materials and closed niti springs. elastomers have been used more frequently, as they are more practical and less costly. however, force degradation studies have proven that the elastomers loose a large part of their force generating capacity with the passage of time1-2,9-15. the environment and temperature in which the elastic acts also interfere considerably in maintaining 168 braz j oral sci. 10(3):167-170 the properties of this type of material. it is known that elastics conserved in a humid environment and exposed to higher temperatures suffer greater reduction on their force generating capacity5. however, niti springs with shape memory may generate lower and continuous forces and be less influenced by the humidity and ph of the environment 11-12,16-20. synthetic orthodontic elastics with memory chain have been launched to the dental market. these elastics are used for intra-arch space closure and are low cost in comparison with the niti spring used for the same purpose. thus, the aim of this study was to evaluate the degradation force suffered by conventional synthetic elastics, synthetic elastics with memory chains and niti springs during 28 days when exposed to an environment similar to the oral cavity. material and methods fifty samples were obtained of each of the three materials to be tested: conventional elastomeric chain (plastic chain, american orthodontics, sheboygan, wi, usa), elastomeric chain with memory (memory chain, american orthodontics) and closed niti spring (gac, new york, usa). to simulate the mechanism of orthodontic retraction, 15 plates (3 mm thick x 30 mm wide x 150 mm long) were made of resin, divided into 3 groups, each of which was composed of 5 plates according to the material to be tested. into these plates, 10 pairs of stainless steel orthodontic wire pins measuring 1.2 mm in diameter (morelli, sorocaba, sp, brazil) were inserted. the pins were arranged in parallel pairs. the samples were inserted into each pair in such a way that their tips were distended during the evaluated test periods (figure 1). fig. 1 test specimen (plastic chain) stretched on plate. the force of the stretched samples was measured in a universal test machine (instron corp., canton, ma, usa). the initial distention force of these samples was standardized close to 200 gf, the force recommended for retraction of the anterior segment of the arches4,7. the amount of material in each tested elastomeric segment was also standardized at 6 links, so that the distension stress would be similar in all elastomeric segments. the samples were carefully transferred to the resin plates while they were still distended. to simulate the oral conditions, the plates containing the elastomeric chains and the niti springs were immersed in artificial saliva (saliform; fórmula e ação, são paulo, sp, brazil) and maintained at 37oc for 28 days. at the time intervals of 1 h, 2 h, 18 h, 24 h, 48 h, 7 days, 14 days, 21 days and 28 days the samples were removed from the resin plates, which in turn were immersed in artificial saliva and kept in an oven. after this, the retraction force of each sample was measured. data (gf) were analyzed statistically using friedman and kruskal-wallis tests at 5% significance level. results table 1 shows the means obtained as a function of the groups under study and the periods of time. the three experimental groups were compared (kruskal-wallis test) according to each period of time, and the samples of each group were compared separately (friedman test) at the experimental time intervals. the forces of niti spring, memory chain and plastic chain are presented in table 1. niti spring showed significantly higher force than synthetic orthodontic elastomeric in all periods. there was no significant difference between memory chain and plastic chain up to 24 h. moreover, no significant difference (p>0.05) was found between memory chain and plastic chain up to 24 h (p<0.05). however, from 48 h to 21 days, memory chain showed force significantly higher (p<0.05) than plastic chain. figure 2 shows the force of the space closure materials over time (28 days). it was found a significant decrease on force of niti spring after 18 h, but the force remained constant up to 21 days. for memory chain and plastic chain synthetic elastomeric materials, it was verified a significant force reduction after 2 h, with a gradual decrease up to 28 days. force (gf) times niti spring memory chain plastic chain initial 228.5 a,a 206.0 b,a 195.0 b,a 1 h 221.5 a,a 152.5 b,a,b 147.5 b,a,b 2 h 220.5 a,a 144.0 b,b,c 132.0 b,b,c 18 h 202.5 a,b 112.5 b,b,c 105.0 b,c,d 24 h 197.0 a,c 98.0 b,c 98.0 b,d 48 h 196.5 a,b,c 102.0 b,c 88.0 c,d,e 7 days 195.0 a,c 94.0 b,c,d 80.0 c,e,f 14 days 198.5 a,b,c,d 90.0 b,d,e 75.8 c,f,g 21 days 205.5 a,b,d 86.5 b,d,e 71.5 c,g 28 days 188.0 a,c,e 76.5 b,e 71.5 b,g table 1 median (1st and 3rd quartiles) of force (in gf) as a function of the groups under study and the two periods of time means followed by different lowercase letters in the line, and capital letters in the column, differ statistically among them, by the friedman and kruskal-wallis tests at a level of significance of 0.05%. discussion in order to obtain increasingly effective tooth movements, that is, with maximum tooth movement associated with anchorage control; tooth inclinations and vertical and rotational forces control, and at the same time, force degradation of different elastomeric chains and nickel titanium closed springs braz j oral sci. 10(3):167-170 169 ensuring the health of the teeth and periodontal tissues3,6-8,12, this study compared the force degradation suffered by three materials: conventional elastomeric chain (plastic chain), elastomeric memory chain (memory chain) and closed niti springs. the initial force of all samples was standardized at close to 200 gf3-4,7. in addition to standardization of force, the stretching of the materials was standardized in the case of the elastics, so that there would be no difference in the stress produced by the distention21. the results of this study pointed out that the closed niti springs were more resistant to force degradation than the two types of elastomeric chains: conventional or with memory, according to figure 1 and table 1. these results are similar to those of previous studies11,16-18,20,22. this characteristic of force maintenance is important, as it is related to a more physiological movement, with maximum amplitude and minimal aggression to the dental and periodontal tissues. the three retraction mechanisms lost force; however, force degradation in the spring was much lower than in the two types of elastics tested. another notable difference between the closed niti spring and the two types of elastics was the period in which there was the highest force degradation. in the niti spring, in addition to the degradation being lower, it was relatively constant during the 28 days of the experiment. whereas, in both elastics there was expressive force degradation, with an approximate value of 100 gf in the first 24 h of the study, corroborating the results of other investigations1,9,13,23, while the niti spring presented force degradation of approximately 25 gf in the same period. this is another result that allows to point the niti spring as the most appropriate device for exerting the characteristic of continuous “optimal force”, as the one that produces rapid tooth movement, without discomfort to the patient, and without side effects on tissues3. regarding the elastomeric chains, both types behaved in a very similar manner, suffering a remarkable initial degradation in the first 24 h, and maintaining a low, constant force degradation from 24 h up to 28 days of the experiment. it was noted that in this same period, from 48 h to 28 days of the experiment, plastic chain showed force approximately fig. 2 force distribution (gf) of space closure materials over 28 days of evaluation. 15 gf lower than the memory chain. nevertheless, the levels of force of these two types of elastics were equal at 28 days of the experiment. during the literature review, no study about the force degradation of the elastomeric memory chain was found. despite the lower force showed by synthetic elastomeric materials in comparison to niti spring, memory chain showed lower force degradation than plastic chain. therefore, the force of memory chain to tooth movement was closer than the clinical recommended (200 gf). from a clinical aspect, this results in faster tooth movement for memory chain. the closed niti springs were shown to have the best properties and best action for attaining more effective and less traumatic tooth movement, when compared with the two types of elastics tested. however, these springs are not used on a larger scale, probably due to their higher cost and the possibility, although remote, of causing soft tissue injuries in patients12. the preferential use of niti springs is suggested in patients who have a fully healthy periodontal status, and assiduously attend clinical appointments. in patients of this type, one could also use preferentially memory elastomeric chains; however, with a force from 30 to 60% greater than necessary, in order to compensate for the rapid initial degradation suffered by these chains24. should the orthodontist be faced with a patient whose periodontitis has been stabilized, but with clinical attachment loss; or with a patient who does not attend consultations as frequently, the use of elastomeric chains calibrated at 200 gf for the anterior segment should be considered the most indicated device. thus, smaller, but biologically acceptable movement will be achieved, without tissue damage to the tooth or the periodontium. in conclusion, the three space closure system showed force degradations over 28 days; however, the force reduction of niti spring was the lowest among the materials. in the first day, force degradation of the elastomeric materials was even more significant, attaining the level of almost 26%, whereas the spring suffered a degradation of only 2.8%. after 28 days, a mean reduction of 16.1%, 63.2% and 64.0%, for the niti spring, memory chain and plastic chain, respectively. the closed niti springs were shown to be the most appropriate for orthodontic space closure treatment. comparing elastomeric chains, memory chain should be preferred to plastic chain. references 1. araújo fbc, ursi wjs. estudo da degradação da força gerada por elásticos ortodônticos sintéticos. rev dental press ortodon ortop facial. 2006; 11: 52-61. 2. ash jl, nikolai rj. relaxation of orthodontic elastomeric chains and modules in vitro and in vivo. j dent res. 1978; 5: 685-90. 3. burstone cj. the mechanics of the segmented arch techniques. angle orthod.1966; 36: 99-120. 4. cabrera mc, cabrera cag, henriques jfc, freitas mr, janson g. elásticos em ortodontia: comportamentos e aplicação clínica. rev dental press ortodon ortop facial. 2003; 8: 115-29. force degradation of different elastomeric chains and nickel titanium closed springs 170 braz j oral sci. 10(3):167-170 5. hwang cj, cha jy. mechanical and biological comparison of latex and silicone rubber bands. am j orthod. 2003; 124: 379-86. 6. begg pr. differential force in orthodontic treatment. am j orthod. 1956; 42: 481-510, 7. reitan k. some factors determining the evaluation of forces in orthodontics. am j orthod. 1957; 43: 32-45. 8. proffit wr, fields jr, henry w. ortodontia contemporânea. rio de janeiro: guanabara kogan; 1995. 9. bishara se, andreasen ge. a comparison of time related forces between plastics alastiks and látex elastics. angle orthod. 1970; 40: 319-28. 10. ferretier jp, meyers jr ce, lorton l. the effect of hydrogen ion concentration on the degradation rate of orthodontic polyurethane chain elastics. am j orthod orthop. 1990; 98: 404-10. 11. samuels rha, rudge sj, mair lh. a comparison of the rate of the space closure using a nickel titanium spring and an elastic module: a clinical study. am j orthod dent orthop.1993; 103: 464-7. 12. ryan a. superelastic nickel titanium coil springs. br j orthod. 1995; 22: 370-6. 13. nattrass c, ireland aj, sherriff m. the effect of environmental factors on elastomeric chain and nickel titanium coil springs. eur j orthod. 1998; 20: 169-76. 14. russell ka, milne ad, khanna ra, lee jm. in vitro assessment of the mechanical properties of látex and nonlátex orthodontic elastics. am j orthod dent orthop. 2001;120: 361-44. 15. santos ac, tortamano a, naccarato srf, dominguez-rodrigues gc, vigorito jw. an in vitro comparison of the force reduction generated by different commercially available elastomeric chains and niti closed coil springs. braz oral res. 2007; 21: 51-7. 16. andreasen g. a clinical trial of alignment of teeth using a 0.019 inch thermal nitinol wire with a transition temperature range between 31°c. and 45°c. am j orthod. 1980; 78: 528-37. 17. miura f, mogi m, ohura y, hamanaka h. the superelastic property of the japanese ni-ti alloy wire for use in orthodontics. am j orthod dentofac orthop.1986; 90: 1-10. 18. von fraunhofer ja, bonds pw, johnson be. force generation by orthodontic coil springs. angle orthod. 1993; 63: 145-8. 19. tripolt h, burstone cj, bantleon p, manschiebel w. force characteristics of nickel-titanium tension coil springs. am j orthod dentofacial orthop.1999; 115: 498-507. 20. ruellas aco, bolognese am. mola de níquel-titânio x mola de aço inoxidável comparação do movimento dentário. j bras ortodon ortop facial. 2000; 5: 45-50. 21. ferreira neto jj. influência do tamanho e do pré-estiramento sobre a degradação de força de elásticos em cadeia [master’s thesis]. rio de janeiro: universidade federal fluminense; 2001. 90p. 22. melsen b, topp lf, melsen hm, terp s. force system developed from closed coil springs. eur j orthod. 1994; 16: 531-9. 23. martins mm, mendes am, almeida mao, goldner mta, ramos, vf, guimarães ss. estudo comparativo entre as diferentes cores de ligaduras elásticas. rev dental press ortodon ortop facial. 2006; 11: 81-90. 24. kanchana p, godfrey k. calibration of force extension and force degradation characteristics of orthodontic látex elastics. am j orthod dent orthop. 2000; 118: 280-7. force degradation of different elastomeric chains and nickel titanium closed springs oral sciences n3 braz j oral sci. 11(3):422-427 original article braz j oral sci. july | september 2012 volume 11, number 3 functional activity of neutrophils and systemic inflammatory response of down’s syndrome patients with periodontal disease isabelle rodrigues freire1, sandra maria herondina coelho ávila aguiar2, sandra helena penha de oliveira3 1graduate student in pediatric dentistry, school of dentistry of araçatuba, unesp, são paulo, sp, brazil 2professor, department of pediatric and community dentistry, school of dentistry of araçatuba, unesp, são paulo, sp, brazil 3professor, department of basic sciences, school of dentistry of araçatuba, unesp, são paulo, sp, brazil correspondence to: sandra maria herondina ávila coelho aguiar pediatric dentistry and social department school of dentistry of araçatuba, unesp rua josé bonifácio, 1193 vila mendonça cep: 16015-050, araçatuba-sp, brasil phone: +55 18 36363315 fax: +55 18 36363332 e-mail: saguiar@foa.unesp.br abstract periodontal disease (pd) is characterized as an inflammatory process that compromises the support and protection of the periodontium. patients with down’s syndrome (ds) are prone to develop pd. neutrophils (ne) are the first line of defense against infection and their absence sets the stage for disease. aim: to compare the activity and function of ne in the peripheral blood from ds patients with and without pd, assisted at the center for dental assistance to patients with special needs affiliated with the school of dentistry of araçatuba, brazil. methods: purified ne were collected from peripheral blood of 22 ds patients. ne were used to detect the 5-lypoxigenase (5-lo) expression by rt-pcr. plasma from peripheral blood was collected to measure tumor necrosis factor-a (tnf-α) and interleukin-8 (il-8) by elisa and nitrite (no 3 ) using a griess assay. results: data analysis demonstrated that ds patients with pd present high levels of tnf-a and il-8 when compared with ds patients without pd. however, there was no statistically significant difference in the levels of no 3 production between the groups. the levels of the inflammatory mediator 5-lo expression increased in ds patients with pd. conclusions: according with these results, it was concluded that tnf-α and il-8 are produced by ds patients with pd. furthermore, ds patients with pd presented high levels of 5-lo expression, suggesting the presence of leukotriene b 4 (ltb 4 ) in pd, thus demonstrating that the changes in ne function due to the elevation of inflammatory mediators contribute to pd. keywords: down syndrome, periodontal disease, 5-lypoxigenase, tnf-a. introduction down syndrome (ds) is the most common chromosomal aberration resulting from trisomy of the chromosome 21. this trisomy is present in 95% of the phenotypic expression of the ds patients. the remaining cases are due translocation, mosaicism and partial trisomy of the chromosome 21. this phenomenon occurs during spermatogenesis, resulting in three copies of the chromosome 21. several functional disorders and physical stigmata, such as mental abnormalities, susceptibility to infections, and hypotonic muscle function are associated with this syndrome1-2. ds patients also present a t cell immunodeficiency causing functional defects of polymorphonuclear leukocytes, reduced chemotaxis, diminished phagocytic ability, defective oxidative response and abnormal bactericidal activity3. braz j oral sci. 11(3):422-427 423423423423423 ds individuals have an increased prevalence of periodontal disease (pd) compared with otherwise normal, age-matched control groups and other mentally handicapped patients of a similar age4. signs of alveolar bone loss can be detected in a high percentage of children with ds5-6. the severe periodontal destruction cannot be explained by poor oral hygiene alone7. meyle and gonzales8 (2001) described the influence of ds on periodontitis in children and adolescents. it has been suggested that endogenous factors might contribute to the rapid progression of periodontal breakdown such as inappropriate regulation of enzymes, lipid mediators, collagen biosynthesis or t cell immunodeficiency. pd is highly prevalent and can affect up to 90% of the worldwide population 9. periodontitis results in loss of connective tissue and bone support and is a major cause of tooth loss in adults. the oral cavity is continually exposed to bacteria, their endotoxins and exototoxins, as well as physical stress. this results in a complex microenvironment within the periodontium, consisting of immune surveillance response, cellular damage and repair, and the production of cytokines, chemokines and other inflammatory mediators10. in general, the host response to bacterial stimuli leads to a cascade of inflammatory mediators such as cytokines (tnf-α), chemokines (il-8) as well as prostaglandins and leukotrienes, which are metabolites from arachidonic acid. the polymorphonuclear leukocyte-neutrophil is a key cell type and an essential part of the host’s inflammatory response. the explanation for the advanced periodontal destruction may include the disturbance in ne chemotaxis. its protective functions have been studied extensively in relation to the pathogenesis of pd. these functions can be categorized as adherence, chemotaxis, phagocytosis and microbicidal activity3,11-12. during the inflammatory response, resident cells can release chemotactic mediators that induce neutrophil accumulation and ne mediators’ expression such as cytokines, chemokines, lipids-derived mediators and reactive nitrogen species. these molecules are potent stimulants of ne chemotaxis and amplify ne-mediated tissue injury and vascular permeability. the objective of this study was to compare the activity and function of ne in the systemic inflammatory response of ds patients with pd and the levels of tnf-α, il-8 and no 3 as well as the expression of 5-lo activity released in peripheral blood from ds patients with and without pd, assisted at a center for dental care to special needs patients. material and methods participants peripheral blood was collected from 22 patients with ds (10 male and 12 females, mean age ± 25.6 ears), being 14 with pd and 8 without, recruited from the patient population of the center for dental assistance to patients with special needs (caoe-unesp-foa), affiliated with the school of dentistry of araçatuba, unesp, araçatuba, sp, brazil, in accordance with the protocol approved by the ethics committee for human subjects. the patients who participated of this study were previously selected according to an accurate diagnosis of ds by the medical staff, psychologists and therapists who recorded all information regarding systemic health and intellectual level of these patients. the dental team performed the oral examinations and found only a small number of ds patients with periodontal alterations because most of them attended the caoe-unesp-foa and present excellent oral conditions. the peripheral venous blood was collected from patients only after their parents or caregivers receive the necessary information and signed a consent form. the general health of subjects was good and care was taken to ensure that none of them was under anti-inflammatory medication for systemic conditions over the 12 months prior to the study or exhibited any inflammatory process. the patients were clinically diagnosed to evaluate the pd by the dentists of the caoe-unesp-foa. ne isolation peripheral blood ne were isolated by ficoll-paquetm premium (invittogen brl, life technologies, rockville, md, usa) gradient. in brief, 20 ml of peripheral blood were collected and diluted 1:1 in saline. subsequently, the diluted whole blood was laid on 15 ml of ficoll-paque tm premium and centrifuged at 450 g for 15 min at 4oc. afterwards, plasma was collected and contaminating erythrocytes were lysed and ne were suspended in trizol â (invittogen brl, life technologies). determination of il-8 and tnf-α by enzyme linked immuno sorbent assay (elisa) plasma from peripheral blood was collected for elisa in order to measure il-8 and tnf-α production. each assay kit (r & d systems inc., minneapolis, mn, usa) contained 96 wells with a microtiter plate coated in monoclonal antibody to tnf-α or il-8 in the base. samples were added to the wells and after 2 h, unbound proteins were washed away and an enzyme-linked polyclonal antibody was added to the wells; this antibody acted as a link between the cytokine or chemokine and a dying agent. a color change proportional to the amount of tnf-α or il-8 was observed. this was quantified by comparing the optical densities of the samples to those of known dilutions using a plate reader at 450 nm. the concentration of each tnf-α and il-8 was calculated from a standard curve (from 4 to 4000 pg/ml). expression of 5-lo by real-time reverse transcription-polymerase chain reaction (rt-pcr) cells were homogenized with 1 ml of trizolâ. rna was extracted with chloroform and centrifuged at 1300g at 4°c for 15 min; before being washed with isopropanol (500 ml) and ethanol (500 ml) following protein precipitation according to the manufacturer’s instructions. complementary dna (cdna) was synthesized using 3 mg of rna through a reverse transcription reaction. real-time pcr quantitative mrna analyses were performed in a rotor gene 6 using the functional activity of neutrophils and systemic inflammatory response of down’s syndrome patients with periodontal disease 424424424424424 sybr-green fluorescence quantification system (corbett research, mortlake, australia) for quantification of amplicons. the standard pcr conditions were 950c (15 min), and then 40 cycles of 95oc (20 s), 55oc (30 s), 72oc (30 s), and 72oc (1 min), followed by the standard denaturation curve. the sequences of the primers and the predicted amplicon sizes used were as follows: 5-lo sense ccc ggg gca tgg aga gca, antisense gcg gtc ggg cag cgt, which results in a 561 base pair (bp) amplification product; b-actin sense ggc gac gag gcc cag a, antisense cga ttt ccc gct cgg c, which results in a 463 base pair (bp). pcr conditions for each target were thoroughly optimized with regard to primer concentration, absence of primer dimer formation and efficiency of amplification of target genes and housekeeping gene control. sybr green pcr master mix (cobertt research, mortlake, australia), 400 nm specific primes and 2.5 ng cdna were used in each reaction. the positivity of real-time pcr was determined based on negative controls. the relative levels of gene expression were calculated according to the instructions by referring to the b-actin in the sample, using the cycle threshold (ct) method. briefly, ct is the point at which the exponential increase in signal (fluorescence) crosses a somewhat arbitrary signal level (usually 10 times background). the mean ct values from duplicate measurements were used to calculate the expression of the target gene, with normalization to bactin, and then compared with the target-internal control subjects to calculate the fold increase expression, using the 2dct formula. negative controls without rna and without reverse transcriptase were also performed. the results show one of three representative experiments. determination of production of no 3 plasma from peripheral blood was collected for nitrate reduction assay in order to measure no 3 production. serum samples (40 ml) from ds patients with and without pd were pipetted into the wells of flat-bottomed 96-well microtitration plates, followed by the addition of 40 ml of freshly mixed (500 ml) nadph (5 mg/ml), 1000 ml kh 2 po 4 , 50 ml nitrate reductase (10 u/500 ml) and 450 ml h 2 o and left overnight at room temperature. after that, an equal part of griess reagent was added to the well plates. the plates were shaken for 5 min at room temperature, after which a purple color developed in positive plates. the plates were read in a microplate reader at 540 nm. the concentration of no 2 and no 3 was determined from a standard curve (200 to 0.78 mm) produced using 8 different concentrations of nano 2 and nano 3 . the results were expressed as mm in triplicate samples. chemotactic migration ne were suspended in rpmi supplemented with 0.01% bovine serum albumin at a concentration of 1x106 cells/ml. the cell suspension was placed in the upper compartment of a modified boyden chamber separated by a 5mm pore-size micropore filter, while the lower compartment was loaded with either the buffer solution or chemoattractant solutions of fmlp (10-7m), ltb 4 (10-8m) and il-8 (10-9 m). the cell migration response was evaluated by enumeration of cells on the distal surface of the filter after one-hour incubation in a 37oc humidified air chamber. ten representative highpower microscopic fields (x1,000) were counted for each of triplicate filters. the chemotactic migration of ne from ds patients with pd was expressed as an average of the calculated percentage of the chemotactic migration of ne from matched healthy subjects. statistical analyses student’s independent t test (two-tailed) was used to compare the means between the groups as well as the correlation within the group. a general linear anova test was used when testing the correlation coefficients between ds patients with pd and controls. the statistical program prisma 3.0 was used. statistical significance was considered as a two tailed p< 0.05. results the subjects were divided into 2 groups and they were classified by sex, age and intellectual disability. periodontal status was evaluated by the clinical parameters (table 1 and 2). in table 1, it was observed that ds patients with pd are predominantly females, with a mean age of 25 years. however, the patients without pd represented in table 2, are predominantly males with a mean age of 24 years. as far as the inflammatory mediators’ production in the present study, it was observed that patients with ds and pd have a significantly higher tnf-α production when compared with ds patients without pd (figure 1). the same pattern of il-8 production was observed when comparing with ds patients without pd (figure 2). the 5-lo expression by purified peripheral ne was evaluated using real time rt-pcr in order to investigate the participation of lipid mediators in the pd of patients with and without ds. it was observed that ds patients with pd showed a higher level of 5-lo expression than ds patients fig. 1levels of tnf-α in plasma of peripheral blood of down’s syndrome patients with and without periodontal disease. the concentration of tnf-α was evaluated by elisa. the results are expressed as means ± standard error of means of the values of at least two experiments. *statistically significant difference (p< 0.05). functional activity of neutrophils and systemic inflammatory response of down’s syndrome patients with periodontal disease braz j oral sci. 11(3):422-427 425425425425425 table 1. data from down’s syndrome patients with periodontal disease fig. 4: nitrate levels in plasma of peripheral blood from down’s syndrome patients with and without periodontal disease. nitrate concentration was determined by the griess method. data are reported as means ± standard error of means of the assays performed in triplicate and representative of three different experiments. table 2data from down’s syndrome patients without periodontal disease without pd (figure 3). it has been demonstrated 11 that leukotrienes are involved in the inflammatory process in periodontitis. in the next step, nitric oxide (no) production by nitrate level was assessed and it was found that no significant difference was detected when comparing the no 3 level in ds patients with and without pd (figure 4). the chemotaxis assay was used to analyze the functional activity of ne in the ds patients, and it was observed that il-8, fmlp and ltb 4 were chemotactic to ne migration in ds patients in comparison with the control group rpmi (figure 5). fig. 2 levels of il-8 in plasma of peripheral blood of down’s syndrome patients with and without periodontal disease. the concentration of il-8 was evaluated by elisa. the results are expressed as means ± standard error of means of the values of at least two experiments. *statistically significant difference (p< 0.05). fig. 3 expression of the 5-lo enzyme by purified neutrophils in peripheral blood of down’s syndrome patients evaluated by real-time rt-pcr. the figure shows the amplification of 5-lo mrna in relation to β-actin mrna. *statistically significant difference (p< 0.05). discussion an increase in susceptibility and prevalence of pd in ds patients has been the object of intense study in functional activity of neutrophils and systemic inflammatory response of down’s syndrome patients with periodontal disease braz j oral sci. 11(3):422-427 426426426426426 recent years, as can be observed in current literature12. some clinical and laboratory studies have suggested that the genetic abnormalities of patients with ds can modify the systemic response. it includes functional defects of the polymorphonuclear and mononuclear leukocytes, which contribute to the prevalence of pd2. oral hygiene is often used as a predictor of patients with pd. poor oral hygiene directly correlates to the degree of mental retardation as do increased rates of oral diseases among those populations. limited access to care, limited manual dexterity and lowered efficacy of self homecare, all of these factors contribute to raising levels of gingivitis3. in the present study, we evaluated ds patients of both sexes, ages and degrees of mental abnormalities. our results support the hypothesis that ds patients with pd present high levels of tnf-α in the peripheral plasma when compared with ds patients without pd. these data strongly suggest that ds patients with periodontitis present an inflammatory disease in due course. these findings are consistent with those of previous studies that reported that high levels of tnf-α were observed in the serum samples of ds patients without periodontal13. tnf-α is a pluripotent cytokine that plays a central pathogenic role in inflammatory disease. this cytokine is associated with stimulated bone resorption, fibroblast proliferation as well as the production of matrix metalloproteinases and prostaglandin e 2 14. its possible significance in inflammatory pd has been subject of intense research15. as far as il-8 is concerned, it was observed that ds patients with pd have a significantly higher level when compared with ds patients without periodontitis. these data are in accordance with literature showing that the mean concentration of il-8 was higher in the ds group than in the control group14. il-8 is a chemokine produced by a variety of tissues and blood cells and is a potent inducer of ne chemotaxis and activation16. this cytokine is produced early in inflammation as a result of the interaction of host cells fig. 5 chemotactic migration function of neutrophils from down syndrome patients with periodontal disease. the figure represents emigrated neutrophils in response to rpmi (random migration) or to chemotactic stimulation with ltb 4 (10-8 m), fmlp (10-7m) and il-8 (10-9 m). *statistically significant difference (p< 0.05). with bacterial stimuli such as lipopolysaccharides (lps). in inflammatory lesions, the interaction between il-8 and other inflammatory mediators leads to extravasation and recruitment of ne. an increase in il-8 expression in periodontal tissue has been previously observed, thus confirming its participation in the periodontitis process17. in the present study, it was found that il-8 is potentiated in ds patients with pd when compared with literature data. no is a molecule that is involved in vascular regulation, homeostasis, bone formation and resorption, neurotransmission and immune function. in recent years, there has been an increasing interest in no in the pathogenesis of oral and pds. in the present study, there was no statistically significant difference between the levels of no in ds patients with pd compared with patients without periodontitis. in the data reported in literature, it is observed that when no production in the salivary and gingival tissue from patients with periodontitis was measured, it was found to be lower compared with that in healthy samples18. no production in pd is controversial since the production may be downregulated by arginase19. overproduction of no can contribute to tissue damage in periodontal tissues and has been implicated in pd pathogenesis. no in periodontal tissue is generated mostly via inos which catalyze the oxidation of guanidine nitrogen associated with l-arginine. arginase also uses l-arginine and can down-regulate no production in saliva and in periodontal tissue17. since a large amount of no is toxic to periodontal tissues, arginase may prevent overproduction of no. the results of the present study showed that ne from peripheral blood of ds patients with pd significantly express 5-lipoxygenase when compared with patients without periodontitis. the data suggest that the ne from these patients are able to produce leukotriene b 4 . ltb 4 production during the progression of periodontitis has been observed in literature. gingival tissue from ds subjects presents a high level of this lipid mediator when compared with the control group20. ltb 4 is a proinflammatory mediator that plays an important role in pd. recently, ltb 4 has been implicated as having a role in host defense against microbial infection21. this lipid mediator induces recruitment and activation of ne, monocytes and eosinophils. it also stimulates the production of a number of proinflammatory cytokines and mediators indicating ability to augment and prolong tissue inflammation22. the involvement of ltb 4 in pd has been observed in gingival crevicular fluid from subjects with periodontitis23-24. defects in ne chemotaxis have also been identified in ds patients25. in addition to the direct involvement of ne in defense against invading pathogens, the ne role in mediating tissue injury plays an important role in the exacerbation of diseases. under this new paradigm, comparing with our data, the polymorphonuclear leukocyte is not “hypofunctional” or “deficient”, but rather “hyperfunctional”. the increased activity and the release of toxic products from the cell are responsible, in part, for the tissue destruction in chronic periodontal inflammation26. in our study, we observed that the ne chemotactic activity of il-8, ltb 4 and fmlp is higher functional activity of neutrophils and systemic inflammatory response of down’s syndrome patients with periodontal disease braz j oral sci. 11(3):422-427 427427427427427 when compared with control group. this may suggests that ne is hyper-functional and activated in these ds patients. during the inflammatory cell migration, the chemotactic factors are essential for the beginning and regulation of the increased inflammatory/immune response. in our study, tnfá and il-8 production as well as 5-lo mrna expression are more pronounced in ds patients with pd when compared with ds patients without pd in literature data. ne function may be altered in patients with pd. the improvement in the activity of peripheral blood ne observed in some study is consistent with observed associations between periodontitis and overall health indicators. intrinsic defects in ne could result in compromised immune conditions, as found in some syndromes. the ne in peripheral blood from patients with pd are defective regarding the production of some mediators of inflammation. the alteration in function refers to an up-regulation of ne, a hyperfunctional activity that may be responsible for the periodontal chronic destruction. in conclusion the changes in ne function due to the elevation of inflammatory mediators contribute to pd. according to the results of the present research, it may be concluded that: • the ne chemotactic activity of il-8, ltb 4 and fmlp was higher when compared with the control group; • the ne is hyper-functional and activated in ds patients; • the tnf-β and il-8 production as well as 5-lo mrna expression are more pronounced in ds patients with pd. acknowledgements this project was supported by grants from the são paulo state research foundation (fapesp2000/08506-0). the authors are indebted to giuliana bertozi for her helpful technical assistance and express their gratitude to dr. carlos f santos for his critical reading and suggestions to this manuscript. we also acknowledge yara regina bianchini ávalos, alba valéria rodrigues mantovani and marlene aparecida costa for taking care of the patients and collecting peripheral blood samples at the center for dental assistance to patients with special needs (caoe-unesp-foa). references 1. forrester mb, merz rd. epidemiology of down syndrome (trisomy 21), hawaii, 1986-97. teratology. 2002; 65: 207-12. 2. khoshnood b, wall s, pryde p, lee ks. maternal education modifies the age-related increase in the birth prevalence of down syndrome. prenat diagn. 2004; 24: 79-82. 3. frydman a, nowzari h. down syndrome associated periodontitis: a critical review of the literature. compendium. 2012; 33: 356-61. 4. amano a, kishima t, akiyama s, nakagawa i, hamada s, morisaki i. relationship of periodontopathic bacteria with early-onset periodontitis in donws’s syndrome. j periodontol. 2001; 72: 368-73. 5. modéer t, barr m, dahllöf g. periodontal disease in children with down’s syndrome. scand j dent res. 1990; 98: 228-34. 6. saxén l, aula s. periodontal bone loss in patients with down’s syndrome: a follow-up study. j periodontol. 1982; 53: 158-62. 7. ulseth jd, hestnes a, stavner lj, storhaug k. dental caries and periodontitis in persons with down’s syndrome. spec care dent. 1991; 11: 71-3. 8. meyle j, gonzales, j.r. influence of systemic disease on periodontitis in children. periodontology 2000. 2001: 92-112. 9. albandar jm, rams te. periodontol 2000 global epidemiology of periodontal diseases 29. copenhagen, denmark: munksgaard .blackwells; 2002. 10. khocht a, yaskell t, janal m, turner bf, rams te, haffajee ad et al. subgingival microbiota in adult down syndrome periodontitis. j periodontal res. 2012; 47: 500-7. 11. carneiro mv, bezerra ac, guimarães md, muniz-junqueira mi. effects of periodontal therapy on phagocytic of peripheral blood neutrophilsevidence for an extrinsic celular defect. oral health prev dent. 2012; 10: 195-203. 12. scott da, krauss j. neutrophils in neutrophils inflammation. front oral biol. 2012; 15: 56-83. 13. komatsu t, kubota m, sakai n. enhancement of matrix metalloproteinase (mmp)-2 activity in gingival tissue a culture fibroblasts from down’s syndrome patients. oral dis. 2001; 7: 47-55. 14. górska dt, gregorek h, kowalski j, laskus-perendyk a, syczewska m, madaliñski k. relationship between clinical parameters and cytokine profiles in inflamed gingival tissue and serum samples from patients with chronic periodontitis. j clin periodontol. 2003; 30: 1046-52. 15. yucel-lindberg t, twetman s, skold-larsson k, modeer t. effect of an antibacterial dental varnish on the levels of prostanoids leukotriene b4, and interlekin-1 beta in gingival crevicular fluid. acta odontol scand. 1999; 57: 23-7. 16. galbraith gmp, hagan c, steed rb, sanders jj, javed t. cytokine production by oral and peripheral blood neutrophils in adult periodontitis. j. periodontol. 1997; 68: 832-8. 17. nelson pg, kuddo t, song ye, dambrosia jm, kohler s, satyanarayana g, et al. selected neutrophils, neuropeptides, and cytokines: developmental trajectory and concentrations in neonatal blood of children with autism or down syndrome. int j dev neurosci. 2006; 24: 73-80. 18. shimauchi h, takayama s, narikawa-kiji m, shimabukuru y, okada h. production of interleukin-8 and nitric oxide in human periapical lesions. j endod. 2001; 27: 749-52. 19. garlet gt , martins jr. w, ferreira br , milanezi cm, silva js. patterns of chemokines and chemokine receptors expression in different forms of human periodontal disease. j. periodont res. 2003; 38: 210-17. 20. ugar-çankal d,ozmeric n. a multifaceted molecule, nitric oxide in oral and periodontal disease. clin chim acta. 2006; 366: 90-100. 21. güllü c, ozmeric n, tokman b, elgün s, balos k. effectiveness of scaling and root planning versus modified wildman flap on nitric oxide synthase and arginase activity in patients with chronic periodontitis. j periodont res. 2005; 40: 168-75. 22. tsilingaridis g, yucel-lindberg t, modéer t. enhanced levels of prostaglandin e2, leukotriene b4, and matrix metalloproteinase-9 in gingival fluid from patients with down syndrome. acta odontol scand. 2003; 61: 154-8. 23. bailie mb, standiford tj, laichalk ll, coffey mj, strieter r, petersgolden m. leukotriene-deficient mice manifest enhanced lethality from klebsiella pneumonia in association with decreased alveolar macrophage phagocytic and bactericidal activities. j immunol. 1996; 157: 5221-4. 24. crooks ws, stockley ar. leukotriene b4. int j biochem cell biol. 1998; 30: 173-8. 25. emingil g, cinarcik s, baylas h, coker i, hüseyinov a. levels of leukotriene b4 in gingival crevicular fluid and gingival tissue in specific periodontal disease. j periodontol. 2001; 72: 1025-30. 26. bäck m, airila-mansson s, jogestrand t, söder b, söder po. increased leukotriene concentrations in gingival crevicular fluid from subjects with periodontal disease and atherosclerosis. atherosclerosis. 2007; 193: 389-94. functional activity of neutrophils and systemic inflammatory response of down’s syndrome patients with periodontal disease braz j oral sci. 11(3):422-427 oral sciences n3 braz j oral sci. 10(2):109-112 original article braz j oral sci. april | june 2011 volume 10, number 2 civil liability of dentists based on reports filed to the regional council of dentistry neusa barros dantas neta1, priscila ferreira torres1, luciana tolstenko nogueira2, carmem milena rodrigues siqueira carvalho3 1undergraduate dental student, school of dentistry, federal university of piauí, brazil 2substitute professor, school of dentistry, federal university of piauí, brazil 3professor, school of dentistry, federal university of piauí, brazil correspondence to: neusa barros dantas neta quadra 196 casa 06 – conjunto dirceu arcoverde 2, 64078120 teresina, pi, brasil e-mail: nbdn2@msn.com abstract civil liability is the obligation to repair the damage that one causes someone else. dentists must respect legal precepts and comply with the consumer protection and defense code. in order to prevent legal action, it is important to communicate with the patients and/or their caregiver. aim: the goal of this study was to assess the number of dentists living in the city of teresina-pi and in some other capitals of the northeastern brazil as well as the number of reports filed against dentists to the regional council of dentistry (cro) between 2002 and 2009. methods: this was a cross-sectional descriptive study conducted in the city of teresina, located in northeastern brazil, with an estimated population of 802,537 inhabitants. in 2009, 1,938 dentists were registered at the cro piauí, 1,239 of whom belonged to the city of teresina. the number of reports filed against dentists is presented, using a comparative description of the data by dental specialty and states in northeastern brazil. results: data collection showed that 70 complaints were registered. the majority were against orthodontists (n=16), regarding advertising (n=15) and regarding administrative, ethical or service violation (n=15, 11 of whom were dentists who worked in a nonregistered practice). conclusions: the number of reports filed against dentists has decreased in piauí, unlike other states. however, professionals must be aware about communication with the patient and archiving of dental charts and treatment-related documents or exams. keywords: civil liability, code of ethics, code of consumer protection, obligation of the dentist. introduction civil liability is the obligation to repair the damage that one causes someone else. this is a topic of growing importance in the brazilian law1 because of the obligation to repair the damage to someone else by the person who has the duty to ensure or enforce a certain order2. as dentists provide a service to consumers, they must respect legal precepts and comply with the brazilian consumer protection and defense code. they must have care and concern for their work and, whenever a mistake is made, they must have liability to repair the damage, respond to lawsuits and are forced to reimburse the patient3. in order prevent these civil actions, it is important to communicate with the patients and/or their legal guardian, as well as prepare, approve and safeguard all documentation, describing the treatment plan and the risks, benefits and costs of the treatment that was carried out4. received for publication: october 19, 2010 accepted: may 09, 2011 110 braz j oral sci. 10(2):109-112 motivation of reports/dental specialty piauí rio gde do norte orthodontics 16 67 endodontics 4 6 illegal advertising 15 67 oral and maxillofacial surgery 9 9 implants 4 22 management / ethical/ treating infraction 15 19 prosthodontics 1 19 restorative dentistry 0 13 medical certificate 0 4 radiology 1 0 total 52 164 table 1.table 1.table 1.table 1.table 1. number of reports against dentists in northeastern states of piauí and rio grande do norte distributed by motivation of reports and involved dental specialties, corresponding to the period between 2002 and 2009, brazil. the aim of this study was to assess the number of dentists living in the city of teresina and in some other capitals of the northeastern brazil as well as to identify the number of reports filed against dentists to the regional council of dentistry (cro) between 2002 and 2009. material and methods this study was a cross-sectional descriptive investigation conducted in the city of teresina, capital of the state of piauí (pi), located in northeastern brazil, with an estimated population of 802,537 inhabitants5. in 2009, 1,938 dentists were registered at the cro piauí, 1,239 of whom belonged to the city of teresina6. the number of reports filed against dentists is presented using a comparative description of the data by dental specialty and states of the northeastern region of brazil. these data were collected by the regional council of dentistry (cro) in the states of piauí (pi), ceará (ce), rio grande do norte (rn) and paraíba (pb) regarding the period between 2002 and 2009. this study complied with the guidelines of brazilian national health council’s resolution no. 196/96 and was approved by the ethics committee of the federal university of piauí. results there were records of 70 reports against dentists between 2002 and 2009. among these, most were against orthodontists (16 reports), followed by irregular advertising (15 reports) and management /ethical/service infraction (15 reports, 11 dentists working at unregistered clinics) (table 1). table 1 shows a comparison of data from cro-pi and cro-rn regarding the motivation of reports and involved dental specialties, revealing that most complaints referred to orthodontics, illegal advertising and management /ethical/ service infraction. according to data provided by the cro-ce and crorn, there were 205 and 232 reports, respectively between 2002 and 2009. fifty-one reports were filed against dentists to the cro-pb between 2002 and 2007 (it was not possible to collect the data from 2008 and 2009). discussion the dentist is a professional whose obligation is to relieve the patient’s pain, treat and restore teeth as well as to provide full information on preventive measures and behaviors in order to prevent future diseases. unrestorable teeth should be extracted and an adequate treatment plan should be outlined. however, the dentist must still respect the patient’s esthetics and only carry out such a procedure after proper communication. the dentist undertakes the obligation to be effective and achieve a result without harming the patient7. patients wish for a comfortable and warm interaction with their dentist, and believe that the dentist is competent and keeps them informed about their oral conditions and the treatment to be carried out. when these wishes are not met, the patient becomes disappointed, discontented, and uncooperative with the dentist’s decisions8. it is therefore important for the dentist to know the patient’s expectations in order to achieve the best possible outcomes9. for a better understanding of civil liability, one must first understand the type of obligation that such professionals undertake when providing services. there are two types of obligations: obligations of means and obligation of outcome. the obligations of means are those in which the dentist must use of their technical skills and personal ability to achieve a specific goal. however, this obligation is not related to the final outcome of the activity, i.e., the dentist is committed to act with fairness, correctness, prudence, dedication and technique, but he/she cannot guarantee the outcome10. the dental specialties traumatology and oral and maxillofacial surgery, endodontics, pediatric dentistry, periodontics, orthodontics, oral and maxillofacial prosthetics, stomatology, temporomandibular dysfunction and orofacial pain, labor dentistry, special care dentistry, geriatric dentistry and functional orthopedics of jaws are considered obligation of means. however, specialties such as restorative dentistry, community health dentistry, forensic dentistry, oral pathology, and radiology are seen more as obligation of outcome. the dentist is not liable only in cases of harmful acts that occur due to a fortuitous event or force majeure, excusable error, state of science, patient’s fault, or if the dentist acted without fault and in accordance with all standards that rule the profession11. however, when the dentist commits an unlawful act, then he/she will be subject to sanctions imposed by the code of dental ethics12 in the criminal code, the civil code13, consumer protection and defense code14 and the regulations of governing bodies or institutions where they are working15. the dentist cannot be assumed guilty without there being a correlation between the injuries suffered by the patient. there must be a proof of professional liability16. one can civil liability of dentists based on reports filed to the regional council of dentistry braz j oral sci. 10(2):109-112 then conclude that if the professional fails to inform the patient about the risks of a surgery, the dentist will be obliged to compensate for the patient in the case of any sequela regardless of whether or not being diligent in carrying out the surgical procedures because knowing the risk in anticipation could contribute to the patient’s decision on undergoing or not surgery17. in brazil, there are 222,203 dentists, according to the federal council of dentistry. therefore, they are all subject to liability, which consists of the duty to repair the damage done to the patient, and compensation for the caused damage. currently, there has been an increase in brazil in the number of people who seek their rights by filing reports against dentists at the cros and even at law courts. this was the reason why we decided to assess the number of reports filed against dentists to the cro in the northeastern states of piauí, ceará, paraíba and rio grande do norte, obtaining number of 1,825; 4,677; 3,087 and 2,558 respectively. figure 1 shows that the number of lawsuits has increased, but not only because of dentists. this increase was also due to the changes in patient’s behavior. formerly, dentists were considered as the holders of knowledge and their ability was above suspicion. nowadays, people better knowledge of their rights due to the advent of the consumer protection and defense code and civil code, and they understand that the dentist is a human being capable of making mistakes. orthodontics was the dental specialty with the largest number of reports in this study. this is because orthodontic treatment corrects malocclusion and aims to improve the function and esthetics of patient’s teeth. however, not all of patient’s expectations are known by the dentists, and this sometimes precludes proper planning based on the wishes of the patient and on the real treatment possibilities. illegal advertising was the second most frequent reason for complaints. it can cause a dentist trouble if he/she does not clearly inform the patient about the possible outcomes fig. 1: number of reports against dentists in some cities of the northeast regions of brazil for the period between 2002 and 2009. analysis of the liability of dentists based on reports filed to the regional councils of dentistry. of treatment. the consumer protection and defense code provides a penalty of 1 to 3 years imprisonment and fines for misleading advertising or omission when the dentist fails to report essential information about the product or service13. another specialty with a large number of lawsuits was prosthodontics. one of the reasons may be the fact that the patient attends only the appointment to receive the prosthesis and does not return for reassessments. on the basis of the obtained results, it may be concluded that although the number of reports against dentists is still large, it is currently in decline, especially in teresina-pi when compared with the other states surveyed. it is known that dentists have obligations and must respond legally for their errors. thus, to prevent possible problems, dentists must take responsibility for their actions from the beginning of service: before reaching a diagnosis, a form should be filled out with the patient’s identification, the anamnesis (a questionnaire about the patient’s condition), the general physical examination and complementary dental examinations in order to have an overall assessment and discover any conditions which might interfere with the treatment. after doing this, a final diagnosis can be reached and the patient should receive an explanation about the treatment options and the prognosis. communication between the professional and the patient is of paramount importance and therefore a relationship based on confidence and trust must be established for the success of treatment. dentist must also be aware of the patient’s expectations and create the most appropriate treatment plan. some advices are worth following: • information about treatment should be passed on to the patient, always in writing, supported with technical reports and with the consent (signature) of the patient. • dentists must file and record any procedure that is carried out on the patient. • dentists should invest in their education and improvement of skills, keep themselves well informed and always bear in mind their role as a health professional. 111 civil liability of dentists based on reports filed to the regional council of dentistry 34 braz j oral sci. 10(2):109-112 references 1. willemann c. a responsabilidade civil do cirurgião dentista não-autônomo nas situações de emergência das atividades hospitalares. teresina: rev jus navigandi. 2002; 6(58). available from: http://jus.uol.com.br/revista/ texto/3129/a-responsabilidade-civil-do-cirurgiao-dentista-nao-autonomo-nassituacoes-de-emergencia-das-atividades-hospitalares. 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[access 2009 jan]. 112civil liability of dentists based on reports filed to the regional council of dentistry oral sciences n3 braz j oral sci. 10(3):204-207 original article braz j oral sci. july | september 2011 volume 10, number 3 effect of powder/liquid ratio of glass ionomer cements on flexural and shear bond strengths to dentin hassan torabzadeh1, amir ghasemi2, soudeh shakeri3, alireza akbarzadeh baghban4, sara razmavar5 1dmd, msc, phd, associate professor, iranian center for endodontic research, iran center for dental research, school of dentistry, shahid beheshti university of medical sciences, tehran, iran 2dds, msd, associate professor, iran center for dental research, school of dentistry, shahid beheshti university of medical sciences, tehran, iran 3dds, iran center for dental research, shahid beheshti university of medical sciences, tehran, iran 4bsc, msc, phd, assistant professor in biostatistics, iranian center for endodontic research, iran center for dental research, shahid beheshti university of medical sciences, tehran, iran 5dds, iranian center for endodontic research, iran center for dental research, shahid beheshti university of medical sciences, tehran, iran correspondence to: amir ghasemi 5th floor, iran center for dental research, dental school, shahid beheshti university of medical sciences, evin, tehran-iran 1983963113 phone: 0098-21-22413897/ fax: 0098-21-22427753 e-mail: icdr.tgr@gmail.com abstract aim: this study evaluated the effect of varying the powder/liquid (p/l) ratio on the shear bond to dentin (sbs) and flexural strength (fs) of glass ionomer cements (gic)s. methods: three types of gics (fuji ii, fuji ii lci and fuji ix gp) were mixed using the following p/l ratios: 20% lower than the manufacturer’s ratio, manufacturer’s ratio, 20% higher than the manufacturer’s ratio (9 groups). sbs (mpa) was evaluated and the mode of failure checked under stereomicroscope. fs (mpa), of the specimens (25×2×2mm) assessed using a universal testing machine at a crosshead speed of 1 mm/min. the data were subjected to two-way anova and tukey’s test for analysis (p< 0.05). results: the highest sbs and fs (mpa) obtained for fuji ii, fuji ii lci and fuji ix were 6.12±2.11 and 16.96±2.73; 11.60±3.19 and 49.58±8.75; 7.39±2.77 and 20.32±2.09, respectively. the interaction between materials and p/l ratios had no significant effect on the properties tested in this study. fuji ii lci exhibited significantly higher sbs and fs than the other two gics in all p/l ratios. conclusions: no significant differences were observed between fuji ii and fuji ix. twenty percent variation in p/l ratio had no significant effect on sbs and fs of gics. keywords: conventional glass ionomers, resin-modified glass ionomers, powder/liquid ratio, bond strength, flexural strength. introduction glass ionomer cements (gics) are recognized due to their remarkable advantages, such as chemical bonding ability to enamel and dentin and longterm fluoride release1-2. however, these cements have limited applications, especially on stress areas, which is attributed to their inferior mechanical properties2-4. new types of gics have been introduced by the addition of resins, such as hema, to the conventional gic formulation. the so-called resin-modified gic (rmgic) relatively possess the same shortcomings5-6. received for publication: may 09, 2011 accepted: july 26, 2011 braz j oral sci. 10(3):204-207 product fuji ii fuji ii lc improved fuji ix gp lot no. 0502261 0612051 0511121 curing method self cure light cure self cure recommended p/l ratio 2.7/1 3.2/1 3.6/1 table 1 glass ionomer cements used in this study powder/liquid (p/l) ratio is one of indicative factors in altering mechanical properties of gics; the higher the amount of powder, the higher the mechanical properties5,7-8. this correlation has been explained through the particles of the powder that remain unchanged due to lower level of acid. it has been proposed that these unreacted particles appear like filler in such a way that they prevent crack propagation within the cement5. the powder amount of the cement has a direct correlation with compressive strength, porosity and wear resistance and an indirect correlation with solubility, setting and working time9-10. accordingly, this is obvious that p/l ratio can significantly affect the mechanical properties of gics and their clinical performance as well. the p/l ratio for clinical use may vary from one manufacturer to another because of several factors. visual scaling and careless use of scaling scoop can affect this ratio adversely7,10-12. the liquid drop can also vary in volume due to bottle inclination during dispensing, shape of the bottle outlet, as well as presence and size of air bubble in the bottle7,10. nevertheless, even an accurate use of the scoop and bottle leads to 2-8% of variation in p/l ratio13. variation in p/l ratio (approximately 6%) in capsulated gics has also been reported14. therefore, the aim of this study was to evaluate the effect of different p/l ratios on the shear bond strength to dentin and flexural strength of three types of gics. material and methods in this experimental study, three types of gics from the same manufacturer (gc corporation, tokyo, japan) were used (table 1). these gics were mixed manually with three different p/l ratios as follow: group 1: 20% lower than the manufacturer’s instruction (subgroups: 1, 4, 7) group 2 : same as the manufacturer’s instruction (subgroups 2, 5, 8) group 3 : 20% higher than the manufacturer’s instruction (subgroups 3, 6, 9) prior to mixing, the powder and liquid were weighted according to the mentioned protocol at room temperature with an accuracy of 0.001 g using a digital scale (aculabal-104). shear bond strength test forty-five extracted, sound (i.e. caries-free, restorationfree and with fully formed crowns), erupted and unerupted human third molars were used. this study was carried out according to the protocol approved by the ethics committee of the iran center for dental research (icdr), shahid beheshti medical university, tehran, iran. soft tissue remnants and other debris were removed from tooth surfaces and the teeth were placed in 0.5% chloramine solution for 3-4 days. subsequently, they were washed thoroughly and kept in distilled water at 4°c for less than two months. teeth were then sectioned sagitally with a microtome to eliminate the occlusal enamel and expose the underneath dentin. smooth surfaces were formed on the dentin surface when the specimens were lapped on 600-grit abrasive paper. under a stereomicroscope, complete removal of the enamel and the smoothness of dentine surface were confirmed. the specimens were then rinsed off thoroughly with distilled water for at least 30 s. twenty-one gauge (1.6 mm) scalp vein tube (shan chuan, zibo, china) with 1 mm height were overfilled with gic and their clean ends were placed over the conditioned dentin. for the purpose of more condensation and also removing the excess material, a mylar strip and a glass plate were slightly pressed over the specimens. the specimens were then kept in 37°c and 80% relative humidity for 15 min and were stored in distilled water for 24±1 h. specimens of fuji ii lci were light cured (arialux, tehran, iran) for 20 s. ten specimens were prepared for each study group. using a scalpel, the surrounding cylindrical tube of each specimen was removed under stereomicroscope then the specimens were transferred to the microtensile tester machine (bisco inc., schaumburg, il, usa). the bond strength (mpa) was measured through the failure load divided by cross sectional area of each specimen. the surfaces were examined under stereomicroscope (x20) to assess the failure mode. flexural strength test from each group, five specimens with dimensions of 2×2×25 mm were prepared using a stainless steel mold (iso 4049)14. the molds were placed on glass plates, overfilled with gic and then, another glass plate was used with slight pressure on the mold in order to remove the excess cement. two mylar strips were used to prevent adherence of specimens to the plates. fuji ii lci specimens were light cured in five overlapping areas from the core part to the sides and were then stored in an incubator (37°c, 80% relative humidity) for 15 min immediately after curing while self cured specimens transferred to the incubator after 3 min. specimens were separated from the molds and stored in distilled water at 37°c for 24±1 h. before transferring the specimens to the universal testing machine, their dimensions were measured with a digital caliper accurate to the nearest 0.01 mm and then loaded at a crosshead speed of 1 mm/min. the fracture load of specimens was recorded to be used for calculating the flexural strength (mpa) as follows: σ = 3fl/2bh² (l: distance between 2 jigs, b: specimen’s width, h: specimen’s height and σ: flexural strength). data were analyzed using two-way anova and tukey’s test. the significant level was set at α=0.05. 205effect of powder/liquid ratio of glass ionomer cements on flexural and shear bond strengths to dentin 206 braz j oral sci. 10(3):204-207 results means and standard deviations of shear bond and flexural strength are shown in table 2. for both conventional gics, the lowest and highest means of bond strength were observed in groups 1 and 3, respectively; for fuji ii lci, these values belonged to groups 1 and 2, respectively. fuji ii showed the lowest mean flexural strength in group 2 and the highest mean flexural strength was found in group 3. for fuji ix gp, the lowest flexural strength was recorded in group 1 and the highest in group 3. the lowest and highest means of flexural strength for fuji ii lci was observed in group 3 and 1, respectively. two-way anova showed that the bond and flexural strengths were not influenced by the p/l ratio, but they were significantly altered by the type of gic. in this respect, bond and flexural strengths of fuji ii lci were significantly higher than those of the two other materials. however, no significant difference was observed between fuji ii and fuji ix gp (p>0.05). failure modes revealed the increase of p/l ratio resulted in more adhesive failures for fuji ii and fuji ix gp specimens. for fuji ii lci, adhesive failures were predominant; however, the effect of p/l ratio was different from that of the other materials. accordingly, the lowest incidence of adhesive failures for fuji ii lci was observed using the p/l ratio recommended by the manufacturer (group 2). results also showed that, in general, the increase in the mechanical strength of cement will increase the bond strength. discussion variation in the p/l ratio is an important factor in optimizing gic properties. nevertheless, the amount of powder used under clinical conditions is often overlooked. billington et al.13 (1990) showed that this amount was 27% less than that recommended by the manufacturer. this finding was confirmed by beher et al.7 (2006) who pointed out a 17% variation range. in addition, some studies showed that the more the amount of powder, the better the mechanical and physical properties7,15-16 and have suggested to use the most possible powder amount the strength of gic is required12. the results of the present study indicated that regardless of the variation in p/l ratio, shear bond and flexural strengths of rmgic are higher than those of conventional gic. this is in accordance with previous studies2,4-5,17. variation in p/l ratio of each material showed no significant difference in bond and flexural strengths, which is in contrast with other investigations5,10,16. this can be explained by some differences including no report of p/l ratio variation, no exact statistical analysis16, differences in the tested materials and studied mechanical properties5,10,16. as mentioned before, some researchers believe that greater powder amount can improve the mechanical properties due to the filler-like function of unreacted powder particles5. in fact, in a recent study, it was shown that the increase in p/l ratio of a rmgic luting cement by 30 percent resulted in an increase in compressive, flexural and bond strengths18. however, no increase was observed when p/l ratio increased by only 20 percent, which was confirmed in the present investigation. in addition, fonseca et al.19 (2010) found that a 50% alteration in p/l ratio produced a weaker rmgic in diametral tensile strength. it should be noted that this reduction was only observed in rmgics, and it could be attributed to a suppressed acidbase reaction, which, in turn, may lead to a compromised interaction between matrix and powder particles 5,15,19. consequently, the strength of the materials does not increase. this way, the type of material, the investigated property and also the amount of unreacted particles are critical factors to be considered. these factors may explain the difference between the present study and that of fleming et al.9 (2003), in which the compressive strength of chemfil was investigated and significant differences were achieved9. lund et al.20 (2007) it was also reported that the effect of p/l ratio on the diametral tensile strength of rmgic is brand dependent. however, it is noteworthy that diametral test is only recommended for brittle materials. due to the vast disparity of p/l ratios and the results reported, it is very challenging to make a definite conclusion concerning the ideal p/l ratio. for instance, mitsuhashi et al.15 (2003) found no significant difference in fracture strength and mechanical properties of rmgics by reducing the amount of powder by 33%. furthermore, a recent study indicated no significant difference in flexural strength of rmgic after 17 and 25% decrease and 33 and 50% increase in powder level7. the majority of the previous studies have considered the reduction in powder amount11,15-16, whereas this study investigated both decrease and increase of powder/liquid ratio. problems attributed to the incorporation of high powder content during mixing procedure have encouraged investigations on the decreased p/l ratio15. this study also evaluated the effect of p/l ratio on the bonding strength; this has not been examined before, which can be due to difficult specimen preparation10. in the present study, mixing the cement with greater powder amount was difficult, but the handling was improved due to less adhesiveness, except for fuji ii lci in which the increase in powder amount resulted in a sticky material that effect of powder/liquid ratio of glass ionomer cements on flexural and shear bond strengths to dentin table 2 means and standard deviations for shear bond and flexural strength (mpa) shear bond strength flexural strengthmaterial group p/l ratio fuji ii group 1 2.16/1 4.75±1.26 15.75±2.84 group 2 2.70/1 5.50±1.94 14.57±1.91 group 3 3.24/1 6.12±2.11 16.96±2.73 fuji ii lci group 4 2.56/1 11.06±3.52 9.58±8.75 group 5 3.20/1 11.60±3.19 47.97±2.85 group 6 3.84/1 11.59±2.85 44.96±5.86 fuji ix gp group 7 2.90/1 5.57±1.64 17.66±1.99 group 8 3.60/1 6.29±1.88 18.99±2.46 group 9 4.40/1 7.39±2.77 20.32±2.09 207 braz j oral sci. 10(3):204-207 immediately lost homogeneity and made the application difficult and almost impossible. under clinical conditions, as the increase in powder amount did not show adverse effects on mechanical properties, it can be recommended to increase the powder amount to achieve better handling properties. in this case, the clinical longevity of gic restorations will probably increase. based on the experimental design of the present study, it was verified that: 1. twenty percent variation in p/l ratio had no significant effect on the shear bond and flexural strengths. 2. shear bond and flexural strengths of rmgic was significantly higher than those of conventional gics; however, no significant difference was observed between the two conventional gics. 3. when the powder amount is increased, adhesive failures most frequently occur between dentin and cement rather than within the cement. acknowledgements this study was financially supported by iran center for dental research, shahid beheshti medical university. the authors are grateful to dr. s. moayedi for her helpful assistance during the study. references 1. mount gj. glass ionomer cements: past¡ present and future. oper dent. 1994; 19: 82-90. 2. iazzetti g, burgess jo, gardiner d. selected mechanical properties of fluoride-releasing restorative materials. oper dent. 2001; 29: 21-6. 3. kleverlaan cj, van duinen rnb, feilzer aj. mechanical properties of glass ionomer cements affected by curing methods. dent mater. 2004; 20: 45-50. 4. xie d, brantley wa, culbertson bm, wang g. mechanical properties and microstructures of glass-ionomer cements. dent mater. 2000; 16: 129-38. 5. yap auj, mudambi s, chew cl, neo jcl. mechanical properties of improved visible light-cured resin-modified glass ionomer cement. oper dent. 2001; 26: 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gj. an atlas of glass-ionomer cement. london: martin dunitz; 1990. p.105-114. 13. billington rw, williams ja, pearson gj. variation in powder/liquid ratio of restorative glass-ionomer cement used in dental practice. br dent j. 1990; 169: 164-7. 14. azillah ma, anstice hm, pearson j. long-term flexural strength of three direct aesthetic restorative materials. j dent. 1998; 26: 177-82. 15. mitsuhashi a, honaoka k, teranaka t. fracture toughness of resin modified glass-ionomer restorative material: effect of powder/liquid ratio and powder particle size reduction on fracture toughness. dent mater. 2003; 19: 747-57. 16. crisp s, lewis bg, wilson ad. characterization of glass-ionomer cements 2. effect of the powder: liquid ratio on the physical properties. j dent. 1976; 4: 287-90. 17. thean hpy, mok byy, chew cl. bond strengths of glass ionomer restoratives to primary vs. permanent dentin. j dent child. 2000; 67: 112-6. 18. lihua e, masao irie, nagaoka n, yamashiro t, suzuki k. mechanical properties of a resin-modified glass ionomer cement for luting: effect of adding spherical silica filler. dent mater j. 2010; 29: 253-61. 19. fonseca rb, branco ca, quagliatto ps, gonçalves l, soares cj, carlo hm et al. influence of powder/liquid ratio on the radiodensity and diametral tensile strength of glass ionomer cements. j appl oral sci. 2010; 18: 577-84. 20. lund rg, ogliari f, lima gs, del-pino fab, petzhold cl, piva e. diametral tensile strength of two brazilian resin-modified glass ionomers cements: influence of the powder/liquid ratio. braz j oral sci. 2007; 21: 1353-6. effect of powder/liquid ratio of glass ionomer cements on flexural and shear bond strengths to dentin 1http://dx.doi.org/10.20396/bjos.v18i0.8657267 volume 18 2019 e191648 original article 1 graduate program in dentistry, school of dentistry, university of vale do taquari, lajeado, rio grande do sul, brazil 2 graduate program in dentistry, department of pediatric dentistry, school of dentistry, federal university of pelotas, pelotas, pelotas, rio grande do sul, brazil 3 graduate program in dentistry, department of restorative dentistry, school of dentistry, federal university of pelotas, pelotas, rio grande do sul, brazil corresponding author: luiz alexandre chisini, dds, msc, phd student professor university of vale do taquari school of dentistry address: 171, avelino talini st. lajeado rs brazil 95914-014 phone/fax: +55 51 3714 7000 / 5454 or 55 53 981121141 e-mail: alexandrechisini@gmail.com https://orcid.org/0000-0002-3695-0361 received: may 03, 2019 accepted: september 10 2019 desire of university students for esthetic treatment and tooth bleaching: a cross-sectional study luiz alexandre chisini1,*, mariana gonzalez cademartori2, kauê collares3, ana luiza cardoso pires3, marina sousa azevedo2, marcos britto corrêa3, flávio fernando demarco3 aim: the aim of this study was to investigate the prevalence of the desire of university students for esthetic treatment and tooth bleaching, and associated factors. methods: a cross-sectional study was performed in 2016 with first-semester university students in pelotas. data was collected via a self-administered questionnaire including demographic, socioeconomic and psychosocial characteristics. oral impact on daily performance (oidp) was assessed and poisson regression models were used to evaluate the association between the following outcomes: prevalence of desire for esthetic treatment and the tooth bleaching. a p-value of ≤ 0.05 was considered significant. results: a total of 2,058 students participated in the study. refusals represented a mere 1.4% of the total sample. of the individuals interviewed, 16.1% underwent tooth bleaching and 74.4% reported a desire for esthetic dental treatment. models showed students with higher family income and with the intention to use private dental services had 65% and 47% higher prevalence of tooth bleaching, respectively. individuals satisfied with dental color and appearance exhibited a higher prevalence of tooth bleaching, as did those who smoked. on the other hand, students satisfied with dental color (pr= 0.77, 95% ci: 0.73-0.80) and with their dental appearance (pr= 0.82, 95% ci 0.78-0.87) demonstrated less desire to undergo esthetic dental treatment. moreover, individuals who suffered some impact in oidp reported a greater desire for esthetic treatment (pr= 1.07, 95% ci: 1.02-1.13). conclusion: general and psychosocial characteristics were associated with the desire for esthetic treatment and tooth bleaching in university students. keywords: tooth bleaching. quality of life. esthetics, dental. https://orcid.org/0000-0002-3695-0361 2 chisini et al. introduction dental appearance is an important component of facial attractiveness. this characteristic is considered a complex, subjective perception influenced by cultural, contextual, individual and environmental factors1,2. teeth that are perfectly aligned and whiter are often considered important requirements for satisfaction with dental appearance1-5. in recent years, the demand for esthetic procedures has increased significantly in dentistry, not only through orthodontic treatment, but also by means of tooth bleaching5. in view of this, a symmetrical, harmonious, white smile has become associated with a better quality of life3,5. moreover, a significant level of social construction in respect of esthetics can be observed in brazil, which attributes an important role to the smile and, consequently, dental color6. a recent study in brazil showed that individuals with significant dental caries displayed a higher prevalence of desire for tooth bleaching compared with the low caries group2. the authors have explained these findings through the social construction of the brazilian population that frequently reports esthetics as a proxy for dental health2. change in tooth color may occur due to a natural process of dental aging or may be caused by oral diseases, dental trauma or by the use of dental material such as mineral trioxide aggregate7-10. moreover, tooth discoloration may also be due to extrinsic pigmentation, which occurs with the consumption of highly pigmented foods and also tobacco use11. tooth discoloration is highly prevalent and may affect self-esteem5,7,8,12. these conditions could increase the desire for bleaching treatments. dissatisfaction with tooth color is one of the main reasons associated with dissatisfaction with dental appearance5. one possible impact of tooth bleaching on the oral health-related quality of life (ohrqol) has been discussed, but is still not entirely clear and may vary according to the age of the individual, since younger people attribute greater value to white teeth. while clinical studies on young adults3 show that individuals reported greater satisfaction with their smiles after tooth bleaching, different findings have been observed in a similar design study with older individuals13 failing to show an improvement in ohrqol after tooth-bleaching treatment. other studies have demonstrated that tooth bleaching increases subjects’ self-esteem, and it could improve social interaction5,7,8,12. accordingly, dental esthetics has been associated with oral health-related quality of life in young university students14. small alterations in dental esthetics significantly influenced the ohrqol of these students14. the university is an environment composed mostly of young people, permeated by a high degree of social interaction and, in this context, facial attractiveness tends to play an important role in the acceptance of individuals by their colleagues and friends15. considering that adverse dental appearance has a negative impact on facial attractiveness, a high demand by university students for esthetic dental procedures might be expected, but this issue has not yet been investigated. therefore, this study aimed to investigate the prevalence of the desire of university students for esthetic treatment and tooth bleaching, and associated factors. 3 chisini et al. materials and methods this study was reported in accordance with the strobe guideline (strengthening the reporting of observational studies in epidemiology). full details concerning the methods of the present study have been published previously16. design study and participants a cross-sectional study (baseline) nested within a prospective cohort study16 was performed on a cohort of university students entering federal university of pelotas (ufpel) in the first semester of the 2016 academic year. all newcomers to the first semester of 2016 (baseline) were invited to participate and signed a consent form. in terms of eligibility criteria, we included only individuals able to self-answer the questionnaire. individuals who were not able to self-answer the questionnaire (such as those with visual impairment or motor deficiency) and those who did not enter university in 2016, were excluded from the final sample. prior to the data collection, all academic units of the university were informed about the study, which consented to its performance. data collection data were collected using a self-administered questionnaire including 74 questions related to demographic and socioeconomic characteristics, psychosocial characteristics, habits and health-related behavior. participants were addressed by trained graduates (n=5) and undergraduate students (n=20). initially, four hours of theoretical training was carried out and the questions were discussed. a pilot study was carried out on 100 university students not eligible for the study, from five different academic units, randomly selected by lots. independent variables: age of participants in years was collected, being categorized as follows: a) less than 18 years of age; b) 18 to 24 years of age; c) 25 to 34 years of age; and d) over 34 years of age. family income data were collected in the currency of brazil (brl) (1 dollar = 3.92 brl) and classified into a) ≤ r$1,000; b) r$1,001 to r$5,000; and c) ≥ r$5,001. in relation to harmful habits, the habit of smoking and its frequency of use was investigated using a validated, brazilian version of the alcohol, smoking and substance involvement screening test (assist) recommended by the world health organization (who). individuals were considered smokers if they reported smoking at least once a month. satisfaction with dental color was assessed by way of the question: are you satisfied with the color of your teeth? 17. answers were dichotomized into satisfied (very satisfied / satisfied) and dissatisfied (very dissatisfied / dissatisfied). satisfaction with dental appearance was assessed using the statement: “i do not like to see my teeth when i look at myself in the mirror, in photographs or in videos”6. the individuals were instructed to choose one of the following options, a) i do not agree; b) i agree a little; c) i agree; d) i agree a lot; and e) i totally agree. alternatives were dichotomized into satisfied (i do not agree) and dissatisfied (the other alternatives). 4 chisini et al. self-perception of oral health was obtained via the following question: compared to other people of your age, how do you consider the health of your teeth, mouth and gums? response options were dichotomized into: a) good (very good / good) and b) poor (regular / bad / very bad). the oral healthcare system data were collected through the following question: “if you need to go to the dentist, which type of dental service would you choose?”, dichotomized into public dental service (public service/ school of dentistry) and private dental service (private health insurance/ private dental office). oral health-related quality of life was assessed using the brazilian version of the oral impact on daily performance (oidp)18. this tool comprises questions including three domains (physical, psychological and social), with the alternatives being presented on a six-point likert scale (a) never; b) less than once a day; c) once or twice a month; d) once or twice a week; e) 3-4 times per week; and f) every or almost every day). a cut-off point was established using the median. oral health status was assessed through self-reported dental caries experience. the participants answered (yes/no) if he/she had at least one decayed, filled or extracted tooth due to dental caries. individuals that answer yes to this question were considered to have experience of caries. dependent variables (outcomes) the desire for esthetic dental treatment was assessed using the question: “would you like to have esthetic treatment to improve the appearance of your teeth?” tooth bleaching was assessed via the question: “have you ever had your teeth bleached using trays or in a dental environment?”. both questions had yes (reference categories) and no as the response alternatives. ethical aspects this study was approved by the ethics committee of the faculty of medicine/ufpel under protocol number 49449415.2.0000.5317. statistical methods statistical analyses were performed using the software stata 12.0 (stata corporation, college station, tx, usa). for the descriptive analysis, relative and absolute frequencies were estimated. multivariate poisson regression models have been proposed to estimate prevalence ratios of binary outcomes in cross-sectional studies19. poisson regression models were used to assess the association between variables of interest (the desire for esthetic treatment and tooth bleaching ) and exposure variables. this strategy permitted the estimate of the prevalence ratio (pr) as the measure of effect and a 95% confidence interval (ci) was adopted. variables with p values of ≤ 0.20 in the crude analyses were included in the model fit. a forward-stepwise procedure was used to include or exclude explanatory variables in the model fit. for the final model, the variables were considered significant if they had a p-value of ≤ 0.05, after adjustment. results a total of 3,237 students entered university in the first semester of 2016, of which 2,058 (63.6%) participated in this study. twenty-nine students (1.4%) declined to participate. more than half of participants were women (52.3%), with ages ranging from 5 chisini et al. 18 to 24 (66.1%) and with family income in the second tertile (61.6%). desire for dental esthetic treatment was reported by 74.4% of the university students. of the participants, 16.1% had already undergone tooth bleaching. table 1 displays characteristics according to the desire for dental esthetic treatment and for tooth bleaching. table 1. sample characteristics according to tooth bleaching and to desire for dental esthetic treatments. federal university of pelotas, pelotas/brazil (n=2,071 university students). 2016. variable/category dental bleaching n (%) desire for dental esthetic treatments n (%) sex 2,061 2,063 male 159 (16.16) 681 (69.21) female 172 (15.97) 858 (79.52) age (years) 2,061 2,063 16 17 53 (17.15) 209 (67.42) 18 24 214 (15.67) 1,032 (75.66) 25 34 36 (16.90) 162 (75.35) 35 or more 30 (17.34) 133 (76.44) family income 1,702 1,704 ≤ r$1000 32 (11.68) 220 (79.42) r$1001 to r$5000 152 (14.49) 796 (75.88) ≥ r$5001 85 (22.43) 271 (71.69) satisfaction with dental color 2,068 2,070 dissatisfied 91 (10.92) 763 (91.60) satisfied 243 (19.68) 777 (62.81) satisfaction with dental appearance 2,048 2,049 dissatisfied 129 (13.03) 864 (87.10) satisfied 204 (19.28) 660 (62.44) caries experience 2,069 2,071 no 97 (14.72) 476 (72.34) yes 237 (16.81) 1,065 (75.37) self-perception of oral health 2,067 2,069 good 256 (17.32) 1,069 (72.38) bad 77 (13.07) 471 (79.56) dental service payment mode 1,747 1,749 public 61 (11.87) 389 (75.24) private 225 (18.25) 903 (73.3) smoking 2,009 2,010 no 263 (15.26) 1,293 (74.96) yes 60 (21.05) 203 (71.23) oral health-related quality of life 2,002 2,003 no impact 202 (17.34) 791 (67.78) with impact 119 (14.22) 698 (83.49) 6 chisini et al. a crude analysis showed that family income, satisfaction with dental color, satisfaction with dental appearance, self-perception about oral health, the oral healthcare system, smoking habits and oral health-related quality of life were associated with having undergone tooth bleaching. after adjustments, family income, satisfaction with dental color, satisfaction with dental appearance, the oral healthcare system and smoking habits remained associated with the outcome. university students with high family income and those with intention to use private dental services equated to 65.0% (pr= 1.65, 95% ci: 1.07–2.54) and 47.0% (pr= 1.47, 95% ci: 1.10–1.97), greater prevalence of tooth bleaching, respectively. similarly, individuals who were satisfied with their dental color (pr= 1.44, 95% ci: 1.08-1.93) and their dental appearance (pr= 1.29, 95% ci: 1.00-1.68) also presented high prevalence of tooth bleaching. student smokers (pr= 1.49, 95% ci: 1.11-2.01) reported a higher prevalence of having undergone tooth bleaching when compared to non-smokers (table 2). when the desire for dental esthetic treatment was considered, a crude analysis showed a positive association with sex, age, family income, satisfaction with dental color, satisfaction with dental appearance, self-perception about oral health, tooth bleaching and oral health-related quality of life. after adjustments, women students (pr= 1.09, 95% ci: 1.09-1.21) aged between 18 and 24 (pr= 1.11, 95% ci: 1.02-1.19) and those aged between 25 and 34 (pr= 1.11, 95% ci: 1.00-1.23) were more likely to crave dental esthetic treatment. students satisfied with their dental color (pr= 0.77, 95% ci: 0.73-0.80) and with their dental appearance (pr= 0.82, 95% ci: 0.78-0.87) were less likely to desire dental esthetic treatment. it was observed that students who suffered an impact on oral health-related quality of life reported a greater desire for esthetic treatment (pr= 1.07, 95% ci: 1.02-1.13) (table 3). discussion this study showed that general and psychosocial characteristics are associated with the desire of university students in southern brazil to undergo esthetic treatment and tooth bleaching. we observed that students that reported a negative impact on ohrqol were associated with a greater desire for esthetic treatment. moreover, students who reported being smokers presented an almost 50% higher prevalence of tooth bleaching. esthetics is a very important concept to the general population and has been considered a key component of social interaction. this media pressure for a better look has increased the demand for esthetic procedures, especially in women and young people5,20-23. women are more concerned about health and more dissatisfied with their appearance5 and are more likely to turn to esthetic procedures20,21, such as plastic surgery23. in dentistry, people are concerned about tooth alignment and color2. an analysis of the printed media in the state of espírito santo showed an excessive appeal to esthetic issues linked to the capitalist philosophy of treating oral health as a commodity rather than as a health issue24. thus, the overt publication of esthetic issues in the media reflects social values related to desire and vanity, which contribute to the production of a utopian-style esthetic benchmark24. 7 chisini et al. beyond the influence of the media, satisfaction with appearance is intimately related to the social interaction that this subjective condition provides, especially in young individuals who want to be accepted into a social group25. our results corroborate the findings of previous studies, in which women and younger subjects show greater table 2. crude (c) and adjusted (a) prevalence ratio (pr) for independent variables for tooth bleaching. poisson regression model. federal university of pelotas, pelotas/brazil (n=1,422 universities students). 2016. variable/category prc (ci95%) p-value pra(ci95%) p-value sex 0.907 male 1 female 0.99 (0.81 – 1.20) age (years) 0.840 16 and 17 1 18 to 24 0.91 (0.69 – 1.20) 25 to 34 0.99 (0.67 – 1.45) 35 or more 1.01 (0.67 – 1.52) family income < 0.001 0.005 ≤ r$1000 1 1 r$1001 to r$5000 1.24 (0.87 – 1.77) 1.16 (0.77 – 1.75) ≥ r$5001 1.92 (1.32 – 2.80) 1.65 (1.07 – 2.54) satisfaction with dental color < 0.001 0.012 dissatisfied 1 1 satisfied 1.80 (1.44 – 2.25) 1.44 (1.08 – 1.93) satisfaction with dental appearance < 0.001 0.050 dissatisfied 1 1 satisfied 1.48 (1.21 – 1.81) 1.29 (1.00 – 1.68) caries experience 0.232 no 1 yes 1.14 (0.92 – 1.42) self-perception of oral health 0.020 good 1 bad 0.76 (0.60 – 0.96) dental service payment mode 0.001 0.010 public 1 1 private 1.54 (1.18 – 2.00) 1.47 (1.10 – 1.97) smoking 0.012 no 1 1 0.008 yes 1.38 (1.07 – 1.77) 1.49 (1.11 – 2.01) oral health-related quality of life 0.062 no impact 1 with impact 0.82 (0.67 – 1.01) -2 log likelihood (empty model) = 1,886.5 -2 log likelihood (final model) = 1,253.6 8 chisini et al. table 3. crude (c) and adjusted (a) prevalence ratio (pr) for independent variables for the desire for esthetic treatments. poisson regression model. federal university of pelotas, pelotas/brazil (n=1,422 universities students). 2016. variable/category prc (ci95%) p-value pra(ci95%) p-value sex < 0.001 < 0.001 male 1 1 female 1.15 (1.09 – 1.21) 1.15 (1.09 – 1.21) age (years) 0.042 0.423 16 and 17 1 1 18 to 24 1.12 (1.03 – 1.22) 1.11 (1.02 – 1.19) 25 to 34 1.12 (1.00 – 1.25) 1.11 (1.00 – 1.23) 35 or more 1.13 (1.01 – 1.27) 1.04 (0.94 – 1.15) family income 0.021 ≤ r$1000 1 r$1001 to r$5000 0.96 (0.89 – 1.02) ≥ r$5001 0.90 (0.83 – 0.98) satisfaction with dental color < 0.001 < 0.001 dissatisfied 1 1 satisfied 0.69 (0.65 – 0.72) 0.77 (0.73 – 0.80) satisfaction with dental appearance < 0.001 < 0.001 dissatisfied 1 1 satisfied 0.72 (0.68 – 0.76) 0.82 (0.78 – 0.87) caries experience 0.150 no 1 yes 1.04 (0.99 – 1.10) self-perception of oral health < 0.001 good 1 bad 1.09 (1.04 – 1.16) dental service payment mode 0.391 public 1 private 0.97 (0.91 – 1.03) smoking 0.204 no 1 yes 0.95 (0.87 – 1.03) tooth bleaching 0.001 0.110 no 1 1 yes 0.87 (0.80 – 0.94) 0.94 (0.87 – 1.01) oral health-related quality of life < 0.001 0.007 no impact 1 1 with impact 1.23 (1.17 – 1.30) 1.07 (1.02 – 1.13) -2 log likelihood (empty model) = 3,995.1 -2 log likelihood (final model) = 3,702.0 9 chisini et al. desire for esthetic treatment in plastic surgery23 and dentistry5,20-22. similarly, our study demonstrated that individuals satisfied with their dental color and dental appearance reported less desire for dental esthetic treatment. however, those individuals who did report some impact on their oral health-related quality of life tend to be more desirous of dental esthetic treatment, probably because they believe that undergoing esthetic dental procedures improves their oral health. in fact, similar observations were found in a birth cohort in southern brazil2. individuals with a history of dental caries over the course of their lives had 9% greater desire for tooth bleaching2, highlighting the narrow relationship between the perception of esthetics and health in this population. so, social construction of esthetics and the excessive pressure of these parameters in the media can affect individuals’ behavior concerning the demand for dental treatment, such as tooth bleaching6 and replacement with non-white dental restoration26, thus causing possible overtreatment. this desire for highly esthetic standards can be observed both in patients and in dentists. in fact, in brazil, which has twice as many dentists than recommended by the world health organization, largely confined to the larger cities27 recommends the total substitution of amalgam restorations for esthetic reasons, mainly in caucasian patients. facial and dental attraction represents an important element in the quality of life. individuals who perceive themselves to be “less attractive” tend to have lower self-esteem than those who consider themselves esthetically attractive. similarly, individuals who find themselves more attractive and have a better health-related quality of life tended to perform orthodontic treatment and tooth bleaching. tooth color is one of the most common components of the smile that causes dissatisfaction amongst people6, and studies have demonstrated that individuals who underwent tooth bleaching state they are more satisfied with their dental color5,28. similarly, we found an association between tooth bleaching and both satisfaction with dental color and satisfaction with dental appearance. this relationship highlights the significant contribution of tooth bleaching in the self-perception of dental appearance, explaining the increasing desire for tooth bleaching5,28. conversely, as previously mentioned, our study showed that students satisfied with their dental appearance and their dental color exhibited less desire for esthetic treatment. tooth bleaching is the most craved after esthetic procedure5 and the cost is lower when compared with composite or ceramic veneers. however, tooth bleaching is still very costly and available only through private dental services. brazil’s unified health system (sus) provides free dental services to the population, focusing mainly on a preventive approach and on treatment of dental diseases29. thus, the costs of esthetic procedures, including tooth bleaching, are not covered by the public system. this fact may partly explain the higher prevalence of tooth bleaching among those subjects with higher family income. it should be noted that brazil has a large number of dentists27 and the prevalence of dental caries is in decline, in all age groups. the market has become very competitive and there is increased demand for esthetic and cosmetic training in continuing education courses for dentists and, as a consequence, an increase in the prevalence of esthetic dental treatment provided by private dental offices. it indicates that the use of private services can be a decisive factor for performing tooth bleaching. on the other hand, a birth 10 chisini et al. cohort study only observed an association between prevalence of tooth bleaching and type of dental service used in crude models. individuals that paid for their own treatment using private services, exhibited a greater prevalence of tooth bleaching, despite the association being discarded in the final model2. moreover, the marketing industry has encouraged individuals to desire esthetic treatment and also constructed a utopian, esthetic demand in brazilian society without fully appreciating the value of health24. our findings also showed a higher prevalence of tooth bleaching among student smokers. changes in tooth color can also occur due to extrinsic pigmentations provided by food and tobacco11. previous studies reported a higher dissatisfaction with dental appearance in smokers12,30. therefore, these individuals tend to undergo more tooth bleaching than non-smokers. a recent clinical trial concerning the effectiveness of at-home bleaching by smokers and non-smokers observed darker teeth in smokers after a year11. however, after dental prophylaxis, the authors observed that this darkening would disappear. this emphasizes, therefore, that the darker tooth color of smokers, even if these pigmentations are only extrinsic in nature, could lead these individuals to be more dissatisfied with dental color12,30, and seek to carry out tooth bleaching to improve their dental appearance. however, a number of limitations should be mentioned. studies that use a cross-sectional design conceal the causal inferences. thus, it is impossible to infer if students that reported a negative impact on ohrqol had a greater desire for esthetic treatment or if this esthetic desire manifested itself prior to the impact on the quality of life (ohrqol). in addition, the use of self-reported measures could be overestimated, as this is a subjective measurement. however, studies have shown individuals perceive the need for treatment in a similar way to the normative question, demonstrating that there is a similarity in these two evaluations and that, in most cases, there is no overestimation of the need for treatment based on self-perception31. moreover, self-reported measurements have been considered as reliable, valid tools, being widely used in epidemiologic studies4,5,32. lastly, the participant may have answered “yes”, interpreting this option as a possible offer to perform some dental esthetic procedure. however, the prevalence was similar to that reported in other studies5. furthermore, some points should be highlighted from the present study. to the best of our knowledge, this is the first study to investigate the desire for dental esthetic procedures and tooth bleaching in a large sample of university students. a tiny minority of losses and refusals render our results consistent, which can be extrapolated to populations with similar characteristics. moreover, few studies have evaluated the association between quality of life and tooth bleaching, usually in studies with small sample sizes3,13. thus, the present findings show that general and psychosocial characteristics are associated with the desire for esthetic treatment and tooth bleaching in university students, including satisfaction with dental color and satisfaction with dental appearance. compliance with ethical standards conflict of interest: the authors declare that there is no conflict of interest. 11 chisini et al. ethical approval: all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 helsinki declaration and its subsequent amendments, or comparable ethical standards. informed consent: informed consent was obtained from all individuals participating in the study. references 1. dong jk, jin th, cho hw, oh sc. the esthetics of the smile: a review of some recent studies. int j prosthodont. 1999 jan-feb;12(1):9-19. 2. silva fbd, chisini la, demarco ff, horta bl, correa mb. desire for tooth bleaching and treatment performed in brazilian adults: findings from a birth cohort. braz oral res. 2018 mar 8;32:e12. doi: 10.1590/1807-3107bor-2018.vol32.0012. 3. meireles ss, goettems ml, dantas rv, bona ad, santos is, demarco ff. changes in oral health related quality of life after dental bleaching in a double-blind randomized clinical trial. j dent. 2014 feb;42(2):114-21. doi: 10.1016/j.jdent.2013.11.022. 4. isiekwe gi, sofola oo, onigbogi oo, utomi il, sanu oo, dacosta oo. dental esthetics and oral health-related quality of life in young adults. am j orthod dentofacial orthop. 2016 oct;150(4):627-636. doi: 10.1016/j.ajodo.2016.03.025. 5. tin-oo mm, saddki n, hassan n. factors influencing patient satisfaction with dental appearance and treatments they desire to improve aesthetics. bmc oral health. 2011 feb 23;11:6. doi: 10.1186/1472-6831-11-6. 6. boeira gf, salas mms, araújo dc, masotti 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10.1590/0103-6440201802132. 11. de geus jl, de lara mb, hanzen ta, fernandez e, loguercio ad, kossatz s, et al. one-year follow-up of at-home bleaching in smokers before and after dental prophylaxis. j dent. 2015 nov;43(11):1346-51. doi: 10.1016/j.jdent.2015.08.009. 12. alkhatib mn, holt r, bedi r. prevalence of self-assessed tooth discolouration in the united kingdom. j dent. 2004 sep;32(7):561-6. 13. bruhn am, darby ml, mccombs gb, lynch cm. vital tooth whitening effects on oral health-related quality of life in older adults. j dent hyg. 2012 summer;86(3):239-47. 12 chisini et al. 14. klages u, bruckner a, zentner a. dental aesthetics, self-awareness, and oral health-related quality of life in young adults. eur j orthod. 2004 oct;26(5):507-14. 15. sarwer db, spitzer jc. body image dysmorphic disorder in persons who undergo aesthetic medical treatments. aesthet surg j. 2012 nov;32(8):999-1009. doi: 10.1177/1090820x12462715. 16. chisini l, cademartori m, collares k, tarquinio s, goettems m, demarco f, et al. methods and logistics of an oral health cohort of university students from pelotas, a brazilian southern city. braz j oral sci. 2019;18:e191460. doi: 10.20396/bjos.v18i0.8655316. 17. samorodnitzky-naveh g, geiger s, levin l. patients’ satisfaction with dental esthetics. j am dent assoc. 2007 jun;138(6):805-8. 18. cortes mi, marcenes w, sheiham a. impact of traumatic injuries to the permanent teeth on the oral health-related quality of life in 12-14-year-old children. community dent oral epidemiol. 2002 jun;30(3):193-8. 19. barros aj, hirakata vn. alternatives for logistic regression in cross-sectional studies: an empirical comparison of models that directly estimate the prevalence ratio. bmc med res methodol. 2003 oct 20;3:21. 20. odioso ll, gibb rd, gerlach rw. impact of demographic, behavioral, and dental care utilization parameters on tooth color and personal satisfaction. compend contin educ dent suppl. 2000;(29):s35-41; quiz s43. 21. hassel aj, wegener i, rolko c, nitschke i. self-rating of satisfaction with dental appearance in an elderly german population. int dent j. 2008 apr;58(2):98-102. 22. chisini la, noronha tg, ramos ec, dos santos-junior rb, sampaio kh, faria esal, et al. does the skin color of patients influence the treatment decision-making of dentists? a randomized questionnaire-based study. clin oral investig. 2019 mar;23(3):1023-30. doi: 10.1007/s00784-018-2526-7. 23. edmonds a, sanabria e. medical borderlands: engineering the body with plastic surgery and hormonal therapies in brazil. anthropol med. 2014;21(2):202-16. doi: 10.1080/13648470.2014.918933. 24. cavaca a, gentilli v, marcolino e, emmerich a. as representações da saúde bican na mídia impressa. interface. 2012;16(43):1055-68. 25. henderson-king d, henderson-king e. acceptance of cosmetic surgery: scale development and validation. body image. 2005 jun;2(2):137-49. 26. chisini la, noronha tg, ramos ec, dos santos-junior rb, sampaio kh, faria esal, et al. does the skin color of patients influence the treatment decision-making of dentists? a randomized questionnaire-based study. clin oral investig. 2019 mar;23(3):1023-1030. doi: 10.1007/s00784-018-2526-7. 27. san martin a, chisini l, martelli s, sartori l, ramos e, demarco f. distribution of dental schools and dentists in brazil: an overview of the labor market. rev abeno. 2018;18(1):63-73. 28. al-zarea bk. satisfaction with appearance and the desired treatment to improve aesthetics. int j dent. 2013;2013:912368. doi: 10.1155/2013/912368. 29. pucca ga, jr., gabriel m, de araujo me, de almeida fc. ten years of a national oral health policy in brazil: innovation, boldness, and numerous challenges. j dent res. 2015 oct;94(10):1333-7. doi: 10.1177/0022034515599979. 30. alkhatib mn, holt rd, bedi r. smoking and tooth discolouration: findings from a national cross-sectional study. bmc public health. 2005 mar 24;5:27. 13 chisini et al. 31. kragt l, wolvius eb, jaddoe vwv, tiemeier h, ongkosuwito em. influence of self-esteem on perceived orthodontic treatment need and oral health-related quality of life in children: the generation r study. eur j orthod. 2018 may 25;40(3):254-61. doi: 10.1093/ejo/cjx054. 32. silva ae, menezes am, assuncao mc, goncalves h, demarco ff, vargas-ferreira f, et al. validation of self-reported information on dental caries in a birth cohort at 18 years of age. plos one. 2014 sep 9;9(9):e106382. doi: 10.1371/journal.pone.0106382. oral sciences n3 braz j oral sci. 11(1):1-9 received for publication: june 05, 2011 accepted: december 07, 2011 original article braz j oral sci. january | march 2012 volume 11, number 1 peri-orbital bone dimensional analysis using computed tomography for placement of osseointegrated implants clemente maia s. fernandes1, beatriz silva câmara mattos2, marcelo de gusmão paraíso cavalcanti3, luciana cardoso fonseca4, mônica da costa serra5 1dds, ll.b student, msc, phd, post-doctorate student, department of social dentistry, faculty of dentistry of araraquara, são paulo state university unesp, brazil 2dds, msc, phd, department of maxillofacial surgery, prosthodontics and traumatology, school of dentistry, university of são paulo, brazil 3dds, msc, phd, post-doctor, department of stomatology, school of dentistry, university of são paulo, brazil 4dds, msc, phd, department of oral diagnosis, school of dentistry, catholic university of minas gerais, brazil 5dds, ll.b, msc, phd, post-doctor, department of social dentistry, faculty of dentistry of araraquara, são paulo state university unesp, brazil correspondence to: mônica da costa serra departamento de odontologia social, faculdade de odontologia de araraquara unesp rua humaitá 1680 araraquara sp brasil 14801-903 phone: + 55 16 33016353 fax: + 55 16 33016343 e-mail: mcserra@foar.unesp.br abstract craniofacial osseointegrated implants enabled producing implant-retained facial prosthesis, namely the orbital prosthesis. aim: to evaluate the length and width of the bone structure of the peri-orbital region and to present the method validation. methods: computed tomography scans of 30 dry human skulls were obtained in order to register linear length and width measurements of the periorbital region. two examiners made the measurements twice with intervals of at least 7 days between them. data were analyzed by descriptive statistics and the paired student’s t-test was used as inferential technique (sas, α=0.05). results: in most cases, the intraand inter-examiner variations were not significant (p>0.05). therefore, the method proposed was considered as precise and valid for the measurement of the peri-orbital region. the measured points correspond to the hours of a clock. the major lengths were observed at 1 h (18.32 mm) for the left peri-orbital bone and at 11h (19.28 mm) for the right peri-orbital bone, followed by the points situated at 2h (13.05 mm) and 12h (11.37 mm) for the left side and at 10 h (12.34 mm) and 12 h (11.56 mm) for the right side. it was verified that the three points with lowest values followed the same anatomical sequence in the supraorbital rim for the right and left orbits, showing compatibility with the insertion of the intraoral osseointegrated implants. the medial wall of both orbits did not present sufficient length to allow the insertion of intraoral or craniofacial implants. conclusions: the largest width points were observed in the supraorbital rim and in the infralateral region of both orbits and those of smallest width were found in the supralateral region of both orbits. keywords: orbit, ct scan, craniofacial rehabilitation, osseointegrated implants, measurement. introduction oral and maxillofacial prosthodontics is the dental specialty responsible for the fabrication of intraoral and extraoral prostheses, indicated when maxillofacial regions are lost due to diseases, cancer surgery or trauma. the application of the osseointegration principles for the restoration of craniofacial defects using facial prostheses retained by implants, enabled by the emergence of osseointegrated 22222 braz j oral sci. 11(1):1-9 implants, has given rise to significant advances, overcoming the limitations of conventional retention methods, by either elastic, adhesive or eye glass frames1-4. implant-retained dentures provide a better quality of life, which ultimately, is the ideal for any rehabilitation process5-6. for the construction of implant-supported orbital prostheses, the implants may be placed in the supra-orbital margin of the frontal bone in the infra-orbital rim, in the zygomatic process of the frontal bone and frontal process of the zygomatic bone7-8. it is crucial to know the bone length in the different peri-orbital regions. the actual indication for placing the implants and the length that they should have derives from this information. regions with a greater length allow for the use of longer implants, and the greater the length of the implant, the greater the success rate9. planning the installation of osseointegrated implants requires the use of computed tomography (ct) to determine the preferential bone sites to install the implants, as they are highly predictable and reliable in the linear and volumetric quantification of the bone structure as well as in bone density evaluation10-11. ct scans in preoperative examinations to check the bone thickness at the recipient sites of implants have been widely indicated10,12-13. this study evaluated the length and width dimensions of the right and left peri-orbital bone, measured on ct scans, seeking to determine the more favorable peri-orbital bone areas for the installation of osseointegrated implants. the validation of the method was also performed, ensuring reliability and safeguard in its application in the planning of rehabilitation by means of osseointegrated implants. material and methods this work used 30 dry adult human skulls of both genders and no age limitations obtained from the department of anatomy of the health sciences center of the federal university of pernambuco, brazil. approval for the study was granted from the ethics committee of the school of dentistry of the university of são paulo, brazil (protocol # 187/03). with the help of reduced copies of a 4-cm-diameter protractor cut and pasted to a white cardboard and placed at every 30 degrees in both orbits of each skull, a clock and number of hours was prepared. the 12 h point corresponded fig. 1 adapted locator in the orbits. points of reference coinciding with the hours of a clock. fig. 2 skull and computed tomography scan of the left orbit. a skull with the demarcated right and left peri-orbital regions; radiopaque points determined in accordance with the marking provided by the locator; b location and delineation of the anatomical points; c scale showing the sequence of reconstructions (from 1 to 60) with the respective correspondence of the radiopaque images of the repair points fixed in each skull. to 90o, 3 h to 0o, 6 h to 270o and 9 h to 1800. in each orbit, the 12 sites that corresponded to the hours of a clock were demarcated, so that point 3 h in the right orbit was always coincident with point 9 h in the left orbit (figure 1). for the ct recording, repair points were set, using a copper wire of 1 mm in diameter and 1 mm in length (figure 2 a and b). the skulls were placed on the elscint select-sp compact spiral ct system (elscint, haifa, israel) and a lowpower laser (645-660 nm wavelength) was applied to assure the right position of the skull on the ct table its red light was coincident with the coronal and sagittal sutures of the skull to be studied. in the workstation, the image protocol was: software dentct; coronal cut; thickness of cut: 1.5 mm; increment: 1.0 mm; pitch: 1.0 mm; gantry angulation: 00. the software was applied to the peri-orbital rim, producing transversal images, obtaining 60 reconstructions. the radiopaque images of the previously established 12 points of repair were recorded on a scale (figure 2c). from the images in the coronal sections of each ct, 2 peri-orbital bone dimensional analysis using computed tomography for placement of osseointegrated implants 33333 braz j oral sci. 11(1):1-9 films were obtained for each orbit. the scale covered a range of all the reconstructions, with the images of the peri-orbital regions arranged from point 12 h to 1 h (figure 3a-d). however, for a correlation of the anatomical areas in the left and right orbits, the transverse section sequence of cuts was set reverse or anticlockwise to the right orbit. the delineations were designed corresponding to the length (longitudinal direction in relation to the radiopaque image of the repair section) and the width (perpendicular direction to the longitudinal delineation length). the evaluation of the left orbits began with the 12 h point, and followed a clockwise direction, while the analysis sequence for the right orbits, also starting at the 12 h point was reversed, in other words, following the anticlockwise direction. in the 7 h, 8 h, 9 h, 10 h and 11 h points, only the measures of length were performed, because the width ones did not have their limits precisely accurate. no measurements were made on point 6, because the region where it was located was close to the infra-orbital foramen, which is of no interest for implant placement. the regions of points 7 h to 11 h and points from 1 h to 5 h, corresponding to the medial wall of the left and right orbits, in which the relative width values were not recorded due to the lack of precision of the ct limits in the transverse cutting section, showed large width dimensions. we therefore consider that the width of this region does not pose any problem for osseointegrated implants. the length and width linear measurements of each point fig. 3 computed tomography scan of the left orbit. images corresponding to the cross-sectional cuts of the peri-orbital. a cuts 13 to 24 b; cuts 25 to 36; c cuts 37 to 48 d; cuts 48 to 60. were performed by two previously calibrated examiners, following a time interval of at least 7 days between the first and second measurement, both for examiner 1 and examiner 2. both ct scans of each orbit were placed on a light box of 1m x 1m, where it was possible to observe the location and delimitation of the anatomical points defined in this study. in each ct image there was a scale corresponding to the cuts made, where the radiopaque images of the 12 previously set repair points were verified. sixty cuts were recorded. the cross section cut to be evaluated was defined by the coincidence of the point of repair with the sequential numbering established in the scale constant in the ct. after defining the cross-section cut, a transparency was placed on the ct using a transparent adhesive tape. then, the tracings corresponding to the length (longitudinal direction in relation to the radiopaque image of the repair point) and width (perpendicular to the longitudinal tracing of the length) were drawn. each measurement was performed using a needle-point compass; the first measurement performed was the length, and the second one was the width. a database consisting of 120 spreadsheets was prepared, with the data obtained by the two examiners, in addition to the two measurements performed by each examiner. data were analyzed by descriptive statistics and the paired student’s t-test was used as inferential technique. the software used to obtain the statistical calculations was sas version 8 (statistical analysis system; sas inc., cary, nc, usa). the significance level was set at 5%. peri-orbital bone dimensional analysis using computed tomography for placement of osseointegrated implants 44444 braz j oral sci. 11(1):1-9 results the results are presented in tables 1 to 5. considering the values for the length of the left periorbital, it was found that the highest averages were recorded at the point corresponding to 1 h, and the lowest at the point corresponding to 10 h, for both examiners and both measurements. the averages of the length measurements obtained in points corresponding to 12 h, 2 h, 3 h, 4h and 5h were higher than those found in points 7 h, 8 h, 9 h, 10 h and 11 h. overall, no significant intra-examiner differences examiner section measure 1 2 value of p (1) average ± dp (1) average ± dp 12 h first 11.18 ± 5.12 11.40 ± 5.11 p = 0.2554 second 11.53 ± 5.16 11.38 ± 4.87 p = 0.3194 value of p p = 0.1007 p = 0.9422 1 h first 18.63 ±5.48 17.92 ±5.08 p = 0.0003* second 18.70 ± 5.45 18.03 ± 5.14 p =0.0007* value of p p = 0.6871 p = 0.5487 2 h first 13.33 ±4.62 12.97 ± 4.65 p = 0.0205* second 13.05 ± 4.44 12.83 ±4.35 p = 0.1411 value of p p = 0.0354* p = 0.5228 3 h first 9.28 ± 2.20 9.18 ± 1.99 p = 0.5407 second 9.20 ± 2.10 8.97 ± 1.97 p = 0.0698 value of p p = 0.6163 p = 0.0850 4 h first 9.30 ± 2.78 9.23 ± 2.59 p = 0.7238 second 9.43 ± 2.93 9.28 ± 2.97 p = 0.3795 value of p p = 0.5171 p = 0.7709 5 h first 9.13 ± 4.64 8.95 ± 4.79 p = 0.2031 second 9.43 ± 4.80 9.23 ± 4.81 p = 0.0563 value of p p = 0.0592 p = 0.0168* 6h 7 h first 3.45 ± 1.33 3.57 ± 1.26 p = 0.4773 second 3.47 ± 1.36 3.65 ± 1.12 p = 0.1176 value of p p = 0.8790 p = 0.5015 8 h first 3.83 ± 1.06 3.98 ± 1.15 p = 0.0951 second 3.83 ± 1.26 4.05 ± 1.22 p = 0.1192 value of p p = 1.0000 p = 0.5362 9 h first 3.97 ± 0.96 3.95 ± 0.98 p = 0.9038 second 3.80 ± 1.13 3.87 ± 0.98 p = 0.5803 value of p p = 0.2383 p = 0.5618 10 h first 3.30 ± 1.06 3.27 ± 0.74 p = 0.7450 second 3.18 ± 1.02 3.13 ± 0.83 p = 0.6300 value of p p = 0.0897 p = 0.2228 11 h first 4.27 ± 2.46 4.32 ± 2.65 p = 0.7354 second 4.17 ± 2.75 4.25 ± 2.68 p = 0.3619 value of p p = 0.3389 p = 0.5256 general first 8.15 ± 5.77 8.07 ± 5.58 p = 0.0673 second 8.16 ± 5.83 8.06 ± 5.58 p = 0.0143* value of p p = 0.8123 p = 0.9212 table 1.table 1.table 1.table 1.table 1. measurements in millimeters and standard deviation of the length of the left peri-orbital bone according to the measurement site, measurement, and examiner. (*) – significant difference at 5.0% level. (1) – paired student’st-test. were observed, as well as inter-examiners in the first measurement, although significant inter-examiner differences occurred at the 5% level in the second measurement (table 1). the highest averages regarding the width of the left peri-orbital bone occurred in point 12 h, and the lowest in point 2 h, with points 1 h, 3 h, 4h and 5h showing intermediate values. intra-examiner differences were only recorded in points 4h and 5 h, for examiner 1. there were significant inter-examiner differences in points 12 h, 4h and 5 h, in general, for the 1st measurement. overall, significant differences were found at level 5%, between the 1st and 2nd peri-orbital bone dimensional analysis using computed tomography for placement of osseointegrated implants braz j oral sci. 11(1):1-9 55555 measurement for examiner 1 and inter-examiners during the 1st measurement (table 2). with regard to the length of the right peri-orbital, the highest mean occurred in the point corresponding to 11 h, and the lowest in point 2h. except for point 10 h, there was no statistically significant intra-examiner difference. however, there were significant inter-examiner differences (p<0.05) for both measurements in points 11 h, 3 h and 1 h, and in the second measurement in the points corresponding to 9 h, 8 h, 5 h and 4 h. in general, there was a significant intraexaminer difference at 5% level for both examiners, but there is no statistical significance in the inter-examiner variation for both measurements (table 3). for the width of the right peri-orbital, the highest mean values were observed for the evaluations of point 8 h, and the lowest for the evaluation of point 10 h. there were statistically significant intraexaminer differences (p<0.05) in the measurement of points 12 h and 8 h, and in the overall average for both examiners. in the inter-examiner variation, there was a significant difference (p<0.05) in both measurements in point 12 h, in the 1st measurement of point 9 h, and in the 2nd measurement of point 8 h. considering the general values, there were also significant inter-examiner differences(p<0.05) in both 1st and the 2nd measurements (table 4). examiner section measure 1 2 value of p (1) average ± dp average ± dp 12 h first 8.18 ± 2.77 8.50 ± 2.64 p = 0.0234* second 8.60 ± 2.62 8.63 ± 2.54 p = 0.8252 value of p p = 0.0929 p = 0.6183 1 h first 6.83 ± 2.29 6.97 ± 2.30 p = 0.2927 second 7.02 ± 1.97 7.23 ± 2.22 p =0.2054 value of p p = 0.3665 p = 0.2329 2 h first 6.03 ± 1.58 5.92 ± 1.54 p = 0.3791 second 6.15 ± 1.54 6.08 ± 1.72 p = 0.6839 value of p p = 0.4282 p = 0.3305 3 h first 7.22 ± 1.79 7.27 ± 1.87 p = 0.6875 second 7.30 ± 1.55 7.23 ± 1.55 p = 0.6139 value of p p = 0.5776 p = 0.8881 4 h first 7.08 ± 1.88 7.30 ± 1.97 p = 0.0509* second 7.47 ± 2.13 7.58 ± 2.08 p = 0.3944 value of p p = 0.0193* p = 0.1432 5 h first 7.03 ± 2.33 7.40 ± 2.38 p = 0.0063* second 7.38 ± 2.32 7.50 ± 2.10 p = 0.4954 value of p p = 0.0421* p = 0.6675 general first 7.06 ± 2.21 7.32 ± 2.16 p = 0.0040* second 7.21 ± 2.26 7.38 ± 2.18 p = 0.3486 value of p p = 0.0005* p = 0.0729 table 2.table 2.table 2.table 2.table 2. measurements in millimeters and standard deviation of the width measurements of the left peri-orbital bone according to the measurement site, measurement, and examiner. (*) – significant difference at 5.0% level. (1) – paired student’s-t test. in this work, the greatest bone length was observed in the points corresponding to 1h (18.32 mm) 11h (19.28 mm), with an average of 18.80 mm, considering the left and right orbits, respectively. for the left orbit, the points 2 h and 12 h, which had an average of 13.05 mm and 11.37 mm, respectively, the same anatomical sequence was observed for the right orbit, with values of 12.34 mm and 11.56 mm, respectively (table 5). discussion few works have been conducted to verify the length and thickness measurement of facial regions, focusing on the installation of implant-supported facial prostheses2,14-18. jensen et al.15 (1992) assessed the available bone volume in 16 places of the skull-facial skeleton of 15 human skulls using a caliper in two places in the supra-orbital rim; 2 in the lateral margin of the orbit, and 1 on the infra-orbital edge, showed that the mean bone length in these places were of 4.4 mm and 4.6 mm; 5.9 mm and 6.1 mm; and 5.4 mm, respectively. in the study of klein et al.10 (1997), the use of ct scan indicated the following values: medial infra-orbital: 2 mm; lateral infra-orbital: 6 mm; medial supra-orbital: 3 mm; lateral supra-orbital: 8 mm; medial region of the orbit: peri-orbital bone dimensional analysis using computed tomography for placement of osseointegrated implants braz j oral sci. 11(1):1-9 66666 examiner section measure 1 2 value of p (1) average ± dp average ± dp 12 h first 11.75 ± 4.99 11.55 ± 5.27 p = 0.1668 second 11.57 ± 5.09 11.37 ± 5.02 p = 0.1953 value of p p = 0.1254 p = 0.2156 11 h first 19.50 ± 3.95 19.08 ± 3.70 p = 0.0086* second 19.50 ± 3.81 19.03 ± 3.92 p =0.0100* value of p p = 1.0000 p = 0.8260 10 h first 12.65 ±4.94 12.45 ± 4.57 p = 0.3514 second 12.30 ± 4.81 11.97 ±4.51 p = 0.0503 value of p p = 0.0101* p = 0.0094* 9 h first 8.70 ± 2.00 8.60 ± 1.93 p = 0.3854 second 8.72 ± 1.76 8.40 ± 1.81 p = 0.0234* value of p p = 0.9038 p = 0.2061 8 h first 10.43 ± 3.12 10.17 ± 3.11 p = 0.1219 second 10.35 ± 2.98 9.82 ± 2.78 p = 0.0022* value of p p = 0.6460 p = 0.1007 7 h first 8.62 ± 4.75 8.68 ±4.54 p = 0.6453 second 8.77 ± 4.75 8.52 ± 4.49 p = 0.1694 value of p p = 0.2221 p = 0.3781 6h 5 h first 3.90 ± 1.33 3.88 ±1.38 p = 0.8973 second 3.82 ± 1.41 4.10 ± 1.40 p = 0.0168* value of p p = 0.4844 p = 0.0677 4 h first 3.45 ± 0.72 3.63 ± 0.67 p = 0.0777 second 3.38 ± 0.67 3.62 ± 0.60 p = 0.0457* value of p p = 0.5803 p = 0.8759 3 h first 3.90 ± 1.00 4.22 ± 1.20 p = 0.0234* second 3.75 ± 1.06 4.08 ± 1.16 p = 0.0245* value of p p = 0.2638 p = 0.3974 2 h first 3.25 ± 0.97 3.32 ± 0.79 p = 0.6078 second 3.07 ± 1.05 3.18 ± 0.70 p = 0.3791 value of p p = 0.0858 p = 0.1033 1 h first 3.52 ± 1.98 3.83 ± 1.84 p = 0.0277* second 3.45 ± 1.84 3.77 ± 1.68 p = 0.0046* value of p p = 0.5642 p = 0.5362 general first 8.15 ± 5.86 8.13 ± 5.66 p = 0.6095 second 8.06 ± 5.84 7.95 ± 5.52 p = 0.0504 value of p p = 0.0212* p = 0.0018* table 3.table 3.table 3.table 3.table 3. measurements in millimeters and standard deviation of the length of the right peri-orbital bone according to the measurement site, measurement, and examiner. (*) – significant difference at 5.0% level. (1) – paired student’st-test. 2 mm; side of the orbit: 10 mm. olate et al.14 (2011) measured the peri-orbital bone region of 40 dry skulls, using the data obtained from cone beam cts virtually reconstructed. the authors divided the orbit in superior and inferior, and each one in lateral, intermediary and medial regions. the thickness data verified, for the superior orbit, were: lateral region:7.5 mm, intermediary region: 8.31 mm, medial region: 9.23 mm. for the inferior orbit, the obtained data were: lateral region: 7.62 mm, intermediary region: 6.51 mm, medial region: 6.11 mm. studies comparing linear or volumetric measures of craniometrical sections using ct, and performing manual measures in dry skulls, cadaverous heads, including imaging data of patients, showed no statistically significant differences between the manual direct measurement method and the method that uses cts19-21. in this study, the greatest bone lengths were observed in the points corresponding to 1 h (18.32 mm), for the left orbit, and to 11 h (19.28 mm), for the right orbit, with a mean value of 18.80 mm. these findings are compatible with those of matsuura et al.16 (2002), who also found the greatest lengths in the point 1 h, with mean value of 16.00 mm. for the left orbit, the second and third major lengths found in this study were verified in the points 2 h and 12 h, which showed 13.05 mm and 11.37 mm, respectively. the same anatomical sequence was observed for the right orbit, with values of 12.34 mm and 11.56 mm, respectively. in the present work, peri-orbital bone dimensional analysis using computed tomography for placement of osseointegrated implants 77777 braz j oral sci. 11(1):1-9 examiner section measure 1 2 value of p (1) average ± dp average ± dp 12 h first 7.33 ± 2.29 7.62 ± 2.33 p = 0.0125* second 7.82 ± 2.20 8.20 ± 2.33 p = 0.0032* value of p p = 0.0056* p = 0.0169* 11 h first 6.97 ± 2.09 7.10 ± 2.04 p = 0.4591 second 7.23 ± 1.60 7.47 ± 1.77 p =0.1334 value of p p = 0.2142 p = 0.0617 10 h first 5.88 ± 1.42 5.83 ± 1.51 p = 0.6757 second 6.02 ± 1.37 6.10 ± 1.52 p = 0.5378 value of p p = 0.2838 p = 0.1256 9 h first 6.93 ± 1.36 7.22 ± 1.30 p = 0.0072* second 7.15 ± 1.35 7.38 ± 1.23 p = 0.0947 value of p p = 0.1671 p = 0.3305 8 h first 7.73 ± 2.22 7.85 ± 2.30 p = 0.3791 second 8.27 ± 2.17 8.77 ± 2.27 p = 0.0016* value of p p = 0.0067* p = 0.0001* 7 h first 6.77 ± 2.39 7.03 ± 2.33 p = 0.1105 second 6.97 ± 2.27 7.07 ± 2.10 p = 0.6338 value of p p = 0.3115 p = 0.9045 general first 6.94 ± 2.06 7.09 ± 2.11 p = 0.0065* second 7.24 ± 1.97 7.50 ± 2.07 p < 0.0001* value of p p < 0.0001* p < 0.0001* table 4.table 4.table 4.table 4.table 4. measurements in millimeters and standard deviation of the width of the right peri-orbital bone according to the measurement site, measurement, and examiner. (*) – significant difference at 5.0% level. (1) – paired student’st-test. table 5. table 5. table 5. table 5. table 5. measurements of length and width of the right and left periorbital bone * means in millimeters sections left peri-orbital bone right peri-orbital bone average length width length width length width 12 h – 12 h 11.37 8.48 11.56 7.74 11.47 8.11 1 h – 11 h 18.32 7.01 19.28 7.19 18.80 7.10 2 h – 10 h 13.05 6.05 12.34 5.96 12.70 6.01 3 h – 9 h 9.16 7.25 8.60 7.17 8.88 7.21 4 h – 8 h 9.31 7.36 10.19 8.15 9.75 7.75 5 h – 7 h 9.19 7.32 8.64 6.96 8.92 7.14 6 h – 6 h 7 h – 5 h 3.53 3.22 3.38 8 h – 4 h 3.92 3.52 3.72 9 h – 3 h 3.90 3.99 3.94 10 h – 2 h 3.22 3.20 3.21 11 h – 1 h 4.25 3.64 3.95 point 12 h presented one of the greatest lengths, what corroborates with the findings of matsuura et al.16 (2002), that employed cadaveric heads in their study. thus, it is observed that the three points with the greatest lengths are located in the supra-orbital rim, which is also substantiated by matsuura et al.16 (2002). olate et al.14 (2011) found the greatest thickness in the medial region of the superior orbit – 9.23 mm. the shift towards the side wall of the orbit, which corresponds to points 3 h-9 h, 4 h-8 h and 5 h-7 h, in the left and right orbits, shows mean values of 8.88 mm, 9, 70 mm and 8.92 mm, respectively. although lower, these mean values are compatible with the use of intraoral osseointegrated implants. these points correspond to the lateral and infraperi-orbital bone dimensional analysis using computed tomography for placement of osseointegrated implants braz j oral sci. 11(1):1-9 lateral wall of the orbit. it is important to consider that longer intraoral implants have been used in the orbital region to support orbital prosthesis1,22. in the same way as in the present study, klein et al.10 (1997) found that the points with the greatest length are located in the lateral-top and orbit lateral regions. the lowest values reported by jensen et al.15 (1992) for the supra-orbital rim lengths (4.4 mm and 4.6 mm) and the lateral wall of the orbit (5.4 mm, 5.9 mm and 6.1 mm) are probably due to the methodology used by those authors (they measured directly the skull using a caliper, while the measurements performed in the present work were made in cts). a large variability in the bone depth around the frontal zygomatic suture has been reported18. reher and duarte 17 (1994), concerned with the appearance of the bone depth, recommend the use of 5-mm and 7-mm-long screws, in mini osteosynthesis plates, above and below the frontal zygomatic suture. this region, which is located on the lateral wall of the orbit, in this paper, show consistent mean values using 7mm-long osseointegrated implants. the point with the lowest length was coincident in both orbits, corresponding to 10 h – 2 h and presenting values of 3.22 mm and 3.20 mm, respectively. these findings are close to those of matsuura et al.10 (2002), who found the lowest bone length (3.0 mm) for point 10 h, as well as those of klein et al.10 (1997) for the medial infra orbital region. with less bone depth, this region represents a less important area in the planning of rehabilitating implant-supported prosthetics in ocularpalpebral prosthesis. in cases of extreme need, this region must receive lesser-length osseointegrated implants. with regard to the width, the points with the greatest values were not coincident between the orbits, corresponding to 12 h for the left orbit (8.48 mm) and 8 h for the right orbit (8.15 mm). however, the immediate inferior values are located in the same correspondent points for the left and right orbits the left orbit showed a mean of 7.36 mm at 4 h (point corresponding to the 8 h point in the right orbit), and the right orbit, a mean of 7.74 mm at 12h. generally, the mean width in point 12 h was of 8.11 mm, greater than those presented by points 4 h and 8 h (7.75 mm). these points correspond to the supra-orbital edge and to the lateral inferior region of the orbit, which as a result of these measurements do not constitute risk locations for the placement of osseointegrated implants. in this work, the width values ranged from 5.96 mm to 8.48 mm, with the lowest values for points 2h (6.05) and 10 h (5.96 mm). matsuura et al.16 (2002) reported values that ranged from 6.8 mm to 11.1 mm, with the lowest value for point 2 h (6.8 mm). the variation of the orbital margin width in this work (3.52 mm) is lower than that reported by matsuura et al.16 (2002) (4.3 mm) and klein et al.10 (1997) (5 mm). the regions of points 7 h to 11 h and points 1 h to 5 h, corresponding to the medial walls of the left and right orbits, for which no width values were recorded due to the lack of accuracy on the ct limits in cross-section cut, showed large width dimensions. thus, we believe that the width of this region does not pose any difficulty for indicating osseointegrated implants. the intraand inter-examiner reading differences in tables 1-4, it appears that, considering a significant difference at the level of 5.0%, there were few differences, given the readings made. these results indicate good accuracy in the data readings between both measurements or between both examiners. we assume that the importance of ct in verifying the bone measurements is indisputable, in light of craniofacial implant placement. the measurement method of peri-orbital regions in ct, proposed in this study, is valid and can be clinically used in the planning of osseointegrated implants in the peri-orbital region. it is essential that the mean values observed in this work are not seen as determinant in defining the length of the implant to be used. the absolute values should serve only as an indication of the bone receptor sites that are more favorable to the installation of osseointegrated implants, keeping in mind the individuality of each patient. the planning of implant-retained prosthetics rehabilitation in the cranial-facial region requires the implementation of cts in all cases, the reason for assessing this method, hence providing information to help in the planning of prosthetic surgery. the determination of the sites depends not only on the bone conditions of the receptor site of the implants, but also on the prosthetic planning, keeping in mind that prosthetic rehabilitation is ultimately the greater goal for the treatment proposed. only the perfect integration of a multidisciplinary team enables the development of a successful rehabilitation process, which is ultimately reflected in safeguarding our patients’ quality of life. acknowledgements to prof. marcos frazão, oral and maxillofacial radiologist and to the boris berenstein diagnosis center, recife, pe. brazil, for the computerized tomographies. to the department of anatomy of the health sciences center – pernambuco federal university, for the skulls used in this work. references 1. goiato mc, santos dm, dekon sfc, pellizzer ep, santiago jr. jf, moreno a. craniofacial implants success in facial 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vannier mw. validation of spiral computed tomography for dental implants. dentomaxillofacial radiol. 1998; 27: 329-33. 20. cavalcanti mg, rocha ss, vannier mw. craniofacial measurements based on 3d-ct volume rendering: implications for clinical applications. dentomaxillofacial radiol. 2004; 33: 170-6. 21. fonteles sms. estudo comparativo da mensuração em cortes tomográficos lineares por métodos informatizados e manual [thesis]. piracicaba: faculdade de odontologia de piracicaba, universidade estadual de campinas; 2002. 22. kovacs af. a follow-up study of orbital prostheses supported by dental implants. j oral maxillofac surg. 2000; 58: 19-23. peri-orbital bone dimensional analysis using computed tomography for placement of osseointegrated implants oral sciences n3 original article braz j oral sci. january | march 2011 volume 10, number 1 comparison of root canals obturated with protaper gutta-percha master point using the active lateral condensation and the single-cone techniques: a bacterial leakage study anizabele milet do amaral mercês1, carlos menezes aguiar2, neide kazue sakugawa shinohara3, andréa cruz câmara4, jose antonio poli de figueiredo5 1private practice, recife, brazil 2associate professor, department of prosthodontics and oral and maxillofacial surgery, dental school, federal university of pernambuco, brazil 3adjunt professor, keizo asami immunopalogy laboratory, federal university of pernambuco, brazil 4substitute professor, department of prosthodontics and oral and maxillofacial surgery, dental school, federal university of pernambuco, brazil 5postgraduate program in dentistry, pontifical catholic university of rio grande do sul, pucrs, brazil correspondence to carlos menezes aguiar rua aristides muniz, 70/501, boa viagem, recife pe 51020-150 brazil. phone: (+55) 81 3467 6821. e-mail: cmaguiar.ufpe@yahoo.com.br received for publication: august 26, 2010 accepted: december 16, 2010 abstract aim: the aim of the present study was to assess the recontamination time of root canals filled with protaper gutta-percha master point using the active lateral condensation and the single-cone techniques. methods: fifty premolars, after biomechanical preparation, were randomly divided into six groups according to the obturation technique and the diameter of the gutta-percha point. for leakage evaluation, the roots were mounted in a dual-chamber leakage model system using pseudomonas aeruginosa, enterococcus faecalis, escherichia coli, and staphylococcus aureus. the kaplan-meier and long-rank tests were employed at the .05 level of significance. results: the recontamination was detected between the 13th and 80th days. the active lateral condensation produced less infiltration than the single cone technique, though without statistically significant differences between them. conclusions: it may be concluded that there was no difference in bacterial penetration between the two root canal filling techniques. the mean root canal contamination time was 67 days. keywords: gutta-percha, microleakage, protaper gutta-percha master point, protaper universal, single-cone technique. introduction successful root canal treatment requires proper cleaning and shaping of root canal, as well as hermetic sealing of root canal space with an inert, dimensionally stable, and biologically compatible material1. when the coronal portion of the root canal is exposed to the oral environment, the root canal is a potential route for microorganisms to gain access to the periapical tissue. this situation may lead to endodontic failure2. from a clinical standpoint, coronal exposure of the root canal obturation might be considered an indication for endodontic retreatment3. braz j oral sci. 10(1):37-41 38 the influence of the quality of the coronal restoration is an important factor in achieving success in root canal treatment. the teeth with a satisfactory coronal restoration displayed significantly better periapical healing than those with an unsatisfactory restoration4-5. radiographically, the technical quality of endodontic treatment is significantly more important than the technical quality of the coronal restoration when the periapical status of the endodontically treated teeth is evaluated 6. the biomechanical preparation with nickel-titanium (niti) rotary system was considerably quicker than the hand instrument series7. it would appear that rotary niti instruments have the potential to prepare root canals more efficiently, and to shape root canals with greater safety8, achieving a three-dimensional obturation of the root canal over its whole length without the time spent on lateral condensation9. cleaned and shaped root canals must be filled threedimensionally, eliminating the empty space, which has the potential to be infected or reinfected10 . the aims of the root canal sealer are to prevent penetration of bacteria and eliminate the risks of infection or reinfection of the root canal system3. given the importance of the root canal filling and the control and prevention of endodontic infection, several studies have been published on the subject 10-12. coronal leakage has been evaluated by different tests, among which is the analysis of bacterial penetration through the filling material. this method has been used because it has reached results similar to those obtained in daily practice13. the purpose of this study was to investigate the time required for recontamination of root canals sealed with protaper gutta-percha cones after obturation with two different techniques and exposure to a simulated oral environment. material and methods specimen selection and preparation and root canal filling fifty intact, caries-free, human mandibular premolars with single roots and mature apices (mean length of 21 mm) were used. the teeth were obtained from the tooth bank of the department of prosthodontics and oral and maxillofacial surgery of the federal university of pernambuco, after approval of the research ethics committee of the university’s center of health sciences. buccolingual and mesiodistal were radiographs were taken to confirm the presence of a single canal. once coronal access had been established, a k-file #10 (dentsply/maillefer, ballaigues, switzerland) was inserted into the root canal until the tip was just visible beyond the apex. the working length (wl) was determined at 1 mm short of this length. the root canals were prepared by the protaper universal™ rotary system (dentsply/maillefer, ballaigues, switzerland). in groups 1 and 2, the root canals were instrumented up to instrument f4, and in the groups 3 and 4 the canals were prepared up to instrument f5. a single operator instrumented all root canals. the root canals were irrigated with a freshly prepared 1% sodium hypochlorite solution (roval, recife, brazil) in conjunction with ethylenediaminetetraacetic acid (biodinâmica, paraná, brazil) as a chelating agent for irrigating the root canals, as well as the 3-ml fcf syringe system (fcf, sao paulo, brazil) with a 30-gauge needle (injecta, diadema, brazil). the irrigation was performed at the beginning of the instrumentation, between the changes of instrument, and at the end of the biomechanical preparation. the specimens were stored in glass test tubes and were individually sterilized in an autoclave at 121°c for 30 minutes. ten samples were randomly chosen and immersed totally in bottles containing 10 ml autoclaved brain heart infusion (bhi; acumedia, lansing,mi). they were kept in an incubator at 37°c for 96 hours to check the sterilization’s efficacy. after preparation, the specimens were randomly divided into six groups as follows: group 1: 10 specimens were filled using the single cone technique with the gutta-percha master point f4 (dentsply/maillefer, ballaigues, switzerland) and ah plus sealer (dentsply detrey, konstanz, germany). group 2: 10 specimens were filled using the active lateral condensation technique with the gutta-percha master point f4 and 10 ff accessory cones (dentsply/maillefer, ballaigues, switzerland) and ah plus sealer . group 3: consisted of 10 specimens filled using the single cone technique with the gutta-percha master point f5 and ah plus sealer. group 4: 10 specimens were filled using the active lateral condensation technique with the gutta-percha master point f5 and 10 ff accessory cones and ah plus sealer. group 5 (positive control group): consisted of 5 specimens that did not have the canal filled. group 6 (negative control group): consisted of 5 specimens that did not have the canal filled but was rendered impermeable by receiving an epoxy resin coating (araldite,brascola, são bernardo do campo, brazil) along its entire length. three blinded endodontists evaluated the root fillings on radiographs in accordance with the criteria of eckerbom and magnusson14. leakage apparatus preparation the apparatus used to evaluated saliva leakage was prepared as previously described by gomes et al.11, with modifications. glass vials with rubber stoppers were adjusted for use in this experiment. using a heated instrument, a hole was made through the center of every rubber stopper in which each tooth was inserted under pressure up to its cementoenamel junction, so that its crown lay outside and its root inside the vial. the teeth were sealed with three layers: a layer of cyanoacrylate (henkel ltda, são paulo, brazil), a layer of epoxy resin (pulvitec s.a. indústria e comércio, são paulo, brazil) and a final layer of cyanoacrylate were applied at the interface between the tooth and rubber. thereafter the roots were sealed with two layers of cyanoacrylate, except for the apical 2 mm around the apical foramen. the cylinders prepared from 5-ml plastic syringes comparison of root canals obturated with protaper gutta-percha master point using the active lateral condensation and the single-cone techniques: abacterial leakage study braz j oral sci. 10(1):37-41 39 (plascap, são paulo, brazil) were adapted on the external surface of the stoppers to create a chamber around the crown of the tooth. the junction between the plastic cylinders and glass tube was sealed as described above. the apparatus was then sterilized with ethylene oxide gas (galdi produtos hospitalares, paulista – brazil) for 12 h. the chamber of each apparatus was filled with bhi, so that approximately 2 mm of the root apex was immersed in the broth. to ensure sterilization, no growth was observed after the whole apparatus was incubated at 37ºc±1 for four days. any test apparatus that showed signs of turbidity in the bhi broth was discarded. bacterial leakage test the bacterial stains used for analysis were pseudomonas aeruginosa (american type culture collection – atcc 9027), enterococcus faecalis (atcc 19433), escherichia coli (atcc 8739) and staphylococcus aureus (atcc 6538). the microorganisms used in this study were selected because of their clinical importance and association with the endodontic infection10,15. the chamber of each apparatus was filled with 3 ml of sterile artificial human saliva (farmácia roval de manipulações, recife, brazil) and mixed in bhi broth in a 3:1 (v/v) ratio. the saliva was replenished every 3 days for 3 months. the turbidity of the inocula was adjusted to approximately 108 ufc/ml, this concentration being obtained using the neubauer chamber technique16. the apparatus was incubated at 35°c ± 1 and checked daily for the appearance of turbidity in the bhi broth over a period of 90 days. the number of days it took for bacterial growth to appear was indicative of the total recontamination of the root canal by bacteria from the saliva. following the appearance of broth turbidity, aliquots were removed and sowed in bhi, agar blood, agar levine, agar macconkey and agar chapman, and the plates were incubated in a cabinet at 35oc ± 1 for 24 hours. the bacterial identification was based on colony features, gram-staining and cell morphology: catalase production test, triple sugar iron agar test (tsi), growth in 6.5% nacl bhi broth, esculin bile agar, and hugh-leifson’s test, constituting the complementary identification tests17. statistical analysis the statistical analysis was performed using the kaplan– meier test for survival analysis, which includes calculation of the median time leakage and pairwise comparisons of groups. the long-rank test was used to compare the survival curves of the groups. a level of significance of .05 was adopted. results all specimens in the positive control group showed broth turbidity within 13 days of incubation. by contrast, none of the negative control specimens leaked during the experimental period (90 days), confirming the efficacy of rendering the whole root surface waterproof (figure 1). four specimens in group 1 showed contamination at 36 days. the first specimen that showed microbial leakage was observed at 13 days. in group 2, 1 sample showed contamination at 61 days. two specimens in group 3 showed contamination at 41 days. the first specimen that showed microbial leakage was observed at 34 days. in group 4, 2 samples showed contamination at 46 days. the first specimen that showed microbial leakage was observed at 34 days. there was no significant statistical difference among the tested techniques. the results of the kaplan-meier test are shown in table 1. group mean contamination standard error confidence interval time (days) 1 67 10 (47; 88) 2 87 3 (82; 92) 3 79 7 (66; 93) 4 80 7 (67; 93) 5 11 0 (11; 12) 6 90 0 table 1 contamination time of each group. the long-rank test revealed no statistically significant differences between the root canal filled techniques and the diameters of the gutta-percha master points employed. discussion methods to measure microleakage have included the use of dyes18, human saliva19, microorganisms20 and bacterial toxins21. the inadequacies of each of these methods have been highlighted and their clinical significance questioned. although the use of dyes, radioisotopes, fluid filtration, bacteria, and endotoxins penetration techniques to evaluate sealing capacity of endodontic materials, the bacteria leakage model has proved to be the most clinically important one12,22. facultative anaerobic and aerobic microorganisms have comparison of root canals obturated with protaper gutta-percha master point using the active lateral condensation and the single-cone techniques: abacterial leakage study braz j oral sci. 10(1):37-41 fig. 1number of teeth contaminated at 90 days. been used, although they are found in only 10 to 20% of root canals with a chronic periapical reaction. such microorganisms are found interacting with strict anaerobes, causing changes in nutritional relationships, shifts in redox potential and oxygen tension, thereby establishing the microbial survival relationship23. determining the sealing capacity of endodontic materials against these microorganisms is essential. the endodontic sealers showed degradation with oral fluids. ah plus showed the smallest weight loss in water and artificial saliva with different ph values24. this is because ah plus is an epoxy resin-based sealer, and provides a tight seal of the root canal system1. these properties justified its choice and use in this study, corroborating the findings of tronstad et al.6. complete obturation of the root canal system is an important factor in achieving success in root canal therapy. ideally, root canal filling should be a complete, homogenous mass that fills the prepared root canal completely. the conventional root-filling technique taught in most dental schools25 and used by dentists26 has been the cold lateral compaction technique using the .02 tapered gutta-percha point as the master cone, supplemented with accessory cones. however, several practitioners regard it as time-consuming and difficult to master. the alternative to root canal filling is the single-cone technique9. this tendency to optimize endodontic treatment led to the evaluation of the sealing capacity of these techniques, using the protaper gutta-percha master points. the teeth obturated using the vertical compaction technique exhibited a dense fill throughout the canal space with the aim of preventing the leakage of microorganisms20. the results of the present study showed no statistically significant differences between the methods employed. although the cold lateral compaction of gutta-percha has been regarded as the gold-standard obturation technique, the results of the research were in agreement with those of britto et al.27, which showed microleakage of microorganisms. yücel et al.3 demonstrated that all samples obturated with cold lateral compaction showed bacterial penetration after 60 days of observation. their results are at odds with those observed in the present study, in which 15% of the specimens showed bacterial leakage after 60 days. the reason for the discrepancies between the two studies was determined by enlarging the apices. at 90 days, bacterial penetration was observed in 22.5% of all the specimens, in contrast with the results of ricucci and bergenholtz28, who demonstrated that well-prepared filled root canals resist bacterial penetration even after long-standing oral exposure to caries, fracture or loss of restoration. although west29 stated that apical enlargement with #40 or #50 files ensures maximum cleaning of the foraminal constriction and increases apical sealing, the results of the present study showed no significant differences between apical enlargement and apical seal. the present study demonstrated that there is no filling material or root canal filling technique able to completely prevent the entry of microorganisms into the root canal space when the teeth are in an oral environment. these results are in agreement with the findings of timpawat et al.1, britto et al.27, yücel et al.3, and monticelli et al.22. this study showed that an increased apical diameter did not hinder bacterial penetration, neither did it minimize root canal recontamination time. the cold lateral compaction and single-cone technique appears to be effective in prevent the leakage of microorganisms. it may be concluded that there was no difference in bacterial penetration between the root canal filling techniques. the mean root canal contamination time was 67 days. acknowledgements this study was supported by grants from conselho nacional de desenvolvimento científico e tecnológico-cnpq-brazil. the authors wish to express their special thanks to professor josé luiz de lima filho and professor maria elizabeth cavalcante chaves, from lika (laboratório de imunopatologia keizo asami) for their valuable technical support. references 1. timpawat s, amornchat c, trisuwan wr. bacterial coronal leakage after obturation with three root canal sealers. j endod. 2001; 27: 36-9. 2. wolanek ga, loushine rj, weller rn, kimbrough wf, volkmann kr. in vitro bacterial penetration of endodontically treated teeth coronally sealed with a dentin bonding agent. j endod. 2001; 27: 354-7. 3. yücel aç, güler e, güler au, ertas e. bacterial penetration after obturation with four different root canal sealers. j endod. 2006; 32: 890-3. 4. shipper g, teixeira fb, arnold rr, trope m. periapical inflammation after coronal microbial inoculation of dog roots filled with gutta-percha or resilon. j endod. 2005; 31: 91-6. 5. ng yl, mann v, rahbaran s, lewsey j, gulabivala k. outcome of primary root canal treatment: systematic review of the literature – part 2. influence of clinical factors. int endod j. 2008; 41: 6-31. 6. tronstad l, asbjørnsen k, doving l, pedersen i, eriksen hm. influence of coronal restorations on the periapical health of endodontically treated teeth. endod dent traumatol. 2000; 16: 218-21. 7. gluskin ah, brown dc, buchanan ls. a reconstructed computerized tomographic comparison of ni–ti rotary gt™ files versus traditional instruments in canals shaped by novice operators. int endod j. 2001; 34: 476-84. 8. câmara ac, aguiar cm, figueiredo jap de. assessment of the deviation after biomechanical preparation of the coronal, middle, and apical thirds of root canals instrumented with three hero rotary systems. j endod. 2007; 33: 1460-3. 9. gordon mpj, love rm, chandler np. an evaluation of .06 tapered guttapercha cones for filling of .06 taper prepared curved root canals. int endod j. 2005; 38: 87-6. 10. siqueira jf, jr, favieri a, gahyva smm, moraes sr, lima kc, lopes hp. antimicrobial activity and flow rate of newer and established root canal sealers. j endod. 2000; 26: 274-7. 11. gomes bpfa, sato e, ferraz ccr, teixeira fb, zaia aa, souza-filho fj. evaluation of time required for recontamination of coronally sealed canals medicated with calcium hydroxide and chlorhexidine. int endod j. 2003; 36: 604-9. 12. saleh m, ruyter ie, haapasalo m, ørstavik d. bacterial penetration along different root canal filling materials in the presence or absence of smear layer. int endod j. 2008; 41: 32-40. 40comparison of root canals obturated with protaper gutta-percha master point using the active lateral condensation and the single-cone techniques: abacterial leakage study braz j oral sci. 10(1):37-41 13. shipper g, ørstavik d, teixeira fb, trope m. an evaluated of microbial leakage in roots filled with thermoplastic synthetic polymer-based root canal filling material. j endod. 2004; 30: 342-7. 14. eckerbom m, magnusson t. evaluation of technical quality of endodontic treatment – reliability of intraoral radiographs. endod dent traumatol. 1997; 13: 259-64. 15. gomes bpfa, pinheiro et, gadê-neto cr, sousa el, ferraz cc, zaia aa et al. microbiological examination of infected dental root canals. oral microbiol immunol. 2004; 19: 71-6. 16. stecher b, robbiani r, walker aw, westendorf am, barthel m, kremer m et al. salmonella enterica serovar typhimurium exploits inflammation to compete with the intestinal microbiota. plos biol. 2007; 5: 2177-89. 17. koneman ew, allen sd, janda wm, schreckenberger pc, winn wc, editors. color atlas and textbook of diagnostic microbiology. philadelphia: lippincott williams & wilkins; 2005, p.1773. 18. barthel cr, moshonov j, shuping g, ørstavik d. bacterial leakage versus dye leakage in obturated root canals. int endod j. 1999; 32: 3705. 19. siqueira jf, rôças in, lopes hp, uzeda m de. coronal leakage of two root canal sealers containing calcium hydroxide after exposure to human saliva. j endod. 1999; 25: 14-6. 20. gilbert sd, witherspoon de, berry cw. coronal leakage following three obturation techniques. int endod j. 2001; 34: 293-9. 21. williamson ae, dawson dv, drake dr, walton re, rivera em. effect of root canal filling/sealer systems on apical endotoxin penetration: a coronal leakage evaluation. j endod. 2005; 31: 599-4. 22. monticelli f, sadek ft, schuster gs, volkmann kr, looney sw, ferrari m et al. efficacy of two contemporary single-cone filling techniques in preventing bacterial leakage. j endod. 2007; 33: 310-3. 23. leonardo mr, silva lab da, tanomaru filho m, bonifácio kc, ito iy. in vitro evaluation of antimicrobial activity of sealers and pastes used in endodontics. j endod. 2000; 26: 391-4. 24. schäfer e, zandbiglarit t. solubility of root-canal sealers in water and artificial saliva. int endod j. 2003; 36: 660-9. 25. qualtrough aje, whitworth jm, dummer pmh. preclinical endodontology: an international comparison. int endod j. 1999; 32: 406-14. 26. hörsted-bindslev p, andersen ma, jensen mf, nilsson jh, wenzel a. quality of molar root canal fillings performed with the lateral compaction and the single-cone technique. j endod. 2007; 33: 468-71. 27. britto lr, grimaudo nj, vertucci fj. coronal microleakage assessed by polymicrobial markers. j contemp dent pract. 2003; 15: 1-10. 28. ricucci d, bergenholtz g. bacterial status in root-filled teeth exposed to the oral environment by loss of restoration and fracture or caries – a histobacteriological study of treated cases. int endod j. 2003; 36: 787-802. 29. west j. progressive taper technology: rationale and clinical technique for the new protaper universal system. dent today. 2006; 25: 66-9. 41 comparison of root canals obturated with protaper gutta-percha master point using the active lateral condensation and the single-cone techniques: abacterial leakage study braz j oral sci. 10(1):37-41 oral sciences n3 braz j oral sci. 11(4):475-480 original article braz j oral sci. october | december 2012 volume 11, number 4 clinical status of permanent first molars in children aged seven to ten years in a brazilian rural community lara jansiski motta1*, joyce garcia dos santos2*, thays almeida alfaya3*, carolina cardoso guedes4*, camila haddad leal de godoy5*, sandra kalil bussadori6* 1phd, professor of pediatric dentistry, department of rehabilitation sciences graduation program, nove de julho university (uninove), são paulo, sp, brazil 2dds, private practice, department of rehabilitation sciences post graduation program, nove de julho university (uninove), são paulo, sp, brazil 3dds, student of dental clinic graduation program, fluminense federal university (uff), niterói, rj, brazil 4ms, professor of pediatric dentistry, braz cubas university, são paulo, sp, brazil 5dds, student of rehabilitation sciences graduation program, nove de julho university (uninove), são paulo, sp, brazil 6phd, professor of rehabilitation sciences post graduation program, nove de julho university (uninove) , são paulo, sp, brazil correspondence to: thays almeida alfaya rua doutor calandrine 235 a, cep: 24755-160, são gonçalo, rj, brasil phone: +55 21 99515428 e-mail: thalfaya@gmail.com received for publication: august 05, 2012 accepted: december 11, 2012 abstract aim: to evaluate the clinical status of permanent first molars and associations with dental caries, gingival bleeding, dental fluorosis and malocclusion. methods: an observational study was carried out in a rural community denominated morro do saboó in the city of são roque, state of são paulo, brazil. a total of 194 children aged seven to ten years were examined for dental caries using the index proposed by the world health organization. other conditions were determined using the gingival alterations index, dean’s index and dental aesthetic index. the chi-squared test was used for the statistical analysis of the data. results: a total of 85.5% of the sample exhibited gingival bleeding and 69.9% exhibited malocclusion. a total of 53.6% had a clinical aspect of normality with regard to dental fluorosis. there was a predominance of sound teeth in the upper arch and teeth with carious lesions in the lower arch. no significant differences were found between sexes regarding gingival bleeding, dental fluorosis or malocclusion. significant associations were found between tooth status and oral alterations (gingival bleeding, malocclusion and fluorosis) in teeth 16, 26 and 46 and between tooth status and gingival bleeding in tooth 36 (p<0.001). conclusions: caries activity in the permanent first molars was mainly associated with dental fluorosis and malocclusion. strategies aimed at health promotion should be adopted on a large scale to minimize the prevalence of oral diseases. keywords: dental caries, molar, fluorosis, dental, gingivitis, malocclusion. introduction oral health status is mainly analyzed by the occurrence of dental caries and periodontal disease. dental caries is estimated to affect 60 to 90% of children in industrialized countries and constitutes the most prevalent oral condition in asia and latin america1. the establishment of the “brazil smiling program” in 2003 led to the expansion of public dental services in brazil. this greater access to oral health care (including prevention actions and specialized treatment) has 476476476476476 braz j oral sci. 11(4):475-480 contributed to an increase in the number of children who are free of caries (44%)2. permanent first molars play a fundamental role in the maintenance of the masticatory function. the development of carious lesions is common during the eruption of the first molars due characteristics that are favorable to the buildup of bacterial plaque3, such as pits and fissures4. in the absence of preventive measures, these teeth may experience all the repercussions stemming from the development of caries5-6. gingivitis is the most common form of periodontal disease in children and adolescents1 and is related to the presence of gingival bleeding. the early onset of aggressive periodontal disease affects approximately 2% of young patients and can lead to tooth loss1. the increase in the prevalence of gingivitis from childhood to puberty may be attributed to the number of sites at risk, with the buildup of bacterial plaque and inflammatory changes related to the process of exfoliation and eruption as well as hormonal factors related to age7. while dental caries and periodontal disease are the most common oral manifestations1, other factors should also be taken into consideration in the analysis of oral health status. dental fluorosis is a formation disorder in which the developing enamel is hypomineralized due to a change in the secretion of ameloblasts caused by an excess of fluoride ions8. malocclusion is a growth and development disorder that affects the occlusion (bite) of the teeth9 and can have a considerable impact on affected individuals, causing discomfort as well as functional and social limitations10. these conditions can have both functional and esthetic repercussions, with an impact on quality of life11-12, and should therefore be identified so that proper intervention steps can be taken. the aim of the present study was to evaluate the clinical status of permanent first molars and associations with dental caries, gingival bleeding, dental fluorosis and malocclusion. material and methods an observational study was carried out involving the analysis of permanent first molars with regard to the presence of dental caries and other oral problems, such as gingival bleeding, dental fluorosis and malocclusion, in 194 children aged seven to ten years at a school located in a rural community denominated morro do saboó in the city of são roque, state of são paulo, brazil, during a collective action. the study received approval from the human research ethics committee of the universidade federal de são paulo, brazil, under project number 1374/07. parents/guardians of the participants signed a statement of informed consent allowing their children to participate in the study. the evaluations were performed by a single, calibrated operator, using a mouth mirror and periodontal probe under natural light with the child seated and the examiner standing. the findings of the exam were recorded on an individual clinical chart for each child. assessment of dental caries the assessment of dental caries was performed on the permanent first molars (teeth 16, 26, 36 and 46). the following codes and criteria were used for the classification of the teeth: (0) sound; (1) with carious lesion; (2) restoration with carious lesion; (3) restoration without carious lesion; (4) loss due to caries; (5) loss for other reasons (6); presence of occlusal sealant; (7) bridge support or crown; (8) non-erupted; (9) excluded tooth (applied to any tooth that could not be examined); (t) trauma/fracture. this assessment was based on the criteria proposed by the world health organization13 and employed in brazilian oral health surveys14. assessment of gingival bleeding gingival bleeding was determined with the use of a periodontal probe, which was lightly inserted in the entrance of the gingival sulcus and run along its length – gingival alterations index15. assessment of dental fluorosis dental fluorosis was determined based on dean’s index and coded as follows: (0) normal; (1) questionable; (2) very mild; (3) mild; (4) moderate; and (5) severe13. assessment of malocclusion malocclusion was determined based on the abnormal positioning of the teeth using the index proposed by cons et al. (1989) denominated the dental aesthetic index (dai)16. the following criteria were used for the classification of normal occlusion: class i relationship of permanent first molars; canines in normal relationship (upper canine in the space between the lower canine and lower first premolar or primary first molar); absence of anterior or posterior crossbite; vertical overjet not surpassing 3 mm; horizontal overjet not surpassing 2 mm; presence or absence of inter-incisor diastema and primate spaces; absence of crowding; absence of tooth anomalies in shape and number; and absence of tooth rotation. statistical analysis data analysis was performed using the spss 17 program (ibm corp., chicago, il, usa). for such, the chi-squared test was employed, with the level of significance set to 5% (p < 0.05). results the sample was made up of 194 children aged seven to ten years; 51.03% (n = 99) of the children were female. table 1 displays the distribution of the children with regard to gingival bleeding, malocclusion and fluorosis according to sex. no significant difference between sexes was found regarding these oral conditions. a total of 84.5% of the participants (n = 164) exhibited gingival bleeding and 69.9% exhibited malocclusion (n = 135). with regard to fluorosis, 53.6% (n = 104) had a clinical aspect of normality, 32% (n = 62) were classified with questionable fluorosis, 10.3% (n = 20) were classified with questionable very mild fluorosis and 4.1% (n = 8) were classified with questionable mild fluorosis. clinical status of permanent first molars in children aged seven to ten years in a brazilian rural community 477477477477477 braz j oral sci. 11(4):475-480 tooth 16 status n % sound 68 35.1 carious 60 30.9 restored with caries 14 7.2 restored without caries 51 26.3 lost due to caries 1 0.5 total 194 100.0 tooth 26 status n % sound 72 37.1 carious 68 35.1 restored with caries 27 13.9 restored without caries 26 13.4 lost due to caries 1 0.5 total 194 100.0 tooth 36 status n % sound 56 28.9 carious 70 36.1 restored with caries 21 10.8 restored without caries 41 21.1 lost due to caries 6 3.1 total 194 100.0 tooth 46 status n % sound 60 30.9 carious 61 31.4 restored with caries 13 6.7 restored without caries 53 27.3 lost due to caries 7 3.6 total 194 100.0 table 2. clinical status of permanent first molars in children analyzed, são paulo, 2011. table 2 displays the results of the classification of dental caries status of the permanent first molars. there was a predominance of sound teeth in the upper arch and teeth with carious lesions in the lower arch. tooth loss was recorded in both the upper and lower arches. significant associations were found between caries activity and oral alterations (gingival bleeding, malocclusion and fluorosis) in teeth 16, 26 and 46 and between caries activity and gingival bleeding in tooth 36 (table 3) (p < 0.001). discussion the present results demonstrate no statistically significant associations between sex and gingival bleeding, malocclusion or fluorosis. however, tooth status was associated with all these conditions in teeth 16, 26 and 46 and with gingival bleeding in tooth 36. there was a predominance of sound teeth in the upper arch and carious teeth in the lower arch. data from the national brazilian oral health survey demonstrate an increase in the number of children who are free of caries2. however, variables related to social context remain predictors of caries, which underscores the need for integrated health actions, as occurs with other childhood diseases17. the present study revealed a large number of carious lesions in the sample analyzed, which may be attributed to the rural region in which the school is located, which has no access to public health care or fluoridated water. a previous study involving children and adults in a rural community in the state of minas gerais, brazil states that deficient oral health is to be expected in this population given the fact that preventive and curative care hinges on access to dental treatment18. another study carried out in a different region of the same state reports a greater prevalence of dental caries and lesser access to dental services as well as evidence of greater social deprivation among rural schoolchildren in comparison to their urban counterparts19. the findings described in a study carried out by de abreu et al. (2004) suggest that underprivileged populations as well as families with a slightly higher income and greater schooling are more prone to adopt behavior that leads to dental caries20. however, a study conducted abroad states that children who participate in preventive oral health programs made available at school have less caries experience21. the molars play a fundamental role in the maintenance of the stomatognathic system16. the first molars are the first clinical status of permanent first molars in children aged seven to ten years in a brazilian rural community gingival bleeding malocclusion fluorosis n o yes n o yes normal questionable very mild mild male n (%) 17 56.7% 78 47.6% 25 42.4% 70 51.9% 53 51.0% 28 45.2% 10 50.0% 4 50.0% female n (%) 13 43.3% 86 52.4% 34 57.6% 65 48.1% 51 49.0% 34 54.8% 10 50.0% 4 50.0% total 30 (100.0%) 164 (100.0%) 59 (100.0%) 135 (100.%) 104 (100.0%) 62 (100.0%) 20 (100.0%) 8 (100.0%) p-value 0.236 0.224 0.911 *chi-squared test table 1. distribution of participants with regard to gingival bleeding, malocclusion and fluorosis according to sex, são paulo, brazil, 2011. 478478478478478 braz j oral sci. 11(4):475-480 permanent teeth to erupt and, due to their anatomy, which includes pits and fissures, these teeth have increased odds of developing carious lesions4. in the present study, the lower molars had a greater frequency of caries, whereas the upper molars were, for the most part, sound. the permanent molars are the teeth most affected by caries22. while the location of the tooth (lower or upper) has no influence on the risk to dental caries, a number of factors may be related to the development of this disease in general, such as dietary habits23, caries experience in the deciduous tooth24 and parents/guardians’ schooling25. tooth loss was found in the sample in both the upper and lower arches. according to gonzalez and manrrique (2001), the loss of permanent first molars can lead to a reduction in local function, the continuous eruption of the antagonist teeth and tooth deviations26. considering the consequences of the absence of such teeth to the stomatognathic system, oral health promotion programs and campaigns aimed at the prevention of oral health problems should be established for the population as a whole and schoolchildren in particular. when performed early, interventions tend to minimize the impact of treatment and costs27. no significant association was found between dental fluorosis and sex. however, the prevalence of fluorosis was relatively low, which may have been due the fact that the participants lived in a rural environment, where some communities without access to a fluoridated water supply make use of artesian wells, mines, rivers, ponds, etc. fluorosis has been related to a reduction in caries28-29. however, a significant association was found between these conditions clinical status of permanent first molars in children aged seven to ten years in a brazilian rural community tooth 16 gingival bleeding malocclusion fluorosis tooth 26 gingival bleeding malocclusion fluorosis tooth 36 gingival bleeding malocclusion fluorosis tooth 46 gingival bleeding malocclusion fluorosis n o yes n o yes normal questionable very mild mild n o yes n o yes normal questionable very mild mild n o yes n o yes normal questionable very mild mild n o yes n o yes normal questionable very mild mild 29 (96.7%) 90 (54.9%) 46 (78.0%) 73 (54.1%) 54 (51.9%) 42 (67.7%) 16 (80.0%) 7 (87.5%) 23 76.7% 75 45.7% 46 78% 73 54.1% 43 41.3% 40 64.5% 10 50.0% 5 62.5% 26 86.7% 71 43.3% 33 55.9% 64 47.4% 47 45.2% 32 51.6% 14 70.0% 4 50.0% 26 86.7% 87 53.0% 76 56.3% 37 62.7% 53 51.0% 38 61.3% 14 70.0% 8 100% 1 3.3% 74 45.1% 13 22.0% 62 45.9% 50 48.1% 20 32.3% 4 20.0% 1 12.5% 7 23.3% 89 45.1% 13 22% 62 45.9% 61 58.7% 22 35.5% 10 5.0% 3 37.5% 4 13.3% 93 56.7% 26 44.1% 71 52.6% 57 54.8% 30 48.4% 6 30.0% 4 50.0% 4 13.3% 77 47.0% 59 43.7% 22 45.9% 51 49.0% 24 38.7% 6 30.0% 0 37.5% 30 (100.0%) 164 (100.0%) 59 (100.0%) 135 (100.0%) 104 (100.0%) 62 (100.0%) 20 (100.0%) 8 (100.0%) 30 (100%) 164 (100%) 59 (100%) 135 (100%) 104 (100%) 62 (100%) 20 (100%) 8 (100%) 30 (100.0%) 164 (100.0%) 59 (100.0%) 135 (100.0%) 104 (100.0%) 62 (100.0%) 20 (100.0%) 8 (100.0%) 30 (100.0%) 164 (100.0%) 59 (100.0%) 135 (100.0%) 104 (100.0%) 62 (100.0%) 20 (100.0%) 8 (100.0%) <0.001* = 0.001* = 0.017* =0.002* = 0.001* = 0.032* <0.001* = 0.175 =0.238 tooth 46 <0.001* =0.001* =0.025* * chi-squared test no n (%) caries lesions table 3. presence of gingival bleeding, malocclusion and fluorosis according to carious lesions in permanent upper first molars, são paulo, brazil, 2011. yes n(%) total p-value braz j oral sci. 11(4):475-480 in three of the four teeth evaluated in the present study. changes in diet have been suggested as an explanation for the increase in the prevalence of caries in groups that also have contact with fluoride ions30. therefore, a greater frequency of visits to the dentist and supervision with regard to sugar intake and brushing should be warrant21. a statistically significant association was found between gingival bleeding and caries activity. a previous study reports that the prevalence of gum disease is high among individuals aged seven to 14 years and is directly related to deficient oral hygiene31. in a systematic review of studies addressing periodontal disease, gjermo et al. (2000) report that gingivitis is the most common periodontal condition among children and adolescents, especially among males and individuals belonging to lower socioeconomic strata32. the findings of the present study are in agreement with these statements, except with regard to sex, for which no significant association was found. the prevalence of malocclusion in the present study was high, likely due to the absence of public promotion measures regarding this aspect of oral health. according to dimberg et al. (2011), the early treatment of malocclusion may be unnecessary if spontaneous correction occurs during the transition from the primary to the mixed dentition33. however, the present study involved the mixed dentition and the findings demonstrate treatment needs in this subgroup of the population. thus, basic epidemiological surveys are fundamental to the assessment of the current situation regarding malocclusion and the drafting of treatment plans for groups at risk. prevention programs should be directed at the population in both rural and urban environments. the findings of a longitudinal study demonstrate that children without caries in the primary dentition tend to remain caries free during the mixed dentition phase34. strategies aimed at health promotion should be adopted on a large scale to minimize the prevalence of oral diseases. moreover, for cases in which such conditions have already developed, early detection and treatment can ensure a future generation of healthy adults from the dental standpoint. references 1. who. world health organization. what is the burden of oral disease? [cited 2012 mar 7available from: http: //www.who.int/oral_health/ disease_burden/global/en/. 2. brasil. ministério da saúde. brasil sorridente: a saúde bucal levada a sério. [cited 2012 mar 28] available from: http: //dab.saude.gov.br/cnsb/ sbbrasil/arquivos/apresentacao_abbrasil_2010.pdf. 3. carvalho jc, ekstrand kr, thylstrup a. dental plaque and caries on occlusal surfaces of first permanent molars in relation to stage of eruption. j dent res. 1989; 68: 773-9. 4. pine cm, pitts nb, nugent zj. british association for the study of community dentistry (bascd) guidance on sampling for surveys of child dental health. a bascd coordinated dental epidemiology programme quality standard. community dent health. 1997; 14(suppl 1): 10-7. 5. feldens ca, kramer pf, abreu me, rosso ed, ferreira sh, feldens eg. association between caries experience in deciduos molars and first molar permanents. pesq bras odontoped clin integ. 2005; 5: 157-63. 6. quaglio jm, sousa mb, ardenghi tm, mendes fm, imparato jc, pinheiro sl. association between clinical parameters and the presence of active caries lesions in first permanent molars. braz oral res. 2006; 20: 358-63. 7. jenkins wm, papapanou pn. epidemiology of periodontal disease in children and adolescents. periodontol 2000. 2001; 26: 16-32. 8. fejerskov o, manji f, baelum v. the nature and mechanisms of dental fluorosis in man. j dent res. 1990; 69(spec n.): 692-700; discussion 21. 9. de oliveira cm, sheiham a. orthodontic treatment and its impact on oral health-related quality of life in brazilian adolescents. j orthod. 2004; 31: 20-7; discussion 15. 10. anosike an, sanu oo, da costa oo. malocclusion and its impact on quality of life of school children in nigeria. west afr j med. 2010; 29: 417-24. 11. chankanka o, levy sm, warren jj, chalmers jm. a literature review of aesthetic perceptions of dental fluorosis and relationships with psychosocial aspects/oral health-related quality of life. community dent oral epidemiol. 2010; 38: 97-109. 12. marques ls, ramos-jorge ml, paiva sm, pordeus ia. malocclusion: esthetic impact and quality of life among brazilian schoolchildren. am j orthod dentofacial orthop. 2006; 129: 424-7. 13. who. world health organization. oral health surveys: basic methods. 4 ed. geneva: orh/epid; 1997. 14. brasil. sb pesquisa nacional de saúde bucal. projeto técnico. secretaria políticas de saúde, departamento de atenção básica, área técnica de saúde bucal. – brasília: ministério da saúde; 2010. 15. brasil. projeto sb 2000: condições de saúde bucal da população brasileira no ano 2000: manual do examinador / secretaria políticas de saúde, departamento de atenção básica, área técnica de saúde bucal. – brasília: ministério da saúde; 2001. 16. cons nc, jenny j, kohout fj, songpaisan y, jotikastira d. utility of the dental aesthetic index in industrialized and developing countries. j public health dent. 1989; 49: 163-6. 17. melo mm, souza wv, lima ml, braga c. factors associated with dental caries in preschoolers in recife, pernambuco state, brazil. cad saude publica. 2011; 27: 471-85. 18. pallos d, loberto jcs, cortelli jr, cortelli sc, souza dm, ricardo lh. periodontal disease in a rural community in minas gerais, brazil. braz j oral sci. 2005; 4: 659-63. 19. mello tr, antunes jl. prevalence of dental caries in schoolchildren in the rural area of itapetininga, sao paulo state, brazil. cad saude publica. 2004; 20: 829-35. 20. de abreu mh, pordeus ia, modena cm. dental caries in schoolchildren from rural communities in itauna (mg), brazil. rev panam salud publica. 2004; 16: 334-44. 21. amalia r, schaub rm, widyanti n, stewart r, groothoff jw. the role of school-based dental programme on dental caries experience in yogyakarta province, indonesia. int j paediatr dent. 2012; 22:203-10. 22. marthaler tm. changes in dental caries 1953–2003. caries res. 2004; 38: 173-81. 23. ohlund i, holgerson pl, backman b, lind t, hernell o, johansson i. diet intake and caries prevalence in four-year-old children living in a lowprevalence country. caries res. 2007; 41: 26-33. 24. li y, wang w. predicting caries in permanent teeth from caries in primary teeth: an eight-year cohort study. j dent res. 2002; 81: 561-6. 25. ismail ai, sohn w. the impact of universal access to dental care on disparities in caries experience in children. j am dent assoc. 2001; 132: 295-303. 26. gonzalez j, manrrique r. proyecto anaco-u.c.v. estudio epidemiológico sobre la pérdida prematura del primer molar permanente en niños con edades comprendidas entre 6 y 10 años. acta odontol venez. 2001; 39: 42-6. 27. singh m, saini a, saimbi cs, bajpai ak. prevalence of dental diseases in 5to 14-year-old school children in rural areas of the barabanki district, uttar pradesh, india. indian j dent res. 2011; 22: 396-9. 28. mascarenhas ak. risk factors for dental fluorosis: a review of the recent literature. pediatr dent. 2000; 22: 269-77. 479479479479479clinical status of permanent first molars in children aged seven to ten years in a brazilian rural community braz j oral sci. 11(4):475-480 480480480480480 29. mcdonagh ms, whiting pf, wilson pm, sutton aj, chestnutt i, cooper j, et al. systematic review of water fluoridation. bmj. 2000; 321: 855-9. 30. sampaio fc, hossain an, von der fehr fr, arneberg p. dental caries and sugar intake of children from rural areas with different water fluoride levels in paraiba, brazil. community dent oral epidemiol. 2000; 28: 307-13. 31. chambrone l, macedo sb, ramalho fc, trevizani filho e, chambrone la. [prevalence and severity of gingivitis among scholars (7-14 years): local conditions associated to bleeding on probing]. cien saude colet. 2010; 15: 337-43. 32. gjermo p, rösing ck, susin c. periodontal diseases in central and south america. periodontol. 2000; 29: 70-8. 33. dimberg l, lennartsson b, soderfeldt b, bondemark l. malocclusions in children at 3 and 7 years of age: a longitudinal study. eur j orthod. 2011; oct 31. [epub ahead of print]. 34. greenwell al, johnsen d, disantis ta, gerstenmaier j, limbert n. longitudinal evaluation of caries patterns form the primary to the mixed dentition. pediatr dent. 1990; 12: 278-82. clinical status of permanent first molars in children aged seven to ten years in a brazilian rural community oral sciences n3 braz j oral sci. 11(1):47-51 received for publication: november 11, 2011 accepted: february 08, 2011 original article braz j oral sci. january | march 2012 volume 11, number 1 clinical effects of supragingival plaque control on uncontrolled type 2 diabetes mellitus subjects with chronic periodontitis andrea son1, claudia pera2, paulo ueda2, renato corrêa viana casarin3, suzana peres pimentel3, fabiano ribeiro cirano3 1dds student, paulista university, brazil 2dds, msc student, periodontics division, paulista university, brazil 3dds, msc, phd, professor, periodontics division, paulista university, brazil correspondence to: fabiano ribeiro cirano periodontics division, paulista university av dr bacelar,1212, vila clementino, são paulo, são paulo cep 04026-002 phone/fax: +55 11 55864000 e-mail: cirano@unip.com.br abstract aim: to determine the clinical changes occurred in chronic periodontitis patients presenting uncontrolled type 2 diabetes mellitus after a supragingival plaque control period. methods: subjects presenting generalized chronic periodontitis were divided into two groups: non-diabetics (n=20) – healthy subjects presenting chronic periodontitis; and diabetics (n=14) – subjects with uncontrolled type 2 diabetes mellitus presenting chronic periodontitis. all subjects went through 28 days of supragingival plaque control st (including prophylaxis, calculus removal, extraction of hopeless teeth and oral hygiene instructions) and were evaluated at baseline and after 28 days by the following parameters: full-mouth plaque score (fmps) and full-mouth bleeding scores (fmbs), periodontal probing depth (ppd), gingival recession (gr) and clinical attachment level (cal). anova/tukey’s test and student’s t test were used for data analysis. results: no statistically significant differences (p>0.05) between groups were observed at baseline for any parameter. both groups presented a significant reduction in fmps and fmbs after 28 days (p<0.05), but no statistically significant difference was found (p>0.05) between groups. clinically, only the non-diabetic group showed a significant improvement after st, in ppd of initially deep pockets (p<0.05). however, no change in the clinical parameters was observed in the diabetic subjects (p>0.05). conclusions: it may be concluded that uncontrolled diabetes mellitus reduces periodontal changes in the supragingival plaque control regimen of subjects presenting with chronic periodontitis. keywords: diabetes mellitus, plaque control, chronic periodontitis. introduction chronic periodontitis results from the presence of complex microbial communities in the subgingival sulcus1, and diabetes mellitus, especially if poorly controlled, increases significantly risk for development of extensive and severe diseases2. hyperglycemia and resultant advanced glycation end product formation, which is one of several pathways thought to lead to the vascular complications with diabetes, are also involved in the pathophysiology of periodontitis in diabetic subjects3, leading to an imbalanced release of proand anti-inflammatory cytokines4-6 and osteoclastogenesis-related factors7. braz j oral sci. 11(1):47-51 4848484848 despite the differences in pathogeneses, biofilm still remains the primary etiologic factor for the development of a destructive periodontal disease2. thus, the primary goal of periodontal therapy is to target the subgingival biofilm present in periodontally diseased sites that are associated with the progressive destruction of the supportive periodontal tissues. it is well documented that conventional therapy, i.e., subgingival scaling and root planning, is effective in achieving this goal. however, supragingival plaque control appears to have a significant effect on clinical and microbiological characteristics of periodontal pockets, which could be associated with the close relationship between those environments8. previous studies have evaluated the relationship between supragingival plaque control and clinical and microbiological effects on subgingival areas, reporting a positive effect in systemically healthy subjects with periodontitis, i.e., a reduction in probing depth and some periodontal pathogens and preventing re-colonization 9-11. however, conflicting results of the impact of supragingival dental biofilm control on clinical features in untreated periodontal sites are found in the literature12-14. in this context, there is an interest in the possible effect of supragingival biofilm control on the subgingival environment in untreated periodontitis sites in diabetic patients, since, in previous studies, these patients presented with some altered biofilm compositions, with a higher prevalence of periodontal pathogens. thus, the aim of the present study was to determine clinical changes in type 2 diabetic patients after 28 days of strict supragingival plaque control compared with non-diabetic patients. material and methods population screening initially, manuscript design was approved by the institutional ethics committee (protocol number 014/09). eligible patients were selected from those referred to the graduate clinic of paulista university, brazil. all patients received a complete periodontal examination, including full mouth periodontal probing, radiographic examination, and complete clinical interview. moreover, type 2 diabetic patients were sent to the clinic by vila mariana health center (hcvm), são paulo, brazil after being diagnosed using the fasting plasma glucose (fpg) > 110 mg/dl and the glycated hemoglobin (hba1c) > 7% in two different examinations. all diabetic subjects were followed by a physician at hcvm. subjects who did not have diabetes but who presented with periodontitis were also selected in order to compare of the clinical response of both types of patients. the study inclusion criteria were the following: diagnosis of chronic periodontitis, according to the criteria of the 1999 international classification15; at least 8 teeth with a periodontal probing depth (ppd) > 5 mm and bleeding on probing; presence of at least 20 teeth; and good general health. patients who were pregnant or lactating, required antimicrobial premedication for the performance of periodontal examination and treatment, received a course of periodontal treatment within the last 6 months, smokers, those under use of longterm antiinflammatory drugs, suffered from any other systemic diseases (cardiovascular, pulmonary, liver, and cerebral diseases), or had received antimicrobial treatment in the previous 3 months were excluded from the study. the sample size was determined after considering data in the literature and was aimed at obtaining a minimum power value of 0.8 to detect a difference and 0.8 mm between groups in clinical attachment level (cal) (primary variable). a blinded and calibrated examiner was used (intra-class correlation for cal) = 94% in a parallel design. supragingival plaque control therapy (st) after full mouth examination and participants’ informed consent, the patients in both groups received a full mouth prophylaxis, supragingival calculus, and biofilm removal using gracey curettes, ultrasonic scaler, bicarbonate spray, and dental floss. also, condemned teeth were extracted and biofilm retentive factors were removed. moreover, the patients were individually instructed on how to perform oral selfcare, including the bass technique, inter-dental flossing, and tongue brushing. all subjects received a standard fluoride dentifrice, toothbrushes, and dental floss as necessary and were asked to perform complete oral self-care hygiene at least twice a day. a week after this first instruction session, patients returned for reinforcement of the oral self-care instructions. twenty-eight days after st, clinical re-evaluation was performed. groups subjects were distributed to the following groups: diabetics (n=14): composed of individuals presenting with uncontrolled type 2 diabetes mellitus and generalized chronic periodontitis and non-diabetics (n=20): composed of individuals presenting with generalized chronic periodontitis. clinical parameters the following clinical parameters were assessed immediately before and 28 days after plaque control therapy using a pcp-15 periodontal probe (hu-friedy, chicago, il, usa): full-mouth plaque index (fmpi)16 and full-mouth bleeding score (fmbs)17; represented by the percentage of positive sites; ppd – distance from the bottom of the pocket to the gingival margin); gingival recession (gr – distance from the gingival margin to cement-enamel junction); cal – distance from the bottom of the pocket to cement-enamel junction) glycemic status a single laboratory performed the glycated hemoglobin (hba1c) and fasting plasma glucose (fpg) tests in order to confirm diabetes mellitus status. hba1c (%) was measured using high-performance liquid chromatography, and fpg was performed using the glucose oxidase method. for clinical effects of supragingival plaque control on uncontrolled type 2 diabetes mellitus subjects with chronic periodontitis 4949494949 braz j oral sci. 11(1):47-51 uncontrolled cut-off, hba1c should be higher than 7% and fpg > 120 mg/dl. all patients were under physician monitoring and taken oral hypoglycemic pills. data management and statistical analysis for clinical parameters, a repeated-measures analysis of variance (anova) was used to detect intra-group differences in clinical parameters (gr, ppd, cal), considering the patient as a statistical unit. when a statistical difference was found, the analysis of the difference was determined using the tukey’s test. the friedman test was used to detect intragroup differences and kruskal-wallis was used for the intergroup analysis of the full mouth plaque and bleeding index among all periods. the level of significance was set at 5%. results table 1 displays the demographic, clinical, and diabetic status of the population included in the present study. no difference was found regarding gender, age, and clinical parameters (ppd, cal). moreover, the fpg and hba1c levels of diabetic subjects evidenced their poor glycemic control. table 2 shows changes in plaque and bleeding indices after supragingival plaque control. both groups exhibited a reduction in plaque and bleeding (p<0.05) and no significant difference between them was observed at baseline or after the supragingival plaque control period (p>0.05). table 3 shows the clinical changes of shallow (ppd<4mm), moderate (ppd = 5-6 mm), and deep pockets (ppd>7mm) after 28 days of strict plaque control. at shallow and moderate pockets, no statistically significant change could be seen regarding any clinical parameter (p>0.05). however, in relation to deep pockets, a significant reduction in ppd was observed in non-diabetic individuals, although no statistical changes were observed regarding cal. at the same time, no significant change in ppd and cal was detected in the diabetic participants, indicating that supragingival plaque control did not promote significant changes in these subjects. discussion periodontal disease and diabetes mellitus belong to a pathologic condition in which both diseases could negatively age (mean±sd) (minimum-maximum) gender (n) glycemic status male female fasting plasma glucose (mean±sd) glycated hemoglobin hba1c (mean±sd) non-diabetics 43.4 ± 6.45 (35 55) 13 7 diabetics 56.14 + 11.7 (41 80) 9 5 145±46.5 mg/dl 8.1±1.1 % table 1. table 1. table 1. table 1. table 1. demographic characteristics of subjects. non-diabetics diabetics fmps baseline 84.2±7.5 a 83.5±12.4 a 28 days 72.2±12.2 b 69.8±18.0 b fmbs baseline 56.1±12.4 a 47.5±20.2 a 28 days 45.0±17.9 b 34.3±17.8 b table 2.table 2.table 2.table 2.table 2. full mouth plaque (fmps) and bleeding scores (fmbs) in diabetic and non-diabetic subjects before and after supragingival therapy. different letters in column indicate a statistically significant difference within the group (anova/tukey, p<0.05) table 3.table 3.table 3.table 3.table 3. clinical parameters at shallow (ppd<4mm), moderate (5<ppd<6mm), and deep (ppd>7mm) pockets or shallow (cal<4mm), moderate (5<cal<6mm), and deep (cal>7mm) cal levels in diabetic and non-diabetic subjects before and after supragingival therapy. non-diabetics diabetics ppd < 4 baseline 3.1±0.4 a 3.5±0.7 a ppd < 4 28 days 3.0±0.2 a 3.2±0.2 a 5 < ppd < 6 baseline 5.4±0.2 a 5.3±0.2 a 5 < ppd < 6 28 days 5.4±0.2 a 5.2±0.2 a ppd > 7 baseline 8.2±1.0 a 8.4±0.8 a ppd > 7 28 days 7.9±1.0 b 8.4±0.8 a cal < 4 baseline 3.2±0.4 a 3.4±0.9 a cal < 4 28 days 3.3±0.5 a 3.2±0.2 a 5 < cal < 6 baseline 5.4±0.2 a 5.3±0.2 a 5 < cal < 6 28 days 5.4±0.4 a 5.4±0.5 a cal > 7 baseline 8.5±1.0 a 8.2±1.2 a cal > 7 28 days 8.4±1.1 a 8.5±1.1 a different letters in column indicate a statistically significant difference within the group (anova/tukey, p<0.05). periodontal probing depth (ppd), clinical attachment level (cal). interfere with each other, constituting a bidirectional relationship 2. diabetes mellitus, especially when uncontrolled, appears to be an important risk factor for periodontal destruction, since an alteration in host-response and microbiological aspects occurs in diabetic subjects. periodontal disease led to an increase in insulin resistance, which could impair glycemic control. this way, the control of periodontal disease is necessary for better systemic health in these individuals. periodontal treatment relies on biofilm disruption and plaque control to prevent recolonization and recurrence of the disease. for this, an essential phase of this clinical effects of supragingival plaque control on uncontrolled type 2 diabetes mellitus subjects with chronic periodontitis 5050505050 braz j oral sci. 11(1):47-51 treatment is supragingival plaque control. so, the present study evaluated the periodontal changes in type 2 diabetic subjects and non-diabetic subjects after a supragingival plaque control regimen. diabetics did not show significant changes in their clinical aspects, although in non-diabetic subjects, a statistically significant change could be seen, especially in deep pockets. diabetic subjects with poor glycemic control present an altered release of pro and anti-inflammatory cytokines4. moreover, some previous studies have shown that the microbiota associated with diabetes does not appear different from the microbiota of non-diabetic patients18-22. glucose concentration in gingival crevicular fluid has been correlated with a high glucose concentration in the serum23. elevated glucose levels in saliva and gingival crevicular fluid could induce an increase in the number of saccharolytic bacteria associated with dental caries in the saliva and in the supragingival and subgingival plaque of diabetic patients24, which could modify the biodiversity in diabetic subjects. moreover, glycemic control could contribute to this harboring-microbiota profile, since different levels of plasma glucose may also indicate different levels of this carbohydrate in the subgingival area, probably altering environmental and microbiota characteristics. these patterns together make uncontrolled diabetes mellitus an important modifier of periodontal tissues and could impair periodontal response to treatment and be associated with this absence of significant changes after a supragingival plaque control regimen. at the same time, clinical improvement after supragingival plaque control was observed in non-diabetic subjects. corroborating with our study, previous clinical and microbiological trials showed that supragingival plaque control are able to promote significant changes in periodontal conditions. ribeiro et al.11 observed a gingival inflammation reduction, corroborating other studies that show a positive influence in shallow and moderate pockets9-10, although some studies also showed a benefit in deep pockets 25-26. in sequential studies, gomes et al.26-27 identified some clinical and microbiological improvement when supragingival plaque control was performed for 6 months. although no statistically significant reduction has been observed regarding specific periodontal pathogens (aggregatibacter actinomycetemcomitans, porphyromonas gingivalis and tannerella forsythia), the total number of subgingival bacteria were reduced when supragingival was performed27. more recently, melmann et al.28, evaluate the impact of supragingival plaque control, following the same protocol used in the present study, a microbiological positive effect was observed, using a 16s cloning technique for biodiversity analysis. in this study, a reduction in some phylotypes was observed in higher frequency in non-smokers than smokers, especially those genera known as periodontal pathogens or associated to sites presenting periodontal destruction28. it could indicate that some systemic modifiers, such as smoking in melmann’s study and type 2 diabetes mellitus in our study, could depreciate the periodontal and microbiological changes occurring after supragingival plaque control, once in both studies normal subjects presented better results. it could be seen when considering subgingival periodontal treatment, when both smokers and type 2 diabetic subjects have a worse periodontal response than healthy individuals2930. although gomes et al.26-27 showed similar response of smokers, it is important to have in mind that those authors used a 3-month period of supragingival plaque control with reinforcement in oral hygiene instruction, while in melmann’s study28 and in the present study a 28-day supragingival plaque control regimen was applied. it could represent a possible effect of supragingival plaque control protocol on clinical and microbiological results, what should be addressed in future trials. in the present study, the removal of plaque retentive factors and oral hygiene instructions was carried out in one session. plaque control during the next 28 days was the responsibility of the patient, with reinforcement in oral hygiene methods at 15 days. the protocol of one session of supragingival plaque control was chosen because of the difficulty in making patients return 3 times per week for professional plaque control. this way, further evaluation in diabetic subjects employing different plaque control regimens should be done. considering the results of the present study, it may be concluded that uncontrolled diabetes mellitus reduces periodontal changes in the supragingival plaque control regimen of subjects presenting with chronic periodontitis. acknowledgements this study was supported by national council for scientific and technological development (pibic/cnpq grant 154857/2010-6). references 1. socransky ss, haffajee ad. periodontal microbial ecology. periodontol 2000. 2005; 38: 135-87. 2. mealey bl. periodontal disease and diabetes. a two-way street. j am dent assoc. 2006; 137 (suppl): 26s-31s 3. mealey bl, rose lf. diabetes mellitus and inflammatory periodontal diseases. curr opin endocrinol diabetes obes. 2008; 15: 135-41. 4. vieira ribeiro f, de mendonça ac, santos vr, bastos mf, figueiredo lc, duarte pm. cytokines and bone-related factors in systemically healthy patients with chronic periodontitis and patients with type 2 diabetes and chronic periodontitis. j periodontol. 2011; 82: 1187-96. 5. santos vr, ribeiro fv, lima ja, napimoga mh, bastos mf, duarte pm. cytokine levels in sites of chronic periodontitis of poorly controlled and well-controlled type 2 diabetic subjects. j clin periodontol. 2010; 37: 1049-58. 6. shin ds, park jw, suh jy, lee jm. the expressions of inflammatory factors and tissue inhibitor of matrix metalloproteinase-2 in human chronic periodontitis with type 2 diabetes mellitus. j periodontal implant sci. 2010; 40: 33-8. 7. duarte pm, de oliveira mc, tambeli ch, parada ca, casati mz, nociti fh-jr. overexpression of interleukin-1beta and interleukin-6 may play an important role in periodontal breakdown in type 2 diabetic patients. j periodontal res. 2007; 42: 377-81. 8. listgarten ma, mayo he, tremblay r. development of dental plaque on epoxy resin crowns in man. a light and electron microscopic study. j periodontol. 1975; 46: 10-26. clinical effects of supragingival plaque control on uncontrolled type 2 diabetes mellitus subjects with chronic periodontitis braz j oral sci. 11(1):47-51 5151515151 9. hellström mk, ramberg p, krok l, lindhe j. the effect of supragingival plaque control on the subgingival microflora in human periodontitis. j clin periodontol. 1996; 23: 934-40. 10. westfelt e, rylander h, dahlén g, lindhe j. the effect of supragingival plaque control on the progression of advanced periodontal disease. j clin periodontol. 1998; 25: 536-41. 11. ribeiro edel p, bittencourt s, nociti-júnior fh, sallum ea, sallum aw, casati mz. the effect of one session of supragingival plaque control on clinical and biochemical parameters of chronic periodontitis. j appl oral sci. 2005; 13: 275-9. 12. kho p, smales fc, hardie jm. the effect of supragingival plaque control on the subgingivalmicroflora. j clin periodontol. 1985; 12: 676-86. 13. beltrami m, bickel m, baehni pc. the effect of supragingival plaque control on the composition of the subgingival microflora in human periodontitis. j clin periodontol. 1987; 14: 161-4. 14. mcnabb h, mombelli a, lang np. supragingival cleaning 3 times a week. the microbiological effects in moderately deep pockets. j clin periodontol. 1992; 19: 348-56. 15. armitage gc. development of a classification system for periodontal diseases and conditions. ann periodontol. 1999; 4: 1-6. 16. ainamo j, bay i. problems and proposals for recording gingivitis and plaque. int dent j. 1975; 25: 229-35. 17. mühlemann hr, son s. gingival sulcus bleeding—a leading symptom in initial gingivitis. helv odontol acta. 1971; 15: 107-13 18. katz pp, wirthlin mr jr, szpunar sm, selby jv, sepe sj, showstack ja. epidemiology and prevention of periodontal disease in individuals with diabetes. diabetes care. 1991; 14: 375-85. 19. yuan k, chang cj, hsu pc, sun hs, tseng cc, wang jr. detection of putative periodontal pathogens in non-insulin-dependent diabetes mellitus and non-diabetes mellitus by polymerase chain reaction. j periodontal res. 2001; 36: 18-24. 20. tervonen t, oliver rc, wol lf, bereuter j, anderson la, aeppli dm. prevalence of periodontal pathogens with varying metabolic control of diabetes mellitus. j clin periodontol. 1994; 21: 375-9. 21. sbordone l, ramaglia l, barone a, ciaglia rn, tenore a, iacono vj. periodontal status and selected cultivable anaerobic microflora of insulindependent juvenile diabetics. j periodontol. 1995; 66: 452-61. 22. zambon jj, reynolds h, fisher jg, shlossman m, dunford r, genco rj. microbiological and immunological studies of adult periodontitis in patients with noninsulin-dependent diabetes mellitus. j periodontol. 1988; 59: 23-31. 23. ficara aj, levin mp, grower mf, kramer gd. a comparison of the glucose and protein content of gingival fluid from diabetics and non diabetics. j periodontal res. 1975; 10: 171-5. 24. hintao j, teanpaisan r, chongsuvivatwong v, ratarasan c, dahlen g. the microbiological profiles of saliva, supragingival and subgingival plaque and dental caries in adults with and without type 2 diabetes mellitus. oral microbiol immunol. 2007; 22: 175-81. 25. nogueira moreira a, luna davila g, bianchini h, alonso c, piovano s. effect of supragingival plaque control on subgingival microflora and gingivoperiodontal tissues. acta odontol latinoam. 2000; 13: 73-86. 26. gomes sc, piccinin fb, susin c, oppermann rv, marcantonio ra. effect of supragingival plaque control in smokers and never-smokers: 6month evaluation of patients with periodontitis. j periodontol. 2007; 78: 1515-21. 27. gomes sc, nonnenmacher c, susin c, oppermann rv, mutters r, marcantonio ra. the effect of a supragingival plaque-control regimen on the subgingival microbiota in smokers and never-smokers: evaluation by real-time polymerase chain reaction. j periodontol. 2008; 79: 2297-304. 28. meulman tb, casarin rc, peruzzo dc, giorgetti ap, barbagallo a, casati mz, et al. impact of supragingival therapy on subgingival microbial profile in smokers versus non-smokers with severe chronic periodontitis. j oral microbiol. 2012; 4: doi: 10.3402/jom.v4i0.e8640. 29. nassarawin na. effect of smoking on the response to nonsurgical periodontal therapy. east mediterr health j. 2010; 16: 162-5. 30. kudva p, tabasum st, garg n. evaluation of clinical and metabolic changes after non-surgical periodontal treatment of type 2 diabetes mellitus patients: a clinico biochemical study. j indian soc periodontol. 2010; 14: 257-62. clinical effects of supragingival plaque control on uncontrolled type 2 diabetes mellitus subjects with chronic periodontitis oral sciences n3 literature review braz j oral sci. october | december 2012 volume 11, number 4 effects of bleaching agents and adhesive systems in dental pulp: a literature review maria antonieta veloso carvalho de oliveira1, paulo sérgio quagliatto2, denildo magalhães3, joão carlos gabrielli biffi 4 correspondence to: maria antonieta veloso carvalho de oliveira departamento de endodontia faculdade de odontologia universidade federal de uberlândia av. pará, 1720, bloco 2bs/25, umuarama cep: 38405-382 uberlândia, mg,brasil e-mail: antocassia@hotmail.com 1dds, undergraduate student, dental school, federal university of uberlândia, mg, brazil 2dds, ms, phd, professor of dentistry, dental school, federal university of uberlândia, mg, brazil 3dds, ms, phd, professor of periodontics, dental school, federal university of uberlândia, mg, brazil 4dds, ms, phd, professor of endodontics, dental school, federal university of uberlândia, mg, brazil abstract the dental pulp may be exposed to several irritants that are potentially noxious to the health and functions of this tissue. each type of irritant or injury has different effects on the pulp, which are generally characterized by acute inflammation, chronic inflammation or necrosis. common examples of irritants are dental caries, cavity preparation procedures, traumatic injuries, and chemical substances like bleaching agents and adhesive systems. the present study aimed to review the current knowledge about the effect of bleaching agents and adhesive systems in the human dental pulp. the review covered literature from 2004 to 2009, and only relevant manuscripts were included. hand search of the references completed the review. based on literature review, it may be concluded that all dental procedures associated with bleaching agents or adhesive systems involve risks to cause pulp damage. however, these risks can be minimized if the causal factors were known and avoided. keywords: adhesive systems, bleaching agents, external dental bleach, pulp response. introduction the dental pulp may be exposed to wide array of irritants that are noxious to the health and functions of this tissue. irritants can be either short-term and longterm irritating agents or trauma. each type of irritant or injury will have different effects on the pulp, which are characterized by acute inflammation, chronic inflammation or necrosis1. the pulp develops inflammation as a basic protective biologic defense mechanism in response to any type of injury1-2. common examples of irritants are dental caries, cavity preparation procedures, traumatic injuries, and chemical substances, like bleaching agents and adhesive systems1. the effect of chemical substances or restorative procedures on dentin and pulp is the result of a complex interaction among many factors such as, health of the underlying pulp, thickness and permeability of the intermediate dentin layer, mechanical injury during tooth preparation, toxicity of the restorative material and microleakage2. the present study aimed to review the current knowledge about the effect of bleaching agents and adhesive systems in the human dental pulp. the review covered literature from 2004 to 2009 and only relevant papers were included. hand search of the references completed the review. the search used pubmed braz j oral sci. 11(4):428-432 received for publication: april 06, 2012 accepted: july 13, 2012 429429429429429 electronic full-text database with the terms “bleaching agents”, “external dental bleach”, “adhesive systems” and “pulp response”. bleaching agents tooth bleaching is one of the most common procedures currently performed in contemporary dentistry. some controversy still exists on the safety of the tooth bleaching. several studies have defended that this procedure is innocuous to dental tissues, but there is evidence in the literature that the chemicals used for tooth bleaching have deleterious effects on the pulp-dentin complex3. the most commonly used chemicals for tooth bleaching usually contain some form of hydrogen peroxide (h 2 o 2 ) at different concentrations. h 2 o 2 is an oxidant agent that, with the aid of some chemical or physical activators (i.e., enzymes, light or heat), dissociates in perhydroxyl and oxygen radicals. these free radicals, known as reactive oxygen species, have low molecular weight and are able to denaturize proteins3-4, creating porosities on the enamel structure that can permit the diffusion through the organic matrix of dentine5-7. the diffusion of h 2 o 2 through dentin depends on the original concentration of the bleaching agent and the length that the agent is in contact with the dentin3-4,8. other factors such as positive pulp pressure and osmotic pressure of the gels, and application of heat during bleaching procedure might have influence on the diffusion of peroxides to the pulp8. the presence of dentinal tubules within the tooth architecture could allow these free radicals to reach the pulp tissue and generate adverse effects3-5. a recent study simulated a professionally applied vital tooth bleaching procedure with 35% h 2 o 2 bleaching gel, and showed that the diffusion of the bleaching gel components through enamel and dentin caused severe toxic effects to the cultured pulp cells5. the potential adverse effects of h 2 o 2 on the oral tissues have been widely investigated, especially because it is a thermally unstable chemical agent with a high oxidative power, which may dissociate into water, oxygen and some free radical species, such as hydroxyl radicals (oh). the oxidative reactions and consequently the cell damage caused by free radicals are the main mechanisms responsible for the toxicity of peroxide-containing compounds9. chemical reactions can be increased by increasing the temperature, where a 10°c rise can double the rate of reaction10. the use of light and/or heat-activated tooth bleaching systems generates an increased reaction of h 2 o 2 with an accelerated release of free radicals, which is clinically translated into a faster whitening effect3. however, there is a significant increase in the intrapulpal temperature and in the diffusion of bleaching gel components to the pulp, causing an increase in pulpal inflammatory reaction3,9. previous studies using different light sources demonstrated that differences in temperature changes depend on the type of light employed11 and may be critical12 or not11 for pulpal health. the bleaching gel usually applied may act as an isolator reducing intrapulpal temperature rise as compared to activated bleaching performed without gel application11-13. this means that laser activation without the use of bleaching gel results in an intrapulpal temperature increase of about 16oc, whereas a 8.7oc temperature increase was recorded when a gel was applied during activation. temperature increase may also depend on amount and type of color pigments included on the gels11. nowadays, an intrapulpal temperature increase of 5.5æ%c is regarded as the threshold value, which should not be exceeded to avoid irreversible thermal pulp damage11-14. most bleaching lamps do not raise the pulpal temperature to dangerous levels. however, care should be taken when using laser lamps at high power settings11 (e.g. above 2w12). composite resins are the most common material used for esthetic restoration of anterior teeth and most patients seeking for whither teeth have restoration. the studies that investigated the effect of bleaching agents on the restorative materials were developed mainly to evaluate if bleaching materials could exert any influence on the physicochemical properties, and on the release of monomers from the composite materials. polydouro et al.15 observed that bleaching of composite fillings can result, in some cases, in less monomer release from composite materials. therefore, the contact of bleaching agents with composite materials may not have any detrimental results for the human health due to an increased monomer release caused by bleaching15. the capacity of h 2 o 2 to diffuse through the pulp chamber in vitro was evaluated in teeth following restorative procedures4. restored teeth had significantly higher h 2 o 2 concentrations in the pulp chamber that intact teeth. according to the authors, this occurs due to the microleakage around the restorative materials. moreover, restorations margins can be considered a possible pathway for h 2 o 2 penetration into the pulp chamber4. the penetration of peroxides into the pulp chamber of restored teeth depends of the type of the restorative material, being higher in teeth restored with resin-modified glass ionomer cements7. the majority of studies that evaluates tooth whitening process involve the use of either h 2 o 2 or carbamide peroxide. in contact with water, carbamide peroxide breaks down into urea and h 2 o 2 10. urea may contribute to the structural changes and roughness of enamel. however, it can promote beneficial side-effects because the alkaline property of urea raises the hydrogen ion concentration (ph) of the bleaching solution and reduces the adverse effects in pulp tissue6. today, tooth bleaching has moved out of the dental office into the patient’s home. generally, a soft, custom-made, plastic tray filled with a bleaching gel is used for home bleaching. recently, a new vital tooth whitening technique, using flexible strips that are applied directly to the tooth, was introduced. these strips contain various concentrations of h 2 o and eliminate the need for custom-made trays for each patient. this bleaching technique was studied to evaluate the h 2 o 2 penetration containing 6.5% and 14% h 2 o 2 in the pulp chamber of the teeth. on the basis of the results and within the limitations of the in vitro study, the authors concluded that h 2 o 2 from whitening strips readily penetrates into the pulp chamber at various amounts and further studies are necessary to evaluate its clinical performance8. effects of bleaching agents and adhesive systems in dental pulp: a literature review braz j oral sci. 11(4):428-432 430430430430430 although the findings of in vitro studies have clinical relevance, the results of laboratory h 2 o 2 diffusion experimental models might not predict the real penetration capacity of this chemical agent in vivo10. in laboratory experimental models, the absence of the outward intradentinal fluid flow might contribute to the rapid diffusion of bleaching gel components through dentin. the presence of a dentinal fluid flow produced by intrapulpal pressure in vital teeth is sufficient to substantially reduce the diffusion of chemicals into the dentinal tubules to reach the pulp4-5,8. therefore, as stated by gokay et al.8, it is possible to assume that the levels of h 2 o 2 or the byproducts of its degradation that reach the pulp tissue in vivo may actually be lower than those observed in ex vivo experiments. adhesive systems contemporary dental adhesive systems are used to improve the contact between composite restorative materials and the walls of the prepared tooth cavity. as these materials come in close and prolonged contact with vital dentin, their influence on pulp tissue is critical16. histopathology studies have indicated that adhesive resins elicit transient, inflammatory changes and irregular dentin formation of the dental pulp at short-term evaluations. these pulp inflammatory responses are most likely due to irritation by diffusible dentin bonding ingredients released following light curing, especially when the remaining dentin thickness is thin or pulp exposure is noted during cavity preparation2. the composition of dentin adhesive is one of the factors that influence pulp response to dentin adhesives2,17-18. polymerized adhesives materials could release residual monomers that may interact with pulp tissues16. some monomers are associated to cytotoxic effects on fibroblast and odontoblastslike cells, such as bis-gma, hema, udma, tegdma18-20. all these components, individually or in combination, ultimately compromise the pulp healing. the main constituents of primer and adhesive solution are hema and bis-gma, respectively. in a ranking of toxicity, hema is the least toxic substance compared to other monomers such as bis-gma, udma and tegdma after 24 and 72 h of exposure18. the high percentage of hema in the primer, its lack of polymer conversion due to the absence of photoinitiators, its low molecular weight and hydrophilic features, facilitate its diffusion through pulp tissue and consequently avoid pulp healing18-19. moreover, hema diffusion was found in young and old primary teeth17. bisgma shows higher cytotoxic effects than hema. consequently, one may suggest that the high concentration of hema and bis-gma present in the primers and adhesive resins may cause remarkable cytopathic effects and elicit severe inflammatory response when applied in contact to connective tissue21. after acid etching, a significant increase in dentin permeability due to smear layer removal and opening of dentinal tubules can facilitate the permeation of resin monomers towards pulp, mainly in deep dentin17,19. odontoblasts are typical pulp cells and are the first cells to be damaged by potentially cytotoxic compounds released from dental materials that diffuse through enamel and dentinal tubules18,20,22. injury to the odontoblasts results in loss of the capacity of these cells to secrete reactionary dentin, and secretion of dentin bridge by a new generation of odontoblasts-like cells22-23. the cytotoxic potential demonstrated by the dental adhesives might be of clinical relevance, since all dental adhesives disturb the cellular redox state of pulp cells in monolayer cultures. demirci et al.16 suggested that the concentrations of biologically active ingredients of some of the bonding agents may be high enough to modify pulp cell metabolism when the materials are used into deep cavities or even in directly contact with the pulp tissue. teixeira et al.21 evaluated in vivo the biocompatibility of adhesive systems and the bonding agent that contained udma on its chemical composition, presented high cytopathic effects interfering with the cellular cycle in pulp tissue. a chronic inflammatory response may be directly related to the cytotoxic effects of the bonding agent’s components, the accumulation of its fragments in the pulp during the procedure may evoke this persistent reaction24. self-etching priming solutions contain larger amounts of acidic monomers to produce an etching effect for enamel and dentin. the acidic characteristics of the adhesive may be retained and, thus, continue to affect dentin and consequently the pulp. onestep self-etch adhesive systems induce inflammatory reactions of the pulp. it seems evident that application of this adhesive on prepared teeth has a potential to interfere with early stage of inflammatory response in dental pulp2. the acid etchants of the total-etch three-step adhesive systems contain other components, which are shared between the primer and adhesive bottles. all different components present in the total-etch threestep adhesive system, individually or in combination, may result in degenerative pulp alterations when placed directly onto pulp exposure sites19. an evaluation of the cytotoxic effect of total-etch two-step adhesive systems showed that both the acidic and non-acidic components of unpolymerized adhesive resins were responsible for high cytotoxic effects on odontoblast-like cells25. in another study where two self-etch adhesive systems were evaluated, the pulp tissue exhibited moderate to severe inflammatory cell infiltrate involving the coronal pulp with chronic abscesses26. the difference in toxic behavior between the total-etch and self-etch systems has been attributed to the fact that the total-etch systems they involve the use of phosphoric acid, which completely remove the smear layer and smear plugs, while enlarging the lumen of the dentinal tubules by the dissolution of the peritubular dentin, favoring the transdentinal diffusion of various substances, including those undesirable from the toxicological perspective20. also, self-etch agents are less acidic than the phosphoric acid, reducing their deleterious potential over the pulp tissue27. furthermore, the use of selfetch adhesives systems is indicated for young, deep, permeable dentin because self-etch adhesives often leave some residual smear plug material in the tubules which limits the diffusion of uncured monomers toward the pulp18. the degree of cytotoxicity depends on the concentration of the material used and the monomers may have synergistic effects of bleaching agents and adhesive systems in dental pulp: a literature review braz j oral sci. 11(4):428-432 431431431431431 or antagonistic cytotoxic effects. resinous monomers are cytotoxic at high concentrations, and they seem to provoke specific effects on pulp cells at low concentrations. the nontoxic concentrations of monomers may reduce the mitochondrial activity of pulp macrophages and the process of mineralization23. according to lanza et al.20, it could be suggested that the toxicity of adhesive materials depends on their chemical characteristics and the concentrations at which their components reach the pulp tissue rather than their capacity to dissolve the smear layer and demineralize the subjacent dentin. photoactivation of monomers in the bonding agents applied to dentin may neutralize their negative effects on the differentiation of human pulp cells into odontoblasts in vitro23. when monomers are photoactivated, the amount of unreacted products leached out is reduced and thus their cytotoxic effects to the cells are dramatically diminished18,26. this is of key importance in restorative dentistry particularly in the restoration of deep cavity where odontoblastic differentiation from pulp fibroblasts is a critical step in hard tissue secretion and pulp healing23. it is important to take into consideration that the residual water present in the hybrid layer and the dentin tubules may result in poor monomer-topolymer conversion and increase of monomer release17. another factor that has some impact on the severity of pulp response is the remaining dentin thickness or proximity of the injury stimulus to the pulp tissue: the thinner the dentin, the more severe the pulp inflammation16,18,23,28. a remaining dentin thickness of more than 0.5 mm is necessary to avoid causing any evidence of pulp injury17,28 about et al.23 concluded in their study that dentin bonding agents do not affect the cytodifferentiation of secondary odontoblasts when the remaining dentin thickness is 0.7 mm and when the materials are properly polymerized. on the other hand, the authors showed that the direct contact of uncured monomers affect the differentiation of odontoblasts from pulp fibroblasts and the process of mineralization23. the monomers in deep cavities may possibly reach the dental pulp in concentrations that disturb the redox state of pulp cells. the production of reactive oxygen species by these monomers has been related to the induction of cell death via apoptosis. the dentin works as an effective barrier, preventing cell damage from a great variety of materials and chemicals16. the advent of adhesive dentistry has broadened the range of possibilities of using resin-based materials for a wide variety of clinical applications in dentistry. amongst them, the possibility of direct pulp capping with bonding agents has increased19. dentin bonding agents have been examined as potential direct capping materials because of their superior ability to adhere to both demineralized enamel and dentin tissues24,29. hybridization of these agents with demineralized intertubular collagen as well as the diffusion of adhesives into the dentin tubules may seal the vital dentin against bacterial leakage and reduce secondary pulpal inflammation. however, in vitro studies have shown that acid etchants, monomers, and other ingredients in the composition of various adhesive systems can be toxic29 to nerves, cytotoxic to cultured cell lines, and mutagenic to microorganisms and mammalian cells24. according to an in vivo study30, when dentin adhesive systems are applied in direct contact with mechanically exposed pulp of healthy dog teeth acceptable repair of the dentin-pulp complex is not expected, such as wound healing with tertiary dentin bridge formation. therefore adhesive components (primer or adhesive) and composite resin should be avoided for pulp capping19,24,26,30. adverse tissue reactions to adhesive systems placed in direct contact with the pulp tissue might also be attributed to the presence of resin particles in the pulp space, triggering a foreign body response, or to the heat originated during light curing30. ca(oh) 2 should be the first choice for pulp capping19,29, which demonstrated biocompatibility in cement and dry powder forms29. final considerations the dental pulp is a unique tissue and its role is of great importance in the long-term prognosis of the teeth. clinicians must have a thorough understanding of the biological consequences of treatment interventions in this tissue. although the literature reports limited to reversible effects of defensive mechanisms of pulp against irritation caused by bleaching agents, careful manipulation is needed in the use of this material. another consideration is that although recent studies indicate that the risks of acute pulp toxicity to adhesive systems are unlikely to occur, it is clear that contemporary existing tests are not adequate to predict long-term clinical biological risks. based on this literature review including papers published over the last five years, it may be concluded that all dental procedures with bleaching agents or adhesive systems involve risks to cause pulp damage, but these risks can be minimized if the causal factors are known and avoided. references 1. yu c, abbott pv. an overview of the dental pulp: its functions and responses to injury. aust dent j endod. 2007; 52(1 suppl): s4-16. 2. bagis b, atilla p, cakar n, hasanreisoglu u. immunohistochemical evaluation of endothelial cell adhesion molecules in human dental pulp: effects of tooth preparation and adhesive application. arch oral biol. 2007; 52: 705-11. 3. caviedes-bucheli j, ariza-garcia g, restrepo-méndez s, rios-osorio n, lombana n, muñoz hr. the effect to tooth bleaching on substance p expression in human dental pulp. j endod. 2008; 34: 1462-5. 4. benneti ar, valera mc, mancini mng, miranda cb, baldicci i. in vitro penetration of bleaching agents into the pulp chamber. int endod j. 2004; 37: 120-4. 5. trindade fz, ribeiro apd, sacono nt, oliveira cf, lessa fcr, hebling j et al. trans-emanel and trans-dentinal cytotoxic effects of 35% h2o2 bleaching gel on cultured odontoblast cell lines after consecutive applications. int endod j. 2009; 42: 516-24. 6. cavalli v, arrais cag, giannini m, ambrosano gmb. high-concentration carbamide peroxide bleaching agents on enamel surface. j oral rehabil. 2004; 31: 155-9. 7. camargo sea, valera mc, camargo chr, mancini mng, menezes mm. penetration of 38% hydrogen peroxide into the pulp chamber in bovine and human teeth submitted to office bleach technique. j endod. 2007; 33: 1074-7. effects of bleaching agents and adhesive systems in dental pulp: a literature review braz j oral sci. 11(4):428-432 432432432432432 8. gökay o, mü jdeci a, algin e. peroxide penetration in to the pulp from whitening strips. j endod. 2004; 30: 887-9. 9. kawamoto k, tsujimoto y. effect of the hydroxyl radical and hydrogen peroxide on tooth bleaching. j endod. 2004; 30: 45-50. 10. joiner a. the bleaching of teeth: a review of the literature. j dent. 2006; 34: 412-9. 11. sulieman m, addy m, rees js. surface and intra-pulpal temperature rises during tooth bleaching: an in vitro study. br dent j. 2005; 199: 37-40. 12. sulieman m, addy m, rees js. surface and pulp chamber temperature rises during tooth bleaching using a diode laser: a study in vitro. br dent j. 2006; 200: 631-4. 13. buchalla w, attin t. external bleaching therapy with activation by heat, light or laser – a systematic review. dent mater. 2007; 23: 586-96. 14. zach l, cohen g. pulp response to externally applied heat. oral surg oral med oral pathol. 1965; 19: 515-30. 15. polydorou o, beiter j, könig a, hellwig e, kümmerer k. effect of bleaching on the monomers from modern dental composite material. dent mater. 2009; 25: 254-60. 16. demirci m, hiller k-a, galler k, schmalz g, schweikl h. the induction of oxidative stress, cytotoxicity, and genotoxicity by dental adhesives. dent mater. 2008; 24: 362-371. 17. çetingüç a, ölmez s, vural n. hema diffusion from dentin bonding agents in young and old primary molars in vitro. dent mater. 2007; 23: 302-7. 18. bouillaguet s. biological risks of resin-based materials to the dentin-pulp complex. crit rev oral biol med. 2004; 15: 47-60. 19. accorinte mlr, loguercio ad, reis a, muench a, araújo vc. adverse effects of human pulp after direct pulp capping with the different components from a total-etch, three-step adhesive system. dent mater. 2005; 21: 599-607. 20. lanza crm, costa cas, furlan m, alécio a, hebling j. transdentinal diffucion and cytotoxicity of self-etching adhesive systems. cell biol toxicol. 2009; 25: 533-43. 21. teixeira hm, do nascimento abl, hebling j, costa cas. in vivo evaluation of the biocompatibility of three current bonding agents. j oral rehabil. 2006; 33: 542-50. 22. goldberg m, smith aj. cells and extracellular matrices of dentin and pulp: a biological basis for repair and tissue engineering. crit rev in oral biol med. 2004; 15: 13-27. 23. about i, camps j, burger a-s, mitsiadis wt, franquin j-c. polymerized bonding agents and the differentiation in vitro of human pulp cells into odontoblast-like cells. dent mater. 2005; 21: 156-63. 24. sübay rk, demirci m. pulp tissue reactions to a dentin bonding agent as a direct capping agent. j endod. 2005; 31: 201-4. 25. costa cas, vaerten ma, edwars ca, hanks ct. cytotoxic effects of current dental adhesive systems on immortalized odontoblast cell line mdpc-23. dent mater. 1999; 15: 434-41. 26. accorinte mlr, loguercio ad, reis a, costa cas. response of human pulps capped with different self-etch adhesive systems. clin oral invest. 2008; 12: 119-27. 27. kenshima s, reis a, uceda-gomez n, tancredo l de l, filho le, nogueira fn, loguercio ad. effect of smear layer thickness and ph of self-etching adhesive systems on the bond strength and gap formation to dentin. j adhes dent. 2005; 7: 117-26. 28. murray pe, smith aj, windsor lj, mjör ia. remaining dentine thickness and human pulp responses. int endod j. 2003; 36: 33-43. 29. cavalcanti bn, rode sm, marques mm. cytotoxicity of substances leached or dissolved from pulp capping materials. int endod j. 2005; 38: 505-9. 30. koliniotou-koumpia e, tziafas d. pulpal responses following direct pulp capping of healthy dog teeth with dentine adhesive systems. j dent. 2005; 33: 639-47. effects of bleaching agents and adhesive systems in dental pulp: a literature review braz j oral sci. 11(4):428-432 oral sciences n3 original article braz j oral sci. july/september 2010 volume 9, number 3 received for publication: march 16, 2010 accepted: august 02, 2010 shear bond strength of dental ceramics to cast commercially pure titanium rodrigo galo1, deborah ganga frizzas1, renata cristina silveira rodrigues2, ricardo faria ribeiro3, maria da gloria chiarello de mattos3 1dds, phd, post-doctoral student, department of prosthodontics and dental materials, ribeirão preto dental school, university of são paulo, brazil 2dds, ms, phd, professor, department of prosthodontics and dental materials, ribeirão preto dental school, university of são paulo, brazil 3 dds, ms, phd, full professor, department of prosthodontics and dental materials, ribeirão preto dental school, university of são paulo, brazil correspondence to: maria da gloria chiarello de mattos department of prosthodontics and dental materials forp/usp. av. do café s/n, monte alegre, 14040-904 ribeirão preto, brasil phone: +55 16 3602-4098 fax : +55 16 3602-4780 e-mail: gloria@forp.usp.br abstract aim: the objective of this study was to evaluate the bond strength of four dental ceramics to commercially pure titanium. methods: to measure the resistance of metal-ceramic bonding, ceramic rings (noritake ti22®, triceran®, ips®, noritake ex-3®) were made around metal rods fused to commercially pure titanium. the area of metal-ceramic union was measured and, after mounting in type iii plaster, the rings were subjected to a shearing force in a universal testing machine at a crosshead speed of 2 mm/s until failure occurred. the metal-ceramic shear bond resistance was calculated in mpa. results: the shear bond strength means for the ceramics triceram and noritake ti22 (42.50 mpa and 61 mpa, respectively) were higher than the minimum value required by the din 13927 standard (25 mpa). the ceramics ips and noritake ex3, although not specifically formulated for titanium, also had shear bond strength means above the iso-recommended value (38.47 mpa and 29.04 mpa, respectively); however, there cracks in some specimens after burning and detachment of the ceramic from the metal. conclusions: the ceramic noritake ti22 should be indicated for the commercially pure titanium casting due to its higher mean bond resistance compared to other ceramics utilized. keywords: shear bond strength, titanium ceramic system, low firing ceramic. introduction the use of metal-ceramic restorations began in the late 1950’s, allowing the development of prosthetic rehabilitation with better cosmetic results. however, the actual mechanism of adhesion of ceramic to metal is complex and not fully understood mainly due to differences in thermal expansion and formation of oxide layer on the surface of dental metal alloys. these factors make the chemical union complicated, acting as an adhesion blocker associated with adherence reduction1-4. several metal alloys have been introduced to the fabrication of fixed partial dentures covered with ceramics. recent developments in casting techniques have enabled the construction of dentures using titanium, as they present excellent biocompatibility, good corrosion resistance and acceptable physical properties, and this have increased the application of titanium and its alloys in dentistry5-6. however, the bonding of ceramic to titanium is still a problem in the current use of metal-ceramic restorations6. one of the characteristics of titanium is that in the presence of oxygen an oxide layer is formed and adheres to titanium surface. while this oxide layer confers corrosion resistance, it decreases considerably the braz j oral sci. 9(3):362-365 363 bond strength at the metal ceramic interface. this layer is sometimes formed by oxides from the investment that react with titanium surface at high temperatures and interferes with the metal-ceramic7. thus, many titanium surface treatments have been proposed to minimize the problem, such as blasting or metal surface treatment with acid, among others2-8. there are still some unfavorable characteristics of titanium, such as the presence of porosities, problems with soldering and bonding to the dental ceramics9. some studies have shown that the adhesiveness of titanium to ceramics is comparable to the adhesion of ceramic to ni-cr alloys10, so the ratio of adhesiveness of titanium to ceramic materials needs further study to answer the question of high reactivity of titanium with certain elements11. considering all these aspects, the objective of this study was to test different metal-ceramic restorations, analyzing the bond strength of four dental ceramics to commercially pure titanium (cp ti). material and methods the four commercial ceramics used in study are listed in table 1. group ceramic manufacturer a noritake ti22® noritake super porcelain, noritake, nagoya, japan b triceran® triceram; esprident gmbh, ispringen, germany c ips® ivoclar-vivadent, schaan, liechtenstein d noritake ex-3® noritake superporcelain ex -3; noritake, nagoya, japan table 1. description of ceramic materials used in study. forty specimens of cp ti (tritan, grade 1; dentaurum, pforzheim, germany) were prepared. for obtaining the titanium rods, brass rods (3.0 mm in diameter and 75.0 mm in length) were included in titanium coating (rematitan plus, dentaurum ispringen jp kg, pforzheim, germany). after setting of the coating, the rods were removed from the refractory mold and subjected to thermal cycling in the oven (edg-7000 3p edg equipment and controls ltd, brazil), following the manufacturer’s instruction. titanium casting was performed on the rematitan machine (dentaurum, pforzheim, germany) by means of voltaic arc and injection through positive pressure of argon gas in the upper portion of the cylinder, and negative through the vacuum in the lower cylinder. after casting, the cp ti rods were divested and cleaned with carbide burs (702l; kg sorensen ind. com. ltda., barueri, sp, brazil) and airborne al 2 o 3 abrasion, which is a standard procedure recommended by composite manufacturers, was performed with particles of 110 µm in size for 20 s, under pressure of 30 to 40 psi and a distance of 3 to 5 cm12-13. after this treatment, the specimens were taken to the oven to 400° c to 750° c, where they remained for 10 min at 750° c without vacuum for pre-oxidation of the metal. during all these procedures the specimens were touched only by the opposite end where they would receive the ceramic ring, thus avoiding contamination of the site of ceramic application. the area to receive the ceramic was drawn with the help of two silicone cursors (optosil, heraeus kulzer south america ltda, brasil) and an acrylic spacer 2.5 mm thick (plexiglass; swedlow inc., gardengrove, ca, usa) to obtain a default in making the porcelain specimen, about 6.0 mm in diameter x 2.0 mm thick. in all specimens, ceramic application was performed by the same investigator. after the application of ceramics, the excess was removed with the aid of abrasive silicone polishers. the specimens were numbered and measured with a digital caliper as follows: two perpendicular measurements from the rod diameter immediately above and below the ring, and four equally spaced measurements from the thickness of the ceramic ring. the mean of the measurements represents, respectively, the rod diameter and thickness of the ceramic ring. these mean values were used to calculate the area of the metal-ceramic bonding using the following equation (eq. 1): s = π.ø.e, where = bonding area; ø = diameter of the rod; e = thickness of the ceramic ring12. to determine the resistance of the metal-ceramic bonding, the ceramic rings were embedded in stone cylinders (vigodent sa, ind. com., brazil), previously isolated with petroleum jelly and placed on a vibrator for plaster. the opposite end of the titanium rod was then attached to the liner and maintained centrally through the support resin with depression in the central of the pvc ring. after setting of the plaster and removing the pvc rings, the specimens were kept at room temperature for seven days so the plaster could dry, before the metal-ceramic bond resistance test. the specimens were subjected to a shearing force in a universal testing machine (emic mem 2000, emic equipment and systems testing ltd, brazil) at displacement speed of 2.0 mm/min and load cell 200 kgf. after failure, the peak load recorded was used to calculate the rupture tension as an indicator of bond strength of the metal-ceramic union using the following equation (eq.2): t = f / s x 9.8 mpa, where t = tension break; f = critical rupture load, s = area of the metal-ceramic union13. all data were analyzed by anova and tukey test to determine the adhesion of ceramics to cp ti. statistical analysis was performed with spss software for windows, version 12.0 (spss inc., chicago, il, usa). in each group, the interface between the ceramic and titanium were examined by scanning electron microscopy (sem). results the shear bond strength means of the titanium-ceramic bonding systems of each group are presented in table 2. there was a statistically significant difference among the groups (p<0.05) (table 2 and 3). group a presented the highest shear bond strength value (61.46 mpa) and groups b and c showed higher bond strength (42.50 and 38.47 mpa) than group d (29.04 mpa). sem micrographs (figure 1) of the surface area of the tested specimens showed unique characteristics of the reaction zone of each bonding system. the sem micrographs of the shear bond strength of dental ceramics to cast commercially pure titanium braz j oral sci. 9(3):362-365 364 sum of squares df mean square f p-value between groups 8342.3926 3 2780.7976 23.53 0.000* within groups 6617.2090 56 118.1644 total 14959.6016 59 table 2. comparisons of the mean of metal-ceramic bond strength values (anova). fig. 1. sem images of commercially pure titanium specimens with different ceramic materials used in study: noritake ti22® (a), triceran® (b), ips® (c) and noritake ex-3® (d). cross section of the tested titanium ceramic specimens demonstrated that the interface had porosities. large gaps were present in the titanium-ceramic bonding interfaces of group d, suggesting the poorest bond in this region. the sem micrographs of group b suggest that minimal differences existed at the metal ceramic interface compared to the other groups. it was observed that the bonding agent bonded to both the ceramic and titanium had no gap, but occupied a width of approximately 30 to 40 µm at the interface. this could be considered an excessive width for an optimal metal ceramic bond (figure 1). the titanium-ceramic specimens of group a showed similar bonding characteristics as observed. the interface produced with the bonding ceramic in the group a was more definite and regular than for the other titanium ceramic groups. however, there was less interfacial porosity for the ceramictitanium systems of groups b and c (figure 1). discussion the use of titanium for dental crown and bridge applications has increased the clinical importance of assessing their compatibility with ceramic systems. this study evaluated the bonding characteristics of four commercially available ceramic designed for use with titanium, by the shear bond strength test and sem analysis. shear bond strength refers to the force required for separating two parts, and it consists of two factors: mechanical bonding and chemical adhesion. mechanical bonding is an anchoring effect that is related to the surface roughness of the alloy surface, and chemical bonding compatibility implies formation of a strong bond during porcelain firing. therefore, both mechanical and chemical factors are essential to create stable bonding. the results of those tests showed that the shear bond strength of titanium ceramic in group a was higher than those others titanium ceramic systems used in this study. the excellence of metal-ceramic adherence was exhibited by the presence of a dentin ceramic layer on specimen surface after the shear bond strength test. as a result, higher shear bond strength was detected on the ceramic titanium in all specimens because the titanium ceramic groups exceeded the lower limit of 25 mpa established in the din 13927 standard14. the ceramic-titanium systems of group b showed higher results than the minimum established by the international standard (42.50 mpa), which may be due to low temperature burn, reducing the risk of overgrowth of metallic oxides which would result in a gain of adhesion, since one of the greatest problems of adhesion of ceramic to metal is to control the formation of the oxide layer7. the sem analysis showed that the titanium-ceramic systems of groups c and d had the poorest metal-ceramic bonds (figs. 1). the titanium ceramic-system of group a showed the best bonding and interfacial characteristics (fig. 1) because no definite oxide layer was observed at the interface between ceramic and titanium. however, it has been reported15 that failures in the titanium-ceramic system occurred at the oxide metal interface, suggesting poor oxide metal adherence. adachi et al. 16 reported complete delimitation of the ceramic from the titanium surfaces, after testing in a constant strain flexure apparatus, with the amount of the remaining ceramic being less than 1%, as occurred at the titanium ceramic interface in the other groups. moreover, lower shear bond strength between titanium and different low fusing ceramic, compared to other alloys and conventional ceramic, has been reported previously5. pröbster et al.17 found that the bond strengths of the titanium shear bond strength of dental ceramics to cast commercially pure titanium braz j oral sci. 9(3):362-365 groups mean (sd) a 61.46 (± 13.50) a b 42.50 (± 12.27) b c 38.47 (± 9.00) bc d 29.04 (± 7.67) c table 3 metal-ceramic mean shear bond strength values (mpa) for the four experimental groups. different letters indicate statistically significant difference at 5% (anova and tukey’s test). ceramic specimens ranged from 38-58% of the strength of the ni-cr ceramic control specimens. however, several studies10,18-19 have reported bond strength values between traditional prosthetic alloys and ceramic to be twice or even three times higher than the corresponding values of cast titanium with low fusing porcelains. only the results obtained in this study have proven to be much higher than the standard requires (42.50 mpa)14. it is likely that factors others than the efficient protection of the castings from contamination from phosphate-bonded investment materials may interfere with the metal ceramic bonding to titanium. one of them could be the high dissolution of oxygen within titanium and consequently its diffusion from the surface into the bulk material, as temperature increases. this diffusion may reduce the number of oxides, creating the conditions for a stronger metal ceramic bond, as previously reported20. it is claimed that at the firing temperature of the low fusing titanium ceramic (720o to 750o c) a dissociation of the superficial native titanium oxides takes place, followed by dissolution of elements within the titanium mass, accompanied by diffusion of the ceramic material elements, increasing the shear bond strength between titanium and ceramic in all groups. many researchers1,10,20-22 have reported mean values of shear bond strength different to those of the present study. more specifically, shear bond strength mean values of 27.79 mpa and 32.2 mpa has been given for noritake ti22 ceramic, when a magnesia investment was used1,21. in other studies1022 shear bond strength mean values different from those of the present study have been documented, even though different ceramics were used. when analyzing these results, it is important to examine the effects of these variables on both experimental groups separately because the ceramic properties and the fusion temperature are very different. however, from a clinical perspective, it was desirable to “rank” or arrange the effects of the interface variables on titanium ceramic adherence on the basis of the shear bond strength values regardless of the type of ceramic used. it is also important to realize that even though this is an in vitro study, the clinical implications of the results may be important. the authors are aware of the differences that exist between the ceramic on which this experiment was conducted and the oral environment. in conclusion, this study demonstrated that the type of ceramic affects the shear bond strength to commercially pure titanium. noritake ti22 should be indicated for commercially pure titanium casting due to its higher mean bond resistance compared to other ceramics used. references 1. atsü s, berksun s. bond strength of three porcelains to two forms of titanium using two firing atmospheres. j prosthet dent. 2000; 84: 567-74. 2. cai z, bunce n, nunn me, okabe t. porcelain adherence to dental cast cp titanium: effects of surface modifications. biomaterials. 2001; 22: 979-86. 3. oshida y, reyes mj. titanium-porcelain system. part iv: some mechanistic considerations on porcelain bond strengths. biomed mater eng. 2001; 11: 137-42. 4. könönen m, kivilahti j. fusing of dental ceramics to titanium. j dent res. 2001; 80: 848-54. 5. hautaniemi ja, hero h, juhanoja jt. on the bonding of porcelain on titanium. j mater sci. 1992; 3: 186-91. 6. wang rr, fenton a. titanium for prosthodontics applications: a review of the literature. quintessence int. 1996; 27: 401-8. 7. kimura h, hong cj, okazaki m, takahashi j. oxidation effects on porcelaintitanium interface reactions and bond strength. dent mater j. 1990; 9: 91-9. 8. al hussaini i, al wazzan ka. effect of surface treatment on bond strength of low-fusing porcelain to commercially pure titanium. j prosthet dent. 2005; 94: 350-6. 9. ida k, togaya t, tsutsumi s, suzuki m. mechanical properties of pure titanium and titanium alloysevaluation for dental casting metals. j jpn dent mater. 1983; 2: 765-71. 10. yilmaz h, dincer c. comparison of the bond compatibility of titanium and an ni-cr alloy to dental porcelain. j dent. 1999; 27: 215-22. 11. hanawa t, kon m, ohkawa s, asaoka k. diffusion of elements in porcelain into titanium oxide. dent mater j. 1994; 13: 164-73. 12. tarozzo lsa, de mattos mdc, ribeiro rf, semprini m. comparison of retentive systems for composites used as alternatives to porcelain in fixed partial dentures. j prosthet dent. 2003; 89: 572-8. 13. galo r, ribeiro rf, rodrigues rc, pagnano vo, mattos mg. effect of laser welding on the titanium composite tensile bond strength. braz dent j. 2009; 20: 403-9. 14. german standard institution. din 13927 metal-ceramic system. berlin: beuth verlag; 1990. 15. white sn, ho l, aa caputo, goo e. strength of porcelain fused to titanium beams. j prosthet dent. 1996; 75: 640-8. 16. adachi m, mackert j, parry e, fairhurst c. oxide adherence and porcelain bonding to titanium and ti–6al–4v alloy. j dent res. 1990; 6: 1230-5. 17. pröbster l, maiwald u, weber h. three-point bending strength of ceramics fused to cast titanium. eur j oral sci. 1996; 104: 313-9. 18. derand t, herø h. bond strength of porcelain on cast vs wrought titanium. scand j dent res. 1992; 100: 184-8. 19. lenz j, schwarz s, schwickerath h, sperner f, schäfer a. bond strength of metal–ceramic systems in three-point flexural bond test. j appl biomater. 1995; 6: 55-64. 20. könönen m, kivilathi j. bonding of low-fusing dental porcelain to commercially pure titanium. j biomed res. 1994; 28: 1027-35. 21. yoda m, konno t, takada y, iijima k, griggs j, okuno o. bond strength of binary titanium alloys to porcelain. biomaterials. 2001; 22: 1675-81. 22. troia m, henriques g, nobilo m, mesquita m. the effect of thermal cycling on the bond strength of low-fusing porcelain to commercially pure titanium and titanium–aluminum–vanadium alloy. dent mater. 2003; 19: 790-6. 365 shear bond strength of dental ceramics to cast commercially pure titanium braz j oral sci. 9(3):362-365 oral sciences n3 original article braz j oral sci. 10(2):152-157 original article braz j oral sci. april | june 2011 volume 10, number 2 caries experience associated to social and preventive factors in children of a pastoral community from limeira-sp maria julia pereira coelho ferraz1, dagmar de paula queluz2, marcelo corrêa alves3, carla cristina gonçalves dos santos4, miriam yumi matsui5 1ms, phd, post-doctorate researcher, department of community dentistry, piracicaba dental school, state university of campinas, brazil 2msph, phd, professor, department of community dentistry, piracicaba dental school, state university of campinas, brazil 3ms, phd student, department of morphology, piracicaba dental school, state university of campinas, brazil 4speech therapist, limeira, sp, brazil 5ms student, são josé dos campos school of dentistry, unesp – univ. estadual paulista, brazil correspondence to: dagmar de paula queluz departamento de odontologia social faculdade de odontologia de piracicaba unicamp avenida limeira, 901 cep 13414-900 piracicaba sp br phone: +55 19 21065277 fax: +55 19 21065218 e-mail:dagmar@fop.unicamp.br mjcoelhoferraz@hotmail.com abstract aim: to evaluate caries experience and prevalence associated to social and preventive factors in 3-6-year-old children of a pastoral community from limeira, brazil. methods: a cross-sectional study was carried out through an epidemiologic research about the oral conditions of 110 children registered at the children’s pastoral community from the city of limeira, sp, brazil. intraoral examination was performed by a trained dentist and the parents/caregivers filled out a questionnaire in order to outline the children’s profile with respect to gender, parental education and oral health. results: differences related to gender were not detected (p= 0.3404). the most frequent periods of breastfeeding were 0 to 2 months (36.36%) and 2 to 6 months (35.45%), corresponding to 71.81% of the cases. the time bottle feeding pointed to two most frequent categories: 1) more than 12 months (35.45%) and 2) between 2 and 6 months (22.63%). regarding parental education, the majority of the sample has not completed primary school (38.32%). it was observed a larger number of decayed teeth in male children (63.39%). caries experience was significantly higher in children who were breastfed for only 2 months of life (41.96%). children that did not use baby bottle or did not use a baby bottle for more than 1 year presented a smaller dmft, corresponding to 29.17% and 28.33%, respectively. time of use of bottle and pacifier presented a significant association (p<0.05). conclusions: the implementation of adequate strategies and actions is needed to reinforce oral health conditions in risk groups. keywords: dental caries, epidemiology, dental health surveys, school health services, dmft index, health promotion, oral health. introduction with the advent of the unified health system unified health system unified health system unified health system unified health system (sus) in the end of the 1980´s, a new challenge to the brazilian public systems of oral health assistance was introduced, since the implementation of initiatives was recommended to follow models based on epidemiologic information of oral health1. in order to monitor changes and trends in dentistry, emerich and castiel received for publication: february 03, 2011 accepted: june 16, 2011 153 braz j oral sci. 10(2):152-157 (2009)2 emphasized the new perspective for the dentist in the family health program. the challenge is to prepare engaged professionals with a comprehensive view: the objective of their practice must be a collective goal, taking in account their social context and innumerable aspects of life and not just the detection of restricted signals and symptoms. in brazil, few studies concerning the prevalence of caries in primary dentition have been done in the past years, though the importance of the detection of decayed teeth for the evaluation of oral condition in children3. dental caries is an important factor for the development of pain and functional disorders, which compromises esthetically the mouth4-5. the most recent epidemiological survey of oral health promoted by the ministry of health confirmed the trend of decline of caries in brazilian schoolchildren6.therefore, it is important to understand the diversity related to needs in the field of dental care arises as a fundamental task for the planning of oral health services, aiming at greater equity7. the multifactorial etiology of caries disease allows various types of interpretations about changes in caries prevalence according to time and level of development of the country. some shifts would have originated from alterations in feeding habits (especially sugar consumption), alterations in oral hygiene, increased contact with fluoride, alteration in ecology and/or virulence of bacterial plaque microflora and change in protection mechanisms, including the immunological condition8-9. regarding the access to dental services, the difficulty in obtaining proper treatment results in accumulated experience of decayed, missing or filled permanent teeth (dmft). the most disadvantaged strata of the society is characterized by the need of treatment, while the strata of society with higher income level is characterized by executed treatment10-11. the association between social and economic conditions (especially family income and level of education of household head) and caries prevalence has been observed in several studies, and children from families with highhighhighhighhigh socioeconomic socioeconomic socioeconomic socioeconomic socioeconomic status often have less decayed teeth than children from families with lower socioeconomicsocioeconomicsocioeconomicsocioeconomicsocioeconomic status12, pointing to the pointing the need for comprehensive measures to address other common childhood health problems13. this study evaluated dental caries experience and prevalence associated with social and preventive factors in 3 to 6 year-old children in limeira, sp, brazil. material and methods a cross-sectional study of prevalence with a convenient sample was undertaken with 110 children aged 3 to 6 years old, with complete primary dentition, who were registered at the children’s pastoral of the city of limeira from august 2007 to december 2009, from a total of approximately 1,000 children distributed in 24 communities. the research started after approval by the research ethics committee of the school of dentistry of piracicaba, university of campinas (protocol no. 144/2007). after the establishment of protocol activities, authorization from the coordinator of the children’s pastoral of the city of limeira was required. contact with the leaders of communities was made, explaining the study proposes and also requesting their support for development of the study. the parents were asked to sign an informed consent form..... children between 3 and 6 years old (both genders) were selected from the list of presence obtained from the celebration of life day (a monthly event, when all children are weighed). children that refused to participate, even with parental permission, as well as children who were not present in this event were not examined. in order to outline the children’s profile with respect to gender, parental education and oral health prevention, data were collected by means of questionnaire that was filled out the children’s parents/caregivers. intraoral clinical examination of each child was done by a single dentist. this examination was conducted under natural light, through visual inspection, with the child sitting in a chair facing the examiner. gloves, head covers, masks, wooden spatulas, disposable gauze, as well as properly sterilized dental mirrors and periodontal probes (community periodontal index – cpi) were used. dental caries experience was evaluated by the dmft index according to world health organization’s diagnosis criteria14. descriptive statistical analysis was calculated from measures from oral health and socioeconomic data. the dependent variable dmft was divided in dmft <3 and dmft > = 3, using as a reference descriptive statistics, which pointed this number as higher than average. the independent variables used in this study were: gender, duration and type (breastfeeding and bottle-feeding) of nursing, pacifier use, finger sucking and parental education. the significant association between the dependent and independent variables was tested by bivariate analysis (chi-square), with a significance level of 0.05. regarding the strength and direction of association, the odds ratio (or) was used. in order to find association between the variables supposedly related to caries experience, fisher’s test, with significance level of 0.01, was used to analyze the possible association between: duration and type (breastfeeding and bottle-feeding) of nursing,; the presence and absence of pacifier habit or sucking habits; and the dmft. sas (sassassassassas/ stat® software, campus drive cary, nc, usa) was the program used for the statistical analysis15. results the final sample consisted of 110 children from six communities of the children’s pastoral of the city of limeira. strong evidence was identified (p <0.01) for the existence of average higher than zero for number of cavities and dmft per child, besides the evidence (p <0.05) of the existence of difference between average zero and number of fillings. no evidence was found (p = 0.3195) between average zero and number of extractions (table 1). table 2 provides an analysis of demographic caries experience associated to social and preventive factors in children of a pastoral community from limeira-sp 154 braz j oral sci. 10(2):152-157 indicator mean standard deviation limits of the confidence interval (95%) t testh0: m=0p-value superior inferior decayed 2.036 3.202 2.642 1.431 0.0001 missing 0.036 0.381 0.108 -0.036 0.3195 filled 0.109 0.531 0.209 0.009 0.0333 dmft: 2.182 3.299 2.805 1.558 0.0001 table 1table 1table 1table 1table 1. mean value, standard deviation, limits of the confidence interval and p value calculated from student t-test to test the hypothesis that the mean value is 0 (n=110). factors factors frequency (n) percentage (%) p-value gender female 50 45.45 male 60 54.55 total 110 100.00 0.3404 how long did you breastfeed your child 0 months 11 10.0 0—| 2 months 40 36.36 2 —| 6 months 39 35.45 6 —| 12 months 19 17.27 > 12 months 1 0.91 total 110 100.00 0.0001 time of bottle use it was not used 18 16.36 0—| 2 months 15 13.64 2 —| 6 months 25 22.73 6 —| 12 months 13 11.82 > 12 months 39 35.45 total 110 100.00 0.0001 time of pacifier use 0 67 60.91 0—| 1 year 4 3.64 1 —| 2 years 12 10.91 2 —| 3 years 8 7.27 > 3 years 19 17.27 total 110 100.00 0.0001 time of finger sucking he/she did not have this habit 100 91.74 0—| 1 year 4 3.67 2 —| 3 years 5 4.59 total 110 100.00 0.0001 educational level of the person who filled out the questionnaire none 1 0.93 incomplete primary school 41 38.32 complete primary school 21 19.63 incomplete secondary school 19 17.76 complete secondary school 25 23.36 total 110 100.00 0.0001 table 2table 2table 2table 2table 2. frequencies, percentages and p value calculated by chi-square test for the hypothesis of equal proportions. characteristics used as determinants of oral health in this study. no evidence of differences between the children in relation to gender is present (p: 0.3404). strong evidence that the differences between the true proportions of children according to all other factors (duration of breastfeeding, bottle feeding time, duration of pacifier use, thumb sucking time and parents’ schooling level) was found. regarding the duration of breastfeeding, it was observed that the periods of 0 to 2 months (36.36%) and 2 to 6 months (35.45%) are the most frequent, together accounting for 71.81% of the cases. the time of baby bottle use can be divided into two most frequent groups: those who used it for more than 12 months (35.45%) and those who used it for a period lasting between 2 and 6 months (22.63%). the second group showed a lower frequency than the first, but still stands out in relation to others that ranged between 11.82 and 16.63%. caries experience associated to social and preventive factors in children of a pastoral community from limeira-sp 155 braz j oral sci. 10(2):152-157 regarding the duration of pacifier use, most children (60.91%) did not use it, with a relatively even distribution within the periods. in relation to finger sucking, this habit can be considered as a rare practice since only 8.26% of the sample acquired the habit against 91.74% who did not. finally, in terms of education of the person that filled out the questionnaire (parent/caregiver), the majority of the sample did not complete primary school (38.32%) while the rest were roughly evenly distributed: 17.76% did not complete secondary school, 19.63% completed the primary school and 23.36% completed secondary school. only one person did not attend any formal education. table 3 presents the comparison of the frequency and proportions of oral health indicators: number of cavities (d), number of missing teeth (m) and number of filled teeth (f), which together comprise the indicator of caries experience in the primary dentition (dmft). it can be initially observed that there were a larger number of decayed teeth in male children (63.39%), a proportion that is significantly higher than in females (36.61%). interestingly, the quantification of missing teeth according to gender showed that all four extractions happened in girls (100.0%). the proportion of filled teeth did not differ significantly between the genders, while the dmft, influenced by the component number of decayed teeth, is significantly higher in male children. in relation to breastfeeding, it was observed that caries experience was significantly higher in children who were breastfed up to the age of 2 months 41.96%). children who did not use baby bottle or who did not carry this habit for more than 1 year had a smaller percentage of dmft: 29.17% and 28.33% respectively. pacifier use (76.79%) and finger sucking (84.2%) suggested that they do not interfere in the caries experience, since those with decayed teeth did not have these habits. table 4 shows the results of bivariate analysis for dmft. the duration of bottle feeding and pacifier use showed a significant association (p <0.05). or (odds ratio) indicated that the bottle use for less than two months is not harmful and that it is associated with a higher probability (rate of 1.753) of having a dmft value lower than 3. similarly, pacifier use for less than 1 year was not harmful either and increased by 2.941 times the probability of having a dmft value lower than 3. discussion developing oral health strategies directed to groups of population requires knowledge, which can be obtained from epidemiology. the basic condition for this is the use of various instruments according to the characteristics of each disease or condition, in order to obtain a better approximation to the real conditions of oral health16. in order to access the prevalence and severity of caries according to who recommendations, this study used the dmft index. this index counts the primary teeth somehow affected by caries (decayed, missing or filled). based on this information, it was possible to evaluate the oral health and particularly caries experience in children from the city of limeira associating them with oral habits and parental level of education. for evaluations at the individual level, the variable was defined by the involvement of at least one primary tooth with untreated caries (component “d” of the dmft > 3), and evaluations at the collective level, the variable was defined by the proportion of children presenting this condition in each area. this study confirmed the existence of average significantly higher than 0 for the number of caries and dmft per child, not reaching the goals recommended by who for the year 2000 or corroborating the last survey sponsored by the ministry of health in 20106. furthermore, this provides information for dental caries prevention and the attention about access to dental treatment, since it can be observed that children who need treatment present few filled teeth16. in relation to habits associated with caries experience, it was found that pacifier use was not related to caries experience in this study since 60.91% did not use a pacifier. this finding disagree with those of yonezu and yakushiji (2008)17 and vázquez-nava et al. (2008)18 who found that pacifier use for 18 months of life is a risk factor for caries development and that this risk is two times greater in children who use pacifiers compared to those who do not have this habit. anyway, considering the aspect of craniofacial growth (open bite, crossbite, inadequate posture of the tongue, articulation alterations), pacifier use can be related to dentofacial changes and depends on the duration, frequency and intensity of habit19-20. in relation to finger sucking, 91.74% of children did not have acquired this habit, which is consistent with castilho and rocha (2009)20 who reported that the pacifier is used not just to calm down, but can also be offered in order to prevent the habit of finger sucking. regarding breastfeeding, only 35.45% of children were breastfed during the period of 2-6 months of life, emphasizing the importance of encouraging this healthy practice. breastfeeding favors growth and development of children, not only for their nutritional, immunological and psychological aspects but also because it enables the harmonious growth of the face, promoting the maturation of the stomatognathic system functions 21-22. in this study, however, it was found that breastfeeding in a period from 6 to 12 months suggested a protection against caries. children who used the bottle for a period longer than 12 months presented a higher dmft value. according to the literature the use of the bottle is a very common practice in 62.8% of children under the age of 1 year in brazil7. data from the latest research of oral health in brazil23, which showed that dmft was 2.80, can be compared to the finding of the present study (average of 2.18). it can also be observed an inequality in the distribution of decayed component (2.03), filled (0.109) and missing teeth (0.03). it is suggested that the access to dental services for the population represented by this sample is not satisfactory since a small number of restored teeth were observed, confirming gomes, et al. (2004)24. it is important to emphasize that the impact of health services in disease prevention is still controversial. defenders of the role of services argue that access to effective treatments caries experience associated to social and preventive factors in children of a pastoral community from limeira-sp 156 braz j oral sci. 10(2):152-157 factors decayed (d) missing (m) filled (f) total (d+m+f) n % n % n % n % gender female 82 36.61 4 100 54 1.67 91 37.92 male 142 63.39 0 0,00 7 58.33 149 62.08 p-value 0.0001 0.5637 0.0002 how long did you breastfeed your child? 0 months 34 15.18 0 0,00 34 14.17 0—| 2 months 94 41.96 4 100 6 50.00 104 43.33 2 —| 6 months 76 33.93 0 0,00 3 25.00 79 32.92 6 —| 12 months 20 8.93 0 0,00 3 25.00 23 9.58 p-value 0.0001 0.4724 0.0001 time of bottle use did not use 70 31.25 0 0.00 0 0.00 70 29.17 0—| 2 months 38 16.96 0 0.00 5 41.67 43 17.92 2 —| 6 months 24 10.71 0 0.00 0 0.00 24 10.00 6 —|12 months 26 11.61 4 100 5 41.67 35 14.58 > 12 months 66 29.46 0 0.00 2 16.67 68 28.33 p-value 0.0001 0.4724 0.0001 time of pacifier use did not use 172 76.79 4 100 11 91.67 187 77.92 0—| 1 year 6 2.68 0 0.00 0 0.00 6 2.50 1 —| 2 years 12 5.36 0 0.00 0 0.00 12 5.00 2 —| 3 years 14 6.25 0 0.00 1 8.33 15 6.25 > 3 years 20 8.93 0 0.00 0 0.00 20 8.33 p-value 0.0001 0.0039 0.0001 time of finger sucking 0 187 84.23 4 100 10 83.33 201 84.45 0—| 1 year 15 6.76 0 0,00 0 0.00 15 6.30 2 —| 3 years 20 9.01 0 0,00 2 16.67 22 9.24 p-value 0.0001 0.0209 0.0001 educational level of the person who filled out the questionnaire incomplete primary school 84 39.44 4 100 8 66.67 96 41.92 complete primary school 46 21.60 0 0,00 2 16.67 48 20.96 incomplete secondary school 41 19.25 0 0,00 1 8.33 42 18.34 complete secondary school 42 19.72 0 0,00 1 8.33 43 18.78 p-value 0.0001 0.0101 0.0001 table 3table 3table 3table 3table 3. frequency and percentage of occurrence of decayed, missing and filled teeth (dmft) in different strata defined for the survey. factors dmft < 3 > 3 or (ic 95%) n % n % p female 38 4872 12 37.5 0,632 (0.272 – 1.466) male 40 51.28 20 62.5 1.000 0.2849 how long did you breastfeed your child? < 2 months 33 42.31 18 56.25 1.753 (0.764 – 4.022) > 2 months 45 57.69 14 43.75 1.000 0.1851 time of bottle use < 2 months 18 23.08 15 46.88 2.941 (1.230 – 7.031) > 2 months 60 76.92 17 53.13 1.000 0.0153 time of pacifier use < 1 year 45 57.69 26 81.25 3.178 (1.175 – 8.594) > 1 year 33 42.31 6 18.75 1.000 0.0227 time of finger sucking < 1 year 74 96.10 30 93.75 0.608 (0.097 – 3.824) > 1 year 3 3.90 2 6.25 1.000 0.5959 educational level of the person did not attend who filled out the questionnaire secondary school 56 73.68 26 83.87 0.538 (0.182 – 1.593) attended secondary school 20 26.32 5 16.13 1.000 0.2634 table 4table 4table 4table 4table 4. frequency and percentage of the number of dmft caries experience associated to social and preventive factors in children of a pastoral community from limeira-sp braz j oral sci. 10(2):152-157 can reduce the level of disease25. however, the effectiveness of preventive interventions, even when proven in controlled clinical trials under ideal conditions, is not always confirmed in the day-to-day services, under realistic conditions26-27. only 3% of the decline of caries between the 1970s and 1980s in developed countries can be attributed to dental services, while 65% were explained by large improvements in socioeconomic conditions28. this study shows that children assisted by the children’s pastoral of limeira belong to poor families, being educated by parents with incomplete schooling. considering that oral health education should begin early in life, it an integrated approach is important including social communication improvement and professionals of general and oral health training, aiming to educate families, especially mothers13,29-30. dental caries experience in communities of the pastoral of the city of limeira emphasizes the importance of maintenance and continuity of the potential capacity of local agents. in general, the obtained results suggest an association of social issues with inequities in oral health, as indicated actions integrating public health care policies. in addition, this study reflects the importance of referral of the most vulnerable and/or more exposed groups to risk factors to services focused on oral health promotion. acknowledgments the authors thank the children’s pastoral of the city of limeira for their support in developing this study. references 1. roncalli ag, frazão p, pattussi mp, araújo ic, ely hc, batista sm. projeto sb2000: uma perspectiva para a consolidação da epidemiologia em saúde bucal coletiva. rev bras odontol saude coletiva. 2000; 1: 9-25. 2. emmerich a, castiel ld. jesus tem dentes metal-free no país dos banguelas? odontologia dos desejos e das vaidades. hist cienc saudemanguinhos. 2009; 16: 95-107. 3. noro lra, roncalli ag, mendes júnior fir, lima kc. a utilização de serviços odontológicos entre crianças e fatores associados em sobral, ceará, brasil. cad saude publica. 2008; 24: 1509-16. 4. mcgrath c, broder h, wilson-genderson m. assessing the impact of oral health on the life quality of children: implications for research and practice. community dent oral epidemiol. 2004; 32: 81-5. 5. antunes jlf, peres ma, mello trc. determinantes individuais e contextuais da necessidade de tratamento odontológico na dentição decídua no brasil. cienc saude coletiva. 2006; 11: 79-87. 6. ministério da saúde. projeto sb brasil 2010: pesquisa nacional de saúde bucal 2010. brasília: ministério da saúde; 2010. available from: http:// www.sbbrasil2010.org. 7. lucas sd, portela mc, mendonça ll. variações no nível de cárie dentária entre crianças de 5 e 12 anos em minas gerais, brasil. cad saude publica. 2005; 21: 55-63. 8. peres kg, bastos jr, latorre mr. severidade de cárie em criançase relação com aspectos sociais e comportamentais. rev saude publica. 2000; 34: 402-8. 9. selwitz rh, ismail ai, pitts nb. dental caries. lancet. 2007; 369: 51-9. 10. pinto vg. saúde bucal coletiva. 4.ed. são paulo: santos; 2000. 11. maltz m, silva bb. relação entre cárie, gengivite, fluorose e nível socioeconômico em escolares. rev saude publica. 2001; 35: 170-6. 12. galindo emv, pereira jac, feliciano kvo, kovacs mh. prevalência de cárie e fatores associados em crianças da comunidade do vietnã, recife. rev bras saude mater infant. 2005; 5: 199-208. 13. melo mmdc, souza wv, lima mlc. fatores associados à cárie dentária em pré-escolares do recife, pernambuco, brasil. cad. saude publica. 2011; 27: 471-85. 14. world health organization. oral health surveys, basic methods. 4th ed. geneve: who; 1997. 15. sas. institute inc. the sas system, release 9.2 ts level 2m0. sas institute inc., cary: nc; 2008. 16. narvai pc, biazevic mgh, junqueira sr, pontes ercj. diagnóstico da cárie dentária: comparação dos resultados de três levantamentos epidemiológicos numa mesma população. rev bras epidemiol. 2001; 4: 72-80. 17. yonezu t, yakushiji m. longitudinal study on influence of prolonged nonnutritive sucking habits on dental caries in japanese children from 1.5 to 3 years of age. bull tokyo dent coll. 2008; 49: 59-63. 18. vázquez-nava f, vázquez re, saldivar ga, beltrán gf, almeida av, vázquez rc. allergic rhinitis, feeding and oral habits, toothbrushing and socioeconomic status. effects on development of dental caries in primary dentition. caries res. 2008; 42: 141-7. 19. silva el. hábitos bucais deletérios. rev para med. 2006; 20: 47 50. 20. castilho sd; rocha mam. uso de chupeta: história e visão multidisciplinar. j pediatr. 2009; 85: 480-9. 21. palmer b. influence of breastfeeding on the development of the oral cavity. j hum lact. 1998; 14: 93-8. 22. coelho ferraz mjp. respirador bucal uma visão multidisciplinar. são paulo: lovise; 2004. 23. brasil. ministério da saúde. secretaria de atenção à saúde. departamento de atenção básica. coordenação nacional de saúde bucal. projeto sb brasil 2003. condições de saúde bucal da população brasileira 20022003. resultados principais. brasília: ministério da saúde; 2004. 24. gomes pr, costa sc, cypriano s, sousa mlr. dental caries in paulínia, são paulo state, brazil, and who goals for 2000 e 2010. cad saude publica. 2004; 20: 866-70. 25. andrulis dp. access to care is the centerpiece in the elimination of socioeconomic disparities in health. ann intern med. 1998; 129: 412-6. 26. rychetnik l, frommer m, hawe p, shiell a. criteria for evaluating evidence on public health interventions. j epidemiol community health. 2002; 56: 119-27. 27. victora cg, habicht jp, bryce j. evidence-based public health: moving beyond randomized trials. am j public health. 2004; 94: 400-5. 28. nadanovsky p, sheiham a. relative contribution of dental services to the changes in caries levels of 12-year-old children in 18 industrialized countries in the 1970s and early 1980s. community dent oral epidemiol. 1995; 23: 331-9. 29. sheiham a. the role of the dental team in promoting dental health and general health through oral health. int dent j. 1992; 42: 223-8. 30. freire mcm; macedo ra.; silva wh. conhecimentos, atitudes e práticas dos médicos pediatras em relação à saúde bucal. pesq odont bras. 2000; 14: 39-45. 157 caries experience associated to social and preventive factors in children of a pastoral community from limeira-sp oral sciences n3 original article braz j oral sci. july/september 2010 volume 9, number 3 bone mineral density on conventional and digitized images under different parameters of digitization and storage matheus lima de oliveira1, frab norberto bóscolo2, guilherme monteiro tosoni3 1 dds, msc, graduate student, department of oral diagnosis, piracicaba dental school, state university of campinas, brazil 2 dds, msc, phd, professor, department of oral diagnosis, piracicaba dental school, state university of campinas, brazil 3 dds, msc, phd, associate professor, department of diagnosis and surgery, araraquara dental school, sao paulo state university, brazil received for publication: november 19, 2009 accepted: august 25, 2010 correspondende to: matheus lima de oliveira department of oral diagnosis, piracicaba dental school, state university of campinas unicamp 13414-903, piracicaba, sp, brazil phone / fax : 55-19-21065327 e-mail: matheusoliveira@hotmail.com abstract aim: to assess the bone mineral density on conventional and digitized images, comparing whether different parameters of digitization and storage change these values. methods: twenty radiographs were taken from five partially dentulous dry mandibles with an aluminum 7-mm stepwedge placed on the superior edge of the film. after processing, the films were digitized with a resolution of 600 and 2,400 d.p.i. and saved as tiff and jpeg files. on every conventional and digitized image, circular regions of interest were selected for densitometry and radiographic contrast analysis. results: pearson’s correlation coefficient showed a significant and strong mean gray values association between digitized and conventional images, differing from radiographic contrast that did not show a significant association. anova did not reveal a statistically significant difference in bone density and radiographic contrast among the four digitized image groups, but the conventional image contrast was significantly lower. conclusions: bone mineral density did not differ in both conventional and digitized images. the parameters of image compression and resolution, tested in this study, did not change the results of densitometry and digitization process increased the radiographic contrast. keywords: bone density, densitometry, dental radiography, digital dental radiography. introduction despite the large number of studies and the development of several direct digital dental systems in the last 20 years, conventional radiography is still being used by dentists, considering that film is reliable and inexpensive when compared with direct digital radiography1-4. although this scenario tends to be changed in the near future, converting the existing records of film-based images to digital is one important issue to be currently addressed. digitized radiography has preceded direct digital radiography and can be useful for quantitative analysis of images5-6. while conventional image is permanent and cannot be changed, digitized image can be post processed by the examiner7-8. besides these advantages, digitized image presents many additional options, such as digital storage, compression and exchange of radiographic information2,4. radiographic images have been used to evaluate object density such as bone tissue9 and dental materials10-13. density on film-based images is quantified by braz j oral sci. 9(3):398-401 399 measuring the optical density. after digitization process, the digitized image also can be used for this purpose using imaging analysis software. however, the analog conversion of film-based image into digital files involves many parameters, and some loss of information may occur, hindering the bone density analysis2. the aim of this study was to assess the bone mineral density on conventional and digitized images, comparing whether different parameters of digitization and storage change these values. material and methods this study was designed according to the local institutional research ethics committee and carried on after its approval (protocol #33/05). five partially dentulous dry mandibles, presenting teeth both on anterior or posterior region, were selected. the teeth were radiographed using the ge 1000 unit (general electric, milwaukee, wi, usa), operating with 70 kvp, 3 mas and 40 mm source-to-film distance. in all 20 radiographic acquisition, insight kodak #2 films were vertically placed in parallel position to the tooth, fixed with adhesive tape and utility wax. a custom-made aluminum 7mm-stepwedge was placed on the superior edge of the film and a 24-mm-thick acrylic plate simulated the soft tissue scatter. automatic film processing was performed in a darkroom with the dent-x 9000 unit (elmsford, ny, usa), set in 6 min. the film-based images were digitized with agfa snapscan 1236s flatbed scanner transparency unit (mortsel, belgium), using dedicated software (agfa fotosnap 32v3.00.05), which allowed digital conversion and storage. the digitization process was performed in duplicate. primarily, the films were scanned with 2,400 d.p.i. (dots per inch), the maximum scanner resolution, and subsequently, with 600 d.p.i. each digitized image was saved into two different file formats, tiff (tagged image file format) and jpeg (joint photographic experts group). the tiff file format corresponded to the raw image, with no compression, while the jpeg corresponded to compressed image format. thus, the film-based image yielded four different groups of digitized images (table 1). group resolution (d.p.i.) format mean size (mb) i 2,400 tiff 2.66 ii 600 tiff 0.174 iii 2,400 jpeg 0.874 iv 600 jpeg 0.0929 table 1. digitized image groups according to file resolution, format and mean size. all images were analyzed using imagej version 1.32j (http://rsb.info.nih.gov/ij/docs/), a public domain software developed by the national institutes of health (nih-usa), and none of them was post processed. on each image, nine circular regions of interest (roi) with 2 mm in diameter were selected using appropriate tool of the software. roi (a) was randomly selected on trabecular alveolar bone, away from adjacent anatomical structures such as lamina dura, mandibular canal, mental and lingual foramen, mental ridge and etc. roi (1) was chosen as background image and represented the most radiolucent region of the image. rois (2-8) were selected on each step of the aluminum 7-mm stepwedge. to have reproducible rois in all digitized image groups, the coordinate values of each roi were registered and this value was used as reference to select the same roi. mean gray values measurements were performed. the mra cq-01 densitometer unit (ribeirão preto, sp, brazil), with a 2-mm diameter circular aperture, measured optical density on each roi of the film-based image. the measurements were performed in triplicate and the mean value of optical density was registered. to insure that the corresponding rois were measured on the densitometer with the same diameter and position previously selected, the digitized images, with all rois marked on it, were printed on a sheet of cellulose acetate. this acetate was cropped and overlapped the film, helping as a template. in order to convert optical density values into mean gray values, all optical density measurements of roi 1 to roi 8 were plotted against the correspondent measurements of mean gray value using microsoft excel 2007 for windows. the equation of the trend line between these values (y = -105.67x + 207.12 and r2 = 0.9912) was used for the conversion, as demonstrated in figure 1. the mean gray values measured on roi 4 and roi 5 were chosen to establish the radiographic contrast of the conventional and digitized images due to their uniformity and similarity to the bone density, using the following equation: radiographic contrast = roi (5) roi (4). pearson’s correlation coefficient was calculated between bone mineral density on conventional and digitized images under different parameters of digitization and storage braz j oral sci. 9(3):398-401 fig. 1 – conversion of optical density values into mean gray values. (y = -105,67x + 207,12 and r2 = 0.9912) 400 group i i i i i i iv ii r 0.990* iii r 1.000* 0.990* iv r 0.985* 0.993* 0.985* conventional r -0.953* -0.936* -0.953* -0.930* table 2. pearson’s correlation coefficient for mean gray value of all the image groups (n=20 per group). *p<0.001 group i i i i i i iv ii r 0.845* iii r 1.000* 0.845* iv r 0.843* 0.998* 0.843* conventional r -0.151 -0.151 -0.154 -0.164 p 0.525 0.524 0.516 0.491 table 3. pearson’s correlation coefficient for mean values of radiographic contrast among all image groups. *p<0.001 mean gray values and contrast measurements of all image groups. anova for differences between image groups and tukey’s test were performed. the significance level was set at 0.05. results pearson’s correlation coefficient showed a negative, significant and strong mean gray values association between digitized and conventional images (table 2). a significant and strong correlation could be observed in the radiographic contrast among the four digitized image groups, which differs of the conventional image group that did not show a significant association when compared with the four digitized image groups (table 3). anova did not reveal a statistically significant difference in mean gray values (figure 2) and radiographic contrast (figure 3) among the four digitized image groups, but the contrast of conventional images was significantly lower (p<0.001) than the digitized images, as can be observed in figure 3. discussion the final quality of the radiographic image may be modified by the digitization process14-16. it includes factors inherent to the digitization process such as file resolution and format. in this study, an obsolete flatbed scanner captured the images in resolutions of 600 and 2400 d.p.i. the former corresponds to a fair image size and a short scan time and the latter corresponds to the maximum scanner resolution. as it is known that the spatial resolution contributes to image quality17, it was chosen to compare a widely used resolution with the maximum possible resolution, and although 2,400 d.p.i. had been selected, the images presented an actual resolution of 1,200 d.p.i.. moreover, modern digital radiographic imaging systems allow high resolution image acquisition and the file size is very close to the file sizes found in this study. in addition, each digitized image was saved in two different file formats, tiff and jpeg. the tiff file corresponded to the original file format of the image, with no compression, while the jpeg format corresponded to a compressed image, with a smaller file size. the image compression in jpeg is sufficient even for the challenging task of radiographic detection of non-cavitated proximal carious lesions18. however, the jpeg compression can introduce potentially deleterious variations to radiodensity data19. it is hard to compare the degree of information loss related to image compression in jpeg because its compression scale is not standardized and software applications have different or even opposing compression scales19-20. thus, four digitized images with different features of resolution and format were assessed. although it could be expected that different resolutions and file formats would yield different results of densitometry21, this was not found in the present study. this might have happened due to the minimum resolution used in this study (600 d.p.i.), considering that lower resolution such as 300 d.p.i. is considered good enough even for dental caries diagnosis15. bone mineral density on conventional and digitized images under different parameters of digitization and storage braz j oral sci. 9(3):398-401 fig. 2 – comparison of mean gray values (± standard deviation) among all image groups. fig. 3 – comparison of mean values of radiographic contrast (± standard deviation) among all image groups. 401 the digitized images showed significant and strong correlation between the bone density values, with no statistical difference. it means that none of the parameters tested in this study (digitized image resolution and compression) interfered in the bone mineral density measured in mandibular alveolar bone. thus, under experimental conditions established in this study, it can be recommend the combination of 600 d.p.i. and jpeg file as a parameter for digitized images in studies of densitometry (group iv); since this group of image presented a 0.0929 mb mean file size, which is nearly 28 times smaller than the image group of maximum resolution (group i), and represents a short time scan, small storage size and a faster transmission. bone mineral density values of conventional and digitized images were statistically similar. the negative correlation is due to density values being inversely proportional when measured in conventional and in digitized images. our findings demonstrated that the results of densitometry are similar when it is conducted on radiographic films using a densitometer device or when it is conducted on digitized image, using image analysis software. therefore, the digitization process can be used as an indirect method to measure bone mineral density, replacing the densitometer and presenting benefits such as different storage and transmission possibilities. the results of image contrast were also very similar among the digitized images but they were significantly higher than conventional image. these findings were expected and they are in agreement with parissis et al.16. digitizing process with 8-bit expands automatically the grayscale resolution to fill the scale from 0 to 255 resulting in increased image contrast. nevertheless, this feature in the displayed image does not interfere with densitometry results. the limitation of this study is that other parameters of measuring image quality, such as noise, have not been tested. it has been reported in the literature22 a trade-off between noise and resolution. as regards this parameter, since our study captured the images with two resolutions (600 and 2,400 d.p.i.), and based on our results of densitometry, it can be inferred that noise does not seem to have an effect on bone mineral density measurements in digitized images, if this latter is captured using these two resolutions. in conclusion, bone mineral density did not differ in conventional and digitized images. the parameters of image compression and resolution, tested in this study, did not change the results of densitometry, and the digitization process increased the radiographic contrast. acknowledgments the authors gratefully acknowledge financial support from fapesp, a research foundation of the state of sao paulo – brazil (process # 2005/04615-3). references 1. baksi bg, ermis rb. comparison of conventional and digital radiography for radiometric differentiation of dental cements. quintessence int. 2007; 38: 532-6. 2. christensen gj. why switch to digital radiography? j am dent assoc. 2004; 135: 1437-9. 3. farman ag, farman tt. a status report on digital imaging for dentistry. oral radiol. 2004; 20: 9-14. 4. parissis n, angelopoulos c, mantegari s, karamanis s, masood f, tsirlis a. a comparison of panoramic image quality between a digital radiography storage phosphor system and a film-based system. j contemp dent pract. 2010; 11: 9-16. 5. güneri p, lomçali g, boyacioðlu h, kendir s. the effects of incremental brightness and contrast adjustments on radiographic data: a quantitative study. dentomaxillofac radiol. 2005; 34: 20-7. 6. versteeg ch, sanderink gc, van der stelt pf. efficacy of digital intra-oral radiography in clinical dentistry. j dent. 1997; 25: 215-24. 7. haiter-neto f, casanova ms, frydenberg m, wenzel a. task-specific enhancement filters in storage phosphor images from the vistascan system for detection of proximal caries lesions of known size. oral surg oral med oral pathol oral radiol endod. 2009; 107: 116-21. 8. van der stelt pf. better imaging: the advantages of digital radiography. j am dent assoc. 2008; 139: 7-13. 9. erdogan o, incki kk, benlidayi me, seydaoglu g, kelekci s. dental and radiographic findings as predictors of osteoporosis in postmenopausal women. geriatr gerontol int. 2009; 9: 155-64. 10. braun ap, grassi soares c, glüer carracho h, pereira da costa n, bauer veeck e. optical density and chemical composition of microfilled and microhybrid composite resins. j appl oral sci. 2008; 16: 132-6. 11. erdogan o, incki kk, benlidayi me, seydaoglu g, kelekci s. dental and radiographic findings as predictors of osteoporosis in postmenopausal women. geriatr gerontol int. 2009; 9: 155-64. 12. goga r, chandler np, love rm. clarity and diagnostic quality of digitized conventional intraoral radiographs. dentomaxillofac radiol. 2004; 33: 103-7. 13. graziottin lf, da costa np, da silveira id, veeck eb. measurement of the optical density of packable composites: comparison between direct and indirect digital systems. pesqui odontol bras. 2002; 16: 299-307. 14. attaelmanan a, borg e, grõndahl hg. digitisation and display of intra-oral films. dentomaxillof radiol. 2000; 29: 97-102. 15. janhom a, van ginkel fc, van amerongen jp, van der stelt pf. scanning resolution and the detection of approximal caries. dentomaxillof radiol. 2001; 30: 166-71. 16. parissis n, kondylidou-sidira a, tsirlis a, patias p. convencional radiographs: image quality assessment. dentomaxillof radiol. 2005; 34: 353-6. 17. wenzel a, kirkevang ll. high resolution charge-coupled device sensor vs. medium resolution photostimulable phosphor plate digital receptors for detection of root fractures in vitro. dent traumatol. 2005; 21: 32-6. 18. schulze rk, richter a, d’hoedt b. the effect of wavelet and discrete cosine transform compression of digital radiographs on the detection of subtle proximal caries. roc analysis. caries res. 2008; 42: 334-9. 19. gürdal p, hildebolt cf, akdeniz bg. the effects of different image file formats and image-analysis software programs on dental radiometric digital evaluations. dentomaxillofac radiol. 2001; 30: 50-5. 20. fidler a, likar b, skaleric¡ u. lossy jpeg compression: easy to compress, hard to compare. dentomaxillofac radiol. 2006; 35: 67-73. 21. berkhout we, verheij jg, syriopoulos k, li g, sanderink gc, van der stelt pf. detection of proximal caries with high-resolution and standard resolution digital radiographic systems. dentomaxillofac radiol. 2007; 36: 204-10. 22. bushberg jt, seibert ja, leidholdt em jr, boone jm. image quality. in: bushberg jt, seibert ja, leidholdt em jr, boone jm, editors. the essential physics of medical imaging. philadelphia, pa: lippincott williams & wilkins; 2002. p. 255-91. bone mineral density on conventional and digitized images under different parameters of digitization and storage braz j oral sci. 9(3):398-401 404 not found oral sciences n3 original article braz j oral sci. october|december 2010 volume 9, number 4 antimicrobial efficacy of fruit extracts of two piper species against selected bacterial and oral fungal pathogens kamal rai aneja1, radhika joshi2, chetan sharma2, ashish aneja3 1phd, professor and chairman, department of microbiology, kurukshetra university, kurukshetra, india 2msc, research scholar, department of microbiology, kurukshetra university, kurukshetra, india 3mbbs, medical officer, haryana government, posted tarori (karnal), india correspondence to: radhika joshi department of microbiology, kurukshetra university, kurukshetra136119, haryana, india. phone: 09355566163 email: joshi_radhika31282@yahoo.com radhikasharma31282@gmail.com received for publication: february 7, 2010 accepted: june 22, 2010 abstract aim: to assess the antimicrobial efficacy of five solvent extracts of two piper species commonly used in diet and traditional medicine, p. cubeba and p. longum, against selected bacterial and oral fungal pathogens i.e. streptococcus mutans, staphylococcus aureus, candida albicans and saccharomyces cerevisiae. methods: the antimicrobial activity of five extracts of cubeb berries and indian long pepper fruits was determined by the agar well diffusion method. the minimum inhibitory concentration (mic) for the acetonic, methanolic and ethanolic extracts was determined by the modified agar well diffusion method. results: of the 5 fruit extracts evaluated, acetone, ethanol and methanol extracts of both the piper spp. were found to have variable antimicrobial activities against all the four oral pathogens. the acetonic fruit extract of p. cubeba was the most effective against both the yeasts with the highest zone of inhibition (15.31 mm) against c. albicans followed by the methanolic (12.31 mm) and ethanolic (11.94 mm) extracts. c. albicans was found to be most sensitive pathogen, which survived up to 6.25 mg/ml in the acetonic extract (mic = 12.5 mg/ml) followed by the methanolic and ethanolic extracts (mic = 25 mg/ml). the acetonic, methanolic and ethanolic extracts of p. longum fruits showed almost equal inhibition zones of both yeasts, ranging between 10.64 and 14 mm. c. albicans survived up to 12.5 mg/ml (mic= 25 mg/ml) while s.cerevisiae survived up to 25 mg/ml (mic = 50 mg/ml). conclusions: the crude extracts obtained from the fruits of the two piper spp. may be used to treat oral fungal species, especially c. albicans, as they produced larger inhibition zones than antifungal drugs often used to treat these pathogens. keywords: oral pathogens, piper cubeba, piper longum, antibacterial or antifungal activity, minimum inhibitory concentration (mic). introduction oral diseases continue to be a major health problem worldwide1. dental caries and periodontal diseases are among the most important global oral health problems2. streptococcus mutans is the major organism implicated with dental caries and oral infections3. candida albicans is the most common yeast isolated from the oral cavity, and is associated with oral fungal infections, endocarditis and septicemia4. staphylococcus aureus, a major human pathogen, is responsible for a number of hospital-acquired infections and propagates mainly in mouth and hands in the hospital environment5-6. saccharomyces cerevisiae considered to be an opportunistic pathogen in the oral cavity, may induce significant oral braz j oral sci. 9(4):421-426 risks by acting as a tertiary colonizer in the progress of dental caries thus causing both superficial and invasive infections7. microbial resistance to most of the antibiotics commonly used to treat oral infections (penicillins and cephalosporins, erythromycin, tetracycline and derivatives and metronidazole) has been documented8. the resistance of microorganisms against the traditional antibiotics needs urgent attention for the development of the new drug molecules. it is well documented from ancient times that active principles from plant origin have been used as medicines for various diseases and microbial infections9. a wide variety of medicinal plants used traditionally have not yet been systematically investigated against various microbial pathogens10. the genus piper of family piperaceae, with over 1,000 species, is distributed in both hemispheres. piper cubeba linn., commonly known as cubeb, tailed pepper (due to the stalks attached), java pepper (in java) and kemukus (in indonesia), is a climbing perennial plant11. the fruits of this plant are used as a spice and have medicinal value, being often used for the treatment of abdominal pain, asthma, chronic bronchitis, diarrhea, dysentery, gonorrhea, enteritis and syphilis and reported to have an inhibitory effect on hepatitis c virus protease12-13. the dried fruits contain up to 10% essential oil composed of monoterpenes (sabinene 50%, carene, α-thujene, 1,4-cineol and 1,8-cineol) and sesquiterpenes (copaene, αand β-cubebene, δ-cadinene, caryophyllene, germacrene, cubebol)14. p. longum linn. (javanese, indian long pepper, pippali), another medicinal plant belonging to the genus piper, is a small shrub characterized by fruits called berries borne in fleshy spikes, oblong, blunt and blackish green in color15. the fruits contain 1% volatile oil, resin, a waxy alkaloid, a terpenoid substance and alkaloids piperine and piperlongumine16. it is widely used as a folk medicine to cure diseases such as leprosy, tuberculosis, gonorrhea, paralysis of the tongue, diarrhea, cold, palsy, gout, rheumatism, lumbago, insomnia, epilepsy, anorexia, piles, dyspepsia, leucoderma cholera, scarlatina, chronic malaria, viral hepatitis, bronchitis, cough, asthma, stomachache, spleen diseases and tumors17-18. a literature search reveals that piper spp. have been used in traditional medicine since long for several ailments15,1820 however, not much work has been done on the antimicrobial activity of their fruits. since p. cubeba and p. longum have been used traditionally in medicine and their fruits have been used as food material, the biological evaluation of the fruits of these plants may lead to development of safer therapeutic agents21. therefore, the present study has been designed to assess the antimicrobial efficacy of fruit extracts of p. cubeba and p. longum against selected bacterial and oral fungal pathogens. material and methods fruits/catkin of p. cubeba and p. longum were collected from the local market of delhi, india. dr. b.d. vashishta (botany department) kurukshetra university, kurukshetra confirmed the identification of the specimens. extraction the samples were carefully washed under running tap water followed by sterile distilled water, and were air dried at room temperature (40oc) for 5 days and pulverized to a fine powder using a sterilized mixer grinder and stored in air-tight bottles. four different solvents, namely ethanol, methanol, acetone and aqueous solvent (hot and cold), often used for the extraction of plant material were used to obtain the extracts22. an amount of 10 g of pulverized fruit was separately soaked in 100 ml of acetone, ethanol, methanol, and cold sterile distilled water for 24 h. the same amount of pulverized fruit (10 g) was immersed in 100 ml of hot sterile distilled water (100oc) and allowed to stand for 30 min in a water bath with occasional shaking, and then left undisturbed for 24 h. each preparation was filtered through a sterilized whatman no.1 filter paper, and the filtered extract was concentrated under vacuum below 40oc using heidolph, ve11 rotaevaporator23-25. the obtained dried extract was exposed to uv rays for 24 h and checked for sterility on nutrient agar plates and stored in labeled sterile bottles in a freezer at 4oc until further use26. test microorganisms two oral pathogenic bacteria s. mutans (mtcc*497) and s. aureus (mtcc 740) and two oral pathogenic yeasts c. albicans (mtcc 227) and s. cerevisiae (mtcc 170) were obtained from microbial type culture collection, imtech, chandigarh. the microorganisms were subcultured on the specific media recommended for different microorganisms such as brain heart infusion agar (s. mutans), nutrient agar (s. aureus), malt yeast agar (c. albicans and s. cerevisiae) and incubated aerobically at 37oc. the media were procured from himedia laboratory pvt. ltd., bombay, india. identification of all the strains was confirmed by standard biochemical and staining methods27-29. screening for antimicrobial activity antimicrobial activity of the 5 extracts of cubeb berries and indian long pepper fruits was determined by following the agar well diffusion method of okeke30. in this method, pure isolate of each microbe was subcultured on the recommended specific medium for each microorganism at 37oc for 24 h. a plate of each microorganism was taken and a minimum of 4 colonies were touched with a sterile loop and transferred into normal saline (0.85%) under aseptic conditions. density of each microbial suspension was adjusted equal to that of 106cfu/ml (standardized by 0.5 mcfarland standard) and used as the inoculum for performing agar well diffusion assay. one hundred microliters of inoculum of each test organism was spread onto the specific media plates so as to achieve a confluent growth. the agar plates were allowed to dry and 8-mm-diamater wells were made with a sterile borer in the inoculated agar plates. the lower portion of each well was sealed with a little specific molten agar medium31. the dried fruit extracts were reconstituted in 20% dimethylsulfoxide (dmso) for the bioassay analysis30. 422422422422422antimicrobial efficacy of fruit extracts of two piper species against selected bacterial and oral fungal pathogens braz j oral sci. 9(4):421-426 a 100 µl volume of each extract was propelled directly into the wells (in triplicates) of the inoculated specific media agar plates for each test organism. the plates were allowed to stand for 10 min for diffusion of the extract to take place and incubated at 37oc for 24h32-34. sterile dmso served as the negative control, and ciprofloxacin (for bacteria) and amphotericin-b (for fungi) served as the positive controls. the antimicrobial activity, indicated by the formation of an inhibition zone surrounding the well containing the extract, was recorded if the inhibition zone was greater than 8 mm. the experiments were performed in triplicates and the mean values of the diameter of inhibition zones with ± standard deviation were calculated35-36. determination of minimum inhibitory concentration (mic) mic is defined as the lowest concentration of a compound/extract/drug that completely inhibits the growth of the microorganism in 24 h35. the mic for the acetonic, methanolic and ethanolic fruit extracts was determined by following the modified agar well diffusion method29. a twofold serial dilution of each extract was prepared by first reconstituting the fruit extract in 20% dmso followed by dilution in sterile distilled water to achieve a decreasing concentration range of 50 mg/ml to 0.39 mg/ml. a 100 µl volume of each dilution was introduced into wells (in triplicate) in the specific media agar plates already seeded with 100 µl of standardized inoculum (106cfu/ml) of the test microbial strain. all test plates were incubated aerobically at 37oc for 24 h and observed for the inhibition zones. the lowest concentration of each extract showing a clear zone of inhibition (>8 mm) (in triplicate), considered as the mic, was recorded for each test organism33. statistical analysis the results are presented as mean ± standard deviation. one-way anova followed by dennett’s t-test for multiple comparisons were used for statistical evaluation. p values less than 0.05 were considered significant. acetone 12.64*±0.57† 10±0 18.96±1 10.64±0.57 15.31±0.57 11.31±0.57 14±0 10.93±1 methanol 12.31±0.57 10±0 17.65±0.57 10±0 12.31±0.57 10.64±0.57 12.94±1 11.31±0.57 ethanol 13±0 10.31±0.57 17.32±0.57 11.31±0.57 11.94±1 11.64±0.57 13.95±1 11.64±0.57 h a c a ciprofloxacin 27.32±0.57 27.32±0.57 34.66±0.57 34.66±0.57 nt nt nt nt amphotericin-b nt nt nt nt 13±0 13±0 11.94±1 11.94±1 20%dmso s.mutans s.aureus c.albicans s.cerevisiae p.c. p.l. p.c. p.l. p.c. p.l. p.c. p.l. diameter of zone inhibition ( mm)solvent extracts ( m g / m l ) table 1. antimicrobial activity of p. cubeba and p. longum fruit extracts against oral pathogens determined by the agar well diffusion method on specific media for each test microorganism. (-) = no activity, nt = not tested, p.c. = piper cubeba, p.l. = piper longum, ha= hot aqueous extract, ca= cold aqueous extract, data shown as * values, including diameter of the well (8 mm), are means of three replicates ± † standard deviation, p = 0.001 indicates significantly different from control; dennett’s t -test after analysis of variance. results and discussion the results of antimicrobial properties of ethanol, methanol, acetone and aqueous (hot and cold) fruit extracts of p. cubeba and p. longum, the positive control ciprofloxacin (for bacteria) and amphotericin-b (for fungi), and the negative control (dmso), are presented in table 1 and values of mic of these extracts against the test pathogens are presented in table 2. the antimicrobial activity of p .cubeba and p. longum extracts on the agar plates varied for the different solvents. both positive controls produced significantly larger inhibition zones against the test bacteria (ciprofloxacin) and yeasts (amphotericin-b). however, the negative control produced no observable inhibitory effect. of the 10 fruit extracts of p. cubeba and p. longum screened for antibacterial and antifungal activity, acetone, methanol and ethanol extracts showed activity against both the bacteria (s. mutans and s. aureus) and both yeasts (c. albicans and s. cerevisiae). however, aqueous extracts, both hot and cold, showed no activity against the test strains (table 1). a perusal of the data (table 1) reveals that the acetonic fruit extract of p. cubeba was the most effective against both yeasts. it showed the largest zone of inhibition (15.31 mm) against c. albicans (figure 1a) followed by the methanolic (12.31 mm) and ethanolic extract (11.94 mm). c. albicans was found to be most sensitive pathogen, which survived up to 6.25 mg/ml in the acetonic extract (figure 2 i) (mic = 12.5 mg/ml) followed by the methanolic and ethanolic extracts (25 mg/ml). the inhibition zones produced by the 3 solvents against s. cerevisiae ranged between 14 and 12.94 mm. s. cerevisiae was found to be comparatively more resistant than c. albicans, as it survived up to 12.5 mg/ml (mic = 25 mg/ml) in all 3 extracts tested. interestingly, the acetonic fruit extract of p. cubeba showed comparatively greater activity against both yeasts, c. albicans (15.31 mm) and s. cerevisiae (14 mm), than that of the standard drug amphotericin-b (13 mm), which indicates a great potential against oral fungal pathogens. among the tested fruit extracts of p. cubeba, the acetonic extract showed greater antibacterial antimicrobial efficacy of fruit extracts of two piper species against selected bacterial and oral fungal pathogens423423423423423 braz j oral sci. 9(4):421-426 fig. 1. zone of antifungal inhibition against c. albicans shown by acetonic extract of p. cubeba (a) and ethanolic extract of p. longum (b), determined by agar well diffusion method and the control. activity against s. aureus, the highest inhibition zone being 18.96 mm followed by the methanolic (17.65 mm) and ethanolic (17.32 mm) extracts (table 1). s. aureus survived up to 12.5 mg/ml, thus having a mic of 25 mg/ml (table 2, figure 2 ii). the inhibition zones produced by the 3 solvents against s. mutans ranged from 13 to 12.64 mm, and this microorganism survived up to 25 mg/ml (mic = 50 mg/ ml). the aqueous (hot and cold) extracts did not show any activity against the tested yeast and bacteria (table 1). a literature search reveals that among the piper spp., the fruits of p. cubeba have received less attention on their antimicrobial activity. the berries contain essential oil consisting of monoterpenes, sesquiterpenes, the oxides 1.4and 1, 8-cineole, kadsurin a, piperenone and the alcohol cubebol 37-38 in addition to two groups of secondary metabolites, i.e., alkaloids (piperine being the most abundant alkaloid39), and lignans (cubebin, though lesser in amounts in berries as compared to other lignans like yatein and hinokinin)11,39-40. the antibacterial and antifungal activity shown by the fruit extracts of p. cubeba against all the 4 test strains in this study may be due to the presence of piperine and cubebin in the berries. cubebin has also been found to possess antiinflammatory, analgesic and trypanocidal activities14, 41-42. the data presented in the table 1 reveal that acetonic, methanolic and ethanolic extracts of p. longum produced almost equal inhibition of both the yeasts, ranging between 10.64 and 14 mm (figure 1b). c. albicans survived up to 12.5 mg/ml thus having an mic of 25 mg/ml while s. cerevisiae survived up to 25 mg/ml thus having an mic of 50 mg/ml (table 2). the zone of inhibition produced against the fig. 2. mic shown by acetonic extract of p. cubeba fruits against c. albicans (i) and s .aureus (ii) determined by modified agar well diffusion method using twofold serial dilutions of the extracts: 50 mg/ml (a), 25 mg/ml (b), 12.5 mg/ml (c), 6.25 mg/ml (d), 3.125 mg/ml (e) and 1.56 mg/ml (f). 424424424424424antimicrobial efficacy of fruit extracts of two piper species against selected bacterial and oral fungal pathogens braz j oral sci. 9(4):421-426 acetone 50 25 12.5 25 12.5 50 methanol 50 25 25 25 12.5 50 ethanol 50 25 25 25 12.5 50 s.mutans s.aureus c.albicans s.cerevisiae p.c. p.l. p.c. p.l. p.c. p.l. p.c. p.l. minimum inhibitory concentration ( mg/ml)solvent extracts table 2. mic of p.cubeba and p.longum fruit extracts against oral pathogens on specific media for each microorganism determined by modified agar well diffusion method. (-) = no activity, p.c. = piper cubeba, p.l. = piper longum. bacterial pathogens by the three solvent extracts of p. longum fruits was mild that ranged between 10 and 11.31 mm without much variation in the different solvents activity (table 1). however, the bacteria were found to be comparatively resistant as they survived up to 50 mg/ml, thus having an mic of 100 mg/ml. the fruit of p. longum contains a large number of alkaloids and related compounds, the most abundant of which is piperine, together with methyl piperine, pipernonaline, piperettine, asarinine, pellitorine, piperundecalidine, piperlongumine, piperlonguminine, retrofractamide a, pergumidiene, brachystamide-b, a dimer of desmethoxypiplar-tine, n isobutyl-decadienamide, brachyamidea, brachystine, pipercide, piperderidine, longamide, dehydropipernonaline piperidine and tetra hydro piperine43. piperine, an alkaloid in the fruits of p.longum is responsible for the pungency of long pepper and has been shown to possess antiinflammatory, antiamoebic, antiasthmatic, anticonvulsant and antibacterial activities44-45. thus, the antibacterial and antifungal activity of the fruits of p. longum may be due to the presence of the alkaloid piperine. it may, therefore, be concluded from the above investigation that the crude extracts obtained from the fruits of the two piper spp. may be used to treat oral fungal species, especially c. albicans, as they produced larger inhibition zones than the antifungal drugs often used to treat these pathogens. however, isolation of pure compounds and their toxicological analysis and clinical investigation in animal models are to be made before their trials on human. acknowledgements we are grateful to our honorable vice chancellor, kurukshetra university, kurukshetra for providing us the infrastructure in the department of microbiology. we would like to thank dr. b.d.vashishta, department of botany, kurukshetra university, kurukshetra, for helping with the identification of the fruit samples. we are thankful to dr. tapan chakrabarti, institute of microbial technology, chandigarh, for providing the microbial cultures. references 1. petersen pe, bourgeois d, ogawa h, estupinan-day s, ndiaye c. the global burden of oral diseases and risks to oral health. bull world health organ. 2005; 83: 661-9. 2. palombo ea. traditional medicinal plant extracts and natural products with activity against oral bacteria: potential application in the prevention and treatment of oral diseases. ecam advance access. 2009; 1-15. 3. lee ss, zhang w, li y. the antimicrobial potential of 14 natural herbal dentifrices: results of an in vitro diffusion method study. j am dent assoc. 2004; 135: 1133-41. 4. bagg j. essentials of microbiology for dental students. new york: oxford university press; 1999. 5. knighton ht. study of bacteriophage types and antibiotic resistance of staphylococci isolated from dental students and faculty members. j dent res. 1960; 39: 906-11. 6. piochi bj, zelante f. contribution to the study of staphylococcus isolated in the mouth.iii. staphylococcus isolated from dental plaque. rev fac odontol sao paulo. 1975; 13: 91-7. 7. bonjar ghs. anti yeast activity of some plants used in traditional herbal medicine of iran. j biol sci. 2004; 4: 212-5. 8. bidault p, chandad f, grenier d. risk of bacterial resistance associated with systemic antibiotic therapy in periodontology. j can dent assoc. 2007; 73: 721-5. 9. borris rp. natural products research: perspective from a major pharmaceutical company. j ethnapharmacol. 1996; 51: 29-38. 10. farrukh a, iqbal a. broad spectrum antibacterial and antifungal properties of certain traditionally used indian medicinal plants. world j microbiol biotechnol. 2003; 19: 653-7. 11. koul jl, koul sk, taneja sc, dhar kl. oxygenated cyclohexanes from piper cubeba. phytochem. 1996; 41: 1097-9. 12. eisai pt. medicinal herb index in indonesia. 2. ed. jakarta: dian rakyat; 1995. 13. junqueira apf, perazzo ff, souza ghb, maistro el. clastogenicity of piper cubeba (piperaceae) seed extract in an in vivo ma mmalian cell system. genet mol biol. 2007; 30: 656-63. 14. borsato mlc, grael cff, souza gep, lopes np. analgesic activity of the lignans from lychnophora ericoides. phytochem. 2000; 55: 809-13. 15. ali ma, alam nm, yeasmin ms, khan am, sayeed ma. antimicrobial screening of different extracts of piper longum linn. res j agri biol sci. 2007; 3: 852-7. 16. wu s, sun c, pei s, lu y, pan y. preparative isolation and purification of amides from the fruits of piper longum l. by upright counter-current chromatography and reversed phase liquid chromatography. j chromatogr. 2004; 1040: 193-204. 17. warrier pk, nambiar vpk, raman kc. piper longum linn: indian medicinal plants. madras, india: orient longman; 1995. 18. reddy ps, jamilk k, madhusudhan p, anjani g, das b. antibacterial activity of isolates from piper longum and taxus baccata. pharma biol. 2001; 39: 236-8. 19. young-cheol y, sang guei l, hee-kwon l, moo-key k, sang-hyun l, hoi-seon l. a piperidine amide extracted from piper longum l. fruit shows activity against aedes aegypti mosquito larvae. j agri food chem. 2002; 50: 3765-7. 20. li x, zhou j, li h, du s, li y, huang l et al. study on proliferation effect of extracts of piper longum on mesenchymal stem cells of rat bone marrow and the relationship to chemical functional groups. zhong-yao-cai. 2005; 28: 570-4. 21. catalano s, cioni pl, panizzi l, morelli i. antimicrobial activity of extracts of mutisia acuminate var acuminate. j ethnopharmacol. 1998; 59: 207-9. 425425425425425 antimicrobial efficacy of fruit extracts of two piper species against selected bacterial and oral fungal pathogens braz j oral sci. 9(4):421-426 22. cowan mm. plant products as antimicrobial agents. clin microbiol rev. 1999; 12: 564-82. 23. lokhande pd, gawai kr, kodam km, kuchekar bs, chabukswar ar, jagdale sc. antibacterial activity of extracts of piper longum. j pharmacol toxicol. 2007; 2: 574-9. 24. bag a, bhattacharya sk, bharati p, pal nk, chattopadhyay rr. evaluation of antibacterial properties of chebulic myrobalan (fruit of terminalia chebula retz.) extracts against methicillin resistant staphylococcus aureus and trimethoprim-suphamethoxazole resistant uropathogenic escherichia coli. afr j plant sci. 2009; 3: 25-9. 25. ogundiya mo, okunade mb, kolapo al. antimicrobial activities of some nigerian chewing sticks. ethnobot leaflts. 2006; 10: 265-71. 26. aneja kr, joshi r. antimicrobial activity of amomum subulatum and elettaria cardamomum against dental caries causing microorganisms. ethnobot leaflts. 2009; 13: 840-9. 27. aneja kr. experiments in microbiology plant pathology and biotechnology. 4. ed. new delhi, india: new age international publishers; 2003. 28. benson hj. microbiological applications: laboratory manual in general microbiology. new york: mcgraw hill; 2004. 29. cappuccino jg, sherman n. microbiology. a laboratory manual. california: benjamin/cummings publishing company; 1995. 30. okeke mi, iroegbu cu, eze en, okoli as, esimone co. evaluation of extracts of the root of landolphia owerrience for antibacterial activity. j ethnopharmacol. 2001; 78: 119-27. 31. nkere ck, iroegbu cu. antibacterial screening of the root, seed and stem bark extracts of picralima nitida. afr j biotechnol. 2005; 4: 522-6. 32. rajasekaran c, meignanam e, vijayakumar v, kalaivani t, ramya s, premkumar n et al. investigations on antibacterial activity of leaf extracts of azadirachta indica a. juss (meliaceae): a traditional medicinal plant of india. ethnobot leaflts. 2008; 12: 1213-7. 33. aneja kr, joshi r. evaluation of antimicrobial properties of fruit extracts of terminalia chebula against dental caries pathogens. jundishapur j microbiol. 2009; 2: 105-11. 34. khokra sl, prakash o, jain s, aneja kr, dhingra y. essential oil composition and antibacterial studies of vitex negundo linn.extracts. ind j phar sci. 2008; 70: 522–6. 35. aneja kr, joshi r, sharma c. antimicrobial activity of dalchini (cinnamomum zeylanicum bark) extracts on some dental caries pathogens. j pharm res. 2009; 2: 1387-90. 36. aneja kr, joshi r, sharma c. in vitro antimicrobial activity of sapindus mukorossi and emblica officinalis against dental caries pathogens. ethnobot leaflts. 2010; 14: 402-12. 37. saleem m, kim hj, ali ms, lee ys. an update on bioactive plant lignans. nat prod rep. 2005; 22: 696-716. 38. thongson c, davidson pm, mahakarrchanakul w, weiss j. antimicrobial activity of ultrasound-assisted solvent-extracted spices. lett appl microbiol. 2004; 39: 401-6. 39. parmar vs, jain sc, bisht ks, jain r, taneja p, jha a et al. phytochemistry of the genus piper. phytochem. 1997; 46: 597-673. 40. usia t, watabe t, kadota s, tezukay. potent cyp3a4 inhibitory constituents of piper cubeba. j nat prod. 2005; 68: 64-8. 41. bastos jk, carvalho jct, de souza ghb, pedrazzi ahp, sarti sj. antiinfla mmatory activity of cubebin, a lignan from the leaves of zanthoxyllum naranjillo griseb. j ethnopharmacol. 2001; 75: 279-82. 42. de souza va, da silva r, pereira ac, royo vd, saraiva j, montanheiro m et al. trypanocidal activity of (-)-cubebin derivatives against free amastigote forms of trypanosoma cruzi. bioorgan med chem lett. 2005; 15: 303-7. 43. atal ck, zutshi u, rao pg. scientific evidence on the role of ayurvedic herbals on bioavailability of drugs. j ethnopharmacol. 1981; 4: 229-32. 44. sunila es, kuttan g. i mmunomodulatory and antitumor activity of piper longum linn. and piperine. j ethnopharmacol. 2004; 90: 339-46. 45. mishra p. isolation, spectroscopic characterization and computational modelling of chemical constituents of piper longum natural product. int j pharma sci rev res. 2010; 2: 78-86. 426426426426426antimicrobial efficacy of fruit extracts of two piper species against selected bacterial and oral fungal pathogens braz j oral sci. 9(4):421-426 oral sciences n3 braz j oral sci. 9(2):94-97 original article braz j oral sci. april/june 2010 volume 9, number 2 prevalence of tt virus in patients with chronic periodontitis, patients with aggressive periodontitis and healthy controls a pilot study gurumoorthy kaarthikeyan1, n.d.jayakumar2, ogoti padmalatha3, sheeja varghese3, khalefathullah sheriff4 1mds, senior lecturer, department of periodontics, saveetha dental college and hospitals, chennai, india 2mds, principal and h.o.d, department of periodontics, saveetha dental college and hospitals, chennai, india 3mds, professor, department of periodontics, saveetha dental college and hospitals, chennai, india 4phd, department of virology , king institute, chennai, india correspondence to: gurumoorthy kaarthikeyan saveetha dental college &hospitals, 162, p.h road, chennai-600077 india. e-mail: drkarthik79@yahoo.co.in received for publication: november 04, 2009 accepted: june 08, 2010 abstract torque teno virus (ttv), a novel dna virus resides in peripheral blood mononuclear cells and replicates when these cells get activated. the ttv replication shifts the immunobalance. aim: to determine the presence of ttv in the gingiva of patients with aggressive periodontitis, patients with chronic periodontitis, and healthy controls, and to correlate the presence of ttv with probing pocket depth and clinical attachment level. methods: forty-two subjects (22 males and 20 females) aged 21 to 55 years were recruited for this study. subjects were stratified into aggressive periodontitis (group i), chronic periodontitis (group ii) and healthy controls (group iii). gingival tissue biopsy was taken from all the subjects and the presence of ttv was analyzed using pcr and 2% agarose gel electrophoresis. results: ttv was identified in half of the subjects and more number of subjects with periodontitis have tt virus compared to controls. there was significant association between presence of tt virus and pocket depth, clinical attachment level. conclusions: the findings from the present study shows that there was no significant association between tt virus and periodontitis, even though it was isolated from more number of subjects with aggressive periodontitis, and ttv was associated with pocket depth and clinical attachment level. these findings need to be investigated in further studies. keywords: clinical attachment level, periodontitis, pocket depth, polymerase chain reaction, torque teno virus. introduction since 1997, groups of novel nonenveloped dna viruses with a circular, singlestranded (negative sense) dna genome of 3.6-3.9 kb, 3.2 kb, or 2.8-2.9 kb in size have been discovered and designated torque teno virus (ttv), torque teno midi virus (ttmdv), and torque teno mini virus (ttmv), respectively, in the floating genus anellovirus1. ttv belongs to the circoviridae family and it was first isolated from the serum of japanese patients with post transfusion hepatitis2. initially the mode of transmission of this virus was considered to be through blood and blood products and hence it was known as transfusion transmitted virus3. but other modes of transmission like nasal secretions, sexual transmission and oral fecal route also exist45. the anelloviruses frequently and ubiquitously infect humans, and the infections are characterized by lifelong viremia and great genetic variability. however, the 95 braz j oral sci. 9(2):94-97 level of virus replication may vary among different individuals and this may represent an important marker of pathogenic role for ttv6. ttv viral loads have been shown to increase in human immunodeficiency virus (hiv)-infected patients who are progressing toward aids, and a high ttv viral load was associated with a low cd4 cell count, indicating a potential role of the immune system in controlling ttv replication7. the frequent identification of ttv in peripheral mononuclear blood cells (pmbc) may suggest that the virus replicates in lymphoid cells8. ttv has been associated with many chronic diseases like asthma, bronchiectasis, and hepatic failure. the previous study9 has shown association between periodontitis and tt virus, but there was no clear cut distinction between chronic and aggressive periodontitis, and their association with tt virus. hence, the purposes of the present study were to determine the presence of ttv in the gingiva of patients with aggressive periodontitis, patients with chronic periodontitis, and healthy controls, and to correlate the presence of ttv with probing pocket depth and clinical attachment level. material and methods study population the study population consisted of 42 subjects who reported to the department of periodontics, saveetha dental college and hospitals, chennai. subjects included 22 males and 20 females belonging to the southern indian population. fourteen subjects were periodontally healthy and 28 subjects were diagnosed with periodontitis. patients with periodontitis were stratified into an aggressive periodontitis group (group i) and a chronic periodontitis group (group ii) based on the criteria of aap 199910. group iii included the healthy controls. subjects taking any medications, those who underwent any periodontal treatment within past six months, smokers, systemically ill subjects like diabetes mellitus, hypertensive subjects, patients with cardiac problem, immunocompromised subjects, patients under medications for any systemic diseases and pregnant, lactating women were excluded from the study. the periodontal parameters observed were plaque index of silness and loe11, bleeding on probing, pocket probing depth, clinical attachment loss, mobility, furcation involvement and gingival recession. the study was approved by the institutional review board of saveetha university and informed consent was obtained from all the subjects enrolled in the study. gingival tissue biopsies were taken from the deepest pocket region in the case of periodontitis subjects. for the control group, tissue samples were collected during crown lengthening procedures or during extractions for orthodontic purposes. the gingival tissue was then washed with sterile saline solution, put into a sterile centrifuge tube containing earles balanced salt solution (ebss) viral transport medium and kept in a freezer at -20° c. tt viral dna extraction was carried out on weighed tissue samples using qiagen dna extraction kit according to manufacturer’s protocol. extracted viral dna was eluted in 50 µl te buffer and stored at -80ºc until pcr amplification. pcr reaction mix (takahashi et al., 1998)12 25 µl reaction volume containing 12.5 µl of 2 x pcr master mix, 0.5 µl of taq 1.0 µl of forward and reverse primers 5 ìl of nuclease free water and 5 µl of dna was taken into the reaction mix. the forward primer sequence 5’-gctacgtcactaaccacgtg-3’ (t 801, sense primer) and the reverse primer 5’-ctccggtgtgtaaactcacc3’ (t935,antisense primer) were used for pcr amplification. reaction cycle the reaction mix was kept at 95 °c for 10 min for reverse transcriptase inactivation and was subjected to the following thermo cycling profile in a thermocycler (perkin elmer cetus, usa).denaturation at 94ºc for 20 s, annealing at 60ºc for 20 s and template extension at 72ºc for 30 s .the cycle was repeated 55 times. the pcr amplified products were analyzed on a 2 % agarose gel with intercalating ethidium bromide dye. 10 µl of the amplified product was mixed with 1 µl of 10x loading dye and loaded into the wells along with 1 µl of molecular weight marker (50 bp ladder). the electrophoresis was run at 100 volts in 0.5x tbe buffer. the gel was visualized under uv transillumination and the products were compared with molecular weight marker as shown in figure 1. fig. 1 gel documentation of ttv-positive samples lane 1 mol wt marker – 50 bp. lane 2 positive control. lane 3 neg control. lane 4 – 8 positive samples. lane 9 negative sample statistical analysis the descriptive statistic analysis was expressed as a mean ± standard deviation. anova and post hoc tukey test were performed to test for significance of means between groups. unpaired t-test was performed to test for the difference between two means. statistically significant values were set at p<0.05. results of the 42 subjects, 20 had positive results for the presence of ttv in gingival tissue. descriptive statistical analyses are reported in table 1 and the analysis of presence of ttv with mean pocket depth and mean clinical attachment prevalence of tt virus in patients with chronic periodontitis, patients with aggressive periodontitis and healthy controls a pilot study 96 group i (n=14) 30.10±2.80 8 6 3.84±0.73 3.96±0.78 9 group ii (n=14) 44.70±5.40 6 8 3.34±0.78 3.36±0.78 7 group iii (n=14) 29.30±6.70 6 8 1.74±0.31 0.32±0.57 4 groups age gender mean pd number of subjects with torque teno virus*mean calfemale male table 1. study population characteristics (n=42) * x2 test ; group i vs group iix2 = 0.146, df=1; not sig. (p > 0.05). group ii vs group iiix2 = 0.599, df=1; not sig. (p > 0.05). group i vs group iiix2 = 2.297, df=1; not sig. (p > 0.05). pd: probing pocket depth. cal: clinical attachment level. present absent present absent 20 22 20 22 7.15±2.540 4.64±2.920 6.60±3.218 3.73±3.508 .005 .009 4.56±0.78 3.32±0.56 4.32±0.82 3.26±0.78 p<0.05 significant p<0.05 significant p d c a l torque teno virus n mean± sd of biopsied sites sig mean± sd of all sites sig pd: probing pocket depth. cal: clinical attachment level. table 2. mean pd and mean cal by tt virus presence or absence in subjects level is shown in table 2. even though the number of subjects with ttv was larger in the aggressive periodontitis group (group i), there was no statistically significant difference between the groups. there was significant association between the presence of ttv and the mean pocket depth and clinical attachment level between the groups. there was also a significant association between the presence of ttv and the mean clinical attachment level of the sites where gingival biopsies for ttv analysis were performed. discussion although the gram negative bacteria is essential in initiating and perpetuating the periodontal destruction, the viruses have been shown to have a role in the etiology of periodontitis, especially the viruses belonging to the herpetoviridae family like herpes simplex, epstein barr virus and cytomegalovirus play an important role in periodontal destruction13-15. the main pathogenic mechanism of the above mentioned viruses tends to be the cytopathic effect on macrophages and monocytes. ttv has been associated with many diseases. for instance, infection with ttv coincided with mild rhinitis in neonates and children hospitalized with acute respiratory disease or with bronchiectasis showed higher ttv viral loads than controls16-17. in addition, children with high ttv loads in nasal specimens were shown to have worse spirometric values, and ttv was suggested to contribute to the pathogenesis of asthma18.ttvgenotype 1 plays a role in the pathogenesis of non-a, -b, or –c fulminant hepatic failure (fhf)19. the possible pathogenic mechanism of ttv in the above mentioned disease includes the following, ttv infects hematopoietic cells but only replicate when these cells are activated8,20. the ttv replication could twist the immunobalance towards the t helper 2 cell (th2) response. this shift in immunobalance is known to have a role in the pathogenesis of asthma18.the other possible pathogenic mechanisms would be the open reading frame protein orf 2 of ttv interferes with the activity of nf-êb, a well-characterized intracellular signal transcription factor known to play a myriad of roles in inflammation and immunomodulation21. hence, the aim of the present study was to determine whether there was any association between the presence of ttv and periodontitis. the present study did not find statistically significant association between gender and presence of ttv, which agrees with the study of masia et al. 200122. overall the results of the present study shows that half of the subjects examined including test and control group harbored ttv in gingiva. also, the ttv was isolated from a larger number of subjects with periodontitis compared to healthy controls. the number of subjects with ttv was larger in the aggressive periodontitis group compared to chronic periodontitis and control groups. this is an important finding and further research is required to analyze the role of ttv in the etiopathogenesis of aggressive periodontitis. it was also observed that the presence of tt virus correlated with pocket depth and clinical attachment level (p =0.005 and p=0.009). within the groups, the ttv was also isolated in more subjects from deeper pocket regions (p=0.015). a possible explanation for this can be ascribed to the ability of ttv to replicate in locally stimulated resident lymphoid cells. it is also possible that the periodontal inflammatory process attracts in situ peripheral lymphocytes that contain ttv. thus, the presence of ttv correlated with increasing pocket depth and clinical attachment level and also ttv was isolated from more subjects with aggressive periodontitis. the major limitation of this study was the smaller sample size and hence further research using larger sample sizes are required. in conclusion, the findings of the present study show that there was significant association between tt virus and pocket depth and clinical attachment level in ttv-positive subjects, even though there was no significant association between patients with periodontitis and healthy subjects. further studies using larger populations and genotyping of ttv in periodontitis are needed. references 1. de villiers, em, zur hausen h, editors. tt viruses; the still elusive human pathogens. berlin: springer; 2009. braz j oral sci. 9(2):94-97 prevalence of tt virus in patients with chronic periodontitis, patients with aggressive periodontitis and healthy controls a pilot study 97 2. nishizawa t, okamoto h, yoshizawa h, miyakawa y, mayumi m. a novel dna virus (ttv) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology. biochem biophys res commun. 1997; 241: 92-7. 3. davidson f, macdonald d, mokili jlk, prescott le, graham s, simmonds p. early acquisition of tt virus (ttv) in an area endemic for ttv infection. j infect dis. 1999; 179: 1070-6. 4. fornai c, maggi f, vatteroni ml, pistello m, bendinelli m. high prevalence of tt virus (ttv) and ttv-like minivirus in cervical swabs. j clin microbiol. 2001; 39: 1-3. 5. inami t, konomi n, arakawa y, abe k. high prevalence of tt virus dna in human saliva and semen. j clin microbiol. 2000; 38: 2407-8 6. bendinelli m, pistello m, maggi f, fornai c, freer g, vatteroni ml. molecular properties, biology, and clinical implications of tt virus, a recently widespread infectious agent of humans. clin microbiol rev. 2001; 14: 98-113. 7. thom k, petrik j .progression towards aids leads to increased torque teno virus and torque teno minivirus titers in tissues of hiv infected individuals. j med virol. 2007; 79: 1-7. 8. maggi f, fornai c, zaccaro l, morrica a, vatteroni ml, isola p. et al. tt virus (ttv) loads associated with different peripheral blood cell types and evidence for ttv replication in activated mononuclear cells. j med virol. 2001; 64: 1-6. 9. rotundo r, maggi f, nieri m, muzzi l,bendinelli and pini prato g.p. tt virus infection of 10. periodontal tissues: a controlled clinical and laboratory pilot study .j periodontol. 2004; 75; 1216-20. armitage gc. development of a classification system for periodontal diseases and conditions. ann periodontol. 1999; 4: 1-6. 11. silness j, loe h. periodontal disease in pregnancy. ii. correlation between oral hygiene and periodontal condition. acta odontol scand. 1964; 22: 121-35. 12. takahashi k., hoshino h, ohta y, yoshida n, mishiro s. very high prevalence of tt virus (ttv) infection in general population of japan revealed by a new set of pcr primers. hepatol. res. 1998; 12: 233-9. 13. amit r, morag a, ravid z, hochman n, ehrlich j, zakay-rones z. detection of herpes simplex virus in gingival tissue. j periodontol. 1992; 63: 502-6. 14. contreras a, slots j. herpesviruses in human periodontal disease. j periodontal res. 2000; 35: 3-16. 15. slots j, contreras a. herpesviruses: a unifying causative factor in periodontitis? oral microbiol immunol. 2000; 15: 277-80. 16. maggi f, pifferi m, fornai c, andreoli e, tempestini e, vatteroni m. et al. tt virus in the nasal secretions of children with acute respiratory diseases: relations to viremia and disease severity. j virol. 2003; 77: 9081-3. 17. pifferi m, maggi f, caramella d, de marco e, andreoli e, meschi s. et al. high torquetenovirus loads are correlated withbronchiectasis and peripheral airflow limitation in children. pediatr infect dis j. 2006; 25: 804-8. 18. pifferi m, maggi f, andreoli e, lanini l, marco ed, fornai c et al. associations between nasal torquetenovirus load andspirometric indices in children with asthma. j infect dis. 2005; 192: 1141-8. 19. shibata m, morizane t, baba t, inoue k, sekiyama k, yoshiba m et al. tt virus infection in patients with fulminant hepaticfailure. am j gastroenterol. 2000; 95: 3602–6 20. mariscal lf, lopez-alcorocho jm, rodriguez-inigo e, ortiz-movilla n, de lucas s, bartolomé j. et al. tt virus replicates in stimulated but not in nonstimulated peripheral blood mononuclear cells. virology. 2002; 301: 121-9. 21. zheng h, ye l, fang x, li b, wang y, xiang x et al torque teno virus (sanban isolate) orf2 protein suppresses nf-kappab pathways via interaction with ikappab kinases. j virol. 2007; 81: 11917-24. 22. masia g, ingianni a, demelia l, faa g, manconi pe, pilleri g et al. tt virus infection in italy: prevalence and genotypes in healthy subjects, viral liver diseases and asymptomatic infections by parenterally transmitted viruses. j viral hepat. 2001; 8: 384-90. braz j oral sci. 9(2):94-97 prevalence of tt virus in patients with chronic periodontitis, patients with aggressive periodontitis and healthy controls a pilot study oral sciences n3 392392392392392 received for publication: december 07, 2009 accepted: august 27, 2010 original article braz j oral sci. october|december 2010 volume 9, number 4 risk factors for oral candidiasis in brazilian hiv-infected adult patients mariela dutra gontijo moura1, soraya de mattos camargo grossman1, linaena méricy da silva fonseca2, maria letícia ramos-jorge3, ricardo alves mesquita4 1msc, graduate students, department of oral pathology, dental school, federal university of minas gerais, belo horizonte, mg, brazil 2phd, assistant professor, school of health, faculdades pitágoras, belo horizonte, mg, brazil 3phd, professor. department of dentistry, federal university of vales jequitinhonha and mucuri, diamantina, mg, brazil 4phd, associate professor, department of oral pathology, dental school, federal university of minas gerais, belo horizonte, mg, brazil correspondence to: ricardo alves mesquita faculdade de odontologia da ufmg disciplina de patologia bucal, sala 3202-d av. antônio carlos, 6627 pampulha 31270-901 belo horizonte mg, brasil phone: +55-31-3409-2499 e-mail: ramesquita@ufmg.br abstract aim: the goals of this study were: 1) to estimate the prevalence of oral candidiasis (oc) in a sample of brazilian hiv-infected adult patients, and 2) to investigate the risk factors for hivassociated oc in this sample. methods: this case-control study included 112 hiv-infected patients treated between 2002 and 2004 at a clinic for sexually transmitted diseases. data were collected from medical records and clinical examinations. diagnosis of oc was performed in accordance with the international classification system and cytological features. seventeen clinical and laboratorial variables were registered. univariate analyses were performed on all variables. multiple logistic regression techniques were used to develop a model and identify the set of variables that may predict risk factors in hiv-infected adult patients with oc. results: prevalence of oc was 31.3%. oc was associated with oral hairy leukoplakia (ohl) [p<0.001; odds ratio (or) = 10.2 (95%ci: 4.0-26.0)], previous use of fluconazole [p<0.001; or=27.4 (95%ci: 8.1-92.0)] and viral load [p=0.042; or=2.3 (95%ci: 1.0-5.3)]. conclusions: these results are important for the development of strategies to eliminate these risk factors and significantly reduce oc in hiv-infected patients. keywords: aids, candidiasis, haart, hiv infection, prevalence ratio, risk factors. introduction oral candidiasis (oc) is the most frequent hiv infection-associated oral disease, and can also act as a marker for immunosuppression1-6. the prevalence and incidence of hiv infection in brazil are 7.5% and 1.39%, respectively5. the literature supports the position that systemically applied antifungal drugs have the greatest efficacy for the treatment of oc. however, these therapies must be prescribed following a thorough assessment of the risk for developing drug-induced toxicities6. oc responds to antifungal therapy, but eradication is rarely achieved unless the underlying immune-compromised state is resolved2,7. data about risk factors for hiv infection-associated oral lesions in the south american population are insufficient1,3,8. some studies have identified potential risk factors for development of hiv-associated oral diseases3-4,7,9-11. moura et al.4 demonstrated statistically significant associations between oral hairy leukoplakia (ohl) and hiv-1 viral load, oc, previous use of fluconazole and systemic acyclovir in brazilian hiv-infected adult patients. therefore, the goals of this study were: 1) braz j oral sci. 9(4):470-474 to estimate the prevalence of oc in a sample of brazilian hiv-infected adult patients, and 2) to investigate the risk factors for hiv-associated oc in this sample. material and methods between 2002 and 2004, 112 hiv-infected adult volunteers were recruited from the orestes diniz treatment center of parasitic and infectious diseases (ctr-dip) (belo horizonte, mg, brazil) to participate in this case-control study, approved by the ufmg’s bioethics research committee (protocol number 339/03). all patients were first diagnosed with hiv-infection by enzyme-linked immunosorbant assay (elisa) as the primary detection test, and the diagnoses were subsequently confirmed by the western blot test. the diagnosis for hiv-infection had already been established during the period of the first exam for all patients. a single, validated examiner, trained in oral medicine, conducted the oral clinical exam in accordance with the world health organization standards12. oc diagnosis was based on the published standard presumptive clinical criteria of international classification systems13. also, cytological features were considered in the diagnosis of oc: morphologic microscopic observation of fungal mycelial filaments from a non-cultured specimen scraped from the oral mucosa by periodic acid schiff (pas) staining. patients with a confirmed diagnosis of oc were included in the case group; those without clinical features of oc were included in the control group. the following variables were obtained from the case and control groups: age, gender, race, route of transmission, cd4 t lymphocytes count, viral load, platelets count, salivary flow, xerostomia, ohl, previous use of fluconazole, previous use of systemic acyclovir, use of highly active anti-retroviral therapy (haart), use of zidovudine (azt), intravenous drug use, smoking, and alcohol consumption. the cd4 t lymphocyte count was divided into <200 cells/mm3 and >200 cells/mm3. the viral load was divided into <3,000 copies/µl and >3,000 copies/µl. platelet count was divided into <150,000/mm3 and >150,000/mm3,4,14-16. the measurement of the salivary flow was performed through the collection of stimulated saliva, over the course of five minutes, in accordance to tárzia17. the salivary flow was identified as normal (> 0.70ml/min), moderately low (0.50 to 0.70 ml/min), low (0.30 to 0.49 ml/min), or severely low (0 to 0.29 ml/min)17. xerostomia was identified when the patient complained of dry mouth. the diagnosis of ohl was established according to clinical features and exfoliative cytology18-19. ohl was treated with topical applications of either podophyllin resin (25%) (prepared at ufmg’s pharmacy), or podophyllin resin (25%) together with acyclovir cream (5%) (ems-genéricos®, são bernardo do campo, sp, brazil)20. smoking individuals were identified as those who had smoked >100 cigarettes over their lifetime and smoked at the time of the study. non-smoking individuals were identified as those who had not smoked >100 cigarettes in their lifetime16. alcohol consumption was considered when the patient consumed alcohol on a daily basis. statistical analysis of data was performed using the statistical package for social service (spss) software program (version 16.0, spss inc., chicago, il, usa). univariate analyses were performed on all variables of this study using the fisher’s and chi-squared tests (2-sided tests). statistical significance was at a level of 0.05. the results of this analysis were expressed as an odds ratio (or) with a 95% confidence interval (ci). variables with p<0.25 were identified and included in the multivariate analyses. multiple logistic regression techniques were then used to develop a model and identify the set of variables that may predict risk indicators in hiv-infected adult patients with oc. results the sample included 35 (31.3%) hiv-infected adult patients with oc and 77 (68.7%) patients who did not have oc. the majority of patients, 82 (73.2%), were men. sixtyfive patients (58.0%) were caucasian and 47 (42.0%) were black. age varied from 20 to 59 years (mean age of 39.5 years). regarding the route of transmissions, the sample included 4 (3.6%) intravenous drug users, 13 (11.6%) not informed, 51 (45.5%) heterosexuals, 40 (35.7%) men who have sex with men (msm) and 4 (3.6%) bisexuals. of the 35 patients with oc, 20 (57.1%) were heterosexual, 8 (22.8%) were msm, 2 (5.7%) were bisexual, 1 (2.8%) was intravenous drug user, and 4 (11.4%) did not inform. oc was erythematous in 19 cases (54.3%), pseudomembranous in 10 cases (28.6%), and both in 06 cases (17.1%). angular cheilitis (9 cases) was also identified in our study, but all cases were in association with erythematous oc. table 1 summarizes the proportional prevalence and univariate analyses. statistically significant association was identified between the viral load of 3,000 copies/µl or greater (p=0.042; or=2.3), the ohl (p<0.001; or=10.2) and the previous use of fluconazole (p<0.001; or=27.4) with oc. platelets count (<150,000/mm3), haart (patients that did not take), gender (men), reduction of salivary flow, previous use of systemic acyclovir, use of azt (patients that did not take) and intravenous drug use (patients that did not use) were more frequently present in association with oc, though without a significant relationship (table 1). table 2 demonstrates the logistic regression models. multiple logistic regression tests confirmed the statistically significant association between the previous use of fluconazole and ohl with oc, regardless of the use of haart. adjusted results showed that hiv-infected patients with ohl, and those who had previously used fluconazole, were 3.6 times (95% ci=1.1-12.3) and 14.3 times (95% ci=3.8-53.6) more likely to present oc, respectively, regardless of the use of haart. discussion it has been suggested that oc represents a relevant marker of immune system status in hiv-infected patients, 471471471471471 risk factors for oral candidiasis in brazilian hiv-infected adult patients braz j oral sci. 9(4):470-474 472472472472472 variable level or unadjusted (95% ci) p or adjusted (95% ci) p haart yes 1 0.173 1 0.823 no 2.2 (0.7-6.5) 0.84 (0.2-4.0) oral hairy leukoplakia no 1 <.001 1 0.041 yes 10.2 (4.0-26.0) 3.6 (1.1-12.3) previous use of fluconazole yes 1 <.001 1 <.001 no 27.4 (8.1-92.0) 14.3 (3.8-53.6) viral load (copies/µl) <3,000 1 0.042 >3,000 2.3 (1.0-5.3) nam table 2. unconditional simple and multiple logistic regression analysis between independent variables and oral candidiasis (final model) among 112 hiv/aids patients attended at the ctr-dip, belo horizonte, mg, brazil, during 2002 to 2004 haart: highly active anti-retroviral therapy; or: odds ratio; ci: 95% confidence interval; nam: non adjusted to the model and is also a clinical predictor of aids progression3-4,7,11,14. in the present study, the prevalence of oc was 31.3%. most studies have reported prevalence rates of oc between 17.2% and 58.6%3,7,11,21. it is intuitive to expect an increase of oc in patients with low cd4 t lymphocyte count (<200 cells/mm3)3,7,910,14,22-23. campisi et al.24, ghate et al.9 and shiboski et al.25 did not find any relation between oc and low cd4 t lymphocyte count in hiv-infected adults, as is reflected in the current study. in our study, this may be attributed to the fact that haart is easily accessible in brazil, especially in ctr-dip, which contributes to the improvement of patient immunity, favoring an increase in the cd4 t lymphocyte count (>200 cells/mm3)4. previous studies have reported a heterogeneous division of viral load, ranging from 3,000 to 30,000 copies/µl3,14,2223,25. the high viral load presented a significant association with oc, as reported in the findings of other studies3,14,23,25. variable level pp patients with oc (n=35) patients without oc (n=77) p cd4 t lymphocyte count (cells/mm3) <200 33.3 8 16 0.804f >200 30.7 27 61 viral load (copies/µl) <3,000 23.4 15 49 0.042* >3,000 41.7 20 28 platelets count (mm3) <150,000 50.0 2 2 0.370f >150,000 30.6 33 75 haart yes 28.9 28 69 0.140f n o 46.6 7 8 gender men 32.9 27 55 0.527* women 26.7 8 22 reduction of salivary flow yes 34.1 14 27 0.615* n o 29.6 21 50 xerostomia yes 33.3 12 24 0.743* n o 30.3 23 53 oral hairy leukoplakia yes 66.7 22 11 <.001 n o 16.5 13 66 previous use of fluconazole yes 84.0 21 4 <.001 n o 16.1 14 73 previous use of systemic acyclovir yes 50.0 5 5 0.160f n o 29.4 30 72 use of azt yes 28.7 21 52 0.438* n o 35.9 14 25 intravenous drug use yes 22.2 2 7 0.424f n o 32.0 33 70 smoking yes 32.0 16 34 0.878* n o 30.6 19 43 alcohol consumption yes 30.0 3 7 0.619f n o 31.4 32 70 table 1. proportional prevalence and univariate analyze for oral candidiasis among 112 hiv/aids patients attended to at the ctr-dip, belo horizonte, mg, brazil, during 2002 to 2004 *chi-square test; ffisher’s exact test; pp: proportional prevalence; oc: oral candidiasis; haart: highly active anti-retroviral therapy risk factors for oral candidiasis in brazilian hiv-infected adult patients braz j oral sci. 9(4):470-474 473473473473473 mercante et al.10 and coogan et al.26 suggested that viral load may be a more important predictor for oropharyngeal candidiasis than cd4 t lymphocyte count. oc was more frequent in patients with platelet count <150,000, though without a significant association. patton et al.27 observed that platelet count <150,000 may predispose hiv-infected patients to the development of oral manifestations, as verified in 15.5% of 516 patients. one possible explanation for our results is the fact that the number of individuals with platelet count <150,000 was small (1.7%). oc can be considered a measure of assessment of the need to begin antiretroviral medication11,7-8,11,14,25,28. in contrast, we did not find statistically significant association between haart and oc. tappuni and fleming28 found no association between antiretroviral medication and the presence of oc. thompson et al.1 confirmed that oc remains a significant infection in advanced aids, even with haart. gender, salivary flow, xerostomia, previous use of systemic acyclovir, use of azt, intravenous drug use, smoking, and alcohol consumption do not represent risk indicators for oc7,14,24-26,29. although it was not statistically significant, in the present study, oc was proportionally more frequent in men, in patients with a reduced salivary flow, with previous use of systemic acyclovir, who were not taking azt and who did not use intravenously drug. although our study was directed towards oc, the presence of ohl and oc was also verified, simultaneously, as being the two most common oral lesions in hiv-infected adults. the association between the presence of oc and ohl verified in this study has also been observed in many other studies in the us and europe3,7,11,27,29. the previous use of fluconazole was a strong indicator of risk for oc. schmidt-westhausen et al.30 found different results, and they concluded that the presence of oral lesions associated with hiv-infection did not have a correlation with the use of fluconazole. it is possible that candida resistance to fluconazole is responsible for our finding. the high incidence of mucosal and deep seated forms of candidiasis in hiv-infected patients has resulted in the use of fluconazole. their widespread use has been followed by an increase in antifungal resistance and candida resistance. various factors may contribute to fluconazole resistance, such as the degree of immunosuppression of the patients, the chemotherapeutic use of drugs and the intrinsic resistance of candida species2. fluconazole used for prolonged periods can select for less susceptible species candida2. the resistance of candida, isolated to currently available antifungal drugs, is a highly relevant factor because it presents important implications for morbidity and mortality1,6. systemically applied antifungal drugs have the greatest efficacy for the treatment of oral candidiasis. however, these therapies must be prescribed following a thorough assessment of the risk for developing drug-induced toxicities, the likelihood of candida species resistance, and the cost-effectiveness of medications. fluconazole prophylaxis should be reserved for patients at high risk for recurrence of fungal infections, and not for routine prophylaxis6. additionally, another explanation for the association of oc with previous use of fluconazole could be the fact that patients with hiv infection can have recurrent oc and that are treated with fluconazole1. these results are important for the development of strategies to eliminate these indicators of risk and significantly reduce oc in brazilian hiv-infected adult patients. acknowledgements this study was supported by grants from the the national council for scientific and technological development (cnpq #301490/2007-4). mesquita ra is research fellows of the cnpq. references 1. thompson gr, patel pk, kirkpatrick wr, westbrook sd, berg d, erlandsen j, et al. oropharyngeal candidiasis in the era of antiretroviral therapy. oral surg oral med oral pathol oral radiol endod. 2010; 109: 488-95. 2. chunchanur sk, nadgir sd, halesh lh, patil bs, kausar y, chandrasekhar mr. detection and antifungal susceptibility testing of oral candida dubliniensis from human immunodeficiency virus-infected patients. indian j pathol microbiol. 2009; 52: 501-4. 3. miziara id, weber r. oral candidosis and oral hairy leukoplakia as predictors of haart failure in brazilian hiv-infected patients. oral dis. 2006; 12: 402-7. 4. moura mdg, grossmann smc, fonseca lms, senna mib, mesquita ra. risk factors for oral hairy leukoplakia in hiv-infected adults of brazil. j oral pathol med. 2006; 35: 321-6. 5. merçon m, tuboi sh, batista sm, telles sr, grangeiro jr, zajdenverg r, et al. risk-based assessment does not distinguish between recent and chronic hiv-1 infection in rio de janeiro, brazil. braz j infect dis. 2009; 13: 272-5. 6. ship ja, vissink a, challacombe sj. use of prophylactic antifungals in the immunocompromised host. oral surg oral med oral pathol oral radiol endod. 2007; 103(suppl.1): s6.e1-s6.e14. 7. chattopadhyay a, caplan dj, slade gd, shugars dc, tien hc, patton ll. risk indicators for oral candidiasis and oral hairy leukoplakia in hivinfected adults. community dent oral epidemiol. 2005; 33: 35-44. 8. hodgson ta, greenspan d, greenspan js. oral lesions of hiv disease and haart in industrialized countries. adv dent res. 2006; 19: 57-62. 9. ghate m, deshpande s, tripathy s, nene m, gedam p, godbole s, et al. incidence of common opportunistic infections in hiv-infected individuals in pune, india: analysis by stages of immunosuppression represented by cd4 counts. int j infect dis. 2009; 13: e1-8. 10. mercante de, leigh je, lilly ea, mcnulty k, fidel pl. assessment of the association between hiv viral load and cd4 cell count on the occurrence of oropharyngeal candidiasis in hiv-infected patients. j acquir immune defic syndr. 2006; 42: 578-83. 11. pinheiro a, marcenes w, zakrzewska jm, robinson pg. dental and oral lesions in hiv infected patients: a study in brazil. int dent j. 2004; 54: 131-7. 12. world health organization. oral health survey: basic methods. 4.ed. geneva: who health organization; 1997. 13. ec-clearinghouse on oral problems related to hiv infection and who collaborating centre on oral manifestations of immunodeficiency virus. classification and diagnostic criteria for oral lesions in hiv infection. j oral pathol med. 1993; 22: 289-91. risk factors for oral candidiasis in brazilian hiv-infected adult patients braz j oral sci. 9(4):470-474 474474474474474 14. chattopadhyay a, caplan dj, slade gd, shugars dc, tien hc, patton ll. incidence of oral candidiasis and oral hairy leukoplakia in hiv-infected adults in north carolina. oral surg oral med oral pathol oral radiol endod. 2005; 99: 39-47. 15. patton ll. hematologic abnormalities among hiv-infected patients: associations of significance for dentistry. oral surg oral med oral pathol oral radiol endod. 1999; 88: 561-7. 16. tomar sl, asma s. smoking-attributable periodontitis in the united states: findings from nhanes iii. national health and nutrition examination survey. j periodontol. 2000; 71: 743-751. 17. tarzia o. importância do fluxo salivar com relação à saúde bucal. cecade news. 1993; 1: 13-7. 18. fraga-fernandes j, vicandi-plaza b. diagnosis of hairy leukoplakia by cytologic methods. am j clinic pathol. 1992; 97: 262-6. 19. epstein jb, fatahzadeh m, matisic j, anderson g. exfoliative cytology and electron microscopy in the diagnosis of hairy leukoplakia. oral surg oral med oral pathol oral radiol endod. 1995; 79: 564-9. 20. moura mdg, guimaraes trm, fonseca lms, pordeus ia, mesquita ra. a random clinical trial study to assess the efficiency of topical applications of podophyllin resin (25%) versus podophyllin resin (25%) together with acyclovir cream (5%) (pa) in the treatment of oral hairy leukoplakia. oral surg oral med oral pathol oral radiol endod. 2007; 103: 64-71. 21. lourenço ag, figueiredo lt. oral lesions in hiv infected individuals from ribeirão preto, brazil. med oral patol oral cir bucal. 2008; 13: e281-6. 22. fidel pl. candida-host interactions in hiv disease: relationships in oropharyngeal candidiasis. adv dent res. 2006; 19: 80-4. 23. gautam h, bhalla p, saini s, uppal b, kaur r, baveja cp, et al. epidemiology of opportunistic infections and its correlation with cd4 tlymphocyte counts and plasma viral load among hiv-positive patients at a tertiary care hospital in india. j int assoc physicians aids care. 2009; 8: 333-7. 24. campisi g, pizzo c, mancuso s, margiotta v. gender differences in human immunodeficiency virus-related oral lesions: an italian study. oral surg oral med oral pathol oral radiol endod. 2001; 91: 546-51. 25. shiboski ch, wilson cm, greenspan d, hilton j, greenspan js, moscicki ab. hiv-related oral manifestations among adolescents in a multicenter cohort study. j adolesc health. 2001; 29(suppl.3): 109-14. 26. coogan mm, fidel pl, komesu mc, maeda n, samaranayake lp. candida and mycotic infections. adv dent res. 2006; 19: 130-8. 27. patton ll, mckaig rg, strauss rp, eron jjjr. oral manifestations of hiv in a southeast usa population. oral dis. 1998; 4: 164-9. 28. tappuni ar, fleming gjp. the effect of antiretroviral therapy on the prevalence of oral manifestations in hiv-infected patients: a uk study. oral surg oral med oral pathol oral radiol endod. 2001; 92: 623-8. 29. nittayananta w, chanowanna n, sripatanakul s, winn t. risk factors associated with oral lesions in hiv-infected heterosexual people and intravenous drug users in thailand. j oral pathol med. 2001; 30: 224-30. 30. schmidt-westhausen am, priepke f, bergmann fj, reichart pa. decline in the rate of oral opportunistic infections following introduction of highly active antiretroviral therapy. j oral pathol med. 2000; 29: 336-41. risk factors for oral candidiasis in brazilian hiv-infected adult patients braz j oral sci. 9(4):470-474 429 too many requests error 429 too many requests too many requests guru meditation: xid: 26736949 varnish cache server oral sciences n3 original article braz j oral sci. july | september 2015 volume 14, number 3 one-year clinical evaluation of the retention of resin and glass ionomer sealants on permanent first molars in children kamila prado graciano1, marcos ribeiro moysés2, josé carlos ribeiro2, camila a. pazzini1, camilo aquino melgaço1, joana ramos-jorge3 1universidade vale do rio verde unincor, school of dentistry, department of pediatric dentistry and orthodontics, três corações, mg, brazil 2universidade vale do rio verde unincor, school of dentistry, department of restorative dentistry, três corações, mg, brazil 3universidade federal dos vales do jequitinhonha e mucuri ufvjm, school of dentistry, department of pediatric dentistry and orthodontics, diamantina, mg, brazil correspondence to: joana ramos-jorge rua arraial dos forros, 215 cep: 39.100-000, diamantina, mg, brazil phone/fax: +55 38 3531-1415 e-mail: joanaramosjorge@gmail.com abstract aim: to compare the retention of glass ionomer cement (gic) used as fissure sealant with a resinbased sealant. methods: sixto nine-year-old children (n=96) with all permanent first molars in occlusion were examined and assigned to two groups: gic sealant or resin-based sealant. the sealants were applied according to the manufacturers’ recommendations. the assessment of sealant retention was performed at two-month interval sessions (n=6), when each sample was scored according to the following criteria: complete retention, partial retention or complete loss. the visual and tactile examinations were carried out with a who probe, mouth mirror, air syringe and artificial light. the data were submitted to descriptive statistics and survival analysis. results: a total of 384 occlusal surfaces were analyzed. independent of the tooth and evaluation time, slightly better results were achieved by the resin-based sealant, but the difference was not statistically significant. conclusions: gic and resin-based sealants achieved similar results with regards to retention during a one-year follow-up period. keywords: pit and fissure sealants; resin cements; glass ionomer cements. introduction dental caries is one of the most common oral health problems in childhood1. in recent years, guidelines and systematic reviews of the literature have recommended the use of pit and fissure sealants for populations at high risk for dental caries2-7. a brazilian study conducted in sevento ten-year-old children from the rural community showed that caries activity in the permanent first molars was associated with dental fluorosis and malocclusion8. accordingly, strategies aimed at health promotion should be adopted in a large scale to minimize the prevalence of oral diseases. a systematic review of randomized clinical trials reports that pit and fissure sealants on permanent molars reduce the incidence of dental caries among children and adolescents9. this is in agreement with the findings of a previous study, in which the authors stated that preventive strategies involving the use of a sealant have significantly contributed to the reduction of dental caries 10. occlusal caries is most prevalent among children because of the morphology of pits and fissures, which are stagnation areas11. thus, dental sealant acts as a mechanical barrier to the accumulation of biofilm12. received for publication: may 13, 2015 accepted: august 11, 2015 braz j oral sci. 14(3):190-194 http://dx.doi.org/10.1590/1677-3225v14n3a03 191191191191191 resin-based and glass ionomer sealants stand out among the materials employed as pit and fissure sealants13. in a review of the literature, niederman14 stated that resin-based and glass ionomer sealants have a similar preventive effect. the fact that glass ionomer cement (gic) recharges the fluoride in fluoridated agents, such as toothpastes and mouthwashes, contributes to the maintenance of adequate levels of fluoride in saliva15. however, the retention of gic is reported to be insufficient13, whereas resin-based sealants exhibit satisfactory retention16. before broadening the use of sealants as a preventive measure in the public healthcare system, it is fundamental to establish the effectiveness of this procedure. thus, the clinical follow up of teeth sealed with different materials is important to strengthen the scientific evidence on this issue. as the effectiveness of a sealant is associated with the retention of the material in the tooth17, the aim of the present study was to compare the retention of gic used as a fissure sealant and a resin-based sealant in sixto nine-year-old children for a one-year follow-up period. material and methods study design and sample this study involved the clinical follow up of children treated at the pediatric dentistry clinic of the vale do rio verde university, três corações, brazil. sixto nine-yearsold children (n=96), who have met the inclusion criteria after being submitted to clinical examination, were indicated to have their first permanent molars sealed (n=384). a statement of informed consent was obtained from the legal guardians prior to the procedures. the inclusion criteria were high risk of dental caries (consumption of carbohydrates three or more times a day between meals), inadequate access to fluoride (water or toothpaste), low socioeconomic level, presence of all four permanent first molars in occlusion and history of carious lesions in the primary dentition. the exclusion criteria were cavitated carious lesions, hypomineralization, restoration or sealant on the occlusal face of any of the permanent first molars and use of a removable or fixed orthodontic appliance. in addition, children who presented any systemic alteration or troublesome behavior were excluded. one group of children received a gic sealant (fuji vii, gc corporation, tokyo, japan) and the other group received a resin-based sealant (delton, dentsply, petrópolis, rj, brazil). the sealant was applied after any curative dental treatment required, such as restoration, root canal or extraction was concluded. the type of sealant was selected based on the needs of each child. children who exhibited active initial dental caries (even after the conclusion of the curative treatment) received gic. all children received oral hygiene instructions and dietary counseling. application of sealant the occlusal sealants were applied by a dentist experienced in pediatric dentistry in a university setting. prior to application, the teeth were cleaned with water and pumice stone paste using pointed bristle brushes at low speed and then rinsed thoroughly to remove the debris. isolation was performed using cotton rolls, with adequate suctioning to manage the salivation flow. the teeth that received the resin-based sealant were dried with an air syringe and etched with 37% phosphoric acid (atacktec, rio do sul, sc, brazil) for 30 s, followed by rinsing for 30 s and drying with an air syringe for 15 s. isolation was replaced before application of the resin-based sealant, which was submitted to a conventional light polymerization method (elipar free light, 3m espe, st. paul, mn, usa; 1200 mw/ cm2 for 20 s). all the sealed surfaces were tested for defects and additional material was added if necessary. for application of the gic sealant, a shallow measure of powder and one drop of liquid were used for each tooth (blending time: 1 min). after drying for 15 s with an air syringe, the material was applied and four minutes were allowed for curing. high points were checked using articulating paper and corrected. the adjustments were performed using composite polishing burs in a single visit. the gic sealant applications were made by three dentists experienced in pediatric dentistry. evaluation of retention the evaluation of sealant retention was performed at two-month intervals (2nd, 4th, 6th, 8th, 10th and 12th) over the course of one year. for such, visual and tactile examinations were carried out with the aid of a who probe, mouth mirror, air syringe and artificial light. the status of the sealant was checked and scored as complete retention (all pits and fissures filled with material), partial retention (loss of some material) or complete loss (absence of material from the entire occlusal surface). this evaluation was performed by a trained dentist, who did not participate in sealant application sessions. statistical analysis data analysis was conducted with the statistical package for social sciences (spss version 20.0 for windows, spss inc, chicago, il, usa). the data were submitted to descriptive statistics, followed by survival analysis. a kaplanmeier plot was constructed for the analysis of sealant retention at the different evaluation times. the level of significance was set to 5% (p<0.05). ethical considerations this study received approval from the human research ethics committee of vale do rio verde university (brazil) under protocol number 30609814.8.0000.5158. all research subjects underwent dental treatment at the school of dentistry and provided written informed consent. results among the children who completed the study (98%), 46 received the gic sealant and 50 received the resin-based sealant. the distribution of children in relation to their one-year clinical evaluation of the retention of resin and glass ionomer sealants on permanent first molars in children braz j oral sci. 14(3):190-194 type of material characteristic of child sex female male age 6 years 7 years 8 years 9 years glass ionomer cement n (%) 25 (54.3) 21 (45.7) 6 (13.0) 9 (19.6) 15 (32.6) 16 (34.8) resin-based sealant n(%) 26 (52.0) 24 (48.0) 6 (12.0) 9 (18.0) 15 (30.0) 20 (40.0) table 1:table 1:table 1:table 1:table 1: distribution of sealants according to children’s characteristics type of material retention of sealant tooth 16 complete loss partial retention complete retention tooth 26 complete loss partial retention complete retention tooth 36 complete loss partial retention complete retention tooth 46 complete loss partial retention complete retention glass ionomer cement n (%) 4 (8.7) 25 (54.3) 17 (37.0) 3 (6.5) 18 (39.1) 25 (54.4) 6 (13.0) 17 (37.0) 23 (50.0) 9 (19.6) 15 (32.6) 22 (47.8) resin-based sealant n(%) 4 (8.0) 24 (48.0) 22 (44.0) 3 (6.0) 14 (28.0) 33 (66.0) 6 (12.0) 17 (34.0) 27 (54.0) 6 (12.0) 15(30.0) 29 (58.0) table 2: table 2: table 2: table 2: table 2: retention of sealant according to tooth and type of material co-variables type of material tooth 16 glass ionomer cement resin-based sealant tooth 26 glass ionomer cement resin-based sealant tooth 36 glass ionomer cement resin-based sealant tooth 46 glass ionomer cement resin-based sealant number of losses 29 28 21 17 23 23 24 21 mean survival 7.91 (6.96-8.87) 8.40 (7.48-9.32) 8.96 (7.94-9.98) 9.68 (8.74-10.62) 9.00 (8.08-9.92) 9.24 (8.37-10.11) 8.48 (7.44-9.52) 9.40 (8.51-10.29) p-value log rank test 0.453 0.264 0.707 0.213 table 3:table 3:table 3:table 3:table 3: survival analysis according to tooth and material characteristics (sex and age) was similar between groups. however, the number of nine-year-old children was higher in the group that received a resin-based sealant (table 1). all occlusal surfaces of their permanent first molars were sealed and included in the study (n=384). the descriptive analysis demonstrated a slightly higher percentage of complete retention of the resin-based sealant in comparison to gic after one year. complete retention rate was higher on the tooth 26 in both groups (table 2). however, independent from the analyzed tooth, this difference did not achieve statistical significance (table 3). the data are confirmed by the kaplanmeier plot, which demonstrates similar sealant retention in both groups at any given evaluation time (figure 1). discussion in the present study, a sample of children was followed up for one year for the evaluation of sealant retention on permanent first molars, since pits and fissures on these teeth are the most susceptible sites to dental caries in the permanent dentition18. in a recent review it was found that the use of resinbased sealants was effective in reducing dental caries 16. however, no conclusion could be reached regarding the relative effectiveness of different types of sealants9,19. in another review it was found that the risk of complete loss of sealant retention was associated with the risk of caries occurrence for resin-based sealants, but not for ionomer-based sealants20. thus, although the performed meta-analysis has pointed out a low retention rate for glass ionomer cement sealants16, the retention of at least part of this material has a protective effect, unlike what occurs with resin-based sealants. these findings underscore the need to evaluate sealant retention with regard to partial retention, as performed in the present study. independent of the analyzed tooth, no significant difference in retention was found between the two types of material used as sealant. however, at follow-up sessions any loss of material present was considered as partial retention. the descriptive analysis demonstrated that the rate of partial retention was similar in both groups. moreover, no significant differences were found at any given evaluation time. although pretreatment with a suitable acid is essential to obtain adequate penetration of a sealing material 21, the 192192192192192one-year clinical evaluation of the retention of resin and glass ionomer sealants on permanent first molars in children braz j oral sci. 14(3):190-194 193193193193193 authors believe that the penetration did not influence the sealants retention since this pretreatment was not performed for the application of gic sealants. the initial loss of sealant may be due to technical flaws and subsequent loss is likely due to occlusal forces22. the largest loss of sealant was found at the six-month evaluation, which is in agreement with data described by subramaniam et al.23. however, the retention rates in the present study were higher, which may be due to the improvements in the quality of the materials with time. moreover, one factor may have contributed to this difference between the studies. the afore-mentioned study has a more robust design, in which the researchers applied resin-based sealants on teeth 16 and 46 and pink-colored gic sealants on teeth 26 and 36. since adding color to the sealant improves perception of the dentist, it is possible that this difference of methods also contributed to the divergent results of studies. the present findings should be confirmed by studies with more robust designs for corroborating the comprehensive use of gic sealants in public oral healthcare services, since resin-based sealants are technique-sensitive and influenced by patient cooperation, operator variability and contamination of the operating field24. moreover, further studies are required with a longer follow-up period and the evaluation of the effects of such sealants on caries prevention and the control of lesions in the early stage. during a one-year follow-up period, considering time intervals of two months, gic and resin-based sealants achieved similar results with regards to retention. references 1. benzian h, hobdell m, holmgren c, yee r, monse b, barnard jt, et al. political priority of global oral health: an analysis of reasons for international neglect. int dent j. 2011; 61: 124-30. fig. 1: kaplan-meier plot comparing two types of sealant 2. griffin so, oong e, kohn w, vidakovic b, gooch bf, cdc dental sealant systematic review work group, et al. the effectiveness of sealants in managing caries lesions. j dent res. 2008; 87: 169-74. 3. beauchamp j, caufield pw, crall jj, donly k, feigal r, gooch b, et al. evidence-based clinical recommendations for the use of pit-and-fissure sealants: a report of the american dental association council on scientific affairs. j am dent assoc. 2008; 139: 257-68. 4. ahovuo-saloranta a, hiiri a, nordblad a, mäkelä m, worthington hv. pit and fissure sealants for preventing dental decay in the permanent teeth of children and adolescents. cochrane database syst rev. 2008; 8: cd001830. 5. gooch bf, griffin so, gray sk, kohn wg, rozier rg, siegal m, et al. preventing dental caries through school-based sealant programs: updated recommendations and reviews of evidence. j am dent assoc. 2009; 140: 1356-65. 6. hiiri a, ahovuo-saloranta a, nordblad a, mäkelä m. pit and fissure sealants versus fluoride varnishes for preventing dental decay in children and adolescents. cochrane database syst rev. 2010; 17: cd003067. 7. mickenautsch s, yengopal v. caries-preventive effect of glass ionomer and resin-based fissure sealants on permanent teeth: an update of systematic review evidence. bmc res notes. 2011; 4: 22. 8. motta lj, santos jg, alfaya ta, guedes cc, godoy chl, bussadori sk. clinical status of permanent first molars in children aged seven to ten years in a brazilian rural community. braz j oral sci. 2012; 11: 475-80. 9. ahovuo-saloranta a, forss h, walsh t, hiiri a, nordblad a, mäkelä m, et al. sealants for preventing dental decay in the permanent teeth. cochrane database syst rev. 2013; 3: cd001830. 10. vieira al, zanella nl, bresciani e, barata tde j, da silva sm, machado ma, et al. evaluation of glass ionomer sealants placed according to the art approach in a community with high caries experience: 1-year followup. j appl oral sci. 2006; 14: 270-5. 11. kidd ea, smith bg, pickard hm. pickard’s manual of operative dentistry. 6th ed. oxford university press; 1990. 12. feldens eg, feldens ca, araujo fb, souza ma. invasive technique of pit and fissure sealants in primary molars: a sem study. j clin pediatr dent. 1994; 18: 187-90. 13. seppä l, forss h, øgaard b. the effect of fluoride application on fluoride release and the antibacterial action of glass ionomers. j dent res. 1993; 72: 1310-4. one-year clinical evaluation of the retention of resin and glass ionomer sealants on permanent first molars in children braz j oral sci. 14(3):190-194 194194194194194 14. niederman r. glass ionomer and resin-based fissure sealants equally effective? evid based dent. 2010; 11: 10. 15. carvalho as, cury ja. fluoride release from some dental materials in different solutions. oper dent. 1999; 24: 14-9. 16. kühnisch j, mansmann u, heinrich-weltzien r, hickel r. longevity of materials for pit and fissure sealing results from a meta-analysis. dent mater. 2012; 28: 298-303. 17. bhatia mr, patel ar, shirol dd. evaluation of two resin based fissure sealants: a comparative clinical study. j indian soc pedod prev dent. 2012; 30: 227-30. 18. sheiham a, sabbah w. using universal patterns of caries for planning and evaluating dental care. caries res. 2010; 44: 141-50. 19. deery c. strong evidence for the effectiveness of resin based sealants. evid based dent. 2013; 14: 69-70. 20. mickenautsch s, yengopal v. validity of sealant retention as surrogate for caries prevention a systematic review. plos one. 2013; 8: e77103. 21. markovic d, petrovic b, peric t, miletic i, andjelkovic s. the impact of fissure depth and enamel conditioning protocols on glass-ionomer and resin-based fissure sealant penetration. j adhes dent. 2011; 13: 171-8. 22. messer lb, calache h, morgan mv. the retention of pit and fissure sealants placed in primary school children by dental health services, victoria. aust dent j. 1997; 42: 233-9. 23. subramaniam p, konde s, mandanna dk. retention of a resin-based sealant and a glass ionomer used as a fissure sealant: a comparative clinical study. j indian soc pedod prev dent. 2008; 26: 114-20. 24. karlzen-reuterving g, van dijken jw. a three-year follow-up of glass ionomer cement and resin fissure sealants. asdc j dent child. 1995; 62: 108-10. one-year clinical evaluation of the retention of resin and glass ionomer sealants on permanent first molars in children braz j oral sci. 14(3):190-194 oral sciences n3 case report braz j oral sci. april/june 2010 volume 9, number 2 desmoplastic ameloblastoma a report of two clinical cases anusha rangare laxmana 1, subhas babu gogineni 2, priya sara thomas 1, shishir ram shetty3 1mds, postgraduate student, department of oral medicine and radiology, a.b shetty memorial institute of dental sciences, mangalore, karnataka, india 2mds, professor and head of the depto of oral medicine and radiology, a.b shetty memorial institute of dental sciences, mangalore, karnataka, india 3mds, assistant professor, department of oral medicine and radiology, a.b shetty memorial institute of dental sciences, mangalore, karnataka, india correspondence to: anusha rangare laxmana department of oral medicine and radiology a.b shetty memorial institute of dental sciences, mangalore 575018, karnataka, india. e-mail: dr.anusharl@gmail.com received for publication: april 15, 2010 accepted: june 16, 2010 abstract desmoplastic ameloblastoma is a relatively rare histological variant of ameloblastoma with specific clinical, radiological, and histological features. although radiographic examination of ameloblastomas usually reveals unilocular or multilocular radiolucency, desmoplastic ameloblastoma may appear as a mixed radiopaque-radiolucent lesion resembling benign fibro-osseous lesions. histologically, desmoplastic ameloblastoma is characterized by small nests and strands of “compressed” odontogenic epithelium supported by pronounced collagenized stroma. this report describes two cases of desmoplastic ameloblastoma in the anterior maxilla of a female patient and the anterior mandible of a male patient, mimicking clinically as an odontogenic cyst. keywords: ameloblastoma, desmoplastic, multilocular, anterior maxilla, anterior mandible. introduction ameloblastomas are tumors arising from the odontogenic epithelium and most commonly encountered odontogenic tumors. despite their locally destructive nature, they are considered benign1. histologically, ameloblastoma has been classified as follicular, plexiform, acanthomatous, granular cell, desmoplastic, and basal cell1-3. in 1984, eversole et al. discovered a new and unusual histologic variant known as desmoplastic ameloblastoma4, but only in 1992, the world health organization (who) recognized this entity as a variant of ameloblastoma3. till date, 145 cases of desmoplastic ameloblastoma have been reported in japanese, chinese, malaysian, western, and african populations, with very few cases described in indians5. this report describes two cases of desmoplastic ameloblastoma in the anterior maxilla of a female patient and the anterior mandible of a male patient, mimicking clinically as an odontogenic cyst. case 1 a 47-year-old female patient came to the department of oral medicine with complaints of swelling in the right anterior region of the upper jaw with 6 months of duration. she revealed a history of insidious onset as a small nodule, gradually reaching the present extent without any paresthesia, discharge but was associated with mild, intermittent type of pain. no other associated symptoms were reported by the patient. intraoral examination revealed a well defined, non-tender, hard swelling in the braz j oral sci. 9(2):137-141 braz j oral sci. 9(2):137-141 right anterior maxilla with intact mucosa causing bicortical expansion (figure 1). the maxillary right canine was vital with grade i mobility. the provisional diagnosis of a fibroosseous lesion of right maxilla was made. intraoral periapical radiograph revealed a diffuse, illdefined, radiolucent lesion interspersed with radiopaque septae, producing a multilocular appearance with widening of the periodontal ligament space and loss of lamina dura of the right canine (figure 2). computed tomography (ct) (figure 3) revealed that the lesion was extending on buccal and palatal aspects from the right lateral incisor to the right second molar, and the walls of the maxillary antrum were intact and not involved. the overall clinical and radiographic features were suggestive of an odontogenic tumor, probably an ameloblastoma, with differential diagnosis of monostotic fibrous dysplasia, or adenomatoid odontogenic tumor. fig. 2. intraoral periapical radiograph showing multilocular appearance fig. 3. axial ct scan showing that the lesion extending on the buccal and palatal aspects from the right lateral incisor to right second molar. incisional biopsy was performed and the histopathologic evaluation of the specimen (figure 4) showed irregular, bizarrely shaped odontogenic epithelial islands and cords in a moderately cellular fibrous connective tissue with abundant thick collagen fibers that appear to compress the odontogenic islands giving them a stellate or an “animallike” configuration and the diagnosis of desmoplastic ameloblastoma was established. excision of the lesion with extraction of right lateral incisor and right canine was done, followed by reconstruction of the defect with buccal pad of fat and primary mucosal fig. 4. histological image showing irregular, bizarrely shaped odontogenic epithelial islands and cords in a moderately cellular fibrous connective tissue with abundant thick collagen fibers that appear to compress the odontogenic islands giving them a stellate or an “animal-like” configuration. 138 fig. 1. photograph showing bicortical expansion on maxilla with intact mucosa. desmoplastic ameloblastoma a report of two clinical cases braz j oral sci. 9(2):137-141 closure under general anesthesia with acrylic splint placement on maxillary arch. patient was recalled after one week, showing ongoing healing of the site. the patient was reviewed after 1 month, and complete healing of the lesion was noticed. the patient was kept on periodic recall every 6 months during 1 year. however, she could not keep up the recall appointments. case 2 a 37-year-old male patient reported with complaints of swelling in the anterior region of the mandible for two months. patient had history of trauma to this region (biting on hard food), after which he noticed the swelling a week later. swelling was initially smaller in size and gradually progressed to the present size. there was no associated pain, pus discharge, paresthesia or other symptoms. intraoral examination revealed a well defined solitary, nontender swelling in right mandibular anterior region crossing midline and causing bicortical expansion (figure 5). on palpation, swelling was soft to firm in consistency with areas of erosion noticed labially with intact mucosa. the mandibular right and left central incisor and mandibular right lateral incisor were vital with grade ii mobility. panoramic radiograph (figure 6) showed multilocular lesion involving left parasymphysis area causing root fig. 6. orthopantomogram showing multilocular lesion with root resorption of left mandibular premolars and canine. fig. 5. photograph showing bicortical expansion on anterior mandible with intact mucosa resorption of left canine and premolars. the anterior extension of the lesion couldn’t be traced out because of superimposition of cervical spine. mandibular occlusal view showed bicortical expansion and thinning of the buccal cortex (figure 7). intraoral periapical radiograph (figure 8) revealed an illdefined multilocular radiolucency superiorly causing destruction of alveolar crestal bone with respect to left mandibular lateral incisor and mandibular canine. the inferior of the radiolucency showed scalloping and the interior of the radiolucency showed remnants of trabeculae. there was displacement of the roots of left mandibular lateral incisor and mandibular canine. the provisional diagnosis of odontogenic cyst was proposed with differential diagnosis of traumatic bone cyst, odontogenic tumor, and central giant cell granuloma. aspiration yielded blood tinged straw colored fluid. incisional biopsy was performed and the histopathologic evaluation of the specimen (figure 9) showed dense collagen stroma with odontogenic epithelial cells arranged in the form of long, thin strands that gives an animal-like pattern configuration. the epithelial cells were hyperchromatic. this proliferation seemed to be compressed by a dense stroma, and a final diagnosis of desmoplastic ameloblastoma was established. peripheral osteotomy of anterior mandible with removal from the mandibular right first molar to the left lateral incisor was done under general anesthesia, and the patient was put on regular periodic recall check up till date (figure 10). the patient has currently been on follow up for the past 8 months. discussion in the who classification, desmoplastic ameloblastoma is considered as a rare variation of ameloblastoma. it accounts for 4% to 5% of all ameloblastoma6. a retrospective study was done to correlate the clinical and radiographic features of 115 cases of desmoplastic ameloblastoma reported in fig. 7. mandibular occlusal view showing thinning of buccal cortex 139desmoplastic ameloblastoma a report of two clinical cases fig. 8. intraoral periapical radiograph showing multilocular appearance with divergence of the roots. fig. 9. histological image showing dense collagen stroma with odontogenic epithelial cells arranged in the form of long, thin strands, giving an animal-like pattern configuration. the epithelial cells are hyperchromatic. these proliferations seem to be compressed by dense stroma. fig. 10. postoperative orthopantomogram showing the wide excision of the lesion. literature from 1984 to 2008, and concluded that it presents distinct clinical, radiographic and histologic features when compared to “conventional ameloblastomas”7. this entity occurs most commonly in the third to fifth decades of life, with an equal male to female ratio2,5. our cases also showed the same age and sex predilection. more than 70% of the desmoplastic ameloblastoma arose within the anterior or premolar regions of the jaws as seen in our both cases, and roughly half of the tumors occurred in the maxilla1,8-9. clinically, maxillary lesions are more dangerous than mandibular ones as they can invade the adjacent sinus and orbit and involve vital structures. additionally, the thin maxillary bone is a weak natural barrier for tumors as compared to the thicker mandible10. radiologically, the desmoplastic variant exhibits atypical and varied radiographic features as: localized irregular multilocular radiolucency with indistinct borders as seen in our second case, or a radiopaque/ radiolucent appearance with ill defined margins similar to fibro-osseous lesion as seen in our first case, or a massive expansible osteolytic lesion with honeycomb, mottled or multilocular appearance4,8. tooth displacement was a common feature for desmoplastic ameloblastoma which was seen in our second case. root resorption was discovered in only 33% of the cases which was seen in our both cases8. ct scan can delineate the internal structure of the lesion more accurately and is particularly helpful in determining its margins and extension into adjacent structures10. mri shows heterogeneous low to intermediate signal intensity on t1w images, heterogeneous high signal intensity on t2w images, and strong enhancement on post-gadolinium t1w images11. histologically, it is characterized by abundant collagenous proliferation of the stroma, loss of cell rich connective tissue, absence of capsule, and existence of some ameloblastoma like structures with peripherally compressed ameloblasts4,12. a few cases of hybrid lesion of ameloblastoma have been reported in the literature in which histologically, areas of follicular and plexiform patterns are found together with characteristic desmoplastic areas. this type of variant was first described by waldron2. the tumor cells of desmoplastic ameloblastoma have shown positive immunoreactivities for cytokeratin (ck), ck 8, 13, 19, filaggrin and ameloblastoma antibodies, retaining the odontogenic epithelial characteristics13. various facts about this lesion may suggest aggressiveness: a potential to grow to a large size; the common location in the maxilla that may produce an early invasion of adjacent structures; the diffuse radiographic appearance. finally, it is almost impossible to find the exact interface of the lesion with normal bone, making it especially difficult to treat surgically9. enucleation or curettage alone of the lesion may lead to recurrence14, as there is indistinct boundary between the tumor and normal tissue. therefore complete resection and regular follow up is recommended4. in conclusion the desmoplastic ameloblastoma reveals unique radiological and histological features. further investigation of a larger number of more cases must be carried 140 braz j oral sci. 9(2):137-141 desmoplastic ameloblastoma a report of two clinical cases out in an attempt to predict the behavior and prognosis of this entity. references 1. kishino m, murakami s, fukuda y, ishida t. pathology of the desmoplastic ameloblastoma. j oral pathol. 2001; 30: 35-40. 2. saini r, samsudin ar. desmoplastic ameloblastoma of the mandible: a case report. int med j. 2008; 7: 1. 3. sivapathasundharam b, einstein a, syed ri. desmoplastic ameloblastoma in indians: report of five cases and review of literature. indian j denta res. 2007; 18: 218-21. 4. eversole lr, hansen ls, leider as. ameloblastomas with pronounced desmoplasia. j oral maxillofac surg. 1984; 42: 735-40. 5. shashikanth mc, neetha mc, ali im, shambulingappa p. desmoplastic ameloblastoma in the maxilla: a case report and review of literature. indian j dent res. 2007; 18: 214-7. 6. manuel s, raghavan n. desmoplastic ameloblastoma: a case report. j oral maxillofac surg. 2002; 60: 1186-8. 7. sun zj, wu yr, cheng n, zwahlen ra, zhao yf. desmoplastic ameloblastoma a review. oral oncol. 2009; 45: 752-9. 8. kaffe i, buchner a, taicher s. radiologic features of desmoplastic variant of ameloblastoma. oral surg oral med oral pathol. 1993; 76: 525-9. 9. kurumaya h, miyata m, okabe k, sukashita h. desmoplastic ameloblastoma in maxilla. j oral surg. 1998 ; 56: 783-6 10. rastogi r, jain h. case report: desmoplastic ameloblastoma. head neck radiol. 2008; 18: 53-5. 11. minami m, kaneda t, yamamoto h, ozawa k, itai y, ozawa m. ameloblastoma in the maxillomandibular region: mr imaging. radiology. 1992; 184: 389-93. 12. mintz s, velez i. desmoplastic variant of ameloblastoma: report of two cases and review of the literature. j am dent assoc. 2002; 133: 1072-5. 13. kumamoto h, kamakura s, ooya k. desmoplastic ameloblastoma in the mandible: report of a case with an immunohistochemical study of epithelial cell markers. j oral med pathol. 1998; 3: 45-8. 14. russel l, steven a. desmoplastic ameloblastoma: a case report and literature review. oral surg oral med oral pathol. 1993; 75: 479-82. 141 braz j oral sci. 9(2):137-141 desmoplastic ameloblastoma a report of two clinical cases oral sciences n3 original article braz j oral sci. october | december 2012 volume 11, number 4 factors involved in dentistry absenteeism since the foundation of the labor court from 1986 to 2008 elaine justino santos1, dagmar de paula queluz2 1dds, graduate student, piracicaba dental school, unicamp university of campinas, piracicaba, sp, brazil 2dds, msph, phd, professor, department of community dentistry, piracicaba dental school, unicamp university of campinas, piracicaba, sp, brazil correspondence to: dagmar de paula queluz departamento de odontologia social faculdade de odontologia de piracicaba unicamp avenida limeira, 901, cep: 13414900 piracicaba, sp, brasil phone: +55 19 21065277 e-mail: dagmar@fop.unicamp.br abstract aim: to identify the most important factors involved in dentistry absenteeism since the foundation of the labor court in campinas, sp, brazil, until december 2008; to verify if there was a decrease in dentistry absenteeism after the introduction of the dental service in the headquarters of the court; to verify if dentistry absenteeism is greater in the others cities of the region compared with the city of campinas. methods: the information was collected from the dental statements of the archives of the health department of the labor court. collected information included: international classification of diseases (icd-10), gender, age, function, origin of the statements, period of the absenteeism and year when the absence occurred. results: 3,084 health files from server group (2,741) and judges (343) were reviewed. the results indicated: higher prevalence of female gender, higher frequency in the age group from 30 to 40 years old and the smallest frequency in the age group upper from 60 years old. it was found an average sick-leave period of 1.93 days. according to the international classification of diseases (icd)-10, the most prevalent dental problems found in this study were: k01 (25.78%), k04 (10.57%) and k05 (6.37%). conclusions: the results of this study pointed to lower rates of absenteeism than the results found in the literature. the introduction of the dental service did not have influence on the rates of absenteeism of the area; however it influenced the absenteeism rates in the city of campinas (headquarters of the court). keywords: dental absenteeism, worker health, occupational health. introduction the occupational health is a technical area of public health that seeks to intervene in the relationship between the production system and health, to promote work that dignifies instead of denigrating the man. its mission is to assist in structuring a society that promotes health through workspaces. regarding the consequences of working conditions for the oral structures, it is known that, given the location and functions of the worker, they are vulnerable to the action of toxic agents in the environment and can lead to alterations. thus, the field of oral health worker, whose principles are close to the worker’s health, focuses on the relationship between oral health and work, trying to promote, preserve and restore the oral health of populations included in the various work processes, thus contributing to their quality of life1,2. inside of health worker, health promotion in companies is an aspect that has been great emphasis in recent years3. braz j oral sci. 11(4):492-504 received for publication: august 12, 2012 accepted: december 14, 2012 braz j oral sci. 11(4):492-504 the dental care (curative procedures mostly) has brought considerable benefits to the employee, but there is still the need of surveillance activities of the risks present in the workplace and on occupational diseases with oral manifestations. the public and private sectors have sought to provide dental care to workers in order to reduce absenteeism on grounds through dental procedures is mainly curative services on their own or through agreements contracted to provide this service. although the rate of absenteeism for dental reasons is not as relevant4 in relation to absenteeism for medical reasons, the most worrying fact is that presenteeism exists for dental reasons, it is difficult to measure and can cause accidents related causes dental and decrease in profitability of companies. presenteeism is an even worse organizational problem because the worker is physically present at work, but he/she is not producing due to multiple variables and factors such as: dissatisfaction, pessimism, discouragement and high stress level, making it harder to identify the problem, and causing harm to the industry and to other workers who are often contaminated by this apathy and lack of productivity. various epidemiological studies have demonstrated that the prevalence of absenteeism resulting from dental reasons varies from 10 to 35%, and the average number of lost working hours varies from 1.24 to 6.20 working hours/workers/ years5-11. ferreira4 (1995) reported that tooth decay and other oral complications are responsible for 20% of the service and lack of production by the fall, and generate other types of organic complications such as generalized infections. recently, evidence has been found linking periodontal disease in pregnant women and the birth of babies with low birth weight and the relationship between diabetes mellitus and periodontal disease has been extensively studied. this is because microorganisms enter the bloodstream can cause serious health problems. therefore, a proper care of oral hygiene can reduce the risk of atherosclerosis, a major cause of heart disease and other complications as mentioned above. sickness absenteeism reflects workers’ health status, besides being an economic and social problem is an increase in spending on social security12-13 through insurance paid to temporary incapacity caused by illness or accident at work. besides, aspects directly related to health, diverse factors determine work absences, such as the organizational culture, lack of employee appreciation strategies, burnout and stress, unfavorable psychosocial environment, dissatisfaction with work, workers’ socioeconomic status, lack of control over work and low social support at work12,14-17. employee absence takes a heavy toll on worker productivity2,18-25. research in the united states charged that change in sleep patterns and mood affects productivity at work, which may lead to an increase in chronic diseases and absenteeism26. getting to a single cause for the absenteeism is problematic because there is a complexity of causes, which is evident in the literature, is that the organization in the workplace can lead to the stressor as a major cause of high rates of absenteeism. the prevalence of mental disorders peaks during working age, which makes them a major cause of sickness absence27-30. employed people with mental disorders lost three times more work days in a 12-month period compared with people without mental disorders28. the study of dental absenteeism in brazil is difficult, as there is not sufficient documentary material, nor statistical data representative at the municipal, state and federal able to draw a profile of lost days at work and its impact on both economic and degree of satisfaction worker31. programs addressing the oral health of workers should be stimulated and developed based on epidemiologic knowledge, in order to promote disease reduction and improve quality of life among workers. the objective of the present study was to identify the most important factors involved in dentistry absenteeism since the foundation of the labor court in campinas, sp, brazil until december 2008; to verify if there was a decrease in dentistry absenteeism after the introduction the dentistry service in the headquarters of the court; to verify if at the others cities of the region area the dentistry absenteeism has larger frequency than in campinas city. material and methods study area the study was conducted in labor court. with the internalization of development initiated in 1960s, and resurfaced in the 1970s with special relevance to the state of são paulo, there was an increase in labor actions and the need to break the hitherto single labor court with jurisdiction over the entire state são paulo. thus, on july 14, 1986 his excellency president josé sarney signed into law creating the regional labor court of the 15th region with headquarters in campinas32. when it was installed on december 5, 1986, there were only 38 joint conciliations and trial under the jurisdiction of this court, in 2008 the 15th region reaches 599 counties, making up 95% of the territory of the state, covering a population of approximately 20 million people, one of the largest among the 24 regions in which is divided the labor court of the country. 153 labor courts are installed. with 22 years of existence, the regional labor court of the 15th region holds the rank of 2nd largest regional labor nationwide procedural motion in 2008. officials of the labor court, as well as all federal employees, were named servers from the law no. 8112 of 11 december 199033 establishing the legal regime of public civil union of municipalities and public foundations feds. the filling of positions is done by public tender with statutory employment scheme. the staff in 2008 consists of 343 judges and 2741 servers (analysts and technical college). the dental service was created in 1991 with the goal to offer a comprehensive health care to the server because the medical and psychological existed. in 2008 the health department includes medical, dental, psychological, physiotherapy and social work in 15th region. the dental care provided by quality always excelled, but within an assistencialist philosophy. preventive care has always been practiced individually, not with collective measures. in 2008, 493493493493493factors involved in dentistry absenteeism since the foundation of the labor court from 1986 to 2008 494494494494494 braz j oral sci. 11(4):492-504 the dental service had six dentists (five clinicians and one endodontist) and five dental auxiliary. procedures performed include prevention, dentistry, periodontics, endodontics, prosthodontics, surgery, laser therapy, among others. study design this study is a cross-sectional, descriptive and quantitative, addressing absenteeism and prevalence of factors of absenteeism in the regional labor court of the 15th region between december 1986 and december 2008, including servers group (analysts and technical college) and judges. data collection the study population consisted of all dental certificates, duly approved the medical records of servers and judges, issued from december 1986 through december 2008 (22 years). medical records are filed with the health department of the regional labor court of the 15th region located in campinas sp. there was no identification of individuals in the medical records. only servers and judges active frame participated in this study. the collection date used following the variables: * with respect to the worker: function, gender, age * with regard to absenteeism: origin of certificate: own or accompaniment duration of absenteeism (lost days) causes of absenteeism (icd-10) date (month, year) of absenteeism type of certificate: external or internal city of origin certificate data on sick leaves were collected from medical records of servers and judges filed with the secretary of health. these data were recorded in excel spreadsheets and incorporated into sociodemographic information obtained with frame personnel, to perform the statistical analyzes. *with regard to international classification of diseases – 10th revision ( icd-10) the causes of absenteeism were grouped by icd-10 (international classification of diseases), and we used the chapter xi of the icd-1034-35 that groups all diseases of the digestive system (k00-k93), and the groups’ k00-k14 “concerning diseases of the oral cavity, salivary glands and jaws. these groups were the responsibility of the dentist used in this study. the icd was established by who for the health professionals could communicate without describing the treatment given, not breaking thus the secrecy. some institutions and companies do not accept dental and medical certificates without the icd, but it was not the case for the period covered by this study, so all certificates were included regardless of whether or not described the icd. the cells of the spreadsheet for the icd that were left blank means that the dentist who issued the statement did not specify the reason of absence (icd), yet they were included in the study due to its high prevalence and because they were approved without this information. data analysis data were statistically analyzed by descriptive analysis (frequency, percentage) and estimate (chi-square, fisher’s exact test). the analyses were performed using sas software version 9.1, using a significance level of 5% (p<0.05). we adopted the concept of medical cause of absenteeism from the international labor organization36, i.e., “the period of absence from work which is accepted as attributable to an inability of the individual, except for that derived from normal pregnancy or prison.” it is understood by the absence labor uninterrupted period of absence from work, told from the very beginning, regardless of its duration. we used the recommendations of the subcommittee on absenteeism of the international society for occupational health, addressing the indices of frequency, severity, and percentage rate of absenteeism, average length of absences, as indicators of absenteeism, represented by the formulas36: frequency index = σ total number of cases / number of employees severity index = σ total lost days / number of workers percentage rate of absenteeism = σ total lost days x 100 / total days worked x number of workers average length of absences = σ total lost days / total number of cases all variables were compared by checking the existence of association. two tests were used to check for association between variables: the test x2 (chi-square) and fisher’s exact test. trend lines are used to graphically display trends in data and analyze problems of prediction. this analysis is also called regression analysis. using regression analysis, you can extend a trend line in a figure beyond the actual data to predict future values. in our study we used three figures with trend lines; percentage rate of absenteeism, the number of certificates and the number of servers and judges, all of these variables in relation to time. the closer to 1 the result of equation (r²), the greater the reliability index of the trend line. ethical considerations this research was approved by the ethics committee of the dental school of piracicaba, unicamp, under approval protocol number 019/2009. results the server group (n= 2741) and judge (n=343) studied were homogeneous regarding sociodemographic characteristics. the education level is above the high school, because that is the minimum required for technicians’ judiciary. analysts and judges have completed university level. the socioeconomic factors provide ease of access to health services both medical and dental and reflect on good oral health status of this population. in december 2008, the staff was 2741 servers (1618 females and 1123 males) and 343 judges (195 males and 148 females), totaling 3084 staff. from december 1986 until december 2008, 1381 factors involved in dentistry absenteeism since the foundation of the labor court from 1986 to 2008 braz j oral sci. 11(4):492-504 495495495495495 certificates were approved in dental medical records of servers and judges (n = 3084), totaling 2676 days of work lost due to dental reasons in 22 years. a random sampling of 1072 certificates verified that the technicians had higher judiciary certificates number, 79.38% (n = 851) than analysts, 20.62% (n = 221). some of these servers and judges are since the foundation; others have been incorporated throughout this period of 22 years. according to table 1, from 1381 dental certificates analyzed, 58.65% belong to females (n = 810) and 41.35% were from males (n = 571). of all the servers, 59.03% (n = 1618) represents females and 40.97% (n = 1123) males. among the judges the proportion is reversed, 43.15% (n = 148) were from females and 56.85% (n = 195) were from males. regarding the function performed, 94.71% (n = 1308) of dental certificates belonged to servers and 5.29% (n = 73) judges (table 1). considering a random sample of 1072 certificates of servers showed that 20.61% (n = 221) were analysts and 79.38% (n = 851) were technicians. according to table 2, 851 of the certificates presented by the sample positions, 20.62% (n = 221) was from analysts, with 48 belonging to males and 173 females. female gender was also responsible for 240 days of sick leave (n = 63.83%), i.e. the majority. for technicians (79.38% of certificate), females accounted for most certificates (n = 440) and also with most days of absence (n = 941). as much as 2676 days were lost for dental reasons along 22 years (table 3) in the studied population (n = 3084). with regard to the days of absence, most frequently found (65.46%, n = 904) refers to one day of absence followed by two days (18.97%, n = 262) and three days (6, 66%, n = 92) (table 4). the average number of days apart by certificate is 1.93 day, being six certificates (table 11), clearance was more than 20 days. regarding the frequency according to lost days in certificates, we found replenished with spacing of 60 days (n = 1), 45 days (n = 1) and 30 days (n = 1) (table 4). these extended absences refer to periods of recovery after major surgery. so 95.28% of the leaves are up to 5 days and only 4.72% are clearances over 5 days (table 4). researching the reasons that led dental absences, using the icd-10 found that 566 (40.98%) did not report the cause of separation (table 5). day lost frequency (n) percentage (%) total lost days 1 day 904 65.46% 904 2 days 262 18.97% 524 3 days 92 6.66% 276 4 days 31 2.24% 124 5 days 27 1.95% 135 10 days 11 0,79% 110 15 days 6 0,43% 90 20 days 1 0.08% 20 30 days 1 0.08% 30 45 days 1 0.08% 45 60 days 1 0.08% 60 others 42 3.04% 358 not informed 2 0.14% total 1381 100.00% 2676 table 4. frequency according to lost days in certificates factors involved in dentistry absenteeism since the foundation of the labor court from 1986 to 2008 male female total 1123 (40.97%) 1618 (59.03%) 2741 (100%) 538 (38.96%) 770 (55.75%) 1308 (94.71%) 195 (56.85%) 148 (43.15%) 343 (100%) 33 (2.39%) 40 (2.89%) 73 (5.29%) 571 (41.35%) 810 (58.65%) 1381 (100%) table 1. distribution of certificates according to function and gender gender servers judges total certificate n(%) certificate n(%) certificate gender male female total n certificate lost days 407 48 136 (36.17%) 569 173 240 (63.83%) *976 221 (20.62%) 376 (100.00%) n certificate lost days 1058 411 599 (38.89%) 710 440 941 (61.11%) *1768 851 (79.38%) 1540 (100.00%) analysts technicians table 2. distribution of certificates to servers according to a sampling of positions gender number of day lost total servers judges n (%) male 924 (n=1123) 142 (n=195) 1066 (39.84%) female 1424 (n=1618) 186 (n=148) 1610 (60.16%) total 2348 (87.74%) 328 (12.26%) 2676 (100%) * (n=2741) * (n=343) table 3. distribution lost days by gender and function braz j oral sci. 11(4):492-504 496496496496496 as for the annual frequency of dental certificates, we found that from 1987 to 1999 it ranged from 0.29% (1987) to 2.81% (1999), peaking in 1997 at the rate of 4.10% . from the year 2000, the rate increased significantly, always above 6.43% to 9.54% of total certificates (figure 1). as for the monthly frequency of the number of statements there were no significant differences, except the lowest frequency in december (4.43% of total) (figure 2). april and october were the months when there are more dental certificates (figure 2). looking at the figures 1 and 3, we find that as you increase the number of servers and judges there is a proportional increase in the number of certificates. since 2005, there has been a considerable increase in the number of servers and thereby the number of certificates even decreased. the age groups that had higher frequencies of the number of certificates were 30 to 40 years (37.43%) and 40-50 years (32.30%) (table 6). for all the people who requested the absence in relation to the number of certificates, there was repetition of statements for the same user. proportionally, ages 40-50 years, 50-60 years and above 60 years had a greater number of certificates than other age groups (respectively an average of 1.59; 1.47 and 1.61 per user certificates). regarding the type of certificate (internal issued by the court’s dentists; or external – issue by other dentists) it was found that 77.84% of the total (n = 1075) were external and 22.16% (n = 306) internal. among the cities inserted by the 15th region, campinas was the one with the highest frequency (41.11%), then came jaboticabal (6.34%), bauru (3.53%), ribeirão preto (3.10 %) and limeira (2.95%) (table 7). analyzing the certificates themselves or own escort, we note that the total of 1381 certificates, 98.78% were dental treatments certificate to the server itself or judge and 1.23% for accompaniment (table 8). it was identified that 58 judges or servers moved away from work for dental reasons above 4 occasionsin 22 years producing various events, so there repetitions that resulted in 512 certificates. thus 6.04% (n = 58) showed 37.07% (n = 512) of all certificates (table 9). the results from the association between variables were factors involved in dentistry absenteeism since the foundation of the labor court from 1986 to 2008 reason for absence icd frequency percentage of total certificates percentage of total reported not informed 566 40.98% 0 k01impacted and impacted teeth 356 25.78% 43.68% k04 diseases of pulp and periapical tissues 146 10.57% 17.91% k05 gingivitis and periodontal diseases 88 6.37% 10.80% *k08 other disorders of teeth and supporting structures 87 6.31% 10.67% *k07 dento-facial anomalies 68 4.92% 8.34% others 70 5.07% 8.60% total 1381 100.00% 100.00% table 5. distribution of icd-10 by gender * the temporomandibular disorders were included in the icd k07 and surgeries for implants were included in k07 and k08 icd by most dentists. fig. 3. evolution of the number of servers and judges. no. fig. 1. frequency of annual certificates. evolution of the number of certificates from the foundation. no. fig. 2. monthly frequency of certificates no. 497497497497497 braz j oral sci. 11(4):492-504 age group *number of persons number of certificates frequency 20 30 years 177 228 16.51% 30 40 years 380 517 37.43% 40 50 years 280 446 32.30% 50 60 years 110 169 12.24% 60 years over 13 21 1.52% total 960 1381 100.00% table 6. frequency of the number of certificates in relation to age group * number of persons requiring absence by age group cities * number of persons frequency percentage of total certificates campinas 441 571 41.11% jaboticabal 8 88 6.34% bauru 30 49 3.53% ribeirão preto 29 43 3.10% limeira 21 41 2.95% others 431 589 42.97% total 960 1381 100.00% table 7. frequency as the cities with the highest prevalence * number of persons requiring absence repetitions certificates *number of persons (n) % number of certificates (n) % certificates up to 3 repetitions 902 93.96% 869 62.63% certificates above 4 repetitions 58 6.04% 512 37.07% total 960 100.00% 1381 100.00% table 9. according to the frequency of repetitions certificates * number of people who presented certificates. performed using sas version 9.1, using a significance level of 5% (table 10). regardless of the type of certificate (external or internal) with certificate origin (own or accompaniment) there was no statistical association. regarding variables function, age and gender was significantly associated with the type of certificate. the following variables showed statistically significant association between gender: type certificate, age and function. regarding the certificate origin and lost days, no statistically significant association was found. the variables that showed statistical association with the function performed were: gender and type of certificate. there was no statistical association in relation to the certificate origin. the variable age showed statistical association between gender, lost days and the type of certificate. the absenteeism indicators are shown in table 11. it was calculated the percentage rate of absenteeism for the period 2004 to 2008 to verify the behavior of these latest indicators. the percentage rate of absenteeism for the period 2004 to 2008 remained between 0.030 to 0.037, with some variations (table 12). it was observed the evolution of absenteeism during the 22 years of the 15th region. we begin with the year 1987 and ended in 2008 with 5-year intervals for the calculation of these indices years (figure 4). one can see that the index has increased from the year 1990 reaching its peak in 2005 and decreasing after this year but has remained at values below the averages found in the literature. as seen in figures 3 and 4 there were peaks in 1995 and 2005 due to high input servers and judges over the years which also happened regarding campinas (figure 5). bases in table 13, the large number of certificates in campinas was probably due to the considerable number of servers and judges who have their homes in this city (n = 1260). observe that only 5 cities accounted for 57.03% (n = 792) of all certificates. the other cities had 42.97% (n = 589) of all certificates. based on the management of personal data for the period 2004 to 2008, we calculated the dental absenteeism for this period (table 14). according to table 14, the 5 cities showed similar rates, but jaboticabal although presenting the smaller number of servers and judges in all years (2004 to 2008) exhibited indices frequency, severity and rate of absenteeism far above other cities in virtually every year. in figure 6 shows that the percentage rate of absenteeism is stable and expressed a declining trend. in figure 7 shows that the number of certificates continues to grow and this growth trend to continue in the future. figure 8 shows that the number of servers and judges continues to grow and the trend is that it remains so. factors involved in dentistry absenteeism since the foundation of the labor court from 1986 to 2008 origin of the certificate frequency percentage of total certificates o w n 1364 98.77% accompaniment 17 1.23% total 1381 100.00% table 8. frequency according to the origin of the certificate. braz j oral sci. 11(4):492-504 fig. 5. number of certificates in campinas over the years. *observe the figure 5 repeats the pattern of figure 3 and 4. variables test type p-value association type of certificate x certificate origin fisher. 0.1418 no gender x type of certificate x ² < 0.001 yes gender x certificate origin x ² 0.6064 no gender x icd x ² 0.56 no gender x function x ² < 0.001 yes type of certificate x function x ² < 0.001 yes certificate origem x function fisher 0.18 no gender x age group x ² < 0.0064 yes age group x icd x ² < 0.001 yes age group x type of certificate x ² < 0.0040 yes lost days x gender x ² 0.61 no lost days x age group x ² 0.04 yes lost days x type of certificate x ² 0.16 no table 10. association between variables fig. 6. percentage rate of absenteeism from the year 2004 to 2008. discussion the profile and the factors associated with dentistry absenteeism in labor court since its foundation represent the socio-economic factors provide ease of access to health services both medical and dental and reflect on good oral health status of this population, suggesting that this is a relevant factor for the low rates of absenteeism dental found. fig. 4. evolution of the index of absenteeism *percentage rate of absenteeism over the years: 1987-0.003; 1990-0.018; 19950.018; 2000-0.029; 2005-0.038; 2008-0.030 fig. 7. evolution of the number of certificates from the foundation of 15th region labor court fig. 8. evolution of the number of servers and judges from the foundation of 15th region labor court. 498498498498498 factors involved in dentistry absenteeism since the foundation of the labor court from 1986 to 2008 no. no. braz j oral sci. 11(4):492-504 499499499499499 number total of certificate 1381 number of days lost 2676 number of cases 1381 total days worked 11020900 number of servers and judges 3084 number of servers and judges 3084 number of days lost 2676 total of days lost 2676 index of frequency 0.447 index of severity 0.867 average duration of absences 1.93 percentage rate of absenteeism 0.024 table 11. indices of average severity, frequency and average duration of absences and percentage rate of absenteeism in the 15th region labor court (december 1986 to december 2008) * due to the long term averages were used year number of servers and judges index of frequency index of severity average duration of absences percentage rate of absenteeism 2004 2865 0.041 0.09 2.12 0.036 2005 3047 0.043 0.087 2.00 0.037 2006 3070 0.034 0.079 2;35 0.034 2007 3078 0.035 0.084 2.35 0.035 2008 3084 0.037 0.073 1.94 0.030 table 12. indices of frequency, severity, average duration of absences and percentage rate of absenteeism from 2004 to 2008 in the 15th region labor court. table 13. frequency as the cities with the highest prevalence cities number of person who presented certificates frequency percentage of total certificates campinas 44 571 41.11% jaboticabal 8 88 6.34% bauru 30 49 3.53 ribeirão preto 29 43 3.10% limeira 21 41 2.95% others 431 589 42.97% total 960 1381 100.00% frequency according to gender considering the absolute numbers there is a higher prevalence of certificates for females (agreeing with mazzilli37, 2003 and martins38, 2005) while the results are proportionally equivalent for both genders. among the judges, where males predominated, most certificates also belonged to females (table 1). diacov and lima39 (1988) studied absenteeism due to dental problems in 701 employees of the municipal government of são josé dos campos, and hito40 (2007) in his dissertation studying dental absenteeism in a meat packing company (probably because there was predominance among male employees) found a higher prevalence in males. this result is contrary to our study and the majority of the literature. lima 41 (1997) analyzed comparatively 1774 certified dental city headquarters and the national institute of social security assistance medical (inamps) são josé dos campos sp/br and concluded that, for the range of 10 to 20 years old, was statistically significant the percentage rate of absenteeism in male; if compared with other age groups there were no significant differences, which agrees with our study because it includes age groups above 20 years. martins38 (2005) researching dental absenteeism in public and private companies reported that there was a female predominance in both the public company and in private enterprise. salvador and tonha 42 (2006), addressing absenteeism in union court also found that the annual rate of absence by gender and general causes was higher in females than males, in agreement with our results although we have only studied dental absenteeism. calle43 (1971) after studying absenteeism and illness came to the conclusion that at 42349.5 lost days from work due to illness, 30000.5 (70.8%) occurred among female workers which agrees with our study in relation to gender in relation to disagree more often (58.59% in this study). frequency second days of absence (lost days) the data suggest that technicians have turned away more than analysts during the period covered in this study, agreeing with salvador and tonha42 (2006) in their study in the court of audit (table 2). the award criteria and protocols used to approve the certificate seem to influence the incidence of absenteeism and its duration. silva and marziale44 (2000) found that the certificate dispensed medical expertise (1 and 2 days) accounted for 79% of certificates issued in the institution studied. in this study it was found that most leaves (up to 5 factors involved in dentistry absenteeism since the foundation of the labor court from 1986 to 2008 braz j oral sci. 11(4):492-504 500500500500500 year city lost days number of cases index of frequency index of severity average duration percentage rate of absenteeism of absences 2004 campinas 107 48 0.041 0.092 2.23 0.039 jaboticabal 42 17 0.548 1.354 2.47 0.581 r. preto 8 3 0.030 0.081 2.66 0.035 bauru 0 0 0 0 0 0 limeira 4 2 0.058 0.117 2.00 0.050 2005 campinas 163 51 0.041 0.132 3.19 0.057 jaboticabal 14 14 0.500 0.500 1 0.215 r.preto 3 3 0.029 0.029 1 0.012 bauru 6 6 0.084 0.084 1 0.013 limeira 1 1 0.030 0.030 1 0.036 2006 campinas 155 49 0.039 0.125 3.13 0.053 jaboticabal 6 6 0.210 0.210 1 0.092 r. preto 0 0 0 0 0 0 bauru 3 3 0.042 0.042 1 0.018 limeira 2 2 0.060 0.060 1 0.026 2007 campinas 121 53 0.042 0.096 2.28 0.041 jaboticabal 1 1 0.034 0.034 1 0.014 r. preto 12 7 0.065 0.112 1.71 0.047 bauru 7 5 0.069 0.097 1.40 0.041 limeira 3 3 0.085 0.085 1 0.036 2008 campinas 93 47 0.035 0.069 1.98 0.029 jaboticabal 3 3 0.107 0.107 1 0.044 r. preto 5 5 0.045 0.045 1 0.019 bauru 5 5 0.040 0.040 1 0.017 limeira 5 5 0.035 0.035 1 0.056 table 14. absenteeism in five cities most prevalent from 2004 to 2008. days) did not require expertise. according to decree 7003 of november 9, 200945 for departures up to 5 days are necessary medical expertise. the average duration of absences found of 1.93 (table 11) is compatible with the number found by martins38 (2005) for a private company (1.93) and well below that found by him to the public company (3.82), and can be explained by some causes such as surgical that elevate the arithmetic mean due to the number of days required for recovery of the patient. according to salvador and tonha42 (2006) all studies, even the most pessimistic, dealing with the cost-effectiveness of prevention of disease and promotion of health and safety at work, has shown a return of at least 20% on invested capital. however, he said, who emphasizes that in most optimistic studies for every r$ 1.00 invested, you can save up to r$ 16.00. in a study addressing the absenteeism causes covering all servers in the court of audit42 departures a day were the majority (36%) while the leaves above 5 days occurred in 20% of the total days away. requests for absences were 80% up to 5 days. this study, though it includes only causes dental, agrees with these results: 95.28% of total absence are up to five days, 65.46% are license with 1 day of absence and only 4.72% are departures over 5 days (table 4). frequency over function being larger number of servers in positions of technicians (n = 1768) than analysts (n = 973) agree with those obtained by salvador and tonha42 (2006) in his study on absenteeism in the court of audit, ie, technicians deviate more than analysts. considering the number of lost days, the servers moved away 2348 days (87.74%) and judges 328 (12.26%) (table 3). frequency according to icd saliba and garbin46 (2000) found in their study that 64.4% of dentists indicated in the certificate the act performed, suggesting lack of confidentiality, or icd, which was established to ensure that health professionals could communicate without describing the treatment performed, not breaking thus the secrecy. a study by silva and marziale44 (2000), with nursing staff of a university hospital revealed that 31% of the certificates did not contain diagnosis or international classification of diseases (icd), alleging as a worker’s right to not be exposed. similar results with our study, we found that 40.98% (n = 566) of the certificates did not inform the icd and not the procedure performed (table 5). annual and monthly frequency of dental certificates from the year 2000 the rate increased significantly the annual frequency of dental certificates, probably due to the entry of servers and judges within the labor court from that year (figure 1). factors involved in dentistry absenteeism since the foundation of the labor court from 1986 to 2008 braz j oral sci. 11(4):492-504 501501501501501 the monthly frequency of the number of statements there were no significant differences probably due to the withdrawal of the judiciary that happens from december 20 to january 6 that makes this month with less days worked for the other months of the year (figure 2). frequency in relation to age martins38 (2005) found that the age group between 30 to 39 years was the most missed the public service and the range 20 to 29 accounted for the largest fault in private, in a comparative study between these services. our results agree with those of martins for public service (table 6). lima41 (1997) analyzed comparatively 1774 certified dental city headquarters and the national institute of social security assistance medical (inamps) são josé dos campos and concluded that, for the range of 10 to 20 years old, was statistically significant index male and absenteeism compared with other age groups there were no significant differences, the prevalence of absenteeism due to dental problems occurred in the group of workers bureaucratic functions in the ranges of 20 to 30 years, 40-50 years and over 50 years for workers not bureaucratic function. frequency according to the type of certificates regarding the type of certificate: internal (issued by the labor court of dentists) or external (for other dentists) found that: 77.84% of the total (n = 1075) were external and 22.16% (n = 306) internal. the large number of certificates can be justified by external procedures that are not offered by the dental service of the court, and therefore must be conducted in private. the small number of internal certificates also justified because most appointments are made before, during or after work hours, in the offices of the court and certificates are provided only when requested by the patient or his superior. furthermore, servers and judges have the option of being cared for his private dentist of choice. these may be the reasons that explain the low number of statements internal in relation to external. reisine47 (1984) found in their research that preventive visits entail greater episode of work absenteeism (62.8%), though they result in fewer lost hours. the author also reported that the prevalence and magnitude of losses for productive reasons oro-dental suffer interaction of several factors, which have social determinants. she concluded that a preventive service can promote a favorable cost-benefit ratio, resulting in the reduction of indirect costs of lost work time and enabling the use of data recorded as adjuncts indicators for dental research. it also revealed that when the individual in the case of oral diseases, participates in a preventive treatment, it decreases their faults with respect to curative treatments, i.e., the preventive service causes less damage to the economy. according to the author, the preventive visits entail greater episode of absenteeism from work (62.8%), though they result in fewer lost hours. according to medeiros and bijella 48 (1970) the opportunity to care for a service installed in the workplace reduces losses to a minimum of hand labor because rejoins the worker as soon as possible in their activities. the dental service is expected to be possible with the medical clinic, in order to contribute exams, as well as serving up the same in cases of emergencies. probably the low percentage of internal certificates was due to the ease of access to servers and judges to use the service according to their needs and schedule availability. campinas is favored in this respect because it is the headquarters of dental care. frequency of 5 cities more prevalent as absence the large number of certificates in campinas was probably due to the considerable number of servers and judges who have their homes in this city (n = 1260). observe that only 5 cities accounted for 57.03% (n = 792) of the total certificates. the other cities had 42.97% (n = 589) of all certificates (table 7). proportionally, it can be seen in table 7 that campinas despite having the highest frequency of dental certificates due to its large cadre of servers, certificates has averaged 1.30 per person while jaboticabal, limeira, ribeirão preto and bauru respectively, 11.0, 1.95, 1.63 and 1.48 on average. these data suggest that the presence of dental care in the city of campinas can influence the low rates of absenteeism presented by the city. frequency of certificate origin analyzing the certificates own or accompaniment, we note that the total of 1381 certificates, 98.78% were dental treatments certificate to the server itself or judge and 1.22% for accompaniment (table 8). the data suggest that followup visits are usually done outside of working hours. it is noteworthy that the court of dental service not only provides care to dependents servers and judges (table 8). frequency as the repetitions it was identified that 58 servers or judges moved away from work for dental reasons above 4 times in 22 years producing various events, so there repetitions that resulted in 512 certificates (table 9). mazzilli49 (2004) also found repetitions of individuals who had two or more times (n = 276; 23.55%) certificates and represented 44.96% of total cases. the majority (76.45%; n = 896) showed a single request absence work in 4 years. association between variables based on the associations of the variables (table 10), it may be concluded that gender is an important variable, the female gender is predominant in most public and private enterprises and the social, emotional, physical, ergonomic and others involved with dental absenteeism should be analyzed by human resources considering the particularities of each gender to obtain the desired results. indicators of absenteeism according to flippo50 (1970), the percentage rate of absenteeism is considered normal around 3%. already forssman 51 (1956) argues that the notion of “normal factors involved in dentistry absenteeism since the foundation of the labor court from 1986 to 2008 502502502502502 braz j oral sci. 11(4):492-504 absenteeism”, applied to a group, a profession, or a country is debatable. the correct is that one should have the firm intention to reduce it to the fullest. possibly for this reason we did not find any reference on the porch ideal for these indices. according to couto52 (1987), it can be classified as absenteeism excessive values of frequency indices greater than 0.10 per month. danatro53 (1997) found in a public institution of montevideo, in uruguay, in one year, compared with medical absenteeism, index of frequency of 1.08, index of severity of 6.84 and average duration of absences of 6.28 days. the state government of são paulo in 2007 has taken steps to combat the high rate of absenteeism of the state. the numbers are alarming, the number of days missed, the main indicator of the problem has been growing in recent years. days were 5.09 million in 2003, rising to 5.4 million in 2004, 5.5 million in 2005 and reaching 6.3 million days in 2006, i.e. an increase of about 25% in three years. only between 2005 and 2006 the increase was 15.44%. the total state is 9.81% of faults on the total number of days that should be worked out by the servers. bispo54 (2002) argues that according to some human resource consultants, an acceptable index of absenteeism stands at around 2.7%, but that few companies can reach levels well below this. this study sought to draw a profile of dental absenteeism in the regional labor court over 22 years of existence by analyzing the factors that affect these absences and calculation of indices recommended by the subcommittee of the international society for occupational health (index of frequency, index of severity, percentage rate of absenteeism and average duration of absences). found absenteeism indicators are shown in table 11. the long period of this study made it difficult to obtain a mean absenteeism. for the calculation of these indices (frequency, severity and percentage rate of absenteeism) was used to calculate indices of 6 (1987, 1990, 1995, 2000, 2005 and 2008) and subsequently obtained the arithmetic average of these indices. this calculation method was selected for the index was not directed to very low values if the number of servers was that of december 2008. we calculated the rates of absenteeism for the period 2004 to 2008 to verify the behavior of these latest indicators. the indices showed up, mostly lower than those found by martins38 (2005), but we emphasize that medical and dental causes were researched while we studied only dental causes. the percentage rate of absenteeism for the period from 2004 to 2008 remained between 0.030 to 0.037 with some variations (table 12). according to ferreira4 (1995), dental causes represent 20% of all leaves. considering the absence of indicators of absenteeism specifically geared to the dental field, following a logical reasoning, we suggest that an acceptable percentage rate of absenteeism for dental 15th region labor court would be up to 0.5%. so the indices are found significantly below this level. only the city of jaboticabal had an absentee rate above 0.5%. we believe that this kind of simplistic reasoning, but of considerable practical application can be used for both the public sector as the private sector. when a company or institutions in the dental absence are above 20% of total absence, some measures related to the oral health of their workers should be deployed aiming to reduce this rate. so the formula used by the subcommittee on absenteeism international society for occupational health can be used both to analyze the index at a given moment and for the projection of goals for the future of an acceptable index of dental absenteeism. absenteeism in campinas according to what has been observed in relation to the 15th region as a whole, considering only the city of campinas, the same phenomenon is repeated: as it increases the number of servers and judges, increases the number of absence requests in the dental field. the dental care was deployed at the headquarters of court in 1991. by the year 1987 the number of statements found in this study was very low (n = 12) even with a considerable number of employees ranged from 332 in 1981 to 669 in 1987. these data suggest that the time servers and judges had no certificates approved because the procedures probably did not follow the current formalities. figure 5 suggests that what really influences dental absenteeism in the city of campinas is the entry of new servers and judges and not other variables. the installation of offices in 1991 probably influences the maintenance of low rates of absenteeism in this city though she has the larger number of servers and judges from across the 15th region. probably the figures would be higher if there were no dental care, emergency care because they are performed in the dental service itself would have to be made out and it would involve a greater number of leaves. as seen in figures 3 and 4 there were peaks in 1995 and 2005 due to high input servers and judges over the years which also happened regarding campinas (figure 5). absenteeism more prevalent in 5 cities among the cities encompassed by the 15th region, campinas was the one with the highest frequency (41.11%) of the total certificates, then came jaboticabal (6.34%), bauru (3.53%), ribeirão preto (3.10%) and limeira (2.95%). the large number of certificates in campinas was probably due to the considerable number of servers and judges who have their homes in this city (n = 1260). observe that only 5 cities accounted for 57.03% (n = 792) of all certificates. the other cities had 42.97% (n = 589) of all certificates (table 13). returned by the management of personal data for the period from 2004 to 2008, and we calculate the dental absenteeism for this period. according to table 14, the 5 cities showed similar rates but jaboticabal although the smaller number of servers and judges in all years (2004 to 2008) got some indices frequency, severity and percentage rate of absenteeism far above other cities in virtually every year. do not investigate the cause of these high rates. the duration of absences, campinas had the highest rates for the years 2005, 2006, 2007 and 2008. only for the factors involved in dentistry absenteeism since the foundation of the labor court from 1986 to 2008 braz j oral sci. 11(4):492-504 year 2004 the city with the longest duration of absences was ribeirão preto. for the frequency index, jaboticabal showed higher rates for the years 2004, 2006 and 2008. in 2005, bauru was the city that had the highest index and for the year 2007 was ribeirão preto. as for the severity index, a city that had the highest rates for 2004, 2006 and 2008 was jaboticabal, for 2005 was bauru and 2007 was ribeirão preto. draws attention to the fact the city jaboticabal stand out with high levels even with a relatively small number of servers and judges. the city of campinas showed the highest duration of absences, noting the spreadsheet data collection verified that the orthognathic surgery and implants reflect the increase in this index, as seen in campinas in 2005 and 2006. jaboticabal showed almost half (46%; n = 41) of total certificates along 22 years (n = 88). we see that the percentage rate of absenteeism is stable and expressed a declining trend. probably this is because the servers and judges make their dental visits during inverse to work (figure 6). the number of certificates continues to grow and this growth trend to continue in the future (figure 7). although the number of certificates is increasing the rate of absenteeism shows fall because its calculation involves the lost days are few considering the number of servers and judges. the number of servers and judges continues to grow and the trend is that it remains so (figure 8). these figures are useful for the planning of strategic actions in an attempt to reduce rates when necessary and design goals for short, medium and long term. one of the difficulties encountered in this study was the lack of national parameters as an index of absenteeism recommended as acceptable, especially when related to dentistry that is new in this field. hopefully with this specialty of dentistry labor 55 grow interest in the research and so that other researchers have increased availability and access to data. for the study, it was concluded that: * absenteeism dental regional labor court in the 15th region had absenteeism rates lower than those found in the dental literature and frequencies of variables similar to most reported studies. it was found that the entry of new servers and judges is one factor that reflects the frequency of dental certificates, the data suggest that sociodemographic characteristics of this population contributes to good levels of oral health and low rates of absenteeism. * it was found that the installation of dental care did not affect the drop rates of absenteeism in the 15th region as a whole, probably due to the difficulty of access to the servers and judges domiciled in other cities. * the dental installed in campinas, probably due to the ease of access and availability of schedules offered, contributed significantly to the maintenance of low rates of absenteeism (even with the large number of servers and judges), below the rates of other cities with fewer servers (for example jaboticabal). some cities had zero indices (bauru2004 and ribeirão preto-2006); demonstrating low levels of absenteeism dental this federal institution. references 1. araújo me, marcucci g. study of the prevalence of oral manifestations caused by chemical agents in the electroplating process: its importance to the area of oral health worker. odont soc. 2000; 2: 20-5. 2. macedo cg, queluz dp. quality of 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probst tm. safety and insecurity: exploring the moderating effect of organizational safety climate. j occup health psychol. 2004; 9, 3-10. 21. queluz dp. labour dentistry: a new specialty in dentistry. braz j oral sci. 2005; 4: 766-72 503503503503503factors involved in dentistry absenteeism since the foundation of the labor court from 1986 to 2008 braz j oral sci. 11(4):492-504 504504504504504 22. nardi a, michel-crosato e, biazevic mgh, crosato e, pizzatto e, queluz dp. relationship between orofacial pain and absenteeism among workers in southern brazil. braz j oral sci. 2009; 8: 50-4. 23. macedo cg, queluz dp. medical and dental absenteeism in workers from a furniture industry in itatiba, sp, brazil. braz j oral sci. 2010; 9: 443-8. 24. tausig m, fenwick r. work and mental health in a social context. new york, ny: springer; 2011. 25. shoss mk, penney lm. the economy and absenteeism: a macro-level study. j appl psychol. 2012; 97(4): 881-9. 26. anderson ra. 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[monofigurey] brasília: faculdade de economia, administração, contabilidade e ciências da informação, universidade de brasília; 2006. 43. calle rovirego bel trabajo de la mujer. in: instituto nacional de previdenciên. tratado de hygiene y seguridad del trabajo. madrid; 1971. p.219-26. 44. silva d m p p, marziale m h p. absenteeism of nursing staff in a university hospital. rev. latinoam. enfermagem. 2000; 8: 44-51. 45. brasil, 2012. decree number 7003, dated 9 november 2009. regulates the license for health care. available from: http: //www.planalto.gov.br/ccivil_03/ _ato2007-2010/2009/decreto/d7003.htm. 46. saliba ta, garbin aio . the dentist and dental issuing certificates. odontol. soc. 2000: 2: 89-92. 47. reisine st. dental disease and work loss. j dent res. 1984; 63: 1158-61. 48. medeiros ep, bijella vt. bases for organizing programs for dental workers. rev.bras.odontol. 1970; 27: 303-11. 49. mazzilli len. analysis of absences from work due to dental municipal public servants são paulo undergoing occupational expertise in the period from 1996 to 2000 [dissertation]. são paulo: faculdade de odontologia da universidade de são paulo; 2004. 50. flippo e.b. principles of personnel management. são paulo: atlas; 1970. 51. forssman s. el absentismo en la industria. bol. oficina sanit.panam: 1956; 41: 526-30. 52. couto h a. occupational health issues: collection of notebooks ergo. belo horizonte: ergo; 1987. 432p. 53. danatro d. ausentismo laboral de causa médica en una institución pública. rev. med.uruguay: 1997; 13: 101-9. 54. bispo p. company achieves low level of absenteeism; 2002. available from: http://carreiraseempregos.com.br/comunidades//rh/fiquepordentro/ 311002_rh_casaverde.shtm. [access 2012 jun]. 55. federal dentistry council. available from: http://www.cfo.org.br.[access 2012 jun]. factors involved in dentistry absenteeism since the foundation of the labor court from 1986 to 2008 oral sciences n3 braz j oral sci. 11(2):81-83 received for publication: september 22, 2011 accepted: march 22, 2012 original article braz j oral sci. april | june 2012 volume 11, number 2 odontogenic cysts in children and adolescents: a 21-year retrospective study vinicius gomes serra1, daniele meira conde2, rogério vera cruz ferro marques2, cláudio vanucci silva de freitas2, fernanda ferreira lopes3, maria carmen fontoura nogueira da cruz3 1 dds, federal university of maranhão, brazil 2msc student, graduate program in dentistry, federal university of maranhão, brazil 3professor, graduate program in dentistry, federal university of maranhão, brazil; phd in oral pathology, federal university of rio grande do norte, brazil correspondence to: maria carmen fontoura nogueira da cruz rua dos rouxinóis, condomínio alphaville, bloco i, apto 102 renascença ii cep: 65075-630, são luís ma brazil phone: +55 98 32273770 e-mail: ma.carmen@uol.com.br abstract aim: to investigate the distribution of odontogenic cysts in patients aged 0 to 18, referred to department of pathology, university hospital of the federal university of maranhão, brazil, to determine the most common types of lesions and their distribution according to gender and anatomical site involved. methods: histopathological data were collected from a database of lesions classified as odontogenic cysts that were indicated for surgical removal and histopathological analysis. data were subjected to descriptive analysis. results: thirty cases of odontogenic cysts were identified, and dentigerous cysts were the most frequent (n=17). most occurrences were in males (66.7%) and the most frequent site was the posterior mandible (73.3%). conclusions: odontogenic cysts in children and adolescents are mostly developmental cysts, especially dentigerous cysts, occurring predominantly in males, with a predilection for the posterior mandible. keywords: odontogenic cysts, child, adolescent. introduction odontogenic cysts are pathological entities that correspond to cavities lined with epithelial tissue, containing in its interior semi solid or liquid material, whose formation is associated with proliferation of epithelial remnants of the enamel organ or even the development of embryonic processes maxillomandibular1. these cysts constitute an important group of lesions in the maxillofacial complex, whose frequency varies from 7 to 13% of lesions diagnosed in this anatomic region2-3, affecting mainly the adult population2,4. these lesions are characterized by having slow growth and tendency to expand, despite the biological behavior of benign entities. however, odontogenic cysts can reach a considerable size if not diagnosed early and properly treated5. because they are generally asymptomatic and some behave aggressively, this group of lesions has required special attention from dentists6. odontogenic cysts can be classified as to their etiology in inflammation and development, and are differentiated by histopathology, mainly because the clinical and radiographic characteristics are very similar. the differential diagnosis of these lesions includes cystic ameloblastoma, adenomatoid odontogenic tumor, odontogenic calcification cyst7. it draws attention to the fact that several developmental processes occur in the maxillofacial area during childhood, including bone growth and odontogenesis, 8282828282 braz j oral sci. 11(2):81-83 type number of cases % dentigerous cyst 17 56.7 periapical cyst 06 20.0 eruption’s cyst 02 6.7 odontogenic keratocystic tumor 01 3.3 cysts of odontogenic nature 04 13.3 total 30 100 table 2 distribution of cases of odontogenic cysts, in order of frequency. são luís, ma-2010. source: department of pathology, university hospital unit dutra / ufma anatomical location number of cases % posterior mandible 22 73.3 anterior maxillary 5 16.7 posterior maxillary 2 6.7 anterior mandible 1 3.3 total 30 100 table 4 distribution of cases of odontogenic cysts according to anatomical location. são luís, ma-2010. source: department of pathology, university hospital unit dutra / ufma gender number of cases % male 20 66.7 female 10 33.3 total 30 100 table 1 distribution of cases of odontogenic cysts according to gender. são luís, ma-2010. source: department of pathology, university hospital unit dutra / ufma which could be associated with cyst formation8. nevertheless, the literature on odontogenic cysts includes studies mainly in the adult population, with few studies in children and adolescents. considering these aspects, the purpose of this study was to investigate the distribution of odontogenic cysts in children and adolescents aged 0 to 18, referred to department of pathology of the university hospital of the federal university of maranhão-ufma, in são luis, ma, brazil, to determine the most common types of lesions and their distribution according to gender and anatomic site involved, since the occurrence of these lesions is poorly known in the study population. material and methods this study followed the fundamental scientific and ethical requirements of resolution 196/96 (standards for research involving human subjects) of the brazilian national health council, and was approved by the institutional ethics committee (protocol #33104-649/2007; approval #407/2007). this research consisted of a retrospective, cross-sectional, and descriptive study of medical records of children and adolescents referred to the department of pathology, university hospital presidente dutra / ufma, são luis, ma, brazil. the sample included all medical records with a histopathologic diagnosis of odontogenic cyst, as defined by the medical subject headings mesh, of patients aged 0 to 18 referred to the service above, diagnosed during the period from 1990 to 2010. as a tool for collecting data, we used a database of lesions classified as odontogenic cysts that were indicated for surgical removal and histopathological analysis. data regarding gender, patient age at the time of diagnosis, anatomical site involved and histopathologic diagnosis were investigated. data were subjected to descriptive statistics and presented in tables. results thirty cases of odontogenic cysts were found in children and adolescents aged 2 to 16. the males were the most affected (66.7%), as shown in table 1. the predominant histopathological type was the dentigerous cyst (56.7%), followed by periapical cyst (20%), eruption cyst (6.7%) and odontogenic keratocystic tumor (3.3%), as shown in table 2. regarding the origin, 66.7% were developmental cysts while 20% were inflammatory cysts. in addition, 13.3% of cysts could not be identified according to the methodology of this study (table 3). concerning the anatomic site involved, the posterior mandible was the most frequently affected region (73.3%), followed by the anterior maxilla (16.7%), posterior maxilla (6.7%), and anterior mandible (3.3%) (table 4). discussion in the present study, the predominant histopathological type was the dentigerous cyst (56.7%), followed by periapical cyst, eruption cysts and odontogenic keratocystic tumor. the higher incidence of dentigerous cysts was also reported by some authors8-10. prockt et al.11 reinforced these data showing a frequency of 42.46% of dentigerous cysts in patients aged 10 to 19. this frequency can be explained by the large number of impacted third molars and canines in this age group, which are teeth associated with the etiology of these cysts. regarding the origin, the fact that most cysts were developmental cysts (66.7%) is in accordance with the literature, which shows a lower incidence of inflammatory cysts in children and adolescents 8-9, unlike the adult population, which is more affected by cystic lesions of inflammatory origin5,12-13. the difference in the distribution of odontogenic cysts among adults, children and adolescents, in particular developmental cysts, is probably due to the fact that the odontogenic cysts in children and adolescents: a 21-year retrospective study origin number of cases % development cyst 20 66.7 inflamatory cyst 06 20.0 not identified 04 13.3 total 30 100 table 3 distribution of cases of odontogenic cysts according to the origin. são luís, ma-2010. source: department of pathology, university hospital unit dutra / ufma 8383838383 braz j oral sci. 11(2):81-83 origin of these lesions occurs during infancy and is associated with continued growth and subsequent changes in teeth, going to the end of puberty12. the occurrence of inflammatory cysts in permanent teeth are also infrequent in children and adolescents, since their pathogenesis starts from the pulp necrosis14, and recently erupted permanent teeth are generally healthy, not showing the condition necessary for the development of cystic lesion. in primary teeth, the inflammatory stimulus would not have enough time to act as a chronic irritant15-16. males were more affected (66.7%) and this result corroborates with the literature 9-10. this finding can be explained by the fact that boys are usually more prone to trauma and have poorer oral hygiene than girls17. in contrast, bodner8 found no significant differences between genders in a sample of pediatric patients. regarding the anatomic site, the posterior mandible was the most frequently affected region, with 73.3% of cases, followed by the anterior maxilla (16.7%), as also observed by godoy et al.9. low incidence of lesions in the anterior mandible is probably due to the fact of the anterior teeth present less fissures and grooves than the posterior teeth, being less susceptible to caries and, consequently, to periapical inflammatory lesions9. from the obtained results, it was possible to conclude that odontogenic cysts in children and adolescents are mostly developmental cysts, particularly dentigerous cysts, with a predominance of male patients and mostly located in the posterior mandible. the importance of the early diagnosis of these lesions by dentists is providing a better treatment for patients. further research on this nature is needed, considering the scarcity of studies in this age group. references 1. nanami r, sampaio c, olivete j, pizzatto e, moresca r, giovanini af. prevalence of cysts of the jaws diagnosed on a brazilian center of reference. rsbo. 2009; 6: 143-6. 2. jones av, craig gt, franklin cd. range and demographics of odontogenic cysts diagnosed in a uk population over a 30-year period. j oral pathol med. 2006; 35: 500-7. 3. ledesma-montes c, hernández-guerrero jc, garcés-ortýz m. clinicopathologic study of odontogenic cysts in a mexican sample population. arch med res. 2000; 31: 373-6. 4. koseoglu bg, atalay b, erdem ma. odontogenic cysts: a clinical study of 90 cases. j oral sci. 2004; 46: 253-7. 5. ochsenius g, escobar e, godoy l, peñafiel c. odontogenic cysts: analysis of 2.944 cases in chile. med oral patol oral cir bucal. 2007; 12: 85-91. 6. pereira jvp, figueiredo du, souza ea, holmes tsv, gomes dqc, cavalcanti al. prevalence of odontogenic cysts and tumors in patients treated at the paraíba health assistance foundation: a retrospective study. arq odontol. 2010; 46: 75-81. 7. pozzer l, jaimes m, chaves neto hdm, olate s, barbosa jra. the odontogenic cyst in children: analysis of the surgical decompression in 2 cases. rev. cir traumatol buco-maxilo-fac. 2009; 9: 17-22. 8. bodner, l. cystic lesions of the jaws in children. int j pediatr otorhinolaryngol. 2002; 62: 25-9. 9. godoy pg, silveira ejd, gordón-núñez ma, queiroz lmg, gomes dmd. jaw cysts in children: a clinical analysis. rev adm. 2007; 64: 226-9. 10. iatrou i, theologie-lygidakis n, leventis m. intraosseous cystic lesions of the jaws in children: a retrospective analysis of 47 consecutive cases. oral surg oral med oral pathol oral radiol endod. 2009; 107: 485-92. 11. prockt ap, schebela cr, maito fdm, sant’ana filho m, rados pv. odontogenic cysts: analysis of 680 cases in brazil. head and neck pathol. 2008; 2: 150-6. 12. tay ab. a 5 year survey of oral biopsies in an oral surgical unit in singapore: 1993 -97. ann acad med singapore. 1999; 28: 665-71. 13. 13 mosqueda ta, irigoyen-camacho me, diaz-franco ma, torres-tejero ma. odontogenic cysts. analysis of 856 cases. med oral. 2002; 7: 89-96. 14. mehgji s, qureshia w, hendersona b, harrisa m. the role of endotoxin and cytokines in the pathogenesis of odontogenic cysts. archiv oral biol. 1996; 41: 523-31. 15. mass e, kaplan i, hirshberg a. a clinical and histopathological study of radicular cysts associated with primary molars. j oral pathol med. 1995; 24: 458-61. 16. lustig jp, schwartz-arad d, shapira a. odontogenic cysts related to pulpotomized deciduos molars: clinical features and treatment outcome. oral surg oral med oral pathol oral radiol endod. 1999; 87: 499-503. 17. meningaud jp, oprean n, pitak-arnnop p, bertrand j-c. odontogenic cysts: a clinical study of 695 cases. j oral sci. 2006; 48: 59–62. odontogenic cysts in children and adolescents: a 21-year retrospective study oral sciences n3 braz j oral sci. 11(2):120-124 original article braz j oral sci. april | june 2012 volume 11, number 2 long-term maxillary behavior in treated skeletal class ii malocclusion ana de lourdes sá de lira1, margareth maria gomes souza2, ana maria bolognese2 1dds, phd, department of orthodontics, school of dentistry, federal university of rio de janeiro (ufrj), brazil 2dds, phd, full professor, department of orthodontics, school of dentistry, federal university of rio de janeiro (ufrj), brazil correspondence to: ana de lourdes sá de lira departamento de ortodontia, faculdade de odontologia, universidade federal do rio de janeiro ufrj av. professor rodolpho paulo rocco, 325 cep: 21941-617 ilha do fundão rio de janeiro rj brasil e-mail: anadelourdessl@hotmail.com abstract aim: to evaluate the post-treatment and long-term anteroposterior and vertical maxillary changes from the use of kloehn cervical headgear in treated skeletal class ii division 1 malocclusion. methods: using a longitudinal prospective study design, 90 lateral radiographs of 30 treated patients (12 male gender and 18 female gender) were taken at the beginning of the treatment (mean age = 10 years and 9 month), at the end of the treatment (mean age = 14 years and 6 months), and in the post-retention phases (mean age = 26 years and 2 months). lateral radiographs of 30 adults patients with class ii malocclusion, as control group (mean age = 24 years and 1 month) were compared with lateral radiographs of patients in the post-retention phase in order to quantify the cephalometric measurements (5 angular and 2 linear) representing the maxillary position in the anteroposterior and vertical direction. results: under the effect of treatment, forward displacement of the maxilla was redirected in a downwards and backwards direction. when the means of the female measurements were compared between the two groups, only anb was greater in the control group. when the male measurements were compared, they presented a mean value of anb greater in the control group, and the mean of snpp greater in the treated group. conclusions: in the post-retention phase, maxillary displacement reverted in a downward and forward direction, confirming the transitory effect of the extra-oral action on maxillary displacement. keywords: skeletal class ii malocclusion, maxillary displacement, cervical extra-oral traction, post-retention. introduction the treatment of class ii malocclusion, a subject of great interest to orthodontists, uses various strategies, extra-oral forces being one whose benefits have been unanimously recognized1. it restricts anterior displacement of the maxilla and thus contributes to correction of the anteroposterior discrepancy between the maxilla and mandible2-3. silas kloehn4 designed the kloehn extra-oral device, used since 1947, and experimentally verified that traction retarded the forward displacement of the maxilla in patients with class ii malocclusion, and in some cases, moved the maxillary teeth in the distal direction. the best results have been obtained when treatment is applied during the growth spurt because while the anterior displacement of the maxilla is retained, the mandible is displaced in a forward direction until a favorable relationship with it was obtained. received for publication: january 31, 2012 accepted: may 14, 2012 121121121121121 braz j oral sci. 11(2):120-124 table 1 mean and standard deviation (sd) of ages (in years) at time intervals t 0 , t 1 and t 2 for both genders in the treated and control groups. time intervals: before treatment (t 0 ), at the end of treatment (t 1 ) and with a minimum of 5 years post-retention (t 2 ). treated group control group n t 0 t 1 t 2 n mean ± sd mean ± sd mean ± sd mean ± sd male 12 11.9 ± 1.33 15.8 ± 1.34 26.6 ± 3.10 13 23.56±5.59 female 18 10.9 ± 1.22 14.6 ± 1.48 26.2 ± 4.78 17 24.43±3.62 studies have shown that cervical traction used in the correction of class ii is effective in redirecting maxillary displacement downwards and backwards5-6. the maxillary permanent first molars are maintained or moved in the distal direction, so that the premolars, permanent canines and second molars are oriented in the same direction7. the most adequate period for evaluating these results is after the removal of retention. thus, the efficacy of the therapy on the cranial and facial structures and the effects of late residual growth can be evaluated8-10. the aim of this study was to evaluate the post-treatment and long-term anteroposterior and vertical maxillary changes from the use of kloehn cervical headgear in skeletal class ii division 1 malocclusion, compared with untreated patients. material and methods a selection was made of 30 teleradiographs of 30 untreated adult patients with class ii malocclusion, as the control group (cg) and 90 teleradiographs of 30 patients submitted to complete fixed corrective orthodontic treatment with the standard edgewise system and extra-oral cervical traction appliance (kloehn), as the treated group at the orthodontic graduate program clinic of the federal university of rio de janeiro, brazil, at different time intervals: before treatment (t 0 ), at the end of treatment (t 1 ) and with a minimum of 5 years post-retention (t 2 ). the research protocol was approved by the ethics committee, number caae 0045.0.239.000-09. the treated group consisted of 30 individuals (18 females and 12 males, and the control group consisted of 30 individuals (17 females and 13 males with class ii malocclusion) (table 1). at the beginning of treatment all patients in the treated group were in the pubertal growth spurt stage, according to the hand and wrist radiograph, with skeletal class ii malocclusion (anb e”5 degrees) and angle sngogn d” 36 degrees. the measurements obtained from the cephalometric tracings at t 0 , t 1 and t 2 were organized in tables for evaluation and statistical analysis, with the angular measurements being taken to the nearest whole degree whenever there was a fraction involved. the changes in maxillary displacement were measured in relation to the base of the skull by means of angles basn, anb, nsena, nsenp and snpp. linear measurements were used to describe maxillary displacement: s’-ena, which corresponded to the distance from the perpendicular to the sela-nasio line passing through point s to point anterior nasal spine. s’-enp, determined by the distance from the perpendicular to sela-nasio line passing through point s to point posterior nasal spine. for each cephalometric measurement the mean and standard deviation at t 0 , t 1 and t 2 time intervals was calculated the behavior of the measurements between the time intervals (t 0 x t 1 ) and (t 1 x t 2 ) was tested for significance using the paired student’s-t test, with significance level set at 5%. the same measurements were obtained for the non-treated group (control). the behavior of measurements was compared between the control and treated groups at time interval t 2 using non paired student’s-t test, with a level of significance of 5%. error of the method thirty cephalograms of 10 randomly selected patients were traced on two separate occasions, totaling 60 tracings. no significant difference was found using the paired t test. the degree of reliability of the measurements was calculated using the dahlberg’s11 formula of method error. for the angular measurements the error of the method did not exceed 0.375 degrees and for linear measurements it did not exceed 0.345 mm. results table 2 shows the means, standard deviations of the angular and linear measurements at time intervals t 0 , t 1 and t 2 of the treated and control groups and the p-values between t 0 x t 1, between t 1 x t 2 and between t 2 x cg. table 3 shows the data obtained only in women, and table 4 shows the reference values for men in the treated and control groups, with p-values. when the genders in the control group were compared, the mean value of anb was 2 degrees greater for the female gender (p < .001), while the mean value of s’ena was 4.45 mm higher for the male gender (p = .04). in the treated group, between t 0 x t 1 , the male gender presented mean values of nsena and snpp 2.5 degrees (p = .003) and 2.84 degrees (p = .006) greater, respectively, showing also mean value of anb 2.34 degrees (p< .001) lower than the female gender. between t 1 x t 2 anb decreased in both genders, while nsena and snpp decreased 0.56 degrees (p = .004) and 0.73 degrees (p= .01) respectively, only in females (tables 3 and 4). when the means of the female measurements were compared between the two groups, only anb was 5.80 degrees greater (p < .001) in the control group (table 3). long-term maxillary behavior in treated skeletal class ii malocclusion braz j oral sci. 11(2):120-124 treated group control group t 0 t 1 t 2 mean ± sd p p p mean ± sd mean ± sd mean ± sd t 0 x t 1 t 1 x t 2 t 2 x cg basn (o) 131.11 ± 5.08 131.67 ± 5.08 131.64 ± 4.69 131.94±5.22 .29 ns .54 ns .77 ns anb (o) 5.61 ± 1.14 3.0 ± 1.45 2.56 ± 1.42 8.35±1.22 < .001 .007 <.001 nsena (o) 35.89 ± 2.11 38.06 ± 2.10 37.50 ± 1.94 36.94±2.46 < .001 .004 .46 ns nsenp (o) 68.78 ± 2.36 71.44 ± 2.99 71.39 ± 3.05 70.05±5.58 < .001 .87 ns .39 ns snpp (o) 6.44 ± 2.59 8.67 ± 2.11 7.94 ± 2.38 8.11±3.78 < .001 .01 .87 ns s’ena (mm) 68.78 ± 3.57 68.83 ± 3.41 70.78 ± 3.11 70.47±6.05 .91 ns < .001 .85 ns s’enp (mm) 17.22 ± 2.31 15.28 ± 2.67 15.61 ± 3.01 15.52±5.10 < .001 .21 ns .95 ns table 3 mean and standard deviation of angular and linear measurements for the female gender in the treated group (n=18) at time intervals t 0 , t 1 and t 2 and in the control group (n=17). time intervals: before treatment (t 0 ), at the end of treatment (t 1 ) and with a minimum of 5 years post-retention (t 2 ). p = 5% of significance. tg cg p value t 0 t 1 t 2 mean sd p p p mean sd mean sd mean sd t 0 x t 1 t 1 x t 2 t 2 xgc basn (o) 134.92 ± 8.27 134.92± 7.34 134.83± 6.52 130.23±4.47 1.0 ns .83 ns .06 ns anb (o) 6.42 ± 1.31 4.08 ± 1.37 3.42 ± 1.62 6.30±1.54 < .001 .02 <.001 nsena (o) 37.92 ± 1.83 40.42 ± 3.08 40.42 ± 3.45 37.60±2.25 .003 1 n.s .06 ns nsenp (o) 71.08 ± 2.46 72.25 ± 4.11 72.75 ± 3.57 71.07±3.04 .19 ns .29 ns .21 ns snpp (o) 9.33 ± 3.02 12.17 ± 3.68 12.0 ± 4.04 6.38±3.59 .006 .50 ns .001 s’ena (mm) 68.17 ± 4.01 69.75 ± 6.21 72.50 ± 6.09 74.92±5.12 .12 ns .001 .29 ns s’enp (mm) 15.25 ± 2.59 14.92 ± 3.65 15.33 ± 3.28 15.52±5.10 .60 ns .47 ns .95 ns table 4 mean and standard deviation of angular and linear measurements for male gender in the treated group (n=12) at time intervals t 0 , t 1 and t 2 and in the control group (n=13).. time intervals: before treatment (t 0 ), at the end of treatment (t 1 ) and with a minimum of 5 years post-retention (t 2 ). p = 5% of significance. table 2 mean and standard deviation (sd) of angular and linear measurements in the treated group (n=30) at time intervals t 0 , t 1 and t 2 and in the control group (n=30). time intervals: before treatment (t 0 ), at the end of treatment (t 1 ) and with a minimum of 5 years post-retention (t 2 ). p = 5% of significance. treated group control group p value t 0 t 1 t 2 mean ± sd p p p mean ± sd mean ± sd mean ± sd t 0 x t 1 t 0 x t 1 t 2 x cg basn (o) 132.63 ± 6.68 132.97 ± 6.18 132.80± 5.64 131.20 ±4.90 0.41 ns .53 ns .24 ns anb (o) 5.93 ± 1.25 3.43 ± 1.50 2.90± 1.53 7.46 ± 1.69 < .001 < . 001 <.001 nsena (o) 36.70 ± 2.21 39.00 ± 2.75 38.67± 2.95 37.20 ± 2.36 < .001 .023 .08 ns nsenp (o) 69.70 ± 2.62 71.77 ± 3.44 71.93± 3.27 70.50 ± 4.61 < .001 .54ns .17 ns snpp (o) 7.60 ± 3.08 10.07 ± 3.29 9.57± 3.69 7.36 ± 3.74 < .001 .01 .02 s’ena (mm) 68.53 ± 3.70 69.20 ± 4.65 71.47± 4.53 72.40 ± 6.00 .19 ns < .001 .050 ns s’enp (mm) 16.43 ± 2.58 15.13 ± 3.04 15.50± 3.07 16.26 ± 4.39 .002 .17 ns .43 ns when the male measurements were compared, they presented a mean value of anb 2.9 degrees (p <.001) greater in the control group, and the mean of snpp 5.62 degrees greater in the treated group (p < .001) (table 4). discussion according to kloehn4, the growth spurt is essential for the correction of class ii malocclusion, while kloehn’s extraoral appliance restricts forward displacement of the maxilla, and orients eruption of the posterior permanent teeth in a more distal direction. as regards angle basn, no significant alteration in this angle was verified, both in the period corresponding to the use of the extra-oral appliance and in the post-retention phase (tables 2, 3 and 4, figure 1), suggesting that growth at the base of the skull is inherent to the individual, without the influence of any orthodontic mechanics12. in treated groups, between t 0 x t 1 , a significant reduction in anb, significant increase in snpp and nsena and inexpressive increase in s’ena were observed (table 2, figures 1 and 2), and also when the genders were observed separately (tables 3 and 4). as previously observed by other authors1,13-14, the action of extra-oral force and orthodontic mechanics during the growth spurt promoted downward and backward displacement of the maxilla. between t 0 x t 1, the mean value of angles nsena and 122122122122122long-term maxillary behavior in treated skeletal class ii malocclusion braz j oral sci. 11(2):120-124 123123123123123 fig. 1 values of basn, nsena, nsenp and snpp angles in the treated group at the time intervals before treatment (t 0 ), at the end of treatment (t 1 ) and with a minimum of 5 years post-retention (t 2 ) and in the control group. fig. 2. values of anb value and of the linear measurements s’ena and s‘enp in the treated group at the time intervals before treatment (t 0 ), at the end of treatment (t 1 ) and with a minimum of 5 years post-retention (t 2 ) and in the control group. snpp diminished and that of s’-ena increased significantly, suggesting that growth was not altered but redirected.15-19. a similar result was observed when the genders were analyzed separately (tables 3 and 4). the results of this research confirm previous conclusions1,14,20-21 that the reduction in facial convexity is a common effect of mechanics in class ii malocclusion. the increase in nsenp and reduction in s’-enp means (table 2, figures 1 and 2) were probably due to displacement of the posterior nasal spine in the posterior direction, by bone apposition at the tuberosity13,22. when the means of the female and male groups were analyzed separately (table 3 and 4), angle nsenp did not increase significantly in the latter group (table 4). that is to say, there was no relevant change in point enp, either in the vertical or horizontal direction in relation to the base of the skull, a phenomenon mentioned as being due to extrusion of the molars during the use of extra-oral mechanics, making it impossible for point enp to be lowered19,23-24 . between t 1 xt 2 , the significant reduction in angle anb resulted from residual mandibular growth, since no orthodontic mechanics were being applied. the means of angles nsena (except in male gender) and snpp diminished and that of s’ena increased significantly, the influence of cervical traction on the maxillary growth pattern being temporary (tables 2, 3 and 4, figures 1 and 2)25. no significant changes were observed in the mean of nsenp and s’-enp due to the posterior nasal spine being maintained in a stable position both in the vertical and horizontal directions (tables 2, 3 and 4, figures 1 and 2), as there was balance of the masticatory muscles and occlusal forces25. in addition, the growth in the posterior region of the maxilla had been expressed in the anterior region with the more forward positioning of point ena26-27. when the treated group was compared with the control group at t 2, no significant difference between the groups was observed as regards growth at the base of the skull. in addition, the maxilla was no longer protruded in the control group, since there was no significant difference with regard to the measurements nsena, nsenp, s’ena and s’enp (tables 2, 3 and 4, figures 1 and 2). this was observed by bishara et al.28, when they pointed out that class ii is not a result of excessive growth of the maxilla, but it is basically due to excessive retrusion of the mandible. hunter et al.29 had confirmed that the maxilla is normally well related to the base of the skull in class ii malocclusion. the skeletal difference between the maxilla and mandible (anb) was significantly greater in the control group. the maxilla was observed to be ahead of the mandible, suggesting that all the growth that occurred from childhood to the adult phase was genetically determined, and that class ii malocclusion is not self-corrected in growing patients30-31. angle snpp was significantly greater in the treated than in the control group, due to the effect of kloehn’s extra-oral mechanics on the maxilla21-22. it may be concluded that during orthodontic treatment combined with the use of kloehn’s extra-oral appliance, displacement of the maxilla was directed downwards and backwards. in the post-retention phase, however, maxillary displacement was reverted in a downward and forward direction, confirming the transitory effect of the extra-oral action on maxillary displacement. references 1. hass a. headgear therapy: the most efficient way to distalize molars. semin orthod. 2000; 6: 79-90. 2. tulloch j, phillip c, koch g, wr p. the effect of early intervention on skeletal pattern in class ii malocclusion: a randomized clinical trial. am j orthod. 1997; 111: 391-400. 3. ghafari j, king gj, tulloch jf. early treatment of class ii, division 1 malocclusion—comparison of alternative treatment modalities. clin orthod res. 1998; 1: 107-17. long-term maxillary behavior in treated skeletal class ii malocclusion 124124124124124 braz j oral sci. 11(2):120-124 4. kloehn s. guiding alveolar growth and eruption of teeth to reduce treatment time and produce a more balanced denture on face. angle orthod. 1947; 17: 10-33. 5. poulton d. the influence of extraoral traction. am j orthod. 1967; 53: 8-18. 6. baumrind s, korn el, molthen r, west ee. changes in facial dimensions associated with the use of forces to retract the maxilla. am j orthod. 1981; 80: 17-30. 7. armstrong mm. controlling the magnitude, direction, and duration of extraoral force. am j orthod. 1971; 59: 217-43. 8. pluger w. a study of the stability of orthodontic treatment. am j orthod. 1961; 47: 229-33. 9. simon m, joondeph d. changes in overbite. a ten years post-retention study. am j orthod. 1973; 64: 349-67. 10. singer j. posttreatment change: a reality. am j orthod. 1975; 67: 277-89. 11. dahlberg g. statistical methods for medical and biological students. london: allen & unwin; 1940. 12. ricketts rm. the influence of orthodontic treatment on facial growth and development. angle orthod. 1960; 30: 103-33. 13. cattaneo p, dalstra m, melsen b. the transfer of occlusal forces through the maxillary molars: a finite element study. am j orthod. 2003; 123: 367-73. 14. schiavon gandini mr, gandini lg jr., da rosa martins jc, del santo m jr. effects of cervical headgear and edgewise appliances on growing patients. am j orthod dentofacial orthop. 2001; 119: 531-9. 15. harris eh, gardner rz, vaden jl. a longitudinal cephalometric study of postorthodontic craniofacial changes. am j orthod dentofacial orthop. 1999; 115: 77-82. 16. bergersen eo. the male adolescent facial growth spurt: its prediction and relation to skeletal maturation. angle orthod. 1972; 42: 319-38. 17. bishara s, zaher a, cummins d, jakobsen j. effects of orthodontic treatment on the growth of individuals with classii division 1 malocclusion. angle orthod. 1994; 64: 221-30. 18. harris e, vaden j, dunn k, behrents r. effects of patient age on postorthodontic stability in class ii, division 1 malocclusion. am j orthod. 1994; 105: 25-34. 19. graber t. extraoral force-fallacies. am j orthod. 1955; 41: 490-505. 20. baumirind s, molten r, west e, miller d. distal displacement of the maxilla and the upper first molar. am j orthod. 1979; 75: 630-40. 21. fidler bc, artun j, joondeph dr, little rm. long-term stability of angle class ii, division 1 malocclusions with successful occlusal results at end of active treatment. am j orthod dentofacial orthop. 1995; 107: 276-85. 22. melsen b. effects of cervical anchorage during and after treatment: an implant study. am j orthod. 1978; 73: 526-40. 23. klein p. an evaluation of cervical traction on the maxilla and the upper first permanent molar. angle orthod. 1957; 27: 61-8. 24. king e. cervical anchorage in class i, division i treatment. a cephalometric appraisal. angle orthod. 1957; 27: 98-104. 25. lima filho r, lima a, de oliveira ruellas a. longitudinal study of anteroposterior and vertical maxillary changes in skeletal class ii patients treated with kloehn cervical headgear. angle orthod. 2003; 73: 187-93. 26. hanes r. bony profile changes resulting from cervical traction compared with those resulting from intermaxillary elastics. am j orthod. 1959; 45: 353-64. 27. ringenberg q, butts w. a controlled cephalometric evaluation of single arch cervical traction therapy. am j orthod. 1970; 57: 179-85. 28. bishara s, jacobsen j, vorhies b, bayati p. changes in dentofacial structures in untreated class ii division i and normal subjects: a longitudinal study. angle orthod. 1997; 67: 55-66. 29. hunter w. the vertical dimensions of the face and skeletodental retrognatism. am j orthod. 1967; 53: 586-95. 30. ngan pw, byczek e, scheick j. longitudinal evaluation of growth changes in class ii division 1 subjects. semin orthod. 1997; 3: 222-31. 31. garbui iu, nouer pr, nouer df et al. cephalometric assessment of vertical control in the treatment of class ii malocclusion with a combined maxillary splint. braz oral res. 2010; 24: 34-9. long-term maxillary behavior in treated skeletal class ii malocclusion 404 not found braz j oral sci. 13(3):209-212 original article braz j oral sci. july | september 2014 volume 13, number 3 body mass index, dental caries and sugar intake in 2-5 year-old preschoolers ronald jefferson martins1, suzely adas saliba moimaz1, mirelli ramiro silva1, orlando saliba1, clea adas saliba garbin1 1universidade estadual paulista – unesp, faculty of dentistry of araçatuba, department of social and preventive dentistry, araçatuba, sp, brazil correspondence to: ronald jefferson martins nepesco núcleo de pesquisa em saúde coletiva departamento de odontologia infantil e social faculdade de odontologia do campus de araçatuba rua jose bonifácio, 1193, vila mendonça cep: 16015-050 araçatuba, sp, brasil phone: +55 18 3636-3250 e-mail: rojema@foa.unesp.br received for publication: june 22, 2014 accepted: september 02, 2014 abstract aim: to determine the association between dental caries, body mass index (bmi) and sugar intake in children attending primary school. methods: a cross-sectional study was conducted with 91 children, with mean age of 3.9 years (sd = ±1.0), considering anthropometric measurements, according to standardized methodology of the ministry of health and occurrence of dental caries was recorded from clinical examinations (dmft). the sugar intake level was measured with a questionnaire that was applied to parents/caretakers of the children. the association of the variables was checked using a correlation with the contingency c coefficient test. results: 33 (36.3%) children were at risk for overweight, overweight or obese. of these, 10 (30.3%) had dmft >1. an association was found between sugar added to the bottle and child dmft (coeff. c = 0.5853 and p <0.0001) and also with bmi (coeff. c = 0.5693 and p <0.0001). there was no association between bmi and caries (chisquare = 0.1447 and p-value = 0.7036). conclusions: it was concluded that there was a correlation between sugar intake and dmft and bmi, but not between caries and bmi. keywords: child, preschool; body mass index; dental caries; obesity. introduction the world health organization (who) considers obesity one of the ten major public health problems in the world, classifying it as an epidemic of the 21st century1. there are 300 million obese people worldwide, and among them 43 million children under five years of age who are overweight2. obesity in childhood is an alert to the public health, a serious and emergency issue because it tends to persist into adulthood, constituting a risk factor for the occurrence of several chronic diseases3-4. according to who, the high sugar consumption is a major cause of weight gain1. as for obesity, the high frequency of intake of sugar-rich foods is closely related to the etiology of dental caries. eating habits are key factors in these two diseases5. it is essential to establish healthy practices and to monitor food and nutrition, especially in children 2 to 5 years of age, as in this period he or she starts adopting habits and behaviors, and a healthy diet is essential to ensure adequate growth and development of children4-6. although obesity theoretically is associated with tooth decay because these diseases present an etiological factor in common, the documentation of such associations is scarce and seemingly inconsistent. population studies on childhood obesity associated with dental caries are scarce in brazil. these surveys provide information about the actual state of health of the child population, providing subsidies to chart new directions to the brazilian health system. braz j oral sci. 13(3):209-212 210 in this context, the objective was to determine the association between dental caries, body mass index (bmi) and sugar intake in children attending primary school in the countryside of araçatuba, sp, brazil. material and methods the research protocol was approved by the ethics committee in human research, araçatuba dental school/unesp (process foa-01411/2011). this was a population-based cross-sectional study and the sample consisted of all children aged 2 to 5 years enrolled in a charity institution that serves as a nursery and kindergarten in araçatuba, sp, brazil. none of the children presented either systemic or neurological conditions that could prevent them from taking part in the study. only children whose parents/caretakers did not agree with the survey were excluded. the codes and criteria for measuring dmft followed the guidelines of the who7. in order to evaluate anthropometric assessment the team used the children’s body mass index (bmi). weight and height taken from each child were transformed in the corresponding z scores by age and sex. complying with the recommendations of the brazilian pediatric association, thinness, normal weight, risk for overweight, overweight and obesity were defined according to the criteria percentile for weight and height related to each sex and age group8. the data on sugar intake were obtained by a questionnaire administered to the parents/caregivers, with the following questions: (a) do you put sugar in your child’s milk? giving a possibility to answer “yes” or “no”. (b) what do you put in the bottle, besides milk? giving a possibility to answer “the child does not use a bottle,” “nothing” or if so, asked to specify the food that was added. (c) do you put some sugary snack in the child’s pacifier? giving a possibility to answer “the child does not use a pacifier”, “nothing” or if so also requested to specify what food was added. a pilot study was performed with 10% of the research universe in a different primary school of the city that presented the same characteristics of the school where the research was made in order to train and calibrate the examiner and recorded the codes and criteria of the used indexes, in addition to the adequacy of the questionnaire. only one trained and calibrated researcher conducted the dental caries exams and took the height and weight of the children. the researcher was also responsible for applying the questions to the children’s parents/caretakers. the quality control of the data was performed by analysis of reproducibility of observations by the examiner and the calibration results were verified by analysis of agreement kappa examiner, obtaining the value k = 0.86, indicating excellent agreement. the bioestat® software, version 5.3, was used to determine the association between the study variables using the nonparametric chi-square test of independence and contingency coefficient c for correlation, with decision level of α= 0.05. results in the analyzed period the institution had a total of 125 preschool children aged 2 to 5 years. the population of the study consisted of 91 (72.8%) children, 45 (49.5%) girls and 46 (50.5%) boys, whose parents/caretakers returned the filled-out survey forms as well as the consenting term of agreement. with respect to age, 4 (4.4%) children were 2 years old, 37 (40.7%) 3 years old, 11 (12.1%) 4 years old and 39 (42.8%) 5 years old. the average age was 3.9 years old (sd = ±1.0). the average dmtf for this group was 0.80, with a total of 30 (33%) children with teeth affected by dental caries (table 1). out of the studied children, 33 (36.3%) were found in the bmi stratification (risk of overweight, overweight or obese) of most concern. of these, 10 (30.3%) had teeth with caries. however, the bmi classification was independent of the presence of dental caries, and no statistically significant association was found, as demonstrated by the chi-square test of independence for association (table 2). regarding the survey, 16 (17.6%) parents/caregivers stated they used sugar in their children’s milk. of the 50 (54.9%) children who used pacifiers, only 5 (10%) had sugar or honey added to them. of the 76 (83.5%) children who were bottle-fed, 45 (59.2%) had sugary food added to milk. the contingency c correlation coefficient test was used to verify the magnitude of the associations between the variables of the survey, the anthropometric data and the clinical results. it was found that there was an association between sugary food added to the children’s bottle and the ceo-d (coeff. c = 0.5853 and p <0.0001) and bmi indexes (coeff. c = 0.5693 and p <0.0001). there was also an association between sugar added to milk and dental caries (coeff. c = 0.5500 and p <0.0001), but no association between sugar and bmi. in addition, no association between pacifier with sugar or honey and bmi or caries was found. discussion dental caries is a multifactorial disease that can be prevented, controlled, or even reversed, by means of prevention programs in the community, educational institutions and in the households. for prevention it is essential that the risk factors be known in order to be controlled. the disease is able to cause great destruction to the teeth, or even lead to teeth loss and may result in local, systemic, psychological and social complications9-10. data from the national survey of oral health showed a dmft average of 2.43 for 5 year-olds, and 46.6% of children in brazil were free of caries11. a dmft of 1.46 in average was detected in a survey conducted in another brazilian city12. in the present study body mass index, dental caries and sugar intake in 2-5 year-old preschoolers dmft number (n) percentage (%) 0 61 67 1 12 13.2 2 8 8.8 3 5 5.5 4 3 3.3 6 1 1.1 12 1 1.1 total 91 100 table 1number and percentage of preschoolers with respect to the degree of dmft index. araçatuba, são paulo, brazil, 2012 211 braz j oral sci. 13(3):209-212 was found a lower value of the index and a higher percentage of caries-free children, probably because children under 5 years of age were included and consequently had their teeth exposed to the oral environment for a shorter time. excess weight and obesity are found with great frequency from the age of 5 up, in all socio-economic groups and regions of brazil. overweight affects more than 30% of children between 5 and 9 years of age and about 20% of the population between 10 and 19 years of age13. in another brazilian study conducted in recife, pe, brazil in 1999, was observed a high frequency of weight disorders in children and adolescents. the prevalence of overweight and obesity in children was 26.2% and 8.5%, respectively14. these data corroborate the present work, because 1/3 of the studied children, albeit in a younger age group, were in the most concerning bmi stratification for quality of life. eating habits are an important etiologic factor for both obesity and tooth caries, since the amount of sugar intake and frequency of ingestion are factors involved in its etiology15. different studies (cross-sectional16 and prospective cohort17) in different countries showed positive correlation between dental caries and bmi. in contrast, a cross-sectional study of iranian children showed no association between weight, height and dental caries18. this result corroborates the finding of the present study where no association was found between bmi and dental caries. a possible explanation is that although both obesity and dental caries are often attributed to the high intake of carbohydrates and sugar, the true etiology of these diseases is much more complex and multifactorial9-10,19. an example of that may be the xavier et al.20 study which demonstrated that people living in low socioeconomic conditions have the worst oral health conditions due to the exposure to risk factors interfering with their quality of life. another possible cause is the rapid bmi changes in children as they grow and these changes do not relate strictly to body fat19. also the different educational and preventive measures undertaken to deal with dental caries in brazil, and the small sample size may have led to lack of association between these diseases10. in other studies21-22 findings the association was inverse, where individuals with higher caries experience had a lower body mass index (bmi). malnutrition may be associated with increased susceptibility to dental caries by the impaired composition and saliva secretion21. ayhan et al.22 observed association between nutritional status and caries in early childhood, where children with low-birth weight were more likely to have serious problems of dental caries compared to children with normal weight. a systematic review23 analyzed evidence for the association between obesity and dental caries, but the study did not find sufficient evidence for the association and did not clarify the possible role of diet and other possible factors of this association. in brazil, further research should be carried out with larger samples in order to try to elucidate the possible relationship between these diseases. currently, it is also critical that dental professionals be aware of the risk that overweight brings to patient’s health and seek to sensitize and motivate them to adopt healthy eating habits in order to prevent the occurrence of dental caries as much as obesity. it is also necessary to implement intervention programs and create a system of food and nutrition surveillance for children at this age, since this period is the most conducive to acquiring good habits and behaviors. references 1. world health organization (who). obesity: preventing and managing the global epidemic: report of a who consultation. geneva: world health organization; 2000 [cited 2013 apr 26]. available from: http://whqlibdoc. who.int/trs/who_trs_894.pdf?ua=1. 2. world health organization (who). obesity and overweight, 2012 [cited 2013 apr 26]. available from: http://www.who.int/mediacentre/factsheets/ fs311/en. 3. mello ed, luft vc, meyer f. childhood obesity: towards effectiveness. j pediatr (rio de janeiro). 2004; 80: 173-82. 4. silva cm, basso df, locks a. feeding in infancy: approach for oral health promotion. rev sul-bras odontol. 2010; 7: 458-65. 5. souza filho md, carvalho gdf, martins mcc. consumption of sugarrich foods and dental caries in preschool children. arqu odontol. 2010; 46: 152-9. 6. ramos m, stein lm. development of children’s eating behavior. j pediatr. (rio de janeiro). 2000; 76: s229-37. 7. world health organization (who). oral health surveys: basic methods. 4th ed. geneva: world health organization; 1997 [cited 2013 apr 26]. available from: http://www2.paho.org/hq/dmdocuments/2009/ oh_st_esurv.pdf. 8. brazilian pediatric society. nutritional evaluation of children and teenagers – orientation manual. são paulo: brazilian pediatric society. nutrology department; 2009 [cited 2013 apr 26]. available from: http:// www.sbp.com.br/pdfs/manual-aval-nutr2009.pdf. 9. losso em, tavares mc, da silva jy, urban ca. severe early childhood caries: an integral approach. j pediatr (rio de janeiro). 2009; 85: 295300. 10. cypriano s, hugo fn, sciamarelli mc, tôrres lhn, sousa mlr, wada rs. factors associated with the incidence of dental caries among schoolchildren living in a municipality with low prevalence of dental caries. cienc saude colet. 2011; 16: 4095-106. 11. brazil. ministry of health department of health care. department of health surveillance. department of primary care general coordination of oral health. sb brazil 2010: national survey on oral health: key findings. brasília: ministry of health; 2011 [cited 2013 apr 26]. available body mass index, dental caries and sugar intake in 2-5 year-old preschoolers thinness ideal risk of overweight obesity total weight overweight n % n % n % n % n % n % dmft = 0 1 1.1 37 40.7 12 13.2 7 7.7 4 4.4 61 67 dmft > 1 0 0 20 22 5 5.5 3 3.3 2 2.2 30 33 total 1 1.1 57 62.7 17 18.7 10 11 6 6.6 91 100 table 2 association between body mass index (bmi) and dental caries in preschoolers. araçatuba, são paulo, brazil, 2012 chi-square = 0.1447; p = 0.7036 212 braz j oral sci. 13(3):209-212 from: http://bvsms.saude.gov.br/bvs/publicacoes/pesquisa_nacional_saude_bucal.pdf. 12. amaral rc, batista mj, meirelles mpmr, cypriano s, sousa mlr. dental caries trends among preschool children in indaiatuba, sp, brazil. braz j oral sci. 2014; 13: 1-5. 13. brazilian institute of geography and statistics (ibge). pof 2008-2009: malnutrition falls and brazilian children’s weight exceeds international standard, 2012 [cited 2013 apr 26]. available from: http://www.ibge.gov. br/home/presidencia/noticias/noticia_visualiza.php?id_ noticia=1699&id_ pagina=1. 14. balaban g, silva gap. overweight and obesity prevalence in children and adolescentes from a private school in recife. j pediatr (rio de janeiro). 2001; 77: 96-100. 15. traebert j, moreira eam, bosco vl, almeida, ics. changing from breastfeeding to family feeding: a common problem for both obesity and dental caries. rev nutr. 2004; 17: 247-53. 16. honne t, pentapati k, kumar n, acharya s. relationship between obesity/ overweight status, sugar intake and dental caries among adolescents in south india. int j dent hyg. 2012; 10: 240-4. 17. alm a, fahraeus c, wendt lk, koch g, andersson-gare b, birkhed d. body adiposity status in teenagers and snacking habits in early childhood in relation to approximal caries at 15 years of age. int j paediatr dent. 2008; 18: 189-96. 18. sadeghi m, lynch cd, arsalan a. is there a correlation between dental caries and body mass index for age among adolescents in iran? community dent health. 2011; 28: 174-7. 19. world gastroenterology organization (wgo). world gastroenterology organization global guideline: obesity. munich: world gastroenterology organization; 2009 [cited 2013 apr 26]. available from: http://www. worldgastroenterology.org/assets/export/userfiles/obesity-master%20 document%20for%20website.pdf. 20. xavier a, carvalho fs, bastos rs, caldana ml, bastos jrm. dental caries-related quality of life and socioeconomic status of preschool children, bauru, sp. braz j oral sci. 2012; 11: 463-8. 21. johansson i, lenander-lumikari m, saellstrom ak. saliva composition in indian children with chronic protein-energy malnutrition. j dent res. 1994; 73: 11-9. 22. ayhan h, suskan e, yildirim s. the effect of nursing or rampant caries on height, body weight and head circumference. j clin pediatr dent. 1996; 20: 209-12. 23. silva aer, menezes amb, demarco ff, vargas-ferreira f, peres ma. obesity and dental caries: systematic review. rev saude publica. 2013; 47: 799-812. body mass index, dental caries and sugar intake in 2-5 year-old preschoolers oral sciences n3 original article braz j oral sci. july | september 2012 volume 11, number 3 sealing ability of gutta-percha/nano ha versus resilon/epiphany after 20 months using an electrochemical model – an in vitro study salma b. abdo1, aziza al darrat2, sam’an malik masudi3, norhayati luddin4, adam husien4 1bds, msc, phd student, department of restorative dentistry, school of dental sciences, university sains malaysia, malaysia 2bds, msc, phd, department of restorative dentistry, college of dentistry, university of sharjah, united arab emirates 3 dds, ms, school of dental sciences, department of restorative dentistry, university sains malaysia, malaysia 4bds , department of restorative dentistry, school of dental sciences, universiti sains malaysia, malaysia correspondence to: salma b abdo tawam dental centre tawam hospital office p.o. box: 15258 abu dhabi, united arab emirates phone: (+971-3)7070719 mobile (+971-50)7850020 e-mail: salma114@hotmail.com abstract aim: to evaluate the sealing ability of gutta-percha-nano-ha and resilon-epiphany by electrochemical method and micro-computed tomography (ct) scan at 48 h and 20 months using three different obturation techniques (cold lateral condensation technique, warm vertical condensation system b, and warm vertical condensation with vibration down-pak). methods: 150 human mandibular single-rooted premolars were prepared and randomly allocated into 6 groups of 25 specimens each, and filled with either gutta-percha-nano ha or resilon-epiphany with the three different obturation techniques (cold lateral, warm vertical system b, and warm vertical with vibration down-pak). electrochemical microleakage method was used to measure the microleakage after 48 h and after 20 months, and a micro-ct scanner 1072 was used to evaluate the quality of obturation after 48 h. results: group 6 (resilon-epiphany/down-pak technique) had the highest microleakage value, followed by group 2 (gutta-percha-nano ha/ system b technique), group 4 (resilon-epiphany/lateral condensation technique), group 3 (gutta-percha-nano ha/down-pak technique), group 5 (resilon-epiphany/system b technique), and group 1 (gutta-percha-nano ha/lateral condensation technique) with the values of 4.69 (6.06) kù, 3.88 (2.92) kù, 3.77 (3.75) kù, 3.38 (3.92) kù, 2.64 (2.90) kù, and 2.44 (4.34) kù, respectively. no significant difference in the quantity of leakage was observed for each root in each group between the two tested filling materials and their sealers (p=0.143). micro ct scan investigations revealed more micro-voids in the resilon-epiphany group obturated with downpak technique. conclusions: nano-hydroxyapatite sealer with gutta-percha filling material provided a reasonable seal compared with epiphany sealer and resilon filling material. keywords: resilon-epiphany, gutta-percha-nano ha sealer, root canal filling, electrochemical test, micro-ct scan. introduction obturation of the root canal is the final step of the endodontic treatment. the success of this step depends mainly on the root filling materials and techniques, which should have the ability to eliminate leakage from the oral cavity and periodontal tissues after cleaning and shaping. a number of obturation received for publication: may 18, 2012 accepted: september 13, 2012 braz j oral sci. 11(3):387-391 braz j oral sci. 11(3):387-391 388388388388388 techniques have been used over the years1-7. it is believed that the excellence of obturation seal is primarily related to the cohesive and adhesive interaction of filling materials to the root canal walls8-10. recently, a new sealer has been introduced by the school of dental sciences, universiti sains malaysia, malaysia, known as nano-hydroxyapatite (nanoha) sealer11. the nano ha crystals, which range from 40-60 nm in size, are synthesized by wet chemical method using calcium hydroxide [ca(oh) 2 ] and phosphoric acid (h 3 po 3 ) as ca and p precursors, respectively. the sealer is composed of nano-hydroxyapatite as a filler, bismuth (iii) oxide as a radiopacity component and hexamethylenetetramine as a polymerization activator, and the liquid is bisphenol a diglycidyl ether12. previous investigations have shown no significant difference between the sealing ability of nano ha compared with ah 26, using dye penetration technique11-12. on the basis of the in-vitro and in-vivo data available, several leakage studies have been conducted comparing resilon-epiphany with gutta-percha and conventional sealers. onay et al.13 (2009) using the fluid filtration method and kaya et al.14(2007) using glucose penetration method have shown that the quality of apical seal achieved with guttapercha and ah plus or ah26 in combination is similar to that of epiphany-resilon. however, other investigators have shown, using the same techniques, that the better quality of apical seal is achieved with epiphany-resilon combination due to superiority of this bonding system14-16. as the bonding systems have improved, so has the resistance to bacterial and fluid penetration of the materials. however, no study has yet been conducted to evaluate the sealing ability of different materials and obturation techniques using electrochemical and micro-computed tomography (ct) scan. therefore, the aims of this study were to evaluate the sealing ability of gutta-percha-nano-ha and resilon-epiphany by electrochemical method and micro-ct scan after 48 h and 20 months, using three different obturation techniques (cold lateral condensation technique, warm vertical condensation system b, and warm vertical condensation with vibration down-pak). material and methods selection of teeth extracted human single-rooted mandibular premolars were obtained and stored at room temperature in sealed vials containing saline immediately after extraction. the teeth were examined under digital stereomicroscope (motic digital microscope, ltd, france) to discard those with any preexisting root fractures. a sample of 150 teeth were selected and sectioned at the cementoenamel junction using a diamond disc at a high-speed handpiece under continuous water spray coolant, according to the following criteria: • the same root curvature; between 0º-5º (using schneider technique) • diameter of apical foramen equals to k-file size 15 • dentin thickness standardized between 2.5-3.5 mm from the apical foramen to the cervical orifice. preoperative periapical digital radiographs from buccolingual and mesiodistal directions were taken to ensure that root samples had normal canal shape and enough thickness of dentinal walls. then the samples were randomly divided into 6 groups of 25 roots and kept in separate plastic containers. each sample was given a unique number. sample preparation working length for each root canal was established using a size 15 k flex file (dentsply tulsa dental specialties, tulsa, ok, usa). file was placed and advanced into the root canal until its tip was visualized at the apical foramen. the working length was set at 1 mm shorter of the apical foramen and it was equal to 15 mm. all canals were instrumented with pro file ni-ti (dentsply-maillefer, ballaigues, switzerland) rotary instruments up to a size 35 master apical file following manufacturer’s instructions and using crown down pressureless technique. during preparation and between each profile file, the canals were irrigated with 2 ml of 5.25% naocl (farmácia amazon, são carlos, sp, brazil). after instrumentation, the canals were rinsed initially with 5 ml of 17% edta to remove the smear layer and followed by 5 ml of distilled water to remove any residues of naocl. sample obturation group 1 (n=25): the prepared root canals were obturated with gutta-percha and nano-ha endodontic sealer using cold lateral condensation technique. a master cone size 35 profile was fitted to the working length and presence of tug-back was confirmed. nano-ha sealer was mixed according to manufacturer’s instructions to a creamy consistency and applied into the canals using lentulo spiral (dentsply-caulk, milford, de, usa) which was inserted within 2 mm short of the working length. after placement and condensation of the master cone at the appropriate working length, accessory cones were placed and condensed using a finger spreader (miltex, inc., york, pa, usa). the excess of guttapercha was removed with a heated instrument and condensed vertically with plugger (nordent, usa) to the level of the canal orifice. group 2 (n=25): similar to group 1, the prepared root canals were obturated with gutta-percha and nano-ha endodontic sealer using vertical condensation technique system b (analytic, sybron dental specialties, orange, ca, usa). group 3 (n=25): similar to group 1, the prepared root canals were obturated with gutta-percha and nano-ha endodontic sealer using vertical and vibration condensation technique down-pak (ei-endo, hu-friedy, chicago, il, usa). group 4 (n=25): the prepared root canals were obturated with resilon/epiphany self-etching primer and epiphany sealer (pentron clinical technologies llc, wallingford, ct, usa). first, the primer was inserted into the root canals and excess was removed with paper point (dentsply tulsa dental specialties). subsequently, epiphany sealing ability of gutta-percha/nano ha versus resilon/epiphany after 20 months using an electrochemical model – an in vitro study 389389389389389 braz j oral sci. 11(3):387-391 sealer was mixed according to the manufacturer’s instructions and inserted into the root canals with a lentulo spiral. resilon master cone size 35 was placed into the root canal. following the application of the sealer, root canal filling was completed by inserting accessory cones dipped in epiphany sealer and laterally condensing with a finger spreader. excess resilon cones were removed with a heated instrument and condensed vertically with a plugger to the level of the canal orifice. group 5 (n=25): similar to group 4, the prepared root canals were obturated with resilon and epiphany using vertical condensation technique system b (analytic, sybron dental specialties, ca, usa). group 6 (n=25): similar to group 4, the prepared root canals were obturated with resilon and epiphany using vertical and vibration condensation technique down-pak (eiendo, hu-friedy). postoperative radiographs were taken to ensure complete and void-free obturation. all the samples were wrapped in wet piece of gauze to assure 100% humidity and were stored individually in screw-capped glass vials in an incubator at 37°c for 48 h and 20 months. iii) quantitative microleakage measurement apical leakage of the obturated root canals was assessed by ac-impedance technique at 48 h and 20 months. a pvcinsulated copper wire with a 5.0mm bared end was inserted coronally into the obturated canal of each root and sealed in position with sticky wax. thereafter, the coronal 2/3 of the external roots surfaces as well as the root/wire junctions were sealed with three layers of nail varnish. each root was immersed in an electrolyte solution (0.9% sodium chloride solution) at room temperature. then the ac-current was applied between the electrodes. the current flow, denoting onset of leakage, was measured by ir drop across a 10-ohm resistor placed in series with the electrodes and power source. then after 20 months, 2 samples from each group were securely placed and fixed into the sample holder of the skyscan micro-ct scanner 1072 (micro photonics inc., allentown, pa, usa) at 100 kv and source current at 120 ma mps, beam hardening set to 10. each sample was placed for roughly 2 h to produce 1000 projections in tiff. these images were then converted to tomograms (cross sections) saved in bmp, using nrecon (version 1.4.3; skyscan). next, image were examined for microleakage using image analysis programs provided by skyscan (t-view and ct-an) using ant for the 3d reconstruction for creating the 3d model. results the results from this study seem to suggest that, group 6 had the highest microleakage value, followed by group 2, group 4, group 3, group 5, and group 1 with the values of 4.69 (6.06) kù, 3.88 (2.92) kù, 3.77 (3.75) kù, 3.38 (3.92) kù, 2.64 (2.90) kù, and 2.44 (4.34) kù, respectively. no statistically significant difference in the quantity of leakage was observed for each root in each group between the two tested filling materials and their sealers (p=0.143), as shown in table 1. down-pak technique group exhibited the highest groups n median (iqr) kù p value group 1 25 2.44 (4.34) 0.143 group 2 25 3.88 (2.92) group 3 25 3.38 (3.92) group 4 25 3.77 (3.75) group 5 25 2.64 (2.90) group 6 25 4.69 (6.06) table 1. comparison of microleakage values among the groups after 20 months of evaluation (kruskal-wallis test). group 1= gutta-percha-nano ha/lateral condensation technique; group 2= guttapercha-nano ha/system b technique; group 3= gutta-percha-nano ha/down-pak technique; group 4= resilon-epiphany/lateral condensation technique; group 5= resilon-epiphany/system b technique; group 6= resilon-epiphany/down-pak technique. technique n mean(sd) kù p value lateral condensation 50 3.96 (2.36) .296 system b 50 3.60 (1.88) down-pak 50 4.33 (2.61) table 2. comparison of microleakage values (one-way anova test). microleakage value (4.33 ± 2.61) kù, followed by the lateral condensation technique group (3.96 ± 2.36) kù, and system b group (3.60 ± 1.88) kù. in addition, no significant difference was observed among all experimental groups based on the obturation technique used (p=0.296). as shown in table 2. the results of comparison between microleakage measured at 48 h and 20 months was not statistically significant (p<0.05) as shown in figure 1. figure 2 (a, b and c) depict micro-computed tomography reconstructions of root canal geometry of samples obturated with nano-ha and gp using cold lateral condensation technique, system b and down-pak technique. while, micro-computed tomography reconstructions of root canal geometry of samples obturated with resilon-epiphany system using the same three obturation techniques are shown in figure 3 (a, b and c). micro ct scan investigations showed micro-voids were observed more in the resilonepiphany group obturated with the down-pak technique. discussion the results of the present study indicate that there was no significant difference in the sealing ability between the two tested filling materials and their sealers (p=0.143) using the three obturation techniques. this may be due to nanoha epoxy resin, which has been shown to have a reasonable sealing ability12,17-18. in addition, it was reported that nanoha sealer was not affected by heat application during sealing ability of gutta-percha/nano ha versus resilon/epiphany after 20 months using an electrochemical model – an in vitro study 390390390390390 fig. 1. mean of microleakage of each group after 48 h and 20 months. continuous waves of condensation technique19-20 unlike the resilon material. the present experimental work indicated that resilon-epiphany root canal fillings when compared with gutta-percha/nano-ha have no significant difference in the sealing ability when the whole length of root canal was filled with lateral, vertical and vibration techniques measured after 20 months of obturation. however, shemesh et al.21 (2008) found that, the apical 4 mm of resilonepiphany root canal fillings allowed more glucose penetration than gutta-percha. shipper et al.16 (2004) detected faster bacterial leakage in gutta-percha and ah26 filling when compared with resilon/epiphany during a period of 31 days for the entire root length. this study showed that there is no significant difference in the sealing ability of resilon-epiphany using 3 three different obturation techniques compared with gutta-perchananoha within 48 h. however, after 20 months, sealer dissolution resulted in gap formation between root dentin and root filling material. according to de munck et al.22 (2005), after 3 months, all dentin adhesives under investigation exhibited mechanical and morphological evidence of degradation. as the bond degrades, interfacial microleakage increases, which resembles the in-vivo ageing effect. in the present study, the ageing effect through storage of the specimens over 20 months possibly evokes the current limitations of dentin bonding in the root canal system. in addition, the polymerization shrinkage of the epiphany sealer and water sorption and solubility play an important role concerning the increased microleakage in the long term23. the solubility values a reported by versiani et al.24 (2006) were 3.41% for epiphany and 0.21% for ah plus. bonding is further compromised in sclerotic dentin, which is found more often in the apical area of permanent teeth25. electrochemical leakage tests offer advantages in terms of speed, accuracy and efficiency, as well as, its ability to fig. 3. micro-computed tomography reconstructions of root canal geometry of samples filled with resilon-epiphany system using cold lateral condensation technique (a), system b (b) and down-pak technique (c). fig. 2. micro-computed tomography reconstructions of root canal geometry of samples filled with nano-ha and gp using cold lateral condensation technique (a), system b (b) and down-pak technique (c). sealing ability of gutta-percha/nano ha versus resilon/epiphany after 20 months using an electrochemical model – an in vitro study braz j oral sci. 11(3):387-391 391391391391391 perform longitudinal studies. the results of the present study corroborate those of previous investigations, even though, those studies used different methods for measuring microleakage, such as dye penetration26, bacterial leakage27, fluid filtration method17 and glucose penetration method21. all of these studies were short-term experiemnts of no more than 3 months, except for one study with duration of 16 months25. the present study had duration of 20 months. microct scan investigation of samples showed more micro-voids for resilon groups obturated by down-pak technique than in gutta-percha groups. sealing ability of both nano-ha sealer and epiphany sealer was similar by using three techniques: cold lateral, heat applied by system b or heat and vibration applied by down pak pressure. as it can be seen clearly in micro-ct reconstructions in figures 1 and 2, none of the techniques was superior in creating a hermetic seal of root canals. generally, the micro ct scan view of root obturated with resilon-epiphany down-pak system showed poor adaptation of resilon-epiphany with a massive amount of voids, which coincide with the result of microleakage using electrochemical test. the electrochemical test showed that this group has the highest amount of leakage, 4.69 kù. however, statistically it cannot be compared between the two tests because the sample size in the microct scan analysis was too small, and the machine is very expensive and time consuming test. nano-hydroxyapatite sealer with gutta-percha filling material provides a reasonable seal as compared to epiphany sealer and resilon filling material. as such, it could be used as an alternative to the commercial available sealer whereas techniques and duration have no effect on the results. none of the filling materials and techniques used in the present study provided a complete tight seal in a three-dimensional manner. further in vitro studies should be conducted to evaluate the effect of increasing temperature and vibration of downpak obturation technique on adhesion, dimensional stability and setting time properties of the tested obturation materials (nano-ha with gutta-percha or epiphany with resilon). references 1. gulsahi k, cehreli zc, kuraner t, dagli ft. sealer area associated with cold lateral condensation of gutta-percha and warm coated carrier filling systems in canals prepared with various rotary niti systems. int endod j. 2007; 40: 275-81. 2. schilder h. filling root canals in three dimensions. j endod. 2006; 32: 281-90. 3. kurtzman gm, von fraunhofer ja. leakage resistance of a self-etch sealercone obturation system. compend contin educ dent. 2008; 29: 246-8. 4. boussetta f, bal s, romeas a, boivin g, magloire h, farge p. in vitro evaluation of apical microleakage following canal filling with a coated carrier system compared with lateral and thermo-mechanical gutta-percha condensation techniques. int endod j. 2003; 36: 367-71. 5. buchanan ls. the continuous wave of obturation technique: ‘centered’ condensation of warm gutta-percha in 12 seconds. dent today. 1996; 15: 60-2, 64-7. 6. gencoglu n, garip y, bas m, samani s. comparison of different guttapercha root filling techniques: thermafil, quick-fill, system b, and lateral condensation. oral surg oral med oral pathol oral radiol endod. 2002; 93: 333-6. 7. marciano ma, bramante cm, duarte mah, delgado rjr, ordinolazapata r, garcia rb. evaluation of single root canals filled using the lateral compaction, tagger’s hybrid, microseal and guttaflow techniques. braz dent j. 2010; 21: 411-5. 8. nunes vh, silva rg, alfredo e, sousa-neto md, silva-sousa ytc. adhesion of epiphany and ah plus sealers to human root dentin treated with different solutions braz dent j. 2008; 19: 46-50. 9. williams c, loushine rj, weller rn, pashley dh, tay fr. a comparison of cohesive strength and stiffness of resilon and gutta-percha. j endod. 2006; 32: 553-5. 10. carvalho-junior jr, guimaraes lf, correr-sobrinho l, pecora jd, sousaneto md. evaluation of solubility, disintegration, and dimensional alterations of a glass ionomer root canal sealer. braz dent j. 2003; 14: 114-8. 11. alshakhshir j. the apical sealing ability evaluation of a new experimental nano hydroxyapatite-filled epoxy resin based endodontic sealerin vitro study. malays j med sci. 2010; 17: 110-5. 12. farea m, masudi s, wan bakar wz. apical microleakage evaluation of system b compared with cold lateral technique: in vitro study. aust endod j. 2010; 36: 48-53. 13. onay eo, ungor m, unver s, ari h, belli s. an in vitro evaluation of the apical sealing ability of new polymeric endodontic filling systems. oral surg oral med oral pathol oral radiol endod. 2009; 108: e49-54. 14. kaya bu, kececi ad, belli s. evaluation of the sealing ability of guttapercha and thermoplastic synthetic polymer-based systems along the root canals through the glucose penetration model. oral surg oral med oral pathol oral radiol endod. 2007; 104: 66-73. 15. kim yk, grandini s, ames jm, gu ls, kim sk, pashley dh, et al. critical review on methacrylate resin-based root canal sealers. j endod. 2010; 36: 383-99. 16. shipper g, orstavik d, teixeira fb, trope m. an evaluation of microbial leakage in roots filled with a thermoplastic synthetic polymer-based root canal filling material (resilon). j endod. 2004; 30: 342-7. 17. 17-wedding jr, brown ce, legan jj, moore bk, vail m. m. an in vitro comparison of microleakage between resilon and gutta-percha with a fluid filtration model. j endod. 2007; 33: 1447-9. 18. sousa-neto md, passarinho-neto jg, carvalho-junior jr, cruz-filho am, pecora jd, saquy pc. evaluation of the effect of edta, egta and cdta on dentin adhesiveness and microleakage with different root canal sealers. braz dent j. 2002; 13: 123-8. 19. tagger m, tagger e, tjan ah, bakland l. k. measurement of adhesion of endodontic sealers to dentin. j endod. 2002; 28: 351-4. 20. wu mk, van der sluis lw, wesselink pr. fluid transport along guttapercha backfills with and without sealer. oral surg oral med oral pathol oral radiol endod. 2004; 97: 257-62. 21. shemesh h, souza em, wu mk, wesselink pr. glucose reactivity with filling materials as a limitation for using the glucose leakage model. int endod j. 2008; 41: 869-872. 22. de munck j, van landuyt k, peumans m, poitevin a, lambrechts p, braem m et al. a critical review of the durability of adhesion to tooth tissue: methods and results. j dent res. 2005; 84: 118-32. 23. tay fr, loushine rj, lambrechts p, weller rn, pashley dh. geometric factors affecting dentin bonding in root canals: a theoretical modeling approach. j endod. 2005; 31: 584-9. 24. versiani ma, carvalho-junior jr, padilha mi, lacey s, pascon ea, sousa-neto md. a comparative study of physicochemical properties of ah plus and epiphany root canal sealants. int endod j. 2006; 39: 464-71. 25. paque f, sirtes g. apical sealing ability of resilon/epiphany versus gutta-percha/ah plus: immediate and 16-months leakage. int endod j. 2007; 40: 722-9. 26. kamalini r, mithra nh, priyadarshini h. apical sealing ability of newer resinbased pulp spacesealers an in vitro study. endodontology. 2008; 16-21. 27. baumgartner g, zehnder m, paque f. enterococcus faecalis type strain leakage through root canals filled with gutta-percha/ah plus or resilon/ epiphany. j endod. 2007; 33: 45-7. sealing ability of gutta-percha/nano ha versus resilon/epiphany after 20 months using an electrochemical model – an in vitro study braz j oral sci. 11(3):387-391 404 not found oral sciences n3 braz j oral sci. 10(3):171-174 original article braz j oral sci. july | september 2011 volume 10, number 3 received for publication: march 26, 2011 accepted: may 31, 2011 comparison of tooth crown discoloration with epiphany and ah26 sealer in terms of chroma and value: an in vitro study fatemeh shahrami 1, mina zaree 1, arash poorsattar bejeh mir2, mojtaba abdollahi-armani 1, abbas mesgarani 3 1 dds, ms endodontics department, dentistry school, mashad university of medical sciences, iran 2 student research committee, dental material research center, dentistry school, babol university of medical sciences, iran 3 dds, ms endodontics department, dentistry school, babol university of medical sciences, iran correspondence to: abbas mesgarani dentistry school, babol university of medical sciences, ganj afrooz ave, babol, iran phone: +989117132710 / fax: +981513216383 e-mail: a.mesgarani@mubabol.ac.ir abstract tooth crown discoloration may possess a heavy emotional burden and esthetic concern, especially when the anterior teeth are affected. residue of sealers within the pulp chamber is a major contributor to the occurrence of tooth discoloration. aim: the aim of this study was to evaluate the degree of crown discoloration when recently introduced sealer, epiphany, is used. methods: forty human incisors were examined in vitro. fifteen teeth were sealed with either ah26 or epiphany sealer as experimental groups and root canals of the remaining 10 teeth with distilled water. digital photographs of the buccal aspect of teeth were then examined in terms of brightness (value) and saturation (chroma) using photoshop software at the beginning and consecutive 3, 6 and 9 months. degree of brightness and saturation changes was defined as follows: less than 5% as slight, 10% to 15% as moderate and 15% to 20% as severe. results: fifteen, 14 and 10 teeth entered in epiphany, ah26 and control groups, respectively. significant brightness deterioration was noticed by time in both experimental groups [f (2, 8) =29.16, p<0.001], with no differences in epiphany compared with ah26 (p=0.086). saturation differed neither by time [f (2, 8) =0.129, p=0.881, nor by sealer type (p=0.136) during 9-month observation. at 9th month, crown segments (incisal, middle and cervical thirds) were not contrasted by the sealer type (p=0.982) or discoloration type (either brightness or saturation) (p=0.50). all changes in the experimental groups were significantly higher than the control group (p<0.001). conclusions: epiphany was equal to ah26 sealer in terms of relative long-term crown discoloration. one clinical correlation learned from the results of the present study is that epiphany may safely be used interchangeably with the traditional ah26 sealer. keywords: sealer, discoloration, esthetic, epiphany, ah26, brightness. introduction mainly, interaction of light with inner dentin and enamel structure reflective characteristics constitutes the color of the tooth. hence, any change in the composition of enamel, dentin or coronal pulp may cause the modification in translation of light through the tooth, and consequently tooth discoloration1. actually any modification in hue, chroma (saturation, hue intensity or color) or 172 braz j oral sci. 10(3):171-174 value (brightness, hue quality) of the tooth is discussed as tooth discoloration2. determining the origin of tooth discoloration is an essential step for further diagnostic and remedial procedures, in order to guarantee a successful outcome. the causes of tooth discoloration are simply classified as two main categories: external or internal. there may be a combination of both in some cases3. sedimentation of colored materials such as coffee, tea carrot, chocolate, mouthrinse or dental plaque is among the frequent reasons for external discolorations4-5. in addition, various factors, either systemic (e.g., drugs, fluorosis, cystic fibrosis, hyperbilirubinemia, dentinogenesis imperfecta) or local (e.g., pulp necrosis, pulp internal hemorrhage, pulp tissue remnant after pulpectomy, endodontic materials, crown restorative materials, aging or root resorption) may contribute to the changes in color of the tooth crown2,5. epiphany is a recently introduced root canal sealer that has been developed specifically for use with resilon cones. epiphany is a composite resin sealer with dual setting action that is composed of a mixture of bis-gma, ethoxylated bisgma, udma and dual hydrophilic methacrylate with calcium hydroxide fillers, barium sulfate, barium glass and silica6. to date, there are few studies that evaluated tooth discoloration after epiphany application as an endodontic sealer. the aim of this experimental in vitro study was to evaluate of degree of crown discoloration when epiphany is used for sealing, comparing the results with those of a traditionally used sealer, ah26. material and methods in this in vitro experimental study, 40 intact extracted human maxillary incisors were used. teeth were free of caries, crown restorations, cracks or any apparent coronal discoloration when selected. the teeth were cleaned with an ultrasonic device to dislodge gross debris, with additional rubber cap and pumice powder cleansing to remove remaining debris and stains from the coronal surfaces. then teeth were randomly assigned to experimental and control groups. randomization performed via permuted block method with 4 intervals and a final 1:1 teeth numbers. fifteen teeth in each arm were assigned to the experimental allocations and the 5 remaining as the controls. experimental sealers were either ah26 (dentsply detrey, switzerland) and epiphany (sybronendo, orange, ca usa). controls were filled with distilled water. tooth preparation started with cross sectioning from the crown to 3 mm below the cementoenamel junction with the apical root discarded pulps were extirpated and the pulp chambers were instrumented with k-files and no 2, 3 gates glidden drills. the canals were irrigated with 1 ml of 2.5 % sodium hypochlorite and 17% edta7. the experimental sealers were placed into pulp chambers via an apical access and the internal walls were thoroughly coated with the sealers. excess sealer was removed with small pieces of cotton pellet from the orifices of the canal and chamber walls. the apical access was then sealed with a sticky wax. the prepared teeth were placed on a gray background allowed the most accurate visualization of coronal discoloration, and the digital images were captured using a digital 12 mega pixel camera (canon ixus, japan) in a dark room and two 60 w light sources from sides on a gray background. thereafter, the images were saved as jpeg images. digital images of all samples were viewed in the photoshop software version cs8. first the full length crown of the tooth was selected. then, the background of each sample was deleted with the tooth crown was divided into three equal segments: incisal, middle and cervical. brightness and saturation of each segment were determined and recorded with the eye dropper option and the correlated histograms. afterward, teeth were then stored in the individually vials in an incubator at 37º and 90 º humidity to simulate intra oral condition. teeth were removed in subsequent 3, 6 and 9 months and examined in the same way. we scored (defined) brightness and saturation changes as follows: 1 for less than 5% (slight), 2 for 10% to 15% (moderate) and 3 for 15% to 20% (severe). all analyses were performed with spss version 17 (spss inc, chicago, il, usa). data were expressed as mean and standard deviation. data calculated for final scoring of tooth discoloration were tested with repeated measures anova to detect pattern of changes regarding time with sealer interaction. an anova test was also applied to determine the segment differences accounted for either brightness or saturation and sealer types with their interactions at 9th month with further post-hoc between-group analyses. a two tailed p<0.05 was considered statistically significant. results a total 15 teeth in epiphany group and 14 teeth in ah26 group (one tooth excluded because of apparent coronal fracture after canal preparation) were evaluated as experimental group. also ten teeth served as control group. obtained scores were adjusted for number of teeth in these groups. basic characteristics of calculated scores for teeth crown color changes by time in ah26 and epiphany sealer are displayed in table 1. brightness was significantly increased by time [f (2, 8) =29.16, p<0.001]. epiphany and ah26 group was not differed in brightness changes during the 9 months of the study (p=0.084). in addition, saturation changes was not neither statistically significant [f (2, 8) =0.129, p=0.881], nor different in experimental groups (p=0.136) (table 1). at 9th month, no differences were observed in different parts of the crown measured for epiphany sealer [f (2, 3.38) = 0.118, p=0.892] and for ah26 sealer [f (2, 6) = 0.06, p=0.936] (table 2). in addition, no interaction of segment*sealer (p=0.50), segment* discoloration (p=0.7), sealer*discoloration (p=0.33) and segment* discoloration* sealer (0. 83) was noticed. changes in the control group were not discernible through the study phases. all changes in experimental groups were significantly higher than the control group (p<0.001). changing trends of crown colors differed at different trimester comparison of tooth crown discoloration with epiphany and ah26 sealer in terms of chroma and value: an in vitro study braz j oral sci. 10(3):171-174 173 sealer month 3mean score(sd) month 6mean score(sd) month 9mean score(sd) significance † brightness epiphany 0.68(0.15) 0.99(0.23) 1.41(0.38) f(2,4)= 21.886, p=0.007 ah26 1.18(0.24) 1.47(0.04) 1.68(0.23) f(2,4)=9.181, p=0.032 significany†† t (4)= -2.63, p=0.58 t (4)= -3.41, p=0.27 t (4)= -1.02, p=0.36 0.084††† saturation epiphany 0.98(0.51) 1.11(0.42) 1.15(0.3) f(2,4)= 0.258, p=0.758 ah26 0.64(0.12) 0.64(0.12) 0.66(0.4) f(2,4)=0.015, p=0.985 significany†† t (4)= 2.72, p=0.53 t (4)= 1.82, p=0.142 t (4)= 0.85, p=0.44 0.136††† table1. changing trends of saturation and brightness during the study in epiphany and ah26 sealers calculated for whole crown. † within group analysis by repeated measurement anova test, †† statistical significant difference between two groups compared for changing trends during study calculated by t-test, ††† between group comparison of total change-trend by repeated measurement anova test. incisal third mean score(sd) middle third mean score(sd) cervical third mean score(sd) significance† brightness epiphany 0.71 (0.59) 0.59 (0.37) 0.55 (0.04) f (2,3.38)= 0.118, p=0.892 ah26 0.69 (0.24) 0.61 (0.21) 0.76 (0.66) f (2,6)= 0.016, p=0.936 significany†† t (4)= 0.03, p=0.97 t (4)= 0.06, p=0.95 t (2.01)= -0.53, p=0.64 saturation epiphany 0.50 (0.21) 0.62 (0.4) 0.62 (0.20) f (2,2.04)= 1.13, p=0.467 ah26 0.37 (0.48) 0.49 (0.31) 0.52 (0.08) f (2,6)=0.138, p= 0.857 significany† t (4)= 0.42, p=0.69 t (4)= 0.42, p=0.69 t (4)= 0.78, p=0.47 table 2. comparison of different segment changes at 9th month in epiphany and ah26 sealers. † statistically significany calculated by one-way anova†† statistically significant segmental difference between two groups calculated by t-test. intervals, with more highlighted changes occurred during first 3 months (figure 1). discussion in the present in vitro study, crown discoloration after filling the root canals with either epiphany or ah26 was investigated during 9 months. recently, epiphany was introduced as an ideal endodontic sealer6. this is the first study that investigated the crown discoloration induced by epiphany. tooth crowns were deteriorated in terms of brightness in both sealer groups. however, saturation increased by time, it was not remarkable for both epiphany (achieved power=0.48) and ah26 (achieved power=0.05). this finding is in agreement with parson et al.6 and partovi et al.8 researches, which concluded that tooth crown progressively discolored with application of endodontic sealers by time6,8. in addition, measured at 9th month, different crown segments were not contrasted by either saturation or brightness (achieved power=0.26). this similarity was present when compared for sealer types and interaction of either segment with discoloration or segment with sealer type. nine-month duration was chosen since it was proposed that most severe crown discoloration may occur during the first 9-month period after sealers were used6. it is postulated that brightness or value is the most important determinant of the color. higher values (i.e., darker shades) were observed in ah26 group when compared to epiphany group, in all study points. although it did not reach significant level, regarding achieved power of 25%, larger scale studies may better reveal this difference. the superiority of epiphany with this regard may be explained by the silver (ag) particles in ah26 sealer9-10. as one limitation of the study, we missed the immediate measurements of saturation and brightness. hence, changing trends and exact amount of changes, when adjusted for the baseline scores of filled teeth, are somehow hampered by this deficit. although not statistically significant, most prominent changes in saturation in the ah26 group occurred in the cervical third. this is in agreement with previous studies, which attributed this pattern to the thinner layer of enamel over this region6,9. surprisingly, as another remarkable characteristic of epiphany, it exhibited least value changes in the cervical third as compared to other regions. many earlier investigators studied color changes of posterior teeth (premolars)6,8,10, mean while, anterior teeth concern the patients mostly with a high socio-emotional burden. hence, we opted to examine the central teeth in the present research. collectively, average brightness and saturation changes occurred faster during the early phase of study (months 0-3), probably due to higher tubular penetration capacity at the beginning. this finding is contrasted from the result of parson et al.6 visual protocol that those authors6 applied in their research, which is acceptable considering the time at which study performed, may rationalize the apparent differences from our findings. nevertheless, our findings are fig. 2 average changes of saturation and brightness during the study phases. comparison of tooth crown discoloration with epiphany and ah26 sealer in terms of chroma and value: an in vitro study 174 braz j oral sci. 10(3):171-174 in agreement with those of partovi et al.8, who clarified more significant changes during early phase of study using a digital system. based on our results, it is speculated that the resinbased ah26 sealer and the composite-based epiphany sealer are similar in penetration through dentin tubules and epiphany is even superior to the ah26. currently methods for evaluation of tooth crown discoloration could be classified into two major categories: visual method that is a subjective method which applies a guiding color shade. this method is very difficult, since many interference variations may adversely influence the interpretation (e.g., eye fatigue and light position)7. alternatively, color analysis with a device is a quantitative objective method that includes colorimetric and spectro-photometric analyses1. however, these techniques have their own limitations, such as limited access of clinicians to such devices. more recently, along with introduction of the computer to the dentistry, we have one ideal method for analyze tooth discoloration. previously, a computer-aided method to discriminate chroma and value changes was used and analyzed with photoshop software11. this may help the observer lessen the measurement bias and simple visual observation flaws. we did not calculate completecomponent-color changes using either munsell (nickerson formula) or cie systems12. using nickerson and cie systems, calculated i or äe changes of more than 5 and 3.5 is considered clinically significant, respectively. ah26 sealer was selected because it is commonly used in endodontics. according to the various previous researches on the older generations of sealer, we did not include those, to be compared with in the present study. we did not attempt to remove the smear layer. the use of 17% edta may lessen the inhibitory effect of smear layer for sealers to be penetrated into the adjacent dentine tubules. this may rationalize the 4day discoloration of the crown when ah26 was used and the smear layer was eliminated with 17% edta9. briefly, both saturation and brightness were increased by time. low achieved powers in our study indicate that higher sample sizes may better distinguish the corresponding changes. a larger scale study with histological examination for depth of enamel and dentin penetration is recommended. external validation with more extended reliable results may be obtained with in-vivo studies. additively, shorter-interval observations can reveal the timely pattern of color changes more precisely. in conclusion, although it might be difficult to eliminate every trace of sealer, it is still recommended that the materials should be carefully removed from the pulp chamber immediately after filling the canal. newer epiphany sealer is a suitable substitute for ah26 sealer. overly, most changes occurred during the first 3 months with minimal gradual increments for the rest of the phases of the study and brightness changes represented the main portion of these differences in the present study. references 1. joiner a. tooth colour: a review of the literature. j dent. 2004, 32: 3-12. 2. ingle i, john i, backland lk, baumgartner jc. ingle’s endodontics. london: bc decker; 2008. 3. ten bosch jj, coops jc. tooth color and reflectance as related to light scattering and enamel hardness. j dent res. 1995; 74: 374-80. 4. watts a, addy m. tooth discoloration and staining: a review of the literature. br dent j. 2001; 190: 309-16. 5. hattab fn, qudeimat ma, al-rimavi hs. dental discoloration: an overview. j esthet dent. 1999; 11: 291-300. 6. parsons j, walton r, ricks-williamson l. in vitro longitudinal assessment of coronal discoloration from endodontic sealers. j endod .2001; 27: 699702. 7. cohen s, burns rc. pathways of the pulp. london: mosby; 2006. 8. partovi m, abdol h, soleimani b. in vitro computer analysis of crown discolouration from commonly used endodontic sealers. aust endod j. 2006; 32: 116-9. 9. van der burgt tp, plasschaert aj. tooth discoloration induced by dental materials. oral surg oral med oral pathol. 1985; 60: 666-9. 10. van der burgt t, mullaney tp: tooth discoloration induced by endodontic sealers, oral surg oral med oral pathol. 1986; 61: 84-9. 11. cal e, sonugelen m, guneri p. application of a digital technique in evaluation of the reliability of the shade guide. j oral rehabil. 2004; 31: 483-91. 12. obrien w. dental materials and their selection. chicago: quintessens; 1997 comparison of tooth crown discoloration with epiphany and ah26 sealer in terms of chroma and value: an in vitro study oral sciences n3 braz j oral sci. 11(4):458-462 original article braz j oral sci. october | december 2012 volume 11, number 4 shear bond strength of nanofilled flowable resins used for indirect bracket bonding carolina ferreira leite moreira da silva1, marcelo alves correa2, lourenço correr sobrinho3, alexandre moro4, ricardo cesar moresca4, gisele maria correr4 1graduate student, masters program in clinical dentistry, positivo university, curitiba, pr, brazil 2researcher, department of statistics, university of são paulo – esalq, piracicaba, sp, brazil 3professor, department of restorative dentistry, dental material area, piracicaba dental school, university of campinas, piracicaba, sp, brazil 4professor masters program in clinical dentistry, positivo university, curitiba pr, brazil correspondence to: gisele maria correr program in clinical dentistry, positivo university rua pedro viriato parigot de souza, 5300, campo comprido, cep: 81280-330 curitiba, pr, brasil phone: +55 41 33173403 fax: +55 41 33173082 e-mail: giselenolasco@up.com.br abstract aim: to evaluate the bond strength of brackets fixed with different materials (two light-cured nanofilled resins transbond supreme lv and flow tain lv, a light-cured resin transbond xt (control) and two chemically cured resins for indirect bonding sondhi rapidset and custom i.q.) using the indirect bonding technique after 10 min and 24 h, and evaluate the type of failure. methods: one hundred premolars were selected and randomly divided into groups (n=10) according to the material and fixation period. the brackets were bonded through the indirect technique following the manufacturer’s instructions and stored in deionized water at 37oc for 10 min or 24 h. after, the specimens were submitted to a shear bond strength (sbs) test (instron) at 0.5 mm/min and evaluated for adhesive remnant index (ari). the data were submitted to anova and tukey’s test (p<0.05) and the ari scores were submitted to the chi-square test. results: it could be observed a significant difference among the materials (flow tain lv = transbond supreme lv = transbond xt> sondhi rapid-set > custom i.q.). there was no significant difference in resistance values between 10 min and 24 h, regardless of the materials. most groups showed adhesive remaining adhered to the enamel (scores 2 and 3) without statistically significant difference (p>0.05). conclusions: it was concluded that the light-cured nanofilled materials used in indirect bonding showed greater resistance than the chemically cured materials. the period of fixation had no influence on the resistance for different materials. keywords: orthodontics, indirect bonding, orthodontic brackets. introduction the success of orthodontic treatment with fixed appliances depends, within other factors, on an accurate bracket positioning and long-term retention of these accessories.¹ the time spent during the bracket bonding is an important factor in the treatment cost and the necessity of rebonding brackets can retard the progress of treatment. to avoid errors during bracket positioning and facilitate bracket bonding, the indirect bond technique was introduced by silverman et al. (1972) 2 and progressed with thomas (1979)3. this technique has been practiced for many years in various forms4-7 and consists of the pre-positioning of appliances on a working model and the use of a transfer tray to capture the appliances and convey them to the patient’s mouth4. the indirect bonding is essential in lingual received for publication: august 09, 2012 accepted: september 28, 2012 459459459459459 orthodontics because of the difficulty of positioning the brackets directly to tooth and also by the variation of the anatomy of the lingual anterior teeth6. bracket positioning is facilitated by indirect bonding because it provides a direct and better view of the model teeth in all planes6. the indirect bonding has some advantages such as shorter clinical time, greater patient comfort, greater accuracy in positioning of brackets6,8 and reduced plaque accumulation around braces9. however, this technique also has disadvantages such as laboratory work time8 higher cost, more phases (laboratorial and clinical) and presence of remaining adhesive in the bracket base, which may impair adhesion10. bracket bonding to etched teeth using chemicalor lightcure adhesives can be considered a standard clinical practice11. advances in the adhesive procedures had occurred over the years, especially regarding the bonding materials. these materials have evolved in their composition and manipulation technique, aiming at sharing the efficient adhesion to the individual needs of the orthodontic patients. several materials are available in the market for bracket bonding, such as resin-modified glass ionomer cements, resin composites, orthodontic adhesives, flowable resins and more recently introduced nanofilled materials12-13. flowable resins have some advantages compared with traditional resin composites such as no stickiness and fluid injectability 14. these materials can have reduced filler content, increased diluents monomers or altered rheology to reduce the viscosity of the mixture. however, this may eventually weaken the mechanical properties of the flowable resins, which are influenced by filler loading within the resin matrix15. flowable resins have been applied for orthodontic use by many clinicians11,13,16. however, there are still few studies available that evaluate the bonding properties of these materials11,13,17 with contradictory reports on the shear bond strength14,16-17. based on the interest in flowable restoratives for orthodontic use, especially for indirect bonding, a nanofilled low-viscosity light cure indirect bonding adhesive was introduced by 3m unitek, monrovia, ca, usa transbond supreme lv. the adhesive contains a dimethacrylate polymer that modifies the rheology, allowing the material to flow under pressure, yet hold its shape after placement until light cured4. this characteristic is beneficial for indirect bonding, since the material will not slump, run, or drift from the bracket base prior to placement in the patient’s mouth. also, this material is a nanofilled resin that shows a reduction on filler size and increase on filler content (nanoclusters), allowing better mechanical properties18. however, little is known about the bonding characteristics of this material, especially for indirect bonding. as observed, there are various materials used for bracket bonding, however, most studies evaluated the bond strength of these new systems by direct bonding technique. another important factor is the post-fixation time that can influence the brackets bond strength and is important for the installation of the arches or accessories in the oral cavity. thus, the aim of the present study was to evaluate the shear bond strength (sbs) of brackets fixed with different materials (two lightcured nanofilled low viscosity resins transbond supreme lv and flow tain lv, a light-cured resin transbond xt (control) and two chemically cured resins for indirect bonding sondhi rapidset and custom i.q.) using the indirect bonding technique after 10 min and 24 h. the hypothesis of this study is that the sbs values of nanofilled resins will be higher than those of the other materials and there will be no influence of post-fixation time on sbs values. material and methods one hundred sound extracted human premolars were stored in a 0.5 chloramine t solution at 4oc for a maximum of 6 months after extraction. exclusion criteria included previously restored premolars and premolars with enamel defects or cracking and delamination of the enamel. this study was carried out after approval of the institutional review board (protocol # 168/09). teeth were randomly assigned into 10 groups (n=10) according to the material (transbond xt – 3m/unitek, transbond supreme lv – 3m/unitek, sondhi rapid set – 3m/unitek, custom i.q. – relience and flow tain lv relience) and the post-fixation time (10 min or 24 h). for the indirect bonding technique, the teeth were mounted in wax (orto central, poá, sp, brazil) to simulate a dental arch, totaling 10 teeth per arch. after, working models in orthodontic stone were obtained from accurate alginate impressions. the working models were prepared with careful trimming, removal of bubbles and filling of small voids, to avoid any problem in fitting of the bonding tray. the models were numbered according to the respective group. reference lines (long axis of the tooth) were drawn on the models to facilitate placement of the brackets. a thin layer of separating medium (cel-lac, sswhite, rio de janeiro, rj, brazil) was applied to the models and allowed to dry for approximately 1h. transbond xt light cure adhesive was placed on the mesh pad of individual metallic brackets (mini dyna-lock “roth” .022 3m/unitek, monrovia, ca, usa) that were positioned on the model and pressed firmly with a hollenback carver to expel the excess adhesive. each bracket was subjected to a 300-g compressive force using a force gauge (correx co, berne, switzerland) for 10 s, after which excess bonding resin was removed using a sharp scaler. the position of the bracket was carefully checked with a bracket gauge. the adhesive was light cured for 20 s from the occlusal edge and 20 s from the gingival bracket edge. the bonding adhesive was light cured with a light curing unit (xl300, 3m/unitek), with a light intensity of 1000 mw/cm2 measured with a built-in radiometer, which was calibrated every 10 min to ensure consistent light intensity. after bracket bonding, the bonding trays were made using a vacuum unit (plastivac, bioart, são carlos, sp, brazil) to vacuum-form a 0.9-mm-thick flexible silicone layer (soft tray sheets, ultradent, indaiatuba, sp, brazil), overlayered with a 1 mm thick rigid pvc crystal layer (bioart). a jet of braz j oral sci. 11(4):458-462 shear bond strength of nanofilled flowable resins used for indirect bracket bonding 460460460460460 material sbs at 10 min sbs at 24 h transbond xt 4.12 (0.51) aa 4.57 (1.00) aa transbond supreme lv 5.61 (2.09) aa 4.24 (2.25) aa flow tain lv 5.44 (1.29) aa 5.09 (0.99) aa sondhi rapid-set 2.40 (1.30) ba 2.86 (0.95) ba custom i.q. 1.22 (1.07) ca 1.53 (0.86) ca table 1 – mean (mpa) and standard deviation of shear bond strength (sbs) at 10 min and 24 h for the different materials. different capital letters in columns and small letters in rows indicate statistically significant difference (tukey’s test p<0.05). an oil-free silicone spray (3m, sumaré, sp, brazil) was applied on models before lamination of the flexible layer, and applied again before the lamination of the rigid layer. this set was submerged in water for 1 h to facilitate the separation of materials. the trays were outlined and excess material was trimmed with crown and bridge scissors and scalpel. the bonding tray’s hard outer shell was trimmed away from all heights of contour for patient comfort and closer fit because it only permits firm seating of the soft tray. then, they were cleaned using bicarbonate/water jet and rinsed in water. following, the brackets basis was sandblasted with aluminum oxide (45 µm, polidental,cotia, sp, brazil), for about 1 min on each tray, in order to increase retention, but without removing the resin transbond xt, and cleaned with acetone. the buccal enamel surface of each premolar was cleaned with fluoride-free pumice slurry, etched with 37% phosphoric acid gel (etch-37, bisco, schaumburg, il, usa) for 30 s, rinsed for 15 s, and dried with oil-free and moisturefree air for 20 s until the enamel had a faintly white appearance. after, the different materials (transbond xt, transbond supreme lv, flow tain lv, sondhi rapid-set and custom i.q.) were applied following the manufacturer’s instructions and the trays were positioned over the teeth. for transbond xt, transbond supreme lv and flow tain lv a small amount of the adhesive was applied to the bracket basis on the bonding tray that was brought into position and firmly hold. each tooth was was light cured for 20 s from the occlusal edge and 20 s from the gingival bracket edge. then, the trays were removed. for sondhi rapid-set a thin layer of resin a was applied to each tooth surface and a thin layer of resin b was applied at each bracket basis. the bonding tray was brought into position and pressed for 30 s, after two min the trays were removed. for custom i.q. a thin layer of part a was applied to each tooth surface and a thin layer of part b was applied at each bracket basis. the set was placed in position and pressed for 30 s, after four min the trays were removed. the bonding adhesives (transbond xt, transbond supreme lv and flow tain lv) were light cured with a lightcuring unit (xl300, 3m/unitek) with light intensity of 1000 mw/cm2 measured with a built-in radiometer, which was calibrated every 10 min to ensure consistent light intensity. after bracket bonding the trays were carefully removed with the aid of a scaler. the teeth were removed from the wax and the roots were embedded in self-cured acrylic resin (vipi flash, pirassununga, sp, brazil) using pvc cylinders (tigre s.a. tubos e conexões, castro, pr, brazil) as moulds. the specimens were then immersed in deionized water and maintained in an oven at 37oc during 10 min or 24 h, and the shear bond strength (sbs) test was performed. the specimens were secured in a jig attached to the base plate of a universal testing machine (model 4411, instron corp, canton, ma, usa). a chisel-edge plunger was mounted in the movable crosshead of the testing machine and positioned so that the leading edge was aimed at the enamelcomposite interface before being brought into contact. a crosshead speed of 0.5 mm/min was used. after debonding, each specimen was examined under a stereoscopic zoom microscope (olimpus szx9, são paulo, sp, brazil) to identify the location of the bond failure. the residual composite remaining on the premolar was assessed by using the adhesive remnant index (ari), where each specimen was scored according to the amount of material remaining on the enamel surface as follows: 0 no adhesive remaining, 1less than 50% of the adhesive remaining, 2 more than 50% of the adhesive remaining, and 3 all adhesive remaining with a distinct impression of the bracket base. descriptive statistics including means (mpa) and standard deviations (sd) were calculated for the sbs analysis. a two-way analysis of variance (anova) and tukey’s multiple-comparison tests were used to determine the statistical significance of any difference in mean sbss among groups. the ari was analyzed for percentage and frequency of fracture type and submitted to a chi-square test. significance for all statistical tests was predetermined at p>0.05. all statistical analyses were performed using bioestat 5.0. results shear bond strengths mean bond strengths and standard deviations for each group are given in table 1. there was a statistically significant difference among the materials regardless of the post-fixation time (flow tain lv = transbond supreme lv = transbond xt > sondhi rapid-set > custom i.q.) (p<0.05). the lightcured materials (transbond xt, transbond supreme lv and flow tain lv) showed higher values compared with the chemically cured materials (sondhi rapid-set and custom i.q.). there was no statistically significant difference between 10 min and 24 h regardless of the materials (p>0.05). ari the distribution of failure modes, as expressed by ari scores (%), is given in figure 1. according to statistical analysis (chi-square analysis) of the ari scores, all of the test groups exhibited similar bracket failure modes (p>0.05). regardless of the bond material, bond failure occurred partly at the bracket-adhesive (resin) interface but mainly within the adhesive (resin) (scores 1, 2 and 3). enamel fractures were not observed in any of the tested specimens. braz j oral sci. 11(4):458-462 shear bond strength of nanofilled flowable resins used for indirect bracket bonding 461461461461461 fig.1 – distribution of failure modes, ari scores (%), found after the sbs test. discussion the material and technique of bracket bonding should promote sufficient adhesion between the brackets and teeth, supporting the application of forces during orthodontic treatment. with the introduction of the indirect bonding technique, several materials have been developed and investigated1,4,19, but the effectiveness of the increase in the bond strength of these materials at the tooth/bracket interface remains unclear. the minimum shear bond strength of an adhesive should be between 5.9 and 7.9 mpa to be considered adequate for clinical needs20. when the results of this study were compared with these reference values, it was found that all the adhesives showed lower resistance values, but it could be noticed that these reference values were recommended based on the direct bonding technique. in the present study, three light-cured resins and two chemically cured resins for indirect bonding were selected and applied according to the manufacturer’s instructions. it could be observed that the light-cured materials showed higher bond strengths than the chemically cured ones. this finding was similar to other studies that also found lower bond strength values for chemically cured materials compared with light-cured ones19,21. nanofilled resins that present smaller and uniformly distributed filler particles, might present greater cohesive strength to penetrate into the etched enamel and also in the bracket base resin, increasing the bond strength18,21. however, the results of this study showed no significant difference among the sbs values of the nanofilled resins and transbond xt resin. thus, the hypothesis that the nanofilled resins would have a better performance than the traditional resins could not be validated. this could have occurred because the materials were applied in the resin previously attached to the bracket base, which would have hindered a complete penetration of the material on this region. uysal et al. (2010)12 did not found lower values for nanofilled materials (resin and ionomer) compared to a conventional orthodontic composite (transbond xt). for the chemically cured resins, which showed lower sbs values, it can be suggested that because these materials have no manipulation prior to its use, the catalyst and the base pastes are mixed only by the pressure at the time of bracket placement. this procedure could lead to incomplete polymerization of some portions of the material, which compromises their resistance21. constant changes are being made in the bonding materials and also on brackets bases in order to increase the bond strength, aiming at an improvement of clinical results. however, increased adherence difficult removal of the accessories in the end of treatment, which may cause cracks and fractures on the enamel surface22. thus, fractures at the adhesive/bracket interface or within the adhesive, leaving the material adhered to the tooth surface, are favorable because these failures avoid enamel loss and the remaining adhesive can be safely removed with appropriate rotary instruments23. regarding the adhesive remnant index, it was observed that the failure of light-cured resins after 10 min and 24 h occurred more frequently at the adhesive/bracket interface and all the material remained adhered to the enamel (score 3). for sondhi rapid – set group, after 10 min to 24 h, the score was predominantly 2, where half of adhesive remained adhered to the tooth. this result indicates that there was a poor adhesion between the material and the resin on the bracket base, which could be related to the indirect bonding technique used in the study. for the custom i.q. group, failure was predominantly score 1 (less than half of the adhesive adhered to the enamel), confirming the results found in other study24. regarding the post-fixation time, there was no significant braz j oral sci. 11(4):458-462 shear bond strength of nanofilled flowable resins used for indirect bracket bonding 462462462462462 difference between 10 min and 24 h tests, regardless of the materials, as observed in other studies25-26. thus, the hypothesis that it will be no influence of post-fixation time on sbs values could be validated. however, other studies have found higher sbs values for groups tested after 24 h27-28. this could be related to the materials selected in other studies, i.e., glass ionomer cements and resin-modified glass ionomer cements that reach their final resistance after 24 h. in this study, only resin based materials (light-cured or chemically cured) were used, demonstrating that after 10 min the polymerization of the materials allowed the material to reach an adequate resistance to be tested. for orthodontists, it is important to know the properties of resins used in bracket bonding procedures because the resin must have the ability to maintain orthodontic accessories firmly adhered to the teeth during the treatment, resisting to masticatory forces and the forces generated by orthodontic mechanics. as shown in this study, many materials of different properties and characteristics can be used for indirect bonding technique. however, light-cured resins were more effective. the indirect bonding technique was found to be simple, and with little ability, any professional can run it safely. this technique is very efficient, provides less wear to the orthodontist at the time of bonding, shortening the chairtime for the complete assembly of the appliance, and also offers the patient greater comfort. based on the results of this study, it could be concluded that light-cured adhesives showed the higher shear bond strength values compared with chemically cured adhesives; the storage period had no influence on shear bond strength values, and the failures were predominantly at the bracket/ resin interface, where most of the material staid on the enamel (scores 1, 2 and 3). references 1. sondhi a. efficient and effective indirect bonding. am j orthod dentofacial orthop. 1999; 115: 352-9. 2. silverman e, cohen m, gianelly aa, dietz vs. a universal direct bonding system for both metal and plastic brackets. am j orthod. 1972; 62: 236-44. 3. thomas rg. indirect bonding: simplicity in action. j clin orthod. 1979; 13: 93-105. 4. cinader dk, james ds. transbond tm supreme lv low viscosity light cure adhesive: suitable for indirect bonding. orthodontic perspectives. exploring treatment options. 2009; 16: 1. 5. cortesi r, molinari l. a simple and efficient procedure for indirect bonding. prog orthod. 2010; 11: 180-4. 6. hedge t, dattada h, jaiswal rk. an avant-garde indirect bonding technique for lingual orthodontics using the first complete digital “tad” (torque angulation device) and “bpd” (bracket positioning device). j indian orthod soc. 2010; 44: 9-16. 7. mezomo m, de lima em, de menezes lm, weissheimer a. indirect bonding with thermal glue and brackets with positioning jigs. prog orthod. 2011; 12: 180-5. 8. swetha m, pai vs, sanjay n, nandini s. indirect versus direct bonding a shear bond strength comparison: an in vitro study. j contemp dent pract. 2011; 12: 232-8. 9. dalessandri d, dalessandri m, bonetti s, visconti l, paganelli c. effectiveness of an indirect bonding technique in reducing plaque accumulation around braces. angle orthod. 2012; 82: 313-8. 10. zachrisson bu, brobakken bo. clinical comparison of direct versus indirect bonding with different bracket types and adhesives. am j orthod. 1978; 74: 62-78. 11. soo-byung p, woo-sung son, ching-chang ko, garcía-godoy f, migyoung p, hyung-ii kim, kwon yh. influence of flowable resins on the shear bond strength of orthodontic brackets. dent mater j. 2009; 28: 730-4. 12. uysal t, yagci a, uysal b, akdogan g. are nano-composites and nanoionomers suitable for orthodontic bracket bonding? eur j orthod. 2010; 32: 78-82. 13. albaladejo a, montero j, gómez de diego r, lópez-valverde a. effect of adhesive application prior to bracket bonding with flowable composites. angle orthod. 2011; 81: 716-20. 14. d’attilio m, traini t, di iorio d, varvara g, festa f, tecco s. shear bond strength, bond failure, and scanning electron microscopy analysis of a new flowable composite for orthodontic use. angle orthod. 2005; 75: 410-5. 15. salerno m, derchi g, thorat s, ceseracciu l, ruffilli r, barone ac. surface morphology and mechanical properties of new-generation flowable resin composites for dental restoration. dent mater. 2011; 27: 1221-8. 16. pick b, rosa v, azeredo tr, cruz filho ea, miranda wgjr. are flowable resin-based composites a reliable material for metal orthodontic bracket bonding? j contemp dent pract. 2010; 11: 17-24. 17. uysal t, sari z, demir a. are the flowable composites suitable for orthodontic bracket bonding? angle orthod. 2004; 74: 697-702. 18. mitra sb, wu d, holmes bn. an application of nanotechnology in advanced dental materials. j am dent assoc. 2003; 134: 382-90. 19. polat o, karaman a i, buyukyilmaz t. in vitro evaluation of shear bond strengths and in vivo analysis of bond survival of indirect bonding resins. angle orthod. 2004; 74: 405-9. 20. reynolds ir, von fraunhofer ja. direct bonding in orthodontics: a comparison of attachments. br j orthod. 1977; 4: 65-9. 21. eliades t. orthodontic materials research and applications: part 1. current status and projected future developments in bonding and adhesives. am j orthod dentofacial orthop. 2006; 130: 445-51. 22. lemke k, xu x, hagan jl, armbruster pc, ballard rw. bond strengths and debonding characteristics of two types of polycrystalline ceramic brackets. aust orthod j. 2010; 26: 134-40. 23. muguruma t, yasuda y, iijima m, kohda n, mizoguchi i. force and amount of resin composite paste used in direct and indirect bonding. angle orthod. 2010; 80: 1089-94. 24. hocevar ra, vincent hf. indirect versus direct bonding: bond and failure location. am j orthod dentofacial orthop. 1988; 94: 367-71. 25. bryant s, retief dh, russel cm, dennys fr. tensile bond strength of orthodontic bonding resins and attachments to etched enamel. am j orthod dentofacial orthop. 1987; 92: 225-31. 26. klocke a, shi j, vaziri f, bärbel kn, ulrich b. effect of time bond strength in indirect bonding. angle orthod. 2006; 76: 289-94. 27. correr sobrinho l, correr gm, consani s, sinhoreti ma, consani rl. [influence of post-fixation time on shear bond strength of brackets fixed with different materials]. pesqui odontol bras. 2002; 16: 43-9. 28. minick gt, oesterle lj, newman sm, shellhart wc. bracket bond strengths of new adhesive systems. am j orthod dentofacial orthop. 2009; 135: 771-6. braz j oral sci. 11(4):458-462 shear bond strength of nanofilled flowable resins used for indirect bracket bonding oral sciences n3 original article braz j oral sci. october/december 2009 volume 8, number 4 braz j oral sci. 8(4):166-170 mandibular behavior in the treatment of skeletal class ii malocclusion: a 5-year post-retention analysis ana de lourdes sá de lira1 ;antonio izquierdo1 ; sávio prado1 ; lincoln issamu nojima2 ; matilde nojima2 1dds, ms, doctorate student, department of orthodontics, dental school, federal university of rio de janeiro (ufrj), rio de janeiro, rj, brazil. 2dds, phd, professor, department of orthodontics, dental school, federal university of rio de janeiro (ufrj), rio de janeiro, rj, brazil. correspondence to: ana de lourdes sá de lira departmento de ortodontia, faculdade de odontologia, universidade federal do rio de janeiro av. brigadeiro trompowsky s/n ilha do fundão rio de janeiro rj cep: 21941-590 e-mail: anadelourdessl@hotmail.com abstract aim: this study aimed to assess mandibular behavior in class ii subjects subjected to full orthodontic treatment with standard edgewise appliance and cervical headgear (kloehn appliance) during the pubertal growth spurt period. methods: lateral cephalometric radiographs of 40 patients (21 females and 19 males) were performed at the beginning of the treatment ( t 0 ), at its end ( t 1 ) and at 5-year post-retention phase (t 2 ) in order to quantify the cephalometric measurements (8 angular and 3 linear), representing the mandibular behavior in the anteroposterior and vertical senses. the mean age of female patients at t 0 , t 1 and t 2 was 11.4, 15 and 26 years, respectively, and for male patients it was 12.2, 16.7 and 28 years, respectively. all patients were treated in just one phase without extractions and not associating class ii intermaxillary elastics. results: the effective treatment of skeletal class ii malocclusion with conventional edgewise fixed appliance and kloehn cervical headgear did not interfere in the direction and amount of mandibular growth as well as remodeling at it is inferior border, with no influence in anti-clockwise rotation of the mandible. the mandibular growth was also observed after the orthodontic treatment, suggesting that it is influenced by genetic factors. conclusion: these observations may lead to the speculation that growing patients with skeletal class ii malocclusion and low mandibular plane are conducive to a good treatment and long-term stability. keywords: skeletal class ii malocclusion, mandibular behavior, kloehn cervical headgear, post-retention introduction class ii malocclusion is an abnormal anteroposterior relationship between the dental arches in which mandible and mandibular arch are distally positioned in relation to maxilla and maxillary dentition1. the resulting convex profile involves maxillary protrusion, mandibular retrusion or combination of both2. magnitude and direction of craniofacial growth, particularly the mandibular growth, are factors influencing the treatment of class ii malocclusion. the capacity of foreseeing the mandibular displacement might help both planning and orthodontic treatment mechanics3. the child’s facial growth from year to year is not regular in terms of amount and direction, and the vertical growth components are crucial in the anteroposterior displacement of the mandible1. a successful treatment of class ii malocclusion in young people depends on the proper orthodontic mechanics, patient cooperation and how satisfactorily growth spurt occurs at the age ranges of 10-13 in girls and 11-14 years in boys4. during the normal craniofacial growth, the mandible suffers a translational movement when the condylar growth is the same like to the maxillary sutures and alveolar processes. on the other hand, a greater condylar growth will result in anterior displacement of the mandible2. ricketts5 reported that condylar growth towards antero-superior direction will increase the facial depth and the brachiocephalic pattern. however, a condylar growth towards a posteriorsuperior direction will result in an increase in the facial height with dolichocephalic trends. kloehn6 has suggested that class ii malocclusions should be treated with cervical traction received for publication: march 30, 2009 accepted: january 19, 2010 167 braz j oral sci. 8(4):166-170 during mixed dentition followed by fixed orthodontic appliance without tooth extractions because of mandibular alveolar processes and tooth shift forward during normal growth. if the mandible grows normally and the maxillary growth is restrained, it will be achieved a good relationship between the anatomical structures6. there are several negative effects of orthodontic treatment, as follow : decrease in anticlockwise rotation of the mandible and pogonion, increase in yaxis angle and mandibular plane, as well as increase in anterior facial height, probably due to excessive extrusion of upper molars7-8. however, these data are not corroborated by hubbard et al.9, who reported that one cannot presume that the negative effects of cervical traction will occur in whole class ii patients treated, because there are several variables involved in it, such as angulation of mandibular plane, techniques for using and adjusting the kloehn appliance, besides the patient age. bjork10 has shown that the increase in y-axis angle and mandibular plane is related to the fact that the lower border of the mandible is frequently remodeled, thus camouflaging its anti-clockwise rotation, which is directly associated to amount and direction of condylar growth5. patients with normal vertical facial proportion, with undergo orthodontic treatment during the growth spurt phase, have tendency to present more favorable results and long-term stability. the clockwise rotation of the mandible resulting from cervical traction therapy is transient in most growing individuals, returning to anti-clockwise rotation after treatment because of the residual growth11-13. the objective of the present study was to assess the changes in mandibular behavior on patients subjected to full orthodontic treatment with standard edgewise appliance and cervical traction headgear during the pubertal growth spurt period by analyzing the data obtained in the active phase of the orthodontic treatment and after at least 5 years of retention. material and methods the ufrj’s ethics committee approved the development of this study under the protocol number (caae 54/2009 – 0050.0.339.000/09). this clinical research was based on 40 brazilian caucasian individuals, 21 girls and 19 boys, who underwent full edgewise appliance and cervical-pull headgear treatment during 48 months in the postgraduate orthodontic program of the federal university of rio de janeiro. all patients were treated in just one phase without extractions and not associating class ii intermaxillary elastics. the cervical headgear was applied during 12 hours/day with an average force of 400 g, being used in a mean of 24 months during the pubertal growth spurt period. each patient was evaluated three times by lateral cephalometric radiographs: at the beginning of the treatment (t0), at the end of the active orthodontic treatment (t1), and after at least 5 years of retention (t2). all the subjects were in the pubertal growth spurt period at the beginning of the orthodontic treatment, with skeletal pattern of class ii evidenced by anb angle > 5o and wits >0 mm. the skeletal maturity stage of all individuals was analyzed in hand and wrist radiographs. th e d ental rel ationship was of cl ass ii, a cco rding t o ang l e’s classification. the individuals also exhibited sngogn angle < 35o. the age interval for female patients at t 0 was 10-13 years (mean = 11.4 years; sd = + 0.64); at t 1 was 12.9-17.6 years (mean = 15 years; sd = + 1.42), and at t 2 was 20.5-29.6 years (mean = 26 years; sd = + 3.91). the range interval for male patients at t 0 was 11.2-14 years (mean = 12.2 years; sd = + 0.9); at t 1 was 14.5-19.9 years (mean = 16.7 years; sd = + 2.12), and at t 2 was 20.5-29.6 years (mean = 28 years; sd = + 5.23). the cephalograms were obtained by delimitating skeletal, dental and tegumentary structures. the measurements from cephalometric tracings regarding t 0 , t 1 and t 2 were tabulated for statistical analysis, with angular measurements being rounded up whenever decimal fraction existed. changes in mandibular displacement were measured in relation to skull base by the following angles: snb, snd, sngogn, sngome, cdgogn, y-axis, facial angle, and fma (figure 1). the linear measurements were used to describe, separately, the mandibular components: cdgo (height of mandibular ramus); cdpog (total mandibular length) and gopog (mandibular body length) (figure 2). fig. 1. cephalogram illustrating angular measurements used in the study: snb, snd, sngogn, sngome, cdgogn, y-axis, facial angle and fma. fig. 2. cephalogram showing linear measurements (mm) used in the study: cdgo, cdpog, and gopog. mandibular behavior in the treatment of skeletal class ii malocclusion: a 5-year post-retention analysis 168 braz j oral sci. 8(4):166-170 the error of the method was evaluated by 30 radiographs chosen at random, traced and digitized by the same investigator on 2 separate occasions at least 2 months apart. the dahlberg formula was used: me =\/σd2/2n, where n is the number of duplicate measurements. random errors varied between 0.26 and 0.92mm for linear measurements and between 0.28o and 1.1o for angular measurements. means and standard deviations were calculated for each cephalometric measurement at t 0 , t 1 , and t 2 . the statistical treatment of the data between t 0 x t 1 as well as between t 1 x t 2 was analyzed using the paired student’s t-test with 5% significance level. results table 1 showed means and standard deviations for angular and linear measurements at t 0 , t 1 , and t 2 as well as the p values between t 0 x t 1 and t 1 x t 2 . tables 2 and 3 present data on female and male patients, respectively. figure 3 illustrates total superimposition at t 0 , t 1 , and t 2 for sn, while figure 4 represents the partial superimposition at t 0 , t 1 , and t 2 for ar. table 1. means and standard deviations for angular and linear measurements regarding the study group at the beginning of treatment (t 0 ), at the end of treatment (t 1 ), and at the 5-year post-retention period (t 2 ). table 2. means and standard deviations for angular and linear measurements regarding female patients at the beginning of treatment (t 0 ), at the end of treatment (t 1 ), and at 5-year the post-retention period (t 2 ). sd = standard deviation **= 1% significant level n.s= no significant discussion the skeletal changes resulting from facial growth, which occurs during the transition from deciduous to permanent dentition, do not correct the class ii malocclusion established at an earlier age. it probably happens due to the morphological characteristics of the class ii malocclusion, justifying a therapeutic intervention during growth spurt14-15. by assessing the mandibular behavior of the study group, it was observed that conventional edgewise fixed appliance and kloehn cervical headgear mechanics used for orthodontic treatment did not interfere with mandibular growth and displacement, since the mean values for snb angle had a statistically significant increase in the t 0 x t 1 interval. this demonstrated a favorable mandibular growth in relation to the skull base during the phase of active orthodontic treatment, which was confirmed by the expressive increase in snd angle. similar conditions were observed in the t 1 x t 2 interval regarding the mean snb and snd angles, which might be the result of residual mandibular growth after the active orthodontic treatment period (table 1)14. sd = standard deviation **= 1% significant level n.s= no significant snb (o) 76.25 ± 2.67 77.95±2.76 79.00 ± 2.84 < .001** < .001** snd (o) 73.25 ±2.67 74.90 ± 2.82 76.00 ± 2.86 < .001** < .001** sngogn (o) 31.85± 2.08 30.65 ± 2.00 29.60 ± 1.98 < .001** < .001** sngome (o) 32.90± 2.10 31.35 ± 2.32 30.30 ± 2.36 <.001** < .001** cdgogn (o) 124.25±5.48 121.45± 4.65 119.35 ± 4.71 < .001** < .001** eixo y (o) 58.25± 4.94 57.80 ± 4.28 57.95 ± 4.26 .20 n.s .18 n.s facial (o) 83.75± 3.91 85.50 ± 3.39 86.60 ± 3.39 < .001** < .001** fma (o) 26.05± 5.36 24.20 ± 4.42 23.20 ± 4.12 .001** < .001** cdgo (mm) 5.0 ± 0.41 5.67 ± 0.40 6.01 ± 0.49 < .001** < .001** cdpog(mm) 10.68± 0.56 11.66 ± 0.41 12.34 ± 0.36 < .001** < .001** gopog mm) 7.23 ± 0.47 7.89 ± 0.41 8.41 ± 0.39 < .001** < .001** p between t 0 x t 1 p between t 1 x t 2 t 2 mean sdt1mean sdt0mean sd snb (o) 76.82± 2.99 78.36 ± 3.13 79.55± 3.17 < .001** < .001** snd (o) 73.82± 2.85 75.18 ± 3.02 76.36± 3.07 < .001** <.001** sngogn (o) 31.45± 2.38 30.36 ± 2.37 29.35 ± 2.36 < .001** < .001** sngome (o) 32.82± 2.63 31.55± 2.62 30.82 ± 2.56 <.001** <.001** cdgogn (o) 123.18± 3.92 120.55 ±4.08 118.36±4.38 .004** .001** eixo y (o) 58.73± 3.60 58.18± 1.99 58.36± 2.11 .40 n.s .34 n.s facial (o) 83.27± 3.31 85.55± 2.33 86.73± 2.19 .004** .001** fma (o) 26.91± 4.01 24.36 ± 2.06 23.45 ± 2.20 .007** .005** cdgo (mm) 4.92± 0.42 5.53 ± 0.39 5.79 ± 0.38 <.001** .018** cdpog(mm) 10.6 ± 0.65 11.50± 0.43 12.10± 0.41 <.001** <.001** gopog(mm) 7.24 ± 0.48 7.83 ± 0.40 8.40 ± 0.37 < .001** <.001** p between t 0 x t 1 p between t 1 x t 2 t 2 mean sdt1mean sdt0mean sd mandibular behavior in the treatment of skeletal class ii malocclusion: a 5-year post-retention analysis 169 braz j oral sci. 8(4):166-170 with regard to the profile, it was found a mean reduction of the facial convexity in the time intervals, which was confirmed by a significant increase in the facial angle. this fact can be supported by the anterior positioning of the mandible during facial growth (tables 1-3; figures 3 and 4) as well as bone apposition in the region of pogonion5,16. the cephalometric evaluation showed a trend to the decrease of the angles related to the mandibular plane during growth due to the intrinsic morphogenetic characteristic of the studied cases17-18. all fig. 3. total superimposition of tracings for sn at t 0 ( ____ ), t 1 ( _ _ _ ), and t 2 ( _ . _ .). fig. 4. partial superimposition of tracings for ar at t 0 ( ____ ), t 1 ( _ _ _ ), and t 2 ( _ . _ .). patients subjected to orthodontic treatment presented low mandibular plane, which is crucial factor for using cervical traction as cited elsewhere2,5,16. the mean values for sngogn, sngome, cdgogn and fma angles showed a significant reduction in the time interval, suggesting that rotation of the mandible is governed by the direction and amount of condylar growth and remodeling at the inferior border of the mandible (table 1, figures 3 and 4)14,18-19. according to the structural analysis established by björk3 the mandibular rotation depends on the morphogenetic pattern, that is determined by mandible’s morpholog y. the vertical growth of mandibular condyles should be greater than that of posterior alveolar processes, being an important factor in the anticlockwise rotation of the mandible20. nevertheless, the changes observed in the y-axis angle revealed the harmonic pattern of facial growth in male and female patients during orthodontic treatment and post-retention phases (tables 2 and 3)11,21-22. analysis of the linear measurements cdgo, cdpog, and gopog (table 1) showed a significant increase in t 0 x t 1 and t 1 x t 2 intervals. these data also suggest that mandibular growth occurs during the active orthodontic treatment as well as post-retention period, including an increase in both mandibular ramus and body. according to the literature, the mandibular growth is more prominent than maxilla table 3. means and standard deviations for angular and linear measurements regarding male patients at the beginning of treatment (t 0 ), at the end of treatment (t 1 ), and at the 5-year post-retention period (t 2 ). sd = standard deviation *= 5% significant level **= 1% significant level n.s= no significant snb (o) 75.56± 2.18 77.44 ± 2.29 78.45± 2.27 <.001** <.001** snd (o) 72.56± 2.40 74.56 ± 2.69 75.57 ± 2.65 <.001** < .001** sngogn (o) 32.33± 1.65 31.00 ± 1.50 29.89± 1.45 <.001** .001** sngome (o) 33.0 ± 1.32 31.11 ± 2.02 29.67± 2.06 .020* .001** cdgogn (o) 125.56±6.98 122.56± 5.29 120.56 ±5.07 .012** .002** eixo y (o) 57.67± 6.40 57.33 ± 6.18 57.44± 6.08 .081 n.s .347 n.s facial (o) 84.33± 4.69 85.44 ± 4.50 86.44± 4.61 < .001** < .001** fma (o) 25.0 ± 6.78 24.0 ± 6.40 22.89± 5.84 < .001** <.001** cdgo(mm) 5.1 ± 0.41 5.84 ± 0.37 6.28 ± 0.46 <.001** .002** cdpog mm) 10.78± 0.44 11.85 ± 0.32 12.50± 0.20 <.001** <.001** gopog(mm) 7.21± 0.48 7.95 ± 0.44 8.41 ± 0.45 .001** < .001** p between t 0 x t 1 p between t 1 x t 2 t 2 mean sdt1mean sdt0mean sd mandibular behavior in the treatment of skeletal class ii malocclusion: a 5-year post-retention analysis 170 braz j oral sci. 8(4):166-170 growth, continuing for an additional period of time11,21,23. when the mandibular displacement was evaluated separately for males and females, it was observed that the mean values for snb, snd and facial angles were significantly increased in both genders between t 0 x t 1 and between t 1 x t 2 , thus supporting the genetic influence on mandibular growth and displacement (tables 2 and 3)5,16-18. amount and direction of mandibular growth are genetically determined. the lower border of the mandible influences the mandibular plane angle because of its bone remodeling (tables 2 and 3)10. the mean values for sngogn, sngome, cdgogn, and fma angles were reduced in both genders patients between t 0 x t 1 , thus demonstrating favorable anticlockwise rotation. it was confirmed by the significant reduction in cdgogn and fma angles2,4. between t 1 x t 2 , all the angular measurements cited above were found to be significantly decreased for all patients, thus suggesting that both growth and displacement of the mandible are determined by genetic factors (tables 2 and 3)3,10. by analyzing the mean values regarding linear measurements cdgo, cdpog, and gopog (tables 2 and 3), it was found a significant increase in both time intervals for both genders. this emphasized the mandibular growth observed during and after the active orthodontic treatment phase. similar results were also found by other authors, who reported a residual mandibular growth11,21,23. bone apposition in the region of pogonion occurs continuously even after active treatment has finished16-18. full corrective orthodontic treatment, using standard edgewise technique and cervical headgear (kloehn appliance), was considered effective in patients with skeletal class ii malocclusions and low mandibular plane. the treatment did not interfere on mandibular growth, which happened during the active treatment as well as it had finished. these observations are in agreement to the tendency that growing patients with skeletal class ii malocclusion and low mandibular plane are conducive to better results of orthodontic treatment and long-term stability. full corrective orthodontic treatment using standard edgewise technique and cervical headgear (kloehn appliance) was considered effective in patients with skeletal class ii malocclusions and low mandibular plane. the treatment did not interfere on the mandibular growth, which happened during the active treatment as well as it had finished. these observations may lead to the speculation that growing patients with skeletal class ii malocclusion and low mandibular plane are conducive to better results of orthodontic treatment and long-term stability. references 1. angle e. classification of malocclusion. dental cosmos. 1899; 41: 248-64. 2. isaacson rj, zapfel rj, worms fw, erdman ag. effects of rotational jaw growth on the occlusion and profile. am j orthod. 1977; 72: 276-86. 3. bjork a. prediction of mandibular growth rotation. am j orthod. 1969; 55: 585-99. 4. evans c. anteroposterior skeletal change: growth modification. semin orthod. 2000; 6: 21-32. 5. ricketts rm. planning treatment on the basis of the facial pattern and an estimate of its growth. angle orthod. 1957; 27: 14-37. 6. kloehn s. evaluation of cervical anchorage force in treatment. angle orthod. 1961; 31: 91-104. 7. baumrind s, molthen r, west ee, miller dm. mandibular plane changes during maxillary retraction. am j orthod. 1978; 74: 32-40. 8. derringer k. a cephalometric study to compare the effects of cervical traction and andresen therapy in the treatment of class ii division 1 malocclusion. part 1-skeletal changes. br j orthod. 1990; 17: 33-46. 9. hubbard gw nrs, currier g f. a cephalometric evaluation of non extraction cervical headgear treatment in class ii malocclusion. angle orthod. 1994; 64: 359-70. 10. bjork a. variations in the growth pattern of the human mandible: longitudinal radiographic study by the implant method. j dent res. 1963; 42: 400-11. 11. fidler bc, artun j, joondeph dr, little rm. long-term stability of angle class ii, division 1 malocclusions with successful occlusal results at end of active treatment. am j orthod dentofacial orthop. 1995; 107: 276-85. 12. melsen b. effects of cervical anchorage during and after treatment: an implant study. am j orthod. 1978; 73: 526-40. 13. schudy f. post treatment craniofacial growth: its implication in orthodontic treatment. am j orthod. 1974; 65: 39-57. 14. you zh, fishman ls, rosenblum re, subtelny jd. dentoalveolar changes related to mandibular forward growth in untreated class ii persons. am j orthod dentofacial orthop. 2001; 120: 598-607; quiz 676. 15. solow b. the dentoalveolar compensatory mechanism: background and clinical implications. br j orthod. 1980; 7: 145-61. 16. odegaard j. growth of the mandible studied with the aid of metal implant. am j orthod. 1970; 57: 145-57. 17. ngan pw, byczek e, scheick j. longitudinal evaluation of growth changes in class ii division 1 subjects. semin orthod. 1997; 3: 222-31. 18. carter ne. dentofacial changes in untreated class ii division 1 subjects. br j orthod. 1987; 14: 225-34. 19. kim j, nielsen la. a longitudinal study of condilar growth and mandibular rotation in untreated subjects with class ii malocclusions. angle orthod. 2002; 72: 105-11. 20. klocke a, nanda rs, kahl-nieke b. skeletal class ii paterns in the primary dentition. am j orthod. 2002; 121: 596-601. 21. karlsen a, krogstad o. morphology and growth in convex profile facial patterns: a longitudinal study. angle orthod. 1999; 69: 334-44. 22. kirjavainen m, hurmerinta k, kirjavainen t. facial profile changes in early class ii correction with cervical headgear. angle orthod. 2007; 77: 960-7. 23. sinclair pm, little rm. dentofacial maturation of untreated normals. am j orthod. 1985; 88: 146-56. mandibular behavior in the treatment of skeletal class ii malocclusion: a 5-year post-retention analysis oral sciences n3 braz j oral sci. 10(4):272-276 original article braz j oral sci. october | december 2011 volume 10, number 4 influence of calcium hydroxide on marginal leakage of endodontically treated teeth maria antonieta veloso carvalho de oliveira1, sara teodoro marra2, paulo dos santos batista3, joão carlos gabrielli biffi4 1ds, undergraduate student, dental school, federal university of uberlândia, brazil 2dds, uberlândia, mg, brazil 3dds, ms, phd, institute of chemistry, laboratory of photochemistry, federal university of uberlândia, brazil 4dds, ms, phd, professor of endodontics, dental school, federal university of uberlândia, brazil correspondence to: maria antonieta veloso carvalho de oliveira departamento de endodontia, faculdade de odontologia, universidade federal de uberlândia av. pará, 1720, bloco 2bs/25 umuarama 38.405-382 uberlândia, mg – brasil phone: +55-34-3232-2466 e-mail: antocassia@hotmail.com abstract aim: to evaluate the influence of residual calcium hydroxide (ch) intracanal medication considering two dye leakage locations (apical foramen and middle root canal third) by quantifying the diffusion of india ink in length and depth. methods: after biomechanical preparation, 72 single-rooted bovine teeth were divided into two groups in which half was filled with a ch and saline paste. after 7 days, the medication was removed and the canals in both groups were filled. half of the samples had the apical third and part of middle third removed for infiltration of india ink at the middle third (mti), while the rest were infiltrated at the apical foramen (ai). the following experimental groups were formed (n = 18 each): a1 – ch-medicated + ai, a2 – ch-medicated + mti, b1 – non-medicated + ai, b2 – non-medicated + mti. for evaluation of dye leakage, the experimental specimens were cross-sectioned, photographed and had the infiltration perimeter measured using the image tool 3.00 software. the mann-whitney u-test was used for statistical analysis (p<0.05). results: the lowest values of dye penetration in length and depth were found in the groups receiving intracanal medication (a1 and a2). conclusions: lower dye penetration in length and depth at the two different locations was observed in the canals with residual ch intracanal medication. keywords: dental leakage, calcium hydroxide, root canal obturation. introduction the use of the medication for root canal system disinfection has been supported to improve the treatment outcome1-2, as the complexity of the root anatomy makes more difficult their cleaning and shaping3. intracanal medications such as calcium hydroxide (ch) are used to reduce or eliminate bacteria located in the root canal system and prevent their proliferation between sessions1-3. regardless of the vehicle of the paste, the instrument or the solution used, ch dressing is not completely removed from the root canal, especially in the apical area1-2,4,6-14. attempts to remove ch remnants have been made using irrigating solutions such as saline solution5,7, sodium hypochlorite1,3,5-6,8-14, edta1,6,10,12,14, acid citrus10, phosphoric acid8 and chlorhexidine6, alone and/or combined with manual1,3-4,6,8-9,12-13 and rotary instruments14 or ultrasound4,11-12. received for publication: september 15, 2011 accepted: november 30, 2011 braz j oral sci. 10(4):272-276 273 the remnants of ch within the radicular dentinal tubules may interfere on the properties of endodontic sealers, especially their sealing ability 1,3,6,8-10,12-21. typically, the sealing ability of filling materials is evaluated by leakage assays because they are easy to perform and do not require sophisticated materials1,3,6,8,12-13,21. however, leakage studies show a variety of assessment methods and parameters, which may be the main reason for the different results achieved with these tests1,6,8,22. early leakage studies that evaluated the effect of ch dressing on apical seal concluded that their presence reduced the infiltration and determined a significant improvement in the quality of marginal sealing of root canal filling17,19-20. all these studies checked the infiltration of teeth after filling using methylene blue, which loses its color when in contact with some filling materials, such as ch23-24. for this reason, when the studies used the fluid transport model or rhodamine b to quantify the infiltration of teeth with or without previous use of ch medication showed no statistically significant differences among groups1,23-24. when india ink was employed, the highest values of infiltration were found in the groups receiving the ch previous to root canal filling3,8. to date, no study has evaluated both the length and the depth of leakage after the use of ch in samples with and without the apical root canal third. evaluation of the length and depth of leakage allows a three-dimensional analysis of dye penetration. removal of the apical dye that can be accumulated during infiltration eliminates a variable that can affect leakage measurement within the root canal20,22. therefore, it seemed important to conduct an in vitro leakage study to evaluate whether the presence of the remaining ch interferes with the leakage of endodontically treated teeth. two different locations of dye leakage, apical foramen and middle root canal third, were used to quantify the diffusion of india ink in length and depth. the tested hypothesis was that the ch remnants in the root canal walls could favor dye penetration. material and methods seventy-eight bovine teeth of adult animals with a single straight root, apex with narrow anatomic diameter compatible with a size 40 k-file and foraminal opening coinciding with the end of the apex were selected for the study. tooth crowns were removed by standardizing the length of the roots to 20.0 mm. all teeth were prepared, maintaining the patency of the foramen with a size 15 k-file and irrigating the canal with 2.0 ml of 1% sodium hypochlorite (farma, serrana, sp, brazil) between each file throughout instrumentation. thereafter, the root canals were dried with paper points (tanari, manacapuru, am, brazil) and two experimental groups of 36 teeth each were formed (a and b). the other 6 teeth were used as the control group. only group a received a paste of pure ch powder (biodinâmica, ibiporã, pr, brazil) mixed with normal saline solution (ariston, são paulo, sp, brazil) at a powder to liquid ratio of 1:1.5. the medication paste was taken to the root canals with the aid of a plastic syringe with 27-gauge needle (endo eze; ultradent products inc., south jordan, ut, usa) introduced to the full working length (19.0 mm). a radiograph was taken to confirm the complete filling of the canal. temporary sealing of the cervical region was performed with zinc oxide-eugenol sealer (irm; dentsply ind. e com. ltda., petrópolis, rj, brazil.), and thereafter the specimens were stored in 100% relative humidity at 37ºc for 7 days. thereafter, the temporary dressing was removed with copious normal saline solution combined mild filing with memory instrument at the working length. irrigation was performed until the reflux of irrigating solution was clear and the root canals underwent aspiration and drying with paper points. the canals in groups a and b were filled following the lateral condensation technique with zinc oxide-eugenol sealer (endofill, dentisply, petrópolis, rj, brazil), dispensed and handled according to the manufacturer’s instructions. after placement of the temporary coronal restoration, the roots were maintained in 100% relative humidity at 37ºc for 72 h. for the marginal leakage evaluation at two different root locations, via the apical foramen and at middle third of the root canal, half of specimens from each group a and b (n = 18) was sectioned at the middle third of the root in a precision cutting machine (buehler ltd., lake bluff, il, usa) (figure 1a). then, groups were divided according to the local of dye infiltration at the root: groups a1 and b1 (n = 18 each) for apical leakage, groups a2 and b2 (n = 18 each) for middle third leakage (figure 2). the teeth in the four groups (a1, a2, b1 and b2) were coated before the dye infiltration with two layers of nail polish (colorama, são paulo, sp, brazil) (figure 1b and 1c). specimens that had been prepared, but received no intracanal medication or filling (n = 6) served as control of the effectiveness of coated with nail polish and dye leakage. half (n = 3) was completely coated with polish even in the apical third (negative control group) and half received no coated (positive control group). all specimens were subjected to vacuum for 10 min. after complete immersion of the roots, horizontally, in indian ink (faber-castell, são carlos, sp, brazil) were subjected fig. 1a. root cross-section, b. nail polish coating to apical leakage (groups a1 and b1), and c. nail polish coating to the middle canal third leakage (groups a2 and b2). influence of calcium hydroxide on marginal leakage of endodontically treated teeth 274 braz j oral sci. 10(4):272-276 fig. 3quantification of dye infiltrated perimeter. fig. 2experimental groups: a – calcium hydroxide-medicated group (chm), b – non-medicated group (nm), a1 and b1 – apical leakage groups (ai), a2 and b2 middle third leakage groups (mti). again to vacuum for 10 min. after 72 h in 100% relative humidity at 37ºc, the roots were washed; polish coating had removed and the specimens were included in self-curing resin (am 190, aerojet, são paulo, sp, brazil) to form blocks. the resin blocks glued to acrylic plates were attached to the precision cutting machine to obtain cross-sections (1.0 to 1.5 mm) from the root apex, using double-sided diamond disk (buehler ltd., lake bluff, il, usa). the number of sections obtained in each subgroup was determined by the presence of dye leakage in root canal walls, viewed with a stereoscopic microscope. photographs of all cross-sections were obtained with a digital camera (nikon d60, tokyo, japan) to quantify dye leakage (figure 3) in the uthscsa image tool 3.0 software (the university texas health science center, san antonio, tx, usa). the mann-whitney u-test25 was used for statistical analysis. the infiltrated perimeters of the first four sections of each sample were used to estimate the length of dye leakage. the total number of sections of each sample that showed the presence of indian ink was used to estimate the depth of infiltration. the significance level was set at 5%. results the mean percentages of dye infiltration in length (sections 1, 2, 3 and 4) and depth of the experimental groups are presented in table 1. comparison of the results of dye leakage among the groups is presented in tables 2 and 3. the specimens subjected to apical leakage (a1 and b1) showed statistically significant differences (p<0.05) among all comparisons of length and depth, with the highest values in the non-medicated group (b1). the groups subjected to middle third leakage (a2 and b2) showed statistically significant differences (p<0.05) between the sections 2, 3 and 4 and in depth measurements, and the highest values were found in the non-medicated group (b2). the negative control demonstrated no dye penetration, whilst the positive control showed leakage of the dye along the length and depth of the root canal. groups section 1 section 2 section 3 section 4 depth a1 51.81 24.61 15.74 9.07 26.04 a2 19.68 3.13 0 0 7.22 b1 55.10 57.69 60.32 56.67 93.05 b2 55.48 33.67 28.96 23.88 78.33 table 1 means of dye infiltration (%) in the experimental groups. variables probabilities section 1 0.001* section 2 0.000* section 3 0.002* section 4 0.008* depth 0.000* table 2 comparison of groups a1 and b1. (*) p < 0.05 table 3 – comparison of groups a2 and b2. (*) p < 0.05. variables analyzed probabilities section 1 0.819 section 2 0.000* section 3 0.000* section 4 0.000* depth 0.011* discussion the results of the present study showed that the ch remnants influenced on leakage unlike we expected in our influence of calcium hydroxide on marginal leakage of endodontically treated teeth 275 braz j oral sci. 10(4):272-276 initial hypothesis, as less infiltration occurred in the presence of residual medication. this result has been described by çaliskan, türkün and türkün21 (1998). other studies using india ink leakage with the same goal showed contrary results to those of the present study3,8. in the studies of kim and kim3 (2002) and contardo et al.8 (2007), the highest values of dye leakage were found in the groups that received the ch prior to filling. according to those authors, the presence of ch residues was not the only factor in reducing apical leakage, since the type of sealer used is also important20. by analyzing the steps of these methodologies, one can justify the confronting results due to differences in sample preparation for infiltration and/or quantification of this infiltration. kim and kim3 (2002) observed the india ink infiltration through cross sections by counting the number of samples with dye infiltration. contardo et al.8 (2007), cleared the samples allowing visualization of the infiltration of india ink only in depth. in the present study, the use of cross sections allowed a three-dimensional analysis of dye penetration not only in depth, but also along the whole root length. dye penetration was measured in millimeters along the root canal using image tool 3.00 software. furthermore, the use of specimens without the apical third in groups a2 and b2 provided an assessment of dye penetration without the variation of the apical delta area20,22. it is noteworthy that the same results as the values of infiltration were found both in specimens with total length (20.0 mm) and root sections (6.0 mm). the results of this study cannot be extrapolated to the clinical situation because of the complex interrelationship between the multiple factors affecting endodontic treatment, which makes it impossible to study the correlation between in vitro dye leakage and in vivo treatment failures 22. however, the ch remnants in the root canal found in our results may represent a problem for the prognosis of endodontic therapy. ch, in the long term, can affect treatment prognosis due to leakage or to the presence the apical area resorption. the ch reacts with tissue fluids to form calcium carbonate, which is resorbable and can create space-filling in the interface wall dentin over time5,26. in addition, the properties of endodontic sealers can be affected by the presence of calcium hydroxide. several in vitro studies have proven that the presence of ch residues within the dentinal tubules reduces the bond strength of different sealers zinc oxide and eugenol based4 and resin based6,13,15. it also reduces the apical sealing ability of zinc oxide and eugenol3, glass ionomer7 and silicone-based sealers8, and prevents the penetration of sealer into the dentinal tubules9-10 and lateral canals16. ch reacts chemically with the sealer, resulting in reduced flow capacity, shorter working and setting times, and increased sealer film thickness9. the reduction in flow and working time results in an inability to work effectively with a material, increasing the chances of creating voids1,3,9, while the thicker sealer film is believed to make sufficient condensation of the root canal filling difficult to achieve9. however, there are studies in which incomplete removal of ch medication did not affect the adhesion or sealing ability of root canal sealers1,12 or resulted in an improvement in this ability17-21, suggesting that the effect of ch on the apical seal of the root canal filling depends on the type of sealer used12,21. new studies are required in order to develop vehicles for ch or auxiliary substances that chemically interact with this medication and permit its complete removal from the root canal system. under the tested conditions, it may be concluded that the remnants of ch intracanal medication led to lower dye penetration in length and depth at the two different locations evaluated in the study. references 1. kontakiotis eg, tsatsoulis in, papanakou si, tzanetakis gn. effect of 2% chlorhexidine gel mixed with calcium hydroxide as an intracanal medication on sealing ability of permanent root canal filling: a 6-month follow-up. j endod. 2008; 34: 866-70. 2. mohammadi z, dummer pmh. properties and applications of calcium hydroxide in endodontics and dental traumatology. int endod j. 2011; 44: 697-730. 3. kim sk, kim yo. influence of calcium hydroxide intracanal medication on apical seal. int endod j. 2002; 35: 623-8. 4. balvedi rpa, versiani ma, manna ff, biffi jcg. a comparison of two techniques for the removal of calcium hydroxide from root canals. int endod j. 2010; 43: 763-8. 5. kwon ty, fujishima t, imai y. ft-raman spectroscopy of calcium hydroxide medicament in root canals. int endod j. 2004; 37: 489-93. 6. böttcher de, hirai vhg, da silva neto ux, grecca fs. effect of calcium hydroxide dressing on the long-term sealing ability of two different endodontic sealers: an in vitro study. oral surg oral med oral pathol oral radiol endod. 2010; 110: 386-9. 7. chung a, titley k, torneck cd, lawrence hp, friedman s. adhesion of glass-ionomer cement sealers to bovine dentin conditioned with intracanal medications. j endod. 2001; 27: 85-8. 8. contardo l, de luca m, bevilacqua l, breschi l, di lenarda r. influence of calcium hydroxide debris on the quality of endodontic apical seal. min stomatol. 2007; 56: 509-17. 9. hosoya n, kurayama h, iino f, arai t. effects of calcium hydroxide on physical and sealing properties of canal sealers. int endod j. 2004; 37: 178-84. 10. rödig t, vogel s, zapf a, hülsmann m. efficacy of different irrigants in the removal of calcium hydroxide from root canals. int endod j. 2010; 43: 519-27. 11. van der sluis lw, wu mk, wesselink pr. the evaluation of removal of calcium hydroxide paste from an artificial standardized groove in the apical root canal using different irrigation methodologies. int endod j. 2007; 40: 52-7. 12. wang cs, debelian gj, teixeira fb. effect of intracanal medicament on the sealing ability of root canals filled with resilon. j endod. 2006; 32: 532-6. 13. wuerch rmw, apicella mj, mines p, yancich pj, pashley dh. effect of 2% chlorhexidine gel as an intracanal medication on the apical seal of the root-canal system. j endod. 2004; 30: 788-91. 14. kuga mc, tanomaru-filho m, faria g, só mvr, galletti t, bavello jrs. calcium hydroxide intracanal dressing removal with different rotary instruments and irrigating solutions: a scanning electron microscopy study. braz dent j. 2010; 21: 310-4. 15. barbizam jvb, trope m, teixeira ecn, tanumaru-filho m, teixeira fb. effect of calcium hydroxide intracanal dressing on the bond strength of a resin-based endodontic sealer. braz dent j. 2008; 19: 224-7. influence of calcium hydroxide on marginal leakage of endodontically treated teeth braz j oral sci. 10(4):272-276 16. goldberg f, artaza lp, de silvio ac. influence of calcium hydroxide dressing on the obturation of simulated lateral canals. j endod. 2002; 28: 99-101. 17. holland r, murata ss, saliba o. efeito a curto e médio prazos dos resíduos de hidróxido de cálcio na qualidade do selamento marginal após a obturação do canal. rev paul odontol. 1995; 17: 12-5. 18. holland r, murata ss, dezan jr e, garlipp o. apical leakage after root canal filling with an experimental calcium hydroxide gutta-percha point. j endod. 1996; 22: 71-3. 19. moraes ig, nunes e, berbert a, duarte mah, betti lv. influência do hidróxido de cálcio e do edta na marcação do azul de metileno em obturação de canais radiculares. rev fob. 2000; 8: 37-44. 20. porkaew p, retief dh, barfield rd, lacefield wr, soong sj. effects of calcium hydroxide paste as an intracanal medicament on apical seal. j endod. 1990; 16: 369-74. 21. çaliskan mk, türkün l, türkün ls. effect of calcium hydroxide as an intracanal dressing on apical leakage. int endod j. 1998; 31: 173-7. 22. plotino g, grande nm, manzulli n, chiaradia g, la torre, somma f. influence of reduced air pressure methods on dye penetration in standardized voids. oral surg oral med oral pathol oral radiol endod. 2007; 103: 289-94. 23. moraes ig, moraes fg, mori gg, gonçalves sb. influence of calcium hydroxide on dyes of dentin labeling analyzed by means of a new methodology. j appl oral sci. 2005; 13: 218-21. 24. wu m-k, kontakiotis eg, wesselink pr. descoloration of 1% methylene blue solution in contact with dental filling materials. j dent. 1998; 26: 585-9. 25. siegel s. estatística não-paramétrica, para as ciências do comportamento. trad. alfredo alves de farias. são paulo: mcgraw-hill do brasil; 2008. 150p. 26. tronstad l, barnett f, flax m. solubility and biocompatibility of calcium hydroxide containing root canal sealers. endod dent traumatol 1988; 4: 152-9. 276 influence of calcium hydroxide on marginal leakage of endodontically treated teeth oral sciences n3 braz j oral sci. 10(4):236-240 original article braz j oral sci. october | december 2011 volume 10, number 4 gunshot injuries in the maxillofacial region: a retrospective analysis and management leandro lauriti1, sandra kalil bussadori2, kristianne porta santos fernandes3, manoela domingues martins4, raquel agnelli mesquita-ferrari4, joão gualberto de cerqueira luz5 1master’s degree student in rehabilitation sciences; professor, surgery sector, nove de julho university, brazil 2 phd professor, master’s degree program in rehabilitation sciences, nove de julho university, brazil 3phd in immunology; professor, master’s degree program in rehabilitation sciences, nove de julho university, brazil 4professor, master’s degree program in rehabilitation sciences, nove de julho university, brazil 5professor, surgery sector, university of são paulo, brazil. received for publication: april 11, 2011 accepted: october 11, 2011 correspondence to: leandro lauriti rua waldemar martins, 116, apto 57 parque peruche cep: 02535-000, são paulo, sp, brasil e-mail: leandrolauriti@ig.com.br abstract aim: to analyze gunshot wounds to the face, assessing the characteristics, immediate treatment, late treatment, complications and after effects. methods: a retrospective observational study was carried out involving 75 cases of victims of gunshot wounds to the face treated at the oral and maxillofacial traumatology unit of the dr. arthur ribeiro de saboya hospital in the city of são paulo (brazil).data analysis used the chi-square test with the level of significance set at 5% (p dd 0.05). results: there was a predominance of the 21-to-30-year-old age group (38.7%), male gender (92%) and wounds occurring due to assaults (37.3%). there was a predominance of entry wounds on the left side of the face (58.5%). the most affected sites were the mandible (50.7%), maxilla (18.3%), zygomatic region (7.0%), eye socket (4.2%) and nose (1.4%). comminuted fractures (88.2%) and simple fractures (10.3%) were recorded. no fracture occurred in 1.5% of the cases. the predominant treatment was rigid internal fixation (rif) (57.2%), followed by exploratory surgery (23.2%) and conservative treatment (19.6%). among the cases in which the rif system was used, there was predominance in the mandible (64.0%). the chi-square test revealed a significant correlation between the 2.4-mm rif system and the mandible in 48.0 % of cases. conclusions: in conclusion, gunshot wounds tended to pierce the face, mainly affecting the mandible and caused comminuted fractures treated with rigid internal fixation. immediate complications occurred in 25% of cases and after effects occurred in 11.7%. keywords: gunshot wounds, comminuted fractures, internal fixation of fractures. introduction gunshot wounds result from the transmission of kinetic energy from the bullet to the tissue with which it collides, with greater projectile speed leading to greater damage1-3. the initial wound depends on the impact of the bullet, with the occurrence of an air pressure wave within two milliseconds that distends the tissue, forming a temporary spindle-like pulsating cavity fourfold larger than the bullet3-4. the pulsation of the temporary cavity aspirates bacteria from the skin to its interior, characterizing an additional source of infection5. to penetrate the skin, the bullet needs to be traveling at a velocity of 50 to 70 m/s, which causes braz j oral sci. 10(4):236-240 237 reason age group aggression assault homicide suicide total 0-20 years 4(5.3%) 4(5.3%) 2(2.7%) 0(.0%) 10(13.3%) 21-30 years 7(9.3%) 13(17.3%) 9(12.0%) 0(.0%) 29(38.7%) 31-40 years 9(12.0%) 4(5.3%) 5(6.7%) 1(1.3%) 19(25.3%) 41-60 years 5(6.6%) 7(9.3%) 4(5.3%) 1(1.3%) 17(22.7%) total(n) 25(33.3%) 28(37.3%) 20(7%) 2(2.7%) 75(100.0%) table 1 distribution of patients with gunshot wounds to the face according to age group and reason. abrasion to the dermis and epidermis. axon degeneration occurs in the nerve tissue, giving rise to anesthesia, paresthesia and paralysis. the rigid bone is fragmented; fracture velocity of the bullet is 65 m/s2-4,6-7. a study on ballistic impacts in the face established greater resistance to impacts to the frontal region (6.0 kn), whereas the mandible and zygomatic region are considerably more fragile (1.9 kn and 1.6 kn, respectively)8. knowledge on factors such as impact velocity, release rate of kinetic energy, retardant effect, bullet design, bullet mass, type of weapon and ballistic coefficient is essential to proper treatment6,9. clinically, patients having suffered gunshot injuries may exhibit signs of shock, neurological impairment, rapidly expanding hematoma and obstruction of the airways. thus, immediate priority treatment is the control of bleeding and unblocking the airways 2,10. in the face, tooth and bone fragments act as secondary projectiles, causing damage far from the original entry wound, which is difficult to diagnose11. analysis with both profile and anterior-posterior radiographs allows the adequate localization of projectiles11. therapeutic conduct regarding facial damage is based on the analysis of the projectile and the treatment of bone factures. the removal of the projectile is only indicated in cases of pain, functional limitation and signs of migration5,11-12. the treatment of comminuted fractures of the mandible by projectiles was once based on the use of kirschner wire, which was believed to be viable treatment to avoid the displacement of the periosteum 5. rigid internal fixation (rif) in comminuted fractures common in gunshot injuries is currently the conduct of choice, as it returns form and function with minimal complications and leads to the revasculation of the comminuted segments. locking plates are the most indicated for comminuted fractures in the mandible due to the locking of the screw in the plate, which allows a space between the bone surface and plate, maintaining the perimeter of the arch13-15. the bone reconstruction period ranges from three to 12 months following the revitalization of the injured tissues and maturation of the scars2,5,9. the decisive factors for successful bone reconstruction are the severity of the injury, destruction of the soft tissue and degree of bone fragmentation. the donor area is determined by the degree of bone loss and age group; there is a preference for autogenous grafts from the iliac crest or ribs (costochondral) and free grafts from the fibula2,16-17. costochondral grafts in the zone of traction and torsion forces, as in the symphysis, are contraindicated, as are thick grafts with little irrigation17-18. in cases of mandibular loss with the proximal and distal stump stabilized by the plate, the iliac crest is used. for young patients with loss of the distal stump, including the temporomandibular joint, a costochondral graft is indicated, whereas a free fibula graft is indicated for this type of loss in adults17. with the increasing violence in urban centers, the number of victims of gunshot wounds has been on the rise. however, few studies have analyzed the characteristics and treatment conduct involved in such injuries. thus, the aim of the present study was to retrospectively evaluate cases of gunshot wounds to the face in order to understand the clinical characteristics, treatment and complications in a brazilian population. materials and methods this study received approval from the local ethics committee under process nº 010/06. a retrospective observational study was carried out involving 75 cases of victims of gunshot wounds to the face treated at the oral and maxillofacial traumatology unit of the dr. arthur ribeiro de saboya hospital in the city of são paulo (brazil) over an 8-year period. information was collected from the charts of each patient, including demographic data, reason for the gunshot wound (aggression, assault, attempted homicide, attempted suicide), entry and exit wounds, clinical aspects and fracture site, considering three large areas of the face: zygomatic region, maxilla and mandible. gunshot wounds in the mandible were subdivided into two groups: condyle, ramus or coronoid process (crc) fractures and angle, body or symphysis (abs) fractures. data were also collected on the type of fracture, immediate conduct (such as procedures for the permeabilization of the upper airways, control of bleeding, suturing, debridement and surgical cleaning) and further conduct (conservative treatment, exploratory surgery and rif) as well as complications and after effects. data analysis involved the chi-square test, with the level of significance set at 5% (p<0.05). results in the present sample, there was a predominance of the 21-to-30-year age group (38.7%) and the male gender (92%). the gunshot wounds were caused mainly by assaults (37.3%), followed by aggression (33.3%), attempted homicide (26.7%) and attempted suicide (2.7%) (table 1). there was a predominance of entry wounds on the left side (58.5%) and exit wounds on the right side (53.7%). the most common clinical signs were pain, edema and trismus. with mandible fractures, paresthesia, increased salivation, malocclusion, bone exposure, deviation of mouth opening and premature contact were noted. when the zygomatic region was affected, otorrhagia, epistaxis, diplopia and paresthesia were noted. in maxillary fractures, oroantral and oronasal communication predominated. of the 75 patients, 4 died and analyzed data from only gunshot injuries in the maxillofacial region: a retrospective analysis and management 238 braz j oral sci. 10(4):236-240 fracture site gender(n=71) no fracture mandible maxilla zygom. region socket nose associated site total female 0(0%) 2(2.8%) 2(2.8%) 0(.0%) 0(.0%) 0(.0%) 2(2.8%) 6(8.5%) male 4(5.6%) 34(47.9%)* 11(15.5%) 5(7.0%) 3(4.2%) 1(1.4%) 7(9.8%) 65(91.5%) total(n) 4(5.6%) 36(50.7%) 13(18.3%) 5(7.0%) 3(4.2%) 1(1.4%) 9(12.6%) 71(100.0%) table 2 distribution of patients with gunshot wounds to the face according to gender and fracture site. there was a statistically significant incidence of mandible fractures in the male gender (p = 0.003) 71 cases as shown in table 2. the most affected sites were the mandible (50.7%), maxilla (18.3%), zygomatic region (7.0%) and eye socket (4.2%). there were associations between the maxilla and zygomatic region (4.2%), mandible and maxilla (2.8%), mandible and zygomatic region (2.8%), maxilla and eye socket (2.8%). fractures in more than one site totaled nine cases (12.6%). the nose was affected in 1.4% of cases. no fractures occurred in 5.6% of cases. there was a statistically significant incidence of mandible fractures in the male gender (p = 0.003) (table 2). of the 71 patients evaluated, only 68 had completed their data records in full form, others were transferred to other services. a total of 88.2% of the patients had comminuted fractures (44.1% in the mandible), whereas 10.3% had simple fractures and 1.5% had no fractures. associated sites (maxilla/ mandible, maxilla/zygomatic region, maxilla/nose, maxilla/ eye socket and mandible/zygomatic region) were grouped on a single line in table 3. there was a statistically significant incidence (p value) of comminuted fractures in the mandible (table 3). of the 68 patients studied, 56 received some form of type of treatment fracture site conservative treatment exploratory surgery rigid internal fixation total no fracture 1(1.8%) 1(1.8%) 0(.0%) 2(3.6%) mandible 9(16.1%) 4(7.1%) 19(33.9%) 32(57.1%) maxilla 1(1.8%) 7(12.5%) 1(1.8%) 9(16.1%) zygomatic 0(.0%) 0(.0%) 5(8.9%) 5(8.9%) socket 0(.0%) 0(.0%) 1(1.8%) 1(1.8%) associate site 0(.0%) 1(1.8%) 6(10.8%) 7(12.5%) total (n) 11(19.6%) 13(23.2%) 32(57.2%) 56(100.0%) table 4 distribution of patients with gunshot wounds to the face according to fracture site and type of treatment. the chi-square test revealed a statistically significant incidence (p = 0.018) of mandible fractures treated with rigid internal fixation. type of fracture fracture site no fracture simple comminuted total no fracture 1(1.5%) 0(.0%) 0(.0%) 1(1.5%) mandible 0(.0%) 6(8.8%) 30(44.1%) 36(52.9%) maxilla 0(.0%) 0(.0%) 13(19.1%) 13(19.1%) zygomatic 0(.0%) 0(.0%) 5(7.4%) 5(7.4%) socket 0(.0%) 1(1.5%) 2(2.9%) 3(4.4%) associated site 0(.0%) 0(.0%) 10(14.7%) 9(13.2%) total (n) 1(1.5%) 7(10.3%) 60(88.2%) 68(100.0%) table 3 distribution of patients with gunshot wounds to the face according to site and type of fracture. there was a statistically significant incidence of comminuted fractures in the mandible (p value). treatment of our specialty as conservative treatment, surgical exploration or rigid internal fixation (rif). the others were referred to other specialties such as neurology and orthopedics. rif was the predominant type of treatment (57.2%), followed by exploratory surgery (23.2%) and conservative treatment (19.6%). the fractures occurred in the nasal region associated with the jaw line associate site grouped in table 4. the chi-square test revealed a statistically significant incidence (p = 0.018) of mandible fractures treated with rif (table 4). of the 56 patients evaluated for the type of treatment system and internal fixation used only 25 showed in their records the data specified. the 1.5-mm system predominated in the zygomatic region in 16% of cases. the system of rigid internal fixation on the face was the predominant 2.4mm used in 56.0% of cases. among the cases in which the rif system was used, there was predominance in the mandible (64.0%). the chi-square test revealed a significant correlation between the 2.4-mm rif system and the mandible in 48.0 % of cases (p = 0.039) (table 5). in the fractures that occurred in the mandible gunshot injuries in the maxillofacial region: a retrospective analysis and management braz j oral sci. 10(4):236-240 type of rif fracture site 1.5 2.0 2.4 2.0+2.4 total mandible 0(.0%) 3(12.0%) 12(48.0%) 1(4.0%) 16(64.0%) zygomatic 4(16.0%) 0(.0%) 0(.0%) 0(.0%) 5(20.0%) socket 1(4.0%) 0(.0%) 0(.0%) 0(.0%) 1(4.0%) associated site 2(8.0%) 0(.0%) 2(8.0%) 0(.0%) 3(12.0%) total 7(28.0%) 3(12.0%) 14(56.0%) 1(4.0%) 25(100.0%) table 5 distribution of patients with gunshot wounds to the face according to fracture site and type of rigid internal fixation (rif). the chi-square test revealed a significant correlation between the 2.4-mm rif system and the mandible (p = 0.039). (abs+crc), rif was the predominant treatment (61.8%), followed by conservative treatment (26.5%) and exploratory surgery (11.8%). immediate complications occurred in 25% of the cases. the main complications were postoperative infection in the mandible, ear canal and eye socket in seven cases (9.3%). paresis occurred in four patients (5.2%). motor deficit of the facial nerve occurred in three cases (4%). sinusitis occurred in two cases (2.6%). soft tissue fibrosis and loss of substance occurred in two cases (2.6%). optic nerve injury occurred in one case (1.3%). among the 75 patients analyzed, there were 4 deaths (5.3%). after effects occurred in 11.7% of the cases. there were two cases of paresthesia (2.6%) – one in the infra-orbital nerve and one in the inferior alveolar nerve. facial asymmetry occurred in two cases (2.6%). dysphagia and speech difficulties occurred in two cases (2.6%). there was one case (1.3%) of paralysis stemming from an injury to the facial nerve. there was one case (1.3%) of a maxillary injury with subsequent formation of oronasal and oroantral fistula. there was one case (1.3%) of pseudo-arthrosis caused by loose osteosynthesis material. discussion the individuals with gunshot wounds to the face in the present study exhibited different clinical characteristics and underwent different forms of treatment. with regard to gender and age group, the findings are in agreement with those reported in the literature, revealing a predominance of the male gender and young adults17-25. a retrospective study on gunshot wounds and explosions reports 1,155 injuries, 36% of which were gunshot wounds; the male gender was affected in 71% of the cases (84% of gunshot injuries); 53% of the sample was between 15 and 29 years of age (59% of whom received gunshot wounds); and there were greater proportions of open wounds (63%) and fractures (42%)5. interpersonal violence, alcohol, drugs and poverty have been reported as the main reasons for gunshot wounds24. in a study on gunshot wounds in children and adolescents between 0 and 19 years of age, the mortality rate was 19.7% and the main cause was assault (78.7%)26. in the present study, there was a predominance of assaults, followed by aggression and attempted homicide, which corroborates the findings reported by cowey et al.27. there was a predominance of entry wounds on the left side and exit wounds on the right side, configuring a piercing pattern for gunshot wounds to the face. this predominance of the left side suggests a connection with the assaults, and this side of the face exposed to the assaults on drivers likely. the face is a common site for gunshot wounds; a previous study reports a prevalence of 33.33% in the neck and face23. in another retrospective study, entry wounds in the right temporal bone were prevalent20. the sites most affected by gunshots in the present study were the mandible, maxilla and zygomatic region, which is in agreement with previous studies27. other studies found a prevalence of gunshot wounds in the maxilla, followed by the mandible7. hollier et al. retrospectively assessed 84 patients and found fractures mainly in the zygomatic region (34.52%), mandible (29.76%) and eye socket (26.19%)21. regarding mandible fractures, the angle, body and symphysis were the most affected sites. similar data are reported by other authors12,27. comminuted fractures predominated, which is in agreement with a review study13. the predominant treatment modality in the present study was rif. comminuted fractures lead to a preference for rif due to the insufficient amount of bone to establish vectors of force13-15,28. in the present sample, rif was the predominant form of treatment for gunshot wounds in the mandible, agreement with other studies19. however, a previous study reports the treatment of such wounds with maxillomandibular block and osteosynthesis with steel wire, with the use of rif in the angle, body and symphysis of the mandible12. immediate complications occurred in 25% of the patients in the present study, four of which ended in death. a number of studies report that the complications that lead to the death of the patient are generally stroke, hypovolemia, sepsis, pneumonia, aneurism, osteomyelitis, abscesses and meningitis10,19. the cases of osteomyelitis were resolved with culture/antibiogram and specific antibiotic therapy followed by drainage. tracheotomy and exploratory surgery of intracranial injuries are other immediate complications10,21,29. ellis et al. report a significant correlation between the degree of comminuted fractures and the development of complications in a study on treatment methods for patients with comminuted fractures of the mandible over a 10-year period; 35.2% of those with more serious wounds treated with external fixation, 17.1% of those treated with maxillomandibular block and 10.3% of those treated with open reduction and rif suffered complications13. however, the primary treatment of injuries to bone and soft tissues may be carried out at the time of debridement, thereby minimizing the rates of hospitalization, procedures and complications29. one study on gunshot wounds to the mandible reports the following after effects: infection, deviation of mouth opening, malocclusion, bone loss and reduced motor activity16. the treatment of the after effects resulting from gunshot wounds involves multidisciplinary therapy, with procedures 239gunshot injuries in the maxillofacial region: a retrospective analysis and management 240 braz j oral sci. 10(4):236-240 such as rhinoplasty, blepharoplasty and orthognathic surgery7,21,30. for mandible reconstruction in adults having suffered gunshot wounds, osteogenic distraction causes the simultaneous expansion of the bone and soft tissues, thereby accelerating rehabilitation with bone-integrated implants30. the present retrospective study found that males between 21 and 30 years of age were most affected by gunshot wounds to the face, with assaults as the main reason for the injuries. the entry wound was mainly on the left side and the exit wound was mainly on the right side of the face. the mandible was the most affected site and associated to comminuted fractures. rigid internal fixation was the predominant form of treatment. paresis, paralysis, infection, sinusitis and soft tissue fibrosis were the main immediate complications in the sample studied. the percentage of after effects was low and characterized by paresthesia, paralysis, oroantral and oronasal fistula, pseudo-arthrosis and loss of vision. references 1. stuehmer c, blum ks, kokemueller h, tavassol f, bormann kh, gellrich nc et al. influence of different types of guns, projectiles, and propellants on patterns of injury to the viscerocranium. j oral maxillofac surg. 2009; 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comminuted mandibular fractures. j oral maxillofac surg. 2003; 61: 861-70. 14. smith br, johnson jv. rigid fixation of comminuted mandibular fractures. j. oral maxillofac surg. 1993; 51: 1320-6. 15. smith br, teencier tj. treatment of comminuted mandibular fractures by open reduction and rigid internal fixation. j oral maxillofac surg. 1996; 54: 328-31. 16. johnson jv. gunshot wounds to the face. j trauma. 2007; 62: s87. 17. motamedi mhk. primary management of maxillofacial hard and soft tissue gunshot and shrapnel injuries. j oral maxillofac surg. 2003; 61: 1390-8. 18. schreuder wh, hein g, goslings jc, kroon fhm, kariem g. poster 75: civilian firearm injuries to the face in an urban hospital: demographics, injury pattern, management and outcome. j oral maxillofac surg. 2011; 69: e95-e96. 19. vayvada h, menderes a, yilmaz m, mola f, kzlkaya a, atabey a. management of close-range, high-energy shotgun and rifle wounds to the face. j craniofac surg. 2005; 16: 794-804. 20. goren s, subasi m, tirasci y, kemaloglu s. firearm-related mortality: a review of four hundred-forty four deaths in diyarbakir, turkey between 1996 and 2001. tohoku j exp med. 2003; 201: 139-45. 21. hollier l, grantcharova ep, kattash m. facial gunshot wounds: a 4year experience. j oral maxillofac surg. 2001; 59: 277-82. 22. mäkitie i, pihlajamäk h. fatal firearm injuries in finland: a nationwide survey. scand j surg. 2002; 91: 328-31. 23. maguire k, hughes dm, fitzpatrick ms, dunn f, rocke lg, baird cj. injuries caused by the attenuated energy projectile: the latest less lethal option. emerg med j. 2007; 24: 103-5. 24. meel bl. firearm fatalities in the transkei region of south africa, 19932004. s afr med j. 2005; 95: 963-7. 25. peleg k, aharonson-daniel l, stein m, michaelson m, kluger y, simon d et al. gunshot and explosion injuries: characteristics, outcomes, and implications for care of terror-related injuries in israel. ann surg. 2004; 239(3): 311-8. 26. nance ml, denysenko l, durbin dr, branas cc, stafford pw, schwab cw. the rural-urban continuum: variability in statewide serious firearm injuries in children and adolescents. arch pediatr adolesc med. 2002; 156: 781-5. 27. cowey a, mitchell p, gregory j, maclennan i, pearson r. a review of 187 gunshot wound admissions to a teaching hospital over a 54-month period: training and service implications. ann r coll surg engl. 2004; 86: 104-7 28. shawi aa. open-packing method for the severely comminuted fractured mandible due to missile injury. br j oral maxillofac surg. 1995; 33: 36-9 29. danino am, hariss pg, servant jm. early management, with a minimal initial hospitalization length, of major self-inflicted rifle wounds to the face by a single latissimus dorsi free musculocutaneous flap: a 10year experience. eplasty. 2009; 9: e23. 30. labbé d, nicolas j, kaluzinsky e, soubeyrand e, sabin p, compère jf et al. gunshot wounds: reconstruction of the lower face by osteogenic distraction. plast reconstr surg. 2005; 116: 1596-603. gunshot injuries in the maxillofacial region: a retrospective analysis and management oral sciences n3 braz j oral sci. 11(1):10-13 received for publication: july 04, 2011 accepted: january 03, 2012 original article braz j oral sci. january | march 2012 volume 11, number 1 preventive effect of an orthodontic compomer against enamel demineralization around bonded brackets alan rafael martins savariz1, mariana marquezan2, maurício mezomo3 1graduate student in orthodontics, school of dentistry, centro universitário franciscano, brazil 2master’s degree in orthodontics, visiting professor, graduate program in dentistry, school of dentistry, centro universitário franciscano, brazil 3master’s degree in orthodontics, professor, department of orthodontics, school of dentistry, centro universitário franciscano, brazil correspondence to: maurício mezomo rua alberto pasqualini – 70 / 809 zip code: 97015-010 santa maria-rs, brazil phone: + 55 55 9144-6660 fax: + 55 55 3026-3701 e-mail: mezomo@ortodontista.com.br abstract orthodontic appliances predispose to the accumulation of plaque due to the great number of retentive sites, which might lead to enamel demineralization adjacent to the accessories. aim: to assess the effectiveness of a compomer for orthodontic bonding in preventing the formation of white spots around orthodontic brackets. methods: forty extracted human premolars were divided into two groups: control group (cg), in which conventional resin transbond™ xt light cure (3m unitek™) was used to bond the brackets; and experimental group (eg), in which the compomer transbond™ plus color change (3m unitek™) was used. ph cycling was performed for 17 days to induce the demineralization process. enamel on the buccal face was photographed under a stereomicroscope (at 10x magnification) before (t0) and after (t1) ph cycling. the images were used to compare demineralization between the groups by using a visual scale. results: a statistically significant difference between control and experimental groups was found (p=0.004) showing that the compomer was more efficient than the conventional resin in preventing white spots. conclusions: the compomer transbond plus color change was capable of inhibiting enamel demineralization adjacent to the bonding area of brackets. however, the inhibition halo did not exceed 1 mm. keywords: brackets, fluoride, orthodontic adhesives, tooth demineralization. introduction the metabolic activity of bacteria that colonize the tooth surface causes alterations in ph, which results in an intermittent process of loss and gain of mineral of the dental tissue. caries lesions are formed by an imbalance in the deremineralization process, when there is more frequent ion output from the dental mineral tissue, leading to the destruction of enamel1. orthodontic treatment with fixed appliances has been associated with an increased risk for the development of caries lesions2, since the placement of fixed appliances in the oral cavity creates new plaque-retention sites3-4, increasing significantly the number of streptococcus mutans immediately after their placement5-6. therefore, enamel demineralization around orthodontic accessories is a possible adverse effect of orthodontic treatment for patients with poor oral hygiene7-9. white spots are formed after approximately 21 days of cariogenic challenge braz j oral sci. 11(1):10-13 and they may be reverted by the presence of fluoride and biofilm disorganization10. initial demineralization may be clinically detected when white opaque and rough spots appear. these spots can evolve to cavitated lesions or they can be inactivated. inactive white spots remain as “scars” of shinny and smooth white spots11 and may compromise esthetics because they remain visible for up to 5 years after the removal of the brackets12. fluoride remains as the best-known cariostatic agent13. it must be constantly present in the oral cavity throughout entire orthodontic treatment to be effective in reducing demineralization around orthodontic brackets14. the use of fluoride mouthrinses with daily toothbrushing has shown positive results11,15. however, these measures have limited effectiveness since they depend on the patient’s cooperation2. therefore, the use of restorative16-18 and bonding materials1923 that release fluoride has been an alternative to prevent caries lesions. fluoride has been added to the composition of resin composites, which are widely used for bracket bonding. several commercial brands have introduced fluoridated resins and compomers on the market, but further studies are needed to prove and validate their effectiveness in preventing enamel demineralization adjacent to orthodontic accessories24. the aim of this study was to assess in vitro the capacity of a compomer for orthodontic bonding to prevent enamel demineralization around brackets after the first days of bonding. material and methods the sample was composed of 40 human premolars, previously cleaned, and stored in a glucose-free physiological solution, which were provided by the human tooth bank (htb) of the centro universitário franciscano. teeth with cracked or damaged enamel surface were excluded. the specimens were sectioned 10 mm below the cementoenamel junction with a diamond disk (h22gk-314-016™ – komet, usa) under water cooling. the root portion was embedded in pvc tubes and filled with autopolymerizing acrylic resin. the teeth were then randomly distributed into a control group (cg), in which transbond™ xt light cure resin (3m unitek, monrovia, ca, usa) was used to bond the brackets, and an experimental group (eg), in which the compomer transbond™ pluscolor change (3m unitek, usa) was applied (table 1). the tooth crowns were cleaned with a rubber cup and pumice for 10 s. then they were washed with jets of water/air and dried with compressed air for 10 s. bracket bonding was preceded by etching with 37% phosphoric acid gel for 15 s (3m unitek, usa), rinsed with a air/water spray for 30 s, and dried until a characteristic frosty white etched area was observed (about 60 s). metallic table 1table 1table 1table 1table 1. division of control and experimental groups. group no of sample bonding agent fluoride release c g 20 transbond xt light cure eg 20 transbond plus color change + premolar brackets (kirium™ 3m abzil, sumaré, sp, brazil) were bonded on the buccal surface, parallel to the long axis of the tooth. the same amount of resin composite was used in all groups and excesses were removed with a #5 explorer with rhomboid tip. light-polymerization was performed during for 40 s with a led light-curing unit (radii-cal™; sdi, bayswater, victoria,. austrália) with 1,200 mw/cm2 of light intensity, maintaining a constant distance of 3 mm from material surface and angulation of 45º in relation to the tooth surface. the whole bonding procedure was performed by a single trained operator according to the manufacturers’ specifications. after bonding, a 2-mm window was delimited in the enamel adjacent to the brackets. the remaining buccal face was rendered waterproof with colorless nail polish (168™ – extase, brazil). next, the specimens were photographed under a stereomicroscope (emf, meiji techno co., ltd, japan) coupled to a camera (coolpix e4500™; nikon, tokyo, japan) under artificial light at 10x magnification (figure 1a). this experimental time was considered as zero (t0). fig. 1. photomicrograph before ph cycling (a) and after ph cycling (b) showing the area exposed to demineralization (window). original magnification 10x. afterwards, the samples were submitted to a ph cycling regimen at room temperature during 17 days to promote the demineralization process. first, the teeth were submerged in an artificial saliva solution [kcl 0.12%; mgcl2 0.0052%; nipagin 0.18%; naf 0.01%; nacl 0.0084%; cacl2 0.0146%; kdp 0.0342%; cmc 1%; xilitol c 4%; naoh 30%] at a neutral ph (7.4) for 20 h per day. they were then submerged in a demineralizing solution [kcl 0.12%; mgcl2 0052%; nipagin 0.18%; naf 0.01%; nacl 0.0084%; cacl2 0.0146%; kdp 0.0342%; cmc 1%; xilitol c 4%; c6h8o7 50%] with ph adjusted to 4.4 for four h. after this period, the specimens returned to the neutral artificial saliva solution, giving sequence to the cycle. at change of solution, the teeth were washed with deionized water. the solutions were changed every 4 days. after the last cycling period, all specimens were washed with deionized water and dried with compressed air. this experimental time was considered as t1. the window working areas were photomicrographed again (figure 1b) and the images at t0 and t1 were compared by a blinded and calibrated examiner (k=0.7). a visual scale for enamel demineralization was created for comparison between the groups, score 0 was 1111111111 preventive effect of an orthodontic compomer against enamel demineralization around bonded brackets braz j oral sci. 11(1):10-13 1212121212 score 0 score 1 p c g 8 (40%) 12 (60%) 0.004* eg 17 (85%) 3 (15%) total 20 20 n o *statistically significant at a level of 0.05% (chi-square tests) table 2.table 2.table 2.table 2.table 2. result of analysis with visual scale for enamel demineralization. attributed when the demineralization process was not identified in the enamel adjacent to the bonding material; and score 1 was attributed when a white spot was found adjacent to the orthodontic accessories. the data obtained were tabulated and subjected to the chi-square test. a significance level of 5% was established. results according to the visual scale for enamel demineralization, eg, in which the brackets were bonded with transbond™ plus color change, showed a larger number of specimens with score 0, which means without areas of demineralization around the brackets (p = 0.004) (table 2). discussion the results of this study showed that the compomer transbond™ plus color change was superior to the conventional resin transbond™ xt ligth cure with regard to the capacity of inhibiting the formation of white spots around orthodontic brackets when the specimens were subjected to ph cycling. however, the inhibition halo was small in amplitude, not exceeding 1 mm in any of the test specimens. previous studies that used fluoridated materials during bracket bonding, such as fluoridated varnishes associated with conventional resins 22, conventional glass ionomer cements7, resin-modified glass ionomer cements13,25 and polyacid-modified resin composites13, showed lower mineral loss around the accessories when compared with the use of conventional resins. on the other hand, other studies have shown that fluoridated adhesives do not seem to differ from conventional adhesives with regard to the capacity of preventing areas of enamel demineralization adjacent to the brackets26. although conventional glass ionomer cements are capable of releasing fluoride and preventing enamel demineralization adjacent to the orthodontic accessories, their bond strength is limited and they are not recommended for clinical use24,27. thus, resin-modified glass ionomer cements and polyacid-modified resins (compomers) have been more frequently used in orthodontic clinics and research studies. when the capacity for preventing enamel demineralization around brackets of these two materials is compared, resinmodified cements have shown greater effectiveness13 because they release more fluoride13,23. fluoride-releasing dental materials, such as resins, cements and elastomeric ligatures, act as a reservoir of fluoride in the oral cavity and may increase the levels of fluoride in saliva, bacterial plaque and hard dental tissues. fluoride increases enamel remineralization28 and reduces the growth of streptococcus mutans29. the values of fluoride release from orthodontic materials vary in the literature due to methodological differences. it is still unclear which is the minimum amount of fluoride necessary to prevent demineralization around orthodontic brackets30 and the need to use fluoride in orthodontic bonding materials is controversial18. it is known that fluoride release from dental materials occurs more markedly in the first 24 h after placement in the oral cavity, followed by an accentuated decline and tendency to stabilize over time16,25. nevertheless, fluoridated materials present in the oral cavity can be recharged with fluoride through other means of exposure, such as toothpastes, mouthrinses and professional topical applications20. the exact mechanism of fluoride recharge is unknown 17. the permeability of the material, form and concentration of fluoride are factors that might be involved in the process. even if fluoride-releasing orthodontic materials are used, patients should be instructed and motivated to perform oral hygiene to disorganize biofilm and use fluoridated toothpaste. oral hygiene measures are the most effective and established methods to prevent dental caries and periodontal disease, although they depend on the patient cooperation2. moreover, it is known that the effectiveness of fluoridereleasing orthodontic materials is limited, as demonstrated in the present study, and fluoride recharge depends on other sources of exposure, including toothpastes. in vitro studies allow greater control of variables and are the first research tool used when a new material and its properties are tested. however, one cannot extrapolate their findings to clinical practice. randomized controlled clinical trials are needed to confirm the relationship between compomers and lower enamel demineralization adjacent to orthodontic accessories. in conclusion, the compomer transbond plus color change was capable of inhibiting enamel demineralization adjacent to the bonding area of brackets. however, the inhibition halo did not exceed 1 mm. references 1. maltz m, carvalho j. diagnóstico de doença cárie. in: kriger l. aboprev promoção de saúde bucal. são paulo: artes médicas; 1997. 2. mitchell l. decalcification during orthodontic treatment with fixed appliances—an overview. br j orthod. 1992; 19: 199-205. 3. larmas m. saliva and dental caries: diagnostic tests for normal dental practice. int dent j. 1992; 42: 199-208. 4. pender n. aspects of oral health in orthodontic patients. br j orthod. 1986; 13: 95-103. 5. corbett ja, brown lr, keene hj, horton im. comparison of streptococcus mutans concentrations in non-banded and banded orthodontic patients. j dent res. 1981; 60: 1936-42. 6. mattingly ja, sauer gj, yancey jm, arnold rr. enhancement of streptococcus mutans colonization by direct bonded orthodontic appliances. j dent res. 1983; 62: 1209-11. preventive effect of an orthodontic compomer against enamel demineralization around bonded brackets 1313131313 braz j oral sci. 11(1):10-13 7. geiger am, gorelick l, gwinnett aj, griswold pg. the effect of a fluoride program on white spot formation during orthodontic treatment. am j orthod dentofacial orthop. 1988; 93: 29-37. 8. lundstrom f, krasse b. caries incidence in orthodontic patients with high levels of streptococcus mutans. eur j orthod. 1987; 9: 117-21. 9. ogaard b, rolla g, arends j. orthodontic appliances and enamel demineralization. part 1. lesion development. am j orthod dentofacial orthop. 1988; 94: 68-73. 10. wiltshire wa, janse van rensburg sd. fluoride release from four visible light-cured orthodontic adhesive resins. am j orthod dentofacial orthop. 1995; 108: 278-83. 11. benson pe, parkin n, millett dt, dyer fe, vine s, shah a. fluorides for the prevention of white spots on teeth during fixed brace treatment. cochrane database syst rev. 2004: cd003809. 12. ogaard b. prevalence of white spot lesions in 19-year-olds: a study on untreated and orthodontically treated persons 5 years after treatment. am j orthod dentofacial orthop. 1989; 96: 423-7. 13. chin my, sandham a, rumachik en, ruben jl, huysmans mc. fluoride release and cariostatic potential of orthodontic adhesives with and without daily fluoride rinsing. am j orthod dentofacial orthop. 2009; 136: 547-53. 14. dijkman ge, de vries j, lodding a, arends j. long-term fluoride release of visible light-activated composites in vitro: a correlation with in situ demineralisation data. caries res. 1993; 27: 117-23. 15. chadwick bl, roy j, knox j, treasure et. the effect of topical fluorides on decalcification in patients with fixed orthodontic appliances: a systematic review. am j orthod dentofacial orthop. 2005; 128: 601-6; quiz 70. 16. aboush ye, torabzadeh h. fluoride release from tooth-colored restorative materials: a 12-month report. j can dent assoc. 1998; 64: 561-4, 68. 17. preston aj, higham sm, agalamanyi ea, mair lh. fluoride recharge of aesthetic dental materials. j oral rehabil. 1999; 26: 936-40. 18. wiegand a, buchalla w, attin t. review on fluoride-releasing restorative materials—fluoride release and uptake characteristics, antibacterial activity and influence on caries formation. dent mater. 2007; 23: 343-62. 19. passalini p, fidalgo tks, caldeira em, gleiser r, nojima mcg, maia lc. preventive effect of fluoridated orthodontic resins subjected to high cariogenic challenges. braz dent j. 2010; 21: 211-5. 20. ahn sj, lee sj, lee dy, lim bs. effects of different fluoride recharging protocols on fluoride ion release from various orthodontic adhesives. j dent. 2011; 39: 196-201. 21. benson pe, shah aa, millett dt, dyer f, parkin n, vine rs. fluorides, orthodontics and demineralization: a systematic review. j orthod. 2005; 32: 102-14. 22. behnan sm, arruda ao, gonzalez-cabezas c, sohn w, peters mc. in-vitro evaluation of various treatments to prevent demineralization next to orthodontic brackets. am j orthod dentofacial orthop. 2010; 138: 712 e1-7; discussion 712-3. 23. rix d, foley tf, banting d, mamandras a. a comparison of fluoride release by resin-modified gic and polyacid-modified composite resin. am j orthod dentofacial orthop. 2001; 120: 398-405. 24. rogers s, chadwick b, treasure e. fluoride-containing orthodontic adhesives and decalcification in patients with fixed appliances: a systematic review. am j orthod dentofacial orthop. 2010; 138: 390 e1-8; discussion 90-1. 25. paschos e, kleinschrodt t, clementino-luedemann t, huth kc, hickel r, kunzelmann kh, et al. effect of different bonding agents on prevention of enamel demineralization around orthodontic brackets. am j orthod dentofacial orthop. 2009; 135: 603-12. 26. leizer c, weinstein m, borislow aj, braitman le. efficacy of a filledresin sealant in preventing decalcification during orthodontic treatment. am j orthod dentofacial orthop. 2010; 137: 796-800. 27. millett dt, mccabe jf. orthodontic bonding with glass ionomer cement— a review. eur j orthod. 1996; 18: 385-99. 28. forss h, seppa l. prevention of enamel demineralization adjacent to glass ionomer filling materials. scand j dent res. 1990; 98: 173-8. 29. friedl kh, schmalz g, hiller ka, shams m. resin-modified glass ionomer cements: fluoride release and influence on streptococcus mutans growth. eur j oral sci. 1997; 105: 81-5. 30. hellwig e, lussi a. what is the optimum fluoride concentration needed for the remineralization process? caries res. 2001; 35 suppl 1:57-9. preventive effect of an orthodontic compomer against enamel demineralization around bonded brackets oral sciences n3 case report braz j oral sci. october | december 2012 volume 11, number 4 angioneurotic edema: report of two cases chaitra .t.r.1, ravishankar .t.l.2 , triveni mohan nalawade3 1senior lecturer, department of pediatric and preventive dentistry, kothiwal dental college & research centre, kanth road , moradabad, india 2 reader, department of community dentistry, kothiwal dental college & research centre, kanth road , moradabad-, uttar pradesh, india 3senior lecturer, department of pediatric dentistry, mannubhai patel dental college, vadodara, india correspondence to: chaitra t.r. department of pedodontics and preventive dentistry kothiwal dental college & research centre kanth road, moradabad-244001 uttar pradesh, india e-mail: chaitu4363@yahoo.co.in abstract pediatric angioedema exhibits a different cause and clinical manifestations than does adult angioedema. unlike angioedema in adults, pediatric angioedema is caused mostly due to food, followed by insect bites, infection and antibiotics. reactions to insect stings, both allergic and toxic, are commonly seen in medical pediatric practice but uncommonly encountered by pediatric dentists. here we present two cases of angioedema involving the face mainly in children who presented insect bite in the affected region. treatment and case resolution are described. keywords: giant urticaria, angioneuroticedema, allergy, pruritis. introduction angioedema is a rather common form of edema occurring in both hereditary and non-hereditary form. it appears to be closely related to general urticaria1. angioedema denotes similar but larger swellings of the deep dermal, subcutaneous and submucosal tissues2. it is also referred to as “giant urticaria”, “quincke’s edema” and also “angioneurotic edema”1. urticaria and angioedema are important components of systemic anaphylaxis which is an acute life threatening condition2. reactions to insect stings, both allergic and toxic, are seen commonly in medical practice3, but uncommon in dental practice. large local reactors are more frequent but rarely dangerous. the chance of a systemic reaction to a insect sting is low (5% to 10%) in large local reactors and in children with mild (cutaneous) systemic reactions.4 herein, we present a 7-year-old boy and a 6-year-old boy with giant urticaria in whom the disease appeared due to insect bite on their forehead followed by swelling of face. case report case 1 a 7-year-old boy reported to the department of pediatric and preventive dentistry, with diffuse facial swelling involving forehead and both the eyes (figure 1). his history of present illness revealed, a millipede bite on the forehead one day before was the cause of initial swelling that involved the eyelid and gradually increased over 2 h to involve the entire face. the swelling remained throughout the day with no diurnal variations and no history of fever. general examination revealed normal vital signs and the boy weighed about 18 kg. on clinical examination, a diffuse swelling involving the whole of the forehead, upper and lower eyelid with bridge of the nose, right and left cheek region, was seen. the edema was non-pitting in nature with no local rise of temperature. swelling was received for publication: april 25, 2012 accepted: june 23, 2012 braz j oral sci. 11(4):505-508 tender on palpation and there was no pus or serous discharge. bite mark of the millipede was seen on the right fore head region (figure 2). intraoral examination revealed generalized dental fluorosis and initial caries lesions in teeth 16, 26, 36, and 46. based on history and clinical features a provisional diagnosis of angioedema was made. fig. 1. a 7-year-old boy exhibiting diffuse facial swelling involving forehead and both the eyes. fig. 2. site of millipede bite on the forehead with diffuse swelling involving the entire forehead, upper and lower eyelid with bridge of the nose. the patient was treated with combination of drugs. injection of avil (pheniramine maleate; 1cc iv) and hydrocortisone (100 mg iv) were given immediately (stat), which are believed to ameliorate delayed effects of anaphylactic reactions and may limit biphasic anaphylaxis. the combination of antihistamines and corticosteroids are routinely used in emergency treatment of anaphylaxis. injection of lasix (furosemide; 20 mg iv stat) was given to reduce the edema by reducing fluid retention and also attenuate the symptoms of troubled breathing. tetanus toxoid injection was given to maintain active immunity as tetanus spores might have entered the body through insect bite. injection of benzyl penicillin 7.5 lakh given three times daily (tid) empirically to prevent the bite area from secondary infection and also condition bear a resemblance to cellulites and lyser d (combination of serratiopeptidase and diclofenac sodium) (tid) was given to reduce inflammation and alleviate the symptoms of pain. tab rantac (ranitidine) 75 mg was given twice daily (bid). h 2 blocking drugs were used concurrently with h 1 antihistamine to reduce gastric secretion, and injection of dexona (dexamethasone; 1.5 cc tid) was also given. iv fluid dns (dextrose normal saline) 10 drops/min was given. iv fluids were continued for 2 days. after 3 days, the edema reduced considerably with slight persisting in the lower eyelids and finally after a period of 8 days of intensive treatment, the swelling subsided completely (figure 3). fig. 3. recovery of the 7-year-old boy after 8 days. case 2 a 6-year-old boy reported to the department of pediatric and preventive dentistry with diffuse facial swelling mainly involving the philtrum area and the left lower eyelid region (figure 4). the patient’s parents suspected that the reason for the swelling was tooth decay and hence reported to our institution for check up. history of the disease revealed that the swelling was sudden in onset and the parents reported immediately the next day morning. no episode attributable to food or drug allergy was present in history. swelling had been diffuse, angioneurotic edema: report of two cases 506506506506506 braz j oral sci. 11(4):505-508 tender on palpation and non-pitting in nature with a small injury towards the left ala of nose, which was suspected to be an insect bite mark, but the patient’s father did not reveal any history of insect bite or fever. general examination revealed normal vital signs and the boy weighed about 15 kg. intraoral examination showed no caries involvement of any tooth. based on history and clinical examination, a provisional diagnosis of angioedema was made. it later responded to antihistaminic therapy. patient was treated with antihistaminic allercet (cetrizine; 5 mg once daily) and was asked to report in case of any aggravation of symptoms. unfortunately the patient could not come for recall visit as he was from a distant place, but gratefully he called us up to let us know of his complete recovery. discussion clinics in previous decades predominantly consisted of children with allergic rhinitis, asthma and eczema, the proportion of children presenting with acute severe allergic reactions has progressively increased. clinics are now dominated by acute food, insect venom and drug allergy, and there is evidence that these problems are increasing5. angioedema is a variant of urticaria where there is involvement of the subcutaneous tissue6. skin lesions may appear on the eyelids, lips, genitalia, tongue and pharynx. sudden onset appears to be characteristic. skin lesions last for few hours to few days7. angioedema affects males and females equally, usually during the 3rd and 4th decade of life. pediatric angioedema exhibits a different cause, severity and clinical manifestations than does adult angioedema8. probable causes of angioedema are food (40%), insect bites (30%), infection (20%) and antibiotics (10%)5. reactions to insect stings, both allergic and toxic are seen commonly in pediatric practice3. papular urticaria occurs through the bite of insects most commonly ‘mosquitos’ and bed bugs. on the contrary bee or wasps stings may produce severe acute urticaria or anaphylaxis, which may be lifethreatening conditions. hereditary angioedema usually manifests during childhood5 but it is rare and is transmitted as an autosomal dominant trait. another common form occurring in young children is allergy to food including multiple food allergies, allergy to nuts and cow’s milk 2,5,8. in case 1, angioedema affected a 7-year-old boy with major manifestations on the face involving forehead and eyelids due to a millipede bite, which, to the best of our knowledge, is the first of its kind to be published. in case 2, angioedema affected a 6-year-old boy with mild manifestations on left side of the face, which responded to antihistamine therapy. no history of insect bite could be elicited; which is common9. the main difficulties arise around a lack of agreed definition for what constitutes an anaphylactic reaction. however, this then excludes patients who have developed angioedema and urticaria, which is appropriately treated prior to its evolution into a more severe and potentially life-threatening reaction. conversely, if patients presenting just with angioedema and urticaria are incorporated into the definition, then this will include many individuals who will not have any progression of symptoms5. typical reactions following an insect sting include erythema, itching, pain, swelling and indurations localized to the area of the sting. these local reactions usually last only several hours and may respond to application of cool compresses. large local reactions also occur frequently involving more extensive areas of the skin typically with swelling 5-10 cm in diameter (sometimes more) that is contiguous to the site of the sting. the swelling generally reach its peak in 24-48 h, but reactions can last up to 7-10 days. children exhibited predominantly facial (80 %) and lip (40%) edema. although systemic reactions to insect stings are exception (less than 1% occurrences in children), they can be life-threatening conditions3. angioedema may present as an emergency and needs to be treated aggressively. understanding the various possible causes is the first step in assessing angioedema. allergic and drug-induced angioedema responds to removal of cause.2 a stinger that remains in the skin after an insect sting should be removed. the area should be cleansed with soap and water. the immediate management of systemic reactions focuses on the treatment of anaphylaxis, for which administration of subcutaneous epinephrine (0.3 ml of a 1:1,000 dilution) is the treatment of choice3,8. it is primarily used in the emergency treatment of non-hereditary angioedema involving larynx, it can be injected intramuscularly, subcutaneously or inhaled depending upon the severity of the reaction2. an antihistamine, such as diphenhydramine (benadryl), or hydroxyzine (atarax, vistaril) may be given after fig. 4. six-year-old boy showing diffuse facial swelling mainly involving the philtrum area and the left lower eyelid region. angioneurotic edema: report of two cases507507507507507 braz j oral sci. 11(4):505-508 epinephrine has been administered to reduce pruritus and inflammation 3,8. when the conventional h1 and h2 antihistamine failed, other drugs like nifedipine is used as an adjunct to antihistamines. in resistant cases a brief course of systemic corticosteroids should be avoided because of significant adverse effects. topical steroids also have been found to be effective2. some authors suggest the use of corticosteroids depending on clinical presentation3 while others mention corticosteroid therapy using intravenous dexamethasone sodium phosphate or hydrocortisone remains the main treatment for angioedema. intravenous fluids for intravascular volume repletion and diuresis or ventilatory support for treatment of pulmonary edema have also been used8. beta-agonists, oxygen histamine 2 blockers and vasopressors may also be useful depending on the clinical presentation3. after 8 days of intensive drug therapy, the first patient described in this paper showed complete reduction in facial swelling and pain and was discharged. reactions to insect stings are seen commonly in pediatric practice, but seldom in pediatric dentistry practice. however, in some cases, history of insect bite cannot be elicited, as observed in our young patients. in addition, facial swellings similar to that presented in these cases also occur due to space infections following tooth decay. in such diagnostic dilemmas, it is our responsibility as health professionals to rule out dental causes and give appropriate treatment or refer the patients to pediatricians. as angioedema can also manifest fatally, dentists need to know, diagnose and treat such rare conditions promptly as “the eyes do not see what the mind does not know!!“ acknowledgements the authors would like to acknowledge the generous contributions of dr. guruprasad, professor in pediatrics in the management of this case. he is associated with the bapuji child health institute, dept of pediatrics, jjm medical college, davangere, karnataka. references 1. shafer wg, hine mk, levy bm. physical and chemical injuries of the oral cavity. a textbook of oral pathology .5th ed. philadelphia: w.b. saunders; 2006. p.719-87. 2. prasad ps. urticaria. indian j dermatol venereol leprol. 2001; 67: 11-20. 3. booker gm, adam hm. insect stings. pediatr rev. 2005; 26: 388-9 4. golden db. insect sting anaphylaxis. immunol allergy clin north am. 2007; 27: 261-72. 5. warner jo. anaphylaxis; the latest allergy epidemic. pediatr allergy immunol. 2007; 18: 1-2. 6. greaves mw, lawlor f. angioedema; manifestations and management. j am acad dermatol. 1991; 25: 155-61. 7. champion rh, roberts sob, carpenter rg, roger jh. urticaria and angioedema: a review of 554 patients. br j dermatol. 1969; 81: 588-97. 8. shah uk, jacobs in. pediatric angioedema: ten year’s experience. arch otolaryngol head neck surg. 1999; 125: 791-5. 9. quercia o, emiliani f, foschi fg, stefanini gf. unusual reaction to hymenoptera sting: a case of schonlein-henoch purpura. allergy. 2007; 62: 333-4. angioneurotic edema: report of two cases 508508508508508 braz j oral sci. 11(4):505-508 oral sciences n3 braz j oral sci. 10(3):208-212 original article braz j oral sci. july | september 2011 volume 10, number 3 do self-ligating brackets favor greater bacterial aggregation? matheus m. pithon1, rogério l. dos santos2, leonard euller nascimento2, amanda osorio ayres2, daniela alviano3, ana maria bolognese4 1professor of orthodontics, university of southwestern bahia (uesb), bahia, brazil 2phd student in orthodontics; federal university of rio de janeiro, rio de janeiro, rj, brazil 3 adjunct professor of microbiology, federal university of rio de janeiro, brazil 4full professor of orthodontics, federal university of rio de janeiro, brazil correspondence to: ana maria bolognese universidade federal do rio de janeiro faculdade de odontologia departamento de ortodontia e odontopediatria av. professor rodolpho paulo rocco, 325 ilha do fundão cep:22245-100 rio de janeiro, rj brazil phone: (21) 25902727 e-mail: anabolognes@yahoo.com.br abstract aim: to verify the hypothesis that self-ligating brackets favor greater aggregation of microorganisms when compared with conventional brackets. methods: four types of self-ligating metal brackets were evaluated. initially, 50 brackets were divided into five groups (n=10): morelli conventional, gac (in-ovation r, dentsply caulk), aditek (easy clip), ormco (damon system) and 3m unitek (smart clip). an in vivo evaluation was carried out in which the brackets were bonded to the mandibular teeth of five healthy individuals who had not undergone previous orthodontic treatment. the right hemiarch brackets were used for bacterial plaque collection and those on the left side were examined by scanning electron microscopy (sem). before bracket bonding, the bacterial plaque material aggregated to the tooth surfaces was collected, with the areas of choice being the cervical-buccal and mesial and distal interproximal regions. after 21 days had elapsed since bonding, the plaque adhered to the winglet, channel and cervical regions of the bracket bases was collected. the materials collected were diluted and seeded on petri dishes onto mitis salivarius medium specific for s. mutans and non-specified bhi culture medium. colony forming unit (cfu) counts were performed visually after 24, 48 and 72 h of incubation. results: greater bacterial accumulation was observed on the winglets of 3m brackets, with statistical statistically significant differences from the other types (p<0.05). as regards the channel regions, most microorganisms accumulated in the ormco group (p<0.05), and in the cervical region of aditek brackets. in all evaluated regions, those with least bacterial accumulation were the conventional brackets. conclusions: the hypothesis was confirmed, as the self-ligating brackets were shown to have greater bacterial accumulation when compared with the conventional brackets. keywords: orthodontic brackets, microbiology, dental plaque, biofilms. introduction self-ligating brackets have been introduced to orthodontics several decades ago. harradine1 mentioned that the concept of self-ligation is as old as that of the edgewise appliance. nevertheless, over the last two decades, there has been increasing production and dissemination of these accessories with active and passive modes of ligation2-4. one of the most favorable aspects with the use of self-ligating brackets, received for publication: may 29, 2011 accepted: september 14, 2011 braz j oral sci. 10(3):208-212 209 according to the literature, would be elimination of the elastomers and steel ligature wires5-6. two basic advantages are achieved with this procedure5-6: eradication of cross contamination that may occur accidentally during ligature placement, and improvement in oral hygiene by the patients. the latter advantage would be attributed to the fact that without the ligature, the bracket surface would be freer for cleaning purposes5-6. there is a rich ecosystem in the oral cavity, with a countless number of microorganisms. although both periodontal disease and dental caries are considered multifactorial diseases, the bacteria in dental plaque are the main factor in their onset and progression. however, there are situations that comprise so-called “ecological stress”, with reference to the displacement of microbiological equilibrium, creating conditions that favor the growth and appearance of caries and/or periodontopathogenic bacteria7,8. the different accessory components of fixed orthodontic appliances contribute to the change in the equilibrium of oral ecology9. the presence of brackets and ligatures has been shown to be related to in gingival inflammation and increased risk of tooth surface decalcification, which ultimately results in the appearance of white spots and caries10, 11. in spite of the literature guiding the thought that the self-ligating bracket system favors less aggregation of mutans group microorganisms, up to now, there are no consistent clinical evaluations in this sense4,12. based on this premise, the aim of this study was to investigate the hypothesis that selfligating brackets favor greater aggregation of s. mutans and other microorganisms when compared with conventional brackets. material and methods brackets fifty brackets were evaluated, being 40 of the selfligating type of four different commercial brands and 10 of the conventional type (n=10). the brackets were divided into five groups, described as follows: conventional (morelli, sorocaba, sp, brazil), gac (in-ovation r, dentsply caulk, milford, de, usa), aditek (easy clip, cravinhos, sp, brazil), ormco (damon system, orange, ca, usa), and 3m unitek (smart clip, monrovia, ca, usa) (figure 1). the bracket channels (passive type) were filled with segments of round 0.020" wire (tp ortho, tokyo, japan). the wire received a gray colored elastic ligature (morelli, sorocaba, sp, brazil) on the conventional brackets and on the others, it was fixed by the specific system of each type of bracket (figure 2). subjects the sample was constituted by 5 male individuals with mean age of 25.3 years, with complete permanent dentition, normal occlusion and no previous orthodontic treatment. the brackets were bonded in the mandibular arch, the right side being used for microbial count and the left for scanning electron microscopy (sem) analysis (figure 2). this study was approved by the ethics committee (protocol 125/2008). fig. 1. brackets evaluated: a) conventional; b) gac; c) aditek; d) ormco and e) 3m. fig. 2. a) intraoral frontal view of one of the patients, in whom brackets were bonded; b) right side view (brackets used for bacterial plaque collection); c) left side view (brackets used for sem evaluation). methods initially, the individuals had their oral hygiene calibrated, according to the modified bass-type technique, do self-ligating brackets favor greater bacterial aggregation? 210 braz j oral sci. 10(3):208-212 and also received oral hygiene kits containing a toothbrush (procter & gamble/oral b, são paulo, brazil), dental floss “super floss” (procter & gamble/oral b, são paulo, brazil) and toothpaste (colgate-palmolive, são paulo, brazil). one week after receiving oral hygiene instructions, the bacterial plaque material aggregated to the tooth surfaces was collected with no. 20 absorbent paper cones (dentsply ind. e com. ltda., petrópolis, rj, brazil), obtained from the colonized sites on the canines, mandibular first and second premolars and mandibular first and second molars, of which the supragingival areas of choice were as follows: a) cervical-buccal; b) mesial interproximal and c) distal interproximal areas. immediately after the initial collection of microorganisms, the brackets were bonded to the tooth surfaces according to the bonding technique proposed by the orthodontic adhesive manufacturer (transbond xt, monrovia, ca, usa). after 21 days had elapsed since bonding, the second bacterial plaque collection was performed on the brackets, in the cervical-distal regions of the winglets, channel and in the cervical region of the bracket base. after plaque collection, initial dilution and homogeniza-tion were performed in a mechanical vibrator, using 1 ml of sterile saline solution composed of 0.85% sodium chloride and 1% sodium thioglycollate as reducing agent for each 1mg of plaque collected. after this 0.1 ml aliquots of each dilution were seeded in petri dishes containing the culture media mitis salivarius (ms) specific for of s. mutans and bhi broth, non-specific for bacteria and fungi. the dishes were incubated in an oven for bacterial growth for periods of 24, 48 and 72 h. colonies formed in all the culture media were macroscopically identified and counted by visual inspection. the valuation periods were 24, 48 and 72 h. statistical analysis data were analyzed with the spss 13.0 program (spss inc., chicago, il, usa). the number of colonies found in ms and bhi media was analyzed statistically by application of the wilcoxon test (p-value <0.05). descriptive statistics included the means of the evaluated groups. sem analysis after removal from the left side of the arches, the brackets were fixed in an ascending ethanol series and thereafter any residual water was eliminated by critical point drying (cpd030 critical point dryer; bal-tec ag, balzers, liechtenstein). the dehydrated parts were fixed on stubs with a silver-based adhesive, sputter-coated with gold, placed on acrylic well plates and examined with a scanning electron microscope (scanning microscope jeol-sm 5310, tokyo, japan). results the cfu counts obtained from the plaque collected from tooth surfaces before bracket placement are shown in table1. the quantitative results of the number of bacterial ufc s mutans ufc bhi groups mean cervical-buccal 5.4 10.4 mesial interproximal 3.6 14.2 distal interproximal 3.4 19.4 table 1 mean colony forming unit values before bracket placement. colonies formed in the plaque adhered to the winglets of brackets revealed that group 3m provided adherence of the largest number of colonies (p<0.05) followed by aditek, gac, ormco and the conventional group. when the collection site was the channels, the brackets showing the greatest accumulation were the ormco brand (p<0.05) and those that formed the fewest colonies were the conventional type. in turn, in the cervical region the aditek brackets were those that formed most colonies, followed by gac, as shown in table 2. it is worth pointing out that the values found were similar for s. mutans and for the non-specific microorganisms. the analysis of the brackets removed from the oral cavity under sem revealed larger plaque accumulation on fig. 3. sem micrograph of brackets with adhered microorganism (35x magnification); a) conventional; b) gac; c) aditek; d) ormco and e) 3m. do self-ligating brackets favor greater bacterial aggregation? braz j oral sci. 10(3):208-212 groups f i n slot cervical ufcs mutans ufc bhi ufcs mutans ufc bhi ufcs mutans ufc bhi mean* stat.** mean* stat.** mean* stat.** mean* stat.** mean* stat.** mean* stat.** conventional 6.2 a 1.4 a 628.8 a 610.6 a 1039.8 a 989.8 a g a c 7.4 a 3.6 a 1696 b 1283.8 c 1927 c 1214.4 c aditek 20.4 b 65.4 b 1500 b 100.8 b 2224.8 d 1764.2 d ormco 7 a 1.8 a 2447.6 c 1361.8 c 1772 bc 1198 bc 3 m 563.4 c 432.6 c 1397.8 c 901.4 b 1696.4 bc 1082.6 ab table 2 mean colony forming unit values and statistical analysis for the different groups of brackets. * mean colony forming unit values; **same letters indicate no statistically significant difference (p>0.05) the winglets of 3m brackets, followed by aditek, gac, ormco and the conventional type. the channels of ormco brackets were those that accumulated greater amount of plaque, followed by gac, aditek and 3m. in the cervical region the largest plaque accumulation could be seen on the aditek brackets, followed by the gac, 3m, and ormco types (figure 3). in all the evaluated regions, the conventional brackets had the least plaque accumulation. discussion in the present study, the proposal was to make a quantitative evaluation of the number of cfu present on conventional and self-ligating brackets of different designs. the evaluation was made in vivo in a model that individuals who had never undergone orthodontic treatment were used as hosts to the brackets. initially, the oral hygiene of all patients was standardized to prevent individual variations from compromising the results. the use of any medications during the experimental period was not allowed in order to avoid alteration of the microbiota. bacterial plaque was collected before bracket bonding. the purpose of this procedure was to quantify the number of cfu present on the tooth surface. the regions of choice were the cervical-buccal, mesial and distal interproximal regions, as these areas are the most difficult to clean from an anatomical point of view. the obtained results showed that in spite of the tooth surfaces being free for correct cleaning, they housed bacteria. for s. mutans counts the cervical-buccal region (5.4 cfu) was where microorganisms most concentrated, followed by the mesial (3.6 cfu) and distal (3.4 cfu) interproximal regions. as regards the non-specific bacterial counts, the interproximal regions were those with more cfu (19.4 cfu in the distal and 14.2 cfu in the mesial) followed by the cervical-buccal (10.4 cfu) regions. the culture media used for seeding petri dishes were ms, specific for growing s. mutans, while bhi broth, a nonspecific medium for bacteria and fungi. the choice for ms was made because of the well known role of s. mutans in dental caries development. the literature has stated that one of the major problems resulting from the use of an orthodontic appliance is the greater susceptibility to the development of dental caries13-15. the non-specific bhi medium was used to evaluate the presence of other microorganisms in order to determine whether the brackets favor the accumulation of s. mutans only or other microorganisms as well. there was a significant increase in both s. mutans and non-specific microorganisms that grew in the bhi medium in the three regions of the brackets: the winglet, channel and the cervical region of the bracket base as observed in previous works16-17. the winglet region was chosen because this was the easiest area to clean; the channel, because it was the most difficult; and the base, because access to this area is most difficult, and it is here that the largest number of carious lesion routinely occur after removal of the orthodontic appliance18. the winglets were the regions with the least presence of microorganisms when compared with the channel and cervical regions. in the comparison among the brackets, the conventional type presented lower mean values, however, without statistical differences from the winglets of the gac and ormco brackets (p>0.05). these results occurred in the evaluation of s. mutans and non-specific microorganisms, and can be attributed to the winglet design of the 3m and aditek brackets, which have larger grooves, favoring greater bacterial accumulation within them. in the channel and cervical regions, the number of colonies increased significantly, in some ways an expected result, due to the difficulty of access during cleaning. ormco brackets were those that presented the greatest contamination, followed by gac and aditek, which did not differ significantly from each other (p<0.05). these results are closely related to the designs of the brackets, which have a system for holding the wire, in such a way that they become tubes, preventing correct cleaning. the ormco brackets represent this well, because when they are closed, they prevent mechanical cleaning; the gac and aditek brands have a small opening in their cover, providing less plaque accumulation when compared with the ormco type. the brackets that showed the cleanest channels were the conventional type followed by the 3m brand. the design of the 3m brackets is very similar to that of the conventional type, providing closer results, in spite of being significantly different (p<0.05). in the cervical region, which was chosen for being a critical area for the appearance of white spots and caries18, the higher number of bacterial colonies was counted on the aditek brackets, with means of 2224.8 cfu for s. mutans and 211do self-ligating brackets favor greater bacterial aggregation? 212 braz j oral sci. 10(3):208-212 1764.2 for non-specific colonies. this result could be attributed to the angulation of the bracket body with the base, creating niches difficult to access with a toothbrush. the ormco brackets that presented many cfus in the channels showed far fewer cfu in the cervical region, presenting more colonies only than the conventional brackets. it is worth mentioning that in all the regions evaluated, the conventional brackets were shown to be more hygienic than the self-ligating type, a fact that is intimately related to their design these results are consistent with those found mota et al. 200813. in conjunction with the quantitative evaluation of the bacterial colonies, the brackets were submitted to sem to evaluate their design and the locations of greater bacterial plaque adherence. the microbiological results could be perfectly related to the morphological aspect of these brackets. when the winglets were verified, the 3m brackets presented greater irregularities, providing greater bacterial accumulation, as previously mentioned. in the ormco bracket channels, because they behave as a tube when they are closed, there was great accumulation of debris and thus a larger number of cfus. in the cervical region of aditek brackets, difficult access by toothbrush was shown as a result of the accentuated inclination from the base to the cervical region. the results of the present study differ from those of other works in the literature. pandis et al.9 performed s. mutans count in the saliva of patients using brackets of the conventional and self-ligating types and found no statistically significant differences. the differences found between pandis’ study and the present investigation might be due to differences in methodology, as in their study cfu were counted in saliva and not on bracket surfaces, as was proposed in the present study. one of the advantages that the literature mentions in using self-ligating brackets would be the reduction in cross-infection which, according to some authors would occur at the time of placing elastic or metal ties. the results of the present study suggest that concern should be focused elsewhere, considering that cross infection is completely controllable, depending on the dental team, whereas the significant increase in cfus adhered to the brackets is something over which the orthodontist has no control. it may be concluded that: conventional brackets favored less bacterial accumulation; selfligating brackets of different designs presented differences among the areas of less and greater bacterial accumulation; and the hypothesis that self-ligating brackets favor greater aggregation of microorganisms was proved. references 1. harradine nw. self-ligating brackets: where are we now? j orthod. 2003; 30: 262-73. 2. miles pg. self-ligating vs conventional twin brackets during en-masse space closure with sliding mechanics. am j orthod dentofacial orthop. 2007; 132: 223-5. 3. pandis n, polychronopoulou a, eliades t. self-ligating vs conventional brackets in the treatment of mandibular crowding: a prospective clinical trial of treatment duration and dental effects. am j orthod dentofacial orthop. 2007; 132: 208-15. 4. rinchuse dj, miles pg. self-ligating brackets: present and future. am j orthod dentofacial orthop. 2007; 132: 216-22. 5. de moura ms, de melo simplicio ah, cury ja. in-vivo effects of fluoridated antiplaque dentifrice and bonding material on enamel demineralization adjacent to orthodontic appliances. am j orthod dentofacial orthop. 2006; 130: 357-63. 6. ogaard b, rolla g, arends j, ten cate jm. orthodontic appliances and enamel demineralization. part 2. prevention and treatment of lesions. am j orthod dentofacial orthop. 1988; 94: 123-8. 7. marsh pd. dental diseases—are these examples of ecological catastrophes? int j dent hyg. 2006; 4 suppl 1: 3-10; discussion 50-12. 8. marsh pd. are dental diseases examples of ecological catastrophes? microbiology. 2003; 149: 279-94. 9. pandis n, papaioannou w, kontou e, nakou m, makou m, eliades t. salivary streptococcus mutans levels in patients with conventional and self-ligating brackets. eur j orthod. 2010; 32: 94-9. 10. gorelick l, geiger am, gwinnett aj. incidence of white spot formation after bonding and banding. am j orthod. 1982; 81: 93-8. 11. naranjo aa, trivino ml, jaramillo a, betancourth m, botero je. changes in the subgingival microbiota and periodontal parameters before and 3 months after bracket placement. am j orthod dentofacial orthop. 2006; 130: 275 e17-22. 12. reicheneder ca, gedrange t, berrisch s, proff p, baumert u, faltermeier a et al. conventionally ligated versus self-ligating metal brackets—a comparative study. eur j orthod. 2008; 30: 654-60. 13. mota sm, enoki c, ito iy, elias am, matsumoto ma. streptococcus mutans counts in plaque adjacent to orthodontic brackets bonded with resin-modified glass ionomer cement or resin-based composite. braz oral res. 2008; 22: 55-60. 14. svanberg m, ljunglof s, thilander b. streptococcus mutans and streptococcus sanguis in plaque from orthodontic bands and brackets. eur j orthod. 1984; 6: 132-6. 15. pramod s, kailasam v, padmanabhan s, chitharanjan ab. presence of cariogenic streptococci on various bracket materials detected by polymerase chain reaction. aust orthod j. 2011; 27: 46-51. 16. peros k, mestrovic s, anic-milosevic s, slaj m. salivary microbial and nonmicrobial parameters in children with fixed orthodontic appliances. angle orthod. 2011; 81: 901-6. 17. rego ro, oliveira ca, dos santos-pinto a, jordan sf, zambon jj, cirelli ja et al. clinical and microbiological studies of children and adolescents receiving orthodontic treatment. am j dent. 2011; 23: 317-23. 18. chaussain c, opsahl vital s, viallon v, vermelin l, haignere c, sixou m et al. interest in a new test for caries risk in adolescents undergoing orthodontic treatment. clin oral investig. 2010; 14: 177-85. do self-ligating brackets favor greater bacterial aggregation? oral sciences n3 braz j oral sci. 11(1):52-55 received for publication: august 26, 2011 accepted: january 06, 2012 original article braz j oral sci. january | march 2012 volume 11, number 1 effect of irrigants using ultrasonics on intracanal calcium hydroxide removal – an in vitro comparative evaluation s. anitha rao1, n. manasa2 1m.d.s, assistant professor, department of conservative dentistry and endodontics mamata dental college, khammam (a.p), india 2graduate student, department of conservative dentistry and endodontics mamata dental college, khammam (a.p), india correspondence to: s. anitha rao department of conservative dentistry and endodontics, mamata dental college giriprasad nagar, khammam 507002 andhra pradesh india phone: 011 91 9866957163 / 011 91 9849490529 fax: 011 91 08742255545 e-mail: anidental@yahoo.com abstract aim: to evaluate the efficacy of various irrigating solutions on calcium hydroxide (apex cal and rc cal) removal with the use of ultrasonics. methods: the root canals of 120 single-rooted maxillary central incisors were prepared using the stepback technique. the teeth were decoronated and split longitudinally. after filling, the two halves of roots were reassembled with sticky wax and each group was further divided into four subgroups according to the irrigating solution: smear clear, 10% citric acid, 5% edta and 3% naocl. evaluation for cleanliness was done under a microscope with ×12.5 magnification. statistical analysis was done with kruskal wallis and mann whitney tests at 5% level of significance. results: there was no statistically significant difference (p>0.05) for calcium hydroxide (apex cal and rc cal) removal by different irrigants. there were more residues in the apical groove than in the coronal groove (p<0.05). conclusions: when the different irrigants were compared at coronal and apical levels, smear clear and citric acid were more effective in calcium hydroxide removal than edta and naocl. keywords: smear clear, calcium hydroxide, root canal irrigants. introduction calcium hydroxide was introduced by herman in 1920. its clinical success is due to its alkaline ph and ability to rapidly disassociate into hydroxyl ions and calcium ions1. its clinical application includes use for root canal disinfection, induction of calcification response and promotion of apexification2. because of its antimicrobial activity, it has been recommended as an intracanal medication between sessions to eliminate persistent microorganisms, particularly in teeth with pulp necrosis and periradicular bone loss3. in vitro studies have shown that calcium hydroxide remnants can prevent the penetration of sealers into dentinal tubules, hinder the bonding of resin sealer adhesion to dentin, markedly increase the apical leakage of endodontically treated teeth, and potentially interact with zinc oxide eugenol sealers and make them brittle and granular1. removal of an intracanal dressing is usually performed by irrigation in combination with hand instrumentation up to working length. in straight root canals, recapitulation with the master apical file in combination with irrigation showed better removal of calcium hydroxide than an irrigant flush alone. rotary niti instruments facilitated calcium hydroxide removal from curved root canals without changing root canal anatomy4. 5353535353 stomas et al. (1987)5 compared cleanliness of canals and isthmuses prepared with hand, sonic, and ultrasonic instrumentation and concluded that the ultrasonic method provided better debridement than either the hand or sonic methods at 1 mm level. lev et al. (1987)6 compared the debridement achieved by hand instrumentation to the debridement achieved by using ultrasonics for 1 and 3 min following hand instrumentation. their results indicated that both ultrasonic groups were cleaner that the hand-instrumented group and that 3 min were better than 1 min in removing debris. the objective of the study was to evaluate the efficacy of removing calcium hydroxide with various root canal irrigating solutions in combination with ultrasonics. material and methods one hundred and twenty single-rooted maxillary central incisors with a straight root were selected. after preparing the canals by the stepback technique the teeth were decoronated with a diamond disc leaving a root length of 13 mm. apical enlargement was done to size 50 k file (dentsply maillefer, tulsa, ok, usa). the specimens were split longitudinally into two halves allowing subsequent reassembling. two standardized grooves were cut into root dentin with dimensions of 3.0 mm length, 0.5 mm width and 0.5 mm depth as checked with a caliper. one groove was placed coronally in one half and the other groove was placed apically in the other half (figure 1). the teeth were assigned to two groups based on the calcium hydroxide pastes: apex cal (calcium hydroxide, 400% al; ivoclar vivadent, andheri west, mumbai, india) and rc cal (calcium hydroxide, barium sulfate; prime dental products, mumbai, maharastra, india). after filling the grooves with the calcium hydroxide pastes with the aid of a spreader to ensure proper loading of the material in both grooves, the two halves were reassembled with sticky wax. the root canals were completely filled with calcium hydroxide paste and sealed coronally with cavit temporary filling (3m espe, st. paul, mn, usa). the teeth were incubated for 7 days at 37oc and 100% humidity. for removal of the calcium hydroxide paste, size #25 h file (dentsply maillefer, ballaigues, switzerland) was introduced into the root canal up to working length and fifteen up-anddown strokes were performed to loosen the medication. a #25 k ultrasonic file mounted on a piezoelectric handpiece was passively activated for 3 min with 10 ml of the respective solutions. the teeth were hand held at the time of ultrasonic irrigation. during the irrigation the #25 k ultrasonic file was moving in the root canal. each group was further divided into four subgroups (n=15) based on the solutions used during ultrasonic irrigation: smear clear (sybronendo, orange, ca, usa), 10% citric acid, 5% edta and 3% naocl. ten milliliter of each solution was used (figure 1). the root canals were dried with size 30 paper points and the roots were separated. the amount of remaining calcium hydroxide in the two longitudinal grooves was scored under the microscope with ×12.5 magnification using a scoring system described by van der sluis et al (2007)7. score 0: cavity is empty; score 1: less than half of the cavity is filled with calcium hydroxide, score 2: more than half of the cavity is filled with calcium hydroxide, score 3: cavity is completely filled with calcium hydroxide. data were analyzed statistically by the kruskal wallis and mann whitney test at 5% level of significance. pairwise comparison was performed separately for coronal and apical sections. results the scores were tabulated, subjected to analysis and the results showed that there were more residues in the apical groove than in the coronal groove (table 1 and 2). when the different irrigants were compared at coronal and apical levels, smear clear and citric acid were more effective in calcium hydroxide removal than edta and naocl (table 1 and 2). discussion calcium hydroxide paste is used as an intracanal medication due to its antimicrobial efficacy. it can reduce canal permeability by promoting the formation of calcium effect of irrigants using ultrasonics on intracanal calcium hydroxide removal – an in vitro comparative evaluation braz j oral sci. 11(1):52-55 fig. 1. composite figure of grooves and apica/coronal sections. (i). schematic presentation of size and location of grooves; (ii) microscopic images of apex cal coronal sections after irrigating with a) smear clear, b) edta, c) citric acid and d) naocl; (iii) microscopic images of apex cal apical sections after irrigating with a) smear clear, b) edta, c) citric acid and d) naocl; (iv) microscopic images of rc cal coronal sections after irrigating with a) smear clear, b) edta, c) citric acid and d) naocl; (v) microscopic images of rc cal apical sections after irrigating with a) smear clear, b) edta, c) citric acid and d) naocl. braz j oral sci. 11(1):52-55 5454545454 table 1.table 1.table 1.table 1.table 1. mean and standard deviations of apical sections of apex cal and rc cal groups. irrigant mean standard deviation 5% edta 1.1333 0.50742 10% citric acid 0.4333 0.62606 smear clear 0.3333 0.54667 3% naocl 2.2000 0.40684 total 1.0250 0.91176 irrigant mean standard deviation 5% edta 0.7667 0.62606 10% citric acid 0.2000 0.40684 smear clear 0.2000 0.40684 3% naocl 1.2000 0.40684 total 0.5917 0.62840 table 2.table 2.table 2.table 2.table 2. mean and standard deviations of coronal sections of apex cal and rc cal groups. carbonate particles and interfering with the sealing ability of endodontic sealer. therefore, several root canal irrigants have been tested for the complete removal of calcium hydroxide from root canals2. numerous solutions have been used as root canal irrigants. in the present study, the efficacy of commonly used irrigating solutions such as citric acid, edta, naocl and smear clear was compared. there are many studies reporting on the activity of smear clear on smear layer removal but only few studies are available on removal of calcium hydroxide. according to gutarts et al.8 (2005), the use of the ultrasonic needle after hand/rotary instrumentation resulted in significantly cleaner canals and isthmuses in the mesial roots of mandibular molars. ultrasonic irrigation was also more effective than syringe irrigation in removing artificially created dentin debris9. according to cameron et al.10 (1983), 1 min of ultrasound removed the superficial smear layer but left the dentinal tubules sealed off; 3 min of ultrasound removed the entire superficial smear layer and most of the dentinal tubule plug layer, and 5 min of ultrasound removed all debris in instrumented and uninstrumented areas except for a few dentin chips. the surface tension of an irrigating solution can be reduced with the addition of surfactants. smear clear is a 17% edta solution with 2 additional proprietary surfactants. the reason for the better efficacy of smear clear was that the reduction of surface tension of an endodontic irrigating solution by addition of surfactants should improve its efficacy in the narrow apical region of root canal. reducing surface tension of endodontic solutions improves their dentin wettability9. smear clear and citric acid were significantly more effective than naocl and edta. the results for the coronal groove were superior to those for the apical groove in terms of cleanliness because there could be a large number of chelating molecules in coronal root canal that were able to bind calcium ions. according to sen et al. 11, lower concentrations of 5% edta can be recommended for clinical use to avoid excessive erosion of root canal dentin. few authors reported a decreased efficacy of edta and citric acid at the apical level owing to the reduced quantity of solution contained in a smaller canal volume11-13. the groove model has an advantage of standardized size and location of the grooves which allows a standardized evaluation. the major disadvantage of this model is that it does not reproduce the complexity of a natural root canal system. hence, the removal of calcium hydroxide from artificial grooves may be easier than from isthmuses or oval extensions in vivo. digital imaging has been used for evaluation of calcium hydroxide removal but the main disadvantage is that only superficial layer of the calcium hydroxide could be quantified. evaluation of cleanliness is performed under the microscope allowing a three dimensional view of variations in depth of focus4. the basis for the passive ultrasonic irrigation is the transmission of energy from an ultrasonically oscillating instrument to the irrigant inside the root canal. it showed that an irrigating solution in addition with ultrasonic vibration is directly associated with the removal of organic or inorganic debris from the root canal walls14. comparing the root canal thirds, the results on the apical third are typically worse than the coronal third, proving the difficulty of cleaning this region15. calcium hydroxide when interacts with eugenol inhibits zinc oxide eugenol chelates formation. the interaction between calcium hydroxide and eugenol is rapid and kinetically dependent, leading to residual eugenol in the set product. the set zinc oxide eugenol cement and zinc oxide eugenol sealers in contact with calcium hydroxide are brittle in consistency and granular in structure13. when calcium hydroxide and zinc oxide eugenol sealer are mixed, a calcium hydroxide-eugenol compound is reported to be produced, with more solubility, less sealing ability, thicker film and higher water absorption value than the original sealer16. in another study, it was reported that calcium hydroxide and zinc oxide eugenol reacts to form calcium eugenol or calcium bonds to eugenol by an ionic bond which can be broken when water is present17. ultrasonic instrumentation of the root canals has been widely advocated as an effective modality for cleaning pulpal remnants and dentinal debris from canals and isthmuses. the mechanical agitation provided by ultrasonic instrumentation or a rotary file in conjunction with irrigation may also enhance removal of calcium hydroxide18. in conclusion, there were more calcium hydroxide residues in the apical groove than in the coronal groove. smear clear and citric acid performed better than edta and naocl in both coronal and apical grooves. in the coronal groove, naocl had the worst result. limitations for this study are that none of the irrigants were able to completely remove the calcium hydroxide. further development of new irrigants for the complete removal of calcium hydroxide is needed. references 1. nandini s, velmurugan n, kandaswamy d. removal efficiency of calcium hydroxide intracanal medicament with two calcium chelators: volumetric analysis using spiral ct, an in vitro study. j endod. 2006; 32: 1097-1. effect of irrigants using ultrasonics on intracanal calcium hydroxide removal – an in vitro comparative evaluation 5555555555 braz j oral sci. 11(1):52-55 2. pacios mg, de la casa ml, de los angeles bulacio m, lópez me.. calcium hydroxide association with different vehicles: in vitro action on some dentinal components. oral surg oral med oral pathol oral radiol endod. 2003; 96: 96-1. 3. de souza cas, teles rp, souto r, chaves mae, colombo apv. ntic therapy associated with calcium hydroxide as an intracanal dressing: microbiologic evaluation by the checkerboard dna-dna hybridization technique. j endod. 2005; 31: 79-83. 4. rodig. t, vogel. s, zapf. a, hulsmann.m. efficacy of different irrigants in the removal of calcium hydroxide from root canals. int endod j. 2010; 43: 519-7. 5. stomas d. sadeghi e, haasch g, gerstein h. an in vitro comparison study to quantitate the debridement ability of hand, sonic and ultrasonic instrumentation. j endod. 1987; 13: 434-40. 6. lev r, reader a, beck m, meyers w. an in vitro comparison of the step back technique versus step back/ultrasonic technique for 1 and 3 minutes. j endod. 1987; 13: 523-30. 7. van der sluis lw, wu mk, wesselink pr. the evaluation of removal of calcium hydroxide paste from an artificial standardized groove in the apical root canal using different irrigation methodologies. int endod j. 2007; 40: 52-7. 8. gutarts r, nusstein j, al reader, beck m. in vivo debridement efficacy of ultrasonic irrigation following hand rotary instrumentation in human mandibular molars. j endod. 2005; 31: 166-70. 9. lui jn, kuah hg, chen nn. effect of edta with/without surfactants or ultrasonics on removal of smear layer. j endod. 2007; 33: 472-5. 10. cameron ja. the use of ultrasonics in the removal of the smear layer: a scanning electron microscope study. j endod. 1983; 9: 289-92. 11. sen bh, ertürk o, piºkin b. the effect of different concentrations of edta on instrumented root canal walls. oral surg oral med oral pathol oral radiol endod. 2009; 108: 622-7. 12. hennequin m, pajot j, aviqnant d. effects of different ph values of citric acid solutions on the calcium and phosphorus contents of human root dentin. j endod. 1994; 20: 551-4. 13. hulsmann m, heckendorff, lennon a. chelating agents in root canal treatment: mode of action and indications for their use. int endod j. 2003; 36: 810-30. 14. teixeira cs, felippe mcs, felippewt. the effect of application time of edta and naocl on intracanal smear layer removal: an sem analysis. int endod j. 2005; 38: 285-90. 15. balvedi rpa, versiani ma, manna ff, biffi jcg. a comparison of two techniques for the removal of calcium hydroxide from root canals. int endod j. 2010; 43: 763-8. 16. salgado rj, moura-netto c, yamazaki ak, cardoso ln, de moura aa, prokopowitsch i. comparison of different irrigants on calcium hydroxide medication removal: microscopic cleanliness evaluation. oral surg oral med oral pathol oral radiol endod. 2009; 107: 580-4. 17. kenee dm, allemang jd, johnson jd, hellstein j, nichol bk. a quantitative assessment of efficacy of various calcium hydroxide removal techniques. j endod. 2006; 32: 563-5. 18. margelos j, eliades g, verdelis c, palaghias g. interaction of calcium hydroxide with zinc oxide eugenol type sealers: a potential clinical problem. j endod. 1997; 23: 43-8. effect of irrigants using ultrasonics on intracanal calcium hydroxide removal – an in vitro comparative evaluation oral sciences n3 original article braz j oral sci. january/march 2010 volume 9, number 1 longitudinal study on skeletal changes during and after bionator therapy using metallic implants andré da costa monini1, luiz gonzaga gandini júnior2, lídia parsekian martins3, dirceu barnabé raveli3 1 dds, ms. graduate student, department of orthodontics and pediatric dentistry, araraquara dental school, são paulo state university, brazil 2 dds, ms, ph d., professor, department of orthodontics and pediatric dentistry, araraquara dental school, são paulo state university, brazil; adjunct clinical professor, baylor college of dentistry, dallas, tx, usa and saint louis university, saint louis, mo, usa. 3 dds, ms, ph d. professor, department of orthodontics and pediatric dentistry, araraquara dental school, são paulo state university, brazil correspondence to: luiz gonzaga gandini júnior av. casemiro perez, 560, vila harmonia araraquara, são paulo, brasil. cep 14802-600 e-mail: luizgandini@uol.com.br received for publication: november 26, 2009 accepted: march 8, 2010 abstract aim: to demonstrate the magnitude and direction of skeletal changes in the maxilla and mandible during and after the use of bionator, as well as their rotations. methods: partial superimposition on the maxilla and mandible on the metallic implants and total superimposition on the cranial base were performed at three periods, t1 before bionator therapy, t2 after bionator therapy, and t3 5.68 years after t2. results: there was total clockwise maxillary rotation and counterclockwise mandibular rotation, in the north american technique, throughout the study period, as well as extensive remodeling on the condylar region, especially in vertical direction and on the gonial region in horizontal direction. conclusions: the total maxillary rotation seemed to be significantly affected by therapy than the mandible. there was a clear change in the direction of condylar remodeling compared to the period of bionator therapy and posterior bionator therapy. considering the entire study period, it was observed that intra-matrix rotation of the maxilla and mandible masked their total rotation, causing minimum changes in the matrix rotation. keywords: angle’s class ii malocclusion, activator appliances, maxillofacial development. introduction in the class ii malocclusion, mandibular retrusion seems to be a common characteristic and the major factor contributing to the problem1. therefore, the best therapeutic approaches should promote forward mandibular positioning. although these appliances are able to redirect the condylar growth, there is no consensus on their capacity to increase the amount of condylar growth2-7. in the maxilla, these appliances may promote some restriction of sagittal growth3,8. after correction of the distal occlusion, there is a tendency to return to the original condition, both dental and skeletal5-6. cephalometric studies with superimposition on metallic implants were demonstrated to be the most effective method for longitudinal evaluation of craniofacial growth on cephalograms9-11 and thus are also valid to evaluate the treatment changes. no study has longitudinally analyzed the maxillary and mandibular remodeling in patients submitted to treatment of class ii malocclusion with bionator by partial superimposition on metallic implants. this study evaluated the magnitude and direction of skeletal changes occurring in the maxilla and mandible during and after bionator therapy, as well as their rotations, using lateral cephalograms of patients with metallic implants. material and methods this study was approved by research ethic committee of the foar-unesp braz j oral sci. 9(1):33-38 34 (no. 0006.0.199.000-07) and informed consent was obtained from the parents of all patients. the treated sample comprised 25 patients who used bionator, who participated in a previous study2. the design and treatment approach is described in the previous study2. all patients presented clinically observed skeletal class ii malocclusion with mandibular retrusion, erupted or erupting maxillary and mandibular incisors, deep bite, no missing teeth, absence of crowding, and/or posterior crossbite. the patients also presented four metallic implants placed in the maxilla and three in the mandible, as suggested by björk12-13. from the original sample of 25 patients, it was possible to obtain long-term cephalograms of 13 patients (table 1 and 2), nine males and four females with 9.34 years at t1, 11.21 at t2 and 16.89 at t3. eleven of these patients (84.6%) received complementary treatment with fixed appliance and class ii elastics after the bionator therapy. the other patients could not be contacted. lateral cephalograms were obtained at three periods: t1, at the onset of bionator therapy; t2, at the completion of bionator therapy; and t3, 5.68 years after t2 in the average. the cephalograms were manually traced, the cephalometric points were digitized twice on the software spss version 10.0 (spss inc., chicago, il, usa) by a single examiner, and the mean of digitizations was used for the cephalometric measurements. the cephalometric points analyzed are described in table 3. a reference system composed of a horizontal line (hl) and a perpendicular vertical line (pvl) was defined on the cephalogram t1 and transferred to the other cephalograms based on the partial superimposition on maxillary and mandibular metallic implants. initially, three fiducial points were identified on the tracing t1, the first in front of the maxilla and mandible, the second behind, and the third above this (figures 1 and 2). the hl and pvl were defined as follows: horizontal line (hl): line contacting the anterior fiducial point and the posterior fiducial point. the line corresponds to the x axis of the reference system. perpendicular vertical line (pvl): line perpendicular to hl through the upper fiducial point. the line corresponds to the y axis of the reference system (figures 1 and 2). fig. 1. partial superimposition on the maxilla, transference of the three fiducial points (1, posterior fiducial point-pfp; 2, anterior fiducial point-afp; 3upper fiducial point-ufp), and construction of hl(horizontal line) and pvl(posterior vertical line). fig. 2. partial superimposition on the mandible, transference of the three fiducial points, and construction of hl(horizontal line) and pvl(posterior vertical line). males 9 9.25 1.39 11.08 1.28 16.99 1.62 females 4 9.55 1.01 11.52 1.7 16.68 2.9 total 13 9.34 1.25 11.21 1.36 16.89 1.72 s d mean (years) s d mean (years) s d mean (years)nindividuals t1 t2 t3 table 1 characteristics of the study sample t1 (treatment onset); t2 (completion of bionator therapy); t3 (final evaluation). the changes occurring in cephalometric points during and after treatment were evaluated in relation to the reference system. for example, the horizontal change in the position of the gonion (go) was measured parallel to hl, and the vertical change was measured parallel to pvl (figure 2). negative values correspond to backward and/or upward displacement. positive values were assigned to forward and/ or downward displacement. the displacement of all cephalometric points was measured. to enhance the observation of direction of displacement of cephalometric points, each point was followed by a reference. for example, the horizontal displacement of go is represented by go h, the vertical displacement of point go is represented by go v, and the total displacement of point go is represented by go t. the radiographic magnification was corrected using a correction coefficient of 0.91. the total and matrix rotations of the mandible and maxilla11 were defined by the angular change of hl transferred from t1 to the others by total superimposition on the cranial base11 in relation to the implant line (total rotation) and the mandibular and palatal planes (matrix rotation), respectively, braz j oral sci. 9(1):33-38 longitudinal study on skeletal changes during and after bionator therapy using metallic implants s n a 82.92º 4.0 81.53º 3.9 81.26º 4.4 s n b 76.75º 3.5 77.56º 3.9 78.20º 4.3 a n b 6.17º 1.9 3.96º 2.3 3.05º 2.6 sn gome 32.91º 5.3 33.57º 5.9 31.73º 6.4 f m a 23.49º 3.8 23.86o 4.4 22.33º 5.4 sn-ans-pns 5.98º 2.7 6.78º 4.2 6.43º 3.5 mean sd mean sd mean sd t1 t2 t3measurements table 2 sagittal and vertical angular cephalometric measurements. 35 cephalometric points description 1. pfp posterior fiducial point 2. afp anterior fiducial point 3. ufp upper fiducial point 4. pns (posterior nasal spine) most posterior point of the posterior nasal spine 5. ans (anterior nasal spine) most anterior point of the anterior nasal spine 6. a (subspinale) most posterior point on the maxillary anterior concavity 7. b (supramentale) most anterior point on the mandibular anterior concavity 8. pg (pogonion) most anterior point on the anterior symphyseal contour 9. gn (gnathion) most anterior and inferior point on the anterior symphyseal contour 10. me (menton) most inferior point on the symphyseal contour 11. ag (antegonial) most superior point on the antegonial champher 12. infgo (inferior gonion) most inferior point of the gonial angle 13. go (gonion) most posterior and inferior point of the gonial angle 14. postgo (posterior gonion) most posterior point of the gonial angle 15. ar (articulare) intersection between the posterior cranial base and the posterior condylar surface 16. co (condylion) most posterior and superior point of the mandibular condyle 17. cosup (superior condylion) most superior point of the mandibular condyle table 3 digitized cephalometric points. in the north american technique. the intra-matrix rotation11 (remodeling) was determined by the angular change in the implant line in relation to the mandibular and palatal planes, respectively. for the total and matrix rotation, positive values indicated clockwise rotation and negative values represented counterclockwise rotation. concerning the intra-matrix rotation, positive values indicated that the mandibular or palatal plane exhibited clockwise rotation in relation to the implant line, whereas negative values indicated counterclockwise rotation. statistical analysis the mean and standard deviation were calculated for each variable. all the variables submitted a normality test (skewness and kurtosis) and showed normal distribution. the differences among the three periods (t2–t1, t3–t2, and t3– t1) were calculated and the one-sample t-test was used to evaluate the significance of changes in those differences. the significance level was set at 5%. all calculations were performed using spss for windows (version 10.0,spss inc., chicago, il, usa.). to evaluate the error in the location of cephalometric points and digitization procedures, all tracings were redigitized after 2 weeks by the same examiner. the casual method error (dahlberg formula) did not exceed 0.33º or 0.88mm. the paired t-test (systematic errors) revealed statistically significant systematic error for only 5 of the 112 measurements performed (postgo v at t1 and t2, me v at t3, infgo h at t3, and pns v at t1). the variation of all measurements was between -0.17 and 0.34 mm for linear measurements and -0.13º and 0.03º for angular measurements. results superimposition on the maxilla during bionator therapy, only the pns exhibited significant change in horizontal direction (mean = -1.52 mm; p = 0.006). evaluation of the total displacement revealed that all points exhibited statistically significant change. after bionator therapy, point a presented significant vertical change (mean 1.78 mm; p=0.007) and the pns in the horizontal direction (mean -2.65 mm; p=0.000). observation of the entire study period revealed significant horizontal change in ans and pns and significant vertical change in point a (figure 3 and table 4). pns v 0.42 (0.9) 0.142 n 0.48 (1.8) 0.354 n 0.91 (1.8) 0.097 n ans v -0.10 (1.4) 0.806 n 0.51 (1.4) 0.233 n 0.41 (1.4) 0.320 n a v -0.06 (1.2) 0.863 n 1.78 (1.9) 0.007* 1.72 (2.0) 0.011* pns h -1.52 (1.6) 0.006 * -2.65 (1.3) 0.000 * -4.18 (2.0) 0.000* ans h 0.41 (1.8) 0.435 n 1.28 (2.9) 0.136 n 1.70 (2.5) 0.035 * a h -0.24 (0.7) 0.292n -0.23 (1.1) 0.493 n -0.47 (1.4) 0.249 n pns t 2.10 (1.2) 0.000 * 3.20 (1.3) 0.000 * 4.77 (1.6) 0.000 * ans t 1.82 (1.4) 0.001 * 2.88 (1.9) 0.000 * 2.95 (1.5) 0.000 * a t 1.33 (0.6) 0.000 * 2.23 (1.8) 0.001 * 2.70 (1.3) 0.000 * table 4 maxillary growth (vertical, horizontal and total) and remodeling (in mm). * = statistically significant values (p < 0.05); n = non statistically significant values. mean (sd) “p” mean (sd) “p” mean (sd) “p” t2 t1 t3-t2 t3-t1variable braz j oral sci. 9(1):33-38 longitudinal study on skeletal changes during and after bionator therapy using metallic implants fig. 3. maxillary remodeling between t1 and t2 (upper data) and between t2 and t3 (lower data). the horizontal/vertical components are proportional in scale. each square represents 0.5 mm. 36 fig. 4. condylar remodeling between t1 and t2 (upper data) and between t2 and t3 (lower data). the horizontal/vertical components are proportional in scale. each square represents 1 mm. fig. 5. remodeling of the gonial angle between t1 and t2 (upper data) and between t2 and t3 (lower data). horizontal/vertical components are proportional in scale. each square represents 1 mm. b v 0.09 (1.3) 0.807 n -1.40 (2.1) 0.038 * -1.30 (2.0) 0.045 * pg v 0.85 (1.3) 0.038 * 0.45 (1.7) 0.376 n 1.31 (2.0) 0.043 * gn v 0.47 (1.1) 0.169 n 0.97 (1.3) 0.019 * 1.45 (1.6) 0.008 * me v 0.23 (0.8) 0.329 n 1.36 (0.8) 0.000 * 1.60 (1.1) 0.000 * a go v -2.68 (2.5) 0.003 * -2.68 (1.8) 0.000 * -5.37 (2.1) 0.000 * inf go v -2.96 (3.0) 0.004 * -4.06 (1.9) 0.000 * -7.03 (3.4) 0.000 * go v -3.32 (4.5) 0.022 * -6.34 (2.5) 0.000 * -9.67 (5.7) 0.000 * post go v -3.20 (5.1) 0.044 * -9.19 (3.7) 0.000 * -12.39 (6.4) 0.000 * ar v -5.71 (2.4) 0.000 * -13.28 (3.1)0.000* -18.99 (3.6) 0.000 * co v -5.77 (3.4) 0.000 * -14.22 (4.5)0.000* -20.00 (4.0)0.000 * co sup v -5.48 (3.1) 0.000 * -14.13 (4.0) 0.000* -19.62 (3.6)0.000 * b h 0.12 (0.4) 0.329 n 0.06 (0.6) 0.705 n 0.19 (0.6) 0.279 n pg h -0.46 (0.9) 0.096 n -0.29 (0.6) 0.145 n -0.76 (1.2) 0.046 * gn h -0.49 (1.2) 0.169 n -0.39(0.8) 0.112 n -0.89 (1.6) 0.083 n me h -0.26 (1.2) 0.468 n -0.69 (1.0) 0.031 n -0.96 (1.8) 0.089 n a go h -3.57 (2.6) 0.000 * -3.96 (2.7) 0.000 * -7.55 (1.8) 0.000 * inf go h -3.17 (3.0) 0.002 * -5.32 (1.7) 0.000 * -8.50 (2.3) 0.000 * go h -2.10 (2.1) 0.005 * -6.02 (1.6) 0.000 * -8.12 (1.7) 0.000 * post go h -1.71 (1.6) 0.003 * -5.09 (1.6) 0.000 * -6.81 (1.5) 0.000 * ar h -1.42 (2.2) 0.039 * -0.56 (2.2) 0.386 n -1.99 (3.7) 0.080 n co h -1.06 (2.8) 0.201 n 0.71 (2.9) 0.405 n -0.35 (4.9) 0.802 n co sup h -1.23 (2.9) 0.160 n 1.24 (2.9) 0.150 n 0.01 (4.7) 0.993 n b t 1.19 (0.7) 0.000 * 1.92 (1.7) 0.002 * 1.84 (1.7) 0.002 * pg t 1.40 (1.2) 0.001 * 1.57 (1.1) 0.000 * 2.08 (1.9) 0.002 * gn t 1.50 (0.9) 0.000 * 1.52 (1.0) 0.000 * 2.32 (1.7) 0.000 * me t 1.19 (0.9) 0.001 * 1.81 (0.9) 0.000 * 2.44 (1.4) 0.000 * a go t 4.81 (3.2) 0.000 * 4.91 (3.1) 0.000 * 9.37 (2.4) 0.000 * inf go t 4.87 (3.5) 0.000 * 6.85 (2.0) 0.000 * 11.21 (3.6) 0.000 * go t 4.94 (3.9) 0.001 * 8.93 (2.3) 0.000* 12.97 (5.1) 0.000 * post go t 5.26 (3.6) 0.000 * 10.69 (3.4) 0.000 * 14.63 (5.3) 0.000 * ar t 6.31 (2.3) 0.000 * 13.44 (3.2) 0.000 * 19.41 (3.7) 0.000 * co t 6.56 (3.2) 0.000 * 14.54 (4.4) 0.000 * 20.52 (4.3) 0.000 * co sup t 6.37 (2.9) 0.000 * 14.52 (3.8) 0.000 * 20.11 (3.8) 0.000 * mean (sd) “p” mean (sd) “p” mean (sd) “p” t2 t1 t3-t2 t3-t1variable table 5 mandibular growth (vertical, horizontal and total) and remodeling (in mm). * = statistically significant values (p < 0.05); n = non statistically significant values. superimposition on the mandible during the bionator treatment period, there was statistically significant vertical change in the following cephalometric points: pg, ag, infgo, go, postgo, ar, co, and cosup. in the horizontal direction, the following cephalometric points presented a statistically significant change: ag, infgo, go, postgo, and ar. after bionator therapy, statistically significant vertical change was observed for the cephalometric points ag, infgo, go, postgo, ar, co, cosup, me, gn, and point b. in the horizontal direction, only the points related to the gonial angle exhibited significant change. no significant change was revealed in a horizontal direction only for the points b, gn, me, ar, co, and cosup during observation of the entire study period (figures 4 and 5 and table 5). maxillary and mandibular rotation: table 6 shows that during the period of bionator therapy, only the total mandibular rotation presented significant change (mean = -1.74º; p = 0.047). after bionator therapy, the total mandibular rotation (mean= -3.96º; p= 0.004) and mandibular intra-matrix rotation (mean= 2.78º; p=0.004) exhibited significant change. during the study period, no significant change was observed in matrix rotations for both the maxilla and the mandible. discussion despite the small sample size and lack of control group, this study may be considered of interest to the scientific community because of the presence of metallic implants and longitudinal follow-up of the same patients. this allowed a detailed analysis of maxillary and mandibular growth in patients with class ii malocclusion submitted to orthopedic treatment using the balters functional appliance. braz j oral sci. 9(1):33-38 longitudinal study on skeletal changes during and after bionator therapy using metallic implants 37 matrix maxilla 0.29 (1.8) 0.583n 0.20 (2.2) 0.753n 0.49 (2.7) 0.524n matrix mandible 0.04 (2.3) 0.950n -1.21 (3.3) 0.213n -1.17 (3.2) 0.214n total maxilla 1.36 (2.5) 0.075n 1.49 (4.2) 0.225n 2.85 (4.6) 0.045* total mandible -1.74 (2.8) 0.047* -3.96 (3.9) 0.004* -5.7 (4.9) 0.001* intra-matrix maxilla -1.30 (3.5) 0.208n -1.17 (3.9) 0.307n -2.47 (3.8) 0.038* intra-matrix mandible 1.79 (4.6) 0.189n 2.78 (2.8) 0.004* 4.57 (5.2) 0.008* mean (sd) “p” mean (sd) “p” mean (sd) “p” t2 t1 t3-t2 t3-t1 variable table 6 . maxillary and mandibular rotation. * = statistically significant values (p < 0.05); n = non statistically significant values. there was progressive closure of the angle of the mandibular implant line (counterclockwise rotation), that is, the total mandibular rotation 9,11,14-15. in addition to the treatment period, there was counterclockwise rotation throughout the study period. araújo et al.2 demonstrated that the bionator was able to inhibit the total counterclockwise mandibular rotation. the rotation observed in the present study (1.74º) was smaller than that observed by araújo et al. 2 (2.53º) in the control group and higher than the counterclockwise rotation of only 0.17º of the treated group. this confirmed that the appliance has the same influence, that is, inhibiting the total counterclockwise mandibular rotation6,16. björk and skieller11 published a case treated with headgear and observed inhibition of counterclockwise mandibular rotation during treatment, which returned after treatment. this is in agreement with the results of melsen17 using the same appliance, and pancherz et al.6 using the herbst appliance. the difference in the quantity of rotation between this study and the findings of araújo et al.2 may be explained by the longer treatment period of this study, that is, 2 years compared to 1 year. moreover, as the need of utilization of the appliance is reduced, its influence is also reduced, allowing the natural rotation expected by growth to occur. the 5.7º mandibular rotation evaluated from t1-t3 was greater than that reported by buschang and gandini jr14, and kim and nielsen18, 2º to 3.3º and 3.5º, respectively. this is smaller than the values observed by lavergne and gasson19, 12.8º, björk and skieller11, 8.6º, and very close to the value observed by lee et al. 20, 5.8º. this could probably be because of the longer follow-up period of this study when compared to other studies that had follow-up periods of 7 years14 and 5 years18. however, it is shorter than the 12-year follow-up by lavergne and gasson19 and 15-year follow-up by björk and skieller 11, and similar to that of lee et al. 20 several studies9,14,16,19,21 have revealed that patients with greater counterclockwise rotation exhibit more vertical condylar growth. analysis of the rotation occurring per year reveals a mean of 0.77o/year, close to the values observed by other authors11,16,20-22 (table 7), demonstrating that the influence of bionator is reduced with time. in the maxilla, the total rotation occurred in clockwise direction and in lower intensity. the most variable behavior of maxillary rotation, which may occur in two directions and in lower intensity compared to the mandibular rotation, is reported in the literature10,15,23 and agrees with the present findings, revealing a variation of -4.6º to 10.8º. the results of this study also revealed clockwise maxillary rotation during and after bionator therapy, which was greater during treatment, 0.8º/year, than after treatment, 0.23º/year. the total counterclockwise maxillary rotation is the most common910,15,24 and the clockwise rotation might be an outcome of treatment using headgear17 and the activator7. clockwise maxillary rotation was also observed after bionator therapy, considering that these patients received fixed appliances and used class ii elastics at some period, the influence of the entire class ii mechanics on the total clockwise maxillary rotation may be inferred as melsen17 observed inversion in the direction of total maxillary rotation after treatment of the class ii malocclusion. table 8 demonstrates that the smaller total maxillary rotations are related to studies whose samples also included treated patients, highlighting the influence of the mechanotherapy for class ii malocclusion in most cases, thus promoting clockwise maxillary rotation. solow and iseri24 observed 2.5º of posterior maxillary intra-matrix rotation from 8 to 15 years. this study also observed nearly 2.5º rotation in the anterior direction, probably as a remodeling to compensate for the total clockwise rotation occurring as a result of treatment10,12,23. as previously mentioned in the literature9-12,16, because there is total counterclockwise rotation and the matrix rotation is very subtle, there is remodeling of the mandibular base and palatal plane to compensate for the rotation. the maxilla exhibited clockwise total and matrix rotation, and remodeling occurred more by vertical displacement of the pns, compared with the ans. this is in agreement with the treated group of baumrind et al.25, suggesting that the orthodontic treatment may interfere with the maxillary remodeling, as untreated groups24-25 present greater vertical displacement of the ans than of the pns. the final outcome was an increase in the maxillary matrix rotation of only 0.5º in the present study and 1º in the study of solow and iseri24, revealing minimum braz j oral sci. 9(1):33-38 longitudinal study on skeletal changes during and after bionator therapy using metallic implants º gu, mcnamara21 i and ii a 20 -0.78º 9 -15.6 lavergne, gasson19 several a 26 -1.07º 7-19 araújo et al.2 ii t 14 -0.17º 9.5-10.5 araújo et al.2 ii u t 11 -2.53º 9-10 gasson, lavergne15,22 several a 22 -0.81º 7-16 björk, skieller9 i, ii and iii u t 21 -1º 6-year follow up björk, skieller11 i u t 9 -0.78º 4 -19.7 lee et al.20 several a 28 -0.74º 8.5-16.2 odegaard 16 several a 25 -0.78º 2.5-year follow up present study ii t 13 -0.77º 9.34-16.89 *t= treated patients; ut= untreated patients; a=both; + indicates clockwise rotation / indicates counterclockwise rotation. table 7. yearly changes in the mandibular total rotation in studies on metallic implants – available data. authorsreference type of malocclusion patients* n degrees/ year interval in years gasson, lavergne15,22 several a 22 -0.21º 7-16 solow, iseri24 several u t 14 -0.38º 8.5-12.5 björk, skieller9 i, ii and iii u t 19 -0.42º 6-year follow up björk, skieller11 i u t 9 -0.41º 10 -19.7 doppel et al.23 i, ii and iii a 50 -0.13º 12-16 present study ii t 13 +0.36º 9.34-16.89 authorsreference type of malocclusion patients* n degrees/ year interval in years * t = treated patients; ut = untreated patients; a = both; + indicates clockwise rotation / indicates counterclockwise rotation. table 8. yearly changes in the maxillary total rotation in studies on metallic implants – available data. 38 change in the palatal plane angle, in agreement with other authors12,15,23, as also observed in table 2. the results demonstrate nearly 4.6º of opening of the angle between the implant line and the mandibular base. this intra-matrix rotation occurred because of upward remodeling of the gonial angle and downward displacement of the me, as presented in table 5. during the treatment period, the mandibular matrix rotation remained unchanged (0.04º) and during the second period there was a slight closure (-1.21º), also identified by reduction in the angles sngome and fma (table 2). throughout the study period and also during the treatment, there was extensive backward and upward remodeling of the gonial region, especially of the go and postgo points, agreeing with previous reports.11-14,21,26 figure 5 reveals that this region did not exhibit changes in the direction of growth between the study periods, maybe because it is less susceptible to the environment14. at the symphyseal region, there was apposition on the lower part and upward repositioning of point b and downward repositioning of points pg, gn, and me, in agreement with other studies14,26. in the horizontal direction, there was stability of cephalometric points at the me region, agreeing with the studies of björk11,13. the condylar region exhibited the greatest vertical changes in the mandible14,26. during the treatment period, there was backward condylar growth2-6,8. after bionator therapy, the condylar growth was redirected in anterior direction, returning almost totally to the initial anteroposterior condylar positioning. studies11-13,26 revealed that the condyle presents upward and forward growth of nearly 6o in relation to the mandibular posterior border in untreated patients and forward growth of 10º in patients with total counterclockwise mandibular rotation. this information suggests that the backward growth obtained by treatment might be statistically significant when compared with a control group. some of the patients in the present sample were followed for 1 year without treatment in a previous study2 and presented anterior condylar growth during this period. after use of the bionator, faltin et al.4 demonstrated stability of condylar redirectioning in patients treated during the peak growth spurt. the results of this study do not support these findings3-4, probably because they were treated before the peak growth. however, the results agree with other studies5-6. the posterior displacement of point a observed in this study was nearly 0.5mm, probably because of the natural resorption occurring on the maxillary anterior region24-25 combined with the class ii mechanics employed25. in the vertical direction, the differences occurred because of the intra-matrix rotation, which presented a different direction in this study compared to earlier reports. concerning the pns point, the authors observed downward and backward displacement24. the pns always presents backward growth, both in the treated and untreated groups, and also exhibits greater displacement of the ans, which is the primary mechanism of maxillary growth25. in conclusion, the total maxillary rotation seemed to be significantly affected by therapy than the mandible. there was a clear change in the direction of condylar remodeling compared to the period of bionator therapy and posterior bionator therapy. considering the entire study period, it was observed that intramatrix rotation of the maxilla and mandible masked their total rotation, causing minimum changes in the matrix rotation. references 1. mcnamara jr ja. components of class ii malocclusion in children 8-10 years of age. angle orthod. 1981; 51: 177-202. 2. araújo am, buschang ph, melo acm. adaptive condylar growth and mandibular remodelling changes with bionator therapy -an implant study. eur j orthod. 2004; 26: 515-22. 3. croft rs, buschang ph, english jd, meyer r. a cephalometric and tomographic evaluation of herbst treatment in the mixed dentition. am j orthod dentofacial orthop. 1999; 116: 435-43. 4. faltin jr k, faltin rm, baccetti t, franchi l, ghiozzi b, mcnamara jr ja. long-term effectiveness and treatment timing for bionator therapy. angle orthod. 2003; 73: 221-30. 5. pancherz h, fischer s. amount and direction of temporomandibular joint growth changes in herbst treatment: a cephalometric long-term investigation. angle orthod. 2003; 73: 493-501. 6. pancherz h, ruf s, kohlhas p. “effective condylar growth” and chin position changes in herbst treatment: a cephalometric roentgenographic long-term study. am j orthod dentofacial orthop. 1998; 114: 437-46. 7. williams s, melsen b. the interplay between sagittal and vertical growth factors. an implant study of activator treatment. am j orthod. 1982; 81: 327-32. 8. melo acm, santos-pinto a, martins jcr, martins lp, sakima mt. orthopedic and orthodontic components of class ii, division 1 malocclusion corrrection with balters bionator: cephalometric study with metallic implants. world j orthod. 2003; 4: 237-42. 9. björk a, skieller v. facial development and tooth eruption. am j orthod. 1972; 62: 339-83. 10. björk a, skieller v. growth of the maxilla in three dimensions as revealed radiographically by the implant method. br j orthod. 1977; 4: 53-64. 11. björk a, skieller v. normal and abnormal growth of the mandible. a synthesis of longitudinal cephalometric implant studies over a period of 25 years. eur j orthod. 1983; 5: 1-46. 12. björk a. facial growth in man, studied with the aid of metallic implants. acta odontol scand. 1955; 13: 9-34. 13. björk a. variations in growth pattern of the human mandible: longitudinal radiographic study by the implant method. j dent res suppl. 1963; 42: 400-11. 14. buschang ph, gandini jr lg. mandibular skeletal growth and modeling between 10 and 15 years of age. eur j orthod. 2002; 24: 69-79. 15. gasson n, lavergne j. maxillary rotation during human growth: annual variation and correlations with mandibular rotation. a metal implant study. acta odontol scand. 1977; 35: 13-21. 16. odegaard j. mandibular rotation studied with the aid of metal implants. am j orthod. 1970; 58: 448-54. 17. melsen b. effects of cervical anchorage during and after treatment: an implant study. am j orthod. 1978; 73: 526-40. 18. kim j, nielsen il. a longitudinal study of condylar growth and mandibular rotation in untreated subjects with class ii malocclusion. angle orthod. 2002; 72; 105-11. 19. lavergne j, gasson n. a metal implant study of mandibular rotation. angle orthod. 1976; 46: 144-50. 20. lee rs, daniel fj, swartz m, baumrind s,korn el. assessment of method for prediction of mandibular rotation. am j orthod dentofacial orthop. 1987; 91: 395-402. 21. gu y, mcnamara jr ja. mandibular growth changes and cervical vertebral maturation: a cephalometric implant study. angle orthod. 2007; 77: 947-53. 22. gasson n, lavergne j. the maxillary rotation: its relation to the cranial base and the mandibular corpus.an implant study. acta odontol scand. 1977; 35: 89-94. 23. doppel dm, damon wm, joondeph dr, little rm. an investigation of maxillary superimposition techniques using metallic implants. am j orthod dentofacial orthop. 1994; 105: 161-8. 24. solow b, iseri h. maxillary growth revisited: an update based on recent implant studies. in: davidovitch z, norton la. biological mechanisms of tooth movement and craniofacial adaptation. boston: harvard society for the advancement of orthodontics; 1996. p.507-27. 25. baumrind s, korn el, ben-bassat y, west ee. quantitation of maxillary remodeling. 1.a description of osseous changes relative to superimposition on metallic implants. am j orthod dentofacial orthop. 1987; 91: 29-41. 26. baumrind s, ben-bassat y, korn el, bravo la, curry s. mandibular remodeling measured on cephalograms. 1.osseous changes relative to superimposition on metallic implants. am j orthod dentofacial orthop. 1992; 102: 134-42. braz j oral sci. 9(1):33-38 longitudinal study on skeletal changes during and after bionator therapy using metallic implants oral sciences n3 original article braz j oral sci. 8(4):206-209 fluoride content of bottled water commercialized in two cities of northeastern brazil consuelo fernanda macedo de souza1, suyene de oliveira paredes2, franklin delano soares forte3, fábio correia sampaio3 1 graduate student, health sciences center, federal university of paraiba, joao pessoa, brazil 2graduate program in dentistry, master’s degree program in preventive and pediatric dentistry, federal university of paraíba, joão pessoa, brazil 3phd, graduate program in dentistry, master’s degree program in preventive and pediatric dentistry, federal university of paraíba, joão pessoa, brazil correspondence to: consuelo fernanda macedo de souza universidade federal da paraíba, centro de ciências da saúde campus i, departamento de odontologia clínica e social. laboratório de biologia bucal. castelo branco; 58051900 joao pessoa, pb brasil e-mail: consuelofernanda79@hotmail.com abstract aim: the objectives of this study were to evaluate the fluoride content of bottled water commercialized in two cities of northeastern brazil and to compare the fluoride values measured in the water to the ones printed on the bottle label, considering risks (dental fluorosis) and benefits (caries control) of systemic fluoride exposure. methods: fifty-six water samples were collected from 20 brands available in several supermarkets with high turnover in different regions of the municipalities of são luís (state of maranhão) and joão pessoa (state of paraíba) in 2009. fluoride concentrations were determined by triplicate analysis using an ion-specific electrode. results: the measured mean fluoride content varied from 0.001 to 0.270 ppmf with a mean (±sd) of 0.037 (±0.041) for the 56 samples. the majority of samples were found to contain less than 0.043 ppmf (92%). conclusion: these results emphasize the importance of controlling the fluoride levels in bottled water enforced by the brazilian sanitary surveillance agency. concerning the risks and benefits, fluoride concentrations in the evaluated bottled water samples were below the suggested concentration (0.7 mg f/l), having neither preventive effect nor the potential for causing dental fluorosis. keywords: fluoride, bottled water, product labeling, health surveillance introduction the replacement of public water with bottled water for daily intake has been observed as a common trend among consumers in several countries1-7. in brazil, between 1974 and 2003 there was an increase of 5.694% in the consumption of bottled water. the southeastern region of the country is responsible for 56.4% of the production of bottled water followed by the northeastern region, holding 23.2% of the national production8. according to data from the national department of mineral production (dnpm) there was a total investment of r$ 44,644,273 in bottled water in the year of 2005 in brazil8. such an investment reflects the growing interest of consumers who use bottled water as their primary source of drinking water. reasons for this preference include concern about the purity of public tap water, avoidance of chemicals such as chlorine, taste preferences and convenience4,5,9-11. water fluoridation is a community health measure that is recognized worldwide for its role in preventing dental caries12. therefore, attention must be given not only to public drinking water, but also to bottled water, since nowadays bottled water is no longer regarded as a privilege, being widely consumed by people who have a healthier lifestyle as a priority7. some studies have shown several top selling brands containing a fluoride content above the recommended level, contributing to an increase in the incidence of dental fluorosis3,6,13-14. on the other hand, some products may have low fluoride content in their composition. consequently, if these bottled waters are used as the primary source of drinking and cooking water, they might not be providing a preventive measure for dental caries5,7,9. braz j oral sci. october/december 2009 volume 8, number 4 received for publication: october 16, 2009 accepted: january 15, 2010 207 braz j oral sci. 8(4):206-209 jp1 0.02 0.04 ± 0.002 0.029 0.042 jp2 ni* 0.01 ± 0.007 0.001 0.012 jp3 0.02 0.03 ± 0.001 0.023 0.041 jp4 0.04 0.02 ± 0.001 0.021 0.036 jp5 0.02 0.01 ± 0.001 0.011 0.017 jp6 0.02 0.03 ± 0.004 0.025 0.030 jp7 0.05 0.04 ± 0.002 0.036 0.040 jp8 0.21 0.23 ± 0.005 0.180 0.270 jp9 0.05 0.06 ± 0.001 0.059 0.080 jp10 0.01 0.03 ± 0.001 0.024 0.027 jp11 ni* 0.01 ± 0.001 0.010 0.016 sl1 0.01 0.03 ± 0.005 0.022 0.031 sl2 0.03 0.04 ± 0.010 0.033 0.053 sl3 0.01 0.01 ± 0.000 0.011 0.020 sl4 0.02 0.02 ± 0.005 0.010 0.024 sl5 0.03 0.04 ± 0.002 0.035 0.043 sl6 0.02 0.02 ± 0.003 0.010 0.022 sl7 0.05 0.04 ± 0.000 0.035 0.039 sl8 ni* 0.02 ± 0.008 0.021 0.036 sl9 0.01 0.02 ± 0.001 0.014 0.016 code fluoride label information (ppm) fluoride concentration (mean± sd) m i n i m u m (ppm) m a x i m u m (ppm) table 1. local of water fountains, labeled fluoride content, minimum fluoride concentration, maximum fluoride concentration and average fluoride content found on analysis. * not informed in the northeastern region of brazil, temperatures can range from 28 to 35ºc and higher consumption of water is observed. studies have revealed an increasingly greater incidence of dental fluorosis in this region13,15, which highlights the need for strict regulation and rigorous surveillance of the fluoride content in bottled water for the region. evidence of fluoride concentration in bottled water consumed in the northeastern region of brazil is scarce. hence, this study aimed at analyzing the concentration of fluoride in bottled waters commercialized in the cities of são luís, capital of the state of maranhão (ma), and joão pessoa, capital of the state of paraíba (pb), comparing the obtained values to the information given on the bottle labels, considering risks (dental fluorosis) and benefits (caries control) of systemic fluoride exposure. material and methods several brands of bottled water were purchased from supermarkets in the cities of joão pessoa (pb) and são luís (ma). whenever possible, 3 bottles of each brand, each with a different batch number and date of bottling, were purchased. all brands available on the market at the moment of purchase were analyzed for this research, except for brands of carbonated water. all samples were stored in 15 ml plastic vials at 10°c in the refrigerator until the moment of analysis. they were assigned an arabic number as a code so that those undertaking the analysis would be blind to the source. a fluoride-ion-specific electrode (model 9409 bn, orion, cambridge, ma, usa) and a potentiometer (model 720a, orion) were used for fluoride measurements. before starting the analysis, a calibration curve was made using known standard samples containing between 0.05 and 1.60 mg/l of fluoride, which were also used to construct standard curves. both standard solutions and water samples were prepared by mixing 1.0 ml of each sample to 1.0 ml of total ionic strength adjusting buffer ii (tisab ii), a substance used to adjust the total ionic strength and the ph of the sample. all samples, including the standard solutions, were mixed using a vortex and kept at room temperature (25oc) at the moment of reading. the calibration was repeated after every ten-sample reading. finally, millivolt readings were converted to fluoride ion concentration using the standard correlation curve. the reading was compared to the fluoride standard curve (r2>0.99). the data was entered into an excel spreadsheet (microsoft excel®) where mean and standard deviations were calculated. the correlation curve was used, as well as the correlation coefficient (r2 > 0.999). the data were statistically analyzed using a one-way anova with a tukey’s post-hoc multiple comparisons test. results fifty-six water samples from twenty brands were analyzed. thirtytwo of those were purchased in joão pessoa and 24 were bought in são luís between december 2008 and january 2009. the label fluoride content (when given), the measured mean fluoride content (±sd) and the minimum and maximum values are shown in table 1. the measured mean fluoride content varied from 0.001 to 0.270 mg f/l with a mean (±sd) of 0.037 (±0.041) for the fifty-six samples. the majority of samples were found to contain less than 0.043 mg f/l (92%) (table 1, figure 1). regarding the quality of the labeling of bottled waters, seventeen 200150100500 num ber of samples 0,30 0,25 0,20 0,15 0,10 0,05 0,00 f lu o ri d e c o n te n t (m g /l ) f lu o ri d e c o n te n t (m g /l ) fig. 1. variation of fluoride content (mg/l) in the bottled water brands commercialized in joão pessoa pb and são luís ma. (85%) of the twenty brands surveyed showed the fluoride content on the labels. upon analysis, ten brands (50%) of bottled water presented fluoride content higher than the value displayed on the label, whereas 4 (20%) brands showed a value lower than the one displayed on the label. three (15%) of the 20 brands did not exhibit any fluoride content on their labels. nevertheless, upon analysis, these brands were found fluoride content of bottled water commercialized in two cities of northeastern brazil 208 braz j oral sci. 8(4):206-209 jp1 0.04 0.04 0.03 <0.0001 jp2 0.01 0.01 0.01 0.01 jp3 0.03 0.04 0.02 <0.0001 jp4 0.04 0.02 0.03 <0.0001 jp5 0.01 0.01 0.02 0.01 jp6 0.03 0.01 0.01 <0.0001 jp7 0.04 0.04 0.03 0.002 jp8 0.27 0.19 <0.0001 jp9 0.06 0.07 0.06 0.008 jp10 0.03 0.02 0.02 0.97 jp11 0.01 0.02 0.01 <0.0001 sl1 0.03 0.03 0.04 0.002 sl2 0.04 0.04 0.871 sl3 0.01 0.01 0.02 0.461 sl4 0.02 0.02 0.01 0.097 sl5 0.04 0.04 0.03 0.004 sl6 0.02 0.02 0.02 0.554 sl7 0.04 0.04 <0.0001 sl8 0.03 0.02 0.02 0.103 sl9 0.02 0.02 0.66 batch #1 batch #2 batch #3 code fluoride concentration (mg/l) p value table 2 variation in fluoride content among batches and the p value. to have a small amount of fluoride content (ranging from 0.006 to 0.025). only 3 brands (15%) presented concordance between the fluoride content measured and that shown on the product label (table 1). of all the brands studied in this research, 85% were commercialized as fluoridated, even though they presented a fluoride content varying from 0.010 to 0.074 ppm f. discussion the majority of the world’s population is replacing public water with bottled water for the daily water intake. according to data from the dnpm, during the last thirty years there has been an increase in the consumption of bottled water, from 0.3 kg to 18.5 kg per capita per year. a comparable increase has also been observed in other countries8. there is a belief by the population that bottled water is healthier than tap water and free of impurities. this may be leading to the increase in the consumption of bottled water as opposed to public tap water4. children who have bottled water as the primary source of drinking water may have its oral health affected in three ways: (1) they could be getting the appropriate amount of fluoride content, (2) they could be getting an amount of fluoride content below that necessary for dental caries prevention, or (3) they could be getting a dose above the recommended level, leading to the risk of developing dental fluorosis1,16. bottled water brands commercialized in joão pessoa and são luís analyzed in this research presented fluoride content below the limit recommended to have a preventive effect on dental caries. note that, only public water in são luís is artificially fluoridated. even if bottled water consumers are not using public water as their primary source of drinking water, they end up using the tap water for cooking and for the reconstitution of aliments. this leads to an additional intake of fluoride. therefore, the population of joão pessoa may not be getting enough fluoride from the consumption of water as neither the public water nor the bottled water have enough fluoride content to prevent dental caries. several studies have reported a large variation in the fluoride content of different bottled water brands1,3-4,6,9-11. furthermore, studies of bottled water brands available on the national or international market have shown that the fluoride content of the product is inadequate. they have demonstrated either a fluoride concentration below the necessary level to be effective for the prevention of dental caries3-4,7,9,11 or a fluoride content above the concentration approved by law, which may increase the risk of development of dental fluorosis2,10,14. the results of the present study are consistent with other surveys in the northeastern brazil, which report average fluoride concentrations varying from 0.06 to 0.26 mg/l9. furthermore, in research performed in australia by cochrane et al. 4, 100% of the samples tested demonstrated fluoride concentrations below 0.08 mg f/l. similar results were also observed in the northeastern region of england where the authors found a mean of 0.08 mg f/l11. these investigations show that, if bottled water is used as the primary source of drinking water, then consumers are at a higher risk of not receiving any benefit regarding dental caries prevention. in contrast, grec et al.6, surveying bottled water in the state of são paulo, recorded fluoride levels of as much as 2.04 mg f/l, which is above the fluoride content level approved by state law6. villena et al.10 gave a broader view studying bottled water brands commercialized throughout brazil and found fluoride concentrations of up to 4.4 mg f/l. all these studies point out to the necessity of more rigorous control when it comes to the composition of bottled water. the present study demonstrated that 85% of the analyzed bottled water brands are being commercialized as fluoridated, while only one brand sold in joão pessoa, presenting fluoride content over 0.1 mg/l, could be classified as such. this lack of concordance shown on these product labels has also reported by villena et al.10. according to the brazilian law, the label of bottled water has to display the 8 most predominant minerals as well as classify the water as fluoridated if it contains more than 1.5 mg f/l. seventeen of the twenty brands studied in this research showed the fluoride content on the product label, only 3 brands showed concordance between the values found on analysis and the values stated on the label. another common mistake occurs when regarding the classification of the product as fluoridated water. this study found that most products display on their label the fluoridated classification, despite presenting less than 1.0 mg/l fluoride. the majority of the studies realized around the world present results consistent with the ones found in the present study regarding the labeling of products1-4,6-7,9-11, 17. the inclusion of the actual fluoride content on the label would allow the consumer to be aware of the presence or absence of fluoride in his/her drinking water. this way, the consumer would be able to know the amount of ingested fluoride and then make an informed decision about the choice of drinking water4. the analysis of fluoride content of different batches (when available) (table 2) of the same brand of bottled water showed that there was not variation among some brands of bottled water. similar studies performed in mexico and australia showed some deviation between batches4,18. it is possible to postulate that seasonal variability in the volume of rainfall could result in fluctuations in the content of fluoride of the bottled waters. therefore, it is necessary to carry out more studies aimed at verifying the reason for the variation between batches of the same brand. the maximum fluoride concentration that may be consumed fluoride content of bottled water commercialized in two cities of northeastern brazil 209 braz j oral sci. 8(4):206-209 daily without incurring the risk of developing enamel fluorosis is estimated at 0.05 to 0.07 mgf/kg/ day19-20. the brands bottled water analyzed in this research demonstrated low fluoride content, thus would not represent a risk for developing fluorosis or an acute intoxication. given the large variability in fluoride content among batches and overall low levels of fluoride across all the bottled water brands surveyed, it is important to consider the need of more accurate surveillance of bottled water commercialized in joão pessoa and são luís with respect to the fluoride content and to the accuracy of the information given on the labels. acknowledgments we are thankful to the national council of technological and scientific development (cnpq) for the scholarship granted to the first author as part of the institutional scientific initiation program (pibic). references 1. ahiropoulos v. fluoride content of bottled waters available in northern greece. int j paediatr dent. 2006; 16: 111-6. 2. bottenberg p. fluoride content of mineral waters on the belgian market and a case report of fluorosis induced by mineral water use. eur j pediatr. 2004; 163: 626-7. 3. brandão img, valsecki a, junior. análise da concentração de flúor em águas minerais na região de araraquara, brasil. rev panam salud publica. 1998; 4: 238-42. 4. cochrane nj, saranathan s, morgan m, dashper s. fluoride content of still bottled water in australia. aust dent j. 2006; 51: 242-4. 5. dobaradaran s, mahvi ah, dehdashtia s. fluoride content of bottled drinking water available in iran. fluoride. 2008; 41: 93-4. 6. grec rhdc, moura pgd, pessan jp, ramires i, costa b, buzalaf mar. concentração de flúor em águas engarrafadas comercializadas no município de são paulo. rev saude publica. 2008; 42: 154-7. 7. ramires i, grec rhc, cattan l, moura pg, lauris jrp, buzalaf mar. avaliação da concentração de flúor e do consumo de água mineral. rev saude publica. 2004; 38: 459-65. 8. brasil. ministério de minas e energia. secretaria nacional de minas e metalurgia. brasília-df. anuário mineral brasileiro. 2006; 20. 9. santos lm, barbosa kmm, xavier shc, forte fds, sampaio fc, reis jil. concentração de flúor em diferentes marcas de água mineral comercializadas em alagoas. rev bras odontol. 2006; 63: 104-6. 10. villena rs, borges dg, cury ja. avaliação da concentração de flúor em águas minerais comercializadas no brasil. rev saude publica. 1996; 30: 512-8. 11. zohouri fv, maguire a, moynihan pj. fluoride content of still bottled waters available in the north-east of england, uk. br dent j. 2003; 195: 515-9. 12. cdc. centers for disease control and prevention. achievements in public health, 1900-1999: fluoridation of drinking water to prevent dental caries. morbidity and mortality weekly report; 1999. 13. carvalho t, kehrle hm, sampaio f. prevalence and severity of dental fluorosis among students from joão pessoa, pb, brazil. braz oral res. 2007; 21: 198-203. 14. lima ybo, cury ja. ingestão de flúor por crianças pela água e dentifrício. rev saude publica. 2001; 35: 576-81. 15. forte fds, freitas chsm, sampaio fc, jardim mcam. fluorose dentária em crianças de princesa isabel, paraíba. pesq odontol bras. 2001; 15: 87-90. 16. sampaio f, von der fehr f, arneberg p, petrucci gigante d, hatloy a. dental fluorosis and nutritional status of 6 to 11 year old children living in rural areas of paraíba, brazil. caries res. 1999; 33: 66-73. 17. jiménez-farfán md, hernández-guerrero jc, loyola-rodriguez jp, ledesmamontes c. fluoride content in bottled waters, juices and carbonated soft drinks in mexico city, mexico. int j paediatr dent. 2004; 14: 260-6. 18. loyola-rodríguez jp, pozos-guillén adj, hernández-guerrero jc. bebidas embotelladas como fuentes adicionales de exposición a flúor. salud publica mexico. 1998; 40: 438-41. 19. galagan dj, vermillion jr. determining optimum fluoride concentrations. public health reports. 1957; 72: 491-3. 20. burt ba. the changing patterns of systemic fluoride intake. j dent res. 1992; 71(spec issue): 1228-37. fluoride content of bottled water commercialized in two cities of northeastern brazil oral sciences n3 original article braz j oral sci. july/september 2009 volume 8, number 3 evaluation of palatal arches made from low-nickel stainless steel wire rogério lacerda dos santos1, matheus melo pithon1, margareth maria gomes de souza2 ana maria bolognese3, mônica tirre de souza araújo2 1 dds, ms, phd student in orthodontics, federal university of rio de janeiro, brazil 2 dds, ms, phd, assistant professor, department of orthodontics, federal university of rio de janeiro, brazil 3 dds, ms, phd, chair professor, department of orthodontics, federal university of rio de janeiro, brazil received for publication: august 5, 2009 accepted: november 9, 2009 correspondence to: rogério lacerda dos santos praça josé batista de freitas, 78, sala 102, centro nova serranamg brazil cep 35519-000 e-mail: lacerdaorto@hotmail.com or lacerdaorto@bol.com.br abstract aim: to test the hypothesis that there is no difference between stainless steel wires and low-nickel stainless steel ones regarding their mechanical behavior. force, resilience and elasticity modulus produced by coffin, “w” arch, and quad-helix appliances made of 0.032-inch and 0.036-inch wires were evaluated. methods: fifteen appliances of each type (coffin, “w” arch, and quad-helix) were made according to metal alloy and wire thickness. all arches (12 groups of 15 appliances each) were submitted to mechanical compression test by using an emic dl-10000 machine simulating activations of 5, 8, 10, and 12 mm. analysis of variance with multiple comparisons and tukey’s test were used (p < 0.05) for analyzing statistically force and resilience data. results: the results showed that mechanical properties depended on shape of the appliance, diameter of the wire, amount of activation, and metal alloy. conclusions: appliances made from lownickel stainless steel alloy had higher release of force, resilience and elasticity modulus compared to those made of stainless steel alloy. keywords: orthodontics, malocclusion, nickel, hypersensitivity. introduction in daily orthodontic practice, a variety of metallic alloys, such as stainless steel, cobaltchromium, nickel titanium and beta-titanium, are used, and the majority of these contain nickel1. the percentage of nickel in the appliances, auxiliaries, and accesories used in orthodontics ranges from 8% (as in stainless steel) to more than 50% (as in the nickeltitanium alloys2. the most common types of stainless steel alloys used in orthodontics are the types 302 and 304 according to the american institute of steel and iron (aisi), consisting of approximately 18% of chrome and 8% of nickel, and being represented by the 18-8 stainless steel group. leaching of these metallic components may be a potential trigger to an allergic reaction. nickel is a strong immunologic sensitizer and may result in contact hypersensitivity3. tissue reactions may consist of intraoral diffuse red zones, blisters and ulcerations extending to the perioral area, and eczematic and urticarial reactions of the face or more distant skin areas4. in order to solve this problem, low-nickel-content stainless steel alloys have been increasingly used for making orthodontic appliances, including the expanders usually employed to correct posterior crossbite in those cases of deciduous, mixed, and permanent dentitions as an alternative treatment for those patients who are potentially allergic to nickel. coffin, “ w” arch, and quad-helix arches are among the most common appliances used to correct the crossbite5-7 in deciduous and mixed. the coffin appliance, designed by walter coffin in 1881, was originally used in removable plates aimed at expanding the constricted dental arches, and its clinical application is still often recommended7-8. the “ w ” arch is braz j oral sci. 8(3):149-153 indeed an evolution of the coffin appliance that resulted in changes in shape and dimension, besides being used with fixed anchorage. in order to improve the flexibility and to release the force slightly and continuously, coil springs had been initially placed at the posterior segment of the palatal arch (double-helix). later, two more coil springs were also placed at the anterior part of the arch, thus resulting in the quad-helix appliance5,8-9. graber10 have stated that 400 g is the lowest orthopedic force needed to achieve an effect on the maxillary arch. conversely, the correction of posterior crossbite requires orthodontic forces, and jarabak and fizzel11 recommend ideal force levels for each group of teeth, that is, 250 g to move the upper molar. the aim of the present study was to test the hypothesis that there is no difference between stainless steel wires and low-nickel stainless steel wires regarding their mechanical behavior. for such purpose, force, resilience and elasticity modulus produced by three different types of expander appliances employed for crossbite correction were assessed and the ideal levels of activation regarding each appliance determined. material and methods a total of 180 appliances were tested using two sizes of wire in 3 configurations, including two types of stainless steel alloys, one with nickel and chrome (morelli, sorocaba, sp, brazil) and another with low-nickel content (less than 0.2%) (morelli). fifteen coffin appliances were made of 0.032-inch stainless steel wire and 15 more were made of 0.036-inch stainless steel wire using each type of metal alloy. the coffin appliance had two 25-mm legs and a posterior loop that measured 10 mm (figure 1a). fifteen porter-w appliances were made of 0.032-inch stainless steel wire and more 15 were made of 0.036-inch stainless steel wire using each type of metal alloy. the porter-w arch (figure 1b) had two 40-mm outer legs, two 35-mm inner legs, and the anterior section was 10 mm. fifteen quad-helix appliances were made of 0.032-inch stainless steel wire and 15 were made of 0.036-inch stainless steel wire using each type of metal alloy. the quad-helix had the same dimensions as the porter-w arch, but had four 1.5-mm diameter helices that were incorporated in the arches (figure 1c). the specimens were manufactured by the same professional, using a template with standardized intercanine and intermolar distances. a split mandrel adapted for the experiment was mounted upon the upper part of the machine in order to allow an application of force to the central part of the appliance external leg. another device served as a base for fixing the other leg outside and keeping it in the same perpendicular position during the tests, thus avoiding momentum creation (figure 2). each sample was first activated at 12 mm and then submitted in sequence to compression trials in the emic dl 10000 testing machine (são josé dos pinhais, pr , brazil) (figure 2), using the mtest program 1.0 version, at a speed of 5 mm/min12. the mtest program provided the force and resilience means produced by 5, 8, 10, and 12 mm activation, as well as the mechanical behavior graph of each appliance in this activation index. the elasticity modulus was calculated based on the arch dimensions, the wire diameter, and the force means obtained for each appliance shape. analysis of variance with multiple comparisons and tukey’s test were used (p < 0.05) for analyzing statistically force and resilience data. figure 1. palatal arches evaluated in the study: (a) coffin appliance, (b) “w” arch, and (c) quad-helix. evaluation of palatal arches made from low-nickel stainless steel wire150 braz j oral sci. 8(3):149-153 figure 2. w-arch subjected to 12-mm compression in the emic dl 10000 testing machine. results force (tables 1-3), resilience and elasticity modulus (table 4) increased proportionally to the activation increases. in addition, the groups using the 0.036-inch wire presented statistically (p < .05) higher levels of force and resiliency when compared to the arches using the 0.032inch wire. on average, the coffin (table 1) arch produced the highest levels of force, followed by the “w” arch (table 2), and the quad-helix showed the lowest values (table 3). coffin appliances also showed higher resilience comparing to the others (table 4). the “w” arches showed higher force and resilience than the quad-helix using the same diameter wire. it was observed statistically significant differences between the wire thicknesses for the same type of appliance (p < 0.05) and same metal alloys. of the metallic alloys wire diameter 5 mm 8 mm 10 mm 12 mm stainless steel 0.032 235.3 ± 29a 326.6 ± 31a 382.8 ± 28a 432.0 ± 26a stainless steel 0.036 326.6 ± 9b 467.1 ± 20b 558.5 ± 22b 653.3 ± 28b stainless steel with low-nickel content 0.032 237.3 ± 0a 330.1 ± 7a 389.8 ± 14a 435.5 ± 14a stainless steel with low-nickel content 0.036 354.7 ± 22b 526.8 ± 27c 628.7 ± 33b 730.6 ± 34b activation rate n=15, for each shape and wire diameter combination. different letters indicate statistically significant difference (p < 0.05). table 1. mean forces (g) produced by the coffin appliance. metallic alloys wire diameter 5 mm 8 mm 10 mm 12 mm stainless steel 0.032 140.5 ± 0a 193.1 ± 12a 224.8 ± 14a 263.4 ± 21a stainless steel 0.036 193.1 ± 12b 284.5 ± 14b 340.7 ± 9b 400.4 ± 14b stainless steel with low-nickel content 0.032 158.0 ± 12a 235.3 ± 23a 251.9 ± 3a 305.5 ± 44a stainless steel with low-nickel content 0.036 214.2 ± 7b 330.1 ± 22c 403.9 ± 4b 477.7 ± 49b “w” arch activation rate n=15, for each shape and wire diameter combination. different letters indicate statistically significant difference (p < 0.05). table 2. mean forces (g) produced by the “w” arch appliance. metallic alloys wire diameter 5 mm 8 mm 10 mm 12 mm stainless steel 0.032 122.9 ± 17a 158.0 ± 17a 214.2 ± 39a 221.2 ± 34a stainless steel 0.036 147.5 ± 9a 224.8 ± 14b 270.4 ± 26a 291.5 ± 34a stainless steel with low-nickel content 0.032 124.9 ± 12a 165.0 ± 15a 216.7 ± 22a 224.2 ± 19a stainless steel with low-nickel content 0.036 149.0 ± 7a 226.7 ± 12b 271.9 ± 22a 305.1 ± 35a activation ratequad-helix table 3. mean forces (g) produced by the quad-helix appliance n=15, for each shape and wire diameter combination. different letters indicate statistically significant difference (p < 0.05). table 4. mean resilience and elasticity modulus values produced by the 3 palatal arches metallic alloys stainless steel 0.032 285.9 ± 19a 55.4 165.27 ± 12a 78.2 144.8 ± 16a 83.8 0.036 410.1 ± 20b 64.8 255.65 ± 9b 90.9 193.92 ± 14b 85.5 stainless steel with low-nickel content 0.032 289.2 ± 7a 56 196.3 ± 22a 92.8 147.7 ± 16a 85.5 0.036 458.1 ± 21b 72.3 299.1 ± 24b 106.3 199 ± 12b 88.19 wire diameter resilience ( g . c m ) resilience ( g . c m ) resilience ( g . c m ) elasticity modulus (g/cm2) elasticity modulus (g/cm2) elasticity modulus (g/cm2) c o f f i n “w” arch quad-helix shape n=15, for each shape and wire diameter combination. statistical difference (p < 0.05) is indicated by different full-size lowercase letters; same letters indicate no significant different mean elasticity modulus values were higher in the coffin group, followed by the “ w ” arch and then the quad-helix. the larger the diameter of wire, the larger was the elasticity modulus, even in appliances with the same shape (table 4). with respect to the wire thickness, no statistically significant difference (p >0.05) was observed between metal alloys regardless of the type of appliance. however, porter-w and quad-helix appliances showed statistically significant differences regarding 0.032-inch and 0.036-inch wires for both types of metal alloy. discussion one can observe statistically significant differences between coffin and “ w” arch appliances regarding the wire thickness using both evaluation of palatal arches made from low-nickel stainless steel wire 151 braz j oral sci. 8(3):149-153 types of metal alloy in all activations (5, 8, 10, and 12-mm), except for 8mm activation in appliances made of 0.036-inch wire (tables 1 and 2). quad-helix appliances showed statistically significant difference regarding the wire thickness for an 8-mm activation using both types of metal alloy, but no statistically significant differences were observed for 5, 10, and 12-mm activations between stainless steel and lownickel stainless steel alloys (table 3). the low-nickel stainless steel alloy used for making these appliances can promote forces different from those of stainless steel alloy. therefore, knowing the behavior of both these alloys allows us to determine the optimal activation levels for orthodontic or orthopedic movement regarding each type of appliance. the mean force values increased proportionally to the activation, corroborating that the appliances worked in the elastic phase, in which deformation is proportional to force. resilience is the property associated with the capacity of absorbing and releasing energy; thus, the higher the resilience, the more continuous the force13. studies have reported different shapes of palatal arches to correct posterior dental crossbites7,9,14. in the present study, different appliances provided distinct mechanical properties, indicating the need for acquiring knowledge of the performance of the appliance to be chosen. in addition, it is important to identify the etiology of such malocclusion and to determine the ideal force for each treatment15. the movement of a single molar might use 250 g11, but orthopedic effects are noted in primary and mixed dentition with forces higher than 400 g10. orthodontic force is capable of moving teeth, whereas orthopedic forces cause bone movement. for orthodontic movement, light and continued forces are preferable because they promote direct, frontal absorption that results consequently in dental movement, whereas greater forces can stimulate indirect absorption at a given distance. as w-arch and quad-helix appliances are made of a greater amount of wire, they become more flexible and consequently are more indicated for orthodontic movement. on the other hand, the coffin appliances incorporates fewer amount of wire compared to other ones and as a result are less flexible, being usually employed for bone movement associated with dental movement. according to adams7, the amount of activation in the coffin appliance depends on the length and diameter of the arch and the number of teeth to be moved. an activation range from 2 to 4 mm using a 0.050-inch wire has been proposed as being sufficient at the beginning. if new adjustments are necessary, they can be made afterwards. in the present study, 5-mm activation yielded results similar to those from adams, who used 4-mm activations. however, the need to use a thinner wire diameter is also suggested when the etiology is a dental abnormality. proffit6 suggested that the success of removable appliances depends on the patient’s co-operation and on controlling the force of the appliance. he analyzed the use of the “ w” arch and recommended 3 or 4 mm of activation as adequate levels of force when using a 0.036-inch wire. other studies such as that by urbaniak, et al.14 showed that the force produced by the quad-helix activation is influenced by the size and wire diameter of the appliance. this study reported that the amount of wire used in the palatal arch and the force are inversely proportional, whereas the wire diameter is directly proportional to the force. other studies referring to the quad-helix appliance5,8-9 suggested the use of a force of approximately 400 g for an activation of 8 mm. according to the present study, the most appropriate force was the one obtained with a “w” arch using a 0.036-inch wire with 12-mm activation for stainless steel alloy and 10-mm activation for low-nickel stainless steel alloy. such a difference might be caused by differences in the size of the appliances, wire diameter, and metal alloy used14. in face of the results obtained from this study, the crossbite of a single molar or a group of a few teeth is appropriately treated with a 0.036-inch quad-helix or a 0.032-inch “w” arch using 10 to 12-mm activation. a 0.032-ich quad-helix with 8-mm activation for both metal alloys provides a very light force and can be used to correct the crossbite of a single tooth. in this way, the results suggest that w-arch and quad-helix appliances using these activations would be appropriate to obtain forces usually indicated to induce orthodontic tooth movement. when a mild orthopedic effect is expected during the deciduous and mixed dentition, a 0.036-inch “w” arch with 12-mm activation for stainless steel alloy and 10-mm activation for low-nickel stainless steel alloy appears to be the best choice. the coffin appliance had the highest forces released, thus suggesting that the orthopedic effects can be reached in co-operative patients or in fixed appliances using this arch shape in either 0.036inch wires with 8-m to 10-m activation for stainless steel alloy or with 5-mm to 8-mm activation for low-nickel stainless steel alloy. nickel is known to be a common cause of contact allergies and hypersensitivity reactions16-18. it is estimated that 4.5% to 28.5% of the population have hypersensitivity to nickel3,16. females have been reported to have a much higher prevalence than males (10:10)16. even though, the presence of metal ions, such as nickel, has been associated with hypersensitivity reactions in orthodontics19. the clinical manifestations of nickel hypersensitivity are easy to diagnose, any intraoral or extraoral appliances containing nickel must be removed until after the dermal or mucosal signs of adverse reactions have healed completely2. history of allergy should be considered a predictive factor of clinical manifestations of nickel hypersensitivity20. instead of using intraor extraoral appliances containing nickel, it is suggested that such devices be replaced by brackets and wires made of stainless steel alloys with no or low-nickel content. the wires made of stainless steel alloys with low-nickel content are an option the orthodontist can consider for patients with history of nickel hypersensitivity, however attention should be paid to the activation to be used for appliances made from this wire. in the present study the appliances made of low-nickel stainless steel wire showed high force released and resilience, and this can influence significantly the treatment as a greater dental response can be obtained. in conclusion, appliances made of low-nickel stainless steel alloy had higher release of force, resilience and elasticity modulus compared to those made of stainless steel alloy. the three types of appliances evaluated can produce adequate forces for orthodontic treatment as long as their clinical application is correctly planned. references: 1. grimsdottir mr, gjerdet nr, hensten-pettersen a. composition and in vitro corrosion of orthodontic appliances. am j orthod dentofacial orthop. 1992; 101: 525-32. 2. eliades t, athanasiou ae. in vivo aging of orthodontic alloys: implications for corrosion potential, nickel release, and biocompatibility. angle orthod. 2002; 72: 222-37. 3. janson gr, dainesi ea, consolaro a, woodside dg, de freitas mr. nickel hypersensitivity reaction before, during, and after orthodontic therapy. am j orthod dentofacial orthop. 1998; 113: 655-60. evaluation of palatal arches made from low-nickel stainless steel wire152 braz j oral sci. 8(3):149-153 4. hensten-pettersen a, jacobsen n, grímssdóttir mr. allergic reactions and safety concerns: in: brantley wa, eliades t, editors. orthodontic materials: acientific and clinical aspects. stuttgart: thieme; 2001. p.287-99. 5. bell ra, lecompte ej. the effects of maxillary expansion using a quad-helix appliance during the deciduous and mixed dentitions. am j orthod. 1981; 79: 152-61. 6. proffit wr. contemporary orthodontics. st louis: mosby; 1986. p.341-9. 7. adams cp. the design and construction of removable orthodontic appliances. bristol: john wright and sons; 1969. 8. chaconas sj, de alba y levy ja. orthopedic and orthodontic applications of the quad-helix appliance. am j orthod. 1977; 72: 422-8. 9. chaconas sj, caputo aa. observation of orthopedic force distribution produced by maxillary orthodontic appliances. am j orthod. 1982; 82: 492-501. 10. graber tm, editor. current orthodontics. concept and technique. philadelphia: saunders; 1969: 919-88. 11. jarabak jr, fizzell ja. aparatología del arco de canto com alambres delgados. buenos aires: mundi; 1975. 12. martinelli fl, couto ps, ruellas ac. three palatal arches used to correct posterior dental crossbites. angle orthod. 2006; 76: 1047-51. 13. phillips rw. skinner’s science of dental materials. philadelphia: saunders; 1973. p.23-7. 14. urbaniak ja, brantley wa, pruhs rj, zussman rl, post ac. effects of appliance size, arch wire diameter, and alloy composition on the in vitro force delivery of the quad-helix appliance. am j orthod dentofacial orthop. 1988; 94: 311-6. 15. graber tm, editor. orthodontics. current principles and prattice. saint louis: mosby; 1985. p.60-2. 16. peltonen l. nickel sensitivity in the general population. contact dermatitis. 1979; 5: 27-32. 17. schubert h, berova n, czernielewski a, hegyi e, jirasek l, kohanka v et al. epidemiology of nickel allergy. contact dermatitis. 1987; 16: 122-8. 18. jacobsen n, hensten-pettersen a. occupational health problems and adverse patient reactions in orthodontics. eur j orthod. 1989; 11: 254-64. 19. bass jk, fine h, cisneros gj. nickel hypersensitivity in the orthodontic patient. am j orthod dentofacial orthop. 1993; 103: 280-5. 20. genelhu mc, m arigo m, alvesoliveira lf, m alaquias lc, gomez rs. characterization of nickel-induced allergic contact stomatitis associated with fixed orthodontic appliances. am j orthod dentofacial orthop. 2005; 128: 378-81. evaluation of palatal arches made from low-nickel stainless steel wire 153 braz j oral sci. 8(3):149-153 oral sciences n3 received for publication: april 08, 2010 accepted: august 23, 2010 original article braz j oral sci. july/september 2010 volume 9, number 3 cytotoxicity of orthodontic elastics: in vitro investigation with on l929 mouse fibroblasts rogério lacerda dos santos1, matheus melo pithon1, fernanda otaviano martins2, maria teresa villela romanos3 1specialist in orthodontics, federal university of alfenas, minas gerais, brazil master in orthodontics, federal university of rio de janeiro, brazil phd student in orthodontics, federal university of rio de janeiro, brazil 2graduated in biological sciences: microbiology and immunology federal university of rio de janeiro-ufrj 3assistant professor of microbiology, federal university of rio de janeiro, brazil correspondence to: rogério lacerda dos santos rua ipatinga, 170, planalto divinópolismgbrazil – cep 35501-191 e-mail: lacerdaorto@hotmail.com; lacerdaorto@bol.com.br abstract aim: to test the hypothesis that there is no difference in the cytotoxicity among natural latex elastics of different manufacturers using a l929 cell line culture. methods: different latex intraoral elastics (i.d. = 5/16", 4.5 oz.) were tested. the sample was divided into 7 groups of 15 elastics each: group ao (american orthodontics), group gac (gac international), group tp (tp orthodontics), group ad (aditek), group ab (abzil), group mo (morelli) and group un (uniden). cytotoxicity assays were performed by using cell culture medium containing l-929 line cells (mouse fibroblast). the cytotoxicity was evaluated by using the “dye-uptake” test, which was employed at two different moments (1 and 24 h). data were compared by anova and tukey’s test (p < 0.05). results: the results showed a significant difference (p < 0.05) between all groups and the group cc (cell control) at 1 and 24 h. groups ad, ab, mo and un were noticeably more cytotoxic than the groups ao, gac and tp at 1 h. after 24 h, a significant decrease in cell viability was observed in all groups. conclusions: intraoral elastics from american orthodontics, gac and tp orthodontics trademarks induced less cell lysis than aditek, abzil, morelli and uniden trademarks. keywords: cytotoxicity, elastics, biocompatibility, orthodontics. introduction the biocompatibility of dental materials has been subject of great speculation and uncertainty. there are, particularly in orthodontics, several materials keeping direct contact with organic tissues for long periods. recent studies have been concerned with the biocompatibility of different types of orthodontic materials1,2. prevulcanized latex is produced by mixing pure natural latex3, with stabilisers such as zinc oxide and chemically vulcanized materials. the resulting mixture is then heated up to 70oc4. although zinc is known to be neurotoxic5, the amount released by orthodontic elastics can be ingested as research studies show no evidence of harm6. anti-ozone and anti-oxidant agents are also added to latex during the manufacture of orthodontic elastics3. this process has the advantage of producing latex with higher mechanical properties, thus increasing its strength and elasticity4,6. however, natural latex is not in the category of materials known to be entirely inoffensive7-8. braz j oral sci. 9(3):366-370 367 allergy caused by latex proteins has been well documented9, including immediate hypersensitivity reactions10. amongst the allergic reactions caused by orthodontic elastics, swelling and stomatitis, erythematous oral lesions, respiratory reactions, and even anaphylactic shock, the most severe form of allergy11-12, can be cited. latex allergy occurs in 3-17% of the cases13. the use of cell cultures for testing the toxicity of dental products is a valid way of understanding the cytotoxic behavior of such materials7. the aim of this in vitro study was to test the hypothesis that there is no difference in the cytotoxicity among natural latex elastics of different manufacturers on l929 mouse fibroblasts. material and methods latex intraoral elastics of different manufacturers (i.d. = 5/16", 4.5 oz.) were selected for studying their cytotoxicity in cell culture (table 1). the samples were divided into 7 groups of 15 elastics each: group ao (american orthodontics, sheboygan, wi, usa), group gac (gac international, bohemia, ny, usa), group tp (tp orthodontics, lodi, ca, usa), group ad (aditek, cravinhos, sp, brazil), group ab (abzil, são josé do rio preto, sp, brazil) group mo (morelli, sorocaba, sp, brazil) and group un (uniden, sorocaba, são paulo, brazil) (figure 1). table 1. elastic and control groups used for the assays. groups trademark main composition reference number a o american orthodontics natural latex 000-113 g a c gac natural latex 11-101-08 tp tp orthodontics natural latex 360-012 a d aditek natural latex 0080-203 a b abzil natural latex 464-402 m o morelli natural latex 60-01-205 u n uniden natural latex 000-1204 p c tween 80 (polyoxyethylene-20-sorbitan, sigma, st. louis, missouri, usa) n c pbs solution (phosphate-buffered saline, cultilab, campinas, são paulo, brazil) fig. 1. latex intraoral elastics evaluated in this study: ao (american orthodontics), gac (gac international), tp (tp orthodontics), ad (aditek), ab (abzil), mo (morelli) and un (uniden). the elastics used in this study belonged to the same production line for each trademark. to verify the cell response in extreme situations, 3 additional groups were included in the study: group cc (cell control), consisting of l-929 cells not exposed to supernatants from the elastics; group c+ (positive control), consisting of tween 80 (polyoxyethylene20-sorbitan, sigma, st. louis, mo, usa); group c(negative control), consisting of phosphate-buffered saline (pbs) solution (table 1). the cell culture model used was the monolayer containing l-929 line cells (mouse fibroblast) (american type culture collection atcc, rockville, md, usa), which were maintained in eagles’ minimum essential medium (mem; cultilab, campinas, sp, brazil) by adding 0.03 mg/ ml of glutamine (sigma, st. louis, missouri), 50 µg/ml of garamicine (schering plough, kenilworth, nj, usa), 2.5 mg/ ml of fungizone (bristol-myers-squibb, new york, ny, usa.), 0.25% sodium bicarbonate solution (merck, darmstadt, germany), 10 mm of hepes (sigma), and 10% bovine fetal serum (cultilab) for growth medium or no bovine fetal serum for maintenance medium only. next, the cell culture medium was incubated at 37oc for 48 h. the cells were reseeded twice a week to ensure exponential growth of the cell line. for standardization of samples, the powder coating of the elastics was removed. the elastics were washed for 15 s with deionized water by using a milli-q purification system (millipore, bedford, ma, usa) and their surfaces were slightly dried with disposable paper. before testing, all elastics were sterilized by exposure to ultraviolet light (labconco, kansas, mo, usa) for 30 min for each surface14-16. the “dye-uptake”17 test was used for evaluating the cytotoxicity. this method is based on neutral red dye incorporated into live cells. it was used in this experiment only at two periods of evaluation: 1 and 24 h. these elastics are usually maintained in the oral cavity for up to 24 h. the 1-h period represents the maintenance of the elastic in the cell culture medium for 1 h after removal, whereas the 24-h period represents the maintenance of the elastic in the cell culture medium for 24 h after removal. dye-uptake aliquots of 100 µl of l-929 line cells were distributed into 96-well microplates. after 48 h, the growth medium was replaced with 100 µl of mem obtained following incubation in the different types of elastics at 1 and 24 h. cytotoxicity of orthodontic elastics: in vitro investigation with on l929 mouse fibroblasts braz j oral sci. 9(3):366-370 368 groups time 1 h 24 h mean median viable cells(%) s. d.(%) mean median viable cells(%) s. d.(%) c c 0.882 0.810 100.0 0.00 0.995 0.910 100.0 0.00 n c 0.874 0.698 99.0 0.01 0.988 0.802 99.2 0.01 p c 0.079a 0.075 9.02 0.88 0.119a 0.101 11.98 0.38 a o 0.818a 0.640 92.79 2.99 0.545a 0.390 54.86 1.00 g a c 0.804a 0.645 91.18 2.12 0.522a 0.440 52.53 0.77 tp 0.811a 0.728 91.99 1.00 0.547a 0.412 55.06 0.74 a d 0.340ab 0.245 38.55 1.85 0.313ab 0.259 31.55 1.20 a b 0.358ab 0.248 40.61 2.29 0.299ab 0.252 30.14 0.66 m o 0.334ab 0.240 37.98 1.42 0.308ab 0.268 31.05 0.80 u n 0.327ab 0.239 37.18 1.72 0.297ab 0.249 29.93 0.87 values followed by same letters are not significantly different (p>0.05) for the same time. sd: standard deviation. pc: positive control/ tween 80. nc: negative control/pbs solution. a(p < 0.05) compared to cell control. b(p < 0.05) compared to groups ao, gac or tp. table 2. descriptive statistics for optical density of latex elastics. mem was used because it is the same type of medium used for cell maintenance, thus not influencing the results. positive and negative control groups consisted of culture medium put in contact with tween 80 and pbs, respectively. after 24 h incubation, 100 µl of 0.01% neutral red dye (sigma, st. louis, mo, usa) were added to the culture medium in the 96-well microplates, which were incubated again for 3 h at 37oc so that the red dye could penetrate the live cells. following this period of time, 100 µl of 4% formaldehyde solution (vetec, rio de janeiro, rj, brazil) in pbs (130 mm of nacl; 2 mm of kcl; 6 mm of na 2 hpo 4 2 h 2 o; 1 mm of k 2 hpo 4 1 mm; ph 7.2) were added to promote cell attachment to the plate. after 5 min, 100 µl of 1% acetic acid (vetec) and 50% methanol (vetec) were added in order to remove the dye. after 20 min, a spectrophotometer (biotek, winooski, vt, usa) at 492 nm wavelength (λ=492 nm) was used for data reading (figure 2). data were compared by anova and tukey’s multiplecomparison test was used for identifying differences between the groups. significance level was set at p<0.05. results a significant difference (p < 0.05) was noted between all groups and group cc (cell control) at 1 h. groups ad, fig. 2. plate with cell culture of group ao (american orthodontics) being analyzed with a spectrophotometer for reading of the optical density (bio tek®). ab, mo and un were noticeably more cytotoxic than the other groups. group un produced the lowest value (37.18% ± 1.72%) and group ao produced the major viability (92.79% ± 2.99%), whereas the viability of the tween 80 (positive cytotoxicity control) was 9.02% ± 0.88% (table 2). after 24 h, a significant decrease in cell viability was observed in all groups. viability ranged from 29.93% to 55.06%, relative to the cell control. the lowest viability (29.93% ± 0.87%) corresponded to group un, whereas the viability of the tween 80 (positive cytotoxicity control) was 11.98% ± 0.38% (table 2). the results showed statistically significant differences between all groups tested with the group cc (cell control) (p<0.05) at 24 h. a significant difference (p < 0.05) was noted between the groups ao, gac, tp and the groups ad, ab, mo, un at 1 and 24 h (table 2). discussion the cell culture model used in the present study was the monolayer18-19. this model was used together with the dyeuptake technique17 because the cytotoxicity of the materials can be determined by spectrophotometry. the spectrophotometric assay allows rapid and reliable evidence for cell viability to be obtained based on the use of vital stain incorporated by viable cells. in this study, neutral red dye was used because it is largely employed for identification of l-929 cell viability. dead or damaged cells cannot incorporate vital stain, thus not being recognized on optical reading. therefore, spectrophotometry does not allow dead cells to be distinguished from the damaged ones. the amount of dye incorporated into the cells is directly proportional to the number of cells with intact membrane, which allows distinguishing the cytotoxicity of each elastic. l-929 mouse fibroblasts were used because they have results comparable to those of primary human gingival fibroblasts20-21, the cell culture results cannot be interpreted as a human response. the percentage of viable cells was obtained by comparing the mean optical density (od) in the control group (cells with no contact with elastics) to that obtained from supernatants of cell cultures that had been in contact with elastics. cytotoxicity of orthodontic elastics: in vitro investigation with on l929 mouse fibroblasts braz j oral sci. 9(3):366-370 369 as sterilization is a prerequisite for cytotoxicity essays, ultraviolet radiation14,16 was used in this study for 30 min for each elastic surface. it was observed that all elastics exhibited the same color aspect and malleability following sterilization with uv light. because natural latex rubber has been increasingly used as dental material, many cytotoxicity issues have been reported as well15,22. a comparison was made among different latex intra-oral elastics. preservatives such as sulfur and zinc oxide as well as antioxidants such as di-thio-carbohydrates, n-nitrosodibutylamine, and n-nitrosopiperidine are all known to be cytotoxic substances22. holmes et al.8 have verified whether the colorants used in the fabrication of colored latex could have some toxic effect. their results showed that these colorants exhibited low toxicity. however, such an effect is clinically inoffensive. allergic reactions23 have been related to the use of orthodontic elastics24, which is characterized by the presence of small vesicles or acute edema and complaints of itching and burning. allergy to natural latex occurs because of the presence of many types of proteins, and the powder covering the orthodontic elastics works as a transporter for these proteins. therefore, the development of non-latex elastics has become increasingly important for clinical use. however, the objective of this study was to evaluate the cytotoxic effect of latex elastic in cell culture. the most serious consequence of natural rubber latex allergy commonly takes place during mucosal absorption of natural rubber latex proteins during intraoperative medical or dental procedures when health care workers or others already sensitized become patients25. the safety biocompatibility of silicone has been well proved through the use of mouth guards in dentistry26. however, hwang and cha22 observed that silicone rubber bands were found to exhibit a low cytotoxicity. however, in terms of the initial force level and the abrupt loss of remaining force with an increase in the extension length, great improvements in the silicone rubber band’s physical properties are required. evidence of this cytotoxic feature was shown following exposition of the elastics to cell culture medium. it was used in this experiment only two times of evaluation 1 and 24 h. these elastics are usually changed every 24 h. natural latex elastics from aditek, abzil, morelli and uniden trademarks induced more cell lysis at 1 and 24 h compared to those from american orthodontics, gac and tp orthodontics trademarks. however, all natural latex elastics were found to cause more cell lysis at 24 h. it has been shown evidence of cytotoxicity in natural latex elastics compared to the silicone elastics6,22. in this study, the elastics from tp orthodontics and american orthodontics trademarks caused lower cell viability at 24 h compared to previous studies6,22. as the powder covering the elastics of all manufacturers was removed before performing the in vitro assays, it was not possible to know whether this powder would have produced any effect. according to schmalz7, the great danger is that potentially cytotoxic intraoral elastics could release substances that might be ingested by the patient over time, thus causing diseases resulting from a cumulative effect. it is known that latex is not entirely biocompatible as it may interact with foods13,27 and medications 28. american orthodontics, gac and tp orthodontics trademarks intraoral elastics evaluated in this study showed over 90% cell viability in the experimental period of 1 h and over 50% at 24 h. hanson and lobner6 evaluated latex and non-latex 3/16-inch interior lumen (medium) elastics and found cell lysis to be 50% higher for latex elastics compared to non-latex ones. however, the authors considered both types of elastics appropriate for orthodontic use. therefore, it is suggested that elastics with cell viability less than 50% should be avoided or used with caution in order to prevent cumulative effects of the cytotoxic components released from these elastics into the organism7. further studies assessing the mechanism of cell lysis can contribute to provide more details on the cytotoxic behavior of these materials. as these materials are widely used in clinical orthodontics, care regarding the cytotoxicity of orthodontic elastics should be taken, mainly with regard to elastics as they have a very close contact with gingiva. thus, clinically proven biocompatible materials should be acquired whenever possible. it may be concluded that intraoral elastics from american orthodontics, gac and tp orthodontics trademarks induced less cell lysis than aditek, abzil, morelli and uniden trademarks at 1 and 24 h. references 1. kao ct, ding sj, he h, chou my, huang th. cytotoxicity of orthodontic wire corroded in fluoride solution in vitro. angle orthod. 2007; 77: 349-54. 2. vande vannet bm, hanssens jl. cytotoxicity of two bonding adhesives assessed by three-dimensional cell culture. angle orthod. 2007; 77: 71622. 3. weiss me, hirshman ca. latex allergy. can j anaesth. 1992; 39: 528-32. 4. perrella fw, gaspari aa. natural rubber latex protein reduction with an emphasis on enzyme treatment. methods. 2002; 27: 77-86. 5. lobner d, asrari m. neurotoxicity of dental amalgam is mediated by zinc. j dent res. 2003; 82: 243-6. 6. hanson m, lobner d. in vitro neuronal cytotoxicity of latex and nonlatex orthodontic elastics. am j orthod dentofacial orthop. 2004; 126: 65-70. 7. schmalz g. use of cell cultures for toxicity testing of dental materials— advantages and limitations. j dent. 1994; 22 suppl 2: s6-11. 8. holmes j, barker mk, walley ek, tuncay oc. cytotoxicity of orthodontic elastics. am j orthod dentofacial orthop. 1993; 104: 188-91. 9. palosuo t, alenius h, turjanmaa k. quantitation of latex allergens. methods. 2002; 27: 52-8. 10. wakelin sh, white ir. natural rubber latex allergy. clin exp dermatol. 1999; 24: 245-8. 11. everett fg, hice tl. contact stomatitis resulting from the use of orthodontic rubber elastics: report of case. j am dent assoc. 1974; 88: 1030-1. 12. tomazic vj, withrow tj, fisher br, dillard sf. latex-associated allergies and anaphylactic reactions. clin immunol immunopathol. 1992; 64: 89-97. 13. turjanmaa k, alenius h, makinen-kiljunen s, reunala t, palosuo t. natural rubber latex allergy. allergy. 1996; 51: 593-602. 14. santos rl, pithon mm, mendes gs, romanos mtv, ruellas aco. cytotoxicity of intermaxillary orthodontic elastics of different colors: an in vitro study. j appl oral sci. 2009; 4: 326-9. cytotoxicity of orthodontic elastics: in vitro investigation with on l929 mouse fibroblasts braz j oral sci. 9(3):366-370 370 15. santos rl, pithon mm, oliveira mv, mendes gs, romanos mtv, ruellas aco. cytotoxicity of intraoral orthodontic elastics. . braz j oral sci. 2008; 24: 1520-5. 16. dos santos rl, pithon mm, martins fo, romanos mt, de oliveira ruellas ac. evaluation of the cytotoxicity of latex and non-latex orthodontic separating elastics. orthod craniofac res. 2010; 13: 28-33. 17. neyndorff hc, bartel dl, tufaro f, levy jg. development of a model to demonstrate photosensitizer-mediated viral inactivation in blood. transfusion. 1990; 30: 485-90. 18. tomakidi p, koke u, kern r, erdinger l, kruger h, kohl a et al. assessment of acute cytoand genotoxicity of corrosion eluates obtained from orthodontic materials using monolayer cultures of immortalized human gingival keratinocytes. j orofac orthop. 2000; 61: 2-19. 19. rose ec, jonas ie, kappert hf. in vitro investigation into the biological assessment of orthodontic wires. j orofac orthop. 1998; 59: 253-64. 20. schedle a, samorapoompichit p, rausch-fan xh, franz a, fureder w, sperr wr et al. response of l-929 fibroblasts, human gingival fibroblasts, and human tissue mast cells to various metal cations. j dent res. 1995; 74: 1513-20. 21. franz a, konig f, skolka a, sperr w, bauer p, lucas t et al. cytotoxicity of resin composites as a function of interface area. dent mater. 2007; 23: 1438-46. 22. hwang cj, cha jy. mechanical and biological comparison of latex and silicone rubber bands. am j orthod dentofacial orthop. 2003; 124: 379-86. 23. snyder ha, settle s. the rise in latex allergy: implications for the dentist. j am dent assoc. 1994; 125: 1089-97. 24. neiburger ej. a case of possible latex allergy. j clin orthod. 1991; 25: 559-60. 25. sussman gl, beezhold dh, liss g. latex allergy: historical perspective. methods. 2002; 27: 3-9. 26. chauvel-lebret dj, pellen-mussi p. evaluation of the in vitro biocompatibility of various elastomers. biomaterials. 1991; 20: 291-9. 27. carey ab, cornish k, schrank p, ward b, simon r. cross-reactivity of alternate plant sources of latex in subjects with systemic ige-mediated sensitivity to hevea brasiliensis latex. ann allergy asthma immunol. 1995; 74: 317-20. 28. towse a, o’brien m, twarog fj, braimon j, moses ac. local reaction secondary to insulin injection. a potential role for latex antigens in insulin vials and syringes. diabetes care. 1995; 18: 1195-7. cytotoxicity of orthodontic elastics: in vitro investigation with on l929 mouse fibroblasts braz j oral sci. 9(3):366-370 oral sciences n3 original article braz j oral sci. july | september 2015 volume 14, number 3 failure of prosthetic screws on 971 implants carolina schäffer morsch1, caroline freitas rafael1, juan felipe montero dumes1, gabriella mercedez peñarrieta juanito1, joão gustavo oliveira de souza1, marco aurélio bianchini1 1universidade federal de santa catarina – ufsc, department of dentistry, area of implantolology, florianópolis, sc, brazil correspondence to: carolina schäffer morsch rua ana luiza vieira street, 143, apt 305 cep 88063-640 campeche florianópolis, sc, brazil phone: +55 48 96940280 e-mail: dracarolmorsch@gmail.com abstract aim: to evaluate the presence of failures in prosthetic screws and whether these failures are related to the type of prosthesis, abutment angulation and presence or not of intermediate. methods: two-hundred and sixty-seven patients were evaluated at the federal university of santa catarina, totaling 971 external hexagonal implants in place for at least one year. a tobit regression model for censored variables was used and the explanatory variables were subjected to percentage analysis. results: the results demonstrated a relationship between the failure in prosthetic screws and the investigated factors with a significance of f=0.003 and p<0.05. the percentage analysis showed that the prosthetic screw failed in 8.82% of 238 unitary prostheses and 10.1% of 773 multiple prostheses. among 912 abutments, 9.43% failed and in the 59 angled abutments, failures appeared in 15.25%. a total of 200 prostheses were placed over the implant platform and 13% failed. a total of 771 prostheses were made over abutment and 8.95% of these screws failed. conclusions: through multiple linear regression it could be concluded that the type of prosthesis showed no significant influence on the prosthetic screw failure, but other variables were related to this mechanical failure. there was a significant relationship among the variables, demonstrating the need for greater attention in choosing the type of prosthesis, the abutment angulation and whether the prosthesis will be placed directly on the implant platform or on the abutment. keywords: dental implant-abutment design; /complications; prosthesis failure; dental prosthesis, implant-supported. introduction it is evident that due to the high survival rates that the implant-supported prostheses are fully accepted today as a reliable treatment option for the replacement of single or multiple missing teeth1. however, some complications are mentioned in several studies including screw loosening and screw, veneer, abutment, framework and implant fractures, and fracture of the luting cement2. screw loosening is recognized as one of the most evident complications3, like fractures of the veneering material4. the reasons for screw loosening include fatigue, inadequate tightening torque, inadequate prosthesis fit, poorly machined components, vibrating micro-movement and excessive loading2,5. external hexagon connection systems have been considered more prone to screw loosening as a result of their mechanical properties under dynamic load. to reduce some of these complications, internal connection systems were developed to improve the biomechanical properties of implant-abutment joints and have shown significant biomechanical advantages over external-hex connection2. the biomechanical behavior of implant-retained restorations, including the screw, may be compromised by some factors6, such as type of prosthesis, the abutment angulation and if the prostheses are placed directly on the implant braz j oral sci. 14(3):195-198 received for publication: may, 17 2015 accepted: september, 17 2015 http://dx.doi.org/10.1590/1677-3225v14n3a04 196196196196196 platform or on the abutment. regarding the type of prosthesis, it is known that a single prosthesis allows for higher patient satisfaction and also facilitates hygiene; however it presents more possibilities of screw loosening 7. concerning the abutment angulation, when a compensating strategy is required this abutment may be used where aesthetics and quantity of soft tissue allow. however, this abutment can present more screw failures due to the small thread area that often loses retention and produces cracks due to the low amount of metal. placing a prosthesis connected directly to the implant has advantages, including low cost, possibility to overcome problems such as limited interocclusal spaces and the possibility of correction of implant angulation error. however, this abutment requires laboratory steps that may cause implant/abutment misfit, which may result in screw loosening and/or fracture6. the aim of this research was to investigate if there is a relationship between the screw failure and the type of prosthesis, the abutment angulation and the presence or not of intermediate. material and methods the present study was approved by the research ethics committee of the federal university of santa catarina (ufsc, florianópolis, sc, brazil) under number 367.077. all the participants were briefed about the purpose and process of the study and patient’s written consent was obtained. data was obtained from a 10-year retrospective study. the researcher was calibrated. a total of 297 patients with implantsupported prostheses, 1001 implants in total, were invited to attend the dentistry department with the purpose of being evaluated. the patients were rehabilitated with external hexagon connection dental implants and with prosthesis in function for at least 1 year. the screw torque ranged from 10 to 32 ncm according to the manufacturer’s instructions and the type of abutment. the cemented restorations were cemented with zinc phosphate cement (vigodent coltene, rio de janeiro, rj, brazil). the inclusion criterion was patients with implant reconstruction in function for at least 1 year that attended the recall visits. patients rehabilitated with internal hexagon connection implants and morse tapper, as well as implants supporting overdentures were excluded from the sample. out of 297 patients evaluated, 267 were selected according to the inclusion and exclusion criteria, totaling 971 implants. all the patients were enrolled in a follow-up program. this provided the opportunity to check the patient every 6 months in the first year and then annually. the following explanatory variables were assessed: amount of implants time in function prosthesis type (single or multiple) presence or absence of the intermediate straight or angulated abutment screw failure (fracture or loosening) statistical analysis: the collected data were submitted to frequency analysis, for the evaluation of prosthetic screw failure percentage (explained variable) in each explicative variable. multiple linear regression was applied to correlate the screw failure with the explained variable and to confirm results the tobit regression method for censored variables. results frequency analysis from the 971 implants, 238 were rehabilitated with single prostheses and 733 with multiple prostheses, including partial arch (168) and total arch (69). from this amount, 21 showed prosthetic screw failure (8.82%) and 74 (10.1%), respectively. from the evaluated 912 straight abutments, 86 (9.43%) presented screw failure. from the 59 angled abutments, 9 (15.25%) failed. two-hundred prostheses were made directly on the implant platform, and from those 200, 26 (13%) had screw failures. a total of 8 (8.95%) screw failures were observed among the 771 prostheses placed on the abutment. multiple linear regression and tobit method the explicative variable ‘type of prosthesis’ had p=0.20, the ‘presence or not of intermediate’, p=0.00020 and ‘angulation’ had p=0.00023 as may be seen in the table 1. by multiple linear regression, it may be stated that the type of prosthesis did not show statistically significant differences on the prosthetic screw failure, but other explicative variables were related with this mechanical failure (p<0.05). both linear regression and the tobit method showed one f of significance lower than 5% (0.003), which prevents rejecting the null hypothesis and turns the model valid. the explanation percentage of the model was close in both methods (1.8% in the linear regression and 1.76% in tobit), indicating that new variables can be incorporated to the model to facilitate more learning. the results demonstrated relationship between the explained variable and the explanatory variables with a significance of f 0.003 and p<0.05. discussion regarding the type of prosthesis, multiple prostheses showed more screw failures (10.1%) compared with the single variables failure percent p regression type of prosthesis single prosthesis 8.95% 0.20383892 multiple prosthesis 10.10% intermediate straight abutments 9.43% 0.000205617 angled abutments 15.25% angulation directly on the platform 13% 0.000232897 over the abutment 8.95% table 1:table 1:table 1:table 1:table 1: overview of screw failures percentage analysis and multiple linear regression failure of prosthetic screws on 971 implants braz j oral sci. 14(3):195-198 197197197197197 prosthesis (8.82%), contradicting what is found in the literature. this may be explained since the number of multiple prostheses (773) was larger than the single ones (238). pjetursson et al.1, showed in a systematic review that 5.6% of single crowns and 4% of fixed dental prostheses (fdps) presented screw loosening and 0.3% to 0.8% screw fractures in 5 years, respectively. glauser et al.7, observed 5.55% of screw loosening in single crowns and bambini et al.8, 9.37%, close to the results of this study. the third most common technical complication of fdps found by pjetursson et al.4 was abutment or occlusal screw loosening. the cumulative complication rate after 5 years follow-up was 5.3%. the angled abutments had more screw failures (15.25%) than the straight ones (9.43%), probably due to the small area of contact between the screw thread and the abutment. another possibility described in the literature is the masticatory force, which is not distributed on the long axis of the implant, but can be questioned due to the occlusal force exerted axially on the prosthetic screw. when compressive occlusal loads are applied along the implant axis, a torsional force that may increase the risk of screw loosening or fracture is created. compared with a straight abutment, a 15º to 25º abutment angulation increased the micromotion level by 30 percent. this micromotion may explain the screw failure9. however, not a single screw failure occurred in a study with 2261 implants evaluated for 96 months10. the restorations placed directly on the implant generated more failures (15,25%) compared with the 8.95% on abutments. these results are confirmed by montero et al.6 that connected 10.8% of screw failure of reconstructions to ucla castable abutments. these findings may be explained by the reduction of the torque values after casting procedures and the presence of roughness and irregularities on the contact surface2. the misfit between implant and abutment increases stress on the screw and results in metal fatigue failure and screw loosening10-11. even the smallest misfit could result in changes in screw geometry and cause incidence of strain on the screws12. however, junqueira’s et al study demonstrated that mechanical cycling reduced the torque of abutments without significant difference between cast or pre-machined ucla abutments (p=0.908)13. the statistical analysis indicated that new variables may be included in the model for further learning. this mechanical failure may be related with other variables like the length of the crown, type of antagonist, initial preload or torque value, time in place, veneering material and overload. the relationship between these variables and the prosthetic screw failure should be availed. the change of screw material from titanium to gold, the use of defined screw fixation torques and implants with internal connections led to significant lowering of screw loosening 1,9. another alternative to reduce the screw loosening is the conical spring washer that extends its resistance to loosening14-16. it was concluded that the presence or absence of intermediate and the abutment angulation are directly related to prosthetic screw failure in implant-supported prostheses, and the type of prosthesis has no significant relation with the presence of faults. considering the outstanding need of similar studies with sample quantities and control over external factors, these results are considered highly significant. due to the consequences generated by mechanical failures it is important to consider these factors in the prognosis of rehabilitation treatment. acknowledgements the authors declare that there is no financial support by any agency. references 1. pjetursson be, dent m, perio mas, asgeirsson ag. improvements in implant dentistry over the last decade/ : comparison of survival and complication rates in older and newer publications bjarni. int j oral maxillofac implants. 2014; 29: 308-24. 2. ha c-y, lim y-j, kim m-j, choi j-h. the influence of abutment angulation on screw loosening of implants in the anterior maxilla. int j oral maxillofac implants. 2011; 26: 45-55. 3. barbosa gs, silva-neto jp da, simamoto-júnior pc, neves fd das, mattos mdgc de, ribeiro rf. evaluation of screw loosening on new abutment screws and after successive tightening. braz dent j. 2011 [cited 2014 oct 15]; 22: 51-5. available from: http://www.scielo.br/pdf/bdj/v22n1/ v22n01a09.pdf. 4. pjetursson be, thoma d, jung r, zwahlen m, zembic a. a systematic review of the survival and complication rates of implant-supported fixed dental prostheses (fdps) after a mean observation period of at least 5 years. clin oral implants res. 2012; 23(suppl 6): 22-38. 5. kim e-s, shin s-y. influence of the implant abutment types and the dynamic loading on initial screw loosening. j adv prosthodont. 2013 [cited 2015 mar 10]; 5: 21-8. available from: http://www.ncbi.nlm.nih.gov/pmc/ articles/pmc3597922/pdf/jap-5-21.pdf. 6. montero j, manzano g, beltrán d, lynch cd, suárez-garcía m-j, castillo-oyagüe r. clinical evaluation of the incidence of prosthetic complications in implant crowns constructed with ucla castable abutments. a cohort follow-up study. j dent. 2012; 40: 1081-9. 7. glauser r, sailer i, wohlwend a, studer s, schibli m, schärer p. experimental zirconia abutments for implant-supported single-tooth restorations in esthetically demanding regions: 4-year results of a prospective clinical study. int j prosthodont. 2004; 17: 285-90. 8. bambini f, muzio l lo, procaccini m. retrospective analysis of the influence of abutment structure design on the success of implant unit. clin oral implants res. 2001; 12: 319-24. 9. kao h-c, gung y-w, chung t-f, hsu m-l. the influence of abutment angulation on micromotion level for immediately loaded dental implants: a 3d finite element analysis. int j oral maxillofac implants. 2007; 23: 623-30. 10. sethi a, kaus t, sochor p. the use of angulated abutments in implant dentistry: five-year clinical results of an ongoing prospective study. int j oral maxillofac implants. 2000; 15: 801-10. 11. barbosa gas, bernardes sr, das neves fd, fernandes neto aj, de mattos mdgc, ribeiro rf. relation between implant/abutment vertical misfit and torque loss of abutment screws. braz dent j. 2008 [cited 2014 dec 5]; 19: 358-63. available from: http://www.scielo.br/pdf/bdj/v19n4/ v19n4a13.pdf. 12. burguete rl, johns rb, king t pe. tightening characteristics for screwed joints in osseointegrated dental implants. j prosthet dent. 1994; 71: 592-9. failure of prosthetic screws on 971 implants braz j oral sci. 14(3):195-198 13. junqueira mc, ribeiro rf, faria acl, macedo ap, almeida rp. screw loosening of different ucla-type abutments after mechanical cycling. braz j oral sci. 2013 [cited 2014 aug 22]; 12: 228-32. available from: http:// www.scielo.br/pdf/bjos/v12n3/a14v12n3.pdf. 14. jamiyandorj o, kim s, shim j-s, lee k-w. effect of using a titanium washer on the removal torque of an abutment screw in the external connection type of dental implant. implant dent. 2012; 21: 156-9. 15. tom w. p. korioth, antonio c. cardoso av. effect of washers on reverse torque displacement of dental implant gold retaining screws. j prosthet dent. 1999; 82: 312-6. 16. versluis a, korioth tw, cardoso a c. numerical analysis of a dental implant system preloaded with a washer. int j oral maxillofac implants. 1999; 14: 337-41. 198198198198198failure of prosthetic screws on 971 implants braz j oral sci. 14(3):195-198 404 not found 1http://dx.doi.org/10.20396/bjos.v18i0.8657266 volume 18 2019 e191643 original article 1 department of restorative dentisty, school of dentistry, pontifical catholic university of rio grande do sul, porto alegre, rio grande do sul, brazil. corresponding author: deise caren somacal school of dentistry – pontifical catholic university of rio grande do sul avenida ipiranga, 6681 90616-900 porto alegre, rs, brazil. phone/fax.: +55-51-3320.3538 e-mail address: deisecaren@gmail.com https://orcid.org/0000-0003-0861-3189 received: april 30, 2019 accepted: september 14 2019 surface roughness of monolithic zirconia ceramic submitted to different polishing systems deise caren somacal1,*, júlia willers dreyer1, patrícia danesi1, ana maria spohr1 aim: the objective was to evaluate, quantitative and qualitative, the abrasive effect of three polishing systems on the monolithic zirconia ceramic. methods: thirty disk-shaped samples of  yttria tetragonal zirconia polycrystal (y-tzp) were randomly distributed in three groups (n = 10) according to polishing system: g1komet system (ko); g2 ceragloss system (cg); g3 eve diacera system (ev). the surface roughness (ra) was obtained with rugosimeter in four different moments: a) initial glaze sample (ra0); b) after occlusal adjustment with diamond burs (ra1); c) after polishing with the abrasive systems (ra2); d) after polishing with felt disc and diamond paste (ra3). four additional samples were observed in scanning electron microscopy (sem). results: according to the generalized estimating equation followed by the bonferroni test (α = 0.05), the cg provided the lowest ra2 (0.63 μm), not differing significantly from the ko (0.78 μm). the highest ra2 was obtained with the ev (0.97 μm), which did not differ significantly from the ko. there was no statistical difference in ra between the polishing with the abrasive systems (ra2) and the final polishing with diamond paste (ra3). sem images showed that the polishing systems did not completely remove the grooves caused by the diamond burs during the occlusal adjustment. conclusion: it was concluded that cg promoted smoother surface of the monolithic zirconia ceramic compared to ev, and intermediate smoothness was obtained with ko. keywords: dental polishing. surface properties. ceramics. https://orcid.org/0000-0003-0861-3189 2 somacal et al. introduction there are different types of dental ceramics for tooth restoration nowadays. the choice of the material depends on some factors, such as aesthetics and the fracture strength. metal-free ceramic restorations have become popular among patients and dentists. these restorations can be obtained through laboratory-based methods which have been described as time-consuming, sensitive in technique and unpredictable due to many variables1. another alternative for dentists and laboratories is the computer-aided design/computer-aided manufacturing (cad-cam) technology. cad-cam system presents greater precision from industrially manufactured blocks, besides the optimization of time and reduction of clinical appointments2. advances in the technique of making metal-free ceramic restorations have allowed the creation of monolithic restorations. they can be obtained in a single moment by the cadcam method, reducing manufacturing time, and using only one type of ceramic. within a wide range of monolithic materials available for cad-cam, monolithic high translucent ytria tetragonal zirconia polycrystals (y-tzp) stands out due to its favorable mechanical and aesthetic properties. this material has characteristics of color stability, hardness, wear resistance and translucency. in addition, it has low thermal conductivity, high flexural strength and phase transformation capacity, preventing the propagation of cracks in areas of greater occlusal stress3-5. the technique of obtaining this monolithic restoration is simpler than porcelain-veneered y-tzp crown cores, favoring the strength of the restoration6. despite the high precision of the restorations made by the cad-cam system, adjustments are often necessary, before or after luting. finishing or even polishing the surface of the y-tzp after sintering can create a layer of compressive stress due to the occurrence of phase reversal. the y-tzp in the tetragonal phase converts to the monoclinic phase (t/m) causing a decrease in its mechanical properties7. therefore, this adjustment requires precaution so that there is no change in the y-tzp structure or excessive roughness of the restoration which can compromising its longevity8,9. in case of restoration adjustment after cementation, the only possibility of restoring the surface smoothness again is through the mechanical polishing procedure. polishing is a process characterized by the production of smoothness and surface gloss by the use of abrasive instruments. the objective of these procedures is to eliminate the roughness caused by the diamond burs, in order to increase patient comfort, decrease bacterial plaque accumulation and avoid excessive wear of the antagonist tooth10,11. there are several ceramic finishing and polishing systems on the market, and it is important to verify the ability of these instruments to polish the ceramic. the aim of this study was to evaluate quantitatively and qualitatively the surface roughness obtained with three polishing systems on the monolithic zirconia ceramic. the study followed the null hypothesis that there is no significant difference in surface roughness among the different polishing systems. material and methods the materials used in this study are described in table 1. 3 somacal et al. obtaining the ceramic disks thirty disk-shaped samples of y-tzp with 7 mm diameter and 3 mm height were obtained. the samples were made by a prosthetic laboratory that has the computerized system. in this system, a pre-sintered zirconia blocks were milled into the desired sample shape and sintered according to the manufacturer’s recommendations. subsequently, the surface was finished with # 400, # 600 and # 1200 with silicon carbide paper with running water in a polishing machine (panambra, são paulo, sp, brazil) for surface standardization. the samples were also ultrasonically cleaned (odontobrás, ribeirão preto, sp, brazil) in distilled water for 10 min to ensure a contaminant-free ceramic surface, followed by drying and glaze in prosthetic laboratory. surface preparation of sample the 30 y-tzp samples were randomly divided into three groups (n = 10) according to the polishing system used: group 1komet system (ko); group 2 – ceragloss system (cg) and group 3 eve diacera system (ev). the diamond bur 4138, in high speed handpiece with refrigeration, was applied for 3 s on the y-tzp surface to simulate the occlusal wear. this procedure was followed by the 4138f and 4138ff diamond burs, which were changed every five samples. after the wear process, the ceramic surface was polished with one of the polishing systems that consisted of different tips. each tip was applied in low speed for 30 s according to the following sequence: a. ko system first step: blue abrasive called a pre-polisher; second step: gray abrasive for final polish. b. cg system – first step: green abrasive used for finishing and rapid wear of the material; second step: blue abrasive to smooth the surface; third step: yellow abrasive for final polish. c. ev system – first step: green abrasive used for finishing; second step: pink abrasive for final polish. in order to complete the polishing, a felt disc was used with a diamond polishing paste for 30 s. after polishing, the specimens were ultrasonically cleaned in distilled water for 10 min. all procedures were done by the same operator. table 1. materials used in this study. material fabricante komet system zr komet, são paulo, brazil. ceragloss system edenta ag, aubonne, switzerland. eve diacera system eve ernst vetter gmbh, keltern, germany. diamond burs (4138, 4138f, 4138ff) kg sorensen, são paulo, brazil. felt discs (diamond flex) fgm, santa catarina, brazil. diamond paste (diamond excel) fgm, santa catarina, brazil. ceramic disks (zircônia lava frame) 3m espe, st. paul, united state of america. 4 somacal et al. quantitative analysis of surface roughness the surface roughness of all ceramic samples was measured with a rugosimeter (model sj-210, mitutoyo, kanagawa, japan) at four different moments: glaze sample (ra0), after occlusal adjustment with diamond burs (ra1), after polishing with the polishing systems (ra2), after polishing with felt disc and diamond paste (ra3). three consecutive measurements were performed in three different regions of the sample (one central, one to the right and one to the left), with a cut-off of 0.25, and the average of the three measurements was obtained. qualitative analysis of surface roughness four additional samples were obtained, one sample being glazed and the other representing each of the polishing systems. the samples were ultrasonically cleaned with distilled water for 15 min, dried in a dehumidifier with silica gel for three days and sputter-coated. then, the surface topography of these samples was observed by scanning electron microscopy (sem) (phillips xl 30, philips electronic instruments inc., mahwah, nj, eua) at 500× magnification. statistical analysis the surface roughness values were submitted to the shapiro-wilk normality test. after the values were analysed by the generalized estimating equation followed by the bonferroni test. the significance level was 5%. results quantitative analysis there was no significant difference in surface roughness among the samples at ra0 (glazed samples). the diamond burs (ra1) increased significantly the surface roughness of all samples when compared with ra0. cg system provided the lowest ra2 (0.63 μm), not differing significantly from ko system (0.78 μm). the highest ra2 was obtained with ev system (0.97 μm), which did not differ significantly from ko system. the polishing with the diamond paste (ra3) reduced the surface roughness, but there was no significant difference in relation to the polishing systems (ra2) (table 2). table 2. surface roughness values (μm) and standard-deviations of the different groups. surface treatment komet system (ko) ceragloss system (cg) eve diacera system (ev) initial – glazed sample (ra0) 0.61ab (±0.24) 0.48ab (±0.16) 0.41ac (±0.25) after abrasion with diamond burs (ra1) 1.03aa (±0.44) 0.96aa (±0.34) 1.22aa (±0.48) after polishing systems (ra2) 0.78abab (±0.31) 0.63bb (±0.24) 0.97aab (±0.34) after felt disc with diamond paste (ra3) 0.72abb (±0.16) 0.56bb (±0.21) 0.87ab (±0.28) means followed by the same capital letter in the lines and the same lowercase letter in the columns do not differ statistically from each other by the bonferroni test. significance level α=0,05. 5 somacal et al. qualitative analysis figure 1 shows the surface topography of y-tzp after the different treatments. it is observed that the polishing systems did not completely remove the grooves caused by the diamond burs during the occlusal adjustment (figures 1b, 1c and 1d). it also did not reproduce the smooth surface of the glazed y-tzp (figure 1a). more grooves remained on the surface after polishing with ev system (figure 1d). discussion according to the results, the surface roughness of monolithic zirconia was influenced by polishing systems. therefore, the null hypothesis was rejected. the dental market offers different polishing systems. each polishing system is composed of a primary abrasive, which determines the polishing effect, as well as figure 1. sem images of y-tzp surface (500x). (a): glazed surface; (b): polished with ceragloss system; (c): polished with komet system; (d): polished with eve diacera system. black arrows indicate grooves caused by the diamond burs. a b c d det etd hv 20.00 kv mag 500 x spot 4.0 wd 11.5 mm 300 µm det etd hv 20.00 kv mag 500 x spot 4.0 wd 11.2 mm 300 µm det etd hv 20.00 kv mag 500 x spot 4.0 wd 11.6 mm 300 µm det etd hv 20.00 kv mag 500 x spot 4.0 wd 11.6 mm 300 µm 6 somacal et al. a complementary abrasive and a binder material to hold these abrasive particles and shape the instrument12. mohs scale quantifies the hardness of the minerals in values from 1 to 10. y-tzp has an absolute hardness value of 9, requiring an abrasive material composed of diamond, silicon carbide, aluminum oxide or zirconium oxide12. goo et al.13 stated that diamond-impregnated polishing systems were more effective than silica carbide-impregnated ones in reducing the surface roughness of y-tzp. according to the manufacturers’ catalogs, the three polishing systems used in the present study are impregnated with diamond particles and are suitable for y-tzp restorations. cg system showed the lowest surface roughness on y-tzp, not differing from ko system. these results corroborate with another study14. al-haj husain et al.5 reported that the effectiveness of cg system is related to minor loss of abrasive particles during the polishing on y-tzp surface. the authors stated that roughness of the polishing instrument and the integrity of its surface are important for polishing effectiveness. ev system promoted higher surface roughness without significant difference from ko system. not all diamond-impregnated polishing systems work alike on the y-tzp, since the effectiveness of the polishing systems is related to the percentage of diamond abrasive particles and the percentage of binder13. in general, none of the polishing systems tested in the present study created a smoother surface than the glazed y-tzp, which corroborates with another study15. the increase of surface roughness on dental ceramics after occlusal adjustment causes wear on the surface of antagonist teeth, especially in sound teeth16,17. dental ceramics present greater hardness than dental surface, causing cracks on dental enamel and loss of its surface structure13. however, y-tzp hardness is double than other dental ceramics18, tending a greater wear of the antagonist tooth and detrimental effects. in the present study, the specimens were glazed before the simulation of occlusal wear and y-tzp surface may also be polished instead of glazed. according to janyavula et al.19 polished y-tzp is wear-friendly to the opposing tooth and glazed y-tzp causes more wear of antagonist tooth. therefore, the glazing of y-tzp should be avoided unless there is a high esthetic demand in which it should be polished and then glazed19. a systematic review analyzed the abrasion effect on the mechanical behavior of y-tzp. it concluded that necessary adjustments are possible on ceramic surface without causing damage to the y-tzp strength. to avoid jeopardizing the y-tzp restoration, a protocol which introduces the least possible surface defects should be chosen. thus, it is suggested using a rotating instrument at low speed, allowing movement control, besides instruments with abrasive particles up to 50 μm20. these requirements were followed in the present study. it was also evaluated the polishing with a diamond paste as the final step in the sequence. this procedure is indicated to increase the smoothness of ceramic surfaces15. based on the results, surface roughness was reduced after diamond paste polishing, but there was no significant difference when compared to polishing systems. these results showed that the diamond paste had little effect in obtaining a smoother surface on y-tzp surface. 7 somacal et al. the surface roughness also influences biofilm formation; when higher than 0.2 µm it favours bacterial adhesion in restorative materials21,22. the surface roughness values were 3 to 5 times higher than 0.2 µm after the use of polishing systems in the present study. happe et al.23 obtained surface roughness of 0.17 µm for ko system on y-tzp surface. however, no diamond bur was used prior to the polishing system, which could justify the difference in surface roughness values between the studies. the sem images showed that the three polishing systems were not able to completely remove the grooves created by the diamond burs. the smooth surface observed on the glazed y-tzp was not reproduced by the polishing systems, which corroborates with another study24. ev system obtained the highest surface roughness value and more grooves were observed on the y-tzp surface. therefore, the quantitative analysis of the surface roughness agreed with the qualitative analysis of the ceramic surface by sem. although there are different results in the literature, most of the studies evaluating polishing procedures is unanimous in pointing out the advantages of a smooth surface. this is also important for the aesthetic viewpoint as well as for patient’s comfort. jones et al.25 determined a threshold of detection values for surface roughness of restorations by patients using their tongue. it was observed that the volunteers were able to distinguish between roughness values of 0.5 µm or less with their tongue. so, it is expected that patients may detect differences on surface roughness between the diamond burs and the polishing systems. probably, patients would not detect differences on surface roughness among the polishing systems. in addition, the authors of a clinical study claimed that polished y-tzp is a versatile restorative material for its aesthetic properties and high strength, being promissory26. the limitation of the present study is the polishing procedures performed on flat surfaces samples. these flat surfaces differ from the clinical reality, as the occlusal surfaces of molars and premolars have anatomical features that could make the polishing procedure harder. the following conclusions can be drawn: • the polishing systems used in this study were able to reduce the roughness of the monolithic zirconia ceramic caused by the diamond burs. however, they did not remove the grooves, presenting greater roughness when compared to the glazed surface. • ceragloss system promoted smoother surface of the monolithic zirconia ceramic compared to eve diacera system, and intermediate smoothness was obtained with komet system. • the polishing with diamond paste did not significantly reduce the surface roughness. acknowledgements this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior – brasil (capes) – finance code 001. 8 somacal et al. conflicts of interest the author does not have any financial interest in the companies whose materials are included in this article. references 1. senyilmaz dp, canay s, heydecke g, strub jr. influence of thermomechanical fatigue loading on the fracture resistance of all-ceramic posterior crowns. eur j prosthodont restor dent. 2010;18(2):50-4. 2. li rw, chow tw, matinlinna jp. ceramic dental biomaterials and cad/cam technology: state of the art. j prosthodont res. 2014 oct;58(4):208-16. doi: 10.1016/j.jpor.2014.07.003. 3. kim hk, kim sh, lee jb, ha sr. effects of surface treatments on the translucency, opalescence, and surface texture of dental monolithic zirconia ceramics. j prosthet dent. 2016 jun;115(6):773-9. doi: 10.1016/j.prosdent.2015.11.020. 4. gui j, xie z. phase transformation and slow crack growth study of ytzp dental ceramic. mater sci eng a. 2016 sep;676:531-5. doi: 10.1016/j.msea.2016.09.026. 5. al-haj husain n, camilleri j, özcan m. effect of polishing instruments and polishing regimens on surface topography and phase transformation of monolithic zirconia: an evaluation with xps and xrd analysis. j mech behav biomed mater. 2016 dec;64:104-12. doi: 10.1016/j.jmbbm.2016.07.025. 6. johansson c, kmet 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roughness of zirconia for full-contour crowns after clinically simulated grinding and polishing. int j oral sci. 2014 dec;6(4):241-6. doi: 10.1038/ijos.2014.34. 11. rashid h. the effect of surface roughness on ceramics used in dentistry: a review of literature. eur j dent. 2014 oct;8(4):571-9. doi: 10.4103/1305-7456.143646. 12. koudi ms. dental materials: prep manual for undergraduates. new delhi: harcourti-india-private; 2007. 13. goo cl, yap a, tan k, fawzy as. effect of polishing systems on surface roughness and topography of monolithic zirconia. oper dent. 2016 jul-aug;41(4):417-23. doi: 10.2341/15-064-l. 14. park c, vang ms, park sw, lim hp. effect of various polishing systems on the surface roughness and phase transformation of zirconia and the durability of the polishing systems. j prosthet dent. 2017 mar;117(3):430-7. doi: 10.1016/j.prosdent.2016.10.005. 15. steiner r, beier us, heiss-kisielewsky i, engelmeier r, dumfahrt h, dhima m. adjusting dental ceramics: an in vitro evaluation of the ability of various ceramic polishing kits to mimic glazed dental ceramic surface. j prosthet dent. 2015 jun;113(6):616-22. doi: 10.1016/j.prosdent.2014.12.007. 9 somacal et al. 16. delong r, douglas wh, sakaguchi rl, pintado mr. the wear of dental porcelain in an artificial mouth. dent mater. 1986 oct;2(5):214-9. 17. jagger dc, harrison a. an in vitro investigation into the wear effects of unglazed, glazed, and polished porcelain on human enamel. j prosthet dent. 1994 sep;72(3):320-3. 18. raigrodski aj. contemporary materials and technologies for allceramic fixed partial dentures: a review of the literature. j prosthet dent. 2004 dec;92(6):557-62. 19. janyavula s, lawson n, cakir d, beck p, ramp lc, burgess jo. the wear of polished and glazed zirconia against enamel. j prosthet dent. 2016 aug 1. pii: s0022-3913(16)30197-4. doi: 10.1016/j.prosdent.2016.04.026. 20. pereira gkr, fraga s, montagner af, soares fzm, kleverlaan cj, valandro lf. the effect of grinding on the mechanical behavior of y-tzp ceramics: a systematic review and meta-analyses. j mech behav biomed mater. 2016 oct;63:417-442. doi: 10.1016/j.jmbbm.2016.06.028. 21. bollen cm, lambrechts p, quirynen m. comparison of surface roughness of oral hard materials to the threshold surface roughness for bacterial plaque retention: a review of the literature. dent mater. 1997 jul;13(4):258-69. 22. teughels w, van assche n, sliepen i, quirynen m. effect of material characteristics and/or surface topography on biofilm development. clin oral implants res. 2006 oct;17 suppl 2:68-81. 23. happe a, röling n, schäfer a, rothamel d. effects of different polishing protocols on the surface roughness of y-tzp surfaces used for custom-made implant abutments: a controlled morphologic sem and profilometric pilot study. j prosthet dent. 2015 may;113(5):440-7. doi: 10.1016/j. prosdent.2014.12.005. 24. mohammadi-bassir m, babasafari m, rezvani mb, jamshidian m. effect of coarse grinding, overglazing, and 2 polishing systems on the flexural strength, surface roughness, and phase transformation of yttrium-stabilized tetragonal zirconia. j prosthet dent. 2017 nov;118(5):658-665. doi: 10.1016/j.prosdent.2016.12.019. 25. jones cs, billington rw, pearson gj. the in vivo perception of roughness of restorations. br dent j. 2004 jan 10;196(1):42-5. 26. esquivel-upshaw jf, kim mj, hsu sm, abdulhameed n, jenkins r, neal d, et al. randomized clinical study of wear of ebamel antagonists against polished monolithic zirconia crowns. j dent. 2018 jan;68:19-27. doi: 10.1016/j.jdent.2017.10.005. oral sciences n3 case report braz j oral sci. january | march 2013 volume 12, number 1 admission dental examination: protocol and its importance in the diagnosis of oral pathology mário marques fernandes1,2, mara rosângeles de oliveira2, rafael bender carpena de menezes de oliveira2,3, talita lima de castro4,5, luiz renato paranhos6, eduado daruge júnior4 1department of community dentistry, faculty of dentistry, são paulo university, são paulo, sp, brazil 2biomedical service of public prosecution of rio grande do sul state, rs, brazil 3oral and maxillofacial surgery service of irmandade santa casa de misericórdia, porto alegre, rs, brazil 4department of community dentistry, piracicaba dental school, university of campinas, piracicaba, sp, brazil 5forensic dentist, civil police of rondônia state, ro, brazil 6oral biology, sacred heart university, bauru, sp, brazil correspondence to: mário marques fernandes rua andrade neves 106, 12ºandar serviço biomédico do ministério público do estado do rio grande do sul centro, cep 90010-210 porto alegre, rs, brasil e-mail: mfmario@mp.rs.gov.br received for publication: november 04, 2012 accepted: february 09, 2013 abstract the aim of this paper is to describe an efficient clinical protocol used in admittance dental examinations, highlighting the importance of this evaluation in the diagnosis of oral pathologies. two admission case reports in which gingival abscess and radiopaque apical lesion were found during the examination are presented for illustration purposes. a clinical examination in the admission inspection is the main factor for a good selection of treatment and prevention of future difficulties. the admission dental examination by means of a standardized clinical approach including clinical interview, physical examination and complementary exams, avoids the person to be admitted to the public service while having some pathology that might compromises his/her capacity of handling the selected function, preventing absenteeism for dental reasons and consequently a cost to the state and functional repercussion to the server. keywords: occupational health, occupational dentistry, routine diagnostic tests, diagnosis, oral, absenteeism. introduction according to the law 5081/661, which regulates the professional practice of dentistry in brazil, the dental examination is a prerogative of the dentist. this examination also finds support in the articles 63 and 64 of the cfo resolution 63/20052, which comprise the technical expertise in administrative and labor courts as functions of the specialist in forensic dentistry, and in articles 67 and 68, the dental examinations for labor ends are competence of the specialist in occupational dentistry. as part of the admission examinations, it is important not only to diagnose oral problems that affect or may affect directly the examinees, specifically investigating the epidemiology and pathology of these problems, but also to study the impact that may result in the individuals’ quality of life. such diseases can affect the work routine of an individual, causing physical and emotional damages as well as decrease their productive capacity. it must also be considered the workers’ unawareness of oral health problems, the health professionals’ lack of interest and knowledge regarding the study and correct interpretation of oral health braz j oral sci. 12(1):46-51 4747474747 problems that affect workers, and the need for a firm and efficient worker’s health policy3. the admission health examinations performed and the exams required from candidates for military public positions are published in the selection announcement of their contest that describes the routines and stomatognathic disabling criteria used4-5. the candidate’s physical and mental health is a requirement to join the public service6, and the entrance is preceded by the performance of health inspection conducted by the official organ of expertise. patients with diseases such as active tuberculosis, cancer, leprosy, parkinson’s disease, ankylosing spondyloarthrosis, later stages of paget’s disease (osteitis deformans), acquired immunodeficiency syndrome (aids), among others, may enter the public service provided that the expert entry assessment and the probation period, present ability to exercise the function, demonstrate clinical attendance and adherence to appropriate treatment in scientific indication standards approved by health authorities7. considering the possibility of clinical findings with systemic origin in addition to the specific conditions of oral health in the admission examinations carried out, there are several studies that support the importance of knowledge of oral manifestations of diseases such as tuberculosis8, leprosy9, parkinson’s disease10, ankylosing spondyloarthrosis11, later stages of paget’s disease12 and aids8,13. this article aims to describe an efficient protocol used in admittance dental examinations, highlighting the importance of this evaluation in the diagnosis of oral pathologies. two admission case reports are presented for illustration purposes. protocol the study was approved by the research ethics committee of the piracicaba dental school, university of são paulo, brazil (protocol number 144/2008). in reviewing the literature on the subject, using the descriptors of this study and related, little was found published in indexed journals. the recommended routines to perform the dental examination for entrance into public service were similar to those used in clinical management. when presenting the cases of a service of occupational medicine in a large hospital in sao paulo, santos et al.14 affirmed that the pre-admission examination requires a rigorous assessment of the conditions of oral infection, aesthetics and other diseases that originate in the mouth and may have systemic repercussions. the authors highlighted that the criteria adopted in oral evaluation and to release the candidate for performing his/her functions can interfere with the amount of emergency care and the number of later removals8,14. the protocol presented here was developed by dentists based on clinical experience and scientific literature for use in admission dental evaluations in public institutions at a first moment, with possibility of being extended to the private sphere. considering the expert nature of the service, the protocol brings elements that help in the description and characterization of the individuals and the morbid events that they can present during the examination. these elements may be useful in future evaluations as identification, redeployment functional exams, removal, granting permission for health care, etc. as in all clinical investigations to establish a diagnosis, the protocol involves anamnesis, physical examination and complementary tests. the anamnesis is obtained mainly through the filling of the term of declaration of health for the purpose of entry, an instrument used by the service staff for the entrant answer questions on general health and thus give his/her views on issues related to current and past illnesses, medication use, alcohol use and use of illegal drugs. the term must be dated and signed by the entrant, who is responsible for the accuracy of the information provided. in the case of inaccuracy or omission of any of the questions raised, the corresponding admission report can be canceled. on the day of expertise, as complementary exams, the following radiographic examinations must be done: a panoramic radiograph, for evaluation of the bone arcades, adjacent structures and possible pathological changes, and bitewing radiographs of bilateral molars and premolars, aiming at the evaluation of caries, marginal adaptation of restorations and the alveolar bone crest. it is oriented that the radiographs are recent dated up to 3 months prior to the expertise. imaging or biochemical tests may also be required as well as reports of various specialties for information and clarification as necessary, through formal acquiescence of the patient15. pregnant women, regardless of gestational age, are exempted from submission of radiographs because of the risks of the procedure, although there is controversy about this subject in literature. on physical examination, it is evaluated general extraoral characteristics such as facial anthropometry (if the examinee has facial skeletal pattern as dolichofacial, brachyfacial or mesofacial type), the temporomandibular joint (if there is clicking, crepitation, limitation of mouth opening, lateral deviation or pain), and the intraoral characteristics such as occlusion, the relationship between the dental arches, transverse, anteroposterior and molar relation, presence of oral parafunction and passive lib seal, stomatological evaluation, observing the lips, tongue, mouth floor, vestibule, palate and oral mucosa, in addition to accurate intraoral inspection of the teeth and periodontal evaluation, and the execution of the odontogram. table 1 illustrates the protocol proposed and table 2 contains the basic materials required. it is worth noting the importance of assessing the immunity of employees of hospitals and insalubrious centers. the collection of oral fluids as a source of material for noninvasive diagnostic tests should be performed by a dental professional as a routine procedure during pre-admissional physical examination14. the entrants who present, at the time of expertise, conditions that compromise the ability to exercise the function must be referred for treatment before the tenure ceremony, and must be submitted to a new assessment after the procedure. in these situations, it is delivered to the examinees the technical report routing, which describes admission dental examination: protocol and its importance in the diagnosis of oral pathology braz j oral sci. 12(1):46-51 4848484848 clinical sequence: 1. general (physical defects, abnormal walking, body asymmetries) 2.extraoral 2.1. visual inspection (asymmetries, scars, hemangiomas) 2.2. analysis of mouth opening trajectory 2.3. facial muscle palpation (changes in the muscles) 2.4. examination of temporomandibular joint (auscultation and palpation external and intra-auricular in the three open positions) 2.5. palpation of ganglionic chains 2.6. paresthesias search 2.7. other evaluations (low salivary flow, altered taste, halitosis) 3. intraoral 3.1. dental examination (odontogram with legend) 3.2. periodontal examination (visible plaque, gingival bleeding and probing depth by sextants) 3.3. evaluation of mobility of teeth, implants and prostheses 3.4. examination of bottom of sulcus, buccal mucosa, lips, palate, floor of mouth and tongue 3.5. examination of the occlusion (positions and movements) 3.6. examination of the muscles (intraoral) 3.7. angle’s classification 4. request additional exams or reports as needed table 1: clinical protocol suggested for admission dental examination materials and instruments for the admission dental examination: 1. personal protective equipment (apron, gloves, mask, glasses and bonnets) 2. dental probe explorer no 5, mirror and clinical tweezers 3. periodontal probe 4. articulating paper tweezer (müller tweezer) 5. articulating paper 6. tongue depressor sticks 7. dental floss 8. dental aspirator, cotton rolls, gauze 9. plaque disclosing solution 10. lip retractors 11. stethoscope 12. camera / camcorder 13. lightbox and magnifying glass 14. spray for pulp sensitivity testing table 2: list of basic instruments and materials suggested for the examinations the pathology, requests the appropriate treatment and emphasizes the need to return for final evaluation. when the deadlines for tenure are scarce, the expert dentist can declare the newcomer temporarily unfit, and then the newcomer has up to 30 days for new examination and subsequent act of tenure on the job7. all findings from the clinical examination are recorded in the newcomer’s medical enchiridion, as well as additional exams and specialty reports. the newcomers who present any clinical changes that do not compromise their occupational ability are qualified and classified as “capable but with dental needs”. the dentist must issue a conclusive report regarding the ability or inability to work, support technically this decision, and also guide the examinee on the need for treatment, when the candidate is not performing15. on this basis, the pre-admission physical examination is of key importance, which, besides its main function of analyzing the individual’s labor capacity, it still plays the role of detecting and diagnosing oral disease and advising the newcomer about the most suitable treatment. case report 1: alterations in the maxilla the candidate was a 38-year-old caucasian male with history of extraction of the maxillary canines nearly 20 years before the admittance dental examination. he mentioned drug therapy, 2 months ago, due to a gingival abscess between the maxillary right lateral incisor and first premolar. clinical examination of the vestibule revealed the presence of mild edema and hyperemia in the gingival mucosa located between the teeth 12 and 14. on the occasion of the expert’s examination, an attest issued by the assistant dentist was delivered, proving to be the newcomer in dental treatment for the pathology found. the periapical radiographs showed divergent roots in the region between teeth 12 and 14 (figure 1), and between teeth 22 and 24 (figure 2), with a radiopaque area surrounded by a less radiopaque halo bilateral. fig. 1 radiopaque lesion in the maxilla periapical radiographic image in the right side. admission dental examination: protocol and its importance in the diagnosis of oral pathology braz j oral sci. 12(1):46-51 4949494949 case report 2: alterations in the mandible a 32-year-old caucasian male presented for dental expertise with the radiograph examinations requested. the analysis of the panoramic radiograph (figure 3) revealed diffuse radiopaque area in the root region of tooth 46. palpation of the vestibule (as oriented in protocol) showed no volume increase in the region, and the pulp sensitivity test was positive. the newcomer reported having knowledge of the injury for 10 years since the time he was an undergraduate dental student, and he believed that the lesion had increased a little. new periapical and occlusal radiographs were requested for a more accurate evaluation. the radiologist’s report indicated radiopaque image of dense aspect associated with the mesial root of tooth 46, extending apically and to the inter-root space, with obscuration of the periodontal ligament space and lamina dura of apical third of the mesial root presenting irregular form and no marginal radiolucent line (figure 4). the occlusal radiograph did not show changes in vestibular or lingual cortical (figure 5). the radiologist’s report suggested pathology compatible with idiopathic osteosclerosis. discussion given the issues mentioned above, the physical examination before employment is of great relevance, which, fig. 2 radiopaque lesion in the maxilla periapical radiographic image in the left side. fig. 3 dense radiopaque lesion in the mandible panoramic radiographic image. fig. 4 radiopaque lesion associated with the mesial root of tooth 46, extending apically to inter-root space periapical radiographic image. fig. 5 lateralized occlusal radiographic image, without changes in vestibular or lingual cortical. besides its main function to analyze the work capacity of the individual, still plays the role of detecting and diagnosing oral diseases, sending the entrant to appropriate treatment. in case 1, calcifying odontogenic cyst (gorlin’s cyst) in one of its variants and odontoma16 were possible differential diagnoses for the lesion. a review of the current classification of odontogenic tumors showed that odontomas are considered by some as developmental disorders and not neoplasias17. considering that the newcomer’s activities did not include continuous exposure to any risk agent (biological, chemical or physical), and knowing about the injury, its treatment and prognosis, the expert opted for approving the applicant’s ability. the examinee was oriented to continue with clinical follow-up with the dental specialist. in case 2, a possible diagnosis for the lesion included condensing osteitis, focal sclerosing osteomyelitis, periradicular osteosclerosis, sclerosing osteitis and sclerotic bone18. as similar changes to that related to the pathology, are described sclerosing osteitis and benign cementoblastoma, admission dental examination: protocol and its importance in the diagnosis of oral pathology braz j oral sci. 12(1):46-51 the latter being a rare case, which is evident before the age of 25, and the most affected tooth is the mandibular first molar, with pulp vitality, with slow growth and may expand bone corticals16. radiographically the tumor has adhered to the tooth and appears as a dense radiopaque mass well circumscribed surrounded by a thin uniform radiolucent halo, and the root outline is usually unseen. it is worthwhile making the differential diagnosis of odontoma, osteoblastoma, hypercementosis and focal sclerosing osteitis19. given this context, the radiopaque radiographic finding, the clinical characteristics at the time of examination (noting the absence of signs and symptoms), the prognosis of the injury, and even if the newcomer’s duties comprised in the public office were not about oral or physical effort or planned exposure to any agent of risk, the dental expert opted to classify the candidate as being capable, believing that the labor ability of the examinee at the time of examination was not hampered by the lesion. the examinee was oriented to maintain clinical and radiographic follow-up with the assistant dentist. both patients agreed to the publication of their cases by signing an informed consent form. it is highlighted here the importance of a detailed and carefully done pre-admission examination, following the standards and protocols, in order to detect possible oral alterations. oral diseases are often chronic and may cause physical and emotional distress, impaired concentration, decrease in employee productivity, or even work accidents14. these disorders are entirely avoidable if the examiners are aware during the clinical examination, about the function of realizing and diagnosing the presented oral pathologies. an excellent clinical examination in the admission inspection is a major factor of a good selection and prevention of future difficulties. it extends and maintains the operational training of selected personnel, prevents loss of financial and human resources in the recovery of easily identifiable deficiencies, and is of paramount importance in the prevention of future disabling causes in the worker career20. the dentist, being an official expert, performs single expertise, hospital and domiciled exams, in administrative processes and procedures, in order to assess the work condition of the examinee in circumstances that are within field of dentistry. the dentist must act impartiality and neutrality, not admitting any kind of pressure, imposition, constraint or restriction, and may even refuse to continue the examination if that happens15. there are few public institutions that have a dentist working in sectors that perform admittance examinations or related to occupational issues exams, but it is noted that the brazilian law has evolved to regulate this practice21. on this matter, it is emphasized the presence of dentists in the staffs of public ministry of the state of rio grande do sul, located in the “biomedical services”, the official organ of expertise of that institution. this department is responsible for the entrance exams, granting of licenses, and other removals provided in law6. the tasks of dentist’s office are listed as, among others, carrying out inspections within dentistry, signing attests and reports for license granting, in addition to performing or assisting expertise, including the admission or entrance examinations22. considering the admittance examinations for entry into public service, the expert who conducts the assessment shall keep in mind that the concept of physical ability is linked to individual’s capability to develop his work activities, included in the tasks relevant to the position for which he/she is being admitted at that time. the evaluator must also list the diseases encountered and the possible need for withdrawal to treatment, both aesthetic and functional, thus avoiding damage to work after the candidate’s entry. in the dental field, the most common needs of treatment found in the admittance examinations performed by the dental service are linked to the signs and symptoms of dental cavities (including in this context its evolution for periapical pathologies ) and periodontal problems, as well as any pathology related to cysts and tumors, as shown in the case reports presented in this paper. the studies found in literature about the dentist’s work in occupational activities are scarce and the dental criteria used for fitness in admission are varied4-5. according to the survey, there are few places that perform the dental entrance exam in public and private areas, for different reasons3. studies on the importance of information obtained from the admissions exams reinforce the idea that the inclusion of dentistry in the medical control program for occupational health, normalized as pcmso23, is important to attest to oral health within the system of occupational health, and to create a database for the dental field. consequently, it is seems clear the importance of the collaboration of dentistry with medicine in the production of admission and periodic medical examinations (occupational), being inserted, together with the various public and private sectors, in the concept of life quality at the workplace3. therefore, the admission dental exam prevents the person from entering the office with any condition that can lead to problems at the workplace and even to removals, with expenses to the state and functional consequences to the server. references 1. brazil. law n.º 5.081, 1996 august 24. available from: http:// www.planalto.gov.br/ccivil_03/leis/l5081.htm. 2. federal council of dentistry. resolution n.º 63/2005, 2005 april 19. available from: http://cfo.org.br/servicos-e-consultas/ato-normativo/ ?id=986. 3. araujo me, gonini jr a. worker oral health: the admissional and periodical examinations as an information health system. odontol soc. 1999; 1: 15-8. 4. brazil. ministry of defense. air force command. health direction. bulletin n.º 200, 2006 october 27. 5. brazil. ministry of defense. navy command. education direction. announcement of the selection process for entry into the marine health corps, annex v, 2007 december 30. 6. brazil. rio grande do sul state. complementary law n.º 10.098, 1994 february 03. available from: http: //www.al.rs.gov.br/legis/normas/ 10098.pdf. 7. brazil. rio grande do sul state. complementary law n.º 11.836,2002 october 21. available from: http: //www.al.rs.gov.br/legis/arquivos/ 11.836.pdf. admission dental examination: protocol and its importance in the diagnosis of oral pathology5050505050 braz j oral sci. 12(1):46-51 5151515151 8. petruzzi mnmr, salum fg, cherubini k, figueiredo maz. epidemiological characteristics and hiv-related oral lesions observed in patients from a southern brazilian city. rev odonto cienc. 2012; 27: 115-20. 9. ghosh s, gadda rb, vengal m, pai km, balachandran c, rao r et al. oro-facial aspects of leprosy: report of two cases with literature review. med oral patol oral cir bucal 2010; 15: e459-62. 10. debowes sl, tolle sl, bruhn am. parkinson’s disease: considerations for dental hygienists. int j dent hyg. 2012; 11: 15-21. 11. arakeri g, kusanale a, zaki ga, brennan pa. pathogenesis of posttraumatic ankylosis of the temporomandibular joint: a critical review. br j oral maxillofac surg. 2012; 50: 8-12. 12. garg k, devi p, thimmarasa vb, mehrotra v. osteomyelitis of the mandible as a complicating factor in paget’s disease: a case report. indian j dent. 2012; 3: 110-7. 13. girardi fm, scroferneker ml, gava v, pruinelli r. head and neck manifestations of paracoccidioidomycosis: an epidemiological study of 36 cases in brazil. mycopathologia. 2012; 173: 139-44. 14. santos pss, pinto mf, guimarães neto ja. occupational dentistry in the hospital setting. rev odonto cienc. 2008; 23: 307-10. 15. federal council of dentistry. resolution n.º 87/2009. available from: http://cfo.org.br/servicos-e-consultas/ato-normativo/?id=1356. 16. bishop ja. atlas of oral and maxillofacial histopathology. am j surg pathol. 2013; 37: 152. 17. costa do, maurício as, faria pa, silva le, mosqueda-taylor a, lourenço sq. odontogenic tumors: a retrospective study of four brazilian diagnostic pathology centers. med oral pathol oral cir bucal. 2012; 17: e389-94. 18. capelas j a, albuquerque b, paulo s, ferreira mm, ginjeira a. glossary of endodontic terms.– part ii. rev port estomatol cir maxilofac. 2008; 49: 57-62. 19. neelakandan rs, deshpande a, krithika c, bhargava d. maxillary cementoblastoma a rarity. oral maxillofac surg. 2012; 16: 119-21. 20. velloso lfn, ferreira lacn, perro filho mjt, caiafa js. health investigation for admittance to active navy service: importance of systematized clinical examination and repercussion at short, medium and long term of selection. arq bras med nav. 1989; 50: 151-60. 21. brazil. law n.º 11.907, 2009 february 2. available from: http:// www.planalto.gov.br/ccivil_03/_ato2007-2010/2009/lei/l11907.htm. 22. brazil. rio grande do sul state. law n.º 10.559, 1995 october 19. available from: http://www.al.rs.gov.br/legiscomp/arquivos/ lei%20n%c2%ba%2010.559.pdf. 23. brazil. ministry of labour and employment department of safety and health at work. regulatory standard 7 (nr 7), 1978 july 06. available from: http://www.mte.gov.br/legislacao/normas_regulamentadoras/ nr_07_at.pdf. admission dental examination: protocol and its importance in the diagnosis of oral pathology braz j oral sci. 12(1):46-51 original articlebraz j oral sci. april/june 2009 volume 8, number 2 thermoplasticity of microseal and tc gutta-percha (tanaka de castro) and three gutta-percha cones mário tanomaru-filho1, roberta bosso2, carlos alexandre souza bier2, juliane maria guerreiro-tanomaru1 1 dds, ms, phd, professor, department of restorative dentistry, faculdade de odontologia de araraquara, universidade estadual paulista “júlio de mesquita filho” (unesp), araraquara (sp), brazil 2 graduate student, faculdade de odontologia de araraquara, unesp, araraquara (sp), brazil received for publication: march 31, 2009 accepted: june 17, 2009 correspondence to: mário tanomaru filho departamento de odontologia restauradora, faculdade de odontologia de araraquara, unesp rua humaitá, 1.680 – centro cep 14801-903 – araraquara (sp), brazil phone: +55 (16) 3301-6390 fax: +55 (16) 3301-6392 e-mail: tanomaru@uol.com.br abstract aim: the aim of this study was to assess the thermoplasticity of materials used in root canal filling. methods: specimens with standardized dimensions were fabricated using tanari, roeko and activ point gutta-percha cones, as well as microseal and tc gutta-percha. after 24 hours, the specimens were placed in water at 70 ºc for 60 seconds and positioned between two glass slabs. each set was compressed by a 5 kg weight. digital images of the specimens before and after compression were obtained and analyzed. the thermoplasticity was evaluated based on the difference between the final and initial areas. data were statistically analyzed using anova and tukey’s tests at a 5% significance level. results: tc and microseal gutta-percha presented the highest thermoplasticity (p < 0.05). among the gutta-percha cones, tanari and roeko presented the highest thermoplasticity and differed when compared to activ point (p < 0.05). conclusions: the results of the present study showed that tc and microseal gutta-percha filling systems present better thermoplastic properties. keywords: endodontics, root canal filling materials, gutta-percha. introduction the main objective of endodontic treatment, after the cleaning and shaping of the root canals, is the complete filling of the pulp cavity in three dimensions1,2. two factors are important in the three-dimensional filling of the root canal system, namely, the filling technique1 and the properties of the material employed3. since root canal sealers may dissolve over time and shrink during setting4, it has been suggested that the ideal root canal filling should consist of a sufficient amount of guttapercha covered by a thin layer of sealer4-6. as a result, several filling techniques employing thermoplastic gutta-percha have been evaluated with regard to their effectiveness in filling irregular or lateral canals5-11. gutta-percha is available in two different crystalline forms and may be converted from one form into another. when extracted, gutta-percha is in its natural, alpha form; beta is the processed form, ready for use in endodontics12. however, some current filling techniques also employ gutta-percha in the alpha form, which is characterized by a lower melting point, lower viscosity and higher flow when compared to the beta form13. 82 tanomaru-filho m, bosso r, bier cas, guerreiro-tanomaru jm braz j oral sci. 8(2): 81-3 the clinical advantages associated w ith the alpha form of g utta-percha resulted in the development of the microseal system (sybron endo, orange, ca, usa). in brazil, a similar process of thermoplastic root canal f illing, the tc system (tanaka de castro & minatel ltda., cascavel, pr, brazil) was developed and patented in 2002, aiming at mak ing this technique more accessible to the clinicians while preser v ing the main properties of the microseal system. in thermoplastic or thermomechanical root canal filling methods, assessing the properties of gutta-percha when submitted to heat is of paramount importance12,14-17. the thermoplasticity of gutta-percha cones is affected by their chemical composition14,16-19, as well as by thermal changes resulting from the manufacturing process and use of these materials16,18. although several endodontic techniques have been evaluated with respect to their ability to adequately fill the root canal and its irregularities5-11, few studies have focused on the thermoplasticity of different brands of gutta-percha cones used in thermomechanical methods3,20,21,22. the aim of the present study was to assess the thermoplastic properties of gutta-percha from microseal and tc system, and three gutta-percha cones, namely tanari, roeko, and activ gp. material and methods five specimens (1.5 mm thick, 10 mm in diameter) were fabricated from each of the brands of gutta-percha listed in table 1, i.e. three different brands of gutta-percha cones as well as microseal and tc gutta-percha. material samples were kept in water at 70 ºc for 60 seconds, using a thermometer-controlled heating apparatus (righetto e cia., campinas, sp, brazil). the heated materials were placed into standardized rings with the above-mentioned dimensions (1.5 mm thick, 10 mm in diameter) and pressed between two glass slabs under controlled and constant force (0.5 n) for one minute. thereafter, the specimens were removed from the molds, the excess material was trimmed and dimensions were checked using a digital caliper. specimens were kept at a temperature between 25 and 30 oc for 24 hours and then returned to the heating apparatus at 70 oc for 60 seconds. next, each sample was again positioned between two glass slabs and a 5 kg weight was placed on the top of the slabs to produce a compressive force for 2 minutes. digital images of each specimen were obtained before and after compression and examined using an image processing and analysis software (uthscsa image tool for windows version 3.0, san antonio, tx, usa) to determine sample areas in mm2. the thermoplasticity of each material was determined by the difference between the final and initial areas obtained for each specimen. data statistical analysis was carried out by means of analysis of variance (anova) and multiple comparisons among the experimental groups were done by tukey’s post-hoc test. significance level was set at 5%. results means and standard deviation for the differences observed between final and initial areas for each specimen are shown in figure 1. statistical analysis revealed that tc and microseal gutta-percha had a similar behavior (p > 0.05) and reached the highest thermoplasticity results among all assessed samples (p < 0.001). among the gutta-percha cones, tanari and roeko presented statistically similar results (p > 0.05) and had a better performance (p < 0.05) in comparison to activ point. discussion almost 50 years ago, schilder1 suggested the need for three-dimensional filling of the root canal system. this method aimed at adequately filling the root canal and its irregularities. while the ability of different techniques to fill root canal irregularities has been extensively assessed, few studies published so far3,20,21,22 have investigated the thermoplastic properties of different brands and types of gutta-percha. due to the low number of studies focusing on the differences between commercially available brands of gutta-percha, no specific methodology for the assessment of the thermoplasticity of these materials has been described. in the present study, the american dental association (ada) specification no 57 (referring to endodontic sealing materials) and the iso 6876/2002 standard (dental root canal sealing materials) were adapted for the testing of gutta-percha samples, as previously described22. using a similar methodology, material manufacturer tanari tanariman ind. ltda., macapuru, am, brazil roeko roeko, langenau, germany activ gp brasseler, savannah, ga, usa microseal analytic endodontics, orange, ca, usa tc system tanaka de castro ltda., cascavel, pr, brazil table 1. endodontic filling materials experimental groups fi n al a re a i n iti al a re a (m m 2 ) 250 200 150 100 50 0 b b c a a tanari roeko active microseal tc figure 1. means (±sd) in mm2 of the differences between final and initial areas for each filling material. means with the same letter did not differ significantly from each other (p > 0.05). 83thermoplasticity of microseal and tc gutta-percha (tanaka de castro) and three gutta-percha cones braz j oral sci. 8(2): 81-3 tanomaru-filho et al.22 have shown that resilon presents adequate thermoplastic properties; the authors concluded by endorsing its use in thermoplastic root canal filling techniques. according to gutmann and witherspoon2, gutta-percha phase transitions occur when the material is heated or cooled. at a temperature of 46 to 52 ºc, the crystalline structure changes from the beta to the alpha phase; at higher temperatures (56 to 62 ºc), the material changes from the alpha phase into an amorphous form. according to schilder et al.15, a minimum temperature of 64 ºc is necessary to plasticize gutta-percha and allow its use in thermoplastic techniques. venturi et al.21 investigated three commercial brands of gutta-percha for the filling of accessory canals at different temperatures and observed flow >1.2 mm only when temperatures above 60 ºc were employed. based on these authors’ findings, the temperature of 70 ºc was chosen for the present study. the results of the present study showed differences among the assessed samples. tc and microseal gutta-percha (alpha phase) presented the highest thermoplasticity results when considering all samples. among the gutta-percha cones (beta phase), the tanari and roeko samples presented higher thermoplasticity means, with significant differences when compared to activ point. the highest thermoplasticity results obtained with tc and microseal systems are probably related to the thermal treatment that the material is submitted to during the manufacturing process12, resulting in alpha gutta-percha. gutta-percha is a natural polymer that undergoes industrial processing and incorporation of other substances before its application in dentistry. the thermoplastic properties of gutta-percha have been shown to depend directly on its composition, and they are more pronounced in its pure form than in the industrialized version17. other studies have also reported that the amount of inorganic fillers added to the material, as well as the thermal changes induced during cone manufacturing, may affect its properties16,18,19. gurgel filho et al.19 chemically and radiographically assessed five commercially available brands of gutta-percha and found differences in material composition that could potentially affect their plasticity. brands with higher amounts of gutta-percha in their composition showed better results while filling simulated accessory canals20. it is important to take into consideration that activ gutta-percha cones present a glass ionomer coating whose aim is to provide increased bonding to activ gp sealer, a glass-ionomer filling material recommended by the manufacturer. the presence of a layer of glass ionomer on activ gutta-percha cones might have been, therefore, responsible for the lower thermoplasticity results observed with this brand. the present study was carried out using a previously described methodology22, considered as an initial model for thermoplasticity evaluation. further research is required to increase the accuracy and standardization of the analysis of the thermoplastic properties of gutta-percha and similar root canal filling materials. in conclusion, the microseal and tc gutta-percha presented more adequate thermoplastic properties when compared to the other brands of gutta-percha evaluated in the study, thus endorsing their use in endodontic procedures. references 1. schilder h. filling root canals in three dimensions. dent clin north am. 1967;11:72344. 2. gutmann jl, witherspoon de. obturation of the cleaned and shaped root canal system. in: cohen s, burns rc. pathways of the pulp. eighth ed. st louis: mosby inc. 2002. p. 295-9. 3. tagger m, gold a. flow of various brands of gutta-percha cones under in vitro thermomechanical compaction j endod. 1988;14:115-20. 4. kontakiotis eg, wu m-k, wesselink pr. effect of sealer thickness on long-term sealing ability: a 2-year follow-up study. int endod j. 1997;30:307-12. 5. wu m-k, kastáková a, wesselink pr. quality of cold and warm gutta-percha fillings in mandibular premolars. int endod j. 2001;34:485-91. 6. jarrett is, marx d, covey d, karmazin m, lavin m, gound t. percentage of canals filled in apical cross sections – an in vitro study of seven obturation techniques. int endod j. 2004;37:392-8. 7. gencoglu n, yildririm t, garip y, karagenc b, yilmaz h. effectiveness of different gutta-percha techniques when filling experimental internal resorptive cavities. int endodo j. 2008,41:836-42. 8. goldberg f, massone ej, esmoris m, alfie d. comparison of different techniques for obturating experimental internal resorptive cavities. endod dent traumatol. 2000;16:116-21. 9. goldberg f, artaza lp, de silvio a. effectiveness of different obturation techniques in filling of simulated lateral canals. j endod. 2001;27:362-4. 10. venturi m. an ex vivo evaluation of a gutta-percha filling technique when used with two endodontic sealers: analysis of the filling of main and lateral canals. j endod. 2008;43:1105-10. 11. bowman c, baumgartner jc. gutta-percha obturation of lateral grooves and depressions. j endod. 2002;28:220-3. 12. goodman a, schilder h, aldrich w. the thermomechanical properties of guttapercha ii. the history and molecular chemistry of gutta-percha. oral surg oral med oral pathol. 1974;37:954-61. 13. ingle ji, newton cw, west jd, gutmann jl, glickman gn, korzon bh, et al. obturation of the radicular space. in: ingle ji, bakland lk. endodontics. fifth ed. hamilton: bc decker inc.; 2002. 14. schilder h, goodman a, aldrich w. the thermomechanical properties of guttapercha. iii. determination of phase transition temperatures for gutta-percha. oral surg oral med oral pathol. 1974;38:109-14. 15. schilder h, goodman a, aldrich w. the thermomechanical properties of guttapercha. v. volume changes in bulk gutta-percha as a function of temperature and its relationship to molecular phase transformation. oral surg oral med oral pathol. 1985;59:285-96. 16. marciano j, michailesco p, charpentier e, carrera lc, abadie mjm. thermomechanical analysis of dental gutta-percha. j endod. 1992;18: 263-70. 17. kolokuris i, arvanitoyannis i, blanshard jmv, robinson c. thermal analysis of commercial gutta-percha using differential scanning colorimeter and dynamic mechanical thermal analysis. j endod. 1992;18:4-9. 18. combe ec, cohen bd, cummings k. alpha and beta forms of gutta-percha in products for root canal filling. int endod j. 2001;34:447-51. 19. gurgel-filho ed, feitosa jpa, teixeira fb, monteiro de paula rc, araújo silva jb, souza filho fj. chemical and x-ray analyses of five brands of dental gutta-percha cone. int endod j. 2003;36:302-7. 20. gurgel-filho ed, feitosa jpa, gomes bpfa, ferraz ccr, souza filho fj, teixeira fb. assessment of different gutta-percha brands during the filling of simulated lateral canals. int endod j. 2006;39:113-8. 21. venturi m, di lenarda r, breschi l. an ex vivo comparison of three different guttapercha cones when compacted at different temperatures: rheological considerations in relation to the filling of lateral canals. int endod j. 2006; 39:648-56. 22. tanomaru filho m, faccio gs, tanomaru jmg, bier cas. evaluation of the thermoplasticity of different gutta-percha cones and resilon. aust endod j. 2007;33:23-6. oral sciences n3 original article braz j oral sci. january | march 2011 volume 10, number 1 influence of the respiratory mode and nonnutritive sucking habits in the palate dimensions luana cristina berwig1, márlon munhoz montenegro2, rodrigo agne ritzel3, ana maria toniolo da silva4, eliane castilhos rodrigues corrêa5, carolina lisbôa mezzomo4 1speech-language pathologist, master, speech-language pathology department, federal university of santa maria, santa maria, rio grande do sul, brazil 2dentist, master student, college of odontology, federal university of rio grande do sul, porto alegre, rio grande do sul, brazil 3otorhinolaryngologist, master, speech-language pathology department, federal university of santa maria, santa maria, rio grande do sul, brazil 4 speech-language pathologist, phd, professor, speech-language pathology department, federal university of santa maria, santa maria, rio grande do sul, brazil 5 physical therapist, phd, professor, physiotherapy department, federal university of santa maria, santa maria, rio grande do sul, brazil correspondence to: luana cristina berwig rua tamanday, 533, cep: 97060-540 santa maria/rio grande do sul, brasil e-mail: luanaberwig@gmail.com received for publication: september 01, 2010 accepted: february 03, 2011 abstract aim: to verify the influence of the respiratory mode and nonnutritive sucking habits in the transverse and vertical dimensions of the palate. methods: seventy-seven children aged 7 to 12 years, were divided, according the diagnosis of the respiratory mode and the presence of prolonged nonnutritive sucking habits. models of the upper dental arc were obtained of all children for evaluation of the measures of the palate in the region of the canines, first and second premolars and first molars. these measures were analyzed by the student’s t-test and analysis of variance. tukey’s test was used for the multiple comparisons. the significance level was set at p<0.05. results: it was verified that the mouth-breathing children showed smaller width and higher depth at the more posterior region of the palate. the children with prolonged nonnutritive sucking habits presented narrower and deeper palate at the anterior region of the palate. the canine distance was smaller in children who present mouth breathing associated to nonnutritive sucking habits and the depth at the second premolar was higher in mouth-breathers associated or not to prolonged nonnutritive sucking habits. conclusions: the results suggest that the respiratory mode and prolonged nonnutritive sucking habits influence in the transverse and vertical palate dimensions in the children evaluated in this study. keywords: thumb-sucking, sucking behavior, palate hard, measures, mouth breathing child. introduction the craniofacial growth and development were genetically determined. however, these processes can be affected by environmental factors such as sucking habits and mouth breathing, which change the muscular balance. this hypothesis is based on the functional matrix theory, according to which the soft parts acting on the different bone parts that compose the face would be the determinant factor of its growth and development1. based on the matrix functional concept, a previous study with rabbits in braz j oral sci. 10(1):42-49 43 phase of facial growth and development demonstrated the importance of the facial musculature on the functional matrix for the determination of development of the skeletal facial of those rabbits2. the nasorespiratory function and its relationship with the craniofacial growth has been a subject of great interest for over one hundred years. the modification of this function by a nasal airway obstruction or simply by mouth breathing habit can be, sometimes, associated to changes in the craniofacial complex3. depending on the frequency, intensity, duration and facial type, nonnutritive sucking habits also cause disturbances on the stomatognathic system due to the unbalance of the forces that naturally act in the oral cavity. these forces can favor the installation of the mouth breathing4-5. frequent adaptations of the orofacial complex commons to mouth breathing and prolonged nonnutritive sucking habits are: malocclusion, narrow maxilla, atresic palate, open lips at the rest position. tongue lowered on the floor of the mouth or protruded between the arches, flaccid orofacial musculature and atypical swallowing4,6-7. the present study was carried out considering that the muscular and functional unbalance of the stomatognathic system due mouth breathing and pacifier and thumb sucking for a prolonged time could influence palate growth and development. the aim of this study was to verify the influence of the respiratory mode and nonnutritive sucking habits in the transverse and vertical dimensions of the palate. material and methods the present study was approved by the ethics in research committee of the federal university of santa maria – cep/ufsm protocol number 220.0.243.000-8. a total of 273 children were screened from four public schools of santa maria city, rio grande do sul state, brazil. children participation was possible after their parents/ guardians received full explanation about the research and expressed their agreement by signing a written consent form. the exclusion criteria were: history of speech-language pathology and/or orthodontic treatment; evident signs of neurological impairment and/or syndromes; craniofacial malformations. the inclusion criteria were: to be 7 and 12 years old and caucasian. from the 273 screened children, 77 were selected, 37 boys and 40 girls. the selected children were subjected to the otorhinolaryngologic evaluation, which consisted of an interview with the children’s parents and oroscopy, anterior rhinoscopy, otoscopy and nasofibrolaryngoscopy exams. from the diagnosis obtained in this evaluation, the children were classified as nose breathers (nb), when they breathed predominantly by the nose and mouth breathers (mb), when they breathed predominantly by the mouth. the children were also classified according to the nonnutritive sucking habits by a speech-language pathologist, who interviewed the parents and asked them about the presence and duration of pacifier and thumb sucking habits. these habits were considered prolonged when present for three years or more. based on this information, the children were distributed in two groups: without habit (who) and with habit (wh). the children were also grouped according to the respiratory mode and the nonnutritive sucking habits in: nose breathing without habit (nbwoh) group nose breathing with habit (nbwh) group mouth breathing without habit (mbwoh) group mouth breathing with habit (mbwh) group children were clinically examined by a dentist, who made alginate impressions and obtained cast models from the upper arch of each one of the 77 participants. next, reference points were marked in the models for the transversal (width) and vertical (depth) measurements of the hard palate. the points were marked in the most apical palatal points of the maxillary canines, first and second premolars at the junction of the tooth and gingival margin8. in the first molars, the marked point corresponded to the union of the gingival margin with the palatal groove9 (figure 1). in case of one tooth or both teeth had not erupted, these points were not marked and the measurements were not made in the respective level of the teeth. these measurements were made using a digital caliper (western®) with 0.01mm of resolution and ± 0.02 mm of precision. for transversal measurements, the internal measuring faces of the instrument were used. for vertical measurements, a 0.05 mm stainless steel wire was cut with orthodontic pliers in the corresponding length to the transversal measurement and fixed with dental wax between the points in the level of each one of the considered tooth. after fixing the wire, the depth was measured with the depth measuring blade. the following measurements of the palate were taken, obeying this order: a) canine distance: transversal distance in millimeters between the points of the gingival margin of the maxillary canine (figure 2) influence of the respiratory mode and nonnutritive sucking habits in the palate dimensions braz j oral sci. 10(1):42-49 fig. 1. landmarks used to determine palate dimensions 44 b) canine depth: vertical measurements in millimeters from the midpalatal line to the stainless wire that linked the gingival margin of the maxillary canine (figure 3). c) first premolar distance: transversal distance in millimeters between the points of the gingival margin of the maxillary first premolars (figure 4). d) first premolar depth: vertical measurement in millimeters from the midpalatal line to the stainless wine that linked the gingival margin of the maxillary first premolars (figure 5). fig. 2. canine distance e) second premolar distance: transversal distance in millimeters between the points of the gingival margin of the maxillary second premolars (figure 6). f) second premolar depth: vertical measurement in millimeters from the midpalatal line to the stainless wire that linked the gingival margin of the maxillary second premolars (figure 7). g) molar distance: transversal distance in millimeters between the points of the gingival margin of maxillary first molars (figure 8). fig. 4. first premolar distance fig. 5. first premolar depth influence of the respiratory mode and nonnutritive sucking habits in the palate dimensions braz j oral sci. 10(1):42-49 fig. 3. canine depth fig. 8. molar distance fig. 7. second premolar depth fig. 6. second premolar distance h) molar depth: vertical measurement in millimeters from the palatine middle line to the stainless wire that linked the gingival margin of maxillary first molars (figure 9). after tabulation of the aforementioned measurements, the value of 0.05mm, corresponding to the diameter of the stainless wire was subtracted from the four depth measurements. after 30 days, 30% of the models were randomly selected and reexamined by the same examiner to confirm the reproducibility of the palate measurements and verify the agreement between the first and second measurements fig. 9. molar depth by intraclass correlation coefficient. it was verified significant agreement between all measurements. all variables presented normal distribution. the student’s t-test was used for the comparison between the palate dimensions between nb and mb, as well as between the who and wh groups. anova and tukey’s multiple-comparison test were used for the comparison of the palate dimensions between nbwoh, nbwh, mbwoh and mbwh. a significant level of 5% was set for all analyses. data were analyzed using the spss statistical software version 10.0 (2006). 45 influence of the respiratory mode and nonnutritive sucking habits in the palate dimensions braz j oral sci. 10(1):42-49 46 results the means, standard deviations, minimal and maximal values of the measurements and the comparison of the palate dimensions among the studied groups are presented in tables 1-3. table 1 shows statistically significant differences (p<0.05) in the mean values of the first and second premolars and molar distances and in the second premolar depth between the nb and mb groups. table 2 shows statistically significant differences (p<0.05) in the mean of the canine distance and the canine and first premolar depth between the woh and wh groups. comparing the mean values of the palate dimensions among nbwoh, nbwh, mbwoh and mbwh (table 3), statistically significant differences were observed in the canine distance and second premolar depth. table 4 presents the results of the multiple comparisons. in the canine distance, there were statistically significant differences between the mean values of the nbwoh and mbwh. in the second premolar depth, there were statistically significant differences between the mean values of the nbwoh and mbwoh, as well as between nbwoh and mbwh. discussion according to reviewed literature, few studies were found concerning the palate morphology or using a similar methodology to that of the present investigation. therefore, the results of this study were compared to others with different methodologies of palatal measurements as: measurements in the mouth with three-dimensional korkhaus compass10-11; with millimeter ruler12; measurements of cast models with threepalate dimensions p table 1: comparative results of mean and standard-deviation ( ± s), minimal and maximal values (x min –x max ) of transversal (t) and vertical (v) palate dimensions in the mouth and nasal breathers, regardless the presence the nonnutritive sucking habits. * student t-test (p< 0.05); nb=nose breathers; mb=mouth breathers. nb mb n=24 n=53 groups canine distance (t) canine depth (v) first premolar distance (t) first premolar depth (v) second premolar distance (t) second premolar depth (v) molar distance (t) molar depth (v) 26.92 ± 2.25 23.41 30.81 6.87 ± 2.08 3.84 10.71 28.35 ± 2.26 25.61 34.71 11.54 ± 1.57 8.26 14.33 31.91 ± 2.24 27.47 36.67 11.84 ± 1.67 8.06 15.20 36.45 ± 2.34 32.90 42.22 10.19 ± 1.86 5.39 14.20 25.98 ± 2.38 22.05 31.98 7.25 ± 2.73 2.63 13.20 27.23 ± 2.26 22.49 31.88 12.27 ± 1.55 9.66 16.34 30.69 ± 2.14 26.00 35.60 12.67 ± 1.5 210.15 16.75 35.24 ± 2.39 29.67 40.25 10.74 ± 1.8 17.27 16.03 0.145 0.590 0.048* 0.063 0.027* 0.035* 0.042* 0.232 dimensional korkhaus compass13. no previous study has investigated the relationship of between non-nutritive sucking habits and the palatal morphology, as analyzed by objective measurements. for this reason, the results of this study were compared to those of studies related to the dimensions of the upper dental arch. the analysis of the palate dimensions, considering the respiratory mode, showed that all means of the transversal measurements were smaller and the mean of vertical measurements were larger in the mouth-breathing children than in the nose breathers. these differences were significant between the groups in the transversal distance in the region of the first premolars, second premolars and first molars and in the second premolar depth (table 1). these results suggests that there is a trend to palate narrowing in the posterior region in mouth breathers, as the mean of the transversal distance were significantly smaller at the first and second premolar and first molar regions in this group (table 1). likewise, feres et al.13 (2009) verified significant difference between nose and mouth breathing groups in the measurements at the second premolar region. mouth-breathing children present narrower palate because the reduction of the air flow passage through the nasal cavity, which affect the lateral growth of the maxilla14. for this reason, the mouth-breathing patients frequently present posterior crossbite15-16. the lack of significant difference between the groups in the mean of canine distance (table 1) is in accordance with feres et al.13 (2009),who compared mouth and nose breathers, and with freitas et al.10 (2001) and ghasempour, mohammadzadeh and garakani et al.11 (2009), who compared influence of the respiratory mode and nonnutritive sucking habits in the palate dimensions braz j oral sci. 10(1):42-49 47 w o h n=38 groups w o h n=39 table 2: comparative results of mean and standard-deviation ( ± s), minimal and maximal values (x min –x max ) of transversal (t) and vertical (v) palate dimensions in the children with and without nonnutritive sucking habits, regardless the respiratory mode. *student’s t-test (p< 0.05); woh=without prolonged habits; wh=with prolonged habits. palate dimensions canine distance (t) canine depth (v) first premolar distance (t) first premolar depth (v) second premolar distance (t) second premolar depth (v) molar distance (t) molar depth (v) 27.02 ± 2.13 22.44 30.81 6.43 ± 2.41 2.63 12.24 27.99 ± 2.68 22.49 34.71 11.64 ± 1.60 8.26 16.34 31.36 ± 2.36 27.47 36.67 12.14 ± 1.74 8.06 16.75 36.16 ± 2.78 30.40 42.22 10.51 ± 1.92 5.39 14.35 25.48 ± 2.37 22.05 31.98 7.84 ± 2.53 4.13 13.20 27.17 ± 1.82 23.42 30.66 12.43 ± 1.48 9.22 16.12 30.78 ± 2.09 26.00 34.32 12.66 ± 1.43 10.37 15.77 35.10 ± 1.93 29.67 38.24 10.62 ± 1.77 7.27 16.03 p 0.007* 0.024* 0.119 0.026* 0.261 0.157 0.059 0.809 palate dimensions canine distance (t) canine depth (v) first premolar distance (t) first premolar depth (v) second premolar distance (t) second premolar depth (v) molar distance (t) molar depth (v) n b w o h n=17 27.54±2.04 24.41-30.81 6.30±1.89 3.84-9.21 28.83±2.48 25.61-34.71 11.22±1.40 8.26-13.28 32.10±2.50 27.47-36.67 11.39±1.62 8.06-14.04 36.71±2.61 33.10-42.22 9.80±1.83 5.39-13.65 n b w h n=7 24.58±1.30 23.41-25.90 9.03±1.23 7.80-10.71 27.18±0.96 25.90-28.38 12.34±1.77 9.22-14.33 31.49±1.60 29.59-34.15 12.94±1.31 11.72-15.20 35.81±1.46 32.90-37.28 11.06±1.75 9.37-14.20 m b w o h n=21 26.58±2.16 22.44-29.69 6.55±2.8 22.63-12.24 27.31±2.70 22.49-31.88 11.98±1.70 9.66-16.34 30.79±2.14 27.66-35.60 12.76±1.61 10.15-16.75 35.70±2.90 30.40-40.25 11.08±1.84 7.78-14.35 m b w h n=32 25.60±2.47 22.05-31.98 7.68±2.63 4.13-13.20 27.17±1.97 23.42-30.66 12.46±1.44 10.55-16.12 30.62±2.18 26.00-34.32 12.60±1.47 10.37-15.77 34.95±2.012 9.67-38.24 10.52±1.78 7.27-16.03 p 0.025* 0.104 0.087 0.066 0.154 0.024* 0.112 0.183 groups table 3: comparative results of mean and standard-deviation ( ± s), minimal and maximal values (x min –x max ) of transverse (t) and vertical (v) palate dimensions considering the presence the nonnutritive sucking habits and respiratory mode. *anova test (p< 0.05); nbwoh= nose breathing without prolonged habits; nbwh= nose breathing with prolonged habits; mbwoh= mouth breathing without prolonged habits; mbwh= mouth breathing with prolonged habits. allergic and non-allergic patients. based on these findings, it can be suggested that the respiratory mode is not related to the narrowing of the palate in its anterior region. mouth-breathing children presented mean significantly higher in the depth of the palate at the second premolars region when compared to the nose breathers (table 1). this result agrees with those of other studies that compared the palate depth in the region of these tooth between different respiratory modes and between children with and without allergic rhinitis10-13. the increase of the vertical dimension can occur due to the amplification of the pressure in the oral cavity in relation to the nasal cavity, which can justify the greater depth in the palate in the mouth-breathing group. however, the inverse relationship cannot be disposable, that is, patients with genetic tendency to a pattern of vertical facial growth and influence of the respiratory mode and nonnutritive sucking habits in the palate dimensions braz j oral sci. 10(1):42-49 48 deep palate can develop the mouth breathing10-11. the analysis of the palate dimensions according to the nonnutritive sucking habits revealed that wh children also presented all transversal dimensions smaller and vertical dimension larger than who children. however, this difference was significant, unlike the previous table, between the mean of the canine distance and canine depth and in the first premolar depth (table 2). these results indicate that prolonged nonnutritive sucking habits are related with the narrowing and the greater depth in the anterior region of the palate, probably due to a higher pressure exerted in this region by the pacifier or the finger. it has been reported that the prolonged nonnutritive sucking habits influence the dimension of the dental arches, with the decrease in the maxillary intercanine distance being frequently observed17-19. warren and bishara20 (2002) verified that thumb and pacifier suction are associated with anterior open bite. particularly, thumb sucking associated with increase of overjet and reduction in maxillary arch width and the pacifier to the increase of the mandibular arch width and with the posterior crossbite. in the present study, the palate dimensions were not analyzed according to the type of the nonnutritive sucking habits due to the low occurrence of the thumb sucking habit, present only in five participants of the study. according to larsson21 (1994), the prolonged use of pacifier is frequently associated with posterior crossbite due to the lowered posture of the tongue. this reduces the arch width and increases the risk of a transverse malrelationship between the upper and lower arches. the low tongue position widens the lower jaw in the same area thus enhancing the probability of the development of a posterior crossbite. comparing the mean of the palate dimensions between nbwoh, nbwh, mbwoh and mbwh (table 3), there were statistically significant differences in the canine distance between nbwoh versus mbwh groups, as well as in the second premolar depth between nbwoh versus mbwoh and nbwoh versus mbwh (table 4). in order to compare the canine distance between nbwoh and mbwh (table 3), the mean value was significantly smaller in the second group, suggesting that mouth breathing associated with prolonged nonnutritive sucking habits can cause narrowing of the anterior region of the palate. the mean values of the second premolar depth were significantly greater in the mbwoh and mbwh compared to the nbwoh (table 3). again, these findings reveal that mouth breathing associated or not with the prolonged nonnutritive sucking habits can be related to the greater depth of the posterior region of the palate. the increase of the palate depth and the decrease of the palate width, due to mouth breathing and the prolonged sucking habits can occur by the open lips at the rest position and the tongue lowered on the floor of the mouth or protruded between the arches. under these conditions, there is not the external restraint by the lips and the tongue does not exert its function of expanding and modeling the palate9,22. it can be concluded that the respiratory mode and the prolonged nonnutritive sucking habits had influence on the vertical and transverse dimensions of the palate in the children evaluated in the present study. when the respiratory mode was considered alone, it was observed smaller width and greater depth in the more posterior region of the palate. when the prolonged nonnutritive sucking habits were evaluated, it was observed a narrowing and greater depth of the anterior region of the palate. when the respiratory mode and the prolonged nonnutritive sucking habits were analyzed together, it was observed that the canine distance was smaller in the mouthbreathing children with nonnutritive sucking habits and the second premolar depth was greater in the mouth-breathing children with and without habits. in view of the adverse manifestation in the craniofacial complex as a result of mouth breathing and prolonged nonnutritive sucking habits, a multidisciplinary team work is required. parents should be oriented to search otolaryngologic treatment and to restrain the nonnutritive sucking habits as earlier as possible. furthermore, orthodontic treatment is frequently needed for occlusal adjustments and adequacy of the facial musculature and the stomatognathic functions by means of myofunctional therapy. this way, the harmonic facial growth can be controlled and morphological changes can be reduced even in individuals with genetic predisposal. acknowledgements supported by “coordenação de aperfeiçoamento de pessoal de nível superior” capes, brazil. references 1. moss ml, salentijn l. the primary role of functional matrices in facial growth. am j orthod. 1969; 55: 566-77. 2. mateus ar, dolci je, costa ho, sousa fc, di biase n. experimental study on the influence of facial muscle activity on the facial mesostructure bones in rabbits. braz j otorhinolaryngol. 2008; 74: 685-90. 3. vianna-lara ms, caria ph. electromyographic analysis of the upper lip in nose and mouth breathers. braz j oral sci. 2006; 5: 1203-8. 4. degan vv, puppin-rontani rm. removal of sucking habits and myofunctional therapy: establishing swallowing and tongue rest position. influence of the respiratory mode and nonnutritive sucking habits in the palate dimensions braz j oral sci. 10(1):42-49 table 4: difference between the mean values ( a b ) and significance level in the canine distance and second premolar depth measurements in the multiple comparisons considering the presence the nonnutritive sucking habits and respiratory mode. nbwoh x nbwh 2.97 0.100 1.55 0.119 nbwoh x mbwoh 0.96 0.616 1.37 0.037* nbwoh x mbwh 1.94 0.042* 1.22 0.048* nbwh x mbwoh 1.24 0.380 0.18 0.993 nbwh x mbwh 1.02 0.828 0.33 0.954 mbwoh x mbwh 0.98 0.473 0.16 0.984 comparisons ( a b) ( a b)p p canine distance second premolar depth palate dimensions *tukey’s test (p< 0.05); nbwoh = nose breathing without prolonged habits; nbwh= nose breathing with prolonged habits; mbwoh = mouth breathing without prolonged habits; mbwh = mouth breathing with prolonged habits. 49 pró fono. 2005; 17: 375-82. 5. almeida fl, silva am, serpa eo. relação entre má oclusão e hábitos orais em respiradores orais. rev cefac. 2009; 11: 86-93. 6. degan vv, puppin-rontani rm. terapia miofuncional e hábitos orais infantis. rev cefac. 2004; 6: 396-404. 7. cattoni dm, fernandes fd, di francesco rc, latorre mr. characteristics of the stomatognathic system of mouth breathing children: anthroposcopic approach. pró fono. 2007; 19: 347-52. 8. laine t, alvesalo l, lammi s. palatal dimensions in 45, x-females. j craniofac genet dev biol. 1985; 5: 239-46. 9. oliveira mo, vieira mm. influência da respiração bucal sobre a profundidade do palato. pró-fono. 1999; 11: 13-20. 10. freitas fc, bastos ep, primo ls, freitas vl. evaluation of the palate dimensions of patients with perennial allergic rhinitis. int j paediatr dent. 2001; 11: 365-71. 11. ghasempour m, mohammadzadeh i, garakani s. palatal arch diameters of patients with allergic rhinitis. iran j allergy asthma immunol. 2008; 8: 63-4. 12. perea pn, quiñones já, lópez am. determinácion de la profundidad del paladar em niños com respiración bucal de 6-8 años de edad. rev estomatol hered. 2005; 15: 50-3. 13. feres mf, enoki c, sobreira cr, matsumoto ma. dimensões do palato e características oclusais de crianças respiradoras nasais e bucais. pesq bras odontoped clin integr. 2009; 9: 25-9. 14. cappellette m, carlini d, pignatari ss, cruz ol, weckx ll. rinometria acústica em crianças submetidas à disjunção maxilar. rev dent press ortod ortop facial. 2006; 11: 84-92. 15. kiliç n, oktay h. effects of rapid maxillary expansion on nasal breathing and some naso-respiratory and breathing problems in growing children: a literature review. int j pediatr otorhinolaryngol. 2008; 72: 1595-601. 16. souki bq, pimenta gb, souki mq, franco lp, becker hm, pinto ja. prevalence of malocclusion among mouth breathing children: do expectations meet reality?. int j pediatr otorhinolaryngol. 2009; 73: 76773. 17. warren jj, bishara se, steinbock kl, yonezu t, nowak aj. effects of oral habits’ duration on dental characteristics in the primary dentition. j am dent assoc. 2001; 132: 1685-93. 18. zardetto cg, rodrigues cr, stefani fm. effects of different paciûers on the primary dentition and oral myofunctional structures of preschool children. pediatr dent. 2002; 24: 552-60. 19. aznar t, galán af, marín i, domínguez a. dental arch diameters and relationships to oral habits. angle orthod. 2006; 76: 441-5. 20. warren jj, bishara se. duration of nutritive and nonnutritive sucking behaviors and their effects on the dental arches in the primary dentition. am j orthod dentofacial orthop. 2002; 121: 347-56. 21. larsson e. artificial sucking habits: etiology, prevalence and effect on occlusion. int j orofacial myology. 1994; 20: 10-21. 22. moreira m, lino ap. evaluation of palatal depth and width in mouth breathers with primary dentition. int j orofacial myology. 1989; 15: 19-24. influence of the respiratory mode and nonnutritive sucking habits in the palate dimensions braz j oral sci. 10(1):42-49 oral sciences n3 braz j oral sci. 11(2):84-87 original article braz j oral sci. april | june 2012 volume 11, number 2 a methodology to standardize the evaluation of root canal instrumentation using cone beam computed tomography cláudia bohrer flores1, patrícia machado1, francisco montagner2, brenda paula figueiredo de almeida gomes3, gustavo nogara dotto4, marcia da silva schmitz4 1dds, msc student, federal university of santa maria, brazil 2dds, msc, phd, adjunct professor, endodontic division, department of conservative dentistry, federal university of rio grande do sul, brazil 3dds, msc, phd, head professor, endodontic division, department of conservative dentistry, piracicaba dental school, university of campinas, brazil 4dds, msc, phd, adjunct professor, endodontic division, department of stomatology, federal university of santa maria, brazil correspondence to: francisco montagner universidade federal do rio grande do sul, faculdade de odontologia rua ramiro barcelos 2492, cep: 90035-003 bairro bom fim porto alegre, rs brazil phone/fax: +55 51 33085430 e-mail: francisco.montagner@ufrgs.br abstract the evaluation of root canal instrumentation is important to observe the action of endodontic instruments in the root canal walls. aim: this study introduces a method to standardize the acquisition of images before and after preparing root canals by using cone-beam computed tomography (cbct). methods: sixteen mandibular molars were included in acrylic resin blocks. samples were inserted in a stable wood box, which was filled with plaster and served as a guide to reinsert the samples. the apparatus was used for the cbct examination before and after cervical flaring of root canals. the software icatvision® was used to equalize the images before and after instrumentation with two computers operating at the same time. the measurements between root canal center and the furcation area were determined. the statistical analysis was performed using the t-test for paired samples (á=0.05). results: the values for dentin thickness in the risk zone before and after root canal flaring with gates-gliden drills were 1.096 (± 0.27) mm and 0.742 (± 0.24) (p<0.01), respectively. conclusions: the proposed method assures the same positioning of the samples before and after root canal preparation. it is extremely important, as any mesiodistal or buccolingual movement can produce a different topogram for comparison procedures. keywords: cone-beam computed tomography, anatomy cross-sectional, image processing computer-assisted, endodontics. introduction the evaluation of root canal instrumentation is necessary to determine the action of the instruments on the original canal shape1-2 and to observe if the principles of canal preparation had been followed3. the thin area in the root canal wall (risk zone), vulnerable to stripping by injudicious filing4, has been analyzed using microscopic analyses5-6, silicone impressions7, muffle system8-14, endodontic cube15-16, multi-slice computed tomography17 and cone-beam computed tomography (cbct)18. cbct evaluation has shown that the exact location and anatomy of the root canal system can be assessed19-21 and it has been validated as a tool to explore root canal anatomy22. furthermore, it has been successfully used for measurements received for publication: november 25, 2011 accepted: april 25, 2012 braz j oral sci. 11(2):84-87 8585858585 before and after instrumentation of root canals and for determining the amount of dentin removed during cleaning and shaping of root canals18. however, a method has not been proposed to standardize the positioning of samples for obtaining images or topograms before and after root canal preparation by cbct. it is extremely important because any displacement of the sample in both buccollingual and mesiodistal directions will not provide preand post-instrumentation image superimposition. therefore, the aim of this study was to develop a methodology to standardize specimen positioning before and after image acquisition by cbct. material and methods this study was approved by the ethics committee of the santa maria university, santa maria, rs, brazil. sixteen extracted periodontally involved mandibular molars with complete root formation were radiographed and stored in 0.1% thymol solution at 9º c. teeth were placed under tap water for 12 h prior to use to eliminate residues of the storage solution. the teeth that exhibited previous endodontic manipulation, internal or external resorption, were excluded from the study. coronal access was performed using a #1014 diamond bur (kg sorensen cotia, sp, brazil) followed by a endo-z drill (dentsply, maillefer, ballaigues, switzerland). the root canal of each tooth was flushed with 2.5% naocl (manipulation pharmacy, nova derme, santa maria, rs, brazil) and explored using a size 10 k-ûle (dentsply maillefer) until the apical foramen was reached. root canal length was determining through the visualization of the #10 file tip. the entire root apex was covered with colorless nail polish (colorama, procosa produtos de beleza ltda, são paulo, sp, brazil), to avoid the penetration of acrylic resin into the root canal system. a plastic box (1.5 cm x 1.5 cm x 1.5 cm) was used as guide where the teeth were inserted. the plastic boxes were lubricated with solid vaseline (rioquímica, são josé do rio preto, sp, brazil). each tooth was placed inside the plastic box and the self-curing acrylic resin was inserted to position the teeth, without covering the crown (figures 1a and 1b). after 24 h, the acrylic resin was polished (arotec, cotia, sp, brazil) with 200-, 400and 600-grit abrasive paper for 30 s each (alcar abrasivos, vinhedo, sp, brazil). the specimen’s surface were covered with a thin layer of separating medium (cel-lac; s.s. white, rio de janeiro, rj, brazil). a 10 cm x 10 cm x 3 cm wood box was made to serve as a template to insert the type iii plaster (polidental ltda., cotia, sp, brazil) (figure 1c). the internal surfaces of the wood box were covered with plastic film (wyda, sorocaba, sp, brazil) to favor cast dislodgement after setting period. the plaster was then prepared and inserted in the box. the specimens were placed inside the apparatus as shown in figure 1d. after the plaster setting, the specimens were removed. both specimens and the spaces that were formed in the casts received a code to identify them (figure 1e). after 24 h, the specimens were replaced in the cast fig.1. sample preparation and imaging acquisition: a) plastic cube; b) specimen embedded in acrylic resin; c) wood mould; d) specimen embedded in gypsum; e) numbering of specimens in the corresponding depression; f) mould of gypsum aligned through the rotation tool. according to the codes and 3-dimensional cbct images (gx cb-500 powered by i-cat) were obtained with exposure time of 26 s, operating at 120 kvp and 5 ma. cbct imaging was performed with the 0.25-mm voxel size. the reconstruction of the sections was performed. cervical instrumentation of the root canals was performed with size 1 and 2 gates-glidden i (dentsply, maillefer) under irrigation with 2.5% naocl. the specimens were then replaced on the apparatus and a new cbct imaging acquisition was performed as previously described. icatvision® software (dental imaging system, salt lake city, ut, usa) was used to equalize the images before and after instrumentation. the images were aligned, observing the axial vision, through the rotation tool (figure 1f). the mpr screen was selected for measuring. the “zoom” tool was applied to allow a better visualization of the tooth. the vertical (blue and red) and horizontal (green) bars were used as reference to align the images. the tool “distance” (on coronal section) was employed to determine the measure from the highest point of the furcation area up to 2 mm apically. then, the horizontal bar was adjusted 2 mm from furcation a methodology to standardize the evaluation of root canal instrumentation using cone beam computed tomography braz j oral sci. 11(2):84-87 8686868686 area (figure 2a), generating an image in the axial section. the distance from the center of the root canal to the furcation area were determined (figures 2b and 2c). statistical analysis was carried out in the bioestat 5.0 (fundação mamirauá, belém, pa, brazil), and the t-test for paired samples was used for the comparison between groups (p<0.01). fig. 2. data acquisition and processing: a) measurement from the highest point of the furcation area to 2 mm apically; b) measurement of the risk zone before cervical preparation; c) measurement of the risk zone after cervical preparation. results the values for dentin thickness in the risk zone before and after cervical flaring with gates-gliden drills were 1.1373 (± 0.2851) mm and 0.7573 (± 0.2663), respectively. there was statistically significant difference between groups. the values showed a small dentin thickness after cervical flaring with gates-gliden drills (figure 3). discussion cbct allows evaluating root canal preparation without cutting off the specimens and loosing the root canal structure fig. 3. mean values and standard deviations for the distances between the root canal center and the furcation area for group 1 (before gates-glidden preparation) and for group 2 (after gattes-glidden preparation) samples. material during sectioning18-23. the methodology proposed in this study permits standardizing specimen positioning for acquisition of cbct images before and after cervical flaring. we used squared gypsum pattern in order to align the samples through the horizontal and vertical bars of the software icatvision®. the images before and after preparation were superimposed. in addition, we used two computers to evaluate the preand post-instrumentation images, which were standardized and measured in the software. thus, there were no other image manipulation programs for generating distortion attempt to superimpose images of the same size before and after preparation. in addition, the “zoom” tool was employed in order to best measure the specimens. dentin thickness in the risk zone after the action of instruments for cervical flaring can be evaluated by different methodologies. methods that use cross-sections of the sample do not provide an adequate stabilization for the tooth structure16. this study suggested a standardizing methodology for acquisition of cbct images. assessment of the risk zone could be carried out by micro-computed tomography (µct), but it is time-consuming, expensive and not always available in all research centers. several methods have been proposed for standardization of imaging acquisition. bramante (1987)24 suggested a method in which images obtained from root slices were superimposed to detect alterations that were produced after root canal preparation. zaia et al. (2000)25, wu (2005)26 and sauáia et al. (2010)23 modified the previously described methodology and suggested to include the teeth in acrylic resin. coutinho filho et al. (2008)14 used a plastic tube and a metallic strip to guide the slices repositioning. our study also adopted this protocol in order standardize the position of each section to evaluate the same sample before and after root canal preparation. however, the squared-shaped samples inside acrylic resin blocks were placed in a box with plaster to guarantee their perfect repositioning. cbct was also adopted to avoid sample sectioning, ensuring that a significant loss of dental structure did not occur. it is feasible to conclude that the use of the present methodology allows comparing specimens before and after a methodology to standardize the evaluation of root canal instrumentation using cone beam computed tomography 8787878787 braz j oral sci. 11(2):84-87 root canal instrumentation, since image superimposition was obtained after acquisition of cbct images. references 1. weine fs, pasiewicz ra, rice rt. canal configuration of the mandibular second molar using a clinically oriented in vitro method. j endod. 1988; 14: 207-13. 2. leeb j. canal orifice enlargement as related to biomechanical preparation. j endod. 1983; 9: 463-70. 3. schilder h. cleaning and shaping the root canal. dent clin north am. 1974; 18: 269-96. 4. abou-rass m, frank al, glick dh. the anticurvature filing method to prepare the curved root canal. j am dent assoc. 1980; 101: 792-94. 5. kessler jr, peters dd, lorton l. comparison of the relative risk of molar root perforations using various endodontic instrumentation techniques. j endod. 1983; 9: 439-47. 6. montgomery s. root canal wall thickness of mandibular molars after biomechanical preparation. j endod. 1985; 11: 6257-63. 7. abou-rass m, jastrab rj. the use of rotary instruments as auxiliary aids to root canal preparation of molars. j endod. 1982; 8: 78-82. 8. mccann jt, keller dl, labounty gl. a modification of the muffle model system to study root canal morphology. j endod. 1990; 16: 114-5. 9. isom tl, marshall jg, baumgartner jc. evaluation of root thickness in curved canals after flaring. j endod. 1995; 21: 368-71. 10. coutinho-filho t, de deus g, pinto tg, gurgel-filho ed, maniglia-ferreira c. a computer evaluation of the dentin remaining after cervical preparation in curved canals: gates-glidden drills vs. orifice shaper. braz j oral sci. 2002; 1: 116-20. 11. zuckerman o, katz a, pilo r, tamse a, fuss z. residual dentin thickness in mesial roots of mandibular molars prepared with lightspeed rotary instruments and gates-glidden reamers. oral surg, oral med, oral pathol, oral radiol and endod. 2003; 96: 351-55. 12. wu mk, van der sluis lwm, wesselink pr. the risk of furcal perforation in mandibular molars using gates-glidden drills with anticurvate pressure. oral surg, oral med, oral pathol, oral radiol and endod. 2005; 99: 378-82. 13. constante igt, davidowicz h, barletta fb, de moura aam. study of the areas and thicknesses of mesiobucal root canals prepared by three endodontic techniques. braz oral res. 2007; 21: 118-26. 14. coutinho-filho t, de deus g, gurgel-filho ed, rocha-lima ac, dias krc, barbosa ca. evaluation of the risk of a stripping perforation with gatesglidden drills: serial versus crown-down sequences. braz oral res. 2008; 22: 18-24. 15. kuttler s, garala m, perez r, dorn so. the endodontic cube: a system designed for evaluation of root canal anatomy and canal preparation. j endod. 2001; 27: 533-6. 16. garala m, kuttler s, hardigan p, steiner-carmi r, dorn s. a comparison of the minimum canal wall thickness remaining following preparation using two nickel-titanium rotary systems. int endod j. 2003; 36: 636-42. 17. mahran ah, aboel-fotouh mm. comparison of effects of protaper, heroshaper, andgates glidden burs on cervical dentin thickness and root canal volume by using multislice computed tomography. j endod. 2008; 34: 1219-22. 18. sanfelice cm, da costa fb, só mvr, vier-pelisser f, bier cas, grecca fs. effects of four instruments on coronal pre-enlargement by using cone beam computed tomography. j endod. 2010; 36: 858-61. 19. cotton tp, geisler tm, holden dt, schwartz as, schindler wg. endodontic application of cone beam volumetric tomography. j endod. 2007; 9: 1121-32. 20. patel s, dawood a, ford tp, whaites e. the potential applications of cone beam computed tomography in the management of endodontic problems. int endod j. 2007; 40: 818-30. 21. patel s, dawood a, whaites e, ford tp. new dimensions in endodontic imaging: part 1. conventional and alternative radiographic systems. int endod j. 2009; 42: 447-62. 22. michetti j, maret d, mallet jp, diemer f. validation of cone beam computed tomography as a tool to explore root canal anatomy. j endod. 2010; 36: 1187-90. 23. sauáia ts, gomes bpfa, pinheiro et, zaia a a, ferraz ccr, souzafilho fj, valdrighi l. thickness of dentine in mesial roots of mandibular molars with different lengths. int endod j. 2010; 43: 555-9. 24. bramante cm, berbert a, borges rp. a methodology for evaluation of root canalinstrumentation. j endod. 1987; 13: 223-5. 25. zaia aa, ferraz ccr, yoshinari gh, souza filho fj. a simple method for the analysis of rootcanal preparationj endod. 2000; 26: 172-4. 26. wu mk, van der sluis wm, wesselink pr, the risk of furcal perforation in mandibular molars using gates-gliddendrills with anticurvature pressure. oral surg oral med oral pathol oral radiol endod. 2005; 99: 378-82. a methodology to standardize the evaluation of root canal instrumentation using cone beam computed tomography oral sciences n3 original article braz j oral sci. january | march 2011 volume 10, number 1 braz j oral sci. 10(1):1-6 permeability, roughness and topography of enamel after bleaching: tracking channels of penetration with silver nitrate ludmila c. mendonça2, lucas zago naves1,2, lucas da fonseca r. garcia3, lourenço correr-sobrinho1, carlos j. soares2, paulo sérgio quagliatto2 1department of restorative dentistry, dental materials division, piracicaba dental school, unicamp university of campinas, brazil 2department of operative dentistry and dental materials, dental school, federal university of uberlândia, brazil 3department of dental materials and prosthodontics, ribeirão preto dental school, university of são paulo, brazil correspondence to: paulo sergio quagliatto department of operative dentistry and dental materials, dental school, federal university of uberlândia, brazil av. pará, 1720 room 2b 24 cep 38 408 902 uberlandia, mg, brazil tel: +55 34 9121 6946 e mail: psquagliatto@ufu.br received for publication: march 26, 2010 accepted: march 15, 2011 abstract aim: this study evaluated the surface roughness, topography and permeability of bovine enamel by profilometry and scanning electron microscopy (sem) with and without silver nitrate solution, after exposure to different bleaching agents. methods: fifty-two enamel samples were randomly divided into four groups (n=13): cp16% –16% carbamide peroxide whiteness perfect; hp6% 6% hydrogen peroxide white class; hp35% 35% hydrogen peroxide whiteness hp maxx; and control not bleached and kept in artificial saliva. for roughness analysis, average surface roughness (ra) and flatness coefficient (rku) parameters were used. the topography and permeability were examined by sem. for permeability evaluation, the samples were immersed in a 50% silver nitrate solution and analyzed using a backscattered electron and secondary electron mode. results: for the roughness (ra) evaluation, kruskal-wallis and wilcoxon signed ranks test were used, showing an increase on the surface roughness in all bleached groups. the rku parameter suggested changes on enamel integrity. the sem micrographs indicated changes on enamel topography and different levels of silver nitrate penetration in the samples of the bleached groups. in the overall analysis, the bleaching agents promoted surface changes and higher silver nitrate penetration when compared to the control group. conclusions: it may be concluded that different bleaching agents might alter the topography and roughness of enamel surface. moreover, the higher infiltration of silver nitrate suggests an easier penetration path for the oxygen molecules into the dentin substrate. keywords: enamel, dentin, silver nitrate. introduction the change in tooth color is the result of a complex physical and chemical interaction between the tooth and the pigmentation factor1. bleaching treatment for vital teeth is a conservative technique obtaining suitable results when compared to more invasive procedures used in aesthetic and cosmetic rehabilitation2. usually bleaching gels contains carbamide peroxide (cp) or hydrogen peroxide (hp). since cp dissociates into urea and hp, the action of cp is produced in the same way as bleaching agents based on hp3. although their chemical action mechanism of bleaching agents is not entirely 2 braz j oral sci. 10(1):1-6 understood, its known they must diffuse through the dental structure for the bleaching procedure to be effective4. hp has low molecular weight chain and is decomposed into h 2 o and o-, the later is a free radical associated with high permeability and diffusibility in the tooth structure2,5. omolecules attack the long-chained, dark-color macromolecules of pigments and split them into smaller, less colored and more diffusible molecules, which are easier to be removed from the structure, producing the bleaching effect4,6. the free radicals (o-) released from hp permeates into the enamel subsurface through interprismatic regions and may reacts not only with pigmented organic molecules, but also with the organic enamel matrix. as the organic phase is mainly distributed in the inter-zones of inorganic structures, i.e. prisms, the removal of organic material increases the surface irregularity7-8. the diffusibility depends on factors such as the composition and concentration of the penetrating product, area, exposed surface location, exposure time, temperature and nature of the substrate to be penetrated5. a model was proposed to assess the penetration through dental hard tissues, including enamel, using silver nitrate9. this methodology may track the channels used by oto penetrate into the enamel structure. questions are still raised about the possible deleterious effects caused by products containing hp and cp. studies have revealed mild to severe alterations3,7,10-17. while others have found no significant changes to the surface area with at-home or in-office bleaching techniques18-25. although several studies have been focused on morphological changes on dental surfaces after bleaching, this seems to be one of first studies reporting on the jump-start approach using silver nitrate penetration to track and evaluate the possible channels of openetration. in this context, the aims of this study were to evaluate by profilometry and scanning electron microscopy (sem) the effect of two gels for at-home use, cp at 16% and hp at 6%, and one gel for in-office use, hp at 35%, on the surface of bovine enamel, and to determine the correlation of these two variables with the ammoniacal silver nitrate penetration onto enamel surface. the hypothesis tested is that the use of at-home and in-office bleaching agents increases enamel roughness by changing its topography. material and methods enamel specimens – bleaching procedures fifty-two freshly extracted bovine central incisors were cleaned and stored in a 0.2% aqueous thymol solution (pharmacia biopharma ltda., uberlândia, mg, brazil) until use. using a double-face diamond disc (kg sorensen, barueri, sp, brazil) in a low-speed handpiece (kavo, biberach, germany) under copious water spray, the root portion were cut and eliminated. then, the coronal pulp tissue was removed and the pulp chamber filled with light-activated composite resin (filtek z250, 3m espe, st. paul, mn, usa). the buccal surfaces were then flattened as parallel as possible without exposing dentin with 600-, 1200and 1500-grit sic paper (norton, são paulo, sp, brazil), and sectioned in a high-precision cutting machine (isomet 1000; buehler, lake bluff, il, usa) to obtain square-shaped samples (5 mm x 5 mm) of the middle third of the buccal surface. all samples were checked with a digital caliper and under 40× magnification to eliminate those with flaws, irregularities or dimensional alterations were eliminated. enamel thickness was standardized to 1.5 mm, and an elastomeric matrix was used to control thickness and flow of the bleaching agent. the samples were embedded into cylindrical polystyrene molds (cristal, piracicaba, sp, brazil) and polished with 6, 3, ½ and ¼ µm diamond grit (arotec, são paulo, sp, brazil). the samples were randomly divided into 4 groups (n = 10): cp at 16% (cp16% whiteness perfect, fgm, joinville, sc, brazil; hp at 6% (6% hp white class, fgm); hp at 35% (35% hp whiteness hp maxx, fgm), which were treated following the manufacturers’ instructions (table 1 and 2). the control group was not bleached and was kept in artificial saliva. all samples were stored in artificial saliva for 14 days. group clinical sessions frequency applications per session exposure time per application total time of exposure hp35% 2 7 days 3 9 min 54 min table 2: bleaching gel treatment for the in-office technique protocol. table 1: bleaching gel treatment for the at-home technique protocol. group number of exposure frequency total exposure applications time cp16% 14 4 h daily 56 h hp6% 14 2 h daily 28 h an approximately 1-mm-thick bleaching gel layer was applied on enamel surface. the thickness was controlled using an elastomeric matrix. after each gel application, the samples were rinsed with deionized water and stored in artificial saliva at 37°c, which was renewed daily. in order to simulate a nightguard situation, hp6% and cp16% groups, samples were kept in plastic containers with small amount of artificial saliva, over the time of gel application. the 35% hp based product, used in in-office bleaching techniques, was activated by a diode-argon emitted light laser unit (whitening laser, dmc, são carlos, sp, brazil) for 3 min, placed 10 mm far from the surface. after the last session of each group, the samples were rinsed and stored in deionized water. surface roughness test after the bleaching procedures, specimens were rinsed with the water spray and air-dried. surface roughness was measured using a surface profilometer (sj-301 surface roughness tester – mitutoyo, tokyo, japan). the surface permeability, roughness and topography of enamel after bleaching: tracking channels of penetration with silver nitrate 3 roughness was measured five times for each specimen, and the average values obtained were defined as ra (arithmetic average height) and rku (flattening coefficient) of each specimen. the measurements were made before and after the bleaching and control procedures. the arithmetic average height parameter (ra), also known as the centre line average (cla), is the most universally used roughness parameter for general quality control/analysis. rku, called the kurtosis coefficient, is the fourth central moment of profile amplitude probability density function, measured over the assessment length. it describes the sharpness of the probability density of the profile. an area normally distributed is presented by kurtosis equal to 3. a distribution of centrally distributed topography height is presented by kurtosis more than 3, while well scattered distributions of height are presented by kurtosis less than 3. therefore, if rku is less than three, the surface area presents few high peaks and few deep troughs; if rku is more than three, the area presents many high peaks and many deep troughs. the rku parameter is also used to differentiate areas that have different topographies, but have the same value of ra. data from initial and final roughness (ra) were subjected to normality kolmogorov-smirnov and shapiro-wilk tests, and the analysis showed that data were not normal. therefore, kruskal-wallis test was used for comparison of roughness (ra) among the groups in the first and last periods. the wilcoxon signed ranks test was used to compare initial and final ra values in each group (p < 0.05). sem analysis two samples were used for sem analysis. after vacuum sputtering with gold (med 010, balzers union, balzers, liechtenstein), the samples were analyzed under ×20,000 magnification (leo 435 vp, leo electron microscopy ltd., cambridge, uk). a representative sample of each group had all sides protected by nail polish, except for the enamel surface. the enamel blocks were then immersed into 50% ammoniacal silver nitrate (aldrich chemical co., milwaukee, wi, usa) solution for 24 h, and then transferred to a developer solution (kodak professional d-76 developer, kodak rochester, ny, usa). the samples remained in the developer solution under a “day-light” fluorescent lamp positioned 15 cm away from the surface of the liquid, during 8 h. after the silver nitrate crystals developing process, the samples were submitted to 5 one-hour deionized water baths, renewed at every bath. the blocks were then cut lengthwise with diamond disc (kg sorensen) mounted on a low-speed handpiece (kavo) under water spray and polished with 1200and 2000-grit sic papers. the samples were then sputtered with carbon under highvacuum ambient (med 010) and examined by sem at ×150400 magnification using the backscattering electron detector and also on the secondary electron mode. results surface roughness analysis showed no significant differences (p = 0.333) in the initial values of ra among saliva 0.080 ± 0.035 141.04 a 0.084 ± 0.023 102.87 b hp6% 0.069 ± 0.023 124.83 a 0.124 ± 0.038 171.26 a cp16% 0.065 ± 0.023 112.78 a 0.088 ± 0.049 97.30 b hp35% 0.071 ± 0.037 118.69 a 0.102 ± 0.039 126.80 b group initial average ± standard deviation initial (mean rank) final average ± standard deviation final (mean rank) similar letters indicate no statistically significant difference (p = 0.333) table 3: results of the ra roughness parameter comparison among groups at initial kruskal-wallis test (p<0.05). group initial final p saliva 25.28 a 21.74 a 0.201 hp6% 6.50 a 25.63 b 0.001 cp16% 19.27 a 25.81 b 0.001 hp35% 16.00 a 26.20 b 0.001 different letters indicate statistically significant difference in lines (p <0.05). table 4: results of the ra roughness parameter comparison of each group in the initial vs. final periods, by the wilcoxon signed ranks test (p<0.05). the groups (table 3). after treatment, all bleached groups showed increased surface roughness, with difference between initial and final roughness values within each group p=0.001 (table 5), confirming the tested hypothesis. however, the hp6% group showed significantly higher ra than the other groups (table 4). tables 3 and 4 show the mean results and standard deviation. the statistical analysis revealed data distribution as non-normal and so the average values are also provided along the result of the statistical analysis. the initial rku readings for all groups was lower than three (<3); therefore the areas are presented with few high peaks and few deep troughs. at the end of treatment (final readings) rku had values greater than three (>3) in all groups, except for the control group, showing that the area began to present many high peaks and many deep troughs, which suggests changes in enamel integrity induced by the mineral loss. non-uniform changes in enamel surfaces exposed to bleaching agents were detected in sem analysis (figure 1), while the sem micrographs of the control group were flat. such changes were also more evident in the group bleached with hp6% (figure 1c), which had more acute pores, irregularities and depressions, and noticeable boundaries of enamel prisms. the sem micrographs of silver nitrate-infiltrated samples (figure 2) revealed different penetration gradients. the qualitative analysis showed that there was silver nitrate penetration in all groups, but the penetration through the interprismatic region occurred mainly in the experimental groups. in the control group (fig. 2: a.1, a.2 and a.3), the detected penetration was more evident through enamel cracks and microcracks (see arrows in fig. 2). sem micrographs of the hp6% bleached group suggests higher penetration when compared to hp35% samples, with the hp6% group showing permeability, roughness and topography of enamel after bleaching: tracking channels of penetration with silver nitrate braz j oral sci. 10(1):1-6 4 group initial average final average saliva 2.91 2.95 hp6% 2.95 3.99 cp16% 2.95 3.28 hp35% 2.93 3.37 table 5: comparison of the rku roughness parameter value in each group during the initial and final periods. rku < 3: few high peaks and deep troughs; rku > 3: several high peaks and deep troughs higher penetration among the prisms (see asterisks in fig. 2). the cp16% group showed a distinct penetration pattern. although low amount of silver nitrate was detected, there was still slight penetration through the prisms. discussion studies are controversial on the actual effects of bleaching agents onto dental enamel surface due to a variety of methodologies employed, use of different products and trademarks, concentration, ph, dosage, application protocols, criteria for analysis of results, and also the difficulty on linking surface changes to a bleaching agent in particular, due to enamel natural morphology and composition variation16. in this study, the tested hypothesis was confirmed. after bleaching treatment, all groups clearly showed an increase in surface roughness (ra), in the same way as reported elsewhere10-11,15. the initial rku parameter for all groups was lower than three, this confirms that the areas were presented with few high peaks and few deep troughs. at the end of the bleaching treatment, rku was higher than three, showing that the surface presented many high peaks and many deep troughs. the enamel surfaces exposed to bleaching agents analyzed by sem showed changes, characterized as pores, irregularities and depressions. however they were not uniform, while the images of representative samples of the control group were presented as flat. however, only the group bleached with hp6% showed statistically higher values of ra and also more evident changes in sem analysis, when compared to the control group. the use of ammoniacal silver nitrate to assess the rate of penetration in the tooth structures subjected to treatment with bleaching agents has been reported by iwamoto et al.9. fig. 1: enamel surfaces after exposure to different bleaching agents. a. nonbleached enamel surface morphology in the control group (c). flat and unchanged surface; b. exposed enamel surface to whiteness perfect (cp16%) 16% carbamide peroxide. discrete change can be observed, superficial and discontinued channels (arrows); c. exposed enamel surface to white class (hp6%) 6% hydrogen peroxide. obvious pores, irregularities and depressions, with clear evidence of enamel prisms boundaries (arrows); d. exposed enamel surface to whiteness hp maxx (hp35%) – 35% hydrogen peroxide. moderate porosity can be observed (asterisk). (all micrographs at 20.000x) fig. 2: profile cuts of superficial enamel (e) and dentin (d), after the infiltration with silver nitrate (white and outshining points). a.1: representative image of c (artificial saliva) group samples 150x infiltrated with silver nitrate; a.2: image with backscattering electron detector. image suggests penetration through the cracks and micro-cracks (see the narrows) present in enamel (400x); a.3: same image of a.2 but in secondary electron mode (400x); b.1: representative image of the cp16% samples group 150 x infiltrated with silver nitrate; b.2: image with back-scattering electron detector. image suggests slight penetration through prisms (400x); b.3: same image of b.2 but in secondary electron mode (400x); c.1: representative image of hp6% group samples 150x infiltrated with silver nitrate; c.2: image with back-scattering electron detector. image suggests great penetration through prisms (see asterisk) (400x); c.3: same image of b.2 but in secondary electron mode (400x); d.1: representative image of the hp35% group samples 150x infiltrated with silver nitrate; d.2: image with back-scattering electron detector (400x); d.3: same image of b.2 but in secondary electron mode (400x). image suggests moderate penetration through prisms (400x). permeability, roughness and topography of enamel after bleaching: tracking channels of penetration with silver nitrate braz j oral sci. 10(1):1-6 5 the fact that these authors have not found enamel penetration may be related to the shorter exposure time to silver nitrate, 1 h. in this study, after 24 h of immersion26-29, two distinct paths can be noticed when the silver nitrate particles penetrated through the enamel and reached dentin: through the prism and through the microcracks typical of the enamel structure30. the fact that silver nitrate penetrated the area suggests an easy oxygen penetration, since the atomic weight of oxygen is approximately 7 times less than silver. the analysis of silver nitrate penetration in the control group when correlated with the findings of roughness and topography of the area examined by sem, suggests that the smaller the surface change, the lower the enamel penetration. a possible explanation for this would be the lower surface energy of this group compared to the other bleached groups. this reduction in energy surface could be responsible for the lower moistening of the enamel and consequently lower action of the bleaching gel31. the opposite can also be hypothesized for the hp6% group, which had significant changes in enamel topography and roughness, coinciding with the highest nitrate penetration. daily applications (30 min during 14 days) of hp gels with low concentration were measured by pinto et al.15, who found no significant increase in roughness when compared to the control group stored in artificial saliva. a similar finding was obtained in the present study with the hp6% group, with daily application of the gel of 2 h, which is the maximum time recommended by the manufacturer. the group bleached with hp at 35%, despite being about 6 times more concentrated showed lower roughness when compared to hp6%. however, the total period of application was 54 min within 14 days, while in the hp6% group it was 28 h. the period of direct contact of the bleaching agent in the tray with the tooth surface makes the enamel more prone to suffer topographic changes. this situation was not observed with the high concentration of agents used in in-office techniques, which accelerates the peroxide decomposition due to activation by light source or heat32-33. according to bitter and sanders34, it is believed that peroxide concentration and the contact time of the agent with the tooth surface, are directly proportional to the changes caused by the agent in the bleached substrate. the group bleached with cp at 16%, which is composed of approximately 5.7% hp, induced minor changes in enamel surface, after daily applications for 4 h. this result is conflicting with the result of the hp6% group, which showed the greatest superficial change. this finding could be justified by the buffering capacity of saliva35-36 and the presence of urea, which is a cp degradation product. urea is degraded into ammonia and carbon dioxide; the ammonia reacts with moisture and produces ammonia dioxide that has the ability to raise the ph3. in addition, home-use bleaching gels based on hp, do not have urea as a decomposition product and so they do not raise the ph leading to non critical levels of demineralization. the oxidative process and the ph of bleaching agent are regarded as the main causes of the adverse effect of dental enamel after the bleaching treatment. the capacity of the oxidative process to create irregularities on the surface of bleached enamel is questionable as the peroxide activation nature and the interaction with the various bleaching gel components need to be determined37. according price et al.38, it is unclear whether the ph of products containing cp or hp undergoes similar changes in the oral cavity or whether these changes can affect the dental tissues during the bleaching process and the intraoral temperature can affect the ph. however, regarding the effects of acidic and basic solutions, it is important to consider the time of exposure and frequency of use of the product. although the artificial saliva used in laboratory studies can present some remineralization capacity39, it is important to note that the dynamics of saliva/enamel interaction is a difficult factor to fully replicate in laboratory research. it has been demonstrated that the saliva remineralization effect may prevent the demineralization effect on bleaching treatment of human enamel in situ, and that the amount of calcium loss was 2.5 times higher in vitro than in situ40. although this does not invalidate the results of this study, it must be observed and analyzed carefully. clinical studies evaluating the propriety and mechanisms of action of bleaching agents are also necessary to guide professionals in the selection of the bleaching technique that presents the least dielectric effect onto enamel surface40. according to the employed methodology and the obtained results, it may be concluded that: • various bleaching agents promote superficial changes in enamel structure surface; • the fact that silver nitrate has permeated the area suggests an easy penetration by the oxygen produced by bleaching agent decomposition, and also suggests a subsuperficial damage to enamel tissue. acknowledgements authors are indebted to: dr. e.w. kitajima, dr. f.a.o. tanaka and r.b. salaroli (nap/mepa-esalq/usp, brazil) for sem equipment support; fgm, joinville, sc, brazil for materials supply; lipo foufu (laboratório integrado de pesquisa em odontologia da faculdade de odontologia da universidade federal de uberlândia). references 1. nathoo sa. the chemistry and mechanisms of extrinsic and intrinsic discoloration. j am 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decomposition rate of hydrogen peroxide bleaching agents under various chemical and physical conditions. j prosthet dent. 1993; 69: 46-8. 33. reyto r. laser tooth whitening. dent clin north am. 1998; 42: 755-62, xi. 34. bitter nc, sanders jl. the effects of four bleaching agents on the enamel surface: a scanning electron microscopic study. quintessence int 1993; 24: 817-24. 35. leonard rh jr, austin sm, haywood vb, bentley cd. change in ph of plaque and 10% carbamide peroxide solution during nightguard vital bleaching treatment. quintessence int. 1994; 25: 819-23. 36. leonard rh jr, bentley cd, haywood vb. salivary ph changes during 10% carbamide peroxide bleaching. quintessence int. 1994; 25: 547-50. 37. li y. biological properties of peroxide-containing tooth whiteners. food chem toxicol. 1996; 34: 887-904. 38. price rb, sedarous m, hiltz gs. the ph of tooth-whitening products. j can dent assoc. 2000; 66: 421-6. 39. yeh st, su y, lu yc, lee sy. surface changes and acid dissolution of enamel after carbamide peroxide bleach treatment. oper dent. 2005; 30: 507-15. 40. justino lm, tames dr, demarco ff. in situ and in vitro effects of bleaching with carbamide peroxide on human enamel. oper dent. 2004; 29: 219-25. permeability, roughness and topography of enamel after bleaching: tracking channels of penetration with silver nitrate braz j oral sci. 10(1):1-6 oral sciences n3 braz j oral sci. 11(2):125-129 original article braz j oral sci. april | june 2012 volume 11, number 2 oral and maxillofacial surgery helmet and maxillofacial trauma: a 10-year retrospective study maximiana cristina de souza maliska1, marcia borba2, luciana asprino3, márcio de moraes4, roger willian fernandes moreira3 1dds, master student of oral and maxillofacial surgery, department of oral diagnosis, oral and maxillofacial surgery division, piracicaba dental school, university of campinas, brazil 2dds, ms, phd, collaborative researcher, department of oral diagnosis, oral and maxillofacial surgery division, piracicaba dental school, university of campinas, brazil 3dds, ms, phd, associate professor, department of oral diagnosis, oral and maxillofacial surgery division, piracicaba dental school, university of campinas, brazil 4dds, ms, phd, associated professor and coordinator of the postgraduate program in oral and maxillofacial surgery, department of oral diagnosis, oral and maxillofacial surgery division, piracicaba dental school, university of campinas, brazil correspondence to: maximiana cristina de souza maliska faculdade de odontologia, unicamp, av. limeira, 901 areião cep 13414-903 cp 52, piracicaba sp brazil phone: + 55 19 21065326 / + 55 19 82221777 e-mail: maximaliska@fop.unicamp.br abstract aim: the aim of the present study was to retrospectively evaluate the epidemiologic characteristics of the prevalence, type and treatment modalities of maxillofacial trauma according to use of helmets by motorcyclists in traffic accidents. methods: data was collected from patients during a 10-year period (1999-2009). data recorded included demographic, etiology, diagnosis, type of fracture, use of helmet, associated facial and general trauma, soft tissue lesions and treatment methods. data analysis included a descriptive analysis, chi-square test and kruskal-wallis test. results: from 376 motorcycle crash victims, 260 had maxillofacial fractures with a male/female ratio of 4:1 and a mean age of 26.1. considering the helmet as a security device, 89 patients were not wearing a helmet during the crash against 287 patients that were wearing it. one hundred and sixteen patients had soft tissue lesions, 80 of them wore a helmet at the moment of the crash and 36 did not (p<0.05). the most frequently fractured facial bone was the zygoma (24%) followed by the mandible. conclusions: motorcycle accidents represented almost one third of all maxillofacial injuries seen at this oral and maxillofacial surgery division, causing high morbidity. educational campaigns, defensive driving and use of adequate helmets are necessary to decrease the number of facial injuries in such accidents. keywords: maxillofacial injuries, epidemiology, motorcycles. introduction motorcycle injuries continue to be a major public health problem worldwide, especially in developing countries. these accidents increase dramatically the health costs involved in treatment and rehabilitation of the injured patients, and have interrupted the life of thousands of people, especially in the economically active age range (adolescents and young adults)1. received for publication: february 23, 2012 accepted: may 03, 2012 braz j oral sci. 11(2):125-129 126126126126126 if motorcyclists are wearing a helmet at the time of the accident, their risk of death is reduced by 42%, while nonhelmeted riders have a 3.1 fold increased risk of head injuries and death2-3 . research has also shown the effectiveness of laws requiring the use of helmets by two wheel motor vehicle occupants for the reduction of fatalities and injuries in this user category4-5. with the changes in the brazilian traffic code issued in 1998, security devices were made mandatory, alcohol abuse was forbidden, and driving without a license or under the age of 18 became serious offenses6. although the impact of helmet use on the reduction of head injuries during motorcycle accidents is well known, the effect of their use on facial injuries is not well documented7-8. the purpose of this study was to review retrospectively the first 10 years after the new traffic law in brazil. the patterns of the maxillofacial trauma in motorcycle accidents relating the use of helmets were evaluated and discussed. material and methods this 10-year retrospectively study was undertaken by the graduate oral and maxillofacial surgery division, university of campinas unicamp at 7 hospitals located in the cities of piracicaba, limeira and rio claro, são paulo state. the research protocol was approved by the human research ethics committee from the same institution, under the protocol number 131/2008. information was obtained from clinical notes and surgical records for each patient using a standardized data collection form that was specifically developed to investigate the epidemiologic features of maxillofacial trauma. the data recorded only patients sustaining maxillofacial injuries after motorcycle accidents. it included patient age, gender, diagnosis, use of helmet, soft tissue lesions, associated facial and general trauma and type of treatment. the exclusion criteria were incomplete information on the trauma in the medical chart. the injury sites were classified into three thirds of the face: upper (frontal bone), middle (maxilla, zygoma, zygomatic arch naso-orbit-ethmoidal, nasal) and inferior (mandible). mandible fractures were divided into specific sites: condyle, ramus, angle, body, parasynphysis, synphysis and dentoalveolar. abrasion and laceration were classified as soft tissue injuries. data were entered into microsoft excel software. simple descriptive statistical, chi-square test and kruskal-wallis test were applied as appropriate. statistical significance was inferred at p<0.05. results a total of 2,785 trauma patients were evaluated from april 1, 1999, to december 31, 2009. of these patients, 772 were traffic accidents victims, 376 were motorcycle crash victims with maxillofacial trauma, and 260 had maxillofacial fractures. of the 376 patients with maxillofacial trauma, 307 were males and 69 females, with male/female ratio of 4:1. patient age at the time of injury was 10 to 65 years (mean 26.1 years). among the total of 376 patients, 29 (8%) were 10 to 17 years old, 257 (68%) were in the 18-30-yearold age group (being 211 males and 46 females), 83 (22%) were 31 to 50 years old, and 7 (2%) were older than 50 years. the kruskal-wallis test did not show a statistically significant association between age groups and use of helmet (p=0.103). table 1 describes absolute and relative distribution of maxillofacial trauma according to age and gender groups correlating the use of helmet. use of helmet female male total groups of age without with without with 10-17 2 6 10 11 29 18-30 11 35 43 168 257 31-50 2 11 19 51 83 >50 1 1 1 4 7 total 16 53 73 234 376 table 1. distribution of facial trauma according to gender and year groups. k-w= 1.33; p= 0.248 number of fractures security device 0 1 2 3 4 total without helmet 36 29 17 3 4 89 with helmet 80 136 55 15 1 287 total 116 165 72 18 5 376 k-w= 6.98; p= 0.136 table 2. distribution of maxillofacial fractures according to the use of helmet. considering the helmet as a security device, 89 patients were not wearing a helmet at the moment of the crash, against 287 patients that were wearing, of which 234 were males. the chi-square test did not show a statistically significant association between gender and wearing security device. of the 287 patients that were wearing helmets, 136 suffered maxillofacial fracture and 80 did not have any facial bone fractured (table 2). four patients had 4 different fractures even wearing helmet, against 1 patient that was not wearing a security device. the kruskal-wallis test did not show a statistically significant association between number of fractures and wearing security device (p=0.136). patients that wearing helmets (212), were treated conservatively, while 75 patients had open treatment. of the 89 patients that were not wearing a security device, 65 had closed treatment and 24 had open treatment (table 3). the x² test did not show a statistically significant difference between types of treatment and wearing a security device (p=0.985). facial fractures were observed in 260 patients and 116 had soft tissue lesions that include laceration, abrasion and oral and maxillofacial surgery helmet and maxillofacial trauma: a 10-year retrospective study braz j oral sci. 11(2):125-129 closed treatment open treatment total without helmet 65 24 89 with helmet 212 75 287 total 277 98 376 table 3. distribution of treatment according to the use of helmet. x² = 0.024; p= 0.985 without helmet with helmet total general trauma (hole body injuries) n o 38 99 137 yes 51 188 239 x² = 1.973; p=0.201 encephalic trauma associated with use of helmet. n o 67 260 327 yes 22 27 49 x² = 14.052; p<0.05 thoracic trauma associated with use of helmet. n o 71 254 325 yes 18 33 51 x² = 4.412; p<0.05 upper members trauma associated with use of helmet n o 64 164 228 yes 25 123 148 x² = 6.207; p<0.05 total 89 287 376 table 5. distribution of associated general trauma and the use of helmet hematoma. of the patients with these lesions, 80 wore helmets at the moment of the crash and 36 did not. patients that were wearing a helmet and had fractures account to 207 against 53 that did not. the chi-square test showed a statistically significant difference between type of trauma and wearing security device (p<0.05). a tota of 382 different types of fractures were found in these 260 patients. the zygoma was the most frequently fractured bone (24%), followed by the mandible: dentoalveolar trauma (14%), condyle and parasynphysis (11%). one one case of panfacial trauma was observed and this patient was not wearing helmet (table 4). overall trauma evaluation revealed 239 patients with concomitant corporeal lesions. encephalic trauma (260 patients), chest trauma (254) and upper members (164) were not found in patients that were wearing helmet, as seen in table 5. the chi-square test showed statistically significant association between associated trauma and wearing security device. type of fracture without helmet with helmet total lower third mandible condile 4 38 42 ramus 0 3 3 angle 6 17 23 body 9 27 36 parasinphisis 14 27 41 sinphisis 1 3 4 dento-alveolar 6 47 53 midle third maxila le fort i 5 18 23 le fort ii 4 8 12 le fort iii 0 1 1 z i g o m a 18 74 92 zigomatic arch 2 8 10 nasal 10 20 30 noe 1 4 5 upper third frontal 4 2 6 panfacial 1 0 1 total 85 297 382 table 4. distribution of maxillofacial fractures according to anatomic sites and the use of helmet. discussion in developing countries, traffic deaths are projected to be the third most important health problem by 2020, and a large proportion of these deaths involve motorcycles9. motorcycles are particularly dangerous vehicles. motorcycle riders are at an increased risk of accident because the small size of the motorcycle makes them more prone than automobile drivers to experience serious injuries10. assuming that a motorcyclist attains a riding license at the age of 17 and rides 13,500 km per year until retirement lifetime, the risk of death or serious injury approaches 100%11. it is clear that the use of helmets decreases the severity of injury and the probability of deaths, reducing health care costs12-14. the population evaluated in this retrospective study, 849,269 inhabitants from the cities of limeira, piracicaba and rio claro, brazil, is young, has great mobility, lives in urban areas and is economically active. in a 7-year period, the number of inhabitant per motorcycle had a great increase in these three cities, in 2002 there were 14.24 inhabitants per motorcycle and this number in 2009 doubled to 7.52 inhabitants per motorcycle 15. the users affirm that motorcycles are affordable, fuel-efficient, have cheaper maintenance than a car and facilitate locomotion in the overcrowding traffic, characteristics of the são paulo state. the results of this study demonstrated that motorcycle accidents are common among young male patients. gopalakrishna et al.13 reported a prevalence of maxillofacial injury in males (89.7%) with a mean age of 28.7 years. according to oginni et al.16, the male-female ratio was 3.5:1, the peak age incidence for males was 20 to 29 years, whereas female had a peak age incidence of 10 to 19 years in motorcycles accidents. in accordance with the present study, other brazilians studies investigated the prevalence of young males involved in two wheel vehicle accidents17-18. our 127127127127127 oral and maxillofacial surgery helmet and maxillofacial trauma: a 10-year retrospective study braz j oral sci. 11(2):125-129 128128128128128 results are similar tose of other studies worldwide, representing similar behavior of motorcyclists in different countries, as discussed herein. motorcyclists are in disadvantage against car drivers when the collision occurs because this two wheel vehicle do not offer a security device like a seat belt or an air bag, that keeps the body of the patient inside the car. the pillions often are heedless far from the motorcycle and the only device that can prevent a worst injury in the head or face is the helmet. helmeted riders have been shown to have 70% reduction in injury severity and 40% reduction in mortality compared to unhelmeted riders in collisions19. ten years after the introduction of the new traffic code in brazil, helmeted motorcyclists represents 76.3% of all patients involved in motorcycle accidents against 31.2% of patients before the introduction of the new law, as demonstrated by liberatti et al. in a study of the brazilian population20. ferrando et al.5 analyzed the mortality rate before and after law changes in spain and found a 25% reduction of fatal accidents among two wheel vehicle occupants, with a reduced incidence of head trauma. another study that assessed motorcycle and maxillofacial trauma among nigerian intracity road users16 reached different results, as none of the riders or passengers used a helmet at the time of the road accident, and soft tissue lesions were prevalent among facial injuries. the number of motorcyclists presenting maxillofacial fractures using helmets in this study was higher than motorcyclists using helmets that sustained facial contusions. as motorcycle crashes are high-energy trauma, it is possible that the impact of the cranium and face during the accident in patients without helmet promote more seriously injury, as immediate death or severe encephalic traumatism. the incidence of associated body traumatism with maxillofacial fractures can vary widely. this study presented a large number of concomitant traumas in patients that were wearing helmets during the crash. this could be explained by the inexistence of a security device that protects the whole body of motorcyclists. another factor that can influence this result would be the increase of riders that were using helmets. brasileiro and passeri21 demonstrated a prevalence of 24.1% in upper limbs associated trauma, this data corroborates with this study. ramli et al.22 affirm that the use of helmet during an accident reduces the incidence of skull injures while the motorcyclist becomes more susceptible to thoracic and orthopedic trauma. alvi et al.23 disagree with this study, concluding that encephalic trauma is commonly associated with facial trauma ranging from 5.4 to 85%. gopalakrishna et al.13 observed that the incidence of mandibular fractures is significantly higher for motorcyclists wearing helmets than for those who do not. the data presented in this study and in the national studies by maliska et al.24 and scariot et al.25 agree with this statement. the prominence of the mandible and the dissipation of impact forces along the architectural framework of the middle third can explain this prevalence of fractures. the data collected in this work did not account the deaths, neither the patients that did not have maxillofacial trauma, considering only the patients that survived for oral and maxillofacial specific examination. further studies must be developed to include these data, probably collected at hospital emergency rooms. high-energy trauma caused by motorcyclists is responsible for higher percentages of soft tissue lacerations and facial fractures. motorcycle accidents represented almost one third of all maxillofacial injuries seen in this division of oral and maxillofacial surgery, causing high morbidity. educational campaigns, defensive driving and the use of adequate helmets are necessary to decrease the number of facial injuries in such accidents. facial fractures in motorcyclists occur primarily among men under 30 years of age in the studied population and the majority of patients sustaining maxillofacial fractures wore helmets at the moment of the crash. overall, the most common fractured sites on the face are the zygomatic complex followed by the mandible. patients that wore helmets had more conservative treatments, and encephalic trauma is related with the use of helmet. young adults are more severely injured and are more frequently involved in accidents. references 1. salvarani cp, colli bo, carlotti júnior cg. impact of a program for the prevention of traffic accidents in a southern brazilian city: a model for implementation in a developing country. surg neurol. 2009; 72: 6-14. 2. liu bc, ivers r, norton r, boufous s, blows s, lo sk. helmets for preventing injury in motorcycle riders. cochrane database syst rev. 2008; 23(1): cd004333. 3. lin mr, kraus jf. a review of risk factors and patterns of motorcycle injuries. accid anal prev. 2009; 41: 710-22. 4. derrick aj, faucher ld. motorcycle helmets and rider safety: a legislative crisis. j public health policy. 2009; 30: 226-42. 5. ferrando j, plasència a, orós m, borrell c, kraus jf. impact of a helmet law on two wheel motor vehicle crash mortality in a southern european urban area. inj prev. 2000; 6: 184-8. 6. queiroz ms, oliveira pc. traffic accidents: a qualitative approach from campinas, são paulo, brazil. cad saude publica. 2002; 18: 1179-87. 7. shapiro aj, johnson rm, miller sf, mccarthy mc. facial fractures in a level i trauma centre: the importance of protective devices and alcohol abuse. injury. 2001; 32: 353-6. 8. mallikarjuna sk, krishnappa p. prevalence of maxillofacial injuries by motorized two wheeler road traffic accidents in bangalore city. dent traumatol. 2009; 25: 599-604. 9. crompton jg, bone c, oyetunji t, pollack km, bolorunduro o, villegas c, et al. motorcycle helmets associated with lower risk of cervical spine injury: debunking the myth. j am coll surg. 2011; 212: 295-300. 10. bogerd cp, brühwiler pa. heat loss variations of full – face motorcycle helmets. appl ergon. 2009; 40: 161-4. 11. hinds jd, allen g, morris cg. trauma and motorcyclists; born to be wild, bound to be injured? injury. 2007; 38: 1131-8. 12. houston dj. the case for universal motorcycle helmet laws. south med j. 2010; 103: 1-2. 13. gopalakrishna g, peek-asa c, kraus jf. epidemiologic features of facial injuries amongst motorcyclists. ann emerg med. 1998; 32: 425-30. 14. yates jm, dickenson aj. helmet use and maxillofacial injuries sustained following low speed motorcycle accidents. injury. 2002; 33: 479-83. 15. fundação sistema estadual de análise de dados: state foundation for data analysis of the são paulo state – brazil [cited 2010 oct 22] available from: http://www.seade.gov. br/produtos/imp. oral and maxillofacial surgery helmet and maxillofacial trauma: a 10-year retrospective study 129129129129129 braz j oral sci. 11(2):125-129 16. oginni fo, ugboko vi, ogundipe o, adegbehingbe bo. motorcyclerelated maxillofacial injuries among nigerian iintracity road users. j oral maxillofac surg. 2006; 64: 56-62. 17. chcarnovic br, freire-maia b, souza ln, abreu mhng. facial fractures: a 1-year retrospective study in a hospital in belo horizonte. braz oral res. 2004; 18: 322-8. 18. gomez pp, passeri la, barbosa jra. a 5-year retrospective study of zygomatico-orbital complex and zygomatic arch fractures in sao paulo state, brazil. j oral maxillofac surg. 2006; 64: 63-7. 19. 19.world health organization. world report on road traffic injury prevention, 2004. [cited 2010 apr 15] available from: http://www.who.int/ violence_injury_ prevention/publications/road_traffic/world_report/en/ index.html. 20. liberatti clb, andrade sm, soares da. the new brazilian traffic code and some characteristics of victims in southern brazil. inj prev. 2001; 7: 190-3. 21. brasileiro bf, passeri la. epidemiological analysis of maxillofacial fractures in brazil: a 5-year prospective study. oral surg oral med oral pathol oral radiol endod. 2006; 102: 28-34. 22. ramli r, abdul rahman r, abdul rahman n, abdul karim f, krsna rajandram r, mohamad ms. pattern of maxillofacial injuries in motorcyclists in malaysia. j craniofac surg. 2008; 19: 316-21. 23. alvi a, doherty t, lewen g. facial fractures and concomitant injuries in trauma patients. laryngoscope. 2003; 113: 102-6. 24. maliska mcs, lima junior sm, gil jn. analysis of maxillofacial fracture in the state of santa catarina, brasil. braz oral res. 2009; 23: 268-74. 25. scariot r, de oliveira ia, passeri la, rebellato nl, müller pr. maxillofacial injuries in a group of brazilian subjects under 18 years of age. j appl oral sci. 2009; 17: 195-8. oral and maxillofacial surgery helmet and maxillofacial trauma: a 10-year retrospective study oral sciences n3 original article braz j oral sci. october|december 2010 volume 9, number 4 comparative isolation protocols and characterization of stem cells from human primary and permanent teeth pulp leliane macedo de souza1, juliana duarte bittar2, izabel cristina rodrigues da silva3, orlando ayrton de toledo4, marcelo de macedo brígido5, marcio josé poças-fonseca6 1dds, ms, department of dentistry, school of health sciences, university of brasilia, brasília, df, brazil 2dds, department of dentistry, school of health sciences, university of brasilia, brasília, df, brazil 3bsc, ms, phd, department of cellular biology, institute of biological sciences, university of brasilia, unb, brasília, df, brazil 4dds, ms, ds, phd, professor, department of dentistry, school of health sciences, university of brasilia, brasília, df, brazil 5bsc, ms, ds, phd, associate professor, department of cellular biology, institute of biological sciences, university of brasilia, unb, brasília, df, brazil. 6bsc, dds, ms, ds, associate professor, department of genetics and morphology, institute of biological sciences, university of brasilia, unb, brasília, df, brazil correspondence to: marcio josé poças-fonseca universidade de brasília instituto de ciências biológicas departamento de genética e morfologia campus universitário darcy ribeiro 70910-900 brasília df, brazil. phone: + 55 (61) 3107-3090 fax: +55 (61) 3107-2923 e-mail: mpossas@unb.br received for publication: march 02, 2010 accepted: july 19, 2010 abstract aim: this study was developed to compare the morphological, proliferative and immunophenotypic profiles of pulp cells from permanent and primary teeth, obtained by two isolation methods. methods: normal human impacted third molars and exfoliated primary teeth were collected and cut around the cementoenamel junction. pulp cells cultures were established by two approaches: enzyme digestion (3 mg/ml type i colagenase and 4 mg/ml dispase), or culture of the tissue explants in cell culture dishes. morphological and proliferative analyses, as well as immunophenotype characterization with monoclonal antibodies against cd117, cd34 and cd45 surface receptors were performed. results: for the permanent teeth, on the 4th day of culture, the cell number was significantly higher for the outgrowth method. by the end of the studied period (14th day), the enzymatic method was more efficient in promoting culture growth. on the other hand, for primary teeth, enzymatic digestion always promoted a higher cell proliferation. the immunophenotypic profiles were cd117+/ cd34-/ cd45and cd117+/ cd34+/ cd45for cells from permanent and primary teeth, respectively. conclusions: the findings of this study indicate that both isolation methods can be efficiently used. the cell population displayed an immunophenotype compatible to the one of stem cells, with remarkable positive expression of cd117. keywords: stem cells, cell culture techniques, dental pulp, primary dentition, permanent dentition. introduction several studies have addressed the regeneration of pulp tissue and the induction of reparative dentin based on the biology of stem cells and tissue engineering approaches1-4. adult mesenchymal stem cells have been identified in the pulp tissue of human permanent teeth dpsc5-6 , in exfoliated primary teeth -shed7-8, but also in the periodontal ligament – pdlsc9-12 they are described as multipotent stem cells, capable to self-renew and to differentiate into various cell types, such as in braz j oral sci. 9(4):427-433 osteocytes, adipocytes, chondrocytes, cementoblasts, odontoblasts and neuronal cells5-6,13-14. dental pulp cells have been isolated by enzymatic digestion5,15-17 and the outgrowth method8,14,18-19. enzymatic digestion is a common method to obtain single cell suspensions from primary tissues and consists of exposing the tissue to enzymes for a minimal period of time in order to preserve maximum cell viability. the outgrowth method is considered simpler and faster and consists of placing pulp fragments directly into the culture plate so that cells outgrow from the pulp tissue explants. stem cells hardly express some specific markers. in this view, they are very difficult to characterize. moreover, there is no rigid pattern of gene expression that could be associated with the stemness state8,17,20. few studies have addressed the possible differences in the immunophenotypic, morphological and proliferative patterns between permanent and primary teeth pulp cells as a function of different isolation protocols. in this regard, this is a pioneer study in comparing the isolation of primary teeth stem cells by enzymatic digestion and by the outgrowth method. this study aimed at performing an in vitro characterization of human dental pulp cells from primary (dpcd) and permanent (dpcp) teeth. we compared the morphological, immunophenotypic and proliferative profiles of cells isolated by enzymatic digestion (c/d) and by the outgrowth method (og). material and methods primary dental pulp cell culture human dental pulps were obtained from 10 freshly extracted impacted third molar teeth (donors aged 9-15 years) and 10 human primary teeth (donors aged 7-12 years), presenting at least two thirds of the root, extracted for orthodontics reasons at the maxillofacial ltda institute brasília (brazil), under the approval of the ethical committee of the medical school of the university of brasília, brazil (process number 034/2007). all experiments were undertaken after patients had signed informed consent forms according to the 196/96 brazilian health department resolution and to the world medical association’s declaration of helsinki. in the proximity of a burning flame, in order to minimize fig. 1. permanent tooth immediately after extraction, manipulated with sterile gloves and close to a burning flame. longitudinal groove made using a diamond disk without reaching the pulp tissue (a). tooth sectioning using a dentinal excavator (b). pulp tissue removal from tooth crown and root (c). the risk of contamination, tooth surfaces were cleaned and a longitudinal groove was made using sterilized diamond discs (kgsorensen, ref.7020, zenith dental aps, agerskov, denmark), without reaching the pulp. the pulp tissue was gently separated from the crown and root with a sterile dentinal excavator (fig.1). the pulp tissue was transported to the laboratory in polystyrene microtubes (corning incorporated, corning, ny, usa) containing dulbecco’s modified eagle’s medium dmem (gibcotm, grand island, ny, usa) supplemented with 10% fetal bovine serum (fbs) (gibcotm) and with 100 units/ml penicillin and 100 mg/ml streptomycin (gibcotm). before culture establishment, the pulp tissue was washed three times with hank’s balanced salt solution (hyclone, logan, ut, usa). pulp cell cultures were established by two distinct approaches: enzyme digestion (c/d) or explant culture outgrowth (og). for the enzyme digestion, pulp tissue was treated with a solution of 3 mg/ml type i collagenase (gibcotmand 4 mg/ml dispase (gibcotm) for 60 min at 37ºc. after enzymatic digestion, the cell suspension was washed three times by centrifugation for 10 min at 750 x g in culture medium (dmem), and single cell suspensions (104 cells) were seeded into 6-well cell culture plates (corning incorporated). for the outgrowth method procedure, the explant fragments were transferred to a new plate once a week until no further outgrowth was observed (up to 5 transfers). for all methods, the culture was maintained in dmem supplemented with 20% fbs and 100 units/ml penicillin and 100 mg/ml streptomycin (gibco tm) in a 80% humidity, 5% co 2 atmosphere at 37ºc. cell proliferation in order to compare the proliferative potential of cells obtained by enzymatic digestion and by the outgrowth method, permanent dental pulp cells (5 x 104), obtained by enzymatic digestion (colagenase type i plus dispase) were seeded after 5 culture passages in 12-well plates with dmem supplemented with 20% fbs, 100 units/ml penicillin and 100 mg/ml streptomycin. cell growth was assessed in triplicate by the trypan blue (sigma chemical co.) exclusion assay after 2, 4, 6, 8, 12 and 14 days of culture. culture medium was changed at every 2 days. growth curves were performed to compare the proliferation 428428428428428comparative isolation protocols and characterization of stem cells from human primary and permanent teeth pulp braz j oral sci. 9(4):427-433 of permanent dental pulp cells obtained by enzymatic digestion and by the outgrowth method. the same experimental approach was employed for primary dental pulp cells. to assess the time of duplication or doubling time (dt), the following equation was applied: dt = t x ln2x1 / [ln (n0 n)], where n= final number of cells, n0= initial number of cells. this formula derives from the calculation of growth curves in n = n0 x 2t / t. student´s t test was used to compare the proliferation rate of cells obtained by the two methods. statistical significance was assumed at a value of p <0.05. all statistical tests were performed with minitab® release 14.20 (mininc, state college, pa, usa) and the plots were obtained through the microsoft office excel® software (microsoft corporation, redmond, wa, usa). morphological analysis for morphological analysis by light microscopy, permanent and primary dental pulp cells (5x104) were grown over microscopy cover slips (18 x 18 mm), previously treated for 30 min with poly-l-lysine (0.1 mg/ml), and placed into 6-well culture plates. after 48 h, adhered cells were fixed for 10 min with 2% paraformaldehyde in phosphate buffer saline, washed and stained with 4% giemsa in methanol (sigma,) for 12 min. another group of cells was analyzed without staining. light microscopy analysis was performed with the nikon diaphot tmd m250 microscope (kogaku, japan). immunophenotypic characterization by flow cytometry after 3 culture passages, 2x106 cells from primary and permanent teeth were pellet (10 min at 750 x g) and resuspended in phosphate buffer saline (pbs) with 0.1% sodium azide and 1% bovine serum albumin (bsa) as a marking solution. cells were then incubated for 15 min with 10 ml of antibody as indicated by the manufacturer. stem cell markers employed were the mouse anti-human antibodies anti-cd117 or c-kit (invitrogen corporation, carlsbad, ca, usa), a membrane tyrosin-kinase receptor that interacts with the stem cell factor (scf); anti-cd34 (fk-biotec, porto alegre, rs, brazil), a marker for endothelial precursors and hematopoietic stem cells; and anti-cd45 ra (fk-biotec), a marker for immature t leukocytes. subsequently, 104 cells were acquired and analyzed at the facscalibur cell analyzer (becton-dickinson) running with the cell quest software (becton dickinson). nonprobed cells were used as controls for each antibody. the statistical analysis was performed by student’s ttest (spss® version 17.0; spss inc., chicago, il, usa) and the significance level was set at 5%. the statistical analysis used for comparison of the cell types obtained with the distinct isolation methods was generated considering the normalized values. results pulp cells obtainment with the two methods (fig. 1) was effective for both crown and root tissues. sample contamination with microorganisms was only rarely observed. considering the proliferation of permanent dental pulp cells obtained by the two methods, growth curves (fig. 2) showed that, on the 4th day of culture, the cell number was significantly higher for the outgrowth isolation method (p=0.015). on the other hand, on the 14th day, culture cell number was statistically higher (p=0.046) for the enzymatic digestion method. growth curves (fig. 3) comparing the proliferation of primary dental pulp cells obtained by the two different methods showed that cell number was statistically higher after 2 (p=0.002), 4 (p=0.000), 6 (p=0.000), 8 (p=0.004) and 12 (p=0.008) days of culture when the cells were isolated by enzymatic digestion. the calculated doubling times were: 18.54 h for dpcpc/d; 19.61 h for dpcp-og; 16.87 h for dpcd-c/d and 17.13 h for dpcd-og. the primary cultures of all experimental groups were very heterogeneous at the morphological level. for both isolation methods, cells presented spindle-shape appearance and polygonal contours with cytoplasmic extensions, displaying a fibroblast-like morphology (figures 4a, 4b, 4c). for cell cultures obtained by enzymatic digestion, cells with fig. 3. primary dental pulp cells growth curves comparing different methods, outgrowth and enzymatic digestion using colagenase type i plus dispase, cultured with d-mem supplemented with 20% fbs. fig. 2. permanent dental pulp cells growth curves comparing different methods, outgrowth and enzymatic digestion using colagenase type i plus dispase, cultured with d-mem supplemented with 20% fbs. 429429429429429 comparative isolation protocols and characterization of stem cells from human primary and permanent teeth pulp braz j oral sci. 9(4):427-433 fig. 4. primary culture of dpcp and dpcd. (a) dpcd-c/d (100x); (b) dpcp-c/d (40x): cells exhibiting polygonal shape, with cytoplasmic extensions and predominantly fibroblast-like morphology. (c) dpcd-c/d (20x) showing spindle-shape appearance and polygonal contours with cytoplasmic extensions. (d) dpcp-c/d (40x) and (e) dpcd-c/d (20x) after 3 culture passages: cells expanded and formed condensed multilayer stripes of cell masses. (f) dpcd-c/d cells (20x) with spherical shapes and epithelial or endothelial-like cells contours. (g) dpcd-og cells (20x) migrating from the tissue fragment (arrow). (h) and (i): confluent cultures of dpcd-c/d (20x and 40x, respectively) showing colony-forming units. (a) and (d ) stained with giemsa. scale bars: 50 µm. spherical shape and epithelialor endothelial-like contours were also observed (fig. 4f). after 3 weeks of culture, for both isolation methods, cells appeared aligned, presenting long extensions of cytoplasm, establishing cell connections and organizing monolayers, thus forming a tissue-like structure (fig. 4d and 4e). after successive culture passages, no more spherical cells were observed for the enzymatic digestion method. in the outgrowth method, cell migration from the pulp fragment was observed throughout the culture plate. cells closer to the fragment showed heterogeneous shapes, but a predominance of polygonal or elongated morphologies was observed (fig. 4g). cell cultures obtained by enzymatic digestion showed, from 7 to 15 days, the formation of clusters of cell colony-forming units, with spherical cells at the center (fig. 4h and 4i). the immunophenotypic analysis (fig. 5) showed that, in both cell groups, cd117 was the most expressed marker: dpcp c/d (100%); dpcp og (99.69%); dpcd c/d (98.95%) and dpcd og (98.73%), at p = 0.05. statistical analysis showed that cells isolated by the distinct methods differently expressed cd34 (p<0.05). results showed that dpcd also presented a high expression of cd34 (dpcd c/d =53.91%; dpcd og =35.59%) (fig. 5a, 5b). both cell groups showed no expression of cd45, for both isolation methods. regardless of the isolation method, the mean frequency of dpcd expressing cd117 (p c/d = 0.00; p og = 0.00) and cd34 (p c/d = 0.01; p og = 0:04) was different from the control group (nonproubed cells). however, the cd117marker was the only one significantly expressed in dpcp (p c/d = 0.00; p og = 0.00) (fig. 5c). the cd45marker was not expressed by dpcd (p c/d = 0.22; p og = 0.33) or by dpcp (p c/d = 0.20; p og =0.30). discussion isolation and proliferation of stem cells are the first step for tissue engineering. our objective was to compare different methods for obtainment of permanent and primary dental pulp cells aiming at the isolation of stem cells within the facilities of the university hospital (hub) of the university of brasilia (unb), brazil. the comparison between the enzymatic digestion and the outgrowth methods had not been employed for primary teeth cells before. although several methods for collecting pulp tissue are mentioned in different studies, they are not described in details. some studies use carbide burs to reach the pulp tissue from extracted teeth5 or from vital teeth, removing the pulp with endodontic files (pulpectomy) 17. other studies use forceps to break extracted teeth18. pulp heating and the 430430430430430comparative isolation protocols and characterization of stem cells from human primary and permanent teeth pulp braz j oral sci. 9(4):427-433 fi.g 5. immunophenotype analyses of dpcd and dpcp by flow cytometry. frequencies of cells expressing the markers cd117, cd34 and cd45. (a): dpcp colagenase/dispase (c/d) versus dpcd (c/d), and dpcp outgrowth (og) versus dpcd (og). the mean values of the control group are deduced from the mean values of the marked cells. (b): comparison of dpcd with the respective control group concerning the two different methods. (c): comparison of dpcd with the respective control group concerning the two different methods. different letters indicate statistically signiicant differences. increase of microbial contamination are some of the disadvantages of these methods. we used a diamond disk to create a furrow without reaching the pulp tissue in order to reduce pulp heating, which occurs when carbide burs are used. tooth breakage was achieved with little pressure in the furrow using a dentinal excavator (fig 1). we did not use forceps to avoid irregular tooth breakage, which could make the pulp separation difficult and increase the contamination risk. the primary teeth included in our study presented at least two thirds of the root. some studies have reported the use of naturally exfoliated primary teeth for the establishment of pulp stem cell culture7. however, these naturally exfoliated teeth barely present pulp tissue anymore. 431431431431431 comparative isolation protocols and characterization of stem cells from human primary and permanent teeth pulp braz j oral sci. 9(4):427-433 both the enzymatic digestion and the outgrowth methods have proved to be effective for obtaining cells. during the subsequent culture passages, cells obtained by the different methods proliferated differently. primary dental pulp cells obtained by the enzymatic digestion method presented a higher proliferative potential when compared to the ones isolated by the outgrowth method, along the entire period of experimentation. unpublished data from our group indicated that d-mem supplemented with 20% fbs promoted for pulp cells a superior proliferative response, when compared to d-mem 10 fbs%, ham-f12 10% and 20% fbs. the doubling times calculated for primary (dpcd) and permanent (dpcp) pulp cells were similar to those observed in other studies of human dental pulp cells (hdpcs)21 and point out to a very high clonogenic capability, particularly for cells obtained from primary teeth. the major advantage of the outgrowth method is its convenience and low cost8,17. the digestion method may completely dissociate cells from the tissue, but it is technically difficult due to a certain level of cell damage and loss. kerkis et al.8 postulated that the cultivation of pulp fragments, prior to the initial cell passage, probably prevents the stem cells premature differentiation. these authors have suggested that the outgrowth method promotes the selective proliferation of the dental pulp immature stem cells (idpscs). they also demonstrated that idpscs obtained from primary teeth expressed several mesenchymal stem cell markers, and they speculated that these cells could represent the multipotent precursors of both dpsc and shed. under the tested experimental conditions, few morphological differences were verified between cells obtained from primary and permanent teeth (fig. 4). at the beginning of the culture (7 days), cells were morphologically heterogeneous, with spherical and elongated shapes. this was particularly evident for the enzymatic digestion method (fig. 4a, 4b, 4c). after a period ranging from 15 to 22 days, cells obtained through both methods showed mainly polygonal morphology, with expansion of the cytoplasm. the nucleus was polarized, as observed in several other stem cells studies14-15,17,19. cells with spherical morphology (fig. 1f) have been related to epithelial or endothelial-like precursor cells22-23. according to sonoyama et al.24, these cells contaminate mesenchymal stem cells cultures when immunomagnetic separation is not efficient. endothelial-like cells usually disappear after successive passages24, as it was also observed in the present experiments. polarized cells were observed in dental pulp cultures and they are considered odontoblasts precursors19,23,25. with the enzymatic digestion method, we could identify cell clusters scattered over the culture plate (fig. 4h, 4i). these clusters were described as cell colony-forming units15,25. these colonies have been identified in stem cells cultures from bone marrow and also from human dental pulp, and they are considered a prerequisite for odontoblasts differentiation14,17,26. considering the immunophenotypic characterization, our results showed that different methods of isolation did not influence the cells immunophenotypic profiles. dpcp and dpcd showed phenotypes compatible to stem cells, with remarkable expression of cd117 (fig. 2). several studies have shown that stem cells from adult human dental pulp present a variety of surface antigens associated with neural crest cells precursors (cd117 or ckit)16,20,26-27. some studies report that these primary cultures do not express hematopoietic markers like cd45, while the cd34 expression remains controversial5,8,16,24. the expression of cd117 and cd34 reinforces the immunophenotypic profile of stem cells. cd117 is an early stem cells marker, since it interacts with the stem cell factor (scf), an important growth factor related to the proliferation, differentiation and recruitment of progenitor cells in various biological systems27. we identified significant difference in the expression profile of cd34, an hematopoietic stem cells marker28, among the distinct cells populations. dpcp were cd117+/cd34-/ cd45-, in accordance to studies that describe dental pulp stem cells as cd34-5-6,8,15. on the other hand, dpcd were cd117+/cd34+/cd45-, in accordance to studies that describe dental pulp stem cells as cd34+ (fig. 5)16,18,20,26. in this perspective, our data reinforce the controversy described in the literature. the cd34+ phenotype was not previously described for primary teeth cells. in conclusion, our results indicate that both isolation methods can be efficiently used aiming at the isolation of human dental pulp stem cells in a clinical environment. in addition, we could demonstrate that stem cells obtained from primary teeth also present the cd34+ phenotype, compatible to hematopoietic precursor cells. we believe that the findings of this study can contribute to the discussion of reliable and reproducible protocols for the isolation and propagation of dental pulp stem cells, aiming at future regenerative therapies not only for dental, but also for other oral structures. acknowledgements this work was supported by finep/mct and fub brazil references 1. murray pe, garcia-godoy f, hargreaves km. regenerative endodontics: a review of current status and a call for action. j endod. 2007; 33: 377-90. 2. cordeiro mm, dong z, kaneko t, zhang z, miyazawa m, shi s, et al. dental pulp tissue engineering with stem cells from exfoliated deciduous teeth. j endod. 2008; 34: 962-69. 3. nakashima m. tissue engineering in endodontics. aust endod j. 2005; 31: 111-3. 4. nakashima m, akamine a. the application of tissue engineering to regeneration of pulp and dentin in endodontics. j endod. 2005; 31: 711-8. 5. gronthos s, mankani m, brahim j, robey pg, shi s. postnatal human dental pulp stem cells (dpscs) in vitro and in vivo. proc natl acad sci usa. 2000; 97: 13625-30. 432432432432432comparative isolation protocols and characterization of stem cells from human primary and permanent teeth pulp braz j oral sci. 9(4):427-433 proved to be 6. gronthos s, brahim j, li w, fisher lw, cherman n, boyde a, et al. stem cell properties of human dental pulp stem cells. j dent res. 2002; 81: 531-5. 7. miura m, gronthos s, zhao m, lu b, fisher lw, robey pg, et al. shed: stem cells from human exfoliated deciduous teeth. proc natl acad sci usa. 2003; 100: 5807-12. 8. kerkis i, kerkis a, dozortsev d, stukart-parsons gc, gomes msm, pereira lv, et al. isolation and characterization of a population of immature dental pulp stem cells expressing oct-4 and other embryonic stem cell markers. cells tissues organs. 2006; 184: 105-16. 9. chen sc, marino v, gronthos s, bartold pm. location of putative stem cells in human periodontal ligament. j periodontal res. 2006; 41: 547-53. 10. kamata n, fujimoto r, tomonari m, taki m, nagayama m, yasumoto s. immortalization of human dental papilla, dental pulp, periodontal ligament cells and gingival fibroblasts by telomerase reverse transcriptase. j oral pathol med. 2004; 33: 417-23. 11. kramer pr, nares s, kramer sf, grogan d, kaiser m. mesenchymal stem cells acquire characteristics of cells in the periodontal ligament in vitro. j dent res. 2004; 83: 27-34. 12. seo bm, miura m, gronthos s, bartold pm, batouli s, brahim j, et al. investigation of multipotent postnatal stem cells from human periodontal ligament. lancet. 2004; 364: 149-55. 13. batouli s, miura m, brahim j, tsutsui tw, fisher lw, gronthos s, et al. comparison of stem cells mediated osteogenesis and dentinogenesis. j dent res. 2003; 82: 976-81. 14. couble ml, farges jc, bleicher f, perrat-mabillon b, boudeulle m, magloire h. odontoblast differentiation of human dental pulp cells in explants cultures. calcif tissue int. 2000; 66: 129-38. 15. huang gt, shagramanova k, chan sw. formation of odontoblast-like cells from cultured human dental pulp cells on dentin in vitro. j endod. 2006; 32: 1066-73. 16. laino g, carinci f, graziano a, d’aquino r, lanza v, de rosa a, et al. in vitro bone production using stem cells derived from human dental pulp. j craniofac surg. 2006; 17: 511-5. 17. huang gt, sonoyama w, chen j, park sh. in vitro characterization of human dental pulp cells: various isolation methods and culturing environments. cell tissue res. 2006; 324: 225-36. 18. pierdomenico l, bonsi l, calvitti m, rondelli d, arpinati m, chirumbolo g, et al. multipotent mesenquimal stem cells with immunosuppressive activity can be easily isolated from dental pulp. transplantation. 2005; 80: 836-42. 19. stanislawski l, carreau jp, pouchelet m, chen zh, goldberg m. in vitro culture of human dental pulp cells: some aspects of cells emerging early from the explants. clin oral investig. 1997; 1: 131-40. 20. laino g, d’aquino r, graziano a, lanza v, carinci f, naro f, et al. a new population of human adult dental pulp stem cells: a useful source of living autologous fibrous bone tissue. j bone miner res. 2005; 20: 1394-1402. 21. suchanek j, soukup t, visek b, ivancakova r, kucerova l, mokry j. dental pulp stem cells and their characterization. biomed pap med fac univ palacky olomouc czech repub. 2009; 153: 31-5. 22. ikeda e, hirose m, kotobuki n, shimaoka h, tadokoro m, maeda m, et al. osteogenic differentiation of human dental papilla mesenchymal cells. biochem biophys res commun. 2006; 342: 1257-62. 23. freshney ir, stacey gn, auerbach jm. culture of human stem cells: culture of specialized cells. new york: wiley; 2007. 24. sonoyama w, yamaza t, gronthos s, shi s. multipotent stem cells in dental pulp. in: freshney ir, stacey gn, aurebach jm. culture of human stem cells: culture of specialized cells. new york: wiley; 2007. p.187206. 25. castro-malaspina h, gay re, resnick g, kapoor n, meyers p, chiarieri d, et al. characterization of human bone marrow fibroblast colony-forming cells (cfu-f) and their progeny. blood. 1980; 56: 289-301. 26. papaccio g, graziano a, d’aquino r, graziano mf, pirozzi g, menditti d, et al. long-term cryopreservation of dental pulp stem cells (sbpdpscs) and their differentiated osteoblasts: a cell source for tissue repair. j cell physiol. 2006; 208: 319-25. 27. gagari e, rand mk, tayari l, vastardis h, sharma p, hauschka pv, et al. expression of stem cell factor and its receptor, c-kit, in human oral mesenchymal cells. eur j oral sci. 2006; 114: 409-15. 28. gangenahalli gu, singh vk, verma yk, gupta p, sharma rk, chandra r, et al. three-dimensional structure prediction of the interaction of cd34 with the sh3 domain of crk-l. stem cells dev. 2005; 14: 470-7. 433433433433433 comparative isolation protocols and characterization of stem cells from human primary and permanent teeth pulp braz j oral sci. 9(4):427-433 oral sciences n3 braz j oral sci. 11(4):481-485 original article braz j oral sci. october | december 2012 volume 11, number 4 mapping of proximal enamel thickness in permanent teeth flávio vellini-ferreira1, flávio augusto cotrim-ferreira2, josé alaor ribeiro3, rívea inês ferreira-santos2 1dds, msc, phd, professor and chairman, department of pediatric dentistry and orthodontics, university of são paulo city (unicid), são paulo, sp, brazil 2dds, msc, phd, associate professor, department of pediatric dentistry and orthodontics, university of são paulo city (unicid), são paulo, sp, brazil 3dds, msc in orthodontics, department of pediatric dentistry and orthodontics, university of são paulo city (unicid), são paulo, sp, brazil correspondence to: rívea inês ferreira-santos universidade cidade de são paulo (unicid) pós-graduação (mestrado em ortodontia) rua cesário galeno, 448 – bloco a, cep: 03071-000, tatuapé, são paulo, sp, brasil phone / fax: +55 11 21781310 e-mail: riveaines@yahoo.com received for publication: october 11, 2012 accepted: december 11, 2012 abstract aim: knowledge of enamel thickness is relevant to perform stripping during orthodontic treatment. thus, proximal enamel measurements of human permanent teeth were compared in this study. methods: the measurements were previously obtained on cut sections of mandibular central (n = 30) and lateral (n = 30) incisors, canines (n = 20), first (n = 40) and second (n = 40) premolars; maxillary central (n = 20) and lateral (n = 20) incisors, canines (n = 20), first (n = 40) and second (n = 42) premolars. comparisons between thicknesses by arch side and proximal surface were carried out using student’s t-tests (α = 0.05). teeth were compared according to the mesial and distal thicknesses by anova and tukey’s test. results: no significant differences were found between right and left teeth. for the mesial surface, the mandibular second premolar presented the highest mean value (1.376 mm ± 0.198; p<0.001). the mandibular central incisor had the smallest thickness in relation to the other teeth (0.675 mm ± 0.144), although not significant compared with the mandibular lateral incisor and canine (0.734-0.781 mm). the mandibular second premolar also presented the higher distal thickness in relation to the others (1.450 mm ± 0.172), although not significant compared with the maxillary first premolar (1.322 mm ± 0.195). mandibular incisors had the lowest means for distal thickness (0.872-0.879 mm), although not statistically different compared with maxillary incisors and mandibular canine (1.002-1.015 mm). distal thickness was greater than mesial (p<0.001). conclusions: interproximal stripping should be less marked in incisors and mesial surfaces. keywords: dental enamel, dentition, permanent, orthodontics, corrective. introduction several treatment options for mild to moderate tooth crowding are available in orthodontics and, after diagnosis and clinical planning, one suitable alternative is the indication of interproximal stripping in permanent teeth1-9. stripping has long been indicated in cases of tooth-size discrepancy to avoid extractions, reshape tooth morphology and create contact surfaces, which would improve posttreatment stability4,6-7,10. some authors even advocate enamel removal and dentin exposure in selected patients7. however, it is generally recommended that only half of the proximal enamel layer should be removed, yet without registration of quantitative data related to enamel thickness11-13. in spite of the well-documented applicability of interproximal stripping1-18, professionals who are willing to practice this option should be aware of the enamel thickness estimates for the most frequently eligible teeth. there are many studies 482482482482482 braz j oral sci. 11(4):481-485 with distinct methodological approaches and for determined teeth3,5,8-9,19-21. an experimental study in 1973 succeeded in mapping the enamel and dentin thicknesses of different tooth groups by microscopy19. however, since then, there is lack of complete studies on this subject. thus, the aim of the present study was to comparatively assess proximal enamel thicknesses of human incisors, canines and premolars. material and methods before the beginning of the study, the research protocol was approved by an ethical institutional review board under the protocol #13525722, in compliance with the helsinki declaration. data collection mapping of enamel thickness was based on analysis of primary data obtained from experimental studies conducted on sound human teeth from tooth banks8-9. the teeth previously analyzed were as follows: mandibular right central incisor (n = 15), mandibular left central incisor (n = 15), mandibular right lateral incisor (n = 15), mandibular left lateral incisor (n = 15), mandibular right canine (n = 10), mandibular left canine (n = 10), mandibular right first premolar (n = 20), mandibular left first premolar (n = 20), mandibular right second premolar (n = 20), mandibular left second premolar (n = 20), maxillary right central incisor (n = 10), maxillary left central incisor (n = 10), maxillary right lateral incisor (n = 10), maxillary left lateral incisor (n = 10), maxillary right canine (n = 10), maxillary left canine (n = 10), maxillary right first premolar (n = 20), maxillary left first premolar (n = 20), maxillary right second premolar (n = 22) and maxillary left second premolar (n = 20). previous experimental procedures to estimate the proximal enamel thickness, the teeth were placed in plastic flasks using utility wax and then embedded in orthophthalic resin (arazyn 1.0; redelease®, são paulo, sp, brazil). the specimens were removed from the flasks and two points were marked in the resin surface (one incisal/occlusal and another apical), which served as reference to trace the long axis of the block. subsequently, the specimens were positioned in a low-speed saw lab cut 1010 (extec® corp., enfield, nh, usa), so as the tooth long axis and contact surfaces coincided with the cutting plane of the diamond disc (diamond wafering 12205 4" x 0.012 x 0.5; extec® corp.), with the incisal or occlusal aspect turned toward the operator8-9. with the aid of the micrometer in the saw, preestablished cuts were made for achievement of central sections with thickness ranging from 0.7 mm to 1 mm, of proximal contact aspects. the specimens were trimmed under water cooling to avoid fractures and loss of enamel components. after obtaining the central section of the specimens, a trained operator performed the measurements of enamel thickness on the proximal surfaces using a perfilometer profile projector® pj 300 (mitutoyo®, kawasaki, japan) with thousandth precision. tooth sections were manipulated to allow standardization of measurements by alignment of cartesian axes (x and y) of the perfilometer with the tooth long axis. the y axis was mesially displaced up to the most external enamel point, determining the “point a”. the perfilometer’s caliper was then zeroed and parallelly displaced up to the most external dentin point, achieving the “point b”. in this way, the mesial enamel thickness was read and expressed in thousandths of millimeters. for achieving of measurements on the distal aspect, the y axis was displaced to the most external dentin point, achieving the “point c”. then, it was parallelly displaced to the most external enamel point on the distal surface, named “point d”, estimating the enamel thickness on this surface. statistical analyses means and standard deviations of proximal enamel thickness measured on the perfilometer were calculated. data on the enamel thicknesses on proximal surfaces of right and left teeth were compared by the unpaired student’s t-test. the mesial and distal thickness measurements were compared by analysis of variance (anova) considering the tooth, side of dental arch and proximal surface as main factors and the double interactions between them. since significant interaction was observed between tooth and thickness (p = 0.030), mesial and distal measurements were compared for each type of tooth by the paired student’s t-test. subsequently, the enamel thickness on mesial and distal surfaces of human incisors, canines and premolars were compared by the anova and tukey’s test. the analyses were performed at a significance level of 5%, using the statistical software minitab® 14 (minitab inc., state college, pa, usa). results mean enamel thicknesses on mesial and distal surfaces of right and left teeth are shown in figure 1. data of right and left teeth presented similar distribution. no statistically significant differences were found between right and left teeth (table 1). therefore, the analyses progressed considering the mean enamel thicknesses of right and left teeth, for both mesial and distal surfaces. figure 2 presents the mapping of proximal enamel fig. 1. mean mesial and distal enamel thicknesses for right and left teeth (central incisor: c-i, lateral incisor: l-i, canine: c, first premolar: fp, second premolar: sp). mapping of proximal enamel thickness in permanent teeth 483483483483483 braz j oral sci. 11(4):481-485 teeth mean (sd)* mesial thickness** mandibular second premolar (n = 40) 1.376 (0.198) a maxillary first premolar (n = 40) 1.220 (0.173) b maxillary second premolar (n = 42) 1.101 (0.176) c mandibular first premolar (n = 40) 1.051 (0.166) c maxillary canine (n = 20) 1.027 (0.126) c maxillary lateral incisor (n = 20) 0.860 (0.129) d maxillary central incisor (n = 20) 0.854 (0.174) d mandibular canine (n = 20) 0.781 (0.106) de mandibular lateral incisor (n = 30) 0.734 (0.139) de mandibular central incisor (n = 30) 0.675 (0.144) e table 2. comparative analysis of mean values in millimeters (mm) for the mesial enamel thicknesses according to the studied teeth. (*) sd: standard deviation. (**) means followed by the same letter in the same column are not significantly different by the tukey’s test at 95% confidence interval. teeth mesial surface distal surface t-value p-value t-value p-value maxillary arch central incisor -1.618 0.124 -0.808 0.430 lateral incisor 0.109 0.914 -1.440 0.172 canine 0.652 0.524 -0.202 0.843 first premolar 0.072 0.943 -1.407 0.168 second premolar -1.222 0.230 -0.971 0.338 mandibular arch central incisor 0.735 0.469 0.619 0.541 lateral incisor -0.370 0.714 -1.374 0.181 canine -0.434 0.671 -1.064 0.301 first premolar -0.984 0.331 -1.268 0.213 second premolar 0.708 0.484 0.456 0.652 table 1. comparative analysis of proximal enamel thickness measurements obtained for right and left teeth. teeth mean (sd)* distal thickness** mandibular second premolar (n = 40) 1.450 (0.172) a maxillary first premolar (n = 40) 1.322 (0.195) ab mandibular first premolar (n = 40) 1.266 (0.187) bc maxillary canine (n = 20) 1.220 (0.145) bc maxillary second premolar (n = 42) 1.155 (0.149) cd maxillary central incisor (n = 20) 1.015 (0.173) de mandibular canine (n = 20) 1.014 (0.118) de maxillary lateral incisor (n = 20) 1.002 (0.176) de mandibular lateral incisor (n = 30) 0.879 (0.158) e mandibular central incisor (n = 30) 0.872 (0.276) e table 3. comparative analysis of mean values in millimeters (mm) for the distal enamel thicknesses according to the studied teeth. (*) sd: standard deviation. (**) means followed by the same letter in the same column are not significantly different by the tukey test at 95% confidence interval. teeth mesial versus distal difference between means (mm) p-value ** maxillary arch central incisor <0.001 0.161 lateral incisor <0.001 0.142 canine <0.001 0.193 first premolar 0.001 0.102 second premolar <0.001 0.054 mandibular arch central incisor <0.001 0.197 lateral incisor <0.001 0.145 canine <0.001 0.233 first premolar <0.001 0.215 second premolar <0.001 0.074 table 4. comparative analysis of mesial and distal enamel thicknesses. (**) highly significant (p<0.001). thickness regardless of the mesial or distal surface. there was an increasing order for enamel thicknesses on the mesial and distal surfaces (tables 2 and 3). for the mesial surface, the mandibular second premolar presented the greatest mean thickness compared with all other teeth (p<0.05). conversely, the mandibular central incisor exhibited the smaller thickness in relation to the other teeth. however, there were no statistically significant differences between the mean mesial thickness of the mandibular central incisor, lateral incisor and canine (table 2). the mandibular second premolar also exhibited greater mean distal enamel thickness. nevertheless, no significant difference was found in comparison to the maxillary first premolar (table 3). the mandibular incisors presented the smallest means of distal enamel thickness, yet not statistically different from the maxillary incisors and mandibular canine (table 3). the average mesial enamel thickness was significantly smaller than distal (table 4). for most teeth, the difference between distal and mesial thicknesses was nearly 0.1 mm, with the smaller difference between means for the maxillary and mandibular second premolars. however, for the mandibular canine and first premolar the difference is up to 0.2 mm (table 4). discussion a space gain of at least 7 mm in the dental arch may be expected by interproximal stripping1,14,16. even though the extent of stripping is related to the severity of tooth-size discrepancy10-12, planning of this procedure should consider that each tooth has different enamel thicknesses on the proximal surfaces5,8,12,19. the results of the present study corroborate the variation in proximal enamel thickness of different human teeth. it should be emphasized that tooth banks do not provide information on the age range, gender or race of donors. however, the validity of outcomes may be demonstrated by the absence of statistically significant differences between mean enamel thicknesses for right and left teeth (table 1), thus evidencing the symmetry of characteristics analyzed. similarly, this kind of symmetry was observed in a clinical study that recorded radiographic measurements of enamel thickness of mandibular incisors3. the enamel thickness is not significantly different between males and females, even though male teeth are larger mapping of proximal enamel thickness in permanent teeth braz j oral sci. 11(4):481-485 due to the dentin layer21-23. based on computer-assisted measurements of proximal enamel thickness of central and lateral incisors, obtained on periapical radiographs of 80 patients, no gender dimorphism was observed3. some authors24-25 have been concerned about the racial influence on tooth dimensions. a study revealed that mandibular central and lateral incisors are not significantly different between caucasoid and african individuals. however, the other teeth were greater in africans compared with caucasians, both for males and females25. the admixture is a marked trait in the brazilian population, which impairs the identification of possible effects of factors related to race on the odontogenesis5. considering the data in tables 2 and 3, as well as the schematic presentation in figure 2, the mandibular second premolar had the greatest average proximal enamel thicknesses (mesial: 1.376 mm ± 0.198, distal: 1.450 mm ± 0.172). however, the smallest mean values of proximal enamel thickness were observed for the mandibular incisors. according to the results of this histological study, there were no significant differences between the mean enamel thicknesses of mandibular incisors, for both mesial and distal surfaces. nevertheless, a radiographic study reported that the mandibular lateral incisors had significantly thicker enamel in both proximal surfaces compared with the mandibular central incisors3. presumably, this difference may be attributed to the employment of distinct measuring methods. fig. 2. overall distribution of mean enamel thicknesses for maxillary (mx) and mandibular (md) teeth (central incisor: c-i, lateral incisor: l-i, canine: c, first premolar: fp, second premolar: sp). it is recommended that the amount of enamel to be removed during interproximal stripping should vary from 0.4-0.5 mm per proximal surface17. the findings of this study, based on measurements obtained using a highly accurate instrument (0.001 mm), suggest that orthodontists should be careful when interpreting that kind of clinical advice. even though the mandibular incisors present the highest frequency of crowding, they also had the smallest values of proximal enamel thickness (tables 2 and 3, figure 2). therefore, removal of 0.5 mm from the mesial surface of a mandibular central incisor may be excessive in some cases. it would be advisable to follow the guidelines for stripping and though limit the interproximal reduction to 50% of the enamel thickness5,11-14. taking into to account the means shown in tables 2 and 3, it was possible to suggest a chart with reference numbers that might be used as parameters for professionals during enamel stripping on mesial and distal surfaces. chart 1 presents the sum of mesial and distal thicknesses for maxillary and mandibular anterior and posterior teeth (in this case, only premolars). these data may yield an indirect reference of the amount of enamel to be removed and hence the maximum achievable space. however, the values in chart 1 should be interpreted with some caution, as they represent only numerical data and should not be used without analyzing the malocclusion, the patient, and each tooth individually. for instance, in a pre-selected patient with class i malocclusion, showing mild anterior crowding in the mandibular arch and having harmonious profile, the orthodontist could easily obtain a 4.0-mm-wide space by removing half the mesial and distal enamel of the anterior teeth. the enamel layer was significantly thicker on the distal surfaces of all teeth (table 4). this is in agreement with two radiographic studies on the enamel thickness of mandibular3 and maxillary21 incisors. the present findings also agree with another brazilian study that registered the dimensions of maxillary first premolars by computer-assisted analysis of histological sections5. in radiographic studies, the authors observed that enamel was significantly thicker on the distal surfaces of mandibular and maxillary central and lateral incisors, with a mean difference of 0.1 mm3,21. the differences between means shown in table 4 are in agreement with previous studies3,21. therefore, it may be suggested that stripping should be less marked on mesial surfaces and in some teeth, such as the mandibular incisors. interproximal stripping has not been related to an increase in the incidence of proximal caries lesions or periodontal disease2,7,13,15,18. however, not all teeth are eligible for interproximal stripping. mandibular incisors with parallel proximal surfaces should not be submitted to this procedure7. to avoid any damage to the adjacent tissues, orthodontists should estimate the amount of enamel removal in accurate radiographs, since the proximal enamel thickness may vary between and within individuals3. the use of fan-beam20 and cone-beam8 computed tomography has also been suggested for assessing proximal enamel thickness, whenever these imaging modalities are needed. moreover, after interproximal stripping, it is useful to insert accurate thickness devices proximal surface group of teeth sum of mean enamel thicknesses per hemiarch mesial maxillary anterior teeth 2.741 mm mandibular anterior teeth 2.190 mm maxillary premolars 2.321 mm mandibular premolars 2.427 mm distal maxillary anterior teeth 3.237 mm mandibular anterior teeth 2.765 mm maxillary premolars 2.477 mm mandibular premolars 2.716 mm chart 1. sum of mean enamel thicknesses on proximal surfaces of maxillary and mandibular anterior teeth and premolars. 484484484484484 mapping of proximal enamel thickness in permanent teeth 485485485485485 braz j oral sci. 11(4):481-485 between teeth to record the magnitude of spaces and enamel reduction5,8,15. the described mapping indicated that mandibular second premolars presented the greatest mean mesial thickness. mandibular central incisors had the smallest thickness in relation to the other teeth, though without significant differences compared with the mandibular lateral incisors and canines. mandibular second premolars also showed the greater distal thickness, albeit without statistically significant difference from maxillary first premolars. mandibular incisors had the smallest average distal thickness, but without significant differences compared with the maxillary incisors and mandibular canines. distal enamel was significantly thicker than mesial. references 1. stroud jl, english j, buschang ph. enamel thickness of the posterior dentition: its implications for nonextraction treatment. angle orthod. 1998; 68: 141-6. 2. zachrisson bu, nyoygaard l, mobarak k. dental health assessed more than 10 years after interproximal enamel reduction of mandibular anterior teeth. am j orthod dentofacial orthop. 2007; 131: 162-9. 3. hall ne, lindauer sj, tüfekçi e, shroff b. predictors of variation in mandibular incisor enamel thickness. j am dent assoc. 2007; 138: 809-15. 4. germeç d, taner tu. effects of extraction and nonextraction therapy with air-rotor stripping on facial esthetics in postadolescent borderline patients. am j orthod dentofacial orthop. 2008; 133: 539-49. 5. macha ac, vellini-ferreira f, scavone-junior h, ferreira ri. mesiodistal width and proximal enamel thickness of maxillary first bicuspids. braz oral res. 2010; 24: 58-63. 6. germec-cakan d, taner tu, akan s. arch-width and perimeter changes in patients with borderline class i malocclusion treated with extractions or without extractions with air-rotor stripping. am j orthod dentofacial orthop. 2010; 137: 734.e1-7. 7. zachrisson bu, minster l, ogaard b, birkhed d. dental health assessed after interproximal enamel reduction: caries risk in posterior teeth. am j orthod dentofacial orthop. 2011; 139: 90-8. 8. fernandes sa, vellini-ferreira f, scavone-junior h, ferreira ri. crown dimensions and proximal enamel thickness of mandibular second bicuspids. braz oral res. 2011; 25: 324-30. 9. munhoz lo, vellini-ferreira f, cotrim-ferreira fa, ferreira ri. evaluation of proximal enamel thickness and crown measurements in maxillary first premolars. braz j oral sci. 2012; 11: 30-5. 10. peck h, peck s. an index for assessing tooth shape deviations as applied to the mandibular incisors. am j orthod. 1972; 61: 384-401. 11. tuverson dl. anterior interocclusal relations. parts i and ii. am j orthod. 1980; 78: 361-93. 12. demange c, françois b. measuring and charting interproximal enamel removal. j clin orthod. 1990; 24: 408-12. 13. jarjoura k, gagnon g, nieberg l. caries risk after interproximal enamel reduction. am j orthod dentofacial orthop. 2006; 130: 26-30. 14. sheridan jj. air rotor stripping. j clin orthod. 1985; 19: 43-59. 15. crain g, sheridan jj. susceptibility to caries and periodontal disease after posterior air-rotor stripping. j clin orthod. 1990; 24: 84-5. 16. rossouw pe, tortorella a. enamel reduction procedures in orthodontic treatment. j can dent assoc. 2003; 69: 378-83. 17. chudasama d, sheridan jj. guidelines for contemporary air-rotor stripping. j clin orthod. 2007; 41: 315-20. 18. grippaudo c, cancellieri d, grecolini me, deli r. comparison between different interdental stripping methods and evaluation of abrasive strips: sem analysis. prog orthod. 2010; 11: 127-37. 19. shillingburg ht jr, grace cs. thickness of enamel and dentin. j south calif dent assoc. 1973; 41: 33-52. 20. spoor cf, zonneveld fw, macho ga. linear measurements of cortical bone and dental enamel by computed tomography: applications and problems. am j phys anthropol. 1993; 91: 469-84. 21. harris af, hicks jd. a radiographic assessment of enamel thickness in human maxillary incisors. arch oral biol. 1998; 43: 825-31. 22. alvesalo l, tammisalo e, townsend g. upper central incisor and canine tooth crown size in 47,xxy males. j dent res. 1991; 70: 1057-60. 23. stroud jl, buschang ph, goaz pw. sexual dimorphism in mesiodistal dentin and enamel thickness. dentomaxillofac radiol. 1994; 23: 169-71. 24. bishara se, jakobsen jr, abdallah em, fernandez garcia a. comparisons of mesiodistal and buccolingual crown dimensions of the permanent teeth in three populations from egypt, mexico, and the united states. am j orthod dentofacial orthop. 1989; 96: 416-22. 25. merz ml, isaacson rj, germane n, rubenstein lk. tooth diameters and arch perimeters in a black and a white population. am j orthod dentofacial orthop. 1991; 100: 53-8. mapping of proximal enamel thickness in permanent teeth oral sciences n3 original article braz j oral sci. october|december 2010 volume 9, number 4 received for publication: may 10, 2010 accepted: december 07, 2010 knowledge, practice and awareness of oral hygiene among three different age populations of same ethnic group – a community based study mohd. abdullah1, md. hassan ali2, shaik rahiman3 1m.sc, department of microbiology, college of medicine, al-jouf university 2 m.d, department of anatomy, college of medicine, al-jouf university 3 m.sc, department of biochemistry, college of medicine, al-jouf university correspondence to: mohd. abdullah department of microbiology, college of medicine, al-jouf university, sakaka, al jouf, kingdom of saudi arabia, po box 2014, sakaka, al-jouf, saudi arabia. phone: 00966535912208 e-mail: gulshanmicro@gmail.com abstract aim: the objective of this study is to compare the knowledge, practice and awareness of oral hygiene among three different age groups (15-30, 30-45 and 45-60-year-olds) of the saudi population in sakaka. methods: 629 subjects aged between 15-60 years were randomly selected. data were collected by self-administered structured questionnaires and analysis was done using the spss statistical software. the subjects were divided in to a group (>30 years), b group (<31 years >45 years) and c group (<46 years). results: 30.7% (a group) of subjects preferred commercial bush to clean their teeth (p<0.001), while 5.9% (b and c groups) choose miswak for cleaning. group b people preferred brushing only in the morning (24.5%), while 16.5% of group a respondents preferred brushing twice a day. 23.1% of group a subjects responded that brushing regularly was the ideal way to avoid caries (p<0.05). 51.8% of all age groups had no idea on the effects of fluoride on caries disease. the reason to visit dentist was mainly for extraction (30.5%) followed by pain relief (28.9%). 62.62% (p<0.05) subjects had carious lesions, whereas 56.6% subjects had fractured teeth. the use of tobacco (16.7%) was highly prevalent in group b subjects (p<0.05). tea consumption among a group subjects (49.6%) more than other age groups (p<0.001). 81.2% subjected were free from gingival bleeding (p<0.05). most of the people (48.6%) preferred frequent dental camps in response to their opinion to improve dental facilities in sakaka, al-jouf. conclusions: the present study shows that poorer knowledge and oral health conditions among 30-45-year-old subjects (b group) compared to the other two groups. generally, these data may be important in the evaluation of the past and planning of future oral health prevention and treatment programs targeting the high-risk populations. keywords: knowledge, habits, dental caries, tooth brushing saudi arabia, ethnic group. introduction the dental profession has limited resources to overcome barriers to oral health such as lack of oral health knowledge, access problem, and dental anxiety. members of other health care professions are often in an appropriate position to give oral health counseling and help overcoming at least some of the barriers1. oral diseases are the most common public health problems worldwide2. their braz j oral sci. 9(4):481-487 482482482482482 impact on individuals and communities in terms of pain and suffering, functional impairment and reduced quality of life is considerable and are the fourth most expensive to treat in most industrialized countries3. one of the most common of the oral diseases is dental caries. dental caries is an infectious and transmissible disease that results in the gradual destruction of enamel, dentin and cementum by acid forming biofilm microorganisms in the presence of sucrose4. it is one of the most common oral diseases showing marked geographical variation and severity of distribution all over the world5. the prevalence is reported to be on the increase as a result of excessive sugar consumption especially in the developing countries6. in socioeconomically developing countries, the change from a traditional lifestyle to a western lifestyle has, among other things, led to an increase in sugar consumption from food and beverages, and in the form of chocolate and candies7. oral diseases are clearly related to behavior, and the prevalence of dental caries and periodontal disease has decreased with improvements in oral hygiene and a decrease in the consumption of sugar products. this general favorable trend in reducing dental caries, however, has not been seen in several developing countries8 or in the middle east9. while twice-a-day tooth brushing seems to be an established practice in several industrialized countries such as the united kingdom10 and norway11, this goal is still very far from being achieved in several other countries, including saudi arabia9, and turkey12. numbers of factors have been put forward to explain the variation in prevalence and severity of dental caries and periodontal disease that can be found between developing and technically developed countries, and also between rural and urban communities. however, this relationship appears to be reversed in the developing countries13. a number of studies have been documented on the oral health status and awareness towards oral health knowledge among the saudi arabian population14. oral health knowledge is considered to be an essential prerequisite for health-related behavior15, although only a weak association seems to exist between knowledge and behavior in crosssectional studies16. nevertheless, studies have shown that there is an association between increased knowledge and better oral health. the present study evaluated the knowledge, practice and awareness about oral hygiene among three different age populations of same ethnic group (15-30, 30-45 and 45-60year-olds) of the saudi population in sakaka. sakaka, is the capital city of the al-jouf province is located north east of al jouf province. the total population of sakaka is 122,686 (as per last 2004 survey records). it is famous for agriculture and it is a central hub for commercial market in al-jouf province. material and methods a sample size of 850 male saudi subjects aged between 15 and 60 years were chosen by simple random sampling technique. there age groups were formed as follows: group a: 15-30 years old, group b: 30-45 years old; and group c: 45-60 years old. the age intervals between the groups were 15±1 years. the instruments for collecting the data required for this study were a self-structured questionnaire and face to face interviewed method for those who were not educated. the content of the questionnaire was guided by review of literature on ways of maintaining dental health and reviewed by subject experts and dental specialists and medical specialists in al-jouf university, sakaka, al-jouf , kingdom of saudi arabia. the participation was voluntary and the answers were anonymous. the questionnaire was pre-tested on a convenience sample of 50 subjects, selected studied population. the questionnaire was modified based upon responses and the survey format was finalized. the questionnaire was organized into 4 sections. section a had 4 questions elicited information on demographic attributes of respondents (age, marital status, profession and education level). section b elicited information on dental health knowledge, practice and attitude. in this section, 10 questions were used to elicit their awareness towards oral-hygiene and the response followed by selection of one of the multiple answers. section c had 5 questions related to their daily habits and the responses followed by selection of one of the multiple answers. section d also had 5 questions to elicit information towards dental current health history and other health problems with a yes / no response format. the questionnaires were administered with the assistance of three faculty members of the college of medicine, al-jouf university, sakaka, aljouf, saudi arbia. on the spot administration and collection of questionnaire was adopted. the research work was carried out between january and march 2010. at the end of the survey, we collected 629 questionnaires with all filled questions. the remaining questions were incomplete, so we excluded those questionnaires for the sample. data analysis the data were analyzed using the statistical software spss for windows version 17.0. the chi square (x2) test was used to examine the association between two categorical variables. z test was used to assess the difference between the two proportions. analysis of variance (anova) was used for evaluation of the statistical significance. a p-value of 0.05 was used as a cut-off level for statistical significance. the multiple logistic regression analysis was performed to study the factors independently associated with the toothbrushing frequency. results a total of 629 subjects responded this survey, most of the respondents belonging to b group (38%), while groupa had the smallest number participants (29.1%). the basic demographic data of respondents showed that 37.8% (238) knowledge, practice and awareness of oral hygiene among three different age populations of same ethnic group – a community based study braz j oral sci. 9(4):481-487 483483483483483 of total were married subjects, and most of the respondents were aged between 30 and 45 years (x2 test, p<0.001). most respondents had graduate level education 42.3% (x2 test, p<0.001) and only 4% were illiterates. own business people responded more (18.1%) than private (15.1%) and government employees (9.2%) in the 3 age groups (x2 test, p<0.001). table 2 summarizes the knowledge and oral hygiene among the three age groups in our sample. as regards practice to oral hygiene, 30.7% (b group; p<0.001) of total 74.9% (471) subjects preferred commercial brushes to clean their teeth; 5.9% (b and c groups) of 16.4% (103) choose miswak for cleaning. only 5.6% (35) used toothpicks in all age groups. most of the b group people brushed their teeth only in the morning (24.5%). regarding brushing twice a day, a group subjects were in higher ranking (16.5%). only 1.3% of the subjects brushed only after dinner from all age groups. 23.1% (145) of a group subjects responded that brushing regularly was the ideal way to avoid caries (p<0.05). it is followed by 8.4% (53) c group subjects chose fluoride use as an ideal way to avoid caries. most of the people (48.6%) preferred frequent dental camps in response to their opinion to improve dental facilities in al-jouf. 25.4% (160) subjects wished to have better dentist. only 7.5% (47) people selected the variable of more dental hospitals and 3.2% (20) subjects liked to have easy access of dental hospitals. table 3 presents the current dental health history of the respondents. among the 62.62% (394) that reported having carious lesions, most were b-group subjects (28.9%) (p<0.05), while 37.4% subjects had no carious lesions. 81.2% (511) of the respondents were free from gingival bleeding (p<0.05); whereas only 16.5% (104) reported this problem. 43.4% (273) had fractured teeth and 56.6% (356) did not have. table 4 shows that 60.9% (383) subjects were nonsmokers, whereas 22.9% (144) respondents were smokers from all 3 age groups. in response to preferable drink question, a group b group c group 15-30 years %(n) 30-45 years %(n) 45-60 years %(n) total %(n) age groups 29.1(183) 38(239)* 32.9(207) 100(629) what is your educational level graduate (college) 22.6(142) 10.2(64) 9.5(60) 42.3(266)* schooling (high school) 2.7(17) 12.2(77) 17.2(108) 32.1(202) some education only 3.7(23) 12.7(80) 5.2(33) 21.6(136) no education 0.2(1) 2.9(18) 1(6) 4(25) what is your marital status married 9.7(61) 37.8(238) 37.8(207) 80.4(506) not-married 19.4(122) 0.2(1) 0(0) 19.6(123) what is your profession private service 15.1(95) 16.2(102) 9.9(62) 41.2(259) own business 1.6(10) 18.1(114) 13.7(86) 33.4(210)* government service 6.4(40) 0.6(4) 9.2(58) 16.2(102) no job 6(38) 3(19) 0.2(1) 9.2(58) table 1. basic demographic characters of the study population (n =629) *p value >0.05 most of the respondents selected tea as the favorite drink (49.6%, p<0.001) and it was followed by the second preferable drink as soft drink (18.6%). 45.6% (287) of the subjects disliked chocolates whereas 34% (214) respondents desired to taste chocolates sometimes only. regarding last food taste in close succession, 60.3% (379) subjects preferred sweet food whereas 39.7% (250) of the respondents choose salty food. discussion caries was a disease of the economically developed countries with their refined carbohydrate consumption, and was of relatively insignificant in the poorer developing countries that subsisted mainly on natural farming products1718. caries experience with age advancement might be due to mere exposure of teeth to the oral environment19. the presence of caries in the b group subjects was significant (28.9%; p<00.5) compared to the other two age groups respondents. the use of tobacco (16.7%) and the smoking habit (11.3%) was highly prevalent in group b subjects (p<0.05). in addition to the effect of smoking on the oral hygiene, it is also associated with a variety of changes in the oral cavity and related to dis in complete denture wearers. cigarette smoke has effect on saliva, oral commensal microorganisms and opportunistic fungal infection in the oral cavity20. as much as 23.7% of b group respondents selected sweet food as their last food taste in close succession (p<0.05). this showed a close relationship between their caries in teeth and their habits (p<0.001). there was in fact a more pronounced increase in tea consumption among 3045 age group subjects than other aged groups (p<0.001). when looking at the response concerning periodontal health, it is interesting to note that most of the respondents reported absence of gingival bleedings (81.2% ), mainly at the age between 30-45 years old, which is supported by previous studies21. this might be due to their habit of tea consumption. knowledge, practice and awareness of oral hygiene among three different age populations of same ethnic group – a community based study braz j oral sci. 9(4):481-487 484484484484484 a group b group c group 15-30 years %(n) 30-45 years %(n) 45-60 years %(n) total %(n) what method you use to clean your teeth commercial brush 22.9(144) 30.7(193)* 21.3( 134) 74.9(471) m i s w a k 4.6(29) 5.9(37 ) 5.9(37 ) 16.4(103) mouth wash 1.6(10) 1( 6) 0.6( 4) 3.2(20) toothpicks 0.5( 3) 5.1( 32) 5.6(35) how many times you clean your teeth only in the morning 9.5(60 ) 24.5(154 ) 12.4( 78) 46.4(292) twice a day 16.5(104) 9.4(59 ) 13.2(83 ) 39.1(246) after every meal 3(19 ) 3.5( 22) 6.7(42) 13.2(83) only after dinner 0.6(4 ) 0.6(4) 1.3(8) what are the ideal way to avoid caries brush regularly 23.1(145)* 8.4(53) 13.7(86) 45.2(284) minimize sweets 0.5(3) 1.3(8) 0.3(2) 2.1(13) visit regularly dentist 0.5(3) 0.8(5) 0.2(1) 1.4(9) use fluoride 0.3(2) 4.5(28) 8.4(53) 13.2(83) all of the above 2.7(17) 8.3(52) 1(6) 11.9(75) don’t know 2.1(13) 14.8(93) 9.4(59) 26.2(165) where would you like to go for dental problems private hospitals 10.8(68) 8.7(55) 23.4(147) 41.2(259) governments hospitals 6(38) 28.6(180) 0.6(4) 33.4(210) traditional expert 5.9(37) 0.3(2) 0.2(1) 16.2(102) managed at home 6.4(40) 0.3(2) 8.7(55) 9.2(58) reason to visit dentist filling 1(6) 5.4(34) 0.6(4) 7(44) r c t 5.6(35) 6(38) 5.6(35) 17.2(108) extraction 6.8(43) 10(63) 13.7(86) 30.5(192) cleaning 7.9(50) 7.3(46) 0.2(1) 15.4(97) pain relief 7.2(45) 9.1(57) 12.7(80) 28.9(182) orthodontic 0.6(4) 0.2(1) 0.2(1) 1(6) how regularly you visit a dentist regularly for check up 0.2(1) 0.5(3) 8.4(53) 9.1(57) every 6 months 0.5(3) 0.5(3) 0.3(2) 1.3(8) when i am in pain 3.2(20) 12.6(79) 14.1(89) 29.9(188) i do not go 25.3(159) 24.5(154) 10(63) 59.8(376) what are the ideal way to avoid caries brush regularly 23.1(145) 8.4(53) 13.7(13.7) 45.2(284) minimize sweets 0.5(3) 1.3(8) 0.3(2) 2.1(13) visit regularly dentist 0.5(3) 0.8(5) 0.2(1) 1.4(9) use fluoride 0.3(2) 4.5(28) 8.4(53) 13.2(83) all of the above 2.7(17) 8.3(52) 1(6) 11.9(75) don’t know 2.1(13) 14.8(93) 9.4(59) 26.2(165) do you know fluoride prevents tooth decay yes 14.9(94) 10(63) 4.9(31) 29.9(188) n o 12.6(79) 4.8(30) 1(6) 18.3(115) don’t know 1.6(10) 23.2(146) 27(170) 51.8(326) does too much sugar in your food causes tooth decay yes 13.4(84) 17.2(108) 10.7(67) 41.2(259) n o 6(38) 2.5(16) 0.2(1) 8.7(55) don’t know 9.7(61) 18.3(115) 22.1(139) 50.1(315) what is your opinion to improve dental facility in al-jouf better dentist 2.4(15) 12.4(78) 10.7(67) 25.4(160) more dental hospital 5.9(37) 8.9(56) 0.5(3) 15.3(96) more dental colleges 6.5(41) 1(6) 7.5(47) frequent dental camps 12.7(80) 14.6(92) 21.3(134) 48.6(306) easy to access dental hospitals 1.6(10) 1.1(7) 0.5(3) 3.2(20) table 2. subjects responses to the question used as measures of the variable “ knowledge, attitude, and practice”. (n=629) * p value >0.05 since, tea contains several naturally occurring substances that help prevent plaque and thus gum disease. the significance of broken teeth also highly reported in age group between 30-45-year-olds, while it was less reported in people aged 15-30 years. this paper focuses on describing differences among male subjects of three age groups in relation to their knowledge, practice and habits toward oral health. concerning knowledge, most of subjects knew that the use of toothbrush help preventing periodontal disease. the findings on oral hygiene habits must be viewed with caution. given the general state of living as observed during the study, the high knowledge, practice and awareness of oral hygiene among three different age populations of same ethnic group – a community based study braz j oral sci. 9(4):481-487 485485485485485 a group b group c group 15-30 years %(n) 30-45 years %(n) 45-60 years %(n) total %(n) do you have any caries in your tooth yes 15.6(98) 28.9(182) 18.1(114) 62.6(394)* n o 13.5(85) 9.1(57) 14.8(93) 37.4(235) are you suffering from gum bleeding yes 1.1(7) 10.8(68) 4.6(29) 16.5(104) n o 26.4(166) 27.2(171) 27.7(174) 81.2(511)* i don’t know 1.6(10) 0.6(4) 2.2(14) do you have any broken teeth yes 7.9(50) 21.5(135) 14(88) 43.4(273) n o 21.1(133) 16.5(104) 18.9(119) 56.6(356) are you a diabetics patient yes 0.5(3) 4.8(30) 5.2(33) n o 29.1(183) 37.5(236) 28.1(177) 94.8(596) are you suffering from any chronic disease yes 0.2(1) 1(6) 4.1(26) 5.2(33) n o 26.4(166) 36.2(228) 28.5(179) 91.1(573) i don’t know 2.5(16) 0.8(5) 0.3(2) 3.7(23) table 3. subjects responses to their current health history (n=629) * p value >0.05 a group b group c group 15-30 years %(n) 30-45 years %(n) 45-60 years %(n) total %(n) are you using any tobacco yes 7.2(45) 16.7(105) 9.9(62) 33.7(212) n o 20.8(131) 20.2(127) 10(63) 51(321) some time 0.8(5) 0.8(5) 4.1(26) 5.7(36) occasionally 0.3(2) 0.3(2) 8.9(56) 9.5(60) do you smoke yes 1.4(9) 11.3(71) 10.2(64) 22.9(144) n o 21.3(21.3) 23.5(135) 18.1(114) 60.9(383) some times 6.2(39) 5.2(33) 0.2(1) 11.6(73) occasionally 0.2(1) 4.6(29) what drink you prefers soft drink (pepsi,mirinda etc.) 2.9(18) 1.9(12) 13.8(87) 18.6(177) tea 2.9(18) 32.8(206) 14(88) 49.6(312)* coffee 6.4(40) 1.1(7) 7.5(47) flavored milk 0.5(3) 0.8(5) 0.3(2) 1.6(10) fruit juice 16.5(104) 1.3(8) 0.6(4) 18.4(116) energy drinks 0.2(1) 4.1(26) 4.3(27) do you like to taste of chocolates yes 2.2(14) 2.4(15) 8.6(54) 13.2(83) very much 6.7(42) 0.3(2) 0.2(1) 7.2(45) some time 8.7(55) 18.3(115) 7(44) 34(214) n o 11.4(72) 17(107) 17.2(108) 45.6(287) what is your last food test in close succession sweet food 21.8(137) 23.7(149) 14.8(93) 60.3(379) salty food 7.3(46) 14.3(90) 18.1(114) 39.7(250) table 4. subject responses to the questions about their habits related to oral hygiene. * p value >0.05 response rate (74.9%, p>0.001) of those reporting using a toothbrush to clean their teeth in this study might not be accurate due to other external factors as suggested by tijmstra22. he states that other factors may have an influence in the findings of social sciences research. amongst these factors is the problem of social desirability whereby respondents tend to answer questions on dental health and dental health behavior in a socially desirable way. a clinical oral examination may have been a useful tool to validate such findings. however, this concept of toothbrush method was also shared by others23. the present study revealed that most of the 15-30-year-old subjects performed the recommended practice of brushing twice a day. this level is similar to that observed in some industrialized countries of east europe24, but low as compared with most western industrialized countries25. the same age group subjects responded that brushing regularly was the ideal way to avoid dental caries. the importance of regular brushing as a means of prevention is well documented26. on the other hand age group c respondents had lack of knowledge towards oral hygiene practice. this is due to poor awareness of dental caries. only 16.4% respondents were practicing miswak as knowledge, practice and awareness of oral hygiene among three different age populations of same ethnic group – a community based study braz j oral sci. 9(4):481-487 486486486486486 their second method for tooth cleaning from all age groups. because of the scientific merit of using miswak and the emphasis of using miswak as a cultural and religious belief among the saudi population, the right method of using miswak as a cleaning technique to achieve maximum benefits should be stressed through various interventions27. however, only 7% of subjects had filled teeth. this finding suggests that extraction of teeth (30%) was the main reason to visit dentist. it was supported by previous studies28. such basic treatment procedures were reported in other studies in developing countries where few dental personnel and resources were available29. generally most of the respondents that visited dentists for extraction of teeth belonged to the age group of 45 60 years old. this was statistically significant (p<0.005). dental pain was found to be the second reason for visiting the dentist among all the older groups, but it was the main reason among those aged 15-30 years old, which is supported by previous studies30. most of the c group subjects preferred private hospitals (23.4%) when they feel pain only. but b group respondents preferred government hospitals when they had the same problem. this denoted that the respondents tended to be satisfied with their own dental health and the appearance of their teeth, which may partly explain why most of them visited a dentist only when they felt a real need and not for regular examinations. this finding is similar to that reported in earlier studies31. only 1.3% respondents visited for dental check up every 6 months although dental check-ups every 6 months are commonly recommended in many countries, there is no scientific evidence to justify this high frequency of dental visits, which can sometimes lead to unnecessary dental treatment32-33. although oral health knowledge does not necessarily relate to better health behavior, people who have assimilated this knowledge and feel a sense of personal control over their oral health are more likely to adopt self-care practices34. this study revealed that only 14.9% of age group a respondents (14.9%) had an idea that fluoride prevent tooth decay, whereas knowledge of sugar in food causing dental caries was only 17.2% among 35-40 years old respondents. interestingly half of the respondents in this survey had lack of knowledge of the effects of fluoride and sugar on dental caries (p<0.001). the low level of knowledge about fluoride use found among respondents might be the result of the limited emphasis placed on this preventive method by dental professionals35. in this survey, 48.6% of total studied population responded dental camps to improve their dental health; the major of respondents for this option was aged between 45-30 years age old, followed by necessity of better dentists (25.4%), while the least option selected by study population was easier access dental hospitals (3.2%). the present study shows that a poorer knowledge and oral health conditions among 30-45 years aged subjects (bgroup) compared to the other two groups. generally, it might be due to the economic status, family and social environment, educational level, and also a change from a traditional lifestyle to a western lifestyle along with the country development. an increase in the knowledge of risk factors for oral disease is important in oral health campaigns that aim to promote healthy habits. this will be achieved only if we have well aware subjects with number of oral health awareness camps and provide easy access to the advanced dental facilities. these data may be uttermost important in the evaluation of the past and planning of future oral health prevention and treatment programs targeting the high risk groups. further studies are necessary to assess more effective modalities of controlling dental caries in this population. acknowledgement the authors are thankful to dr. abdul haleem, md ministry of interior, sakaka, saudi arabia, dr. hemant bashkar, asst. prof, college of dentistry, jamia millia islamia, new delhi, india who assisted in the preparation of questionnaire and helps in the evaluation of survey data and all the participants for their cooperation in this study. references 1. hamilto fa, davis ke, blinkhorn as. an oral health promotion programme for nursing caries. int j paediatr dent. 1999; 9: 195-200. 2. petersen pe, bourgeois d, ogawah, estupinan-day s, ndiaye, c. the global burden of oral diseases and risks to oral health. bull world helath organ, 2005; 83: 661-9. 3. petersen pe. improvement of oral health in africa in the 21st century-the role of the who global oral health program. dev dent. 2004; 5: 9-20. 4. ingraham jl, ingraham ca, prentiss h. introduction to microbiology: a case-history study approach. 3rd ed. belmont: wadsworths; 2001. p.558-9. 5. talaro kp, talaro a. foundations in microbiology. 4th ed. new york: mcgraw hill; 2002. p.654-8. isbn: 0-07-112275-3. 6. patro bk, ravi kumar b, goswami a, mathur vp, nangkynrih b. prevalence of dental caries amoung adult and elderly in an urban resettlement colony of new delhi. indian j dent res. 2008, 19: 95-8. 7. murray cjl, lopez ad. global mortality, disability and the contribution of risk factors: global burden of disease study. lancet. 1997, 349: 1498-1504. 8. sheiham a. changing trends in dental caries. int j epidemiol. 1984; 13: 142-7. 9. al-tamini s, petersen pe. oral health situation of schoolchildren, mothers and schoolteachers in saudi arabia. int dent. 1998, 48: 180-6. 10. bradnock g, white da, nuttall nm, morris aj, treasure et, pine cm. dental attitudes and behaviours in 1998 and implications for the future. br dent j. 2001; 190: 228-32. 11. åstrøm an, samdal o. time trends in oral health behaviors among norwegian adolescents. acta odontol scand. 2001, 59: 193-200. 12. kulak-özkan y, ozkan y, kazazoglu e, arikan a. dental caries prevalence, tooth brushing and periodontal status in 150young people in istanbul: a pilot study. int dent j. 2001, 51: 451-6. 13. milen a, havsen h, heinonen op, paunio i. caries in primary dentition related to age, sex, social status and country of residence in finland. community dent oral epidemiol. 1981; 9: 83-6. 14. al khateeb tl, o’mullan dm, whelton h, sulaiman mi. periodontal treatment needs among saudi arabian adults and their relationship to the use of miswak. community dent health. 1991; 8: 323-8. 15. ashley fp. role of dental health education in preventive dentistry. in: murray jj, editor. prevention of dental disease. oxford: oxford university press; 1996. p.406-14. knowledge, practice and awareness of oral hygiene among three different age populations of same ethnic group –a community based study braz j oral sci. 9(4):481-487 487487487487487 16. freeman r, maizels j, wyllie m, sheiham a. the relationship between health related knowledge, attitudes and dental health behaviours in 14– 16-year-old adolescents. community dent health. 1993, 10: 397-404. 17. axelsson p. an introduction to risk prediction & preventive dentistry. the axelsson series on preventive dentistry. chicago: quintessence; 1999. vol.1, p.65, 109. 18. brian ab, stephen ae. dentistry, dental practice and the community. 5th ed. philadelphia: saunders; 1999. p.217-24. 19. burt da, eklund sa. dentistry, dental practice and the community. 4th ed. philadelphia: saunders; 1992. p.96-7, 15. 20. soya ns, ellepola anb. the impact of cigarette / tobacco smoking on oral candidosis: an overview. oral dis. 2005; 11: 268-73. 21. berey, p; mevrisse, jb; lambert, ml; et al. periodontal health and care need in a sample of belgian population. rev belge med dent. 2002; 57: 206-14. 22. tijmstra t. how successful are surveys on the relationship between caries and sociocultural variables? community dent oral epidemiol. 1981; 9: 230-5. 23. corbin sb, mass wr, kleinman dv, backing cl. 1985 nhis findings on public knowledge and attitudes about oral diseases and preventive measures. public health rep. 1987; 102: 53-60. 24. king a, wold b, tudor-smith c, harel yossi. dietary habits, dental care and body image (pp39-55). in: world health organization. the health of youth: a cross-national survey. copenhagen: who regional office for europe; 1995. 25. petersen pf, danila i, samoila a. oral health behavior, knowledge, and attitudes of children, mothers, and school teachers in romania in 1993. acta odontol scand. 1995; 53: 363-8. 26. tewari a, gauba k, goyal a. evaluation of existing status of knowledge practice and attitude towards oral health of rural communities of horyana india. j indian soc pedod prev dent. 1991; 9: 21-30. 27. lissau i, holst d, feiis-hasche e. dental health behaviours and periodontal disease indicators in danish youth: a 10-year epidemiological follow-up. j clin periodontol. 1990; 17: 42-7. 28. petersen pe, mzee mo. oral health profile of schoolchildren, mothers and schoolteachers in zanzibar. community dent health. 1998; 15: 256-62. 29. ogawa h, soe p, myint b, sein k, kyaing mm, maw kk et al. a pilot study of dental caries status in relation to knowledge, attitudes and practices in oral health in myanmar. asia pac j public health. 2003; 15: 111-7. 30. rajab ld, petersen pe, bakeen g, hamdan ma. oral health behaviour of school children and parents in jordan. int j paediatr dent. 2002; 12: 168-76. 31. rimondini l, zolfanelli b, bernardi f, bez c. self-preventive oral health behavior in an italian university student population. j clin periodontol. 2001; 28: 207-11. 32. elderton rj. overtreatment with restorative dentistry: when to intervene? int dent j. 1993; 43: 17-24. 33. elderton rj, nuttall nm: variation among dentists in planning treatment. br dent j. 1983; 154: 201-6. 34. freeman r, maizels j, wyllie m, sheiham a the relationship between health related knowledge, attitudes and dental health behaviours in 14– 16-year-old adolescents .community dent health. 1993, 10: 397-404. 35. gift hc, larach dc, brunelle ja. public knowledge of fluoride status and purpose. j den res. 1991; 70: 489. knowledge, practice and awareness of oral hygiene among three different age populations of same ethnic group – a community based study braz j oral sci. 9(4):481-487 oral sciences n3 original article braz j oral sci. july/september 2010 volume 9, number 3 received for publication: may 26, 2010 accepted: august 27, 2010 antimicrobial activity of chlorhexidine in root canals instrumented with the protaper universal™ system andréa cruz câmara1, miracy muniz de albuquerque2, carlos menezes aguiar3, ana cristina regis de barros correia4 1phd student in pharmaceutical sciences, department of pharmaceutical sciences, school of pharmaceutical sciences, federal university of pernambuco, brazil 2associate professor, department of pharmaceutical sciences, school of pharmaceutical sciences, federal university of pernambuco, brazil 3associate professor, department of prosthodontics and oral and maxillofacial surgery, dental school, federal university of pernambuco, brazil 4msc student in fungal biology, department of pharmaceutical sciences, federal university of pernambuco, brazil correspondence to: andréa cruz câmara rua nadir de medeiros, 51, piedade, jaboatão dos guararapes pe 54410-110 brasil. phone: (+55) 81 3361 5269. e-mail: andreaccam@yahoo.com.br abstract aim: the purpose of the present study was to evaluate the antimicrobial activity of 0.2%, 1%, and 2% chlorhexidine in root canals instrumented with the protaper universal™ system. methods: fifty human mandibular premolar teeth were infected with a mixture of candida albicans, pseudomonas aeruginosa, enterococcus faecalis and staphylococcus aureus. the specimens were randomly divided into 5 groups with 10 root canals according to the irrigant used. all root canals were instrumented with the protaper universal™ system. assessment of the antimicrobial action of the irrigant was performed before, during, and after instrumentation. data were analyzed statistically by chi-squared test and the fisher exact test at 5% significance level. results: the 0.2% chlorhexidine solution was ineffective against all test microorganisms. the 1% chlorhexidine solution was effective in eliminating p. aeruginosa and c. albicans after the use of the f1 and f3 instruments, respectively. the 2% chlorhexidine solution was effective at killing s. aureus, p. aeruginosa and c. albicans after the use of the s1 instrument. there were statistically significant differences (p<0.05) between the concentrations of chlorhexidine and the instruments used. conclusions: the 0.2% chlorhexidine solution in combination with rotary instrumentation was ineffective against all test microorganisms. the 1% chlorhexidine solution was ineffective against s. aureus and e. faecalis. the 2% chlorhexidine solution was not sufficient to inactivate e. faecalis. keywords: antimicrobial activity, chlorhexidine, protaper universal™, root canal irrigant, rotary instrumentation. introduction the contribution of microorganisms to the development of pulpal and periapical disease has been well documented. enterococcus faecalis, candida albicans, pseudomonas aeruginosa, and staphylococcus aureus are considered by many to be the most resistant species in infected root canals and they are often associated with endodontic treatment failures1-3. root canal system disinfection may be accomplished by using irrigating braz j oral sci. 9(3):402-409 403 solutions during mechanical instrumentation. antibacterial irrigating solutions may reach canal ramifications and inaccessible areas and permeate completely through the dentinal tubules. therefore, several irrigating solutions in different concentrations with antibacterial activity have been recommended for use to irrigate and disinfect root canals in combination with root canal preparation3. a large number of substances have been used as root canal irrigants. although several irrigants have been proposed over the years, sodium hypochlorite (naocl) remains the most widely used1. it is an effective antimicrobial agent and an excellent organic solvent for vital, necrotic and fixed tissues. however, it is highly irritating to periapical tissues, especially at high concentrations4. the biocompatibility problems associated with the use of concentrated naocl have led to the use of substances with known antimicrobial properties and less toxicity, such as chlorhexidine (chx). this substance has been used in endodontics as an irrigating solution and intracanal medicament on account of its broad antimicrobial-spectrum2, substantive properties5, relatively low cytotoxicity6, and ability to inhibit adherence of certain bacteria7. however, it does not dissolve organic tissue. in the last decade, several rotary nickel-titanium (niti) instruments with different configurations and designs have been developed with the aim to reduce the preparation time and to simplify the preparation procedure8. the protaper universal™ rotary system is one of the most popular endodontic niti systems currently on the market9-11. as the advent of rotary techniques for root canal preparation shortened the working time in comparison to manual instrumentation, the irrigant should be one that exerts its antimicrobial activity quickly against the microorganisms found in the root canal and dentinal tubules. therefore, the purpose of this in vitro study was to evaluate the antimicrobial activity of 0.2%, 1%, and 2% chx against some endodontic pathogens in root canals instrumented with the protaper universal™ system. material and methods sample selection and preparation fifty freshly intact human mandibular premolar teeth (length 20-21 mm), straight, with radiographically confirmed single root canal and fully formed apices, were obtained from the human tooth bank of the prosthodontics and oral and maxillofacial surgery, dental school, federal university of pernambuco, brazil, for this study after approval by the research ethics committee of the university’s center of health sciences. the teeth were stored in 10% formalin until use. the coronal access was performed. to determine the working length (wl), a #10 k-file (dentsply-maillefer, ballaigues, switzerland) was inserted into the root canal until it was visible at the apical foramen. the wl was calculated to be 1 mm less than the length obtained with this initial file. the specimens were stored in glass test tubes and were individually sterilized in an autoclave at 121 °c for 30 min. ten samples were randomly chosen and immersed totally in bottles containing 10ml of autoclaved brain heart infusion (bhi, acumedia, lansing, michigan, usa). they were kept in an incubator at 37°c for 96 h to check the sterilization’s efficacy. experiment preparation the methodology used was described previously by câmara et al.12. glass vials with rubber stoppers were adjusted for use in this experiment (figure 1, a). the experimental systems were sterilized in an autoclave at 121°c for 30 min, and inside a laminar flux chamber (veco, piracicaba, brazil), they were filled with bhi (acumedia). the experimental systems were kept in an incubator at 37ºc for 96 h and no turbidity of the medium was observed. fig. 1. experiment preparation. (a) experimental system; (b) turbidity of the medium indicating bacterial growth. bacterial strains the microorganisms strains used in this experiment were obtained from the american type culture collection™ (atcc, rockville, md, usa): candida albicans (atcc 10231), pseudomonas aeruginosa (atcc 9027), enterococcus faecalis (atcc 19433) and staphylococcus aureus (atcc 6538). bacterial cultures and root canal infection the following procedures were performed inside a laminar flux chamber (veco, piracicaba, brazil) using sterilized instruments and materials. isolated 24 h colonies of pure cultures of c. albicans, p. aeruginosa, e. faecalis and s. aureus grown on 10% sheep blood plus bhi (newprov, paraná, brazil) agar plates were suspended in a sterile 0.85% nacl solution. the suspensions antimicrobial activity of chlorhexidine in root canals instrumented with the protaper universal™ system braz j oral sci. 9(3):402-409 of the microorganisms had the optical density adjusted spectrophotometrically to approximately 3.0 x 108 colonyforming units (cfu) ml-1 (equivalent to 1.0 mcfarland scale). from each experimental suspension, 1 ml was removed and a mixture of the four selected microorganisms was prepared. the sterilized experimental systems were then opened. the root canals were infected, except for the negative control, with 10µl of the suspension containing the microorganisms using an automatic micropipette (gilson, villiera-le-bel, france) placed into the access cavity of each tooth. after introduction of the suspension, sterile #10 k-files (dentsplymaillefer) were used to carry the bacterial suspension to the wl. the infected teeth were incubated at 37 ºc for 48 h. the turbidity of the medium during the incubation period indicated bacterial growth (figure 1, b). the purity and the identification of the cultures were confirmed by gram staining, the colony morphology and the growth on petri dishes with the media: cetrimide agar (acumedia) to verify the presence of p. aeruginosa, vogel and johnson agar (acumedia) to verify the presence of s. aureus, saboraud dextrose agar (acumedia) and candida elective agar according to nickerson (merck, darmstadt, germany) to verify the presence of c. albicans, blood agar (newprov, pinhais, pr, brazil) to verify the hemolytic activity, and chromocult enterococci agar (merck) to verify the presence of e. faecalis. if the 4 microorganisms were not identified, the experimental system would be discarded. the efficiency of the method for the infection of the root canal was observed in a pilot study. all assays were conducted in triplicate under aseptic conditions. root canal biomechanical preparation freshly prepared 0.2%, 1%, and 2% chx solutions (farmácia escola carlos dumont de andrade, recife, pe, brazil) were used for root canal irrigation. the infected teeth were removed from the experimental systems with the contamined medium and transferred to glass vials without the medium, in order for the teeth to remain fixed at the beginning of the instrumentation. the specimens were randomly divided into 3 experimental groups and 2 control groups with 10 root canals each according to the irrigant used during root canal preparation as follows: group 1: 0.2% chx; group 2: 1% chx; group 3: 2% chx; group 4 (positive control): 0.85% sterile saline; group 5 (negative controlwithout microorganisms): 0.85% sterile saline. for irrigation of the root canal, the 3-ml fcf syringe system (fcf, são paulo, sp, brazil) with a 30-gauge needle (injecta, diadema, sp, brazil) was used. irrigation was performed at the beginning of instrumentation, between changes of instruments, and at the end of biomechanical preparation. all root canals were instrumented with the protaper universal™ using an electric motor (driller endo-pro torque, sao paulo, brazil) at a speed of 300 rpm as follows: (1) sx file was used to one half of the wl; (2) s1 file was used up to 4 mm short of the apex; (3) s1 and s2 files were used to the full wl; and (4) f1, f2 and f3 files were used to the full wl. a single operator instrumented all root canals. after the instrumentation with each instrument and before drying the root canal, in groups 1, 2, and 3, irrigation with 0.5% tween 80 + 0.07% lecithin was used to neutralize chx. the shaping time was recorded, excluding the assessment of the antimicrobial action of the irrigants. assessment of antimicrobial action the irrigants: to assess the antimicrobial action of the irrigants, sterile paper points were consecutively placed in the root canal. each paper point was left in the root canal for 1 min, as follows: 0-initial (before biomechanical preparation), 1-after instrumentation with s1 file to the full wl, 2-after instrumentation with s2 file, 3-after instrumentation with f1 file, 4-after instrumentation with f2 file, and 5-after instrumentation with f3 file. the paper points were transferred to petri dishes containing the media: cetrimide agar, vogel and johnson agar, saboraud dextrose agar, candida elective agar according to nickerson, chromocult enterococci agar, and blood agar. the plates were then incubated at 37ºc for 48 h. after incubation, bacterial growth was assessed with light microscopy at 400×. all assays were conducted in triplicate under aseptic conditions to ensure the reliability of this study. statistical analysis the categorical data were summarized by means of absolute frequency and relative percentage and the numeric data by means of the usual descriptive statistics of location and dispersion. the results were statistically analyzed using the chi-squared test and the fisher exact test were used when the conditions for the use of the chi-squared test were not verified (statistical inference). a level of significance of 0.05 was adopted. the spss software (statistical package for the social sciences, version 13, chicago, usa) was used. results irrigant antimicrobial efficacy microbial growth was found in all initial samples (0before biomechanical preparation), except for the negative control group, demonstrating that the contamination was effective in all root canals of all groups. all positive controls showed microbial growth before the biomechanical preparation and after the instrumentation with s1 (1), s2 (2), f1 (3), f2 (4), and f3 (5) files (figures 2a and b), while negative controls showed no microbial growth (figure 2c) before the biomechanical preparation and after the instrumentation with s1, s2, f1, f2, and f3 files. the irrigants’ antimicrobial efficacy against s. aureus, p. aeruginosa, c. albicans and e. faecalis is shown in tables 1, 2, 3, and 4, respectively. table 1 presents the antimicrobial efficacy of 0.2%, 1%, and 2% chx in root canals infected with s. aureus. in the 0.2% concentration, the frequency of positive samples was 404antimicrobial activity of chlorhexidine in root canals instrumented with the protaper universal™ system braz j oral sci. 9(3):402-409 405 higher in s1 (with 5 specimens), equal to 3 in s2 and equal to 2 specimens in f1, f2 and f3. in the 1% concentration, the frequency of positive samples was higher in s1 and s2 (with 3 specimens), equal to 2 in f1 and f2 and equal to 1 specimen in f3. in the 2% concentration all the samples were negative for s. aureus. there was no statistically significant difference between the concentrations of chx for any of the instruments used (p>0.05). table 2 presents the antimicrobial efficacy of 0.2%, 1%, and 2% chx in root canals infected with p. aeruginosa. in the 0.2% concentration, the frequency of positive samples was higher in s1, s2 and f1 (with 6 specimens), equal to 4 in f2 and equal to 1 specimen in f3. in the 1% concentration, the frequency of positive samples was equal to 6 in s1, equal to 3 in s2 and there was no bacterial growth in f1, f2 and f3. in the 2% concentration, all the samples were negative for p. aeruginosa. with the exception of the f3 instrument, there were statistically significant differences between the concentrations of chx and the instruments used (p <0.05). the antimicrobial efficacy of 0.2%, 1%, and 2% chx in root canals infected with c. albicans is shown in table 3. in the 0.2% concentration, the frequencies of positive samples were 6, 5, 3, 2 and 1 respectively for s1, s2, f1, f2 and f3. in the 1% concentration, the frequency of positive samples was equal to 7 in s1, equal to 3 in s2, equal to 1 in f1 and there was no microbial growth in f2 and f3. in the 2% concentration, all samples were negative for c. albicans. statistically significant difference was found only between the concentrations of chx and the instruments used was observed in s1 (p <0.05). the antimicrobial efficacy of 0.2%, 1%, and 2% chx in root canals infected with e. faecalis is shown in table 4. instrument chx positive neg ative total p value n % n % n % � s1 0.2% 5 50.0 5 50.0 10 100.0 p (1) = 0.053 1.0% 3 30.0 7 70.0 10 100.0 2.0% 10 100.0 10 100.0 total 8 26.7 22 73.3 30 100.0 � s2 0.2% 3 30.0 7 70.0 10 100.0 1.0% 3 30.0 7 70.0 10 100.0 p(1) = 0.195 2.0% 10 100.0 10 100.0 total 6 20.0 24 80.0 30 100.0 � f1 0.2% 2 20.0 8 80.0 10 100.0 1.0% 2 20.0 8 80.0 10 100.0 p(1) = 0.507 2.0% 10 100.0 10 100.0 total 4 13.3 26 86.7 30 100.0 � f2 0.2% 2 20.0 8 80.0 10 100.0 1.0% 2 20.0 8 80.0 10 100.0 p(1) = 0.507 2.0% 10 100.0 10 100.0 total 4 13.3 26 86.7 30 100.0 � f3 0.2% 2 20.0 8 80.0 10 100.0 1.0% 1 10.0 9 90.0 10 100.0 p(1) = 0.754 2.0% 10 100.0 10 100.0 total 3 10.0 27 90.0 30 100.0 table 1. antimicrobial efficacy of 0.2%, 1%, and 2% chx in root canals infected by s. aureus n=number of specimens (1): fisher exact test all samples were positive in s1 and s2 in all test irrigants. in the 0.2% concentration, all the samples were positive in f1, equal to 8 in f2 and equal to 7 in f3. in the 1% concentration, the frequencies of positive samples were 9, 7 and 5 respectively for f1, f2 and f3. in the 2% concentration, the frequency of positive samples was equal to 5 in f1, equal to 2 in f2 and equal to 1 in f3. there were statistically significant differences between the concentrations of chx and the instruments f1, f2 and f3 (p <0.05). figure 3 shows the antimicrobial efficacy of 1% chx against (a) c. albicans, (b) e. faecalis, (c) s. aureus, (d) e. faecalis, (e) p. aeruginosa, and (f) c. albicans, respectively. root canal preparation time the mean preparation time for the protaper universal™ system was 4.0 ± 1.0 min. discussion the methodology used to assess the antimicrobial activity of endodontic irrigants in this study was the artificial infection of extracted teeth with the selected microorganism and in situ irrigation with the test antimicrobial agents, which according with some authors is an effective method to evaluate anti-fungal as well as antibacterial properties of any solution12-13. after 48 h of incubation, all initial samples were recovered with pure cultures of viable microorganisms confirming the efficiency of the infection methodology adopted for the present research. microbial sampling is another important step that varies among the different methodologies. in this research, the microbial samples collected within the root canals with sterile paper points were obtained before, during and after the antimicrobial activity of chlorhexidine in root canals instrumented with the protaper universal™ system braz j oral sci. 9(3):402-409 406 instrument chx positive neg ative total p value n % n % n % � s1 0.2% 6 60.0 4 40.0 10 100.0 p(1) = 0.003* 1.0% 7 70.0 3 30.0 10 100.0 2.0% 10 100.0 10 100.0 total 13 43.3 17 56.7 30 100.0 � s2 0.2% 5 50.0 5 50.0 10 100.0 p(1) = 0.053 1.0% 3 30.0 7 70.0 10 100.0 2.0% 10 100.0 10 100.0 total 8 26.7 22 73.3 30 100.0 � f1 0.2% 3 30.0 7 70.0 10 100.0 p(1) = 0.286 1.0% 1 10.0 9 90.0 10 100.0 2.0% 10 100.0 10 100.0 total 4 13.3 26 86.7 30 100.0 � f2 0.2% 2 20.0 8 80.0 10 100.0 p(1) = 0.754 1.0% 10 100.0 10 100.0 2.0% 10 100.0 10 100.0 total 2 20.0 28 80.0 30 100.0 � f3 0.2% 1 10.0 9 90.0 10 100.0 p(1) = 1.000 1.0% 10 100.0 10 100.0 2.0% 10 100.0 10 100.0 total 1 10.0 29 90.0 30 100.0 table 3. antimicrobial efficacy of 0.2%, 1%, and 2% chx in root canals infected by c. albicans (*): statistically significant difference at 5% n=number of specimens (1): fisher exact test instrument chx positive neg ative total p value n % n % n % � s1 0.2% 6 60.0 4 40.0 10 100.0 p (1) = 0.005* 1.0% 6 60.0 4 40.0 10 100.0 2.0% 10 100.0 10 100.0 total 12 40.0 18 60.0 30 100.0 � s2 0.2% 6 60.0 4 40.0 10 100.0 p(1) = 0.016* 1.0% 3 30.0 7 70.0 10 100.0 2.0% 10 100.0 10 100.0 total 9 30.0 21 70.0 30 100.0 � f1 0.2% 6 60.0 4 40.0 10 100.0 p(1) = 0.001* 1.0% 10 100.0 10 100.0 2.0% 10 100.0 10 100.0 total 6 20.0 24 80.0 30 100.0 � f2 0.2% 4 40.0 6 60.0 10 100.0 p(1) = 0.023* 1.0% 10 100.0 10 100.0 2.0% 10 100.0 10 100.0 total 4 13.3 26 86.7 30 100.0 � f3 0.2% 1 10.0 9 90.0 10 100.0 p(1) = 1.000 1.0% 10 100.0 10 100.0 2.0% 10 100.0 10 100.0 total 1 3.3 29 96.7 30 100.0 table 2. antimicrobial efficacy of 0.2%, 1%, and 2% chx in root canals infected by p. aeruginosa (*): statistically significant difference at 5% n=number of specimens (1): fisher exact test biomechanical preparation with the protaper universal™ system in order to evaluate the antimicrobial activity of 0.2%, 1%, and 2% chx. the role of bacteria and their by-products in the initiation and perpetuation of pulpal and periapical disease has been well established. the pathogens used in this study were selected because of their clinical importance and association with the endodontic infection. microorganisms such e. antimicrobial activity of chlorhexidine in root canals instrumented with the protaper universal™ system braz j oral sci. 9(3):402-409 407 fig. 2. positive control showing: (a) s. aureus growth; (b) p. aeruginosa growth. negative control showing: (c) no microbial growth. instrument chx positive negative total p value n % n % n % � s1 0.2% 10 100.0 10 100.0 ** 1.0% 10 100.0 10 100.0 2.0% 10 100.0 10 100.0 total 30 100.0 30 100.0 � s2 0.2% 10 100.0 10 100.0 p(1) = 1.000 1.0% 10 100.0 10 100.0 2.0% 10 100.0 10 100.0 total 30 100.0 30 100.0 � f1 0.2% 10 10.0 10 100.0 p(1) = 0.027* 1.0% 9 90.0 1 10.0 10 100.0 2.0% 5 50.0 5 50.0 10 100.0 total 24 80.0 6 20.0 30 100.0 � f2 0.2% 8 80.0 2 20.0 10 100.0 p(1) = 0.020* 1.0% 7 70.0 3 30.0 10 100.0 2.0% 2 20.0 8 80.0 10 100.0 total 17 56.7 13 43.3 30 100.0 � f3 0.2% 7 70.0 3 30.0 10 100.0 p(1) = 0.035* 1.0% 5 50.0 5 50.0 10 100.0 2.0% 1 10.0 9 90.0 10 100.0 total 13 43.3 17 56.7 30 100.0 table 4. antimicrobial efficacy of 0.2%, 1%, and 2% chx in root canals infected by e. faecalis (*): statistically significant difference at 5% (**): could’t be determined because of the absence of one of the categories (1): fisher exact test faecalis, c. albicans, p. aeruginosa, and s. aureus have been associated in cases of persistent periradicular lesions1-3,14. several irrigating solutions might be used during the treatment of infected root canals. these require, among other properties, antimicrobial activity15-17. chx was developed in the late 1940s in the research laboratories of imperial chemical industries ltd. (macclesfield, england). it is widely used as a root canal irrigant or intracanal medication5,18. this cationic bisbiguanide is highly efficacious against several gram-positive and gram-negative oral bacterial species as well as yeasts19. chx was chosen as the irrigating solution to be used in this study because it has been recommended as an alternative or potentially preferred irrigant for endodontic treatment when there are reports of hypersensitivity to naocl by the patient, in cases of open apex where there is great risk of apical leakage of the chemical solution and in cases of microorganisms considered to be resistant to endodontic therapy20. when the antimicrobial activity of chx was compared with naocl, siqueira júnior et al.19 reported that 0.12% chx and 2.5% naocl presented comparable results relative to bacterial elimination from infected root canals in vivo. the present study was unable to confirm the in vitro findings of ohara et al.21 and d’arcangelo et al.22, in which 0.2% chx eliminated all microorganisms, including e. faecalis, because 0.2% chx did not eliminate the microorganisms evaluated. in the present research it was noted that 1% chx was effective in eliminating p. aeruginosa and c. albicans, but ineffective against s. aureus and e. faecalis, disagreeing with sassone et al.23 where the 1% chx showed antimicrobial activity against s. aureus and e. faecalis. there has been increasing concern regarding the antimicrobial activity of chlorhexidine in root canals instrumented with the protaper universal™ system braz j oral sci. 9(3):402-409 408 fig. 3. antimicrobial efficacy of 1% chx. (a) candida elective agar according to nickerson; (b) blood agar; (c) vogel and johnson; (d) chromocult enterococci agar; (e) cetrimide; (f) saboraud dextrose agar. insufficient antimicrobial efficacy of chx against e. faecalis even after prolonged contact of the medication in the root canal5,16. these findings were observed in this study where the antimicrobial activity of 0.2%, 1%, and 2% chx solutions in combination with the niti rotary instrumentation were not effective in eliminating e. faecalis. however, other studies have reported contradictory results regarding the efficacy of 2% chx in eliminating e. faecalis2,6,20,22. ruff et al. 24 found 2% chx to be effective against c. albicans. additionally, huth et al.25 observed that 2% chx was able to eliminate c. albicans and p. aeruginosa, which is consistent with the results of this study, where the 2% chx was effective against c. albicans and p. aeruginosa. because chemomechanical preparation is a short procedure, it would appear that the antibacterial effectiveness of the irrigant inside the root canal might be highly dependent on both, the concentration and type of irrigant used as well as the microbial susceptibility. gomes et al.26 and vianna et al.20 demonstrated that 0.2%, 1% and 2% chx showed antimicrobial activity, and the time required by the 0.2% chx to produce a negative culture was 30 s and less than 30 s for the 1% chx and 2% chx. however, athanassiadis et al.7 reported that when chx is used as an irrigating solution it has relatively short effective exposure time in the root canal and this does not allow the medication to exert its full antibacterial action. as a result, a large number of bacteria may persist within the dentinal tubules and remain viable. this occurred in the present research, since the mean instrumentation time for the protaper universal™ system was 4.0 ± 1.0 min and the chx did not exhibit its full antibacterial action when applied for a short exposure time in the root canal during irrigation. the present study did not investigate the residual effect of the chx as it was neutralized by the addition of tween 80 plus 0.07% lecithin. this study reinforces the importance of using irrigants with antimicrobial activity during the biomechanical preparation because in the positive control group, a 0.85% saline was used and showed no antibacterial activity, in accordance with previous works12,18. however, berber et al.27 found that saline was able to remove microorganisms from the main root canal. in recent years, niti rotary root canal preparation systems, such as the protaper universal™, along with several others, have altered the techniques of canal instrumentation10. the use of rotary niti files for root canal preparation helped shortening significantly the time required to instrument canals with minimal deviation from the original canal path compared with hand instrumentation8,9. rollison et al.28 reported that only instrumentation with rotary systems without irrigating solutions was unable to remove all the bacteria from the root canal. in the same way as observe din the present study, chuste-guillot et al.29 evaluated the bacterial reduction of in vitro infected root canals after instrumentation by niti rotary files and showed that despite extensive instrumentation and antiseptic irrigation, bacteria could remain in the root canal, maintaining the endodontic infection. in this research, the 0.2% chx solution in combination with the protaper universal™ system was ineffective against all test microorganisms. the 1% chx solution was effective in eliminating p. aeruginosa and c. albicans at the end of instrumentation after the use of the f1 and f3 instruments, respectively, but was ineffective against s. aureus and e. faecalis. the 2% chx solution was effective at killing s. aureus, p. aeruginosa and c. albicans at the antimicrobial activity of chlorhexidine in root canals instrumented with the protaper universal™ system braz j oral sci. 9(3):402-409 beginning of the instrumentation after the use of the s1 instrument, but was not sufficient to inactivate e. faecalis. in this study, due to anatomic variations and with the purpose of standardizing the final apical instrument, the root canals were instrumented up to instrument f3 to achieve size #30 for the apical preparation, which, according to khademi et al.30, is the minimum instrumentation size needed for penetration of irrigants into the apical canal third. further research should be carried out to evaluate if by increasing the final apical instrument diameter, microorganisms considered to be resistant to endodontic therapy would completely eliminated. this study investigated in vitro the antimicrobial activity of different chx concentrations against some endodontic pathogens in root canals instrumented with the protaper universal™ system. the 0.2% chx solution in combination with rotary instrumentation was ineffective against all test microorganisms. the 1% chx solution was ineffective against s. aureus and e. faecalis. the 2% chx solution was not sufficient to inactivate e. faecalis. the conclusions of the present work are limited to the in vitro conditions of the study and should be confirmed by further in vivo investigations. acknowledgments this study was supported by grants from coordenação de aperfeiçoamento de pessoal de nível superior – capes brazil. references 1. siqueira júnior jf, rôças in. clinical implications and microbiology of bacterial persistence after treatment procedures. j endod. 2008; 34: 12911301. 2. vianna me, gomes bpfa. efficacy of sodium hypochlorite combined with chlorhexidine against enterococcus faecalis in vitro. oral surg oral med oral pathol oral radiol endod. 2009; 107: 585-9. 3. tirali re, turan y, akal n, karahan zc. in vitro antimicrobial activity of several concentrations of naocl and octenisept in elimination of endodontic pathogens. oral surg oral med oral pathol oral radiol endod. 2009; 108: e117-20. 4. pelka m, petschelt a. permanent mimic musculature and nerve damage caused by sodium hypochlorite: a case report. oral surg oral med oral pathol oral radiol endod. 2008; 106: e80-3. 5. lee jk, baik je, yun ch, lee k, han sh, lee w, et al. chlorhexidine gluconate attenuates the ability of lipoteichoic acid from enterococcus faecalis to stimulate toll-like receptor 2. j endod. 2009; 35: 212-5. 6. williamson ae, cardon jw, drake dr. antimicrobial susceptibility of monoculture biofilms of a clinical isolate of enterococcus faecalis. j endod. 2009; 35: 95-7. 7. athanassiadis b, abbot pv, walsh lj. the use of calcium hydroxide, antibiotics and biocides as antimicrobial medicaments in endodontics. aust dent j. 2007; 52: s64-82. 8. câmara ac, aguiar cm, figueiredo jap de. assessment of the deviation after biomechanical preparation of the coronal, middle, and apical thirds of root canals instrumented with three hero rotary systems. j endod. 2007; 33: 1460-3. 9. aguiar cm, câmara ac. radiological evaluation of the morphological changes of root canals shaped with protaper™ for hand use and the protaper™ and race™ rotary instruments. aust endod j. 2008; 34: 115-9. 10. aguiar cm, mendes d de a, câmara ac, figueiredo jap de. evaluation of the centreing ability of the protaper universal? rotary system in curved roots in comparison to nitiflex? files. aust endod j. 2009; 35: 174-9. 11. vaudt j, bitter k, neumann k, kielbassa am. ex vivo study on root canal instrumentation of two rotary nickel-titanium systems in comparison to stainless steel hand instruments. int endod j. 2009; 42: 22-3. 12. câmara ac, albuquerque mm de, aguiar ca, correia acr de b. in vitro antimicrobial activity of 0.5%, 1%, and 2.5% sodium hypochlorite in root canals instrumented with the protaper universal™ system. oral surg oral med oral pathol oral radiol endod. 2009; 108: e55-61. 13. mercade m, duran-sindreu f, kuttler s, roig m, durany n. antimicrobial efficacy of 4.2% sodium hypochlorite adjusted to ph 12, 7.5, and 6.5 in infected human root canals. oral surg oral med oral pathol oral radiol endod. 2009; 107: 295-8. 14. paz lec de, bergenholtz g, svensäter g. the effects of antimicrobials on endodontic biofilm bacteria. j endod. 2010; 36: 70-7. 15. prabhakar j, senthilkumar m, priya ms, mahalakshmi k, sehgal pk, sukumaran vg. evaluation of antimicrobial efficacy of herbal alternatives (triphala and green tea polyphenols), mtad, and 5% sodium hypochlorite against enterococcus faecalis biofilm formed on tooth substrate: an in vitro study. j endod. 2010; 36: 83-6. 16. arias-moliz mt, ferrer-luque cm, gonzález-rodríguez mp, valderrama mj, baca p. eradication of enterococcus faecalis biofilms by cetrimide and chlorhexidine. j endod. 2010; 36: 87-90. 17. aubut v, pommel l, verhille b, orsière t, garcia s, about i, camps j. biological properties of a neutralized 2.5% sodium hypochlorite solution. oral surg oral med oral pathol oral radiol endod. 2010; 109: e120-5. 18. zehnder m. root canal irrigants. j endod 2006; 32: 389-98. 19. siqueira júnior jf, rôças in, paiva ssm, guimarães-pinto t, magalhães km, lima kc. bacteriologic investigation of the effects of sodium hypochlorite and chlorhexidine during the endodontic treatment of teeth with apical periodontitis. oral surg oral med oral pathol oral radiol endod. 2007; 104: 122-30. 20. vianna me, gomes bpfa, berber vb, zaia, aa, ferraz ccr, souzafilho fj de. in vitro evaluation of the antimicrobial activity of chlorhexidine and sodium hypochlorite. oral surg oral med oral pathol oral radiol endod. 2004; 97: 79-84. 21. ohara p, torabinejad m, kettenring jd. antibacterial effects of various endodontic irrigants on selected anaerobic bacteria. endod dent traumatol. 1993; 9: 95-100. 22. d’arcangelo c, varvara g, de fazio p. an evaluation of the action of different root canal irrigants on facultative aerobic-anaerobic, obligate anaerobic, and microaerophilic bacteria. j endod. 1999; 253: 351-3. 23. sassone lm, fidel r, fidel s, vieira m, hirata júnior r. the influence of organic load on the antimicrobial activity of different concentrations of naocl and chlorhexidine in vitro. int endod j. 2003; 36: 848-52. 24. ruff ml, mcclanahan sb, babel bs. in vitro antifungal efficacy of four irrigants as a final rinse. j endod. 2006; 32: 331-3. 25. huth kc, quirling m, maier s, kamereck k, alkhayer m, paschos e, et al. effectiveness of ozone against endodontopathogenic microorganisms in a root canal biofilm model. int endod j. 2009; 42: 3-13. 26. gomes bpfa, ferraz ccr, vianna me, berber vb, teixeira fb, souzafilho fj. in vitro antimicrobial activity of several concentrations of sodium hypochlorite and chlorhexidine gluconate in the elimination of enterococcus faecalis. int endod j. 2001; 34: 424-8. 27. berber vb, gomes bpfa, sena nt, vianna me, ferraz ccr, zaia aa, et al. efficacy of various concentrations of naocl and instrumentation techniques in reducing enterococcus faecalis within root canals and dentinal tubules int endod j. 2006; 39: 10-7. 28. rollison s, barnett f, stevens rh. efficacy of bacterial removal from instrumented root canal in vitro related to instrumentation technique and size. oral surg oral med oral pathol oral radiol endod. 2002; 94: 366-71. 29. chuste-guillot mp, badet c, peli jf, perez f. effect of three nickel-titanium rotary file techniques on infected root dentin reduction. oral surg oral med oral pathol oral radiol endod. 2006; 102: 254-8. 30. khademi a, yazdizadeh m, feizianfard m. determination of the minimum instrumentation size for penetration of irrigants to the apical third of root canal systems. j endod. 2006; 32: 417-20. 409 antimicrobial activity of chlorhexidine in root canals instrumented with the protaper universal™ system braz j oral sci. 9(3):402-409 original articlebraz j oral sci. january/march 2009 volume 8, number 1 dmft index assessment and microbiological analysis of streptococcus mutans in institutionalized patients with special needs marcia h. tanaka1, karina bocardi2, kátia yukari kishimoto2, paula jacques3, denise madalena palomari spolidorio4, elisa maria aparecida giro5 1 undergraduate student at faculdade de odontologia de araraquara, universidade estadual paulista “júlio de mesquista filho” (unesp), araraquara (sp), brazil 2 dds, faculdade de odontologia de araraquara, unesp, araraquara (sp), brazil 3 dds, ms, phd, assistant professor of the dentistry course, universidade de fortaleza (unifor), fortaleza (ce), brazil 4 dds, ms, phd, assistant professor at the department of physiology and pathology, faculdade de odontologia de araraquara, unesp, araraquara (sp), brazil 5 dds, ms, phd, assistant professor at the department of orthodontics and pediatric dentistry, faculdade de odontologia de araraquara, unesp, araraquara (sp), brazil received for publication: september 12, 2008 accepted: december 16, 2008 correspondence to: elisa maria aparecida giro faculdade de odontologia de araraquara da unesp rua humaitá, 1.680 – centro cep 14801-903 – araraquara (sp), brazil e-mail: egiro@foar.unesp.br abstract aim: to assess the dmft (d = decayed; m = missing; f = filled) index of institutionalized patients with mild and moderate physical and mental disabilities and to correlate it with the streptococcus mutans (s. mutans) counts in the supragingival bacterial biofilm. methods: dental examination of 28 patients aged 15 to 25 years was conducted to determine the dmft index (number of decayed, missing and filled teeth). supragingival plaque samples were collected from the buccal surfaces of all teeth. the samples were inoculated in sb20 medium and incubated at 37 °c for 48 hours. spearman’s correlation test was applied (p = 0.05) to evaluate the correlation between the dmft index and the amount of s. mutans. results: the mean dmft recorded was 7.68 and a large mean number of s. mutans colony-forming units (cfu > 106) was found. no statistically significant correlation was found between the dmft index and the number of s. mutans. conclusions: under the conditions of this study, no correlation was found between the dmft index and the number of s. mutans cfu in institutionalized patients with mental retardation and physical disabilities. keywords: streptococcus mutans, disabled persons, dental caries. introduction dental caries and periodontal disease appear earlier in patients with physical and mental disabilities than in non-disabled patients1,2. the inability to perform adequate oral hygiene may explain the high incidence of the oral diseases found in this population3-5. however, other conditions must be added to the intellectual deficit and impaired motor skills, such as mouth breathing, occlusion abnormalities, bruxism, cariogenic diet, mastication and deglutition dysfunction, abnormal tension of facial muscles, reduced salivary flow and effects from medications1. furthermore, these individuals usually have low socioeconomic level, which aggravates the situation2,6. not only oral hygiene but also dietary habits have been known to influence the dental health. a significant association has been found between the frequency of consumption of sweets and high levels of dental caries7. concerned about disabled persons, parents and caregivers are more likely to allow consumption of sweets and a smaller interval between meals, 10 tanaka mh, bocardi k, kishimoto ky, jacques p, spolidorio dmp, giro ema braz j oral sci. 8(1): 9-13 thus creating an environment that promotes the growth and prevalence of cariogenic microorganisms, such as streptococcus mutans (s. mutans). because of the difficulty in carrying out motor activities, including mastication and toothbrushing, individuals with neuropathies prefer a pureed or pasty diet, which is more cariogenic8. in most cases, proper oral hygiene is done by parents or caregivers, but they report difficulties because disabled persons are usually non-cooperative with this activity2,9. a lthough the basic health, social, psychological and educational needs of these disabled patients are identical to those of non-disabled indiv iduals, the oral health of patients w ith special needs is still ver y poor. the main reasons that lead to this situation are the absence of specialized centers w ith trained dentists10,11 and, most of all, the family’s lack of education, motivation and interest regarding oral homecare. this fact is aggravated by the low socioeconomic and cultural level of the family 2. in order to achieve collaboration through good oral hygiene, it is necessar y to establish a solid relationship between dentist, patient and their parents or caregivers. this interchange that involves motivation, education and especially w illpower, is harder to be achieved for institutionalized patients, because there is no emotional involvement w ith the caregiver. knowledge of the clinical and microbiological characteristics of caries disease in disabled patients may allow the rational establishment of educational and preventive measures that contribute to improve oral health and, consequently, the general health of this population. therefore, the purposes of this study were to assess the dmft (d = decayed; m = missing; f = filled) index of institutionalized patients with mild and moderate physical and mental disabilities and to correlate the dmft index with the s. mutans counts in the supragingival bacterial biofilm. material and methods subjects after approval of the research project by the research ethics committee of the dental school of araraquara, universidade estadual paulista “júlio de mesquita filho” (unesp), 28 patients aged 15 to 25 years were enrolled in this study. written informed consent was obtained from at least one of their parents or legal guardians, before enrollment in the study. participants were individuals institutionalized in an institution for mentally disabled persons who presented diagnosis of mild to moderate physical and mental disabilities and were taking anticonvulsant, antipsychotic, neuroleptic and antidepressant medication (table 1). the participants had not made use of antibiotics for at least three month before the investigation. dental examination dental examinations were conducted after teeth were air-dried, under artificial light and with the aid of a dental mirror and explorer. the number of teeth was recorded for each patient. all erupted teeth were evaluated according to the criteria recommended by the world health organization (who)12 using the dmft index for permanent teeth. plaque samples supragingival plaque samples were collected from buccal surfaces from all maxillary and mandibular teeth using a sterile swab before patient medication cfu decayed missing filled dmft 1 anticonvulsant and antipsychotic 4.0 x 105 0 0 6 6 2 7.2 x 104 2 4 7 13 3 7.6 x 104 1 0 3 4 4 2.8 x 106 2 0 3 5 5 2.8 x 105 1 1 12 14 6 8.0 x 105 0 3 5 8 7 anticonvulsant and neuroleptic 6.0 x 105 9 0 0 9 8 antipsychotic and neuroleptic 1.2 x 106 0 2 3 5 9 anticonvulsant 7.0 x 104 1 1 3 5 10 2.0 x 106 1 5 0 6 11 anticonvulsant and antidepressant 2.08 x 105 0 3 9 12 12 anticonvulsant 6.6 x 105 4 1 0 5 13 4.4 x 106 5 0 0 5 14 3.6 x 104 1 0 0 1 15 anticonvulsant 1.48 x 107 2 6 0 8 16 4.0 x 105 0 0 6 6 17 2.8 x 105 1 1 3 5 18 neuroleptic 2.14 x 106 1 2 13 16 19 anticonvulsant 7.4 x 106 1 0 0 1 20 anticonvulsant 7.2 x 105 1 2 5 9 21 neuroleptic 2.4 x 106 1 1 4 6 22 neuroleptic 3.6 x 106 1 4 11 16 23 6.6 x 107 3 6 1 10 24 anticonvulsant 9.4 x 105 3 0 6 9 25 anticonvulsant, antipsychotic and neuroleptic 5.64 x 107 2 0 9 11 26 1.28 x 106 13 0 0 13 27 1.12 x 106 2 0 6 8 28 6.8 x 104 0 0 0 0 table 1. medication in use, number of s. mutans colony-forming units (cfu), number of decayed, missing and filled teeth and the dmft index for each patient 11dmft index assessment and microbiological analysis of streptococcus mutans in institutionalized patients with special needs braz j oral sci. 8(1): 9-13 toothbrushing. immediately after sample collection, the swab was placed in a sterile tube containing 1 ml saline. the plaque samples of each patient were dispersed by vortexing with sterile 3.5 to 4.5mm diameter glass beads for 30 seconds to disperse bacterial segregates and were diluted in decimal series from 10 -1 to 10 -4 in 0.15 m saline. aliquots of each dilution were inoculated in bacitracin sucrose agar/sb-20 for s. mutans and then incubated at 37 °c for 48 hours. after this period, the colonies with s. mutans characteristics were counted using a stereoscopic microscope (model citoval, rda, carl zeiss jena, germany) with 10x magnification and a digital colony counter (phoenix cp 600 plus; phoenix indústria e comércio de equipamentos científicos ltda., araraquara, sp, brazil). s. mutans were identified following the standards described for the sb-20 medium: opaque and firm colonies that do not disintegrate when touched with a platinum needle, easily displaced, surrounded by a milky white halo and with a scintillating droplet of polysaccharide on the top frequently present. spearman’s correlation was used to evaluate the correlation between dmft index and the s. mutans counts in the supragingival bacterial biofilm. significant level was set at 5% for all analyses. results the data obtained in the clinical examination and in the microbiological analysis are presented in table 1. the mean dmft of the study population was 7.68, with a mean number of decayed, missing and filled teeth of 2.07, 1.5 and 4.11, respectively (table 2). the f component was therefore the one that most contributed to the high dmft. table 2 also shows that the individuals presented a high s. mutans colony-forming units count (cfu > 106). there was no correlation between the dmft index and the number of s. mutans cfu. there was a weak positive correlation (r = 0.389; p = 0.041) between the number of s. mutans cfu and the number of decayed teeth (d) (table 3). discussion the beginning and progression of dental caries are influenced by several risk factors, including bacterial, dietary, environmental and socioeconomic factors. the most significant indicators of caries risk are past caries experience, concentration of s. mutans and lactobacilli, and the presence of protective factors like the buffering capacity of saliva13. an unbalance between protective and risk factors results in growth of specific microorganisms (s. mutans and lactobacilli), which are part of the human dental biofilm14 and are considered the main acidogenic and aciduric organisms associated with dental caries14-16. the dmft index is one of the most widely used indices for presenting epidemiological data about the caries experience of a population. however, this index relates to past signs of the disease, since it allows verifying the incidence or prevalence of decayed, missing and filled teeth, but does not reveal if the caries disease is active or not. in this study, the mean dmft was 7.68 and we considered for analysis the active white spot lesions. rodríguez-vázquez et al.5 found a mean dmft of 5.97 among 20 to 40-year-old institutionalized adult patients with mild to moderate mental retardation; the majority of whom (70.4%) participated in a preventive program that included weekly mouthrinses with a 0.2% fluoride solution and use of a fluoride dentifrice. unfortunately, it has been extensively demonstrated over time and worldwide, including in brazil, that there is a great lack of dental care to patients with special needs1,11,17-19. these studies reported a dmft of 17.4 in patients aged 17 to 24 years, in which 90% of them needed restorations and presented poor oral hygiene and periodontal disease11; dmft of 4.4 in patients aged 11 to 14 years, almost 74% of whom presented carious lesions17; dmft of 7.92 in 25-year-old adults, with the d component reflecting many untreated decayed teeth18 and 88% of the patients needing conservative treatment19. rodrigues dos santos et al.1 studied dental caries in brazilian patients with cerebral palsy and observed high dmft and biofilm indices. these outcomes suggest that this population belong to a group that is at high caries risk and require preventive oral health measures. in the present study, since the patients attended a dental care program, the component that most contributed to the high dmft was filled teeth (f). several investigations have tried to associate s. mutans colonization levels with dental caries incidence, but there are few studies with institutionalized disabled persons. in patients with mental retardation, caries incidence and the amount of bacteria in the dental biofilm seem to be higher than the average for the general population. sánchez-pérez et al.13 verified an association between the s. mutans counts from dental biofilm and the dmft index and surfaces with active caries. the authors reported that, 18 months after the initial examination, 86% of the children at high risk developed multiple carious lesions, while 94% of the children at low risk developed few or no lesions. linear regression analysis identified s. mutans from cfu decayed missing filled dmft mean 6.1 x 106 2.07 1.5 4.11 7.68 standard deviation 1.6 x 107 2.85 1.93 3.98 4.3 table 2. mean number of s. mutans colony-forming units (cfu), mean number of decayed, missing and filled teeth and mean dmft index for the studied population (n = 28) decayed missing filled dmft correlation coefficient 0.389* 0.175 -0.057 0.245 significance 0.041 0.372 0.775 0.209 table 3. correlation between the number of s. mutans colony-forming units (cfu) and the number of decayed, missing and filled teeth and the mean dmft index for the studied population (n = 28) * statistically significant correlation (p < 0.05). 12 tanaka mh, bocardi k, kishimoto ky, jacques p, spolidorio dmp, giro ema braz j oral sci. 8(1): 9-13 the dental biofilm as the most significant bacteriological indicator for dmft. matee et al.20 found a significant relationship between s. mutans levels and dental caries index, but they also observed high levels of this microorganism in children who did not present carious lesions, which suggests that the presence of cariogenic bacteria does not necessarily mean high caries activity as this is a multifactorial pathology. in accordance with the findings of matee et al.20, this study also found high s. mutans cfu counts in patients with low or absent dmft. there was significant correlation between s. mutans cfu and the number of decayed teeth, but no correlation was found between s. mutans cfu and the dmft index. likewise, llena puy et al.21, while studying the relationship between dental caries and s. mutans and lactobacilli cfu, buffering capacity of the saliva and salivary flow in school children, did not find a statistically significant correlation between the dmft index and bacterial count. according to several authors13,22-25, the presence of decayed teeth increases significantly s. mutans counts in saliva and dental biofilm. petti et al.24 pointed out that when these teeth are restored, the concentration of these microorganisms fall to levels similar to those found in health individuals, reducing the risk of infecting other teeth. since the number of restored teeth was the component that most contributed to the high dmft found in this study, the absence of correlation between s. mutans cfu and the dmft index can be explained. in the present study, all patients had s. mutans in their dental biofilm and the number ranged from 3.6x104 to 6.6x107. half of the study population had s. mutans cfu levels higher than 106. spearman’s rank correlation coefficient (r = 0.389) revealed a significant but weak positive correlation between s. mutans and decayed teeth (p < 0.05). this can be explained by the fact that s. mutans has been strongly associated with the beginning of the caries process26. even though this study did not find a significant correlation between the dmft index (caries experience) and the number of s. mutans cfu, several studies have shown a highly significant relationship between these two factors, supporting the infectious nature of caries disease15,21,27,28. studies such as this, in which only one collection of dental biofilm was done to determine the number of microorganisms present in the mouth, reveal a single moment of a multifactorial and dynamic disease that takes some time to develop. in addition, the bacteria involved in dental caries vary in response to changes in the oral environment. however, such studies are important as a base to develop preventive programs and to assess their success. under the tested conditions, a correlation between the dmft index and the number of s. mutans cfu in institutionalized patients with mental retardation and physical disabilities was not found. references 1. rodrigues dos santos mt, masiero d, novo nf, simionato mr. oral conditions in children with cerebral palsy. j dent child. 2003;70:40-6. 2. pezzementi ml, fisher ma. oral health status of people with intellectual disabilities in the southeastern united states. j am dent assoc. 2005;136:903-12. 3. whyman ra, treasure et, brown rh, macfadyen ee. the oral health of long-term residents of a hospital for the intellectually handicapped and psychiatrically ill. n z dent j. 1995;91:49-56. 4. velasco e, machuca g, martinez-sahuquillo a, rios v, lacalle j, bullón p. dental health among institutionalized psychiatric patients in spain. spec care dentist. 1997;17:203-6. 5. rodríguez-vázquez c, garcillan r, rioboo r, bratos e. prevalence of dental caries in an adult population with mental disabilities in spain. spec care dentist. 2002;22:65-9. 6. schultz st, shenkin jd, horowitz am. parental perceptions of unmet dental need and cost barriers to care for developmentally disabled children. pediatr dent. 2001;23:321-5. 7. moynihan pj. dietary advice in dental practice. br dent j. 2002;193:563-8. 8. sayegh a, dini el, holt rd, bedi r. oral health, sociodemographic factors, dietary and oral hygiene practices in jordanian children. j dent. 2005;33:37988. 9. martens l, marks l, goffin g, gizani s, vinckier f, declerck d. oral hygiene in 12-year-old disabled children in flanders, belgium, related to manual dexterity. community dent oral epidemiol. 2000;28:73-80. 10. leviton fj. the willingness of dentists to treat handicapped patients: a summary of eleven surveys. j dent handicap. 1980;5:13-7. 11. pieper k, dirks b, kessler p. caries, oral hygiene and periodontal disease in handicapped adults. community dent oral epidemiol. 1986;14:28-30. 12. organização mundial de saúde. levantamento epidemiológico básico em saúde bucal: manual de instruções. 4 ed. genebra: oms, 1997. 13. sánchez-pérez l, acosta-gío ae, méndez-ramírez i. a cluster analysis model for caries risk assessment. arch oral biol. 2004;49:719-25. 14. loesche wj. clinical and microbiological aspects of chemotherapeutic agents used according to the specific plaque hypothesis. j dent res. 1979;58: 2404-12. 15. zickert i, emilson cg, krasse b. streptococcus mutans, lactobacilli and dental health in 13-14 year old swedish children. community dent oral epidemiol. 1982;10:77-81. 16. kingman a, little w, gomez i, heifetz sb, driscoll ws, sheats r, et al. salivary levels of streptococcus mutans and lactobacilli and dental caries experiences in a u.s. adolescents population. community dent oral epidemiol. 1988;16: 98-103. 17. vyas ha, damle sg. comparative study of oral health status of mentally subnormal, physically handicapped, juvenile delinquents and normal children of bombay. j indian soc pedod prev dent. 1991;9:13-6. 18. rudolph mj, chikte um. dental caries experience and periodontal disease in institutionalized male psychiatric patients. j dent assoc s afr. 1993;48(8):451-4. 19. kenkre am, spadigam ae. oral health and treatment needs in institutionalized psychiatric patients in india. indian j dent res. 2000;11:5-11. 20. matee mi, mikx fh, maselle sy, van palestein helderman wh. mutans streptococci and lactobacilli in breast-fed children with rampant caries. caries res. 1992;26:183-7. 21. llena-puy mc, montañana-llorens c, forner-navarro l. cariogenic oral flora and its relation to dental caries. asdc j dent child. 2000;67:42-6. 22. pollard ma, curzon me. dental health and salivary streptococcus mutans levels in a group of children with heart defects. int j pediatr dent. 1992;2:81-5. 23. zoitopoulos l, brailsford sr, gelhier s, ludford rw, marchant sh, beighton d. dental caries and caries-associated microorganisms in the saliva and plaque of 3and 4-year old afro-caribbean and caucasian children in south london. arch oral biol. 1996;41:1011-8. 24. petti s, pezzi r, cattaruzza ms, osborn jf, d’arca as. restoration-related salivary streptococcus mutans level: a dental caries risk factor? j dent. 1997;25:257-62. 25. seibert w, farmer-dixon c, bolden t, stewart jh. streptococcus mutans levels and caries prevalence in low-income schoolchildren. j tenn dent assoc. 2002;82:19-22. 26. krishnakumar r, singh s, subba reddy vv. comparison of levels of mutans streptococci and lactobacilli in children with nursing bottle caries, rampant caries, healthy children with 3-5 dmft-dmft and healthy caries free children. j indian soc pedod prev dent. 2002;20:1-5. 13dmft index assessment and microbiological analysis of streptococcus mutans in institutionalized patients with special needs braz j oral sci. 8(1): 9-13 27. gábris k, nagy g, madléna m, dénes z, márton s, keszthelvi g, et al. associations between microbiological and salivary caries activity tests and caries experience in hungarian adolescents. caries res. 1999;33:191-5. 28. hedge pp, ashok brk, ankola va. dental caries experience and salivary levels of streptococcus mutans and lactobacilli in 13-15 years old children of belgaum city, kanataka. j ind soc pedod prev dent. 2005;23:23-6. oral sciences n3 original article braz j oral sci. july | september 2012 volume 11, number 3 effect of cetylpyridinium chloride with xylitol on the formation of biofilm and development of gingivitis bruna ghiraldini1, erika tie furushima2, renato corrêa viana casarin3, karina teixeira villalpando4, suzana peres pimentel3, fabiano ribeiro cirano3 1dentist, unip, são paulo, sp, brazil 2undergraduate student, unip, são paulo, sp, brazil 3msc, phd, professor of periodontics, unip, são paulo, sp, brazil 4msc, phd, professor of periodontics and dental clinic, puc, campinas, sp, brazil correspondence to: fabiano ribeiro cirano professor of periodontics, universidade paulista (unip) rua dr. bacelar, 1212 cep: 04026-002 vila clementino, são paulo, sp brasil phone: +55 11 50849950 fax: +55 11 50849948 e-mail: cirano@unip.br abstract aim: to assess the effect of the combination of cetylpyridinium chloride and xylitol on the formation of dental biofilm and development of experimental gingivitis. methods: a crossover, double-blind, placebo-controlled study was conducted and divided into two phases of 21 days each with a time interval of 10 days between them. a modified experimental gingivitis model was used and 31 volunteers were randomly divided into 2 groups. the volunteers performed daily mouthwashes twice a day with the test solution containing cetylpyridinium combined with xylitol or a placebo solution. on day 0 and day 21 of each phase the plaque index (pi) and gingival index (gi) of each volunteer were measured. during this phase, the volunteers brushed their teeth with standard toothbrushes and dentifrice, protecting the third quadrant with a toothshield. after brushing, the toothshield was removed and the mouthwash was used. results: the pi values observed in the test group at baseline and on day 21 were 0 (0.00 – 0.03) and 0 (0.22-0.48) respectively, and in control group 0 (0.00 – 0.03) and 1 (0.45 – 0.81) (inter-group analysis mcnemar test, p<0.05). for gi, the values obtained in the test group were 0 (0.00 – 0.03) and 1 (0.48 – 0.71), at baseline and day 21 and in control group 0 (0.00 – 0.03) and 1 (0.58 – 0.84) (inter-group analysis mcnemar test, p>0.05). conclusions: the test solution had a positive effect on dental biofilm control. however, it was not capable of preventing the development of experimental gingivitis. keywords: cetylpyridinium chloride, xylitol, gingivitis. introduction mechanical control of dental biofilm is an important factor for preventing gingival inflammation and dental caries. however, the daily use of a toothbrush and interdental cleaning devices are not adequately performed by most individuals. therefore, the use of substances with a potential of chemical control of dental biofilm may be indicated1-2. in this connection, mouthwashes are frequently recommended as well as several products containing different active ingredients. received for publication: june 26, 2012 accepted: september 18, 2012 braz j oral sci. 11(3):392-395 393393393393393 among the different types of substances, two cationic antiseptics called chlorhexidine and cetylpyridinium chloride (cpc) deserve to be pointed out. with a view to reducing the quantity and virulence of biofilm, these substances promote a reduction in the inflammatory response3. chlorhexidine has been considered the gold standard regarding chemical control of dental biofilm, presenting the highest values of plaque reduction within oral antiseptics (plaque reduction of 58.3% to 92.9%) 4-5. however, chlorhexidine causes pigmentation of teeth and restorations, has an unpleasant flavor, leads to taste alterations, increases the formation of supragingival calculus and, it may be associated with mucosal desquamation. therefore, the longterm daily use of chlorhexidine is not recommended and alternative substances that present efficacy in biofilm control and reduced adverse effects could be considered an important therapeutic approach. cpc is capable of reducing biofilm formation4-12, reducing plaque index 34.5% to 70.9%13, and although presents same side effects, such as dental pigmentation, they are much less intense than chlorhexidine4-13. although the use of cpc has been shown to be a feasible option as an adjuvant in controlling biofilm, its potential could be increased by the association of other substances that could contribute towards this purpose. in this regard, xylitol has shown to be effective in preventing dental caries. clinical studies that combined xylitol with the use of fluoridated dentifrices, dietary and behavioral changes have shown the efficacy of this concomitant therapy5,14. studies have indicated that xylitol might act in the reduction of caries incidence15 by decreasing the number of streptococcus mutans with its prolonged use16, indicating that xylitol might decrease the ability of bacteria to multiply in its presence. furthermore, hildebrant and sparks17 (2000) showed that chlorhexidine mouthwash reduces s. mutans levels and long-term use of xylitol is capable of maintaining these levels low. in view of the above, the aim of this study was to evaluate the effect of a cpc and xylitol solution on supragingival plaque formation and development of experimental gingivitis. material and methods thirty-one volunteers participated in this study. the research subjects were selected after signing an informed consent form and the research was approved by the research ethics committee of the paulista university under protocol #492/09. the following inclusion criteria were adopted: be in the age group between 18 and 28 years, have no medical history of systemic diseases, and have at least 20 teeth in the mouth. the following exclusion criteria were considered: be a smoker, have used systemic antibiotics in the 3 months previous to the study, have used chemical agents to control plaque 15 days before the study, be allergic to cpc and/or xylitol, be pregnant, be a permanent drug user, wear prosthesis or orthodontic appliances, and have probing depth greater than 3 mm. a crossover, double-blind, placebo-controlled study was conducted in accordance with the modified experimental gingivitis model. the volunteers were randomly divided into two groups: test group (0.5 % cpc + 12.5% xylitol solution) and control group (placebo) and the study consisted of an experimental phase composed of two periods of 21 days, with intervals of 10 days between them (wash out period). impressions were made of the left mandibular hemi-arch of the volunteers with alginate and the resulting model was used for preparation of a toothshield using polyvinyl acetate lamina prepared in a vacuum plasticizer and cut to cover the entire area of teeth 34, 35, 36 and 37 and 2 mm beyond the gingival margin both in the vestibular and lingual surfaces, which was used until the end of the experiment. samples containing cpc combined with xylitol (atco pharma, são paulo, sp, brazil) and a placebo agent (solution with the same flavor and coloring of the test solution, but with no cpc or xylitol) were given to the volunteers in identical bottles so that neither the examiner nor the volunteer could identify them. both the test and placebo solutions were properly codified and the secrecy of the codes was revealed only at the end of the study. all the participants tested the chemical agent in alternating periods in accordance with the proposed crossover study. before the beginning of each study period, professional removal of the supragingival dental biofilm was performed. afterwards, during each period of 21 days, the volunteers performed normal oral hygiene using the acetate toothshield so that the area selected did not receive mechanical control of biofilm. each research subject was instructed to use a standard toothbrush and dentifrice (professional colgate toothbrush and mfp colgate dentifrice, colgate-palmolive, são bernardo do campo, sp, brazil), and the dentifrice used did not have any active ingredient besides fluoride. twice a day (every 12 h) the volunteers performed mouthwashing with 20 ml of the determined solution for 1 min, without acetate toothshield18-19. the bottles of the solutions used and the ones that were not used were returned at the end of each period to prevent them from being reused. new bottles with the new solution were provided after the time interval of 10 days, which means that the group that used the test solution started using the placebo solution and the other group the opposite (figure 1). the toothshields were assessed by the researcher and replaced when damaged and the reusable ones were washed and disinfected between the experimental periods. in the washout period the volunteers practiced conventional oral hygiene with a dentifrice and toothbrush in the entire oral cavity. the assessments of the clinical parameters performed on day 0 and day 21 of each period were dichotomously done, at six sites per tooth, at teeth covered by toothshield, using visible plaque20 and gingival indices21. the indexes used were codified as follows: plaque index (pi): 0 = absence of plaque and 1 = presence of plaque; gingival index (gi): 0 = absence of bleeding on probing and 1 = presence of bleeding on probing. effect of cetylpyridinium chloride with xylitol on the formation of biofilm and development of gingivitis braz j oral sci. 11(3):392-395 394394394394394 baseline 21 days test group 0 (0.00 – 0.03) aa 0 (0.22 – 0.48) ab control group 0 (0.00 – 0.03) aa 1 (0.45 – 0.81) bb table 1 – plaque index values (median (95% confidence interval)), before and after the use of control and test solutions. different capital letters indicate the difference between the groups, while lowercase letters indicate the difference between time intervals (mcnemar test, p<0.05). baseline 21 days test group 0 (0.00 – 0.03) aa 1 (0.48 – 0.71)ab control group 0 (0.00 – 0.03) aa 1 (0.58 – 0.84)ab table 2 – gingival index values (median (95% confidence interval)), before and after use of control and test solutions. different capital letters indicate the difference between the groups, while lowercase letters indicate the difference between time intervals (mcnemar test, p<0.05). fig. 1. study design and flowchart of the present crossover study. all clinical examinations were performed by a previously calibrated clinician (bg – kappa index = 0.85). for statistical analysis, the non-parametric mcnemar test was used and a level of significance of 5% was adopted. results all the volunteers (65% female, mean age 21.1±2.2) accepted the research conditions in a satisfactory manner and they all completed the study until the end. it was observed a statistically significant increase in the pi in the test and control group (table 1). however, inter-group analysis showed higher pi in the control group (p<0.05). in addition, an increase in gi in the two groups (p<0.05) was observed. nevertheless, in the inter-group analysis no statistically significant differences were observed on day 21 (table 2). discussion as biofilm is considered the primary etiologic factor of periodontal diseases, prevention approached focus on different forms to inhibit its formation and development. in this effort, mechanical control and chemical agents, alone or in association has been used for obtain periodontal health. within several chemical agents, chlorhexidine is considered the gold standard, although it has very intense side effects. thus, cpc appears as an alternative agent, present antibacterial action and lesser side effects. moreover, some agents could be added to cpc solution, promoting other oral benefits. recently, a combination of cpc and xylitol, a recognized anti-caries agent, has been produced, but its effect on plaque and gingivitis control is yet unknown. therefore, the present study was conducted to analyze the effect of a cpc and xylitol solution on the formation of supragingival biofilm and development of experimental gingivitis in comparison with a placebo solution. the findings of the present crossover randomized study indicate that cpc+xylitol mouthwash has potential in controlling supragingival dental biofilm, as inter-group differences were seen regarding plaque accumulation after the experiment. however, the potential of this mouthwash in controlling dental biofilm did not result in benefits on the development of experimental gingivitis. comparing the bleeding on probing after use of cpc+xylitol and placebo solutions, no inter-group differences were observed (p<0.05). these results indicate that the cpc+xylitol solution has a significant benefit on plaque control, although it did not lead to benefits in controlling the development of gingivitis. similar results were observed by several studies that showed a significant reduction in pi when cpc was used in comparison with a placebo solution5,7-10,12,22-24. the antiplaque effect of cpc is due to its ability to decrease the surface tension of water and alter bacterial cell permeability that causes the output of enzymes and essential metabolites25. this action allows cpc to penetrate the bacterial cell membrane causing destruction of cellular components, disruption of bacterial metabolism and inhibition of cell growth, ultimately leading to cell death and its interference with bacterial adherence26. albert-kiszely et al.26 (2007) found that its use, in the form of mouthwash, caused a reduction in the number of bacteria adhered to epithelial cells of the oral mucosa. the present study showed that the test solution was not capable of promoting actual beneficial effects in gi, despite having promoted a statistically significant reduction in pi. however, ayad et al.9 (2011) and silva et al.24 (2009) found a statistically significant difference in gi, showing that cpc was superior in comparison with the placebo solution. it is worth mentioning that both studies had different methodologies from that of the present study because they did not use the modified experimental gingivitis model2, the research subjects used chemical control in combination with mechanical control, and longer evaluation periods, namely 6 weeks in the study of silva et al.24 (2009), and 3 and 6 months in the study of ayad et al.9 (2011). in the other hand, rioboo et al.27 (2012) showed limited benefits of the cpc as adjuncts effect of cetylpyridinium chloride with xylitol on the formation of biofilm and development of gingivitis braz j oral sci. 11(3):392-395 395395395395395 to unsupervised oral hygiene in reducing plaque accumulation, and no effect on gingivitis, corroborating to our results. clinical studies have shown that the combination of xylitol with the use of fluoridated dentifrices and dietary and behavioral changes reduces dental biofilm and cariogenic bacteria14. this action may be the result of the decrease in the number of s. mutans with the use of the substance or the potential of xylitol in penetrating the biofilm by diffusion and reducing adhesiveness of the bacteria16,28-29. perhaps these characteristics of xylitol may contribute to explain the results of the present study, which are in agreement with other studies that have shown the potential for reducing dental biofilm30. the results of this study showed the potential of the combination of cpc and xylitol in preventing the formation of dental biofilm, but these are short-term results and with no mechanical control. it is important to consider that this model of experimental gingivitis allows evaluating the effect of chemical agents on plaque formation and gingivitis development, removing the effect of mechanical control. in our opinion, this represents one of the best forms to determine the efficacy of mouthwash alone. however, sometimes, it did not represent the usual clinical condition and, further long-term studies combining the solution with mechanical control should be made seeking greater effectiveness in controlling gingival inflammation. within the limitations of this study, it may be concluded that the combination of cpc and xylitol has the potential to control the formation of dental biofilm, but it does not have any effect on the development of experimental gingivitis. references 1. hull ps. chemical inhibition of plaque. j periodontol. 1980; 7: 431-42. 2. lindhe j, koch g. the effect of supervised oral hygiene on the gingivae of children. j periodontol. 1967; 2: 215-20. 3. axelsson p, albandar jm, rams te. prevention and control of periodontal diseases in developing and industrialized nations. periodontol 2000. 2002; 29: 235-46. 4. quirynen m, soers c, desnyder m, dekeyser c, pauwels m, van steenberghe d. a 0.05% cetylpyridinium chloride/0.05% chlorhexidine mouth rinse during maintenance phase after initial periodontal therapy. j clin periodontol. 2005; 32: 390-400. 5. paula va, modesto a, santos kr, gleiser r. antimicrobial effects of the combination of chlorhexidine and xylitol. br dent j. 2010 dec 18; 209: e19. 6. witt jj, walters p, bsoul s, gibb r, dunavent j, putt m. comparative clinical trial of two antigingivitis mouthrinses. am j dent. 2005; 18: 15a-7a. 7. charles ca, mcguire ja, sharma nc, qaqish j. comparative efficacy of two daily use mouthrinses: randomized clinical trial using an experimental gingivitis model. braz oral res. 2011; 25: 338-44. 8. garcía v, rioboo m, serrano j, o’connor a, herrera d, sanz m. plaque inhibitory effect of a 0.05% cetyl-pyridinium chloride mouth-rinse in a 4day non-brushing model. int j dent hyg. 2011; 9: 266-73. 9. ayad f, prado r, mateo lr, stewart b, szewczyk g, arvanitidou e et al. a comparative investigation to evaluate the clinical efficacy of an alcoholfree cpc-containing mouthwash as compared to a control mouthwash in controlling dental plaque and gingivitis: a six-month clinical study on adults in san jose, costa rica. j clin dent. 2011; 22: 204-12. 10. rao d, arvanitidou e, du-thumm l, rickard ah. efficacy of an alcoholfree cpc containing mouthwash against oral multispecies biofilms. j clin dent. 2011; 22: 187-94. 11. williams mi. the antibacterial and antiplaque effectiveness of mouthwashes containing cetylpyridinium chloride with and without alcohol in improving gingival health. j clin dent. 2011; 22: 179-82. 12. barnes vm, arvanitidou e, szewczyk g, richter r, devizio w, cronin m et al. evaluation of the antiplaque efficacy of two cetylpyridinium chloridecontaining mouthwashes. j clin dent. 2011; 22: 200-3. 13. he s, wei y, fan x, hu d, sreenivasan pk. a clinical study to assess the 12-hour antimicrobial effects of cetylpyridinium chloride mouthwashes on supragingival plaque bacteria. j clin dent. 2011; 22: 195-9. 14. takahashi n, washio j. metabolomic effects of xylitol and fluoride on plaque biofilm in vivo. j dent res. 2011; 90: 1463-8. 15. macek md. xylitol-based candies and lozenges may reduce caries on permanent teeth. j evid based dent pract. 2012; 12: 71-3. 16. tanzer jm. xylitol chewing gum and dental caries. int dent j. 1995; 45: 65-76. 17. hildebrant gh, sparks bs. maintaining mutans streptococci suppression with xylitol chewing gum. j am dent assoc. 2002; 131: 909-16. 18. nogueira-filho gr, toledo s, cury ja. effect of 3 dentifrices containing triclosan and various additives. an experimental gingivitis study. j clin priodontol. 2000; 27: 494-8. 19. nogueira-filho gr, duarte pm, toledo s, tabchoury cp, cury ja. effect of triclosan dentifrices on mouth volatile sulphur compounds and dental plaque trypsin-like activity during experimental gingivitis development. j clin periodontol. 2002; 29: 1059-64. 20. ainamo j, bay i. problems and proposals for recording gingivitis and plaque. int dent j. 1975; 25: 229-35. 21. mühlemann hr, son s. the effect of human supragingival calculus formation of acetohydroxamic acid. helv odontol acta. 1971; 15: suppl 7: 158-9. 22. lotufo r, calil cm, feng hs, sekiguchi rt, stewart b, de vizio w et al. clinical investigation of a commercial mouthrinse containing 0. 05% cetylpyridinium chloride in preventing dental plaque. j clin dent. 2009; 20: 50-4. 23. hernandez-cott pl, boneta ae, stewart b, devizio w, proskin hm. clinical investigation of the efficacy of a commercial mouthrinse containing 0.05% cetylpyridinium chloride in reducing dental plaque. j clin dent. 2009; 20: 39-44. 24. silva mfa, santos nb, stewart b, de vizio w, proskin hm. a clinical investigation of the efficacy of a commercial mouthrinse containing 0.05% cetylpyridinium chloride to control established dental plaque and gingivitis. j clin dent. 2009; 20: 55-61. 25. rane vs, davison g, borutell p, gallitschke n. 1092 changes in gumline plaque pathogenicity: 3 weeks cpc treatment. j dent res. 2006; a: 626. 26. albert-kszely a, pjetursson be, salvi ge, witt j, hamilton a, persson gr et al. comparison of the effects of cetylpyridinium chloride with an essential oil mouth rinse on dental plaque and gingivitis – a six – month randomized controlled clinical trial. j clin periodontol. 2007; 34: 658-67. 27. rioboo m, garcía v, serrano j, o’connor a, herrera d, sanz m. clinical and microbiological efficacy of an antimicrobial mouth rinse containing 0.05% cetylpyridinium chloride in patients with gingivitis. int j dent hyg. 2012; 10: 98-106. 28. maguire a, rugg-gunn aj. xylitol and caries prevention – is it a magic bullet?. br dent j. 2003; 194: 429-36. 29. söderling em. xylitol, mutans streptococci, and dental plaque. adv dent res. 2009; 21: 74-8. 30. badet c, furinga a, thebaud n. effect of xylitol on an in vitro model of oral biofilm. oral health prev dent. 2008; 6: 337-41. effect of cetylpyridinium chloride with xylitol on the formation of biofilm and development of gingivitis braz j oral sci. 11(3):392-395 429 too many requests error 429 too many requests too many requests guru meditation: xid: 67821623 varnish cache server oral sciences n3 original article braz j oral sci. 8(4):171-174 evaluation of the in vitro biocompatibility of orthodontic elastics correspondence to: mônica tirre de souza araújo universidade federal do rio de janeiro ufrj faculdade de odontologia departamento de ortodontia av. prof. rodolpho paulo rocco325, ilha do fundão rio de janeiro rj brasil cep: 21941-617 e-mail: monicatirre@gmail.com abstract aim: latex has been extensively used in orthodontics since the advent of the specialty. natural latex does not fall into the category of materials known to be entirely inoffensive. the objective of the present in vitro study is to test the hypothesis that there is no difference in the cytotoxicity between natural latex elastics of different colors. methods: the present article compared different latex intra-oral elastics (5/16 = 7.9 mm, mean load). the samples were divided into six groups of 15 elastics according to their manufacturer: groups n, y, v, r, g and p (uniden, natural latex elastics and colored elastics, namely, yellow, violet, red, green and pink, respectively). cytotoxicity assays were performed by using cell culture medium containing cells from mouse fibroblast cell line l929. the cytotoxicity was evaluated by using the “dye-uptake” test, which was employed at two different moments (1 and 24 h). data were compared by anova and tukey’s test (p < 0.05). results: there was statistically significant difference (p < 0.05) between the groups n, y, v, r, g, p and the cell control at 1 h. after 24 h, a decrease in cell viability was observed in all groups. there was statistically significant difference (p < 0.05) between all test elastics groups and the cell control at 24 h. no statistically significant difference (p >0.05) was found among the test elastics groups at 24 h. conclusion: latex elastics from natural, yellow, violet and red colors induced a lowest amount of cell lysis compared to the elastics green and pink colors at 1 h, all latex elastics were found to be highly cytotoxic, regardless of their color at 24 h. keywords: cytotoxicity, elastics, biocompatibility, orthodontics. introduction latex elastic has been extensively used in orthodontics since the advent of the specialty. however, natural latex does not fall into the category of materials known to be entirely inoffensive1-2. allergy caused by latex proteins has been well documented3, including immediate hypersensitivity reactions4. among the allergic reactions caused by orthodontic elastics, swelling and stomatitis, erythematous oral lesions, respiratory reactions, and even anaphylactic shock, the most severe form of allergy5-6, can be cited. latex allergy occurs in 3-17% of the cases7. prevulcanized latex is produced by mixing pure natural latex, which has the highest molecular weight8, with stabilizers such as zinc oxide and chemically vulcanized materials. the resulting mixture is then heated until 70o c9. although zinc is known to be neurotoxic10, the amount released by orthodontic elastics can be ingested as research studies show no evidence of harm11. anti-ozone and anti-oxidant agents are also added to latex during the manufacture of orthodontic elastics8. this process has the advantage of producing latex with higher mechanical properties, thus increasing its strength and elasticity9,11. the use of cell culture medium for testing the toxicity of dental products is a valid way of understanding the biological behavior of such materials1. the objective of the present in vitro study was to test the hypothesis that there is no difference in the cytotoxicity between natural latex elastics of different colors. braz j oral sci. october/december 2009 volume 8, number 4 received for publication: may 27, 2009 accepted: november 11, 2009 matheus melo pithon1; rogério lacerda dos santos1; fernanda otaviano martins2; maria teresa villela romanos3; mônica tirre de souza araújo4 1 dds, ms, phd student in orthodontics, federal university of rio de janeiro, brazil 2 bs, federal university of rio de janeiro, brazil 3 bs, assistant professor of microbiology, federal university of rio de janeiro, brazil 4 dds, ms, phd, assistant professor of orthodontics, federal university of rio de janeiro, brazil 172 braz j oral sci. 8(4):171-174 material and methods latex intra-oral elastics of different colors (5/16" = 7.9 mm, mean load), were selected for studying their cytotoxicity in cell culture (table 1). the samples were divided into six groups of 15 elastics according to their manufacturer: groups n, y, v, r, g and p (natural latex elastics and colored elastics, namely, yellow, violet, red, green and pink, respectively, uniden, sorocaba, sp, brazil) (figure 1). groups trademark main composition c o l o r reference number n uniden natural látex natural 000-1204 y uniden natural látex yellow 000-1206 v uniden natural látex violet 000-1206 r uniden natural látex red 000-1206 g uniden natural látex green 000-1206 p uniden natural latex pink 000-1206 c+ tween 80 (polyoxyethylene-20-sorbitan, sigma, st. louis, mo, usa) cpbs solution (phosphate-buffered saline, cultilab, campinas, sp, brazil) table 1. elastic and control groups used for the assays. fig. 1: latex intraoral elastics evaluated in this study: n, y, v, r, g and p (uniden, natural latex elastics and colored elastics, namely, yellow, violet, red, green and pink, respectively). all samples had recent manufacturing dates, from the same production lot and came in sealed plastic packages. the elastics of experimental and control groups had their both sides previously sterilized with ultraviolet light (labconco, kansas city, mo, usa) during 30 minutes12. to verify the cell response to extreme situations, other three groups were included in the study: group cc (cell control), consisting of cells not exposed to any material; group c+ (positive control), consisting of tween 80 and group c(negative control), consisting of pbs solution in contact with the cells (table 1). cells from mouse fibroblast cell line l929 (american type culture collection atcc, rockville, md) were cultured in eagles’ minimum essential medium (mem; cultilab, campinas, sp, brazil) by adding 0.03 mg/ml of glutamine (sigma, st. louis, mo, usa), 50 µg/ml of garamicine (schering plough, kenilworth, new jersey, usa), 2.5 mg/ ml of fungizone (bristol-myers-squibb, new york, usa), 0.25% sodium bicarbonate solution (merck, darmstadt, germany), 10 mm of hepes (sigma), and 10% bovine fetal serum (cultilab) for growth medium or no bovine fetal serum for maintenance medium only. next, the cell culture medium was incubated at 37oc for 48 h. the method for evaluating the cytotoxicity was the “dye-uptake”13 test. this method is based on neutral red dye incorporated into live cells. it was used in this experiment only at two periods of evaluation: 1 and 24 h. the 1-h period represents the maintenance of the elastic in the cell culture medium for 1 h after removal, whereas the 24-h period represents the maintenance of the elastic in the cell culture medium for 24 h after removal. dye-uptake volumes of 100 µl of l-929 cells were distributed into 96-well microplates. after 48 h, the growth medium was replaced with 100 µl of mem obtained following incubation in the different types of elastics and positive and negative control at 1 and 24 h. positive and negative control groups consisted of culture medium put in contact with 100 µl of tween 80 and 100 µl pbs solution, respectively. after 24-h incubation, 100 µl of 0.01% neutral red dye (sigma) were added to the culture medium in the 96-well microplates, which were incubated again for 3 h at 37oc so that the red dye could penetrate the live cells. following this period of time, 100 µl of 4% formaldehyde solution (vetec, rio de janeiro, rj, brazil) in pbs (130 mm of nacl; 2 mm of kcl; 6 mm of na 2 hpo 4 2 h 2 o; 1 mm of k 2 hpo 4 1 mm; ph 7.2) were added in order to promote cell attachment to the plate. after 5 minutes, 100 µl of 1% acetic acid (vetec) and 50% methanol (vetec) were added in order to remove the dye. after 20 minutes, a spectrophotometer (biotek instruments, winooski, vt, usa) at 492 nm wavelength (λ492 nm) was used for data reading. this test was repeated 3 different times. dat a w ere co mpared by anova, an d tukey ’s multipl e comparison test was used for identifying differences between the groups. significance level was set at p<0.05. results the results showed significant difference (p< 0.05) between the groups n, y, v, r, g, p and the cell control at 1 h (table 2). group g produced the lowest value (34.8% ± 2.9%) and group n produced the more viability (49.8% ± 10.5%), whereas the viability of the tween 80 was 8.8% ± 10.5% (table 2). the results showed statistically significant differences (p<0.05) between the groups n, y, v, r and the groups g and p at 1 h. no significant difference was found between the groups n and y, n and v, n and r, y and v, y and v, y and r, v and r, and g and p tested (p>0.05) at 1 h (table 2). evaluation of the in vitro biocompatibility of orthodontic elastics 173 braz j oral sci. 8(4):171-174 after 24 h, a decrease in cell viability was observed in all groups. viability ranged from 15.9% to 18.9%, relative to the cell control (table 2). the lowest viability (15.9% ± 5.2%) corresponded to group g, whereas the viability of the tween 80 (positive cytotoxicity control) was 9.0% ± 12.9%. the results showed statistically significant differences (p<0.05) between all test elastics groups and the cell control at 24 h. on the other hand, no statistically significant difference (p>0.05) was found among the test elastics groups at 24 h (table 2). discussion although case reports on latex allergy is not so frequently seen in the literature, allergic reactions have been relatively prevalent as latexbased products become commercially available. most of the allergic reactions14 have been related to the use of orthodontic elastics15, which is characterized by presence of small vesicles or acute edema and complaints of itching and burning. the most serious consequence of natural rubber latex allergy commonly takes place during the mucosal absorption of natural rubber latex proteins during intraoperative medical or dental procedures when health care workers or others already sensitized become patients16. because natural latex rubber has been increasingly used as dental material, many cytotoxicity issues have been reported as well17. preservatives such as sulfur and zinc oxide as well as antioxidants such as di-thio-carbohydrates, n-nitrosodibutylamine, and nnitrosopiperidine are all known to be cytotoxic substances18. allergy to natural latex occurs because of the presence of many types of proteins, and the powder covering the orthodontic elastics works as a transporter for these proteins. therefore, the development of non-latex elastics has become increasingly important for clinical usage17. as sterilization is a prerequisite for cytotoxicity assay, ultraviolet light was used in the present study for sterilizing both sides of the elastics1,12 during 30 minutes. all the elastics were found to have the same color and malleability following uv light sterilization. it was demonstrated by franz et al.19 and schedle et al.20 that l929 mouse fibroblasts show comparable results to primary human gingival fibroblasts and therefore might represent a model for gingival toxicity in vitro21-22. the percentage of viable cells was obtained by comparing the mean optical density (od) of control cells (no contact with the materials) with that of cell cultures put in contact with different elastics, resulting in 50% toxicity for the cell cultures (cc 50 ) (table 2). evidence of this cytotoxic feature was shown following exposition of the elastics to cell culture medium. it was used in this experiment only two times of evaluation 1 and 24 h, a time that, usually these elastics are changed to each 24 h for the patient. natural latex elastics from all colors induced a greater amount of cell lysis at 24 h compared to the time of 1 h. latex elastics from natural, yellow, violet and red colors induced a lowest amount of cell lysis compared to the elastics green and pink colors at 1 h. after 24 h, a lowest viability was observed in all groups regardless of their color, which is accordance with the findings of santos, et al.12,23. this suggests a greater release of toxic ingredients at 24 h, due to a possible latex degradation and release of allergenic proteins, which was not shown in 1 h. holmes, et al.2 have verified whether the colorants used in the fabrication of colored latex could have some toxic effect. their results showed that these colorants had low toxicity. however, such an effect is clinically inoffensive. variations occur in the composition of the latex elastics and this could explain the different results between the elastics. according to schmalz1, the great danger is that potentially cytotoxic intra-oral elastics could release substances that might be ingested by the patient over time, thus causing diseases resulting from a cumulative effect. it is known that latex is not entirely biocompatible as it may interact with foods7,24 and medications25. as these materials are widely used in clinical orthodontics, care regarding the cytotoxicity of orthodontic elastics should be taken, mainly with regard to intraoral elastics as they have a very close contact with gingival and mucosa. thus, clinically proven biocompatible materials should be used whenever possible. it is important for the practitioner to know how to manage patients presenting latex allergy and how to deal with this problem26. the safety biocompatibility of silicone has been well proved through the use of mouth guards in dentistry27. an alternative for patients with allergy to latex is to use non-latex elastics, which can be used in orthodontics without jeopardizing the orthodontic treatment28. it may be concluded that natural, yellow, violet and red latex orthodontic elastics induced less cell lysis than green and pink elastics at 1 h. at 24 h, latex elastics were found to be highly cytotoxic, regardless of their color. evaluation of the in vitro biocompatibility of orthodontic elastics c c 0.874ª 0.910 0.062 100.0 0.610ª 0.642 0.049 100.0 c0.866 0.898 0.007 99.1 0.603 0.634 0.012 98.9 c+ 0.076 0.084 0.008 8.8 0.054 0.058 0.007 9.0 n 0.435b 0.458 0.046 49.8 0.115b 0.134 0.008 18.9 y 0.403cb 0.442 0.042 46.2 0.102b 0.127 0.014 16.8 v 0.434dbc 0.452 0.013 49.7 0.106b 0.129 0.007 17.5 r 0.379ebcd 0.402 0.011 43.4 0.101b 0.119 0.006 16.7 g 0.304f 0.338 0.009 34.8 0.096b 0.108 0.005 15.9 p 0.329gf 0.346 0.008 35.7 0.100b 0.114 0.007 16.4 mean median s. d. viable cells (%)mean median s. d. viable cells (%) 1 h 24 h time groups table 2. descriptive statistics for optical density of latex elastics. n=15.values followed by same letters are not significantly different (p>0.05) for the same time. sd: standard deviation. 174 braz j oral sci. 8(4):171-174 references 1. schmalz g. use of cell cultures for toxicity testing of dental materials— advantages and limitations. j dent. 1994; 22 suppl 2: s6-11. 2. holmes j, barker mk, walley ek, tuncay oc. cytotoxicity of orthodontic elastics. am j orthod dentofacial orthop. 1993; 104: 188-91. 3. palosuo t, alenius h, turjanmaa k. quantitation of latex allergens. methods. 2002; 27: 52-8. 4. wakelin sh, white ir. natural rubber latex allergy. clin exp dermatol. 1999; 24: 245-8. 5. everett fg, hice tl. contact stomatitis resulting from the use of orthodontic rubber elastics: report of case. j am dent assoc. 1974; 88: 1030-1. 6. tomazic vj, withrow tj, fisher br, dillard sf. latex-associated allergies and anaphylactic reactions. clin immunol immunopathol. 1992; 64: 89-97. 7. turjanmaa k, alenius h, makinen-kiljunen s, reunala t, palosuo t. natural rubber latex allergy. allergy. 1996; 51: 593-602. 8. weiss me, hirshman ca. latex allergy. can j anaesth. 1992; 39: 528-32. 9. perrella fw, gaspari aa. natural rubber latex protein reduction with an emphasis on enzyme treatment. methods. 2002; 27: 77-86. 10. lobner d, asrari m. neurotoxicity of dental amalgam is mediated by zinc. j dent res. 2003; 82: 243-6. 11. hanson m, lobner d. in vitro neuronal cytotoxicity of latex and nonlatex orthodontic elastics. am j orthod dentofacial orthop. 2004; 126: 65-70. 12. santos rl, pithon mm, oliveira mv, mendes gs, romanos mtv, ruellas aco. cytotoxicity of intraoral orthodontic elastics. braz j oral sci. 2008; 24: 1520-5. 13. neyndorff hc, bartel dl, tufaro f, levy jg. development of a model to demonstrate photosensitizer-mediated viral inactivation in blood. transfusion. 1990; 30: 485-90. 14. snyder ha, settle s. the rise in latex allergy: implications for the dentist. j am dent assoc. 1994; 125: 1089-97. 15. neiburger ej. a case of possible latex allergy. j clin orthod. 1991; 25: 559-60. 16. sussman gl, beezhold dh, liss g. latex allergy: historical perspective. methods. 2002; 27: 3-9. 17. hwang cj, cha jy. mechanical and biological comparison of latex and silicone rubber bands. am j orthod dentofacial orthop. 2003; 124: 379-86. 18. fiddler w, pensabene j, sphon j, andrzejewski d. nitrosamines in rubber bands used for orthodontic purposes. food chem toxicol. 1992; 30: 325-6. 19. franz a, konig f, skolka a, sperr w, bauer p, lucas t et al. cytotoxicity of resin composites as a function of interface area. dent mater. 2007; 23: 1438-46. 20. schedle a, samorapoompichit p, rausch-fan xh, franz a, fureder w, sperr wr et al. response of l-929 fibroblasts, human gingival fibroblasts, and human tissue mast cells to various metal cations. j dent res. 1995; 74: 1513-20. 21. franz a, konig f, lucas t, watts dc, schedle a. cytotoxic effects of dental bonding substances as a function of degree of conversion. dent mater. 2009; 25: 232-9. 22. schmid-schwap m, franz a, konig f, bristela m, lucas t, piehslinger e et al. cytotoxicity of four categories of dental cements. dent mater. 2009; 25: 360-8. 23. santos rl, pithon mm, mendes gs, romanos mtv, ruellas aco. cytotoxicity of intermaxillary orthodontic elastics of different colors: an in vitro study. j appl oral sci. 2009; 4: 326-9. 24. carey ab, cornish k, schrank p, ward b, simon r. cross-reactivity of alternate plant sources of latex in subjects with systemic ige-mediated sensitivity to hevea brasiliensis latex. ann allergy asthma immunol. 1995; 74: 317-20. 25. towse a, o’brien m, twarog fj, braimon j, moses ac. local reaction secondary to insulin injection. a potential role for latex antigens in insulin vials and syringes. diabetes care. 1995; 18: 1195-7. 26. hain ma, longman lp, field ea, harrison je. natural rubber latex allergy: implications for the orthodontist. j orthod. 2007; 34: 6-11. 27. chauvel-lebret dj, pellen-mussi p. evaluation of the in vitro biocompatibility of various elastomers. biomaterials. 1991; 20: 291-9. 28. gandini p, gennai r, bertoncini c, massironi s. experimental evaluation of latexfree orthodontic elastics’ behaviour in dynamics. prog orthod. 2007; 8: 88-99. evaluation of the in vitro biocompatibility of orthodontic elastics oral sciences n3 braz j oral sci. 10(3):175-179 original article braz j oral sci. july | september 2011 volume 10, number 3 received for publication: march 31, 2010 accepted: july 13, 2011 dimensional accuracy of stone casts made by a monophase impression technique using different elastomeric impression materials rafael pino vitti1, lourenço correr-sobrinho2, mário alexandre coelho sinhoreti 3 1msd, graduate student, dental materials division, department of restorative dentistry, piracicaba dental school, university of campinas, piracicaba, sp, brazil 2msd, dds, phd, full professor, dental materials division, department of restorative dentistry, piracicaba dental school, university of campinas, piracicaba, sp, brazil 3msd, dds, full professor, dental materials division, department of restorative dentistry, piracicaba dental school, university of campinas, piracicaba, sp, brazil correspondence to: mário alexandre coelho sinhoreti piracicaba dental school dental materials department av. limeira, 901. p.o. box 52. piracicaba sp brazil zip code 13414-903 phone: 55-19-2106-5348 e-mail: sinhoret@fop.unicamp.br abstract impression taking is a critical step in the process of producing successful crowns and fixed partial dentures in oral rehabilitation, and the impression material is an important factor related to clinical success. aim: the aim of this in vitro study was to assess and compare the dimensional accuracy of stone casts made from a monophase technique using 10 elastomeric impression materials. methods: first, a stainless steel model with reference points in the teeth 33, 43, 37, and 47 was used to obtain the impressions. the distances were measured among teeth 33-43, 37-47, 33-37, and 43-47. for the impression technique, acrylic resin trays were made with an internal relief of approximately 2 mm. specific adhesives for each material were used in the custom trays. tray detachment movement was standardized by pneumatic equipment. after the impression procedures and obtaining of samples, the stone casts were observed in a measuring microscope at 30x magnification. data recorded for each distance were analyzed statistically by one-way analysis of variance and tukey’s test at 5% significance level. results: stone casts made with elastomeric impression materials showed statistically significant (p<0.05) differences when the dimensional accuracy values were compared. the order for the highest to lowest accuracy for the types of impression materials was as follws: polyvinylsiloxane (pvs), polyether, polysulfide and polydimethylsiloxane (pdms). conclusions: pvs were the most dimensionally accurate impression materials, and the pdms showed the worst results of dimensional accuracy. keywords: impression technique, dimensional accuracy, dental materials. introduction high accuracy impression materials (elastomeric impression materials) appeared in dentistry in the 1950s1-2. nowadays, four different elastomeric impression materials are used namely polysulfide, polyether, polydimethylsiloxane (pdms) and polyvinylsiloxane (pvs), and each one of them has specific chemical reactions and setting characteristics. the elastomeric impression materials made with a silicone base are found in four different viscosities: putty (type 0), heavy-body (type 1), regular or medium-body (type 2) and light-body (type 3). polyether and polysulfide are already available in all consistencies, except putty1-6. the elastomeric impression materials possess elastic behavior after the set reaction; in other words, they resemble an rubber7-8. these materials are polymers 176 braz j oral sci. 10(3):175-179 brand names manufacturers batch number clonage (pdms) dfl, rio de janeiro, rj, brazil 08010080 oranwash l (pdms) zhermack, rovigo, italy 107165 xantopren vl plus (pdms) heraeus kulzer gmbh, hanau, germany r330335 silon 2 aps (pdms) dentsply ind. e com. ltda., petrópolis, rj, brazil 349629 futura ad (pvs) dfl, rio de janeiro, rj, brazil 462216 express regular set (pvs) 3m unitek, monrovia, ca, usa 387100 elite hd+ normal setting (pvs) zhermack, rovigo, italy 110577 aquasil ultra regular set (pvs) dentsply gmbh, konstanz, germany 0811003044 impregum soft (polyether) 3m unitek, monrovia, ca, usa 1026300114 permlastic (polysulfide) kerr corporation, romulus, mi, usa 0-1088 rubber base adhesive kerr corporation, romulus, mi, usa 8-1099 polyether adhesive 3m unitek, monrovia, ca, usa 0003061 universal adhesive heraeus kulzer gmbh, hanau, germany 280023 table 1 materials (brand names) used and manufacturers. formed by large molecular chains. when tension is applied, these chains are uncoiled, elastically recovering after the load removal1,6. impression materials should reproduce hard and soft tissues in order to obtain biologically, mechanically, functionally and esthetically acceptable restorations9-10. however, dimensional changes in the molds inherent to the impression materials can occur, such as: wettability, handling properties11, viscosity and thickness of the material existing between the oral structures and tray, fixation method of impression material to tray12-13, time elapsed for cast pouring13, material’s hydrophilicity11, byproduct loss, polymerization shrinkage, thermal shrinkage due the temperature change (from the mouth to room temperature), incomplete elastic recovery, and, in some cases, soak1. other factors, such as tray selection, impression technique and preparation design can also influence the impression quality14. there are several brand names and categories of impression materials that can be used in dentistry. dimensional stability of impression materials has been widely discussed in the dental literature10,15, revealing significant differences in the properties of products of the same type. some dentists still finds unclear which category of impression materials is best for clinical uses to obtain success of prosthodontic procedures10. the use of an appropriate impression material can reduce considerably the likelihood of inaccuracies in the molds7. new materials have been developed and subjected to continuous modifications with the aim of improving the impression quality, but these modifications do not gua rantee maintenance of their properties16. then, it is important to evaluate the dimensional accuracy of recently developed materials. the aim of this in vitro study was to evaluate the dimensional accuracy of stone casts made with different elastomeric impression materials by a monophase impression technique. the hypothesis tested in the present study was that there are differences on dimensional accuracy in stone casts among the elastomeric impression materials. material and methods table 1 shows the materials used in the study. stainless steel model evaluation at first, a stainless steel model of the mandibular arch partially edentulous with reference points in the teeth 37, 47, 33, and 43 was made17. the transversal distances among teeth 33-43 and 37-47, and anteroposterior among teeth 3337 and 43-47 (figure 1), were measured by a measuring microscope at 30x magnification (olympus® measuring microscope stm, olympus optical co., japan). fig. 1. distances considered in the measurements. monophase impression technique all elastomeric impression materials were handled following the manufacturers’ instructions, and impressions procedures were made in a room with temperature and relative humidity controlled (23°c ± 2°c and 50% ± 10%)2,4,18. custom acrylic resin trays (vipi flash, vipi, pirassununga, sp, brazil) were made with an internal relief of approximately 2 mm18-19 to provide an adequate and standard thickness to the impression material20-21. a 2-mm-thick polypropylene spacer was used on the stainless steel model. then, the acrylic resin was placed on the set polypropylene dimensional accuracy of stone casts made by a monophase impression technique using different elastomeric impression materials braz j oral sci. 10(3):175-179 177 spacer/model to obtain the custom trays with 2 mm of relief. initially, an adhesive layer was applied to each elastomeric impression material, throughout the internal surface of all trays, which left on a bench for 5 min for adhesive drying18,22. afterwards, the light-body elastomeric impression materials were handled following the manufacturer’s instructions and used to cover the whole internal surface of the tray, being careful with possible excesses. the set tray/impression material was positioned and seated manually on the stainless steel model, from posterior to anterior. after the setting time recommended by the manufacturer, the tray was attached to the pneumatic equipment and detached from stainless steel model by a movement standardized, in order to avoid distortions in the mold (figure 2)18 due to material expansion that occur by tension release after the impression removal20. the detachment pressure was 3 bar. this procedure was repeated five times for each impression material brand name (n=5). following the manufacturers’ instructions, a ratio of 150 g of dental stone type iv (durone iv, dentsply, são paulo, sp, brazil) and 28.5 ml of water was used. each of five elastomeric casts was poured after 30 min of the tray detachment, in order to allow a correct elastic recovery. in the same way as in the stainless steel model evaluation, three measurements were made by a single calibrated operator in each one of the four distances among the teeth (33-43, 33-37, 43-47, and 37-47), and the respective means were recorded. statistical analysis data recorded for each distance were analyzed statistically by one-way analysis of variance and tukey’s test at 5% significance level. results tables 2 and 3 show that there was a significant difference in the dimensional change means when the impression materials were compared for all distances: 33-43 fig. 2. mold removal with single movement. after setting of the impression material, the tray was attached to the pneumatic equipment and the device actuated. then, the tray was detached from the stainless steel model by upright movement. (p<0.0001), 33-37 (p<0.0001), 43-47 (p<0.0001) and 3747. in general, the pvs showed the best results, followed by polyether. on other hand, polysulfide and pdms had the worst results. the stone casts made with express presented the smallest dimensional change means among all impression materials. silon 2 aps and clonage produced casts with the largest dimensional change means. discussion the hypothesis tested in the present study was accepted, as the results showed that, in general, pvs provided greater accuracy in the stone casts and greater reliability in impression structures than polysulfide, polyether and pdms. these results can be attributed to the excellent physical and mechanical properties of this type of material, such as good dimensional stability and elastic recovery (approximately 99%)10, in addition to an appropriate tear strength7. pvs materials possess a set reaction by the terminal group ethylene or vinyl with hydride groups1-2, without the formation of byproducts and with non-occurring impression material shrinkage, allowing that these materials stay dimensionally stable after impression removal1,23. in general, pvs materials showed results that did not differ significantly among themselves. the small differences found in the dimensional accuracy among the pvs materials table 2 dimensional change means and sd (%) of stone casts made with the elastomeric impression materials (transversal distances). means followed by different lowercase letters differ significantly (p<0.05) -0.29 (0.02) a -0.20 (0.02) b -0.19 (0.03) bc -0.17 (0.01) bcd -0.15 (0.01) cd -0.13 (0.01) d -0.04 (0.01) e -0.03 (0.01) e -0.03 (0.01) e -0.01 (0.01) f 37-47 distance silon 2 aps clonage permlastic xantopren vl oranwash l impregum futura ad elite hd+ aquasil ultra express 33-43 distance silon 2 aps clonage permlastic xantopren vl oranwash l impregum futura ad elite hd+ aquasil ultra express -0.33 (0.02) a -0.23 (0.02) ab -0.21 (0.03) b -0.20 (0.03) b -0.19 (0.02) b -0.17 (0.02) b -0.06 (0.02) c -0.06 (0.02) c -0.05 (0.02) cd -0.02 (0.01) d -0.43 (0.02) a -0.42 (0.03) a -0.38 (0.01) ab -0.37 (0.02) ab -0.30 (0.01) bc -0.24 (0.02) cd -0.19 (0.02) de -0.18 (0.03) de -0.17 (0.01) de -0.13 (0.02) e -0.45 (0.03) a -0.44 (0.03) a -0.40 (0.02) ab -0.39 (0.03) ab -0.32 (0.02) bc -0.26 (0.02) cd -0.25 (0.02) cd -0.23 (0.04) cd -0.22 (0.02) d -0.18 (0.02) d 43-47 distance silon 2 aps clonage oranwash l xantopren vl permlastic impregum elite hd+ futura ad aquasil ultra express 33-37 distance silon 2 aps clonage oranwash l xantopren vl permlastic impregum futura ad elite hd+ aquasil ultra express table 3 dimensional change means and sd (%) of stone casts made with the elastomeric impression materials (anteroposterior distances). means followed by different lowercase letters differ significantly (p<0.05) dimensional accuracy of stone casts made by a monophase impression technique using different elastomeric impression materials 178 braz j oral sci. 10(3):175-179 can be attributed to the variability in the composition of each brand name, mainly in the matrix-filler ratio, which can provide the material with different levels of shrinkage polymerization and elastic recovery4,24. the stone casts made with the polyether material behaved, statistically, in the same manner as stone casts made from pvs in the anteroposterior measures (43-47 and 33-37) and as the pdms oranwash l (zhermack, rovigo, italy) and xantopren vl (heraeus kulzer gmbh, hanau, germany) in the distance 37-47. for the other transversal distance, 33-43, the polyether did not differ among oranwash l, xantopren vl, and permlastic (kerr corporation, michigan). these results differ from those found results in other studies7,25, in which polyether presented better dimensional accuracy than condensation silicone-based materials and polysulfide. however, these results corroborate those of another study9 in which polyether had an intermediate behavior between pdms and pvs. a possible explanation for these conflicting results is that the behavior of this material is easily influenced by the room humidity as this material has a hydrophilic nature1,6. in laboratorial studies, the material stays in a dry room and does not absorb water from the room. it is speculated that, under clinical conditions, water sorption could compensate partly for the shrinkage observed in the laboratorial tests, as seen in this study. besides, the polyether has inferior tear strength than pvs, so this may avoid its indication for use in interproximal and subgingival prepared tooth areas26. pdms materials, specifically silon 2 aps and clonage, were the materials that created stone casts with the largest dimensional change values, as found in other studies26-27. the worst performance for that material class is due to the continuous polymerization that occur after setting of the impression material and is more accentuated than in other materials, which causes the evaporation of volatile byproducts, such as ethyl alcohol, and affects the dimensional stability and the accuracy of the pdms2-3,26,28. polysulfide polymerization occurs by the condensation reaction between the lead oxide and the pending and terminal groups with the mercaptan groups2,6. in that reaction, as in pdms polymerization, there is byproduct formation (water), which can evaporate and distort the mold. therefore, polysulfide had similar behavior to that of pdms. furthermore, the elastic recovery of this material is smaller and more incomplete than in other elastomeric materials2. other factors, such as short handling time, prolongable time of polymerization, high sensitivity to temperature and humidity and higher tear strength can affect the dimensional accuracy26. the iso 4823 specification admits that dimensional changes less than 1.5% to elastomeric impression materials are clinically acceptable. within the limitations of this study, despite the statistical differences found among the elastomeric impression materials, when poured in 30 min after impressions in a room with temperature and relative humidity controlled, all the stone casts made with those impression materials showed satisfactory dimensional accuracy. future studies are needed to verify the use of the elastomeric impression materials with others impression techniques and the clinical relevance. the choice of a product for a particular clinical application should be based on material’s properties rather than on the type and class of impression material. the dental professionals should be informed about the advantages and disadvantages of each material to adequately use them in clinical practice and provide adequate clinical longevity to the prostheses. acknowledgements the authors thank capes for the financial support. references 1. shen c. impression materials in: anusavice kj. dental materials. 11th ed. rio de janeiro: elsevier; 2005. p.193-238. 2. giordano r 2nd. impression materials: basic properties. gen dent. 2000; 48: 510-6. 3. international organization for standardization. dentistry. elastomeric impression materials. n° 4823:2000(e); third edition. 4. ciesco jn, malone wfp, sandrik jl, mazur b. comparasion of elastomeric impression materials used in fixed prosthodontics. j prosthet dent. 1981; 45: 89-94. 5. braga as, braga srs, catirse abceb, vaz lg, mollo júnior, fa. quantitative analysis of lead in polysulfide-based impression material. mater res. 2007; 10: 161-3. 6. craig rg. restorative dental materials. 9th ed. saint louis : mosby; 1993. p.298. 7. lacy am, fukui h, bellman t, jendresen md. time-dependent accuracy of elastomer impression materials part ii: polyether, polysulfides, polyvinylsiloxane. j prosthet dent. 1981; 45: 329-333. 8. stackhouse junior ja. the accuracy of stones die made from rubber impression materials. j prosthet dent. 1970; 24: 377-86. 9. perakis n, belser uc, magne p. final impressions: a review of material properties and description of a current technique. int j periodontics restorative dent. 2004; 24: 109-17. 10. christensen gj. what category of impression material is best for your practice? j am dent assoc. 1997; 128: 1026-8. 11. pratten dh, novetsky m. detail reproduction of soft tissue: a comparison of impression materials. j prosthet dent. 1991; 65:188-91. 12. johnson gh, craig rg. accuracy of addition silicones as a function of technique. j prosthet dent. 1986; 55: 197-203. 13. reisbick mh. effect of viscosity on the accuracy and stability of elastic impression materials. j dent res. 1973; 52: 407-17. 14. nam j, raigrodski aj, townsend j, lepe x, mancl la. assessment of preference of mixing techniques and duration of mixing and tray loading for two viscosities of vinyl polysiloxane material. j prosthet dent. 2007; 97: 12-7. 15. craig rg. review of dental impression materials. adv dent res. 1988; 2: 51-64. 16. franco eb, cunha lf, benetti ar. effect of storage period on the accuracy of elastomeric impressions. j appl oral sci. 2007; 15: 195-8. 17. yoder jl, thayer ke. rubber-base impression technique for fixed partial dentures. j prosthet dent. 1962; 12: 339-46. 18. gilmore wh, schnell rj, phillips rw. factors influencing the accuracy of silicone impression materials. j prosthet dent. 1959; 9: 304-14. 19. al-bakri ia, hussey d, al-omari, wm. the dimensional accuracy of four impression techniques with the use of addition silicone impression materials. j clin dent. 2007; 18: 29-33. 20. faria acl, rodrigues rcs, macedo ap, mattos mgc, ribeiro rf. accuracy of stone casts obtained by different impression materials. braz oral res. 2008; 22: 293-8. dimensional accuracy of stone casts made by a monophase impression technique using different elastomeric impression materials 179 braz j oral sci. 10(3):175-179 21. donovan te, chee ww. a review of contemporary impression materials and techniques. dent clin north am. 2004; 48: 445-70. 22. custer f, updegrove l, ward m. accuracy and dimensional stability of a silicone rubber base impression material. j prosthet dent. 1964; 14: 111521. 23. hung sh, purk jh, tira de, eick jd. accuracy of one-step versus twostep putty wash addition silicone impression technique. j prosthet dent. 1992; 67: 583-9. 24. carlo hl, fonseca rb, soares, cj, correr ab, correr-sobrinho l, sinhoreti mac. inorganic particle analysis of dental impression elastomers. braz dent j. 2010; 21: 520-7. 25. shah s, sundaram g, bartlett d, sherriff m. the use of a 3d laser scanner using superimpositional software to assess the accuracy of impression techniques. j dent. 2004; 32: 653-8. 26. pereira jr, murata ky, valle ac, ghizoni js, shiratori fk. linear dimensional changes in plaster die models using different elastomeric materials. braz oral res. 2010; 24: 336-41. 27. mccabe jf, wilson hj. addition cure silicone rubber impression materials. br dent j. 1978; 145: 17-20. 28. camarinha smlb, pardini lc, garcia lfr, consani s, pires-de-souza fcp. cast metal core adaptation using two impression materials and intracanal techniques. braz j oral sci. 2009; 8: 128-31. dimensional accuracy of stone casts made by a monophase impression technique using different elastomeric impression materials oral sciences n3 braz j oral sci. 10(1):124-129 original article braz j oral sci. april | june 2011 volume 10, number 2 influence of ceramic primers on microshear bond strength between resin cements and zirconia-based ceramic valéria bisinoto gotti1, saturnino calabrez filho2, marcos massao shimano3, gilberto antônio borges3, luiz henrique borges3, luciano de souza gonçalves3 1dds, graduate student, dental school, university of uberaba, brazil 2dds, ms, phd, professor, restorative dentistry division, dental school, university of uberaba, brazil 3dds, ms, phd, professor, biomaterials division, dental school, university of uberaba, brazil received for publication: december 08, 2010 accepted: april 29, 2011 correspondence to: luciano de souza gonçalves laboratório de biomateriais, bloco h sala 207 campus aeroporto. av. nenê sabino, 1801 bairro universitário, uberaba mg cep: 38.055-500 phone: +55 (34) 9155 9083 e-mail: luciano.goncalves@uniube.br goncalves1976@yahoo.com.br abstract aim: this study evaluated the effect of a coupling agent ceramic primer (cp) on the microshear bond strength (ìsbs) between luting cements and zirconia-based ceramic. methods: zirconia discs (cercon) were made and finished with silicon carbide paper and submitted to air abrasion using 50 µm aluminum oxide particles (al2o3) under 4 bar pressure. zirconia disks were randomly assigned to four groups (n=10): (g1) resicem luting cement + porcelain primer, (g2) resicem luting cement only, (g3) clearfil esthetic cement + clearfil ceramic primer and (g4) clearfil esthetic cement only. the luting cements were prepared according to manufacturers’ recommendations and inserted in an elastomeric mold positioned onto a zirconia disc. each specimen was photoactivated by 20 s. the specimens were stored at 100% relative humidity and 37ºc for 24 h. the ìsbs test was performed using universal testing machine at a crosshead speed of 0.5 mm/min. an optical microscope was used to analyze the failure modes and illustrative images were captured with a scanning electron microscope. the ìsbs data were analyzed by two-way anova (p<0.05). results: no significant difference was found among the four groups g1 (17.4±6.8), g2 (17.1±5.5), g3 (15.6±5.5) and g4 (14.2±3.5), all of which showed 100% of adhesive failures. conclusions: cp showed no increase in the ìsbs between zirconia-based ceramic and resin luting cements. keywords: zirconia, dual luting cement, bond strength. introduction zirconia-based ceramics present optimal mechanical properties, which are important for the long-term performance of these restorative materials1-3. the clinical success of all-ceramic restorations is dependent on the cementation process. zirconia restorations can be cemented to the tooth with non-adhesive cements. however, the choice of a resin-cement agent would be justified due to the bond to the dental structure, with higher retention and better marginal adaptation4-6. as there is no silica in its composition and it lacks a vitreous phase, conventional hydrofluoric acid etching and silanization procedures are incapable of modifying the zirconia surface6-9. previous studies have recommended the use 125 braz j oral sci. 10(1):124-129 of airborne particle abrasion or silica coating for improving the bond strength to zirconia10-11. however, controversial results have been found12-15 with regard to the efficacy of these alternative treatment procedures. it has been shown that airborne particle abrasion alone does not provide adequate bond strength to zirconia-based ceramics because it promotes only mechanical retention on the surface16. silica coating combined with silanization provides a chemical interaction with the resin luting cement and appears to be a more appropriate treatment17-18. however this treatment is expensive for the prosthetic laboratories and dentist. different resin luting cements with various blends of monomers have been developed to improve these materials and their chemical affinity with the ceramic surface. higher chemical affinity would be attained with the use of resin cements containing phosphate monomers, such as 10methacryloyloxydecyl dihydrogen phosphate (mdp), promoting higher bond strength4, or by using additional bond agents, called primers19-20, which also have these monomers in their composition. nevertheless, another study has shown that the use of the primers without airborne particle abrasion promoted a non-durable bond to zirconia21. therefore, the combination of airborne particle abrasion with primers requires further investigation. according to previous studies4,20-21, the association of resin luting cements with primers promoted a better interaction with the ceramic surface due to the increase in cement wetting. this wetting favors the adhesion process and improves the chemical interaction between resin cement and the zirconia surface. these ceramic primers usually contain silane and a functional phosphated monomer. clearfil esthetic cement (kuraray) is composed of the functional monomer 10-mdp, 3-methacryloxypropyltrimetoxisilano (3mps) as silane and ethanol. conventional silane is not effective on zirconia due to the absence of silica in its composition10,13,19. however, when a silane primer (3-mps) reacts with 10-mdp, the interaction of the primer with the substrate and resin cement is promoted, forming cross links resin luting cement resicem clearfil esthetic cement characteristics of cement udma, tegdma, bx-ema, filler of f-al-si glass, acryl adhesive monomer, filler consisting of silanized amorphous silica and camphorquinone bis-gma, tegdma, aromatic hydrophobic dimethacrylates,aliphatic hydrophilic dimethacrylates, silanized ba glass, silanized colloidal silica and camphorquinone characteristics of primers* ethanol (95%), maleic anhydride (<1%), others (non-specified) ethanol (>80%), 3trimethoxysilylpropyl methacrylate (<5%) and mdp manufacturer shofu inc, kyoto, japan kuraray medical inc, sakazu, kurashiki, okayama, japan photo-activation time 20 s 20 s table 1.table 1.table 1.table 1.table 1. resin luting cements and respective primers. bis-gma (bisphenol a glycidyl dimethacrylate), bx-ema (2-hydroxyethyl methacrylate), udma (urethane dimethacrylate), tegdma (triethylene glycol dimethacrylate), mdp (10-methacryloyloxydecyl dihydrogen phosphate). *information obtained from the msds. with the oh groups from ceramic and cement methacrylates. this reaction can be induced and sustained by the acidity of the ceramic treated with the coupling solution4. primers containing other phosphate monomers, such as 6-mhpa (6methacryloyloxyhexyl phosphonoacetate)9 and mtu-6 (6methacryloyloxyhexyl 2-thiouracil-5-carboxylate)22 have been evaluated with less favorable results when compared with 10-mdp. addition of multifunctional methacrylate acids directly to cements that do not have primers, called self-adhesive cements, can also promote this higher interaction between the material and substrate 4,23. however, resicem’s manufacturer, shofu inc., does not disclose the full composition of the primer, omitting some components. in addition, no phosphate monomer is specified in the cement or primer formulations stated in the material safety data sheet (msds) (table 1). therefore, the aim of this study was to investigate the effect of primers on the bond strength between resin luting cements and zirconia-based ceramic. the failure mode analysis was also examined after the test. the hypothesis tested in this study was that the application of a ceramic primer on airborne particle abraded zirconia surfaces increases the bond strength of resin cements. material and methods the materials used in this study are presented in table 1. forty zirconia discs (20 mm in diameter and 2 mm thick) were made from pressed blocks (cercon, degudent gmbh, hanau/wofgang, germany). after sintering, the finishing and polishing procedures were performed with 320-, 400-, 600and 1200-grit silicon carbide papers. after the trimming, the zirconia discs were submitted to airborne particle abrasion with 50 µm (al2o3), performed perpendicularly to the ceramic surface for 10 s at a distance of 10 mm. after airborne particle abrasion, the zirconia discs influence of ceramic primers on microshear bond strength between resin cements and zirconia-based ceramic 126 braz j oral sci. 10(1):124-129 were cleaned in an ultrasonic bath (odontobras ind. & com. de equipamentos médico odontológicos ltda., ribeirão preto, sp, brazil) immersed in ethanol for 5 min, immersed in distilled water for 5 min and dried with oil-free air blast. pretreated zirconia discs were then divided into four groups (n=10) according to the bond procedure and the resin luting cement (table 2). adhesion procedures were performed at room temperature and controlled humidity (23 ± 2ºc and 50 ± 5%, respectively) according to the iso/ts 11405/2003 standard. customized 0.5-mm-thick polyvinyl siloxane molds (vigodent s.a. indústria e comércio, rio de janeiro, rj, brazil) with five cylinder-shaped orifices (0.8 mm in diameter) were placed on the ceramic discs to allow delimitation of the bond area. resin luting cements were prepared according to the manufacturers’ instructions and inserted in the mold orifice with a #5 explorer (duflex ss white do brasil, rio de janeiro, rj, brazil). excess cement was removed with a #24 spatula (duflex). the orifices were filled with each of influence of ceramic primers on microshear bond strength between resin cements and zirconia-based ceramic resin luting cement bond strength with primer without primer resicem 17.40 (6.8) aa 17.08 (5.5) aa clearfil esthetic cement 15.61 (5.5) aa 14.15 (3.5) aa table 2.table 2.table 2.table 2.table 2. means (standard deviations) for microshear bond strength (mpa) of the resin luting cements to zirconia-based ceramic. same uppercase letters in line and lowercase letters in column indicate statistical similarity (p>0.05). the resin luting cements, and a transparent polyester strip was placed over the filled orifices. before polymerization, a constant and uniform 0.454 kgf load was applied for 1 min, using a custom-made device. samples were photoactivated in continuous mode with a led radii cal (sdi. victoria®, australia) appliance with 1,400 mw/cm2 irradiance, as verified with a power meter (ophir optronics ltda.®, jerusalém, israel) for 20 s for each orifice individually. after activation, samples were stored at 37 ºc and 100% relative humidity for 24 h, protected from light. the µsbs test was performed in a universal testing machine (emic dl 3000®; (emic, são josé dos pinhais, pr, brazil), using a knife-edged blade at a crosshead speed of 0.5 mm/min until fracture. the µsbs of each zirconia disc was obtained by calculating the mean value of five repetitions on the surface of each disc, as shown in figure 1. this procedure was performed to submit a larger area of the disc to bond strength test, increasing the reliability of the values of each specimen. the µsbs data were submitted to two-way anova (p=0.05). failure mode analysis was performed with a stereomicroscope at 40x magnification and classified as: cohesive in cement (c), adhesive (a) and mixed (m). in addition, representative fractured specimens were sputter-coated with gold and examined with a scanning electron microscope (jsm5600lv; jeol inc., peabody, ma, usa). results the µsbs test results are shown in table 2. two-way anova showed that no significant differences in bond fig. 1. experimental set-up for the µsbs test: (1) polyvinyl siloxane mold and (2) al2o3 airborne particle abrasion of all zirconia discs. (3) polyvinyl siloxane mold with cylinder-shaped orifices positioned over the zirconia surface (4) and filled with luting agent without ceramic primer or (4’) with previous application of ceramic primer. (5) polyester strip placed between the mold and the glass slab (6). (7) cementation load (454 gf) applied for one minute. (8) photoactivation (20 s) of the resin cement through the glass slab for each orifice. (9) microshear bond strength test. 127 braz j oral sci. 10(1):124-129 influence of ceramic primers on microshear bond strength between resin cements and zirconia-based ceramic strength were detected. the studied factors revealed no statistically significant difference among the resin luting cements used in this study (p = 0.179). the same occurred with regard to the ceramic surface treatments with primer (p = 0.609) and the interaction between the factors (p = 0.742). failure mode analysis showed 100% of adhesive failures, sem micrographs of fractured specimens are shown in figure 2. discussion the influence of different surface treatments and ceramic primers on the bond strength of zirconia-based ceramics has been extensively investigated. the majority of studies have demonstrated that chemical or mechanical modification was shown to positively influence bond strength to resin luting cements12-15,24. however the increase in bond strength was not always achieved, with contradictory results being shown 2,6,12-15,20,25. in the present study, no statistically significant difference was found in the bond strength between the resin luting cements and zirconia discs, irrespective of the bond agent applied to the ceramic (table 2). the similarity between the bond strength values of the two agents, fig. 2. sem micrographs. in images a and b adhesive failures can be seen in the clearfil esthetic cement with ceramic primer (g3) and resicem (g2), respectively. the circle shows the original position of specimens. images a’ and b’ (higher magnification) show details of the adhesive failure on the zirconia surface after the ìsbs test. resicem (shofu) and clearfil esthetic cement (kuraray), probably occurred because of the absence of phosphate monomers in their compositions as reported in the manufacturer’s msds of the respective products. both resin cements have only inorganic particles in the organic matrix of methacrylates, as shown in table 1. it is known that monomers, such as mdp or 4-methacryloxyethyl trimellitic anhydride (4-meta), present chemical bond to metal oxides13,26. previous studies have shown that the chemical interaction of these functional monomers can improve the bond strength of crystalline ceramics such as zirconia and alumina, as well as the long-term bond stability, when compared with conventional methacrylates26-27. a previous study reported that the functional monomers are able to form chemical bonds with the metal oxides of zirconia, secondary bonds such as van der waals, or hydrogen bonds at the ceramic-resin interface, increasing the surface wettability21. this increase in bond strength is more evident when functional monomers are used in combination with mechanical retention by means of surface pretreatment, such as airborne particle abrasion or silica coating4. in the present study, the application of primer to ceramic had no influence on the µsbs of the cementation agents to braz j oral sci. 10(1):124-129 128influence of ceramic primers on microshear bond strength between resin cements and zirconia-based ceramic the zirconia discs treated with 50 µm airborne particle abrasion. nevertheless, the majority of studies have shown better bond strength results when associated with primer application4,12,20. however, previous studies have shown that even in the presence of functional monomers, it is important to use different surface treatments to increase resin cement bond strength to zirconia-based ceramics4,9. the combination factors, such as the use of resin cements containing adhesive phosphate monomers, airborne particle abrasion pressure and primers containing mdp, could promote a durable long-term bond to zirconia20-22,28. the use of primers without alumina airborne particle abrasion resulted in no long-term interlocking with the zirconia surface20-21. in a previous study, when airborne particle abrasion was combined with primers containing mdp, higher tensile bond strength values allied to low percentages of adhesive failures were observed21. therefore, the absence of phosphate monomers in the composition of the cement and primer of resicem may have not affected the bond strength. perhaps, there should be phosphate monomer in the resin cement and primer. the combined action of the two materials would increase the presence of phosphate-radicals, and could lead to a better bond between the zirconia and resin cement. therefore, in the present study, the effect of primers, irrespective of the presence of mdp probably had less influence than retentions created by the al2o3 airborne particle abrasion, resulting in similar µsbs values for both groups. the failure mode analysis also indicated that the bond between the resin cement and zirconia surface was not improved by the action of the primers. all specimens showed adhesive failures, and no increase in µsbs values was found. the appearance of resin cement debris (e.g. mixed failure) would indicate a better bond of cement to parts of the zirconia surface, forcing the stress into the cement bulk, inducing points of cohesive failure. figure 2 shows the adhesive failure mode for clearfil esthetic cement with ceramic primer (a and a’) and resicem without the use of primer (b and b’). at higher magnification, details of the adhesive failure show the zirconia surface with grooves formed by the al2o3 airborne particle abrasion without residual cement in the bond area (dotted line). some authors have pointed out that the use of primer for ceramic, especially those that contain mdp, promoted a more resistant bond in the short term. silica coating associated with application of the primer would be the treatment of choice to increase bond stability. however, degradation of this bond was found even in the presence of the functional monomer with a long-chain carbonyl contained in clearfil ceramic primer4. the bond to zirconia remains an insightful and challenging procedure to the clinician. therefore, with the increase in clinical use of ceramics in dentistry, more evidence related to the adhesive cementation of zirconiabased ceramic restorations is necessary to establish the most reliable technique, since the zirconia surface cannot be conditioned by conventional pretreatment methods. the present study showed that primers do not necessarily improve the bond strength to zirconia. therefore, further studies are required to develop more efficient primers and cements, or more reliable alternative methods to improve the bond strength to zirconia-based ceramics. the aging process of resin cements must be analyzed to establish a more durable and reliable chemical and mechanical bond strength on the cementation process. based on the limitations of this study it may be concluded that: no difference in µsbs was found between the resin luting cements used in this study; application of the ceramic primer to the ceramic did not improve the bond strength of the resin cements to zirconia-based ceramic surfaces. 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influences zirconia ceramic bonding. j dent res. 2009; 88: 817-22. 21. yang b, barloi a, kern m. influence of air-abrasion on zirconia ceramic bonding using an adhesive composite resin. dent mater. 2010; 26: 44-50. 22. yun jy, ha sr, lee jb, kim sh. effect of sandblasting and various metal primers on the shear bond strength of resin cement to y-tzp ceramic. dent mater. 2010; 26: 650-8. 23. miragaya l, maia lc, sabrosa ce, de goes mf, da silva em. evaluation of self-adhesive resin cement bond strength to yttria-stabilized zirconia ceramic (y-tzp) using four surface treatments. j adhes dent. 2010 nov 8. doi: 10.3290/j.jad.a19820. 24. menezes fch, borges ga, valentino ta, oliveira mahm, turssi cp, correr-sobrinho l. effect of surface treatment and storage on the bond strength of different ceramic systems. braz j oral sci. 2009; 8: 119-23. 25. della bona a, borba m, benetti p, cecchetti d. effect of surface treatments on the bond strength of a zirconia-reinforced ceramic to composite resin. braz oral res. 2007; 21: 10-5. 26. leung y, morris md. characterization of the chemical interactions betwen 4met and enamel by raman spectroscopy. dent mater. 1995; 11: 191-5. 27. blatz mb, sadan a, martin j, lang b. in vitro evaluation of shear bond strengths of resin to densely-sintered high-purity zirconium-oxide ceramic after long-term storage and thermal cycling. j prosthet dent. 2004; 91: 356-62. 28. mirmohammadi h, aboushelib mn, salameh z, feilzer aj, kleverlaan cj. innovations in bonding to zirconia based ceramics: part iii. phosphate monomer resin cements. dent mater. 2010; 26: 786-92. oral sciences n3 braz j oral sci. 10(3):213-216 original article braz j oral sci. july | september 2011 volume 10, number 3 how the anterior, middle and posterior portions of the temporalis muscle work during mastication mirian nagae1, fausto bérzin2, marcelo corrêa alves3, maria da graça rodrigues bérzin4 1speech-language pathologist, msc, phd, faculty of medical science department, university of campinas, campinas, sp, brazil 2dds, msd, phd, professor, morphology department, piracicaba dental school, university of campinas, brazil 3it analyst, msc, phd, superior school of agriculture luiz de queiroz, university of são paulo, brazil 4psychologist, msc, phd, electromyography center, piracicaba, sp, brazil correspondence to: mirian nagae rua tessália vieira de camargo 126 bairro barão geraldo campinas/sp cep: 013.084-971 phone: (11)83250331 (11) 32624944 e-mail: mnagae@fcm.unicamp.br abstract aim: the aim of this study was to investigate mean electrical activity and how the anterior, middle, and posterior portions of the temporalis muscle work during mastication. methods: the sample consisted of 16 healthy male college freshmen trichotomized, aged between 18 and 25 years, with angle’s class i and no temporomandibular disorders. electromyographic (emg) recordings were made in anterior, middle and posterior portions of the temporalis muscle during mastication for 5 s. results: it was found a significantly lower rms value in the posterior portion (rms: 1243.92) compared with those of the anterior (rms: 2149.77) and middle (rms: 2531.38) portions. conclusions: there is an association between the portions of the temporalis muscle. it was found a significantly lower rms value in the posterior portion showing that the anterior and middle portions of the muscle have a predominant function of maintaining movement during mastication. keywords: electromyography, mastication, temporal muscle. introduction the temporalis muscle is capable of performing different functions in the stomatognathic system and can have an agonist, antagonist and synergistic action depending on the activity involved 1. this muscle has a complex pennate architecture2-4 that allows wide movements and great range of ajustment5 for maintaining the stability of the mandible. it is divided into anterior, middle and posterior portions, which protrudes, elevates and retracts the mandible, respectively2. the wide movements of the mandible produced during mastication allow studying the integrated actions of the different portions of the temporalis muscle. however, little is known on how the anterior, middle and posterior portions work together to produce movement3-4. most electromyography (emg) studies have focused on maximum voluntary contraction (cvm), showing greater activity of the posterior portion of the temporalis muscle6. mastication studies have reported inconsistent results showing similar actions of the portions4 activity only of the anterior and posterior portions7 and significant activity of the anterior and middle portions only3. received for publication: may 26, 2011 accepted: september 14, 2011 braz j oral sci. 10(3):213-216 214 standard sigma portion mean deviation interval estimate c v upper lower median mad anterior 2149.8 a 882.4 41.1 2683.0 1616.5 1918.0 469.0 695.3 middle 2531.4 a 1408.3 55.6 3382.4 1680.4 1884.0 580.0 859.9 posterior 1243.9 b 1018.0 81.8 1859.1 628.7 619.0 384.0 569.3 analysis of variance: f: 9.75 – p-value=0.0008 95% confidence table 1 basic statistics and tukey’s tests comparing mean rms values of the temporalis muscle. cv: coefficient of variation mad: median absolute deviation means followed by same letters do not differ by tukey’s test (α=0.05). several studies8-9 have reported the clinical importance of the temporalis muscle for mandibular stability and its high susceptibility in patients with morphological deviations10 such as malocclusion and intermaxillary disproportion11-13. emg findings have been widely applied in dental practice 14 for diagnosis, orthodontic care planning and assessment of devices such as occlusal splints2. however, it is not yet clear how the portions of the temporalis muscle work together in healthy individuals. this knowledge can allow comparisons in individuals with dental and skeletal deviations. the present study aimed to investigate how the anterior, middle, and posterior portions of the temporalis muscle work during mastication and to assess mean electrical activity of each portion in angle’s class i individuals with the use of surface emg. the study hypothesis is that there is an association between the muscle portions and that mean electrical activity decreases anteroposteriorly. material and methods the study was approved by the ethics committee of the school of medical sciences of the university of campinas (unicamp) (protocol # 315/2011) the sample consisted of 16 healthy male college freshmen trichotomized, aged between 18 and 25 years, with angle’s class i and no temporomandibular disorders. emg recordings were performed using myosystem br1, myosystem software version 2.52 (datahominis tecnologia ltda.) with signal conditioning, 12-bit resolution, 112 db and 60 hz common mode rejection ratio (cmrr) and a/d myosystem converter (model pci-das 1200, prosecon ltda.). the reference electrode was positioned on the manubrium of the sternum bone of a volunteer. signals were captured using disposable passive bipolar surface ag/agcl electrodes (noraxon usa inc., model 272), of 1 cm diameter and positioned with a fixed 1-cm interelectrode distance, connected to a preamplifier (lynx tecnologia eletrônica ltda., model pa 1010-va) with gain of 20 times forming a differential circuit. in the temporalis muscle, anterior electrodes were positioned 1 cm above the anterior portion of the zygomatic arch and the upper margin of the coronoid process near the zygomatic-temporal suture and lateral margin of the supraorbital ridge, and vertically arranged slightly forward15. in the muscle’s middle portion, electrodes were positioned slightly oblique at a 2 cm distance from the external ear canal16. posterior electrodes were positioned about 1 cm away from the middle portion of the temporalis muscle and arranged at a 15-degree angle in the frankfurt horizontal plan6. a test of muscle function17 was performed after electrodes had been attached to ensure its adequate positioning. all emg recordings were made during regular mastication for 5 s. the mastication cycles were during normal right and left chewing. as it was not known which side was determinant for the differences, the right side was chosen at random. the sample capture for signal was of 2 khz. all subjects were given a chewing gum to assess mastication. emg recordings obtained were band pass filtered at 20-500 hz using a butterworth filter. the mean electrical activity of the cycles was estimated using the root mean square18 (rms) approach. analysis of variance was performed to compare mean rms values of the three portions of the temporalis muscle. tukey’s studentized range (hsd) test for significance (p < 0.05) was used for simultaneous multiple comparisons. since data from the three muscle portions were collected from the same individual, repeated measures analysis was performed using sas glimmix procedure as it allows to adjust data from populations with different distributions19 (sas software; sas institute inc., cary, nc, usa). results the f-test provides strong evidence (p<0.01) of differences between actual mean rms values in at least two levels of the “portion” effect (figure 1). the analysis of data indicate that the mean rms of the posterior portion of the temporalis muscle is significantly lower than rms values of the anterior and middle portions (table 1). in addition, means and standard deviations as well as median and median absolute deviation (mad) values can be used as reference values for healthy people. means and standard deviations are more meaningful when data with normal or gaussian distribution are analyzed, which was not observed in this data set. in view of non-normal distribution of data, we suggest the use of robust indicators such as median and mad that can replace mean and standard deviation, respectively, in cases where outliers or deviations from normality may occur. how the anterior, middle and posterior portions of the temporalis muscle work during mastication braz j oral sci. 10(3):213-216 215how the anterior, middle and posterior portions of the temporalis muscle work during mastication fig. 1. raw emg activity of the anterior (ta), middle (tm) and posterior (tp) portions of the temporalis muscle during mastication. it is of note the simultaneity and balance among the muscle portions and mastication cycles and lower electrical activity in the posterior portion. standard sigma portion mean deviation interval estimates c v upper lower median mad anterior 2149.77 a 882.38 41.05 2682.99 1616.55 1918.00 469.00 695.34 middle 2531.38 a 1408.2 55.63 3382.41 1680.36 1884.00 580.00 859.91 9 posterior 1243.92 b 1018.0 81.84 1859.09 628.75 619.00 384.00 569.32 0 95% confidence table 2 basic statistical tests and tukey’s comparison test of mean rms values of the temporalis muscle cv: coefficient of variation mad: median absolute deviation means with same letters do not have any difference by tukey’s test (α = 0.05) graphic presentation of data distribution shows the lowest estimate of central tendency of the posterior portion of the temporalis muscle during mastication and the greatest suitability of the log-normal distribution for comparison of group means. discussion in the present investigation on how the anterior, middle and posterior portions of the temporalis muscle work during mastication, it was found they all work together but with different mean electrical activity (rms) (figure 1). although the temporalis muscle is considered unique single unit, the analysis of variance evidenced differences between mean rms values in at least two of the three portions. tukey’s test for multiple comparisons of means showed that the posterior portion had a significantly lower activity than all other portions (table 1). several studies20-21 have described the ability of the central nervous system to activate subsets of motoneurons in the same muscle so that specific functions can be carried out. regional differences in the histochemical composition of the fibers of the temporalis muscle have also been reported5. neither the muscle nor its portions can function separately. by integrating the portions, depending on the motor unit firing rate and amplitude, they can have different functions. rms values in the anterior, middle and posterior portions were 2149.77, 2531.38 and 1243.92, respectively, demonstrating that they work together with predominance of the anterior and middle portions11. however, another study found inconsistent results including similar values for the three portions4 and increased activity in the anterior and posterior portions7. these inconsistencies can probably be attributed to different methodological approaches and use of wire7 or needle4 electrodes in the tenuous and delicate fibers of the temporalis muscle, which may cause discomfort and inaccurate recording, compromising data reliability2. the highest rms values were found in the anterior (2149.77) and middle (1243.92) portions of the muscle, which indicates that during mastication the temporalis muscle activity is predominantly continuous 20 focused on maintaining movement. there is a predominance of type i fibers characterized by tonic contraction5 that contrast with type ii fibers found in the posterior portion of the temporalis muscle that are activated during fast phasic contraction20 showing significantly lower rms values in the present study (table 2). the present results are supportive to the findings of other studies, which stated the importance of skeletal and occlusal morphological aspects of muscle activity11-13. in angle’s class i individuals who have adequate occlusal stability and balance intermaxillary the main function of the temporalis muscle is to maintain movement. in contrast, in retrognathic individuals22, maximal voluntary contraction occurs predominantly in the posterior portion of the temporalis muscle probably to produce an antagonistic action to that of the pterygoid muscle and thereby ensure the stability of the mandible3. in conclusion, there is an association between the portions of the temporalis muscle. it was found a significantly lower rms value in the posterior portion showing that the anterior and middle portions of the muscle have a predominant function of maintaining movement during mastication. references 1. moyers r.e. an electromyographic analysis of certain muscles involved in temporomandibular movement. am j orthod. 1950; 36: 481-515. 216 braz j oral sci. 10(3):213-216 how the anterior, middle and posterior portions of the temporalis muscle work during mastication 2. ahlgren j, sonesson b, blitz m. an electromyographic analysis of the temporalis function of normal occlusion. am j orthod. 1985; 87: 230-9. 3. pruzansky s. the application of electromyography to dental research. j am dent. 1952; 44: 49-68. 4. vitti m, basmajian jv. integrated actions of masticatory muscles: simultaneous emg from eight intramuscular electrodes. anat rec. 1976; 187: 173-90. 5. eriksson po, thornell le. histochemical and morphological muscle-fibre characteristics of the human masseter, the medial pterygoid and the temporal muscle. arch oral biol. 1983; 28: 781-95. 6. scott bjj, mason ag, cadden sw. voluntary and reflex control of the human temporalis muscle. j oral rehabil. 2002; 29: 634-43. 7. ahlgren j, ingervall b, thilander b. muscle activity in normal and postnormal occlusion. am j orthod. 1973; 64: 445-55. 8. landulpho ab, silva wabe, silva fae, vitti m. electromyographic evaluation of masseter and anterior temporalis muscles in patients with temporomandibular disorders following interocclusal appliance treatment. j oral rehabil. 2004; 31: 95-8. 9. shinozaki eb, santos mbf, okasaki lk, marchini l, brugnera junior a. clinical assessment of the efficacy of low-level laser therapy on muscle pain in women with temporomandibular dysfunction, by surface electromyography. braz j oral sci. 2010; 9: 434-8. 10. pasinato f, souza ja, corrêa ec, silva amt. temporomandibular disorder and generalizes joint hypermobility: electromyographic analysis of the masticatory muscles. braz j oral sci. 2011; 10: 146-51. 11. ahlgren j. emg pattern of temporalis in normal occlusion. europ j orthod. 1986; 8: 185-91. 12. subtelny jd. malocclusions, orthodontic correction and orofacial muscle adaptation. angle orthod. 1970; 40: 170-201. 13. saifuddin m, miyamoto k, ueda hm, shikata n, tanne k. an electromyographic evaluation of the bilateral symmetry and nature of masticatory muscle activity in jaw deformity patients during normal daily activities. j oral rehabil. 2003; 30: 578-86. 14. hugger a, hugger s, schindler hj. surface electromyography in the assessment of the masticatory muscles for application in dental practice. current evidence and future development. int j comput dent. 2008; 11: 81-106. 15. cram jr, kasman gs, holtz j. introduction to surface electromyography. gaithersburg maryland: aspen publicationâ; 1998. p.257-8. 16. fernandes mr. estudio electromiográfico del músculo temporal. rev cub estomat. 1985; 22: 177-86. 17. de luca cj. the use of surface electromyography in biomechanics. j appl biomech. 1997; 13: 135-63. 18. de luca cj. electromyography. in: webster jg, editor. encyclopedia of medical devices and instrumentation. 2nd ed. hoboken: john wiley and sons; 2006. p.98-109. 19. sas institute inc. the sas system, release 9.2. sas institute inc., cary: nc.; 2008 20. blanksma ng, van eijden tmgj. electromyographic heterogeneity in the human temporalis muscle. j dent res. 1990;19: 1686-90. 21. ter haar romeny bm, denier van der gon jj, gielen ccam. changes in recruitment order of motor units in the human biceps muscle. exp neurol. 1982; 78: 360-8. 22. lindauer sj, gay t, rendell j. effecto of jaw opening on masticatory muscle emg force characteristics. j dent res. 1993; 71: 51-5. oral sciences n3 literature review braz j oral sci. january/march 2010 volume 9, number 1 braz j oral sci. 9(1):1-6 aging and the periodontium simone guimarães farias gomes1, carolina beraldo meloto1, william custodio1, célia marisa rizzatti-barbosa2 correspondence to: simone guimarães farias gomes faculdade de odontologia de piracicaba unicamp av. limeira 901 c.p. 52, cep 13414-903, piracicaba, sp, brasil e-mail: monegfg@hotmail.com received for publication: november 11, 2009 accepted: march 20, 2010 abstract the geriatric population has been growing fast over the last decades in brazil and all over the world, changing demographics. additionally, increased knowledge and the advances of modern dentistry have led the old population to retain more natural teeth, needing specialized dental services for a longer time. changes in biochemical and physiological processes occur with aging in all body tissues, including the periodontium. the association between periodontal and systemic diseases has also been widely discussed, suggesting the need of a multidisciplinary care, especially in older adults, who are frequently affected by chronic systemic conditions and multiple drug therapy. these features lead to a highly complex management of the geriatric population, challenging the dental care providers. the aim of this study is to review the age-related changes and the consequences of other factors, such as systemic diseases and drugs, on the periodontium of aged patients. keywords: aging, periodontium, disease. introduction population of older adults has been growing in a non preceding rhythm all over the world. in brazil, individuals aged 60 to 64 years are the most prevalent within the elderly, representing 30% of this population. however, the most significant increase has been observed for individuals over 80 years, whose number increased from 560 thousand people in 1980 to almost 2 million people in 20061. nowadays, more people retain their natural teeth throughout life. comparison of five nursing home populations from 1988 to 2004 indicates improvement in dental status among the elderly population. the rate of edentulism decreased from 71 to 43% and the percentage of subjects retaining 20-28 teeth increased from 3 to 23%. moreover, the mean number of teeth for each participant was 3.1 in 1988 compared with 8.4 in 20042. with the longer retention of teeth, older adults remain at risk for periodontal disease. in a population composed by 89 individuals, being 87% over 80 years of age, none presented healthy periodontium. all subjects presented bleeding on probing, 93% presented calculus and 65% had periodontal pockets of 4 mm depth or more2. gingival recession and loss of periodontal attachment are also very prevalent among the older adults. fifty-six to 92% of individuals aged 45 years and older present periodontal attachment loss of 3 mm or more in at least one oral site3. this condition worsens with time4, most likely because of the increase in gingival recession rather than probing depth5. in spite of the high incidence of moderate attachment loss in middle-aged and geriatric subjects, severe loss is confined to a minority and is present in only a few sites. generalized severe attachment loss is experienced by approximately one fifth of older individuals3. this damage to the periodontal tissues may be due to various changes on biochemical, immunological and physiological processes during aging3. however, it is not known whether and to what extent the changes are due to the aging process or consequences of other factors, such as systemic diseases, medications or 1dds, ms, graduate student, department of prosthodontics and periodontology, piracicaba dental school, university of campinas, brazil 2dds, ms, phd, professor, department of prosthodontics and periodontology, piracicaba dental school, university of campinas, brazil 2 braz j oral sci. 9(1):1-6 social and environmental modifications6. this paper reviews the changes in the periodontal tissues due to the aging process, systemic medications and medical conditions, in order to guide the clinician to a better multidisciplinary understanding, which is imperative for the treatment of older adults. age-related changes in periodontal tissues morphological alterations the thinning and decreasing in the keratinization of the gingival epithelium may occur with aging and may indicate an increase in tissue permeability and a decrease in its resistance to infection and trauma7. moreover, flattening of the epithelial invaginations toward the conjunctive tissue7, a high vacuolization of interstitial spaces and the invasion of fat cells are also observed with aging8. however, there is no consensus in the literature regarding this issue. the apical migration of the junctional epithelium, with consequent gingival recession, has been discussed. although such a migration is associated with aging, the loss of insertion caused by aging alone may not seem to have clinical significance3. gingival recession progression may occur due to several factors, such as passive eruption caused by physiological wear of teeth, a consequence of anatomically thin tissues and toothbrushing trauma. apparently, gingival recession is not an avoidable physiological process caused by aging, but a cumulative and progressive effect from periodontal disease or trauma over time7. there is a reduction in the number of total cells8-9 in the connective tissue and a consequent reduction in collagen synthesis. however, a higher collagen amount can be found due to an increase in the conversion of soluble collagen to insoluble collagen, as well as to an increase in the denaturation temperature and mechanical resistance of fibers7. there is a reduction in the number of fibroblasts in the periodontal ligament and also in its functional activity, which includes the mitotic and synthesis ability. there is also a reduction in the organic matrix production and in vascularization, and an increase in the number of elastic fibers6-7,9-10. the thickness of the periodontal ligament varies and may reduce due to the reduction in the force applied by masticatory muscles along the time in subjects with complete dentition or having dental elements with no antagonist7-8. on the other hand, when several elements are missing, there might be an overload on the existing remaining teeth, with consequent periodontal ligament thickening8-9. therefore, it can be said that any alteration in the periodontal ligament width reflects the functional condition of teeth7. a continuous deposition of cementum was observed, which leads to an increase in thickness of this tissue with age, mainly in the teeth apical region to compensate for the physiological wear of teeth10. radiographic exams revealed that the mean alveolar bone height was reduced in older patients in a population ranging from 20 to > 80 years of age11. however, data should be analyzed with caution once some alterations in the bone tissue in the oral cavity are similar to those in other parts of the body. this reduction in height may be due to systemic factors instead of being due to age-related alterations7. alterations in disease progression the induction of gingivitis in subjects of different age groups and with healthy periodontium showed a similar plaque accumulation in old and young adults. nevertheless, old adults developed a more severe gingivitis and with a greater number of inflammatory cells12, demonstrating an infiltrate with a greater proportion of b cells and lower density of polymorphonuclear cells13. this situation reveals possible effects of aging on the quality of the immune response. modifying factors of periodontal disease periodontal changes during increasing age are not sufficient to account for all damage experienced by elderly patients. the clinical appearance of periodontal tissues in this population reflects age-related changes and accumulation of disease and trauma over time14-15. association between periodontium and systemic diseases the influence of oral condition on general health has been well documented in the last decades. infectious diseases, such as periodontitis, cause inflammation and contribute to levels of overall infection and inflammation in the body and may trigger the beginning and/or the progression of other diseases such as diabetes and arteriosclerosis16. there are two mechanisms through which infection and inflammation apparently located in periodontal pockets may harm general health: the passage of periodontal pathogens and their products into circulation (bacteremia), and the passage of locally produced inflammatory mediators into circulation. both mechanisms can contribute to inflammatory systemic diseases17. diabetes mellitus it is suggested that the potential interactions between diabetes and periodontitis seem to enhance the morbidity of these two diseases18. the chronic hyperglycemic condition of diabetes is associated with damage, dysfunction, or failure of various organs and tissues, including the periodontium, due to the increased risk for infections in patients with diabetes, impairment of the synthesis of collagen and glycosaminoglycans by gingival fibroblasts, and increased crevicular fluid collagenolytic activity19. it has been demonstrated by a meta-analysis study20 that patients with types 1 and 2 diabetes had worse oral hygiene and higher severity of gingival and periodontal diseases, compared to nondiabetic subjects. on the other hand, periodontal therapy with or without adjunctive antibiotic therapy in patients (mean age: 53 years) may influence the systemic conditions of patients with type 2 diabetes, leading to improvements in glycemic control and in the reduction of inflammatory markers21. there is still much to know about the relationship between periodontitis and diabetes mellitus, and its mechanisms. clinically, the decrease of periodontal pathogens as well as the improvement of the diabetic aging and the periodontium 3 condition should be sought to slow down the progression of the disease or promote tissue repair. coronary heart disease (chd) periodontal and cardiovascular diseases are common inflammatory conditions in the human population, atherosclerosis being the major component of the latter. in the atheroma formation, inflammation plays a continuous role from endothelial cell expression of adhesion molecules to the development of the fatty streak and established plaque, which is surrounded by a fibrous cap and narrows the lumen of the affected vessels, compromising blood flow. the rupture of the atherosclerotic plaque may lead to thrombus formation and to the major clinical sequelae of atherosclerosis: coronary thrombosis, acute myocardial infarction and stroke22. a meta-analysis study indicated that individuals with periodontitis had 1.14 times higher risk of developing coronary heart disease23. the more severe the periodontal disease the easier the periodontal pathogens could enter the circulation, reaching blood vessels and atherosclerotic lesions. the chronic and cyclic nature of periodontal diseases provides multiple opportunities for repeated bacteremia and affection of endothelial integrity 24-25, perpetuating inflammatory events in atherosclerosis22. another linkage between periodontal disease and chd is the level of c-reactive protein, which is an acute-phase reactant in response to infection or trauma and its high sustained level was associated with advanced periodontitis26. additionally, it is suggested that high levels of c-reactive protein are also related to chd. ridker et al. 27 demonstrated that c-reactive protein levels predict the risk of coronary events, although, there is still no consensus weather this protein contributes to atherogenesis or is a marker of atherosclerosis26. there are many traditional risk factors for cardiovascular disease, such as cigarette smoking, hypertension, high lowdensity lipoprotein cholesterol level, low high-density lipoprotein cholesterol level, diabetes mellitus, family history of premature chd, age, obesity, physical inactivity and an atherogenic diet. however, ridker et al.28 reported that individuals with low low-density lipoprotein cholesterol levels, but high c-reactive protein levels were at greater risk for cardiovascular events than those with high low-density lipoprotein cholesterol levels but low c-reactive protein levels. this may highlight the attention for other risk factors, in addition to the traditional ones, as periodontal disease to the onset or progression of chd. chronic renal disease chronic renal disease and renal replacement therapy can affect oral tissues and can greatly influence the dental management of the renal patient. otherwise, chronic adult periodontitis is suggested to contribute to overall systemic inflammatory burden, with possible consequences in the management of the end stage renal disease patient on hemodialysis maintenance therapy29. additionally, some of the most common causes for end-stage renal disease are diabetes mellitus and chronic hypertension, which are also associated with periodontal disease. moderate to severe periodontitis appears to be highly prevalent in the renal hemodialysis population, and was associated to overt nephropathy and end-stage renal disease in subjects with type 2 diabetes30 and to death of end-stage renal patients from cardiovascular causes31. thus, it is suggested that periodontal therapy, which may reduce systemic inflammation, could be a treatment consideration for this population. other conditions depression – the association between periodontitis and depression may be related to psychoneuroimmunologic changes, damaging behavior, or a combination of both32. atrisk behavior includes a disinterest in performing appropriate oral hygiene techniques, a cariogenic diet, reduced salivary flow, rampant dental caries, advanced periodontal disease and oral dysesthesias. additionally, several medications used for the treatment may intensify xerostomia and increase the incidence of dental disease33. people with chronic diseases are at high risk of developing depression, once this psychological condition was present in 40% of the patients with chd33, and a positive relation between psychosocial factors and periodontal disease was found by 57.1% of the studies analyzed in a systematic review34. thus, it is suggested that depression therapy may be an important procedure for periodontal health maintenance32. immunosuppression – it is suggested that immunocompromising conditions do not predispose the subject to periodontal disease; however, it has been reported that the prevalence of periodontal disease is increased in hiv-positive when compared to hiv-negative subjects35. additionally, immunocompromised hiv-positive patients with preexisting periodontitis may have their oral condition exacerbated36. regarding the treatment improvement and the increase in life span for hiv patients, a higher prevalence of this condition may be found in the elderly. basic periodontal therapy using mechanical technique improved gingival condition in 78.2% of hiv-positive and aids patients after 6 months of therapy, showing that even under immunosupression, mechanical therapy is important for maintenance of periodontal health37. neurodegenerative disease – dementia, as well as reduction in cognitive ability, are common in the elderly and have been related to a higher index of periodontal disease in patients over 60 years of age38. it has been suggested that inflammation within the brain may play a role in the etiology and pathogenesis of alzheimer’s disease. as peripheral infection/inflammation, such as periodontitis, may affect inflammatory systemic state, including the central nervous system, chronic periodontal disease has been also associated to this type of dementia39. subjects with parkinson’s disease also present poorer oral health comparing to controls, including more missing teeth, caries, dental plaque, and poorer periodontal health, due to the lack of muscular control40. it is of great importance that neurodegenerative patients and their caregivers must be oriented about the need for monitoring oral hygiene procedures in these individuals38 and professional periodontal health maintenance. braz j oral sci. 9(1):1-6 aging and the periodontium 4 obesity an association between obesity and periodontal disease has been presented, which cause could be related to biological or behavioral determinants in common41. increased food intake and reduced physical activity may be accompanied by other changes in health related behaviors resulting in poorer periodontal health, as well as an elevated risk of cardiovascular disease and type 2 diabetes42. association between the measures of body fat and periodontal disease was found among younger adults, but not middle or older adults43. this result was not confirmed neither by ylostalo et al.41, who found this association among non-diabetic, non-smoking subjects aged 30-49, nor by linden et al.42, who found that obesity (bmi>30) was associated with low-threshold periodontitis, once obese older adults from 60 to 70 years of age, presented a greater number of sites with periodontal pockets deeper than 5 mm42. studies were adjusted for confounders as smoking, diabetes, education, oral hygiene and dental attendance41,42. it has been suggested that focus on prevention in individuals at risk of developing obesity may benefit both general and dental health42. osteoporosis – a systematic review about oral implication of osteoporosis showed that individuals with reduced systemic bone density presented high periodontal bone loss, tooth loss and temporomandibular joint bone loss44. in addition, older adults with osteoporosis had higher periodontal attachment loss45, while subjects aged 65 years and older, who took calcium and vitamin d supplementation presented less tooth loss than subjects taking no supplementation over a-5-year follow-up46. despite these findings, the relationship between systemic and oral bone loss, and periodontal disease is not well established. however, it is suggested that findings in clinical and radiographic exams, may help dentists identifying patients with undetected low bone mineral density and refer them to medical professionals44. drugs montenegro et al.47 reported 46 oral side effects after evaluating the 8,635 commercial drugs most used by the elderly, and stated that oral side effects present great incidence and cause many difficulties to this population (figure 1). despite this, 21.6% of the drugs did not present any information concerning oral side effects. it is believed that these and some other important data that should been found in the prescribing information might have been neglected, rather than only the absence of side effects47. among gingival and periodontal problems observed, gingival overgrowth with false periodontal pocket formation is included. this condition promotes bacterial plaque accumulation and makes oral hygiene procedures more difficult. calcium channel-blocking and immunosuppressive drugs used in the treatment of angina, arterial hypertension and cardiac arrhythmias and also in the management of transplanted patients, respectively, show such characteristics36. indirect damage to oral tissues may also be promoted by reduced salivary flow. it is well known that saliva is an important fluid for oral health and its reduction may cause gingivitis and a decrease in antimicrobial activity48, among other disorders, which may affect health and quality of life. according to nagle and hershkovic49, elderly people present a reduction of most salivary components, as well as salivary flow decreased by 62% when compared with younger people, limiting its protective capacity, and turning saliva into a highly concentrated fluid, which compromises its lubrication and moistening abilities. there was no difference either in salivary flow or in saliva composition comparing older adults who used and did not use medications. however, although individuals who used drugs presented more extensive oral sensorial complaints, including xerostomia, it was suggested that salivary alterations were directly related to age rather than disease and/or drug use49. systemic diseases as well as the use of medicines like antihypertensive and antiparkinsonian drugs, which are most used by the elderly, are pointed as the main causes of xerostomia. in a previous study, patients aged 67 to 96 years with poor health were examined. almost 53% of them presented very low salivary flow, and this condition was strongly related to endocrine diseases, and the use of ophthalmologic and respiratory drugs, as well as potassium chloride. it was not possible to identify the effect of each drug. however, the number of drugs used daily, which ranged from 0 to 14, appear to be a key factor to the reduced salivary flow50. as many chronic diseases are found and many drugs are usually used by older adults even in healthy conditions, prescribing a drug is of major importance and responsibility, and involves an interdisciplinary focus, in order to reduce damage and improve patient’s quality of life47. behavioral and environmental modifiers tobacco smoking – smokers are at a higher risk of developing periodontal disease, once tobacco may affect vascularization, the immunologic and inflammatory systems36, attachment and function of fibroblasts, and increase the prevalence of periopathogens51. in a previous study, smokers over 50 years of age showed more sites with bacterial plaque, deeper probing depth and higher periodontal attachment loss when compared to former smokers or to nonsmokers of the same age. additionally, smokers had a braz j oral sci. 9(1):1-6 aging and the periodontium fig. 1. percentage of drugs presenting oral side effects reported in prescribing information, medicine guides and textbooks from a total of 8,635 drugs used by the brazilian elderly population (adapted from montenegro et al. 47). induced 5 diminished response to periodontal therapy and present 4.8 times more risk of developing periodontitis than nonsmokers52. the amount of cigarette consumption is also associated with higher risk for having moderate and severe periodontitis52. as quitting smoking reduced the risk of having periodontal disease52, dental professionals should be more active in smoking cessation counseling, in order to achieve positive effects in patient’s health51. stress genco et al.53 observed that emotional distress was a significant risk factor for individuals from 25 and 74 years of age having financial problems, once differences for stress were related to different levels of periodontitis. stressed individuals showed higher pro-inflammatory cytokine level 54, this psychological condition being a possible positive influence on chronic periodontal disease34. additionally, stress may affect the host immune response, making the individual more susceptible to the development of unhealthy conditions34, which are commonly found in elderly. thus, stress might exacerbate periodontal disease, especially in older patients. in addition, stressed individuals may adopt harmful habits, such as, altering oral hygiene habits, intensification of smoking and changing eating habits, worsening the periodontal condition53. discussion age-related periodontal alterations do not seem to be responsible for the high incidence of periodontal disease in the elderly population. the deterioration of periodontal tissues found in older adults may seem to be the result of the accumulation of diseases and traumas acquired throughout the lives of these individuals14-15. older patients are more susceptible to systemic diseases, their associations and, consequently, to the use of systemic medical therapies. moreover, the elderly population is exposed to environmental and social factors for a longer time than younger people, increasing the susceptibility of the elderly population to possible changes in these factors. according to seymour et al.16, infectious diseases, such as gastrointestinal, respiratory and periodontal diseases, may contribute, alone or in combination, to the overall infection state of the individual. in the same way, infectious diseases and systemic conditions, such as obesity, emotional distress, autoimmune diseases, as well as social factors like smoking, are related to the increase in inflammatory markers. they may also contribute to the overall inflammation state of the body, worsening periodontal disease and other important diseases, such as arteriosclerosis and diabetes mellitus. type-2 diabetes is frequently observed as part of a multifactorial syndrome that includes obesity and arteriosclerotic cardiovascular disease55. periodontal disease has also been related to such conditions, which shows that it may be part of a systemic net, contributing to the general inflammatory state of the individual16. despite these findings, more studies are needed to clarify the relationship between periodontal disease and diabetes mellitus, mainly on the treatment effects on this condition. clinically speaking, the reduction in periodontal pathogens and the control of diabetes should be achieved in order to reduce the speed of disease progression and promote tissue repair. as for cardiovascular diseases, there are several traditional risk factors, such as smoking, arterial hypertension, high levels of low-density cholesterol (ldl), low levels of high-density cholesterol (hdl), diabetes mellitus, family history among relatives of premature coronary disease, age, obesity, physical inactivity and an atherogenic diet22. ridker et al.28 suggested that the level of c-reactive protein is a stronger predictor of cardiovascular events than the ldl cholesterol, popularly known as a risk factor. this is an important finding, which suggests that the clinical interest must focus beyond traditional risk factors and must also consider other factors, such as the periodontal disease as a contributor to the coronary disease onset and progression. drugs commonly used by the elderly may cause oral side effects, such as gingival overgrowth and salivary flow reduction47. whenever possible, these drugs should be replaced in agreement with the patient’s physician, but the dentist must be aware of the medical history and orient the patient about the importance of periodical visits for controlling bacterial plaque. measures for the induction or replacement of salivary flow have to be considered, when necessary, in order to minimize the effects of hyposalivation and xerostomia. when treating the elderly, the dentist has to take into account the physiological alterations in the oral cavity, as well as obtain accurate information on medical problems, drug regimen and possible limitations of patients of this age group, such as limited motor skills to keep good oral hygiene15. according to duval and leport56, simple everyday habits like toothbrushing, flossing and chewing may cause bacteremia. therefore, to avoid the contribution of the cumulative and spontaneous effect of microorganisms entering the bloodstream to overall generalized infection and inflammation, it is necessary to adopt a preventive strategy to reduce the source of oral bacteria by means of adequate oral hygiene and regular professional care. once periodontal disease is a consequence of lifetime damages, the best conduct for patients is to seek professional advice in order to prevent the disease since an early age. it is possible then, to reduce the risk of developing, worsening or perpetrating some systemic conditions at old age. in conclusion, only age-related periodontal alterations may not seem to have clinical significance. associations among such alterations and systemic diseases, medicines and behavioral and environmental factors may significantly compromise the well-being of the elderly. the relation between periodontal disease and some systemic diseases seems to have a 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infect dis. 2008; 8: 225-32. oral sciences n3 braz j oral sci. 10(4):241-245 original article braz j oral sci. october | december 2011 volume 10, number 4 knowledge, attitude and practice on hepatitis b prevention among dental professionals in india r. sudhakara reddy1, l. a. swapna2, t. ramesh3, k. pradeep4 1professor and h.o.d, department of oral medicine and radiology, vishnu dental college, bhimavaram, andhra pradesh 2graduate student, department of oral medicine and radiology, vishnu dental college, bhimavaram, andhra pradesh 3associate professor, department of oral medicine and radiology, vishnu dental college, bhimavaram, andhra pradesh 4assistant professor, department of periodontics, sri sai college of dental sciences vikarabad received for publication: july 05, 2011 accepted: september 15, 2011 correspondence to: l. a. swapna department of oral medicine and radiology vishnu dental college, bhimavaram, west godavari district, pin code:534202. andhra pradesh phone: +91 9573201334 e-mail: laswapna_123@rediffmail.com/ laswapna123@gmail.com abstract aim: to obtain comprehensive information about the knowledge, attitude and practices towards hepatitis b infection by dental health care professionals, and their effort to prevent the transmission among the patients. methods: a cross sectional survey conducted among 540 dental health care workers in a dental college and private practitioners in and around bhimavarm, india. a selfassessment questionnaire with queries on levels, namely knowledge and practices, and protective measures to prevent transmission of hepatitis b was recorded and statistically analyzed. results: frequency distribution scores of knowledge, attitude and practice in relation to hepatitis b infection revealed that the faculty members, the students under training along with, nurses, hygienists and lab technicians were relatively aware of hepatitis b vaccination, precautions and principles droplet isolation procedures to prevent transmission of hepatitis b infection. conclusions: the results showed that the dental professionals had good knowledge and attitude regarding hepatitis b and its transmission, but that the infection control measures among the health care professionals are moderately poor and an educational program on isolation precautions can further enhance these levels and thereby, reducing the risk of infection transmission . keywords: dental health care professionals, hepatitis-b, protective and preventive measures. introduction dental health care professionals are said to be at a risk of infections caused by various microorganisms including m. tuberculosis, hepatitis b, hepatitis c viruses, streptococci, staphylococci, herpes simplex virus type 1, hiv, mumps, influenza, and rubella1-3. in a dental office, infections can be expedited through several routes, including direct contact with blood, oral fluids or other secretions; indirect contact with contaminated instruments, operatory equipment or environmental surroundings; or contact with airborne contaminants present in either droplet splatter or aerosols of oral and respiratory fluids4. hepatitis-b virus (hbv) is a major worldwide cause of acute and chronic liver infection, cirrhosis, and primary hepatocellular carcinoma. there are more than 300 millions carriers of the virus globally, and about 90% of these live in developing countries, among the world’s carriers, 75% are from the asian continent, where between 8% and 15% of the population carries the virus5. the majority of the infections are sub242 braz j oral sci. 10(4):241-245 clinical, so that approximately 80% of all hbv infections are undiagnosed. it has been established that patient medical histories are unreliable in identifying exposure to hbv infection6. regardless of the medical history, all patients should therefore be regarded as potential hbv carriers. infection control practices in developing countries have not been widely indexed. most of the hospitals have no infection control programmer due to indigence of awareness about the disease or penury of trained personnel’s. so a need exists for proper practice of infection control by both dental staff, dental nurses and hygienists6-9. center for disease control and prevention (cdc) has suggested the guidelines that include precautions and transmission based isolation safety measures that aid in safe working environment and eliminate the spread of infections9. the aim of this study was to contrive comprehensive information about the knowledge, attitude and practice with regard to hepatitis b by dental health care professionals, and their effort to prevent the transmission of hepatitis among the patients. materials and methods a cross sectional survey was conducted among 540 dental health care workers. a structured questionnaire was used to assess 3rd year and 4th year b.d.s. bachelor of dental surgery students, house surgeons, graduate students, m.d.s. master of dental surgery , hygienists, medical lab technicians and nurses of vishnu dental college, bhimavaram, andhra pradesh, india and local private dental practitioners in and around the state about the “methods to control cross infection, attitude towards protection and prevention of transmission of hepatitis infection , their approach towards sterilization of instruments and knowledge of immunization against hepatitis b virus. the questionnaire was designed and the ethical clearance was obtained from the ethical committee of vishnu dental college and hospital, bhimavaram. the questionnaire comprised of queries on knowledge, attitude towards protective and preventive measures with respect to hepatitis b vaccination and droplet isolation precautions as advocated by the cdc. the content authenticity was pretested on a random sample of population to ascertain practicability, cogency and rendition of responses. a visit was made to the faculty, students, lab technicians, hygienist and nurses of the dental college in bhimavaram and the private dental practitioners in and around the state. the questionnaire was handed over with exigent instructions for the same. the data collected were maintained under strict confidentiality. only valid responses were used for analysis. the age, sex and qualification & years of experience were also determined. completed questionnaires were collected on the same day and prospectively analyzed. all responses were entered into a computer database and analyzed using a statistical package (spss; spss inc., chicago, il, usa). simple frequencies were calculated for all variables. all the frequency variables had percentages, cumulative percentages and corresponding related statistics. in the knowledge questions having 10 questions , if given a response yes =1 and no=0, total maximum score will be 10, percentage of obtained score for each individual computed and compared according to age and other variables, similarly protective and preventive categories of questions were calculated. descriptive statistical analysis has been carried out in the present study. results on continuous measurements are presented on mean ± sd (min-max) and results on categorical measurements are presented in number (%). significance level was set at 5 % level. analysis of variance (anova) was used to find the significance of study parameters between three or more groups of study subjects, student’s t test (two tailed, independent) was used to find the significance of study parameters on continuous scale between two groups (inter-group analysis) on metric parameters. results out of the 540 respondents (table 1), 53 individuals were mds staff, 17 were bds staff, 73 graduate students, 98 house surgeons, 186 were the students of 3rd and 4th year, 15 nurses,7 hygienists, 5 labtechnicians in the dental college and 86 private dental practitioners in and around the state of andhra pradesh. (table 2) represents the percentage of responses on knowledge, attitude and practice regarding the protection and prevention of transmission of hepatitis-b infection. mean score for knowledge of mds staff was 56.42 ± 8.11; for protective measures was 88.04 ± 22.03; for attitude to prevent transmission was 54.93 ± 4.58 (figure 1).hygienists showed minimum knowledge with mean score of 5.71 ± 7.87, the protective and preventive measures followed were with a mean score of 66.7 ± 0.00 & 41.27 ± 5.42 respectively. none of them scored positive answer to the question -have you attended any workshop regarding hepatitis transmission (or) prevention. frequency of correct answer was higher for the question “do you believe that hepatitis can transmit through saliva from patients?”, showing 81.3% positive response. almost of 77.2% participants got the vaccination against hepatitis b. only qualification number of persons % 3rd year 96 17.8 4th year 90 16.7 bds staff 17 3.1 house surgeon 98 18.1 hygienist 7 1.3 technicians 5 0.9 mds staff 53 9.8 nurse 15 2.8 graduate students 73 13.5 private practitioners 86 15.9 total 540 100.0 table 1 number of persons in each category divided as per their qualification. knowledge, attitude and practice on hepatitis b prevention among dental professionals in india braz j oral sci. 10(4):241-245 qualification total score (%) knowledge protective measures preventive measures 3rd year 17.29 ± 12.44 34.17 ± 6.57 44.44 ± 0.00 4th year 37.89 ± 9.30 37.75 ± 9.30 39.5 ± 5.55 bds staff 52.35 ± 4.37 48.02 ± 26.94 47.71 ± 6.54 house surgeon 43.67 ± 6.32 33.47 ± 1.69 33.44 ± 1.12 hygienist 5.71 ± 7.87 66.7 ± 0.00 41.27 ± 5.42 technicians 38.00 ± 16.43 33.3 ± 0.00 6.67 ± 6.09 mds staff 56.42 ± 8.11 88.04 ± 22.03 54.93 ± 4.58 nurse 17.33 ± 4.58 33.3 ± 0.00 48.15 ± 5.43 pg students 46.85 ± 12.12 36.51 ± 15.13 35.61 ± 9.07 private practitioners 49.77 ± 16.09 35.24 ± 12.64 28.29 ± 14.73 significance f=87.563; p<0.001** f=103.803; p<0.001** f=74.888; p<0.001** table 2 correlation of total score (%) of positive response of knowledge, protective and preventive measures adopted. 14.4% thoroughly evaluated the patient to rule out any signs related to hepatitis. nearly 65.9% of respondents were willing to perform any treatment procedures on patients known positive with hepatitis. as much as 53% of the dental health care professionals advised routinely for their patients to undergo blood investigations to rule out hepatitis before doing any invasive procedures. under the category of questions relating to protective and preventive measures to avert transmission of hepatitis the results showed that about 89.8% of the participants responded that they change gloves for every patient. only 15% of the individuals used protective eye wear for all procedures on patients or while assisting. 22.2% of the participants advise pre-procedural oral rinses (betadine/ chlorhexidine etc) to the patients. only 1.1% participants used disposable kits for patients suspected or proved positive for hepatitis. and merely 2.2% of the participants used rubber dam where required to prevent atmospheric viral contamination. though the knowledge of private dental practitioners fig. 1. knowledge, protective and preventive measures followed by dhps was fair, their protective and preventive measures to avoid hepatitis transmission were not up to the mark, probably because of a small set up of clinics and other financial constraints. there was an increase in knowledge of dental health care professionals as their years of experience in dental clinic was increased from 1-5 years to 6-11 years (figure 2). there was not much change in the knowledge, protective and preventive measures between dental health care professionals who had 6-11 and 11-15 years of experience. discussion though there is substantial literature regarding the knowledge and attitude of dentists towards other infectious diseases, no study assessed the different categories of dental health care professionals and their attitude towards hepatitis b infection and very few studies have been conducted in india in this regard. this study showed that b.d.s staff had a mean knowledge of 52.35, which was close to the knowledge 243knowledge, attitude and practice on hepatitis b prevention among dental professionals in india 244 braz j oral sci. 10(4):241-245 fig. 2. knowledge and protective and preventive measures varying with the years of clinical experience. showed by m.d.s staff (mean value of 56.42). however, the protective and preventive measures adopted by b.d.s staff (mean values of 48.02 and 47.71, respectively) was lower than the protective and preventive measures adopted by m.d.s staff (mean values of 88.04 and 54.93, respectively). though the private practitioners showed a score of 49.77 as the mean value in knowledge, their measures followed to protect and prevent the transmission of hepatitis was only 35.24 and 28.29 respectively. when the results were compared between the female and the male dental health care professionals, it was found that the female subjects were appropriately following the protective and preventive measures to avert the transmission of hepatitis. several other similar studies have also been conducted to investigate the infection control knowledge, attitude and practice of dental health care professionals. previous study among nigerian investigators found that nearly 97.5% dentists routinely used gloves and 70.6% used face masks and 61.3% dentists reported to use protective gowns and eye wear during the procedures10. in our study, nearly up to 89.8% of the respondents changed their gloves for every patient and 81.1% of participants used protective clothing, but only 16.5% of participants used protective eye wear during procedures. previously, the wearing of gloves before examining patients was hardly practiced regularly as an essential part of cross infection control. however, 25% of dental health care professionals in this study routinely wore gloves before patient examination. whether routine wearing of gloves would prevent cross infection of blood borne viruses has not been resolutely established nevertheless, it would protect minor cuts, and abrasions from contamination and so reduce the transmission of hbv from carrier to p ractitioners 10-13. only 20.2% practitioners in this study frequently scrubbed their hands with disinfectant before and after gloving, there is evidence of a reduction in skin flora when hands are scrubbed with soap and this should be encouraged in practice14-16. viral hepatitis caused by hbv is a disease that has no oral manifestation but is of great concern to the dental profession due to ease of transmission of the virus from patients with the condition. it may be difficult to identify those capable of transmitting hbv for several reasons. many patients infected with hepatitis b virus may be unaware of their carrier status or they may be asymptomatic. others may not want to disclose their infectious status10-12. in a previous study, only 32% of the dentists took vaccination against hepatitis, where as 77.2% of the dental health care professionals in our study were vaccinated against hepatitis10. similar study conducted to assess the hepatitis b awareness and attitudes among dental health care workers in riyadh saudi arabia, results showed that nearly 43% did not have their antibody titre measured after completion of the full course to assess the efficacy of the vaccine. on the contrary, 87.9% of the dental health care professionals in our study did not check their antibody titre measured after completion of course of vaccination4. these findings are somewhat similar to those of several other western-based studies in which post-vaccination testing had not been carried out in between 38-54% of dental health care professionals17-21. saheeb et al in a similar study found that nearly 19.5% of the dental health care professionals reused syringes, which was less when compared to our study where nearly 56.5% dental professionals reused syringes10. blood is very often found in the aerosols produced by dental equipment like an ultrasonic scaler or other highspeed equipment ultrasonic scaling was obviously associated with increased air contamination levels confirming the results knowledge, attitude and practice on hepatitis b prevention among dental professionals in india braz j oral sci. 10(4):241-245 245 reported by several other studies showing that this procedure is the main executor of airborne contaminants in dentistry. previous research demonstrated that rinsing with an antiseptic mouthwash produced a 94.1% reduction in airborne contaminants compared to the non-rinsed controls. hence, high volume suction evacuators and preprocedural oral rinses would prevent the air contamination22-25. a survey was conducted to assess the extent of awareness regarding transmission of hepatitis among the dhps in bhimavaram, andhra pradesh, india. this study also threw light on the knowledge, attitude and behavior of dental health care professionals regarding the protective and preventive measures to avert the transmission of hepatitis b, and also made them seriously think about the risks that their patients and they themselves face during the treatment procedures. as we approached the participants to collect the filled questionnaire, the respondents were curious to know the correct protocol to follow during the dental treatment procedures to prevent the transmission of hepatitis infection and the information about the vaccination against hepatitis and its importance. one of the limitations of this study was that we could not supervise the respondent’s practice, so we had to rely on their subjective self-assessment. therefore, the responses might not have accurately reflected the true levels of knowledge, attitude and behavior, and thus, the reported level of practice might be lower than the real level. it is important for any hospital or a dental clinic to set up cdc protocol to prevent the spread of infectious and transmissible diseases. for this purpose, it is important that the dental health care professionals be aware of the risks and the seriousness of infections. educational programs on infection control isolation precautions for all the health care workers, especially the dental health care professionals, and the facilities to allow compliance with the infection policies are necessary to lessen the infection hazards among dental health care professionals and their patients. acknowledgements we wish to thank all the participants who took part in our study. references 1. smith aj, cameron so, bagg j, kennedy d. management of needlestick injuries in general dental practice. br dent j. 2001; 23: 12-5. 2. araujo mw, andreana s. risk and prevention of transmission of infectious diseases in dentistry. quintessence int. 2002; 33: 376-82. 3. takahama aj, tatsch f, tannus g, lopes ma. hepatitis c: incidence and knowledge among brazilian dentists; community dent health. 2005; 22: 184-7. 4. askarian m, mirzaei k, honarvar b, etminan m, araujo mw. knowledge, attitude and practice towards droplet and airborne isolation precautions among dental health care professionals in shiraz, iran. j public health dent. 2005; 65: 43-7. 5. paul t, maktabi a, almas k, saeed s. hepatitis b awareness and attitudes amongst dental health care workers in riyadh, saudi arabia. odontostomatol trop. 1999; 22: 9-12. 6. goebel wm. reliability of medical history in identifying patients likely to place dentists at an increased hepatitis risk. j am dent assoc. 1979; 98: 90713. 7. kiselova a, ziya d. cross-infection in dentistry and its control ohdmbsc. 2005; 1: 24-9. 8. qudeimat ma, farrah ry, owais ai. infection control knowledge and practices among dentists and dental nurses at a jordanian university. teaching center. am j infect control. 2006; 34: 218-22. 9. garner js. guideline for isolation precautions in hospitals. part i.evolution of isolation practices, hospital infection control practices advisory committee. am j infect control. 1996; 24: 24-31. 10. saheeb bdo, offor e, okojie oh. cross infection control methods adopted by medical and dental practitioners in benin city, nigeria. ann afr med. 2003; 2: 72–6. 11. utomi il. attitudes of nigerian dentists towards hepatitis b vaccination and use of barrier techniques. west afr j med. 2005; 24: 223-6. 12. yaacob hb, samaranayake lp. awareness and acceptance of the hepatitis b vaccine by dental practitioners in malaysia. j oral pathol med. 1989; 18: 236-9. 13. reingold al, kame ma, hightower aw. features of gloves and other protective devices to prevent transmission of hepatitis b. virus to oral surgeons. j am med assoc. 1988; 259: 2558–60. 14. lowbury ejl, lilly ba, ayliffe g aj. preoperative disinfection of surgeons’ hands: use of alcoholic solutions and effects of gloves on skin flora. br med j. 1974; 4: 369-72. 15. ugbam ga. comparative study of different scrubbing agents in surgical practice. west afr j med. 1988; 5: 13-9. 16. field ea, martin mv. hand washing: soap or disinfectant? br dent j. 1986; 160: 278-80. 17. ashish b, manoj ag, pallav r, shubham g, sreenivas v, jacob mp. calculating prevalence of hepatitis b in india: using population weights to look for publication bias in conventional meta-analysis. indian j pediatr. 2009; 76: 1247-57. 18. barlean l, ianc ls, minea ml, danila i, baciu d. airborne microbial contamination in dental practices in iasi, romania. ohdmbsc. 2010; 9: 16-20. 19. saravanan s, velu v, kumarasamy n, shankar em , nandakumara s, murugavel kg et al .the prevalence of hepatitis b virus and hepatitis c virus infection among patients with chronic liver disease in south india. int j infect dis. 2008; 12: 513-8. 20. mathews rw, scully cm, dowell tb. acceptance of hepatitis b vaccination by auxiliary dental personnel in the united kingdom. health trends. 1987; 9: 371-3. 21. mccartan be, samaranayake lp. awareness and acceptance of hepatitis b vaccine by irish dental practitioners. j ir dent assoc. 1988; 33: 33-6. 22. samaranayake lp, scully c, dowell tb, lamey pj, macfarlane tw, matthews rw et al. new data on the acceptance of the hepatitis b vaccine by dentalpersonnel in the united kingdom. br dent j. 1988; 164: 74-7. 23. harrel sk, barnes jb, rivera-hidalgo f. aerosol and splatter contamination from the operative site during ultrasonic scaling. j am dent assoc. 1998; 129: 1241-9. 24. timmerman mf, menso l, steinfort j, van winkelhoff aj, van der weijden ga. atmospheric contamination during ultrasonic scaling. j clin periodontol. 2004; 31: 458-62. 25. azari mr, ghadjari a, nejad mrm, nasiree nf. airborne microbial contamination of dental units. tanaffos. 2008; 7(2): 54-7. knowledge, attitude and practice on hepatitis b prevention among dental professionals in india oral sciences n3 braz j oral sci. 10(4):277-281 received for publication: september 26, 2011 accepted: december 07, 2011 original article braz j oral sci. october | december 2011 volume 10, number 4 effect of post length on endodontically treated teeth: analyses of tensile strength jefferson ricardo pereira1, accácio lins do valle2, fábio kenji shiratori3, janaina salomon ghizoni4 1phd, department of prosthodontics, dental school, university of southern santa catarina – unisul, brazil 2phd, department of prosthodontics, bauru dental school, university of são paulo, brazil 3msc, department of prosthodontics, bauru dental school, university of são paulo, brazil 4msc, department of prosthodontics, dental school, university of southern santa catarina – unisul, brazil correspondence to: jefferson ricardo pereira rua rio grande do sul 1901 apart 303 mar grosso laguna – sc cep: 88790-000 brazil phone: +55 (48) 36471571. fax: +55 (48) 36264088 e-mail: jeffripe@rocketmail.com abstract aim: this study compared the tensile strength of endodontically treated teeth restored with different posts and cores with different post lengths. methods: sixty extracted intact canines were randomly divided into 6 groups. groups cp1, cp2 and cp3 were restored with custom cast postand-core and groups pf1, pf2 and pf3 were restored with prefabricated post and composite resin core, with different combinations of post length of 5,0 mm, 7.5 mm and 10 mm, respectively (n = 10). all teeth were restored with a total metal crown. a tensile loading was applied at a 180degree angle to the long axis until failure. results: the 2-way analysis of variance (á=0.05) showed statistically significant difference (p<0.001) among the groups. however, when the mean fracture forces for the groups were compared (groups 1, 2, 3, 4, 5 and 6: 134.5 n (34.2), 178.9n (40.1), 271,5 n (55.9), 161.7 n (22.0), 216.1 n (42.0) and 257.9 n (41.0), respectively), no significant differences could be detected among the groups restored with prefabricated post and cast post-and-core. it was found significant differences when it was compared the different lengths for each type of post (p<.05). conclusions: this study showed that increasing post length significantly increased the tensile strength of prefabricated posts and cast post-and-core used in endodontically treated teeth. on the other hand, significant differences were not found when comparing endodontically treated teeth restored with custom cast post-and-cores or pre-fabricated posts and composite resin cores with the same post length. keywords: post and core technique, composite resin, tensile strength. introduction when coronal tooth structure is missing, custom cast post-and-core has been regarded as the “gold standard” in post-and-core restorations due to its higher success rate 1-2. alternatives to cast post-and-core have been developed. prefabricated post systems simplify the restorative procedure because all steps can be completed chairside, and a clinical success can also be expected1-10. the intact teeth are known to present a lower risk of biomechanical failure than endodontically treated teeth11. the substantial loss of tooth structure during endodontic access, post-space preparation and cavity preparation are generally accepted explanations for the increased failure rates of endodontically treated teeth12. posts are necessary to allow the clinician to rebuild enough tooth structure to retain restorations13, but the ‘price’ for additional retention, may be an increased risk to damage tooth structure. 278 braz j oral sci. 10(4):277-281 an unresolved problem using posts and cores is the length of a post relative to root length14. laboratory studies have shown that increasing the length of the post in teeth with post-and-core systems results in a more favorable stress distribution along the post15-17 and an increased post length improves the resistance of the restored tooth to fracture10-18. furthermore, in a clinic study an increased survival rate has been correlated with increasing post length11. however, a previous study showed a minimal difference in stress distribution between varying post lengths 19, while other authors observed that an increase in post length as such will not necessarily increase the fracture resistance of the tooth8,20. the pattern of failure was changed since the introduction of pre-fabricated post and, because of this, the irreversible fracture of roots has become a rare occurrence2-10. however, adhesive failure, which is clinically expressed as decementation of the post, has become the main failure mode when pre-fabricated posts are used21-23. it is important to notice that it may not always possible to use a long post, especially when the remaining root is short or curved. it has been suggested that it is important to preserve 3 to 5 mm of apical gutta-percha to maintain the apical seal24. the purpose of this study was to evaluate the relative effect of post length (length of the vertical dentinal overlap of the crown) and type of post and core on the tensile strength when it is used to restore endodontically treated teeth. the research hypotheses were that there was a significant difference in the effect of post length on the fracture resistance; and there was a significant difference between the types of posts. material and methods sixty recently-extracted maxillary canines with similar root sizes (between 15 mm and 16 mm (measured with a millimeter ruler from the apex until the cementoenamel junction cej) were selected from 173 maxillary canines extracted for periodontal reasons and stored in distilled water at 37º c during the course of the study. the inclusion criteria for tooth selection were teeth without cracks, caries, restoration, and/or roots longer than 15 mm. the roots were scaled wet with periodontal curettes. the crowns were reduced perpendicular to the root axis with double-faced diamonds discs (kg sorensen, barueri, sp, brazil), leaving a standardized root length of 15 mm. the endodontic treatment was done using a standard master apical file international standardization organization (iso) 20 (dentsply ind. e com. ltda., petrópolis, rj, brazil) that extended 1 mm beyond the apex and the preparation took place with a conventional step-back technique to an iso 35 file (dentsply ind. e com. ltda.) at the apical constriction. the teeth were obturated by lateral condensation using gutta-percha points (tanari, tanariman industrial ltda., manaçapuru, am, brazil) and an iso 35 primary gutta-percha master cone (tanari, tanariman industrial ltda.). root canal cement (endometazone ivory; septodont brasil, barueri, sp, brazil) was used as the sealer. after this, the teeth were randomly divided into 6 groups of 10 teeth each by drawling lots. different post preparations were standardized using a #5 reamer (largo; dentsply ind. e com. ltda.). five millimeters of gutta-percha were removed (apical to the cementoenamel junction (cej)) from each filled canal in groups cp1 and pf1, it was removed 7.5 mm in groups cp2 and pf2, and it was removed 10 mm in groups cp3 and pf310. for the groups cp1, cp2 and cp3 the tooth was restored with a custom cast post-and-core. impressions of the root canals were made with acrylic resin (duralay, reliance dental mfg. co. chicago, il, usa). the cores were standardized using a core-forming matrix (tdv dental, pomerode, sc, brazil). the patterns were invested (cristobalite, whip-mix corporation, louisville, ky, usa) and cast in cu-al alloy (npg, aalbadent, cordelia, ca, usa). after casting, small nodules were removed if present. the post-cores were sectioned and seated to their corresponding teeth. resin modified glass ionomer cement (rely x luting, 3m espe, st. paul, mn, usa) was used to cement all posts. the cement mix was prepared according to the manufacturer’s instructions and inserted into the canal with a lentulo spiral (dentsply ind. e com. ltda.) mounted on a low-speed handpiece. cement was placed on the post and seated under 9 kg of pressure. after 2 min, the excess of the cement was removed. even under pressure, the setting time of the cement was completed (5 min) and after 10 min the pressure was released and each specimen was returned to storage in distilled water. for the groups pf1, pf2 and pf3 the canals were restored with prefabricated stainless steel, parallel-sided, serrated posts with a tapered end (number 5317, screw-post, euro-post anthogyr s.a., sallanches, france). in these groups the teeth were cemented with the same material and the same technique as used in the other groups. the coronal portion was made with composite resin material (filtek z250; 3m espe).the root surfaces and cervical dentin was etched with 37% phosphoric acid for 30 s, rinsed, and air dried. two layers of bonding agent (prime&bond 2.1; dentsply ind. e com. ltda.) was applied to the cervical dentin and the coronal portion of the post and were light-polymerized for 20 s using a halogen light-curing unit (ultraled, dabi atlante, ribeirão preto, sp, brazil) with 450 mw/cm2 light intensity. cores were fabricated in a standardized form, using the same coreforming matrix as used in the other groups. five increments of the composite resin were applied to complete the coronal core, each requiring 40 s of photo-activation. the tip of the light guide of the light-curing unit was positioned on the top of the core at a distance of 1 cm from the specimens. all teeth were prepared with a round diamond bur (#3216; kg sorensen) in a high-speed handpiece with water spray cooling (super torque 625 autofix; kavo do brasil ind. com. ltda., joinville, sc, brazil) to simulate a crown preparation with 1.5 mm of facial reduction with a chamfer finish line and 0.5 mm of chamfered lingual reduction and receive a total metal crown. all the finish lines, for all specimens and groups, were placed at the cej level. crown wax (kerr corporation, orange, ca, usa) patterns were then made for the specimens with the aid of the impression made using a vinyl polysiloxane impression material (aquasil, dentsply, effect of post length on endodontically treated teeth: analyses of tensile strength braz j oral sci. 10(4):277-281 279 konstanz, germany) of the tooth prior to preparation. a lingual ledge was added to create a standard loading point. the wax patterns were sprued, invested (cristobalite; whipmix corporation) and cast in a ni-cr alloy (durabond, são paulo, sp, brazil) (figure 1). crowns were cemented using the same material used with the posts. teeth were embedded in acrylic resin (clássico, artigos odontológicos s/a, são paulo, sp, brazil) poured into molds made of same material (30 mm in height, and diameter of 22 mm and a internal space, located in the center of the mold, with diameter of 10 mm and 20 mm in height) along their long axes using a surveyor (bio art equipamentos odontológicos ltda., são carlos, sp, brazil). the specimens were tested in a universal testing machine (kratus k2000 mp, dinamometros kratos ltda, são paulo, sp, brazil (figure 2) 7 days after post cementation. each specimen was affixed in a apparatus that allowed it to be positioned at 180 degrees to the buccolingual long axis (figure 2). the testing machine was set at a crosshead speed of 0.5 mm/ min. the load was measured in n. failure threshold was defined as the point at which a specimen could no longer withstand increasing load and the dislodgment of the crown or the post or fracture of the post-core complex, or root occurred. the mode of failure was recorder after the test using a x4 binocular loupe (bio art equipamentos odontológicos ltda.). two-way analysis of variance (anova) was used to determine the overall differences among the mean values of the groups and the overall variability within the groups. tukey’s multiple comparison test was used to establish intergroup differences (α= 0.05). fig. 1. specimens embedded in acrylic resin before tensile strength testing in a universal testing machine. groups mean (n) s d cp1 134.5a (34.2) pf1 161.7ab (22.0) cp2 178.9bc (40.1) pf2 216.1c (42.0) pf3 257.9d (41.0) cp3 271.5d (55.9) table 1. resistance to failure values of test specimens, means (n) of test groups (standard deviation) and statistical comparisons groups with same superscripted letters are not significantly different at p< 0.05 (tukey’s test). fig. 2. specimens subjected to tensile load at 180o in a universal testing machine. results table 1 summarizes the mean tensile strength for the 6 tests groups. anova showed that 1 or more of the conditions were significantly different from each other (p<0.001). the tukey’s test confirmed that the mean tensile strength for the group pf3 was significantly higher (p<0.001) than those of groups pf2 and pf1 while the mean tensile strength for the group cp3 was significantly higher (p<0.001) than those of groups cp2 and cp1. no statistically significant difference (p>0.05) was found when comparing the mean tensile strengths of the groups with the same post length (table 1). discussion although this study was an in vitro experiment, it was observed that shorter posts were the main cause of post decementation independently if cast post-and-core or preeffect of post length on endodontically treated teeth: analyses of tensile strength 280 braz j oral sci. 10(4):277-281 fabricated post had been used. this result suggests that it is important to use posts as longer as possible to achieve the treatment success. the limitations of this study include the facts that in vitro design does not replicate oral conditions and that a single load was used to test the tensile strength of different posts used to restore endodontically treated teeth. for more meaningful results, future studies should incorporate thermal cycling of the specimens and fatigue loading. the present study accepted the hypothesis that there is a significant difference in the effect of post length on the fracture strength, it rejected the hypothesis that there is a significant difference between the types of posts. some studies observed that teeth restored with cast postand-core systems present higher fracture strength when post length is increased10,15,17. otherwise, others authors10,20 showed that increasing of post length in teeth restored with prefabricated posts and composite resin core did not increase significantly the fracture strength of endodontically treated teeth. however, post decementation has been pointed as the main cause of failure when pre-fabricated posts are used21-23. not only does the material of the root canal posts affect the retention of the restorations, but also the properties of the luting agents may influence the bonding strength. this study used glass ionomer cement in accordance with the manufacturer’s instructions to cement all posts. glass ionomer cements adhere to dentin via chemical and micromechanical retention25. it is well know that these materials set by means of two distinct reactions25. the first reaction consumes all of the available water in its composition, and a second reaction occurs only when water is available from the dentin26-27. there is an initial contraction due to the setting reaction 28 and then cement maturation leads to a hygroscopic expansion29. this can result in a more intimate adaptation between cement and dentin30, which increases resistance to displacement of the post29. frictional retention is directly proportional to the contact area (the larger the contact surfaces, the better the retention). the hygroscopic expansion and larger contact areas could explain the results of this study, in which a statistically significant difference was found for post length (p<0.001), with the 10-mm-long posts of both post systems requiring greater force to dislodge than 5-mm and 7.5-mmlong posts. in addition to the contact area, closer contact between cement type and dentin is also important in order to improve the frictional retention of the post31. it has been demonstrated that the cementation procedure is a highly significant factor affecting the retention of cast post-and-core systems. in the present study, the cement was inserted into the canal with a lentulo spiral at low speed, which has been shown to produce significantly greater values of retention32-35. this can be justified by taking into account the most critical problem encountered in cementation, which is air entrapment through the liquid cement to create voids, thereby compromising the physical properties of the cement film36. turner37 (1981) suggested that those voids are responsible for the unexpected low retentive values for dowels. the lentulo spiral can ensure complete coating of the post space walls without inclusion of air bubbles, and results in an even layer of luting cement around the post, which will result in increase of the retention in the cement-tooth interface and cement-post interface32. these considerations, together with hygroscopic expansion and higher contact areas produced by the cement, could explain the lack of significant difference in retention between cast post-and-cores and pre-fabricated posts when the same post length was compared in the present study. salvi et al.38 (2007) showed that after 4 years of follow up, from 166 pre-fabricated posts and 82 cast post-and-cores only 4 posts lost retention, all of them were cast ones. although the tensile strength of pre-fabricated posts and cast post-and-cores were statistically similar, the pre-fabricated posts presented higher strength, when they are restored with 5-mm and 7.5-mm-long posts. gómez-polo et al.39 (2010) found no significant difference between pre-fabricated posts and cast post-and-cores after a mean follow up period of 10 years. in conclusion, this study showed that increasing post length significantly increased the tensile strength of prefabricated posts and cast post-and-core used in endodontically treated teeth. on the other hand, significant differences were not found when comparing endodontically treated teeth restored with 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during setting. j dent res. 2000; 79: 818-23. 29. cury ah, goracci c, de lima navarro mf, carvalho rm, sadek ft, tay fr, et al. effect of hygroscopic expansion on the push-out resistance of glass ionomer-based cements used for the luting of glass fiber posts. j endod. 2006; 32: 537-40. 30. huang c, kei lh, wei sh, cheung gs, tay fr, pashley dh. the influence of hygroscopic expansion of resin-based restorative materials on artificial gap reduction. j adhes dent. 2002; 4: 61-71. 31. 31.macedo vc, faria e silva al, martins lr. effect of cement type, relining procedure, and length of cementation on pull-out bond strength of fiber posts. j endod. 2010; 36: 1543-6. 32. al-omari wm, zagibeh am. the retention of cast metal dowels fabricated by direct and indirect techniques. j prosthodont. 2010; 19: 58-63. 33. reel dc, hinton t, riggs g, mitchell rj. the effect of cementation method on the retention of anatomic cast post and cores. j prosthet dent. 1989; 62: 162-5. 34. fakiha z, al-aujan a, al-shamrani s. retention of cast post cemented with zinc phosphate cement using different cementing techniques. j prosthodont. 2001; 10: 37-41 35. goldestein gr, hudis sl, weintraub de. comparison of four techniques for the cementation of posts. j prosthet dent. 1986; 55: 209-11. 36. assif d, bitenski a, pilo r, oren e. effect of post design on resistance to fracture of endodontically treated teeth with complete crowns. j prosthet dent. 1993; 69: 36-40. 37. turner ch. cement distribution during post cementation. j dent. 1981; 9: 231-9. 38. salvi, ge, siegrist guldener b e, amstad t, joss a, lang np. clinical evaluation of root filled teeth restored with or without post-and-core systems in a specialist practice setting. int endod j. 2007; 40: 209-15. 39. gómez-polo m, llidó b, rivero a, río j, celemin a. a 10-year retrospective study of the survival rate of teeth restored with metal prefabricated posts versus cast metal posts and cores. j dent. 2010; 38: 916-20. 281effect of post length on endodontically treated teeth: analyses of tensile strength oral sciences n3 braz j oral sci. 11(1):14-18 original article braz j oral sci. january | march 2012 volume 11, number 1 received for publication: july 12, 2011 accepted: january 03, 2012 atraumatic restorative treatment used for caries control at public schools in piracicaba, sp, brazil cristina gibilini1, janice simpson de paula1, regina marques2, maria da luz rosário sousa3 1phd student, graduate program in dentistry, sub-area dental public health, department of community dentistry, piracicaba dental school, university of campinas, brazil 2phd in public health and oral health coordinator of the municipality of são paulo, brazil 3professor, department of community dentistry, piracicaba dental school, university of campinas, brazil correspondence to: maria da luz rosário sousa faculdade de odontologia de piracicaba avenida limeira 901, bairro areão. cep 13414-903, piracicaba, sp, brazil. phone: +55 19 2106-5209 e-mail: luzsousa@fop.unicamp.br abstract aim: the aims of this study were (1) to evaluate the atraumatic restorative treatment (art) as a measure to control caries at public schools in piracicaba, sp, brazil, discussing the importance of the promotion of individual and collective health and (2) to evaluate the longevity of art restorations in the first six months after placement. methods: the sample consisted of 173 schoolchildren aged 5 to 10 years from public schools in piracicaba, treated according to art approach and reassessed after six months. results: it could be observed that after art, a large part of the students initially fitted into risk e classification (high caries risk) were reclassified into risk b classification (moderate caries), characterized by the presence of restored teeth only (caries free teeth; c = 0). in addition, 81.78% success rate was obtained after six months of art. conclusions: art was considered effective in health promotion programs, given its importance as a measure to control caries in schoolchildren. keywords: dental atraumatic restorative treatment, dental caries, public health. introduction although dental caries prevalence has declined significantly over the last few years, it is still considered a public health problem in brazil and worldwide. this fact is confirmed when the data of the last national epidemiologic survey are analyzed, indicating that among 5-year-old children approximately 60% presented at least one primary tooth with caries experience, and 70% of the 12year-olds presented caries in their permanent dentition1. in view of this situation, dental control and follow-up are necessary during childhood and adolescence for preventing or attenuating the affections caused by caries disease. with the goal of encouraging health promotion measures required for caries control and prevention, the world health organization (who) proposed the program of atraumatic restorative treatment (art) for countries with economically underprivileged or marginalized communities2. art has been considered a feasible treatment option, as it is based on the contemporary philosophy of minimum intervention and maximum preservation of the tooth, 1515151515 braz j oral sci. 11(1):14-18 and can be applied to the entire population. art consists of a simplified, low-cost technique that uses only hand instruments in the removal of carious tissues to reduce contamination and viability of microorganisms3, and requires immediate restoration of the cavity with and adhesive restorative material, namely glass ionomer. moreover, the art approach causes less anxiety in children than the traditional restorative approach2,4. it is worth mentioning that the use of high-viscosity glass ionomer cements has been outstanding, as it facilitates the technique and reduces the technical difficulties, especially in public services5. in this context, studies have pointed out the clinical performance of the glass ionomer cement ketac molar® (3m espe)6-7. other studies have found better results for glass ionomer over conventional amalgam in restorations in primary teeth8-10. because of properties such as continuous fluoride release and fluoride ion recharging capacity, glass ionomer cements act as a constant reservoir of fluoride in the oral cavity, and participate in the deand remineralization processes, contributing to the good clinical performance of the restorations8. various longitudinal studies have been conducted to assess the efficacy of art. frencken et al.11 (1996) and holmgren et al.12 (2000) evaluated the longevity of restorations in class i and ii cavities for a period of 3 years with satisfactory results in 88.3% and 92% of them, respectively, in primary and permanent teeth. other longitudinal studies using art have shown various long-term success rates using ketac molar® (3m espe), with results that reach up to 21% of success within 36 months in permanent teeth12. van germert-schriks et al.13 (2007) found 43.4% success for oneface restorations and 12.2% for two-face restorations, using the same restorative material and same period of evaluation. research studies have indicated art as an effective method in the control of caries disease, with low operating costs, but further studies are necessary to evaluate its application in public health, including a more comprehensive assessment of the participants’ oral health. it should be noted that collective environments such as schools are ideal spaces for o ral health promotion actions, as they allow the development of group activities and contribute to the control and evaluation of interventions. in this context, the verification of caries activity is emphasized. the early diagnosis of incipient caries lesions at a reversible stage not only permits evaluating the determinant factors, but also helps determining the caries risk, which makes the treatment simpler, less invasive and less expensive based on the use of fluoride and changes in the patient’s behavior with respect to diet and oral hygiene14. thus, careful determination of caries risk is necessary so that preventive actions and specific control may be directed towards high-risk individuals even before the lesions develop15. caries risk assessment can be made both in the clinical environment and in other collective spaces16. the use of art in schoolchildren may help fulfilling the demand in public services as this approach allies a faster treatment outcome compared with conventional treatment1719 with caries control, which is of particular interest for children at higher risk for developing caries. thus, the aim of the present study was to evaluate art as a measure to control caries in public schools in piracicaba, são paulo, considering the longevity of restorations in the first six months after treatment, as well as discussing the importance of promoting public health. material and methods the present study was first sent to the research ethics committee of the piracicaba school of dentistry (university of campinas) for appreciation, and was approved under protocol no. 106/2009. the children’s and parents’/ guardians’ consent was obtained. the convenience sample analyzed 1,167 children in the age range between 5 and 10 years old. a total of 173 schoolchildren with indication for art were included, totalizing 330 teeth. the children came from six public schools (olívia caprânico, josé pousa de toledo, benedito de andrade, francisco a. kronka, antônio rodrigues domingues and melita l. brasiliense) in the municipality of piracicaba, são paulo, which participate in prevention programs. the municipality of piracicaba is located 160 km from são paulo, in the southwestern region of the state, and has 368,029 inhabitants20. it has had fluoridated public water supplies since 197121 and at present 99.38% of residents are favored with water supply20. the human development index (hdi) of the municipality was 0.836 in 200019-20. the mean dmft index (“d”-decayed; “m”-missing;”f”-filled; “t”-teeth) among preschool children was 1.30 in 200622 and 1.32 in 201023. before treatment, an epidemiologic survey of dental caries was conducted to obtain the children’s dmft and caries risk classification, as well as to verify the caries cavities with indication for art. the dmft index was determined in accordance with the who criteria24 and the caries risk evaluation was obtained in accordance with the criteria of the state secretariat of health16, according to which the schoolchildren are classified into five different categories: a (healthy teeth), b (presence of restoration), c (presence of chronic caries), d (presence of plaque, gingivitis or white spot lesion), e (presence of acute caries) and f (urgency) (table 1). the criterion used for the indication of art followed the criteria described by frencken et al.2 (1996), with evaluation of primary teeth only. thus, teeth with cavited dentin caries lesion that could be accessed with a 0.9-mmdiameter excavator were included. teeth with evident pulp involvement or painful symptomatology were excluded from the sample. a trained dentist performed art in accordance with the protocol proposed by frencken et al.2 (1996). the treated caries cavities were restored with glass ionomer ketac molar® (3m espe), in accordance with the manufacturer’s instructions. the longevity of art restorations at 6 months was atraumatic restorative treatment used for caries control at public schools in piracicaba, sp, brazil braz j oral sci. 11(1):14-18 classification group individual situation low risk a absence of caries, plaque, gingivitis and/or white spot moderate risk b history of restored teeth, without plaque, gingivitis, and/or white spot c one or more cavities in a situation of chronic carious lesions, but no plaque, no gingivitis, and/or white spot high risk d absence of caries and/or restored teeth, but with presence of plaque, gingivitis and/or white spot e one or more cavities in a situation of caries f presence of pain or abscess table 1.table 1.table 1.table 1.table 1. table used as a reference for the classification of individuals according to the risk of caries. health department of são paulo state, (2000) age (years) gender total dmft (mean) decayed component “d” (mean) m f 5 4 6 10 3 2.80 6 23 20 43 4.05 3.56 7 18 24 42 3.31 2.69 8 16 21 37 3 2.00 9 16 16 32 3.34 2.28 10 4 5 9 3.60 2.00 total 81 92 173 3.38 2.55 table 2.table 2.table 2.table 2.table 2. distribution of children by age, gender, mean dmft and “decayed” component in the173 schoolchildren participating in the study. risk classification before art classification after art % n % a b 81 46.9 c d 35 20.2 e 173 100 57 32.9 f total 173 100 173 100 table 3.table 3.table 3.table 3.table 3. distribution of the risk evaluation among schoolchildren before and after art evaluated according to the criteria proposed by taifour et al.8 (2002), with the use of a flat oral mirror and cpi probe by a single calibrated examiner, with intra-examiner agreement of 84.2%. the criteria used for evaluating the restorations were described and grouped into 3 categories8,25: a) success (restorations present with a maximum of 0.5 mm marginal defect); b) failure (restorations with a marginal defect >0.5mm, fracture of the restoration or tooth, restoration completely lost); and c) not evaluated (absent due to natural exfoliation of the tooth or replacement of the glass ionomer restoration by another type of restorative material) or excluded (children who missed the evaluation visit, moved to another school or completed primary schooling in the period between baseline and reevaluation). the data were tabulated and analyzed by descriptive statistics, using the spss software (spss inc., shicago, il, usa) version 17.0. results it could be observed that among the 173 schoolchildren subjected to art, 81 (46.82%) were boys and 92 (53.18%) were girls. the mean age was 7.36 years (sd 1.36), ranging between 5 and 10 years. the descriptive data are presented in table 2. at the time of the epidemiologic survey (baseline) the children selected for treatment according to art presented a dmft equal to 3.38, with mean number of decayed teeth equal to 2.55. after treatment, the children were re-evaluated and presented a mean of the ‘decayed’ component of dmft equal to 0.72. in addition, in the previous survey, the schoolchildren were classified with regard to the criterion of risk16 and reclassified after art had been performed. in the first evaluation, all participants fitted into the risk e classification, due to the presence of carious teeth. after art, a large part of the schoolchildren were re-classified into risk b, characterized by the presence of restored teeth only (no caries lesions). nevertheless, it is worth pointing out that even with the reclassification, 53.1% of the schoolchildren presented teeth without indication for art, but with the need for curative and/or preventive treatment. thus, 20.2% of the participants still presented white spot lesions, plaque or gingivitis in some teeth, fitting into risk d classification. the remainder of the schoolchildren (32.9%) still presented carious teeth with contra-indication for art, keeping them in risk e (presence of acute cries), as demonstrated by the distribution in table 3. six months after the initial treatment, a new evaluation/ follow-up was performed (table 4). thus, 36 (10.9%) of the 1616161616atraumatic restorative treatment used for caries control at public schools in piracicaba, sp, brazil 1717171717 braz j oral sci. 11(1):14-18 table 4.table 4.table 4.table 4.table 4. re-evaluation 6 months after art. no. of teeth % success 220 81.78 failure 49 18.21 total 269 100.0 teeth re-evaluated presented another type of restorative material, or had exfoliated naturally, and 25 teeth (7.6%) were not evaluated because the children had moved to another school, making longitudinal evaluation of the restoration impossible. therefore, 269 (81.51%) of the 330 teeth initially treated were re-evaluated 6 months after treatment with a success rate of 81.78%. discussion from the 173 schoolchildren subjected to art, 67% shifted from caries risk classification e (acute caries) to risk classification b (restoration) or d (white spot lesion, plaque and/or gingivitis). these data demonstrate that art per se improved the oral status of a large percentage of the children treated, as described by frencken et al.5 (2010). nevertheless, among the schoolchildren that shifted from risk e to risk d (20.2%), implementation of educational measures allied with motivation to perform oral hygiene is still necessary to decrease the prevalence of biofilm, white spot lesions and gingivitis. thus, the children fitted into risk d could, by means of simpler actions than art, shift into risk b with improved oral health, and consequently, quality of life. therefore, art must be considered a measure for promoting public health and should be performed in conjunction with practices in health education and preventive programs to be effective in controlling the etiologic factors of caries26-27. furthermore, in spite of art, 32.95% of the schoolchildren remained in the risk e classification, that is, continued to have caries, which could be justified by two situations: 1) teeth with cavities with pulp involvement or painful symptomatology (contraindicated for art); 2) presence of carious permanent teeth, not included in the indications for art in this study. in spite of the mentioned factors, an improvement in the schoolchildren’s oral health was observed by the reduction in the mean of the ‘decayed’ component of dmft from 2.55 to 0.72, proving the effectiveness of this type of treatment. art improves the oral status, preventing caries progression to more complex levels, such as the need for endodontic treatment and extractions. this occurs because art fits into the concept based on the prevention and early interception of the caries process, with the goal of performing interventions that are as conservative as possible2,11,28. in addition, the treatment allows a reduction in the level of streptococcus mutans in the oral cavity, and consequently, the risk of new carious lesions3. considering the large number of children that wait for treatment at the family health units or basic health units, art can collaborate significantly in fulfilling the demand of these pediatric patients, being an important form of caries control and prevention in the family health program5,29-30. it is also worth emphasizing that as a low-cost, simple and fast (mean of 6 min per restoration) technique art may optimize the time and human resources that would be required, for example, to transport these children to a location outside the school environment during treatment, as has been described by figueiredo et al. 29 (2004); schriks and amerongen17 (2003) and pellegrinetti et al.18 (2005). however, a limiting factor as far as minimal dental intervention is concerned, is the overall difficulty of acceptance by dentists. rios et al.31 (2006) evaluated professionals from the public service in goiânia and found that less than half of the professionals (42.8%) believed in the efficacy of the technique, and that more information would be required about the technique, its efficiency and its use in public service. in spite of the difficulties encountered, mainly in following up the restorations, art continues to provide an important scope for covering schoolchildren as well as the community in general, taking into account the shorter time for performing restorations and its low cost5. in this context, mickenautsch et al.32 (2002) reported that the introduction of art into a mobile dental assistance program led to a significant reduction in the percentages of amalgam restorations and extractions. according to the results of the present study, art appeared as a feasible alternative for caries treatment, particularly in collective health, with proven efficacy, low cost and broad improvement in covering dental care to schoolchildren. these factors may be observed by the technique simplicity and speed of the procedures and contribution to the problem of the huge repressed demand by patients with treatment needs. it could therefore be concluded from this six-month evaluation that art was effective within health promotion programs as an important measure to control caries disease in schoolchildren. acknowledgements the authors thank the “coordenação de aperfeiçoamento de pessoal de nível superior – capes” for granting scholarships and for constantly supporting research in graduation programs at the piracicaba school of dentistry (fop/unicamp). references 1. brazil. ministry of health. secretary of health care. department of primary care. project “sb brasil” 2003. oral health status of the brazilian population 2002-2003: principal results. brasília: ministry of health, 2004. 2. frencken je, pilot t, songpaisan y, phantumvanit p. atraumatic restorative treatment (art): rationale, technique and development. j public health dent 1996; 56:135-40. 3. roshan nm, anand ls, deshpande sd. microbiological evaluation of salivary streptococcus mutans from children of age 5-7 years, preand post-atraumatic restorative treatment. contemporary clinical dentistry 2010; 1(2): 94-7. 4. almeida neves a, cutinho e, cardoso mv, lembrechts p, van meerbeek b. current concepts and techniques for caries excavation and adhesion to residual dentin. j adhes dent 2011 feb; 13(1): 7-22 atraumatic restorative treatment used for caries control at public schools in piracicaba, sp, brazil braz j oral sci. 11(1):14-18 1818181818atraumatic restorative treatment used for caries control at public schools in piracicaba, sp, brazil 5. frencken je. the art approach using glass ionomers in relation to global oral health care. dental materials, 2010, 26: 1-6. 6. menezes jpl, rosenblatt a, medeiros e. clinical evaluation of atraumatic restorations in primary molars: a comparison between 2 glass ionomer cements. journal of dentistry for children 2006; 73:2: 91-7. 7. silva rc, zuanon acc, spolidorio dmp, campos jadb. antibacterial activity of four ionomer cements used in atraumatic restorative treatment. j mater sci: mater med, 2007; 18: 1859-1862. 8. taifour d, francken je, beiruti n, van´t hof ma, truin gj. effectiveness of glass ionomer (art) and amalgam restorations in the deciduous dentition: results after 3 years. caries res 2002;36:437–444. 9. frencken je, van’t hof ma, taifour d, al-zaher i. effectiveness of art and traditional amalgam approach in restoring single-surface cavities in posterior teeth of permanent dentitions in school children after 6.3 years. community dent oral epidemiol 2007; 35: 207–214. 10. yu c, gao xj, deng dm et al. survival of glass ionomer restorations placed in primary molars using atraumatic restorative treatment (art) and conventional cavity preparations: 2year results. int dent j 2004; 54:42-46. 11. frencken je, makoni f, sithole wd. art restorations and glass ionomer sealants in zimbabwe: survival after 3 years. community dent oral epidemiol 1998; 26: 372-81. 12. holmgren cj, lo ecm, hu dy, wan hc: art restorations and sealants placed in chinese school children – results after three years. community dent oral epidemiol 2000; 28: 314–20 13. van gemert-schriks mcm, van amerongen we, ten cate jm, aartman iha. three-year survival of singleand two-surface art restorations in a high-caries child population. clin oral invest 2007; 11:337–343. 14. couto gbl, vasconcelos mmvb, melo mmc, camelo cac, valença pam. prevalence of caries, white spot and visible plaque in children 036 months, assisted by the family health program in the city of camaragibe pe. odontol clín-cient 2005; 4:19-28. 15. sant´anna, g.r.; bonecker, m.j.s.; diarte, d.a.; suga, s.n. book of pediatric dentistry; sonetos ed. 2002 apud martins, a.a., santos, a.a.; barreto, m.a.c.; oliveira, c.c.c. table of risk factors for decay applicable in public health.odontologic . clín. científ., recife, 2003; 2(1): 37-42, jan/abr. 16. ministry of health. ss-95 resolution, 27/06/2000. recommendations about the use of fluoride products in the “sus” / sp according to the risk of dental caries. [on line] available in url: http://www.saude.sp.gov.br/ fr_sbucal.html. 17. schriks mcm, van amerongen we. atraumatic perspective of art: psychological and physiological aspects of treatment with and without rotary instruments. community dent oral epidemiol 2003; 31: 15–20. 18. pellegrinetti mb, imparato jcp, bressan mc, pinheiro sl, echeverria s. evaluation of the glass ionomer cement retention in cavities restored by atypical atraumatic restorative technique. pesq bras odontoped clin integr 2005; 5(3): 209-13. 19. ibge. brazilian institute of geography and statistics. data de piracicaba. available in: http://www.ibge.gov.br. 20. são paulo (state). ministry of economy and planning. foundation state system of data analysis. municipality of piracicaba profile [cited in 2011 nov, 21]. available em: http://www.seade.sp.gov.br/produtos/perfil/ perfil.php. 21. basting rt, pereira ac, meneghim mc. evaluation of the dental caries prevalence in schoolchildren from piracicaba sp, brasil, after 25 years of fluoridation of public water supply. rev odontol univ são paulo 1997; 11(4):287-92. 22. cortellazzi, kl, tagliaferro eps, assaf av, ambrosano gmb, bittar to, meneghim mc, pereira ac. influence of socioeconomic, demographic and clinical experience of dental caries in preschool children in piracicaba, sp. rev bras epidemil 2009; 12 (3): 490-500. 23. guerra lm, pereira ac, pereira sm, meneghim mc. assessment of socioeconomic variables in the caries and fluorosis prevalence in cities with and without water supply fluoridation. rev odontol unesp. 2010; 39(5): 255-262. 24. word health organization. oral health surveys, basics methods. geneva: word health organization; 1997. 25. sacramento pa, borges afs, puppin rontani rm. actuarial analysis in a comparative study of two glass ionomer cements used in the technique of art: direct and indirect assessment after 3 and 6 months. tev odontol unesp 2009; 38(5):291-300. 26. lima dc, saliba na, moimaz sas. atraumatic restorative treatment and it aplication in public health. rgo 2008; 56:71-9. 27. navarro mfl, modena kcs, freitas mcca, fagundes tc. transfering art research into education in brazil. j appl oral sci. 2009; 17(sp issue): 99-106. 28. smales rj, yip hk. the atraumatic restorative treatment (art) approach for primary teeth; review of literature. pediatric dentistry 2000; 22(4):2948. 29. figueiredo ch, lima fa, moura ks. atraumatic restorative treatment: assessment of their suitability as a strategy for controlling dental caries in public health. rbps 2003; 17(3);109-18. 30. bresciani e. clinical trials with atraumatic restorative treatment (art) in deciduous and permanent teeth. j appl oral sci 2006; 14: 14-9. 31. rios le, essado rep, freire mcm. tratamento restaurador atraumático: knowledge and attitudes of dentists in the public service of goiânia-go. rev odontol unesp 2006; 35(1): 75-80. 32. mickenautsch s, munshi i, grossman es. comparative cost of art and conventional treatment within a dental school clinic. sadj. 2002; 57(2): 52-8. revista fop n 13 967 antimicrobial activity of two brazilian commercial propolis extracts giovanna pires da silva ribeiro de rezende1 fabiana cristina pimenta2 luciane ribeiro de rezende sucasas da costa3 1health sciences program sponsored by university of brasilia, df, brazil 2tropical pathology and public health institute, federal university of goias, go, brazil 3faculty of dentistry, federal university of goias, go, brazil received for publication: august 05, 2005 accepted: february 07, 2006 correspondence to: luciane ribeiro de rezende sucasas da costa rua j-34, qd. 60, lt.12, setor jao, goiânia-go, brazil, 74673-520 phone.: +55 62 32044075 fax: +55 62 32044075 e-mail: lsucasas@odonto.ufg.br abstract the antimicrobial activity of propolis extracts is well documented, but little is known about the antimicrobial properties of commercial products containing propolis, since these vary according to the geographical region in which the propolis is obtained. this study evaluated the antimicrobial activity of two samples of commercial propolis on 26 species of microorganisms obtained from atcc and some wild strains: gram-positive cocci and bacilli, and gram-negative rods and yeasts. the tested products were two samples of brazilian commercial propolis from apis flora™: 11.0% ethanolic extract of propolis (eep) and propomax™ 11.0% extract of propolis without alcohol (ep). antimicrobial activity was determined by the agar diffusion technique, well method. mic was determined for staphylococcus sp. and streptococcus mutans using the method of broth dilution with the propolis extract in serial concentrations. eep and ep showed antimicrobial activity against all tested bacteria and yeasts, having a more pronounced action against gram-positive bacteria and candida albicans atcc 10231, and a less evident activity against gram-negative and candida albicans ft2010. for s. mutans, the eep mic ranged from 8.8 to 4.4 ml of propolis, and the ep mic, from 4.4 to <1.1 ml. for staphylococcus sp., the mic of both extracts was <1.1. key words: microorganisms, propolis, antimicrobial activity braz j oral sci. january-march 2006 vol. 5 number 16 968 introduction the antimicrobial activity of propolis against gram-positive bacteria and yeasts is well documented1. however, this antimicrobial activity depends on the chemical composition of propolis2, which in turn seems to vary depending on the geographical region where it is extracted3-5. the main source of brazilian propolis is baccharis dracunculifolia dc, but its antimicrobial efficiency is controversial; that is, brazilian propolis may promote better or worse effects than that from other countries6. thus, while commercial products containing propolis from various regions of brazil are sold, their efficacy is not clear. the aim of this study was to evaluate the antimicrobial activity of two samples of commercial propolis on different groups of microorganisms including oral pathogens. material and methods twenty-six sample species of microorganisms obtained from the american type culture collection (atcc) and some wild strains from the laboratory of medical bacteriology, tropical pathology and public health institute, federal university of goias, brazil, were selected. they included gram-positive cocci and bacilli, and gram-negative rods and yeasts (table 1). the tested products were two samples of brazilian commercial propolis from apis flora™: 11.0% ethanolic extract of propolis (eep) and propomax™ 11.0% extract of propolis without alcohol (ep). the antimicrobial activity was determined using the agar diffusion technique, well method7. sterile mueller hinton (mh) agar or brain heart infusion, according to the requirements of the microorganisms, was poured into 20 (20 ml) petri dishes and left to set. then, 10 ml of agar was inoculated with 1 ml of the microorganism inoculum poured on top. the inoculum was prepared with an overnight culture of test microorganism and the size was adjusted to 0.5 mcfarland standard turbidity, approximately 108 colony forming units (cfu/ml). equidistant wells of 5 mm in diameter and 4 mm in depth were bored into the agar using a sterile cork borer and the wells were completely filled with the tested products. the plates were left at room temperature for two hours and then incubated at 37oc for 24 hours. antimicrobial activity was determined by measuring the diameters of the zone of inhibition of eep and ep. controls were maintained with methanol and penicillin g 10 mg/ml (for gram-positive) and erythromycin 15 mg/ml (for gram-negative). the minimal inhibitory concentration (mic) was determined for eep and ep by the agar dilution method in mh agar medium (nccls 2003). staphylococcus sp (21 strains) were grown on mh agar plates (difcoò) and suspended in mh broth (difcoò). streptococcus mutans (21 strains) were grown on brain heart infusion (bhi) broth (difcoò) and the assay done with bhi agar. the inoculum suspensions were prepared with an overnight culture of test microrganism and the size was adjusted to 0.5 mcfarland standard turbidity, approximately 108 colony forming units (cfu/ml). serial 10-fold dilutions were made that furnished a concentration range from 1.1 to 17.6 mg/ml for eep and ep. before gelling, the mh agar was added to each of the petri dishes containing the dilutions and swirled carefully until the agar began to set. the bacterial suspensions were inoculated using a steers replicator on the muller hinton agar surface and incubated at 37oc for 24 hours. the mic was defined as the lowest concentration able to inhibit any visible bacterial growth. control cultures, containing only the mh/bhi agar, were also prepared. tests were performed in duplicate. results and discussion in the present study, commercial propolis with or without ethanol showed in vitro antimicrobial activity against bacteria and yeasts (table 1). the standard strains tested were chosen table 1. antimicrobial activity of 11% ethanolic extract of propolis (eep) and 11% extract of propolis without alcohol (ep) obtained from brazilian commercial products. micrococcus luteus atcc9341 14 15 streptococcus mutans 41* 18 18 streptococcus mutans 28* 17 27 streptococcus mutans 29.2* 13 17 streptococcus mutans 30.1* 10 10 streptococcus mutans 35.1* 14 14 streptococcus mutans 39.1* 15 12 staphylococcus aureus atcc25923 15 13 staphylococcus aureus atcc6538 17 22 staphylococcus aureus atcc29213 11 11 enterococcus faecalis 06 08 bacillus stearotermophylus atcc1262 17 16 bacillus subtilis atcc6633 14 06 bacillus cereus atcc14579 12 14 pseudomonas aeruginosa atcc27853 06 11 pseudomonas aeruginosa atcc 9027 06 06 escherichia coli atcc 11229 08 12 escherichia coli atcc 25922 06 07 escherichia coli atcc 8739 06 06 proteus vulgaris atcc 13315 07 10 serratia marcescens atcc 14756 06 10 salmonella choleraisuis atcc 10708 08 10 salmonella typhimurium atcc 14028 10 12 enterobacter cloacae ft505 08 08 candida albicans ft2010 07 09 candida albicans atcc 10231 16 15 diameter of the zone of inhibition (mm) e e p e p microorganisms *wild bacteria isolated from children saliva braz j oral sci. 5(16):967-970 antimicrobial activity of two brazilian commercial propolis extracts 969 160 17.6 80 8.8 40 4.4 20 2.2 10 1.1 <10 <1.1 160 17.6 80 8.8 40 4.4 20 2.2 10 1.1 <10 <1.1 product µl propolis µg mic number of strains inhibited e e p e p 21 21 19 2 6 15 staphylococcus sp n= 21 streptococcus mutans n= 21 microorganisms table 2. minimum inhibitory concentration (mic) of 11% ethanolic extract of propolis (eep) and 11% extract of propolis without alcohol (ep) obtained from brazilian commercial products. according to a screening protocol including gram-positive cocci and bacilli, gram-negative bacteria and yeasts. in the first step of an antimicrobial activity screening, the product should be tested against these standard strains, which represent microorganisms associated with important infections. commercial propolis products had a more pronounced activity against gram-positive bacteria and candida albicans ft2010, and a less evident action against gram-negative bacilli and candida albicans atcc 10231. the controversial result concerning candida albicans ft2010 and candida albicans atcc 10231 could be explained by the inherent virulence of each strain. that is one reason to employ different microbial strains of a same species. this efficient antimicrobial action, mainly towards gram-positive bacteria, was also observed in other studies which tested non-commercial extract of propolis8-10. probably, the antibacterial activity of propolis is greater on grampositive bacteria due to flavonoids, acids and aromatic esters found in the resin, which would act on the cell wall through an unknown mechanism11. this study is in accordance with sforcin et al.12, who verified that the growth of gram-positive bacteria is inhibited by low propolis concentrations (0.4%) whereas gram-negative bacteria were less susceptible to this substance, with the mic ranging from 4.5 to 8.0%. drago et al.8 also observed that in low concentrations propolis shows bacteriostatic rather than bactericidal activity. among the yeasts, this study showed that the c. albicans was more susceptible to propolis than other species. this result is supported by ota et al.13, who found the following order of susceptibility to hydroalcoholic propolis: c. albicans > c. tropicalis > c. krusei > c. guilliermondii. another study has shown that a commercial 20% ethanol propolis extract inhibited candida albicans strains collected from hiv-positive patients with oral candidiasis14. the results of this study have to be interpreted carefully as far as its methodological procedures are concerned. it is reported that the best microbiological method to evaluate the activity of propolis extracts against species of candida is agar dilution in plates9. otherwise, serial dilution in tubes is the best method for the evaluation of the bactericidal activity of propolis samples. however, agar plate diffusion tests are strongly influenced by the solubility of the components of propolis in agar, leading to incorrect results. this method should not be used for the comparison of samples of different hydro-solubility nor for the evaluation of poorly hydro-soluble samples9. after the evidence of in vitro antimicrobial activity against all tested strains in the screening diffusion test, the minimum inhibitory concentration (mic) was established using the agar serial dilution method for 21 staphylococcus sp and 21 streptococcus mutans isolated from saliva. both products tested contained 11.0% of propolis (ethanolic extract of propolis-eep and propomax™-ep). the products were measured in µl and the propolis in µg. table 2 illustrates the mic obtained for s. mutans and staphylococcus sp., showing an extremely low concentration, especially against staphylococcus sp. the eep mic for streptococcus mutans ranged from 80 to 40ml (8.8mg to 4.4mg of propolis) and the ep mic for streptococcus mutans ranged from 40 to <10ml (4.4 to <1.1mg of propolis). for staphylococcus sp, the mic of the two extracts ranged from <10ml (<1.1mg of propolis). on the other hand, gebara et al.2 showed a greater mic for propolis ethanolic extract (14 µg/ml) against s. aureus. however, it should be borne in mind that the determination of mic values depends on technical details that may vary between laboratories and the bacteria’s inherent virulence and susceptibility. the results of this study do not corroborate the statement that one of the limitations to propolis use is its variability in composition and action as a consequence of variations in the flora of the region where it is produced, since the commercial propolis studied consist of a mixture of various propolis collected in brazil. future in vitro and in vivo research must be conducted to analyze the biological effects and the viability of using different propolis formulations in various oral infections. it is important to remember that in vitro tests do not reflect the real conditions found in clinical infections, because they do not take into account biofilm formation. therefore, this finding can hypothetically permit a more comprehensive clinical use of propolis after further in vivo studies prove its efficacy in the treatment of oral infections, since preliminary braz j oral sci. 5(16):967-970 antimicrobial activity of two brazilian commercial propolis extracts 970 antimicrobial propolis activity against cariogenic microorganisms3 and periodontopathogens15 has already been demonstrated. another potential field for propolis research is endodontics. references 1. uzel a, sorkun k, onçag o, cogulu d, gençay o, salih b. chemical compositions and antimicrobial activities of four different anatolian propolis samples. microbiol res. 2005; 160: 189-95. 2. gebara ece, lima la, mayer mpa. propolis antimicrobial activity against periodontopathic bacteria. braz j microbiol. 2002; 33: 365-9. 3. koo h, rosalen pl, cury ja, park yk, ikegaki m, sattler a. effect of apis mellifera propolis from two brazilian regions on caries development in desalivated rats. caries res. 1999; 33: 393-400. 4. miorin pl, levy junior nc, custodio ar, bretz wa, marcucci mc. antibacterial activity of honey and propolis from apis mellifera and tetragonisca augustula against staphylococcus aureus. j appl microbiol. 2003; 95: 913-20. 5. popova m, silici s, kaftanoglu o, bankova v. antibacterial activity of turkish propolis and its qualitative and quantitative chemical composition. phytomedicine. 2005; 12: 221-8. 6. orsi ro, sforcin jm, funari sr, bankova v. effects of brazilian and bulgarian propolis on bactericidal activity of macrophages agains salmonella typhimurium. int immunopharmacol. 2005; 5: 359-68. 7. nccls national committee for clinical laboratory standars. wayne, pa, usa; 2003. documents m7-a6 and 100-s13: nccls 20 (2). 8. drago l, mombelli b, de vecchi e, fassina mc, tocalli l, gismondo mr. in vitro antimicrobial activity of propolis dry extract. j chemother. 2000; 12: 390-5. 9. sawaya achf, souza ks, marcucci mc, da silva cunha ib, shimizu mt. analysis of the composition of brazilian propolis extracts by chromatography and evaluation of their in vitro activity against gram-positive bacteria. braz j microbiol. 2004; 35: 104-9. 10. sawaya ac, palma am, caetano fm, marcucci mc, da silva cunha ib, araujo ce, et al. comparative study of in vitro methods used to analyze the activity of propolis extracts with different compositions against species of candida. lett appl microbiol. 2002; 35: 203-7. 11. marcucci mc, ferreres f, garcía-viguera c, bankova vs, de castro sl, dantas ap, et al. phenolic compounds from brazilian propolis with pharmacological activities. j ethnopharmacol. 2001; 74: 105-12. 12. sforcin jm, fernandes a, lopes ca, bankova v, funari sr. seasonal effect on brazilian propolis antibacterial activity. j ethnopharmacol. 2000; 73: 243-9. 13. ota c, unterkircher c, fantinato v, shimizu mt. antifungal activity of propolis on different species of candida. mycoses. 2001; 44: 375-8. 14. martins rs, pereira es, lima sm, senna mi, mesquita ra, santos vr. effect of commercial ethanol propolis extract on the in vitro growth of candida albicans collected from hivseropositive and hiv-seronegative brazilian patients with oral candidiasis. j oral sci. 2002; 44: 41-8. 15. santos fa, bastos em, maia ab, uzeda m, carvalho ma, farias lm, et al. brazilian propolis: physicochemical properties, plant origin and antibacterial activity on periodontopathogens. phytother res. 2003; 17: 285-9. braz j oral sci. 5(16):967-970 antimicrobial activity of two brazilian commercial propolis extracts oral sciences n3 original article braz j oral sci. january/march 2010 volume 9, number 1 human teeth versus bovine teeth: cutting effectiveness of diamond burs laiza maria grassi fais1, caroline canhizares marcelo1, regina helena barbosa tavares da silva2, dalton geraldo guaglianoni3, lígia antunes pereira pinelli2 correspondence to: lígia antunes pereira pinelli faculdade de odontologia de araraquara unesp rua humaitá, 168014801-903 araraquara-sp, brasil phone: +55 16 3301-6413 / fax: +55 16 3301-6406 e-mail: ligia@foar.unesp.br received for publication: november 26, 2009 accepted: march 8, 2010 abstract aim: in this study, a mass-loss method was used to verify whether bovine enamel has the same wear pattern as human enamel in cutting efficiency tests of diamond burs. methods: seventy-two teeth were used: 36 human molars (ht) and 36 bovine mandibular central incisors (bt). the enamel of the teeth was cut using diamond bur #1092 attached to a high-speed handpiece under controlled pressure (50-80 g). each bur (n=12) cut for a total of 72 min, divided into 6 periods of 12 min each (12-min, 24-min, 36-min, 48-min, 60-min, and 72-min). the amount of enamel removed was determined by the difference between preand post-cut tooth masses. results: the mean amounts (g) of enamel removed were: ht12-min=0.11; 24-min=0.12; 36-min=0.11; 48-min=0.11; 60-min=0.10; 72-min=0.12; bt12-min=0.12; 24-min=0.15; 36-min=0.15; 48min=0.13; 60-min=0.16; 72-min=0.14. data were analyzed using anova followed by tukey’s test, and the results showed statistically significant differences between human and bovine teeth (p<0.001) and among the cutting periods (p<0.001).conclusions: it was concluded that the cutting efficiency of the burs was different between the tested substrates, and that bovine enamel underwent greater mass loss than did human enamel. keywords: teeth; dental enamel; cattle; efficiency, diamond. introduction the development of dental materials and technologies over time has facilitated the use of new clinical techniques, including rotary instruments. a new metallic binder between crystals and adhesion systems of diamond burs of different sizes and shapes has been introduced to increase their efficiency1. in this context, studies have been conducted to analyze the cutting efficiency of diamond burs, and particularly, their service life cycle when submitted to frequent use and sterilizing processes2-3. several studies have been performed on extracted human teeth to analyze different dental materials and instruments4-8. however, the use of human teeth in laboratory research has been restricted due to ethical limitations, difficulty in obtaining the appropriate sample size and impossibility of standardization9-10. in an endeavor to find an ideal substitute for human teeth, enamel and dentin from different animal species, such as bovine and swine, have been used as substitutes for human substrates, since mammalian and human teeth are morphohistologically similar to each other, but the constant use of bovine teeth in dental research has stimulated a number of studies seeking to certify their suitability1112. some of these studies have focused on enamel and dentin demineralization13braz j oral sci. 9(1):39-42 1msc, department of dental materials and prosthodontics, araraquara dental school; são paulo state university, brazil 2phd, associate professor, department of dental materials and prosthodontics, araraquara dental school; são paulo state university, brazil 3msc, professor of statistics, mathematics and statistics department, araraquara science and language school, são paulo state university, brazil 40 15. other investigations have compared various properties, such as microhardness16 and tensile strength6, and several studies have been performed to examine whether bovine teeth could be used as substitutes for human teeth when testing different bonding methods or bonding substrates5,7-9,17-20. there has been extensive research into analyzing the effectiveness of rotary cutting instruments21-25, but limited work has been done as regards comparisons between human and bovine teeth as substrates for these types of experiments. therefore, considering the difficulty in obtaining human teeth for laboratory research, the aim of this study was to evaluate, by a mass-loss method, whether bovine enamel has the same wear pattern as human enamel in cutting efficiency tests of diamond burs. the null hypothesis was that bovine enamel has the same resistance to abrasion as human enamel. material and methods specimen preparation in this study, approved by the research ethics committee, araraquara dental school,são paulo state university, under the certificate #9102, freshly extracted human molars (n = 36) and bovine central incisors (n = 36) were used. the recently extracted human teeth were cavity-free, and the patients’ permission and authorization were obtained to use them. the recently extracted bovine teeth were obtained from a slaughterhouse. both human and bovine teeth were stored in 10% formaldehyde for 24 h for disinfection26. afterwards, they were stored in distilled water under refrigeration to prevent dehydration. the remaining periodontal ligament and calculus were removed with periodontal curettes, and the teeth were submitted to prophylaxis with pumice-stone and water using a robinson brtistle brush (kg sorensen, barueri, sp, brazil). the teeth were analyzed under a zeiss stereoscopic magnifying glass (×10) (model 475200/9901, jena, west germany, germany) in order to detect possible cracks or structural alterations that could lead to experimental errors. the areas that were not involved in the experiment were coated with nail varnish to avoid any alteration in the real mass of teeth. after this phase, the teeth were kept in distilled water at 37ºc. experimental groups twelve cylindrical shaped diamond burs (kg sorensen # 1092) were used: 6 for the human teeth (ht) and 6 for the bovine teeth (bt). the human tooth crown was divided into six abrasion zones: mesiobuccal (zone 1), distobuccal (zone 2), distal (zone 3), distolingual (zone 4), mesiolingual (zone 5) and mesial (zone 6) (figure 1). because the bovine teeth were central incisors, only the buccal surface was divided into six parts (figure 1). statistics indicated the need for division of the tooth surface, so that the cut by the diamond burs would be similar in human and bovine teeth. for both, ht and bt, six repeated cutting periods were evaluated in accordance with the time of abrasion of 12, 24, 36, 48, 60 and 72 min. six teeth were used for each period. abrasion test and mass-loss method a mass-loss method was used to measure the amount of enamel abraded by the diamond burs. immediately before the cutting, each tooth was cleaned under running water and dried with a 30 s air stream and weighed on an analytical scale (sartorius-werke ag, gottingen, germany) with capacity of 200 g and accurate to 0.0001 g; thus obtaining the initial mass. a sensitive cutting pressure device was used during the cutting of the teeth because it allows a standardized pressure during the test. this machine is equipped with an alarm that would go off if the cutting pressure was outside the range from 50 to 80 gf27. the diamond burs were attached to a high speed handpiece (dabi ms 350, gnatus medical and dental products ltd., ribeirão preto, são paulo, brazil) at 350,000 rpm, and applied for 2 min in each of the tooth zones, according to the following order and sequence: bur #1 was applied for 2 min in zone 1 of tooth 1, then for 2 min in zone 1 of tooth 2, 2 min in zone 1 of tooth 3, and so on, until the six teeth of the 12-min period had been abraded, totalizing 12 min of use of bur #1. thereafter, bur #2 was applied for 2 min in zone 2 of tooth 1, then for 2 min in zone 2 of tooth 2, 2 min in zone 2 of tooth 3, and so on, until the six teeth of the 12-min period had been abraded, totalizing 12 min of use of the bur #2. these procedures were repeated with burs #3, 4, 5, and 6. the teeth were then cleaned, dried and reweighed. the difference between the final and initial mass represented the amount of enamel removed during the 12-min cutting period. all burs were then cleaned with steel brush under running water and applied to other the six teeth, which were selected for the 24-min cutting period, using the same order and sequence described above. after that, these teeth were cleaned, dried and reweighed, thus obtaining the amount of enamel removed during the 24-min cutting period. braz j oral sci. 9(1):39-42 human teeth versus bovine teeth: cutting effectiveness of diamond burs fig. 1. zones for cutting in human teeth (ht) and bovine teeth (bt). variation source ss df m s f value p cutting periods 0.005257 5 0.001051 22.59825 <.001 teeth 0.032213 1 0.032213 692.3923 <.001 interaction 0.013671 5 0.002734 58.76903 <.001 residue 0.006141 132 4.65e-05 total 0.474374 503 table 1. two-way anova for mass loss. *p< 0.01 – highly significant difference. these procedures were repeated until all burs had been used to abrade all the 36 teeth (human and bovine), so that the amount of enamel removed during each cutting period, as well as the total amount of wear (12-min + 24-min + 36min + 48-min + 60-min + 72-min), could be obtained. data were entered to the excel software and analyzed by 2way anova and tukey’s post-hoc tests for statistical comparisons (α =0.05). results table 1 shows the results of anova for the effect of the type of teeth and cutting period on enamel mass loss all p values of probability were lower than 0.05 (p<0.05), showing that the human dental enamel differs from bovine dental enamel, and that the amount of cut varies over time. the p value for interaction between the two main factors (tooth type and cutting period) was also lower than 0.05 (p<0.05). the total mass loss of human and bovine enamel after 72-min cutting was 0.6725 g and 0.8520 g, respectively. the mean mass loss values for the 12-min cutting periods are shown in figure 2. for the factor cutting period, there were statistically significant differences between the 12-min and 24-min, 12-min and 36-min, 12-min and 60-min, 12-min and 72-min, 24-min and 48-min, 36-min and 48-min, 48-min and 60-min, and 48-min and 72-min periods. 41 discussion intensive research has been conducted to verify the cutting efficiency and durability of the diamond burs using human enamel and dentin28-30. it has been difficult to conduct such studies due to the limited amount of the available material used to prepare the samples. several articles have reported that bovine teeth are an alternative for replacing human teeth6,20 because they have a similar microscopic structure5,17 and are easily obtained in large number31, and because the size of bovine mandibular incisors allows more than one sample to be prepared from one tooth15. furthermore, there are ethical concerns involving the use of human teeth31, which have led many investigators to search for materials that can be used as a substitute for human teeth in several tests, without compromising the quality of the results. however, other studies have pointed out some discrepancies between the values obtained in adhesion 18, shear bond strength7 and tensile bond strength19 tests applied to human and bovine teeth7,18-19. in this study, a highly significant statistical difference was observed when the mass loss of human and bovine enamel was compared (table 1). bovine enamel showed greater mass loss than human enamel, which suggests that the former is less resistant than the latter. human enamel, which is the hardest tissue in the body, is composed of 92-96% inorganic matter, 1-2% organic material and 3-4% water by weight32. most of the inorganic matter is ca 10 (po 4 ) 6 (oh) 2 hydroxyapatite. at a microstructural level, enamel consists of mineral-rich prisms of 3-6 mm cross-sectional diameter embedded in a matrix of inorganic and organic components33. human enamel has a slightly lower density, and a lower vickers hardness, but shows a slightly higher calcium and phosphorus content when compared with bovine enamel13. although there are some minor quantitative differences with respect to calcium content, an analogous behavior of calcium distribution from outer to inner levels of human and bovine enamel has been observed. bovine enamel reveals a higher porosity with larger crystals than human enamel13, and this aspect might explain why, in this study, bovine enamel was more easily abraded than the human enamel. according to fonseca et al.12 human enamel shows low interprismatic substance and distinct prisms while bovine enamel has a larger amount of interprismatic substance with an indicative appearance of the presence of fibrils. according to the authors, this difference would be explained by the possibility of the collagen fibrils not having been removed from enamel in the course of mineralization and maturation. therefore, this different microstructure may be the reason for the difference in microhardness, and could explain the differences between human and bovine groups found in this study. with regard to the effect of cutting time, irrespective of the type of tooth, the results indicated that there was no gradual decrease in the cutting power of the burs. after the first cutting period (12-min) there was a slight increase in the amount of wear, which remained constant up to 36-min, reduced between 36-min and 48-min and increased again and remained constant up to 72-min this pattern of increases in the amount of wear of enamel, interspersed with periods of stabilization, may be attributed to the loss of diamond particles, which could compromise the cutting power of the bur, or to an increase in the area of contact between the bur and the tooth, when the diamond particles are broken instead of being pulled out, thus increasing the cutting power. a photomicrographic analysis of the burs used in the present study demonstrated that during cutting human and bovine enamel, a gradual alteration on the bur surface was noted26; nevertheless, the most significant alterations for the burs used on human enamel were seen after 48 min of cutting while for bovine enamel, the most significant alterations only occurred after 60 min of cutting26. this was explained by the result obtained in an adjustment of the linear regression equation, which have indicated that the speed of wear of the diamond tips was 7% higher when cutting human teeth compared to cutting bovine teeth26. the results of the present study also demonstrated that the interaction between the factors tooth type and cutting period was significant. this is illustrated in figure 2, which demonstrates that the increases and decreases in the amount of enamel wear produced by the burs were not equivalent for the two types of teeth in the evaluated periods. from the 24to the 36-min period, the amount of wear of bovine teeth remained the same, while for the human teeth there was a decrease in the amount of enamel wear. from the 36to the 48-min period, the amount of wear of human teeth remained braz j oral sci. 9(1):39-42 human teeth versus bovine teeth: cutting effectiveness of diamond burs 42 the same, while for the bovine teeth there was a decrease in the amount of enamel wear. the amounts of enamel wear for the human teeth decreased from 48-min to 60-min and then increased at the 72-min cutting period. by contrast, an opposite trend was observed for the bovine teeth when these three cutting periods are considered. these different patterns were responsible for the interaction between the evaluated factors. as already mentioned, the reasons for such differences may be the differences in the microstructure and/or hardness of the substrates evaluated, which might have influenced the alterations on the surface of the burs (loss of diamond particles or increase in the area of contact with the tooth). although the use of the bovine teeth as substitute for human teeth has been used successfully for in vitro studies that evaluate microleakage5, in the present investigation the null hypothesis was rejected, thus demonstrating that the use of bovine teeth instead of human teeth may not be indicated for studies that analyze the cutting efficiency of diamond burs by the mass-loss method. in spite of the differences in the wear pattern between human and bovine teeth, the results of the present study must be interpreted with caution, since no structural analyses of the teeth used were performed. moreover, in addition to diamond burs, other types of rotary cutting instruments must also be evaluated using the same methodology. it may be concluded that the cutting efficiency of the burs was different on the tested substrates, and that bovine enamel underwent greater mass loss than did human enamel. acknowledgements to the são paulo state research foundation (fapesp, grant # 02/10781-5) and prointer unesp internationalization program. references 1. jackson mj, sein h, ahmed w. diamond coated dental bur machining of natural and synthetic dental materials. j mater sci mater med. 2004; 15: 1323-31. 2. 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belanger gk. the use of bovine enamel in bonding studies. am j orthod dentofacial orthop. 1998; 114: 514-9. 21. taira m, wakasa k, yamaki m, matsui a. effects of the diamond grit sizes of the commercial dental diamond points on the weight-load cutting of bovine enamel and glass-ceramic typodont teeth. dent mater j. 1990; 9: 173-80. 22. tanaka n, taira m, wakasa k, shintani h, yamaki m. cutting effectiveness and wear of carbide burs on eight machinable ceramics and bovine dentin. dent mater. 1991; 7: 247-53. 23. miyawaki h, taira m, wakasa k, yamaki m. dental high-speed cutting of four cast alloys. j oral rehabil. 1993; 20: 653-61. 24. ohmoto k, taira m, shintani h, yamaki m. studies on dental high-speed cutting with carbide burs used on bovine dentin. j prosthet dent. 1994; 71: 319-23. 25. watanabe i, ohkubo c, ford jp, atsuta m, okabe t. cutting efficiency of air-turbine burs on cast titanium and dental casting alloys. dent mater. 2000; 16: 420-5. 26. pinelli lap, faria ir, marcelo cc, pita apg, silva rhbt, dg g. estudo fotomicrográfico do desgaste de pontas diamantadas em diferentes substratos. pós-grad rev. 2005; 7: 60-6. 27. fontana uf, dinelli w, gabrielli f, fontana rhbts, lmdf a. estudo comparativo da eficiência de instrumentos rotatórios de carbeto de tungstênio e diamante. análise gravimétrica. efeito de tempo e procedência do instrumento. rev assoc paul cir dent. 1985; 39. 28. siegel sc, von fraunhofer ja. assessing the cutting efficiency of dental diamond burs. j am dent assoc. 1996; 127: 763-72. 29. siegel sc, von fraunhofer ja. dental cutting with diamond burs: heavyhanded or light-touch? j prosthodont. 1999; 8: 3-9. 30. siegel sc, von fraunhofer ja. cutting efficiency of three diamond bur grit sizes. j am dent assoc. 2000; 131: 1706-10. 31. reeh es, douglas wh, levine mj. lubrication of human and bovine enamel compared in an artificial mouth. arch oral biol. 1995; 40: 1063-72. 32. gwinnett aj. structure and composition of enamel. oper dent. 1992; suppl 5: 10-7. 33. braly a, darnell la, mann ab, teaford mf, weihs tp. the effect of prism orientation on the indentation testing of human molar enamel. arch oral biol. 2007; 52: 856-60. braz j oral sci. 9(1):39-42 human teeth versus bovine teeth: cutting effectiveness of diamond burs oral sciences n3 original article braz j oral sci. january | march 2012 volume 11, number 1 profile and scientific production of brazilian researchers in dental materials daniela araújo veloso popoff1, raquel conceição ferreira2, daniella reis b. martelli3, eduardo araújo de oliveira4, joão robson vieira júnior3, hercílio martelli-júnior3 1department of dentistry, state university of montes claros – unimontes; medical school, pitágoras integrated college, fip-moc, brazil 2department of dentistry, state university of montes claros – unimontes, brazil 3graduate program in health sciences, state university of montes claros – unimontes, brazil 4department of pediatrics, medical school, federal university of minas gerais – ufmg, brazil correspondece to: daniela araújo veloso popoff rua amapá, 108, ibituruna, cep: 39401-287, montes claros, mg phone: +55 38 99859911 e-mail: danielavelloso@yahoo.com.br abstract aim: to describe the demographic characteristics and academic production of the national council for the development of science and technology (cnpq) researchers whose areas of expertise focus on dental materials. methods: secondary data were obtained from the details of dentistry researchers registered as receiving a cnpq grant for scientific productivity. variables such as gender, category of scientific productivity grant, time of completion of phd program, geographical distribution and affiliated institution, scientific production and human resources training were analyzed. data from 2007-2009 triennium were considered. mann-whitney and chisquare tests compared the variables. results: researchers in categories 1a + 1b are those who completed their phd program more than 14 years ago. all (100%) of dental material researchers are dentists and affiliated to universities, being approximately 92% of those affiliated to public institutions. male gender (62.2%) and category 2 grant (40.5%) are prevalent, and a high concentration of cnpq grantees in the southeastern region (75.7%) was observed. in general, there was a predominance of publications in capes qualis b journals (59.0%), but a predominance of publications by new doctors in capes qualis a2 and b1 journals was found. a higher mean of publications of book chapters (0.97) compared with full publications of books (0.43) per researcher was observed. annually, researchers supervised more scientific initiation grants (median = 0.45), followed by doctoral (mean = 0.36) and master students (median = 0.30). conclusions: there is a high concentration of researchers in the southeastern region, predominance of male gender and scholars in grant category 2. publications are mainly in capes qualis b journals. and researchers in grant categories 1c and 1d and with less time doctoral produce more publications. the publication of books chapters is larger than entire books. the researchers supervised more undergraduate students, followed by phd and master’s degree students annually. keywords: scientific production indicators, dental materials, health sciences. introduction increasingly, science and technology and educational qualifications are seen as fundamental pillars of economic, technological and industrial development of established and recently developed nations, and the essential foundation of a virtuous cycle that underpins the growing socioeconomic progress of these countries1. the development of infrastructure in science and technology has braz j oral sci. 11(1):56-61 received for publication: september 21, 2011 accepted: february 28, 2012 braz j oral sci. 11(1):56-61 5757575757 boosted quantitative indicators in science, technology and innovation since 20002-3. in general, the rise of brazilian scientific production constitutes a high percentage compared with other countries, as confirmed by data from isi (institute for scientific information) and scielo (the scientific electronic library online), and national scientific production ranks thirteenth in the world4, accounting for approximately 2% of total world scientific production, surpassing countries such as switzerland (1.42%), taiwan (1.72%) and netherlands (1.99%) and approaching the output of the australia (2.72%) and south korea (2.56%)5. in brazil there are two main federal science funding agencies, capes (coordination for the improvement of higher education personnel, part of the ministry of education) and cnpq (national council for the development of science and technology, part of the ministry of science and technology). cnpq is the funding agency that evaluates and funds researchers, and capes evaluates and supports graduate programs. cnpq funds research based on peer evaluation of the merits of the candidate and their proposals. cnpq also provides a particular form of funding for research, scientific productivity fellowships. researchers are currently classified in two main categories for this fellowship: researcher category 1 and researcher category 2, in a decreasing order of value and prestige. category 1 includes only researchers of notorious scientific productivity and is subdivided into four levels: 1a, 1b, 1c and 1d, category6. state foundations for research support in the brazilian states were created from the constitution of 19887. currently, these foundations have been organized into a national council of research foundations. these state articulations and their interaction with the ministries and agencies at the federal level have integrated programs, added resources, and catalyzed actions countrywide. all this creates an environment of encouragement that can produce significant changes that place brazil in a position to keep up with global competition8. as the profile of these researchers has begun to interest the scientific community in recent years, several studies have examined the profile and the scientific production of researchers from the cnpq in various knowledge areas9-13. recent studies, especially in health sciences, have revealed the profile of medical researchers, comparing it with various other areas4,9-13. apart from the study by cavalcanti et al. (2008)11 data for dentistry are scarce. this cross-sectional study describes the demographic characteristics and the academic production of cnpq researchers whose areas of expertise are primarily focused on dental materials. the hypothesis was that cnpq researchers are geographically concentrated in the southeastern region of brazil, are linked to educational institutions and have completed a phd in the last two decades. material and methods study design this study, used cross-sectional and descriptive secondary data, obtained from details of cnpq researchers in health sciences and dentistry and the lattes curriculum vitae platform available on the cnpq website14. population and sample first, 187 researchers registered as receiving a cnpq scientific productivity grant were included in the database, according to the list provided by the cnpq in september 201015. the researcher’s specific area as described on the lattes16 platform was used to determine the area of expertise focus. when such information was lacking, the researcher’s scientific production in the 2007-2009 triennium was assessed and the predominating area was determined based on the journals the authors has published, the main topic of the research and/or supervised themes at-tributed to the researcher. this methodology identified 37 researchers in the area of dental materials. inclusion and exclusion criteria the researchers in this study were those whose cnpq scientific productivity grant was still valid when the data were collected for dental materials researchers. not included in the analysis were researchers who had their scholarship suspended, as in cases of post-doctoral students living abroad. data collection protocol: the researchers’ area of action was identified by reading the text supplied by the author in the lattes curriculum, also publicly available on the electronic site in september 2010. if, however, the information contained in the text was not enough to provide identification of the area in which there was a predominance of published and/or supervised issues, the researcher’s productions during the last five years was assessed. variables of interest the following variables for the cnpq researchers were analyzed: gender, category of scientific productivity grant (2, 1a, 1b, 1c, 1d and senior), time of completion of phd program, geographical distribution and category of institution to which the researchers were affiliated, scientific productions (scientific papers, book chapters, entire books and patents) and human resources training. analysis of scientific production included only publications of scientific papers in journals and supervisions were restricted to the 2007-2009 triennium. to classify the papers, the qualis system17 was adopted a1, a2, b1, b2, b3, b4, b5 or c, taking into account the impact factor of the journal citation reports15,18. lattes curricula were consulted between september and november 2010. statistical analysis after construction of the database using the statistical software spss 18.0 for windows (spss, chicago, il, usa), a descriptive and univariate statistical analysis of the data obtained was performed. continuous data were reported with median and interquartile range between 25 and 75 percentiles. profile and scientific production of brazilian researchers in dental materials braz j oral sci. 11(1):56-61 5858585858 variables n % gender male 23 62.2 female 14 37.8 grant category 1a 3 8.1 1b 5 13.5 1c 4 10.8 1d 10 27.0 2 15 40.5 affiliated institution usp 11 29.7 unicamp 10 27.0 unesp 5 13.5 ufpel 2 5.4 uepg 2 5.4 sl mandic 1 2.7 ufpa 1 2.7 ufsc 1 2.7 ufsm 1 2.7 ufu 1 2.7 ulbra/rs 1 2.7 upf 1 2.7 brazilian region of origin southeastern 28 75.7 south 8 21.6 north 1 2.7 brazilian state são paulo 27 73.0 rio grande do sul 5 13.5 paraná 2 5.4 minas gerais 1 2.7 pará 1 2.7 santa catarina 1 2.7 institution category state 28 75.7 federal 6 16.2 private 3 8.1 table 1.table 1.table 1.table 1.table 1. distribution of researchers according to gender, grant category, affiliated institution, brazilian region of origin, brazilian state and institution category. the nonparametric mann-whitney test was used to compare these variables. dichotomous or nominal variables were compared by chi-square tests. results there was a predominance of male gender (62.2%) and grant category 2 (40.5%) (table 1). the distributions of the 37 researchers by gender and grant category are summarized in table 2, where a significant association (p= 0.01) with the time of completion of phd program can also be observed: most researchers in categories 1a and 1b are those who completed their phd program more than 14 years ago. also, in relation to the time of completion of the phd program, dental materials researchers mean time was 14.49 years (standard deviation= 7.17), with a maximum of 42 years and a minimum of 5 years. the median time was 13 years (interquartile range = 9.5 to 17.5 years). all (100%) dental materials researchers are dentists. table 1 also shows the geographical origin of cnpq dental materials researchers. two brazilian states accounted for approximately 86% of the researchers (são paulo and rio grande do sul), with the majority in são paulo state (73%). about 75% of these researchers are affiliated to state institutions, being 16.2% to federal institutions and only 8.1% to private institutions. regarding affiliation, the dental materials researchers are distributed over 12 different institutions in the country and three institutions accounted for approximately 70% of them: usp (29.7%), unicamp (27%) and unesp (13.5%). table 3 presents the scientific production and supervisory work of cnpq dental materials researchers. as regards their academic career, the researchers supervised 209 scientific initiation grants, with a median of six (iq = 2-7) for researchers, 199 master’s dissertations (median of four, iq = 2.5 6) and 192 phd theses (median of five, iq = 3.5 7.5). the median of the values set by the time of completion of phd program shows that researchers supervised 0.45 bic (iq =0.22-0.64), 0.30 master’s dissertations (iq = 0.16 0.48) and 0.36 phd theses (iq= 0.25 053) annually are also presented in table 3. in terms of the 2007-2009 triennium, the cnpq dental materials researchers published 1110 papers, with a median of 26 papers/researcher (iq = 19-38). the median of the values adjusted by the time from completion of the phd program showed researchers published 2.21 (iq = 1.14 4.02) papers annually (table 3). overall, there was a predominance of publications in qualis b journals, compared with those in qualis a and qualis c, respectively (table 4). among the stratifications, there was a predominance of grant category 1a + 1b 1c + 1d 2 p value gender female 2 (25.0) 6 (42.9) 6 (40.0) 0.69 male 6 (75.0) 8 (57.1) 9 (60.0) brazilian region of origin southeastern 5 (62.5) 10 (71.4) 13 (86.7) 0.30 southern 2 (25.0) 4 (28.6) 2 (13.3) northern 1 (12.5) 0 (0.0) 0 institution category state public 5 (62.5) 11 (78.6) 12 (80.0) 0.91 federal public 2 (25.0) 2 (14.3) 2 (13.3) private 1 (12.5) 1 (7.1) 1 (6.7) time since completion of phd program 1 to 13 years 1 (12.5) 11 (78.6) 8 (53.3) 0.01 more than 14 years 7 (87.5) 3 (21.4) 7 (46.7) table 2. table 2. table 2. table 2. table 2. bivariate analysis between grant categories and gender, brazilian region of origin, affiliated institution and time since completion of phd program. profile and scientific production of brazilian researchers in dental materials 5959595959 braz j oral sci. 11(1):56-61 total (min-max) mean (dp) median (iq) absolute set by absolute set by absolute set by scientific productions total number of publications (papers) 1110 (4-67) 98.2 (0.1-8.0) 30.0 (15.39) 2.6 (1.8) 26.0 (19.0-38.0) 2.2 (2.8) a1 174 (0-14) 15.7 (0-1.7) 4.7 (3.81) 0.4 (0.4) 4.0 (2.0-7.5) 0.3 (0.4) a2 271 (0-17) 26.3 (0-3.0) 7.3 (4.66) 0.7 (0.6) 7.0 (4.0-9.5) 0.5 (0.6) b1 204 (0-17) 17.4 (0-1.1) 5.5 (4.43) 0.4 (0.3) 5.0 (2.0-7.5) 0.3 (0.6) b2 142 (0-13) 12.8 (0-2) 3.8 (3.40) 0.3 (0.4) 3.0 (1.0-6.0) 0.2 (0.4) b3 126 (0-17) 10.4 (0-1.3) 3.4 (4.49) 0.2 (0.3) 2.0 (0.5-4.5) 0.1 (0.3) b4 143 (0-11) 11.7 (0-1.0) 3.8 (3.11) 0.3 (0.2) 4.0 (1.0-6.0) 0.2 (0.3) b5 37 (0-9) 2.6 (0-0.6) 1.0 (1.76) 0.0 (0.1) 0.0 (0.0-1.5) 0.0 (0.1) c 8 (0-3) 0.6 (0-0.2) 0.2 (0.58) 0.0 (0.0) 0.0 (0.0-0.0) 0.0 (0.0) other 5 (0-4) 0.3 (0-0.3) 0.1 (0.67) 0.0 (0.0) 0.0 (0.0-0.0) 0.0 (0.0) total number of published book chapters 36 (0-9) 2.5 (0-0.4) 0.9 (2.08) 0.0 (0.1) 0.0 (0.0-1.0) 0.0 (0.1) total number of published books 16 (0-5) 1.5 (0-0.6) 0.4 (1.14) 0.0 (0.1) 0.0 (0.0-0.0) 0.0 (0.0) human resources training scientific initiation grants supervisions 209 (0-20) 19.9 (0-4) 5.6 (3.8) 0.5 (0.6) 6.0 (2.0-7.0) 0.4 (0.4) master dissertation supervisions 199 (0-38) 18.6 (0-4.2) 5.3 (6.7) 0.5 (0.8) 4.0 (2.5-6.0) 0.3 (0.3) doctoral thesis supervisions 192 (0-12) 14.8 (0-0.8) 5.1 (2.9) 0.4 (0.0) 5.0 (3.5-7.5) 0.3 (0.2) table 3.table 3.table 3.table 3.table 3. descriptive analysis of scientific production and supervisions of cnpq dental material researchers in absolute numbers and set by year of completion of phd program. a1 a2 b 1 b 2 b3 b4 b 5 c outras total grant category 1a + 1b 19.3 18.3 20.0ab 20.3ab 24.0 21.5 24.7a 18.2 18.0 21.6ab 1c + 1d 21.2 21.0 25.0b 24.2b 19.9 17.7 21.1ac 18.5 20.6 23.8b 2 16.7 17.4 12.8a 13.40 15.4 18.7 13.9b 19.8 18.0 13.0a p value 0.53 0.65 0.01 0.02 0.16 0.73 0.02 0.84 0.19 0.02 time from completion phd program 1 to 13 years 19.6 22.5 22.1 21.0 19.4 18.4 18.6 19.6 18.9 22.1 more than 14 years 18.2 14.8 15.3 16.5 18.5 19.6 19.3 18.2 19.0 15.3 p value 0.71 0.03 0.056 0.22 0.79 0.75 0.85 0.73 0.99 0.05 institution category state 19.6 19.7 20.8 18.9 17.6 17.8 17.5 18.6a 19.3 19.2 federal 18.7 20.4 14.3 19.5 27.4 19.5 23.5 16.0ac 18.0 20.0 private 13.8 9.1 11.0 18.1 15.0 28.5 23.5 28.0b 18.0 14.6 p value 0.68 0.25 0.16 0.98 0.09 0.26 0.27 0.04 0.72 0.76 brazilian region of origin southeastern 17.8 18.7 20.2 19.4 17.9 18.7 18.0 18.6 19.3 18.6 southern 25.1 21.9 17.5 19.3 23.3 21.8 20.5 20.5 18.0 22.6 northern 2.5 1.5 2.0 3.5 13.5 4.0 33.5 16.0 18.0 1.0 p value 0.07 0.20 0.24 0.34 0.39 0.28 0.25 0.73 0.72 0.16 table 4.table 4.table 4.table 4.table 4. number of published papers by year of completion of phd program and grant categories, time since completion of phd program, affiliated institution and brazilian region of origin. publications in qualis a2 journals, with a median of seven papers/researcher (iq = 4 9.5), and qualis b1 journals, with a median of five papers/researcher (iq = 2 7.5) (table 3). table 4 compares the number of papers published for variables such as grant category, time since completion of phd program, affiliated institutions and region of origin. it is possible to observe that researchers in category 1c and 1d (p = 0.02) who completed their phd program more recently (p = 0.056) produce more papers and their publications are mainly in qualis a2 journals (p = 0.03) and b1 (p = 0.056). publication rate in qualis c journals is higher at private institutions (p = 0.04). also, as regards indicators of scientific output, there was an upper mean of publications of book chapters (0.98, sd= 2.08) compared with full publications of books/ researcher (0.43, sd =1.14) (table 3). there was a statistically significant difference in the number of books (p = 0.00) and book chapters (p = 0.05) published for institutions to which the cnpq researchers are affiliated, with more publications by researchers working in private institutions than those in public institutions (table 5). as regards the geographical region where the institutions are located, profile and scientific production of brazilian researchers in dental materials braz j oral sci. 11(1):56-61 6060606060 book chapter books grant category 1a + 1b 17.5 18.3 1c + 1d 20.0 19.8 2 18.7 18.5 p value 0.82 0.83 time since completion of phd program 1 to 13 years 19.5 19.5 more than 14 years 18.3 18.3 p value 0.75 0.75 institution category state 17.9a 16.5a federal 17.8ab 22.6ab private 31.3b 34.1b p value 0.05 0.00 brazilian region of origin southeastern 19.0 16.5a southern 17.5 27.8b northern 28.5 16.0ab p value 0.52 0.00 table 5.table 5.table 5.table 5.table 5. number of published book chapters and entire books and grant categories, time since completion of phd program, affiliated institution and brazilian region of origin. differences were observed in the production of books between the southern and southeastern regions, with the largest number of books published in the southern region (p = 0.00) (table 5). discussion this cross-sectional study, focusing on cnpq researchers in dental materials, showed that there is a significant geographic concentration of research in this area of knowledge: the southeastern region accounts for over seventy-five percent of researchers in this area. in particular, the são paulo state, with its three state public schools and one private school, accounts for 73% of them. the current findings also show a large imbalance between the types of institution to which these researchers are affiliated, 75.7% being linked to state institutions, 16.2% to federal institutions and only 8.1% to private institutions. the concentration of dental research described here is also recognized in similar studies where other subject areas were surveyed 4,12-13. even in terms of geographical and institutional distribution, it was observed that all researchers investigated are linked to universities and none of them are linked to research institutes, hospitals or foundations. these features corroborate the observations of contini and séchet (2005)19 about the large gap that still exists between science and technology in brazil, although it is clear from the survey that there has been some progress in brazilian scientific production and the growth of private institutions participation4. considering the number of grants of scientific productivity in the area of dental material per million inhabitants, the national mean was 0.19. the southern and southeastern regions showed the top means, 0.36 and 0.29, whereas the northern region showed a mean much lower than the national mean (0.06). these indicators are also similar to those collected by martelli-junior et al. (2010)4 and santos et al. (2010)12, who found that the national mean is lower compared with the means of the southeastern and southern regions. another important feature of scientific activity refers to the formation of qualified human resources, especially masters and doctors. this research found a significant involvement of the cnpq researchers in training new researchers. the medians of supervision set by the time since completion of phd programs were 0.36 and 0.30 per year for masters’ dissertations and phd theses, respectively. these values are similar to those found by martelli-junior et al. (2010)4 in an analysis of cnpq researchers in the medical field. when, however, we considered the production of the cnpq researchers in the 2007-2009 triennium, the means of supervisions were 5.19 and 5.38 for dissertations and theses respectively. these values, compared with the results of cavalcante et al.11 in their study on the profile of cnpq researchers in the area of dentistry in 2003-2005 (2.2 phds and 3.6 masters) show a significantly increased productivity, possibly reflecting the impact of capes and other funding agencies on the production of qualified human resources at postgraduate level in recent years4. according to santos et al. (2009) 13, bibliometric indicators highlight the dramatic changes in the panorama of scientific research in the last 10 to 15 years. although on the one hand more than 70% of the world belongs to the axis united states/european community/japan, on the other there has been a dramatic growth in countries like ireland and china and other impacts such as the decline in britain. invariably, the expansion in international and brazilian scientific production has led to a considerable increase in competition for research funding and reduced public resources for this purpose20. despite some dental material researchers have published some of their articles together, thus incurring in a doublecounting of papers, events like these constitute limitations of the this study that reveals 1110 articles published in journals in the three years from 2007 to 2009, with a median of 26 articles per researcher in total or articles 2.21 annually, which shows that scholarship holders in the category 1c and 1d who completed their phd more recently produce more articles and publications, mainly in qualis a2 and qualis b1 journals. similar findings are presented in other studies that have looked more broadly at the areas of dentistry, public health and physical therapy9-13,21-23. this quantitative increase in scientific production in the area of dental materials accompanies the general increase in scientific production in brazil and possibly reflects the various inductor mechanisms established by various national funding agencies. among these, the system for evaluation of graduate students stands out, which, through an analysis by capes, prioritizes the number and quality of published papers to conceptualize national programs24. in this context, this research also shows profile and scientific production of brazilian researchers in dental materials braz j oral sci. 11(1):56-61 6161616161 that the production of papers in qualis c journals is higher among grantees affiliated to private institutions, as well as most of the publications of books and book chapters. the own scientific productivity grant signals another induction mechanism that promotes competition among peers, and encourages the training of new researchers as well as encourages the search for impact publications. the result is a qualitative increase of brazilian scientific production, gaining greater international integration of production, and is proof of the superiority of publications in qualis a2, b1 and a1 journals in the three years evaluated by this research. for these reasons, this study suggests that knowing the profile of researchers can define more effective strategies to encourage scientific production and thus the demand for funds to support research projects. in the present study, it was observed a high concentration of researchers with cnpq productivity grants in the southeastern region of the country. although distributed in 12 states of the federation, an absolute predominance of the são paulo state was observed, which could be explained due to the actions of its state foundation for research support, as well as due to the higher number of state universities. also, there was a quantitative predominance of males and scholars in category 2. all researchers are affiliated to universities, and are graduates in dentistry who completed their phd program 14.49 years ago on average. the scientific production indicators point to a predominance of publications in qualis a2 and b1 journals, and the total dominance of stratification in qualis b. although it was observed that the stock categories 1c and 1d and less time doctoral produce more papers and other publications, these are mainly in qualis a2 journals. even on indicators of scientific production, the publication of books chapters by the researchers showed to be about three times larger than the publication of entire books. as regards the training of human resources, the researchers supervised more undergraduate students, followed by phd and master’s degree students annually. researchers in category 1c and 1d, and with less time from completion of phd program have done more supervisions. references 1. guimarães ja. the medical and biomedical research in brazil: comparisons with the brazilian and international scientific performance. cienc saude colet. 2004; 9: 303-27. 2. leta j, glanzel w, thus b. science in brazil. part 2: sectoral and institutional research profiles. scientometrics. 2006; 67: 87-105. 3. mugnaini r, jannuzzi pm, quoniam l. bibliometric indicators of the brazilian scientific production: an analysis from the pascal base. cienc inf. 2004; 33: 123-31. 4. martelli junior h, martelli dr, quirino ig, oliveira mcl, lima ls, oliveira ea. cnpq researchers in medicine: comparison of areas. rev assoc med bras. 2010; 56: 478-83. 5. scimago. sjr — scimago journal & country rank. 2007 [cited 2012 jan]. available from: http://www.scimagojr.com. 6. arruda d, bezerra f, neris va, toro pr, wainer j. brazilian computer science research: gender and regional distributions. scientometrics. 2009; 79: 651-65. 7. brazil. constitution (1988). constitution of the federal republic of brazil. brasília, df: federal senate; 1988. 8. barreto fcs, borges mn. new policies for post-graduation courses: the case of fapemig-capes. ensaio: aval pol públ educ. 2009; 17: 599-612. 9. mendes phc, martelli drb, souza wp, filho sq, martelli junior h. profile of the medical researchers with scientific productivity grants from the brazilian national research council, brazil. rebm 2010; 34: 53541. 10. barata rb, goldbaum m. a profile of researchers in public health with productivity grants from the brazilian national research council (cnpq). cad saude publica 2003; 19: 1863-76. 11. cavalcante ra, barbosa dr, bonan prf, pires mbo, martelli-junior h. profile of the dentistry researchers of the national council for scientific and technological development (cnpq). rev bras epidemiol 2008;11: 106-13. 12. santos ncf, candido lfo, kuppens cl. cnpq research productivity: analysis of the profile of the chemistry researchers. quim nova. 2010; 33: 489-95. 13. santos smc, lima ls, martelli drb, martelli junior h. profile of the public health researchers in the national council for the development of science and technology. physis 2009; 19: 761-75. 14. national council for the development of science and technology [cited 2010 sept]. available from: http://www.cnpq.br. 15. national council for the development of science and technology [cited 2010 sept]. available from: http://plsql1.cnpq.br/divulg/resultado_pq_102003.curso. 16. national council for the development of science and technology [cited 2010 sept]. available from: http://lattes.cnpq.br/. 17. coordination for the improvement of higher education personnel [cited 2010 sept]. available from: http://qualis.capes.gov.br/webqualis/. 18. science gateway [cited 2010 sept]. available from: http:// sciencewatch.com/dr/sci/10/nov7-10_1/. 19. contini e, séchet p. there is still a long way in science and technology in brazil? rbpg. 2005; 2:30-9. 20. coimbra jr, cea. forum: scientific production and public health evaluation. cad saude publica. 2003; 19: 845-6. 21. zorzetto r, razzouk d, dubugras mtb, gerolin j, schor n, guimarães ja, et al. the scientific production in health and biological sciences of the top 20 brazilian universities. braz j med biol res. 2006; 39: 1513-20. 22. petherick a. high hopes for brazilian science. nature. 2010; 465: 674-5 23. moed hf. new developments in the use of citation analysis in research evaluation. arch immunol ther exp warsz. 2009; 57:13-8. 24. deheinzelin d, caramelli b. produção científica, pós-graduação e a ramb. rev assoc med bras. 2007; 53: 471-2. profile and scientific production of brazilian researchers in dental materials oral sciences n3 literature review braz j oral sci. july/september 2009 volume 8, number 3 clinical trials with nanoparticle composite in posterior teeth: a systematic literature review ana karina maciel andrade1, rosângela marques duarte2, fábia danielle sales da cunha medeiros e silva3, andré ulisses dantas batista2, kenio costa lima4, marcos antonio japiassú resende montes5 1dds, msc, assistant professor, department of restorative dentistry, federal university of paraíba, brazil; doctorate student university of pernambuco, brazil. 2dds, msc, phd, associate professor, department of restorative dentistry, federal university of paraíba, brazil. 3dds, msc, assistant professor, department of restorative dentistr y, federal university of paraíba, brazil 4dds, msc, phd, associate professor, department of dentistry, federal university of rio grande do norte,brazil 5dds, msc, phd, associate professor, department of restorative dentistry, university of pernambuco, brazil received for publication: may 4, 2009 accepted: september 16,2009 correspondence to: marcos antonio japiassú resende montes faculdade de odontologia de pernambuco –upe, departamento de odontologia restauradora / materiais dentários, av. gen. newton cavalcanti, 1650, cep: 54753-220 camaragibe, pe, brazil. phone: + 55-81-3184-7667; fax: + 55-81-3184-7659. e-mail: majrm@uol.com.br abstract aim: the aim of this article is to evaluate clinical trials with nanoparticle composite in posterior teeth through a systematic review of the literature. methods: this analysis includes controlled clinical trials with nanoparticle composites, with at least 6 months of evaluation, published in the english language between 1997 and 2009. in vitro and retrospective studies and studies on anterior teeth were excluded. articles were retrieved from the following full-text electronic journal databases: medline, lilacs and the cochrane library. results: the largest number of articles was found in the medline database, but only 4 of them met the inclusion and exclusion criteria. no articles were found in the lilacs database; only two articles were selected from the cochrane library databases and they coincided with those already included in medline. conclusions: the nanoparticle composites give a satisfactory performance for use in posterior teeth for at least 2 years of functional activity, but their performance was not superior to that of the other composites. long-term studies must be conducted to evaluate the performance after 2 years of functional activity. new controlled and randomized clinical trials are necessary. keywords: controlled clinical trials, composite resins introduction restorative composites have undergone continuous improvement in dental practice for over 40 years, since bowen1 incorporated inorganic particles into a resinous matrix. composites comprise a blend of hard, inorganic particles bound together by a soft, resin matrix, and generally encompass three main components: (1) the resin matrix comprising: (i) a monomer system, (ii) an initiator system for free radical polymerization, and (iii) stabilizers to maximize the storage stability of the uncured composite resin and the chemical stability of the cured composite resin; (2) the inorganic filler consisting of particulates such as glass, quartz, and/or fused silica; and (3) the coupling agent, usually an organo-silane, that chemically bonds the reinforcing filler to the resin matrix2. the first formulations contained large load particles of up to 150 µm (macrofilled composites), and had several unsatisfactory physical and mechanical properties3. investigations conducted in the 1970s and early 1980s concluded that the properties of macrofilled composites, particularly wear, color stability and marginal leakage, were clinically unacceptable for posterior teeth. research indicated that the inorganic content, geometry and load particle dimensions greatly influenced the final properties of the material4-5. during the last few decades, new composite formulations have appeared and the mean size of the particles has been drastically reduced. sub-micrometric particles have been used to braz j oral sci. 8(3):114-118 improve the physical and mechanical properties, namely reduction of the linear thermal expansion coefficient, greater dimensional stability and greater resistance to abrasion and wear3,6-7. microfilled composites with a mean filler particle size of 0.04 µm maintain the gloss produced after polishing. unfortunately, because of their low tensile strength and fracture toughness, microfilled composites are contraindicated for class iv and stress-bearing restorations8-9. hybrid composites are materials that contain a blend of prepolymerized and inorganic fillers. microhybrid composite has been successful to replace missing tooth structure even though hybrids are less polishable than microfilled composite; they have excellent mechanical properties10-11. according to peutzfeldt2, and walker and burgess10, most improvements in the properties of composites are due to the changes in the inorganic particles. these aspects have encouraged the use of various nanometric particles in resin-based materials. nanotechnology, also known as molecular nanotechnology or molecular engineering, is the production of functional materials and structures in the range of 0.1 to 100 nm (the nanoscale) by various physical or chemical methods12. the use of nanoparticles is useful for many applications including industry, transport, packaging, high-performance coatings, electronics, biomedicals, where nanoparticles improve the mechanical properties of materials. in dentistry, posterior class i or ii restorations require composites with high mechanical properties; anterior restorations need composites with superior esthetics. the composite resin that meets all the requirements of both posterior and anterior restorations has not yet emerged13. therefore, nanotechnology is of great interest in composite resin research13. the nanocomposite contains a unique combination of two types of nanofillers (5–75 nm) and nanoclusters. nanoparticles are discrete nonagglomerated and nonaggregated particles of 20–75 nm in size. nanocluster fillers are loosely bound agglomerates of nanosized particles. the agglomerates act as a single unit enabling high filler loading and high strength. due to the reduced dimension of the particles and the wide size distribution, an increased filler load can be achieved with the consequence of reducing polymerization shrinkage and increasing the mechanical properties, such as tensile strength and compressive strength to fracture. these seem to be equivalent or even sometimes higher than those of hybrid composites and significantly higher than microfilled composites. as a consequence, manufacturers now recommend the use of nanocomposites for both anterior and posterior restorations11,13–15. there are also composites on the market which are not exclusively nanoparticles, but contain nanometric and micrometric particles, and this has led to better performance. these materials are considered the precursors of nanoparticle composites and some refer to them as nanohybrids13. within the context of nanotechnology, the aim of this study was to analyze, by means of a systemic review of the literature, studies that conducted clinical trials with nanoparticle composite in posterior teeth in order to verify the performance of these composites in real conditions. material and methods the systematic review of the literature was conducted using the following full-text electronic journal databases: medline (international literature in health sciences), lilacs (latin american and caribbean literature in health sciences) and the cochrane library. for inclusion in this review, an article had to meet the following specifications: it should be a controlled clinical trial investigating nanoparticle composite in posterior teeth, with at least 6 months of evaluation, published in the english language between 1997 and 2009. articles with the following characteristics were excluded: in vitro studies, retrospective studies and studies on anterior teeth. the search strategy for the medline and lilacs databases was: nanofiller or (nanocomposite) or (nanofill) or (nanofilled) [words] and “clinical trial” or “controlled clinical trial” or “clinical trial phase i” or “clinical trial phase ii” or “clinical trial phase iii” or “clinical trial phase iv ” or “randomized controlled trial” or “multicenter study ” or “evaluation studies” [publication type] and “english ” [language]. in the cochrane library, the search was conducted as follows: nanofiller or (nanocomposite) or (nanofill) or (nanofilled). after aplication of the search strategy, two examiners reviewed the titles and abstracts of the articles and performed the selection by consensus. with the objective of complementing the database searches, non-automated manual searches were also conducted on the references within the selected articles. results and discussion nanoparticle composites were developed with the aim of combining high mechanical properties and maximum polishing. laboratory tests provide useful information on material performance and its manipulation characteristics, but they cannot provide answers about its clinical longevity. only controlled and randomized clinical trials can provide conclusions on the use of composites16. after application of the search strategy, 79 articles were found in the medline database, but only four of them met the pre-established criteria; no studies were found in lilacs. thirteen articles were found in the cochrane library, but only two met the inclusion criteria, and these coincided with those selected from medline database. the searches conducted from within the references of the selected articles yielded no additional articles fulfilling the inclusion requirements. table 1 explains the objectives, methodology, results and conclusions of the selected articles. although controlled and randomized clinical trials have a reference pattern, only controlled clinical trials were included in this review, except for dresch et al.17 who indicated that randomization was used and explained how it was accomplished. with regard to the sample size, all articles included at least 30 restorations per group, which is considered as a satisfactory number. in the study by ernst et al.18, a split mouth design was used and 56 restorations were performed in each group, but the total number of subjects was 50 because some individuals received more than one restoration per group. this contradicts the recommendations of hickel et al.19 who affirmed that each research subject must have, at most, one sample unit per group. in the research by efes et al.16 each patient received only one restoration, but the ideal condition of all the groups in the same mouth was not followed. dresch et al.17 designed 4 groups in the same patient, giving a total of 148 restorations and 37 per 115clinical trials with nanoparticle composite in posterior teeth: a systematic literature review braz j oral sci. 8(3):114-118 title (author, year) clinical evaluation of a nanofilled composite in posterior teeth: 12month results (dresch et al., 2006)17 a i m s to compare the clinical performance of a nanoparticle composite with two microhybrid composite and one compactable composite in posterior restorations after 12 months methodology thirty-seven patients with at least four class i or ii cavities and with normal occlusion were selected. a total of 148 restorations were performed, 25% of each material (filtek supreme/3m espe, st paul, mn, usa; pyramid/ bisco, schaumburg, il, usa; esthetx/dentsply, konstanz, germany or tetric ceram/ivoclar-vivadent, brendererstrasse liechtenstein, germany). two calibrated operators performed the treatment, in accordance with the manufacturer’s instructions. the adhesive systems used were from the same manufacturers as the resin, and in the case of deep cavities, calcium hydroxide cement and/or glass ionomer cement was applied. finishing and polishing were done 1 week later. two independent examiners assessed the restorations at baseline and 12 months later according to the united states public health service (usphs) modified criteria results and conclusions all the patients were assessed 12 months later. at baseline, postoperative sensitivity was observed in seven restorations, which disapeared at the 12-month evaluation. no secondary caries, marginal discoloration or lack of retention was observed after 1 year. color match and marginal adaptation were the items that received the highest number of bravo scores (11 and 12, respectively). only four restorations were classified as bravo in the anatomic form item. six restorations showed poor surface texture after 12 months. no statistically significant difference was observed between materials, and their performance at baseline and after 1 year was statistically similar. the authors concluded that the filtek supreme, pyramid, esthet-x and tetric ceram composites exhibited excellent clinical performance after 1 year clinical evaluation of an ormocer, a nanofill composite and a hybrid composite at 2 years (efes et al., 2006 during a period of 2 years evaluate the clinical performance of an ormocer, a nanoparticle composite, and as control, a hybrid composite, in small class i cavities in permanent molars ninety class i cavities were prepared in 90 patients, who had primary caries. the teeth were restored incrementally, in oblique layers with ormocer (admira/ voco, cuxhaven, germany), nanoparticle (filtek supreme/3m espe) or hybrid (renew/bisco) composite by a single operator. the restorations were examined using the ryge criteria (usphs modified) at baseline, after 6 months, 1 years and 2 years at the follow-up examinations, 100%, 100%, and 97% of 90 restorations were evaluated at 6 months, 1 year and 2 years, respectively. the scores for all the performance criteria were either alpha or bravo. none of the restorative materials presented secondary caries or postoperative sensitivity at 6 months, 1 year or 2 years. after 2 years, there were no clinically unacceptable criteria, except in one admira restoration (ormocer) which failed. according to the cavosurface marginal discoloration and the surface texture criteria, there were no significant differences between the restorative materials. the marginal adaptation rate was 100% for the nanofill composite and hybrid composite and 97% for the ormocer at both 1 and 2 years. there were no significant differences between the restorative materials. the composites studied resulted in high clinical performance after 2 years two-year clinical evaluation of ormocer, nanohybrid and nanofill composite restorative systems in posterior teeth (mahmoud et al., 2008)20 to evaluate and compare the 2-year clinical performance of an ormocer, a nanohybrid, and a nanofill resin composite with that of a conventional microhybrid composite in restorations of small occlusal cavities made in posterior teeth forty dental students from the faculty of dentistry at mansoura university were enrolled in this study. the criteria for their inclusion were the presence of primary caries or replacement of existing amalgam for esthetic reasons. each patient received at least 4 occlusal restorations. a total of 140 restorations was carried out, 25% for each material: ormocer-based, admira (voco); a nanohybrid resin composite, tetric evoceram (ivoclar-vivadent); a nanofill resin composite, filtek supreme (3m espe); and a microhybrid resin composite, tetric ceram (ivoclarvivadent). two operators carried out all restorations according to the manufacturers’ instructions. two independent examiners made all evaluations according to the usphsmodified ryge criteria immediately after placement of restorations and after 6 months, 1 year and 2 years all patients attended the 2-year recall visit. the scores for all the performance criteria were either alpha or bravo. only one ormocer and one microhybrid composite restoration had failed after 2 years. the color match of 13 microhybrid composite restorations was scored as bravo at the baseline examination. this criterion did not change during the 2-year period. regarding clinical performance, there were no statistically significant differences among the materials used. after 2 years, the ormocer, nanohybrid, and nanofill composites showed acceptable clinical performance similar to that of the microhybrid resin composite table 1 – title, author, year, aims, methodology, results and conclusion of articles 116 clinical trials with nanoparticle composite in posterior teeth: a systematic literature review braz j oral sci. 8(3):114-118 group. mahmoud et al.20 placed 140 restorations, 35 for each restorative composite, with each patient receiving at least 4 occlusal restorations. the studies addressed here were not unanimous with regard to the type of adhesive system used. the manufacturers of the composites recommend the use of an adhesive system from the same manufacturer. this rule was followed by efes et al.16, dresch et al.17 and mahmoud et al.20. however, ernst et al.18 decided to vary only the composite and used the same adhesive for both groups. they reported that the influence of the adhesive on the result of the clinical studies can first be seen in marginal discoloration or in the presence of open margins. these aspects were not verified in this study, which corroborates the clinical evidence that composite performance might not be compromised when used with an adhesive other than the one recommended. all restorations were assessed blindly, since the examiners did not know the restorative material. agreement among the examiners was high in all the articles (> 0.87). the experimental designs of the three studies had some differences, shown in table 2, but these particularities did not result in distinct outcomes among the articles. the restorations of the articles selected were evaluated by the modified usphs (united states public health service) criteria, which is a long-established method used in clinical trials. the criteria evaluated in common in the 4 articles were color match, retention, marginal adaptation, anatomic form, surface roughness, marginal staining, sensibility and secondary caries. the restorations were classified in alpha, bravo, charlie and delta. alpha and bravo scores mean excellent and clinically acceptable results, while charlie and delta scores mean clinically not acceptable, an indication to replace the restoration to prevent future damage or to repair present damage18. in the research by dresch et al.17, no differences were found between the nanoparticle composite, filtek supreme, the compactable pyramid and two microhybrids (esthet-x and tetric ceram) for the study periods. the majority of the scores were alpha and bravo, that is, all the materials exhibited excellent clinical performance after 1 year. no restorations failed. efes et al.16 ratified these results as they reported that the scores for color match, marginal discoloration, anatomical form, marginal adaptation and surface texture for all the restorative materials, changed from alpha to bravo at most. for the retention criteria, except for one admira (ormocer) restoration considered clinically unacceptable (score delta, restoration was partially or totally missing); all the others were alpha. there was no secondary caries, or postoperative sensitivity in the patients assessed. consequently, admira filtek supreme (nanoparticle) and renew (hybrid) resulted in high clinical performance after 2 years. study dresch et al, 200617 efes et al., 200616 ernst et al., 200618 mahmoud et al., 200820 number of professionals w h o performed the restorations 2 1 6 2 sample per g r o u p 37 30 56 35 total n u m b e r of restorations 148 90 112 reason for performing the restoration the majority w a s replacement of existing amalgam for esthetic reasons primary caries primary caries or deficient restoration primary caries or replacement of existing amalgam for esthetic reasons patient age not given 18–48 years mean 35.7 years (sd 11.3) not given isolation rubber dam cotton rolls and suction rubber dam cotton rolls and suction type of cavity classes i and ii class i minimally invasive class ii class i adhesive system one per group, from the same composite manufacturer one per group, from the same composite manufacturer one for all the groups one per group, from the same composite manufacturer pulp capping adhesive system (as). in deep cavities: calcium hydroxide and/ or glass ionomer cement before the adhesive system adhesive system adhesive system adhesive system. in cavities extending for more than 2mm into the dentin, a glass-ionomer cement before as followu p period 1 year 2 years 2 years 2 years final return rate (%) 100 97 100 100 number of failures with nanofill composite 0 0 1 0 table 2 – particular characteristics of the selected articles. 117clinical trials with nanoparticle composite in posterior teeth: a systematic literature review braz j oral sci. 8(3):114-118 in the study of ernst et al.18, the two composites used (tetric ceram-hybrid ( fine particles) and filtek supreme (nanoparticles) gave acceptable clinical performance at the end of 2 years. according to the usphs criteria, global clinical success (alpha and bravo scores) was 98%. with regard to color match, good performance was 95% for the nanoparticle composite and 98% for the hybrid. two fractures of restorations were observed within the observation period of 2 years: one chipping fracture (cohesive-type fracture) of a distal marginal ridge in a filtek supreme restoration placed in a mandibular molar and one bulk fracture in the mesial part of a tetric ceram restoration placed in a mandibular premolar. the study by mahmoud et al.20 also corroborates the findings of the articles mentioned above. the scores for all the performance criteria were either alpha or bravo. only one ormocer and one microhybrid composite restoration had failed after 2 years, showing secondary caries. although all the articles included in this review obtained good results, the authors16–18 warned about the need for longitudinal followup studies in order to obtain long-term answers about this new type of composite because the trials described were of short duration. notwithstanding the fact that all the articles included in this review were, in general, well designed, there is the need for additional controlled and randomized clinical trials that could shed light on some of the questions still not completely resolved. the following conclusions may be drawn: the performance of nanoparticle composites is satisfactory for use in posterior teeth for at least 2 years of functional activity; nnanoparticle composites can be used in posterior teeth, although their performance was not superior to that of the other composites studied; longer-term studies must be conducted to evaluate performances after 2 years of functional activity.; new controlled and randomized clinical trials are necessary to further evaluate some of the issues and questions that have not been fully addressed by current studies. references 1. bowen rl. properties of a silica-reinforced polymer for dental restorations. j am dent assoc. 1963; 66: 57-64. 2. peutzfeldt a. resin composites in dentistry: the monomer systems. eur j oral sci. 1997; 105: 97-116. 3. leinfelder kf. posterior composite resin: the materials and their clinical performance. j am dent assoc. 1995; 126: 663-76. 4. adabo gl, cruz cas, fonseca rg, vaz lg. the volumetric fraction of inorganic particles and the flexural strength of composites for posterior teeth. j dent. 2003; 31: 353-9. 5. willems g, lambrechts p, braem m, celis jp, vanherle g. a classification of dental composites according to their morphological and mechanical characteristics. dent mater. 1992; 8: 310-9. 6. bryant rw. direct posterior composite resin restorations: a review 1—factors influencing case selection. aust dent j. 1992; 37: 81-7. 7. lu h, lee yk, oguri m, powers jm. properties of a dental resin composite with a spherical inorganic filler. oper dent. 2006; 31: 734-40. 8. lambrechts p, ameye c, vanherle g. conventional and microfilled composite resins. part ii. chip fractures. j prosthet dent. 1982; 48: 527-38. 9. fortin d, vargas ma. the spectrum of composites: new techniques and materials.j am dent assoc. 2000; 131: 26s-30s. 10. walker r, burgess jo. comparing resin-based composites. compend contin educ dent. 2004; 25: 424-35. 11. mitra sb, wu d, holmes bn. an aplication of nanotechnology in advanced dental materials. j am dent assoc. 2003; 134: 1382-90. 12. kirk re, othmer df, kroschwitz j, howe-grant m. encyclopedia of chemical technology. 4th ed. new york: wiley; 1991. p.397. 13. beun s, glorieux t, devaux j, vreven j, leloup g. characterization of nanofilled compared to universal and microfilled composites. dent mater. 2007; 23: 51-9. 14. condon jr, ferracane jl. reduced polymerization stress through non-bonded nanofiller particles. biomaterials. 2002; 23: 3807-15. 15. yap auj, yap sh, teo ck, ng jj. comparison of surface finish of new aesthetic restorative materials. oper dent. 2004; 29: 100-4. 16. efes bg, dörter c, gömeç y. clinical evaluation of an ormocer, a nanofill composite and a hybrid composite at 2 years. am j dent. 2006; 19 :236-40. 17. dresch w, volpato s, gomes jc, ribeiro nr, reis a, loguercio ad. clinical evaluation of a nanofilled composite in posterior teeth: 12-month results. oper dent. 2006; 31: 409-17. 18. ernst cp, brandenbusch m, meyer g, canbek k, gottschalk f, willershausen b. two-year clinical performance of a nanofiller vs a fine-particle hybrid resin composite. clin oral investig. 2006; 10: 119-25. 19. hickel r, roulet jf, bayne s, heintze sd, mjör ia, peters m et al. recommendations for conducting controlled clinical studies of dental restorative materials. clin oral investig. 2007; 11: 5-33. 20. mahmoud sh, el-embaby ae, abdallah am, hamama hh. two-year clinical evaluation of ormocer, nanohybrid and nanofill composite restorative systems in posterior teeth. j adhes dent. 2008;10: 315-22. 118 clinical trials with nanoparticle composite in posterior teeth: a systematic literature review braz j oral sci. 8(3):114-118 oral sciences n3 case report braz j oral sci. october | december 2012 volume 11, number 4 complex odontoma: report of two unusual cases santosh patil1, d.n.s.v ramesh2, a. r kalla3 1mds, senior lecturer, dept of oral medicine and radiology, jodhpur dental college, jodhpur national university, jodhpur (raj), india 2mds, dean and head, department of oral medicine and radiology, navodaya dental college and hospital, raichur (kar), india 3md, associate professor, dept of pathology, s. n. medical college, jodhpur (raj), india correspondence to: santosh patil, dept of oral medicine and radiology, jodhpur dental college, jodhpur national university, jhanwar road, naranadi, jodhpur (raj). 342001. india. phone: +919887779845 / fax: +912931281416 e-mail: drpsantosh@yahoo.com abstract odontomas are the most common benign, slow-growing and nonaggressive odontogenic tumors of the jaws. they are usually clinically asymptomatic and are diagnosed on routine radiological examination in the second decade of life. the eruption and infection of odontomas are uncommon and very few cases are reported in the literature. this paper reports two cases of complex odontomas with such unusual features. keywords: odontoma, erupted odontoma, odontogenic tumors. introduction the term odontoma was first coined by broca in 1866, who defined it as a tumor formed by overgrowth of complete dental tissue1. they are composed of enamel, dentin, cementum and occasionally pulp tissue2. the exact etiology of odontomas is uncertain, different factors such as local trauma, infection, growth pressure, hereditary and developmental influences may be implicated3. according to the 2005 world health organization (who) classification of odontogenic tumors, there are two types of odontomas, composite and complex odontomas4. odontomas have also been classified as central odontoma (occur inside the bone), peripheral odontoma ( occur in the soft tissue covering the tooth-bearing portions of the jaws, which tends to exfoliate) and erupted odontoma5. though odontomas are common, eruption into the oral cavity and getting infected are exceptionally rare. two cases of complex odontomas exhibiting unusual features are reported in this paper. clinical cases case 1 a 25-year-old apparently healthy female was referred to our department of oral medicine and radiology with a complaint of a slow-growing painless mass in the left side of maxilla with 8 months of evolution. intraoral examination revealed a yellowish white mass on the alveolar ridge distal to left maxillary first premolar (figure 1). the mass was non-tender and hard in consistency. all left maxillary molars were absent and expansion of the buccal cortex was observed in the region of the left first and second molars. orthopantomography showed the presence of a radiopaque lesion distal to the maxillary left premolar above the alveolar bone level, measuring about 50 mm. an impacted molar was noted inside the lesion (figure 2). received for publication: june 23, 2012 accepted: november 12, 2012 braz j oral sci. 11(4):509-512 fig. 1. clinical photograph showing the odontoma erupting in the oral cavity. fig. 2. panoramic radiograph showing radiopaque mass distal to maxillary second premolar. on the basis of clinical and radiological findings, a diagnosis of erupting complex odontoma was established. the lesion was excised surgically under general anesthesia and sent for histopathological examination. fig. 3. photomicrograph showing disorganized mass of dentin, cementum and pulp (decalcified he; 20× magnification). the specimen was decalcified and processed in the usual manner. the paraffin embedded, hematoxylin and eosin stained, decalcified section of the hard tissue specimen revealed microscopically the presence of disorderly arranged mature hard tissues, the dentin and cementum and soft tissue pulp. the pulp tissue was surrounded by disordered dentin and mass of cementum. clear empty areas representing enamel lost during decalcification were present. these disorganized areas of dentin, cementum and pulp tissue with empty spaces representing enamel confirmed the diagnosis of complex odontoma (figure 3). case 2 a 22-year-old male patient was referred to us with a complaint of swelling on the right mandibular body with 1 year of evolution and recurrent pus discharge from the same site since 6 months. there was associated dull aching, localized intermittent pain. extraorally, a solitary fluctuant swelling near the angle of the mandible along with a healed sinus tract was noted (figure 4). fig. 4. diffuse extraoral swelling with sinus formation. intraoral examination revealed absence of all right mandibular molars. a panoramic radiograph showed a homogeneous radiopaque mass distal to the tilted mandibular second premolar, overlying the disto-inferiorly displaced crown of the mandibular right first molar. the primary mandibular right second molar was displaced inferiorly near to the lower border of mandible. there was no evidence of second and third molars. a uniform well-defined radiolucent halo was seen surrounding the radiopacity except in the coronal surface. the size of the lesion was approximately 40 mm (figure 5). based on the clinical and radiological findings, the lesion was tentatively diagnosed as infected complex odontoma. the mass was excised surgically along with the impacted complex odontoma: report of two unusual cases braz j oral sci. 11(4):509-512 510510510510510 fig. 5. orthopantomograph showing odontoma displacing permanent mandibular first molar and primary second molar. teeth under general anesthesia via intraoral approach with pre and post operative antibiotic and analgesic coverage (amoxicillin 500 mg and clavulanic acid 125 mg, three times a day for 7 days along with diclofenac sodium 50 mg, three times a day for 5 days). histopathological examination of the decalcified hard tissue mass revealed an ill-organized structure of dentin, cementum and pulp tissue. dentin and cementum were present in relatively larger quantities and formed the bulk of the tumor; enamel matrix and pulp were present in smaller quantities. spherical basophilic masses representing cementum were seen scattered in the decalcified section and the dentin was evident in the form of osteodentin or dysplastic dentin (figure 6). fig. 6. disordered arrangement of dentin, cementum and pulp. (decalcified he; 20× magnification). discussion odontomas are considered as hamartomas of aborted tooth formation rather than true neoplasms6. according to the histopathological perspective, odontomas can be grouped as: (a) complex odontomas, in which the dental tissues are well formed but exhibit a more or less disorderly arrangement and (b) composite odontomas, in which the dental tissues are normal, but their size and conformation are altered giving rise to multiple small tooth-like structures called denticles4,7. the complex odontomas are usually located in the posterior mandible, while composite odontomas are more often found in the anterior maxilla1. there have been isolated reports of odontomas in the maxillary sinus8. an infrequent situation is when the odontoma has erupted, i.e., when it becomes exposed through of the soft tissues. rumel et al. in 1980 described the first case of erupted odontoma9. since then only 20 cases were reported in the literature10. in general, most of these lesions are diagnosed in patients of less than 40 years of age. complex odontomas are less common in comparison with composite variety in the ratio 1:22. most odontomas are asymptomatic and literature reports only few cases of swelling, delayed eruption and in severe cases, infection or lymphadenopathy11,12. ferrer et al.13 reported a case of a young female with recurrent infection associated with erupted odontoma in the second quadrant. odontoma was surgically resected along with maxillary left second molar after administration of broad spectrum antibiotics and antiinflammatory drugs13. similar features were found in case two and the patient responded very well to broad spectrum antibiotics. the radiographic characteristics of odontomas always validate the diagnosis. the lesion consists of welldefined radiopacity surrounded by a radiolucent halo, which represents an enlarged cystic follicle. in composite odontoma are seen multiple teeth-like structures of varying size and shape. complex odontomas are seen as irregular radiodense masses with no resemblance to dental structures. three different development stages can be radiographically identified depending on the degree of odontoma calcification. in the first stage the lesion appears radiolucent due to the lack of calcification; the intermediate stage is characterized by partial calcification; and in the final stage the odontoma appears radiopaque surrounded by a radiolucent halo2. since odontomas are well capsulated lesions and have less chances of recurrence, the management comprises of a conservative surgical excision6. histologically, odontomas comprise varying amount of enamel, pulp tissue, enamel organ and cementum. the connective tissue capsule is similar to that of dental follicle. ghost cells are often seen along with spherical dystrophic calcification, enamel concretions and sheets of dysplastic dentin1. in conclusion, since odontomas represent a large proportion of jaw tumors, adequate knowledge of their characteristics is necessary for establishment of proper diagnosis and management. in this paper, two cases of complex odontomas with unusual features were presented. references 1. cohen dm, bhattacharyya i. ameloblastic fibroma, ameloblastic fibroodontoma, and odontoma. oral maxillofacial surg clin n am 2004;6:375384. 2. vengal m, arora h, ghosh s, pai km. large erupting complex odontoma: a case report. j can dent assoc 2007;73:169-172. 3. hitchin ad. the etiology of calcified composite odontoma. br dent j 1971;130:475-482. 4. barnes l, eveson jw, reichart p, sidransky d. (eds.). world health organization classification of tumours. pathology and genetics of e558 head and neck tumours. lyon: iarc press; 2005. p. 310. complex odontoma: report of two unusual cases braz j oral sci. 11(4):509-512 511511511511511 5. junquera l, de vicente jc, roig p, olay s, rodriguez-recio o. intraosseous odontoma erupted into the oral cavity: an unusual pathology. med oral patol oral cir bucal 2005;10:248-251. 6. shekar se, roopa sr, gunasheela b, supriya n. erupted compound odontoma. j oral maxillofac pathol 2009;13(1):47-50. 7. amailuk p, grubor d. erupted compound odontoma: case report of a 15 year old sudanese boy with a history of traditional dental mutilation. br dent j 2008;204:11-14. 8. mupparapu m, singer sr, rinaggio j. complex odontoma of unusual size involving the maxillary sinus: report of a case and review of ct and histopathologic features. quintessence int 2004; 35(8):641–645. 9. rumel a, de fritas a, birman e, tannous l, chacon p, borkas s. erupted complex odontoma. report of a case. dentomaxillofac radiol. 1980;9:5-9 10. serra-serra g, berini-aytes l, gay-escoda c. erupted odontomas: a report of three cases and review of the literature. med oral patol oral cir bucal. 2009 jun 1;14 (6):e299-303. 11. hidalgo-sanchez o, leco-berrocal mi, martinez-gonzalez jm. metaanalysis of the epidemiology and clinical manifestations of odontomas. med oral patol oral cir bucal. 2008 nov 1;13(11):e730-734. 12. al-sahhar w, putrus s. erupted odontoma. oral surg oral med oral pathol. 1985;59:225-226. 13. ferrer ramírez mj, silvestre donat fj, estelles ferriol e, grau garcía moreno d, lópez martínez r. recurrent infection of a complex odontoma following eruption in the mouth. med oral. 2001 augoct;6(4):269-275. complex odontoma: report of two unusual cases braz j oral sci. 11(4):509-512 512512512512512 oral sciences n3 original article braz j oral sci. july/september 2010 volume 9, number 3 received for publication: april 09, 2010 accepted: july 26, 2010 effect of post length on endodontically treated teeth: fracture resistance jefferson ricardo pereira1, accácio lins do valle2, talita magro juvêncio3, thais maria freire fernandes3, janaina salomon ghizoni4, marcus vinícius de reis só5 1dds, msc, phd, department of prosthodontics, dental school, university of south of santa catarina, brazil 2dds, msc, phd, department of prosthodontics, bauru dental school, university of são paulo, brazil 3dds, department of prosthodontics, bauru dental school, university of sao paulo, brazil 4dds, msc, phd, department of periodontology, dental school, university of south of santa catarina, brazil 5dds, msc, phd, department of endodontics, dental school, federal university of rio grande do sul. brazil correspondence to: jefferson ricardo pereira rua rio grande do sul, 1901 apto 303 mar grosso laguna – sc cep: 88790-000 brazil phone: +55 (48) 36471571 fax: +55 (14) 36264088 e-mail: jeffripe@rocketmail.com abstract aim: this study compared the fracture resistance of endodontically treated teeth restored with posts and cores systems with different post lengths. methods: sixty extracted intact canines were randomly divided in 6 groups of 10 teeth each, as follows: groups 1, 2 and 3 were restored with custom cast post-and-core, and groups 4, 5 and 6 were restored with prefabricated post and composite resin core, with different post lengths (5.0, 7.5 and 10 mm, respectively). a compressive load was applied at a 45-degree angle to the long axis until failure occurred. results: two-way analysis of variance (á=0.05) showed statistically significant difference between the groups (p<0.001). however, when the mean fracture forces for the groups were compared (group 1: 254.4 n; groups 2, 3, 4, 5, 6 – 331.7 n, 434.7 n, 405.4 n, 395.6 n and 393.8 n, respectively), no significant differences could be detected among the three groups restored with prefabricated post and group 3. conclusions: this study showed that an increased post length in teeth restored with prefabricated posts does not significantly increase the fracture resistance of endodontically treated teeth. on the other hand, endodontically treated teeth restored with custom cast-post and core showed significant increase on fracture resistance when the post length is increased. keywords: post and core technique, composite resin, fracture strength. introduction numerous techniques to restore endodontically treated teeth have been advocated with criteria for success depending on variations in length1, shape and surface configuration2, amount of dentin structure3-5, materials and techniques used in construction6. in prosthodontic practice, the task of restoring endodontically treated teeth is encountered almost daily. the pulpless tooth is known to present a higher risk of biomechanical failure than vital teeth7. the generally accepted explanation for the increased failure rate is the substantially loss of tooth structure during endodontic access, dowel space preparation, and cavity preparation8. the posts are necessary to allow the clinician to rebuild enough tooth structure to retain restorations9. the price for added retention, however, may be an increased risk to damage tooth structure. braz j oral sci. 9(3):371-375 372 the custom cast post and core system has been regarded as the restoration of choice for endodontically treated teeth when there is no coronal dentin. however, the use of prefabricated post systems are increasing because all steps can be completed chairside, and fair clinical success can be expected, simplifying the restorative procedures 3. some authors 3,10 advocated that roots restored with cast posts exhibited significantly higher internal stresses than prefabricated posts. the length of a post relative to root length is an unresolved problem in post design11. with recent improvements in the bonding of composite resin to dentin, true internal retention may assist with treatment success12-14. laboratory studies have shown that increasing the length of the post in teeth with post and core results in a more favorable stress distribution along the post 2,15-16 and an increased post length improves the resistance of the restored tooth to fracture17. a previous clinical study showed an increased survival rate has been correlated with increase of post length7. on the other hand, another study showed a minimal difference in stress distribution between varying post lengths 18, while other authors observed that an increase in post length as such will not necessarily increase the fracture resistance of the tooth19. it is important to notice that it may not always be possible to use a long post, especially when the remaining root is short or curved. several studies have suggested that it is important to preserve 3 to 5 mm of apical gutta-percha to maintain the apical seal20. the purpose of this study was to evaluate the relative effect of post length (length of the vertical dentinal overlap of the crown) and type of post and core (custom cast post and core or prefabricated post and composite resin core) on the resistance of endodontically treated teeth. the research hypotheses were that there is a significant difference in the effect of post length on the fracture resistance, and that there is a significant difference between the types of posts. material and methods sixty human maxillary canines freshly extracted for periodontal reasons were selected and stored in distilled water at 37ºc during the course of the study. teeth with cracks, caries, restoration, and/or roots shorter than 15 mm (measured with a millimeter ruler from the apex until the cementoenamel junction cej) were discarded. the roots were scaled with periodontal curettes and water and were sectioned with double-faced diamonds discs (kg sorensen, barueri, sp, brazil) to a standardized length of 15 mm. the endodontic treatment was done using a standard master apical file #20 (dentsply maillefer, ballaigues, switzerland) that extended 1 mm beyond the apex and the preparation took place with a conventional step-back technique to an international standardization organization (iso) file #35 (dentsply maillefer) at the apical constriction. the teeth were obturated by lateral condensation using gutta-percha points (tamari, tamariman industrial ltda, macaçaruru, am, brazil) and an iso 35 primary gutta-percha master cone (tamari, tamariman industrial ltda). root canal sealer (endomethasone ivory; septodont brasil, barueri, sp, brazil) was used as the sealer. after this, the teeth were randomly divided into 6 groups of 10 teeth each. the randomization was accomplished by drawling lots. different post preparations were standardized using a #5 reamer (largo; dentsply maillefer). five millimeters of gutta-percha (apical to cej) from each filled canal in groups 1 and 4, 7.5 mm in groups 2 and 5, and 10 mm in groups 3 and 6, as showed in figure 1. fig.1. post lengths of 10.0 mm (groups 3 and 6), 7.5 mm (groups 2 and 5) and 5.0 mm (groups 1 and 3) from left to right. in groups 1, 2 and 3 the tooth was restored with a custom cast post and core. impression of the root canals was made with acrylic resin (duralay, reliance dental mfg. co. chicago, ill). the cores were standardized using a coreforming matrix (tdv dental, pomerode-sc, brazil). the patterns were invested (cristobalite, whip-mix corporation, louisville, ky,) and cast in cu-al alloy (npg, aalbadent, cordelio, ca). after casting, small nodules were removed if present. the post-cores were sectioned and seated to their corresponding teeth and rely x luting cement (3m dental products division, st. paul, mn, usa) was used to cement them. the material was prepared according to the manufacturer’s instructions and taken to the canals with a spiral file (lentulo, dentsply maillefer, ballaigus, switzerland) at low speed. cement was placed on the post and seated under 9 kg of pressure during 10 min. after this time, the pressure was released and the post was held in place until final setting of the cement. excess cement was removed and each specimen was returned to storage in distilled water. in groups 4, 5 and 6 the canals were restored with prefabricated stainless steel, parallel-sided, serrated posts with a tapered end (number 5317, screw-post, euro-post anthogyr s.a., sallanches, france). in these groups, the teeth were cemented with the same material and the same technique as used in the other groups. the coronal portion was made with composite resin material (z250; 3m dental products division).the root surfaces and cervical dentin was etched with 37% phosphoric acid for 30 s, rinsed, and air dried. two coats of bonding agent (prime & bond 2., dentsply ind. e com ltda., petropolis, rj, brazil) was applied to the cervical dentin and the coronal portion of the post and were lightcured for 20 s (ultraled, dabi atlante, ribeirão preto, sp, brazil; 110 w). cores were fabricated in a standardized form, using the same core-forming matrix as used in the other effect of post length on endodontically treated teeth: fracture resistance braz j oral sci. 9(3):371-375 373 groups. five increments of the composite resin were applied to complete the coronal core, each requiring 40 s of light curing (ultraled, dabi atlante, ribeirão preto-sp, brazil) to complete the coronal core. the tip of the light guide of the curing unit was positioned 2 cm from the specimens on top of the core. all teeth were prepared with a #3216 diamond bur (kg sorensen, barueri, sp, brazil) coupled to a high-speed handpiece (super torque 625 autofix, kavo do brasil ind. com. ltda., joinville, sc, brazil) with water spray coolant to simulate a crown preparation with 1.5 mm of facial reduction with a chamfer finish line and 0.5 mm of chamfered lingual reduction, and receive a pfm crown. all the finish lines, for all specimens and groups, were placed at the cej level. crown wax (kerr corporation, ca, usa) patterns were then made for the specimens using vinyl polysiloxane (aquasil, dentsply, konstanz, germany) impressions obtained from the teeth prior to preparation. a lingual ledge was added to create a standard loading point. the wax patterns were sprued, invested (cristobalite, whip-mix corporation, louisville-ky, usa) and cast in a ni-cr alloy (durabond, são paulo, brazil). crowns were cemented using the same material used with the posts. root surfaces were covered with a 0.6-mm-thick foil (adapta foil; bego, bremen, germany) to a depth 2 mm below the cej to produce a layer approximately equal to the average thickness of the periodontal ligament. teeth were embedded in acrylic resin (clássico, artigos odontológicos s/a, são paulo, sp, brazil) poured into molds made of same material (30 mm in height, and diameter of 22 mm and a internal space, located in the center of the mold, with diameter of 10 mm and 20 mm in height) along their long axes using a surveyor (bio art equipamentos odontológicos ltda, são carlos, sp, brazil). each tooth was removed from the resin block, by moving rods in an upward direction, when the first signs of polymerization were observed. the adapta spacers were removed from the root surfaces. aquasil was injected into the acrylic resin blocks, and the teeth were reinserted into them. a standardized silicone layer that simulated periodontal ligament thus was created3. the specimens were tested in a universal testing machine (kratus k2000 mp, dinamometros kratos ltda, são paulo, sp, brazil (figure 2). each specimen was affixed in a custommade apparatus (fabricated by the authors) that allowed it to be positioned at 45 degrees to the buccal/lingual long axis (figure 2). the testing machine was set at a crosshead speed of 0.5 mm/min. the load was measured in newtons (n). failure threshold was defined as the point at which a specimen could no longer withstand increasing load and fracture of the post-core complex or root occurred. the mode of failure was recorder after the test using x4 binocular magnifier (bio art equipamentos odontológicos ltda, são carlos, sp, brazil). data were analyzed statistically by 2-way anova to determine the overall differences among the means of the test groups and the overall variability within the test groups. tukey’s multiple-comparison test was conducted to determine which test groups were statistically different from the others. all testing was done with alpha equal to 0.05. results table 1 presents the mean fracture resistance values (in n) standard deviations obtained for each studied group. significant differences were detected by anova (table 2) (p<0.001). the tukey’s test confirmed that the mean fracture resistance for group 3 was significantly greater than groups 1 and 2 and fracture resistance for the group 6 was not significantly greater than groups 5 and 6 (table 1). the fracture patterns of all groups are presented in table 3. the majority of the failures in the groups restored with custom cast and post occurred due to root fracture. however in the groups restored with prefabricated posts the failure occurred due to core fracture. groups mean (n) s d 1 254.4a (35.6) 2 331.7ab (77.3) 6 393.8bc (81.1) 5 395.6bc (57.8) 4 405.4bc (71.4) 3 434.7c (75.1) table 1. resistance to failure values of test specimens, means (n) of test groups (standard deviation) and tukey’s comparisons groups with same superscripted letters are not significantly different at p< 0.05 (tukey test). sd standard deviation fig. 2. specimens were subjected to load at 45 degrees on universal testing machine. effect of post length on endodontically treated teeth: fracture resistance braz j oral sci. 9(3):371-375 374 effect dfeffect mseffect dferror mserror f p type of restoration 1 529.3728 54 48.49611 10.91578 .001697 post length 2 369.3319 54 48.49611 7.61570 .001220 interaction 2 477.6714 54 48.49611 9.84968 .000225 table 2. two-way analysis of variance. discussion the present study accepted the hypotheses that there is a significant difference in the effect of post length on the fracture resistance and the hypotheses that there is a significant difference between the types of posts. group 3 presented higher fracture resistance (p<0.05) than groups 1 and 2. these findings are believed to be related to the higher strength of the nickel-chromium alloy, and the higher modulus of elasticity of this material10. this is in agreement with standlee et al.2 and holmes et al.16, who showed that increasing the post length in the tooth results in a more favorable stress distribution along the post. on the other hand, the results of the present study showed that the increase of post length in teeth restored with prefabricated posts and composite resin core did not increase significantly the fracture resistance of endodontically treated teeth. studies have shown that post preparation weakens the root considerably15. furthermore cementation of a post can regain some of the root’s original strength15. in other words, the use of a post may weaken the root more than it reinforces it. this may explain why increasing post length in these groups did not consistently increase the fracture resistance of these roots in the present study. our results are in agreement with those of isidor et al.19, who showed that increasing post length in teeth restored with prefabricated posts did not increase the fracture resistance of endodontically treated teeth. we found no significant difference on fracture resistance was found when the group restored with custom cast post and core was compared to the group restored with prefabricated post and composite resin core with 5 mm of post length each one. this could be explained because the size and shape of the composite resin matrix particles account for 66% of its volume13, and this higher amount of inorganic particles corresponds to the maximum resistance of compressive load, surface hardness and wear resistance13. furthermore, it has been demonstrated by some authors that roots restored with custom cast post and core presented significantly higher internal stress than prefabricated posts10. core construction using prefabricated posts and composite resin is a viable technique for endodontically treated teeth3-6. composite resin core fracture occurring when occlusal force is applied may be a positive occurrence because it could be protective of the supporting root12. the most common cause of failure when the choice is the direct technique (prefabricated post and composite resin) is fracture of the restorative material12. the results of this study are in agreement with another study that concluded that composite resin fracture can occur at a lower force than that required to yield root fracture14. when the cast post-andcore was used the most common failure is the fracture of the root12 as was observed in this study (table 3). groups location of failure composite resin only root only crown dislodged 1 5 (50%) 5 (50%) 2 8 (80%) 2 (20%) 3 10 (100%) 4 10 (100%) 5 10 (100%) 6 9 (90%) 1 (10%) table 3. types of failure (number and percentage of the teeth) this investigation demonstrated that roots restored by individual cast posts with 10 mm of post length exhibited higher fracture resistance than those restored by prefabricated post and composite resin core. despite its lower resistance, the technique using prefabricated posts and composite resin may be appropriate because there were no root fractures. hence, the direct method appeared to protect the tooth structure12. the limitations of this study include that this was an “in vitro” experiment, which did not replicate oral conditions, and a single load to fracture test was used to test the fracture resistance of endodontically treated teeth. for more meaningful results, future studies should incorporate thermal cycling of the specimens and fatigue loading. acknowledgements this study was supported by fapesp. references 1. shillingburg ht, fisher dw, dewhirst rb. restoration of the endodontically treated posterior teeth. j prosthet dent. 1970; 24: 401-9. 2. standlee jp, caputo aa, collard ew, pollack mii. analysis of stress distribution by endodontic posts. oral surg 1972; 33: 952-60. 3. pereira jr, de ornelas f, conti pc, do valle al. effect of a crown ferrule on the resistance of endodontically treated teeth restored with prefabricated posts. j. prosthet dent. 2006; 95: 50-4. 4. pereira jr, mendonça neto t, porto v de c, pegoraro lf, valle al. influence of the remaining coronal structure on the resistance of teeth with intraradicular retainer. braz dent j. 2005; 16: 197-201. 5. pereira jr, valle al, shiratori fk, ghizoni js, melo mp. influence of intraradicular post and crown ferrule on the fracture strength of endodontically treated teeth. braz dent j. 2009; 20: 297-302. 6. zogheib lv, pereira jr, valle al, oliveira ja, pegoraro lf. fracture resistance of weakened roots restored with composite resin and glass fiber post. braz dent j. 2008; 19: 329-33. 7. hammerle ch, ungerer mc, fantoni pc, bragger u, burgin w, lang np. long-term analysis of biologic and technical aspects of fixed partial dentures with cantilevers. int j prosthodont. 2000; 13: 409-15. 8. panitvisai p, messer hh. cuspal deflection in molars in relation to endodontic and restorative procedures. j endod. 1995; 21: 57-61. effect of post length on endodontically treated teeth: fracture resistance braz j oral sci. 9(3):371-375 9. caputo aa, standlee jp. pins and posts-why, when and how. dent clin north am. 1976; 20: 299-311. 10. fraga rc, chaves gsb, mello jf, siqueira jr. fracture resistance of endodontically treated roots after restoration. j oral rehabil. 1998; 25: 80913. 11. fernandes as, shetty s, coutinho i. factors determining post selection: a literature rewiew. j prosthet dent. 2003; 90: 556-62. 12. abdalla al, alhadainy ha. 2-years clinical evaluation of class i posterior composites. am j dent. 1996; 9: 150-2. 13. bex rt, parker mw, judkins jt, pelleu gb. effect of dentinal bonded resin post-core preparation on resistance to vertical fracture. j prosthet dent. 1992; 67: 768-72. 14. bowen rl, cobb en. a method for bonding to dentin and enamel. j am dent assoc. 1983; 107: 1070-6. 15. leary jm, aquilino, sa, svare cw. an evaluation of post length within the elastic limits of dentin. j prosthet dent. 1987; 57: 277-81. 16. holmes dc, arnold am, leary jm. influence of post dimensions on stress distribution in dentin. j prosthet dent. 1996; 75: 140-7. 17. trabert kc, caputo aa, abou rass m. tooth fracture – a comparison of endodontic and restorative treatment. j endod. 1978; 4: 341-5. 18. burns da, krause wr, douglas hb, burns dr. stress distribution surrounding endodontic posts. j prosthet dent. 1990; 64: 412-8. 19. isidor f, brondum k, ravnholt g. the influence of post length and crown ferrule on the resistance to cyclic loading of bovine teeth prefabricated titanium post. int j prosthodont. 1999; 12: 79-82. 20. kvist t, rydin e, reit c. the relative frequency of periapical lesions in teeth with root canal –retained posts. j endod. 1989; 15: 578-80. 375 effect of post length on endodontically treated teeth: fracture resistance braz j oral sci. 9(3):371-375 oral sciences n3 braz j oral sci. july/september 2009 volume 8, number 3 original article clinical performance of indirect esthetic inlays and onlays for posterior teeth after 40 months regina helena barbosa tavares silva1, ana paula dias ribeiro2, alma blacida conception elisaur catirze 3 lígia antunes pereira pinelli2, laisa maria grassi fais2 1 dds, ms, phd, professor, department of dental materials and prosthodontics, são paulo state university, araraquara school of dentistry, araraquara, sp, brazil. 2 dds, ms, graduated student, department of dental materials and prosthodontics, são paulo state university, araraquara school of dentistry, araraquara, sp, brazil. 3 dds, ms, phd, professor, department of dental materials and prosthodontics, são paulo state university, ribeirão preto school of dentistry, ribeirão preto, sp, brazil. received for publication: june 22, 2009 accepted: november 19, 2009 correspondence to: regina helena barbosa tavares da silva rua humaitá, 1680 araraquara-sp brazil phone: +55-163301-6409. fax: +55-163301-6406 e-mail: reginats@foar.unesp.br abstract aim: searches for biocompatible restorative materials with better clinical properties, longevity and esthetics have resulted in the development of several ceramic types. the aim of this study was to evaluate the performance of ceramco inlays and onlays over 40 months. methods: thirty ceramic indirect restorations were placed in 10 patients and all were adhesively cemented with a dual resin cement. the clinical performance was evaluated by a calibrated examiner who attributed scores adapted from the cvar and ryge criteria: color, marginal adaptation, abrasion, caries recurrence, fracture and postoperative pain. these assessments were performed after cementation of the restorations (t 0 =baseline) and after 4 periods: t 1 (10 months), t 2 (20 months), t 3 (30 months) and t 4 (40 months). photographs were made in t 0 and t 4 to illustrate the general condition of each restoration. data were analyzed statistically by kruskal-wallis h statistics (p=0.05) and results were presented using percentage values. results: clinical evaluation revealed no color alteration or abrasion (100%); a success rate of 96.7% for caries, fractures and postoperative pain; and 76.7% of failure for marginal adaptation. conclusion: the ceramic restorations did not show alterations that could result in their replacement, although there was a moderate failure in the marginal adaptation. keywords: dental materials, inlays, onlays, clinical trial introduction the search for an ideal restoration has led to the development of several restorative techniques and materials. although metallic alloys were used due to their favorable physicomechanical properties1-2, much effort has been made to develop materials with better performance to fulfill clinical requirements3-5 and meet the patients’ esthetic expectations5. restorations with similar characteristics to those of the dental structures, such as color, brightness, and superficial texture, associated to low cost and high durability, are often the patients’ desire. the most recent results of this evolution are the composite materials and the non metallic ceramic restorations4,6-7. ceramic materials are brittle, with relatively high compressive strength, but present low flexural strength and fracture toughness8-9. nevertheless, the main disadvantage of this material is the high potential of wearing the enamel or resin restoration of the antagonist teeth. the new ceramic compositions have demonstrated an abrading potential of dental enamel similar to that of natural teeth, which means that they have less aggressive behavior. moreover, they are biocompatible with the pulp and periodontal tissues, present less biofilm accumulation than the enamel, and transfer less heat and electric current when compared to metallic alloy restorations3. the restorative technique with dental ceramics has also been enhanced significantly and had its acceptance increased due to the advancement of adhesive systems. the advent of dual braz j oral sci. 8(3):154-158 inlay/onlay characteristics color marginal adaptation abrasion caries recurrence fracture postoperative pain a compatible color great marginal adaptation no abrasion no secondary caries no fracture no postoperative pain b changes on color within an acceptable range of tooth color changes on marginal adaptation: no visible evidence of a crevice into which the explorer could penetrate presence of abrasion: missing material is not sufficient to expose dentin base presence of secondary caries presence of marginal fracture presence of postoperative pain c severe defects on color that is outside the acceptable severe defects on marginal adaptation: explorer penetrates into a crevice that is of a depth that exposes dentin or base presence of abrasion*: sufficient material lost to expose dentin or base _________________ presence of body fracture* _________________ table 1. criteria used for the clinical evaluation of the ceramic inlays/onlays *severe defects that indicate the replacement of the restoration. cure resin cements associated with recent dental adhesives represent a significant improvement in cement adhesion, thus optimizing the retention and stability of ceramic restorations3,5,8,10-11. despite of all this advantages, the longevity and success of this type of restoration depend on the correct indication, clinical experience of the operator and an accurate work of the laboratory technician12-14. since only few long-term clinical studies exist under controlled conditions 15-17, it seemed interesting to assess the longitudinal performance of ceramic inlays/onlays restorations over 40 months using a direct evaluation. this study is expected to contribute to a better indication of dental ceramics aiming to contribute to return teeth to a normal condition and promote their reintegration in the stomatognathic system. material and methods ten undergraduate dental students from the araraquara school of dentistry, são paulo state university aged 18 to 21 years were select for this study. the volunteers should have almost similar buccal and dental conditions, habits, diet, age and indication for an adhesive restoration. patients with parafunctional habits, poor hygiene and periodontal conditions were excluded from the selected group. the patients were informed of the research protocol and agreed to attend a recall program of 40 months. thirty ceramic restorations (12 onlays and 18 inlays) (dentsply ceramco, york, pa, usa) were placed in 6 maxillary premolars, 8 maxillary molars, 4 mandibular premolars and 12 mandibular molars. the cavities included o (12), mo (4), od (3), mod (6), mol (1), modv (2) and modl (2) preparations. a calibrated professional prepared the posterior teeth for indirect ceramic inlay/onlay restorations using tapered and round diamond burs (kg sorensen indústria e comércio ltda., barueri, sp, brazil). the carious tissue was removed with excavator and the dentin-pulp complex was protected with calcium hydroxide (hydro c; dentsply, rj, brazil) and glass ionomer cements (vitrebond; 3m espe, sumaré, sp, brazil) in deep cavities. all cavities were prepared according to the established principles for adhesive inlays/onlays, which included an occlusal reduction of 1.5 to 2.0 mm, with a wide isthmus rounded internal angles and the axial wall with 1.5 mm of thickness. gingival margins were prepared entirely in enamel whenever possible at the cementoenamel junction18. custom trays were used for the 1-step full-arch impressions using vinyl polysiloxane (reprosil dentsply, petrópolis, rj, brazil) and the antagonist impression was made with alginate compound ( jeltrate; indústria e comércio ltda.). the casts were placed on an adjustable articulator and all the inlays and onlays fabricated by a dental technician according to the manufacturer’s instructions. th e resto rations w ere e valu at ed a nd , w h en n ece ssa r y, adjustments were made with diamonds burs (kg sorensen indústria e comércio ltda.) and fine-grain diamond discs. the adjusted restorations were polished with aluminum oxide discs (sof-lex, 3m, sumaré, sp, brazil), enhance abrasive rubbers (denstply indústria e comércio ltda.) and pumice slurry. under rubber dam, the enamel was etched with 37% phosphoric acid gel (scotchbond; 3m espe) for 30 s and the remaining dentin was etched for 10 s. afterwards, syntac primer (ivoclar vivadent, são paulo, sp, brazil) was a applied for 15 s on the dentin and the excess was gently removed with air syringe. then, syntac adhesive was applied for 10 s on the cavity and dried thoroughly with blown air. the restorations were cemented with dual cement a ccording to the manufacturer’s instructions (ivoclar vivadent). polymerization was performed by the application of a halogen light (heliomat-vivadent, são paulo, sp, brazil) for 20 s in each tooth face with a fluence rate of 500 mw/cm2. the occlusal contacts were checked and, when necessary, teeth were adjusted using diamond rotary cutting instruments. final polishing was conducted using f and ff diamond finishing rotary cutting instruments (kg sorensen indústria e comércio ltda.), and rubbers and felt discs with polishing diamond slurry. all restorations were clinically evaluated on four recalls during 40 months. the periods were: t1 (10 months), t2 (20 months), t3 (30 months) and t4 (40 months). all the restorations of t0 (baseline) were considered satisfactory and received high scores for each variable. the photographs were made with a dental eye ii camera (yashica, sorocaba, sp, brazil) with the maximum approach only to illustrate the general condition of each restoration. clinical evaluations were made by a calibrated professional using an explorer and plain clinical mirror, with the tooth and the restoration previously dried. the variables studied were: color, marginal adaptation, abrasion, caries recurrence, fracture and postoperative pain. all restorations received an adapted score following the description of cvar and ryge criteria19 (table 1). clinical performance of indirect esthetic inlays and onlays for posterior teeth after 40 months 155 braz j oral sci. 8(3):154-158 evaluation period m e d i a n t 1 24.8 t 2 27.5 t 3 29.8 t 4 39.9 h statistics: 6.41 p-value: 0.0933 table 2. kruskal-wallis h statistics results. a b c a b c a b c a b c color 30 0 0 30 0 0 30 0 0 30 0 0 % 100 0 0 100 0 0 100 0 0 100 0 0 marginal adaptation 7 23 0 7 23 0 7 23 0 7 23 0 % 23.3 76.7 0 23.3 76.7 0 23.3 76.7 0 23.3 76.7 0 abrasion 30 0 0 30 0 0 30 0 0 30 0 0 % 100 0 0 100 0 0 100 0 0 100 0 0 caries recurrence 29 1 0 29 1 0 29 1 0 29 1 0 % 96.7 3.3 0 96.7 3.3 0 96.7 3.3 0 96.7 3.3 0 fracture 29 1 0 29 1 0 29 1 0 29 1 0 % 96.7 3.3 0 96.7 3.3 0 96.7 3.3 0 96.7 3.3 0 postoperative pain 24 6 0 25 5 0 27 3 0 29 1 0 % 80 20 0 83.3 16.7 0 90 10 0 96.7 3.3 0 t1 t2 t3 t4 table 3. clinical evaluation of the ceramco restorations. results the 40-month recall rate was 100% on clinical re-evaluations. in order to evaluate the clinical performance of ceramco inlays/onlays during the 40 months, kruskal-wallis h statistics were applied (p=0.05). no statistically significant difference was observed between the periods (t1, t2, t3, t4) (table 2). the outcomes observed in clinical evaluations for the ceramco inlays/onlays during t1, t2, t3 and t4 periods are presented in table 3. during the 40-month evaluation period, no color alteration or abrasion occurred. fracture and secondary caries occurred in only 1 restoration, corresponding to 3.3%, which maintain in 96.7% the success index. postoperative pain was present in 20% of the cases, in t1 period of evaluation. however, postoperative pain was not present on t4, which represents a success index of 100% on that period. the same success level was not achieved for the marginal adaptation. twenty-three restorations (76.7%) presented alterations in the restoration/tooth interface. these alterations occurred in the first months of use, and the same level was maintained after the first time of evaluation (t1) until the t4 analysis. figures 1 and 2 show the general condition of restorations in t0 and t4. discussion the evaluation of the clinical performance of the ceramco restorative ceramic system within a 40-month period showed a high success rate for most analyzed variables (color, marginal adaptation, abrasion, secondary caries, fracture and postoperative pain), which is in accordance with the literature20-21. although this system has a relatively complex technique and high cost, it has excellent esthetic results and mechanical resistance, in addition to presenting biocompatibility when compared to other restorative materials14,22. in recent years, these features have contributed to the high-quality performance of ceramic restoration, which leads to good acceptance by the professionals. figure 2: ceramco inlay photograph evaluated after 40 months (t4). the restoration maintained high scores for each assessed category and continued satisfactory figure 1: ceramco inlay photograph taken immediately after cementation (t0). the restoration was considered satisfactory and received high scores color, marginal adaptation, abrasion, caries recurrence, fracture and postoperative pain. clinical performance of indirect esthetic inlays and onlays for posterior teeth after 40 months156 braz j oral sci. 8(3):154-158 in addition to these successful rates, the longevity of ceramic restorations is subjected to many factors that can act in a positive or negative form and should be carefully controlled. on these concerns, factors that have the major occurrence were fracture of the restorations, hypersensitivity, loss of retention and fracture of the restored tooth15. molin and karlson21 reported that these factors can be originated from misfits, occlusal forces, incorrect cavity designs and imperfections in the cementation technique. among the 30 (33%) restorations evaluated in this clinical study, fracture occurred in only one during the analyzed period. friability is an inherent characteristic of dental ceramics and it has direct influence on their durability 21,23. ceramic fractures have been related to inadequate tooth preparation, occlusal adjustments disrupting the surface and faulty material. tagtekin et al.24 (2009) found that fractures in ceramic restorations usually occur during the first 6 or 8 months, and the results of the present study agree with this evidence. the fracture observed in our study could possibly be due to the occlusal adjustments done after cementation, although the other restorations were also adjusted. these adjustments might have caused disruption of the superficial glazed layer, generating micro-fractures that became points of crack propagation9,15-16,25. some special care should be necessary to deal with this problem as an occlusal adjustment of the restoration integrated with a balanced occlusion, an adequate preparation and adhesion technique for cementation. the use of resin cement provided a better integration between tooth and restoration, transferring the external forces to the dentin. therefore, an increase in ceramic resistance occurred, probably resulting in enhanced clinical durability of these restorations5. the results of this study clearly demonstrate that the major problem is situated at the margin between the restoration and dental structure. kramer and frankenberger12 reported that every clinical trial assessing ceramic inlays revealed a certain deterioration of marginal quality. this might be caused by insufficient bonding to enamel or degradation of the luting agent caused by fatigue. therefore, it is necessary an adaptation with the dental structure as good as possible for these restorations, including edges and external cavosurface margins5. the negative results observed for marginal adaptation occurred in the first months, and showed a tendency to keep the same level (76.7%) until the final analysis (40 months). this fact clearly demonstrates that the main concern with this type of restoration must be the initial adaptation and the friability of ceramic material at the cementation moment and the first months of use, as well as for the material and technique used to cement these restorations26. these results were also observed in other clinical studies21,27 where the marginal adaptation was the factor that presented more alteration for indirect ceramic restoration. however, lange and pfeiffer28 showed that 93% of the c eramic inlays received score “a” for marginal adaptation after 57 months of evaluation, and the other 7% did not need replacement. the union imperfection can be explained by the incomplete polymerization of the resinous cement, by the low resistance of some cements, lack of acid and silane treatment on the internal surface of the restoration, absence of an adhesive agent, and/ or by the possible fatigue that occurs in the adhesive agent after long periods of clinical use and action of occlusal loads, mainly in patients with bruxism3. postoperative pain was the second more incident alteration. ceramic postoperative hypersensitivity has been initially reported to be problematic due to incomplete sealed of dentin or detachment between material and dentin12. this was a common occurrence in the majority of the cases, is a transitory characteristic and it is directly related to the wet technique of dentin hybridization, which was used in this study. the need of restoration replacement is rare, similar to what occurred in this study29. one limitation of the present study may be the fact that probably the period of 40 months was not sufficient for the appearance of significant clinical alterations. however, in recent studies18,24,28 a great clinical performance of ceramic inlays/onlays was observed at longer periods of evaluation. finally, it might be considered that the good results were positively influenced by the selected patients. nevertheless, it can be noticed that, although there were not statistically significant results, there was a small tendency of deterioration of the restorations with time. this shows that long-term analyses could probably indicate critical alterations that require the restoration substitution15,27. in spite of the positive results obtained in the present study, long-term clinical investigations are needed to obtain in situ information on the performance of the ceramic materials22,30-31. it is also necessary to have studies and improvements on the tooth/ restoration interface, as the clinical success with ceramic inlays/onlays depends on the ability to develop a reliable bond of the composite to dental tissues. this study evaluated 30 ceramco ceramic inlays and onlays and none of them showed any alteration that could indicate their replacement, although there was a moderated failure of marginal adaptation. within the limitations of the design and the evaluation time of this investigation, this restoration technique seems clinically acceptable as an esthetic and conservative treatment method for molar and premolar restorations. references 1schulte ag, vöckler a, reinhardt r. longevity of ceramic inlays and onlays luted with a solely light-curing composite resin. j dent. 2005; 33: 433-42. 2wirz j, jaeger k. modern alternatives to amalgam: cementable restorations and inlays. quintessence int. 1999; 30: 551-6. 3ferrari m, dagostina a, fabianelli a. marginal integrity of ceramic inlays luted with a self-curinrg resin system. dent mater. 2003;19: 270-6. 4ozturk an, usumez a. influence of different light sources on microtensile bond strength and gap formation of resin cement under porcelain inlay restorations. j oral rehab. 2004; 31: 905-10. 5rosentritt m, behr m, lang r, handel g. influence of cement type on the marginal adaptation of all-ceramic mod inlays. dent mater. 2004; 20: 463-9. 6al-hiyasat a, saunders wp, smith gm. three-body wear associated with three ceramics and enamel. j prosthet dent. 1999; 82: 476-81. 7rosenblum mh, schulman aj. a review of all-ceramic restoration. j am dent assoc. 1997; 128: 297-307. 8abdalla al, davidson cl. marginal integrity after fatigue loading of ceramic inlay restorations luted with three different cements. am j dent. 2000; 13: 77-80. 9smales rj, etemadis s. survival of ceramic onlays placed with and without metal reinforcement. j prosthet dent. 2004; 91: 548-53. 10bott b, hannig m. effect of different luting materials on the marginal adaptation of class i ceramic inlay restorations in vitro. dent mater.2003; 19: 264-9. 11dalpino ph, francischone ce, ishikiriama a, franco eb. fracture resistance of teeth directly and indirectly restored with composite resin and indirectly restored with ceramic materials. am j dent. 2002; 15: 389-94. 12krämer n, frankenberger r. clinical performance of bonded leucite-reinforced glass ceramic inlays and onlays after eight years. dent mater. 2005; 21: 262-71. 13magne p, belser uc. porcelain versus composite inlays/onlays: effects of mechanical loads on stress distribution, adhesion, and crown flexure. int j periodontics restorative dent. 2003; 23: 543-55. 14metzler kt, woody rd, miller aw, miller bh. in vitro investigation of the wear of human enamel by dental porcelain. j prosthet dent. 1999; 81: 356-64. clinical performance of indirect esthetic inlays and onlays for posterior teeth after 40 months 157 braz j oral sci. 8(3):154-158 15bergman ma. the clinical performance of ceramic inlays: a review. aust dent j. 1999; 44: 157-68. 16hayashi m, tsuchitani y, miura m, takeshige f, ebisu s. eight-year clinical evaluation of fired ceramic inlays. oper dent. 2000; 25: 473-81. 17scheibenbogen a, manhart j, kunzelmann kh, hickel r. one-year clinical evaluation of composite and ceramic inlays in posterior teeth. j prosthet dent. 1998; 80: 410-6. 18galiatsatos aa, bergou d. six-year clinical evaluation of ceramic inlays and onlays. quintessence int. 2008; 39: 407-12. 19cvar jr, ryge g. criteria for the clinical evaluation of dental materials. san francisco: us department of health, education, and welfare; 1971. publication no. 7902244. 20friedl kh, schmalz g, hiller ka, saller a. in vivo evaluation of a feldspathic ceramic system: 2-year results. j dent. 1996; 24: 25-31. 21molin m, karlsson s. a 3-year clinical follow-up study of a ceramic (optec) inlay system. acta odont. scand. 1996; 54:145-9. 22fradeani m, barducci g. versatility of ips empress restorations. part ii: veneers, inlays, and onlays. j esthet dent. 1996; 8: 170-6. 23van dijken jw, ormin a, olofsson al. clinical performance of pressed ceramic inlays luted with resin-modified glass ionomer and autopolymerizing resin composite cements. j prosthet dent. 1999; 82: 529-35. 24tagtekin da, ozyöney g, yanikoglu f. two-year clinical evaluation of ips empress ii ceramic onlays/inlays. oper dent. 2009; 34: 369-78. 25frankenberger r, petschelt a, kramer n. leucite-reinforced glass ceramic inlays and onlays after six years: clinical behavior. oper dent. 2000; 25: 459-65. 26thordrup m, iisidor f, horstedbindlev p. a 5-year clinical study of indirect and direct resin composite and ceramic inlays. quintessence int. 2001; 32: 199-205. 27odén a, andersson m, krystek-ondracek i, magnusson d. five-year clinical evaluation of procera allceram crowns. j prosthet dent. 1998; 80: 450-6. 28lange rt, pfeiffer p. clinical evaluation of ceramic inlays compared to composite restorations. oper dent. 2009; 34: 263-72. 29qualtrough aje and piddock v. ceramics update. j dent. 1997; 25: 91-5. 30canay r, hersek ne, uzun g, ercan mt. autoradiographic determination of marginal leakage of a pressed glass ceramic inlay. j oral rehabil. 1997; 24: 705-8. 31rosenstiel sf, land mf, crispin bj. dental luting agents: a review of the current literature. j prosthet dent. 1998; 80: 280-301. clinical performance of indirect esthetic inlays and onlays for posterior teeth after 40 months158 braz j oral sci. 8(3):154-158 1http://dx.doi.org/10.20396/bjos.v18i0.8657265 volume 18 2019 e191638 original article 1 department of restorative dentistry, faculty of nova esperança , pb, brazil. 2 department of clinic dentistry, federal university of pernambuco, pe, brazil. 3 department of restorative dentistry, federal university of paraíba, pb, brazil. corresponding author: renally bezerra wanderley lima faculty of nova esperança av. frei galvão, 12 58067-698, joão pessoa, pb, brazil phone: 55 19-982156352 e-mail: renallywanderley@gmail.com https://orcid.org/0000-0003-4477-7850 conflict of interest: the authors declare that they have no conflict of interest. received: april 26, 2019 accepted: june 30 2019 impact of radiotherapy on the bond strength of different adhesive systems to human dentin: one-year in vitro evaluation renally bezerra wanderley lima1,*, maria luiza pontual2, raquel venâncio fernandes dantas3, sônia saeger meireles3, ana karina maciel andrade3, rosângela marques duarte3 aim: the aim of this study is to evaluate the effect of radiotherapy on the bond strength of resin-based composite restorations to dentin, performed either 24 h or 1 year before or after radiation. methods: ninety-six posterior teeth were randomly distributed into the following groups: ib (n = 16), irradiated teeth were restored 1 year after x-ray application; nb (n = 16), not irradiated teeth were stored for 1 year and then restored. ia (n = 32), teeth were restored and irradiated at 24 h or 1 year after the restoration. na (n = 32), teeth were restored, not irradiated, and tested as ia. eight samples from each group were randomly assigned to either the three-step or two-step etch-and rinse adhesive system procedure. the irradiated specimens were subjected to 60 gy of x-ray radiation fractionally. the restored teeth were vertically sectioned, and 1-mm2 resin–dentin sticks were obtained and submitted to the microtensile bond strength test. the bond strength data were analyzed by twoway analysis of variance (anova) followed by tukey’s test (p < 0.05). failure modes were examined by optical microscopy and scanning electron microscopy. results: the ib group showed lower bond strength values compared to the nb group. the bond strength values between the adhesive systems were not statistically different. conclusion: the application of radiation dose decreased the bond strength of the adhesive restorations to dentin when the bonding procedure was conducted 1 year after in vitro radiotherapy. keywords: dentin. tensile strength. radiotherapy. head and neck neoplasms. https://orcid.org/0000-0003-4477-7850 2 lima et al. introduction radiotherapy is the most common modality of treatment for malignant tumors in the head and neck regions. this treatment uses high-energy x-rays at doses of 40–60 gy, and even low doses may cause changes in normal tissue located within the irradiation field, drastically diminishing the quality of life of irradiated patients1. regarding radiation-induced damage to the orofacial tissues, severe alterations in these tissues have been documented, such as mucositis, candidosis, hyposalivation, radiation caries, dysgeusia, trismus, and osteoradionecrosis2. radiation caries is one of the principal consequences of radiotherapy that results in severe destruction of human dentition3,4. according to some studies, the development of radiation caries is related to indirect or direct radiogenic damage to dental hard tissues. the indirect effects include salivary changes, hyposalivation, changes in the oral microbiota, limitations in performing adequate oral hygiene, and adoption of a soft diet due to swallowing difficulties4-7. with respect to the direct effects, there is a direct alteration in biological molecules, which appear to have a negative effect on the dentinoenamel junction, enamel, dentin, and pulp components of teeth, increasing the severity of dentition breakdown7-13. morphological alterations of the dentin structure such as degeneration of the odontoblast processes and obliteration of the dentin tubules also have been observed14. in addition, previous studies have indicated the presence of x-rays-induced damage to collagen present in the main peptide chains of dentin15,16. the protocol for dental restorations in oral cancer patients is still under controversial discussion. hence, the choice of the best restorative material for dental restorations in patients undergoing radiotherapy seems to be based on the clinical experience of the professional6. recent studies have recommended the use of adhesive restorative techniques for the treatment of irradiated patients6,17-21. in these studies, irradiation treatment did not affect the bond strength of the adhesive restorations to the dentin and enamel structure, when the restoration was carried out before radiotherapy. on the other hand, when the restoration protocol was performed after the application of radiation, lower bond strength values for irradiated teeth were observed compared to teeth that had not been irradiated19,21. however, there is limited information regarding the direct effects of x-rays on the bond strength of resin-based composite restorations to enamel and dentin as well as whether the restorative procedure should be carried out before or after irradiation. as we wanted to develop a restorative protocol based on adhesive materials for irradiated patients and to determine whether the restorative procedure should be carried out before or after irradiation, the aim of this study was to evaluate the effect of radiotherapy on the bond strength of resin-based composite restorations to dentin, using a three-step etch-and-rinse and a two-step etch-and-rinse adhesive, performed either 24 h or 1 year before or 1 year after the radiation treatment. the null hypotheses tested in this study were as follows: 1) the bond strength of the resin-based composite restorations to dentin would not be affected by radiotherapy either before or after restoration placement. 2) there would be no difference in the dentin bond strength 3 lima et al. between the three-step etch-and-rinse and two-step etch-and-rinse adhesive systems for all study conditions. materials and methods ninety-six intact, non-carious, unrestored posterior human maxillary/mandibular molars extracted over the course of three months, were obtained under a protocol approved (0394/11) by the institutional review board of federal university of paraiba, brazil. the teeth were stored in 0.2% thymol solution. sample preparation all tooth roots were embedded using self-curing acrylic resin. the teeth were randomly distributed into the following groups: ib (n = 16), irradiated teeth were restored 1 year after x-ray application; nb (n = 16), not irradiated teeth were stored for 1 year and then restored; ia (n = 32), teeth were restored and irradiated at 24 h or 1 year after the restoration. na (n = 32), teeth were restored, not irradiated, and tested as ia. eight teeth from each subgroup were randomly assigned to one of two adhesive system protocols: three-step etch-and-rinse adhesive (adper™scotchbond mp plus, 3m/espe, st. paul, mn, usa sc) and two-step etch-and-rinse adhesive (adper™ single bond 2, 3m/espe, st. paul, mn sb). compositions and batch numbers of each material are shown in table 1. for restorative treatment after or before the application of x-rays, the occlusal enamel surface was removed using a diamond disc mounted in a low-speed laboratory cutting machine (labcut 1010, extec, enfield, ct, usa) under cooling conditions. the superficial dentin was exposed and finished with 600-grit silicon carbide abrasive paper for 60 s in polishing machine (politriz erios – 27000, são paulo, sp, brazil), table 1. composition and batch numbers of materials used. product (batch number) manufacturer composition manufacture’s instructions adper™scotchbond mp plus lot. n133000 3m/espe, st. paul, mn, usa primer: water, hema. adhesive: bis-gma, hema, dimethacrylates, polyalkenoic acid copolymer, photoinitiator. apply the phosphoric acid 37 % for 15 s, following rinse for 30 s. apply 1 coat of primer and gently air dry for 5 s. apply two consecutive coats of adhesive and applying a gentle stream of air for 10 s. light-cured for 10 s. adper™ single bond 2 lot. n 30077 3m/espe, st. paul, mn, usa bis-gma, hema, dimethacrylates, polyalkenoic acid copolymer, photoinitiator, water, ethanol apply the phosphoric acid 37 % for 15 s, following rinse for 30 s. after apply two consecutive coats of adhesive and applying a gentle stream of air for 10 s. light-cured for 10 s. 37% phosphoric acid etching gel fgm, joinville, sc, br cobalt aluminate blue spinel apply the phosphoric acid 37 % for 15 s, following rinse for 30 s. optilux plus gnatus gnatus, ribeirão preto, sp, br led light cure light-cured using a irradiance ³400 mw/cm2 4 lima et al. and a flat dentin surface was obtained. the adhesives were applied according to the manufacturers’ instructions (table 1). after the adhesion process, the resin block was built up using three layers of z350 composite resin (3m/espe, st. paul, mn, usa), resulting in a height of 4.5 mm. each layer was light cured for 40 s with an intensity of 400 mw/cm2 (optilux plus gnatus, ribeirão preto, são paulo, brazil). radiotherapy the simulated radiotherapy was performed using a primus k linear accelerator (siemens healthineers, usa) with an energy of 6 mev, a source–surface distance of 100 cm, and a field size of 18 cm × 23 cm. the specimens were placed in individual containers, which were filled with distilled water above the resin blocks, in order to provide dose homogeneity. radiation was applied perpendicular to the surface of the specimen, and a total dose of 60 gy, in fractions, was delivered. all groups of specimens were stored in distilled water, changed daily, at 37 °c. microtensile bond strength test for the bond strength test, the restored teeth were sectioned longitudinally in the mesio-distal and buccal-lingual directions across the bonded interface, using a slow-speed diamond saw (isomet, 1000 buehler ltd., lake bluff, il, usa) to obtain 15–30 resin–dentin sticks with a cross-sectional area of approximately 1 mm2 (±0.1 mm2). the resin–dentin bonded sticks were fixed to a testing jig with cyanoacrylate glue (super bond gel, loctite brazil ltd.) and subjected to the tensile load at a crosshead speed of 0.5 mm/min until failure (shimatzu, kyoto, japan). the microtensile bond strength was expressed in mpa and derived by dividing the imposed force (n) at the time of fracture by the bond area (mm2). failure mode analysis the fractured surfaces of all specimens were observed by using an optical microscope (xjm-400, kozo, nanjing, china) at a magnification of 100×. the fracture mode was classified as follows: (i) cohesive failure in the adhesive, (ii) cohesive failure in the dentin, (iii) cohesive failure in the hybrid layer, or (iv) mixed failure, cohesive failure in the adhesive and cohesive failure in the hybrid layer. representative fractured surfaces of each tested group exhibiting the most frequently observed failure mode were analyzed by scanning electron microscopy (jsm-5600, jeol, tokyo, japan) operating at 15 kv and a working distance of 15 mm. statistical analysis bond strength data were submitted to two-way (adhesive system vs. moment of radiation) analysis of variance (anova) followed by tukey’s test at a significance level of p < 0.05. results anova revealed that the material, radiation, and all possible interactions between the factors resulted in statistically significant differences (p < 0.005) for the two storage times (24 h and 1 year). there was no statistical difference in the bond strength values https://www.google.com.br/url?sa=t&rct=j&q=&esrc=s&source=web&cd=8&cad=rja&uact=8&ved=0ahukewin9f3r65trahwjipakhabpca4qfggymac&url=https%2525252525253a%2525252525252f%2525252525252fusa.healthcare.siemens.com%2525252525252fradiation-oncology%2525252525252fupgrades-and-options-for-your-linac%2525252525252ftreatment-data-management&usg=afqjcnf6iia6zjejashrx0q_ee166vakeq 5 lima et al. between the control group (not irradiated) and irradiated group restored before radiotherapy, using both adhesive systems, after shortand long-term storage (24 h and 1 year) (tables 2 and 3). when the adhesive restorations were carried out one year after radiation application, the irradiated group presented lower bond strength values compared to the control group (table 4). there was no statistical difference in the bond strength values between the adhesive systems used for all study conditions. no pretesting failures were recorded for any group. table 5 and 6 show the mode of failure. the predominance of cohesive failure in the adhesive was detected for the control and irradiated groups restored 24 h and 1 year before radiotherapy. on the other hand, mixed failures (cohesive failure in the adhesive and cohesive failure in the hybrid layer) predominated when the specimens were restored 1 year after radiotherapy (fig. 3). table 2. means values of bond strength (mpa) and standard deviation of adhesive restorations performed before the radiation in 24 hours storage. treatment group adhesive system single bond scotch bond irradiated 39,64 ± 4,9 aa 38,28 ± 7,9 aa control (no irradiated) 41,67 ± 7,6 aa 41,43 ± 10,06 aa groups identified with different upper case letter superscripts (analysis in rows) and lower case letters (analysis in columns) represent statistical significant differences (p<0.05). table 3. means values of bond strength (mpa) and standard deviation of adhesive restorations performed before the radiation in 1year storage. treatment group adhesive system single bond scotch bond irradiated 39,65 ±5,44 aa 38,15 ± 9,19 aa control (no irradiate) 40,67 ±5,67 aa 40,34 ± 7,90 aa groups identified with different upper case letter superscripts (analysis in rows) and lower case letters (analysis in columns) represent statistical significant differences (p<0.05). table 4. means values of bond strength (mpa) and standard deviation of adhesive restorations performed after the radiation in 1year storage. treatment group adhesive systems single bond scotch bond irradiated 35,39 ± 7,47 ab 32,68 ± 7,45 ab control (no irradiated) 41,33 ±5,09 aa 41,01 ± 7,63 aa groups identified with different upper case letter superscripts (analysis in rows) and lower case letters (analysis in columns) represent statistically significant differences (p<0.05). 6 lima et al. discussion according to the results of this study, the bond strength of the adhesive systems tested to dentin decreased when the restorations were performed 1 year after radiation application. moreover, no difference in the dentin bond strength between those adhesive systems was observed for all study conditions. consequently, both hypotheses of this study were rejected. therefore, the restorative treatment for oral cancer patients who have dental cavities should be carried out before initiating head and neck radiotherapy. because irradiation doses may modify human tissues structure, as enamel and dentin, impairing the formation of an adequate bond between adhesive treatment and tooth structure. the ionizing radiation used as a treatment for oral cancer patients has a short wavelength and a high energy, which may induce micro-morphological alterations in dentin and enamel7,8,14,22,23. the results of this study showed that radiotherapy caused a (1) (3)(2) 15 kv x500 50 µm h a h 15 kv x500 50 µm15 kv x500 50 µm a figure 1. sem photo illustrating the mode of type i cohesive fracture in the adhesive (1). type iv, fracture at the base of the hybrid layer (a) with filled dental tubules and resin and cohesive fracture in the hybrid layer (h) (2) and presence of filled (a) and unfilled (b) dental tubules (3). table 5. percentage (%) of specimens according to the fracture mode of restorations performed before radiation. groups failure mode (%) sb sc i ii iii iv i ii iii iv control group/24h 68 7 5 20 50 10 30 10 radiation group/24 h 60 5 10 25 65 5 10 20 control group/ 1 year 60 4 18 18 55 7 18 20 radiation group/ 1year 55 2 15 28 40 12 20 28 table 6. percentage (%) of specimens according to the fracture mode of restorations performed after radiation. groups failure mode (%) sb sc i ii iii iv i ii iii iv control group/1 year 20 5 20 55 20 8 28 44 radiation group/1 year 21 12 25 42 23 16 20 41 7 lima et al. significant detrimental effect on the bond strength of adhesive systems to dentin when the adhesive restorations were carried out 1 year after radiation application to the teeth, using twoor three-step adhesives. this fact may be explained by a direct effect of high-dose radiation (60 gy) on the dentin structure. it is well known that radiation reacts with water, forming hydrogen and hydrogen peroxide free radicals24. dentin contains a considerable amount of water in its composition; therefore, x-rays act through the formation of free radicals, which may have a negative effect on the secondary and tertiary structures of dentin proteins, causing the loss of collagen fiber hydration and leaving the tissue dry and friable7,24. as a result of this process, some micro-morphological alterations in the dentin structure can occur, including collagen fiber fragmentation7,15 and obliteration of dentin tubules, which is preceded by degeneration of odontoblast processes14. this damage may impair formation of the hybrid layer, producing a permeable adhesive interface between the adhesive system and the irradiated dentin. moreover, alteration of the structural organization of collagen may occur because some chemical bonds are broken by free radicals during radiation and reorganization of the chemical components may happen, thus altering the structure15. in this study, a self-adhesive system was not assessed and further investigation is required to evaluate if that adhesive system will produce a strong and durable adhesive interface with the irradiated dentin, since self-adhesive systems containing an acidic monomer, as mdp, is capable to bond chemically to dentin and enamel structure25. the results of this in vitro study corroborate with previous findings, which showed the lowest bond strength values to dentin when the adhesive restoration was carried out after radiotherapy19,21,26. however, in these studies, the adhesive procedure was performed immediately or 24 h after radiation therapy. in clinical conditions, dental bonding procedures are not performed immediately after finishing radiotherapy. thus, in our study, the restorative procedure was carried out 1 year after the application of radiation to the teeth. this is the first study to evaluate the long-lasting effect of radiation on the dentin structure and its consequences on the bonding effectiveness of adhesive systems to irradiated dentin. a previous report has hypothesized that in vivo high-dose radiotherapy causes induction and activation of enzymes that degrade collagens over a period of months or years27. the effect of radiation (60 gy) on the dentin microstructure could be observed by the mode of failure for the irradiated group restored after radiotherapy (table 6). the predominant fracture modes were mixed failures (cohesive failure in the adhesive and cohesive failure in the hybrid layer), which showed failure of the formation of a stable and strong hybrid layer (figure 1). regarding the group in which the restorative procedure was carried out before the radiation application, there were no significant differences for the bond strength values between the irradiated and nonirradiated restored teeth after storage for 24 h or 1 year. this finding is in accordance with others studie.6,19,28 which allege that when hybridization is obtained prior to irradiation, the alterations in the substrate might not be great enough to affect the behavior of the pre-existing hybrid layer and to compromise the bonding effectiveness between dentin and the adhesive materials. for all groups in which the teeth were restored before radiation, the predominant failure mode was cohesive failure in the adhesive (table 5). it is possible that radiation applied after 8 lima et al. the restorative procedure did not influence the bond quality of the adhesive material to dentin; consequently, adequate bond strength values could be obtained. as observed in the present investigation, the application of x-rays had a harmful effect on the bond strength to human dentin when the adhesive restorations were placed 1 year after radiotherapy. nevertheless, this scenario could be different in clinical practice considering the dry mouth of patients, increased viscosity, decreased salivary ph, dietary changes, and deficiencies in oral hygiene during and after radiotherapy. therefore, future research should be conducted to simulate intraoral conditions in order to validate the findings of this study. within the limitations of the current study, it was concluded that the application of x-rays decreased the bond strength of the tested adhesive restorations to dentin when the bonding procedure was conducted 1 year after in vitro radiotherapy. in addition, the different adhesive systems used in this study (twoor three-step etch-and-rinse procedure) showed similar bond effectiveness to dentin, regardless of the time period after the restoration was introduced. acknowledgements the work was supported department of restorative dentistry, federal university of paraíba, state of paraiba, brazil. references 1. al-nawas b, al-nawas k, kunkel m, grötz ka. quantifying radioxerostomia: salivary flow rate, examiner’s score, and quality of life questionnaire. strahlenther onkol. 2006 jun;182(6):336-41. 2. sulaiman f, huryn jm, zlotolow im. dental extractions in the irradiated head and neck patient: a retrospective analysis ofmemorial sloan-kettering cancer center protocols, criteria, and results. br j oral maxillofac surg. 2003 oct;61(10):1123-31. 3. beumer j, curtis tha, marunick mt. maxillofacial rehabilitation, prosthodontic and surgical considerations. saint louis: medico dental media international; 1996. p.43-71. 4. kielbassa am, hinkelbein w, hellwig e, meyer-lückel h. radiation-related damage to dentition. lancet oncol. 2006 apr;7(4):326-35. 5. walker mp, wichman b, cheng a-l, coster j, williams kb. impact of radiotherapy dose on dentition breakdown in head and neck cancer patients. pract radiat oncol. 2011;1(3):142-8. 6. galetti r, santos-silva ar, antunes an, alves fa, lopes ma, de goes mf. radiotherapy does not impair dentin adhesive properties in head and neck cancer patients. clin oral investig. 2014 sep;18(7):1771-8. doi: 10.1007/s00784-013-1155-4. 7. goncalves lm, palma-dibb rg, paula-silva fw, oliveira hf, nelson-filho p, silva la et al. radiation therapy alters microhardness and microstructure of enamel and dentin of permanent human teeth. j dent. 2014 aug;42(8):986-92. doi: 10.1016/j.jdent.2014.05.011. 8. davis wb. reduction in dentin wear resistance by irradiation and effects of storage in aqueous media. j dent res. 1975 sep-oct;54(5):1078-81. 9. franzel w, gerlach r, hein hj, schaller hg. effect of tumor therapeutic irradiation on the mechanical properties of teeth tissue. z med phys.2006;16(2):148-54. 9 lima et al. 10. kielbassa am, beetz i, schendera a, hellwig e. irradiation effects on microhardness of fluoridated and non-fluoridated bovine dentin. eur j oral sci. 1997 oct;105(5 pt 1):444-7. 11. thiagarajan g, vizcarra b, bodapudi v, reed r, seyedmahmoud r, wang y, et al. stress analysis of irradiated human tooth enamel using finite element methods. comput methods biomech biomed engin. 2017 nov;20(14):1533-42. doi: 10.1080/10255842.2017.1383401. 12. lu h, zhao q, guo j, zeng b, yu x, yu d, et al. direct radiation-induced effects on dental hard tissue. radiat oncol. 2019 jan 11;14(1):5. doi: 10.1186/s13014-019-1208-1. 13. marangoni-lopes l, rovai-pavan g, steiner-oliveira c, nobre-dos-santos m. radiotherapy reduces microhardness and mineral and organic composition, and changes the morphology of primary teeth: an in vitro study. caries res. 2019;53(3):296-304. doi: 10.1159/000493099. 14. grötz ka, duschner h, kutzner j, thelen m, wagner w. [new evidence for the etiology of the so-called radiation caries. proof for direct radiogenic damage of the dento-enamel junction]. strahlenther onkol. 1997 dec;173(12):668-76. german. 15. fisher bv, morgan re, phillips go, wardale hw. radiation damage in calcium phosphates and collagen: an interpretation of esr spectra. radiat res. 1971 may;46(2):229-35. 16. cheung dt, perelman n, tong d, nimni me. the effect of gamma-irradiation on collagen molecules, isolated alpha-chains, and crosslinked native fibers. j biomed mater res. 1990 may;24(5):581-9. 17. odlum o. preventive resins in the management of radiationinduced xerostomia complications. j esthet dent. 1991 nov-dec;3(6):227-9. 18. mcomb d, erickson rl, maxymiw wg, wood re. a clinical comparison of glass ionomer, resin modified glass ionomer and resin composites restorations in the treatment of cervical caries in xerostomic head and neck radiation patients. oper dent. 2002 sep-oct;27(5):430-7. 19. naves lz, novais vr, armstrong sr, correr-sobrinho l, soares cj. effect of gamma radiation on bonding to human enamel and dentin. support care cancer. 2012 nov;20(11):2873-8. doi: 10.1007/s00520-012-1414-y. 20. bernard c, villat c, abouelleil h, gustin mp, grosgogeat b. tensile bond strengths of two adhesives on irradiated and nonirradiated human dentin. biomed res int. 2015;2015:798972. doi: 10.1155/2015/798972. 21. rodrigues rb, soares cj, simamoto junior pc, lara vc, arana-chavez ve, novais vr. influence of radiotherapy on the dentin properties and bond strength. clin oral investig. 2018 mar;22(2):875-883. doi: 10.1007/s00784-017-2165-4. 22. açil y, springer i, niehoff p, gabling v, warnke p, açmaz s et al. proof of direct radiogenic destruction of collagen in vitro. strahlenther onkol. 2007 jul;183(7):374-9. 23. mellara ts, palma-dibb rg, oliveira hf, paula-silva fwg, nelson-filho f, silva rab et al. the effect of radiation therapy on the mechanical and morphological properties of the enamel and dentin of deciduous teeth – an in vitro study. radiation oncology. 2014 jan 22;9:30. doi: 10.1186/1748-717x-9-30. 24. cole t, silver as. production of hydrogen atoms in teeth by x-irradiation. nature. 1963 nov 16;200:700-1. 25. van landuyt kl, snauwaert j, de munck j, peumans m, yoshida y, poitevin a, et al. systematic review of the chemical composition of contemporary dental adhesives. biomaterials. 2007 sep;28(26):3757-85. 26. madrid troconis cc, santos-silva ar, brandão tb, lopes ma, de goes mf. impact of head and neck radiotherapy on the mechanical behavior of composite resins and adhesive systems: a systematic review. dent mater. 2017 nov;33(11):1229-43. doi: 10.1016/j.dental.2017.07.014. https://www.ncbi.nlm.nih.gov/pubmed/29063816 https://www.ncbi.nlm.nih.gov/pubmed/30635005 https://www.ncbi.nlm.nih.gov/pubmed/?term=marangoni-lopes l%5bauthor%5d&cauthor=true&cauthor_uid=30317232 https://www.ncbi.nlm.nih.gov/pubmed/?term=rovai-pavan g%5bauthor%5d&cauthor=true&cauthor_uid=30317232 https://www.ncbi.nlm.nih.gov/pubmed/?term=steiner-oliveira c%5bauthor%5d&cauthor=true&cauthor_uid=30317232 https://www.ncbi.nlm.nih.gov/pubmed/?term=nobre-dos-santos m%5bauthor%5d&cauthor=true&cauthor_uid=30317232 https://www.ncbi.nlm.nih.gov/pubmed/?term=van landuyt kl%5bauthor%5d&cauthor=true&cauthor_uid=17543382 https://www.ncbi.nlm.nih.gov/pubmed/?term=snauwaert j%5bauthor%5d&cauthor=true&cauthor_uid=17543382 https://www.ncbi.nlm.nih.gov/pubmed/?term=de munck j%5bauthor%5d&cauthor=true&cauthor_uid=17543382 https://www.ncbi.nlm.nih.gov/pubmed/?term=peumans m%5bauthor%5d&cauthor=true&cauthor_uid=17543382 https://www.ncbi.nlm.nih.gov/pubmed/?term=yoshida y%5bauthor%5d&cauthor=true&cauthor_uid=17543382 https://www.ncbi.nlm.nih.gov/pubmed/?term=poitevin a%5bauthor%5d&cauthor=true&cauthor_uid=17543382 https://www.ncbi.nlm.nih.gov/pubmed/17543382 10 lima et al. 27. mcguire jd, mousa aa, zhang bj, todoki ls, huffman nt, chandrababu kb et al. extracts of irradiated mature human tooth crowns contain mmp-20 protein and activity. j dent. 2014 may;42(5):626-35. doi: 10.1016/j.jdent.2014.02.013. 28. gernhardt cr, kielbassaam, hahn p, schaller hg. tensile bond strengths of four different dentin adhesives on irradiated and non-irradiated human dentin in vitro. j oral rehabil. 2001 sep;28(9):814-20. original article braz j oral sci. april/june 2009 volume 8, number 2 cytotoxicity of polycarbonate orthodontic brackets matheus melo pithon1, rogério lacerda dos santos1, fernanda otaviano martins2, antônio carlos de oliveira ruellas3, lincoln issamu nojima3, matilde gonçalves nojima3, maria teresa villela romanos4 1 specialist in orthodontics and master in orthodontics, universidade federal do rio de janeiro (ufrj), rio de janeiro (rj), brazil; doctorate student in orthodontics, ufrj, rio de janeiro (rj), brazil 2 graduate student in microbiology and immunology, ufrj, rio de janeiro (rj), brazil; trainee at the professor paulo de goés institute of microbiology, ufrj, rio de janeiro (rj), brazil 3 phd in orthodontics, ufrj, rio de janeiro (rj), brazil; adjunct professor, ufrj, rio de janeiro (rj), brazil 4 phd in microbiology and immunology, ufrj, rio de janeiro (rj), brazil; adjunct professor, ufrj, rio de janeiro (rj), brazil received for publication: april 3, 2009 accepted: july 24, 2009 correspondence to: matheus melo pithon centro odontomédico doutor altamirando da costa lima avenida otávio santos, 395, sala 705 – recreio cep 45020-750 – vitória da conquista (ba), brasil e-mail: matheuspithon@bol.com.br abstract aim: to assess the cytotoxicity of polycarbonate orthodontic brackets. methods: polycarbonate brackets from two different manufacturers, namely, composite bracket (morelli™) and silkon plus bracket (american orthodontics™), were assessed. in addition to these two experimental groups, other three control groups were included: positive control group (c+) consisting of amalgam cylinders, negative control group (c-) consisting of glass rods, and cell control group (cc) consisting of cells not exposed to any material. all brackets were previously sterilized under ultra-violet light (uv ) and, then, immersed in eagle’s minimum essential media (mem) for 24 hours, after which the supernatants were removed and placed into contact with l929 fibroblast cells. cytotoxicity was evaluated at 24, 48, 72 and 168 hours. after contact with mem, the cells were further incubated at 37oc for 24 hours and 100 ml of 0.01% neutral red dye were added. the cells were incubated again at 37oc for three hours to incorporate the dye. after this period, the cells were fixed and viable cell counting was performed by spectrophotometry at 492 nm wavelength. results: no statistically significant difference was found between the experimental groups (1 and 2) and the negative and cell control groups (p > 0.05). the positive control group exhibited high cytotoxicity throughout experimental period are differed significantly from the other groups (p < 0.05). conclusions: polycarbonate orthodontic brackets were found not to be cytotoxic within the evaluated experimental period. keywords: orthodontic brackets, cytotoxicity tests, immunologic, cell culture techniques. introduction dentistry has the main purpose of keeping or improving the patient’s quality of life by preventing diseases, relieving pain, and improving the masticatory efficacy, phonetics and/or esthetics. most of these objectives require replacement or alteration of the existing dental structure as well as changes in tooth positioning. developing and selecting biocompatible materials have been one of the major challenges in dentistry1. metals, ceramics, polymers and composites are the four groups of materials that are currently employed2. little scientific information on these materials was available until the middle of the last century. toxic, inflammatory, allergic or mutagenic reactions are the possible biological responses to these materials3-6. toxicity is one of the main parameters for evaluating the biological response and the potential damage to cells and tissues related to the use of such materials7,8. 85cytotoxicity of polycarbonate orthodontic brackets braz j oral sci. 8(2): 84-7 in dentistry, various kinds of materials are used for transitory restoration (wires, bands, brackets and resins) during medium and long periods of time, and the orthodontic appliance consists of a series of these materials. in attempt to make the orthodontic fixed devices esthetically more acceptable, the manufacturers are producing lingual brackets and accessories mimicking tooth colors. polycarbonate is a material for application in a wide variety of areas. this material is formed with small molecules called monomers or giant molecules called polymers. they are produced through chemical reactions that may be reversible or not, spontaneous or stimulated (by heat or reagents), in which the monomers combine chemically to form long ramified molecules with the same centesimal composition9. the polymers usually employed in the orthodontic materials may be divided into three groups with distinct characteristics: 1) finished material to be used in its original shape; 2) cast polymers to be used for structuring a variety of removable or functional artifacts, and 3) polymeric materials for impression, adhesion and sealing. esthetic plastic orthodontic brackets are comprised in the first group. these accessories are not chemically resistant when in contact with solvents and, under high temperatures, allow migration of monomers away from the original products6. it is important to point out that, once inside the mouth, any of these materials creates a dynamic interface whose interactions may cause changes, thus leading to either an active biological reaction to the material (i.e. biocompatibility) or degradation or corrosion of the material itself 10,11. therefore, the biocompatibility depends on the release of elements from these materials. in addition, composition, pretreatment, and manipulation of these apparatuses influence on the release of such elements3,4. under these circumstances, it is of crucial importance to evaluate the cytotoxicity of polycarbonate brackets to be clinically used in orthodontics in order to detect any possible harmful effect of the materials to the oral cavity. material and methods cell culture the cell line used for this study was mouse l929 f ibroblasts obtained from the american ty pe culture collection (tcc, rockv ille, md, usa) and cultivated in eagle’s minimum essential medium (mem) (cultilab, campinas, sp, brazil). the cell culture was supplemented w ith 2 mm of l-glutamine (sigma, st. louis, missouri, usa), 50 µg/ml of gentamicin (schering plough, kenilworth, nj, usa), 2.5 µg/ml of fungizone (bristol-myers-squibb new york , n y, usa), 0.25 mm of sodium bicarbonate solution (merck ™, darmstadt, germany), 10 mm of hepes (sigma), and 10% of fetal bov ine serum (fbs) (cultilab), then being kept at 37oc in a 5%-co 2 env ironment. orthodontic brackets the sample consisted of polycarbonate brackets from two different manufacturers, which were divided into two groups: group 1, composite bracket (morelli, sorocaba, são paulo, brazil) and group 2, silkon plus bracket (american orthodontics, sheboygan, wi, usa). controls to verify the cell response to extreme situations, other three groups were included in the study: group c+ (positive control), consisting of amalgam cylinders; group c(negative control), consisting of glass rods in contact with the cells; group cc (cell control), consisting of cells not exposed to any material. cytotoxicity assays the materials were previously sterilized by exposing them to ultra-violet light (labconco corporation, kansas city, mo, usa) for one hour. next, three samples of each material were placed in 24-well plates containing eagles’ mem (cultilab). the culture medium was replaced with fresh medium every 24 hours, and the supernatants were collected after 24, 48, 72, and 168 hours (7 days) for analysis of the toxicity to l929 cells. the supernatants were placed in a 96-well plate containing a single layer of l929 cells and then incubated at 37oc for 24 hours a in 5%-co 2 environment. after the incubation period, cell viability was determined using the “dye-uptake” technique described by neyndorff et al.12, but slightly modified. after the 24-hour incubation period, 100 µl of 0.01% neutral-red staining solution (sigma) were added to the medium within each well of the plates, and these were incubated for 3 hours at 37oc to allow the dye to penetrate the living cells. after this period, the cells were fixed using 100 µl of 4% formaldehyde solution (reagen, rio de janeiro, rj, brazil) in pbs (130 mm nacl; 2 mm kcl; 6 mm na 2 hpo 4 2h 2 o; 1 mm k 2 hpo 4 , ph = 7.2) for 5 minutes. next, 100 µl of 1% acetic acid solution (vetec, rio de janeiro, rj, brazil) with 50% methanol (reagen) were added to the medium to remove the dye. absorption was measured after 20 minutes by using a spectrophotometer (biotek instruments inc., winooski, vt, usa) at 492 nm wavelengths. statistical analysis statistical analyses were performed by using a spss version 13.0 software (spss inc., chicago, il, usa), and means and standard deviations were calculated for descriptive statistical analysis. the values for the amount of viable cells were submitted to anova and tukey’s test to determine whether statistically significant differences existed between the groups. significance level was set at 5% for all analyses. results the results of the cytotoxicity of polycarbonate orthodontic brackets are listed in table 1. 86 pithon mm, santos rld, martins fo, ruellas aco, nojima li, nojima mg, romanos mtv braz j oral sci. 8(2): 84-7 groups 1st day 2nd day 3rd day 7th day m. cel./sd statistics m. cel./sd statistics m. cel./sd statistics m. cel./sd statistics 1 503.5 (50.05) a 502.6 (113.7) ac 328.4 (25.23) a 958.7 (70.29) a 2 496.4 (112.0) a 457.4 (66.0) a 284.7 (27.33) b 922.3 (62.53) a c+ 274.6 (62.21) b 251.6 (50.23) b 212.6 (37.8) c 581.6 (62.74) b c539 (6.06) a 513.3 (35.6) ac 318 (26.84) ab 951.6 (55.53) a cc 566 (21.3) a 579 (126.2) c 319.6 (18.86) ab 1041.3 (81.03) a table 1. statistical analysis with means and standard deviations for the studied groups m. cel.: mean values for the amount of viable cells; sd: standard deviation; same letters indicate no statistically significant differences. in the first day, no statistically significant differences were found between the experimental groups in relation to positive control and cell control groups. on the second day, however, a statistically significant difference was observed between group 2 and cell control group. on the third day, groups 1 and 2 differed significantly from each other, but there were no statistically significant differences between the positive control and cell control groups. at the end of the experiment (seventh day), the brackets exhibited low cytotoxicity, with no statistically significant differences between them and between the control groups. group c+ showed high level of cytotoxicity during the whole period of the study. discussion the development and selection of biocompatible materials have been one of the greatest challenges in the area of health care. toxic, inflammatory, allergic or mutagenic reactions are the possible biological responses to these materials, and cytotoxicity is one of the main parameters for biological evaluation. the goal of orthodontic treatment is to promote tooth movements by using a series of materials, including the brackets. these accessories, available in metallic, plastic, and ceramic compositions, are attached to tooth surface and, consequently, are in direct contact with oral tissues and saliva. changes in the properties of these materials may cause harmful effects on the surrounding tissues, leading to the development of inflammatory processes. based on these premises, the present study assessed the cytotoxicity of polycarbonate brackets, as they represent a great demand due to their esthetic appearance and low cost in comparison to ceramic brackets. the method in which vital dye neutral red is used was employed to evaluate cell viability. analysis of neutral red is a cell survival/ viability essay based on the capacity of viable cells to incorporate and process the neutral red within their lysosomes. this is normally performed by adherent cells. the neutral red is a weak cationic dye that penetrates the cell membrane and accumulates within the lysosomes (lysosomic ph < cytoplasmatic ph), where it combines with the anionic part of the lysosomic matrix13. the changes in either cell surface or lysosomic membrane result in lysosomal membrane fragility and other changes that become gradually irreversible. such alterations resulting from the action of xenobiotics decrease the absorption and process of neutral red dye. therefore, it is possible to distinguish viable, damaged or dead cells, which is the basis of this essay. the amount of dye incorporated in the cells is measured by spectrometry, being directly proportional to the number of cells with intact membrane. this method was firstly used by pithon et al.14, who compared it to the agar diffusion method for evaluating the cytotoxicity of orthodontic materials and found that both methods provide adequate cytotoxic evaluation. the results obtained in the first, second, third days and at the end of the experiment are in accordance with the findings of costa et al.15, who assessed the cytotoxicity of stainless steel brackets with nickel in the composition. under these conditions, the polycarbonate brackets showed no toxic effects on cells for all study periods. this may have occurred due to the fact that only new brackets were evaluated, so none of them were exposed to chemical, thermal or mechanical agents, which, in turn, would release residual monomers, as previously suggested6. although four experimental periods were used (24, 48, 72, and 168 hours), these periods are very short in comparison to the length of time in which the brackets remain within the oral cavity, 30 months on average. in face of the initial results herein obtained, further researches with longer experimental periods are necessary. the evaluation of cytotoxicity is important to clarify the biological mechanism by which the cytotoxic effect is produced16, as well as the action mechanism of different materials during the material-tissue interaction17,18. the presence of in vitro cytotoxic effect does not mean that the material is toxic for in vivo application. on the other hand, the absence of cytotoxic effect is guarantee of good clinical response18. therefore, the results from in vitro studies should be judiciously compared to clinical outcomes as there is still controversy on this issue19. based on the findings of the present study, it may be concluded that polycarbonate brackets were not cytotoxic within the experimental period of zero to seven days. references 1. jorge jh, giampaolo et, pavarina ac. cytotoxicity of the dental materials. a literature review. rev odontol unesp 2004;33:65-8. 2. phillips rw. materiais dentários. são paulo: elsevier; 2005. 3. morais ls, serra gs, muller ca, palermo efa, andrade lr, meyers ma, et al. in vivo metal ion release from ti-6al-4v orthodontic mini-implants. revista matéria 2007;12:290-7. 4. gioka c, bourauel c, hiskia a, kletsas d, eliades t, eliades g. light-cured or chemically cured orthodontic adhesive resins? a selection based on the degree of cure, monomer leaching, and cytotoxicity. am j orthod dentofacial orthop 2005;127:413-9; quiz 516. 87cytotoxicity of polycarbonate orthodontic brackets braz j oral sci. 8(2): 84-7 5. hanson m, lobner d. in vitro neuronal cytotoxicity of latex and nonlatex orthodontic elastics. am j orthod dentofacial orthop 2004;126:65-70. 6. howdeshell kl, peterman ph, judy bm, taylor ja, orazio ce, ruhlen rl, et al. bisphenol a is released from used polycarbonate animal cages into water at room temperature. environ health perspect 2003;111:1180-7. 7. siqueira goncalves t, minghelli schmitt v, thomas m, lopes de souza ma, macedo de menezes l. cytotoxicity of two autopolymerized acrylic resins used in orthodontics. angle orthod 2008;78:926-30. 8. kao ct, ding sj, min y, hsu tc, chou my, huang th. the cytotoxicity of orthodontic metal bracket immersion media. eur j orthod 2007;29:198-203. 9. anusavice kj. phillips, materiais dentários. 11ª ed. rio de janeiro: elsevier; 2005. 10. oh kt, kim kn. ion release and cytotoxicity of stainless steel wires. eur j orthod 2005;27:533-40. 11. jonke e, franz a, freudenthaler j, konig f, bantleon hp, schedle a. cytotoxicity and shear bond strength of four orthodontic adhesive systems. eur j orthod 2008;30:495-502. 12. neyndorff hc, bartel dl, tufaro f, levy jg. development of a model to demonstrate photosensitizer-mediated viral inactivation in blood. transfusion 1990;30:485-90. 13. griffon g, marchal c, merlin jl, marchal s, parache rm, bey p. radiosensitivity of multicellular tumour spheroids obtained from human ovarian cancers. eur j cancer 1995;31a:85-91. 14. pithon mm, santos rl, ruellas aco, fidalgo tk, romanos mt v, mendes gs. citotoxicidade in vitro de elásticos ortodônticos: comparação entre duas metodologias. rev saúde com 2008;4:19-26. 15. costa mt, lenza ma, gosch cs, costa i, ribeiro-dias f. in vitro evaluation of corrosion and cytotoxicity of orthodontic brackets. j dent res 2007;86:441-5. 16. schmalz g. use of cell cultures for toxicity testing of dental materialsadvantages and limitations. j dentistry 1994;22:6-11. 17. santos rl, pithon mm, oliveira mv, mendes gs, romanos mt v, ruellas aco. cytotoxicity of introral orthodontic elastics. braz j oral sci 2008;7:1520-5. 18. pizzoferrato a, ciapetti g, stea s, cenni e, arciola cr, granchi d, et al. cell culture methods for testing biocompatibility. clin mater 1994;15:175-90. 19. wennberg a, mjor ia, hersten-pettersen a. biological evaluation of dental restorative materials a comparison of different test methods. j biomed mater res 1983;11:23-36. oral sciences n3 original article braz j oral sci. july | september 2011 volume 10, number 3 ultrasonic irrigation in the removal of smear layer and enterococcus faecalis from root canals letícia maria menezes nóbrega1, cícero romão gadê-neto2, fábio roberto dametto2, carlos frederico de m. sarmento2, rejane andrade de carvalho2 1msc, integrated clinical dentistry, department of dentistry, potiguar university (laureate international universities), natal, rn, brazil 2phd, integrated clinical dentistry, department of dentistry, potiguar university (laureate international universities), natal, rn, brazil correspondence to: letícia maria menezes nóbrega rua tiradentes, 537, apto 43. centro, piracicaba/sp – brazil 13400-760 phone: +55 19-8223-4545 / 84-9908-7318 e-mail: letnobrega@hotmail.com abstract aim: this study evaluated both smear layer removal and reduction of enterococcus faecalis after instrumentation with ultrasonic irrigation. methods: root canals were experimentally inoculated with e. faecalis for 20 days and microbiological samples were collected before and after chemomechanical preparation by using sterilized absorbent paper points. the irrigation solutions used were naocl 2.5% and edta 17%. in group 1 (g1), conventional irrigation was used, whereas in group 2 (g2) ultrasonic irrigation was performed. in group 3 (control), root canals were irrigated with distilled water. the samples were inoculated in bhi broth and turbidity was observed after 48 h to evaluate the reduction in the number of bacteria. residual smear layer was examined by scanning electron microscopy (sem). results: the results showed no significant differences between ultrasonic and conventional irrigation. conclusions: it was concluded that the level of disinfection and cleanliness of root canals achieved with ultrasonic irrigation is comparable to that obtained by conventional methods. keywords: e. faecalis, endodontic irrigation, smear layer, ultrasonic. introduction when endodontic infection occurs, pathogenic bacteria may spread throughout the entire root canal system1-3. the host immunological system cannot reach the bacteria present within the root canal due to an absence of local vascularization4. elimination of infection, however, is possible via proper chemomechanical preparation (instrumentation and irrigation) and, when necessary, the use of intracanal medication5-8. sodium hypochlorite is one of the most commonly used agents for root canal therapy and has been proven an excellent irrigation solution due to its tissue-dissolving capacity and antibacterial action9-11. among the methods recommended by the current literature for smear layer removal, the use of chelating solutions, laser application and ultrasonic irrigation can be cited12-15. a number of protocols have been used in an attempt to efficiently remove smear layer, thus exposing the dentinal tubules of the root canal wall. in theory, this allows the action of intracanal medications and adequate seal by the filling material and root canal obturation. in fact, the presence of significant numbers of residual bacteria and smear layer may compromise the success of endodontic therapy16-17. several reports have demonstrated good results when ultrasonic irrigation is received for publication: may 21, 2011 accepted: september 20, 2011 braz j oral sci. 10(3):221-225 braz j oral sci. 10(3):221-225 222 used to remove smear layer. in the mechanism being proposed, endodontic files mounted on an ultrasonic endodontic handpiece vibrate and produce streaming of the irrigation solution, which enhances elimination of debris from the root canal wall18-21. other studies, however, have not shown statistically significant differences between conventional and sonic or ultrasonic irrigation in the removal of both smear layer and microorganisms present in the apical third of root canals22-24. scanning electron microscopy (sem) analysis allows observing the cleanliness of root canal walls and has been frequently used to evaluate the cleaning efficiency of different chemomechanical preparation techniques8,11,25-28. microbiological methods have also been used for such purpose. the aim of this study was to evaluate in vitro the capacity of both smear layer removal and reduction of e. faecalis from root canals after instrumentation with ultrasonic irrigation. material and methods sample preparation fifty-two freshly extracted human teeth with complete apex formation and similar anatomical features (single root without curvature and with uniform width and length) were divided into two groups. all root canals were initially instrumented with manual k-files (dentsply-maillefer) to a size #25 master apical file and irrigated with distilled water to remove pulp tissue and to enlarge the canal for subsequent microbiological contamination. in order to eliminate the smear layer produced during this initial preparation, all roots were submitted to a bath under agitation for 10 min in 17% edta followed by 10 min in a 5.25% naocl29. the roots were then abundantly washed with distilled water to remove the edta and naocl and the apical foramens sealed with resin (natural flow-dfl). the roots were immersed in glass tubes containing 5 ml of brain heart infusion broth (bhi-difco) and sterilized at 121°c for 20 min. they were then kept at 37°c for 24 h to verify the success of the sterilization protocol. pure cultures of e. faecalis (atcc 29212) were cultivated in bhi agar for 24 h and a suspension was prepared with turbidity corresponding to 1.0 mcfarland standard (3x108 cfu/ml). the glass tubes with the sterilized roots and bhi broth were opened in a laminar flow chamber and sterile pipettes used to add 1 ml of the bacterial suspension. the tubes were kept at 37°c for 20 days, with replacement of 2.0 ml of contaminated bhi by 2.0 ml of freshly prepared bhi every 2 days to avoid medium saturation. the turbidity of the medium during the incubation period indicated bacterial growth. the purity of the cultures was confirmed by gram-staining and colony morphology on bhi agar. root canal preparation and experimental groups all samples were shaped with protaper rotary files to size f5 (dentsply-maillefer), with the working length being determined at 1 mm from the foramen. in group 1 (n = 19), after each instrument used, irrigation with 2 ml of 2.5% naocl was performed by using a disposable syringe coupled with a 27-g needle. for smear layer removal, irrigation was carried out by initial application of 1 ml of 17% edta for 3 min, followed by irrigation with 2 ml of 2.5% naocl and a final washing with 5 ml of distilled water. in group 2 (n = 20), after each instrument used, irrigation with 2 ml of 2.5% naocl was followed by passive irrigation for 15 s by using an endo l ultrasonic handpiece (dabi atlante) with a size #10 k-file on a profi ii as ceramic unit (dabi atlante). passive ultrasonic irrigation is performed by applying vibrations to shake only the irrigant solution without affecting the dentin walls (15, 30). this passive irrigation was also performed after irrigation with edta 17%. in group 3 (n = 13), the control group, distilled water was used for irrigation and no protocol for smear layer removal was performed. microbiological samples microbiological samples obtained with sterile paper points were collected from contaminated root canals before and after instrumentation. after chemomechanical preparation in groups 1 and 2, irrigation with 0.6% sodium thiosulfate solution was used to neutralize the naocl, followed by irrigation with 2 ml of distilled water. after collection of each sample, the paper points were transferred to tubes containing 1 ml of bhi broth, vortexed for 1 min and incubated at 37°c for 48 h. each group had ten roots. tubes were then examined to investigate the presence of turbidity of the medium, which was classified according to the mcfarland standard scale for estimation of the number of bacteria before and after chemomechanical preparation. due to the great standard deviation of the turbidity value, a rank transformation was indicated for this study. it is a statistical tool that produces a table containing the ordinal rank of each value in a data set, in other words, the rank transforms the dependent variables. the kruskal-wallis test (biostat 5.0 software, cnpq 2000, brasília-df, brazil), a nonparametric test, was applied with a level of significance set at 5% (p < 0.05). sem analysis immediately after root canal preparation and microbiological sampling, each root was fixed in 2.5% glutaraldehyde for 48 h. by using a diamond disc at low speed and a wedge to expose the prepared canals, the roots were then divided into two halves in a buccolingual orientation. after dehydration with increasing concentrations of alcohol, the halves selected were coated with a thin layer of gold and submitted to sem analysis. photographs of the coronary, middle and apical thirds of the canals were taken at magnifications of 700x and 1,000x. the amount of smear layer was scored as follows: score 0 = all dentinal tubules were open and no smear layer was present; score 1 = smear layer was covering some dentinal tubules, but the majority was open; score 2 = smear layer was covering the majority of dentinal tubules; and score 3 ultrasonic irrigation in the removal of smear layer and enterococcus faecalis from root canals 223 braz j oral sci. 10(3):221-225 = all dentinal tubules were covered by smear layer. these photographs were analyzed by three examiners (who had no prior knowledge on the treatment applied to each root canal) and kappa test showed high intra and inter-examiner agreement values. data (scores obtained) were also submitted to statistical evaluation by using the non-parametric kruskalwallis test, and p values were computed and compared for statistical significance at the p < 0.05. results microbiological samples the level of turbidity of the samples before and after chemomechanical preparation is shown in table 1. kruskalwallis analysis showed a significant decrease in the turbidity, indicating reduction in the number of e. faecalis in groups 1 and 2 (p < 0.05) regarding the samples collected before and after chemomechanical preparation. however, there was no difference between these two protocols of irrigation. differently from groups 1 and 2, group 3 (control group) had only a slight decrease in the turbidity. sem analysis with respect to the smear layer removal capacity, there were no significant differences between groups 1 and 2 (p > 0.05). the conventional and passive ultra-sonic irrigation protocols resulted in a similar satisfactory cleaning. the presence of smear layer was significantly greater in the control group (p < 0.05) and in the apical third, when compared with cervical and middle thirds. it was observed that score 3 was more frequent in group 3 (control group), showing the least capacity to remove the smear layer. the scores for each group are presented in figure 1. the distribution of the smear layer and their corresponding scores are depicted in representative sem micrographs (figure 2). discussion there is a continuous search for optimal instrumentation table 1level of turbidity in the bhi broth containing samples before and after chemomechanical preparation according to mcfarland’s standard scale. samples 1 2 3 4 5 6 7 8 9 10 g1 before 6 6 7 6 7 7 6 7 7 7 g1 after 1 1 0 0 3 2 2 2 0 0 g2 before 7 7 7 7 6 7 7 7 7 7 g2 after 0 0 0 0 0 0 0 0 2 0 g3 before 8 7 7 7 7 7 7 7 8 7 g3 after 7 7 7 6 6 7 7 6 7 7 g1 before = g1 after; g2 before = g2 after; g3 before = g3 after (kruskal-wallis / p < 0.05). technique and irrigation solution. a number of studies have evaluated the antimicrobial capacity of irrigation solutions and their potential advantages when used in association with other techniques. other studies have focused on the efficacy of different instrumentation techniques and their capacity to remove debris and smear layer. the ultimate goal of such studies is to establish protocols that allow root canal walls to be as clean as possible6-8,11,13,27-31. indeed, ultrasonic instrumentation is not recommended because of the limited control allowed to the operator. endodontic files do not support the propagation of vibratory energy along their central axis, which increases the risk of file breakage. ultrasonic technology, however, may be useful for endodontic irrigation32. a few studies have shown that ultrasonic root canal irrigation produces agitation of irrigation solutions, improving both their antimicrobial actions and smear layer removal capacity20,30. the aim of this study was to evaluate in vitro the effectiveness of passive ultrasonic irrigation during chemomechanical preparation regarding removal of smear layer and root canal decontamination. the apical foramina of all samples were sealed to prevent extravasation of the solution and to allow it to flow optimally. root canals were contaminated with e. faecalis not only because it is present in most endodontic infections, but also because it is routinely resistant to conventional chemomechanical preparation2-4,10,25,33. fig. 1 – number of scores attributed to each group. ultrasonic irrigation in the removal of smear layer and enterococcus faecalis from root canals 224 braz j oral sci. 10(3):221-225 fig. 2 – representative sem micrographs: score 0, 700x (a1) and 1000x (a2) magnification; score 1, 700x (b1) and 1000x (b2); score 2, 700x (c1) and 1000x (c2); score 3, 700x (d1) and 1000x (d2). ultrasonic irrigation should be carried out passively without the file touching the root canal walls. free-oscillating files are preferably used because they promote adequate agitation of the irrigation solution13,34. thus, a k-file was adapted onto an endo l ultrasonic handpiece (dabi atlante) for the present study. cameron 12,35 evaluated ultrasonic activation for different time lengths (30 s, 1 s, 3 and 5 min). jensen et al.14 used it for 3 min, whereas weber et al. 30, gutarts et al. 20 and lui et al.21 for 1 min. it is not easy to maintain the ultrasonic file in a stationary position during an extended time period without touching the surrounding walls. in addition, this procedure can cause significant operator fatigue. therefore, in the present study, ultrasonic irrigation was performed for 15 s after each instrumentation. according to abbot et al.13 and lopes et al.7, the action of edta is more effective when it remains in contact with the surface for a certain period of time. edta should not be agitated. thus, in group 2, ultrasonic irrigation was carried out only after a 3 minute period of contact between edta and the root canal walls. in contrast, lui et al.21 obtained better results when edta was a constituent of the irrigation solution used. in the present study, ultrasonic irrigation did not clean the root canal better than conventional irrigation. both disinfection and smear layer removal seem to be due to the use of sodium hypochlorite and edta as irrigant solutions, and not because of the agitation promoted by the ultrasonic irrigation13,15-17,24,36. a number of reports have shown that the mechanical actions of irrigation alone can reduce the amount of bacteria inside the root canal5,9-10,37. in the present study, however, this was not the case. in the control group, a significant number of bacteria were still present following preparation. this may be explained by the evaluation methods used here. we carried out a qualitative analysis by assessing the bacterial growth in bhi broth and observing the medium turbidity instead of using dilution and quantification of cfus (used in other studies). some studies have shown that the use of ultrasonic irrigation significantly improved disinfection and cleaning of root canal walls14,17-19,27-28,37. the results of the present study, however, did not indicate significant differences between ultrasonic and conventional irrigation. mayer et al.22 reported similar findings. in conclusion the results of this study suggest that the level of disinfection and cleanliness of root canals achieved with ultrasonic irrigation is comparable to that obtained by conventional methods. references 1. siqueira-junior jf, uzeda m. influence of different vehicles on the antibacterial effects of calcium hydroxide. j endod. 1998; 24: 653-65. 2. dametto fr, ferraz cc, gomes bp, zaia aa, texeira fb, souza-filho fj. in vitro assessment of the immediate and prolonged antimicrobial action of chlorhexidine gel as na endodontic irrigant against enterococcus faecalis. oral surg oral med oral pathol oral radiol endod. 2005; 99: 768-72. 3. gomes bp, pinheiro et, sousa el, jacinto rc, zaia aa, ferraz cc et al. enterococcus faecalis in dental root canal detected by 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evaluation. j endod. 1987; 13: 541-5. 13. abbot pv, heijkoop ps, cardaci sc, hume wr, heithersay gs. an sem study of the effect of different irrigation sequences and ultrasonics. int endod j. 1991; 24: 308-16. 14. jensen sa, walker tl, hutter jw, nicoll bk. comparison of cleaning efficacy of passive sonic activation and passive ultrasonic activation after hand instrumentation in molar root canal. j endod. 1999; 25: 735-8. 15. karadag ls, tinaz c, mihçioglu t. influence of passive ultrasonic activation on penetration depth of different sealers. j contemp dent pract. 2004; 5: 1-7. 16. de-deus g, gurgel-filho ed, maniglia-ferreia c, coutinho-filho tauby. influence of the filling technique on depth of tubular penetration of root canal sealer: a scanning electron microscopy study. braz j oral sci. 2004; 3: 433-8. 17. moretti ag, pantoja cams, moreira dm, zaia aa, almeida jfa. effect of the smear layer on the filling of artificial lateral canals and microleakage. braz j oral sci. 2011; 10: 55-9. 18. kahn fh, rosenberg pa, gliksberg j. an in vitro evaluation of the irrigant characteristics of ultrasonic and subsonic handpieces and irriganting needles and probes. j endod. 1995; 21: 277-80. 19. lee sj, wu mk, wesselink pr. the effectiveness of syringe irrigation and ultrasonics to remove debris from simulated irregularities within prepared root canal walls. int endod j. 2004; 37: 672-8. 20. gutarts r, nusstein j, reader a, beck m. in vivo debridement efficacy of ultrasonic irrigation following hand-rotatory instrumentation in human mandibular molar. j endod. 2005; 31: 166-70. 21. lui jn, kuah hg, chen nn. effect of edta with or without surfactants or ultrasonics on removal of smear layer. j endod. 2007; 33: 472-5. 22. mayer be, peters oa, barbakow f. effects of rotary instruments and ultrasonic irrigation on debris and smear layer scores: a scanning electron microscopic study. int endod j. 2002; 35: 582–9. 23. huffaker sk, safavi k, spangberg lsw, kaufman b. influence of a passive sonic irrigation system on the elimination of bacteria from root canal systems: a clinical study. j endod. 2010; 36: 1315-8. 24. pascon fm, kantovitz kr, puppin-rontain rm. influence of cleanser and irrigation om primary and permanent root dentin permeability: a literature review. braz j oral sci. 2009; 5: 1063-9. 25. yamashita jc. avaliação, por microscopia eletrônica de varredura, da capacidade de limpeza de algumas soluções irrigadoras empregadas em endodontia [master’s thesis]. araraquara, sp: state university of são paulo; 2000. 26. scelza mf, pierro v, scelza p, pereira m. effect of three different time period of irrigation with edta-t, edta, and citric acid on smear layer removal. oral surg oral med oral pathol oral radiol endod. 2004; 98: 499-503. 27. yamashita jc, duarte mah, valim fa, almeida jm, kuga mc, fraga sc. evaluation of the surface of root canal walls after utilization of endodontic rotator systems: a sem study. j appl oral sci. 2005; 13: 78-82. 28. marque aaf, marchesan ma, sousa-filho cb, silva-sousa ytc, sousaneto md, cruz-filho am. smear layer and chelanted calcium ion quantification of three irrigating solutions. braz dent j. 2006; 17: 306-9. 29. ferraz cc, gomes bp, zaia aa, teixeira fb, souza-filho fj. in vitro assessmento of the antimicrobial action and the mechanical ability of the chlorhexidine gel as an endodontic irrigant. j endod. 2001; 27: 452-5. 30. weber cd, mcclanahan sb, miller ga, diener-west m, johnson jd. the effect of passive ultrasonic activation of 2% chlorhexidine or 5,25% sodium hypochlorite irrigant on residual antimicrobial activity in root canal. j endod. 2003; 29: 562-4. 31. sabins ra, johnson jd, hellstein jw. a comparison of the cleaning efficacy of short-term sonic and ultrasonic passive irrigation after hand instrumentation in molar root canals. j endod. 2003; 29: 674-8. 32. nabeshima ck, machado mlb. avaliação da resistência de limas durante preparo ultra-sônico. rev assoc paul cir dent. 2007; 61: 473-8. 33. gomes bp, souza sf, ferraz cc, teixeira fb, zaia aa, valdrighi l, souza-filho fj. effectiveness of 2% chlorhexidine gel and calcium hydroxide against enterococcus faecallis in bovine root dentine in vitro. int endod j. 2003; 35: 1-9. 34. sluis lw, shemesh h, wu mk, wesselink pr. passive ultrasonic irrigation of root canal: a review of the literature. int endod j. 2007; 40: 415-26. 35. cameron ja. the use of ultrasonic in the removal of the smear layer: a scanning electron microscope study. j endod. 1983; 9: 289-92. 36. chopra s, murray p, namerow k. a scaning electron microscope evaluation of the effectiveness of th f-files versus ultrasonic activation of k-file to remove smear layer. j endod. 2008; 34: 1243-5. 37. gurgel-filho ed, vivacqua-gomes n, gomes bp, ferraz cc, zaia aa, souza-filho fj. in vitro evaluation of the effectiveness of the chemomechanical preparation against enterococcus faecalis after singleor multiple-visit canal treatment. braz oral res. 2007; 21: 308-13. ultrasonic irrigation in the removal of smear layer and enterococcus faecalis from root canals oral sciences n3 impact on quality of life of removable partial denture wearers after 2 years of use alessandra oliveira barreto1, luana maria martins de aquino2, ana rafaela luz de aquino1, ângelo giuseppe roncalli3, bruna aguiar do amaral1, adriana da fonte porto carreiro4 1 doctoral student in oral pathology, department of dentistry, federal university of rio grande do norte, brazil 2 doctoral student in clinical dentistry, department of periodontics and dental prosthetics, piracicaba dental school, university of campinas, brazil 3 phd in community dentistry, department of dentistry, federal university of rio grande do norte, brazil 4 phd in oral rehabilitation, department of dentistry, federal university of rio grande do norte, brazil correspondence to: alessandra oliveira barreto rua jacarandá 227, cond. campos do cerrado, bl. e, apto 1401, cidade verde. cep: 59152-210. natal-rn, brasil. phone: +5584-36082371 / +5584-96651188 e-mail: alle.barreto@hotmail.com received for publication: october 07, 2010 accepted: march 03, 2011 abstract the use of clinical indicators of satisfaction (ohip) can be applied to evaluate the impact of denture use on patient quality of life, since dental problems and disorders interfere in the normal life of individuals. aim: this study aimed at evaluating the satisfaction level of patients rehabilitated with removable partial dentures (rpd) after 2 years of use. methods: an observational study was carried out on 28 patients with a mean age of 45 years, treated with rpd at the department of dentistry of the federal university of rio grande do norte in 2005. patients signed informed consent and answered the oral health impact profile (ohip) questionnaire on three occasions: prior to rehabilitation and at 3 months and 2 years of denture use. repeated-measures anova was applied for data analysis. results: a difference was found between data obtained at the moment of fitting and three months after denture use (p<0.001). however, no variation was observed when comparing data from 3 months and 2 years of use (p>0.05). the variables of gender and age did not interfere in the result (p>0.05). conclusions: the degree of patient satisfaction after rpd installation was significant at the moment of fitting and 3 months after denture use, but no significant difference was found between 3 months and 2 years of denture use. keywords: oral health, removable partial denture, quality of life. introduction reestablishing and maintaining stomatological health in edentulous patients through different types of prostheses is aimed at providing biopsychosocial balance. removable partial denture (rpd) is a common treatment alternative to restore edentulous areas because it requires conservative preparations and offers rapid resolution and more accessible costs1. oral rehabilitation can have a positive impact on the physical, social and psychological well-being of patients. it may hence prevent problems such as concentration difficulties, anxiety and even social exclusion2-3. with this in mind, dental surgeons have become increasingly concerned about the influence of clinical outcomes on quality of life in their patients2. according to ruffino netto4, good quality of life provides minimum means for individuals to fully develop their potential. these include living, feeling or loving, working to produce assets or services, making art or science, being useful citizens or simply existing. quality of life is also understood as a standard that original article braz j oral sci. january | march 2011 volume 10, number 1 braz j oral sci. 10(1):50-54 51 braz j oral sci. 10(1):50-54 society itself defines and attempts to achieve, consciously or subconsciously5. substantial interest in quantifying the consequences of disease on quality of life prompted the development of several instruments indicating the impact of oral health on quality of life. among these, the ohip (oral health impact profile) is a powerful tool in oral health assessment related to quality of life3. the questionnaire was developed by the researchers slade and spencer6. its original version presents 49 items and is considered a subjective indicator as it reveals individual expectations in relation to oral health. the ohip is based on locker’s7 conceptual model of oral health and includes seven dimensions: functional limitation, physical pain, psychological discomfort, physical disability, psychological disability, social disability and incapacity8. in a study evaluating oral health issues, or specific health issues, the ohip was considered the optimal questionnaire in accordance with the eq-5d+ (european quality of life indicator or euroqol instrument)9. this is due to the wide scope of its seven dimensions. pires et al.10 validated the questionnaire after translation into portuguese and adaptation to the brazilian culture. results showed that the questionnaire is valid in assessing the impact of oral conditions on the quality of life of brazilians. in most people, oral health changes such as tooth loss affect quality of life 11. the fully edentulous condition negatively impacts oral health-related quality of life (ohrqol)12, including the inability to chew, poor speech, pain, and dissatisfaction with appearance13. biazevic et al.14 used the ohip to evaluate the impact of oral condition on quality of life of elderly peoples. the authors concluded that the need for prosthesis was related to the impact on quality of life. variables such as type of denture use, gender, age, education level and area of residence are also assessed by the ohip questionnaire15. only type of prosthesis was found to influence quality of life. each population has different perceptions of their oral health status and quality of life16, depending on their lifestyle, socioeconomic status and access to the health system. the aim of the present study was to assess the impact of oral health on quality of life in patients with rpd after 2 years of use. material and methods the present intervention study was carried out in the department of dentistry at the federal university of rio grande do norte, with patients from the removable partial denture and integrated clinic disciplines and approved by the ufrn research ethics committee (protocol number 11/ 05). the initial sample included all patients in the rpd fitting phase in 2005, making randomization unnecessary. all of the 56 patients who received rpds in 2005 had previously used prostheses. however, 2 were lost to follow up for reasons such as transfer to another state and lack of interest in participating. this represents a loss of 3.57%, totaling 54 patients. of these, 38 were female and 16 were male, with an age range between 26 and 66 years and a mean age of 45 years. patients were rehabilitated by undergraduate dental students under the supervision of prosthodontics professors. all participants received prior oral treatment and mouth p r e p a r a t i o n s p e c i f i c t o e a c h c a s e , p l a n n e d w i t h t h e assistance of a delineator. every care was taken to maintain the state of health of biological structures. in addition, patients were instructed on care and cleaning procedures for the dentures. the ohip questionnaire was applied before fitting the new removable partial denture (baseline) to evaluate the impact of oral health on quality of life in wearers. it was reapplied 3 months (time 1), time needed for patients to adapt to new prosthesis, and 2 years (time 2) after fitting, allotted time for patients able to identify possible changes in quality of life, totaling 3 assessment times. at time 2, there was a 56% loss of baseline patients due to transfer to another state, disinterest in participation or loss of contact (address/telephone), totaling a final sample of 28 patients. of these, 16 were female and 12 male, with a mean age of 46 years. data were then compiled into a databank on microsoft office excel 2003 and spss 13.0 software was used for descriptive statistical analysis. the three assessment times were compared by analyzing the mean (m) and standard deviation (sd) of the ohip dimensions. a normality test was applied (kolmogorov-smirnov) followed by repeatedmeasures anova. student’s t-test was performed to determine whether the variables of gender and age interfered in results. results in relation to the duration of rpd use: before fitting (baseline), after 3 months (time 1) and after 2 years (time 2) of wearing the new rpd, a statistically significant difference was found from baseline (mean: 125.75) to time 1 (mean: 88.61) (p<0.001). however, no such difference was recorded from time 2 (mean: 88.61) to time 3 (mean: 83.82), (p>0.05) (table 1). both the mean ohip value and its dimensions decreased. the former fell by 33%, from the baseline (mean: 125.75) to time 2 (mean: 88.61) (figure 1), as well as its seven dimensions: incapacity (baseline:10.75 and time 2: 9.09), disability social (baseline: 9.61 and time 2: 7.57), disability psychological (baseline:14.57 and time 2: 4.43), disability physical (baseline: 22,89 and time 2: 5.37), discomfort psychological (baseline: 17.14 and time 2: 8.3), physical pain (baseline: 24.46 and time 2: 10.04) and limitation functional (baseline: 26.32 and time 2: 5.4) (figure 2). the mean variables gender, of non-evaluated patients, at baseline (male: 118,7 and female: 131.0) , time 1 (male: 86.3 and female: 90.3) and time 2 (male: 92.42 and female: 77.4) as well as the variable age, at baseline (up to 46: 135.9 and over 46:115.6) , time 1 (up to 46: 90.4 and over 46: 86.8) and time 2 (up to 46: 96.8 and over 46:70.9) did not interfere in the results obtained for ohip values (p>0.05) (table 2). impact on quality of life of removable partial denture wearers after 2 years of use 52 braz j oral sci. 10(1):50-54 table 1: ohip values of the respective dimensions in relation to time: before, after 3 months and after 2 years of rpd use. * repeated-measures anova test. same letters, no significant difference; different letters, significant difference. dimension n mean s.d mean s.d mean s.d p * functional limitation 28 26.3 a 8.8 17.7 b 7.6 16.0 b 5.4 < 0.001 physical pain 28 24.5 a 6.1 18.3 b 7.2 18.1 b 10.0 0.004 psychological discomfort 28 17.1 a 4.8 11.7 b 4.9 12.1 b 8.3 0.002 physical disability 28 22.9 a 8.7 16.8 b 5.6 13.9 b 5.4 < 0.001 psychological disability 28 14.6 a 6.4 9.5 b 4.2 8.5 b 4.4 < 0.001 social disability 28 9.6a 5.3 6.5 b 2.7 6.9 b 7.6 0.081 incapacity 28 10.8 a 4.5 8.1 a 4.1 8.3 a 9.1 0.225 ohip 28 125.8 a 35.0 88.6 b 30.0 83.8 b 41.3 <0.001 before 3 months 2 years variable n mean s.d p * mean s.d p * mean s.d p * gender male 12 118.7 38.3 0.382 86.3 37.2 0.751 92.42 56.3 0.403 female 16 131.0 32.6 90.3 24.5 77.4 25.4 age up to 46 years 14 135.9 37.6 0.129 90.4 33.8 0.755 96.8 54.8 0.107 over 46 years 14 115.6 30.2 86.8 26.9 70.9 13.29 before 3 months 2 years table 2. ohip values in relation to gender (female/male) and age (up to 46 years/over 46 years). * student’s t-test discussion based on a recent systematic review, there is a trend in the literature indicating that validated instruments, such as ohip, are being increasingly applied to investigate the influence of prosthodontic and dental implant treatment on patient satisfaction and ohrqol17-18. the present study found that prior to fitting the new rpd (baseline) and following 3 months of use, ohip values decreased by approximately 33%, representing a statistically significant difference (p<0.001). impact on quality of life of removable partial denture wearers after 2 years of use fig. 2. mean ohip dimension values in relation to time: before (baseline), after 3 months (time 1) and after 2 years (time 2) of removable partial denture use. fig. 1. mean ohip value in relation to time: before (baseline), after 3 months (time 1) and after 2 years (time 2) of removable partial denture use. 53 braz j oral sci. 10(1):50-54 these results show that patients were already in a better quality of life after 3 months using the new dentures. john et al.19 investigated the minimal important difference (mid) in ohip scores in prosthodontic patients. in corroboration with data from our study, these authors assessed a consecutive sample of 224 adult patients who answered the ohip questionnaire twice before treatment and 4 to 6 weeks after completion of the prosthodontic treatment. the mean baseline and follow-up ohip differences (score change) were calculated for subjects (n = 47). a slight improvement was reported in the amount of 6 ohip units (95% confidence interval). similarly, jokovic and locker20 evaluated the dissatisfaction with oral health in 907 50-year-old canadians and found that edentulous patients are more unsatisfied than dentate subjects. comparable findings were reported by smith, baysan and fenlon21 in a sample of 216 patients aged 18-83 years, recruited from the implant assessment clinic. these authors concluded that the impact of oral health on quality of life in these subjects was strongly associated with general well-being. biazevic et al.14 also observed that quality of life was affected in patients requiring prosthesis. the type of prosthesis may also impact the life quality of wearers, with complete dentures obtaining worse results22. when analyzing the influence of denture use times on quality of life, we found that wearers maintained the same quality of life after 3 months (time 1) and 2 years (time 2) of rpd use, since levels of the ohip dimension were not statistically different between times 1 and 2 (p>0.05), even with a reduction of 50% of the participants can observe similarity in mean ohip. these results show that professors and students are careful when making the prosthesis, thereby helping to achieve patient satisfaction with their denture over 2 years. zlataric22 confirms that greater dissatisfaction about rpd is related to esthetics (50%), which depends on the dental surgeon for success. chewing ability, a common and important oral health indicator among the elderly23-24 has been reported to affect general health and quality of life when unsatisfactory. kim et al.25 investigated the association between chewing capacity and oral health-related quality of life (ohrqol) by applying the oral health impact profile14 (ohip-14). this cross-sectional study comprised a sample of 307 community-dwelling and 102 institutionalized people over the age of 60, using a cluster sampling procedure. a significant association was found between chewing ability and ohrqol measured by the ohip-14 score. amelioration of chewing ability might independently contribute to improving ohrqol in elderly patients. in an effort to clarify the exact nature and use of ohrqol instruments, several researchers have explored comprehensive relationships between these and other potentially associated factors such as: demographic26-27 and socioeconomic factors28, clinical measurements of oral health26-29 health behaviors including patterns of dental attendance28, need for dental treatments27, self-perceived health, differences in target population age, types of treatment provided, cultural and different expectations. this may indicate that the minimal important difference (mid) for ohip instruments is not constant across settings19. in the present study, the variables gender and age were analyzed to determine whether they interfered with results for time of denture use in relation to quality of life. both variables were found to have no influence on results. gonçalves et al.30 reported similar findings as they observed no difference in the impact on oral health between men and women. john et al.15 assessed the influence of variables: type of prosthesis, gender, age, education and area of residence on quality of life. only the type of prosthesis was found to influence quality of life, with the rpd achieving better satisfaction from wearers. however, john et al.19 observed slight to moderate differences in ohip mean scores between gender and age groups. differences in mean baseline scores were usually slight, indicating no subgroup differences in the level of ohrqol impairment. score changes (baseline and follow-up) varied somewhat more in female subjects, younger individuals and patients with removable dentures experienced marginally larger changes than the remaining subjects. no attempt was made to assess the statistical significance of these differences since the minimal important difference (mid) was not expected to vary between subgroups. similarly, gilbert et al.16 observed the gender and age range associated with some disadvantages and found that women were more likely to report avoiding tough foods. thus, these factors should to be taken into account when investigating the possible relationship between oral conditions and well-being21. the obtained results indicate that oral rehabilitation with rpd reestablishes and maintains health in the stomatological system and can therefore improve patient quality of life. references 1. de fiori sr. atlas de prótese parcial removível. 4 ed. são paulo: pancast; 1993. 525p. 2. zlataric dk, celebic a, valentic-peruzovic m, jerolimov v, panduric j. a survey of treatment outcomes with removable partial dentures. j oral rehabil. 2003; 30: 847-54. 3. vargas amd, paixão hh. perda dentária e seu significado na qualidade de vida de adultos usuários de serviço público de saúde bucal do centro de saúde boa vista, em belo horizonte. cienc saude colet. 2005; 10: 1015-24. 4. ruffino netto a. qualidade de vida: compromisso histórico da epidemiologia. saude debate. 1992; 35: 63-7. 5. minayo mcs, hartz zna, buss p. qualidade de vida e saúde: um desafio necessário. cienc saude colet. 2000; 5: 25-33. 6. slade gd, spencer aj. development and evaluation of the oral health impact profile. community dent 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szentpétery ag, john mt, slade gd, setz jm. problems reported by patients before and after prosthodontic treatment. int j prosthodont. 2005; 18: 124-31. 13. walton jn, macentee mi. choosing or refusing oral implants: a prospective study of edentulous volunteers for a clinical trial. int j prosthodont. 2005; 18: 483-8. 14. biazevic mgh, michel-crosato e, iagher f, pooter ce, correa sl, grasel ce. impact of oral health on quality of life among the elderly population of joaçaba, santa catarina, brazil. braz oral res. 2004; 18: 85-91. 15. jonh mt, koepsell td, hujoel p, miglioretti dl, leresche l, micheelis w. demographic factors, denture status and oral health-related quality of life. community dent oral epidemiol. 2004; 32: 125-32. 16. gilbert gh, duncan rp, heft mw, dolan ta, vogel wb. multidimensionality of oral health in dentale adults. med care. 1998; 36: 988-1001. 17. strassburger c, heydecke g, kerschbaum t. influence of prosthetic and implant therapy on satisfaction and quality of life: a systematic literature review: part 1. characteristics of the studies. int j prosthodont. 2004; 17: 83-93. 18. strassburger c, kerschbaum t, heydecke g. influence of implant and conventional prostheses on satisfaction and quality of life: a literature review: part 2. qualitative analysis and evaluation of the studies. int j prosthodont. 2006; 19: 339-48. 19. john mt, reißmann dr, szentpétery a, steele j. an approach to define clinical significance in prosthodontics. j prosthodont. 2009; 18: 455-60. 20. jokovic a, locker d. dissatisfaction with oral health status in an older adult population. j public health dent.1997; 57: 40-7. 21. smith b, baysan a, fenlon m. association between oral health impact profile and general health scores for patients seeking dental implants. j dent. 2009; 37: 357-9. 22. zlataric dk, celebic a. factors related to patients’ general satisfaction with removable partial dentures. int j prosthodont. 2008; 21: 86-8. 23. sheiham a, steele jg, marcenes w, tsakos g, finch s, walls awg. prevalence of impacts of dental and oral disorders and their effects on eating among older people; a national survey in great britain. community dent oral epidemiol. 2001; 29: 195-203. 24. locker d. changes in chewing ability with ageing: a 7-year study of older adults. j oral rehabil. 2002;29: 1021-9. 25. kim hy, jang ms, chung cp, paik di, park yd, patton ll et al. chewing function impacts oral health-related quality of life among institutionalized and community-dwelling korean elders. community dent oral epidemiol. 2009; 37: 468-76. 26. mcgrath c, bedi r. population based norming of the uk oral health related quality of life measure (ohqol-uk). br dent j. 2002; 193: 521-4. 27. mariño r, schofield m, wright c, calache h, minichiello v. self-reported and clinically determined oral health status predictors for quality of life in dentate older migrant adults. community dent oral epidemiol. 2008; 36: 85-94. 28. lawrence hp, thomson wm, broadbent jm, poulton r. oral healthrelated quality of life in a birth cohort of 32-year-olds. community dent oral epidemiol. 2008; 36:305-16. 29. pallegedara c, ekanayake l. effect of tooth loss and denture status on oral health related-quality of life of older individuals from sri lanka. community dent health. 2008; 25: 196-200. 30. gonçalves jr, wassall t, vieira s, ramalho as, flório fm. impacto da saúde bucal sobre a qualidade de vida entre homens e mulheres. rgo. 2004; 52: 240-2. impact on quality of life of removable partial denture wearers after 2 years of use oral sciences n3 original article braz j oral sci. october | december 2012 volume 11, number 4 dental caries-related quality of life and socioeconomic status of preschool children, bauru, sp angela xavier1, fábio silva de carvalho1, roosevelt da silva bastos2, magali de lourdes caldana3, josé roberto de magalhães bastos4 1phd student, department of pediatric dentistry, orthodontics and community health dentistry, bauru dental school, university of são paulo, bauru, sp, brazil 2phd, professor, bauru dental school, university of são paulo, bauru, sp, brazil 3associate professor, bauru dental school, university of são paulo, bauru, sp, brazil 4professor, bauru dental school, university of são paulo, bauru, sp, brazil correspondence to: angela xavier departamento de pediatria, ortodontia e saúde coletiva, faculdade de odontologia de bauru, universidade de são paulo alameda otávio pinheiro brisolla n° 9-75 cep: 17012-901, bauru, sp,brasil phone: +55 14 96068223 / +55 14 30212176 / +55 14 32358256 e-mail: dra.axavier@gmail.com received for publication: july 29, 2012 accepted: november 28, 2012 abstract aim: to evaluate oral health-related quality of life of preschool children of bauru, state of são paulo, brazil, and associate it with socioeconomic profile of households. methods: the sample consisted of 229 preschool children between 3 and 5 years and the dmft (decayed, missing due to caries, filled teeth) index was adopted for assessment children’s dental caries in accordance with the standards recommended by the world health organization. questionnaires were used for evaluation oral health-related quality of life (early childhood oral health impact scale) and socioeconomic profile of parents or guardians of the preschool children. statistical analysis was performed descriptively by relative and absolute frequencies and by spearman’s correlation and kruskal-wallis test (p <0.05). results: a dmft of 1.65 (± 2.87) and a sic index 4.88 (± 3.20) were found, indicating the polarization of dental caries in the studied group. it was verified low influence of oral health on quality of life of the children examined. with respect to socioeconomic classification, 66.38% of families were in the lower middle class. linear and statistically significant correlation was found between dmft and oral health-related quality of life for the overall score and domains of the questionnaire (p<0.001). conclusions: it was found low influence of oral health on quality of life of the preschool children and the assessment of socioeconomic conditions of the children’s families may guide practices aiming to reducing inequalities in the distribution of dental caries in the population. keywords: quality of life, dental caries, oral health. introduction the association between social and economic conditions and dental caries prevalence has been observed in several studies1. researchers have found that people living in precarious socioeconomic conditions are more favorable to be exposed to risk factors that influence oral health conditions, and this is directly related to quality of life, not only in functional domains, but also in its social and psychological ones2. the environment in which children live and grow up has also been reported as influencing their health behaviors and their perception of oral health3. braz j oral sci. 11(4):463-468 464464464464464 braz j oral sci. 11(4):463-468 oral health problems has been increasingly recognized as important factors causing a negative impact on daily performance and quality of life because they influence how people grow, enjoy life, speak, chew, taste food, and socialize4 most studies on evaluation oral health status were carried out using only clinical measures, however, oral health-related quality of life (ohrqol) instruments should be used in conjunction with them5. adults’ and children’s perception of health conditions takes place in a different way and in the case of children that accuracy varies with cognitive capacity for each age group. this ability may vary according to the stage of emotional development, language or social environment of the child. moreover, the socioeconomic and cultural conditions in which children were born and grew up may also influence their perception6. thus, there may be a child’s difficulty in answering questions concerning the events in their health during a previous period of time depending on the age, where a questionnaire directed to 12-year-old children should not be answered by a 5-year-old child. the capacity for abstraction as well as comparisons of physical characteristics and personality starts from 6 years of age, and temporal concepts from 8 years of age. this mean that very young children as those in preschool age have difficulties in providing accurate information on the influence of oral health conditions on quality of life and, thus, questionnaires geared to this age group are answered by parents or guardians of them7. based on the foregoing considerations, the main purpose of this study was to evaluate the influence of oral healthrelated quality of life of preschool children of bauru, state of são paulo, brazil, and associate it with socioeconomic profile of households. material and methods the research protocol was approved by the institutional review board (irb) of bauru dental school, university of são paulo, brazil, (process no 156/2009), and the authorization for conduction of study was obtained from the municipal secretary education and directors of kindergartens. also, written informed consent was obtained from the parents/ legal representatives prior to enrolment of the children. to perform this cross-sectional observational research the target population consisted of preschool children of public kindergartens in bauru, state of são paulo, brazil, totaling 6502 preschool children. the municipality of bauru has 60 public kindergartens8 and the city was divided according to five regions described as follows: north, south, east, west and central region. thus, the districts of each region were grouped to perform a raffle which would be the research participant. likewise, schools of each district were grouped and randomly selected for the survey. this way, six public kindergartens were randomly selected to be part of the research. sample calculation was made based on the error level á = 0.05 and â error level of 0.20. for this calculation, was established a correlation coefficient (r) of 0.20 finding an amount of 198 children to be examined. six hundred thirty (630) consent forms were sent to the five public kindergartens that were part of this research, and there was a response rate of 36.35% allowing children to participate in the study. the participating children were those who were in kindergarten at the time of the survey and who were allowed to participate in research through the informed consent signed by parents/guardians, and who permitted clinical examination. therefore, 229 preschool children of both genders between 3 and 5 years of age were examined. however, this sample is not representative for preschool from public kindergarten population. clinical examinations were performed by a calibrated examiner and a recorder. an intra-examiner agreement of 0.92 was found. the preschool children were examined seated on chairs under natural light and the materials used for the clinical examinations were dental mirrors and cpi (community periodontal index) probes in order to remove biofilm and to confirm visual evidence of caries. the clinical examinations used for observation of the mean number of decayed, missing due to caries or filled teeth (dmft index) were performed according to the criteria established by the world health organization (who)9. to assess oral health-related quality of life of the preschool children was used the early childhood oral health impact scale (ecohis). the ecohis was developed by researchers of north carolina university by the selection of 13 items from the 36 that comprise the questionnaire child oral health quality of life instrument (cohqoli). the brazilian version of the questionnaire was transculturally adapted in brazil and this version has 14 questions, being 10 issues relating to the child subscale and 4 in the family’s subscale. these items were considered most relevant to measure the impact of oral health on quality of life of preschool children and consider the experiences of the children’s oral diseases and dental treatment10. the ecohis is answered by the parents or guardians of the children, assessing their perceptions about the influence of oral health on quality of life of the children in preschool age. the responses options are listed in codes ranging from 0 to 5, where code 0 = never, 1 = almost never, 2 = sometimes 3 = frequently, 4 = very frequently 5 = do not know. the amount scores and domains were calculated from the sum of the reply codes. the responses “not know” were counted, but were excluded from the sum to calculate the amount score and by domain of each patient. the minimum score obtained in the questionnaire was zero corresponding to no influence of oral health on quality of life and the maximum was 56 where there was strong influence of oral health on quality of life of children. it was proposed a classification of the questionnaire’s results in different impacts aiming to verify the impact of oral health on quality of life of preschool children. the total score of the questionnaire ranges from 0 to 56 points. for the classification, score = 0 was considered with no impact and the score between 1 and 56 was divided into three equal ranges, being weak impact, when the sum score of the questionnaire is greater than zero and less than or equal to dental caries-related quality of life and socioeconomic status of preschool children, bauru, sp 465465465465465 braz j oral sci. 11(4):463-468 18.67, impact medium when the result is greater than 18.67 and less than or equal to 37.34 and strong impact, when the score of the questionnaire is greater than 37.34 and less than or equal to 56. for the socioeconomic classification of the family was used the methodology proposed by graciano (1980) which assess five factors that received a score system. the sum of points allowed determination of an individual score and hence the ranking of the participants in one of the six classes propose11. each of these factors has a specific goal, as follows: factor 1: the economic situation of the family. seeks to identify the level of family income. factor 2: number of family members. it is considered the number of family members who participate and/or depend on the economic situation. factor 3: statement of the head of the family. with the evaluation of grade school to expand the possibilities for social mobility. seeks to identify the education level of the medium in which the child lives. factor 4: housing. seeks to identify the status of ownership of their living. factor 5: occupation of the head of the family. seeks to identify through the profession and at the same time, social cultural and economic level of household head. depending on the sum of points from the evaluation is calculated socioeconomic classification itself, being: 0 to 5 points lower low class, 6 to 11 pointsupper low class, 12 to 20 points lower middle class, 21 to 29 pointsmiddle class, 30 to 38 pointsupper middle class and over 39 high class. percentages of dmft and caries-free children were used to describe dental caries distribution among preschool children. significant caries index (sic index), gini coefficient and care index were adopted to assess the unequal distribution of dental caries and oral health care. sic index was calculated by taking the mean dmft of the one third of the individuals having the highest of dmft values in a given population, and was used to measure the polarization of the dental caries occurrence among preschool children12. the care index was calculated using the means dmft without caries-free. the component “f” (filled teeth) was divided by the dmft and multiplied by 10013. the gini coefficient was used to assess inequality of caries distribution in this study14. the kruskal-wallis test was used for comparison of dmft according to age. spearman’s correlation test was used to relate the mean dmft with the oral health-related quality of life and socioeconomic classification as well as to relate the results of oral health-related quality of life with the socioeconomic conditions. a significance level of 5% was adopted. the tests were calculated using statistica 9.1 software. results as much as 229 preschool children were recruited for the study, being 50.66% boys and 49.34 girls. these 229 children were part of the survey because consent forms were distributed beyond the amount calculated for the sample, due to losses that occur when distributing the consent forms of those mothers who do not want to allow the participation of children in research, being examined all children whose parents consented to participate. table 1 summarizes the distribution of dental caries of preschool children examined according to age. there was a higher dmft in 5-year-olds than in 3-year-olds, whereas at 3 years it was found a dmft of 1.07 (2.12) and at 5 years a dmft of 1.98 (2.95). it was found a significant caries index of 4.88 (3.20), nearly three times higher than the average dmft. the gini coefficient shows a change from zero to one, where zero is related to absence of inequality and one demonstrates the presence of the same. in this population was observed a gini coefficient greater expressiveness in 4-year-olds (0.83), demonstrating the unequal distribution of dental caries. the care index was used to assess the capacity of health services in meeting the needs of the population, which revealed a low percentage of care for this population (30.00%). in the 4-year-old group there was the lower percentage of care (22.00%), as described in table 1. with respect to socioeconomic status, it was found that 66.38% of the families of preschool children examined were classified in the lower middle class, 21.83% were classified in the middle class, 11.35% in the upper low, 0.44% in the upper middle class and there was no family in the lower low and high class. according to the responses of the different domains of the questionnaire oral health-related quality of life according to age, was verified that symptoms and functional limitations presented the highest means in subscale of children and anguish of parents demonstrated the highest mean in subscale of family. in the domains symptoms, psychological aspects and anguish of parents there was an increase in average with increasing age. it was verified statistically significant difference in the domains’ symptoms (p=0.016) and selfimage (p=0.040), as shown in table 2. for the development of this research, it was proposed the assessment of the impact of oral health status in quality of life of preschool children, where it was observed 55.90% dental caries-related quality of life and socioeconomic status of preschool children, bauru, sp age dmft (sd) caries free sic índex (sd) gini coefficient care index (%) 3 years 1.07 (±2.12) 68.97 3.44 (±2.55) 0.80 36.00 4 years 1.33 (±2.94) 69.74 4.04 (±3.94) 0.83 22.00 5 years 1.98 (±2.95) 50.00 5.34 (±2.95) 0.71 32.00 amount 1.65 (±2.87) 58.95 4.88 (±3.20) 0.76 30.00 table 1inequality on dental caries distribution of preschool children examined according to age, bauru, sp, 2010. 466466466466466 braz j oral sci. 11(4):463-468 3 yearsaverage (sd) 4 yearsaverage (sd) 5 yearsaverage (sd) amount kruskal wallis (p) symptoms 0.31 (±0.71) 0.46 (±0.93) 0.84 (±1.12) 0.65 (±1.04) 0.016* functional limitations 1.28 (±2.80) 0.89 (±1.76) 2.42 (±3.58) 0.30 (±0.12) 0.180 ns psychological aspect 0.28 (±0.88) 0.30 (±0.95) 0.63 (±1.34) 0.24 (±0.03) 0.241 ns self-image 0.21 (±1.11) 0.01 (±0.11) 0.27 (±0.88) 0.13 (±0.04) 0.040* anguish of parents 0.28 (±1.03) 0.55 (±1.31) 1.06 (±2.07) 0.41 (±0.01) 0.454 ns family function 0.17 (±0.47) 0.11 (±0.56) 0.35 (±0.95) 0.12 (±0.05) 0.366 ns amount 2.34 (±6.37) 1.82 (±3.29) 4.71 (±6.73) 3.49 (±6.09) 0.247 ns table 2average responses of oral health related quality of life of preschool children according to age, bauru, sp, 2010. * statistically significant difference. nsno statistically significant difference dmft (sd) sic índex (sd) caries free (%) gini coefficient care index (%) no impact 0.49 (±1.14) 1.50 (±1.58) 78.13 0.85 39.00 weak impact 2.95 (±3,64) 7.23 (±3.23) 36.26 0.63 28.00 medium impact 4.60 (±3,40) 8.33 (±0.58) 20.00 0.39 26.00 strong impact 0.00 (±0.00) 0.00 (±0.00) 0.00 0.00 0.00 kruskal-wallis p p<0.001* table 3 inequality on dental caries distribution according to different impacts of quality of life, bauru, sp, 2010. * statistically significant difference of children with no impact of oral health on quality of life, 39.74% with weak impact, 4.36% of the preschool children with medium impact, and no children with strong impact. assessing the dmft of children according to the different impacts of oral health on quality of life, it was found that children who showed no impact presented a dmft of 0.49 (1.14) and those who had a medium impact presented a dmft of 4.60 (3.40), where it was found a statistically significant difference between the different impacts (p<0.001). moreover, it was found a reduction in the percentage of caries free children and the care index in accordance with the increasing impact of oral health on quality of life as described in table 3. assessing the correlation between dmft and oral healthrelated quality of life was observed a linear and significant ratio for the overall score and domains of the questionnaire (p<0.001). relating oral health-related quality of life with socioeconomic conditions of the families, it was observed a significant inverse relationship (r =0.183, p=0.006), i.e., with higher influence of oral health on quality of life of preschool children, lower the socioeconomic condition of households, as well as between oral health-related quality of life and income components (r =0.196, p= 0.003) and housing (r =0.166; p= 0.011). however, no relationship was found with the educational component of the parents or guardians (r =0.113, p= 0.086). relating dmft with socioeconomic conditions of the families, it was found a significant inverse relationship (r = 0.190, p= 0.003). discussion in order to evaluate the prevalence and severity of dental caries according to who recommendations, this study used the dmft index. it was possible to evaluate the caries experience in preschool children from the city of bauru, state of são paulo. it could be observed polarization of dental caries in the studied group, with a sic index of 4.88 (+3.20) and it was found low care index, demonstrating the limited access of this population to health services and the low restorative care to which they have been exposed. the findings of this study are consistent with the goals of who for 2000 of 50% of caries-free children among 5-year-olds15, but are not consistent with the goals of who for 2020 in increasing the proportion of caries free children16. it has been reported that when there is a large number of cases concentrated in a small group of individuals exist a phenomenon known as polarization17. this phenomenon is expressed in the concentration of greater burden of disease and treatment needs in a small portion of the population (2040%), whereas most the children presents caries-free (40-60%), may be reflecting the measures of prevention and control of dental caries, based on solid population strategy, in which moved from a situation of high prevalence of the disease for a large percentage of caries-free individuals18. in this sense, the greater vulnerability to injury is associated with intense exposure to risk factors and social deprivation. in some studies, it was emphasized that the prevalence of dental caries decreased as socioeconomic level increased, even in areas without the addition of fluoride to public water supply19. in order to quantify the extent which oral health problems interfere on daily life and well-being of people, were developed instruments of oral health-related quality of life to assess the impact of oral health in the physical and psychosocial development. children, as well as young adults are also affected by several oral health problems, which have the potential to compromise the well-being and quality of life of them 20-21. the ecohis was developed for use in epidemiological dental caries-related quality of life and socioeconomic status of preschool children, bauru, sp 467467467467467 braz j oral sci. 11(4):463-468 studies aiming to evaluate the influence of oral diseases and treatment on preschool children’s quality of life. it considers the experience of oral diseases and dental treatment of the child’s lifetime with the answers provided by parents22. there are few studies in the literature regarding the influence of oral health on quality of life of children in preschool age. this research found a greater influence of oral health on quality of life in the domains’ symptoms and anguish of parents and lower means on self-image and family function. the maximum score obtained in the questionnaire was 33 points. in this study the domains with the highest means were symptoms and functional limitations, which demonstrate that the influence of oral health on quality of life of children can be perceived by parents/guardians, when there are symptoms such as pain and limitations in daily activities such as speech and feeding. these results highlight the need to promote health education activities with parents or guardians of preschool children in order to raise awareness about the importance of maintaining a healthy primary dentition both for oral health and general health of children in this age group. similarly, pahel et al. (2007) found that the highest average of the influence of oral health on quality of life domains were registered in symptoms, followed by functional limitations and emotional well-being. children who had higher caries experience reported greater influence on quality of life that children who had lower caries experience7. in a research conducted by abanto et al. (2010)22 with preschool children using the ecohis, parents reported greater impact related to the child’s subscale (69.30%) than with family’s subscale (30.70%). parents reported no influence of oral health on quality of life in 40.10% and in 59.90% of children in child’s subscales and family’s subscale respectively. the maximum score of 30 was recorded at child’s session and 12 on family’s session. a recent study conducted in the city of diamantina, mg, brazil showed that in the child impact section, “pain in the teeth, mouth or jaws” was the most frequently reported item by the parents (21.5%) and in the family impact section the most frequently reported item was “felt guilty” (14.2%)23. this research also aimed to assess the perception of parents of oral health-related quality of life using a scale with different impacts from preschool children with no impact, weak impact, medium impact and strong impact, in order to facilitate their use in public health, where it was observed that 55.9 % of the children had no impact, i.e., a score of zero, and 39.74% were weak impact of oral health on quality of life. by linking the different impacts to the prevalence of dental caries, it was observed that children with medium impact showed a dmft of 4.60 (3.40) and those with no impact a dmft of 0.49 (1.14). likewise, the study by li et al. (2008) revealed that the majority of parents reported a weak impact of oral health on quality of life of their children before they perform dental treatment, and according to the parents, the same children had dental problems that required treatment 24. according to baldani et al. (2002)15, the assessment of socioeconomic conditions allows to consider possible etiologic factors of social inequalities such as income, educational attainment and housing conditions. in this sense, it was found that 66.38% of the families evaluated in this study were in the lower middle class, 21.83%, in middle class, 11.35% in upper low and 0.44% in the upper middle class. knowledge of these data allows a reorientation of health care and public spending on prevention and care activities, enabling a fair distribution of available resources, providing more resources to those groups with the greatest needs. epidemiological studies have been conducted to evaluate the relationship between oral health and socioeconomic conditions and have been observed that low socioeconomic status is related to higher prevalence of dental caries. the reason for the association between oral health and socioeconomic status is reasoned on the fact that socioeconomic status determines access to resources that determine the distribution of oral health, as well as, behavioral factors and consumption of sugar among them: toothbrushing, preventive activities and regular dental visits25. socioeconomic data were collected from parents in order to allow inequalities in oral health-related quality of life to be examined. an inverse and significant relationship was found between dmft and socioeconomic conditions (p=0.003) as well as the education level (p=0.046) and housing (p=0.010). likewise meneghim et al. (2007)26 showed that income, education level, housing conditions and socioeconomic status have a significant relationship with higher prevalence of dental caries27. similarly, a study conducted in piracicaba, sp, brazil, in 2009 found an inverse association between dental caries with income and with education level of the father and the mother. the present study found inverse relation between oral health-related quality of life and socioeconomic conditions where children from low socioeconomic conditions demonstrated higher influence of oral health conditions on quality of life. these results indicate that people living in low socioeconomic conditions have worst oral health conditions due to exposure to risk factors interfering with their quality of life. similarly, a study conducted with brazilian schoolchildren found that higher impacts on cohrqol were observed for children presenting with untreated dental caries. socioeconomic factors were also associated with cohrqol, as poorer scores were reported by children whose mothers had not completed primary education (rr 1.31; 95% ci 1.17-1.46) and those with lower household income (rr 1.17; 95% ci 1.05-1.31).2 likewise, a study developed in canada with schoolchildren demonstrated that in children from higher income backgrounds, mean cpq 11-14 scores were low, close to the minimum score of 10, irrespective of the presence or severity of oral diseases and disorders. for children from lower income backgrounds, those free of oral diseases and disorders also had relatively low scores. however, scores increased significantly in the presence of oral disease. this suggests that oral health problems have less perceived impact on high income children, but a more marked impact on dental caries-related quality of life and socioeconomic status of preschool children, bauru, sp braz j oral sci. 11(4):463-468 children from low income environments3. the questionnaires to evaluate oral health-related quality of life of preschool children can be a valuable instrument to demonstrate the perception of parents about the oral health of their children and to guide the oral health attention of this population group. the present study identified low impact of oral health on quality of life of preschool children examined from the perspective of parents and verified socioeconomic inequalities associated with oral health related quality of life of the children. the results indicate the need of planning educational activities with parents about the importance of taking care of the primary teeth as well as the low capacity of the health system to treat people of this age group. the assessment of perceived needs by the use of quality of life questionnaires as well as socioeconomic parameters can assist the planning of oral health programs aiming the reduction of unnecessary and unavoidable inequalities in the distribution of dental caries in populations of different socioeconomic conditions. acknowledgements the authors thank the children of public kindergartens of the city of bauru for their support in developing this study. references 1. ferraz mjpc, queluz dpq, alves mc, santos ccg, matsui my. caries experience associated to social and preventive factors in children of a pastoral community from limeira-sp. braz j oral sci. 2011; 10: 152-7. 2. piovesan cjlf, guedes rs, ardenghi tm. impact of socioeconomic and clinical factors on child oral health-related quality of life (cohrqol). qual llfe res. 2010; 19: 1359-66. 3. locker d. disparities in oral health-related quality of life in a population of canadian children. community dent oral epidemiol. 2007; 35: 348-56. 4. piovesan c, batista a, ferreira fv, ardenghi tm. oral health-related quality of life in children: conceptual issues. rev odont cienc. 2009; 24: 81-5. 5. koposova n, widström e, eisemann m, koposov r, eriksen hm. oral health and quality of life in norwegian and russian school children: a pilot study. stomatologija, bdm j. 2010; 12: 10-6. 6. jokovic a, et al. validity and reliability of a questionnaire for measuring child oral-health-related qual life j dent res. 2002; 81: 459-63. 7. pahel bt, rozier rg, slade gd. paternal perceptions of children’s oral health: the early childhood oral helath impact scale (ecohis). health qual life outcomes. 2007; 5(6). [cited 2010 sept 10] available from: www.hqlo.com/content/5/1/6. 8. municipal department of education. the municipal government of bauru. [cited 2012 aug 20] available from: http://www.bauru.sp.gov.br/ secretarias/sec_educacao/infantil.aspx. 9. word health organization. oral health surveys, basics methods. 4th ed. geneve: who; 1997. 10. tesch fc, oliveria bh, leão a. semantic equivalence of the brazilian version of the early childhood oral health impact scale. cad saude publica. 2008; 24: 1897-909. 11. graciano mig. evaluation criteria for socioeconomic classification. serv social soc. 1980; 1: 81-103. 12. nishi m, stjernswärd j, carlsson p, bratthall d. caries experience of some countries and areas expressed by the significance caries index. community dent oral epidemiol. 2002; 30: 296-301. 13. smith ge. tooth decay in the developing world. n z med j. 1987; 100: 669-70. 14. armfield jm, spencer aj, slade gd. changing inequalities in the distribution of caries associated with improving child oral health in australia. j. public health dent. 2009; 69: 125-34. 15. bastos rs, silva rp, maia-junior af, carvalho fs, merlini s, caldana ml et al. dental caries profile in monte negro, amazonian state of rondônia, brazil, in 2008. j appl oral sci. 2010; 18: 437-41. 16. hobdell m, petersen pe, clarkson j, johnson n. global goals for oral health 2020. int dent j. 2003; 53: 285-8. 17. narvai pc, frazão p, roncalli ag, antunes jlf. dental caries in brazil: decline, polarization, inequality and social exclusion. pan am j public health. 2006; 19: 385-93. 18. sales peres shc, carvalho fs, carvalho cp, bastos jrm, lauris jrp. polarization of dental caries in teen-agers in the southwest of the state of são paulo, brazil. cien saude colet. 2008; 13(sup 2): 2155-62. 19. baldani mh, narvai pc, antunes jlf. dental caries and socioeconomic conditions in the state of paraná, brazil, 1996. cad saude publica. 2002; 18; 755-63. 20. barbosa ts, gavião mbd. oral health-related quality of life in children: part ii. effects of clinical oral health status. a systematic review. intern j dent hyg. 2008; 6: 100-7. 21. peres kg, peres ma, araujo clp, menezes amb, hallal pc. social and dental status along the life course and oral health impacts in adolescents: a population based birth cohort. health qual life outcomes. 2009; 7: 95. 22. abanto j, et al. impact of oral diseases and disorders on oral health-related quality of life of preschool children. community dent oral epidemiol. 2011; 39: 105-14. 23. martins-júnior pa, ramos jorge j, paiva sm, marques ls, ramosjorge ml. validations of the brazilian version of the early childhood oral health impact scale (ecohis). cad saude publica. 2012; 28: 367-74. 24. li s, malkinson s, veronneau j, allison pj. testing responsivenss to change for early childhood oral health impact scale (ecohis). community dent oral epidemiol. 2008; 36: 542-8. 25. marmot m. social determinants of health inequalities. the lancet. 2005; 365; 1099-104. 26. cortellazzi kl et al. influence of socioeconomic, clinical and demographic variables on caries experience of preschool children in piracicaba, sp. rev bras epidemiol. 2009; 12: 490-500. 27. meneghim mc, kozlowski fc, pereira ac, ambrosano gmb, meneghim zmap. a socioeconomic classification and the discussion related to prevalence of dental caries and dental fluorosis. cien saude colet. 2007; 12: 523-9. 468468468468468 dental caries-related quality of life and socioeconomic status of preschool children, bauru, sp oral sciences n3 original article braz j oral sci. october|december 2010 volume 9, number 4 clinical assessment of the efficacy of low-level laser therapy on muscle pain in women with temporomandibular dysfunction, by surface electromyography ewerson bertolini shinozaki1,2 , mateus bertolini fernandes dos santos 3, luiz kioaki okazaki 2 leonardo marchini 2,4,5, aldo brugnera junior1 1 dds, msd, phd, dental laser center, institute for research and development, universidade do vale do paraíba, são josé dos campos, sp, brazil. 2 dds, msd, phd, school of dentistry, university of vale do paraíba, são josé dos campos, sp, brazil. 3 dds, msd, department of prosthodontics and periodontics, piracicaba dental school, state university of campinas, piracicaba, sp, brazil. 4 dds, msd, phd, dentistry department, university of taubaté, taubaté, sp, brazil 5 dds, msd, phd, são josé dos campos school of dentistry, são paulo state university– unesp, são josé dos campos, sp, brazil correspondence to: mateus bertolini fernandes dos santos rua engº joão fonseca dos santos, 123/34, são josé dos campos 12243-620, sp, brazil phone +55-19-8830-9867 fax +55-12-39221555 e-mail: mateusbertolini@yahoo.com.br received for publication: march 17, 2010 accepted: september 10, 2010 abstract the use of low-level laser (lll) may be an useful tool to promote reduction of muscular pain caused by tmd. aim: this study evaluated the immediate efficacy of low-level laser therapy on women reporting pain and diagnosed with temporomandibular dysfunction (tmd). methods: diode laser (gaalas) at 790 nm wavelength (infrared spectrum) was applied as experimental treatment. irradiations of 1.5 j/cm² were made at 4 points of the temporomandibular joint (tmj) and of 3 j/cm² at 3 points in the temporal muscle. an electromyographic (emg) evaluation of the masseter and anterior temporal was done at the following intervals: before, immediately after, 5 min and 20 min after laser application. results: comparison of the electrical activity at the times of measurement revealed a statistically significant difference in masseter muscles before (p=0.025) and immediately after (p=0.013) lllt. conclusions: both masseter and temporal muscles showed a reduction in the measured emg activities at all times after lllt, and the temporal muscle showed higher emg activity than the masseter muscle at all the evaluation times. lllt caused significant immediate relaxation of the masseter muscles. keywords: temporomandibular dysfunction, low-level laser, electromyography. introduction general clinicians and even specialists in orthodontics, prosthetics and oral and maxillofacial surgery have been treating a growing number of patients complaining of pain in the region of the temporomandibular joint (tmj), which may be diagnosed as temporomandibular dysfunction (tmd). very often, these patients have already consulted doctors of other specialties in an attempt to solve painful symptoms in the muscles of the head, face, neck, and shoulders. orofacial braz j oral sci. 9(4):434-438 pain is particularly stressful because this area is powerfully represented in the cerebral cortex1. tmd is a term used to define the clinical joint and muscle disorders affecting the maxillofacial region. these dysfunctions are characterized firstly by pain, articular noise and irregular or limited mandibular functions. they are the main cause of nondental-related pain in the orofacial region2. the correct diagnosis is crucial in planning tmd treatment, since its etiology is multifactorial and the treatment of these dysfunctions covers a wide range of interdependent variables that are often interdisciplinary2-4. the treatment of tmd basically consists of occlusal corrections involving teeth drilling (occlusal adjustments), use of occlusal splints, physiotherapy, psychological advisory, orthodontic or prosthetic rehabilitation2,5-6. low-level laser therapy (lllt) acts as an adjuvant in the treatment of the dysfunction, promoting the reduction of muscular pain caused by muscle hyperactivity and/or mandibular displacement2. the markedly analgesic, anti-inflammatory and stimulating effects of lllt are characterized by increased production of â-endorphins and control of the production of prostaglandins7-8. the application of laser beams diminishes pain while simultaneously reducing muscle contraction. when a local effect is desired, laser is effective by stimulating microcirculation and local cell tropism or, when a general therapeutic effect is needed, by promoting pain relief for a variety of etiologies, including irradiation of trigger points in microfacial pain, acting on tissue repair, reduction of edema and hyperemia9-10. this work involved a clinical assessment of the immediate efficacy of low-level laser therapy on women displaying signs and symptoms of tmd, using surface electromyography. we hypothesized that lllt promotes muscle relaxation. material and methods subjects the sample consisted of thirteen 18 to 36-year-old women presenting spontaneous facial pain at rest, exacerbated during the function; limited mandibular opening (<45 mm) and alterations in the trajectory of movements evidenced by lateral deviations; moderate to severe sensitivity to manual palpation of the masseter (superficial) and temporal (anterior bundle) muscles; muscular weakness and fatigue. all patients answered a questionnaire regarding about tmd and only the subjects diagnosed with tmd were assigned to this study. the pain was also assessed by the same questionnaire, which was applied before and after lllt. during the experiment, each patient remained in the same room and was attended by the same operator. this study was evaluated and approved by the ethical committee of the university of vale do paraíba for the use of human subjects (protocol number l170/2004/cep). all subjects signed an informed consent form before participating in this study. electromyographic (emg) measurements emg measurements were taken with a surface electromyograph (model mp100, biopac system, bioresearch assoc. inc., milwaukee, usa) with 8-channel amplifier (model 800); this equipment present a common mode rejection ratio (cmmr) of 110 db (50/60 hz), low (500hz) and high (20hz) pass filters and 16 bits of resolution. surface silver/silver chloride electrodes of 10 mm diameter (duotrodes; myotronics co.) were used to measure the electrical activity of the right and left temporal (anterior bundle) muscles and the right and left masseter (superficial) muscles, it was used a low pass filter in order to retain signals in the range of 10 to 350 hz for surface electrodes11. the protocol proposed by macaluso and delaat in 1995 was followed for the electrodes location12. before their placement, the skin of the patients was cleaned with liquid soap suitable for skin cleansing and soon after with ethyl alcohol 70ºgl. the electrodes were placed on the muscles in the region of highest volume and parallels to the long axis with a distance of 21mm between them. a single monopolar electrode with a neutral point function (grounding cable) was also used in the neck region. the data were carefully recorded simultaneously and bilaterally. before starting the measurements the equipment was always calibrated with a trial test, which is necessary to verify the correct state of the electrodes and connections. the first recording was performed during 10-ms. the electrical activity of the four muscles simultaneously appears reflected. thus, we obtain a reference value of the state of the muscles at rest which can be achieved by the patient by one’s own means by simply responding when being asked to relax13. for this, we calibrated the screen reticule to a range of 30 ìv/div and a speed of 1 ms/div. each patient underwent emg tests at resting position of the superficial masseter and anterior temporal muscles. the record of the emg activity at rest was measured for a period of 10-ms with five-fold amplification. after recording the emg activity of the right and left masseter (superficial) and temporal (anterior bundle) muscles, the electrodes were removed and their exact location marked so that they could be put back on exactly the same spot after lllt. immediately after application of the laser (as described below), the electrodes that had been removed were placed back on the marked locations and the entire above described procedure was repeated to record the electrical activity of the aforementioned muscles after the laser therapy, thus obtaining the second emg record. the third and fourth records were obtained after 5 and 20 min, respectively. the patient was allowed to rest, seated comfortably but keeping the electrodes in place, and the muscles were then monitored following the above described protocol. all data of the electrical activity of the muscles in rest, prior to the low-level laser therapy, immediately after, 5 and 20 min after the therapy were recorded automatically by the 435435435435435 clinical assessment of the efficacy of low-level laser therapy on muscle pain in women with temporomandibular dysfunction, by surface electromyography braz j oral sci. 9(4):434-438 computer. the emg tests were performed on the side that the patient reported increased pain on muscles, the other side of the patients were used as a placebo treatment. lllt the lllt was applied using a bio-wave device (kondortech) with 40 mw power, gallium aluminum arsenide (gaalas) diode laser, 790 nm wavelength, beam delivery system by direct contact with the skin, and a beam area of 0.2 cm2. the low-level laser irradiations were applied pointwise on standardized tmj points: head, anterior, posterior, superior and inferior portions of the mandible, each of which received an application of 1.5 j/cm2, and posterior to the tmj at the height of the outer ear, the region through which the auriculotemporal nerve and bilaminar zone pass, where 2.5 j/cm2 was applied. the masseter muscle (superficial and anterior bundle) received 3 j/cm2 of laser energy pointwise on three predetermined points of the muscle, superior, median and inferior in case of superficial and anterior, and median and posterior in the anterior bundle. data analysis the data, average of rectified signals, were processed with the software biopak for windows, and then examined by one-way anova to check possible correlations between emg results and the lllt. whenever anova indicated a difference, scheffe’s multiple-comparison method was used to identify the existing correlations. student’s paired t-test was also applied to analyze each muscle. all the tests were performed at a p<0.05 level of significance. results emg tracings obtained acquisition values before and five minutes after the lllt is presented in figures 1 and 2. the mean and standard deviation of the results obtained by electromyography at the abovementioned times are shown in table 1. a statistical difference was found among the times in masseter muscle (p=0.025). therefore, scheffe’s multiple-comparison method was applied, indicating a 0.025*0.236p-value 0.540.250.350.390.861.040.801.05 standard deviation 1.581.461.381.681.952.091.952.39average after 20 minafter 5 min imme d. after beforeafter 20 minafter 5 min immed. after before masseter muscletemporal musclelaser table 1 – average of electrical (ìv/ms) activity of the masseter and temporal muscles subjected to laser therapy, measured before, immediately after, 5 minutes and 20 min after lllt. the asterisk (*) indicates statistically significant difference (anova). fig. 2. emg tracings obtained acquisition values before the application of the lllt for right and left temporal muscles (ta-r and ta-l, respectively) and for right and left masseter muscles (mm-r and mm-l, respectively) 5 min after lllt. fig. 1. emg tracings obtained acquisition values before the application of the lllt for right and left temporal muscles (ta-r and ta-l, respectively) and for right and left masseter muscles (mm-r and mm-l, respectively) before lllt. statistically significant difference between the emg activity before and immediately after lllt application (p=0.013). 436436436436436clinical assessment of the efficacy of low-level laser therapy on muscle pain in women with temporomandibular dysfunction, by surface electromyography braz j oral sci. 9(4):434-438 discussion emg as well as clinical examinations and application of questionnaires are efficient methods for evaluating and diagnosing tmd3,14. the severity of pain cannot be reflected in emg activity, but since limited mandibular movement heightens muscle tonus activity, tmd patients present a slight increase in basal muscle tonus which is detected electromyographically, confirming that emg is an effective tool for evaluation and diagnosis in the treatment of these dysfunctions3,7. in a comparison of normal and tmd patients, gervais et al.14 demonstrated that individuals presenting tmd have increased emg activity in the painful muscles and, hence, a shortening of the fibers of these muscles. the results of the present study demonstrated the effectiveness of lllt in promoting relaxation of the masseter muscles, resulting in the relief of pain produced by muscle contraction in women suffering from tmd. nunez et al.8 also found an improvement in the mandibular motion of patients with tmd immediately after lllt. however, we should consider some of the limitations of the present study, such as the limited sample size. other important limitations for clinical application are the tmd diagnostic criteria, instrumentation, electrodes and equipment used in this study, and the fact of only women were evaluated. according to simunovic et al.15, the muscle relaxation and the analgesic effects of lllt are due to factors such as the increase of endorphin production and elevation of pain threshold through an electrolytic blocking mechanism of the nerve fibers. on the other hand, bertolucci and gray16-17 believe that these effects are due to increased cell metabolism accompanied by a gain in energy due to the transformation of adp into atp in the cells exposed to the laser beam, reducing the painful symptoms. we agree with the latter authors insofar as it concerns the mechanism of laser action in producing muscle relaxation and analgesia. lllt can help removing metabolites and increase blood flow to muscles in painful conditions through local vasodilatation18-19. hunter et al.20 stated that there is a cell response to the application of lllt through increased intracellular calcium. lllt promoted immediate relaxation of the masseter muscles in a single session. conti21, in turn, stated that laser therapy increased mandibular mobility, reducing and stabilizing pain after the third day of application. the rapid reduction of intra-articular and muscle inflammation achieved with infrared laser contributes toward stabilization of the occlusion and to symmetry of the muscle functions, which influence reparative processes22. similarly, bertolucci and grey16-17 reported significant improvement in articular noise, in limitation of oral opening, and in the masticatory function through reduction of muscle contraction and of intra-articular inflammation by lllt4. conti21, on the other hand, reported relief of painful symptoms only in cases of myogenic dysfunction, while patients with arthrogenic pain showed improvement only of their mandibular functions. kulekcioglu et al.23 stated that the effects of the therapy on cases of myogenic and arthrogenic dysfunction were similar. in view of the noninvasive action of lllt, it can be considered a useful tool in the treatment of tmd, although further studies are required to define reliable clinical parameters21. the correct diagnosis and an appropriate application protocol are key factors to enable lllt to produce positive effects24. this study also proposes the use of an gaalas laser emitted at a wavelength of 790 nm and power of 40 mw. the sessions should vary according to the severity of the pathology and the painful symptoms. the laser beam should be applied to the tmj at four points around the joint, using an ed of 1.5 j/cm2 on each point, and at a fifth point in the region of the outer ear, through which the auriculotemporal nerve passes, with an ed of 2.5 j/cm2. muscles with painful symptoms should be treated with the laser beam applied pointwise with an ed of 3 j/cm2 at three predetermined points on the masseter and temporal muscles. within the limitations of this study, it is possible to conclude that women with tmd subjected to laser therapy present a reduction of the emg activity in both the masseter and temporal muscles. the proposed lllt promoted significant immediate relaxation of the masseter muscles, measured electromyographically. references 1. bradley p, groth e, gursoy b. lasers in medicine and dentistry: basic science and up-to-date clinical applications of low-energy-level laser therapy (lllt). croatia: vitagraf; 2000. p.385-402. 2. okeson jp. the clinical management of temporomandibular disorders and occlusion. 6th eed. saint louis: mosby; 2008. 3. pinho jc, caldas fm, mora mj, santana-penin u. electromyographic activity in patients with temporomandibular disorders. j oral rehabil. 2000; 27: 985-90. 4. gray rj, quayle aa, hall ca, schofield ma. physiotherapy in the treatment of temporomandibular joint disorders: a comparative study of four treatment methods. br dent j. 1994; 176: 257-61. 5. fikackova h, dostalova t, navratil l, klaschka j. effectiveness of lowlevel laser therapy in temporomandibular joint disorders: a placebo-controlled study. photomed laser surg. 2007; 25: 297-303. 6. wexler gb, steed pa. psychological factors and temporomandibular outcomes. cranio. 1998; 16: 72-7. 7. cetiner s, kahraman sa, yucetas s. evaluation of low-level laser therapy in the treatment of temporomandibular disorders. photomed laser surg. 2006; 24: 637-41. 8. nunez sc, garcez as, suzuki ss, ribeiro ms. management of mouth opening in patients with temporomandibular disorders through low-level laser therapy and transcutaneous electrical neural stimulation. photomed laser surg. 2006; 24: 45-9. 9. schindl a, schindl m, pernerstorfer-schon h, schindl l. low-intensity laser therapy: a review. j investig med. 2000; 48: 312-26. 10. schawlow al. principles of lasers. j clin laser med surg. 1995; 13: 127-30. 11. soderberg gl, knutson lm. a guide for use and interpretation of kinesiologic electromyographic data. phys ther. 2000; 80: 485-98. 12. macaluso gm, de laat a. the influence of the position of surface recording electrodes on the relative uptake of the masseteric and temporal m-responses in man. eur j oral sci. 1995; 103: 345-50. 437437437437437 clinical assessment of the efficacy of low-level laser therapy on muscle pain in women with temporomandibular dysfunction, by surface electromyography braz j oral sci. 9(4):434-438 13. ardizone i, celemin a, aneiros f, del rio j, sanchez t, moreno i. electromyographic study of activity of the masseter and anterior temporalis muscles in patients with temporomandibular joint (tmj) dysfuction: comparison with the clinical dysfunction index. med oral patol oral cir bucal. 2010; 15: 14-9. 14. gervais ro, fitzsimmons gw, thomas nr. masseter and temporalis electromyographic activity in asymptomatic, subclinical, and temporomandibular joint dysfunction patients. cranio. 1989; 7: 52-7. 15. simunovic z, trobonjaca t, trobonjaca z. treatment of medial and lateral epicondylitis—tennis and golfer’s elbow—with low level laser therapy: a multicenter double blind, placebo-controlled clinical study on 324 patients. j clin laser med surg. 1998; 16: 145-51. 16. bertolucci le, grey t. clinical comparative study of microcurrent electrical stimulation to mid-laser and placebo treatment in degenerative joint disease of the temporomandibular joint. cranio. 1995; 13: 116-20. 17. bertolucci le, grey t. clinical analysis of mid-laser versus placebo treatment of arthralgic tmj degenerative joints. cranio. 1995; 13: 26-9. 18. basford jr. low intensity laser therapy: still not an established clinical tool. lasers surg med. 1995; 16: 331-42. 19. tullberg m, alstergren pj, ernberg mm. effects of low-power laser exposure on masseter muscle pain and microcirculation. pain. 2003; 105: 89-96. 20. coombe ar, ho ct, darendeliler ma, hunter n, philips jr, chapple cc, et al. the effects of low level laser irradiation on osteoblastic cells. clin orthod res. 2001; 4: 3-14. 21. conti pc. low level laser therapy in the treatment of temporomandibular disorders (tmd): a double-blind pilot study. cranio. 1997; 15: 144-9. 22. hansson tl. infrared laser in the treatment of craniomandibular disorders, arthrogenous pain. j prosthet dent. 1989; 61: 614-7. 23. kulekcioglu s, sivrioglu k, ozcan o, parlak m. effectiveness of lowlevel laser therapy in temporomandibular disorder. scand j rheumatol. 2003; 32: 114-8. 24. pinheiro al, cavalcanti et, pinheiro ti, alves mj, miranda er, de quevedo as, et al. low-level laser therapy is an important tool to treat disorders of the maxillofacial region. j clin laser med surg. 1998; 16: 223-6. 438438438438438clinical assessment of the efficacy of low-level laser therapy on muscle pain in women with temporomandibular dysfunction, by surface electromyography braz j oral sci. 9(4):434-438 oral sciences n3 original article braz j oral sci. january | march 2011 volume 10, number 1 effect of chlorhexidine gel containing saccharin or aspartame in deaf children highly infected with mutans streptococci frederico silva de freitas fernandes1, juliana de kássia braga fernandes2, sirlei garcia marques3, rubenice amaral da silva4 1dds, msc, phd, department of prosthodontics and periodontology, piracicaba dental school, state university of campinas, sp, brazil 2dds, msc, department of oral physiology, piracicaba dental school, state university of campinas, sp, brazil 3dds, msc, phd, university hospital, federal university of maranhão, ufma, são luís, ma, brazil 4dds, msc, department of dentistry ii, faculty of dentistry, federal university of maranhão, ufma, são luís, ma, brazil correspondence to: frederico silva de freitas fernandes rua cel. barbosa n.333. apto. 51 13416-381 piracicaba, sp (brazil) phone: +55 19 3302-8624 e-mail: fredfernandes@fop.unicamp.br received for publication: may 12, 2010 accepted: december 20, 2011 abstract aim: since deaf children are unable to comprehend or cooperate with dental treatment due to lack of communication, preventive measures may be an important way to control the high prevalence of dental caries in these patients. the aim of the this study was to evaluate the effect of an intensive treatment with chlorhexidine (chx) gel, containing either saccharin or aspartame, in deaf children highly infected with mutans streptococci (ms). methods: eighteen children were randomly divided into two groups, according to the sweetener used to improve the chx gel bitter taste: saccharin or aspartame. before chx treatment, saliva samples were collected to establish baseline microbial data for ms. chx gel was applied on two consecutive days, four times the first day and three times the second day. saliva samples were then taken after 7, 30, 60, 90 and 120 days to evaluate ms oral recolonization. results: chx gel containing saccharin was not effective on the reduction of ms levels, while the gel containing aspartame decreased significantly ms levels after treatment (p<.05). conclusions: although a new chx application may be necessary after 60 days to control caries risk and ms levels, chx treatment should be individually controlled because of variations in the response of subjects. keywords: chlorhexidine gel, saccharin, aspartame, mutans streptococci, saliva, deaf children introduction dental caries results from the interaction of specific bacteria with constituents of the diet within a biofilm termed ‘dental plaque’1. among the cariogenic bacteria, mutans streptococci (ms) are most closely associated with the development of caries disease2-3 and, therefore, salivary ms levels have been used as an indicator of caries risk4-5. therefore, one way of controlling cariogenic activity would be to reduce the ms oral levels by the use of antimicrobial agents6-7. among the antimicrobial agents available for dental use, chlorhexidine (chx) is the most effective and most widely documented substance in controlling cariogenic activity6,8-10. chx reduces the proportions of some microorganisms, especially ms, which are particularly sensitive to this substance4,11-13. the major braz j oral sci. 10(1):7-11 8 advantage of chx over most other compounds lies on its oral substantivity14, as it is a cationic substance that adsorbs to soft and hard tissues of the mouth15 as well as to bacterial cell walls16. because chx has an extremely bitter taste, it is often necessary to flavor and sweeten gel products. however, when formulations are prepared the availability of chx can be impaired. regarding this, there are few studies17 evaluating the effect of sweeteners commonly used to improve the gel taste on the antimicrobial activity of chx. studies with highly ms-infected children have demonstrated that an accentuated reduction of ms levels occurs after an intensive treatment with chx gel13,18-19, with ms levels returning gradually to those verified initially within 2 to 6 months after treatment6,20-21. however, there is no report on the effect of this antimicrobial agent in subjects with disabilities, such as deaf children. for special needs patients, the use of chx gel may be an important method to control the high prevalence of caries disease and poor oral hygiene22-23, since dental care is often neglected in those children because of poor cooperation with dental treatment due to lack of communication24. thus, the aims of this study were: (a) to compare the effect of intensive treatment with chx gel, containing saccharin or aspartame, in decreasing the ms levels in deaf children and (b) to evaluate the reappearance pattern of ms over time after the chx applications in those subjects. material and methods experimental design this prospective study involved a randomized, doubleblinded (regarding patients and examiners) design approved by the research ethics committee of the university hospital of the federal university of maranhão (protocol n.º 3310400320/2002). the parents or the children’s legal tutors read and signed an informed consent form, in which all procedures, possible discomforts or risks, and benefits were fully explained. eighteen patients, aged 5-10 years, who fulfilled the inclusion criteria were selected from a special school for deaf children, located at the city of são luis, in the northeast of brazil. inclusion criteria involved healthy deaf children of both genders, highly infected with ms (1.0 x 106 cfu ml-1 of saliva)4-5. exclusion criteria excluded those using any type of antimicrobials or even chemical products for mouthrinsing or topical use, during a minimum interval of 6 months before the beginning of the experiment. all children received restorative dental care at the public dental health service prior to the start of the experiment. children were randomly divided into two groups, according to the sweetener used to improve the chx gel bitter taste: saccharin (n=9) or aspartame (n=9). the number of subjects in each test group was determined by comprehensive preliminary tests that demonstrated that the sample size yielded an adequate power (0.80) to detect statistically significant differences. the treatment with 1% chx gel was performed according to the protocol established by maltz et al. (1981)18. before chx treatment, saliva samples were collected to establish baseline microbial data for ms. after the application of chx, saliva samples were taken in the following periods: 7, 30, 60, 90 and 120 days to evaluate ms oral recolonization. microbiological analysis of the saliva was made using a dip slide test for ms. saliva sample collection children underwent the saliva sample collection between 9.30 and 11.00 a.m., at least 2 h after breakfast and before the mid-morning snack. then, a paraffin-stimulated saliva sample of approximately 2.0 ml was collected. the saliva produced in the first 30 s was discarded, beginning the collection after this time. the saliva was picked up in sterile test tubes, containing 10 glass pearls. the tube was conserved in ice25. microbiological analysis of the saliva saliva samples were microbiologically analyzed using a dip slide test for ms, caritest-sm (herpo; dental products ltda, rio de janeiro, rj, brazil), which is similar to the test proposed by jordan et al. (1987) 26, marketed commercially as cariescreen sm (automated diagnostic documentation, grand haven, mi, usa). the test tubes containing the saliva samples were homogenized individually in a vortex mixer, during 1 min. soon after, using an automatic micropipette with sterile plastic tips, 1.5 ml was poured into the tube with buffer solution and bacitracin previously added. this tube was slightly agitated and the dip slide was immersed in it. meanwhile, a co 2 generating tablet was placed into the empty tube with 2 drops of distilled water. the dip slide was immediately removed from the solution and immersed in the tube with co 2 , which was sealed. all tubes containing the dip slides were incubated at 37° c for 48 h and remained at controlled room temperature at 23° c for 24 h. a single examiner underwent a single pre-study calibration session to be familiar with the codes for converting the appearance of the incubated dip slide to an approximation of the number of colony-forming units per ml of saliva (cfu ml-1 of saliva). the colony density chart was supplied by the manufacturer, which showed 6 cfu scores: 10,000; 50,000; 100,000; 250,000; 500,000 and 1,000,000 cfu ml1 of saliva. in terms of bacterial count, children with an ms level e” 100,000 cfu ml-1 of saliva were classified as high caries risk4-5. application of chx the 1% chx gel (facial pharmacy, são luís, ma, brazil) was prepared containing saccharin or aspartame as sweetener to improve the chx gel bitter taste. prior to the chx application, children received reinforcement of basic oral hygiene, and the parents or the children’s their legal tutors were instructed to restrict the children’s consumption of sucrose. after professional prophylaxis, a disposable tray was used for chx application, in accordance with the protocol established by maltz et al. (1981)18 – first day with four 5min applications and 5-min intervals between each effect of chlorhexidine gel containing saccharin or aspartame in deaf children highly infected with mutans streptococci braz j oral sci. 10(1):7-11 9 application; and second day (24 h after) with three 5-min applications and 5-min intervals between each application. during these intervals, the children mouthrinsed with water, and the trays were prepared for the next application. statistical analysis the non-parametric wilcoxon test was applied to the intragroup data. statistical analysis was done using bioestat software (version 3.0, 2003; brazil) with the significance level set at 5%. results the results obtained after the application of the chx gel containing saccharin can be observed in table 1. the chx gel was not effective on the reduction of ms levels in saliva, as no statistically significant difference (wilcoxon test, p>.05) was observed between the data presented after and before chx application (1.0 x 106 cfu ml-1 of saliva). it was verified that the ms levels remained elevated during the whole analysis period after chx application. the results obtained after the application of the chx gel containing aspartame are shown in table 2. chx was effective on reducing ms in saliva, with a statistically significant difference between the baseline values (1.0 x 106 cfu ml-1 of saliva) and values observed in all sample collection periods after the chx application (wilcoxon test, p<.05). considering that children with an ms level e” 1.0 x 105 cfu ml-1 of saliva are classified as high caries risk4-5, our study compared these ms levels with those observed after the application of chx gel containing aspartame (table 2); after 60 days, there was no statistically significant difference was found between the ms levels in this period and the high caries risk ms level. *statistically significant difference compared to baseline (p<.05). † no statistically significant difference compared to the high caries risk ms level (1.0 x 105 cfu ml-1 of saliva) (p.05). table 2: means and standard deviations of salivary ms levels (cfu ml-1 of saliva) before (baseline) and 7, 30, 60, 90 and 120 days after application of the chx gel containing aspartame (n = 9 subjects). periods of collection of saliva samples baseline after 7 days after 30 days after 60 days after 90 days after 120 days mean 1.0 x 106 6.7 x 103*† 2.6 x 104*† 8.6 x 104* 1.6 x 105* 4.2 x 105* s.d. 0 1.7 x 104 3.4 x 104 9.8 x 104 1.5 x 105 3.6 x 105 discussion although epidemiological studies have reported higher caries prevalence and poorer oral hygiene in deaf subjects than in their normal counterparts22-23, little attention has been paid to caries preventive measures for those patients. this is why the present evaluated the effect of chx in special care children highly infected with ms, classified as high caries risk patients4-5. our study have shown that chx gel containing aspartame as sweetener was effective in reducing ms in saliva, being thus important to control the cariogenic activity in deaf children due to their cooperative behavior. however, it is important to highlight that other established evidence-based prevention methods should not be excluded for those children, such as diet modifications and good oral hygiene practices, since dental caries is a disease with a multifactorial etiology. chx gel containing saccharin as sweetener was not effective on the reduction of ms in saliva. similar results were found by rocha et al. (2003)25 after application of the chx gel containing saccharin in volunteers using removable dentures. these findings may be explained by the results obtained in vitro by cury et al. (2000)17, who demonstrated that the antibacterial activity of chx may be reduced depending on the concentration of saccharin used to make its taste acceptable. for chx gel formulations, 1% saccharin is commonly added to make the gel acceptable, especially for children. however, cury et al. (2000)17 observed that at concentrations above 0.5% saccharin significantly reduced the anti-mutans activity of 1% chx gel. therefore, when saccharin is used, the detrimental effect of concentration on chx activity should be considered or another non-complex sweetener should be used, such as aspartame. the aspartame, added to improve the taste of the chx gel, became it acceptable by the deaf children, without inhibiting the antibacterial activity of 1% chx gel, since there was a reduction in the children salivary ms after the gel application. although ms had been decreased to low or undetectable levels by the gel, as observed 7 days after chx application, the treatment was not able to completely eliminate these bacteria from the mouth. this suggests that there must be reservoirs or retention sites in the dentition hardly affected or not affected at all by chemotherapy, and from which the ms recolonize tooth surfaces after the antimicrobial pressure is removed6,20. in this study, chx treatment was followed by a gradual reappearance of the organisms, where saliva samples obtained after the gel application has shown that ms levels increased with time. periods of collection of saliva samples baseline 7 days after chx 30 days after chx application application mean 1.0 x 106 8.9 x 105 1.0 x 106 s.d. 0 2.2 x 105 0 no statistically significant difference compared to baseline (p>.05). table 1: means and standard deviations of salivary ms levels (cfu ml-1 of saliva) before (baseline) and 7 and 30 days after application of the chx gel containing saccharin (n = 9 subjects). effect of chlorhexidine gel containing saccharin or aspartame in deaf children highly infected with mutans streptococci braz j oral sci. 10(1):7-11 10 however, even 120 days after chx treatment the ms levels were significantly different from the baseline values. these results corroborate those of previous studies6,13,18,20-21 which observed a gradual ms re-appearance during the period of 180 days, after these bacteria had been suppressed to undetectable levels by the treatment with chx. although ms levels was significantly different from the baseline values during the whole evaluation period, a new chx treatment may be necessary 60 days after the application of the chx gel containing aspartame, since ms levels observed in this period did not differ significantly from caries risk ms level (e”1.0 x 105 cfu ml-1 of saliva)4-5. this short reapplication period reinforces the opinion that other studies are necessary to improve and develop techniques that promote more lasting reduction periods for deaf children, therefore enabling a better caries-preventive effect of the chx gel. similar results were recorded by previous studies with nonhandicapped subjects, which found that chx applications must be repeated within 60 to 90 days6. however, the findings of the present study should be interpreted with care, since there was a large intra-individual variation in the time of ms recolonization among the children. therefore, the effect of chx treatment must be monitored, given sharp individual variability in response to this treatment. this large individual variation in response to treatment may be due several reasons, and explain the high standard deviation values found in the present study. emilson et al. (1987)21 and wallman (1998)27 verified that highly ms-infected dental faces before the chx therapy were more quickly recolonized, even when ms was suppressed to undetectable levels. this is in agreement with the findings of studies showing that teeth of individuals with several fillings were more rapidly recolonized with ms after chx than those of subjects with few fillings18,28-29. barkvoll et al. (1989)30 verified that polysaccharides synthesized from sucrose interfere with the chx effect, inhibiting its antibacterial activity, suggesting restriction of sucrose consumption during treatment with chx, for more longstanding effects. within the limitations of this study, it can be concluded that chx gel containing saccharin was not effective in reduction ms levels, while the gel containing aspartame decreased significantly the ms levels in deaf children during the whole evaluation period. although a new chx application may be necessary after 60 days to control caries risk ms level, chx treatment should be individually controlled because of variations in response of the subjects. acknowledgements the authors thank pibic/cnpq for the scholarships granted for the first author. references 1. bowen wh. do we need to be concerned about dental caries in the coming millennium? crit rev oral biol med. 2002; 13: 126-31. 2. marsh pd. are dental diseases examples of ecological catastrophes? microbiology. 2003; 149: 279-94. 3. van houte j. role of micro-organisms in caries etiology. j dent res. 1994; 73: 672-81. 4. petti s, hausen h. caries-preventive effect of chlorhexidine gel applications among high-risk children. caries res. 2006; 40: 514-21. 5. kopycka-kedzierawski dt, billings rj. a longitudinal study of caries onset in initially caries-free children and baseline salivary mutans streptococci levels: a kaplan-meier survival analysis. community dent oral epidemiol. 2004; 32: 201-9. 6. emilson cg. potential efficacy of chlorhexidine against mutans streptococci and human dental caries. j dent res. 1994; 73: 682-91. 7. twetman s. antimicrobials in future caries control? a review with special reference to chlorhexidine treatment. caries res. 2004; 38: 223-9. 8. joharji rm, adenubi jo. prevention of pit and fissure caries using an antimicrobial varnish: 9 month clinical evaluation. j dent. 2001; 29: 247-54. 9. araujo am, naspitz gm, chelotti a, cai s. effect of cervitec on mutans streptococci in plaque and on caries formation on occlusal fissures of erupting permanent molars. caries res. 2002; 36: 373-6. 10. baca p, munoz mj, bravo m, junco p, baca ap. effectiveness of chlorhexidine-thymol varnish in preventing caries lesions in primary molars. j dent child. 2004; 71: 61-5. 11. emilson cg. susceptibility of various microorganisms to chlorhexidine. scand j dent res. 1977; 85: 255-65. 12. koo h, pearson sk, scott-anne k, abranches j, cury ja, rosalen pl et al. effects of apigenin and tt-farnesol on glucosyltransferase activity, biofilm viability and caries development in rats. oral microbiol immunol. 2002; 17: 337-43. 13. ribeiro lg, hashizume ln, maltz m. the effect of different formulations of chlorhexidine in reducing levels of mutans streptococci in the oral cavity: a systematic review of the literature. j dent. 2007; 35: 359-70. 14. adams d, addy m. mouthrinses. adv dent res. 1994; 8: 291-301. 15. rolla g, melsen b. on the mechanism of the plaque inhibition by chlorhexidine. j dent res. 1975; 54(spec n.b): b57-62. 16. jones cg. chlorhexidine: is it still the gold standard? periodontol 2000. 1997; 15: 55-62. 17. cury ja, rocha ep, koo h, francisco sb, del bel cury aa. effect of saccharin on antibacterial activity of chlorhexidine gel. braz dent j. 2000; 11: 29-34. 18. maltz m, zickert i, krasse b. effect of intensive treatment with chlorhexidine on number of streptococcus mutans in saliva. scand j dent res. 1981; 89: 445-9. 19. zickert i, emilson cg, ekblom k, krasse b. prolonged oral reduction of streptococcus mutans in humans after chlorhexidine disinfection followed by fluoride treatment. scand j dent res. 1987; 95: 315-9. 20. autio-gold j. the role of chlorhexidine in caries prevention. oper dent. 2008; 33: 710-6. 21. emilson cg, lindquist b, wennerholm k. recolonization of human tooth surfaces by streptococcus mutans after suppression by chlorhexidine treatment. j dent res. 1987; 66: 1503-8. 22. al-qahtani z, wyne ah. caries experience and oral hygiene status of blind, deaf and mentally retarded female children in riyadh, saudi arabia. odontostomatol trop. 2004; 27: 37-40. 23. jain m, mathur a, kumar s, dagli rj, duraiswamy p, kulkarni s. dentition status and treatment needs among children with impaired hearing attending a special school for the deaf and mute in udaipur, india. j oral sci. 2008; 50: 161-5. 24. hennequin m, faulks d, roux d. accuracy of estimation of dental treatment need in special care patients. j dent. 2000; 28: 131-6. 25. rocha ep, francisco sb, del bel cury aa, cury ja. longitudinal study of the influence of removable partial denture and chemical control on the levels of streptococcus mutans in saliva. j oral rehabil. 2003; 30: 131-8. 26. jordan hv, laraway r, snirch r, marmel m. a simplified diagnostic system for cultural detection and enumeration of streptococcus mutans. j dent res. 1987; 66: 57-61. effect of chlorhexidine gel containing saccharin or aspartame in deaf children highly infected with mutans streptococci braz j oral sci. 10(1):7-11 11 27. wallman c, krasse b, birkhed d, diacono s. the effect of monitored chlorhexidine gel treatment on mutans streptococci in margins of restorations. j dent. 1998; 26: 25-30. 28. sandham hj, brown j, phillips hi, chan kh. a preliminary report of long-term elimination of detectable mutans streptococci in man. j dent res. 1988; 67: 9-14. 29. sandham hj, brown j, chan kh, phillips hi, burgess rc, stokl aj. clinical trial in adults of an antimicrobial varnish for reducing mutans streptococci. j dent res. 1991; 70: 1401-8. 30. barkvoll p, rolla g, svendsen k. interaction between chlorhexidine digluconate and sodium lauryl sulfate in vivo. j clin periodontol. 1989; 16: 593-5. effect of chlorhexidine gel containing saccharin or aspartame in deaf children highly infected with mutans streptococci braz j oral sci. 10(1):7-11 oral sciences n3 received for publication: may 07, 2010 accepted: december 05, 2010 original article braz j oral sci. october|december 2010 volume 9, number 4 chronological table of third molar mineralization in a survey in the state of alagoas, brazil ana paula cavalcante carneiro1, joão alfredo tenório lins guimarães2, roseli mayumi ikeda e silva3, adriana paula costa e silva santiago4, josé rodrigues laureano filho5 1dds, undergraduate student, dental school, university of pernambuco, recife, pe, brazil 2dds, ms, assistant professor, forensic dentistry, state university of alagoas, maceió, al, brazil 3dds, institute of forensic medicine, maceió, al, brazil 4dds, ms, phd, professor, forensic dentistry, dental school, federal university of pernambuco, recife, pe, brazil 5dds, ms, phd, associate professor, forensic dentistry, dental school, university of pernambuco, recife, pe, brazil correspondence to: ana paula cavalcante carneiro rua: escritor antônio saturnino de mendonça júnior, nº 99, apto 702 jatiúca, maceió, alagoas, brazil, cep: 57036-420 e-mail: analegista@hotmail.com abstract aim: the aim of this study was to develop an age estimation table based on mineralization stages of third molars in a survey in the state of alagoas, brazil. forensic dentistry has largely contributed to solve age estimation cases. radiographic study on tooth mineralization stages has shown major importance on age estimation with forensic application. methods: this investigation was a crosssectional study developed at the clinic of the local branch of the brazilian dental association in alagoas. three hundred and twelve patients of both genders aged 9 to 21 years were enrolled. three calibrated examiners (kappa=0.789, p<0.001) determined the mineralization stages of each third molar from radiographs collected in the survey. the age of each patient was also recorded. results: a third molar chronological mineralization table based on the study’s survey was developed. there was correlation (spearman=0.869; p<0.01) between real and estimated ages, mainly when both upper and lower third molars were considered together. conclusions: it is important to develop regional age estimation tables based on local surveys mainly considering ages of forensic interest. keywords: age determination by teeth, molar third, radiography, panoramic. introduction over the last few years, age evaluation of live subjects has become very important in forensic sciences1. there are several situations in which age estimation procedures are required. some specific ages present legal importance worldwide. in the brazilian legal system, age is commonly mentioned as a requirement for some civil acts or characterization of facts of legal interest. concerning civil situations, subject’s legal capacity may be determined by age estimation. also, people with incorrect birth certificates often need age estimation procedures to obtain regular documents. in adoption cases, it is frequently necessary to develop age estimation procedures to fulfill the purpose of the process. in criminal circumstances, lack of documents to confirm chronological age may have important consequences. in brazil, the most common are the determination of criminal age (18 years old), age limit of 14 years old to establish the crime “rape of vulnerable”, and also to determine whether a subject involved on a braz j oral sci. 9(4):488-492 489489489489489 crime or violent situation is a child (less than 12years old) or an adolescent (more than 12 and less than 18 years old). still concerning criminal situations, there is a requirement of age estimation to help identifying non-identified corpses. a number of methods to achieve age estimation have been described2. studies have distinguished evaluation of dental mineralization stages3-4, cervical vertebra maturation index5, carpal maturation5, and tooth eruption chronology1. frequently tooth mineralization methods have been used for age estimation, mainly among children6. thus dental radiology plays an important role as a complementary specialty, helping forensic dentistry to perform age estimation procedures7. kurita8 has demonstrated that age estimation through tooth mineralization methods present strong correlation with chronological age and there is no statistically significant difference from bone methods. almeida9 mentions that human tooth development takes place up to 21 years old. the same author assumes that teeth are the best body structures for age estimation because they are poorly affected by systemic and nutrition alterations that can influence bone development and maturity. an age estimation method based on a canadian survey that has been largely used worldwide through tooth mineralization study was first described in 1973 by demirjian et al.10. over the years, the applicability of demirjian’s method has been tested in various countries all over the world11-16. some of these studies11-13,15 have suggested the need of local adjustment to the method development. studies have shown that global differences concerning demirjian’s method are due to differences in population composition14. in brazil, a tooth mineralization table developed by nicodemo et al.17 in brazilian southern region survey has been used as a reference pattern in many age estimation situations. it would be useful to develop specific tooth mineralization patterns for each region, considering population differences in a continental country such as brazil8. the purpose of this study was to develop an age estimation table based on mineralization stages of third molars in a survey conducted in the brazilian northeastern state of alagoas, brazil. material and methods this study was developed following ethical principles for research involving human subjects and was evaluated and approved by the ethics committee of university of alagoas (protocol number 927/08). this investigation was a cross-sectional study developed at the clinic of the local branch of the brazilian dental association in alagoas (bda/al), brazil. all preoperative panoramic radiographs from the records of all patients undergoing orthodontic treatment at bda/al were reviewed. three hundred and twelve panoramic radiographs from subjects aged 9 to 21 years were included. exclusion criteria were: history of chronic systemic disease, dentofacial syndromes, history of long-term medication use, extracted or missing third molars, and individuals born in other brazilian states but alagoas. these data were accessed from patients’ files. three examiners, forensic dentists from the forensic medicine institute of maceio, alagoas were calibrated to detect the different tooth mineralization stages. first, the examiners evaluated third molar mineralization stages on 10 previously selected panoramic radiographs. the same radiographs were reevaluated after a 15-day interval, showing substantial agreement18 among examiners (kappa=0.789; p<0.001). panoramic radiographs were evaluated with the aid of light-boxes and magnifiers without direct incidence of ambient light. the mineralization stage for each tooth was scored ranging from 1 to 8, according to the mineralization stages described by nicodemo et al.17. additional data included gender and chronological age for each subject. besides, age estimation for each subject was performed based on mineralization values for third molars proposed by nicodemo et al.17. data were stored in the statistical package for the social sciences (spss, version 15.0 for win, spss inc., chicago, il, usa). age means, standard deviations and 95% confidence interval were calculated for each mineralization stage. linear regression graphs were built and spearman’s correlation test (p<0.01) was applied to determine the strength of correlation between chronological and estimated ages. independent student’s t-test was applied (p<0.05) to measure differences of chronological age means of each mineralization stage between male and female groups. results and discussion from the 312 panoramic radiographs evaluated, 118 (37.8%) were from male and 194 (62.2%) from female individuals. tables 1 and 2 show age means (in months), confidence interval and standard deviations for each mineralization stage of upper and lower third molars, respectively. concerning ages of forensic interest¸ according to the confidence intervals presented in our survey, it can be stated that, when the third molar is classified in the 5th to 8th stage of mineralization, there is no doubt that the analyzed subject is above 12 years of age. to solve doubts concerning the criminal age of 14 years old, third molars in the 6th stage of mineralization are required at least. on the other hand, concerning majority determination, it can just be affirmed that the individual is 18 years old or older if third molars in the 8th stage of mineralization are present (closed apex). unlike reported in previous studies, this survey did not show significant differences (t-test, p>0.05) between the age means and the mineralization stages concerning gender (tables 3 and 4), except for stage 7 of upper third molars (table 3), which showed a higher mean for female chronological table of third molar mineralization in a survey in the state of alagoas, brazil braz j oral sci. 9(4):488-492 490490490490490 table 1. tooth mineralization chronology of upper third molars*. *data from 282 subjects. mineralization stages n (%) age means (months) 95% confidence interval standarddeviation 1 16 (5.7) 116.31 107.33 – 125.28 16.84 2 20 (7.1) 127.40 120.97 – 133.82 13.72 3 34 (12.0) 132.14 124.45 – 139.84 22.05 4 52 (18.4) 142.07 136.91 – 147.23 18.53 5 57 (20.2) 160.77 155.39 – 166.15 20.18 6 38 (13.7) 183.21 176.27 – 190.14 21.10 7 39 (13.8) 210.51 203.37 – 217.64 22.01 8 26 (9.2) 235.84 228.57 – 243.11 18.00 mineralization stages n (%) age means (months) 95% confidence interval standarddeviation 1 18 (6.0) 117.94 109.39 – 126.49 17.19 2 20 (6.7) 116.40 111.24 – 121.55 11.00 3 42 (14.2) 133.00 127.43 – 138.56 17.87 4 64 (21.6) 142.31 138.26 – 146.35 16.18 5 53 (17.9) 163.75 157.35 – 170.15 23.22 6 41 (13.8) 184.53 178.86 – 190.21 17.98 7 38 (12.8) 212.42 205.14 – 219.69 22.13 8 20 (6.7) 236.80 227.62 – 245.97 19.60 table 2. tooth mineralization chronology of lower third molars*. *data from 296 subjects. table 3. tooth mineralization chronology of upper third molars according to sex. sd: standard deviation *different values at p<0.05 (t test) stages male(n=106) female(n=177) n (%) age means (months±sd) n (%) age means (months±sd) 1 5 (4.7) 120.60±9.47 11 (6.2) 114.36±19.40 2 7 (6.6) 129.00±6.95 13 (7.3) 126.53±16.48 3 18 (17.0) 132.00±16.16 16 (9.0) 132.31±27.82 4 21 (19.8) 144.61±21.79 31 (17.5) 140.35±16.11 5 20 (18.8) 159.25±19.51 38 (21.4) 161.57±16.11 6 12 (11.3) 180.75±17.76 26 (14.7) 184.34±22.72 7 9 (8.5) 197.88±16.55* 30 (16.9) 214.30±22.25* 8 14 (13.2) 234.42±19.00 12 (6.8) 237.50±17.43 subjects. despite these results, correlation between chronological and estimated ages was stronger in female lower third molars (spearman=0.883; p<0.01) and weaker in male lower third molars (spearman=0.832; p<0.01) (table 5). compared to those values suggested by nicodemo et al.17, this survey’s general age means were higher until the 4th mineralization stage and lower concerning the other stages. stages male(n=115) female(n=159) n (%) age means (months±sd) n (%) age means (months±sd) 1 6 (5.2) 127.83±16.04 5 (3.1) 113.00±16.11 2 12 (10.4) 120.08±10.43 4 (2.5) 109.85±10.33 3 18 (15.6) 135.66±17.56 16 (10.0) 131.00±18.20 4 25 (21.7) 145.32±18.86 36 (22.6) 140.38±14.13 5 21 (18.2) 158.28±21.98 32 (20.1) 167.34±23.65 6 11 (9.5) 183.18±17.08 30 (19.1) 185.03±18.55 7 12 (10.4) 207.00±20.46 26 (16.3) 214.92±22.80 8 10 (8.7) 232.40±23.11 10 (6.2) 241.20±15.28 table 4. tooth mineralization chronology of lower third molars according to sex. sd: standard deviation kurita et al.19 (2007), studying a sample of fortaleza (another northeastern brazilian state), demonstrated that nicodemo’s method underestimated the chronological ages of both genders. therefore, this method does not present a variation pattern, being below or above the chronological age depending on the age of the considered subjects6. figures 1-3 show correlations between chronological chronological table of third molar mineralization in a survey in the state of alagoas, brazil braz j oral sci. 9(4):488-492 491491491491491 female nicodemo male nicodemo upper lower general upper lower general sperman’s correlation 0.856* 0.883* 0.873* 0.856* 0.832* 0.859* table 5. chronological and tooth age correlation according to sex. *significant correlation (p<0.01) and estimated ages for upper and lower third molars according to nicodemo’s data17. correlation coefficients (spearman’s test) were 0.853 (upper teeth), 0.867 (lower teeth) and 0.869 (both upper and lower teeth), suggesting that the combination of upper and lower tooth values leads to a discrete increase of spearman’s coefficient between the estimated and chronological ages. in this survey, although all values have suggested significant correlations (p<0.01), it is evident that there are significant discrepancies between the chronological and estimated ages. the practical importance of these differences comprises the responsibility that expert dentists assume when apply age estimation methods in order to formulate reports that can determine the future of examined individuals concerning criminal issues. the use of age estimation methods based on mineralization stages of third molars is very profuse in the specialized literature. in our sample, it was evident that there are substantial differences between estimated and chronological ages in the various mineralization stages. in this study, a chronological mineralization table based on third molars was developed. according to the obtained results, we suggest the development of regional tables of tooth mineralization chronology that can decrease the discrepancies between the estimated and chronological ages in dental examinations of forensic interest. references 1. olze a, peschke c, schulz r, schmeling a. studies of the chronological course of wisdom tooth eruption in a german population. j forensic leg med. 2008; 15: 426-9. 2. schmidt cm. estimativa da idade e sua importância forense [dissertation]. unicamp/fop; 2004. 3. costa fe. estimativa de idade em radiografias panorâmicas através dos estágios de calcificação de nolla [dissertation]. unicamp/fop; 2001. 4. fereira ae, fereira j, céspedes m, barrios f, ortega a, maldonado y. empleo de la edad dental y la edad osea para el cálculo de la edad cronológica com fines forenses, en niños escolares com alteraciones em el estado nutricional, en maracaibo, estado zulia – estudo preliminar. acta odontol venez. 2007; 45: 3-10. chronological age (months) estimated age (months) spearman = 0.853 (p<0.01) fig. 1. linear regression curve correlating estimated and chronological ages. (upper third molar mineralization evaluation). spearman = 0.867 (p<0.01) chronological age (months) estimated age (months) fig. 2. linear regression curve correlating estimated and chronological ages. (lower third molar mineralization evaluation). spearman = 0.869 (p<0.01) estimated age (months) chronological age (months) fig. 3. linear regression curve correlating estimated and chronological ages. (third molar mineralization evaluation). chronological table of third molar mineralization in a survey in the state of alagoas, brazil braz j oral sci. 9(4):488-492 492492492492492 5. camargo gtl, cunha gte. estudo do sincronismo entre índice de maturação das vértebras cervicais, idade dentária e idade carpal com a idade cronológica. sotau rev virtual odontol. 2007; 2: 2-7. 6. gonçalves acs, antunes jlf. estimativa da idade em crianças baseada nos estágios de mineralização dos dentes permanentes, com finalidade odontolegal. odontol soc. 1999; 1: 55-62. 7. gruber j, kameyama mm. o papel da radiologia em odontologia legal. pesq odontol bras. 2001; 15: 263-8. 8. kurita lm. aplicabilidade de métodos de estimativa de idade óssea e dentária em brasileiros, cearenses [thesis]. unicamp/fop; 2004. 9. almeida csl. estimativa da idade por radiografias panorâmicas em indivíduos melanodermas. piracicaba, sp: [s.n.], 2002. 171p. 10. demirjian a, goldstein h, tanner jm. a new system of dental age. hum biol. 1973; 45: 211-7. 11. koshy s, tandon s. dental age assessment: the applicability of demirjian’s method in south indian children. forensic sci int. 1998; 94: 73-85. 12. landeira ac, argote jl, rodríguez mm, calvo msr, otero, xl, concheiro, l. dental age estimation in spanish and venezuelan children. comparision of demirjian and chaillet’s scores. int j legal med. 2010; 124: 105-12. 13. leurs ih, wattel e, aartman iha, etty e, andersen pb. dental age in dutch children. eur j orthod. 2005; 27: 309-14. 14. liversidge hm, chaillet n, mörnstad h, nyström m, rowlings k, taylor j et al. timing of demirjian’s tooth formation stages. ann hum biol. 2006; 33: 454-70. 15. qudeimat ma, behbehani f. dental age assessment for kuwaiti children using demirjian’s method. ann hum biol. 2009; 36: 695-704. 16. wykänen r, espeland l, kvaal si, krogstad o. validity of the demirjian method for dental age estimation when applied to norwegian children. acta odontol scand. 1998; 56: 238-44. 17. nicodemo ra, moraes lc, médici fe. tabela cronológica da mineralização dos dentes permanentes entre brasileiros. rev fac odontol são josé dos campos. 1974; 3: 55-6. 18. landis jr, koch gg. the measurement of observer agreement for categorical data. biometrics. 1977; 33: 159-74. 19. kurita lm, menezes av, casanova ms, haiter-neto f. dental maturity as an indicator of chronological age: radiographic assessment of dental age in a brazilian population. j appl oral sci. 2007; 15: 99-104. chronological table of third molar mineralization in a survey in the state of alagoas, brazil braz j oral sci. 9(4):488-492 404 not found oral sciences n3 braz j oral sci. 11(2):88-93 original article braz j oral sci. april | june 2012 volume 11, number 2 sensitivity and accuracy of panoramic radiography in identifying calcified carotid atheroma plaques janaína sens bastos¹, thalita queiroz abreu¹, sebastião barreto de brito filho², kelston paulo felice de sales³, fernanda ferreira lopes4, ana emília figueiredo de oliveira5 1msc student, graduate program in dentistry, school of dentistry, federal university of maranhão, brazil 2msc, professor, department of medicine ii, school of medicine, federal university of maranhão, brazil 3msc student in vascular surgery, school of medicine, federal university of são paulo, brazil 4phd, professor, discipline of semiology, department of dentistry i, school of dentistry, federal university of maranhão, brazil 5phd, professor, department of dentistry i, school of dentistry, federal university of maranhão, brazil correspondence to: janaína sens bastos rua 01, lote 08, condomínio village intermares, casa 31 planalto vinhais i cep: 65074-856 são luís – ma, brasil phone: +55 98 32464787 e-mail: janaina100s@gmail.com abstract atherosclerosis is a chronic inflammatory disease that can cause death and physical and/or mental disabilities. it represents a serious public health problem owing to the high healthcare costs involved in rehabilitating patients. among the different methods available for diagnosing atherosclerotic disease, color doppler examination may be considered the “gold standard.” on the other hand, panoramic radiography of the jaws (prj) is a diagnostic resource commonly used by dentists, and the obtained images allow examining other structures apart from the maxilla and the mandible. aim: to evaluate the sensitivity and accuracy of panoramic radiography in identifying calcified carotid atheroma, and compare the results to those obtained with color doppler examination. methods: forty-two cervical regions were evaluated in panoramic radiographs and color doppler scans. the kappa test (p < 0.05) was used to assess agreement between the examinations. data were tested for sensitivity, specificity and negative predictive value, as well as positive and negative likelihood ratios. results: the kappa value was 0.11. the sensitivity and specificity of prj were 73.9 and 36.8%, respectively, and the positive and negative likelihood ratios were 1.2 and 0.7, respectively. conclusions: although a low correlation between prj and color doppler examination was observed, as shown by the kappa test, the sensitivity of prj in identifying calcified carotid atheroma was considered acceptable. keywords: stroke, carotid artery diseases, radiography, panoramic. introduction atherosclerosis is an inflammatory disease that produces circulatory changes caused by fat deposits on the walls of the arteries. when the carotid arteries are involved, it can cause stroke – a cerebral vascular accident (cva) – that can be ischemic or hemorrhagic1. the formation of atheromatous plaques in the carotid artery – also called calcified carotid atheromatous plaques (ccap) – is not a simple and inevitable degenerative process resulting from advanced age, but rather a chronic inflammatory disease that can develop into an acute clinical condition owing to plaque rupture, rendering the patient susceptible to thromboembolism or stroke2. received for publication: november 29, 2011 accepted: april 18, 2012 8989898989 braz j oral sci. 11(2):88-93 stroke is the third leading cause of death in the united states, accounting for over 150,000 deaths/year, ranking only behind cardiovascular disease and cancer3-4. by 2009, it was the first cause of death in brazil, claiming over 300,000 deaths/ year5. it represents a serious public health problem because it causes death and physical and/or mental disabilities – approximately 60% of survivors suffer from such disabilities6 – and have high costs involved in treating and rehabilitating patients. the main risk factors said to favor or accelerate the atherosclerotic process are: heredity, dyslipidemia, smoking, hypertension, diabetes, viral infections, elevated homo-cysteine (an amino acid derived from eating meat and dairy products) in the blood, obesity, sedentary lifestyle and stress7. preventive measures and early diagnosis are key factors to reduce the prevalence of the disease. however, a non-invasive, inexpensive and reliable diagnostic method still lacks. angiography, which considered the “gold standard” for diagnosing this disease, is an invasive method that can cause major complications8, and color doppler (also known as laser doppler flowmetry or duplex scan), although fast, accurate, noninvasive and painless9, is not yet widely accessible to all strata of society because of its high costs (figures 1a, 1b). fig. 1b color doppler – ccap fig. 1a color doppler – ccap since 1981, the presence of radiopaque images on panoramic radiographs has been described as a sign of the presence of ccap10-11, indicating that this examination could contribute to the early diagnosis of stroke risk. there are studies reporting that partially calcified atherosclerotic lesions can be observed in panoramic radiographs of the jaws (prj)12-13. the image of the calcification is described as a nodular, radiopaque mass in one or more areas, either elongated or triangular, of various sizes, measuring from 1.5 to 4.0 cm, and as observable below and posteriorly to the angle of the mandible, adjacent to the intervertebral spaces between c2, c3, and c48,14-15 (figures 2a, 2b). fig. 2a panoramic radiography of the jaws fig. 2b panoramic radiography of the jaws ccap some studies have reported calcifications in dental panoramic radiographs between the second, third and fourth cervical vertebrae, which were later confirmed by color doppler examination as atheromatous plaques16-17. because panoramic radiography may be able to detect ccap during routine examinations, thus enabling proper referral of patients for appropriate treatment, it has the potential to promote a significant reduction in the morbidity rate caused by this disorder, and a substantial reduction in the healthcare costs associated with atherosclerosis18. nevertheless, because panoramic radiographs cover several different anatomical areas, developing the ability to perform differential diagnosis of the structures that comprise the carotid region is of utmost importance. this ability involves comprehensive anatomical knowledge of structures such as the hyoid bone, epiglottis, calcified submandibular and styloid ligaments, and triticeous sensitivity and accuracy of panoramic radiography in identifying calcified carotid atheroma plaques braz j oral sci. 11(2):88-93 cartilage, as well as of pathological processes such as sialoliths, phleboliths, and calcified lymph nodes19. in view of these considerations, it is reasonable to assume that prj has a great potential in identifying ccap, or should at least be further investigated as a diagnostic tool for atherosclerosis. nevertheless, there is still controversy in the literature about its use as a reliable method for this purpose, and great divergence in results. this could be explained by the different methodologies used and because the methods used for comparative analysis of panoramic radiographs in these studies are not considered the “gold standard,” and are based solely on radiographic interpretation for diagnostic conclusion. based on the methodological limitations of the existing studies, on the fact that dentists use panoramic radiographs routinely, and on the fact that early detection of patients at risk for developing atherosclerosis is an important public health goal, the purpose of the present study was to evaluate the sensitivity and accuracy of prj in identifying ccap compared with color doppler examination. material and methods sample in this study, the accepted ethical principles governing human experimentation were followed closely, after approval was obtained from the ethics committee of the federal university of maranhão (protocol n. 2311-005753/2009-57). the sample was calculated based on the estimated prevalence of calcification in the carotid artery, approximately 2%. the following parameters were used for calculating sample size: significance level of 0.05 and maximum permissible error of ± 5%, thus producing n = 31. in the period from august 2009 to june 2010, panoramic two dental radiologists evaluated prj of patients of both genders in a clinic commissioned by the public health system in the city of são luis, ma, brazil. radiographic diagnosis of ccap was considered conclusive when there was agreement between both examiners regarding the presence of ccap and the side in which its presence was detected. twenty-one panoramic radiographs were considered by the examiners as undoubtedly positive for the diagnosis of ccap, on at least one side, thus leading to an analysis of 42 cervical regions (n = 42). the inclusion criteria for all participants of this study was the detection of an image suggestive of ccap on at least one side of the prj in patients who were at least 40 years of age, and who agreed to undergo color doppler angiography. radiographic examination prj was performed using the standard positioning technique, orthophos 3 ceph x-ray equipment (sirona dental systems gmbh, germany) and 15 x 30 cm panoramic film (eastman kodak co., rochester, ny, usa). the films were processed automatically (revell-xtec ltd., são paulo, sp, brazil), with appropriate developer and fixer solutions (kodak brazilian trade and industry ltd., sao josé dos campos, sp, brazil). interpretation of the radiographic images was performed by two dentists, with less than three years of experience as radiology specialists, and in an environment with dimmed light and using a light box. the presence of ccap could be unilateral or bilateral. both sides of each one of the 21 patients were evaluated, for a total of 42 cervical exams (n = 42). doppler examination all 21 patients with positive radiographic exams for ccap on at least one side of the prj were subjected to doppler examination at the presidente dutra university hospital, são luis, ma, brazil. a dupplex scan equipment (general electric, new york, usa) was used owing to its reliability in demonstrating blood flow in the artery under study. a physician specialized in angiology had previously determined the presence of stenosis and/or obstruction caused by an atheromatous plaque. in the same way as done in the radiographic examination, both sides of each exam were evaluated, also totaling 42 cervical analyses (n = 42). statistical methods data were analyzed statistically using bioestat 5.0 software, according to criteria adopted by the related literature20. the kappa agreement test (p < 0.05) was used to assess and compare sensitivity, specificity, negative predictive value (npv), accuracy (acc), positive likelihood ratio (plr) and negative likelihood ratio (nlr) for the different examinations. the kappa values and their interpretations were < 0 (no agreement), 0 to 0.19 (very weak agreement), 0.20 to 0.39 (poor agreement), 0.40 to 0.59 (moderate agreement), 0.60 to 0.79 (substantial agreement), and 0.8 to 1.0 (excellent agreement), following criteria previously approved 21. the sensitivity test was used to match the prj’s ability to detect the disease when its presence was confirmed, i.e., to assess how many positive and negative results were found using prj for the cases found positive in the color doppler examination (“gold standard”)18. sensitivity was calculated as follows: sensitivity (%) = true positives / (true positives + false negatives) x 100. the specificity test was used to identify the true negatives determined by prj in individuals who were genuinely healthy, as revealed by the doppler examination. it was calculated as follows: specificity = true negatives / (true negatives + false positives) x 100. the npv for prj was calculated to assess how many test results among those that proved negative were actually negative according to the doppler examination. it was calculated as follows: npv (%) = true negatives / (true negatives + false negatives) x 100. prj’s accuracy was defined as the proportion of successful examinations, i.e., the total number of true 9090909090sensitivity and accuracy of panoramic radiography in identifying calcified carotid atheroma plaques braz j oral sci. 11(2):88-93 9191919191 positives and true negatives, as confirmed by the doppler examination. it was calculated as follows: acc = (true positives + true negatives) / total. the likelihood ratio is a means of describing the performance of a diagnostic test. it expresses how many times the result of a given test is more likely (or less likely) to occur in affected subjects compared to unaffected subjects. if a test is dichotomized (positive/negative), two types of likelihood ratio describe its ability to separate those affected from those not affected: one associated with a positive test and another associated with a negative test, respectively calculated as follows: plr = sensitivity / (1 specificity), and nlr = (1 sensitivity) / specificity. results of the 42 cervical analyses (l and r sides of 21 patients), 17 images visualized on the prj were confirmed by the doppler examination (true positives), with 6 non-visualized sides (false negatives). prj identified 12 images of ccap that were not confirmed by the doppler examination (false positives), with 7 sides that were truly without disease (true negatives) (table 1). doppler present absent total prj + 17 (a) 12 (b) 29 (a + b) prj 6 (c) 7 (d) 13 (c + d) total 23 (a + c) 19 (b + d) 42 (n) table 2 accuracy (acc) of the panoramic radiography in identifying calcified carotid atheroma plaques (n, %). prj: panoramic radiography of the jaws acc = [(a + d)/n]*100 acc = 57.1% table 1 assessment of the agreement between panoramic radiography and color doppler in identifying calcified carotid atheroma plaques (n, %). kappa = 0.11 / p = 0.2265 prj: panoramic radiography of the jaws doppler sick (%) healthy (%) total prj + 17 (73.9) 12 (63.2) 29 (69.0) prj 6 (26.1) 7 (36.8) 13 (31.00) total 23 (100) 19 (100) 42 (100) we found a kappa value of 0.11 (p = 0.2265), indicating that the agreement between the doppler and the prj examinations was weak and not significant (table 1). the sensitivity calculated for prj was 73.9%, indicating that 73 out of 100 patients with the disease were correctly identified and 26 were considered erroneously healthy. the calculated specificity was 36.8%, indicating that 36 out of 100 patients with the disease were identified as healthy and 63 were considered erroneously sick. the npv of prj was found to be 53.8%, indicating that 53 out of 100 patients who had a negative result in the prj examination actually did not have ccap, or, stated differently, 53.8% of the diagnoses found to be negative by prj were correct, and 46.2% of the diagnoses initially considered negative by prj were actually positive in the color doppler examination. the likelihood ratios were as follows: plr = 1.2 and nlr = 0.7. the higher the plr, the better the test, and, in order to be considered a good test, the plr should be significantly higher than 1. this would mean that a positive outcome is more likely to be a true-positive than a false-positive. the lower the nlr (closer to 0), the better the test. this would mean that a negative result is more likely to be a real negative than a false negative. the accuracy found for prj was 57.1%, indicating that this examination provided a correct diagnosis – a true positive or a true negative – in 57 out of 100 patients. the sensitivity and accuracy results obtained with the use of prj for the identification of calcifications in the carotid artery indicate that this test could contribute to reducing the risk of patients suffering a stroke (table 2). discussion when atheromatous plaques of the carotid bifurcation region are identified, the first feature to note is the size of the obstruction. if it blocks less than 60% of the artery, simply inhibiting platelet aggregation associated with thrombus formation can treat the patient. when the obstruction is greater than 60%, invasive procedures are required to remove the plaque22. panoramic radiography is limited to identifying ccaps, and cannot evaluate its exact location, or the degree of the obliteration13. the main limitation of this study was that the population evaluated consisted of a selective group (patients with an image suggestive of ccap detected in panoramic radiographs), rather than the general population. nonetheless, this bias can also be considered one of its strengths, from a methodological standpoint, because it compared the sensitivity and accuracy of prj to that of the “gold standard” method (doppler) in identifying the presence of atherosclerotic plaques, considering that prj is an exam commonly requested by dentists, in which the occasional presence of ccap is a radiographic finding. the npv found in this study was not substantial (63.2%), considering that atherosclerotic diseases are among the major causes of death worldwide. however, the sensitivity of prj in identifying ccap was significant (73.9%) when compared to angiographic examination (color doppler imaging), which is considered the “gold standard” (figure 3) although the sensitivity of prj in identifying ccap was 73.9%, its accuracy was only 57.1% and its specificity was 36.8%, a trend confirmed by the kappa test (0.11). thus, prjs should not be suggested as a diagnostic imaging examination for the identification of ccap, as proposed by some authors. the results of the present study sensitivity and accuracy of panoramic radiography in identifying calcified carotid atheroma plaques braz j oral sci. 11(2):88-93 9292929292 corroborate those of previous studies19, which stated that a radiopacity between c2, c3 and c4 in a panoramic radiograph does not always represent a ccap. our results differ from those of a previous study23 that found prj sensitivity and specificity values of 22.2% and 90.0%, respectively, for identifying ccap using ct as the “gold standard.” the authors also concluded that panoramic radiographs could be considered a diagnostic method with moderate accuracy and low sensitivity for the detection of ccap. this difference may be explained by the different “gold standards” adopted for comparison with prj. in a previous study24, prj presented low sensitivity and no accuracy for detecting calcification or stenosis. only one dental radiologist interpreted the panoramic radiographs in that study, and the authors used two different radiographic xray apparatuses (siemens corp., iselin, new jersey, usa or gendex corp., milwaukee, wisconsin, usa) for image acquisition. in contrast, the authors also mentioned that the doppler images were obtained at various locations, and interpreted by different radiologists. a study assessing the performance of digital prj in forty patients with carotid atherosclerotic disease diagnosed by angiography found npv and sensitivity values of 46% and 60%25, which differ from those found in our study (53.8% and 73.9%, respectively). one possible explanation for this divergence in results is the different characteristics of the projection devices used, particularly the rotation system, the section thickness and the apparatus calibration19. a survey8 conducted at the health department of the city of valença, rj, brazil, reviewed panoramic radiographs and doppler images of 16 patients to investigate whether there was agreement between the diagnosis of ccap in the doppler color and in the prj images. the results indicate substantial agreement between these tests (kappa = 0.78), contrasting with those of the present study. this difference may be explained by the different sample sizes and by the different number of evaluators used to assess the panoramic radiographs. in another study8, calcifications were observed in 9.4% of prjs, but not confirmed by the color doppler examination, and were considered false positives; in the same study, 3.1% of results were considered false negatives, meaning that the color doppler examination revealed the presence of calcifications that went undetected in the prjs. in contrast, fig. 3 assessment of the agreement between panoramic radiography and color doppler in identifying calcified carotid atheroma plaques (n, %). the present study showed even higher values for false positives (63.2%) and false negatives (36.8%). it is noteworthy that the identification of ccap in panoramic radiographs may not be a simple task. published articles8,23, and ours alike, have used dental radiologists in their methodology, leading one to believe that false results, whether positive or negative, could be even greater if the radiographic analysis were performed by general dentists, less trained to visually identify pathologies. a previous study8 showed that a general dentist, who is more familiar with evaluating the maxilla, mandible and temporomandibular joint cannot adequately perform the identification of ccap in panoramic radiography. in that study8, dental radiologists found evidence of calcification in the carotid artery in 9 out of 1,818 panoramic radiographs examined, whereas the general dentist, examining the same panoramic radiographs, failed to identify any of these calcifications. misinterpretation could be linked to the various anatomical structures existing in the cervical region studied, representing differential diagnoses. in 1981, when the panoramic radiograph was described for the first time as an aid in identifying patients at risk for stroke, calcification in the region of the carotid artery bifurcation was identified in 2% of cases, of which 88% were actually carotid artery calcifications and the remaining 12% were calcified lymph nodes or salivary calculi11. although prj may not be considered a reliable method to detect calcifications in the carotid artery, it should be used as a referral for the doppler25 examination (the “gold standard”) and other specialists8. merely detecting calcifications or stenosis of the carotid artery in prj is of limited value in assessing the risk of stroke, insofar as it does not take into account plaque vulnerability26-28. according to a recommendation of the american heart association, physicians should routinely assess the vulnerability of all major arterial plaques 27, including their morphology, as well as biochemical and molecular factors, none of which can be identified by means of panoramic radiography. dental clinicians should refer all patients with atheromalike lesions to a physician, since early aggressive treatment has been shown to reduce strokes, fatal or otherwise11. on the other hand, owing to the wide divergence of results observed, it remains unclear whether dentists could be adequately trained to reduce the number of false positive diagnoses to an acceptable level. otherwise, unnecessary referrals to physicians could occur, leading to unnecessary tests and increased healthcare spending 29. we believe that knowledge of this topic should be generalized, and general dentists should be trained in identifying carotid artery alterations. within the limitations of the present study, prj presented good sensitivity and only moderate accuracy in identifying calcified carotid atheroma plaques compared with color doppler examination. these results were confirmed by the observed positive likelihood and negative likelihood ratios. these results support previous findings and point to enhancing the prospects of an interdisciplinary approach, sensitivity and accuracy of panoramic radiography in identifying calcified carotid atheroma plaques 9393939393 braz j oral sci. 11(2):88-93 where dentists can refer their patients to other specialists, who, in turn, may reach a definitive diagnosis based on specific tests and prescribe the proper treatment. references 1. manual merck. 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[cited 2012 feb 6] .available from: http:// boasaude.uol.com.br/lib/showdoc.cfm?libdocid=3118& return catid=1770. 8. romano-sousa cm, krejci l, medeiros fmm, graciosa-filho rg, martins mff, guedes vn, et al. diagnostic agreement between panoramic radiographs and color doppler images of carotid atheroma. j appl oral sci. 2009; 17: 45-8. 9. frauchiger b, schimid hd, roedel c, moosmann p, staub d. comparison of carotid arterial resistive indices with intima-media thickness as sonographic markers of atherosclerosis. stroke. 2001; 32: 836-41. 10. levy ba, mandel dds. calcified carotid artery imaged by computed tomography. j oral maxillofac surg. 2010; 68: 218-20. 11. friedlander ah, lander a. panoramic x-ray identification of carotid arterial plaques. oral surg oral med oral pathol. 1981; 51: 102-4. 12. friedlander ah. identification of stroke prone patients by panoramic and cervical spine radiography. dentomaxillofac radiol. 1995; 24: 160-4. 13. manzi fr, tuji fm, almeida sm, neto fh, bóscolo fn. panoramic radiography as an auxiliary in detecting patients at risk for cerebrovascular accident. rev assoc paul cir dent. 2001; 55: 131-3. 14. senosiain-oroquitea a, pardo-lópez b, de carlos-villafranca f, gonzálezmontoto, cobo-plana j. detección de placas de ateroma mediante radiografias dentales. rcoe. 2006; 11: 297-303. 15. pornprasertsuk-damrongsri s, thanakun s. carotid artery calcification detected on panoramic radiographs in a group on thai population. oral surg oral med oral pathol oral radiol endod. 2006; 101: 110-5. 16. freymiller eg, sung ec, friedlander ah. detection of radiation-induced atheromas by panoramic radiography. oral oncology. 2000; 36: 175-9. 17. almog dm, tsimidis k, moss me, gottilieb rh, carter lc. evaluation of a training program for detection of carotid artery calcifications on panoramic radiographs. oral surg oral med oral pathol oral radiol endod. 2007; 90: 111-7. 18. ramesh a, pabla t. panoramic radiographs: a screening tool for calcified carotid atheromatous plaque. j mass dent soc. 2007; 56: 20-1. 19. kamikawa rs, pereira mf, fernandes a, meurer mi. study of the localization of radiopacities similar to calcified carotid atheroma by means of panoramic radiography. oral surg oral med oral pathol oral radiol endod. 2006; 102: 579-81. 20. ayres m, ayres jr m, ayres dl, santos as. bioestat versão 5.0.belém: sociedade civil mamirauá, mct-cnpq; 2007. 21. teixeira ac, cruvinel dl, roma fr, luppino lf, resende lhp, sousa t, et al. evaluation of the agreement between clinical and laboratorial exams in the diagnosis of leprosy. rev soc bras med trop. 2008; 41: 48-55. 22. guimarães go, oliveira ie, nobile junior d, messina calderón jc, saddy ms. radiografia panorâmica: identificação de pacientes suscetíveis ao acidente vascular cerebral por meio da detecção de ateromas na bifurcação da carótida. conscientiae saúde 2005; 4: 97-104. 23. yoon sj, yoon w, kim os, lee js, kang bc. diagnostic accuracy on panoramic radiography in the detection of calcified carotid artery. dentomaxillofac radiol. 2008; 37: 104-8. 24. madden rp, hodges js, salmen cw, rindal db, tunio j, michalowicz bs, et al. panoramic radiography does not reliably detect carotid artery calcification nor stenosis. oral surg oral med oral pathol oral radiol endod. 2007; 103: 543-8. 25. damaskos s, griniatsos j, tsekouras n, georgopoulos s, klonaris c, bastounis e, et al. reliability of panoramic radiograph for carotid atheroma detection: a study in patients who fulfill the criteria for carotid endarterectomy. oral surg oral med oral pathol oral radiol endod. 2008; 106: 736-42. 26. nighoghossian n, derex l, douek p. the vulnerable carotid artery plaque: current imaging methods and new perspectives. stroke. 2005; 36: 2764-72. 27. naghavi m, libby p, falk e, casscells sw, litovsky s, rumberger j, et al. from vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: part i. circulation. 2003; 108: 1664-72. 28. bhatia v, bhatia r, dhindsa s, dhindsa m. imaging of the vulnerable plaque: new modalities. south med j. 2003; 96: 1142-7. 29. farman ag, farman tt, khan z, chen z, carter lc, friedlander ah. the role of the dentist in detection of carotid atherosclerosis. sadj. 2001; 56: 549-53. sensitivity and accuracy of panoramic radiography in identifying calcified carotid atheroma plaques oral sciences n3 received for publication: april 02, 2011 accepted: july 13, 2011 braz j oral sci. 10(3):180-183 original article braz j oral sci. july | september 2011 volume 10, number 3 fracture strength of ceramic brackets submitted to archwire torsional strain fernando rayes manhães2 , rosilene divino1 , adriana simone lucato3 , sílvia amélia scudeler vedovello3, cristian correa2 , heloísa cristina valdrigh3 1undergraduate student in dentistry, fundação hermínio ometto-uniararas, araras, sp, brazil 2dds, master student in orthodontics, fundação hermínio ometto-uniararas, araras, sp, brazil 3phd, professor in orthodontics, fundação hermínio ometto-uniararas, araras, sp, brazil correspondence to: heloísa cristina valdrigh centro universitário hermínio ometto uniararas departamento de ortodontia av. dr. maximiliano baruto, 500 jardim universitário araras sp brasil cep 13607-339 phone: +55 19 3543 1474 fax: +55 19 3543 1412 e-mail: vedovelloorto@terra.com.br abstract aim: to evaluate the fracture strength of ceramic brackets submitted to archwire torsional strain. methods: different types of maxillary central incisor ceramic brackets from four different commercial brands, roth prescription (0.022" x 0.028"), were evaluated, namely: mystique (gac), invu (tp orthodontics), clarity (3m unitek) and luxi ii (rmo ortohodontics). to evaluate the fracture strength, 0.019"x 0.025" stainless steel orthodontic wires were inserted into the bracket channels and submitted to torsion until they fractured. results: the invu brackets showed a significantly higher fracture strength than the other brackets (p<0.05), which did not differ significantly among them (p>0.05). conclusions: among the brackets tested, the traditional ceramic bracket invu showed the highest fracture strength, while the luxi ii bracket, obtained the lowest value. keywords: braces, ceramics, orthodontics, biomechanics. introduction as patients who seek orthodontic treatment attribute great importance to esthetics, particularly the growing number of adult patients, esthetic brackets has been increasingly used instead of metal brackets. among the esthetic brackets, those made of polycarbonate and ceramic are outstanding at present1. ceramic brackets are more frequently used than those made of polycarbonate, due to their better physical and mechanical properties2, better color stability, esthetics and wear resistance than polycarbonate brackets. nevertheless, ceramic brackets are more friable, generate more friction with the orthodontic wire, and may cause enamel wear in the antagonist tooth when coming into contact with it3-5. ceramic brackets are composed of aluminum oxide (al 2 o 3 ) and may be monocrystalline (a single aluminum oxide crystal) or polycrystalline (several aluminum oxide crystals fused at high temperatures)6. the major difference between polycrystalline and monocrystalline brackets is translucence. monocrystalline brackets are more translucent than the polycrystalline type, which tend to be opaque4. in addition, monocrystalline ceramic brackets are more resistant to fracture than the polycrystalline type due to the greater tensile strength of the monocrystalline 181 braz j oral sci. 10(3):180-183 alumina7. nevertheless, polycrystalline brackets are more frequently used because they are easier to produce and have a lower cost1. over the last few years, studies have been conducted to compare the fracture strength of ceramic brackets during second and third order activations, as ceramic brackets present high friability and low tensile strength8-9. the moment of torque normally applied to anterior teeth is one of the causes of ceramic bracket fractures10-11. the site of major ceramic bracket fracture is at the base of the winglets, probably because this area concentrates a greater deal of stress12 and is the most handled area by the orthodontist when inserting and removing wires and accessories, thus creating microscratches that contribute to the formation and propagation of cracks12-14 and increase bracket susceptibility to fracture1,15. thus, the aim of this study was to evaluate the fracture strength of four different types of polycrystalline ceramic brackets submitted to archwire torsion or torque. material and methods forty maxillary right central incisor brackets, roth prescription (0.022" x 0.028"), of four different commercial brands (n=10) were evaluated. among the brackets (figure 1), there were four polycrystalline types, being traditional ceramic semi-twin mystique (gac international, bohemia, ny, usa), traditional ceramic twin invu (tp orthodontics, laporte, in, usa), ceramic semi-twin with gold-reinforced channel luxi ii (rocky mountain orthodontics, denver, co) and ceramic twin with stainless steel-reinforced channel clarity (3m unitek, st. paul, mn, usa). each bracket was fixed in a pvc cylinder (tigre do brasil, joinville, sc, brazil) filled under vibration with chemically activated acrylic resin (jet set; clássico artigos odontológicos ltda., são paulo, sp, brazil), at the sand stage, proportioned and prepared in accordance with the manufacturer’s recommendations. after polymerization and smoothing the resin surface with 220-grit silicon carbide abrasive papers (norton s.a., são paulo, sp, brazil), the brackets were fixed onto the surface of the cylinders using cyanoacrylate adhesive (super bonder, loctite, são bernardo do campo, sp, brazil). forty segments of 0.019" x 0.025" 30-mm-long stainless fig. 1 – front and side views of ceramic brackets. steel (gac) arches were bent into u-shapes with 6 mm of width and height, using 139 pliers (dentaurum, ispringen, germany). the bends were made in juxtaposition to the bracket wings. a vertical bend was made at a distance of 6 mm from the bracket so that it could be used as a support for the test machine chisel (figure 2). this distance of 6 mm from the bracket up to the point of support of force was used because it is the mean inter-bracket distance. for the mechanical test, the wire was inserted into the bracket channel through the base of the “u” pre-contoured with 0.019" x 0.025" wire and fixed with 0.008" tying wire (figure 3). fig. 2 – steel wire fig. 3 – steel wire fixed onto bracket mechanical test the pvc cylinder (tigre do brasil) was adapted to a universal testing machine (instron 4411) so that the cervical winglets were positioned facing downwards, and so that the machine chisel would be supported on the wire rods, where the chisel had the vertical bends of the wire as a stop so that it would not slide during the test. in this test, the wire was pushed downwards by the universal test machine chisel, simulating a vestibular torque, this being the reason why fracture strength of ceramic brackets submitted to archwire torsional strain 182 braz j oral sci. 10(3):180-183 the bracket was positioned with the cervical winglets facing down. the test was performed at a crosshead speed of 1.0 mm/min until bracket fracture occurred. data (kgf) were transformed into g·mm and submitted to anova and tukey’s test at a level of significance of 5%. results one-way anova showed that there was significant difference for the fracture strength of brackets submitted to torsion strain (p<0.001). among the polycrystalline brackets, the highest fracture strength (g·mm) was obtained with the traditional ceramic invu (8113,2 ± 1755,2 g·mm) followed in a decreasing order by the traditional ceramic mystique (6022.2 ± 1740.9 g·mm), ceramic with metal channel clarity (5676.6 ± 983.6 g·mm) and ceramic with gold channel luxi ii (4885.8 ± 1043.0 g·mm). the results of tukey’s test (table 1) revealed that the invu brackets presented a statistically higher fracture strength (p<0.05) than the mystique, clarity and luxi ii brackets, which did not differ significantly among them (p>0.05). discussion evaluation of the fracture strength of ceramic brackets submitted to archwire torsion strain was held due to the high friability and low tensile strength of ceramic brackets, particularly during the second and third order activations8-9. design and manufacturing process are factors that determine the strength of ceramic brackets, and the channel and winglet designs are critical for the strength of the accessory. the winglets may easily fracture due to the high flexural strength exerted by the rectangular arches, and this process is facilitated by the presence of defects on the surface and within the ceramic brackets, which may lead to crack propagation when the bracket it submitted to the action of forces14. in the present study, invu brackets presented a significantly higher fracture strength than the mystique, clarity and luxi ii brackets (p<0.05), which did not differ from each other (p>0.05). these results corroborate those of a previous study7, in which the twin ceramic brackets invu presented higher fracture strength of the winglets under traction than the twin ceramic brackets clarity and semitwin luxi ii brackets. already nishio11, testing the strength of esthetic brackets with torsion forces of the arch, found that the clarity brackets showed higher fracture resistance to the brackets luxi ii. one of the factors responsible for the higher fracture strength of the twin ceramic bracket invu may be due to its fabrication method. for these brackets, the fabrication process is molding by injection, which makes it possible to obtain uniform surfaces with fewer irregularities when compared with brackets fabricated by the machining method16. injection molded brackets have a smoother finish than machined brackets, thus reducing the number of surface defects14. thus, these brackets have greater fracture strength under traction when compared with twin ceramic brackets fabricated by machining, which show damage and defects caused by the equipment during fabrication, and serve as foci for fractures12-13. the lowest fracture strength was obtained with the goldreinforced ceramic bracket luxi ii (rmo, orthodontics). this may have occurred due to the thinness of the wings in the occlusogingival and buccolingual directions, when compared with the other ceramic brackets. another characteristic of the luxi ii brackets is their rougher surface morphology, making the ceramic material more susceptible to crack propagation and fracture7,12. this might have occurred as a result of the method of fabricating this bracket, capable of promoting defects and roughness on the surface of these materials, and significantly reducing the ceramic fracture strength5,13,17. of the brackets reinforced with a metal channel, gold reinforcement luxi ii was less efficient than stainless steel reinforcement clarity, though without statistically significant difference. one of the hypotheses for this result was raised in a previous scanning electron microscopy study11, which stated that the sealant material between the gold channel and the bracket appeared to be thicker than the sealant material between the stainless steel channel and bracket. this sealant material could affect the channel rigidity and fracture strength. the traditional semi-twin ceramic bracket mystique presented the second highest fracture strength, but without statistically significant difference from the clarity and luxi ii brackets. this might have occurred due to the morphological characteristics of the brackets, as in the semitwin brackets the mesial and distal winglets could be seen as a single unit. in this bracket configuration, there is a point of ceramic connected to the winglets, which in turn, has the effect of a transverse stabilizer. the mesial and distal winglets are not designed independently of the bracket base as occurs in twin brackets7. however, in the present study it was verified that the twin bracket invu obtained the highest fracture strength, suggesting that the fabrication method was more determinant to the fracture strength than the winglet configuration of the bracket. insertion of the metal channel with either stainless steel (clarity) or gold (luxi ii) had no influence on the fracture strength during torque simulation, and presented the lowest values. ceramic reinforcement on the semi-twin mystique brackets had no influence on the fracture strength either, when compared with the twin ceramic brackets. in this study the 0.019"x 0.025" stainless steel arch was bracket fracture strength (gm·mm) invu 8113.2 (1755.2) a mystique 6022.2 (1740.9) b clarity 5676.6 (983.6) b luxi ii 4885.8 (1043.0) b table 1 fracture strength (gm·mm) of brackets submitted to arch wire torsion force means followed by different letters differ statistically among them (tukey’s test; p<0.05). fracture strength of ceramic brackets submitted to archwire torsional strain 183 braz j oral sci. 10(3):180-183 used because it is the most commonly used type of arch by orthodontists during the stages of control and torque incorporation in orthodontic treatment. the variation in force necessary for the orthodontic movement of inclination (torque) of the maxillary central incisor is high, starting with a minimum of 941 g·mm up to a maximum of 3500 g·mm, has been reported by nikolai10. the fracture strength means during the torque movements in this study ranged from 4885.8 g·mm (luxi ii) to 8113.2 g·mm (invu). therefore, after simulation of torque, the brackets evaluated in this study presented higher fracture strength than that necessary for torque movement reported in the literature. thus, they showed the strength to bear the forces required for dental movement without fracturing. nishio11 also reported that the ceramic brackets clarity, luxi ii and 600 transcend (3m unitek, monrovia, ca, usa) bear the force necessary for movement of torque and did not find no correlation between the dimensions of the brackets and fracture resistance. ceramic bracket fracture at the moment of torque may therefore be due to the excess of force incorporated during torsion of the archwire, and not due to lack of bracket strength, this being an important parameter for the prevention of undesirable effects, such as dental resorptions, for example, commonly found in these types of movements. in the present study, the fracture site of the bracket and the arch angulation were not verified during the mechanical test, and thus complementary studies are necessary. it may be concluded that the traditional twin ceramic bracket invu (tp orthodontics) presented the highest fracture strength during torque simulation in comparison with the mistyque, clarity and luxi ii brackets, probably due to the method of fabrication. references 1. maltagliati la, feres r, figueiredo ma, siqueira df. braquetes estéticos – considerações clínicas. rev clin ortod dent press. 2006; 5: 89-95. 2. faltermeier a, behr m, müssig d. in vitro colour stability of aesthetic brackets. eur j orthod. 2007; 29: 354-8. 3. viazis ad, delong r, bevis rr, douglas wh, speidel tm. enamel surface abrasion from ceramic orthodontic brackets: a special case report. am j orthod dentofacial orthop. 1989; 96: 514-8. 4. viazis ad, cavanaugh g, bevis rr. bond strength of ceramic brackets under shear stress: an in vitro report. am j orthod dentofacial orthop. 1990; 98: 214-21. 5. karamouzos a, athanasiou ae, papadopoulos ma. clinical characteristics and properties of ceramic brackets: a comprehensive review. am j orthod dentofacial orthop. 1997; 112: 34-40. 6. viazis ad, chabot ka, kucheria cs. scanning electron microscope (sem) evaluation of clinical failures of single crystal ceramic brackets. am j orthod dentofacial orthop. 1993; 103: 537-44. 7. johnson g, walker mp, kula k. fracture strength of ceramic bracket tie wings subjected to tension. angle orthod. 2005; 75: 95-100. 8. aknin pc, nanda rs, duncanson mgjr, currier gf, sinha pk. fracture strength of ceramic brackets during arch wire torsion. am j orthod dentofacial orthop. 1996; 109: 22-7. 9. holt mh, nanda rs, duncanson mgjr. fracture resistance of ceramic brackets during arch wire torsion. am j orthod dentofacial orthop. 1991; 99: 287-93. 10. nikolai rj. bioengineering analysis of orthodontic mechanics: lea & febiger, philadelphia; 1985. p.299-305. 11. nishio c, mendes am, almeida ma, tanaka e, tanne k, elias cn. evaluation of esthetic brackets’ resistance to torsional forces from the archwire. am j orthod dentofacial orthop. 2009; 135: 42-8. 12. lohbauer u, amberger g, quinn gd, scherrer ss. fractographic analysis of a dental zirconia framework: a case study on design issues. j mech behav biomed mater. 2010; 3: 623-9. 13. scoot gej. fracture toughness and surface cracks – the key to understanding ceramic brackets. angle orthod. 1988; 58: 5-8. 14. russel js. aesthetic orthodontic brackets. j orthod. 2005; 32: 146-63. 15. gibbs sl. clinical performance of ceramic brackets: a survey of the british orthodontist’s experience. br j orthod. 1992; 19: 191-7. 16. tp orthodontics. available from: www.invubrackets.com. 17. anuavice kj. phillips science of dental materials. philadelphia, penn: saunders; 2003. p.85-6. fracture strength of ceramic brackets submitted to archwire torsional strain oral sciences n3 braz j oral sci. 10(2):79-82 received for publication: january 01, 2011 accepted: april 19, 2011 original article braz j oral sci. april | june 2011 volume 10, number 2 antimicrobial activity of chlorhexidine in patients with fixed orthodontic appliances nakas enita1, vildana dzemidzic1, alisa tiro1, enes pasic2, sanja hadzic2 1dds, msc, senior research assistant, department of orthodontics, school of dentistry, university of sarajevo, sarajevo, bosnia and herzegovina 2dds, msc, senior research assistant, department of oral medicine and periodontology, school of dentistry, university of sarajevo, sarajevo, bosnia and herzegovina correspondence to: nakas enita department of orthodontics, school of dentistry, university of sarajevo bolnièka 4a , 71 000 sarajevo, bosnia and herzegovina phone: ++ 387 61 10 02 29 e-mail: enitta@gmail.com abstract aim: oral environment of orthodontic patients undergoes changes, such as ph reduction, larger number of sites available for streptococcus mutans collection, and increased accumulation of food particles, which may lead to an increased number of s. mutans colony-forming units (cfu) in saliva. chlorhexidine gluconate (chx) is the most potent documented antimicrobial agent against mutans streptococci and dental caries. the aim of this work was to assess efficiency of chx-based mouthwashes in patient with fixed orthodontic appliances. methods: twenty patients with fixed appliances were selected for this study. they were undergoing full-bonded non extraction treatment with metal brackets (roth 0.22, discovery dentaurum) on their teeth and bands on their molars. each patient was provided with a toothbrush and toothpaste. the mouthwashes were used by patients according to the manufacturers’ directions 15 min after toothbrushing: 5 ml of 0.2% chx (corsodyl, glaxosmithkline) was applied for 60 s in the morning and before bedtime for 2 weeks. results: the data were analyzed according to the nonparametric wilcoxon signed rank test (spss software). statistical significance level was set at p<0.05. conclusions: the use of chx-based mouthwashes in patients with fixed orthodontic appliances led to reduction in the level of s. mutans. keywords: chlorhexidine, streptococcus mutans, orthodontic treatment. introduction bonded orthodontic appliances increase the number of plaque retention sites, reduce the possibility self-cleaning on vestibular areas and impede oral hygiene. as of result of this, the oral cavity of orthodontic patients undergoes changes, such as ph reduction, larger number of sites available for streptococcus mutans collection, and increased accumulation of food particles, which may lead to an increased number of s. mutans colony-forming units (cfu) in saliva1. dental plaque is a multi-species biofilm consisting of more than 700 species of oral bacteria on tooth surfaces, and mutans streptococci (ms) are the major group of early primary colonizers2. ms are microorganisms that primarily colonize dental surfaces. at concentrations of 104-105 cfu/ml in saliva, s. mutans is able to colonize clean, smooth surfaces of teeth. the presence of ms on tooth surfaces increases the possibility of caries development3. due to some of their vital characteristics, ms are regarded potentially highly cariogenic4. therefore, preventive efforts in these risk groups have concentrated on direct suppression of the cariogenic microflora by chemotherapeutics as an adjunct to improved oral hygiene. chlorhexidine gluconate (chx) is the most potent documented antimicrobial agent against ms and dental caries5. mouth rinsing with a chemical agent could 80 braz j oral sci. 10(2):79-82 be a useful clinical adjunct for reducing the bacterial plaque accumulation during the active phase of orthodontic treatment. chemical agents also help orthodontic patients who have difficulties in maintaining plaque control by mechanical means alone. the aim of this work was to assess efficiency of chx-based mouthwashes in patients with fixed orthodontic appliances. material and methods twenty patients with fixed appliances were selected for this study. the following criteria were used for selecting patients who were undergoing full-banded non extraction treatment with metal brackets (roth 0.22, dentaurum gmbh & co., ispringen, germany) on their teeth and bands on their molars who: were at least 13 and no more than 18 years old with complete permanent dentition including second molars; had no evidence of decalcification on their teeth; had no active caries lesion; had no known medical problem or current antibiotic therapy; had anterior composite fillings; had no known hypersensitivity to chx. each patient was provided with a toothbrush, a toothpaste and a leaflet with brushing instructions; all patients were instructed to brush for a minimum of 3 min. the same protocol was followed for each patient. first, the gingiva of individual teeth was examined and scoring was performed, according to the following scoring system: 0: normal gingiva; 1: slight inflammation a minor change in color, consistency (no bleeding on probing); 2: average inflammation redness, swelling (bleeding on probing); 3: severe inflammation strong redness and swelling (spontaneous bleeding). final score was obtained when the total score was divided by the number of examined teeth. after collection of stimulated saliva sample for the purpose of establishing the quantities of s. mutans and lactobacillus spp.. saliva samples were collected at two timelines for each patient: 1. before use of chx (at least 1 month after bonding); 2. after 2 weeks of use of chx. for such purpose, colony counting test kit (crt bacteria, ivoclar vivadent, liechtenstein) was used, according to manufacturer’s instruction. patients were given a piece of (factory-made) wax, which they chewed for 5 min in order to collect enough stimulated saliva. while chewing, they collected saliva into disposable sterile plastic cups. after 5 min, disposable pipettes were used to take saliva and seed it on slides for streptococci mutans and lactobacilli spp. the protective coat from sterile slides was removed, 5 drops were neatly dripped on the slide (until its entire surface was covered in saliva), which was held under a slight angle, taking special care not to damage the surface of the slide with the sharp edge of pipette. once the procedure was over, a tablet of nahco3 was put in slide boxes, and then the boxes were closed and placed into an incubator at 37°c. forty-eight hours later, the results were read as indicated in the manufacturer’s scheme, according to the following scale: class 1= <104 cfu/ml saliva; class 2=104-105 cfu/ml saliva; class 3=105-106 cfu/ml saliva; class 4=>106 cfu/ml saliva (figure 1.) the mouthwashes were used by patients according to the manufacturer’s directions 15 min after toothbrushing: 5 ml of 0.2% chx (corsodyl, glaxosmithkline) was applied for 60 s in the morning and at bedtime for 2 weeks. the patients were instructed not to consume any liquid or food or at least 30 min after using the prescribed mouthwash. all patients are asked to bring the mouthwash bottle, so we could determine patient compliance based on how much liquid was left. the data were analyzed using the nonparametric wilcoxon signed rank test (spss software, spss inc., chicago, il, usa). statistical significance level was set at p <0.05 results the results of the statistical analysis of ms levels, lactobacillus spp. levels and gingival tissue status before and after use of chx mouthwash are presented in tables 1, 2 and 3. the distribution of s. mutans before and after use of chx mouthwash is given in table 4. the findings of the present study indicate that the use of chx mouthwashes reduced s. mutans levels (p = 0.002) in patients with fixed orthodontic appliances. thirteen patients presented lower s. mutans while in 6 patients s. mutans counts remained the same regardless of use of the antimicrobial agent. chx-based mouthwash had no significant impact on lactobacillus spp. levels, as 5 patients presented higher counts, 5 presented lower counts while in 10 patients lactobacillus spp. counts remained the same regardless of use of the antimicrobial agent. antimicrobial activity of chlorhexidine in patients with fixed orthodontic appliances fig. 1 mutans streptococci (cfu/ml saliva) 81 n mean rank sum of ranks strept2 strept1 negative ranks 13 7.58 98.50 positive ranks 1 6.50 6.50 tied ranks 6 s u m 20 z -3.116 asymp. sig. (2-tailed) .002 table 1.table 1.table 1.table 1.table 1. statistical analysis of ms levels before and after use of chx mouthwash. n mean rank sum of ranks lakt2 lakt1 negative ranks 5 5.50 27.50 positive ranks 5 5.50 27.50 tied ranks 10 s u m 20 z .000 asymp. sig. (2-tailed) 1.00 table 2.table 2.table 2.table 2.table 2. statistical analysis of lactobacillus spp. levels before and after use of chx mouthwash. n mean rank sum of ranks ging2 ging1 negative ranks 5 3.00 15.00 positive ranks 0 .00 .00 tied ranks 15 s u m 20 z -2.032 asymp. sig. (2-tailed) .042 table 3.table 3.table 3.table 3.table 3. statistical analysis of gingival tissue status before and after use of chx mouthwash. streptococcus mutans baseline after chlorhexidine class (%) (%) no detected 0 10 class 1= <104 cfu/ml saliva 0 15 class 2=104-105 cfu/ml saliva 20 10 class 3=105-106 cfu/ml saliva 15 40 class 4=>106 cfu/ml saliva 65 25 table 4.table 4.table 4.table 4.table 4. distribution of s. mutans before and after use of chx mouthwash discussion the findings of the present study regarding the effect of the chx mouthwash on the microbial counts are in accordance with those of other authors. oltramari-navaro et al. (2009)6 suggested that the use of dentifrices with lower chx concentration can reduce the risk of tooth s t a i n i n g w i t h o u t c o m p r o m i s i n g i t s e f f e c t i v e n e s s i n controlling gingivitis and bleeding in orthodontic patients. m a s e k e t a l . ( 2 0 0 8 ) 7 f o u n d t h a t c e r v i t e c ® c a u s e d a reduction of s. mutans and lactobacillus spp. saliva counts in patients with previously identified high level of these bacteria. sari (2007)8 reported that 0.2% chx mouthwash decreased s. mutans levels, but had no effect on lactobacilli levels. olympio et al. (2006)9 observed that the use of dentifrices containing chx seems to be effective for the treatment of gingivitis in orthodontic patients. beyth (2003)10 found a significant reduction of s. mutans cfu in 10 patients after use of chx oral rinse10. according to eldridge (1998)11, the use of chx mouthwash reduces the levels of s. mutans, gingival index and gingival bleeding. anderson (1997)12 found that the use of chx oral rinse contributes to improving oral hygiene in patients with fixed orthodontic appliances12. in the present study, 6 patients reported discoloration of teeth as a result of the treatment, which is a possible s i d e e f f e c t o c h x . e l d r i d g e ( 1 9 9 8 ) 1 1 d e s c r i b e d t h e appearance of brown color tongue that spontaneously resolved after cessation of the use of chx, which, however, was not observed in the present study. anderson (1997)12 observed discoloration of teeth and increase in dental c a l c u l u s f o r m a t i o n , b u t t h e y d i d n o t d e t e r m i n e t h e significance of these phenomena. our results corroborate previous findings about the influence of chx on s. mutans levels, oral hygiene and gingival condition13-14. in the present study, the use of chx-based mouthwashes reduced s. mutans counts and improved the gingival index, but had no significant impact on lactobacillus counts. braz j oral sci. 10(2):79-82 antimicrobial activity of chlorhexidine in patients with fixed orthodontic appliances 82 references 1. mota sm, enoki c, ito iy, elias am, matsumoto ma. streptococcus mutans counts in plaque adjacent to orthodontic brackets bonded with resin-modified glass ionomer cement or resin-based composite. braz oral res. 2008; 22: 55-60. 2. yanti, rukayadi y, lee kh, hwang jk. activity of panduratin a isolated from kaempferia pandurata roxb. against multi-species oral biofilms in vitro. j oral sci. 2009; 51: 87-95. 3. nakas e, zukanovic a.the prevalence of cariogenic salivary microorganisms in children of various ages. bosn j basic med sci. 2007; 7: 166-70. 4. gibbons rj, cohen l, hay di. strains of streptococcus mutans and streptococcus sobrinus attach to different pellicle receptors. infect immun. 1986; 52: 555-61. 5. attin r, ilse a, werner c, wiegand a, attin t. antimicrobial effectiveness of a highly concentrated chlorhexidine varnish treatment in teenagers with fixed orthodontic appliances. angle orthod. 2006; 76: 1022-7. 6. oltramari-navarro pv, titarelli jm, marsicano ja, henriques jf, janson g, lauris jr et al. effectiveness of 0.50% and 0.75% chlorhexidine dentifrices in orthodontic patients: a double-blind and randomized controlled trial.. am j orthod dentofacial orthop. 2009; 136: 651-6. 7. i. mašek, d. matoševic, h.juric, s.meštrovic antimicrobial effects of chlorhexidine in orthodontic patients ascro. acta stomatol croat. 2008; 42: 41-8. 8. sari e, birinci i. microbiological evaluation of 0.2% chlorhexidine gluconate mouth rinse in orthodontic patients. angle orthod. 2007; 77: 881-4. 9. olympio kp, bardal pa, de m bastos jr, buzalaf ma effectiveness of a chlorhexidine dentifrice in orthodontic patients: a randomized-controlled trial. j clin periodontol. 2006; 33: 421-6. 10. beyth n, redlich m, harari d, friedman m, steinberg d. effect of sustained-release chlorhexidin digluconate varnish on streptococcus mutans and actinomyces viscosus in orthodontic patients.; am j orthod dentofacial orthop. 2003; 123: 345-8. 11. eldridge kr, finnie sf, stephens ja, mauad am, munoz ca, kettering jd. efficacy of an alcohol-free chlorhexidin digluconate mouthrinse as an antimicrobial agent. j prosthet dent. 1998; 80: 685-90. 12. anderson gb, bowden j, morrison ec, caffesse rg.: clinical effects of chlorhexidin digluconate mouthwashes on patients undergoing orthodontic treatment.; am j orthod dentofacial orthop. 1997;111: 606-12. 13. schaeken mj, van der hoeven js, hendriks jc. effects of varnishes containing chlorhexidine on the human dental plaque ûora. j dent res. 1989; 68: 1786-9. 14. lundstrom f, krasse b. streptococcus mutans and lactobacilli frequency in orthodontic patients: the effect of chlorhexidine treatments. eur j orthod. 1987; 9:109-16. braz j oral sci. 10(2):79-82 antimicrobial activity of chlorhexidine in patients with fixed orthodontic appliances oral sciences n3 original article braz j oral sci. october | december 2012 volume 11, number 4 morse taper implants at different bone levels: a finite element analysis of stress distribution marcelo bighetti toniollo1, ana paula macedo2, daniel palhares3, paulo linares calefi4, danilo balero sorgini5, maria da gloria chiarello de mattos6 1specialist in prosthodontics; master of science and phd student of oral rehabilitation at the dental school of ribeirão preto – university of são paulo, department of dental materials and prosthodontics, ribeirão preto, sp, brazil 2master of science and phd student at the school of medicine of ribeirão preto – university of são paulo; technical responsible of the metrology laboratory of dental school of ribeirão preto – university of são paulo, department of dental materials and prosthodontics, ribeirão preto, sp, brazil 3specialist in prosthodontics; master of science student of health sciences – implantology – unifeb, barretos, sp, brazil 4specialist in prosthodontics; master of science student of oral rehabilitation at the dental school of ribeirão preto – university of são paulo, department of dental materials and prosthodontics, ribeirão preto, sp, brazil 5specialist in prosthodontics; master of science and phd student of oral rehabilitation at the dental school of ribeirão preto – university of são paulo, department of dental materials and prosthodontics, ribeirão preto, sp, brazil 6full professor at the dental school of ribeirão preto – university of são paulo, department of dental materials and prosthodontics, ribeirão preto, sp, brazil correspondence to: marcelo bighetti toniollo departamento de materiais dentários e prótese, universidade de são paulo av. josé adriano arrobas martins, 130, nova aparecida, cep: 14883-298 jaboticabal, sp, brasil phone: + 55 16 32356643 / +55 16 97096643 e-mail: martoniollo@yahoo.com.br abstract aim: to explore the biomechanical effects of the different implantation bone levels of morse taper implants, employing a finite element analysis (fea). methods: dental implants (titamaxcm) with 4x13 mm and 4x11 mm, and their respective abutments with 3.5 mm height, simulating a screwed premolar metal-ceramic crown, had their design performed using the software ansysworkbench10.0. they were positioned in bone blocks, covered by 2.5 mm thickness of mucosa. the cortical bone was designed with 1.5 mm thickness and the trabecular bone completed the bone block. four groups were formed: group 11cbl (11 mm implant length on cortical bone level), group 11tbl (11 mm implant length on trabecular bone level), group 13cbl (13mm implant length on cortical bone level) and group 13tbl (13 mm implant length on trabecular bone level). oblique 200 n loads were applied. von mises equivalent stresses in cortical and trabecular bones were evaluated with the same design program. results: the results were shown qualitatively and quantitatively by standard scales for each type of bone. by the results obtained, it can be suggested that positioning the implant completely in trabecular bone brings harm with respect to the generated stresses. its implantation in the cortical bone has advantages with respect to better anchoring and locking, reflecting a better dissipation of the stresses along the implant/bone interfaces. in addition, the search for anchoring the implant in its apical region in cortical bone is of great value to improve stabilization and consequently better stress distribution. conclusions: the implant position slightly below the bone in relation to the bone crest brings advantages as the best long-term predictability with respect to the expected neck bone loss. keywords: biomechanics, bone, dental implants, finite element analysis. introduction the use of dental implants in contemporary dentistry has become a reality, bringing many solutions, but also problems related to various factors (both clinical and biomechanical)1-2. because the proper functioning of implants is based on received for publication: june 17, 2012 accepted: september 18, 2012 braz j oral sci. 11(4):440-444 osseointegration, the relationship between biomechanical implants and the surrounding hard tissue is of great importance, as well as its spatial positioning and implantation insertion depth. the quality and type of bone involved in implantology have been extensively studied3-7, and is clear that there is importance and relevance of these factors in effective and appropriate force dissipation generating or not a favorable prognosis to the implants. the basic biomechanical difference between cortical and trabecular bones is linked to their different modulus of elasticity, as well as their different types, and this is where studies involving finite elements differ in their analyzes5,7-10. the higher the modulus of elasticity in simulated analysis, the greater the simulated bone density (type i to type iv). de almeida et al.7 obtained results with the highest maximum principal stress in bones of type iii and iv. three unilateral posterior loads of 150 n were used (perpendicular to the prefabricated bar; 30 degrees in a buccolingual direction and 30 degrees in a linguobuccal direction). they also concluded that the bone type should not exclusively be the only determining factor in stress distribution. these authors also affirmed that there are various other factors that influence the pattern of stress distribution, such as implant design, length and diameter, applied forces, implant insertion depth and type of internal connection. the morse taper implant has been highlighted on several positive features, such as its ability to decrease bacterial contamination between implant and prosthesis, more aesthetic predictable and biological quality of the peri-implant tissue, in addition to reducing the risk of loosening the prosthetic screw1112. quaresma et al.13 also showed that the morse taper connection dissipates less stress to the implant surrounding bone than the internal hexagonal connection. moreover, some manufacturers’ recommendation is that the best positioning of morse taper implants has to be slightly below the bone in relation to the bone crest; however if it is placed too deep, it can bring too many differences regarding the stresses distribution. evidence found by akça and cehreli14 revealed that the gradual loss of marginal bone around the implant led to considerable increase of stress in trabecular bone in contact with the cervical region of the implant. this simulated effect of bone resorption would be equivalent to the deeper implantation of the implant, since the loss of cortical bone would result in only trabecular bone support of the implant, which has different characteristics from the first one. similar findings were found in okumura et al.15 study. the aim of this study was to explore the biomechanical effects in the peri-implant bone of the different implantation bone levels of morse taper implants. material and methods the implants used in this study (profile projector nikon model 6c and stereomicroscope leica model s8ap0) were measured so that it could have higher degree of fidelity. the implants and respective abutment used are shown in table 1 and figure 1. it was standardized only 4 mm for implant diameter in order not to create another variable and only evaluate the correlation between implant length and different implantation bone levels. the implant prostheses were designed to be screwed premolar metal-ceramic crowns. three-dimensional finite element graphic models reproduction of all prosthetic elements and implants required for this study, as well as a bone block in which the implants were inserted, were performed using the program ansysworkbench10.0 (swanson, analysis systems, inc., houston, tx, usa). morse taper implant length(mm) abutment height(mm) trademark titamax cm 13 11 pilar cm 3.5 table 1. type and lengths of 4 mm implants diameter and abutment height neodent fig. 1. abutments (pilar cm 3.5mm) positioned with the respective implants (titamax cm – 13 mm and 11 mm length). the implants were positioned in bone blocks, “covered” of 2.5 mm thickness of mucosa. the cortical bone was designed with 1.5 mm thickness and the trabecular bone completed the bone block, both configuring a 1,742 mm3 total volume. four groups were formed: group 11cbl (11 mm implant length on cortical bone level), group 11tbl (11 mm implant length on trabecular bone level), group 13cbl (13 mm implant length on cortical bone level) and group 13tbl (13 mm implant length on trabecular bone level) (figure 2). the results were analyzed by a color scale, where each tone corresponds to an amount of stress generated in the structures, and how they were distributed over the analyzed structures (implants, abutments, bone, or any other object of 441441441441441morse taper implants at different bone levels: a finite element analysis of stress distribution braz j oral sci. 11(4):440-444 group 11cbl group 11tbl group 13cbl group 13tbl proximal view of the groups morse taper implants at different bone levels internal view of the vestibular face cortical legend cortical bone trabecular legend trabecular bone fig. 2. groups formed with morse taper implants at different bone levels and vmes for cortical and trabecular bones structure modulus of elasticity/young (mpa) poisson’s ratio (v) references trabecular bone 1,370 0.30 ko et al., 199223; sertgoz, 199725 cortical bone (1.5 mm) 13,700 0.30 ko et al., 199223; papavasiliou et al., 199626; assif et al., 199627 mucosa (2.5 mm) 19,6 0.30 ko et al., 199223; reinhardt et al., 198324 implant (ti) 110,000 0.35 ciftci & canay, 200018; pierrisnard et al, 200328; monteith, 199329; cocr structure 218,000 0.33 anusavice, 200330; craig, 199731 resin 7,000 0.2 craig, 198932 porcelain 82,800 0.35 sertgoz, 199725 table 2. mechanical properties of materials analysis) in the three directions of space (x, y and z). oblique loads (approximately 45o) in the linguobuccal direction with 200 n of intensity were applied and von mises equivalent stresses (vmes) on cortical and trabecular bones were evaluated. all the specific properties of each structure involved in the simulations (modulus of elasticity/young and poisson’s ratio) are presented in table 2. standard scales for each type of bone were constructed to analyze qualitatively and quantitatively. it was considered the internal face of the buccal side for stress analysis because of their greater relevance considering the direction of oblique forces. in the absence of information about the precise organic properties of the cortical and trabecular bones and mucosa, they were assumed to be homogeneous, isotropic, and linearly elastic as were the other materials used in the analysis. results the vmes were evaluated, which represents the mean of the stresses in all directions, in different groups. quantitative comparisons were made between the different groups to determine the generated stresses in cortical and trabecular bones (figure 2). the vmes values ranged from 0 to 40 mpa in cortical bone, and from 0 to 7 mpa in trabecular bone, approximately. in general, the absolute values of stress on cortical bone were the same, but with greater width in the groups with cortical bone level implant (cbl). for the trabecular bone stress, it was higher in the groups with trabecular bone level implant (tbl) and reaching higher absolute values than cbl. 442442442442442 morse taper implants at different bone levels: a finite element analysis of stress distribution braz j oral sci. 11(4):440-444 discussion the objective of this study, using three-dimensional finite element analysis (fea), was not to replicate exact in vivo stresses, but rather to illustrate a possible difference of the stress distribution on different bone levels of morse taper implants. fea allows a better understanding of implants’ biomechanical aspects and how such stress occurs on the surrounding bone. long-term clinical research is required to determine the influence of observed stress levels on implants and surrounding bone16. bone quality has been considered the most critical factor for implant success at both surgical and functional stages, and it is therefore suggested that occlusal overload in poor quality bone can be a clinical concern for implant longevity3. in studies n human patients, higher failures of implants were observed in bone with poor quality 8-9. tada et al.5 also confirms the importance of bone quality and its pre-surgical diagnosis for implant long-term prognosis. the results of their study suggest that trabecular bone with higher bone density might ensure a better biomechanical environment for implants. rubo and souza17 concluded in fea that stresses tended to be concentrated at the cortical bone around the cervical region of the implant closest to the load, whereas stresses in trabecular bone were considered low. contrary to the findings of baggi et al.18 and the results in this study, chou et al.19 concluded that, evaluating the biomechanical response of the jaw bone with wide-diameter and short implants versus narrow-diameter and long implants, using fea, there was more even and higher strain distribution in the peri-implant bone at the wide-diameter and short implant as compared with the narrow-diameter and long implant, apart from the fact that stress levels in peri-implant bone were reduced as the insertion depth of the implant was increased. these findings may be due to the comparative association just of the wide-diameter and short implants to the narrow-diameter and long implants. the short length of the implants may have been superimposed on the beneficial effect of large diameter, also interfering with the stress in relation to the insertion depth. undue tensions, originated from a possible occlusion maladjusted, poorly positioned implant or even due to the presence of poor quality bone, can lead to injury or bone resorption20. according to isidor6, occlusal forces affect an oral implant and the surrounding bone, and according to the bone physiology theories, bones carrying mechanical loads adapt their strength to the load applied on it by bone modeling/remodeling. the phenomenon of bone resorption in the form of a saucer around the cervical region of implants called saucerization may arise from both exacerbated tensions in the region or even local biological factors, such as bone loss is observed also in non-loaded implants21. another possible cause of this bone loss is related to the low stresses acting on the peri-implant bone. an equivalent stress of 1.6 mpa has been deemed sufficient to avoid crestal bone loss from disuse atrophy in the mandibular canine-premolar region22. akça and cehreli14 simulated by fea the gradual loss of peri-implant bone. the authors concluded that this loss is highly prejudicial to the biomechanical system. the presence of cortical bone contacting a load-carrying implant, even in a bone defect, improves the biomechanical performance of implants in comparison with only trabecular bone support as a sequel of progressive marginal bone loss. these findings are totally in favor of the results present in this study. pierrisnard et al.23 specifically studied the bicortical anchorage effect on the transferred stress to implant components, the implant proper, and the surrounding bone. such as in this study, the authors showed that the stress concentrated to the cortical bone, at the cervical area, and affirmed that the use of long implants (more than 10 mm) is a positive factor in osseointegration. however, this does not always result in better stress distribution to the implant components and bone, as if the cervical portion of the cortical anchoring of the implant is good, the influence of implant length becomes less important. in this study, stresses were higher in cortical bone, but this should not represent a risk factor because it was expected due to its higher modulus of elasticity. this finding was also found by baggi et al.18, and that implant biomechanical behavior greatly improves efficiently if bone is preserved at the crest. the literature reports various values of the elastic and plastic boundary deformation, which may be mentioned an average of 140 mpa for cortical bone and 10 mpa for trabecular bone24-25. trabecular bone stresses were higher when the implants were at this level (tbl), and better distributed for greater implant length and anchored with its apex in the cortical bone. qian et al.26 investigated the interactions between diameter, insertion depth, and load angle applied on the implant by three-dimensional finite element analysis. the authors concluded that a narrow-diameter implant, when inserted into jawbone with a shallow insertion depth and loaded with an oblique loading angle, is most unfavorable for stress distribution in both bone and implant. this result may have been found because with greater depth of implant installation the lever arm portion of the prosthesis has less effect. however, it has also to be considered that the implant in the cortical bone tends to increase its stability and locking, as reported by pierrisnard et al.23. okumura et al.15 performed a fea to investigate the effect of maxillary cortical bone thickness, implant design and diameter on stress around implants and the von mises stresses were calculated. regardless of load direction, implant design and diameter, cortical and trabecular bone stresses increased with the decrease of crestal cortical bone thickness. in the absence of crestal cortical bone, trabecular bone stresses were highest and, under axial load, were transferred to the sinus floor. from a biomechanical viewpoint, to improve implant success odds in the posterior maxilla, rather than implant selection, careful preoperative evaluation of the cortical bone at the planned implant site is recommended. if this cortical bone is too thin or even lacking, implant treatment 443443443443443morse taper implants at different bone levels: a finite element analysis of stress distribution braz j oral sci. 11(4):440-444 should be carried on with caution by progressive loading in the range of functional loads. thus, just as observed by akça and cehreli14, okumura et al.15 and pierrisnard et al.23, the loss of cortical bone at the cervical region of the implant can cause biomechanical harm at the implant/bone interface. within the limitations of this in vitro study, the following conclusions can be drawn: • according to the literature, the implant position slightly below the bone in relation to the bone crest brings advantages already known as the best long-term predictability with respect to the expected bone loss around its cervical region, but also the best behavior of soft periimplant tissue; • however, by the results obtained, it can be suggested that the positioning of the implant completely in trabecular bone brings harm with respect to the generated stresses. its implantation in the cortical bone has advantages with respect to better anchoring and locking, reflecting the better dissipation of the stresses along the implant/bone interfaces. • in addition, the search for anchoring the implant in its apical region in cortical bone is of great value to improve stabilization and consequently better stress distribution. references 1. goodacre cj, bernal g, rungcharassaeng k, kan jyk. clinical complications with implants and implant prostheses. j prosthet dent. 2003; 90: 121-32. 2. davarpanah m, martinez h, tecucianu jf, celletti r, lazzara r. smalldiameter implants: indications and contraindications. j esthet dent. 2000; 12: 186-94. 3. misch ce. density of bone: effect on treatment plans, surgical approach, healing, and progressive loading. j oral implant. 1990; 6: 22-31. 4. akagawa y, sato y, teixeira er, shindoi n, wadamoto m. a mimic osseointegrated implant model for three-dimensional finite element analysis. j oral rehabil. 2003; 30: 41-5. 5. tada s, stegaroiu r, kitamura e, miyakawa o, kusakari h. influence of implant design and bone quality on stress/strain distribution in bone around implants: a 3-dimensional finite element analysis. int j oral maxillof implants. 2003; 18: 357-68. 6. isidor f. influence of forces on peri-implant bone. clin oral impl res. 2006; 17: 8-18. 7. de almeida eo, rocha ep, freitas acjr, martin mjr. finite element stress analysis of edentulous mandibles with different bone types supporting multipleimplant superstructures. int j oral maxillofac implants. 2010; 25: 1108-14. 8. jaffin ra, berman cl. the excessive loss of branemark fixtures in type iv bone: a 5-year analysis. j periodontol. 1991; 62: 2-4. 9. becktor jp, eckert se, isaksson s, keller ee. the influence of mandibular dentition on implant failures in bone-grafted edentulous maxillae. int j oral maxillofac implants. 2002; 17: 69-77. 10. guan h, staden r, loo yc, johnson n, ivanovski s, meredith n. incluence of bone and dental implant parameters on stress distribution in the mandible: a finite element study. int j oral maxillofac implants. 2009; 24: 866-76. 11. sutter f, weber h, sorensen j. the new restorative concept of the iti dental implant system: design and engineering. int j periodontics restorative dent. 1993; 13: 409-31. 12. binon pp, sutter f, beaty k, brusnki j, gulbransen h, weiner r. the role of screws in implant systems. int j oral maxillofac implants. 1994; 9(suppl): 48-63. 13. quaresma set, cury pr, sendyk wr, sendyk c. a finite element analysis of two different dental implants: stress distribution in the prosthesis, abutment, implant, and supporting bone. j oral implantol. 2008; 34: 1-6. 14. akça k, cehreli mc. biomechanical consequences of progressive marginal bone loss around oral implants: a finite element stress analysis. med bio eng comput. 2006; 44: 527-35. 15. okumura n, stegaroiu r, kitamura e, kurokawa k, nomura s. influence of maxillary cortical bone thickness, implant design and implant diameter on stress around implants: a three-dimensional finite element analysis. j prosthodont res. 2010; 54: 133-42. 16. tawil p, tawil g. short implants in deficient posterior jaws: current knowledge. implant dent. 2009; 46: 9-16. 17. rubo jh, souza eac. finite element analysis of stress in bone adjacent to dental implants. j oral implantol. 2008; 34: 248-255. 18. baggi l, cappelloni i, girolamo md, maceri f, vairo g. the influence of implant diameter and length on stress distribution of osseointegrated implants related to crestal bone geometry: a three-dimensional finite element analysis. j prosthet dent. 2008; 100: 422-31. 19. chou hy, muftu s, bozkava d. combined effects of implant insertion depth and alveolar bone quality on periimplant bone strain induced by a wide-diamenter, short implant and a narrow-diameter, long implant. j prosthet dent. 2010; 104: 293-300. 20. erkmen e, meriç g, kurt a, tunç y, eser a. biomechanical comparison of implant retained fixed partial dentures with fiber reinforced composite versus conventional metal frameworks: a 3d fea study. j mech behav biomed mater. 2010; 4: 107-16. 21. consolaro a, carvalho rs, francischone jr ce, consolaro mfmo, francischone ce. dental implants saucerization and orthodontic clinical cases. dent press j orthod. 2010; 15: 19-30. 22. geng j, tan kbc, liu g. application of finite element analysis in implant dentistry: a review of the literature. j prosthet dent. 2001; 85: 585-98. 23. pierrisnard l, renouard f, renault p, barquins m. influence of implant length and bicortical anchorage on implant stress distribution. clin implant dent relat res. 2003; 5: 254-62. 24. martens m, audekercke rv, delport p, meester pd, mulier jc. the mechanical characteristics of cancellous bone at the upper femoral region. j biomech. 1983; 16: 971-83. 25. jensen nc, madsen lp, linde f. topographical distribution of trabecular bone strength in the human os calcanei. j biomech. 1991; 24: 49-55. 26. qian l, todo m, matsushita y, koyano k. effects of implant diameter, insertion depth, and loading angle on stress/strain fields in implant/jawbone systems: finite element analysis. int j oral maxillofac implants. 2009; 24: 877-86. 444444444444444 morse taper implants at different bone levels: a finite element analysis of stress distribution braz j oral sci. 11(4):440-444 oral sciences n3 braz j oral sci. 11(2):130-134 original article braz j oral sci. april | june 2012 volume 11, number 2 laser fluorescence on dental caries detection under epidemiological setting renato pereira da silva1, andréa vieira assaf2, marina vannucci mena romeiro3, aline sampieri tonello bennazzi4, gustavo antônio martins brandão5, antonio carlos pereira6 1dds, msc, phd, collaborator professor, department of community dentistry, piracicaba dental school, university of campinas, brazil 2dds, msc, phd, professor, department of community services, fluminense federal university, brazil 3dds, piracicaba dental school, university of campinas, piracicaba, sp, brazil 4dds, msc, phd, substitute professor, department of public health, federal university of maranhão, brazil 5dds, msc, phd, professor, department of orthodontics, federal university of pará, brazil 6dds, msc, phd, professor, department of community dentistry, piracicaba dental school, university of campinas, brazil correspondence to: antonio carlos pereira faculdade de odontologia de piracicaba, unicamp av limeira, 901, areião cep: 13414-903 piracicaba, sp, brasil phone: +55 19 21065209 e-mail: apereira@fop.unicamp.br abstract aim: to evaluate reproducibility of visual exam under artificial lighting (vi) and diagnodent™ (dd) exams, their validity and their association (vi+dd) in examining occlusal dental surfaces under epidemiological setting. methods: one hundred and sixty five 12-year-old schoolchildren from public schools from piracicaba, sp, brazil, were examined under epidemiological setting using different diagnostic criteria (d1, d1+d3 and d3). kappa statistics was adopted for reproducibility study, while sensibility, specificity and accuracy were adopted for validity study. results: the intra/interexaminer kappa for vi and dd exams at d1, d3 and d1+d3 criteria were 0.88/0.82 and 0.04/0.18, 0.91/0.85 and 0.36/0.35, 0.88/0.83 and 0.30/0.32, respectively. the vi was the most accurate exam at d1 (a=87.38%) and d1+d3 (a=82.45%) criteria, while vi+dd was the most accurate exam at d3 criterion (a=75.29%). conclusions: initial caries lesions can be satisfactorily detected by vi exam, while dd exam improved the vi exam sensitivity at d1 and d3 diagnostic criteria under epidemiological setting. keywords: dental caries, epidemiology, dental health surveys, diagnosis; lasers. introduction dental caries is currently seen as the reflex of signs of a past or ongoing disease, resulting from the dynamic balance between de-/re-mineralization of hard dental tissues, being reversible provided it is detected early. dental caries is a process beginning with subclinical manifestations of enamel demineralization at atomic level, culminating in spontaneous cavitation of dental surface. the closest dentists can come to measuring the carious process itself is by the clinical detection of evidence of past occurrence of this process on the dental surfaces1. one of the challenges of modern dentistry is the early diagnosis of dental caries in occlusal tooth surfaces, particularly in children and young adults. dentists are faced with a new epidemiological profile of this disease, characterized by a considerable portion of the world’s child population being free of dental caries, with less prevalence of cavited carious lesions and greater prevalence of initial carious lesions in dental enamel, an increasing prevalence of ‘hidden caries’ lesions received for publication: april 12, 2012 accepted: may 04, 2012 braz j oral sci. 11(2):130-134 131131131131131 visual exam diagnodent™ bitewing (benchmark examiner) code classification code (reading values) classification code classification 0 sound 0 (0 – 10) sound surface 0 absent surface il enamel initial carious lesions 1 (11 – 20) enamel carious lesions r 1 radiolucency in enamel 1 decayed 2 (21 – 99) dentin carious lesions r 2 radiolucency in enamel/outer dentin 1il decayed + initial lesions r 3 radiolucency in dentin 2 filled, with decay 4 filled 2il filled, with decay + initial lesions 5 sound 3 filled r 6 impossible diagnosis 3il filled + initial lesions 4 missed by caries 5 missed by other reason 6 sealant 6il sealant + initial lesions 7 bridge abutment, special crown or veneer/implant 8 unerupted tooth t trauma 9 not recorded table 1. codes for dental exams in dentin, and polarization of dental caries through underprivileged socioeconomic groups2-5. new methodologies and diagnostic adjunct technologies have been suggested to improve the precision of the diagnosis of initial carious lesions5-8. laser fluorescence measurement of the hard dental tissues has shown to be quite promising for this purpose9-10. the aim of this study was to evaluate the reproducibility and validity of dental occlusal caries exams under epidemiological setting in a 12-year-old schoolchildren group. material and methods this study was approved by the ethics committee of piracicaba dental school/unicamp (protocol #082/2006), and conducted in accordance with resolutions 196/96 from the national health council of the brazilian ministry of health, and 179/93 of dental professional code of ethics from brazilian dental council. the study was conducted under a cross-sectional design in two stages. participation was voluntary and written informed consent was obtained from all children’s parents/caregivers. stage 1 the reproducibility of the visual exam under artificial lighting (vi) and the laser fluorescence exam performed with diagnodent™ 2095 (kavo, biberach, germany) (dd) on posterior tooth occlusal surfaces, under epidemiological conditions, was verified. one benchmark examiner and 3 experienced examiners participated. the examiners were submitted to nine 4-hour training/calibration sessions conducted by the benchmark examiner. the sample included thirteen 12-year-old randomly selected children enrolled in a public school of piracicaba, sp, brazil, presenting low to high prevalence of dental caries, who had not participated in the examiners’ previous clinical training. children with fixed orthodontic appliances, severe fluorosis and enamel hypoplasia or serious systemic disease were replaced by children free of such conditions. this number of participants and the interval between the calibration sessions was based on assaf et al. (2006)5. vi was performed with a flat buccal mirror, cpi probe (debris removal) and artificial lighting. all children performed supervised toothbrushing for 2 min (bass modified with fluoridated toothpaste) before the dental examination. each dental surface was dried for 5 s5 with a compressed air jet from a portable dental unit (proquest delivery system, model 4010; compressor technologies ltd, englewood, nj, usa) before the exams. at d1 diagnostic criterion, the tooth condition was codified on ‘with initial lesions on enamel’ and ‘without such condition’. at d1+d3 diagnostic criterion carious lesions (‘cavited or not’ and ‘none’) in enamel and/ or dentin were registered. at d3 diagnostic criterion, only dental caries in dentin or none, according to conventional world health organization (who) diagnostic criterion11 and suspected ‘hidden caries’ lesions were registered. the criteria and codes adopted as d1+d3 and d3 criteria were based on assaf et al. (2006)5 (table 1). the dd exam was accomplished in accordance with manufacturer’s recommendations. however the scale for interpreting and classifying its readings were defined by zanin et al. (2005)12 (table 1). the interexaminer agreement was obtained at first calibration session. for intraexaminer agreement, a second calibration session was performed after a 10-day interval to avoid bias memorization5. the weighted kappa statistics was adopted. the data obtained were not dichotomized. stage 2 the sample for this stage included 165 randomly selected 12-year-old schoolchildren with the same clinical laser fluorescence on dental caries detection under epidemiological setting braz j oral sci. 11(2):130-134 visual exam 1st calibration 2nd calibration d 1 d 3 d1+d3 d 1 d 3 d1+d3 intra – – – 0.88 (0.89) 0.91 (0.93) 0.88 (0.89) – – – [0.87 – 0.89] [0.86 – 0.95] [0.88 – 0.89] inter 0.82 0.85 0.83 0.83 0.83 0.84 [0.81 – 0.83] [0.81 – 0.87] [0.82 – 0.84] [0.81 – 0.87] [0.80 – 0.86] [0.82 087] diagnodent 1st calibration 2nd calibration d 1 d 3 d1+d3 d 1 d 3 d1+d3 intra – – – 0.04 (0.32) 0.36 (0.50) 0.30 (0.46) – – – [0.01 – 0.06] [0.23 – 0.52] [0.23 – 0.39] inter 0.15 0.35 0.32 0.18 0.77 0.60 [0.07 – 0.21] [0.23 – 0.54] [0.26 – 0.39] [0.04 – 0.30] [0.75 – 0.80] [0.56 – 0.66] table 2. reproducibility for dental exams ( ) benchmark examiner’s kappa value [ ] examiners’ kappa value intervals characteristics of the stage 1. a total of 2,187 occlusal surfaces were examined. the examiner who had the highest intraexaminer kappa values in stage 1 was selected to conduct the exams. the validity of vi, dd and vi+dd exams was evaluated. the data were dichotomized in ‘with’ and ‘without caries’. the sas system for windows™ 9.1 (sas institute inc., cary, nc, usa) was used at both stages. at d1 diagnostic criterion, the validation standard was the vi exam performed by benchmark examiner, while for d1+d3 diagnostic criterion, it was vi exam combined with bitewing radiography exam (vi+bw) performed by benchmark examiner. the vi+bw exam enables a more refined detection and evaluation of occlusal carious lesion depth, especially when the carious lesion is confined to dentin13. although the value of bitewing radiograph has been questioned in epidemiological surveys14, it has been proven important in studies in which histological validation or tooth drilling is unfeasible and unethical 13, especially when suspicious carious lesions are confined in enamel. the sample dmf-t index was 1.00 (sd=1.65) according to who diagnostic criterion11. when enamel initial carious lesions were detected, the dmf-t index was 5.74 (sd=4.29). the difference among the dmf-t index obtained by dental exams under all diagnostic criteria was evaluated by anova and tukey’s test. significance level was set at 5%. results the reproducibility for vi and dd exams is presented in table 2. the intraexaminer agreement was ‘almost perfect’ for the vi exam at all diagnostic criteria. however, the intraexaminer agreement for the dd exam was ‘slight’ at d1 criterion and ‘fair’ for the others criteria. the intraexaminer agreement was ‘fair’ at d1 and ‘moderate’ at d3 and d1+d3 criteria for the benchmark examiner performing dd exam (table 2). the interexaminer agreement was ‘almost perfect’ for vi exam at all diagnostic criteria. for the dd exam, the interexaminer agreement was ‘slight’ at d1 and ‘fair’ at the others criteria (table 2). the number of carious lesions detected by vi, dd, vi+dd examinations, and by the benchmark examiner (vi+bw exam) is expressed in table 3. the vi+dd exam detected the largest number of occlusal caries for all diagnostic criteria. a trend to overestimate caries detection was observed for vi+dd exam under d1 and d1+d3 criteria in relation to benchmark examiner (table 3). the validity of the vi, dd and vi+dd examinations is presented in table 4. at d1 criterion, the highest sensitivity was found by vi+dd exam. at d3 criterion, the highest sensitivity was found by dd exam; the highest accuracy was found for dd and vi+dd exams at this diagnostic criterion. at d1+d3 criterion, the highest value for sensitivity was found for the vi+dd exam, however the best accuracy was observed for vi exam (table 4). differences among the dmf-t indexes obtained by vi, dd and vi+dd exams, at d3 and d1+d3 criteria were revealed by the tukey’s test (table 5). discussion in view of the current dental caries development profile, seeking new methodologies and diagnostic methods that can identify its earliest stages is the intention of dentists in order to accomplish more conservative and effective treatment plans. likewise, researchers can use different study designs and conduct studies that can prove or refute their hypotheses6-8,15-18. the d 1 d 3 d1+d3 vi 721 30 729 d d 345 337 682 vi+dd 870 341 980 vi+bw 755 652 953 table 3. number of carious lesions detected by the dental exams vi: visual exam under artificial lighting; dd: diagnodent™; bw: bitewing radiography exam. 132132132132132laser fluorescence on dental caries detection under epidemiological setting 133133133133133 braz j oral sci. 11(2):130-134 exams d 1 d 3 d1+d3 s n s p a s n s p a s n s p a vi 79.47 91.55 87.38 3.85 99.67 71.21 68.10 93.52 82.45 d d 26.16 88.37 66.10 36.10 92.04 75.24 57.10 85.71 73.03 vi+dd 83.31 83.17 83.22 34.67 92.45 75.29 80.48 82.74 81.75 table 4. validity of the dental exams vi: visual exam under artificial lighting; dd: diagnodent™. sn = sensitivity; sp = specificity; a = accuracy exams d3 d1+d3 dmf-t* sd** dmf-t sd vi 0.46a 0.80 4.93b 3.70 d d 2.12b 2.00 4.13a 2.93 vi+dd 2.71c 2.18 6.60c 3.71 table 5. dmf-t index for dental exams vi: visual exam under artificial lighting; dd: diagnodent™ * different letters indicate statistically significant difference (p<0.05) ** standard deviation d1+d3 criterion is more refined than the conventional who diagnostic criteria and has shown to be successful in this task2,5. in the present study, the d1+d3 criterion was responsible for a 574% increase in the dmf-t index at d3 criterion for the sample on stage 2. although the diagnostic complexity increases with d1+d3 in relation to d3, an increase on the accuracy of vi and vi+dd exams was verified. a further step searching for a more accurate dental caries diagnostic is the addition of diagnostic adjuncts to the traditional visual clinical exam. the present study evaluated the reproducibility and validity of caries detection by laser fluorescence according to the d1, d3 and d1+d3 diagnostic criteria, under epidemiological setting. this adjunct technology is based on the principle that demineralized dental surfaces colonized by bacteria and exposed to their metabolites exhibit an increased fluorescence in comparison with sound enamel when excited by a diode laser beam (655 nm) emitted by diagnodent™. because of its high sensitivity, but low specificity, the combination of diagnodent™ with traditional diagnostic methods, such visual exam, is justified10,19. however a great deal of the studies evaluating this technology is performed under laboratorial and/or clinical conditions, not under epidemiological conditions. this way, the preliminary comparisons made in this study, as well its design, require caution in both interpretation and generalization. the intraexaminer reproducibility of dd examination concerning all diagnostic criteria was lower than in in vitro studies16 and in vivo clinical trials12,17. this can reflect the difficulty to keep an adequate oral hygiene level in epidemiological conditions and that the presence of plaque on dental occlusal surfaces worsens diagnodent™ accuracy 20-21. although diagnodent’s™ manufacturer recommends previous professional prophylaxis, the previous supervised toothbrushing adopted in this study can be implemented with similar success20. after that, vigorous oral rinse was performed to remove any toothpaste residues, which could interfere on diagnodent™ readings20-21. drying of dental surface to be examined was also performed as recommended5,22. the kappa values of the second calibration session for interexaminer agreement were higher than those of the first session, suggesting that the oral hygiene level improved during this period. the reproducibility of dd exam contraindicates its use on epidemiological surveys. the reproducibility was more influenced by the examination setting than the examiners’ ability to operate the diagnodent™ device. an accuracy of at least 80% was obtained by vi and vi+dd exams at d1 and d1+d3 criteria. the lower values of sensitivity and accuracy for dental examinations at d3 criterion are due to the validation method adopted (vi+bw exam), which is more sensitive than the dental examinations adopted, detecting dentin carious lesions under sound enamel, which are not detectable by the visual exam alone. so, the sensitivity was extremely low for vi examination at d3 criterion. the assertive that dd exam should be used as an adjunct diagnostic tool6,8,10,18 is corroborated by the sensitivity values for vi+dd exam under d1 and d1+d3 criteria. however the accuracy values of vi+dd exam were slightly inferior to the accuracy of vi exam at d1 and d3 criteria. although an increasing vi exam sensitivity can be seen, the good results of the dd exam found in clinical setting17 were not found in the present study. as in a previous study by pereira et al.9, data available from multiple diagnostic methods did not improve the accuracy of examiners, but it influenced the number of surfaces indicated for operative treatment. for d1 and d1+d3 criteria, the combination of dd with vi decreased its accuracy. however, the number of surfaces indicated to treat was overestimated with vi+dd exam in relation to benchmark examiner. the dd exam increased the accuracy of vi for d3 criteria. discrepant amounts of caries detected between dental exams and benchmark examiner for d3 criterion was due to validation method. for d3 criterion, vi+bw exam allowed detecting more dental caries than the other diagnostic methods. the financial costs of the inclusion of the bitewing radiograph and diagnodent™ examinations in dental caries surveys are limitations to the use of these adjuncts on such surveys. however, when the public health services support financially those epidemiological surveys such limitations can be solved. in epidemiological dental caries surveys, the bitewing radiograph must be used for suspicious cases only due to the ethical concerns. another limitation of this study was the validation process. facing to the actual prevalence of initial carious laser fluorescence on dental caries detection under epidemiological setting braz j oral sci. 11(2):130-134 134134134134134 lesions in enamel, treatable by preventive proceedings, other ways to validate diagnostic methods, that are not histological (extracting teeth) or biopsy (drilling suspicious surfaces) are necessary. the vi+bw exam adopted in this study, although it is far from the ideal method, was one of them. more and more, detection of initial carious lesions is necessary for planning actions in oral public health services3. a meticulous visual examination using d1 criterion was sufficient for this purpose, corroborating with heinrich-weltzien et al.17. for d3 and d1+d3 criteria, the diagnodent™ showed its potential to supplement the vi exam on epidemiological surveys18,23. however, caution and additional in vivo studies, under epidemiological settings, are needed to corroborate or refute those results. the results of the present study reveal that a meticulous visual exam is sufficient to detect initial carious lesions in enamel using d1 diagnostic criterion. it was also observed that the combination of laser fluorescence and visual exam improved its sensitivity according to d1 and d3 diagnostic criteria under epidemiological setting. acknowledgements the authors would like to thanks the volunteers for their collaboration, the proex/capes-unicamp for the first author scholarship and the fapesp for the financial support (grants #06/58881-9). references 1. featherstone jdb. the continuum of dental caries – evidence for a dynamic disease process. j dent res. 2004; 83 (spec iss c): c39-42. 2. ricketts d, kidd e, weerheijm ê, soet h. hidden caries: what is it? does it exist? does it matter? int dent educ. 1997; 47: 259-65. 3. vehkalahti m, tarkkonen l, varsio s, heikkilä p. decrease in and polarization of dental caries occurrence among child and youth populations, 1976-1993. caries res. 1997; 31: 161-5. 4. kassawara abc, assaf av, meneghim mc, pereira ac, topping g, levin k, et al. comparison of epidemiological evaluations under different caries diagnostic thresholds. oral health prev dent. 2007; 5: 137-44. 5. assaf av, meneghim mc, zanin l, cortelazzi kl, pereira ac, ambrosano gmb. effect of different diagnostic thresholds on dental caries calibration. j public health dent. 2006; 66: 17-22. 6. da silva rp, assaf av, pereira sm, mialhe fl, ambrosano gm, meneghim mde c, et al. validity of caries-detection methods under epidemiological setting. am j dent. 2011; 24: 363-6. 7. da silva rp, assaf av, pereira sm, ambrosano gm, mialhe fl, pereira ac. reproducibility of adjunct techniques for diagnosis of dental caries in an epidemiological situation. oral health prev dent. 2011; 9: 251-9. 8. duruturk l, ciftçi a, baharoglu s, oztuna d. clinical evaluation of diagnodent in detection of occlusal caries in newly erupted noncavitated first permanent molars in caries-active children. oper dent. 2011; 36: 348-55. 9. pereira ac, eggertsson h, martinez-mier ea, mialhe fl, eckert gj, zero dt. validity of caries detection on occlusal surfaces and treatment decisions based on results from multiple caries-detection methods. eur j oral sci. 2009; 117: 51-7. 10. bader jd, shugars da. a systematic review of the performance of a laser fluorescence device for detecting caries. j am dent assoc. 2004; 135: 1413-26. 11. world health organization. oral health surveys, basic methods. 4th ed. geneve: who; 1997. 12. zanin l, meneghim mc, assaf av, pardi v, pereira ac, mialhe fl. depth of occlusal caries assessed clinically by fluorescence laser, conventional and digital radiographic methods. braz j oral sci. 2005; 4: 735-40. 13. wenzel a. bitewing and digital bitewing radiography for detection of caries lesions. j dent res. 2004; 83 (spec iss c): c72-5. 14. bloemendal e, de vet hcw, bouter lm. the value of bitewing radiographs in epidemiological caries research: a systematic review of the literature. j dent. 2004; 32: 255-64. 15. chesters rk, ellwood rp, biesbrock ar, smith sr. potential modern alternative designs for caries clinical trials (ccts) and how these can be validated against the conventional model. j dent res. 2004; 83 (spec iss c): c122-4. 16. souza-zaroni wc, ciccone jc, souza-gabriel ae, ramos rp, corona sam, palma-dibb rg. validity and reproducibility of different combinations of methods for occlusal caries detection: an in vitro comparison. caries res. 2006; 40: 194-201. 17. heinrich-weltzien r, weerheijm kl, kühnisch j, oehme t, stösser l. clinical evaluation of visual, radiographic, and laser fluorescence methods for detection of occlusal caries. ascd j dent child. 2002; 69: 127-32. 18. rando-meirelles mp, de sousa mda l. using laser fluorescence (diagnodent) in surveys for the detection of noncavitated occlusal dentine caries. community dent health. 2011; 28: 17-21. 19. matos r, novaes tf, braga mm, siqueira wl, duarte da, mendes fm. clinical performance of two fluorescence-based methods in detecting occlusal caries lesions in primary teeth. caries res. 2011;45: 294-302. 20. lussi a, reich e. the influence of toothpastes and prophylaxis pastes on fluorescence measurements for caries detection in vitro. eur j oral sci. 2005; 113: 141-4. 21. diniz mb, sciasci p, rodrigues ja, lussi a, cordeiro rc. influence of different professional prophylactic methods on fluorescence measurements for detection of occlusal caries. caries res. 2011;45: 264-8. 22. pinelli c, loffredo lde c, serra mc. effect of drying on the reproducibility of diagnodent to detect caries-like lesions. braz dent j. 2010; 21: 405-10. 23. rodrigues ja, hug i, neuhaus kw, lussi a. light-emitting diode and laser fluorescence-based devices in detecting occlusal caries. j biomed opt. 2011; 16: 107003. laser fluorescence on dental caries detection under epidemiological setting oral sciences n3 braz j oral sci. 11(3):396-400 original article braz j oral sci. july | september 2012 volume 11, number 3 influence of two methods of additional activation on composite resins surface hardness alessandro ribeiro gonçalves1, caroline de deus tupinambá rodrigues2, carlos henrique de carvalho e souza3, leilane ferraz moreira de sousa4, pedro henrique de souza lopes4 1phd in oral rehabilitation – foar/unesp, assistant professor, department of restorative dentistry, federal university of piauí (ufpi), teresina, pi, brazil 2 phd in restorative dentistry – foar/unesp, assistant professor, department of restorative dentistry, federal university of piauí (ufpi), teresina, pi, brazil 3dentistry master’s student, department of restorative dentistry, federal university of piauí (ufpi), teresina, pi, brazil 4scientific initiation student, department of restorative dentistry, federal university of piauí (ufpi), teresina, pi, brazil correspondence to: alessandro ribeiro gonçalves rua visconde de parnaíba 2315, apt 1602, cep: 64049-570 horto florestal teresina, pi – brasil phone: +55 86 32313842 e-mail: argoncalves@yahoo.com abstract aim: to evaluate the influence of two methods of additional activation on the surface hardness of composite resins. methods: two types of composites were tested: filtek p60 and filtek p350. for each material, 48 specimens were prepared and divided into four groups: group 1 (control) conventional activation, using a halogen light for 40 s; group 2 conventional activation and additional activation with a halogen lamp for 60 s; group 3 conventional activation and additional activation with an autoclave at 127°c for 6 min at 1.7 kg/cm3 pressure; and group 4 conventional activation and additional activation with an autoclave at 134 °c for 15 min at 2.1 kg /cm3 pressure. the use of autoclave has been suggested for being a standard equipment at dental offices, and thus, even at locations far from dental laboratories, it would be possible to have simple techniques that allow access to indirect restorations at lower costs. data obtained in the study were analyzed statistically by analysis of variance followed by tukey’s test at a 5% level of significance. results: for z350, there was a significant increase in hardness for all groups of additional activation (groups 2, 3 and 4), compared with the control group. for p60, a significant increase in surface hardness was found compared with the control group for the groups that used additional activation with an autoclave (groups 3 and 4). conclusions: additional activation with an autoclave increased the surface hardness of the tested resins to a greater degree than additional activation with a halogen light. keywords: polymerization, composite resins, hardness. introduction technological evolution in dentistry is driven by the constant attempt to improve materials and techniques in line with the market’s demands. such advances mean that it is increasingly possible for clinicians to produce outcomes that combine function with good aesthetics. at the moment, the most widely used restorative materials in dentistry are composite resins, mainly because they adhere well to the tooth structure, have suitable mechanical properties and are available in a wide range of shades and translucencies that produce good aesthetic results1. received for publication: may 12, 2012 accepted: september 18, 2012 braz j oral sci. 11(3):396-400 since their introduction as restorative materials for posterior teeth, composite resins have significantly improved in terms of their physical and mechanical characteristics. the composition of their organic and inorganic matrix has changed, and there are currently various types of resins that can be used in posterior teeth, such as microhybrid resins and composites with nanoparticles2-3. composite resins are usually recommended for direct restorations in posterior teeth when cavities are small and medium in size. to restore large cavities and extensively destroyed teeth, dentists generally opt for amalgam fillings or indirect laboratory-made resin, porcelain or metallic restorations4-5. amalgam can be used for direct restorations, but it is limited by its unaesthetic outcome. the main limiting factor of indirect restorations is their high cost. one viable alternative (published in the norms of the manufacturers of resins indicated for use in posterior teeth) is the use of these resins in indirect restorations such as crowns, veneers, inlays, onlays and so on. however, these indirect restorations require additional costs and special equipment for activation. various studies have shown that by means of simple technical changes, such as additional activation, direct-use resins can achieve mechanical properties that are similar to laboratory-made restorations6-8. different methods of extraoral additional activation, including activation by light, dry heat and autoclaving, were proposed in order to improve physical and mechanical properties, and enable the use of direct-use composite resins in indirect restorations. the purpose of this additional treatment is to broaden the indication of resins and the clinical longevity of restorations7-9. however, there are few studies that investigate which of these are the best method. the autoclave has been suggested because it is a standard equipment at dental offices. therefore, even at locations far from dental laboratories, it would be possible to develop simple techniques that could allow access to indirect restorations at lower costs. the surface hardness of composite resins depends mainly on their microstructure and composition, but there is a correlation with the degree of conversion of monomers2,10. the increase in hardness is related to a higher degree of conversion and improved mechanical properties of polymeric materials. thus, the study of the microhardness of materials consists of an indirect method to evaluate the effect of different treatments on the properties of dental composites1012. the vickers hardness test is commonly used to investigate improvement in material’s mechanical properties2,13-16. using this test as a parameter, the aim of this study was to evaluate the microhardness of two direct resin composites after additional activation with light or using an autoclave. material and methods specimen preparation to obtain the resin specimens, a circular split-ring matrix with an outer diameter of 2 cm and an inner diameter of 1 cm was used. it was held in position by a circular metal matrix with a 2-cm diameter perforation and 3-cm outer diameter (figure 1 and 2). fig. 2: metal matrices assembled. fig. 1: circular split-ring metal matrices. the composite resins used were: 1) filtek p60 (3m espe, st. paul, mn, usa) – a hybrid composite, for direct and indirect restorations in posterior teeth and 2) filtek z350 (3m espe) – a nanoparticulate composite, for direct anterior and posterior restorations and for indirect inlay, onlay and veneer restorations (table 1). p60 was used in this study to an extensively researched material, with a large number of published papers, and because it is a material with an indication for use in posterior teeth, region usually requires partial indirect restorations. z350 composite was selected due to its recent launch in the dental market and for representing the newest class of composite resins with an indication for anterior and posterior teeth. the matrix was placed on a glass plate and the inner space was filled with one of the studied composite resins in a single increment. a polyester strip was placed on the top surface and activated for 60 s using a kondertech activation device, model cl-k200 (kondortech, são carlos, sp, brazil) having a light intensity of 500 mw/cm2. the light intensity was checked with a digital radiometer (dabi-atlante, são paulo, sp, brazil). ninety-six specimens were prepared, 48 for each restorative material, divided into the following groups: group 1 (control) – conventional activation, using a 397397397397397influence of two methods of additional activation on composite resins surface hardness halogen light for 60 s; group 2 – conventional activation and additional activation with a halogen light for an additional 60 s; group 3 – conventional activation and additional activation with an autoclave at 127°c for 6 min and 1.7 kg/cm3 pressure; group 4 – conventional activation and additional activation with an autoclave at 134°c for 15 mins at 2.1 kg/cm3 pressure. hardness test after they had been prepared, the specimens were stored in distilled water in a bacteriological oven at 37°c for 7 days in a light proof container. the vickers microhardness tests were carried out with the aid of a mmt-3 microhardness tester (buehler, lake bluff, il, usa) under a load of 50 gf for 30 s. three microhardness impressions were carried out per sample, one in the center and two at the periphery, as follows: traced an imaginary line dividing the sample in half, a central impression was made and the other between the center and right and left edges. statistical analysis the data obtained from the means were subjected to analysis of variance (anova) followed by tukey’s test at a 5% level of significance. results the tested resins had different means of hardness, according to the type of material and the type of treatment of the samples, as shown in table 2. discussion the results of this study indicate that additional activation methods significantly improved the microhardness of the composite resins evaluated. these results are consistent with the study of dickerson and hastings17 (1995), which reported polymerization rates of approximately 50% to 60% for self-activated resins and 55% to 65% for photoactivated resins, and reported that these resins reached a degree of material classification indication composition manufacturer p60 microhybrid posterior bisgma, udma, bisema 3m espe z350 nanoparticulate anterior and posterior bisgma,udma, tegdma, bisema 3m espe table 1: specifications of composite resins used filtek p60 filtekz350 mean standard deviation mean standard deviation group 1 49.9 (a) 3.1 40.2 (a) 3.6 group 2 51.6 ( a) 3.9 44.7 (b) 4.1 group 3 55.6 (b) 1.8 46.2 (bc) 2.0 group 4 56.1 (b) 3.6 48.4 (c) 2.9 table 2: mean and standard deviation of vickers hardness of experimental groups according to the type of composite resin. different letters indicate a statistically significant difference (á=0.05) according to the t-test. conversion of 80-85% when subjected to a temperature of 125°c. the use of an autoclave for additional activation (groups 3 and 4) significantly increased the microhardness compared with the control group, for both materials tested, probably because the autoclave generates greater amounts of energy in the form of heat and pressure, which increases the conversion rate. other studies that evaluated the influence of heat treatment also found improvements in the mechanical properties of materials, which may be related to a reduction in the amount of residual double carbon bonds in the polymer18-21. in another study of the behavior of resins and additional activation, bagis and rueggeberg6 (2000) reported that heat treatment increases the conversion rate, and this increase would not be possible if it was only photoactivated. table 2 confirms this statement, and even the use of additional photoactivation time did not result in a greater hardness of filtek p60. perhaps this difference is due to the composition of the resins. trujillo, newman and stansbury22 (2004) reported that exposure of composites to additional heat treatment, limited to a biologically compatible time period, significantly affects the kinetics of activation, and increases the conversion rate of resins and improves their properties. the increase in hardness can be explained by the fact that the temperatures used in the heat treatment were close to the glass transition temperature, which increases the kinetic energy of the resinous monomers and the quantity of free radicals. the greater mobility within the polymer chain enables new reactions of the activated radicals and a greater number of crosslinks in the organic matrix. the continuation of the activation process leads to greater stability and hardness of the composite7-8,21,23. with filtek z350 resin, as well as the additional conversion using heat and pressure, the longer light exposure (exceeding the amount recommended by the manufacturer) affected significantly the surface hardness. this fact suggests that, although the microhardness of this material is suitable for dental needs, it is possible to produce a higher conversion of residual monomers through greater exposure to halogen 398398398398398 influence of two methods of additional activation on composite resins surface hardness braz j oral sci. 11(3):396-400 399399399399399 light and thereby increase the restoration’s durability24. another advantage is the greater biocompatibility of the material. increased activation results in a significant decrease in the amount of non-activated monomers and, consequently, lower levels of leachable materials that promote oral cytotoxicity6. the use of heat also causes a similar effect: it results in more monomers linked to the polymer chain and some of the unreacted monomers are volatilized during the heating process25. the effect of additional activation and the different microhardness values of the resinous materials depend mainly on their composition. bis-gma has a low degree of conversion because of its characteristics of high molecular weight, high viscosity and low flexibility. the addition of diluent monomers with higher flexibility, such as egdma or tegdma, enhances bis-gma’s mobility and its polymerization conversion rate. another alternative to bisgma is the monomer udma, which has a molecular weight similar to bis-gma, but a lower viscosity18,26. it has been shown that the monomer tegdma creates a thicker polymer chain, but it is the most flexible and has a greater rate of water absorption. bis-gma forms a more rigid chain and absorbs less water; however, it absorbs more water than the udma/bis-ema combination. hydrolysis of intermolecular bonds weakens the polymer. in udma-based composites, hydrogen bonds increase the conversion rate and improve mechanical properties. when tegdma is replaced by udma and/or bis-ema (during co-polymerization with bis-gma), the absorption of water is decreased. such characteristics influence the conversion rate and the mechanical properties of composites27-28. the abovementioned information explains the different behaviors between the z350 and p60 composites after additional activation: z350 contains bis-gma, udma, tegdma and bis-ema, while p60 contains bis-gma, udma and bis-ema. from the above discussion, it is possible to state that additional activation by means of thermal treatments improved the hardness of the composite resins tested, regardless of their composition. the use of an autoclave as an additional method of activation is very effective and provides better results. by using a routinely found equipment at dental offices, such as autoclave, it is possible to develop a simple, low cost technique, especially in cases of difficult access to special equipment for the production of laboratory restorations. it can be concluded that the additional activation using an autoclave increased the surface hardness of the tested resins more than additional activation with halogen light. references 1. anfe te, caneppele tm, agra cm, vieira gf. microhardness assessment of different commercial brands of resin composites with different degrees of translucence. braz oral res. 2008; 22: 358-63. 2. hosseinalipour m, javadpour j, rezaie h, dadras t, hayati an. investigation of mechanical properties of experimental bis-gma/tegdma dental composite resins containing various mass fractions of silica nanoparticles. j prosthodont. 2010; 19: 112-7. 3. monteiro gqm, montes majr. evaluation of linear polymerization shrinkage, flexural strength and modulus of elasticity of dental composites. mat. res. 2010; 13: 51-5. 4. christensen gj. considering tooth-colored inlays and onlays versus crowns. j am dent assoc. 2008; 139: 617-20. 5. krämer n, garcía-godoy f, reinelt c, feilzer aj, frankenberger r. nanohybrid vs. fine hybrid composite in extended class ii cavities after six years. dent mater. 2011; 27: 455-64. 6. bagis yh, rueggeberg fa. the effect of post-cure heating on residual, unreacted monomer in a commercial resin composite. dent mater. 2000; 16: 244-7. 7. lombardo ghl, carvalho cf, galhano g, souza roa, nogueira júnior l, pavanelli ca. influence of additional polymerization in the microhardness of direct composite resins. cienc odontol bras. 2007; 10: 10-5. 8. santana il, lodovici e, matos jr, medeiros is, miyazaki cl, rodriguesfilho le. effect of experimental heat treatment on mechanical properties of resin composites. braz. dent. j. 2009; 20: 205-10. 9. coelho lfb, herbstrith srm, mota eg, oshima hms, balbinot ce, bondan jl. influence of different secondary cure techniques on hardness of composite resins. rev odonto cienc. 2007; 22: 317-20. 10. camargo ej, moreschi e, baseggio w, cury ja, pascotto rc. composite depth of cure using four activation techniques. j appl oral sci. 2009; 17: 446-50. 11. price rb, fahey j, felix cm. knoop hardness of five composites cured with single-peak and polywave led curing lights. quintessence int. 2010; 41: e181-91. 12. albino lg, rodrigues ja, kawano y, cassoni a. knoop microhardness and ft-raman evaluation of composite resins: influence of opacity and photoactivation source. braz oral res. 2011; 25: 267-73. 13. bhamra gs, fleming gj, darvell bw. influence of led irradiance on flexural properties and vickers hardness of resin-based composite materials. dent mater. 2010; 26: 148-55. 14. ceballos l, fuentes mv, tafalla h, martínez a, flores j, rodríguez j. curing effectiveness of resin composites at different exposure times using led and halogen units. med oral patol oral cir bucal. 2009; 14: e51-6. 15. lucey s, lynch cd, ray nj, burke fm, hannigan a. effect of preheating on the viscosity and microhardness of a resin composite. j oral rehabil. 2010; 37: 278-82. 16. marchan sm, white d, smith wa, raman v, coldero l, dhuru v. effect of reduced exposure times on the microhardness of nanocomposites polymerized by qth and second-generation led curing lights. oper dent. 2011; 36: 98-103. 17. dickerson wg, hastings jh. indirect composite restorations. curr opin cosmet dent. 1995; 1: 51-6. 18. soares cj, pizi ec, fonseca rb, martins lr.. mechanical properties of light-cured composites polymerized with several additional post-curing methods. oper dent. 2005; 30: 389-94. 19. poskus lt, latempa ama, chagas ma, silva em, leal mps, guimarães jga. influence of post-cure treatments on hardness and marginal adaptation of composite resin inlay restorations: an in vitro study. j appl oral sci. 2009; 17: 617-22. 20. da silva gr, simamoto-júnior pc, da mota as, soares cj. mechanical properties of light-curing composites polymerized with different laboratory photo-curing units. dent mater j. 2007; 26: 217-23. 21. busato als, arossi ga, ogliari f, samuel smw. the effect of post-cure heating in autoclave, microwave oven and conventional oven on direct composite resin. rev odonto cienc. 2007; 22: 177-80. 22. trujillo m, newman sm, stansbury jw. use of near-ir to monitor the influence of external heating on dental composite photoactivation. dent mater. 2004; 20: 766-77. 23. santana il, gonsalves lm, lage lm, lima dm, pereira afv, rodrigues filho le. inlays/onlays in resin composites for direct use heat treated part i: description of technique. rev bras pesq saude. 2010; 12: 76-81. influence of two methods of additional activation on composite resins surface hardness braz j oral sci. 11(3):396-400 400400400400400 24. nomoto r, asada m, mccabe jf, hirano s. light exposure required for optimum conversion of light activated resin systems. dent mater. 2006; 22: 1135-42. 25. bagis yh, rueggeberg fa. mass loss in urethane/ tegdma and bisgma/ tegdma based resin composites during post cure heating. dent mater. 1997; 13: 377-80. 26. reinhardt jw, boyer db, stephens nh. effects of secondary curing on indirect posterior composite resins. oper dent. 1994; 19: 217-20. 27. peutzfeldt a. resin composites in dentistry: the monomer systems. eur j oral sci. 1997; 105: 97-116. 28. sideridou i, tserki v, papanastasiou g. study of water sorption, solubility and modulus of elasticity of light-cured dimethacrylate-based dental resins. biomaterials. 2003; 24: 655-65. influence of two methods of additional activation on composite resins surface hardness braz j oral sci. 11(3):396-400 original article braz j oral sci. january/march 2009 volume 8, number 1 higher prevalence of periodontal disease among patients with predialytic renal disease rosamma joseph1, rajaratnam krishnan2, vivek narayan3 1 mds, professor and head, department of periodontics, calicut, kerala, india 2 dm, consultant in nephrology at nirmala hospital; former professor and head, department of nephrology and hypertension, government medical college, calicut, kerala, india 3 bds, post-graduate student, department of periodontics, government dental college, calicut, kerala, india received for publication: november 10, 2008 accepted: march 19, 2009 correspondence to: rosamma joseph department of periodontics government dental college medical college campus calicut, kerala, india 673008 e-mail: drrosammajoseph@gmail.com abstract aim: periodontal diseases can have a significant effect on the systemic health. chronic systemic diseases such as renal disease may also influence progression of periodontal disease. the present study assessed the prevalence of periodontal disease among a group of patients with renal disease and compared their periodontal status to that of healthy controls. methods: 77 patients with different forms of renal disease and 77 healthy controls were examined for oral hygiene status, gingival inflammation, probing pocket depth and clinical attachment loss. the subjects were grouped into three as no/mild, moderate and severe periodontitis. results: all periodontal parameters were significantly elevated in the case group as compared to controls (p < 0.001). the prevalence and severity of periodontal disease was also significantly higher in the case group (p < 0.001). conclusions: this study provides evidence for a greater prevalence and severity of periodontal disease among patients with renal disease. the periodontal health of all patients with renal disease needs to be carefully monitored. keywords: periodontal disease, periodontitis, nephrology, renal disease introduction periodontal diseases comprises of a group of inflammatory diseases affecting the supporting tissues of the teeth resulting from a complex interplay between specific gram-negative microorganisms, their by products and the host-tissue response. this results in progressive destruction of the periodontal ligament and alveolar bone. earlier, periodontitis had been considered as a disease confined to the oral cavity. however, in the past several years, substantial scientific data have emerged to indicate that the localized infections characteristic of periodontitis can have a significant effect on the systemic health. it is now recognized that the periodontopathic gram-negative bacteria and bacterial products, such as lipopolysaccharides, activate the host immune response significantly and their actions have consequences beyond periodontal tissues. pro-inflammatory cytokines, such as α2-macroglobulin, α1-antitrypsin and c-reactive protein are significantly elevated during the destructive phase of periodontitis1-4. these inflammatory mediators may have a profound influence in the pathogenesis of many systemic diseases. during the past decade, numerous studies, mostly cross-sectional and few longitudinal, have been carried out and they provide evidence for the link between periodontal and cardiovascular diseases, like atherosclerosis and myocardial infarction5-10. recently, several studies have been published in the literature, providing evidence for an increased prevalence of periodontal disease in patients with renal disease, especially in dialysis patients, and renal transplant recipients11-19. 15higher prevalence of periodontal disease among patients with predialytic renal disease braz j oral sci. 8(1): 14-8 however, conflicting results regarding the periodontal status of these patients are also available20-24 and further studies are warranted in this regard. the aim of the present study was to know the prevalence of periodontal disease among a group of patients with predialytic renal disease from a south indian population. furthermore, we wanted to compare their periodontal status to that of healthy controls. materials and methods study population this study was designed as a matched case-control study. cases were identified as patients attending the outpatient clinic at the department of nephrology, medical college, calicut, kerala, india, over a period of six months, from july 2007 to december 2007. only patients who were diagnosed with renal disease were included. these diseases include chronic kidney disease of varied etiology which includes diabetic nephropathy, lupus nephritis, glomerular disorders, and nephrotic syndrome. systemically healthy individuals who accompanied patients to government dental college, calicut, during the same period were selected as control subjects. the controls were matched for age, sex, and socioeconomic status. subjects who had previously undergone dialysis or renal transplantation were excluded from the study. subjects with history of smoking, those who had received periodontal therapy or systemic antibiotic therapy within a period of six months prior to the examination and subjects with any acute condition that contraindicated a periodontal examination were also excluded. to assess the periodontal status, all subjects were required to have at least six natural teeth. a written informed consent was obtained from all participants in the study. the study was conducted by the joint efforts of the departments of nephrology, medical college, and the department of periodontics, government dental college. the study was approved by the institutional review board for human ethics, government medical college, calicut. data collection all subjects were required to answer a detailed questionnaire. the information collected included demographic characteristics like name, age, address, sex, occupation etc. a detailed medical and dental history was also collected from all subjects. oral and dental examination the dental and periodontal examination in all subjects was carried out by a single, trained examiner. the dental status was determined by visual examination under direct and indirect illumination, using a plane dental mirror and a dental explorer. oral hygiene indexsimplified (ohi-s) (greene and vermillion)25 for assessing the oral hygiene status. the index was calculated using six index teeth: 16, 11, 26, 36, 31, 46. modified gingival index (mgi) (lobene et al.)26, for the entire dentition, was calculated as a measure of gingival inflammation. periodontal examination the periodontal examination was carried out with calibrated periodontal probes with william’s markings. the periodontal status was determined using measurements of probing pocket depth (ppd), gingival recession and clinical attachment level measurements (cal) from four sites on each tooth (buccal, mesial, lingual/palatal, distal). ppd was taken as the distance from the gingival margin to the base of the gingival sulcus/ periodontal pocket. gingival recession was measured as the distance from the cementoenamel junction to the gingival margin. these scores were then added up to indirectly obtain the values for cal. all the subjects were categorized into three groups (mild/no periodontitis, moderate periodontitis and severe periodontitis) based on cal and ppd measurements, using the criteria proposed by the joint working group of the centre for disease control and prevention in collaboration with the american academy of periodontology in 2003 which are depicted in table 127. statistical analysis descriptive statistics including mean values for ohi-s, modified gingival index, ppd and cal were calculated. for comparisons between the case and control groups, the student’s t-test and chisquare tests were used for quantitative and qualitative variables respectively. the difference in proportions in both groups was tested using chisquare test. all statistical analyses were carried out using statistical package for the social sciences package for windows, version 13. the 95% confidence intervals were taken (p-value < 0.05). results a total of 154 patients were included in the study (77 in each group). the mean age of patients was 40.38 + 7.47 years. there were a total of 71 males and 83 females in the study. the group wise distribution of age and gender of the subjects is depicted in table 2. no significant difference was found between the distribution of age and gender among the groups. table 3 shows the distribution of different forms table 1. clinical case definitions proposed by the cdc working group for use in population-based surveillance of periodontitis27 category clinical attachment level (cal) probing pocket depth (pd) severe periodontitis > 2 interproximal sites with cal > 6 mm and (not on same tooth) > 1 interproximal site with pd > 5 mm moderate periodontitis > 2 interproximal sites with cal > 4 mm or (not on same tooth) > 2 interproximal sites with pd > 5 mm no or mild periodontitis neither ‘‘moderate’’ nor ‘‘severe’’ periodontitis 16 joseph r, krishnan r, narayan v braz j oral sci. 8(1): 14-8 table 2. group wise distribution of age and gender cases controls p-value age (in years) 40.63 + 7.92 40.14 + 7.04 *0.6834 males n (%) 34 (44.2%) 37 (55.8%) #0.6276 females n(%) 43 (48.1%) 40 (51.8%) *chi-square test; # student’s t-test. table 3. distribution of renal disease in case group disease frequency (%) chronic kidney disease diabetic kidney disease 34 (44) chronic tubulo-interstitial disease 15 (19) hypertensive nephrosclerosis 9 (12) other chronic glomerulopathies 2 (3) lupus nephritis 1 (1) total 61 (79) nephrotic syndrome 16 (21) total (n) 77 (100) table 5. distribution of periodontal disease severity in case group severity frequency (%) mild/no periodontitis 6 (7.8) moderate 34 (44.2) severe 37 (48.0) total 77 (100.0) table 6. distribution of periodontal disease severity in control group severity frequency (%) mild/no periodontitis 66 (85.7) moderate 8 (10.4) severe 3 (3.9) total 77 (100.0) table 4. mean values for periodontal parameters in both groups case group control group #p-value ohi-s 2.49 + 0.57 1.37 + 0.48 < 0.001 mgi 1.58 + 0.39 0.86 + 0.53 < 0.001 ppd 2.48 + 0.40 1.37 + 0.49 < 0.001 cal 2.76 + 0.66 1.50 + 0.53 < 0.001 student’s t-test. of renal disease among the case group. mean values for ohi-s, mgi, ppd and cal are given in table 4. all these values were significantly elevated in the case group as compared to controls (p < 0.001). table 5 indicates the severity of periodontal disease in the case group; 71 patients (92.3%) of the total 77 had moderate to severe periodontitis and the remaining patients (6, 7.7%) belonged to the category mild/ no periodontitis. table 6 shows the severity of periodontitis in the control group. whereas 66 of the 77 subjects (85.7%) belonged to the category of mild/no periodontitis, only 11 (14.3%) subjects had moderate to severe periodontitis. when the proportion of moderate to severe periodontal disease between the groups were compared using a chisquare test, it was observed that the prevalence and severity of periodontal disease was significantly higher in the case group as compared to the controls (p < 0.001). discussion periodontal disease results from the interaction between specific bacteria existing in the dental plaque biofilm with components of host immune response in susceptible individuals. the inflammatory lesion in periodontitis extends from the gingiva to deeper connective tissues resulting in periodontal pockets and loss of alveolar bone. the periodontal pocket serves as a portal of entry for pathogenic bacteria and their products into the systemic circulation. the large surface area of the aggregate periodontal lesion thus serves as a significant source of inflammation in patients with moderate or severe periodontitis28. a large body of epidemiological evidence provides proof that the systemic chronic inflammatory burden of periodontal disease contributes to endothelial injury and atherosclerosis, perhaps mediated by the acute phase reactants29-34. previous studies have shown that chronic inflammation contributes to progressive atherosclerosis in patients with end-stage renal disease (esrd) undergoing hemodialysis35-41. available data suggest that pro-inflammatory cytokines and the acute phase response play a central role in the genesis of both malnutrition and cardiovascular complications in these patients42. emerging evidence also suggest that periodontal disease may provide a covert source of systemic inflammation in these patients28,36 and it may, in fact, predict the development of esrd and the development of overt nephropathy in diabetic patients43. a recently conducted longitudinal study demonstrated that periodontal disease is a significant nontraditional risk factor for chronic kidney disease4 4. our study compared the periodontal health status of patients with different forms of renal disease to that of healthy controls from a south indian population. the results of the present study indicate that a greater prevalence and severity of periodontal disease exists in patients with renal disease. although many previous authors have obtained similar results11-19, conflicting reports are also available and they have failed to detect any difference in the periodontal health in patients undergoing hemodialysis.20-24 in the present study, all the periodontal parameters (ohi-s, mgi, ppd and cal) were elevated in the case group as compared to the control group and the results were statistically significant. the periodontal destruction as indicated by elevated ppd and cal levels is significantly worse in the case group (2.4 + 0.40 and 2.76 + 0.66) as compared to the controls (1.3 + 0.49 and 1.50 + 0.72). the prevalence of moderate to severe periodontitis in the case group (92%) is very high as compared to that in controls (14.3%). while earlier authors14, 18, 22 have conducted similar studies in dialysis populations, our study population included only predialytic patients. the prevalence obtained in the present study is greater than that observed by previous studies in hemodialysis patients 17higher prevalence of periodontal disease among patients with predialytic renal disease braz j oral sci. 8(1): 14-8 (58.914 and 25.9%22) and in chronic ambulatory peritoneal dialysis patients (67.3%)18. therefore, our study result confirms our hypothesis that a greater prevalence and severity of periodontal disease exists in patients with renal disease as compared to systemically healthy controls. it might be possible that severe periodontal inflammation in these patients could have also contributed to the level of their renal disease burden. the systemic disease burden could have also influenced the progression of periodontal disease in these patients. it has been already established that the chronic renal condition could have significant effects on the prevalence and severity of periodontal disease24,45. a recent study conducted in a japanese population suggests that the increased incidence of chronic renal failure that occurs with age might increase the probability of severe periodontal disease in community-dwelling elderly subjects45. the authors also postulate that periodontal disease is influenced by chronic renal failure because of insufficient bone metabolism. earlier studies provide evidence that vitamin d polymorphisms may predispose to both chronic kidney disease and periodontitis46. hence it is possible that periodontal disease and chronic kidney disease might share common risk factors. our study, being cross-sectional in design, does not establish a cause and effect relationship. therefore, further studies are necessary to elucidate the complex relationship between these chronic diseases. to conclude that the development of periodontal disease preceded the onset or progression of the renal condition, further studies with longitudinal study designs are necessary. another limitation of this study is that the case group included subjects with diabetic kidney disease. this may be regarded as a confounder as diabetes mellitus is a risk factor for both, renal and periodontal disease. the studies that have assessed the effect of periodontal therapy on systemic inf lammation prov ide promising results. several reports have indicated that periodontal therapy, consisting of scaling and root planning and microbial plaque control, results in decreased levels of markers of systemic inf lammation47,4 8 and may contribute to improved endothelial function49, 50. however, further research w ith well controlled randomized clinical trials is necessar y to establish whether early detection of periodontal disease followed by effective periodontal therapy w ill actually result in decreased in atherosclerotic complications in patients w ith renal disease. this study prov ides ev idence for a greater prevalence and severity of periodontal disease among predialy tic patients w ith renal disease. as periodontal evaluation is not performed as part of routine medical assessment in these patients, the periodontal source of inf lammation may be overlooked. we hope that the results of our study prov ide emphasis for the fact that the periodontal health of all patients w ith renal disease needs to be carefully monitored. references 1. ebersole jl, machen rl, steffen mj, willmann de. systemic acute-phase reactants, c-reactive protein and haptoglobin in adult periodontitis. clin exp immunol. 1997;107:347-52. 2. loos bg, craandijk j, hoek fj, wertheim-van dillen pm, van der velden u. elevation of systemic markers related to cardiovascular diseases in the peripheral blood of periodontitis patients. j periodontol. 2000;71:1528-34. 3. noack b, genco rj, trevisan m, grossi s, 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endothelial dysfunction in patients with periodontitis and its improvement after initial periodontal therapy. j periodontol. 2004;75:1694-700. 50. tonetti ms, d’aiuto f, nibali l, donald a, storry c, parkar m et al. treatment of periodontitis and endothelial function. n engl j med. 2007;356:911-20. oral sciences n3 original article braz j oral sci. july/september 2010 volume 9, number 3 trends in dental caries in 12and 13-year-old schoolchildren from florianópolis between 1971 and 2009 helena mendes constante1, joão luiz bastos2, marco aurélio peres2 1 undergraduate dental student, federal university of santa catarina, brazil 2 dds, msc, phd, professor, department of public health, federal university of santa catarina, brazil correspondence to: marco aurélio peres departamento de saúde pública, centro de ciências da saúde universidade federal de santa catarina campus universitário – trindade 88010-970 florianópolis sc phone: +55-48-37219046 fax: +55-48-37219542 e-mail: mperes@ccs.ufsc.br received for publication: may 19, 2010 accepted: august 24, 2010 abstract aim: to estimate the prevalence and severity of dental caries in schoolchildren from the city of florianópolis, sc, brazil, in 2009 and to compare these results with data from previous studies carried out in the same institution since 1971. methods: all 145 12and 13-year-old schoolchildren enrolled in a public school were eligible for the study. dental caries was assessed with the dmfs/ dmft indexes, the significant caries index (sic) and their 95% confidence intervals (ci 95%). results: the response rate was 68.2%. the dmft ranged from 9.2 in 1971 to 0.78 in 2009, although different diagnostic criteria were adopted to estimate these counts. the prevalence of dental caries declined from 98.0% (ci 95% 96.0-100.0) in 1971 to 43.5% (ci 95% 33.7-53.4) in 2009. there was a reduction of 47% in the mean sic index between 2002 and 2009, i.e. from 3.4 (ci 95% 3.0-3.8) to 1.8 (ci 95% 1.5-2.1). the mean dmfs index was 2.0, and it was composed of an average of 1.8 and 0.2 decayed and filled surfaces, respectively. conclusions: even though different diagnostic criteria were adopted during the study period, an effective decline in the prevalence and severity of dental caries in adolescents was observed throughout 38 years of monitoring. keywords: dental caries, dmf index, oral health, trends. introduction the world health organization (who) holds epidemiological data showing a decline in the levels of caries experience in 12-year-old children in several countries in recent decades, including some from south america, such as argentina, chile, uruguay, paraguay, and brazil. according to the who1, in 2004, the global mean dmft (an index measuring counts of decayed, missing, and filled permanent teeth) was 1.61. in brazil, the mean dmft was 2.8 in 2002-2003, although almost 70% of the brazilian children aged 12 years and nearly 90% of adolescents aged between 15-19 years presented at least one permanent tooth with dental caries experience, according to the brazilian ministry of health2 and the who1. a bibliographic search carried out in pubmed and scielo using “dental caries” and “trends” as descriptors, limited to the period between 2004 and 2009, showed that several studies in brazil documented declining trends in the mean dmft in 12-year-old adolescents. in the city of bauru, sp, bastos et al.3 observed that the dental caries decline was of 84.5% between 1976 and 2001. gushi et al.4, using braz j oral sci. 9(3):410-414 data from oral health surveys carried out with adolescents between 12 and 18 years of age in the state of são paulo, observed a reduction of 32.3% in the mean dmft between 1998 and 2002. in the city of palhoça, sc, traebert et al.5 observed a reduction of 41.9% in the mean dmft index for adolescents in the same age group between 1997 and 2003. in the city of goiânia, go, the decline in dental caries was even greater, reaching 70.6% between 1988 and 2003, as reis et al.6 reported. studies carried out in the same school in 1971-19977, 20028, and 20059 in florianópolis, sc, also demonstrated an expressive reduction in the severity (91.3%) and in the prevalence (58.2%) of dental caries. worldwide, very few studies have monitored dental caries prevalence and severity in the same population group for a long period. in australia, armfield et al.10 conducted a study with schoolchildren enrolled in public and private schools from 1977 to 2002 and observed a reduction of 79% in the experience of dental caries throughout 25 years. in somerset, united kingdom, anderson11 reported that the mean dmft declined by 80% in 12-year-old adolescents from two schools, between 1963 and 1988. the monitoring of dental caries over time is a part of oral health surveillance system; it is important to subsidize health planners to implement and evaluate preventive programs and dental care. the aim of this study was to evaluate the prevalence and severity of dental caries in 12and 13year-old schoolchildren from florianópolis, sc, brazil, in 2009 and to compare these data to the results obtained in previous studies conducted in the same school in 1971, 1997, 2002, and 2005. material and methods the sample comprised all 145 students 12 and 13 years of age, enrolled in a public school (escola de educação básica padre anchieta) in the city of florianópolis, state of santa catarina, southern brazil, in 2009. the same methodological procedures used in previous studies7-9 were adopted in the present investigation, including the collection of clinical data under natural light and using plane dental mirrors and periodontal probes (ball-end probes), following the diagnostic criteria recommended by the who12. the fieldwork team was composed of two undergraduate dental students; the first performed all dental examinations, whereas the second transferred clinical records to appropriate forms. both were supervised and trained by an experienced dental epidemiologist (map) to ensure standardization of dental caries diagnosis, as well as to avoid discrepancies in interpretation of dental examinations. this calibration training consisted of repeated dental examinations on 15 subjects, followed by discussion on the dental caries diagnostics. this process was ended when the discrepancies were eliminated, and consistency in dental caries diagnosis was achieved among dental examiners. for this phase of the fieldwork, intraand interexaminer reliabilities were not estimated. because a remarkable decline in dental caries prevalence over the past decades has been observed, the dmfs index (number of decayed, missing, or filled permanent dental surfaces) was used as a diagnostic criterion, in addition to the dmft index. a tooth surface was considered as “decayed” when a lesion in a pit or fissure or a smooth tooth surface presented an unmistakable cavity, undermined enamel, or a detectable softened floor or walls. a filled but still decayed surface was also included in this category. this index, recommended by the who12, also considered further areas with past experience of dental caries, recording those that received restorative dental treatment and those that have been extracted because of caries. according to the methodology proposed by pinto13, the 28 permanent teeth were divided into 128 surfaces, as follows: molars and premolars with five surfaces (occlusal, mesial, distal, buccal, and lingual) and incisors and canines with four surfaces (mesial, distal, vestibular, and lingual). to record clinical data, the following sequence was respected: upper right (2nd molar to the central incisor), upper left (central incisor to 2nd molar), lower left (2nd molar to central incisor), and lower right (central incisor to 2nd molar). to analyze tooth surfaces, the examination started in the occlusal surface (o), followed by mesial (m), buccal (b), distal (d), and lingual (l) surfaces. data were typed into a microsoft® excel spreadsheet. statistical analysis was performed with stata, v.9. the prevalence of dental caries, the mean dmfs index, the mean dmft index, and their components were also estimated with 95% confidence intervals. the chi-square test was used to compare caries prevalence according to sex, and the mannwhitney u test was used to compare the distributions of dmfs according to sex and age. the significant caries index (sic) was calculated based on the mean dmft index of the highest dmft tertile in the studied population14. a letter was sent to parents explaining the aims and importance of the study and asking permission for their children’s participation. when the survey was completed, another letter was sent to acknowledge the participation of adolescents, to report any dental treatment needs, and to provide advices on oral health. the project was approved by the ethics committee for human research of the federal university of santa catarina (protocol number 126249/2008-3). results from a total of 148 eligible schoolchildren, 101 took part in the study (response rate of 68.2%). losses were due to refusals (18.2%) and transferences to other schools, during data collection period. the distribution of the studied adolescents by age and sex in the present and previous studies is shown in table 1. in 1971, 1997, 2002, and 2005, most of the schoolchildren were aged 12 years; this proportion ranged from 62.9% in 1971 to 45.5% in 2009. however, in 2009, 13-year-old children were more frequent (54.5%), when taking the 1971 study as a reference. the differences in caries prevalence by sex and age were not statistically significant (p = 0.4 and p = 0.1, respectively). consequently, the results are shown for the sample as a whole. 411411411411411 trends in dental caries in 12and 13-year-old schoolchildren from florianópolis between 1971 and 2009 braz j oral sci. 9(3):410-414 variables 1971 1997 2002 2005 2009 n % n % n % n % n % age 12 years 127 62.9 101 57.7 88 52.1 118 69.0 46 45.5 13 years 75 37.1 74 42.3 81 47.9 53 31.0 55 54.5 gender male * * 90 51.4 77 45.6 81 47.4 48 47.5 female * * 85 48.6 92 54.4 90 52.6 53 52.5 total 202 100.0 175 100.0 169 100.0 171 100.0 101 100.0 * unavailable information table 1. distribution by age and sex of 12and13-year-old schoolchildren enrolled in escola de educação básica padre anchieta in 1971, 1997, 2002, 2005 and 2009. florianópolis, santa catarina, brazil, 2009. table 2 displays the mean dmft and its components, as well as 95% confidence intervals. in the present study, the mean d component was 0.65 (ci 95% 0.45-0.83), followed by the f component with 0.12 (ci 95% 0.03-0.22) and by the m component with 0.01 (ci 95% 0.000.03). the mean dmft index was 0.78 (ci 95% 0.56-1.00). in the later period of the present study (2009), the d component contributed most to the mean dmft value, corresponding to 83.3% of the index. when compared to the percent composition of the dmft index in the first study year (1971), there was a greater reduction of the m component (97.43%), followed by the d (91.71%) and f (87.09%) components in the present study. caries prevalence, i.e. the number of participants with dmfte”1 divided by all examined schoolchildren, declined from 98.0% (ci 95% 96.0-100.0) in 1971 to 93.7% (ci 95% 90.0-98.0) in 1997, when the klein and palmer (1938) diagnostic criteria were adopted. between 1997 and 2002, and taking into account the who 1987 and the who 1997 diagnostic criteria, respectively , the caries prevalence decreased from 80.0% (ci 95% 74.0-86.0) to 57.4% (ci 95% 50.0-65.0). in the penultimate period of observation (20022005), caries prevalence declined from 57.4% (ci 95% 50.065.0) to 40.9% (ci 95% 33.5-48.4), according to the criteria set forth by the who12 in 1997. in the later period of the year d m f dmf-t % mean 95% ci % mean 95% ci % mean 95% ci % mean 95% ci 19711 85.7 7.85 * 4.2 0.39 * 10.1 0.93 * 100.0 9.17 * 19971 90.7 5.67 4.57–6.77 0.5 0.03 0.02–0.04 8.8 0.55 0.22–0.88 100.0 6.25 5.15–7.35 19972 2.42 3.00 2.67–3.33 20023 46.8 0.65 0.63–0.67 5.7 0.08 0.03–0.10 47.5 0.66 0.46–0.86 100.0 1.39 1.14–1.64 20053 56.8 0.50 0.30–0.60 4.9 0.04 0.01–0.06 38.3 0.3 0.20–0.40 100.0 0.84 0.60–1.00 20093 83.3 0.65 0.45–0.83 1.3 0.01 0.00– 0.03 15.4 0.12 0.03–0.22 100.0 0.78 0.56–1.00 table 2. mean dmft index and its components decayed (d), missing (m) and filled (f) teeth in 12and 13-year-old schoolchildren enrolled in padre anchieta basic school in 1971, 1997, 2002, 2005 and 2009. florianópolis, sc, brazil, 2009. *unavailable information 1klein and palmer (1938)21 diagnostic criteria 2who diagnostic criteria (1987)22 3who diagnostic criteria (1997)12 present study (2009), the dental caries prevalence reached 43.5% (ci 95% 33.753.4). the average of dmft index was 9.2 (ci 95% not available) in 1971, declining to 6.2 (ci 95% 5.2-7.4) in 1997, according to the klein and palmer (1938) diagnostic criteria. the mean reached 3.0 (ci 95% 2.7-3.3) and 1.4 (ci 95% 1.11.6) in 2002, when the who 1987 and the who 1997 diagnostic criteria were adopted, respectively. in 2005, the mean dmft index was 0.8 (ci 95% 0.6-1.0), a value also observed in the present study (figure 1). in 2002, the sic index was 3.4 (ci 95% 3.0-3.8), decreasing to 2.2 (ci 95% 1.82.5) in 2005 and to 1.8 (ci 95% 1.5-2.1) in the present study. this corresponds to a reduction of 47% (figure 2). table 3 shows that the mean decayed and filled surfaces in 2009 was 1.8 (ci 95% 1.1-2.5) and 0.2 (ci 95% 0.0-0.4), respectively. seventy-five percent (75th percentile) of the adolescents showed a mean of decayed and filled surfaces (d and f) below 2. in 2009, the mean dmfs index was found to be 2.0, with an average of decayed surfaces of 2.3 in males and 1.7 in females, a nonsignificant difference. at the age of 13 years, an average of 2.7 decayed and filled surfaces was observed; at the age of 12 years, this average was 1.2. however, this difference was not statistically significant. 412412412412412trends in dental caries in 12and 13-year-old schoolchildren from florianópolis between 1971 and 2009 braz j oral sci. 9(3):410-414 table 3. descriptive statistics of decayed and filled surfaces of 12and 13-year-old schoolchildren enrolled in escola de educação básica padre anchieta, florianópolis santa catarina, brazil, 2009. decayed surfaces (ds) filled surfaces (fs) decayed and filled surfaces (dfs) mean 1.8 0.2 2.0 ci 95% 1.1-2.5 0.0-0.4 1.2-2.8 median 0.0 0.0 0.0 minimum 0 0 0 maximum 20 8 20 percentile 75 1.5 0.0 2.0 discussion since 1971, when the first of this series of studies was conducted, until 2009, an effective decline in dental caries prevalence and severity was observed in the studied population even though different diagnostic criteria were used along the studied period, a reduction of 91.4% in the caries severity was found in the 2009 study, when compared with the 1971 study. compared to the 2002 study, which adopted the same diagnostic criteria, a reduction of 43.4% in the caries severity was observed in 2009. despite the fact that the mean dmft did not change between 2005 and 2009, its components presented contradictory proportions when compared with those observed in previous studies. the d component showed a reduction of 93.6% between 1971 and 2005, represented 83.3%of the dmft in 2009, and showed a relative increase of 30% over the last studied period (2005-2009). these data may hypothetically indicate a decrease in dental care utilization in the region, a paradox considering that, for this period, the number of dentists in the city increased, achieving a ratio of 1 dentist for every 250 inhabitants in the city of florianópolis in june 2009. a less invasive approach by dentists in the city may be another explanation for such a decline in the proportion of d component. the 2009 f component showed a reduction of 60% as compared with the previous study (2005) and comprised 15.4% of the dmft index in 2009. the m component showed an even greater reduction (75%) since the last year of study, although it represents only 1.3% of the mean dmft index in the current study. the mean dmft was 0.78, reaching one of the who goals for the year 2010, that is, a mean dmft below 1.0 for the age of 12 years, according to gomes et al.15. it is noteworthy that there are few studies conducted in the same place for such a long period and under the supervision of the same research team. marcenes et al.16 and souza et al.9, suggested that the reduction observed in dental caries experience may be partially attributed to changes in diagnostic criteria for dental caries over time. in brazil, similar decline in dental caries was demonstrated by other researchers. as stated by martins et al.17 the city of in bilac, sp, a similar reduction in the mean dmft index was achieved, despite the lack of water fluoridation supply during the study period. according to bastos et al.3 (2005), fluoridated water supplies contributed to the remarkable reduction in the mean dmft in bauru, sp. in fact, the results from the “sb brazil: oral health in the brazilian population” held in 2003, produced by ministry of health2, revealed that in the south region an area with high water fluoridated coverage presented 37.7% of cariesfree adolescents aged 12 years, in contrast to only 12.9% of caries-free adolescents in non-fluoridated regions. worldwide, similar reductions in caries experience have been documented. armfield and spencer10 (2008) analyzed data from dental caries in adolescents enrolled in public and private schools between 1977 and 2002 in australia and found a reduction of 79% for the age of 12 years. in this same study, an annual reduction of 2.6% the sic index between 1977 and 2002 for the same age was observed. these values were lower than the ones found in the present study (annual reduction of 6.7%). in lithuania (1993-2001), aleksejûnienë et al.18 (2004) reported that the decline in dental caries rates may be explained by an improvement in oral hygiene because 413413413413413 trends in dental caries in 12and 13-year-old schoolchildren from florianópolis between 1971 and 2009 braz j oral sci. 9(3):410-414 fig.1 mean dmft index and its decayed, missing, and filled components in the years 1971, 1997, 2002, 2005, and 2009 of 12and 13-year-old schoolchildren enrolled in escola de educação básica padre anchieta in 1971, 1997, 2002, 2005, and 2009. florianopolis, santa catarina, brazil, 2009. * unavailable information; 1klein and palmer (1938)21 diagnostic criteria 2who diagnostic criteria (1987)22; 3who diagnostic criteria (1997)12 fig. 2 trend of mean significant caries index (sic) of 12and 13-year-old schoolchildren enrolled in escola de educação básica padre anchieta from 2002 to 2009. florianopolis, santa catarina, brazil, 2009. products for this purpose began to be sold in 1991 and had an increase of 95% in their use in 2001 in that country. the us centers for diseases control and prevention19 declared that the fluoridation of water supplies is a the most effective mass intervention to prevent dental caries, and it was considered one of the most important public health achievements in the last century. in addition to water fluoridation, the major decline in dental caries prevalence and severity has been attributed to the access to other fluoride sources, especially to the widespread use of fluoridated toothpaste. moreover, an improvement in socioeconomic indicators and the boost of initiatives in dental public health also have been listed, by bastos et al.3, ministry of health20, martins et al.17, as important to dental caries reduction over time. as stated by bastos et al.3 and martins et al.17, the use of fluoridated toothpaste in brazil was intensified from 1989, when the ministry of health regulated the addition of fluoride in these products and mouthrinses. possibly, according to freysleben et al.7, bastos et al.8 and souza et al.9, these factors contributed to a higher rate of annual reduction in the dental caries severity over the periods that followed the studies through the 1990s in florianópolis. however, the design of this study does not allow an assessment of the possible reasons for the decline in dental caries prevalence. in the first period (1971 to 1997), in which the lowest annual reduction rate was observed compared with other years, this could be explained, as noted by freysleben et al.7, by the shorter period of public exposure to fluoridated water, implemented in florianópolis in 1982, and the availability of use of fluoride toothpaste from the late 1980’s. in 1971, the school was located in a suburban area of the city, which became, in 1997, a middle class region with a residential profile and close to the city’s downtown with better living conditions. in the second studied period (1997 to 2002), the caries decline can be attributed to the increased availability of fluoridated toothpaste and to the implementation of fluoridated water supply. additionally, florianópolis in 2003 occupied the third position among the capitals with the lowest level of social exclusion in brazil. in 2008, bastos et al.8 reported that florianópolis appeared as the city with the highest human development index (hdi of 0.86) among 33 other brazilian metropolitan areas and cities and presented the highest average income among brazilian cities. despite the observed declining trend in dental caries over the studied period, different authors10-11,18 found that the disease rate is likely to stabilize because of the low value already reached. some limitations of this study must be mentioned. the 2009 study had a relatively low response rate, lower than those observed in the previous phases. we can hypothesize that the mean dmft could be higher, assuming that individuals who were not examined presented higher dental caries attack than those who effectively took part in the study. in conclusion, despite the fact that different dental caries diagnostic criteria were used, there was an actual decrease in the mean dmft over the 38-year study period. it should be noted that these results cannot be readily generalized to the entire population of 12 and 13 years of age from florianópolis, sc. acknowledgments helena mendes constante was supported by a scholarship granted from the conselho nacional de desenvolvimento científico e tecnológico (cnpq) of brazil from november 2008 to july 2009 (number 126249/2008 3). references 1. world health organization. oral health country profiles. available from: http://www.whocollab.od.mah.se/index.html. acessed 2009 aug. 2. brasil. ministério da saúde. secretaria de atenção à saúde. departamento de atenção básica. projeto sb brasil 2003: condições de saúde bucal da população brasileira 2002-2003: resultados principais. brasília: ministério da saúde; 2004. 3. bastos rs, olympio kpk, bijella vt, buzalaf mar, bastos jrm. trends in dental caries prevalence in 12-year-old schoolchildren between 1976 and 2001 in bauru, brazil. public health. 2005; 119: 269-75. 4. gushi ll, rihs lb, soares mc, forni tib, vieira v, wada rs, et al. cárie dentária e necessidades de tratamento em adolescentes do estado de são paulo, 1998 e 2002. rev saúde pública. 2008; 42: 480-6. 5. traebert j, lacerda jt, fischer tk, junbo y. dental caries and orofacial pain trends in 12-year-old school children between 1997 and 2003. oral health prev dent. 2005; 3: 243-8. 6. reis scgb, freire mcm, higino masp, batista smo. declínio de cárie em escolares de 12 anos da rede pública de goiânia, goiás, brasil, no período de 1988 a 2003. rev bras epidemiol. 2009; 12, 1: 92-8. 7. freysleben gr, peres maa, mercenes w. prevalência de cárie e cpod médio em escolares de doze a treze anos de idade nos anos de 1971 e 1997, região sul, brasil. rev saúde pública. 2000; 34: 304-8. 8. bastos jld, nomura lh, peres ma. trends in dental caries rates in schoolchildren 12 and 13 years old in florianópolis, santa catarina, brazil, 1971-2002. cad saúde pública. 2004; 20: 117-22. 9. souza ml, bastos jld, peres ma. trends in dental caries rates in 12and 13year old schoolchildren from florianópolis (brazil) between 1971 and 2005. oral health prev dent. 2006; 4: 187-92. 10. armfield jm, spencer aj. quarter of a century of change: caries experience in autralian children, 1977-2002. aust dent j. 2008; 53: 151-9. 11. anderson rj. the changes in dental caries experience of 12-year-old schoolchildren in two somerset schools. a review after an interval 25 years. br dent j. 1989; 167: 321-4. 12. world health organization. oral health surveys: basic methods. 4th ed. geneva: world health organization; 1997. 13. pinto vg. saúde bucal e coletiva. 4. ed. são paulo: santos; 2000. 14. bratthall d. introducing the significant caries index together with a proposal for a new global oral health goal for 12-year olds. int dent j. 2000; 50: 378-84. 15. gomes pr, costa sc, cypriano s, sousa mlrp, são paulo, brasil: situação da cárie dentária com relação às metas oms 2000 e 2010. cad saúde pública 2004; 20: 866-70. 16. marcenes w, freysleben gr, peres ma. contribution of changing diagnostic criteria toward reduction of caries between 1971 and 1997 in children attending the same school in florianopolis, brazil. community dent oral epidemiol 2001; 29: 449-55. 17. martins rj, garbin cas, garbin aji, moimaz sas, saliba o. declínio de cárie em um município da região noroeste do estado de são paulo, brasil, no período de 1998 a 2004. cad saúde pública. 2006; 22: 1035-41. 18. aleksejûnienë j, holst d, balciûnienë i. factors influencing the caries decline in lithuanian adolescents—trends in the period 1993-2001. eur j oral sci. 2004; 112: 3-7 19. centers for diseases control and prevention (1999). achievements in public health, 1900-1999. fluoridation of drinking water to prevent dental caries. morb mortal wkly rep. 44(rr-13): 1-40. 20. brasil. ministério da saúde. secretaria nacional de vigilância sanitária. portaria n. 22, de 20/12/1989. diário oficial união, seção i. available from: http: //dtr2004.saude.gov.br/dab/docs/legislacao/portaria22_20_12_89.pdf. acessed 2009 aug. 21. klein h, palmer ce. dental caries in american indian children. washington dc: government printing; 1938. 22. world health organization. oral health surveys: basic methods. 3rd ed. geneve: world health organization; 1987. 414414414414414trends in dental caries in 12and 13-year-old schoolchildren from florianópolis between 1971 and 2009 braz j oral sci. 9(3):410-414 oral sciences n3 braz j oral sci. 9(1):7-10 original article braz j oral sci. january/march 2010 volume 9, number 1 intravenous procedural sedation: an alternative in the treatment of patients with intellectual disability cíntia megid barbieri1, alessandra cristina gomes2, taís elisabete crivellaro de menezes2, sandra maria herondina coelho ávila de aguiar3 received for publication: june 24, 2009 accepted: march 03, 2010 correspondence to: sandra m. h. c. ávila de aguiar faculdade de odontologia de araçatuba unesp rua josé bonifácio, 1193 cep: 16.015-050 araçatuba sp brasil e-mail: saguiar@foa.unesp.br abstract conscious sedation has become established as an important alternative to general anesthesia (ga) in dental treatment of patients with intellectual disability (id). aim: to investigate dental patients undergoing sedation using a mean dose of 0.6 mg/kg intravenous midazolam and the adverse events of sedation in patients with id. methods: this study analyzed the records of 163 dental patients with id aged between 2 and 76 years who had undergone conscious intravenous sedation (civ) using a mean dose of 0.61 mg/kg of midazolam at araçatuba dental school, são paulo state university, brazil. the efficacy and complications induced by civ were evaluated in each subject. results: civ was effective for dental treatment in 80% of the cases. a total of 626 dental procedures were performed. the mean treatment time was 33.9 minutes. there was statistically significant difference (p<0.05) between absence and presence of adverse reactions. adverse reactions were observed in 21.47% of the cases. conclusions: the results of this study showed that civ is a useful method for dental treatment of patients with id and these patients can need higher doses of sedative to reach an adequate level of sedation. keywords: dental anesthesia, dental care, intellectual disability, sedation. introduction it has been acknowledged that the population with disabilities has higher rates of poor oral hygiene, gingivitis, and periodontitis than the general population. moderate or severe gingivitis has been found almost ordinarily, with degree and extent increasing with age and severity of mental retardation1-2. moreover, special needs patients tend to have more decayed and missing teeth compared to nondisabled patients3. an extended series of reports on the use of dental services by children and adults with disabilities focuses on limited physical access to buildings, limited practitioner willingness to provide care, and associated insurance and financial difficulties resulting in longer periods between dental visits and a tendency to have had extractions at the last visit4-5. sedation is a useful method for relieving dental fear and anxiety in patients, and it is often essential for people with id undergoing dental treatment 6. benzodiazepines are currently the pharmacological group of choice due to their greater effectiveness provided by their efficacy and safety margin. in addition, muscular relaxant effect of these drugs helps calming the patient down. in this group, midazolam has been efficient in controlling the anxiety and maintaining blood pressure and oxygen saturation within normal levels7-12. the pharmacological characteristics that made midazolam a drug of choice for clinical sedation are: quick action, short half life and suitable sedation duration. 1dentist, special care clinic of the araçatuba dental school, unesp – univ estadual paulista, brazil 2postgraduate student, department of pediatric dentistry, araçatuba dental school, unesp – univ estadual paulista, brazil 3professor, department of pediatric dentistry, araçatuba dental school, unesp – univ estadual paulista, brazil 8 braz j oral sci. 9(1):7-10 its anxiolytic, hypnotic and amnesiac effects compose the main objectives to be reached with its utilization. moreover, the diversity of administration routes (oral, rectal, intramuscular, endovenous and intranasal) offers alternatives that better suits a great number of patients7-8,12-13. conscious intravenous sedation (civ) is frequently provided for dental patients as an alternative to ga in the center of dental care to special needs patients of araçatuba dental school, são paulo state university (caoe/foa/ unesp), brazil. however, the decision about the required dose for an adequate level of sedation is difficult because the effect of sedation cannot be adequately assessed in patients with severe id. it has been reported that a high dose of sedative is required for patients with id to obtain an adequate level of sedation9,14, but an appropriate sedative dose has not been satisfactorily established for dental patients with id. thus, the authors investigated dental patients undergoing sedation using a medium dose of 0.6 mg/kg intravenous midazolam and the adverse events of sedation in patients with intellectual disability (id). material and methods one hundred and sixty three mentally handicapped patients were treated under civ at the caoe/foa/unesp, brazil between january and july 2003 (figure 1). this treatment was chosen because each patient had previously exhibited combative behavior sufficiently negative to dental treatment using routine behavior management techniques or presented muscular spasticity (cerebral palsy patients). the research ethics committee of araçatuba dental school, são paulo state university, brazil, approved the study (protocol #foa 876/2003). the age of the patients undergoing civ ranged from 2 to 76 years. only patients with asa i or asa ii physical status were eligible for the study. civ was induced with a bolus intravenous administration of midazolam ranging 0.3 to 1.2 mg/kg for a mean dose of 0.61 mg/kg and all patients were immobilized with bands to control involuntary movements that might occur during the dental procedure. the dental treatment started 2 min after midazolam administration. once the patients were sufficiently sedated, local anesthesia with 2% lidocaine with 1/100,000 adrenaline was delivered before the clinical procedures. the maximum fig. 1. classification of patients according to the handicapping condition. figure 2. number of dental procedures performed under sedation. dose of local anesthetic was limited strictly to 4.4 mg/kg for all patients. resuscitation equipment was available if required and a medical team followed each treatment session. the efficacy of sedation and the duration of the dental treatment were investigated. the efficacy of sedation was evaluated by judgment of the patient cooperation to the dental treatment and the determination of the dose to be administered to each patient was accomplished by the cardiologist responsible for the sedation at caoe/foa/ unesp, according to the patient’s body weight, physiological, nutritional and pathological evaluation. in the present study, the mean effective dosage was 0.61 mg/kg. according to the observations made by the senior researcher, the sedation was considered to be effective when the patient was classified as “cooperative” (the patient was sedated and remained awake, sleeping or sleepy during the treatment session, but was cooperative). the sedation was considered not-effective when the patient was classified as “non-cooperative” (the patient was awake and uncooperative, not allowing the procedures to be done, even after sedation. the adverse effects of civ were also evaluated in each subject. the incidence of preand postciv complications, including vomiting, respiratory depression and others (tremor, tachycardia), were also evaluated. data were analyzed statistically by the chi-square test. statistical differences were considered significant at p<0.05. results a total of 626 dental procedures, such as amalgam, resin and glass ionomer cement restorations, sealants, extractions, scaling and root planing, root canal therapy, and others (frenectomy, ulectomy and biopsy), were performed (3.8 procedures per patient on the average) (figure 2). the mean treatment duration was 33.9 min (table 1). in 56% of the patients, all necessary procedures were completed in a single session. civ was effective for dental treatment in 80% of the subjects with id. there was accentuated resistance in 20% of the patients during the dental procedures, and these patients were subjected to ga in another day. there was no statistically significant association (p>0.05) between midazolam dose (mg/kg) and patient response (table 2). there was statistically significant difference (p<0.05) between the absence and the presence of adverse reactions intravenous procedural sedation: an alternative in the treatment of patients with intellectual disability 9 duration of procedures (min) number of subjects 00 – 20 45 (27.6%) 21 – 40 73 (44.8%) 41 – 60 40 (24.5%) 61 – 80 4 (2.4%) 81 – 100 1 (0.7%) total 163 (100%) table 1 number of subjects according to the duration of procedures in minutes. side effects number of subjects respiratory depression 27 (55%) vomiting 11 (22.4%) fluttering 7 (14.2%) others 4 (8.4%) total 49 (100%) table 3 distribution of side effects in the subjects during dental treatment in this study. there were 49 episodes of adverse reactions in 35 patients (only 21.47%), in which 16.5% were related to respiratory depression, 6.7% to vomiting, 4% to flutter and 2.4% to other reactions (table 3). in these cases, patients received appropriate medical assistance and medication. there was no difference regarding to the dosage per body weight of medication administered to the patients who developed adverse reactions (table 2). discussion over the last few years several management modalities have been developed to overcome difficulties encountered during complex dental procedures for treatment of special needs patients. various methods of sedation and sedatives have been described for dental treatment for patients with id as preferred alternative to full ga6,9-10. it is widely accepted that civ is safer than ga11-13. however, poorly controlled conscious sedation may result in “deep sedation” or even ga with all its attendant risks13. since the possibility of an exaggerated level of central nervous system depression always exists, the drugs and techniques employed must carry a wide margin of safety, and the physician should be particularly careful to render the loss of reflexes unlikely. this is especially true in some of the disable patients who are chronically medicated, which could alter their response to certain sedative drugs 12. however, it is necessary to emphasize that the highest interaction chances are reported, mainly through the use of oral midazolam, with the following drugs: carbamazepine, phenytoin, rifampicin, roxithromycin, erythromycin, fluconazole and verapamil 7. it is important that during intravenous sedation vital signs including blood pressure, pulse rate, and arterial blood oxygen saturation are continuously and properly monitored and recorded15. currently, there is divergence among the dose, drugs and administration route used in civ. the present study describes the use of civ as an alternative to ga in dental treatment of patients with id at caoe/foa/unesp, brazil. a bolus administration of midazolam was performed, which has been reported to be useful in dental treatment16-18. the subjects in this study had previously exhibited behavior sufficiently negative to dental treatment using routine behavior management techniques. however, the intravenous technique resulted in a sedative effect. carrying out dental care by this method, the patient’s rejection and evasive behavior patterns were reduced as compared with the controls (previous treatment without intravenous sedation). the rate of behavior evaluated as positive were 80% of cases, although the other 20% that exhibited negative behavior were subjected to ga in another day. it was found that intravenous midazolam is a very effective form of sedation for patients with id. the dose used in intravenous sedation in this study for each patient was obtained by analysis of weight, pathology, behavior and physiology. in this analysis a medium dose was 0.61 mg/kg. the doses used were higher than that used in other studies with patients without id17-18. in accordance with this result, others studies have also demonstrate that dental patients with id need higher doses of sedative to obtain an adequate level of sedation9,19. the reason that dental patients with id required a higher dose of midazolam than other patients is unclear. variations among individuals are thought to result from pharmacokinetic and pharmacodynamic factors9. in this study, the blood concentration of midazolam was not measured, but it is possible that in subjects with id it does not increase proportionally to the administrated dose17. in this way, pharmacokinetic pharmacodynamic factors are thought to be involved in the variations of the required dose of sedative for intravenous sedation. the differences in the required doses of midazolam between subjects with id and other subjects might derive from differences in sensitivity to the sedative because the success will be dependent on the individual response of each patient9. adverse events are rare in dental anesthesia, and a definitive evaluation of safety requires a long history of treatment using a given technique. while a trial of this size cannot assess the frequency of possible adverse events, the present results indicate a safe technique. the conscious sedation techniques practiced in the present ensured braz j oral sci. 9(1):7-10 intravenous procedural sedation: an alternative in the treatment of patients with intellectual disability dose of midazolam mg/kg positive behavior negative behavior presence of side effects absence of adverse reactions 0.1 – 0.60 kg 63 16 12 68 0.61 – 1.20 67 17 23 60 total 130 33 35 128 table 2 distribution of behavior and side effects of subjects according to the dose of midazolam (mg/kg) during dental treatment. 10 cooperation and consciousness throughout the procedure and full control of protective reflexes. this procedure presents minimal risk in contrast to ga11. the subjects of this study were able to receive the expected dental care, but complications like vomiting, respiratory depression, flutter, tremor and tachycardia could be noted. despite these reactions, there were not significant differences for the presence of adverse reaction associated to drug dose (mg/kg). it was defined as a true decrease of oxygen saturation when a pulse oxymeter reading of spo2 below 92 percent. it was found 27 episodes of true decrease of oxygen saturation in this study. in accordance with others studies, the respiratory depression was the most predominant adverse reaction occurred during conscious sedation15,20-24. the literature shows that children, elderly, obese, and patients with cerebral palsy, autism or medically compromised who were treated with conscious sedation were more susceptible to respiratory problems15,25-26. supplemental oxygen has been recommended for all patients undergoing civ27 and in the present study supplementation was used in some cases, which presented respiratory depression. the reported incidence of nausea and vomiting after sedation and local anesthesia is less than that observed after ga (7% versus 14%). moreover, the time to discharge was shorter for patients receiving sedation28. this study observed a very low complication rate with the use of oral midazolam in dental treatment for patients with id. in conclusion, the results of this study showed that civ is a useful method for providing dental care to patients with id and produced fewer adverse reactions in comparison to ga. the dose used indicated that dental patients with id need higher doses of sedative to obtain adequate levels of sedation. in spite of being considered an important matter, there is insufficient evidence of the health care for subjects with id, and further studies are necessary for offering better dental care to these patients. references 1. waldman hb, perlman sp, swerdloff m. what if dentists did no treat peolpe with disabilities? asdc j dent child. 1998; 65: 96-101. 2. glassman p, miller c, wozniak t, jones c. a preventive dentistry trainning program for caretakers of persons with disabilities residing in community residential facilities. spec care dent. 1994; 14: 137-43. 3. nunn jh, murray jj. the dental health of handicapped children in newcastle and northumberland. br dent j. 1987; 162: 9-14. 4. leviton fj. the willingness of dentists to treat handicapped patients: a summary of eleven surveys. j dent handicap. 1980; 5: 13-7. 5. ferguson fs, berentsen b, richardson ps.dentists’ willingness to provide care for patients with developmental disabilities. spec care dentist. 1991; 11: 234-7. 6. manley mc, skelly am, hamilton ag. dental treatment for people with challenging behaviour: general anaesthesia or sedation? br dent j. 2000; 188: 358-60. 7. ramos tc, ramos mesp, azoubel mcf, soares ar, alves etp, bezerra f. midazolam: aspectos clínicos e farmacológicos em sedação consciente. estudo retrospectivo avaliando 258 casos clínicos. innov implant j. 2009; 4(1): 39-45. 8. pèrez y, pardo a, sequera r, crespo jd. sedación y analgesia en pacientes pediátricos para procedimientos fuera del área quirúrgica. rev venez anestesiol. 2001; 6: 86-100. 9. miyawaki t, kohjitani a, maeda s, egusa m, mori t, higuchi h et al. intravenous sedation for dental patients with intellectual disability. j intellect disabil res. 2004; 48: 764-8. 10. fukuta o, braham rl, yanase h, kurosu k. the sedative effects of intranasal midazolam administration in the dental treatment of patients with mental disabilities. part 2: optimal concentration of intranasal midazolam. j clin pediatr dent. 1994; 18: 259-65. 11. averley pa, girdler nm, bond s, steen n, steele j. a randomised controlled trial of paediatric conscious sedation for dental treatment using intravenous midazolam combined with inhaled nitrous oxide or nitrous oxide/ sevoflurane. anaesthesia. 2004; 59: 844-52. 12. malamed sf. sedation. a guide to patient management, 3rd edn. boston: mosby; 1995. 13. chaushu s, gozal d, becker a. intravenous sedation: an adjunct to enable orthodontic treatment for children with disabilities. eur j orthod. 2002; 24: 81-9. 14. morton ns, oomen gj. development of a selection and monitoring protocol for safe sedation of children. paediatr anaesth. 1998; 8: 65-8 15. coté cj, notterman da, karl hw, weinberg ja, mccloskey c. adverses sedation events in pediatrics: a critical incident analysis of contributing factors. pediatrics. 2000; 105: 805-14. 16. milnes ar, maupomé g, cannon j. intravenous sedation in pediatric dentistry using midazolam, nalbuphine and droperidol. pediatr dent. 2000; 22: 113-9. 17. runes j, ström c. midazolam intravenous conscious sedation in oral surgery. a retrospective study of 372 cases. swed dent j. 1996; 20: 29-33. 18. richards a, griffiths m, scully c. wide variation in patient response to midazolam sedation for outpatient oral surgery.oral surg oral med oral. pathol. 1993; 76: 408-11. 19. roelofse ja, van der bijl p.propofol for sedation in a mentally retarded dental patient. anesth prog. 1994; 41: 81-2. 20. bailey pl, pace nl, ashburn ma, moll jw, east ka, stanley th. frequent hypoxemia and apnea after sedation with midazolam and fentanyl. anesthesiology. 1990; 73: 826-30. 21. kupietzky a, houpt mi. midazolam: a review of its use for conscious sedation of children. pediatr dent. 1993; 15: 237-41. 22. litman rs, berkowitz rj, ward ds. levels of consciousness and ventilatory parameters in young children during sedation with oral midazolam and nitrous oxide. arch pediatr adolesc med. 1996; 150: 671-5. 23. rohlfing gk, dilley dc, lucas wj, vann wf jr. the effect of supplemental oxygen on apnea and oxygen saturation during pediatric conscious sedation. pediatr dent. 1998; 20: 8-16. 24. blumer jl. clinical pharmacology of midazolam in infants and children. clin pharmacokinet. 1998; 35: 37-47. 25. hardeman jh, sabol sr, goldwasser ms. incidence of hypoxemia in the postanesthetic recovery room in patients having undergone intravenous sedation for outpatient oral surgery. j. oral maxillofac surg. 1990; 48: 942-4. 26. zacharias m, hunter km, parkinson r. respiratory effects of intravenous midazolam. n z dent j.1996; 92: 76-9. 27. manara ar, smith dc, nixon c. sedation during spinal anaesthesia: a case for the routine administration of oxygen. br j anaesth. 1989; 63: 343-5. 28. brandt sk, bugg jl jr. problems of medication with the pediatric patient. dent clin north am. 1984; 28: 563-7. braz j oral sci. 9(1):7-10 intravenous procedural sedation: an alternative in the treatment of patients with intellectual disability oral sciences n3 braz j oral sci. 10(4):246-249 original article braz j oral sci. october | december 2011 volume 10, number 4 received for publication: july 19, 2011 accepted: october 03, 2011 perception of dental surgeons on the ethical and legal aspects of exercising their profession as personal and corporate entities mariana mourão de azevedo flores pereira1, rhonan ferreira da silva1, luíza valéria de abreu maia2, ricardo henrique alves da silva3, eduardo daruge junior4, luiz renato paranhos5 1dds, msc, department of community dentistry, school of dentistry, university of campinas (unicamp), brazil 2dds, department of community dentistry, school of dentistry, university of campinas (unicamp), brazil 3dds, msc, phd, professor, forensic dentistry, school of dentistry of ribeirão preto, university of são paulo (forp/usp), brazil 4dds, msc, phd, professor, department of community dentistry, school of dentistry, state university of campinas (unicamp), brazil 5dds, msc, phd, professor, department oral biology, school of dentistry, sacred heart university (usc), brazil correspondence to: luiz renato paranhos rua padre roque, 958. centro. mogi mirim/sp/ brasil. cep: 13800-033. phone: (19) 3804-4002 e-mail: paranhos@ortodontista.com.br abstract aim: to evaluate the level of knowledge of dentists of the ethical and legal aspects of exercising their profession as individual and corporate entities; to observe the level of knowledge of professionals on the main taxes inherent to their existence as individual and corporate entities; and to highlight the positive and negative aspects of each entity. methods: a questionnaire was prepared containing structured and open questions, which was sent to dentists working in the cities of betim and contagem (belo horizonte metropolitan area) in letters containing self-addressed stamped envelopes for return to the research team. the data were analyzed using descriptive statistics. results: 77.9% of professionals work as individual entities, 19.7% as individual and corporate entities, and only 2.5% act solely as corporate entities. of the professionals who work as corporate entities, 88.8% reported that the reason that led them to act as individual and/or corporate entities was a requirement by health plans. with regard to taxes imposed on each type of entity, 67.4% of individual entity professionals declared not knowing the main taxes related to their profession. conclusions: the level of knowledge of dentists regarding the ethical and legal implications of performing their profession as individual and corporate entities is insufficient; the interviewed professionals are not properly trained on the pertinent ethical and legal aspects of their profession, thus becoming vulnerable to litigation; and these professionals do not have knowledge on the main taxes related to each type of legal entity. keywords: insurance, health, damage liability, ethics. introduction the law is as old as humanity itself, existing since the dawn of civilization and adapting over time to different community contexts. along with this evolution in law, people became liable for their actions, both as individual and corporate entities. specifically, civil liability has evolved from personal retribution, incorporated into the juridical domain and implemented by the state1-2. 247 braz j oral sci. 10(4):246-249 form of professional activity frequency percentage individual entity 95 77.9 corporate entity 3 2.5 both 24 19.6 total 122 100.0 table 1 legal nature of the entity adopted by professionals, betim and contagem, minas gerais state, brazil, 2010. reason frequency percentage own choice 6 22.2 taxation 3 11.1 health plan requirements 24 88.8 table 2 reasons for professionals to work as corporate entities, betim and contagem, minas gerais state, brazil, 2010. advantages percentage ta x e s 61.5 administrative advantage 61.5 working alone 44.0 legislation 21.0 does not know 10.7 other 1.0 table 3 advantages for dental surgeons to act as individual entities, betim and contagem, minas gerais state, brazil, 2010. the term liability can be employed both in the ethical and juridical senses3. civil liability deals with the obligation by agents to answer for their professional actions and withstand their consequences. for liability to be constituted, five elements have to be present: agent, professional act, existence of damages, absence of malice, and causal nexus (link between the professional action and damage)4. thus, in the scope of civil liability, the greatest advance in consumer relations in brazil took place with the enactment of the consumer defense code5 and the adoption of the objective theory with regard to suppliers and service providers, including corporate entities. for liberal professionals, the code clearly adopts the benefit of proof of fault in the framework of the subjective theory6-9. this code, encompassing all consumer relations, depicts the patient as a consumer and the dentist as a provider and can obligate professionals, under civil jurisdiction, to provide a private service10-11. due to the facts exposed and the requirement by some healthplans that professionals change their entity status, the aim of the present study was to examine the level of knowledge of dental surgeons (dss) of the main taxes imposed on individual and corporate entities; to evaluate their knowledge level of the ethical and legal aspects of exercising the ds profession as individual or corporate entities; and to highlight the positive and negative aspects of each entity. material and methods this research was approved by the ethics committee of the piracicaba school of dentistry/unicamp (piracicaba, sp, brazil), and carried out by mailing 392 questionnaires to dentists working in the cities of betim and contagem, located in greater belo horizonte, mg, brazil, whose addresses were provided by the regional dentistry council of minas gerais, brazil. of the 392 questionnaires sent, 122 were returned and analyzed using descriptive statistics. the questionnaire contained questions on whether the professionals took in patients from health plans, attended ethics and dentistry legislation courses, which type of entity was adopted for their professional activities and why, the advantages and disadvantages of each entity and the main taxes imposed on each type of entity. each participant of the research received the questionnaire along with an informed consent form that explained the methodology of the study, possible risks and benefits, as well as a sealed self-addressed stamped envelope to return the documents. the confidentiality of the information given was assured, as well as its use exclusively for research purposes. the questionnaires were not individually identified. results the affirmatives below were demonstrated from the results obtained in the 122 questionnaires returned by the participants, comprising 31.1% of the mailed sample. of the interviewed professionals, 71.1% alleged taking in patients from healthplans, and 40.2% had not undergone any training on ethics or dentistry legislation in the last 5 years. when asked about the legal entity adopted in their professional activity, 77.9% were individual entities, while 19.6% acted simultaneously as individual and corporate entities, and only 2.5% acted solely as corporate entities (table 1). table 2 shows the reasons that led professionals to act as corporate entities in their professional activities. in the question dealing with the advantages of acting as an individual entity, the main advantages cited were simpler administration and taxation (61.5%), and 10.7% of interviewees reported not knowing what the advantages are, as shown in table 3. however, when consulted on their knowledge of the legal and ethical implications when acting as a corporate entity, only 23.8% declared knowing those advantages, whereas 76.2% did not. with regard to the advantages of working as corporate entities, 65.6% of professionals reported the ease in obtaining health plan accreditations as the main advantage (table 4). when professionals who act as individual entities were inquired on the main taxes involved in their professional activity, 67.4% reported not knowing the main taxes to which they are subjected, while 32.6% did. in turn, when professionals who act as corporate entities and/or both were inquired on the main taxes involved in their professional activity, 77.8% reported not knowing the main taxes to which they are subjected, and only 22.2% did know. perception of dental surgeons on the ethical and legal aspects of exercising their profession as personal and corporate entities braz j oral sci. 10(4):246-249 advantages percentage accreditation 65.6 does not know 41.0 working in corporation 24.0 ta x e s 15.0 table 4 advantages for dental surgeons to act as corporate entities, betim and contagem, minas gerais state, brazil, 2010 discussion the number of lawsuits involving dental professionals has been growing in brazil in both the administrative and judicial spheres. thus, with the social development of the activities of dentists, ethical and legal precepts come into effect, as well as conditions for the professional exercise of service rendering, which obligate these professionals to become more aware of these aspects. in this way, it is verified that in general dentistry, the knowledge of dentists regarding the civil liabilities and other legal aspects of professional practice are considered superficial12-13. an important change in the scope of civil liability took place with the application of the rules of the consumer defense code7. among the guidelines of this document is the principle of consumer vulnerability (the weaker part in consumer relations) and the reversal of the burden of proof2,14. as a result of this legislation, traditional care in dental practice (proper and correct filling of the documentation, such as records) take on a superlative importance in defending the professional11,15,16,17, but are often neglected in clinical routine11,18. the rules set by the consumer defense code5 afforded a greater balance in the legal relations between consumers and product/service providers, leading to important changes in brazil’s legal scene. these changes took place in the different health services, favoring good professional-patient relations, transforming into a more commercial relationship and leaving aside the previous confidence. currently, patients affiliated with a health service provider are sent to a given professional through a mere referral by their health plan’s administrative department. therefore, patients seek more information on their rights and complain when dissatisfied with the quality of the services rendered. this often leads to poor professional/patient relationships, leading to lawsuits. however, dental surgeons are generally unfamiliar with lawsuits and do not bother to learn more about civil law, the brazilian civil code and the consumer defense code, all of which determine the civil liability of professionals while in the exercise of their profession, as shown by the obtained results. this demonstrates a lack of preparedness by professionals – efforts are not made to learn more about these legal questions, even when aware of the existence of administrative and judicial suits. the brazilian consumer defense code clearly adopts the objective theory with regard to suppliers and service providers, including corporate entities such as clinics. all that is needed is to prove the act, damage and causal nexus in order to award damages8,19-22, without the need to establish fault. liberal professionals, however, are exempt from this condition, and are given the benefit of the need to establish fault in the framework of the subjective theory. nevertheless, the fact of whether a professional is an individual or corporate entity does not change the liability from subjective to objective20,23, as a liberal professional may set up a professional partnership only to better organize revenue and expenses, still acting as a liberal professional. what effectively annuls the liberal professional status is not the existence of a corporate entity, but rather the establishment of a corporate entity that explores a liberal profession activity specifically to exploit the advantages of such status. however, there is no consensus in the jurisprudence on this topic. with regard to the liability of health plans, our courts, based on article 14 of the consumer defense code, have been deciding that the liability of health plans, in addition to being objective, must be solidary (joint and several)14,24-27. dentists, as the liberal professionals they are, can act as individual entities (self-employed professionals), corporate entities, or both. the great majority (77.9%) of interviewed professionals act as individual entities in their profession – that is, act as individually considered individuals, capable of contracting rights and obligations within the law. a minority (19.6%) acts as corporate entities or both, in this case consisting of a grouping of persons whom the law affords the ability to bear rights and obligations28. the civil liability of liberal professionals, as individual entities, is generally subjective, thus requiring fault to be established in order to award damages. the liability of health plans, according to the prevailing doctrine and jurisprudence is objective, whether because these companies perform a public service or as service providers, in the framework of the consumer defense code. the civil liability of dental clinics is generally objective. however, with regard to damages resulting from professional/clinical practice, regular professional action would sever the causal nexus, requiring an evaluation of the professional’s conduct prior to faulting the clinic. when these professionals perform their duties as individual entities, they are subject to certain municipal and federal taxes, which are different from taxes for corporate entities29. the main positive aspects of working as an individual entity are the use of the subjective liability theory and lower tax burden compared to corporate entities. this corroborates the opinion of most (61.5%) interviewed professionals, who affirm that taxation is one of the main advantages of acting as individual entities. the negative aspect involves the difficulty in being included in health plans. nevertheless, an expressive minority (13%) reported not knowing the advantages of acting as individual entities. however, with regard to knowledge on the ethical and legal implications of working as corporate entities, the results lead us to reflect on the lack of attention paid by dentistry professionals on the legal knowledge related to their professional actions, ignoring its importance in preventing 248 perception of dental surgeons on the ethical and legal aspects of exercising their profession as personal and corporate entities 249 braz j oral sci. 10(4):246-249 eventual litigation and making the best possible choice of legal entity. among the advantages of acting as a corporate entity is the ease of accreditation with health plans; however, some professionals (41%) still cannot specify what these advantages are. the negative aspects cited included the tax burden and civil liability, for which there is no consensus in the doctrine or jurisprudence and can therefore be interpreted as objective or subjective. thus, these data clearly show the lack of preparation by professionals regarding the issue, effectively demonstrating that most professionals change their legal entity status without even knowing the advantages and disadvantages of doing so. the most often mentioned taxes on individual entities are30: individual income tax (in brazil, original acronym: irpf); federal pension contribution (in brazil, original acronym: inss); union dues; municipal service tax (in brazil, original acronym: issqn); corporate contribution (in brazil, original acronym: cro regional dentistry council of the respective district). for corporate entities, the main taxes are: federal income tax (in brazil, original acronym: irpj); social contribution; social security financing contribution (in brazil, original acronym: cofins); social integration program (in brazil, original acronym: pis); federal pension contribution; municipal service tax (in brazil, original acronym: issqn) and corporate contribution (in brazil, original acronym: cro regional dentistry council of the respective district). it may be concluded that the knowledge level of dental surgeons regarding the ethical and legal implications of performing their profession as individual and corporate entities is insufficient; the interviewed dental surgeons are not properly trained on the pertinent ethical and legal aspects of their profession, thus becoming vulnerable to litigation; and these professionals do not have knowledge of the main taxes related to each type of legal entity. references 1. diniz mh. curso de direito civil brasileiro. responsabilidade civil. 22. ed. são paulo: saraiva; 2008. v.7. 2. prux oi. a responsabilidade civil do profissional liberal no código de defesa do consumidor. belo horizonte: del rey; 2007. 3. frança gv. medicina legal. 6. ed. rio de janeiro: granabara koogan; 2002. 4. daruge e, massini n. direitos profissionais na odontologia. são paulo: saraiva; 1978. 5. brasil. lei n. 8.078 de 11 de setembro de 1990. código de proteção e defesa do consumidor. 1990. [cited 2008 jun 4]. available from: url: http://www.planalto.gov.br/ccivil_03/leis/l8078.htm. 6. alvim ea, cheim fj. a responsabilidade civil no código de proteção e defesa do consumidor e o transporte aéreo. rev direito consumidor. 1996; (19): 144-7. 7. bittar ca. direitos do consumidor. 3. ed. são paulo: forense universitária; 1991. 8. khouri pr, roque a. contratos e responsabilidade civil no cdc. brasília: brasília jurídica; 2002. 9. nery junior n. os princípios gerais do código brasileiro de defesa do consumidor. rev direito consumidor. 1992; (3): 52. 10. silva aa, malacarne gb. responsabilidade civil do cirurgião dentista perante o código de defesa do consumidor. j bras ortod ortop facial. 1999; 4: 305-10. 11. paranhos lr, salazar m, torres fc, pereira ac, silva rf, ramos al. avaliação do perfil dos profissionais da área de ortodontia quanto às condutas legais. dental press j orthod. 2011; 16: 127-34 12. coelho mp, vaz de melo ls, carvalho cm. aspectos odontolegais do exercício profissional entre recém-formados. rev odontol ufes. 2008; 10: 4-8. 13. garbin cas, garbin aji, gonçalves pe, gonçalves sh. o conhecimento do cirugião-dentista frente à responsabilidade civil da sua especialidade. rev assoc paul cir dent. 2007; 61: 458-61. 14. cavalieri filho s. programa de responsabilidade civil. 8. ed. são paulo: atlas; 2008. 15. saquy pc, pécora jd, silva rg, souza-neto md. o código de defesa do consumidor e o cirurgiäo dentista. rev paul odontol. 1993; 15: 4-5. 16. benedicto en, lages lhr, oliveira of, silva rha, paranhos lr. a importância da correta elaboração do prontuário odontológico. odonto. 2010; 18: 41-50. 17. malacarne gb, silva aa. gestão de riscos: decisões dos tribunais quanto a obrigação dos profissionais da área da saúde. ortho sci orthod sci pract. 2010; 3: 166-74. 18. garbin cas, garbin aji, saliba na, zina lg, gonçalves pe. el consentimiento informado en la clínica odontológica. acta odontol venez. 2007; 45: 37-43. 19. benjamin ahv. comentários ao código de proteção ao consumidor. são paulo: saraiva; 1991. 20. nunes lar. comentários ao código de defesa do consumidor. são paulo: saraiva; 2000. 21. oliveira mll. responsabilidade civil odontológica. belo horizonte: del rey; 1999. 22. silva m. compêndio de odontologia legal. rio de janeiro: guanabara koogan; 1997. 23. borges i. responsabilidade civil médica. 29/08/2007. [cited 2008 nov 21]. available from: url: http://www.sindimed-ba.org.br/conteudo.php?id=652. 24. baú mk. o contrato de assistência médica e a responsabilidade civil. rio de janeiro: revista forense; 1999. 25. kfouri neto m. responsabilidade civil do médico. 6. ed. são paulo: revista dos tribunais; 2007. 26. schaefer f. responsabilidade civil dos planos e seguros de saúde. curitiba: juruá; 2003. 27. seguin e. plano de saúde e o código de defesa do consumidor. in: lana rl, figueiredo am, coordenators. temas de direito médico. rio de janeiro: espaço jurídico; 2003. 28. farah ee, ferraro l. responsabilidade civil: guia prático para dentistas, médicos e profissionais da saúde. são paulo: quest; 1998. 29. diniz mh. curso de direito civil brasileiro. responsabilidade civil. 4. ed. são paulo: saraiva; 1998. 30. cassone v. direito tributário: fundamentos constitucionais da tributação, definição de tributos e suas espécies, conceito e classificação dos impostos, doutrina, prática e jurisprudência. 19. ed. são paulo: atlas; 2008. perception of dental surgeons on the ethical and legal aspects of exercising their profession as personal and corporate entities oral sciences n3 braz j oral sci. 10(1):130-135 original article braz j oral sci. april | june 2011 volume 10, number 2 cumulative trauma disorders, overweight and obesity among brazilian dentists evelyne pessoa soriano1, marcus vitor diniz de carvalho1, ana beatriz vasconcelos lima araújo2, humberto gomes vidal3, kátia maria diniz de carvalho3, clóvis césar de mendoza3 1adjunct professor, msc in forensic sciences, faculty of dentistry, university of pernambuco, brazil 2msc student in public health, faculty of dentistry, university of pernambuco, brazil 3msc student in forensic sciences, faculty of dentistry, university of pernambuco, brazil corespondence to: evelyne pessoa soriano avenida general newton cavalcanti, 1650 tabatinga camaragibe -pe cep: 54753-901 phone: (81) 9960 6674 e-mail: evelynesoriano@yahoo.com.br abstract aim: this study aimed to assess the prevalence of cumulative trauma disorders (ctd), overweight and obesity and the association between them, among public health dentists. methods: the study included 150 dentists working at various public health system units in recife, pe, brazil. a brazilian version of the nordic musculoskeletal symptoms questionnaire was used. obesity and overweight were observed across the bmi (body mass index) recommended by the world health organization. a descriptive analysis was carried out using spss 13.0 software. results: among respondents, 79.3% were female with a mean age of 44.42 years. the presence of musculoskeletal symptoms was reported by 138 (92%) of participants, 129 of whom linked the symptoms to their work activity. the cervical region was the most involved, accounting for 104 (14.3%) of the total 723 affected areas. overweight was observed in 32% of the professionals and obesity in 13%. of the 150 participants, 30 (20%) received a medical diagnosis of ctd and of these, 13 (43.3%) had a high bmi. the 40-49-year-old age group was most affected by ctd and females were more affected than males, accounting for 22.7% of professionals interviewed. there was no significant association between the presence of ctd and overweight/obesity (p >0.05). conclusions: however, given the multifactorial nature of occupational diseases, it is likely that overweight and obesity may act as a predisposing factor in these diseases, interacting and enhancing the effects of other important risk factors for the occurrence of work-related musculoskeletal disorders. keywords: occupational health, cumulative trauma disorders, overweight, obesity. introduction recently there have been reports of an increase in the prevalence of musculoskeletal conditions among dental professionals, with an etiology of repetitive movements, injuries due to cumulative trauma or work-related musculoskeletal disorders1-2. cumulative trauma disorders (ctd) are a group of diseases that affect muscles, tendons, nerves and vessels of the upper limbs (fingers, hands, wrists, forearms, arms, shoulder, neck and spine) and lower limbs (knee and ankle, especially) that are directly related to the demands of work tasks, physical environments and with the organization of work. they include clinical manifestations characterized by the occurrence of various symptoms, concomitant or not, such as pain, numbness, heaviness and fatigue and are often causes of temporary or permanent employment disability3-4. received for publication: december 14, 2010 accepted: june 16, 2011 131 braz j oral sci. 10(2):130-135 there are several risk factors associated with these disorders, which can be divided into individual risk factors and occupational risk factors. the following are considered to be the most likely individual risk factors: age, gender, body mass index, muscular imbalance, muscle strength, socioeconomic conditions and the presence of other pathologies. overweight and obesity may act as a permissible factor in musculoskeletal disease, interacting and enhancing the effects of other risk factors, as well as the alignment of the skeleton and the condition of muscles5-6. in a swedish study that compared 6,328 obese individuals with 1,135 individuals from the general population of either gender, it was found that neck pain, along with pain in the back, hip, knee and ankle, was more common in obese people than in the general population7. although they are not recent illnesses, ctd are undoubtedly taking on an epidemic character, and some of their pathologies are chronic and recurring, difficult to treat and are creating disability that is not restricted to the workplace but also found in other normal activities of personal life8. a high prevalence of musculoskeletal complaints was found in 430 greek dentists participating in a study where a questionnaire was applied. the survey results showed that 62% reported at least one painful complaint9. in another study, 98.6% of dentists of caxias do sul, rs, brazil, had some symptoms of musculoskeletal disorder that were associated with certain demographic, clinical and laboratorial characteristics. pain was the primary symptom of disorder, affecting mainly the regions of the cervical spine, the elbows, lumbar spine and shoulders10. in a study carried out with 227 brazilian dental students in recife and camaragibe, pe, brazil, 176 (76.2%) reported experiencing pain during or after clinical work11. musculoskeletal disorders have a major impact on quality of life of the population in many countries, not to mention the financial costs. although not exclusively caused by work, they represent a third of the occupational diseases in countries such as japan, the united states and the scandinavian countries12. in brazil, the total of benefits provided by social security during the months from january to august 2009 was 1,162,818; of these, 18.97% were related to diseases of the musculoskeletal system and connective tissue13. in order to draw attention to occupational health and to contribute to the quality of the working lives of these professionals, the aim of this study was to assess the prevalence of musculoskeletal symptoms and the association with obesity and overweight among brazilian public sector dentists. material and methods this is a cross-sectional study that investigates the factor and effect at the same historical moment. the study was carried out from april to july 2010 and included 150 dentists (drawn from a population of 243) who worked in various units that are part of the public health system in the city of recife, pe, brazil, selected by the calculation for finite population, from a list provided by the municipality of the city of recife. of the 283 professionals who originally appeared in the list, 33 were excluded because they were managers and did not perform clinical activities in health units, two had died and five were removed (two professionals for maternity leave, one for an ongoing long-term out of state, and two due to health problems). thus, the estimated prevalence was 50%, since no data were available concerning the prevalence for this population, and 5% error and confidence interval of 95%, resulting, then, in 150 subjects. a modified version of the nordic musculoskeletal symptoms questionnaire (nmsq) was used, which was validated in brazil by pinheiro14. the aim was to investigate the occurrence of musculoskeletal symptoms in the previous 12 months, the anatomical regions most affected, the frequency of symptoms, the participant’s demographic profile (gender, age, education and marital status) and the characterization of the work process (activity time and work schedule). pain intensity was assessed from the clinical stages of ctd as advocated by oliveira (1989)15, using an increasing scale of symptoms reported by the interviewee. the questionnaire was given to the dentists in their work environment. after filling it out, their anthropometric measurements were taken to assess if they were overweight or obese. obesity and overweight were observed across the bmi (body mass index), with the objective of determining if there is an association between this variable and the occurrence of ctd in this sample. to record the bmi, dentists were weighed using a digital scale with a maximum capacity of 150 kg and precision of 0.1 kg. height was measured in meters, using a flexible measuring tape fixed to the wall. the participants were asked to remove their shoes, their heels and shoulders touching the wall, after which his head was positioned so that the occlusal plane were parallel to the ground. so, with the help of a frame of wood, the individual’s height was marked on the tape, making it easier to read. in population studies, the bmi, defined as the weight in kilograms divided by height in meters squared, is a useful measure to assess excess body fat, and it is generally agreed that, regardless of gender and age, adults with a bmi equal to or greater than 30 kg/m2 are classified as obese16. data was analyzed through statistical tests, using the statistical package for social sciences (spss 13.0). initially, a descriptive analysis was carried out to evaluate the frequency of the distribution of variables and to check if there were errors in data entry, seeking thereby to characterize the sample. the bivariate relationships between the dependent and independent variables were examined using tests of association (chi-square and fisher’s exact test). a statistical significance was considered when p <0.05 and confidence interval was determined at 95%. this research was duly approved by the ethics committee of the university of pernambuco, according to the resolution cns 196/96, via protocol 212/09. results data analysis revealed that 119 (79.3%) of the 150 participants were female and their mean age was 44.42 years. cumulative trauma disorders, overweight and obesity among brazilian dentists 132 braz j oral sci. 10(2):130-135 among respondents, 77 (51.3%) reported working an average of eight hours per day and 78 (52%) reported performing some physical activity. the presence of musculoskeletal symptoms was reported by 138 (92%) of participants and 129 (86%) of respondents linked these symptoms to their work activity. the cervical region was identified by respondents as the most affected by painful symptoms, accounting for 104 (14.3%) of the total of 723 affected areas, followed by wrists, hands and fingers (13.4%), lower back (13.4%) and shoulders (13.1%). using the bmi calculations, it was found that 48 individuals (32%) were overweight and 20 (13%) were obese. among the 68 respondents who were overweight, the most affected age group was the 50 to 59 years one and the gender had a significant association with overweight/obesity (p <0.05) using the pearson chi-square test, in which 26 (83.9%) of the total male respondents were overweight. the number of years doing the same activity was also significantly associated with overweight/obesity, and professionals with 21 or more years of activity as dentists had a greater increase in bmi. physical activity was carried out by 35 (51.4%) of the dentists who were overweight or obese (table 1). of the 150 participants, 30 (20%) were diagnosed with ctd in the previous 12 months. among these, 14 (46.7%) reported that their pain symptoms corresponded to the first stage of clinical ctd, as described by oliveira (1989)15, feeling of heaviness, discomfort or mild and transient localized pain and without irradiation, that worsens with work and improves with rest. the most affected age group by ctd was the 40 to 49 years one and females were more affected than males, accounting for 22.7% of professionals interviewed. of the 30 professionals diagnosed with ctd, 13 (43.3%) were overweight or obese. there was no significant association (p <0.05) between the presence of ctd and overweight/obesity (table 2). discussion one of the main objectives of the results of this study was to describe and analyze how dentists are affected by overweight/obesity variable yes n o group total p value or (95% ic) n % n % n % total 68 45.3 82 54.7 150 100.0 ·age group up to 39 22 38.6 35 61.4 57 100.0 p(1) = 0.182 1.00 40 to 49 17 39.5 26 60.5 43 100.0 1.04 (0.46 a 2.34) 50 to 59 21 58.3 15 41.7 36 100.0 2.23 (0.95 a 5.21) 60 or over 8 57.1 6 42.9 14 100.0 2.12 (0.65 a 6.94) ·gender female 42 35.3 77 64.7 119 100.0 p(1) < 0.001* 1.00 male 26 83.9 5 16.1 31 100.0 9.53 (3.41 a 26.66) ·time since graduation up to 10 10 29.4 24 70.6 34 100.0 p(1) = 0.077 1.00 11 to 20 17 44.7 21 55.3 38 100.0 1.94 (0.73 a 5.16) 21 or more 41 52.6 37 47.4 78 100.0 2.66 (1.12 a 6.29) ·time in the same profession up to 10 12 27.9 31 72.1 43 100.0 p(1) = 0.013* 1.00 11 to 20 17 44.7 21 55.3 38 100.0 2.09 (0.83 a 5.27) 21 or more 39 56.5 30 43.5 69 100.0 3.36 (1.48 a 7.62) ·work load 6 hours 12 40.0 18 60.0 30 100.0 p(1) = 0.260 1.00 8 hours 32 41.6 45 58.4 77 100.0 1.07 (0.45 a 2.52) more than 8 hours 24 55.8 19 44.2 43 100.0 1.89 (0.74 a 4.88) ·carry out physical exercise yes 35 44.9 43 55.1 78 100.0 p(1) = 0.906 1.00 n o 33 45.8 39 54.2 72 100.0 1.04 (0.55 a 1.98) table 1 -table 1 -table 1 -table 1 -table 1 assessment of overweight/obesity according to age, gender, time since graduation, time in the same occupation, working hours and physical exercise. (*): significant difference at a level of 5.0% (1): using the pearson’s chi square test cumulative trauma disorders, overweight and obesity among brazilian dentists braz j oral sci. 10(2):130-135 work-related musculoskeletal symptoms. despite the occurrence of musculoskeletal disorders found in the study group was considered high, 129 of professionals reported some work-related painful symptoms in the previous 12 months, only 30 were diagnosed with ctd. this suggests that even though the presence of musculoskeletal pain corresponding to the first clinical stage of the ctd condition, dentists trend towards minimization of signs pointing to musculoskeletal changes that somehow relate to their laboring. the symptoms of ctd vary during the working activity and tend to relieve through the rest. over time, they may become frequent throughout the workday, and also in the performance of other activities. people tend to seek medical help only when the pain becomes unbearable or their movements are restricted17. on the other hand, treatment is often based only on prescription of antiinflammatory drugs and physical therapy sessions, which will only mask the problem without reaching its causes, neglecting the changes in conditions of work18. the study of santos filho and barreto19, which assessed all dentists of the public health service in belo horizonte/ brazil (n = 358) also observed a large number of professionals with symptoms (58%). the same situation occurred in the state of santa catarina, where regis filho et al.20 reported that among 771 dentists registered at the regional state council of dentistry, 437 (56.68%) had some painful musculoskeletal manifestation that was work-related, which is in line with our study. these data reinforce the observation that the practice of dentistry favors, by its own nature, the occurrence of disorders affecting primarily the musculoskeletal 133 cumulative trauma disorders, overweight and obesity among brazilian dentists ctd variable present absent group total p value or (95% ic) n % n % n % total 30 20.0 120 80.0 150 100.0 • age group up to 39 10 17.5 47 82.5 57 100.0 p(1) = 0.724 1.00 40 to 49 11 25.6 32 74.4 43 100.0 1.62 (0.61 a 4.25) 50 to 59 6 16.7 30 83.3 36 100.0 0.94 (0.31 a 2.85) 60 or more 3 21.4 11 78.6 14 100.0 1.28 (0.30 a 5.45) • gender female 27 22.7 92 77.3 119 100.0 p(1) = 0.107 2.74 (0.77 a 9.71) male 3 9.7 28 90.3 31 100.0 • time since graduation up to 10 5 14.7 29 85.3 34 100.0 p(1) = 0.628 1.00 11 to 20 9 23.7 29 76.3 38 100.0 1.80 (0.54 a 6.03) 21 or more 16 20.5 62 79.5 78 100.0 1.50 (0.50 a 4.48) • time in the same profession up to 10 5 11.6 38 88.4 43 100.0 p(1) = 0.227 1.00 11 to 20 10 26.3 28 73.7 38 100.0 2.71 (0.83 a 8.83) 21 or more 15 21.7 54 78.3 69 100.0 2.11 (0.71 a 6.30) • work load 6 hours 6 20.0 24 80.0 30 100.0 p(1) = 0.270 1.00 8 hours 12 15.6 65 84.4 77 100.0 0.74 (0.25 a 2.19) more than 8 hours 12 27.9 31 72.1 43 100.0 1.55 (0.51 a 4.72) • carry out physical exercise yes 14 17.9 64 82.1 78 100.0 p(1) = 0.513 1.00 n o 16 22.2 56 77.8 72 100.0 1.31 (0.59 a 2.91) • age group up to 39 40 to 49 13 19.1 55 80.9 68 100.0 p(1) = 0.806 1.00 50 to 59 17 20.7 65 79.3 82 100.0 1.11 (0.49 a 2.48) 60 or older table 2 -table 2 -table 2 -table 2 -table 2 evaluation of diagnosed ctd in the past 12 months according to age, gender, time since graduation, time in the same occupation, working hours, physical exercise and overweight/obesity (1): using the pearson’s chi square test. braz j oral sci. 10(2):130-135 134 system of dental surgeons. in this study, the body region that was most affected by the symptoms was the cervical spine, followed by wrists, hands and fingers, lower back and shoulders. these results are similar to those of a study of 421 dentists who graduated between the years 1986 and 1997 in british columbia, canada, which revealed that approximately 18% reported experiencing shoulder pain, 24% had pain in the neck and the lumbar region, with 19% in the upper part of the spine, 30% in the middle and 17% in the lower part21. in a survey of 268 polish dentists22 symptoms were also mainly related to the thoracolumbar region (60.1%), neck (56.3%), lower limbs (47.8%) and wrist and hand (44.0%). a study that was carried out with all 115 dentists that were members of the danish society of craniomandibular disorders23 also found that two thirds of respondents had some type of problem, such as pain or discomfort in the neck and/or shoulder with similar frequency in relation to the problems present in the lumbar region. with respect to ctd, women were more affected in the present study. this is in accordance with the study by regis filho et al.20 in which the statistical analysis indicated the presence of a significant association between the two genders for the presence of ctd, with women showing more pathologies related to ctd than men. the mean age obtained in this study was 44.42 years, and the most affected age group was the 40 to 49 years one. this is in contrast with the study by santana et al.24, which surveyed 100 dentists in salvador, ba, brazil, and the most affected age group was the 27 to 34 years one. however, our study did agree with these authors regarding the higher prevalence of painful symptoms of ctd in females and those professionals who worked 8 hours per day. in the present study, 30 (20%) participants were diagnosed with ctd in recent months. of these, 14 (46.7%) reported that they had painful symptoms corresponding to the first clinical stage of the condition as advocated by oliveira (1989)15. these results are similar to those obtained in the study of 227 dental students from recife and camaragibe, pe, brazil, where 42 (18.5%) had a diagnosis of ctd11, emphasizing the fact that since their academic training, dentists are already prone to work-related risk factors. this fact is of great importance and should be taken into account on the establishment of preventive practices. our study also found a prevalence of overweight in 48 (32%) professionals and obesity in 20 (13%), similar to the study that measured the prevalence of overweight and obesity in 435 employees of the state university of feira de santana in the state of bahia, brazil25. the prevalence of overweight and obesity was 31.95% and 10.34%, respectively25. among those interviewees who were overweight, the most affected age group was the 50 to 59 years group and 26 (83.9%) of the total of male respondents were overweight. this is in contrast with the study by abrantes et al. (2003)26, in which the prevalence of overweight and obesity was higher among females aged 40 to 79 years. the study that compared estimates of the household budget survey with data from the national health and nutrition survey, found that the prevalence of overweight and obesity is higher in the male population27, as in this study. as overweight and obesity are consistently identified as risk factors for the development of a number of diseases, evaluation of their prevalence among various population groups is needed, especially when it comes to individuals who develop occupational activities. the illness among workers may lead to dismissal or even partially or totally unable to work, as can occur with people who develop workrelated musculoskeletal disorders. some authors report that given the multifactorial nature of musculoskeletal disorders, it is likely that overweight and obesity may act as a permissible factor in musculoskeletal disease, interacting and enhancing the effects of other risk factors, as well as the alignment of the skeleton and the muscular condition6. one study from sweden, which compared 6,328 obese individuals with 1,135 individuals from the general population of both genders, found that pain in the neck, lumbar region, hip, knee and ankle was more common in obese people than in the general population (odds ratio or ranging from 1.7 to 9.9, p<0.001)7. in the present study, the statistical analysis showed no significant association between ctd and overweight and obesity. however, although there was no association, one cannot overlook the fact that overweight and obesity are present in these professionals and can cause, in addition to musculoskeletal problems, other health problems, such as hypertension, diabetes and vascular problems, which can interfere with the work quality of these people. the evaluation of the prevalence of overweight and obesity, which act as risk factors for the onset of work-related diseases, should be continued, since it is known that the population, not only in brazil but in many countries, has increasingly showing an increase of body mass. because dentists are part of a category rather prone to developing diseases related to work due to the ergonomic features and overload of dental activity, this study had the scope to contribute to an initial analysis of productive working health professionals to highlight the need for preventive measures that should be implemented since the academic training of dentists. this way, they are less likely to have problems with their working life quality and suffer impediments in exercising their professional activity. acknowledgements special thanks to the national council for scientific and technological development (cnpq) for the financial support. references 1. santos filho sb, barreto sm. occupational activity and prevalence of upper-limb and back pain among dentists in belo horizonte, minas gerais state, brazil: a contribution to the debate on work-related musculoskeletal disorders. cad saude publica. 2001; 17: 181-93. cumulative trauma disorders, overweight and obesity among brazilian dentists 135 braz j oral sci. 10(2):130-135 2. brasil. ministries of welfare, of health, of labour and employment. national policy on occupational health and safety. brasilia: ministries of welfare, of health, of labour and employment; 2004. 3. merlo rc, jacques mgc, hoefel mgl. groups activity with cumulative trauma disorders workers: experience report. psicol reflex. crit. 2001; 14: 253-8. 4. chiavegato filho l, pereira jr a. work-related osteomuscular diseases: multifactorial etiology and explanatory models. interface. 2003; 8: 149-62. 5. mota r, dutra dsg, fabiano s. barbosa fs. estudo da prevalência de algias na coluna vertebral em colhedores de café do município de vieiras mg. rev ponto vista. 2008; 5: 99-110. 6. wearing sc, hennig em, byrne nm, steele jr, hills ap musculoskeletal disorders associated with obesity: a biomechanical perspective. obesity reviews. 2006; 7: 239-50. 7. peltonen m, lindroos ak, torgerson js. musculoskeletal pain in the obese: a comparison with a general population and long-term changes after conventional and surgical obesity treatment. pain. 2003; 104: 549-57. 8. salim ca. doenças do trabalho exclusão, segregação e relações de gênero. sao paulo perspectiva. 2003; 17: 11-24. 9. alexopoulos ec, stathi ic, charizani f. prevalence of musculoskeletal disorders in dentists. bmc musculoskeletal disorders. 2004; 5: 16. 10. gazzola f, sartor n, ávila sn. prevalence of musculoskeletal disorders in caxias do sul’s dentists. rev cienc saude. 2008; 1: 50-6. 11. carvalho mvd, soriano ep, caldas jr af, campello ric, miranda hf, cavalcanti fid. cumulative trauma disorders among brazilian dental students. j dent educ. 2009; 73: 624-30. 12. punnett l, wegman d. work-related musculoskeletal disorders: the epidemiologic evidence and the debate. j electromyogr kinesiol. 2004; 14: 13-23. 13. araujo abvl; soriano ep; carvalho mvd; caldas junior af; coelho junior lgtm; vidal hg. prevalência de doenças de caráter ocupacional em cirurgiões-dentistas. int j dent. 2009; 8 suppl 2: 82. 14. pinheiro fa. aspectos psicossociais dos distúrbios osteomusculares relacionados ao trabalho – dort/ler [thesis]. brasília: universidade de brasília; 2002. 15. oliveira cr. terminais e saúde: as explicações devidas. proteção. 1989; 1:30-44. 16. who. world health organization. obesity: preventing and managing the global epidemic. report of a who consultation on obesity. geneva: who; 1998. 17. álvares tt, lima, mea. fibromialgia: interfaces com as ler/dort e considerações sobre sua etiologia ocupacional. cienc saude coletiva. 2010; 15: 803-12. 18. oliveira rmr. a abordagem das lesões por esforços repetitivos/distúrbios osteomusculares relacionados ao trabalho ler / dort no centro de referência em saúde do trabalhador do espírito santo crst/es [dissertation]. rio de janeiro: escola nacional de saúde pública, fundação oswaldo cruz; 2001. 19. santos filho sb, barreto sm. atividade ocupacional e prevalência de dor osteomuscular em cirurgiões-dentistas de belo horizonte, minas gerais, brasil: contribuição ao debate sobre os distúrbios osteomusculares relacionados ao trabalho. cad saude publica. 2001; 17: 181-93. 20. regis filho gir, michels g, sell i. lesões por esforços repetitivos/ distúrbios osteomusculares relacionados ao trabalho em cirurgiões-dentistas. rev bras epidemiol. 2006; 9: 346-59. 21. rucker lm, sunell s. ergonomic risk factors associated with clinical dentristry. j calif dent assoc. 2002; 30: 139-48. 22. szymanska j. disorders of the musculoskeletal system among dentists from the aspect of ergonomics and prophylaxis. ann agric environ med. 2002; 9: 169-73. 23. finsen l, christensen h, bakke m. musculoskeletal disorders among dentists and variation in dental work. appl ergon. 1998; 29: 119-25. 24. santana ejb, rocha lefl, calmon trv, alves il. estudo epidemiológico de lesões por esforços repetitivos em cirurgiões-dentistas em salvadorbahia. rev fac odontol univ fed bahia. 1998; 17:67-74. 25. caires nfr. sobrepeso e obesidade entre os funcionários da universidade estadual de feira de santana [dissertation]. feira de santana: universidade estadual de feira de santana; 2004. 200p. 26. abrantes mm, lamounier ja, colosimo ea. prevalência de sobrepeso e obesidade nas regiões nordeste e sudeste do brasil. rev assoc med bras. 2003; 49: 162-6. 27. sousa rmrp, sobral dp, da paz smrs, martins mcc. prevalência de sobrepeso e obesidade entre funcionários plantonistas de unidades de saúde de teresina, piauí, brazil. rev nutr. 2007; 20: 473-82. cumulative trauma disorders, overweight and obesity among brazilian dentists oral sciences n3 braz j oral sci. 11(1):19-24 original article braz j oral sci. january | march 2012 volume 11, number 1 ethics in the provision of removable partial dentures arcelino farias-neto1, renata suellen galvão da silva2, alexandre da cunha diniz2, andré ulisses dantas batista3, adriana da fonte porto carreiro4 1dds, msc, phd, professor, department of dentistry, potiguar university, brazil 2graduate student, department of dentistry, federal university of rio grande do norte, brazil 3dds, msc, phd, professor, department of restorative dentistry, federal university of paraíba, brazil 4dds, msc, phd, professor, department of dentistry, federal university of rio grande do norte, brazil correspondence to: arcelino farias-neto faculdade de odontologia de piracicaba – departamento de prótese e periodontia av. limeira, 901 caixa postal 52 piracicaba sp cep: 13414-903 phone: + 55 19 2106-5211 fax: + 55 19 2106-5218 e-mail:saudeoral@fop.unicamp.br abstract aim: to investigate the construction of cobalt-chromium removable partial dentures by commercial private dental laboratories. methods: ninety master casts for fabrication of cobalt-chromium removable partial dentures were obtained from three commercial laboratories randomly selected. casts were assessed for dental arch treated, kennedy classification, cast surveying, denture design information provided by the dentist, and mouth preparation (rest seat, guiding plane and retentive area). dental technicians answered a questionnaire regarding qualification of assisted dentists, monthly number of framework castings, and use of dental surveyor. mouth preparation was compared among laboratories using kruskal-wallis test (α=0.05). results: the percentage of kennedy class i was 16%, class ii 19%, class iii 56%, and class iv 9%. the majority of master cats (51%) examined was sent to dental laboratories without any design information and did not comply with ethical guidelines in the provision of rpd. approximately half of the casts were considered “inappropriate” for guiding planes and retentive areas. one of the laboratories presented all casts “inappropriate” for rest seat distribution (p<0.001). conclusions: mouth preparation frequently failed for guiding planes, retentive areas and distribution of rest seats. it is necessary to provide students with adequate clinical experience at the dental school environment, which will actually be carried into the practice of dentistry. keywords: dental technician, denture design, dentist’s attitudes, mouth preparation. introduction according to data from the last epidemiological assessment of brazilians’ oral health, accomplished by the ministry of health in 2010, sixty-nine percent of the brazilian adult population needs some kind of dental prosthesis1. it was also found that need for prosthetic treatment is markedly associated with socioeconomic factors. in the united states, prospective analysis has shown that the substantial growth in the u.s. population and extended life expectancy may contribute to increase the prevalence of partially dentate adults by the year 20202. the authors argue that adults are retaining more of their teeth throughout life and a larger proportion of partially edentulous patients will require removable and/or fixed partial dentures. removable partial denture (rpd) is an appropriate treatment received for publication: october 03, 2011 accepted: december 22, 2011 braz j oral sci. 11(1):19-24 2020202020 for partial edentulism, and may be suitable for a wide range of clinical situations3. patients with one missing tooth as well as those with at least one remaining abutment may be rehabilitated with this treatment modality. additionally, rpd offers a less expensive option of prosthetic treatment than implant assisted restorations and fixed partial dentures, and implicates lower biological cost (tooth preparation) if the patient cannot afford or is not sure about implant treatment at that moment. the dentist is responsible for all phases of a rpd service in the strict sense of the word, although the dental laboratory technician is requested to perform certain technical phases of the service3. the creation of an optimal rpd design is dependent on the following factors: clinical knowledge and training; a thorough assessment of the patient; appropriate treatment planning including surveyed diagnostic casts and mouth preparation; and technical expertise and knowledge of laboratorial procedures and properties of materials. clearly the dentist’s contribution is related primarily to the first three aspects while the technician’s contribution is concerned with the fourth4. if inappropriately designed, planned, or placed, rpds may have deleterious effects on oral health and supporting structures, as caries and periodontal disease5. unfortunately, studies have reported that dentists are often negligent with fundamental principles for the construction of rpd and frequently fail to comply with ethical and legal requirements6-12. impressions and master casts for rpds are frequently sent to dental laboratories without mouth preparation and written instructions of design information. this problem seems to be worldwide, as it has been reported in developing countries such as south africa9 and kingdom of bahrain12, as well as developed countries such as the united kingdom6, canada7, sweden8, ireland11 and the united states10. however, no scientific data about rpd prescription in south america has been indexed on medline/pubmed. therefore, the purpose of this study was to investigate the construction of rpd by commercial private dental laboratories in natal, brazil. material and methods this cross-sectional survey was approved by the research ethics committee of the federal university of rio grande do norte (protocol nº 095/09). full name list and contact details of dental laboratory technicians regularly classification completely appropriate partially appropriate inappropriate rest seat distribution adjacent to edentulous areas in tooth-supported rpd, except when molar tooth is mesially inclined. mesial rest for distal extension rpd. auxiliary rests must provide indirect retention. one or two rest seats are inadequate, but it does not disrupt the biomechanics. there is no rest seat preparation, or location of rest seats is completely inadequate. retentive areas vertical and horizontal retentions are satisfactory. horizontal or vertical retention is satisfactory. vertical and horizontal retentions are unsatisfactory. guiding planes vertical and horizontal extensions are satisfactory. horizontal or vertical extension is satisfactory. vertical and horizontal extensions are unsatisfactory. table 1.table 1.table 1.table 1.table 1. criteria for assessment of mouth preparation. registered and working in the city of natal, brazil were obtained from the regional dental council. a telephone contact revealed that only 8 of the 87 commercial laboratories in the city of natal had the facility to cast cobalt-chromium rpd frameworks. three of them were randomly selected to join the study and denominated lab i, ii and iii. the laboratories were visited and the laboratory technicians were asked to answer a questionnaire. questionnaires were structured to collect data about the qualification of the dentists assisted by the laboratories, monthly number of framework castings, and use of dental surveyor. anonymity and confidentiality of participants’ personal information as well as of their responses to questions were assured. thirty master casts for fabrication of cobalt-chromium rpds were obtained from each commercial laboratory, in a total of 90 casts. all master casts were photographed (finepix a900; fujifilm, valhalla, usa) and evaluated by the same examiner. the photographic technique was standardized, enabling evaluation of the casts as well as the design of the dentures13. data were collected regarding dental arch treated, kennedy classification, cast surveying, and denture design information provided by the dentist. with the cast positioned on the dental surveyor, mouth preparation was assessed for retentive areas, guiding planes and rest seat distribution. retentive areas were evaluated by the magnitude of the angle of cervical convergence below the point of convexity (horizontal retention), and the depth at which the clasp terminal is placed in the angle (vertical retention). undercuts of 0.01 inch and clasp terminals placed at least 2 mm depth in the angle of convergence were considered satisfactory3. guiding planes were evaluated by measurement of vertical and horizontal lengths. as a rule, proximal guiding plane surface should extend horizontally about one third of the buccal lingual width of the tooth, and vertically about two thirds of the length of the enamel crown portion from the marginal ridge cervically3. rest seat distribution was evaluated according to the classical biomechanical principles for rpd design proposed in the available literature3,14-15. according to the above mentioned criteria, retentive areas, guiding planes and rest seat distribution were classified as “completely appropriate”, “partially appropriate” or “inappropriate” (table 1). classes were converted into scores and means were compared among laboratories using kruskal-wallis test (α=0.05). ethics in the provision of removable partial dentures braz j oral sci. 11(1):19-24 results according to the answers to the questionnaires, lab i and ii have a monthly number of framework castings that ranges from 100 to 200, and serve private general practitioners as well as prosthodontists. additionally, lab ii also assists dental schools and graduate programs/courses. lab iii assists primarily private general practitioners, and has a monthly number of framework castings that ranges from 50 to 100. sixty percent of the master casts were from maxillary arches, while 40% were from mandibular arches. kennedy class iii was the most common type of partially edentulous arch (56%). the percentage of kennedy class i rpds was 16%, class ii 19%, and class iv 9%. sixty-five percent of the casts exhibited at least one modification. fifty-one percent of the casts were sent to dental laboratories without any design information, and this function was delegated to the dental technician. the design was accomplished by dentists in 49% of the cases, and communicated to the dental technician by different manners: as the outline of the framework on the cast (35%), written instructions (2%), and just by rest seats (12%). all laboratories assured to use the dental surveyor. assessment of guiding planes, retentive areas and rest seats are shown on figures 1 and 2. approximately half of the casts presented “inappropriate” for guiding planes and retentive areas. one of the laboratories presented all casts “inappropriate” for rest seats. rest seat distribution was significantly different among laboratories (p<0.001), while no significant difference was found for guiding planes and retentive areas. discussion this study evaluated the construction of cobaltchromium rpds by three commercial private dental laboratories in the city of natal, brazil. a representative sample of commercial laboratories that have the facility to cast cobalt-chromium rpd frameworks was randomly selected (37.5%). three laboratories were visited, dental technicians interviewed, and 90 master casts photographed and evaluated on dental surveyor. therefore, it was possible to observe the practices of dentists by assessment of the master casts sent to dental laboratories, instead of asking them about their attitudes. this is important because it has been previously shown that dentists’ practices are different from their knowledge16. kennedy class iii was the most common type of partially edentulous arch (56%). it may result from the fact that adults are retaining more of their teeth throughout life. for guiding planes, almost half of the casts were considered “inappropriate” (49%), and no cast was “completely appropriate”. this trend has also been shown by schwarz and barsby17, who found that only 6% of the general dental practitioners frequently prepare guiding planes. adequate guiding planes should be planned to establish the path of placement and dislodgement. properly prepared guiding surfaces contribute to the retention and stability of the rpd, and may improve masticatory efficiency up to 40%.18 although no statistically significant difference was found for guiding planes among dental laboratories (p=0.229), the percentage of “partially appropriate” guiding planes was higher for lab ii (64%). a possible explanation is that lab ii works for dental schools and graduate programs/ courses, which means that treatments are developed under the supervision of a prosthodontics professor. lab ii also presented the highest percentage for “completely appropriate” retentive areas (44%), and the reason may be the same mentioned above. interestingly, although questionnaires have revealed that all dental laboratories assured to use the dental surveyor, no cast presenting evidence of surveying was found. this may explain why 53% percent of the casts were considered “inappropriate” for retentive areas. it is impossible to select a site on dental surface with adequate undercut gauge by visual examination. a dental surveyor must be used to locate the exact undercut that retentive clasp terminals will occupy and mark it on the master cast. undercuts of 0.01 inch are adequate for retention by cobalt-chromium cast retainers3. dentists using insufficient or too deep undercuts for clasps in their rpds have experienced poorly fitting clasps to abutments, poorly fitting rpds in patients’ mouth, pain in the abutment and events of deformed, bent and broken clasps19. philosophies of rpd support are based on principles of broad or selective distribution of occlusal forces3,14-15. occlusal and incisal rests are important supporting elements that serve to transmit vertical forces to abutment teeth and to direct those forces along the long axes of teeth20. in this study, 63% of the casts were “inappropriate” for rest seat distribution. of these, 57% had no rest seat preparation. recently, it was found that just 30% of the master casts with prescribed occlusal and cingulum rests had an obvious rest seat preparation. in many instances, rest seats were overprepared or under-prepared, and the interocclusal clearance available for the planned rest was inappropriate21. rest seats must provide adequate space for rest thickness, be strong enough to endure functional stress and prevent premature contact20. other common faults observed in this study were the placement of a distal rest in free-end rpds (10%) and inadequate indirect retention (17%). as a general rule, while rest seats are commonly adjacent to edentulous areas in tooth supported rpds, there are theoretical advantages for positioning a mesial occlusal rest in extension base rpds: anterior position of the fulcrum line, reduction in need of indirect retention, and increased resistance to distal displacement of the denture3,14-15. rest seat distribution was significantly different among laboratories (p<0.001). lab iii presented 100% of the casts “inappropriate”, while lab i and ii, 52% and 36%, respectively (p<0.001). this difference may be explained by the qualification of dentists assisted by lab iii, which is primarily composed of general practitioners, in contrast to lab i and ii, which also assist prosthodontics specialists, dental schools and postgraduate courses. the tendency for an association between dentist’s knowledge and denture 2121212121 ethics in the provision of removable partial dentures 2222222222 braz j oral sci. 11(1):19-24 fig. 1. general assessment of master casts for mouth preparation. inappropriate (in), partially appropriate (pa), completely appropriate (ca). service quality is also supported by the lowest percentage of “inappropriate” casts presented by lab ii for all items assessed. various studies have shown that most general practitioners neglect fundamental rpd principles and transfer the responsibility for planning the prosthesis to dental laboratory technicians6-12. the reason for this is not clear, but two hypotheses may explain this issue: dentists may disregard the acquired knowledge in order to save time and expedite the treatment; or dentists may receive inadequate orientation from dental schools during graduation 22. a previous study found that educational factors seem to have a more significant effect on this issue than financial factors23. another study revealed that the majority of dentists are aware that success will be positively influenced if they design the rpd, but only half reported that they did this in their practice16. a recent study showed that only 12% of senior dental students from a representative number of dental schools in the state of são paulo, brazil, were capable of accomplishing completely appropriate mouth preparations and rpd designs22. the authors suggested that immediate fig. 2. assessment of mouth preparation according to dental laboratories. inappropriate (in), partially appropriate (pa), completely appropriate (ca). changes in the teaching of rpd are necessary with emphasis on the treatment planning, mouth preparation and survey and design principles. inappropriately designed rpds may have potentially harmful effects, such as gingival irritation, tooth mobility, root caries, tooth loss, and low patient satisfaction5. previous studies have shown that the number of cobalt-chromium rpds constructed by dental laboratories without any written communication of the design may be higher than 90%6-12. in the present study, 51% of the casts were sent to dental laboratories without any design information. when the design was communicated to the dental technician, it was done in different manners, as the outline of the framework on the cast (35%), written instructions (2%), and only by rest seats (12%). it is suggested that a satisfactory work authorization for a rpd design takes the form of an annotated diagram of the design accompanied with written instructions. alternatively, a photocopy of the working cast may be taken and the design drawn on it. the design of the rpd framework also should be drawn on the study cast to transmit this ethics in the provision of removable partial dentures braz j oral sci. 11(1):19-24 2323232323 fig. 3. rpd framework drawn on the study cast to transmit this information to the prosthodontic technician. information to the technician, since subsequent transfer of two-dimensional information by the technician from the paper diagram to the three-dimensional cast can lead to errors of interpretation (figure 3)4. clarification of the design diagram may be achieved by using a color code to identify different rpd components or functions. although all investigated dental laboratories affirmed to use a dental surveyor, no evidence of cast surveying was found. surveying avoids unnecessary removal of tooth substance, while identifying the optimal path of insertion. in many dental offices, this most important phase of dental diagnosis is delegated to the dental laboratory because the dental surveyor is absent or because the dentist is apathetic3. this situation places the technician in the role of diagnostician, which is illegal and unethical. according to the brazilian code of dentistry ethics, diagnosis, prognosis, treatment planning and treatment are dentist’s responsibilities24. in the usa, the legislation states that the dentist has ultimate responsibility for all dental treatment, including the design and material of any prosthesis produced by dental laboratories. state laws require a written work authorization order to accompany all work sent by a dentist to a dental laboratory 4. the work authorization must be made in duplicate and both the dentist and dental laboratory technician must retain a copy for a specified period. thus documents are available to substantiate or refute claims and counterclaims that concern the illegal practice of dentistry or to aid in the settlement of misunderstandings between a dentist and a dental laboratory technician3. the distinction between the clinician’s and dental technician’s responsibilities, as well as the risks posed by poor-quality denture designs to oral health, should be clearly understood for the ethical provision of rpds with no harmful effects to patients. in summary, this study demonstrated that the construction of cobalt-chromium rpds by commercial private dental laboratories in the northeastern region of brazil presents the same issues already found in other countries6-12. the majority of master cats examined was sent to dental laboratories without any design information and did not comply with ethical guidelines in the provision of rpd. mouth preparation frequently failed for guiding planes, retentive areas and rest seat distribution. these findings reinforce the fact that distinction between the clinician’s and dental technician’s responsibilities should be clearly understood. available evidence suggests an association between dentist’s knowledge and denture service quality. therefore, it is necessary to provide students with adequate clinical experience in the dental school environment that will in turn carry into the practice of dentistry. the growing population and extended life expectancy, both associated with the reduction in tooth loss, may contribute to increase the prevalence of partially dentate adults and the need for rpd. references 1. brazil. ministry of health. brazilian oral health project: oral health status of brazilian population 2002-2003 – main results. brasília; 2004. 2. douglass cw, shih a, ostry, l. will there be a need for complete dentures in the united states in 2020? j prosthet dent. 2002; 87: 5-8. 3. carr ab, glen pm, brown dt. partially edentulous epidemiology, physiology, and terminology. mccracken’s removable partial prosthodontics. saint louis: elsevier mosby; 2004. p.3-10. 4. davenport jc, basker rm, heath jr, ralph jp, glantz po, hammond p. communication between the dentist and the dental technician. br dent j. 2000; 189: 471-4. 5. owall b, budtz-jorgensen e, davenport j et al. removable partial denture design: a need to focus on hygienic principles? int j prosthodont. 2002; 15: 371-8. 6. basker rm, harrison a, davenport jc, marshall jl. partial denture design in general dental practice – 10 years on. br dent j. 1988; 165: 245-9. 7. woolfardt jf, han-kuang t, basker rm. removable partial denture design in alberta dental practices. j can dent assoc. 1996; 62: 637-44. 8. von steyern pv, widolf-kroon r, nilner k, basker rm. removable partial denture design in general dental practice in sweden. swed dent j. 1995; 19: 205-11. 9. dullabh hd, slabbert jcg, becker pj. partial denture prosthodontics procedures employed by practising graduates of the university of witwatersrand, johannesburg. j dent assoc s afr. 1993; 48: 129-34. 10. taylor td, mathews ac, aquilino sa, logan ns. prosthodontic survey. part i: removable prosthodontic laboratory survey. j prosthet dent. 1984; 52: 598-601. 11. lynch cd, allen pf. a survey of chrome-cobalt rpd design in ireland. int j prosthodont. 2003; 16: 362-4. 12. radhi a, lynch cd, hannigan a. quality of written communication and master impressions for fabrication of removable partial prostheses in the kingdom of bahrain. j oral rehabil. 2007; 34: 153-7. 13. owall g, bieniek kw, spiekermann h. removable partial denture production in western germany. quintessence int. 1995; 26: 621-7. 14. kratochvil fj. influence of occlusal rest position and clasp design on movement of abutment teeth. j prosthet dent. 1963; 13: 114-24. 15. mccracken wl. contemporary partial denture designs. j prosthet dent. 2004; 92: 409-17. 16. allen pf, jepson nj, doughty j, bond s. attitudes and practice in the provision of removable partial dentures. br dent j. br dent j. 2008; 204: e2. epub 2007. 17. schwarz wd, barsby mj. a survey of the practice of partial denture prosthetics in the united kingdom. j dent. 1980; 8: 95-101. 18. manly rs, vinton p. factors influencing denture function. j prosthet dent. 1951; 1: 578-86. ethics in the provision of removable partial dentures 2424242424 braz j oral sci. 11(1):19-24 19. fayyaz m, ghani f. appropriateness of knowledge and practices of dentists relating to using clasps in removable partial dentures. j ayub med coll abbottabad. 2008; 20: 52-5. 20. principles, concepts, and practices in prosthodontics – 1994. academy of prosthodontics. j prosthet dent. 1995; 73: 73-94. 21. rice ja, lynch cd, mcandrew r, milward pj. tooth preparation for rest seats for cobalt-chromium removable partial dentures completed by general dental practitioners. j oral rehabil. 2011; 38: 72-8. 22. neto af, duarte arc, shiratori fk, leite phas, rizzatti-barbosa cmr, bonachela wc. evaluation of senior brazilian dental students about mouth preparation and removable partial denture design. j dent educ. 2010; 74: 1255-60. 23. lynch cd, allen pf. why do dentists struggle with removable partial denture design? an assessment of financial and educational issues. br dent j. 2006; 200: 277-81. 24. federal council of dentistry. code of ethics for dentists. rio de janeiro: federal council of dentistry; 2002. ethics in the provision of removable partial dentures oral sciences n3 braz j oral sci. 10(4):282-287 received for publication: september 26, 2011 accepted: december 06, 2011 original article braz j oral sci. october | december 2011 volume 10, number 4 comparison of the centering ability of the protaper universal, profile and twisted file rotary systems daniela de andrade mendes1, carlos menezes aguiar2, andréa cruz câmara3 1bds, msc student in integrated clinical dentistry, department of prosthodontics and oral and facial surgery, dental school, federal university of pernambuco, brazil 2bds, msc, associate professor, department of prosthodontics and oral and facial surgery, dental school, federal university of pernambuco, brazil 3bds, msc, phd, department of prosthodontics and oral and facial surgery, dental school, federal university of pernambuco, brazil correspondence to: daniela de andrade mendes rua professor augusto lins e silva, 383, apt. 1002, boa viagem, recife pe cep 51130-030 brazil phone: (+55) 81 3461 1591 e-mail: mendes_dam@hotmail.com abstract aim: to determine the centering ability of twisted file™ rotary system compared with protaper universal™ and profile™ rotary systems by evaluating preand postoperative cross-sectional images of the apical root canals third. methods: thirty mesiobuccal canals of human mandibular first molars were divided into three groups with 10 root canals each according to the instrument used: group 1, protaper universal™ rotary system; group 2, profile™, and group 3, twisted file™. preand postoperative images of the apical thirds were viewed with a stereoscopic magnifier with ×10 magnification and were captured digitally for further analysis using the image tools software. the results were analyzed statistically by the kruskal-wallis test and the mannwhitney test. a level of significance of 0.05 was adopted. results: the means of the buccolingual measurement ranged from 0.79 to 1.5. the largest deviation was registered to instrument 25.06 in group 2. the means of the mesiodistal measurement ranged from 0.86 to 1.52, with the largest deviation being registered to instrument 25.04 in group 3; however, there were no statistically significant differences (p>0.05) among the three groups or among the instruments in the same group in terms of centering ability. conclusions: none of the rotary systems evaluated in this study was totally effective in performing biomechanical preparation of the root canals. keywords: instruments, protaper universal, root canal, root canal preparation, twisted file. introduction root canal preparation is one of the major components of endodontic treatment, and it is directly related to subsequent disinfection and filling1. the aim of root canal preparation is to form a continuously tapered shape with the smallest diameter at the apical foramen and the largest at the orifice to allow effective irrigation and filling2 without deviating from the original trajectory3-4. when curvature is present, endodontic preparation becomes more difficult, and there is a tendency for all preparation techniques to divert the prepared canal away from the original axis5. nickel-titanium (niti) rotary instruments, due to their superelastic behavior and shape-memory properties, are able to maintain the original canal shape without braz j oral sci. 10(4):282-287 283 significant transportation or creation of irregularities such as zipping, ledges, perforations, or danger zones, in curved canals6-7. many types of rotary root canal instruments have been introduced, varying in cross-section, blade and pitch design, and taper8-10. the profile™ (dentsply, maillefer, ballaigues, switzerland) rotary system is a three-fluted file of constant taper, with three radial lands, a u-shaped cross-section, and noncutting safety tip11. the protaper™ niti rotary system has been upgraded to the protaper universal™ system, which includes shaping, finishing, and retreating instruments. it incorporates a shallow, u-shaped groove at each of its convex triangular sides in cross-section, supposedly to improve flexibility in the larger instruments1,12-14. recently, a completely different manufacturing process has been developed by sybronendo to create a new rotary file for root canal preparation called the twisted file™ (sybronendo, orange, ca, usa). these files have a triangular cross section with constant tapers of .04, .06, .08, .10, and .12. they are available in five tip sizes from 25 to 50. the manufacturer claims that the three new manufacturing processes of these files, namely r-phase heat treatment, twisting of the metal, and special surface conditioning, significantly increase the instrument’s resistance to cyclic fatigue and flexibility, even with .06-, .08-, .10-, and .12tapered instruments, maintaining the original canal center and minimizing canal transportation even in severely curved root canals5,15-17. there have been few studies published on the ability of the twisted file™ rotary systems to maintain root canal morphology. by evaluating preand postoperative crosssectional images of the apical third of root canals, the present study set out to determine the centering ability of the twisted file™ rotary system compared with the protaper universal™, and profile™ systems. material and methods selection and preparation of the samples thirty mesiobuccal canals of extracted human mandibular first molars (length, 20-21 mm) obtained from the human tooth bank of the department of prosthodontics and oral and facial surgery of the federal university of pernambuco, brazil, were selected with the approval of the ethics committee of the center of health sciences of the same university. the mesiobuccal roots had completely formed apices and severely curved root canals whose curvature ranged from 50° to 60° according to the canal access angle (caa) technique18. after coronal access, the distal root was separated from the mesial root with a carborundum disk (kg sorensen, barueri, brazil). the distal root was returned to the tooth bank, and the mesial root was washed in running water for 2 min and left to dry at room temperature. a #10 senseusflexofile (dentsply/maillefer) was inserted into the mesiobuccal canal until its tip was visible at the apical foramen and the working length (wl) was calculated to be 1 mm less than the length obtained with this initial file. obtaining the preoperative images the specimens were embedded in autopolymerizing resin acrylic blocks (artigos odontológicos clássico ltda., são paulo, sp, brazil) according to a previously described method3. after polymerization, the acrylic blocks were removed from the molds and sectioned transversely 3 mm from the apex for standardization purposes with the aid of a double-faced diamond disk (kg sorensen). preoperative images of the apical thirds were viewed with a ×10 stereoscopic magnifier (ramsor, são paulo, sp, brazil) at the biomaterials clinical research unit of the federal university of pernambuco and captured digitally. the specimens were remounted in their molds and biomechanical preparation was performed. biomechanical root canals preparation the specimens were randomly divided into three groups with 10 root canals each. all instrumentation was performed according to each manufacturer’s instructions. random distribution of the groups considered the degree of canal curvature, allowing the average curvature, as well as the more severe cases, to be evenly allocated to each group: group 1: protaper universal™ rotary system. the canals were instrumented at a rotational speed of 300 rpm (driller endo-pro torque, sao paulo, brazil) as follows: (a) the sx file was used to one half the of the wl, (b) the s1 file was used up to 4 mm short of the apex, (c) the s1 and s2 files were used to the full wl, and (d) the f1 and f2 files were used to the full wl. group 2: profile™ rotary system. the canals were instrumented at a rotational speed of 300 rpm as follows: (a) #20.08 and #25.08 files were used up to the coronal one third of the root canal; (b) #20.06 and #25.06 files were used up to 4 mm short of the apex; and (c) #20.04, #25.04, and #25.06 files were used up to the full wl. group 3: twisted file™ rotary system. the canals were instrumented at a rotational speed of 300 rpm as follows: (a) #25.08 file was used up to the coronal one third of the root canal, (b) #25.06 file was used up to 4 mm short of the wl, and (c) #25.04 and #25.06 files were used up to the full wl. after the use of each file, the root canals were irrigated with 3 ml of a freshly prepared 1% sodium hypochlorite solution (roval, recife, brazil). glyde™ (dentsply/ maillefer) was used as a lubricant during instrumentation. a single operator experienced in rotary systems prepared all root canals. each instrument was changed after five canals. instruments were examined after every use to record and reject deformed or fractured instruments. obtaining the postoperative images after instrumentation with files f1 and f2 (group 1), #25.04 and #25.06 (group 2), and #25.04 and #25.06 (group 3), the specimens were removed from the molds and the apical third section was viewed again in the stereoscopic magnifier with ×10´ magnification, the postoperative images being captured by a computer. comparison of the centering ability of the protaper universal, profile and twisted file rotary systems 284 braz j oral sci. 10(4):282-287 evaluation of centering ability using the image tool software (university of texas health science center, san antonio, tx, usa), the preoperative and postoperative images were compared (figure 1). according to a previously described method19, the following equation was devised to evaluate centering capacity (figure 2): d1: x1/ x2 = (x1 “ x2 1)/(x2 “ x2 2) d2: y1/ y2 = (y1 “ y2 1)/(y2 “ y2 2) where d1= the buccolingual measurement and d2 = the mesiodistal measurement. according to this equation, a result of 1 indicates perfect centering. statistical analysis of the data the data regarding d1 and d2 were calculated from the usual location measurements (mean and average) and dispersion (standard deviation, minimum and maximum) at the 95% confidence interval. the results were statistically analyzed using the student’s t-test and the mann-whitney test (statistical inference). a level of significance of 0.05 was adopted, using the statistical package for the social sciences, version 13 (spss inc., chicago, il, usa). fig. 2. schematic presentation of the image used in the evaluation. fig. 1. preand postoperative images showing the centering ability of the protaper universal™ (a and b), profile™ (c and d), and twisted file™ (e and f) rotary. results table 1 presents the main descriptive statistics of the buccolingual measurement (d1) and mesiodistal measurement (d2) according to the diameter of the instrument used and the group to which the instruments (protaper universal™, profile™, or twisted file™ rotary systems) belong. this table shows that the means of d1 ranged from 0.79 to 1.5. the largest deviation was registered to instrument #25.06 in group 2. the means of d2 ranged from 0.86 to 1.52, with the largest deviation being registered to instrument #25.04 in group 3; however, there were no statistically significant differences (p>0.05) among the three groups nor among the instruments in the same group in terms of centering ability. discussion root canal shaping comprises one of the fundamental stages of endodontic treatment2. however, the presence of curvatures may pose difficulty in root canal instrumentation. the results of shaping curved root canals is influenced by several factors, such as flexibility and diameter of the endodontic instruments, instrumentation techniques, location of the foramen, and hardness of the dentin. ledge formation, blockages, perforations, and apical transportation are undesirable accidents that have occurred during preparation of curved root canals20. the introduction of niti instruments allowed a safer and easier preparation of canals with complex anatomic characteristics21. several niti rotary instrument systems have been introduced to endodontics9. these instruments offer greater flexibility and more resistance to torsional separation than stainless steel files17. because of these features, they are better able to maintain curvature even in severely curved canals. by preserving the original canal as far as possible, iatrogenic complications arising from cleaning and shaping can be avoided22. to reduce canal aberrations, new niti instruments have been developed, such as the systems investigated in this study. an increasing number of niti rotary systems have been marketed by various manufacturers. the choice of the instruments used for this study took into account several factors that make them different from other systems, such as comparison of the centering ability of the protaper universal, profile and twisted file rotary systems braz j oral sci. 10(4):282-287 instrument n mean median s d m i n max ci p value1 p value2 ptu f1 d1 10 1.09 1.05 0.57 0.25 1.92 (0.69 1.50) 0.631 0.853 ptu f1d2 10 1.25 1.09 0.58 0.42 2.13 (0.83 1.66) 0.694 0.971 ptu f2d1 10 0.99 1.04 0.40 0.29 1.74 (0.70 1.27) 0.665 0.853 ptu f2d2 10 1.14 1.07 0.30 0.77 1.84 (0.93 1.36) 0.080 0.971 pf 25.04 d1 10 1.03 1.02 0.35 0.30 1.65 (0.78 1.28) 0.548 0.481 pf 25.04 d2 10 1.45 0.86 1.68 0.06 5.52 (0.24 2.65) 0.272 0.315 pf 25.06 d1 10 1.50 0.47 2.72 0.08 9.05 (-0.44 3.45) 0.651 0.481 pf 25.06 d2 10 0.86 0.33 1.09 0.04 3.07 (0.08 1.65) 0.088 0.315 tf 25.04 d1 10 0.79 0.76 0.53 0.17 1.42 (0.41 1.18) 0.548 0.529 tf 25.04 d2 10 1.52 1.20 1.25 0.36 4.35 (0.63 2.42) 0.272 0.481 tf 25.06 d1 10 1.04 1.20 0.62 0.18 2.13 (0.59 1.48) 0.651 0.529 tf 25.06 d2 10 1.05 0.78 0.78 0.37 3.00 (0.49 1.61) 0.088 0.481 table 1main descriptive statistics of the buccolingual and mesiodistal measurements according to the instrument used systems. 1kruskal-wallis test ; 2mann-whitney test; n=number of specimens; sd=standard deviation; ci=confidence interval; ptu= protaper universal™;pf= profile™;tf= twisted file™. cutting blades, body taper, and configuration of the file tip. the profile™ rotary system has been available for some years, having become a system with which other niti enginefiles are compared. the same manufacturer has introduced another instrument of different design, the protaper™ system, purportedly to enhance cutting efficiency and improve flexibility of the instrument23. the protaper™ niti rotary system has been upgraded to the protaper universal™ system, which differs essentially in the cutting blade designed for increased flexibility, cutting, and efficiency with a low proportion of apical deviation as demonstrated in the present and previous studies1,12-14. ünal et al.24 has evaluated whether changes in the protaper™ system contributed to their shaping ability in terms of the morphology of curved canals. the authors observed that the protaper™ modifications did not create any discrepancy in the shaping abilities of the instruments. these findings are in contrast to previous studies25-27 in which the protaper universal™ showed a greater tendency to produce apical transportation. a number of methods for investigating the effectiveness of endodontic instruments in shaping root canals have been used1,3-6,8-9,11-12,19,22-25. one of these is the use of the radiographic platform22,28-29. it merely provides, however, a two-dimensional image, precluding observation of the three-dimensional conformation of root canals. in the present research, as with previous studies3,30-31, the preoperative and postoperative images of the sectioned root canals were viewed by using a stereoscopic magnifier with ×10´ magnification and were compared using the image tool software, in which the centering ability of the niti rotary systems was assessed. this method allows a relatively easy and repeatable comparison of preand postinstrumented canals so as to analyze the action of the instruments on the root canal walls32. another method of analysis is computed tomography, a noninvasive method for analyzing canal geometry and the efficiency of shaping techniques5,10,26,33-34. with this technique, it is possible to compare the anatomic internal structure of the canal before and after instrumentation, but it was not used in this study. human teeth were used in the present study, as in previous ones1,3,5,9,11-12,22,28,31,33-34. the main reason for choosing human teeth is that they simulate clinical conditions better than do acrylic blocks. acrylic resin is not an excellent material for testing rotary instruments because it does not reproduce the microhardness of dentin and the frequently encountered anatomic variations (enlargements, oval root canals, etc), which cannot be easily simulated35. mesiobuccal root canals of extracted human mandibular molars were used herein because they usually present an accentuated curvature36. several studies have used the schneider method37 to determine root canal curvature5,11,22,33,36. in the present study, curvature was measured by the caa method because it is as effective as the schneider angle in evaluating root canal curvature and is better to measure the centering ability of root canal instruments18. although the protaper universal™ rotary system supplies instruments with larger apical diameters, this study was limited to the f1 and f2 instruments in order to standardize the final apical preparation diameter to size 25 for the three groups. we showed that the three different rotary systems with distinct designs produced similar results in terms of centering ability. analysis of the d1 measurement, in the present research revealed the largest deviation to be registered to instrument #25.06 of the profile™ rotary system when compared with the other systems; this is in agreement with the results demonstrated by vanni et al.38. nevertheless, al-sudani and al-shahrani11 demonstrated that profile™ produced centered preparations. this distinct performance could be attributed to the different designs of this instrument. profile™ instruments use a u-shaped file design with radial land areas, and have a neutral or slightly negative rake angle. ersev et al.8 have shown profile™ to be significantly superior to other systems in terms of centering ability and yamashita et al.39 reported that profile™ had the best cleaning ability compared with quantec and pow-r systems. in the d2 measurement, the highest value of deviation 285comparison of the centering ability of the protaper universal, profile and twisted file rotary systems braz j oral sci. 10(4):282-287 286 was registered to instrument #25.04 of the twisted file™ rotary system. gergi et al.5 were the first to compare the centering ability of twisted file™, pathfile-protaper™ and conventional stainless steel k-files. they observed that the best centering was achieved with the twisted file™ rotary instruments. el batouty et al.40 reported that twisted file™ produced significantly less transportation and preserved the original canal to a greater degree than did the k3 system. although none of the instruments evaluated in this study was totally effective in performing biomechanical preparation of the root canals, because each of them produced morphological changes, the protaper universal™, profile™, and twisted file™ rotary systems demonstrated an acceptable capacity to shape curved root canals. there were no significant differences among the three groups or among the instruments in the same group in terms of the centering ability. considering the limitations of this study and the fact that the twisted file™ is new to the arsenal of endodontic tools, further investigations are required to provide more information about this new rotary system. although none of the instruments evaluated in this study was totally effective in performing biomechanical preparation of the root canals, because each of them produced some morphological changes, protaper universal™, profile™, and twisted file™ rotary systems demonstrated an acceptable capacity to shape curved root canals. references 1. aguiar cm, mendes d de a, câmara ac, figueiredo jap de. assessment of canals walls after biomechanical preparation of root canals instrumented with protaper universaltm rotary system. j appl oral sci. 2009; 17: 590-5. 2. schilder h. cleaning and shaping the root canal. dent clin north am. 1974; 18: 269-96. 3. câmara ac, aguiar cm, figueiredo jap de. assessment of the deviation after biomechanical preparation of the coronal, middle, and apical thirds of root canals instrumented with three hero rotary systems. j endod. 2007; 33: 1460-3. 4. hata gi, uemura m, kato as, imura n, novo nf, toda t. a comparison of shaping ability using profile, gt file and flex-r endodontic instruments in simulated canals. j endod. 2002; 28: 316-21. 5. gergi r, rjeily ja, sader j, naaman a. comparison of canal transportation and centering ability of twisted files, pathfile-protaper system, and stainless steel hand k-files by using computed tomography. j endod. 2010; 36: 904-7. 6. madureira rg, navarro lf, llena mc, costa m. shaping ability of nickel-titanium rotary instruments in simulated s-shaped root canals. oral surg oral med oral pathol oral radiol endod. 2010; 109: e136-44. 7. mercês am do a, aguiar cm, shinohara nks, câmara ac, figueiredo jap de. comparison of root canals obturated with protaper gutta-percha master point using the active lateral condensation and the single-cone techniques: a bacterial leakage study. braz j oral sci. 2011; 10: 37-41. 8. ersev h, yilmaz b, çiftçioðlu e, özkarsli ªf. a comparison of the shaping effects of 5 nickel-titanium rotary instruments in simulated s-shaped canals. oral surg oral med oral pathol oral radiol endod. 2010; 109: e86-93. 9. karabucak b, gatan aj, hsiao c. a comparison of apical transportation and length control between endosequence and guidance rotary instruments. j endod. 2010; 36: 123-5. 10. alencar ahg de, dummer pmh, oliveira hcm, pécora jd, estrela c. procedural errors during root canal preparation using rotary niti instruments detected by periapical radiography and cone beam computed tomography. braz dent j. 2010; 21: 543-9. 11. al-sudani d, al-shahrani s. a comparison of the canal centering ability of profile, k3, and race nickel titanium rotary systems. j endod. 2006; 32: 1198-1201. 12. aguiar cm, mendes d de a, câmara ac, figueiredo jap de. evaluation of the centreing ability of the protaper universal? rotary system in curved roots in comparison to nitiflex? files. aust endod j. 2009; 35: 174-9. 13. grazziotin-soares rg, barato filho f, vanni jr, almeida s, oliveira epm de, barletta fb, limongi o. flexibility of k3 and protaper universal instruments. braz dent j. 2011; 22: 218-22. 14. martins r de c, bahia mg de a, buono vtl. geometric and dimensional characteristics of simulated curved canals prepared with protaper instruments. j appl oral sci. 2010; 18: 44-9. 15. gambarini g, grande nm, plotino g, somma f, garala m, de luca m, testarelli l. fatigue resistance of engine-driven rotary nickel-titanium instruments produced by new manufacturing methods. j endod. 2008; 34: 1003-5. 16. kim hc, yum j, hur b, cheung gsp. cyclic fatigue and fracture characteristics of ground and twisted nickel-titanium rotary files. j endod. 2010; 36: 147-52. 17. larsen cm, watanabe i, glickman gn, he j. cyclic fatigue analysis of a new generation of nickel titanium rotary instruments. j endod. 2009; 35: 401-3. 18. günday m, sazak h, garip y. a comparative study of three different root canal curvature measurement techniques and measuring the canal access angle in curved canals. j endod. 2005; 31: 796-8. 19. gambill jm, alder m, del rio ce. comparison of nickel-titanium and stainless steel hand-file instrumentation using computed tomography. j endod. 1996; 22: 369–75. 20. jain n, tushar s. curved canals: ancestral files revisited. indian j dent res. 2008; 19: 267-71. 21. walia hm, brantley wa, gerstein h. an initial investigation of the bending and torsional properties of nitinol root canal files. j endod. 1988; 14: 346–51. 22. guelzow a, atamm o, martus p, kielbassa am. comparative study of six rotary nickel-titanium systems and hand instrumentation for root canal preparation. int endod j. 2005; 38: 743–52. 23. shen y, cheung gsp, bian z, peng b. comparison of defects in profile and protaper systems after clinical use. j endod. 2006; 32: 61-5. 24. ünal gç, maden m, savgat a, onur orhan e. comparative investigation of 2 rotary nickel-titanium instruments: protaper universal versus protaper. oral surg oral med oral pathol oral radiol endod. 2009; 107: 886-92. 25. kunert gg, fontanella vrc, moura aam de, barletta fb. analysis of apical root transportation associated with protaper universal f3 and f4 instruments by using digital subtraction radiography. j endod. 2010; 36: 1052–5. 26. hartmann msm, fontanella vrc, vanni jr, fornari vj, barletta fb. ct evaluation of apical canal transportation associated with stainless steel hand files, oscillatory technique and protaper rotary system. braz dent j. 2011; 22: 288-93. 27. silva kt da, grazziotin-soares r, limongi o, irala led, salles aa. wear promoted in the apical third of simulated canals after instrumentation with protaper universal system. j appl oral sci. 2009; 17: 501-7. 28. aguiar cm, câmara ac. radiological evaluation of the morphological changes of root canals shaped with protaper™ for hand use and the protaper™ and race™ rotary instruments. aust endod j. 2008; 34: 1159. 29. sydney gb, batista a, melo ll. the radiographic platform: a new method to evaluate root canal preparation in vitro. j endod. 1991; 17: 570-2. 30. jeon is, spangberg lsw, yoon tc, kazemi rb, kum ky. smear layer production by 3 rotary reamers with different cutting blade designs in straight root canals: a scanning electron microscopic study. oral surg oral med oral pathol oral radiol endod. 2003; 96: 601-7. 31. schäfer e. shaping ability of hero 642 rotary nickel-titanium instruments and stainless steel hand k-flexofiles in simulated curved root canals. oral surg oral med oral pathol oral radiol endod. 2001; 92: 215-20. comparison of the centering ability of the protaper universal, profile and twisted file rotary systems 287 braz j oral sci. 10(4):282-287 32. veltri m, mollo a, pini pp, ghelli lf, balleri p. in vitro comparison of shaping abilities of protaper and gt rotary files. j endod. 2004; 30: 1636. 33. versiani ma, pascon ea, sousa cja de, borges mag, sousa-neto md. influence of shaft design on the shaping ability of 3 nickel-titanium rotary systems by means of spiral computerized tomography. oral surg oral med oral pathol oral radiol endod. 2008; 105: 807-13. 34. paqué f, balmer m, attin t, peters ao. preparation of oval-shaped root canals in mandibular molars using nickel-titanium rotary instruments: a micro-computed tomography study. j endod. 2010; 36: 703-7. 35. kum k, spängberg l, cha by, il-young j, seung-jang l, chan-young l. shaping ability of three profile rotary instrumentation techniques in simulated resin root canals. j endod. 2000; 26: 716-23. 36. hartmann msm, barletta fb, fontanella vrc, vanni jr. canal transportation after root canal instrumentation: a comparative study with computed tomography. j endod. 2007; 33: 962-5. 37. schneider sw. a comparison of canal preparations in straight and curved canals. oral surg oral med oral pathol. 1971; 32: 271-5. 38. vanni jr, albuquerque ds de, reiss c, baratto fiflo f, limongi o, della bona a. apical displacement produced by rotary nickel-titanium instruments and stainless steel files. j appl oral sci. 2004; 12: 51-5. 39. yamashita jc, duarte mah, valim fa, almeida jm de, kuga mc, fraga s de c. evaluation of the surface of root canal walls after utilization of endodontic rotary systems: sem study. j appl oral sci. 2005; 13: 78-82. 40. el batouty km, elmallah we. comparison of canal transportation and changes in canal curvature of two nickel-titanium rotary instruments. j endod. 2011; 37: 1290-2. comparison of the centering ability of the protaper universal, profile and twisted file rotary systems oral sciences n3 braz j oral sci. 11(4):486-491 original article braz j oral sci. october | december 2012 volume 11, number 4 dentistry for babies: caries experience vs. assiduity in clinical care letícia vargas freire martins lemos1, terezinha esteves de jesus barata2, silvio issáo myaki3, luiz reynaldo de figueiredo walter4 1dds, ms, phd student, department of pediatric dentistry, araraquara dental school, unesp – univ estadual paulista, araraquara, sp, brazil 2dds, ms, phd, professor, department of preventive dentistry and oral rehabilitation, dental school, federal university of goiás (ufg), goiânia, go, brazil 3dds, ms, phd, professor, department of pediatric and social dentistry, são josé dos campos dental school, unesp – univ estadual paulista, são josé dos campos, sp, brazil 4dds, ms, phd, full professor, department of pediatric dentistry, dental school, state university of londrina (uel), londrina, pr, brazil correspondence to: letícia vargas freire martins lemos faculdade de odontologia de araraquara unesp departamento de clínica infantil rua humaitá, 1680, cep: 14801-903 araraquara, sp, brasil phone: +55 16 33016328 e-mail: letvargas@uol.com.br received for publication: august 15, 2012 accepted: december 14, 2012 abstract aim: this study analyzed and compared the experience of dental caries in 300 children aged 0 to 48 months, who were participants and non-participants of a preventive program ‘dentistry for babies’, as well as the correlation between assiduity of dental visits and experience of dental caries. methods: the subjects were randomly selected and divided into two groups: g1 ‘non participant children of the program’ (n=100) and g2 ‘participant children of the program’ (n=200). each group was subdivided in two subgroups: 0-24 months and 25-48 months. the collected data from g2 were analyzed, relating the variation of the dmft index (dmft refers to primary teeth: d = decayed, m = missing/extracted due to caries, f = filled, t = teeth) (c) and dental caries prevalence (p) with the influence of assiduity factor in each subgroup. to collect data, clinical examinations were performed using tactile and visual criteria by a single calibrated examiner. the data were statistically analyzed using the ‘paired t-test’, ‘mann-whitney’ and ’chi-squared’ tests (p<0.05). results: it was found that prevalence and dmft index were statistically significant (p=0.0001) with the greatest values observed in g1 (p=0.0001). the values were: pg1 (73%), pg2 (22%), cg1 (3.45±3.84), cg2 (0.66±1.57). assiduity was significant in g2 (p=0.0001). the values observed were: p-assiduous (2%), p-non-assiduous (42%), c-assiduous (0.04±0.31), and c-non-assiduous (1.29±2.01). conclusions: the participation in the program had a positive influence on the oral health of babies. complete assiduity to the program resulted in the lowest rates and prevalence of dental caries. keywords: pediatric dentistry, dental caries, epidemiology, referral and consultation, dental care. introduction early childhood caries (ecc) are to be considered any stage of caries lesion in any surface of primary teeth in babies and pre-school children up to 71 months of age1 in which there is an association with feeding before sleeping, sugar intake and inadequate hygiene2. as the development of caries is a dynamic process3, evaluation at every dental visit is necessary4-5. assiduity to the oral health program means that the families attended regularly the program, being diligent in compliance with the instructions of the program. the brazilian public health department provided information about the 487487487487487 braz j oral sci. 11(4):486-491 prevalence of dental caries6, which reveals that almost 27% children aged 18-36 month have at least one primary carious tooth. at the age of five, the rate of children with carious teeth reaches almost 60%6. thus, the ecc being considered a significant public health problem3,6-7. according to the study of walter and nakama8, the peak time for dental caries prevalence occurs in early childhood between the 13th-24th months. therefore, the ideal age to start preventive dental care is before the 12th month8. the inclusion of the initial stages of caries in epidemiological surveys is important because of the possibility of lesion reversal. its identification is socially relevant, as it allows for early intervention9-10. fraiz and walter11 evaluated 200 children, aged 24 to 48 months, who participated in a preventive oral health program for babies for at least 12 months. the population studied presented a low prevalence of dental caries. the results showed that this program positively influenced oral health of those children11. in order to diminish the ecc index, an oral health program tailored for babies named ‘baby clinic’ was created in 1996 in jacareí, sp, brazil. the city has a fluoridated water supply (0.7 ppm) since 198112. ‘baby clinic’ focuses on health promotion. it was organized to educate the population to seek for oral health assistance in the first year of a baby’s life. later, parents and family were invited to attend speeches and individual dental visits to motivate them to learn the preventive measures and to carry out the preventive treatment in the clinic and at home. a great obstacle to the success of the oral health program for babies was the lack of commitment with the program4,13-14. compliance with the preventive/educational program is a determinant factor for success15. however, it is affected by factors such as level of satisfaction, motivation and results expected by the individuals2. in order to obtain effective health promotion and prevention of oral health diseases, it is essential to be assiduous to the program16. after 13 years of the implementation of the baby clinic in jacareí, brazil, a pilot study showed an important decrease in dental caries in the children who participated on the program ‘dentistry for babies’16. the goals of this study was to analyze and compare the experience of dental caries in 300 children aged 0 to 48 months, who were participants and non-participants of a preventive program ‘dentistry for babies’, as well as the correlation between assiduity of dental visits and experience of dental caries. based on the facts reported, the hypothesis of the present study was that participation in the preventive program ‘dentistry for babies’ positively affects the oral health of children between the ages of 0 to 48 months, and assiduity to dental visits positively impacts the oral health of the participants of this program. material and methods a cross-sectional observational study was carried out at a public health facility in the city of jacareí, sp, brazil. parent consent was obtained prior to each survey. the study was approved by the ethics in research committee of the são josé dos campos dental school – unesp (#088/2007). three hundred brazilian children participated of the study. all participants were in good health. they were randomly divided into two groups, g1, named ‘nonparticipant babies of the program’ (n=100), children that have never participated in preventive oral health program and g2, ‘babies of the program’ (n=200), children who received early oral health care in preventive program for babies. both groups (g1 and g2) were divided into two age groups between 0 and 24 months and between 25 and 48 months (figure 1). assiduity to the program was considered in which the families attended the program regularly, and were diligent in compliance with the instructions. so, babies assiduous to the program are the ones who attended the recalls and had no absences. non-assiduous babies had at least one absence. to analyze the influence of assiduity in dental caries fig. 1. flowchart of methodological sequence used for group division dentistry for babies: caries experience vs. assiduity in clinical care braz j oral sci. 11(4):486-491 prevalence and the dmft index (dmft refers to primary teeth: d = decayed, m = missing/extracted due to caries, f = filled, t = teeth) and modified dmft index (considering white spots) in the oral health program for babies, we analyzed all the babies of the program, aged between 0 and 48 months (g2, n=200) (figure 1). in addition, g2 non-assiduous (n=100) was subdivided according to the number of absences to the recalls: g2-a: 1 to 2 absences, g2-b: 3 to 5 absences and g2-c: 6 or more absences (table 3). children of group g2 received routine dental visits, in compliance with the program: orientation on oral hygiene and education about feeding habits, professional cleaning with pumice cup and topical application of 5% sodium fluoride varnish, according to the patient’s caries risk13. it is the protocol used in oral health program for babies. the eligibility criteria for this study were: inclusion criteria’! children (0 to 48 months) with no distinction of gender or ethnicity. group g1: those who never participated in any oral health programs and, for g2: those who participated in the program for at least three months and had at least the maxillary and mandibular central incisors present in the oral cavity. exclusion criteria’! presence of systemic disease, and/or syndromes leading to stained dental enamel. eligible children were randomly selected using a random method (face-crown). clinical examinations were carried out using a visual and tactile method with the aid of a blunt probe and dental mirror by a single trained and calibrated operator (k=0.98). children aged up to 24 months were examined on a ‘macri’ (a special stretcher used in children)16. children aged from 25 to 48 months were examined on a dental chair. all of the examinations were carried out with the aid of an artificial light reflector. if curative treatment was required, the treatment was carried out in the baby clinic or in the basic unities of health. the paired t-test, mann-whitney and chi-squared test at 5% significance level (bioestat software, 5.0 version, 2008, belém/pa, brazil) were used to analyze the data. results there was a statistical difference for the prevalence of dental caries in the population with and without early dental care (g1 and g2) for the 0-48 months age group (p<0.0001) (table 1). both groups presented statistically significant differences, and in both cases, the greatest values observed were for g1 [dmft g1 (3.45±3.84) and dmft g2 (0.66±1.57)]. g1 g2 g1 (0 to 24m) g2 (0 to 24 m) g1 (25 to 48 m) g2 (25 to 48 m) dental caries prevalence * (%) 73 22 60.24 11.34 90.67 32.03 dmft index (average) 3.45 0.66 1.65 0.32 5.92 0.9 modified dmft index (average) 6.53 2.01 4.31 1.25 9.59 2.72 groups table 1. data referent to dental caries prevalence and to dmft index and modified dmft in all groups * chi-squared test (p<0.05) it was also found a difference between the dmft index and modified dmft index, within each group, according to a chisquared test (g1 and g2, p<0.0001) (table 2 and figure 2). assiduity was significant for the g2 group (p<0.0001). data obtained in the present study confirmed the null hypothesis that assiduity to dental visits positively contributed to the oral health of individuals (figure 2). to analyze the influence of assiduity in dental caries prevalence and dmft index and modified dmft, babies who were between 0-48 months in age that were not assiduous to the program was further analyzed (g2 non-assiduous) (table 3). table 3 shows an increasing prevalence of dental caries in relation to assiduity factor. there is a direct relation for the number of absences and the prevalence. discussion in this study g1 and g2 corresponding to ages 0-48 months were subdivided into two groups according to age. the first group was 0-24 months in age while the second was 25-48 months. this division was based on the time of primary teeth appearance since there is a direct relationship between child’s age and exposure to cariogenic challenges11,16. in addition, there is a window for the initial acquisition of streptococcus mutans (between 19 and 31 months) termed the ‘window of infectivity’17. however vertical contamination (mother-child) and horizontal contamination (day care center) can occur earlier during the baby’s first year of life, suggesting that the period of occurrence of this ‘window’ may occur earlier for some children18-19. the presence of streptococcus mutants in children aged six month is related to habits of finger sucking, salivary contact, sharing utensils such as nipples and spoons, and by having their food tasted fig. 2. dental caries prevalence and dmft index of g2, age group between 0 and 48 months 488488488488488 dentistry for babies: caries experience vs. assiduity in clinical care braz j oral sci. 11(4):486-491 489489489489489 groups no. of individuals (n) dental caries prevalence (%) dmft index (average) modified dmft index(average) g2 assiduous 100 2.00 0.04 0.14 g2 non assiduous 100 42.00 1.29 3.89 table 2. dental caries prevalence (%), dmft index and modified dmft in g2 groups (assiduous and non assiduous) * chi-squared test (p<0.05) assiduity factor (nº absences) dmft index modified dmft index dental caries prevalence (%) assiduous non assiduous 0 0.04 0.14 2.00 group a 1 to 2 0.30 2.06 16.27 group b 3 to 5 5.15 1.89 56.52 group c 6 or more 5.72 2.72 81.81 table 3. assiduity factor in assiduous program (g2 assiduous, n=100) and non assiduous (g2 non assiduous, n= 100) participant babies and dmft index and modified dmft to dental caries prevalence and/or cooled (blowing it) previously by an adult20. after 13 months of life, children may have already been contaminated depending on habits and level of cariogenic contamination of the family18-19. in age groups between 0-48 months, 0-24 months and 25-48 months in g1, highest levels of caries prevalence, modified dmft and dmft index described in this study were found (table1). in these groups, the results corroborated with the literature5,7,21-22 and clearly showed the need for early oral treatment on health maintenance and disease prevention11,13. these findings are supported by a national survey carried out in 20036 which showed that in children between 18 and 36 months, the average is 1.0 carious teeth per child (without including white spot lesions). according to kidd and fejerskok9, it is incorrect not to consider white spot lesions as neglecting this type of lesion causes an underestimation of caries prevalence in some populations. white spots deriving from enamel demineralization are early signs of caries disease, and when not treated, may develop cavited lesions in a period of 6 months to 1 year23. it is important to emphasize that, in the present study, the positive influence of preventive actions implemented by the program were recorded when compared g2 to g1 (table 1). these results were similar to obtained by fracasso et al.24 who concluded that a program of preventive care for babies is more effective than spontaneous demand. the effectiveness of the program developed by the baby clinic of the state university of londrina was already proven11,22, and is supported by our findings. in the present study, the results showed a statistically significant manifestation of dental caries in g1, compared with g2. the prevalence was proportional to age, as seen between groups g1 0-24 months (60.24%, dmft 1.65) and g1 25-48 months (90.67%, dmft 5.92) and g2 0-24 months (11.34%, dmft 3.2) and g2 25-48 months (32.03%, dmft 0.98). these data also agreed with the literature7,21-22. these results confirm that the most susceptible age for dental caries occurrence is in the first three years of life2,7,21-22. the results suggest that prevention and control measures of dental caries must be reinforced in the program, mainly in the period between the ages of one to three. caries lesions of primary teeth are perceived as normal by many mothers, since they do not know this condition. however, dental caries is an infectious and transmissible but preventable disease, which may be controlled by avoiding contamination between mothers and their children18-19. parents’ previous education, is one of the most important factors for caries prevention in children2,23. access to these programs at the time of birth will minimize disparities between education levels, and efforts should be directed toward socioeconomic, behavioral and community determinants of oral health5. based on the differences between the experience of dental caries in this study, educational programs directed toward parents are needed to establish healthy habits to avoid contamination of the child’s oral cavity18-19. from the data collected, baby oral care is recommended to maintain his/her health, through education in public health programs as an effective way to establish healthy behavior25. the dentistry for babies is a practical, simple, comprehensive, low-cost and efficient possibility to make parents aware of the cariogenic risks in children at early ages. therefore parents must be instructed about the necessary dental care. indeed, it is known that the appropriate time to provide health education is during pregnancy5. in addition, tiano et al. 26 confirmed that the high prevalence of dental caries in children (d” 36 months of age) is associated with lower socio-economical levels, lower educational levels of mothers, increased time of breast feeding and delay of oral hygiene. the results of this study suggest that the presence of dental caries in some participants may explain that a few inadequate habits have persisted. it is assumed that this is due to a lack of compliance to the dental program. although total prevention of the disease was not reached, the reduction of its occurrence and severty constituted an important milestone, which corroborates the findings of fraiz and walter11. it is suggested that behavioral factors (difficulties in following orientation suggested by the program, parents’ awareness about the disease development) deserve to be reevaluated. these findings support the conclusion and confirm the dentistry for babies: caries experience vs. assiduity in clinical care 490490490490490 braz j oral sci. 11(4):486-491 hypothesis that assiduity factor positively influenced the dental caries prevalence. in fact, “babies assiduous to the program”, presented lower index of caries than ‘babies not assiduous to the program’ (table 3). in this sense it is essential, in every dental visit, to asses the risk of the child to develop dental caries and highlight to the parents the importance on the correct hygiene of the baby’s mouth. it is believed that it is important to carry out studies to evaluate the reasons why information provided to mothers was not sufficient to make them sensitive to the problem. low cultural and socioeconomic levels may be factors to be evaluated. to maximize interest, trust, and success among participating parents, educational and treatment programs must be tailored to the social and cultural norms within the community being served. recruiting mothers during pregnancy improves the likelihood that they will participate in the assessment program26. the findings of present study reinforce the importance of motivation. each dental visit should provide new information regarding the developmental phase of the child. it is essential to reinforce the need of follow-up visits in order to parents be aware of the importance of dental visits. therefore, establishing a relationship with the family, understanding and respecting their cultural aspects, the parents’ feelings, respecting the child and re-examining the mother-child relationship is important2. based on the results of the present study, it is suggested a follow up regarding the number of times a child aged from 0 to 48 months have dental visits during childhood. in the first year of a baby’s life, dental visits should be in the 1st month (if necessary evaluation of anomalies/ breastfeeding), 4th, 8th and 12th month. in the 2nd year of life, an additional four dental visits should occur (15th, 18th, 21st and 24th month). in the 3rd year of life, four dental visits are recommended. between 36 months and five years of age, two dental visits per year are suggested. the authors agree that in it of the follow up dental visits, the evaluation of a child’s risk category, needs assessments and oral examinations must be carried out26, and if necessary, an individualized preventive planning for the child should be done. in conclusion, the participation in the program had a positive influence on the oral health of babies. complete assiduity to the program resulted in the lowest rates and prevalence of dental caries. references 1. hallett kb, o’rourke pk. pattern and severity of early childhood caries. community dent oral epidemiol. 2006; 34: 25-35. 2. fraiz fc. supervisão de saúde bucal durante a infância. pesq bras odontoped clin integr. 2010; 10: 7-8. 3. american academy of pediatric dentistry. policy on early childhood caries (ecc): classifications, consequences, and preventive strategies. pediatric dent 2011; 33(sp issue): 47-9. 4. kowash mb, pinfield a, smith j, curzon mej. effectiveness on oral health of a long-term health education programme for mothers with young children. br dent j. 2000; 188: 201-5. 5. mouradian we, huebner ce, ramos-gomez f, slavkin hc. beyond access: the role of family and community in children’s oral health. j dent educ. 2007; 71: 619-31. 6. brazil. public health department. national coordination of oral health. sb brazil 2003 project. oral health conditions of the brazilian population 20022003. mainly results [text on the internet]. brasília; 2004 [cited 2011 jul 28]. available from: http: //bvsms.saude.gov.br/bvs/publicacoes / projeto_sb2004.pdf. 7. bönecker m, ardenghi tm, oliveira lb, sheiham a, marcenes w. trends in dental caries in 1-to 4-years-old children in a brazilian city between 1997 and 2008. int j paediatr dent. 2010; 20: 125-31. 8. walter lrf, nakama rk. prevention of dental caries in the first year of life. j dent res. 1994; 73: 773. 9. american academy of pediatric dentistry. guideline on periodicity of examination, preventive dental services, anticipatory guidance/ counseling, and oral treatment for infants, children, and adolescents. pediatric dent. 2009; 33: 102-8. 10. fraiz fc, walter lrf. study of the factors associated with dental caries in children who receive early dental care. pesqui odontol bras. 2001; 15: 201-7. 11. alves rx, fernandes gf, razzolini mtp, frazão p, marques raa, narvai pc. evolução do acesso à água fluoretada no estado de são paulo, brasil: dos anos 1950 à primeira década do século xxi. cad saúde pública. 2012; 28(sp issue): s69-s80. 12. ramos-gomez f, crystal yo, ng mw, tinanoff n, featherstone jd. caries risk assessment, prevention, and management in pediatric dental care. gen dent. 2010; 58: 505-17. 13. maltz m, jardim jj, alves ls. health promotion and dental caries. braz oral res. 2010; 24(sp. issue): 18-25. 14. hashim r, williams s, thomson wm. severe early childhood caries and behavioural risk indicators among young children in ajman, united arab emirates. eur arch paediatr dent. 2011; 12: 205-10. 15. lemos lvfm, barbosa dl, ramos cj, myaki si. influência do fator assiduidade na prevalência de cárie dentária em indivíduos atendidos na bebê clínica da prefeitura do município de jacareí, sp, brasil. pesq bras odontoped clin integr. 2008; 8: 203-7. 16. caufield pw, cutter gr, dasanayake ap. initial acquisition of mutans streptococci by infants: evidence of a discrete window of infectivity. j dent res. 1993; 72: 37-45. 17. karn ta, o’sullivan dm, tinanoff n. colonization of mutans streptococci in 8-to 15-month-old children. j public health dent. 1998; 58: 248-9. 18. mohan a, morse de, o’sullivan dm, tinanoff n. the relationship between bottle usage/content, age, and number of teeth with mutans streptococci colonization in 6-24-month-old children. community dent oral epidemiol. 1998; 26: 12-20. 19. wan akl, seow wk, purdie dm, bird ps, walsh lj, tudehope di. oral colonization of streptococcus mutans in six-month-old predentate infants. j dent res. 2001; 80: 2060-5. 20. mattos-granner ro, pupin-rontani rm, gavião mbd, bocatto harc. caries prevalence in 6-36-month-old brazilian children. community dent health. 1996; 13: 96-8. 21. morita mc, walter lrf, guillain m. prévalence de la carie dentaire chez des enfants brésiliens de 0 à 36 mois. j odonto-stomatol pediatr. 1993; 3: 19-28. 22. weinstein p, harrison r, benton t. motivating mothers to prevent caries: confirming the beneficial effect of counseling. j am dent assoc. 2006; 137: 789-93. 23. fracasso mlc, rios d, provenzano mga, goya sj. efficacy of an oral health promotion program for infants in the public sector. j appl oral sci. 2005; 13: 372-6. 24. pinto lmcp, walter lrf, percinoto c, dezan cc, lopes mb. dental caries experience in children attending an infant oral health program. braz j oral sci. 2010; 9: 345-50. dentistry for babies: caries experience vs. assiduity in clinical care 491491491491491 braz j oral sci. 11(4):486-491 25. tiano avp, moimaz sas, saliba o, saliba na. dental caries prevalence in children up to 36 months of age attending daycare centers in municipalities with different water fluoride content. j appl oral sci. 2009; 17: 39-44. 26. feldens ca, giugliani er, vigo á, vítolo mr. early feeding practices and severe early childhood caries in four-year-old children from southern brazil: a birth cohort study. caries res. 2010; 44: 445-52. dentistry for babies: caries experience vs. assiduity in clinical care oral sciences n3 175 braz j oral sci. 8(4):175-180 original article effect of light-activation on the antibacterial activity of dentin bonding agents cristiane duque1 ; thais de cássia negrini2 ; denise madalena palomari spolidorio3 ; josimeri hebling4 1 dds, phd, professor, school of dentistry, federal fluminense university (uff), nova friburgo, rio de janeiro, brazil. 2 dds, ms, doctorate student, depar tment of clinical analysis, school of pharmaceutical sciences, univ. estadual paulista unesp, araraquara, são paulo, brazil 3 dds, phd, professor, department of physiology and pathology, araraquara school of dentistry, univ. estadual paulista unesp, araraquara, são paulo, brazil 4 dds, phd, professor, department of orthodontics and pediatric dentistry, araraquara school of dentistry, univ. estadual paulista unesp, araraquara, são paulo, brazil correspondence to: josimeri hebling department of orthodontics and pediatric dentistry, univ. estadual paulista unesp rua humaitá, 1680, cep: 14801-903 araraquara, sp, brazil. e-mail: jhebling@foar.unesp.br abstract aim: this study evaluated the effect of light-activation on the antibacterial activity of dentin bonding systems. methods: inocula of streptococcus mutans and lactobacillus casei cultures were spread on the surface of bhi agar and the materials were applied and subjected or not to light-activation. zones of bacterial growth inhibition around the discs were measured. results: excite, single bond and the bond of clearfil se bond (se) and clearfil protect bond (cp) did not show any antibacterial activity. the strongest inhibitory activity was observed for the primers of cp and prompt (pr) against s. mutans and the primers of se and pb against l. casei. conclusion: light-activation significantly reduced or suppressed the antibacterial activity of the initially active uncured dentin bonding systems. keywords: dentin bonding systems, antibacterial activity, streptococcus mutans, lactobacillus casei introduction in spite of recent advances in restorative dentistry, the formation of microgaps at toothrestoration interfaces has been considered a significant problem related to the polymerization shrinkage of composite resins, especially at enamel-free margins1. the influx of bacteria and their byproducts through these microgaps may be involved in the development of secondary caries which has been considered the most frequent reason of failures in resin composite restorations2-3. in addition, after the removal of carious dentin, residual microorganisms can remain on the cavity floor or inside the dentinal tubules and cause pulp inflammation4. an impervious sealing of the cavity margins would be ideally desirable to prevent these bacteria from receiving nutrients and maintaining their metabolism and proliferation. composite-enamel bonding has been proven predictable, adequate and reliable by the application of the adhesive technique. however, since dentin adhesive systems are incapable of providing an unfailing and consistent dentinal sealing, despite showing high bond strengths1, it is enviable that these materials present shortand long-term antibacterial effect. it has been shown that some adhesive systems present inhibitory activity against different oral bacteria. such activity seems to be dependent on their acidity and chemical composition5-6. however, this activity can be suppressed after light-activation7. the incorporation of an antibacterial monomer such as 12-methacryloylixydodecylpyridinium bromide (mdpb) has been proven effective in granting antibacterial effect to a dentin primer before and after the curing process5,8-9. the ability of a mdpb-containing primer to penetrate artificially demineralized lesions and to kill bacteria in dentin, preventing the progression of root-surface caries has been recently demonstrated10-11. since tooth-restoration interfaces do not provide a hermetic sealing against the diffusion braz j oral sci. october/december 2009 volume 8, number 4 received for publication: may 27, 2009 accepted: january 15, 2010 braz j oral sci. 8(4):175-180 of microorganisms and/or their byproducts, it would be beneficial if the restorative materials could exert some antibacterial activity as long as the restoration is in function in the oral cavity. therefore, the aim of this study was to evaluate the effect of light-activation on the inhibitory activity of five contemporary dentin bonding systems against streptococcus mutans and lactobacillus casei. the null hypothesis was that light-activation does not affect the antibacterial performance of the dentin bonding systems. materials and methods the following dentin bonding systems were evaluated in this study: single bond (sb 3m espe), excite (ex vivadent) – two-step totaletch systems; clearfil se bond (se kuraray), clearfil protect bond (cp kuraray) – two-step self-etch systems; adper prompt l-pop (pr – 3m espe) – one-step self-etch system. their composition is shown in table 1. for clearfil se bond and protect bond, the two components (primer and bond) were tested separately. however, since the primer of clearfil protect bond does not contain photoinitiators, 10 µl of the bond were also applied. the antibacterial activity of each material was evaluated against streptoccocus mutans (atcc # 25175) and lactobacillus casei (atcc materi als manuf acturer composition ph batch no. s i n g l e b o n d ( s b ) 3 m e s p e , s t . p a u l , m n , u s a b is g m a , h e m a , d i m e t h a c r y l a te s , w a te r , e th a n o l, p o ly a l k e n o i c a c i d , a c i d c o p o l y m e r , p h o t o in it i a to r 5 . 0 1 1 0 5 e x c i t e ( e x ) v i v a d e n t e ts , s c h a a n , l i e c h t e n s te i n b i s g m a , h e m a , p h o s p h o r ic a c id a c r y l a te , g l y c e r i n d im e t h a c r y l a t e , i n i ti a t o r s , s t a b i l i z e r s , e th a n o l , h i g h d i s p e r s e d s i li c a 1 . 7 e 3 0 1 0 8 c l e a r f i l s e b o n d ( s e ) k u r a r a y m e d i c a l i n c . , o k a y a m a , j a p a n p r i m e r : m d p , h e m a , h y d r o p h i l i c d i m e th a c r y l a t e s , c a m p h o r q u i n o n e , n , n d i e th a n o l p to lu i d i n e , w a t e r . b o n d : m d p , h e m a , b i s g m a , h y d r o p h o b i c d i m e th a c r y l a t e s , c a m p h o r q u in o n e , n , n d i e t h a n o l p to lu id i n e , s i la n a te d c o l l o i d a l s i l i c a 1 . 9 2 . 8 0 0 4 1 6 a 0 0 5 5 5 a c l e a r fi l p r o t e c t b o n d ( c p ) k u r a r a y m e d i c a l i n c . , o k a y a m a , j a p a n p r i m e r : h e m a , h y d r o p h i li c d im e t h a c r y la te s , m d p , m d p b , w a t e r . b o n d : s i l i n a te d c o l l o i d a l s il i c a , s o d i u m fl u o r i d e , b i s g m a , h e m a , h y d r o p h i li c d i m e t h a c r y l a t e s , m d p , c a m p h o r q u in o n e , n , n d i e t h a n o l p to l u i d i n e . 1 . 9 2 . 8 0 0 0 0 2 a 0 0 0 0 5 a a d p e r p r o m p t l p o p ( p r ) 3 m e s p e , s t . p a u l , m n , u s a m e th a c r y la te d p h o s p h o r i c a c i d e s te r , w a t e r , p h o to i n i ti a t o r ( b i s 2 ,4 , 6 t r i m e th y lb e n z o y l ) p h e n y lp h o s p h i n e o x i d e ( b a p o ) , s t a b i l i z e r , f l u o r i d e c o m p l e x w i th z i n c , p a r a b e n e s 0 . 8 ( m i x e d ) 1 6 8 5 3 5 abbreviations: bis-gma: bisphenol a diglycidylmethacrylate; hema: 2-hydroxyethyl methacrylate; mdp: 10-methacryloyloxydecyl dihydrogen phosphate; mdpb: 12-methacryloyloxydodecylpyridinium bromide table 1. chemical composition of the dentin bonding systems used in this study. #193) using the agar plate diffusion test. chlorhexidine gluconate at 0.2% was used as the control group. indicator strains were grown in brain heart infusion broth (bhi™, difco laboratories, detroit, mi) for 48 h at 37oc, according to the physiological characteristics of each microorganism. the resultant inoculum was again placed in 5 ml bhi for 24 h at 37oc. the turbidity of the bacterial suspension was adjusted to a 0.5 mcfarland standard (approximately 108 cfu/ml, according to previous bacterial count). in petri dishes, base layers containing 15 ml of bhi agar were prepared and 250 µl of each inoculum was spread on their surface. sterilized paper discs with 5-mm diameter and 1.5-mm thickness were impregnated with 20 µl of each material and subjected to one of the following conditions of light-activation: 1 without light-activation, 2 direct light-activation (dla) after placement of the uncured specimens on the culture medium, or 3 indirect light-activation (ila) performed previously to the placement of the specimens on the culture medium. each material was irradiated for 10 seconds using a pre-calibrated (400 mw/cm2) light-activation unit (2500 curing light, 3m espe, st. paul, ct, usa). tests were performed six times for each material, bacteria and condition of light-activation. the plates were kept for 2 h at room temperature for diffusion of the materials and then incubated at 37o c for 24 hours. 176 effect of light-activation on the antibacterial activity of dentin bonding agents 177 braz j oral sci. 8(4):175-180 zones of bacterial growth inhibition around the discs were measured (in millimeters) using a digital caliper (mitutoyo, sp, brazil). measurements were taken at the greatest distance between two points at the outer limit of the inhibition halo formed around the discs. this measurement was repeated three times and the mean was computed for each disc. for statistical analysis of the results, kruskal-wallis and mann-whitney non-parametric tests were used at a significance level of 5%. results the median values and ranges of the inhibition zones for each material according to the bacteria strain and light-activation condition are shown in tables 2 and 3. among the tested materials, no inhibition zones were observed for sb, ex and the bond component of se and cp, for both bacteria regardless of light-activation. for the uncured dentin bonding agents, the greatest inhibitory effect against streptococcus mutans was observed for the primer component of cp and pr, with no statistical difference between them (p>0.05), followed by se primer. for lactobacillus casei, both primer components of cp and se showed the largest inhibitory zones, without light-activation material without direct light-activation (dla) indirect light-activation (ida) single bond (sb) 0 0 0 excite (ex) 0 0 0 primer 15.5 (15.0-17.5) a,a 15.5 (12.0-17.0) a,a 0 clearfil se bond (se) adhesive 0 0 0 primer 20.0 (17.0-23.5) b,a 18.8 (15.5-26.0) b,a 12.0 (8.0-14.5) a,b clearfil protect bond (cp) adhesive 0 0 0 adper prompt (pr) 18.0 (16.0-23.0) b,a 19.5 (14.0-20.0) b,a 0 clorhexidine (ch) 12.5 (12.0-14.0) c,a 12.5 (12.0-13.0) a,a 11.3 (10.0-13.0) a,a table 2. median and range of the inhibition zones (mm) obtained against streptococcus mutans. * values followed by the same lowercase letter in columns and uppercase letters in the rows are not statistically different (mann-whitney, p>0.05) l i gh ta ctiv a tio n m a te ria l w ith o u t d ire ct lig h t -a ct iva t io n (d l a ) i n d ire ct lig h t a c tiva t io n ( id a ) s in g le b o n d (s b ) 0 0 0 e xc ite (e x ) 0 0 0 p rim e r 2 3 . 5 ( 20 .0 -3 0 . 0 ) a ,a 2 3 . 5 (2 0 .0 2 7 .0 ) a , a 0 c le a r fil s e b o n d (s e ) a d h e sive 0 0 0 p rim e r 2 0 . 3 ( 19 .0 -2 1 . 5 ) a ,a 2 0 .8 (2 0 . 0 -2 4 . 0 ) a b ,a 1 5 . 5 ( 1 4 .5 -1 7 .0 ) b , bc le a rf il pro te ct b o n d (c p ) a d h e sive 0 0 0 a d p e r p ro m p t (p r ) 2 1 . 5 ( 16 .0 -2 5 . 0 ) a ,a 2 0 . 0 (1 5 .0 2 2 .0 ) b , a 9 . 3 ( 7 .5 -1 6 .0 ) a , b c lo r h e xid in e ( c h ) 1 5 . 5 ( 15 .0 -1 7 . 0 ) b ,a 1 6 . 0 ( 0 -0 ) c, a 1 6 . 0 ( 1 4 .0 -1 6 .0 ) b , a table 3. median and range of the inhibition zones (mm) obtained against lactobacillus casei. * values followed by the same lowercase letter in the columns and uppercase letters in the rows are not statistically different (mann-whitney, p>0.05) statistical difference (p>0.05), followed by pr. chlorhexidine gluconate at 0.2% presented the weakest antibacterial activity. direct light-activation of the uncured materials on the culture medium did not interfere with the immediate inhibitory activity of the dentin bonding systems against bacterial growth. however, when these materials were light-cured before being placed on the culture medium, the antibacterial activity was suppressed, except for the primer component of cp, for both bacteria and pr only against lactobacillus casei, although the inhibitory effect of these materials had been significantly reduced. discussion since most restorations are performed due to caries, microorganisms are present in the cavity walls, left behind intentionally (incomplete caries removal) or not. it is desirable that materials for direct application on the contaminated dentin present some antimicrobial activity to accelerate the inactivation of such microorganisms, mainly represented by s. mutans and lactobacillus. it is also desirable that this antimicrobial activity could last as long as the restoration is in function in the oral cavity to prevent or at least minimize the negative effect of light-activation on the antibacterial activity of dentin bonding agents (ila) 178 braz j oral sci. 8(4):175-180 effects of bacteria in the restorative material-dental structure interface. adhesive systems may act as a antimicrobial material due to some characteristics such as ph, ions release (e.g. fluoride)5-6 or the inclusion of specific monomers (mdpb)8,12. however, it is interesting to investigate if this antimicrobial property has only an immediate effect or a longterm action. a direct relationship between material acidity and growth inhibition of s. mutans has been reported13. however, some oral bacteria, including s. mutans and l. casei, produce organic-acids as the endproduct of glycolysis and are able to function at the low ph generated in the surrounding environment. as a consequence, acidogenicity and acidurance are the major physiological traits associated with the virulence of these microorganisms14. the sudden exposure of strains of oral streptococci and lactobacilli to ph values between 6.0 and 3.5 results in the induction of an acid tolerance response (atr) that enhances the survival of these strains at or below ph 3.514. the most rapid adaptative response is exhibited by s. mutans, involving a process that requires protein synthesis within 30 minutes of acid shock15. bacteria that are forewarned by mild acidification can prepare through the induction of a wide range of protective measures, including systems that alter cell membrane composition, extrude protons (h+), protect macromolecules, alter metabolic pathways and generate alkalis16. based on the above mentioned, it should be considered the possibility that the use of mildly acidic materials could, instead of having a detrimental effect on bacterial growth, be stimulating the generation of more resistant strains. complete lack of bacterial growth inhibitory activity was seen for sb. this system, in addition to the absence of a specific antibacterial component, has a ph of 5.0, which is not acidic enough to prevent s. mutans and l. casei from maintaining their metabolism. this observation is in line with the results reported by imazato et al.10 (2002), atac et al.17 (2001) and baseren et al.18. further studies are necessary to investigate the induction of an acid tolerance response by dentin bonding systems and other dental materials, which enhances the survival of some cariogenic bacteria. although presenting a low ph (1.7) due to the presence of phosphoric acid acrylates in its composition, ex did not show inhibitory activity against both bacteria strains for all light-curing conditions. based on this fact we believe that probably some physical characteristics of the material are important in modulating the antibacterial activity. although ex has alcohol as solvent, it is presented as a viscous fluid due to the inclusion of filler particles into its composition, which could have prevented this material from diffusing properly in the agar medium. as a consequence, the ph drop was not intense enough to reach the lethal ph values reported for s. mutans (3.5-3.0) or l. casei (2.3)15. despite the negative results obtained for excite in the present investigation, bacterial growth inhibition using this same filled dentin bonding agent was reported by çehreli et al.19 the bond components of se and cp are also highly viscous since they do not contain solvents. these substances also did not show inhibitory activity against the cariogenic bacteria used in this study. in addition, when compared to the corresponding primer components, they are considered less hydrophilic despite the presence of mdp and he ma which are an ionic and a neutral hydrophilic monomer, respectively. hence, viscosity and hydrophilicity are important characteristics of dentin adhesives concerning their inhibitory effect against bacterial growth6-7. despite the fact that sb and ex did not inhibit bacterial growth, it is relevant to remember that these two-step etch-and-rinse adhesive systems are used after the previous application of phosphoric acid as a way to superficially demineralize the substrate. it has been demonstrated that phosphoric acid has antibacterial activity20 and causes a significant immediate reduction in the number of microorganisms in carious dentin21. in the present study, pr and the primer component of se and cp had an acidic ph (<2.0) and were antibacterially active even against l. casei, which are more acid tolerant22. this means that the bacteria were not able to counter the negative impact of a sudden reduction in cytoplasmatic ph, even possessing constitutive and inducible strategies to survive and function in acid environments. these results may be explained by the entrance of high levels of h+ protons in the cell cytoplasm that result in loss of activity of the relatively acid-sensitive glycolytic enzymes (which severely affects the ability to produce atp) and structural damage to the cell membrane and macromolecules such as dna and proteins culminating with cell death16. the low ph of self-etch primers/adhesives could be the result of the presence of polymerizable acidic monomers which are esters originating from the reaction of a bivalent alcohol with methacrylic acid and phosphoric/ carboxylic acid23. se, cp and pr contain large amounts of acidic monomers22 that can interfere on the survival of oral bacteria and could be implicated in the antibacterial activity18. monomers, such as triethylene glycol dimethacrylate (tegdma) and 2-hydroxyethyl methacrylate (hema), are major components used in dentin bonding systems as amphiphilic substances to enhance chemical compatibility between the hydrophilic dentin and the hydrophobic composite resin base monomers24. however, hema, which is a neutral hydrophilic monomer included in the composition of all dentin bonding systems used in this study, does not present inhibitory activity against cariogenic bacteria such as s. mutans, s. sobrinus and l. acidophilus24. this fact is sustained by the results of this study since several of the tested adhesive materials, which contain hema in their composition, did not show any antibacterial activity. the resin monomer 12-methacryloyloxydodecylpyridinium bromide (mdpb) has been incorporated in the composition of some bonding systems to enhance the antibacterial effect of these materials. the bacterial growth inhibitory activity of this monomer has been demonstrated in several studies4-5,8-11,25-27. the progression of root-surface caries lesions in vitro was completely prevented after the application of a mdpb-containing primer through a combination of its antimicrobial activity and sealing of the demineralized dentin28 moreover, in vitro8 and in vivo12 studies have demonstrated that bonding resins containing mdpb could inhibit bacterial growth, without adversely affecting its bonding characteristics. mdpb is a compound of an antibacterial agent quaternary ammonium with a methacryloyl group, which copolymerizes with other monomers immobilizing the antibacterial agent into the polymer matrix9. among the bonding systems investigated in the present study, this antibacterial monomer is incorporated in the primer component of cp which was active against both s. mutans and l. casei. however, although the inhibitory activity of this particular bonding system was comparable to that of the uncured primer component of se and pr, it maintained this effect even after polymerization. the polymerization process negatively affects the antibacterial activity of resinous materials4. in this present study, antibacterial activity of dentin bonding systems was evaluated analyzing the influence of photo-activation on this property. the studies using agar effect of light-activation on the antibacterial activity of dentin bonding agents 179 braz j oral sci. 8(4):175-180 diffusion plate test, including recent publications25-26,29 that used the same materials tested in our study, clearfil protect bond (cp) and clearfil se (se), did not compare the inhibitory activity of adhesive systems against cariogenic bacteria, with or without photo-activation, only photo-actived. when cured specimens were placed on the agar medium, only the primer component of cp maintained its antibacterial activity against both s. mutans and l. casei, while pr prevented bacterial growth only of l. casei. however, this activity was significantly reduced when compared to the uncured specimens. a dentin primer incorporating mdpb could show antibacterial activity before and after curing against oral bacteria such as s. mutans5,9. the antibacterial agent is immobilized in the polymer network by polymerization of mdpb conferring to the materials which incorporate this monomer strong bacteriostatic and small bactericidal effect against cariogenic bacteria5. however, since the antibacterial agent is not released from the material, it has been demonstrated that the bacterial growth inhibitory activity of a mdpb-containing material after cured is exerted by direct contact with its surface4. this information is conflicting with the results of the present study, since a clear inhibition halo was seen for the primer component of cp, comparable to the inhibition zones observed for chlorhexidine. this indicates that the antibacterial effect exerted by this material occurred even without the direct contact of the microorganisms. as the amount of unpolymerized mdpb released from a cured bis-gma-based composite resin was confirmed to be less than the minimum inhibitory concentration (mic) value for s. mutans5, it could be speculate that maybe a different component other than the antibacterial agent was leached from the cured material. wang and spencer30 reported the ability of pr, which utilizes bisacylphosphine oxide (bapo) as a photoinitiator, in demineralizing the underlying dentin even after polymerized. based on the microraman spectroscopy results obtained in that study, it was suggested that due to the incomplete polymerization, the acidic characteristics of this self-etching system were retained in the water-presence environment. transferring this information to the present study, it may be speculated that unconverted acidic monomers present in the oxygen-inhibited layer after light-activation were capable of ionizing when in contact with the hydrophilic culture medium. it has been demonstrated that the layer of air-inhibited, poorly polymerized oligomers produced by pr is unusually thick31. this fact could explain the antibacterial effect exerted by pr even after cured, although in a smaller extent. the same was not noticed for the primer component of se which has a lower concentration of acidic monomers. the incorporation of fluorides in the composition of dentin bonding systems is an attempt to mainly augment the demineralization protective effect of these materials. in addition to enhancing remineralization in the cyclic demineralization-remineralization caries process, fluoride can act on cariogenic microorganisms by altering their physiological status. the three main microorganism-growth inhibitory mechanisms of fluoride are direct binding of f-/hf to enzymes and other bacterial proteins, binding of metal f complexes and action as a transmembrane proton carrier32. high concentrations of fluoride ranging from 0.16 to 0.3 mol/l can inhibit bacteria growth33. fluoride release from resinous materials has been proven inferior to that from glass-ionomer cements, and appears to play limited role in exhibiting substantial antibacterial effect4,24. in the present study, fluoride is incorporated in the composition of pr and the bond component of cp. despite that, the latter did not show any antibacterial activity, which is in line with the results reported by özer et al.4. since tooth-restoration interfaces do not provide a hermetic sealing against the diffusion of microorganisms and/or their byproducts, it would be beneficial if the restorative materials could exert some antibacterial activity as long as the restoration is in function in the oral cavity. unfortunately, the results of the present study demonstrated that adhesive systems do not fulfill that requirement since their antimicrobial activity is significantly suppressed or completely inhibited after curing. exception should be made to the mdpb-containing adhesive system clearfil protect bond. the positive results for this adhesive indicate that the inclusion of specific monomers in the composition of these materials is an interesting approach to lengthen the inhibitory effect against microorganisms that can infiltrate the tooth-restoration interface, preventing, for instance, the installation of recurrent caries lesions which represent the main factor responsible for the failure of resin restorations. in conclusion, the null hypothesis was rejected since lightactivation reduced significantly or suppressed the antibacterial activity of the initially active dentin bonding systems. moreover, the inhibitory effect on bacteria growth is material dependent. some investigators are using other methods to evaluate antibacterial activity of dental materials, such as direct contact test (dct) that could be important to confirm the results of agar diffusion test28. references 1. ferrari m, garcia-godoy f. sealing ability of new generation adhesiverestorative materials placed on vital teeth. am j dent. 2001; 15: 117-28. 2. hickel r, manhart j. longevity of restorations in posterior teeth and reasons for failure. j adhes dent. 2001; 3: 45-64. 3. mjör ia, moorhead je, dahl je. reasons for replacement of restorations in permanent teeth in general dental practice. int dent j. 2000; 50: 361-6. 4. özer f, karakaya s, ünlü n, kav k, imazato s. comparison of antibacterial activity of two dentin bonding systems using agar well technique and tooth cavity model. j dent. 2003; 31: 111-6. 5. imazato s, imai t, ebisu s. antibacterial activity of proprietary self-etching primers am j dent. 1998; 11: 106-8. 6. meiers jc, miller ga. antibacterial activity of dentin bonding systems, resinmodified glass ionomers, and polyacid-modified composite resins. oper dent. 1996; 21: 257-64. 7. prati c, fava f, di gioia d, selighini m, pashley dh. antibacterial effectiveness of dentin bonding systems. dent mater. 1993; 9: 338-43. 8. imazato s, kinomoto y, tarumi h, ebisu s, tay fr. antibacterial activity and bonding characteristics of an adhesive resin containing antibacterial monomer mdpb. dent mater. 2003; 19: 313-9. 9. imazato s, kinomoto y, tarumi h, torii m, russell rrb, mccabe jf. incorporation of antibacterial monomer mdpb into dentin primer. j dent res. 1997; 76: 768-72. 10. kuramoto a, imazato s, walls awg, ebisu s. inhibition of root caries progression by an antibacterial adhesive j dent res. 2005; 84: 89-93. 11. imazato s, kuramoto a, kaneko t, ebisu s, russell rrb. comparison of antibacterial activity of simplified adhesive systems. am j dent. 2002; 15: 356-60. 12. imazato s, tay fr, kaneshiro a, takahashi y, ebisu s. an in vivo evaluation of bonding ability of comprehensive antibacterial adhesive system incorporating mdpb. dent mater. 2006; 23: 170-6. 13. veermeersch g, leloup g, delmee m, vreven j. antibacterial activity of glassionomer cements, compomers and resin composites: relationship between acidity and material setting phase. j oral rehabil. 2005; 32: 368-74. 14. svensäter g, larsson u-b, greif ecg, cvitkovitch dg, hamilton ir. acid tolerance reponse and survival by oral bacteria. oral microbiol immunol. 1997; 12: 266-73. 15. hamilton ir, svensäter g. acid-regulated proteins induced by streptococcus mutans and other oral bacterial during acid shock. oral microbiol immunol. 1998; 13: 292-300. 16. cotter pd, colin hill. surviving the acid test: responses of gram-positive bacteria to low ph. microbiol molec biol rev. 2003; 67: 429-53. 17. atac as, çehreli zc, sener b. antibacterial activity of fifth-generation dentin bonding systems. j endod. 2001; 27: 730-3. 18. baseren m, yazici ar, ozalp m, dayangac b. antibacterial activity of different generation dentin-bonding systems. quintessence int. 2005; 36: 339-44. effect of light-activation on the antibacterial activity of dentin bonding agents braz j oral sci. 8(4):175-180 19. çehreli zc, stephan a, sener b. antimicrobial properties of self-etching primer-bonding system. oper dent. 2003; 28: 143-8. 20. settembrini l, boylan r, strassler h, scherer w. a comparison of antimicrobial activity of etchants used for a total etch technique. oper dent. 1997; 22: 84-8. 21. wicht mj, haak r, kneist s, noack mj. a triclosan-containing compomer reduces lactobacillus spp. predominant in advanced carious lesions. dent mater. 2005; 21: 831-6. 22. harper ds, loesche wj. growth and acid tolerance of human dental plaque bacteria. arch oral biol. 1984; 29: 843-8. 23. yoshida y, nagakane k, fukuda r, nakayama y, okazaki m, shintani h, et al. comparative study on adhesive performance of functional monomers. j dent res. 2004; 83: 454-8. 24. schmalz g, ergücü z, hiller ka. effect of dentin on the antibacterial activity of dentin bonding agents. j endod. 2004; 30: 352-8. 25. feuerstein o, matalon s, slutzky h, weiss ei. antibacterial properties of selfetching dental adhesive systems. j am dent assoc. 2007; 138: 349-54. 26. gondim jo, duque c, hebling j, giro em. influence of human dentine on the antibacterial activity of self-etching adhesive systems against cariogenic bacteria. j dent. 2008; 36: 241-8. 27. imazato s, kuramoto a, takahashi y, ebisu s, peters mc. in vitro antibacterial effects of the dentin primer of clearfil protect bond. dent mater. 2006; 22: 527-32. 28. slutzky h, matalon s, weiss ei. antibacterial surface properties of polymerized single-bottle bonding agents: part ii. quintessence int. 2004; 35: 275-9. 29. paradella tc, koga-ito cy, jorge ao. in vitro antibacterial activity of adhesive systems on streptococcus mutans. j adhes dent. 2009; 11: 95-9. 30. wang y, spencer p. continuing etching of an all-in-one adhesive in wet dentin tubules. j dent res. 2005; 84: 350-4. 31. pashley el, agee ka, pashley dh, tay fr. effects of one versus two applications of an unfilled, all-in-one adhesive on dentine bonding. j dent. 2002; 30: 83-90. 32. marquis re, clock sa, mota-meira m. fluoride and organic weak acids as modulators of microbial physiology. fems microbiol rev. 2003; 26: 493510. 33. bowden ghw. effects of fluoride on the microbial ecology of dental plaque. j dent res. 1990; 69(spec issue): 660-7. 180 effect of light-activation on the antibacterial activity of dentin bonding agents oral sciences n3 braz j oral sci.9(1):43-47 original article braz j oral sci. january/march 2010 volume 9, number 1 periodontal status of an indigenous population at the xingu reserve luana pinho de mesquita1, pablo natanael lemos2, lucila brandão hirooka1, selma aparecida chaves nunes3, soraya fernandes mestriner4, mario taba jr.5, wilson mestriner júnior6 1dds, undergraduate student, ribeirão preto dental school, university of são paulo, brazil 2dds, federal university of são paulo, project xingu, brazil 3dds, ms, graduate student, ribeirão preto dental school, university of são paulo, brazil 4dds, ms, phd, professor, department of pediatric clinic, preventive and community dentistry, ribeirão preto dental school, university of são paulo, brazil 5dds, ms, phd, associate professor, department of oral surgery and periodontology, ribeirão preto dental school, university of são paulo, brazil 6dds, ms, phd, associate professor, coordinator of project huka-katu-usp, department of pediatric clinic, preventive and community dentistry, ribeirão preto dental school, university of são paulo, brazil correspondence to: wilson mestriner júnior department de clínica infantil, odontologia preventiva e social, faculdade de odontologia de ribeirão preto, universidade de são paulo avenida do café s/n ribeirão preto, cep 14040-904 sp, brasil e-mail: mestri@forp.usp.br received for publication: september 09, 2009 accepted: march 15, 2010 abstract aim: to describe the prevalence of periodontal disease in the indigenous population of the middle and lower xingu compared to the non-indigenous brazilian population. methods: the evaluated indigenous population of the xingu reserve had oral and dental examinations performed by calibrated examiners assisted by oral health indigenous agents. from a sample of 2,299 indigenous subjects, epidemiological investigations were conducted in 1,911 individuals, using the methodology recommended by the world health organization. comparative periodontal data from the nonindigenous population were obtained from the brazilian ministry of health’s national epidemiological survey on oral health conditions (“sb brasil” project). the periodontal data of 508 indigenous individuals were presented by age intervals of 15-19 (n=219), 35-44 (n=128) and 65-74 (n=161) years. results: in the non-indigenous population, the periodontally healthy individuals were 46.2%, 21.9% and 7.9% for each age group, respectively, and in the xingu population they were 28.76%, 3.12% and 0% for each age group, respectively. the most frequent finding in the xingu population was the presence of calculus in 62.55% of younger people, 82.03% of adults and 45.45% of the elderly. the analysis by sextants demonstrated the presence of calculus in 25.04%, 44.79% and 18.18% for young, adults and elderly respectively. conclusions: despite the higher prevalence of calculus, in all age groups of the indigenous population, tooth loss does not seem to follow the same pattern observed in the non-indigenous brazilian population, suggesting differences in susceptibility, habits or conditions. keywords: epidemiology, periodontal disease, risk factor, indigenous health. introduction indigenous populations usually have primitive living conditions and limited health care due to their unprotected interaction with the predatory economic exploration. it has marked negatively the history of colonization of brazil and is an example of social exclusion of large portion of the brazilian population. the epidemiology has provided the basis for the study of the healthy and diseased populations and the main causal factors involved. furthermore, it has provided the basis for identifying the population segments with higher risks and evaluating the effectiveness of the services, programs and public health policies. arantes analyzing the oral health of indigenous populations affirmed that indigenous population studies are rare in brazil, and only transversal surveys with 44 braz j oral sci. 9(1):43-47 small samples are found. this means that there are not sufficient quantitative and qualitative information about the indigenous oral health in brazil1. recently, interdisciplinary studies, especially in public health, have been developed aiming at the social determination of disease. in oral health, there are epidemiological studies about caries and periodontal disease in different indigenous groups2. in 2000, the brazilian ministry of health initiated the discussion on the implementation of a project for the most comprehensive national epidemiological survey on oral health conditions to assess the main problems in different age groups including both urban and rural populations. this project, called as “sb brasil” project oral health conditions in the brazilian population, began in 1999 with the creation of the subcommittee responsible for developing and implementing the project3. among all types of diseases of oral diseases, periodontal disease stands out, especially when there is involvement with a concomitant systemic condition. even having a different time scale and other human, social, economic and environmental factors, current indigenous groups, once in contact with domestic companies, also experience socioeconomic and ecological changes with strong potential to change their oral health conditions4. in order to integrate the national oral health policy and national policy for indians health care, as well as to develop actions of oral health for the interest of the indigenous communities, the ribeirão preto dental school of the university of são paulo established a partnership approach with the federal university of são paulo (unifesp) and the ministry of health (funasa)5. this model of oral health care aimed at reducing the prevalence of oral diseases in the middle and lower xingu. as an initial stage of the process, we have oral health diagnosis that enables the development of strategies for implementation and evaluation of oral health actions, since groups may have different prevalence of periodontal disease and may be influenced by conditions, susceptibility or habits. the aim of this study was to evaluate the prevalence of periodontal disease in the indigenous population of the middle and lower xingu compared to the clinical periodontal findings in the brazilian population3. material and methods created by a federal government act in 1961, the xingu indigenous park is located in the north of the state of mato grosso, brazil, with a length of 2.8 million hectares and a perimeter of 920 km. located in an area of ecological transition, formed by tropical forests and savannah from north to south, the region presents great complexity in ecological, social and cultural questions. it is inhabited by 14 ethnic groups kuikuro, kalapalo, matipu, nahukuá, mehinaku, wavre, aweti, kamaiurá, trumai, yawalapiti, suiá, kaiabi, ikpeng and yudjá who speak different languages and are distributed in 49 villages and posts, with a total population of around 4,700 individuals6. a cross-sectional study was done with the entire indigenous population of the xingu indigenous park – covering an area of middle and lower xingu, of both sexes. from a sample of 2,299 subjects, epidemiological investigations were conducted in 1,911 individuals because of lack of authorization or failure of showing at the place of examination in time. the clinical examinations were performed in 35 villages by 5 calibrated examiners and assisted by oral health indigenous agents (aisb), with natural light, using oral mirror and world health organization (who) probe (hu-friedy, chicago, usa), using the methodology recommended by the who. data were processed in an electronic formed in the software sb data3. community periodontal index (cpi) was used considering the highest cpi score per individual, according to the age group. the periodontal data of 508 indigenous individuals were presented by age intervals of 15-19 (n=219), 35-44 (n=128) and 65-74 (n=161) years, as recommended by who7. comparative periodontal data from the non-indigenous population were obtained from the epidemiological data collected from the “sb brasil” project. the results of the calibration were measured by percentage of agreement and kappa coefficient for the periodontal condition in the age groups reached 0.82% of reliability8. in accordance with the guidelines of the brazilian national health council 196 resolution (1996)9, the research project was approved by the research ethics committee of the ribeirão preto dental school of the university of são paulo and by the brazilian research ethics commission (process 2008.1.166.587). the enrollment of the study subjects was authorized by the members of the indian council of xingu. after receiving explanations about the study design and warrants about their privacy and confidentiality of their information, all participants or their legal representatives signed an informed consent form. clinical data were grouped and stratified by age groups. comparisons among the groups and sextants were performed using anova and student’s t-test with significance level set at 5%. results all clinical periodontal data recorded from the middle and low xingu population are presented in the table 1 (individual means) and table 2 (sextants). in general, the non-indigenous population had better periodontal health than the middle and lower xingu population, as demonstrated by the clinical indicators gingivitis and calculus. in the sbbrasil project3, the percentage of periodontally healthy individuals the 15-19, 35-44 and 65-74-year-old age groups was, respectively, 46.2%, 21.9% and 7.9%, for the nonindigenous population, and 28.76%, 3.12% and 0.00%, respectively, for the middle and lower xingu population (table 1). the most frequent finding in the xingu was the presence of calculus with 62.55% of young people, 82.03% of adults and 45.45% of the elderly (p<0.05). the analysis by sextants demonstrated the presence of calculus in 25.04%, 44.79% and 18.18% for young, adults and elderly periodontal status of an indigenous population at the xingu reserve respectively (table 2). the prevalence of 4-5-mm-deep periodontal pockets, which indicates poor periodontal condition, was higher among the elderly, with 4.48% in the non-indigenous population and 9.09% in xingu population. discussion brazilian indigenous populations are experiencing a process of epidemiological transition marked by the coexistence of high rates of infectious and parasitic diseases, which remain as major causes of death, and the emergence 45 braz j oral sci.9(1):43-47 periodontal status of an indigenous population at the xingu reserve periodontal condition (cpi) healthy * p<0.001 bleeding p=0.06 calculus * p<0.001 periodontal pocket of 45 mm periodontal pocket of 6 e + mm excluded not examinated population n % n % n % n % n % n % n % xingu 4.03a 67.12 0.44 7.38 1.50a 25.04 0 0 0 0 0 0 0.03 0.4615 a 19 years brazil 4.15 69.17 0.87 14.5 0.88 14.67 0.02 0.33 0 0 0.08 1.33 0 0 xingu 1.12b 18.62 0.32 5. 34 2.69b 44.79 0.11 1.82 0 0 1.58 26.30 0 035 a 44 years brazil 1.87 31.11 0.53 8.82 1.31 21.8 0.15 2.5 0.04 0.67 2. .11 35 1 0 0 xingu 0.09c 1.52 0.06 1.01 1.09a 18.18 0.09 1.52 0 0 3.67 61.11 1.00 16.6765 a 74 years brazil 0.42 7 0.12 2 0.50 8.33 0.88 1. .33 0 03 0.50 4.85 80.84 0 0 table 2 average number of sextants and the respective shares for each cpi scores according to age and population. brazil (2004)4 and middle and low xingu, 2006. *anova p<0.05; different letters indicate statistically significant difference periodontal condition (cpi) healthy bleeding calculus periodontal pocket of 4 5 mm periodontal pocket of 6 e +mm excluded without information population n % n % n % n % n % n % n % xingu 63 28.76 18 8.21 137 62.55 0 0 0 0 1 0. . 45 0 0 15 a 19 years brazil 7772 46.18 3160 18.77 5622 33 4 0 0 0 0 53 0 31 0 0 xingu 4 3.12 4 3. . 12 105 82.03 9 7.03 0 0 6 4.68 0 035 a 44 years brazil 2947 21.94 1339 9.97 6279 46.76 1056 7.86 0 0 1525 11.35 0 0 xingu 0 0 0 0 15 45.45 3 9.09 0 0 11 33 33 4 12.1265 a 74 years brazil 422 7 89 175 3.27 1.163 21.74 238 4. . 48 99 1,85 3252 60.8 0 0 . table 1number and percentage of people, according to the highest degree of periodontal condition observed in the individual, age and population of study. brazil (2004)4 and middle and lower xingu, 2006. of chronic diseases later. the emergency of this group of diseases, which affect their morbidity and mortality profile, is directly related to a sociocultural and economic change that this population has been facing. it is relevant to highlight that there is a large number of excluded sextants, when one considers the percentage of poor scores or the mean of sextants affected in the 35-44 and 6574-year-old groups. on that last track, for example, over 80% of sextants examined were excluded in the non-indigenous population. in the xingu population over 60% of sextants examined were excluded, or did not have any tooth present or only one functional tooth. this fact reflects a low prevalence of severe periodontal disease in these age groups. as for any index that imposes categorical scales, in a biological process, there are limitations to be identified and recognized. many of the restrictions are outside the proposals for what was designed or by recent advances in understanding the process of periodontal disease. in this context, considering the need to overcome the limitations listed above and based on the comparability of data on national and international level, it is suggested the use of the cpi index, which is current recommended by the who to address the needs for treatment10. in relation to periodontal disease, it is interesting to note that despite the absence of a systematic and widespread oral hygiene routine among xavante indians in pimentel barbosa village, the cpitn index suggests low occurrence of periodontal disease in advanced stages 4. it was also observed by niswander (1967) in xavantes of the simões lopes village10. in pimentel barbosa village, gingivitis was more common among women, while calculus was more common among mean. however, the small sample sizes do not permit definitive conclusions about the differences between genders for these parameters. it is little expressive the occurrence of periodontal pockets and teeth with mobility in both sexes, even among the elderly. this profile may reflect the greater resistance of local irritating factors (plaque, calculus) and these results come from areas not investigated in this study, such as oral flora or possible beneficial effects of stimulating the tissue of support, promoted in part by diet, which consists of hard and fibrous foods4. the distribution of gingivitis in any population is important since current theory holds that the gingival lesion is the precursor of periodontitis11. historically, the brazilian indigenous societies have undergone accelerated process of sociocultural and economic changes, once in contact with the national society. these changes impact directly on the dietary habits and nutritional status of these groups, especially during the initial process of integration into the regional economic market. as a result, basic activities of subsistence tend to be partially or completely abandoned, leading to a reduction in variability and to increase the dependence on manufactured food and products12. furthermore, the literature have shown evidence that macronutrients and micronutrients may modulate proinflammatory and anti-inflammatory host responses, which, in turn, contribute to the individual baseline inflammatory status13 and also predispose to other systemic conditions14. modern concepts of periodontal disease etiology, pathogenesis and natural history clearly classify the disease into different types within individuals of varying risk, who all possess sites that are at variable risk from destructive disease15. since the creation of the xingu’s indigenous park in the early 1960s up to the mid-1980s, its inhabitants lived in a state of isolation from the outside world and counted on a strong and protective presence of the brazilian state. however, the presence and assistance from the brazilian state decreased significantly after that and the indians of the xingu’s indigenous park started taking account of the vulnerability of their territorial limits and sustainability of its natural resources. they have witnessed the spread of the fires originated in farms that had been illegally installed in their lands, the intermittent intrusion of hunters and fishermen, the silting of the rivers due to increasing deforestation, the risk of water contamination by the defensive use of chemical activities agricultural and intense illegal exploitation of timber resources. as a consequence of these events, it has been observed in recent years a change in the behavior of young people with regard to the rituals of incarceration and their sexuality. a major risk factor that has been presented in a more significant way is the increased mobility of the indians, and the exits to the city are much more frequent, which has collaborated to the change in dietary habits. the introduction of nursing bottle and powdered milk has generated changes in the pattern of morbidity. the first case of diabetes mellitus was diagnosed, and other cases of hypertension have emerged. children who have been bottle fed have presented problems more frequent such us diarrhea and/or malnutrition16. the model of health care that has been consolidating is a product of a long journey, in which the unifesp has collaborated since the beginning. it seeks to provide answers to key problems arising from social and historical process of contact between indians and settlers, prioritizing intersetcorial actions and the growing involvement of xingu’s population. it is guided to the construction of a new intercultural dialogue that enhances the citizenship in contrast to relations historically marked by paternalism and dependency. it is characterized by an integral assistance to the individual and the communities, and the adoption of standardized clinical protocols, health programs and team support by local actions have been its main instruments. there is no conflict between the causal and clinical approaches of biomedicine and traditional medicine. the actions of prevention and intervention on the health-disease dyad are organized according to the complexity of each case and the local reality. prevailing systems of different etiology, prevention and cure of diseases, there is a major concern of both the indians as the team health to maintain intercultural dialogue as a way to better understand the health needs of the xingu’s indians17. despite the higher prevalence of calculus, in all age groups of the indigenous population, tooth loss does not seem to follow the same pattern observed in the nonindigenous brazilian population, suggesting differences in susceptibility, habits or conditions. 46 braz j oral sci. 9(1):43-47 periodontal status of an indigenous population at the xingu reserve 47 funding: this work was supported by the national foundation of health (funasa) [partnership funasa-usp 1419/04]. acknowledgments the authors want to thank the support of the federal university of são paulo team and the other partners, specially the national foundation of health (funasa) and the oral health indians agents of xingu’s indigenous park. references 1. arantes r. saúde bucal dos povos indígenas no brasil: panorama atual e perspectivas in: coimbra ce, ventura rs, escobar al, org. epidemiologia e saúde dos povos indígenas no brasil. rio de janeiro: abrasco; 2003. p.49. 2. oliveira, ma. representações e práticas em saúde bucal entre os guaranis mbyá da aldeia boa vista no município de ubatuba, são paulo [master, s thesis]. são paulo: secretaria de estado da saúde; 2006. 84p. 3. brasil. ministério da saúde. secretaria de atenção à saúde. departamento de atenção básica. projeto sb brasil 2003: condições de saúde bucal da população brasileira 2002-2003: resultados principais. brasília: ministério da saúde; 2004. 68p. 4. arantes r, santos rv, coimbra jr cea. saúde bucal na população indígena xavante de pimentel barbosa, mato grosso, brasil. cad saúde pública. 2001; 17: 375-84. 5. mestriner jr w, mestriner sf, bulgarelli a, mishima sm. o desenvolvimento de competências em atenção primária à saúde: a experiência no “projeto huka-katu”. ciênc saúde coletiva [online]. 2009. available from: http:// www.abrasco.org.br/ cienciaesaudecoletiva/artigos/artigo_int.php ?id_artigo=3503. 6. instituto socioambiental (isa). available from: http://www.socioambiental.org/ prg/xng.shtm. accessed feb 12 2009. 7. world health organization. oral health surveys: basic methods. 4.ed. geneva: orh/epid; 1997. 8. world health organization. calibration of examiners for oral health epidemiological surveys. geneva: orh/epid; 1993. 9. brasil. ministério da saúde. conselho nacional de saúde. comissão nacional de ética em pesquisa. resoluções do conselho nacional de saúde sobre pesquisas envolvendo seres humanos 1999. available from: http:// www.datasus.gov.br/ conselho/comissoes/etica/resolucoes.htm 10. niswander jd. further studies on xavante indians vii. the oral status of the xavantes of simões lopes. am j hum genet. 1967; 19: 543-53. 11. lang np, schätzle ma, löe h. gingivitis as a risk factor in periodontal disease. j clin periodontol. 2009; 10: 3-8. 12. coimbra jr, carlos ea, santos rv. an assessment of the nutritional status in a context of socioeconomic change: the suruí indians from the state of rondônia, brazil. cad saúde pública. 1991; 7: 538-62. 13. chapple ilc. potential mechanisms underpinning the nutritional modulation of periodontal inflammation. j am dent assoc. 2009; 140: 178-84. 14. lamster ib, depaola dp, oppermann rv, papapanou pn, wilder rs. the relationship of periodontal disease to diseases and disorders at distant sites, j am dent assoc. 2008; 139: 1389-97. 15. chapple ilc. periodontal disease diagnosis: current status and future developments. j dent. 1997; 25: 3-15. 16. baruzzi rg, junqueira c. parque indígena do xingu: saúde, cultura e história. são paulo: terra virgem; 2005. 17. mestriner sf, lemos pn, hirooka lb, nunes sac, arantes r, mestriner jr w. o modelo de atenção a saúde bucal no médio e baixo xingu: parcerias, processos e perspectivas. ciênc saúde coletiva. 2008 (1651) [online]. 2008. available from: http://www.abrasco.org.br/cienciaesaudecoletiva/artigos/ artigo_int.php?id_artigo=1 braz j oral sci.9(1):43-47 periodontal status of an indigenous population at the xingu reserve original article braz j oral sci. april/june 2009 volume 8, number 2 comparison of cutaneous incisions to approach the infraorbital rim and orbital floor juliana de melo crosara1, everton luis santos da rosa2, micena roberta miranda alves e silva3 1 dds, oral and maxillofacial surgeon, private practice –uberlândia-(mg) brazil 2 dds, msc, oral and maxillofacial surgeon hospital de base -brasília–(df), brazil 3 dds, msc, anatomist, federal university of minas gerais (ufmg), belo horizonte (mg), brazil received for publication: april 8, 2009 accepted: june 22, 2009 correspondence to: everton luis santos da rosa sepn 504, edifício mariana, sala 10, subsolo – asa norte cep: 70730-523 – brasília (df), brazil e-mail: zazai547@terra.com.br abstract aim: the aim of this article was to retrospectively analyze and compare the esthetic outcomes achieved after the use of 20 subciliary incisions, 22 subtarsal incisions and 16 infraorbital incisions to approach the infraorbital rim and orbital floor in orbital fractures. methods: the sample consisted of 58 patients (37 males and 21 females) with orbital trauma (floor and infraorbital rim) treated with open reduction and internal rigid fixation in the department of oral and maxillofacial surgery at “hospital de base do distrito federal”, brazil, between september 1996 and august 2003. the following aspects were evaluated: (1) the average distance of the scars measured from the ciliary margin caudally, (2) the esthetic appearance of the scars, (3) chronic lid edema, (4) scleral show, (5) ectropion. results: subciliary incision demonstrated better surgical results when compared to the non-subciliary incisions. no statistically significant difference in chronic lid edema rates was found between the three groups of incisions (fisher, p>0.217 in all cases). there was no statistically significant difference in ectropion, scleral show and chronic edema rates between the three groups of incisions. conclusions: the subciliary and subtarsal incisions offer better esthetic results than the infraorbital incision, keywords: facial injuries, orbital fractures, eyelid, incisions. introduction several types of incisions have been used to approach the infraorbital rim and orbital floor, such as conjunctival, subciliary, subtarsal and infraorbital incisions, in addition to the endoscopically assisted intraoral approach1. the subciliary incision is placed about 2 mm caudal to the ciliary line. dissection may proceed in three different ways: the skin flap (in which the orbicularis muscle is divided at the level of the infraorbital rim), the non-stepped skin-muscle flap (in which the orbicularis muscle is divided at the same level of the skin incision) and the stepped skin-muscle flap (in which the orbicularis muscle is divided 2 to 3 mm below the level of the skin incision), keeping the pretarsal fibers of the orbicularis muscle attached to the tarsal plate, and assisting in the maintenance of the eyelid position and its contact with the globe; in other words, it presumably helps preventing ectropion and scleral show2. the subtarsal incision, also termed mid-lower eyelid incision, is placed in a natural subtarsal lid crease, about 5 to 7 mm below the ciliary margin. the dissection must be done in 89comparison of cutaneous incisions to approach the infraorbital rim and orbital floor braz j oral sci. 8(2): 88-91 a stepped skin-muscle flap fashion, dividing the orbicularis fibers a few millimeters below the level of the skin incision2. the infraorbital incision is typically placed in a skin crease at the junction of the thin eyelid skin and the thicker cheek skin, overlying the inferior orbital rim. the orbicularis muscle is divided at this same level3. although several studies have compared conjunctival and subciliary incisions, there are only two reports comparing cutaneous incisions. holtmann et al.3 compared the esthetic results achieved after the use of 45 subciliary incisions, 36 subtarsal incisions and 37 infraorbital incisions and found that the scar appearance was similar among the three groups. in fact, the only statistically significant difference mentioned in this study was the higher incidence of ectropion in the subciliary incision group. on the other hand, bähr et al.4 compared 16 subciliary incisions, 91 subtarsal incisions and 23 infraorbital incisions and found not only an incidence of ectropion, but also scleral show. chronic edema and noticeable scar rates were statistically different between groups. face to these controversies, the present study compared the cutaneous incisions with respect to the scar appearance and ectropion, scleral show and chronic edema rates, in order to determine which type of incision offers the best esthetic result. the aim of this article was to retrospectively analyze and compare cutaneous incisions to approach the infraorbital rim and orbital floor in orbital fractures in terms of scar appearance and ectropion, scleral show and chronic edema rates in order to determine which type of incision offers the best esthetic outcome. material and methods a retrospective study was developed with 58 patients (37 males and 21 female; age range at time of surgery: 17-50 years; mean age: 31 years) treated at the department of oral and maxillofacial surgery of hospital de base do distrito federal, brazil, between september 1996 and august 2003. the ethics committee of the university of brasília approved this study. only cases of fractures in which the orbital floor and infraorbital rim were surgically exposed for open reduction and internal rigid fixation were considered. all enrolled patients had at least six months of follow-up and the cases that presented postoperative infection were excluded of the study. the subciliary incision had been used 20 times, the subtarsal incision had been used 22 times, and the infraorbital incision had been used 16 times. the following aspects were evaluated: (1) the average distance of the scars measured from the ciliary margin caudally, (2) the esthetic appearance of the scars, (3) chronic lid edema, (4) scleral show, (5) ectropion. the scars were characterized as noticeable or unnoticeable. scleral show was considered when there was an increased visibility of the sclera below the lower margin of the iris compared to the opposite side. if the ciliary margin presented caudal draw and had lost contact with the bulbar conjunctiva, the change was characterized as ectropion. each patient was evaluated by two of the authors ( jmc and elsr) statistical analysis two categories were defined for each evaluated parameter (tables 1 and 2). tabulated data were analyzed by fisher’s exact test, with sigmastat® 3.1 statistical software (spss inc., chicago, il, usa). significance level was set at p<0.05. results the average distance between the scar and the ciliary margin was 2.4 mm for the subciliary incision group, 6.2 mm for the subtarsal incision group and 9.6 mm for the infraorbital incision group. regarding the scar appearance, both authors characterized all scars of the subciliary group as unnoticeable. in the subtarsal incision group, the rates of unnoticeable scars were 68 and 63%, while in the infraorbital incision group, the rates of unnoticeable scars were 31 and 19%. the higher unnoticeable rate for each surgical technique was used to test statistical differences among them. the subciliary incisions showed a higher rate of unnoticeable scars in comparison to the subtarsal incisions (fisher, p = 0.009) and infraorbital incisions (fisher, p < 0.001). the subtarsal incision demonstrated a tendency (border-line significance) to give better esthetic outcomes when compared to infraorbital incisions (chi-squared, p = 0.055). the subciliary incisions also demonstrated better surgical results when compared to the non-subciliary incisions (subtarsal and infraorbital incisions together) (fisher, p < 0.001). the rate of scleral shown was 25% in the subciliary incision group, 8% in the subtarsal incision group and 19% in the infraorbital incision group, though without statistical significance. no cases of ectropion were found in the subciliary incision group. the rate of ectropion in the subtarsal incision group was 17%, and in the infraorbital incision group was 6%. there were no statistically significant differences between the incision groups (fisher, p > 0.108 in all cases). scleral show was not different between type of incision total incisions unnoticeable scar (1)* % unnoticeable scar (2)** % subciliary 20 20 100 20 100 subtarsal 22 15 68 14 63 infraorbital 16 5 31 3 19 table 1. unnoticeable scar rates associated with each type of incision * unnoticeable scar rate according to the evaluation of author jmc; ** unnoticeable scar rate according to the evaluation of author elsr. type of incision total incisions ectropion % scleral show % chronic edema % subciliary 20 0 0 4 20 0 0 subtarsal 22 4 18 31 3 0 0 infraorbital 16 1 6 3 19 2 12,5 table 2. ectropion, scleral show and chronic edema rates associated with each type of incision 90 crosara jm, rosa els, silva mrma braz j oral sci. 8(2): 88-91 the incision groups (fisher, p > 0.681 in all cases). only two cases of chronic lid edema were observed, both in the infraorbital incision group (12.5%). in spite of this, no statistically significant differences in chronic lid edema rates were found between the three incision groups (fisher, p > 0.217 in all cases). the number of impairments associated with each type of incision is presented in table 2. discussion this study suggests that the average distance between the scar and the ciliary margin was 2.4 mm for the subciliary incision group, 6,2 mm for the subtarsal incision group and 9.6 mm for the infraorbital incision group. bähr et al.4 found an average distance of 1.5 mm for the subciliary incisions, 3.5 mm for the subtarsal incisions and 9,5 mm for the infraorbital incisions. regarding the scar appearance, the findings of the present study suggest that the rate of unnoticeable scars is significantly higher when higher incisions (subciliay and subtarsal) are used instead of the infraorbital incision. however, no statistically significant difference was observed with respect to the scar appearance between the subciliary and the subtarsal incisions groups. a retrospective study of 16 subciliary incisions, 91 subtarsal incisions and 23 infraorbital incisions4 also demonstrated that, considering the craniocaudal placement of the incisions, the esthetic appearance of the scar deteriorates from the subciliary margin downwards. the authors found that the rate of noticeable scars was 17.4% in the infraorbital incision group, 2.2% in the subtarsal incision group and 0% in the subciliary incision group. however, holtmann et al.3 did not find any statistically significant difference of imperceptible scars rate among the 45 subciliary incisions, 36 subtarsal incisions and 37 infraorbital incisions evaluated in their research. as far as it could be ascertained, there are no other studies comparing the esthetic appearance of the scars among these three types of incisions, but the superiority of the scar appearance when subciliary incisions are used is also corroborated by heckler et al.5, who analyzed 154 subciliary incisions and found that in 100% of the cases, the scar appearance was considered to be excellent. although there is not a consensus in the literature, it seems to be reasonable to consider that better esthetic results are reached, regarding the scar appearance, when higher incisions are used instead of the infraorbital incision. this establishment has its foundation on the anatomic bases of the eyelid region, and may be justified by the progressive increase of skin thickness and also by the progressive increase of excursion extent of the orbicularis’ fibers so far as the incision is more inferiorly placed4. the present study did not found any statistically significant difference of chronic lid edema rates among the three different types of incision, although the only two cases observed belonged to the infraorbital incision group. bähr et al.4 reported that the occurrence of chronic edema is an approach-dependent phenomenon based on the observation of a higher incidence of chronic lid edema as far as the incision was more inferiorly placed. a possible explanation for this edema distribution is that lower incisions interrupt larger lymphatic vessels, thus being responsible for a greater amount of chronic edema4. the present study did not found statistically significant difference of eyelid vertical shorting rates between the three groups. on the other hand, holtmann et al.3 observed a significantly higher rate of ectropion (42%) in the subcilliary incision group, and bähr et al.4 found that both ectropion and scleral show rates were significantly higher the more superiorly was the incision. to the best of our knowledge, there is no other study comparing these three types of incisions regarding the incidence of vertical shortening of the lower eyelid. the literature shows that the incidence of ectropion and scleral show after subciliary incisions largely varies from one study to another. netscher et al.6, in a prospective study of 20 subciliary incisions, found a scleral show rate of 70%. heckler et al.5 revised 154 subciliary incisions and did not find any cases of permanent ectropion or scleral show. conversely, smith and wood-smith7 reported that with an incision placed below the tarsus, avoiding the orbicularis’ pretasal fibers, a vertical shortening of the lid is less likely than with the subciliary incision, since the vertical shortening deformity seems to be causally related to the tonus of the orbicularis oculi muscle in its tarsal portion. however, still following this line of thought, the occurrence of ectropion and scleral show seems to be more related to the kind of flap rather than to the kind of incision, since subciliary and subtarsal incisions, when used in association with the stepped skin-muscle flap, also preserve the pretarsal portion of the orbicularis attached to the tarsal plate. moreover, there are many other factors that may contribute to the occurrence of vertical shortening, such as preexisting lid laxity, hypoplastic zygoma and relative globe protrusion8,9. in addition, there are many factors related to the surgical technique that may prevent the occurrence of ectropion and scleral show, such as avoidance of deep lateral dissection of the orbicularis muscle9, meticulous attention to hemostasis9, correct incision of the periosteum on the anterior surface of the rim, away from the orbital septum10; avoidance of wide dissection of the anterior periosteum11; use of the suspensory suture or frost suture12,13, which may reduce in 50% the incidence of ectropion, according to lacy and pospisil’s study12 and, of course, of main importance, the use of a stepped skin-muscle flap. certainly, no approach offers absolute protection against the possibility of eyelid retraction or ectropion; except for the conjunctival approach without lateral canthotomy, all of them leave an external scar that may be visible, even though each author advocates a different type of incision according to the results of their respective studies. according to bähr et al.4 the subtarsal incision combines the advantages of the infraorbital incision regarding the low risk of vertical shortening of the lower eyelid with the advantages of the subciliary incision regarding the formation of unnoticeable scars. holtmann et al.3 advocated the use of either the subtarsal or infraorbital incision instead of the subciliary incision, since their study demonstrated no statistically significant difference of unnoticeable scar rates between the three types of incision, but observed a significant higher rate of ectropion associated with the subciliary incision. an extensive liter91comparison of cutaneous incisions to approach the infraorbital rim and orbital floor braz j oral sci. 8(2): 88-91 ature review2 stated that the superiority of one type of incision over another has not been clearly demonstrated. ellis iii and zide13, supported by their vast clinical experience, have suggested the use of the subciliary incision because of the unnoticeable scar that generally results from it, associated with the stepped skin-muscle flap, in order to prevent the occurrence of ectropion. the present study suggests the superiority of the subciliary and subtarsal incisions, and advocates their use instead of the infraorbital incision, since the subciliary and subtarsal incisions showed rates of unnoticeable scars higher than the infraorbital incision and no statistically significant difference in ectropion, scleral show or chronic edema rates were found between the three types of incision. in order to prevent vertical shortening of the lower eyelid, the use of a stepped skin-muscle flap or any other preventive measure, such as the use of a frost suture, is suggested. references 1. chen ct, chen yr. endoscopically assisted repair of orbital floor fractures. plast reconstr surg. 2001;108:2011-8 2. rohrich rj, janis je, adams wp. subciliary versus subtarsal approaches to orbitozygomatic fractures. plast reconstr surg. 2003;111:1708-14 3. holtmann b, wray rc, little ag. a randomized comparison of four incisions for orbital fractures. plast reconstr surg. 1981;67:731-7 4. bähr w, bagambisa fb, schlegel g, schilli w. comparison of transcutaneous incisions used for exposure of the infraorbital rim and orbital floor: a retrospective study. plast reconstr surg. 1992;90:585-91 5. heckler fr, songcharoen s, sultani fa. subciliary incision and skin-muscle eyelid flap for orbital fractures. ann plast surg. 1983;10:309-13 6. netscher dt, patrinely jr, peltier m, polsen c, thornby j. transconjunctival versus transcutaneous lower eyelid blepharoplasty: a prospective study. plast reconstr surg. 1995;96:1053-60 7. converse jm, smith b, wood-smith d. orbital and naso-orbital fractures. in: converse, john marquis. reconstructive plastic surgery. 2. ed. filadélfia: saunders; 1972. p. 768-70. 8. werther jr. cutaneous approaches to the lower lid and orbit. j oral maxillofac surg. 1998;56:60-5 9. carraway jh, mellow cg. the prevention and treatment of lower lid ectropion following blepharoplasty. plast reconstr surg. 1990;85:971-81 10. manson pn, clifford cm, su ct, iliff nt, morgan r. mechanisms of global support and posttraumatic enophthalmos: i. the anatomy of the ligament sling and its relation to intra-muscular cone orbital fat. plast reconstr surg. 1986;77:193-202 11. appling wd, patrinely jr, salzer ta. transconjunctival approach vs subciliary skin-muscle flap approach for orbital fracture repair. arch otolaryngol head neck surg. 1993;119:1000-7 12. lacy mf, pospisil oa. lower blepharoplasty post-orbicularis approach to the orbit – a prospective study. br j oral maxillofac surg. 1987;25:398-401 13. ellis iii e, zide mf. periorbital approaches. in: ellis iii e, zide mf. surgical approaches to the facial skeleton. media: williams & wilkins; 1995. p. 9-37. oral sciences n3 original article braz j oral sci. july/september 2009 volume 8, number 3 effect of surface treatment and storage on the bond strength of different ceramic systems fernando carlos hueb de menezes1, gilberto antônio borges1, thiago assunção valentino1, maria angélica hueb de menezes oliveira1, cecília pedroso turssi1, lourenço correr-sobrinho2 1dds, msc, phd, associate professor, department of dental materials, university of uberaba, brazil 2 dds, msc, phd, full professor, dental materials area, piracicaba dental school, state university of campinas, brazil received for publication: april 13, 2009 accepted: october 1, 2009 correspondence to: fernando carlos hueb de menezes, departament of dental materials, faculty of dentistry of uberaba, university of uberaba, av. nenê sabino, 1801, uberaba, mg, 38055-500, brazil. phone: +55-34-3319-8884. e-mail: fernando.menezes@uniube.br abstract aim: the aim of this study was to evaluate the micro shear bond strength of different ceramic systems ips empress 2, cergogold, in-ceram alumina and cercon and a dual luting agent. methods: twelve specimens of each ceramic were fabricated and divided according different surface treatments: group 1: no additional treatment was applied to the ceramic surface; group 2: ceramics were etched with 9.5% hydrofluoric acid; group 3: specimens treated with airborne particle abrasion for each ceramic system in accordance with manufacturer’s instructions (n=20). the tests were performed after 24 h or after water storage for 6 months. data were then assessed statistically using the 3-way anova and the tukey’s test (p<0.05). results: for cergogold and ips empress 2 systems, the treatments performed with airborne particle abrasion and hydrofluoric acid showed no significant differences from each other, and both were superior to the groups without treatment. for cercon and in-ceram ceramics, no differences were found among the groups (p<0.05). when the surface was treated with hydrofluoric acid, the highest bond strength was found to ips empress 2 in the 6-month storage period (p<0.05). conclusion: lower bond strength values were only observed with ips empress 2 ceramic for the control group in the 6-month storage (p<0.05). keywords: ceramics, cementation, surface treatment, micro shear, bond strength. introduction ceramic has been used in dentistry as a restorative material since the 18th century. their clinical use has oscillated throughout history, being widely used in some periods and almost abandoned in others1. the association with a metallic substructure assured ceramic success, combining metal resistance with the excellent esthetics of the ceramic material2. however, dentistry has always sought for eliminating the use of metal to improve esthetics and this esthetic demand has stimulated the research with new ceramic systems and mechanisms to increase their attachment to the luting agent and tooth structure3. with the development of adhesive dentistry and improvement of the resins, the use of metal-free restorations has increased. in the recent past decades, the development of ceramic materials has increased significantly and their use has been more and more frequent. this material presents features, such as translucence, fluorescence, thermal-linear expansion coefficient close to dental structure, biological compatibility, chemical stability and compression and abrasion resistance. these properties enable it for being used as a substitute of natural tooth 3. the bonding between ceramics and dental structure is a relevant factor for the longevity of restorations and, depending on the ceramic material used, the cementation can be carried out by conventional or adhesive technique. either glass ionomer or zinc phosphate cements can be used for the conventional luting, although adequate frictional area is necessary to provide retention4. however, when retentive areas are small or even absent, friction may be inadequate and a resin based luting agent is needed3. when using adhesive technique, both dental and braz j oral sci. 8(3):119-123 ceramic surfaces must be treated. acid etching is performed on enamel and dentin, followed by hydrophilic adhesive application 4. polymerization of monomers at the demineralized regions provides a micromechanical bonding and the formation of the hybrid adhesive layer. likewise, inner surfaces of ceramic restorations must be susceptible to treatments that provide micromechanical retentions between the ceramic and the resin-based material. the frequently applied technique for feldspathic ceramic has been hydrofluoric acid etching, which provides irregular surface formation by removing the vitreous and crystalline phase5. another pre bonding treatment for ceramic surfaces is airborne aluminum oxide particle abrasion and the airborne abrasion changes the microstructure of ceramic, similarly. in addition, the use of chemical substances such as silane, allows for a chemical bonding between the inorganic phase of ceramic and the organic phase of the resin material, since ceramic presents components that are susceptible to be bonded to silane5. however, the surface of some ceramics is not likely to be modified by hydrofluoric acid etching. thus, restorations with usual etching procedures and silanization, used for silicate-based ceramic, are not efficient for all types of ceramic materials6. the treatment using airborne particle abrasion and silane agent application has shown to be effective for ceramic restorations reinforced with aluminum and zirconia7-8. the literature is controversial with relation to the type of treatment for the different metal-free ceramic systems3,7-9 in order to obtain an effective and long-term bonding with the luting agents used. furthermore, the hydrolytic degradation of adhesive systems should be considered. therefore, the present study aimed to evaluate the bond strength of different ceramic systems, according to the types of surface treatments applied and the time of restoration storage. the null hypothesis was that the ceramic surface inner treatments and the time of storage are not influence the bond strength of the restorations. material and methods compositions of the resin cement, the ceramic systems, and the porcelain primer are listed in table 1. twelve rectangular ceramic specimens were fabricated for each ceramic system in accordance with manufacturer’s instructions, as follows: a) ips empress 2 (ivoclar-vivadent, schaan, liechtenstein): wax patterns of 15 mm in length, 10 mm in width, and 1 mm in thickness were sprued and invested in ips empress 2 speed investment. the wax was eliminated in a burnout furnace (700-5p; edg equipments ltda, são carlos, brazil). following, the investment, plunger, and 2 ingots of ips empress 2 (shade 300) were transferred to a furnace (ep 500; ivoclar-vivadent) and automatically pressed in accordance with manufacture’s instructions. after cooling to room temperature, the specimens were divested with 50-mm glass beads at 2-bar pressure, ultrasonically cleaned in a special liquid (invex liquid; ivoclarvivadent), washed in running water, and dried. they were then treated with airborne particle abrasion with 100-mm aluminum oxide at 1bar pressure. b) cergogold (degussa dental, hanau, germany): wax patterns 15 mm in length, 10 mm in width, and 1 mm in thickness were invested (cergofit investment; degussa dental) and allowed to set. it was then placed in a burnout furnace to eliminate the wax. the cergogold ingots (shade a3) were pressed in an automatic press furnace (cerampress qex, ney dental inc, bloomfield, conn.). after cooling, the specimens were divested using 50-mm glass beads at 4bar pressure, followed by airborne particle abrasion with 100-mm aluminum oxide at 2-bar pressure, to remove the refractory material. the specimens were then treated with airborne abrasion with 100mm aluminum oxide at 1-bar pressure. c) in-ceram alumina: (vita zahnfabrik, seefeld, germany) a model of stainless steel (30 x 20 x 5 mm) with a rectangular central depression (15 x 10 x 1 mm) was obtained. an impression of this model was made with polyvinyl siloxane, and then duplicated in a plaster (special plaster ; vita zahnfabrik). the aluminum oxide powder was mixed with a special liquid as instructed by the manufacturer. the slurry mixture was then painted into the depression in the special plaster die and fired at 1120o c in the furnace (inceramat ii; vita zahnfabrik) for 10 h. glass infiltration was achieved by coating the aluminum oxide framework with glass powder (silicate-aluminum-lanthanum) mixed with distilled water, and fired for 4 h at 1100o c. the excess glass was removed by use of a fine-grained diamond (renfert, hilzingen, germany) followed by airborne particle-abraded with 100-mm aluminum oxide at a pressure of 3-bar. d) cercon (degudent): wax patterns of 15 mm in length, 10 mm in width, and 1 mm in thickness were obtained. the wax model was placed in the cercon brain unit for scanning. the confocal laser system measured the wax to an absolute precision of 10 mm and reproducibility of < 2 mm, scanning was accomplished in 4 min. a cercon base blank of presintered zirconia was milled and then sintered to fully dense structure in the cercon heat at 1350o c for 6 h. the specimens were finished by use of a fine-grained diamond (renfert, hilzingen, germany) under refrigeration, followed by airborne particleabrasion with 100-mm aluminum oxide at a pressure of 3-bar. the tablets of ceramic system were ground with al 2 o 3 sandpapers with decreasing granulation of 320, 400 and 600. they were then randomly divided into 3 groups, according to the surface treatments: group 1: no additional treatment was applied to the ceramic surface after the treatment with al 2 o 3 sandpapers; group 2: ceramics were etched with 9.5% hydrofluoric acid (utradent, south jordan, utah). the etching time protocol was considered for each ceramic type (20 seconds for ips empress 2, 60 seconds for cergogold, and 2 min for in-ceram alumina and cercon). after etching, ceramics were washed in tap water for 1 min and cleaned ultrasonically for 10 min; group 3: specimens treated with airborne particle abrasion according to described for each ceramics systems previously (1-bar for ips and cergogold; 3-bar for in-ceram and cercon). the distance of the tip from the ceramic surface was approximately 4 mm. these specimens were washed under running tap water for 1 minute, ultrasonically cleaned in a water bath for 10 min, and air-dried. for panavia f (kuraray co., osaka, japan) (table 1), the ceramic surface was first treated with a primer-acid mixture and silane agent (clearfil porcelain bond activator, kuraray co., osaka, japan) for 20s and then the adhesive system was applied (clearfil sebond; kuraray co.), being light-cured for 20s. the same amount of the cement universal and catalyst pastes were mixed for 10 s. the cement mixed was used to fill the plastic microtubule (tyg-030, small parts inc., miami lakes, fl) with inner diameter of 0.75 mm and 0.50 mm in height, which were bonded at ten different locations of the ceramic tablet surface (n=20) and light-cured for 40 s. test specimens were left at room temperature (23º ± 2º c) for 1 h before the plastic microtubule removal. later, half the specimens was stored in distilled water at 37º c during 24 h, and the other half stored in distilled water effect of surface treatment and storage on the bond strength of different ceramic systems120 braz j oral sci. 8(3):119-123 at 37º c for 6 months. after the storage, the test specimens were subjected to the microshearing bonding test. before the test, all of the specimens were verified under optical microscope at a 20x magnitude in order to check for bonding interface. microtubules showed interfacial opening formation; bubble inclusion or any other relevant defects were excluded from the study and replaced. the ceramic tablet was bonded to a metallic device treated with aluminum oxide sandblasting of 120 micrometers (especially developed) with cyanoacrylate-based adhesive (superbond, loctite, são paulo, brazil). this set was positioned into a test machine (emic dl3000, são josé dos pinhais, pr, brazil) so that the microshearing test could be performed. a thin steel blade was gently placed in the interface ceramic/resin. the load was applied upon each test specimen at a speed of 0.5 mm/min until the failure occurred. the test interface, the blade and the load cell were gently aligned to assure the test force direction. additionally, after the micro shear bond strength was carried out, samples were examined with a scanning electron microscope (leo 435 vp; cambridge, england) at 100x magnification to asses the fracture pattern and at 1000x to obtain better visualization of the most characteristic regions of the fracture patterns. the bonding interface fractures were ranked according to the predominance of the surface observed as mixed, cohesive in the resin cement, cohesive in the ceramic, and adhesive10. data were analyzed by a 3-way anova to check significant effect of factors under study and their interactions. tukey’s test was applied to run the post-hoc comparisons. significance level was set at a=0.05. results anova revealed interactions among ceramics x treatment (p<0.0001), ceramics x storage (p=0.0120), and treatment x storage (p=0.0156) were observed, followed by tukey’s test (table 2). the cergogold and ips empress 2 ceramics groups, that received either etched with hydrofluoric acid or airborne particle abrasion, presented higher bond strength values when compared to the control groups, for both immediate and after 6-month storage. with regard to cercon and in-ceram ceramics systems, for bond strength between surface treatments, no differences were found among the groups (figure 1). considering the treatment groups, no differences were found between the control and treated with airborne particle abrasion groups. however, when the surface was treated with hydrofluoric acid, the highest bond strength was found for ips empress 2 in the 6-month fig 2. sem of ips empress 2: cohesive fracture pattern (arrow) (aoriginal magnification x 100; boriginal magnification x 1000). fig 1. shear bond strength (mpa) of ceramics in accordance with surface treatment and storage time. fig 3. sem of cergogold: cohesive fracture pattern (arrow) (aoriginal magnification x 100; boriginal magnification x 1000). fig 4. sem of in-ceram alumina: adhesive fracture pattern (arrow) (aoriginal magnification x 100; boriginal magnification x 1000). fig 5. (a) sem of cercon: adhesive fracture pattern (arrow) (aoriginal magnification x 100; boriginal magnification x 1000). storage period. the cercon ceramic system surface treated with hydrofluoric acid showed lower bond strength values in comparison to the ips empress ceramic system for immediate storage group. for the 6-month storage period, decrease of the bond strength was only observed in ips empress 2 ceramic for the control group (figure1). predominance of the cohesive fracture pattern was observed in the sem analysis of the bonding interface, i.e., rupture of the bonding interface between the resin cement and the ceramic systems ips empress 2 (68%) and cergogold (73%) (figs. 2a, 2b, 3a and 3b). for in-ceram and cercon, adhesive fracture pattern was foremost (75% and 78% respectively) (figs. 4a, 4b, 5a and 5b). the sem analysis also showed that the treatment with airborne particle abrasion and hydrof luoric acid modified the surface topography, increasing the irregularities of cergogold and ips empress 2 ceramics. effect of surface treatment and storage on the bond strength of different ceramic systems 121 braz j oral sci. 8(3):119-123 discussion the null hypothesis that both the surface treatments and storage time do not interfere in the ceramic bond strength was rejected. the results showed that bond strength can be modified, influenced not only by surface treatment, but also by storage time and composition of the ceramic used. when a comparative study was carried out, it was observed that there are variables that can be used in the methodology in order to reach the objective formerly proposed in the investigation. nevertheless, it is sometimes difficult to compare results obtained due to the lack of standardization of the techniques and materials used in the literature. within the limits of the present study, the tested specimens were all treated and cemented by the same investigator in an attempt to standardize the procedures. thus, considering that the methodology used to standardize the size of tested specimens is quite sensitive, results obtained can provide important information for the application of the materials studied here. micro shear bond strength test was used in this present study, through which the surface area is significantly reduced; hence, it leads to a safer and more accurate assessment of the bonding interface11. although several investigations have used a myriad of bond strength methods, microshearing test has been found to be rather popular, providing satisfactory results12-13. it is believed that the tensions caused by the shearing test are important for the occurrence of restorative systems bonding failure14. in the present study, some bond strength values obtained with micro shear bond test showed to be comparable to the results obtained by shimada et al.15, demonstrating coherence in the methodology used. nevertheless, several difficulties were found, mainly during the insertion of the cement in the microtubules, as well as in controlling the overflow of the material on its base. bond failures between ceramics and resin cements may lead to premature loss of restorations. considering this statement, several papers have been conducted in order to investigate the relationship between ceramic materials and composites16-17. the cementation technique is vital for the success of ceramic restorations, which depending on the clinical situation and the composition of the ceramic; it is possible to use cements that do not bond micromechanically to the ceramic restoration and to the tooth, such as zinc phosphate cement. however, if preparations without frictional retention are used, a closer relationship among cement, restoration, and dental structure is necessary. this relationship is provided by the formation of a hybrid layer between the resinous material and dental structure by means of an adhesive bond system7. on the other hand, the ceramic material also needs to have micro-retention and an excellent relationship with the cementation material16. according to the results obtained in the present study, treatment with airborne particle abrasion with 50-mm aluminum oxide caused morphological change that favored the material retention in ips empress 2 and cergogold ceramic systems. results are in accordance with those found by kamada et al.18. treatment with 50-mm aluminum oxide airborne particle abrasion produced morphological conditions with surface aspect susceptible to mechanical retention through the formation of irregularities with uniform distribution in these ceramics systems8. however, for cercon and in-ceram systems, the airborne particle abrasion altered the surface but did not increase the bond strength. these results disagree with a previous study that used the same treatment19. the present investigation verified the efficiency of panavia f bonding agent in the adhesion of ceramics treated with airborne particle abrasion, which was already observed in a former study3. the hydrofluoric acid etching changed significantly the surface morphology of ips empress and cergogold ceramics. this process can be explained by the preferential reaction of the hydrofluoric acid with t h e s i l i c a p h a s e o f t h e f e l d s p a t h i c c e r a m i c t o f o r m h e x afluorosilicates8. these silicates are removed by rinsing with water. the final result is a surface rich in irregularities for micromechanical retention18. however, for cercon and in-ceram, the etching treatment did not interfere, probably due to the absence of glass phase (sio 2 ) in these systems, which did not influence the results of bond strength, as demonstrated by borges et al.19. although there are similarities between the results of bond strength, it is also important to observe the fracture pattern occurred. for cercon and in ceram, the pattern of fracture was predominantly adhesive, which features more weakness at the interface. as for the ips and cergogold, the predominant pattern was cohesive in the cement, suggesting a greater bonding strength at the interface and greater weakness in the bulk of the material. storage time can also be considered an influence factor in adhesive restorations bond strength. recent publications showed that the bonding interface degradation is an ordinary phenomenon when the clinician uses composite materials20-21. however, in the present study, lower bond strength values were observed only for ips empress 2 without treatment, after 6 months of storage. it could be suggested that the interface degradation during the storage is more intense with less surface area interaction between the luting agent and the ceramic19. due to the different ceramics available in the market, as well as to the different luting agents and surface treatments, the present paper aimed to reach the best relationship among these materials, minimizing failures of the restorative system. former and traditional procedures in cementation techniques have been questioned with the availability of state-of-the-art adhesive products, which provide promising perspectives, regarding that the professional has basic knowledge about them22. within the limitations of the present investigation, it may be concluded that the bond strength of cergogold and ips empress 2 ceramics was superior when the systems were treated with airborne particle abrasion with 50-mm aluminum oxide or etched with hydrofluoric acid. however these treatments did not influence in the bond strength of in-ceram and cercon systems. storage for 6 months only interfered on the ips empress 2 when this system was tested without treatment. the literature is controversial regarding to the durability of th e c erami c / re sin r e st orat iv e sy st e m cl i ni c al ly. th e s ur fa c e treatment of ceramics is dependent on their composition and dictates the relationship between the ceramic and the cement s y s t e m . t h e r e f o r e , t h e k n o w l e d g e o f t h e c e r a m i c m a t e r i a l composition is vital for the correct application of the ideal surface treatment and obtains an appropriate adhesive cementation to achieve a better longevity. acknowledgments this study was supported in part by fapemig – fundação de amparo a pesquisa de minas gerais and pape – programa de apoio à pesquisa – university of uberaba. effect of surface treatment and storage on the bond strength of different ceramic systems122 braz j oral sci. 8(3):119-123 references 1. jones dm. development of dental ceramics: an historical perspective. dent clin north am. 1985; 29: 621-44. 2. brecker sc. porcelain baked to gold – a new medium in prosthodontics. j prosth dent. 1956; 6: 801-10. 3. blatz mb, sadan a, kern m. resin-ceramic bonding: a review of the literature. prosthet dent. 2003; 89: 268-74. 4. fusayama t, nakamura m, kurosaki n, iwaku m. non-pressure adhesion of a new adhesive restorative resin. j dent res. 1979; 58: 1364-70. 5. holand w, schweiger m, frank m, rheinberger v. a comparison of the microstructure and properties of the ips empress 2 and the ips empress glass-ceramics. j biomed mater res. 2000; 53: 297-303. 6. atsu ss, kilicarslan ma, kucukesmen hc, aka ps. effect of zirconium-oxide ceramic surface treatments on the bond strength to adhesive resin. j prosthet dent. 2006; 95: 430-6. 7. blatz mb, oppes s, chiche g, holst s, sadan a. influence of cementation technique on fracture strength and leakage of alumina all-ceramic crowns after cyclic loading. quintessence int. 2008; 39: 23-32. 8. derand t, molin m, kleven e, haag p, karlsson s. bond strength of luting materials to ceramic crowns after different surface treatments. eur j prosthodont restor dent. 2008; 16: 35-8. 9. kato h, matsumura h, atsuta m. effect of etching and sandblasting on bond strength to sintered porcelain of unfilled resin. j oral rehabil. 2000; 27: 103-10. 10. clark dj, sheets cg, paquette jm. definitive diagnosis of early enamel and dentin cracks based microscopic evaluation. j esthet restor dent. 2003; 15:391-401. 11. shimada y, kikushima d, tagami j. microshear bond strength of resin-bonding systems to cervical enamel. am j dent. 2002; 15: 373-7. 12. chung ch, brendlinger ej, brendlinger dl, bernal v, mante fk. shear bond strengths of two resin-modified glass ionomer cements to porcelain. am j orthod dentofacial orthop. 1999; 115: 533-5. 13. ozden an, akaltan f, can g. effect of surface treatments of porcelain on the shear bond strength of applied dual-cured cement. j prosthet dent. 1994; 72: 85-8. 14. söderholm kj. correlation of in vivo and in vitro performance of adhesive restorative materials: a report of the asc md156 task group on test methods for the adhesion of restorative materials. dent mater. 1991; 7: 74-83. 15. shimada y, yamaguchi s, tagami j. micro-shear bond strength of dual-cured resin cement to glass ceramics. dent mater. 2002; 18: 380-8. 16. barghi n, berry t, chung k. effects of timing and heat treatment of silanated porcelain on the bond strength. oral rehabil. 2000; 27: 407-12. 17. chen jh, matsumura h, atsuta m. effect of different etching periods on the bond strength of a composite resin to a machinable porcelain. j dent. 1998; 26: 53-8. 18. kamada k, yoshida k, atsuta m. effect of ceramic surface treatments on the bond of four resin luting agents to a ceramic material. j prosthet dent. 1998; 79: 508-13. 19. borges ga, spohr am, correr sobrinho l, de goes mf, chan d. effect of etching and airborne particle abrasion on the microstructure of different dental ceramics. j prosthet dent. 2003; 89: 479-88. 20. osorio r, pisani-proenca j, erhardt mc, osorio e, aguilera fs, tay fr et al. resistance of ten contemporary adhesives to resin-dentine bond degradation. j dent. 2008; 36: 163-9. 21. garcía-godoy f, tay fr, pashley dh, feilzer a, tjäderhane l, pashley el. degradation of resin-bonded human dentin after 3 years of storage. am j dent. 2007; 20: 109-13. 22. pazin mc, moraes rr, gonçalves ls, borges ga, sinhoreti ma, correr-sobrinho l. effects of ceramic thickness and curing unit on light transmission through leucite-reinforced material and polymerization of dual-cured luting agent. j oral sci. 2008; 50: 131-6. effect of surface treatment and storage on the bond strength of different ceramic systems 123 braz j oral sci. 8(3):119-123 oral sciences n3 clinical case braz j oral sci. july/september 2009 volume 8, number 3 complication of local dental anesthesia a broken needle in the pterygomandibular space bruno ramos chrcanovic1, djalma cordeiro menezes junior1, antônio luis neto custódio2 1 dds, department of oral and maxillofacial surgery, pontifical catholic university of minas gerais, brazil 2dds, ms, phd, oral and maxillofacial surgeon, depar tment of oral and maxillofacial surgery, pontifical catholic university of minas gerais, brazil received for publication: august 19, 2009 accepted: november 11, 2009 correspondence to: bruno ramos chrcanovic av. raja gabaglia, 1000/1209 – gutierrez – belo horizonte, mg – cep 30441-070 – brazil. phone: +55 31 91625090 , +55 31 32920997. e-mail: brunochrcanovic@hotmail.com abstract broken dental needles are a rare event. they are difficult to find and remove. this paper reports a case of broken needle in the pterygomandibular space. the needle was localized using plain radiograph and removed under local anesthesia and venous sedation. preventing needle breakage is important, as it can be a traumatic experience for the patient. practitioners should routinely inspect dental needles before administering injections and minimize the number of repeated injections using the same needle. a meticulous injection technique is imperative. if breakage occurs, immediate referral to an oral and maxillofacial surgeon is necessary. it is strongly recommended that only an oral and maxillofacial surgeon may indicate and/or perform surgery to remove the broken needle. keywords: local anesthesia, complications, broken dental needle, pterygomandibular space. introduction metal instruments used in clinical practice may be subjected to considerable wear and may subsequently fracture. the removal of the broken instrument may be simple if the fractured part remains accessible, but this is not always the case. before the advent of the disposable spiral-constructed dental needle, breakage of needles during the administration of local anesthetics was not an uncommon event. since the introduction of disposable dental needles in the early 1960s, the frequency of needle breakage has been minimal, compared with the first quarter of this century during which time rigid, inflexible, nondisposable needles were used. blum1 reported 100 cases of broken needles over a 14 year period from 1914 to 1928. the occurrence of needle breakage has decreased as a result of the development of stainless, flexible alloys used in modern, disposable dental needles2. scientific advances in metallurgy and manufacturing, as well as better training of dental practitioners in how to administer anesthetic also have reduced breakage frequency3. this article reports a case of broken needle in the pterygomandibular space and discusses the modalities of localization and preventive measures. clinical case an 18-year-old male patient was referred by his general dentist to the department of oral and maxillofacial surgery of the pontifical catholic university of minas gerais for evaluation and removal of a fragment of 30-gauge long needle that broke during an inferior alveolar nerve block. the patient had moved his head quickly because of the sensation of shock when receiving the injection. the fragment disappeared into the tissues and the dental practitioner was unable to retrieve it. the patient was healthy with no significant medical history. physical examination showed a moderate trismus. the patient complained of localized pain in the left pterygomandibular braz j oral sci. 8(3):159-162 region and the ability to feel the broken needle during mandibular movements. clinical intraoral examination revealed bruising in the right pterygomandibular region, but no bleeding or visible punctures wounds. a lateral skull radiograph was made (figure 1). the lateral skull view helped us determine the needle’s anterior-posterior location and also showed us its vertical relation to the teeth. it also showed that the needle was located just below the mandibular lingula. under local anesthesia and venous sedation, a vertical incision parallel and medial to the anterior border of the ramus was made and a periosteal elevator was used to reflect the masseter and medial pterygoid musculature. during blunt dissection of the medial pterygoid muscle, the broken needle was removed (figure 2 and 3). the postoperative period was uneventful with no evidence of trismus. figure 1. lateral skull radiograph showing the needle under the lingula. figure 2. retrieval of the broken needle. figure 3. retrieved broken needle. discussion needle breakage during administration of a nerve block was a complication more frequent prior to the 1960s. this was thought to be at least partly due to the use of more rigid, non disposable needles which were subjected to repeated sterilization cycles, with attendant alterations to their physical properties during this time4. needle fracture is now a rare complication. this is probably due to the use of modern flexible alloys in their fabrication. when fracture occurs it is usually due to the inappropriate use of short, narrow gauge needles inserted to the hub or bending before use as well as poor operator technique. any sudden or unexpected movement by the patient and redirection of the needle against tissue resistance during administration of the injection are considered to be contributing factors to needle breakage3. the most common site for loss of a fractured needle is the pterygomandibular space during an inferior dental nerve block5-6. there is a degree of controversy over management of broken dental needles. different authors7-9have mentioned that that removal is not necessary unless the patient developed symptoms such as pain, infection, numbness and swelling. no author has cited the possibility of formation of fibrosis in the tissues around the needle over time. retrieval of the needle in itself can lead to neurological and tissue damage during removal9. on the other hand, many other authors6,10-14 suggested removal, fearing that the needle might migrate toward large blood vessels in the head and neck. we agree with these authors, which state that because of the fear of needle migration and also because of the medico-legal considerations, removal of the broken needle is important. it is obvious that presence of active symptoms such as pain, trismus and infection that are not alleviated by standard treatments necessitate needle removal15. there are in the literature no contraindications to perform the surgical removal under local anesthesia. a further argument in favor of removal is the possible psychological trauma to the patient that may result from the knowledge that a needle has been retained “somewhere in the throat”16. every effort should be made to retrieve the needle immediately, if the tip is visible, using fine haemostatic tool4. prompt retrieval is advocated to minimize symptoms of pain, dysphagia, trismus and to prevent migration of the needle and potential damage to vital complication of local dental anesthesia a broken needle in the pterygomandibular space160 braz j oral sci. 8(3):159-162 structure6,9. if its tip is not visible, attempts by the general practitioner in order to recover it should be discouraged, because the needle could be pushed deeper into the tissue. it is strongly recommended that only an oral and maxillofacial surgeon may indicate and/or perform surgery to remove the broken needle. the most important aspect of the surgical technique is accurate localization of the needle. determining the position of a broken needle in the pterygomandibular space in an anaesthetized patient is a difficult task. several different techniques have been described including the use of metal detectors. these have been used for localization of metallic foreign bodies in the floor of the mouth17 and have been successfully used to find a broken needle in the pterygomandibular space18, although crouse3 had a long time ago employed a metal detector but found no response on both ferrous and non-ferrous settings of the instrument and thus found it of no value. metal detectors though are not readily available and the probe must be small enough to use in the mouth17,19. the more common method is the use of intraoperative radiographs with localizing needles20-21. intraoperative radiographs though are difficult to obtain and prolong the procedure14. plain radiographs are useful in confirming the presence, dimensions and approximate position of the needle. they are, however, unable to provide the accurate position of the needle and its relationship to adjacent structures. the incision and site of exploration can be determined from the information available on the ct scan. the use of 3d reformatting is especially useful. if dental restorations are present, beam-hardening artifacts can interfere with the quality of the image obtained4. the lateral skull view, used in this case, helps to determine the needle’s anterior-posterior location and also shows us its vertical relation to the teeth22, although not precise in providing accurate position of the needle. despite the availability and ease of preoperative radiograph exams, they are not sufficiently accurate because of the time period between obtaining the images and performing the surgery15. thompson et al.14 used a simple stereotactic technique using an image intensifier and two 19-gauge venepuncture needles under general anesthesia. they stated that image intensifiers are usually readily available in theatres as they required for orthopedic and urological surgery. nezafati and shahi15 used c-arm digital fluoroscopy. the rapid taking and immediate reviewing of images at various angles without disturbing the reference needle, reducing radiation dose by using intensifiers and excellent image quality are the advantages of this technique, according to nezafati and shahi15. however, as plain radiographs, intensifiers only shows two-dimensional images, and are unable to provide the accurate position of the needle. the radiation dose increases, as one must review the image in three directions (antero-posterior, latero-lateral, cranio-caudal) in order to locate the needle precisely in the space. the use of stents has been described. intra-operative x-rays with positioning stents are often time consuming and the discrimination between small changes in position between the two (three) localizing needles can often be poor21. magnets are no longer used, because the manufacturing process has eliminated ferrous compounds4. most reports have suggested the use of a vertical mucosal incision often on the medial aspect of the mandible in the area penetrated by the needle, followed by blunt supra-periosteal dissection to identify the needle6,9,13,23-24. but an initial subperiosteal dissection can help identify bony landmarks (lingula), which can be used as reference during exploration and also provides greater protection to the inferior alveolar and lingual nerves4. the inferior alveolar and lingual nerves can be injured in case of extensive dissection in a surgical retrieval of broken needles in the pterygomandibular space. the extensive dissection can also cause local pain and trismus for a considerable period. kennett et al.16 described postoperatively considerable swelling and trismus but the patient was fit for discharge on the third postoperative day. the swelling subsided over the next week, but normal jaw opening was not present until three weeks postoperatively. other authors reported uneventful postoperative recovery4,6,13-15,17, as also occurred in the present case. examination of the needle before administering the injection should be standard practice among dental practitioners. if a practitioner notes any needle defects, he or she should discard the needle6. repeated injections with the same needle should be minimized, as needle fragility and susceptibility to breakage with repeated injections has been documented12,15,21. needles should not be bent. using fine and short needles for inferior alveolar nerve block demands the insertion of the needle up to the hub. long needles should be used. it is recommended to not penetrate the needle to its hub, as this is where the needle is the weakest and sight of the needle can be lost when it is buried to the hub6,16. pietruszka et al.20 suggested that a 30-gauge needle should not be used for nerve blocking injections because it is the most narrow, least rigid needle available and also the most susceptible to breakage. though most dentists use a 27 gauge (35 mm long) needle for administration of an inferior alveolar nerve block in an adult, there is occasionally a perception that the use of a thinner needle (30 gauge) is associated with less discomfort4. it has however, been shown that there is little difference in the pain perception between the use of 27 and 30 gauge needles25. flanagan et al.26 also showed that there is no statistically significant difference in perceived injection pain based on needle gauge when analyzed for injection location (mandibular, maxillary posterior, maxillary anterior, and palatal), injection side, patient gender, treating dentist, or overall. these results indicate that when it comes to injection pain and needle gauge, size does not matter. safer et al.27 assessed and compared the chemical composition, microstructure and compositional homogeneity in the alloy used by different manufacturers to make dental anesthesia needles. they found that aluminum and niobium were present in the alloy from which the needles least likely to break had been manufactured, but not in the alloy from which the most brittle needles were made. the concentrations of chromium and nickel were higher in the better quality needles. greater needle deflection may contribute to needle breakage. previous studies of dental needle penetration in vitro have demonstrated that the amount of deflection was inversely proportional to needle gauge; that is, thicker needles deflect less than thinner ones28. the study of jeske and boshart29, however, has demonstrated that a 28-gauge needle with a specially modified, nondeflecting tip produces less needle deflection than a 25-gauge needle with a conventional tip. this would suggest that a thicker needle per se will not minimize deflection (at least in the range of needle gauges used in dentistry), and that tip design is perhaps more important than gauge in reducing deflection. in this connection, another study 30 demonstrated significantly less force required to insert a smaller gauge needle. this improved penetrability may contribute to decreased deflection if it allows the needle to penetrate through, rather than deflect around, various tissues encountered during a dental injection. the use of a complication of local dental anesthesia a broken needle in the pterygomandibular space 161 braz j oral sci. 8(3):159-162 bidirectional rotation insertion technique minimized needle deflection, resulting in a straighter tracking path for 30-, 27-, and 25-gauge dental needles, in 3 different tissue-like substances tested in the study of hochman and friedman31. comparing the resistance to penetration of two types of disposable injection needles, lehtinen30 showed that the 30-gauge needle required significantly less force (69 mn) than the 27-gauge needle (139 mn). being required significantly less force to penetrate the tissue, the 30-gauge needle is less prone to breakage. a further consideration is the use of adequate preoperative sedation in the nervous patient, thereby minimizing the possibility of his sudden movement during the injection16. despite the rarity of this complication and the improvement in needles, there is no room for complacency and a meticulous injection technique is imperative16. surgeons performing the removal must not only be skilled and well-trained, but be familiar with the anatomy involved. immediate referral to an oral and maxillofacial surgeon is necessary, as is thorough and complete documentation of the events that led to the breakage. in conclusion, preventing needle breakage is important, as it can be a traumatic experience for the patient. practitioners should routinely inspect dental needles before administering injections and minimize the number of repeated injections using the same needle. a bidirectional rotation insertion technique should be used in order to minimize the needle deflection. it is recommended to not penetrate the needle to its hub, as this is where the needle is the weakest and sight of the needle can be lost when it is buried to the hub. a meticulous injection technique is imperative. the use of adequate preoperative sedation in the nervous patient may be considered, thereby minimizing the possibility of his sudden movement during the injection. needle breakage rarely occurs nowadays, but when it occurs, the situation must be managed appropriately. every effort should be made to retrieve the needle immediately, if the tip is visible. if it is not visible, the required steps include immediate referral to a maxillofacial unit, imaging to identify the position of the fragment, and surgery to remove the needle. the imaging exam should be the one available at the clinical facility, but a combination of two or more could be of great help in locating the broken needle. surgeons performing the removal must not only be skilled and well-trained, but be familiar with the anatomy involved. it is strongly recommended that only an oral and maxillofacial surgeon may indicate and/or perform surgery to remove the broken needle. although there is a degree of controversy over management of broken dental needles, it is the authors’ opinion that every broken needle should be withdrawn. acknowledgements we like to thank mr. m. ethunandan for providing us his article. references 1. blum t. a report of 100 cases of hypodermic needles broken during administration off oral local anaesthesia. dent cosmos. 1928; 70: 865-74. 2. stafne ec. oral roentgenographic diagnosis. 3rd ed. philadelphia: saunders; 1969. p.283. 3. crouse v. migration of a broken anesthetic needle: report of a case. s c dent j. 1970; 28: 16-9. 4. ethunandan m, tran al, anand r, bowden j, seal mt, brennan pa. needle breakage following inferior alveolar nerve block: implications and management. br dent j. 2007; 202: 395-7. 5. archer wh. oral and maxillofacial surgery. 5th ed. philadelphia: saunders; 1975. p.1540-680. 6. bedrock rd, skigen a, dolwick mf. retrieval of broken needle in the pterygomandibular space. j am dent assoc. 1999; 130: 685-7. 7. brown lj, meerkotter va. an unusual experience with a broken needle. j dent assoc s afr. 1963; 18: 74. 8. cawson ra. essentials of dental surgery and pathology. 2nd ed. london: churchill; 1968. 9. faura-solé m, sánchez-garcés ma, berini-aytes l, gay-escoda c. broken anesthetic injection needles: report of 5 cases. quintessence int. 1999; 30: 461-5. 10. amies abp. broken needles. aust dent j. 1951; 55: 403-6. 11. fraser-moodie w. recovery of broken needles. br dent j. 1958; 105: 79. 12. fitzpatrick b. the broken dental needle. aust dent j. 1967; 12: 243-5. 13. marks rb, carlton dm, mcdonald s. management of a broken needle in the pterygomandibular space: report of a case. j am dent assoc. 1984; 109: 263-4. 14. thompson m, wright s, cheng lh, starr d. locating broken dental needles. int j oral maxillofac surg. 2003; 32: 642-4. 15. nezafati s, shahi s. removal of broken dengtal needle using mobile digital c-arm. j oral sci. 2008; 50: 351-3. 16. kennett s, curran jb, jenkins gr. management of a broken hypodermic needle: report of a case. anesth prog. 1973; 20: 48-50. 17. moore uj, fanibunda k, gross mj. the use of a metal detector for localization of a metallic foreign body in the floor of the mouth. br j oral maxillofac surg. 1993; 31: 191-2. 18. mcdonogh t. an unusual case of trismus and dysphagia. br dent j. 1996; 180: 465-6. 19. abe k, nakamatsu k, beppu k, ariji e, oka m. use of intraoperative ultrasonography to detect a small foreign body in the soft tissues of the upper lip. br dent j. 1994; 177: 292-4. 20. pietruszka jf, hoffman d, mcgivern be jr. a broken dental needle and its surgical removal: a case report. ny state dent j. 1986; 52: 28-31. 21. ho hk. a simple technique for localizing a broken dental needle in the pterygomandibular region. aust dent j. 1988; 33: 308-9. 22. dudani ic. broken needles following mandibular injections. j indian dent assoc. 1971; 43: 14-7. 23. bhatia s, bounds g. a broken needle in the pterygomandibular space: report of a case and review of the literature. dent update. 1998; 25: 35-7. 24. zelter r, cohen c, casap n. the implications of a broken needle in the pterygomandibular space: clinical guidelines for prevention and retrieval. paediatr dent. 2002; 24: 153-6. 25. fuller np, menke ra, meyers wj. perception of pain to three different intraoral penetration of needles. j am dent assoc. 1979; 99: 822-4. 26. flanagan t, wahl mj, schmitt mm, wahl ja. size doesn’t matter: needle gauge and injection pain. gen dent. 2007; 55: 216-7. 27. safer a, behbehani e, al haddad a, rafique m. dental anaesthesia needles: an eds study. annals of first african and middle-east iadr federation conference. jabriya, kuwait. september 27-29; 2005. 28. aldous ja. needle deflection: a factor in the administration of local anaesthetics. j am dent assoc. 1968; 77: 602-4. 29. jeske ah, boshart bf. deflection of conventional versus nondeflecting dental needles in vitro. anesth prog. 1985; 32: 62-4. 30. lehtinen r. penetration of 27and 30-gauge dental needles. int j oral surg. 1983; 12: 444-5. 31. hochman mn, friedman mj. in vitro study of needle deflection: a linear insertion technique versus a bidirectional rotation insertion technique. quintessence int. 2000; 31: 33-9. complication of local dental anesthesia a broken needle in the pterygomandibular space162 braz j oral sci. 8(3):159-162 oral sciences n3 original article braz j oral sci. july | september 2015 volume 14, number 3 influence of different water types on the physical and mechanical properties of gypsum juliana dos santos proença1, marcos massahiro suzuki1, silvano cesar da costa2, bruno shindi hirata1, murilo baena lopes1, edwin fernando ruiz contreras1 1 universidade estadual de londrina – uel, school of dentistry, department of restorative dentistry, londrina, pr, brazil 2 universidade estadual de londrina uel, department of statistics, londrina, pr, brazil correspondence to: edwin fernando ruiz contreras departamento de odontologia restauradora clínica odontológica universitária universidade estadual de londrina uel rua pernambuco 540, centro cep: 86020-120 londrina, pr, brasil phone: +55 43 3371-6750 fax: +55 43 3371-6755 e-mail: edwin@uel.br abstract aim: to evaluate if gypsum mixed with different water types, with their different compositions influence the dimensional change, surface roughness and compressive strength of type iv and v gypsum specimens. methods: sixty specimens were fabricated from metal matrices and divided into six groups (n=10) according to the used type of gypsum and water: g1, g2 and g3 – type iv gypsum and tap, mineral and distilled water, respectively; g4, g5 and g6 – type v gypsum and tap, mineral and distilled water, respectively. water/powder ratio followed the manufacturer’s recommendations (19 ml/100 g) in all groups. the same specimens were used for all tests. a micrometer dial evaluated the dimensional change. the surface roughness of three random points was determined with a rugosimeter; a universal testing machine performed the compressive strength test. the data were subjected to analysis of variance (anova). results: there was no statistically significant difference (p>0.05) between the three types of water in the studied variables. there was statistically significant difference (p<0.05) between the gypsum mixes for the dimensional change variable. conclusions: the different water types, with their different compositions, did not influence the analyzed physical and mechanical properties. keywords: calcium sulfate; water; compressive strength; dental prosthesis. introduction mold and model are essential to prosthetic rehabilitation and connect the clinical and the laboratorial phases required for dental prosthesis manufacture1. for dental treatment success, gypsum models should copy, as faithfully as possible, the desired structures contained in the mold2. model accuracy is a critical factor in prostheses and indirect restorative manufacture. hence, the tooth preparation must be reproduced accurately to obtain a correct marginal adaptation of these restorations. the gypsum model must have dimensional stability over time, abrasion resistance, fracture resistance1 and hardness3, properties that are very important during the manufacturing process1,3. type iv and v gypsum are widely used in fixed prostheses manufacture because they have strength and hardness needed during the sculpture wax pattern procedure. type v gypsum has high setting expansion, therefore, it is indicated for material models that have high solidification contraction, such as basic metals, in order to offset this contraction4. some studies5-8 evaluated if the addition of certain substances to gypsum (calcium sulfate hemihydrate powder) or in water could change the setting expansion, surface hardness, setting time and compressive strength of gypsum braz j oral sci. 14(3):199-203 http://dx.doi.org/10.1590/1677-3225v14n3a05 received for publication: may, 28 2015 accepted: september, 18 2015 200200200200200 specimens. brukl et al.9 (1984) evaluated the effect of using different types of water in setting time and setting expansion of gypsum. different water types can be used to manipulate gypsum, such as tap, mineral and distilled water. most dentists use tap water to manipulate gypsum powder. however, it may have mineral content variation depending of the city and of the sources within the same city, and water consumption of the population. mineral water differs among commercial brands in its level of minerals10. on the other hand, distilled water is standardized, independent of commercial brand and has no mineral salts11. the main differences between the three types of water of interest to clinical dentistry are the mineral content and the cost, since tap water is cheaper than mineral11 and distilled water. the research hypothesis was that the mineral salts in tap and mineral water react with the calcium sulfate hemihydrate powder, influencing the physical and mechanical properties of gypsum. thus, distilled water would provide better results: less dimensional change and surface roughness, and higher compressive strength; improving the quality of the gypsum models and, consequently, of the final product. the aim of this study was to evaluate if the mixture of gypsum powder with different water types, with their different mineral compositions, influence the dimensional change, surface roughness and compressive strength of type iv and v gypsum specimens. material and methods gypsum specimens manufacture type iv and v gypsum (durone, dentsply ind. comp. ltda, rio de janeiro, rj, brazil) were weighed on a digital scale (actlife, balmak, santa bárbara d’oeste, sp, brazil) and mixed according to the ratio recommended by the manufacturer (19 ml/100 g). tap (sanepar, londrina, pr, brazil), mineral (cristal safira, maringá, pr, brazil) and distilled water (ssplus, maringá, pr, brazil) were dosed using a 20 ml syringe. gypsum and water were mechanically manipulated (polidental, model number 2191/06, cotia, sp, brazil) for 30 s to reach a homogeneous and smooth mix, avoiding possible air bubbles. the mix was poured into five identical metal matrices placed on a vibrator (vh equipamentos, araraquara, sp, brazil). the same calibrated operator produced all specimens. group division sixty specimens were obtained and divided according to the type of gypsum and water (n=10). the same specimens were used for all tests: · group 1: type iv gypsum and tap water · group 2: type iv gypsum and mineral water · group 3: type iv gypsum and distilled water · group 4: type v gypsum and tap water · group 5: type v gypsum and mineral water · group 6: type v gypsum and distilled water tests the dimensional change was evaluated by a micrometer dial (digimess instrumentos de precisão, são paulo, sp, brazil). the metal matrices used to make the specimens had a movable piece attached at the end, where the active tip of the micrometer touched. the needle of the micrometer was set on zero and the matrix was positioned against a concrete wall. when any change in gypsum volume occurred, the micrometer showed this change. the expansion was analyzed at 10, 20, 30 and 40 min; after this, the specimen was separated from the matrix. the surface roughness of three random points was determined by a rugosimeter (mitutoyo surftestsj-400 series, mitutoyo, kawasaki, kanagawa, japan). the test was performed on the left specimen face that was in direct contact with the metal matrix, which provided a smooth standardized surface. the average of the three measurements was considered for statistical analysis. the unit and parameter selected were micrometers (µm) and ra, respectively. the arithmetic average of the existing peaks and valleys in a measurable length sets the ra variable, which characterizes the average roughness of a surface12. in the present study, a 7.5 mm specimen length was measured. the compressive strength was evaluated using a universal testing machine (emic dl2000, emic equipamentos e sistemas de ensaio, são josé dos pinhais, pr, brazil). after 7 days of fabrication, the dry strength was tested at crosshead speed of 1 mm/min with the specimens placed vertically between two compression plates. the results were obtained in kilogram force (kgf). statistical analysis the data were subjected to two-way anova, using gypsum factor in two levels (iv and v) and water factor in three levels (tap, mineral and distilled water), totalizing 6 treatments with 10 repetitions each. for the dimensional change variable, the result was observed at four different times 10, 20, 30 and 40 min thus, longitudinal analysis with autoregressive correlation structure of 1st order (ar1) was required for time modeling. all assumptions for anova validation according to the shapiro-wilk test for error normality were checked and met and bartlett test for variance homogeneity. differences were considered statistically significant at p<0.05. results the mean values of dimensional change of type iv and v gypsum specimens can be observed in figure 1. there was no statistically significant difference between the specimens prepared with the same gypsum type; on the other hand, the two gypsum types differ from each other (p<0.05). the expansion values were lower for type iv than for type v gypsum (table 1). for dimensional change, the analysis of variance showed no statistically significant difference (p>0.05) between the water types and for the water-gypsum interaction. influence of different water types on the physical and mechanical properties of gypsum braz j oral sci. 14(3):199-203 201201201201201 gypsum iv gypsum v tap water 0.07aa 0.11ab mineral water 0.06aa 0.11ab distilled water 0.07aa 0.13ab table 1.table 1.table 1.table 1.table 1. mean dimensional change of type iv and v gypsum (mm). small letters compare the rows in each column. capital letters compare the columns in each row. p<0.05 (anova with autoregressive correlation structure of 1st order). group (g) surface roughness (ra) g 1 1.59a g 2 1.69a g 3 1.59a g 4 1.61a g 5 1.56a g 6 1.51a table 2.table 2.table 2.table 2.table 2. mean surface roughness of type iv and v gypsum (ra). means followed by same small letters in each column do not differ statistically by two-way anova (p>0.05). group (g) compressive strength (kgf) g 1 990.28a g 2 120.18a g 3 1135.18a g 4 1171.68a g 5 1308.42a g 6 1384.84a table 3.table 3.table 3.table 3.table 3. mean compressive strength of type iv and v gypsum (kgf). means followed by same small letters in each column do not differ statistically by two-way anova (p>0.05). the surface roughness analysis showed no statistically significant difference (p>0.05) between the water types, gypsum types and their interaction (table 2). the same was observed for the compressive strength variable (table 3). discussion the gypsum (calcium sulfate hemihydrate powder) was mixed with three different types of water: tap, mineral and distilled. the tap water used in the research meets all requirements for drinking water. mineral water has chemical, physical or physicochemical properties different from tap water and is enriched by certain minerals, such as calcium, fluoride, magnesium, chloride, carbonate, sodium, among others10. on the other hand, distilled water has no mineral salts11. since the three types of water have different compositions, it was assessed if it could affect the dimensional change, surface roughness and compressive strength of gypsum specimens. the expansion was analyzed at 10, 20, 30 and 40 min after manipulation of gypsum. according to anusavice4 (2003), the final setting time gilmore test shows that this occurs up to 20 min after gypsum and water mix and reported that, technically, the gypsum model is ready for use 30 min after manipulation. clinically, mold and model are separated about 40 min after manipulation. moreover, according to marquezan et al.13 (2012), contact times of alginate with gypsum longer than 1 h damage the model surface, reducing detail reproduction and microhardness. therefore, the present study reproduced and evaluated the dimensional change that is clinically more important. a possible reaction between the mineral water salts and calcium sulfate hemihydrated powder could cause an increase in model surface roughness, damaging the prosthetic work. prostheses produced over rough models cannot accurately reproduce the details, which can cause clinically a misfit of the prosthesis. thus, surface roughness is an important property to be evaluated in researches with gypsum models. the present research results led to the conclusion that the study hypothesis was rejected, i.e. the mineral salts in tap and mineral water did not react with the calcium sulfate hemihydrate powder in order to influence the dimensional change, surface roughness and compressive strength of gypsum. no significant differences between the studied water types for dimensional change was found for gypsum types iv and v. similar behavior was observed by brukl et al.9 (1984) who analyzed the influence of four water types, including tap and distilled water, in a type iii gypsum with additives for setting time and setting expansion control. according to the authors, the additives minimize the variances caused by the different types of water in setting time and in setting expansion. hiraguchi et al.14 (2003) investigated the effect of rinsing alginate impression using acidic electrolyzed water for mold disinfection, on the dimensional change of gypsum models. there were no statistically significant differences in dimensional change of gypsum models obtained by alginate molds rinsing with tap water and acidic electrolyzed water, for the same rinsing time. in a previous study15 it was evaluated the dimensional accuracy of gypsum models (durone iv) obtained from molds of three alginate impression materials that were disinfected by spraying and storage for 15 min, with three solutions (2% sodium hypochlorite, 2% chlorhexidine digluconate and 0.2% peracetic acid). there was no statistically significant difference between the disinfectant solutions and the alginate impression materials combination or for independent factors in the dimensional change of gypsum models. another research13 showed that the time of contact between alginate and gypsum did not alter the model dimensions. thus, the type of water, some methods of alginate mold disinfection, the alginate impression material and the time of alginate/ gypsum contact are factors that do not influence the dimensional change of gypsum. in this study, the behavior of the gypsum type iv and v was different over time for the dimensional change variable. in the first 20 min both gypsum types had a growing expansion, which stabilized at this time on type iv gypsum. however, the type v gypsum changed remarkably until the final 40 min (figure 1). the expansion values were lower for influence of different water types on the physical and mechanical properties of gypsum braz j oral sci. 14(3):199-203 group (g) 10 min 20 min 30 min 40 min g 1 0.02 0.07 0.08 0.09 g 2 0.01 0.07 0.08 0.08 g 3 0.04 0.09 0.09 0.09 g 4 0.00 0.07 0.16 0.23 g 5 0.00 0.07 0.15 0.21 g 6 0.00 0.10 0.19 0.24 table 4.table 4.table 4.table 4.table 4. mean dimensional change per group, in mm, to each measurement time. fig. 1. dimensional change variable: the setting expansion increased over time for type v gypsum, showing a linear behavior. unlike the type iv gypsum which showed a quadratic behavior, the setting expansion was more evident in the first 20 min, stabilizing at this time. p<0.05 (anova with autoregressive correlation structure of 1st order). type iv than for type v gypsum, as expected, because this material has high setting expansion4. the mean dimensional change to durone iv was 0.032 mm in a study that analyzed the effect of dry gypsum specimens at room temperature (25±4 ºc) for 2 h16. in the present research, approximate values were also found to durone iv specimens after 10 min of water/gypsum mix at room temperature. after the final setting time (40 min), the mean dimensional change ranged from 0.08 to 0.09 mm (table 4). it could be assumed that the difference between the mean dimensional change values occured due to the present study specimens being 3 times larger than previous study specimens, and to some variations in the measurement methods. in this research, there was no significant difference between the studied water and gypsum for surface roughness specimens. moura et al.12 (2010) found that the alginate molds disinfection with sodium hypochlorite steam (5.25%) for 10 min produced type iii and iv gypsum specimens with dimensional stability similar to the control (untreated molds). this disinfection method did not influence either the surface roughness, which indicates that it is independent of the water type and alginate molds disinfection. there was no statistically significant difference in compressive strength between the specimens prepared with tap, mineral and distilled water. alsadi et al.5 (1996) evaluated if the use of gum arabic and calcium hydroxide would alter the compressive strength of improved die stone, but did not find differences between the specimens with or without this compound. twomey et al.8 (2003) analyzed the type v gypsum mix with water containing different calcium hypochlorite disinfectant concentrations and found, in general, a decrease in compressive strength and a higher water demand to produce a material with the same consistency as the control, which resulted in specimens with greater porosity. this can be a cause for the decrease gypsum strength. however, the mix of type v gypsum and water containing 0.5% calcium hypochlorite increased dry strength and proved to be a good disinfectant that can be used in dental clinics8. the type of water and the addition of gum arabic and calcium hydroxide to the gypsum powder are factors that neither improve nor decrease the compressive strength of gypsum. on the other hand, the type v gypsum mix can modify this variable with water containing calcium hypochlorite. the three types of water showed no difference among themselves and did not alter the properties for both type iv and v gypsum, which means economic advantages to dentists, to prosthetics and even to patients, because there is no cost increase in gypsum models manufacture, and consequently, in the final product. thus, good quality models can be obtained with tap, mineral and distilled water, provided that the technique is appropriate and the manufacturer’s recommendations are followed. the different water types, with their different mineral compositions, did not influence the analyzed physical and mechanical properties. we suggest that, despite different compositions, there is something common between them and their pureness that could influence these research results. therefore, more studies are required to investigate the water purity, in order to determine whether there is something common between the tap, mineral and distilled water. acknowledgements the authors would like to thank rubens nisie tango, ricardo danil guiraldo and ricardo alves matheus for providing the apparatus necessary for the development of this research. references 1. dias sc, moysés mr, agnelli jam, ávila gb, ribeiro jcr, pereira lj. impact fracture strength applied to dental modeling materials. braz j oral sci. 2007; 6: 1349-52. 2. rudd kd, morrow rm, brown ce jr., powell jm, rahe aj. comparison of effects of tap water and slurry water on gypsum casts. j prosthet dent. 1970; 24: 563-70. 3. harris pe, hoyer s, lindquist tj, stanford cm. alterations of surface hardness with gypsum die hardeners. j prosthet dent. 2004; 92: 35-8. 4. anusavice kj. phillips’ science of dental materials. philadelphia: saunders; 2003. 202202202202202influence of different water types on the physical and mechanical properties of gypsum braz j oral sci. 14(3):199-203 203203203203203 5. alsadi s, combe ec, cheng ys. properties of gypsum with the addition of gum arabic and calcium hydroxide. j prosthet dent. 1996; 76: 530-4. 6. diakoyanni in, kaloyannides am, panagiotouni eg. dental stone and improved dental stone surface hardness: the effect of the addition of potassium salts. eur j prosthodont restor dent. 1992; 1: 79-85. 7. zakaria mr, johnston wm, reisbick mh, campagni wv. the effects of a liquid dispersing agent and a microcrystalline additive on the physical properties of type iv gypsum. j prosthet dent. 1988; 60: 630-7. 8. twomey jo, abdelaziz km, combe ec, anderson dl. calcium hypochlorite as a disinfecting additive for dental stone. j prosthet dent. 2003; 90: 282-8. 9. brukl ce, mcconnell rm, norling bk, collard sm. influence of gauging water composition on dental stone expansion and setting time. j prosthet dent. 1984; 51: 218-23. 10. azoulay a, garzon p, eisenberg mj. comparison of the mineral content of tap water and bottled waters. j gen intern med. 2001; 16: 168-175. 11. environmental protection agency (epa). bottled water basics. washington: epa; 2005 sep. 9p. (water health series). 12. moura cdvs, moura wl, frança fmg, martins gas, nogueira lblv, zanetti rv. disinfection of irreversible hydrocolloid impressions with sodium hypochlorite steam: assessment of surface roughness and dimensions of gypsum models. j dent sci. 2010; 25: 276-81. 13. marquezan m, jurach em, guimarães vd, valentim rga, nojima li, nojima mcg. does the contact time of alginate with plaster cast influence its properties? braz oral res. 2012; 26: 197-201. 14. hiraguchi h, nakagawa h, uchida h, tanabe n. effect of rinsing alginate impressions using acidic electrolyzed water on dimensional change and deformation of stone models. dent mater j. 2003; 22: 494-506. 15. guiraldo rd, borsato tt, berger sb, lopes mb, gonini-jr a, sinhoreti mac. surface detail reproduction and dimensional acuraccy of stone models: influence of disinfectant solutions and alginate impression materials. braz dent j. 2012; 23: 417-21. 16. silva mab, vitti rp, consani s, sinhoreti mac, mesquita mf, consani rlx. linear dimensional change, compressive strength and detail reproduction in type iv dental stone dried at room temperature and in a microwave oven. j appl oral sci. 2012; 20: 588-93. influence of different water types on the physical and mechanical properties of gypsum braz j oral sci. 14(3):199-203 oral sciences n3 case report braz j oral sci. january | march 2013 volume 12, number 1 differential diagnosis between post-polio syndrome symptoms and temporomandibular disorder – clinical case gustavo augusto seabra barbosa1, maria helena de siqueira torres morais1 1department of dentistry, federal university of rio grande do norte, natal, rn, brazil correspondence to: gustavo augusto seabra barbosa av. salgado filho, 1787, lagoa nova, cep: 59056-000 natal, rn, brasil phone: 55 84 32154135 e-mail: gustavoseabra@hotmail.com received for publication: march 19, 2012 accepted: august 11, 2012 abstract post-poliomyelitis syndrome (pps) is characterized by the delayed appearance of new neuromuscular symptoms in patients several years after their acute poliomyelitis paralysis. clinical features of pps include fatigue, joint and muscle pain, new muscular weakness and bulbar symptoms. the diagnosis is essentially clinical after excluding other neurological, orthopedic or rheumatologic problems. temporomandibular disorders (tmd) are usually diagnosed by means of comprehensive review of patient history and clinical examination and the symptoms are pain/ discomfort in the jaw, mainly in the region of the temporomandibular joints (tmjs) and/or masticatory muscles, limitation of mandibular function and/or tmj sounds. in the same way as pps, the diagnosis of tmd is challenging. this study reports the case of a patient that presented the symptoms of both conditions in the stomatognathic system, and discusses how to achieve the differential diagnosis for proper management of the cases. keywords: diagnosis differential, post-poliomyelitis syndrome, temporomandibular disorders. introduction in the first half of the 20th century, poliomyelitis was greatly feared. it often struck without warning, was highly contagious, and affected large, young populations, causing prolonged or permanent flaccid paralysis or death 1. transmission can occur from person-to-person through nasopharyngeal secretions or of objects, food and water, contaminated with feces of patients or carriers2. this disease is caused by viral damage of motor neurons in the spinal cord. loss of muscle fibers is furthermore compensated through hypertrophy of the remaining muscle fibers3. the onset of spinal poliomyelitis is associated with myalgia and severe muscle spasms, with the subsequent development of an asymmetrical, predominantly lower limb, flaccid weakness that becomes maximal after 48 h2. after the introduction of vaccination in the 1950s, the epidemics disappeared from most countries and polio became a rare disease2-3. patients with polio may experience progression with new symptoms decades after the acute disease. these late symptoms are termed post-polio syndrome (pps)3-5 and this designation was introduced by halstead and rossi in 1985 apud ramaj (2007)6. the nature of the condition remains controversial and diagnosis is essentially clinical after excluding other neurological, orthopedic or rheumatologic braz j oral sci. 12(1):57-60 5858585858 problems. weakness with generalized fatigue is the most common symptom, but joint pain, muscle pain, atrophy, cold intolerance, respiratory insufficiency and dysphagia may also be present1,3,5-6. pps occurs 30 to 40 years after an acute poliomyelitis attack and is observed in approximately 25 to 28% of patients and according to the visual analogue scale (vas) the intensity of pain is relatively high 5. current diagnosis is based on thorough clinical examinations in order to eliminate other possible diagnosis7 but it is still unclear at this point time if the occurrence of pps increases with age1,34,6,8. to date, several risk factors for pps development have been reported, although the etiology of this disorder remains elusive9. the early detection of correctable and treatable causes of late-onset weakness and pain may help to reduce the functional declines of polio survivors10. the term “temporomandibular disorders” (tmd) has been used as a collective term that involve the masticatory musculature, the tmjs and associated structures, or both. these disorders have been identified as a major cause of nondental pain in the orofacial region, and are considered to be a subclassification of musculoskeletal disorders11-13. in general, tmd mostly appear in the adult popution14. orofacial pain and tmd can be associated with pathologic conditions or with disorders related to somatic and neurologic structures, such as primary headache disorders and rarely have a solitary cause and numerous factors have been implicated15. this is further compounded by the patient habitually reporting several problems during history taking and clinical examination.15. a complete understanding of the associated medical conditions with symptoms common to tmd and orofacial pain is necessary for a proper diagnosis13. in view of difficult of diagnosis and the similar symptoms between pps and tmd, this paper reports the case of a patient that presented symptoms of both entities in the stomatognathic system, and discusses how to achieve the differential diagnosis for proper management of the cases. clinical case a 42-year-old caucasian female patient sought the department of dentistry of the federal university of rio grande do norte (ufrn, brazil) complaining of fatigue in facial muscles, clicking sounds and pain in the region near the tmj, and “an unbearable weight on the face”. by exclusion of orthopedic, neurological and rheumatic problems, these symptoms were attributed to pps, as the patient that had contracted polio at the age of 1 year and 10 months. during the clinical interview, the patient reported tooth clenching, which was confirmed by the present of wear facets on several teeth (figure 1). the clinical examination also revealed a clicking sound on mouth opening/closure at the left tmj and tenderness to palpation of the lower portion of the left lateral pterygoid muscle and masseter muscles of the both sides. the diagnosis was suggestive of disk displacement with reduction in the left tmj accompanied by myofascial pain. a visual analogue scale for pain (vas), usually used fig. 1 frontal view of occlusion. to check the effectiveness of the proposed treatment, was applied and the patient reported pain level 5 on the scale from zero to 10. we suspected that the parafunctional habit was causing the muscle and tmj pain instead of the pps, and this was informed to the patient. in order to clarify this point, the patient wore an anterior bite jig during 3 intercalated days for neuromuscular deprogramming (figure 2). the patient reported immediate relief after installation of the device (vas=0), and thus the use of a stabilization occlusal splint associated with behavioral therapy was prescribed. fig. 2 anterior bite jig installed. upper and lower plaster models (durone iv, dentsply) were obtained from alginate impressions (jeltrate plus, dentsply) and mounted in semi adjustable articulator. the upper model was mounted using the facial arc. using the neuromuscular deprogramming device16, registration was taken with vinyl polysiloxane (occwfast, zhermack) in centric relation to mount the lower model. the oclusal splint was waxed and fabricated using conventional thermally activated differential diagnosis between post-polio syndrome symptoms and temporomandibular disorder – clinical case braz j oral sci. 12(1):57-60 5959595959 acrylic resin (classico, art. odontológicos classico). the contacts of teeth with the oclusal splint were adjusted in centric relation occlusion. the lateral and anterior excursive movements were adjusted with the canine and anterior guides, respectively. the patient was instructed to use the stabilization occlusal splint during sleep and avoid the parafunctional habit while awake (figure 3). the supposed tdm etiology was explained to the patient in order to reduce the repetitive strain of the masticatory system (tooth clenching), to encourage relaxation, and to control the amount of the masticatory activity. there was a significant improvement in painful symptoms 1 week after installation of the occlusal splint. the patient reported pain level zero in vas, decreased fatigue and relief in the tmj tenderness. after 1-year of treatment, the patient was free of symptoms in the facial region. fig. 3 occlusal splint installed. discussion pps presents as a new onset of weakness, fatigue, fasciculations, and pain with additional atrophy of the muscle group involved during the initial paralytic disease 20 or more years earlier. this syndrome is more common among women and after longer time of the acute disease1,3-7. the pathogenesis of pps is not completely understood. the most widely accepted hypotheses suggests a dysfunction of surviving motor neurons that causes a progressive loss of the terminals on single axons or a dysfunction of motor units that are already weak, owing to forced exercise1-2,10. the current diagnosis for pps was first described by mulder et al.4 in 1972 apud lin. these criteria are 1) a prior episode of poliomyelitis with residual motor neuron loss; 2) a period of at least 15 years of neurological and functional stability after recovery from the acute illness; 3) gradual or, rarely, abrupt onset of new weakness or abnormal muscle fatigue; and 4) the exclusion of other conditions that could cause similar manifestations. when no alternative explanation can be found, this late onset weakness is referred to as pps1-2,4. the combined effect of aging, overwork, weight gain, other medical comorbidities, and muscular overuse or disuse play a role in new weakness, pain and fatigue. it is important to note that there is no reactivation of the original poliomyelitis virus or reinfection. this is often a specific anxiety in pps patients and needs to be addressed3,11. prospective studies have focused mainly on the progression of neurologic deficit, in terms of loss of muscle strength. although results are sometimes conflicting, they do not suggest a rapid loss of muscle force over time. in contrast, relatively little attention has yet been paid to functional assessment in pps, which is surprising since the loss of functional abilities is probably a major concern to the involved patients6. there is no way for preventing late symptoms and little is known to which extent rehabilitation influences the longterm outcome4. symptoms usually appear earlier in patients who have a lot of residual weakness, early bulbar respiratory difficulty during the acute illness, and those who were older when they contracted acute polio. patients suffer from pain in the joints, bones and muscles. they also have fatigue with muscle wasting, weakness, cramps and fasciculation. there is a severe deterioration in functional abilities including mobility and activities of daily living4,6. the tmd symptoms are well known. they include pain/ discomfort in the jaw, mainly in the region of the tmjs and/ or muscles of mastication, limitation of mandibular function and/or tmj sounds11,17 however, a correct diagnosis is frequently very difficult. our patient presented both the pps symptoms 40 years after polio and the tmd symptoms. behavioral therapy is generally considered as a first conservative approach for the treatment of tmd patients18. there is an application of behavioral science theories and methods to change the perception and appraisal of pain and to attenuate or eliminate the personal suffering and psychosocial dysfunction that often accompanies persistent pain conditions. the rationale for choosing behavioral therapy arises from the idea that parafunctional activity and psychosocial factors play a role in the pathogenesis of musculoskeletal pain. this method has proved to be effective in tmd management18-19. furthermore the patient compliance is of fundamental importance. compare to others treatments, the occlusion splint is much less demanding on the patients and some of them prefer this modality20. the underlying concept of the anterior bite jig is to deprogram the memorized pattern of muscle activity by preventing tooth contacts at the moment of swallowing. it is presumed to work through a relaxation effect, leading to a reduction in the muscle activity at postural position13. this device was used for differential diagnosis. the patient reported pain level zero in vas=0 using it. splint therapy is a non-invasive and reversible biomechanical method of managing pain and dysfunction of the craniomandibular articulation and its associable musculature20-24. why occlusal splints are effective in reducing symptoms is not entirely clear, but several theories have been differential diagnosis between post-polio syndrome symptoms and temporomandibular disorder – clinical case braz j oral sci. 12(1):57-60 proposed to accountant for their mechanisms of action21,23. dylina (2001)25 reported that the splints have at least 6 functions, including the following: to relax the muscles, to allow the condyle to seat in the centric relation occlusion, to provide diagnostic information, to protect teeth and associated structures from bruxism, to mitigate periodontal ligament proprioception, and to reduce cellular hypoxia levels25. the characteristics of a successful splint should include occlusal stability, equal intensity stops on all teeth; immediate posterior disocclusion during excursion movements; smooth transitions in lateral, protrusive, and extended lateral excursions (crossover); comfort during wear; and reasonable esthetics. patient compliance also contributes to splint therapy success25. splint therapy can be an important diagnostic tool to determine tmd status. if a patient rapidly becomes comfortable with a splint, it may be an indication that the disorder is of muscular origin. if symptoms worsen with permissive splint wear, this may indicate an internal derangement (disk) problem (perhaps caused by free reign of the condylar head back to the retrodiscal tissues without housing by the disk) or an error in the initial diagnosis25. using the splint, our patient remained without symptoms. it can assign the absence of pain due to possible effects of the splint as muscle relaxation21, changes in impulses to the central nervous system (cns) 26 and cognitive theory 1 1 (according to this theory, the presence of splint as a foreign object in the mouth would likely change the oral tactile stimuli, decrease the oral volume and space for the tongue, and make the patients conscious about the position and potentially harmful use of their jaw). as described, the clinical diagnosis of pps is made by exclusion of options and requires differentiation with other diseases that may present similar characteristics; however, in the present case, there was probably no exclusion of tmd for symptoms in the facial region. pps and tmd are difficult to diagnose because the symptoms are non-specific. pps is essentially clinical after excluding other neurological, orthopedic or rheumatologic problems. in most cases, the diagnosis of tmd is based on careful review of patient history and clinical examination, which depends on patient report of levels of pain/discomfort at the tmjs and associated muscles. without a differential diagnosis the two entities may be confused due to their similarities. in conclusion, this clinical report describes an association, but not necessarily a cause-effect relationship between pps and tmd symptoms. more specific investigations are necessary for a better understanding of a possible link between pps and tmd. references 1. howard rb. poliomyelitis and the postpolio syndrome. bmj. 2005; 330: 1314-8. 2. vranjac, a. poliomyelitis and the postpolio syndrome. sao paulo: state department of health; 2006. 3. nollet f, beelen a, prins mh, de visser m, sargeant aj, lankhorst gj et al. disability and functional assessment in former polio patients with and without postpolio syndrome. arch phys med rehabil. 1999; 80: 136-43. 4. lin kh, lim yw. post-poliomyelitis syndrome: case report and review of the literature. ann acad med singapure. 2005; 34: 447-9. 5. werhagen l, borg k. impact of pain on quality of life in patients with postpolio syndrome. j rehabil med. 2013: 45: 161-3. 6. ramaraj r. post poliomyelitis syndrome: clinical features and management. br j hosp med. 2007; 68: 648-50. 7. gonzalez h, khademi m, borg k. intravenous immunoglobulin treatment of the post-polio syndrome: sustained effects on quality of life variables and cytokine expression after one year follow up. j neuroinflammation 2012; 9: 1-12. 8. matyja e. post-polio syndrome. part i. the “legacy” of forgotten disease, challenges for professionals and polio survivors. neurol neurochir pol. 2012; 46: 357-71. 9. bertolasi l, acler m, dall’ora e, gajofatto a, frasson e, tocco p et.al. risk factors for post-polio syndrome among an italian population: a casecontrol study. neurol sci. 2012; 33: 1271-5. 10. lim jy, kim ke, choe g. myotonic dystrophy mimicking postpolio syndrome in a polio survivor. am j phys med rehabil. 2009; 88: 161-5. 11. suvinen t, reade pc, kemppainen p, könönen m, dworkin sf. review of aetiological concepts of temporomandibular pain disorders: towards a biopsychosocial model for integration of physical disorder factors with psychological and psychosocial illness impact factors. eur j pain. 2005; 9: 613-33. 12. al-ani z,gray r. tmd current concepts: 1. an update. dent update. 2007; 34: 278-88. 13. auvenshine rc. temporomandibular disorders: associated features. dent clin n am. 2007; 51: 105-27. 14. niemela k, korpela m, raustia a, ylöstalo p, sipilä k. efficacy of stabilisation splint treatment on temporomandibular disorders. j oral rehabil. 2012; 39: 799-804. 15. jerjes w. muscle disorders and dentition-related aspects in temporomandibular disorders: controversies in the most commonly used treatment modalities int arch med. 2008; 23: 1-13. 16. lucia vo. a technique for recording centric relation. j dent. 1964; 14: 492505. 17. pimentel mj, gui ms, martins de aquino lm, rizzatti-barbosa cm. features of temporomandibular disorders in fibromyalgia syndrome. cranio. 2013; 31: 40-5. 18. michelotti a, wijer ,steenks m, farella m. home-exercise regimes for the management of non-specific temporomandibular disorders. j oral rehabil. 2005; 32: 779-85. 19. dworkin s. behavioral and educational modalities. oral surg oral med oral pathol. 1997; 8: 128-33. 20. de felicio cm, melchior mo, silva mam. effects of orofacial myofunctional therapy on temporomandibular disorders. cranio. 2010; 28: 250-61. 21. pertes ra, gross sg. clinical management of temporomandibular disorders and orofacial pain. chicago: quintessence; 2005. p. 197-209. 22. major pw, nebbe b. use and effective of splint appliance therapy: review or literature. cranio. 1997; 15: 159-66. 23. nelson sj. principles of stabilization bite splint therapy. dent clin north am. 1995; 39: 403-21. 24. lindfors e, magnusson t, teglberg a. interocclusal appliancesindications and clinical routines in general dental practice in sweden. swed dent j. 2006; 30: 123-34. 25. dylina tj. a common-sense approach to splint therapy. jprosthet dent. 2001; 86: 539-45. 26. okeson jp. management of temporomandibular disorders and occlusion. 6th ed. saint louis: mosby; 2008. p. 377-99. differential diagnosis between post-polio syndrome symptoms and temporomandibular disorder – clinical case 6060606060 braz j oral sci. 12(1):57-60 oral sciences n3 case report braz j oral sci. october | december 2012 volume 11, number 4 florid cement-osseous dysplasia of maxilla and mandible: a rare clinical case chandramani b. more 1, rajan shirolkar2, chhaya adalja2, mansi n tailor3 1mds,phd, professor and head, department of oral medicine and radiology, k.m.shah dental college and hospital, sumandeep vidyapeeth, piparia, vadodara, gujarat state. india 2mds, professor, department of oral medicine and radiology, k.m.shah dental college and hospital, sumandeep vidyapeeth, piparia, vadodara, gujarat state. india 3post graduate student (part ii), department of oral medicine and radiology, k.m.shah dental college and hospital, sumandeep vidyapeeth, piparia, vadodara, gujarat state. india correspondence to: chandramani b. more department of oral medicine and radiology, k.m.shah dental college and hospital, sumandeep vidyapeeth, piparia, vadodara, gujarat state. india. phone: +91 9974900278 / +91 9867111478. fax: +91 2668-245292 e-mail: drchandramanimore@rediffmail.com abstract cemento-osseous dysplasia is a group of disorders known to originate from periodontal ligament tissues. florid cemento-osseous dysplasia clearly appears to be a form of bone and/or cementoid tissues restricted to jaw bones. this lesion is usually asymptomatic and is incidentally detected during routine radiographic examination. the diagnosis is made by clinical and radiographic examinations, and biopsy is not necessary. the patient must manifest the typical changes in at least two quadrants. an asymptomatic individual requires only a periodic follow-up to ensure that there is no change in the disease behavior. surgical management is indicated only for a progressive lesion that produces orofacial disfigurement. this paper presents a rare case of a 60-year-old female who was clinically and radiographically diagnosed as having florid cemento-osseous dysplasia in the maxilla and mandible. discussion is presented with emphasis on clinical and radiographic manifestations. keywords: fibro-osseous lesion, florid cemento-osseous dysplasia, maxilla, mandible. introduction the term florid cement-osseous dysplasia (flcod) was first proposed by melrose et al in 1976 to describe a condition of exuberant multi quadrant masses of cementum and/or bone in both the jaws and in some cases, simple bone cavity like lesions in affected quadrant. this condition has been interpreted as a dysplastic lesion or developmental anomaly arising in tooth bearing areas. the word ‘florid’ was introduced to describe the widespread, extensive manifestations of the disease in the jaws 1. the world health organization (who) workshop group on head and neck tumors classified osseous dysplasia into four subtypes based upon the extent and radiographic appearances periapical osseous dysplasia in the anterior mandible, focal osseous dysplasia or focal cemento-osseous dysplasia (fcod) in a posterior region of the jaws, florid osseous dysplasia (fod) involving many quadrants of the jaws, and familial gigantiform cementoma 2. florid-cemento-osseous dysplasia was a term proposed in the 2nd edition of the who “international histological classification of odontogenic tumors’’ to replace the gigantiform cementoma of the 1st edition 3. received for publication: august 09, 2012 accepted: september 10, 2012 braz j oral sci. 11(4):513-517 flcod lesions are usually asymptomatic and detected incidentally during routine radiographic examination 2. the diagnosis of this lesion is based on clinical and radiographic examination, and biopsy is not necessary 4, 5. the patient must manifest the typical changes in at least two quadrants for the diagnosis of flcod. a four quadrant disease may be suggestive of a familial nature. this article reports the case of a 60-year-old woman presenting flcod of the maxilla and mandible. discussion is presented with emphasis on clinical and radiographic manifestations. clinical case a 60-year-old female patient (fig.1a) presented to the department of oral medicine and radiology, k. m. shah dental college and hospital, vadodara, with a complaint of pain of varying intensity at different sites of upper and lower jaw with 5 to 6 months of evolution. the patient was under analgesics prescribed by the local dentist sin then. the dentist extracted the teeth 45, 46 and 47 assuming that they were the culprits for pain. as there was no relief, she was referred to our department for further evaluation. the pain was episodic and dull to severe at different sites of maxilla and mandible. her medical history was significant only for hypertension, which, according to her, was under control with amlodipine for the last 10 years. the family history had no significant associations to the dental case. the patient was responsive and well oriented as to time and place. her vital signs were within normal limits. on extraoral examination, tenderness was noticed from left ramus to right ramus of the mandible and from left to right maxillary bone. the submandibular lymph nodes were enlarged, palpable and tender bilaterally. the intraoral examination revealed presence of all teeth except for 45, 46 and 47. the labial, buccal, palatal and lingual gingiva was firm, fibrotic, hyperplastic, non tender and had generalized pseudo-pockets (fig 1b and 1c). also, mild cortical plate expansion was noted throughout the jaws. crowding was present in the maxillary and mandibular anterior area. generalized attrition and grade iii mobility was present in 31, 32, 33, 34, 35, 36, 37, 38 and 41. all the remaining teeth had grade i mobility. the clinical diagnosis of drug (amlodipine) induced gingival enlargement was considered with differential fig. 1a. extraoral view, b and c: intraoral views showing gingival enlargement. diagnosis of idiopathic gingival enlargement and pseudo gingival enlargement. on viewing the periapical fig. 2a) and panoramic (fig.2b) radiographs, we noted multiple ill defined, varying size radiopaque areas of calcification in the premolar and molar regions of all the four quadrants. in the maxilla, the lesion extended bilaterally, from the distal root of first premolar to the mesial root of third molar. bilaterally, the lesion extended mesiodistally from the distal root of canine to the anterior surface of mandible ramus. the right mandibular canal appeared to be displaced inferiorly. the radiopaque calcification was more severe in the mandible. the radiopaque lesion had well-defined borders. the bone adjoining the lesion borders was sclerosed at multiple sites with a pseudocorticated appearance. the radiopaque calcifications appeared like cotton-wool structures. generalized bone loss was severe in 31, 32, 33, 34, 35, 36, 37, 38 and 41. the cross-sectional mandibular occlusal view (fig.2c) showed multiple radiopaque foci with minor cortical plate expansion. the plain computed tomography scan of maxilla and mandible (fig. 2d and 2e) showed bilateral multiple hyperdense areas in the premolar and molar regions. multiple lobulated calcified masses were symmetrically distributed in the posterior segment of maxilla and mandible. the routine hematological investigations along with serum calcium and serum alkaline phosphatase levels were within normal limits. in spite of explaining the complications of extraction, patient consistently insisted on the extraction of mobile 36, 37 and 38 with minimal injury to the tissues. these teeth were extracted and biopsy specimens were collected from the extraction sockets. the histopathological analysis of the specimen showed presence of multiple structures including metaplastic osteoid and cementoid structures in a collagenous spindle-shaped background and plump fibroblasts. a granulation tissue surrounding the lesion showed hemorrhagic areas and neo-vascularization with chronic inflammatory cell infiltration suggestive of flcod. based on the radiographic and histopathological findings, the final diagnosis of flcod was made. florid cement-osseous dysplasia of maxilla and mandible: a rare clinical case braz j oral sci. 11(4):513-517 514514514514514 fig. 2a. periapical view; b: panoramic view; c: occlusal view showing multiple ill-defined, varying size radiopaque areas of calcification in premolar and molar region; d and e: axial computed tomography scan showing bilateral multiple hyperdense areas in the premolar and molar region. discussion flcod is a benign, non-neoplastic and self-limiting jaw lesion. many authors use several synonyms, such as multiple enostoses, multiple cemento-ossifying fibromas, multiple periapical osteo-fibromatosis, florid osseous dysplasia and gigantiform cementoma. this amalgamates flcod with other bone diseases, thereby confounding the literature. who classifies flcod as a type of fibro-osseous lesion2. fibroosseous lesions are a group of bone disorders in which the normal bone is replaced with fibrous connective tissue containing abnormal bone or cementum. the initial radiographic appearance of this group of lesions is radiolucent. over time, the lesion evolves to a mixed radiolucent radiopaque stage before progressing to a completely radiopaque stage. the normal trabecular pattern of bone is not seen in the lesion. each lesion has its characteristic texture, a fact that can aid in the differential diagnosis6. the etiology of flcod remains unknown. waldron et al.10 have proposed that reactive or dysplastic changes in the periodontal ligament might cause the disease. these lesions are characterized by replacement of bone by connective tissue matrix. the matrix displays varying degrees of mineralization in the form of woven bone or cementum-like round basophilic acellular structures. the affected area undergoes changes from vascular bone into cementum-like lesion. the term cementoosseous dysplasia is a histopathological term, yet the diagnosis can be made by clinical and radiographic findings1. florid cement-osseous dysplasia of maxilla and mandible: a rare clinical case braz j oral sci. 11(4):513-517 515515515515515 although the disease may be totally asymptomatic, some patients present pain, swelling, purulent discharge and sequestrum formation2,7,8. it can be hypothesized that the etiological factors such as wearing dentures, tooth extraction make the vascular tissue of the lesion exposed to the oral cavity. traumatic episodes of this calcified tissue cause retrograde infection and subsequent cellulitis, sequestration or even osteomyelitis2. the hypovascular nature of the disease increases in severity when infection of the lesion occurs2,8. in severe cases, mandibular nerve paresthesia and/or jaw expansion, albeit rare, may be found2. flcod is discovered most frequently in the mandible of middle aged females (the mean age being 42 years), although the age range is broad. the condition shows a marked predilection for blacks and asians2,6-8. often flcod produces no symptoms and is found incidentally during radiographic examination. occasionally patient complains of intermittent, poorly localized pain in the affected bone. if the lesions become secondarily infected, features of osteomyelitis may develop, including mucosal ulceration, fistulous tracts with suppuration, sequestrum formation and pain2,7,8. clinically, it may appear as cortical expansion, particularly of the mandible. the expansion may be sufficient pronounced to cause the professional to suspect a neoplasm of paget’s diseases of the bone. infection may be absent, but dull aching sensation of intermittent nature may be the fundamental feature in the mandibular molar region. teeth in the involved bone are vital unless other dental disease coincidentally affects them. flcod lesions are lobulated masses of dense, lightly mineralized almost acellular cementoosseous tissue typically occurring in several parts of the jaw 3. flcod may have a familial nature, hence familial flcod. in 1953, agazzi and belloni1 described an italian family in which several members manifested four-quadrant disease that had begun at an early age and resulted in facial disfiguration. radiographically, flcod usually presents as a diffuse distribution of lobular irregularly shaped radiopacities throughout the alveolar process 7. the epicenter is apical to the teeth, within the alveolar process and usually posterior to the cusp. in the mandible, lesions occur above the alveolar canal. the periphery usually is well defined and has a sclerotic border. the soft tissue capsule may not be apparent in mature lesions. the internal structure can vary from an equal mixture of radiolucent and radiopaque lesions to almost completely radiopaque 8. its mineralization increases with time and the same lesion may appear in various stages depending upon the degree of calcification. immature flcod usually presents densely sclerotic lobular or irregular-shaped radiopacities with a radiolucent halo which separates the lesion from the surrounding bone. with maturation, flcod involves multiple sites throughout the jaw. occasionally, a radiopaque lesion blends with the adjacent bone 2. some prominent radiolucent lesions may be present, which usually represent the development of a simple bone cyst. the radiopaque lesions can vary from small oval and circular regions (cottonwool appearance) to large, irregular, amorphous areas of calcification7,8. large flcod lesions can displace the inferior alveolar nerve canal in an inferior direction. the lesion can also displace the floor of the maxillary antrum in a superior direction and can cause enlargement of the alveolar bone by displacement of the buccal and lingual cortical plates. the roots of associated teeth may have a considerable amount of hypercementosis, which may fuse with the abnormal surrounding cemental tissue of the lesion. extraction of these teeth may be difficult 8. these variable features of flcod militate against the diagnosis of fibrous dysplasia, cementoossifying fibroma and to a lesser extent, paget’s disease of the bone. a solitary bone cyst (traumatic bone cavity) coexists in some flcod cases. a possible explanation for this occurrence may be that the fibro-osseous proliferation obstructs the interstitial fluid drainage, resulting in cystic degeneration within the flcod lesion. this collision phenomenon necessitates surgical exploration and histopathological examination to establish a definite diagnosis. in a patient without solitary bone cyst formation, the diagnosis of flcod is clinical or radiographic. biopsy is not necessary. a patient must manifest the typical changes in at least two quadrants for a clinical diagnosis of flcod to be made. malignant fibrous histiocytoma developing in flcod was also reported in one patient, but the prognosis was good 2. histopathologically the tissue consists of fragments of cellular mesenchymal tissue composed of spindle-shaped fibroblasts and collagen fibers with numerous small vessels. free hemorrhage is typically noted interspersed throughout the lesion. within this fibrous connective tissue background is a mixture of woven bone, lamellar bone and cementum-like particles. the proportion of each mineralized material varies from lesion to lesion and from area to area in the individual site of involvement. as the lesion matures and become more sclerotic, the ratio of fibrous connective tissue to mineralized material decreases. with maturation, the bone trabeculae become thick, curvilinear structures that have been said to resemble the shape of ginger roots. with progression to the final radiopaque stage, the individual trabeculae fuse and form lobular masses composed of sheets or fused globules of relatively acellular and disorganized cemento-osseous material2, 9. when the diagnosis of flcod is suspected, an incisional biopsy and/or elective tooth extraction in the unaffected (asymptomatic) area of the lesion is best avoided because they may cause acute (and possibly serious) infection in that region 7. hence, complete surgical removal of the entire disease, which may cause discontinuity of the jaw, is absolutely unnecessary. an asymptomatic individual requires only a periodic follow-up to ensure that there is no change in the disease behavior2,4,8. surgical management is indicated only for a progressive lesion that produces orofacial disfigurement. it is not normally justified to surgically remove these lesions as this often requires extensive surgery. since the tissue diffusion of the lesion is poor, antibiotic treatment may be ineffective for an infected lesion2. the main differential diagnoses for flcod are chronic diffuse sclerosing osteomyelitis (cdso), periapical cemental dysplasia, fibrous dysplasia and paget’s disease of bone (pd) florid cement-osseous dysplasia of maxilla and mandible: a rare clinical case braz j oral sci. 11(4):513-517 516516516516516 2, 7, 8. cdso usually produces signs and symptoms of infection and it is usually limited in extent, whereas flcod is usually asymptomatic and affects at least two or more quadrants of the jaw. in periapical cemental dysplasia lesion will always be associated with root of mandibular anterior teeth whereas in flcod lesions are not contacting the teeth. fibrous dysplasia is common in children and flcod is more common in adults. pd is often a polyostotic lesion involving several bones of the body apart from the jaw and produces biochemical changes such as elevated alkaline phosphatase 2. an accurate differential diagnosis is important. notable, progressive bone atrophy under a denture can also create significant clinical problems due to exposure of the lesion. good oral care to prevent tooth loss and denture maintenance is therefore, pivotal 2. in conclusion, flcod is a benign, non-neoplastic and self-limiting jaw lesion and discovered most frequently in the mandible of middle aged females. this lesion is usually asymptomatic and detected incidentally during routine radiographic examination. it affects at least two or more quadrants of the jaws. the diagnosis is made by clinical and radiographic examination, and biopsy is not necessary. an asymptomatic individual requires only a periodic follow-up of the treatment. surgical management is indicated only for a progressive lesion that produces orofacial disfigurement. acknowledgements the authors are thankful to dr. j. r. patel, faculty dean, sumandeep vidyapeeth university, pipariya, vadodara for his constant support and encouragement for preparing this clinical case. references 1. mangala m., ramesh dn, surekha ps, santosh p. florid cementoosseous dysplasia : review and report of two cases; ijdr 2006, volume 17, issue 3, page 131-134. 2. pitak-arnnop p., dhanuthai k., chaine a., bertrand j., bertolus c. florid osseous dysplasia: report of a case presenting acute cellulitis; med oral pathol oral cir bucal. 2009 sep 1;14 (9):e461-4. 3. c ogunsalu, d miles. cemento-osseous dysplasia in jamaica. review of six cases; west indian med j 2005; 54 (4): 264-267 4. dagistan s., tozoðlu u., göregen m., çakur b. florid cemento-osseous dysplasia: a case report; med oral pathol oral cir bucal 2007;12: e348-50. 5. mupparapu m., singer s., milles m., rinaggio j. simultaneous presentation of focal cemento-osseous dysplasia and simple bone cyst of the mandible masquerading as a multilocular radiolucency; dentomaxillofacial radiology. 2005; 34: 39-43. 6. singer s., mupparapu m., rinaggio j. florid cemento-osseous dysplasia and chronic diffuse osteomyelitis: report of a simultaneous presentation and review of the literature; j am dent assoc 2005;136;927-931 7. wood n., goaz p. differential diagnosis of oral and maxillofacial lesions. fifth edition; page 509-511, 515-517. 8. white s., pharoh m. oral radiology principles of interpretation. fifth edition. page 495-498. 9. neville b., damm d., allen c., bouquot j. oral and maxillofacial pathology; 2nd edition:558-561. 10. waldron ca. fibro-osseous lesions of the jaw. j oral maxillofac surg 1993; 51: 828-835. florid cement-osseous dysplasia of maxilla and mandible: a rare clinical case braz j oral sci. 11(4):513-517 517517517517517 original articlebraz j oral sci. january/march 2009 volume 8, number 1 evaluation of identification cases involving forensic dentistry in the city of pelotas, rs, brazil, 2004-2006 fernanda nedel1, ana paula nedel2, ricardo henrique alves da silva3, rafael guerra lund4 1undergraduate student, dental school, universidade federal de pelotas (ufpel), pelotas (rs), brazil 2dds, msc, postgraduate student in social politics, universidade católica de pelotas (ucpel), pelotas (rs), brazil 3dds, msc, phd, professor, department of pediatric clinic, community and preventive dentistry, dental school of ribeirão preto, universidade de são paulo (usp), ribeirão preto (sp), brazil 4dds, msc, phd, assistant professor, department of biochemistry, institute of chemistry and geosciences, ufpel, pelotas (rs), brazil received for publication: december 12, 2008 accepted: march 25, 2009 correspondence to: rafael guerra lund universidade federal de pelotas, faculdade de odontologia rua gonçalves chaves, 457 cep 96015-560 – pelotas (rs), brasil e-mail: rafael.lund@gmail.com abstract aim: this study investigated the use of dental arch examination as a forensic technique for body identification at the institute of forensic medicine (iml, acronym in portuguese) of pelotas (rs), a city located in the south of brazil. methods: the data collected for the study referred to the period between 2004 and 2006, when a forensic dentist was part of the iml staff. the post-mortem records with regard to the entry of unidentified bodies that had undergone dental identification by the forensic dentists were analyzed quantitatively. results: ten unidentified bodies entered the iml and all of them were submitted to dental arch examination for body identification. however, the conclusive identification was based on the analysis of dna, because the victims’ dental records were not accurate, complete and updated. only five assessed bodies had been referred from pelotas police station, two from pedro osório police station, one from capão do leão police station, one from arroio grande police station and one had no information about its origin. conclusions: the current configuration of pelotas iml staff does not include a forensic dentist, and the presence of this professional is needed, for the city is reference for referral of forensic cases from the surrounding region. keywords: forensic dentistry; human identification; tooth; forensic anthropology. introduction forensic dentistry is the specialty that investigates psychological, physical, chemical and biological phenomena that can reach human beings (alive, dead or body fragments), comprehending aspects of human identification; criminal, civil, labor and administrative forensic investigation; forensic tanatology; legal documents; forensic traumatology; image examinations (e.g.: x-ray, tomography); saliva analysis and other aspects involving a multidisciplinary approach1. recent events have emphasized the important role that forensic dentistry plays in the identification of victims of air and industrial accidents, natural disasters and terrorist attacks2,3. furthermore, forensics dentistry also uses its expertise to identify the relation between aggressor and victim in crimes involving rape, murder, body injury and child abuse in which damage has occurred by leaving remaining bite marks2. the identification of human remains is usually made by photographs, comparison of radiographic images, fingerprints and, more recently, dna-based techniques. however, these 56 nedel f, nedel ap, silva rha, lund rg braz j oral sci. 8(1): 55-8 methods of identification have some limitations and may become ineffective when the investigated bodies are decomposed, skeletonized, fragmented or mutilated, preventing its recognition in expert investigations4-6. such cases are referred to forensic dentistry specialists. dental identification is the most commonly used technique in these cases. the application of forensic dentistry techniques may be very useful in human identification, for the dental tissue may resist extreme conditions of degradation, such as exposure to high temperatures, humidity and excessive pressure. the high mineral content of dental tissues, especially enamel, is responsible for their hardness and resistance, making the dental examination a key component for identification of human bodies5. the dental characteristics were proved to be particularly important for the identification of carbonized victims in mass disasters, when visual recognition and identification by means of fingerprints and dna analyses are not possible. the unique characteristics of a person’s teeth can make a positive identification of human corpus2. since restorations and dentures also have high durability, the combination of restored, non-restored, absent and temporary teeth can be exceptional as a fingerprint, and it is very unlikely for two dentitions to be completely equal4,7. the importance and acting fields of the forensic dentist at institutes of forensic medicine (iml, acronym in portuguese), outside them and in congener institutions, range from working as an expert or technical assistant (in cases of civil, criminal and labor area) to being the head of an administrative headquarter. furthermore, the different methods of human identification used in forensic dentistry (dental arch exams, palatal rugae and dna extraction of the dental pulp) must be exclusiveness of the forensic dentist because this professional is the only one who has the proper skills to intervene in the cadaver’s encephalic biotype. therefore, deep forensic knowledge from the dentist is required to work in this field because it implies several attributions and liabilities, such as being legally nominated to undertake several kinds of expertise in the civil, criminal and labor areas and acting in the administrative council8,9. this study investigated the use of dental arch examination as a forensic technique for body identification in the iml of pelotas (rs), a city located in the south of brazil. material and methods the research protocol was reviewed and approved by the research ethics committee of the universidade federal de pelotas, brazil. the data collected for the study referred to the period between 2004 and 2006, when a forensic dentist was incorporated in the staff of the iml of pelotas (rs), brazil. the post-mortem records referring to the entry of unidentified bodies that had undergone dental identification by the forensic dentist were analyzed quantitatively. the records comprised a complete tooth diagram (odontogram), detailed description of the upper and lower dental arches and information about age, skin color and gender. results data analysis revealed that, between 2004 and 2006, ten unidentified bodies entered the iml and were subjected to dental arch examination for body identification. from these, only five had been referred to the iml from pelotas police station, two from pedro osório police station, one from capão do leão police station, one from arroio grande police station and one case had no information about the body’s origin (figure 1). several methods of corpse identification are used in the expertise routine at pelotas’ iml. firstly and immediately, the clothes are carefully examined, photographed, described in the necropsy report and stored. within the clothes, it is possible to find, for example, an identity card, a driver’s license and personal objects in the pockets. these things give information about the possible places that the corpse had been to or where it had passed by, the social status of the victim, etc. all these data are extremely valuable to reach initial conclusions about corpse identification. after the necropsy, the human identification department searches for fingerprints in order to compare them to those registered in their files and identify the corpse by the datiloscopic method. this is when the forensic dentist may help in the identification of the corpse. the forensic dentist has to fill in a form with the corpse data, such as skin color, age, odontogram and the description of the upper and lower dental arches. discussion modern society was characterized by the high concentration of people in urban areas and consequent agglomerations in buildings, schools and restaurants, increasing the possibility of mass accidents, as seen in the attacks to the world trade center occurred on september 11, 20015,10. forensic dentistry has been requested in these scenarios, helping in the identification of bodies in major accidents. teeth have been widely used as source of information for human identification, especially when soft tissues cannot provide reliable information5. however, according to the brazilian law 5081/66, dentists acting in the field figure 1. cases that used dental identification referred from the police stations of surrounding cities. 10% 20% 10% 10% 50% pelotas police station no information of the bodys origin arroio grande police station capão do leão police station pedro osório police station legend source: iml, 2007. 57evaluation of identification cases involving forensic dentistry in the city of pelotas, rs, brazil, 2004-2006 braz j oral sci. 8(1): 55-8 of forensic dentistry have access to neck and head region and are not limited to the dentition. by means of an anthropometric study of elements of the skull and teeth, the forensic dentist can estimate height, sex, racial group (extremely questionable because of miscegenation) and age11-13. forensic techniques are used worldwide. it is important to have in mind that, in cases of major accidents, the traumatic forces of the accident are so intense that the fragmentation and conflagration permit only the preservation of the most resistant post-mortem tissues of the victims, which are those derived from the human dentition. in these cases, the teeth are the main source for identification of the corpses14,15. forensic dentistry can be very helpful on the identification of victims of mass disasters and, sometimes, in the differentiation of human remains of people victimized by situations like natural catastrophes, such as the tsunamis occurred in 200416,17, bus accidents involving body carbonization18,19, plane crashes20-22, firings23,24, train accidents25, military accidents and wars26. in brazil, there were two airplanes mass disasters in less than ten months with national flight companies. the first one happened on september 29th 2006, in mato grosso state, when a 737-800 new generation boeing struck against a n600l legacy jet, resulting in 154 victims27. the second accident was on july 17th 2007, in congonhas airport, são paulo, when an airbus 320 lost control while landing, resulting in 199 casualties28. in this context, the cooperation of forensic dentistry in the processes of post-mortem human identification has an irrefutable value, developing a fundamental status in the identification of bodies that cannot be identified by visual inspection or other traditional means29-31. therefore, dentists must have better working conditions to provide a most specialized contribution in cases of mass accidents and other types of collective disasters in the future15. furthermore, the identification of human remains depends on the availability of antemortem information collected from records and family members, and the existence of sufficient post-mortem material3,19. however, the process of dental identification is limited due to a number of reasons such as lack of standardization of dental documentation necessary for human identification, difficulty in gathering complete dental information for each individual, the incompleteness or inaccuracy of dental information, which compromises the quality of identification, and limitation of qualified personnel32. other conditions such as nature of the accident, nationality and home country of the victims, previous dental treatment, and severity of dental injury can determine the success of identification18. in a recent study, the relevance of dental records in the ‘tsunami mass disaster’ in thailand was evaluated. this study was interesting because the large number of tourists involved in that event, especially from europe, allowed a comparison of dental records from different countries. ninety percent of the victims from europe, north america, oceania and africa were identified mainly by means of dental records, indicating not only the existence, but the quality of these records. in contrast, 90% of the dental records from thailand were useless for identification of the victims33. according to the brazilian federal council of dentistry (cfo), dental record should contain the professional data (full name and the inscription number of the regional council) and the patient’s identification data (full name, sex, date of birth, home address etc), anamnesis (main reason to seek dental service, medical history and dental history), clinical examination (extraoral and intraoral exams and the use of an odontogram before and after the dental treatment), treatment planning, development and intercurrence of the treatment, prescriptions, complementary tests34. dental treatment records offer a valuable resource for establishing the identification of deceased people by means of dental comparison, as required for forensic purposes. the creation, maintenance, storage and custody of such records are legal and ethical duties of each dentist. they are also required by law to record and report evidence of child abuse observed in the course of treatment. furthermore, when dental records are required for forensic purposes, certain procedures should be followed for their release and collection35. the data collected in the present survey showed that all bodies that entered the iml of the city of pelotas between years 2004 and 2006, period in which the institute had a forensic dentist in its team, were subjected to dental examination for identification purposes. however, the conclusive identification was based on dna analysis because the victims’ dental records were not accurate, complete and updated. in contrast, dental identification has been used around the world with promising results. for example, in a bus accident that occurred in spain, 28 passengers lost their lives and dental identification was established in 57% of the cases18. also, in 1994, an m/s estonia ferry sunk, 94 victims were recovered and 60% could be identified by comparing antemortem and post-mortem dental data36. the lyon-strasbourg airline disaster in 1992 had a positive dental identification in 52% of cases and a partial match was achieved in 14% of cases25. currently, the iml of pelotas does not have a forensic dentist and so the unidentified bodies have to be transferred to the iml of porto alegre (capital of rio grande do sul state) or a forensic dentist has to be brought to pelotas, which is a disturbing situation. in the present survey, 40% of the cases were from areas surrounding pelotas, a reference for smaller cities. this situation illustrates that, in brazil, the forensic dentist is not appreciated as an important member of the forensic staff and that relevant information for human identification is not obtained. on the other hand, there is a demand for this technique since the iml received ten cases in only two years, which aggravates the problem of the current absence of a hired forensic dentist for the city of pelotas. according to meléndez37, the unsatisfactory presence of forensic dentist in forensic institutes and the negligence of general dentists with forensic matters can be justified by the few number of theoretical and practical hours in the discipline of forensic dentistry during graduation, the small number of forensic dentistry teachers with expertise in this field, lack of a phd programs in the area. however, a promising future can be anticipated for forensic dentistry since most latin american imls have already recognized the importance of having a forensic dentist in their staff. 58 nedel f, nedel ap, silva rha, lund rg braz j oral sci. 8(1): 55-8 it is extremely important that the imls have a forensic dentist in their team of specialists, for this professional can aggregate knowledge and contribute remarkably with cases in which the identification of human remains is difficult or in mass disasters. however, it is also of paramount importance that dentists keep complete, accurate and updated their patient’s records for providing reliable information so it can be actually used by forensic dentists. in the iml of pelotas, the presence of this professional is necessary because this city is the reference referral from smaller cities of the surrounding region. references 1. brasil. conselho federal de odontologia. resolução cfo 22/2001. baixa as normas sobre anúncio e exercício das especialidades odontológicas e sobre cursos de especialização, revogando as redações do capítulo viii, título i; capítulos i, ii e iii, título iii, das normas aprovadas pela resolução cfo-185/93, alterada pela resolução cfo-198/95. conselho federal de odontologia: brasília, df; 2001. 2. bushick rd. forensic dentistry: an overview for the general dentist. gen dent. 2006;54(1):48-52. 3. sakoda s, zhu bl, ishida k, oritani s, fujita mq, maeda h. dental identification in routine forensic casework: clinical and postmortem investigations. leg méd. 2000;2(1):7-14. 4. fereira jl, fereira ae, ortega ai. methods for the analysis of hard dental tissues exposed to high temperatures. forensic sci int. 2008;178(2-3):119-24. 5. ramenzoni ll, line sr. automated biometrics-based personal identification of the hunter-schreger bands of dental enamel. proc biol sci. 2006;273(1590):1155-8. 6. silva rh, sales-peres a, oliveira rn, oliveira ft, sales-peres sh. use of dna technology in forensic dentistry. j appl oral sci. 2007;15(3):156-61. 7. bush ma, miller rg, prutsman-pfeiffer j, bush pj. identification through x-ray fluorescence analysis of dental restorative resin materials: a comprehensive study of noncremated, cremated, and processed-cremated individuals. j forensic sci. 2007;52(1):157-65. 8. granjeiro jam. padronização de conduta na realização de perícias civis, criminais, trabalhistas e administrativas direcionados à odontologia legal. [master’s thesis]. piracicaba: faculdade de odontologia da unicamp; 2007. 9. cintra jaa. a importância da odontologia legal no exame de corpo delito [master’s thesis]. piracicaba: faculdade de odontologia da unicamp; 2004. 10. prieto jl, tortosa c, bedate a, segura l, abenza jm, mariscal de gante mc, et al. the 11 march 2004 madrid terrorist attacks: the importance of the mortuary organisation for identification of victims. a critical review. int j legal méd. 2007;121(6):517-22. 11. brasil. conselho federal de odontologia, lei 5081/66, artigo 6º, item 1, rj (1966 ago 24). 12. keiser-nielsen s, strom f. the odontological identification of eva braun hitler. forensic sci int. 1983;21(1):59-64. 13. miyajima f, daruge e, daruge-júnior e. a importância da odontologia na identificação humana: relato de um caso pericial. arq odontol. 2001;37(2):133-42. 14. gould ga. forensic odontology: a global activity. j calif dent assoc. 2004;32(5):410-5. 15. li g. [the role dental profession can play in mass casualty and disaster events]. [article in chinese]. hua xi kou qiang yi xue za zhi. 2008;26(4):347-51. 16. lau g, tan wf, tan ph. after the indian ocean tsunami: singapore’s contribution to the international disaster victim identification effort in thailand. ann acad med singapore. 2005;34(5):341-51. 17. morgan ow, sribanditmongkol p, perera c, sulasmi y, van alphen d, sondorp e. mass fatality management following the south asian tsunami disaster: case studies in thailand, indonesia, and sri lanka. plos med. 2006;3(6):e195. 18. valenzuela a, martin-de las heras s, marques t, exposito n, bohoyo jm. the application of dental methods of identification to human burn victims in a mass disaster. int j legal med. 2000;113(4):236-9. 19. valenzuela a, marques t, exposito n, martã n-de las heras s, garcã a g. comparative study of efficiency of dental methods for identification of burn victims in two bus accidents in spain. am j forensic med pathol. 2002;23(4):390-3. 20. ludes b, tracqui a, pfitzinger h, kintz p, levy f, disteldorf m, et al. medicolegal investigations of the airbus, a320 crash upon mount ste-odile, france. j forensic sci. 1994;39(5):1147-52. 21. ferreira ra. reconhecendo pela boca. rev assoc paul cir dent. 1996;50(6): 464-73. 22. nambiar p, jalil n, singh b. the dental identification of victims of an aircraft accident in malaysia. int dent j. 1997;47(1):9-15. 23. campobasso cp, falamingo r, vinci f. investigation of italy’s deadliest building collapse: forensic aspects of a mass disaster. j forensic sci. 2003;48(3):635-9. 24. pretty, ia. forensic dentistry: 1. identification of human remains. dent update. 2007;34(10):621-2, 624-6, 629-30 passim. 25. dumancić j, kaić z, njemirovskij v, brkić h, zecević d. dental identification after two mass disasters in croatia. croat med j. 2001;42(6):657-62. 26. brannon rb, morlang wm. the uss iowa disaster: success of the forensic dental team. j forensic sci. 2004;49(5):1067-8. 27. o globo. em menos de 10 meses os dois piores acidentes aéreos do país. são paulo, 18 de jul. 2007. [homepage on the internet]. [cited may 25, 2009]. available from: <http://oglobo.globo.com/sp/mat/2007/07/17/em_menos_ de_10_meses_os_dois_piores_acidentes_aereos_do_pais-296831801.asp>. o globo. são paulo. 28. folha on line. justiça suspende todos os indiciamentos do acidente da tam. são paulo, 24 nov. 2007. [homepage on the internet]. [cited may 25, 2009]. available from: <http://www1.folha.uol.com.br/folha/cotidiano/ult95u471173. shtml>. 29. calabrez mct, saldanha ph. a pesquisa de dna em odontologia forense. in: silva m. compêndio de odontologia legal. são paulo: medsi; 1997. p. 167-221. 30. sweet, d. why a dentist for identification? dent clin north am. 2001;45(2): 237-51. 31. glass, rt. forensic dentistry in a terrorist world. n y state dent j. 2005;71(3):21-5. 32. chomdej t, pankaow w, choychumroon s. intelligent dental identification system (idis) in forensic medicine. forensic sci int. 2006;158(1):27-38. 33. petju m, suteerayongprasert a, thongpud r, hassiri k. importance of dental records for victim identification following the indian ocean tsunami disaster in thailand. public health. 2007;121(4):251-7. 34. brasil. conselho federal de odontologia. prontuário odontológico: uma orientação para o cumprimento da exigência contida no inciso viii do art. 5° do código de ética odontógica. conselho federal de odontologia: rio de janeiro; 2004. 35. brown ka. procedures for the collection of dental records for person identification. j forensic odontostomatol. 2007;25(2):63-4. 36. soomer h, ranta h, penttilä a. identification of victims from the m/s estonia. int j legal med. 2001;114(4-5):259-62. 37. meléndez bvdlc. o perfil do ensino da odontologia legal na américa latina [master’s thesis]. piracicaba: faculdade de odontologia da unicamp; 2003. oral sciences n3 received for publication: may 03, 2010 accepted: august 23, 2010 original article braz j oral sci. october|december 2010 volume 9, number 4 shear bond strength test using different loading conditions – a finite element analysis rodivan braz1, mário alexandre coelho sinhoreti2, aloísio oro spazzin3, sandro cordeiro loretto4, arine maria viveros de castro lyra1, agenor dias de meira-júnior5 1dds, ms, phd, professor, department of restorative dentistry, school of dentistry, university of pernambuco, recife, pe, brazil 2dds, ms, phd, professor, department of restorative dentistry, dental materials division, piracicaba dental school, state university of campinas, piracicaba, sp, brazil 3dds, research scientist, department of restorative dentistry, dental materials division, piracicaba dental school, state university of campinas, piracicaba, sp, brazil 4dds, ms, phd, research scientist, department of restorative dentistry, universitary nucleus of pará, belém, pa, brazil 5ms, phd, professor, department of mechanical engineering, school of engineering and architecture, university of passo fundo, passo fundo, rs, brazil correspondence to: aloísio oro spazzin departmento de odontologia restauradora, área materiais dentários, fop/unicamp, piracicaba, sp, brazil. av. limeira 901, vila rezende, 13414-903, piracicaba, sp, brazil; phone: +55 (19) 2106 5345, fax: +55 (19) 2106 5211 e-mail: aospazzin@yahoo.com.br abstract aim: this study evaluated the stress distribution at the bond interface during shear bond strength testing for three loading conditions. methods: a three-dimensional model was created of a specimen for evaluation by the shear bond strength test, using three cylindrical volumes representing the dentin, adhesive system and composite resin. a linear analysis was performed to calculate the stress distribution at the dentin-adhesive interface. three models simulating different loading conditions were prepared: chisel, orthodontic-looped wire and stainless steel tape. results: chisel presented severe stress concentrations near the loading site (-10681 to 637 mpa). wire presented stress concentrations along the radial loading axis (-382 to 216 mpa). tape presented more uniform stress distribution (-83 to 21 mpa). conclusions: the loading with stainless steel tape allowed more uniform stress distribution at the bond interface, and was a more reliable way to evaluate the bond with regard to the aim of the shear bond strength test. keywords: shear bond strength, stress distribution, loading. introduction in-vitro mechanical tests of dental restorative materials provide dental practitioners with guidance as regards material selection criteria and identifying patterns of optimal clinical use of material. the quality of adhesive material bonding is frequently verified by various laboratory tests, using shear and tensile efforts under certain limitations1. in 1997, a study evaluated 50 studies that used laboratory tests to quantify the bond strength at the bond interface, and observed that 80% used the shear bond strength (sbs) test in its several forms2. today, use of the microtensile bond strength (µtbs) and microshear bond strength (µsbs)3 tests have increased considerably. however, several recent studies still use the sbs test to evaluate adhesive material bonding4-10. in some situations, sectioning of the specimen for µtbs induces its loss due to failure before the test, and µsbs cannot be used braz j oral sci. 9(4):439-442 because of the difficulty of making specimens with some materials. in these cases, the sbs test may be used to evaluate adhesive material bond strength. it is important to consider the changes in the test procedures commonly applied in different investigations that have the same aim: to determine the bond strength. for this reason, analyses of the same material inevitably produce different bond strength data1,11-14. a factor concerns the stress created at the bond interface by the load applied. sinhoreti et al. 15 compared the morphological characteristics of the fractured compositedentin interface, using an iso specified test (loading applied with chisel), and non-specified tests (loading applied with stainless steel tape and orthodontic-looped wire). the authors found that the failures were fractures between the adhesive and the dentine when the stainless steel tape was used, suggesting that this loading condition was not subject to the complexity of stress produced by a loading test1. however, no information was found in the literature on how the stress distribution occurs at the bond interface during the sbs test under these three conditions. the simultaneous interaction of the many variables affecting a restorative system can be studied using simulation in a computerized model. the finite element analysis (fea) consists of dividing a geometric model into a finite number of elements, each with specific physical properties. the variables of interest are approximated with some mathematical functions. stress distributions in response to different loading conditions can be simulated with the aid of computers with dedicated software16. the aim of this study was to compare the effects of different loading conditions (chisel, orthodontic-looped wire and stainless steel tape systems) used in shear bond tests on stress distribution at the dentin-adhesive interface using fea. the hypothesis tested was that the loading condition simulating a stainless steel tape creates a more uniform stress distribution at the bond interface. materials and methods a 3-d model was created of a specimen for evaluation by the shear bond strength test, using three cylindrical volumes representing the dentin (6 mm diameter and 1 mm thick), adhesive system (4 mm diameter and 10 µm thick) and composite resin (4 mm diameter and 6 mm thick). the study model presented the configurations and dimensions presented in figure 1. the fea was performed with the fe software program (ansys rel 5.2, ansys inc., houston, tx, usa). the model components were assumed to be isotropic. the elastic constants used in the calculations were obtained from the literature (table 1)17-18. the solid92 element was used, dentin and composite resin (the solid corpus), with 10 nodes and three degrees of freedom per node. the following assumptions were made: there is complete bonding between dentin, adhesive and composite resin; dentin was assumed to contain elastic isotropic material. the volumes were meshed, finally resulting in a 3-d fe model with 15,436 elements and 23,835 nodes. components young’s modulus(e) poisson’s ratio(í) reference dentin 18 gpa 0.31 18 adhesive system 4 gpa 0.35 17 composite resin 10 gpa 0.25 17 table 1. mechanical properties considered for fea fig. 1 (a) components involved in the investigated model; (b) dimensions (mm) of investigated model all of the nodes on the external dentin surface were constrained in all directions. a linear static structural analysis was performed to calculate the stress distribution at the dentinadhesive interface, under a total load of 200 n. this load produced a mean shearing stress of approximately 16 mpa. three experimental models simulating different loading conditions were performed (figure 2): chisel group – punctual loading at the adhesive-dentin interface, simulating the load applied on the specimen with a chisel; wire group – application of a radial loading 0.5 mm from adhesive-dentin interface, simulating the load applied on the specimen with orthodontic-looped wire; tape group – application of a radial pressure with 5 mm of width, simulating the load applied on the specimen with a stainless steel tape; accuracy of the model was checked using convergence tests. particular attention was given to the refinement of the mesh resulting from the convergence tests at the interfaces. the results were qualitative and quantitatively analyzed with regard to shearing stress distribution at the dentin-adhesive interface. fig. 2 schematic illustrations of the different loading conditions: (a) chisel simulation; (b) orthodontic-looped wire simulation; (c) stainless steel tape simulation 440440440440440shear bond strength test using different loading conditions – a finite element analysis braz j oral sci. 9(4):439-442 fig. 3 shearing stress distribution in the different loading simulations: (a) chisel; (b) orthodontic-looped wire; (c) stainless steel tape. arrows indicate the direction of load incidence results the stress distribution at the bond interface for the different loading conditions is shown in figure 3. for the chisel group, the bond interface presented high stress levels concentrated next to the point of load application. the shearing stress values ranged from -10681 to 637 mpa. for the wire group, the bond interface presented stress concentration along the radial loading axis, but it was considerably lower than that for the chisel. the shearing stress values ranged from -382 to 216 mpa. for the tape group, the bond interface did not present peaks of stress concentration, showing more uniform stress distribution. the shearing stress values ranged from -83 mpa to 21 mpa. discussion the results of the current study showed more homogenous stress distribution at the bond interface during the shear bond strength test using stainless steel tape, supporting the hypothesis of the study. the larger area of contact between the stainless steel tape and the specimen created stress distribution over the entire bond interface. therefore, the lower stress concentration along the bond interface explains that using tape, sliding occurs between the components of the specimens, characterizing a shear bond strength test. as regards the loading using the chisel, severe stress concentrations were presented near the loading site, caused by the simulation of the small area of contact between the chisel and the specimen (punctual load). this loading condition creates stresses of complex nature, involving cleavage, traction and compression1,19. cohesive failures in dentin are commonly found, with portion of the substrate being literally pulled away1,20. the strength values obtained must be ignored when these cohesive failures occur in the dentin, once they do not represent the mean strength measured at the bond interface, and but the cohesive strength of the substrate19-20. the loading simulating the orthodontic-looped wire also showed stress concentrations, although considerably lower compared with the chisel. a region showing the presence of red color near the radial load observed in figure 3 indicates the effect of the stress concentrations due to the small area of contact between the wire and specimen. the stresses are not distributed along of whole the interface as when using tape. sinhoreti et al.1 considered the failure using wire caused by flexional stress, promoting cohesive fracture of the composite or cohesive fracture of the adhesive. the wire has circular transversal section so the force cannot be applied joint to interface, compatible with the simulation using wire in the current study, in which the radial load was applied 0.5 mm from the bond interface. this fact could increase the flexion pattern at the bond interface21-22. in addition, the data of the present study explained the results found by sinhoreti et al.1. among the three loading conditions tested, they found the lowest bond strength values for loading using tape, and interfacial failures between dentin and adhesive, suggesting that tape creates the best condition for establishing the true shear bond strength test. the bond failure occurs due the sliding between the surfaces of composite resin and dentin, as result concentration of tangential force. moreover, this load produces no fulcrum point or flexion of the composite cylinder, or superficial cleavage, as observed for the load with orthodontic-looped wire and chisel, respectively1. della-bona et al. 23 suggested that the sbs test is 441441441441441 shear bond strength test using different loading conditions – a finite element analysis braz j oral sci. 9(4):439-442 inadequate as a means of assessing the quality of the adhesive bond of resin composite to ceramic. however, in some situations, sectioning the specimen for the µtbs induces its loss by failure before of the test. in these cases, the sbs test using the stainless steel tape may decrease the tensile and compressive stresses during the test to evaluate the bond of these friable materials. considering the results obtained with 3-d fea and literature available, it may be concluded the loading with stainless steel tape allows more uniform stress distribution along the bond interface. therefore, loading using a tape is a more reliable method and must be used to evaluate the bond strength of adhesive materials concerning the aim of the sbs test. acknowledgements the authors thank the dr cezar augusto garbin (in memoriam) for his generous help, essential for carrying of study. the faculty of mechanical engineering of the engineering and architecture school, university of passo fundo, rs, for its help with the element finite analysis. references 1. sinhoreti ma, consani s, de goes mf, sobrinho lc, knowles jc. influence of loading types on the shear strength of the dentin-resin interface bonding. j mater sci mater med. 2001; 12: 39-44. 2. al-salehi sk, burke fj. methods used in dentin bonding tests: an analysis of 50 investigations on bond strength. quintessence int. 1997; 28: 717-23. 3. placido e, meira jb, lima rg, muench a, de souza rm, ballester ry. shear versus micro-shear bond strength test: a finite element stress analysis. dent mater. 2007; 23: 1086-92. 4. kanehira m, finger wj, ishihata h, hoffmann m, manabe a, shimauchi h, et al. rationale behind the design and comparative evaluation of an allin-one self-etch model adhesive. j dent. 2009; 37: 432-9. 5. czarnecka b, deregowska-nosowicz p, limanowska-shaw h, nicholson jw. shear bond strengths of glass-ionomer cements to sound and to prepared carious dentine. j mater sci mater med. 2007; 18: 845-9. 6. ercan e, erdemir a, zobra yo, eldeniz au, dalli m, ince b, et al. effect of different cavity disinfectans on shear bond strength of composite resin to dentin. j adhes dent. 2009; 11: 343-6. 7. dos santos pa, garcia pp, palma-dibb rg. shear bond strength of adhesive systems to enamel and dentin. thermocycling influence. j mater sci mater med. 2005; 16: 727-32. 8. aida m, odaki m, fujita k, kitagawa t, teshima i, suzuki k, et al. degradation-stage effect of self-etching primer on dentin bond durability. j dent res. 2009; 88: 443-8. 9. phark jh, duarte s, jr., kahn h, blatz mb, sadan a. influence of contamination and cleaning on bond strength to modified zirconia. dent mater. 2009; 25: 1541-50. 10. erickson rl, barkmeier ww, kimmes ns. bond strength of self-etch adhesives to pre-etched enamel. dent mater. 2009; 25: 1187-94. 11. pecora n, yaman p, dennison j, herrero a. comparison of shear bond strength relative to two testing devices. j prosthet dent. 2002; 88: 511-5. 12. van noort r, cardew ge, howard ic, noroozi s. the effect of local interfacial geometry on the measurement of the tensile bond strength to dentin. j dent res. 1991; 70: 889-93. 13. katona tr, long rw. effect of loading mode on bond strength of orthodontic brackets bonded with 2 systems. am j orthod dentofacial orthop. 2006; 129: 60-4. 14. oilo g, olsson s. tensile bond strength of dentin adhesives: a comparison of materials and methods. dent mater. 1990; 6: 138-44. 15. iso/tr-11405. guidance on testing of adhesion to tooth structure. international standardization organization. 1994. 16. spazzin ao, galafassi d, de meira-junior ad, braz r, garbin ca. influence of post and resin cement on stress distribution of maxillary central incisors restored with direct resin composite. oper dent. 2009; 34: 223-9. 17. dehoff ph, anusavice kj, wang z. three-dimensional finite element analysis of the shear bond test. dent mater. 1995; 11: 126-31. 18. kamposiora p, papavasilious g, bayne sc, felton da. finite element analysis estimates of cement microfracture under complete veneer crowns. j prosthet dent. 1994; 71: 435-41. 19. sudsangiam s, van noort r. do dentin bond strength tests serve a useful purpose? j adhes dent. 1999; 1: 57-67. 20. tantbirojn d, cheng ys, versluis a, hodges js, douglas wh. nominal shear or fracture mechanics in the assessment of composite-dentin adhesion? j dent res. 2000; 79: 41-8. 21. van noort r, noroozi s, howard ic, cardew g. a critique of bond strength measurements. j dent. 1989; 17: 61-7. 22. versluis a, tantbirojn d, douglas wh. why do shear bond tests pull out dentin? j dent res. 1997; 76: 1298-307. 23. della bona a, van noort r. shear vs. tensile bond strength of resin composite bonded to ceramic. j dent res. 1995; 74: 1591-6. 442442442442442shear bond strength test using different loading conditions – a finite element analysis braz j oral sci. 9(4):439-442 oral sciences n3 braz j oral sci. 10(1):55-59 original article braz j oral sci. january | march 2011 volume 10, number 1 effect of the smear layer on the filling of artificial lateral canals and microleakage andréa gonçalves moretti1, carlos augusto de morais souto pantoja2, danna mota moreira2, alexandre augusto zaia3, josé flávio affonso de almeida3 1undergraduate student, department of restorative dentistry, endodontics division, piracicaba dental school, university of campinas, sp, brazil 2graduate student, department of restorative dentistry, endodontics division, piracicaba dental school, university of campinas, sp, brazil 3associate professors, department of restorative dentistry, endodontics division, piracicaba dental school, university of campinas, sp, brazil correspondence to: josé flávio affonso de almeida department of restorative dentistry, endodontics division, piracicaba dental school, university of campinas. av. limeira, 901, 13.414-903 piracicaba,sp, brazil. phone: +55 19 2106 5344 fax: +55 19 2106 5218 e-mail: jfalmeida@fop.unicamp.br received for publication: november 10, 2010 accepted: february 23, 2011 abstract aim: to evaluate the influence of the smear layer on the filling and prevention of microleakage in artificial lateral canals after use of different irrigating solutions. methods: two lateral canals were produced in 44 human dental roots with drills of 0.1 mm in diameter. the roots were divided in 4 groups according to the irrigation protocol: gi 0.9% saline solution (control); gii 2.5% sodium hypochlorite + 17% edta; giii 2% chlorhexidine gel + 17% edta; giv 2% chlorhexidine solution + 17% edta. four roots were used as negative (n=2) and positive (n=2) controls. lateral condensation technique and ah plus were used to fill the root canals. digital buccolingual radiographs were exposed and after the sealer had set, the roots were immersed in indian ink dye and then cleared in methyl salicylate. the extent of filling and microleakage were measured and the values analyzed statistically. results: no difference was found in the percentage of filling and microleakage among the groups, regardless of the location of the artificial lateral canals and the irrigation protocol used. twenty percent of lateral canals produced no radiographic images, but their filling was confirmed using the clearing technique. conclusions: the presence or absence of the smear layer did not affect the filling ability and the prevention of microleakage in artificial lateral canals. keywords: endodontics, root canal obturation, smear layer. introduction the smear layer consisting of dentin shavings, cell debris and pulp remnants1-5, includes two separate strata: a loose superficial deposit and an attached stratum that extends into the dentinal tubules and forms occluding plugs6. several studies have shown the effect of smear layer on the filling and adhesive procedures7-9. the removal of the smear layer promotes an increase in dentin permeability, thereby allowing greater penetration of filling materials inside the root canal system10-12. considering that lateral canals have small diameters, equal to or higher than 0.1 mm ranging from of 43.6%13 to 69.3%14, the removal of smear layer could enrich the filling of these structures. the use of different irrigating regimens during endodontic treatment can improve both antimicrobial activity and the removal of the smear layer from the dentin walls. according to goldberg et al.15 (1986), removal of the smear layer using 15% edta can improve the filling of lateral canals, when compared to the braz j oral sci. 10(1):55-59 use of 5% naocl and distilled water. however, villegas et al.16 (2002) evaluated the filling of accessory canals using cleared teeth and the warm obturation technique. different irrigating solutions were used and the authors observed no differences between 6% naocl alone or in association with 15% edta. this result was similar to that of bertacci et al.17 (2007) who found no influence of edta on the filling of lateral canals. although a warm gutta-percha filling can cross over the smear layer, there is still no consensus on whether the smear layer can affect lateral condensation, one of the most commonly used filling techniques18-19, or ah plus in the filling and prevention of microleakage in lateral canals. thus, the aim of this study was to evaluate the ability of lateral condensation and ah plus to fill and prevent microleakage in the presence or absence of the smear layer in artificial lateral canals. material and methods fifty-two recently extracted human maxillary anterior teeth were selected after the approval of the human research ethics committee of the piracicaba dental school, university of campinas, brazil. the crowns were removed using a lowspeed diamond disc under running water and a standard length of 15 mm was achieved for each root. digital buccolingual radiographs (digora, orin corp., soredex, helsinki, finland) was obtained and teeth with resorption, fracture or incompletely formed apices were excluded from the study. the artificial lateral canals were prepared, one in the middle and the other in the apical third, perpendicular to the longitudinal axis of the teeth using 0.10 mm cylindrical drills (unio tool europe s.s., marin, neuchâtel, switzerland) according to almeida et al.20. the penetration of the drill into the root canal was checked by introducing a size 06 kfile (dentsply maillefer, ballaigues, switzerland) into the perforations and another digital radiograph was exposed. if the file did not penetrate into the main root canal, the tooth was discarded. initially, the teeth were instrumented with largo drills #3-2 and gates glidden drills #5-2 (maillefer, ballaigues, switzerland). the working length was established 1 mm short of the apex. all teeth were instrumented at the working length to a #40 file and the apical patency was checked with of a # 25 file. the irrigation protocols were carried out as follows (n=10): group i irrigation with 3 ml of 0.9% saline solution after the use of each instrument. group ii –the root canal was filled with 3 ml of 2.5% sodium hypochlorite (naocl), instrumented, and finally irrigated with 3 ml of saline after each instrument. the root was refilled with naocl. finally, 3 ml of 17% edta was used to remove the smear layer for 3 min, and 3 ml of 0.9% saline solution were used as the final irrigant. group iii the canals were filled with 2% chlorhexidine gel (clx gel), instrumented and finally irrigated with 3 ml of 0.9% saline solution after the use of each instrument. then, 3 ml of 17% edta was used to remove the smear layer for 3 min, and 3 ml of 0.9% saline solution were used as the final irrigant. group iv as in group iii, but using 2% chlorhexidine solution (clx sol) instead of 2% chlorhexidine gel (clx gel). before root canal filling, the teeth were mounted in alginate blocks (dentsply, petrópolis, rj, brazil) to confine the sealer after it had flowed through the artificial lateral canals, in an attempt to simulate the periodontal ligament according to almeida et al.20. the root canals were dried using paper points (tanari, manacapuru, am, brazil) and were obturated by lateral condensation of the gutta-percha and ah plus sealer mixed according to the manufacturer’s instructions and placed in the canal roots with a size 40 spiral lentulo (maillefer). a standardized gutta-percha cone #40 (tanari) was fitted as the master cone. lateral condensation was performed using a size 30 endodontic finger spread (dyna, bourges, france) and b7 and b8 accessory gutta-percha cones (tanari). following obturation, the excess gutta-percha was removed with a heated instrument (3 mm inside the canal), and cold pluggers (duflex-s.s. white; rio de janeiro, rj, brazil) were used to vertically condense the coronal gutta-percha. the coronal access was filled with temporary cement (coltosol; vigodent sa, rio de janeiro, rj, brazil). the filled teeth were stored at 100% humidity at 37 °c for 48 h to allow the sealers to set. the digital radiographs were taken and the images were assessed using the image manager program (im50; leica microsystems, wetzlar, germany). the total length of the lateral canal and its filling was measured and the values obtained were converted to the percentage of canal filled. the external root surface was sealed with two layers of red nail varnish, except for the area over the lateral canals. four unfilled teeth were used as positive and negative microleakage controls. the positive control was sealed like the other teeth and the negative control was sealed completely including the lateral canal orifices. images of two additional teeth for each group were taken in a scanning electronic microscope (jeol; model jsm 5600 lv, tokyo, japan) to examine the cleanness of the root canal walls in the middle and apical third illustratively. the roots were then immersed in indian ink (royal talens, apeldoorn, the netherlands), submitted to vacuum for 30 min and stored in the ink for 24 h. following exposure to the dye, the roots were rinsed in tap water and the nail varnish was removed completely. the teeth were decalcified in 5% hydrochloric acid for 72 h and cleared in methyl salycilate according to the protocol reported by zaia et al.21. the extent of filling, dye penetration and the total length of the lateral canals were measured on the buccal and lingual root surfaces, under a magnifying microscope (×34) (leica microsystems, wetzlar, germany). the obturation of the lateral canals was measured linearly, using a scale in millimeters. the values obtained were divided by the total length of the lateral canal and multiplied by 100 to obtain the percentage of obturation after the use of each irrigant solution. similarly, the leakage of dye between the sealer 56effect of the smear layer on the filling of artificial lateral canals and microleakage 57 braz j oral sci. 10(1):55-59 and the wall of the lateral canal was measured and the values were divided by the amount of sealer in the lateral canal. the kruskal-wallis test was used to compare the values of dye microleakage obtained in the middle and apical third of the root canal with different irrigating solutions. anova and post hoc tukey’s test was used to evaluate the values of filling obtained between experimental groups considering the same third. the wilcoxon test was used to compare the values obtained from the different thirds of the root canal after the use of each irrigation protocol. results the radiographic analysis failed to identify filled lateral canals in 20% of specimens, although these were visible using the clearing technique (figure 1). thus, the parameters for establishing the filling results were based on the clearing technique and dye microleakage, when partially filled or unfilled lateral canals were shown by x-rays. the percentage of filled lateral canals in each group was statistically similar, regardless of the location of the lateral canals or the presence/absence of the smear layer (figure 2, table 1). sem micrographs revealed smear layer along almost all root canal walls and covering the artificial lateral canal openings in group i (saline solution without edta). the other groups (with edta) presented root canal walls almost clean and artificial lateral canals partially or totally opened (figure 3). the linear analysis of microleakage between the sealer and dentinal walls revealed variability among the groups of auxiliary chemical substances and the presence/absence of the smear layer (figure 2). however, no statistically significant differences were found (p = 0.1283 kruskal-wallis test table 2). discussion although some studies have shown that lateral condensation results in less filling of lateral canals when effect of the smear layer on the filling of artificial lateral canals and microleakage fig. 1 (a) radiographic image showing absence of filling of the lateral canal in the middle third (arrow); (b) cleared tooth image showing the middle and apical lateral canals completely filled (×34 magnification). 0.9% ss 100 a# 0–100 100 a 0–100 0.79 2.5% naocl + 17% edta 100 a 0–100 100 a 0–100 1.00 2% chx gel + 17% edta 100 a 43–100 100 a 0–100 0.10 2% chx sol + 17% edta 100 a 14–100 61 a 0–100 0.08 median range median range (%) (%) apical third middle third groups p table 1: medians of filling percentages for each irrigation protocol and p-values between root canal thirds for each group. #comparison between medians for apical and middle thirds considering the same irrigation protocol is presented in the same row (wilcoxon test, α=5%). medians followed by the same letter in each column do not differ significantly (anova post hoc tukey, p>0.05). group median range (%) 0.9% ss 0a 0-83 2.5% naocl + 17% edta 0a 0-85 2% chx gel + 17% edta 0a 0-36 2% chx sol + 17% edta 0a 0-100 table 2:median of dye microleakage percentages for each irrigation protocol. medians followed by the same letter in each column do not differ significantly (kruskal-wallis test, p>0.05). fig. 2 images of the filling of artificial lateral canals and microleakage occurring in the apical and middle third of the artificial lateral canals: (a) group i microleakage in the lateral canal of the middle third (arrow) and absence of microleakage in the apical lateral canal; (b, c, d) groups ii, iii, iv abscence of microleakage in both lateral canals. 58 braz j oral sci. 10(1):55-59 compared with the thermoplasticized gutta-percha22-23, others have shown no significant differences between the two techniques 24-25, or that lateral condensation is able to efficiently fill the natural root ramifications15,20,26. analysis of the specimens via digital radiography, clearing and dye leakage showed no significant differences in the filling of artificial lateral canals in the presence or absence of the smear layer, regardless of the location of the lateral canals. according to villegas et al.16 (2002) irrigation with 2.5% sodium hypochlorite alone or combined with edta gives better sealing of natural accessory canals compared with instrumentation without irrigation or with irrigation with distilled water. these results are attributed to the fact that edta in combination with other irrigating solutions removes inorganic tissues during irrigation, resulting in better penetration of the filling material15-16. the ability to remove the smear layer presented by sodium hypochlorite alone or associated with edta can be observed with the use of 2% chlorhexidine gluconate gel. this auxiliary chemical substance is able to eliminate anaerobic microorganisms and decrease the formation of smear layer in the root canals walls27. the divergence of the results of this study from those described above can be explained by the fact that the smear layer formed during instrumentation, especially in group i, was not able to totally obliterate the previously empty artificial lateral canals, or even to prevent the compression of filling materials. it should be emphasized that the smear layer is not an organized structure28 and does not offer any resistance when the filling material is compressed into the natural ramifications, which are much larger than the dentinal tubules. kasahara et al.29 (1990) showed that about 90% of ramifications present on 510 maxillary central incisors had thick less or equal than 150 µm. in this study, the diameter of artificial lateral canals was 100 µm. bertacci et al.17 (2007) reported that the thickness of the endodontic smear layer is probably 1-5 µm and can easily be pushed with enough pressure by warm gutta-percha inserted inside the root canal. like warm gutta-percha, lateral condensation and ah plus could cross over the smear layer into artificial lateral canals. although group iv presented higher rates of dye penetration, the results of this study showed no significant differences after the use of different irrigation protocols. similarly, after the use of different irrigants such as saline solution, 5.25% sodium hypochlorite and 0.12% chlorhexidine digluconate to prepare the root canal, there was no difference in microleakage in the apical region of the root canal30. however, removal of the smear layer using 5.25% naocl associated with 17% edta had a positive effect on reducing apical and coronal leakage5. it is likely that in artificial lateral canals the thickness of the smear layer is not sufficient to obstruct the penetration and reduce the sealing ability of filling materials. the physical and chemical properties of sealers or the root filling techniques may be more important for filling and preventing microleakage than the presence of smear layer in artificial lateral canals. according to wu et al. 31 (1994) some endodontic sealers with a thinner film thickness, between 0.25 to 0.05 mm, can seal tightly the root canal filling. in this study, the ah plus sealer and the diameter of ramifications were able to prevent microleakage. twenty-nine natural ramifications were found in 13 teeth, comprising lateral, secondary and accessory canals. almeida et al.20 (2007) used lateral condensation, as in this study, and found 18 ramifications in 13 teeth. other studies showed a total of 100 filled lateral canals in 84 teeth32, 130 filled lateral canals in 40 teeth17 and 318 filled ramifications in 64 teeth33. these divergent results are probably due to the group of teeth analyzed or the filling technique used in each study. in conclusion, the presence or absence of the smear layer did not affect the ability of lateral condensation and ah plus to fill and prevent microleakage in artificial lateral canals. the radiograph images were not able to demonstrate artificial lateral canal filling in approximately 20% of cases, but their filling was confirmed using the clearing technique. references 1. mccomb d, smith dc. a preliminary scanning electron microscopic study of root canals after endodontic procedures. j endod. 1975; 1: 238-42. 2. goldman m, goldman lb, cavaleri r, bogis j, lin ps. the efficacy of several endodontic irrigating solutions: a scanning electron microscopic study: part 2. j endod. 1982; 8: 487-92. 3. mader cl, baumgartner jc, peters dd. scanning electron microscopic investigation of the smeared layer on root canal walls. j endod. 1984; 10: 477-83. 4. sen bh, safavi ke, spangberg ls. antifungal effects of sodium hypochlorite and chlorhexidine in root canals. j endod. 1999; 25: 235-8. 5. cobankara fk, adanr n, belli s. evaluation of the influence of smear layer on the apical and coronal sealing ability of two sealers. j endod. 2004; 30: 406-9. effect of the smear layer on the filling of artificial lateral canals and microleakage fig. 3 sem micrographs from the root canal walls adjacent to the lateral canals in the middle third (×500 magnification): (a) group i 0.9% saline solution: presence of smear layer obliterating the dentinal tubules and artificial lateral canal; (b) group ii 2.5% sodium hypochlorite + 17% edta: lateral canal and dentinal tubules free of artificial smear layer; (c) group iii 2% chlorhexidine gel + 17% edta: dentinal tubules and lateral channel free smear layer; (d) group iv – 2% clorexidine solution + 17% edta: some dentinal tubules exposed and partial obstruction of the canal side by smear layer. 59 braz j oral sci. 10(1):55-59 6. cameron ja. the use of ultrasonics in the removal of the smear layer: a scanning electron microscope study. j endod. 1983; 9: 289-92. 7. kouvas v, liolios e, vassiliadis l, parissis-messimeris s, boutsioukis a. influence of smear layer on depth of penetration of three endodontic sealers: an sem study. endod dent traumatol. 1998; 14: 191-5. 8. serafino c, gallina g, cumbo e, ferrari m. surface debris of canal walls after post space preparation in endodontically treated teeth: a scanning electron microscopic study. oral surg oral med oral pathol oral radiol endod. 2004; 97: 381-7. 9. kokkas ab, boutsioukis ach, vassiliadis lp, stavrianos ck. the influence of the smear layer on dentinal tubule penetration depth by three different root canal sealers: an in vitro study. j endod. 2004; 30: 100-2. 10. pashley dh, kehl t, pashley e, palmer p. comparison of in vitro and in vivo dog dentin permeability. j dent res. 1981; 60: 763-8. 11. lloyd a, thompson j, gutmann jl, dummer pm. sealability of the trifecta technique in the presence or absence of a smear layer. int endod j. 1995; 28: 35-40. 12. hülsmann m, heckendorff m, lennon a. chelating agents in root canal treatment: mode of action and indications for their use. int endod j. 2003; 36: 810-30. 13. kasahara e, yasuda e, yamamoto a, anzai m. root canal system of the maxillary central incisor. j endod. 1990; 16: 158-61. 14. miyashita m, kasahara e, yasuda e, yamamoto a, sekizawa t. root canal system of the mandibular incisor. j endod. 1997; 23: 479-84. 15. goldberg f, massone je, spielberg c. effect of irrigation solutions on the filling of lateral root canals. endod dent traumatol. 1986; 2: 65-6. 16. villegas jc, yoshioka t, kobayashi c, suda h. obturation of accessory canals after four different final irrigation regimes. j endod. 2002; 28: 534-6. 17. bertacci a, baroni c, breschi l, venturi m, prati c. the influence of smear layer in lateral channels filling. clin oral investig. 2007; 11: 353-9. 18. gopikrishna v, parameswaren a. coronal sealing ability of three sectional obturation techniques—simplifill, thermafil and warm vertical compaction— compared with cold lateral condensation and post space preparation. aust endod j. 2006; 32: 95-100. 19. kontakiotis e, chaniotis a, georgopoulou m. fluid filtration evaluation of 3 obturation techniques. quintessence int. 2007; 38: 410-6. 20. almeida jf, gomes bp, ferraz cc, souza-filho fj, zaia aa. filling of artificial lateral canals and microleakage and flow of five endodontic sealers. int endod j. 2007; 40: 692-9. 21. zaia aa, nakagawa r, de quadros i, gomes bp, ferraz cc, teixeira fb, et al. an in vitro evaluation of four materials as barriers to coronal microleakage in root-filled teeth. int endod j. 2002; 35: 729-34. 22. dulac ka, nielsen cj, tomazic tj, ferrilo pj jr, hatton jf. comparison of the obturation of lateral canals by six techniques. j endod. 1999; 25: 376-80. 23. goldberg f, artaza lp, de silvio ac. effectiveness of different obturation techniques in the filling of simulated lateral canals. j endod. 2001; 27: 362-4. 24. clark ds, el deeb me. apical sealing ability of metal versus plastic carrier thermafil obturators. j endod. 1993;19: 4-9. 25. reader cm, himel vt, germain lp, hoen mm. effect of three obturation techniques on the filling of lateral canals and the main canal. j endod. 1993; 19: 404-8. 26. pécora jd, ribeiro rg, guerisoli dm, barbizam jv, marchesan ma. influence of the spatulation of two zinc oxide-eugenol-based sealers on the obturation of lateral canals. pesq odontol bras. 2002; 16: 127-30. 27. ferraz ccr, gomes bpfa, zaia aa, teixeira fb, souza-filho fj. in vitro assessment of the antimicrobial action and mechanical ability of chlorhexidine gel as an endodontic irrigant. j endod 2001; 27: 452-5. 28. sen bh, wesselink pr, türkün m. the smear layer: a phenomenon in root canal therapy. int endod j. 1995; 28: 141-8. 29. kasahara e, yasuda e, yamamoto a, anzai m. root canal system of the maxillary central incisor. j endod. 1990; 16: 158-61. 30. marley jt, ferguson db, hartwell gr. effects of chlorhexidine gluconate as an endodontic irrigant on the apical seal: short-term results. j endod. 2001; 27: 775-8. 31. wu mk, de gee aj, wesselink pr. leakage of four root canal sealers at different thickness. int endod j. 1994; 27: 304-8. 32. venturi m, breschi l. evaluation of apical filling after warm vertical guttapercha compaction using different procedures. j endod. 2004; 30: 436-40. 33. barbosa fo, gusman h, pimenta de araújo mc. a comparative study on the frequency, location, and direction of accessory canals filled with the hydraulic vertical condensation and continuous wave of condensation techniques. j endod. 2009; 35: 397-400. effect of the smear layer on the filling of artificial lateral canals and microleakage oral sciences n3 braz j oral sci. 11(1):62-66 original article braz j oral sci. january | march 2012 volume 11, number 1 trends in dental caries experience and fluorosis prevalence in 12-year-old brazilian schoolchildren from two different towns aline sampiere tonello benazzi1, renato pereira da silva2, marcelo de castro meneghim3, antonio carlos pereira3, glaucia maria bovi ambrosano3 1phd, department of community dentistry, piracicaba dental school, university of campinas, brazil 2graduate student of program in dentistry, department of community dentistry, piracicaba dental school, university of campinas, são paulo, brazil 3professor, department of community dentistry, piracicaba dental school, university of campinas, são paulo, brazil correspondence to: marcelo de castro meneghim departamento de odontologia social, faculdade de odontologia de piracicaba, universidade estadual de campinas av. limeira 901, cep: 13414-903, piracicaba, sp, brasil phone: + 55 19 21065209 fax: +55 19 21065218 e-mail: meneghim@fop.unicamp.br abstract aim: to describe the prevalence of dental caries and fluorosis in schoolchildren from two different towns in são paulo state, brazil, 2007 town a (water fluoridation since 1971) and town b (water fluoridation since 1997) and to compare current prevalence rates with previous surveys, in town a, for dental caries (1971-2005) and for dental fluorosis (1991-2001), and in town b, for dental caries and dental fluorosis (1991-2004). methods: the sample consisted of 724 schoolchildren aged 12 years from public and private schools (town a) and 197 schoolchildren from public schools (town b). the schoolchildren were examined under natural light by a dentist, using cpi probes and oral mirrors. the mean number of decayed, missing and filled permanent teeth (dmft), and significant caries (sic) index were determined for dental caries and the thylstrup and fejerskov index (t-f) for fluorosis. results: the dmft was 0.85 and 1.02; sic index was 2.52 and 2.83 in towns a and b, respectively. fluorosis prevalence was 29.4% (town a) and 25.4% (town b). in both towns, a significant dental caries reduction has been observed. concerning fluorosis prevalence, an increase of 44.1% was noted in town a and 1170% in town b. conclusions: results show continuous decrease in dental caries experience in both towns. regarding fluorosis prevalence, stabilization trends were observed in town a. in town b, however, a constant increase was noted. keywords: dental caries, fluorosis, dmf index, oral health, epidemiology. introduction trends in caries experience have been reported throughout the world1-4. caries decline has been also observed in brazil in both fluoridated and non-fluoridated areas, mainly in schoolchildren in the southern and southeastern regions5-6. the most recent epidemiological survey of oral health promoted by the ministry of health confirmed the trend of decline of caries in brazilian schoolchildren7. although, a series of clinical consequences have been observed over the last decades, such as the reduction in disease progression speed8, and the polarization phenomenon in which a minority of individuals presents the highest caries scores9-11. the minority of individuals, the so-called high-caries risk individuals12, usually belongs to a family with low monthly income13. received for publication: august 23, 2011 accepted: february 28, 2012 braz j oral sci. 11(1):62-66 on other hand, an increase of fluorosis prevalence has also been observed throughout the world14-15. some findings suggest that risk of fluorosis development is associated with regular use of fluoride supplements16-17. despite of the use of fluoride toothpaste by young children can be considered a risk factor for dental fluorosis18, a recent review of the literature showed that the evidence pointing to the conclusion that starting the use of fluoride toothpaste in children under 12 months of age may be associated with an increased risk of fluorosis is weak and unreliable, and, even for older children, the evidence is equivocal19. although the present study is regarded as a local epidemiological survey, it presents 36 years of longitudinal data. taking into account all these factors, it is essential that studies are carried out to monitor these tendencies and plan actions for public oral health. the aims of this research were to describe the prevalence of dental caries and dental fluorosis in 12-year-old schoolchildren from two different towns in são paulo state, brazil, 2007 town a (water fluoridation since 1971) and town b (water fluoridation since 1997) and to compare the current prevalence rates with those from previous surveys, developed in town a (1971-2005), for dental caries and for dental fluorosis (1991-2001), and in town b, for dental caries and dental fluorosis (1991-2004). material and methods ethical aspects this study was approved by the ethics committee of piracicaba dental school, university of campinas, protocol number 089/2006. characteristics of the towns both towns are located in the são paulo state, brazil. town a has 358,108 inhabitants20. fluoride has been added to water supply since 1971 (0.7 ppmf). town b has 18,026 inhabitants20. fluoride has been added to water supply since 1997 (0.7 ppm f). population studied in town a, the sample size was calculated on the basis of caries experience reported in previous studies. a cluster sampling method was used admitting a sampling error of 0.2, mean number of decayed, missing and filled permanent teeth (dmft) and, design error of 2, mean of 1.32 dmft, standard deviation (sd) of 1.92, non-reply rate (loss of sampling elements) of 20%, and confidence level of 95%, 850 schoolchildren aged 12 years were selected in 2007. public and private schools were randomly selected. thus, 18 public and 6 private schools were selected, totalizing 24 schools, and 12 year-old children were chosen at random in each school (n=850). the inclusion criteria were: children whose parents had given consent for participation, were present on the examination day, did not present severe dental hypoplasia, and did not use fixed orthodontic appliance. the final sample in 2007 was composed of 724 12-year-old schoolchildren of both genders, out of which, 613 were from public schools and 111 from private schools, achieving a response rate of 85%. in town b, considering that exist only three public schools, all 12-year-old schoolchildren, were invited to participate in this study, totalizing 244 children. the inclusion criteria were the same for town a. the final sample in 2007 was composed of 197 12-year-old schoolchildren, achieving a response rate of 80.7%. diagnostic criteria and codes dental caries was registered using the dmft index according to world health organization caries diagnostic criteria21 and the significant caries (sic) index that was determined for the onethird of the sample with the highest caries scores22. fluorosis prevalence was measured by the t-f index23. calibration a benchmark dental examiner, skilled in epidemiological surveys, conducted the calibration process in 2007. in the practical activities with clinical examinations and data analyzes, the mean kappa was 0.89 for dental caries and 0.88 for dental fluorosis. approximately 10% of the sample was re-examined in order to verify the intra-examiner reproducibility. kappa values of 0.95 for dental caries and 0.89 kappa for dental fluorosis were observed. examination methodology the results of the present study were compared with the results of previous surveys carried out in town a, (19712005) for dental caries and (1991-2001) for dental fluorosis, and in town b (1991-2004), for dental caries and dental fluorosis. all epidemiological surveys reported for both towns were conducted following the same protocol. epidemiological exams in this study were carried out in 2007, and performed by one previously calibrated dentist in outdoor setting, under natural light, using cpi probes and mirrors #521. before examination each child brushed their teeth under the supervision of a dental hygienist. statistical procedures the dmft and sic indexes, the proportion of cariesfree children and the percentage of children with dental fluorosis were calculated. the variation of dmft index over time was assessed by analysis of regression, and fluorosis prevalence was compared over time by the chi-square test at 5% significance level. results in 2007, the mean value of dmft and sic index were 0.85 (sd=1.54) and 2.52 (sd = 1.72), respectively, in town a and 1.02 (sd=1.61) and 2.83 (sd=1.60), respectively, in town b. the results show that 65.61% and 59.39% of children were caries-free for town a and town b, respectively. table 1 summarizes the results of dental caries 6363636363 trends in dental caries experience and fluorosis prevalence in 12-year-old brazilian schoolchildren from two different towns braz j oral sci. 11(1):62-66 6464646464 experience obtained in all surveys in both towns. in town a, the studies carried out between 1971 and 2007 and showed a reduction of 90.12% in the dmft index, out of which 57.5% was in the last 11 years, in the 1996-2007 period. in town b, the six surveys carried out between 1991 and 2007, showed a reduction of 85.1% in the dmft index. after 10 years of fluoridation of the water supply (1997-2007), caries experience decreased by 65.5%, while in the 1991-1997 period, with no fluoride in drinking water, the percentage of caries reduction was 56.7%. table 2 shows the prevalence of fluorosis (t-f ³ 1) in both towns between 1991 and 2007. in town a, 29.4% of the individuals presented fluorosis. a total of 70.6%, 13.95%, 14.78% and 0.67% of the children were scored as t-f=0, tf=1, t-f=2 and t-f=3, respectively, in 2007. according to the data collected in 1991, out of the 211 children examined, 20.4% presented fluorosis, result that remained nearly the same when data was collected in 1995, when 17.6% presented the same condition, not showing significant difference (p<0.05). in the 1997-2001 period, the increase was only 5.1%, not showing significant difference either (p<0.05). when comparing the data collected in 1991 and in 2007, a 44.1% increase of fluorosis prevalence was observed. in town b, between 1991 and 2007, an increase of 1170% of fluorosis prevalence was noted. in 2007, 25.4% of the sample presented fluorosis (table 2). a total of 74.6% of the schoolchildren were fluorosis free (t-f=0), and 7.64%, 16.25% and 1.51% of the sample presented fluorosis t-f=1, t-f=2 and t-f=3, respectively. a significant decline of dmft in the 12-year-old schoolchildren could be demonstrated over a 36-year period of evaluation by analysis of regression with r2 = 0.9916, (p<0.01) for of town a and over a 16-year-period of evaluation by analysis of regression with r2 = 0.9898 for town b, showing linear effect for dmft and year of survey (figures 1 and 2). fig. 1. dmft variation for 12 year-old schoolchildren over time in town a, brazil (p<0.01). year of survey town a sample mean dmft % reduction in consecutive surveys % reduction in relation to 1971 year of survey town b sample mean dmft % reduction in consecutive surveys % reduction in relation to 1991 197124 204 8.60 199128 200 6.7 197724 188 7.41 13.84 13.84 199528 160 3.9 41.8 41.8 198024 144 6.17 16.73 28.25 199728 314 2.9 25.7 56.7 199225 123 3.47 43.76 59.65 200126 244 2.1 27.6 68.7 199526 142 2.70 22.19 68.60 20045 236 1.2 42.9 82.1 199627 189 2.00 25.92 76.74 2007* 197 1.0 16.6 85.1 200126 824 1.70 15.00 80.23 20056 939 1.32 22.35 84.65 0.85 35.60 90.12 65.5 57.5 polynomial regression (p<0.01) of mean of dmft according to year of survey. *present study table 1.table 1.table 1.table 1.table 1. mean dmft and reduction (%) of caries experience for 12-year-old schoolchildren in town a and town b, brazil, according to year of survey. fig. 2. dmft variation for 12 year-old schoolchildren over time in town b, brazil (p<0.01). trends in dental caries experience and fluorosis prevalence in 12-year-old brazilian schoolchildren from two different towns braz j oral sci. 11(1):62-66 6565656565 town year of survey and authors sample fluorosis prevalence % increase in consecutive surveys % increase in relation to 1991 town a 199128 211 20.4 b 199528 142 17.6 b 13.7 13.7 199728 190 3 1.0 a 76.1 52.0 200126 824 31.4 a 1.3 54.0 2007* 724 29.4 a 6.4 44.1 town b 199128 200 2.0 c 199528 160 4.4 c 120 120 199728 314 10.2 b 132 410 200126 244 12.7 b 25 535 20045 236 15.7 b 24 685 2007* 197 25.4 a 62 1170 149 5.1 table 2.table 2.table 2.table 2.table 2. percentage of individuals with tf ³ 1 and variation (%) of fluorosis prevalence for 12-year-old schoolchildren in town a and b, brazil, according to year of survey. different letters indicate statistically significant difference at 5% significance level (chi-square test ). *present study discussion results show constant decrease in caries prevalence in both towns over time (figures 1 and 2). the decline in caries prevalence detected in the present study is an event also observed worldwide29. in comparison to national data30, 12year-old schoolchildren from town a and b presented lower caries experience (0.85 and 1.0 dmft respectively). recent international reported data have shown that the dmft for 12-year-old children is also low ranging from 0.80 in dublin, ireland31. however, higher results were observed in a survey conducted with non-indigenous schoolchildren living in the amazon basin of ecuador, showing a dmft of 5.2532. in town a, taking into account caries prevalence in studies carried out in 1980, dmft was found to be nearly three times higher than the one found in 1992, which lead us to infer that schoolchildren examined in this last year benefited from fluoridated water, other forms of caries control and prevention, which may have caused this reduction. even though the experimental design of the present study did not supply data for the evaluation of the causes for this reduction, one can conclude that the wide use of dentifrice fluoridated, which became available in brazil in 1989, interacted in such a way that promoted a decline in caries prevalence. as for the sic index, 2.52 and 2.83 were found in town a and b in 2007, respectively. these values are over two times higher than the mean dmft for the entire sample in both towns. these findings are in line with some studies reported33, demonstrating that caries experience in those individuals more affected by the disease is over two times higher34. regarding dental fluorosis, reports in scientific literature have demonstrated an increase in prevalence rates35-36, which could be confirmed in this research in town b, when comparing data from 2007 with those from 1991 to 2004 (table 2). however, in town a, results show that this index remained nearly the same for 10 years (1997 to 2007), presenting a tendency to stabilize in most part of the period, showing a small reduction of 6.4% between 2001 and 2007, without statistical significance (p<0.05). considering the increase of fluorosis prevalence comparing both towns from 1997 (year that began the process of water fluoridation in town b) to 2007, it was observed that town a presented an increase of only 5.1%, whereas in town b, the increase was 149%. one can suggest that others studies could be carried out to monitor fluorosis prevalence in town b. in relation to fluorosis severity, the lowest score for both towns was the component t-f 3, and the highest was the tf 2. however, in a research carried out in nigeria, the most severe form was t-f 6 and t-f 537. according to the epidemiological surveys discussed in this study, a continuous decline of dental caries experience could be verified after 36 years of water supply fluoridation in town a, from 1971 to 2007, and in town b from 1991 to 2007. regarding dental fluorosis, stabilization trends were observed in town a. however, in town b, a constant increase was noted. it is possible that concomitant use of fluoridated dentifrice and water are directly related with the increase in trends in dental caries experience and fluorosis prevalence in 12-year-old brazilian schoolchildren from two different towns 6666666666 braz j oral sci. 11(1):62-66 the prevalence of dental fluorosis. future epidemiological surveys should be carried out to evaluate and monitor dental caries and fluorosis trends over time. acknowledgements the authors would to acknowledge the financial support of the fapesp (grants #06/50788-0 and #06/58881-9). we also give special thanks to the principals of the schools and all the children, who contributed to the accomplishment of the survey. references 1. pieper k, schulte ag. the decline in dental caries among 12-year-old children in germany between 1994 and 2000. community dent health. 2004; 213: 199-206. 2. tagliaferro eps, meneghim mc, ambrosano gmb, pereira ac, salesperes shc, sales-peres a, et al. distribution and prevalence of dental caries in bauru, brazil, 1976-2006. int dent j. 2008; 58: 75-80. 3. parker ej, jamieson lm, broughton j, albino j, lawrence hp, thomson kr. the oral health of indigenous children: a review of four nations. j paediatr child health. 2010; 46: 483-6. 4. micheelis w. oral health in germany: an oral epidemiological outline. bundesgesundheitsblatt gesundheitsforschung gesundheitsschutz. 2011; 54: 1022-6. 5. meneghim mc, tagliaferro eps, tengan c, pedroso zma, pereira ac, ambrosano gmb, et al. trends in caries experience and fluorosis prevalence in 11to 12-year-old brazilian children between 1991 and 2004. oral health prevent dent. 2006; 4: 193-8. 6. pereira sm, tagliaferro eps, ambrosano gmb, cortellazzi kl, meneghim mc, pereira ac. dental caries in 12-year-old schoolchildren and its relationship with socioeconomic and behavioral variables. oral health prevent dent. 2007; 5: 299-306. 7. brazil. health ministry of brazil. sb brazil 2010 project: national oral health research, 2010. brasília: health ministry ministério da saúde. available from: http://www.sbbrasil2010.org. 8. moberg skold u, birkhed d, borg e, petersson lg. approximal caries development in adolescents with low to moderate caries risk after different 3-year school-based supervised fluoride mouth rinsing programmes. caries res. 2005; 39: 529-35. 9. narvai pc, frazão p, roncalli ag, antunes, jlf. dental caries in brazil: decline, polarization, inequality and social exclusion. rev panam salud publica. 2006; 19: 385-393. 10. namal n, can g, vehid s, koksal s, kaypmaz a. dental health status and risk factors for dental caries in adults in istanbul, turkey. east mediterr health j. 2008; 14: 110-8. 11. ditmyer m, dounis g, mobley c, schwarz e. a case-control study of determinants for high and low dental caries prevalence in nevada youth. bmc oral health. 2010; 10: 18. 12. tagliaferro eps, ambrosano gmb, meneghim mc, pereira ac. risk indicators and risk prediction of dental caries in schoolchildren. j appl oral sci. 2008; 16: 408-13. 13. piovesan c, mendes fm, antunes, jlf, ardenghi tm. inequalities in the distribution of dental caries among 12-year-old brazilian schoolchildren. braz oral res. 2011; 25: 69-75. 14. kukleva mp, kondeva vk, isheva av, rimalovska si. comparative study of dental caries and dental fluorosis in populations of different dental fluorosis prevalence. folia med (plovdiv). 2009; 51: 45-52. 15. anuradha b, laxmi gs, sudhakar p, malik v, reddy ka, reddy sn, et al. prevalence of dental caries among 13 and 15-year-old school children in an endemic fluorosis area: a cross-sectional study. j contemp dent pract. 2011; 12: 447-50. 16. pendrys dg, haugejorden o, bårdsen a, wang nj, gustavsen, f. the risk of enamel fluorosis and caries among norwegian children: implications for norway and the united states. j am dent assoc. 2010; 141: 401-4. 17. levy sm, broffitt b, marschall ta, eichenberger-gilmore jm, warren jj. associations between fluorosis of permanent incisors and fluoride intake from infant formula, other dietary sources and dentifrice during early childhood. j am dent assoc. 2010; 141: 1190-1201. 18. mascarenhas ak. risk factors for dental fluorosis: a review of 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[cited 2009]. available from: http: //www.ibge.gov.br/cidadesat. 21. world health organization: oral health surveys basic methods. 4th ed. geneva: who; 1997. p.66. 22. bratthall d. introducing the significant caries index together with a proposal for a new global oral health goal for 12-yearolds. int dent j. 2000; 50: 37884. 23. thylstrup a, fejerskov o. clinical appearance of dental fluorosis in permanent teeth in relation to histologic changes. community dent oral epidemiol. 1978; 6: 315-28. 24. moreira bhw, tuma aj, guimarães lo. incidence of dental caries in students of piracicaba, sp after 6 and 9 years of water supply fluoridation. rev bras odontol. 1983; 40: 11-4. 25. pereira ac, biscaro sl, moreira bhw. oral conditions of 7–12-year-old schoolchildren, after 20 years of fluoridation of the public water supply in piracicaba. rev paul odontol. 1995; 17: 30-6. 26. kozlowski fc. prevalence and severity of fluorosis and dental caries related to socioeconomic factors [thesis]. piracicaba (sp): school of dentistry, state university of campinas –unicamp; 2001. 27. basting rt, pereira ac, meneghim mc. evaluation of dental caries prevalence in students from piracicaba, sp, brazil, after 25 years of fluoridation of the public water supply. rev odontol univ sao paulo. 1997; 11: 287-92.. 28. pereira ac, mialhe fl, bianchini flc, meneghim mc. prevalence of dental caries and dental fluorosis in schoolchildren of towns with different fluoride concentrations in water supply. rev bras odontol saude colet 2001; 2: 34-9. 29. campus g, sacco g, cagetti m, abati s. changing trend of caries from 1989 to 2004 among 12-year old sardinian children. biomed central public health. 2007; 7: 1-6. 30. brazil. health ministry of brazil. sb brasil 2003 project oral health conditions of the brazilian population 2002-2003. national division of oral health. brasília: health ministry; 2004. 31. sagheri d, mcloughlin j, clarkson jj. the prevalence of dental caries and fissure sealants in 12 year old children by disadvantaged status in dublin (ireland). community dent health. 2009; 26: 32-7. 32. medina w, hurtig ak, san sebastián m, quizhpe e, romero c. dental caries in 6-12-year-old indigenous and non-indigenous schoolchildren in the amazon basin of ecuador. braz dent j. 2008; 19: 83-6. 33. ditmyer m, dounis g, mobley c, schwarz e. inequalities of caries experience in nevada youth expressed by dmft index vs. significant caries index (sic) over time. bmc oral health. 2011;11: 12. 34. marthaler t, menghini g, steiner m. use of the significant caries index in quantifying the changes in caries in switzerland from 1964 to 2000. community dent oral epidemiol. 2005; 33: 159-66. 35. browne d, whelton h, o’mullane d. fluoride metabolism and fluorosis. j dent. 2005; 33: 177-86. 36. buzalaf ma, levy sm. fluoride intake of children: considerations for dental caries and dental fluorosis. monogr oral sci. 2011; 22: 1-19. 37. akosu tj, zoakah ai, chirdan oa. the prevalence and severity of dental fluorosis in the high and low altitude parts of central plateau, nigeria. community dent health. 2009; 26: 138-42. trends in dental caries experience and fluorosis prevalence in 12-year-old brazilian schoolchildren from two different towns oral sciences n3 braz j oral sci. 10(1):12-16 original article braz j oral sci. january | march 2011 volume 10, number 1 in vivo evaluation of different techniques for establishment of proximal contacts in posterior resin composite restorations paula de carvalho cardoso1, alessandra reis bastos de oliveira2, letícia vilela lopes3, sabrina caetano cabral2, maria beatriz r. g. oliveira4 1phd in operative dentistry at dental school of federal university of santa catarina; coordinator of the operative dentistry graduate program of funorte.go.brazil 2specialist in operative dentistry graduate program of funorte.go. brazil 3msc in operative dentistry at sagrado coração university; professor at operative dentistry graduate program of funorte.go.brazil 4.professor at operative dentistry graduate program of funorte.go.brazil correspondence to: paula de carvalho cardoso rua 3, n. 691 setor oeste cep: 74115-050, goiânia, go, brazil. email: paulaccardoso@hotmail.com received for publication: may 30, 2010 accepted: march 03, 2011 abstract aim: to evaluate the re-establishment of proximal contacts on class ii resin composite restorations by means of 4 different in vivo techniques. methods: restorations were divided into 4 groups (n=10): group 1 (preformed metallic matrix + wooden wedges + separating ring); group 2 (preformed metallic matrix + elastic interdental wedges+ separating ring); group 3 (preformed metallic matrix + wooden wedges + translucent contact spatula); group 4 (preformed metallic matrix + wooden wedges + resin inserts). initially, two previously calibrated examiners conducted training on models representing three different proximal contact situations: tight (t); satisfactory (s) and unacceptable (u). two evaluations were accomplished: immediately and 30 days after the restorative procedures. fisher’s exact test was used to verify differences among the techniques for establishment of proximal contacts in posterior resin composite restorations. results: there was no statistically significant differences (p>0.05) among the 4 groups. conclusions: all techniques produced satisfactory proximal contacts. different restorative techniques for class ii resin composite cavity preparations enabled satisfactory proximal contacts. keywords: composite resins, definitive restorations, interdental matrix, dentistry. introduction the evolution of dental materials properties and adhesive systems along with patients’ esthetic requirements has increased the use of resin composite restorations for posterior teeth1-5. however, resin composite restorative procedures present some technical difficulties, such as the establishment of proximal contacts6-9. a correct proximal contact enables balanced mesiodistal forces and provides resistance against food impaction at marginal interproximal ridges10-11. one of the limitations of posterior resin composite restorations is the establishment of an effective correct proximal contact12. some techniques have been proposed for resolution of this problem: the use of pre-polymerized resin composite inserts 1,10-11 or ceramic inserts1,13 within restorations, the use of a special instrument for matrix pressuring against the adjacent tooth (contact +, tdv, são paulo, sp, brazil), special polymerization tips (lighttip) 7 and even the development of compactable resin composites14. in addition, preformed matrices and separating rings combined with wedges have produced good results, with adequate contour and form to the restoration15. proximal contact strengths can be measured using: the tooth pressure meter, analysis of proximal contact strength during dental floss passage and analysis of mesiodistal tooth diameter15-19. due to the limitations of in vitro studies by the lack of clinical periodontal behavior evaluation, in vivo studies seem more relevant. the aim of this work was to evaluate the re-establishment of proximal contacts on class ii resin composite restorations by means of 4 different in vivo techniques. the tested null hypothesis is that there is no difference among resin composite restorative techniques for the establishment of proximal contacts. material and methods after ethics committee approval (federal university of goiás, #017/2009), patients were included in this study, receiving all necessary information and signing an informed consent for their participation. selection of patients restorative requirements based on inclusion/exclusion criteria (table 1) were used for the selection of patients. cavity preparation forty class ii restorations were prepared in human permanent premolars or molars for this study (mo or do). cavity dimensions were accessed with a periodontal probe. occlusal box isthmus was limited to 3 mm, with 2.5 mm in deepness. proximal box were limited to 6 mm buccolingual width and 1.5 mm mesiodistal width, with a 5 mm-deep gingival wall (table 1). one expert operator performed all preparations after an anesthetic shot. a #329 carbide bur (kg sorensen, barueri, sp, brazil) was used at high-speed rotation under constant air-water spray. internal angles were all rounded. dental caries were removed with spherical steel burs in low speed. a new bur was used after every 5 preparations. if an old restoration was to be replaced, a cavity preparation was created after its removal. the following clinical standardized protocol was employed: table 1: inclusion and exclusion criteria. inclusion exclusion age: 18 to 42-year old patients. absence of sound tooth adjacent to the tooth to be restored. poor class ii (mo or do) restorations in premolars or molars. periodontal disease. presence of aligned teeth in occlusal function physical or mentally affected patients. maximum occlusal box: width= 5 mm, depth= 2.5mm. maximum proximal box: buccolingual width= 6 mm, mesiodistal extension= 1.5 mm. parafunctional habits. maximum gingival wall at proximal box: depth= 5mm tooth cuspal integrity and cervical preparation in enamel posterior teeth with diastemata enough time to participate in the research. 1. pumice and water slurry with rubber cup in low speed. 2. resin composite color selection. 3. rubber dam placement. 4.cavity preparation. 5. insertion of matrix wedges and separating ring according to each experimental group. 6. acid etching with 37% phosphoric acid (etchant gel; coltene/whaledent, altstatten, switzerland) for 30 s (enamel) and 15 s (dentin). 7. water spray, gentle dry, 2% chlorhexidine application. 8. primer application, gentle air drying for 5 s and adhesive system application (scotchbond multi-purpose, 3m espe, st. paul, mn, usa). 9. light-polymerization for 20 s. 10. insertion and polymerization of resin composite with an incremental technique. 11. finishing and polishing after final polymerization. description of the experimental groups ten restorations per group were evaluated (n=10, figure 1 and table 2). group 1 (g1) (n=10): preformed metallic matrix (unimatrix, tdv dental ltda, pomerode, sc, brazil), kept into position with a wooden edge (cunhas anatômicas, tdv dental ltda, pomerode, sc, brazil) combined with a 13 in vivo evaluation of different techniques for establishment of proximal contacts in posterior resin composite restorations braz j oral sci. 10(1):12-16 fig. 1. illustration of the of experimental groups. 14 table 2: description of experimental groups. group description group 1 preformed metallic matrix + wooden wedges+ separating ring group 2 preformed metallic matrix + elastic interdental wedges+ separating ring group 3 preformed metallic matrix + wooden wedges + translucent contact spatula group 4 preformed metallic matrix + wooden wedges + resin inserts separating ring (unimatrix kit, tdv dental ltda, pomerode, sc, brazil). group 2 (g2) (n=10): preformed metallic matrix (unimatrix), kept into position with elastic interdental wedges (danville materials inc., san ramon, ca, usa) combined with a separating ring (unimatrix kit). group 3 (g3) (n=10): preformed metallic matrix (unimatrix), kept into position with a wooden edge (cunhas anatômicas). before light polymerization of the first resin composite increment, a translucent contact spatula (contact +, tdv dental ltda) was pressed over the matrix, against the adjacent tooth. group 4 (g4) (n=10): preformed metallic matrix (unimatrix), kept into position with a wooden edge (cunhas anatômicas). during the first light polymerization cycle of the resin composite, a resin composite insert (3m espe, st. paul, mn, usa) was inserted within restoration. restorative procedures forty cavities were restored according to described techniques. the nanoparticle resin composite filtek z350 and the adhesive system scotchbond multi-purpose were used (3m espe). restorative procedure for group 1 a preformed metallic matrix was inserted in the interdental space with the convex surface in contact with the adjacent tooth, kept into position with a wooden edge. for all restorations the wooden wedge was prepared with a #12 surgical blade according to the interdental space. with the exception of restorations that involved the distal surface of superior premolars or mesial surface of inferior first molars, wedge insertion was performed through the lingual side. after acid etching with 37% phosphoric acid (scotchbond etchant, 3m espe, st. paul, mn, usa) for 30 s (enamel) and 15 s (dentin), cavities were sprayed with water for 60 s, gently dried with absorbent paper and received 2% chlorhexidine application. then, the primer was applied, following a gently drying and adhesive application (scotchbond multi-purpose), which was light polymerized for 20 s. light polymerization was performed with optilux (demetron research corporation, danbury, ct, usa) at 450 mw/cm2, as checked with an internal radiometer. three 2-mm-thick resin increments (filtek z350) were inserted at the proximal box with metallic spatula (hu-friedy mini 4 goldstein flexi, hu-friedy do brazil ltda, rio de janeiro, rj, brazil), according to the following scheme: first horizontal increment and second plus third ones in an oblique direction. each increment was light polymerized by 10 s. the occlusal box was restored with 2-mm-thick increments until completion of the occlusal anatomy. after that, an additional light exposure of 40 s was performed in the entire restoration. restorative procedure for group 2 the restorative procedure for group 2 was similar to group 1, with the exception that a green elastic interdental wedge was inserted after placement of the metallic matrix in the occlusogingival direction. after completion of the restoration, the elastic wedge was removed by cutting it with a scissor and lateral removal in separate portions. restorative procedure for group 3 a preformed metallic matrix was inserted in the interdental space with the convex surface in contact with the adjacent tooth, kept into position with a wooden edge. next, a contact creator translucent spatula (contact +, tdv, sp brazil) was pressed against the adjacent tooth during the first layer insertion. after 20 s light polymerization, the spatula was removed and restoration completed. restorative procedure for group 4 a preformed metallic matrix was inserted in the interdental space with the convex surface in contact with the adjacent tooth, kept into position with a wooden edge. next, resin composite insert was pressed against the adjacent tooth during the first layer insertion. the insert was made with z-350 (3m, espe), a 2 shade. a resin composite sphere corresponding to 4d 10 of proximal box width was created on a glass slab. a resin composite increment was applied to the proximal box and after that the resin insert was positioned over it and pressed with a metallic spatula against the adjacent tooth until the end of the 20 s polymerization cycle. the spatula was removed after that and restoration completed. occlusal adjustment was performed with 16and 30blade carbide burs following finishing with sandpaper strips (3m espe) at the proximal surfaces and abrasive tips at restoration/tooth interfaces (jiffy polisher, ultradent products). after 30 days, the final polishing was performed with jiffy regular brushes (ultradent products). subjective evaluation of proximal contact re-establishment six metallic inlays were made on dental models (poclusal produtos odontológicos ltda, são paulo, sp, brazil) representing one of the following types of proximal contacts: tight (t) for heavy contacts; satisfactory (s) for acceptable ones; and unacceptable (u) for the absence of proximal contact16. the subjective evaluation or restored proximal surfaces was performed by two previously calibrated examiners, through a direct comparison with standard patterns defined on metallic inlays. after calibration, evaluation was accomplished by passing a dental floss (expasion plus, in vivo evaluation of different techniques for establishment of proximal contacts in posterior resin composite restorations braz j oral sci. 10(1):12-16 15 johnson & johnson, sp, brazil) between each restored tooth and the adjacent one, following the same direction during insertion and removal. the classification was guided by the previously adopted criteria for metallic inlays at tooth models [tight (t); satisfactory (s) and unacceptable (u)]. a consensual decision was done in cases where doubts impaired a direct conclusion. all evaluations were performed immediately after restoration (l 0 ) and 30 days after it (l 1 ). before each clinical evaluation, teeth were cleaned with water spray, dried with air and visually examined with the aid of a #5 clinical probe explorer. additionally, an interproximal radiographic examination was performed before and after the restorative procedure. fisher’s exact test was used to verify differences among the techniques for establishment of proximal contacts in posterior resin composite restorations. statistical significant differences were computed with pd”0.05. results figure 2 shows that the performance of the four groups was statistically similar. table 3 shows paired comparisons for the groups (fisher’s exact test at p > 0.05). at the second evaluation (l 1 ), results were similar to the first evaluation l 0 . intergroup comparisons p g 1 x g 2 0.244 g 1 x g 3 0.418 g 1 x g 4 0.395 g 2 x g 3 0.244 g 2 x g 4 0.135 g 3 x g 4 0.395 table 3: comparison among the groups. fisher’s exact test. discussion direct resin composite restorations in posterior teeth represent a clinical challenge due to the difficult in establishing an effective proximal contact, which is an important parameter for periodontal health, mesiodistal stability, absence of food impaction and patient comfort20-21 however, few studies have investigated the influence of different restorative techniques on the re-establishment of adequate proximal contacts. several techniques have been proposed to enable better proximal contacts, e.g., special contact creation instruments22, use of resin or ceramic inserts23, and use of different resin viscosities19,24-25. in vivo study seems more meaningful, being used in the present study for the clinical evaluation of the proximal contact re-establishment with four restorative techniques using matrices and wedges, eventually analyzing the difficulties and facilities of each technique. two evaluations were performed in the present study: immediate after restoration (lo) and 30 days after restoration (l1). the reason for the 2 evaluations is related to the period necessary for periodontal accommodation since during restorative procedures teeth are subjected to lateral forces at placement of wedge, claps and matrices26. according to loomans et al. 2006, the restoration of class ii cavities tend to alter the original proximal contact due to tooth extractions, mesiodistal tooth movements under forces produced by progressive eruption of third molars, or unbalanced occlusal forces. however, they did not found any alteration during 18-month follow up in patients with restored class ii cavities16. results from figure 2 and table 3 show that the null hypothesis was accepted since no differences were found among all clinical techniques. although group 4 showed a larger number of acceptable contacts there was no statistically significant difference in relation to the other groups. no tight proximal contact was observed in any restoration of all groups. it was not found any scientific explanation for this observation. in face of the obtained results, the use of elastic wedges and special contact creator instruments seems questionable since dentists would spent more money on new devices and practicing lessons, with no significant benefits. according to the employed methodology, it is possible to conclude that there was no difference among techniques for proximal contact reconstruction with composite resin. references 1. applequist ea, meiers j c. effect of bulk insertion, prepolymerized resin composite balls, and beta-quartz inserts on microlekage of class v resin composite restorations. quintessence int. 1996; 27: 253-8. 2. baratieri ln, monteiro sjr, correa m, ritter av. posterior resin composite restorations: a new technique. quintessence int. 1996; 27: 7338. 3. baratieri ln, ritter av, perdigão j, felipe la. direct posterior resin composite restorations: current concepts for the technique. pract periodontics aesthet dent. 1998; 10: 875-86. 4. bernadon j. avaliação clínica de uma resina composta em dentes posteriores [thesis]. florianópolis: universidade federal de santa catarina; 2008. 115p. 5. doukoudakis s. establishing approximal contacts in class 2 composite resin restorations. oper dent. 1996; 21: 182-4. 6. eli i, weiss e, kozlovsky a, levi n. wedges in restorative dentistry: principles and applications. j oral rehabil. 1991; 18: 257-64. 7. jordan re, suzuki .m. posterior composite restorations: where and how they work best. j am dent assoc. 1991; 122: 131-7. 8. lopes gc, ferreira rs, baratieri ln, vieira lcc, monteiro sjr. direct posterior composite resin restorations: new techniques and clinical possibilities. quintessence int. 2002; 33: 337-46. in vivo evaluation of different techniques for establishment of proximal contacts in posterior resin composite restorations braz j oral sci. 10(1):12-16 fig. 2. percent score distribution of the subjective evaluation of the experimental groups. values within each bar indicate the number of observed scores in each group. 16 9. lopes gc, vieira lcc, araujo e. direct posterior composite resin restorations: a review of some clinical procedures to achieve predictable results in posterior teeth. j esthet restor dent. 2004; 16: 19-32. 10. 10.lacy am. an effective technique for extended proximal contacts in composite resin restorations. pract periodontics aesthet dent. 1996; 8: 287-93. 11. lacy am. a critical look at posterior composite restorations. j am dent assoc. 1987; 114: 357-62. 12. lopes gc. cunhas elásticas: uma nova alternativa para resolver o problema de contato interproximal nas restaurações diretas de resina composta em dentes posteriores. clin int j braz dent. 2007; 2: 321-432. 13. liebenberg wh. the proximal precinct in direct posterior composite restorations: interproximal integrity. pract proced aesthet dent. 2002; 14: 587-94. 14. 14.loomans bac, opdam njm, roeters fjm, bronkhorst em, burgersdijk rcw. a randomized clinical trial on proximal contacts of posterior composites. j.dent. 2006; 34: 292-7. 15. 15.lowe e. restoring form, function, and aesthetics in class ii direct composite restorations. dent today. 1998; 17: 72-6. 16. prakki a, cilli r, saad jo, rodrigues jr. clinical evaluation of proximal contacts of class ii esthetic direct restorations. quintessence int. 2004; 35: 785-9. 17. rau pj, pioch t, staehle hj, dörfer ce. influence of the rubber dam on the proximal contact strengths. oper dent. 2006; 31: 171-5. 18. 18.stolf sc. avaliação in vitro da influência de diferentes sistemas de matrizes e cunhas no contato proximal de restaurações diretas de resina composta em dentes posteriores [dissertation]. florianópolis: universidade federal de santa catarina; 2008. 115p. 19. loomans ba, opdam nj, roeters fj, bronkhorst em, burgersdijk rc. comparison of proximal contacts of class ii resin composite restorations in vitro. oper dent. 2006; 31: 688-93. 20. hancock eb, mayo cv, schwab rr, wirthlin mr. influence of interdental contacts on periodontal status. j periodontol. 1980; 51: 445-9. 21. jernberg gr, bakdash mb, keenaan km. relationship between proximal tooth open contacts and periodontal disease. j periodontol. 1983; 54: 529-33. 22. 22.el-badrawy wa et al. evaluation of proximal contacts of posterior composite restoration with 4 placement techniques. j can dent assoc. 2003; 69: 162-7. 23. 23.dunnwj. establishing proximal contacts with pre-polymerized composite insert. oper dent.2004; 29: 473-6. 24. klein f, keller ak, staehle hj, dörfer ce. proximal contact formation with different restorative materials and techniques. am j dent. 2002; 15: 232-5. 25. peumans m, van meerbeek b, asscherickx k, simon s, abe y, lambrechts p et al. do condensable composites to achieve better proximal contacts? dent mater. 2001; 17: 533-41. 26. loomans ba, opdam nj, roeters fj, bronkhorst em, plasschaert aj. the long-term effect of a composite resin restoration on proximal contact tightness. j dent. 2007; 35: 104-8. in vivo evaluation of different techniques for establishment of proximal contacts in posterior resin composite restorations braz j oral sci. 10(1):12-16 oral sciences n3 braz j oral sci. 9(2):108-114 original article braz j oral sci. april/june 2010 volume 9, number 2 effect of platelet-rich plasma and bioactive glass in the treatment of intrabony defects a split-mouth study in humans manmeet kaur1, t. ramakrishnan2, n.amblavanan2, pamela emmadi2 1mds, graduate student, department of periodontology and implantology, meenakshi ammal dental college and hospital, india 2mds, professor, department of periodontology and implantology, meenakshi ammal dental college and hospital, india correspondence to: manmeet kaur, mds department of periodontology and implantology meenakshi ammal dental college and hospital, maduravoyal, chennai95, india email: meetsandhu80@gmail.com received for publication: december 15, 2009 accepted: may 04, 2010 abstract aim: to compare the efficacy of platelet rich plasma (prp) associated with bioactive glass (bg) and bg alone in the treatment of periodontal intrabony defects. methods: ten patients participated of the study. using a split-mouth design, interproximal bony defects were surgically treated with either prp/bg or bg alone. the clinical parameters plaque index, gingival index, gingival bleeding index, pocket probing depth (ppd), clinical attachment level (cal) and gingival recession were recorded, and the defect fill was evaluated radiographically at baseline and 3 and 6 months after surgery. results: at 6 months after therapy, the prp/bg group showed mean ppd reduction of 3.4±1.4 mm, cal mean gain of 4.3±1.3 mm, and defect fill of 3.5±1.0 mm. the bg group showed mean ppd reduction of 2.6±1.1 mm, mean cal gain of 3.3±1.3 mm, and defect fill of 3.1±1.2mm. there was statistically significant greater ppd reduction at 3 months and cal gain at 6 months for prp/bg compared to bg alone, but no significant difference was observed in defect fill. conclusions: both therapies resulted in significant ppd reduction, cal gain and defect fill. the association of prp with a bg graft material seemed to add some benefits to the improvement of the clinical parameters in the treatment of intrabony defects. keywords: intra-bony defect, periodontal regeneration, platelet-rich plasma, bioactive glass. introduction the goal of any periodontal therapy is the control of active inflammation, the arrest of disease progression and the reconstruction of structures lost to disease, where appropriate1. although the effectiveness of scaling and root planing or surgical access for root planing plus regular maintenance care in moderate to severe periodontal disease cases has been well established, the efficacy is judged based on the ability of the therapy to improve osseous lesions2. periodontal regeneration is a multifactorial process and requires an orchestrated sequence of biological events including cell adhesion, migration, multiplication and differentiation which involves recruitment of locally derived progenitor cells to the site3. therapies capable of achieving this goal include guided tissue regeneration (gtr), autografts, allografts, alloplasts, growth factors and combination of these techniques as well as osseous resective surgery4-6. although observations from histological studies in humans and data from controlled clinical trials have demonstrated that some of the available grafting procedures may result in periodontal regeneration in intrabony defects, but complete reconstruction on a regular predictable basis has been difficult to achieve7-8. 109 braz j oral sci. 9(2):108-114 a u t o g e n o u s b o n e g r a f t t a k e n f r o m i n t r a o r a l o r extraoral donor sites is a well accepted treatment option in the periodontal community, but it has disadvantages of limited availability of donor sites and requirement for an additional surgical site to obtain the graft material. t h e u s e o f a n a l l o g r a f t m a y h a v e r i s k o f d i s e a s e transmission and questions have been raised to their osteogenic potential 9-10. this has renewed interest in evaluating alloplastic bone substitutes in treatment of intraosseous defects. bioactive glass (bg) is a synthetic material composed of sodium and calcium salts, phosphates and silicon dioxide. it has been suggested that bg has advantages of forming strong bond with both bone and soft connective tissue and to having modulus of elasticity similar to that of bone thus preventing the formation of intervening f i b r o u s c o n n e c t i v e t i s s u e i n t e r f a c e 1 1. b g h a s a n osteostimulatory effect in addition to its osteoconductive properties12, and it has also shown to have antibacterial effect against subgingival and supragingival bacteria 13. low et al.9 and zamet et al.14 reported good clinical results in intrabony defects in sites treated with a bg when compared to debridement. a s t h e k n o w l e d g e o f t h e f u n d a m e n t a l s o f b o n e regeneration has increased, marx et al.15 studied the growth factors contained within platelets in relation to bone regeneration. several growth factors are deposited in the extracellular matrix where they are released during matrix degradation and they act as a part of a complex network of signals with mutual effects during tissue remodeling and regeneration. platelet derived growth factor (pdgf), transforming growth factor-β (tgf-β) and insulin-like growth factor (igf) may be available for this purpose3,1516. p l a t e l e t r i c h p l a s m a ( p r p ) i s a n a u t o l o g o u s concentration of platelets in plasma developed by gradient density centrifugation and contains many growth factors, such as pdgf, tgf-β, vascular endothelial growth factor, and others. prp also contains proteins known to act as cell adhesion molecules for osteoconduction and as a m a t r i x f o r b o n e , c o n n e c t i v e t i s s u e , a n d e p i t h e l i a l migration15-17. one major criterion for periodontal regeneration is the maintenance of a wound space for the periodontal ligament cells to migrate into. for growth factors to exert their potential, they require a medium that can provide this space, and thus cell induction and differentiation can be obtained by combining a bone graft with prp in the wound space,. marx et al.15 demonstrated a 1.62-2.16-fold increase in bone maturation using autologous platelet preparation in combination with a bone autograft compared t o b o n e a u t o g r a f t a l o n e i n l a r g e h u m a n m a n d i b u l a r continuity defects. however, few studies have focused on the application of prp in combination with alloplastic graft materials, evaluating the treatment outcomes in intrabony defects18-20. therefore, the purpose of this study was to compare clinically and radiographically the regenerative potential of a combination of prp and bg to that of bg alone in the treatment of periodontal intrabony defects. material and methods ten systemically healthy 25-45-year-old patients with moderate to advanced chronic periodontitis were selected from the department of periodontology and implantology of the meenakshi ammal dental college and hospital, chennai, india. the study design was approved by the university institution review board. informed consent was obtained from each patient following the information about the treatment plan in the form of a duly signed document prior to the surgical phase. the oral hygiene conditions of the patients were evaluated 2 weeks after the initial therapy (oral hygiene instructions and four quadrants of scaling and root planing, and occlusal adjustment if required). criteria for inclusion in the study were (1) having a good level of oral hygiene [plaque index (pi) <1 loe 1967], (2) probing pocket depth (pd) > 6mm (3) radiographic evidence of at least one vertical bony defect in bilateral sites of mandibular posterior teeth. the exclusion criteria comprised systemic disease, compromised immune system, tobacco smoking, history of antibiotics usage 6 months prior to surgery and treatment for periodontitis during the last 6 months and molars with furcation defects. the selected sample included 20 sites (premolars and molars) in 10 patients where two interproximal sites were randomly (toss of a coin) assigned to the prp/bg or bg groups in a split-mouth design. ten sites in group i were treated with prp and bg graft (perioglas®; us biomaterials corporation, alachua, fl, usa) and 10 sites in group ii were treated with bg alone. clinical recordings customized acrylic stents were fabricated with guiding grooves to provide reproducible alignment for a periodontal probe and the cemento-enamel junction was used as a reference point (figure 1). the outcome variables included pi (silness and loe 1964)21, gingival index (gi) (loe and silness 1963)22, gingival bleeding index (gbi) (ainamo and bay 1975)23, clinical attachment level (cal), probing pocket fig. 1. customized acrylic stent with guiding grooves. effect of platelet-rich plasma and bioactive glass in the treatment of intrabony defects a split-mouth study in humans depth (ppd) and gingival recession (gr). all measurements were made at six sites per tooth (mesio-facial, mid-facial, disto-facial, mesio-lingual, mid-lingual, distolingual). measurements were performed with a williams probe to the nearest millimeter. all clinical and radiographic measurements were performed by a single examiner. radiographic examination a commercially available film holder device with putty impression material was used on bite blocks to index the dentition. standardized reproducible digital radiographs using a standardized paralleling cone technique with positioning aids were taken at each treated site and imported into sopro® imaging software (satelec), which yields an accuracy of 0.1mm. defects were radiographically evaluated to measure defect fill: distance from the cementoenamel junction to base of the defect (cej-bd), alveolar crest height: distance from the cementoenamel junction to the alveolar crest (cej-cd). all clinical and radiographic measurements were performed by a single examiner at baseline, and 3 and 6 months after the surgical procedure. percentage of defect resolution was calculated as preoperative minus postoperative defect depth/ preoperative defect depth×100. preparation of prp with bg nine milliliters of whole blood was drawn by venipuncture of the antecubital vein and collected into two 4.5ml blood collection tubes. these tubes contained 0.105mol/l buffered sodium citrate. the tubes were initially centrifuged at 200g for 10 min. and the plasma part was separated from the red blood cells (rbcs). the whole plasma portion and the top layer of the rbcs, which include fresh platelets, were transferred to a second sterile tube. second centrifuge procedure was performed at 250 g for 10 min leaving the prp at the bottom of the tube. the upper portion of the plasma, namely platelet-poor plasma (ppp), was discarded. finally, 0.5 ml of prp, 0.3 ml 0.025 m cacl 2 and the bovine thrombin were mixed in the vial containing the bg graft and left for gelation. surgical procedure a single surgeon performed all surgeries. after proper isolation of the surgical field, the operative sites were anaesthetized using 2% xylocaine hydrochloride with adrenaline (1:200,000). intra-crevicular incisions were performed, extending to the neighboring teeth and full thickness flaps were raised retaining sufficient tissue to attain primary closure. no conditioning of the root surfaces was performed. defect debridement and root planning were carried out carefully with hand and ultrasonic instruments to remove subgingival plaque, calculus and inflammatory granulation tissue. the prp preparation for group i (prp/bg) started 30 min before surgery. after debridement and pre-suturing, the combination of prp and bg was placed in sites treated as group i(figure 2), while bg alone was mixed with 4 to 6 drops saline, according to the manufacturer’s instructions, and was placed in sites treated as group ii (figure 3). graft was condensed to adapt the particles to configuration of defect taking care not to over fill the defect. every effort was made to avoid contamination of debrided root surface with saliva and blood until the graft material was applied. after grafting, flaps were repositioned to their original levels and periodontal dressing was placed (coe pak)tm. patients were instructed to avoid chewing in the surgical area during the first postoperative day and were recommended to refrain from mechanical plaque control, in and around the surgical area for 2 weeks. systemic antibiotics (amoxicillin 500 mg 3 times a day for 5 days) and (ibuprofen 400 mg 3 times a day for 3 days) were prescribed to achieve an analgesic and antiinflammatory effect with instructions to rinse the mouth twice daily with a solution of 0.2% chlorhexidine digluconate for 2 weeks to aid in plaque suppression. sutures were removed 2 weeks following surgery. recall appointments were scheduled once in 10 days for the 1st month and every 4 weeks thereafter. at every recall appointment oral hygiene was checked and reinforced. all clinical and radiographic measurements were repeated at 3 months and 6 months after the initial surgery. fig. 3. surgical procedure in group ii (bg alone). a. preoperative clinical view; b. preoperative radiograph with intrabony defects on mesial and distal sides of tooth 46; c. intraoperative view depicting graft placement after full-thickness flap elevation; d. postoperative view (after flap closure); e. postoperative view (6 months); f. postoperative radiograph (6 months) fig. 2. surgical procedure in group i (prp/bg). a. preoperative clinical view; b. preoperative radiograph with intrabony defects on the mesial and distal sides of tooth 36; c. intraoperative view depicting graft placement after full-thickness flap elevation; d. postoperative view (after flap closure); e. postoperative view (6 months); f. postoperative radiograph (6 months). 110 braz j oral sci. 9(2):108-114 effect of platelet-rich plasma and bioactive glass in the treatment of intrabony defects a split-mouth study in humans baseline 3 months 6 months 0-3 month 0-6 months 3-6 months pi 0.7±0.1 0.4±0.1 0.4±0.1 0.28±0.09 0.28±0.1) 0.0±0.03 gi 1.33± 0.32 0.78± 0.11 0.71± 0.12 0.55± 0.22 0.62± 0.22 0.07± 0.04 gbi** 73.8± 15.4 37.5± 2.2 28.7± 4.1 36.3± 15.6 45.1± 16.7 8.8± 4.1 p-value 0.005(s) p-value 0.004(s) p-value 0.31(ns) p-value 0.004(s) p-value 0.004(s) p-value 0.004(s) p-value 0.004 (s) p-value 0.004 (s) p-value 0.004(s) table 1 changes in plaque index (pi), gingival index (gi), gingival bleeding index (gbi) scores*(full mouth). *mean ± standard error of the mean; **% of bleeding sites; sig, significant; ns, not significant. statistical analysis mean values (± standard deviations) were reported for the clinical and radiographic parameter. taking into account the nature of changes from baseline to 6 months in each group, the wilcoxon signed ranked test was employed to test mean and standard deviation of clinical and radiographic parameters for both groups at baseline, 3 months and 6 months. the mann whitney test was used to assess the significance of mean differences between groups from baseline to 3 months, baseline to 6 months, and 3 months to 6 months. results all 10 patients completed the study. healing was uneventful in all cases with no noted side effects or unusual complaints. oral hygiene level and infection control a mean plaque index at baseline was 0.7±0.1, with significant reduction to 0.4±0.1 at 3 months and 6 months, thus demonstrating good compliance with oral hygiene instructions. mean gi and bleeding on probing values showed significant reduction at both measurement points compared to baseline (table1). probing pocket depth, clinical attachment level and gingival recession the differences between groups at baseline for ppd and gr parameters were not statistically significant (p>0.05); however, both groups showed significant changes for ppd, cal and gr at different time intervals compared to baseline. changes in ppd are reported in table 2. the mean reduction in ppd was 3.7±1.4 mm and 3.0±1.2 mm from baseline to the 3rd month (p=0.10), and 3.4±1.4 mm and 2.6±1.1 mm up to the 6th month (p=0.10) for prp/bg and bg group, respectively, with no statistically significant difference. there was a slight increase in mean ppd from 3 to 6 months for both groups (p=0.74). cal gain for prp/bg and bg groups was 3.9±1.4 mm and 3.5±1.2 mm, respectively, from baseline to the 3rd month, and 4.3±1.3 mm and 3.3±1.3 mm up to 6th month, with statistically significant difference (p=0.03). at 6 months, there was no significant change between study groups for ppd however there was significant gain in cal in prp/bg group compared to bg group (4.3 mm versus 3.3 mm), which was consistent with slightly higher preoperative cal (9.7 mm versus 9.1 mm). both groups showed a statistically significant reduction in gr with coverage of 0.9±0.2 mm for prp/bg group and 0.7±1.0 mm bg group from baseline to 6th month, though without significant difference between among the groups (p=0.7). radiographic measurements table 3 reports changes in defect fill and alveolar crest resorption for both groups. both groups presented with resorption of alveolar crest adjacent to the defect (-0.2±0.07 mm for prp/bg group and -0.2±0.08 mm for bg group from baseline to the 6th month), but the difference between the two groups was not statistically significant (p=0.90). the mean defect fill was similar for both groups at all time intervals. from baseline to the 6th month, prp/bg and bg groups showed a mean defect fill of 3.5±1.0 mm and 3.1±1.2 mm, respectively, with no statistically significant difference (p=0.72). the mean percentage of defect resolution was slightly greater for prp/bg group (50.3±9.8% and 52.0±11.2%) compared to the bg group with mean defect resolution of 45.1±14.0% was 46.3±13.7 from baseline to the 3rd month and baseline to the 6th month, respectively. though the mean percentage of defect resolution was slightly higher for the prp/bg group, but it was not statistically significant (p=0.44). discussion a wide array of new materials has been used for promoting periodontal regeneration in intraosseous defects. the bone replacement grafts provide regeneration through conductive or inductive processes and in combination with growth factors, have the potential to optimize the outcome of periodontal regeneration. proliferation and migration of periodontal ligament cells and synthesis of extracellular matrix as well as differentiation of cementoblasts and osteoblasts is a prerequisite for obtaining periodontal regeneration and growth factors (prp) may represent a 111 braz j oral sci. 9(2):108-114 effect of platelet-rich plasma and bioactive glass in the treatment of intrabony defects a split-mouth study in humans baseline 3 months 6 months 0-3month 0-6 month 3-6 month prp+bg 8.1±1.4 4.4±0.5 4.7±0.8 3.7±1.4 0.005(s) 3.4±1.4 0.005(s) -0.3±0.7 0.18(ns) ppd bg 8.0±1.2 5.0±0.5 5.4±0.5 3.0±1.2 0.004(s) 2.6±1.1 0.004(s) -0.4±0.5 0.046(s) comparison p-value 1.00 (ns) 0.01 (s ) 0.07 (ns) 0.10 (ns) 0.10 (ns) 0.74 (ns) prp+bg 9.7±1.3 5.8±1.3 5.4±1.1 3.9±1.4 0.004(s) 4.3±1.3 0.004(s) 0.4±0.8 0.16 (ns) cal bg 9.1±1.4 5.6±1.2 5.8±1.0 3.5±1.2 0.005(s) 3.3±1.3 0.005(s) -0.2±0.8 0.41 (ns) comparison p-value 0.03 (s) 0.61(ns) 0.21 (ns) 0.34(ns) 0.03 (s) 0.10(ns) prp+bg 1.6±1.0 1.4±0.9 0.7±0.8 0.2±0.9 0.15(ns) 0.9±0.2 0.034(s) 0.7±0.2 0.059(ns) gr bg 1.1±1.2 0.6±1.0 0.4±1.2 0.5±0.2 0.059(ns) 0.7±1.0 0.02(s) 0.2±1.2 0.41(ns) comparison p-value 0.24(ns) 0.06(ns) 0.51(ns) 0.3(ns ) 0.7(ns) 0.41(ns) table 2 changes in probing pocket depth (ppd), clinical attachment level (cal) and gingival recession (gr) in millimeters*. *mean ± standard deviation; s, significant; ns, not significant; prp, platelet-rich plasma; bg, bioactive glass defect fill alveolar crest prp+bg bg comparison p-value prp+bg bg comparison p-value baseline 6.7±1.0 6.5±1.3 0.72(ns) 3.6±0.6 3.7±0.5 0.42(ns) 3 months 3.3±0.6 3.4±0.9 0.81(ns) 3.7±0.6 3.9±0.5 0.40(ns) 6 months 3.2±0.8 3.4±1.1 0.61(ns) 3.8±0.5 4.0±0.8 0.54(ns) 0-3months 3.4±1.0 0.005(s) 3.1±1.3 0.005(s) 0.68(ns) -0.1±0.04 0.004(s) -0.1±0.07 0.004(s) 0.42(ns) 0-6 months 3.5±1.0 0.005(s) 3.1±1.2 0.005(s) 0.72(ns) -0.2±0.07 0.004(s) -0.2±0.08 0.004(s) 0.90(ns) 3-6 months 0.1±0.3 0.26(ns) 0.02±0.2 0.71(ns) 0.44(ns) -0.1±0.07 0.009(s) -0.09± 0.07 0.014(s) 0.53(ns) table 3 changes in defect fill and in alveolar crest resorption in millimeters* *mean ± standard deviation; s, significant; ns, not significant; df, defect fill; ac, alveolar crest height potential aid in attempts to regenerate the periodontium15. the present study was designed to evaluate the efficacy of associating prp with a bg material. the lack of adverse reactions, abscesses, or rejection of implanted materials, suggested that bg and prp used were tolerated well and in line with observation from previous studies14,18-19,24 that failed to show any foreign body reaction during initial healing and thereafter in the 6-month evaluation period. prp is an autogenous preparation, and is inherently safe and free from concerns over transmissible diseases. in the present study, each patient was placed on monthly recall visits, including supragingival scaling and patient motivation to maintain the hygienic conditions which led to significant change for pi, gi and gbi at 3 and 6 months after treatment. cortellini et al.25 suggested that monthly periodic controls enhanced patient cooperation and infection control. the results of this study have demonstrated that treatment of deep intrabony defects with both, the combination of prp/ 112 braz j oral sci. 9(2):108-114 effect of platelet-rich plasma and bioactive glass in the treatment of intrabony defects a split-mouth study in humans bg and bg alone demonstrated significant reduction in various periodontal parameters. froum et al.26 compared bg graft material and open flap debridement in the treatment of 59 intrabony defects in a 12-month reentry study, and observed 4.26 mm ppd reduction, 2.96 mm cla gain, and 3.28mm of defect fill. lovelace et al.27 observed that bg is capable of producing similar results to dfdba in short term of 6 months in moderate to deep periodontal osseous defects. however, there is no histological evidence in humans that bg may promote true periodontal regeneration10. nevins et al.28 confirmed the new attachment at only one tooth out of five human intrabony defects that were treated with bg. there are only a few studies comparing the clinical effects of prp/graft combination with a graft material alone. in one of the study by ouyang xy et al.29 and hanna et al.30 revealed additional effectiveness of prp+bpbm to bpbm in cal gain in the treatment of intrabony defects. okuda et al.18 compared a combination of prp and ha with a mixture of ha and saline in a 1-year study. their study revealed a statistically significant cal gain of 4.7 mm in prp + ha group versus 3.7 mm with ha alone. however, the benefit of prp to ha in reduction of bony defect depth measured on radiographs was not found in their study which was comparable to results of our study. the results of the present study demonstrated a mean ppd reduction of 3.4±1.4mm, cal gain of 4.3±1.3 mm, gr coverage of 0.9±0.2 mm, and a mean defect fill of 3.5±1.0 mm in the prp/bg group; and ppd reduction of 2.6±1.1 mm, cal gain of 3.3±1.3 mm, gr coverage of 0.7±1.0 mm, and a mean defect fill of 3.1±1.2 mm for the bg group after 6 months. there was statistically significant reduction in ppd at 3 months and gain in cal from baseline to 6 months with prp/bg. gr coverage was statistically significant from baseline to 6 months in both the groups. in contrast to this, demir et al.20 compared the same graft materials and found a mean cal gain of 3.1±0.5 mm and a mean defect fill of 3.5±0.5mm in the prp/bg group and cal gain of 2.9±0.4mm and mean defect fill of 3.4±0.6 in bg group, which failed to show significant difference in any of the investigated parameters. gr was 0.47±0.19mm for the prp/bg group and 0.43±0.25 mm for the prp/bg group, both of which were not statistically significant (p>0.05). using a similar study design, harnack et al.31 demonstrated no additional effect of prp/ beta-tcp over beta-tcp alone in 22 patients with contralateral intrabony defects in a 6-month reentry study. this may be attributed to the use of a different study design. the explanation for slightly higher mean gain in cal for prp/bg could be the potential of prp to contribute in soft tissue healing32. arikan et al.33 and caceres et al.34 suggested the ability of prp to stimulate gingival fibroblast and to modulate several cell responses potentially involved in wound healing, such as cell adhesion, cell migration, and myofibroblastic differentiation. the method of prp preparation is an important aspect to be considered. different techniques of preparation have been known to yield substantially different amounts of cells, i.e., platelets and leukocytes as well as different levels of growth factors35-36. as periodontal defects are small in size, obtaining a large amount of blood from patients for prp preparation is unnecessary, hence only 9 ml of venous blood was withdrawn. according to the technique suggested by landesberg et al.37, two centrifugal forces of 200 g for 10 min provide the maximum increase at platelet number. dugrillon et al.38 stated that an increased amount of g force decreases the amount of growth factor instead of increasing it. the number of platelets in prp is another important issue in the literature about prp, though many studies including a recent study by creeper et al.39 observed the effect of prp on osteoblasts and periodontal ligament cell function to be concentration specific, christgau et al.40 showed only a weak correlation between the platelet counts or the growth factor levels and the clinical and radiographic regeneration outcomes. it should also be kept in mind that prp, as used in this study, may affect wound healing not only by a release of pgfs from platelets, but also due to its other physical and chemical properties. according to obarrio et al. 41, prp preparation assumes a sticky consistency, due to its high fibrin content, which works as a haemostatic and stabilizing agent and may aid the blood clot and bone graft immobilization in the defect area. in conclusion, the present results indicate that at 6 months after surgery both therapies resulted in significant ppd reduction, cal gain, gr coverage and defect fill. the association of prp and bg group provided good soft tissue response with an additional effect on the cal for intrabony defects. future long-term clinical and histological studies should be undertaken to determine the efficacy of bioactive ceramics in combination with prp in the treatment of intrabony defects. acknowledgements the authors would like to thank dr. n.s jagadeeswari, professor, department of biochemistry (meenakshi ammal dental college and hospital) for the excellent assistance in lab preparation of prp. 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sciences n3 original article braz j oral sci. july/september 2010 volume 9, number 3 orofacial injuries in sports and use of mouthguards among university students maria gabriela haye biazevic1, edgard michel-crosato1, ângelo detoni2, renato klotz2, érika regina de souza3, dagmar de paula queluz4 1dds, ms, phd, assistant professor, department of community dentistry, dental school, university of são paulo, brazil 2dds, undergraduate student, university of west santa catarina, brazil 3dds, master’s degree student, department of community dentistry, dental school, university of são paulo, brazil 4dds, msph, phd, professor, department of community dentistry, piracicaba dental school, sate university of campinas, brazil received for publication: may 19, 2010 accepted: june 21, 2010 correspondence to: edgard michel crosato departamento de odontologia social da faculdade de odontologia da usp avenida professor lineu prestes, 2.227 – cidade universitária cep 05508-000 são paulo (sp), brazil e-mail: michelcrosato@usp.br abstract aim: to evaluate the prevalence of orofacial injuries during practice of sports and the use of mouthguards by university students in the southern region of brazil. methods: in this crosssectional study, 700 undergraduates from the university of west santa catarina, brazil, filled out a questionnaire with multiple-choice questions addressing social and economic status, education level of parents, orofacial injuries from sport practice, knowledge, importance and use of mouthguards. the collected data were entered into the epidata 3.2 program, and were analyzed by descriptive statistics and the chi-square test using stata 8.0 statistical software package with a significance level set at 5%. results: out of the total number of students surveyed, 37.02% claimed to have already sustained some kind of orofacial injury. out of these, only 9.90% sought dental assistance after trauma. the most common injuries were: bleeding 54.55%, swelling 23.72%, and dental fractures 16.21%. among the interviewees, 19.24% had already used mouthguard and 44.89% considered its use as important. conclusions: the prevalence of orofacial injuries during sport activities was high in the studied sample and only few of the interviewed university students used individual protection measures to avoid these injuries. keywords: epidemiology, mouth health, dental traumas. introduction health promotion in dentistry has been developing in various specific areas. one of them is sports dentistry, which focuses on assuring oral health for those practicing sports as well as avoiding accidents that might cause orofacial injuries during training or a sports competition1. the prevention and treatment of orofacial trauma is now considered a very important part of the general practice. children and adults are participating more in events where the probability of trauma is significant. increase in orofacial injuries comes along with the increase in sports engagement. the general population is taking its health more seriously2. mouthguards have been used by athletes who recognize the need for oral protection during their sports activities; however the frequency of mouthguard usage is still limited3. reasons for not wearing a mouthguard are mainly the discomfort and the difficulty in breathing as well as in speaking4. to increase mouthguard use, properly fitted mouthguards should be fabricated and provided braz j oral sci. 9(3):380-383 381 by dental professionals as indicated in the academy for sports dentistry position statements5. aspects such as the prevalence and incidence of orofacial injuries occurred in sports and the prevalence of mouthguard use are topics often addressed in sports dentistry6. dental trauma is a very prevalent orofacial injury in sport practice and differs from other traumas in that it can be prevented7, with the possibility of drastically reducing its occurrence by means of mouth devices that promote the protection of all dental and periodontal structures8. several studies on individual or team sports for different age groups and competition levels have been published. research suggests that the dental community be more alert to the dental trauma risk. in contact sports, such as basketball, soccer or boxing, where there is a real possibility for direct dental trauma, the use of mouthguards to prevent sports dental injuries is highly indicated9 sport practice and orofacial injuries are prevalent at schools and universities10, where sports are traditionally practiced. the objective of this study was to investigate the prevalence of orofacial injuries from sport practice and the use of mouthguards among university students in the southern region of brazil. material and methods the study was approved by the research ethics committee of the university of west santa catarina, joaçaba, brazil, under the protocol #114/2005. this study had a cross-sectional design. the study population was composed of university students from the university of west santa catarina’s campus of joaçaba. the university of west santa catarina is a community university distant 400 km from the santa catarina state capital, florianópolis, which has 14,000 students attending its several courses at different campi. the sampling system was made up of conglomerates in two stages. in the first phase, the courses of business administration, accounting, social communication/ advertising, social communication/radio and tv, law, physical education, civil engineering, electric engineering, mechanical engineering, physiotherapy, languages, dentistry, pedagogy, psychology, medicine, tourism and hotel management were selected. in the second phase, students from the first and last periods of each course described above were asked to fill out the proposed questionnaire. in order to meet the goals of the present study, information about the prevalence of orofacial injuries and the use of mouthguards in sport practice and associated factors was collected. the pre-test and the pilot-study were carried out with students taking the last semester of dental school. the examining team took a 2-h training session in order to have a standardization of interviewers, and the data were collected in june, 2005. the data were typed into and analyzed on stata 10.0 statistical software package. frequency distribution and measures of central tendency and dispersion, and specific statistical tests to test the association of pain with associated variables were done. the significance level was set at 5% for all the tests. results out of 21 regular courses at university of west santa catarina, 17 courses made up the study population. the initial and final periods of each course were used, totaling 34 participating classes. the total number of university students involved in the study was 700. the distribution of participants in relation to the courses was as follows: business administration was the course that provided the largest number of participants (n=72;10.29%), and pedagogy was the course the provided the smallest number of participants (n=14; 2%). the initial periods of all the courses were also accountable for the largest proportion of results, with 461 interviewees (65.86%) in comparison to the final phases, with 239 interviewees (34.14%) (table1). as for gender, women had a larger participation, corresponding to 53.14% of the sample, while men corresponded to 46.86% of the sample (table 1). as far as housing is concerned, 65.57% of the interviewees lived with their parents and/or relatives, 20.71% lived with friends and, finally, 13.71% live alone (table 1). as for socioeconomic status, 459 (65.57%) of the interviewees had a family income to live on, and 241 (34.43%) relied on their own income. the great majority (n=530; 75.72%) fit into the pattern of the brazilian middleclass family, 20.14% into low-income family, and 4.14% into high-income family. the parental education level of parents of the 700 interviewees varied a lot: 9.43% of parents had a high-school degree, 23.57% had a college degree, and 23.14% had dropped out of elementary school (table 1). as for sport practice, 84.65% of the university students practiced sports regularly and 15.35% reported they were not engaged in any kind of sport activity. the most popular individual sports were track and field, martial arts and tennis. the most popular team sports were soccer, volleyball, handball and basketball. out of the total of students practicing sports, 90.74% were amateurs and 9.26% did it professionally (table 1). in relation to orofacial injuries resulting from sport practice, 37.02% of students reported having already sustained some kind of injury. out of these, only 9.90% sought dental assistance. out of the total, 62.98% had never had accidents while practicing sports. the most reported injuries were bleeding (54.55%), swelling (23.72%) and dental fractures (16.21%) (table 2). as much as 44.89% of the interviewees reported being aware of the importance of using mouthguards in sport practice, and 55.11% reported that they were not. out of the 44.89%, 125 (39.56%) got the information from dentists, 109 (34.49%) from television programs, and 18 (5.70%) from other means (table 2). as for use of mouthguards, 80.76% did not use them and 19.24% reported using them constantly (table 2). gender (p=0.01) and team sport practice (p=0.01) orofacial injuries in sports and use of mouthguards among university students braz j oral sci. 9(3):380-383 variable categories n % phase initial 461 65.86 final 239 34.14 gender male 328 46.86 female 372 53.14 h o u s i n g lives alone 96 13.71 lives with parents/relatives 459 65.57 lives with friends 145 20.71 i n c o m e f a m i l y 459 65.57 o w n 241 34.43 social class l o w 141 20.14 middle 530 75.72 high 29 4.14 education level of parents incomplete elementary school 162 23.14 elementary school degree 70 10.00 incomplete high school 66 9.43 high-school degree 146 20.86 college dropout 91 13.00 college degree 165 23.57 practice or has practiced a sport? yes 590 84.65 n o 107 15.35 individual sport practice track and field 73 72.28 marcia art 14 13.86 motocross 8 7.92 tennis 6 5.29 team sport practice soccer 262 53.36 volleyball 146 29.74 handball 48 9.78 basketball 35 7.13 sports level amateur 539 90.74 professional 55 9.26 table 1. distribution of university students according to period of courses, gender, housing, income, social class and education level of parents, and profile of sport practice. joaçaba, sc, brazil, 2005. variable categories n % knows the importance of the use of mouthguards in sport practice n o 383 55.11 yes 312 44.89 got information about mouthguards from dentist 125 39.56 television 109 34.49 sport team 42 13.29 teammates 22 6.96 others 18 5.70 has already used mouthguard in sport practice n o 256 80.76 yes 61 19.24 remembers having hit or being hit on the mouth during sport practice n o 439 62.98 yes and didn’t look for help 189 27.12 yes and looked for help 69 9.90 result of hit on the mouth during sport practice bleeding 138 54.55 swelling 60 23.72 dental fracture 41 16.21 dental avulsion 8 3.16 dental extrusion 4 1.58 dental intrusion 2 0.79 table 2. distribution of university students according to the use of mouthguards. joaçaba, sc, brazil, 2005. showed a statistically significant association with the prevalence of orofacial injuries. males showed 2.78 (2.00-5.24) more chances of having injuries than female. sport practitioners had 3.45 more chances of being injuried (table 3). discussion the uneven distribution of university students in the different courses is due to the fact that there are more students 382orofacial injuries in sports and use of mouthguards among university students braz j oral sci. 9(3):380-383 383 in certain courses, such as business administration. another fact is the larger number of interviewees in the initial periods of the courses, since several students end up dropping out of college for different reasons. the university student’s profile in the present research is similar to the one found in the brazilian setting11. most students interviewed for this survey, almost 85% of them, practiced sports. it has been reported that the sport practice is prevalent at schools and universities10. the prevalence of orofacial injuries resulting from sports was 37.02%. though high, it was similar to that of other studies12-13. the prevalence of dental injuries, even dental trauma, was associated with the gender and with the practice of team sports1,6,14. in the present study, only 19.24% of the university students used mouthguards and 44.89% found their use important, which reveals a huge lack of information on the importance of mouthguards. likewise, kvittem et al.15 found that only 6% of sportspeople at the university of minnesota (usa) used mouthguards and 50% found their use important. although the use of mouthguards is important and necessary16, newsome et al.17 explain that their use alone is not the most efficient way to prevent orofacial injuries, mainly the mouthguards commercially sold. this study comes to show the importance of setting up effective programs to promote mouth health in the age groups which most practice sports amateurishly. the limitation of this study has to do with the fact that it is a cross-sectional study in which the analytical power is low; to go further into this subject, it would be interesting to do some research with a qualitative approach18-19. in light of the obtained results, it may be concluded that the prevalence of orofacial injuries during sport activities was high in the studied sample and only few of the interviewed university students used individual protection measures to avoid these injuries. references 1. raghoebar gm, bos rr, vissink a. sports and orofacial injuries. ned tijdschr tandheelkd. 2005; 112: 141-6. 2. padilla rr. a technique for fabricating modem athletic mouthguards. hawaii dent j. 2009; 40: 4, 6-12. 3. maeda y, kumamoto d, iagi k, ikebe k. effectiveness and fabrication of mouthguards. dent traumatol. 2009; 25: 556-64. 4. gardiner dm, ranalli dn. attitudinal factors influencing mouthguard utilization. dent clin north am. 2000; 44: 53-65. 5. academy for sports dentistry [internet]. farmersville, il. available from: http://www.academyforsportsdentistry.org/organization/positionstatement/ tabid/58/default.aspx. 6. echlin p, mckeag db. maxillofacial injuries in sport. curr sports med rep. 2004; 3: 25-32. 7. knobloch k, rossner d, jagodzinski m, zeichen j, gossling t, martinschmitt s et al. prevention of school sport injuries—an analysis of ball sports with 2234 injuries. sportverletz sportschaden. 2005; 19: 82-8. 8. ferrari ch, ferreria de mederios jm. dental trauma and level of information: mouthguard use in different contact sports. dent traumatol. 2002; 18: 1447. 9. kumamoto dp, maeda y. a literature review of sports-related orofacial trauma. gen dent. 2004; 52: 270-80. 10. kvittem b, hardie na, roettger m, conry j. incidence of orofacial injuries in high school sports. j public health dent. 1998; 58: 288-93. 11. porto c, régnier k. o ensino superior no mundo e no brasil – condicionantes, tendências e cenários para o horizonte 2003-2025: uma abordagem exploratória. brasília: ministério da educação; 2003. 173p. 12. yamada t, sawaki y, tomida s, tohnai i, ueda m. oral injury and mouthguard usage by athletes in japan. endod dent traumatol. 1998; 14: 84-7. 13. flanders ra, bhat m. the incidence of orofacial injuries in sports: a pilot study in illinois. j am dent assoc. 1995; 126: 491-6. 14. sang-cohen hd, megnagi g, jacobi y. dental trauma and its association with anatomic, behavioral, and social variables among fifth and sixth grade schoolchildren in jerusalem. community dent oral epidemiol. 2005; 33: 174-80. 15. kvittem b, hardie na, roettger m, conry j. incidence of orofacial injuries in high school sports. j public health dent. 1998; 58: 288-93. 16. newsome pr, tran dc, cooke ms. the role of the mouthguard in the prevention of sports-related dental injuries: a review. int j paediatric dent. 2001; 11: 396-404. 17. ranalli dn, demas pn. orofacial injuries from sport: preventive measures for sports medicine. sports med. 2002; 32: 409-18. 18. rothaman kj, yankauer a confidence intervals vs. significance tests: quantitative interpretation. am j public health. 1996; 76: 587-8. 19. uchoa e, rozemberg b, porto mfs. entre a fragmentação e a integração: saúde e qualidade de vida de grupos populacionais específicos. informe epidemiol sus. 2002; 11: 115-28. table 3. distribution of university students according to gender, team sport practice and presence of orafacial injuries. joaçaba, sc, brazil. 2005. variable categories presence of injuries absence of injuries odss (ic 95%) p gender male 162 166 1 1 female 96 273 2.78(2.00-3.86) 0.01 practice team sport yes 219 272 1 1 n o 39 167 3.45(2.30-5.24) 0.01 orofacial injuries in sports and use of mouthguards among university students braz j oral sci. 9(3):380-383 oral sciences n3 braz j oral sci. 11(2):94-99 received for publication: january 12, 2012 accepted: april 18, 2012 original article braz j oral sci. april | june 2012 volume 11, number 2 measurements of the mandibular canal by multidetector computed tomography frederico sampaio neves1, daniel fernandes tourinho2, manoela carrera3, iêda crusoé-rebello4, thereza bittencourt5, miguel setúbal2 1department of oral diagnosis, piracicaba dental school, state university of campinas, piracicaba, sp, brazil 2department of oral and maxillofacial surgery, school of dentistry, bahiana foundation for the development of sciences, salvador, ba, brazil 3department of oral pathology, piracicaba dental school, state university of campinas, piracicaba, sp, brazil 4department of oral radiology, school of dentistry, federal university of bahia, salvador, ba, brazil 5department of animal development, veterinary school, federal university of bahia, salvador, ba, brazil correspondence to: frederico sampaio neves departamento de diagnóstico oral faculdade de odontologia de piracicaba – unicamp av. limeira, 901, cx postal 52, cep: 13414-903 piracicaba sp brazil phone: + 55 19 21065327 fax: + 55 19 21065218 e-mail: fredsampaio@yahoo.com.br abstract aim: the aim of this study was to investigate the measurements of the mandibular canal in different patterns of reabsorbed alveolar ridges, using multidetector computed tomography in order to evaluate the relationship of the mandibular canal with the cortex of the mandible remains. methods: central cross-sectional slice of 30 edentulous alveolar ridges in the mandibular first molar region of otherwise healthy patients using multidetector computed tomography were analyzed. horizontal and vertical lines were performed tangent to the corticals of the mandible and mandibular canal. fisher’s exact test, spearman test and linear regression were used for statistical analysis. significance level for all statistical tests was 95%. results: the height of the mandible and the distance of mandibular canal to superior cortical in males were significantly higher when compared with females (p<0.05). when the height of the mandibular bone was correlated to the classification of edentulous jaws, significant differences were observed when comparing the three types (iii, iv and v) as well as between iii and iv type. conclusions: the results of the present study show that the measurements for dental implant placement in the posterior region of the mandible are affected by the different patterns of bone resorptions. it was also demonstrated the importance of the computed tomography in the process of planning dental implant placements. keywords: bone, mandibular canal, computed tomography, mandible, measurements. introduction for preoperative planning of mandibular implant placement, precise evaluation of bone size and morphology is important. the size of the selected implant depends on the height and width of available bone and the location of the mandibular canal1. physiological absorption of the edentulous alveolar ridge will reduce the distance between the bone crest and the cortical of the mandibular canal. the measures obtained in panoramic and periapical radiographs do not accurately correspond to the reality. the mandibular canal could be lateralized and not necessarily in the center of the mandible body, requiring a specific preoperative radiologic examination for better management of the case1-2. placement of endosseous implant in the appropriate position to allow creation of an aesthetic prosthesis depends on bone volume, height and density. bone 9595959595 braz j oral sci. 11(2):94-99 density and height can both be estimated by radiographic evaluation, using conventional tomography and periapical or panoramic radiographs2. computed tomography (ct) scan by image reconstruction has been used to investigate periapical injuries and its relation to the mandibular canal3 and due to its property to trustily reconstruct bone anatomy, represents an excellent adjunct to the evaluation of the actual position of the mandibular canal within the mandible4. multidetector ct (mdct) is considered one of the most valuable imaging modalities for preoperative procedures because it allows the acquisition of fast, reliable and reproducible images. each anatomical structure can be viewed in the three orthogonal planes (axial, coronal, sagittal), overlapping of surrounding anatomical structures can be eliminated and a three-dimensional reconstruction of the evaluated structure can be done. in comparison with cone beam computed tomography, the greatest advantage of mdct is the optimal contrast resolution, allowing differentiation between hard and soft tissues5. ct was considered a highdose technique for a long time, but with the development of mdct and low-dose protocols tailored for diagnosis, doses below 0.15 msv are achievable6. it is important to measure bone volume to avoid injuries in the alveolar inferior nerve resulting from implant surgery. the trajectory of the mandibular canal at the site of implant placement may alter sensation of the lower lip due to inferior alveolar nerve injury and it is one of the most serious complications of mandibular implant surgery7-8. the aim of this study was to investigate the measurements of the mandibular canal in different patterns of resorbed alveolar ridges, using mdct in order to evaluate the relationship of the mandibular canal with the cortex of the mandible remains. material and methods data were collected from the data bank of a radiology center. all patients enrolled at this evaluation were conducting mdct examination as a procedure of their treatment planning for rehabilitation with titanium implants. therefore, no radiation was imposed to the patient to obtain the study data. all patients gave written informed consent for the examination and use of data for the research protocols. a retrospective study was conducted from more central cross-sectional slice of 30 edentulous alveolar ridges in the mandibular first molar region of otherwise healthy patients using mdct. the mandibles were evaluated using noncontrasted exams performed in high resolution helical ct device (ct synergy helicoidal; general eletric company, milwaukee, wi), the examination was performed with the patient in supine position (slice thickness 0.625 mm and increment of 0.625 mm, field of view of 15.8cm, bone filter, 120 kv and 200 ma). each coronal slice was analyzed on the ct workstation monitor (advantage workstation 3.1 ultra 10sm ge medical systems, usa), under dim lighting conditions, by one oral and maxillofacial radiologist with more than 10 years experience in ct. all mdct images of the bone sections were categorized according the criteria of cawood and howell (1988)9 edentulous jaws classification: i dentate, ii immediately post extraction, iii well-rounded ridge form, adequate in height and width, iv knife-edge ridge form, adequate in height and inadequate in width, v flat ridge form, inadequate in height and width and vi depressed ridge form, with some basilar loss evident. horizontal and vertical lines were performed tangent to the cortical plates of the mandible and mandibular canal. the outcome variables were the linear distances (cm) between mandible width and height, mandibular canal width and height, distance of the mandibular canal to buccal, lingual, superior and inferior cortical plates of the mandible and the distance of the mandibular canal to the higher point of the alveolar ridge (figure 1). images were re-evaluated after a four-month interval and intraobserver agreement was calculated, demonstrating high reproducibility (kappa index = 0.95). fig. 1. central coronal mdct slice showing the measurements of the mandibular canal and mandibular bone. data were analyzed using the sas 9.1 software (sas institute, cary, nc, usa). descriptive parameters were given as mean and standard deviation (sd). significant differences between comparable measurements were tested for gender and classes of the edentulous jaws with fisher’s exact test. spearman test was used to evaluate the correlation between variables with biological plausibility. linear regression was used to determinate the best fit between the variables. significance level for all statistical tests was set at 95%. results table 1 shows the classification of the edentulous jaws according to cawood and howell (1988)9. the most prevalent classes were iii, iv and v. the mean and standard deviation (sd) of the measurements are summarized in the table 2. measurements of the mandibular canal by multidetector computed tomography braz j oral sci. 11(2):94-99 9696969696 table 1. distribution of the bone sections studied according cawood and howell (1988)9 edentulous jaws classification. class number of cases (%) i 0 (0%) ii 0 (0%) iii 12 (40%) iv 7 (23.3%) v 11 (36.4%) vi 0 (0%) (*) sd, standard deviation. table 2. mean and sd of the measurements in mdct images. measurements mean (sd) width of the mandible (wm) 1.680 (±0.05) height of the mandible (hm) 2.133 (±0.06) width of the mandibular canal (wmc) 0.250 (±0.01) height of the mandibular canal (hmc) 0.250 (±0.01) distance of the mandibular canal to buccal cortical (mc-bc) 0.610 (±0.03) distance of the mandibular canal to lingual cortical (mc-lc) 0.820 (±0.04) distance of the mandibular canal to superior cortical (mc-sc) 1.253 (±0.05) distance of the mandibular canal to inferior cortical (mc-ic) 0.623 (±0.03) distance of the mandibular canal to the higher point of the alveolar ridge (mc-hpar) 1.493 (±0.06) the differences between males and females regarding mandible width and height, mandibular canal width and height, distance of the mandibular canal to buccal, lingual, superior and inferior cortical plates of the mandible and the distance of the mandibular canal to the higher point of the alveolar ridge are presented in figure 2. the height of the mandible in males was significantly higher when compared to females (p<0.05). however, the mandibular canal width in females was significantly higher when compared to males (p<0.05). the width of the mandible and the distance of the mandibular canal to the higher point of the alveolar ridge were higher in females, but not statistically significant. fig. 2. stratification of the measurements used in this study according to gender: mandible width (a) and height (b), mandibular canal width (c) and height (d), distance of the mandibular canal to buccal (e), lingual (f), superior (g) and inferior (h) corticals of the mandible and the distance of the mandibular canal to the higher point of the alveolar ridge (i). when the height of the mandibular bone was correlated to the classification of edentulous jaws, there were significant differences when comparing the three types (iii, iv and v) as well as between iii and iv types. there was no significant difference between types iv and v’s edges height. type iii mandibular bone height had a higher value (mean = 2.383 cm), followed in a descending order by type v (mean = 2.0 cm) and type iv (mean = 1.914 cm) (figure 3). comparing the three types (iii, iv and v) with all measurements performed in this study, statistically significant differences were found in the mandibular bone height and the distance of the mandibular canal to the superior cortical (alveolar ridge). the other measurements and types of edentulous jaws showed no statistical differences (figure 4). it was found that the greater the horizontal diameter of the mandibular bone, the greater the vestibular (r2=0.646; fig. 3. association between the classification of edentulous jaws iii, iv and v and the measures of mandibular bone height. fig. 4. association between the types of edentulous jaws (iii, iv, v) and the measurements of the mandible width (a) and height (b), mandibular canal width (c) and height (d), distance of the mandibular canal to buccal (e), lingual (f), superior (g) and inferior (h) corticals of the mandible and the distance of the mandibular canal to the higher point of the alveolar ridge (i). measurements of the mandibular canal by multidetector computed tomography 9797979797 p<0.05) and lingual (r2=0.675; p<0.05) width. linear regression analysis also demonstrated that there was a linear interaction between the horizontal diameter and vestibular width (r2=0.417) and lingual ridge width (r2=0.455). although positive correlation was observed between the mandibular bone horizontal diameter and the distance to the highest point of the ridge (r2=0.188), the interaction between these two variables was weaker (figure 5). fig. 5. association between the width of the mandibular bone and the distance from the mandibular canal to lingual cortical (mb-lc), the distance from the mandibular canal to buccal corticals (mc-bc) and the distance from the mandibular canal to the higher point of the alveolar ridge (mc-hpar). a positive correlation could be noticed between the increased vertical distance and the increment of the superior (r2=0.821; p<0.05) and inferior heights (r2=0.423; p<0.05), as well as the distance of highest point of the alveolar ridge (r2=0.634; p<0.05). linear regression between the vertical diameter and the distance to the highest point of the alveolar ridge (r2=0.402) and the superior height (r2=0.673) showed a strong positive association (figure 6). fig. 6. association between the height of the mandible and the measurements of the distance from the mandibular canal to superior cortical (mc-sc), the distance from the mandibular canal to inferior cortical (mc-ic), and the distance from the mandibular canal to the higher point of the alveolar ridge (mc-hpar). the distance of the highest point on the alveolar ridge is directly correlated with the superior height (r2=0.897; p<0.05) and the lingual weight (r2=0.537; p<0.05). a strong linear interaction was observed between the distance to the highest point of the alveolar ridge and the superior height (r2= 0.804), though, a weak positive association between the first and the last variable (r2=0.288) (figure 7). fig. 7. association between the distance from the highest point on the alveolar ridge and the distance from the mandibular canal to superior cortical (mc-sc) and the distance from the mandibular canal to lingual cortical (mc-lc). discussion an adequate dental treatment is based on comprehensive planning, which includes the use of imaging studies to assist on diagnosis. imaging examinations have an important role to a successful treatment. the preoperative evaluation for implant therapy should consider the height and width of the bone, degree of corticalization, density of mineralization and amount of cancellous bone. panoramic radiography is a the supplementary examination initially requested to implant surgery, however, because the two-dimensional image, this does not provide specific diagnostic information about the relationship of anatomical structures, thus, are necessarily a more accurate exam to a provide a higher operative safety2. the mandibular canal is an anatomical structure used as reference to surgical approaches in the jaws. extractions of third molars, implant placement, orthognathic surgery, reduction and fixation of fractures in various areas of the mandible are examples of procedures performed in close proximity to this canal, what increases the risk of injury to the inferior alveolar nerve10. the use of ct for dental implant planning has been strongly advocated over the past decades2,11 due to its superiority to conventional radiographs12 and even when compared with conventional tomography. this can be attributed to three-dimensional visualization of the bone structures in ct scans13. lindh (1995)14 compared the accuracy of measurements of two panoramic devices and three tomographic techniques. in all image modalities the distance between the superior border of the mandibular canal and the alveolar crest, the height of the mandibular canal and the distance between the mandibular base and the inferior border of the mandibular canal were measured. the values from the tomographic images deviated less from the panoramic images measurements. the observers underestimated the distances related to the mandibular canal, except for hypocycloidal tomography. the mean height of the mandibular canal was measurements of the mandibular canal by multidetector computed tomography braz j oral sci. 11(2):94-99 9898989898 3.0 mm, with a standard deviation of 0.7 mm. height values of the mandibular canal were similar to those found in the present study. it has been observed in dentate individuals, using ct, that the mean distance from the mandibular canal to the alveolar crest was 17.4 mm15. in similar and recent studies, watanabe et al. (2010)16 and de oliveira-junior et al. (2010)17 measured the height of the mandible and distance of the mandibular canal to the superior cortical of the mandible in dentate and edentulous mdct cross-sectional scans in a inferior first molar region. comparing these studies with our findings, the distances were higher than in the present study, which is justified by the presence of teeth, not occurring the physiological bone resorption. paes et al. (2007)18 evaluated the accuracy of relative measurements from the roof of the mandibular canal to the alveolar crest in mdct and single-slice ct. mdct has a more accurate method and a higher reproducibility. it helps in the analysis of important anatomical landmarks for the planning of dental implants, namely the mandibular canal pathway and alveolar crest height. according to the degree of bone resorption, significant changes may occur in relation to facial muscles and facial height, but the distance between the mandibular canal to mandibular cortex remains stable19. in the present research, performed in 30 mandibles, the distance of the mandibular canal to the inferior bone cortical diminished when the level of ridge resorption became more severe. in type iii, the mean was 0.68 cm, followed by 0.62 cm in type iv and type v at 0.58 cm. when anatomic structures and ridge resorption limit the placement of a standard implant, the clinician can use short implants or bone graft augmentation. in edentulous alveolar bone ridges less than 0.5 mm wide bone graft augmentation is required prior to implant placement. crestal ridge bone augmentation is an alternative bone expansion technique that can be used to augment the atrophic maxilla and mandible prior to implant placement20. short implants can also be placed to avoid the use of grafts, but it must always be taken into account when using these implants that a minimum bone height of 7-8 mm is needed anyway and that bone quality is a critical factor21. inferior alveolar nerve injury can result from traumatic local anesthetic injections, during dental implant site preparation or placement, or poor surgical technique. during implant surgery, damage to the inferior alveolar nerve can occur when the twist drill or implant encroaches, transects, or lacerates the nerve. the insertion of implants close to the canal can compromise the success of the surgery. the contact with the neurovascular bundle can actually cause the nonintegration of the implant or lead to sensory dysfunction. it is essential to obtain, especially in ct images, as much information as possible from the anatomic appearances and variations for a safer surgical procedure22. mdct provides an excellent visualization and delineation of mandibular anatomy, which, in turn, allows establishing the buccolingual position and height of neurovascular bundle, as well as, evaluating the amount of bone available for correct placement of implant fixtures. the results of the present study showed that the measurements for dental implant placement in the posterior region of the mandible are affected by the different patterns of bone resorption. therefore, the surgeon and radiologist should be aware of the correct bone measurements in the preoperative planning for dental implant placement, being ct extremely important in this process to avoid intraor postoperative complaints. acknowledgements we are grateful to delfin clinic for the support in this study. references 1. garg ak, morales mj, navarro i, duarte f. autogenous mandibular bone grafts in the treatment of the resorbed maxillary anterior alveolar ridge: rationale and approach. implant dent. 1998; 7: 169-76. 2. schwarz ms, rothman sl, chafetz n, rhodes m. computed tomography in dental implantation surgery. dent clin north am. 1989; 33: 555-97. 3. velvart p, hecker h, tillinger g. detection of the apical lesion and the mandibular canal in conventional radiography and computed tomography. oral surg oral med oral pathol oral radiol endod. 2001; 92: 682-8. 4. jacobs sg. radiographic localization of unerupted mandibular anterior teeth. am j orthod dentofacial orthop. 2000; 118: 432-8. 5. rydberg j, liang y, teague sd. fundamentals of multichannel ct. radiol clin north am. 2003; 41: 465-74. 6. koizumi h, sur j, seki k, nakajima k, sano t, okano t. effects of dose reduction on multi-detector computed tomographic images in evaluating the maxilla and mandible for pre-surgical implant planning: a cadaveric study. clin oral implants res. 2010; 21: 830-4. 7. kiyak ha, beach bh, worthington p, taylor t, bolender c, evans j. psychological impact of osseointegrated dental implants. int j oral maxillofac implants. 1990; 5: 61-9. 8. bartling r, freeman k, kraut ra. the incidence of altered sensation of the mental nerve after mandibular implant placement. j oral maxillofac surg. 1999; 57: 1408-12. 9. cawood ji, howell ra. a classification of the edentulous jaws. int j oral maxillofac surg. 1988; 17: 232-6. 10. xie q, wolf j, soikkonen k, ainamo a. height of mandibular basal bone in dentate and edentulous subjects. acta odontol scand. 1996; 54: 379-83. 11. cavalcanti mg, yang j, ruprecht a, vannier mw. accurate linear measurements in the anterior maxilla using orthoradially reformatted spiral computed tomography. dentomaxillofac radiol. 1999; 28: 137-40. 12. dantas ja, montebello filho a, campos psf. computed tomography for dental implants: the influence of the gantry angle and mandibular positioning on the bone height and width. dentomaxillofac radiol. 2005; 34: 9-15. 13. todd ad, gher me, quintero g, richardson ac. interpretation of linear and computed tomograms in the assessment of implant recipient sites. j periodontol. 1993; 64: 1243-9. 14. lindh c, petersson a, klinge b. measurements of distances related to the mandibular canal in radiographs. clin oral implants res. 1995; 6: 96-103. 15. levine mh, goddard al, dodson tb. inferior alveolar nerve canal position: a clinical and radiographic study. j oral maxillofac surg. 2007; 65: 470-4. 16. watanabe h, mohammad abdul m, kurabayashi t, aoki h. mandible size and morphology determined with ct on a premise of dental implant operation. surg radiol anat. 2010; 32: 343-9. 17. oliveira júnior mr, saud al, fonseca dr, de-ary-pires b, pires-neto ma, ary-pires r. morphometrical analysis of the human mandibular canal: a ct investigation. surg radiol anat. 2010; 33: 345-52. measurements of the mandibular canal by multidetector computed tomography braz j oral sci. 11(2):94-99 18. paes ada s, moreira cr, sales ma, cavalcanti mg. comparative study of single and multislice computed tomography for assessment of the mandibular canal. j appl oral sci. 2007; 15: 220-4. 19. sutton dn, lewis br, patel m, cawood ji. changes in facial form relative to progressive atrophy of the edentulous jaws. int j oral maxillofac surg. 2004; 33: 676-8. 20. demetriades n, park ji, laskarides c. alternative bone expansion technique for implant placement in atrophic edentulous maxilla and mandible. j oral implantol. 2011; 37: 463-71. 21. romeo e, bivio a, mosca d, scanferla m, ghisolfi m, storelli s. the use of short dental implants in clinical practice: literature review. minerva stomatol. 2010; 59: 23-31. 22. juodzbalys g, wang hl, sabalys g, sidlauskas a, galindo-moreno p. inferior alveolar nerve injury associated with implant surgery. clin oral implants res. 2012. doi: 10.1111/j.1600-0501.2011.02314.x. 9999999999 measurements of the mandibular canal by multidetector computed tomography braz j oral sci. 11(2):94-99 oral sciences n3 original article braz j oral sci. october|december 2010 volume 9, number 4 received for publication: july 15, 2010 accepted: december 07, 2010 short implants with single-unit restorations in posterior regions with reduced height – a retrospective study jamal hassan assaf1, agenor montebello filho2, fabricio batistin zanatta3 1dds, ms, são leopoldo mandic research center, dental school, campinas, sp, brazil 2 phd, professor, são leopoldo mandic research center dental school, campinas, sp, brazil 3dds, ms, professor, franciscan university center , santa maria, rs, brazil correspondence to: jamal hassan assaf rua pinheiro machado, 2380/310 cep: 97050-600 santa maria rs brazil phone: +55 55 32221088 fax: +55 55 30270927 e-mail: jamal.assaf@hotmail.com abstract aim: previous studies have shown excellent prognosis with short implants. however, evidence of short implants rehabilitated with single-unit restorations is scarce. the purpose of this study was to evaluate the percentage of success of short implants in posterior maxillary and mandibular regions with single-unit restorations. methods: this study comprised a retrospective analysis of data of 41 patients that received 54 short implants between 2004 and 2009. all implants were placed by the same surgeon and all restorations were performed by experienced professionals. all implants were placed in sites with reduced bone volume; 48 implants were evaluated by computed tomography and 6 implants by radiography. failures and biological complications were analyzed. results: it was observed a success rate of 96.3% in the osseointegration period and 100% in the load period that ranging from 7 to 38 months. two complications were observed, which were successfully treated without risk of implant loss. conclusions: short implants in posterior regions rehabilitated with single-unit restorations are a treatment option with high success rate and thus may be indicated in posterior regions with reduced bone height. keywords: short implants, single-unit restorations, success, retrospective study. introduction the clinical use of several types of dental implants has been highly predictable in the last decades. however, its use may be restricted in the presence of limitations related to the morphology and volume of the bone ridge. these limitations are usually more common in the posterior maxillary and mandibular regions1. when the morphology and volume of bone ridge are insufficient for the placement of conventional implants, the treatment of choice comprises anatomical modification of the surgical site by the use of surgical techniques for bone augmentation. in the mandible, in some cases, the alveolar nerve must be displaced to allow the subsequent placement of long implants. this approach requires more complex surgical procedures with high morbidity rate, higher cost and longer time for final treatment2. within this context, the placement of short implants in existing anatomical sites may be considered an appropriate option. the rationale for the use of short implants is that the osseointegrated interface braz j oral sci. 9(4):493-497 contacting the bone tissue distributes a greater amount of occlusal load to the most coronal portion of the implant, compared to the middle and apical thirds1. some studies on short implants demonstrated greater occurrence of failure compared to long implants, with lower success rates for short implants3-5. other studies revealed adequate success rates of short implants after 1 to 5 years of follow up6-9, ranging from 90 to 94.7%. these studies demonstrated that the posterior maxillary region exhibited the highest percentage of failures. however, no study described if single-unit restorations had been placed and the type of antagonist tooth. this information would be important, since there are doubts on whether short implants may support occlusal loads; if the implant is joined to another short or long implant, evaluation of the short implant as an isolated prosthetic unit is not possible10. regardless of that, it should be mentioned that the highest percentage of failure of short or long implants occur during the osseointegration period, in addition to a small percentage during the first year of load1,5-6,9,11-12. this study retrospectively analyzed short implants with single-unit restorations in posterior maxillary and mandibular regions with insufficient bone volume for the placement of long implants. material and methods using a retrospective design, this study included all patients attending a private dental office between june 2004 and june 2009, who were submitted to clinical and radiographic examination and required single-unit prosthetic rehabilitation in posterior maxillary and mandibular regions using short implants, due to reduced bone quantity in the sites of implant placement. all such cases were considered eligible for inclusion in the sample. additionally, the following inclusion criteria were considered: no history of severe cardiac disorders, immune diseases, coagulation deficiencies, psychiatric and neurological disorders, severe metabolic diseases, bleeding on probing in any site of the mouth; radiotherapy and/or chemotherapy, smoking, or alcohol or drug addiction. p a t i e n t s w h o d i d n o t a t t e n d t h e c o n s u l t a t i o n s a f t e r placement of the prostheses were excluded. the research protocol reviewed and approved by the institutional review board (irb) of são leopoldo mandic research center, campinas, sp, brazil (in accordance with resolution n. 196/96 of the ministry of health national council), under protocol #2008/0295. data of 41 patients (17 males and 24 females), who attended the dental office of the investigator jha, were reviewed by the analysis of records clinical interviews and clinical examinations had been conducted by the investigator. all patients evaluated were operated by the investigator with the aid of the same team, and all followup consultations were performed by the same investigator. implants the study analyzed clinical and radiographic data of osseotite® 54 implants with treated surfaces (3i implant innovations, inc., palm beach gardens, fl,usa), being 17 implants in the maxilla and 37 in the mandible. data on the characteristics of implants were obtained from individual forms containing identification of the series number, batch and specifications of all implants. surgical stage and healing stage all cases in the sample were performed according to the general surgical guidelines established by branemark et al.13, with specific indications as recommended by buser et al.14, summers15-17 and martinez et al.18. all surgeries were performed by an experienced surgeon (jha) and the healing period ranged from 3 to 6 months. prosthetic stage all prosthetic reconstructions were performed by experienced professionals. the antagonist teeth were either restored or intact natural teeth, except for 6 mandibular implants whose antagonist teeth were conventional removable dentures and implant-supported dentures. radiographic examinations the pre and postoperative radiographic examinations were conducted in radiology centers, and the computed tomography scans of preoperative examinations were obtained with a hospital tomography machine (phillips®, aura model, pembroke pines, fl, usa) and analyzed by the software dentascan®, ge medical systems milwaukee, wi, usa). the measurements were obtained from the bone crest of the maxillary sinus floor in the maxilla, and from the bone crest of the upper portion of the mandibular canal in the mandible. in some cases, measurements in the mandible were obtained from the bone crest of the submandibular notch. clinical and radiographic evaluation evaluation of the success of implants by analysis of the records followed the success criteria suggested by esposito et al.19, including the following clinical and radiographic criteria: a) absence of pain and sensitivity during placement of healing and prosthetic abutments, ruling out the possibility of pain due to the presence of gingival or mucous tissue between the implant platform and these devices; absence of pain during mastication and absence of pain and/or permanent sensitivity due to aggression to the inferior alveolar nerve; b) absence of horizontal and vertical mobility. the occurrence of sensitivity during manual placement of provisional or healing abutments was carefully considered and the healing period was extended. in these cases, the provisional restorations were performed after this sensitivity had disappeared. in these cases, the final torque was applied after 6 months of function with the provisional restorations. the case was considered as successful when there was no 494494494494494short implants with single-unit restorations in posterior regions with reduced height – a retrospective study braz j oral sci. 9(4):493-497 pain or sensitivity during this period, and also when rotation mobility did not occur after final torque (20 to 30 n) of the definitive restoration; c) maintenance of osseointegration during the load period; d) absence of periimplantitis, or periimplantitis diagnosed and successful treated. in the presence of periimplantitis, the same investigator performed the treatment following the diagnostic and therapeutic guidelines established by mombelli and lang20. mucositis was not included in the criteria for definition of success or failure of the implants analyzed. results the sample comprised 41 patients with 54 implants. among these, 17 were placed in the maxilla and 37 in the mandible. table 1 describes the characteristics of the sample. the mean age was 52.1 years, ranging from 18 to 78 years (table 1). table 2 describes the number and distribution of gender number (%) mean age (standard deviation) male 17 (41.4%) 51.3 (±12.8) female 24 (58.5%) 53.7 (±11.9) total 41 (100%) 52.1 (± 12.26) table 1. demographic characteristics of the sample. regions 4x7.0 4x8.5 5x7.0 5x8.5 6x7.0 6x8.5 total per site maxillary right second molar maxillary right first molar 1 1 2 4 maxillary right second premolar 1 1 maxillary right first premolar 1 1 2 maxillary left second molar 1 1 maxillary left first molar 2 2 2 6 maxillary left second premolar 1 1 2 maxillary left first premolar 1 1 mandibular left second molar 1 4 1 6 mandibular left first molar 5 1 1 7 mandibular left second premolar 3 1 1 5 mandibular left first premolar 1 1 mandibular right second molar 3 2 2 7 mandibular right first molar 2 1 5 8 mandibular right second premolar 1 2 3 mandibular right first premolar total 1 19 14 11 6 3 54 table 2. characteristics of implants used in different areas of the dental arches (width x length). posterior regions number of implants mean bone height available analyzed by computed tomography (variation) upper jaw 11 6.53 mm (4.0-9.0) 7.63 mm (7.0-8.5) lower jaw 37 9.28 mm (7-11.2) 7.89 mm (7.0-8.5) mean length of implants employed (variation) table 3. mean bone height (mm) x length of implants (mm) on the 48 sites analyzed by computed tomography. time interval in months of load (longitudinal evaluation) number of implants loss to follow-up loaded implants failuresin the period percentage of success (%) 7-12 52 1 51 0 100% 13-24 43 43 0 100% 25-38 35 35 0 100% table 4. percentage of success of loaded implants implants placed in posterior maxillary and mandibular regions. it is observed that 39 implants were placed in the region of first and second molars, 11 in sites of second premolars and 4 in regions of first premolars. table 3 presents the mean height of implant sites assessed by topographic examination, with the minimum and maximum height, related to the mean length of implants placed, as well as to the minimum and maximum length of implants. in the maxilla, the mean length of implants was 1.10 mm greater than the mean bone height of implant sites, i.e. the height of the alveolar ridge was extremely affected, requiring use of the summers technique in some cases. in the mandible, the mean length of implants was 1.39 mm smaller than the mean bone height available on the implant sites, i.e. the implants were placed very close to the inferior alveolar nerve. the success in the osseointegration period was 96.3%. two implants were lost from the total 54 implants evaluated (one in the mandible and one in the maxilla). both losses occurred during the healing period, and no loss occurred during the load period. failure in the maxilla was detected after 6 months, on reopening. in the preoperative evaluation, the alveolar ridge at this region exhibited bone height of 4.7 mm (evaluated on the tomography) and low bone density, as evaluated during drilling by the surgeon. this site received another implant, which presented successful osseointegration and constitutes one of the short implants in the sample. the 495495495495495 short implants with single-unit restorations in posterior regions with reduced height – a retrospective study braz j oral sci. 9(4):493-497 other failure occurred in the mandible. this site was submitted to immediate implant placement. due to the anatomical characteristics, the surgical site was damaged after tooth extraction. the implant had low primary anchorage obtained manually. after 3 months, on reopening, the implant did not present osseointegration. this site received another implant, which had successful osseointegration and is one of the short implants in the sample. table 4 presents the percentage of success of loaded implants in different periods. there was no loss to followup. however, due to different periods of implantation and prostheses, of the duration of follow-up was not similar for all implants. fifty-one implants were followed from 7 to 12 months, 43 implants from 13 to 24 months and 35 implants from 25 to 38 months. there was no failure of the 51 implants followed up to 12 months. the implants evaluated in this period presented few complications. two implants exhibited periimplantitis, which was treated and responded favorably. discussion the present results revealed a high success rate after the use of short implants. previous studies evaluated the success of short implants and reported favorable outcomes21-24. however, these studies did not describe the presence of antagonist teeth, and the number of short implants with single-unit restorations was small. one of the limitations of retrospective studies is the possibility of biases, which may impair the validity of the results. in the present study, the outcome analyzed was the implant loss, which reduces the possibility of measurement errors by the examiner. the method for data collection should also be highlighted, which allowed the achievement of results from all patients considered as eligible during the study period. there are studies that reported excellent prognosis with short implants1,25-29. these investigations were conducted on posterior regions rehabilitated with single-unit restorations with follow-up periods of 32 months to 6 years, revealing success rates of 94.5% to 100%. however, even though these studies reported alveolar ridges with reduced height, they did not specifically describe the extent of damage of the alveolar ridges. concerning the presence and type of antagonist tooth, fugazzoto et al.27 described the antagonist teeth, which yet were highly variable, including restored or intact natural teeth, fixed dentures, conventional complete dentures, removable partial dentures and implant-supported dentures. all implants in this study received single-unit restorations and were placed in the posterior region. also, data on the bone height available before implant placement are also presented, evidencing that the alveolar ridge was actually impaired. in the posterior maxillary region, the mean bone height assessed on the tomographic examination was 6.53 mm, ranging from 4.0 to 9.0 mm; many implants were placed using the summers technique15-17. in the posterior mandibular region, the mean bone height was 9.28 mm, ranging from 7 to 11.2 mm. the mean length of implants was 7.63 mm (7.0-8.5) in the maxilla and 7.89 mm (7.0-8.5) in the mandible. most implants occluded with restored or intact natural teeth, and only 6 implants occluded with removable acrylic dentures. considering the present results, it may be concluded that short implants constitute a treatment option for partially edentulous patients with need of single-unit restorations in sites with reduced bone height, with a success of 96.3% in the osseointegration period and 100% in the load period. these results corroborate those of previous studies1,25-27,29. it should be highlighted that all implants in this study presented surface treatment, which enhances the quality of contact between bone and implant, increasing the resistance to removal30. when planning the placement of implants in posterior regions with reduced bone height, professionals should consider a systematic review31, in which the success of long implants in regions submitted to traumatic maxillary sinus lift ranged from 61.7 to 100% (mean of 91.8%), similar to the success of short implants. considering these outcomes, it may be stated that the use of short implants in posterior regions may be considered an option, even when the short implant is indicated for single-unit restorations. it may also be assumed that these results are similar to those observed in long implants placed in these surgical sites (posterior maxillary and mandibular region), with success rates ranging from 93 to 98% using implants longer than 10 mm2,8,1011,32. it should be noticed that, in these studies, long implants were placed in regions with adequate bone volume and height that were not submitted to bone augmentation techniques. comparison of the success of single-unit short and long implants in posterior maxillary and mandibular regions by a randomize clinical trial would not be the most adequate experimental design, since it should include sites that might receive either long or short implants, which would not reproduce the clinical situation with better indication of short implants, i.e. sites with reduced bone height. conversely, it would be interesting to compare short and long implants with single-unit restorations (in impaired sites submitted to bone augmentation techniques), analyzing the percentage of success obtained in surgeries or bone augmentation associated with the percentage of success of long implants placed in these sites, to allow comparison of both treatment options. although further research is necessary, the use of short implants should be considered a viable option with good prognosis; besides reducing the cost and time required for the procedures, it also involves a much lower human cost. according to the present clinical and radiographic findings, it may be concluded that short implants with treated surfaces presented adequate success for single-unit restorations in posterior maxillary and mandibular regions in nonsmoking, systemically health patients. acknowledgements the authors wish to thank the radiography center of dix (caridade hospital, santa maria, rs, brazil) for the support related to tomography exams. dr. assaf, montebello 496496496496496short implants with single-unit restorations in posterior regions with reduced height – a retrospective study braz j oral sci. 9(4):493-497 filho and dr. zanatta report no conflicts of interest related to this study. references 1. misch ce, steigenga j, barboza e, misch-dietsh f, cianciola lj, kazor c. short dental implants in posterior partial edentulism: a multicenter retrospective 6-year case series study. j periodontol. 2006; 77: 1340-7. 2. renouard f, nisand d. impact of implant length and diameter on survival rates. clin oral impl res. 2006; 17(suppl 2) : 35-51. 3. weng d, jacobson z, tarnow d, hürzeler mb, faehn o, sanavi f et al. a prospective multicenter clinical trial of 3i machined-surfaced implants: results after 6 years of follow-up. int j oral maxillofac implants. 2003; 18: 417-23. 4. winkler s, morris hf, ochi s. implant survival to 36 months as related to length and diameter. ann periodontol. 2000; 5: 22-31. 5. herrmann i, lekholm u, holm s, kultje c. evaluation of patient and implant characteristics as potential prognostic factors for oral implant failures. int j oral maxillofac implants. 2005; 20: 220-30. 6. van steenberghe d, lekholm u, bolender c, folmer t, henry p, herrmann i et al. the applicability of osseointegrated oral implants in the rehabilitation of partial edentulism: a prospective multicenter study on 558 fixtures. int j oral maxillofac implants. 1990 ; 5: 272-81. 7. jemt t, lekholm u. implant treatment in edentulous maxillae: a 5-year follow-up report on patients 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10(2):83-87 original article braz j oral sci. april | june 2011 volume 10, number 2 cytokine profiles in the sera of egyptian patients with oral pemphigus vulgaris hossam eid1, basiouny el-gamal2 1phd, oral medicine, oral diagnosis and periodontology department, faculty of dentistry, suez canal university, egypt 2phd, clinical biochemistry department, college of medicine, king khalid university, abha, saudi arabia correspondence to: basiouny el-gamal department of clinical biochemistry college of medicine, king khalid university, abha, saudi arabia phone: +966 543175779 e-mail: basiouny_el_gamal@hotmail.com abstract cytokines have been suggested to play an important role in the pathogenesis of various inflammatory and autoimmune diseases, including the potentially fatal blistering disease, oral pemphigus vulgaris (pv). no data are currently available on the cytokine levels in the sera of egyptian patients with oral pv. aim:the aim of this study was to measure the serum levels of some proinflammatory and antiinflammatory cytokines in egyptian patients with pv. methods: using highly sensitive elisa kits, the levels of tnf-α, il-2, il-4 and il-6 were measured in the sera of 10 patients affected with oral pv and 10 healthy subjects. results: serum levels of tnf-α and il-6 were found to be significantly higher in patients with oral pv than in healthy controls (p<0.001). on the other hand, no significant differences were observed in the levels of il-2 and il-4 between oral pv and control sera (p<0.05). conclusions: these data showed that tnf-α and il-6 levels were significantly increased in the sera of egyptian patients with oral pv and this might suggest its role in the pathogenesis of this disease. keywords: pemphigus vulgaris, oral lesions, cytokines. introduction pemphigus vulgaris (pv) is a chronic, vesiculobullous, mucocutaneous autoimmune fatal disease, characterized by the presence of antibodies against adhesion molecules (desmoglein, dsg3) present on the surface of keratinocytes, leading to the loss of cellular adhesion or acantholysis and is typically associated with oral lesions1-5. histologically, pv can be detected by indirect immunofluorescence assay of anti-dsg3 antibodies in the sera6 and blister fluid7. in fact, the oral mucosa is the first site to be involved in up to 70% of cases, and it is the only site to be affected in over 50% of patients8. most patients exhibit oral lesions at some time of the disease8. clinically, the most common sites of oral pv lesions are the labial and buccal mucosa or the edentulous ridges. oral lesions are commonly characterized by the presence of vesiculobullous and ulcerative lesions. recently, there has been increasing interest in the role of cytokines in the pathogenesis of various inflammatory and autoimmune diseases, including the potentially fatal blistering disease, pv1-2,4,8-13. cytokines are regulatory compounds produced by cells of the immune system [t(h)1 and t(h)2] and act as intracellular mediators and control the immune and inflammatory reponses9. t(h)1 control the cell-mediated response and produce a number of proinflammatory cytokines, e.g. il-1, il-2, il-6 and tnf-α which are counterbalanced by a number of received for publication: january 01, 2011 accepted: april 20, 2011 84 braz j oral sci. 10(2):83-87 antiinflammatory cytokines, e.g. il-4 and il-10 that are produced by t(h)2 that participate in humoral response and antibody production. recent studies point out at proinflammatory cytokines such as tnf-α, il-1, or il-6 as strong players involved in this process 13. moreover, experimental studies revealed that synergistic cooperation of pemphigus antibodies with il-6 and tnf-α in the pathogenesis of pv. there is a fairly strong genetic background to pemphigus vulgaris with linkage to hla class ii alleles. certain ethnic groups, such as ashkenazi jews and those of mediterranean and south asian origin are especially liable to pv14-15. no data are currently available on the cytokine levels in the sera of egyptian patients with oral vp. in the present study, we measured the serum levels of some proinflammatory cytokines (il-2, il-6, tnf-α) and the antiinflammatory cytokine il-4 in egyptian patients with oral pv, in comparison to healthy controls. material and methods all participants were recruited from the outpatient clinics of the department of skin and venereal diseases, faculty of medicine, and the department of oral medicine, oral diagnosis, and periodontology, faculty of dentistry, suez canal university, egypt. ten patients with oral pv and 10 healthy controls were enrolled in the study after ethical approval by the suez canal university. a written informed consent was taken from all participants prior to enrollment. the group of patients with oral pv consisted of 6 males and 4 females with mean age of 50.6 years (range: 45-60 years). the selection and diagnosis of patients was based on the history, clinical characteristics of oral pv lesions, the histopathological specimens and the indirect immunofluorescence testing6 of pv. the duration of oral pv lesions ranged from 1-3 years. none of the patients had received any topical and/or systemic treatment for the present illness at least 1 month prior to study. the control group consisted of 6 males and 4 females with mean age of 48.4 years (range: 40-53 years). they were healthy volunteers completely free from any local or systemic diseases who were not taking any medication or contraceptives (in females). serum samples were collected from the individuals of the two groups and used to measure the levels of the cytokines tnf-α, il-2, il-4 and il-6 using commercially available elisa kits (quantikine, r & d, minneapolis, mn, usa), according to manufacturer’s procedure. all results were expressed as mean ± sd. differences between two means were analyzed by student’s t-test. p values equal or less than 0.05 were considered as significant. results the most common sites of oral pv lesions found in the present study were the lip, buccal mucosa, palatal, ventral surface of tongue, and gingival. examples of palatal mucosa and buccal mucosa with oral lesions in patients with pv are shown in figures 1 and 2, respectively. direct immunofluorescence testing revealed circulating pemphigus antibodies in all patients with oral pv. figure 3 shows an example of pv with intercellular deposition of antibodies in stratum spinosum. there was no statistically significant difference (p>0.05) between patients with oral pv and controls with respect to the mean age. table 1 shows serum cytokines levels (pg/ml) of tnfα, il-2, il-4, and il-6 in patients with oral pv and controls. in comparison with controls, patients with oral pv had 640% and 179% higher mean tnf-α and il-6 levels (p<0.001 and p<0.001), respectively. on the other hand, there were no significant differences in the levels of il-2 and il-4 of patients with oral pv compared with controls (p>0.05 and p>0.05), showing only 1% and 4% increase, respectively. fig. 1: pemphigus vulgaris oral lesion (palatal mucosa) fig. 2: pemphigus vulgaris oral lesion (buccal mucosa) cytokine profiles in the sera of egyptian patients with oral pemphigus vulgaris cytokines group range (pg/ml) mean ± s.d. (pg/ml) student’s t test tnf-α control group 2.86-5.33 3.83 ± 0.81 *p<0.001 pv group 19.69-36.90 28.35 ± 5.91 il-6 control group 12.70-21.11 17.52 ± 2.80 *p<0.001 pv group 37.90-63.74 48.91 ± 8.35 il-2 control group 7.32-12.51 9.90 ± 1.80 p>0.05 pv group 7.56-12.66 10.0 ± 1.83 il-4 control group 14.81-23.60 18.93 ± 2.81 p>0.05 pv group 15.20-23.70 19.66 ± 2.86 table 1:table 1:table 1:table 1:table 1: cytokines levels in the sera of patients with pemphigus vulgaris (pv) (n=10) and healthy controls (n=10). *statistically significant difference between patients with pv and healthy control. fig. 3: direct immunofluorescence examination of pemphigus vulgaris showing intercellular deposition of antibodies in stratum spinosum. discussion pv is a chronic, antibody mediated autoimmune disease that affects the skin and oral mucous membrane with distinct clinical, histopathological, and immunological features16. the exact cause of oral pv is unknown. several studies support the immunological basis for the disease. of importance to dentist is the frequency with which oral lesions were the initial presenting feature of oral pv as they often precede the skin lesions by several months or may be the major, if not the only, manifestation in some patients8. serum cytokine levels were recently investigated in many autoimmune disorders such as lichen planus (lp) and rheumatoid arthritis6, and a relationship between serum cytokine levels and the clinical appearance of these diseases has been reported. previous studies6,17 have reported that, the increase of some cytokines levels, such as il-1, il-6, il8 and tnf-α in the serum of patients with inflammatory and autoimmune diseases is well known, and these elevated cytokine levels seem to be important mediators restricting each disease6,17. in the present study, the serum levels of some proinflammatory cytokines (il-2, il-6, tnf-α) and the antiinflammatory cytokine il-4 were measured in egyptian patients with oral pv and compared with healthy controls. cytokine generation is regulated by other cytokines and also by itself6. in cytokine network with autocrine and paracrine control, cytokine actions are usually balanced; however, cytokine imbalance occurs under pathological conditions and large amounts of cytokines are generated, which may be beneficial or harmful to the body17. unbalanced cytokine actions are considered to be one of the immunopathogenesis mechanisms of autoimmune disorders6. elevation of cytokine activities and unbalanced cytokine network may induce oral mucosal lesions6. the identification of cytokine activities in patient’s tissues and sera seems to be advantageous for pathological analysis of oral diseases6. the present study revealed a 640% increase in serum levels tnf-α of patients with oral pv compared to controls. increased serum levels of tnf-α were reported by many authors. thus, alecu et al.18 found increased tnf-α levels in the sera and blister fluid obtained from patients with pv. similar results were obtained by d’auria et al.1,13 who showed increased levels and in situ expression of tnf-α. also, narbutt et al.2 showed 72% increase in tnf-α levels in the sera of patients with pv compared to control. this indicates that, tnf-α may play a role in the disease process of pv. like il-1, tnf-α is also a key stone in the cytokine network. tnf-α is a cytokine involved in the majority of inflammatory processes, and its increased activity is found in many skin diseases including psoriasis, sle, or systemic sclerosis19-20. tnf-α is released by cells under various stimuli including bacterial infections or ultraviolet radiation. it plays a role in many biological processes, enhances phagocytosis, cytotoxicity, and modulates activity of other cytokines such as il-1 and il-421. tnf-α is generated from macrophages, t and b cells and endothelial cells17. the important inducers of tnf-α production include viruses, il-1, immune complex, endotoxin and tnf-α itself6. the tnf-α plays a major role in cell mediated cytotoxicity being able to induce cytotoxic t-cell differentiation, enhances monocyte cytotoxicity, and stimulates lymphokine activated natural killer cells22. tnf-α has marked effects on epithelial cells. it is cytotoxic at high concentration and anti-proliferative at lower concentration22. prolonged release of tnf-α has been implicated in epithelial cell damage. so in the view of the enhanced cytotoxicity 85 braz j oral sci. 10(2):83-87 cytokine profiles in the sera of egyptian patients with oral pemphigus vulgaris 86 and epithelial cell lysis reported in pv, tnf-α seems to be implicated in the pathologic process of pv. the increased serum levels of tnf-α may be attributed to the activation of macrophages during recognizing the antigenic epitopes and presenting them to t-lymphocytes. moreover, mononuclear leukocytes might be stimulated to migrate into the submucosal area to produce cytokines including tnf-α23. alternatively, the predominant cellular sources of tnf-α are the mast cells, macrophages, monocytes and endothelial cells. these cells can secrete tnf-α into the circulation, thus the tnf-α serum levels reach an appreciable amount that could play a significant role in the immunopathogenesis of pv, and in the same time gets reflected in the patient’s serum22. consequently, it seems that, in pv the antigen-antibody complex formation within the epithelial layer may induce increased cytokine release including tnf-α, which enhances the epithelial cell damage. the il-6 is a multifunctional cytokine which is an important mediator in host response to injury and infection. it stimulates the production of acute phase protein by liver cells24. previous studies24-25, reported that, the production of il-6 may be a response to many inducers such as tnf-α, il4, il-3, and some viruses. moreover, the production of il-6 by epithelial cells is increased in many inflammatory autoimmune diseases such as lichen planus and psoriasis19,26. it seems likely therefore that, il-6 plays an important role in immunopathogenesis of a number of immunoinflammatory skin diseases, and since several inflammatory skin diseases also affect the oral mucous membrane such as lp, sle, and pv, it is reasonable to detect high serum level of il-6 in patients with active pv oral lesions24. the results of the present study revealed a marked increase of il-6 serum levels (179%) in patients with oral pv compared with those of the control group. as much as 72% increase in serum il-6 levels was reported by others in active stage of pv2. the elevated il-6 serum levels in patients with active pv oral lesions seem to be compatible with the high levels of il-6 generated and released in acute inflammatory conditions. the high il-6 serum levels in patients with pv may be attributed to the elevated serum levels of tnf-α which is one of the strong inducers for generation and release of il-624. there are wide variety of cells that are responsible for generation and release of il-6 including lymphocytes, endothelial cells and epithelial cells. therefore, il-6 seems to be generated and released into the circulation in acute and chronic immunoinflammatory as well as autoimmune disorders 17,24. since pv is a chronic inflammatory mucocutaneous disease, the higher il-6 serum levels in patients with oral pv seem to be a reasonable finding. the findings of the present study revealed a slight, nonsignificant elevation of il-4 serum levels in patients with oral pv compared with those of control. however, satyam et al.9 reported significantly higher levels of serum il-4 in patients with pv compared to healthy controls and suggested that this increase shows the induction of t(h)2 cells in the pathogenesis of pv. also, keskin et al.11 reported elevated levels of il-4 in serum of patients with pv and showed reduced levels of this cytokine to the control values following treatment with high-dose, long term systemic corticosteroids with or without immunoglobulins. the il-4 has a wide range of biological functions that include activation of immunoglobulin synthesis, t-cell proliferation, and t-cell adhesion to endothelial cells. generation of il-4 in some patients with autoimmune disorders seems to be likely in these diseases. in the present study, it could not be confirmed any significant changes in il-2 serum levels in patients with oral pv when compared to the healthy controls. however, satyam et al.9 reported decreased levels of il-2 in the sera of patients with pv. in conclusion, the present findings showed that tnf-α and il-6 levels were significantly increased in the sera of egyptian patients with oral pv, probably suggesting its role in the pathogenesis of this disease. acknowledgements we acknowledge the help and guidance of prof mohmoud kandeel of oral diagnosis, oral medicine & periodontology department, faculty of oral & dental medicine, cairo university, egypt. references 1. d’auria l, bonifati a, mussi a, d’agosto g, de simone c, giacalone et al. cytokines in the sera of patients with pemphigus vulgaris: interleukin-6 and tumour necrosis factor-alpha levels are significantly increased as compared to healthy subjects and correlate with disease activity. eur cytok netw. 1997; 8: 383-7. 2. narbutt j, lukamowicz j, bogaczewicz j, sysa-jedrzejowska a, torzecka jd, leslak a. serum concentrations of interleukin-6 is increased both in active and remission stages of pemphigus vulgaris. mediators inflamm. 2008; 2008: 875-94. 3. stern jnh, keski n db, barteneva n, zuniga j, yunis ej, ahmed ar. possible role of natural killer cells in pemphigus vulgaris preliminary observations. clin exp immunol. 2008; 15: 472-81. 4. raychaudhuri sp, smriti k. biologics: target-specific treatment of systemic and cutaneous autoimmune diseases. ind j dermatol. 2009; 54: 100-9. 5. femiano f. pemphigus vulgaris: recent advances in our understanding of its pathogenesis. minerva stomatol. 2007; 56: 215-23. 6. yamamoto t, yoneda k, ueta e. serum cytokine levels in patients with oral mucous membrane disorders. j oral pathol med. 1991; 20: 275-9. 7. shanmugasekar c, ram gaesh vr, jayaramun a, srinivas cr. blister fluid immunofluorescence in case of pemphigus vulgaris. ind j dermatol. 2010; 55: 188-9. 8. mignogna md, lorenzolo m, robert c. oral pemphigus: long term behaviour and clinical response to treatment with deflazacort in sixteen cases. j oral surg pathol med. 2000; 29: 145-52. 9. satyam a, khandpur s, sharma vk, sharma a. involvement of t(h)1/ t(h)2 cytokines in the pathogenesis of autoimmune skin diseasepemphigus vulgaris. immunol invest. 2009; 38: 498-509. 10. baroni a, perfetto b, ruocco e, greco r, criscuolo d, ruocco v. cytokine pattern in blister fluid and sera of patients with pemphigus. dermatology. 2002; 205: 116-21. 11. keskin db, stern jn, fridkis-hareli m, razzaque ahme a. cytokine profiles in pemphi-gus vulgaris patients treated with intravenous igs as compared to controlled immunosuppressive therapy. cytokine. 2008; 4: 15-21. braz j oral sci. 10(2):83-87 cytokine profiles in the sera of egyptian patients with oral pemphigus vulgaris 87 12. feliciani c, toto p, amerio p, pour sm, coscione g, shivji g et al. in vitro and in vivo expression of interleukin-1-alpha and tumor necrosis factor-alpha mrna in pemphigus vulgaris: interleukin-1-alpha and tumor necrosis factor-alpha are involved in acantholysis. j invest dermatol. 2002; 114: 71-7. 13. lopez-robles e, avalos-diaz e, vega-memije e, et al. tnfá and il-6 are mediators in the blistering process of pemphigus. int j dermatol. 2001; 40: 185-8. 14. ahmed ar, yunis ej, kharti k, wagner r, notani g, awdeh z et al. major histocompatibility complexhaplotype in ashkenazi jewish patients with pemphigus vulgaris. proc natl acad sci. 1990; 87: 7658-62. 15. brenner s, tur e, shapio j, ruocco v, a’avino m, ruocco e et al. pemphigus vulgaris: environmental factors, occupational, behavioral, medical and frequently quetionnaire . int j dermatol. 2001; 40: 562-9. 16. john c, robinson ma, lozada f. oral pemphigus vulgaris. oral surg oral med oral pathol. 1997; 84: 349-55. 17. tetsuya y, kazunori y, osaki t. serum cytokines, interleukin-2 receptor, and soluble intercellular adhesion molecule-1 in oral disorders. oral surg oral med oral pathol. 1994; 78: 727-35. 18. alecu m, alecu s, coman g, galatescu e, ursaciuc c. tnf-á and il6 in serum and blister liquid of pemphigus vulgaris patients. roumanian. arch microbiol immunol. 1999; 58: 121-30. 19. gomi t, shiohara t, munakata t, imanishi k, nagashima m. interleukin 1á, tumor necrosis factor á, and interferon ã in psoriasis. arch dermatol. 1991; 127: 827-30. 20. kantor tv, friberg d, medsger ta jr, buckingham rb, whiteside tl. cytokine production and serum levels in systemic sclerosis. clin immunol immunopathol. 1992; 65: 278-85. 21. kock a, schwarz t, kimbauer r, urbanski a, perry p, ansel jc et al. human keratinocytes are a source for tumor necrosis factor á: evidence for synthesis and release upon stimulation with endotoxin or ultraviolet light. j exp med. 1990; 172):1609-14. 22. sugermann nw, savage gj, seymour l. is there a role for tumor necrosis factor-alpha (tnf-á) in oral lichen planus? j oral pathol med. 1996; 25: 219-24. 23. takeuchi y, tohani i, kaneda t. immuno-histochemical analysis of cells in mucosal lesions of lichen planus. j oral pathol med. 1988; 17: 367-71. 24. farthing pm, thornhill mh. il-1 á and il-6 production by oral and skin keratinocytes: similarities and differences in response to cytokine treatment in vitro. j oral pathol med. 1996; 25: 157-62. 25. wilson m, reddi, henderson b. cytokine-inducing components of periodonto-pathogenic bacteria. j priodont res. 1996; 31: 393-407. 26. yamamoto t, osaki t, ueta k. cytokine production by keratinocytes and mononuclear infiltrates in oral lichen planus. j oral pathol med. 1994; 23: 309-15. braz j oral sci. 10(2):83-87 cytokine profiles in the sera of egyptian patients with oral pemphigus vulgaris oral sciences n3 braz j oral sci. 10(3):184-188 original article braz j oral sci. july | september 2011 volume 10, number 3 received for publication: april 27, 2010 accepted: july 13, 2011 detection of single and mixed colonization of candida species in patients with denture stomatitis gustavo davi rabelo1, elisângela noborikawa2, carla silva siqueira2, fernando ricardo xavier da silveira3, mônica andrade lotufo4 1 dds, msc, department of stomatology, school of dentistry, university of são paulo, são paulo, sp, brazil 2 dds, department of stomatology, school of dentistry, university of são paulo, são paulo, sp, brazil 3 dds, phd, associate professor, department of stomatology, school of dentistry, university of são paulo, são paulo, sp, brazil 4 dds, phd, professor, department of stomatology, school of dentistry, guarulhos university, são paulo, sp, brazil correspondence to: gustavo davi rabelo faculdade de odontologia da universidade de são paulo – departamento de estomatologia av. prof. lineu prestes, 2227 são paulo sp cep: 05508-000 phone: 11-30917902. e-mail: drgustavorabelo@yahoo.com.br e frxsilve@usp.br abstract aim: to evaluate the profile of the colonization by candida spp. using presumptive identification to classify the patients with denture stomatitis as having single or mixed colonization, correlating with oral and systemic status. methods: the chromagar candida™ medium (cc) for yeast culture and exfoliative cytology was used to identify colonization by candida spp. and distinguish the different species of the candida genus from patients with denture stomatitis (ds) and denture wearers without ds (control group). in addition, colonization was correlated with specific habits, such as tobacco and alcohol use, as well as with the use of systemic drugs. results: direct swabbing of whole unstimulated saliva (wus) and palatal mucosa revealed colonization in 97.3% of the patients with ds. in the control group, 55.0% patients presented colonization. the presumptive identification found c. albicans as the most prevalent between both groups, respectively in 89.4% of the ds group and 40.0% from the control group. regarding the nonalbicans species in the ds group, the most frequent were c. krusei (31.5%), c. glabrata (21.0%) c. tropicalis (15.7%) and candida spp (2.6%). smokers presented 90% of mixed isolates, and no c.albicans single colonization in the ds group, with statistically significant difference between smokers and non-smokers (p=0.0051). in the control group, the non-albicans species were c.glabrata (23.0%) and c.tropicalis (23.0%). the results of cytology from the ds group showed positive results in 22.2% of the cases. conclusions: the use of cc was effective as a complementary method for the diagnosis of colonization by candida spp. and ds, with the additional advantage of enabling a rapid presumptive identification of the specie. smoking seemed to play a role in the colonization of oral mucosa by mixed albicans and non-albicans species. mixed colonization seems to be more prevalent between patients with ds. keywords: culture media, candida albicans, candidiasis, denture stomatitis. introduction candida infections are common and often recurrent, and represent a significant clinical problem1. host factors play a more important role than organism virulence in the pathogenesis of oral candidosis, and intraoral environment conditions, such as the presence of dentures, also play a crucial role in the disease2. 185 braz j oral sci. 10(3):184-188 candidosis is most commonly caused by candida albicans3. other species including c. tropicalis, c. glabrata, c. parapsilosis and c. krusei have also been isolated from denture wearers4. in these patients, an important infection described as candida-associated denture stomatitis (ds) occurs in about 50-60% of them5. the role of non-albicans species has become increasingly important, especially in high-risk subjects, such as hivpositive patients6. another recent study with patients with hematological malignancies and head and neck solid tumors revealed that the majority of cases of oral candidiasis were caused by c. albicans, but almost one third of patients harbored non-albicans strains, such as c. glabrata, which were often more resistant to anti-fungal agents7. these facts underline the importance of an appropriate sampling method, for a more accurate diagnosis. cytology is the most frequently used technique in dental clinics because it is easy to perform and les time consuming when compared with other diagnostic techniques. for the culture techniques, the usual collecting methods to obtain yeasts are whole unstimulated saliva (wus) collection for swabbing, swabbing the oral mucosa, scraping it with a spatula or rinsing the mouth8. the identification of yeasts is crucial to ensure an effective antifungal treatment. several chromogenic media have been developed to aid the rapid identification of candida species. these media contains chromogenic substrates that react with yeast enzymes, resulting in colored colonies 9. chromagar candida™ medium (cc) is a differential culture medium that allows the presumptive identification of yeasts species, with sensitivity and specificity higher than 99% to c. albicans10. the aim of this study was to evaluate cc and cytology methods to identify colonization and candidiasis as well as to distinguish the presumptive species on candida genus from denture wearers with and without ds, correlating single and mixed colonization with some specific oral conditions namely denture wearing and predisposing factors to the installation of infection, such as tobacco and alcohol habits and the use of systemic drugs. material and methods fifty-eight patients undergoing dental treatment at the school of dentistry of university of são paulo were selected for this study. informed consent was obtained from all subjected and the study protocol was approved by the university’s ethics committee (fr 268407 – pr 95/2009). exclusion criteria were age under 18 and over 95 years, use of antibiotics and/or steroids up to 1 month before the study, use of antifungal drugs (systemic or topical use) in the 3 months before the study and complaints of dry mouth sensation. the patients were divided in two groups: ds group (patients with denture stomatitis; n=38) and control group (denture wearers without ds; n=20). clinical examination clinical examination was performed according to the criteria proposed by newton (1962) for ds classification in types i, ii, iii11. type i showed localized inflammation or pinpoint hyperemia; type ii showed a generalized erythema and type iii comprised papillary hyperplasia of the palate. all patients who met these criteria were included in the test group. denture evaluation was done by direct examination and the patients were questioned about their age, gender, smoking and alcohol use, systemic health status, use of medicines (class, dose and frequency), oral health and denture wearing. the criteria to establish smoking and alcohol habits were the following: smoker was the individual who had the habit of daily use of tobacco at the time of enrolment in the study and alcohol habit was the use of distilled and/or fermented beverage two or more times a week at the time of enrolment in the study. samples and techniques wus was collected during 3 min in a sterile tube, under standard conditions: between 8 am and 11 am, no feeding, drinking, smoking or hygienic habits allowed for 60 min before the test section. after this step, a sterile swab was inserted in the tube and was direct sampled in half of a cc plate (plastlabor, rio de janeiro, rj, brazil). on the other half of the plate, an oral swab from palatal mucosa were taken by passing this sterile cotton several times across the mucosal surface and then seeding onto the cc plate. exfoliative cytology was performed following the swabbing. the technique consisted in passing a cytobrush several times across the palate surface and disposed the collected material on two glass slides and fixed in absolute alcohol. these slides were sent to the oral pathology department and stained by periodic acid schiff (pas) and papanicolaou techniques. the fungal presence was searched by analyzing the presence of hyphal outgrowths (abundant, moderate, scarce and absent). after 48 h of incubation at 37ºc, the cc plates were photographed and the digitized images were entered into the imagej software (imagej 1.44f, wayne rasband, bethesda, md, usa) for analysis of colonies. the presumptive identification of candida species was based on the criteria proposed by odds and bernaerts (1994), who described the species by the color of the colony. c. albicans colonies are described by their green color, c. tropicalis colonies based on their dark-blue to blue-gray color, surrounded by a dark/ pink halo, c. glabrata by their white/dark pink/purple range of colors and c. krusei colonies based on their pale pink color and downy/rough appearance with pale edges. the colonies presenting other colors were classified as candida ssp.12. the identification of the colors of the colonies in the digitalized images was made by two calibrated dentists. patients who had moderate and abundant proportions of hyphal outgrowths in the cytological exams, along with positive clinical signs and positive culture tests, were considered as having candidiasis. the statistical tests were made in the ds group only, with comparison between single and mixed colonization when considering the tobacco and alcohol habits and use of systemic drugs, by the fisher’s exact test. detection of single and mixed colonization of candida species in patients with denture stomatitis braz j oral sci. 10(3):184-188 186 results the patients with ds had mean age of 58.1 years (sd 11.6) being 28 females and 10 males. in the control group, the mean age was 62.1 years (sd 6.9), with 18 females and 2 males. direct swabbing from the wus and palatal mucosa revealed colonization in 37 (97.3%) of patients with ds. in the control group, 11 (55.0%) patients presented colonization from wus, and swabbing of palatal mucosa was positive to reveal colonization in 6 (30.0%) patients. in the ds group, 22 patients were using medicines and the major group of drugs taken was osmotic diuretics (10 patients). in the control group, 13 patients were using medicines and angiotensin-converting enzyme (ace) inhibitors were the most frequently taken drugs, used by 8 patients. direct examination of the denture prosthesis revealed the majority of them with some inadequacy in shape, contour and surface as well as inadequate hygiene. the mean time of denture wearing was 11.6 years (sd 12.3). the isolates showed c. albicans as the most prevalent between both groups, respectively in 89.4% from the patients with ds and 40.0% from the control group. about the nonalbicans species in the ds group, the most encountered were c. krusei (31.5%), followed by c. glabrata (21.0%), c. tropicalis (15.7%) and candida spp (2.6%). in the control group, the non-albicans species encountered were c. glabrata (23.0%) and c. tropicalis (23.0%). to obtain the prevalence of colonization, when a patient harbored two or more species, these were counted as separate events, i.e., if a patient had colonization by c. albicans and c. krusei, they were counted as two species. the wus swabbing from ds patients revealed mixed colonization in 20 of them, c. albicans only in 12, nonalbicans in 3 and no colonization in 3 patients. the swabbing of palatal mucosa revealed mixed colonization from 15 patients, 17 by c. albicans only, 3 of non-albicans and 3 with no colonization. cytology showed positive results only in 8 (22.2%) patients from the ds group, with 4 in the moderate range colonization smoking alcohol use use of systemic drugs yes n o yes n o yes no c. albicans only 0 14 3 11 8 6 c. albicans and non-albicans species 9 12 7 14 12 9 p value 0.0051* 0.7041 1.0000 table 1 distribution of primary colonization of candida species in patients with denture stomatitis. *significant difference (p < 0.05). colonization smoking alcohol use use of systemic drugs yes n o yes n o yes no c. albicans only 0 6 0 6 4 2 c. albicans and non-albicans species 0 2 0 2 2 0 table 2 distribution of primary colonization of candida species in patients without denture stomatitis (control group). and 4 in the scarce range. in the control group, only 1 patient (7.6%) presented positivity to the test, in the scarce range. all patients with positive exfoliative cytology results were also positive for cc. regarding the presumptive identification, the data were distributed with all patients allocated as shown in tables 1 and 2. the patient was considered as harboring mixed yeast population when presented mixed colonization by swabbing of wus and/or palatal mucosa. when the patients were allocated in different groups, smokers revealed an increased proportion in mixed colonization, with a significant difference between the patients with and without the tobacco habit (p=0.0051). four patients with positivity to the tests and clinical indication of therapeutics received treatment for candidiasis with nystatin topical and all individuals were sent to receive new dentures. discussion ds is a frequent finding among denture wearers. although its etiology is unknown, the influence of the colonization by candida spp. has already been described. at the same time, not all patients with ds present colonization by candida spp., although some of them were not classified even as carriers. in the present study, only 1 patient with ds showed absence of colonization. abaci et al. (2010), demonstrated that the number of yeast cells in the saliva of patients with ds was <400 cfu/ ml in 11 individuals and >400 cfu/ml in 19 individuals. in addition, all patients with ds, who were complete denture and removable partial denture users, presented colonization by candida spp. from saliva samples. however, one had no colonization from swabs taken from the palatal mucosa13. no patient, in either the ds and or control group, complained about symptoms like continuous burning sensation or taste disorder. although these symptoms are relatively common in patients with ds, it has been reported that there is no association between burning mouth syndrome and prevalence of candida spp.14. in the same way, an increase detection of single and mixed colonization of candida species in patients with denture stomatitis 34 braz j oral sci. 10(3):184-188 187 in oral yeasts is not necessarily associated with changes in mouth sensation alone15. the direct seeding without saliva dilution or any other procedure for sampling is preferable for clinical use, as performed in our study, bringing advantages such spending less time to carry out the technique and achievement of satisfactory presumptive results that can guide the choice of therapeutic support. the time elapsed for presumptive identification with cc, as used in the present study, was about 48 h. the identification of yeasts in a mycology laboratory with conventional media is time-consuming and cumbersome as it requires between 4 and 6 days16. cytology showed positive results in 22.2% of the cases in the ds group, demonstrating the inefficiency of this method for the diagnosis of oral candidosis when the material is obtained from scraping and/or swabbing. although the method is valuable to distinguish yeasts from hyphal forms, it is less sensitive than culture methods17, as seen in the present study. colonization by candida spp. in both groups represented the importance of the denture use on oral yeast carriage. old age, clinical signs of oral dryness, denture wearing and reduction in whole unstimulated salivary flow increase the prevalence of oral yeasts15. also, if the balance of the normal flora is disrupted or the patient’s immune defenses are compromised, candida spp. can invade mucosal surfaces and cause disease manifestations18. in addition, the high incidence of colonization in the patients with ds (97.3%) corroborates with the long-term period of the denture use (11 years on the average). this may have positively influenced the colonization and predisposed to the installation of ds. regarding the mixed yeast populations from patients with ds, non-albicans species have been found in only 14.7% individuals in a previous investigating candida spp. incidence in denture wearers13. in the present study, the frequency of colonization by non-albicans and c. albicans plus non-albicans was higher. these findings suggest that our patients presented more mixed fungal populations because their clinical and/or systemic conditions might have predisposed them to the candida infection. in addition, when the patients with ds were evaluated by groups, which means separation of the patients according to their specific conditions, smokers presented 90% of mixed colonization and no c. albicans colonization alone. a recent study19 found a frequency of 54.8% of c. albicans and 45.2% of non-albicans species in healthy smokers, demonstrating a possible influence of smoking habit on colonization, although nonsmokers presented 35.1% of non-albicans colonization. in addition, they concluded that there was a marginally significant positive correlation between the number of cigarettes smoked per day and the density of candidal growth from oral rinse cultures. figueiral et al. 20 (2007) indentified c. albicans, c. glabrata and c. tropicalis in a group of patients with ds, and c. tropicalis appeared always together with one of the other species. those authors concluded that yeasts, particularly c. albicans, are associated with ds. in the present study, only 2 patients in the control group were smokers and neither of them presented colonization of c. albicans only. our results revealed a higher colonization in patients using diuretics, which could explain the reduced salivary flow, favoring the presence of yeasts. however, it is important to emphasize that there were no dryness complains. torres et al.21 reported a frequency of 28% of mixed colonization in patients with xerostomia, being 88.4% users of medications and 58% of these patients diagnosed with hyposalivation. the data obtained in the present study and reported in the literature revealed the influence of some variable conditions on yeast carriage and the presence of infection. when we separated the patients into different groups, we intended to evaluate the influence of each variable in colonization, aiming at determining whether some clinical aspects and habits could result in a difference between groups in an important rate. smoking showed a high difference when the patients were allocated as smokers and non-smokers, with rates of 90% of mixed colonization. it is worth mentioning that our study used the presumptive identification, but our objective was focused on sampling techniques. cc can be extremely helpful in a clinical stomatology service routine, facilitating the detection of mixed cultures of yeasts and allowing direct identification of c. albicans, as well as rapid presumptive identification of other species like c. glabrata, c. tropicalis, candida krusei and even sacharomyces cerevisiae22. however, tintelnot et al.23 (2000) described cc as an insufficiently selective medium for isolation of c. dubliniensis as it can produce dark green in primary cultures. our findings also reinforces the need to employ other common tests, such as germ tube observation and carbohydrate assimilation, to institute an appropriate therapy for cases of undiagnosed candida spp., recurrent infections or lack of response to antifungal drugs. it has been reported that there is increasing evidence that more than one candida spp. may simultaneously colonize mucosal habitat. the need to investigate the factors that play a role in the initial attachment and subsequent colonization of denture-base materials and the oral mucosa of patients subjected to candida infection should be known, controlled and may prevent the disease24. differentiation between species is also important for the identification of infection. cc was used as an easy and rapid technique, reaching the target to an appropriated diagnosis and institution of adequate therapy. the findings of the present study also revealed the prevalence of infections by c. albicans followed by c. krusei and c. glabrata. in an extensive review, samaranayake and samaranayake25 (2001) reported that c. glabrata emerges as the second most prevalent species, frequently isolated from acrylic denture surface and the palatal mucosa. our results led us to encourage the use of cc in stomatology daily practice. it enables better knowledge of the most prevailing species of the genus candida in the oral mucosa, resulting in a better management and control of infections, and, even for screening purposes, with faster and less expensive results. finally, it is important to diagnose patients with ds, recommending the adequate treatment for each case. even asymptomatic patients should be oriented and treated if detection of single and mixed colonization of candida species in patients with denture stomatitis braz j oral sci. 10(3):184-188 necessary, due to the importance of the oral mucosa as a primary source of reinfection, mainly in the event of an eventual hospitalization, which would affect substantially their quality of life. in conclusion, the use of cc was an effective subsidiary technique for the diagnosis of oral candidosis in patients with ds, with the additional advantage of enabling presumptive species identification within the 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s, abdallah s, gray g, stubbings h, gow i, baker p et al. epidemiology of oral yeast colonization and infection in patients with hematological malignancies, head neck and solid tumors. j oral pathol med. 2011; 40: 83-9. 8. rautemaa r, rusanen p, richardson m, meurman jh. optimal sampling site for mucosal candidosis in oral cancer patients is the labial sulcus. j med microbiol. 2006; 55: 1447-51. 9. sendid b, françois n, standaert a, dehecq e, zerimech f, camus d et al. prospective evaluation of the new chromogenic medium candiselect 4 for differentiation and presumptive identification of the major pathogenic candida species. j med microbiol. 2007; 56: 495-9. 10. bernal s, martín mazuelos e, garcía m, aller ai, martínez ma, gutiérrez mj. evaluation of chromagar candida medium for the isolation and presumptive identification of species of candida of clinical importance. diagn microbiol infect dis. 1996; 24: 201-4. 11. newton av. denture sore mouth: a possible aetiology. br dent j. 1962: 112-357. 12. odds fc, bernaerts r. chromagar candida, a new differential isolation medium for presumptive identification of clinically important candida species. j clin microbiol. 1994; 32: 1923-9. 13. abaci o, haliki-uztan a, ozturk b, toksavul s, ulusoy m, boyacioglu h. determining candida spp. incidence in denture wearers. mycopathologia. 2010; 169: 365-72. 14. cavalcanti dr, birman eg, migliari da, silveira frx. burning mouth syndrome: clinical profile of brazilian patients and oral carriage of candida species. braz dent j. 2007; 18: 341-5. 15. shimizu c, kuriyama t, williams dw, karasawa t, inoue k, nakagawa k et al. association of oral yeast carriage with specific host factors and altered mouth sensation. oral surg oral med oral pathol oral radiol endod. 2008; 105: 445-51. 16. willinger b, manafi m. evaluation of chromagar candida for rapid screening of clinical specimens for candida species. mycoses. 1999; 42: 61-5. 17. williams dw, lewis ma. isolation and identification of candida from the oral cavity. oral dis. 2000; 6: 3-11. 18. mardegan rc, foglio ma, gonçalves rb, höfling jf. candida albicans proteinases. braz j oral sci. 2006; 5: 944-52. 19. darwazeh am, al-dwairi zn, al-zwairi aa. the relationship between tobacco smoking and oral colonization with candida species. j contemp dent pract. 2010; 11: 17-24. 20. figueiral mh, azul a, pinto e, fonseca pa, branco fm, scully c. denture-related stomatitis: identification of aetiological and predisposing factors a large cohort. j oral rehabil. 2007; 34: 448-55. 21. torres sr, peixoto cb, caldas dm, silva eb, akiti t, nucci m, et al. relationship between salivary flow rates and candida counts in subjects with xerostomia. oral surg oral med oral pathol oral radiol endod. 2002; 93: 149-54. 22. bouchara jp, declerck p, cimon b, planchenault c, de gentile l, chabasse d. routine use of chromagar candida medium for presumptive identification of candida yeast species and detection of mixed fungal populations. clin microbiol infect. 1996; 2: 202-8. 23. tintelnot k, haase g, seibold m, bergmann f, staemmler m, franz t, naumann d. evaluation of phenotypic markers for selection and identification of candida dubliniensis. j clin microbiol. 2000; 38: 1599-608. 24. pereira-cenci t, del bel cury aa, crielaard w, ten cate jm. development of candida-associated denture stomatitis: new insights. j appl oral sci. 2008; 16: 86-94. 25. samaranayake yh, samaranayake lp. experimental oral candidiasis in animal models. clin microbiol rev. 2001; 14: 398-429. 188 detection of single and mixed colonization of candida species in patients with denture stomatitis oral sciences n3 braz j oral sci. 11(2):135-140 original article braz j oral sci. april | june 2012 volume 11, number 2 cytotoxic response of two cell lines exposed in vitro to four endodontic sealers camilla christian gomes moura1, natássia cristina martins oliveira2, cláudia renata bibiano borges3, maria aparecida de souza4, joão carlos gabrielli biffi5 1dds, msc, phd, postdoctoral student, school of dentistry, federal university of uberlândia, uberlândia, mg, brazil 2dds, msc, phd student, piracicaba dental school, unicamp – university of campinas, piracicaba, sp, brazil 3msc, phd student in pathology, department of general pathology, federal university of triângulo mineiro, uberaba, mg, brazil 4msc, phd, researcher, department of applied immunology and parasitology, federal university of uberlândia, uberlândia, mg, brazil 5dds, msc, phd, professor, department of endodontics, school of dentistry, federal university of uberlândia, uberlândia, mg, brazil correspondence to: camilla christian gomes moura departamento de endodontia faculdade de odontologia da universidade federal de uberlândia av. pará 1720 bloco 2b jardim umuarama cep: 38400-902 uberlândia, mg brasil phone: +55 34 32182731 e-mail: camillahistologia@yahoo.com.br abstract aim: to investigate the cytotoxicity of four endodontic sealers with different bases – epiphany (eph), ah plus (ahp), sealer 26 (s26) and endofill (enf) – on human foreskin fibroblasts (hff) and mouse macrophages (j774/g8). methods: cells were placed in direct contact with freshly prepared endodontic sealers in polypropylene tubes. the cells were incubated for 24, 48 and 72 h. cytotoxicity was assessed using the mtt assay (cell viability) and griess reagent (no release). results: on the hff cultures, eph showed the lowest viability levels of all four sealers at 24 h (p<0.05), but over time (72h), eph lessened its toxic levels in a similar pattern as the other three materials (p>0.05). the viability of all four sealers on the macrophage cultures showed no statistically significant difference over time, except between eph and ahp at 72 h (p<0.05). although uniformity was not detected in macrophage and fibroblast release of no in response to sealers over time, a trend of increased no levels for eph (p<0.05) was observed. conclusions: the response pattern varied depending on time and type of cell line used for analysis, although the results indicate a higher cytotoxicity for eph in short-term tests. keywords: cell culture, cytotoxicity, fibroblast, macrophage, root canal sealers. introduction a root canal sealer should be chosen based on its biological 1 and physicochemical characteristics, ability to adhere to and seal the root canal system, dimensional stability, nonabsorbability, radiopacity, and adequate working time2-3. although root-filling materials are designed for use only within the canal space, leakage through the apical constriction may occur, allowing the periradicular tissues to come in contact with the toxic components of the sealer4. furthermore, the induction of cell death caused by these materials, which is associated with the release of proinflammatory mediators, leads to a persistence of periapical inflammatory reaction and increases the time required for wound healing5. hence, it is important to comprehensively evaluate the biocompatibility of different sealers. this type of analysis can be conducted both in vitro and in vivo. however, in vitro studies are faster and less expensive than in vivo tests; furthermore, factors and variables may be controlled in vitro6. in spite of the advantages of the in vitro tests, they are not able to mimic the orchestrated role of cells present in received for publication: april 02, 2012 accepted: june 19, 2012 braz j oral sci. 11(2):135-140 sealer ah plus epiphany sealer 26 endofill composition paste a: bisphenol-a epoxy resin, bisphenol-f epoxy resin, calcium tungstate, zirconium oxide, silica, iron oxide pigmentspaste b: dibenzyldiamine, aminoadamantane, tricyclodecane-diamine, calcium tungstate, zirconium oxide, silica, silicone oil. mixture of resins (bis-gma, udma, pegdma, ebadma), barium sulphate, bismuth, calcium hydroxide, silica, silane-treated barium borosilicate glass, colouring pigment, dual-cured initiators, stabilizer, peroxide. powder: calcium hydroxyde, bismuth oxide, methenamine, titanium dioxide. resin: bis-gma powder: zinc oxide, staybelite resin, bismuth subcarbonate, barium sulphate, sodium borate anhydrate. liquid: eugenol manufacturer dentsply de trey gmbh, konstanz, germany pentron clinical technologies, llcc, wallingford, ct, usa dentsply, petropolis, rj, brazil dentsply, petropolis, rj, brazil table1. main composition of evaluated endodontic sealers periradicular region and the long-term cytotoxicity presented by the sealers. cytotoxicity assays are the initial screening tests used to evaluate the biocompatibility of materials7. genotoxicity/mutagenicity/carcinogenicity and microbial effects are the other parameters that characterize biocompatibility8. the cytotoxic responses to different root canal sealers vary considerably, depending on the sealer’s chemical composition4, namely zinc oxide eugenol, calcium hydroxide, mineral trioxide aggregate9, glass-ionomer or polymers (i.e., epoxy resins, polydimethylsiloxane and methacrylates). although the cytotoxicity of endodontic sealers has been extensively investigated, most previous studies have used fibroblast cell lines. 4,6,8,10-11 the behavior of other inflammatory cells present in the periapical region has not been widely assessed, although they also contribute to the intensity of the biological response to these materials. in order to take a new approach to previous in vitro studies on the cytotoxicity of endodontic materials, it would be interesting to evaluate the response of fibroblasts and macrophages, which are cell lines involved in periapical inflammation and repair. macrophages are the prevalent cells in inflammatory infiltrates that respond to sealers12-13. they also play a key role in defense and repair by producing a myriad of substances with inflammatory activity12-14. nitric oxide (no) is a pro-inflammatory compound that plays an important role in the investigation of a sealer’s cytotoxicity13,15 because it is produced by various cells others than macrophages, such as fibroblasts16. given that these two cell lines are widely distributed on periradicular tissues and that their response can be influenced by the stage-setting of the sealing material, the aim of this study was to investigate the cytotoxicity of four endodontic sealers – a multi-methacrylate resin-based (epiphany), an epoxy resin-based (ah plus), an epoxy resin and calcium hydroxide-based (sealer 26) and a zinc oxide eugenol-based (endofill) – with respect to the cell viability and no release in immortalized cell cultures of macrophages and fibroblasts. material and methods root canal sealers and sample preparation four classes of endodontic sealers were evaluated in this study (table 1). sealers were prepared according to the manufacturers’ instructions, under aseptic conditions, prior to insertion into a sterile 1-mm-diameter urethral polypropylene tube (medsonda, arapoti, pr, brazil). the tube was cut into 10-mm-long segments. the experiments were performed at two different days on four tubes per day for a total of eight samples per group in each experimental time. sealers were prepared immediately before they were introduced into cell cultures in order to simulate fresh conditions. cell cultures two cell lines were used to evaluate the endodontic sealers: human foreskin fibroblasts (hff, cell bank of rio de janeiro, rio de janeiro, rj, brazil) and mouse macrophages (j774/g8, system biosciences, mountain view, ca, usa). the two cell lines were cultivated separately. the cells were routinely maintained in dulbecco’s modified eagle’s medium (dmem) (vitrocell, campinas, sp, brazil), supplemented with a 5% fetal calf serum (laborclin, pinhais, pr, brazil), 100 u/ml penicillin, 100 µg/ml streptomycin and 2 mm lglutamine (cambrex bio science, verviers, belgium) at 37ºc in a humidified atmosphere of 5% co 2 and 95% air. the cells were plated at 2x104 cells/well in a 48-well plate that was constantly in contact with the culture medium in which the polypropylene tubes containing the endodontic sealers were placed. after incubation for 24, 48, and 72 h, cell viability assays and no quantification within the supernatant were performed on each cell line at each experimental time. culture wells containing empty tubes were filled with the culture medium. the median absorbance of these wells was subtracted from the samples in cell viability assays. cell viability cell viability was evaluated using a mtt [3-(4,5136136136136136cytotoxic response of two cell lines exposed in vitro to four endodontic sealers 137137137137137 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. succinate dehydrogenase activity was determined by adding 40 µl of a 5 mg/ml mtt salt (m-2128, sigmaaldrich, st louis, mo, usa) to each well and incubating the cells at 37°c for 4 h. after incubation, the resulting formazan crystals were dissolved by adding 400 µl of dmso (labsynth, diadema, sp, brazil). then, a 100 ìl aliquot of this solution was transferred to separate wells of a 96-well elisa plate (corning costar, corning, ny, usa), and the absorbance was measured at 570 nm using a microplate reader (instrutherm spectrophotometer uv-2000a, são paulo, sp, brazil). the optic density (od) of each well was proportional to the amount of coloring. determination of no levels the production of no was determined by measuring the accumulation of nitrite (no 2 ¯), a stable metabolite of no, in culture supernatants by using a colorimetric reaction with griess reagent (0.1% n-(1-naphthyl) ethylenediamine dihydrochloride, 1% sulfanilamide and 2.5% h 3 po 4 ). in each well, an equal volume of culture supernatant and griess reagent were mixed and incubated in the dark for 10 min at room temperature. absorbance was measured with a microplate reader at 570 nm (instrutherm spectrophotometer uv-2000a). the concentration of nitrite in the samples was determined from a sodium nitrite (nano 2 ) standard curve (200 µmol). statistical analysis the statistical analysis was performed with the graphpad software (graphpad inc., san diego, ca, usa). the kruskalwallis test and post hoc dunn’s multiple-comparison test were performed to compare data from cell viability. the oneway analysis of variance and tukey’s multiple-comparison test were used to compare data from no release. differences were considered significant at p<0.05. results cell viability the mtt analysis of cell viability within 24 h showed a statistically significant difference between the hff fibroblasts that were cultivated in contact with eph and the fibroblasts that were in contact with other sealers (ahp p<0.01; s26 p<0.001; enf p<0.001, figure 1a). at 48 h, only the fibroblasts that were cultured in contact with eph and ahp showed statistically significant differences for viability (p<0.05, figure 1b). at 72 h, there were no significant differences between the fibroblasts that were cultivated in contact with any of the four root canal sealers (p>0.05, figure 1c). the j774/g8 macrophage showed no significant differences in viability when cultured with any of the four sealers at 24 h (p=0.1202, figure 1d) or 48 h (p=0.6098, figure 1e). in these cells, the absorbance levels regarding cell viability only showed significant differences at 72h in comparison to the cells that were cultivated in contact with eph and ahp (p=0.020, figure 1f). determination of no levels the nitrite levels that were produced by the hff cells in contact with the four sealers showed eph to be the most cytotoxic. at 24 h, eph caused a greater release of nitrite by hff, which was significant when compared with the cells that were cultivated with ahp (p<0.001, figure 2a). however, no significant difference was found between the other two sealers in the same period (p>0.05). at 48 h, there were significant differences in the nitrite levels produced by the fibroblasts that were cultured in contact with eph and ahp (p<0.05) and ahp and enf (p<0.01), while the enf produced more of this metabolite (figure 2b). at 72 h, the cells cultured with eph released nitrite levels that were significantly higher than the levels released by the cells cultured with the other sealers (ahp p<0.001; s26 p<0.001; enf p<0.05, figure 2c). the levels of nitrite that were released by macrophages showed a different pattern of hff with significant differences for the different sealers at each experimental time (p<0.05). at 24 h, eph showed higher average levels of nitrite than the other sealers. during this period, differences were statistically significant (p=0.0002, figure 2d) in the levels of nitrite produced by macrophages that were cultivated in contact with eph and s26 (p<0.001) and eph and enf (p<0.01). at 48 h, enf caused the j774/g8 cells to release less nitrite (p=0.0016, figure 2e). there were significant differences between eph and enf (p<0.05) as well as between s26 and enf (p<0.05). at 72 h, the average levels of nitrite were higher in eph (p=0.0001, figure 2f), with braz j oral sci. 11(2):135-140 cytotoxic response of two cell lines exposed in vitro to four endodontic sealers fig. 1: cell viability of fibroblasts (a, b, c) and macrophages (d, e, f) cultivated with epiphany (eph), ah plus (ahp), sealer 26 (s26) and endofill (enf) at different periods of time. *statistically significant differences between groups. 138138138138138 significant differences between eph and ahp (p<0.001) and eph and s26 (p<0.001). discussion the complex response of periapical tissues that come into contact with sealant materials is the result of individual reactions of each cellular group involved as well as the reactions of the extracellular matrix. in this way, the present research aimed to evaluate the effect of four groups of freshly manipulated sealers on the viability of two types of cells over time as well as evaluate the release of nitric oxide, which is considered a cytotoxic mediator. immortalized cell cultures of fibroblasts and macrophages were evaluated for cytotoxicity in the periods of 24-72 h in order to closely mimic the initial cell response and simulate the worst scenario, wherein sealers are periapically extruded during obturation. most in vitro studies use a fibroblast cell line (l929, balb c 3t3, v79) to analyze the biological response to root canal sealers.4,6,8,10-11 although macrophages are used less often, they represent the prevalent cells in inflammatory infiltrates that respond to the sealers12,17. they also play a key role in defense and repair by producing a myriad of substances with inflammatory activity12. the present study used two methods to evaluate cytotoxicity: cell viability through an mtt assay, an approach that has been widely used in many previous studies6,8,11,13,17 due to its simplicity, rapidity and reliability; and no release by griess reaction, which constitutes an important proinflammatory mediator13,15-16. the cytotoxic responses of cells vary depending on the chemical composition of the sealer in a given experimental set-up. this study compared cell responses to freshly prepared resin-based, calcium hydroxide-based and zinc oxide eugenol-based sealers. most products exert some toxic effect when they are fresh, and the effect lessens over time as the concentration of leachable components decreases10. the present study observed this same phenomenon in the fibroblasts cultures. epiphany was the most cytotoxic of the sealers at 24 h. however, over time (72 h), epiphany’s toxic levels lowered to a similar pattern as the ones observed in the other 3 materials (p>0.05), with ah plus having even smaller viability levels than epiphany at 48h (p<0.05). similar to the present result, susini et al., when using a root model, found that epiphany was most cytotoxic at days 1 and 2, after which cytotoxicity decreased18. on the other hand, these results contradict previous studies, which found that epiphany became more toxic with the time of exposure to cells4,19, while ah plus had no or minimal cytotoxicity at 48h8. these conflicting results may be attributed to differences in the cell lines, as well as to the experimental design and methodology for cell viability analysis. studies enrolling the cytotoxicity of endodontic sealer may differ regarding the use of fresh or set materials4, direct contact between sealers and cells, use of filter technique11 or teflon molds to contain the sealers19, and treatment of cells with sealer extracts obtained after elution4,8. in contrast to fibroblasts, the viability of macrophage cultures showed no statistically significant difference among the groups over time. the exception was between eph and ahp at 72 h, with the former being more cytotoxic than the latter (p<0.05). this result suggests a late effect of toxic compounds, such as the residual monomers released by cured epiphany on this cell line, occurring within the first 7 days of placement20. another explanation for the cytotoxicity of epiphany might be the leaching of the sealer’s filler particles as a result of degradation8,10. although the methodology used in this study does not allow researchers to identify which components are responsible for the cell response, the current result suggests that hff may be more sensitive to epiphany at initial periods than j774/g8 cells. in relation to the production of nitrite by cells that were in contact with the sealers, a trend of increased production of this mediator was observed within the fibroblasts in contact with epiphany. this pattern was truly evident at 72 h. these results indicate that the components of epiphany activate a pro-inflammatory cascade that involves nitric oxide. although the present study did not evaluate the influence of other mediators, such as tnfá and il-1â21, on regulation of no release, it is possible that those mediators will increase upon contact with this material. this could be related in vivo to an increase in the initial inflammatory reaction after inadvertent leakage that could occur during filling. in the macrophage culture, the pattern of no release was different from the pattern that was observed in the fibroblasts cultures. different sealers changed this secretion braz j oral sci. 11(2):135-140 cytotoxic response of two cell lines exposed in vitro to four endodontic sealers fig. 2: comparison of nitric oxide levels released by fibroblasts (a, b, c) and macrophages (d, e, f) cultivated with epiphany (eph), ah plus (ahp), sealer 26 (s26) and endofill (enf) at different periods of time. *statistically significant differences between groups 139139139139139 profile over time. however, it is possible to observe high levels of no produced by cells in contact with epiphany for 24 h and 72 h. contrary to previous studies17,22, which observed several toxic alterations to materials containing zinc oxide eugenol, the present research found that endofill presented the smallest absorbance levels of no release at 48 h (p<0.05). endofill was also the least cytotoxic of the four sealers on macrophage cultures at initial times. however, in agreement with the present study, queiroz et al.15 found that the no levels were significantly smaller for endofill than for sealer 26 at 48 h, possibly due to the components from the epoxy resin and formaldehyde release during the polymerization of the latter5. reports using relatively short-term in vitro tests (d”72 h) suggest that newer materials have varying degrees of initial cytotoxicity depending on the testing conditions6,11,19. a major limitation of current in vitro data is that the shortterm evaluations are likely to be inadequate for the prediction of biological responses of materials that remain in direct contact with periradicular tissues for as long as decades23. recently, some investigators have extended in vitro test intervals to better simulate clinical conditions by using in vitro “aging” of the materials23. the results of these studies showed that many endodontic sealers remain severely cytotoxic for 5 or 6 weeks after they were mixed, but longerterm responses to other materials are unknown. the results of this study raise a question about cytotoxicity and the induction of release of proinflammatory mediators by epiphany. conversely, the results in literature suggest different responses, which this study confirmed by using two different cell lines. more studies that use primary cell cultures and co-cultures should be conducted in order to evaluate these sealers in the short-, medium-, and long-term, as well as when sealers are newly prepared under fresh conditions. thus, it would approximate to what is observed in vivo and it would be possible to compare the observations to those findings obtained within immortalized cultures. the findings of the present study showed that the pattern of response to each sealer varied depending on time6,24 and cell line6,25, as previously demonstrated. souza et al.25 reported that cfu-gm, which are macrophage progenitor cells, were more sensitive to endodontic sealers than fibroblast line. in general, the cytotoxic effects of root canal sealers may be considerably less intense over time6. however, some materials present a persistent inflammatory reaction after setting periods26. although this study has only evaluated the toxicity of compounds released by fresh sealer (unset), the major cytotoxicity for eph in short-term tests (<72h) deserves consideration. accordingly, further in vitro and in vivo studies must be carried out for a more detailed evaluation of eph behavior compared with other traditional endodontic sealers. acknowledgements this research was supported by fapemig. references 1. figueiredo jap, pesce hf, gioso ma, figueiredo maz. the histological effects of four endodontic sealers implanted in the oral mucosa: submucous injection versus implant in polyethylene tubes. int endod j. 2001; 34: 37785. 2. marciano ma, guimarães bm, ordinola-zapata r, bramante cm, cavenago bc, garcia rb, et al. physical properties and interfacial adaptation of three epoxy resin-based sealers. j endod. 2011; 37: 141721. 3. cecchin d, souza m, carlini-júnior b, barbizam jv. bond strength of resilon/epiphany compared with gutta-percha and sealers sealer 26 and endo fill. aust endod j. 2012; 38: 21-5. 4. karapinar-kazandag m, bayrak öf, yalvaç me, ersev h, tanalp j, sahin f, et al. cytotoxicity of 5 endodontic sealers on l929 cell line and human dental pulp cells. int endod j. 2011; 44:626-34. 5. queiroz ces, soares ja, leonardo rt, carlos iz, dinelli w. evaluation of cytotoxicity of two endodontic cements in a macrophage culture. j appl oral sci. 2005; 13: 237-42. 6. brackett mg, messer rlw, lockwood pe, bryan te, lewis jb, bouillaguet s, et al. cytotoxic response of three cell lines exposed in vitro to dental endodontic sealers. j biomed mater res b appl biomater. 2010; 95: 380-6. 7. dahl je. toxicity of endodontic filling materials. endod topics. 2005; 12: 39-43. 8. al-hiyasat as, tayyar m, darmani h. cytotoxicity evaluation of various resin based root canal sealers. int endod j. 2010; 43: 148-53. 9. bin cv, valera mc, camargo se, rabelo sb, silva go, balducci i, camargo ch. cytotoxicity and genotoxicity of root canal sealers based on mineral trioxide aggregate. j endod. 2012; 38: 495-500. 10. eldeniz au, mustafa k, orstavik d, dahl je. cytotoxicity of new resin, calcium hydroxideand silicone-based root canal sealers on fibroblasts derived from human gingiva and l929 cell lines. int endod j. 2007; 40: 329-37. 11. lodiene g, morisbak e, bruzell e, orstavik d. toxicity evaluation of root canal sealers in vitro. int endod j. 2008; 41: 72-7. 12. mendes sto, sobrinho apr, carvalho at, côrtes mis, vieira lq. in vitro evaluation of the cytotoxicity of two root canal sealers on macrophage activity. j endod. 2003; 29: 95-9. 13. da silva pt, pappen fg, souza em, dias je, bonetti filho i, carlos iz, et al. cytotoxicity evaluation of four endodontic sealers. braz dent j. 2008; 19: 228-31. 14. soares ja, queiroz ces. patogenesia periapical aspectos clínicos, radiográficos e tratamento da reabsorção óssea e radicular de origem endodôntica. jbe. 2001; 2: 124-35. 15. queiroz ce, soares ja, leonardo rde t, carlos iz, dinelli w. evaluation of cytotoxicity of two endodontic cements in a macrophage culture. j appl oral sci. 2005; 13: 237-42. 16. takeichi o, saito i, hayashio m, tsurumachi t, saito t. production of human-inducible nitric oxide synthase in radicular cysts. j endod. 1998; 24: 157-60. 17. silva pt, leonardo rt, carlos iz, bonetti-filho i. avaliação da citotoxicidade de cimentos endodônticos em relação aos reativos intermediários do oxigênio e do nitrogênio em culturas de macrófagos peritoneais de camundongos. rev odontol unesp. 2005; 34: 17-23. 18. susini g, about i, tran-hung l, camps j. cytotoxicity of epiphany and resilon with a root model. int endod j. 2006; 39: 940-4. 19. bouillaguet s, wataha jc, tay fr, brackett mg, lockwood pe. initial in vitro biological response to contemporary endodontic sealers. j endod. 2006; 32: 989-92. 20. ruyter ie. physical and chemical aspects related to substances released from polymer materials in an aqueous environment. adv dent res. 1995; 9: 344-7. braz j oral sci. 11(2):135-140 cytotoxic response of two cell lines exposed in vitro to four endodontic sealers 140140140140140 21. bose m, farnia p. proinflammatory cytokines can significantly induce human mononuclear phagocytes to produce nitric oxide by a cell maturation dependent process. immunol lett. 1995; 48: 59-64. 22. schwarze t, leyhausen g, geurtsen w. long-term cytocompatibility of various endodontic sealers using a new root canal model. j endod. 2002; 28: 749-53. 23. brackett mg, marshall a, lockwood pe, lewis jb, messer rl, bouillaguet s, et al. cytotoxicity of endodontic materials over 6-weeks ex vivo. int endod j. 2008; 41: 1072-8. 24. ma j, shen y, stojicic s, haapasalo m. biocompatibility of two novel root repair material. j endod. 2011; 37: 793-8. 25. souza nja, justo gz, oliveira cr, haun m, bincolleto c. cytotoxicity of materials used in perforation repair tested using the v79 fibroblast cell lines and the granulocyte-macrophage progenitor cells. int endod j. 2006; 39: 40-7. 26. brackett mg, marshall a, lockwood pe, lewis jb, messer rl, bouillaguet s, et al. inflammatory suppression by endodontic sealers after aging 12 weeks in vitro. j biomed mater res part b: appl biomater. 2009; 91: 839-44. braz j oral sci. 11(2):135-140 cytotoxic response of two cell lines exposed in vitro to four endodontic sealers oral sciences n3 braz j oral sci. 10(4):250-253 original article braz j oral sci. october | december 2011 volume 10, number 4 influence of gutta-percha cone disinfection on leakage: comparison of two sealability assessment methodologies roberta kochemborger scarparo1, fabiane canali2, vinicio hidemitsu goto hirai3, ulisses xavier da silva neto4 , carolina bender hoppe5, fabiana soares grecca6 1dds, msc, department of conservative dentistry, school of dentistry, federal university of rio grande do sul, brazil 2 dds, department of conservative dentistry, school of dentistry, federal university of rio grande do sul, brazil 3 msc, department of endodontics, school of dentistry, pontifical catholic university of paraná, brazil 4dds, phd, department of endodontics, school of dentistry, pontifical catholic university of paraná, brazil 5undergraduate student, department of conservative dentistry, school of dentistry, federal university of rio grande do sul, brazil 6 dds, phd, department of conservative dentistry, school of dentistry, federal university of rio grande do sul, brazil correspondence to: fabiana soares grecca ramiro barcelos, 2492 porto alegre, rs, brazil cep 90035-003 phone: +55 (51) 33085191 / +55 (51) 3737-5220 / fax: +55 (51) 33085010 e-mail: fabiana.grecca@ufrgs.br abstract aim: to clarify the influence of 5.25% sodium hypochlorite disinfection of gutta-percha cones on leakage and to compare the outcomes of two sealability assessment methodologies (fluid filtration and dye penetration methods). methods: thirty teeth were prepared and filled using the guttapercha lateral condensation technique. group i was filled with gutta-percha cones disinfected with sodium hypochlorite 5.25%, while group ii was filled with cones that were not disinfected. apical leakage was measured using: (a) fluid filtration technique and (b) linear measurement of dye penetration after clearance. results: the student’s t-test showed no significant difference between the groups for both techniques (p<0.05). conclusions: both methodologies employed, even with the inherent limitations, were capable of identifying sealing failures. the disinfection of gutta-percha cones with 5.25% sodium hypochlorite did not alter apical leakage. keywords: disinfection, gutta-percha, dental leakage, sodium hypochlorite, endodontics. introduction the hermetic endodontic sealing is considered a major objective for achieving root canal treatment success. on this regard, much emphasis has been placed on the improvement of material sealing ability1-2 and, therefore, it is of paramount importance to investigate of the quality of root canal filling2-8. several studies using leakage tests have been developed with this purpose2-8. on the other hand, the results of leakage studies have been seen as polemical and controversial. the great diversity of methodologies, the lack of studies approaching the comparison among different methods and the difficulty of extrapolating the scientific findings related to the in vitro sealing ability to the clinical field have been pointed out9. received for publication: february 14, 2011 accepted: december 12, 2011 251 braz j oral sci. 10(4):250-253 another important aspect is that most of the available studies have investigated the sealing capacity of endodontic sealers1,5-7, although the major portion of endodontic fillings is comprised of a solid material, i.e., the gutta-percha cones. as a matter of fact, chemical disinfection of gutta-percha cones has been suggested10-11, and its influence on leakage has yet to be properly addressed. even so, the assumption that surface deformation and physical alterations on this material may influence its sealing ability has been raised10,12. several different substances have been used for cone disinfection, the most common of which are 2% chlorhexidine and sodium hypochlorite, at concentrations that vary from 0.5 to 5.25% 10,13-14. chlorhexidine has demonstrated antimicrobial potential for disinfecting gutta-percha cones, but it is ineffective against bacillus subtillis spores even after immersion for 72 h10,15. sodium hypochlorite offers an excellent antimicrobial capacity when in contact with this material, even inhibiting sporogenous forms10,13. on the other hand, it is an oxidative solution which breaks down polymer chains, and causes structural deformations to a wide range of materials12,16-18. in view of the importance of comparing various sealability assessment methodologies and clarifying the relevance of disinfection on leakage19-20, the purpose of this study was to investigate the influence of 5.25% sodium hypochlorite cone disinfection on the endodontic sealing and to compare the results obtained with the fluid filtration and dye penetration methods. material and methods this study was approved by the institutional review board (irb) and research ethics committee of the school of dentistry of the federal university of rio grande do sul, brazil. thirty human canines with a straight root canal were selected. roots with cracks, caries, open apices, or resorption were excluded. instrumentation and filling of the canals the coronal portion of all teeth was removed with diamond disks, so that each specimen was 20-mm long. a #15 k-flexofile (dentsply maillefer, ballaigues, switzerland) was introduced into the root canal until the tip was just visible at the apical foramen. working length was determined by subtracting 1 mm from this length. apical patency was confirmed by inserting a #15 file through the apical foramen before and after root canal preparation. the teeth were prepared with hand instrumentation according to a crown-down technique. the last instrument was a # 35 file so that the enlargement of all canals was standardized. canals were irrigated with 1% sodium hypochlorite (biodinâmica, ibiporã, pr, brazil) using a syringe and a 27-gauge needle, and were dried with paper points. the prepared roots were randomly divided into 2 groups of 15 roots each. group i was obturated with 5.25% sodium hypochlorite disinfected gutta-percha cones for 5 min and washed with distilled water, while group ii was obturated with non-disinfected cones. a standardized gutta-percha cone (dentsply ind. e com. ltda., petrópolis, rj, brazil) was fitted with a “tugback” at working length. the canals were obturated using gutta-percha lateral condensation and a zinc oxide and eugenol based sealer (endofill; dentsply ind. e com. ltda.). radiographs were taken to evaluate the quality of root filling regarding homogeneity and apical extension. canal obturation was complemented if voids were detected to obtain well-compacted fillings. excess material was removed with a heated plugger at coronal level and the remaining filling material was vertically condensed using schilder pluggers 3 and 4 (dentsply maillefer). thereafter, the specimens were stored at 100% humidity and 37°c. fluid filtration two layers of nail polish were applied on the entire extent of the root (except on the apical surface of the root canal). the apical surfaces of the roots were cemented onto 2x2x0.7-cm pieces of plexiglass with a cyanoacrylate adhesive (loctite henkel ltda., itapevi, sp, brazil). the pieces of plexiglass had 18-gauge stainless steel tubes placed through their centers, ending flush with the upper surfaces. the apical openings of the tooth segment were then positioned over the tubes to permit a direct communication between the root canal and the micropipette/microsyringe system. the apical portion of the specimens was filled with water through the 18-gauge needle, taking care to remove all air bubbles. the roots were mounted in the fluid filtration device designed to measure leakage, assembled in a similar way to that described by derkson et al.19, and later adapted for endodontic leakage studies according to wu et al.3 and da silva neto et al1. the leakage of the 2 groups was quantified by following the progress of a tiny air bubble traveling within a 25-ì micropipette (microcaps, fisher scientific, philadelphia, pa, usa). the entire tubing pipette and the syringe were both filled with distilled water under the pressure of 10 psi. the fluid filtration volume was measured by observing the air bubble movement. the air bubble displacement was recorded as the fluid filtration result, expressed in ì/min–1.10 psi. fluid movement measurements were made at 2-min intervals for 8 min and then averaged. after all connection points had been sealed, the system was allowed to equilibrate for 4 min. one sample was excluded during the measurement procedures because a crack was verified. dye penetration after the fluid filtration measurements, the specimens were kept in india ink for 10 days (john faber castel, são paulo, sp, brazil) and then washed with tap water to remove the excess of ink. the nail polish layers were then removed with a scalpel and the roots were cleared. the teeth were demineralized in 5% nitric acid until a milky color was achieved. the solution was renewed every influence of gutta-percha cone disinfection on leakage: comparison of two sealability assessment methodologies braz j oral sci. 10(4):250-253 24 h until the process ended. the specimens were then dehydrated in 80º ethanol for 12 h, followed by 1 h in 90º ethanol and 1 h in 96º ethanol. this last procedure was repeated three times. all ethanol solutions were obtained by the dilution of 100% ethanol in distilled water. the specimens were then allowed to dry naturally for 10 min and were put into glass vials with methyl salicylate (c 8 h 8 o 3 ). after 2 h, the internal structure of the tooth could be visualized. the ink leakage was measured three dimensionally under stereoscope microscopy (x40) (meiji techno co., ltd., tokyo, japan). for clearance assessment, the examiner was calibrated using the intra-class correlation coefficient test. the data were subjected to statistical analysis using a student’s t-test. the confidence level was pre-set at 5% for both evaluation methods. results for clearance evaluation, the examiner was calibrated and the icc test scored 0.939. there were no statistically significant differences (p>0.05) between the study groups by means of the fluid filtration and clearance test, as shown on tables 1 and 2. group n min m a x mean sd t p i 15 0 8 2.33 1.88 1.774 0.087 i i 14 0 4 1.29 1.20 table 2 mean of trace penetration measure in mm after clearance (p<0.05). group n mean s d t p i 15 0.997 0.47 0.499 0.622 i i 14 0.910 0.46 table 1mean of fluid movements measures and statistical analysis for the groups (µ/min–1.10 psi) (p<0.05). discussion the present study has attempted to elucidate two important aspects: the comparison of different sealability assessment methodologies and the influence of cone surface alterations caused by disinfection on leakage. several methods have been described aiming at evaluating the sealing quality of root canals fillings. the most common is the dye tracer penetration test21. this technique, associated with clearance, makes possible the three-dimensional observation of the dental structure and the identification of the dye penetration extension. fluid filtration is based on the principle that no fluid movement will be detected if the root canal system is completely sealed. measurements of fluid transportation are carried out by observing an air bubble and its movement along a capillary. further, the fluid filtration technique quantifies microleakage and allows repeated measurements because it is nondestructive9,20,22-23. for this reason, in the present study, it was possible to evaluate the same sample using two different in vitro methodologies. since so far there is no leakage method able to effectively assess clinically relevant aspects, comparison between different materials and techniques used in endodontic practice must still be carried out based on preliminary tests such as those described in this study. the efforts to reduce artifacts and improve methodologies have not generated conflicting results for sealing capacity, since both methods employed in the present study have not shown significant differences between the groups. however, the statistical analysis shows some variations that should be further explored. fluid filtration presented a p value of 0.662, and die penetration tests a p value of 0.087. these values show a smaller difference between the groups when fluid infiltration is used, which may indicate that dye penetration tests produce some confounding factors that might be increasing the variability of measurements. on the other hand, despite of the technique used, leakage could be detected. although it has been currently documented that dye penetration measurements yield some level of variation, in the present study, icc data showed that this fact is not related to the variability in the operator’s experience. the variation in the analysis of the dye penetration is probably influenced by some factors: (a) gaps between the root filling and the canal wall may contain air and/or liquid, which would prevent the penetration of the tracers and influence the results; (b) the potential of the tracer to react or affect the filling material itself might make the results from some of these tests unreliable (c) because of dentinal tubule permeability, different leakage tracers could penetrate through root dentin and not through the canal4. another problem for extrapolating the findings related to the dye penetration tests to the clinical field is that there is no equivalence between the ink particles and the bacteria and their toxin sizes. also, this technique does not provide any information about the volume of tracer that penetrates the dental structure2. due to these aspects, dye penetration tests have their relevance related to the identification of visible gaps in the sealing. generally, in vivo microleakage is expected to be not as severe as the ones observed in in vitro methodologies because it is accepted that a root canal filling should be bacteria tight. on the other hand, the importance of bacterial toxins should not be neglected, and further investigations are necessary to determine the clinically relevant amount of leakage that can be considered as pathological2. considering the influence of disinfection on sealing, this procedure did not cause alterations on leakage. one can infer that the possible changes in the structure and surface of gutta-percha cones do not jeopardize the seal of root canal system. according to pang et al. 11, chemical disinfection promotes alterations on the tensile strength and elongation rate of the gutta-percha cones. valois et al.12 observed significant surface alterations in gutta-percha cones after disinfection in sodium hypochlorite solutions, particularly 252 influence of gutta-percha cone disinfection on leakage: comparison of two sealability assessment methodologies 253 braz j oral sci. 10(4):250-253 at elevated concentrations, using atomic force microscopy. furthermore, some studies have confirmed the sodium hypochlorite capacity to cause alterations during the process of disinfecting gutta-percha cones11. the aim of the present research did not encompass the analysis of structural and surface alterations. even though, it is presumed that these occurred in the disinfected gutta-percha cones used in this study. short et al.17 proved that crystals are formed on the surface of gutta-percha cones after the immersion of them in sodium hypochlorite. however, washing the material in alcohol, saline or distilled water efficiently removes these crystals10,17. in the present study, after disinfection, the cones were washed with distilled water, thus avoiding the influence of the presence of crystals in analysis of the results. microbiological studies have confirmed the efficiency of disinfection of gutta-percha cones using 5.25% sodium hypochlorite for 1 mine10,14, but in the present study the immersion time chosen was 5 min. a longer period was used in order to increase the contact time of the cones with the solution. it is important to note that, even exposing guttapercha cones for 5 min, disinfection did not lead to higher infiltration levels, when compared to non-disinfected cones. the influence of the disinfection of gutta-percha cones in endodontic sealing is one of the most important aspects in the determination of protocols. the results of the present study do not indicate negative effects, in this sense. further investigations are necessary to determine whether material alterations caused by disinfection could impair long-term outcomes of endodontic sealing, causing, for instance, a faster onset of components degradation. according to maniglia-ferreira et al.24, the oxidation reaction of gutta-percha components is a slow process, but significant degradation can be accelerated by some factors, such as the presence of periapical lesions. in conclusion, both methodologies employed in this study, even with the inherent limitations, were capable of identifying sealing failures in the root canal system. the disinfection of gutta-percha cones with 5.25% sodium hypochlorite did not cause changes in apical leakage. acknowledgements the authors deny any conflicts of interest. the authors are grateful to the propesq/ufrgs for their financial support. the authors gratefully acknowledge capes for the scholarship granted to carolina bender hoppe. references 1. da silva neto ux, de moraes ig, westphalen vpd, menezes r, carneiro e, fariniuk lf. leakage of 4 resin-based root canal sealers used with a single-cone technique. oral surg oral med oral pathol oral radiol endod. 2007; 104: e53-7. 2. wu mk, wesselink pr. endodontic leakage studies reconsidered. part1. methodology, applicationand relevance. int endod j. 1993; 26: 37-43. 3. wu mk, de gee aj, wesselink pr, moorer wr. fluid transport and bacterial penetration along root-canal fillings. int endod j. 1993; 26: 203-8. 4. wu mk, kontakiotis eg, wesselink pr. decoloration of 1% methylene blue solution in contact with dental filling materials. j dent. 1998; 26: 585-9. 5. mannocci f, ferrari m. apical seal of roots obturation with latterally condensed gutta-percha, epoxy resin cement, and dentin bonding agent. j endod. 1998; 24: 41-4. 6. shipper g, orstavik d, teixeira fb, trope m. an evaluation of microbial leakage in roots filled with a thermoplastic synthetic polymer-based root canal filling material (resilon). j endod. 2004; 30: 342-7. 7. kqiku l, städtler p, gruber hj, baraba a, anic i, miletic i. active versus passive microleakage of resilon/epiphany and gutta-percha/ah plus. aust endod j. 2011; 37:141-6. 8. farea m, masudi s, bakar wzw. apical microleakage evaluation of system b compared with cold lateral technique: in vitro study. aust endod j. 2010; 36: 48-53. 9. camps j, pashley dh. reliability of the dye penetration studies. j endod. 2003; 29: 592–4. 10. gomes bpfa, vianna me, matsumoto cu, rossi vde p, zaia aa, ferraz cc et al. disinfection of gutta-percha cones with chlorhexidine and sodium hypochlorite. oral surg oral med oral pathol oral radiol endod. 2005; 100: 512-7. 11. pang ns, jung iy, bae ks, baek sh, lee wc, kum ky. effects of short-term chemical desinfectation of gutta-percha cones: identification of affected microbes and alterations in surface texture and physical properties. j endod. 2007; 33: 594-8. 12. valois cr, silva lp, azevedo rb. effects of 2% chlorhexidine and 5,25% sodium hipochlorite on gutta-percha cones studied by atomic force microscopy. int endod j. 2005; 38: 425-9. 13. da motta pg, de figueiredo cb, maltos sm et al. efficacy of chemical sterilization and storage conditions of gutta-percha cones. int endod j. 2001; 34: 435-9. 14. souza re, souza ea, sousa-neto md, pietro rc. in vitro evaluation of different chemical agents for the decontamination of gutta-percha cones. pesq odontol bras. 2003; 17: 75-7. 15. siqueira jr jf, da silva ch, cerqueira md, lopes hp, de uzeda m. effectiveness of four chemical solutions in eliminating bacillus subtilis spores on gutta-percha cones. endod dent traumatol. 1998; 14: 124-6. 16. yamauti m, hashimoto m, sano h, ohno h, carvalho rm, kaga m et al. degradation of resin-dentin bonds using naocl storage. dent mater. 2003; 19: 399-405. 17. short rd, dorn so, kuttler s. the crystallization of sodium hypochlorite on gutta-percha cones after the rapid-sterilization technique: a sem study. j endod. 2003; 29: 670-3. 18. berutti e, angelini e, rigolone m, migliaretti g, pasqualini d. influence of sodium hypochlorite on fracture and corrosion of protaper rotary instruments. int endod j. 2006; 39: 693-9. 19. derkson gd, pashley dh, derkson me. microleakage measurement of selected restorative materials: a new in vitro method. j prosthet dent. 1986; 56: 435-40. 20. kececi ad, kaya bu, belli s. coronal and apical leakage of various root filling materials using two different penetration models – a 3-month study. j biomed mater res b appl biomater. 2010; 92: 261-7 21. robertson d, leeb ij, mckee m, brewer e. a clearing technique for study of root canal systems. j endod. 1980; 6: 421-4. 22. onay eo, ungor m, unver s, ari h, belli s. an in vitro evaluation of the apical sealing ability of new polymeric endodontic filling systems. oral surg oral med oral pathol oral radiol endod. 2009; 108: e49-54. 23. mahera f, economides n, gogos c, beltes p. fluid-transport evaluation of lateral condensation, protaper gutta-percha and warm vertical condensation obturation techniques. aust endod j. 2009; 35: 169-73. 24. maniglia-ferreira c, silva jr jba, de paula rcm, feitosa jp, zaia aa, ferraz cc et al. degradation of trans-polyisoprene over time following the analysis of root fillings removed during conventional retreatment. int endod j. 2007; 40: 25-30. influence of gutta-percha cone disinfection on leakage: comparison of two sealability assessment methodologies oral sciences n3 original article braz j oral sci. july | september 2012 volume 11, number 3 temporomandibular dysfunction and cervical posture and occlusion in adolescents lara jansiski motta1, kristianne porta santos fernandes1, raquel agnelli mesquita-ferrari1, daniela aparecida biasotto-gonzalez1, sandra kalil bussadori1 1phd, rehabilitation sciences department, nove de julho university (uninove), são paulo, sp, brazil correspondence to: lara jansiski motta av. getúlio vargas, 386 apt. 53 cep: 18130-430 jd. lourdes,são roque, sp – brasil phone: + 55 11 47128358 e-mail: larajmotta@terra.com.br abstract aim: to evaluate the effect to the degree of temporomandibular dysfunctions (tmd) in adolescents and occlusal classes in the cervical posture. methods: a cross-section, observational study was carried out, in which 296 adolescents took part. for the evaluation, the patients were divided into groups according to the presence and severity of the tmd, using the helkimo questionnaire and occlusal angle classification. the posture analysis was carried out using photogrammetry and the software alcimage® to measure the predefined angle based on the protuberances of the spinous process of the 7th cervical vertebra (c7), manubrium of the sternum and mentum vertex. results: 48% (n= 142) of the 296 adolescents evaluated presented no dysfunction, while 52% (n= 154) presented some degree of tmd. of the different degrees of tmd, the highest average cervical angulation observed in the group with moderate dysfunction was 97.59º ±7.40, followed by a mild degree, of 96.32º± 9.36, and the lowest average was 93.01º±10.08 in the patients with a severe degree in the different occlusal classes. in relation to occlusal class, higher values for this angulation were observed in class ii (96.77º± 8.79), compared with class i (90.64º±8.80) and class iii (94.67º± 10.70), a difference which was statistically significant. conclusions: the subjects with tmd presented a greater alteration in head posture, compared with those without tmd. in particular, the class ii angle was correlated with tmd and alterations in cervical posture. keywords: temporomandibular disorders, head posture, adolescents, cervical column. introduction the characteristic symptoms of temporomandibular dysfunctions (tmd) are: muscular and/or joint pain on touch, mandibular function impairment, and joint noises, the overall prevalence of these symptoms affecting over 75% of the population and this condition is not limited to adults1. epidemiological studies such as those conducted by egermark et al.2 and thilander et al.3 have shown that signs and symptoms of tmd can be found in all age groups. however, its prevalence in children is considered low, increasing with age in adolescents and young adults. the masticatory system is a regulatory or perturbing element of the postural system. therefore, a disequilibrium induced by a temporomandibular joint (tmj) disorder can lead to postural decompensation, just as disequilibrium of the postural system alters the masticatory system4. the association between head posture, cervical posture, mandibular posture and equilibrium of the stomatognathic system has been studied and discussed for years, and has been a source of divergent opinions5. authors affirm that patients with tmd present more alterations in head positioning than patients with no received for publication: may 15, 2012 accepted: september 18, 2012 braz j oral sci. 11(3):401-405 402402402402402 braz j oral sci. 11(3):401-405 dysfunction5-11. the ideal head position in the space depends on three planes: the optical plane, the transverse occlusal (masticatory) plane and the auriculo-nasal plane. these three planes together maintain a horizontal and parallel relationship which ensures cranial postural stability. this position is assured by the mechanoreceptors of the upper part of the cervical column8,10-11. in an ideal head posture, its highest volume is found slightly anterior to the cervical column12. numerous aspects of stomatognathic system conditions have been found to be associated with head posture alterations. among these aspects are: mandible position, dental or skeletal malocclusion, and tmd12-13. correct dental occlusion is important for maintaining the equilibrium of the muscles which form part of the mandible10. with this occlusion, it is possible to verify the vertical dimension of occlusion, which corresponds to the vertical dimension of the face. if an alteration to this dimension occurs, the growth of the muscles inserted in and passing through the cranium, via the hyoid bone and shoulders, is altered. therefore, alterations in head position could influence the cervical column and stomatognathic system, altering the distribution of occlusal stress and affecting the craniofacial morphology8,11-12. patients with malocclusion or tmj disorders habitually report dysfunction and pain in their neck muscles14. the cervical muscles are directly related to the tmj by an interconnected neuromuscular system. changes in the cervical column tmd and the opposite is also correct. since head and cervical muscles are closely related to the stomatognathic system, studies have been carried out to confirm that postural changes of the head and the body could have an adverse biomechanical effect on the tmj and lead to tmd12-13. clinical studies showed influences of head and body posture on the mandibular rest position, range of functional movements and the jaw and neck muscles work together during rhythmic movements12. the relationship between tmd and cervical posture involves the relationship between tmj, painful cervical symptoms, and posture alterations of the cervical column and the head. the position of the cervical column could be the initial cause of some occlusal discrepancies and alterations in neuromuscular harmony. the position of the head and neck lead to modifications in the mandibular trajectory, with future impairment of the tmj, consequently affecting the entire posture9,11-12. due to the possibility of dysfunctions of the tmj originating at the beginning of craniofacial growth, a high percentage of adolescents present signs and symptoms associated with tmd13. the study of tmd in adolescents is important for determining, at an early stage, the problems that may predispose to abnormalities of craniofacial growth, pain or muscular dysfunction in the adult phase14. the aim if this study was to evaluate the effect to the degree of tmd in adolescents and occlusal classes in the cervical posture. material and methods this study was approved by the human research ethics committee, under no. 82622/08, and those responsible for the participants signed an informed consent form, authorizing their participation in the study. this is an observational, cross-sectional study for the evaluation of the students of a municipal school in the countryside of the state of são paulo (brazil). a total of 454 adolescents were evaluated. as criteria for inclusion, age group and the presence of first permanent molars were used for evaluation of the occlusal class. individuals undergoing orthodontic treatment, those with absent first molars, and cases where it was impossible to clinically evaluate the occlusion were excluded. of the 454 students, 301 were in this age group, 3 used braces and 2 had lost their first permanent molars, therefore a total 296 adolescents took part in the study. the participants were divided into groups according to the degree of tmd and occlusal angle classification. the evaluation of signs and symptoms of tmd was obtained from the helkimo questionnaire15, which includes information regarding difficulties on opening the mouth and movement of the mandible, pains in the head, nape of the neck, neck or joint regions, noise in the tmd, and the habit of clenching or grinding the teeth. a large number of indices are found in the international literature for the classification of patients with tmd, such as the widely used helkimo index 15, which is a pioneering questionnaire aimed at measuring the severity of tmd. in an epidemiological study, an index subdivided into anamnestic, clinical disorder and occlusal disorder was developed to assess the prevalence and degree of severity of tmd in patients with severe mandibular pain both on the individual level and in the general population16,17. in an attempt to define diagnostic criteria for the classification of patients with tmd, dworkin and le resche17 developed a detailed questionnaire. however, as this assessment tool is long and complex, it may not be applicable to adolescents. moreover, the questionnaire was validated for a different culture and therefore requires validation in the portuguese language for possible administration in brazilian studies. fonseca evaluated the index for the assessment of tmd adapted to the brazilian population that would be easy to understand and administer and could be more successfully applied to the younger population. studies have found a strong positive correlation (r = 0.95) between the fonseca translation and the helkimo index18. the questionnaire is comprised of 10 questions with possible answers of yes (10 points), sometimes (5 points) and no (0 points). for each question, only one answer can be checked. the total score is used to classify the severity of the tmd as severe (70 to 100 points), moderate (45 to 65), mild (20 to 40) and no dysfunction (0 to 15). in order to correlate the data for tmd and occlusal class, the students were divided into subgroups, according to the occlusal angle classification obtained from the analysis temporomandibular dysfunction and cervical posture and occlusion in adolescents braz j oral sci. 11(3):401-405 403403403403403 relating to the first permanent molars. the clinical assessment of head posture was conducted using a postural grid. each subject was asked to remain in his/her normal posture, in a standing position. the selfbalance position was used to standardize the posture of each subject, asking him/her to look straight ahead, parallel to the floor, keeping the gaze horizontal19. to study the posture, three different points were marked with painted semi-spheres (1.5 cm), which were attached to the volunteer’s skin with double-sided adhesive tape. the spinous process of the seventh cervical vertebra, the manubrium of the sternum, and the mentum vertex, were chosen for this evaluation based on rocabado’s20 description. the subject’s posture was also photographed (right side) using a digital camera (kodak™ z740 7.1). the camera was placed on a height-adjustable tripod with a standard distance of 1.5 m between the camera and the subjects. all the clinical examinations and the pictures were carried out by the same person, who was blind to which group the subjects were in. the measurements in the photographs were performed using a computer software (alcimage®) to quantify the posture, using angular calculus. the angle was calculated automatically by the software, so the examiner was unaware of the angle value until the end of the analysis. in this study, three reference points were established for the head posture analysis: the spinous process of the seventh cervical vertebra, manubrium of the sternum and mentum vertex. the average of the three measurements, for each volunteer, was used to obtain greater reliability of the angles collected. according to the classification, the volunteers were compared in terms of degree of tmd and posture (mean angles). the data obtained were tabulated and submitted to chi-squared statistical calculations for the qualitative variables, shapiro-wilk w normality test (p>0.05) and analysis of variance for the quantitative variables. a level of significance of 0.05 was considered in all the analyses, using the spss12.0 for windows statistical software (spss inc., chicago, il, usa) . age total 10 11 12 13 14 15 16 17 18 19 20 5 28 42 22 11 4 9 1 14 3 3 142 0 23 52 31 32 13 3 0 0 0 0 154 5 51 94 53 43 17 12 1 14 3 3 296total no tmd with tmd table 2. distribution of the participants by age and the presence of tmd. sex m f total no tmd n 97 45 142 % 68.3% 31.7% 100.0% with tmd n 46 108 154 % 29.9% 70.1% 100.0% total n 143 153 296 % 48.3% 51.7% 100.0% table 1. distribution of the participants by sex and the presence of tmd results of the 296 adolescents evaluated, 143 (48.3%) were male and 153 (51.7%) female. the mean age was 13 years (±2.02). in relation to the presence of tmd, it was observed that 48% (n= 142) presented no dysfunction, while 52% (n= 154) presented some degree of tmd. of the 142 patients that did not have any tmd, 68.3% (n=97) were male while 31.7% (n= 45) were female. of the group of individuals classified as having tmd, a higher frequency of females, 70.1% (n=108) was observed, compared with 29.9% (n=46) males (table 1). evaluating the relationship between sex and the presence of tmd statistically, the association between the female sex and the presence of the dysfunction was significant (p<0.001). in relation to age and the presence of tmd, a higher frequency was observed in the 11 to 13 year age group, but in this age group, the number of participants was larger (table 2) and when evaluated statistically, there was no significant difference. in relation to the cervical angle, higher mean values were found for the group of adolescents with tmd: 96.37° ±9.01, while the group with no tmd presented mean values of 91.80 ± 9.37. the distribution of the adolescents by degree of tmd shows a more frequent occurrence of the mild degree (36.5%) among the patients with tmd. within the different degrees of tmd, a greater cervical angulation for the group with moderate dysfunction (97.59º ±7.40), and the mild degree (96.32º± 9.36) were observed compared with the values obtained for the group with no tmd (91.80 ± 9.37), these differences being statistically significant. the group with severe dysfunction presented values of 93.01º±10.08, with no statistically significant difference between it and the other groups (table 3). table 4 shows the distribution of the participants in relation to the occlusal angle classification. in relation to temporomandibular dysfunction and cervical posture and occlusion in adolescents braz j oral sci. 11(3):401-405 404404404404404 tmd tmd mean difference standard error sig. no tmd mild -4.51774(*) 1.17326 p<0.001 moderate -5.78504(*) 1.73416 0.001 severe -1.20673 2.87599 0.675 mild moderate -1.26730 1.78733 0.479 severe 3.31101 2.90835 0.256 moderate severe 4.57831 3.17636 0.151 table 3. significance between the differences in means for cervical angles in the different groups. i ii iii total n n n n m i l d 38 (35.2%) 56 (51.9%) 14 (13.0%) 108 (100%) moderate 10 (28.6%) 17 (48.6%) 8 (22.9%) 35 (100%) severe 3 (27.3%) 3 (27.3%) 5 (45.5%) 11 (100%) total 51 (33.1%) 76 (49.4%) 27 (17.5%) 154 (100%) tmd table 4. classification of occlusal angle for the different degrees of tmd. occlusal class i ii occlusal class ii iii iii mean difference -6.12947(*) -4.02632 2.10314 std. error 1.12968 1.74929 1.69967 sig. p<0.001 0.057 0.432 lower bound -8.7906 -8.1471 -1.9007 upper bound -3.4683 .0945 6.1070 95% confidence interval table 5. significance between the means for cervical angles in the different occlusal classes. * the mean difference is significant at a level of 0.05. occlusal class, higher values were seen for this angle in class ii in comparison to class i and class iii, this difference was statistically significant (p<0.005) for a confidence interval of 95% (table 5). discussion in the present study, alterations in cervical posture in adolescents with tmd and malocclusion were observed. in the evaluation between tmd and cervical posture among the adolescents, a greater cervical angle was observed in those with tmd. this cervical alteration is related to the forward leaning positioning of the head in patients with tmd, and has been discussed in various studies5,21-24. this finding agrees with other authors, such as armijo-olivo et al.9, who state that the forward position of the head results in mandible function and positioning disturbances. this outcome would increase the tension in the masticatory muscles and, consequently, cause tmd10. sonnesen et al.25 observed that tmj disorder was associated with a marked forward inclination of the upper cervical spine and increased craniocervical angulation. however, this relationship is investigated mainly in adults26,27,28 and the number of works with children and adolescents is scrace29-31. in the detailed analysis of the relationship between degree of tmd and cervical posture, a greater cervical angulation was observed in this study, i.e. a greater forward leaning posture of the head in patients with mild and moderate tmd, compared with adolescents with severe tmd. this result could be because in this study, the severe tmd group had a proportionally larger number of individuals with class iii malocclusion, which commonly presents the head and neck in a more backward leaning posture to compensate for the forward protruding mandible4,32. the relationship between malocclusions and head posture has been described between features of skeletal class ii malocclusions, retruded mandibular position and reduced mandibular length on the sagittal plane and increased cervical lordosis. the head and neck angulation variation in patients with and without tmd problems was associated with malocclusion in the literature32,33, which was observed in this research, in which the greatest average cervical angulation was observed in the class ii malocclusion group. there was no difference in posture between the class i and class iii patients. this data is in accordance with aspects described in the literature, which affirm that patients with class i occlusion maintain the position of the head in equilibrium while class ii patients alter the position of the head and the shoulders, leaning forward to compensate for the retracted mandibular position, and those with class iii malocclusion position the head backwards or adjust the cervical column4,7,12,31,34. the hypothesis of the relationship between malocclusion and cervical posture is that the position of the mandible may influence the muscles, causing the change of the neck and spinal column position. the postural evaluation in many studies is carried out in the form of a clinical examination6 and at other times, a cephalometric x-ray is used 27. this work used photogrammetry, which is a non-invasive method that can quantify the postural alterations without the inconvenience of radiation. the use of software for quantitative evaluation of cervical posture prevents examiner subjectivity. thus, it was demonstrated that the study of tmd in adolescents is important for determining, at an early stage, the problems that predispose to abnormalities of craniofacial growth, pain or muscular dysfunction in the adult phase23. bearing in mind the close relationship established by the dental occlusion, oral functions, and head and body posture, temporomandibular dysfunction and cervical posture and occlusion in adolescents 405405405405405 braz j oral sci. 11(3):401-405 the need arises for establishing a new standard for the evaluation and treatment of patients21 on the basis of a multidisciplinary approach. based on the results obtained from the analysis in this study, it can be concluded that there is a relationship among tmd, cervical posture and occlusal class in adolescents, this effect being evident in the participants with angle class ii. references 1. hirsch c, hoffmann j, türp jc. are temporomandibular disorder symptoms and diagnoses associated with pubertal development in adolescents? an epidemiological study j orofac orthop. 2012; 73: 6-18. 2. egermark-eriksson i, carlsson ge, magnusson t. a 20-year longitudinal study of subjective symptoms of temporomandibular disorders from childhood to adulthood. acta odontol scand. 2001; 59: 40-8. 3. thilander b, rubio g, pena l, de mayorga c. prevalence of temporomandibular dysfunction and its association with malocclusion in children and adolescents: an epidemiologic study related to specified stages of dental development. angle orthod. 2002; 72: 146-54. 4. ghessa g, capobianco s, lai v. stabilometria e disturbicranio-cervicomandibolari. minerva stomatol. 2002; 51: 167-71. 5. armijo-olivo s, rappoport k, fuentes j, gadotti ic, major pw, warren s, et al. head and cervical posture in patients with temporomandibular disorders. j orofac pain. 2011, 25: 199-209. 6. arntsen t, sonnesen l. cervical vertebral column morphology related to craniofacial morphology and head posture in preorthodontic children with class ii malocclusion and horizontal maxillary overjet. am j orthod dentofacial orthop. 2011; 140: 1-7. 7. d’atitilio m, caputi s, epifania e, festa f, tecco s. evaluation of cervical posture in skeletal class i, ii and iii. cranio. 2005, 23: 219-28. 8. ferraz-junior aml, guimarães jp, rodrigues mf, lima rhm. evaluation of postural changes prevalence in patients with temporomandibular disorders: a therapeutic proposal. rev serviço atm. 2004, 4: 24-32. 9. armijo-olivo s, warren s, fuentes j, magee dj. clinical relevance vs. statistical significance: using neck outcomes in patients with temporomandibular disorders as an example. man ther. 2011; 16: 563-72. 10. armijo-olivo s, silvestre ra, fuentes jp, da costa br, major pw, warren s, et al. patients with temporomandibular disorders have increased fatigability of the cervical extensor muscles. clin j pain. 2012; 28: 55-64. 11. olivo sa, fuentes j, major pw, warren s, thie nm, magee dj. the association between neck disability and jaw disability. j oral rehabil. 2010; 37: 670-9. 12. manfredini d, castroflorio t, perinetti g, guarda-nardini l. dental occlusion, body posture and temporomandibular disorders: where we are now and where we are heading for. j oral rehabil. 2012; 39: 463-71. 13. 13.thilander b, rubio g, pena l, de mayorga c. prevalence of temporomandibular dysfunction and its association with malocclusion in children and adolescents: an epidemiologic study related to specified stages of dental development. angle orthod. 2002; 72: 146-54. 14. emodi-perlman a, eli i, friedman-rubin p, goldsmith c, reiter s, winocur e. bruxism, oral parafunctions, anamnestic and clinical findings of temporomandibular disorders in children. j oral rehabil. 2012; 39: 126-35. 15. helkimo m. studies on function and dysfunction of the masticatory system iii. analysis of anamnestic and clinical recordings of dysfunction with the aid of indices. swed dent j. 1974; 67: 165. 16. he ss, deng x, wamalwa p, chen s correlation between centric relation; maximum intercuspation discrepancy and temporomandibular joint dysfunction. acta odontol scand. 2010; 68: 368-76. 17. dworkin sf, leresche l. research diagnostic criteria for temporomandibular disorders: review, criteria, examinations and specifications, critique. j craniomandib disord. 1992; 6: 301-55. 18. fonseca dm, bonfante g, valle al, de freitas sft. diagnosis of the craniomandibular disfunction through anamnesis. rev gauch de odontol. 1994; 4:23-32. 19. costa jr, pereira sra, mitri g, motta jc, pignatari ssn, weckx llm. relationship between dental occlusion, the head and cervical spine-position in mouth breathing children. rev paul pediatr. 2005; 23: 88-93. 20. rocabado m. biomechanical relationship of the cranial, cervical, and hyoid regions. j craniomandibular pract. 1983; 1: 61-6. 21. solow b, sandham a. cranio-cervical posture: a factor in the development and function of the dentofacial structures. eur j orthod. 2002; 24: 447-56. 22. strini pj, machado na, gorreri mc, ferreira ade f, sousa gda c, fernandes neto aj. postural evaluation of patients with temporomandibular disorders under use of occlusal splints. j appl oral sci. 2009; 17: 539-43. 23. perinetti g, contardo l, biasati as, perdoni l, castaldo a. dental malocclusion and body posture in young subjects: a multiple regression study. clinics. 2010; 65: 689-95. 24. conti pb, sakano e, ribeiro ma, schivinski ci, ribeiro jd. assessment of the body posture of mouth-breathing children and adolescents. j pediatr. 2011; 87: 357-63. 25. sonnesen bake m, solow b. temporomandibiular disorder in relation to craniofacial dimensiones, head posture and bite force in children select for orthodontic treatment. eur j orthod. 2001, 23: 179-92. 26. maluf sa, moreno bg, crivello o, cabral cm, bortolotti g, marques ap. global postural reeducation and static stretching exercises in the treatment of myogenic temporomandibular disorders: a randomized study. j manipulative physiol ther. 2010; 33: 500-7. 27. olivo sa, bravo j, magee dj, thie nmr, flores-mir r. the association between head and cervical posture and temporomandibular disorders: a systematic review. j.orofacial pain. 2006; 20: 9-23. 28. matheus ra, ramos-perez fm, menezes av, ambrosano gm, haiter-neto f, bóscolo fn, et al. the relationship between temporomandibular dysfunction and head and cervical posture. j appl oral sci. 2009; 17: 204-8. 29. barbosa ts, miyakoda ls, poctztaruk rl, rocha cp, gavião mbd. temporomandibular disorders and bruxism in childhood and adolescence: review of the literature.int j pediatr otorhinolaryngol. 2008, 72: 299-314. 30. maluf sa, moreno bg, crivello o, cabral cm, bortolotti g, marques ap. global postural reeducation and static stretching exercises in the treatment of myogenic temporomandibular disorders: a randomized study. j manipulative physiol ther. 2010; 33: 500-7. 31. torii k, chiwata i. occlusal adjustment using the bite plate-induced occlusal position as a reference position for temporomandibular disorders: a pilot study. head face med. 2010; 6: 5. 32. fink m, wähling k, stiesch-scholz m, tschernitschek h.the functional relationship between the craniomandibular system, cervical spine, and the sacroiliac joint: a preliminary investigation. cranio. 2003; 21: 202-8. 33. yamamoto t, nishigawa k, bando e, hosoki m. effect of different head positions on the jaw closing point during tapping movements. j oral rehabil. 2009; 36: 32-8. 34. chaves tc, de andrade e silva ts, monteiro sa, watanabe pc, oliveira as, grossi db. craniocervical posture and hyoid bone position in children with mild and moderate asthma and mouth breathing. int j pediatr otorhinolaryngol. 2010; 74: 1021-7. temporomandibular dysfunction and cervical posture and occlusion in adolescents oral sciences n3 braz j oral sci. 10(2):136-139 original article braz j oral sci. april | june 2011 volume 10, number 2 apical and cervical displacement produced by hand and engine-driven stainless steel and nickel-titanium instruments in simulated curved root canal benito andré silveira miranzi1, gilberto antônio borges1, almir josé silveira miranzi4, fernando hueb de menezes2, mário alfredo silveira miranzi3, luis henrique borges1 1dds, phd, department of biomaterials, dental school, university of uberaba, brazil 2dds, phd, department of biopathology, dental school, university of uberaba, brazil 3dds, phd, department of community medicine, federal university of triângulo mineiro brazil 4dds, phd, department of dental prosthodontics, university of uberaba, brazil correspondence to: benito andré silveira miranzi universidade de uberaba uniube, departamento de endodontia av. nenê sabino, 1801, bloco h, sala nº 207 3055-500 uberaba, mg, brazil. phone: (34)84069382 e-mail: bmiranzi@mednet.com.br abstract aim:aim:aim:aim:aim: the purpose of this study was evaluate the occurrence of displacement on the apical and cervical thirds of artificial curved root canals with 30° of curvature, comparing the use of stainless steel hand and oscillatory files with nickel-titanium (ni-ti) hand, rotary and oscillatory files. methods:methods:methods:methods:methods: seventy artificial curved root canals were divided into 7 groups (n=10). all canals were prepared with sizes 15 to 40 instruments at the working length according to a crown-down technique. the amount of material removed in the curved portion of the simulated canals at 2 mm level outside and 11 mm inside was measured. data were analyzed statistically by anova and tukey’s test (p<0.05). results: results: results: results: results: there was statistically significant differences (p<0.05) at 2 mm level between ni-ti and steel stainless groups. at 11 mm, statistically significant differences (p<0.05) were observed between the groups that used rotary systems (5 and 6) and the remaining groups. conclusions:conclusions:conclusions:conclusions:conclusions: the most centered preparations were those belonging to groups 5 and 6, at both levels. root canal preparation with ni-ti files yielded better results than the stainless steel files especially when mechanically activated. keywords:keywords:keywords:keywords:keywords: root canal preparation, endodontic instruments, root canal therapy. introduction the proper preparation of curved root canals is directly related to the capacity of providing uniform removal of intracanal dentin without resulting in undesirable canal aberrations. the use of stainless steel files for dilatation of curved canals may cause problems such as zipping, elbow, and perforations especially when the canals have to be enlarged above a size 25 file1. the introduction of nickel-titanium (ni-ti) files made possible to provide received for publication: january 24, 2011 accepted: may 31, 2011 137 braz j oral sci. 10(2):136-139 more regular preparations because these instruments have high flexibility, low elasticity modulus and shape memory, which lead these files to producing fewer mistakes during instrumentation of narrow and curved canals1-6. it has been shown that instrumentation with stainless steel hand files yield a larger number of aberrations compared with ni-ti rotary files4-5,7-8. nevertheless, neither stainless steel files nor ni-ti instruments (either manual or engine-driven) are able to yield complete cleaning and shaping of canal walls9-11. the purpose of this study was evaluate the occurrence of displacement on the apical and cervical thirds of artificial curved root canals with 30° of curvature, comparing the use of stainless steel hand and oscillatory files with ni-ti hand, rotary and oscillatory files. material and methods seventy artificial curved root canals (tecnodon, uberaba, mg, brazil) were fabricated following the methodology proposed by dummer et al.12 with gradual curvatures of about 30º, measured according to the schneider’s method13. the working length (wl) was determined 1 mm short of the apical portion of the artificial root canals using size 10 k-files (union broach, montgomeryville, pa, usa). three reference points were made in the acrylic blocks to allow further precise superimposition of the images obtained before and after preparation of the simulated root canals. the blocks were positioned always in the same direction and photographed with a nikon f-2 camera (nikon, tokyo, japan) using standard object/film distance. in order to quantify the possible distortions produced by instrumentation, two measured sections were put close to the resin blocks. after preparation, the blocks were photographed again, maintaining the initial position and the previously established object/film distance. after digitization of the photographs with a flatbed scanner genius hr5 scsi (kye dcom systems, china) images were superimposed using adobe photoshop 6.0 image-editing software and image tool 3.0 image-analysis software for possible provoked changes. the 70 canal-resin blocks were randomly assigned to 7 groups (n=10) and instrumented by a single operator. in all groups, the straight portion of the canals was prepared with size 1 to 3 gates-glidden drills (union broach, montgomeryville, pennsylvania usa), according to a crown-down instrumentation technique, and root canal preparation was complemented using sizes 15 to 40 files at the wl. in group 1, the canals were manually prepared with flex-r stainless steel files (union broach) using balanced force. in group 2, the canals were manually prepared with onix-r ni-ti files (union broach) as in group 1. in group 3, the simulated canals were prepared with flex-r stainless steel files activated by endo-gripper oscillatory system (union broach). in group 4, the canals were prepared with onix-r ni-ti files (union broach) activated by endo-gripper oscillatory system. in group 5, the canals were prepared with pow-r niti files (union broach) activated by anthogyr (micromega, france) rotary pneumatic engine using a handpiece with 1:64 reduction. in group 6, the canals were prepared with pow-r ni-ti files activated by endo-plus motor (vk driller, brazil) with controlled speed of 250 rpm. in group 7, the canals were prepared with flex-r files activated by endo-plus motor and endo-gripper handpiece allowing constant oscillation of 250 rpm. at each change of instrument, the canals were copiously irrigated with 2 ml of distilled water together with bi-distilled glycerin (farmax, são paulo, sp, brazil) to lubricate the canals and facilitate instrumentation. the blocks with root canals were fixed on a mini lathe (western, l/c,t/t, china china china china china) to facilitate instrumentation. the superimposed images were magnified 4 times and evaluated using image tool 3.0 software, which allows measuring distances, angles and areas in digitized images. initially, it was calibrated in millimeters as unit of measure having as reference to calibration the measured sections placed close to the blocks. then, the software calculated by means of a drawning, the amount of material removed in the curved portion of the simulated canals at 2 mm level outside (figure 1) and 11 mm inside. the normality lilliefors was applied to the two levels analyzed and it showed normal curve which allowed using one-way anova and tukey’s post-hoc test. the significance level was set at 5%. fig. 1. superimposed preand post-instrumentation images at 2 mm level. results results are shown in tables 1 and 2. there were statistically significant differences (p<0.05) at 2 mm level between ni-ti and steel stainless groups (table 1). on the other level, at 11 mm, statistically significant differences (p<0.05) were observed between groups 5 and 6 (rotary systems) when compared with the remaining groups (table 2). in group 1, it was observed loss of the wl and blockage, mainly after the use of size 25 file. difficulties were found on moving from size 30 to 35 file and from size 35 to 40 file. there was zipping and elbow formation. a size 35 file fractured during instrumentation. apical and cervical displacement produced by hand and engine-driven stainless steel and nickel-titanium instruments in simulated curved root canal braz j oral sci. 10(2):136-139 group n mean ± sd g1 10 0.3790 ± 0.0296a g2 10 0.2170 ± 0.0427b g3 10 0.2960 ± 0.0450a g4 10 0.1522 ± 0.0757b g5 10 0.1260 ± 0.0425b g6 10 0.1670 ± 0.0585b g7 10 0.2850 ± 0.0774a table 1 table 1 table 1 table 1 table 1 amount of material emoved at 2 mm level (measured from outside). statistically significant differences are expressed by different letters (p<0.05). group n mean ± sd g1 10 0.4150 ± 0.0838a g2 10 0.4220 ± 0.0961a g3 10 0.4350 ± 0.0597a g4 10 0.3880 ± 0.0639a g5 10 0.2520 ± 0.0666b g6 10 0.2390 ± 0.0545b g7 10 0.3530 ± 0.0618a table 2 -table 2 -table 2 -table 2 -table 2 amount of material emoved at 11 mm (measured from inside) statistical differences expressed by different letters (p<0.05) in group 2, there was difficulty in moving from size 20 to 25 file and from size 30 to 35 file. there was loss of wl but no instrument breakage was observed. in group 3, files up to size 25 were flexible enough to accompany the curvature without resistance. however, from size 30 on there was loss of the wl and need for using greater force with the handpiece. in spite of zipping and elbow formation, there was neither fracture nor distortion of any instrument. on the simulated canals in group 4, the file had to be first prepared manually, until reaching the wl and then the mechanical instrumentation system could be used. sizes 35 and 40 files did not reach the wl. there was neither fracture nor distortion of any instrument. the files in group 5 had no difficulty in penetrating the canals. the wl was reached without finding resistance. there was not blockage and a size 25 file fractured. likewise, the canals in group 6 were enlarged up to size 40 files without any difficulty. there was no instrument breakage and only a size 35 file presented tip distortion. no instrument fracturing or distortion was observed in group 7. it was noticed transport starting from size 30 file with loss of the wl, blockage and zipping and elbow formation. these findings are similar to those of group 3, which used the same hand piece but without speed control. discussion several methodologies have been used to evaluate root canal preparation7-8,10-11. simulated canals allow standardization of length, radius, diameter and curvature angle, while natural teeth present variations of canal shape and diameter. dentin hardness may not be identical to that of the resin used for preparation of the blocks containing the artificial canals7-8,12. in this study, both hand and engine-driven (oscillatory or rotary) stainless steel and ni-ti files had similar design configuration not to interfere with the results. the use of pow-r is justified because the study evaluated the most effective preparation techniques and not the file design. even though they are not used anymore, the pow-r files are similar to the conventional (stainless steel) instruments used in hand instrumentation. tan and messer14 indicate files with small taper to finish the preparation in the work length, to improve the intracanal cleaning and filling. it was observed that the ni-ti instruments were able to shape the simulated curved canals more appropriately, as observed in previous studies7-8,11. it was also verified that rotary ni-ti instrumentation using pneumatic or electric engines was more effective than manual instrumentation with stainless steel and ni-ti files. these findings are consistent with those of previous studies1,11. however, rasquin et al.15 found better shaping and cleaning ability for aet (endoeze®) files (oscillatory) compared with race rotary system (fkg® dentaire) in the cervical third. the authors did not found significant differences in the apical part. in all groups, it was observed that root canal preparation selected areas on the curved portion of the canal; in the apical region of the curvature the selected areas occurred on the external side while in the cervical region of the curvature they occurred in the internal side. similar outcomes have been reported2,7,16. canals prepared with ni-ti instruments driven by rotary or oscillatory systems showed more centered preparations17. there was greater impaction of resin and, consequently, loss of the wl for the stainless steel instruments used either manually or activated by the endo-gripper oscillatory motion. similar observations with less extrusion and impaction using of ni-ti files coupled to rotary systems have been reported18-19. preparation of curved root canals up to size 40 instrument confer a better cleaning and facilitate filling1,20. therefore, all root canals were prepared up to size 40 files at the wl, aiming to comply with the basic requirements for good root canal shaping. however in the present results, the stainless steel instruments should be used up to file 20, followed by rotary ni-ti instruments. the investigation analyzed the hypothesis that the oscillatory instrumentation with controlled speed and stainless steel files would be an alternative to decrease deformations during preparation of curved and strait root canals. the results showed that no significant difference was found for the tested hypothesis, resulting in similar deformation and blockage (table 1). the crown-down technique combined with apical enlargement determine apical foramen diameter, as well as prevent aberrations of curved canals1,18,21-22. as the instrumentation technique used in all groups was the same, it could be assumed that the type of metal and movement of instruments are crucial to keep the artificial root canal curvature. further studies should be carried out on natural teeth to assess root canal cleaning because, as previously mentioned, simulated 138apical and cervical displacement produced by hand and engine-driven stainless steel and nickel-titanium instruments in simulated curved root canal braz j oral sci. 10(2):136-139 139 root canal methodology analyzes better other requirements. in conclusion, the most centered preparations were those belonging to groups prepared with ni-ti (groups 5 and 6) files and rotary systems, at both levels analyzed. mechanical root canal preparation with ni-ti instruments yielded the best results. references 1. pécora jd, capelli a. shock of paradigms on the instrumentation of curved root canals. braz dent j. 2006; 17: 3-5. 2. gergi r, rjeily ja, sader j, naaman a. comparison of canal transportation and centering ability of twisted files, pathfile-protaper system, and stainless stell hand k-files by using computed tomography. j endod. 2010; 36: 904-7. 3. sadeghi s. shaping ability of niti rotary versus stainless stell hand instruments in simulated curved canals. med oral patol oral cir bucal. 2010; 15: 505-8. 4. thompson sa. an overview of nickel-titanium alloys used in dentistry. int endod j. 2000; 33: 297-10. 5. vanni jr, albuquerque ds, reiss c, barato filho f, limongi o, della bona a. apical displacement produced by rotary nickel-titanium instruments and stainless steel files. j appl oral sci. 2004; 12: 51-5. 6. walia h, brantley wa, gerstein h. an initial investigation of the bending and torsional properties of a nitinol root canal files. j endod. 1988: 14: 346-51. 7. schäfer e, lohmann d. efficiency of rotary nickel-titanium flexmaster instruments compared with stainless steel hand k-flexofiles – part 1. shaping ability in simulated curved canals. int endod j. 2002; 35: 505-13. 8. schäfer e, florek h. efficiency of rotary nickel-titanium k3 instruments compared with stainless steel hand k-flexofiles. part 1. shaping ability in simulated curved canals. int endod j. 2003; 36: 199-207. 9. baumann ma. nickel-titanium: options and challenges. dent clin north am. 2004; 48: 55-67. 10. chen jl, messer hh. a comparison of stainless steel hand and rotary nickel-titanium instrumentation using a silicone impression technique. aust dent j. 2002; 47: 12-20. 11. schirrmeister jf, strohl c, altenburger mj, wrbas k-t, hellwig e. shaping ability and safety of five different rotary nickel-titanium instruments compared with stainless steel hand instrumentation in simulated curved root canals. oral surg oral med oral pathol oral radiol endod. 2006; 101: 807-13. 12. dummer pmh, alodeh mha, al-omari mao. a method for the construction of simulated root canals in clear resin blocks. int endod j. 1991; 24: 63-6. 13. schneider sw. a comparison of canal preparation in straight and curved root canals. oral surg oral med oral pathol. 1971; 32: 271-5. 14. tan bt, messer hh. the quality of apicaql canal preparation using hand and rotary instruments with specific criteria for enlargement based on initial apical file size. j endod. 2002; 28: 658-64. 15. rasquin lc, carvalho fb, lima rkp. in vitro evaluation of root canal preparation using oscillatory and rotary systems in flattened root canals. j appl oral sci. 2007; 15: 65-9. 16. miranzi bas, miranzi mas, costa wf, rezende lc. estudo comparativo in vitro da instrumentação de canais artificiais mediante o emprego das limas flex-r, valendo-se de dois tamanhos de preparos apicais. rev usf. 1999; 17: 43-9. 17. setzer fc, kwon tk, karabucak b. comparison of apical transportation between two rotary files systems and two hybrid rotary instrumentation sequences. j endod. 2010; 36: 1226-9. 18. al-omari mao, dummer pmh. canal blockage and debris extrusion with eight preparation techniques. j endod. 1995; 21: 154-8. 19. mckendry dj. comparison of balanced forces, endosonic, and step-back filing instrumentation techniques: quantification of extruded apical debris. j endod. 1990; 16: 24-7. 20. card sj, sigurdsson a, orstavik d, trope m. the effectiveness of increased apical enlargement in reducing intracanal bacteria. j endod. 2002; 28: 779-83. 21. barroso jm, guerisoli dmz, capelli a., saquy pc, pécora jd. influence of cervical preflaring on determination of apical file size in maxillary premolars: sem analysis. braz dent j. 2005; 16: 1-9. 22. vanni jr, santos r, limongi o, guerisoli dmz, capelli a, pécora jd. influence of cervical preflaring on determination of apical file size in maxillary molars: sem analysis. braz dent j. 2005; 16: 181-6. apical and cervical displacement produced by hand and engine-driven stainless steel and nickel-titanium instruments in simulated curved root canal oral sciences n3 literature review braz j oral sci. april/june 2010 volume 9, number 2 braz j oral sci. 9(2):70-76 palatally impacted canine: diagnosis and treatment options marcelo aires vilarinho1, ana de lourdes sá de lira2 1dds, specialization student, department of orthodontics, dental school, federal university of piauí, brazil 2dds, ms, professor, department of orthodontics and orthodontics specialization program, dental school, federal university of piauí, brazil correspondence to: ana de lourdes sá de lira rua motorista gregório, 2530 planalto ininga cep: 64052-140 teresinapiauí e-mail: anadelourdessl@hotmail.com received for publication: march 08, 2010 accepted: june 16, 2010 abstract canines contribute significantly to the esthetic and chewing functions. orthodontists should diagnose canine ectopic eruption early, trying to prevent retention of these teeth. its multifactorial etiology involves general and local factors and the correct diagnosis depends on clinic, radiographic and/ or tomographic exams. several therapeutic procedures depend on factors such as relationship between canine and adjacent structures, possibility of orthodontic movement and patient age. orthodontic and surgical treatment with canine traction is very much effective, with time of treatment being shorter than in patients under the age of 25. keywords: impacted canine, diagnosis, treatment. introduction dental impaction is a condition in which tooth cannot erupt because it may be retained by either adjacent bone or tooth. following the third molars, upper canines are among the most frequently impacted teeth, with prevalence ranging from 1% to 3%. therefore, impacted canines are defined as being those teeth not erupted within 6 months of their complete root formation or when they are not present in the arch during the eruption phase1. impacted upper canines are found in approximately 2.0% of the general population, occurring more than twice as frequently in women (1.17%) as in men (0.51)2-4 and moving more palatally (60-80%) than unilaterally (75-95%)2. impacted teeth are particularly more difficult to treat in adults. success rate among patients older than 30 years was 41%, whereas patients aged 20-30 years old achieved 100% success4. prognosis depends on the canine position in relation to adjacent teeth and its alveolar height. however, it should be considered the possibility that they cannot be orthodontically moved, thus requiring extraction, rehabilitation with prosthesis or implant, or space closing with orthodontic appliance5. previous planning based on reliable risk estimates, length of orthodontic treatment, and success probability can be useful in the decision-making process for patients. an adequate diagnosis should be supported by clinical and complementary examinations for evaluating the sites of impacted canines and their relationship with adjacent teeth and anatomical structures (nasal fossae and maxillary sinus)4. few studies have focused on traction period and related factors. it is supposed that such a period is shorter in younger than in older patients. this literature review aimed at addressing diagnostic means and therapeutic procedures with emphasis on traction duration of palatally impacted canine. 71 braz j oral sci. 9(2):70-76 this study was approved by the local research ethics committee under the protocol (caae number: 0028.0.045.00010). studies selected for literature review were retrieved from medline database (national library of medicine, usa, entrez pub med, www.ncbi.nim.nih.gov ), ovid, cochrane (www.cochrane.org ), lilacs, web of science, google scholar beta, embase, extenza, evidence-based medicine, and bbo (brazilian dentistry library), within the 1992-2009 period. only clinical case reports and controlled human clinical studies were addressed, including a book chapter on topics discussed in the present literature review. etiology upper canine has the longest period of development at the top portion of the canine fossae, being the most difficult eruption trajectory among all teeth. they usually emerge in the dental arch between the ages of 11 and 12 years old4, and due to their complex eruption trajectory they become more susceptible to factors that can interfere with such a process1. its impaction can be the result of sequelae from endocrine anomalies, fever, vitamin deficiency, and irradiation 5. however, local etiologic factors may also be involved, such as, discrepancy between dental arch length and tooth size, prolonged retention or early loss of primary canine, abnormal position of the tooth germ, ankylosis, cystic, neoplastic formation, root dilaceration, presence of alveolar fissure and traumatic factors1. studies have shown that there is no significant relationship between lack of arch space and palatally impacted canine6. in general, palatal displacement occurs regardless of dental arch space. other factors may be involved such as excessive growth in the base of maxillary bone, agenesis or cone-shaped lateral incisors, thus making root orientation difficult or even impossible during the initial eruption phases7. becker and chaushu8 have found that delayed dental development was present in at least half of the patients with palatally impacted canines, but with no impairment of eruption orientation. they also suggested that palatal impaction may be preponderantly related to genetic factors or familial inheritance pattern1,6-8. diagnosis the diagnosis of palatally impacted canine consists in evaluating its relationship with the adjacent teeth. it is necessary an association between clinical (i.e. inspection and palpation) and radiographic examinations. it is important to correlate the patient’s age to a chronological sequence of tooth eruptions and also investigate about the family history of agenesis or prolonged retention of primary teeth3. some events may indicate the presence of impaction during the clinical examination, such as delayed eruption of one or more canines following the age of 14 years, insufficient amount of arch space for permanent canine, and prolonged retention of primary teeth associated with palatal elevation of soft tissue (figure 1)6-7. fig. 1: occlusal photograph with impacted canines. in general, the palatally impacted permanent canine exerts a buccal pressure on the lateral incisor root, displacing the crown palatally. there is also a horizontal orientation in close relationship to nasal fossa and their crowns, thus increasing the contact with central or lateral incisors. palatal depth and curvature provide a false radiographic image of these teeth, showing them closer to the bone surface9. mobility of the primary canine can indicate normal root resorption by the permanent successor. however, mobility of the permanent lateral incisor can be the result of root resorption caused by the pressure exerted by the impacted canine10. annual clinical examination of the alveolar process by palpating the alveolar process where the permanent canine will erupt is necessary starting from the age of 8 years old10. palpation is possible in 70% of the cases3. absence of elevation of gingival mucosa at early age should not be indicative of impaction. in case of deviation of the normal eruption patterns at the ages from 10 to 12 years old, clinical evaluation should be associated with radiographic examinations10. in most cases, periapical radiographs are sufficient to evaluate the canine position, based on the clark’s rule (mesial or distal displacement of the x-ray cone) (figure 2)11-12. occlusal films also help to determine whether canine is fig. 2: clark’s rule to evaluate canine position. palatally impacted canine: diagnosis and treatment options 72 braz j oral sci. 9(2):70-76 buccally or palatally located as well as to identify the transverse position of its long axis (figure 3)11. panoramic radiography is used to determine the position of non-erupted canines in two spatial planes, besides offering a satisfactory indication of canine height, its relationship with median sagittal plane, and information on its inclination. however, no diagnostic indication of buccal-palatal position is given (figure 4)3. fig. 3: occlusal film to identify canine position. fig. 4: panoramic radiograph with bilateral impacted canines. when the cusp tip of the canines is mesially positioned, along the axis of the erupted lateral incisor, palatal retention of these teeth occurs. if the cusp tip overlaps in the middistal root of the lateral incisor, palatal retention can occur as well. however, when the cusp tip is distally positioned in relation to lateral incisor (i.e., with no overlap), the great majority of canines will erupt normally in the dental arch1. lateral and frontal cephalometric teleradiographs (figure 5) help to identify the long canine axis in relation to the palatal plane as well as incisors in the antero-posterior sense and the vertical inclination of its crown. in addition, facial structures surrounding the canine, such as maxillary sinus and floor of the nasal cavity, are also related11. computed tomography (ct) not only accurately determines the position of impacted canine and its relationship with adjacent structures in the three planes, but also defines the real extension of possible resorption, fig. 5: lateral cephalometric teleradiograph with impacted canines. occurrence of ankylosis, and dilacerations12. this is possible because ct eliminates any superposition of other structures obscuring the image visualized in traditional radiography (figures 6, 7)13. this method is widely used to identify its exact position, mainly when root resorption of lateral canine is suspected14. fig. 6: maxillary computed tomography image. prevention of dental impaction selective extractions of primary canines have been suggested for normalization of its eruption trajectory13. ericsson and kurol apud bishara 14 have suggested that removal of primary canine before the age of 11 years old can normalize the position of ectopic permanent canines in 91% of the cases if the crown is located distally to midline of lateral incisor. however, the success rate will be palatally impacted canine: diagnosis and treatment options 73 braz j oral sci. 9(2):70-76 fig. 7: maxillary computed tomography image on transversal slice. significantly reduced if canine’s crown is mesial to midline of the lateral incisor, and angulation of the long canine axis exceeds 31o in relation to the mid-sagittal plane12. this type of preventive intervention, however, is contraindicated in cases of very horizontal eruption trajectory, apical movement of permanent canine or evidence of root resorption of permanent incisor3. risks of dental traction the possibility of palatal traction depends on the position of the retained tooth in relation to adjacent teeth, angulation of its long axis, height of alveolar ridge, presence of ankylosis or dilaceration, presence of enough space arch, and correlation between chronological age and dental eruption sequence13. orthodontic traction involves risks such as ankylosis, discoloration, loss of vitality, and root resorption of retained tooth and adjacent teeth, gingival regression, and loss of keratinized mucosa2. therapeutic procedures for palatally impacted canines several treatment options can be considered such as: radiographic follow-up of the impacted tooth should be performed as any pathological change may result; canine auto-transplantation; extraction of impacted canine and movement of premolar towards the space left; extraction of canine and osteotomy for moving posterior segment in order to close the residual space; reestablishment of occlusion with prosthesis, and finally, the most recommended option, surgical exposure with orthodontic treatment for moving the tooth to occlusal line14-15. before surgical exposure, orthodontic treatment should be performed to obtain enough space in the dental arch for accommodation of permanent tooth. also, teeth should be leveled and aligned until a rectangular stainless steel wire can be placed in order to avoid the adverse effects of traction, such as intrusion of adjacent teeth, constriction of dental arch, or change on occlusal plane, all impairing the movement control4. there are various surgical methods, but the most recommended ones are the traditional surgical exposure allowing natural eruption and the surgical exposure with attachment of an orthodontic accessory on to the impacted canine’s crown for traction1,16. in those cases of surgical exposure with spontaneous eruption, incision in the tissue covering the impacted tooth should be made in such a way that part of the crown remains exposed. canine will erupt spontaneously towards where enough space exists, a phenomenon attributed to the force of periodontal tissues which will guide the exposed crown towards the area where the tissues were sectioned3. levis apud bishara14 proposed other technique, which is performed in two steps. firstly, the canine is surgically exposed and then protected with surgical cement. after healing and cement removal, the orthodontic accessory is bonded to the non-erupted tooth. the crown bone of the impacted tooth has to be minimally removed, allowing the tip of the crown to be exposed. otherwise, the tooth will not be able to reabsorb the bone efficiently during crown movement, since crown enamel will be in contact with bone and therefore no cells will exist within the enamel to reabsorb it15. palatally ectopic canines which have been surgically exposed and aligned orthodontically exhibit clinically insignificant reduction in periodontal support compared to contra-lateral canine. the current literature shows no study proving that closed-eruption surgical technique has some advantage on the open-eruption technique at long-term regarding periodontal health. in fact, there is scientific evidence that the amount of bone removal and type of orthodontic movement needed to align the canines can be more important than the variables influencing the periodontal health17. methods of attachment for many years the “loop technique” was used, which consisted in placing a ligature wire around the neck of the impacted tooth during surgical exposure. this procedure required very extensive bone removal, which resulted in many cases of ankylosis and external tooth resorption caused by the mechanical trauma exerted on the periodontal cells2,18. the method of transfixation was widely used despite using incisal perforations of the crowns of the teeth to be submitted to traction by ligature wires. due to the possibility of pulp injuries and dental destruction during access procedures as well as to the difficulty in keeping the surgical site dry for bonding the orthodontic accessory, this method has no longer been recommended3,18. with the emergence of acid attack and adhesive systems, the technique of directly bonding the orthodontic accessory (bracket, button, mesh) to the retained tooth’s enamel is now being employed. osteotomy is enough only for exposing portion of the enamel for bonding procedure, thus allowing palatally impacted canine: diagnosis and treatment options 74 braz j oral sci. 9(2):70-76 determined local to be chosen according to the movement and direction desired by the orthodontist. as the more horizontal the impacted canine is, the more incisal the accessory should be bonded in order to promote its verticalisation1. ligature wire or elastomeric ligature may be attached around the orthodontic accessory before the flap is repositioned and sutured, thus exposing part of the wire or ligature for immediate traction3. ideal system of forces palatally impacted canines need to follow a buccal trajectory to obtain adequate positioning in the dental arch. application of traction force promotes, consequently, an intrusive force on and anterior inclination of the posterior segment, thus keeping the system in equilibrium. on the transverse plane, canine tends to erupt palatally, with posterior teeth shifting buccally. after eruption, the tooth should be orthodontically moved in the buccal direction so that it becomes aligned with other teeth in the arch. the force applied buccally to canine generates a lingual force to the molar, which in turn undergoes a mesio-palatal rotation. this explains, therefore, the use of a posterior rigid wire segment during traction in association with transpalatal bar (figures 8, 9) in order to avoid undesirable movements in the posterior segment of the dental arch11. bishara18 recommends that traction force should not be greater than 60 gf. fig. 8: lateral photograph of cantilever activation. fig. 9: occlusal photograph of cantilever activation. traction systems the “ballista spring” is a system in which the impacted tooth undergoes continuous traction force released by a spring made of 0.14", 0.16" or 0.18" round stainless wires. the tip of this spring being inserted into the molar tube should be attached to it by using a 0.25-mm ligature wire, thus avoiding rotation of the tube. the tip of the spring will be occlusally rigid and once placed next to the impacted canine, in association with the ligature wire, will exert a force linking the device to the tube. this traction system has some advantages such as application of continuous force as well as control of force magnitude and direction without requiring complete appliance assembly. in addition, this method not only reduces the final time for alignment and leveling of the impacted tooth, but also provides less traumatic surgical intervention on a conservative basis16. extraction of impacted canine extraction of canine should only be performed in the following situations: if impacted tooth is ankylosed and transplantation impossible; if internal or external root resorption exists; if root is severely dilacerated; if impaction is severe (e.g. canine situated between the roots of central and lateral incisors, which can affect the orthodontic movement of these teeth); if occlusion is acceptable, that is, first premolar in the canine position exhibits functional occlusion while other teeth are well aligned; if pathological changes occur (e.g. cystic formation, infection); or if the patient desires no orthodontic treatment14. auto-transplantation of impacted canine auto-transplantation is only recommended when intervening measures are inappropriate or failed, or when impaction is severe enough to compromise the orthodontic alignment. a favorable prognosis is possible if the canine is atraumatically removed, ankylosis is absent, and dental arch space is enough17. palatally impacted canine: diagnosis and treatment options 75 braz j oral sci. 9(2):70-76 traction duration for palatally impacted canine duration of the traction for malocclusion involving impacted canine is greater than that involving erupted permanent teeth. this occurs as a result of the initial need for space to accommodate the impacted tooth in the dental arch. the other teeth in the same dental arch and sometimes the lower ones should be well anchored to resist the forces applied during traction10. the treatment difficulty and probability of complications, which interfere with duration of the traction, are related to age, occlusal movement, apical movement, angulation and mesiodistal location of the impacted canine’s crown, complex relationship between canine’s crown and midline, close contact between canine’s incisal facet and adjacent lateral incisor, and presence of transposed lateral incisor or first premolar2. zuccati et al.2 have observed that if the impacted canine cusp tip is located mesially to the root of adjacent lateral incisor, the mean number of visits will be 10 times greater than that if the cusp tip is located distally to or on the root of lateral incisor. patients aged 25 years or older need, on average, 30 visits or more compared to younger patients. discussion although canines are one of the last teeth to erupt, the dentists are not accustomed to prevent ectopic eruption from occurring. on the other hand, early diagnosis and preventive intervention are possible, thus reducing the need for more complex treatment of permanent dentition. removal of primary canines can influence positively the eruption of the succeeding permanent tooth that is palatally displaced12. early diagnosis can minimize the problems caused by impaction, such as root resorption of canines and lateral incisors, ankylosis or infections resulted from impactation3. orthodontic traction in permanent dentition is aimed at positioning the canines in the dental arch without causing periodontal damage, since they plays an important aesthetic and functional roles in the development of a normal occlusion1. traction of teeth used to be a great challenge for orthodontists, mainly in those cases involving palatally impacted permanent canines, because such a treatment was frequently unsuccessful due to the surgical techniques employed at the time. today, with the advances in surgery, dental traction is performed with great odds of success17. surgical exposure for traction is the most commonly used treatment today, but some complications can occur such as loss of pulp sensibility, root resorptions, bone loss, and gingival recession10. in periodontal aspects, the esthetics, the establishment of normal function and periodontal health at the end of treatment are fundamental, because the patients, mainly adults, have the expectation regarding the probability of successful of treatment13. bishara14 has emphasized that two methods are largely used: surgical exposure for spontaneous eruption and surgical exposure for attachment of auxiliary accessory for application fig.10: osteotomy and bonding the orthodontic accessory. of orthodontic forces (figure 10). impacted canines with chances for spontaneous eruption within 6 months can follow a more anterior and palatal trajectory, thus requiring further orthodontic movement. however, if orthodontic traction is applied soon after the surgical exposure, the canine will be directly brought into its correct position and time will be consequently saved. one can argue that the time elapsed between exposure and application of orthodontic traction is the most important factor 16. both soft and hard tissues, which form a barrier impeding the natural course of eruption, are removed during the surgical procedure. it is important to avoid excessive and unnecessary bone removal as well as to preserve the gingiva as much as possible4. fig. 11: canine (left side) after open-eruption method the closed eruption method preserves the periodontal tissues (figure 11). in this way, eruption occurs more naturally despite the lack of scientific evidence confirming the advantage of this surgical technique over the open-eruption method in terms of periodontal health at long-term16. although a retrospective comparative study have found no difference in treatment duration after surgical exposure using either open-eruption or closed-eruption method 2. langlade apud zuccali et al.2 have reported that the maximum age for traction of an impacted tooth is 45 years old, provided that only small apical movements are to be palatally impacted canine: diagnosis and treatment options 76 braz j oral sci. 9(2):70-76 performed, being the treatment duration significantly longer in patients aged 25 years or older. it is thought that residual eruptive potential and good bone density in younger patients might have made traction of impacted canines easier. location of the impacted canine also plays an important role in determining both treatment duration and probability of failures and complications. if canine is impacted more than 14 mm from the occlusal plane, the mean duration of the treatment is 31.1 months18. the distance between canine tip and occlusal plane, long axis inclination of the lateral incisor, and mesiodistal location are the most strongly correlated variables with the treatment duration2. there is a significant difference in the treatment duration between cases of unilateral and bilateral impaction. the mean treatment duration for those cases of bilateral palatally impacted canines is more than the mean value for unilateral cases because, in general, bilaterally impacted canines are in more unfavorable positions, with less space than the unilateral cases18. the traction mechanics consist of verticalization, palatal positioning, and extrusion, with posterior teeth, premolars, and first molars serving as anchorage, whereas tooth leveling and space recovery are obtained by using the conventional orthodontic mechanics. this allows traction to be controlled without risk of root resorption of adjacent teeth or loss of rigidity in the support tissues. a statistically determined system of forces enables the canine movement to be controlled with smaller anchorage loss and less side effects of adjacent teeth. by using the segmented arch technique, with insertion of a beta-titanium cantilever into the molar tube to the impacted canine (figures 8, 9), the intrusive force occurring on lateral incisors would be null and the canine traction would be more effective. in addition, the spring could be further activated and orthodontic force slightly disseminated for a longer period of time16. in conclusions: 1. initial diagnosis is carried out by inspection, palpation, and radiographic examination. in the majority of cases, periapical radiography (with clark’s rule) is sufficient to determine whether canine is palatally impacted or not. computed tomography is a more precise diagnostic method as the relationship between canine and adjacent structures is determined three-dimensionally; 2. treatment of palatally impacted canine depends mainly on its location and patient’s age. in the cases of early diagnosis, the best option is ortho-surgical treatment for later traction; 3. the traction mechanics consist on verticalization, palatal positioning, and extrusion, with posterior teeth, pre-molars, and first molars serving as anchorage, has been controlled without risk of root resorption of adjacent teeth or loss of rigidity in the support tissues. references 1. mesotten k, naert i, van steenberghe d, willems g. bilaterally impacted maxillary canines and multiple missing teeth: a challenging adult case. orthod craniofacial res. 2005; 8: 29-40. 2. cati g, ghobadlu j, nieri m, clauser c. factors associated with the duration of forced eruption of impacted maxillary canines: a retrospective study. am j orthod dentofacial orthop. 2006; 130: 349-56. 3. ngan p, hornbrook r, weaver b. early timely management of ectopically erupting maxillary canines. semin orthod. 2005; 11: 152-63. 4. suri s, utreja a, rattan v. orthodontic treatment of bilaterally impacted maxillary canines in an adult. am. j orthod dentofacial orthop. 2002; 429: 429-37. 5. brin i, solomon y, zilberman y. trauma as a possible etiologic factor in maxillary canine impaction. am j orthod dentofacial orthop. 1993; 104:132-7. 6. peck s, peck l, kataja m. the palatally displaced canine as a dental anormaly of genetic origin. angle orthod. 1994; 64: 249-56. 7. jacobs sg. palatally impacted canines: a etiology of impaction and the scope for interception: report of cases outside the guidelines for interception. aust dent j. 1994; 39: 206-11. 8. becker a, chaushu s. dental age in maxillary canine ectopia. am j orthod dentofacial orthop. 2000; 117:657-62. 9. jacobs sg. localization of the unerupted maxillary canine: how to and when to. am j orthod dentofacial orthop. 1999; 115: 314-22. 10. becker a, chaushu s. success rate and duration of orthodontic treatment for adult patients with palatally impacted maxillary canines am j orthod dentofacial orthop. 2003; 124: 509-14. 11. mason c, papadakou p, roberts gj. the radiographic localization of impacted maxillary canines. eur j orthod. 2001; 23:.25-34. 12. shapira y, kuftinec m. early diagnosis and interception of potential maxillary canine impaction. j am dent assoc. 1998; 129: 1450-4. 13. bishara se. clinical management impacted of maxillary canines. semin orthod. 1998; 4: 87-98. 14. bishara se. impacted maxillary canines: a review. am j orthod dentofacial orthop. 1992; 101: 159-71. 15. kokich vg. surgical and orthodontic management of impacted maxillary canines. am j orthod dentofacial orthop. 2004; 126: 278-83. 16. burden dj, mullally bh, robinson sn. palatally ectopic canines: closed eruption versus open eruption. am j orthod dentofacial orthop. 1999; 115: 634-9. 17. mcsherry pf. the ectopic maxillary canine: a review. br j orthod. 1998; 25: 209-16. 18. stewart ja, heo g, glover ke, williamson pc, lam wn, major pw. factors that relate to treatment duration for patients with palatally impacted maxillary canines. am j orthod dentofacial orthop. 2001; 119: 216-25. palatally impacted canine: diagnosis and treatment options xix aboprev meeting/2015 braz j oral sci. 14(1) 85-99 85 xix aboprev meeting/2015 braz j oral sci. 14(1) 85-99 86 001 relationship between degree of fluorosis and enamel hypomineralization marín, l.m.*; castellanos, j.e.; tenuta, l.m.a.; martignon, s.; cury, j.a. background: the clinical manifestations seen in fluorotic teeth have been associated with the histological changes provoked in the subsurface hypomineralyzed areas, but the degree of hypomineralization has not been measured and associated with the level of fluorosis. aim: to determine enamel subsurface hypomineralization in teeth with different degrees of fluorosis. methods: forty-nine third unerupted human teeth were visually classified according to the thylstrup & fejerskov (tf) index in sound (tf0) and fluorosed (tf1 to 4). teeth were cut transversely through the middle third of the crown and one enamel block (4x2x4 mm) was obtained. enamel blocks were embedded in acrylic resin, flattened and polished. cross-sectional microhardness (csmh) was used as indicator of hypomineralization because there is a high correlation between csmh and percentage of mineral in enamel. three rows of 20 indentations each and above this. the set of data were curved-fitted to graph the csmh profile. hypomineralization area (δshypomineralization) was determined from the difference between the area under the curve of sound and hypomineralized enamel. the data obtained were grouped as tf0, tf1-2 and tf3-4 for the statistical analysis: anova and tukey test (p<0.05). results: the khn values through-out enamel were lower with increasing fluorosis severity. the fluorosis severity showed an effect on δshypomineralization, being the data obtained from tf3-4 statistically higher than the other groups (p<0.05). conclusions: the severity of fluorosis is directly associated with enamel subsurface hypomineralization found in fluorotic teeth. 002 evaluation the role of dental schools in the school environment: a qualiquantitative study simpriano, d.c.a.b.*; kubo, f.m.m.; mialhe, f.b. background: the health promotion should be multidisciplinary and intersectoral. teachers of elementary school, when well grounded and empowered, can become multipliers of health workers. aim: this study investigated using collective subject discourse methods (dsc), such as dental schools could help elementary school teachers working with students the oral health issue across the board. methods: the sample consisted of 80 teachers from first to fourth grade of elementary school in the city of indaiatuba-sp. the responses were analyzed by qualitative research technique of dsc. results: presented to the quantiqualitativos results. the synthesis of the central ideas were guiding students through lectures, videos, jokes, games and other activities (n 32), train teachers on the subject oral health (n 11), guide parents and / or community about the importance to adopt healthy habits regarding oral health (n 28), the need of oral / dental evaluation in schools and, if necessary, make referrals to health agencies (n 9), provide (more) apprenticeship positions for dental students can act in schools (n 8), develop teaching materials on the subject oral health (n 23), advise on the importance of oral health lectures, events and activities (without further elaboration) (n 17) , response discarded for not answering the question (n 5). conclusions: for the participating teachers, the best way colleges help in the development of oral health theme would be guiding students, parents and the community about the importance of oral health, developing educational materials and empowering teachers on the subject. 003 the dental caries experience of inhabitants of manaus and são paulo related to the fluoridation in the public water supply system olivati, f.n.*; meirelles, m.p.m.; marques, t.cn.; franco, d.h.; sousa, m.l.r. background: the fluoridation in the public water supply systems part of the health improvement proposal, being considered an effective and consolidated measure as well as having low costs for the prevention and control of dental caries. aim: this study aimed at comparing the dental caries experience of two cities in brazil: são paulo, which adds fluoride to its public water supply system and manaus, which does not add fluoride to its public water supply system. methods: this study was based on the data collected by sb brasil 2010 project and those data were evaluated and compared with dtmf, dmft and sic index. among the 2.176 individuals examined, there were children, adolescents, adults and elders. the dental examinations followed the methods proposed by who. results: were showed that the dtmf, dmft as well as sic were higher in manaus compared to são paulo, at the ages of 5 and 12 years old (p<0.001). the percentage of decayed and missed teeth was higher and the restored ones was lower at the ages of 15 and 19 years old and the percentage of free decay among adults and elders in those cities was not significant. conclusions: from the results, it was identified that são paulo showed a better oral health condition in children and adolescents, while there was no difference among adults and elders, compared to manaus, demonstrating that part of the population with access to fluoridated water benefitted from this method. 004 impact of oral conditions on oral health-related quality of life of children liver transplant candidates – a pilot study vidigal, e.a.*; abanto, j.; alves, f.a.; bönecker, m. background: preschool children with end-stage liver disease tend to suffer of dental problems, which can negatively affect their oral health-related quality of life (ohrqol). aim: the purpose of this study was to assess the prevalence of oral diseases and their impact on ohrqol in preschool children candidates for liver transplant. methods: seventeen preschool children aged 12 to 36 months with biliary liver atresia who were in program to liver transplant at the ac camargo cancer center were examined. presence of gingivitis, dental plaque (ohi-s), dental caries (dmf-t), dental hypoplasia (ded-index) and hyperbilirrubinemic stain of teeth were evaluated by one trained and calibrated examiner prior to liver transplantation. parents answered the brazilian version of the early childhood oral health impact scale (b-ecohis). descriptive analyses were performed. results: hyperbilirrubinemic stain (70,6%), presence of dental plaque (47,1%) and dental caries (41,2%) showed the higher rates. the mean (standard deviation) b-ecohis score was 6.52(4.74). parent’s distress domain showed the highest mean score (3.41(2.26)). conclusions: hyperbilirrubinemic stain was the most frequent oral problem in preschool children liver transplant candidate and may be responsible for a negative impact on the ohrqol. 005 oral health in a programmed children’s initiative: indicators and objectives of a primary health care service faustino-silva, d.d.*; schwendler, a.; famer rocha, c.m.f. background: early childhood is the ideal time to introduce good habits and begin an educational/preventive oral health program. given the importance of prioritizing educational initiatives aimed at this population within primary health care, the community health service of the grupo hospitalar conceição (ssc-ghc) implemented a programmed oral health initiative for children under the age of 4 years. aim: assess compliance with goals and indicator distribution for the children’s programmed oral health initiative at health care units (us) of the sscghc. methods: this is a cross-sectional analytical study using a quantitative approach to analyze the coverage of dental visits in the service’s 12 health care units. a total of 660 children born in 2010 were included in the study, based on information collected from the institution’s information system. results: in regard to coverage of dental visits, the health care units failed to meet the objectives established by the service. most of the children studied (41%) had their first dental visit in their first year of life. with respect to the total number of visits, 22% had never seen a dentist and 8% had completed all four expected consultations. there was a positive relationship between the ratio of the total population and 0 to 4-yearolds enrolled from the area with the number of oral health team (esb) members and coverage in the first year of life for each us (p0.001 and 00.008, respectively). conclusions: although few children are properly monitored in terms of the objectives established, coverage percentages were higher than those found in the literature, indicating that programmatic and surveillance initiatives can be effective in children’s oral health care. 006 simplifying the aesthetic and functional full-mouth rehabilitation in child with structurally abnormal teeth ferreira, f.r.*; saito, w.a.; reyes, a.; alvarez-vidigal, e.; kohara, e.k.; wanderley, m.t.; mendes, f.m.; braga, m.m. dental anomalies, as imperfect dentinogenesis and other teratogenic defects characterized by intense dental discolouration and possible association with worsening factors, may lead to severe aesthetic, functional and psychosocial impact on children’s lives. due to that, measures to minimize these effects are desirable since early ages. describing a simplified purpose to rehabilitate strongly discoloured and excessively attrition in primary dentition in order to return her aesthetic and oral functions. a 6-year-old girl sought the dental clinic for treatment due to her dental appearance and difficult to chewing. although the dental abnormalities diagnosis has not been concluded until the moment, dental procedures to return aesthetics and oral function had to be performed. aiming to simplify the process, we opted for reconstructing teeth in a model and using a vacuform matrix loaded with composite resin and applied to the unprepared etched teeth. the restorative treatment lasted seven 2-hour-clinical sessions and was well accepted by the child. besides the aesthetical smile, the restorations permitted increasing the vertical dimension and improved child´s oral function. the child and her mother declared to be very satisfied with the final result of the proposed treatment. this simplified technique for full-mouth rehabilitating reduced the clinical hours with child and reached a satisfactory result considering both aesthetics and oral function. consequently, it could be expected an improvement in this child´s psychosocial condition and quality of life. xix aboprev meeting/2015 braz j oral sci. 14(1) 85-99 87 007 esthetics and functional oral rehabilitation: case report moreira, k.m.s.*; cardoso, m.; inagaki, l.t.; martins, l.f.b.; pascon, f.m.; galvão, m.b.d.; rontani, r.m.p. despite all the existing means for prevention, dental caries is still a public health problem. a big part of the population has not access to guidance, prevention and treatment of this disease. the study aimed to report the clinical case of a patient with five years of age, male gender and early childhood caries. the clinical examination revealed total destruction of upper deciduos teeth and partial of the lower teeth involvement. the radiographic examination confirmed the indication to pulpectomias in 75, 84 and 85 teeth. we prepared the treatment plan: oral middle adequate, removing up focus of infection through of extractions, root canal treatment and restoration. topical applications of fluoride varnish was performed and after restoration in the 85, 83, 75 and 73 teeth with composite and steel crowns in the 74 and 84 teeth. dietary and oral hygiene guidance was performed to family and child. upper total prosthesis was made. the child was transferred for fonaudiologia. preservation of the total prosthesis was 7 and 14 days after the installed. treatment success was observed, since the masticatory and aesthetic functions were reestablished, returning to child the self-esteem. 008 evaluation of a protocol to simulate in vitro the decreased absorption of f from toothpaste due to gastric content lima-arsati, y.b.o.*; andrade, h.k.; gomes, a.r.l.f. background: in vivo studies show that fluoride (f) absorption from toothpaste is decreased after meals due to gastric content. the studies that estimate f dose from diet and toothpaste do not take in account this f inactivation, leading to overestimated doses. aim: to evaluate a protocol to simulate in vitro the decreased absorption of f from toothpaste due to gastric content. methods: samples of duplicate-diet (24h) were collected from 20 children aged 1 to 3 years. their brushing habits were determined in our previous study, showing that they ingested ~ 0.35g of toothpaste per brushing. f was determined in samples of diet and diet+toothpaste (standard amount of 967.8 μg f/g toothpaste), after incubation in 0.01m hcl/2 hours, centrifugation and buffering with tisab ii. f analysis were performed using ion-specific electrode. original fluoride (of) was calculated by adding the amount of f in diet to the amount of f in toothpaste samples. recovered fluoride (rf) was the amount of f in samples diet+toothpaste. results: of15.954±1.730 μg f rf15.212±1.805 μg f. rf was statistically lower than of (p0.011 t-paired test), but this reduction of 4.5% is not consistent with the inactivation of f from toothpaste by gastric content reported in the literature by in vivo studies. conclusions: the evaluated protocol did not reproduce in vitro the decreased absorption of f from toothpaste due to gastric content. however, it is an interesting method for f analysis in diet samples, due to its simplicity and low cost. 009 dental microabrasion and at-home whitening: an effective association for aesthetic problems. coelho, c.s.s.*; brito, m.h.s.f.; sousa, g.p.; silva, m.a.f.; mendes, r.f.; moura, l.f.a.d.; lima, m.m.; de moura, m.s. enamel microabrasion is an efficient method for the removal of superficial enamel white spots caused by caries or dental fluorosis. this study reports two cases of the combined use of enamel microabrasion and at-home tooth whitening. case 1: a 9 year-old female was diagnosed by a clinical dentist as having enamel white spots due to caries. she had been prescribed daily mouth washes with 2% sodium fluoride solution. on clinical examination at the universidade federal do piauí (ufpi), the permanent first molars and incisors presented a tf 4 (moderate) fluorosis level. eight years later, when the patient presented the complete permanent dentition, she was diagnosed with fluorosis tf 4 level and tf 5 (loss of hard tissue) in some teeth. case 2: a 10 year-old male with poor oral hygiene habits and non-compliant with the recommended preventive oral hygiene procedures during orthodontic treatment. at the end of treatment, he had widespread caries white spot lesions. after removal of the braces, the hygiene recommendations were reinforced by the dentist for two months. afterwards, enamel microabrasion was carried out on the buccal surfaces of the aesthetically important teeth. in both reported cases, enamel microabrasion was carried out using a 37% phosphoric acid gel and pumice paste on a 1:1 (v/v) ratio and the tooth whitening was carried out using 16% carbamide peroxide gel due to the yellowish appearance of the teeth after microabrasion. conclusion: the combination of the methods was successful to recover the aesthetic appearance of the smile. 010 evaluation of the daily intake of coffee, cola and guaraná soft drinks on bone mineral density and blood mineral levels costa, e.d*; alonso, m.b.c.c.; silva, a.i.v.; ambrosano, g.m.b.; marcondes, f.k.; watanabe, p.c.a.; hait, f. background: nutrition is an important modifying factor in the development and maintenance of bone mass. aim: to determine the effects of the daily intake of coffee, cola and guaraná soft drinks on bone mineral density and blood levels of calcium (ca), phosphorus (pi) and magnesium (mg) in male and female rats. methods: sixty-days-old wistar rats were divided into four groups according to the test drink, namely control (water), cola, guaraná and coffee. after 48 days, all animals were sacrificed, had their blood collected for biochemical analyzes and their femora evaluated for bone mineral density (bmd) and bone mineral content (bmc). results: all animals gained weight during the experiment. consumption was highest for cola and lowest for coffee. changes in bmd appeared only in females of the coffee group. as for bmc, males showed higher values across groups, while coffee intake provoked significant bmc reduction in females. regarding blood biochemistry, males showed higher serum ca levels across groups. while pi levels were similar across groups for females, males in the guaraná group showed significantly lower pi levels. coffee intake produced a significant increase in mg levels regardless of gender. conclusions: taken together, our data suggest that daily coffee intake can lead to decreased bmd and bmc in female rats, and that habitual consumption of coffee, cola and guaraná soft drinks may induce changes in the levels of blood minerals essentially related to bone metabolism. 011 saddle seat: new ergonomic concept in the clinical practice of the dentists literature review gouvêa, g.r.*; silva, m.a.v.; sofia poletti; uemura,s.t.; zanatta, j.; bulgareli, j.v.; pereira, a.c. the implementation of an ergonomic sitting posture is necessary, once that the majority of the dentists complains about musculoskeletal injuries. those can be caused by the seat used in the clinical practice. investigate the produced knowledge in scientific literature about the new ergonomic equipment of mobile sustention in sitting posture of dentists, through the saddle seat. it is a literature review based on specialized literature through the consult of selected scientific articles in data base of scopus and medline in the years of 2014 and 2015. it was found 6 articles and 1 thesis that addressed this theme. the saddle seat differs from other kinds of seat especially because of the angle of the knee that can vary between 120° to 130°, differently of the 90° angle that is commonly used by the dentists. with a bigger angular amplitude between pelvis and knees an improvement of the lymphatic and peripheral circulation of the lower extremities, prevents pathologies in the circulatory system, the tension in hamstrings are relived, enabling lumbar, thoracic and cervical vertebras to get in an anatomic position keeping postural control and the prevention of the work related musculoskeletal disorders (wmsd). the saddle seat can be a strategy to prevent, stabilize and treat postural problems that affects dentists, allowing them to adopt an anatomic posture and keep an ergonomic posture in clinical practices. its necessary more longitudinal experimental studies that evaluate the impact of this kind of seat in posture health of the dentists. 012 water fluoridation: an approach based on the string literature. leite, c.n.*; narvai, p.c. fluoridation of public water supplies is among the most important public health measures for control of dental caries. the aim of this paper is to present the history of water fluoridation in brazil as well as around the world, with the intention of showing the importance of this process to the reduction of tooth decay. as a strategy the popular literature of cordel was chosen, considering its efficiency and usefulness in oral communication process, and also in the understanding that it could be an important tool in the process of dissemination of different fields of scientific knowledge. as a result, we have noticed that the cordel that we have produced has significantly contributed to the dissemination of the issue discussed in this paper. what shows us that literature of cordel can really contribute to science, demystifying the wrong understanding of important data to be traditional, popular and assessable. xix aboprev meeting/2015 braz j oral sci. 14(1) 85-99 88 013 assessment of oral health knowledge of community health workers linked to family health strategy silva, m.a.v.*; gouvêa, g.r.; guerra, l.m.; mialhe, f.l. background: in 2013, over 250 thousand community health workers (chw) and more than 22 thousand oral health teams (oht) were working in brazil in order to collaborate in health promotion and disease prevention. aim: to compare the work process and the oral health knowledge of chw who work with and without oht. methods: a total of 162 subjects participated in the study, with 81 chw linked to the family health unit with oht and 81 chw linked to the family health unit without oht. we used a form containing data on income, education and length of service, 7 items on the work process, behavior, self-perception of oral health and household access to dental services, as well as 12 questions on validated knowledge of the health-disease. results: the results showed that the differences observed between the chw groups were statistically significant (p<0,05) when analyzing the total knowledge of questionnaire scores on oral health-disease (p<0,0021). regarding the other study variables, only the questions that focused on training in oral health (p<0,0002) and household access to dental care (p<0,0001) had statistically significant differences when compared the two groups of chw. conclusions: knowledge about oral health / disease chw linked to the family health unit with oht process is best, and that the number of chw who has had training in oral health was significantly higher in the group with oht. 014 inequalities on oral health among adults batista, m.j.*; sousa, m.l.r. aim:the objective of this study was to evaluate social inequalities in oral health among adults. methods: in piracicaba, brazil, a cross-sectional study selected by a probabilistic sampling, 248 adults aged 20 to 64 years. caries (dmft) and periodontal disease (cpi) were evaluated according to who criteria. ohip-14 was used to evaluate quality of life related to oral health. health inequalities were assessed by social class, education, family income and type of dental service obtained from a questionnaire. analyzes were performed for each outcome (clinical status) with socioeconomic variables by binary logistic regression or multinomial logistic regression adjusted by age and sex. results: among adults, 35.4% (29.741.4) presented untreated caries 43.0% (32.7-53.9) had periodontal pockets over 4 mm 19.5% (20.3-24.4) have lost any teeth due to dental diseases and 48.1% (41.654.7) reported severe impacts on ohip (fairly/very often). impact of oral health on quality of life, decayed teeth and tooth loss varied according to the gradients of social class, family income and education (p<0.05). having decayed teeth was associated with type of dental service. in the middle social class 46.1% had missing teeth and 1.8% lost more than 13 teeth in the lower social class, this percentage was 17.2% and 32.9% respectively (p<0.05). conclusions: clinical attachment loss was associated with social class e income. social inequities could be reflected in these adults oral health, including the impact of oral health on quality of life independently of age and sex. these results showed the determinants of health role play among this adult population. 015 caf2 formation in sound and carious enamel by fluoridated mouthrinse or gel lima, c.v.*; noronha, m.s.; oliveira, b.e.c.; caldarelli, p.g.; souza, j.g.s.; tabchoury, c.p.m.; tenuta, l.m.a.; cury, j.a. background. it has been reported that the reactivity of fluoride with carious enamel is higher than sound, however this fact has not been widely explored in the literature. in addition, the presence of other ingredients in commercial formulations can interfere with this reactivity. aim. to compare the reactivity of two commercial products, a f-mouthrinse and a f-gel, with sound and carious enamel. methods. for this in vitro, randomized and blind study, bovine enamel slabs, sound and with provoked incipient caries lesions, were divided into 5 groups (n6): 1) negative control (purified water) 2) naf solution containing 225 ppm f (control of commercial mouthrinse) 3) colgate plax®, 225 ppm f 4) naf solution containing 9,000 ppm f (control of commercial f-gel) and 5) neutral gel 9,000 ppm f (f-gel), dfl®. the duration of exposure to the treatment was 4 minutes. loosely bound fluoride ("caf₂") was extracted with koh and fluoride concentration was determined with ion specific electrode, being the results expressed in µg f/cm². the data were analyzed by two-way anova followed by tukey test at 5% of significance. results: there was a larger formation of "caf₂" on carious enamel than on sound (p<0.0001). all treatments significantly increased "caf₂" concentration in enamel compared with the negative control (p<0.0001). the commercial f-gel formed greater "caf₂" concentration on enamel than fmouthrinse (p<0.0001), but they did not differ from their respective controls (p>0.05). conclusions: incipient caries lesions can benefit more than sound enamel by either professional application of fluoride or the daily use of mouthrinse. 016 antimicrobial use in early childhood and its association with dental enamel defects in primary health care faustino-silva, d.d.*; rocha, a.f.; rocha, b.s. background: the dental enamel is a highly mineralized tissue and without metabolic activity after formed, which means that the disturbances during development may manifest as permanent defects in erupted teeth. a change of great importance is the molar-incisor hypomineralization (mih), which has systemic etiology and affects the first permanent molars and the permanent incisors. it has been pointed out in the literature a relationship of antimicrobial use with mih, especially amoxicillin. aim: to determine whether there is an association between the use of antibiotics early in life and the development of dental enamel hypoplasia (deh) in permanent teeth of 6-12 years children in primary health care. methods: this is a cross-sectional study, in which were included children examined who had at least one visit in your health unit in the first months of life and during the first four years and excluded all those who did not have regular monitoring in their health units throughout this period, as well as children whose files were not found. of the 228 children examined by trained and calibrated dentists, 144 remained in evaluation of the records of the antimicrobial use and infections in early childhood. results: amoxicillin was the most prescribed drug in all age groups assessed, followed by sulfamethoxazole+trimethoprime. of the 144 children, 43 (29.9%) had normal dentition and 101 had deh (70.1%). 17 patients had prescriptions of antibiotics at least once in their lives in the first 4 years of life, and of these, 13 had some type of deh. among patients who had deh, amoxicillin has been the most widely prescribed drug, at least 6 patients using more than 6 times over the first 4 years. there was no statistically significant association for either cases. conclusions: this study concludes that there was a high prevalence of children with deh and that amoxicillin was the most prescribed drug in the first 4 years of life of these patients and this may be related to the development of opacities and hypoplasia, even if they are necessary and longitudinal studies with sample more representative to prove this relationship. 017 relationship between asthma, malocclusion and mouth breathing in primary health care children faustino-silva, d.d.*; santos, n.m.l.; rezende, g.; hugo, f.n.; hilgert, j.b. background: asthma is a chronic inflammatory disease of high prevalence in children and possible oral effects in children and adolescents. aim: to assess the possible relationship between asthma and mouth breathing and malocclusion in the primary health care context. methods: cross-sectional study with a sample of 228 children between 6 and 12 years, divided into a group of asthmatic (n 112) and other non-asthmatic (n 116), in two basic health units, located in the city of porto alegre, rio grande do sul, brazil. the evaluation consisted of oral examination conducted by two trained dentists and calibrated (kappa 0.85 to 1.00), structured interviews with parents and data records. data were analyzed with spss, using the poisson regression, the statistical significance level of p <0.05. results: the most common type of asthma was intermittent, in 62 children (55.4%), followed by mild persistent, in 38 children (33.9%). the most used drug was salbutamol (a shortacting bronchodilator) in 95 children (84.8%), followed by 35 children (31.3%) who used budesonide (an inhaled corticosteroid), 33 children (29.4%) who used systemic corticosteroids and 28 children (25%) who used beclomethasone (inhaled corticosteroid). asthma [pr 2.12 (95% ci: 1.46-3.08), p<0.001] and the use of pacifiers [pr 1.98 (95% ci: 1.27-3.07), p<0.001] were associated with mouth breathing, in the final multivariate model (table 2). age [pr 1.02 (95% ci: 1.00– 1.03), p0.039] and thumb sucking [pr 1.08 (95% ci: 1.03–1.13), p0.001] were associated with malocclusion in the final multivariate model. conclusions: there is a need to qualify the comprehensive care of asthmatic children and adolescents because this approach to asthmatic people’s oral health is the way to effectively practice interdisciplinary and complete care. 018 evaluation of dental caries previous knowledge from undergraduate dental students junior, m.f.s.*; santos, g.f.; gavi, r.s.; gomes, m.j.; daroz, l.g.d.; claudia batitucci dos santos daroz, c.b. background: the previous knowledge of the causal factors, of the development and of the evolution aspects from a disease, such as caries, should be points of discussion throughout the individual’s academic formation as part of health education. aim: to evaluate dental caries previous and acquired knowledge from undergraduate dental students from espírito santo federal university (ufes). methods: undergraduate dental students (n121) answered a questionnaire about the previous and the acquired knowledge of dental caries. a pilot study concluded in another dental college validated the questionnaire. results: respondents (38.8%) stated that most of the knowledge about dental caries disease was acquired after the entrance in the dental college while 28.1% and 13.2% stated from home/family and from private dentist, respectively. the knowledge about the disease improved oral health daycare of 95% of the respondents. no significant difference about dental caries’ knowledge between the students from the middle of the course and from the last year of the course could be observed although when asked about dental restoration and about the consequences of dental caries, significant differences were found among the students in the beginning of the course (chi-square,p<0.05). significant difference about the knowledge acquired after the entrance in the dental college compared to before was observed among the students from the middle of the course (mcnemar test,p <0.05). conclusions: dental caries real knowledge was acquired after students started dental college, becoming evident in the middle of the course. there was relevance from the collected responses with the oral health daycare. xix aboprev meeting/2015 braz j oral sci. 14(1) 85-99 89 019 rondon project: awakening looks for oral health in the city of pirpirituba/pb in health program actions in school aguiar, b.m.*; perin, p.c.p.; leite, c.n.; bergamaschi, m. background: the oral health promotion is the process of producing favorable general living conditions to a healthy development of the mouth, in view of the biological and social role that it represents. aim: given these considerations, this study aimed to promote partnership with professional dentists and public school system educators so that it includes the practice of supervised brushing and guidance in oral and general health in schools. methods: it was conducted in january and february 2015, in the city of pirpirituba, state of paraíba, rondon operation “porta do sol” project, linked to brazil’s ministry of defence. the methods employed with the 119 local teachers of kindergarten, elementary, youth and adult education (eja) and health professionals was through the use of visual aids such as banners, brochures, lectures and guides from the ministry of health and education. results: in the course of the lectures and meetings, it was realized the need for health activities in schools, and from this finding we could stimulate greater interest from educators and professional dentists to seek a viable and more effective alternative to inclusion of such activities. conclusions: as a result, we can see that health education as a mean of health promotion in the school environment is done by building partnerships between health and education professionals, highlighting the long-term social change potential entering this theme in the reality of students, who will be the future trainers and processors of that local reality. 020 oral health of the elderly treated in the family health strategy: reports and perceptions on tooth loss silva, m.a.s.*; lauris, j.r.p.; tomita, n.e. background: this study focuses on the oral health conditions of the elderly since, with aging, some changes occur in the mouth and other changes happen due to cultural, care, economic and social factors, such as tooth loss. aim: to obtain selfperceptual information on the oral health conditions of older people living in the city of bauru, são paulo state, treated in the family health strategy. methods: this cross-sectional study consisted of interviews made with subjects aged 60 or over, of both genders, living in a neighborhood known as bairro santa edwirges, north of bauru, an area under the responsibility of a family health center (2012). we collected information on the self-perception of the general condition of the teeth, presence of teeth, use of dental prosthesis and interference in mastication. the instrument used was the structured questionnaire brazil old age schedule (boas). the descriptive statistical analysis shows the relative and absolute frequencies. results: we interviewed 122 old people, 41 male and 81 female, aged 60 to 89 years old. the self-referred oral conditions were: 58.2% had a negative perception on their teeth general condition 96.7% reported tooth absence 76.2% claimed to use some type of dental prosthesis and 59% believe that tooth absence does not interfere in mastication. conclusions: having a significant percentage of selfreferred edentulism, this social stratum understands the precariousness of their objective condition of oral health, marked by tooth loss. however, the fact that tooth loss is considered natural in aging produces speeches that denote a functional adaptation to the absence of teeth, which is a reflection of the scarcity of information and access to treatment that marks the groups located in the periphery of society (geographic, social and cultural). 021 techniques of sealant application: clinical guideline on case report moreira, k.m.s.*; cardoso, a.a.; iwamoto, a.s.; pascon, f.m.; kantovitz, k.r.; rontani, r.m.p. dental sealants are used as additional prevention of dental caries on oclusal surfaces, since they act as protective barrier. the objective of this study was to present a clinical report focused on the different sealant techniques of resin and ionomer sealant and their main indications, in order to facilitate dental clinical practice. m.e.m.o. aged of 6y and 8mo., female gender, with presence of active white spot lesions on the erupted first permanent molars, high biofilm index (80%), frequent cariogenic ingestion and previous history of caries on primary teeth. bitewing radiographs were performed from the focused teeth, oral hygiene instruction and guidance of the diet, emphasizing the importance of the patient and family core compliance. the conventional encapsulated ionomer sealant was applied on the tooth 16, resin sealant with intermediary adhesive layer on the 46 and resin sealant on the tooth 36. the tooth 26 was not present in the oral cavity, yet. the type of sealant was indicated according to the dental eruption stage and clinical techniques followed the specifications of each sealer material. due to greater difficulty of humidity control in the partially erupted teeth (16 and 46), two step by step of different types of sealants were described. application of sealant requires appropriate indication and technique to contribute in the reducing the failure of dental treatment. 022 psychometric properties of an instrument to assess infection control practices in oral radiology costa, e.d.*; pinelli, c.; tagliaferro, e.p.s.; corrente, j.e.; ambrosano, g.m.b. background: infection control is an important issue in dentistry because of concern of transmissible diseases in health care settings. in the intraoral radiographic procedures the contact with saliva and/or blood is common and deserves more attention. aim: to develop an instrument to assess infection control practices in intraoral examinations in oral radiology. methods: a questionnaire with 17 multiple-choice questions divided into three domains, related to hands hygiene frequently, type of radiographic equipment used, protection/disinfection of surfaces of the x-ray equipment and accessories and disinfectant type used for cleaning/disinfecting. the psychometric properties were evaluated by means of reliability (participation of 115 dental students) and validity (participation of 641 dental students, 20 phd students in oral radiology, 15 teachers of oral radiography and 3 radiology technicians). results: the reliability by test-retest indicated adequate reproducibility values (0.722≤icc≥1.000 and 0.662≤kappa≥0.913) and excellent internal consistency (cronbach's alpha 0.88). the validity verified by the content validity and the factor analysis resulted in a reduction of some items of the questionnaire and reunification of the issues in new domains. significant differences were observed when answers were evaluated among the academic group, phd students and teachers. conclusions: the developed instrument was considered reliable for evaluating infection control practices in oral radiology in health settings. 023 use of fluoridated toothpaste by pre-school children silva, m.a.f.*; coelho, c.s.s.; carvalho, n.s.r.; brito, m.h.s.f.; albuquerque, h.c.l.; moura, l.f.a.d.; lima, m.d.m.; moura, m.s. background: based on scientific evidence, the brazilian and american academies of pediatric dentistry and pediatrics recommend the use of fluoridated toothpastes for controlling dental caries after the eruption of the first tooth. aim: this study aimed to evaluate the use of fluoride dentifrice by preschool children. methods: a total of 308 children, 02-05 years of age who attended public and private day care centers in teresina, piauí were evaluated. parents answered a questionnaire on socio-demographic and oral hygiene habits. results: 62.7% of children were from private centers and 71.1% of mothers had above 11 years of schooling. when asked about the first toothpaste used by their children (around the first year of life) 64% reported that their child used toothpaste without fluoride, 59.7% of which had been the parents’ decision to use, 24.0% had been recommended by dentists and 11.3% by the pediatricians. regarding the type of toothpaste used at the time of this study, 37.3% used toothpaste for children without fluoride, 46.1% use fluoride toothpaste for children, and 16.6% use toothpaste for adults. in 41.8% of the cases, the parents reported that the children swallow toothpaste while brushing. when asked what could cause this swallowing, 75.6% of parents said they did not know. when asked about the attitude taken to prevent the ingestion of toothpaste 43.8% reported not taking any action. conclusions: a significant percentage of pre-school children use toothpaste without fluoride and are therefore more susceptible to the development of dental caries. 024 susceptibility of streptococcus mutans to the phytochemical citronellol bezerra, l.m.d.*; sousa, f.o.g.s.a.; cury, a.a.d.b. background: substances derived from plants may represent a promising strategy in preventive dentistry due theirs antimicrobial potencial. the citronellol is a monoterpene found in the essential oils of citronella-grass (cymbopogon winterianus jowitt), lemongrass (cymbopogon citratus) and bushy matgrass (lippia alba), whose antibacterial, antifungal and anticonvulsant activity, have been described in the literature. aim: evaluate in vitro antibacterial activity of citronellol against streptococcus mutans. methods: it was determined the minimum inhibitory concentration (mic) by microdilution technique and minimum bactericidal concentration (mbc) through the subculture of mics. the chlorhexidine digluconate was used as positive control and the assays were performed in triplicate. the reading for mic determination was performed through the visual method after 18 hs, the tct 0.5% dye (2, 3, 5 triphenyl tetrazolium chloride) was inserted in all wells to confirm the presence of viable microorganisms in non-inhibitory concentrations, once such dye reflects the action of dehydrogenase enzymes, involved in the process of cellular breathing. results: the citronellol showed mic of 234,375 µg/ml against s. mutans, the chlorhexidine digluconate, in turn, had mic of 2,34 µg/ml. for mbc it was obtained the values of 7500 µg/ml and 4,68 µg/ml for citronellol and chlorhexidine digluconate respectively. conclusions: the microorganism was susceptible to citronellol. besides that, its primarily bacteriostatic activity could be very important in the maintenance of biofilm's equilibrium, once the microorganism will be inhibited but not extinct, what could lead to the growth of other microorganisms that could be even more pathogenic. xix aboprev meeting/2015 braz j oral sci. 14(1) 85-99 90 025 quality of water fluoridation comparing ordinance 635/bsb/1975 and cecol/usp recommendation bergamo, e.t.p.*; barbana, m.; terada, r.s.s.; cury, a.a.d.b.; cury, j.a.; fujimaki, m. background: an adequate concentration of f in drinking water is necessary for the effect of fluoridation to reach its maximum anticaries benefit with a minimum risk of fluorosis. aim: thus, this study evaluated the concentration of fluoride (f) in water of maringá, pr, brazil, considering a new classification for f concentration based on the anticaries benefit and risk of fluorosis (cecol/usp) compared to the current classification (635/bsb/1975). methods: water samples (n325) were collected monthly during one year from 28 distribution water network, 20 from the treatment plant and 8 from artesian wells. f concentration was determined using specific ion electrode. results: the average f concentration was 0.77 mg f/l (ppm f), ranging from 0.44 to 1.22 mg f/l. considering all water samples analyzed, while 68.7% were within the beneficial range by the ordinance 635/bsb/1975, 97.5% of them were classified by cecol/usp recommendation as adequate in terms of caries control and fluorosis risks. however, this difference of almost 29% was lower (27,4%) when only the water from artesian wells was compared. conclusions: although the classification of the quality of water fluoridation by cecol/usp is more conservative than the current ordinance, it is more appropriate because it takes into account that both caries and fluorosis are chronic disease and the time of duration of the dose is more relevant that occasional variation below or above the optimal range. 026 evaluation of bleached enamel exposed to different mouthwashes vieira-junior, w.f.*; ferraz, l.n.; ambrosano, g.m.b.; aguiar, f.h.b.; lovadino, j.r.; lima, d.a.n.l. background: the effect of mouthwashes exposure after in office dental bleaching is unexplored and unknown. aim: to evaluate, in vitro, the effect of mouthwashes exposure on chromatic and morphological properties of bleached enamel. methods: sixty bovine enamel blocks were bleached with 35% hydrogen peroxide and randomly divided into six groups in accordance with mouthwash exposure (n10): g1)periogard colgate®, g2)plax classic colgate®, g3)plax whitening colgate®, g4)luminous white colgate®, g5)listerine® tartar control e g6)distilled water (control). after bleaching, the specimens were immersed in mouthwash twice daily for 14 days (manufacturer's instructions). the specimens were stored in artificial saliva during the experiment. enamel roughness (ra) and color analysis (δe formula, l, a and b) were performed in the frames: before treatment (t0), after 24 hours of bleaching (t1) and after 14 days of mouthwash exposure (t2). color was assessed by reflectance spectrophotometry and quantification were done using cielab coordinate system. scanning electron microscopy (sem) analysis was used to evaluate enamel morphology. statistical analysis: raanova (proc mixed), color l, a, bfriedman, kruskal-wallis, dunn and δeanova and tukey-kramer (p <0.05). results: ra, g3 and g5 did not recover the initial ra compared to control. g5 showed increasing in roughness (t1xt2). color analysis, g2 presented values of a (a+redness) statistically different between times (t1xt2) and δe value was clinically perceptible (δe3.33). sem, g3 and g5 showed enamel demineralization with mineral loss and porosity. conclusions: listerine® tartar control increased roughness and colgate® plax classic changed the color of bleached enamel. 027 dental hygienists acting in the prevention of dental caries: a literature review uchida, t.h.*; terada, r.s.s.; pascotto, r.c.; fujimaki, m. dental hygienists (dh) are important members of the oral health team,which are inserted in the family health strategy and is one of the tasks significantly contribute to the development of educational activities,working in health promotion and in the control of oral diseases, particularly dental caries. the aim of this study was to review the literature regarding the role of the dh in the oral health team forward to preventive practices for the control of dental caries. the databases were consulted,pubmed,web of science,lilacs, virtual health library and brazilian dental library. there were 18 articles making an approach to the knowledge, practices and opinions of dhs about preventing tooth decay and its early detection.most research found pointed to the importance of the dh work with actions aimed at prevention and health promotion.a study in norway showed that dhs of work in oral health education,providing information on diet,hygiene and fluoride, resulted in 70% of children under 5 years and 66% of young people under 12 with no decayed tooth,and most of these children were only met by dhs.in usa, 14% of 6,723 dhs understand that their main role is to prevent and not in clinical work. international studies have valued the presence of dh within the team, principlamente in children,showing that the number of dhs and the proportion of children receiving preventive care by a dh are increasing. the dh plays an important role in the development of actions aimed at disease prevention and health promotion can significantly contribute to the reduction of dental caries. 028 evaluation of physical properties of a nanocomposite after aging, bleaching, and staining gouveia, t.h.n.*; públio, j.c.; ambrosano, g.m.b.; paulillo, l.a.m.s.; aguiar, f.h.b.; lima, d.a.n.l. aim: the objective of the in vitro study was to assess the effect accelerated artificial aging, bleaching agents and coffee staining on the color, gloss, roughness and microhardness of a nanocomposite. methods: one hundred twenty samples (7 mm x 2 mm) were prepared and randomly divided into 2 groups (n 60) depending on the aging. each group was further subdivided into 6 subgroups (n 10) according to the bleaching and staining as follows: 10% carbamide peroxide (10% cp), 10% cp + staining, 35% hydrogen peroxide (35% hp), 35% hp + staining, without bleaching treatment (wb) and wb + staining. scanning electron microscopy was performed for qualitative analysis of the resin surface. data were submitted to three-way analysis of variance (anova) and tukey’s test for multiple comparisons. as for ∆e, multiple comparisons were performed by using tukey’s and dunnett’s tests (α 0.05). results: the bleaching reduced significantly the microhardness of the nanocomposite in without aging groups. all physical properties were found to be negatively changed after the aging process, with bleaching treatment with 10% cp increasing significantly the roughness and loss of gloss compared to the 35% hp. staining reduced microhardness of both with and without aging nanocomposite as well as the color of the former (∆e > 25), a significantly different result compared to controls. conclusions: the bleaching reduced the microhardness of the nanocomposite, whereas the aging process changed all the properties studied. the bleaching favored extrinsic staining of the with aging nanocomposite. 029 pathogenic microorganisms in the oral cavity and oropharinx of neurological disorders and enteral nutrition patients. cunha-correia, a.s.*; correia, t.m.; pereira, m.f.; ranieri, r.v.; aguiar, s.m.h.c.a.; schweitzer, c.m.; gaetti-jardim jr, e. background: enteral nutrition involves accumulation of dental calculus, and respiratory complications associated with contaminants of the oral biofilm or oropharynx. aim: evaluate, by culture and polymerase chain reaction (pcr), the presence of periodontopathogens and opportunistic pathogens in patients with neurological disorders and enteral nutrition and also propose a biofilm control protocol. methods: the evaluated patients were divided into two groups: (gtg, n 11) gastrostomy group, with neurological disorders and enteral nutrition patients and (cg, n13) control group, with neurological disorders and oral feeding patients. oral clinical indices like gingival bleeding (gbi), and simplified oral hygiene index (ohi-s) were collected. a prevention protocol of monthly dental appointments, daily tooth brushing, daily use of dental-floss, and daily oral cleaning with gauze embedded in chlorhexidine digluconate solution (0.12%) added with xylitol (5%), was established. saliva, mucous and oropharyngeal secretion, subgingival and supragingival biofilm specimens was carried out before and 12 months after introduction of the preventive protocol. the chi-square test, mann-whitney’s test, pearson’s correlation tests, spearman’s test and kendall’s test were used. results: the occurrence of yeasts (89%, p<0.05), e. corrodens (53.6%, p<0.01) and members of the mollicutes class (63.6%, p<0.01) was higher among gtg patients while the enterobacteriaceae family did not differ between groups. clinically, the gtg presented an average 61% reduction of ohi-s and elimination of gingival bleeding. conclusions: patients with neurological disorders and enteral nutrition present alteration in their resident microbiota. a preventive protocol improved oral health, aiding in the prevention of septic events in these patients. 030 doctors and nurses´ knowledge, attitudes and practices in oral health in childcare, in primary health care faustino-silva, d.d.*; reis, m.l.; luvison, i.r. background: the care of the oral health of the future baby should begin during the prenatal period, at which time the pregnant woman is more open to receive information concerning the child's health. care actions in the first year of life should be conducted in a multidisciplinary way, avoiding the creation of specific oral health programs isolated but integrated with the health team. aim: investigate the knowledge, attitudes and practices in oral health in childcare doctors and nurses, contractors and residents of the community health service hospital concept group (ssc-ghc), located in the city of porto alegre, rio grande do sul, brazil. methods: application of a questionnaire with 32 multiple-choice questions, developed by the researchers to assess the knowledge, attitudes and practices (cap) in oral health in childcare doctors and nurses, contractors and residents, in eleven of the twelve health units of the ssc-ghc. the sample was composed of 47 doctors and 27 nurses. data were tabulated and analyzed using the spss software using the chi-square test and t test, with statistical significance level of p <0.05. results: the results show that there is little significant difference between the knowledge, practices and attitudes of doctors and nurses of the ssc-ghc, including in relation to the time of training / professional practice. conclusions: there is still lack of many in a few points on oral health in childcare, requiring permanent educations on the topic. xix aboprev meeting/2015 braz j oral sci. 14(1) 85-99 91 031 dental caries, enamel defects and erosion in asthmatic children in primary health care faustino-silva, d.d.*; rezende, g.; dos santos, n.m.l.; balbinot hilgert, j.; hugo, f.n. background: asthma is a chronic inflammatory disease of high prevalence in children and possible oral effects in children and adolescents. aim: to assess the relationship between asthma disease and the occurrence of dental caries, enamel defects and dental erosion. methods: cross-sectional study with a sample of 228 children between 6 and 12 years, divided into a group of asthmatic (n 112) and other non-asthmatic (n 116), in two basic health units, located in the city of porto alegre, rio grande do sul, brazil. the evaluation consisted of oral examination conducted by two trained dentists and calibrated (kappa 0.69 to 1.00), structured interviews with parents and data records. data were analyzed with spss, using the poisson regression, the statistical significance level of p <0.05. results: most of the children had intermittent asthma and none with severe persistent asthma. regarding the medication used, most used salbutamol, alone or with other medications. oral habits and hygiene frequency were evaluated, and most children performs oral hygiene with toothbrush and fluoride toothpaste. among asthmatic statistically significant differences relate to the number of medications used with caries experience, children who use a greater number of medications have a lower prevalence of dental caries (p 0.01). considering the enamel defects, children with moderate persistent asthma and uses a greater number of drugs have a higher prevalence of enamel defects, but no statistical significance. about dental erosion when compared with the number of medications, there was no statistically significant association. regarding maternal education, there was a statistically significant difference between the higher the prevalence of caries to a lower level of education. conclusions: the oral health of asthmatic children, especially the most serious cases, deserves special attention, both by dentists, for the entire health team to identify, limit and reduce the damage caused by these alterations. 032 motivational interviewing and oral health: lessons learned from a crosssectional study of dentists and oral health tech faustino-silva, d.d.*; hilgert, j.b.; hugo, f.n.; meyer, e. background: motivational interviewing (mi) is an alternative approach to discussing behavior change that encourages a constructive relationship between health care professionals and patients and provides an opportunity for better treatment results. although mi has proved effective in the last two decades, most brazilian health professionals are not familiar with the approach, including dentists and oral health technicians (ohts). the main components of mi could be applied when professionals advise and instruct their patients on oral health. aim: assess the prevalence of motivational statements by dentists and ohts prior to training, within a primary health care (phc) setting. methods: the study was conducted at the community health service of the grupo hospitalar conceição in porto alegre (rio grande do sul state rs), with a total of 59 dentists and ohts who participated in an 8-hour training session for active learning of basic mi principles. this is a cross-sectional study aimed at assessing the prevalence of motivational statements in written answers to pre-established clinical situations as well as the ability to identify basic mi skills, such asking open-ended questions, capacity for reflective listening, providing affirmations and summary statements in a dialogue interview. results: for the “open-ended questions” item, 76% of the sample was able to identify these however, as expected, 65% of participants did not recognize affirmations, 58% did not detect summary statements and could not identify reflective listening. half of the sample did not notice when the patient’s statements might signal resistance to behavior change. finally, the exercises demonstrated that half of the professionals assessed use advice/persuasion or direct confrontation, which are likely to increase resistance and should be avoided. conclusions: training is needed to ensure that oral health teams in phc provide care within the spirit of mi. 033 oral impact on daily performance among brazilian adolescents kaieda, a.k.*; bulgareli, j.v.; vazquez, f.l.; marques, t.c.n.; pereira, a.c.; ambrosano, g.m.b.; cortellazzi, k.l. background: the oral impact on daily performance (oidp) is the instrument used to measured oral problems perceptions through the frequency and severity. aim: this study investigated the relationship between the oral impact on daily performance and individual and contextual factors among brazilian adolescents. methods: an analytical cross-sectional study was conducted in piracicaba, in 2012, with 877 adolescents aged 13-19 years-old, from 21 state schools and 34 family health units. the dependent variable was oidp. the independent variables were classified as individual (components of the daí dental aesthetic index, sex and age) and contextual (social exclusion index) variable.the multilevel regression model was estimated by the proc glimmix (‘‘generalized linear models-mixed’’) procedure, considering the individuals’ variables as being level 1 and the contextual variables as being level 2, and the statistical significance was evaluated at level of significance of 5%. results: the mean age was 15.31(standard deviation 1.09) and 46.86% of the sample was male. male were found to have higher impact of oidp (p<0.0001). there was na increase in oidp with an increase of lost teeth in the upper arch (0.0477) and maxilary overjet in milimeters (p0.0171). the social exclusion index was not significant in the model (p0.0913). conclusions: individual variables were related to the oidp in underprivileged brazilian adolescents. 034 motivational interviewing and oral health: importance attributed by primary health care dental professionals faustino-silva, d.d.*; hilgert, j.b.; hugo, f.n.; meyer, e. background: motivational interviewing (mi) is a simple evidence-based approach that is highly effective in addressing behavior change among patients in health care settings, including the field of oral health within primary health care. the approach is consistent with contemporary theories on behavior change. it is a client-centered communication style that encourages and strengthens patients’ intrinsic motivation to achieve positive change. aim: evaluate the importance and confidence of professional oral health teams in terms of mi. methods: cross-sectional analytical study conducted at the community health service of the grupo hospitalar conceição in porto alegre, rio grande do sul state (rs), within a primary health care setting. a total of 59 dentists and oral health technicians (ohts) participated in an 8-hour training session for active learning of basic mi principles. training was didactic in nature and consisted of theoretical elements and practical activities, such as debates and role-playing. preand post-test results were assessed using objective measures entitled the importance and confidence scale. this visual analog scale asks respondents to indicate how important it is to learn about mi and how confident they would feel about applying it in clinical practice on a scale of 0 to 10, where 0 corresponds to “not important” and 10 “very important”. results: according to the importance scale, before training 25% of the sample attributed an importance level of 10 to mi, rising to 50% after training. on the confidence scale level 8 was selected by 20% of participants prior to training, increasing to 32% once training was complete. participants exhibited a significant positive increase (p<0.005) in measures related to importance and confidence. analysis of posttraining measurements on the confidence scale suggests that some participants their ability to apply mi, while others overestimated it. conclusions: training professionals from phc oral health teams has the potential to increase the importance they attribute to addressing behavior changes in their patients and their confidence in applying these techniques, within the spirit of mi. 035 epidemiological profile of caries in children 12 years of age in são borjars paz, a.h.*; paz, d.s.; brizon, v.s.c.; pereira, a.c.; meneghim, m.c. background: prevalence of caries in schoolchildren of 12 years old in são borja-rs aim: evaluate the dmft index of school 12 years of age in são borja-rs, to determine the prevalence of caries in these children between 2008 and 2014 and also evaluate access of this population to the treatment of disease. methods: this cross-sectional study was conducted on a regular basis every two. data collection was implemented by dentists of the health department in all public schools in the city. dmft were collected from all students 12 years of age who were enrolled in schools. results: participants were 2290 students from 12 years of age, 53.23% were females. in this study, it was found that the prevalence of dental caries among adolescents was 43.27% in permanent teeth. the decay rate decreased over time, as the dmft in 2008 spent 0.9 to 0.7 in 2014. the number of filled teeth exceeded that of decayed in 2014, reducing the number of extracted teeth , showing therefore greater access to treatment of the disease.conclusions: it is essential that crosssectional studies are conducted sequenced way, because it is through them that allow carrying out the services offered and planning future health programs appropriate for the community. 036 molar incisor hypomineralization (mih): a case report. tomo, s.*; correia, t.m.; fortunato, j.b.; sakashita, m.s.; boer, n. p.; cunhacorreia, a.s. molar incisor hypomineralization (mih) describes a clinical statement of dental hypomineralization of systemic origin, which affects one or more first permanent molars (fpms) frequently associated to permanent incisors (pis) affected. etiological associations with systemic conditions or environmental insults during the child's first three years of life have been implicated. this report a ten years old female patient complaining of tooth sensitivity, difficulty eating, speaking, facial muscle pain, and dental caries in posterior teeth. case-report: the clinical and radiographic examination, was observed a discrepancy between the bony bases with apparent mandibular protrusion and dental hypoplastic stains in permanent incisors, as well as extensive injuries in the enamel of the first permanent molars, exposing the dentin. treatment plan was performed treatment with fluoride using varnish, shallow restorations with glass ionomer cement photopolymerized for the adequacy of oral environment. it is evident that molar incisor hypomineralization has important variations in their clinical characteristics such as color, texture and depth of the lesions, which indicate the degree of severity of the change and determine the appropriate choice of treatment. xix aboprev meeting/2015 braz j oral sci. 14(1) 85-99 92 037 association between indicators of vulnerability and oral health status of adolescents lucchette, a.c.t.*; vazquez, f.l.; taglietta, m.f.a.; guerra, l.m.; pereira, a.c.; bulgareli, j.v.; cortellazzi, k.l. background: the worsening of social vulnerability influences in the health-disease, including diseases of the oral cavity. aim: to evaluate the association between social vulnerability and family grant program with oral diseases and socioeconomic factors in adolescents of piracicaba-sp. methods: 1179 individuals aged 15-19 years of age from 34 family health units and 21 state schools. clinical examinations were performed under artificial light, using periodontal probe and dental mirror. data were collected from clinical variables (caries, periodontal disease, fluorosis and caries risk) and socioeconomic (family income, number of people in family, housing, education of father and mother, são paulo state social vulnerability index – idlh and family grant program gmp. to verify the association between the dependent variables (idlh and gmp) with the independent variables (clinical and socioeconomic) was used bivariate analysis by chi-square significance level of 5%. results: it was found that 60.9% of individuals who had not lost tooth, 67.4% of those parents had high school and college, and 58.3% with caries risk a, b, c and d were in the group with low vulnerability. teenagers lack of decayed teeth (81.1%), missing component (79.4%), dmft (83.1%) and ipc (81.1%), with caries risk a, b, c and d (81.3%), higher income (85.6%), fewer people in the home (86.2%), higher educational level of the father (89.9%) and mothers (88.2%) belonged to the group that did not receive the benefit family grant. conclusions: it was concluded that oral health policies with socially oriented promotion and recovery should be focused primarily adolescents with low socioeconomic status. 038 access flow study through health care center of beds macro-regional regulation in the north of parana state bado, f.m.r.*; lucchette, a.c.t.; buges, a.f.c.; meneghim, m.c.; guerra, l.m.; pereira, a.c. background: the unified health system provide universal access, effective care and quality in health care services of the brazilian population. mechanisms such as public health regulation are operated, which controls the balance between the supply of services and the demand of users with the purpose of correcting market failures and promoting social welfare. aim: the present study identified the obstructions to access through a quantitative and qualitative analysis in hospital assistance of medium and high complexity by investigating macroregional macro norte's hospital beds center, and was conducted in two stages. methods: in the quantitative, data registry of hospital beds with soliciting municipality, performing municipality and executing region, used accommodations and performed access, were collected. by analyzing the quantitative data, the second stage of the qualitative was carried out, which consisted of interviews with municipal and state supervisors. results: in the first part, it was verified that 11% of the registered cases had not been regulated, and that, for 36% of them, the access was not provided by routine mechanisms. it was confirmed that performance and bed accommodation were different in each health regulation's region. in the qualitative stage of the research, failures were pointed out, such as deficient attendance infrastructure, frailty in contracts between providers and with , vulnerability of health assistance networks, the role of regulation in promoting access and interference in regulatory actions. conclusions: macro north has been serving the purposes user access at emergency times, even though it does not maximum performance by organizational and structural problems. 039 individual and contextual variables related to periodontal disease: multilevel analysis in underprivileged teenagers oliveira, m.c.*; vazquez, f.l.; cortellazzi, k.l.; guerra, l.m.; zanin, l.; marques, a.c.; ambrosano, g.m.b.; pereira, a.c. background: adolescents are a group with particular characteristics, as they as subject to a range of psychosocial changes and exposure to situations of risk. aim: to investigate the individual and contextual risk indicators of periodontal disease in underprivileged adolescents, and the disparity in distribution of the disease. methods: cross-sectional analytical study, conducted in the city of piracicaba, sp, brazil, in 2012. the probabilistic sample was composed of 1,130 adolescents from 15-19 years of age, randomly selected from 21 state schools and 34 primary health units – family health (phu-fh). the dependent variable studied was presence of periodontal disease (cpi). the independent variables were classified into individual and contextual the multilevel regression model was estimated by the proc glimmix (generalized linear models-mixed) procedure, considering the individuals’ variables as level 2 and the contextual variables of the suburbs as level 3. results: as regards the individual variables, the adolescents who never had toothache, and with a lower impact on oral health, and resided in homes with higher percentage of domestic sewage percentage had less periodontal disease. conclusions: individual and contextual variables were associated with the presence of periodontal disease in underprivileged adolescents, indicating that they must be taken into consideration in the formulation of policies directed towards oral health promotion and prevention activities in this group. 040 interdisciplinary in esthetic and functional rehabilitation of patients with early loss of deciduous teeth giovani, p.a.*; rodrigues, l.p.; montes, a.b.m.; kantovitz, k.r.; magnani, m.b.b.a.; gavião, m.b.d. the early loss of deciduous teeth can be responsible for malocclusion. the case report discusses a multi-disciplinary approach to oral functional and esthetic rehabilitation on two high-caries-risk children due to endodontic complications associated with wide-spread dental caries. both 8-years-old patients were referred to the piracicaba dental school whose main complaint was pain. after intra-oral examination, the first patient was diagnosed with failure of previous endodontic treatment of mandibular left second primary molar with association of bone lyse involving the first premolar germ. in addition, there was an agenesis of mandibular left second premolar. the second patient presented extensive caries lesions of mandibular left first and second primary molar and mandibular right second primary molar, bone lysis, crypt rupture and periodontal infection affecting the germ of a mandibular left first premolar. in both cases, the tooth extractions were indicated following by esthetic and functional appliance to preserve the arch space and avoid early eruption of the successors, which were in the early stages of root formation. after 10-month follow-up, during clinical and radiographic evaluations could notice that the patients did not show phoniatric alterations and mesial movement of first permanent molar, preventing dental arch length. however, accelerated eruption of permanent teeth could be observed. also, parents reported patients’ esthetic satisfaction. it can be concluded that the integrated treatment between pediatric dentistry and orthodontics could avoid malocclusion and improve evidentially the patient’s facial esthetics, allowing better patients’ quality of life. 041 non syndromic oligodontia: a case report tomo, s.*; simonato, l.e. oligodontia is the term used to describe a rare and severe degree of dental agenesis, characterized by the congenital absence of more than six dental elements excluding the third molars, which are the most frequently missing teeth in cases of dental agenesis. the prevalence rates of this condition varies around 0.3% and the most frequently missing teeth are the maxillary lateral incisors and the mandibular second pre-molars. this condition may be associated to a syndrome or occur as a non-syndromic form, in familial traits or sporadic in nature cases. it has been shown that the congenital absence of dental elements are given to defects in specific genes, mainly the pax9 and msx1 genes, which plays key roles on the teeth development. several authors stand up for the hypothesis of the human dental reduction of size and number given to evolutionary factors, specifically, changes in dietary patterns. this report presents a case of a 20 years old male patient diagnosed with oligodontia due to the congenital absence of 11 dental elements, there were no systemic alterations or hereditary data associated. the preventive orthodontic intervention, performed in early age, allowed the patient to develop without any occlusion or phonetic alteration. 042 treating multicystic ameloblastoma in anterior mandible by partial mandibulectomy: a case report lima, k.f.a.*; silva, d.s.; tomo, s.; lúcia, m.b.i.; simonato, l.e. multicystic ameloblastoma is the most common of all odontogenic tumors originating from odontogenic epithelium, and despite it is considered a benign tumor in nature, it shows a wide aggressive behavior, growing through the trabeculae of the spongy bone. according to some authors the diagnosis may be performed by imaginologic exams, nevertheless, others affirm that the golden standard for diagnosing this pathology is the biopsy. the treatment choose for the multicystic ameloblastoma is also controversial, however, it is know that the best treatment is the surgical resection with a margin of 1,5cm of healthy bone. this case report aims to present a rare case of a multicystic ameloblastoma occurring in the anterior mandible of a 55 years old female patient, which the hypothetical diagnosis was based on the history of the lesion associated to imaginologic tests. the chosen treatment for the tumor was the partial mandibulectomy, aiming to avoid recurrence, once the literature points high recurrence rates in ameloblastic tumors conservatively treated. the microscopic evaluation of the tumor confirmed the diagnosis of multicystic ameloblastoma. xix aboprev meeting/2015 braz j oral sci. 14(1) 85-99 93 043 validity and reliability of the quality of masticatory function questionnaire applied in brazilian adolescents mamani, m.h.*; castelo, p.m.; barbosa, t.s. background: proper chewing and swallowing functions have great importance in general health, since it potentially affects food’s digestion and the subject’s nutritional status. aim: the aim was to assess the validity and reliability of the selfapplied quality of masticatory function questionnaire (qmfq) in a convenience (non-referred) sample of brazilian adolescents divided into three groups: control (n57), dental caries (n51) and malocclusion (n54). methods: caries experience and malocclusion were evaluated using dmf-s index (number of decayed, missing and filled primary and permanent surfaces) and index of orthodontic treatment need, respectively. qmfq comprises 26 items regarding frequency and intensity of chewing problems with five domains: food-mastication, habits, meat, fruits and vegetables. data were submitted to descriptive statistics, kolmogorov-smirnov and chi-square tests. psychometric evaluation included measures of reliability (internal consistency cronbach's alpha and matrix of correlation) and discriminant validity (kruskal-wallis/dunn post-test). results: the instrument showed satisfactory internal consistency, with significant positive correlations between the domains scores except between habits and vegetables. total scale cronbach's alpha coefficient was 0.87 and the coefficient did not increase significantly with the removal of each domain. significant differences were found between controls and caries group in food-mastication, meat and fruits scores. caries group also showed higher median values in food-mastication and fruits than the malocclusion group. conclusions: the quality of masticatory function questionnaire showed acceptable properties in terms of internal consistency, reliability, and discriminant validity in evaluating the impact of caries experience on the quality of masticatory function perception of brazilian adolescents. 044 the performance evaluation guiding the quality of attention in oral health (pmaq) in a northeastern brazilian city cavalcante, d.f.b.*; arruda, j.s.t.; meneghim, m.c.; pereira, a.c. background: there are few studies on evaluation in oral health in brazil. this scope the ministry of health since 2011 has been applying the evaluation of family health teams through the pmaq (program for improvement, access and quality). but little attention has been given to the preparation of those teams for this evaluation process. aim: the objective was to analyze the quality of performance indicators in oral health (pmaq-ab) and information about access and link. methods: two instruments were applied: a) comparing the data informed with those of dental records and b) the user informed about access, link and confirmation of registered procedures results: the acess was gotten over a period of up to one week, 43.75% registered showed inconsistency (10.93% subrecords) and 37% of the records of "first dental appointment" were invalid. the teams certified had better indicators. conclusions: it was concluded the population had access and link to the oral health teams and significant part of the records of procedures presented inconsistency. 045 the impact of oral health on quality of life of addicted persons marques, t.c.n.*; sarracini, k.l.m.; cortellazzi, k.l.; mialhe, f.l.; meneghin, m.c.; pereira, a.c.; ambrosano, g.m.b. background: the use of illicit and licit substances has increased worldwide with important health and social consequences, with human lives and productive years of people being lost. aim: the aim of this cross-sectional study was to evaluate the impact of oral health conditions, socioeconomic status and use of specific substances on quality of life of alcohol and drug addicted persons, receiving care at outpatient treatment facilities in brazil. methods: a random sample of 262 participants, mean age 37 years, from psychosocial care centers for alcohol and drugs (caps ad) located in three cities in the state of são paulo, brazil, were clinically examined for caries experience (dmft index) by a calibrated examiner. they were asked to complete a series of questionnaires, including the alcohol, smoking and substance involvement screening test (assist), socioeconomic characteristics, and the world health organization quality of life assessment (whoqol), which were considered the outcome variables of the study. associations between oral health status, socioeconomic characteristics, substance involvement with whoqol were investigated by means of the chi-square test and multiple logistic regression analysis with a level of significance α < 0.05. results: the mean dmf index of the group was 13.0. subjects with dmft >14 (or 2.25 ci 95% 1.303.89) low-income (or 2.41 ci 95% 1.15-3.59 1.22-4.77) and users of cocaine / crack (or 2.02 ci 95% 1.15-3.59) were more likely to have poor general quality of life. conclusions: this study demonstrated that the general quality of life of addicted persons was associated with caries experience, low income and cocaine/crack use. 046 importance of saliva to evaluate the cariogenicity of starch souza, s.*; sampaio, a.; cavalcanti, y.; cury, a.a.d.b.; silva, w.j.; cury, j.a. background: saliva has been used in models of cariogenic biofilm to only simulate acquired pellicle formation but when the cariogenicity of starchy products is evaluated it should be present during biofilm formation to simulate the effect of salivary amylase on starch metabolism. aim: evaluate the suitable exposition of saliva in a biofilm model composed of a. naeslundii, s. gordonii and s. mutans to estimate the cariogenic potential of subproducts of starch. methods: biofilms (n4) were formed on surface of bovine root-dentin slabs of known surface hardness (sh). the biofilms were exposed 8x/day to 1% starch. the biofilms were exposed to human whole saliva in three different steps: group 1 (acquired pellicle), group 2 (acquired pellicle + presence of saliva in the medium culture) and group 3 (acquired pellicle + presence of saliva in the medium culture and before each treatment with starch). the ph of the culture medium was measured daily to assess the biofilms’ acidogenicity. after 96 h of growth, the biofilms were removed from the slabs by sonication and biomass was determined. sh was again assessed in the slabs and percentage of surface hardness loss (%shl) was calculated. results: the groups did not differ in acidogenicity. group 3 showed greater biomass (9.3 mg) and %shl (16.4), which did not differ from the other groups (p>0.05). conclusions: saliva should be incorporated in biofilm models to evaluate the cariogencity of starch since it does not interfere in biofilm formation and is essential for starch metabolization. 047 characterization of novel spx-regulated genes in streptococcus mutans galvão, l.c.c.*; kajfasz, j.k.; miller, j.j.h.; scott-anne, k.; freires, i.a.; franco, g.c.n.; abranches, j.; rosalen, p.l.; a. lemo, j.a. background: two of the major environmental challenges encountered by streptococcus mutans in oral biofilms are acid and oxidative stresses. previously, we showed that the s. mutans transcriptional regulators spxa1 and spxa2 are involved in general stress survival. in particular, phenotypic characterization and microarray profiling indicated that spxa1 and spxa2, play a major role in activation of oxidative stress genes. we reasoned that some of the uncharacterized genes under spxa1/a2 positive control are potentially involved in oxidative stress management. aim: to use spx-regulated genes (srg) as a tool to identify novel oxidative stress genes in s. mutans. methods: quantitative real-time pcr was used to evaluate the responses of selected srg during h2o2 stress in the parent and δspx strains and these srg were selected for downstream mutational analysis. all mutants were characterized in relation to: growth under oxidative stress, iron sensitivity, ability to form biofilms in sucrose, and ability to colonize the teeth of winstar rats.results: among 7 srg tested, transcription of 6 was significantly induced during h2o2 challenge and was strongly dependent on spxa1. mutational analysis revealed that inactivation of selected srg has impact on biofilm formation, growth under oxidative stress and iron sensitivity. in particular, inactivation of smu248 displayed major growth defects in the presence of oxidative stressors, was extremely sensitive to free iron, and showed reduced ability to colonize the teeth of rats. conclusions: our results serve as an entryway into the characterization of novel genes and pathways that allow s. mutans to cope with oxidative stress. 048 concentration and activity of salivary carbonic anhydrase vi and dental caries. lopes, l.m.*; picco, d.c.r.; marques, m.r.; line, s.r.p.; nobre-dos-santos , m. aim: this study aimed to perform a quantitative analysis of the concentration and ca vi activity in saliva and investigate the correlation between these parameters and their relationship with dental caries in 7-9 year-old school children. methods: one hundred school children were divided into two groups (n50): (cf) caries-free group and (c) caries group. clinical examinations were conducted by one examiner (k 0,684) according to who criteria (dmft) + early caries lesions. from each subject, salivary flow rate (sfr), ph and buffering capacity (bc) were analyzed. salivary ca vi concentration and activity were performed by elisa and zymography respectively. the data was submitted to student’s t-test and pearson correlation analysis (α0.05). results: our results showed that there was no difference between c and cf groups respectively, regarding the sfr (0,722±0,05, 0,777±0,05 ml/min), salivary ph (7,209±0,05, 7,257±0,04) and bc (423,7±16,55, 455,6±13,54 mmoles hclph). however, it was observed that the ca vi concentration was significantly higher in cf children (0,8363±0,11 ng/µl) and that the ca vi activity was significantly higher in c children (2,933±0,31). we found no correlation between the ca vi activity and dental caries. also, a moderate negative correlation between the concentration and ca vi activity in saliva of c children (r0,3627, p0,02) and between salivary ph and dental caries (r-0,3635, p0,01) were found. conclusions: a higher concentration of ca vi in caries-free children may suggest a greater protection against dental caries. moreover, is related to lower activity of isoenzyme and these two parameters were negatively associated. xix aboprev meeting/2015 braz j oral sci. 14(1) 85-99 94 049 caries prevalence and treatment needs in eight coastal communities pará, brazil matos, ao*; carvalho, da; sakai, gp; jr, scc; beline, t; ogawa, es; barão, var; amaral, rc. aim: to know the oral health of children and teenager 0-15 years eight coastal communities of pará, located the banks of maró river, tributary of the river arapiuns, pa. methods: we carried out a visit to each community, lasting three days each visit, being the first day realized an epidemiological investigation in relation to dental caries, treatment needs (who criteria). in the second and third days educational, preventive activities were carried out, as well as dental treatment in this population. results: we examined 416 children and teenagers aged 0-15 years, and 183 0-6 years 234 7-15 years, 196 females and 218 males. the average dmft for five years was 5.3 (± 4.5) and the dmf to 12 years was 4.0 (± 3.1). the most prevalent treatments needs has restore cases 1 and 2 on both sides and extractions age. it was observed that this population is at-risk of tooth decay due to the lack of education regarding oral hygiene and the difficulty of access to the assistance provided by the health service in the region. conclusions: there is the need to meet the oral health of coastal communities, so that they can plan health promotion measures, together with the provision of services. 050 spatial analysis of mortality by oral cancer and oropharynx in brazil brizon, vsc*; fonseca, ep; fonseca, sgo, meneghim, mc; pereira, ac. background: the oral and oropharynx cancer is a multifactorial and the main risk factors are: tobacco, alcohol, mechanical trauma, biological agents, genetic predisposition, systemic and general condition of the individual. aim: the aim of this study was to correlate the deaths from oral and oropharyngeal cancer in brazil with the geographical location of people. methods: international statistical classification of diseases and related health problems (icd-10) was used as reference and underlying cause cancer of the mouth and oropharynx. data were retrieved from brazilian mortality information system (sim) and organized in absolute and relative frequency tables to perform the descriptive statistics. results: in 2011, 6973 deaths were recorded for oral and oropharyngeal cancer in brazil. being, 5473 (78,49%) men, 1500 (21,51%) women. the geographical distribution of mortality by region were 3499 (50.18%) southeast, 1533 (21.98%), northeast (17, 97%) south 434 (6.22%) percent west and 254 (3, 64%) north. regarding the spatial distribution of mortality can be said that it occurred heterogeneously with cluster formation in parts of south, southeast and northeast. conclusions: the spatial lisa index query showed that there is spatial autocorrelation for certain states. deaths from cancer of the mouth and oropharynx have high prevalence and are heterogeneously distributed across regions and states. 051 in vitro evaluation of remaining dentine after chemo-mechanical removal of caries in deciduous teeth mastrantonio, sds*; pinto, lams. background: traditionally, caries excavation was performed with use of burs, but today there are alternative methods such as chemo-mechanical for caries removal. aim: this study analyzed the chemical, morphological and physical characteristics of the remaining dentin after chemo-mechanical and mechanical removal of carious tissue. methods: thirty-three extracted carious deciduous molars were sectioned mesiodistally trough the center of the carious lesion into two similar halves. the 66 specimens were divided into 3 groups according to the method used for caries removal: cs group (carisolv™), pc group (papacárie®) and cb group (low speed conventional bur). the specimens were analyzed under confocal laser scanning microscopy (clsm), light microscopy (lm), scanning electron microscopy (sem), energy dispersive analysis of x-rays (eds) and microhardness. results: in clsm analysis, it was observed that the chemo-mechanical method removed less carious tissue. sem/eds analysis showed that ca content was statistically significant higher in the cb group. the cs and pc group showed similarity in relation to dentinal surface characteristics and statistical difference to cb group (heavy smear layer with all tubules obstructed). microhardness of dentine after caries removal was similar regardless of the method. histological analysis under light microscopy showed more preservation of dentin structure in cs and pc groups. conclusions: chemo-mechanical method is more conservative in the removal of carious tissue, leaving a dentine surface with less mineral content and no change in dentine microhardness when compared to the remaining tissue after use of conventional bur. 052 the effectiveness of strategies to oral health promote in primary health care in brazil: a comparative study filho, a.d.s.*; kusma, s.z.; moysés, s.j.; moysés, s.t. background: the oral health services in primary health care (phc) should achieve full shares in health (brazil, 2011). to be effective, strategies to oral health promote (sohp) should be structured based on the pillars equity, participation and sustainability and values governance, autonomy, empowerment, intersectoral, completeness (kusma et all, 2010.). aim: the paper analyzes the effectiveness of strategies to promote oral health (sohp) in primary health care (phc) in 26 brazilian capitals and the federal district, mapped in the national health promotion practices study – e.n.pro.sa. methods: comparison analyzes were performed stratified the final scores of 1,819 sohp-phc. the key informants were dentists working in the aps in 26 brazilian capitals and the federal district. data collection occurred in the second half of 2013, through the application of tool evaluation of the effectiveness of promotion of oral health strategies. after a brief description of sohp, 23 descriptors were answered on a likert pops, where 1 represents complete disagreement and 5 total agreement. the scores ranged from 23 to 115. the sohp with scores from 23 to 74, do not include the pillars and values of oral health promotion in the phc from 75 to 115, come. results: as a result, the 1819 sohp analyzed, 1,004 (55.19%) sohp not include the pillars and values of oral health promotion in the phc and 815 (44.81%) come. the proportion of sohp that include, by municipality is: rio de janeiro (76.9%), joao pessoa (75.6%), palmas (61.5%), são paulo (60.8%), belo horizonte ( 59.0%), fortaleza (57.1%), campo grande (54.7%), recife (53.1%), curitiba (50.3%), florianópolis (50.0%), são luís ( 48.9%), victoria (43.6%), porto velho (42.4%), porto alegre (40.4%), rio branco (38.9%), manaus (38.4%), salvador (37.3%), natal (37.0%), goiânia (36.8%), aracaju (32.5%), teresina (28.1%), cuiabá (21.3%), macapa (19 4%), belém (13.6%), brasilia (9.5%), boa vista (6.7%), maceió (6.5%). conclusions: based on the proposed theoretical model (kusma et al, 2010), concluded that most of the analyzed sohp, do not include the pillars and values of oral health promotion, demonstrating the low effectiveness of these sohp. there is great variability in the results in the brazilian capital, it is important that investments for the qualification of sohp-phc, considering the observed inequalities. 053 sb project brazil 2010: individual and contextual factors that impact on the decay of prevalence anselmo, mg*; battaglia, g; brizon, vsc; coppi, am; ambrosano, gmb; pereira, ac; cortellazzi, kl; silva, rp. aim: the objective of this study is to identify individual and contextual variables associated with the prevalence of caries in 12 614 individuals allocated the ages of 12 and 15 to 19, examined in the latest epidemiological survey of oral health in brazil sbbrasil 2010. methods: the individual independent variables "age", "demography", "harm to oral health," "socioeconomic", "education", "morbidity", "use of dental services" and "self-perception and impact of oral health on health" and contextual "bolsa familia", "index" development of the unified health system (idsus) "," human development index (hdi) "and" interaction gdp / fluoridated water "were raised from secondary data base public and institutional data from the department of primary care, the ministry of health of brazil. results: the results show that self-perceived need for dental treatment and dental impacts on daily performance and the toothache report are directly related variables to the development of caries in this population, while family income higher than r $ 1,500.00, value top idsus 5.76 and the presence of the interaction of gdp / water fluoridation in the municipalities were considered as protective factors to disease. conclusions: it follows that, in addition to fluoridation policies of the public water supply and access to oral health services in the sus, proper planning of oral health practices should also include self-reported subjective questions by the population. 054 botulinum toxin a, a viable form of treatment for gummy smile? machado, lg; souza, jc*. background: the action of the toxin is given by blocking the release of acetylcholine at the neuromuscular junction, which causes a process of muscular inactivity. this inactivity is welcome for treatment of certain muscle disharmony of the face. aim: let based on the manufacturer's protocol (botox ®) and literature to apply the clinical cases that the central complaint is the gummy smile. methods: we use in this clinical study, two female patients who sought the clinic complaining of gummy smile. photographed patients with canon eos rebel t3 camera with macro lenses 100mm and 50mm (normal) with flash macro ring lite yn-14 ex. they were imaged preoperatively (botulinum toxin) and after a week. preparing botulinum toxin a (100 u) diluted in 4 ml of saline solution, the tube we move in circles as recommended by the manufacturer and the hypodermic needle inserted to fill the syringe. choose the point on the face and apply the minimum dose to achieve the therapeutic effect on muscle activity. results: we had resulted in a response a week the effect of botulinum toxin under the gummy smile. we evaluate salivation and gum quality preoperatively. conclusions: efficacy, safety and patient satisfaction will be our criteria for evaluation. botulinum toxin type a was shown to be an option in the treatment of gummy smile for the patient returns a favorable labial position without invasive and costly surgical procedures. xix aboprev meeting/2015 braz j oral sci. 14(1) 85-99 95 055 the role of probiotics in the control of dental caries schroeder, t*; ripplinger, t; pinto, gs; cenci, ms. background: whereas the prevalence of dental caries has decreased worldwide, the disease still represents a global burden and it is considered the most prevalent chronic disease. consequently, new methods of prevention such as the use of probiotic bacteria have been investigated aiming the control of the caries process. aim: the aim of this review is to describe the current knowledge about the use of probiotics in caries prevention and the benefits and possible harms to the oral health. methods: the search strategy was carried out in pubmed and comprised the terms probiotics, caries, streptococcus mutans and oral health, and their combinations. original reports and reviews were included. as the inclusion criteria for clinical studies, the trial should have included the use of probiotics for caries prevention. results: initially found 106 articles, of which, according to the criteria of inclusion and exclusion were selected 94 full texts, included original papers and literature revision. among these 94 papers, 22 were clinical studies. half of those had used milk and dairy products, such as yogurt, cheese and ice cream as a way of probiotics consumption. most clinical studies analysed obtained positive results on reducing the level of cariogenic bacteria on saliva, but the most studies just used surrogates as endpoints. conclusions: the clinical trials on the role of probiotics in the prevention of caries are still very limited and further studies should be conducted to clarify its mechanisms of action and beneficial effects in the oral cavity. 056 erosive potential of tropical fruit juices romão, da*; veras, agc; hara, at; cury, ja; tabchoury, cpm. background: dental erosion has been associated to dietary habits and several studies, mainly in vitro, have evaluated the erosive potential of drinks. aim: evaluate the effect of juices prepared from brazilian tropical fruits on dental erosion. methods: an erosive cycling model, with the duration of 5 days, was conducted and bovine slabs (n5/group), selected by surface hardness and profilometry analysis, were treated 4 times/day with the respective solutions (2.5 ml/mm2 enamel area): purified water (negative control) 1% citric acid ph 3.75 (positive control) araçá-boi juice camu-camu juice cupuaçu juice taperebá juice and between treatments and during the night. artificial saliva was changed twice a day (after the 2nd and 4th treatments). the slabs were evaluated for surface hardness at the end of each day to calculate the percentage of surface hardness loss (%shl) and profilometry analysis was conducted again at the end of the experiment. data were analyzed by anova followed by tukey test. results: araçá-boi, taperebá and umbu juices showed the highest %shl, differing from the positive control and cupuaçu juice. the profilometry analysis showed that araçá-boi (2.3±1.0 µm) and umbu (3.2±1.1 µm) juices resulted in significantly higher surface loss than the positive control (0.5±0.4 µm). conclusions: the data of the present study suggest that some fruit juices have the potential to cause dental erosion. 057 concentration of total and reducing sugars on maltodextrins botelho, jn*; tabchoury, cpm; cury, ja. background: starch is a non-reducing sugar, but it can be enzymatically hydrolyzed in industrial scale, producing maltodextrins with different hydrolysis degrees and reducing by-products. maltodextrins are classified by their dextrose equivalent (de), which is a measurement of the amount of reducing sugars, relative to dextrose (de 100). thus, a high de value of maltodextrins means more reducing products of low molecular weight, including maltose and glucose. maltodextrins are commonly used in industrialized foods and can present cariogenic potential depending on the hydrolysis degree. aim: this study aimed to determine the amount of total and reducing sugars in maltodextrins of distinct de. methods: the solutions of glucose (positive control) and maltodextrins de 5, 20 and 40 were prepared in a concentration of 20%, from which the concentrations of total and reducing sugar of the commercial products were calculated. the concentration of total sugar and reducing sugars of the solutions (preparation in triplicate) were determined using colorimetric methods. results: the percentage (mean±sd) of total sugar found in commercial products was 99.2±0.8, 101.4±1.5, 100.7±0.7 and 102.3±1.8 for glucose, maltodextrins de5, de20 and de40 respectively. the percentage of reducing sugars were 100.4±3.3, 3.8±0.0, 12.0±0.2 and 29.9±0.3. conclusions: the findings showed that the hydrolysis degrees in the maltodextrins analyzed were lower than those reported by the manufacturer, what can influence their cariogenic potential. 058 moisture and presence of streptococcus oralis affect candida albicans biofilm growing on an in vitro model of oral mucosa bertolini, mm*; xu, h; sobue, t; thompson, a; cury, aadb; dongaribagtzoglou, a. background: studies using mice and in vitro mucosal models demonstrated that candida albicans and oral streptococci mixed biofilms present a more pathogenic potential. aim: to evaluate c. albicans biofilm with or without streptococcus oralis regarding its architecture, mucosal invasion ability and tissue damage, under wet (media-submerged) and semidry (media limited to inoculum) conditions. methods: it was used an in vitro model of human oral mucosa with immortalized keratinocytes (okf6-tert2) and 107 cells of c. albicans (reference strain sn425 or a mutant strain with paseudohyphae phenotype ndt80-/-), with or without 106 cells of s. oralis 34. after 16 hours at 37°c and 5% co2 incubation, tissue and supernatant were collected for analysis. biofilm architecture was evaluated by confocal laser scanning microscopy, histology sections stained by hematoxylin and eosin or fluorescent in situ hybridization showed tissue invasion and lactate dehydrogenase was monitored as tissue/cell damage indicator. results: wild type c. albicans formed homogeneous biofilm under wet conditions without invasion and leading slight tissue damage when present, s. oralis grew mostly in contact with mucosal surface. under semidry conditions, c. albicans formed a biofilm with welldefined foci, high tissue invasion and damage s. oralis was co-localized with biofilm foci. mutant ndt80-/formed a defective biofilm without invasion and tissue damage even for semidry condition s. oralis did not co-localized with this mutant. s. oralis presence promoted increase in fungal invasion and tissue damage under all conditions. conclusions: moisture and presence of s. oralis affect architecture and virulence of c. albicans mucosal biofilms. 059 effect of surface polishing on the in vitro formation of carious lesions subsurface soares, al*; iwamoto, as; reis, fmm; rontani, rmp; kantovitz, kr; pascon, fm. background: bovine teeth are considered good substitute for human teeth for conducting studies in vitro and in situ. many of these studies require the polishing of the tooth surface. however, this procedure could influence the demineralization process during formation of the carious lesions subsurface. aim: thus, the aim of this study was to evaluate the effect of surface polishing of bovine deciduous enamel in the formation of carious lesions subsurface. methods: fourteen primary bovine teeth were selected, sectioned in order to discard the roots and from the crowns, enamel blocks were obtained (5x5 mm), which were randomly divided into two groups (n 7): 1) polishing the enamel surface, and 2) absence of polishing the enamel surface. after that, the specimens were submitted to the formation of carious lesions subsurface, immersing them individually in demineralizing solution under saturated with respect to hydroxyapatite, for sixteen hours. the specimens were sectioned again (final thickness of 0.10 ± 0.02 mm) and evaluated concerning the depth of the formed lesion (in µm) using polarized light microscopy. results: the data were submitted to shapiro-wilk and anova tests (p<0.05). absence of significant difference was observed between the groups with and without surface polishing (84.01 ± 45.57 and 66.24 ± 13.88, respectively). conclusions: according to the conditions of this study, it can be concluded that surface polishing of the bovine enamel did not affect the in vitro formation of carious lesions subsurface. 060 occlusal art restorations using three different filling materials: preliminary results costa, ico*; hesse, d; bonifácio, cc; raggio, dp. background: the atraumatic restorative treatment (art) uses more commonly the high viscosity glass ionomer cement (gic) as filling material however some drawbacks related to this material may limit their indication. this draws the question on how a new developed material called glass carbomer cement (car) and compomer (com) influence the survival rate (sr) of occlusal restorations in primary teeth with the use of the art technique. aim: to compare sr of three different fillings materials on artrestorations in primary molarsocclusal cavities . methods: a total of 281 children (4-8 years old) with an occlusal carious lesion were selected and randomly allocated into the three restoration material groups: gic restoration (fuji ix – gc), com restorations (dyract – dentsply) and car restorations using (glass carbomer – gc corp). the children were treated on school premises following art principles. restorations were evaluated after 6 months by a calibrated examiner (cohen's kappa coefficient: к 0.78). restorations sr were evaluated using kaplan-meier survival analysis and log-rank test. rate ratio was calculated and cox regression analysis was used for testing association with clinical factors (α 5). results: the overall sr was 83.9% and the sr per group was gic 90% com81.7% and car81.2%. there was no significant difference among the three materials tested (rr 1.05, ci 0.72-1.53, p 0.79). cox regression analysis showed no influence of any investigated clinical characteristic in the survival rate of restorations. conclusion: there was no difference in the survival rate of the three materials tested in in primary molarsocclusal cavities. xix aboprev meeting/2015 braz j oral sci. 14(1) 85-99 96 061 pit and fissure sealants in permanent teeth: critical analysis of literature moreira, kms*, kantovitz, ks; pestana, jt; pascon, fm; rontani, rmp. background: dental sealants have showed to be effective in the dental caries prevention, especially on the occlusal surface. aim: this study performed a critical analysis of the literature about the occlusal pits and fissures sealant materials in permanent teeth of caries-risk patients, discussing the evidences on the sealants indication. methods: the authors searched the pubmed for papers from 1984-2014. the main search terms were "clinical trial", “sealants "and" permanent tooth". the inclusion criteria were: randomized studies showing the retention and progression of caries lesions of different sealants and techniques in sound and/or early enamel caries lesions occlusal surface. exclusion criteria: sealants fluoride release ability, patient's preference regarding sealing technique, sealing materials used for marginal defects repairing and sealing of dentin lesions. results: initially, 83 articles were identified, 22 studies were included in the project of critical appraisal by titles, abstracts and/or full versions. resin and ionomer sealant are the most commonly used. the efficacy of sealer materials was related to retention rate on the enamel substrate. since resin sealants showed higher retention rate and, consequently, lower increase of caries, they are indicated when the technique can be properly performed. the glass ionomer sealant is an alternative for partially erupted teeth, which preclude the use of rubber dam. conclusions: it can be concluded that resin sealants are the first choice for occlusal pit and fissures sealing. 062 sequelae of upper alveolar ridge trauma in permanent teeth by fall with baby walker kimura, js*; alencar, cjf; yanikian, cv; rosa, pcf; fonoff, rn; suga, ss; wanderley, mt. baby walkers are commonly related to injuries associated with falls downstairs. in canada, baby walker is prohibited since 2007 however, it is still being used in brazil even with pediatricians’ contraindications. to describe a case report of dental trauma in an infant due to the use of baby walker, and its sequelae in the permanent successors. an 8 months-old male patient fell downstairs in a baby walker and had a laceration on the upper alveolar ridge. at that moment he did not present any erupted tooth. at the age of 9 years old, the patient was referred to the clinic and centre of research of dental trauma in primary teeth of the university of sao paulo due to trauma sequelae observed in the upper permanent incisors on the radiographic exam. after clinical exam, we observed enamel hypoplasia on teeth 22/12 and 53/63 and upper labial frenum. on radiographic exam, tooth 21 was intra-osseo and malformed, and tooth 11 presented an image similar to an odontoma. it was performed ulectomy to facilitate the eruption of tooth 21 and upper labial frenectomy in the same appointment after this, orthodontic treatment was performed and with tooth in position it was performed gingivoplasty and restorative treatment with resin composite. an aesthetic functional space mantainer was performed until extraction of tooh 11. baby walkers should not be used by infants due to the possibility of occurrence of dental trauma and sequelae in their permanent teeth. this sequelae lead long term problems on patient’s treatment. 063 indirect composite resin restorations for oral rehabilitation of a child with early childhood caries: a case report huamani, jrs*, freitas, cn; martins, lfb; barbosa, ts; pascon, fm; rontani, rmp; kantovitz, kr. early childhood caries (ecc) is highly prevalent in developing countries. when this disease achieves atypical, progressive, acute, or rampant patterns, it implies serious consequences for the development of the stomatognathic system and for the child’s quality of life. although composite resin has been shown promising alternative to treat extensive carious destruction in posterior teeth. report a case of a child aged 4 years and 9 months, male, with ecc, submitted to oral rehabilitation using indirect composite resin restorations. clinical and radiographic examinations revealed the teeth premature loss (51), residual roots (52, 61, 62), caries lesion (53, 63) and extensive carious coronary destructions (55, 65, 75, 85). diet and oral hygiene instructions were given and supervised tooth brushing was conducted at every clinical appointment. initial procedures for improving the oral environment consisted of provisional restorations and extractions, followed by definitive restorations with composite resin. oral rehabilitation included indirect composite resin restorations, which consisted of clinical (cavities preparations and impressions), laboratorial (plaster models and indirect restorations preparations using an incremental technique) and bonding/finishing procedures. after 4 months, the long-term success of the treatment was confirmed by the retention of the restorations and the abscence of teeth pain, abscess and mobility. the full management, including preventive and curative measures, of a young child with ecc was found successful after 4 months of follow up. this result can encourage the clinicans to use indirect composite resin restorations to reestablish the oral functions and improve the child’s psycho-social development. 064 prevalence of dental trauma associated with risk factors in 6-17 years old schoolchildren in piracicaba sp and region neto, acc*; pereira, a; lima, t; lins, f; monteiro, mr; almeida, jf; zaia, aa; soares, aj. background: dental trauma exhibit significant prevalence worldwide and children and young people are the most affected. aim: the aim of this study was to evaluate the prevalence of traumatic injuries to the permanent teeth of children from public schools in piracicaba and region (sp) and its relationship with the presence of risk factors such as overjet and lip seal. methods: a cross-sectional study among students aged 6 to 17 years was conducted in thirteen public schools in the elementary and high school in piracicaba -sp and region (americana, limeira e campinas) from june 2007 to june 2014. the evaluation was conducted in a school environment and used the diagnostic classification modified o'brien. through clinical examination and completion of a standardized case report form, was analysed the incisal overjet and the lip seal (adequate and inadequate). results: was evaluated 610 male children (51.91%) and 565 female (48.09%), totaling 1.175 students. the prevalence of dental trauma found was 13.36% (n 157). were affected by traumatic injuries 92 school males (15.08%) and 65 females (11.05%) (p>0,05%). the students who had higher than 3 mm overjet and inadequate lip sealing were more susceptible to dental trauma (p <0,01). conclusions: it was concluded that the presence of higher than 3 mm overjet and inadequate lip sealing are risk factors to dental trauma recommending the implementation of preventive and health programs through information to parents, teachers and schoolchildren. 065 effect of cigarette smoke and ph cycling in dental structure theobaldo, jd*; catelan, a; júnior, wfv; mainardi, mcaj; ysnaga, oae; filho, upr; lima, danl; aguiar, fhb. background: it was not found in literature any study that linked the changes in tooth structure caused by smoking with the carious challenge. aim: the aim of this study is to evaluate the influence of exposure to smoke and ph cycling in dental structure. methods: 40 bovine dental fragments were allocated into 4 groups (n10): control (without treatment), cs (exposure to cigarette smoke), pc (ph cycling) and cs-pc (exposure to smoke and ph cycling). the specimens for groups cs and cs-pc were subjected to smoke from 20 cigarettes per day, for 5 days. the ph cycle consisted of specimens immersion in demineralizing solution for 4 h followed by immersion in remineralizing solution for 20 h at 8 days. surface microhardness (smh) was analyzed before and after treatments. cross–sectional microhardness (csmh) and rx-microfluorescence (μ-xrf) were performed after treatments. data were analyzed using anova (smh), split-plot anova (csmh), fisher's exact test (μxrf) and tukey test post-hoc (α 0.05). results: μ-xrf: increase of cd and pb and a low amount of ni and as in dental structure. smh and csmh means of cs was statistically higher than the control group. the samples exposed to ph cycling (pc and cs-pc) showed lower surface microhardness means than the groups not exposed to the cycling (cs and control), did not differ them. conclusions: the exposure to cigarette smoke increased the enamel microhardness without promoting a protective effect on the development of caries in vitro. 066 a biofilm model to evaluate the cariogenic potential of candida albicans – pilot study sampaio, aa*; souza, se; cury, aadb; silva, wj; cury; ja. background: streptococcus mutans are considered the most cariogenic bacteria and it has been suggested that candida albicans could enhance the cariogenicity of this bacteria, but this has not been clearly showed. in addition, there is not validated biofilm model to evaluate the cariogenicity of this fungus or its ability to increase the cariogenic property of s. mutans. aim: the aim of this pilot study was to validate a biofilm model for to evaluate the cariogenic potential of c.albicans. methods: s.mutans ua159, c.albicans atcc 90028 and s.mutans + c.albicans (n4) were formed on bovine root-dentin slabs of known surface hardness (sh), which were suspended in culture medium. the biofilms were formed for 96 h and they were exposed 8x/day to 10% sucrose. the ph of the culture medium was measured twice/day to assess the biofilm’s acidogenicity. after 96 h, dentin surface hardness was again determined and the percentage of loss (%shl) was calculated. data were analyzed by one-way anova, followed by tukey’s test (α0.05). results: the %shl (means±sd n4) for the groups c. albicans, s. mutans and s. mutans + c.albicans biofilms were respectively: 8.7±3.7 36.2±4.0 and 51.4±16.6, but the groups s.mutans + c.albicans and s.mutans did not differ statistically (p>0.05). conclusions: considering the size sampling of this study (n4), the findings suggest that c. albicans may increase the cariogenic potential of s. mutans. xix aboprev meeting/2015 braz j oral sci. 14(1) 85-99 97 067 gain autonomy and efficiency on careful of oral health maintenance during basic educational background, águas de são pedro-sp rochelle, imf*; ferraz, gc; brisolla, mjf; rochelle, abfa. the civil organizational of public interest (pais), in partnership with city office of education, lead a pilot project with, by applicating a didactic material, – appropriate to improve teachers’ knowledge and enable students – integrates on municipal schools in águas de são pedro-sp the oral health plan. despite the city has a high human development index hdi (0,854) presents alarming groups of caries disease in schoolchildren. the childhood is the step of life which allows with more facility the learning by changing habits, making this step the most favorable to introduce the health concept as quality of life. the presence of teacher on daily educational background of students is very strong and it facilitates the incorporation of care oral habit. the project consists on assess the results provided by the application of the didactic material. the teacher’s book provides background to enable the mainstreaming of oral health content in the school curriculum, being possible to avoid oral diseases as well as controlling it. the student´s notebook is a partner in the teaching-learning process, giving to the students specific knowledge about oral health for each school year, level to that, at the end of course, acquire autonomy and efficiency in care with teeth. the data collection will involve the whole school community in gathering information for qualitative analysis and epidemiology. the student, in its passage through elementary school, has the right to be able to keep your teeth in health status and gain autonomy until the end of the course. 068 permanent molar restoration using occlusal replica technique: case report scudine, kgo*; soares, al. the occlusal surface of posterior teeth have complex anatomical characteristics, making it difficult to reproduct in direct resin composite restorations. the occlusal replica is a technique that allows the anatomical and functional reconstruction by prior molding the occlusal surface with autopolymerizing acrylic resin. this technique is well-indicated in cases of hidden caries, characterized as a dentin lesion covered by enamel, demanding better accuracy in the clinical examination associated with radiographic examination.this clinic case presents the restorative technique using oclusal replica to restore the first permanent molar of a 9 year old. after clinical and radiographic examination, associated with transillumination, the tooth 36 was diagnosed with hidden caries. the main advantages of the occlusal replica are the technical ease of use, including its simplicity and its high accuracy in reconstructing occlusal morphology and the reduced clinical time by eliminating the restoration sculpture phase and simplifying the finishing procedure, being well indicated in patients in this age group. however, it is worth noting that for the realization of this technique is required specific material to construct the replica. 069 changes in dental arch dimensions in patients with class ii, division 1 malocclusion treated with headgear scudine, kgo*; siqueira, vcv; magnani, mbba; arbex, a. aim: the aim of this study was to evaluate possible changes in the intermolar distamce, intercanine distance and arch length, through dental casts, of patients undergoing orthodontic treatment with headgear. methods: to that end, we selected 12 pairs of dental casts of patients with class ii, division 1 before and after treatment with headgear, totaling 24 pairs of dental casts. the measures of intermolar and intercanine distances were performed on casts with digital calipers accurate to 0.000 mm. results: the results showed a statistically significant increase in the upper intermolar distances. there were no statistically significant differences in the intercanine distances and dental arches lengths. conclusions: it was concluded that orthodontic treatment with headgear in class ii,division 1 patients, in mixed dentition we can expect a significant increase in the upper intermolar distances. 070 historical series of caries experience among children in jundiaí, sp (brazil) silva, es*; sciamarelli, mc; miyashiro, lc; tonetti, j; pereira, c; batista, mj; armitt, d; meirelles, mp. background: epidemiological surveys of oral health contributes with construction and consolidation of a theoretical framework that enables the municipal management planning actions directed to population. aim: this study have monitored caries experience among children aged 12 through surveys conducted in 1975, 1998, 2003, 2008 and 2014 and its relation to collective actions of prevention and control of dental caries. methods: original reports were analyzed, assigned by the oral health management of municipality of jundiaí, located 50 km from the capital of são paulo, with a population of approximately 398,000 inhabitants. epidemiological exams were carried out among 12 years-old children in the school, following world health organization criteria for caries experience, using dmft index. results: the in 1975, the first study showed a high prevalence of dental caries, with a mean dmft of 10.05. after 1980, when started the water fluoridation in the city, it was observed a marked decrease in the caries index being: 2.33 in 1998, 1.69 in 2003, 1.22 in 2008 and 0.94 (ci95% 0.76-1.12) in 2014. in the last (2014), mean of untreated caries was 0.24. conclusions: the data of this study showed reduction in caries experience that may highlight the effectiveness oral health prevention, developed in this municipality over the 39 years since the first epidemiological survey. 071 pilot project: art as a tool for expanding access to dental treatment in jundiaí, sp marsi, cl; koga, ly; souza, ap; miyashiro, lc; santos, p; zago, c; amaral, rcgpa; silva, es*. the challenge of increasing access to dental treatment in the national health system has led municipalities to resort to strategies like the atraumatic restorative treatment (art) in school spaces in search of cost reduction, query time and organization of demand. through oral diseases for risk classification held at the school health program in jundiaí, it was found that 38% of students surveyed in américo mendes school caries lesions. this school, located in a rural area, was chosen to host the art application pilot project, in order to assess the feasibility of their use to expand access to dental treatment and reduce the risk for dental caries of students in the most vulnerable areas . the team consisted of four dentists and three assistants. the total number of school students is 150, aged between 6 and 9 years, 132 examined, with 12% loss for absences. 51 children at high risk for tooth caries, 5 forwarded directly to treatment in the reference basic health unit for presenting pain of dental origin. 46 students passed through art, and 93 treated teeth of a total of 132 teeth with treatment needs related to dental caries in 10hrs of team care. of treaties, 19 children were referred for treatment in office. even in progress, the project was favorable for increasing access for fast service, low absenteeism and good use of professional hours. 072 dental agenesis with genetic heritance: a case report rebouças, pd*; rodrigues, lp; araújo, ds; martins, l; rontani, rmp; júnior, fhn; kantovitz, kr. tooth agenesis, the congenital absence of one or more teeth, is one of the most relevant abnormalities of human dentition. the prevalence of permanent tooth agenesis, excluding third molars, varies from 2.2% to 10.1%, depending on racial and ethnic backgrounds. molecular studies have shown that more than 300 genes regulate tooth morphogenesis, and that mutations in axin2 (axis inhibition protein 2), msx1 (muscle segment homeobox 1), pax9 (paired box gene 9) and wnt10a (wingless-type mmtv integration site family, member 10a) and complete or partial disruption of the eda (ectodysplasin a) signaling pathway may lead to teeth agenesis. the aim of the present study is to report a case of dental agenesis with potential genetic heritance affecting 4 consanguineous individuals (3 female/1 male). a 10-year-old boy was referred to the piracicaba dental school due to the absence of a number of permanent teeth. intraoral and radiographic examinations indicated the absence of all mandibular incisors and maxillary lateral incisors and pre-molars. family pedigree was analyzed and the affected individuals were clinically and radiographically documented. data analysis strongly suggested that the inheritance might be associated with x-cromossome and family clinical findings, and therefore, eda was chosen as the candidate gene to be sequenced. xix aboprev meeting/2015 braz j oral sci. 14(1) 85-99 98 073 integral approach in pediatric dentistry: a case report cardoso, aa*; moreira, kms; iwamoto, as; pascon, fm; kantovitz, kr; rontani, rmp. the pediatric dentistry is a special area in the dentistry, since it includes the integral care of the baby, child and adolescent, aiming rehabilitation and maintaining oral health in the growing people. the aim of this study was to report the integral approach developed in a pediatric dentistry case. memo, a high caries risk/activity child patient aged 6y and 8mo, was referred to the pediatric dentistry clinic, at fop/unicamp presenting visible biofilm in free smooth surfaces, active carious lesions, early loss of the 85 tooth, fistula related with the 75 tooth region, color change of the 61 tooth and significant loss of posterior vertical dimension. treatment plan consisted in integral approach focused as primary outcome on health promotion, behavior change in relation to oral health, and as secondary outcome, the surgical/restorative approach. the oral hygiene instruction was conducted in all sessions. the conditioning behavior has been driven to dental biofilm (evidence and control of visible dental plaque), prophylaxis and oral environment adequation extraction of 75 tooth, pit and fissure sealing of the 36, 46, 16 teeth, and restorations in the 55 and 65. a lower removable appliance (space maintainer) was made. after 6 months it could be noticed that there was no visible biofilm, the ip was controlled and there was no evidence of new caries lesions. it could be concluded that the success obtained on this clinical case stand out the importance of the integral performance focused on healthy promotion in pediatric dentist clinic. 074 condition observed versus self-perception of oral health of families of pickers of recyclable material of passo fundo-rs cardoso, ac*, corralo, dj; palma, dr; comim, ld; tessaro, t. background: pickers of recyclable material get income from an informal labor market, which reflects the living conditions of these families. aim: this study examined whether the oral health perception of pickers of recyclable material corresponds to the oral health status observed by the oral health professional. methods: individuals were selected from families of pickers of recyclable material (valinhos neighborhood, passo fundo, rs) (15-90 years). data on self-perceived oral health were obtained by interview (questionnaire sbbrasil 2010). clinical data were obtained by dental examination (the world health organization criteria). the study was approved by the ethics committee (university of passo fundo) and the participants agreed to participate in the study. data were analyzed descriptively. results: of the women surveyed (n 20), 95% needed some type of dental treatment of these, 65% reported feeling the need for treatment and 30%, not (a case of no perceived need and observed coincident). of men (n 18), 100% requiring dental treatment 55.6% reported feeling the need for treatment and 38.9% did not. among the interference conditions in the daily activities of individuals who reported treatment need was highlighted the difficulty of eating associated with irritability and difficulty sleeping (75%). conclusions: the oral health perception of pickers of recyclable material differed from that observed by the oral health professional, reported less need for treatment than that observed, indicating that the social environment in which individuals live can influence the oral health status perceived. 075 oral health status of families of pickers of recyclable material of passo fundo-rs corralo, dj*, cardoso, ac; palma, dr; comim, ld; tessaro, t. background: the public health system has not supplied the basic health needs of individuals. socially excluded populations are more affected by system weaknesses. aim: this study aimed to perform an epidemiological survey on the dmft index of pickers of recyclable material of valinhos and bom jesus neighborhoods (passo fundo rs). methods: individuals were selected from families of pickers of recyclable material of valinhos and bom jesus neighborhoods (passo fundo, rs) (15-74 years). the epidemiological survey was conducted following the criteria of the world health organization. it wasn’t held prior oral hygiene and no auxiliary apparatus for lighting the oral cavity. the tests were performed at home with the help of woonder spatula and gauze. the study was approved by the ethics committee (university of passo fundo). the participants agreed to participate (informed consent term). data were analyzed descriptively. results: were included 59 individuals (38, valinhos, 21, bom jesus). the mean dmft was 18.64 (valinhos) and 19.04 (bom jesus). in the age groups analyzed, the dmft observed in valinhos and bom jesus were 8.42 and 7.66 (15-19 years) 11.54 and 10.40 (20-34 years) 18 and 16.4 (35-44) 29.46 and 25.25 (45-64 years) and 31 and 26 (65-74), respectively. the decayed and lost components contributed more than 50% of the dmft observed in the two communities. conclusions: the oral health status of the families of pickers of recyclable material in the city of passo fundo is precarious and reflects the need dental care for these communities. 076 effect of sucrose on streptococcus mutans adhesion to enamel oliveira, bec*; cury, já; filho, apr. background: sucrose is the only dietary carbohydrate that is substrate for extracellular polysaccharides (eps) synthesis and insoluble glucans formed could facilitated the adhesion of cariogenic microorganisms to enamel surface, forming biofilms. aim: therefore, this study evaluated the adhesion of streptococcus mutans to enamel in the presence of sucrose or its constituent monosaccharides, glucose and fructose. methods: cultures of s. mutans ua159 were grown overnight in lmw medium supplemented with 1% glucose. the bacterial inoculum was washed in pbs and bacterial density was adjusted (od 1.6 660 nm). the adhesion was made on bovine enamel slabs (n12) of known surface hardness (sh). after salivary pellicle formation on the slabs, these were immersed in lmw medium containing the bacterial inoculum and 0.525% glucose + 0.525% fructose or 1% sucrose. after incubation for 8 h at 37 oc and 10% of co2, the following analyses were made: percentage of sh loss (%shl, as indicator of demineralization), medium ph (indicator of acidogenicity) and cfu adhered to enamel. eps formation was assessed by confocal laser microscopy. data were analyzed by student's t-test (α 5%). results: the groups did not differ neither for acidogenicity (5.1 ± 0.5 and 5.3 ± 0.3) nor %shl (14.0 ± 5.9 and 10.9 ± 5.0) (p> 0. 05). higher bacteria adhesion (difference of 2 log cfu) was found for sucrose group (p<0.05), which also showed eps formation. conclusions: it was concluded that the adhesion of s. mutans to enamel depends on the metabolization of dietary sugars in eps rather than their fermentation to acids. 077 assessment of psychological factors, sleep and oral health-related quality of life in children with sleep bruxism araujo, ds*, sousa, bs; barbosa, ts. background: the literature suggests an association between psychological factors, such as anxiety and depression, and the presence of sleep bruxism (sb). sb may be associated with negative impacts on the quality and duration of sleep, which may compromise the quality of life. aim: this study aimed to evaluate 36 eightto tenyr-old children (12♂, 24♀), with mixed dentition, divided in two groups (bruxists, n12 controls, n24) matched for gender and age. methods: sb was confirmed by parental report of grinding sounds and the presence of shiny and polish facets on incisors and/or first permanent molars. the index of orthodontic treatment need was used for occlusion evaluation. self applied questionnaires were used to evaluate the quality and duration of sleep, symptoms of anxiety and depression and oral health-related quality of life. data were analyzed using shapiro-wilk, chisquared and unpaired t tests. results: approximately half of the children had slight need of orthodontic treatment (58.3% bruxists 45.8% controls) (p>0.05). controls reported more functional limitations and negative impacts on social well-being than bruxists (p<0.05). the groups did not differed in relation to quality and duration of sleep and symptoms of anxiety and depression. conclusions: in the evaluated sample, while sb was not associated with psychological factors and quality of sleep, children with this parafunction reported better ohrqol than healthy children. 078 work-related musculoskeletal disorders: dentists need to get up soares, acb*; castilho, avs; orenha, es; kaieda, ak; marques, tcn; sarracini, klm; meneghim, mc. background: ergonomics non-compliance of dental equipment is recurrent which contribute for impairs the performance of dental procedures and obliges the dentist and staff to adopt inadequate working postures, producing injuries that can lead to work-related musculoskeletal disorder (wrmsd). methods: this study aimed to evaluate the ergonomic compliance of dental equipment through two methodologies: m1 a proposal of criteria which are consistent with the philosophy currently practiced in the brazilian market and m2, which considers ergonomic criteria proposed by the european society of dental ergonomics (esde), whose adoption has been increasing in the european dental market. results: dental equipment from 39 workplaces was assessed and findings shown that there was great differences of the conformity achieved between method 1 and method 2. on average, by using m1 resulted in 80% of compliance and by using m2 resulted in 57% of ergonomic compliance and it was founded that there was a regular level of ergonomic compliance regarding to newest requirements recommended by esde. conclusions: corrective procedures are necessary once the high prevalence of nonconformity causes injury, reduced efficiency and comfort as well as leads to loss of quality of service. xix aboprev meeting/2015 braz j oral sci. 14(1) 85-99 99 079 assessment of salivary composition, flow and ph, halitosis and taste in asthmatic children freitas, cn*, zanon, an; amato, jn; marquezin, mcs; castelo, pm; barbosa, ts. background: the influence of salivary characteristics, taste perception and halitosis on the physiopathology of asthma is still scarce in the literature. aim: this study aimed to evaluate the salivary composition, flow and ph, halitosis and taste of 59 sevento ten-yr-old children, both genders, selected from policlinic santa teresinha doutor antonio haddad dib (asthmatics, n28 ♂18, ♀10) and from public schools (controls, n29 ♂10, ♀19), piracicaba, sp, brazil. methods: the concentration of volatile sulfur compounds was measured using a gas chromatograph. stimulated and unstimulated saliva were collected to determine salivary flow and ph. the drop test with four different flavors in three different concentrations was used to evaluate the taste perception. salivary concentrations of total protein, amylase, calcium and phosphate were determined by spectrophotometry. the results were analyzed using descriptive statistics, shapiro-wilk, mann-whitney and wilcoxon tests (α0.05). results: halitosis, salivary composition, flow and ph were similar between groups. asthmatics had lower scores of taste perception than controls (8.93±2.16 vs. 10.21±2.35 p<0.01), and the sweet solution (0.05g/ml of sucrose) was the less perceptive (60.71% of asthmatics). when comparing stimulated and unstimulated saliva, both groups had higher phosphate concentrations and total proteinfor the latter than the former (p<0.01). asthmatics also presented higher concentration of calcium in unstimulated saliva (1.36±1.79 vs. 0.64±0.67 p<0.001). conclusions: asthma in children was associated with worse perception of taste and higher concentrations of calcium, phosphate and total protein in unstimulated saliva. 080 oral manifestation caused by acid vapors campos, rmms*; queluz, dp. background: prevention is a health branch and occupational activity that has as main purpose the protection of workers from risks to their health when exposed in their activities. aim: to report that the dentistry occupational health has an important role in preventing occupational poisoning linked to acid vapors. methods: it was to conduct a literature review of key articles related to the topic. results: health damage often begins in the mouth and when diagnosed early by occupational dentist, allowed the worker is protected from disastrous consequences. in the group of chemical agents of occupational diseases, the acid vapors play a role of great importance, under the dental point of view. the occupational dentist recognizes and prevents risks that are causative of oral manifestations of diseases arising from work as well as the correction of these lesions. all of these acids, from a certain concentration in the atmosphere of the workplace (form of vapor) cause intense irritation of the upper airways, which causes employees to breathe through the mouth also. as a result, the incisors are exposed to the action of air and quickly lose their protective cover of saliva, making it so easily attacked by acids. in gold handling, a systemic poisoning occurring initially an oral manifestation known as a reddish tinge on the gums, in more extensive cases it is noted presence of ulcers in the oral mucosa. conclusions: preventive measures should be implemented collectively. in work environments with physical possibility, the ideal is exhaust ventilation where there is no such possibility the use of personal protective equipment being accompanied by occupational dentist. 081 evaluation of burnout syndrome in dentists the city of cuiabá-mt, brazil battaglia, g; anselmo, mg; sguarezi, d; sguarezi, d; pereira, ac; meneghim, mc; possobon, rf; abrosano, gmb. aim: to evaluate the prevalence of burnout syndrome in dentalsurgeons in the city of cuiabá-mt and analyze its association withsociodemographic and economic variables. methods: this was an observational study, cross-sectional, analytical,quantitative in nature. human services survey (mbi-hss) sociodemographic,economic, and the maslach burnout inventory: the sample consisted of 253volunteers dentists two autoaplicativos questionnaires were used. we appliedtwo methods of data collection. the first, by delivering the questionnairespersonally for later pickup. the second, by invitation via e-mail attachmentcontaining the link to access and complete the survey and forward the answersto a database. initially, data were analyzed using frequency tables and chi-square or fisher exact tests. the following associations of each variableindividually were studied, and the syndrome (yes or no) by crude analyzes.variables with p ≤ 0.20 in the crude analysis were tested in a multiple logisticregression model remained in the model with p ≤ 0.10. results: in the associations between sociodemographic characteristics andburnout syndrome, considering each variable individually, without adjusting forother characteristics, it is observed that dentists females (p 0.0552), age ≤ 40years (p 0.0409), family income up to r $ 5,000.00 (p 0.0133), withoutmasters or doctorate (p 0.0523) have more chance to express syndrome. stillconsidering the individual variables (crude analysis), behavioral characteristics,professionals serving more than 10 patients per day (p 0.0266) and non-teaching staff (0.0641) has more chance of presenting syndrome. volunteerswho reported no sleep usually have more chance of having the syndrome (p <0.0001). in the adjusted analysis it was observed that those who stated they didnot sleep normally, those with family income up to r $ 5,000.00 and servingmore than 10 patients per day is 4.88 (ic95%: 2.22 10.73), 2.06 (ic95%: 1.123.80), 1.66 (ic95%: 0.95 2.92) times more likely, respectively, to introduceburnout syndrome. conclusions: the survey showed that 51.4% (95% ci: 45.2% -57.5%) ofdental surgeons of cuiabá-mt have high prevalence for developing burnoutsyndrome. oral sciences n3 braz j oral sci. 11(1):25-29 received for publication: october 15, 2011 accepted: december 12, 2011 original article braz j oral sci. january | march 2012 volume 11, number 1 reproducibility of caries diagnosis in permanent teeth according to who, icdas-ii and nyvad criteria priscila florentino silva1, franklin delano soares forte2, ana maria barros pereira chaves3, isabela albuquerque passos farias4, kaline silva castro5 1graduate program in dentistry, master’s degree program in preventive and pediatric dentistry, federal university of paraíba, joão pessoa, brazil 2phd, graduate program in dentistry, master’s degree program in preventive and pediatric dentistry, federal university of paraíba, joão pessoa, brazil 3phd, graduate program in dentistry, master’s degree program in preventive and pediatric dentistry, federal university of paraíba, joão pessoa, brazil 4professor, department of restorative dentistry, federal university of paraiba, joão pessoa, brazil 5graduate program in dentistry, master’s degree program in preventive and pediatric dentistry, federal university of paraíba, joão pessoa, brazil correspondence to: priscila florentino silva pedro ferreira de freitas st, 48, cep: 58052-755 jardim cidade universitária, joão pessoa pb brazil phone: +55 83 8880-6399 / +55 83 9609-0660 e-mail: priscilafsilva2007@yahoo.com.br abstract aim: to assess inter-examiner reproducibility in the detection of 20 occlusal caries in permanent teeth using three diagnostic codes and criteria: who (1997), nyvad and icdas-ii. methods: three graduate students (g) and two undergraduate dental students (ug) without previous experience in the use of nyvad and icdas-ii were trained by a reference examiner. examiner validity was assessed by consensus agreement between the investigators. two cutoff points were used for icdas-ii and nyvad to represent reproducibility values: the alesion, bcavity lesion. results: according to the examiners consensus, the kappa values ranged from 0.71 to 0.85 for g group and from 0.85 to 0.95 for ug group (icdas-ii). for the nyvad index values varied from of 0.77 to 1.00 (g) and from 0.65 to 0.74 (ug), for the who index, values obtained ranged from 0.66 to 1.00 (ug) and 1.00 (g). using a cutoff a, interexaminers reproducibility (icdas-ii) ranged from 0.73 to 0.87 (g) and 1.00 (ug). according to nyvad criteria, the kappa value ranged from 0.78 to 1.00 (g) and from 0.70 and 0.90 (ug) when compared to the consensus. the kappa values using the cutoff point b ranged from 0.66 to 1.00 (g), 0.76-0.89 (ug) in icdas-ii and the in nyvad criteria varied from 0.87 to 1.00 (g) and from 0.65 to 0.88 (ug). conclusions: reproducibility values ranged from good to perfect. the reproducibility revealed precise answers in the occlusal caries lesions diagnosis according to the criteria used. the best use of reliability tools for examiner training was important regardless of the examiners being undergraduate or graduate students. keywords: dental diagnosis, tooth decay, reproducibility. introduction in recent years, the prevalence of dental caries in children and adults has declined and changed both in industrialized and in developing countries like brazil1-2. the international epidemiological criteria used in brazil, the who standard, do not register lesions caries in pre cavitations stages, such as white spot 2626262626 braz j oral sci. 11(1):25-29 lesions3. thus, the progress of non cavitated caries lesions in populations it is an important evidence of using new indices in a public with new dental profile. treatments that emphasize prevention need diagnostics that reveal the real stage of carious process, from the subtle demineralization changes to the cavitations itself, monitoring the individual routine4. the development of new criteria for detecting initial carious lesions process are followed by studies5-6. among the new caries detection systems are nyvad et al. (1999) 7 and international caries detection and assessment system ii (icdas-ii) created in 20028. proposed by nyvad et al. (1999)7, the nyvad criteria includes initial manifestation of caries in the pre-cavitary stages differencing active and inactive caries in both levels cavitated and non-cavitated. three stages of severity related to the depth of penetration are considered in nyvad index: intact surface, descontinuous surface and cavity in enamel or dentin, avoiding use of an explorer unless the visual examination is not sufficient to choose lesion as active or inactive. the icdas-ii is another set of criteria for non-cavitated caries diagnosis. it recommends the direct visual examination on clean teeth, followed by careful drying of the of the lesion surface preceding the exam, identifying the state of teeth using an ordinal scale from the health surface to cavitated caries. according to studies, epidemiological surveys using these new indices are possible with acceptable reliability in brazil9-10 and other countries11-13. reproducibility is recognized as the ability to obtain similar results on several examination and has been considered of great importance when evaluating a particular method of validation, ensuring uniformity of caries criteria interpretation14-15. the aim of this study was to assess the reproducibility of visual examination of occlusal caries lesions in human permanent teeth using the who diagnostic criteria (1997), nyvad and icdas-ii. graduate and undergraduate students of dentistry course at the federal university of paraíba were selected as examiners for this study. material and methods this research was approved by the ethics committee of lauro wanderley hospital, federal university of paraíba, under protocol #458/10. the procedures for this research complied with the guidelines and regulations involving human subjects, approved by resolution no. 196 of 10 october, 1996 national council of health16. three graduate students (g) and two undergraduate students (ug) concluding the dentistry course were selected as examiners and were trained in who (1997), icdas-ii and nyvad indexes. the examinations were performed in a clinical setting with access to triple syringe and artificial light. approach to other tests performed previously with other codes and criteria was not allowed, also tests performed by another examiner. the dental mirror and who periodontal probe were used without pressure on tooth surface, only to precise questions arising from the visual diagnosis. the teeth were kept immersed in 10% neutral formaldehyde preventing changes in their appearance. drying the tooth was permitted. the examiners were trained with sb brasil 201015 methodology using the consensus method. according to this method, the prevailing consensus of the examiners was used as gold standard. initially, the activities consisted of a lecture of codes and criteria to be used in the subsequent examination, digital pictures of the teeth with occlusal caries lesions were presented and theoretical exercises were performed. the examiners also practiced on icdas elearning17 electronic program with images of dental caries according to the scores and theoretical exercises. clinical training the training phase consisted of clinical examination of 10 teeth with several situations, from healthy teeth, white spot, enamel fracture, cavitation in dentin to teeth with extensive destruction. the examiners reviewed the examination register in their files and discussed disagreements to reach a consensus. following the training methodology, 20 teeth were examined. all examiners completed the tests, recorded the results in their files, discussed to obtain consensus and a common consent filled was used to calculate the kappa coefficient of sample. tests with other criteria were conducted after 1 week to reduce the influence of one system in subsequent examination. the last criteria assessment was oms (1997)3. cutoff two criteria cutoff points were used to analyze the nyvad and icdas-ii results. thus, the cutoff a (lesion) for nyvad and icdas-ii criteria considered caries lesion all present demineralization at enamel or dentin, excluding score “0” (without demineralization). the cutoff b (cavity lesion) classified carious lesions those with discontinuous surface (icdas-ii scores 3, 4, 5 and 6, and nyvad scores 2, 3, 5 and 6), the other scores 0, 1w, 1b, and 2b, 2w (icdas-ii) and 0, 1 and 4 (nyvad) were considered as “not decayed”. interexaminer agreement was expressed by weighted kappa coefficient. the recommended values for agreement were superior to 0.65 (the same adopted in sb 201015 brazil methodology). results the kappa results of inter-examiner reproducibility tests obtained from the cohen’s kappa index and classified according to landis and koch18 are presented (table 1). the interexaminer reproducibility verified according to the consensus icdas-ii ranged from 0.71 to 0.85 for g and 0.85 to 0.95 for ug, nyvad from 0.77 to 1.00 (g) and 0.65-0.74 (ug), oms value 1.00 was obtained for g group and from 0.66 to 1.00 for ug group. for a cutoff a, the reproducibility according to consensus (icdas-ii) ranged from 0.73 to 0.87 for g and 1.00 for ug. reproducibility of caries diagnosis in permanent teeth according to who, icdas-ii and nyvad criteria braz j oral sci. 11(1):25-29 2727272727 table 1 -table 1 -table 1 -table 1 -table 1 inter-examiner reproducibility exam for the contents icdas-ii nyvad, cohen’s kappa index †graduate student *examiner ‡ consensus file ¥ undergraduate student reproducibility pg† criteria ex*2 x cs‡ ex3 x cs ex4 x cs ex2 x ex3 ex2 x ex4 ex3 x ex4 icdas-ii 0.85 0.71 0.79 0.61 0.73 0.70 nyvad 0.77 0.93 1.00 0.72 0.77 0.93 oms 1.00 1.00 1.00 1.00 1.00 1.00 reproducibility g¥ criteria ex2 x cs ex3 x cs ex2 x ex3 icdas-ii 0.95 0.85 0.78 nyvad 0.74 0.65 0.45 oms 0.66 1.00 0.66 reproducibility pg† criteria ex*2 x cs‡ ex3 x cs ex4 x cs ex2 x ex3 ex2 x ex4 icdas-ii 0.87 0.73 0.76 0.57 0.87 nyvad 0.88 0.78 1.00 0.68 0.88 reproducibility g¥ criteria ex2 x cs ex3 x cs ex2 x ex3 icdas-ii 1.00 1.00 1.00 nyvad 0.70 0.90 0.60 †graduate student *examiner ‡ consensus file ¥ undergraduate student table 2 -table 2 -table 2 -table 2 -table 2 inter-examiner reproducibility exams using cutoff a in icdas-ii and nyvad index, cohen kappa. reproducibility pg† criteria ex*2 x cs ‡ ex3 x cs ex4 x cs ex2 x ex3 ex2 x ex4 ex3 x ex4 icdas-ii 1.00 0.66 0.87 0.66 0.87 0.56 nyvad 0.87 1.00 1.00 0.87 0.87 1.00 reproducibility g¥ criteria ex2 x cs ex3 x cs ex2 x ex3 icdas-ii 0.89 0.76 0.66 nyvad 0.65 0.88 0.56 table 3 table 3 table 3 table 3 table 3 inter-examiner reproducibility exams using cutoff b in cdas-ii and nyvad index, cohen kappa. †graduate student *examiner ‡ consensus file ¥ undergraduate student disagreement involving occlusal caries lesions disagreement (%) cavitated non-cavitated active inactive pg† 5.0 33.3 66.6 58.3 41.6 g¥ 20.0 68.8 31.3 40.0 60.0 †graduate student ¥undegraduate student table 4 -table 4 -table 4 -table 4 -table 4 percentage distribution of diagnostic disagreements among examiners. reproducibility of caries diagnosis in permanent teeth according to who, icdas-ii and nyvad criteria 2828282828 braz j oral sci. 11(1):25-29 for nyvad criteria, the kappa value ranged from 0.78 to 1.00 (g) and for g group was 0.70 and 0.90 (table 2). data obtained from the cutoff point b are presented in table 3. in icdas-ii, the kappa value calculated from consensus ranged from 0.66 to 1.00 (g) and from 0.76 to 0.89 (ug), for nyvad criteria cut off b was 0.87 to 1.00 (g) and from 0.65 to 0.88 (ug). the reproducibility values ranged from good to very good agreement, according landis and koch18. table 4 shows the distribution of discordant diagnoses between the graduate and undergraduate examiners. graduate students disagreed at 15% of examinations and 20% of undergraduate examiners tests was discordant. among the disagreements, 33.6% involved cavitated lesions and 66.6% involved non-cavitated lesions (41.6% were inactive lesions and 58.3% active lesions). the graduate examiners obtained 68.75% of discordant diagnoses represented by cavitated lesions and 31.25% non-cavitated lesions (40% active and 60% inactive). disagreement between sound surfaces and non-cavitated lesions was 40% of undergraduate discordant diagnoses and 36% of graduate discordant diagnoses. discussion an accurate and reliable exam of dental caries is an essential stage of epidemiological surveys, which will contribute to the overall quality and reliability of research, as well as support appropriate interventions19-20. the occlusal surface is the most affected by caries among children, adolescents and young adults, due to its complex anatomy, making the detection of non-cavited lesions a difficult task2122. examiners presented different experience levels as different values of reproducibility has been observed20-21. studies that investigate the relationship between examiner calibration and the reproducibility achieved in the use of new methods for caries detection are necessary in order to validate and refine these systems and epidemiological assessment22-23. during data collection, examiner training was important to minimize both the random and systematic error15,24 mainly because an early stage of caries diagnosis was assessed, increasing the reproducibility and validity problems12,25. a substantial decrease of scores values (icdas-ii) after training sessions was observed either enhance of reproducibility before (0.72) and after (0.78) examination17. careful reading of the codes and criteria combined with images of characteristic lesions in training session17,25 emphasize learning effectiveness the enamel caries percentage according to consensus found in the present study confirms the ability of the icdasii and nyvad criteria to detect early changes in the enamel similar to other studies5,21. several studies have shown examination outcomes related to examiners experience20, and previous training17,26. detection of early lesions and detailed features is necessary in new indices which monitor the changes in teeth surface related to patient’s attitudes. in the present study, higher reproducibility values were observed while cutoff point a (lesion) was chosen unlike studies in which examiners with little experience achieved high reproducibility in d3 cut off (cavity lesion)20. braga et al. (2009)22 reported the use of cutoff points for diagnostic reproducibility not altering the sensitivity, specificity and accuracy. differentiating active lesions from inactive induced some classification doubt as reported by examiners 7,24, although in this study values ranged from good to perfect when cutoff was not used. the sensitivity can vary significantly (0.18 to 0.52) between unexperienced and experienced examiners20, with an increased inter-examiner agreement similar to presented in our study. the sensitivity of graduates emerged in the who (1997) index application. the majority of discordant diagnoses between the undergraduate examiners and among the graduate examiners were represented by pre-cavitated active lesions and sound surfaces codes7,22. these findings show effectiveness of new criteria to differentiate active lesions from inactive lesions whether cavitated or not cavitated. according to nyvad et al. (1999)7 the agreement between examiners for non-cavitated active lesions and inactive non-cavitated lesions were 68.7% and 72.5%. according to ismail et al. (1992)27, trained examiners confuse incipient caries 17 times more than cavitated caries surfaces. the icdas-ii and nyvad-ii criteria have several scores to demonstrate pre-cavited lesions with respect to depth, and its relation to the activity, allowing the caries development analysis by examiners22. this is a positive feature in surveys using these indices where the additional objective is treatment decision planning instead of reporting the prevalence of caries only. visual inspection of incipient caries lesions should be performed by trained examiners with new methods and criteria in clean and dry teeth surfaces, increasing sensitivity and specificity apply to epidemiologic surveys. in conclusion, examiner training according to the methodology proposed in this study, with theoretical discussion, discussion and exercise for proper calibration, makes possible the use of different criteria for caries diagnosis, considering active and inactive, pre-cavitated or cavitated caries lesions. acknowledgments this study was financial support by capes. references 1. brazil. ministry of health care/health surveillance. departament of primmary care. coordination of oral health. project sb brazil 2010 – main results. brasilia; 2011. 92 p. 2. bernabé e, sheiham a, sabbah w. income, income inequality, dental caries and dental care levels: an ecological study in rich countries. caries res. 2009; 43: 294-301. 3. world health organization. oral health surveys. basic methods. 4th ed. geneva: who; 1997. 4. assaf av, zanin l, meneghim mc, pereira ac, ambrosano gmb. comparison of reproducibility measurements for calibration in epidemiological surveys of dental caries. cad saude publica. 2006; 22: 1901-7. 5. ismail ai. clinical diagnosis of carious lesions precavitated. community dent oral epidemiol. 1997; 25: 13-23. reproducibility of caries diagnosis in permanent teeth according to who, icdas-ii and nyvad criteria braz j oral sci. 11(1):25-29 6. ekstrand kr, ricketts dnj, kidd 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(icdas) and comparability with world health organization criteria in a cross-sectional study. community dent oral epidemiol. 2010; 38: 398-407. 11. cadavid as, lince cma, jaramillo mc. dental caries in the primary dentition of a colombian population according to the icdas criteria. braz oral res. 2010; 24: 211-6. 12. kuhnisch j, bererger s, goddon i, senkel h, pitts n, heinrich-weltzien r. occlusal caries detection in permanent molars according to who basic methods, icdas ii and laser ûuorescence measurements. community dent oral epidemiol. 2008; 36: 475-84. 13. ismail ai, sohn w, tellez m, willem jm, betz j, lepkowski j. risk indicators for dental caries using the international caries detection and assessment system (icdas). community dent oral epidemiol. 2008; 36: 55-68. 14. begston al, gomes ac, mendes fm, cichello lr, begston ng, pinheiro sl. influence of examiner’s clinical experience in detecting occlusal caries lesions in primary teeth. pediatr dent. 2005; 27: 238-43. 15. brazil. ministry of health. department of primary care. sb brazil 2010 project: manual calibration of examiners. brasilia; 2009. 16. national board of health guidelines and regulating norms for research involving humans. brasília; 1997. 17. diniz mb, lima lm, santos-pinto l, eckert gj, zandoná ag, cássia loiola cordeiro r. influence of the icdas e-learning program for occlusal caries detection on dental students. j dent educ. 2010; 74: 862-8. 18. landis rj, koch gg. the measurement of observer agreement for categorical data. biometrics. 1977; 33: 159-74. 19. assaf av, meneghim mc, zanin l, mialhe fl, pereira ac, ambrosano gmb. assessment of different methods for diagnosing dental caries in epidemiologic surveys. community dent oral epidemiolol. 2004; 32: 418-25 20. fyffe he, deery c, nugent zj, nuttall nm, pitts nb. effect of diagnostic threshold on the validity and reliability of epidemiological caries diagnosis using the dundee selectable threshold method for caries diagnosis (dstm). community dent oral epidemiol. 2000; 28: 42-51. 21. diniz mb, rodrigues ja, hug i, cordeiro rcl, lussi a. reproducibility and accuracy of the icdas ii for occlusal caries detection. community dent oral epidemiol. 2009; 37: 399-404. 22. braga mm, mendes fm, martignon, s, ricketts dnj, ekstrand, dkr. in vitro comparison of nyvad’s icdas-ii system and with lesion activity assessment for evaluation of severity and activity of occlusal caries lesions in primary teeth. caries res. 2009; 43: 405-12. 23. ekstrand kr, martingnon s, ricketts djn, qvist v. detection and activity assessment of primary coronal caries lesions: a methodologic study. oper dent. 2007; 32: 225-35. 24. rodrigues ja, hug i, diniz mb, lussi a. performance of fluorescence methods, examination and radiographics examination and icdas ii on occlusal surfaces in vitro. caries res. 2008; 42: 297-304. 25. zandoná agf, al-shih s, h eggertsson, g. eckert. student versus faculty performance using a new visual criteria for the detection of caries on occlusal surfaces: an in vitro examination with histological validation. oper dent. 2009; 34: 598-604. 26. jablonski-momeni a, stachiss v, ricketts dn, heinzel-gutenbrunner m, pieper k. reproducibility and accuracy of the icdas-ii for detection of occlusal caries in vitro. caries res. 2008; 42: 79-87. 27. ismail ai, brodeur jm, gagnon p, payette m, picard d, hamalian t et al. prevalence of non-cavitated and cavitated carious lesions in a random sample of seven to nine-year-old schoolchildren in montreal, quebec. community dent oral epidemiol. 1992; 20: 250-5. 2929292929 reproducibility of caries diagnosis in permanent teeth according to who, icdas-ii and nyvad criteria oral sciences n3 braz j oral sci. 9(1):11-15 original article braz j oral sci. january/march 2010 volume 9, number 1 a comparative study of microstrain around three-morse taper implants with machined and plastic copings under axial loading celina wanderley de abreu1, luis gustavo oliveira de vasconcellos2, ivan balducci3, renato sussumu nishioka4 correspondence to: celina wanderley de abreu university of campinas, piracicaba dental school, sp. madre cecília street # 1560/11a, centro. postal code:13400490, piracicaba,sp. brazil. e-mail: celwanderley@fop.unicamp.br abstract aim: to evaluate the occurrence of microstrain around morse taper implants in straight configuration under axial load in a cast monoblock framework. methods: three implants were inserted in a polyurethane block and microunit abutments were installed on the implants with 20 ncm torque. plastic and machined copings were adapted on the preset waxing to fabricate the framework (n=5). four strain gauges were attached on the upper surface of the block and then each framework was tightened on the abutments and a vertical load of 30 kg was applied to five points of the framework. results: the data obtained in the strain gauge analysis were subjected to twoway anova and tukey’s test (α=0.05). there was statistically significant difference (p=0.0222) for the factor application point and the mean microstrain values were: application point b 402,04µε, point a 401.21µε, point e 390.44µε , point d 341.76µε and point c 309.19 µε. conclusions: there was no microstrain difference between plastic and machined copings during axial loading. difference in the application point was observed, but remained within bone physiological limits. keywords: dental implant, fixed prosthesis, axial load, strain gauge, morse taper. introduction the use of oral implants for rehabilitation has become a clinical routine. the abutment/implant connection must have the ability to reduce the stress peak and strain at the bone interface. the design of morse tapered implant posts is characterized by the internal walls of the implant and the external walls of the abutment fabricated with an 8° taper. during the abutment threading in the implant body, there is an intimate contact between the two components, creating frictional lock1. this design promotes significant retention and resistance under lateral loads creating frictional adaptation to the internal anchorage or implant body, allowing for an extended duration of function2. the prudent control of biomechanical loading in dental implants is imperative for their extended success3; if the loading is not controlled, implant failures can occur after delivering of the prostheses. although the mechanisms responsible for failures are not completely understood4, a consensus exists that the localization and magnitude of occlusal loading affect the quality and amount of induced strain in all the components of the prostheses/bone/implant complex5-7. received for publication: august 06, 2009 accepted:: march 11, 2010 1dds, msc, phd student in prosthodontics, department of prosthodontics, piracicaba dental school, university of campinas, brazil 2postgraduate student, department of dental materials and prosthodontics, são josé dos campos dental school, são paulo state university, brazil 3professor, department of community dentistry and pediatric clinic, são josé dos campos dental school, são paulo state university, brazil 4professor, department of dental materials and prosthodontics, são josé dos campos dental school, são paulo state university, brazil 12 braz j oral sci. 9(1):11-15 the application of functional load induces stress and strain in the bone/implant complex and affects the periimplant bone remodeling 8-9. the fraction of this occlusal load transmitted to the implants, and its induced stress, is dependent upon where the load is applied to the prostheses7. excessive loading on the bone/implant interface is one of the main factors accounting for marginal loss bone, motivating this current strain study10. the transference of occlusal loading can be influenced by factors related to the precision of the implant/abutment and abutment/prosthesis interfaces. the coping is one of the factors responsible for the precision, and machined copings have higher precision than plastic copings11-12. moreover, these authors reported that the precision of copings is associated with the distribution of stress, demonstrating the importance of comparing the precision between plastic and machined copings. some implant failures can be related to unfavorable stress magnitudes13. when pathologic overload occurs, above 4000 µå, gradients of stress and strain exceed the physiologic bone tolerance and cause micro fractures in the bone/implant interface14. occlusal overload results in an increase of bone resorption around the implant and a decrease in the percentage of mineralized bone tissue15, showing that a remodeling process occurs when the bone is subjected to stress16-17. the aim of this study was test the hypothesis that different application points promote similar microdeformations, but machined copings are preferred over plastic copings to reduce the occurrence of these microdeformations. material and methods an aluminum matrix with internal dimensions of 95 x 45 x 30 mm was developed for this study. identical proportions of base and catalyst of a polyurethane resin (f16 axson, cergy, lle-de-france, france) were mixed until a homogeneous mixture was obtained. after resin polymerization, the surfaces were polished with wet 220to 600-grit abrasive papers to obtain flat surfaces, free of irregularities. a second aluminum matrix was used to standardize the linear placement of three implants in the polyurethane block as well as to standardize the waxing of the frameworks (figure 1). the distance and places for inserting the three cone fig. 1 – matrix: 1base with in line cylinders (3.75mm diameter x 4 mm height). 2: component with central opening level with the height of the cylinders. 3: rectangular bar with 3 holes coinciding with the location of the cylinders. lateral screws to keep the three components stable. morse implants (conexão sistemas de prótese, são paulo, sp, brazil) in the polyurethane block was standardized by fixing component 3 in the block with horizontal screws and rings identified by colors with internal diameters compatible with the diameters of the burs used: white, yellow and blue rings with diameters of 2, 3 and 3.15 mm, respectively. a handpiece with 20:1 reduction (koncept, kavo ind.com ltda, são paulo, sp, brazil) connected to an electric engine (mc 101 omega, dentsclar, ribeirão preto, sp, brazil) was used to perforate and insert the implants. mean speed for inserting the implants was 14 rpm and torque was adjusted to 40 ncm. three morse taper implants (3.75 mm in diameter x 13 mm long; conexão sistemas de prótese) were placed in the polyurethane block. microunit abutments (conexão sistemas de prótese) were screwed into the implants with torque of 20 ncm as measured with a manual torque meter (conexão sistemas de prótese). before adapting the waxing standardizations, the copings were reduced with the aid of a carborundum disk (dentrium, new york, ny, usa) to a height of 10 mm in order to facilitate and level the insertion of the waxing. a heated dropper-type instrument was used to promote peripheral sealing of all copings (pk thomas type waxing set: ss white, rio de janeiro, rj, brazil). then, 10 waxed were cast in co-cr, being 5 for plastic copings (n=5) and 5 for machined copings (n=5). the waxings of the frameworks were standardized by using the base (component 1) and component 2 which, when fixed by vertical screws, resulted in a rectangular compartment that allowed a systematic reproduction of the waxing of all the tested specimens, especially in terms of thickness. cr-co alloy (wirobond sg, bremen, bremen, germany) was used for casting. the frameworks were individually adapted to the polyurethane block, in which the stability of the set was gauged by tightening the screws. the screw tightening sequence was standardized from the center to the edges of the piece, starting with the central implant 2, followed by lateral implants 1 and 318. in order to determine exactly the bonding place of four strain gauges (kyowa electronic instruments co., ltd, tokyo, kanto, japan), a line was drawn with a ruler and a 0.7 mm lead pencil. the four strain gauges were bonded along this line tangential to the abutments with a thin layer of cyanoacrylate adhesive (superbonder, são paulo, sp, brazil) under slight pressure for 3 min. after bonding, each strain gauge was measured by using a multi-meter appliance (minida et 2055: minida são paulo, sp, brazil), and the terminal plates to which the electric connections were adapted, were bonded onto the upper surface of the polyurethane block (figure 2). the linear electric strain gauges were connected to an electric signal conditioning appliance (ads 2000ip; lynx, são paulo, sp, brazil), arranged in a ¼ wheatstone bridge configuration with 120 &! resistance , which is an electric circuit appropriate for detecting minimal alterations in resistance caused by deformation. the signals were interpreted, modified and processed by using a strain-smart computational program. an idealized load application device was connected to a comparative study of microstrain around three-morse taper implants with machined and plastic copings under axial loading 13 fig. 2 strain gauges around the microunit abutments. fig. 3 experimental model in the loading apparatus with load applied at point a. the electrical signal conditioning appliance (model 5100b scanner; system 5000, raleigh, nc, usa) in order to apply the load. the experimental model was placed on the load application appliance (figure 3) with the framework in place, on which axial loads of 30 kg19 were applied for 10 s on the center of each implant and on the mid-point between them, totalizing 5 load application points. the points referred to were designated as: a (center of the retention screw of implant 1), b (mid-point between the orifices of the screws of implants 1 and 2), c (center of the retention screw of implant 2), d (mid-point between the screw orifices of implants 2 and 3) and e (center of the retention screw of implant 3) (figure 4). the measurement of points (b and d) between two implants was checked with a ruler. the microdeformations determined at the five points were recorded by four extensometers and the same procedure was performed for all the frameworks, repeating three loadings per load application point. the data obtained in the strain gauge test were subjected to two-way (coping and application point) repeated-measures analysis of variance (anova), and tukey’s multiplecomparison test was used to determine the occurrence of statistically significant differences. a significance level of 5% was adopted results the two-way anova revealed that the interaction between factors was not significant (p = 0.0699) and the factor coping was not significant either (table 1). but application point factor (p = 0.0222) show significant influence on the microstrain. then, the tukey’s test was realized according table 2. the mean strain values for the interaction factor between cooping and load point are presented in figure 5. discussion since the introduction of osseointegration, dental implants have been widely used in the rehabilitation of partially or completely edentulous patients20, and showing the success of implantology for prosthetic treatments in modern dentistry21. in spite of this, implant failures might occur after delivery of prosthesis, and have been reported to be mainly due to biomechanical complications4. occlusal overload has been identified as the primary cause of loss of peri-implant bone, implants and implant supported dentures10,22. the extensometers used in implant dentistry are based on the use of electrical resistance and the association of equipment promoting measures of strain induced by static and dynamic loads both in vivo23-24 and in vitro7,25. under an applied force, the strain gauge measures the mean dimensional change5,7. bone quality is one of the factors that influence in the result of the treatment with implants. the bone surrounding them does not constitute a homogeneous substratum and its physical properties vary as the age, functional state and systemic factors of the patient13. moreover, in vitro studies have used homogeneous and isotropic materials25-26. in the present study, a homogeneous model with uniform elastic properties was designed11, and a polyurethane block with similar modulus of elasticity to that of the human medullary bone was used to simulate the human bone (polyurethane: 3.6gpa/medullary bone: 4.0 4.5gpa)17. some strain gauge studies used special devices for load application on implants7, but others used universal testing machines26 to apply load. the amount of load used in this experiment, 30 kg (approximately 294n), was based on the study by merick-stern et al.19, who investigated the occlusal force in patients with fixed partial implant-supported dentures, and found a mean value of 30.6 kg (300n) for maximum force in the region of the second molars. placement of extensometers on the surface of the polyurethane block, adjacent to the cervical area of the implant is justified because in this region there is higher stress concentration after load application25. other studies opt for bonding the extensometers onto the implants22 and on the metal structures of the denture24, but bonding on the surface simplifies the procedure. the aim of the methodology used in this study was to eliminate steps that would promote dimensional alterations, such as those resulting from the transfer molding of implants and obtaining the plaster model. the plastic and machined copings were adapted directly onto the implants and joined braz j oral sci. 9(1):11-15 a comparative study of microstrain around three-morse taper implants with machined and plastic copings under axial loading 14 fig. 4 five points of load application (a, b, c, d and e) effect df ss m s f p-value cooping 1 25222 25222.2 1.22 0.3014 residue i 8 165304 20663.0 application point (ap) 4 70280 17570.0 3.32 0.0222* interaction (cooping/ ap) 4 51013 12753.1 2.41 0.0699 residue ii 32 169575 5299.2 total 49 481394 table 1 – results of two-way analysis of variance for conditional experiments. *p<0.05. application point mean homogeneous group b 402.04 a a 401.21 a e 390.44 ab d 341.76 ab c 308.19 b table 2 – results of tukey’s test for the mean microstrain values at five load points. *same superscript letters indicate no statistically significant differences (p>0.05). fig. 5 mean values and standard deviations of the microstrain (µε) for plastic and machined copings in each load point. to the standardized waxing. this method was based on the study by heckmann et al.11, who recorded that metallic structures fabricated on the plaster model produced larger deformations when compared with those made without the molding procedures. the mean microdeformation with reference to the interaction of the coping and the load application point, observed in figure 5, showed that the copings had the same deformation pattern, and showed no significant difference. these results are in agreement with those of previous strain gauge studies, in which the fixed partial implant-supported dentures made from plastic and machined copings produced the same magnitude of microdeformation during retention screw tightening, before ceramic application10-11. based on the physiological balance, clinical and laboratory studies indicate that permanent mechanical stimulation is needed21. deformation intensities above 100 µå are necessary to prevent bone resorption. however, the stimulation values must not exceed the physiological limit of 4000 µε16-17. the reason for casting the structure in a monoblock configuration is based on the study by watanabe et al.27, who verified that monoblock castings do not differ from those made in segments and later welded as regards the distribution of stresses on screwed implant-supported dentures. the structure in this study was made in a flat shape due to the need to evaluate axial loads, because the inclination of the cusps would generate horizontal force and the magnitude of axial loading would be altered3. the data on table 1 shows significant difference (p=0.0222) for the load application point effect and the tukey’s multiple comparison test (table 2) verified that there was no homogeneous distribution of microdeformation among the groups28-29, probably due to the absence of absolute passive seating, suggesting that clinical evaluation to verify passive seating allowed small distortions that were not perceived by the visual method30. casting of the metal structure is a determinant step in the influence of passivity determining a non-homogeneous seating of the structure. this is because the fit obtained in implant 2 could have been different from the one achieved in implant 1 and from the one seen in implant 3, hence justifying stress generation and producing a different microdeformation distribution for the various points of load application. the rationale for the parameters of this investigation, considering the type of coping and place of load application, is based on the idea of choosing the best option when performing a treatment with a three-element fixed partial implant supported denture, to allow long term clinical success. according to the pertinent literature, determining the best option continues to be a vital question for retrospective and prospective clinical studies supported by in vivo and in vitro biomechanical studies. within the limitations of this study, it may be concluded that the type of coping used, plastic or machined, did not interfere in the level of microstrain at the time of axial load braz j oral sci. 9(1):11-15 a comparative study of microstrain around three-morse taper implants with machined and plastic copings under axial loading 15 application. however, the place of axial load application had a direct influence, and axial loads applied on different application points produced a magnitude of bone microstrain within the physiological threshold. acknowledgments the authors wish to thank fapesp (fundação de amparo à pesquisa do estado de são paulo – protocol #07/ 57696-6) for the financial support and conexão sistemas de prótese for providing some materials. the authors also express their gratitude to professor ivan balducci. references 1. solnit gs, schneider rl. an alternative to splinting multiple implants: use of the iti system. j prosthodont. 1998; 7: 114-9. 2. sutter f, schroeder a, buser d. the new concept of iti hollow-cylinder and hollow-screw implants: part 1. engineering and design. int j oral maxillofac implants. 1988; 3: 161-72. 3. çehreli m c, iplikçioglu h, bilir ö g. the influence of the location of load transfer on strains around implants supporting four unit cement-retained fixed prostheses: in vitro evaluation of axial versus off-set loading. j rehabil. 2002; 29: 394-400. 4. taylor td, agar jr, vogiatzi t. implant prosthodontics: current perspective future directions. int j oral maxillofac implants. 2000; 15: 66-75. 5. glantz po, rangert b, svensson a, stanfford gd, arnvidarson b, randow k. et al. on clinical loading of osseointegrated implants. a methodological and clinical study. clin oral implants res. 1993 4: 99-105. 6. richter ej. basic biomechanics of dental implants in prosthetic dentistry. j prosthet dent. 1989 61(5): 602-9. 7. assif d, marshak b, horowitz a. analysis of load transfer and stress distribution by an implant-supported fixed partial denture. j prosthet dent. 1996; 75: 285-91. 8. bidez mw, misch ce. force transfer in implant dentistry: basic concepts and principals. j oral implantol. 1992; 23: 264-74. 9. branemark pi, zarb ga, albrektsson t. tissue-integrated prosthesis. osseointegration in clinical dentistry. chicago: quintessence; 1987. 129p. 10. hekimoglu c, anil n, cehreli m. analysis of strain around endosseous implants opposing natural teeth or implants. j prosthet dent. 2004; 92: 441-6. 11. heckmann sm, karl m, wichmann mg, winter w, graef f, taylor td. cement fixation and screw retention: parameters of passive fit. an in vitro study of three-unit implant-supported fixed partial dentures. clin oral implant res. 2004; 15: 466-73. 12. may kb, edge mj, russell mm, razzoog me, lang br. the precision of fit at the implant prosthodontic interface. j prosthet dent. 1997; 77: 497-502. 13. sahin s, çehreli mc, yalçin e. the influence of functional forces on the biomechanics of implant-supported prostheses – a review. j dent. 2002; 30: 271-82. 14. roberts we. fundamental principles of bone physiology, metabolism and loading. in: naert i, van steenberghe d, worthington p, editors. osseointegration in oral rehabilitation. an introductory textbook. london: quintessence; 1993. p.163-4. 15. hoshaw sj, brunski jb, cochran gvb. mechanical loading of branemark implants affects interfacial bone modeling and remodeling. int j oral maxillofac implants. 1994; 9: 345-60. 16. frost h m. wolff’s law and bone’s structural adaptations to mechanical usage: an overview for clinicians. angle orthod. 1994; 64: 175-88. 17. wiskott h w, belser u c. lack of integration smooth titanium surfaces: a working hypothesis based on strains generated in the surrounding bone. clin oral implants res. 1999; 10: 4429-44. 18. millington nd, leung t. inaccurate fit of implant superstructures. part 1. stresses generated on the superstructure relative to the size of fit discrepancy. int j prosthodont. 1995; 8: 511–6. 19. mericske-stern r, assal p, merickse e, ing wb. oclussal force and oral tactile sensibility measured in partially edentulous patients with iti implants. int j oral maxillofac implants. 1995; 10: 345-54. 20. branemark pi, adell r, breine u, hansson bo, lindstrom j, olsson a. intra-osseous anchorage of dental prostheses. i. experimental studies. scand j plast reconstr surg. 1969; 3: 81-100. 21. smedberg ji, nilner k, rangert b, svensson sa, glantz sa. on the influence of superstructure connection on implant preload: a methodological and clinical study. clin oral implants res. 1996; 7: 55-63. 22. adell r, lekholm u, rockler b, branemark pi. a 15-year study of osseointegrated implants in the treatment of the edentulous jaw. int j oral surg. 1981; 10: 387-416. 23. duyck j, van oosterwyck h, vander stolen j, de cooman m, puers r, naert i. magnitude and distribution of occlusal forces on oral implants supporting fixed prostheses: an in vivo study. clin oral impl res. 2000; 11: 465-75. 24. heckmann sm, karl m, winter w, grael f, taylor td. loading of bone surrounding implants through three-unit fixed partial denture fixation: a finite elements analysis based on in vitro and in vivo strain measurements. clin oral implants res. 2006; 17: 345-50. 25. çehreli m, duyck j, de cooman m, puers r, naert i. implant design and interface force transfer: a photoelastic and strain-gauge analysis. clin oral implants res. 2004; 15: 249-57. 26. akça k, cehreli mc, iplikçioglu h. a comparison of three-dimensional finite element stress analysis with in vitro strain gauge measurements. int j prosthodont. 2002; 15: 115-21. 27. watanabe f, hata y, komatsu s, ramos tc, fukuda h. finite element analysis of the influence implant location, loading position, and load direction on stress distribution. odontology. 2003. 91: 31-6. 28. huang hl, lin cl, ko cc, chang ch, hsu jt, huang js. stress analysis of implant-supported partial prostheses in anisotropic mandibular bone: inline versus offset placements of implants. j oral rehabil. 2006; 33: 501-8. 29. karl m, wichmann mg, winter w, graef f, taylor t, heckmann sm. influence of fixation mode and superstructure span upon strain development of implant fixed partial dentures. j prosthodont. 2008; 17: 3-8. 30. kan jyk, rungcharassaeng k, bohsali k, goodacre cj, lang br. clinical methods for evaluating implant framework fit. j prosthet dent. 1999; 81: 7-13. braz j oral sci. 9(1):11-15 a comparative study of microstrain around three-morse taper implants with machined and plastic copings under axial loading original articlebraz j oral sci. january/march 2009 volume 8, number 1 histomorphometric study of alveolar bone after therapy with immunosuppressant fk506 rogério lacerda dos santos1, marcos farina de souza2, renato torres gonçalves3, marco aurélio martins4, margareth maria gomes de souza5 1 specialist in orthodontics, universidade federal de alfenas (unifal), alfenas (mg), brazil; master of orthodontics, universidade federal do rio de janeiro (ufrj), rio de janeiro (rj), brazil 2 doctor of biological sciences, ufrj; titular professor, ufrj; chief of the biomineralization laboratory, ufrj, rio de janeiro (rj), brazil 3 nephrologist physician in the department of nephrology and responsible for the renal transplantation unit, hospital universitário clemente fraga filho, rio de janeiro (rj), brazil 4 doctor in cell and molecular biology, fundação oswaldo cruz (fiocruz); chief of the inflammation laboratory, fiocruz, rio de janeiro (rj), brazil; post-doctorated, william harvey research institute and imperial college, london, uk 5 doctor of orthodontics, ufrj; adjunct professor, department of pediatric dentistry and orthodontics, ufrj, rio de janeiro (rj), brazil received for publication: december 01, 2008 accepted: march 03, 2009 correspondence to: rogério lacerda dos santos praça josé batista de freitas, 78, sala 102 – centro cep 35519-000 – nova serrana (mg), brazil e-mail: lacerdaorto@hotmail.com or lacerdaorto@bol.com.br abstract aim: several medications affect bone metabolism and the rate of tooth movement. the objective of the present study was to test the hypothesis that treatment with immunosuppressant tacrolimus (fk506) can interfere with bone turnover, decreasing the rate of tooth movement. methods: sixty male wistar rats were divided into four groups of 15 animals each: group 1: rats subjected to orthodontic movement plus treatment with saline solution vehicle; group 2: rats subjected to orthodontic movement plus treatment with fk506; group 3: rats treated with fk506 only; and group 4: rats treated with saline solution vehicle only. the fk506 dose was 2 mg/kg/day. the treatment was initiated 14 days before the appliance installation and then kept for up to 14 days. in addition to the administration of the immunosuppressive drug, 10 mg/kg of oxytetracycline were injected at intervals of three days in order to show osteoblastic activity and bone growth at a histological level. results: histomorphometrical measurements showed greater tooth movement in group 1 than in group 2 at all periods (days 3, 7 and 14), though significant difference (p < 0.05) was observed only on days 7 and 14. conclusions: fk506 significantly influenced the rate of tooth movement in rats subjected to the application of this medication. keywords: tooth/immunology, tooth movement, bone and bones. introduction tacrolimus (fk506) is an immunosuppressive agent derived from streptomyces tsukubaensis1, which is widely used in patients subjected to organ transplantation2. some authors3,4 have suggested that fk506 exerts its anti-inflammatory effect mainly by interfering with the activation of t-cells, by suppressing the production of cytokines, particularly tnf-α, il-1β, il-2, and il-6, modulating the inflammation, and decreasing both tissue turnover and bone resorption. on the other hand, bone destruction and development of severe osteoporosis have been reported5. studies using cell culture6,7 have demonstrated that not only messenger rnas of nfatc1, nfatc2 and nfatc3 are present in the osteoclast-precursor cells, but also fk506 inhibits the final stages of cell cycle through osteoclastic apoptosis. altogether, these findings are in accordance with the idea that the mechanisms by which osteoclastogenesis is inhibited and osteoclastic apoptosis is promoted are similar to those involved in inhibiting the production of transcription factor nfat and inflammatory cytokines within t lymphocytes8. therefore, 20 santos rl, souza mf, gonçalves rt, martins ma, souza mmg braz j oral sci. 8(1): 19-24 the objective of the present study was to test the hypothesis that fk506 treatment can interfere with bone turnover, decreasing the rate of tooth movement. material and methods animal model a total of 60 male wistar rats (rattus norvegicus) aged nine weeks and weighting 220 to 280 g were housed according to the guidelines for animal research as recommended by fundação oswaldo cruz (fiocruz, rio de janeiro, rj, brazil). the immunosuppressive agent fk506 (tacrolimus, prograf, astellas, ireland) was orally administrated at a dose of 2 mg/kg/day as a suspension containing water and 5% dextrose9. the treatment was initiated 14 days before the appliance installation and, then, kept for up to 14 days. in addition to the administration of the immunosuppressive drug, 10 mg/kg of oxytetracycline (ouro fino, cravinhos, sp, brazil) were injected at intervals of three days, in order to show osteoblastic activity10 and bone growth at a histological level. the experiments were reviewed and approved by the ethics committee on animal research of universidade federal do rio de janeiro (ufrj). experimental groups four groups of 15 animals each were formed according to the treatment modality: group 1: rats subjected to orthodontic movement plus treatment with saline solution vehicle; group 2: rats subjected to orthodontic movement plus treatment with fk506 (2 mg/kg/day); group 3: rats treated with fk506 only (2 mg/kg/day); and group 4: rats treated with saline solution vehicle only. appliance design the appliance was made according to description by arias and marquez-orozco11 regarding wire diameter and installation. the initial load to be exerted by the orthodontic appliance was determined before its insertion by means of a ball dynamometer (dentaurum 040-711, ispringen, germany) for measuring of 35 gf11. the rats were anesthetized with an intraperitoneal injection of sodium thiopental (cristália, campinas, sp, brazil; 50 mg/kg body weight). incisor gaps were measured in the cervical region of the tooth and recorded by two investigators using a precision caliper accurate to 0.01 mm (starret, itú, sp, brazil), and the mean values were recorded when the animals were sacrificed. blood count total and differential leukocyte counts (lymphocytes, monocytes, eosinophils and neutrophils) were evaluated on day 0 (before appliance installation) and on sacrifice day in all groups, in order to monitor the immunosuppressive effect of fk506. blood was collected from the tail tip of each rat and leukocyte count was realized with a light microscope (olympus bx40; olympus, hamburg, germany). histomorphometry after 3, 7, and 14 days of orthodontic movement, the rats were sacrificed and then decapitated. their heads were dissected and the premaxillae were removed, fixed in 10% formaldehyde solution for 24 hours and, thereafter, washed with pbs solution. the specimens were dehydrated and embedded in spurr epoxy resin (ted pella inc., san jose, ca, usa), which was dissolved in ethanol at proportions of 1:3, 1:2, 1:1, 2:1, and 100% spurred every 12 hours before polymerization for 24 hours at 70 °c. the specimens were sectioned in the frontal plane at 1 mm from the palatal face of central incisors, resulting in bone blocks of 3 mm. the blocks were placed perpendicular to the diamond saw machine (model 650, south bay technology, inc., san clemente, ca, usa), producing 40-µm-thick cross-sections, which were polished with #400-, #600-, #1200-, #2000and #2500-grit abrasive papers (3m do brasil, sumaré, são paulo, brazil) to obtain a uniform thickness for visualization with a fluorescence microscope (axioplan, zeiss, jena, germany). this histomorphometric analysis focused on the alveolar bone located laterally to the incisors, which was divided into cervical pressure (distal) and traction (mesial) sides to the apical region following the cervical-apical axis of the root (adapted from kale et al.)13. three parallel sections were randomly counted without repetition, from cervical to apical regions of the tooth, regarding pressure and traction sides. a single operator blinded to the groups recorded the data on measurements of the fluorescence lines (perpendicularly interlines distance) regarding bone growth during the 3-day-interval applications of oxytetracycline. image tool 1.0 software (image tool, san antonio, tx, usa) was used for processing the data collected and for intra and intergroup comparisons. the distance between the growth lines was divided by three in order to set the daily speed of growth (osteoblastic activity expressed as µm/day), as described by roberts et al.12. the fields of periodontal ligament at pressure and traction sides were captured by using the axiovision rel 4.5 software with the fluorescence microscope at ×20 magnification. results effect of appliance installation and fk506 treatment on weight gain the orthodontic appliance was well tolerated by the animals during the experimental period and caused no soft tissue irritation. the weight gain regarding each period of time was calculated on the ba21histomorphometric study of alveolar bone after therapy with immunosuppressant fk506 braz j oral sci. 8(1): 19-24 sis of the initial treatment with fk506. both the immunosuppressant and the orthodontic appliance were found to have influenced the weight gain, since the non-treated animals had the highest gains. group 3 had higher weight gain within 28 days (41.0 ± 1.2 g) compared to group 1 (35.3 ± 2.0 g), though without statistical significance (p > 0.05). on the other hand, group 2 had the lowest weight gains on days 3 (15.7 ± 2.3 g), 7 (16.7 ± 2.2 g), and 14 (21.4 ± 1.6 g). there were significant differences (p < 0.05) between group 2 and groups 1 and 3 in all time periods of study. effect of appliance installation and fk506 treatment on leukocyte count immunosuppression was observed in the animals from groups 2 and 3 as leukocyte and lymphocyte counts were shown to have similar and decreasing values during the experiment in all time periods. also, statistically significant differences (p < 0.05) were observed between both groups and their respective controls (day 0). there was statistically significant difference (p < 0.05) between group 2 and group 1 on days 7 and 14. contrary to groups 2 and 3, however, group 1 (rats subjected to orthodontic movement) showed increased values of leukocytes and lymphocytes counts, i.e., there was body response to the stimulus (force) applied to the teeth. group 1 also showed significant difference (p < 0.05) between day 0 and day 14. no statistically significant difference (p > 0.05) was found in neutrophil counts recorded in groups 2 and 3. group 1 showed an increased neutrophils count on day 14, which was significant different compared to the respective control (day 0) (p < 0.05). eosinophil and monocyte counts showed similar patterns between groups 1 and 2, with increased values on day 7 and decreased values on day 14. group 3 had significantly greater (p < 0.05) monocyte count compared to group 1 on day 0, which was kept for other time periods. no significant difference (p > 0.05) regarding this cell types was observed between the groups in all periods of time studied (figure 1). a trend of increase in the number of eosinophils due to the presence of orthodontic appliance could be observed, whereas the presence of fk506 promoted an increase in the number of monocytes. tooth movement before installing the appliances, a lack of measurable space, between the upper incisors, was observed. group 1 showed greater tooth movement compared to group 2 on days 3, 7, and 14 (figure 2), however significant difference (p < 0.05) was observed only on day 7 (2.09 ± 0.10 mm and 1.66 ± 0.09 mm) and day 14 (3.06 ± 0.05 mm and 2.48 ± 0.11 mm), respectively. bone histomorphometry bone growth was lower on the pressure side of the groups subjected to orthodontic movement compared to saline group, and higher in fk506 + appliance (group 2) fk506 (group 3) saline + appliance (group 1) 0 5 10 15 20 25 0 3 7 14 * * * * * * + + * le uk oc yt es / m m 3 a 0 5 10 15 0 3 7 14 * * * * * * + + * ly m ph oc yt es / m m 3 b 0 1 2 3 4 5 0 3 7 14 + * n eu tr op hi ls / m m 3 c 0.06 0.07 0.08 0.09 0.10 0.11 0 3 7 14 eo si no ph ils / m m 3 d time period of appliance installation (days) 0.0 0.1 0.2 0.3 0.4 0.5 0 3 7 14 m on oc yt es / m m 3 e figure 1. total leukocyte count (1a), lymphocytes (1b), neutrophils (1c), eosinophils (1d) and monocytes (1e) in group 1 (rats subjected to orthodontic movement plus treatment with saline solution vehicle) (circle), group 2 (rats subjected to orthodontic movement plus treatment with fk506) (triangle), group 3 (rats treated with fk506 only) (square) on days 3, 7, and 14 following installation of appliance. the values represent mean ± standard deviation regarding five experimental animals. * p < 0.05 compared to respective controls; +p < 0.05 compared to orthodontically-treated animals. 22 santos rl, souza mf, gonçalves rt, martins ma, souza mmg braz j oral sci. 8(1): 19-24 the fk506 group after seven days. there was statistically significant difference (p < 0.05) between the saline (3.40 ± 0.24 µm/day) and saline plus orthodontic appliance (1.69 ± 0.22 µm/day) groups as well as between the saline (3.40 ± 0.24 µm/day) and the fk506 plus orthodontic appliance (2.07 ± 0.27 µm/ day) groups after seven days of orthodontic movement. the traction side of the group subjected to orthodontic movement plus treatment with saline solution had higher bone growth after seven days compared to the group subjected to application of saline solution only. there was statistically significant difference (p < 0.05) between the saline (3.21 ± 0.18 µm/day) and saline plus orthodontic appliance (4.44 ± 0.11 µm/day) groups as well as between the saline (3.21 ± 0.18 µm/day) and the fk506 plus orthodontic appliance groups (4.35 ± 0.19 µm/day) after seven days of orthodontic movement. within 14 days of tooth movement, no statistically significant differences (p > 0.05) were found among the groups on either pressure or traction sides (figures 3, 4 and 5). discussion previous studies have demonstrated that treatment with fk506 (tacrolimus) can induce bone loss in human beings14 as well as in experimental models5,15. in cell cultures, on the other hand, fk506 can also inhibit the final phases of the life cycle of osteoclast-precursor cells through induction of apoptosis6,7. it has been suggested that fk506 inhibits the production of pro-inflammatory cytokines, particularly tng-α, il-1β and il-63, both modulating the inflammation and decreasing tissue destruction and bone resorption4. because fk506 has the capacity to alter bone metabolism, one can suggest that it can interfere with the rate of tooth movement. investigating this possibility was the goal of the present study. although rats have continuous tooth eruption, their periodontal structures are arranged similarly to that of human beings, with the incisors quickly responding to orthodontic forces ranging from 20 to 40 gf16. a load of 35 gf was used in the present study11. it was observed that animals subjected to orthodontics plus treatment with fk506 had a smaller amount of tooth movement. this finding was confirmed by the bone histomorphometry, which showed greater bone growth (µm/day) for the group subjected to orthodontic movement plus treatment with fk506 compared to the group subjected to orthodontic movement plus treatment with saline in the pressure side adjacent to the orthodontic movement. bone is a tissue that grows and maintains its structural integrity by two basic physiological processes: modeling, when size and shape of the skeletal unit change, and remodeling, when there is no change in the shape of skeletal unit in previously existing bone metabolism. histomorphometry is well recognized as an important benchmark for evaluating the lines of bone growth with vital colors, such as those obtained with oxytetracycline, alizarin, and calcein10 on fluorescence microscope. 0 1 2 3 4 * * 3 7 14 saline + appliance (group1) fk506 + appliance (group 2) time period of appliance installation (days) to ot h m ov em en t (m m ) * p < 0.05 compared to vehicle-treated animals. figure 2. tooth movement of rats treated with fk506 (2 mg/kg/day) or saline observed on days 3, 7, and 14 following appliance installation. the values represent mean ± standard deviation regarding five experimental animals. figure 3. bone histomorphometry in the group 1 (rats subjected to orthodontic movement plus treatment with saline solution vehicle), group 2 (rats subjected to orthodontic movement plus treatment with fk506), group 3 (rats treated with fk506 only), and group 4 (rats treated with saline solution) on days 3, 7, and 14 following installation of appliance. the values represent mean ± standard deviation regarding five experimental animals. 0 1 2 3 4 5 * pressure side * b o n e g ro w th ( µ m /d ay ) 7 14 days 0 1 2 3 4 5 traction side * * saline (group 4) fk506 + appliance (group 2) fk506 (group 3) saline + appliance (group 1) b o n e g ro w th ( µ m /d ay ) 7 14 days * p < 0.05 compared to respective controls. 23histomorphometric study of alveolar bone after therapy with immunosuppressant fk506 braz j oral sci. 8(1): 19-24 similar bone growth was observed in the animals subjected to the application of fk506 and saline, which suggests that the immunosuppressive drug did not interfere significantly with bone growth, though an increasing trend was found in the groups treated with the immunosuppressant. as the influence of fk506 on bone metabolism is a dose-dependent event9, in the present experiment, the doses were daily calculated and adjusted according to the weight gain of each animal. some factors such as metabolic and gastrointestinal changes17,18 caused by fk506 can also interferer with weight gain. the presence of an orthodontic appliance itself is another factor that can have an influence on weight changes as it makes feeding more difficult, which might explain why animals receiving either orthodontic treatment or fk506 treatment had low weight gain compared to animals treated with saline solution. in the present study, weight gain observed in fk506-treated animals was higher than that found in previous studies9,19, which used the same daily fk506 dosage. in fact, the use of orthodontic appliance in combination with fk506 treatment resulted in significant decrease in the weight gain, a joint influence of both factors. figure 4. bone histomorphometry: lines of growth with fluorescence marker in the alveolar bone adjacent to the pre-maxillary periodontal ligament (pressure side) of rats. presence of oxytetracycline (arrows) at intervals between the tracks indicating bone growth every three days (µm/day). (a) group 4 (rats treated with saline solution); (b) group 1 (rats subjected to orthodontic movement plus treatment with saline solution vehicle); (c) group 3 (rats treated with fk506 only); (d) group 2 (rats subjected to orthodontic movement plus treatment with fk506) with three applications of oxytetracycline, 0 (first application), 3 (second application) and 6 day (third application). after 14 days of tooth movement (figures e-g), oxytetracycline applications were performed on days 0 (first application), 3 (second application), 6 (third application), 9 (fourth application) and 12 (fifth application); (e) group 1; (f) group 3; (g) group 2. bar-scale: 100 μm. a 3 2 1 * b 3 2 1 * c 3 2 1 * d 3 2 1 * e 3 2 1 * 5 4 f 3 2 1 * 5 4 g 3 2 1 * 5 4 figure 5. bone histomorphometry: lines of growth with fluorescence marker in the alveolar bone adjacent to the pre-maxillary periodontal ligament (traction side) of rats. presence of oxytetracycline (arrows) at intervals between the tracks indicating bone growth every three days (µm/day). (a) group 4 (rats treated with saline solution); (b) group 1 (rats subjected to orthodontic movement plus treatment with saline solution vehicle); (c) group 3 (rats treated with fk506 only); (d) group 2 (rats subjected to orthodontic movement plus treatment with fk506) with three applications of oxytetracycline, 0 (first application), 3 (second application) and 6 day (third application). after 14 days of tooth movement (figures e-g), applications of oxytetracycline were performed on days 0 (first application), 3 (second application), 6 (third application), 9 (fourth application) and 12 (fifth application ); (e) group 1; (f) group 3; (g) group 2. bar-scale: 100 μm. b 3 2 1 * c 3 2 1 * d 3 2 1 * e 3 2 1 * 5 4 f 3 2 1 * 5 4 g 3 2 1 * 5 4 a 3 2 1 * 24 santos rl, souza mf, gonçalves rt, martins ma, souza mmg braz j oral sci. 8(1): 19-24 nevertheless, histological data demonstrated that fk506 can significantly reduce the induction of alveolar bone loss and granulocytes infiltration4, because of its capacity to inhibit osteoclastogenesis in vivo and in vitro7 by means of osteoclastic apoptosis. such an effect would result in inactivation of nfatc1, nfatc2, and nfatc3 (messenger rnas) existing within osteoclast-precursor cells6. osteoclasts are the main cells involved in the process of alveolar bone remodeling and there seems to be a direct relationship between rate of tooth movement and number of osteoclasts20. in the present study, however, the bone growth observed in the animals subjected to orthodontic movement was greater than in those animals subjected to orthodontic movement plus fk506 treatment and significantly greater compared to the control animals (treated with saline), which is in accordance with the findings of previous investigations6,7,21,22. in conclusion, the analysis of tooth movement and the histomorphometrical measurements of the alveolar bone indicate that the therapy with immunosuppressant tacrolimus (fk506) influenced significantly the bone turnover. there was a lower rate of tooth movement in the group subjected to application of the immunosuppressant. acknowledgements the authors would like to thank the fundação de amparo à pesquisa do estado do rio de janeiro (faperj), for the research funding, and the department of pathological anatomy of the ufrj, for the great help in carrying out this research. references 1. kino t, hatanaka h, hashimoto m, nishiyama m, goto t, okuhara m, et al. fk-506, a novel immunosuppressant isolated from a streptomyces. i. fermentation, isolation, and physico-chemical and biological characteristics. j antibiot (tokyo) 1987;40:1249-55. 2. kondo h, abe t, hashimoto h, uchida s, irimajiri s, hara m, et al. efficacy and safety of tacrolimus (fk506) in treatment of rheumatoid arthritis: a randomized, double blind, placebo controlled dose-finding study. j rheumatol. 2004;31:243-51. 3. miyata s, ohkubo y, mutoh s. a review of the action of tacrolimus (fk506) on experimental models of rheumatoid arthritis. inflamm res. 2005;54:1-9. 4. guimarães mr, nassar po, andia dc, nassar ca, spolidorio dm, rossa jr. c, et al. protective effects of tacrolimus, a calcineurin inhibitor, in experimental periodontitis in rats. arch oral biol. 2007;52:882-8. 5. cvetkovic m, mann gn, romero df, liang xg, ma y, jee ws, et al. the deleterious effects of long-term cyclosporine a, cyclosporine g, and fk506 on bone mineral metabolism in vivo. transplantation. 1994;57:1231-7. 6. hirotani h, tuohy na, woo jt, stern ph, clipstone na. the calcineurin/nuclear factor of activated t cells signaling pathway regulates osteoclastogenesis in raw264.7 cells. j biol chem. 2004;279:13984-92. 7. takayanagi h, kim s, koga t, nishina h, isshiki m, yoshida h, et al. induction and activation of the transcription factor nfatc1 (nfat2) integrate rankl signaling in terminal dif ferentiation of osteoclasts. dev cell. 2002;3: 889-901. 8. igarashi k, hirotani h, woo jt, stern ph. cyclosporine a and fk506 induce osteoclast apoptosis in mouse bone marrow cell cultures. bone. 2004;35: 47-56. 9. sabry a, el-agroudy a, sheashaa h, hawas s, el-shahat fb, barakat n. coadministration of ketoconazole and tacrolimus therapy: a transplanted rat model. int urol nephrol. 2006;38:713-8. 10. roberts we, smith rk, zilberman y, mozsary pg, smith rs. osseous adaptation to continuous loading of rigid endosseous implants. am j orthod. 1984;86:95111. 11. arias or, marquez-orozco mc. aspirin, acetaminophen, and ibuprofen: their effects on orthodontic tooth movement. am j orthod dentofacial orthop. 2006;130:364-70. 12. roberts we, helm fr, marshall kj, gongloff rk. rigid endosseous implants for orthodontic and orthopedic anchorage. angle orthod. 1989;59:247-56. 13. kale s, kocadereli l, atilla p, aşan e. comparison of the effects of 1,25 dihydroxycholecalciferol and prostaglandin e 2 on orthodontic tooth movement. am j orthod dentofacial orthop. 2004;125:607-14. 14. cohen a, shane e. osteoporosis after solid organ and bone marrow transplantation. osteoporos int. 2003;14:617-30. 15. abdelhadi m, ericzon bg, hultenby k, sjöden g, reinholt fp, nordenström j. structural skeletal impairment induced by immunosuppressive therapy in rats: cyclosporine a vs tacrolimus. transpl int. 2002;15:180-7. 16. king gj, keeling sd, wronski tj. histomorphometric study of alveolar bone turnover in orthodontic tooth movement. bone. 1991;12:401-9. 17. scott lj, mckeage k, keam sj, plosker gl. tacrolimus: a further update of its use in the management of organ transplantation. drugs. 2003;63:1247-97. 18. plosker gl, foster rh. tacrolimus: a further update of its pharmacology and therapeutic use in the management of organ transplantation. drugs. 2000;59(2):323-89. 19. hayakawa k, hata m, nishiyama t, ohashi m, ishikawa m. maintenance of unresponsiveness by short-term pulse therapy with fk 506 in rat transplantation. transplant proc. 1996;28:1830-1. 20. ren y, kuijpers-jagtman am, maltha jc. immunohistochemical evaluation of osteoclast recruitment during experimental tooth movement in young and adult rats. arch oral biol. 2005;50:1032-9. 21. kirino s, fukunaga j, ikegami s, tsuboi h, kimata m, nakata n, et al. regulation of bone metabolism in immunosuppressant (fk506)-treated rats. j bone miner metab. 2004;22:554-60. 22. nakamura k, sahara n, deguchi t. temporal changes in the distribution and number of macrophage-lineage cells in the periodontal membrane of the rat molar in response to experimental tooth movement. arch oral biol. 2001;46:593-607. oral sciences n3 original article braz j oral sci. january/march 2010 volume 9, number 1 implant-supported titanium framework: photoelastic analysis before and after spark erosion procedure mauricio pompeu cariello1, mauro antonio de arruda nóbilo2, guilherme elias pessanha henriques2, marcelo ferraz mesquita3, rafael leonardo xediek3, andréa fabiana de lira1 1phd level student, department of prosthodontics and periodontology, area of fixed denture, piracicaba dental school, state university of campinas, brazil 2professor, department of prosthodontics and periodontology, area of fixed denture, piracicaba dental school, state university of campinas, brazil 3professor, department of prosthodontics and periodontology, area of complete denture, piracicaba dental school, state university of campinas, brazil correspondence to: mauricio pompeu cariello rua leonardo mota, 620, apto 1101, meireles, fortaleza – ce e-mail: cariello@fop.unicamp.br received for publication: september 09, 2009 accepted: march 13, 2010 abstract aim: this study used a photoelastic analysis to evaluate the passive fit of titanium cast laser welding frameworks before and after spark erosion procedure. methods: a stainless steel cast was used in order to reproduce a human mandible. five multi-unit abutment analogs were attached to this cast and 6 frameworks were produced in commercial pure titanium. the cast was molded and a photoelastic matrix was produced incorporating 5 dental implants with multi-unit abutments. all samples were subjected to a laser welding. the precision of adjustments within a range of 0.5 µm was evaluated under microscope observation. the best fitted framework was selected and subjected to a photoelastic analysis (group i). the tightening of the screws was in 3 predetermined sequences (1,2,3,4,5/ 5,4,3,2,1/ 3,2,4,1,5). then the same framework was subjected to a refinement by spark erosion technique (group ii) and evaluated by photoelastic analysis. results: the sequence (3,2,4,1,5) achieved better results in both groups. a larger number of fringes were observed around the median implants in all sequences in both groups. conclusions: the titanium cast laser welded frameworks processes associated with spark erosion procedure improves significantly the marginal framework adaptation and is effective for its passive fit. keywords: titanium, misfit, implant, framework. introduction the use of implant-supported prostheses has achieved a significant success in edentulous patients, providing retention and prosthetic rehabilitation. the biomechanical aspect of an implant is fundamentally different from that of a natural tooth overloading an implant. there is the possibility to transfer excessive load to the implant and consequently from this to the circumjacent bone, which may exceed the physiologic limit and cause bone loss around the implants1. promoting a passive fit between the implants and the framework is a predictable manner to achieve a reliable prosthetic treatment over implants2-3. a previous review article showed that among several procedures to improve passive fit in implant-supported prostheses, laser-welding4 and electrical discharge machine (edm) refinement gave the best results5. titanium frameworks improved by laser-welding showed better adaptation over the implants when compared to the conventional welding on fine alloys6. edm allows overcoming the issues related to accuracy and passive fit of metallic frameworks and its usage has been justified due to a variety of alloys that braz j oral sci. 9(1):48-53 49 can replace the conventional gold prosthesis, which has excellent passive fit and has been used since 19827-8. many researchers working with microscopic analysis, strain gauges and finite element have introduced other methods to evaluate the passive fit of implant-supported prosthesis8-13. laboratorial procedures are extremely important to evaluate the best technique to be used in clinical situations and the photoelastic analysis is an experimental method that allows verifying the stress caused by the frameworks and their design. this method provides information in colored fringes of visible light viewed through a polariscope which helps to establish the stress level14. in dentistry, the photoelastic analysis has been largely used in order to evaluate the stress level on implant-supported prostheses allowing the global evaluation of stress in a specific component14-15. this procedure enables having a quality analysis of the stress level through the optical effect patterns caused by the frameworks over the photoelastic cast. considering the statements above, the aim of this study was to establish a comparison between frameworks before and after the edm procedure by the sequence of screw tightening. fig.1. master model with implant abutments (frontal view). material and methods a stainless-steel matrix was manufactured to simulate an edentulous mandibular arch. five parallel orifices were made in the matrix based on the design defined by a classic protocol16 and five multi-unit analogs (conexão sistema de próteses, são paulo, sp, brazil) were installed in each orifice. each analog was fixed by one screw located horizontally in the master cast and defined as 1, 2, 3, 4 and 5 (fig 1). six titanium framework groups were produced to fit the analogs (tritan; dentaurum, germany). five multi-unit cast cylinders were linked by a 4mm diameter blue wax profile (dentaurum pforzheim, germany). pk opaque wax was applied between the blue wax (dentaurum pforzheim) and the cast cylinders to provide better connection. a standard 10-mm-long blue wax was left as a cantilever. after waxing the framework it was put in a heatproof lining to the pre-heated electrical machine (vulcan 3-550ndi box furnace degussa ney dental inc yucaipa, ca, usa). after pre-heating, the framework was settled in the casting machine rematitan (dentaurum pforzheim) and a 31 g-titanium block was automatically injected. laser welding was performed using a desktop laser welding machine (dentaurum jp winkelstroeter kg pforzhein, germany). the titanium cylinders were screwed between the multi-unit analogs and the framework, and the framework-cylinder interface was welded. in order to minimize any deviation during the welding procedure one weld point was initially applied to the vestibular, lingual, mesial and distal surfaces. the welding procedure was completed by overlaying the welding points in about 60% (fig 2). after laser welding all samples, the misfit of each framework in both groups was evaluated with an optical microscope (stm digital olympus, japan) with 0.5-µm precision and x30 magnification. a gypsum index standardized the stainless-steel matrix position on the microscope dish. to take measurements, the frameworks were placed on the metallic matrix and the titanium screw (immediate loading prosthetic system; conexão sistema de próteses,) that corresponds to implant “1” was tightened with a 10 ncm torque force measured by a torque machine (immediate loading prosthetic system; conexão sistema de próteses,) and the misfit of the component “5” was evaluated. the same procedure was done on abutment “5” and the misfit in abutment “1” was evaluated17. each abutment was measured three times on the buccal (b) surface and three times on the lingual (l) surface and their averages calculated. the micrometric head ran from the previously marked abutment border up to the line located on the prosthetic cylinder on axis x. only the framework that demonstrated the best fit of each group was used for the photoelastic analysis. the square refinement tools were placed into the multi-unit frameworks on the metallic matrix through a long fit screw to obtain the photoelastic model. in order to produce the transfer mold, the long fit screw was connected to a dental floss and to the acrylic resin (pattern resin; gc corporation, tokyo, japan) using the “nealon” technique to keep the stability of the structure and the position of the abutment. after the acrylic resin polymerization the structure was split and reconnected using the same refinement tools. once all frameworks were connected, the metallic matrix was placed in the middle of a plastic cylinder (10.0 cm diameter x 5.5 cm high) for support for the molding procedure. fig. 2. laser-welded cylinder framework. braz j oral sci. 9(1):48-53 implant-supported titanium framework: photoelastic analysis before and after spark erosion procedure 50 laboratorial silicon (silibor; clássico produtos odontológicos ltda., são paulo, sp, brazil) with 48-hour curing time was used to duplicate the stainless steel matrix. after curing, the long fit screw was removed and the metallic matrix taken from the silicon mold. five multi-unit abutments were connected at 20 ncm tightness measured in a torque machine (clássico produtos odontológicos ltda.) to the connect artm implants (4.1 mm diameter x 13.0mm long; clássico produtos odontológicos ltda.). the whole assembly was installed to the transfer in the mold. the mold was filled with a photoelastic resin (araldite; huntsman llc, salt lake city, ut, usa) that has two components: the gy-279 modified with dilute reactive agent of low and medium viscosity composed of a biphenyla basis and the catalyst hy-2964 composed of cycloaliphatic amine as basis. fig. 3. photoelastic acrylic model. measurements were taken in a test tube and the handling of the resin took a becker. in order to get a uniform mixture, the catalyst was added to the resin in a glass tube and vigorously stirred. the bubbles formed from the stirring were eliminated in a vacuum chamber at 750 mm hg of internal pressure for 20 min. as recommended by the manufacturer, the photoelastic model was covered and left undisturbed for 72 h in order to disassemble the model from the mold (fig 3). the use of polariscope on the stress analysis allows a better view of the stress distribution on the structure. this enables to accurately determine the stress level around the implants when bright colored fringes are observed due to the polarized light dispersion through a photoelastic material. the analysis of stress distribution through photoelastic structure was made taking in account the images generated by the circular polariscope, connected to a digital camera to focus the fringes and to record the images. the pictures were taken for each sequence of screw tightening. the photoelastic analysis by counting the number of fringes around each abutment showed the magnitude of the stress and the distance in between fringes. a predetermined colored order of fringes, classified as lower or higher order, was used to identify the stress of the fringes (fig 4)17-18. this technique transforms the internal mechanical stress, produced in complex geometric structures, into visible light patterns of colors that indicate the areas and the magnitude of stress. to establish a possible connection between the level of framework misfit and the level of stress around the abutments, the frameworks were installed in the following tightening fig. 4. predetermined order of fringes to determine the intensity of stress. fig. 5. copper cable passing around copper analogous. fig. 6. gypsum structure and copper analogous. braz j oral sci. 9(1):48-53 implant-supported titanium framework: photoelastic analysis before and after spark erosion procedure 51 fig. 7. electrical discharge procedure: framework and cupper analogous immersed in dielectric liquid. sequence: abutments 1, 2, 3, 4, 5; abutments 5, 4, 3, 2 , 1; abutments 3, 2, 4, 1, 5. after having shot the pictures of each sequence, the photoelastic model was placed into an oven at 50°c for 20 min to release the stress. the titanium frameworks were placed in the electrical discharge machine (tel med technologies port huron, mi, usa) and high voltage electrical discharges were released with the objective to connect pieces within a precision of 0.01 mm. the transference of abutments of the metallic matrix enabled to obtain a gypsum model with copper analog abutments threaded by a copper cable for the electrical discharge procedure. (figs. 5 and 6). the titanium framework was fixed to the vertical rod using a proper glue (quick lock; tel med technologies port huron), settled to the gypsum mold without connection to the positive pole (red electrode), and plugged into the negative pole (black electrode). in order to start the electrical discharge, the gypsum structure model and the metallic framework were immersed in a dielectric liquid (tel med technologies port huron) to reduce the conductivity, to insulate and cool off the structure18 (fig 7). the vertical rod movements were controlled by the lower surface that also controlled the power intensity and the frequency of the released electrical discharges (250,000/s). the temperatures generated by the power between the copper electrode and the structure ranged from 3000°c to 5000°c, evaporating the metal refining the cervical finishing19. full adjustment of structure was visually shown after 8 h. the photoelastic analysis was performed again in the sequence of screws mentioned before. results from the welded laser structure without electrical discharge refinement, the following results can be considered: photoelastic analysis considering the prosthetic screw tightness: sequence 1/2/3/4/5. the digital pictures showed that after tightening the first screw some stress was released from the apical region of abutment 1. following the sequence and tightening the abutment 2 and 3, respectively, more stress was generated as shown on the photoelastic fringes at abutment distal 2 and abutment mesial 3. the fringes, which appeared around the abutments 2 and 3, reduced to a lower photoelastic order after tightening the screws 4 and 5 and the stress was more homogenous around the apical region of the 5 implants. photoelastic analysis considering the prosthetic screw tightness: sequence 5/4/3/2/1. in this sequence, there was a gradual increase of stress as identified by the photoelastic fringes. the stress was concentrated on the mesial and distal surfaces of the cervical region. stress was greater around abutments 4 and 3 with no change when the screws 2 and 1 were tightened. photoelastic analysis considering the prosthetic screw tightness: sequence 3/2/4/1/5 (fig 8). stress, and therefore fringes, was generated on mesial, distal and cervical regions when tightening the screw on the implant 3. when the screw 2 was tightened, the fringes on implant 3 did not reduce. tightening screw 4, the fringes grouped due to a great concentration of stress between the distal region of implant 3 and the mesial region of implant 4. fringes increased at the apical region of implants 3 and 4. the stress observed after tightening the sequence reduced the concentration of fringes and stress. more homogenous stress distribution was observed at implant 5. laser welding framework after electrical discharge gave the following results: photoelastic analysis on the prosthetic screw tightness: sequence 1/2/3/4/5e. higher stress was observed on the distal region after tightening screw 1. the tightening of screws 2 and 3 showed a concentration of fringes on the mesial, cervical and apical regions of implant 2 and on the mesial and distal regions of implant 3. however, the photoelastic fringes around implant 2 reduced to a lower order after tightening screws 4 and 5 and the stress showed better distribution. photoelastic analysis on the prosthetic screw tightness: sequence 5/4/3/2/1e. the stress was located on the top of implant 5 and reduced to a lower photoelastic order when fig. 8. photoelastic analysis of group i tightening sequence: 3/2/4/1/5 (a, b, c, d, and e, respectively). braz j oral sci. 9(1):48-53 implant-supported titanium framework: photoelastic analysis before and after spark erosion procedure fig. 9. photoelastic analysis of group i tightening sequence: 3/2/4/1/5e (a, b, c, d, and e, respectively). 52 implants 4 and 3 were tightened. colored fringes that indicate less stress could be observed around and on top of implants 4, 3, 2 and 1. after tightening the prosthetic screws (5, 4, 3, 2, 1), the fringes were observed around the implants. photoelastic analysis on the prosthetic screw tightness: sequence 3/2/4/1/5e (fig 9). the screw tightened on implant 3 produced photoelastic fringes around implant 3, on the distal, mesial and apical regions. however, when the screw was tightened on implant 2, the stress located at the apex reduced. on this sequence of tightening the screws, the stress was similar in all implants, with lesser photoelastic fringe formation around all implants homogeneously. less stress was induced on the implants in this sequence. discussion several methods to evaluate the passive fit of implant prosthesis have received great attention from the scientific community20-23. this study used the photoelastic analysis to determine the stress level on the framework, analyzed in the polariscope. however, it could be verified that the stress generated by the tightening of screws on the structure in the photoelastic cast had a different pattern at the apical region from the side region of implants. this demonstrates a nonuniform distribution of stress. the photoelastic analysis is highly indicated to evaluate the distribution and the quality of stress level produced by screw tightening21-23. in this study, the photoelastic analysis qualitatively described stress distribution. it may measure how certain transparent materials show photoelastic fringes when stressed under the polarized light. the larger the number of fringes, the larger the stress produced. however, other colors not strictly related to the fringes can also indicate some stress level21-23. the results of this study showed an important difference from the fringe standards on the photoelastic mold and laser welding when compared to the results on the framework undergone to laser welding and electrical discharge technique. the photoelastic analysis, however, has some limitations. it is necessary to calibrate the method of evaluation of the colored fringes, and then, the examiner will classify the stress generated around the implants. the result may be near to the real, but it is subjective. comparing the results of the photoelastic analysis to other method of stress evaluation, as strain gauges, is a valuable way to confirm the results. within the limitations of this study, the following conclusions were drawn: 1. the stress generated around the implants before or after the electrical discharge application was more evident on mesial implants than on the distal ones. 2. the sequence 3,2,4,1,5 associated with electric discharge application showed better results after all prosthetic screws had been tightened. 3. distal implants clinically overloaded by the cantilever extension in fixed implant-supported prosthesis was less stressed using the sequence of tightening the screws from the medial to distal direction of a framework when associated with laser welding and spark erosion. 4. the laser welding 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analysis of tensions induced over implant superstructures manufactured through cad/cam method, border soldering and one-piece technique [dissertation]. piracicaba: unicamp/ fop; 2007. 93p. 18. jemt t. failures and complications in 391 consecutively inserted fixed prostheses supported by branemark implants in edentulous jaws: a study of treatment from the time of prosthesis placement to the first annual checkup. int j oral maxillofac implants. 1991; 6: 270-6. 19. van roekel nb. electrical discharge machining in dentistry. int j prosthod. 1992; 5: 114-21. 20. gordon te, smith dl. laser welding of prostheses – an initial report. j prosthet dent. 1970; 24: 472-6. 21. lindquist lw, rockler b, carlsson ge. bone resorption around fixtures in edentulous patients treated with mandibular fixed tissue-integrated prostheses. j prosthet dent. 1988; 59: 59-63. 22. evans db. correcting the fit of implant-retained restorations by electric discharge machining. j prosthet dent. 1997; 77: 212-5. 23. bernardes sr, neves fd. tension analysis of a one-piece framework over different connections in implants [dissertation]. uberlândia: dental school of uberlândia; 2004. 192p. 53 braz j oral sci. 9(1):48-53 implant-supported titanium framework: photoelastic analysis before and after spark erosion procedure oral sciences n3 case report braz j oral sci. january | march 2013 volume 12, number 1 apexification with white mta in an immature permanent tooth with dens invaginatus anderson de oliveira paulo1, mário tanomaru-filho1 , renato de toledo leonardo1 gisselle moraima chávez-andrade1 , juliane maria guerreiro-tanomaru1 1 department of restorative dentistry, araraquara dental school, unesp – univ estadual paulista, araraquara, sp, brazil correspondence to: mário tanomaru filho rua humaitá, 1680, caixa postal 331, centro cep: 14801-903 araraquara, sp, brasil phone: +55 16 33016390 fax. +55 16 33016392 e-mail: tanomaru@uol.com.br received for publication: july 06, 2012 accepted: november 11, 2012 abstract dens invaginatus, also known as “dens in dente”, is a developmental dental anomaly resulting in an invagination of the enamel organ into the dental papilla. these cases present technical difficulties to the root canal treatment. apexification using an apical plug of mineral trioxide aggregate (mta) has been indicated as an alternative to long-term intracanal use of calcium hydroxide in immature permanent teeth. it is considered as a simple and rapid technique. this paper reports a case of oehlers’ type 1 dens invaginatus in an immature permanent maxillary right lateral incisor, which presented pulp necrosis secondary to dental trauma and was treated by apexification with white mta apical plugging followed by conventional root canal therapy. the operative procedures are described and the technique is discussed. the physical and biological properties of mta, associated with appropriate instrumentation and obturation techniques, make this material an excellent option in the endodontic therapy of immature permanent teeth with dens invaginatus. keywords: dens invaginatus, dens in dente, apexification, mineral trioxide aggregate, calcium hydroxide. introduction the term dens invaginatus was coined in 1953 by hallet1, who made the first documented attempt to classify invaginated teeth, suggesting the existence of four types of invagination based on both clinical and radiographic criteria. this is a term that applies for all of variants of this developmental dental anomaly, which results from an alteration in the normal growth pattern of the dental papilla during odontogenesis, showing a wide array of anatomical variations2-6. the classification system proposed by oehlers2 in 1957 is probably the most clinically relevant and is by far the most commonly used in clinical studies, case reports and case series6-9. it is based on hallet’s original classification1, but the cases are grouped in three major categories, according to the depth of the invagination and the existence of communication with the pulp tissue or periodontal ligament, regardless of the affected tooth (anterior, posterior, supernumerary). a single tooth can present multiple invaginations, but each one may fall into a different classification: type 1 the invagination is confined to the tooth crown, being limited to enamel and not extending beyond the cementoenamel junction (cej); type 2 the invagination extends apically beyond the cej and may or may not have pathways of communication with the dental pulp; type 3 the invagination extends apically beyond the cej and a second foramen is evident in the periodontal region. there is braz j oral sci. 12(1):61-65 6262626262 no communication with the pulp. new perspectives arose in the practice of dentistry with the development of mineral trioxide aggregate (mta). mta has been indicated in endodontics for treatment of perforations, retrograde fillings, pulpotomy and as an apical barrier in cases of immature non-vital teeth, also has antimicrobial properties 10-12. it has been extensively demonstrated that mta is a biocompatible material that stimulates the formation of new cementum on its surface and provides good seal10,12-16. white and gray mta present similar chemical composition and biological properties12,14,17-18. the use of an apical plug of mta has been indicated as an alternative to long-term intracanal use of calcium hydroxide for root-end closure of non-vital immature permanent teeth19-23. apical plugging with mta is a simple and rapid technique that eliminates the need of successive intracanal dressing changes10,21. some authors, however, have advocated that the use of calcium hydroxide-based intracanal dressing followed by preparation of an apical mta plug should be the treatment of choice for apexification of teeth with incomplete apex formation3,13,18,24. this paper reports a case of oehlers’ type 1 dens invaginatus in an immature permanent maxillary right lateral incisor, which presented pulp necrosis secondary to dental trauma and was treated by apexification with white mta apical plugging followed by conventional root canal therapy. case report a 9-year-old male child presented for treatment 1 day after sustaining a trauma to the maxillary right lateral incisor. the intraoral clinical examination showed no soft tissue lacerations, no crown or root fracture and no mobility. the preoperative pe riapical radiograph (figure 1) showed incomplete root formation with open apices and type 1 dens invaginatus according to oelhers’ classification. no significant periapical alteration was observed. since the tooth did not respond to cold and hot stimuli, the patient was scheduled for reevaluation within 2 weeks, but he did not show up. the patient returned 3 months later with a fistula associated with the apical region of the traumatized tooth. a new periapical radiograph showed the development of apical periodontitis (figure 2). considering the mode of treatment that is commonly adopted for cases of apexification7,13,18,24, the initial treatment plan was apexification mediated by changes of calcium hydroxide-based intracanal pastes followed by definitive obturation. the coronal access was prepared through the invaginated portion. monthly changes of the calcium hydroxide-based intracanal medication were done during 6 months, but no progress in apex closure was observed. therefore, an attempt was made to induce apexification by the placement of an apical plug with white mta (angelus dental solutions, londrina, pr, brazil). after local anesthesia and rubber dam isolation, the provisional restoration was removed access to the root canal was gained. a new determination of root canal length was done with a size 80 fig. 1. preoperative periapical radiograph showing incomplete root formation and oehlers’ type 1 dens invaginatus. fig. 2. periapical radiograph showing the development of apical periodontitis associated with the traumatized tooth 3 months after the first visit. k-file, and the total root length was established as being 20 mm (figure 3). the root canal was instrumented with a size 80 k-file, irrigated with a 1% sodium hypochlorite solution followed by a final flush with edta (byodinamic europe s.l., ibiporã, pr, brazil) and dried with sterile absorbent paper points. the mta powder and liquid components were mixed according to the manufacturer’s instructions and a size 70 k-file was used to prepare a 4-mm-thick apical white mta plug by repeated movements of file insertion and removal in an anticlockwise direction (figure 4). excess mta was removed from the canal walls and the remainder of the canal was obturated by lateral condensation of gutta-percha apexification with white mta in an immature permanent tooth with dens invaginatus braz j oral sci. 12(1):61-65 6363636363 fig. 3. periapical radiograph of root canal length determination with a size 80 k-file. and sealer 26 endodontic sealer (dentsply industry and trade ltda., rio de janeiro, rj, brazil). the coronal portion was restored with composite resin. the final radiographs showed a well-obturated root canal. the 21-month post treatment followup showed clinical and radiographic success (figure 5). discussion the etiology of dens invaginatus remains controversial. most authors2,5-6,9,25 describe its origin in a distortion of the enamel organ causing a protrusion inside the dental papilla, which produces an invagination of the tooth crown before fig. 5. periapical radiograph after 21 months of follow-up. fig. 4. periapical radiograph showing the apical white mta plug. calcification. also, a genetic source may be a significant factor6,8,26. according to the literature, the prevalence of dens invaginatus ranges from 0.03 to 10%4,6,26-27 and there is a femaleto-male predominance of 3:16. the permanent maxillary right lateral incisors are the most commonly affected teeth4,6,26, being bilateral in over 40-43% of the cases5,8-9,25, followed in a decreasing order of prevalence by central incisors, canines, premolars and molars7. the importance of an accurate and early diagnosis is justified because pathways of communication with the oral cavity can be created in invaginated teeth. this permits the penetration of irritating agents and microorganisms into the pulp tissue and leading to potential complications, namely caries disease, internal resorption, pulp necrosis, apical periodontitis and apical cyst formation2,8,26. in the present case, the tooth was classified as having dens invaginatus type 1, according to oehlers’ classification2. depending on the type of malformation and the communication of the invagination with the pulp tissue, the clinician may confine the endodontic therapy to the invaginated portion and, as a result, preserve pulp vitality. however, in most cases, the endodontic treatment must include both the invagination and the root canals. the task can become even more challenging, considering the multiple anatomical variations that a dens invaginatus may present within the root canal system. complete debridement of the root canal system can be compromised by limited access and as a result some areas may remain uninstrumented when a conventional technique is used. in order to overcome these limitations, some researchers have suggested that the invagination is removed under the operating microscope26. in the present case, the access apexification with white mta in an immature permanent tooth with dens invaginatus braz j oral sci. 12(1):61-65 6464646464 to the canals was gained through the invaginated portion. if nonsurgical endodontic therapy fails, a combined approach with apical surgery may be indicated. pulp involvement can occur at an early age when the roots are not completely formed. the large and irregular volume of the root canal system of invaginated teeth is an additional challenge for cleaning, shaping and instrumentation. the endodontic therapy of choice for non-vital immature teeth is apexification, which is the induction of apical closure to produce more favorable conditions for conventional root canal filling. calcium hydroxide is the most commonly advocated medicament because, in addition to a low cost, its efficacy to induce the formation of an apical mineralized barrier is well documented 15,16 even in cases of dens invaginatus with necrotic pulp7,9,27. however, apexification with calcium hydroxide requires successive changes of the intracanal medication that may last for several months, which may increase the risk of coronal leakage and recontamination of the root canal system. in addition, there have been reports on the decrease of the fracture strength of teeth subjected to long-term intracanal use of calcium hydroxide to induce apexification 13,15,20 and considerable interest has been expressed in the use of other materials, such as mta. the use of mta alternatively to calcium hydroxide has become increasingly widespread. several studies 10,22,28-29 showed that pediatric dentists in the world had used mta or arranged for its use in apical barrier formation in non-vital immature permanent teeth. kusgoz et al.10 and erdem and sepet21 reported clinical cases in which mta was used as a filling material in traumatized teeth with necrotic pulp and open apices and concluded that mta can be considered a very effective option for apexification of traumatized and immature permanent teeth with the advantage of shorter treatment time, good sealing ability and high biocompatibility. d’arcangelo and d’amario 18 reported two cases of apexification with apical mta plug after intracanal use of calcium hydroxide, in the same way as used in the present study. the presentation of these cases confirms that mta acts as an apical barrier and can be considered as an effective material to support regeneration of apical tissues in immature teeth with necrotic pulp for both young and adult patients. mta may be used in the treatment of dens invaginatus3,26. bogen and kuttler30 report a type 2 dens invaginatus with periapical periodontitis treated using an apical plug of mta. a paper describes the use of mta in two cases of dens invaginatus which were prepared with ultrasonic instrumentation under magnification26. silberman et al.25 reported a case of dens invaginatus in an immature tooth that was successfully treated with the placement of an apical plug of gray mta and conventional endodontics. in the present case, the apical plug was prepared with white mta, which has similar properties14,23. in conclusion, apical plugging with mta for induction of apexification allows for completing the treatment in a single session, which can be preceded by another session for p l a c e m e n t o f a c a l c i u m h y d r o x i d e b a s e d i n t r a c a n a l medication in teeth with infected root canals. the physical and biological properties of mta, associated with appropriate instrumentation and obturation techniques, make this material an excellent option in the endodontic therapy of immature permanent teeth with dens invaginatus. references 1. hallett ge. incidence, nature, and clinical significance of palatal invaginations in the maxillary incisor teeth. proc r soc med. 1953; 46: 491-9. 2. oehlers fa. dens invaginatus (dilated composite odontome). i. variations of the invagination process and associated anterior crown forms. oral surg oral med oral pathol 1957; 10: 1204-18. 3. paula-silva fw, rocha ct, flores ds, nelson-filho p, silva la, queiroz am. root canal treatment of an immature dens invaginatus with apical periodontitis: a case report. j dent child (chic). 2011; 78: 66-70. 4. gharechahi m, ghoddusi j. a nonsurgical endodontic treatment in openapex and immature teeth affected by dens invaginatus: using a collagen membrane as an apical barrier. j am dent assoc. 2012; 143: 144-8. 5. khan sa, khan sy, bains vk, bains r, loomba k. dens invaginatus: review, relevance, and report of 3 cases. j dent child (chic). 2012; 79: 143-53. 6. kallianpur s, sudheendra u, kasetty s, joshi p. dens invaginatus (type iii b). j oral maxillofac pathol. 2012; 16: 262-5. 7. kalaskar r, kalaskar ar. nonsurgical treatment of periapical lesion associated with type iii dens invaginatus using calcium hydroxide: a case report. j indian soc pedod prev dent. 2008; 26(suppl 2): s72-5. 8. alani a, bishop k. dens invaginatus. part 1: classification, prevalence and aetiology. int endod j. 2008; 41: 1123-36. 9. borges ah, semenoff segundo a, nadalin mr, pedro fl, da cruz filho am, sousa-neto md. conventional treatment of maxillary incisor type iii dens invaginatus with periapical lesion: a case report. isrn dent 2011; 2011: 257609. doi: 10.5402/2011/257609. 10. kusgoz a, yildirim t, tanriver m, yesilyurt c. treatment of horizontal root fractures using mta as apical plug: report of 3 cases. oral surg oral med oral pathol oral radiol endod. 2009; 107: e68-72. 11. tanomaru jm, tanomaru-filho m, hotta j, watanabe e, ito iy. antimicrobial activity of endodontic sealers based on calcium hydroxide and mta. acta odontol latinoam. 2008; 21: 147-51. 12. parirokh m, torabinejad m. mineral trioxide aggregate: a comprehensive literature review—part i: chemical, physical, and antibacterial properties. j endod. 2010; 36: 16-27. 13. moore a, howley mf, o’connell ac. treatment of open apex teeth using two types of white mineral trioxide aggregate after initial dressing with calcium hydroxide in children. dent traumatol. 2011; 27: 166-73. 14. porter ml, berto a, primus cm, watanabe i. physical and chemical properties of new-generation endodontic materials. j endod. 2010; 36: 524-8. 15. trope m. treatment of the immature tooth with a non-vital pulp and apical periodontitis. dent clin north am. 2010; 54: 313-24. 16. stefopoulos s, tzanetakis gn, kontakiotis eg. non-surgical retreatment of a failed apicoectomy without retrofilling using white mineral trioxide aggregate as an apical barrier. braz dent j. 2012; 23: 167-71. 17. da silva ej, andrade cv, tay ly, herrera dr. furcal-perforation repair with mineral trioxide aggregate: two years follow-up. indian j dent res. 2012; 23: 542-5. 18. d’arcangelo c, d’amario m. use of mta for orthograde obturation of nonvital teeth with open apices: report of two cases. oral surg oral med oral pathol oral radiol endod. 2007; 104: e98-101. apexification with white mta in an immature permanent tooth with dens invaginatus braz j oral sci. 12(1):61-65 19. annamalai s, mungara j. efficacy of mineral trioxide aggregate as an apical plug in non-vital young permanent teeth: preliminary results. j clin pediatr dent. 2010; 35: 149-55. 20. bidar m, disfani r, gharagozloo s, khoynezhad s, rouhani a. medication with calcium hydroxide improved marginal adaptation of mineral trioxide aggregate apical barrier. j endod. 2010; 36: 1679-82. 21. erdem ap, sepet e. mineral trioxide aggregate for obturation of maxillary central incisors with necrotic pulp and open apices. dent traumatol. 2008; 24: e38-41. 22. mooney gc, north s. the current opinions and use of mta for apical barrier formation of non-vital immature permanent incisors by consultants in paediatric dentistry in the uk. dent traumatol. 2008; 24: 65-9. 23. stefopoulos s, tsatsas dv, kerezoudis np, eliades g. comparative in vitro study of the sealing efficiency of white vs grey proroot mineral trioxide aggregate formulas as apical barriers. dent traumatol. 2008; 24: 207-13. 24. holden dt, schwartz sa, kirkpatrick tc, schindler wg. clinical outcomes of artificial root-end barriers with mineral trioxide aggregate in teeth with immature apices. j endod. 2008; 34: 812-7. 25. silberman a, cohenca n, simon jh. anatomical redesign for the treatment of dens invaginatus type iii with open apexes: a literature review and case presentation. j am dent assoc. 2006; 137: 180-5. 26. alani a, bishop k. the use of mta in the modern management of teeth affected by dens invaginatus. int dent j. 2009; 59: 343-8. 27. carvalho-sousa b, almeida-gomes f, gominho lf, albuquerque ds. endodontic treatment of a periradicular lesion on an invaginated type iii mandibular lateral incisor. indian j dent res. 2009; 20: 243-5. 28. doyle tl, casas mj, kenny dj, judd pl. mineral trioxide aggregate produces superior outcomes in vital primary molar pulpotomy. pediatr dent. 2010; 32: 41-7. 29. subramaniam p, konde s, mathew s, sugnani s. mineral trioxide aggregate as pulp capping agent for primary teeth pulpotomy: 2 year follow up study. j clin pediatr dent. 2009; 33: 311-4. 30. bogen g, kuttler s. mineral trioxide aggregate obturation: a review and case series. j endod. 2009; 35: 777-90. 6565656565 apexification with white mta in an immature permanent tooth with dens invaginatus braz j oral sci. 12(1):61-65 original article braz j oral sci. april/june 2009 volume 8, number 2 dental arch morphology of mazahua and mestizo teenagers from central mexico edith lara-carrillo1, juan carlos gonzález-pérez2, toshio kubodera-ito3, norma margarita montiel-bastida4, gema isabel esquivel-pereyra1 1 dds, ms, assistant professor, department of orthodontics, research center and advanced studies, school of dentistry, universidad autónoma del estado de méxico (uaem), mexico 2 dds, ms, student, department of orthodontics, research center and advanced studies, school of dentistry, uaem, mexico 3 dds, phd, professor, department of orthodontics, research center and advanced studies, school of dentistry, uaem, mexico 4 dds, phd head professor, research center and advanced studies, school of dentistry, uaem, mexico received for publication: april 13, 2009 accepted: june 22, 2009 correspondence to: edith lara-carrillo facultad de odontología de la universidad autónoma del estado de méxico paseo tollocan esq. jesús carranza, colonia universidad c.p. 50130 – toluca, estado de méxico, mexico e-mail: laracaedith@hotmail.com abstract aim: the aim of this study was to determine the morphologic characteristics of the dental arches in the ethnic group “mazahua” and mestizo teenagers from central mexico. methods: a sample of 80 mazahua and 80 mestizo teenagers with normal occlusion, divided into two age groups, were evaluated. a digital caliper was used to measure in cast models the intercanine width, intermolar width, length and perimeter of the arch, occlusal intermaxillary curve (of spee), overjet, and overbite. a comparative analysis with student’s t-test was applied between gender and population groups. results: comparison of the dental arches between mazahua and mestizo teenagers revealed that statistically significant differences existed with respect to most of the measurements. in most cases, they were greater in males; the mazahua teenagers had intercanine and intermolar widths greater than mestizo teenagers. conclusions: each group has a characteristic dental arch form. the ethnic group mazahua has squared arches, whereas the mestizo teenagers have oval arches, which give them their particular facial characteristics. these findings indicate that population-specific standards are necessary for clinical assessments. keywords: dental arch, odontometry, ethnic groups. introduction evaluation of dental arches is of great importance for definitive diagnosis and optimal craniofacial treatment. the values of the dimensions of the arch include: width, depth and circumference, intercanine and intermolar distances, overjet and overbite, which change during growth in different ways (the width of the teeth remains the same, whereas the lengths of the mandibular and maxillary bones increase)1. the circumference or perimeter is the most important dimension of the dental arch and changes according to age and gender. the increases in the arch are more related to the events underlying tooth development and somewhat less to skeletal growth. the intercanine distance increases significantly in the changeover dentition. the primate spaces allow the eruption of the permanent canines. the intercanine and intermolar widths do not change after 13 years old in females and 16 years old in males2,3. the overjet and overbite can be described in millimeters or in percentage; both go through significant changes during the transition from primary to permanent dentition. the overbite 93dental arch morphology of mazahua and mestizo teenagers from central mexico braz j oral sci. 8(2): 92-6 is related to the facial vertical dimensions, whereas the overjet is related to the skeletal anteroposterior relation and is also affected by the labial function and abnormal tongue. both can modify the skeletal growth of the patient4,5. the occlusal intermaxillary curve (of spee) has been related to overbite, lower arch circumference, lower incisor proclination and craniofacial morphology6. the final form of the arch is obtained by the configuration of the supporting bone, tooth eruption, orofacial muscles and intraoral functional forces7. most studies indicate that normal measurements for one group may not be considered normal for other race or ethnic groups. different racial groups must be treated according to their own characteristics8. however, there is no published study addressing the morphologic characteristics of the dental arches for ethnic and mestizo groups. mexico has enormous racial and sub-racial diversity, which is characterized by particular facial and oral characteristics. the mazahua is one of the most numerous indigenous groups of central mexico and the descendents of the tolteca-chichimeca culture. they live in isolated locations and preserve their own traditions. their craniofacial constitution differs from the settlers of the big cities. few anthropometric and dental studies have been made with the mazahua group. kiyomura9 found similarities of the metric and non-metric dental characteristics between mazahua and african and japanese races, establishing mongoloid characteristics in mazahuas, as determined by other studies on american inhabitants10. the aim of this study was to determine the morphologic characteristics of the dental arches of mazahua and mestizo teenagers from the central region of mexico, with the purpose of establishing similarities or differences between these population groups. material and methods a sample of 80 mazahua teenagers was selected from two schools in the municipality of san felipe del progreso in the central region of mexico, and 80 mestizo cast models obtained from the files of the orthodontics department of the research center at school of dentistry, universidad autónoma del estado de méxico, also in the central region of mexico. the teenagers were divided into groups based on age (12-14 and 15-17 years old) and gender. the inclusion criteria were: 1) for the mazahua group, those of mazahua origin (parents and grandparents from mazahua); 2) for the mestizo group, those of tolucan origin (parents and grandparents from toluca city); 3) 12-17 years old; 4) permanent dentition; 5) no dental crowding; 6) teeth free of visible interproximal decay and clinically visible cavities as well as misadjusted restorations which compromise the integrity of the contact point; 7) class i angle molar relation; and 8) no previous orthodontic, orthopedic or surgical treatment. the procedures followed ethical standards, with prior permission from the authorities and the informed consent from the parents and the subjects. complete dental impressions were obtained from the upper and lower arches, using alginate with an impression tray of rigid plastic that had been previously disinfected. an electrical vibrator was used (dv34, ray dental foster equipment, huntington beach, ca, usa) to fill the impressions. after obtaining the cast models, measurements were made with a digital caliper (ntd12-6”cx, mitutoyo co., utsunomiya, japan) directly from the cast models under natural light. the following parameters were measured (figure 1): 1. intercanine width: the distance in millimeters between the cuspid of the right and left permanent canines, in both arches; 2. intermolar width: the distance in millimeters between the central fossae of the right and left first permanent molars, in both arches; 3. length of the arch: the distance in millimeters from the central line to one point in the half distance between central incisors until a tangent that touches to the distal faces of the permanent second molars; 4. perimeter of the arch: measurement in millimeters from the distal face of the permanent first molar around the arch on the contact points and incisal edges, in a smooth curve to the distal face of the permanent first molar in the other arch side; 5. occlusal intermaxillary curve (of spee): the depth was measured in millimeters as the perpendicular distance between the deepest cusp tip and a flat plane that was laid on the top of the mandibular dental cast, touching the incisal edges of the central incisors and the distal cusp tips of the most posterior teeth in the lower arch. the measurement was made on the right and left sides of the dental arch and the mean value of these two measurements was used as the depth of the occlusal intermaxillary curve (of spee); 6. overjet: the horizontal distance in millimeters between the labial surface of the mandibular central incisors and the incisal tips of the maxillary central incisors; 7. overbite: the vertical distance between the incisal tips of the maxillary and mandibular central incisors. in order to eliminate the variability among examiners, two people measured the models and compared their measurements, repeating all the parameters when the difference between the first and second measurement was ± 1 mm. taking into account, the approximately normal distribution of each studied parameter, a student’s t-test was applied to assess differences in gender and population groups. all analyses were carried out using spss version 12.0 (statistical package for social sciences; spss inc., chicago, il, usa). statistical significance level was determined at p≤0.05. results comparison of the dental arches between mazahua and mestizo teenagers at the ages of 12-14 and 15-17 revealed statistically significant differences with respect to the majority of the measurements (p < 0.05) (tables 1 and 2; figure 2). in the comparative analysis of the dental arches between the mazahua and mestizo males who were 12-14 years old, there were statistically significant differences in most of the measurements. 94 lara-carrillo e, gonzález-pérez jc, kubodera-ito t, montiel-bastida nm, esquivel-pereyra gi braz j oral sci. 8(2): 92-6 measurements gender ethnicity mazahua mestizo p-value upper intercanine width male 38.41 ± 2.31 36.69 ± 1.75 0.02 female 35.76 ± 2.40 34.97 ± 1.84 ns p-value 0.01 0.01 lower intercanine width male 29.11± 2.61 27.66 ± 1.45 0.05 female 26.87 ± 1.86 26.13 ± 1.07 ns p-value 0.01 0.01 upper intermolar width male 48.87 ± 2.91 47.71 ± 2.71 ns female 47.34 ± 2.05 47.71 ± 2.35 ns p-value ns ns lower intermolar width male 43.48 ± 2.52 41.48 ± 2.61 0.02 female 41.24 ± 2.04 41.92 ± 1.68 ns p-value 0.01 ns length of the upper arch male 48.15 ± 2.20 47.01 ± 1.94 ns female 44.86 ± 3.20 44.32 ± 2.04 ns p-value 0.01 0.01 length of the lower arch male 44.49 ± 2.02 43.47 ± 2.05 ns female 41.82 ± 3.20 40.87 ± 2.11 ns p-value 0.01 0.01 perimeter of the upper arch male 11.20 ± 0.39 10.84 ± 0.44 0.02 female 10.65 ± 0.30 10.41 ± 0.42 0.05 p-value 0.01 0.01 perimeter of the lower arch male 10.12 ± 0.49 9.81 ± 0.43 0.05 female 9.58 ± 0.41 9.30 ± 0.34 0.02 p-value 0.01 0.01 curve of spee male 2.28 ± 0.69 1.65 ± 0.53 0.01 female 1.97 ± 0.78 1.56 ± 0.50 0.05 p-value ns ns overjet male 2.76 ± 0.90 2.71 ± 1.07 ns female 2.26 ± 0.74 2.45 ± 0.91 ns p-value ns ns overbite male 1.65 ± 0.76 2.77 ± 0.65 0.01 female 1.72 ± 1.14 2.17 ± 0.61 ns p-value ns 0.01 table 1. comparison of the dental arches between mazahuas and mestizos by genders (12 to 14 years old) data shown as mean ± sd; based on student’s t-test; ns: non significant; n=20 per group. measurements gender ethnicity mazahua mestizo p-value upper intercanine width male 38.01 + 2.0 36.01 + 1.48 0.01 female 36.19 + 2.80 35.03 + 1.81 ns p 0.02 ns lower intercanine width male 28.50 + 1.94 26.49 + 1.18 0.01 female 27.43 + 2.33 26.72 + 1.43 ns p ns ns upper intermolar width male 50.11 + 2.41 48.34 + 1.71 0.02 female 47.62 + 2.42 47.77 + 2.04 ns p 0.01 ns lower intermolar width male 43.89 + 2.23 41.89 + 1.56 0.01 female 41.67 + 1.84 42.12 + 1.87 ns p 0.01 ns length of the upper arch male 46.75 + 3.29 46.80 + 1.71 ns female 45.21 + 2.13 45.37 + 2.01 ns p ns 0.02 length of the lower arch male 43.83 + 2.49 42.38 + 1.40 0.02 female 41.82 + 2.12 42.24 + 2.25 ns p 0.01 ns perimeter of the upper arch male 11.08 + 0.55 10.70 + 0.40 0.02 female 10.66 + 0.50 10.58 + 0.45 ns p 0.02 ns perimeter of the lower arch male 9.92 + 0.54 9.65 + 0.29 ns female 9.52 + 0.56 9.57 + 0.45 ns p 0.02 ns curve of spee male 2.75 + 0.71 1.76 + 0.62 0.01 female 2.25 + 0.77 1.73 + 0.49 0.01 p 0.05 ns overjet male 2.29 + 0.88 2.51 + 0.84 ns female 2.30 + 0.78 2.34 + 0.75 ns p ns ns overbite male 1.85 + 0.98 2.34 + 1.17 ns female 1.67 + 1.07 2.25 + 1.23 ns p ns ns table 2. comparison of the dental arches between mazahuas and mestizos by genders (15-17 years old) data shown as mean + sd; based on student’s t-test; ns: non significant; n=20 per group. figure 1. measurements in cast models: 1. intercanine width. 2. intermolar width; 3. arch length; 4. perimeter of arch; 5. curve of spee; 6. overjet; 7. overbite. 95dental arch morphology of mazahua and mestizo teenagers from central mexico braz j oral sci. 8(2): 92-6 on the other hand, in the statistical analysis of the females between the mazahua and mestizo teenagers from the same age group, there were only significant differences in the perimeter of the upper and lower arches and in the occlusal intermaxillary curve (table 1). the measurements of the dental arches between the mazahua and mestizo males in the 15 to 17-year-old age group showed statistically significant differences in most variables. according to the analysis between the mazahua and mestizo females from this age group, a significant difference was observed in the occlusal intermaxillary curve in which the mazahua group had the greater depth of curve (table 2). the 12 to 14-year-old group showed significant differences between genders in most variables. the males in both ethnicity groups had significant larger values in most measurements than females except for overbite for mazahua population and lower intermolar width for the mestizo population. in the 15 to 17-year-old group, males had values significantly larger than females in most measurements, except for overjet for mazahuas and in lower intercanine and intermolar widths for the mestizo teenagers. in this age group, most measurements with statistically significant differences were observed among mazahuas, while among the mestizo population there was only one, in the length of the upper arch. discussion the analyses of dental size and arch dimensions establish human biological characteristics, such as the genetic relationship between populations and the adaptation of humans to their place of residence. odontometrics is one of the least studied areas of dentistry, so the variations and factors that affect normal growth are not understood11. rivera et al.7 suggested that the dimensions of arch width are genetically determined in a more specific way than the dimensions of arch length. in the present study, the morphologic characteristics of the dental arches of mazahua and mestizo teenagers from the central region of mexico were investigated. the results between genders in both ethnic groups differed with respect to the size of dental arches; males had larger dimensions, which is in accordance to the findings of previous studies4,12-16 that reported statistically significant differences between genders, males having greater dimensions. specifically, after ten years of age, males have a greater growth than females. at the same time, our results differed from those reported by nojima17 and ward18, who concluded that there is no sexual dimorphism in the dental arches and that is not necessary to establish gender groups because there are similar male-to-female ratios in ethnic populations. in the present study, mazahua and mestizo teenagers from the youngest age group showed significant differences between genders in most measurements. in the older age group, the mazahuas also had statistically differences between genders in most variables; but the mestizo population demonstrated some similarities between genders. it is probable that the pubertal growth spurt starts later in males than females in the mestizo population. it is interesting to observe that in both mazahua and mestizo populations, the occlusal intermaxillary curve increased with age. there is a natural tendency of this measurement to deepen with time. a deep curve of spee is usually associated with an increased overbite because the lower jaw’s growth downwards and forwards sometimes is faster and continues longer than that of the upper jaw6. in spite of this, no significant differences in the overbite were observed between age groups. intermolar and intercanine widths increased in the older population, but were more extreme in females, probably because girls finish tooth eruption before boys, except for third molars19. the data obtained in this study indicate that the mazahua group had larger arch dimensions than the mestizo population and, clinically, mazahuas are less likely to exhibit dental crowding. in mazahuas, the arch form differs, being more squared than in the mestizos figure 2. (a) representative mazahua dental arch and (b) representative mestizo dental arch. 96 lara-carrillo e, gonzález-pérez jc, kubodera-ito t, montiel-bastida nm, esquivel-pereyra gi braz j oral sci. 8(2): 92-6 (figure 2a) because they showed greater intercanine and intermolar widths. there was a larger arch perimeter and a steeper occlusal intermaxillary curve, which reflects greater overjet. the mestizo population had an oval arch (figure 2b) because the diameter of the intercanine and intermolar widths was smaller. hence, there are noticeable differences between these two ethnic groups that probably reflect greater miscegenation in the mestizo than in the mazahua population. in this way, it is reaffirmed that variations in the size of the dental arches are influenced by factors such as race, inheritance, and environment, as previously reported13,20-27 burris15 reported similar characteristics in african-americans compared to caucasian americans; african-americans had significantly larger arch lengths and widths. the arch in caucasians was disproportionately narrow in the canine-first premolar area, and defined a more rounded arch form. in contrast, the straighter and less convergent buccal tooth rows in african-americans defined a more squared arch form. some other studies carried out with australians27 or amazonian’s aborigines7 determined a good maxillary width development, as demonstrated by harmonic occlusal relations, little crowding and almost total absence of open bite or crossbite; in accordance to this mazahua sample. in conclusion, the findings of the present study indicate that there are morphologic characteristics of the dental arches of the mazahua and mestizo teenagers that differ between genders; males had larger diameters in both age groups. it was established that there is a characteristic form of the arches for each ethnic group. mazahuas have an arch with a squared form, since they have greater intercanine and intermolar widths, whereas the mestizos have oval arches because they have smaller intercanine and intermolar widths. characteristics in each population should be considered because or their influence on the craniofacial morphology. further studies should be developed to identify correlations between the different parameters measured in this study in order to establish the interactions among them in the human face growth. these ethnic differences should be considered particularly in specialties such as prosthodontics or orthodontics, in which arch shape matters for the treatment. references 1. moyers re. handbook of orthodontics. 4th ed. chicago: year book medical publishers; 1998. 2. alhaija esj, qudeimat ma. occlusion and tooth/arch dimension in the primary dentition of preschool jordanian children. int j paediatr dent. 2003;13:230-9. 3. lee rt. arch width and form: a review. am j orthod dentofac orthop. 1999;115:305-13. 4. abdula hh, al-ghamdi s. tooth width and arch dimensions in normal and malocclusion samples: an odontometric study. j contemp dent pract. 2005;6:36-51. 5. tibana rh, meira w, palagi l, augusto mj. changes in dental arch measurements of young adults with normal occlusiona longitudinal study. angle orthod. 2004;74:618-23. 6. baydas b, yavuz i, atasaral n, ceylan i, metin i. investigation of the changes in the positions of upper and lower incisors, overjet, overbite, and irregularity index in subjects with different depths of curve of spee. angle orthod. 2004;74:349-55. 7. rivera s, triana f, soto l, bedoya a. form and size of the dental arches in a school population of amazonian’s aborigines. [in spanish]. colom med. 2008;39 suppl 1:51-6. 8. uysal t, sari z. intermaxillary tooth size discrepancy and mesiodistal crown dimensions for a turkish population. am j orthod dentofacial orthop. 2005;128:226-30. 9. kiyomura m. affinities of human race populations as viewed from dental morphology. dental anthropology studies of mazahua indians and central mexicans. j meikai univ sch dent. 1996;25:326-34. 10. moreno f, milena ms, diaz ca, bustos ea, rodriguez jv. prevalence and variability of eight dental morphological characteristics of young people of three schools of cali. [in spanish]. colom med. 2004;35 suppl 1:16-23. 11. kieser ja. human adult odontometrics: the study of variation in adult tooth size. series: cambridge studies in biological and evolutionary anthropology. (no. 4) england: cambridge university press; 1990. 12. barrett mj, brown t, macdonald mr. size of dental arches in a tribe of central australian aborigines. j dent res. 1965;44:912-20. 13. bishara es, jakobsen jr, treder je and stasi mj. changes in the maxillary and mandibular tooth size-arch length relationship from early adolescence to early adulthood: a longitudinal study. am j orthod dentofac orthop.1989; 95:46-59. 14. slaj m, jezina ma, lauc t, rajic-mestrovic s, miksic m. longitudinal dental arch changes in the mixed dentition. angle orthod. 2003;73:509-14. 15. burris bg, harris ef. maxillary arch size and shape in american blacks and whites. angle orthod. 2000;70:297-302. 16. defraia e, baroni g, marinelli a. dental arch dimensions in the mixed dentition: a study of italian children born in the 1950s and the 1990s. angle orthod. 2006;76:446-51. 17. nojima k, mclaughlin r, isshiki y, sinclair p. a comparative study of caucasian and japanese mandibular clinical arch form. angle orthod. 2001;71:195-200. 18. ward d, workman j, richmond s. changes in arch width. a 20-year longitudinal study of orthodontic treatment. angle orthod. 2006;76:6-13. 19. harris e. mineralization of the mandibular third molar: a study of american blacks and whites. am j phys anthropol. 2007;132:98-109. 20. moorees cfa, thomsen so, jensen e, yen pk. mesiodistal crown diameters of the deciduous and permanent teeth in individuals. j dent res. 1957;36:39-47. 21. sanin c, savara bs. an analysis of permanent mesiodistal crown size. am j orthod. 1971;59:488-500. 22. arya bs, savara bs, thomas d, clarkson q. relation of sex and occlusion to mesiodental tooth size. am j orthod. 1974;66:479-86. 23. kenee hj. mesiodistal crown diameters of permanent teeth in male american negroes. am j orthod.1979;76:95-9. 24. howe rp, mcnamara ja jr, o’connor ka. an examination of dental crowding and its relationship to tooth size and arch dimension. am j orthod. 1983;83:36373. 25. staley rn, stuntz wr, peterson lc. a comparison of arch widths in adults with normal occlusion and adults with class ii, division 1 malocclusion. am j orthod. 1985;88:163-9. 26. bishara se, jakobsen jr, abdallah em, fernández a. comparisons of mesiodistal and buccolingual crown dimensions of the permanent teeth in three populations from egypt, mexico and the united states. am j orthod dentofac orthop. 1989;96:416-22. 27. merz ml, isaacson rj, germane n, rubenstein lk. tooth diameters and arch perimeters in a black and white populations. am j orthod dentofac orthop. 1991;100:53-8. oral sciences n3 original article braz j oral sci. 8(4):181-184 bonding to wet or dry deproteinized dentin: microtensile bond strength and confocal laser micromorphology analysis aloísio o. spazzin1; daniel galafassi2 ; luciano s. gonçalves3; rafael r. moraes4; bruno carlini-júnior5 1 dds, ms, graduate student; department of restorative dentistry, dental materials division, piracicaba dental school, state university of campinas, brazil 2 dds, ms, graduate student, department of restorative dentistr y, ribeirão preto dental school, university of são paulo, brazil 3 dds, ms, professor, department of dental materials and restorative dentistry, dental school, university of uberaba, brazil 4 dds, ms, phd, professor; department of restorative dentistry, dental school, federal university of pelotas, brazil 5 dds, ms, phd, professor; department of restorative dentistry, dental school, university of passo fundo, brazil correspondence to: aloísio o. spazzin department of restorative dentistry, dental materials division piracicaba dental school, state university of campinas, av. limeira 901 – 13414-903, piracicaba-sp, brazil e-mail: aospazzin@yahoo.com.br abstract aim: to investigate the influence of deproteinization and moisture condition (wet vs. dry) on the bond strength and micromorphology of resin-dentin bonding interfaces. methods: dentin surfaces were etched with 37% phosphoric acid for 15 s and rinsed with water. four groups (n = 10) were tested: wet: dentin was left visibly moist; dry: dentin was dried with compressed air; wet-d: dentin was deproteinized for 60 s using 10% naocl solution and left moist; dry-d: dentin was deproteinized and dried. prime&bond 2.1 adhesive was applied and the teeth were restored with composite resin. microtensile test was carried out after 24 h, and failure modes classified under magnification. data were subjected to two-way anova and tukey’s test (p < 0.05). the bonding micromorphology was analyzed by confocal laser scanning microscopy. results: the group dry showed significantly lower bond strength (p < 0.05) than the other groups, which were similar to each other (p > 0.05). adhesive failures were predominant. analysis of micromorphology showed formation of a collagen-resin hybrid layer only for the non-deproteinized groups. adhesive penetration into the dentinal tubules was deeper for the dry-d compared to the wetd group. conclusion: the bond strength was not dependent on the moisture condition and a more homogeneous hybridization was obtained when dentin was deproteinized. keywords: bonding agents, dentin, microscopy, confocal, sodium hypochlorite, bond strength. introduction when dentin is acid-etched, the moisture condition of the exposed collagen network is critical for achieving optimal bond strengths1-2. dehydration causes shrinkage and collapse of the unsupported mesh, inhibiting efficient wetting and penetration by the bonding solution3. incomplete resin infiltration leaves an exposed, non-infiltrated zone beneath the hybrid layer4. it has been suggested that this exposed collagen network is susceptible to hydrolytic degradation over the course of time5. however, pooled moisture must also be avoided, as excess water can dilute the bonding agent and impair the adhesive procedure6. due to the clinical difficulty in maintaining the appropriate moisture level on the etched dentin, alternatives for reducing the technique sensitivity of bonding strategies have been investigated. gwinnett et al.7 suggested that the adhesive efficiency relies on resin diffusion into the partially demineralized dentin at the basal portion of the substrate rather than on micromechanical interlocking with the collagen fibrils. according to vargas et al.8, the collagen braz j oral sci. october/december 2009 volume 8, number 4 received for publication: june 24, 2009 accepted: november 11, 2009 182 braz j oral sci. 8(4):181-184 layer inhibits penetration of the resin monomers into the dentin, leaving collagen fibrils unprotected and susceptible to degradation. therefore, it has been suggested that adhesion to the mineral substrate of the dentin after deproteinization with sodium hypochlorite (naocl) could produce a more stable bond, as the unprotected collagen areas would be eliminated9-11. naocl is a non-specific proteolytic agent that effectively removes organic compounds at room temperature 12. scanning electron microscopy studies of demineralized dentin treated with naocl showed that the collagen network is removed to reveal an eroded, rough mineral surface with numerous lateral branches, larger than normal tubular orifices13-15. previous studies have reported an increase in bond strength to etched dentin when the bonding agent was applied after deproteinization8,13,16. nonetheless, little is known about the interaction between dentin deproteinization and the moisture condition of the substrate during the bonding procedures. the aim of this in vitro study was to evaluate the influence of deproteinization and moisture condition (wet vs. dry) on the bond strength of an acetone-based bonding agent to etched dentin. the micromorphology of the bonding interfaces created using the different bonding strategies was also evaluated. the hypothesis tested was that collagen removal would increase the bond strength irrespective of the dentin moisture condition. material and methods the study was approved by the research ethics committee of the dental school of the university of passo fundo, brazil (protocol 856/ 2005). extracted sound human third molars were immersed in distilled water at 4oc and used within 4 months after extraction17. preparation of specimens the roots of 40 teeth were embedded in self-curing acrylic resin. the occlusal enamel was removed using a low speed water-cooled diamond saw (isomet 1000; buehler, lake bluff, il, usa) to expose a flat area in medium coronal dentin. the dentin surfaces were wet-polished with 600-grit sic abrasive papers for 30 s using an automatic polisher (metaserv 2000; buehler) to standardize the smear layer17. all dentin surfaces were etched with 37% phosphoric acid gel (dentsply caulk, milford, de, usa) for 15 s and rinsed with water for 15 s. the teeth were randomly divided into four groups (n = 10), according to the dentin treatment after acid-etching and rinsing: wet: the dentin surface was dried with a cotton pellet, leaving the dentin visibly moist; dry: the dentin surface was dried for 15 s using compressed air, leaving the dentin visibly dry ; wet-d: the dentin surface was deproteinized for 60 s using 10% naocl solution, rinsed with water for 15 s, and dried with a cotton pellet, leaving the dentin visibly moist; dry-d: the dentin surface was deproteinized for 60 s using 10% naocl solution, rinsed with water for 15 s, and dried for 15 s using compressed air, leaving the dentin visibly dry. the acetone-based, single-bottle adhesive system prime&bond 2.1 (dentsply caulk) was applied to all groups, according to the manufacturer’s directions. a first layer of the bonding agent was applied using a microbrush and left undisturbed for 20 s, and then another layer was applied and gently air-dried for 5 s. after light-activation for 20 s using a quartz-tungsten-halogen curing unit (xl2500; 3m espe, st. paul mn, usa) with irradiance ~600 mw.cm-2, 4 mm height blocks of resin composite (supreme; 3m espe) were built up in 4 increments, which were light-activated for 20 s each. all bonding procedures were performed by only one operator. specimens were stored in distilled water at 37oc for 24 h. microtensile bond strength (µtbs) test after storage, the specimens were sectioned to the long axis of the tooth into 1 mm-thick slices using the low speed water-cooled diamond saw. the bonding interface of each slice was trimmed to create an hourglass shape as previously described18, with trimmed crosssectional area of approximately 1 mm2. each specimen was fixed to the grips of a microtensile device and tested in tension on a mechanical testing machine (dl2000; emic, são josé dos pinhais, pr, brazil) at a crosshead speed of 0.5 mm.min-1 until failure. after testing, the fractured specimens were carefully removed from the testing device with a scalpel blade and the cross-sectional area at the site of fracture was measured to the nearest 0.01 mm using a digital caliper (starret, itu, sp, brazil). the cross-sectional area was used to calculate bond strength values in mpa. the number of teeth tested was 10 for all groups, as the tooth was considered the experimental unit for the statistical analysis. however, during the trimming procedures, some slabs were lost. therefore, for each specimen from all groups, 2-4 hourglass-shaped specimens were obtained and the average was recorded as the µtbs value for each tooth. data were subjected to two-way anova followed by tukey’s test (p < 0.05). the fractured specimens were analyzed under optical microscopy at 200× magnification. the modes of failure were classified as adhesive failure or mixed failure involving bonding agent and dentin. confocal laser scanning microscopy (clsm) analysis a mass of 0.5% of rhodamine b fluorescent dye was added to the bonding agent prior to application to the dentin surfaces. for each bonding condition, two dentin slices (2 mm in thickness) were obtained, and the same bonding procedures were used. the fluorescent dye was added to the bonding solution only for the clsm analysis, not for the bond strength measurements. therefore, the dye did not have any influence on the bond strength test. the specimens were sectioned longitudinally using the low speed water-cooled diamond saw and embedded in polyester resin. the surfaces were wet-polished with 600 and 1200-grit sic papers and 3 µm alumina paste, and ultrasonically cleansed for 10 min. a cls microscope (tcs-sp2; leica, heidelberg, germany) was used to obtain images of the bonded interfaces, focusing on the thickness of the bonding agent, formation of a collagen-resin hybrid layer, and penetration of the bonding solution into the dentinal tubules. the protocol used to obtain the images was described elsewhere19. results µtbs test results for µtbs for all groups are shown in table 1. the statistical analysis showed significant differences for the dentin treatment (p = 0.002), but not for the moisture condition (p = 0.125). however, the interaction between the two factors was significant (p = 0.025). significantly higher µtbs values (p<0.009) were observed for the groups wet and d ry-d compared to th e g roup d ry. no sig nif icant differences were detected between the wet and dry moisture conditions for the deproteinized samples (p = 0.588), and no significant differences were detected between the dentin treatments when adhesion was obtained using the wet-bonding technique (p = 0.465). results for the failure analysis are also shown in table 1. a predominance of adhesive bonding to wet or dry deproteinized dentin: microtensile bond strength and confocal laser micromorphology analysis 183 braz j oral sci. 8(4):181-184 bon d stre ngth (m p a ) f a ilure mo de s (% a – m *) d e n t in t re a t m e n t w e t d r y w et d r y n o n tre a te d 1 5 . 3 ( 7. 6 ) a ,a 8 . 2 ( 3 .2 ) b ,b 8 5 – 1 5 9 1 – 9 d e p r o te in ize d d e n t in 1 7 . 8 ( 8. 0 ) a ,a 1 9 . 2 ( 7 .2 ) a ,a 8 1 – 1 9 7 6 – 2 4 table 1. means (standard deviations) for microtensile bond strength and failure mode distribution. means followed by different uppercase letters in the same line and lowercase letters in the same column are significantly different at p < 0.05. *percentage of adhesive (a) and mixed (m) failures. failures was observed for all groups. however, the group dry-d presented higher percentage of mixed failures than the other groups. clsm analysis figure 1 shows representative clsm images for all bonding strategies. irrespective of the moisture condition, the specimens not exposed to naocl showed evidence of hybridization11, characterized by encapsulation of the inter-tubular collagen mesh by the adhesive resin forming a collagen-resin hybrid layer beneath the adhesive layer. hybridization was more clearly visible for the wet group compared to the dry group. the wet group also showed deeper penetration of the bonding agent into the dentinal tubules than the dry group. for the non-deproteinized samples, a distinct layer of concentrated bonding agent was observed in the bottom of the adhesive layer, characterized by a highlighted thin line right above the hybrid layer. on the other hand, no hybrid layer formation and bonding agent concentration were observed for the deproteinized groups, irrespective of the dentin moisture condition. a thicker layer of bonding agent and deeper penetration into the dentinal tubules was observed for the dry-d compared to the wet-d group. fig. 1 clsm images of the resin-dentin bonding interfaces (ad = adhesive; hl = hybrid layer; t = resin tag). the specimens not exposed to naocl (a, b) showed formation of a collagen-resin hybrid layer; a distinct layer of adhesive concentration above the hybrid layer (pointers) was also observed. the wet group showed deeper penetration of the bonding agent into the dentinal tubules than the dry group. no hybrid layer formation or bonding agent concentration was observed for the deproteinized groups (c, d). the dry-d group showed a thicker layer of bonding agent and deeper penetration of the bonding solution into the dentinal tubules compared to the wet-d group. discussion the present results showed that the deproteinization increased the bond strength to the dry dentin, while no increase in bond strength was observed for the wet condition. therefore, the tested hypothesis must be partially rejected. the clsm analysis also showed no formation of collagen-resin hybrid layer for the deproteinized groups. previous studies agree that the collagen network might not be required to achieve high bond strengths to dentin1,7-8. deproteinized surfaces show a completely eroded, rough mineral surface with numerous lateral branches, larger than normal dentin orifices. these characteristics may explain the improved bonding performance to dry dentin after naocl treatment, as higher amount of monomers could diffuse into the irregularities for mechanical interlocking. the increased wettability of the collagen-depleted substrate may also facilitate interand intratubular resin infiltration11. regarding the moisture conditions, the dry group showed the lowest bond strength values. it is well-known that dehydration of the demineralized dentin leads to a collapse of the collagen network, impairing infiltration of the bonding agent into the mesh. the clsm analysis, however, showed formation of a hybrid layer and resin tags even for the dry group, indicating diffusion of the bonding solution into the exposed mesh. nonetheless, the wet group showed a more clearly visible hybrid layer, and also deeper penetration of the bonding agent into the dentinal tubules, indicating the wet condition is important for bonding when no deproteinization is carried out. indeed, no significant differences were detected between the dentin treatments when adhesion was obtained using the wet-bonding technique. another aspect to be highlighted is that no significant differences in µtbs were detected between the wet and dry moisture conditions for the deproteinized samples. also, no layer of bonding agent concentration was detected when deproteinization was performed, probably due to the absence of the collagen mesh interfering with the infiltration of the bonding agent into the substrate. these characteristics provide evidence that the adhesion associated with collagen depletion might have advantageous characteristics. one characteristic is that the moisture condition may not affect the bond strength when deproteinization is carried out. in addition, the bonding agent diffuses better within the deproteinized substrate, forming a more homogeneous bonding layer that may be potentially less sensitive to hydrolytic degradation over the course of time20. the dry-d group presented higher percentage of mixed failures than the other groups. the clsm analysis provided evidence to explain this result. the group dry-d showed the deepest penetration of the bonding agent into the dentinal tubules. in corroboration, dayem21 reported deeper penetration depth of a one-bottle adhesive through the acid-conditioned dentin after treatment with 10% naocl. during the µtbs test, the better mechanical interlocking of the adhesive with bonding to wet or dry deproteinized dentin: microtensile bond strength and confocal laser micromorphology analysis braz j oral sci. 8(4):181-184 the dentin may favor the generation of mixed failures involving both substrates. however, the deepest penetration into the dentinal tubules did not provide additional increase in bond strength. a probable explanation for this finding is that the deeper resin tags may have occupied the tubular liners without adhering to the tubular walls22. in conclusion, the bond strength to dentin became independent of the moisture condition and a more homogeneous hybridization (e.g. no adhesive concentration) was observed when deproteinization was carried out. these findings confirm that the collagen layer is not primordial for bonding, and its presence may impair the diffusion of monomers into the substrate. these results suggest that a clinical adhesive procedure combining collagen and moisture removal might be effective, as the control of the dentin moisture is a critical procedure and the deproteinization may compensate for the dehydration by removing the collapsible collagen fibrils. however, other bonding solutions should be tested, as the effect of naocl treatment might be material-dependent23. as the results of this study cannot be directly extrapolated to in vivo situations, clinical data are still required. references 1. gwinnett aj. altered tissue contribution to interfacial bond strength with acid conditioned dentin. am j dent. 1994; 7: 243-6. 2. lima fg, moraes rr, demarco ff, del pino fa, powers j. one-bottle adhesives: in vitro analysis of solvent volatilization and sealing ability. braz oral res. 2005; 19: 278-83. 3. swift ej, jr., perdigao j, heymann ho. bonding to enamel and dentin: a brief history and state of the art, 1995. quintessence int. 1995; 26: 95-110. 4. spencer p, swafford jr. unprotected protein at the dentin-adhesive interface. quintessence int. 1999; 30: 501-7. 5. kato g, nakabayashi n. the durability of adhesion to phosphoric acid etched, wet dentin substrates. dent mater. 1998; 14: 347-52. 6. tay fr, gwinnett ja, wei sh. micromorphological spectrum from overdrying to overwetting acid-conditioned dentin in water-free acetone-based, singlebottle primer/adhesives. dent mater. 1996; 12: 236-44. 7. gwinnett aj, kanca ja, 3rd. micromorphology of the bonded dentin interface and its relationship to bond strength. am j dent. 1992; 5: 73-7. 8. vargas ma, cobb ds, armstrong sr. resin-dentin shear bond strength and interfacial ultrastructure with and without a hybrid layer. oper dent. 1997; 22: 159-66. 9. torres cr, de araujo ma, torres ac. effects of dentin collagen removal on microleakage of bonded restorations. j adhes dent. 2004; 6: 33-42. 10. sauro s, mannocci f, tay fr, pashley dh, cook r, carpenter gh et al. deproteinization effects of naocl on acid-etched dentin in clinically-relevant vs prolonged periods of application. a confocal and environmental scanning electron microscopy study. oper dent. 2009; 34: 166-73. 11. sauro s, mannocci f, toledano m, osorio r, pashley dh, watson tf. edta or h3po4/naocl dentine treatments may increase hybrid layers’ resistance to degradation: a microtensile bond strength and confocal-micropermeability study. j dent. 2009; 37: 279-88. 12. sakae t, mishima h, kozawa y. changes in bovine dentin mineral with sodium hypochlorite treatment. j dent res. 1988; 67: 1229-34. 13. inai n, kanemura n, tagami j, watanabe lg, marshall sj, marshall gw. adhesion between collagen depleted dentin and dentin adhesives. am j dent. 1998; 11: 123-7. 14. montes ma, de goes mf, sinhoreti ma. the in vitro morphological effects of some current pre-treatments on dentin surface: a sem evaluation. oper dent. 2005; 30: 201-12. 15. perdigao j, thompson jy, toledano m, osorio r. an ultra-morphological characterization of collagen-depleted etched dentin. am j dent. 1999; 12: 250-5. 16. saboia vp, rodrigues al, pimenta la. effect of collagen removal on shear bond strength of two single-bottle adhesive systems. oper dent. 2000; 25: 395-400. 17. uceda-gomez n, loguercio ad, moura sk, grande rh, oda m, reis a. longterm bond strength of adhesive systems applied to etched and deproteinized dentin. j appl oral sci. 2007; 15: 475-9. 18. cenci m, demarco f, de carvalho r. class ii composite resin restorations with two polymerization techniques: relationship between microtensile bond strength and marginal leakage. j dent. 2005; 33: 603-10. 19. bitter k, paris s, martus p, schartner r, kielbassa am. a confocal laser scanning microscope investigation of different dental adhesives bonded to root canal dentine. int endod j. 2004; 37: 840-8. 20. wakabayashi y, kondou y, suzuki k, yatani h, yamashita a. effect of dissolution of collagen on adhesion to dentin. int j prosthodont. 1994; 7: 302-6. 21. dayem rn. assessment of the penetration depth of dental adhesives through deproteinized acid-etched dentin using neodymium:yttrium-aluminum-garnet laser and sodium hypochlorite. lasers med sci. 2010; 25: 17-24. 22. prati c, chersoni s, mongiorgi r, pashley dh. resin-infiltrated dentin layer formation of new bonding systems. oper dent. 1998; 23: 185-94. 23. castro ak, amaral cm, ambrosano gm, pimenta la. effect of sodium hypochorite gel on shear bond strength of one-bottle adhesive systems. braz j oral sci. 2004; 3: 465-9. 184 bonding to wet or dry deproteinized dentin: microtensile bond strength and confocal laser micromorphology analysis oral sciences n3 original article braz j oral sci. july | september 2015 volume 14, number 3 elderly caregivers at long-stay institutions: quality of life and temporomandibular dysfunction tânia adas saliba rovida1, rosana leal do prado1, renata colturato joaquim1, luiz fernando tano1, cléa adas saliba garbin1 1universidade estadual paulista – unesp, araçatuba dental school, department of pediatric and social dentistry, araçatuba, sp, brazil correspondence to: tânia adas saliba rovida departmento de odontologia infantil e social universidade estadual paulista – unesp rua josé bonifácio, 1193 cep: 16015-050 araçatuba, sp, brasil phone: +55 18 3636 3249 fax: + 55 18 3636 3332 e-mail: tasalibarovida@foa.unesp.br received for publication: august 02, 2015 accepted: september 22, 2015 http://dx.doi.org/10.1590/1677-3225v14n3a06 abstract aim: to assess the profile, quality of life and the presence of temporomandibular disorders (tmd) in elderly caregivers. methods: thirty-nine caregivers who worked in six long-stay institutions for the elderly in three different municipalities participated in the study. the research instruments were the whoqol-bref, the fonseca questionnaire and the socio-demographic questionnaire. the kruskal-wallis test with the dunn’s correction was performed at a level of 5%. results: females accounted for 94.9% of the sample. the average monthly wage was r$ 832.00. the average working week was 39 hours. the whoqol dominions showed the following averages: 74.25 physical; 70.33 psychological; 65.79 social affairs; 58.38 environment. mild dtm was present in 43.6% of the caregivers, of which 7.7% were moderate, 5.2% severe, 23.0% of the professionals were asymptomatic and 20.5% did not answer. the association between tmd and the quality of life showed p=0.6752. conclusions: it was concluded that there was no relationship between the quality of life and symptoms of temporomandibular dysfunction among the caregivers of elderly in this study. keywords: caregivers; aged; quality of life; temporomandibular joint dysfunction syndrome. introduction currently in brazil, more than 12% of the population is considered elderly. data from the 2010 census showed that the brazilian population consists of about 20 million people aged over 60 years. the percentage of individuals aged over 65 years (7.4%) is higher than the percentage of the group of children 0-4 years of age (7.3%)1. with the increasing elderly population, chronic degenerative diseases that compromise the autonomy of the elderly become evident, requiring the presence of an individual caregiver who can be informal (family member or friend) or formal (professional)2. regardless of the relationship of the caregiver to longevity, their primary function is the provision of care to supplement the temporary or permanent functional impairment3. the most common difficulties faced by these caregivers are work overload, loss of purchasing power, social isolation, physical and psychiatric social burden4. in addition, concerning the interpretation of activities related to the welfare of the elderly, the caregiver might have restrictions, affecting their quality of life5. physical-psychological disorders are commonly found in people who are willing to treat the elderly. some emotional disturbances such as anxiety, irritability, resentment and stress usually are frequent findings in the lives of these employees. braz j oral sci. 14(3):204-208 205205205205205 a consequence of these changes has been studied since the 50s, showing that they may predispose or act as etiological factors for temporomandibular disorders (tmd). such changes may retain or be a consequence of tmd6. tmd refers to a group of chronic orofacial disorders affecting the masticatory muscles, temporomandibular joints (tmj) and the surrounding structures, and may change the performance of certain functions of the stomatognathic system, such as chewing and speaking7-8. the signs and characteristic symptoms of this disorder are mainly pain and tenderness in the tmj and muscles of mastication, asymmetry and restriction of mandibular movements and joint noises7-8. tmd has a multifactorial etiology, including parafunctional habits like bruxism, teeth clenching, occlusal factors, traumatic or degenerative lesions of the tmj, unbalanced action of the masticatory muscles. in addition, some psychological factors such as stress and anxiety may predispose to this dysfunction8-9. people exposed to various stressors, such as caregivers to the elderly commonly show an increase of muscle overactivity, which may predispose to tmd10. among the changes caused by tmd, pain in particular can interfere with the daily activities of the affected individual, leading to a negative effect on social function and emotional health11-13. the present study aimed to investigate the presence of temporomandibular joint dysfunction in caregivers of longstay institutions for the elderly (lsie) and relate it to the quality of life of these professionals. material and methods a cross-sectional survey study in all long-stay institutions for the elderly (lsie), of three municipalities (araçatuba, birigüi and penápolis), totaling six entities was conducted. after prior authorization from their leaders, all professionals who work as caregivers in these institutions were invited to participate in the survey in the following manner: no sampling was performed but a census of the target population. the study included caregivers who agreed to participate, signed the consent form and completed the questionnaire correctly. for the collection of data, the following self-applied instruments were used: sociodemographic questionnaire containing data such as age, sex, marital status, professional training, time on the job in the institution, salary and weekly workload. the whoqol-bref questionnaire (who, 1994, translated into portuguese by fleck, 1998) was employed to investigate quality of life. this instrument was drafted by the study group on the quality of life of the division of mental health of the who (1994). initially, the whoqol100 was developed, containing 100 questions. however, with the need for tools for rapid implementation for large epidemiological studies, the whoqol-bref was developed. it is an abbreviated form containing the 26 questions that provided the best psychometric results of the whoqol-100, meeting the criteria for scientific research. the questionnaire consists of two general questions about the participant’s perception of their quality of life and their health and over 24 questions relating to four domains: physical, psychological, social relationships and environment14. the scores represent a profile of the quality of life of the participants. it has a positive relationship to the quality of life, in other words, the higher the score, the higher the quality of life. this score is converted to a scale ranging from 0 to 10015. a questionnaire developed by fonseca et al.16 was used for evaluating tmd. it allows characterizing the symptoms of tmd and thus classifies patients according to their severity. its main advantage when compared to the research diagnostic criteria for temporomandibular disorders, called drc or the helkimo index, is the lower time of application and therefore a lower cost17. there are three possible answers for each question of the questionnaire, each one with a score. adding the scores, an anamnesis index is obtained, which allows classifying the severity of the symptoms: without tmd (0 to 15 points), mild tmd (20 to 45 points), moderate tmd (50 to 65) and severe tmd (70-100 points). the professionals signed a free consent form, and an explanation on the filling of the instruments was performed. the studied variables were gender, age, marital status, weekly working hours, vocational training (education), expressed compensation, assessment of quality of life and tmd, the latter being the outcome of interest in this research. socioeconomic and demographic variables were used to characterize only the sample, without the inclusion of confounding variables in the analyses. data analysis was performed by applying a kruskalwallis statistical test with dunn’s correction at a significance level of 5% to determine the association between the variables using the biostat, version 5.0 statistical software. the study was approved by the ethics committee of the araçatuba school of dentistry (process 01916-2011), paulista state university, brazil. results thirty-nine elderly caregivers consented to participate in the study. of these, 94.9% were female and 5.1% male. the average age was 38.7 years, ranging between 24 and 60 years. 51.3% were married, 28.2% single, separated and divorced represented 10.3% each. the average monthly wage was r$ 832.00, ranging between r$ 570.00 and r$ 1,190.00. the average weekly working hours were 39.0 hours ± 7.9 sd. nursing technicians represented 28.2% (n=11) of the participants, followed by nursing at 20.5% (n=8). table 1 demonstrates the training of the caregivers. regarding the quality of life domains, figure 1 shows the mean scores. the environment domain presented the worst elderly caregivers at long-stay institutions: quality of life and temporomandibular dysfunction braz j oral sci. 14(3):204-208 professional training n % social worker 1 2.6 nursing assistant 8 20.5 nurse 3 7.7 elementary school 5 12.8 high school 4 10.3 physiotherapist 1 2.6 psychology 1 2.6 nursing technician 11 28.2 no response 5 12.8 total 39 100.0 table 1 table 1 table 1 table 1 table 1 – professional training reported by caregivers of elders of the six long-stay institutions for the aged research participants araçatuba, 2011. fig. 1 – mean scores and standard deviations of the domains of quality of life measured by the whoqol-bref for caregivers of institutionalized elderly araçatuba, 2011. results regarding the quality of life of the caregivers (figure 1). at that moment were exposed the facets that comprise the environmental domain of the whoqol-bref: physical safety and protection, home environment, financial resources, availability and quality of health care and social opportunity to acquire new information and skills, participation and opportunities for recreation/leisure and physical environment (pollution, noise, traffic, weather and transportation). all the facets that make up the four domains of the whoqol-bref are shown in figure 2, with their scores. in general, the caregivers participating in this study had a reasonable quality of life (figures 1 and 2), with lower scores for recreation and leisure activities and remuneration obtained with work (figure 2). out of the caregivers, 43.6% had mild tmd, 7.7% had moderate, 5.2% had severe, 23.0% of the professionals were asymptomatic and 20.5% did not answer, as shown in figure 3. regarding the tmd, the mean score of the assessed quality of life using the whoqol-bref among caregivers who reported no symptoms was 68.8 (±10.3), light tmd 69.5 (±7.9), moderate tmd 70.2 (±15.5) and severe tmd 60.6 (±14.9). the main finding of this study was that there was no significant association between the quality of life and temporomandibular dysfunction. when comparing the tmd outcome variable, regardless of its severity with the quality of life, the p-value was 0.6752. fig. 2 –mean scores of the facets of the four domains of the quality of life measured by the whoqol-bref for caregivers of institutionalized elderly araçatuba, 2011. 206206206206206elderly caregivers at long-stay institutions: quality of life and temporomandibular dysfunction braz j oral sci. 14(3):204-208 207207207207207 discussion in the present study, it was found that the social and demographic characteristics of the study population were similar to those found in other studies of institutionalized caregivers’ profile. the prevalence of women in the practice of elderly care is common in the literature18-19. the increasing integration of women into the labor market, mainly from the second half of the twentieth century can be explained by the deterioration of wages, leading the women to help in the family budget, and their increasing participation in public spaces, especially in nursing which is historically considered a female profession18. the cultural issue that sees the care of women with children, husband and home is also seen as facilitating its adaptation to exercise care for the elderly19. regarding the professional qualifications of workers, there is dissonance in relation to the dominant formation. in the present study, most caregivers have technical training in nursing, which agrees with the study of sampaio et al.20, who found that 57.7% of caregivers also had this training. reis and ceolim18 found this function in 40% of nursing assistants18. carneiro et al.21 observed an unfavorable situation in the institutions studied in the tocantins state, because when it comes to schooling, 9% of the caregivers were illiterate, 27.2% of the caregivers did not complete the first and second grades, and 18.2% did not complete the third grade. thus, in their study, none of the caregivers directed their training for occupations that ordinarily attend to this public21. however, the current brazilian scenario shows a large deficit in the training of professionals to care adequately for the elderly, whether at home, hospital or institutional18. there is no specific regulation for the caregiver profession for the elderly, which hinders the establishment of remuneration for these professionals. there is a gap in the literature that makes it difficult to compare the results obtained in this study, where the average wage was less than two minimum brazilian wages. a low wage may lead to looking for other forms of salary compensation, contributing to the stress of these professionals. fig. 3: percentage distribution of the classification of professionals according to the severity of the symptoms of tmd by the fonseca questionnaire araçatuba, 2011. regarding the workload, the board resolution rdc / anvisa no. 283 of 2005 establishes a minimum to professionals working in the lsies, with the aim to constitute a standard of operations. thus, the qualified technician must have a minimum workload of 20 hours per week, while the number of working hours of caregivers varies with the degree of dependency of the elderly, from 8 hours a day or a working shift22. overall, the caregivers reported a good quality of life (figure 2), which may also be found in the study conducted in portugal (2009), in which, despite being reported as a stressful profession, caregivers did not report poor quality of life23. the separate evaluation of the whoqol facets showed that the environment was the area with the worst average, i.e., demonstrating a worse evaluation than quality of life, and participation in recreation and leisure had the worst average score appointed by the participants in this dominion (figure 2). it is worth mentioning the importance of these activities in people’s health while fighting stress, since it offers opportunities for interaction and social development, contributing to the well being of individuals while enhancing their potential and cannot be dismissed24. dissatisfaction with pay was expressed as the second worst score (figure 2), and may have consequences for job performance, leading to the demotivation of the workers with their work. the dominion of personal relationships was the one that had the second worst ranking. personal relationships, sexual activity and support or social support were assessed in this domain. the way individuals relate to themselves and to the people around them is an important tool to preserve their well being and therefore their quality of life. thus the quality of life can be affected by perception, feelings and behaviors related to daily activities, and is not limited to health status only25. the psychological and physical domains had the best averages, the facets being positive feelings, sleep and rest with the lowest scores within these domains. evidences show that tmd causes an impact on the quality of life, especially when related to pain26. in the present study there was no relationship between the quality of life and tmd, as demonstrated by the results. the mean score obtained by the whoqol among caregivers that presented symptoms or not were similar. this fact could be explained by the small number of professionals with moderate or severe tmd, i.e., the majority of professionals did not present pain symptoms. this can be seen in the positive score of the pain facet in the physical domain (figure 2), which suggests that caregivers had no negative interference of pain related to the general state of quality of life. however, one can observe that those professionals who presented severe tmd had the worst scores in relation to the average quality of life, thus suggesting that the pain could be related to that fact. in relation to the issue of self-rating of the quality of life, the average score was 72.76, i.e., it may be considered a good self-perception, not influencing the development of temporomandibular disorders. the fact is that studies of patients diagnosed with this disorder indicate a worse quality elderly caregivers at long-stay institutions: quality of life and temporomandibular dysfunction braz j oral sci. 14(3):204-208 208208208208208 of life, especially concerning the issue of physical pain as well as sleep quality, when compared with patients without this disorder27-28. in view of the above, it may be concluded that there was no relationship between the quality of life and symptoms of temporomandibular dysfunction among the elderly caregivers participating in this study. references 1. brazilian institute of geography and statistics. [census brazil 2010]. [cited 2011 sep 20]. available from: http://www.censo2010.ibge.gov.br. portuguese. 2. valer db, aires m, fengler fl, paskulin lmg. adaptation and validation of the caregiver burden inventory for use with caregivers of elderly individual. rev lat am enfermagem. 2015; 23: 130-8. 3. rovida tas, peruchini fld, moimaz sas, garbin cas. the meaning of general and oral health on the elderly caregiver’s view. odontol clín cient. 2013; 12: 43-6. 4. nascimento lc, moraes er, silva jc, veloso lc, vale armc. elderly caregiver: the knowledge available in lilacs database. rev bras enferm. 2008; 61: 514-7. 5. fernandes bcw, ferreira kcp, marodin mf, do val mon, fréz ar. influence of physiotherapeutic guidelines in the caregiver’s quality of life and overload. rev fisioter mov. 2013; 26: 151-8. 6. rugh jd, woods bj, dahlström l. temporomandibular disorders: assessment of psychological factors. adv dent res. 1993; 7: 127-36. 7. pihut m, szuta m, ferendiuk e, zeñczak-wiêckiewicz d. differential diagnostics of pain in the course of trigeminal neuralgia and temporomandibular joint dysfunction. biomed res int. 2014 [cited 2015 feb 15]; 2014: 563786. doi: 10.1155/2014/563786. available from: http://www.ncbi.nlm.nih.gov/pmc/ articles/pmc4065756/pdf/bmri2014-563786.pdf. 8. manfredi aps, bortolleto ppb, silva aa, araújo iem, araújo s, vendite ll. environmental stress and temporomandibular disorder (tmd) among members of a public university in brazil. braz j oral sci. 2006; 5: 1074-8. 9. calixtre lb, grüninger bls, chaves tc, oliveira ab. is there an association between anxiety/depression and temporomandibular disorders in college students? j appl oral sci. 2014; 22: 15-21. 10. cestari k, camparis cm. psychological factors: its importance in temporomandibular disorders diagnosis. j bras oclusao atm dor orofac. 2002; 2: 54-60. 11. chen y, williams sh, mcnulty al, hong jh, lee sh, rothfusz ne, et al. temporomandibular joint pain: a critical role for trpv4 in the trigeminal ganglion. pain. 2013; 154: 1295-304. 12. lobbezoo f, drangsholt m, peck c, sato h, kopp s, svensson p. topical review: new insights into the pathology and diagnosis of disorders of the temporomandibular joint. j orofac pain. 2004; 18: 181-91. 13. john mt, dworkin sf, mancl la. reliability of clinical temporomandibular disorder diagnoses. pain 2005; 118: 61-9. 14. fleck mpa, louzada s, xavier m, chachamovic e, vieira g, santos l, et al. application of the portuguese version of the abbreviated instrument of quality life whoqol-bref. rev saude publica. 2000; 34: 178-83. 15. the whoqol group. whoqol-bref: introduction, administration, scoring and generic version of assessment. geneva: world health organization; 1996. 16. fonseca dm, bonfante g, valle al, freitas sft. [diagnosis of the craniomandibular disfunction through anamnesis]. rgo. 1994; 42: 23-8. portuguese. 17. biasotto-gonzalez da, andrade dv, gonzalez to, martins md, fernandes kps, corrêa jcf, et al. [correlation between temporomandibular dysfunction, cervical posture and quality of life]. rev bras crescimento desenvolv hum. 2008; 18: 79-86. portuguese. 18. reis po, ceolim mf. [the meaning attributed to ‘being old’ by workers of long term care nursing institutions]. rev esc enferm usp. 2007; 41: 5764. portuguese. 19. colomé ics, marqui abt, jahn ac, resta dg, carli r, winck mt, et al. [taking care of institutionalized elders: characteristics and difficulties of the caregivers]. rev eletr enf. 2011; 13(2): 306-12. portuguese. 20. sampaio amo, rodrigues fn, pereira vg, rodrigues sm, dias ca. [senior caretakers: perception about aging and its influence on the nursing act]. est pesqui psicol. 2011; 11: 590-613. portuguese. 21. carneiro bg, pires edo, dutra filho ad, guimarães ea. [profile of caregivers of elderly people from long-stay institutions and prevalence of painful symptoms]. conscientiae saude. 2009; 8: 75-82. portuguese. 22. brazil. resolution rdc no. 283 of 26 september 2005. [approves the technical regulation para que define operating standards as long-stay institutions for seniors]. diário oficial; 2005 set 27 . portuguese. 23. pimenta gmf, costa masmc, gonçalves lhtg, alvarez am. [profile of the caregiver of dependent elderly family members in a home environment in the city of porto, portugal]. rev esc enferm usp. 2009; 43: 609-14. portuguese. 24. kanters ma. the health benefits of parks and recreation. illinois parks & recreation; 1996 [cited 2012 jan 30]. available from: http:// www.lib.niu.edu/1996/ip960127.html. 25. carvalho fs, maia júnior af, carvalho cap, sales peres a, bastos jrm, sales peres shc. [quality of life the dentists]. rev odontol unesp. 2008; 37: 65-8. portuguese. 26. oliveira as. evaluation of quality of life and pain in temporomandibular disorders (tmd). braz j oral sci. 2005; 4: 646-50. 27. figueiredo vmg, cavalcanti al, farias abl, nascimento sr. prevalence of signs, symptoms and associated factors in patients with temporomandibular dysfunction. acta sci health sci. 2009; 31: 159-63. 28. biasotto-gonzalez da, mendes pcc, jesus la, martins md. [healthrelated quality of life in patients with temporomandibular disorder: a cross sectional study]. rev inst cienc saude. 2009; 27: 128-32. portuguese. elderly caregivers at long-stay institutions: quality of life and temporomandibular dysfunction braz j oral sci. 14(3):204-208 oral sciences n3 original article braz j oral sci. july/september 2009 volume 8, number 3 apical gaps after apicoectomy procedures performed on teeth filled with gutta-percha or resilontm marco antonio hungaro duarte1, angélica marquezim lopes locci2, ivaldo gomes de moraes1 juliane maria guerreiro tanomaru3, mário tanomaru filho3 1 dds, msc, phd, professor of endodontics, department of dentistry, dental materials and endodontics, bauru school of dentistry, univeristy of são paulo, bauru, sp, brazil 2 dds, postgraduate student in endodontics, deaprtment of dentistry, dental school, university center of nor thern são paulo, são josé do rio preto, sp, brazil 3 dds, msc, phd, professor of endodontics, department of restorative dentistry, dental school of araraquara, são paulo state university, araraquara, sp, brazil received for publication: july 14, 2009 accepted: october 28, 2009 correspondence to: marco antonio hungaro duarte departamento de endodontia faculdade de odontologia de bauru, usp rua alameda otávio pinheiro brisolla, 9-75, bauru, sp, brasil cep: 17012-101 c.p. 13 phone: +55-14-32358344 phone/fax: +55-14-32346147 e-mail: mhungaro@travelnet.com.br abstract aim: this ex vivo study compared, under scanning electron microscopy (sem), the marginal adaptation of root canal obturation with either resilontm or gutta-percha cones following root-end resection. methods: thirty human single-rooted teeth with fully formed apices were collected and decoronated. the root canals were instrumented up to a size 45 taper .04 and obturated with laterally condensed gutta-percha (group 1; n=15) or resilontm (group 2; n=15). ah plus sealer was used in both groups. after 48-h storage in saline, the apical 3 mm of each root were resected with a water-cooled high-speed plain fissure #170l carbide bur. epoxy resin replicas of the resected root ends were examined by sem. the total area of apical gap in each replica was measured using uthscsa imagetool software. data were analyzed statistically by the mannwhitney u-test (α=5%). results: the mean area of apical gap in groups 1 and 2 was 0.0042 mm2 and 0.0015 mm2, respectively, with no statistically significant difference (p = 0.83). conclusions: the type of material did not influence at the apical adaptation of root canal obturation after apicoectomy, and the misfit may be related to anatomic factors. keywords: apicoectomy, resilontm, gutta-percha. introduction periradicular surgery is based on two goals, namely to eliminate the etiologic agents causing infection and to prevent root canal reinfection and recontamination of the periodontal tissues thereafter. basically, the etiologic agents involved in endodontic infections may be classified as intraradicular or extraradicular microorganisms, intraradicular or extraradicular chemical substances and extraradicular physical factors1-3. the root apex surrounded by a periapical lesion presents areas of cemental resorption and harbors microorganisms and bacterial biofilm4-5. resection of the root apical portion may be performed with either highor low-speed rotary instruments under constant saline irrigation. it has been demonstrated that depending on its type, angulation and rotary direction, the bur used for root-end resection may create surface irregularities and expose the dentinal tubules to a greater extent. the use of surgical length fissure burs6, cross-cut fissure burs7 and diamond burs8 has been recommended for root-end resection. a previous scanning electron microscopy (sem) study9 examined root-end resections performed using three bur configurations in both high and low-speed handpieces and observed that the smoothest surface and the least amount of guttapercha disturbance were produced by the #57 plain fissure bur at low-speed. in addition, better fit of the filling material to the canal walls is obtained when root-end resection is performed braz j oral sci. 8(3):141-144 with the handpiece moved across the tooth in a forward direction in relation to the direction of rotation of the bur10. however, the above-mentioned studies9-10 have examined guttapercha root fillings. although gutta-percha is universally accepted as a standard of root canal filling material, it does not have adhesion to root canal dentin and always requires association with an endodontic sealer11. advances in adhesive technology and the search for a material with greater adhesion to the canal walls and to the sealer have resulted in a solid material named resilontm (resilon research llc, madison, ct, usa), which is based on a blend of synthetic thermoplastic polyester polymers and contains bioactive glass and radiopaque fillers. this material performs like gutta-percha, has the same handling properties and is usually used in combination with a dual-cure m eth acr yl at e resin-based s eale r (epiphany ; pentron c lini cal technologies, walling ford, ct, usa) supplied with a self-etching primer12. obturation using the resilontm/epiphany system is reported to create a tight seal with the dentinal tubules within the root canal system; in essence, it is claimed to produce a “monoblock” effect, where the core material (resilontm), sealer and dentinal tubules become a single solid structure12-13. however, a recent study14 has found significantly lower push-out bond strength of the new obturation system to intraradicular dentin compared to gutta-percha/ah 26 sealer. the resilontm/epiphany system has demonstrated good sealing properties when subjected to different leakage tests15-17, though no statistically significant difference has been found when compared to other root filling materials, like gutta-percha/ah plus sealer18-19. some studies 20-22 have shown that resilon tm cones have similar thermoplasticity between gutta-percha and resilon cones. nevertheless, no study has yet evaluated resilontm and guttapercha with respect to their apical fit in apicoectomized teeth. therefore, the purpose of this in vitro study was to compare, under sem, the apical fit of root canal obturation with either resilontm or gutta-percha cones after root-end resection with high-speed #170l carbide burs. material and methods thirty extracted single-rooted human teeth with fully formed apices were selected for the study. the teeth were immersed in 5% sodium hypochlorite (naocl) for 12 h and then stored in saline until use, when they were decoronated at the cementoenamel junction with a double-faced diamond saw at low speed. a size 10 k-file (maillefer, ballaigues, switzerland) was introduced into the canal until its tip was visible at the apical foramen and the working length was established 1 mm short of this length. the root canals were instrumented using the profile rotary system (dentsply/maillefer, ballaigues, switzerland). the cervical preparation was performed with orifice shaper (maillefer) number 2 (30 taper 06), number 3 (40 taper 06) and number 4 (50 taper 07). after cervical preflaring, the apical portion was prepared using the profile 04 size 15 up to a size 45 at the working length. the canals were irrigated with 2.5% naocl at each change of file. when instrumentation was completed, the canals were filled with 1 ml 17% edta during 3 min, received a final flush with 1% naocl and were dried with absorbent paper points. two groups of 15 specimens each were formed at random. in group 1, the root canals were obturated with a fitted size 45/04 gutta-percha master cone (maillefer) and ah plus resin-based sealer (dentsply detrey konstanz, germany) using a lateral compaction technique. the sealer was taken to the canal using a lentulo spiral (maillefer) before the insertion of the gutta-percha cone (maillefer). a finger spreader was placed alongside the master cone and compaction was done to make space for up to three ff accessory gutta-percha points (maillefer). excess material was removed from the pulp chamber and the filling mass was vertically condensed. in group 2, the gutta-percha cone was replaced by a size 45/04 resilontm master cone. the endodontic sealer was taken to the canal in the same way as described for group 1 and resilontm accessory points were also used. the coronal portion of each root canal was sealed with irm (dentsply/caulk, milford, de, usa). the root-filled teeth were stored in saline at 37oc during 48 h for complete setting of the sealer. after this period, the apical 3 mm of each root were resected using a plain fissure #170l carbide bur in a high-speed handpiece under constant water cooling to remove any accumulated debris and to keep the root surface moist. the cutting direction followed the direction of rotation of the bur (clockwise rotation). a new bur was used for each root-end resection and an attempt was made to produce the smoothest possible surface in all specimens. after root-end resection, the filling material was burnished against all root canal walls with a cold #33 burnisher, from the center to margins, and the resected root surfaces were washed and dried with a gentle air stream. impressions were obtained from all faces of the resected apical segments with a condensation silicone impression material (zeta plus/oranwash l; zhermak, badia polesine, rovigo, italy). the heavy-bodied material (zeta plus) was first applied onto the specimen and allowed to polymerize for 7 min. next, the lightbodied material (oranwash l) was used to refine the impression. in both groups, each resected apical segment was paired with its respective impression. thereafter, the impressions were replicated with epoxy resin (rd-6921; redelease, são paulo, sp, brazil) with a hardening agent in positive vacuum, allowed to polymerize within 24 h. care was taken to minimize entrapment of air bubbles. the obtained epoxy resin positive replicas were sputter-coated with gold (hammer vi sputtering system, anatech ltd., alexandria, va), examined with a scanning electron microscope ( jsmt220a, jeol, tokyo, japan) and photographed at ×75 magnification. the sem micrographs of the epoxy resin replicas of the resected apical segments were digitized and analyzed with respect to the area (in mm2) of apical gap using imagetool software version 3.01 (uthscsa, san antonio, tx, usa). after calibration, the measurements of gap space between the obturation and the root canal walls were summed and one value (mm2) was obtained for each specimen. data were analyzed statistically by the mann-whitney utest and fisher’s exact test at 5% significance level. results ta b l e 1 p re s e n t s t h e m e a n a n d m e d i a n g a p a re a ( i n m m 2 ) obtained in each group and show the sum of post and mean post o b t a i n e d b y m a n n w h i t n e y t e s t . t h e r e w a s n o s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s ( p > 0 . 0 5 ) b e t w e e n g r o u p s 1 a n d 2 . comparison of the number of specimens with and without gap between the two groups (table 2) showed no significant differences either (p>0.05). figures 1 and 2 show sem micrographs of teeth subjected to apicoectomy after root canal filling with either gutta-percha or resilontm cones, respectively. apical gaps after apicoectomy procedures performed on teeth filled with gutta-percha or resilontm142 braz j oral sci. 8(3):141-144 group mean m e d i a n sum of posts mean post gutta-percha 0.0042 0.00020 226.5 15.1 resilon 0.0015 0.00044 238.5 15.9 table 1. mean and median (mm2) of the gap area and sum post and mean post obtained by mann-whitney test. group with w i t h o u t total gutta-percha 9 6 15 resilon 11 4 15 table 2. number specimens with and without gap in each group. figure 2. group 2 (resilontm). sem micrograph of a tooth subjected to apicoectomy after root canal filling with resilontm cones. arrow indicates to gap areas (×75 magnification). figure 1. group 1 (gutta-percha). sem micrograph of a tooth subjected to apicoectomy after root canal filling with gutta-percha cones. arrow indicates to gap areas (×75 magnification). discussion in periradicular surgeries, curettage of the pathologic apical lesion and resection of the contaminated root apex are of paramount importance for treatment success. even if the root canal filling is radiographically classified as adequate, the occurrence of apical gap between the obturation and the canal walls and the need for root-end cavity preparation and retrograde restoration should always be assessed after apicoectomy6. studies have compared the action of different rotary instruments and techniques on root apex morphology after apicoectomy8, the refinement of resected root-end surfaces with finishing burs to improve root apex topography23, the use of high-power lasers for apicoectomy2425, the use of ultrason26 and the sealing capacity of several filling materials, such as resilontm cones, gutta-percha cones, epiphany sealer, ah plus sealer16,19,20. however, to the best of our knowledge, no other study has duplicated the present experimental model to evaluate the marginal adaptation of obturations with resilontm and gutta-percha cones in apicoectomized teeth. the type of rotary instrument, the technique23,25-26 and the direction of rotation of the bur12 may produce an irregular surface following root-end resection and gap formation between the filling material and the root canal walls in the apical portion leading to microbial recontamination and treatment failure. in the present study, root-end resections were performed with a water-cooled high-speed #170l multifluted carbide bur because this type of rotary instrument has been shown to produce smoother surfaces9-10. the direction of rootend resection was the same as that of bur rotation in order to minimize tearing, smearing and distortion of the cones onto the root canal walls10. in the present study, comparison between the groups based on the mean values of apical gap demonstrate that the group with root canals filled with resilontm cones presented less gap formation (0.0015 mm2) than the group with root canals filled with gutta-percha cones (0.0042 mm2). this difference was not statistically significant, probably because the filling materials had similar thermoplasticity27. although water-cooling was used in the present study, a temperature rise may occur during root-end resection procedures10. adhesion of the filling material to the root canal walls after apicoectomy is another important factor. the sealer used in the present study, ah plus, has shown better adhesion to the dentin walls when compared to other sealers28. in this sense, although resilontm cones have been developed for use with epiphany sealer, in the present study ah plus was used in both experimental groups because this sealer has demonstrated a good interaction with resilontm cones, and better adhesion to resilontm than epihany when used with cold compaction techniques28. the use of the same sealer in both groups allowed analyzing the influence of the type of cone (gutta-percha or resilontm) without interference of the sealer as an additional variable. the root end was burnished prior to sem analysis to provide a better fit of gutta-percha to the canal walls because, in a previous study29, this procedure reduced significantly the apical leakage after root end resection and glass ionomer cement retroseals. in the present study, the root canals were filled by lateral compaction because it is a widely employed obturation technique that does not require especial instruments or devices. given that the goal of periradicular surgery is to eliminate root canal infection and prevent recontamination, apical gap of the filling material after root-end resection is an important factor that should be taken into account. in the present study, the great majority of specimens presented gap between the obturation and the root canal walls, and the type of cone used for root canal obturation (guttapercha or resilon) did not influence the marginal adaptation after apical gaps after apicoectomy procedures performed on teeth filled with gutta-percha or resilontm 143 braz j oral sci. 8(3):141-144 root-end resection. this indicates that there is material able to avoid gaps at the obturation and very often the misfit is related to root canal anatomy30. in this way, the findings of this sem evaluation reinforce the need of performing root-end cavity preparation and retrograde filling in apicoectomized teeth because the areas of apical gap observed in both groups may serve as niches for microbial recolonization invariably leading to failure of the surgical treatment. acknowledgements the authors would like to thank bauru dental school, university of são paulo, brazil, and mr. edmauro de andrade for undertaking the sem images. references 1. nair pnr, sjogren u, sundqvist g. cholesterol crystals as an etiological factor in non-resolving chronic inflammation: an experimental study in guinea pigs. eur j oral sci. 1998; 106: 644-50. 2. sjogren u, sundqvist g, nair pn. tissue reaction to gutta-percha particles of various sizes when implanted subcutaneously in guinea pigs. eur j oral sci. 1995; 103: 313-21. 3. sjogren u, ohlin a, sundqvist g, lerner uh. gutta-percha-stimulated mouse macrophages release factors that activate the bone resorptive system of mouse calvarial bone. eur j oral sci. 1998; 106: 872-81. 4. leonardo m, rossi m, silva lab, ito iy, bonifácio kc. em evaluation of bacterial biofilm and microorganisms on the apical external root surface of human teeth j endod. 2002; 28: 815-8. 5. nair pnr, henry s, cano v, vera j. microbial status of apical root canal system of human mandibular first molars with periapical periodontitis after “onevisit” endodontic treatment. oral surg oral med oral path oral rad endod. 2005; 99: 231-52. 6. taylor gn, bump r. endodontic considerations associated with periapical surgery. oral surg oral med oral pathol. 1984; 58: 450-5. 7. block rm, bushell a. retrograde amalgam procedures for mandibular posterior teeth. j endod. 1982; 8: 107-12. 8. gutmann jl, harrison jw, editors. surgical endodontics. boston: blackwell scientific publications; 1991. p.230-277. 9. nedderman ta, hartwell gr, portell fr. a comparison of root surfaces following apical root resection with various burs: scanning electron microscope evaluation. j endod. 1988; 14: 423-7. 10. weston gd, moule aj, bartold pm. a scanning electron microscopic evaluation of root surfaces and the gutta-percha interface following root-end-resection in vitro. int endod j. 1999; 32: 450-8. 11. spangberg l, langeland k. biologic effects of dental material. 1. toxicity of root canal filling materials on hela cells in vitro. oral surg oral med oral pathol. 1973; 35: 402-14. 12. shipper g, ørstavik d, teixeira fb, trope m. an evaluation of microbial leakage in roots filled with a thermoplastic synthetic polymer-based root canal filling material (resilon). j endod. 2004; 30: 342-7. 13. skidmore lj, berzins dw, bahcall jk. an in vitro comparison of the intraradicular dentin bond strength of resilon and gutta-percha. j endod. 2006; 32: 963-6. 14. sly mm, moore bk, platt ja, brown ce. push-out bond strength of a new endodontic obturation system (resilon/epiphany). j endod. 2007; 33: 160-2. 15. stratton rk, apicella mj, mines p. a fluid filtration comparison of gutta-percha versus resilon, a new soft resin endodontic obturation system. j endod. 2006; 32: 642-5. 16. tunga u, bodrumlu e. assessment of the sealing ability of a new root canal obturation material. j endod. 2006; 32: 876-8. 17. sagsen b, er o, kahraman y, akdogan g. resistance to fracture of roots filled with three different techniques. int endod j. 2007; 40: 31-5. 18. ungor m, onay eo, orucoglu h. push-out bond strengths: the epiphanyresilon endodontic obturation system compared with different pairings of epiphany, resilon, ah plus and gutta-percha. int endod j. 2006; 39: 643-7. 19. onay eo, ungor m, orucoglu h. an in vitro evaluation of the apical sealing ability of a new resin-based root canal obturation system. j endod. 2006; 32: 976-8. 20. biggs sg, knowles ki, ibanola jl, pashley dh. an in vitro assessment of the sealing ability of resilon/epiphany using fluid filtration. j endod. 2006; 32: 759-61. 21. baumgartner g, zehnder m, paqué f. enterococcus faecalis type strain leakage through root canal filled with gutta-percha/ah plus or resilon/epiphany. j endod. 2007; 33: 45-7. 22. tanomaru-filho m, silveira gf, tanomaru jmg, bier cas. evaluation of the thermoplasticity of different gutta-percha cones and resilon®. aust endod j. 2007; 33: 23-6. 23. morgan la, marshall jg. the topography of root ends resected with fissure burs and refined with two types of finishing burs. oral surg oral med oral pathol oral radiol endod. 1998; 85: 585-91. 24. francischone ce, padovan lapa, padovan lem, duarte mah, fraga sc, curvello vp. apicoectomy with the er:yag laser or bur, followed by retrograde root filling with zinc oxide/eugenol or sealer 26. photomed laser surg. 2005; 23: 395-8. 25. duarte mah, domingues r, matsumoto ma, padovan lem, kuga mc. evaluation of apical surface roughness after root resection: a scanning electron microscopic study. oral surg oral med oral path oral rad endod. 2007; 104: 74-6. 26. bernardes ra, souza-junior jv, duarte mah, gomes de moraes i, bramante cm. ultrasonic chemical vapor deposition-coated tip versus highand lowcarbide speeds burs for apicoectomy: time requerid for resection and scanning electron microscopy analisys of the root-end surfaces. j endod. 1999; 35: 265-8. 27. hsieh kh, liao kh, lai eh, lee bs, lee cy, lin cp. a novel polyurethane-based root canal-obturation material and urethane acrylate-based root canal sealer—part i: synthesis and evaluation of mechanical and thermal properties. j endod. 2008; 34: 303-5. 28. üreyen kaya b, keçeci ad, orhan h, belli s. micropush-out bond strengths of gutta-percha versus thermoplastic synthetic polymer-based systems – an ex vivo study. int endod j. 2008; 41: 211-8. 29. bernardineli n, duarte mah, finishing of glass ionomer retroseals. j endod. 2002; 28: 36-9. 30. hammad m, qualtrough a, silikas n. evaluation of root canal obturation: a three-dimensional in vitro study. j endod. 2009; 35: 541-4. apical gaps after apicoectomy procedures performed on teeth filled with gutta-percha or resilontm144 braz j oral sci. 8(3):141-144 oral sciences n3 original article braz j oral sci. july/september 2009 volume 8, number 3 effect of commercial dental investments at low temperature on the marginal adaptation of cast cp ti inlays rodrigo galo1, ricardo faria ribeiro2, maria da glória chiarello de mattos2, fernanda de carvalho panzeri pires-de-souza3 1dds, msc, phd student, department of dental materials and prosthodontics, ribeirão preto dental school, university of são paulo, brazil 2dds, msc, phd, full professor, department of dental materials and prosthodontics, ribeirão preto dental school, university of são paulo, brazil 3dds, msc, phd, professor, department of dental materials and prosthodontics, ribeirão preto dental school, university of são paulo, brazil received for publication: may 12, 2009 accepted: agust 6, 2009 correspondence to: fernanda de carvalho panzeri pires-de-souza, faculdade de odontologia de ribeirão preto/ usp, departamento de materiais dentários e prótese, avenida do café, s/n, bairro monte alegre, 14040-904 ribeirão preto, sp, brasil. phone: +55-16-3602-3973. fax: +55-016-3633-0999. e-mail: ferpanzeri@forp.usp.br abstract aim: the purpose of this study was to evaluate the adaptation of inlay restorations cast in commercially pure titanium (cp ti) after inclusion of the wax patterns in either a phosphate-bonded investment (rematitan plus®) or a silicon oxide-based investment (termocast®). methods: the wax patterns were prepared over an inlay-type mold. after waxing, 5 measurements of the marginal adaptation were made on the mesial and distal faces. five wax patterns were included in each type of investment under vacuum. the cast specimens were repositioned in the mold and other 5 measurements of the adaptation were made based on the same initial testing conditions. data were analyzed statistically by two-way anova and duncan’s post-hoc (p<0.01) using the spss statistical software package version 12.0 (spss, chicago, il, usa). results: the mean marginal discrepancies of the mod inlays were higher in the termocast® group. termocast® presented significantly greater marginal discrepancy than rematitan plus®. due to the great permeability of the investment refractory material, internal porosity was extremely rare in rematitan plus® and more common in termocast®. conclusion: it may be concluded that termocast® investment should not be indicated for cp ti casting due to poor adaptation and porosity on the casting surface. keywords: marginal adaptation, titanium, investment, inlay, porosity. introduction some properties of commercially pure titanium (cp ti), such as good biocompatibility1-2 resistance to corrosion3-5, low density4 and high mechanical strength4-7, have led to an increasing use of this material for casting inlays and partial crowns. in addition, titanium alloys present as an alternative for patients allergic to ni-containing dental alloys8-9. in theory, the low weight of titanium and its high strength-to-weight ratio allow the design of more functional and comfortable prosthetic restorations10. however, the mechanical properties of cast titanium may be affected by the casting process itself11-12. titanium has an extremely high melting point and react with elements in the air (e.g.: oxygen and nitrogen) and with some investment components (e.g.: magnesium, alumina and calcium) at high temperatures6,12-14. typically, molten titanium (melting temperature = 1660ºc) is forced into a room temperature or preheated (<800ºc) mold6. the reactions between molten metal and investment materials result in the formation of the alpha-case (α-case) layer14-15, which will change the mechanical properties of the surface of titanium castings16. the investments usually used for other alloys are based on silica, but this component is present in a low content in investments indicated for titanium casting, because its chemical braz j oral sci. 8(3): 124-127 affinity with silicon (si) affects negatively its mechanical properties13. sio 2 is more unstable and easily reacts with titanium to form more ti x o y , increasing the oxide content in the composite and resulting in higher microhardness of the surface-reaction layers. this is also the reason why the reaction layers formed in specimens cast with sio 2 based investments are thicker compared to those with al 2 o 3 and mgo-based investments17. the specific molds for titanium castings reduce the production of á-case thickness18, but they are expensive materials and their thermal expansion is not enough to compensate for the titanium casting shrinkage5,18. a possibility to overcome these deficiencies would be using investments with smaller silica content and injecting the material in the molds at a low temperature (430ºc)19. however, the castability of an alloy is often associated with its ability to fill the mold20, and mold filling is dependent on numerous factors other than the metal or alloy, such as mold temperature, superheating of the casting, pressure, type of machine, and chemical stability of the investment against the molten titanium21. the aim of the present study was to evaluate the accuracy of cp ti casting when phosphate-bonded and silicon oxide-based investments at a low temperature were used. the null hypothesis tested was that there is no difference between the investments regarding the marginal adaptation of the cast crowns. material and methods the mod design used in the plastic-die method described in a previous study was employed for the assessment of cast inlay accuracy (figure 1). initially, an inlay wax pattern was annealed at room temperature and a reference line was engraved on the wax pattern aligned with a line on the plastic die. the distance separating the margin of the plastic die was measured at two mesial and distal fixed points. five patterns were made for each investment and a total of 20 inlay specimens were produced, being 10 for rematitan plus® phosphate-bonded investment (dentaurum j.p. winkelstroeter kg, pforzheim, german) (group 1) and 10 for termocast® silicon oxide-based investment (polidental ind. e com. ltda., são paulo, sp, brazil) (group 2). the investments were mixed according to the manufacturers’ instructions under vacuum, and were poured over the patterns previously adapted to silicone casting rings by means of a wax sprue. the molds in group 1 were heated to 150ºc for dewaxing and maintained at this temperature for 90 min, then heated to 250ºc and maintained at this temperature for 90 min. finally, they were heated to 1000ºc for 1 h to ensure thermal expansion. in group 2, the molds were heated to 950ºc for dewaxing and maintained at this temperature for 180 min, after which the molds were slowly cooled inside the furnace to a final temperature of 430ºc. heating was conducted in an electric furnace ((edg 7000; edg equipamentos e controles ltda., são carlos, sp, brazil) in both groups. after the heating period, the investment block was transferred to a vacuum-pressure casting machine with 2 chambers and a voltaic arc (discovery plasma; edg equipamentos e controles ltda), programmed and adjusted for a 22-g ingot of cp ti for each casting (tritan, grade 1; dentaurum j.p. winkelstroeter kg). the upper melting chamber houses a copper crucible and a tungsten electrode, and the lower casting chamber where the invested mold is placed. titanium was placed on the cooper crucible and the centrally aligned tungsten electrode was positioned 5 mm above the titanium surface. an argon gas pressure and a current were chosen and the mold was shifted from the furnace to the casting. after cooling to room temperature, the cp ti castings were divested and airborne-particle abraded with 110-µm aluminum oxide (renfert gmbh, germany) at 80 psi to remove the excess investment. a diamond-coated disc was used to separate the inlays from the sprues and they were radiographed to detect possible casting defects that could contraindicate their use22. a conventional x-ray equipment (spectro x; dabi atlante sa, ribeirao preto, sp, brazil) set to 70 kv (peak) and 8 ma for a 2-second exposure time was used. the films were developed in a dark chamber according to the manufacturer’s instructions and views on the radiograph illuminator. each inlay was seated in the original plastic die and the distances separating the margin of the inlay were measured at the same two fixed points as the wax pattern using an image analysis system (leica microsystems imaging solutions ltd., cambridge, england) (figure 1). the internal porosity of the titanium castings was evaluated from the examination of radiographic films of the castings. porosity was ranked as 0 (without porosity) and 1 (with porosity). data w ere analyzed statistically by two-way anova and duncan’s post-hoc (p<0.01) using the spss statistical software package version 12.0 (spss, chicago, il, usa). results the mean marginal discrepancies of the mod inlays are shown in figure 2. group 2 (termocast®) presented significantly greater marginal discrepancy than group 1 (rematitan plus®) (p<0.01). due to the great permeability of the investment refractory material, internal porosity was extremely rare in group 1 (rematitan plus®) and more common in group 2 (termocast®) (figure 3). fig. 1. dimensional accuracy of a cast crown expressed by the discrepancy measured on its was pattern (dx) and coating (dy) on the same die. fig. 2. marginal discrepancies of the cast mod titanium inlays invested with rematitan plus® and termocast®. each line represents the means and standard deviation before (a) and after (b) casting. effect of commercial dental investments at low temperature on the marginal adaptation of cast cp ti inlays 125 braz j oral sci. 8(3):124-127 discussion although the interest in the use of cp ti and titanium alloys for fabricating restorations increased remarkably in the early 1980s, casting difficulties and structure imperfections are obstacles to be overcome23. one of these difficulties is the high reactivity of titanium with some investment elements at high temperatures, in addition to easy oxidization, especially reducing its mechanical properties. in this study, the high reactivity of titanium with the phosphate-bonded base material was manipulated by lowering the investment temperature when molten titanium got in contact with the mold walls. luo et al.5 suggested reducing the temperature to minimize the contamination area. nevertheless, the marginal discrepancy of cast inlay restorations is still higher than the clinically acceptable levels (50 µm)24, indicating that the reduction of the phosphate-bonded investment temperature does not result in a better adaptation. in the present study, there was a significant difference in titanium inlays cast in different types of investments. smaller marginal discrepancy was obtained with rematitan plus® investment, which could be explained by lower reactivity between this material and the metal. it is suggested that the better castability obtained with titanium is associated with the chemical stability of the investment against molten titanium, which is one of the main factors of mold filling19,21. a greater chemical stability would result in a smaller reaction layer on the titanium surface, resulting in lower fluidity. also, according to taira et al.25, alloying could reduce the detrimental mold reaction because the reactivity of titanium with oxygen could be lowered by the addition of other metallic elements that have a higher affinity for oxygen. previous studies13-14 have reported that titanium restorations cast in phosphate-bonded based investments, such as those in rematitan plus (group 1), resulted in a contaminated surface, with a thickness of 200 µm. indirectly produced cast restorations have a process that induce marginal discrepancy. because of the solubility of the cement in the saliva, a marginal cementation line will develop before long into a marginal gap24. this contamination of the cast restoration occurred due to the presence of elements such as phosphorus, silicon, and oxygen in the investment material6,12,16,25. especially when the liquid titanium fills the mold at high temperatures, it reduces some of the oxides of the investment material and the free elements (mainly si, o, p, and fe) are dissolved into the molten metal; their presence strongly affects the solidification process and, therefore, the final microstructure of the castings13. this phenomenon does not occur in the rematitan plus ® investment due to the presence of magnesium oxide (mgo), calcium oxide (cao), and aluminum oxide (al 2 o 3 ) in its composition, which reduces the interaction of titanium with the investment material26, fig. 3. radiographic images of a titanium inlay cast with rematitan plus® (a) and termocast® (b). minimizing the á-case extension16. guilin et al.17 observed that the type of investment affects the reaction layer and the microhardness on the surface of cast titanium, obtaining better results with an mgobase investment, since it reduces the á-case layer thickness. nevertheless, the formation of this layer does not result from the reaction between molten titanium and investment, but from cooling molten titanium rapidly. sung and kim15 called it the ‘hardening layer’. thus, the á -case layer of titanium castings consists of the reaction layers, which resulted from the oxide layer, the alloy layer, and the hardening layer. the reactivity of the alloy with the investment components27-28, as well as with the gases released5,28, leads to the formation of porous areas in the inlay restorations cast with phosphate-base investment, which contributes to some mechanical properties suffering variations in ductility, tensile strength , elongation, fatigue and corrosion resistance11,29, in addition to contributing to a greater marginal desadaptation due to hard reactive areas on the titanium casting surfaces of the molten titanium inter-diffusion with the investment30 internal porosities are commonly observed defects in titanium castings31 and the technology available for casting titanium also has problems, such as argon pressure affecting the quality of titanium castings. the formation of undesirable porosity affects negatively the mechanical properties of titanium, such as decrease of the tensile strength and elongation11. radiographic analysis of titanium castings with termocast® (group 2) revealed an inconsistency of internal porosity. by using x-ray inspection of titanium castings, wang and boyle22 found porosity to be a common occurrence. results of the present study indicated that rematitan plus® investments result in less porosity for the cast titanium mod inlays. this good castability is probably due to the superior gas permeability caused by the investment32. this strongly suggested that radiographic examination should be used to analyze titanium casting for internal porosity before clinical use. cast restorations produced indirectly show a process-induced marginal discrepancy, being one of the disadvantages of titanium33. successful restoration must have good marginal seal and design because these factors are essential to protect the luting agent from dissolution and prevent microorganism retention34. the primary consequences of the adherence of pathogenic microorganisms to irregular surfaces are the increase of the incidence of oral diseases34 and the acceleration of biocorrosion by providing retentive niches. the null hypothesis of this study was rejected, since there was significant difference in the marginal discrepancy between the groups. the investment material should be carefully selected, so that satisfactory castability and accuracy can be obtained. this study demonstrated that the type of investment affects the marginal adaptation and internal porosity of titanium castings. cp ti castings presented higher marginal discrepancy and increased internal porosity when invested with termocast®. references 1. lautenschlager ep, monaghan p. titanium and titanium alloys as dental materials. int j prosthodont. 1993; 43: 245-53. 2. wang rr, fenton a. titanium for prosthodontics applications: a review of the literature. quintessence int. 1996; 27: 401-8. 3. geis-gerstorfer j. in vitro corrosion measurements of dental alloys. j dent. 1994; 22: 247-51. effect of commercial dental investments at low temperature on the marginal adaptation of cast cp ti inlays126 braz j oral sci. 8(3):124-127 4. king aw, chai j, lautenschlager ep, gilbert j. the mechanical properties of milled and cast titanium for ceramic veneering. int j prosthodont. 1994; 7: 532-7. 5. luo x-p, guo t-w, ou y-g, liu q. titanium casting into phosphate bonded investment with zirconite. dent mater. 2002; 18: 512-5. 6. takahashi j, kimura h, lautenschlager ep, chern-lin jh, moser jb, greener eh. castings pure titanium into commercial phosphate-bonded sio 2 investiment molds. j dent res. 1990; 69: 1800-5. 7. jang ks, youn sj, kim ys. comparation of castability and surface roughness of commercially pure titanium and cobalt-chromium denture frameworks. j prosthet dent. 2001; 86: 93-8. 8. baran gr. the metallurgy of ni-cr alloys for fixed prosthodontics. j prosthet. dent. 1983; 50: 639-50. 9. geurtsen w. biocompatibility of dental casting alloys. crit rev oral biol med. 2002; 13: 71-84. 10. blackman r, barghi b, tran c. dimensional changes in casting titanium removable partial denture frameworks. j prosthet dent. 1991; 65: 309-15. 11. watanabe i, watkins lh, nakajima h, atsuya m, okabe t. effect of pressure difference on the quality of titanium casting. j dent res. 1997; 76; 773-9. 12. ida k, togaya t, tsutsumi s, takeuche m. effect of magnesia investments in the dental casting of pure titanium or titanium alloys. dent mater j. 1982; 1: 8-21. 13. papadopoulos t, zinelis s, vardavoulias m. a metallurgical study of the contamination zone at the surface of dental ti casting, due to the phosphatebonded investiment material: the protection efficacy of a ceramic coating. j mater sci. 1999; 34: 3639-46. 14. koike m, cai z, fujii h, brezner m, okabe t. corrosion behavior of cast titanium with reduced surface reaction layer made by a face-coating method. biomater. 2003; 24: 454-9. 15. sung sy, kim yj. alpha-case formation mechanism on titanium investment castings. mater sci eng a. 2005; 405: 173-7. 16. eliopoulos d, zinelis s, papadopoulos t. the effect of investment material type on the contamination zone and mechanical properties of commercially pure titanium castings. j prosthet dent. 2005; 94: 539-48. 17. guilin y, nan l, yousheng l, yining w. the effects of different types of investments on the alpha-case layer of titanium castings. j prosthet dent. 2007; 97: 157-64. 18. miyakawa o, watanabe k, okawa s, nakano s, kobayachi m, shiokawa n. layered structure of cast titanium surface. dent mater j. 1989; 8: 175-85. 19. oliveira pcg, abado gl, ribeiro rf, rocha ss. the effect of mold temperature on castability of cp ti and ti-6al-4v castings into phosphate bonded investment materials. dent mater. 2006; 22: 1098-102. 20. hero h, waarli m. effect of vacuum and supertemperature on mold filling during casting. scand j dent j. 1991; 99: 55-9. 21. syverud m, okabe t, hero h. casting of ti-6al-4v alloy compared with pure ti in an ar-arc casting machine. eur j oral sci. 1995; 103: 327-30. 22. wang rr, boyle am. a simple method for inspection of porosity in titanium casting. j prosthet dent. 1993; 70: 275-6. 23. bridgeman jt, marker va, hummel sk, benson bw, pace ll. comparison of titanium and cobalt-chromium removable partial denture clasps. j prosth dent. 1997; 79: 187-93. 24. stoll r, fischer c, springer m, stachnis v. marginal adaptation of partial crowns cast in pure titanium and in gold alloy – an in vitro study. j oral rehabil. 2002; 29: 1-6. 25. taira m, moser jb, greener eh. studies of titanium alloys for dental castings. dent mater. 1989; 5: 45-50. 26. hsu hc, kikuchi h, yen sk, nishiyama m. evaluation of different bonded investments for dental titanium casting. j mater sci: mater med. 2005; 16: 821-5. 27. herÿ h, syverud m, waarti m. mold filling and porosity in castings of titanium. dent mater. 1993; 9: 15-8. 28. mesmar hs, morgano sm, mark le. investigation of the effect of three sprue designs on the porosity and the completeness of titanium cast removable partial denture frameworks. j prosthet dent. 1999; 82: 15-21. 29. cai z, bunce n, nunn me, okabe t. porcelain adherence to dental cast cp titanium: effects of surface modifications. biomaterials. 2001; 22: 979-86. 30. hsu hc, kikuchi h, yen sk, nishiyama m. evaluation of different bonded investments for dental titanium casting. j mater sci: mater med. 2007; 18: 605-9. 31. chai t–i, stein rs. porosity and accurancy of multiple-unit titanium castings. j prosthet dent. 1995; 73: 534-41. 32. hung c-c, hou g-l, tsai c-c, huang c-c. pure titanium casting into zirconiamodifed magnesia-based investment molds. dent mater. 2004; 20: 846-51. 33. jang ks, youn sj, kim ys. comparation of castability and surface roughness of commercially pure titanium and cobalt-chromium denture frameworks. j prosthet dent. 2001; 86: 93-8. 34. bollen cm, lambrechts p, quirynen m. comparison of surface roughness of oral hard materials to the threshold surface roughness for bacterial plaque retention: a review of the literature. dent mater. 1997; 13: 258-69. effect of commercial dental investments at low temperature on the marginal adaptation of cast cp ti inlays 127 braz j oral sci. 8(3):124-127 oral sciences n3 braz j oral sci. 10(4):288-293 received for publication: august 28, 2011 accepted: november 29, 2011 original article braz j oral sci. october | december 2011 volume 10, number 4 prevalence of oral mucosal lesions and variations in indian public school children ambika l.1, vaishali keluskar 2, shivayogi hugar3, sudha patil3 1department of oral medicine and radiology, school of dental sciences, krishna institute of medical sciences university, karad, maharashtra, india 2department of oral medicine and radiology, kle vk institute of dental sciences, belgaum karnataka, india 3department of pediatric dentistry , kle vk institute of dental sciences, belgaum karnataka, india correspondence to: ambika l department of oral medicine & radiology, school of dental sciences, krishna institute of medical sciences university, near dhebewadi road malkapur, karad – 415110, satara maharashtra, india phone: +919845545283, +919980056656 e-mail: ambikamds@gmail.com abstract aim: to study the nature and distribution of oral mucosal lesions and variations in children. methods: this investigation was a cross-sectional study. all the study subjects were taken from government primary school. children were in the age group of 4-14years. the children who were attending the department of pediatric dentistry for dental check up were examined. results: a total of 1,003 children were included in the present study,. the mucosal lesions were observed in 643 (64.11%) children, developmental variations of mucosa were evident in 220 (21.93%) children and normal mucosal variations were present in 391 (38.98%) children. the most commonly observed mucosal lesions were gingivitis, gingival abscess and traumatic ulcers. the most frequent developmental mucosal variations were ankyloglossia and high frenal attachment. normal mucosal variations noted were linea alba buccalis and fordyce’s spots. distribution of mucosal lesions/variations were same among both the gender except fordyce’s spots (p=0.018). parotid papillae and pigmented spots had positive relation with age. conclusions: mucosal lesions/variations were present in more than half of the children in this study. only few oral mucosal lesions had gender differences and vary with age. keywords: children, oral mucosal lesions, oral mucosal variations, developmental mucosal variations. introduction the oral cavity is considered as a mirror of general health. the tongue lesions fissured tongue, geographic tongue, median rhomboid glossitis and oral mucosal lesions fordyce’s spots and leukedema are classically considered as developmental lesions and normal conditions rather than having virtual disease characteristics. these lesions may be present at birth or become evident later in life1. mucosal lesions may be discovered during routine dental examinations and vary depending on age, gender and /or race. majority of oral diseases are confined to oral tissues, but numerous underlying systemic conditions may manifest with signs and symptoms within the oral cavity2. shulman3 (2005) studied 10,030 individuals aged between 2 and 17 years, out of which 914 individuals had 976 lesions. the lip was the most frequent site of lesions (30.70%), followed by the dorsum of the tongue (14.70%) and the buccal mucosa (13.60%). lesions were more prevalent in males (11.76%) than females (8.67%). the most prevalent lesions were lip/cheek bite (1.89%), followed braz j oral sci. 10(4):288-293 289 by aphthous stomatitis (1.64%), recurrent herpes labialis (1.42%) and geographic tongue (1.05%). in a study involving turkish children, found that excessive melanin pigmentation (6.9%) was the most common lesion in the study population 4. a crosssectional survey on 1,211 brazilian children by bessa, et al.5 (2004) found mucosal lesions in 27.0% children. the most common lesions were geographic tongue, cheek biting, and melanotic macula. they also concluded that the frequency of mucosal alterations in children was high and increased with age, and some of them were associated with habits and systemic problems. a study in spanish children revealed that 30.92% children had oral mucosal lesions6. studies on oral mucosal lesions in indian subcontinent are limited. a single study from india by mathew, et al.7 (2008) studied prevalence of oral mucosal lesions in patient aged 2-80 years. out of 243 children/adolescent only 41(16.87%) had mucosal lesions. in his study the sample size was too small to generalize the results. the present study was undertaken to highlight the nature of oral mucosal alterations and lesions among 4-14-year-old children and to study the influence of age and gender on oral mucosal lesions. material and methods the study sample consisted of 1,003 children aged 4 to 14 years. ethical clearance was obtained from the institutional ethics committee to examine these children and also permission was obtained from the parents and teachers who were accompanying the children. initially all primary schools children underwent regular screening after which children with oral problems were referred to the department of pediatric dentistry for further dental examination and treatment. children were informed briefly about the procedure involved. children without parent’s and teacher’s consent were excluded from the study. children suffering from any chronic illness were also excluded from the study. the examination was performed by one qualified and two trained dentists using plain mouth mirror under adequate illumination. children’s detailed history was noted. mucosal lesions, normal mucosal variations and developmental abnormalities were recorded in the specially designed proforma. a minimum of 10 children were examined per day over a period of 6 months. the data were compiled and analyzed using statistical package for social sciences software (spss inc., chicago, il, usa) version 14. descriptive statistics, chi-square test and pearson’s correlation tests were used for analysis. for tests of significance p value d” 0.05 was considered as significant. results there were 459 (45.76%) boys and 544 (54.24%) girls. most of the children were in the age group of 6-12 years. only few children were aged 4,5,13 and 14 years old (table 1). as much as 643 (64.11%) children had mucosal lesions. gingivitis was most commonly observed in 268 (26.72%) boys and 298 (29.71%) girls. traumatic ulcer was observed in 15 (1.5%) boys and 17 (1.69%) girls. the most common sites for traumatic ulcers were the buccal mucosa (n=12), lower lip (n=10), upper lip (n=6) and tongue (n=4). the third most common mucosal lesion observed were superficial erosions of the oral mucosa. they were observed in 12 (1.2%) boys and 15 (1.5%) girls and the most common site for occurrence of erosion was the buccal mucosa (n=16), upper lip (n=6), lower lip, palate (n=2) and floor of the mouth (n=1). children were also screened for reactive lesions, but they were not evident among them. mucosal lesions were common in both the sexes with no statistically significant difference. most children, 148(14.78%) boys and 187(18.69%) girls, had uncleaned tongue and associated halitosis. the other mucosal lesions observed are depicted in table 2. developmental oral mucosal variations were observed in 100 (9.97%) boys and 120 (11.96%) girls. high frenal attachment was observed in 39 (3.89%) boys and 40 (3.99%) girls. ankyloglossia was observed in 32 (3.19%) boys and 52 (5.18%) girls. among these, 80 had partial ankyloglossia and 4 had complete ankyloglossia. fissured tongue was observed in 24 (2.39%) boys and 25 (2.49%) girls. other developmental disturbances of oral mucosa are shown in table 3. microglossia, macroglossia, cleft lip and palate were not evident among any subjects. normal oral mucosal variations were also studied. as much as 188 (18.74%) boys and 203 (20.24%) girls had these variations. linea alba buccalis was present in 88 (8.77%) boys and 115(11.47%) girls. fordyce’s spots were present in 61 (6.08%) boys and 47 (4.69%) girls. among all the mucosal lesions/variations, only fordyce’s spots were significantly (p=0.018) common in boys than girls. for rest of the lesions, there were no gender differences. other developmental variations are represented in table 4. data were analyzed to find out any correlation between commonly occurring mucosal lesions/variations with age. developmental variations were not included in the analysis because they are likely to be present since birth and unlikely to change as the age advances. the age (years) boys girls n % n % 4 0 0 1 0.10% 5 3 0.30% 6 0.60% 6 34 3.39% 64 6.38% 7 62 6.18% 68 6.78% 8 61 6.08% 106 10.57% 9 75 7.48% 119 11.86% 10 145 14.46% 127 12.66% 11 52 5.18% 48 4.79% 12 19 1.89% 3 0.30% 13 5 0.50% 1 0.10% 14 3 0.30% 1 0.10% total 459 45.76% 544 54.24% table 1 age distribution of children. total number of patients = 1,003 prevalence of oral mucosal lesions and variations in indian public school children braz j oral sci. 10(4):288-293 290 mucosal lesions male female p n % n % gingivitis 268 26.72% 298 29.71% 0.251 gingival abscess 19 1.89% 13 1.30% 0.116 traumatic ulcer 15 1.50% 17 1.69% 0.861 aphthous ulcer 1 0.10% 0 0 0.458 herpes ulcer 5 0.50% 3 0.30% 0.378 erosions 12 1.20% 15 1.50% 0.269 angular chelitis 2 0.20% 5 0.50% 0.463 inflammatory hyperplasia 2 0.20% 0 0 0.209 tonsillitis 2 0.20% 2 0.20% 1.000 white patch 2 0.20% 1 0.10% 0.596 frictional keratosis 9 0.90% 7 0.70% 0.396 scar 3 0.30% 4 0.40% 0.898 reactive lesions 0 0 0 0 0 cysts 3 0.30% 0 0 0.168 tumours 0 0 0 0 0 coated tongue 148 14.76% 187 18.64% 0.476 table 2 oral mucosal lesions total number of patients = 1,003 total number of patients = 1,003. table 3 developmental variation of oral mucous membrane. developmental anomalies male female p n % n % overall 100 9.97% 120 11.96% 0.917 cleft lip 0 0 0 0 0 cleft palate 0 0 0 0 0 pits 17 1.69% 18 1.79% 0.344 frenal attachment 39 3.89% 40 3.99% 0.503 tongue tie 32 3.19% 52 5.18% 0.299 deep palate 14 1.40% 10 1.00% 0.211 microglossia 0 0 0 0 0 macroglossia 0 0 0 0 0 geographic tongue 8 0.80% 6 0.60% 0.389 fissured tongue 24 2.39% 25 2.49% 0.643 bald tongue 10 1.00% 14 1.40% 0.684 findings are described in table 5. among the studied variables, only parotid papilla (p=0.013) and pigmented spots (p=0.013) had significant positive correlation with age. discussion prevalence in earlier studies varies from 4.1 to 52.6%8. in this study, children refered to the department of pediatric dentistry were examined and 643 (64.11%) had mucosal lesions. as a routine, children are screened for any dental problems and those who require further treatment are sent to the department of pediatric dentistry. the developmental variations of mucosa were evident in 220 (21.93%) children and normal oral mucosal variations were present in 391 (38.98%) children. distribution of mucosal lesions/variations were same among both genders except for fordyce’s spots (p=0.018). parotid papillae and pigmented spots had significant positive relation with age and the rest of the lesions did not vary with age. each type of mucosal lesion is discussed below. 1. mucosal lesions gingival diseases in the present study, gingivitis was seen in 268 (26.72%) boys and 298 (29.71%) girls, thus comprising 56.43% of total population. frequency of gingivitis was difficult to determine because of the lack of agreement on measurement criteria. many studies have concluded that gingivitis begins in early childhood and that 9-17% of children aged 3-11 years present with gingivitis. at puberty, prevalence rises to 70-90%9. in the present study, only 3.2% children had gingival abscess. gingival abscess (dental abscess) is an acute lesion characterized by localization of pus in the structures that surround the teeth10. traumatic ulcers traumatic ulcer was seen in 32 (3.19%) children and prevalence of oral mucosal lesions and variations in indian public school children 291 braz j oral sci. 10(4):288-293 mucosal lesions male female p n % n % overall 188 18.74% 203 20.24% 0.239 leukoedema 12 1.20% 11 1.10% 0.532 linea alba 88 8.77% 115 11.47% 0.440 fordyce spots 61 6.08% 47 4.69% 0.018* parotid papilla 24 2.39% 26 2.59% 0.745 pigmented spots 60 5.98% 72 7.18 0.633 table 4 normal variations of oral mucous membrane total number of patients = 1,003 variables age groups r p 4-7 years(n=238) 8-10 years(n=633) 11-14 years(n=132) overall mucosal lesions 152 (63.87%) 397 (62.72) 94 (71.21%) 0.035 0.274 gingivitis 140 (58.82%) 341 (53.87%) 85 (64.39%) 0.016 0.609 gingival abscess 5 (2.10%) 23 (3.53%) 4 (3.03%) 0.023 0.475 traumatic ulcer 9 (3.78%) 17 (2.69%) 6 (4.55%) -0.006 0.840 erosions 4 (1.68%) 17 (2.69%) 5 (3.79%) 0.026 0.404 coated tongue 78 (32.77%) 207 (32.70%) 50 (37.88%) 0.026 0.408 overall normal variations 82 (34.45%) 252 (39.81%) 57 (43.18%) 0.056 0.077 leukedema 5 (2.10%) 13 (2.05%) 5 (3.79%) 0.027 0.392 linea alba 48 (20.17%) 131 (20.70%) 24 (18.18%) -0.011 0.738 fordyce spots 21 (8.82%) 67 (10.58%) 20 (15.15%) 0.056 0.076 parotid papilla 7 (2.94%) 31 (4.90%) 12 (9.09%) 0.079 0.013* pigmented spots 24 (10.08%) 85 (13.43%) 23 (17.42%) 0.064 0.041* r = pearson’s correlation value; p < 0.05 is considered statistically significant; * statistically significant values. table 5 correlation between mucosal lesions / variations with age. the most frequent sites affected were buccal mucosa in 12 (1.2%) followed by lower lip in 6 (1.0%). traumatic ulcerations are considered to be the most common oral ulceration. in a study by shulman3 (2005), most prevalent lesions were lip/cheek bite (1·89%). these ulcers may be caused by direct physical/ mechanical, thermal or chemical trauma to the vascular compromise, causing tissue damage and ulceration11. aphthous ulcer was seen in 1 (0.1%) child with a history of recurrence once in 6 months. superficial erosions of oral mucous membrane were observed in 2.7% children. sites affected were buccal mucosa 16 (1.6%), upper lip (0.6%), lower lip and palate (0.4%) and floor of the mouth 1 (0.1%). all causes were due to trauma from toothbrush, hot food and dental instruments. herpes simplex infection in the present study, children with lower socioeconomic status had herpetic ulcers. among 8 (0.8%) children, 6 (0.6%) had vesicles and ulcers on the upper lip, 2 (0.2%) had ulcers on lower lip and palate respectively. a previous study reported prevalence of 0.78-5.2%8. all children gave a positive history of prodromal symptoms. most cases of primary hsv-1 infections are subclinical and generally occur in children and teenagers. there is a 1-to 3-day viral prodrome of fever, loss of appetite, malaise and myalgia that may also be accompanied by headache and nausea11. angular cheilitis in the present study, 7 (0.7%) children had angular cheilitis and all had pallor on general physical examination. a study on turkish children reveled that angular cheilitis was the only oral mucosal lesion that had a significant correlation with anemia12. angular cheilitis is infected fissure of the commissures of the mouth, often surrounded by erythema. the lesions are frequently coinfected with both candida and staphylococcus aureus. other factors like vitamin b12 deficiency and iron deficiency have been associated with this disorder. atopy has also been associated with the formation of angular cheilitis13. white lesions in the present study, 3(0.3%) children had homogenous, thick, non-scrapable, white patch on the buccal mucosa suggestive of oral leukoplakia. all children had a habit of chewing gutka (a chewing preparation using areca nut and tobacco) since 2-3 years. history revealed that gutka chewing habit was learnt from their parents. frictional keratosis of buccal mucosa due to of sharp teeth was observed in 16 (1.6%) children. frictional keratosis is defined as a white plaque with a rough and frayed surface that is clearly related to an identifiable source of mechanical irritation and that usually resolve on elimination of irritants. lesions belonging to this category of keratosis include linea alba and cheek, lip and tongue chewing. it is frequently associated with sharp cusps and edges of broken teeth14. leukedema in the present study, 12 (1.2%) boys and 11(1.1%) girls prevalence of oral mucosal lesions and variations in indian public school children 292 braz j oral sci. 10(4):288-293 had leukoedema. the total prevalence was 2.2%. leukoedema was alleged to occur only in adult population until martin and crump15 found this lesion in children and in youths. leukoedema is a common mucosal alteration that represents a variation of the normal condition rather than a true pathologic change. it has been reported in up to 90% of black adults and up to 50% of black teenagers. the most frequent site of leukoedema is the buccal mucosa bilaterally, and may be seen rarely on labial mucosa and soft palate14. linea alba buccalis in this study, 88(8.8%) boys and 115(11.47%) girls had linea alba buccalis. total prevalence of this mucosal variation was 20.27%. it is a normal variation in the buccal mucosa that appears as a white line beginning at the corners of the mouth and extending posteriorly at the level of the occlusal plane. it is a very common finding and it is most likely associated with pressure, frictional keratosis, or suction trauma from the facial surfaces of the teeth. it is usually present bilaterally and may be pronounced in some individuals. these white lines may disappear spontaneously in some people14. fordyce’s spots in the present study, 61 (6.08%) boys and 47 (4.69%) girls had fordyce’s spots and statistically significant difference (p=0.018) was observed among both the gender. these tubuloacinar sebaceous glands found in the vermillion border of the lip, buccal mucosa and occasionally on the palate, gingiva and tongue. the number of fordyce granules increases with age and is not correlated with systemic atherosclerosis and smoking. men usually exhibit more fordyce’s granules than women exhibit16. this finding was consistent with the present study. oral pigmentations in the present study, 132 (13.16%) children had normal physiological pigmentation on the tongue and buccal mucosa. oral pigmentations had significant positive correlation (p=0.041) with age. this may be true as some of the factors like smoking induced, hormone induced and drug related pigmentations are likely to increase as the age advances17. a study by mumcu, et al.4 (2005) in turkish children found that excessive melanin pigmentation (6.9%) was the most common oral mucosal lesion. erica amir, et al.17 (1991) conducted a study to investigate the prevalence of physiologic pigmentation in israeli jewish children of different ethnic origins. a total of 1,300 6-10-year-old children were examined. physiological pigmentation was found in 13.5% of the population studied. children of eastern origin showed a significantly higher prevalence of pigmentation compared with ashkenazi and sephardic groups. because melanin pigmentation can be enhanced by mechanical and chemical stimulation (smoking), this study may serve as a baseline for investigation of melanin pigmentation in various ethnic groups. the melanotic macula in the mouth is equivalent to a freckle or brown pigmented patch of the skin. in children it is most likely racial in origin, in which case it may be called racial pigmentation or physiological pigmentation, and no treatment is necessary. maculae were seen in 10(1.0%) children. petechiae were seen in 22(2.2%) children and all were traumatic in origin. cysts and tumors in the present study, only 3(0.3%) children had mucoceles. the location of these mucoceles was lower lip, floor of the mouth and buccal mucosa. tumors were not noticed in any subjects. mucoceles, which are of minor salivary gland origin, are also referred to as mucus retention phenomenon and mucus escape reaction. data from the third national health and nutrition examination survey (nhanes iii) that included 17,235 adults aged 17 years or older documented an overall prevalence ranking of 44 for the mucocele and a point prevalence of 0.02%. in the same study, which comprised of 10,030 children aged 2-17 years, mucocele had a point prevalence of 0.04%18. oral vascular lesions, like hemangioma, vascular malformation and varix, are common19 but none was noted in our study sample. 2. tongue lesions and anomalies ankyloglossia (tongue-tie) in the present study, 84 (8.37%) children had tongue tie. eighty (7.98%) had partial tongue tie and 4 (0.39%) had complete tongue-tie and children with complete tongue tie had speech problem. in previous studies, ankyloglossia was noted in 0.3%4 and 2.8%6. in our study, because of selective population, the number of children with ankyloglossia was high. messner and lalakea studied speech in children with ankyloglossia and noted that the phonemes likely to be affected due to ankyloglossia include sibilants and lingual sounds such as [t d z s θ ð n l]. messner and lalakea also examined speech and ankyloglossia in another study. they examined 15 patients and speech was grossly normal in all subjects. however, half of the subjects reported that they thought that their speech was more effortful than other peoples’ speech20. in the present study, 79 (7.88%) patients had high frenal attachment and all had midline diastema seeking orthodontic consultation. geographic tongue this is an annular lesion that affects the dorsum and margins of the tongue. the lesion is also known as erythema migrans. it is one of the most prevalent oral mucosal lesions, as prevalence may vary from 0.60-9.8%8. in this study, 14(1.4%) children had geographic tongue with no gender predilection and all were asymptomatic. the prevalence of this seems to decrease with age, which supports spontaneous regression over time11. in the present study, correlation with age was not analyzed as only few patients had geographic tongue. complete bald tongue was seen in 10 (1.0%) boys and 14 (1.4%) girls. nutritional deficiency was thought as one of the predisposing factor since these children did not have any other abnormalities. prevalence of oral mucosal lesions and variations in indian public school children 293 braz j oral sci. 10(4):288-293 fissured tongue in the present study, fissured tongue was noted in 49 (4.9%) children. there were 24 (2.39%) boys and 25 (2.49%) girls and all had shallow grooves. the prevalence of fissured tongue worldwide varies by geographic location and has been reported to vary from 1.49-23%8. yarom, cantony and gorsky21 (2004) studied the prevalence of fissured tongue, geographic tongue and median rhomboid glossitis among israeli adults of different ethnic origins and they found prevalence of ft was 30.5%. the most prevalent, typical fissured tongue, grooves over one third of the dorsal tongue, was noted in 11.5% of the study group. in the present study, when tongue was examined for cleanliness, 335 children had unclean tongue and associated halitosis. for all these children oral hygiene instructions were given after their examination. congenital pits in the present study, 35 (3.5%) children had commissural lip pits and all had bilateral presentation. gorsky, buchner and cohen22 (1985) studied the prevalence of commissural lip pits in a group of 2,462 israeli jews and found a relationship between commissural lip pits and ethnic background. their results showed the presence of commissural lip pits in 17.4% and 9.7% were unilateral and 7.7% were bilaterally located. in this study, more than 50% of the children had mucosal lesions and variations. only few oral mucosal lesions had gender differences and vary with age. some of the lesions were symptomatic and some were not. thus, this study helped revealing all unnoticed findings in children, which can facilitate the diagnosis of oral lesions at an initial stage. routine oral examination is mandatory in children to identify lesions at an initial stage, thus facilitating their further management. references 1. jahanbani j, sandvik l, lyberg t, ahlfors e. evaluation of oral mucosal lesions in 598 referred iranian patients. open dent j. 2009; 3: 42-7. 2. jones av, franklin cd. an analysis of oral and maxillofacial pathology found in children over a 30-year period. int j paediatr dent. 2006; 16: 19-30. 3. shulman jd. prevalence of oral mucosal lesions in children and youths in the usa. int j paediatr dent. 2005; 15: 89-97. 4. mumcu g, cimilli h, sur h, hayran o, atalay t. prevalence and distribution of oral lesions: a cross-sectional study in turkey. oral dis. 2005; 11: 81-7. 5. bessa cf, santos pj, aguiar mc, do carmo ma. prevalence of oral mucosal alterations in children from 0 to 12 years old. j oral pathol med. 2004; 33: 17-22. 6. garcia-pola mj, garcia-martin jm, gonzalez-garcia m. prevalence of oral lesions in the 6-year-old pediatric population of oviedo (spain). med oral. 2002; 7: 184-91. 7. mathew al, pai km, sholapurkar aa, vengal m. the prevalence of oral mucosal lesions in patients visiting a dental school in southern india. indian j dent res. 2008; 19: 99-103. 8. rioboo-crespo mr, planells-del pozo p, rioboo-garcía r. epidemiology of the most common oral mucosal diseases in children. med oral patol oral cir bucal. 2005; 10: 376-87. 9. stephen jm. gingivitis. emedicine [cited 28 oct 2010]. available from: url: http: //emedicine.medscape.com/article/763801-overview. 10. gould jm, cies jj. dental abscess. emedicine. [cited 1 nov 2010]. available from: url: http: //emedicine.medscape.com/article/909373overview. 11. woo sb, greenberg ms. ulcerative, vesicular, and bullous lesions. in: greenberg ms, glick m, ship ja, editors. burket’s oral medicine. 11th ed. hamilton: bc decker; 2008. p.41-75. 12. parlak ah, koybasi s, yavuz t, yesildal n, anul h, aydogan i et al. prevalence of oral lesions in 13to 16-year-old students in duzce, turkey. prevalence of oral lesions in 13to 16-year-old students in duzce, turkey. oral dis. 2006; 12: 553-8. 13. jontell m, holmstrup p. red and white lesions of the oral mucosa. in: greenberg ms, glick m, ship ja, editors. burket’s oral medicine. 11th ed. hamilton: bc decker; 2008. p.77-106. 14. bhattacharyya i, cohen dm, jr silverman s. red and white lesions of the oral mucosa. in: greenberg ms, glick m, editors. burket’s oral medicine. 10th ed. hamilton: bc decker; 2003. p.85-125. 15. martin jl. leukoedema: a review of the literature. j natl med assoc. 1992; 84: 938-40. 16. brightman vj. red and white lesions of the oral mucosa. in: lynch ma, brightman vj, greenberg ms, editors. burket’s oral medicine. 9th ed. philadelphia, new york: lippincott-raven publishers; 2001. p. 51-120. 17. amir e, gorsky m, buchner a, sarnat h, gat h. physiologic pigmentation of the oral mucosa in israeli children. oral surg oral med oral pathol. 1991; 71: 396-8. 18. flaitz cm, hicks mj. mucocele and ranula. emedicine [cited 28 oct 2010]. available from: url: http: //emedicine.medscape.com/article/ 1076717-overview. 19. corrêa ph, nunes lc, johann ac, aguiar mc, gomez rs, mesquita ra. prevalence of oral hemangioma, vascular malformation and varix in a brazilian population. braz oral res. 2007; 21: 40-5. 20. messner ah, lalakea ml, aby j, macmahon j, bair e. ankyloglossia: incidence and associated feeding difficulties. arch otolaryngol head neck surg. 2000; 126: 36-9. 21. yarom n, cantony u, gorsky m. prevalence of fissured tongue, geographic tongue and median rhomboid glossitis among israeli adults of different ethnic origins. dermatology. 2004; 209: 88-94. 22. gorsky m, buchner a, cohen c. commissural lip pits in israeli jews of different ethnic origin. community dent oral epidemiol. 1985; 13: 195-6. prevalence of oral mucosal lesions and variations in indian public school children oral sciences n3 braz j oral sci. 11(1):67-71 original article braz j oral sci. january | march 2012 volume 11, number 1 social and demographic profile of unified health system users in a medium-sized city of southern brazil simone gomes dias de oliveira1, pedro henrique azambuja carvalho2, rafael guerra lund3 1dds, msc student in prosthodontics, school of dentistry, federal university of pelotas, brazil 2undergraduate student, school of dentistry, federal university of pelotas, brazil 3dds, msc, phd in dentistry, school of dentistry, federal university of pelotas, brazil correspondence to: rafael guerra lund programa de pós-graduação em odontologia faculdade de odontologia universidade federal de pelotas (ufpel) rua gonçalves chaves, 457/504 cep: 96015-000 pelotas, rs, brasil phone/fax: +55 53 32226690 ramal 135 e-mail: rafael.lund@gmail.com received for publication: september 28, 2011 accepted: march 12, 2012 abstract aim: the use of health services is a complex behavior and results from a series of determinants that includes the users’ social and demographic characteristics. the current survey interprets the social and demographic profile of unified health system (uhs; sus in portuguese) users from pelotas rs brazil, which was conducted by interviews with 333 users from five health basic units (hbus). methods: the social and demographic characterization was deployed by the variables sex, age, occupation, city/town or district of origin, number of family members, use of continuous medication, diseases and family income. results: hbu users were mostly women (82.4%), within the 30-40-year-old age range (21.4%), housewives (36.3%) from pelotas (96.2%) and from the district of fragata (26.1%). families of users comprise mostly two to four members (66.6%), with an income of one to two minimum wages (66.2%). fifty-one percent of the interviewees do not use continuous medication, while hypertension was the most cited disease (51.8%). conclusions: an understanding of hbu users’ characteristics and their associated social and demographic context may contribute towards a better reception and may confirm the uhs contribution towards universality and equality of public health care. keywords: unified health system, health profile, social conditions, brazil. introduction wealth distribution in brazil is still highly uneven not only among the federal states, but also among the population. the unified health system (uhs sus in portuguese) represents a health program of the brazilian government notorious for its general principles of organization which covers medical expenses of about 80% of the brazilian population¹. the uhs guarantees access to any health service to all the population and warrants citizen participation through their representative entities in the process of health public polices and their execution². moreover, the health services should have a macroeconomic (cost control) and microeconomic (maximization of services, with maximum satisfaction at minimum costs) efficiency³. users’ satisfaction and the use of health services are a complex behavior resulting from a set of determinants, which include the users’ socio-demographic characteristics. in fact, health practices do not have merely a technical dimension; 6868686868 braz j oral sci. 11(1):67-71 they are, at the same time, complex social practices permeated by culture, economy, politics and ideological dimensions². these multiple aspects interact mutually, compete for a set of actions and services availability, as well as compete for emergence of issues to gain access to activities and services4. the health service organization in brazil comprises the unified health system (uhs), which is public and g o v e r n m e n t a l , f e a t u r i n g a u n i v e r s a l a n d e q u i t a b l e orientation, and a complementary system, which is private, made up of health insurance and care through payment. a c c o r d i n g t o d a t a b y t h e w o r l d h e a l t h s e a r c h a n d national research by domiciliary sample 5, the private system in brazil covers about 34.5% of the population, whereas the public health system covers 100% of the brazilian population. moreover, uhs differs from the health systems of other countries in that it is comprises all citizens in brazil and promotes equal and equitable orientation6. the brazilian public health system warrants to all citizens the same rights and promotes total universal access to all free offered services. further, the equity principle guarantees more access to the most needy and thus health democratization is provided7. since its implementation, uhs administrators have been adapting its institutional model through social participation guidelines and through decentralization triggered by the activities of the brazilian state in deploying in-depth modifications in the social field6. however, changes have not reduced inequality and inequities in the brazilian public health system are still disparate8. health care humanization should be analyzed by focusing on professionals and users. the more integrated the uhs assistance, the greater guarantee of a quality and equitable health services. however, this process depends on a critical view by users and professionals9. so that a constant dialogue with the movements may be kept and permanent and sustainable ways of articulation established, the system’s vulnerability must be reduced and the population’s life quality increased. such relationship multiplies the possibilities of solving health problems, which enhance uhs activities10. according to buss and pellegrini filho (2008)8, the determining factor in a country’s general health situation is not total wealth but its distribution. in brazil, besides n o t o r i o u s l y s e r i o u s i n e q u i t i e s , l a r g e s e c t o r s o f t h e population live in poor conditions which, in the main, impair their access to minimum health conditions. population surveys on life and health conditions have been undertaken periodically in a large number of countries to organize public policies and their attributions. colombia, for instance, is conducting several population surveys aimed at building a database for monitoring and assessing public health policies under implementation11. the current survey interprets the socio-demographic profile of the uhs users from the city of pelotas-rs, at the southern region of brazil. material and methods the study was approved by the ethics committee of the school of dentistry of the federal university of pelotas (rs, brazil) (no. 083/2009). this descriptive cross-sectional survey of a convenience sample comprised 333 users of five health basic units (hbus) from the city of pelotas, southern brazil (table 1). semistructured interviews with open and closed questions were carried out for the survey. the socio-demographic profile of uhs users was determined by data collection during a 1year period and all users selected for the study freely accepted to participate in the interviews. pelotas is a medium-sized city (323,034 inhabitants, with 22,082 residents in rural areas) in a relatively affluent area in southern brazil. its economy is based on services and food industries (department of economic development, 2002). the hbus were chosen according to their importance, care diversity and location in the city in such a way that a more diversified population could be covered and the study range expanded. the interviews were carried out by two students from the school of dentistry of the federal university of pelotas, who were properly instructed within a pilot study. the only requirement for the users to be included in the sample was being treated at the hbus. if they accepted the invitation, the participants were interviewed randomly while waiting for treatment, after signing an informed consent form. all eligible uhs users agreed to participate in the survey. after the interviews, data collected were tabulated in software spss 10.0 and analyzed by stata se 10. so that the socio-demographic characterization of the sample population could be analyzed the following variables were taken: sex, age, occupation, city/town or district of origin, number of family members, continuous medication use, diseases with extended treatment, and family income per capita. hbus names freq. percent (%) c u m . centro de especialidades 129 38.7 38.7 simões lopes 113 34 72.7 puericultura 74 22.2 94.9 sansca 16 4.8 99.7 navegantes 1 0.3 100 total 333 100 table 1. table 1. table 1. table 1. table 1. distribution of the users interviewed at the different unified health system’s health basic units (hbus) from pelotas-rs, brazil. results the results for gender, age and occupation of users are shown in table 2. the professional profile characterized most users as housewives (33.93%) and pensioners (15.3%) with monthly 1-2 minimum wages (64.26%) (figure 1). the number of persons per household was between 2 and 4 people (64.56%). data reveal that users from 33 districts of pelotas and social and demographic profile of unified health system users in a medium-sized city of southern brazil braz j oral sci. 11(1):67-71 fig. 1: distribution of the unified health system users according to monthly family income in the city of pelotas-rs, brazil. variable categories freq percent gender female 280 84 male 53 16 occupation housewife 113 34 retired 51 15 cleaner house 39 12 unemployed 20 6 student 19 5.7 trades 19 5.7 trader 15 4.5 public servant 11 3 other 46 13.5 age (years) <20 11 3.3 20 to 30 68 20.4 31 to 40 62 18.6 41 to 50 70 21 51 to 60 62 18.6 than sixty years 60 18 total 333 100 table 2. table 2. table 2. table 2. table 2. characterization of the sample. home users make use of the five selected uhs. most of them were from the neighboring district of fragata (26.6%), and 2.7% were residents in neighboring towns (capão do leão, canguçu, piratini, turuçu and cerrito). a considerable portion of the sample reported to use allopathic medicines (43.84%) (figure 2). hypertension was reported by the uhs users as the most common chronic disease (46.58%) (figure 3). discussion uhs users interviewed were mostly female (84.08%), but it is important to mention that one of the hbus chosen for this study was a children’s clinic. according to ribeiro et al.6, women are more concerned with health and are traditionally responsible for the care to children and other sick family members. this fact explains the discrepant percentage when compared to the number of males in current study. owing to such characterization, high investments must fig. 3: distribution of the unified health system users according to reported chronic diseases in the city of pelotas-rs, brazil. fig. 2: distribution of the unified health system users according to continuous allopathic drugs use in the city of pelotas-rs, brazil. be made so that women have healthier lives. medical doctors should be trained to treat this female public by clear communication and in places where they feel comfortable. comprehensive health facilities with more flexible hours are also of great importance12. moreover, the difference in dealing with men’s health provokes serious reflections. most conventional health indicators show that a gap exists between the sexes and their access to health services. a higher male mortality exists at almost every age and for almost all causes. moreover, life expectancy at birth and in other age ranges is always lower among men. in addition, various diseases affect men more. it is an issue that must be considered when dealing with family health and, by extension, a healthy community. in fact, in general, there are health programs that include children, adolescents, women and the elderly; there are none, however, with the exception of programs for workers’ health, which 6969696969 social and demographic profile of unified health system users in a medium-sized city of southern brazil 7070707070 braz j oral sci. 11(1):67-71 specifically target the adult male13. the income level and age structure of the population should be taken into account to organize the provision of health services and health expenditures for each country according to its model of medical assistance14. in examining the age group which demands services at the uhs, some authors have observed a high prevalence of the elderly and relate this fact to the aging population. about 70% of the elderly in the world health search and national research by domiciliary sample (pnad-2003) reported having at least one chronic illness condition. moreover, the proportion of people with health problems increases according to age in both sexes. thus the elderly population is a great user of health services. in developed countries, the use of these services among people aged 65 years and over is three to four times greater than its proportional size in the population15. in the present study, increased service offer for the elderly was not reported since the elderly population comprised only 18% of the sample. a broader age group in the population sampled was within the 41-50-year-old age range (21.02%), followed by the 20-30-year-old age range (20.42%). the features such as women and low income in this study are consistent with other studies that portray the reality of the users of the uhs. ribeiro et al.6 have also characterized individuals as women (2:1) with low schooling level (52.8% with up to 3 years of study) and a concentration on the first four quintiles of family income with an excess of individuals in the first two quintiles. several studies indicate that the individual’s position in the social structure is an important indicator of health needs, and the pattern of perceived risk tends to be disadvantageous for those individuals belonging to the underprivileged social groups16. in a 2008 study carried out in the city of pelotas, the authors concluded that the socioeconomic status influenced the use and the type of health service, with difficulties in accessing health care17. a marked change occurs when the structure of service consumption among income groups was analyzed: high income groups consume more outpatient services and consumption of hospital services tends to increase as the income rises. moreover, a change occurs in the type of outpatient service used: the higher income groups use predominantly private practices and clinics, whereas the smaller income groups use more clinics and health centers11. such events go contrary to the third principle of the unified health system, which says that every citizen has the right to humane care that is friendly and free from any discrimination. this principle refers to the universal nature of the uhs as a health system for all brazilians, which has yet to happen; this proves, in practice, discrimination and restricted access9. a considerable portion of the studied population claimed to use allopathic medicines regularly. the cost of drugs has a similar pattern with regard to spending money on health care, which means that the poorest people (who have more morbidity and therefore have greater need to consume drugs) tend to spend less on this item. restricting the use of drugs among people with low purchasing power becomes evident when one notes that the weight of that spent on household income is proportionally greater than for those with higher income11. arterial hypertension was the most common pathology, which indicates a prevalence of chronic diseases, with an increasing rate in recent decades, and the prevalence of cardiovascular conditions18. a study by the national research per residence sample revealed similar results for the disease that most affects uhs users. the authors reported that the disease most often cited by study participants was hypertension (43.9%), and the proportion decreased with age in both sexes15. it is very difficult to generalize the results of a convenience sample to any population that has practical relevance. however, convenience sampling provides useful information for other studies involving probability sampling techniques. despite limitations, the present study suggests that the data collected from this convenience sample can represent the socio-demographic profile of uhs users and is an appropriate sampling strategy to provide national population data that will inform and contribute towards the current and future public health policies in brazil. some aspects of the uhs are highly successful. a social basis exists so that the reform may continue; in fact, thousands of workers and millions of brazilians recognize the importance of the uhs. however, impairments exist with regard to both the users and the system19,20. the results obtained in the present survey reveal that understanding the characteristics of hbu users and the associated socio-demographic context may contribute to a better health care for citizens and confirm the uhs contribution towards the universality and the equity of public health care. acknowledgements this project was partially funded by an undergraduate grant provided by the national council for scientific and technological development (cnpq), brazil. references 1. castilla ee, luquetti dv. brazil: public health genomics. public health genomics 2009;12:53-8. 2. oliveira dc, sá cp, gomes amt, ramos rs, pereira na, santos wcr. brazilian national health policy: health professionals’ social representation. cad saude publica. 2008;24:197-206. 3. cesconetto a, lapa js, calvo mcm. evaluation of productive efûciency in the uniûed national health system hospitals in the state of santa catarina, brazil. cad saude publica. 2008;20:2407-17. 4. moretti-pires ro. complexity in the family health and education of future health professionals. interface (botucatu). 2009;13:153-66. 5. brazilian institute of geography and statistics (ibge). national survey by household sample survey: access and use of health services in 2003. rio de janeiro: ibge; 2005. social and demographic profile of unified health system users in a medium-sized city of southern brazil 7171717171 braz j oral sci. 11(1):67-71 6. ribeiro mcsa, barata rb, almeida mf, silva zp. sociodemographic profile and utilization patterns of the public health care system (sus)– pnad 2003. cien saude colet. 2006;11:1011-22. 7. silva zp, ribeiro mcsa, barata rb, almeida mf. sociodemographic profile and utilization pattern of health services of the unified health system (sus). cien saude colet. 2011;16:3807-16. 8. buss pm, pellegrini filho a. inequalities in health in brazil, our most serious disease: comments on the reference document and the work of the national commission on social determinants of health. cad saude publica. 2006;22:2005-8. 9. backes ds, koerich ms, rodrigues acrl, drago lc, klock p, erdmann al. what do the users think and say about the brazilian health system (sus)? an analysis of meanings based on the users’ rights booklet. cien saude colet. 2009;14:903-10. 10. horta nc, sena rr, silva meo, oliveira sr, rezende va. practice of family health teams: challenges for the promotion of health care. rev bras enferm. 2009;62:524-9. 11. travassos c, viacava f, fernandes c, almeida cm. social and geographical inequalities in health services utilization in brazil. cien saude colet. 2000;5:133-49. 12. torres mea, ribeiro pm, machado cj. access to gynecological consultations in belo horizonte, minas gerais, brazil. rev bras estud popul. 2008;25:49-69. 13. schraiber lb, figueiredo ws, gomes r, couto mt, pinheiro tf, machin r et al. health needs and masculinities: primary health care services for me. cad saude publica. 2010;26:961-70. 14. guanais fc. health equity in brazil. bmj. 2010;341:c6542. 15. lima-costa mf, barreto sm, giatti l. health status, physical functioning, health services utilization, and expenditures on medicines among brazilian elderly: a descriptive study using data from the national household survey. cad saude publica. 2003;19:735-43. 16. evans rg, barer ml, marmor tr. why are some people healthy and others not?: the determinants of health of populations. nova york: aldine de gruyter; c1994. p.3-26. 17. dias-da-costa js, gigante dp, horta bl, barros fc, victora cg. pattern of health services utilization by adults of the pelotas birth cohort from 1982 to 2004-5, southern brazil. rev saude publica. 2008;42:51-9. 18. malta dc, cezario ac, moura l, neto olm, silva jr. jb. building surveillance and prevention for chronic non communicable diseases in the national unified health system. epidemiol serv saude. 2006;15:47-65. 19. campos gs. political and organizational challenges in the brazilian unified national health system: twenty years of public policy. cad saude publica. 2008;24:2200-1. 20. gouveia gc, souza wv, luna cf, souza-junior prb, szwarcwald cl. user satisfaction in the brazilian health system: associated factors and regional difference. rev bras epidemiol. 2009;12:281-96. social and demographic profile of unified health system users in a medium-sized city of southern brazil oral sciences n3 original article braz j oral sci. january | march 2011 volume 10, number 1 adenoid hypertrophy and open bite ana de lourdes sá de lira1, antonio de moraes izquierdo1, sávio prado1, margareth maria gomes souza2, sandra regina torres3 1dds, ms, phd student, department of pediatric dentistry and orthodontics, dental school, federal university of rio de janeiro, brazil 2dds, phd, professor, department of pediatric dentistry and orthodontics, dental school, federal university of rio de janeiro, brazil 3dds, phd, professor, department of oral pathology and diagnosis, dental school, federal university of rio de janeiro, brazil correspondence to: ana de lourdes sá de lira. departamento de ortodontia, faculdade de odontologia university federal fo rio de janeiro (ufrj), av. brigadeiro trompowsky s/n ilha do fundão rio de janeiro, cep: 21941-590, rio de janeiro, rj, brasil e-mail: anadelourdessl@hotmail.com received for publication: june 13, 2010 accepted: december 16, 2010 abstract aim: to evaluate if the constricted airway passage, measured by the nasopharyngeal (np) and oropharyngeal (op) width, has a correlation to anterior open bite. methods: lateral cephalometric radiographs of 43 patients with anterior open bite (26 with mixed dentition and 17 with permanent dentition) and 30 patients with overbite (15 with mixed dentition and 15 with permanent dentition) were obtained from de department of pediatric dentistry and orthodontics of the federal university of rio de janeiro for examination. eight patients with syndromes were excluded from study. cephalometric measurements were carried out using dolphin imaging &management solutionstm software (chatsworth, ca, usa). measures of np and op were evaluated according to the mcnamara’s soft tissue analysis and were correlated with the presence of open bite or overbite. student’s t test and chi-square were used to assess statistical differences in continuous and dichotomic variables, respectively. kruskal-wallis test was employed to compare multiple variables. results: open bite patients showed significant decreased mean np and op values compared to overbite patients. lack of labial seal was observed in all open bite patients. when patients with mixed and permanent dentitions were analyzed separately, the mean np value was still significantly smaller in the open bite group. however, the mean op values were smaller in the open bite group in both dentitions analysis, but were not statistically different. conclusions: all patients with anterior open bite had reduced np and op measures compared to overbite patients, in spite of dentition. keywords: anterior open bite, adenoid hypertrophy; nasopharyngeal width; oropharyngeal width. introduction in the child normal growing process, there is an increase in the nasopharyngeal lymphoid tissue and a downward and forward shift of the face to allow airflow passage1. this area is crucial for determining the nasal respiratory strength because of the presence of adenoids2. in general, lymphoid tissues are hypertrophic during childhood, becoming less hypertrophic following puberty and atrophic by adulthood2. however, when lymphoid tissues increase in size, a mechanical obstruction of the airflow passage may be expected, and the child may develop a compensatory mouth-breathing habit3.this may interfere with both growth and development of the face, which eventually results in skeletal open bite1-2. adenoid hypertrophy decreases the nasopharyngeal width, which favors the backward and upward positioning of the head in relation to cervical column, thus increasing the facial height3. posterior rotation or inclination of the mandible and consequent increase in the angle between anterior maxillary/mandibular parts and nasion point (anb) may occur, as well as flaccidity and shortening of upper lip braz j oral sci. 10(1):17-21 18 associated with flaccid perioral muscles3. alterations in the oral structures such as protrusion and lowering of the tongue, high-vaulted arch, v-shaped constriction of upper arch, and anterior open might be observed particularly in those patients with vertical growth pattern4. though anterior open bite is more frequently observed, adenoid hypertrophy may also cause posterior open bite when lingual interposition occurs in the premolar and molar regions1-4. it has been reported that mouth breathing and open bite cause changes in muscular balance, tongue and head posture in addition to obstructive sleep apnoea5. breathing difficulty during sleep also decreases nocturnal secretion of growth hormone, resulting in poor development of the ramus and lower edge of the mandible6-7. nasopharyngeal width tends to decrease if adenoids are hypertrophic8. on the other hand, there has been reported an increasing op width related to adenoid hypertrophy, which could be explained by a compensatory anterior lingual posture when hypertrophy of adenoids are present6-7. our hypothesis was that patients with smaller np and op measurement develop open bite as a compensating mechanism for breathing. the aim of the present study was to evaluate if the constricted airway passage, as measured by the np and op width, has a correlation to anterior open bite. material and methods this study was approved by the local ethics research committee (caae number: 131/2009.0049.0.239.000-09). from 81 patients that started the treatment between march 2004 and march 2008, 8 were excluded for being syndromic patients. after obtaining written informed consent, the sample consisted of 73 patients who had undergone total corrective orthodontic treatment through edgewise system in the orthodontic clinic of the department of pediatric dentistry and orthodontics, dental school, federal university of rio de janeiro (rio de janeiro, rio de janeiro, brazil). the study group consisted of 43 patients with anterior open bite, whereas the control group consisted of 30 patients with overbite. demographic and clinical data regarding all patients were obtained from clinical records, study models and baseline cephalometric x-rays. cephalometric measurements were carried out using dolphin imaging &management solutions tm software (chatsworth, ca, usa). the distance between incisal edges was measured from the lower incisal edge to a parallel line traced along the upper incisor axis. anterior open bite was defined as being the lack of vertical trespass between upper and lower central incisors. overbite was defined by the presence of a vertical trespass. negative values were attributed to anterior open bite, whereas positive values were attributed to overbite. the nasopharyngeal (np) width was obtained by measuring the smallest distance between the posterior wall of soft palate and the posterior wall of the pharynx. the np values greater than 12 mm and 17.4 mm were considered normal for individuals with mixed and permanent dentition, respectively5. the oropharyngeal (op) width was obtained by measuring the smallest distance between the intersection of lingual posterior edge with mandibular lower edge and the posterior wall of the pharynx. normal op values ranging from 10 to 12 mm were considered normal5. in order to determine the consistency of the method, two examiners were calibrated by repetition of the process until the method was considered adequate by a third examiner. random errors in landmark localization were decreased by tracing each lateral cephalogram twice and using the medium values of each measurement. the intraexaminer consistency (icc) was calculated for reliability of tracing, landmark identification and analytical measurement showing a correlation coefficient always greater than 0.94. data were collected and analyzed by using the statistical software spss v.10.0 for windows© (chicago, il, usa). student’s t test and chi-square test were used for assessing the possible differences regarding continuous and dichotomic variables, respectively. kruskal-wallis test was employed for comparing multiples variables. a significant level of 5% was established. results demographic and clinical characteristics of the 73 studied patients were shown in table 1 and figure 1. no statistically significant differences were observed between open bite and overbite groups regarding demographic data, dentition and malocclusion. labial seal was absent in 100% of open bite group and in only 6% of overbite patients (table 1). the mean value for np measurements of patients with open bite (6.8 mm) was significantly smaller than that of the patients with overbite (11.43 mm) (p < 0.001). (table 1). patients in the open bite group had a mean value of the op measurements significantly smaller than that of patients in the overbite group (10.69 mm vs. 11.88 mm) (p = 0.044). (table 1) the mean values for np and op in both groups adenoid hypertrophy and open bite braz j oral sci. 10(1):17-21 fig. 1. mean np and op values for overbite and open bite groups regarding mixed and permanent dentitions. footnote: nfnasopharynge. oforopharynge. opbopen bite. ovboverbite. 19 regarding mixed and permanent dentitions were shown in figure 1. when patients with mixed dentition were analyzed, the mean np value was found to be significantly smaller in the open bite group (6.03 mm) than in the overbite group (10.2 mm) (p < 0.001) (table 2). there was no statistically significant difference between the open bite (10.71 mm) and overbite (11.33 mm) groups regarding the mean op values for mixed dentition (table 2). when the permanent dentition was analyzed separately, the mean np value was found to be significantly smaller in the open bite group (8.17 mm) than in the overbite group (12.6 mm) (p < 0.001) (table 3). there was no statistically significant difference between the open bite (10.67 mm) and overbite (12.43 mm) groups regarding the mean op values for permanent dentition (table 3). no statistically significant differences were found between np and op measurements of mixed and permanent dentitions regarding age and gender in both groups (figure 2). when these values were compared to mcnamara’s normal parameters5, the mean value for np measurement for both groups were below of which is considered normal and op mean values from both groups were within the considered normal range. when mixed and permanent dentitions were analyzed separately, both groups had a mean np value below to the normal and op mean values within the normal parameters. when patients with open bite were analyzed, no statistically significant difference was found between number of patients and type of dentition (p = 0.29). however, a significantly larger number of patients with class i malocclusion was observed (p = 0.003). by associating the np measurement with the type of malocclusion, no statistically significant difference was found for patients with open bite (p = 0.05). on the other hand, a significant reduction in op measurement was observed in patients with open bite with class ii malocclusion (p = 0.05). discussion adenoid hypertrophy causes obstruction of the airflow through np possibly favouring mouth-breathing habit and open bite7. the present study measured the np and op dimensions in 73 orthodontic patients ranging from 10 to 24 years of age. these measures were compared between patients presenting open bite and overbite. confirming our adenoid hypertrophy and open bite braz j oral sci. 10(1):17-21 fig. 2. profile of patients with open bite and overbite regarding gender and age. footnote: opbopen bite. ovboverbite. hypothesis, the mean np and op values were significantly smaller among patients presenting open bite, than in those presenting overbite. in a similar study, mcnamara5 found equivalent results for np measurement only. the results of the present study seem to confirm our hypothesis of the development of an open bite as a compensatory breathing mechanism for the narrowing of the airflow passage. patients with open bite had a decreased mean np value compared to patients with overbite in both dentitions, regardless of the type of malocclusion. the marked np constriction before and during the growth spurt, which occurs in the mixed and permanent dentitions, predisposes the development of anterior open bite6-8. moreover, this np constriction makes nasal breathing difficulty, consequently favoring mouth breathing, posterior teeth extrusion, narrowing of dental arches, and vertical growth of the anterior face8-10. other factors like genetic characteristics, infections or recurrent adenoid inflammations may also be involved in this process1, 6-11. the decreased mean op value observed in the patients with open bite may be explained by the anterior positioning of the tongue as compensatory respiratory mechanism. consequently, there is a greater predisposition to a vertical facial growth8,12. in the same group, the significant reduction of mean op value in patients with class ii malocclusion is possibly due to the more posterior positioning of the mandible in relation to the skull base13,14. lack of labial sealing was observed in all patients with anterior open bite. this result has been found by other authors, correlating adenoid hypertrophy with mouth breathing and vertical growth pattern of the face5,15-20. when patients were analyzed separately by type of dentition, the mean np value was small in open bite patients regarding mixed and permanent dentition. when adenoid hypertrophy persists until permanent dentition, the resulting np narrowing may influence the vertical facial growth and anterior open bite19-20. in both dentitions, the op measurement was also small in the open bite group, but the results were not statistically significant. other authors have shown opposite findings, reporting an increase in op measurement associated with open bite5,15. other studies should be conducted in order to confirm if op measurements have a correlation to open bite. in the present study, anterior open bite was more predominantly found in patients with class i malocclusion, although some authors 13-14,21-22 have reported a higher frequency in cases of class ii malocclusion. those authors1314,21-22 also observed that patients with class i or class ii malocclusion who had a predominance of vertical facial growth, showed greater nasopharyngeal narrowing compared to those patients with horizontal facial growth. it suggests that the type of malocclusion and growth pattern have no influence on np width, despite predisposing to vertical growth of the face and anterior open bite23. all patients with open bite and class iii malocclusion showed a significant decrease in the nasopharyngeal width. according to some authors, anterior open bite seems to be established by mandibular vertical growth and extrusion of posterior teeth, as a result of mouth breathing9-11. the present study also contributes with epidemiologic data for np and op measures. the mean np values for patients with either overbite or open bite were below the normal range established by mcnamara5. the results of the present study suggested that the parameters used by the author5 may not be applicable to the patterns of normality of the brazilian population. the findings of this study may have implications in the clinical practice. adenoid hypertrophy is a problem that should be investigated as an etiologic factor of anterior open bite. further studies correlating np to the growth of facial structures and other measurable parameters of mouth breathing, should be conducted in order to determine the relationship between soft and hard tissues of the face. in conclusion, all patients with anterior open bite showed reduced nasopharyngeal and oropharyngeal measurements compared to those with overbite in both mixed and permanent dentitions. references 1. linder-aronson s, woodside dg, lundstrom a. mandibular growth direction following adenoidectomy. am j orthod dentofacial orthop. 1986; 89: 273-84. 2. trask gm sg, shapiro pa. the effects of perennial allergic rhinitis on dental and skeletal development: a comparison of sibling pairs. am j orthod dentofacial orthop. 1987; 92: 286-93. 3. jones ag, bhatia s. a study of nasal respiratory resistance and craniofacial dimensions in white and west indian black children. am j orthod dentofacial orthop. 1994; 106: 34-9. 4. ung n, koenig j, shapiro pa, shapiro g, trask g. a quantitative assessment of respiratory patterns and their effects on dentofacial development. am j orthod dentofacial orthop. 1990; 98: 523-32. 5. mcnamara ja. influence of respiratory pattern on craniofacial growth. angle orthod. 1981; 51: 269-300. 6. behlfelt k, linder-aronson s, mcwilliam j, neander p, laage-hellman j. cranio-facial morphology in children with and without enlarged tonsils. eur j orthod. 1990; 12: 233-43. 7. subtelny jd. oral respiration: facial maldevelopment and corrective dentofacial orthopedics. angle orthod. 1980; 50: 147-64. 8. linder-aronson s, leighton bc. a longitudinal study of the development of the posterior nasopharyngeal wall between 3 and 16 years of age. eur j orthod. 1983; 5: 47-58. 9. mcnamara jr ja. an orthopedic approach to the treatment of class iii malocclusion in young patients. j clin orthod. 1987; 21: 598-608. 10. sugawara j, mitani h. facial growth of skeletal class iii malocclusion and the effects, limitations, and long-term dentofacial adaptations to chincap therapy. sem orthod. 1997; 3: 244-54. 11. buschang ph, martins j. childhood and adolescent changes of skeletal relationships. angle orthod. 1998; 68: 199-206. 12. vig kw. nasal obstruction and facial growth: the strength of evidence for clinical assumptions. am j orthod dentofacial orthop. 1998; 113: 603-11. 13. paul jl, nanda rs. effect of mouth breathing on dental occlusion. angle orthod. 1973; 43: 201-6. 14. mergen dc, jacobs rm. the size of nasopharynx associated with normal occlusion and class ii malocclusion. angle orthod. 1970; 40: 342-6. 15. cuccia am, lotti m, caradonna d. oral breathing and head posture. angle orthod. 2008; 78: 77-82. 16. cheng mc, enlow dh, papsidero m, broadbent jr bh, oyen o, sabat m. developmental effects of impaired breathing in the face of the growing child. angle orthod. 1988; 58: 309-20. 20adenoid hypertrophy and open bite braz j oral sci. 10(1):17-21 21 17. tourne lp. the long face syndrome and impairment of the nasopharyngeal airway. angle orthod. 1990; 60: 167-76. 18. tourne lp. growth of the pharynx and its physiologic implications. am j orthod dentofacial orthop. 1991; 99: 129-39. 19. yamada t tk, miyamoto k, yamauchi k. influences of nasal respiratory obstruction on craniofacial growth in young macaca fuscata monkeys. am j orthod dentofacial orthop. 1997; 111: 38-43. 20. martin o, muelas l, vinas mj. nasopharyngeal cephalometric study of ideal occlusions. am j orthod dentofacial orthop. 2006; 130: 431-9. 21. kerr wj. the nasopharynx, face height, and overbite. angle orthod. 1985; 55: 31-6. 22. subtelny jd. malocclusions, orthodontic corrections and orofacial muscle adaptation. angle orthod. 1970; 3: 170-201. 23. de freitas mr, alcazar nm, janson g, de freitas km, henriques jf. upper and lower pharyngeal airways in subjects with class i and class ii malocclusions and different growth patterns. am j orthod dentofacial orthop. 2006; 130: 742-5. adenoid hypertrophy and open bite braz j oral sci. 10(1):17-21 original articlebraz j oral sci. january/march 2009 volume 8, number 1 surface morphology alterations in bovine dentin exposed to different bleaching agents juliana nascimento santos1, daniel pinto de oliveira1, fábio roberto dametto1, brenda paula figueiredo de almeida gomes2, alexandre augusto zaia2, josé flávio affonso de almeida2, caio cezar randi ferraz2 1 dds, msc, phd, department of restorative dentistry and endodontics, faculdade de odontologia de piracicaba, universidade estadual de campinas (unicamp), piracicaba (sp), brazil 2 dds, msc, phd, professor, department of restorative dentistry, endodontics, faculdade de odontologia de piracicaba, unicamp, piracicaba (sp), brazil received for publication: november 5, 2007 accepted: april 23, 2009 correspondence to: caio cezar randi ferraz avenida limeira, 901 – areião cep 13414-018 – piracicaba (sp), brazil e-mail: cferraz@fop.unicamp.br abstract aim: this study evaluated the morphological changes caused by internal bleaching agents on dentin surface. methods: twenty crowns of bovine incisors were cut into slabs that were randomly distributed in six experimental groups (n = 5), according to the bleaching agent used: g1 – sodium perborate + water, g2 – sodium perborate + 2% chlorhexidine gel, g3 – sodium perborate + 30% hydrogen peroxide, g4 – 30% hydrogen peroxide, g5 – 37% carbamide peroxide and g6 – gel base without carbamide. two control groups were used: c1 with distilled water and c2 with 2% chlorhexidine gel. the specimens were immersed in the respective test bleaching agent and incubated at 37 °c for seven days. following, they were prepared for scanning electron microscopy and five images from each tooth segment were recorded and analyzed for surface morphological alterations, by three previously calibrated examiners. inter-examiner agreement was verified using the kappa test. the rank averages obtained for the groups were subjected to kruskal-wallis analysis of variance at 5% significance level. results: the analysis of the scores obtained indicated that all tested materials caused some morphological alteration on dentin, except for sodium perborate + water (g1) and control groups 1 and 2. hydrogen peroxide and carbamide peroxide caused significantly more severe alterations (p < 0.05) to dentin structure, than the other bleaching agents evaluated. conclusions: sodium perborate-based pastes seemed to be the most harmless agent to dentin structures in non-vital tooth bleaching, while hydrogen peroxide solutions and carbamide peroxide agents caused the greatest alterations. keywords: tooth bleaching, dentin, dental pulp cavity. introduction color changes may occur in teeth that had undergone endodontic treatment, consisting in an important esthetic problem. bleaching is one of the procedures that can be used to treat discoloration, and it is commonly performed in non-vital teeth1. the walking bleach technique is considered the safest and most accepted intracoronal bleaching technique1-2. it consists on the placement of bleaching agents in the pulp chamber of root-filled teeth. traditionally, intracoronal bleaching is achieved with the use of 30% hydrogen peroxide and sodium perborate, which can be used either separately or in combination3. carbamide peroxide formulations, commonly used for nightguard vital bleaching, have also shown to be effective as an intracoronal bleaching agent4. recently, a gel base containing 2% chlorhexidine was introduced as a vehicle for sodium perborate, being an alternative to water or hydrogen peroxide in order to prevent coronal microleakage during the walking bleach technique5. however, some side effects resulting from the use of internal bleaching agents have been reported. thirty-percent hydrogen peroxide has been associated to the development of ex26 santos jn, oliveira dp, dametto fr, gomes bpfa, zaia aa, almeida jfa, ferraz ccr braz j oral sci. 8(1): 25-9 ternal cervical root resorption6. chng et al.7 found that intracoronal bleaching with 30% hydrogen peroxide and sodium perborate used, either alone or in combination, weakened dentin. a significant alteration on the levels of inorganic components of dentin8 and surface morphological changes9 were reported after treatment with carbamide peroxide. during intracoronal bleaching, the materials are placed in direct contact with dentin, and subsequently the access cavity should be readily sealed to minimize coronal leakage10. being the major constituent of tooth structure, any changes on the surface morphology of dentin are likely to affect the outcomes of dentin bonding2. the aim of this study was to determine in vitro, the effects of different bleaching agents on dentin surface morphology using scanning electron microscopy. material and methods twenty bovine mandibular incisors were obtained from slaughtered cattle at the age of one to two years, and stored in 0.2% thymol to be used within six months after extraction. the coronal portion of each tooth was cut longitudinally from mesial to distal into two equal segments with a water-cooled diamond disk (kg sorensen, barueri, sp, brazil). the buccal segment was sectioned in “x” and “y” directions and serial coronal slabs were obtained. the exposed pulp chamber dentin was cleaned, by immersing the slabs in 5.25% naocl for ten minutes, and sequentially in 17% edta for the same time in order to remove the smear layer formed during sectioning11. the pooled coronary slabs were rinsed in running water for 12 hours, dried, and then randomly assigned to eight groups of five specimens each. each experimental group was treated with one of the following bleaching agents: g1 – sodium perborate (nabo 3 .4h 2 o; proderma, piracicaba, sp, brazil) mixed with water, g2 – sodium perborate mixed with 2% chlorhexidine gel (proderma), g3 – sodium perborate mixed with 30% hydrogen peroxide, g4 – 30% hydrogen peroxide, g5 – 37% carbamide peroxide (whiteness, porto alegre, rs, brazil), and g6 – gel base without carbamide (whiteness). the following control solutions were used: c1– distilled water and c2 – 2% chlorhexidine gel. the bleaching agent was combined with the designated vehicle for each group to maintain a 2:1 ratio in solution. the specimens were immersed in the respective test material and incubated at 37 °c for seven days in 100% humidity, as previously described by zalkind et al.9. the samples were, then, rinsed in an ultrasonic bath during one hour, dehydrated in ascending alcohol concentrations, dried and sputter coated with gold (denton vaccum desk ii, moorestown, nj, usa)9. dentin surface morphology was analyzed with a scanning electron microscope ( jeol – jsm 5,600 lv, noran instruments, tokyo, japan) operated at 15 kv. the five most representative images from different regions of each tooth segment were captured at × 2000 magnification, and the surface morphological alterations were analyzed by three previously calibrated examiners. morphological changes were classified as absent (score 0), moderate (score 1) and severe (score 2), according to pre-established parameters (figure 1). inter-examiner agreement was verified using the kappa test. the rank averages obtained for the groups were subjected to kruskalwallis analysis of variance at 5% significance level. results the effects of the various bleaching agents on dentin surface morphology are expressed in ranks in table 1. the kappa value for interexaminer agreement was 0.9. most tested materials caused some morphological alteration on dentin, except for sodium perborate mixed with water (g1), the control solutions distilled water and 2% chlorhexidine gel (c1 and c2) (figure 2). specimens treated with sodium perborate mixed with 2% chlorhexidine gel (g2) and sodium perborate mixed with 30% hydrogen peroxide (g3) showed more evident surface alterations than groups g1, c1 and c2, as a moderate flattening of dentin surface and a mild erosion of intertubular dentinal matrix could be detected. the dentinal tubules remained opened but not widened (figure 3). severe morphological changes could be observed for the groups treated with 30% hydrogen peroxide (g4), 37% carbamide peroxide (g5) and the gel base without carbamide peroxide (g6), which differed significantly (p < 0.05) from the other groups. these agents gave figure 1. scanning electron microscope micrographs representing score rates of dentin surface alterations. a: score 0 – absent morphological changes. b: score 1 – moderate morphological changes. c: score 2 – severe morphological changes. 27surface morphology alterations in bovine dentin exposed to different bleaching agents braz j oral sci. 8(1): 25-9 to the dentin a flattening and an etching-like appearance on intertubular area. the dentinal tubules were wider, showing demineralization of intratubular dentin (figure 4). discussion the results of this study indicated that the materials commonly used for internal bleaching may have an effect on dentin surface morphology. the alterations observed showed a strong relationship between the morphological changes and the bleaching agent used. it is quite relevant to test the effects of the bleaching materials on dentin, since a close contact occurs during internal bleaching procedures. most morphological studies have evaluated external bleaching agents and their effects on enamel12-13, whilst information concerning the influence of materials used for internal bleaching on dental tissues is rather limited. structural changes of dentin substrate may have an important role on the performance of dental restorations. in fact, a variety of studies have shown a detrimental effect of the bleaching procedures on the bond strength and sealing ability of composite restorations10,14-16 to dentin, thus the authors suggest morphological alterations as one of the reasons for that. the most significant morphological changes were caused by 30% hydrogen peroxide, 37% carbamide peroxide and the gel base without carbamide peroxide. the adverse effects of hydrogen peroxide on dental hard tissues in external and internal bleaching has been previously reported on the literature and include alterations in the chemical structure of dentin17 and a reduction on the calcium/phosphorous (ca/p) ratio8-9, which could be responsible for the alterations observed. in addition, hydrogen peroxide was found to increase dentin solubility and cause protein oxidation of organic matrix8, this may explain the severe etching-like appearance and surface flattening observed in this study. the acidity provided by the low ph of hydrogen peroxide solutions2 might have contributed for the enlargement of dentinal tubules. the above-mentioned complications, allied to a frequent occurrence of external root resorption, made some researchers recommend avoidance of highly concentrated hydrogen peroxide solutions for intracoronal bleaching18-19. bleaching agents containing carbamide peroxide are commonly used in the treatment of discolored vital teeth. these materials started to be considered for intracoronal bleaching since satisfactory esthetic results were reached4 and no association with external root resorption was found20. however, the severe dentin surface alterations observed in this study indicate that carbamide peroxide agents may have an adverse effect on the organic and inorganic components of dentin. moderate surface alterations have already been reported for 10 and 15% carbamide peroxide9,21. since a highly concentrated (37%) formulation was used in the present study, more severe morfigure 2. scanning electron microscope micrographs showing dentin surface without morphological alterations. a: specimen treated with water (control group 1). b: specimen treated with chlorhexidine gel (control group 2). c: specimen treated with sodium perborate mixed with water (group 1). table 1. dentin surface morphological changes caused by different bleaching agents groups bleaching agent ranks g1 sodium perborate + water 55.24c g2 sodium perborate + 2% chlorhexidine gel 73.34bc g3 sodium perborate + 30% hydrogen peroxide 104.98b g4 30% hydrogen peroxide 161.60a g5 37% carbamide peroxide 143.28a g6 gel base without carbamide peroxide 133.36ab c1 distilled water 62.48c c2 2% chlorhexidine gel 69.72c different superscript letters indicate statistically significant differences between the groups. figure 3. scanning electron microscope micrographs showing moderate alterations of dentin surface. a: specimen treated with sodium perborate mixed with chlorhexidine (group 2). b: specimen treated with sodium perborate mixed with hydrogen peroxide (group 3). 28 santos jn, oliveira dp, dametto fr, gomes bpfa, zaia aa, almeida jfa, ferraz ccr braz j oral sci. 8(1): 25-9 phological changes would be expected. another study20 showed a decrease on dentin microhardness similar to that caused by hydrogen peroxide-based solutions. as carbamide peroxide breaks down into urea and hydrogen peroxide, the previously reported adverse effects of hydrogen peroxide bleaching may be applicable for carbamide peroxide. even though an acidic ph is attributed to hydrogen peroxide solutions, the ph value of carbamide peroxide is almost neutral9, indicating that the surface morphological alterations were not strictly related to ph variations among the bleaching agents. another important consideration is the fact that the gel base depleted of carbamide peroxide also caused severe morphological changes on dentin surface. although the effects of carbamide peroxide’s vehicle have not been tested before, these findings indicate that other constituents of the bleaching formulation may also cause some kind of structural alterations on dental hard tissues. specimens treated with sodium perborate mixed with water did not show any morphological changes and were similar to the control groups c1 and c2. this result confirms a previous report of the absence of dentin alterations related to the use of sodium perborate as a bleaching agent9. in addition, it has been stated that the solubility and chemical composition of dentin remained undisturbed3, and the biomechanical properties were reduced only to a small extent7. these findings could be explained by the lower amount of hydrogen peroxide released from this formulation. however, significant surface changes were observed when sodium perborate was mixed with hydrogen peroxide, probably as a result of the increase in hydrogen peroxide concentration. the association of sodium perborate with hydrogen peroxide results in an alkalinization of the ph of the latter, which may explain why the morphological changes were less severe than those found for the specimens treated with hydrogen peroxide alone. a preparation of sodium perborate and 2% chlorhexidine gel was also tested on this experiment. chlorhexidine digluconate in a gel base has a broad antimicrobial activity spectrum and substantivity22. it showed a good potential to increase the antimicrobial properties of the bleaching agents when used as a vehicle23, since the bleaching effect of sodium perborate associated with chlorhexidine gel was not decreased5. the morphological changes caused by this formulation were very slight and occupied an intermediary classification, when compared to the results obtained with the other preparations containing sodium perborate. it is likely that the use of a gel based vehicle – a hydroxyethyl cellulose – would allow a slower ionization of sodium perborate molecule than the other liquid vehicles used in the present study, thus lowering hydrogen peroxide concentration24-25. in the present study, tooth slabs were exposed to bleaching agents for seven days before the analysis of surface morphological alterations. clinically, the bleaching procedure is usually repeated for additional seven days until achieving a satisfactory esthetic outcome. based on the present results, it is possible to anticipate more intense alterations on dentin surface with repeated applications of the bleaching agents. nevertheless, this hypothesis should be further investigated. this study confirmed the general concern about the hazardous effects of 30% hydrogen and carbamide peroxide on dental hard tissues, and indicated that the use of these materials as an intracoronal bleaching agent should be carefully considered. sodium perboratebased formulations caused slight or even absent alterations on dentin surface morphology and seemed to be the safest agent for use in non-vital tooth bleaching. the association of a gel-based chlorhexidine with sodium perborate showed satisfactory results concerning dentin surface integrity and should; therefore, be further investigated. acknowledgements this study was supported, in part, by grant 02/14168-6 from fundação de amparo à pesquisa do estado de são paulo (fapesp). references 1. nutting eb, poe g. a new combination for bleaching teeth. j s calif assoc. 1963;41:289-91. 2. attin t, paqué f, ajam f, lennon am. review of the current status of tooth whitening with the walking bleach technique. int endod j. 2003;36:313-29. 3. rotstein i, lehr z, gedalia i. effect of bleaching agents on inorganic components of human dentin and cementum. j endod. 1992;18:290-3. figure 4. scanning electron microscope micrographs of severe altered dentin surface. a: specimens treated with hydrogen peroxide (group 4). b: specimens treated with carbamide peroxide (group 5). c: specimens treated with the gel base without carbamide peroxide (group 6). 29surface morphology alterations in bovine dentin exposed to different bleaching agents braz j oral sci. 8(1): 25-9 4. perrine ga, reichl rb, baisden mk, hondrum so. comparison of 10% carbamide peroxide bleaching and sodium perborate for intracoronal bleaching. gen dent. 2000;48:264-70. 5. oliveira dp, gomes bpfa, zaia aa, souza-filho fj, ferraz cc. in vitro assessment of a gel base containing 2% chlorhexidine as a sodium perborate’s vehicle for intracoronal bleaching of discolored teeth. j endod. 2006;32:672-4. 6. heithersay gs, dahlstrom sw, marin pd. incidence of invasive cervical resorption in bleached root-filled teeth. aust dent j. 1994;39:82-7. 7. chng hk, palamara jea, messer hh. effect of hydrogen peroxide and sodium perborate on biomechanical properties of human dentin. j endod. 2002;28:62-7. 8. rotstein i, dankner e, goldman a, heling i, stabholz a, zalkind m. histochemical analysis of dental hard tissues following bleaching. j endod. 1996;22:23-5. 9. zalkind m, arwaz jr, goldman a, rotstein i. surface morphology changes in human enamel, dentin and cementum following bleaching: a scanning electron microscopy study. endod dent traumatol. 1996;12:82-8. 10. shinohara ms, rodrigues ja, pimenta laf. in vitro microleakage of composite restorations after nonvital bleaching. quintessence int. 2001;32:413-7. 11. perez f, rochd t, lodter jp, calas p, michel g. in vitro study of the penetration of three bacterial strains into root dentine. oral surg oral med oral pathol. 1993;76:97-103. 12. titley kc, torneck cd, smith dc, adibfar a. adhesion of composite resin to bleached and unbleached bovine enamel. j dent res. 1988;67:1523-8. 13. mcguckin rs, babin jf, meyer bj. alterations in human enamel surface morphology following vital bleaching. j prosthet dent. 1992;68:754-60. 14. teixeira ec, hara at, turssi cp, serra mc. effect of non-vital tooth bleaching on microleakage of coronal access restorations. j oral rehabil. 2003;30:1123-7. 15. elkhatib h, nakajima m, hiraishi n, kitasako y, tagami j, nomura s. surface ph and bond strength of a self-etching primer/adhesive system to intracoronal dentin after application of hydrogen peroxide bleach with sodium perborate. oper dent. 2003;28:591-7. 16. attin t, hannig c, wiegand a, attin r. effect of bleaching on restorative materials and restorations – a systematic review. dent mater. 2004;20:852-61. 17. lai yl, yang ml, lee sy. microhardness and color changes of human dentin with repeated intracoronal bleaching. oper dent. 2003;28:786-92. 18. madison s, walton r. cervical root resorption following bleaching of endodontically treated teeth. j endod. 1990;16:570-4. 19. friedman s. internal bleaching: long-term outcomes and complications. j am dent assoc. 1997;128 suppl:51s-55s. 20. chng hk, yap auj, wattanapayungkul p, sim cp. effect of traditional and alternative intracoronal bleaching agents on microhardness of human dentine. j oral rehabil. 2004;31:811-6. 21. cobankara fk, unlü n, altinöz hc, füsun o. effect of home bleaching agents on the roughness and surface morphology of human enamel and dentine. int dent j. 2004;54:211-8. 22. ferraz cc, gomes bp, zaia aa, teixeira fb, souza-filho fj. in vitro assessment of the antimicrobial action and the mechanical ability of chlorhexidine gel as an endodontic irrigant. j endod. 2001;27:452-5. 23. amaechi bt, higham sm. dental erosion: possible approaches to prevention and control. j dent. 2005;33:243-52. 24. stevens rh, grossman li. evaluation of the antimicrobial potential of calcium hydroxide as an intracanal medicament. j endod. 1983;9:372-4. 25. stuart kg, miller ch, brown ce jr, newton cw. the comparative antimicrobial effect of calcium hydroxide. oral surg oral med oral pathol. 1991;72:101-4. oral sciences n3 original article braz j oral sci. april/june 2010 volume 9, number 2 hyaluronan does not improve bone healing in critical size calvarial defects in rats a radiographic evaluation mariana amade mendes brazão1, beatriz de brito bezerra2, márcio zaffalon casati3, enilson antônio sallum3, antônio wilson sallum4 1dds. graduate student, department of endodontics, são leopoldo mandic dental school 2dds, ms. graduate student, department of prosthodontics and periodontics, division of periodontics, piracicaba dental school, state university of campinas 3 dds, ms, phd. professor, department of prosthodontics and periodontics, division of periodontics, piracicaba dental school, state university of campinas 4 dds, phd, professor, department of prosthodontics and periodontics, division of periodontics, piracicaba dental school, state university of campinas correspondence to: beatriz de brito bezerra departmento de prótese e periodontia, divisão de periodontia fop/unicamp av. limeira, 901 – 13.414-903 caixa postal 52 piracicaba, brazil phone: +55 19 2106-5301 e-mail: beatrizbb@yahoo.com received for publication: march 02, 2010 accepted: june 01, 2010 abstract aim: this study evaluated radiographically the effects of 1% hyaluronan in bone healing using a critical size rat-calvaria defect model. methods: thirty adult male wistar rats were used in this study. two 6-mm-diameter critical-size defects were created and the treatments were randomly distributed as follows: 1) 1% hyaluronan; 2) 1% hyaluronan soak loaded onto an absorbable collagen sponge (acs) carrier; 3) saline; and 4) acs alone. the animals were sacrificed at 4 and 8 weeks when biopsies were collected and radiographs obtained using a direct digital radiograph system and a standardized protocol. a blind examiner evaluated the radiographic density of the images twice and an intraclass correlation was performed to evaluate examiner reproducibility (r2=0.99, p<0.001). comparisons between 4 and 8 weeks of treatment were performed by student’s t test and comparisons between treatments and time by two-way anova at 5% significance level. results: there were no noteworthy differences between 4 or 8 weeks within each treatment group (p>0.05). when treatments were compared no significant differences between groups were found (p>0.05). conclusions: within the limits of this study, it can be concluded that 1% hyaluronan gel alone or its association with a carrier does not improve bone healing. keywords: bone repair, wound healing, hyaluronan. introduction hyaluronic acid (ha) is a high molecular weight polysaccharide ubiquitously distributed in the extracellular space of higher animals; the highest concentrations are found in soft connective tissues. in recent years, ha has been reported to play critical roles in a wide variety of biological events, such as wound healing, chondrogenesis, osteogenesis, the immune response, and migration of rat transformed cells1-3. sasaki and watanabe4 showed that ha is capable of accelerating new bone formation through mesenchymal cell differentiation, in bone created wounds in the animal model. they were able to demonstrate that bone formation had already been induced at day 4 after the application of ha. ha possesses biochemical and physical properties suitable to perform an important role in the early events of braz j oral sci. 9(2):124-127 125 osteogenesis as well as in many other tissues. it is a prominent extracellular matrix component during bone morphogenesis5 and large amounts of ha are present during the transition from mesenchymal cell to cartilage6. in terms of its correlations with wound healing mechanisms and hard tissue development, ha can be thought as a “primer” in cell regeneration. bone maintenance and bone regeneration have become essential concepts in the treatment of periodontal disease, in the healing of tooth extraction sockets and in the utilization of dental implants. currently, much effort has been made not only to maintain and prevent bone loss, but also to augment and regenerate bone around teeth and implants and to rebuild edentulous ridges. these findings suggest that associating ha with devices, such as absorbable collagen sponges and membranes, may help improve bone regeneration. this study evaluated radiographically the effects of 1% hyaluronan in bone healing using a critical size rat-calvaria defect model. material and methods thirty 12-week-old male wistar rats with mean body weight of 300 g. were used in this study. the animals were kept in plastic cages with access to food and water ad libitum. prior to the surgical procedures all animals were allowed to acclimate to the laboratory environment for a 5-day period. the protocol was approved by the state university of campinas institutional animal care and use committee under the protocol #1112-1. general anesthesia was obtained by intramuscular administration of a combination of ketamine chloride (50 mg/kg) and xylazine chloride (15 mg/kg). the surgical site was shaved and washed with iodine. an l-shaped incision was made and a full-thickness flap including periosteum was reflected, exposing the calvaria bone. in each animal, two 6-diameter round defects were created, one on each side of the mid-sagittal suture, with a trephine bur in a dental handpiece under constant irrigation of sterile saline. the trephined bone was removed from the surgical field. each animal was included in two different treatments. four treatments were evaluated: 1) 1% hyaluronan gel (ha); 2) 1% hyaluronan soak-loaded onto an absorbable collagen sponge (acs) carrier; 3) saline; and 4) acs alone. care was taken to have both hyaluronan groups in the same animal so that no carry-over of the gel would occur between defects, avoiding bias to the results. the periosteum and skin were sutured for total coverage with 5-0 nylon suture. this way, 15 animals were assigned for the 4 week time point and another 15 for the 8 week time point. there were 8 animals in treatment groups 1 and 2, and 7 animals treatment groups 3 and 4 at each time point. animals were euthanized by an overdose of anesthesia. radiographs were taken using the direct digital radiograph system sens-a-ray 2000 (regan medical systems ab, sundsvall, sweden) and a dental radiograph unit (dabi atlante, ribeirão preto, brazil) (70kvp, 7ma, 0.1s) (figure 1). the distance between the x-ray source and the digital sensor was 20 mm. the relative bone density was measured as described by ahn et al.7. defect radiodensity was assessed by imagej (version 1.38, national institutes of health, bethesda, md, usa). intra-examiner reproducibility was examined by repeated evaluation of all defect sites (r2=0.99, p<0.001). comparisons between 4 and 8 weeks of treatment were performed by student’s t test and comparisons between treatments and time by two-way anova. the level of significance was set at 5%. fig. 1. radiographs showing calvarial defects in each treatment group. aacs (left defect), saline (right defect) 4 weeks; bacs (left defect), saline (right defect) 8 weeks; cha+acs (left defect), ha (right defect) 4 weeks; dha+acs (left defect), ha (right defect) 8 weeks. results the results of the radiographic analysis are shown in table 1. at 4 weeks post-surgery mean radiodensity (±sd) for saline, acs alone, 1% hyaluronan alone and 1% hyaluronan/acs amounted to 13.36 ± 2.67%, 13.86 ± 2.79%, 14.41 ± 3.50%, and 16.69 ± 3.12%, respectively, with no significant differences among treatments (p>0.05). at 8 weeks post-surgery the corresponding values were 12.79 ± 5.04%, 13.26 ± 2.91%, 11.40 ± 1.94%, and 13.99 ± 3.16%, respectively, and no statistically significant differences among treatments were observed (p>0.05). no statistically significant interaction effects were found between treatment and time (p>0.05). discussion the aim of this study was to evaluate the effects of hyaluronan on bone healing in a critical size calvaria defect braz j oral sci. 9(2):124-127 hyaluronan does not improve bone healing in critical size calvarial defects in rats a radiographic evaluation model. the calvaria defect, compared to other experimental bone defects, is a convenient model for studying bone regenerative materials because of its effective accessibility and the lack of fixation requirements. schmitz and hollinger8 suggested that an 8-mm-diameter defect is suitable to evaluate candidate biomaterials for bone regeneration and constitutes a critical-size defect in the rat. this experimental design has been utilized in previous studies to evaluate the osteoconductive/inductive potential of candidate biologics, biomaterials, and devices for bone reconstruction9-,11. others have defined and successfully used smaller critical-size rat calvarial defects12-13. thus, the geometric and physiological nature of the rat calvaria and the 6 mm trephine defects used in this study appear adequate to investigate the regenerative potential of hyaluronan. radiographic evaluations have been used to evaluate the biologic potential of various devices, as well as osteoconductive/inductive biomaterials and biologics to promote local bone formation in rat calvarial defects. briefly, barrier membranes for guided bone regeneration12-13, autograft bone, and growth and differentiation factors, including purified protein constructs9-11 or combinations thereof, have been evaluated. the radiographic observations in these studies demonstrated limited radiographic bone fill in the control sites. this is in agreement with the findings in this study where complete defect closure was not observed. the results demonstrate that radiographic evaluations of bone formation are associated with significant weaknesses, and, as such, poorly represent actual healing events. radiographic evaluations have been used to evaluate the effect of various treatment concepts on bone formation. it appears from the present study and from other investigations7,14 that radiographic analyses should be viewed with caution, and that observations of bone healing in experimental models such as herein or in more complex models should be confirmed using histologic observations13-16. a histological evaluation will be necessary to verify the real effects of ha on bone healing in this experimental model. a greater evaluation period may also be necessary in this model to observe significant differences when conducting a radiographic evaluation, however, some of the studies which use this model last 8 weeks and they show good results. the only difference is that they perform a qualitative evaluation1718 or use more sensitive radiograph methods such as micro-ct scans4,19. pryor et al.14 pointed out that bone healing in animal models aiming at treatment recommendations for clinical application must not only be determined based on radiographic analysis, but should also be confirmed histologically. previous studies have also attempted to establish ha as an alternative in bone reconstruction. ha was used as raw material for hydrogels, sponges and polymers. in order to increase the bone regenerative effects growth factors, mesenchymal stem cells and demineralized bone matrix have been added to this material20-21. due to the natural presence of ha as major part of the extracellular matrix of many tissues this seemed to be a promising approach. aslan et al.22 examined the effect of autologous bone graft versus autologous bone graft and ha in a defect model in rabbit tibiae. they documented that ha needs an osteoconductive scaffold to be effective because the addition of ha leads to higher scores for bone formation in each time period of the study. wiedmann-al-ahmad et al.23 documented one of the highest proliferation rates for ha when compared to 16 different biomaterials. kim et al.21 showed that a hyaluronic-based hydrogel is suitable as carrier for human mesenchymal stem cells and bmp-2. another explanation for the present results may be that ha did not have the optimal molecular weight. different authors examined the effects of various molecular weights on osteogenesis. pilloni and bernard24 showed that low molecular weight ha accelerated osteogenesis in vitro in a bone marrow ablation model in rat. in contrast to this study, sasaki and watanabe4 described that high molecular ha increases bone formation after bone marrow ablation in comparison to untreated controls. the results of the studies about the optimal molecular weight are therefore inconsistent at best. histomorphometric analyses of the specimens are underway to better understand the healing dynamics of these defects as a result of each treatment evaluated. within the limits of this study, it may be concluded that 1% hyaluronan gel alone or its association with a carrier (acs) was not able to improve bone healing in this experimental model. acknowledgements dr. brazão received financial support by the national council for scientific and technological development (cnpq), brasilia, df, brazil. references 1. fraser jr, laurent tc, laurent ub. hyaluronan: its nature, distribution, functions and turnover. j intern med. 1997; 242: 27-33. 2. jiang d, liang j, noble p. hyaluronan in tissue injury and repair. annu rev cell dev biol. 2007; 23: 435-61. 3. laurent tc, fraser jr. hyaluronan. faseb j. 1992; 6: 2397-404. 4. sasaki t, watanabe c. stimulation of osteoinduction in bone wound healing by high-molecular hyaluronic acid. bone. 1995; 16: 9-15. 5. toole bp, gross j. the extracellular matrix of the regenerating newt limb: synthesis and removal of hyaluronate prior to differentiation. develop biol. 1979; 25: 57-77. 6. handley cj, lowther da. inhibition of proteoglycan biosynthesis by hyaluronic acid in chondrocytes in culture. biochim biophys acta. 1976; 444: 69-74. 4 weeks 8 weeks saline (n=7) 13.36±2.67aa 12.79±5.04 aa acs (n=7) 13.86±2.79 aa 13.26±2.91 aa ha (n=8) 14.41±3.50 aa 11.40±1.94 aa ha+acs (n=8) 16.69±3.12 aa 13.99±3.16 aa table 1. relative bone density (mean ± sd) of treatment groups according to time. distinct uppercase letters in rows indicate statistically significant differences among treatments (two-way anova, p<0.05). distinct lowercase letters in columns indicate statistically significant differences between times (student’s t test; p<0.05). 126 braz j oral sci. 9(2):124-127 hyaluronan does not improve bone healing in critical size calvarial defects in rats a radiographic evaluation 7. ahs sh, kim cs, suk hj, lee yj, choi sh, chai jk et al. effect of recombinant human bone morphogenetic protein-4 with carriers in rat calvarial defects. j periodontol. 2003; 74: 787-97. 8. schmitz jp, holinger jo. the critical size defect as an experimental model for craniomandibulofacial nonunions. clin orthop. 1986; 205: 298-307. 9. dahlin c, alberius p, linde a. osteopromotion for cranioplasty. an experimental study in rats using a membrane technique. j neurosurg. 1991; 74: 487-91. 10. marden lj, hollinger jo, chaudhari a, turek t, schaub rg, ron e. recombinant human bone morphogenetic protein-2 is superior to demineralized bone matrix in repairing craniotomy defects in rats. j biomed mater res. 1994; 28: 1127-38. 11. winn sr, schmitt jm, buck d, hu y, grainger d, hollinger jo. tissueengineered bone biomimetic to regenerate calvarial critical-sized defects in athymic rats. j biomed mater res. 1999; 45: 414-21. 12. dupoirieux l, pourquier d, picot mc, neves m. comparative study of three different membranes for guided experimental study in rabbits. j biomater appl. 2006; 20: 209-20. 13. verna c, bosch c, dalstra m, wikesjö um, trombelli l. healing patterns in calvarial bone defects following guided bone regeneration in rats: a micro-ct scan analysis. j clin periodontol. 2002; 29: 865–70. 14. pryor me, susin c, wikesjö um. validity of radiographic evaluations of bone formation in a rat calvaria osteotomy defect model. j clin periodontol. 2006; 33: 455-60. 15. cacciafesta v, dalstra m, bosch c, melsen b, andreassen tt. growth hormone treatment promotes guided bone regeneration in rat calvarial defects. eur j orthod. 2001; 23: 733–40. 16. kim e-s, park e-j, choung p-h. platelet concentration and its effect on bone formation in calvarial defects: an experimental study in rabbits. j prost dent. 2001; 86: 428-33. 17. hyun s, han d, choi s, chai j, cho k, kim c et al. effect of recombinant human bone morphogenetic protein-2, -4, and -7 on bone formation in rat calvarial defects. j periodontol. 2005; 76: 1667-74. 18. pryor me, yang j, polimeni g, koo kt, hartman mj, gross h et al. analysis of rat calvaria defects implanted with a platelet-rich plasma preparation: radiographic observations. j periodontol. 2005; 76: 1287-92. 19. wong rwk, rabie abm. statin collagen grafts used to repair defects in the parietal bone of rabbits. br j oral maxillofac surg. 2003; 41: 244-8. 20. aebli n, stich h, schawalder p, theis j-c, krebs j. effects of bone morphogenetic protein-2 and hyaluronic acid on the osseointegration of hydroxyapatite-coated implants: an experimental study in sheep. j biomed mater res. 2005; 73a: 295-302. 21. wiedmann-al-ahmad m, gutwald r, gellrich nc, hübner u, schmelzeisen r. search for ideal biomaterials to cultivate human osteoblast-like cells for reconstructive surgery. j mater sci mater med. 2005; 16: 57-66. 22. pilloni a, bernard gw. the effect of hyaluronan on mouse intramembranous osteogenesis in vitro. cell tissue res. 1998; 294: 323-33. 23. kim j, kim is, cho th, lee kb, hwang sj, tae g et al. bone regeneration using hyaluronic acid-based hydrogel with bone morphogenic protein-2 and human mesenchymal stem cells. biomaterials. 2007; 28: 1830-7. 24. aslan m, simsek g, dayi e. the effect of hyaluronic acid-supplemented bone graft in bone healing: bone regeneration of rat cranial defects. int j oral maxillofac surg. 2001; 30: 58-62. 127 braz j oral sci. 9(2):124-127 hyaluronan does not improve bone healing in critical size calvarial defects in rats a radiographic evaluation oral sciences n3 braz j oral sci. 10(1):60-64 original article braz j oral sci. january | march 2011 volume 10, number 1 the operator as a factor of success in art restorations carolina da franca1, viviane colares2, evert van amerongen3 1 phd, postdoctoral student, faculty of dentistry, university of pernambuco, brazil 2 phd, associate professor of paediatric dentistry, faculty of dentistry, university of pernambuco, brazil 3 dds, phd, professor of paediatric dentistry, academic centre for dentistry in amsterdam, the netherlands correspondence to: carolina da franca rua jacobina, 45/2102, graças recife – pe – brazil phone: +55 81 32429708 / +55 81 96066850 e-mail: carolinafbandeira@yahoo.com.br received for publication: november 26, 2010 accepted: march 28, 2011 abstract aim: to evaluate the operator as a factor of success in atraumatic restorative treatment (art) restorations. methods: this was a clinical intervention study. the sample consisted of 271 glassionomer cement (ketac molar, 3m espe) restorations placed in 246 children aged 5 to 9 years attending public schools in recife, brazil. the operators were two senior undergraduate dental students and one dentist, classified as ‘inexperienced’ and ‘experienced’, respectively. two groups of children were formed: one treated by the undergraduate students and the other treated by the dentist. the evaluation of the restorations took place at 1, 4 and 12 months by three senior undergraduate dental students. results: comparing the operators, a significant difference was found in class ii cavities filled by students, who had a lower rate of success. there were no differences between the dentist and undergraduate dental students for class i cavities. conclusions: the operator’s experience makes a difference in the success rate of more complex art restorations when an experienced operator receives the same training as an inexperienced operator. keywords: dental atraumatic restorative treatment, permanent dental restoration, longevity, primary dentition, operators. introduction atraumatic restorative treatment (art) is an approach that was initially developed to provide preventive and restorative care to people in low-income countries. however, in the last few years, the use of art has spread to countries such as the usa, england, scotland and the netherlands1. this technique has the advantages of reducing pain and fear2 and being more cost-effective than the traditional approach using amalgam3. however, the technique is still being studied aiming at increasing the survival of such restorations, especially on multi-surfaces. some reasons for the less than satisfactory results are related to the operator. in the literature, factors such as material4, type of cavity5-6 and cavity size7 have been associated with the longevity of art restorations. in this paper, these factors associated with the operator will be discussed. it is important to bear in mind that, in the literature, the word ‘operator’ indistinctly refers to a dentist, a dental student or to any other oral health worker, each presenting different rates of successful outcomes. some authors cited non-compliance with the protocol for art8, the professional qualification of the operator8, an inappropriate indication for art9, the number of braz j oral sci. 10(1):60-64 operators10, and the lack of a specific training course in art1113, as well as the experience of operator and assistant7, as factors that influence their performance. the training of operators (dentists) in art has led to higher survival rates for these restorations14-15 furthermore, in another study, when trained operators were dental students, longevity was lower16. the operator’s experience is another important factor to be considered in a deeper manner. a dentist has a wider experience of clinical interventions than a student. despite a greater clinical experience, however, the dentist might not have had specific experience with art restorations, or with the working conditions at schools. according to kemoli et al.7 (2009), experienced operators and assistants in art showed the best rate of survival for proximal art restorations. considering dentition, some studies on permanent teeth showed the best results when the operator was a dentist and much less satisfactory results when the operator was a dental student (table 1). in the studies with primary teeth, although the operator was always a dentist, the success rate varied greatly for both class ii (from 12.2 to 83.3%) and class i (from 43.4 to 93.7%). the aim of the present study was to evaluate the operator as a factor of success in art restorations, especially considering clinical experience and specific training in art restorations. material and methods this study was carried out in public schools in recife, capital of pernambuco state, brazil. children aged 5 to 9 years presenting carious lesions involving one or two surfaces in primary molars were included in this study. the examination was performed using a mouth mirror and an explorer under natural light. the simplified world health organization (who) form was used to record information. dental caries was recorded according to who criteria22. the indication of art was considered in cases of: (i) class i or class ii cavities in primary molars, accessible by manual instruments; (ii) absence of abscess or fistula near the carious tooth and no pulp exposure expected if caries was removed. the operators were two senior undergraduate dental students and one dentist. in this study, the dentist was a practitioner with 5 years of experience. the students were senior undergraduate dental students in their final year. the dentist was an experienced and trained practitioner with over two hundred art restorations performed, while the students had training with over thirty art restorations but lacked experience with children and with clinical practice in general. the training consisted of getting informed about the art technique and protocol, watching practical demonstrations of the technique and finally, applying this information in a short, supervised practice. after training, the trainee was able to perform restorations according to standard procedures. although the training was essential for performing the art protocol, it was limited to specific knowledge, while experience provided skills to manage the patient as a whole for both art restorations and other clinical interventions in the school environment. two groups of children selected by random lottery were treated. one group was treated by the dental students and the other by the dentist. treatment was carried out inside classrooms at the schools selected to take part in the trial. patients were positioned on a table. after using cotton rolls to isolate the tooth, cavity opening was performed with a dental hatchet, the soft carious tissue was removed with an excavator, and the cavity and adjacent pits and fissures were filled with a glass-ionomer cement (ketac molar/3m esp/ germany). caries removal followed the recommendations of frencken et al.23, which preserved sound tooth tissue. caries removal after cavity preparation was checked by conventional optical and tactile criteria. the conditioning of the cavity (using the liquid of glass-ionomer cement-ketac molar/3m espe) and adjacent pits and fissures with cotton wool pellets preceded the placement of the glass ionomer. the cavities were cleaned with three moist cotton wool pellets and dried with three other cotton wool pellets before and after the conditioning. the chair-side assistant, a dental student, mixed the glass-ionomer cement according to the manufacturer’s instructions. the dental students who participated in this study had also been trained to mix the glass ionomer, but they were not experienced assistants. class ii cavities were filled after the placement of plastic bands and wedges. no local anesthesia was used. excess material was removed by honkala et al., 2003 14 dentists 22 months 93.7 83.3 taifour et al., 2002 15 dentists 36 months 86.1 48.7 frencken et al., 2006 17 dentists 6.3 years 68.9 gemert-schriks et al., 2007 18 dentists 36 months 43.4 12.2 wang et al., 2004 16 students 36 months 21.0 cefaly et al., 2005 19 dentists 6 months 100.0 96.0 delgado-angulo et al., 2005 20 students 9 months 65.5 lo et al., 2007 21 dentists 6 years 76.0 primary permanent dentition author/year operators follow-up duration class i class ii (%) (%) success table 1: overview of some studies in according to qualification of operator and survival of art. 61 the operator as a factor of success in art restorations 62 braz j oral sci. 10(1):60-64 means of an applier/caver instrument, and the restoration was coated with a layer of petroleum jelly. the evaluation of the restorations took place after 1, 4 and 12 months, according to previously defined criteria by gemert-schriks et al.18 (table 2). three trained and calibrated senior undergraduate dental students evaluated the restorations clinically using a cpitn probe, a mouth mirror and a headlamp. restorations scored code 0 and 10 were considered successful, those scored codes 11-40 were considered failures, while restorations scored codes 50-90 were excluded from the analysis. a kappa test was performed for intraand inter-examiner evaluations. the kappa test result was higher than 0.8. the evaluators were involved neither in the planning of the study nor in its execution. ethical approval was obtained from the research ethics committee of the university of pernambuco, brazil (protocol no. 190/08). the subjects were included in this study only after the parent or guardian had signed the respective informed consent form. results the sample consisted of 271 restorations placed in 246 children of both genders aged 5 to 9 years, who were attending public schools in recife, brazil. the restorations were divided into two groups, 118 class i and 153 class ii restorations. the loss in the sample was around 5.0%. there were no statistically significant differences (p>0.05) between class i and ii restorations performed by the dentist at any of the evaluation periods (1, 4 and 12 months). however, there was a lower success rate in the class ii restorations performed by the students, revealing a statistically significant difference (p<0.05) (table 3). comparing the operators, a significant difference (p<0.05) was found after 4 and 12 months in class ii cavities filled by students, who had a lower rate of success. there were no significant differences (p>0.05) between the performance of the dentist and undergraduate dental students for class i restorations (table 4). type of cavity operator class i class ii p value % (n) % (n) 1 month dentist 88.0 (22) 73.2 (41) p (1) = 0.139 student 94.6 (88) 70.1 (68) p (1) < 0.001* 4 months dentist 85.7 (18) 70.8 (34) p (1) = 0.184 student 77.1 (64) 38.1 (32) p (1) < 0.001* 12 months dentist 66.6 (10) 48.3 (14) p (1) = 0.246 student 51.9 (42) 14.1 (11) p (1) < 0.001* table 3: distribution of success rate according to the type of cavity at 1, 4 and 12 months (*): significant association at 5%. (1): using the pearson chi-square test. operator type of cavity dentist students p value % (n) % (n) 1 month class i 88.0 (22) 95.2 (88) p (1) = 0.364 class ii 73.2 (41) 70.1 (68) p (2) = 0.682 4 months class i 85.7 (18) 77.1 (64) p (1) = 0.553 class ii 70.8 (34) 38.1 (32) p (2) < 0.001* 12 months class i 66.6 (10) 51.9 (42) p (2) = 0.290 class ii 48.3 (14) 14.1 (11) p (2) < 0.001* (*): significant association at 5%. (1): using fisher´s exact test. (2): using the pearson chi-square test. table 4: distribution of success rate according to the operator at 1, 4 and 12 months code evaluation characteristics 00 restoration present, correct. 10 restoration present, slight marginal defect/wear of surface (<0.5 mm). no repair needed. 11 restoration present, gross marginal defect/wear of surface (>0.5 mm). repair needed. 12 restoration present, underfilled (>0.5 mm). repair needed. 13 restoration present, overfilled (>0.5 mm). repair needed. 20 secondary caries, discoloration in depth, surface hard and intact, caries within dentin. repair needed. 21 secondary caries, surface defect, caries within dentin. repair needed. 30 restoration not present, bulk fracture, moving or partially lost. repair needed. 40 inflammation of the pulp; signs of dentogenic infection (abscesses, fistulae, pain complaints). restoration might still be in situ. extraction needed. 50 tooth not present because of extraction. 60 tooth not present because of shedding. 70 tooth not present because of extraction or shedding. 90 patient not present. 91 patient transferred. table 2: evaluation criteria for the art restorations 18 discussion this study considered experience and specific training in art restorations as factors related to operator that the operator as a factor of success in art restorations 63 braz j oral sci. 10(1):60-64 influence the longevity of these restorations. the operator’s experience in dental clinical procedures can give the best results in the survival of art restorations10. the operator’s professional skills, experience and training in the art approach are decisive for treatment success because the clinical procedures will take place at schools, with the children positioned on ordinary tables, under conditions very different from those of the dental office setting. although the professional’s general dental experience has formerly been considered a positive factor in the survival of art restorations24, having specific art training has subsequently been found as more efficient for the longevity of art restorations than the dental professional’s experience25. with regard to the type of restoration and the operator, the results of the class ii restorations showed a significantly lower success rate than those of class i, which is in agreement with a number of other studies4,21,26,28. however, in the present study, there was no difference between class i and ii restorations performed by the dentist. class ii restorations performed by the students showed the worst results (p<0.001). this operator effect has also been observed for two-surface restorations by gemert-schriks et al.18, who reported that more complex restorations need more experienced operators. the suggestion is that inexperienced operators require further practice after they have completed their training. according to kemoli and amerongen27, prior to the operative stage of the study, any operator who had performed 50 art restorations (half being class ii and the rest of any other class) would be classified as ‘experienced’, and any operator who, after being trained, had done fewer than 10 but more than 5 restorations of any class would be classiûed as ‘inexperienced’ in the art technique. in the literature, the experience and training received by operators are not always clearly described, making these factors difficult to analyze. furthermore, there are other number of factors related to the operator that are not clear in most studies such as the operator’s experience with children, with the school environment or with the lack of infrastructure of the dental office. specific training and experience can be summed up as a combination of factors that associate the operator with success in art, such as those cited by frencken et al.24: the correct indication for art, the appropriate mixing of the glass ionomer cement, and the judicious insertion of the material deep into the cavity. it seems that training and experience are complementary; while training helps the operator monitoring the protocol adopted, experience provides the clinical skills to facilitate the implementation of procedures, thus increasing the chances of success. it may be concluded that clinical experience and specific training in art restorations for operators remains a goal that has still to be achieved. the simple recognition that both factors are fundamental for achieving higher rates of survival of art restorations represents per se a major step towards finding a solution to the problem of survival in art restorations. acknowledgements the authors are very grateful to the acta students who participated in this study, some of them as operators and others as evaluators. we also acknowledge the support given by 3m espe in providing the materials used in the study, and capes – brazilian ministry of health’s federal agency for support and evaluation of graduate education – for granting the phd fellowship. references 1. topaloglu-ak a, eden e, frencken je, oncag o. two years survival rate of class ii composite resin restorations prepared by art with and without a chemomechanical caries removal gel in primary molars. clin oral invest. 2009; 13: 325–32. 2. carvalho ts, ribeiro tr, bonecker m, pinheiro ecm, colares v. the atraumatic restorative treatment approach: an “atraumatic” alternative. med oral patol oral cir bucal. 2009; 14: 668-73. 3. seale ns, casamassimo ps. access to dental care for children in the united states. a survey of general practitioners. j am dent assoc. 2003; 134: 1630-40. 4. ercan e, dülgergil t, soyman m, dalli m, yildirim i. a field-trial of two restorative materials used with atraumatic restorative treatment in rural turkey: 24-month results. j appl oral sci. 2009; 17: 307-14. 5. koenraads h, van der kroon g, frencken je. compressive strength of two newly developed glass-ionomer materials for use with the atraumatic restorative treatment (art) approach in class ii cavities. dent mater. 2009; 25: 551-6. 6. van’t hof ma, frencken je, van palenstein helderman wh, holmgren cj. the art approach for managing dental caries: a meta-analysis. int dent j. 2006; 56: 345-51. 7. kemoli am, van amerongen we, opinya g. influence of the experience of operator and assistant on the survival rate of proximal art restorations: two-year results. eur arch paediatr dent. 2009; 10: 227-32. 8. frencken je, holmgren cj. how effective is art in the management of dental caries? community dent oral epidemiol. 1999; 27: 423-30. 9. mickenautsch s, grossman e. atraumatic restorative treatment (art) – factors affecting success. j appl oral sci. 2006; 14(sp. issue): 34-6. 10. cefaly dfg, tapety cmc, mondelli rfl, lauris jrp, phantumvanit p, navarro mfl. three-year evaluation of the art approach in class iii and v restorations in permanent anterior teeth. caries res. 2006; 40: 389-92. 11. holmgren cj, frencken je. painting the future for art. community dent oral epidemiol. 1999; 27: 449-53. 12. bresciani e. clinical trials with atraumatic restorative treatment (art) in deciduous and permanent teeth. j appl oral sci. 2006; 14(sp. issue): 14-9. 13. barata tje, bresciani e, mattos mcr, lauris jrp, ericson d, navarro mfl. comparison of two minimally invasive methods on the longevity of glass ionomer cement restorations: short-term results of a pilot study. j appl oral sci. 2008; 16: 155-60. 14. honkala e, behbehani j, ibricevic h, kerosuo e, al-jame g. the atraumatic restorative treatment (art) approach to restoring primary teeth in a standard dental clinic. int j paediatr dent. 2003; 13: 172-9. 15. taifour d, frencken je, beiruti n, hof ma, truin gj. effectiveness of glass-ionomer (art) and amalgam restorations in the deciduous dentition: results after 3 years. caries res. 2002; 36: 437-44. 16. wang l, lopes lg, bresciani e, lauris jr, mondelli rf, navarro mf. evaluation of class i art restorations in brazilian schoolchildren: three year results. spec care dent. 2004; 24: 28-33. 17. frencken je, taifour d, hof ma. survival of art and amalgam restorations in permanent teeth of children after 6.3 years. j dent res. 2006; 85: 622-6. the operator as a factor of success in art restorations 64 braz j oral sci. 10(1):60-64 18. gemert-schriks mcm, amerongen we, cate jm, aartman iha. threeyear survival of singleand two-surface art restorations in a high-caries child population. clin oral invest. 2007; 11: 337-43. 19. cefaly dfg, barata the, tapety cmc, bresciani e, navarro mfl. clinical evaluation of multisurface art restorations. j appl oral sci. 2005; 13: 15-9. 20. delgado-angulo ek, ortiz eb, sánchez-borjas pc. análisis de supervivencia de sellantes y restauraciones art realizados por estudiantes de pregrado. rev estomatol herediana. 2005; 15: 119-23. 21. lo ecm, holmgren cj, hu d, van palenstein helderman w. six-year follow up of atraumatic restorative treatment restorations placed in chinese school children. community dent oral epidemiol. 2007; 35: 387-92. 22. world health organization. oral health surveys: basic methods. geneva: world health organization; 1997. 23. frencken je, pilot t, songpaisan y, phantumvanit p. atraumatic restorative treatment (art): rationale, technique, and development. j public health dent. 1996; 56: 135-40. 24. frencken je, makoni f, sithole wd. art restorations and glass ionomer sealants in zimbabwe: survival after 3 years. community dent oral epidemiol. 1998; 26: 372-81. 25. frencken je, van’t hof ma, van amerongen we, holmgren cj. effectiveness of single-surface art restorations in the permanent dentition: a meta-analysis. j dent res. 2004; 83: 120-3. 26. ersin nk, candan u, aykut a, önçag o, eronat c, kose t. when primary molars lesions are cavitated into dentin, glass ionomer has similar 2-year survival to resin composite as a restorative material when using the “art” technique. j evid base dent pract. 2008; 8: 24-5. 27. kemoli am, amerongen we. inûuence of the cavity-size on the survival rate of proximal art restorations in primary molars. int j paediatr dent. 2009; 19: 423-30. 28. ersin nk, candan u, aykut a, önçag o, eronat c, kose t. a clinical evaluation of resin-based composite and glass ionomer cement restorations placed in primary teeth using the art approach. j am dent assoc. 2006; 137: 1529-36. the operator as a factor of success in art restorations oral sciences n3 original article braz j oral sci. july/september 2010 volume 9, number 3 dimensional stability of distances between teeth in complete dentures comparing microwave polymerization and conventional cycles wagner araújo de negreiros1; rafael leonardo xediek consani2; marcelo ferraz mesquita2 1phd, professor, department of prosthodontics, school of dentistry, university of fortaleza, brazil 2phd, professor, department of prosthodontics, piracicaba dental school, university of campinas, brazil correspondence to: wagner araujo de negreiros rua jovino guedes 60, apto 1104, aldeota, fortaleza, ce, brasil. cep: 60140-130 phone: +55 (85) 3067 9365 / 9162 9894 e-mail: wnegreiros@fop.unicamp.br received for publication: may 07, 2010 accepted: august 25, 2010 abstract aim: this study investigated the tooth movement of complete dentures processed by microwave activation and conventional processing method in water bath. methods: twenty maxillary complete dentures were fabricated and randomly assigned to 4 groups (n=5): group i: classico conventional heat-curing acrylic resin processed by microwave polymerization; group ii: classico resin processed in water bath at 74°c for 9 h (control-group); group iii: qc-20 fast heat-curing acrylic resin processed in boiling water for 20 min; group iv: onda-cryl microwave acrylic resin processed at the same conditions of group 1. metallic referential pins were placed on the incisal border of the central incisors, buccal cusp of the first premolars, and the mesiobuccal cusp of the second molars. transversal and anteroposterior distances were measured before and after the complete dentures processing with a linear optical microscope (olympus optical co., tokyo, japan) accurate to 0.0005 mm. data were subjected to anova and tukey’s test at 5% significance levels. results: inside each group, dentures showed some tooth movement but without statistical difference before and after the polymerization. conclusions: dentures processed by microwave energy presented similar performance to those subjected to conventional cycles in water bath for most of distances evaluated. keywords: complete denture, tooth movement, artificial tooth. introduction it is clinically important that dentures have accurate occlusal contacts to guarantee normal function. the evaluation of tooth displacement has been important in seeking a more stable occlusal pattern, retention, and functional quality of complete dentures1. studies have shown that typically, the magnitude of dimensional changes is not too large2-3, and mean changes of -0.1% to -0.4% have been reported as having no significant influence on the serviceability of dentures4. the performance of different polymerization cycles, including the microwave technique, was analyzed in some investigations considering the processing alterations5-6. after the nishi’s publication in 19687, microwave processing has been appointed as fast8, clean9 method that promotes an ideal fitting of the denture to the cast10. the use of conventional heat-curing acrylic resins instead of microwave-activated ones and that influence on the dimensional changes also remain unresolved11-12. thus, this study evaluated the magnitude of the linear tooth movement in complete dentures submitted to conventional and microwave braz j oral sci. 9(3):384-387 385 polymerization cycles using different resin types. material and methods a silicone mold (elite double; zhermack, rovigo, italy) was obtained from a metal master edentulous maxillary die without irregularities in the alveolar ridge walls. twenty identical casts were poured from this mold with type iii dental stone (herodent soli-rock; vigodent, rj, brazil) and the water/powder ratio was 30:100. a uniform denture base was made with a 2-mm-thick plate wax (epoxiglass; epoxiglass chemical products, diadema, sp, brazil) measured with a caliper. the height of the occlusion wax rim was 20mm in the buccal sulcus of the cast and 10 mm in the second molar area. the maxillary stone cast was mounted in a mondial 4000 semi-adjustable articulator (bio-art dental products, são carlos, sp, brazil) with the wax rim interocclusal relation according to the mandibular metal cast teeth, with the following references: intercondylar distance in m, bennett angle at 15 degrees, and condylar guide at 30 degrees. the arrangement of the left anterior teeth started with the carved wax rim serving as a guide to the positions of the central and lateral incisors and canines. the same procedure was used on the right side. the posterior teeth were arranged starting with the first premolar up to the second molar. the same procedure was used in the right arch. the arrangement of the teeth for the interocclusal relationship was anterior vertical overlap and posterior in angle class i. after finishing the tooth arrangement of the first denture, a silicone (zetalabor, zhermack, rovigo, italy) matrix was made fitted to all buccal aspects of the denture, comprising the buccal and incisal surfaces of anterior teeth and buccal and occlusal surfaces of posterior teeth. the purpose of this matrix was to guide the standardized arrangement of the teeth in all the samples. metallic reference pins (cadena, coats textil ltda., sp, brazil) were placed with cianoacrylate adhesive (super bonder; loctite, são paulo, sp, brazil) at the incisal border of the central incisors, buccal cusp of the first premolars, and mesiobuccal cusp of the second molars to serve as reference to quantify tooth movement (figure 1). therefore, the following linear distances were considered: rpm-lpm (right premolar to left premolar), rm-lm (right molar to left molar), ri-rm (right incisor to right molar) and li-lm (left incisor to left molar). the distances were measured with a stm microscope (olympus optical co., tokyo, japan), with an accuracy of 0.0005 mm. the casts and wax patterns of the groups i (cla-micro) and iv (onda-cryl) were flasked in the lower part of a glass fiber flask (bmf1, classico dental products, são paulo, sp, brazil) for microwave polymerization with type ii dental stone (pasom; dental products, sp, brazil), and the sets of groups ii (cla-water) and iii (qc-20) were flasked in the lower part of a traditional brass flask (safrany; j safrany dental metallurgy, são paulo, sp, brazil). petroleum jelly (labsynth; labsynth chemical products, diadema, sp, brazil) was used as a separating medium between the plaster in the lower part of the flask and the type iii dental stone used in the upper portion. after 1 hour the flasks were placed in boiling water to soften the baseplate wax. the flasks were separated, the wax removed, and the stone was cleaned with boiling water and liquid detergent (ype; chemical amparo, sp, brazil). two coats of sodium alginate (isolak; classico dental products, são paulo, sp, brazil) were used as a mold separator. the resins were prepared in accordance with the manufacturer’s directions and each sample was packed in accordance with the group assignments: group i (clamicro): classico heat-curing acrylic resin (classico dental products, são paulo, sp, brazil) polymerization for 3 min at 35%, 4 min at 0%, and 3 min at 65% power of the 900 w microwave oven (continental domestic products, manaus, am, brazil); group ii (cla-water): classico heat-curing acrylic resin (classico dental products) polymerization in water bath at 74°c for 9 h (control-group) in the thermocuring unit (thermotron dental products, piracicaba, sp, brazil); group iii (qc-20): qc-20 fast heat-curing acrylic resin (dentsply, dental products, rj, brazil) processed in boiling water for 20 min; group iv (onda-cryl): ondacryl microwave acrylic resin (classico dental products) processed at same conditions of group i. after polymerization, the flasks of the groups i and vi were removed from the microwave oven, and the ones of the groups ii and iii were slowly cooled in the water bath, removed from the thermo-curing unit, and all were bench stored for 3 h. after this period, the dentures were deflasked, polished, and the transverse and anteroposterior distances were measured again. the data collected were subjected to anova and tukey’s test at 5% level of significance. results considering all distances evaluated, some tooth movement occurred but without statistical difference at 5% fig. 1 metallic reference pins. dimensional stability of distances between teeth in complete dentures comparing microwave polymerization and conventional cycles braz j oral sci. 9(3):384-387 386 treatment factor polymerization cycle cla-micro cla-water qc-20 onda-cryl before curing 39.07(0.55) aa 38.70(0.82) aa 38.65(0.59) aa 38.24(0.78) aa after curing 39.01(0.50) aa 38.66(0.87) aba 38.57(0.58) aba 38.10(0.79) ba table 1. means and standard deviations (mm) of tooth movement for the rp-lp distance considering the polymerization cycle and treatment factor. means followed by the same uppercase letters in each row and the same lowercase letters in each column are not significantly different (5%) treatment factor polymerization cycle cla-micro cla-water qc-20 ondacryl before curing 51.23(0.85)aa 51.50 (0.78) aa 50.97 (0.50) aa 51.17(0.59) aa after curing 51.16(0.72) aa 51.39(0.81) aa 50.88(0.46) aa 50.94(0.58) aa table 2. means and standard deviations (mm) of tooth movement for the rm-lm distance considering the polymerization cycle and treatment factor. means followed by the same uppercase letters in each row and the same lowercase letters in each column are not significantly different (5%) treatment factor polymerization cycle cla-micro cla-water qc-20 ondacryl before curing 41.79(0.61) aa 41.70(0.42) aa 41.55(0.26) aa 41.68(0.51) aa after curing 41.63(0.53) aa 41.67(0.45) aa 41.49(0.18) aa 41.51(0.55) aa table 3. means and standard deviations (mm) of tooth movement for the ri-rm distance considering the polymerization cycle and treatment factor. means followed by the same uppercase letters in each row and the same lowercase letters in each column are not significantly different (5%) treatment factor polymerization cycle cla-micro cla-water qc-20 ondacryl before curing 40.62(0.72) aa 40.70(0.53) aa 40.67(0.31) aa 40.68(0.54) aa after curing 40.52(0.64) aa 40.56(0.47) aa 40.53(0.31) aa 40.61(0.49) aa table 4. means and standard deviations (mm) of tooth movement for the li-lm distance considering the polymerization cycle and treatment factor. means followed by the same uppercase letters in each row and the same lowercase letters in each column are not significantly different (5%) of significance before and after the polymerization (tables 1-4). for the rp-lp distance, the magnitude of tooth movement was greater for cla-micro (group i) compared to all other groups after the polymerization (table 1). the rm-lm, ri-rm, and li-lm distances had no significant changes after polymerizing the dentures (tables 2-4). discussion dimensional changes may modify the planned vertical occlusion dimension, and cause traumas in mucosa and bone loss. careful measures have been taken to overcome some inaccuracies such as base distortion and displacement of artificial teeth, factors that lead to loss of stability and retention, and necessity of more difficult occlusal adjustments1. in the present study, tooth movement occurred in all interactions. however, statistically significant difference (p<0.05) was found only in the rp-lp distance after the polymerization, with higher value for group i (table 1). three explanations for this phenomenon may be considered: 1) the denture bases were made with 2 mm thickness and, according to previous studies13-14, this fact may reduce dimensional change in the base; 2) resin polymerization shrinkage may be, in part, compensated by the thermal expansion of the own resin during the processing15; and 3) the restrictive effect of investing plaster on keeping the tooth position when the resin induces polymerization and cooling stresses16. it is also possible that during or after the procedures a great amount of internal stresses was relieved before definitive closure of the flask. therefore the remaining internal tensions were not able to promote statistically significant tooth movement after deflasking process. previous studies reported that the greater degree of base dimensional changes was observed in the denture posterior palatal seal17-19, with changes of posterior tooth position and vertical dimension19-20. only the rp-lp distance presented significant dimensional change and the explanation, as dimensional stability of distances between teeth in complete dentures comparing microwave polymerization and conventional cycles braz j oral sci. 9(3):384-387 suggested elsewhere21, may be related to the complexity of variables that characterize the acrylic resin processing. it is possible that other factors generated a great deal of stress in this region, with displacement of artificial teeth. the long polymerization cycle in water bath was reported as preferable because less dimensional change occurs in the base22. on the order hand, the fast cycle is characterized by the occurrence of incomplete resin polymerization, with temperature peaks and a great deal of exothermic heat23. long and fast cycles in water bath studied in the present study were alike in relation to the linear dimensional stability. to explain this phenomenon it is necessary to consider the complexity of all factors that interact during the complete denture processing24. the dimensional changes expected in the final of the fast cycle were probably insignificant, or minimized by the action of other processing variables. most of distances presented regular dimensional stability after processing, and the microwave polymerization groups had similar behavior than the conventional cycles in water bath ones. last studies advocated in favor to microwave polymerization highlighting the manipulation pattern of resins, its clinical use and dimensional accuracy25-26. other studies reported that basis cured by microwave energy presented the same or a better fit on the cast than others conventionally polymerized27-28. it is possible that the energy emitted by microwave generates a little gradient of temperature between the resin and the cast. that uniform heat result in a fast polymerization and a reduction of stress release29-30, and these aspects would lead to a less dimensional distortion. however, these possible properties of microwaving method had little advantage on maintaining the tooth position when compared to the conventional cycles in the present study, especially for clamicro (group i) in the rp-lp distance. the microwave groups of the study (groups i and iv) were comparable to each other for 3 distances (rm-lm, ri-rm, and li-lm). braun11 also verified this aspect, concluding that the conventional resins showed similar dimensional change than those resins specially designed to cure by microwave activation. from a laboratory point of view, this study showed a similar behavior between microwave method and the conventional cycles in water bath. the use of conventional heat-curing acrylic resin for microwave energy polymerization seems not to strongly influence on tooth position change. references 1. mccartney jw. flange adaptation discrepancy, palatal base distortion, and induced malocclusion caused by processing acrylic resin maxillary complete dentures. j prosthet dent. 1984; 52: 545-53. 2. peyton fa. packing and pressing denture base resins. j am dent assoc. 1950; 40: 520-8. 3. firtell dn, green aj, elahi jm. posterior peripheral seal distortion related to processing temperature. j prosthet dent. 1981; 45: 598-601. 4. mowery we, burns cl, dickson g, sweeney wt. dimensional stability of denture base resins. j am dent assoc. 1958; 57: 345-52. 5. blagojevic v, murphy vm. microwave polymerization of denture base materials. a comparative study. j oral rehabil. 1999; 26: 804-8. 6. keenan pjl, radford dr, clark rkf. dimensional change in complete dentures fabricated by injection molding and microwave processing. j prosthet dent. 2003; 89: 37-44. 7. nishi m. studies on the curing of denture base resins with microwave irradiation; with particular reference to heat-curing resins. j osaka dent univ. 1968; 2: 23-40. 8. hayden wj: flexural strength of microwave-cured denture base plates. gen dent. 1986; 34: 367-71. 9. sanders jl, levin b, reitz pv. porosity in denture acrylic resins cured by microwave energy. quintessence int. 1987; 18: 453-6. 10. takamata t, setcos jc, phillips rw, boone me. adaptation of acrylic resin dentures as influenced by the activation mode of polymerization. j am dent assoc. 1989; 119: 271-6. 11. braun ko, rodrigues garcia rcm, rizzatti-barbosa cm, del bel cury aa. linear dimensional change of denture base resins cured by microwave activation. braz oral res. 2000; 14: 278-82. 12. yannikakis s, zissis a, polyzois g, andreopoulos a. evaluation of porosity in microwave-processed acrylic resin using a photographic method. j prosthet dent. 2002; 87: 613-9. 13. winkler s, ortman hr, morris hf, plezia ra. processing changes in complete dentures constructed from pour resins. j am dent assoc. 1971; 82: 349-53. 14. reeson mg, jepson nja. achieving an even thickness in heat-polymerized acrylic resin denture bases for complete dentures. j prosthet dent. 1999; 82: 359-61. 15. kawara m, komiyama o, kimoto s, kobayashi n, nemoto k. distortion behavior of heat-activated acrylic denture-base resin in conventional and long, low-temperature processing methods. j dent res. 1998; 77: 144653. 16. barco mt, moore bk, swartz ml, boone me, dykema rw, phillips rw. the effect of relining on the accuracy and stability of maxillary complete dentures – an in vitro and in vivo study. j prosthet dent. 1979; 42: 17-22. 17. 17-anthony dh, peyton fa. evaluating dimensional accuracy of denture bases with a modified comparator. j prosthet dent. 1959; 9: 683-92. 18. 18-polyzois gl. improving the adaptation of denture base by anchorage to the casts: a comparative study. quintessence int.1990; 21: 185-90. 19. wesley rc, henderson d, frazier qz, rayson jh, ellinger cw, lutes mr et al. processing changes in complete dentures: posterior tooth contacts and pin opening. j prosthet dent. 1973; 29: 46-53. 20. consani rlx, domitti ss, mesquita mf, consani s. influence of flask closure and flask cooling methods on tooth movement in maxillary dentures. j prosthodont. 2006; 15: 229-34. 21. lorton l, phillips rw. heat-released stress in acrylic dentures. j prosthet dent. 1979; 42: 23-6. 22. stanford jw, paffenbarger gc. processing denture base resins: heat-cure type. j am dent assoc. 1956; 53: 72-3. 23. yau wfe, cheng yy, clark rkf, chow tw. pressure and temperature changes in heat-cured acrylic resin during processing. dent mater. 2002; 18: 622-9. 24. becker cm, smith de, nicholls ji. the comparison of denture base processing techniques. part 1. material characteristics. j prosthet dent. 1977; 37: 450-9. 25. reitz pv, sanders jl, levin b. the curing of denture acrylic resin by microwave energy. physical properties.quintessence int. 1989; 16: 54751. 26. de clerck jp. microwave polymerization of acrylic resin used in dental prosthesis. j prosthet dent. 1987; 57: 650-8. 27. 27-al-hanbali e, kalleway jp, howlett ja. acrylic denture distortion following double processing with microwave or heat. j dent. 1991; 19: 176-80. 28. levin b, sanders jl, reitz pv. the use of microwave energy for processing acrylic resins. j prosthet dent. 1989; 61: 381-3. 29. hogan pf, mori t. development of a method of continuous temperature measurement for microwave denture processing. dent mater. 1990; 9: 1-11. 30. kimura h, teraoka f, ohnishi h, saito t, yato m. applications of microwave for dental technique (part i). dough forming and curing of acrylic resins. j osaka univ dent sch. 1983; 23: 43-9. 387 dimensional stability of distances between teeth in complete dentures comparing microwave polymerization and conventional cycles braz j oral sci. 9(3):384-387 404 not found oral sciences n3 original article braz j oral sci. july | september 2015 volume 14, number 3 child abuse: perception and knowledge by public health dentistry teams in brazil mariana dalledone1, ana paula borges de paola1, gisele maria correr1, eduardo pizzatto1, juliana feltrin de souza1, estela maris losso1 1universidade positivo up, dental school, area of pediatric dentistry, curitiba, pr, brazil correspondence to: estela maris losso rua professor pedro viriato parigot de souza, 5300 campo comprido cep: 81280-330 curitiba, pr, brasil phone: +55 41 33173454 e-mail: lossoem@gmail.com abstract aim: to evaluate the experience, knowledge and attitudes of dentists and oral health technicians (ohts) who work in basic health units in the city of curitiba, pr, brazil, regarding cases of abuse against children and adolescents. methods: in this observational study, three hundred eighty-three questionnaires (validated for brazilian portuguese) were sent to dentists and ohts who work in public health units. the response rate was 38.12% (n = 146) for dentists and 40% (n = 77) for ohts. results: the mean age of the dentists was 40±8.29 years, and 53.43% treated more than 10 children per week. the ohts’ mean age was 45.39±9.71 years, and 62.35% attended to more than 10 children per week. although the dentists perceived 185 suspected cases of maltreatment, only 35.67% were reported to authorities. in the oht group, 22.08% were reported to authorities. sixty-eight dentists (47%) and 11 ohts (14.28%) reported having treated at least one case of orofacial trauma in the previous 6 months, totalizing 166 orofacial traumas. about the mechanism for reporting suspected cases, 83% of the respondents knew how to report. over 50% of the responses concerning why the professionals did not report cases of physical violence against children included fear of retaliation and difficulties in diagnosis. conclusions: suspected cases of maltreatment against children and adolescents are underreported. more information is required to recognize suspected maltreatment cases and notify authorities. keywords: child abuse; dentists; public health dentistry. introduction children and adolescents are adversely affected by maltreatment because of their physical and psychological weakness and reliance on others1. according to the brazilian ministry of health2 107,572 cases of child abuse were reported in 2011. the network of protection for children and adolescents at risk for violence, an organization associated with the city of curitiba, pr, brazil, reported 5,371 suspected cases of child abuse in 20113. among them, 81.8% characterized as intrafamilial, of which 74.8% were cases of neglect and 12.1% were cases of physical aggression. the age of the children who suffered abuse ranged from 5 to 14 years3. child abuse is defined as any action or omission that prejudices the physical and psychological wellbeing, freedom and children’s right to full development. child abuse can be classified as physical, sexual or psychological abuse or neglect1,4-5. neglect was reported as the most common type of maltreatment3-4. in the united kingdom 44% of all children were reported in child protection registers6. this indicates a failure of parents or guardians to meet the children’s basic care braz j oral sci. 14(3):224-229 http://dx.doi.org/10.1590/1677-3225v14n3a10 received for publication: july 14, 2015 accepted: september 26, 2015 and needs, including their oral heath needs, to enjoy adequate function and freedom from pain and infection5. the neglect of children’s oral health may include the absence of managing oral hygiene and seeking dental treatment when needed, which can lead to dental caries, pain, poor dental growth and impaired quality of life. this situation should be recognized in dental offices if it is related to the guardian’s lack of knowledge or neglect7. physical aggression can be considered a non-accidental injury inflicted on a child or adolescent by an individual with superior strength or age. the lesions may be multiple types, such as hematomas, burns, lacerations, cuts, bruises, contusions, and abrasions on the body, face, lips and mouth8. in the mouth, physical violence can be identified by dental trauma and soft tissue injuries that are not consistent with patient reports5,7. physical aggression may be identified in the dental office because 50-67% of physical injuries occur in the head, neck, face and mouth8. these regions are easily visible to the dentist. according to cavalcanti9, the face accounted for 41% of the lesions, 55.6% of them were in the maxilla and 94.8% were in soft tissues. in the brazilian basic health units, public health dentistry teams comprise dentists and oral health technicians (ohts). these professionals, mainly the dentists, have legal, moral and ethical duty to notify the authorities suspected cases of child abuse, according to the federal constitution and brazilian statute of the child and adolescent10. previous studies reported the difficulties that dentists have to diagnose, document and report suspected cases of child abuse to authorities, resulting in underreporting the cases of aggression11-18. an australian study evaluated the level of knowledge and attitudes among dental professionals on the important issue of child abuse. the authors observed under-reporting of suspected child abuse cases. among the specialties, pediatric dentistry was responsible for 75% of the reports19. in denmark, dentists and ohts from public health dentistry reported more cases of maltreatment than private dental offices and all professionals reported the need for further information and training to recognize and report child abuse20. a brazilian study evaluated the perceptions and attitudes of child abuse in pediatric dentistry and underreporting was also found. no significant difference was found between the places of work (public and private offices). over 50% of pediatric dentists provided services at private offices, 25% provided services at public and private offices, 10% provided services at public healthcare offices only, and 10% were university professors17. child abuse is disturbingly common in today’s society21. considering the high proportion of orofacial injuries in victims of child abuse, the public health dentistry team has a strategic position to recognize and report suspected cases. however, the literature lacks data about the knowledge and attitudes of dental teams regarding child abuse. thus, the aim of the present study was to evaluate the level of knowledge, attitudes and interest of public health dentistry teams (dentists and ohts) who work in basic health units in the city of curitiba, pr, brazil, regarding cases of abuse against children and adolescents. material and methods this observational study was conducted after approval by the institutional ethics committee (protocol no. 118/ 2011). according to the regional dental council of paraná, the basic health units of the city of curitiba had 383 dentists and 194 ohts in 2012. all these professionals were invited to participate in this study. russell et al.22 developed the questionnaire22, which was validated for brazilian portuguese by marengo et al.23. this questionnaire has four parts. the first part refers to the social and demographic characteristics of the professionals. the second part refers to prior experience with maltreatment (including six yes/no closed-end questions). the third part refers to the healthcare professional’s knowledge, diagnoses and attitudes, and includes six questions (one yes/no closed-end question, two questions with answers on a 0-10 scale and three open-ended questions). the fourth part refers to involvement with the subject of child abuse and includes three questions (two closed-end questions and one with answers on a 0-10 scale)23. the questionnaire was sent by internal communication to the dentists and ohts in an envelope that contained the survey questionnaire, a letter of instruction for filling it and an informed consent form. responses were anonymous. a deadline was set to complete and return the questionnaire. after this date, an electronic reminder was sent to the participants to increase the response rate. the χ2 and fischer tests were used to analyze the associations between variables (suspected cases of child abuse, notifications and time since graduation). a significance level of 5% was set for the statistical analyses. results the initial response rate of the dentists was 33.15% (n=127). after the electronic reminder, the response rate increased to 38.12% (n=146), among which 78.76% (n=115) were female and 21.24% (n=31) were male. in the oht group, the initial response rate was 34% (n=67); after the electronic reminder, the response rate increased to 40% (n = 77), among which 94.80% (n = 73) were female and 5.20% (n=4) were male. with regard to age, the dentists ranged from 24 to 60 years (mean: 40±8.29 years), with 2-35 years (mean: 18±8.15 years) since graduation. the ohts’ ages ranged from 26 to 63 years (mean: 45.39±9.71 years), with 5-31 years (mean: 17.55±6.09 years) since graduation (table 1). according to their places of work, 69% of the dentists worked at public offices, and 31% worked at both public and private offices. most of the dentists (53.43%) treated more than 10 children per week. the ohts treated from 1 to 20 children per week, and 36.37% (n=28) treated 11-20 children per week (table 2). child abuse: perception and knowledge by public health dentistry teams in brazil225225225225225 braz j oral sci. 14(3):224-229 226226226226226 time since dentists % (n) ohts % (n) graduation (years) 2-10 26.71 (39) 18.18 (14) 11-20 24.67 (36) 45.45 (35) > 20 44.52 (65) 35.08 (27) no answer 4.10 (6) 1.29 (1) table 1. table 1. table 1. table 1. table 1. distribution of respondents according to years since graduation. no significant association was found between the number of children seen per week and suspected cases of child abuse (χ2=2.11, df=2, p>0.10). the number of notifications by dentists was associated with the number of children seen per week (p=0.029). for the ohts, no association was found between the number of children seen and suspected cases of child abuse (χ2=3.46, df=2, p=0.17) or the number of notifications (χ2=0.79, df=2, p=0.67; table 3). for dentists, a significant association was found between the time since graduation and suspected cases of child abuse (χ2=7.35, df=2, p<0.03). most suspected cases of child abuse were found by the group of professionals with over 20 years since graduation (χ2=6.28, df=2, p<0.02; table 3). for ohts (n=76), no association was found between the time since graduation and suspected cases of child abuse (time since graduation: 10 years, p=0.69; 11-20 years, p=0.73; >20 years, p=0.64). the time since graduation was unrelated to notified cases (time since graduation: 10 years, p=0.08; 11-20 years, p=0.08; >20 years, p=1.00; table 3). for prior experience with suspected maltreatment cases against children and adolescents, 52,73% of the dentists (n=77) and 46.75% of the ohts (n=36) examined suspected cases among their patients. although the dentists suspected 185 cases of maltreatment, only 35.67% (n=66) were reported to authorities. in the oht group, 22.08% (n=17) notified authorities about suspected cases of child abuse. sixty-eight dentists (47%) and 11 ohts (14.28%) reported having treated at least one case of orofacial trauma in the previous 6 months, totalizing 166 orofacial traumas. in the self-assessment on a 0-10 scale about their perceived ability to identify the signs and symptoms of child abuse, the medians were six (n=146) and seven (n=74) for dentists and ohts, respectively. as to the ability to diagnose physical aggression, the medians were five (n=143) for dentists and six (n=73) for ohts. for the professionals who reported that the subject of maltreatment was important, the medians were nine (n=144) and eight (n=76) for the dentists and ohts, respectively. with regard to their understanding of notification mechanisms, 117 dentists (63.83%) and 70 ohts 90.90%) reported that they were able to perform the notifications. in response to questions about the reasons why child abuse cases were not reported, the most common reasons were fear and threats of retaliation and lack of knowledge in diagnosing, 26.95% of the ohts and 44.18% of the dentists stated that they did not report such cases because of fear and threats of retaliation (figure 1). other reasons included connivance of the involved family, lack of interest by the professionals, lack of commitment by the professionals, cultural issues, fear of consequences for the victim and/or their family, lack of a relationship with the patients, omission, no quick resolution, lack of time for questioning and socioeconomic factors (figure 1). for possible ways to motivate the professionals’ notification of child abuse cases, the most cited were courses, lectures, campaigns, and establishing clinical guidelines for managing suspected child abuse cases (figure 2). with regard to their interest in obtaining more information about child abuse, 97% of the dentists (n=142) child abuse: perception and knowledge by public health dentistry teams in brazil braz j oral sci. 14(3):224-229 number of children dentists % (n) ohts % (n) treated per week 1-5 10.96 (16) 9.09 (7) 6-10 30.83 (45) 27.27 (21) 11-20 41.79 (61) 36.37 (28) > 20 11.64 (17) 25.98 (20) none 4.10 (6) 1.29 (1) no answer 0.68 (1) — table 2.table 2.table 2.table 2.table 2. number of children treated per week. number of children suspected child abuse % (n) notifications % (n) treated per week dentists ohts dentists ohts 1-10 27.03 (50) a 30.55 (11)a 10.61 (7) b 17.64 (3) a 11-20 36.23 (67) a 33.33 (12)a 43.94 (29) a 35.30 (6) a > 20 36.74 (68) a 36.12 (13)a 45.45 (30) a 47.06 (8) a years since graduation suspected child abuse % (n) notifications % (n) dentists ohts dentists ohts ≤10 17.30 (32)a 14.29 (5) a 7.58 (5) a —— 11-20 18.38 (34) a 48.57 (17) a 18.18 (12) a 41.18 (7) a > 20 64.32 (119)b 37.14 (13) a 74.24 (49) b 58.82 (10) a statistically significant differences are represented by different letters in the columns (fisher exact test and c2 test, p < 0.05). table 3. table 3. table 3. table 3. table 3. distribution of the number of children treated per week by dentists and ohts and time since graduation of the professionals according to suspected maltreatment cases and notifications. 227227227227227 fig. 2. possible methods reported by dentists and ohts for motivating professionals’ notifications of child abuse. fig. 1. primary reasons reported by dentists and ohts for not reporting abuse. and 93.51% of the ohts (n=72) reported they wished to have more information about the management of maltreatment cases and how to identify and report suspected cases, in addition to including the subject of maltreatment in their graduate studies. discussion the initial response rate was 38% for the dentists and 40% for the ohts, which is reasonable for this type of study, considering that the professionals work with time constraints and a high patient demand. similar rate was found by lazenbatt and freeman6, who evaluated health professionals in the united kingdom. the survey revealed a high prevalence of female dentists working at basic health units in the city of curitiba17. in other countries11,16,20,24, studies reported a prevalence of males worked in public health services. the respondents were included in the present study regardless of their time since graduation, but most of the professionals who returned the questionnaire had more than 20 years since graduation, demonstrating that their interest in the subject of child abuse was independent of the time since graduation14,24. with regard to working places, 69% of the respondents worked at public offices only, which was expected because the sample was selected from public health services. moreover, 31% of the dentists worked in private offices. these data are consistent with a previous brazilian study, in which most professional respondents worked at public health services only14. the oht group worked exclusively at public health services in public health dentistry teams. these professionals should be qualified to assist dentists to document suspected cases of child abuse and thus actively participate in the efforts against child maltreatment. most notifications of suspected cases were found by the dentist group that treated more than 11 children per week. however, this association was not found for the oht group. this may be explained by the ability to recognize signs and symptoms of suspected cases and notify authorities. although the professionals suspected cases of child abuse, there was under-reporting25. unfortunately, this is in agreement with substantial data from other countries6,15,17,24,26 .. among the reasons why abuse was under-reported, the professionals frequently mentioned fear, threats of retaliation, uncertainty in the diagnosis, and lack of knowledge about how to report suspected cases. these reasons were also reported in several previous studies, demonstrating the difficulties that professionals have around the world8,20,22. thus, the decision to report a suspected case depends on extant legislation, personal and professional factors and peculiarities of the case27. child abuse: perception and knowledge by public health dentistry teams in brazil braz j oral sci. 14(3):224-229 228228228228228 in the present study, an important issue that the professionals mentioned is the need for anonymity after notification, which is warranted by brazilian guardianship councils. health professionals in brazil have legal duty to report cases of suspected abuse against children and adolescents, based on the brazilian federal constitution and its statute of children and adolescents. moreover, protection of children is obligatory for every citizen10. another important point is the definition of “notification” as described by the brazilian ministry of health 1 “an instrument for ensuring legal and social protection of children and adolescents, allowing professionals from health, education, social assistance, as well as community councils and justice to take immediate action to stop the violence.” some political actions should be instituted to increase notifications by professionals, such as providing more information about child abuse, integration among multidisciplinary health professionals who work at public services, and senior health professionals providing support to health teams. ideally, mutual support among professionals is required, including sharing the knowledge and opinions, and adopting guidelines for protective actions. these actions between colleagues and senior health professionals would make them feel safer to diagnose and report suspected cases of violence. the lack of information about child abuse was reported by luna et al.28 who found that 69% of the health professionals who work at basic health units never received training on violence against children and adolescents. training of dental staff should be routine and thus increase the ability to recognize the signs and symptoms of abuse and provide security for reporting suspected cases. previous studies that were conducted in several countries reported dentists’ difficulties to diagnose, document, and report suspected abuse cases to authorities6,12-13,17,20,24,29. although notifications were higher in the present study compared with russell et al.22, brazilian professionals should be trained to improve their ability to recognize the signs and symptoms of child abuse and increase their confidence in reporting suspected cases of child abuse to authorities. with regard to the respondents’ interest in detecting physical child abuse, the dentists reported a higher interest compared with russell et al.22 , who found that some dentists did not perceive the relevance of child abuse cases and saw it as a cumbersome obligation. more than 50% of cases of physical child abuse involve the head, face, neck and mouth, which are easily visible to dentists and ohts. therefore, these professionals are sometimes the first to attend to such victims. information about child abuse is needed to modify the actions of health professionals when faced with child abuse cases and increase notifications of suspected cases. integral and multidisciplinary actions are needed to face this serious global problem29. it may be concluded that fear and lack of knowledge about diagnosing violence against children and adolescents and reporting such cases are barriers to recognizing and reporting suspected cases, thus resulting in under-reporting. public health dentistry teams showed interest in learning more about and reporting child abuse, thus indicating the importance of establishing political actions to educate and support these teams. acknowledgements the authors thank the oral health professionals in the city of curitiba. references 1. brazil. ministry of health, department of health policy. family violence guidelines for the practice in service. brasília: ministry of health; 2002 [cited 2015 oct 19]. (cadernos de atenção básica, 8). available from: http://bvsms.saude.gov.br/bvs/publicacoes/cd05_19.pdf. 2. brazil. ministry of health. datasus. tabnet linux 2.4: domestic violence, sexual and/or other violence sinan [cited 2015 oct 19]. available from: http://dtr2004.saude.gov.br/sinanweb/tabnet/tabnet?sinannet/ violencia/bases/violebrnet.def. 3. muraro hms. network for the protection of children and adolescents at risk for violence. curitiba: secretary of helth; 2012 [cited 2015 oct 15]. available from: http://www.crianca.mppr.mp.br/arquivos/file/ curso_de_atualizacao/2012/a3_rede_de_protecao_mp_2012.pdf. 4. valente la, dalledone m, pizzatto e, zaiter w, de souza jf, losso em. domestic violence against children and adolescents: prevalence of physical injuries in a southern brazilian metropolis. braz dent j. 2015; 26: 55-60. 5. american academy of pediatrics committee on child abuse and neglect; american academy of pediatric dentistry; american academy of pediatric dentistry council on clinical affairs. guideline on oral and dental aspects of child abuse and neglect. pediatr dent. 2008-2009; 30(7 suppl): 86-9. 6. lazenbatt a, freeman r. recognizing and reporting child physical abuse: a survey of primary healthcare professionals. j adv nurs. 2006; 56: 227-36. 7. tsang a, sweet d. detecting child abuse and neglect—are dentists doing enough? j can dent assoc. 1999; 65: 387-91. 8. cairns am, mok jy, welbury rr. injuries to the head, face, mouth and neck in physically abused children in a community setting. int j paed dent. 2005; 15: 310-8. 9. cavalcanti al. prevalence and characteristics of injuries to the head and orofacial region in physically abused children and adolescents—a retrospective study in a city of the northeast of brazil. dent traumatol. 2010; 26: 149-53. 10. brazil. status of children and adolescents. law no. 8069 of july 13, 1990 [cited 2010 aug 23]. available from: http://www.planalto.gov.br/ccivil_03/ leis/l8069.htm. 11. bsoul sa, flint dj, dove sb, senn dr, alder me. reporting of child abuse: a follow-up survey of texas dentists. pediatr dent. 2003; 25: 541-5. 12. thomas je, straffon l, inglehart mr. knowledge and professional experiences concerning child abuse: an analysis of provider and student responses. pediatr dent. 2006; 28: 438-44. 13. manea s, favero ga, stellini e, romoli l, mazzucato m, facchin p. dentists’ perceptions, attitudes, knowledge, and experience about child abuse and neglect in northeast italy. j clin ped dent. 2007; 32: 19-25. 14. granville-gracia af, silva mjf, menezes va. abuse of children and adolescent: a study in the city of são bento do una, pe, brazil. pesqui bras odontopediatria clin integr. 2008; 8: 301-7. 15. sonbol hn, abu-ghazaleh s, rajab ld, baqain zh, saman r, al-bitar zb. knowledge, educational experiences and attitudes towards child abuse amongst jordanian dentists. eur j dent educ. 2012; 16: e158-65. child abuse: perception and knowledge by public health dentistry teams in brazil braz j oral sci. 14(3):224-229 229229229229229 16. laud a, gizani s, maragkou s, welbury r, papagiannoulis l. child protection training, experience, and personal views of dentists in the prefecture of attica, greece. int j paediatr dent. 2013; 23: 64-71. 17. el sarraf mc, marego g, correr gm, pizzatto e, losso em. physical child abuse: perception, diagnosis, and management by southern brazilian pediatric dentists. pediat dent. 2012; 34: e72-6. 18. thomas je, straffon l, inglehart mr. child abuse and neglect: dental and dental hygiene students’ educational experiences and knowledge. j dental educ. 2006; 70: 558-65. 19. john v, messer lb, arora r, fung s, hatzis e, nguyen t, et al. child abuse and dentistry: a study of knowledge and attitudes among dentists in victoria, australia. aust dent j. 1999; 44: 259-67. 20. uldum b, christensen hn, welbury r, poulsen s. danish dentists’ and dental hygienists’ knowledge of and experience with suspicion of child abuse or neglect. int j paediatr dent. 2010; 20: 361-5. 21. colucci e, hassan g. prevention of domestic violence against women and children in low-income and middle-income countries. curr opin psychiatry. 2014; 27: 350-7. 22. russell m, lazenbatt a, freeman r, marcenes w. child physical abuse: health professionals’ perceptions, diagnosis and responses. brit j community nurs. 2004; 9: 332-8. 23. marengo g, paola ap, ferreira fm, pizzatto e, correr gm, losso em. child abuse: validation of a questionnaire translated into brazilian portuguese. braz oral res. 2013; 27:163-8. 24. owais ai, qudeimat ma, qodceih s. dentists’ involvement in identification and reporting of child physical abuse: jordan as a case study. int j paediatr dent. 2009; 19: 291-6. 25. azevedo ms, goettems ml, brito a, possebon ap, domingues j, demarco ff, et al. child maltreatment: a survey of dentists in southern brazil. braz oral res. 2012; 26: 5-11. 26. cairns am, mok jy, welbury rr. the dental practitioner and child protection in scotland. brit dent j. 2005; 199: 517-20; discussion 512; quiz 30-1. 27. gonçalves hs, ferreira al. health professionals’ reporting of family violence against children and adolescents. cad saude publica 2002; 18: 315-9. 28. luna gl, ferreira rc, vieira ljes. mandatory reporting of child abuse by professionals of family health teams. cienc saude colet. 2010; 15: 481-91. 29. deshpande a, macwan c, poonacha ks, bargale s, dhillon s, porwal p. knowledge and attitude in regards to physical child abuse amongst medical and dental residents of central gujarat: a cross-sectional survey. j indian soc pedod prev dent. 2015; 33: 177-82. child abuse: perception and knowledge by public health dentistry teams in brazil braz j oral sci. 14(3):224-229 revista fop n 13 1673 carrea’s index in dental students at the federal university of paraíba laíse nascimento correia lima1; germana louanne santos neves2; patrícia moreira rabello3 1undergraduate dental student 2dds, graduate student in oral diagnosis, postgraduate program in dentistry 3adjunct professor, forensic and ethical dentistry and dental legislation federal university of paraíba, joão pessoa, pb, brazil received for publication: october 15, 2008 accepted: december 02, 2008 correspondence to: laíse nascimento correia lima rua rosa lima dos santos, 480. bancários, 58051-590 joão pessoa, pb, brasil. e-mail: laiselima@msn.com a b s t r a c t aim: to determine the applicability of carrea’s index in arches with normal tooth position and diastema and to evaluate its use in the maxillary arch. methods: 51 pairs of plaster models made by dental students from the federal university of paraíba were analyzed. the arches and hemiarches were divided according to dental position and measured with a manual caliper and a digital caliper. with these measurements, maximum and minimum heights were estimated, comparing the values to the real height of the subjects. the data were then submitted to pearson’s chi-square test and fischer’s exact test (ci = 95%). results: in the mandibular hemiarches, there was statistically significant difference (p=0.017) between the types of dental position only in women, with crowded teeth obtaining the highest number of hits (95.2%), followed by diastema (50.0%). only the left hemiarch was significantly different (p=0.049) and crowded teeth had the highest index of hits (82.6%), whereas diastema accounted for 40.0%. in the maxillary arch, the error percentage was 100% in the three types of dental position. conclusion: carrea’s index is applicable in normal and crowded teeth. however, it was not efficient in the maxillary arch and in hemiarches with diastema. keywords: forensic anthropology. legal dentistry. dental arch. i n t r o d u c t i o n the identification of human cadavers in official investigations has received innumerable contributions from legal dentistry. the dental arch has uncountable individual variables that make it impossible for two different individuals to have identical dental elements. this fact often allows irrefutable proof of identification for legal purposes1,2. the importance of teeth in identification processes owes to their peculiar characteristics, such as resistance to the effects of time, fire and trauma, which is of great value in large catastrophes and mass disasters. furthermore, teeth can provide information on species, racial group, gender, age, height and individual cadaver data2-5. in the processes of identifying bones, and carbonized or decomposed human remains, estimating height is important as an objective characteristic of identity. height can be estimated by analyzing the long bones of the body. however, there are situations in which only part of the skeleton or a single bone is available. in cases where only the head has been found, height can be estimated by examining the teeth. carrea6 developed a mathematical model that allows for calculating of an individual’s height by measuring the dimensions of some mandibular teeth2,4,7,8. however, carrea’s index has originally been suggested for dental arches with normal tooth position. considering the scarcity of information in the literature and the fact that a large part of the population presents some type of malocclusion, mainly tooth crowding with frequent diastema, the actual applicability of this index must be investigated. some studies have also indicated its use in the maxillary arch7,9-11. the aim of using carrea’s index in the maxilla is to establish a new identification method to aid in cadaver examinations because the skulls are frequently found fragmented and with missing mandible. in addition, because of its anterior and medial location, the mandible is often more damaged in accidents, which would preclude the use of the method12. therefore, the purpose of this study is to determine the applicability of carrea’s index for use in official investigations to identify human remains in arches with normal tooth position and diastema and to evaluate its use in the maxillary arch. material and methods this study was conducted in accordance with the braz j oral sci. october/december 2008 vol. 7 number 27 1674 regulations established by resolution 196/96 and the research protocol was approved by the research ethics committee of the health sciences center at the federal university of paraíba on may 28, 2008 (protocol #. 0061). a pilot study was carried out to calibrate the examiner. twelve plaster models were used (6 mandibular and 6 maxillary). in both the 6 mandibular and 6 maxillary models, 2 were of normally positioned teeth, 2 of crowded teeth and 2 of teeth with diastema. these models provided 24 measurements, since each unit included a right and left hemiarch. the examinations were repeated after 8 days and statistical analysis showed 21 matches (87.5%), kappa’s index of 0.74 and confidence interval of 95% (0.47 to 1.00). the agreement was considered good, according to this index, since it lies in the 0.61 to 0.80 range. a blind cross-sectional study was performed, using a comparative procedure and descriptive statistics. the sample was composed of 102 plaster models (51 maxillary and 51 mandibular) belonging to students from the course of dentistry at ufpb. the subjects were 27 women and 24 men. all participants were submitted to anthropometric analysis. the maxillary and mandibular arches and the right and left hemiarches were evaluated separately, for a total of 204 hemiarches. each hemiarch was then divided, according to tooth position, into three groups: normal, crowded and with diastema. the lower level of crowded teeth and diastema was considered. of the 102 maxillary hemiarches, 60 had normal tooth position, 23 were crowded and 19 had diastema. of the mandibular hemiarches, 41 had normal position, 43 were crowded and 18 had diastema. the arch and the string described by carrea6 were measured. the arch consists of the sum of the mesial-distal diameters of the central incisor, lateral incisor and mandibular canine, measured on the buccal surface of these teeth. the string of this arch corresponds to the measure of the straight line located between the initial and final points, represented by the mesial edge of the central incisor up to the distal edge of the ipsilateral canine, measured on the lingual surface of the aforementioned teeth. individual tooth sizes of the arch were measured with a manual caliper (ice® cajamar, sp, brazil), transferred to a millimeter ruler (angelus®, londrina, pr, brazil) and totaled. the string was measured with a digital caliper (digimess®, são paulo, sp, brazil) by tracing a straight line from the mesial of the central incisor to the distal of the canine. from these measurements, maximum and minimum heights were estimated, according to carrea’s index (figure 1). the resulting values were compared to the real height of the subjects. spss (statistical package for the social sciences, spss inc., chicago, il, usa), version 13.0 was used to obtain statistical calculations and data analysis was performed using pearson’s chi-square and fischer’s exact tests, at a confidence interval of 95.0%. r e s u l t s table 1 shows the distribution of hits and misses obtained in the mandibular arch for normal tooth position, crowded teeth and teeth with diastema, evaluated separately by gender. there was no statistically significant difference in men (p=0.221), different from that observed in women (p=0.017). the crowded group recorded the highest number of hits (95.2%), followed by the diastema group (50.0%). table 2 shows the analysis of different tooth positions according to the hemiarch, in which statistically significant difference was found only on the left side (p=0.049), with the highest percentage of hits in the crowded hemiarches (82.6%), and the lowest in the hemiarches with diastema (40.0%). tables 3 and 4 show that in the maxillary arches the percentage of misses was 100% for all types of tooth position in both genders and hemiarches, respectively. d i s c u s s i o n in contrast to the findings of previous studies7,8,10, which ruled out the use of carrea’s index in crowded dental arches, we found a significant percentage of hits in the hemiarches that exhibit this type of tooth position. indeed, this percentage was even higher than that found in women with normal tooth position (table 1) and on the left side (table 2), where there was statistically significant difference among the three types of position. the high percentage of hits obtained in the crowded hemiarches might have been due to the individual measurement of each tooth with manual caliper, resulting in a more reliable arch value. cavalcanti et al13 compared two forms of measuring teeth in plaster models to estimate the height of 50 dental students using carrea’s index. in the first method, called conventional, the arch was measured with a metric tape measure and the string with a caliper; in the second method, called modified, the string and the arch were measured with a manual caliper, with the value of the arch represented by the sum of the individual measurements of each tooth. the authors13 concluded that the results between the two methods were statistically significant (p<0.01), given that in the modified method the percentage of hits was 96.0% for both sides, while the conventional method obtained 36.0% and 48.0% of hits on the right and left sides, respectively. in the same study13, it was found that, maximum height = arch (in mm) x 6 x 3.1416 x 100 2 minimum height = string-radius (in mm) x 6 x 3.1416 x 100 2 fig. 1 equations to calculate maximum and minimum height source: carrea14 braz j oral sci. 7(27):1673-1677 carrea’s index in dental students at the federal university of paraíba 1675 table 1 distribution of hits and misses for normal tooth position, crowded teeth and teeth with diastema, in the mandibular arch, according to the gender of the study subjects. joão pessoa, brazil, 2008 (*): significant association at 5.0%. fisher’s exact test table 2 distribution of hits and misses for normal tooth position, crowded teeth and teeth with diastema, according to hemiarch, in the mandibular arch of the study subjects. joão pessoa, brazil, 2008 (*): significant association at 5.0%. (1)using fisher’s exact test. (2)using pearson’s chi-square test in the modified method, the percentage of hits was greater for men (100%) than for women (93.3%), with no difference between the right and left sides. however, in the conventional method the number of hits was higher in women on both sides. it should be pointed out that cavalcanti et al.13 included normal and crowded arches, without assessing them separately and did not statistically evaluate the differences between genders, since neither the test value nor the p-value was found. in a study conducted by silva8, the string was measured with a caliper and the arch with a metric tape measure. the author observed that the real height of an individual lay between the maximum and minimum heights in 70% of the cases. however, the author did not assess the genders or the hemiarch and did not consider the plaster models in which the arches displayed crowding or some other anomaly. with respect to the low percentage of hits obtained in hemiarches with diastema, it must be underscored that in the present study, there was a wide range of diastema severity in the analyzed hemiarches. they varied between 0.5 and 3.0 mm and occurred between the canine and lateral incisor and/or lateral and central incisor. large diastemas compromised the string value, given that the spaces between the teeth led to a greater measure than if they had been in contact with one another. braz j oral sci. 7(27):1673-1677 carrea’s index in dental students at the federal university of paraíba 1676 table 3 distribution of hits and misses for normal tooth position, crowded teeth and those with diastema in the maxillary arch, according to the gender of the study subjects. joão pessoa, brazil, 2008 table 4 distribution of hits and misses for normal tooth position, crowded teeth and those with diastema, according to hemiarch, in the maxillary arch of the study subjects. joão pessoa, brazil, 2008 therefore, the string value, which estimates minimum height, was higher than that of the arch, which corresponds to maximum height. in the difference between the heights, the minimum was greater than the maximum because the arch value was not compromised, given that its measurement represents the individual sum of the teeth. these occurrences may have been a determinant for the low percentage of hits for the hemiarches with diastema, since the real height of the subjects lay within the maximum and the misses were observed when obtaining the minimum height. however, based on the methodology used and on the results obtained, it may be inferred that carrea’s index is applicable in mandibular arches for normal and crowded tooth positions. in the mandibular hemiarches that present diastema, the severity of the diastema must be evaluated for the index to be considered useful in estimating height. however, in these cases, the method is not reliable owing to the low percentage of hits in the values obtained. regarding the maxillary arches, the present findings showed that there were no hits in any of the types of tooth position for both men and women (table 3), and on both the left and right sides (table 4), demonstrating the ineffectiveness of the method when maxillary arch teeth are evaluated. moreover, no theoretical reference was found on the use of carrea’s index in maxillary arches to compare the obtained results. height is an essential element when seeking the identity of an individual and determining it is no easy task. in contrast to the adversities found in human identification braz j oral sci. 7(27):1673-1677 carrea’s index in dental students at the federal university of paraíba 1677 processes, especially those involving human fragments or pieces of bone remains, carrea’s index appears as a practical, easy and low-cost method to be used, requiring only the presence of a number of mandibular teeth. a c k n o w l e d g e m e n t s this study received financial support from the institutional program for scientific initiation grants (pibic), coordinated by the national council of scientific and technological development (cnpq). r e f e r e n c e s 1. campello ric, genú pr. o estudo das mordeduras. in: vanrell j. odontologia legal e antropologia forense. rio de janeiro: guanabara koogan; 2002. p.67-72. 2. campos mlb. os arcos dentários na identificação. in: vanrell j. odontologia legal e antropologia forense. rio de janeiro: guanabara koogan; 2002. p.203-11. 3. vanrell j. odontologia legal e antropologia forense. rio de janeiro: guanabara koogan; 2002. 4. freire jjb. estatura: dado fundamental em antropologia forense [dissertação]. são paulo: universidade estadual de campinas; 2000. 83p. 5. fígun me, garino rr. anatomia odontológica: funcional e aplicada. porto alegre: artmed; 2003. 6. carrea ju. ensayos odontométricos [tese]. buenos aires: universidad nacional de buenos aires; 1920. 64p. 7. croce d, croce júnior d. manual de medicina legal. 7. ed. são paulo: saraiva; 2006. 8. silva m. compêndio de odontologia legal, são paulo: medsi; 1997. 9. freitas mr, freitas ds, pinheiro fhsl, freitas kms. prevalência da más oclusões em pacientes inscritos para o tratamento ortodôntico na faculdade de odontologia de bauru-usp. rev fac odontol bauru. 2002; 10: 164-9. 10. galvão, lcc. antropologia forense. in: vanrell j. odontologia legal e antropologia forense. rio de janeiro: guanabara koogan; 2002. 11. marques ls, barbosa cc, ramos-jorge ml, pordeus ia, paiva sm. prevalência da maloclusão e necessidade de tratamento ortodôntico em escolares de 10 a 14 anos de idade em belo horizonte, minas gerais, brasil: enfoque psicossocial. cad saude publ. 2005; 21: 1099-106. 12. andrade filho ef, fadul júnior r, azevedo raa, rocha mad, santos ra, toledo sr, et al. fraturas de mandíbula: análise de 166 casos. rev ass med brasil. 2000; 46: 272-6. 13. cavalcanti al, porto de, maia ama, melo trnb. estimativa da estatura utilizando a análise dentária: estudo comparativo entre o método de carrea e o método modificado. rev odontol unesp. 2007; 36: 335-9. 14. carrea ju. talla individual human en función al radio cuerda. ortodoncia. 1939; out. 6. braz j oral sci. 7(27):1673-1677 carrea’s index in dental students at the federal university of paraíba oral sciences n3 original article braz j oral sci. october|december 2010 volume 9, number 4 medical and dental absenteeism in workers from a furniture industry in itatiba, sp, brazil cristina gomes de macedo1, dagmar de paula queluz2 1dds, master student, piracicaba dental school, state university of campinas (unicamp), piracicaba (sp), brazil 2dds, msph, phd, professor, department of community dentistry, piracicaba dental school, state university of campinas (unicamp), piracicaba (sp), brazil correspondence to: dagmar de paula queluz departamento de odontologia social faculdade de odontologia de piracicaba – unicamp avenida limeira, 901 cep 13414-900 piracicaba sp brazil phone: +55 -19-2106 5277 e-mail: dagmar@fop.unicamp.br received for publication: june 16, 2010 accepted: november 08, 2010 abstract introduction: absenteeism is a matter of growing interest due to the current economic competitiveness and leads companies to seek means to minimize its occurrence in order to increase the profitability and productivity. aim: to identify the major factors involved in medical and dental absenteeism in a furniture industry in the city of itatiba, sp, brazil, between 2005 and 2008. methods: a descriptive cross-sectional study in which all workers’ medical and dental certificates issued from january 2005 to december 2008 were reviewed. data collection was performed in the industry and the variables in the dental and medical certificates were: activity, date of the certificate (year and month), international classification of diseases (icd), absenteeism duration (hours), certificate type (medical or dental), declaration variations, and completeness. questionnaires were also applied to all employees of the industry without exclusion criteria for demographic profile, using the variables focusing on gender, age and education level. results: 111 (65.2%) workers replied to the questionnaire. as much as 84.6% of them were men with mean age of 31.4 years. the education level of most employees was high school (53.2%). 943 certificates were analyzed, being 775 (82.2%) medical and 168 (17.8%) dental certificate. the only icd found in the dental certificates is k 525-8, which refers to “other diseases and disorders of the teeth and supporting structures”. the loss of working time was 7098.5 work hours and 887.3 work days considering an 8-hour working day. there was a statistically significant association (p<0.0001) between the duration of the absence and the cause. conclusions: the absenteeism in the furniture industry was more frequently due to medical rather than dental reasons. there were several certificates lacking the icd codes. the number of lost work hours due to medical and dental problems is a significant economic factor. keywords: absenteeism, workers, sick leave. introduction the term absenteeism is used to designate the absences of workers at work process, either by lack or delay, due to some intervening reason. absenteeism has become a crucial problem for both private and public organizations and their administrators, which causes a quantitative impact on human resources and reflects on the quality of service provided1-3. the individual workers (and their dependents) many times see their income reduced due to absenteeism, especially in long-lasting cases. absenteeism at work also has a negative effect on the national economy as a result of potential loss production due to reduction of the available labor force and the increased costs of medical treatment and social security4. braz j oral sci. 9(4):443-448 work should be considered by the worker not as an obligation, but as a form of growth, interpersonal development, enhancement of skills, abilities, sharing of experiences. worker should feel happy with his/her activity, the working environment and with his/her co-workers. presenteeism is an even worse organizational problem because the worker is physically present at work, but he/she is not producing due to multiple variables and factors such as: dissatisfaction, pessimism, discouragement and high stress level, making it harder to identify the problem, and causing harm to the industry and to other workers who are often contaminated by this apathy and lack of productivity. gaidzinski5 (1994) developed a study to grade the staff services, and classified absences as foreseen and unforeseen. the first class of absences if formed by those that are granted by law and that may be planned in advance, such as vacation, retreats and holidays. unforeseen absences effectively characterize absenteeism due to their unpredictable nature, such as unjustified absences, medical licenses, accidents, maternity and paternity licenses, mourning, specialization courses and other situations that prevent a worker of staying at the work place. in any case, this phenomenon causes not only direct, but also indirect costs, caused by decrease in productivity because fewer workers are active. this will reduce the quality of service since another worker has to cover for the missing one, certainly decreasing the efficiency and effectiveness of the expected results. another classification of absenteeism presented by midorikawa6 (2000) discusses the aspect of lack at work and absenteeism where the worker is only physically present. the first can be measured and has its cost calculated by the absence of the individual. the second, which cannot be measured, is when the worker cannot perform his usual tasks because of any kind of pain. the world health organization (who) recognizes that pain, suffering, psychological and social constraints may result from oral diseases, leading to losses at the individual and collective levels7. oral problems are directly related to lost working days. workers with lower wages and minorities are disproportionately affected. oral health is undeniably important for appropriate standards of quality of life. brazilian dental health indicators of the adult population, indicate a high caries incidence, a significant number of teeth loss in young adults, great need for use of prostheses and severe periodontal problems8. dental absenteeism in brazil is very difficult to evaluate because the relevant documents are not duly stored. there are few statistically processed data to measure the absenteeism for dental reasons1-3. data are scarce about both the economic burden that such losses may entail and the worker’s dissatisfaction by breaking the health-work binomial9. according to nardi10 (2005), although dentists have been empowered to certify dental facts and their consequences since 1966, they were allowed to issue certificates to justify sick leaves only 9 years later. the federal council of medicine resolution nº 1.851/ 2008 regulates the issuing of medical certificates11. according to the law 5.081, art. 6i, dated august 24 1966, it is within the dentist’s scope to attest, in their professional activity and other unwholesome states, including justification of absence to employment. the dental ethics code classifies as infringement of professional ethics to issue a certificate that does not correspond to the facts or in which the dentist did not participate12. thus, from the ethical point of view, it is unacceptable to issue a certificate that is incompatible with the truth, for which only a professional who was involved in patient care can testify and, after the due examination of the real condition, determine whether a certificate is required. a dental certificate may not be refused for any reason, if it obviously meets all the required conditions to ensure that the referenced document can be accepted: who is the dentist that actually treated the patient, if the dentist is legally empowered for dental practice, understood as having professional qualification, possession of a legal document certified by records at the competent offices; not to have his/her practice suspended by judicial decision; not be the dentist suspended the profession by virtue of penalty imposed by the regional council of dentistry12. the dentist can have a printed form especially for the issuance of certificates, or, as commonly practiced use plain paper, which must contain the dentist’s professional qualification, the registration number in regional council of dentistry and address. never should the professional issue a certificate in advance, since the document can be use for purposes other than those mentioned by the requester. the purpose of the certificate must be specific, whether for school, sports activity or to justify absence at work. the term of absence shall be entered in full stating clearly the number of days stipulated by the professional12. in the current context of economic competitiveness, sick leave, mainly if related to oral health, is a cause of growing concern for businesses, since only with a strict control and care of these factors they can reach a significant increase in profitability. studies have shown that caries and other oral diseases are responsible for 20% of work day loss, and decreased productivity and directly affect the worker’s selfesteem and quality of life. the aim of this study was to identify the main factors involved with medical and dental absenteeism between 2005 and 2008 in a furniture industry in the city of itatiba, sp, brazil. material and methods the sample of this study was composed of dental and medical certificates and duly approved presence declarations, issued between january 2005 and 2008, and delivered to the human resources department of a furniture industry, whose purpose was to vouch for sick leave up to 15 days. the research protocol was reviewed and approved by the research ethics committee of the university of campinas (unicamp); protocol number 009/2009. this was a descriptive cross-sectional study in which all medical and dental certificates issued in the proposed time span were reviewed. the data collection was carried 444444444444444medical and dental absenteeism in workers from a furniture industry in itatiba, sp, brazil braz j oral sci. 9(4):443-448 out in the industry and the variables in the dental and medical certificates were: activity, date of the certificate (year and month), international classification of diseases (icd) code, absence duration (hours), certificate type (medical or dental), declaration variations, and completeness. questionnaires were applied to all employees without exclusion criteria to study the demographic profile with variables focusing on: gender, age and education level. the collected data were automatically tabulated using excel software13 and analyzed statistically by sas14. results in the evaluated period, the industry had 170 workers with a variation of 10 to 15 workers per year spread across sectors such as polishing, glaziers, maintenance, carpentry, sorting, warehouse management, human resources, budget, purchases, projects, gatehouse and expedition. one hundred and eleven workers replied to the questionnaire (65.2%), being 84.6% men (n=94) and 15.3% women (n=17) with mean age of 31.4 years. the education level of most employees was high school (53.2%). twentythree (21.5%) workers completed only elementary school, while incomplete elementary school was the education level of 15 (14.0%) workers. the manufacturing activity involves the largest number of workers in the factory (n= 72; 64.82%) and covers the following areas: polishing, maintenance, glass factory, carpentry, sorting, warehouse and management. other areas covered in the study are reception, administration, and projects and correspond to a total of 39 workers (35.18%). the brazilian furniture branch consists predominantly of traditional familiar and national capital enterprises, counting about 50 thousand formal industries. in the 1990’s brazil diversified its commercial ties and this branch was encouraged to modernize the industrial processes and imported machinery and equipment, mainly from italy and germany, with resources made available by the national bank for economic and social development,. the branch had an explosive growth of exportations, and brazil is currently ranked #18 among furniture exporting countries, which represents 1.5% of world exports of that branch15. according to the national classification of economic activities, the furniture industry belongs to the group c-6, wood, national classification of economic activities 36 110, presenting degree of risk 3 in the regulatory standard no.4, in a 1 to 4 scale16. in this study, 943 certificates were reviewed, being 775 (82.2%) medical and 168 (17.8%) dental documents. there was a predominance of males (n= 751, 79.6%) over females (n=192, 20.4%). in 2005, 137 (14.5%) medical and dental certificates were delivered to the human resource department, in 2006 they were 242 (25.6%), in 2007, 325 (34.6%) and in 2008, 239 (25.3%). there was no statistically significant association between the number of certificates and the different months and years examined (p>0.005). table 1. association between the removal duration and cause chi-square= p< 0.001 frequency medical dental total <= 1 hour 227 (33.39%) 77 (46.39%) 304 (35.93%) 1 —| 4h 173 (25.43%) 68 (40.96%) 241 (28.49%) > 4h 280 (41.18%) 21 (12.65%) 301 (35,58%) total 680 (100%) 166 (100%) 846 (100%) the icd code was not mentioned in 618 (65.5%) certificates. the remaining 325 (34.5%) certificates contained the icd code, being 241 (74.1%) medical certificates and 84 (25.9%) dental certificates. among the certificates with the icd code, dentists were more concerned in providing this information (50%) compared to physicians (31.1%). in all dental certificates, a single icd code was used, k-525-8, which means: “other diseases and disorders of the teeth and its support structures.” in the medical certificates four categories of icd presented greater frequency: group m-musculoskeletal system diseases and connective tissue (n=41, 17%), group j – respiratory diseases (n=32, 13.3%), group r – symptoms signs and abnormal in clinical examination and laboratory (n=27, 11.2%) and group k – digestive diseases (n=17, 7%). regarding the duration, most justified absences from work due to dental and medical reasons lasted up to 8 hours (n=846, 89.7%) and only 97 (10.3%) cases lasted over 8 hours to 5 days. the loss of working time was 7098.5 work hours and 887.3 work days considering an 8-hour working day. the types of documents presented to the human resource department to justify sick leaves included consultation, ambulatory service and exam, vouchers (n=284, 30.1%), hospital vouchers (236, 25%), incomplete medical/dental certificates issued by he professionals (199, 21.1%), and recipes with fill full recipes syndicate category complete and incomplete as to fill totaled 224 (23.8%). there was a statistically significant association (p<0,001) between the sick leave duration and its cause. dental certificates prevailed in absence duration up to four hours, whereas leaves of absence over four hours prevailed in the medical certificates (table1). discussion whatever the driving economic activity of a developing country, the instrument for the final objectives pursued depends on the work carried out by humans. this fact reveals the interest of the authorities of the country in relation to the workers’ health, regardless of their level of work. the dental and medical certificates are the only legal instruments that justify sick leaves and ensure full payment of the wages. distribution of absenteeism as regards age, gender, education level is required. other important data are the duration and the cause of absences. the availability of such information is very important for planning the different strategies of prevention. 445445445445445 medical and dental absenteeism in workers from a furniture industry in itatiba, sp, brazil braz j oral sci. 9(4):443-448 in our study, we found in both the social demographic profile and the analysis of certificates, a prevailing population of young males with a high education level for the standards of a developing country. it is clear that the variables gender, age and education level, if considered alone, contribute to the understanding of absenteeism globally, depending on the type and workplace studied. on the other hand, they provide important information when the analysis is restricted to a specific enterprise, providing subsidies for searching solutions to decrease absence at the work place17. grzywacz and butler18 (2005) rank the schooling as the most important component of the socioeconomic condition for determination of health-related behaviors. high levels of schooling are generally associated with better housing and employment, income and socio-economic position. in a study on absenteeism for dental and medical reasons in public and private services, in the city of araçatuba, sp, brazil, the dental certificates had little weight on the total certificates, both in the public (3.3%) and in the private (6.3%) services. due to lack of icd codes in almost all dental certificates, the reason for most worker absences in both public and private services was pain (icd m 54)19. the present study also found a predominance of medical rather than dental certificates, but a greater concern by the dentists relative to placing of the icd code. in medical certificates the prevalent icd codes were from group m, which deals with diseases of the musculoskeletal system and connective tissue. reisine and miller20 (1985) analyzed the effects of dental conditions on social functioning by measuring the incidence of work loss days associated with dental problems and treatments. a longitudinal study of 1992 employed adults in the hartford, connecticut, usa, was conducted. participants were interviewed at baseline to collect data on sociodemographic, health care and health status factors and were followed for 1 year to assess the incidence of dental work loss days. the results showed that 26.4% of the sample reported an episode of dentally-related work, with a mean of 1.26 hours per person per year. the results suggest that work loss days may be a useful population statistic in measuring oral health status because of the high prevalence of dental disease. mazzili and crosato21 (2005) published a survey to investigate the prevalence, incidence and the average absence from work for dental reasons, according to the variables of interest to healthcare, performed by nosologic group and in accordance with the international classification of diseases and related health problems. data collection was held in the medical department of the administration secretary of são paulo between december 1996 and december 2000. the results indicated greater prevalence of females, most often aged 20 to 29 years, and the main reasons were: dental extractions, pulp diseases periodontal diseases, temporomandibular joint disorders and extractions of included or impacted teeth. requests for absence for dental reasons in the studied population were genderand agedependent, according to the study in proportions. acute conditions or post-operative were the most frequent causes. chronic conditions represented, however, the greatest impact on total days of absence. in the conducted study it was not possible to assess the main reasons of absence by dental causes, because most of the evaluated certificates had a single icd (525-8, other diseases and disorders of the teeth and their structures). in cooperation with the fondazione maugeri of and the fondazione ospedale maggiore policlinic, milan, the local health unit in italy, conducted a research project, sponsored in part by the italian ministry of health about damages in work-related upper limb muscle-skeletal system in a specific sector of a manufacturing industry of upholstered furniture. this “upholstered district” is widely represented approximately 14,000 workers in 500 factories over a large geographical area of southern italy. advanced technology of manufacturing process is combined with workers engaged in intensive tasks of hand and arm. the risk groups were: workers of preparation, operators of leather-cutting, sewing and workers assembling upholstery padding. data collected in private companies of different size this extensive industrial area emphasize the importance of prevention through appropriate ergonomics solutions and the need to improve training program covering the entire area22. there was no significant prevalence in the certificate analysis of the respiratory system diseases (n = 32, 13.3%). workers in industries where wood is processed are exposed to mechanical activities employing different types of tools, machines and various chemical substances. an example related to working conditions is the trauma suffered by the mutilation of one of the members or other permanent impairment. often insurance intended for protection of victims and ensuring a fair indemnity as the degree of the problem and results in revolt and exacerbates the feeling of less value because it does not match the actual loss both in their physical and psychological aspects. the loss of one of the fingers, for example, does not imply only in press movement’s inability, for example, but also implies a set of hand signals that can cause social constraints23. taking into consideration the risks in this segment of the industry, the following items are of extreme importance in medical certifications: proximity to rotating elements, noise, effects of chemicals or biological factors, including carcinogenic substances (wood dust). for these reasons the procedures involved in medical/dental examinations are very complex24. although influenza vaccine is considered effective in preventing influenza and its complications, there are controversies about the advantages of its use in healthy adults. this survey was developed to characterize the common occurrence of respiratory problems, particularly the flu, and evaluate a program of immunization against influenza between workers. it was a study of cross-sectional delineation with industry workers. data were collected on sociodemographic and respiratory problems. occurrence of the flu in the past year was characterized and the vaccine was assessed. 446446446446446medical and dental absenteeism in workers from a furniture industry in itatiba, sp, brazil braz j oral sci. 9(4):443-448 among the 446 respondents, 18.3% presented strong incidence of flu and 11.7% were dismissed from work due to influenza. overall, 70% of the employees noticed that there was an improvement in the occurrence of flu after vaccination. the positive assessment of vaccination made by workers and the low occurrence of side effects support the maintenance of the current program against influenza. however, several questions still persist about the economic advantages of vaccine, indicating the need of further studies on this subject25. the developed societies reached the understanding that the preservation of health and physical integrity of workers provides personal well-being, family and social, as well as results in greater productivity for the company and reduce expenses with insurance and indemnities, etc. ensuring the physical and mental health of the worker is no longer seen as an act of charity, and has assumed the international features of social obligation, insofar as the prevention of accidents and occupational diseases decrease absenteeism and collaborates to the increase of national production, reducing spending with social security affairs26. the conducted study revealed that most of the sick leaves were up to 8 hours, which usually is not insufficient time to cure any disease. it can be a work day loss due to a simple lab test or minor procedure, but it can also be “disease caused”. medical and dental licenses are often used by the worker as a form of aggression against the chiefs and the institution. it also represents an escape for social, economic and psychological tensions. it is a critical point to be attacked in the struggle against absenteeism27. the federal council of medicine resolution no. 1.851/ 2008 that regulates the issuance of medical certificates11 has established that the physician should specify the granted leave of absence time required for the recovery of the patient; establish the diagnosis, when expressly authorized by the patient; inform legibly the date and his/her full name, sign the certificate and stamp or put in writing the number of registry in the regional council of medicine11. when the certificate is required by the patient or his legal representative for the purposes of medical expertise it must contain: the diagnosis, the results of complementary examinations, conducted therapy, prognosis; consequences to the patient’s health, the estimated rest time necessary for recovery, legible certificate date, and full identify of the health professional. the employee may leave the service without loss of wage and with term provided for by the law for a period of up to 15 (fifteen) days in the case of absence by illness or accident by a medical certificate and observed the law tax benefit. any type of medical certificates does not require the industry to receive their absence to the service, unless the hypothesis of employed pregnant woman which should be exempted from working hours for the time necessary for holding at least 6 medical consultations and other additional tests, according to art. 392, subparagraph ii of working law code. an expressive number of declaration variations (n=284, 30.1%) in the total of medical and dental certificates in our study were approved by the human resources department. the loss of productivity and competitive capacity of industries are directly linked to the number of working hours lost compared to those that are planned. the absenteeism has long been simply treated by focusing on the certificates that justified the absences of workers. however, we know today that several factors within and outside the industry, affect the absenteeism. some factors are manageable and may, with the due care, reduce significantly the risks in the planning of the companies. the first step is to quantify the losses and identify the factors that generate them. as an example of internal factor causing faults is the lack of clarity in the personnel management policies (what is the benefit and what is legal compliance is often not clear to the worker), the lack of comfort at the desk, valuation of the professional, the pace of work, the ranges, accidents and incidents. the programs of medical control of occupational health, as described in nr-07, promotion and preservation of workers’ health, are based on tracking and prevention, early diagnosis of grievances on the work-related health, in addition to noting cases of occupational disease or irreversible damage to the health of workers. all data obtained during medical examinations and diagnostic findings must be registered in individual clinical records and kept for a minimum of 20 years after the worker leaves the premises, according to the regulatory standard 7.4.5.1 item no. 728. prevention programs of environmental risks – nr-09 – must include the recognition of environmental risks (physical, chemical and biological agents) occurring in working environments and which are capable of causing harm to workers’ health, as well as the implementation of control measures. health promotion at work is valuable in ability to promote well-being and productive in terms of fewer absences on grounds of illness. activities involving the exercise, the quality of life and the ergonomics are potentially effective. and psychological applied alone does not seem effective. health promotion at work should have a joint action between the physical and psychosocial environment29. invest in the well-being of the worker is much more than putting social responsibility; it should be seen as a protection to the business, which may be slowing down due to improper observation of people management. thus, it is understood that the management of human resources is a strategy and may be the great competitive differentiator in most industry branches30. in conclusion, based on the obtained results and considering the applied methodology, is that: 1the absenteeism in the furniture industry was more frequently due to medical rather than dental reasons; the fouls and long duration of absences from work lead to problems arising from the reduced number of employees to complete the tasks at the work place; 2there were several certificates lacking the international classification of diseases; 3the number of lost work hours due to medical and dental problems was found to be a significant economic factor. 447447447447447 medical and dental absenteeism in workers from a furniture industry in itatiba, sp, brazil braz j oral sci. 9(4):443-448 references 1. queluz dp. labour dentistry: a new specialty in dentistry. braz j oral sci. 2005; 4: 766-72. 2. queluz dp. a multidisciplinaridade da odontologia do trabalho colaborando com a saúde bucal dos trabalhadores. in: silva e, martins i. odontologia do trabalho: construção e conhecimento. rio de janeiro: rubio; 2008. p.91-109. 3. queluz dp. odontologia do trabalho. in: pereira ac, organizador. tratado de saúde coletiva em odontologia. nova odessa: napoleão; 2009. p.217-38 4. european research report. fundação européia para a melhoria das condições de vida e de trabalho; 1997. preventing absenteeism at the workplace. 5. gaidzinski, rr. dimensionamento de pessoal de enfermagem segundo a percepção de enfermeiros que vivenciam essa prática [thesis]. são paulo: escola de enfermagem, universidade de são paulo, são paulo; 1994. 258p. 6. midorikawa et. a odontologia como saúde do trabalhador como uma nova especialidade profissional: definição do campo de atuação e funções do cirurgião-dentista na equipe de saúde do trabalhador [dissertation]. 2000. são paulo: faculdade de odontologia da universidade de são paulo; 2000 7. world health organization. who technical report series 916. geneva; 2003. available from: http://www.int/dietphysicalactivity/publications/trs916. [access 2009 nov]. 8. brasil. ministério da saúde. projeto sb brasil: condições de saúde bucal da população brasileira 2002-2003. brasília: df; 2004. 9. lima jrs. absenteísmo por causa odontológica: análise comparativa entre funcionários da prefeitura do município de são josé dos campos e segurados do instituto nacional de previdência socialinamps. available from: http://www.odontologia.com.br. [access 2009 nov]. 10. nardi a. dor orofacial, absenteísmo e qualidade de vida em trabalhadores do sul do brasil [dissertation]. joaçaba: universidade do oeste de santa catarina; 2005. 192p. 11. conselho federal de medicina. available from: http://www.cfo.org.br. [access 2009 nov]. 12. conselho federal de odontologia. available from: http://www.cfo.org.br. [access 2009 nov]. 13. microsoft corp. excel, release 2007. microsoft corp, richmond: ca; 2007. 14. sas institute inc. the sas system, release 9.2 – sp4. sas institute inc, cary: nc; 2002. 15. serviço social da indústriasesi. perfil do trabalhador formal brasileiro, 2005. available from: http://www.sesi.org.br. [access 2009 nov]. 16. brasil. ministério do trabalho e emprego. cbo classificação brasileira de ocupações. available from: http://www.mtecbo.gov.br. [access 2009 nov]. 17. peres shcs, peres as, oliveira ft, adachi a, silva al, morandini acf. absenteeism: a revision of the literature about the absence to the work related to the dentistry. rev odontol araçatuba. 2006; 27: 96-100. 18. grzywacz jg, butler ab. the impact of job characteristics on work-tofamily facilitation: testing a theory and distinguishing a construct. j occupat health psychol. 2005; 10: 97-109. 19. martins rj, saliba ca, garbin aji, moimaz sas. absenteísmo por motivos odontológico e médico nos serviços público e privado. rev bras saude ocupac. 2005; 30: 9-15. 20. reisine s, miller j. a longitudinal study of work loss related to dental diseases. soc sci med. 1985; 21: 1309-14. 21. mazzili len, crosato e. análise dos afastamentos do trabalho por motivo odontológico em servidores públicos municipais de são paulo submetidos à perícia ocupacional no período de 1996 – 2000. rpg-rev pós grad. 2005; 12: 444-53. 22. di leone g, trani g, falco s, attimonelli r, carino m, lagravinese d. from mandatory to participatory prevention: a model applied to the upholstered furniture industry. med lav. 2008; 99: 314-8. 23. cruz rm, maciel sk. perícia de danos psicológicos em acidentes de trabalho. est pesq psicol. 2005; 5: 120-9. 24. romankow j. medical certification in workers involved in logging and wood processing. arch med sadowej kryminol. 2007; 57: 89-94. 25. faria nmx, gianisella jf. prevalence of respiratory diseases and evaluation of the influenza vaccination program for workers. rev bras epidemiol. aug. 2002; 5: 174-84. 26. domingues jmm. exame médico periódico [monografia]. campo grande: sociedade universitária estácio de sá; 2002. available from: http:// www.fundacentro.sc.gov.br. [access 2009 nov]. 27. quick tc, lapertosa jb. análise do absentismo em usina siderúrgica. rev bras saude ocup. são paulo, 1982; 10: 62-7. 28. brasil, ministério do trabalho e emprego. normas regulamentadoras. avalable from: http://www.mte.gov.br. [access 2009 nov]. 29. 29 kuoppala j, lamminpaa a, husman p. workhealth promotion, job well-being, and sickness absences a systematic review and metaanalysis. j occup environ med. 2008; 50: 1216-27. 30. serviço social da indústriasesi. available from: http://www.sesi.org.br. [access 2009 nov] 448448448448448medical and dental absenteeism in workers from a furniture industry in itatiba, sp, brazil braz j oral sci. 9(4):443-448 oral sciences n3 braz j oral sci. 10(3):189-192 original article braz j oral sci. july | september 2011 volume 10, number 3 microhardness of nanofilled composite resin light-cured by led or qth units with different times ana isabelle salvador groninger1, giulliana panfiglio soares2, robson tetsuo sasaki3, glaucia maria bovi ambrosano4, josé roberto lovadino5, flávio henrique baggio aguiar6 1undergraduate student, department of restorative dentistry, piracicaba dental school, university of campinas, brazil 2dds, ms, phd student, department of restorative dentistry, piracicaba dental school, university of campinas, brazil 3dds, ms student, department of restorative dentistry, piracicaba dental school, university of campinas, brazil 4dds, ms, phd, assistant professor, department of community dentistry and statistics, piracicaba dental school, university of campinas, brazil 5dds, ms, phd, chairman, department of restorative dentistry, piracicaba dental school, university of campinas, brazil 6dds, ms, phd, assistant professor, department of restorative dentistry, piracicaba dental school, university of campinas, brazil correspondence to: flávio henrique baggio aguiar departamento de odontologia restauradora, fop/unicamp av. limeira, 901 piracicaba, sp brasil cep 13414-018 phone: 55 19 2106 5340 fax: 55 19 3421 0144 e-mail: aguiar@fop.unicamp.br abstract aim: to evaluate the influence of light-curing units and light-curing time on the microhardness of a nanofilled composite resin. methods: forty-five composite resin (z350 3m) specimens were randomly prepared using teflon ring molds (4.0 mm internal diameter and 2 mm depth) and divided into nine experimental groups (n=5): three polymerization units (conventional 450 mw/ cm2; 2nd generation led 1100 mw/cm2; and 3rd generation led 700 mw/cm2) and three lightcuring times (20 s, 40 s, and 60 s). all specimens were polymerized with the light-curing tip positioned 8 mm far from the top surface of the specimen. after 24 h, knoop microhardness measurements were made on the top and bottom surfaces of the specimen, with a load of 10 g for 10 s. five indentations were made on each surface. all results were analyzed statistically by subdivided parcel anova (split-plot) and tukey’s tests (p<0.05). results: there were no statistically significant differences for the polymerization unit and light-curing time factors in either top or bottom surface. for all experimental conditions, the top surfaces showed greater hardness than the bottom surfaces (p<0.0001). conclusions: the mode of polymerization and the lightcuring time did not affect the hardness of the nanofilled composite resin, and increasing the lightcuring time did not improve the hardness of the bottom surface of the composite resin. keywords: composite resins, hardness. introduction composite resin has been described as an esthetic restorative material with excellent physical and mechanical properties1 when adequate polymerization is obtained2. however, many variables affect the amount of light energy received on the top and bottom surfaces of a composite resin restoration, such as the design and size of the light guide, distance of the light guide tip from the composite resin, power density, exposure duration, shade and opacity of the composite resin, increment thickness, and material composition3-5. if the restoration does not receive sufficient total energy, various problems may arise, e.g., reduced degree of conversion, increased cytotoxicity, reduced hardness, increased pigmentation, decreased dynamic elasticity modulus, increased wear, increased marginal leakage and weak a bond among the tooth, adhesive, and restoration3-4,6. received for publication: july 12, 2010 accepted: august 19, 2011 braz j oral sci. 10(3):189-192 190 clinically, deficient polymerization can occur in deeper cavities due to the dispersion of light energy that occurs because of the distance between the light-curing tip and the first composite resin increment7. in a deeper cavity, the interface between the composite resin and the tooth structure may be less polymerized, and exposure of this interface to the oral environment can generate marginal discolorations, restoration fractures, and composite resin and adhesive solubility, leading to microleakage and secondary caries7. however, few studies have been conducted with the purpose of testing the depth of composite resin curing in situations where the light-curing tip is distant from the filling material, as in the aforementioned clinical situations. thus, it is important to evaluate the minimum light-curing time required for correct polymerization in accordance with the light-curing unit used. the aim of this in vitro study was to evaluate the influence of the light-curing time using led or qth on the hardness of the top and bottom composite resin surfaces in a clinical simulation when the light-curing tip was at distance of 8 mm and the material thickness was 2 mm. material and methods a nanofilled composite resin, z350 (3m-espe dental products, st. paul, mn, usa), was used in this study. fortyfive cylindrical specimens were prepared using teflon ring molds (4.0 mm internal diameter and 2 mm depth) held between two glass slabs separated by mylar matrix strips and then pressed with a 500 g static load. the cavities were randomly filled in one resin increment and polymerized according to the nine experimental groups (n=5): three polymerization units (conventional 450 mw/cm2; 2nd generation led 1100mw/cm2; 3 rd generation led 700mw/cm2) and three light-curing times (manufacturer’s recommended time 20s; twice the manufacturer’s recommended time 40s; and thrice the manufacturer’s recommended time 60s). polymerization was performed with a source-to-specimen distance of 8 mm, checked with a digital caliper (mitutoyo, usa). irradiance of the light curing units was measured using a curing radiometer (demetron research corp., danbury, ct, usa). the energy density was calculated according to the product of the irradiance of the light curing unit (mw/cm2) x exposure duration (s) (table 1). each specimen was removed from the mold and stored in a lightproof container at 37c and 95 ± 5% relative humidity for 24 h. after this period, the specimens were washed and the knoop hardness on the bottom and top surfaces was tested using a knoop hardness indenter (fm future tech corp., japan) under a 10 g load for 10 s. five measurements were made at the approximate center of the specimen4. the values, obtained in micrometers, were converted to knoop hardness number (khn) using computer software (microsoft excel for windows®). the knoop hardness values obtained on top and bottom surfaces were subjected to the subdivided parcel anova (split-plot) test (p=0.05) and tukey’s test at the 5% significance level. the light-curing unit and light-curing time factors were considered in the parcels, and the factor surface (top and bottom surfaces) was considered in the sub-factor. results the anova results showed no statistically significant differences among the light-curing units (p=0.3007) or among the light-curing times (p=0.1819). the interactions of the light-curing mode x light-curing time (p=0.2224), table 2 mean hardness knoop number (khn) according to the light-curing units, the light-curing times and specimen surface. results of the hardness means (khn). mean values with the same uppercase letter were not statistically different (p<0.05) in the comparison of the surfaces. microhardness of nanofilled composite resin light-cured by led or qth units with different times table 1 light-curing units, manufacturers, radiant incidence, light-curing time, and energy density of each experimental group * irradiance was measured using a curing radiometer. 191 braz j oral sci. 10(3):189-192 light-curing mode x surface (p=0.3136), surface x lightcuring time (p=0.1036), and light-curing mode x light-curing time x surface (p=0.0705) also were not significant. there was a significant difference between the surfaces (p <0.0001) in that, for all the experimental conditions, the top surface showed higher hardness values than the bottom surface (table 2). considering the ratio between the bottom and top surface hardness values (table 3), the highest ratio was obtained by bluephase 16i with 40 s light-curing time (@ 0.75). in the other experimental conditions, the ratio was lower than 0.6. discussion the present study evaluated the influence of light-curing units and light-curing times on the microhardness of top and bottom nanofilled composite resin surfaces. the results showed that, for the top and bottom surfaces, there were no statistically significant differences among light-curing units or among lightcuring times. for all experimental conditions, the top surface showed higher hardness than the bottom surface. the depth of cure for composite resin can be affected by several factors associated with the source of light polymerization, including the spectral emission (wavelength distribution), light intensity, exposure period, and irradiation distance4,8-11. among these factors, the radiant incidence of light given out by different light-curing units and the lightcuring times were analyzed in this study. the results showed that these two factors were not able to significantly affect the microhardness of polymerization on the top and bottom surfaces. in other words, when the light-curing time recommended by the manufacturers (20 s) was doubled or tripled, the hardness values did not increase. regarding the light-curing units used (bluephase 16i vivadent, ultra-lume led 5 ultradent, xl 3000 3m espe), there were no statistically significant differences among them, independent of the light-curing time used and of the energy density at the tip of the light-curing units (table 2). energy dispersal can take place when polymerization happens with the tip of the light-curing unit at a distance from the surface of the composite resin, and this approximate irradiance that reaches the surface is independent of the irradiance released by the light-curing unit 12 (table 1). similarly, in a study conducted by aguiar et al.12, there were no statistically significant differences between the top surfaces of the specimens. the study also showed that when the lightcuring time recommended by the manufacturer was tripled, higher hardness values were obtained on the bottom surface. however, gomes et al.13 and awliya14 obtained different hardness values when comparing the light-curing of led and halogen lights. in the present study, there was no significant difference for the studied factors on the bottom surface. the difference in the results between the present study and the studies mentioned above can be explained by two hypotheses: 1) light penetration on the bottom surface of the nanofilled resin might have happened in a similar way, regardless of the light-curing mode or photoactivation time; and 2) nanofilled resin presents better physical properties when compared with other resins15-16 and, due to its high load content (78.5% by weight), the degree of polymerization was not the main factor in the hardness values obtained. in a recent study17, light-curing units with different irradiances did not result in significant hardness differences on the bottom surface when a nanofilled composite with similar translucency was used. in the same study, higher radiant exposure increased the hardness of the bottom surface only for the composite resins with higher opacities (dentin shade). thus, although the hardness of the bottom surface was significantly lower than that of the top surface for all groups in the present study, increased radiant exposure did not improve the hardness of the bottom surface for composite resins with translucent characteristics. in addition, the nanofilled composite resin used in the present study showed an adequate entanglement between the resin components and the nanofillers, which improves the physical properties of the material18. thus, possibly even with a lower degree of conversion, the bottom surface of the specimen may have showed similar hardness due to the filler system used. another finding of this study was that, for all the experimental conditions, the top surface showed higher hardness values than the bottom surface. this could be due to the capacity of the composite resin to reduce light penetration, which causes a reduction in the irradiance and, consequently, the efficiency of polymerization of the bottom surface7,12,19-21. furthermore, on the top surface, high irradiance photoactivation initiated a multitude of growth centers of polymers with higher cross-linking density22-23. low irradiant energy reached the bottom surface, decreasing the crossmicrohardness of nanofilled composite resin light-cured by led or qth units with different times table 3 hardness ratios between the bottom and top surfaces 192 braz j oral sci. 10(3):189-192 linking density and the hardness of the composite resin24-25. yap et al.20 affirmed that the hardness ratio between the bottom and top must be 1 to consider the polymerization completely efficient, but even a proportion of “approximately 0.8” can be considered an appropriate polymerization. in this study, the results showed that the proportion was between 0.50 and 0.74 for groups in which the bluephase 16i lightcuring unit was used, between 0.51 and 0.59 for groups in which the ultra-lume led 5 light-curing unit was used, and between 0.45 and 0.59 for groups in which the xl-3000 lightcuring unit was used (table 3). this shows that, although not presenting a ratio between 0.8 and 1.0, as suggested by yap et al.20, the ratios obtained in the present study were higher in relation to the study of aguiar et al.12 so it may be suggested that the nanofilled composite resins showed higher hardness on the bottom surface than the micro-hybrid resins. it may be concluded that the mode of polymerization and the light-curing time did not affect the hardness of the nanofilled composite resin and that increasing the light-curing time did not improve the hardness of the bottom surface of the composite resin. further studies must be carried out to compare the best efficiency of the nanofilled resin compared with other resins used for subsequent teeth and to determine whether the degree of conversion of the compound nanofilled resin interferes with the superficial hardness. acknowledgements this study was supported by fapesp (grant # 06/61136-3). references 1. della bona a, rosa v, cecchetti d. influence of shade and irradiation time on the hardness of composite resins. braz dent j. 2007; 18: 231-4. 2. knezeviæ a, tarle z, meniga a, sutalo j, pichler g, ristiæ m. degree of conversion and temperature rise during polymerization of composite resin samples with blue diodes. j oral rehabil. 2001; 28: 586-91. 3. yap au. effectiveness of polymerization in composite restoratives claiming bulk placement: impact of cavity depth and exposure time. oper dent. 2000; 25: 113-20. 4. price rb, derand t, loney rw, andreou p. effect of light source and specimen thickness on the surface hardness of resin composite. am j dent. 2002; 15: 47-53. 5. sobrinho lc, goes mf, consani s, sinhoreti ma, knowles jc. correlation between light intensity and exposure time on the hardness of composite resin. j mater sci mater med. 2000; 11: 361-4. 6. ferracane jl, greener eh. fourier transform infrared analysis of degree of polymerization in unfilled resins—methods comparison. j dent res. 1984; 63: 1093-5. 7. prati c, chersoni s, montebugnoli l, montanari g. effect of air, dentin and resin-based composite thickness on light intensity reduction. am j dent. 1999; 12: 231-4. 8. yearn ja. factors affecting cure of visible light activated composites. int dent j 1985; 35: 218-25. 9. atmadja g, bryant rw. some factors influencing the depth of cure of visible light-activated composite resins . aust dent j. 1990; 35: 213-8. 10. mills rw, uhl a, jandt kd. optical power outputs, spectra and dental composite depths of cure, obtained with blue light emitting diode (led) and halogen light curing units (lcus). br dent j. 2002; 193: 459-63. 11. fan pl, schumacher rm, azzolin k, geary r, eichmiller fc. curinglight intensity and depth of cure of resin-based composites tested according to international standards. j am dent assoc. 2002; 133: 29-34. 12. aguiar fh, braceiro a, lima da, ambrosano gm, lovadino jr. effect of light curing modes and light curing time on the microhardness of a hybrid composite resin. j contemp dent pract. 2007; 8: 1-8. 13. gomes gm, calixto al, santos fa, gomes om, d’alpino ph, gomes jc. hardness of a bleaching-shade resin composite polymerized with different light-curing sources. braz oral res. 2006; 20: 337-41. 14. awliya wy. the influence of temperature on the efficacy of polymerization of composite resin. j contemp dent pract. 2007; 8: 9-16. 15. beun s, glorieux t, devaux j, vreven j, leloup g. characterization of nanofilled compared to universal and microfilled composites. dent mater. 2007; 23: 51-9. 16. mota eg, oshima hm, burnett lh jr, pires la, rosa rs. evaluation of diametral tensile strength and knoop microhardness of five nanofilled composites in dentin and enamel shades. stomatologija. 2006; 8: 67-9. 17. albino lgb, rodrigues ja, kawano y, cassoni a. knoop microhardness and ft-raman evaluation of composite resins: influence of opacity and photoactivation source. braz oral res. 2011; 25: 267-73. 18. moraes rr, gonçalves ls, lancellotti ac, consani s, correr-sonbrinho l, sinhoreti ma. nanohybrid resin composites: nanofiller loaded materials or traditional microhybrid resins? op dent. 2009; 34: 551-7. 19. sobrinho lc, de lima aa, consani s, sinhoreti ma, knowles jc. influence of curing tip distance on composite knoop hardness values. braz dent j. 2000; 11: 11-7. 20. yap au, wong ny, siow ks. composite cure and shrinkage associated with high intensity curing light. oper dent. 2003; 28: 357-64. 21. yoon th, lee yk, lim bs, kim cw. degree of polymerization of resin composites by different light sources. j oral rehabil. 2002; 29: 1165-73. 22. schneider lf, pfeifer cs, consani s, prahl sa, ferracane jl. influence of photoinitiator type on the rate of polymerization, degree of conversion, hardness and yellowing of dental resin composites. dent mater. 2008; 24: 1169-77. 23. meng x, yoshida k, atsuta m. influence of ceramic thickness on mechanical properties and polymer structure of dual-cured resin luting agents. dent mater. 2008; 24: 594-9. 24. calheiros fc, daronch m, rueggeberg fa, braga rr. influence of irradiant energy on degree of conversion, polymerization rate and shrinkage stress in an experimental resin composite system. dent mater. 2008; 24: 1164-8. 25. peutzfeldt a, asmussen e. resin composite properties and energy density of light cure. j dent res. 2005; 84: 659-62. microhardness of nanofilled composite resin light-cured by led or qth units with different times oral sciences n3 braz j oral sci. 10(4):254-257 original article braz j oral sci. october | december 2011 volume 10, number 4 surgical dental treatment prior to liver transplantation paulo sérgio da silva santos1, maria izabel sarmento e souza pacheco2, mateus pereira alonso soler3, valtuir barbosa felix4 1dds, md, phd, department of stomatology, bauru school of dentistry, university of são paulo, brazil 2dds, department of oral and maxillofacial surgery, santa casa de misericórdia of são paulo, são paulo, brazil 3dds, department of oral and maxillofacial surgery, hospital regional sul, são paulo, brazil 4dds, md, phd, department of oral and maxillofacial surgery, hospital regional of osasco, são paulo, brazil correspondence to: paulo sérgio da silva santos departmento de estomatologia, fob/usp alameda octávio pinheiro brisolla, 9-75,17012-901 bauru-sp phone: +55 14 3235-8373, fax: +55 14 3235-8000. e-mail: paulosss@fob.usp.br abstract oral-source infections are a potential threat for transplant candidates because oral diseases tend to be more severe and untreated in people who have received transplants. although not yet scientifically proven, evaluation and dental treatment during the pre-transplantation period is recommended in order to prevent infections and resultant odontogenic-origin sepsis during the post-transplant period, when patients receive immunosuppressive therapy. aim: to evaluate the invasive dental procedures for removing dental foci performed in patients scheduled for liver transplantation and its accompanying complications. methods: the medical records of 33 preliver transplant recipients who were undergoing invasive dental procedures were reviewed, including their laboratory tests, special handling needs and resulting complications. results: fifty invasive dental procedures were carried out on the 33 patients. three of them were subjected to basic periodontal treatment and 47 were subjected to multiple or simple extractions. three surgical procedures resulted in postoperative complications. conclusions: surgical intervention to remove dental foci in liver disease patients requires careful clinical evaluation, laboratory tests, knowledge and skills in the use of local and systemic hemostatic procedures, and a partnership approach between dentists and physicians, in order to reduce the risk of complications. keywords: surgery, oral, hemostatic techniques, organ transplantation. introduction dental treatment, including the elimination of oral and mucosal infection foci, is essential for transplant patients during both preparation and post-surgery periods; patients should be under constant dental care and undergo mycological tests, especially in the first months1-5. oral-source infections are a potential threat to transplant candidates because oral diseases tend to be more severe if left untreated in people who have received transplants3,6. although no evidence-based guidelines for dental septic focal treatment exist, patients should be advised to have dental foci removed before undergoing organ transplants in order to avoid post-transplant systemic and local oral complications. in addition, these patients should be organized into a dental or oral maxillofacial surgical follow-up program7. teeth that need to be restored but are impossible to treat, as well as those with advanced periodontal disease, must be extracted. non-vital teeth (at risk of received for publication: may 26, 2011 accepted: december 07, 2011 34 braz j oral sci. 10(4):254-257 255 infection) must be extracted or endodontically treated, and all active caries lesions should be treated and resulting cavities should be restored8. for any type of oral surgery, the patient must have an intact hemostatic process because even the least invasive procedures can lead to severe bleeding, increased risk of infection, poor wound healing and airways may be compromised if there is bleeding in critical fascial spaces9. any liver disease that affects the synthesis of clotting factors may manifest itself by an increase of prothrombin time (pt), which reflects the extrinsic pathway for the coagulation mechanism. the activated partial thromboplastin time (aptt) reflects all clotting factors except platelet factor iii and factors xiii and vii and is indicative of the function of the coagulation mechanism’s intrinsic pathway. therefore, before any surgical procedure, the preoperative evaluation of these patients must include a complete blood count (cbc), pt, international normalized ratio (inr) and aptt9. this research aimed at evaluating invasive dental procedures for removing dental foci in patients scheduled for liver transplantation along with any accompanying complications. material and methods after approval by the research ethics committee for the santa casa de misericórdia de são paulo, this study evaluated 33 prospective patients who were candidates for liver transplants and had been receiving treatment in a dentistry department for patients with special needs, during 3 years, according to the indication/medical records maintained by the liver transplant team. all potential candidates in the liver transplant program from regional hospital of são paulo, brazil, during this period were included in the study. the 33 selected patients consisted of 25 (75.8%) males and 8 (24.2%) females, with a mean age of 53.15 years. they underwent clinical, radiographic (panoramic and periapical x-rays) and laboratory (cbc, pt/inr, aptt) examinations for diagnosis and planning of dental treatment, totalizing 50 procedures. tooth extraction criteria were: presence of residual roots; extensive carious lesions with partial crown destruction and risk of pulpal involvement; teeth with periapical lesions; semi-erupted teeth; teeth with periodontal involvement; as well as the capacity and interest of the patient in caring for and maintaining oral hygiene. the periodontal treatment consisted of scaling and root planing divided into several sections, starting from the supra-gingival area, gingival and subgingival level for cases of mild to moderate periodontitis, and prescription of 5% tranexamic acid mouthwash three times a day during 7 days. patients were subjected to oral surgery for dental extraction to remove odontogenic foci, and received systemic antifibrinolytic therapy according to laboratory parameters inr > 3.0, aptt> 60 sec and platelet count <30,000 cells/µl). local hemostasis techniques, applied to all patients, consisted of: application of a tranexamic acid paste (macerated pill mixed with saline or anesthetic solution) in order to fill the alveolar socket after dental extractions; the placement of fibrin sponge that was properly trimmed and well adapted to the alveolar socket10; suture with maximum coaptation of the gingival edge and application of another layer of tranexamic acid paste applied to the wound11. the systemic hemostasis technique consisted of the administration of blood derivate or tranexamic acid12 (500 mg, 3 times day), starting 2 days before the procedure and ending 3 days after. results fifty invasive dental procedures were carried out on the 33 patients. three of them were subjected to basic periodontal treatment and 47 were subjected to multiple (2 or more teeth, n = 39) or single (1 tooth, n = 8) extractions. three invasive procedures resulted in postoperative complications: 1 (3.03%) single post-extraction infectious alveolitis and 2 (6.06%) cases of postoperative bleeding as a result of a simple extraction and a multiple extraction (table 1). evidence of poor oral hygiene in this group of patients was clearly demonstrated by the number of tooth extractions needed. all patients had altered laboratory results (cbc – platelet count, pt/inr, aptt/rt), consistent with the clinical status of end-stage liver disease. the only infectious complication was a dry socket, which was treated by removing sutures, intra-alveolar curettage and application of zinc oxide and eugenol paste13. the hemorrhagic complications were treated by removal of sutures, intra-alveolar curettage, irrigation with saline and 2% chlorhexidine, application of tranexamic acid paste in the alveolar socket, fibrin sponge placement, suture, application of another layer of tranexamic acid paste and daily monitoring within the first 72 h postsurgery. discussion although there are no evidence-based guidelines for dental septic focal treatment, patients should be advised to have their dental foci removed before undergoing organ transplants and receiving immunosuppressive therapy. meticulous clinical examination, including extraoral and intraoral, and radiographic examinations (panoramic and periapical x-rays) are important to find infectious foci and to establish the diagnosis and the development of an adequate treatment plan. these examinations are all part of the protocol for pre-organ-transplant dental evaluation. laboratory assessment before dental surgery, which includes blood cell count, pt, aptt and platelet count, is mandatory for assessing the risk of bleeding during surgery or in the postoperative period, and provides the dentist and the medical team with guidance parameters that will demonstrate if there is risk of surgical or clinical complications during the monitoring of this group of patients (figure 1). the patients in this study sample had poor oral conditions, with the presence of plaque and calculus, periodontitis, extensive carious lesions, residual roots, surgical dental treatment prior to liver transplantation braz j oral sci. 10(4):254-257 256 infection by candida and xerostomia. radiographic examination revealed periapical lesions, unerupted teeth, and alveolar ridge resorption due to periodontitis. these clinical and radiographic findings are consistent with our research findings, which show that liver disease patients are in large part, consumers of alcohol and tobacco, and have poor oral hygiene5,14. strategies for improving the preventive dental care are needed15 for this group and the role of the multidisciplinary team is essential16. in this study sample, despite the significantly altered laboratory tests and surgical procedures carried out, the rate of complications after dental surgical procedures in the liver transplant candidates (6.06%) was lower than that found by niederhagen et al. (2003)17 (15.4%) and fairly consistent with table 1 data from the gender, diagnosis and laboratory evaluation of liver transplant candidates, and total procedures and complications during the dental management. abreviations: prothrombin time (pt), prothrombin activity (pa), international normalized ratio (inr), activated partial thromboplastin time (aptt) that found by stanca et al. (2010)18 (5%). the few complications were of hemorrhagic and infectious nature and were easily controlled. our results suggest that proper surgical techniques associated with local and systemic haemostatic procedures are effective in preventing complications associated with coagulation disorders that liver disease patients may develop. of the 33 patients in this study, 2 had previously received blood transfusions, and 31 patients were treated only with local hemostatic maneuvers. in two cases, when bleeding during surgery was greater than expected, a successful intravenous administration of vitamin k was carried out. the reduction in salivary flow and the presence of candidiasis found in some patients may be associated with the use of diuretics for the treatment of ascites and edema, surgical dental treatment prior to liver transplantation braz j oral sci. 10(4):254-257 fig. 1. algorithm criteria for dental care to patients scheduled for liver transplantation frequent complications of cirrhosis, which increases the depos ition and retention of plaque as well as the susceptibility of opportunistic infections. this condition requires special attention from the dental professional to prevent and treat opportunistic infections. solid organ transplant patients have a high risk of developing squamous cell carcinoma of the upper digestive tract, which is also associated with prolonged use of immunosuppressant drugs, but it is primarily attributed to smoking5. this condition requires special care when monitoring these patients in the post-transplant period. most patients in this study reported not having received any dental monitoring because dental professionals reported lack of knowledge on management techniques and treatment of liver disease and transplant patients. this fact corroborates the findings of guggenheimer et al. (2007)4, who studied patients undergoing liver transplant5. the need for radical prophylactic dental sanitation should be further studied so that appropriate protocols are established for these patients. in conclusion, the surgical intervention to remove dental foci in liver disease patients requires careful clinical evaluation, laboratory tests, knowledge and skills in the use of local and systemic hemostatic maneuvers, and a well established partnership approach between dentists and physicians in order to maintain a low risk of complications. acknowledgements we thank the santa casa de misericórdia de são paulo for the assistance given to us throughout our research. references 1. wakefield cw, throndson rr, brock t. liver transplantation: dentistry is an essential part of the team. j tenn dent assoc. 1995; 75: 9-16. 2. douglas lr, douglass jb, sieck jo, smith pj. oral management of the patient with end-stage liver disease and the liver transplant patient. oral surg oral med oral pathol oral radiol endod. 1998; 86: 55-64. 3. guggenheimer j, mayher d, eghtesad b. a survey of dental care protocols among us organ transplant centers. clin transplant. 2005; 19: 15-8. 4. guggenheimer j, eghtesad b, close jm, shay c, fung jj. dental health status of liver transplant candidates. liver transpl. 2007; 13: 280-6. 5. golecka m, mierzwiñska-nastalska e, oldakowska-jedynak u. influence of oral hygiene habits on prosthetic stomatitis complicated by mucosal infection after organ transplantation. transplant proc. 2007; 39: 2875-8. 6. ribeiro pm, bacal f, koga-ito cy, junqueira jc, jorge ao. presence of candida spp. in the oral cavity of heart transplantation patients. j appl oral sci. 2011; 19:6-10. 7. rustemeyer j, bremerich a. necessity of surgical dental foci treatment prior to organ transplantation and heart valve replacement. clin oral investig. 2007; 11: 171-4. 8. little jw, rhodus nl. dental treatment of the liver transplant patient. oral surg oral med oral pathol. 1992; 73: 419-26. 9. ziccardi vb, abubaker ao, sotereanos gc, patterson gt. maxillofacial considerations in orthotopic liver transplantation. oral surg oral med oral pathol. 1991; 71: 21-6. 10. medeiros fb, de andrade ac, angelis ga, conrado vc, timerman l, farsky p, et al.. bleeding evaluation during single tooth extraction in patients with coronary artery disease and acetylsalicylic acid therapy suspension: a prospective, double-blinded, and randomized study. j oral maxillofac surg 2011; 69: 2949-55. 11. hewson i, makhmalbaf p, street a, mccarthy p, walsh m. dental surgery with minimal factor support in the inherited bleeding disorder population at the alfred hospital. haemophilia. 2011; 17: e185-8. 12. givol n, goldstein g, peleg o, shenkman b, zimran a, elstein d, et al.. thrombocytopenia and bleeding in dental procedures of patients with gaucher disease. haemophilia. 2011; may 5. doi: 10.1111/j.13652516.2011.02540.x. [epub ahead of print] 13. oginni fo, fatusi oa, alagbe ao. a clinical evaluation of dry socket in a nigerian teaching hospital. j oral maxillofac surg. 2003; 61: 871-6. 14. lins l, bittencourt pl, evangelista ma, lins r, codes l, cavalcanti ar, et al.. oral health profile of cirrhotic patients awaiting liver transplantation in the brazilian northeast. transplant proc. 2011; 43: 1319-21. 15. shiboski ch, kawada p, golinveaux m, tornabene a, krishnan s, mathias r, et al.. oral disease burden and utilization of dental care patterns among pediatric solid organ transplant recipients. j public health dent. 2009; 69: 48-55. 16. gelb b, feng s. management of the liver transplant patient. expert rev gastroenterol hepatol. 2009;3: 631-47. 17. niederhagen b, wolff m, appel t, von lindern jj, bergé s. location and sanitation of dental foci in liver transplantation. transpl int. 2003; 16: 173-8. 18. stanca cm, montazem ah, lawal a, zhang jx, schiano td. intranasal desmopressin versus blood transfusion in cirrhotic patients with coagulopathy undergoing dental extraction: a randomized controlled trial. j oral maxillofac surg. 2010; 68: 138-43. 257surgical dental treatment prior to liver transplantation oral sciences n3 original article braz j oral sci. 9(1):16-19 braz j oral sci. january/march 2010 volume 9, number 1 analysis of mechanical properties and forces produced by transpalatal bars made from low-nickel alloy matheus m. pithon1, rogério l. dos santos1, ana maria bolognese2, eduardo franzotti sant´anna2, margareth maria gomes de souza2 correspondence to: margareth maria gomes de souza universidade federal do rio de janeiro ufrj faculdade de odontologia depto. de ortodontia av. prof. rodolpho paulo rocco, 325 ilha do fundão rio de janeiro cep: 21941-617 rj brasil e-mail: margasouzaster@gmail.com abstract aim: this study evaluated assess the mechanical properties and forces produced by transpalatal bars made from low-nickel alloy. methods: using a template, a single operator made all transpalatal bars from 0.032" and 0.036" wires of two different alloys, thus originating four groups, namely: a8 (0.032" conventional stainless steel), b8 (0.032" low-nickel stainless steel), a9 (0.036" conventional stainless steel), and b9 (0.036" low-nickel stainless steel). the bars were then activated and mounted onto a device developed to serve as a support for mechanical assay in a universal testing machine (emic dl 10.000). the values of resilience and ductility were obtained using the origin 8 software. results: no statistically significant differences (p > 0.05) were observed between groups a8 and b8 neither between a9 and b9 for 0.5-, 1.0-, and 5-mm deformations. however, statistically significant differences (p < 0.05) were found in all groups for 15-mm deformation. groups b8 and b9 showed greater ductility and resilience compared to groups a8 and a9, respectively. conclusions: low-nickel stainless steel transpalatal bars release the same amount of force for activations less than 10 mm compared to those made from conventional stainless steel. mechanically, the low-nickel stainless steel bars are more ductile and resilient. keywords: stainless steel, nickel, orthodontics. introduction first described by goshgarian in 19721, the transpalatal bar has been largely used by orthodontists since then for assisting the orthodontic treatment. its inclusion in the orthodontist’s arsenal was due to its varied array of clinical applications, namely correction of molar rotation, correction of molar mesiodistal inclination, molar distalization (associated with anchorage system), anchorage2-3, control of first molar eruption, relative intrusion, upper posterior segment expansion or contraction, and torque control of molars 4-5.in addition to these clinical applications, this low-cost device is easy to make and use because it is fabricated from stainless steel segments6-7. most metallic appliances used in orthodontic treatment, including the transpalatal bars8, are fabricated from austenitic stainless steel containing 8% nickel and 18% chrome9-10. nickel has often been related to allergic manifestations as this metal causes more reactions than all other metal combinations11. some case reports in have received for publication: october 06, 2009 accepted: march 03, 2010 1dds, msc, doctorate student in orthodontics, federal university of rio de janeiro, brazil 2dd, msc, phd, adjunct professor, department of orthodontics, dental school, federal university of rio de janeiro, brazil 3department of orthodontics, dental school, federal university of rio de janeiro, brazil 17 suggest that orthodontic devices can unchain contact dermatitis in susceptible individuals12-14. the incidence of nickel hypersusceptibility is significant, ranging from 10% to 30% in the population15. women are more often affected than men at a 5:1 ratio. it is thought that the use of certain jewelry pieces can exacerbate the susceptibility to this metal10 11,16. in view of this, the industry of orthodontic products has developed a series of materials to fulfill the needs of nickelintolerant patients one of the products available in the brazilian market is biowire (morelli, sorocaba, são paulo, brazil), an orthodontic wire made from low-nickel stainless steel alloy. the present study evaluated the behavior of the transpalatal bars made from conventional and low-nickel stainless steel alloys when subjected to distalization forces. material and methods the transpalatal bars were fabricated by a single operator using a template (figure 1). the specimens were distributed into four groups, namely: group a8: bars made from 0.032inch conventional stainless stain alloy (crni) (8% ni) wire; group b8: bars made from 0.032-inch low-nickel stainless stain alloy (crmnmoni) (0,2% ni) wire; group a9: bars made from 0.036-inch conventional stainless stain alloy (crni) (8% ni) wire; group b9: bars made from 0.036-inch low-nickel stainless stain alloy (crmnmoni) (0,2% ni) wire. in order to assess the force generated during activation of the transpalatal bars, an acrylic resin device was made and mounted onto the base of a universal testing machine (emic dl 10,000; emic – equipamentos e sistemas de ensaio ltda, são josé dos pinhais, pr, brazil) in order to provide stability during the mechanical tests. this device allowed simulating an upper semi-arch in which the one arm of the bar was attached to the molar tube and the other left free to be moved upward during the tests (figure 2). prior to the tests, the bars were activated in such a way that a 15-mm distalization was achieved for the free arm. next, a hook was adapted to the moving part of the universal fig. 1: template used to fabricate and activate the transpalatal bars under study. fig. 2. device used to attach the bars during the mechanical tests. testing machine to pull the free arm until becoming in parallel to the floor at a speed of 1 mm/min, thus allowing assessing the force generated according to dislocation of 15 to 0 mm. the values obtained were expressed in n and converted into gf, which facilitates the clinical application of the results. based on these data, the ductility and resilience of the bars were calculated using the origin 8 software (originlab corporation, northampton, ma, usa). statistical analysis was done using the spss software version 13.0 (spss inc. chicago, il, usa). descriptive statistical analysis was also performed for the 4 groups assessed, including means and standard deviations. maximum deformation forces obtained in gf unit were subjected to anova and tukey’s test. results the forces generated during the distalization movement using different transpalatal bars are shown in table 1. there was no statistically significant difference between groups a8 and b8 neither between a9 and b9 for 0.5-, 1.0-, and 5.0-mm deformations. as for 10-mm deformation, the bars made from 0.032-inch wire (groups a8 and b8) showed no differences, but statistically significant differences were found in those bars made from 0.036-inch wire (groups a9 and b9). the force generated by all bars increased as a function of dislocation, and groups a9 and b9 showed similar forces for 0.5-, 1.0-, and 5.0-mm dislocations, whereas the bars of braz j oral sci. 9(1):16-19 analysis of mechanical properties and forces produced by transpalatal bars made from low-nickel alloy 18 fig. 3. graph showing forces released by the bars for dislocations ranging from 0.5 to 15 mm. groups a8 and b8 showed force similarity for the 0.5-mm dislocation only (figure 3). the relationship between applied force and bar dislocation (deflexion) is shown in figure 3. the results of resilience and ductility of the bars are presented in table 2. discussion corrosion of the metals composing orthodontic wires, such as nickel, can release metallic ions into the oral cavity and consequently allergic reactions. allergy to nickel is a reaction of the body that manifests as contact dermatitis or even carcinogenic signs17. this hypersusceptibility to nickel can provoke oral manifestations and allergic contact stomatitis, which can mistakenly lead to diagnoses of gingival hyperplasia and oral ulcerations. low-nickel stainless steel wires have been indicated to overcome this problem17 and manufacturers of these orthodontic materials have developed metal alloys with such characteristics, among which is biowire, a stainless steel alloy containing 0.2% nickel. however, the use of these materials has been questioned. do these low-nickel alloys possess the same mechanical characteristics as those of conventional materials, that is, stainless steel alloys containing 8% nickel? based on this premise, the present study aimed at assessing the forces released by transpalatal bars made from conventional and low-nickel stainless steel alloys as well as their mechanical characteristics. specific methods have been developed in which a device simulating the upper semi-arch served as a support for pulling the bars, thus allowing the forces generated by different deformations to be assessed. two types of wires measuring 0.032" and 0.036" in diameters were used as such dimensions are largely employed by orthodontists. the 0.036-inch wire is used for control purposes because it is optimally adjusted in the tube. for standardization purposes, the bars were fabricated by the same operator using a template drawn on graph paper to serve as a model for preparing the samples. only the force generated for molar distalization was assessed because this would be the main function of the transpalatal bar, which requires more activation for releasing the necessary force. after 15-mm activation, the bars were inserted into molar tube and attached to it, and a hook was adapted to pull the bar upward until being in parallel to the floor. the forces generated were continuously recorded as a function of deformation. the bars made from 0.032-inch wire generated less force compared to those made from 0.036-inch wire for all deformations observed. as for 0.5-mm deformation, both bars made from conventional and biowire wires produced similar forces. after 1-mm activation, however, biowire produced ever-increasing forces, but with no statistically significant differences between groups a8 and b8 for deformations of 1.5 and 10 mm. on the other hand, statistically significant differences regarding the force released for 15-mm deformation were observed, with biowire producing more force than other groups. analyzing the forces generated by the transpalatal bars evaluated in the present study, it can be observe a direct relationship to the mechanical properties (resilience and ductility) as obtained with computer software. biowire (groups b8 and b9) wires were found to have more resilience and ductility. these findings were also observed during the preparation of the samples, since biowire wires were considered more difficult to bend properly. however, although low-nickel stainless steel bars (biowire) are difficult to handle, their clinical use is facilitated by their mechanical properties, as they store more energy and consequently allow longer activation time. in terms of clinical importance, this study shows that orthodontists treating nickel-intolerant patients may have to use low-nickel materials. therefore, knowing the specific mechanical behavior of these materials prevents excessive activations of orthodontic devices that might generate nonphysiological forces, which will cause periodontal ligament damage and delay the orthodontic treatment. the following conclusions may be drawn from the results obtained in the present study: 0.036" transpalatal bars braz j oral sci. 9(1):16-19 analysis of mechanical properties and forces produced by transpalatal bars made from low-nickel alloy groups resilience (gf/mm) ductility (%) ration/ductility (%) a8 919.05 6.29 b8 23.5%>a8 b8 1147.38 7.77 a9 70.59% >a8 a9 1966.27 10.73 b9 16.8% >a9 b9 2153.96 12.52 b9 61.13% > b8 table 2. values regarding resilience and ductility of the bars. grupos a8 b8 a9 b9 med./dp 17.59 (0) 52.79 52.79 17.59 (0) med./dp 17.9 24.63 (9.63) 63.35 (9.63) 63.91 (7.23) med./dp 45.75 (9.63) 59.83 (9.63) 109.08 (14.72) 109.11 (14.72) med./dp 80.95 (9.63) 98.55 (9.64) 158.35 (0) 183.03 (15.74) med./dp 105.5 (0) 130.23 (9.63) 211.17 (0) 235.83 (9.63) est. a a b b est. a a b b est. a a b b est. a b c d est. a a b c table 1. means and standard deviations of forces (gf) generated by the bars according to the deformation (mm). mean: mean values of forces generated the bars. sd: standard deviation. stat: statistics, where equal letters indicate no statistically significant difference. 19 generated more force than 0.032" bars in all activations; lownickel stainless steel transpalatal bars produced more force compared to those made from conventional stainless steel wire for the 5-mm deformation; and transpalatal bars made from conventional stainless steel wire had less ductility and resilience compared to those made from low-nickel material. references 1. goshgarian ra. orthodontic palatal arche wires. united states government patent office; 1972. 2. dahlquist a, gebauer u, ingervall b. the effect of a transpalatal arch for the correction of first molar rotation. eur j orthod. 1996; 18: 257-67. 3. garcia-rojas guerra h. a modified transpalatal arch. j clin orthod 2002; 36: 210. 4. kupietzky a, tal e. the transpalatal arch: an alternative to the nance appliance for space maintenance. pediatr dent. 2007; 29: 235-8. 5. tsibel g, kuftinec mm. a bonded transpalatal arch. j clin orthod. 2004; 38: 513-5; quiz 487-518. 6. kojima y, fukui h. effects of transpalatal arch on molar movement produced by mesial force: a finite element simulation. am j orthod dentofacial orthop. 2008; 134: 335 e1-7; discussion 335-6. 7. zablocki hl, mcnamara jr ja, franchi l, baccetti t. effect of the transpalatal arch during extraction treatment. am j orthod dentofacial orthop. 2008; 133: 852-60. 8. counts al, miller ma, khakhria ml, strange s. nickel allergy associated with a transpalatal arch appliance. j orofac orthop. 2002; 63: 509-15. 9. bishara se, barrett rd, selim mi. biodegradation of orthodontic appliances. part ii. changes in the blood level of nickel. am j orthod dentofacial orthop. 1993; 103: 115-9. 10. eliades t, athanasiou ae. in vivo aging of orthodontic alloys: implications for corrosion potential, nickel release, and biocompatibility. angle orthod. 2002; 72: 222-37. 11. peltonen l. nickel sensitivity in the general population. contact dermatitis. 1979; 5: 27-32. 12. bishara se. oral lesions caused by an orthodontic retainer: a case report. am j orthod dentofacial orthop. 1995; 108: 115-7. 13. lowey mn. allergic contact dermatitis associated with the use of an interlandi headgear in a patient with a history of atopy. br dent j. 1993; 175: 67-72. 14. greig dg. contact dermatitis reaction to a metal buckle on a cervical headgear. br dent j. 1983; 155: 61-2. 15. menezes lm, quintao ca, bolognese am. urinary excretion levels of nickel in orthodontic patients. am j orthod dentofacial orthop. 2007; 131: 635-8. 16. thyssen jp, linneberg a, menne t, nielsen nh, johansen jd. the association between hand eczema and nickel allergy has weakened among young women in the general population following the danish nickel regulation: results from two cross-sectional studies. contact dermatitis. 2009; 61: 342-8. 17. kusy rp. types of corrosion in removable appliances: annotated cases and preventive measures. clin. orthod. res. 2000; 3: 230-9. braz j oral sci. 9(1):16-19 analysis of mechanical properties and forces produced by transpalatal bars made from low-nickel alloy oral sciences n3 braz j oral sci. 10(2):88-92 original article braz j oral sci. april | june 2011 volume 10, number 2 comparison of halitosis parameters and sialometry between patients subjected to head and neck radiotherapy and patients with periodontal disease danielle frota de albuquerque1, elen de souza tolentino2, flávio monteiro amado3, cazuo arakawa4, luiz eduardo montenegro chinellato5 1msc in stomatology, department of stomatology, bauru dental school, university of são paulo, brazil 2phd student, bauru dental school, department of stomatology, university of são paulo, brazil 3phd in stomatology, bauru dental school, department of stomatology, university of são paulo, brazil 4 md, radiotherapy specialist, department of radiotherapy, manoel de abreu hospital, bauru, sp, brazil 5phd, professor, bauru dental school, department of stomatology, university of são paulo, brazil correspondence to: elen de souza tolentino faculdade de odontologia de bauru-usp departamento de estomatologia alameda dr. octávio pinheiro brisola, 9 75 vila universitária cep 17012-901 bauru sp brasil phone: (14) 3235-8000 / fax: (14) 3223-4679 e-mail: elen_tolentino@hotmail.com abstract aim: the aim of this study was to evaluate halitosis parameters and sialometry in patients subjected to head and neck radiotherapy compared to patients with periodontal disease, establishing a relationship between oral concentration of volatile sulfur compounds (vscs) and tongue coating presence, salivary flow rate and banatm test. methods: thirty-eight patients were examined and divided into 2 groups: group i: patients with chronic generalized periodontal disease previously diagnosed and not treated; group ii: patients subjected to head and neck radiotherapy. all volunteers were subjected to halitosis measurements through a sulphide monitor, evaluation of tongue coating weight, stimulated and non-stimulated sialometry and banatm test. results: the results were analyzed by analysis of the variance, pearson’s correlation coefficient and student’s t-test, showing that both groups presented halitosis. there was also a relationship between tongue coating presence and vsc levels in both groups and the irradiated patients showed lower salivary flow rates. conclusions: under the tested conditions, it may be concluded that halitosis can be considered as an adverse effect of radiotherapy, associated with low salivary flow and poor oral health, which seems to be the main contribution to bad breath, since patients with periodontal disease also showed halitosis. keywords: halitosis, radiotherapy, head and neck neoplasms, periodontitis. introduction radiotherapy, alone or associated with surgery or chemotherapy, is the therapeutic method indicated in cases of oral cancer and has produced a significant increase in cure rates for several malignancies of the head and neck1. the adverse effects of head and neck radiotherapy are very important for the dental surgeon, who has a key role in preventing and/or minimizing their occurrence. salivary received for publication: january 28, 2011 accepted: may 20, 2011 89 braz j oral sci. 10(2):88-92 glands are radiosensitive and the atrophic and acinar degeneration caused by radiotherapy commonly result in reduction of the saliva production2-5. halitosis is highly associated with the amount of saliva6. it has been shown a relationship between hyposalivation and radiotherapy, as well as between halitosis and periodontal disease. however, the association among these variables is not well known. there is only one study which established a correlation between halitosis and head and neck radiotherapy, through evaluation of halitosis and sialometry in patients who had undergone radiotherapy when compared to healthy and non-irradiated individuals7. howsoever, there are no studies comparing halitosis between irradiated patients and patients with periodontal disease. the aim of this study was to establish a correlation between halitosis and head and neck radiotherapy, through evaluation of halitosis and sialometry in patients who had undergone radiotherapy in comparison to patients with periodontal disease. material and methods this study was approved by the human research ethics committee of bauru dental school, university of são paulo, brazil (process #104/2005) and is in accordance with the helsinki declaration of 1975, revised in 1983. all volunteers signed an informed consent form. the sample was composed of 38 patients, divided into two groups: group i: 13 patients from the screening sector of the bauru dental school , with generalized chronic periodontal disease previously diagnosed but not treated; group ii: 25 volunteers selected among patients from manoel de abreu hospital, referenced for cancer treatment in the city of bauru, sp, brazil. the sample size was justified by the fact of many patients have not finished the radiotherapy treatment at the moment of the consultation and only volunteers with the treatment completed were included in the study. in group i, patients with localized or treated periodontal disease were excluded, which reduced the initial sample size. all patients were selected among a population of each institution, both with spontaneous demand. careful exam and screening of these patients according to the exclusion and inclusion criteria led to the reduction of the sample; however, according to the statistician responsible for this study, the sample size was appropriated. patients of group i presented periodontal pockets > 5 mm in all molars and incisors, bleeding on probing and generalized vertical bone loss observed radiographically. the diagnosis of these patients was chronic generalized periodontal disease. patients of group ii were subjected to radiotherapy as the main or complementary treatment of head and neck tumors. the radiation area covered at least one of the major salivary glands or part of them. the radiotherapy treatment of all patients had already been concluded and they were under continuous monitored from 1 to 6 months after the last radiotherapy session. it is important to emphasize that all patients of group ii were periodontally healthy, that is, they did not present bleeding on probing or periodontal pockets > 4 mm). also, the sequelae of the radiotherapy treatment (e.g.: radiation caries and mucositis) were treated in the period between the radiotherapy and this research. the management of the patients was similar to a previous study7. all patients underwent two appointments conducted by the same examiner. in the first one, the patients received some guidelines for the procedures to be carried out in a further moment: avoid spicy and/ or flavored food 24 h before the clinical appointment; avoid brushing the teeth, using dental floss, chewing gum, drinking alcoholic drinks and smoking 3 h before the clinical appointment and not being using perfume at the moment of the appointment. halimetry the halimetry values in all individuals were obtained using a sulfide monitor (halimeter; interscan corporation, chatsworth, nj, usa). before measurements, patients were kept seated, relaxed and without talking for 1 min, with their mouth closed. a disposable plastic tube connected to the monitor was introduced 4 cm inside the patient´s mouth7. the patients were instructed to leave their mouth slightly opened on the tube without inhaling or exhaling during the analysis. those measures were taken three times, which resulted in a final average. evaluation of tongue coating and new halimetry measurements after removing the excess of humidity of the tongue with gauze, the tongue coating was removed with a tongue cleaner and placed in a previously weighted filter paper. the material was stored for 24 h for drying and weighing. the tongue coating weight was obtained using a digital precision scale (a&d system co., ltd. tokyo, japan). immediately after removing the tongue coating, halimetry was performed again, in order to identify possible variations in the vscs concentrations7. banatm test (benzoyl-dl-arginine-2 napthylamide) the test was performed to identify a possible relation between the presence of microorganisms bana positive and the values of halimetry. a small amount of tongue coating was collected and transferred to the bana test strip (banamet llc, ann arbor, mi, usa). a drop of distilled water was poured on the reagent strip, which was folded and kept this way with a paperclip, so that the reagent would be in contact with the organic material, left undisturbed for 24 h, as indicated by the manufacturer. after that period, the reaction was noted and the result obtained, classified as negative and positive, according to the obtained color7. sialometry after remaining 5 min at rest, with the eyes opened, without stimulating salivation, each patient spit out all saliva obtained in that period in a recipient graded in millimeters (ml) to obtain the non-stimulated salivary flow. after this comparison of halitosis parameters and sialometry between patients subjected to head and neck radiotherapy and patients with periodontal disease braz j oral sci. 10(2):88-92 procedure, each patient was given a piece of sterile hyperboloid which was used to stimulate salivation through mastication for 5 min, and the saliva obtained was also placed in a recipient graded in ml to obtain the stimulated salivary flow7. radiotherapy treatment protocol the radiotherapy treatment protocol was the same for all patients of group ii. all selected patients have had at least one of the major salivary glands involved in the area of radiation and the protocol of radiotherapy was the same used in the patients of a previous study7. statistical analysis the results were analyzed by anova and tukey’s test for the variables that quantified halitosis before and after removing tongue coating. the relationship among oral halimetry, dry weight of tongue coating and stimulated/nonstimulated salivary flow rate were analyzed by pearson’s correlation test. student’s t-test was used to verify the correlation between halimetry and bana test. a significance level of 5% was used for all analyses. the computer software used was microsoft office excel 2007. results group i presented lower initial halimetry average (148.102 ppb) when compared to group ii (143.747 ppb), with no statistically significant difference (table 1). the dry weight of tongue coating in group ii (0.016mg) was smaller than in group i (0.0245 mg), but this difference was not significant (table 1). group ii showed a decrease in salivary flow, in stimulated and non-stimulated sialometry, with a statistically significant difference (p<0.001) (table 1). in the evaluation of the correlation between the studied variables and halitosis, in group i this correlation was noted between initial halimetry and presence of tongue coating ( p = 0 . 0 1 2 ) a n d b e t w e e n i n i t i a l a n d f i n a l h a l i m e t r y (p<0.001) (table 2). group ii, showed a significant correlation between initial halimetry and presence of tongue coating (p=0.043), between initial and final halimetry (p<0.001) and between stimulated and non-stimulated salivary flow (p<0.001) (table 3). in both groups, the number of negative banatm tests table 1 table 1 table 1 table 1 table 1 mean (sd) obtained for the variables ih, fh, tc, nssf and ssf in groups i and ii. *statistically significant difference (p<0.05); ns non statistically significant difference. ih – initial halimetry; fhfinal halimetry – after removing the tongue coating (ppb); tc – dry weight of tongue coating (mg) ; nssf – non-stimulated salivary flow (ml); ssf– stimulated salivary flow (ml) variables group i group ii p value mean±sd mean±sd ih 148.102 60.324 143.747 62.092 ns fh 137.846 57.887 123.453 49.670 ns tc 0.0245 0.009 0.016 0.017 ns nssf 4.26 1.386 1.448 1.015 0.000* ssf 9.085 1.664 2.548 1.660 0.000* variables r p ih x tc 0.407 0.043* ih x nssf 0.021 0.918 ih x ssf 0.197 0.344 ih x fh 0.861 0.000* tc x nssf 0.069 0.740 tc x ssf 0.172 0.411 nssf x ssf 0.882 0.000* table 3 table 3 table 3 table 3 table 3 pearson’s correlation test between the variables and halitosis in group ii. *statistically significant difference (p<0.05). ih – initial halimetry; fhfinal halimetry – after removing the tongue coating (ppb); tc – dry weight of tongue coating (mg); nssf – non-stimulated salivary flow (ml); ssf– stimulated salivary flow (ml) variables r p ih x tc 0.672 0.012* ih x nssf 0.238 0.434 ih x ssf 0.037 0.904 ih x fh 0.971 0.000* tc x nssf 0.132 0.668 tc x ssf 0.269 0.374 nssf x ssf 0.049 0.873 table 2 table 2 table 2 table 2 table 2 pearson’s correlation test between the variables and halitosis in group i. *statistically significant difference (p<0.05). ih – initial halimetry; fhfinal halimetry – after removing the tongue coating (ppb); tc – dry weight of tongue coating (mg); nssf – non-stimulated salivary flow (ml); ssf– stimulated salivary flow (ml) negative positive mean ± sd mean ± sd t p a l l 104.1 51.2 157.3 67.1 -3.10 0.003* group i 100.7 44.1 177.7 50.2 -2.80 0.017* group ii 128.1 45.6 167.1 77.6 -1.58 0.126 table 4 table 4 table 4 table 4 table 4 student’s t-test for comparison between halimetry and positive and negative banatm test groups. *statistically significant difference (p<0.05) (n=25) was greater than the positive results (n=23), with a statistically significant difference among the negative and positive results and halimetry in group i (p=0.017) and when the entire sample was analyzed together (p=0.003) (table 4). 90comparison of halitosis parameters and sialometry between patients subjected to head and neck radiotherapy and patients with periodontal disease 91 discussion regarding oral halimetry, group i presented lower initial average when compared to group ii (table 1), but without statistically significant difference. however, in the present study, the use of the sulfide monitor enabled the evaluation only of the relation between vscs and halitosis. this is a limitation of methodology since bad breath is caused by other volatile organic compounds and other gases8. final halimetry was performed soon after tongue cleaning. a previous study7 demonstrated that the values of halimetry can decrease substantially immediately after the tongue cleaning. it is known that tongue coating is an important etiological factor of halitosis9. waler10 showed that the largest production of vscs is in the tongue dorsum. yaegaki and sanada11 reported that removing tongue coating reduced by 50% the production of vscs. albuquerque et al.7 found a relationship between presence of tongue coating and vscs levels, with a decrease in the values of halimetry after removing tongue coating. boever and loesche12 noticed that the score of oral breath was highly connected to the odor of the tongue, presence and extension of coating. in this work, the values of initial halimetry were greater than final halimetry in both groups. however, there was no statistically significant difference (table 1). since the tongue cleaner shows a higher percentage of reduction of vscs than the tooth brush13, it justifies the use of this tool in the methodology of this research. in our study, the amount of tongue coating was evaluated quantitatively after drying, confirming methodologies used by other studies7,11,14. the dry weight of tongue coating in patients with periodontal disease was greater than in the irradiated patients, but the difference was not statistically significant. this finding is a little surprising because it is well established that the tongue coating is a result of the hypossalivation7. salivary reduction provides the implantation of the tongue coating because the saliva becomes more viscous, with higher amounts of mucin, increasing the adherence of microorganisms and epithelial debris in the tongue. as the bacteria present in the tongue coating and in the periodontal pockets are similar (anaerobic proteolytic), colonization of the tongue can be favored in patients with periodontal disease. however, it does not explain the higher amounts of tongue coating in group i than in group ii, since irradiated patients have an outstanding decrease in salivary flow, the main etiological factor of tongue coating. perhaps the poor hygiene, which is common in patients with periodontal disease, can explain this finding of the present work. regarding salivary flow, there was significant difference between the groups in stimulated and non-stimulated sialometry (table 1). all patients in group ii showed a decrease in the amount of saliva. the value of 0.1 ml/min for salivary flow without stimulus was considered severe glandular hypofunction. there was a decrease in sialometry without stimulus from group ii of 66.03% compared to group i. albuquerque et al.7 found a decrease in sialometry without stimulus in irradiated patients of 56.51% when compared to healthy patients. in group i, correlation between the studied variables and halitosis was noted between initial halimetry and presence of tongue coating and between stimulated and non-stimulated salivary flow (table 2). in group ii, a significant correlation was observed between initial and final halimetry, between initial halimetry and presence of tongue coating and between stimulated and non-stimulated salivary flow (table 3). the banatm test an enzymatic method used as an indicator of the presence of microorganisms responsible for periodontal diseases was performed in groups i and ii. similarly to a previous study7, in both groups the number of negative tests was greater than positive results. in table 4, it is noted that there was a significant difference among the negative and positive results and halimetry in group i and when the entire sample was together. some authors have not noted relation between the levels of vscs and the results of lingual banatm test15. according to monteiro-amado et al.14, there is not a relation between the value of banatm test and the values of halimetry. irradiated patients did not have main complaint of halitosis. however, when they were questioned about oral bad breath, the majority answered that felt it. patients with periodontal disease did not ask for treatment of halitosis, but they had complaints of bad breath and presented indication for periodontal treatment. according to conceição et al.16, halimetry above 100 ppb can be considered as halitosis. in this study, all evaluated patients, of both groups, presented halitosis, since the values of initial and final halimetry were greater than 100 ppb. however, there was no statistically significant difference between the groups, confirming the fact that the oral health condition is an important etiological factor of halitosis, and bad breath is strongly associated with periodontal disease. it is known that saliva incubation of patients with periodontal disease produces vscs quickly, inducing an intense bad breath. the saliva of these patients produces more vscs than the saliva of healthy people. halitosis may occur in any individual, but it is emphasized when inflammatory and degenerative processes are present. for example, gingivitis and periodontitis are almost always associated with halitosis, which corroborates the findings of the present study. the present work is a continuation of a previous study, which detected halitosis and hyposalivation in irradiated patients when comparing them with healthy people7. it is believed that the comparison of periodontally healthy irradiated patients, which recognizably have halitosis,) and patients with periodontal disease can show the true degrees of halitosis in each group as well as demonstrate that both conditions play important roles in bad breath development. it is known that hyposalivation is a definitive sequel of head and neck radiotherapy and that this condition plays a keyrule in halitosis pathogenesis 6. we believe that the significant reduction in the amount of saliva is the most important parameter in the bad breath development in irradiated patients, since, in this research, they had a satisfactory oral condition. on the other hand, the significant differences among the negative and positive results and braz j oral sci. 10(2):88-92 comparison of halitosis parameters and sialometry between patients subjected to head and neck radiotherapy and patients with periodontal disease 92 halimetry of the banatm tests in group i show the substantial role of periodontopathogenic microorganisms in the development of halitosis in patients periodontally affected. additionally, despite the lack of statistical significance, the dry weight of tongue coating in group i was greater than in group ii, showing a close relationship between periodontitis and tongue coating, since the bacteria that colonize the tongue and periodontal pockets are the same. for these reasons, we agree that the most expressive factors that influence the halitosis formation in patients with periodontal disease are tongue coating and presence of periodontopathogenic microorganisms. summarizing our findings, halitosis was detected in patients with periodontal disease and patients subjected to head and neck radiotherapy and that there was relation between presence of tongue coating and vscs levels in the studied groups. there was a significant decrease in the values of halimetry after removing tongue coating in both groups. also, stimulated and non-stimulated salivary flow was extremely reduced in irradiated patients. according to the results obtained under the tested conditions, it is possible to conclude that halitosis is an adverse effect of head and neck radiotherapy and is strongly associated with periodontal disease. acknowledgements the authors would like to thank professor josé roberto lauris for the support in the statistical analysis of this study. this investigation was supported by capes. references 1. otmani n. oral and maxillofacial side effects of radiation therapy on children. clin pract. 2007; 73: 257-61. 2. prott fj, handschel j, micke o, sunderkotter c, meyer u, piffko j. long-term alterations of oral mucosa in radiotherapy patients. int j rad oncol biol phys. 2002; 54: 203-10. 3. ko c, citrin d. radiotherapy for the management of locally advanced squamous cell carcinoma of the head and neck. oral dis. 2009; 15: 121-32. 4. bomeli sr, desai sc, johnson jt, walvekar rr. management of salivary flow in head and neck cancer patients – a systematic review. oral oncol. 2008; 44: 1000-8. 5. sciubba jj, goldenberg d. oral complications of radiotherapy. lancet oncol. 2006; 7: 175-83. 6. möller p, perrier m, ozsahin m, monnier p. a prospective study of salivary gland function in patients undergoing radiotherapy for squamous cell carcinoma of the oropharynx. oral surg oral med oral pathol oral radiol endod. 2004; 97: 173-89. 7. albuquerque df, tolentino es, monteiro-amado f, arakawa c, chinellato lem. evaluation of halitosis and sialometry in patients subjected to head and neck radiotherapy. med oral patol oral cir bucal. 2010; 15: e850-4. 8. goldberg s, cardash h, browning h, 3rd, sahly h, rosenberg m. isolation of enterobacteriaceae from the mouth and potential association with malodor. j dent res. 1997; 76: 1770-5. 9. rosenberg m. clinical assessment of bad breath: current concepts. j am dent assoc. 1996; 127: 475-82. 10. waler sm. on the transformation of sulfur-containing amino acids and peptides to volatile sulfur compounds (vsc) in the human mouth. eur j oral sci. 1997; 105: 534-7. 11. yaegaki k, sanada k. volatile sulfur compounds in mouth air from clinically healthy subjects and patients with periodontal disease. j periodontal res. 1992; 27: 233-8. 12. boever eh, loesche wj. assessing the contribution of anaerobic microflora of the tongue to oral malodor. j am dent assoc. 1995; 126: 1384-93. 13. seemann r, kison a, bizhang m, zimmer s. effectiveness of mechanical tongue cleaning on oral levels of volatile sulfur compounds. j am dent assoc. 2001; 132: 1263-8. 14. monteiro-amado f, chinellato le, de rezende ml. evaluation of oral and nasal halitosis parameters in patients with repaired cleft lip and/or palate. oral surg oral med oral pathol oral radiol endod. 2005; 100: 6827. 15. willis cl, gibson gr, holt j, allison c. negative correlation between oral malodour and numbers and activities of sulphate-reducing bacteria in the human mouth. arch oral biol. 1999; 44: 665-70. 16. conceição md, marocchio l, tarzia o. evaluation of a new mouthwash on caseous formation. braz j otorhinolaryngol. 2008; 74: 61-7. braz j oral sci. 10(2):88-92 comparison of halitosis parameters and sialometry between patients subjected to head and neck radiotherapy and patients with periodontal disease oral sciences n3 received for publication: january 19, 2012 accepted: april 18, 2012 original article braz j oral sci. april | june 2012 volume 11, number 2 perception of orthodontists regarding the management of root resorption – clinical and legal aspects luíza valéria de abreu maia1, renata de castro martins2, mariana mourão de azevedo flores pereira3, maurício de almeida cardoso4, leopoldino capelozza filho4, luiz renato paranhos4 1dds, department of community dentistry, school of dentistry, federal university of minas gerais (ufmg), brazil 2dds, msc, phd, department of community dentistry, school of dentistry, federal university of minas gerais (ufmg), brazil 3dds, msc, professor, brazilian dental association bda/mg, belo horizonte, brazil 4dds, msc, phd, professor, department oral biology, school of dentistry, sagrado coração university (usc), brazil correspondence to: luiz renato paranhos rua padre roque, 958, centro cep: 13800-033 mogi mirim sp brasil phone: +55 19 38044002 e-mail: paranhos@ortodontista.com.br abstract aim: to evaluate the management adopted by orthodontists regarding root resorption caused by orthodontic treatment, relating it to their time of experience and the applicable legal norms. methods: the study population consisted of 56 orthodontists from the city of belo horizonte (minas gerais state), and the tool to collect the data was a questionnaire with open and closed questions. data were subjected to descriptive analysis. the association between the variables was analyzed by the chi-square test with significance level of 5%. results: 57.6% of professionals temporarily interrupt the treatment; in the prevention and/or control, most professionals (80.4%) perform initial and periodical radiographs (every six months). among the professionals with less than 5 years of experience as a specialist, 88.9% usually make periodic radiographic control; 100% affirmed to file the documents. no significant difference was found between dentists with different times of experience regarding the practice of archiving the documents (p = 0.271). among those with 5-15 years of experience, 77.4% perform periodic radiographic control and 45.2% had already detected root resorption. among those with more than 15 years of experience, 71.4% usually perform the periodic radiographic control, and 85.7% had already faced some case of root resorption in the clinical practice. conclusions: although most orthodontists adopt a correct management facing the occurrence, prevention and control of root resorption associated with orthodontic treatment, the majority are unaware about existing legislation in our country. keywords: root resorption, perception, legal norms. introduction external root resorption associated with orthodontic treatment is recognized as a clinical problem and has been a factor of controversy and concern due to the imminent risk for apical root rounding, which occurs in practically all patients under orthodontic treatment in different magnitudes. according to petrelli1, root resorption may occur in any orthodontic treatment with any professional. depending on the associated predisposing and etiologic factors, a resorption may occur with loss of more than one half of root length, being one of the most braz j oral sci. 11(2):100-105 braz j oral sci. 11(2):100-105 101101101101101 request for orthodontic documentation percentage of responses do not request final documentation 8.9% sometimes request final documentation 19.6% always request final documentation 71.5% total 100.0% table 1. distribution of results relative to the request for final orthodontic documentation. undesirable side effect of orthodontic treatment. this attracts considerable interest from orthodontists, especially because of the legal aspects related to indemnities2. the etiology of external root resorption is not yet completely understood3. it has great importance when is consequence and/or complication of certain clinical situations, such as induced tooth movement, and when related to legal implications because it is a borderline phenomenon between cost/benefit and iatrogenesis, particularly in orthodontic practice4. the complexity of orthodontic diagnosis and treatment requires legal and ethical attitudes by the specialists from the very first contact with the patient. with the creation of the code of consumer protection5, the lawsuits have increased significantly in several sectors of consumption, which brought direct consequences for orthodontics. therefore, the need to keep patients’ documentation as complete as possible is unquestionable. before the beginning of the treatment, all patients (or parents/guardians) should be clearly informed that there is a real possibility of one or more teeth suffer root resorption as a consequence of orthodontic treatment6, and sign an informed consent form explaining all associated risks and factors affecting root resorption evolutionarily7-8. the brazilian civil procedure code9 argues in chapter vi – evidences that the record is a document of probative force to defend the dentist, as presented in the art. 332: “all legal means, as well as the morally legitimate, though not specified in this code, are able to prove the veracity of facts which substantiate the action or defense”, being necessary and very important to keep a complete and organized documentation that contains the largest number of diagnostic elements and clinical procedures performed7,10-13. radiographic examination is the most important tool for diagnosis before starting the orthodontic treatment, and periodical radiographs also should be taken during the treatment for monitoring the occurrence of root resorption. this preventive measure has a low financial and biological cost, with a great benefit14. when the resorption is found at the beginning or during the orthodontic treatment, the best practice is to inform the patient, reevaluate the mechanics, control periodically, and warn about a possible extension in treatment time11. in this way, this study investigated the perception of orthodontists regarding the practices adopted with respect to the prevention and/or control of the occurrence of root resorption caused by orthodontic treatment, relating it to the time of experience as specialists and to the applicable brazilian laws. material and methods this study was approved by the ethics committee of fop/unicamp, preserving the patients and researchers of any legal and ethical drawbacks. a questionnaire with open and closed questions was sent to 150 dentists, specialists in orthodontics, from the city of belo horizonte (minas gerais state), with a return ratio of 37.33% (n= 56). it was assured confidentiality of the information and the exclusive use of the data for research purposes. the questionnaire was sent to the participants by reply-paid mail. after receipt of the filled questionnaires, the data were compiled and distributed in percentage by means of graphs and tables that showed the results descriptively. the association between the variables was checked by chi-square test at 5% significance level. the questionnaire was divided into two sections; the first with identification, and the second with questions about the formation, professional documentation, and the practice adopted facing the occurrence of root resorption. in the first section addressed the gender and characteristics relative to the time of experience in orthodontics. in the second section, it was questioned whether they: ask for initial and final patient documentation; perform regular radiographic control and how often this is performed; use an informed consent form before starting the treatment; know the physiological mechanisms of root resorption; had already encountered root resorption of more than a half of root length (if yes, how they managed the case); and usually file the documentation of the patients and for how long. results the tabulation of personal data obtained by the questionnaires characterized the profile of the professionals composing the sample: 56 orthodontists regularly registered in the cro-mg, predominantly male (60.7%), with different times of experience as a specialist [18 (32%) had less than 5 years, 31 (55.3%) had from 5 to 15 years, and only 7 (12.5%) had over 15 years of experience]. concerning the request for initial documentation, 100% of the interviewed professionals affirmed to request the documentation before starting an orthodontic treatment. table 1 shows the result for the request for final documentation. in relation to the regular periapical radiographic follow up during the orthodontic treatment, 80.4% of professionals perform this control, and 19.6% do not always do it. table 2 presents the relationship between periodic radiographic follow up and time of experience as specialist in orthodontics. among the professionals that usually perform periapical radiographic follow up, the majority (61%) makes it every six months. regarding the informed consent form, most interviewees (77%) have included this document, or similar, warning about the imminent risks for root resorption. perception of orthodontists regarding the management of root resorption – clinical and legal aspects 102102102102102 braz j oral sci. 11(2):100-105 experience time as specialist in orthodontics total <5 years 5 15 years > 15 years usually perform periapical radiographic follow up n o yes 2 11.1% 16 88.9% 7 22.6% 24 77.4% 2 28.6% 5 71.4% 11 19.6% 45 80.4% 56 100.0% 7 100.0% 31 100.0% 18 100.0% total p = 0.259 table 2. frequency distribution of orthodontists that usually perform periapical radiographic follow up during orthodontic treatment, stratified by the experience time as a specialist. experience time as specialist in orthodontics total <5 years 5 15 years > 15 years any case of root resorption yes n o 14 45.2% 17 45.8% 31 100.0% 26 46.4% 30 53.6% 56 100.0% total p = 0.034 6 33.3% 12 66.7% 18 100.0% 6 85.7% 1 14.3% 7 100.0% table 3. frequency distribution of orthodontists that had already had a case of root resorption caused by the orthodontic treatment, stratified by the experience time as a specialist. experience time as specialist in orthodontics total <5 years 5 15 years > 15 years archive the documentation n o yes 4 12.9% 27 87.1% 31 100.0% 5 8.9% 51 91.1% 56 100.0% total p = 0.271 .00 0% 18 100% 18 100.0% 1 14.3% 6 85.7% 7 100.0% table 4. frequency distribution of orthodontists that usually archive the documentation, stratified by the experience time as a specialist. time of archiving < 5 years 5 – 10 years 10 -15 years 15 – 20 years > 20 years no answer total percentage of responses 21.4% 23.3% 12.5% 12.5% 21.4% 8.9% 100.0% table 5. period of archiving of dental records after completion the orthodontic treatment. all interviewed orthodontists affirmed to know the physiological mechanisms of root resorption, and 46.4% of them reported that had already encountered a resorption case of more than a half of root length during professional exercise. a significant association was found between the time since graduation as a specialist and the occurrence of a case of root resorption (table 3). of the professionals that had already encountered a root resorption, 57.6% responded that they interrupted the treatment for a while; 19.6% continued the treatment – with lighter forces – and none of them reported to have continued normally the treatment without interrupting. with regard to archiving practices, 91% informed that they usually archive the documentation. no significant association was found between the time since graduation as a specialist and the adoption of a routine of archiving the documentation (table 4) considering the period of archiving the orthodontic documentation, there was a remarkable variation in the responses, as listed in table 5. discussion the present study investigated the perception of orthodontists regarding the ideal practice facing root resorption due to orthodontic treatment. most studies have shown that the professionals know how to prevent the occurrence of root resorption during orthodontic treatment and how control this event when it appears8,15-17. however, perception of orthodontists regarding the management of root resorption – clinical and legal aspects 103103103103103 some professionals are still unaware of the legal provisions relating to dental practice existing in our country. in relation to request of initial documentation, all the professionals recognize its importance because 100% affirmed to do it. in contrast, about the final documentation, 8.9% of orthodontists responded that they do not request it, and 19.6% of them request it only sometimes. these results differ from findings of a previous study17 in which 5.7% of professionals affirmed to request the documentation after completion of treatment, and only 14.2% did not request. the request for final documentation minimizes the chances of legal problems, since it has great relevance in the follow up of orthodontic completion and post-treatment period, providing a favorable legal support15. high-risk patients should be identified through periapical radiographs of all teeth before starting the orthodontic treatment, which are one of the most effective manners for preventing problems associated with root resorption. radiographic control throughout the treatment is also highly recommended. additionally, the diagnosis of oral and dental problems is part of the duty of every dentist4,18. in view of this, we questioned about the regular periapical radiographic control during orthodontic treatment to monitor the occurrence of root resorption. by analyzing the relationship between the accomplishment of a periodic control and the time of experience in orthodontic practice, the percentages were well distributed among the categories of the experience time (88.9% 0-5 years; 77.4% 5-15 years, and 71.4% >15 years), with no correlation between these factors. thus, the routine of performing periapical radiographic control is independent of the experience time as an orthodontist. six months after starting the treatment is the time recommended to reevaluate radiographically the occurrence of root resorption, which may lead to temporary suspension of the treatment, thus reducing the resorption rate at the end of the orthodontic treatment4. a preformed proof is all documentation produced by the professional, i.e., elaborated along the clinical practice for all phases of the performance as a dentist11-12,15. if produced on timely basis, the dental records may be presented by the professional to prove the existence of a fact. however, it is not enough only to have documentation; it has to have quality to safeguard the professional, containing all necessary documents that make up the patient chart7-8. the consent form is an agreement between the professional and patient about a possible and licit object (orthodontic treatment), showing the conditions and limitations of the professional along with the understanding and acceptance from the patient7-8. this document should be prepared in simple language, explaining everything about the service, its indications and limitations, risks and benefits of the treatment, all the treatment options possible within the technical and biological limits, and information about the post-treatment care7-8. most interviewees (77%) reported to include this document in the patient chart, alerting about the possible risk of root resorption as a consequence of the orthodontic treatment. it is known that root resorption during the orthodontic treatment cannot be avoided. nevertheless, preventive measures can avoid, in most cases, important damage to the affected tooth. therefore, the non-adoption of these measures may characterize professional negligence19. in the present study, all the interviewees responded to know the physiological mechanisms of root resorption; and 46.4% reported that had already faced a case of root resorption of more than a half of root length in their clinical practice. among those with over 15 years as a specialist, 85.7% had already encountered this problem in their orthodontic clinic. the proportions of cases of root resorption increased with the increase of the experience time as an orthodontist (33.3% and 45.2%, respectively 0-5 and 5-15 years as specialist). this higher frequency of occurrence among the most experienced professionals is probably due to the performance of a larger number of services, and thus they had been more frequently exposed to the risk of this type of occurrence. among the professionals that had already dealt with a case of resorption of more than a half of root length in their clinical practice, 56.7% responded that they interrupted the treatment for a certain time. this procedure reduces significantly tooth shortening at the end of orthodontic treatment 4; if root resorption is detected by means of intermediate radiographs, the patient is informed, as a change, interruption or termination of the treatment may be necessary. most professionals are not aware of the limitation of time for legal demands in relation to dental treatment. this ignorance leads to an unsafe clinical practice. the participants of the present study were not aware of the exact time that the patient has to complain to justice in relation to performed treatment. in the present study, 91% of the interviewees affirmed to file the orthodontic documentation of their patients. likewise, in similar study16 with 56 dentists 98% of the sample affirmed to keep the documentation on file after completion of the treatment, and only 2% give the documentation to patients without keeping copies on file. in relation to the association between experience time and archiving of dental records, 100% of professionals with less than 5 years of experience as specialist usually archive the documentation; among those with between 5 and 15 years, 87.1% keep documentation of file, and 85.7% of professionals with over 15 years of experience affirmed to do this. regarding the archiving time, an expressive variation of responses was found in the present study (table 5), whereas another study8 showed that most professionals (58.9%) filed the records after completion of treatment throughout the life. barroso et al.17 revealed that 8.3% of professionals filed the documentation for 5 years; 3.8% for 10 years; 12.4% for 15 years; 25.5% for 20 years, and most of them (49.8%) for over 20 years. this difference in the archiving time exposed and characterized the contradictions regarding this theme, emphasizing the need for better professional orientation in relation to the brazilian legal system. moreover, in the brazilian law, there is not such a term “archiving time”, but perception of orthodontists regarding the management of root resorption – clinical and legal aspects braz j oral sci. 11(2):100-105 104104104104104 a limitation time, which is frequently misunderstood. still on the documentation archiving, the code of dental ethics20 recommends that it is the duty of the dentist to prepare and keep current the records of the patients, maintaining them on own file. the civil code21 establishes in three years the limitation time for civil compensation claim, while the consumer defense code5 establishes this limitation in five years from the date of the discovery of the fact. nevertheless, the professionals that responded that file the documentation for more than 20 years have corroborated the recommendation of silva22 that states: “…the clinical records must be kept at least for twenty years…”. the occurrence of external root resorption cannot be avoided, representing a biological cost inherent to any orthodontic movement, with a variable magnitude for each individual 19. this statement is consensual for to the orthodontist, but several clinicians that not work in the orthodontics area may consider this scar as an iatrogeny caused by orthodontic treatment and lead the patient to question the resorption consequent of orthodontic movement. levander and malmgren23, in 1988, suggested a classification of the levels of root resorption during the orthodontic treatment: 1 = minimal resorption (irregular apical contour); 2 = moderate (d” 2 mm); 3 = severe resorption (> 2 mm < 1/3 root); 4 = extreme resorption (> 1/3 root), as illustrated in figure 1. fig. 1. classification of the levels of root resorption during orthodontic treatment, according to levander and malmgren23. the same knowledge allows the orthodontist to have a set of attitudes that permit the early identification of individuals and teeth predisposed to resorption before the treatment, reorientation of orthodontic procedures (once identified the process), post-treatment care of patients that suffered root resorption. this set of actions allows avoiding, in most cases, significant damages to the involved tooth or teeth. given the increased number of legal cases against the professionals, more experienced and newly graduated dentists should increasingly think about a professional development, particularly in deontology to increase their knowledge of legal and ethical aspects that govern the exercise of dentistry. if not properly trained on relevant ethical and legal aspects concerning the professionals, dentists become vulnerable to litigation24. it may be concluded that all professionals interviewed in this study request the documentation before starting an orthodontic treatment – a basic condition for professional practice. most of these professionals perform periodic periapical radiographic control during the orthodontic treatment, but the differences in orthodontic management showed the need for dissemination of a protocol of actions among the professionals when dealing with root resorption. most orthodontists affirm to prepare an informed consent form, indicating concern and zeal for the profession. the range of responses in relation to the archiving period of orthodontic documentation revealed the lack of knowledge about the legal provisions existing in our country, such as the civil code and the consumer defense code. references 1. petrelli ne. aspectos éticos e legais em ortodontia. rev dent press ortod ortop facial. 1998; 3: 6-10. 2. kaley j, philips c. factors related to root resorption in edgewise practice. angle orthod. 1991; 61: 125-32. 3. capps l. reabsorção radicular periapical e o tratamento ortodôntico. in: rielson jac, gonçalves ean. ortodontia e ortopedia funcional dos maxilares. são paulo: artes médicas; 2002. p.179-84. 4. consolaro a. reabsorções dentárias nas especialidades clínicas. maringá: dental press; 2002. 5. brasil. lei no 8.078/90. código de defesa do consumidor. diário oficial da união. 11 mar. 1991. 6. rupp r. root resorption related to orthodontics and other factors: a review of the literature. j gen orthod. 1995; 6: 25-9. 7. paranhos lr, guedes tmp, jóias rp, torres fc, scanavini ma. orientações legais aos ortodontistas: elaboração de contrato de prestação de serviços odontológicos e consentimento esclarecido parte 2. ortodontia spo. 2009; 42: 237-43. 8. paranhos lr, salazar m, torres fc, pereira ac, silva rf, ramos al. avaliação do perfil dos profissionais da área de ortodontia quanto às condutas legais. dent press j orthod. 2011; 16: 127-34. 9. brasil. lei nº 5.869, de 11 de janeiro de 1973. institui o código de processo civil; 1973. 10. rosa fb. dentista x indivíduo ortodôntico: levantamento de problemas jurídicos nas últimas três décadas. j bras ortod ortop facial. 1997; 2: 14-27. 11. silva m. compêndio de odontologia legal. rio de janeiro: editora médica e científica ltda; 1997. 12. calvielli itp. responsabilidade profissional do cirurgião-dentista. in: silva m. compêndio de odontologia legal. rio de janeiro: editora médica e científica ltda; 1997. p.399-411. perception of orthodontists regarding the management of root resorption – clinical and legal aspects braz j oral sci. 11(2):100-105 13. silva rf, chaves p, paranhos lr, lenza ma, daruge júnior e. utilização de documentação ortodôntica na identificação humana. dent press j orthod. 2011; 16: 52-7. 14. brezniak n, wasserstein a. root resorption orthodontic treatment: part i. literature review. am j orthod dentofac orthop. 1993; 103: 62-6. 15. fernandes f, cardozo hf. responsabilidade civil do cirurgião-dentista: o pós-tratamento ortodôntico. rev abo nac. 2004; 12: 298-305. 16. garbin cas, garbin aji, lelis rt. verificação das atitudes de cirurgiõesdentistas quanto à documentação de seus pacientes. rev assoc paul cir dent. 2006; 60: 442-5. 17. barroso mg, vedovello filho m, vedovello sas, valdrighi hc, kuramae m, vaz v. responsabilidade civil do ortodontista após a terapia. rgo. 2008; 56: 67-73. 18. consolaro a. radiografias periapicais prévias ao tratamento ortodôntico. rev dent press ortod ortop facial. 2007; 12: 14-6. 19. capelozza filho l, silva filho og. reabsorção radicular na clínica ortodôntica: atitudes para uma conduta preventiva. rev dent press ortod ortop facial. 1998; 3: 104-26. 20. conselho federal de odontologia (brasil). código de ético odontológico [cited 2007 may 7]. rio de janeiro. available from: http://cfo.org.br/wpcontent/uploads/2009/09/codigo_etica.pdf. 21. brasil. novo código civil. lei no 10.406/2002. diário oficial da união. 10 jan 2002. 22. silva m. a ética e a legislação odontológica aplicadas ao especialista em ortodontia. in: interlandi s. ortodontia bases para a iniciação; 1994. cap. 30. 23. levander e, malmgren o. evaluation of the risks of root resorption during orthodontic treatment: a study of upper incisors. eur j orthod. 1988; 10: 30-8. 24. pereira mmaf, silva rf, maia lva, silva rha, daruge-junior e, paranhos lr. perception of dental surgeons on the ethical and legal aspects of exercising their profession as personal and corporate entities. braz j oral sci. 2011; 10: 246-9. 105105105105105 perception of orthodontists regarding the management of root resorption – clinical and legal aspects braz j oral sci. 11(2):100-105 oral sciences n3 braz j oral sci. 9(2):77-80 original article braz j oral sci. april/june 2010 volume 9, number 2 “in vitro” surface roughness of different glass ionomer cements indicated for art restorations marília gabriela corrêa momesso1 , renata cristiane da silva2, josé carlos pettorossi imparato2, celso molina3, ricardo scarparo navarro4, sidney josé lima ribeiro5 1reserach fellow, department of pediatric dentistry, camilo castelo branco university, brazil 2associate professor, department of preventive dentistry, camilo castelo branco university, brazil 3professor, institute of environmental sciences, chemical and pharmaceutical, university of são paulo, diadema sp, brazil 4associate professor, department of dental materials and restorative dentistry, camilo castelo branco university, brazil 5professor, institute of chemistry of araraquara, são paulo state university, brazil correspondence to: marília gabriela corrêa momesso rua tomé portes, 313 parada inglesa – são paulo – sp – brazil. cep: 02241-010. e-mail: ma_momesso@hotmail.com received for publication: august 04, 2009 accepted: may 12, 2010 abstract aim: the aim of this in vitro study was to evaluate the surface roughness of three glass ionomer cements (gics) indicated for art restorations. methods: ten cylindrical specimens of three commercial glass ionomers cements (vidrion r s.s. white, maxxion r fgm and vitromolar dfl) were prepared (n=30) without surface finishing or protection. twenty-four hours after preparation, the surface roughness measurements were obtained as the mean of three readings of the surface of each specimen by profilometry. the roughness values (ra, µm) were subjected to one-way anova and tukey’s test (p<0.05). results: no statistically significant differences were observed between vidrion r (0.18 ± 0.05) and vitromolar (0.21 ± 0.05), whereas maxxion r presented significantly higher roughness values than those of the other materials. conclusions: it may be concluded that characteristics of particle size and composition of the different gics affected their surface roughness 24 h after preparation. keywords: roughness, profilometry, glass ionomer cements, art, restorative dental materials. introduction when first introduced in the 1970’s, glass ionomer cements (gics) were used as a lining material or as the basis for restorations1. however, alterations to its composition and the powder/liquid ratio affected their mechanical properties, handling time, setting time, consistence and wear, improving the feasibility and application of these conventional fast-setting ionomeric cements in clinical practice. these materials are particularly effective in the atraumatic restorative treatment (art) and in places lacking the conventional infrastructure needed for clinical treatment1-5. the properties of gics, comprise a coefficient of thermal expansion similar to that of dentin2,6-7, lower volumetric contraction during the setting reaction7, chemical adherence to the dental strucutre2,6-8, biocompatibilty with the pulp tissue7,9, fluoride release and cariogenic action2,6-8,10.11, and antimicrobial activity5,11. however, bond strength and resistance to wear are rather limited, especially for conventional restorative gics and fast-setting or high-viscosity gics, in comparison to amalgam and modern resin composite materials. these properties are also affected by their composition and the acid-base reactions between the inorganic portion of the powder and the organic portion of the carboxylic acids used, the size and number of vitreous particles, and the number and size of bubbles present in the material12-14. 78 braz j oral sci. 9(2):77-80 brand manufacturer powder-liquid ratio basic composition particle size vidrion r vitromolar maxxion r ss white artigos dentários ltda(rio de janeiro, rj, brazil) dfl indústria e comércio ltda(rio de janeiro, rj, brazil) fgm produtos odontológicos (joinville, sc, brazil) 1:1 1:1 1:1 sodium fluorosilicate, calcium aluminum, barium sulfate, polyacrylic acid, pigments, tartaric acid, distilled water aluminum and barium silicate, dehydrated polyacrylic acid, ferric oxide, polyacrylic acid, tartaric acid, distilled water fluoraluminosilicate glass, calcium fluoride, water <75µm <10µm ±12.5µm table 1 – brand, manufacturer and powder-liquid ratio of the materials used. *information from manufacturers mair et al.15 defines wear as the last consequence of the interaction between the surfaces, leading to the steady removal of the material. clinically, surface roughness must be observed, as it plays a decisive role in the retention and accumulation of dental biofilm16. surface roughness has been used as a criterion to foresee and evaluate the deterioration of restorations made from different materials. while surface roughness of aesthetic materials in vivo is put down to mechanical abrasion, attrition and erosion, most of the current in vitro studies have evaluated surface roughness after mechanical abrasion and polishing16. bollen et al.17 reported that, on a rough surface, the microorganisms are less exposed to the dislocation forces and have the necessary time to adhere to this structure. the surface and the border of the restorative materials, when colonized by cariogenic bacteria, especially streptococcus mutans, favor the development of caries and future damage to the dentin-pulp complex10-11,18. profilometry is the measurement of the surface height variation of an object. it can be used to determine measurements of surfaces, shape and roughness. this latter requires instruments with both high lateral (x axis) and vertical resolution (z axis). this in vitro study used profilometry to evaluate the surface roughness of a conventional restorative gic and two fast-setting gics, 24 h after preparation of the materials. material and methods the glass ionomer cements used in this study are presented in table 1. ten disc-shaped specimens of each material were fabricated using a matrix with diameter of 6.0 mm and a 4.0-mm-deep cavity. the materials prepared following the manufacturer’s instructions by a previously calibrated operator at room temperature (approximately 23°c) and 50% relative air humidity (humidity/temperature meter – ht – 3003 – lt lutron). the matrix was placed on a glass plate with a polyester strip (k-dent, quimidrol) interposed between the matrix and the glass plate. the materials were mixed and inserted in the matrix cavity using a centrix injector until it was completely filled, and was then covered with another polyester strip and a glass plate18-21. a uniform pressure was applied and excess material was removed, leveling of the cement with the top of the matrix. after 10 min, the polyester strips were removed, and the specimens were stored in 100% humidity, without any surface protection, finishing or polishing. after 24 h of storage under these conditions, surface roughness was evaluated using the form talysurf series 2 profilometer22. the form talysurf series 2 instrument consists of a mechanical profilometer in which a mechanical transducer is dragged across a surface and its movement in a vertical direction is recorded to obtain a surface profile22. for every reading made, the mean roughness value (ra, mm) was represented by the arithmetic mean between the peaks and valleys registered, after the needle of the profilometer had scanned a stretch of 3.1mm in length, with a cut-off of 0.25mm to maximize the filtering and the undulation on the surface. each surface was read three times, always with the needle scanning the geometric center of the specimen, starting from three different points13,21. the mean value of the three readings yielded the mean value of the roughness of each specimen. subsequently, a 3d image (form talysurf series 2 profilometer) of the surface profile of the specimens was obtained. the roughness mean values (ra, µm) were subjected to one-way anova) and tukey’s test at a 5% significance level. results the roughness mean values (ra, µm) and standard deviations obtained for the tested materials were as follows: vidrion r: 0.18 (0.06), vitromolar: 0.21 (0.06), and maxxion r: 0.73 (0.38). the one-way anova and tukey’s showed that maxxion r presented the highest roughness mean values and differed significantly from the other materials (p<0.05). there was no statistically significant difference (p>0.05) between vidrion r and vitromolar. fig. 1. 3d image of the profile of the glass ionomer cement vidrion r. “in vitro” surface roughness of different glass ionomer cements indicated for art restorations 79 braz j oral sci. 9(2):77-80 fig. 3. 3d image of the profile of the glass ionomer cement maxxion r. fig. 2. 3d image of the profile of the glass ionomer cement vitromolar. discussion gics have becoming widely used in dentistry due to their properties of adherence, biocompatibility, aesthetics, fluoride release and similar linear thermal expansion to dentin, and because of their clinical uses in both primary and permanent teeth 1-2,4-9. as a result, the study of their biomechanical properties and clinical applications is important for the evaluation and prediction of the clinical behavior of these cements. according to the methodology adopted in this study, the specimens were kept for 24 h in an environment where the relative humidity of the air was about 100%, without any protection, finishing system or polishing. sidhu et al.23 reported that the cover or finishing used in clinical procedures may veil the characteristics of the material in laboratory experimentations. the best evenness of the surface was attained when the materials were cured in contact with the polyester strip18-21. while the setting reaction of gics is taking place, links are formed between the carboxylic acids (liquid portion) and the alumina cations and/or the inorganic part yielded by the powder (solid portions). these reactions play a role in forming the ionomer, while the others act as reinforcement particles12,24. according to the present study, the surface roughness mean values for the conventional restorative gic (vidrion r) proved to be lower when compared to the other ionomeric cements. according to rios et al. 25, the gics, whose consistence is more fluid during handling and insertion, produce a decreased surface roughness, which may be caused by the greater portion of its gel matrix. mair et al.15 observed that the distribution and morphology of the inorganic particles are an important factor in determining surface roughness. the lack of significant differences between vidrion r and vitromolar might be attributed to the similar size and location of the inorganic particles in these materials, despite the differences in their consistency and mechanical properties4 although vidrion r presented the lowest roughness mean values in the present study, the worse mechanical properties and high solubility of this material restricts its use in the art technique4,25-26. on the other hand, the conventional high-viscosity gic, which present better mechanical properties and art indication, presented higher roughness mean values in this study, especially maxxion r. it is important to point out that, as the surface hardness of gics is inversely proportional to its wear, the conventional highviscosity gics are harder and display reduced surface wear, preserving the initial roughness pattern12,26-27. the exception was observed for maxxion r, suggesting that this behavior may be related to the size and shape of glass particles on its surface21. leitão and hegdahl 28 reported that the surface is considered rough when it bears peaks and valleys of great amplitude with reduced undulation. the value of the surface roughness (ra) considered critical for the retention and adherence of microorganisms is equal to 0.2 µm17. in this study, two gics yielded results aligned with the parameters acceptable for surface roughness: vidrion r (0.18 ± 0.05) and vitromolar (0.21 ± 0.05), showing evidence of a greater susceptibility to biofilm retention, where the value of 0.2 µm is used as a reference. in contrast, the surface roughness of maxxion r (0.73 ± 0.38) was much higher than expected, increasing its potential for the adherence of microorganisms. figures 1-3 show the roughness 3-d images obtained for each material used in this study. it is possible to observe that figures 1 and 2 illustrate a smoother surface than figure 3. these results are in agreement with the roughness values obtained. the study of surface roughness is important due to the fact that this property affects light reflection, color fading, appearance of cracks and aesthetics, in addition to favoring biofilm accumulation13,17. increased surface roughness results in substantial biofilm accumulation, thus aggravating the risk of carious lesion and periodontal disease17,25. since the surface roughness of gic must be carefully observed when it comes to choosing the material, it is vital for the professional to analyze laboratory and clinical requisites such as surface microhardness, mechanical resistance, solubility, setting time and work, ease of handling, in addition to location and extension of the cavities in relation to the chewing load and, finally, the clinical durability of the restoration. references 1. sasaki mt, silva rcsp, araújo mam, krabbe dfm, damião aj. avaliação da rugosidade superficial de cimentos de ionômero de vidro com diferentes sistemas de acabamento e polimento. rev odontol unesp. 2000; 29: 81-92. 2. berg jh. glass ionomer cement. pediatr dent. 2002; 24: 430-8. 3. de witte mjc, maeyer eap, verbeeck rmh. surface roughening of glass ionomer cements by neutral naf solutions. biomaterials. 2003; 24: 19952000. 4. raggio dp, rocha ro, imparato jcp. avaliaçäo da microinfiltraçäo de cinco cimentos de ionômero de vidro utilizados no tratamento restaurador atraumático (tra). j bras odontopediatr odontol bebe. 2002; 5: 370-7. “in vitro” surface roughness of different glass ionomer cements indicated for art restorations 80 braz j oral sci. 9(2):77-80 5. frencken je, van´t hof ma, van amerogen we, holmgren cj. effectiveness of single surface art restorations in the permanent dentition: a metaanalysis. j dent res. 2004; 83: 120-3. 6. cole boi, welbury rr. the atraumatic restorative treatment (art) technique: does it have a place in everyday practice? dent update. 2000; 27: 118-23. 7. hse kmy, leung sk, wei shy. resin-ionomer restorative materials for children: a review. aust dent j. 1999; 44: 1-11. 8. mjör ia, gordan vv. a review of atraumatic restorative treatment (art). int dent j. 1999; 49: 127-31. 9. yip h-k, peng d, smales rj. effects of apf gel on the physical structure of compomers and glass ionomer cements. oper dent. 2001; 26: 231-8. 10. serra mc, cury ja. the in vitro effect of glass-ionomer cement restoration on enamel subjected to a demineralization and remineralization model. quintessence int. 1992; 23: 143-7. 11. van amerongen, we. dental caries under glass ionomer restorations. j public health dent. 1996; 56: 150-4. 12. xie d, brantley wa, culbertson bm, wang g. mechanical properties and microstructures of glass-ionomer cements. dent mater. 2000; 16: 129-38. 13. turssi cp, magalhães cs, serra mc, rodrigues jr al. surface roughness assessment of resin-based materials during brushing preceded by phcycling simulations. oper dent. 2001; 26: 576-84. 14. hurrell-gillingham k, reaney im, miller ca, crawford a, hatton pv. devitrification of ionomer glass and its effect on the in vitro biocompatibility of glass-ionomer cements. biomaterials. 2003; 24: 3153-60. 15. mair lh, stolarski ta, vowles rw, lloyd ch. wear, mechanisms, manifestations and measurement. report of a workshop. j dent. 1996; 24: 141-8. 16. yip h-k, lam wtc, smales rj. surface roughness and weight loss esthetic restorative materials related to fluoride release and uptake. j clin pediatr dent. 1999; 23: 321-6. 17. bollen cml, lambrechts p, quirynen m. comparison of surface roughness of oral hard materials to the threshold surface roughness for bacterial plaque retention: a review of the literature. dent mater. 1997; 13: 258-69. 18. serra mc, navarro mfl, freitas sft, carvalho rm, cury ja, retief dh. glass ionomer cement surface protection. am j dent. 1994; 7: 203-6. 19. pedrini d, gaetti-jardim jr e, mori gg. influência da aplicação de flúor sobre a rugosidade superficial do ionômero de vidro vitremer e adesão microbiana a este material. pesq odontol bras. 2001; 15: 70-6. 20. pedrini d, candido msm, rodrigues jr al. analysis of surface roughness of glass-ionomer cements and compomer. j oral rehabil. 2003; 30: 714-9. 21. silva rc, zuanon acc. surface roughness of glass ionomer cements indicated for atraumatic restorative treatment (art). braz dent j. 2006; 17: 106-9. 22. exploring surface texture: a fundamental guide to the measurement of surface finish. leicester, england: taylor hobson ltd; 2003. 23. sidhu sk, sherriff m, watson tf. in vivo changes in roughness of resinmodified glass ionomer materials. dent mater. 1997; 13: 208-13. 24. pelka m, elbert j, schneider h, krämer n, petschelt a. comparison of two and threee body wear of glass ionomers and composites. eur j oral sci. 1996; 4: 132-7. 25. rios d, honório hm, araújo pa, machado maam. wear and superficial roughness of glass ionomer cements used as sealants, after simulated toothbrushing. braz oral res. 2002; 16: 343-8. 26. raggio dp, bönecker m, imparato jcp, weiner a, de gee a, van amerogen we. dureza knoop de cimentos de ionômero de vidro indicados para o tratamento restaurador atraumático (tra). rev ibero am odontopediatr odontol bebe. 2006; 10: 152-7. 27. peutzfeldt a, garcia-godoy f, asmussen e. surface hardness and wear of glass-ionomer and compomers. am j dent. 1997; 10(1): 15-7. 28. leitão j, hegdahl t. on the measuring of roughness. acta odontol scand. 1981; 39(6): 379-84. “in vitro” surface roughness of different glass ionomer cements indicated for art restorations oral sciences n3 braz j oral sci. 11(3):406-410 original article braz j oral sci. july | september 2012 volume 11, number 3 analysis of canine dimorphism in the estimation of sex yuri trigueiro faustino da costa1, laíse nascimento correia lima2, patrícia moreira rabello3 1dds, federal university of paraíba, joão pessoa, pb, brazil 2specialist in forensic dentistry, msc in forensic dentistry and deontology, piracicaba dental school, university of campinas, piracicaba, sp, brazil 3adjunct professor, forensic and ethical dentistry and dental legislation, federal university of paraíba, joão pessoa, pb, brazil correspondence to: yuri trigueiro faustino da costa rua francisco timóteo de souza, 577 cep: 58052-130 bancários, joão pessoa, pb brasil phone: + 55 83 32350296 e-mail: yuri.trigueiro@yahoo.com.br abstract aim: to evaluate the sexual dimorphism of mandibular and maxillary canines among dental students of the federal university of paraiba, brazil. methods: this was an observational, blind and cross-sectional study with comparative and statistical-descriptive procedure. fifty-one pairs of plaster models belonging to undergraduate dental students aged 18-29 years were analyzed. quantitative data were organized and processed by means of the statistical package for the social sciences (spss) software, version 15.0. this research has followed the guidelines of 196/ 96 brazilian resolution of the national health council, ministry of health. results: all measures were found to show statistically significant differences between sexes (p<0.001) according to student’s t-test. regarding the difference between the four canines for each sex separately, it was found difference only between mandibular and maxillary canines (p<0.001) according to the f test (anova), but with no significant difference between the right and left sides. conclusions: odontometric techniques allowed concluding that canine teeth present statistically significant sexual dimorphism, and that they may be useful in the estimation of sex in complementary methods during body identification. the data obtained in this study were compared with those of other studies to provide information about sexual dimorphism be specific for each population. keywords: canine tooth, forensic anthropology, forensic dentistry, odontometry, sex dimorphism. introduction the estimation of sex is an important step to build the biological profile of unidentified human remains, especially when an accurate outcome is achieved, considering that about half of the population would be automatically excluded during search operations. although dna analysis provides irrefutable evidences on sex identification in human remains, such a technique is relatively prolonged and quite exhaustive if compared to assessment of skeletal parameters1. it is known that morphological and metric aspects of the skeleton allow a more reliable diagnosis of sex. the more measurements and data obtained, the more reliable will be the result. for instance, sex may be estimated by means of bones that constitute the pelvic girdle since it is the skeleton segment which most presents sexual dimorphism. it also may be analyzed the morphology and dimension of long bones of the body. additionally, estimation of sex may still be performed through observation of qualitative or morphological and quantitative or metric aspects present in the skull2. accordingly, estimation of sex does not represent a problem when a complete skeleton is found. nevertheless, if only the mandibular bone along with the teeth received for publication: may 16, 2012 accepted: september 18, 2012 braz j oral sci. 11(3):406-410 407407407407407 is found or mandible fragments or even the teeth by themselves are available in the site, so estimation of sex may be performed with the help of teeth dimensions3. with this approach, numerous authors have studied intensively sexual dimorphism present in teeth by means of odontometric analyses, and most studies have showed statistically significant differences in the permanent dentition4-6.thus, this work aimed to analyze the degree of sexual dimorphism of mandible and maxillary canines among undergraduate dental students of the federal university of paraiba, using odontometric techniques described in literature, as well as to provide arguments for the debate about dimorphism being specific for each population. material and methods this research project was conducted in accordance with the 196/96 brazilian resolution of the national health council, ministry of health, which regulates research involving human beings. it was submitted to and approved by the research ethics committee of lauro wanderley university hospital (protocol #360/10). an observational, blind and cross-sectional study was performed, applying comparative and statistical-descriptive procedure. the procedure method employed was an intensive direct observation by means of examination of plaster models of the upper and lower arches. the sample was composed by plaster models of the upper and lower arches belonging to undergraduate dental students of the federal university of paraiba. fifty-one students, aged 18-29 years, being 26 female, took part of the research, which encompassed a total of 102 plaster models. a digital caliper with thin beaks (digimess®, 150 mm/ 6" 100.250, 0.01 mm/.0005 resolution”, são paulo, sp, brazil) was used to measure the mesiodistal (md) and buccolingual (bl) distances of crowns of the maxillary and mandible canines, as well as their diagonal, mesiobuccal-distolingual (mb-dl) and distobuccal-mesiolingual (db-ml) dimensions. the measurements in the md and bl directions were obtained following the acharya and mainali’s method 7 applied only for the canine teeth, proceeding as follows: md measurements the greatest distance between the proximal surfaces of the tooth crown was registered by means of the caliper beaks incisally positioned along the long axis of the tooth. in cases of bad position or giroversions, measurements should be taken between the points of the proximal surfaces of the crown, where contact with the adjacent tooth would normally occur (figure 1). bl measurements the greatest distance between the buccal and lingual surfaces of the tooth crown was obtained with the caliper located incisally and forming a right angle in relation to the md sense (figure 2). for the measurements in the mb-dl and db-ml senses, fig. 1. sense of mesiodistal measurement. source: direct research. fig. 2. sense of buccolingual measurement. source: direct research. the methodology described by karaman4 was employed, being performed in the canine teeth, proceeding as follows: mb-dl measurements the greatest distance between the mb and dl corners of the tooth crown was obtained with the caliper positioned parallel to the incisal edge (figure 3). db-ml measurements the greatest distance between the db and ml corners of the tooth crown was achieved with the caliper positioned fig. 3. dark line indicating measurements in the mb-dl sense and red line indicating measurements in the db-ml sense. source: direct research. analysis of canine dimorphism in the estimation of sex braz j oral sci. 11(3):406-410 408408408408408 gender measurements tooth male female p-value mean ± sd(1) mean ± sd(1) · mesiodistal 13 8.17 ± 0.49 (a) 7.46 ± 0.36 (a) p(1) < 0.001* 23 8.07 ± 0.38 (a) 7.45 ± 0.36 (a) p(1) < 0.001* 33 6.98 0.40 (b) 6.48 ± 0.36 (b) p(1) < 0.001* 43 6.99 0.40 (b) 6.47 ± 0.34 (b) p(1) < 0.001* p-value p(3) < 0.001* p(3) < 0.001* · buccolingual 13 8.59 ± 0.66 (a) 7.93 ± 0.63 (a) p(1) = 0.001* 23 8.54 0.61 (a) 7.85 ± 0.58 (a) p(1) < 0.001* 33 7.73 0.57 (b) 7.09 ± 0.49 (b) p(1) < 0.001* 43 7.70 0.61 (b) 7.09 ± 0.55 (b) p(1) < 0.001* p-value p(3) < 0.001* p(3) < 0.001* · mesiobuccal-distolingual 13 8.08 ± 0.47 (a) 7.48 ± 0.46 (a) p(1) < 0.001* 23 7.96 0.43 (a) 7.37 ± 0.44 (a) p(1) < 0.001* 33 7.26 0.57 (b) 6.61 ± 0.41 (b) p(1) < 0.001* 43 7.28 0.49 (b) 6.70 ± 0.43 (b) p(1) < 0.001* p-value p(3) < 0.001* p(3) < 0.001* · distobuccal-mesiolingual 13 7.72 ± 0.44 (a) 7.14 ± 0.34 (a) p(1) < 0.001* 23 7.71 0.37 (a) 7.13 ± 0.40 (a) p(1) < 0.001* 33 6.63 0.40 (b) 6.16 ± 0.35 (b) p(1) < 0.001* 43 6.59 0.42 (b) 6.13 ± 0.34 (b) p(1) < 0.001* p-value p(3) < 0.001* p(3) < 0.001* (*): 5% significant difference. (1): student’s t test with equal variances. (2): student’s t test with unequal variances. (3): f test (anova) with repetitive measures. note: if all the letters in brackets are distinct, there is significant difference between the corresponding teeth using multiple (paired) bonferroni’s comparisons. table 1. mean and standard deviation of measurements according to gender and tooth. joão pessoa, pb, brazil. teeth named according to the fdi system. parallel to the incisal edge (figure 3). were excluded from the sample: all models lacking one of the teeth of interest to the research (upper and lower canines, right and left sides) and models exhibiting fillings in the proximal and free surfaces, bad tooth positioning (impossible to obtain the measurement), and damaged or broken teeth. were also excluded the students who: wore orthodontic appliances, felt nausea during impression procedures, and those who expressed the feeling of being withdrawn from the study, after theoretical approach about the subject in question. quantitative data attained in the study were organized and processed by means of the statistical package for the social sciences (spss), version 15.0 (spss inc., chicago, il, usa). it was conducted a descriptive and inferential analysis through specific statistical methods, in which were obtained: mean and standard deviation, student’s t with equal or unequal variances and f (anova) tests for repetitive measures with bonferroni’s comparisons. verification of the equal variances hypothesis was performed using the levene f test. the error margin used in the decision of the statistical tests was 5%. from the results, it was made the distribution of frequencies of all variables addressed in the study, presented in tables, charts or graphs to characterize the sample and data description. results table 1 presents the means and standard deviations for the md, bl, mb-dl and db-ml measurements, according to tooth and sex. in this table, it was verified whether existed or not statistical difference between means of the measurements for each tooth in relation to sex. statistically significant differences indicated by student’s t test were observed for all teeth and in all measurements (p(1) < 0.001 or p (1)=0.001). it was also investigated in that table whether there was significant difference or not among the 4 canines, for each sex separately, in relation to each one o f t h e m e a s u r e m e n t s a n a l y z e d . t o t h i s e n d , f t e s t (anova) was employed for repetitive measures with bonferroni’s comparisons, considering a 5% error margin. no statistically significant difference was found between the right and left sides, either in the upper or lower arches, in none of the measures analyzed. nevertheless, were noted differences between the upper and lower measurements (p(2)<0.001). table 2 expresses the comparison of dimensions of the md and bl measurements in the considered sample in relation to other studies. it is verified that teeth dimensions in the md and bl senses resembles the portuguese population’s measures. analysis of canine dimorphism in the estimation of sex braz j oral sci. 11(3):406-410 409409409409409 discussion numerous odontometric studies performed in human dental arches attest the fact that there are differences in teeth size patterns between sexes, and that those differences vary among populations, reaching significance or not. canines have demonstrated higher degree of sex dimorphism among the populations10-11 and are considered as one the most resistant teeth in the dentition, remaining intact in several post-mortem scenarios12. md and bl distances according to table 1, the md and bl distances were found to show statistically significant differences for all canines, being higher for males than females, which is a finding that corroborates the results verified in other populations. comparing with measurements obtained in other studies (table 2), it was noted that, for both sexes and measures, the present study assimilates with the upper canine teeth of the portuguese6 population sample, but are higher than the indian13 population also for both sexes and measures. nonetheless, it is observed that for other populations under comparison, the means of measurements for both sexes vary a lot, being higher for a given measure and sex and lower for another measure and sex, e.g., when the present investigation is compared with the study by s u a z o e t a l . 1 4, i t i s v e r i f i e d t h a t b r a z i l i a n ’ s c a n i n e dimensions are higher for male and lower for the female gender. carefully, of course, we can assign these apparent d i f f e r e n c e s i n m e a s u r e m e n t s d u e t o s u c h t h i n g s a s differences in techniques of tooth measurement and degree of wear. garn et al.15 report that genetic influence tends to keep low/high magnitude dimorphism in interrelated racial groups originating from the same geographical region. that may explain the differences between tooth size in the diverse populations. however, those differences should also be in part assigned to the degree of miscegenation that some populations present. thus, it makes sense that the sample studied resembles with the sample surveyed in the portuguese population, despite the fact that only the upper teeth were assessed in the present study. regarding the difference between right and left sides (asymmetry), it was found no significant difference for both sexes, evidenced by the minimum difference between the averages of right and left sides (table 1). it is suggested that there is no tendency for the dimensions of the crown being larger on one side than on the other side, so confirming some literature findings16-17. however, pereira et al.6 showed that, for females, in the md measurement, there was statistically significant difference between right and left sides of the upper canine (p <0.005), using the paired t test. analysis of canine dimorphism in the estimation of sex author population male female tooth md bl tooth md bl pettenati-soubayroux, 20028 french 13 e 23 7.79 13 e 23 7.06 33 e 43 7.01 33 e 43 6.70 iscan; kedici, 200310 turks 13 e 23 8.61 13 e 23 7.78 33 e 43 8.04 33 e 43 7.19 ling; wong, 20079 chinese 13 e 23 8.30 8.09 13 e 23 7.92 7.84 33 e 43 7.31 7.20 33 e 43 6.89 7.04 suazo et al., 200814 chilean 13 8.04 8.36 13 7.91 8.05 23 7.92 8.12 23 7.83 7.94 astete; san pedro; suazo, 20095 spanish 13 8.49 8.53 13 8.18 7.84 23 8.60 8.43 23 8.14 7.84 prabhu; acharya, 200913 indian 13 7.65 8.12 13 7.44 7.75 23 7.59 8.06 23 7.39 7.75 33 6.69 7.32 33 6.49 7.00 43 6.61 7.38 43 6.45 7.03 pereira et al., 20106 portuguese 13 8.17 8.76 13 7.77 8.21 23 8.03 8.74 23 7.70 8.21 13 e 23 8.10 8.76 13 e 23 7.74 8.21 present study, 2010 brazilian 13 8.17 8.59 13 7.46 7.93 23 8.07 8.54 23 7.45 7.85 13 e 23 8.12 8.56 13 e 23 7.45 7.89 33 6.98 7.73 33 6.48 7.09 43 6.69 7.70 43 6.47 7.09 33 e 43 6.83 7.71 33 e 43 6.47 7.09 “13”, “23”, “33” and “43” = individual averages; “13 and 23”, “33 and 43” = averages of the sides. table 2. differences between averages of the mesiodistal (md) and buccolingual (bl) dimensions in the sample studied and in other populations. joão pessoa, pb, brazil. 410410410410410 braz j oral sci. 11(3):406-410 mb-dl and db-ml distances according to the results obtained in the analysis of diagonal measures undertaken in the course of this study, males’ teeth were considered higher than females’, and such a difference was statistically significant between sexes (p<0.001). these findings are consistent with other studies described in literature4,18, which affirm canines to be the most dimorphic elements in these diagonal lengths. no statistically significant difference was observed between the right and left sides, in upper and lower arches, similarly to the md and bl measurements. this way, although the completion of odontometric measurements in this field is relatively new, these results corroborate with those found in literature, confirming that these dimensions suggest a high level of reliability, and may be used in conjunction with other measures. the present investigation showed significant differences between dimensions of canines in both sexes, suggesting a high reliability rate for using these teeth in the estimation of sex. references 1. acharya ab, prabhu s, muddapur mv. odontometric sex assessment from logistic regression analysis. int j legal med. 2011 mar.; 125(2): 199204. 2. vanrell j. odontologia legal e antropologia forense. 2º ed, rio de janeiro: guanabara koogan; 2009. 420 p. 3. kavitha b. sex determination in teeth. [dissertation]. chennai: the tamil nadu dr. m.g.r. medical university; 2005. 23p. master of dental surgery. 4. karaman, f. use of diagonal teeth measurements in predicting gender in a turkish population. j forensic sci. 2006 may.; 51(3): 630-635. 5. astete jc, san pedro vj, suazo gi. sexual dimorphism in the tooth dimensions of spanish and chilean peoples. int j odontostomatol. 2009 jan-jun.; 3(1): 47-50. 6. pereira c, bernardo m, pestana d, santos jc, mendonça mc. contribution of teeth in human forensic identiûcation – discriminant function sexing odontometrical techniques in portuguese population. j forensic leg med. 2010 feb.; 17(2): 105-110. 7. acharya ab, mainali s. univariate sex dimorphism in the nepalese dentition and the use of discriminant functions in gender assessment. forensic sci int. 2007 nov.; 173(1): 47-56. 8. pettenati-soubayroux i, signoli m, dutour o. sexual dimorphism in teeth: discriminatory effectiveness of permanent lower canine size observed in a xviiith century osteological series. forensic sci int 2002 may.; 126: 227–232. 9. ling jyk, wong rwk. tooth dimensions of southern chinese. homo 2007 mar.; 58(1): 67-73. 10. iscan my, kedici ps. sexual variation in bucco-lingual dimensions in turkish dentition. forensic sci int. 2003 nov.; 137(2-3): 160-164. 11. lund h, mörnstad h. gender determination by odontometrics in a swedish population. j forensic odontostomatol. 1999 dec.; 17(2): 30-34. 12. acharya ab, aangali pv, prabhu s, nagnu s. validity of the mandibular canine index (mci) in sex prediction: reassessment in an indian sample. forensic sci int. 2011 jan.; 204(1-3): 207.e1-207.e4. 13. prabhu s, acharya ab. odontometric sex assessment in indians. forensic sci int. 2009 nov.; 192(1-3): 129.e1-129.e5. 14. suazo gi, cantín lm, lopez fb, sandoval mc, torres ms, gajardo rp, gajardo rm. sexual dimorphism in mesiodistal and bucolingual tooth dimensions in chilean people. int j morphol. 2008 sep.; 26(3): 609-614. 15. garn sm, lewis ab, swindler dr, kerewsky rs. genetic control of sexual dimorphism in tooth size. j dent res. 1967 sep.; 46(5): 963-972. 16. sherfudhin h, abdullah ma, khan n. a cross-sectional study of canine dimorphism in establishing sex identity. comparison of two statistical methods. j oral rehabil. 1996 sep.; 23(9): 627-631. 17. kalia s. a study of permanent maxillary and mandibular canines and inter-canine arch widths among males and females. [dissertation]. karnataka: rajiv gandhi university of health sciences; 2006. 175 p. master of dental surgery. 18. rai b, anand sc. gender determination by diagonal distances of teeth. the internet journal of biological anthropology [serial on the internet] 2007; 1(1): [about 7 pages]. analysis of canine dimorphism in the estimation of sex oral sciences n3 braz j oral sci. 10(2):140-145 original article braz j oral sci. april | june 2011 volume 10, number 2 association between infant feeding duration and the terminal relationships of the primary second molars ana carla raphaelli nahás-scocate1, paulo xavier de moura2, renata bara marinho3, adriana pinheiro alves2, rívea inês ferreira1, fernanda meister guimarães3 1associate professor, department of orthodontics, university of são paulo city (unicid), brazil 2graduate student, university of são paulo city (unicid), brazil 3research fellow, department of orthodontics, university of são paulo city (unicid), brazil correspondence to: ana carla raphaelli nahás-scocate universidade cidade de são paulo – unicid pós-graduação mestrado em ortodontia rua cesário galeno, 448 – bloco beta 03071-000 – tatuapé – são paulo – sp phone: 11-21781310 / fax: 11-21781310 e-mail: carlanahas@yahoo.com.br abstract aim: this study was developed to assess the effect of prolonged bottle feeding and breastfeeding on the anteroposterior relationship of the dental arches in 3-6-year-old children attending preschools in the eastern zone of são paulo city. methods: the association between infant feeding duration (artificial and natural) and occlusal characteristics were evaluated in 485 children at the stage of complete primary dentition, who attended municipal preschools in the eastern zone of são paulo city. the information related to types and duration of child breastfeeding was investigated by means of questionnaires filled out by parents/guardians. the occlusal characteristics corresponding to the terminal relationships of the primary second molars were classified as vertical plane (vp), mesial step (ms) and distal step (ds). logistic regression analysis (p < 0.05) was applied. results: considering the right and left sides, ds was diagnosed in 9.7% of the cases, ms in 14.2% and vp in 76.1%, without significant differences between sides and genders. the association between the presence of ds and the age of bottle-feeding and breastfeeding cessation was significantly high (p < 0.001). the older the child when bottle-feeding ceased (34 years old) and the shorter the breastfeeding duration (<3 months), the greater the chances of the child presenting ds. conclusions: breastfeeding duration is one of the factors that could influence the development of ds because the longer the breastfeeding duration, the higher the percentage of children who were not bottle fed, ranging from 5.8% (among children who interrupted breastfeeding before the age of 3 months) to 63.8% (after 12 months of age). keywords: bottle feeding, breastfeeding, malocclusion, epidemiology. introduction there are several factors that could lead to malocclusion in primary dentition. in general, these factors may be genetic or environmental, for example, such as nonnutritive sucking habits finger and pacifier-sucking1-3. the key to determining the etiology of malocclusions is to assess the preponderant effect of each factor in particular. nowadays, there has been emphasis on the importance of breastfeeding, since mother’s milk is the best diet from a nutritional point of view, reinforcing received for publication: february 09, 2011 accepted: may 09, 2011 141 braz j oral sci. 10(2):140-145 the child’s immunological system against infectious and allergic diseases. furthermore, the act of breast sucking produces adequate development of the masticatory system structures, providing balance of the internal and external muscular contention forces4-7. lower prevalence and severity of malocclusions in breastfed children, in comparison with those that were bottle fed, have been reported8. the association between nonnutritive sucking habits and the development of malocclusions in the primary dentition has been mentioned in previous investigations1-3,5. however, few studies have addressed the influence of bottle feeding on occlusal characteristics. thus, the aim of this epidemiological study was to assess the effect of prolonged bottle feeding and breastfeeding on the anteroposterior relationship of the dental arches in children aged 3 to 6 years attending to public preschools in the eastern zone of são paulo city. material and methods this cross-sectional study was conducted in agreement with the brazilian national council health’s resolution act 196/96. the sample consisted of children of both genders aged 3 to 6 years from three public preschools located in the eastern zone of são paulo, sp, brazil. the total number of children assessed was 984, and 499 were excluded for not meeting the inclusion criteria: informed consent form signed by the parents/guardians; adequately filled out questionnaires on sucking habits; complete primary dentition, without erupted permanent teeth or teeth in the process of eruption; absence of extensive caries lesions or loss of coronal structure, which would compromise occlusion; absence of early primary tooth loss; absence of any type of trauma; absence of visual and/or mental and/or hearing deficiencies; and no history of orthodontic and/or speech treatment. the children who were assessed did not show characteristics suggestive of cleft lip and palate fissures or any other anomalies that could contribute to the establishment of malocclusions. therefore, the final sample was composed of 485 children, being 248 girls and 237 boys. information on the types and duration of infant feeding and the data about their general health were investigated by means of questionnaires filled out by the parents/guardians. the clinical examinations were performed by a calibrated dentist who was blind to the questionnaire data. the aim of examiner calibration was to explain the main doubts with regard to the clinical data and standardize the method of assessment and registering the information for each individual. the data obtained from calibration were submitted to kappa statistics (k) for reproducibility analysis. a k index higher than 0.81 was obtained, indicating good intraexaminer agreement. the clinical examinations were performed in the school environment, using disposable wooden spatulas, under a suitably directed source of artificial lighting. the children remained comfortably seated while clinical records were filled out. during the examinations the child was asked to perform maximum mouth opening and then to bite in maximum intercuspation (mi) to collect the clinical data. to classify the relationships of the distal surfaces of primary second molars the criteria proposed by baume were applied9: 1. vertical plane (vp) – the distal surfaces of the primary second molars coincide in the same vertical plane; 2. mesial step (ms) – the distal surface of the mandibular primary second molar is located in a more mesial direction in relation to the distal surface of the maxillary primary second molar; 3. distal step (ds) – the distal surface of mandibular primary second molar is located in a more distal direction in relation to the distal surface of the maxillary primary second molar. after clinical assessments and collection of information based on the above-mentioned inclusion criteria, the children were divided into two major groups: group according to the age of interrupting bottle feeding, which was divided into four sub-groups: no bottle feeding habit; bottle feeding interrupted by the age of 2 years; bottle feeding interrupted between the ages of 3 and 4 years; bottle feeding interrupted between the ages of 5 and 6 years. group according to the age of interrupting breastfeeding, which was divided into six sub-groups: not breastfed; breastfeeding for less than 3 months of age; breastfeeding interrupted between 3 and 6 months of age; breastfeeding interrupted between 6 and 9 months of age; breastfeeding interrupted between 9 and 12 months of age; and over than 12 months of age. initially, the frequency of the data with reference to breast and bottle feeding, age and gender were calculated. next, the prevalence of the relationships of the distal surfaces of the primary second molars was obtained, in the complete sample and according to the study group. logistic regression analysis was applied to identify the factors related to breastfeeding and bottle feeding durations that could influence on the development of the relationships of the distal surfaces of the primary second molars. the level of significance was set at 5%. two regression models were constructed simultaneously, one for the presence of ds and the other for the presence of ms, using the group that presented vp as a reference. the co-variables used in the models were: age of bottle feeding cessation/interruption – the reference group did not have the habit; age of breastfeeding interruption – the reference group was the one that interrupted the habit after 12 months of age; gender – the reference group was composed by boys; and side – the reference group was the right side. the odds ratio (or) was estimated based on the co-variables that showed significant association with the types of relationships of the distal surfaces of the primary second molars. results considering the complete sample, the distribution according to chronological age in years was as follows: 13.9% association between infant feeding duration and the terminal relationships of the primary second molars 142 braz j oral sci. 10(2):140-145 table 2. table 2. table 2. table 2. table 2. distribution of the relationships of the distal surfaces of the primary second molars according to the age when bottle feeding was interrupted. vertical plane (vp), mesial step (ms) and distal step (ds). relationships of the distal surfaces of the primary second molars vp ds m s total without the habit up to 2 years between 3 and 4 years between 5 and 6 years % % % % % 79.6 78.7 70.7 76.7 76.1 4.8 10.0 13.9 7.0 9.7 15.6 11.3 15.4 16.3 14.2 100.0 100.0 100.0 100.0 100.0 total sample age when bottle feeding was interrupted (years) relationships of the distal surfaces of the primary second molars vp ds m s total table 1. table 1. table 1. table 1. table 1. distribution of the relationships of the distal surfaces of the primary second molars, expressed in number (n) and percentage (%), according to side and gender. vertical plane (vp), mesial step (ms) and distal step (ds). side gender total sample right left boys girls n % n % n % n % n % 366 75.5 372 76.7 358 75.5 380 76.6 738 76.1 54 11.1 40 8.3 46 9.7 48 9.7 94 9.7 65 13.4 73 15.1 70 14.8 68 13.7 138 14.2 485 100.0 485 100.0 474 100.0 496 100.0 970 100.0 for children aged 3; 37.7% for those aged 4; 37.1% for those aged 5; and 11.3% for children aged 6 years. the percentage of boys (48.9 %) was very close to that of girls (51.1%). distribution of the sample according to the bottle feeding duration showed 25.8% for children without the habit; 32% for children who interrupted the habit at the age of 2; 33.3% interrupted it between the ages of 3 and 4, and 8.9% between the ages of 5 and 6 years. whereas, distribution of the sample according to the breastfeeding duration showed 7% for children who were not breastfed; 21.2% for children who interrupted it below the age of 3 months; 23.9% interrupted breastfeeding between 3 and 6 months of age; 8.7% between the ages of 6 and 9 months; 6.8% between the ages of 9 and 12 months; 30.7% over the age of 12 months, and 1.7% referred to the parents/guardians who did not remember the age when breastfeeding was interrupted. figure 1 shows the fig.1. distribution of the sample according to the bottle feeding duration in the breastfeeding subgroups. distribution of the sample according to the bottle feeding duration in the breastfeeding subgroups. the distribution of the sample according to prevalence of the relationships of the distal surfaces of the primary second molars in age groups presented the following sequence: at the age of 3, 79.1% showed vp; 5.2%, ds and 15.7%, ms. at the age of 4, 73.8% showed vp; 10.9%, ds and 15.3%, ms. at the age of 5, 76.1% had vp; 10.6% had ds and 13.3% had ms. at the age of 6, the respective values were of 80% for vp; 8.2% for ds and 11.8% for ms. table 1 shows the distribution of the terminal relationships of the primary second molars according to side and gender. it can be observed the distributions of the three characteristics are relatively balanced according to the two above-mentioned factors. table 2 shows the frequency of the terminal relationships association between infant feeding duration and the terminal relationships of the primary second molars braz j oral sci. 10(2):140-145 relationships of the distal surfaces of the primary second molars vp ds m s total not breastfed under 3 between 3 and 6 between 6 and 9 between 9 and 12 over 12 months 75.0 73.8 69.4 72.6 84.9 80.9 76.1 13.2 15.1 13.8 10.7 1.5 4.0 9.7 11.8 11.2 16.8 16.7 13.6 15.1 14.2 100.0 100.0 100.0 100.0 100.0 100.0 100.0 total sample age when breastfeeding was interrupted (months) table 3. table 3. table 3. table 3. table 3. distribution of the relationships of the distal surfaces of the primary second molars according to the age when breastfeeding was interrupted. vertical plane (vp), mesial step (ms) and distal step (ds). comparisons ds/vp ms/vp odds ratio p-value odds ratio p-value age when bottle feeding was interrupted up to 2 years/without habit 2.11 0.035 0.73 0.215 between 3 and 4 years/without habit 3.26 0.000 1.11 0.645 between 5 and 6 years/without habit 1.51 0.430 1.08 0.817 age when breastfeeding was interrupted between 9 and 12 months/after 12 months 0.36 0.329 0.86 0.704 between 6 and 9 months/after 12 months 2.96 0.019 1.23 0.542 between 3 and 6 months/after 12 months 3.99 0.000 1.30 0.281 less than 3 months/after 12 months 4.10 0.000 0.81 0.447 not breastfed/after 12 months 3.54 0.007 0.84 0.673 table 4. table 4. table 4. table 4. table 4. logistic regression model of the relationships of the distal surfaces of the primary second molars and the age when breastfeeding and bottle feeding were interrupted. vertical plane (vp), mesial step (ms) and distal step (ds). of the primary second molars in the sub-groups according to the age when bottle feeding was interrupted. from the group without the habit up to the interval between 3 and 4 years of age, the relationship of vp showed successively lower percentage values; the inverse situation occured with ds. table 3 shows the frequency of the relationships of the distal surfaces of the primary second molars in sub-groups with reference to the age when breastfeeding was interrupted. considering the children who were breastfed, the relationship in ds showed successively lower prevalence. the percentage value 1.5 is pointed out for children who interrupted breastfeeding between the ages of 6 and 9 months of life. the logistic regression models are shown in tables 4 and 5 with respect to the possible associations between the analyzed co-variables studied and the types of the relationships of the distal surfaces of the primary second molars. discussion evaluation of the relationships between the distal surfaces of primary second molars is relevant because it is one of the important parameters in establishing occlusion of the permanent first molars9. it is known that the terminal relationship of primary second molars seems to be the first factor that could determine or influence the future relationship between the permanent molars and the subsequent stages in the development of occlusion. therefore, the goals of this study were to assess the prevalence of different terminal relationships of dental arches in primary dentition in a group of children from municipals preschools in the eastern zone of são paulo city and to study the association between the type of infant feeding (breast and artificial) and the development of these terminal relationships. there is a lack of studies related to these variables which also consider specific feeding durations. this study provides scientific evidence that clearly demonstrates the association between longer periods of breastfeeding and lower prevalence of distal step in primary dentition. the results of prevalence of the types of relationships of the distal surfaces of the primary second molars are close to those found in a previous study10 in terms of percentage and sequence of predominance. when compared with other studies11, our results were the same in sequence of predominance, but differed in their percentages. on the other hand, our results disagree with those of other authors12 who observed a predominance of terminal relationship of primary second molars in ms. these divergences might have occurred due to the differences in methodology which involved the sample group and the age group of the children who were examined. as regards the side of the dental arch and gender, no differences in distribution of terminal relationship of primary second molars were verified, which is in agreement with several other studies10-11 children at the age of 3 were those who showed the highest percentage of ms, and the 6-yearolds showed a lower percentage. the percentage of children with ds was lower than that of children with ms at all ages, in agreement with several authors11. in the present study, it was found that the longer 143 association between infant feeding duration and the terminal relationships of the primary second molars 144 braz j oral sci. 10(2):140-145 comparisons ds/vp ms/vp odds ratio p-value odds ratio p-value age when bottle feeding was interrupted up to 2 years/without habit 0.88 0.772 0.64 0.141 between 3 and 4 years/without habit 1.41 0.399 0.97 0.921 between 5 and 6 years/without habit 0.60 0.382 1.09 0.829 age when breastfeeding wasinterrupted between 9 and 12 months/after 12 months 0.39 0.375 0.96 0.930 between 6 and 9 months/after 12 months 2.74 0.046 1.33 0.435 between 3 and 9 months/after 12 months 4.01 0.001 1.49 0.172 less than 3 months/after 12 months 4.05 0.001 0.92 0.786 not breastfed/after 12 months 3.26 0.025 0.94 0.884 gender girls/boys 0.99 0.963 0.93 0.704 side left/right 0.72 0.145 1.10 0.594 table 5.table 5.table 5.table 5.table 5. logistic regression model for the relationships of the distal surfaces of the primary second molars and all studied co-variables. vertical plane (vp), mesial step (ms) and distal step (ds). the breastfeeding duration, the higher the percentage of children who did not have the bottle feeding habit, and the older the child when breastfeeding was interrupted, the lower the chances of presenting ds. this result reaffirms the literature 7,13 because there is greater prevalence of malocclusions in children who received only artificial or mixed feeding. furthermore, the increase in breastfeeding duration is related to the reduced occurrence of undesirable habits in children from 3 to 6 years of age12. according to the logistic regression models, it was evident that the longer the child was bottle fed, the greater the chances of presenting distal step, and this relationship is not self-corrected with the natural development of occlusion. therefore, in many cases, orthopedic and/or early orthodontic intervention will be needed. moreover, the longer the breastfeeding duration, the lower the chances of developing ds between the primary second molars. children who were not breastfed or who were breastfed for less than 3 months presented 3 or 4 times greater chances, respectively, of developing distal steps in comparison with the children who were breastfed for more than 12 months (table 5). when the latter model was adjusted, only the age at which breastfeeding was interrupted presented significance, and the age whose bottle feeding was interrupted became irrelevant. therefore, it is suggested that the duration of breastfeeding has a preponderant influence on the occurrence of distal step. considering the prevalence of distal step based on the results of this study, there was no significant difference between children who stopped breastfeeding between 9 to 12 months and those who were older than 12 months when breastfeeding ceased. scavone et al. 4 reported that breastfeeding beyond 9 months of age was significantly associated to a lower prevalence of non-nutritional sucking habits. considering that these habits have been associated with malocclusions in the primary dentition4, our results corroborate those of scavone et al.4 in that breastfeeding for over 9 months of age has marked beneficial and meaningful consequences on occlusion and avoidance of non-nutritional sucking habits. based on the results of this research, it may be suggested that the longer the duration of breastfeeding, the lower the possibility of the child using a bottle for a long period. furthermore, the shorter the breastfeeding duration, the greater the chances of the child developing distal step. in fact, the duration of breastfeeding seems to have a marked influence on the development of the distal step. references 1. adair sm, milano m, lorenzo i, russell c. effects of current and former pacifier use on the dentition of 24 to 59-month-old children. pediatr dent. 1995; 17: 437-44. 2. farsi nma, salama fs. sucking habits in saudi children: prevalence. contributing factors and effects on the primary dentition. pediatr dent. 1997; 19: 28-33. 3. warren jj, bishara se. duration of nutritive and non-nutritive sucking behaviors and their effects on the dental arches in the primary dentition. am j orthod dentofacial orthop. 2002; 121: 347-56. 4. scavone-jr h, guimarães-jr ch, ferreira ri, nahás acr, vellini-ferreira f. association between breastfeeding duration and non-nutritive sucking habits. community dent health. 2008; 25: 161-5. 5. karjalainen s, ronning o, lapinleimu h, simell o. association between early weaning, non-nutritive sucking habits and occlusal anomalies in 3year-old finnish children. int j pediatr dent.1999; 9: 169-73. 6. legovic m, ostric l. the effects of feeding methods on the growth of the jaws in infants. j dent child. 1991; 58: 253-5. 7. meyers a, hertzberg j. bottle-feeding and malocclusion: is there an association? am j orthod dentofacial orthop. 1988; 93: 149-52. 8. degano mp, degano ra. breastfeeding and oral health. a primer for the dental practitioner. ny state dent j. 1993; 59: 30-2. 9. baume lj. physiological tooth migration and its significance for the development of occlusion. j dent res. 1950; 29: 123-32. 10. farse nma, salama fs. characteristics of primary dentition occlusion in a group of saudi children. int j paediatric dent. 1996; 6: 253-9. association between infant feeding duration and the terminal relationships of the primary second molars braz j oral sci. 10(2):140-145 145 11. kerosuo h. occlusion in the primary and early mixed dentitions in a group of tanzanian and finnish children. asdc j dent child. 1990; 57: 293-8. 12. ferreira midt, toledo oa. relação entre tempo de aleitamento materno e hábitos bucais. rev abo nac. 1997; 5: 317-20. 13. lópez del valle lm, singh gd, feliciano n, machuca mdc. associations between a history of breast feeding, malocclusion and parafunctional habits in puerto rican children. p r health sci j. 2006; 25: 31-4. association between infant feeding duration and the terminal relationships of the primary second molars original article braz j oral sci. january/march 2009 volume 8, number 1 knoop hardness of composites cured with halogen and led light-curing units in class i restorations maria cecília c. giorgi1, luís alexandre maffei sartini paulillo2 1 dds, ms, graduate student, department of restorative dentistry, faculdade de odontologia de piracicaba, universidade estadual de campinas (unicamp), piracicaba (sp), brazil 2 dds, ms, phd, professor, department of restorative dentistry, faculdade de odontologia de piracicaba, unicamp, piracicaba (sp), brazil received for publication: august 29, 2008 accepted: february 10, 2009 correspondence to: luís alexandre maffei sartini paulillo avenida limeira, 901 – areão cep 13414-903 – piracicaba (sp), brazil e-mail: paulillo@fop.unicamp.br abstract aim: to evaluate the effect of light-curing units (lcus) on the microhardness of class i composite restorations at different depths. methods: two light emitting diodes (led) (freelight 2, radii) and one halogen (optilux 501) lcus were evaluated. thirty class i cavities prepared in human third molars were restored with a microhybrid (charisma) and a microfilled (renamel) resin composite. after seven days of water storage, the teeth were decoronated and the crowns were bisected mesiodistally and tested for microhardness under a 25 g load for 20 seconds. fifteen indentations were performed at three depths for each half-crown. results: charisma presented significantly higher knoop hardness number (khn) values than renamel. at the superficial depth, there were no statistically significant differences (p > 0.05) when charisma was cured with both led curing units. however, statistically significant difference (p < 0.05) was found when charisma was light-cured with the halogen lcu. the lowest khn value was obtained by renamel light-cured with both led lcus, regardless of the composite. nevertheless, when the microfilled composite was light-cured with the halogen lcu, hardness was significantly higher compared to those cured with led units at all evaluated depths. conclusions: the effectiveness of polymerization is related not only to the light-curing source, but also to the type of composite and the curing depth. keywords: composite resins, hardness introduction light-activated composite materials polymerize by free radical polymerization when exposed to light at wavelengths in the 400 to 500 nm range. the photoinitiator absorbs light energy emitted from the light-curing unit (lcu), and directly or indirectly initiates polymerization1. camphorquinone (cq) is a commonly used photoinitiator that absorbs energy and reacts with a photo reducer to begin the polymerization process1-3. both the light source and the resin composite play an important role in ensuring adequate polymerization. composites can be easy, moderate or difficult to polymerize because of the differences in their photoinitiator content like shade, filler size and filler load4. while the resin composite composition and shade influence polymerization, light intensity and wavelength are also contributing factors5. if a light-activated composite does not receive sufficient total energy at the correct wavelength from the lcu, several clinical consequences6 will be observed, such as decrease in the mechanical properties7-9; increase in water sorption and solubility, reduced hardness and potential pulpal damage10,11. quartz tungsten halogen (qth) lcus are the most widely sources for composite activation, but light emitting diode (led) lcus are gaining popularity12. the main difference between these light sources is that qth lcus produce a broad wavelength spectrum and need a filter to reduce output of undesired wavelengths, delivering light in the 410 to 500 nm region of the visible spectrum1. halogen light bulbs generate light when electrical energy heats a 31knoop hardness of composites cured with halogen and led light-curing units in class i restorations braz j oral sci. 8(1): 30-3 small tungsten filament to extremely high temperatures1,3, which are responsible for qth light bulb or filter deterioration, decreasing the power density of the curing unit and resulting in a lifetime of 30 to 50 hours. on the other hand, led units produce a narrow band of wavelength, specifically chosen to excite the cq, and last for thousands of hours because they convert electricity into light more efficiently, producing less heat. the use of only a radiometer to compare the curing efficacy of led and qth lcus is not sufficient because these lcus emit light in a different spectrum of the visible light13. tests that evaluate mechanical properties of the cured material are the most indicated method to determinate the light activation potential of these light sources14. the good correlation, between the results of hardness testing and infra-red spectroscopy15,16, has allowed the microhardness to be a frequently used method to investigate the factors which influence the effectiveness of polymerization, since it is relatively easier to perform16. the aim of this study was to investigate the effect of qth and led lcus on the microhardness of microfilled and microhybrid composite resin restorations placed in class i cavities at three different depths. the null hypothesis tested was that composite resin microhardness is not influenced by different lcus. material and methods in the present study, two led lcus (freelight 2; 3m/espe, st. paul, mn, usa and radii; sdi ltd, victoria, australia) and one qth lcu (optilux 501; kerr-demetron, orange, ca, usa) were used. since the radii led lcu operates only in soft-start mode, the other curing units were initially activated for five seconds with increasing intensity and, thereafter; light activation was done for 10, 20 or 40 seconds, according to the manufacturers’ recommendations, in a continuous mode. thus, optilux 501 qth lcu was set in such a way that the first five seconds were in an exponential mode, and the continuous mode was activated immediately afterwards. the corresponding option was selected for elipar freelight 2 led lcu. all lcus were, thus, used for the same time. emission spectra and power output were measured before the experiment to characterize the units. a power meter (ophir optronics inc., wilmington, ma, usa) and an integrating sphere 3a-p-sa (ophir optronics inc.) were used for power measurement. the power density of each lcu was determined by measuring the output power of the entire light guide and dividing the output power by the area of the light guide or lens. output powers of 526, 381 and 960 mw/ cm² were found for optilux 501, radii and freelight 2, respectively. the energy density (mj/cm²) was determined by the product of power density and time. the spectra were measured with a spectrometer usb 2000 (ocean optics inc, dunedin, fl, usa) and are presented in figure 1. a microhybrid (charisma; heraeus-kulzer gmbh, hanau, germany) and a microfilled (renamel; cosmedent inc., chicago, usa), composite resins, both a3 shade, were used in association with a two-step etch-and-rinse adhesive system (single bond; 3m/espe). thirty sound human third molars were selected for this study. the teeth were embedded in pvc molds with polystyrene resin (piraglass, piracicaba, sp, brazil) in such a way that the crown and the 5 mm below the cementoenamel junction remained exposed. after inclusion, the occlusal surfaces of teeth were cut off with a watercooled low-speed double-faced flexible diamond disc (#7020; kg sorensen, são paulo, sp, brazil) and flattened in a polishing machine (south bay technology inc, san clemente, ca, usa) using 180and 360-grit abrasive paper (carborundum, saint-gobain abrasivos ltda, cruz de rebouças/igaraçu, pe, brazil) under water cooling. standardized box-shaped class i cavities were prepared using a precision cavity preparation device. the cavities were outlined with a carbide bur (#fg 245; ss white, rio de janeiro, rj, brazil) operated in a high-speed handpiece (kavo do brasil sa ind. & com., joinvile, sc, brazil) using copious air-water spray. a new bur was used for every five preparations. the final cavities had a mesiodistal width of 4 mm, a buccolingual width of 3 mm and depth of 3 mm. the teeth were randomly divided into six groups of five teeth each and restored with either charisma or renamel composites and light-cured with optilux 501, radii or freelight 2. the exposure time used was 20 seconds for charisma and 40 seconds for renamel, according to manufacturer’s recommendations. for all groups, a 37% phosphoric acid gel (cond ac 37; fgm dental products, joinville, sc, brazil) was applied to the entire cavity for 15 seconds. the acid was rinsed off with water spray for 15 seconds and the excess water was removed with a small damp cotton pellet. single bond adhesive system was applied in accordance with the manufacturer’s instructions to the cavity walls and light-cured. after that, the cavity was restored incrementally in three oblique layers less than 2 mm thick. the increments were light-cured for the recommended time with the light source close to the occlusal surface withfigure 1. specimen prepared for knoop hardness test. 32 giorgi mcc, paulillo lams braz j oral sci. 8(1): 30-3 out touching it. the finishing of restorations was done with flexible discs (sof-lex pop on; 3m/espe). after seven days of storage in water at 37 ± 1° c, the teeth were decoronated at the cementoenamel junction using a water-cooled low-speed saw and the roots were discarded. the crowns were, then, bisected mesiodistally parallel to their long axis resulting providing two halves. each half was embedded in polystyrene resin to facilitate handling and microhardness testing. the included restorations were finished with wet 400-, 600and 1,200-grit al 2 o 3 abrasive paper and then polished with 3 and 1 µm diamond paste (arotec ind. com., são paulo, brazil) using a polishing cloth. microhardness was measured by means of a knoop indenter under 25 g load and 20 seconds dwell time (hmv-2000, shimadzu, japan). fifteen indentations were made in each specimen, five at each depth of 500, 1,500 and 2,500 µm. for depth, the values read, referring to the size of the greater diagonal, were transformed into knoop hardness number (khn) and the average of the values was calculated. data was submitted to a three-way anova (resin composite versus lcu versus depth) followed by a tukey’s multiple comparison test (ls means) at α = 0.05 significance level. results there were statistically significant differences (p < 0.05) between the composite resins and a composite versus lcu versus depth triple interaction (table 1). the highest khn (p < 0.05) was obtained for the microhybrid composite charisma when light-cured with radii at the superficial depth, with no difference (p > 0.05) to radii at the other depths and freelight 2 at all depths. however, there was statistically significant difference (p < 0.05) when charisma was light-cured with the halogen lcu at all depths, compared to the same composite cured with radii at the superficial depth. the microfilled renamel presented significantly lower (p < 0.05) hardness than charisma in spite of the curing unit and depth. the lowest khn (p < 0.05) was obtained when renamel was light-cured with both led lcus, irrespective of the depths. however, when renamel was cured with the qth curing unit, there was a significant increase (p < 0.05) in hardness compared to the led lcus at all depths. the null hypothesis was rejected. discussion radii showed the lowest light irradiance (381.6 mw/cm²) among the tested lcus. since the exposure time was the same for all devices, it also presented the lowest energy density (irradiance versus time). however, there were no significant differences between the mean khn values of charisma cured with radii and the other units, which had light irradiance of 960 mw/cm² (elipar freelight 2) and 526 mw/cm² (optilux 501). the hypothesis that leds could produce a polymerization depth similar to that of qth lcus, in spite of showing lower irradiance, is due to their better overlap between the emission and absorption spectra of lcus and photoinitiators9. the absorption peak of cq in methylmethacrylate resins is 470 nm, which is coincident with the emission peak of the lcus evaluated in the present study (450 to 490 nm). outside this range, however, the wavelength dependence is much stronger and the conversion rate drops rapidly17. no significant differences were observed at the superficial depth of charisma cured by radii and elipar freelight 2 compared to optiliux 501, with leds resulting in higher knh values. considering the energy density applied, one could expect that optilux 501 (13,150 mj/cm²) would behave similarly to radii (9,540 mj/cm²). the superior performance of radii may be justified by its emission spectrum, which is more coincident with the absorption peak of cq. the hardcomposite lcu depth n mean (sd) charisma radii superficial 4 62.92 (1.17)a charisma freelight 2 superficial 4 60.94 (4.13)ab charisma radii medium 4 60.83 (1.04)ab charisma radii deep 4 60.19 (1.07)ab charisma freelight 2 deep 4 60.08 (5.70)ab charisma freelight 2 medium 4 58.48 (3.35)ab charisma optilux 501 medium 5 55.62 (10.3)bc charisma optilux 501 deep 5 54.63 (8.98)bc charisma optilux 501 superficial 5 51.95 (9.61)c renamel optilux 501 medium 5 43.62 (8.42)d renamel optilux 501 superficial 5 42.05 (8.35)d renamel optilux 501 deep 5 40.40 (6.73)d renamel radii deep 5 35.50 (5.35)e renamel radii medium 5 34.79 (5.91)e renamel freelight 2 medium 5 33.22 (6.09)e renamel freelight 2 deep 5 33.14 (5.41)e renamel freelight 2 superficial 5 32.60 (6.31)e renamel radii superficial 5 32.22 (6.43)e table 1. results of knoop hardness test lcu: light-curing unit; different letters indicate statistically significant difference at 5%. 33knoop hardness of composites cured with halogen and led light-curing units in class i restorations braz j oral sci. 8(1): 30-3 ness values obtained by freelight 2 and radii did not differ significantly from each other, although the former showed more than twice the energy density when compared to the latter (24,000 mj/cm² and 9,540 mj/cm², respectively). a possible explanation for this result is that radii does not have a fiber optic tip, but an acrylic structure instead, called lens cap, which may have affected the measurement of irradiance by the radiometer. in all groups, the microfilled composite renamel presented lower hardness values than the microhybrid composite charisma. this is in agreement with the results of previous studies18,19, and can be explained by the fact that microfilled composites are more difficult to light-cure than microhybrid composites20, indicating that adequate polymerization is not only a function of exposure time to the light, but it is also influenced by the material’s composition18. the small filler size of microfilled composites causes light scattering, decreasing the effectiveness of polymerization6,21,22. furthermore, resistance, hardness and other mechanical properties of the composites are influenced not only by the degree of conversion, but also by the nature of the monomer subunits of the polymer. thus, tetraethylene glycol dimethacrylate (tegdma) monomer is more flexible then bis-gma. the flexibility of tegdma is related to the ether linkages of the molecule, giving rise to only slight barriers to free rotation about the bonds. the relative stiffness of bis-gma is related to the bulk, aromatic groups of the central part of the molecule, causing much larger barriers to rotation about the bonds23. as renamel has a higher tegdma content in its composition, its lower hardness can also be credited to the nature of the resin matrix. the highest khn values of renamel were obtained when the material was light-cured with the qth lcu at all depths, while the lowest khn values of renamel at al depths were obtained when it was light-cured with both led lcus, at all depths. due to the broad wavelength spectrum emitted by qth, the light presents a portion of emitted light with higher wavelengths and it also presents better transmittance in microfilled composites with the potential of hitting photoinitiators at deeper depths24. arikawa et al.24 showed that there was a tendency of increase in light transmittance in the material body when an increase in wavelength from 400 nm to 700 nm occurred. the authors explained this result based on the rayleigh equation24, which indicates that higher light scattering occurs at lower wavelengths. consequently, the decrease in light transmittance at lower wavelengths can be caused by higher light scattering in the material. this might have occurred with the led lcus, which have narrower spectra. the use of led lcus may represent a clinical advantage because they undergo minimal degradation of the device. in addition, qth lcus are known to generate heat resulting, which results in degradation of their constituents over time and decrease in light irradiance. however, the findings of the present study showed that, led lcus do not present the same performance for different types of composites. therefore, it is important that clinicians also know the composition of materials, especially regarding their filler particles and photoinitiator, when choosing a lcu to be used in daily practice. references 1. rueggeberg f. contemporary issues in photocuring. compend contin educ dent suppl. 1999;s4-15; quiz s73. 2. hammesfahr pd, o’connor mt, wang x. light-curing technology: past, present, and future. compend contin educ dent. 2002;23:18-24. 3. burgess jo, walker rs, porche cj, rappold aj. light curing – an update. compend contin educ dent. 2002;23:889-906. 4. leonard dl, charlton dg, roberts hw, cohen me. polymerization efficiency of led curing lights. j esthet restor dent. 2002;14:286-95. 5. aravamudhan k, rakowski d, fan pl. variation of depth of cure and intensity with distance using led curing lights. dent mater. 2006;22:988-94. 6. price rb, felix ca, andreou p. evaluation of a second-generation led curing light. j can dent assoc. 2003;69:666. 7. ferracane jl. correlation between hardness and degree of conversion during the setting reaction of unfilled dental restorative resin. dent mater. 1985;1:11-4. 8. yearn ja. factors affecting cure of visible light activated composites. int dent j. 1985;35:218-25. 9. mills rw, jandt kd, ashworth sh. dental composite depth of cure with halogen and blue light emitting diode technology. br dent j. 1999;186:388-91. 10. spagnuolo g, annunziata m, rengo s. cytotoxicity and oxidative stress caused by dental adhesive systems cured with halogen and led lights. clin oral investig. 2004;8:81-5. 11. yap au, saw ty, cao t, ng mm. composite cure and pulp-cell cytotoxicity associated with led curing lights. oper dent. 2004;29:92-9. 12. price rb, felix ca, andreou p. knoop hardness of ten resin composites irradiated with high-power led and quartz-tungsten-halogen lights. biomaterials. 2005;26:2631-41. 13. stahl f, ashworth sh, jandt kd, mills rw. light-emitting diode (led) polymerization of dental composites: flexural properties and polymerisation potential. biomaterials. 2000;21:1379-85. 14. meyer gr, ernst cp, willershausen b. decrease in power output of new lightemitting diode (led) curing devices with increasing distance to filling surface. j adhes dent. 2002;4:197-204. 15. yap au. effectiveness of polymerization in composite restoratives claiming bulk placement: impact of cavity depth and exposure time. oper dent. 2000;25:113-20. 16. dewald jp, ferracane jl. a comparison of four modes of evaluating depth of cure of light-activated composites. j dent res. 1987;66:727-30. 17. jandt kd, mills rw, blackwell gb, ashworth sh. depth of cure and compressive strength of dental composites cured with blue light emitting diodes (leds). dental mater. 2000;16:41-7. 18. leonard dl, charlton dg, roberts hw, hilton tj, zionic a. determination of the minimum irradiance required for adequate polymerization of hybrid and microfill composite. oper dent. 2001;26:176-80. 19. dunn wj, bush ac. a comparison of polymerization by light-emitting diode and halogen-based light curing units. j am dent assoc. 2002;133:335-41. 20. ruyter ie, oysaed h. conversion in different depths of ultraviolet and visible light activated composite materials. acta odontol scand. 1982;40:179-92. 21. ferracane jl, aday p, matsumoto h, marker va. relationship between shade and depth of cure for light-activated dental composite resins. dent mater. 1986;2:80-4. 22. atmadja g, bryant rw. some factors influencing the depth of cure of visible light-activated composite resins. aust dent j. 1990;35:213-8. 23. peutzfeldt a. resin composites in dentistry: the monomer systems. eur j oral sci. 1997;105:97-116. 24. arikawa h, fujii k, kanie t, inoue k. light transmittance characteristics of lightcured composite resins. dent mater. 1998;14:405-11. oral sciences n3 original article braz j oral sci. october | december 2011 volume 10, number 4 received for publication: october 20, 2011 accepted: december 12, 2011 using biomodels for maxillofacial surgeries: 10 years of experience in a brazilian public service gabriela mayrink1, luciana asprino2, roger william fernandes moreira2, gustavo henrique de lima paschoal3, pedro noritomi4, márcio de moraes2 1dds, msc, phd student in oral and maxillofacial surgery, piracicaba dental school, university of campinas, brazil 2dds, msc, phd, associate professor in oral and maxillofacial surgery piracicaba dental school, university of campinas, brazil 3eng., renato archer center of research, product development, brazil 4msc, phd, renato archer center of research, product development, brazil correspondence to: gabriela mayrink fop/unicamp caixa postal 52 13.414-903, piracicaba, sp, brasil phone: 55 19 81367564 e-mail: gabrielamayrink@fop.unicamp.br abstract aim: to evaluate 10 years of experience of use of biomodels at the department of oral and maxillofacial surgery of the piracicaba dental school, university of campinas (unicamp), brazil, showing the difficulties and importance of using biomodels in a public oral and maxillofacial surgery service. methods: the records of all patients treated at the referred department of oral and maxillofacial surgery between january 2000 and december 2010 were reviewed. results: biomodels were used in 63 cases, including pathologies (47%), trauma sequelae (23%), dentofacial deformities (8%), temporomandibular joint anomalies (8%), implant surgery (8%) and maxillofacial prosthesis (6%). these cases were performed in a partnership with renato archer information of technology center – cti, campinas, brazil. conclusions: the partnership with cti enables the use of prototypes for treatment planning of patients of a public health system using selective laser sintering, a cheaper prototyping method. the patients can benefit from this technology, without any costs for them. keywords: technology assessment, biomedical, health planning, prototype. introduction biomodels have been used in treatment planning of oral and maxillofacial surgery since its introduction in 1985 by brix and lambrecht1-3. currently, biomodels have been used in cases involving craniofacial deformities surgeries, extraoral implants, pathologies and trauma sequelae. the first and most common method to acquire biomodels is stereolithography. in this technique, the liquid resin is polymerized by laser light to form a solid material with the desired shape1. the model is created from many thin horizontal contour layers each 0.25 mm thick. these are fused on top of each other to form a 3d model4. the other way to acquire biomodels is from selective laser sintering. this technique produces prototypes with fewer details, but it is less expensive. this article presents the outcome of the evaluation of 10 years of experience of use of biomodels in the department of oral and maxillofacial surgery of the piracicaba dental school, university of campinas (unicamp), brazil, showing braz j oral sci. 10(4):294-296 295 indications number of cases (%) pathology 29 (47%) trauma sequelae 15 (23%) dentofacial deformities 5 (8%) temporomandibular joint anomalies 5 (8%) implant surgery and reconstruction 5 (8%) maxillofacial prosthesis 4 (6%) table 1 – number of cases and indications of using biomodels in the treatment planning over 10 years of experience the difficulties and importance of using biomodels in a public oral and maxillofacial surgery service. material and methods the research protocol was approved by the ethics committee of piracicaba dental school/unicamp, piracicaba, são paulo, brazil. data were collected from the records of patients from the aforementioned department of oral and maxillofacial surgery, who were subjected to surgeries with use biomodels in the treatment planning, between january 2000 and december 2010. computed tomography images that were used in fabrication of the biomodels were collected from the database of the renato archer information of technology center cti, campinas, brazil. results biomodels were used in 63 cases. all biomodels used in these cases were made by the cti. table 1 summarizes the number of cases and indications of using biomodels in the treatment planning over 10 years of experience, which included mostly treatment planing of pathologies, trauma sequelae and others. discussion the importance and advantages of using biomodels in the treatment planning are well defined in the literature. d’urso et al. (1999)4, emphasized this advantages: 1 -enhances interpretation of volumetric image data; 2. optimizes preoperative surgical planning and allows realistic and interactive surgical simulation; 3. improves implant design and fit while reducing operating time and risk; 4. provides patients with a clearer understanding of their pathology and the aims and limitations of surgery; 5. improves teaching demonstrations; 6. facilities team communication; 7. requires no specialized knowledge or equipment for interpretation and use; 8. may be used as a sterile reference intraoperatively. erickson et al.5 emphasized that biomodels can take a mean time saving of 20% in expended operating room and anesthesia time. it could hypothetically minimize additional surgical trauma, blood loss, risk of infection and postoperative complications. different methods can be used to make biomodels. the stereolithography apparatus machine starts with a tank of liquid resin and constructs from bottom to up. a laser beam selectively polymerizes ultraviolet sensitive liquid monomer on a platform suspended in a vat of the liquid and the platform is lowered by increments of 0.25 mm as each slice is polymerized. a multi-layered model is then built up as the contour slices are progressively fused together1. the main advantage of this technique is that the ensuing models can incorporate complete internal structure within a closed skull including sinuses and even intrabony neurovascular canals1. however, stereolithography is a highly expensive method to manufacture biomodels. d’urso et al.6 showed that the cost of the stereolithography in australia is around u$1,000 per case. in brazil, to manufacture a biomodel on a private service, the cost of the stereolithography (case complete: skull base, maxilla and mandible) is around u$3,200. this could preclude its use in a public service in brazil. the department of oral and maxillofacial surgery of piracicaba dental school at the university of campinas, in a partnership with cti, enables the use of prototypes for treatment planning of patients of a public system of health using a less expensive method: selective laser sintering. similar to stereolithography, original ct data are stored in a cd-rom on a dicom format. it is important to obtain images with 1.0 mm reconstructed slice not to lose details at the time of the confection of the biomodels. these data are transferred to the cti for 3d image and laser sintering biomodel. the software invesalius, created by cti, is used to generate 3d images, compensate for dental restoration artifacts and monitor the effect of threshold values for segmentation purposes7. then, the dicom file is converted into stl format and this data is transferred to the selective laser sintering rapid prototyping machine to produce the biomodel. in our cases, the models of the patients are reproduced in cast resin through technology of 3d printer zcorp machine (zp 510, zcorporation, burlington, ma, usa), where layers of 1.0 mm in the axis z are added together by a head printout with accuracy of 4.0 mm in x and y axes (figure 1) the high precision is important to decrease the chances of errors in planning. for example, in surgeries of trauma sequelae and pathologies, the osteotomies are performed in the biomodel and the trans-operative guides are made according to this model surgery to make easier the bone plate install (figure 2). however, the occlusal splints are made in the normal sequence in the casts and the biomodel is useless to this stage. if the prototype is imprecise, the guide will make the bone remain poorly repositioned. accordingly to this, nizam et al.8 made a study to determine the dimensional accuracy of the skull models produced by rapid prototyping technology using stereolithography apparatus. they compared measures in the dry skull and their replicas and using biomodels for maxillofacial surgeries: 10 years of experience in a brazilian public service braz j oral sci. 10(4):294-296 296 fig. 1. processing steps of acquiring biomodels: a) ct is performed on a dicom format with less than 1 mm slices, b) specific software is used to generate 3d images, c) the file is transferred to prototyping machine and, d) the biomodel is produced. fig. 2. plates are bent at the biomodels and trans-operative guides (arrows) are made (a, b) to make easier the bone plate to be installed (c) found that the percent difference was 0.08% with a standard deviation of 1.25%, concluding that biomodels are affordable to using in treatment planning in oral and maxillofacial surgery. in conclusion biomodels are an interesting tool in treatment planning in oral and maxillofacial surgery. in brazil, the difficulties to obtain prototypes can be minimized by using selective laser sintering technique supported by the federal institute cti. it is relevant to select cases in which biomodels are really important in the planning. at our public service, 63 patients have benefited from this technology without any costs to them and with good results, improving surgical planning and allowing the patients and their families figuring the perspective outcomes. references 1. arvier jf, barker tm, yau yy, d’urso ps, arkinson rl, mcdermant gr. maxillofacial biomodelling. br j oral maxillofac surg. 1994; 32: 276-83. 2. brix vf, hebbinghaus d, meyer w. verfahren und vorrichtung fü r den modellbau in rahmen der orthopädischen und traumatologischen operationsplanung. röntgenpraxis. 1985; 38: 290-2. 3. sinn dp, cillo jr je, miles ba. stereolithography for craniofacial surgery. j craniofac surg. 2006; 17: 869-75. 4. d’urso os, barker tm, earwaker wj, bruce lj, atkinson l, lanigan mw et al. stereolithographic biomodelling in cranio-maxillofacial surgery: a prospective trial. j craniomaxillofac surg. 1999; 27: 30-7. 5. erickson dm, chance d, schmitt s, mathisf j. an opinion survey of reported benefits from the use of stereolithographic models. j oral maxillofac surg. 1999; 57: 1040-3. 6. d’urso ps, earwaker wj, barker tm, redmond mj, thompson rg, effeney dj et al. custom cranioplasty using sterolithography and acrylic. br j plast surg. 2000; 53: 200-4. 7. sannomiya ek, silva jvl, brito aa, saez dm, angelieri f, dalben gs. surgical planning for resection of an ameloblastoma and reconstruction of the mandible using a selective laser sintering 3d biomodel. oral surg oral med oral pathol oral radiol endod. 2008;106: e36-40. 8. nizam a, gopal rn, naing l, hakim ab, samsudin ar. dimensional accuracy of the skull models produced by rapid protptyping technology using stereolithography apparatus. arch orofac sci. 2006; 1: 60-6. using biomodels for maxillofacial surgeries: 10 years of experience in a brazilian public service braz j oral sci. 10(4):294-296 oral sciences n3 original article braz j oral sci. july/september 2010 volume 9, number 3 received for publication: june 18, 2009 accepted: june 21, 2010 response of human dental pulp to calcium hydroxide paste preceded by a corticosteroid/ antibiotic dressing agent elisa maria aparecida giro1, juliana oliveira gondim2, josimeri hebling1, carlos alberto de souza costa3 1 dds, msc, phd, professor, department of orthodontics and pediatric dentistry, school of dentistry of araraquara, universidade estadual paulista unesp, araraquara, sp, brazil 2 dds, msc, assistant professor, department of dentistry, school of dentistry, federal university of ceará, sobral, fortaleza, ce, brazil; graduate student, department of orthodontics and pediatric dentistry, school of dentistry of araraquara, universidade estadual paulista unesp, araraquara, sp, brazil 3 dds, msc, phd, professor, department of physiology and pathology, school of dentistry of araraquara, universidade estadual paulista unesp, araraquara, sp, brazil correspondence to : elisa maria aparecida giro departamento de clínica infantil, faculdade de odontologia de araraquara – unesp rua humaitá, 1680 – centro. cep:14801-903 cp: 331, araraquara, são paulo, brasil phone:+ 55 16 33016336; fax:+55 16 33016329 e-mail: egiro@foar.unesp.br abstract aim: to evaluate the treatment with corticosteroid/antibiotic dressing in pulpotomy with calcium hydroxide. methods: forty-six premolars were pulpotomized and randomly assigned into 3 groups. in group i pulpal wound was directly capped with calcium hydroxide, and group ii and group iii received corticosteroid/antibiotic dressing for 10 min or 48 h, respectively, before pulp capping. teeth were processed for histological analysis after 7, 30 or 60 days to determine inflammatory cell response, tissue disorganization, dentin bridge formation and presence of bacteria. attributed scores were analyzed by kruskal-wallis and mann-whitney tests (α=0.05). results: on the 7th day, all groups exhibited dilated and congested blood vessels in the tissue adjacent to pulpal wound. the inflammatory cell response was significantly greater in group iii (p<0.05). on the 30th day, in all groups, a thin dentin matrix layer was deposited adjacent to the pulpal wound and a continuous odontoblast-like cell layer underlying the dentin matrix was observed. on the 60th day, all groups presented a thick hard barrier characterized by an outer zone of dystrophic calcification and an inner zone of tubular dentin matrix underlined by a defined odontoblast-like cell layer. conclusions: within the limitations of present study, considering that the treatment was performed in healthy teeth, it may be concluded that the use of a corticosteroid/antibiotic dressing before remaining tissue protection with calcium hydroxide had no influence on pulp tissue healing. keywords: calcium hydroxide, corticosteroid, dental pulp capping, permanent dentition, pulpotomy. introduction the application of antiinflammatory agents on exposed pulp tissue in an attempt to prevent or minimize inflammatory reaction and to favor healing has been investigated for a long time1-7. corticosteroid can be used as a dressing agent for deep cavities and exposed pulp tissue in order to control the inflammatory pulp response and reduce postoperative pain1,3,8-10. however, there is no consensus in the dental literature concerning the use of this medicament1-5,8. topic corticosteroid application for 5 min on the cavity floor did not significantly change intrapulpal pressure when compared to healthy teeth2. the braz j oral sci. 9(3):337-344 338 therapeutic effect of a corticosteroid agent seems to depend upon its potency, concentration and ability to diffuse into connective tissue. otosporin ®, one of the commercial denominations of hydrocortisone/antibiotic association, presents high displacement capacity in dentin in comparison to other corticosteroid agents available to different therapies6. this product has been reported to prevent intense inflammatory reaction, cause no pulp damage when applied as a dressing agent in exposed pulps and preserve pulp vitality, showing a mild inflammatory reaction restricted to the superficial pulp zone underneath the pulp capping site3. on the other hand, the effects of corticosteroid agents have been questioned when they are applied for a long period because inhibition of collagen synthesis and interference with pulp recovery may occur in this situation11. calcium hydroxide has been indicated as the material of choice to treat exposed pulp tissue because it presents biocompatibility, antibacterial activity and is able to induce mineralized barrier formation7,12-15. studies have shown that the high ph values of this material when in contact with pulp tissue causes coagulation necrosis, which stimulates mineralized tissue formation7,12-13,15-17. the use of calcium hydroxide for direct pulp capping is supported by several studies3,7. however, considering that the mechanical trauma produced during pulpotomy therapy results in mild inflammatory pulp response, few clinical and histopathologic studies have investigated the use of corticosteroid before direct pulp capping3-4. the aim of this in vivo study was to evaluate pulp healing in teeth subjected to corticosteroid/antibiotic dressing agent application prior to direct pulp capping with calcium hydroxide paste. material and methods for this study, 46 intact, caries-free and periodontally healthy premolars scheduled to be extracted for orthodontic reasons (28 maxillary and 18 mandibular) were obtained from 23 patients aged between 12 and 16 years. all patients enrolled in the trial were attending the pediatric dentistry clinic of the dental school of araraquara, brazil, and none of them were using medicines or have any systemic disease. after receiving full explanation about the experimental rationale, clinical procedures and possible risks, parents of all volunteers were asked to carefully read the research protocol before signing the informed consent form. both the consent form and the research protocol were previously submitted and approved by the human research ethics committee of the são paulo state university – unesp, brazil (protocol # 52/99). the teeth were examined clinically and radiographically for the presence of caries or periapical pathology, and were polished with rubber cup and prophylactic paste at low speed. local anesthesia was administrated (2% mepivacaine, dfl ind. e comércio ltda, rio de janeiro, rj, brazil) and rubber dam isolation was provided followed by antisepsis with 70% alcohol. pulp chambers were opened under aseptic conditions with #4 round bur (beavers dental div. of sybron canada, ontario, canada) at high speed and under irrigation with sterile cooled water. after pulp exposure, the coronary pulp tissue was carefully cut with a sharp dentin spoon. saline solution and sterile cotton pellets were used to control hemorrhage. teeth were randomly assigned to 3 groups: group i (control), group ii (10-min otosporin® application) and group iii (48-h otosporin® application). group i (n=14 teeth; 7-day period = 6 teeth, 30day period = 4 teeth and 60-day period = 4 teeth): in this group, calcium hydroxide paste was directly applied onto the pulp wound with a sterile instrument and gently compressed with sterile cotton pellets. this pulpcapping agent was prepared by mixing 120 mg of calcium hydroxide powder (calcium hydroxide p.a.; merck kgaa, darmstadt, germany) with 60 µl of sterile distilled water. no dressing agent was applied before pulp capping. then, a hard-setting calcium hydroxide cement (dycal; dentsply, milford, de, usa) hand-mixed according to the manufacturer’s instruction was applied, followed by placement of a reinforced zinc-oxide and eugenol hard-setting cement base (irm; dentsply indústria e comécio ltda., petrópolis, rj, brazil), and cavity restoration with amalgam (permite; sdi, southern dental industries, australia). group ii (n=16 teeth; 7-day period = 6 teeth, 30day period = 5 teeth and 60-day period = 5 teeth): in this group, a sterile cotton pellet embedded with corticosteroid/antibiotic dressing agent (otosporin®; glaxo wellcome s.a., rio de janeiro, rj, brazil) was applied for 10 min on the pulp wound, and then removed with sterile cotton pliers. the pulp chamber was irrigated with sterile saline and gently dried with a sterile cotton pellet. calcium hydroxide paste was applied onto the pulp wound with a sterile instrument and gently compressed with sterile cotton pellets. then, a hard-setting calcium hydroxide cement was applied, followed by placement of a reinforced zinc-oxide and eugenol hard-setting cement base, and cavity restoration with amalgam, as described in group i. group iii (n=16 teeth; 7-day period = 5 teeth, 30day period = 6 teeth and 60-day period = 5 teeth): in this group, a sterile cotton pellet embedded with otosporin® dressing agent was applied onto the pulpal wound and the cavity was temporarily restored with reinforced zincoxide and eugenol hard-setting cement. after 48 h, the temporary restorative material and the cotton pellet embedded with the dressing agent were removed as previously described. then, the pulpal wound was capped with calcium hydroxide paste and the cavity restored following the clinical sequence described for groups i and ii. in all 3 groups, the pulpotomy procedures were performed by the same experienced operator. after the experimental periods of 7, 30 or 60 days, teeth were extracted under local anesthesia and had their roots sectioned halfway between the cementoenamel junction (cej) response of human dental pulp to calcium hydroxide paste preceded by a corticosteroid/antibiotic dressing agent braz j oral sci. 9(3):337-344 339 and the root tip with a high-speed handpiece under copious water spray cooling. teeth were immersed for 48 h in 10% buffered formalin, decalcified in morse’s solution, dehydrated, and vacuum-embedded in wax paraffin. six-micron-thick serial sections were cut (‘820’ spencer microtome, carson, ca, usa) parallel to the long axis of the tooth. the sections were mounted on glass slides and stained with hematoxylin and eosin, masson’s trichrome and brown and brenn technique for bacterial assessment. all histological sections were evaluated by a previously standard-trained pathologist and the analyses conducted in a blind fashion protocol under light microscope (carl zeiss 62774, oberkachen, west germany). each tooth was independently examined in serial sections and specific scores were attributed according to the established criteria presented in tables 1 to 4. the attributed scores for the histological events were analyzed statistically by kruskal wallis and mann-whitney tests to determine any significant differences (p<0.05) among the ranked groups and periods. results the patients did not report pain or any kind of discomfort during the postoperative periods in any of the groups. the observed scores for the histological events in the different score characterization 1 none or few scattered inflammatory cells adjacent to the pulpal wound 2 mild inflammatory reaction characterized by the presence of polymorphonuclear (pmns) or mononuclear leukocytes (mnls) 3 moderate inflammatory reaction with pmns or mnls comprising two thirds of the remaining radicular pulp tissue 4 severe inflammatory cell infiltrate comprising all the radicular pulp or presence of abscess table 1. scores attributed to the parameter inflammatory cell response table 2. scores attributed to the parameter tissue disorganization score characterization 1 normal tissue 2 mild tissue disorganization adjacent to the pulpal wound or bellow the newly deposited hard tissue barrier 3 moderate tissue disorganization comprising two thirds of the remaining radicular pulp tissue 4 complete tissue disorganization characterizing pulp necrosis or abscess score characterization 1 presence of elongated cells organized in monolayer associated with intense hard tissue barrier deposition beneath the pulpal wound 2 presence of elongated cells organized in monolayer associated with moderate hard tissue barrier deposition beneath the pulpal wound 3 presence of elongated cells organized in monolayer associated with mild deposition of dentin matrix beneath the pulpal wound 4 absence of organized elongated cells associated with absence of hard tissue barrier formation adjacent to the pulpal wound table 3. scores attributed to the parameter reparative dentin formation score characterization 1 absence of stained bacteria 2 presence of stained bacteria along the outer third of the cavity lateral walls 3 presence of stained bacteria along all cavity lateral walls 4 presence of stained bacteria along all cavity walls and within the pulp tissue table 4. scores attributed to the parameter stained bacteria experimental groups and periods are shown in table 5. 7-day period on the 7th day after the procedure, 6 teeth of group i, 6 of group ii, and 5 of group iii were analyzed. in all groups, a thick coagulation necrosis layer was observed adjacent to the pulp-capping agent, with pulp cells remaining within this necrotic tissue. in most specimens, the subjacent pulp tissue exhibited few polymorphonuclear neutrophils and few mononuclear cells. small congested and dilated blood vessels were observed close to the coagulation necrosis or getting into this necrotic tissue (figure 1). in group i (control), pulp response was characterized by a mild inflammatory reaction associated with mild deposition of dentin matrix in all 6 specimens. stained bacteria along the outer third of lateral cavity walls were observed in 1 specimen of this group. group ii presented histological characteristics of mild tissue disorganization. moderate tissue disorganization was seen in only one specimen in which bacteria was evidenced along the outer third of the lateral cavity walls. in 2 specimens, elongated pulp cells (odontoblast-like cells) associated with mild deposition of dentin matrix underneath the coagulation necrosis zone was observed. in the 4 specimens that showed response of human dental pulp to calcium hydroxide paste preceded by a corticosteroid/antibiotic dressing agent braz j oral sci. 9(3):337-344 340 histopathological events scores inflammatory cell response tissue disorganization reparative dentin formation stained bacteriagroups/periods 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 7 days 0 6 0 0 0 6 0 0 0 0 6 0 5 1 0 0 30 days 2 2 0 0 2 2 0 0 0 4 0 0 4 0 0 0group i 60 days 3 1 0 0 3 1 0 0 3 1 0 0 3 1 0 0 7 days 6 0 0 0 0 5 1 0 0 0 2 4 5 1 0 0 30 days 5 0 0 0 4 1 0 0 0 4 1 0 5 0 0 0group ii 60 days 4 1 0 0 3 2 0 0 2 3 0 0 5 0 0 0 7 days 0 4 1 0 0 4 1 0 0 0 3 2 5 0 0 0 30 days 5 1 0 0 5 1 0 0 0 4 2 0 4 2 0 0group iii 60 days 4 1 0 0 4 1 0 0 4 1 0 0 4 1 0 0 table 5. number of teeth for each score according to groups and periods fig. 1 (group ii; 7th day ) pulpotomy zone with large coagulation necrotic area between material and pulp tissue (arrows). presence of few inflammatory cells and small congested and dilated blood vessels in subjacent pulp tissue. hematoxylin-eosin, original magnification 32x. fig. 2 (group iii; 7th day) elongated cells organized in layer (arrows) in the area immediately bellow the coagulation necrotic zone. large number of blood vessels in the subjacent pulp tissue. hematoxylin-eosin, original magnification 125x. disorganized elongated cells no dentin matrix deposition was seen. in group iii, pulp response was similar to that of group ii. however, mild inflammatory response was observed in 4 specimens (figure 2) and moderate inflammatory reaction was seen in 1 specimen. it should be emphasized that no microleakage was evidenced in all evaluated specimens. fig. 3 (group i; 30th day) moderate deposition of hard tissue subjacent to the pulp wound. presence of odontoblast-like cells in monolayer. hematoxylin-eosin, original magnification 280x. 30-day period after 30 days of the procedure 4 teeth of group i, 5 teeth of group ii, and 6 of group iii were analyzed. in group i (control), all specimens showed a monolayer of elongated cells associated with moderate deposition of hard tissue subjacent to pulp wound (figure 3). mild inflammatory reaction associated with mild tissue disorganization was seen in 2 out of 4 specimens. in group ii, all specimens presented dystrophic calcification in the deepest zone of the coagulation necrosis layer. presence of a monolayer of elongated cells (odontoblastresponse of human dental pulp to calcium hydroxide paste preceded by a corticosteroid/antibiotic dressing agent braz j oral sci. 9(3):337-344 341 like cells) underlying the dystrophic calcification zone was noted. these differentiated elongated pulp cells were responsible for the deposition of a thin dentin matrix layer which, associated to the dystrophic calcification, characterized the moderate hard tissue barrier formation. the subjacent pulp tissue exhibited normal histological characteristics (figure 4). delay in the pulp repair occurred in one specimen in which calcium hydroxide remnants were displaced into pulpal space. in this specimen, heterogeneous deposition of dentin matrix associated to mild tissue disorganization was observed. no bacterial contamination in the lateral cavity walls or in the pulp tissue was observed in any of the specimens. in group iii, stained bacteria were seen along the outer third of the lateral cavity walls in 2 specimens, in which mild dentin matrix deposition was observed. in one of these specimens, mild inflammatory response occurred associated with tissue disorganization. however, moderate hard tissue barrier formation was observed in 4 out of 5 specimens in which no inflammatory response was determined (figure 5). 60-day period after 60 days of the procedure 4 teeth of group i, 5 teeth of group ii, and 5 of group iii were analyzed. at this period, an intense deposition of partially calcified dentin matrix underneath the thick layer of dystrophic fig. 6 (group ii; 60th day) presence of a large hard tissue barrier. odontoblastic like cells are organized in layer (arrow) and absence of inflammatory characteristics in the connective tissue. masson trichrome stain, original magnification 125x. calcification was observed in all groups. as a result, the dystrophic calcification associated with the dentin matrix gave rise to a noticeable hard barrier that remained between the capping material and the vital pulp tissue. it could also be seen a homogeneous monolayer of odontoblast-like cells underlying the tubular dentin matrix which was synthesized and deposited by these elongated pulp cells. subjacent pulp tissue exhibited normal histological characteristics (figure 6). one specimen of group i (control) and 1 specimen of group iii presented mild persistent inflammatory pulp response associated with mild tissue disorganization and in these specimens stained bacteria along the outer third of the lateral cavity wall were observed. the kruskal wallis statistical test applied to the attributed scores for the histological parameters showed that the period of evaluation had a significant influence (p<0.05) on tissue disorganization and on hard tissue barrier formation in group ii. in the other groups (i and iii) this test indicated fig. 4 (group ii; 30th day) included cells in the midst of hard barrier tissue underlined by odontoblast-like cells (arrowheads). absence of inflammatory characteristics in the subjacent pulp tissue. masson trichrome stain, original magnification 125x. fig. 5 (group iii; 30th day) hard tissue barrier with embedded cells (arrows). newly-formed odontoblast-like cells organized in layer (arrowheads) and moderate deposition of dentin matrix. hematoxylin-eosin, original magnification 125x. response of human dental pulp to calcium hydroxide paste preceded by a corticosteroid/antibiotic dressing agent braz j oral sci. 9(3):337-344 342 that tissue disorganization, and hard tissue barrier formation were also influenced significantly (p<0.05) by the evaluation period and the inflammatory cell response. there was greater inflammatory cell response (p<0.05) in groups i and iii than in group ii on the 7th postoperative day. however, no significant difference was observed among the three groups on the 30th and on the 60th postoperative days (p>0.05). discussion pulpotomy consists in cutting inflamed or infected coronary pulp and capping the remaining vital pulp tissue with a material that maintains its vitality18. this pulpal therapy has been strongly recommended for young permanent teeth, especially in clinical situations when a large pulp exposure is caused by decay or trauma or when the root is not completely formed 7,12,14,16-18. in the present study, the antiinflammatory effect of otosporin® application before (10 min or 48 h) pulp capping with calcium hydroxide was investigated. calcium hydroxide has been the material of choice recommended for pulpotomy7,12,14,16-20. the application of this material on pulpal wound causes superficial coagulation necrosis, which seems to participate in the pulpal healing process in association with hard barrier formation, although the calcium hydroxide mechanism of action is not yet well understood12-13,16-17,19-22. the dentin bridge may be a sign of healing or mild irritation, and the histological reaction to direct calcium hydroxide application onto pulpal tissue has been shown in several studies12,16-17,20,22. in the present study, 7 days after application of this material, it could be seen the presence of odontoblast-like cells underlying the thick area of coagulation necrosis subjacent to pulp-capping agent, and only a mild inflammatory cell infiltrate. after 30 days, necrotic tissue in a dystrophic calcification process could be noted as well as the start of pulp repair with dentin bridge formation close to this tissue. dentin bridge formation was apparently complete after 60 days of calcium hydroxide application, and the remaining pulp tissue presented normal histological characteristics. the efficacy of calcium hydroxide as a pulp capping agent was reported to depend on its ability in releasing calcium and hydroxyl ions into the subjacent connective pulp tissue. the degree and speed of calcium and hydroxyl ion dissociation as well as the change in tissue ph values which the calcium hydroxide paste was applied to depend on the vehicle used to prepare the paste23 (distilled water, in the present study). at the postoperative period of 7 days, a large zone of coagulation necrosis was observed in contact with pulp-capping material. this was probably due to the vehicle utilized, which allowed a highly alkaline ph since the moment that it was put in contact with the tissue. a previous study on the effects of ph on enzymatic activity in pulp tissue24, have shown that the alkaline phosphatase activity, which is related to mineralization process, is increased by alkaline ph. on the 30th postoperative day, the coagulation necrosis area presented calcification in its inner layer. a new odontoblastic layer was present, and newly formed dentin matrix was deposited nearby the pulpotomy area. this hard tissue barrier was larger on the 60th postoperative day. in all groups, it was characterized by the presence of dystrophic calcified tissue in the superficial layer and dentin deposition in the inner layer. these reactions are similar to those observed on healthy pulp tissue when direct calcium hydroxide application was made12,16-17,20,22. in a previous study13, on the other hand, an irregular dentin bridge formation was seen and no calcification or odontoblastic layer formation were observed on the 90th postoperative day. in a longer postoperative period (6 month), calcification and necrosis underneath the dentin bridge, mild hyperemia and mild chronic inflammation dominated by lymphocytes were observed, but no odontoblastic layer formation was noted13. franz et al.25 in a study in humans and yoshiba et al.26 in monkeys have also observed that the mineralized tissue barrier formed by pulps treated with calcium hydroxide consisted of a superficial osteodentin layer and a tubular dentin layer subjacent to that one, next to the pulp tissue. the thickness of this mineralized barrier increased in direct relationship to the length of the postoperative period25 and its production tended to be made far from the capping material26. corticosteroid can be used as a dressing agent in order to minimize pulp inflammation and consequently to provide relief of postoperative pain and sensisitivity1,4-5,8. however, there is no consensus regarding the benefits of the use of this agent. the results of studies that employ corticosteroids as a cavity liner support that these medications are effective in reducing or preventing postoperative thermal sensitivity1,8,27. on the other hand, when applied alone as direct pulp capping the results are poor3-5. many investigators believe that the treatments are more successful when the products are used in combination with calcium hydroxide or when previous topical application is made before direct pulp capping3,28. researchers have shown that application of corticosteroid/ antibiotic association for short period of time was effective to control inflammation in the pulp tissue without determining changes in the healing process3,18. kakehashi et al.27 have reported that 1-min application of predinisolone onto pulp tissue appeared not to alter the normal healing process in the absence of microorganisms, whereas appeared not to retard the pulpal degeneration process in the presence of bacterial infection. in the present study, the histological analysis showed no inhibition on blood vessels proliferation or on collagen deposition processes after corticosteroid/ antibiotic dressing application for 10 min or 48 h. the absence of significant difference among control and treated groups ii and iii (10 min or 48 h otosporin® dressing, respectively) on different postoperative periods is in agreement with the observation that the corticosteroid/antibiotic association has no effect when applied on healthy dental pulp11. therefore, it can be suggested that previous pulp-capping application of a corticosteroid/antibiotic agent may be response of human dental pulp to calcium hydroxide paste preceded by a corticosteroid/antibiotic dressing agent braz j oral sci. 9(3):337-344 343 unnecessary in routine clinical situations of accidental pulp exposure without inflammation like complicated crown fractures or complicated crown-root fractures in which the search for dental treatment is immediate or in cases of pulpotomies. when pulp tissue is inflamed, souza and holland3 have reported that dressing with corticosteroid/antibiotic association for 48 h produced best results, presenting complete hard barrier deposition with absence of dispersion of inflammatory cells in the remaining tissue. also in inflamed pulp tissue, santini 18 has observed that corticosteroid/ antibiotic association maintained vitality for a longer period without interfering in the healing produced by the use of calcium hydroxide in pulpotomy made in young permanent human teeth. in the present study, significant differences were observed for the scores of tissue disorganization and reparative dentine formation in the postoperative periods in all groups. however, significant difference was detected only between group i (control) and group iii (48-h otosporin® dressing) for inflammatory cell response. in these groups, the intensity of the inflammatory cell response was greater at 7 days than in the other periods. in group ii (10-min otosporin® dressing), no significant difference was found in the inflammatory cell response scores during the course of the experiment. sazak et al.28 have also observed greater inflammation in ledermix and calcium hydroxide association treated group when compared to calcium hydroxide alone on the 7th day. bacterial contamination is an important factor that may influence the response of injured pulp tissue and could be considered the primary cause of pulp death5,12,29. after direct pulp-capping with calcium hydroxide, tissue healing and dentin bridge formation will be promoted by the pulp as long as bacterial microleakage is prevented12. bacteria can arise from the original carious lesion, from saliva, from the margins of the restoration by microleakage, and from the operator and instruments during the treatment5,12,29. in the present study, presence of bacteria in the most superficial third of the lateral cavity walls was detected in only 6 specimens. from these, mild inflammatory response and tissue disorganization were observed in only1 specimen of group iii on the 30th postoperative day. since only noncarious teeth were used and the clinical procedures were performed under aseptic conditions, it may be assumed that bacteria probably reached the tooth-restoration interface through microleakage. within the limitations of present study, considering that the pulpotomy was performed in healthy teeth, it may be concluded that the use of a corticosteroid/antibiotic dressing before remaining tissue protection with calcium hydroxide had no influence on pulp tissue healing. references 1. dachi sf, ross a, stigers rw. effects of prednisolone on the thermal sensitivity and pulp reactions of amalgam restored teeth. j am dent assoc. 1964; 69: 565-71. 2. van hassel hj, mchugh jw. effect of prednisolone on intrapulpal pressure [abstract 499]. j dent res. 1972; 51: 172. 3. souza v, holland r. treatment of the inflamed dental pulp. aust dent j. 1974; 19: 191-6. 4. paterson rc. corticosteroids and the exposed pulp. br dent j. 1976; 140: 174-7. 5. paterson rc. bacterial contamination and the exposed pulp. br dent j. 1976; 140: 231-6. 6. holland r, okabe ja, souza v, saliba o. diffusion of corticosteroidantibiotic solutions through human dentine. rev odontol unesp. 1991; 20: 17-23. 7. qudeimat ma, barrieshi-nusair km, owais ai. calcium hydroxide vs mineral trioxide aggregates for partial pulpotomy of permanent molars with deep caries. eur arch paediatr dent. 2007; 8: 99-104. 8. fry ae, watkins rf, phatak nm. topical use of corticosteroids for the relief of pain sensitivity of dentine and pulp. oral surg oral med oral pathol. 1960; 13: 594-7. 9. mosteller jh. use of prednisolone in the elimination of postoperative thermal sensitivity: a clinical study. j prosthet dent. 1962; 12: 1176-9. 10. schroeder a. combination of antibiotics and cortisone in the treatment of root canals. rev belge med dent. 1965; 20: 291-8. 11. uitto vj, antila r, ranta r. effects of topical glucocorticoid medication on collagem biosynthesis in the dental pulp. acta odontol scand. 1975; 33: 287-98. 12. sübay rk, suzuki s, suzuki s, kaya h, cox cf. human pulp response after partial pulpotomy with two calcium hydroxide products. oral surg oral med oral pathol oral radiol endod. 1995; 80: 330-7. 13. aeinehchi m, eslami b, ghanbariha m, saffar as. mineral trioxide aggregate (mta) and calcium hydroxide as pulp-capping agents in human teeth: a preliminary report. int endod j. 2003; 36: 225-31. 14. el-meligy oa, avery dr. comparison of mineral trioxide aggregate and calcium hydroxide as pulpotomy agents in young permanent teeth (apexogenesis). pediatr dent. 2006; 28: 399-404. 15. pradhan dp, chawla hs, gauba k, goyal a. comparative evaluation of endodontic management of teeth with unformed apices with mineral trioxide aggregate and calcium hydroxide. j dent child. 2006; 7: 79-85. 16. hebling j, giro ema, costa cas. biocompatibility of an adhesive system applied to exposed human dental pulp. j endod. 1999; 25: 676-82. 17. de albuquerque ds, gominho lf, dos santos ra. histologic evaluation of pulpotomy performed with ethyl-cyanocrylate and calcium hydroxide. braz oral res. 2006; 20: 226-30. 18. santini a. assessment of the pulpotomy technique in human first permanent mandibular molars. br dent j. 1983; 155: 151-4. 19. heys dr, cox cf, heys rj, avery jk. histological considerations of direct pulp capping agents. j dent res. 1981; 60: 1371-9. 20. schroeder u. effects of calcium hydroxide-containing pulp capping agent on pulp cell migration, proliferation and differentiation. j dent res. 1985; 64: 541-8. 21. stanley hr, lundy t. dycal therapy for pulp exposures. oral surg oral med oral pathol. 1972; 34: 818-27. 22. mjor ia, dahl e, cox cf. healing of pulp exposures: an ultrastructural study. j oral pathol med. 1991; 20: 496-501. 23. de andrade ferreira fb, silva e souza pdea, do vale ms, de moraes ig, granjeiro jm evaluation of ph levels and calcium ion release in various calcium hydroxide endodontic dressings. oral surg oral med oral pathol oral radiol endod. 2004; 97: 388-92. 24. gordon tm, ranly dm, boyan bd. the effect of calcium hydroxide on bovine pulp tissue: variations in ph and calcium concentration. j endod. 1985; 11: 156-60. 25. franz fe, holz j, baume lj. ultrastructure (sem) of dentin bridging in the human dental pulp. j biol buccale. 1984; 12: 239-46. 26. yoshiba k, yoshiba n, iwaku m. histological observations of hard tissue barrier formation in amputed dental pulp capped with alpha-tricalcium phosphate containing calcium hydroxide. endod dent traumatol. 1994; 10: 113-20. response of human dental pulp to calcium hydroxide paste preceded by a corticosteroid/antibiotic dressing agent braz j oral sci. 9(3):337-344 27. kakehashi s, stanley hr, fitzgerald r. the exposed germ-free pulp: effects of topical corticosteroid medication and restoration. oral surg oral med oral pathol. 1969; 27: 60-7. 28. sazak h, günday m, alatli c. effect of calcium hydroxide and combinations of ledermix and calcium hydroxide on inflamed pulp in dog teeth. j endod. 1996; 22: 447-9. 29. cox cf, bergenholtz g, heys dr, syed sa, fitzgerald m, heys rj. pulp capping of dental pulp mechanically exposed to oral microflora: a 12 year observation of wound healing in the monkey. j oral pathol. 1985; 14: 156-68. 344response of human dental pulp to calcium hydroxide paste preceded by a corticosteroid/antibiotic dressing agent braz j oral sci. 9(3):337-344 oral sciences n3 braz j oral sci. 8(4):185-188 original article prevalence of dental caries and treatment needs in preschool children in a recently fluoridated brazilian town jocianelle maria felix de alencar fernandes1 ; suyene de oliveira paredes1; daniele bezerra de almeida1; fábio correia sampaio1; franklin delano soares forte1 1federal university of paraiba, health science center, postgraduate program in preventive dentistry and pediatric dentistry, campus i, brazil correspondence to: franklin delano soares forte travessa antônio leopoldo batista, 71, apto 402 bancários joão pessoa, pb, brasil e-mail: fdsforte@terra.com.br abstract aim: the aim of this study was to evaluate the prevalence of dental caries and to estimate the treatment needs, in 4-6-year-old preschool children in a brazilian town that has recently started a water fluoridation program. methods: children (n=127) were randomly selected and examined according to the who criteria. the children were examined in schools by calibrated dentists. the intraand inter-examiner reliability of caries scores showed kappa values ranging from 0.81-0.95 and 0.8, respectively. mann-whitney and chisquare tests were used to analyze the results with 5% significance. results: the mean ± sd dmft was 4.45 ± 3.9 (95%ci 3.76-5.41). the dmft index was mainly composed by the decayed component (4.18 ± 3.4 95%ci 3.57-4.79). only 21.3% of the children were “caries-free” (dmft=0) and 62.2% showed dmfte”3. fillings and pulp therapy were the most required treatments. conclusions: the data collected in this study indicate that young children have high caries prevalence and many teeth needing treatment. in addition to the fluoridation program in the public water supply, appropriate health promoting strategies must be implemented to the most vulnerable age groups for controlling dental caries. keywords: dental caries, epidemiology, preschool, oral health. introduction dental caries is one of the most prevalent chronic diseases affecting humans. it is caused by a complex interaction of factors over time: production of acid by microorganisms and fermentation of carbohydrates in the diet, and factors such as saliva and dental susceptibility. in addition, physical, biological, environmental and socioeconomic factors are also considered risk factors for the development and progression of disease1-2. the oral health of children has improved in recent years in developed countries, therefore, increasing the number caries-free individuals3. the goal of the world health organization (who) for the year 2000 was to achieve 50% caries-free 5-year-olds. more than 40% of the global decline of dental caries may be associated with the use of fluoridated toothpastes and other forms of fluoride, such as fluoridation of public water supply4. however, the prevalence and severity of dental caries in some developing countries are still high particularly in primary teeth. it is estimated that more than half of the children have caries in primary teeth. until recently only 43.2% of young children in a chilean community were classified as “caries-free”. in subsequent years the number of children with carious lesions in primary teeth increased considerably reaching almost 70% at the age of five years old5. there is no doubt that information about dental caries development in this age group is relevant. nevertheless, data about dental caries for the 5-6-year-old children are still scarce6. caries in primary teeth can be a good predictor of caries development in permanent dentition7. braz j oral sci. october/december 2009 volume 8, number 4 received for publication: july 13, 2009 accepted: november 11, 2009 186 braz j oral sci. 8(4):185-188 in order to cover up this topic, the national brazilian survey (sb brasil) has included this age group in the brazilian oral health epidemiologic investigation since 20038. analysis of the data of the last brazilian national oral health survey shows that 65% of the 5year-old children in the northeast region showed at least one decayed primary tooth. the mean dmft observed was 3.21 and 90% of this value was due to the decayed component8. in a study conducted in joão pessoa, state of paraíba, brazil, 43.7% of the children at 48 months of age had dental caries. among these, 10.7% presented early childhood caries and 33.0% had severe caries9. in 1996, an epidemiological survey was conducted in the state of paraíba, brazil, comprising 11 cities and totaling 41 public schools in the region. alagoinha was one of the cities involved, and presented an average dmf-t of 3.09 for 6-12 year-old children at the time. regarding the treatment needs, the cities with the highest indexes were araruna (21%), cuitegi (19%) and alagoinha (18%)10. the aim of the present study was to determine the prevalence of dental caries and the related treatment needs in 4-6 year-old children of both genders from public schools in the city of alagoinha, state of paraíba, brazil. material and methods the city of alagoinha is located at 84 km from paraíba capital ( joão pessoa) and has approximately 13,000 inhabitants, mostly located in the urban area. the human development index (hdi) was 0.573 in 2000, which is considered medium development. the city displays fluoridated public water supply since 2006. the autonomous service of water and sewage (asws) is responsible for the supervision, maintenance and distribution of water. according to information obtained from asws and external monitoring held in th e city, th e wat er f ro m public supply h as average f luori de concentrations between 0.2 and 0.7 mg / l. as alagoinha is considered a small city, all children in the target age group (4-6 year olds) who were permanent residents and were in enrolled in public schools and kindergartens in the city were examined, resulting in a study population of 127 children [73 male (57.5%) and 54 female (42.5%)] initially, the parents or guardians were contacted to authorize the study by signing informed consent forms. there were only 2 losses (only 1.6%): one child that refused undergoing examination and one children whose parents did not return the signed informed consent form. the codes and criteria of the who11 and health ministry were adopted12. the examinations were performed by two examiners assisted by a noter under natural light, in public units of children education. the intraexaminer kappa accordance values were 0.81 and 0.95 and the interexaminer value was 0.80, indicating satisfactory male female total n= 73 n= 54 n= 127 caries-free (%) 21.9 20.4 21.3 with caries (%) 78.1 79.6 78.7 table 1 prevalence of dental caries among children according to the gender. alagoinha, paraíba, brazil, 2009. (m ± sd) ic (95%) (m ± sd) ic (95%) (m ± sd) ic (95%) d e c a y * 4.0 ± 3.4 3.19-4.81 4.43 ± 3.5 3.47-5.38 4.18 ± 3.4 3.57-4.79 missing* 0.34 ± 0.8 0.14-0.55 0.33 ± 0.9 0.06-0.60 0.34 ± 0.92 0.18-0.50 filled* 0.05 ± 0.3 0.02-0.13 0.19 ± 1.3 0.19-0.56 0.11 ± 0.91 0.05-0.27 dmft* 4.3 ± 3.8 3.4-5.20 4.65 ± 4.0 3.54-5.76 4.45 ± 3.9 3.76-5.14 male n = 73 female n= 54 total n= 127 table 2 – dmft index and components in children aged to 4 to 6 years old, according to the gender. alagoinha, paraíba, brazil, 2009. *mann-whitney u p>0.05 reproducibility of diagnoses. examinations were performed only after supervised toothbrushing. for the clinical examination, the child was sat in a chair facing the examiner. disposable gloves, hoods, masks and wooden spatulas, and properly sterilized dental mirrors and ballpoint millimeter probes (who standard)11 were used. data were processed in the database using the program spss (statistical package for social science) version 13.0. the descriptive statistics was performed to determine the prevalence of dental caries in the population studied. the data were also subjected to statistical tests of association such as fischer’s exact test and chi-square, in addition to the mann whitneyu test at 0.05 significance level. this research started after obtaining approval from the ethics committee in human research of the center for health sciences, federal university of paraíba, brazil (0491/2008). results the prevalence of dental caries among 4-6-year-old children was 78.7% (table 1). the mean dmf-t and standard deviation found were 4.45 ± 3.9 (95% ci 3.76-5.14) (table 2). the carious component constituted 78.7% of the index while missing and filled components constituted 16.5% and 2.4%, respectively. there was no statistically significant difference when comparing mean dmft data of boys and girls (p > 0.05). the same was observed when taking into account each dmft component versus gender (p > 0.05). only 21.3% of children examined were caries-free and approximately 62.2% had high caries experience with dmft e” 3. the caries experience was slightly higher in females than in males, but this difference was not statistically significant (p> 0.05). examining the type of treatment need during the epidemiological investigation, the highest percentage was the indication of restorations (69%), and restoration of two or more surface was the most predominant (46%), followed by restoration of only one surface, pulp treatment plus restoration and remineralization of white spots. other treatments such as extractions and sealants were also recommended (table 3). more complex treatments, such as metal crowns or esthetics were not indicated. the most commonly decayed teeth were the molars (35%) and central incisors (22%) (figure 1). the primary mandibular second molars and the maxillary central incisor were more affected than the other teeth. prevalence of dental caries and treatment needs in preschool children in a recently fluoridated brazilian town 187 braz j oral sci. 8(4):185-188 type of treatment need n % restoration of one surface 154 23 restoration of two or more surface 299 46 pulp treatment+dental restoration 82 12 exodonty 42 06 remineralization 77 12 pit-and-fissure sealant 07 01 table 3 percentage of teeth according to type of treatment need. alagoinha, paraíba, brazil, 2009. fig. 1. percentage of teeth affected by dental caries. alagoinha, state of paraíba, brazil, 2009. discussion in the same way as observed in the city of alagoinha, other studies have also obtained high mean dmft and prevalence of dental caries above 60%7,13-14. the prevalence of dental caries observed in the present study was higher than in other regions of brazil, as in the city of indaiatuba, são paulo state, where 5-year-old preschoolers presented mean dmft of 1.73 and prevalence of dental caries of 42.6%15. similar data have been found in municipal public schools in the city of recife, pernambuco state with 47% prevalence of dental caries and mean dmft of 2.0616. the results of the present study (21.3% of caries-free children) are far from the goal recommended by the who for 2000 (50% of caries-free children at 5 years of age), although this research covered 4-6-year-old children. this is similar to the findings of cypriano et al.7, who reported that only 38.5% of 6-year old children were cariesfree, but differed from those of studies performed in chile5 and in other regions of brasil15-18. reaching the goals of who for this age group is an important issue for the municipalities, even though it can be observed that for those cities with higher living standards better life conditions and better health conditions are reached in shorter periods19. concerning the sample stratification by gender, it was found that females had a slightly higher caries experience than males, but this difference was not statistically significant. according to declerck et al.20, the caries experience is significantly associated with gender, where female children are less susceptible to develop the disease at the age of 5, which was not observed in this study. taking into consideration the components of dmft index, it was observed that the highest part was composed by the carious component, as in several studies conducted in brasil15,21 or even in other countries13. this is partly due to a lack of organization in the system of oral health and limited access to health services indicating the need for early identification of risk groups17. these data refer to the need for more coverage of dental services in the city and extensive need for care. a cause of concern was the low percentage of children with filled teeth (only 2.4%) in the studied population. one explanation for the lack of treatment in preschool children may be due to the fact that parents and/or professionals do not give the due value to the primary teeth16. moreover, this result can be interpreted as reflecting the difficulty of access of this population to measures of health promotion. some preventive measures that have broad range if adopted in population strategies, such as the development of oral health care models, according to the principles of universality, equity and integrality could provide more appropriate oral health promotion22. attention to oral health should be established in the first year of life, before a more severe or even more difficult and expensive condition to completion of preventive treatment is installed17. the results obtained for the city of alagoinha not only provided relevant information regarding the prevalence of dental caries, but also identified the main needs for treatment in the studied population. there are several requirements to be met, mainly of low complexity, represented by restorations mostly involving two or more surfaces. the benefit of fluoride can be measured in a study conducted in rural areas of paraíba, in which groups living in areas with moderate levels of fluoride showed lower prevalence of dental caries than those living in areas with lower fluoride levels23. the city of alagoinha presents artificial fluoridation of public water supply since 2006. the maximum benefit of fluoridation requires time and the continuity of the method can result in a greater control of disease progression. thus, it is emphasized the importance of maintaining of this measure of health promotion as well as the control performed by asws and the partnership with the ufpb in monitoring water fluoridation. preventive strategies targeting children affected by dental caries, such as gels for topical applications, use of fluoridated dentifrices, supervised toothbrushing 3 to 4 times a year, as well as health education can contribute to significant changes in dental caries process. there is a need to focus on strategies that can mitigate the damage caused by the progression of the disease, in addition to the access of children to restorative treatment for those with caries experience. the results of this study indicate that children in the city of alagoinha have a high prevalence of dental caries with great need of treatment. in addition to the recently implemented fluoridation of public water supply, actions and strategies appropriate to the most vulnerable group should be introduced, with greater investment towards programs for prevention and control of dental caries involving children and parents and/or guardians. acknowledgements to the parents, children, directors and teachers from carlos beltrão school, josé barbosa school and kindergartens in the city of alagoinha. references 1. fejerskov o. changing paradigms in concepts on dental caries: consequences for oral health care. caries res. 2004; 38: 182-91. 2. selwitz rh, ismail ai, pitts nb. dental caries. lancet. 2007; 369: 51-9. 3. petersson hg, bratthall d. the caries decline: a review of reviews. eur j oral sci. 1996; 104: 436-43. 4. narvai pc, frazão p, roncalli ag, antunes jlf. cárie dentária no brasil: declínio, polarização, iniqüidade e exclusão social. rev panam salud publica. 2006; 19: 385-93. 5. lópez iy, bustos bc, ramos aa, espinoza rm, jara mn, smith lp. prevalence of dental caries in preschool children in peñaflor, santiago, chile. rev. odonto ciênc. 2009; 24: 116-9. 6. bonecker m, cleaton-jones p. trends in dental caries in latin america and caribbean 5-6 and 11-13 year-old children: a systematic review. community dent oral epidemiol. 2003; 31: 152-7. prevalence of dental caries and treatment needs in preschool children in a recently fluoridated brazilian town 188 braz j oral sci. 8(4):185-188 7. cypriano s, sousa mlr, rihs lb, wada rs. saúde bucal dos pré-escolares, piracicaba,brasil, 1999. rev saúde pública. 2003; 37: 247-53. 8. brasil. ministério da saúde, secretaria de atenção à saúde, departamento de atenção básica, coordenação nacional de saúde bucal. projeto sb brasil 2003 -condições de saúde bucal da população brasileira 2002–2003: resultados principais. brasília: ms-cnsb; 2004. 9. ribeiro ag, oliveira af, rosenblatt a. cárie precoce na infância: prevalência e fatores de risco em pré-escolares, aos 48 meses, na cidade de joão pessoa, paraíba, brasil. cad saúde pública. 2005; 21: 1695-1700. 10. projeto nordeste. secretaria de saúde. universidade federal da paraíba. relatório final do levantamento epidemiológico de cárie dentária em 3 regionais de saúde. paraíba; 1996. 11. world health organization. oral health surveys, basic methods. 4 ed. geneve: who; 1997. 12. ministério da saúde. projeto sb 2000 condições de saúde bucal da população brasileira no ano 2000. manual do examinador. brasília: secretaria de políticas de saúde, ministério da saúde; 2001. 13. ivankovic a, lukic ik, ivankovic z, radic a, vukic i, simic a. dental caries in post war bosnia and herzegovina. community dent oral epidemiol. 2003; 31: 100-4. 14. jamieson lm, thomson wm, mcgee r. caries prevalence and severity in urban fijian school children. int j paediatric dent. 2004; 14: 34-40. 15. rihs lb, sousa mlr, cypriano s, abdalla nm, guidini ddn, amgarten c. atividade de cárie na dentição decídua, indaiatuba, são paulo, brasil, 2004. cad saúde pública. 2007; 23: 593-600. 16. feitosa s, colares v. prevalência de cárie dentária em pré-escolares da rede pública de recife, pernambuco, brasil, aos quatro anos de idade. cad saúde pública. 2004; 20: 604-9. 17. ferreira sh, beria ju, kramer pf, feldens eg, feldens ca. dental caries in 0to 5-year-old brazilian children: prevalence, severity, and associated factors. int j pediatric dent. 2007; 17: 289-96. 18. leite icg, ribeiro ra. dental caries in the primary dentition in public nursery school children in juiz de fora, minas gerais, brazil. cad saúde pública. 2000; 16: 717-22. 19. lucas sd, portela mc, mendonça ll. variações no nível de cárie dentária entre crianças de 5 e 12 anos em minas gerais, brasil. cad saúde pública. 2005; 21: 55-63. 20. declerck d, leroy r, martens l, lesaffre e, garcia-zattera mj, broucke vs et al. factors associated with prevalence and severity of caries experience in preschool children. community dent oral epidemiol. 2008; 36: 168-78. 21. ogido em, dezan cc, garbelini wt, salomão f, morita mc. prevalence of dental caries in 3and 5-year-old children living in a small brazilian city. j appl oral sci. 2004; 12: 34-8. 22. gushi ll, soares mc, forni tib, vieira v, wada rs, sousa mlr. cárie dentária em adolescentes de 15 a 19 anos de idade no estado de são paulo, brasil, 2002. cad. saúde pública. 2005; 21: 1383-91. 23. sampaio fc, nazmul hossain anm, von der fehr fr, arneberg p. dental caries and sugar intake of children from rural areas with different water fluoride levels in paraíba, brazil. community dent oral epidemiol. 2000; 28: 307-13. prevalence of dental caries and treatment needs in preschool children in a recently fluoridated brazilian town oral sciences n3 braz j oral sci. 11(1):30-35 original article braz j oral sci. january | march 2012 volume 11, number 1 evaluation of proximal enamel thickness and crown measurements in maxillary first premolars leni okamoto munhoz1, flávio vellini-ferreira2, flávio augusto cotrim-ferreira3, rívea inês ferreira3 1msc in orthodontics, department of pediatric dentistry and orthodontics, university of são paulo city (unicid), brazil 2phd, professor and chairman, department of pediatric dentistry and orthodontics, university of são paulo city (unicid), brazil 3phd, associate professor, department of pediatric dentistry and orthodontics, university of são paulo city (unicid), brazil correspondence to: rívea inês ferreira universidade cidade de são paulo (unicid) pós-graduação (mestrado em ortodontia) rua cesário galeno, 448 – bloco a cep 03071-000 – tatuapé, são paulo, brasil phone / fax: +55 11 2178-1310 e-mail: riveaines@yahoo.com received for publication: september 09, 2011 accepted: january 24, 2012 abstract estimating enamel thickness and planning the resultant optimal morphology of premolars are substantial steps before interproximal stripping. aim: to analyze proximal enamel thickness and crown measurements in maxillary premolars. methods: the mesiodistal, buccolingual and cervico-occlusal measurements of 40 human maxillary first premolars (20 right, 20 left) were registered with a digital caliper. the teeth were embedded in acrylic resin and sectioned mesiodistally at the level of the contact areas to obtain 1 mm-thick central sections. enamel thickness on the proximal surfaces was measured using a perfilometer. measurements were compared by the student’s t-test (α = 0.05). results: the mean enamel thickness on the mesial surface was 1.22 mm for the right (± 0.17) and left (± 0.18) sides. on the distal surface, the corresponding values were 1.28 mm (± 0.19) on the right side and 1.39 mm (± 0.17) on the left side. mean values, in millimeters, for the mesiodistal, buccolingual and cervico-occlusal measurements on both sides ranged from 7.03 (± 0.43) to 7.07 (± 0.48), 9.59 (± 0.48) to 9.65 (± 0.58) and 8.65 (± 0.66) to 8.85 (± 0.65), respectively. there were no significant differences between right and left teeth. however, enamel thickness was significantly greater (p<0.05) on the distal surface. conclusions: in maxillary first premolars, considering the greater thickness of distal enamel, interproximal stripping during orthodontic treatment may be more pronounced on this surface. the greatest mean value was observed for the buccolingual crown measurement, followed by the cervicoocclusal and mesiodistal dimensions. keywords: bicuspid, tooth crown, dental enamel, orthodontics. introduction currently, orthodontic patients are searching for esthetics combined to satisfactory functioning of the stomatognathic system in the dental offices. treatments comprising tooth extraction and appliances that cause discomfort and social constraint are avoided and effectively overcome, for example, by interproximal stripping. good occlusion and normal periodontal conditions, with intact gingival papillae between all teeth in the maxillary and mandibular arches, could be observed in follow-up examinations more than 3.5 years after orthodontic treatment1. some authors2 mention that enamel reduction in premolars using rotary instruments and appropriate technique does not damage dental pulp in most cases, even if dentin is slightly exposed and polished. the use of fluoride after enamel stripping was indicated and patients did not complain of any symptoms. braz j oral sci. 11(1):30-35 3131313131 interproximal enamel stripping yields an alternative method to tooth extraction for solving mild to moderate crowding and has also been associated to short treatment duration1-6. moreover, interproximal enamel stripping is not only useful for correcting tooth-arch discrepancies, but it also increases treatment stability7-9, and keeps the transverse dental arch dimensions and anterior inclinations constant1,6,10. although some authors mentioned that about fifty percent of proximal enamel can be safely removed11-12, all professionals who are willing to perform the striping procedure should be aware of the estimates relative to the proximal enamel thickness and crown measurements for avoiding damaging to the dental structures and achieving proper tooth recontouring. thus, the aim of this experimental investigation was to accurately assess the mean values of the proximal enamel thickness, as well as the mesiodistal, cervico-occlusal and buccolingual crown measurements, in maxillary first premolars. the null hypotheses stated no differences between enamel thicknesses according to the side of the dental arch and proximal surface. material and methods this experimental study was approved by the institutional review board, under the protocol number 13260742/2007, and complies with the brazilian resolution regulating research involving humans (resolution 196/96). sample forty sound human maxillary first premolars (20 right and 20 left) were selected from two tooth banks. all donors were brazilians, yet information on the gender, age and race was unavailable. teeth were separated into two groups (right and left sides) and randomly numbered from 1 to 20. achievement of crown measurements mesiodistal (figure 1), buccolingual (figure 2) and cervico-occlusal (figure 3) measurements were obtained with a digital caliper (mitutoyo® sul americana ltda., suzano, sp, brazil) accurate to 0.01 mm, by a well-trained examiner. the mesiodistal measurement was assumed as the distance from the most central point in the contact area on the distal surface to its counterpart on the mesial surface. the values obtained from the midpoint on the buccal surface to the midpoint on the lingual surface of crowns were registered as the buccolingual measurement. the distance between the occlusal edge of the buccal surface and the cementoenamel or dentinoenamel junction at the cervical region of the tooth corresponded to the cervico-occlusal measurement. enamel thickness assessment all teeth were kept in a fixed position in rectangular plastic flasks using utility wax and embedded in acrylic resin (arazyn 1.0 – redelease®, são paulo, sp, brazil). tooth sections were obtained using a diamond disc in a trimmer lab cut® 1010 (extec® corp., enfield, ct, usa), under fig. 1 – measure of the tooth crown mesiodistal width. fig. 2 – measure of the tooth crown buccolingual dimension. fig. 3 – measure of the tooth crown cervico-occlusal height. fig. 4 – resin block sectioned with the tooth 1-mm-thick cut central section. evaluation of proximal enamel thickness and crown measurements in maxillary first premolars braz j oral sci. 11(1):30-35 3232323232 cooling to avoid specimen fracture. the mean speed of the disc was maintained at 350 rpm. the specimens were sectioned longitudinally through the proximal surfaces, parallel to the buccal surface, producing 1-mm-thick central sections (figure 4). each section corresponded to the most central part of the crown, on the proximal surfaces, because it encompassed the greatest mesiodistal dimension and, hence, the area where the proximal enamel is thicker. based on previous research13, the sections were placed in a perfilometer (mitutoyo®, profile projector® pj 300, kawasaki, japan) accurate to 0.001 mm, so as the tooth long axis coincided with the cartesian axes of the device (x and y), which were displaced in horizontal and vertical directions, respectively (figure 5). proximal enamel thickness was measured by displacement of the cartesian axis in mesial and distal directions of the tooth section. considering that the measurements were obtained on the projected images of the cut sections, all dimensions were measured three or six times until the values coincided three times. statistical analyses mean (and the standard deviation), minimum and maximum values and the coefficient of variation were calculated for the enamel thickness on the mesial and distal surfaces, as well as the mesiodistal, buccolingual and cervicoocclusal measurements. comparisons between mean values registered for the right and left teeth, and enamel thickness on the mesial and distal surfaces were carried out using the student’s t-test (α = 0.05). results minimum, maximum and mean values, as well as standard deviations and coefficients of variation for the crown measurements and enamel thicknesses of maxillary first premolars are shown in table 1. the greatest mean value was observed for the buccolingual crown measurement, followed by the cervico-occlusal and mesiodistal dimensions. fig. 5 – projected image on the profilometer for measuring enamel thickness (a and b). aaaaa bbbbb variables minimum (mm) maximum (mm) mean (mm) standard deviation coefficient of variation (%) right side mesiodistal width 6.19 7.85 7.03 0.43 6.12 buccolingual dimension 8.70 10.80 9.59 0.48 5.01 cervico-occlusal height 7.37 9.82 8.65 0.66 7.63 mesial enamel thickness 0.86 1.48 1.22 0.17 13.93 distal enamel thickness 0.73 1.62 1.28 0.19 14.84 left side mesiodistal width 6.02 7.76 7.07 0.48 6.79 buccolingual dimension 8.74 10.84 9.65 0.58 6.01 cervico-occlusal height 7.34 9.84 8.85 0.65 7.34 mesial enamel thickness 0.87 1.64 1.22 0.18 14.75 distal enamel thickness 1.02 1.60 1.39 0.17 12.23 table 1 -table 1 -table 1 -table 1 -table 1 measures of central tendency and dispersion relative to crown dimensions and proximal enamel thickness of maxillary first premolars (right teeth n = 20, left teeth n = 20). mean enamel thickness on the mesial surface was 1.22 mm on the right (± 0.17) and left (± 0.18) sides. on the distal surface, the corresponding values were 1.28 mm (± 0.19) on the right side and 1.39 mm (± 0.17) on the left side (table 1). however, there were no statistically significant differences (p>0.05) between measurements obtained for right and left teeth (table 2). based on the coefficients of variation, there was marked variability of data in relation to the means for enamel thicknesses on the mesial (13.93% to 14.75%) and distal evaluation of proximal enamel thickness and crown measurements in maxillary first premolars braz j oral sci. 11(1):30-35 variables t value* p value significance mesiodistal width -0.220 0.827 not significant buccolingual dimension -0.356 0.723 not significant cervico-occlusal height -0.957 0.344 not significant mesial enamel thickness 0.081 0.936 not significant distal enamel thickness -1.994 0.053 not significant table 2 table 2 table 2 table 2 table 2 comparative analysis of the measurements obtained for right (n = 20) and left (n = 20) maxillary first premolars. *critical value of two-tailed “t” test (38 degrees of freedom), tcritical (0.05;38) = 2.024394. variables mean (sd*) p value significance mesial enamel thickness 1.2195 (0.1730) distal enamel thickness 1.3223 (0.1947) 0.001 highly significant * sd: standard deviation table 3 table 3 table 3 table 3 table 3 comparative analysis of the measurements obtained for mesial (n = 40) and distal (n = 40) mean enamel thicknesses. (12.23% to 14.84%) surfaces of both sides (table 1). conversely, data obtained for the mesiodistal, buccolingual and cervico-occlusal measurements demonstrated homogeneity, since the coefficients of variation were lower than 10%. although the coefficients of variation for proximal enamel thicknesses were higher in comparison to the values obtained for crown measurements, these indices may still be considered relatively low. because no significant differences between mean enamel thicknesses on the mesial and distal surfaces of right and left maxillary first premolars were found, the measurements for both sides were considered for comparison in table 3. the resultant mean value of enamel thickness was significantly higher on the distal surfaces compared to the mesial surfaces of maxillary first premolars. discussion the analyses of tooth crown measurements and proximal enamel thickness is actually useful to establish the diagnosis and adequate orthodontic treatment planning, specifically concerning the decision between tooth extraction and interproximal enamel stripping. the latter treatment modality may avoid extractions, decrease treatment time and provide more favorable outcomes3-6. in addition, the orthodontist should take into account that many patients may not be willing to undergo tooth extractions14. interproximal enamel stripping may be a suitable treatment alternative to solve mild to moderate tooth crowding1,3,5,9. accordingly, this therapeutic option may be used in tooth-size discrepancies occurring in class i malocclusions, class ii malocclusions with mild deviations in cephalometric measurements, especially after the growth period, and bolton discrepancy14, in which the teeth sizes are greater than the space available in the dental arch. around 8.9 mm of space may be gained in the dental arches using interproximal enamel stripping techniques, which may also be applied in posterior teeth15. however, the amount of enamel to be removed should be estimated according to the severity of tooth-size discrepancy5,11,16. enamel reduction may be substantial on teeth with deviating morphology, while incisors with parallel proximal surfaces, screwdriver-shaped teeth and rotated premolars may not be eligible for any stripping1. concerning the crown measurements of maxillary first premolars (table 1), the smallest mean values were observed for the mesiodistal width (7.03 – 7.07 mm). another brazilian study recorded slightly greater values (7.51 – 7.53 mm)9. presumably, this variation may be attributed to methodological differences between studies. in the present experimental research, all measurements were taken directly on the teeth using a digital caliper accurate to 0.01 mm, while the other authors obtained the measurements on images of histological sections of teeth projected on a computer monitor screen. digital images were acquired by a coupled camera and the operator used the software tools for measuring crown width9. this procedure may cause some image magnification, though increasing mesiodistal crown measurement. the greatest mean value was observed for the buccolingual crown dimension (9.59 – 9.65 mm). interestingly, the cervico-occlusal height presented greater values of standard deviation and coefficient of variation, maybe because the crown heights in some teeth were reduced by occlusal wear (table 1). the direct register of crown measurements in the mouth using a caliper revealed that the teeth had smaller mesiodistal and greater buccolingual dimensions 17. this finding corroborates the results shown in table 1. in another study5, an index was designed to evaluate morphological deviations of teeth, adding knowledge for better understanding mandibular incisors crowding. the sample was composed of white young adult females divided into two groups: one group with satisfactory alignment of mandibular incisors and the second group of patients with diagnosis of tooth crowding, which was taken as the control group. measurements were also obtained directly on the patient’s mouth, using a caliper with venier scale. comparison of the mesiodistal and buccolingual dimensions of the same tooth revealed that the former was smaller than the latter measurement in the second group5. conventional and digital intraoral radiographs, as well as computed tomography, are considered proper diagnostic adjuncts for clinical assessments of tooth crown and proximal enamel measurements18-19. nevertheless, one disadvantage of computed tomography is the blurred image on the limits of enamel thickness smaller than 1.1 mm, which precludes determination of the point from which the measurements should be initiated, despite the high image resolution19. this study presented data on the proximal enamel thickness of maxillary first premolars (table 1). mesial (1.22 mm for both sides) and distal (varying from 1.28 mm on the right side to 1.39 mm on the left side) mean values of enamel thickness indicated a possible difference according to the proximal surface. as shown in table 3, the proximal enamel was 3333333333 evaluation of proximal enamel thickness and crown measurements in maxillary first premolars 3434343434 braz j oral sci. 11(1):30-35 significantly thicker on the distal surface in comparison to the mesial surface (p = 0.001). this finding agrees with a brazilian study in which measurements were similarly obtained on human maxillary first premolars, yet using a different methodology9. the authors observed mean values of mesial enamel thickness of 1.08 mm (right side) and 1.19 mm (left side) and distal enamel thickness of 1.29 mm (both sides). a recent study also reported the significantly higher enamel thickness on the distal surface in second mandibular premolars, compared to the mesial surface13. it is worth mentioning that some studies suggest limits for proximal enamel stripping of 0.4-0.5 mm, regardless of the surface6-7,10. tooth banks do not provide information on the age range, gender or race of the donors. however, since this sample included only sound human premolars, it may be assumed that these teeth were donated by adolescents or young adults. even though mastication also influences the reduction of proximal enamel, its greater effect occurs on occlusal wear. moreover, considering that people have adopted a predominantly semi-solid diet since the past century, a significant loss of proximal enamel due to mastication forces is more frequent in elderly individuals20-22. with regard to gender dimorphism, a study revealed that the mesiodistal dimensions of the tooth crown for males were greater compared to the measurements obtained in females23. some authors evaluated the mesiodistal and buccolingual dimensions of tooth crowns in north americans, egyptians and mexicans24. all these populations exhibited significant difference between measurements in males and females24, which confirmed the findings of that previous study23. males presented greater canines and first molars24. a plausible explanation for the gender dimorphism may be related to the fact that the dentin thickness seems to be greater in males10,18. thus, the greater mesiodistal crown measurement in males may probably be a consequence of the thicker dentin layer under the tooth enamel. in the present study, the mesiodistal, buccolingual and cervico-occlusal crown measurements, as well as the proximal enamel thickness, were evaluated irrespective of the gender. furthermore, the maxillary first premolars selected were donated by patients who underwent a certain degree of the so-called miscegenation. however, for all measurements obtained, there was no statistically significant difference between right and left teeth (table 2), demonstrating symmetry in crown morphology and proximal enamel thickness of the maxillary first premolars. radiography and computed tomography, specifically cone-beam computed tomography, can be used in the clinical practice to estimate the amount of proximal enamel that may be safely removed, considering each patient individually9. nonetheless, it would also be clinically relevant to perform experimental studies for assessing crown measurements and proximal enamel thickness, since these values may be taken as parameters during interproximal stripping. in the present study, distal enamel was significantly thicker compared to the mesial enamel. hence, it is suggested that greater interproximal stripping may be performed on the distal surface. after interproximal stripping, the use of accurate measuring devices is advocated to estimate the magnitude of enamel reduction25. as a contribution to orthodontists, this study demonstrated that, in maxillary first premolars, the buccolingual measurement presented the highest mean value, followed by the cervico-occlusal and mesiodistal dimensions, even though the difference between the mean values related to these measurements were lower than 2 mm. acknowledgments the authors are especially thankful to professor washington steagal junior from the restorative dentistry department of the university of são paulo (fousp) for his dedication and collaboration in the statistical analyses of this study. references 1. zachrisson bu, minster l, ogaard b, birkhed d. dental health assessed after interproximal enamel reduction: caries risk in posterior teeth. am j orthod dentofacial orthop. 2011; 139: 90-8. 2. zachrisson bu, mjör ia. remodeling of teeth by grinding. am j orthod. 1975; 68: 545-53. 3. ballard ml. asymmetry in tooth size: a factor in the etiology, diagnosis and treatment of malocclusion. angle orthod. 1944; 14: 67-70. 4. neff cw. tailored occlusion with the anterior coefficient. am j orthod. 1949; 35: 309-13. 5. peck h, peck s. an index for assessing tooth shape deviations as applied to the mandibular incisors. am j orthod. 1972; 61: 384-401. 6. germeç d, taner tu. effects of extraction and nonextraction therapy with air-rotor stripping on facial esthetics in postadolescent borderline patients. am j orthod dentofacial orthop. 2008; 133: 539-49. 7. rossouw pe, tortorella a. enamel reduction procedures in orthodontic treatment. j can dent assoc. 2003; 69: 378-83. 8. zachrisson bu, nyoygaard l, mobarak k. dental health assessed more than 10 years after interproximal enamel reduction of mandibular anterior teeth. am j orthod dentofacial orthop. 2007; 131: 162-9. 9. macha ac, vellini-ferreira f, scavone-junior h, ferreira ri. mesiodistal width and proximal enamel thickness of maxillary first bicuspids. braz oral res. 2010; 24: 58-63. 10. stroud jl, buschang ph, goaz pw. sexual dimorphism in mesiodistal dentin and enamel thickness. dentomaxillofac radiol.1994; 23: 169-71. 11. demange c, françois b. measuring and charting interproximal enamel removal. j clin orthod. 1990; 24: 408-12. 12. jarjoura k, gagnon g, nieberg l. caries risk after interproximal enamel reduction. am j orthod dentofacial orthop. 2006; 130: 26-30. 13. fernandes sa, vellini-ferreira f, scavone-junior h, ferreira ri. crown dimensions and proximal enamel thickness of mandibular second bicuspids. braz oral res. 2011; 25: 324-30. 14. stroud jl, english j, buschang ph. enamel thickness of the posterior dentition: its implications for nonextraction treatment. angle orthod. 1998; 68: 141-6. 15. sheridan jj. air rotor stripping. j clin orthod. 1985; 19: 43-59. 16. tuverson dl. anterior interocclusal relations. parts i and ii. am j orthod. 1980; 78: 361-93. 17. doris jm, bernard bw, kuftinec mm, stom d. a biometric study of tooth size and dental crowding. am j orthod. 1981; 79: 326-36. 18. harris af, hicks jd. a radiographic assessment of enamel thickness in human maxillary incisors. arch oral biol. 1998; 43: 825-31. evaluation of proximal enamel thickness and crown measurements in maxillary first premolars 3535353535 braz j oral sci. 11(1):30-35 19. spoor cf, zonneveld fw, macho ga. linear measurements of cortical bone and dental enamel by computed tomography: applications and problems. am j phys anthropol. 1993; 91: 469-84. 20. begg pr. stone age man’s dentition. am j orthod. 1954; 40: 298-312. 21. molnar s, gantt dg. functional implications of primate enamel thickness. am j phys anthropol. 1977; 46: 447-54. 22. macho ga, berner me. enamel thickness of human maxillary molars reconsidered. am j phys anthropol. 1993; 92: 189-200. 23. ghose lj, baghdady vs. analysis of the iraqi dentition: mesiodistal crown diameters of permanent teeth. j dent res. 1979; 58: 1047-54. 24. bishara se, jakobsen jr, abdallah em, fernandez garcia a. comparisons of mesiodistal and buccolingual crown dimensions of the permanent teeth in three populations from egypt, mexico, and the united states. am j orthod dentofacial orthop. 1989; 96: 416-22. 25. chudasama d, sheridan jj. guidelines for contemporary air-rotor stripping. j clin orthod. 2007; 41: 315-20. evaluation of proximal enamel thickness and crown measurements in maxillary first premolars oral sciences n3 original article braz j oral sci. july | september 2015 volume 14, number 3 morse taper internal connection implants: would abutment reseating influence retention? stenio cardoso rabelo1, sheyla viana omonte2, sarita pires vieira2, wellington correa jansen2, paulo isaias seraidarian2 1private practice, divinópolis, mg, brazil 2pontifícia universidade católica de minas gerais – puc minas, school of dentistry, department of restorative dentistry, belo horizonte, mg, brazil correspondence to: paulo isaias seraidarian departamento de odontologia pontifícia universidade católica de minas gerais avenida dom josé gaspar, 500. prédio 46. sala 101. coração eucarístico. cep: 30535-901 belo horizonte, mg, brasil phone: +55 31 3319 4414 fax: +55 31 3319 4414 e-mail: seraidarian@gmail.com abstract aim: to investigate whether the removal and reseating of the abutment influences the retention of morse taper implant system. methods: two morse taper implant systems were selected: bicon dental implanttm system (bicon, llc, boston, ma, usa) and kopp implant® system (kopp®, curitiba, pr, brazil). in both systems, the abutment is connected to the implant with a locking taper. to seat the abutment, the same vertical force was applied in both systems. it was measured the compressive force necessary to engage effectively the locking taper connection and the tensile force to displace it. results: the compressive force was determined by four activations in each abutment-implant set, and the sum of these forces was 21 n and 17 n in the bicontm and kopp® systems, respectively. next, a tensile test was performed, revealing that the bicontm system presented a 208 n resistance, whereas it was 194 n in the kopp® system. other three rounds of compression and tensile loads were applied, removing and reseating the abutment. we obtained the following tensile values: 367 n, 500 n and 756 n in the bicontm system and 336 n, 360 n and 420 n in the kopp® system. conclusions: when the sets were subjected to repeated rounds of compressive and tensile forces, displacing and reseating the abutment, the tensile value increased. keywords: dental implant-abutment design; dental implants; compressive strength; tensile strength. received for publication: may 20, 2015 accepted: september 22, 2015 http://dx.doi.org/10.1590/1677-3225v14n3a07 introduction the search for an optimal implant configuration has led to various internal geometries with different prosthetic platforms1. the goal is to reduce the fatigue on the setting screws and obtain a better biological seal, mechanical strength and aesthetics2-5. one of the main challenges confronting implant dentistry today is to achieve an implant-abutment connection that satisfactorily meets the principles of biomechanics. this difficulty is often due to loosening or even breaking of the fixation screw6-8. screwless implant systems have several advantages, such as the possibility of appropriate cervical region aesthetics and fewer prosthetic components. it leads to cost reduction, simpler clinical procedures, and greater fracture resistance of the prosthetic component3-4,9-10. moreover, the morse cone design platform offers a greater and stronger implant-abutment interface, yielding a better fit between these elements. it allows gap reduction and increased micromovement resistance. it also provides joint stability5 due to the high frictional force created by the locking taper, which is produced by the pressure of two braz j oral sci. 14(3):209-213 210210210210210 sliding surfaces. as a result, surface oxide layers break down, a phenomenon known as cold welding11. therefore, there is less risk of gaps between the implant and abutment, producing a seal proven to delay or even prevent bacterial invasion12-14 and to be clinically reliable15. a recent study on implant components stability demonstrated that loosening in sets with internal tapered connections is less frequent than in sets with other connection types16. although high success rate in morse taper implant systems has been reported3-4,9,15,17-18, implant treatment is often hindered by complications1,19. abutment displacement may occur 9,17. furthermore, removing the abutment–crown complex may be required. this raises the question of whether the reinsertion procedure will jeopardize the system retention. additionally, doubts were raised regarding quantification of forces applied during installation of frictional systems19. it should be noted that in morse taper systems, the manufacturer’s recommendation is to insert, align and then put the abutment in place by seating taps intermittently applied using a weight load. this load is applied to achieve the required mechanical friction between the external wall of the abutment and the internal wall of the implant, which results in high contact pressure1. in screw-retained systems, the screw is tightened with a torque established by the manufacturer19. to the best of our knowledge, in the morse taper system there is no recommendation as to the compression required to obtain optimal imbrication. given the aforementioned considerations, the following questions arise: a) will the intensity of the applied vertical force modify the tensile strength? b) will the removal and repositioning of the abutments influence their retention? the present study addresses the latter. material and methods in this study, two commercial brands of morse taper implant systems, kopp® (kopp®, curitiba, pr, brazil) (figure 1) and bicontm (bicon inc., boston, ma, usa), were analyzed, both with implant-abutment sets accomplished by mechanical and frictional imbrication. they have a 1.23-1.5 degree tapered post that fits into a mirror-image shaft, with no screw. an implant and an f ii straight abutment model were selected from the kopp® company measuring 4.3 x 13 mm and 4.5 x 13 mm, respectively. similarly, an implant and abutment set was selected from the bicon tm company measuring 4.0 x 11 mm and 4.0 x 6.5 mm, respectively (figure 1). for each set, a rigid base was fabricated consisting of a bolt (m 12 x 1.25 mm) with a hole at the top drilled with a mechanical lathe with the appropriate diameter to fit each implant tightly. to obtain the greatest possible resistance, a metal anaerobic adhesive (three bond 1375 threebond of brazil ltda) was used. the sets were fixed individually in a universal testing machine (dl 500 emic equipment and testing systems ltd., pr, brazil). then, they were connected to a load cell (model cce5kn emic) with a capacity of 5000 n at the machine’s lower section. a weight (bcp, kopp®, curitiba, pr, brazil) was used to generate compressive load. this device consisted of a constant-length (0.065 m) metal rod weighing 0.188 kg. it was set in the upper part of the machine in a position concentric to the impact body trajectory to promote the attachment of the abutment to the implant (figure 2). in the first phase of the experiment, the abutment-implant sets were activated four times each by means of compressive force. briefly, a single operator positioned the abutment over the implant without any pressure. next, engagement between the complementary tapered surfaces of implant and abutment was accomplished by the impact of the body weight, dropped from a known height (h=0.065 m), limited by the instrument. the force was applied along the long axis of the implant. in the second phase of the experiment, the abutment of each set was fixed to a forceps shaped device connected to the same load cell, but now in a reversed position, on the upper part of the universal testing machine (figure 3). next, the sets were subjected to a tensile force in the same machine until the abutment-implant assembly was displaced. the tensile force values required for the removal of abutment were recorded. in the last phase of the experiment, the abutments were removed and reseated again three times, summing four reseating/removal cycles. at each displacement of the abutment, the tensile force necessary to remove was recorded, enabling an evaluation of whether the applied tensile force was greater than, equal to, or less than that obtained the first time. thus, it was checked whether the removal and repositioning of the abutments increased the friction and therefore the retention values, or if there was a friction loss and consequent loss of retention. results in the first phase of the experiment, the compressive load applied by the weight (0.188 kg) on the kopp® and bicontm abutment-implant sets was measured by the load cells. the experiment was performed under identical conditions for both brands. there were four activations in fig. 1: kopp® and bicontm implant-abutment sets fixed to a rigid base. morse taper internal connection implants: would abutment reseating influence retention? braz j oral sci. 14(3):209-213 fig. 2: implant-abutment set positioned in the universal testing machine for compressive test. a: weight in position; b: abutment; c: implant; d: load cell. fig. 3: implant-abutment set positioned in the universal testing machine for tensile test. each set and the total results were 17 n for the kopp® set and 21 n for the bicontm set. in the second phase of the experiment, tensile loads were applied under identical conditions on the two implantabutment sets. it was observed that the bicontm system presented a 208 n resistance and the kopp® system, 194 n. in the last experiment, when new compressive and tensile loads were re-applied three times, it was possible to measure the following tensile values: 367 n, 500 n and 756 n for the bicontm system and 336 n, 360 n and 420 n for the kopp® system. discussion this study aimed to investigate whether the removal and reseating of the abutment influences the morse taper implant system retention. high success rate in morse taper implant systems has been reported9,15,17-18. however, an implant treatment may be hampered by complications, abutment displacement may occur9 and removal of the abutment may be necessary. this raises the question of whether the reinsertion procedure will risk the system retention17. the present study showed that the tensile strength was proportional to the seating force applied to the abutment. in the kopp® system implant-abutment set, in which the impact force was lower (17 n), the force to remove the abutment was 194 n. in the bicontm set, with a greater seating force (21 n), the force required to pull out the abutment was 208 n, in the same conditions. these findings are in agreement with the statement that locking taper systems, when inserted into the implant, require a removal force greater than the insertion force9. in the final test, when the abutments were removed and then inserted three more times, tensile strength values of 367 n, 500 n and 756 n were obtained with the bicontm system and 336 n, 360 n and 420 n with the kopp® system. according to these results, bicontm and kopp® implant systems, when subjected to the same compressive loading conditions, had similar behavior. it means that the greater the applied compression, the greater is the tensile strength, although the obtained values were different. previous research has shown a positive correlation between the number of tappings and the removal forces for a locking taper implant system9, which is consistent with our findings. in fact, these results may be explained by the phenomenon of superficial hardening, which is the increased induration of the outer and inner surfaces of the implant and abutment, varying according to the elasticity of each metal alloy. with greater impact force there is a tendency of the abutment to undergo superficial plastic deformation on its platform (figure 4a). additionally, our results can be explained by the phenomenon of the buckling of the abutment. when compressing a rod with a large diameter, a slight inclination along the axis is produced (figure 4b). finally, these results can also be explained by the fact that the tension between the surfaces tends to be asymptotic, due to their angulation, which creates a non-uniform distribution of the compressive stresses and consequently changes the cross-sectional rigidity (figure 4c). therefore, the tensile strength should increase as the compressive force increases. when these phenomena 211211211211211 morse taper internal connection implants: would abutment reseating influence retention? braz j oral sci. 14(3):209-213 212212212212212 fig. 4: schematic drawings: a) superficial hardening; b) buckling; c) asymptotic tension superficial induration. a b c occur, the individual compressive force may not be concentric, generating a variable compressive load. although this increase in surface rigidity could be interesting, cracks may occur, which is an undesirable effect20. it should be mentioned that in vitro study models might not predict the clinical reality. there are many factors influencing in vivo implant components placement and behavior. as a matter of fact, this in vitro study did not consider bending forces, usual in a clinical situation, which may alter the abutment resistance to displacement. furthermore, to the extent of our knowledge only the two systems analyzed in this work use a tapered interference fit solely. thus, it is difficult to compare our data with other researches. in a recent systematic review about conical abutment connection implants, the majority of the morse taper implant system designs included a screw4. nonetheless, this experimental work sheds light on the influence of abutment reseating on the retention of morse taper implant systems. although only one sample of each implant system was used, the mechanical tests revealed a clear tendency with important practical application. in the present study, we observed that higher compressive forces generated higher tensile strength. the results obtained in this study reveal that the removal of the abutment and subsequent repositioning for a total of four repetitions increased the tensile force required to remove the abutment, thereby increasing its frictional retention. within the limits of this in vitro study, the reseating of abutments in locking-taper implants seems to be a successful procedure. further research is necessary to clarify the clinical relevance of our findings, and to establish the optimum compressive force for each commercial brand that uses exclusively the morse taper fixation system to connect implant and abutment. acknowledgements this work was supported by grants from capes, brazil; cnpq, brazil; fundação de amparo à pesquisa do estado de minas gerais (fapemig), brazil. svo is a capes fellow. references 1. pita ms, anchieta rb, barão va, garcia ir, pedrazzi v, assunção wg. prosthetic platforms in implant dentistry. j craniofac surg. 2011; 22: 2327-31. 2. sannino g, barlattani a. mechanical evaluation of an implant-abutment self-locking taper connection: finite element analysis and experimental tests. int j oral maxillofac implants. 2013; 28: e17-26. 3. mangano f, macchi a, caprioglio a, sammons rl, piattelli a, mangano c. survival and complication rates of fixed restorations supported by locking-taper implants: a prospective study with 1 to 10 years of follow-up. j prosthodont. 2014; 23: 434-44. 4. schmitt cm, nogueira-filho g, tenenbaum hc, lai jy, brito c, döring h, et al. performance of conical abutment (morse taper) connection implants: a systematic review. j biomed mater res a. 2014; 102: 552-74. 5. yamanishi y, yamaguchi s, imazato s, nakano t, yatani h. influences of implant neck design and implant-abutment joint type on peri-implant bone stress and abutment micromovement: three-dimensional finite element analysis. dent mater. 2012; 28: 1126-33. 6. wittneben jg, buser d, salvi ge, bürgin w, hicklin s, brägger u. complication and failure rates with implant-supported fixed dental prostheses and single crowns: a 10-year retrospective study. clin implant dent relat res. 2014; 16: 356-64. 7. junqueira mc, ribeiro rf, faria acl, macedo ap, almeida rp. screw loosening of different ucla-type abutments after mechanical cycling. braz j oral sci. 2013; 12(3): 228-32. 8. jung re, zembic a, pjetursson be, zwahlen m, thoma ds. systematic review of the survival rate and the incidence of biological, technical, and aesthetic complications of single crowns on implants reported in longitudinal studies with a mean follow-up of 5 years. clin oral implants res. 2012; 23(suppl 6): 2-21. 9. chapman rj, grippo w. the locking taper attachment for implant abutments: use and reliability. implant dent. 1996; 5: 257-61. 10. shepherd nj. affordable implant prosthetics using a screwless implant system. j am dent assoc. 1998; 129: 1732-38. 11. keating k. connecting abutments to dental implants: “an engineer’s perspective”. irish dent. 2001: 43-6. 12. assenza b, tripodi d, scarano a, perrotti v, piattelli a, iezzi g, et al. bacterial leakage in implants with different implant-abutment connections: an in vitro study. j periodontol. 2012; 83: 491-7. 13. silva-neto jp, majadas mff, prudente ms, carneiro tapn, penatti mpa, neves fd. bacterial microleakage at the implant-abutment interface in morse taper implants. braz j oral sci. 2014; 13: 89-92. 14. do nascimento c, miani pk, pedrazzi v, gonçalves rb, ribeiro rf, faria ac, et al. leakage of saliva through the implant-abutment interface: in vitro evaluation of three different implant connections under unloaded and loaded conditions. int j oral maxillofac implants 2012; 27: 551-60. morse taper internal connection implants: would abutment reseating influence retention? braz j oral sci. 14(3):209-213 213213213213213 15. nentwig gh. ankylos implant system: concept and clinical application. j oral implantol. 2004; 30: 171-7. 16. feitosa pc, de lima ap, silva-concílio lr, brandt wc, neves ac. stability of external and internal implant connections after a fatigue test. eur j dent. 2013; 7: 267-71. 17. urdaneta ra, marincola m, weed m, chuang sk. a screwless and cementless technique for the restoration of single-tooth implants: a retrospective cohort study. j prosthodont. 2008; 17: 562-71. 18. norton mr. an in vitro evaluation of the strength of an internal conical interface compared to a butt joint interface in implant design. clin oral implants res.1997; 8: 290-8. 19. zielak jc, rorbacker m, gomes r, yamashita c, gonzaga cc, giovanni af. in vitro evaluation of the removal force of abutments in frictional dental implants. j oral implantol. 2011; 37: 519-23. 20. gere jm, timoshenko sp. mechanics of materials. boston: pws pub co.; 1997. morse taper internal connection implants: would abutment reseating influence retention? braz j oral sci. 14(3):209-213 revista fop n 13 braz j oral sci. april/june 2008 vol. 7 number 25 1531 phenotypic characterization of phenotypic characterization of phenotypic characterization of phenotypic characterization of phenotypic characterization of candida candida candida candida candida spp. isolatesspp. isolatesspp. isolatesspp. isolatesspp. isolates from chronic periodontitis patientsfrom chronic periodontitis patientsfrom chronic periodontitis patientsfrom chronic periodontitis patientsfrom chronic periodontitis patients cristiane yumi koga-ito1; edson yukio komiyama2; clélia aparecida de paiva martins2; silvana soléo ferreira dos santos3; ivan balducci4; antonio olavo cardoso jorge1 1phd in microbiology and immunology, department of oral biosciences and diagnosis, dental school of são josé dos campos, são paulo state university, brazil; 2msc in microbiology and immunology, department of oral biosciences and diagnosis, dental school of são josé dos campos, são paulo state university, brazil; 3phd in oral biopathology, basic institute of biosciences, university of taubaté, brazil; 4msc in biostatistics, department of social dentistry, dental school, são josé dos campos, são paulo state university, brazil. received for publication: september 05, 2007 accepted: may 30, 2008 a b s t r a c t aim: several typing methods for candida spp. have been suggested in the literature in order to distinguish isolates for studies about the virulence or infection routes of these microorganisms and, in particular, for epidemiological purposes. the aim of this study was to establish a comparison between the phenotypic profile of oral candida isolates from periodontitis patients and control individuals. methods: the morphotyping and biotyping of 35 c. albicans isolates obtained from chronic periodontitis patients and 48 isolates from control individuals were performed. for morphotyping, the isolates were plated on malt extract agar and incubated for 10 days. sixteen different morphotypes were observed for c. albicans, the most frequently observed being 0000 and 0001. results: biotype 0000 (complete absence of fringe) was most prevalent among the isolates obtained from periodontitis patients compared to those from control individuals, with statistical significance. biotyping revealed 5 different biotypes with higher prevalence of the biotype 357 among the isolates from control and periodontitis groups. conclusions: the results obtained by biotyping of the isolates did not permit to differentiate a characteristic model related to periodontal disease, whilst the morphotype 0000 was most frequently isolated from periodontitis patients. key words: periodontitis, candida albicans, biotyping, morphotyping i n t r o d u c t i o n candida spp. have been correlated to cases of severe and refractory periodontal infections, particularly in immunocompromised patients or individuals under antimicrobial therapy for long periods1-5. several candida spp. typing methods have been suggested in the literature in order to distinguish isolates for studies about the virulence or infection routes of these microorganisms and, in particular, for epidemiological purposes6-10. among the methods based on phenotypic characteristics, serotyping, biotyping, morphotyping and sensitivity to killer toxins are included7. there are also methods based on genotypic characteristics, such as immunobloting11 and dna fingerprinting techniques12. morphotyping is considered an efficient, reproducible and low cost method for c. albicans characterization7,13. moreover this method reveals a good discriminatory power. the variation in the morphology of candida colonies was firstly observed by negroni14. later, based on brownthomsen’s16 observations about the morphological variations of colonies due to alterations in the incubation temperature or medium composition, phongpaichit et al.15 proposed a typing method with codes. this coding system was based on the characteristics of the colonies as well as on their surfaces. hunter et al.17, studying the morphotype distribution among 446 c. albicans strains, suggested a typing method based in the characteristics of colony surfaces, represented by 4 digits. the phenotypic switching and candida morphotypes have been associated to their virulence. different phenotypic expressions in relation to colony growth are related to variations that are considered responsible for the several degrees of virulence7,18. phenotypic switching occurs frequently and causes changes in colony morphology and cell surface properties, such as alteration in the adherence to epithelial cells19. correspondence to: cristiane yumi koga-ito faculdade de odontologia de são josé dos campos/unesp av. eng. francisco josé longo, 777, 12245-000 são josé dos campos, sp, brasil phone: +55-12-3947-9033. e-mail: cristianeykito@directnet.com.br 1532 braz j oral sci. 7(25):1531-1534 phenotypic characterization of candida spp. isolates from chronic periodontitis patients the biotyping technique proposed by odds and abbott20 has been reported as a method with an adequate discriminatory power mainly when associated with the morphotyping method21. considering the adequate discriminatory power of the combination of morphotyping and biotyping methods, the purpose of this study was to establish a comparison between the phenotypic profile of oral candida isolates from periodontitis patients and control individuals, aiming to correlate specific phenotypic features to the occurrence of periodontal disease. material and methods this research project has been independently reviewed and approved by the bioethics research committee of the dental school of são josé dos campos, são paulo state university, brazil (protocol #72/99-ph/cep). candida albicans isolates from chronic periodontitis (n=35) and control individuals (n=48) were included in this study. these strains were previously isolated and belonged to the strain collection of the microbiology laboratory at the dental school of são josé dos campos, são paulo state university. isolates from chronic periodontitis were obtained from 88 individuals aged 25 and 62 years (mean age of 41.33 ± 5.54 years), with at least two 5-mm deep periodontal sites and diagnosed clinically as chronic periodontitis patients. control group isolates were obtained from 68 individuals aged 25 to 55 years (mean age of 34.45 ± 7.93 years) diagnosed as periodontally healthy patients. candida isolates were morphotyped according to the methodology proposed by hunter et al.7 and pongpaichit et al 15. briefly, the isolates were plated on sabouraud dextrose agar and incubated at 25ºc for 48 h. then, yeast suspensions were prepared in sterile distilled water adjusted to the turbidity of tube #3 of mcfarland scale. using sterile swabs, the suspensions were plated on the surface of malt extract agar (difco, detroit, mi, usa). plates were maintained at room temperature in a light-proof environment for 10 days. after this period, the macromorphological aspects of the fringes and surface of the colonies were evaluated. the results of morphotyping were recorded using 4-digit codes. biotyping methods were performed according to odds and abbot22, with a combination of tests of tolerance (to ph 1.4 and nacl), resistance (5-fluorocytosine, safranine and boric acid), enzymatic activity (proteinase) and growth in presence of urea, sorbose and citrate. the isolates were plated on sabouraud dextrose agar and incubated at 37ºc for 24 h. then, saline suspensions adjusted to the turbidity of tube #3 of mcfarland scale were obtained. aliquots of 100 µl of these saline suspensions were deposited in the wells of a steers’ inoculator and plated in the test culture media and the positive control (sabouraud agar). all tests were performed in triplicate. plates were incubated at 37ºc for different periods of time (3 to 4 days for control, safranine, urea, citrate, boric acid and sodium chloride; 6 to 7 days to resistance to ph 1.4 and tests of sorbose, proteinase and 5fluorocytosine). positive tests were considered when the strains grew at ph 1.4, 5-fluorocytosine, sodium chloride, boric acid, urea, sorbose and sodium citrate, as well as for the strains that formed colonies with diameter grater than 2 mm in presence of safranine. the results of biotyping were recorded using 3-digit codes, according to odds and abbott22. values of 1, 2, 3 and 4 were attributed to the positive tests and 0 to the negative tests. statistical analysis the data regarding the occurrence of the morphotypes and biotypes were compared between the periodontitis and control groups by two proportion z test at 5% significance level. r e s u l t s the morphotype 0000 was the most frequently observed (50%) among the oral isolates from control individuals. the morphotype 0001 was observed in 12.5% of the isolates. the morphotypes 1232, 7240, 0004, 2331, 1230 and 7520 were also observed (table 1). among the isolates from chronic periodontitis patients, the morphotype 0000 was also the most frequently found (77.14%) differing significantly from the control group (p=0.007) (figure 1). other 7 morphotypes were observed among the isolates of this group (table 1). biotypes 357 and 757 were the most frequently observed in both groups without statistically significant differences (p=0.522) (table 2). d i s c u s s i o n candida genus yeasts are considered opportunistic microorganisms23,24 and may lead to severe periodontal infections in immunocompromised patients or individuals under antimicrobial therapy for long periods1. under specific situations, such as in immunodepressed patients25, superinfection by candida can be refractory to conventional periodontal treatments. in fact, c. albicans presents virulence factors that can have an important role in the pathogenesis of periodontal disease such as the ability of penetrating the epithelium, inhibiting polymorphonuclear cells and causing lysis of monocytes8,26. also, lu et al.27 have described the hyphal invasion capacity of c. albicans to inhibit the production of antimicrobial peptides by oral epithelium. candida species have been isolated from the subgingival microbiota of the gingival tissues of patients with periodontal abscesses1, periodontitis28,29, and patients with chronic periodontitis under therapy with antibiotics17. morphotyping has been employed in epidemiological and virulence studies. ribeiro et al.30 studying children with down’s syndrome showed that the c. albicans isolates from these patients induced more formation of fringes compared to the isolates obtained from control individuals, and the authors suggested a correlation with increased virulence. in fact, hunter et al.17 had previously suggested a correlation 1533 morphotype n % 0000 24 50.0 7340 1 2.1 7540 1 2.1 5340 2 4.1 0001 6 12.5 1040 1 2.1 1240 2 4.2 7520 1 2.1 7320 1 2.1 7330 1 2.1 7531 1 2.1 0002 2 4.1 0006 2 4.1 1240 1 2.1 7321 1 2.1 3340 1 2.1 0000 27 77.14 1232 1 2.86 7240 2 5.70 0004 1 2.86 2331 1 2.86 1230 1 2.86 7520 1 2.86 7540 1 2.86 control group (n=48) periodontitis group (n=35) table 1 distribution of the morphotypes observed among candida albicans isolates from control individuals and periodontitis patients. control group (n=48) biotype n % 357 36 75.0 757 9 18.7 157 1 2.1 313 1 2.1 353 1 2.1 periodontitis group (n=35) 357 24 68.6 757 5 14.2 777 3 8.6 377 2 5.8 577 1 2.8 table 2 distribution of biotypes observed among candida albicans isolates from control individuals and periodontitis patients fig. 1 statistical comparison of the morphotype 0000 and biotype 357 occurrence in the studied groups. control periodontitis control periodontitis 0 10 20 30 40 50 60 70 80 90 100 biotype 357 z = 2.86; p = 0.007 <0.05 (ci, 95%: 7 .30 to 46 .98) z = 0 .64; p = 0.522>0.05 (ci, 95%: -13 .23 to 26 .09%) morphotype 0000 % between invasive infections and isolates with discontinued fringes. the increased virulence of isolates with fringes was also related by spadari et al.31. these authors observed a higher degree of adherence in these isolates compared to strains without fringes. in the present study, this tendency was not observed, as similar proportion of isolates in the groups showed discontinued fringes. four isolates from the braz j oral sci. 7(25):1531-1534 phenotypic characterization of candida spp. isolates from chronic periodontitis patients control group (8.33%) and 3 from the periodontitis group (8.57%) presented discontinued fringes. these results suggest that no correlation between colonies with discontinued fringes and periodontitis occurrence could be done. the morphotype 0000 (complete absence of fringes) was the most frequently isolated from the oral cavity of control and periodontitis patients, and this result corroborates the absence of correlation between the presence of discontinuous fringes, which is associated to filamentation, and periodontitis occurrence. previous studies6,17,32 also observed the higher incidence of this morphotype among isolates from the oral cavity. this result may suggest that the mycelial form formation might not be an essential feature of candida in the periodontal milieu, and other factors (i.e. proteolytic enzymes and toxins production) might be more important contributors to the pathogenesis of this disease. for more detailed data, further studies on these features could be useful. also, studies on genotyping could provide important results. in fact, several authors have pointed out the importance of combining phenotyping and genotyping methods6,9,33,34. hunter et al.7 have demonstrated the occurrence of the morphotypes 7540 and 7340 among isolates from several sites of the body. these morphotypes were also observed among the isolates of the present study. the great variability of morphotypes in the control group (16 models) has been reported elsewhere17. ribeiro et al.29 did not observe the occurrence of morphotype 0000 and found a high prevalence of morphotype 5530 in down’s syndrome patients. the biotyping method proposed by odds and abbott20 has been considered as a method with adequate discriminatory power. however, in the present study, it was not able to distinguish between periodontitis and control isolates. biotyping permitted the identification of 5 different biotypes among the isolates from the oral cavity of control individuals. the biotypes 357 and 757 were the most frequently observed (75% e 18.8%, respectively). among 1534 braz j oral sci. 7(25):1531-1534 phenotypic characterization of candida spp. isolates from chronic periodontitis patients the isolates from periodontitis patients, the biotypes 357, 757, 777, 377 and 577, and the model 357 (68.6%) were the most frequently observed. in the present study, most isolates assimilated urea and citrate and did not assimilate sorbose. also, these isolates were tolerant to salt and developed in presence of boric acid and safranine. neely et al.35 also verified a high prevalence of isolates with these features among candida isolates from children. in conclusion, the results obtained by biotyping of the isolates did not allow distinguishing a characteristic model related to periodontal disease, whilst the morphotype 0000 was most frequently isolated from periodontitis patients. r e f e r e n c e s 1. hevoluo h, hakkarainen k, paunio k. changes in the prevalence of subgingival enteric rods, staphylococci and yeasts after treatment with penicillin and erythromycin. oral microbiol immunol 1993;8:75-9. 2. slots j, feik d, rams te. age and sex relationshisp of superinfecting microorganisms in periodontitis patients. oral microbiol. immunol 1990; 5:305-8. 3. oliveira lf, jorge aoc, santos s s. in vitro minocycline activity on superinfecting microorganisms isolated from chronic periodontitis patients. braz oral res 2006; 20:202-6. 4. dáhlen g. microbiological diagnostics in oral diseases. acta odontol scand 2006; 64: 164-8. 5. ito cy, de paiva martins ca, loberto jc, dos santos ss, jorge aoc. in vitro antifungal susceptibility of candida spp. isolated from patients with chronic periodontitis and from control patients. braz oral res 2004; 18: 80-4. 6. candido rc. candida albicans : marcadores epidemiológicos em amostras isoladas de diferentes materiais biológicos. são paulo, 1991, 167p. (ph. d. thesis. escola paulista de medicina. epm). 7. hunter pr. a critical review of typing methods for candida albicans and their applications. crit rev microbiol 1991; 17:417-34. 8. pfaller m.a. epidemiological typing methods of mycoses. clin infect disc 1992; 14(suppl. 1): s4-10. 9. maffei cm, paula cr, mazzocato cs, franceschini s. phenotype and genotype of candida albicans strains isolated from pregnant women with recurrent vaginitis. mycopathologia 1997; 137:87-94. 10. manfredi m, mccullough mj, al-karaawi zm, vescosi p, porter sr. in vitro evaluation of virulence attributes of candida spp. isolated from patients affected by diabetes mellitus. oral microbiol immunol 2006; 21:181-9. 11. lee w, burnie j, matthews r. fingerprinting candida albicans. j immunol meth 1986; 93: 177-82. 12. scherer s, stevens da. aplication of dna typing methods to epidemiology and taxonomy of candida species. j clin microbiol 1987; 25: 675-9. 13. giammanco g m, lopes m.m, coimbra rs, pignato s, grimont pad, grimont f et al. value of morphotyping for the characterization of candida albicans clinical isolates. mem instit oswaldo cruz.2005; 100: 483-90. 14. negroni p. variacion hacia el tipo r de mycotorula albicans. rev soc arg biol 1935; 11: 449. 15. phongpaichit s, mackenzie dwr, fraser c. strain differentiation of candida albicans by morphotyping. epidemiol infect 1987; 99: 421-8. 16. brown-thomsen j. variability in candida albicans (robin) berkhout. i. studies in morphology and biological activity. hereditas 1968; 60: 355-98. 17. hunter pr, fraser cam. application of a numerical index of discriminatory power to a comparison of four physiochemical typing methods for candida albicans. j clinmicrobiol 1989; 27: 2156-60. 18. o’connell b, coleman dc, bennett d, sullivan d, mc cann sr et al. an epidemiological study of candida species infection in cancer patients using genetic fingerprinting and morphotyping. j hosp infect 1995; 31: 211-7. 19. mattews rc. pathogenicity determinants of candida albicans: potencial targets for immunotherapy. microbiol 1994; 140: 1505-11. 20. odds fc, abbott ab. modification and extension of tests for differentiation of candida albicans species and strains. sabouraudia 1983; 21: 79-81. 21. otero l, vásquez l, palacio v, vásquez s, carreño f, méndez fj. comparison of seven phenotyping methods for candida albicans. eur j epidemiol 1995; 11: 221-224. 22. odds fc, abbott ab. a simple system for the presumptive identification of candida albicans and differentiation of strains within the species. sabouradia 1980; 18: 301-317. 23. arendorf tm, walker dm. candida albicans: its association with dentures, plaque and oral mucosa. j dasa 1980; 35: 563-569. 24. leung kw, dassanayake rs, yau jyy, jin lj, yam wc, samaranayake l i. oral colonization, phenotypic, and genotypic profiles of candida species in irradiated, dentate, xerostomic nasopharyngeal carcinoma survivors. j clin microbiol 2000; 38:2219-26. 25. brawner dl, cutler je. oral candida albicans isolates from nonhospitalized normal carriers, immunocompetent hospitalized patients, and immunocompromised patients with or without acquired immunodeficiency syndrome. j clin microbiol 1989; 27: 1335-41. 26. barret-bee ky, wilson rg, ryley jf. a comparison of phosphoslipase activitiy cellular adherence and pathogenicity of yeasts. j gen microbiol 1985; 131: 1217-21. 27. lu q, jayatilake jams, samaranayake lp, jin l. hyphal invasion of candida albicans inhibits the expression of human â-defensins in experimental oral candidiasis. j investig dermatol 2006; 126:2049-56. 28. del castilho l, bikandi j, nieto a, quindós g, sentandreu r, pontón j. comparison of morphortypic and genotypic methods for strain delineation in candida. mycoses 1997; 40: 445-59. 29. slots j, rams te, listgarten ma. yeasts, enteric rods and pseudomonas in the subgingival flora of severe adult periodontitis. oral microbiol immunol 1988; 3: 47-52. 30. ribeiro el, scroferneker ml, cavalilaes ms, campos cc, nagato gm, souza na et al. phenotypic aspects of oral strains of candida albicans in children with down’s syndrome. braz j biol 2006; 66: 939-44. 31. spadari e, arosio v, malighetti v, bellotti mg, zambelini a, spadari f, et al. morphotipi de candida albicans e adesività in vitro a cellulle della mucosa orale di pazienti hiv-positivi e com aids dopo esposizione delle blastopore al fluconazolo. parte ii. minerva stomatol. 1998; 47: 293-7. 32. barreto de oliveira mt. leveduras isoladas da mucosa bucal de portadores sadios, pacientes com sida e neoplasias: produção de exoenzimas e tipagem das amostras de candida albicans. são paulo, 1993, 107p. (master’s degree dissertation. institute of biomedical sciences, usp). 33. betremieux p, chevrier s, quindós g, sullivan d, polonelli l, guiguen c. use of dna fingerprinting and biotyping methods to study a candida spp. outbreak in neonatal intensive can unit. pediatr infect dis j 1994; 13: 899-905. 34. maffei cml, paula cr, mazoocato ts, franceschini s. phenotype and genotype of candida albicans strains isolated from pregnant women with recurrent vaginitis. mycopathol 1997; 137: 87-94. 35. neely an, odds fc, basatia bk, holder ia. characterization of candida isolates from pediatric burn patients. j clin microbiol 1988; 26: 1645-9. oral sciences n3 original article braz j oral sci. july/september 2009 volume 8, number 3 cast metal core adaptation using two impression materials and intracanal techniques sônia maria lemos brancato camarinha1, luiz carlos pardini2, lucas da fonseca roberti garcia3, simonides consani4, fernanda de carvalho panzeri pires-de-souza5 1dds, ms, graduate student, department of dental materials and prosthodontics, ribeirão preto dental school, university of são paulo, brazil 2 dds, ms, phd, associate professor, department of morphology, stomatology and physiology, ribeirão preto dental school, university of são paulo, brazil 3 dds, ms, graduate student, department of restorative dentistry, dental materials area, piracicaba dental school, state university of campinas, brazil 4 dds, ms, phd, full professor, department of restorative dentistry, dental materials area, piracicaba dental school, state university of campinas, brazil 5dds, ms, phd, associate professor, depar tment of dental materials and prosthodontics, ribeirão preto dental school, university of são paulo, brazil received for publication: may 22, 2009 accepted: september 9, 2009 correspondence to: lucas da fonseca roberti garcia rua bernardino de campos, 30 apto. 1002 higienópolis 14015-130 ribeirão preto, sp, brasil phone: +55-16-3964-6910/ +55-16-9796-0776. e-mail: drlucas.garcia@gmail.com abstract aim: this study evaluated the adaptation of cast metal cores (cmcs) using two impression materials and two intracanal impression techniques. methods: ten single-rooted human teeth had their root canals prepared to receive a cmc. intracanal impressions were obtained with addition and condensation silicone materials using two types of intraradicular supports: hypodermic needle or prefabricated polycarbonate posts. four impressions were obtained from each tooth totalizing 40 models, on which cmcs were prepared. cmc adaptation to the canal was analyzed radiographically and microscopically. the teeth were radiographed with the cmc positioned and the images were analyzed using an image-analysis software (imagelab). in the second analysis, the teeth were sectioned longitudinally and the cmc adaptation was evaluated with a two-coordinate microscope. in both analyses, cmc adaptation to the canal was determined by calculating the percentage of the prepared canal space that was occupied by the core. results: statistically significant difference (2-way anova bonferroni; p<0.05) was found between the mean values cmc adaptation obtained with the two intraradicular supports. conclusions: the intracanal impression technique using hypodermic needle allowed a more accurate reproduction of the prepared canal space and provided significantly better cmc adaptation when the cores were obtained from addition silicone impressions. keywords: impression technique, cast posts, silicone elastomers, intraradicular support. introduction endodontically treated teeth are usually weakened due to the loss of dental structure by caries, cavity preparation, root canal instrumentation and decrease of dentin moisture, becoming more susceptible to fractures1. the restoration of an endodontically treated tooth should reestablish its form and function, creating resources for anchorage of the restorative material that are capable to avoid displacement and promote an adequate distribution of the forces acting on the restoration2-3. the construction of a cast metal core (cmc) is one of the possible treatment options to increase tooth resistance to fracture and serve as a support for a subsequent prosthetic restorative treatment4. the best possible core adaptation to the root canal should always be pursued because it is a critical factor for achieving an optimal retentive and biomechanical behavior of the fixed denture5. direct intracanal impression for cmc fabrication is a very time-consuming procedure because the dentist needs to have direct access to the root canal during all phases of preparation, adjustment and finishing/polishing of the core6. indirect intracanal impression for cmc fabrication may thus be a viable and time-saving option. in this technique, impression of the root canal is obtained with an elastomeric impression material. a fundamental requirement for a correct cmc adaptation is the accurate reproduction of the prepared canal space shape in all of its braz j oral sci. 8(3):128-131 details in order to distribute forces uniformly within the root7. the success of all subsequent operative steps depends directly on the production of a precise mould8. the use of appropriate impression materials and correct molding techniques reduces considerably the possibility of fails in the molds8. among the elastomeric materials, silicones have as advantages their ease of handling and lack of significant alterations at room temperature8-9. however, byproducts (e.g.: ethyl alcohol) formed in the condensation silicone reaction promotes a slow and small contraction due to its evaporation8-9. addition silicones are considered superior compared to others elastomeric materials because they do not have byproduct formation, which reduces the polymerization shrinkage of the material 10, promoting higher dimensional stability and an appropriate resistance to rupture8-10. due to the anatomic characteristics of the root canal system, the precise reproduction of the end of the prepared canal is difficult, which may result in a cmc with inadequate length and a high degree of distortion from the mould11. therefore, the development of impression techniques that may increase the quality of reproduction and hence improve cmc adaptation to the prepared root canal is necessary 12. thus, the purpose of this study was to compare radiographically and microscopically the adaptation of cmcs fabricated from intracanal impressions obtained using an indirect double-impression procedure with two elastomeric impression materials (addition and condensation silicone) and two intraradicular supports (polycarbonate posts and hypodermic needle). the tested hypothesis was that the use of a hypodermic needle as an intraradicular support allows for the air exiting the canal during injection of the light body impression material promoting a better material flow and more accurate reproduction of the canal space. material and methods this study was conducted after approval of the research protocol by the research ethics committee of ribeirão preto dental school, university of são paulo, brazil (process # 2005.1.1204.58.7). ten single-rooted human teeth were selected from a random collection and were checked for absence of root caries, cracks and structural defects. soft debris was removed with hand curettes. initially, the teeth were radiographed using f-speed periapical x-ray films (insight, eastman kodak company, rochester, ny, usa) with a heliodent 60b x-ray device (siemens, erlangen, germany) operated at 60 kvp and 10 ma, with 0.16 s exposure time, total filtration equivalent to 2 mm al and 18 cm focus-film distance. all films were labeled with lead letters and numbers (konex, são paulo, sp, brazil) and all teeth were radiographed near a 10x32mm aluminum stepwedge with variable thickness ( from 2 to 16 mm, in 2 mm increments) in order to simulate the densities of the oral structures compared to that of the soft and hard tissues, and hence detect possible variations during the radiological procedures. crowns were removed at the cementoenamel junction with a water spray-cooled double-faced diamond disc (kg sorensen, são paulo, sp, brazil). the root canals were instrumented with a series of k-files (dentsply/tulsa dental, tulsa, ok, usa) according to a stepback technique, being irrigated with 0.5 ml of 2.5% sodium hypochlorite at each change of instrument. the canals were obturated by cold lateral compaction of gutta-percha and a zinc-oxide-eugenol-based sealer (endofill, dentsply indústria e comércio ltda., petrópolis, rj, brazil), and the root-filled teeth were stored in distilled water at 37oc during 48 h for complete setting of the sealer. after this period, 2/3 of the filling material was removed with peeso burs (fkg dentaire, la chaux-de-fonds, switzerland) mounted in a low-speed handpiece (dabi atlant e, ribeirão p reto, sp, brazil). in o rder to warrant standardization of the amount of filling material to be removed from each canal, the working length of all roots was measured with a digital caliper (absolute, mitutoyo, são paulo, sp, brazil). next, the roots were radiographed again in the same way as described above. using an indirect double-impression procedure, the prepared root canals were molded with either addition-cure silicone (polyvinyl siloxane adsil; vigodent s/a indústria e comércio, rio de janeiro, rj, brazil) or condensation-cure silicone (speedex; vigodent s/a indústria e comércio) impression materials using two types of intraradicular supports: 0.70 x 30 mm hypodermic needles or prefabricated polycarbonate posts designed for root canal impression (pin-jet; angelus indústria de produtos odontológicos ltda, londrina, pr, brazil). four impressions were obtained from each tooth (one for each type of impression material and intraradicular support) totalizing 40 impressions that were allocated to 4 groups (n=10). an intracanal impression protocol was developed for this study using a delineator. a partial impression tray was adapted to the vertical shaft of the delineator in such a way that it could be loaded with impression material always following the same insertion and removal path. another partial impression tray with a central hole for attachment of the teeth was loaded with autopolymerizing acrylic resin (artigos odontológi cos clássico, são paulo, sp, brazil) and fixed at the delineator’s b ase. two 1-cm-thick resin cylinders fixed at the delineator’s base served as stoppers that limited the insertion of the tray to a standardized depth. manufacturers mixing instructions were followed for both impression materials. a 380 g load was applied during the impression procedures and maintained until complete setting of the materials in order to standardize the force applied to the trays. the double-impression procedure can be summarized as follows. first, a layer of #7 wax (herpo produtos dentários ltda, rio de janeiro, rj, brazil) was applied on the tooth to be molded and an acrylic resin layer (artigos odontológicos clássico) was applied onto the wax. after polymerization, the wax was removed and the acrylic resin was adapted on the tooth to serve as a relief for the intracanal impression with the light body material. an initial impression was obtained with the heavy body material, with care to avoid material flowing into the canal. the acrylic relief was removed together with the heavy body material and the intraradicular support was positioned loosely inside the root canal. next, the light body material was injected into the canal with a syringe in a sufficient amount to fill it completely and reach the working length. after complete setting, the material/ intraradicular support set was removed in a single movement, obtaining a final mould. all moulds were poured with type iv dental stone (durone, dentsply indústria e comércio ltda.), copying the area corresponding to the prepared canal space. acrylic resin cores (duralay, reliance dental mfg company, worth iii, usa) were fabricated on the stone models according to the brush technique described by nealon13, for further inclusion (termocast investment; polidental, cotia, sp, brazil) and casting with cu-al alloy (aje goldent comercial ltda., são paulo, sp, brazil). cast metal core adaptation using two impression materials and intracanal techniques 129 braz j oral sci. 8(3):128-131 the accuracy of the intracanal impression techniques was analyzed radiographically and microscopically. for the radiographic analysis, the roots were radiographed with the cmc in position using the same intraoral film, x-ray equipment and parameters described before. the radiographs were digitized and analyzed using imagelab image-analysis software (softium, são paulo, sp, brazil). for the microscopic analysis, the teeth were embedded in acrylic resin, sectioned longitudinally using a digital precision low-speed diamond saw (syj-150, mti corp., richmond, ca, usa) and the cmc was fitted to the canal and examined with a two-coordinate microscope (nikon measurescope; nippon kogaku k.k., chiyoda-ku, tokyo, japan). in both analyses, cmc adaptation to the canal was determined by calculating the percentage of the prepared canal space that was occupied by the core. in the radiographic analysis, the length of the prepared canal space ( from the coronal third up to the beginning of the residual filling material was initially measured and then the coronal-apical length of the core was measured in the same image. in the microscopic analysis, canal space and core length were measured separately (figure 1). data were subjected to statistical analysis by 2-way anova and bonferroni correction at 5% significance level. figure 1. distance between the apical portion of the cmc and the residual filling material, as observed in the microscopic analysis. results the data obtained from the radiographic and microscopic analyses are graphically presented in figures 2 and 3. similar results were obtained in both analyses. the cmc fabricated from both addition and condensation silicone impressions using hypodermic needles as intraradicular supports presented significantly higher filling percent values, which indicate a better cmc adaptation to the prepared canal (p<0.05). comparing the impression materials (figure 2), there was no statistically significant difference (p>0.05) in the percentage of prepared canal space occupied by the cmc regardless of the type of intraradicular support. comparing the techniques, the use of hypodermic needle as an intraradicular support produced significantly better (p<0.05) cmc adaptation to the canal when addition silicone was the impression material . for the condensation silicone, no figure 3. data from microscopic analysis. percentage of the root canal occupied by the cmc with the intracanal impression techniques (hypodermic needle or polycarbonate post) using either addition or condensation silicone. horizontal lines above the columns indicate results without statistical significance (p>0.05). different letters comparing the same material indicate statistically significant difference (p<0.05). figure 2. data from radiographic analysis. percentage of the root canal occupied by the cmc with the intracanal impression techniques (hypodermic needle or polycarbonate post) using either addition or condensation silicone. horizontal lines above the columns indicate results without statistical significance (p>0.05). different letters comparing the same material indicate statistically significant difference (p<0.05). statistically significant difference (p>0.05) was observed between the impression techniques. discussion the use of adequate impression material techniques reduces significantly the likelihood of inaccuracies, such as cmc misfit, during intracanal impression procedures8. starting from the first impression of the canal, small distortions are successively incorporated during all subsequent stages of up to the definitive installation of the prosthesis14. the challenge is to determine a minimum level of distortion and associated stress that is acceptable and that confers adequate clinical longevity to the prosthesis. impression materials and techniques that are able to reproduce canal anatomy with a high level of reliability are expected to allow the fabrication of prosthetic pieces with the least possible misfit to the canal8. the present in vitro study was designed to analyze radiographically and microscopically the adaptation of cmcs to the root canal of extracted human teeth. endodontically treated human teeth were used to simulate the clinical conditions as close as possible15-17. the radiographic technique is the most suitable for evaluation of cmc cast metal core adaptation using two impression materials and intracanal techniques130 braz j oral sci. 8(3):128-131 adaptation because, in clinical practice, a radiographic image is obtained to certify that the cmc is well adapted, occupying the whole extension of the root canal18. however, due to the limitation imposed by the x-ray diffraction, such as the superposition of structure images and distortions18, the microscopic analysis of the cmc adaptation was also performed in the present study. periapical radiographs are the most commonly used in dental offices to ensure that the core is intimately fitted and occupies the whole extent of the prepared canal space18-19. in the present study, both radiographic and microscopic analyses showed that the intracanal impression technique combining addition silicone and hypodermic needle produced a more accurate reproduction of the canal space as demonstrated by fabrication of cmcs with better adaptation to the canals. when the condensation silicone was used, the intraradicular supports had a similar performance with no statistical significance in cmc adaptation. the difference between the intracanal impression techniques when addition silicone was used can be explained by the highest flowability of the light-body paste, in addition to its capacity of offering better reproduction of details than the condensation silicone810,20. condensation silicones are more susceptible to fail, irrespective of the molding technique. according to stackhouse jr20, the lack of space for adequate flow of the light-body impression material induces the formation of an internal pressure during the impression procedure that leads to air entrapment into the root canal. it is assumed that this phenomenon occurred in the present study in the technique that used polycarbonate posts as intraradicular supports. during injection of the light-body material after placement of the polycarbonate post, the internal pressure formed by the air contained in the canal promoted bubble formation and the impression resulting from this procedure presented inaccuracies that interfered with cmc adaptation20. on the other hand, the use of hypodermic needle as an intraradicular support allowed the air exiting the canal during injection of the light-body impression material, which promoted a better material flow within the canal space. as a result, a more accurate impression was obtained and the cmc was more precisely adapted to the canal. in both analyses, the impression material did not produce significant differences in the results. the impression technique, on the other hand, had a significant influence on core adaptation, with better results when hypodermic needles were used regardless of the impression material. no previous study used hypodermic needles as supports for intracanal impression in the fabrication of cast metal cores. additional in vitro and in vivo studies should be performed to confirm these findings before the clinical use of this technique can be recommended. within the limitations of this study, both radiographic and microscopic analyses showed that: 1. the intracanal impression technique using hypodermic needle as intraradicular support provided significantly better cmc adaptation than the technique using polycarbonate post when the cores were fabricated from addition silicone impressions; 2. the type of impression material did not interfere on cmc adaptation to the canal. references 1. assif d, gorfil c. biomechanical considerations in restoring endodontically treated teeth. j prosthet dent. 1994; 71: 565-7. 2. baldissara p, zicari f, valandro lf, scotti r. effect of root canal treatments on quartz fiber posts bonding to root dentin. j endod. 2006; 32: 985-8. 3. meira jb, espósito co, quitero mf, poiate ia, pfeifer cs, tanaka cb et al. elastic modulus of posts and the risk of root fracture. dent traumatol. 2009; 25: 394-8. 4. dakshinamurthy s, nayar, s. the effect of post-core ferrule on the fracture resistance on endodontically treated maxillary central incisors. indian j dent res. 2008; 19: 17-21. 5. ravanshad s, ghoreeshi n. an in vitro study of coronal microleakage in endodontically-treated teeth restored with posts. aust endod j. 2003; 29: 128-33. 6. morton d, steven aa. fabrication of multiple posts and cores using a thermoplastic material and an indirect technique: a clinical report. j prosthet dent. 1997; 78: 542-4. 7. krammer r. a time-saving method for indirect fabrication of cast posts and cores. j prosthet dent. 1996; 76: 209-11. 8. lacy am, fukui h, bellman t, jendresen md. time-dependent accuracy of elastomer impression materials. part ii: polyether, polysulphides and polyvinylsiloxane. j prosthet dent. 1981; 45: 329-33. 9. myers ge, peyton fa. clinical and physical studies of the silicone rubber impression materials. j prosthet dent. 1959; 9: 315-24. 10. clancy jms, scandrett fr, ettinger rl. long-term dimensional stability of three current elastomers. j oral rehabil. 1983; 10: 325-33. 11. serafino c, gallina g, cumbo e, ferrari m. surface debris of canal walls after post space preparation in endodontically treated teeth: a scanning electron microscopic study. oral surg oral med oral pathol oral radiol endod. 2004; 97: 381-7. 12. sabbak sa. indirect fabrication of multiple post-and-core patterns with a vinyl polysiloxane matrix. j prosthet dent. 2002; 88: 555-7. 13. nealon fh. acrylic restorations by the operative nonpressure procedure. j prosthet dent. 1952; 2: 513-27. 14. tan kb. the clinical significance of distortion in implant prosthodontics: is there such a thing as passive fit? ann acad med singpore. 1995; 24: 138-57. 15. mendoza db, eakle s, kahl ea, ho r. root reinforcement with a resin-bonded preformed post. j prosthet dent. 1997; 78: 10-4. 16. pierrisnard l, bohin f, renaul p, barquins m. corono-radicular reconstruction of pulpless teeth: a mechanical study using finite element analysis. j prosthet dent. 2002; 88: 442-8. 17. newman mp, yaman p, dennison j, rafter m, billy e. fracture resistance of endodontically treated teeth restored with composite posts. j prosthet dent. 2003; 89: 360-7. 18. rubo mhm, el-mowafy o. radiopacity of dual-cured and chemical-cured resin-based cements. int j prosthodont. 1998; 11: 70-4. 19. soares cj, mitsui fh, neto fh, marchi gm, martins lr. radiodensity evaluation of seven root post systems. am j dent. 2005; 18: 57-60. 20. stackhouse jr ja. the accuracy of stones dies made from rubber impression materials. j prosthet dent. 1970; 24: 377-86. cast metal core adaptation using two impression materials and intracanal techniques 131 braz j oral sci. 8(3):128-131 original articlebraz j oral sci. april/june 2009 volume 8, number 2 dental caries experience in preschool children of bauru, sp, brazil fábio silva de carvalho1, cristiane alves paz de carvalho1, roosevelt da silva bastos1, angela xavier2, sabrina pulzatto merlini3, josé roberto de magalhães bastos4 1 dds, ms, graduate student, department of pediatric dentistry, orthodontic and public health, faculdade de odontologia de bauru, universidade de são paulo (usp), bauru (sp), brazil 2 dds, undergraduate student, department of pediatric dentistry, orthodontic and public health, faculdade de odontologia de bauru, usp, bauru (sp), brazil 3 dds, department of pediatric dentistry, orthodontic and public health, faculdade de odontologia de bauru, usp, bauru (sp), brazil 4 dds, ms, phd, professor, department of pediatric dentistry, orthodontic and public health, faculdade de odontologia de bauru, usp, bauru (sp), brazil received for publication: april 29, 2009 accepted: july 24, 2009 correspondence to: fábio silva de carvalho universidade de são paulo department of pediatric dentistry, orthodontic and public health alameda octávio pinheiro brisolla, 9-15 cep 17012-901 – bauru (sp), brasil e-mail: fasicar@usp.br abstract aim: the main purpose of this study was to evaluate dental caries experience and prevalence in three to six-yearold preschool children from three public kindergartens of the city of bauru, sp, brazil. methods: the sample comprised 283 children (142 boys; 141 girls) aged 3 to 6 years old. for data analysis, the dmft (decayed, missing and filled teeth), significant caries index (sic index), percentage of caries-free children, gini coefficient and care index were determined. mann-whitney and kruskal-wallis tests were used with 5% significance. results: the mean (sd) dmft index was 1.40 (2.63). among the examined children, 63.25% were caries-free. the decayed component comprised 77.28% of the dmft. the care index was 19.70%, indicating limited utilization of dental treatment by children. the sic index was 4.11 and the gini coefficient was 0.78 in the survey. the results showed that there were no statistically significant differences in the prevalence of the disease between the ages and between males and females. conclusions: dental caries experience and prevalence in preschool children were considered low in this study and the occurrence of polarization of the disease was identified. keywords: dental caries, child, preschool, oral health. introduction the fluoridation of the public water supply since the 1970s and the massive use of fluoridated dentifrices from the 1980s are scientifically recognized as the main responsible factors for dental caries decline in the last decades1,2. in spite of this, a heterogeneous distribution of dental caries has been observed, in which high disease levels have been identified in a minority of individuals3. studies directed to primary dentition did report the same decline in the prevalence of caries and, additionally, reported an increase in the mean dmft, with high proportions of untreated caries4-8. in brazil, few studies on caries prevalence in primary teeth have been developed over the last years. age is an important factor to be regarded in order to better understand children’s oral health in the primary dentition since past caries experience is considered as the most powerful predictor of the disease9-11. epidemiological dental studies have shown changes in the distribution of dental caries and this trend has been investigated4,6-8,11. epidemiological 98 carvalho fs, carvalho cap, bastos rs, xavier a, merlini sp, bastos jrm braz j oral sci. 8(2): 97-100 data would allow oral health administrators to plan specific measures according to oral health needs. due to the abovementioned considerations, the main purpose of this study was to evaluate dental caries experience and prevalence in three to six-year-old preschool children in three public kindergartens in the city of bauru, sp, brazil. material and methods this epidemiological study was carried out in the city of bauru, located in the state of são paulo, brazil, which had an estimated population of 355,675 in 200812. fluoride has been added to water supply since 1975 in bauru (0.07 ppm). the research protocol was approved by the institutional review board (irb) of bauru dental school, university of são paulo, brazil, (process no 71/2008), and the authorization for conduction of study was obtained from the municipal secretary education and directors of kindergartens. also, written informed consent was obtained from the parents/legal representatives prior to enrolment of the children. sample this study provided epidemiological data for the development of educational and preventive program in oral health in three public kindergartens. information about total number of students in each unit by age was obtained from the directors of the kindergartens. there was a return rate of 77.43% (319 out of 412) of signed consent forms from parents/legal representatives. however, the final sample was reduced, since 36 children were absent on the examination day or refused to be examined despite the parents’ consents. the study was conducted with 283 preschool children, 142 boys and 141 girls, aged 3 to 6 years old, corresponding to 68.69% of the total number of children in the three kindergartens. examination methodology the exams were done with the children seated on chairs under natural light. materials used were dental mirror and cpi (community periodontal index) probe13. the clinical examinations used for observation of the mean number of decayed, missing or filled teeth (dmft index) were performed according to the criteria established by the world health organization (who)13. only one examiner was trained and calibrated for this study. during data collection, duplicate examinations of approximately 10% of the sample were performed to assess intra-examiner variability. intra-examiner agreement for dental caries diagnosis was evaluated by means of the kappa coefficient, showing values of 0.92, whose agreement was considered to be excellent. data analysis percentages of caries-free children and dmft were used to describe dental caries distribution among children. significant caries index14 (sic index), gini coefficient15 and care index16 were adopted to assess the unequal distribution of dental caries and oral health care. sic index was calculated by taking the mean dmft of the onethird of the individuals having the highest of dmft values in a given population14, and was used to measure the polarization of the dental caries occurrence among children. the care index16 was calculated using the means dmft without caries-free. the component “f ” (filled teeth) was divided by the dmft and multiplied by 100. the gini coefficient was used to assess inequality of caries distribution in this study15. mann-whitney and kruskal-wallis non-parametric tests were used to check the statistical association between the studied variables. a significance level of 5% was adopted. results table 1 shows the caries experience according to age and gender. from the group of examined children, 179 (63.25%) were caries-free. homogenous distribution, according to gender, was observed in this sample. the mean dmft was 1.40 to total sample. the boys had dmft greater than girls. the mean dmft was 1.13 at 3 years of age and 1.68 at 6 years of age. no statistically significant differences (p > 0.05) were found in this study with regard to the prevalence of disease among gender and age. the decayed component comprised 77.28% of the dmft and the filling component 19.70%. one third of the sample concentrated 97.47% of the dmft distribution in the respective study. the sic index was 4.11, the care index was 19.70% and the gini coefficient was 0.78. discussion data from the last brazilian oral health survey (sbbrasil 2003)17 showed dmft at 5 years of age of 2.80, which is similar to the results observed in other brazilian cities, such as in bilac18 (2.84), piracicavariables sample caries-free mean dmft (sd) age n % n % 3 32 11.30 21 65.62 1.13 (1.90) 4 97 34.28 63 64.95 1.42 (3.11) 5 104 36.75 64 61.54 1.33 (2.28) 6 50 17.67 31 62.00 1.68 (2.75) total 283 100.00 179 1.40 (2.63) gender boys 142 50.18 87 61.27 1.65 (3.11) girls 141 49.82 92 65.25 1.15 (2.03) total 283 100.00 179 1.40 (2.63) dmft: decayed, missing and filled teeth. table 1. caries experience according to age and gender, bauru, brazil, 2008 99dental caries experience in preschool children of bauru, sp, brazil braz j oral sci. 8(2): 97-100 ba19 (2.68), rio claro20 (2.50), juiz de fora21 (2.40) and teresina22 (2.13). mean dmft was lower in the cities of paulínia23 (1.90), campinas24 (1.68), indaiatuba25 (1.62) and higher in the city of cambira26 (3.51). in the present study, mean dmft was 1.33. breaking the dmft index into decayed (“d”) and filled (“f ”) components revealed greater inequality in the distribution these components. at 5 years old, the decayed and filled represented respectively 78.99% and 18.12% of the dmft index. higher values for the decayed (“d”) component were found in the cities of indaiatuba25 (84.80%), juiz de fora21 (87.60%) and cambira26 (92.30%), while lower values were found in the city of campinas24 (75.60%). the access to health services is not satisfactory to the children that participated of this study yet, as demonstrated by the small number of filled teeth in relation to the number of decayed teeth, as reported elsewhere23. considering the total sample, the care index values reached 19.70%, indicating a low coverage of the oral health service for the studied preschool children, as observed in the cities of indaiatuba25 (12.30%). nevertheless, greater values were observed in piracicaba19 (23.10%), rio claro20 (64.00%) and paulínia23 (65.40%). the results of another investigation carried out in bauru showed an improvement in the oral health service coverage among 12-year-old children in 2006, since care index reached 56.30%27. in this study, at 5 years old, 61.54% of the preschool children were caries-free. lower values were observed in the cities of cambira26 (31.14%), juiz de fora (44.00%), piracicaba19 (44.30%), bilac18 (45.30%), rio claro20 (51.00%), paulínia23 (54.20%), teresina22 (55.70%) and campinas24 (56.17%). as observed in bilac18, juiz de fora21, teresina22 and cambira26, the number of children affected by caries disease increased with age, with a percentage of children with dmft > zero at six years old greater than that at three years old. the results also showed that 97.47% of the dmft distribution was concentrated in one third of the sample, which clearly demonstrated the phenomenon of caries polarization. this fact was not observed in the city of teresina22, where approximately 49% of the children presented caries. sic index was developed in 2000 to bring attention to those children with the highest caries scores in each population28,29. in the present study, sic index was used to identify the group with highest caries rate and the value of 4.11 was found, lower than that recorded in other cities such as bilac18 (5.90), itaí30 (5.08) and água doce31 (9.97). inequalities in caries distribution were measured by the gini coefficient. this index indicate perfect inequality with a coefficient of 1 and perfect equality with a coefficient of 0. it has been used in a way to measure the association between exposure to a risk factor and disease prevalence15. a higher gini coefficient means that the risk of disease is more variable amongst the population. in this study, the gini coefficient was of 0.78, which is similar to that obtained in a previous study carried out in bauru27 involving 12-year-old children (0.76), indicating an unequal distribution of caries in this municipality. this unequal distribution occurs due to different socioeconomic levels of the population according to cypriano et al.32, and some factors, such as local service organization, access to fluoridated water, preventive procedures and investment in public health, may change this trend. acquaintance with inequalities in the distribution of child caries must inevitably invite the question as causes of this phenomenon. there are several risk factors for caries experience, for example, transmissibility of oral bacteria and amount and timing of exposure to cariogenic foodstuffs and drinks, and difficulty of access to dental services. it is important to mention that this study had some limitations. the sample was drawn from a population of three to six-year-old preschool children attending three public kindergartens. therefore, the conclusions are valid only for this population. the educational and preventive program in oral health may change the epidemiological profile of caries in preschool children from these kindergartens. in addition to the dmft index, other approaches for caries evaluation should be considered. the sic index and gini coefficient supply the oral health service epidemiological data that allows the promotion of adequate oral health based on the community needs. the interaction of health professionals, educators and parents, and the planning of strategies may reduce the inequality distribution of dental caries in children to advance the oral health patterns. in conclusion, dental caries experience and prevalence in preschool children were considered low in this study, and the occurrence of the polarization of the disease was identified. references 1. truman bi, gooch bf, sulemana i, gift hc, horowitz am, evans ca, et al. reviews of evidence on interventions to prevent dental caries, oral and pharyngeal cancers, and sports-related craniofacial injuries. am j prev med. 2002;23(1 suppl):21-54. 2. bratthall d, hänsel-petersson g, sundberg h. reasons for the caries decline: what do the experts believe. eur j oral sci. 1996;104:416-22. 3. marthaler t, menghini g, steiner m. use of the significant caries index in quantifying changes in caries in switzerland from 1964 to 2000. community dent oral epidemiol. 2005;33:159-66. 4. lucas sd, portela mc, mendonça ll. variations in tooth decay rates among children 5 and 12 years old in minas gerais, brazil. cad saude publica. 2005;21:55-63. 5. downer m. caries prevalence in the united kingdom. int dent j. 1994;44(4 suppl 1):365-70. 6. pitts nb, boyles j, nugent zj, thomas n, pine cm. the dental caries experience of 5-years-old children in england and wales (2003/4) and scotland (2002/3). community dent health. 2005;22:46-56. 7. cypriano s, pecharki gd, sousa mlr, wada rs. oral health of schoolchildren residing in areas with or without water fluoridation in sorocaba, são paulo state, brazil. cad saude publica. 2003;19:1063-71. 8. hashim r, thomson wm, ayres km, lewsey jd, awad m. dental caries experience and use of dental services among preschool children in ajman, uae. int j paediatr dent. 2006;16:257-62. 9. creedon mi, o’mullane dm. factors affecting caries levels amongst 5-year-old children in country kerry, ireland. community dent health. 2001;18:72-8. 10. li y, wang w. predicting caries in permanent teeth from caries in primary teeth: an eight-year cohort study. j dent res. 2002;81:561-6. 11. skeie ms, raadal m, strand gv, espelid i. the relationship between caries in the primary dentition at 5 years of age and permanent dentition at 10 years of age – a longitudinal study. int j paediatr dent. 2006;16:152-60. 100 carvalho fs, carvalho cap, bastos rs, xavier a, merlini sp, bastos jrm braz j oral sci. 8(2): 97-100 12. ibge. institute brazilian of geography and statistics. estimate of the population residing in brazilians municipalities on july 1, 2008. [internet]. [cited 2009 ago 25]. available from: <www.ibge.gov.br/home/estatistica/populacao/ estimativa2008/pop2008_dou.pdf> 13. word health organization. oral health surveys, basics methods. 4th ed. geneve: who; 1997. 14. nishi m, stjernswärd j, carlsson p, bratthall d. caries experience of some countries and areas expressed by the significance caries index. community dent oral epidemiol. 2002;30:296-301. 15. armfield jm, spencer aj, slade gd. changing inequalities in the distribution of caries associated with improving child oral health in australia. j public health dent. 2009;69(2):125-34. 16. smith ge. tooth decay in the developing world. n z med j. 1987;100:669-70. 17. brazil. health ministry of brazil. sb brazil 2003 project – oral health conditions of the brazilian population 2002-2003. brasília, df; 2004. 18. martins rj, garbin cas, garbin aji, moimaz sas, saliba o. declining caries rate in a municipality in northwestern são paulo state, brazil, 1998-2004. cad saude publica. 2006;22:1035-41. 19. cypriano s, sousa mlr, rihs lb, wada rs. oral health among preschool children in brazil, 1999. rev saude publica. 2003;37:247-53. 20. hoffmann rhs, cypriano s, sousa mlr, wada rs. dental caries experience in children at public and private schools from a city with fluoridated water. cad saude publica. 2004;20:522-8. 21. leite icg, ribeiro ra. dental caries in the primary dentition in public nursery school children in juiz de fora, minas gerais, brazil. cad saude publica. 2000;16:717-22. 22. moura lfad, moura ms, toledo oa. dental caries in children that participated in a dental program providing mother and child care. j appl oral sci. 2006;14:53-60. 23. gomes pr, costa sc, cypriano s, sousa mlr. dental caries in paulínia, são paulo state, brazil, and who goals for 2000 e 2010. cad saude publica. 2004;20:86670. 24. cardoso sv, pereira sm, tagliaferro eps, pereira ac, meneghin mc. oral health conditions in campinas: a critical evaluation. arquivos em odontologia. 2004;40:341-52. 25. ueda emo, dezan cc, frossard wtg, salomão f, morita mc. prevalence of dental caries in 3 and 5-year-old children living in a small brazilian city. j appl oral sci. 2004;12:34-8. 26. peres ma et al. social and biological early life influences on severity of dental caries children aged 6 years. community dent oral epidemiol. 2005; 33:53-63. 27. rihs lb, sousa mlr, cypriano s, abdalla nm, guidini ddn, amgarten c. dental caries activity in primary dentition, são paulo, brazil, 2004. cad saude publica. 2007;23:593-600. 28. tagliaferro eps, meneghim mc, ambrosano gmb, pereira ac, sales-peres shc, sales-peres a, et al. distribution and prevalence of dental caries in bauru, brazil, 1976-2006. int dent j. 2008;58:75-80. 29. bratthall d. introducing the significant caries index together with a proposal for a new global oral health goal for 12-year-olds. int dent j. 2000;50: 378-84. 30. sales-peres shc, carvalho fs, carvalho cap, bastos jrm, lauris jrp. polarization of dental caries in teen-agers in the southwest of the state of são paulo, brazil. cien saude colet. 2008;13(suppl 2):2155-62. 31. biazevic mgh, rissoto rr, michel-crosato e, mendes la, mendes moa. relationship between oral health and its impact on quality of life among adolescents. braz oral res. 2008;22:36-42. 32. cypriano s, hoffmann rhs, sousa mlr, wada rs. dental caries experience in 12year-old schoolchildren in southeastern brazil. j appl oral sci. 2008;16:286-92. oral sciences n3 braz j oral sci. 14(3):182-185 original article braz j oral sci. july | september 2015 volume 14, number 3 received for publication: may 05, 2015 accepted: august 03, 2015 comparison of palatal bone height in different facial morphological patterns by cone beam computed tomography gabriella lopes de rezende barbosa1, laura ricardina ramirez-sotelo1, phillipe nogueira barbosa alencar1, solange maria de almeida1 1universidade estadual de campinas – unicamp, piracicaba dental school, department of oral diagnosis, area of oral radiology, piracicaba, sp, brazil correspondence to: gabriella lopes de rezende barbosa av. limeira, 901 cep 13414-903 bairro areião, piracicaba, sp, brasil phone/fax: +55 19 2106-5327 e-mail: gabriellalopes@live.com abstract aim: to evaluate the relationship between palatal bone height and facial types using cone beam computed tomography (cbct) images. methods: the study sample consisted of 110 cbct images. subjects aged 18 to 35 years old were classified as brachyfacial, mesofacial and dolichofacial, according to sn.gogn angle, measured in cephalometric analysis with nemotec 3d software. three observers evaluated the bone height of the palate in the anterior region, at the level of the maxillary first premolars. the measurements were performed in sagittal and coronal views, in median and paramedian regions of the palate, also using nemotec 3d software. measurements were repeated after 15 days on 25% of the sample to analyse intra and interobserver agreements by cci test. analysis of variance was performed to calculate statistical differences between the bone heights of the three facial types, with a significance level of 5%. results: the correlation level of intra-observer agreement was excellent. no significant differences were observed between the palatal bone height in the region of first premolars and the different facial types (p>0.05). conclusions: based on cbct evaluations, there is no association between palatal bone height and facial morphological patterns. keywords: cone-beam computed tomography; diagnostic imaging; orthodontic anchorage procedures. introduction the facial type of an individual comprises variations of the craniofacial skeleton structures and it is related to genetic and environmental factors1-2. the classification of facial types has a direct relationship with craniofacial growth and is divided in three types: brachyfacial, mesofacial and dolichofacial. in the brachyfacial type, there is a tendency to horizontal facial growth; mesofacial type is characterized by balanced growth of all facial thirds and dolichofacial type has a tendency to vertical facial growth and mouth breathing3-4. these facial types are related to aesthetics and functional features of bones and muscles. they also present significant correlation with dental arch features, which can lead to other complex orofacial changes1,5-6. the proper determination of variations in facial types is important for orthodontic diagnosis and treatment since certain procedures performed during orthodontic therapy may attenuate or enhance facial features. morphological changes can occur in several structures such as the hard palate, a structure of the http://dx.doi.org/10.1590/1677-3225v14n3a01 183183183183183 maxillofacial complex that is involved anatomically and functionally in all stages of the craniofacial development7. according to the facial type, the palate can be, for example, deep and narrow in the dolichofacial individuals while wide and shallow in brachyfacial subjects8-12. according to christie13, features of each facial type may have a significant implication in orthodontic treatment planning, as well as the configuration of dental arches and amount of available bone. this individualized approach allows an adaptation of the orthodontic mechanics, containment procedures and in some cases, even skeletal anchorage procedures allowing greater control of the results14. the amount of available bone in regions for insertion of anchorage implants is a limiting factor for orthodontic treatments that require skeletal anchorage, especially in cases of palatal mini-implants. even temporary, such implants require a satisfactory bone volume for placement and stability, and must be inserted at safe distances from vital structures that cannot be injured14-16. since the region of first premolars has the highest levels of bone height and most clinically satisfactory palatal implants are inserted at this level, this was the site selected for evaluation in the present study14,17. several authors compared the dimensions of the hard palate among facial types by clinical evaluations and plaster model analysis2,18-20. however, in general, such studies assess characteristics like the morphology of palatal vault, palatal depth and thickness. in this sense, few of them have emphasized by quantitative measurements the internal assessment of the palate such the bone height. the study conducted by esteves and bommarito21 (2007) proved the correlation of palatal depth and facial type, leading to the authors’ hypothesis that other palatal features would also be influenced by the facial type. taking into account the lack of reports concerning the relationship between facial types and palatal bone height, and the raised hypothesis, this research aimed to evaluate the relationship between palatal height and the different facial types, brachyfacial, mesofacial and dolichofacial, using cone beam computed tomography (cbct) images. moreover, a positive correlation would lead to a possible predictability of the bone amount in the palatal region of patients classified with a determined facial pattern. material and methods after the approval of this study by the ethics committee in research (protocol number 118/2012), 110 cbct scans were selected from an archive. the sample consisted of images from patients (18-35-year-old) with no evident facial and dental asymmetries, torus palatinus or anomalies in the head and neck region. the patients had complete permanent dentition and were referred to the tomographic examination for different treatment purposes unrelated to this study. cbct scans were obtained by an i-cat unit (imaging sciences international, inc., pennsylvania, usa) with the following parameters: voxel size of 0.3 mm, 120 kvp, 8 ma, fov of 23 x 17 cm, and 40 s scan time. the assessment of the images was done in a secluded room with dim light by expert oral radiologists. first, to classify each subject according to the facial type, raysum multi-planar reformations were created using nemotec software (nemoceph imaging cephalometric and tracing software s.l., spain), based on the cbct volume. this reformatted image, also known as virtual lateral radiograph (vlr), simulates a two-dimensional lateral exam, enabling the acquisition of cephalometric analysis on this exam. the facial type was determined based on the sn.gogn angle that was measured by one of the authors on the vlr images through the computerized module nemoceph 2d in nemotec software (nemoceph imaging cephalometric and tracing software s.l., spain). according to the obtained sn.gogn angle, the subjects were classified according to the following criteria: brachyfacial, ≤27°; mesofacial, between 27°and 37° and dolichofacial, sn.gogn ≥37°22. after determining the facial type of the sample, the amount of available bone was assessed by three expert oral radiologists with at least two years experience in tomographic appraisal. using nemoceph 3d module (nemoceph imaging cephalometric and tracing software s.l., spain) the bone height in the anterior region of the hard palate was assessed at the level of the maxillary first premolars. the regions of interest were determined in multi-planar reconstructions (mpr), using the orientation lines of the planes. first, in the axial view, the mid-sagittal plane of the patient and the maxillary first premolar region were defined, superimposing the sagittal and coronal lines, respectively (figure 1a). the midsagittal plane was created by an imaginary line perpendicular to the frankfurt plane that divided the body into right and left halves oriented by the anterior nasal spine. the maxillary first premolar region was defined by the plane that overlaps the root canals of both right and left first premolars. for bone height assessment, linear measurements were performed in sagittal and coronal planes, using the ruler tool of the software, creating measures superimposed to the orientation line of the planes, predetermined on the axial view as described before. in the sagittal view, the measurement was performed between the outer cortical layer of the nasal floor and the cortical layer of the oral hard palate, superimposed to the coronal orientation line (figure 1b). next, in the coronal view, the palatal bone height of the median and paramedian regions were assessed. the median/central site was assessed superimposing the measurement on the orientation line of the sagittal plane. then, the paramedian sites were defined and evaluated 3 mm and 6 mm bilaterally to the central measurement (figure 1c). after one week interval, the same evaluation was repeated with 25% of the sample to evaluate statistically the intra-observer agreement. the intraand inter-observer agreements were evaluated by the intraclass correlation coefficient (icc) using spss® (statistical package for social sciences) program package version 17.0 (spss inc., chicago, il, usa) for windows. comparison of palatal bone height in different facial morphological patterns by cone beam computed tomography braz j oral sci. 14(3):182-185 184184184184184 facial type midsagittal view coronal view mean s d mean s d brachyfacial 7.32 2.73 7.22 2.35 dolichofacial 6.82 2.72 7.07 2.91 mesofacial 6.97 2.59 6.78 2.07 table 1. table 1. table 1. table 1. table 1. mean and standard deviation (sd) values for palatal bone height of the evaluated facial patterns in midsagittal and coronal views (mm) no statistical difference between the groups. fig. 1. palatal bone height evaluation in multiplanar reconstruction of cbct image. definition of mid-sagittal plane and upper first premolar region (a). linear measurement corresponding to midsagittal palatal bone height superimposed on the orientation line sagittal view (b). median and paramedian measurements 3 mm and 6 mm bilaterally – coronal view (c). the means of the measurements obtained by the three examiners were calculated. the mean of the values obtained in the coronal view (median and paramedian sites) were also obtained. anova was performed to calculate the statistical differences between the bone heights from the three different facial types, with a significance level of 5%, using sas statistical package version 20.0 (sas inc., cary, nc, usa) for windows. results one hundred and ten images from patients aged 18 to 35 years comprised the final sample of the study. the distribution of sample groups was similar in all three divisions: 38 brachyfacial subjects, 38 mesofacial and 34 dolichofacial individuals. regarding the correlation level of the intra-observer agreement according to the intra-class correlation coefficient, the values were close to 1 for the three examiners, which represents an excellent correlation, as interpreted by szklo and nieto23 (2000). the comparison of mean values for palatal bone height in different facial types, according to the analysis of variance, showed no statistically significant differences between the groups in both evaluations, sagittal (p=0.738) and coronal assessments (p=0.695) (table 1). discussion the influence of the facial type on functional, neuromuscular, skeletal and dental functions has been subject of debate in articles, texts and researches for over a century24. the evaluation of craniofacial characteristics and morphology are usually made by conventional lateral radiographs7,9, which are believed by several authors to be effective for the diagnosis and orthodontic planning24. however, recent threedimensional imaging technologies, such as cbct, allow the assessments of craniofacial structures without some disadvantages of the conventional techniques, especially the superimposition of structures25. according to cortes et al., 2012, cbct is an accurate image-acquisition method to provide information regarding bone measurements, which are important, for example, to perform precise surgical planning for implant placement26. taking into account this advantage and the improvement in the visualization of the palatal region anatomy, this study was conducted based on tomographic images. this threedimensional approach allowed the accurate quantitative evaluation of internal features of the region, different from previous studies that assessed distinct features such as the palatal morphology using plaster models2,18-20. the facial typology is given according to anatomical variations that form the craniofacial complex27. establishing the facial pattern of each patient and fit them into a classification is required since each facial type presents different morphological and functional features4. the growth trend of the thirds of the face determines the patient’s facial type and the direction of its evolution, which can be more horizontal (brachyfacial), balanced (mesofacial) or vertical (dolichofacial)3. in this sense, the shape of the palate is one of the individual characteristics that are subject to influence of the facial typology and may have different morphologies21. assessment of the palatal bone height in cases of skeletal anchorage using palatal mini-implants is the key to indicate or contraindicate the procedure, also preventing possible injuries to structures adjacent to implant insertion sites, such as perforation of the nasal floor15,28. based on our results, there were no significant differences between the palatal measurements evaluated in the three different facial patterns. thus, it is not possible to correlate palatal bone height with facial types and neither comparison of palatal bone height in different facial morphological patterns by cone beam computed tomography braz j oral sci. 14(3):182-185 185185185185185 estimate a larger amount of bone in this region as a predictor for surgical procedures. there is lack of studies in the literature that aimed to evaluate bone availability of the palate, as well as to correlate this information with variations in the maxillofacial complex. in this sense, our results can only be confronted with studies that had different approaches but also evaluated the palate according to facial types. the results of the present study disagree with esteves and bommarito reports21 (2007), who stated that dolichofacial individuals have narrow and deep palate while brachyfacial patients have a wider and shallow palate, demonstrating differences in the palatal morphology in each facial type, an outcome not observe d in our study. however, these characteristics were assessed in plastic models, disagreeing with our methodology and the selected palatal feature. our results also differ from previous findings of significant differences between the depths of the palate according to facial types. despite addressing different characteristics, it is believed that in the same way that the depth evaluated in plaster models varies according to the facial pattern, the height palate would also suffer such influences19. however, the lack of correlation between the palatal heights found in the present study disagrees with these findings. the present study evaluated bone height in two different views of the tomographic multiplanar reconstruction in order to avoid the interference of anatomical structures in the midsagittal view. the evaluation was then performed in different sites of the same region to provide additional values, since it is proven that paramedian sites have comparable heights when compared to median measurements29. based on cbct evaluations, it may be concluded that there is no association between palatal bone height and facial patterns. according to the obtained data, it may be observed that there is no predictability of palatal bone height and it is therefore recommended to request appropriate diagnostic imaging modality for planning interventions in the region of the hard palate, reducing the risk of injuries and perforations. references 1. cassidy km, harris ef, tolley ea, keim rg. genetic influence on dental arch form in orthodontic patients. angle orthod. 1998; 68: 445-54. 2. retamoso lb, knop la, guariza filho o, tanaka om. facial and dental alterations according to the breathing pattern. j appl oral sci. 2011; 19: 175-81. 3. mcnamara ja. influence of respiratory pattern on craniofacial growth. angle orthod. 1981; 51: 269-300. 4. kageyasma t, domínguez-rodríguez gc, vigorito jw, deguchi t. a morphological study of the relationship between arch dimensions and craniofacial structures in adolescents with class ii division 1 malocclusions and various facial types. am j orthod dentofacial orthop. 2006; 129: 368-75. 5. osburn rc. the effects of brachycephaly and dolichocephaly on the teeth of man. dent cosmos. 1910; 52: 517-26. 6. bhat m, enlow dh. facial variations related to headform type. angle orthod. 1985; 55: 269-80. 7. faria pt, de oliveira ruellas ac, matsumoto ma, anselmo-lima wt, pereira fc. dentofacial morphology of mouth breathing children. braz dent j. 2002; 13: 129-32. 8. linder-aronson s. the relation between nasorespiratory function and dentofacial morphology. am j orthod. 1983; 83: 443-4. 9. frasson jm, magnani mb, nouer df, de siqueira vc, lunardi n. comparative cephalometric study between nasal and predominantly mouth breathers. braz j otorhinolaryngol. 2006; 72: 72-81. 10. bianchini ap, guedes zc, vieira mm. a study on relationship between mouth breathing and facial morphological pattern. braz j otorhinolaryngol. 2007; 73: 500-5. 11. cattoni dm, fernandes fd, di francesco rc, latorre mdo r. characteristics of the stomatognathic system of mouth breathing children: anthroposcopic approach. pro fono. 2007; 19: 347-51. 12. coelho ar, tanaka o, ribeiro js, machado ma, camargo es. transverse craniofacial dimensions in angle class ii, division 1 malocclusion according to breathing mode. braz oral res. 2010; 24: 70-5. 13. christie te. cephalometric patterns of adults with normal occlusion. angle orthod. 1977; 47: 128-35. 14. cousley r. critical aspects in the use of orthodontic palatal implants. am j orthod dentofacial orthop. 2005; 127: 723-9. 15. wehrbein h, feifel h, diedrich p. palatal implant anchorage reinforcement of posterior teeth: a prospective study. am j orthod dentofacial orthop. 1999; 116: 678-86. 16. feldmann i, bondemark l. orthodontic anchorage: a systematic review. angle orthod. 2006; 76: 493-501. 17. jung ba, wehrbein h, heuser l, kunkel m. vertical palatal bone dimensions on lateral cephalometry and cone-beam computed tomography: implications for palatal implant placement. clin oral implants res. 2011; 22: 664-8. 18. braun s, hnat wp, fender de, legan hl. the form of the human dental arch. angle orthod. 1998; 68: 29-36. 19. de freitas fcn, bastos ep, primo ls, de freitas vl. evaluation of the palate dimensions of patients with perennial allergic rhinitis int j paediatr dent. 2001; 11: 365-71. 20. armando rs, bommarito s, mandetta s, zanelato act. evaluation of palatine depth in patients with different facial types. rev. odonto cienc. 2002; 10: 15-22. 21. esteves a, bommarito s. evaluation of palatine depth and dimensions of the upper dental arch in patients with malocclusion and different facial types. r dental press ortodon ortop facial. 2007; 12: 84-98. 22. riedel ra. the relation of maxillary structures to cranium in malocclusion and normal occlusion. angle orthod. 1952; 22: 142-5. 23. szklo r, nieto fj. epidemiology beyond the basics. 2nd ed. gaithersburg: aspen publishers; 2000. p.343-404. 24. kluemper gt, vig ps, vig kw. nasorespiratory characteristics and craniofacial morphology. eur j orthod. 1995; 17: 491-5. 25. lascala ca, panella j, marques mm. analysis of the accuracy of linear measurements obtained by cone beam computed tomography (cbctnewtom). dentomaxillofac radiol. 2004; 33: 291-4. 26. cortes arg, gomes afam, tucunduva mjaps, arita, es. evaluation of linear tomography and cone beam computed tomography accuracy in measuring ridge bone width for planning implant placement. br j oral sci. 2012; 11: 116-9. 27. sies ml, farias sr, vieira mm. oral breathing: relationship between facial type and dental occlusion in adolescents. rev soc bras fonoaudiol. 2007; 12: 191-8. 28. cousley rr, parberry dj. combined cephalometric and stent planning for palatal implants. j orthod. 2005; 32: 20-5. 29. de rezende barbosa gl, ramírez-sotelo lr, távora dm, almeida sm. comparison of median and paramedian regions for planning palatal miniimplants: a study in vivo using cone beam computed tomography. int j oral maxillofac surg. 2014; 43: 1265-8. comparison of palatal bone height in different facial morphological patterns by cone beam computed tomography braz j oral sci. 14(3):182-185 oral sciences n3 braz j oral sci. 10(1):65-68 original article braz j oral sci. january | march 2011 volume 10, number 1 dental arch morphology in normal occlusions luiz renato paranhos1, will a. andrews2, renata pilli jóias3, fausto bérzin4, eduardo daruge júnior4, tarcila triviño5 1 dds, ms, phd, full professor, department of orthodontics, school of dentistry, methodist university, brazil 2 dds, assistant clinical professor, department of orthodontics, school of dentistry, university of california, usa 3 dds, ms, department of orthodontics, school of dentistry, methodist university, brazil 4dds, ms, phd, department of dental biology, school of dentistry, university of campinas, brazil 5dds, ms, professor, department of orthodontics, school of dentistry, methodist university, brazil correspondence to: luiz renato paranhos rua padre roque, 958 – centro / mogi mirim são paulo – brasil – 13800-033. phone: 55 19 3804-4002 e-mail: paranhos@ortodontista.com.br received for publication: november 14, 2010 accepted: march 23, 2011 abstract aim: the aim of this study was to identify the prevalence of 3 different mandibular dental arch morphologies in individuals with natural normal occlusion. methods: fifty-one mandibular dental casts of caucasian individuals with natural normal occlusion were digitized. each was without a history of orthodontic treatment and presenting at least four of the six keys to normal occlusion described by andrews. twelve orthodontists evaluated the prevalence of the square, oval and tapered arch shapes by analyzing the mandibular digital images. results: the most prevalent dental arch shape was oval (41%), followed by square (39%), and tapered (20%) shapes. conclusions: during leveling and alignment phases, when elastic-alloy-wires are greatly used, the orthodontist could use any of the studied arch shapes (oval, square, tapered), once the prevalence of all of them was similar. keywords: normal occlusion, dental arch morphology, mandibular dental arch, anatomy. introduction dental arch morphology is an important consideration in orthodontic treatment of dentofacial deformities. for over one century dental arch morphology has been studied in hopes of defining proper goals for tooth position, esthetics, function and long-term stability1-4. because the mandibular dental arch is one of the main references for orthodontic treatment planning, many studies have strived to define its ideal size and morphology5-8. according to the literature9, human dental arch morphology shows wide individual variation. descriptions range from elipsoid6, parabolic6,10, a segment of circle joined to lines10, a segment of circle11 caternary curve, etc… diagrams were subsequently designed to aid orthodontists in forming wires to shape the dental arches during the treatment12-13. the use of standardized diagrams has been contested by some authors6,14-16, who suggested instead to use mathematical formulas17 to find a more individualized shape for arches. the aim of this study was to verify the prevalence of three different morphologies of the mandibular dental arch in natural normal occlusions and then correlate those findings to other variables that may help guiding orthodontists customizing orthodontic archwires. 66 braz j oral sci. 10(1):65-68 material and methods this study was approved by the research ethics committee of the school of dentistry of piracicaba, university of campinas (protocol # 149/2008). sample the study sample consisted of 51 mandibular dental casts from caucasian individuals with natural normal occlusion. the ages ranged from 15y2m to 19y4m, with a mean age of 16y6m. there were 21 (41.2%) males and 30 (58.8%) females in the sample. individuals with craniofacial and/or dental anomalies or asymmetries were excluded. all subjects had no previous preventive and/or corrective orthodontic treatment. each cast presented at least four of the six keys to normal occlusion described by andrews18. the first key (molar class i of angle10) bilaterally was required on all casts. all the permanent teeth were in occlusion, except the third molars. plaster model digitizing the mandibular dental casts were individually digitized on a scanner dw5-140 (dental wings®; montreal, quebec, canada), previously calibrated as instructed by the manufacturer. the image acquisition was processed by points (accuracy: 20-50 microns), according to the cast cartesian axes. the images were automatically generated by a software program (dental wings®), generating a volumetric archive (.stl) for each cast. attainment of the images after digitizing the casts the computer “print screen” resource was used to convert the images into figures. these figures had been transferred to the vectorial software coreldraw® x3 software, in which they had been cropped and prepared for evaluation. the control points in each mandibular cast were: the incisal edges of the incisors, the canine cusp tips, and the premolar and molar buccal cusp tips. when connected, these points formed andrews’ “perimeter line”19, which assisted in the determination and evaluation of the dental arch morphology (figure 1). subjective determination of the morphology of the mandibular dental arch the dental arch 3d images were printed on white paper (90 g/m2). each image was individually arranged in the center of a sheet of paper, below a guide, with three different dental arch configurations: square, oval and tapered20 (figure 2). an album containing the 51 images, with one image on each sheet of paper, was distributed to each appraiser. the examiners (5 male and 7 female) were dentists with at least a master’s degree in orthodontics. each examiner was instructed to subjectively pick one, and only one, match for the shape of the arch relative to the 3 given categories. statistical analysis to test the level of agreement of the dental arch subjective classification among the examiners, the modified fig. 2 – album sheet of paper with the mandibular dental arch. morphology classification by mclaughlin & bennett20 (1997): a) square; b) oval; and c) tapered. dental arch morphology in normal occlusions fig.1 – a) the mandibular dental arch image in coreldraw® x3; b) marking of the reference points; c) delimitation of the angle occlusion line; d) final morphology of the evaluated mandibular dental arch. braz j oral sci. 10(1):65-68 kappa statistical test21-22, at 5% significance level was used. the kappa values range from -1 to +1. +1 establishes a perfect agreement. after a 15-day interval, the examiners reclassified all the images to evaluate the reliability of the measurement method and the operator’s calibration. the systematic error was calculated by the paired student’s t test, at a significance level of 5%. dahlberg’s method was used to calculate the casual error23. the inter-examiner level of agreement was tested by the modified kappa statistical test21-22 at a significance level of 5%. to evaluate the measurement method reliability and the operator’s calibration, all the images were reclassified by all examiners after a 15-day interval. the systematic error was calculated by the paired student’s t test, at a significance level of 5%, and to calculate the casual error, it was used the dahlberg’s method23. results it was not verified any systematic or casual error (p<0.05), showing the reliability of the used methods. the kappa results showed statistically significant agreement for the arch configuration between the examiners (table 1). according to landis and koch21 the agreement value (0.55) was “moderate”. the percentages of mandibular dental arches morphology in this sample of natural normal occlusions are shown in figure 3. morphology kappa ic95% p arch 0.55 0.52 0.58 <.001 * table 1:agreement among the 12 examiners about the arch configuration evaluation. *statistically significant difference (p<0.05) discussion the purpose of orthodontic treatment is to correct, intercept and/or prevent incorrect dental positions and dentofacial deformities, so that teeth and bone structures can be in harmony. the changes achieved during treatment should not disrupt the balance between teeth, bone structures and muscles. early orthodontic philosophies advocated expansion of the dental arches, without consideration to the balance among other stomatognathic structures. later philosophies discovered that dental expansion over a certain limit could be unstable1-2. however, some dental arch expansion or contraction might be stable in growing patients if the tooth positions did not significantly modify physiological muscles function1,3,5. since mature patient’s teeth tend to return to their original positions because orthopedics is not possible to be applied, the transverse dental movement should be as minimal as possible during the orthodontic therapy. orthodontic movement will be more stabile if there is a satisfactory balance between the muscles, bone structures and teeth3. some authors claim that some buccolingual tooth movement is necessary during treatment in order to correct buccolingual inclinations and improve occlusal interfacing. such movement is limited by alveolar bone and periodontal tissues1,5,18 called by andrews as the wala ridge19. the new positions of teeth are mainly determined and limited by the mandibular morphology, which is established around 8.5 weeks of intra-uterine life8. it is believed the maxillary arch shape is determined by the mandibular teeth 5,8 due to the similarity between mandibular and maxillary arches morphology and their morphogenesis11. as such, in pursuit of defendable, evidence-based treatment results, orthodontists should first consider the anatomic limits of the mandibular arch. in the beginning of orthodontic treatment, in the leveling and alignment phase, elastic-alloy-wires (such as ni-ti), as a standard, or average shape and size (i.e. oval, which is the most prevalent) could be utilized. after this phase, maintaining the dental arch configuration, as it relates to basal bone, is essential to the success of the orthodontic therapy because of its great influence on stability12,20. it has been suggested5 to use reference models, called “diagrams”, to assist in forming orthodontic wires or in the selection of pre-formed archwires. the diagrams are based on measures of the dental arch elements, mainly, the inter canines distance and can provide parameters about the pretreatment arch wire shape. choosing a personal diagram for the patient, the metallic arches would be contoured in a standard shape and dimension, allowing the maintenance of the transverse dimension during treatment. there are several studies suggesting different methods for the attainment of optimum arch shape. some authors have suggested using photocopies of the occlusal aspect of the dental casts to select the arch configuration, based on prefig. 3 percentages of mandibular dental arch morphology in the natural normal occlusion. 67 dental arch morphology in normal occlusions 68 braz j oral sci. 10(1):65-68 contoured arches4. another option is the application of a cartesian system to the cast photocopies, identifying x and y axes, to facilitate the visual evaluation of the arch morphology among three preselected shapes (square, tapered, oval)17. recently, some authors presented the digitization of natural normal occlusion casts followed by the application of sixth-degree polynomials, establishing the six most preponderant arch configurations, guiding the orthodontist to choose visually, among these shapes, the one best fit to each patient8. thus, it was observed that, independent of the complexity of the methodology used to determine and choose the dental arch shape, the final choice is made by the orthodontist in a visual way, and so, subjectively. searching in literature, it was observed that several authors had found more than one dental arch shape6,20, but there is not a consent about the amount of joined forms. according to a previous study9, the dental arches of natural normal occlusion individuals presented the anterior region in semicircle and the posterior teeth in straight-line, while the malocclusion arches were represented by four distinct forms. however, other authors, evaluating a sample comprised of normal occlusion and malocclusion casts had found five to six8 different geometric configurations to the mandibular dental arch. despite the authors’ agreement about the diversity of morphologies that best described the dental arch, some differences might have occurred because of the kind of methodology used. the use of three dental arch shapes was initially classified in 193413, looking for the improvement, customization, and simplification of treatment planning. the square morphology is indicated to large dental arches in which it is necessary to preserve morphology after rapid or slow expansion treatment. it presents the anterior region of the arch flat, arranging the central and lateral incisors in straight-line and the posteriors teeth almost vertical in the alveolar bone. in this study, this configuration was found in 39% of the sample, data different from other studies4,17-18. the tapered configuration, unlike the previous one, is usually used in atrophic arches, presenting a smaller intercuspal distance. this shape could be proper to adult patients, because it minimizes the relapses and the periodontal problems happening, in special in patients with thin periodontal profile. the present study showed 21% of tapered arch morphology, similar to other study findings4 and different from others17,20. the oval shape, used often by orthodontists, shows a little greater intercuspal distance than the tapered arches. it was the most common arch morphology in the present sample (41%) as in other studies13,20, and when the ovoid shape was not the majority, it showed significant percentages21. in conclusion, the shape of the mandibular dental arch with highest prevalence was the oval (41%), followed by the square shape (39%), which showed very similar in results. the tapered shape was the lowest prevalent (20%). as a clinical application, at the beginning of the orthodontic treatment, during leveling and alignment phases, elasticalloy-wires are greatly used and, in order to facilitated orthodontist work, any of the studied arch shapes could be used, once the prevalence of all of them was similar. after this, the orthodontist should observed the patient’s dental arch morphology and choose an archwire shape that best fits. references 1. housley ja, nanda rs, currier gf, mccune de. stability of transverse expansion in the mandibular arch. am j orthod dentofacial orthop. 2003; 124: 288-93. 2. little rm, riedel ra, stein a. mandibular arch length increase during the mixed dentition: postretention evaluation of stability and relapse. am j orthod dentofacial orthop. 1990; 97: 393-404. 3. steadman sr. changes of intermolar and intercuspid distances following orthodontic treatment. angle orthod. 1961; 31: 207-15. 4. felton jm, sinclair pm, jones dl, alexander rg. a computerized analysis of the shape and stability of mandibular arch form. am j orthod dentofacial orthop. 1987; 92: 478-83. 5. strang rhw. factors of influence in producing a stable result in the treatment of maloclusion. am j orthod oral surg. 1946; 32: 313-32. 6. biggerstaff rh. three variations in dental arch form estimated by a quadratic equation. j dent res. 1972; 51: 1509. 7. interlandi s. new method for establishing arch form. j clin orthod. 1978; 12: 843-5. 8. trivino t, siqueira df, scanavini ma. a new concept of mandibular dental arch forms with normal occlusion. am j orthod dentofacial orthop. 2008; 133: 10 e5-22. 9. weinberger bw. study of normal dental arches and normal occlusion. dent cosmos. 1914; 56: 665-80. 10. angle eh. treatment of malocclusion of the teeth. 7. ed. philadelphia: ss white manufacting company; 1907. 11. burdi ar. morphogenesis of mandibular dental arch shape in human embryos. j dent res. 1968; 47: 50-8. 12. boone gn. archwires designed for individual patients. angle orthod. 1963; 33: 178-85. 13. chuck gc. ideal arch form. angle orthod. 1934; 4: 312-27. 14. sanin c, savara bs, thomas dr, clarkson qd. arc length of the dental arch estimated by multiple regression. j dent res. 1970; 49: 885. 15. begole ea. application of the cubic spline function in the description of dental arch form. j dent res. 1980; 59: 1549-56. 16. pepe sh. polynomial and catenary curve fits to human dental arches. j dent res. 1975; 54: 1124-32. 17. nojima k, mclaughlin rp, isshiki y, sinclair pm. a comparative study of caucasian and japanese mandibular clinical arch forms. angle orthod. 2001; 71: 195-200. 18. andrews lf. the six keys to normal occlusion. am j orthod. 1972; 62: 296-309. 19. andrews lf, andrews wa. syllabus of the andrews orthodontic philosophy. 9. ed. san diego: lawrence f. andrews foundation; 2001. 20. mclaughlin rp, bennett jc. orthodontic management of the dentition with the preadjusted appliance. 3. ed. oxford: isis medical media; 1997. 21. landis jr, koch gg. the measurement of observer agreement for categorical data. biometrics. 1977; 33: 159-74. 22. fleiss jl. measuring nominal scale agreement among many raters. psychol bull. 1971; 76: 378-82. 23. houston wjb. the analysis of errors in orthodontic measurements. am j orthod. 1983; 83: 382-90. dental arch morphology in normal occlusions oral sciences n3 case report braz j oral sci. january/march 2010 volume 9, number 1 computed tomography findings of periostitis ossificans luciana soares de andrade freitas oliveira1, thaís feitosa l. de oliveira2, daniela pita de melo3, alynne vieira de menezes4, ieda crusoé-rebello5, paulo sérgio flores campos3 1department of health sciences, division of pathology, dental school, federal university of bahia, brazil 2department of oral diagnosis, division of periodontology, state university of feira de santana, brazil 3department of oral diagnosis, division of oral radiology, piracicaba dental school, state university of campinas, brazil 4department of oral diagnosis, division of oral radiology, piracicaba dental school, state university of campinas, brazil 5department of oral diagnosis, division of oral radiology, dental school, federal university of bahia, brazil correspondence to: luciana freitas oliveira av. araújo pinho, 62 disciplina de radiologia, faculdade de odontologia da ufba, canela. cep:40110-150, salvador, ba, brasil. e-mail: lucianafreitasoliveira@yahoo.com.br received for publication: november 22, 2009 accepted: march 29, 2010 abstract periostitis ossificans (po) is a type of chronic osteomyelitis, an inflammation of cortical and cancellous bone. in the maxillofacial region, the mandible is most frequently affected. the cause of inflammatory subperiosteal bone production in po is spread of infection from a bacterial focus (e.g.: odontogenic disease, pulpal or periodontal infection, and extraction wounds). this pathology is most common in younger people (mean age of 13 years). conventional radiographs are one of the most useful tools for diagnosis, but in some cases computed tomography (ct) has a key role in both diagnosis and identification of the tissues involved. this paper reports two cases of po in which ct helped establishing the suspicious etiology: a 12-year-old boy with po of pulpal origin and a 14-year-old boy with po of periodontal origin. keywords: osteomyelitis, tomography. introduction periostitis ossificans (po) is a type of chronic osteomyelitis that is more popularly known as garrè’s osteomyelitis. osteomyelitis is defined as an inflammation of cortical and cancellous bone. in the maxillofacial region, the mandible is most frequently affected1-4. most reported cases are unifocal and unilateral3. po represents a periosteal reaction to the presence of inflammation. the affected periosteum forms several rows of reactive vital bone that are parallel to each other and expand to the surface of the altered bone5-6. the cause of inflammatory subperiosteal bone production in po, affecting the mandible or maxilla, is spread of infection from a bacterial focus (e.g. odontogenic disease, pulpal and periodontal infection, extraction wounds, foreign bodies, and infected fractures), perforating the cortex and becoming attenuated, which in turn stimulates bone formation by the periosteum1-2. however, in some cases, the cause of osteomyelitis is not clear1. inflammatory exudates spread subperiosteally, elevating the periosteum and stimulating formation of new bone. high-quality radiographs reveal radiopaque bone laminations that are arranged roughly parallel to each other and to the underlying cortical surface. these laminations vary from 1 to 12 in number, and radiolucent separations are often present between the new bone and the original cortex. periapical, occlusal and panoramic radiographs are often used for diagnosis of po and have a high diagnostic braz j oral sci. 9(1):59-62 value. however, at the initial osteomyelitis stage, there is no radiographic evidence and diffuse radiolucency begins to appear with time. computed tomography (ct) may also be used and it is accurate for detecting not only typical alterations inside the bone, but also periosteal reactions and soft tissue involvement. ct is also helpful for determining the extension of bone involvement and the relationship with adjacent anatomic structures7. however, ct has been used for diagnosis in only a few cases reported in the literature6,89. po is usually caused by a periapical infection secondary to caries or pulp infection, but it might be associated with a periodontal pocket occasionally. this paper reports two cases of po, one of pulpal origin and another of periodontal origin, emphasizing the diagnostic role of ct. case report case 1 a 12-year-old male patient was referred to our clinic with complaint of swelling on the posterior region of the left mandible. on the clinical examination, the patient referred diffuse pain on palpation in the affected area and lymphadenopathy, though without evidence of suppuration. multislice ct (msct) scans showed an increase in the cortical bone and volume of the mandible and periosteal new bone formation. a hyperdense area surrounding the left premolar region up to the ascending ramus of the mandible on the same side was observed, leading to mandible cortical duplication on the buccal surface (figure 1). a hypodense lesion related to the periapical region of the first molar was seen, indicating endodontic involvement (figure 2). a threedimensional volume rendering reconstruction showed facial asymmetry (figure 3). the msct images also showed hyperplasic lymph node causing soft tissue distension and sensitive palpation (figure 4). fig. 1. msct axial images showing periosteal new bone formation. fig. 2. cross-sectional images showing pulpal involvement of the first molar. fig. 3. msct three-dimensional volume rendering reconstruction showing an increase of volume due to new bone formation. fig. 4. msct axial image showing hyperplasic lymph node. a bone biopsy was performed and the material was sent for histological evaluation, confirming the diagnosis of po. case 2 a 14-year-old male patient was referred to our clinic with complaint of swelling on the posterior region of the left mandible. on clinical examination, the patient reported diffuse pain on palpation of the affected area. msct scans revealed a slight increase in the cortical bone and periosteal new bone formation. there were distinct changes of periosteal bone on the left mandible (figure 5), cortex duplication (figure 6) associated with the sound second molar, which was partially erupted and exhibited local bone fenestration (figure 7a and 7b). bone biopsy was performed and the material was sent for histological analysis, confirming the diagnosis of po. discussion proliferative periostitis was first described by karl garrè in 1893. since then, several terms have been used to describe 60 braz j oral sci. 9(1):59-62 computed tomography findings of periostitis ossificans fig. 7. (a) three-dimensional volume rendering image showing a slight increase in volume on the posterior region of the left side of the mandible. (b) opacity filter showing a slight destruction of the vestibular wall of the second molar. fig. 6. coronal slices showing a discrete increase in volume, with cortex duplication, on the left side of the mandible associated with the second molar showing periodontal involvement. this reactive proliferation of periosteum, including proliferative periostitis of garrè, garrè’s osteomyelitis, nonsuppurative ossifying periostitis, osteomyelitis sicca, osteomyelitis with proliferative periostitis, perimandibular ossification and po, as referred to in this paper3,10. the most frequent cause of po is dental caries with associated periapical inflammatory disease. most cases arise in the mandibular premolar-molar area, but buccal cortex involvement is also common6. there are other causes, including recent dental extraction or mild periodontitis, but only a few reports have discussed an association between periodontal infection and po in pediatric dental patients4,11-12. it is generally accepted that removal of the cause results in resolution of the infection and remodeling of the excessive bone4. two cases of po are described in this paper: the first associated with periapical infection of a permanent first molar, and a second case of periodontal origin. this association is not common, but cortical changes and bone perforation can be observed on ct images. teeth close to po usually seem sound, with no signs of pulpal involvement or apical periodontitis. local bone loss can be observed, as reported herein. bone loss was also reported by van den bossche et al.12, who were the first to describe this association. during clinical examination, those authors found a probing pocket depth of 10 mm in the mandibular right first molar. oulis et al.11 also found bone loss on clinical examination and the associated tooth showed a probing pocket depth of 8 mm. both cases presented here affected the mandible. this can be expected because the distribution of blood vessels is poorer in the mandible than in the maxilla, and the mandibular cortical bone is thicker and more compact than the maxillary bone. the typical course of this disease is observed in the mandible rather than in the maxilla9. according to ida et al.13, 76% of cases of po take place between the mandibular premolars and second molars, as seen in the cases reported here. it usually occurs in children and adolescents11 (mean age of 13 years)3-4 because in these individuals the periosteum is loosely attached to the bone surface and has greater osteogenic potential6,14. the patients of the present cases were two male adolescents aged 12 and 14 years, which is consistent with the cases reported in the literature. the clinical appearance may vary widely, but the lesion is usually asymptomatic with no accompanying general or local signs of inflammation. however, both patients of this paper were referred for radiological examination because of pain. there are some case reports of patients who did not experience the acute phase of the disease, evolving directly to a chronic stage of po13. this is why early diagnosis is one of the most important factors for a successful management of this pathology13. modern diagnostic imaging techniques such as ct allow an earlier diagnosis when the bone infection is still in a more localized stage15. case 1 was at a late stage and new bone formation could be observed. in case 2, the diagnosis of po was made at an earlier stage, in which only a discrete bone expansion could be observed. ct examination is very important because of its contribution to the differential diagnosis of po and other known pathologies that also cause bone expansion, such as fibrous dysplasia, ewing sarcoma, osteogenic sarcoma, infantile cortical hyperostosis, callus, exostosis, calcifying hematoma, and osteotomas. ct is very important in the investigation of the possible causes of the disease as well as in determining the growing pattern and nature of the lesion. however, a final diagnosis can only be achieved by lesion biopsy1,4,14. ct scans also allow visualizing all involved and surrounding structures, which is essential for case follow-up8. fig. 5. axial image showing distinct changes on the cortical bone of the left mandible. 61 braz j oral sci. 9(1):59-62 computed tomography findings of periostitis ossificans the two cases of po presented in this paper had different causes: pulpal infection (classic cause) and periodontal infection (unusual cause), and ct had a fundamental role in the diagnosis. references 1. eyrich gkh, baltensperger mm, bruder e, graetz kh. primary chronic osteomyelitis in childhood and adolescence: a retrospective analyses of 11 cases and review of literature. j oral and maxillofac surg. 2003; 61: 561-73. 2. benca pg, mostofi r, kuo pc. proliferative periostitis (garré’s osteomyelitis). oral surg oral med oral pathol. 1987; 63: 258-60. 3. kannan sk, sandhya g, selvarani r. periostitis ossificans (garrè’s osteomyelitis): radiographic study of two cases. int j paediatr dent. 2006; 16: 59-64. 4. tong ack, ng iol, au yeng km. osteomyelitis with proliferative periostitis: an unusual case. oral surg oral med oral pathol oral radiol endod. 2006; 102: e14-9. 5. neville bw, damm dd, allen cm, bouquot je. pulp and periapical diseases. in: neville bw, editor. oral and maxillofacial pathology. 2nd edn. rio de janeiro: w.b. saunders; 2004. p.128-9. 6. zand v, lofti m, vosoughhrosseini s. proliferative periostitis: a case report. j endod. 2008; 34: 481-3. 7. schulze d, blessmann m, pohlenz p, wagner kw, heiland m. diagnostic criteria for the detection of mandibular osteomyelitis using cone-beam computed tomography. dentomaxillofac radiol. 2006; 35: 232-5. 8. ebihara a, yoshioka t, suda h. garrè’s osteomyelitis managed by root canal treatment of a mandibular second molar: incorporation of computed tomography with 3d reconstruction in the diagnosis and monitoring of the disease. int endod j. 2005; 38: 255-61. 9. tanaka r, hayashi t. computed tomography findings of chronic osteomyelitis involving the mandible: correlation to histopathological findings. dentomaxillofac radiol. 2008; 37: 94-103. 10. nakano h, miki t, aota k, sumi t, matsumoto k, yura y. garré’s osteomyelitis of the mandible caused by an infected wisdom tooth. oral science international. 2008; 5: 150-4. 11. oulis c, berdousis e, vadiakas g, goumenos g. garre’s osteomyelitis of an unusual origin in a 8-year-old child. a case report. int j paediatr dent. 2000; 10: 240-4. 12. van den bossche lh, demeulemeester jda, bossuyt mh. periodontal infection leading to periostitis ossificans (‘garrè’s osteomyelitis’) of the mandible. report of a case. j periodontol. 1993; 64: 60-2. 13. ida m, watanabe h, tetsumura a, kurabayashi t. ct findings as a significant predictive factor for the curability of mandibular osteomyelitis: multivariate analysis. dentomaxillofac radiol. 2005; 34: 86-90. 14. lee l. lesões inflamatórias dos maxilares. in: white sc, pharoah mj, editors. radiologia oral. fundamentos e interpretação. 5.ed. rio de janeiro elsevier; 2007. p.370-5. 15. baltensperger m, eyrich g. osteomyelitis of the jaws: definition and classification. in: baltensperger m, eyrich g, editors. osteomyelitis of the jaws. springer; 2009. p. 5-56. 62 braz j oral sci. 9(1):59-62 computed tomography findings of periostitis ossificans oral sciences n3 original article braz j oral sci. january | march 2012 volume 11, number 1 prevalence of dental trauma among 6-7-yearold children in the city of recife, pe, brazil bruno carvalho1, carolina franca2, mônica heimer2, sandra vieira2, viviane colares2 1master’s degree student in pediatric dentistry, dental school, university of pernambuco, brazil 2associate professor of pediatric dentistry, dental school, university of pernambuco, brazil correspondence to: bruno carvalho universidade de pernambuco av. agamenon magalhães, s/n santo amaro cep: 50100-010 recife, pe, brasil phone: +55 81 99258400 fax: +55 81 32223552 e-mail: brunocarvalho@ortodontista.com.br received for publication: october 06, 2011 accepted: march 15, 2012 abstract dental trauma has a great impact on quality of life, affecting children physically, esthetically and psychologically. aim: to assess the prevalence of dental trauma in 6-7-year-old northeastern brazilian children and its correlation with gender. methods: this cross-sectional study was carried out with 1,791 children attending 20 public schools in the city of recife, brazil. data collection included oral examination to identify indicators of traumatic dental injury, such as crown discoloration in incisors, and was conducted by 4 previously trained graduate students (kappa > 0.8). traumatic dental injuries were classified according to andreasen’s criteria. root fracture was not recorded as no radiographs were taken. statistical analysis was performed using pearson’s chi-square at a 5.0% level of significance. results: the prevalence of dental trauma was 9.1% and males were significantly more affected. crown discolorations followed by enamel fracture were, respectively, the injuries indicators more frequently observed in primary and permanent dentition. the most affected teeth were the upper central incisors in both dentitions. conclusions: the prevalence of dental trauma in primary teeth was low among 6-7-year-old children in the city of recife and showed association with gender. keywords: tooth injuries, child, prevalence, preschool, oral health. introduction the decline in the prevalence and severity of dental caries among children in many countries, including brazil, can make dental trauma into one of the most serious public health problems. moreover, dental trauma causes a great impact on quality of life, affecting children physically, esthetically and psychologically1-3. some studies reported a prevalence of traumatic dental injuries ranging from 5.02% to 62.1% (table 1) in primary teeth and from 6.4% to 27.56% in the permanent dentition (table 2). this variation may be caused by a number of factors such as: differences in data collection method, sample selection and place where the study was conduced20,21. regarding age of highest prevalence of trauma in primary teeth, a great discrepancy among findings is observed, as it ranges between 0 and 5 years2,3,6,21-24. at this age range, children’s poor motor skills do not allow precise and safe movements23. as to permanent dentition, a higher incidence is observed in the 910-year-old age group24,25. luxations are more common than fractures in the primary dentition because of the resilient bone and surrounding periodontal tissues. meanwhile, crown fracture is the most frequent dental injury in the permanent dentition22,24,26. the maxillary central incisors have been reported as the most frequently injured teeth in both primary and permanent dentitions. crown discoloration is a complication of dental trauma and it is very common in children1-3,10,12,13,22,24,26. braz j oral sci. 11(1):72-75 some studies claim that boys are more prone to dental trauma than girls1,24,26, which may be explained by male’s higher participation in sports and physical activities.the most frequent etiologic factor for the occurrence of dental trauma is fall from the child’s own height, followed by collision against a hard object 1,2,5,7,22,26. in the literature, there is a shortage of population-based studies investigating the prevalence of dental trauma and its etiological factors in the age range of 6-7 years. therefore, this study was carried out to assess the prevalence of traumatic dental injuries in 6-7-year-old northeastern brazilian children and its correlation with gender. material and methods this study was carried out in the city recife, the capital of pernambuco state, located in the northeastern region of brazil. according to the brazilian institute of geography and statistics, this city has a total area of 220,000 km2. in accordance with data supplied by the department of education of recife, children start elementary education at the age of six. about 144,000 schoolchildren are enrolled in municipal schools, of which almost 100,000 are in elementary education. the city of recife has a total of 214 public schools run by the municipality. the sample size was calculated using the epi-info 6.0 software program. the estimated prevalence for dental trauma was 10.0%, based on a similar population27. the precision index adopted was 3.0% with a margin of error of 5.0%. because the subjects were not selected at random, but per conglomerate, the correction factor of 2.1 was used. minimum sample size for fulfilling the requirements was estimated at 800 children. to compensate for possible losses during the survey of data, the sample size was increased by 20.0%, resulting in a final sample of 960 children. this cross-sectional study was carried out with 1,791 children attending 20 public schools in recife. the data were collected in larger amounts than the minimum required because other factors in this sample were investigated for other study; however we maintain the proportion of children in the schools that have participation in this study. the schools were selected by lottery and following the proportion of children. data collection included an oral examination conducted in classrooms under natural illumination to identify the type of the traumatic injury to the incisors teeth was conducted by 4 dentists (postgraduate students) trained in the calibration and standardization of the criteria used. inter-examiner agreement was satisfactory (kappa > 0.8). the calibration was placed with photographs and compared with a gold standard. traumatic dental injuries were recorded according to the method described by andreasen, andreasen20 for dental trauma. root fracture was not recorded and no radiographs were taken. crown discoloration indicating pulp damage was included in the criteria. the data were subjected to simple descriptive analysis and the statistical analysis was done with spss (statistical package for the social sciences) version 11 and sas (statistical analysis system) version 8. pearson’s chi-square test was used and a 5.0% margin of error was allowed in evaluating the reliability of the statistical tests. author year country sample age dental trauma (%) (n of children) oliveira et al. 4 2007 brazil 892 5-59 months 9.4 feldens et al. 5 2008 brazil 376 first year 14.3 ferreira et al. 6 2009 brazil 3489 < 5 years 14.9 tumen et al.1 2009 turkey 657 5-72 months 5.0 jorge et al. 7 2009 brazil 519 1-3 years 41.6 feldens et al 8 2010 brazil 888 3-5 years 36.4 viegas et al 9 2010 brazil 388 60-71 months 62.1 wendt et al 10 2010 brazil 571 12-71 months 36.6 granville-garcia et al 11 2010 brazil 820 1-5 years 20.1 table 1.table 1.table 1.table 1.table 1. prevalence of traumatic dental injuries in primary teeth in different studies. artun et al 12 2005 kuwaiti 1583 13-14 14.9 fakhruddin et al 13 2008 ontario 2422 12-14 11.4 lin & naidoo 14 2008 south africa 290 10-14 9.3 naidoo et al 15 2009 south africa 1665 11-13 6.4 adekoya-sofowora et al. 16 2009 nigeria 415 12 12.8 cavalcanti et al 17 2009 brazil 448 7-12 21.0 huang et al 18 2009 taiwan 6312 15-18 19.2 navabazam & farahani 19 2010 iran 1440 9-14 27.5 author year country sample age dental trauma (%) (n of children) table 2.table 2.table 2.table 2.table 2. prevalence of traumatic dental injuries in permanent teeth in different studies. 7373737373 prevalence of dental trauma among 6-7-year-old children in the city of recife, pe, brazil braz j oral sci. 11(1):72-75 trauma gender male female total p-value n % n % n % yes 102 11.2 61 6.9 163 9.1 p (1) = 0.002* n o 811 88.8 817 93.1 1628 90.9 total 913 100.0 878 100.0 1791 100.0 table 3.table 3.table 3.table 3.table 3. distribution of children considering the gender and presence or not of dental trauma (*): significant association at 5.0%. (1): using the pearson’s chi-square test. table 4.table 4.table 4.table 4.table 4. prevalence of dental trauma in children considering the dentition and different types or sequelae of trauma type of trauma dentition total primary permanent n % n % n % enamel fracture 49 31.4 3 42.8 52 34.9 enamel-dentin fracture 5 3.2 3 42.8 8 4.1 extrusive luxation 6 3.8 6 3.5 lateral luxation 3 2.0 1 14.4 4 2.3 intrusive luxation 3 2.0 3 2.3 avulsion 1 0.6 1 0.6 crown discoloration 89 57.0 89 52.3 total 156 100.0 7 100.0 163 100.0 the parents or guardians were previously informed of the purpose and methods of this study and their written informed consent was obtained. the study design was in accordance with the principles of the helsinki declaration and was approved by the ethics committee of the university of pernambuco, brazil (protocol # 211/09). results the prevalence of dental trauma in both dentitions was 9.1% (n=163) and male children were significantly more affected by trauma (p>0.05) (table 3). among 1,791 children examined, 163 were affected by trauma. the most common indication of dental trauma was crown discoloration (5.0%), followed by enamel fracture (2.9%), enamel-dentin fracture (0.5%), extrusive luxation (0.3%), lateral luxation (0.2%), intrusive luxation (0.2%) and avulsion (0.0%). the primary dentition (n=156) was more affected than the permanent dentition (n=7) in the age range considered (table 4). in all children, 14,000 teeth were examined, being 8,078 (57.7%) permanent incisors and 5,922 (42.3%) primary incisors. with regard to permanent teeth, only 0.15% (n=7) had dental trauma. the maxillary central incisors were the only affected teeth and the types of trauma were: enamel fracture (n=6), enamel-dentin fracture (n=4) and luxation (n=2). with regard to primary teeth, 3.6% (n=163) were affected by dental trauma. the maxillary teeth were again the only affected, firstly the maxillary central incisors (89.2%) followed by the maxillary lateral incisors. the most common indicator of dental trauma in the teeth of primary dentition were color change (57%) followed by enamel fracture (31.4%). the other 11.6% (n=18) were due to luxation (n=12), enamel-dentin fracture (n=5), and avulsion (n=1). discussion the prevalence of dental trauma among 6-7-year-old children reported in this study was 9.1%. however, the prevalence of children with dental trauma in the primary dentition was 8.7%. this result is similar to the one reported by oliveira et al.4, higher than that found by tumen at al.1 and lower than that reported by other authors5-11. the low prevalence of children with dental injury in permanent teeth (0.4%) can be explained by the fact that the sample consisted of children in the mixed dentition phase, most of them showing permanent incisors in the process of eruption. it is important to bear in mind that the root fracture was not recorded, as no radiographs were taken, which certainly accounted for the low number of traumas. regarding gender, boys had more injuries than girls. many other authors also reported a similar pattern1,15,19,22,27,28. this difference can be attributed to the greater participation of boys in contact sports, fights and more aggressive types of playing activities28. however, others studies indicated an increasing trend of dental trauma among girls, probable because of their increasing participation in sports or activities formerly practiced by boys only29. 7474747474prevalence of dental trauma among 6-7-year-old children in the city of recife, pe, brazil braz j oral sci. 11(1):72-75 for both primary and permanent teeth, dental trauma occurred more frequently in the maxilla involving central incisors. this finding is in accordance with previous studies1-3,6,7,10,11,22,24,26,27. the most common type of injury in the primary dentition was crown discoloration (60.6%) followed by enamel fracture (29.1%). however, in the study by tumen et al.1 these results were inverted: enamel fracture was followed by crown discoloration. other authors2,22,24,28 who also assessed primary dentition found a higher prevalence of the concussion and luxation. in fact, crown discoloration is a complication of the trauma. in the present study, this complication was high in the primary dentition and it can have consequences to the permanent incisors. this result might be due to the fact that this part of the population has some difficulties to access the public dental service and do not seek treatment at the time of the trauma. the other injuries that followed crown discoloration were of small magnitude, although a large number of need of treatments was observed, in the same way as reported by traebert30. regarding the permanent dentition, only 7 of a total of 8,078 teeth presented dental trauma, fracture being the most prevalent type of injury. these results agree with the findings of naidoo et al.15 and navabazam & farahani19, who claim that the permanent dentition is mostly affected by crown fracture than luxation injury. it is possible that primary teeth were more associated with luxation, while permanent teeth were more associated with fracture because skeleton plasticity decreases as the patient gets older. our results indicate that the prevalence of dental trauma in primary teeth was low among 6-7-year-old children in the city of recife and showed association with gender. references 1. tumen ec, adiguzel o, kaya s, uysal e, yavuz i, atakul f. the prevalence and etiology of dental trauma among 5-72 months preschool children in south-eastern anatolia, turkey. j int dent med res. 2009;2:40-4. 2. cardoso m, de carvalho rocha mj. traumatized primary teeth in children assisted at the federal university of santa catarina, brazil. dent traumatol. 2002;18:129-33. 3. cortes mis, marcenes w, sheiham a. impact of traumatic injuries to the permanent teeth on the oral health-related quality of life of 12-14-year-old children. community dent oral epidemiol. 2002;30:193-8. 4. oliveira lb, marcenes w, ardenghi tm, sheiham a, bönecker m. traumatic dental injuries and associated factors among brazilian preschool children. dent traumatol. 2007;23:76-81. 5. feldens ca, kramer pf, vidal sg, faraco junior im, vítolo mr. traumatic dental injuries in the first year of life and associated factors in brazilian infants. j dent child. 2008;75:7-13. 6. ferreira jms, andrade emf, katz crt, rosenblatt a. prevalence of dental trauma in deciduous teeth of brazilian children. dent traumatol. 2009;25:219-23. 7. jorge ko, moysés sj, ramos-jorge ml, zarzar pmpa. prevalence and factors associated to dental trauma in infants 1-3 years of age. dent traumatol. 2009;25:185-9. 8. feldens ca, kramer pf, ferreira sh, spiguel mh, marquezan m. exploring factors associated with traumatic dental injuries in preschool children: a poisson regression analysis. dent traumatol. 2010;26:143-8. 9. viegas cm, scarpelli ac, carvalho ac, ferreira fm, pordeus ia, paiva sm. predisposing factors for traumatic dental injuries in brazilian preschool children. eur j paediatr dent. 2010;11: 59-65. 10. . wendt fp, torriani dd, assunção mc, romano ar, bonow ml, da costa ct et al. traumatic dental injuries in primary dentition: epidemiological study among preschool children in south brazil. dent traumatol. 2010; 26:168-73. 11. granville-garcia af, vieira it, siqueira mj, de menezes va, cavalcanti al. traumatic dental injuries and associated factors among brazilian preschool children aged 1-5 years. acta odontol latinoam. 2010;23:47-52. 12. 12.. artun j, behbehani f, al-jame b, kerosuo h. incisor trauma in a adolescent abab population: prevalence, severity, and occlusal risk factors. am j orthod dentofacial orthop. 2005;128:347-52. 13. 13.. fakhruddin ks, lawrence hp, kenny dj, locker d. etiology and environment of dental injuries in 12to 14-year-old ontario schoolchildren. dent traumatol. 2008;24:305-8. 14. lin h, naidoo s. causes and prevalence of traumatic injuries to the permanent incisors of school children aged 10-14 years in maseru, lesotho. sadj. 2008;152:154-6. 15. naidoo s, sheilham a, tsakos g. traumatic dental injuries of permanent incisors in 11 – to 13-year-old south african schoolchildren. dent traumatol. 2009;25:224-8. 16. adekoya-sofowora ca, adesina oa, nasir wo, oginni ao, ugboko vi. prevalence and causes of fractured permanent incisors in 12-year-old suburban nigerian schoolchildren. dent traumatol. 2009;25:314-7. 17. cavalcanti al, bezerra pkm, de alencar crb, moura c. traumatic anterior dental injuries in 7to 12-year-old brazilian children. dent traumatol. 2009;25:198-202. 18. huang b, marcenes w, croucher r, hector m. activities related to the occurrence of traumatic dental injuries 15to 18-years-olds. dent traumatol. 2009;25:64-8. 19. navabazam a, farahani ss. prevalence of traumatic injuries to maxillary permanent teeth in 9to 14-year-old school children in yazd, iran. dent traumatol. 2010;26:154-7. 20. 20 andreasen jo, andreasen fm. textbook and color atlas of traumatic injuries to the teeth. 3rd ed. copenhagen: munsksgaard; 1994. 21. kramer pf, zembruski c, ferreira sh, feldens ca. traumatic dental injuries in brazilian preschool children. dent traumatol. 2003;19:299-303. 22. de jesus ma, antunes laa, risso pa, freire mv, maia lc. epidemiologic survey of traumatic dental injuries in children seen at the federal university of rio de janeiro, brazil. braz oral res. 2010;24:89-94. 23. do espírito santo jácomo d, campos v. prevalence of sequelae in the permanent anterior teeth after trauma in their predecessors: a longitudinal study of 8 years. dent traumatol. 2009;25:300-4. 24. eyuboglu o, zehir yyc, sahin h. a 6-year investigation into types of dental trauma treated in a pediatric dentistry clinic in eastern anatolia region, turkey. dent traumatol. 2009;25:110-4. 25. andreasen jo, andreasen fm, bakland lk, flores mt. epidemiology of traumatic dental injuries. in: traumatic dental injuries – a manual. 2nd ed. iowa: blackwell munksgaard; 1999. p.8-9. 26. de carvalho rocha mj, cardoso m. traumatized permanent teeth in brazilian children assisted at the federal university of santa catarina, brazil. dent traumatol. 2001;17:245-9. 27. beltrão em, cavalcant al, albuquerque ss, duarte rc. prevalence of dental trauma children aged 1-3 years in joão pessoa (brazil). eur arch paediatr dent. 2007;8:141-3. 28. noori aj, al-obaidi wa. traumatic dental injuries among primary school children in sulaimani city, iraq. dent traumatol. 2009;25:442-6. 29. rocha mjc, cardoso m. traumatized permanent teeth in brazilian children assisted at the federal university of santa catarina, brazil. dent traumatol. 2001;17:245-9. 30. traebert j, marcon kb, lacerda jt. prevalence of traumatic dental injuries and associated factors in schoolchildren of palhoça, santa catarina state. cien saude colet. 2010;15:1849-55. 7575757575 prevalence of dental trauma among 6-7-year-old children in the city of recife, pe, brazil braz j oral sci. 11(1):72-75 oral sciences n3 braz j oral sci. 11(2):141-147 original article braz j oral sci. april | june 2012 volume 11, number 2 relationship between craniocervical posture, mandible and hyoid bone and influence on alimentary functions priscila weber1, eliane castilhos rodrigues corrêa2, geovana de paula bolzan3, fabiana dos santos ferreira4, juliana corrêa soares1, ana maria toniolo da silva5 1physical therapist, msc in human communication disorders, federal university of santa maria, ufsm, brazil 2professor, department of physical therapy, federal university of santa maria, ufsm, brazil 3speech-language pathologist, msc in human communication disorders, federal university of santa maria, ufsm, brazil 4physical therapist, graduate student at program of human communication disorders, federal university of santa maria, ufsm, brazil 5professor, department of speech therapy, federal university of santa maria, ufsm, brazil correspondence to: priscila weber avenida presidente vargas 1855/1101 centro cep: 97015-513 santa maria, rs brasil phone: +55 19 99678173 e-mail: prifisio07@yahoo.com.br abstract aim: to evaluate the anatomical relationship between craniocervical posture and mandibular and hyoid bone position and the influence on the masticatory and swallowing functions. methods: thirty-six women aged 19 to 35 years without temporomandibular disorder diagnosis were evaluated. variables related to the craniocervical posture and mandibular and hyoid bone position were obtained by cephalometric measurements. masticatory and swallowing function evaluations were performed according to a protocol of orofacial myofunctional evaluation with scores. results: significant correlations were observed between craniovertebral angle and the linear distance from the hyoid bone to the mentum (p=0.02) and to the mandible (p=0.03). the angle that measured the forward head position also demonstrated a significant correlation with the linear distance between hyoid bone and jaw (p=0.00). the cervical curvature degree showed a significant correlation with the linear distance from hyoid bone to the third cervical vertebra (p=0.01). modifications of the cranium base inclination in relation to the cervical column at the two levels (nsl/cvt and nsl/opt) were the only variables, which showed a significant correlation with the mandible position. important craniocervical postural changes were observed in the subjects, although there was no association between them and a higher frequency of atypical behaviors evaluated during masticatory and swallowing functions. conclusions: cephalometric findings confirm the anatomical relationship between craniocervical posture, mandible and hyoid bone. however, association between craniocervical posture alterations and masticatory and swallowing function impairments was not detected. such findings suggest that, in the presence of a musculoskeletal imbalance, the body readapts itself ensuring that alimentary functions will not be affected. keywords: posture, mastication, deglutition, cephalometry. introduction considering the human biomechanical principles, it is known that there is interdependence between the structural condition and the action of the movement. in other words, the muscle action, on which the performance of several functions of the organism depends, is determined by the muscle-skeletal balance relationship received for publication: april 25, 2012 accepted: june 15, 2012 braz j oral sci. 11(2):141-147 142142142142142 of the body segments observed in the posture called ideal1. therefore, the influence of the craniocervical posture on the functionality of the stomatognathic system may be based on the concept of the craniocervicomandibular unit, which defines the muscle-skeletal relationship covered by head, neck and mandible2. the hyoid bone, despite not being articulated to the system, it maintains muscle connections with the cranium basis and with the mandibular symphysis, and fascial connections with the cervical column3. a synergism between the tonic-postural cervical, masticatory, supra and infrahyoid muscles is established up to the point that a craniocervical change may alter it and consequently, the mandibular and hyoid bone position as well4. the conditions of mandibular and hyoid bone at rest, in turn, are determinants in the performance of the stomatognathic alimentary functions. the efficient chewing is exerted through a coordinated interaction between the mandible, tongue and hyoid movements with the occlusal plane alignment and the masticatory muscle performance5. during swallowing, the stabilization of the mandible to the cranium is the first physiological event that permits the action of the tongue and hyoid in the food bolus propulsion. the antero-superior displacement of the hyoid bone is also essential in the airway protection against the aspirations and in the food passage through the upper esophageal sphincter6-7. based on the above-mentioned, it can be inferred that the aligned craniocervical posture is necessary for a good performance during alimentary functions. however, no similar study was found aiming to investigate qualitatively the craniocervical posture simultaneous to the clinical evaluation of myofunctional orofacial ability during masticatory and swallowing functions. on the other hand, it was found an increasing number of researchers concerned about elucidating the still unclear relationship between stomatognathic and postural systems89. for this purpose, several mechanical or electronic devices are used such as surface electromyography, kinesiography, postural platforms, among other tools. however, their use in the clinical practice, beyond being far from most professional’s reality, has been criticized because of the absence of normative values controlled for age, sex, weight, and facial morphology. thus, it still remains essential to deepen into this issue, encouraging the application of a functional instrumental assessment in order to answer more specifically some daily clinical questions of the professionals involved in the diagnostic and treatment of patients who suffer from disturbances in any of these systems. therefore, the aim of the present study was to investigating the anatomical relationship between the craniocervical posture, mandible and hyoid bone position, by means of the cephalometric analysis, as well as the influence on the masticatory and swallowing functions. due to the scope of the subject, this study emphasized only these functions, excluding breathing and phonoarticulation, which are also influenced by the craniocervical posture and could be investigated in further studies. material and methods this study was a transversal and observational investigation with a quantitative approach. females aged 19 to 35 years, interested in a free functional evaluation of the orofacial and cervical regions, were invited to take part in this study. the research project was approved by the ethics committee of the federal university of santa maria (ufsm), under protocol number 0048.0.243.000-08. the exclusion criteria of the study were: temporomandibular disorder (tmd) diagnosis, facial trauma, craniomandibular and/or cervical surgical procedures, musculoskeletal deformities, class ii and ii subdivision malocclusion, tooth loss, anterior and posterior open bite, cross bite, level bite and overbite, as well as current use of orthodontics appliance. for subject selection, the tmd diagnosis was evaluated by the same examiner, according to research diagnostic criteria for temporomandibular disorder (rdc/tmd)10. malocclusion was evaluated by an orthodontist by observation of intraoral photographs . from august 2010 and may 2011, 94 subjects contacted the researcher as answer to an invite published in press and electronic media. from these, 58 were excluded and oriented to look for specialized treatment. the subjects with tmd were invited to take part in the project of physical therapy at ufsm. the body mass index (bmi) and age were analyzed to characterize and standardize the volunteers. therefore, the study was carried out with 36 women who agreed in taking part in the study by signing an informed consent form. for the cephalometric analysis, the volunteers underwent a right lateral cranium and cervical column radiograph in orthostatic position. in order to reproduce the natural head positioning, the volunteers were oriented to glance at their eyes in a mirror placed in one-meter distance11. the radiograph was carried out using the orthophos plus x-ray unit (siemens, german) with 1.52 m focus-film distance. the exam was in accordance to the radioprotection protocol (resolution 453 of 06/01/1998, brazilian health ministry). the variables were measured manually by a single examiner. twenty radiographs were randomly selected for a second analysis after 1 week in order to verify the cephalometric trace reliability. the anatomical points used in the cephalometric analysis are described in figure 1. the angles and lines traced from these points are shown in chart 1. chart 2 describes the cephalometric variables: five related to the craniocervical posture3,12-13, one related to the mandibular position12 and three related to the hyoid bone position14. in order to investigate the influence of the craniocervical posture on the mastication and swallowing functions, the cervical curvature and the flexion/extension head position were interpreted based on their reference values. the forward head posture was classified in more or less accentuated. relationship between craniocervical posture, mandible and hyoid bone and influence on alimentary functions 143143143143143 fig. 1 anatomical points used in the cephalometric analysis: ans – anterior nasal spine; pns– posterior nasal spine; n (nasion) –anterior point at fronto-nasal suture; s (sella) – center of sella turcica; h – most anterosuperior point of the hyoid bone; o – basi-occiput; rgn (retrognathion) – the most inferior posterior point at the mandibular symphysis; me (mentum) – most inferior point at the mandibular symphysis; cv2tg the tangent point at the superior posterior extremity of the odontoid process of the second cervical vertebra (c2); cv2ip the most inferoposterior point on the body of the second cervical vertebra; cv2ap – tangent point to the apex of the c2 dente;; cv2ia the most infero-anterior point on the body of the second cervical vertebra; cv3ai – the most anterior inferior point on the body of the third vertebra; cv4ip the most infero-posterior point on the body of the fourth cervical vertebra; cv6ip the most infero-posterior point on the sixth cervical vertebra; ce1 a ce7 – central points of the vertebral bodies from c1 to c7. the relation of the cervical curve was obtained from the hyoid triangle trace. the triangle consists in the union of the cv3ai rgn, cv3ai h, and h – rgn points (chart 1). the curve is determined by the hyoid position in relation to the cv3ai – rgn line: hyoid placed till 5 mm bellow this line refers to the physiological cervical lordosis, hyoid on the line means cervical rectification and above this line it corresponds to curve inversion3. the craniovertebral angle (cva) classifies gradually the anteroposterior cranium position related to the cervical column: cva from 96 to 106° corresponds to the normal head posture, smaller than 96° corresponds to the head extension and greater than 106° corresponds to the head flexion3. based on the cpl angle9, the subjects were classified according to more (cpl < 80°) or less (cpl > 80º) accentuated forward head posture. the evaluation of the masticatory and swallowing functions was based on the orofacial myofunctional evaluation protocol with scores (amiofe)15 and was conducted by a single qualified speech therapist. both functions were tested with bread. particularly in the swallowing function evaluation, water was also offered to the volunteers during the exam. in the swallowing evaluation, the labial occlusion with effort or the absence of the oral cavity sealing was considered atypical, while the labial occlusion with no e f f o r t w a s c o n s i d e r e d t y p i c a l . t o n g u e i n t e r p o s i t i o n between the dental arches corresponds to the atypical pattern. the tongue contained in the oral cavity in contact w i t h t h e h a r d p a l a t e d u r i n g s w a l l o w i n g a c t i o n w a s considered as typical. other evaluated signs of alterations were the presence of the head movement and the facial muscle tension during the function. concerning the masticatory function, the atypical pattern is characterized when it is carried out in both sides of the occlusal surfaces, that is, bilateral vertical mastication. the alternated way characterizes the typical pattern. when the mastication is carried out in only one side, it is classified as unilateral chronic or preferential, considered as atypical or typical pattern, respectively. signs of alteration in the mastication function such as the presence of the head movements and/or altered head posture were also evaluated. the intraclass correlation coefficient (icc) verified the reliability of the cephalometric measurements. the icc values should be greater than 0.75 to indicate good reliability and those below 0.75 indicate poor to moderate reliability16. in order to verify de data normality, the lilliefors test was used in the angular and linear cephalometric variables. the correlation between the cephalometric variables related to the craniocervical posture and the variables related to the mandibular and hyoid position was tested by the pearson’s coefficient (r). based on the coefficient value, the correlation was considered strong for r > 0.7; moderate for 0.3 < r < 0.7, and weak for 0 < r < 0.317. a descriptive statistical verifies the subject percentage in each group, according to the number of craniocervical postural changes and the aspects evaluated in the masticatory and swallowing functions. the braz j oral sci. 11(2):141-147 relationship between craniocervical posture, mandible and hyoid bone and influence on alimentary functions craniocervical postural variables nsl/cvt cranium inclination in relation to the cervical spine (c2 – c4) nsl/opt cranium inclination in relation to c2 cvt/evt cervical lordosis cva head flexion/extension cpl forward head hyoid bone and mandible position nsl/ml cranium basis inclination in relation to the mandible hy/c3 hyoid to third cervical vértebra distance hy/me hyoid to mentum distance hy/ml hyoid to the mandibular plane chart 2 – cephalometric variables related to the craniocervical posture and mandibular and hyoid position line definition nsl nasion-sella line between n and s opt odontoid process tangent between cv2tg and cv2ip c v t cervical vertebra tangent between cv2tg and cv4ip evt lower part of the cervical spine between cv6ip and cv4ip m l mandibular line tangent to the lower border of the mandible through gn angle definition cva intersection of the lines between o anspns and cv2ap cv2ia cpl intersection line between ce1 to ce6 with the horizontal chart 1 – lines and angles of the cephalometric analyisis of the cranicocervical posture and the mandibular position. 144144144144144 chi-square test analyzed the difference of the percentage between the groups. it was admitted a significance level of 5% (p<0.05). results the study evaluated 36 women with mean age of 23.77 ± 3.47 years and bmi of 22.61 ± 4.38 kg/cm2. figure 2 presents the results related to the craniocervical postures based on the cephalometric analysis. it was verified that 86.11% of the subjects presented changes in the cervical curve, 77.78% with cervical lordosis rectification. changes in the head position related to the upper cervical spine were observed in 47.23% of the subjects, 30.56% presenting head hipextension. the forward head was more acentuated in 41.67% of the subjects. icc results demonstrated excelent reliability in all measured angles by the same examiner in two distincts moments, as shown in table 1. correlations between the craniocervical variables and the ones that assessed the mandible and hyoid position are presented in table 2. from these results, it can be inferred that the head hyperextension was correlated to a greater distance from the hyoid bone to the mandible and to the mentum. the more forward head position also influenced the hyoid bone to the mandible distance. a negative correlation between the cervical curve and the distance between the hyoid bone and the third cervical vertebra (c3) was observed. thus, the more rectified the cervical column, the more away the hyoid to the c3. the degree of the cranium base inclination on the cervical column in two levels (nsl/opt and nsl/cvt) presented positive correlation with the distance from the hyoid bone to the mentum, to the mandible and to the c3. these were the only variables that obtained a significant correlation with the mandibular position (nsl/ml). all the subjects presented at least one postural change, both in the flexion/extension and forward head position and in the cervical curve. therefore, it was not possible to analyze the association between the presence or absence of craniocervical postural changes and the masticatory and deglutition functions. for the statistical analysis, the subjects were classified as presenting i) only one or ii) two and three postural changes (table 3). regarding the masticatory function, the unilateral chronic and preferential patterns were grouped, once only one subject presented the chronic pattern. as demonstrated in table 3, there was no association between more accentuated changes in the craniocervical posture and the changes and the masticatory and deglutition functions, evaluated in the myofunctional orofacial evaluation. regarding the deglutition function, the changes more frequently observed were: effort in the lip occlusion (16.66% of the subjects) and facial muscle tension (19.44% of the subjects). in the masticatory function, the bilateral simultaneous pattern was not observed. therefore, the pattern was typical, that is, alternated in all subjects with bilateral pattern. the unilateral pattern was observed in 52.77% of the subjects. measurements nsl/ml hy/me hy/ml hy/c3 nsl/opt 0.38* 0.40* 0.34* 0.32* nsl/cvt 0.42* 0.41* 0.36* 0.33* cvt/evt -0.23 0.30 0.12 -0.40* cva -0.27 -0.48* -0.45* -0.34* cpl -0.23 -0.17 -0.51** -0.11 table 2 correlation between craniocervical posture and the mandible and hyoid bone position cva: craniovertebral angle; cvt/evt: cervical spine curve; cpl: angle related to the forward head posture; nsl/opt: cranium inclination in relation to the c2; nsl/cvt: cranium basis inclination in relation to the cervical spine; hy/c3: hyoid bone to the third cervical vertebra distance; hy/me: hyoid to mentum distance; hy/ml: hyoid to mandibular plane distance. n= 36. results expressed in r (pearson’s coefficient correlation) *p<0.05; **p<0.01 angles i c c confidence interval p nsl/cvt 0.986 0.964 – 0.955 0.00* nsl/opt 0.994 0.986 – 0.998 0.00* cvt/evt 0.979 0.947 – 0.992 0.00* cva 0.995 0.987 – 0.998 0.00* cpl 0.901 0.754 – 0.960 0.00* nsl/ml 0.918 0.797 – 0.967 0.00* hy/c3 0.994 0.984 – 0.997 0.00* hy/ml 0.990 0.976 – 0.996 0.00* hy/me 0.990 0.975 – 0.996 0.00* cva: craniovertebral angle; cvt/evt: cervical spine curve; cpl: angle related to the forward head posture; nsl/opt: cranium inclination in relation to the c2; nsl/cvt: cranium inclination in relation to the cervical spine ; hy/ c3: hyoid bone to the third cervical vertebra distance; hy/me: hyoid to mentum distance; hy/ml: hyoid to mandibular plane distance; nsl/ml: cranium inclination in relation to the mandible; n=20. table 1 intra-examiner reliability of the cephalometric measurements fig. 2 frequency of postural changes found in the study group based on the cephalometric analysis. braz j oral sci. 11(2):141-147 relationship between craniocervical posture, mandible and hyoid bone and influence on alimentary functions 145145145145145 2 and 3 alterations (n = 20) 1 alteration (n = 16) f % f % 13 81.25 17 85 3 18.75 3 15 15 93.75 20 100 1 6.25 0 0 15 93.75 18 90 1 6.25 2 10 13 81.25 16 80 3 18.75 4 20 8 50 9 55 8 50 11 45 16 100 19 95 0 0 1 5 16 100 18 90 0 0 2 10 typical with effort typical interposed absent present absent present bilateral alternate unilateral preferential or chronic absent present absent present lips occlusion during the deglutition tongue posture during the deglutition head moviment during deglutition facial muscles tension during deglutition masticatory pattern head moviment during mastication altered head posture during mastication craniocervical posture changes: forward head position, flexion/extension head and cervical curve altered,(1) presence of one of these changes; (2 to 3) presence of 2 and/or 3 changes. chi-square test * p<0,05 p 0.57 0.42 0.53 0.61 0.54 0.56 0.32 table 3 presence of craniocervical posture changes (1 or 2 and 3) and its relationship with the masticatory and swallowing function evaluations discussion all studied subjects presented at least one postural deviation in the craniocervical segment. the modification of the physiological lordosis curve was the most evident postural alteration, since 86.11% of the subjects presented some type of deviation such as rectification or inversion of the curve. the presence of significant postural changes has been observed, particularly, in working subjects, who are more vulnerable to the postural bad habits and vicious. the decrease in the cervical lordosis was previously described in subjects at the same age of this study and the authors alert for this severe postural deviation expressed in so young subjects18. from the correlation analysis, it was verified that head hyperextension was correlated with a greater distance from the hyoid bone to the mandible and to the mentum. other studies presented similar results, demonstrating significant correlation between the craniovertebral angle with the greater distance in hy/ml19-21 and hy/me19,21. a more accentuated forward posture was also correlated to a greater hy/ml distance, possibly because this is a change that usually follows the cranium hyperextension in order to keep the optical plane in the horizon. however, it puts the mandibular symphysis in a more elevated position. moreover, considering the negative correlation among cpl and cva angles with the hy/ml distance observed in this study, we can infer that in the presence of a forward and extend head posture a major effort by the suprahyoid muscles will be necessary to pull the hyoid bone and larynx upward and forward during swallowing function, allowing the pharyngoesophageal sphincter to open with guarantee of no food aspiraton22. muto et al.21 (2002) showed that the hy/c3 distance stayed constant regardless the cranium position. on the other hand, özbek et al.20 (1998) found a significant correlation between the forward head and the cranium hyperextension with the increase of the hy/c3 distance, similar to the negative correlation between this distance and craniovertebral angle observed in the present study. the relationship between the craniocervical posture and the hyoid bone can be the reflex of the tongue position in the oral cavity. the hyoid bone serves as anchor to the tongue musculature, thus its lower position tractions the tongue to a lower and back position reducing the airway space. in face of the vital need of keeping this space, the craniocervical postural change acts as compensatory mechanism, pulling the hyoid bone far from the posterior pharyngeal wall (increasing the hy/c3 distance) in order to guarantee the airway permeability20. the cranium inclination on the cervical column in two levels (nsl/cvt and nsl/opt) presented a moderate and significant correlation with the hyoid bone distance from the hyoid bone to the mentum, also previously found in the literature21. additionally, there were moderate correlations with the hy/ml and hy/c3 distances, in disagreement with another studies20,23. nsl/cvt and nsl/opt were the only variables to show a significant correlation with the mandibular position (nsl/ ml). such finding illustrates the interdependence between the structures that composed the biomechanical unit represented by the craniocervicomandibular system2. taking into account that the main muscles that displace the hyoid bone (mylohyoid and geniohyoid muscles) originate in the mandible, the length-tension relation of these muscles are affected by the mandible position, interfering directly on the stomatognathic functions7. the changes in hyoid bone position can be also correlated to the cervical curve, once the supra and infrahyoid muscles act as antagonist of the posterior cervical muscles braz j oral sci. 11(2):141-147 relationship between craniocervical posture, mandible and hyoid bone and influence on alimentary functions 146146146146146 that maintain the physiological cervical lordosis. a higher position of the hyoid can be related to the rectification and inversion of this curvature3. in the present study, the positive correlations between cvt/evt and hy/me and hy/ml are in accordance with this reasoning, since curve rectification was correlated with the smaller distance from the hyoid to the mandible and to the mentum. however, such correlations were weak and had no statistical significance. for hellsing19 (1989), the cervical curve rectification was correlated to the lower position of the hyoid bone, nevertheless the correlations, as in the present study, were not significant. similarly, we observed that the hyoid position related to c3 was the same in one group of young subjects, despite the reduction of the cervical lordosis18. in the present study, there was a significant correlation only between cvt/evt and hy/c3, with a trend to the rectification and inversion of the cervical curve in a presence of a greater distance hy/c3. a greater muscle tension in the supra and infrahyoid muscles, together with the other muscles of the anterior chain, tend to produce the cervical rectification. thus, the hyoid bone may be pulled to a more anterior position, increasing the distance hy/c3. kollias and krosgstad24 (1999) observed that the forward head posture was followed by a reduction of the physiological lordosis and in the pharyngeal airway space, probably due to the approximation of the tongue to the posterior pharyngeal wall. thus, in addition to the relation of postural biomechanical, the increase in the hy/c3 may occur in the attempt of opening the airway by the hyoid traction, due to its relation with the tongue base. since the anatomical relation among cranium, cervical column, mandible and hyoid bone was confirmed, we hypothesized that craniocervical posture changes could intervene in the stomatognathic functions. however, no association was found between a greater misalignment in the craniocervical posture and atypical behavior during masticatory and swallowing functions. silva et al.25 (2004) conducted a similar study with 15 subjects evaluated by anthroposcopic analysis. the authors also observed that all subjects presented at least one postural change in the craniocervical segment. additionally, they could not demonstrate a significant association between changes in the craniocervical posture and in the stomatognathic system. nevertheless, 60% of the studied subjects presented alteration in the anterior muscular chain and in the masticatory function. harmony and balance between form and function are determinant features for a healthy condition of the stomatognathic system, which in turn leads to typical orofacial behaviors during alimentary functions performance1. on the other hand, in face of a structural alteration, the functions may be maintained through compensatory actions, a mechanism found by the body to perform the masticatory and swallowing functions. this can, partially, explain the fact that, despite the craniocervical posture changes present in all studied subjects, those were not associated to myofunctional orofacial changes. in summary, to understand how the body can adapt itself in such situations, it must be borne in mind a human body entirely composed by muscular chains, which are dependent one for each other. it has been described models explaining myofascial trains and sequences comprising myofascial connections crossing the entire body. these trains are directly involved in the organization of movements and function as well as muscular force transmission26. it is well known, that a prolonged altered head posture may lead to asymmetric electromyography (emg) activity in the jaw muscles. on the other hand, symmetry of the emg activity of the masticatory system may be a contributing factor for the appropriate development of physiologic functions such as mastication, deglutition, respiration, and speech. thus, to reach a more symmetrical emg activity of these muscles and typical mandible and hyoid movements, patients may continuously adopt postural compensations at the level of the craniocervical segment27. supporting this reasoning, douglas et al.28 (2010) stated that when some kind of disturb intervene on the motor control of the stomatognathic system, the body adapts in order to keep functions performing perfectly. however, it occurs up to the point that the adaptive reactions become insufficient to transpose the system fails. in this case, the functions will suffer the effects of the signals and symptoms of a stomatognathic disorder such as tmd, previously related to the atypical behaviors of the mastication and swallowing29. thus, it is possible that the atypical behaviors were less evidenced in this study because the presence of tmd was an exclusion criterion. in this study, it is assumed that the postural changes not influenced the alimentary functions due to the possibility of the organism adapts itself to the structural conditions. based on this, it must be adverted that the craniocervical posture evaluation is not neglect in the orofacial motricity assessment, and the reciprocal is true as well. such approach provides not only therapeutics but also prophylactic intervention in these cases. it must be mentioned that, despite the important postural changes observed in the subjects of the present study, none of them had undergone physical therapy treatment for postural training. in the same way, they were not previously undergone to speech therapy, even with atypical behavior in the alimentary functions. therefore, this study is relevant in disclosing for theses participants, the need of these therapeutic interventions to improve the performance of the postural and stomatognathic systems. in the studied subjects, it was observed at least one postural deviation on the craniocervical segment, with predominance of rectification and inversion of the cervical curvature. in conclusion, cephalometric findings confirmed the anatomical relationship between craniocervical posture, mandible and hyoid bone. however, the association between craniocervical posture alterations and masticatory and swallowing function impairments was not observed. such findings suggest that, in the presence of a musculoskeletal imbalance, the body readapts itself, ensuring that alimentary functions will not be affected. braz j oral sci. 11(2):141-147 relationship between craniocervical posture, mandible and hyoid bone and influence on alimentary functions references 1. sakagushi k, metha nr, abdallah ef, forgione ag, hirayama t, kawasaki t, et al. examination of the relationship between mandibular position and body posture. j craniomand pract. 2007; 24: 237-49. 2. villanueva p, valenzuela s, santander h, zuniga c, ravera mj, miralles r. efecto de la postura de cabeza em mediciones de la vía aérea. rev cefac. 2004; 6: 44-8. 3. rocabado m. biomechanical relationship of the cranial, cervical and hyoid regions. j craniomandib pract. 1983; 1: 61-6. 4. ritzel ch, dienfenthaeler f, rodrigues am, guimarães acs, vaz ma. temporomandibular joint dysfunction and trapezius muscle fatigability. rev bras fisiot. 2007; 11: 333-9. 5. matsuo k, palmer jb. anatomy and physiology of feeding and swallowing: normal and abnormal. phys med rehabil clin n am. 2008; 19: 691-707. 6. matsuo k, palmer jb. coordination of mastication, swallowing and breathing. jpn dent sci rev. 2009; 45: 31-40. 7. pearson wg, langmore sf, zumwalt ac. evaluating the structural properties of suprahyoid muscles and their potential for moving the hyoid. dysphagia. 2011; 26: 345-51. 8. manfredini d, castroflorio t, perinetti g, guarda-nardini l. dental occlusion, body posture and temporomandibular disorders: where we are now and where we are heading for. j oral rehab. 2012; 39: 463-71. 9. manfredini d, bucci mb, monstagna f, guarda-nardini l. 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protocol of orofacial myofunctional evaluation with scores. int j otorhinolaryngol. 2008; 72: 367-75. 16. gadotti ic, vieira er, magee dj. importance and clarification of measurement properties in rehabilitation. rev bras fisiot. 2006; 10: 137-46. 17. chan vh. biostatistics 104: correlational analysis. singapore med j. 2003; 44: 614-9. 18. andrade av, gomes pf, teixeira-salmela lf. cervical spine alignment and hyoid bone positioning with temporomandibular disorders. j oral rehabil. 2007; 34: 767-72. 19. hellsing e. changes in the pharyngeal airway in relation to extension of the head. eur j orthod. 1989; 11: 359-65. 20. özbek mm, miyamoto k, lowe aa, fleetham ja. natural head posture, upper airway morphology and obstructive sleep apnoea severity in adults. eur j orthod. 1998; 20: 133-43. 21. muto t, takeda s, kanazawa m, yamazaki a, fujiwara y, mizoguchi i. the effect of head posture on the pharyngeal airway space (pas). int j oral maxillofac surg. 2002; 31: 579-83. 22. perry jl, bae 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308-18. 28. douglas cr, avoglio jlv, oliveira h. stomatognathic adaptive motor syndrome is the correct diagnosis for temporomandibular disorder. med hypotheses. 2010; 74: 710-8. 29. felício cm, melchior mo, da silva mamr. effects of orofacial myofunctional therapy on temporomandibular disorders. j craniomand pract. 2010; 28: 249-56. 147147147147147 braz j oral sci. 11(2):141-147 relationship between craniocervical posture, mandible and hyoid bone and influence on alimentary functions revista fop n 13 braz j oral sci. october/december 2008 vol. 7 number 27 1636 effect of cavity preparation design on the fracture resistance of directly and indirectly restored premolars denise sá maia casselli1 ; andré luis faria e silva2 ; henrique casselli3; luis roberto marcondes martins4 1dds, msc, phd professor, department of dentistry, são leopoldo mandic dental school-unit ceará fortaleza, ceará brazil. 2dds, msc, phd professor, department of dentistry, state university of montes claros, montes claros, mg, brazil, 3dds, msc, phd professor, department of prosthetics, são leopoldo mandic dental school-unit ceará, fortaleza, ceará brazil. 4dds, msc, phd professor, department of restorative dentistry, piracicaba dental school, university of campinas piracicaba piracicaba, sp, brazil received for publication: april 01, 2008 accepted: september 09, 2008 correspondence to: denise sá maia casselli r. padre valdevino, 1415b. aldeota fortalezace brasil cep 60.135-040 e-mail: dsmaia@yahoo.com a b s t r a c t aim: the aim of this study was to evaluate the fracture resistance of human premolars with extensive cavity preparations of the inlay and onlay types, for performing direct and indirect composite resin restorations. methods: eight-four premolars were divided into 7 groups (n=12): g1=intact teeth; g2=inlay/filtek z-250 direct; g3=inlay/ filtek z-250 light polymerized indirectly; g4=inlay/ solidex; g5=onlay/ filtek z-250 direct; g6=onlay/ filtek z-250 light polymerized indirectly; g7=onlay/ solidex. indirect filtek z250 restorations were light activated using the edg-lux oven. all samples were submitted to axial compression load in a universal testing machine until fracture. the data were submitted to the one-way anova and tukey’s test (a=0.05). results: the results (n) were: g6-1938a, g5-1570ab, g7-1285b, g1-1215b, g4-1174b, g3-1146b and g2-1112b. the onlay cavities restored indirectly with filtek z-250 presented significantly higher fracture resistance (g6) than the other groups, except for onlay restorations made directly with filtek z-250 (g5), which, in turn, did not differ significantly from the other groups. however, the onlay restorations made with filtek z-250 led to more catastrophic failure modes. conclusions: 1. adhesive inlay restorations, irrespective of the type of composite resin and light-activation technique used, restored the fracture resistance of intact teeth. key words: composite resin, fracture resistance, cavity preparation, restorative technique, indirect restoration. i n t r o d u c t i o n sound teeth rarely fracture during normal masticatory stress1. the presence of the palatal and buccal cusps with intact mesial and distal ridges forms a continuous circle of dental structure, reinforcing and maintaining tooth integrity. however, cuspal fracture can frequently occur in teeth that have been weakened by caries, large cavity preparations and reduction of dental structure as a result of erosion or abrasion2. studies have shown that teeth with cavity preparations become weaker as the occlusal isthmus is widened, and they fracture more easily than intact teeth2,3. therefore, it is important to preserve the integrity of the dental structure to maintain its resistance2-4. recently, there has been increasing use and acceptance of the acid-etch technique to bond various materials to tooth substrate3-5. with the introduction of dental composites and the development of adhesive systems, it was possible to reduce significantly the amount of healthy dental tissue removed during cavity preparation. this has enabled more esthetic restorations to be performed and to reestablish the fracture strength lost due to cavity preparation. composite materials potentially have many applications, such as anterior and posterior restorations, indirect inlay/ onlays and pit-and-fissure sealants4,6. one of the main drawbacks of current composites is the shrinkage during polymerization, which creates stress on the restored tooth and may result in poor marginal integrity, as well as inadequate durability and longevity in the oral environment7. furthermore, it is difficult to obtain proximal contacts, and anatomic reproduction, finishing and polishing are limited in more complex clinical situations. in order to overcome these limitations, the choice for indirect restorations with laboratory-processed resins is a feasible option. the composite resin solidex (shofu, kyoto, japan) is an example of intermediate laboratory-processed resin that is polymerized by light-curing unit of xenon stroboscopic light. this high-intensity light source with intermittent pulses has been reported to improve hardness, compressive strength and flexural strength, and to reduce water 1637 braz j oral sci. 7(27):1636-1640 effect of cavity preparation design on the fracture resistance of directly and indirectly restored premolars g1 _ _ _ g2 inlay direct filtek z-250 g3 inlay indirect filtek z-250 g4 inlay indirect solidex g5 onlay direct filtek z-250 g6 onlay indirect filtek z-250 g7 onlay indirect solidex groups cavity preparation light-activation m o d e material table 1. description of cavity preparations, light activation mode and materials used. absorption and water solubility of composite materials8. despite these advantages, indirect composite resins are approximately twice more expensive than direct composite resins. recently, casselli et al.8 demonstrated that the direct composite resin filtek z-250 (3m/espe, st. paul, mn, usa) presented better diametral tensile strength than the indirect composite resin solidex. in addition, this mechanical property was improved when the composite resin was heat light-polymerized using an edg-lux high-intensity xenon photo-curing unit (edg, são carlos, sp, brazil). therefore, the aim of this study was to evaluate the fracture resistance of human premolars with extensive inlay and onlay cavity preparations, restored with indirect and direct composite resins. the direct composite was used both directly and indirectly. the null hypothesis was that the material, the restorative technique and the type of cavity preparation would not interfere with the fracture resistance. material and methods eighty-four human, single-root maxillary premolars, unrestored, non-carious, free of cracks and defects, extracted due to periodontal or orthodontic reasons and stored in 10% formalin solution (ph = 7) for up 3 months, were used in this study. teeth with similar size and shapes were selected by crown dimensions after measuring the buccolingual and mesiodistal widths, allowing a maximum deviation of 10% from the determined mean. after cleaning with periodontal curettes, the teeth were mounted in polystyrene resin cylinders, with simulated periodontal ligament, exposing 2 mm of root surface below the cementoenamel junction9. to simulate the periodontal ligament, a radiographic film with a centralized circular hole was used to stabilize the teeth during the inclusion procedure. first, the root was covered with wax and then a 25-mm-diameter plastic cylinder was placed over the root. the polystyrene resin was inserted into the cylinder and, after polymerization, the tooth was removed from the resin and the wax removed from the root. the resin cylinders were filled with a polyether material (impregum f; 3m/espe) and the teeth were reinserted and the excess polyether material was removed. the teeth were randomly assigned to seven experimental groups, according to table 1. tooth preparations were made with a standardized preparation machine, which consists of a high-speed handpiece (kavo do brasil, joinvile, sc, brazil) coupled to a mobile base that moves vertically and horizontally. this movement is controlled with aid of a micrometer (mitutoyo, tokyo, japan), accurate to 0.1 mm. a tapered cylinder diamond bur # 4137 (kg sorensen, barueri, sp, brazil) was used under water cooling for the cavity preparation. the isthmus width was 2/3 of the distance between the cusp tips, and the pulpal depth was ½ the crown height (figure 1). cavity width was checked in the occlusal portion and the pulpal depth was measured with a digital caliper in relation to the buccal cusp tip. the cavities were finished with the same diamond bur in a low-speed handpiece. this preparation was classified as inlay. fig.1. inlay and onlay cavity preparations. for direct restorations, each cavity was etched with 35% phosphoric acid (scotchbond etchant; 3m/espe) for 15 s, washed with an air-water spray and dried with absorbent paper. two consecutives layers of the adhesive system single bond (3m/espe) were applied and light cured for 20 s (degulux, degussa hülz, frankfurt, germany) after solvent evaporation with an air stream. the composite resin filtek z-250 was inserted in 2-mm thick increments and polymerized for 40 s with the same halogen light-curing unit. for indirect restorations, impressions were made of the prepared teeth using a polyvinyl siloxane impression material (aquasil, dentsply de trey, konstanz, germany) in a 2-step technique, and the impressions were poured with a type iv stone (durone, dentsply, petrópolis, rj, brazil) after 30 min. the cavity dimensions of the onlay preparation were similar to those of the inlay preparation, with additional removal of the palatal cusp. the impressions of this preparation were also made in the same manner as described before. the restorations were made with z-250 or solidex composite resin inserted in approximately 2mm-thick increments on the isolated stone die. each increment was heat light polymerized for 2 min using the edg-lux unit. after 24 h, the internal surface of the indirect composite resin restorations was airborne-particle abraded with 50µm aluminum oxide for 10 s, washed with an air-water spray for 60 s, air-dried and coated with a layer of the the 1638 silane agent (ceramic primer, 3m/espe) for 60 s. next, single bond adhesive system was applied and light cured for 20 s. the same adhesive procedure described for direct restorations was used for these teeth. the indirect restorations were cemented with a dual-cured resin-based cement (relyx arc; 3m/espe), which was light cured for 40 s from each side of the restoration. the specimens were stored in 100% relative humidity at 37°c for 24 h. after this period, the specimens were submitted to compressive load in a universal testing machine (instron 4411; instron corp, canton, england) with a 6-mm-diameter steel sphere at a crosshead speed of 0.5 mm/min until fracture. the sphere came into contact with the dental structure and restorations on both buccal and lingual cusps the data were converted into n and submitted to one-way anova using a 95% level of significance. the fractured specimens were analyzed under a stereomicroscope to determine the failure modes, and were assigned to 1 of 4 categories, using a modified classification system based on the one proposed by burke et al.10: 1= fracture involving a small portion of the coronal structure; 2= fracture involving a small portion of the coronal structure, but requiring an increase in cavity preparation during the repair procedure; 3= fracture involving the tooth structure with root involvement, which can be restored in association with periodontal surgery; and 4= severe root and crown fracture, which determine tooth extraction. r e s u l t s the one-way anova revealed significant differences among the groups (p<0.001).the results of the tukey’s test are described in figure 2. the onlay preparations restored indirectly with composite resin filtek z250 (g6) presented the highest fracture resistance. however, the onlay preparations restored with filtek z-250, both directly and indirectly (g5 and g6), presented no statistical difference from each other (p>0.05). the onlay preparations restored directly with filtek z250 (g5) did not differ significantly from the other groups either (p>0.05), which presented similar fracture resistance values. the fracture mode data were submitted to the kruskal-wallis non-parametric statistical analysis. the results are shown in table 2. the control group (g1) presented only type 1 failures. the onlay cavities restored with composite resin z-250, both directly and indirectly (g5 and g6), presented mainly type 4 fractures, which are not repairable. d i s c u s s i o n the null hypothesis of this study was rejected since both the material and the cavity preparation type had an effect on fracture resistance. it would appear that the extensive cavity preparations used in this study significantly reduced the fracture resistance of the premolars11. it has been demonstrated that adhesive restorations may recover fracture resistance to values similar to those of sound nonrestored teeth3. in the present study, all restorations presented similar or higher values than those of intact teeth. moreover, in this study a bonding system and a resin agent were used for cementation of the indirect restorations. this procedure increases tooth resistance compared to the use of non-adhesive conventional cements because it allows the formation of a single body between the restorative material and the dental structure3. it may also be considered that the use of an adhesive luting cement would enhance the fracture strength of the restored unit6. burke et al.11 reported that one could consider that teeth restored with indirect composite resin restorations would provide similar resistance to that provided by direct composite resin restorations. in the present study, the highest values fracture resistance were obtained with onlays made with filtek z-250. it was expected that a more extensive preparation, such as onlays, would present lower values because of the greater amount of dental structure removed compared to inlay preparations11. a possible explanation is that the lower elastic modulus of the direct composite resin promoted a greater distribution of stress than the enamel12, which also comes into contact during the test on intact teeth and inlay preparations. the enamel has high elastic modulus and friability13,14. the stress generated during the compressive load is concentrated and could initiate crack braz j oral sci. 7(27):1636-1640 effect of cavity preparation design on the fracture resistance of directly and indirectly restored premolars different letters indicate statistically significant difference (p<0.05). fig. 2. mean fracture resistance (in n) for each group. b b b b b a b a groups type 1 type 2 type 3 type 4 kruskal-wallis t e s t group 1 12 0 0 0 a group 2 0 3 6 3 b c group 3 1 4 5 2 b c group 4 1 3 8 0 b c group 5 1 1 4 6 c group 6 1 2 3 6 b c group 7 4 3 2 3 b table 2. failure modes. different letters indicate statistically significant difference (p<0.05). 1639 formation and propagation, resulting in lower fracture resistance4. in addition to the elastic modulus of the material, the cohesive strength is also important in order to improve the fracture resistance of restorations15-17. the indirect composite resin solidex presents lower filler content (53% v/v) than the direct composite resin filtek z-250 (60% v/ v). this difference in the filler content results in a lower elastic modulus of solidex18, which could be beneficial. on the other hand, the lower filler content reduces the cohesive strength and may compromise the fracture resistance of the restoration19. however, the only difference between solidex and filtek z-250 occurred when the two composite resins were heat light polymerized indirectly. the directly light cured filtek z-250 presented no difference from either solidex or filtek z-250 subjected to indirect light polymerization. this probably occurred because the indirect heat light polymerization process increased the degree of conversion of the composite resins, improving their cohesive strength20-22. in spite of the highest fracture resistance being obtained with onlay restorations made with filtek z-250, these restorations presented more severe failure modes when compared to inlay restorations. this observation is perhaps more important than the fracture strength values because these fractured restorations cannot be repaired. one possible explanation is the less homogeneous stress distribution3-4. the onlay restoration used in this study involved only one cusp, and the other one remained almost intact. this non-homogeneous stress distribution added to more stress absorption by composite resin restoration resulted in a high fracture resistance and predominance of catastrophic failure. although composite resin solidex showed lower fracture strength values than those of the heat lightpolymerized filtek z-250, it presented more favorable failure modes for this type of cavity preparation. perhaps this occurred because the lower cohesive strength of this material allowed the material to fail before fracture of the dental structure. even with all the limitations of an experimental test, it can be observed that direct composite restorations are sufficient for reestablishing the strength of a permanent tooth. however, for more extensive preparations, such as onlays, the composition of the restorative material used was shown to have more influence on the fracture strength than the light activation method used. to restore this type of cavity preparation, the direct composite resin filtek z250 can be used indirectly. this technique presents the possibility of improving the degree of conversion of these materials and costs less than the use of a composite resin indicated exclusively for the indirect technique, such as solidex. nevertheless, the composite resins with higher filler content, resulting in better mechanical properties, presented a larger number of catastrophic failures in this study. this finding is a serious disadvantage, since it leads to indication of tooth extraction. within the limitations of this study, it can be concluded that: 1. adhesive inlay restorations, irrespective of the type of composite resin and light-activation technique used, restored the fracture resistance of intact teeth; 2. onlay restorations made with composite resin filtek z-250 presented the highest fracture resistance, but led to a more catastrophic failure mode. r e f e r e n c e s 1. grisanti lp 2nd, troendle kb, summitt jb. support of occlusal enamel provided by bonded restorations. oper dent. 2004; 29: 49-53. 2. mondelli j, sene f, ramos rp, benetti ar. tooth structure and fracture strength of cavities. braz dent j. 2007; 18: 134-8. 3. soares pv, santos-filho pc, martins lr, soares cj. influence of restorative technique on the biomechanical behavior of endodontically treated maxillary premolars. part i: fracture resistance and fracture mode. j prosthet dent. 2008; 99: 30-7. 4. soares pv, santos-filho pc, gomide ha, araujo ca, martins lr, soares cj. influence of restorative technique on the biomechanical behavior of endodontically treated maxillary premolars. part ii: strain measurement and stress distribution. j prosthet dent. 2008; 99: 114-22. 5. geurtsen w, garcia-godoy f. bonded restorations for the prevention and treatment of the cracked-tooth syndrome. am j dent. 1999; 12: 266-70. 6. signore a, benedicenti s, covani u, ravera g. a 4to 6-year retrospective clinical study of cracked teeth restored with bonded indirect resin composite onlays. int j prosthodont. 2007; 20: 609-16. 7. goracci g, mori g, martinis lc. curing light intensity and marginal leakage of resin composite restorations. quintessence int. 1996; 27: 355-62. 8. casselli ds, worschech cc, paulillo la, dias ct. diametral tensile strength of composite resins submitted to different activation techniques. braz oral res. 2006; 20: 214-8. 9. soares cj, pizi ec, fonseca rb, martins lr. influence of root embedment material and periodontal ligament simulation on fracture resistance tests. braz oral res. 2005; 19: 11-6. 10. burke fjt, wilson nhf, watts dc. fracture resistance of teeth restored with indirect composite resins: the effect of alternative luting procedures. quintessence int. 1994; 25: 269-75. 11. soares cj, martins lr, fonseca rb, correr-sobrinho l, fernandes neto aj. influence of cavity preparation design on fracture resistance of posterior leucite-reinforced ceramic restorations. j prosthet dent. 2006; 95: 421-9. 12. cara rr, fleming gj, palin wm, walmsley ad, burke fj. cuspal deflection and microleakage in premolar teeth restored with resin-based composites with and without an intermediary flowable layer. j dent. 2007; 35: 482-9. 13. baldassarri m, margolis hc, beniash e. compositional determinants of mechanical properties of enamel. j dent res. 2008; 87: 645-9. 14. addison o, marquis pm, fleming gj. resin elasticity and the strengthening of all-ceramic restorations. j dent res. 2007; 86: 519-23. 15. touati b. the evolution of aesthetic materials for inlays and onlays: a review. pract. periodont aesthet dent. 1996; 8: 657-66. 16. touati b, aidan n. second generation laboratory composite resins for indirect restorations. j esthet dent. 1997; 9: 108-18. 17. lehmann f, eickemeyer g, rammelsberg p. fracture resistance of metal-free composite crowns-effects of fiber reinforcement, thermal cycling, and cementation technique. j prosthet dent. 2004; 92: 258-64. braz j oral sci. 7(27):1636-1640 effect of cavity preparation design on the fracture resistance of directly and indirectly restored premolars 1640 18. masouras k, silikas n, watts dc. correlation of filler content and elastic properties of resin-composites. dent mater. 2008; 24: 932-9. 19. kim kh, ong jl, okuno o. the effect of filler loading and morphology on the mechanical properties of contemporary composites. j prosthet dent. 2002; 87: 642-9. 20. correr sobrinho l. correlation between light intensity and exposure time on the hardness of composite resin. j mater sci. 2000; 11: 3361-4. 21. small bw. a review of devices used for photocuring resin-based composites. gen dent. 2001: 457-60. 22. soares cj, pizi ec, fonseca rb, marins lrm. mechanical properties of light-cured composites polymerized with several additional post-curing methods. oper dent. 2005; 30: 389-94. braz j oral sci. 7(27):1636-1640 effect of cavity preparation design on the fracture resistance of directly and indirectly restored premolars oral sciences n3 original article braz j oral sci. january | march 2011 volume 10, number 1 effect of fluoride-containing bleaching agents on bovine enamel microhardness samira padilha gabasso1, cristiane franco pinto2, vanessa cavalli3, adriana franco paes-leme4, marcelo giannini5 1undergraduate student, department of restorative dentistry, piracicaba dental school, university of campinas, piracicaba, sp, brazil 2dds, ms, phd student, department of restorative dentistry, piracicaba dental school, university of campinas, piracicaba, sp, brazil 3dds, ms, phd, assistant professor, department of dentistry, university of taubaté, taubaté, sp, brazil 4dds, ms, phd, researcher at center for structural molecular biology, brazilian synchrotron light laboratory, campinas, sp, brazil 5dds, ms, phd, associate professor, department of restorative dentistry, piracicaba dental school, university of campinas, piracicaba, sp, brazil correspondence to: marcelo giannini department of restorative dentistry operative dentistry division piracicaba school of dentistry, p.o. box 52 university of campinas unicamp av. limeira, 901 – areião piracicaba, sp zip code: 13414-900 brazil phone:55 19 21065340 /fax:55 19 21065218 e-mail:giannini@fop.unicamp.br received for publication: july 07, 2010 accepted: march 28, 2011 abstract aim: the purpose of this study was to evaluate the effect of 10% carbamide peroxide (10%cp) bleaching agents with different fluoride concentrations on enamel microhardness after induction of artificial caries lesions during ph-cycling model. methods: bovine dental enamel blocks with known surface microhardness were subjected to caries lesion induction and another surface microhardness was determined after a demineralization protocol. the enamel blocks were divided into four groups (n=17) and subjected to 12-day ph-cycling. the groups consisted of the following treatments: 1) artificial saliva (control group not subjected to bleaching treatment); 2) 10%cp; 3) 10%cp (with 0.11% fluoride); 4) 10%cp (with 0.5% fluoride). after treatments, the enamel was evaluated using surface microhardness, polarized light microscopy (plm) and scanning electronic microscopy. the percentage of surface microhardness recovery was determined for each group and analyzed by the kruskal wallis and dunn’s tests (a=0.05). the values of lesion depth by plm were analyzed by anova and tukey’s test (a=0.05). results: the enamel treated with bleaching gels containing or not fluoride presented lower mineral recovery and higher caries lesion depth than the control group. conclusions: these data suggest that bleaching procedures on enamel with artificially induced caries lesions should be used with caution even in the presence of fluoride because there was no recovery in the microhardness. keywords: dental enamel, tooth bleaching, fluoride. introduction carbamide peroxide is the active ingredient of most home-use tooth bleaching agents. since the introduction of nightguard vital whitening, concern has been expressed regarding the potential effects of carbamide peroxide solution on dental hard tissues1. as the bleaching technique of vital teeth comprises the direct contact of the whitening gel on the outer enamel surface, the oxidation reaction for an extended period of time can be related to demineralization processes2-3. various effects of carbamide peroxide bleaching on teeth have been reported. braz j oral sci. 10(1):22-26 23 braz j oral sci. 10(1):22-26 scanning electron microscopy (sem) investigations have shown that 10% carbamide peroxide changes enamel, promoting surface dissolution and topographical alterations46. the morphological changes and reduction of enamel microhardness are not limited to the enamel surface; alterations have also been detected in the subsurface enamel layer7-9. moreover, some studies have shown alterations of the histological aspects and composition of sound dental enamel after carbamide peroxide gel application9-10. the bleaching treatment with 10% carbamide peroxide slightly increased caries susceptibility of enamel11-12. however, although most studies have shown the effects of bleaching agents on sound teeth, the effect of carbamide peroxide gels on white spots lesions and early erosions is not known, and neither is the capability of fluoridated carbamide peroxide gels in recovering the enamel microhardness. thus, the objective of this study was to evaluate the effect of one unfluoridated and two fluoridated carbamide peroxide gels on the microhardness of enamel with caries-like lesions induced during ph-cycling model. the null hypothesis was that enamel surface microhardness recovery (smhr) is not influenced by the type of bleaching agent used. material and methods three bleaching agents containing 10% carbamide peroxide were tested: one unfluoridated gel (fgm prod. odont. ltda, joinville, sc, brazil) and two fluoridated carbamide peroxide bleaching agents (opalescence pf with 0.11 % fluoride, ultradent products inc., salt lake city, ut, usa and whiteness with 0.5% fluoride, fgm prod. odont. ltda, joinville, sc, brazil). specimen preparation bovine teeth, stored in saturated thymol at 5oc for up to 1 month were used in this study. eighty bovine dental enamel blocks (4x4x2 mm) were obtained from the buccal surface with the use of double-faced diamond discs (kg sorensen, barueri, sp, brazil). the buccal enamel surfaces were wetpolished with 800-, 1000and 1200-grit sic paper, followed by diamond pastes (3 µm and 1 µm). five microhardness indentations spaced 100 µm from each other were performed on the enamel surface with a microhardness tester (fm-1e, future tech, tokyo, japan), under a 50-g load for 5 s. means of the five indentations were calculated for each block and the samples with surface knoop hardness ranging from 430.9 to 92.6 khn units were selected to standardize the samples among the experimental groups, which had enamel blocks with similar initial knoop microhardness values. half of the enamel block surface (8 mm2) was coated with an acid-resistance varnish and the exposed enamel surfaces were subjected to demineralizing solution containing 0.05 m acetate buffer, ph 5.0, 50% saturated with enamel bovine powder, for 16 h at 37oc using 2 ml of solution to each 1 mm2 of exposed area13. the aim of demineralized solution was to produce artificial caries lesion. afterwards, the enamel blocks with surface microhardness ranging from 72.6 to 172.8 khn units were selected. ph-cycling regimen and experimental groups the enamel blocks were randomly divided into 4 groups (n = 17) and subjected to a 12-day ph-cycling13 consisted of 5 phases: (1) 1-min soak in fluoridated dentifrice (1,100 ppm f as naf)/water slurries three times a day to simulate daily toothbrushing; (2) between the treatments with dentifrice, samples were individually immersed in artificial saliva14 (14.2 mm sodium carboxymethylcellulose, 280 mm xylitol, 13.4 mm potassium chloride, 17.1 mm sodium chloride, 0.004 mm sodium fluoride, 0.2 mm magnesium chloride, 0.4 mm calcium chloride, 2.9 mm potassium phosphate, 0.1 mm potassium thiocyanate, ph 7.2) at 37 oc; (3) to simulate the daily acid challenge, enamel blocks were individually immersed in demineralized solution for 2 h at 37 oc, with the same composition of the solution used in the initial carious lesion procedure; (4) the samples were immersed in human saliva during 2 h at 37oc before bleaching treatment to promote acquire pellicle formation; (5) to simulate daily treatment, the samples were subjected to artificial saliva (control group) or bleaching treatment with 3 whitening gels: 10% carbamide peroxide (fgm prod. odont. ltda); 10% carbamide peroxide with 0.11% fluoride (opalescence pf); and 10% carbamide peroxide with 0.5% fluoride (fgm prod. odont. ltda) for 8 h (nocturnal period) at 37oc. for the bleaching procedure (5), 0.1 ml of the whitening gel was mixture with 0.05 ml of artificial saliva and this mixture was applied on enamel surface and covered with an individual tray. the mixture with artificial saliva tends to increase the decomposition of hydrogen peroxide and release more water, oxygen gas and free radicals, simulating the mouthguard bleaching technique14. during the bleaching, the specimens were placed in 100% relative humidity at 37°c and, after bleaching, the specimens were rinsed with an air/water spray for 10 s and stored in 100% humidity environmental until analysis. surface microhardness and microhardness recovery analyses of control and tested groups. surface microhardness was determined for the enamel blocks after polishing (baseline or sound enamel), after demineralization and after experimental treatments15. the percentage of smhr (%smhr) was calculated as: % hardness recovery = hardness after ph cycling hardness after demineralization x 100 / sound enamel hardness (baseline) hardness after demineralization13. after surface microhardness measurements, all blocks were longitudinally sectioned into 2 halves with a diamond saw (isomet 1000, buehler, lake bluff, il, usa) and one half was used to evaluate the lesion depth by polarized light microscopy (plm) and the other half was used to examine the subsurface of treated enamel by sem. plm analysis slices of 150 ± 10 mm from specimens were polished with 600and 1200-grit aluminum oxide disks under water effect of fluoride-containing bleaching agents on bovine enamel microhardness 24 braz j oral sci. 10(1):22-26 cooling to a thickness of 100 ± 10 µm. the slabs were immersed in distilled and deionized water, mounted in glass slides and the demineralization depth was analyzed in a polarized light microscope (dm lsp, leica microsystems, heerburg, switzerland). the images were transferred to the computer via digital camera. the lesion depth was measured at five points from the enamel surface using computer software (image-pro plus, 4.1 version for windows, media cybernetics, silver spring, md, usa) and the values were expressed in micrometers (µm) to calculate the means. sem analysis the surfaces from the other halves were polished with 600-, 1200and 2000-grit sic papers, followed by diamond pastes (6, 3, 1 and ¼ µm) and dehydrated in ascending ethanol concentrations (30, 50, 70, 90 and 100%). afterwards, they were sputter-coated with gold (med 010, baltec, balzers, liechtenstein) and observed with a scanning electron microscope (jsm-5600, jeol inc., peabody, ma, usa). representative areas of enamel were photographed at 2,500× magnification. statistical analysis the %smhr data were analyzed by the kruskal wallis and dunn’s test (α=0.05). the data from microhardness analysis and demineralization depth were, respectively, analyzed by two-way (bleaching and enamel treatment) repeated-measures analysis of variance and one-way analysis of variance followed by tukey’s test (α=0.05). results the microhardness mean values and standard deviation (baseline, after demineralization and after experimental treatments) for the groups and %smhr data are displayed in table 1. the control and experimental groups showed significant lower microhardness (p<0.01) after demineralization when compared to baseline. after ph-cycling regimen, the surface microhardness after treatment was significantly higher than after demineralization but lower than baseline. the microhardness and microhardness recovery from control group was significantly higher than the experimental groups (p=0.0007). significant differences concerning the %smhr were not observed among the experimental groups (p values = 0.1873, 0.3544 and 0.5363). the analysis of demineralization depth by plm (table 2) treatments baseline after demineralization after treatments %smhr artificial saliva 378.3 ± 45.1 aa 137.3 ± 39.0 ab 230.4 ± 85.5 ac 38.6a 10% cp 388.1 ± 50.3 aa 119.3 ± 59.0 ab 148.7 ± 49.0 bb 10.9b 10% cp + 0.11% f 383.7 ± 48.8 aa 106.8 ± 42.6 ab 151.6 ± 49.6 bb 16.1b 10% cp + 0.5% f 388.4 ± 43.8 aa 128.7 ± 56.2 ab 143.2 ± 64.5 bb 5.5b table 1: microhardness mean values at baseline, after demineralization and after experimental treatments (means ± sd, n=17), and percentage of surface microhardness recovery (%smhr). for microhardness analysis, means followed by different uppercase letters differ statistically vertically and different lowercase letters differ statistically horizontally (tukey test, p<0.05) (cp – carbamide peroxide). for %smhr, values followed by distinct superscript letters differ statistically (kruskal wallis and dunn’s test, p<0.05). artificial saliva 10 % cp 10% cp + 0.11% f 10% cp + 0.5% f 5.7 ± 3.2 a 13.4 ± 3.3 b 16.8 ± 5.6 b 15.9 ± 4.0 b table 2 analysis of caries lesion depth (mm) after phcycling model (means ± sd, n=17). means followed by distinct letters differ statistically (p<0.05) (cp – carbamide peroxide). showed superficial and sub-superficial demineralization areas in enamel for all the groups evaluated (figs 1a, 1b, 1c and 1d). the demineralization depth for specimens immersed in artificial saliva was lower than the bleached groups (p<0.0026). sem micrographs revealed demineralization areas located at sub-superficial region of enamel for all the experimental groups (figs 2a, 2b, 2c and 2d). however, the demineralization seemed to be milder in enamel immersed in artificial saliva (figs 1a and 2a) than in the bleached enamel. discussion a recent study has shown that enamel treatment with either fluoridated or unfluoridated carbamide peroxide gels, at both neutral and acidic ph, yielded enamel more susceptible to demineralization16. however, it is not known if the 10% carbamide peroxide can promote similar effects on enamel with caries lesions. the bleaching agents containing or not fluoride did not show mineral recovery when the enamel microhardness values after demineralization were compared to those after ph-cycling, which is in accordance with previous study2,1720. indeed, even in the presence of daily treatment with a fluoridated dentifrice during the ph-cycling, it was not able to promote remineralization. oliveira et al.21 (2005) evaluated the effect of carbamide peroxide containing calcium (0.05% and 0.2%) or fluoride (0.2% and 0.5%) on enamel and showed the reduction of enamel surface microhardness post-bleaching treatment. also, the bleaching of enamel with carbamide peroxide followed by fluoride treatment with 2,000 ppm fluoride solution four times during 2 min did not improve erosive resistance22. the effect of fluoridated dentifrice and saliva on remineralizing artificial caries lesions has been recognized13,23. however, the lack of fluoride effect either in the bleaching agent or in dentifrice can be explained by the fact that carbamide peroxide could have promoted erosion on enamel surface, such as open enamel prisms, increasing porosities as previously described17, which could impair an accurate surface effect of fluoride-containing bleaching agents on bovine enamel microhardness fig. 2. scanning electron microscopy micrographs of sub-superficial enamel (×2,500): specimens subjected to artificial saliva (2a); 10% carbamide peroxide (2b); 10% carbamide peroxide + 0.11% fluoride (2c) and 10% carbamide peroxide + 0.5% fluoride (2d). figures 2a to 2d show enamel sub-superficial demineralization (de) and the interprismatic matrix partially removed, exhibiting the prism cores (pc) (s – superficial enamel). microhardness measurement. also, attin et al.16 (2003) showed that carbamide peroxide/fluoride mixture did lead to a fluoride uptake less than enamel samples treated with pure fluoride gel. however, the study did not evaluate whether this amount of fluoride uptake had any cariostactic effect. the present study did not show remineralization effect of fluoride-containing bleaching gel when compared to non-fluoridated gel. conversely, pretty et al.24 (2005) showed that tooth bleaching with carbamide peroxide did not increase the susceptibility of enamel to acid erosion or caries. furthermore, remineralization of bleached enamel was improved by application of a high fluoride concentration (2.23% fluoride, duraphat, colgate, piscataway, nj, usa)17. in the present study, a fluoridated dentifrice was used at the same dilution that occurs in the mouth during toothbrushing, but no substantial effect was observed for the bleached groups. another study showed that carbamide peroxide containing or not fluoride promoted mineral loss after demineralization and remineralization cycles, concluding that the treatment with either fluoridated or nonfluoridated carbamide peroxide gels (neutral and acidic gel) rendered enamel more susceptible to demineralization16. this study used bovine teeth, which can be considered a research limitation. based on the fact that some authors reported some differences between human and bovine teeth25-26, the results speculate what would happen with human enamel in the same conditions proposed for this study. induction of demineralization reduced the bovine enamel microhardness approximately from 384.6 (430.9 338.3) to 122.7 (72.6 172.8) khn units. this investigation was based on previous studies, which showed similar surface microhardness after demineralization13,27. it is important to emphasize that although the caries-like lesions were superficial, it was possible to observe differences among the treatments (table 1), i.e., the effect of the artificial saliva (unbleached control group) compared to bleaching treatments. enamel demineralization was observed for all specimens, however, with lower mineral loss for those stored in artificial saliva. sem observations revealed that matrix interprismatic of subsuperficial enamel was removed, exposing the prism cores for all experimental groups (figs. 2a, 2b, 2c and 2d). plm analysis also showed demineralization areas for all specimens (figs. 1a, 1b, 1c and 1d). however, the demineralization seemed to be more intense for enamel treated with bleaching agents in subsuperficial part of enamel and on its surface (figs. 1b, 1c and 1d), as a result of bleaching procedures regardless of the presence of fluoride in its composition. the non-bleached specimens were kept in artificial saliva, which favored the remineralization, according to featherstone et al.28 (1986). thus, artificial saliva and fluoridated dentifrice treatments during ph-cycling seemed to increase the %smhr. if the study was performed with more periods of observation after treatments, the storage of the bleached samples for prolonged time in saliva could increase the remineralization28. the results of the present study showed that the presence of fluoride in the bleaching gels did not increase mineral recovery, suggesting that bleaching procedures on enamel with active caries-like lesions should be avoided or used with caution even with daily use of fluoridated dentifrice and fluoride-containing bleaching agent. acknowledgements this study was supported by grants from pibic-cnpq, sae/prp-unicamp and 02/04135-3 from fapesp, brazil. references 1. li y. biological properties of peroxide-containing tooth whiteners. food chem toxicol. 1996; 34: 887-904. 25 effect of fluoride-containing 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asdc j dent child. 1996; 63: 249-56. 12. ganss c, klimek j, schaffer u, spall t. effectiveness of two fluoridation measures on erosion progression in human enamel and dentine in vitro. caries res. 2001; 35: 325-30. 13. paes leme af, tabchoury cmp, zero dt, cury ja. effect of fluoridated dentifrice and acidulated phosphate fluoride application on early artificial carious lesions. am j dent. 2003,16: 91-5. 14. cavalli v, reis af, giannini m, ambrosano gmb. the effect of elapsed time following bleaching on enamel bond strength of resin composite. oper dent. 2001; 26: 597-602. 15. pinto cf, paes leme, cavalli v, giannini m. effect of 10% carbamide peroxide bleaching on sound and artificial enamel carious lesions. braz dent j. 2009; 20: 48-53. 16. attin t, kocabiyik m, buchalla w, hannig c, becker k. susceptibility of enamel surfaces to demineralization after application of fluoridated carbamide peroxide gels. caries res. 2003; 37: 93-9. 17. attin t, kielbassa am, schwanenberg m, hellwig e. effect of fluoride treatment on remineralization of bleached enamel. j oral rehabil. 1997; 24: 282-6. 18. basting rt, rodrigues al jr, serra mc. the effects of seven carbamide peroxide bleaching agents on enamel microhardness over time. j am dent assoc. 2003; 134: 1335-42. 19. pinto cf, oliveira r, cavalli v, giannini m. peroxide bleaching agent effects on enamel surface microhardness, roughness and morphology. braz oral res. 2004; 18: 306-11. 20. mahmoud sh, elembaby as, zaher ar, grawish ma, elsabaa hm, negoly sar et al. effect of 16% carbamide peroxide bleaching gel on enamel and dentin surface micromorphology and roughness of uremic patients: an atomic force microscopic study. eur j dent. 2010; 4: 175-82. 21. oliveira r, paes leme af, giannini m. effect of a carbamide peroxide bleaching gel containing calcium or fluoride on human enamel surface microhardness. braz dent j. 2005; 16: 103-6. 22. burgmaier gm, schulze im, attin t. influence of pre-treatment with carbamide peroxide on fluoride uptake in enamel. j oral reabil. 2002; 29: 799-804. 23. white dj. reactivity of fluoride dentifrices with artificial caries. effects on early lesions: f uptake, surface hardening and remineralization. caries res. 1987; 21: 126-40. 24. pretty ia, edgar wm, higham sm. the effect of bleaching on enamel susceptibility to acid erosion and demineralisation. br dent j. 2005; 198: 285-90. 25. arends j, christoffersen j, ruben j, jongebloed wl. remineralization of bovine dentine in vitro. caries res. 1989; 23: 309-14. 26. sydney-zax m, mayer i, deutsch d. carbonate content in developing human and bovine enamel. j dent res. 1991; 70: 913-6. 27. queiroz cs, hara at, paes leme af, cury ja. ph-cycling models to evaluate the effect of low fluoride dentifrice on enamel deand remineralization. braz dent j. 2008; 19: 21-7. 28. featherstone jdb, o’reilly mm, shariati m. enhancement of remineralisation in vitro and in vivo. oxford: irl press limited; 1986. effect of fluoride-containing bleaching agents on bovine enamel microhardness braz j oral sci. 10(1):22-26 oral sciences n3 original article braz j oral sci. april/june 2010 volume 9, number 2 mechanical and acid root treatment on periodontally affected human teeth a scanning electronic microscopy priscilla barbosa ferreira soares1, carolina guimarães castro2, carolina assaf branco3, denildo magalhães1, alfredo júlio fernandes neto3, carlos josé soares2 1department of periodontology and implantology, dental school, federal university of uberlândia, brazil 2department of operative dentistry and dental materials, dental school, federal university of uberlândia, brazil 3department of prosthodontics and dental materials, dental school, federal university of uberlândia, brazil correspondence to: carlos josé soares faculdade de odontologia – universidade federal de uberlândia área de dentística e materiais odontológicos av. pará, n° 1720, campus umuarama, bloco 2b, sala 2b-24, cep: 38405-902 uberlândia, mg, brasil phone: 55 34 32182255 / fax: 55 34 32182279 e-mail: carlosjsoares@umuarama.ufu.br received for publication: march 26, 2010 accepted: june 16, 2010 abstract aim: to evaluate the root topography of human teeth affected by periodontitis, after different root surface treatments. methods: forty-two periodontally affected single-rooted human teeth were selected and randomly divided into 7 groups (n=6): contcontrol group, which received no treatment; scaroot surface scaling and root planning with curettes; scaph sca followed by 37% phosphoric acid gel etching for 15 s; scaedtasca followed by 24% edta gel ph 7 for 1 min; scacisca followed by 30% citric acid ph 1.6 for 5 min; scatesca followed by mixture obtained by 500 mg tetracycline capsule dissolved in saline solution for 3 min; scategsca followed by 0.2 g/ml tetracycline gel ph 1.8 for 1 min. the specimens were analyzed by scanning electronic microscopy to verify the presence of calculus, demineralization level and residues of the product. results: calculus deposits were found in all control specimens. scaedta, scaci and scateg removed completely calculus deposits and resulted in adequate demineralization without smear layer and smear plug on root surface. scate produced great tetracycline residues with several demineralization areas on root dentin surface. conclusions: scaedta, scaci and scateg produced clean root surfaces associated with regular dentin demineralization. keywords: scanning electron microscopy; periodontitis; conditioning; dental scaling; root planning and demineralization introduction dental calculus is constituted by mineralized structure with numerous holes, which leading to the accumulation of a larger number of microorganisms1. associated with plaque colonization it is one of the main determinants of periodontal disease. diseased root surfaces are unfavorable to cell attachment probably due to endotoxin adsorption2-3. to regenerate the periodontal structure affected by disease it is necessary to eliminate calculus, bacterial plaque and cytotoxic substances from the contaminated root surface4. in addition, exposed cementum removal by scaling and root planning has been recommended as part of periodontal therapy5-6. these procedures can also effectively to remove the bacterial deposits and endotoxin2. after root planning, the instrumented root surface is invariably covered by a smear layer, which contains remnants of dental calculus, contaminated root cementum, bacterial toxins, and subgingival plaque7. therefore, braz j oral sci. 9(2):128-132 129 the use of additional chemical protocols after scaling and root planning tends to be decisive to achieve successful in periodontal therapy. acid agents produce a demineralization zone that improves periodontal regeneration, which is an important natural phenomenon in the healing process8-9. the use of demineralizing agents increases the degree of connective tissue attachment to denuded roots 10. this procedure exposes dentin collagen fibrils and opens dentinal tubules11, removes cementum-bound proteins11 and eliminates the contaminated smear layer produced by root planning8,12. several agents, such as phosphoric acid7, ethylenediaminetetraacetic acid7,13-14, citric acid7-8,15 and tetracycline hydrochloride6,8,11 have been used to chemically treat the periodontitis affected root surfaces. phosphoric acid, with low ph 1.9416, has been widely used in adhesive dentistry to remove the smear layer, smear plug and to demineralize the peritubular and intertubular dentin16. on the other hand, it is not frequently used in periodontal therapy. edta with neutral ph has recently been used not only to preserve periodontal cell vitality but also to demineralized and remove the smear layer of the dentin14-15. this product also has bactericidal effects4 depending on the concentration6. citric acid has been recommended for removing smear and exposing collagen in order to retard gingival epithelium down-growth 10,17-18. another product usually used on periodontal treatment is tetracycline hydrochloride because of its effective antibacterial action on periodontal pathogens11,19. this product also demineralizes the root surface, removes the smear layer10, promotes fibrin clot stabilization10-11, increases chemotaxis, adhesion, and growth of fibroblasts on the root surface19-20 and inhibits matrix metaloproteinases21-22. tetracycline has been used as root surface conditioning agent to enhance periodontal tissue regeneration19. however, it is not clear the real performance of this protocol on the root dentin, mainly the use of tetracycline capsules, regarding product residues left after use and the smear layer removal capacity. all chemical products used after scaling and planning of the dentin should result in clean surface without calculus remaining and adequate demineralization. scanning electronic microscopy (sem) is an adequate tool used to characterized surface of dental structures13. this methodology can be employed to demonstrate the effect of the different chemical protocols used in association with scaling and root planning over root dentin. the aim of this study was to analyze by sem the effect of hand scaling and planning associated with different acid agents on the roots of periodontally affected teeth, regarding to 3 factors: presence of calculus, product residues after use, and presence of smear layer and smear plug on dentin surface. material and methods forty-two single root human teeth extracted due to severe periodontitis were used in this study after approval by the local research ethics committee (protocol #051/05). the specimens were randomly divided into 7 groups (n=6): cont (control)no root surface treatment was performed; scaroot surfaces were scaled with a gracey curette (hu-friedy, chicago, il, usa) to remove calculus deposits and cementum, thus exposing visual clean dentin (this was the first step for all the others groups); scaphafter sca the dentin was etched with 37% phosphoric acid gel (sdi, victoria, australia) for 15 s. scaedtaafter sca the dentin was etched with 24% edta gel ph 7.0 (biopharma, uberlândia, mg, brazil) for 1 min; scaciafter sca the dentin was etched with 30% citric acid ph 1.6 (biopharma, uberlândia, mg, brazil) for 5 min; scateafter sca the dentin was etched for 3 min with a solution obtained by dissolving one 500 mg capsule of tetracycline (tetraciclina, medquimíca, são paulo, sp, brazil) in 2 ml of saline solution; scategafter sca the dentin was etched 0.2 g/ml tetracycline gel ph 1.8 (biopharma, uberlândia, mg, brazil) for 1 min. after chemical etching, all specimens were rinsed for 1 min with 10 ml of saline solution. the specimen was mounted on aluminum stubs, sputtercoated with gold, and examined with a scanning electron microscope (leo 435 vp, carl zeiss, germany). images of representative areas of each specimen were obtained at 5004000x magnification. the sem micrographs were analyzed by 3 calibrated examiners according to 3 parameters: presence of calculus: 1 – presence of great amount on dentin; 2absence of calculus; presence of smear layer: 1presence of great amount of smear layer and smear plug; 2absence of smear layer and smear plug; presence of the product residues: 1presence of great amount of product residues; 2no product residues left use. data were described by distribution and percentages for 2 parameters on each level, and were data were presented by score distribution frequency. representative sem images of each group were obtained. results the parameter scores for all groups are shown in table groups presence of calculus presence of smear layer presence of product residues 1 2 1 2 1 2 1control 6 n a n a n a n a 2scaling and root planing 6 6 n a n a 3phosphoric acid etching 6 6 6 4edta etching 6 6 6 5citric acid etching 6 6 6 6tetracycline capsule dissolved in saline 6 n a n a 6 7tetracycline gel 6 6 6 table 1. parameter scores evaluated by scanning microscopy analysis for all groups na not applied to this group. braz j oral sci. 9(2):128-132 mechanical and acid root treatment on periodontally affected human teeth a scanning electronic microscopy 1. the sem analysis showed that all dentin specimens of cont group were covered by calculus and debris (figure 1). sca resulted in considerable amount of debris, no residual calculus, irregular root surface and a smear layer and smear plug covering all dentin (figure 2). the sem of the scaph group showed absence of smear layer and smear plug in all specimens, and no remaining of phosphoric acid (figure 3). in the scaedta group was found the complete removal of smear layer, more regularity of the dentin surface and no remaining of the edta (figure 4). the scaci protocol resulted in complete smear layer removal and no remaining of citric acid (figure 5). in the scate group, irregular demineralized dentin with deeper depressions and the presence of a high amount of the residual tetracycline was found on the dentin surface. over all specimens of this group the dentin tubules were completely closed by tetracycline residues (figure 6). the scateg protocol resulted in a uniform demineralization of the dentin tubules, absence of smear layer and smear plug, and no remaining of the tetracycline gel (figure 7). fig. 1 sem image of the cont group showing calculus and debris over root dentin, covering the entire root surface. fig. 2 sem image of the sca group showing the presence of debris, no residual calculus, irregular root surface and smear layer and smear plug covering all root dentin. fig. 3 sem image of the scaph group showing complete removal of smear layer and smear plug and no acid product residues. fig. 4 sem image of the scaedta group showing complete removal of smear layer and no edta residues. fig. 5 sem image of the scaci group showing complete removal of smear layer and no citric acid residues. 130 braz j oral sci. 9(2):128-132 mechanical and acid root treatment on periodontally affected human teeth a scanning electronic microscopy fig. 7 sem image of the scateg showing uniform demineralization of the dentin tubules, absence of smear layer and smear plug, and no tetracycline gel residues. fig. 6 sem image of the scate demonstrating presence of residual tetracycline on dentin surface, and all dentin tubules closed. discussion one of the objectives of periodontal therapy is to convert the root surface affected by periodontitis into a surface biologically compatible with epithelial and connective tissue adherence and attachment11. the presence of dental calculus associated with plaque colonization is one of the main determinants of periodontal disease. this study demonstrated that all teeth (table 1), extracted by the patients with periodontal disease, presented calculus with numerous holes (figure 1). the smear layer and mineralized debris formed after root surface instrumentation may serve as a physical barrier to the development of connective tissue attachment to the root surface11. in the present study, after scaling, the root dentin of all specimens (table 1) was covered by smear layer smear plug (figure 2). however, no calculus deposits were found after mechanical treatment (table 1), demonstrating that this procedure should be the first protocol in all periodontal therapies. since root scaling forms an irregular smear layer along the external root dentin, and it is invariably contaminated by toxins generate by previous disease present5-6, additional chemical treatment is indicated in periodontal therapy. the results of this study confirmed morphological modifications caused by the application of complementary substances. edta and citric acid were applied in the form of gel preparations, which are considered to increase the possibility of controlling the etching agent action15. the 24% edta used in this study was demonstrated by blomlöf et al. 23 to be significantly more effective than the lower concentrations tested regarding to removal capacity of smear layer. the citric acid 30% used in this study is more effective to demineralize dentin than the lower concentrations ones24. citric acid application causes superficial root surface demineralization24, which is capable to eliminate bacterial endotoxinas25. this product is bactericidal26 and capable to partially exposes dentin collagen27. this latter effect has been proven important to increase collagen splicing, improve fibrin linkage, and consequently inhibit epithelial down growth28. although it was not assessed in this study, previous investigations have found that this effect stimulates the fibroblast attachment and migration29, and facilitates new cementum formation17,30. in this study, the use of edta and citric acid after scaling and root planning resulted in effective smear layer and smear plug removal (figures 4 and 5). however, it has recently been demonstrated that edta, which is used in a neutral ph (ph 7.0), is better to maintain the periodontal cell vitality adjacent to the etched surface than the citric acid14 and phosphoric acid14. hand scaled roots treated with phosphoric acid presented dentin demineralization with complete removal of the smear layer and smear plugs (table 1 and figure 3). however, this result should be carefully analyzed because this product has a high capacity of the demineralization. it could be a serious problem if some area was not covered by gingival after surgery, resulting in dentin hypersensitivity31. when the results of the groups that used tetracycline were compared, it could be observed that tetracycline gel removed the smear layer and no tetracycline residue was found. however, the tetracycline capsule dissolved in saline left tetracycline residues on the dentin root surface. the tetracycline from capsules for oral use bought at pharmacies and used to prepare an acid solution resulted in a significant amount of filler and other substances on dentin. this medication should be avoided until the effect of these materials on the root surface has been investigated32. the sem results (table 1) of this study showed a severe demineralization of root dentin substrate with the presence of a high amount of residues on the surface (figure 6), occurred because tetracycline was not completely dissolved. the presence of residues of tetracycline particles during long time may result in continuous demineralization of the root dentin. furthermore, this procedure caused an extremely irregular surface with many depressions after demineralization. for this reason, the application of tetracycline capsule dissolved in saline solution should be avoided. however, the same effect was not found with the tetracycline gel (table 131 braz j oral sci. 9(2):128-132 mechanical and acid root treatment on periodontally affected human teeth a scanning electronic microscopy 132 1 and figure 7). the tetracycline gel tested in the present study presents additional benefits for use on dentin surfaces as the enhanced extracellular matrix glycoprotein fibronectin binding to dentin and stimulated fibroblast growth and attachment19. this substance has potent inhibitory effects on metalloproteinases21-22, osteoclast function21 and has antiinflammatory properties19. furthermore, tetracycline is adsorbed to and subsequently released from dentin, maintaining its antimicrobial activity33. the results of this study suggest that tetracycline gel, edta or citric acids can be indicated to obtain an efficient and uniform demineralization of root dentin surface. moreover, their pharmacological characteristics are important for periodontal disease management. however, other characteristics of these products should not be overlooked. other researchers have suggested testing the viability of cellular fixation and the possibility of soft tissue alterations as a result of the use of these protocols. acknowledgements this study was supported by fapemig (fundação de amparo à pesquisa do estado de minas gerais, mg, brazil). this study was carried out in the lipo-foufu. the authors are indebted to prof. ew kitajima (nap/mepa-esalq/usp) for technical support in scanning electron microscopy. references 1. rohanizadeh r, legeros rz. ultrastructural study of calculus-enamel and calculus-root interfaces. arch oral biol. 2005; 50: 89-96. 2. aleo jj, de renzis fa, farber pa, varboncoeur ap. the presence and biologic activity of cementum-bound endotoxin. j periodontol. 1974; 45: 672-5. 3. higashi t, okamoto h. the effect of ultrasonic irrigation before and after citric acid treatment on collagen fibril exposure: an in vitro sem study. j periodontol. 1995; 66: 887-91. 4. lasho dj, o’leary tj, kafrawy ah. a scanning electron microscope study of the effects of various agents on instrumented periodontally involved root surfaces. j periodontol. 1983; 54: 210-20. 5. babay n. attachment of human gingival fibroblasts to periodontally involved root surface following scaling and/or etching procedures: a scanning electron microscopy study. braz dent j. 2001; 12: 17-21. 6. babay n. sem study on the effect of two different demineralization methods with saturated tetracycline hydrochloride on diseased root surfaces.j contemp dent pract. 2001; 2: 25-35. 7. blomlof j, jansson l, blomlof l, lindskog s. root surface etching at neutral ph promotes periodontal healing. j clin periodontol. 1996; 23: 50-5. 8. babay n. nondiseased dentinal root surface following citric acid or tetracycline hydrochloride conditioning: a scanning electron microscopic study on the effects of ultrasonic irrigation before and after root conditioning. quintessence int. 1997; 28: 93-7. 9. sakallioglu u, acikgoz g, ayas b, kirtiloglu t, sakallioglu e. healing of periodontal defects treated with enamel matrix proteins and root surface conditioning—an experimental study in dogs. biomaterials. 2004; 25: 1831-40. 10. isik ag, tarim b, hafez aa, yalcin fs, onan u, cox cf. a comparative scanning electron microscopic study on the characteristics of demineralized dentin root surface using different tetracycline hcl concentrations and application times. j periodontol. 2000; 71: 219-25. 11. isik g, ince s, saglam f, onan u. comparative sem study on the effect of different demineralization methods with tetracycline hcl on healthy root surfaces. j clin periodontol. 1997; 24: 589-94. 12. baker pj, rotch ha, trombelli l, wikesjo um. an in vitro screening model to evaluate root conditioning protocols for periodontal regenerative procedures. j periodontol. 2000; 71: 1139-43. 13. babay n. the effect of edta on the attachment and growth of cultured human gingival fibroblasts in periodontitis-affected root surface. j contemp dent pract. 2001; 2: 13-23. 14. sampaio je, rached rs, pilatti gl, theodoro lh, batista lh. effectiveness of edta and edta-t brushing on the removal of root surface smear layer. pesq odontol bras. 2003; 17: 319-25. 15. blomlof l, bergman e, forsgardh a, foss l, larsson a, sjoberg b, uhlander l, jonsson b, blomlof j, lindskog s. a clinical study of root surface conditioning with an edta gel. i. nonsurgical periodontal treatment. int j periodontics restorative dent. 2000; 20: 560-5. 16. oliveira ss, marshall sj, habelitz s, gansky sa, wilson rs, marshall gw jr. the effect of a self-etching primer on the continuous demineralization of dentin. eur j oral sci. 2004; 112: 376-83. 17. register aa, burdick fa. accelerated reattachment with cementogenesis to dentin, demineralized in situ. i. optimum range. j periodontol. 1975; 46: 646-55. 18. larjava h, salonen j, hakkinen l, narhi t. effect of citric acid treatment on the migration of epithelium on root surfaces in vitro. j periodontol. 1988; 59: 95-9. 19. seymour ra, heasman pa. tetracyclines in the management of periodontal diseases. a review. j clin periodontol. 1995; 22: 22-35. 20. terranova vp, franzetti lc, hic s, diflorio rm, lyall rm, wikesjo um, baker pj, christersson la, genco rj. a biochemical approach to periodontal regeneration: tetracycline treatment of dentin promotes fibroblast adhesion and growth. j periodontal res. 1986; 21: 330-7. 21. golub lm, ramamurthy ns, mcnamara tf, greenwald ra, rifkin br. tetracyclines inhibit connective tissue breakdown: new therapeutic implications for an old family of drugs. crit rev oral biol med. 1991; 2: 297-321. 22. ingman t, sorsa t, suomalainen k, halinen s, lindy o, lauhio a et al. tetracycline inhibition and the cellular source of collagenase in gingival crevicular fluid in different periodontal diseases. a review article. j periodontol. 1993; 64: 82-8. 23. blomlöf j, blomlöf l, lindskog s. effect of different concentrations of edta on smear layer and collagen exposure in periodontitis-affected root surfaces. j clin periodontal. 1997; 24: 534-7. 24. sterrett jd, bankey t, murphy hj. dentin demineralization. the effects of citric acid concentration and application time. j clin periodontol. 1993; 20: 366-70. 25. fine dh, morris ml, tabak l, cole jd. preliminary characterization of material eluted from the roots of periodontally diseased teeth. j periodontal res. 1980; 15: 10-9. 26. daly cg. anti-bacterial effect of citric acid treatment of periodontally diseased root surfaces in vitro. j clin periodontol. 1982; 9: 386-92. 27. vanheusden aj, goffinet g, zahedi s, nusgens b, lapiere cm, rompen eh. in vitro stimulation of human gingival epithelial cell attachment to dentin by surface conditioning. j periodontol. 1999; 70: 594-603. 28. polson am, proye mp. fibrin linkage: a precursor for new attachment. j periodontol. 1983; 54: 141-7. 29. rompen eh, kohl j, nusgens b, lapiere cm. kinetic aspects of gingival and periodontal ligament fibroblast attachment to surface-conditioned dentin. j dent res. 1993; 72: 607-12. 30. somerman mj, archer sy, hassell tm, shteyer a, foster ra. enhancement by extracts of mineralized tissues of protein production by human gingival fibroblasts in vitro. arch oral biol. 1987; 32: 879-83. 31. haugen e, johansen jr. tooth hypersensitivity after periodontal treatment. a case report including sem studies. j clin periodontol. 1988; 15: 399401. 32. lafferty ta, gher me, gray jl. comparative sem study on the effect of acid etching with tetracycline hcl or citric acid on instrumented periodontallyinvolved human root surfaces. j periodontol. 1993; 64: 689-93. 33. stabholz a, kettering j, aprecio r, zimmerman g, baker pj, wikesjo um. antimicrobial properties of human dentin impregnated with tetracycline hcl or chlorhexidine. an in vitro study. j clin periodontol. 1993; 20: 557-62. braz j oral sci. 9(2):128-132 mechanical and acid root treatment on periodontally affected human teeth a scanning electronic microscopy original article braz j oral sci. april/june 2009 volume 8, number 2 burning mouth syndrome: a discussion about possible etiological factors and treatment modalities paula aparecida nakazone1, andressa vilas boas nogueira2, francisco guedes pereira de alencar júnior3, elaine maria sgavioli massucato4 1 dds, ms, post-graduate student, department of restorative dentistry, faculdade de odontologia de araraquara, universidade estadual paulista “júlio de mesquita filho” (unesp), araraquara (sp), brazil 2 dds, ms, post-graduate student, department of diagnosis and surgery, faculdade de odontologia de araraquara, unesp, araraquara (sp), brazil 3 dds, ms, phd, assistant professor, department of general dental sciences, marquette university school of dentistry, milwaukee, wisconsin, usa 4 dds, ms, phd, assistant professor, department of diagnosis and surgery, faculdade de odontologia de araraquara, unesp, araraquara (sp), brazil received for publication: april 3, 2009 accepted: may 18, 2009 correspondence to: elaine maria sgavioli massucato departamento de diagnóstico e cirurgia, faculdade de odontologia de araraquara, universidade estadual paulista rua humaitá, 1.680, 2º andar cep 14801-903 – araraquara (sp), brazil e-mail: emassucato@yahoo.com.br abstract although several studies discuss the contributing factors associated with the burning mouth syndrome (bms), there is still controversy with regard to its etiology. therefore, in the majority of cases, the establishment of an adequate diagnosis and consequently the best treatment modality is complicated. in order to assist the clinician in the establishment of the correct diagnosis and management of bms, this article reviews the literature, providing a discussion on the various etiologic factors involved in bms, as well as the best treatment modalities for this condition that have showed to be the most effective ones in randomized clinical trials. in addition, the authors discuss some clinical characteristics in the differential diagnosis of bms and other oral diseases. it is important for the clinician to understand that bms should be diagnosed only after all other possible causes for the symptoms have been ruled out. keywords: burning mouth syndrome, xerostomia, mouth diseases. introduction several diseases of the oral mucosa may have burning as a symptom, such as herpes simplex virus, oral lichen planus (under its clinical forms: erosive, atrophic and ulcerative), aphthous stomatitis, candidiasis (mainly under its acute form) and xerostomia. however, patients who refer a burning sensation if the oral mucosa or a chronic pain without any visible alteration of the oral tissues might be diagnosed as having burning mouth syndrome (bms)1. it is very important for clinicians to be able to distinguish a burning mouth symptom caused by a specific disease besides bms. bms is considered to be a pain or burning sensation affecting the clinically normal oral tissues, for which local and systemic causes have been excluded. the term “syndrome”, in this case, is justified because of the simultaneous presence of several subjective symptoms, including feeling of dryness (subjective xerostomia), altered taste, and burning sensation of the oral tissues, comprising or not the tongue2,3. 63burning mouth syndrome: a discussion about possible etiological factors and treatment modalities braz j oral sci. 8(2): 62-6 literature review definition bms is referred as a chronic orofacial pain or burning sensation in the oral mucosa or tissues without any clinically significant lesion or alteration. according to the international headache society, bms is an intraoral burning sensation for which no dental or medical cause is found4. the bms pain may be described as a burning sensation that is often qualitatively compared to a toothache. a wide variety of terms have been reported in the literature to describe bms, which include glossodynia/stomatodynia, glossopyrosis/stomatopyrosis, oral dysesthesia and sore mouth5,6. epidemiology bms may occur in any tissue inside the oral cavity, although most often it is found on the two thirds of the anterior and on the tip of the tongue1. this disease has a prevalence that varies from 0.7 to 15% in the general population7-10 and has an average duration of two to three years11. it predominantly affects middle-aged women in the postmenopausal phase and in a ratio of 7:1 when compared to men12,13. few studies have mentioned the presence of bms in earlier ages7,12,14, which indicates that its prevalence increases with age4. according to lamey and lamb15, the symptomatology associated with bms may be classified in three types16,17, as shown in table 1. pathogenesis and etiology the pathogenesis and etiology of the bms are not completely understood yet. some authors have suggested that there is a multifactorial etiology: local, systemic and psychological factors (table 2)14,18-24. the local factors may include temporomandibular disorders (tmd), oral candidiasis, parafunctional habits (clenching and bruxism), xerostomia (figure 1), salivary glands dysfunction, hypersensitivity reactions (figure 2) and misfited and poorly designed dentures. however, the literature is sometimes conflicting and unclear, because the diagnosis of bms should be confirmed after ruling out other causes to the burning sensation, such as oral candidiasis or tmd. special attention must be given to the dentures, since it has been demonstrated that there is a possible correlation between the problems related to oral dentures (adjustment, design) and the bms for both may cause central and/or peripheral changes in the sensory nerve function, causing atypical oral pains25,26. types bms symptomatology according to lamey and lamb15 factors associated with bms in each type16,17 1 symptoms are not present when the patient wakes up, but they will appear and increase during the day. moderate anxiety disorders. 2 symptoms are present all day and night and strongly associated with anxiety. severe psychiatric disorders. 3 symptoms are not present during some days and are associated with emotional instability or a hypersensitivity reaction to some foods. emotional instability or allergic reactions. table 1. classification of the symptomatology associated with burning mouth syndrome (bms) local systemic psychological neuropathic temporomandibular disorders nutritional deficiencies depression neurogenic abnormalities oral candidiasis diabetes mellitus anxiety parafunctional habits hormonal disturbances psychopathologic distress xerostomia immunomediated diseases salivary glands dysfunction systemic drugs hypersensitiveness reactions misfited or poorly designed dentures table 2. factors associated with the etiology of burning mouth sensation figure 1. patient with xerostomia. figure 2. patient with hypersensitivity reaction. 64 nakazone pa, nogueira avb, alencar júnior fgp , massucato ems braz j oral sci. 8(2): 62-6 concerning hypersensitivity reaction, mott et al.26 mentioned not only allergy to denture base acrylic resins and contouring or fabrication errors, but also the presence of parafunctional habits as important factors in the development of bms. furthermore, gao et al.24 have found that oral parafunctional habits are causative agents in bms, specially tongue thrusting and lip sucking. xerostomia has been identified in almost 65% of the patients with bms7,14,27-29, which demonstrates that these patients are significantly more susceptible to this condition. marques-soares et al.30 have investigated the function of the salivary gland in bms pathogenesis and found divergent results, concluding that it is still not clear whether hyposalivation is a typical sign of this syndrome. those authors have also evaluated the salivary flow rate and found no statistically significant differences. furthermore, it is known that the administration of certain medications as diuretics, anti-hypertensive drugs and mainly psychotropics may influence salivary gland function31. radiotherapy on head and neck regions may produce severe and irreversible damages to the salivary glands, leading to a severe condition of permanent xerostomia, which have to be identified during the clinical interview32. bergdahl and bergdahl32 have stated that psychological factors have an influence on xerostomia, sometimes without hyposalivation, and that they could be intimately related to depression, anxiety and use of antidepressants. in a case-control study that investigated anxiety and salivary cortisol levels in patients with bms, amenábar et al.33 found that 50% of these patients presented a worse level of anxiety than those without bms. these authors associated this bmsanxiety relationship with the salivary cortisol level that is presented in a higher level in patients with these two disorders. cavalcanti et al.13 have found no difference in the presence of candida albicans between bms and control patients. thus, candidiasis has not been confirmed as an associate etiological factor for bms. some systemic factors might also be associated with bms, such as nutritional deficiencies as pernicious anaemia, iron deficiency, vitamin b complex deficiency, folate deficiency and vitamin c deficiency34. yet, systemic diseases, such as diabetes mellitus, hormonal disturbs, immunomediated diseases and psychological disorders could be associated factors21,35. some authors have pointed out a possible relationship between diabetes mellitus and bms, since this syndrome is found in 2 to 10% of diabetic patients15,34,36,37. although sardella et al.38 did not find this relationship, burning sensation could be a symptom of an undiagnosed diabetes mellitus39 and perhaps the control of diabetes leads to the improvement or cure of bms6. deficiency disorders have always been referred as cause of bms. nutritional deficiencies have been claimed to cause bms in 2 to 33% of patients40. however, other studies did not find a high prevalence of nutritional deficiency in patients with bms15,36. bms may also change the individual’s general and psychological well being, reducing the quality of life, even though it is not clear if psychopathologic distress is related to this syndrome or if it is a result of the chronic symptoms that these patients passed through18. some studies have reported that people with bms experience adverse life events more frequently than people without bms, which may be a risk factor for developing bms24,41. there is evidence of a possible relationship between bms and psychogenic factors, as shown by sardella et al.38. more recently, several authors have investigated the trigeminal somatosensory system in order to detect neurogenic abnormalities42-47. these studies suggest peripheral alterations in the function of this system with the presence of abnormal reflex, for example, the blink reflex44. calcitonin gene-related peptide (cgrp) is one of the neurotransmitters found in the nerve fibers of the nervous system that is involved in salivary secretion and plays an important role in the development of pain and hyperalgesia48. supporting this interpretation, some studies showed that the use of neuroprotective/neurotropic drugs improved the symptoms in patients with bms19,49. however, zidverc-trajkovic et al.50 found no elevated levels of cgrp in the saliva of patients with bms, which seems to demonstrate the trigeminal nerve degeneration in this syndrome. psychiatric disorders could be associated with more than 50% of the cases of bms51. several studies in psychiatric literature have associated anxiety and depression with bms symptomatology14,42,52,53. gao et al.24 found anxiety, depression and somatization symptoms to be the most common psychological problems in bms. bergdahl et al.16 demonstrated that patients with bms exhibited low levels of socialization and high levels of anxiety and health status concern when compared to a control group. marques-soares et al.30 identified medications that could have a preventive role with regard to bms, such as systemic drugs for vascular and digestive disorders, analgesics and psychotropic drugs. hugosson and thorstensson53, in a study involving patients with bms and a control group, have observed that 87.5% of the patients with the syndrome were usually taking one or more drugs; 44% were psychotropic drugs, 25% were digestive disorder drugs, 25% were respiratory disorder drugs and 6.2% were vitamins54. according to bergdahl and bergdah7, the chronic use of systemic drugs may be a significant factor for bms. in a study that investigated the clinical basis of this syndrome and a possible association with the oral carriage of candida species, cavalcanti et al.13 found that 80.6% of the patients with bms were chronic users of systemic drugs, among which 35.5% were benzodiazepines, 19.35% were other antidepressants and 38.7% were antihypertensive drugs. some drugs, like antihypertensive agents, are frequently associated with the beginning of symptoms compatible with bms. antihypertensive drugs that act in the renin-agiotensin system are more frequently related to the occurrence of bms55. according to drage and rogers11, over 37% of the patients exhibit more than one factor contributing to oral pain sensation (burning), which must be identified and treated. in contrast, there are cases of spontaneous remission in approximately half of patients with bms, as reported elsewhere5,28,56. sardella et al.38 evaluated a group of patients with bms who received the definitive diagnosis after being submitted to a clinical examination, standard set of examinations (salivary flow rate), lab tests (complete blood cell count, blood glucose levels, serum iron level, transferrin level, serum vitamin b level and folic acid level) and isolation of candida spe65burning mouth syndrome: a discussion about possible etiological factors and treatment modalities braz j oral sci. 8(2): 62-6 cies from oral mucosa scraping. tests were performed for contact allergy as a means of excluding this possibility. after analysis and 18 months of follow-up, spontaneous remission cases occurred in the patients who had not received any type of treatment. therefore, the findings of this study suggest that a spontaneous remission may be expected only in a small portion of patients within five years after the onset of bms. alcohol consumption and smoking must be abolished during the treatment of bms. treatment bogetto et al.57, in a randomized clinical trial (rct), compared amisulpride, paroxetine, clordemetildiazepam and amitriptyline to a placebo and found a statistically significant reduction from baseline in bms symptoms and depression for patients receiving 50 mg/day amisulpride. in a 60-day rct, femiano et al.58 compared alpha-lipoic acid to cellulose (placebo). the results showed that 97% of the patients receiving the treatment had some level of improvement, and only 40% of those receiving placebo had a slight improvement. in a 14-day rct19 with six months of follow-up, clonazepam was compared to placebo. at the end of the treatment period, a statistically significant difference in the mean decrease in pain intensity was observed among the patients who used clonazepam. cognitive behavioral therapy is another type of treatment that showed to be of great value in the management of bms. bergdahl et al.59, in a previous rct, found a statistically significant difference in the reduction of pain intensity for those receiving this type of therapy in comparison to a placebo attention program immediately following the therapy and after six months of follow-up. oral lafutidine showed a significant effect in reducing the intensity of oral burning sensation and may be a viable option for the treatment of bms60. yamazaki et al.61 observed that the use of paroxetine for the treatment of bms reduced the pain in about 80% of patients with bms with minor transient side effects. there is no consensus in the literature concerning the best treatment approach. conservative management, such as low doses of tricyclic antidepressants, benzodiazepines or doxepins and topical clonazepam or gabapentin are some options that have been evaluated32,62,63. however, heckmann et al.64 demonstrated that gabapentin presents few or no effect in bms. there is insufficient evidence to understand the real cause and to provide clear guidance for an effective treatment of bms. most studies are small, uncontrolled investigations, and there are no reports of longitudinal cohort studies. further research is therefore needed. the importance of assessing whether burning mouth is a symptom of other disease or a real distinct syndrome must be highlighted. clinicians should identify the bms and its situation and also be able to give a reliable explanation about this condition and its benign nature to the patient. additionally, an individual approach concerning the assessment and treatment of patients with bms is necessary. in order to reduce patient’s suffering, psychological methods may be helpful for patients to cope with bms symptoms. especially in clinical situations in which there is no consensus about the best treatment approach to be adopted, to be as conservative as possible is a wise choice. in the authors’ experience, the use of combined treatment, such as behavioral modification and psychotherapy associated with the use of gapapentin and/or clonazepan, has been successful. bms could be a kind of neuropathic pain and could respond positively to treatment with gabapentin and other drugs used to manage this condition. definitive diagnosis of bms must be preceded by a thorough clinical examination focusing on the presence of signs such as erythema, candidiasis, xerostomia or any mucosa abnormalities, in addition to review of medical history in a detailed clinical interview. the bms diagnosis will be defined only after excluding all possibilities of oral mucosa diseases, contact allergy reactions and all other possible causes of referred pain or burning sensation. special attention must be given to the prescription of drugs to these patients because most medication can induce xerostomia, which may aggravate bms. due to the multifactorial etiology of this condition (local, systemic, psychogenic and neuropathic), 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med oral patol oral cir bucal. 2008;13:e167-70. 56. gilpin sf. glossodynia. jama. 1936;106:1722-4. 57. bogetto f, bonatto revello r, ferro g, maina g, ravizza l. psychopharmacological treatment of burning mouth syndrome (bms). a study on a sample of 121 patients. minerva psichiatrica. 1999;40:1-10. 58. femiano f, gombos f, scully c, busciolano m, de luca p. burning mouth syndrome (bms): controlled open trial of the efficacy of alpha-lipoic acid (thioctic acid) on symptomatology. oral diseases. 2000;6:274-7. 59. bergdahl j, anneroth g, perris h. cognitive therapy in the treatment of patients with resistant burning mouth syndrome: a controlled study. j oral pathol med. 1995;24:213-5. 60. toida m, kato k, makita h, long nk, takeda t, hatakeyama d, et al. palliative effect of lafutidine on oral burning sensation. j oral pathol med. 2009;38:262-8. 61. yamazaki y, hata h, kitamori s, onodera m, kitagawa y. an open-label, noncomparative, dose escalation pilot study of the effect of paroxetine in treatment of burning mouth syndrome. oral surg oral med oral pathol oral radiol endod. 2009;107:e6-11. 62. drage la, rogers rs. burning mouth syndrome. dermatol clin. 2003;21:135-45. 63. petruzzi m, lauritano d, de benedittis m, baldoni m, serpico r. systemic capsaicin for burning mouth syndrome: short-term results of a pilot study. j oral pathol med. 2004;33:111-4. 64. heckmann sm, heckmann jg, ungethum a, hujoel p, hummel t. gabapentin has little or no effect in the treatment of burning mouth syndrome results of an open-label pilot study. eur j neurol. 2006;13:6-7. oral sciences n3 received for publication: december 07, 2009 accepted: august 27, 2010 original article braz j oral sci. july/september 2010 volume 9, number 3 self-perception of generalized aggressive periodontitis symptoms and its influence on the compliance with the oral hygiene instructions a pilot study renato corrêa viana casarin1, erica del peloso ribeiro2, francisco humberto nociti-jr3, enilson antônio sallum3, antonio wilson sallum3, márcio zaffalon casati3 1dds, ms, phd, assistant professor, university são francisco, brazil 2dds, ms, phd, assistant professor, bahia foundation of science, brazil 3dds, ms, phd, professor, piracicaba dental school, state university of campinas, brazil correspondence to: márcio zaffalon casati departamento de prótese e periodontia fop/unicamp av. limeira 901 13414-018 piracicaba sp phone/fax: 55 19 34125301 e-mail: casati@fop.unicamp.br abstract aim: patient’s adherence to the periodontal treatment is fundamental to the success of the therapy. lack of response to the clinician’s instructions is influenced by various factors, including gender, age and psychosocial profile. the aim of the present study was to evaluate the relationship between self-perceived symptoms of generalized aggressive periodontitis and compliance with the oral hygiene instructions. methods: twenty-six subjects presenting a generalized aggressive form of periodontal disease were selected. the subjects answered a questionnaire to rate the perceived symptoms of periodontal disease with a sensitivity scale, in which a numeric score is attributed to each mentioned symptom. the percentage of sites with pocket probing depth (ppd) e” 5mm as well as the plaque index (pi) and gingival index (gi) were evaluated and the patients received a full mouth prophylaxis. one month later, the patients were re-evaluated for pi, gi, and ppd, and their percent reductions were correlated with the numeric score attributed to the aggressive periodontitis symptoms. spearman’s correlation and wilcoxon’s test were used with a significance level of 5%. results: the greater the self-perception of some of the symptoms, the greater the adherence to the oral hygiene instructions. a positive correlation was observed between the reduction of gi and self perception of bleeding on tooth brushing (p=0.04, r=0.27) and redness and swelling of gums (p=0.04, r=0.26). conclusions: the self-perception of symptoms of generalized aggressive periodontitis could have an influence on the patient’s response to the oral hygiene instructions. keywords: compliance, generalized aggressive periodontitis, periodontal disease symptoms, self perception, oral hygiene instructions. introduction a major aspect of the effective treatment of periodontal disease is adequate oral hygiene1, which consists of a combination of daily tooth brushing, interdental cleaning, and when necessary, use of chemotherapeutic agents (e.g. mouthwash)2. therefore, the success of the treatment ultimately relies on patients’ braz j oral sci. 9(3):388-392 389 compliance with daily dental care, in which satisfactory plaque and inflammation control is fundamental. unfortunately, evidence shows that only a portion of the patients actually comply with the treatment and the level of adherence decreases as the time of treatment is extended over the years3-6. ciancio2 suggested that this lack of compliance would lead to serious consequences for chronic periodontitis, and there is an urgent need to develop and validate effective strategies to improve the performance of patients with periodontitis in applying daily prophylactic dental care and identifying the reasons for their noncompliance. patient adherence depends on believing in the necessity of following preventive and/or treatment recommendations7 and this has been addressed in numerous studies that have investigated patients’ behavioral practices with regard to adherence to the treatment of periodontal disease, especially oral hygiene. some reasons given for non response with respect to oral hygiene include unwillingness to perform oral self-care8, lack of motivation9 and poor dental health beliefs10. an important consideration is that adherence also depends on social and psychological factors11. background factors that influence periodontal disease and beliefs regarding oral health could have a negative influence on the adhesion to supportive periodontal treatment12. age and gender can also negatively modify adherence to the treatment13-14. some theories of human cognitive behavior have also been used to explain non-compliance. the four main theories include the health belief model (hbm); transtheoretical model (tm); theory of reasoned action/ theory of planned behavior (tra); and the social-cognitive theory (sc)15. the hbm focuses on an individual’s perception of the threat posed by a health problem. the tm is concerned with an individual’s readiness to change. the tra focuses on an individual’s intention to perform a behavior. the sc incorporates intra-personal and inter-personal factors and suggests that the benefits of behavior must outweigh the costs. in this context, self perception of the symptoms could have an influence on adherence to periodontal treatment, since the determining factor is that the patient must seek treatment, and this could represent the real importance the patient attaches to oral conditions. this idea increases in importance when considering severe periodontal disease, such as aggressive periodontitis, recognized as a rare condition affecting young subjects, and presents rapid periodontal destruction, also induced and aggravated by biofilm accumulation. thus, the recognized approaches known to contribute to patient adhesion to oral hygiene could be useful tools in therapy to control the disease. the aim of the present study was to evaluate the relationship between self-perception of generalized aggressive periodontitis symptoms and adherence to the oral hygiene instructions. material and methods subjects the patients enrolled in the present study were selected from those initially referred to the periodontal clinic of the piracicaba dental school, state university of campinas, brazil for oral examination and periodontal treatment. patients who presented generalized aggressive periodontitis were selected in accordance with the following inclusion criteria: 1) under 35 years of age; 2) clinical and radiograph attachment loss on incisors and first molars, and in three other teeth; 3) at least 8 sites with pocket probing depth (ppd) > 5 mm, of which 3 of them with ppd > 6 mm; and 4) at least 20 teeth in the mouth. the exclusion criteria were: 1) smoking and pregnancy/nursing mothers; 2) use of mouthrinse or antibiotics 6 months before the study; 3) history of relevant medical condition; and 4) periodontal treatment, including professional prophylaxis, 6 months before the study. ethics the study protocol was previously approved by the institutional clinical research ethics committee of the state university of campinas (unicamp) protocol no. 24/2006. all the selected patients received and signed an informed consent form and thereby agreed to participate in the study. questionnaire initially, the patients received a questionnaire to assess their periodontal status. the patients were questioned about their perception of: pain in the gums, bleeding while brushing, bad breath, spaces between the teeth, mobility of teeth, redness and swelling of gums, dry mouth, bad taste, recent loss of teeth, loss of interproximal gingival tissue and sensitivity when drinking cold or hot drinks. each affirmative was followed by a vas (visual analogue scale) ranging from no feeling to strong feeling. to indicate a recent tooth loss, the patient checked yes or no. the selfevaluation of recent loss was considered without determining the length of time since the loss. each subject was instructed to check on the chart their self-assessment of each symptom listed above. after this, the distance from the origin on the scale to the mark made by the patient, was measured with a caliper. clinical parameters and compliance assessment the measurements were made by a single calibrated examiner (kappa index = 0.91) using a periodontal probe (pcpunc 15-hu-friedy/chicago, il, usa), at the beginning of the study. a dichotomous analysis of the presence of plaque (plaque index – pi16) was made on six surfaces of each tooth, excluding the third molars. the presence of dental plaque accumulation on the buccal, lingual and proximal surfaces of all teeth was assessed using a periodontal probe. to determine the gingivitis status of each patient, a dichotomous self-perception of generalized aggressive periodontitis symptoms and its influence on the compliance with the oral hygiene instructions a pilot study braz j oral sci. 9(3):388-392 390 index was also used (gingival index – gi16). bleeding of the gingival margin within 10 s after probe stimulation was considered positive. pi and gi were evaluated at the beginning and thirty days after the patient received prophylaxis and oral hygiene instructions. reduction in pi and gi was considered to assess each patient’s level of compliance with the oral hygiene instructions. correlation between self-perception and severity of the disease was also studied. initially, the total number of sites with probing depth > 5 mm presenting bleeding on probing was determined in a baseline examination using a periodontal probe. after that, a percentage of these sites per patient was calculated (ppd). prophylaxis and oral hygiene instructions immediately after the first evaluation, professional prophylaxis was performed in each patient, consisting of calculus and plaque removal with an ultrasonic device (profi iii – bios, dabi-atlante, ribeirão preto, sp, brazil) and brushes. after this, patients were given instructions on toothbrushing and interproximal cleaning with dental floss. all the patients received the same protocol of prophylaxis and instructions. the importance of plaque control and instructions on oral care were reinforced within 15 days after the first session17-18. data analysis the biostat® program (version 3.0) was used to determine the relationship between the parameters. spearman’s correlation test was used to test the relationship between the values of self-perception and initial pi and gi values, as well the ppd values. to determine the influence of selfperception of symptoms on adherence to the oral hygiene instructions, the percent reduction in pi and gi was also correlated with these values. wilcoxon’s test was used to evaluate the difference in pi and gi before and after initial treatment. the level of significance was set at 5%. results twenty-six patients were enrolled in this study. the mean age was 27.1±4.9 years. gender distribution revealed a predominance of females (73.1%). the pi and gi values and the results of treatment are shown in table 1. the initial pi was 56.8% and 30 days after the prophylaxis and oral hygiene instructions this value declined to 28.4% (p<0.0001). there was also a statistically significant reduction in gi after the treatment (28.4% to initial 1 month follow-up reduction pi 56.8 ± 17.8 28.4 ± 13.4 28.4 ± 15.8 p<0.0001* gi 28.4 ± 13.4 16.9 ± 11.7 10.3 ± 7.4 p<0.0001* table 1. mean reduction (%) and standard deviation (sd) of pi and gi at baseline and 1 month follow-up. * difference between periods showing a statistical significantly difference at wilcoxon test (level of significance 5%). 16.9%) (p<0.0001). the self-perception values attributed by each patient are shown in table 2. none of the perception values showed a correlation with the initial pi and gi values (table 2), and no correlation was observed between the self-perceived symptoms and the reduction in pi. there was however, a positive correlation between the perception of some of the symptoms and reduction in gi. the greater the perception of bleeding on tooth brushing (p=0.04, r=0.27) and redness and swelling of gums (p=0.04, r=0.26), the greater the reduction in bleeding (statistical significance shown by spearman’s correlation) (table 2). the mean percentage of sites with probing depth > 5mm with bleeding on probing was 63±20%. the spearman’s correlation test also showed no correlation between this percentage and the self-perceived symptom scores (table 2). discussion the efficient inflammation control obtained by means of supragingival plaque control is fundamental to periodontal treatment success, and many psychosocial and psychological characteristics influence the patient’s adherence to the oral hygiene instructions19. the proper perception of oral health could influence adherence by showing the real importance the patient attaches to the treatment20, determining a high or low acceptance of the oral hygiene instructions. this is especially important in young patients, who normally present a low adherence to treatment5. thus, the present study was designed to evaluate the relationship between the selfperception of generalized aggressive periodontitis symptoms and patient compliance with instructions. the results showed that the patients with a greater perception of some of the aggressive periodontitis symptoms adhered to the treatment more firmly, with greater reductions in the gi within 1 month. a positive correlation was found between the perception of bleeding on tooth brushing and redness and swelling of the gums and reductions in the gi. moreover, these symptoms, which recent studies have shown to be normally associated with periodontal disease, were those most perceived by the patients18. this correlation could be involved in a personal definition of disease by patients. patient’s definition of disease differs from that of the professionals’ definition20-21. professionals usually consider the presence or absence of illness whereas patients consider the social and functional problems caused by the disease, such as the capacity to smile, speak and chew22. hence, the social influence of the disease appears to interfere more significantly in the perception than the presence of the actual disease, and to the patients, bleeding and swelling of gums could represent a social impact. this difference regarding the influence based on the definition of the disease was ratified by another result found in the present study. none of the self-perceived symptoms of the disease showed a correlation with the percentage of sites presenting bleeding, and with ppd >5mm. patients appear to perceive the disease differently from the self-perception of generalized aggressive periodontitis symptoms and its influence on the compliance with the oral hygiene instructions a pilot study braz j oral sci. 9(3):388-392 symptoms self perception (mm±sd) pi initial pi reduction gi initial gi reduction ppd > 5mm pain when biting 12.88±13.10 0.21 (0.19) 0.20 (0.16) 0.41(-0.27) 0.86(-0.08) 0.35(-0.21) bleeding on brushing 38.39±21.32 0.27 (0.19) 0.33 (0.03) 0.77 (0.04) 0.04£ (0.26) 0.06(0.36) bad breath 25.26±19.07 0.90 (0.00) 0.25(-0.26) 0.35(-0.09) 0.51(-0.17) 0.19(-0.29) space between teeth 27.22±25.70 0.77 (0.11) 0.95 (0.10) 0.74 (0.01) 0.35(-0.03) 0.68(0.06) mobility of teeth 28.39±24.26 0.79 (0.06) 0.68 (0.05) 0.23(-0.25) 0.86(-0.12) 0.58(-0.09) redness and sweeling of gums 38.41±19.41 0.82 (0.01) 0.49(-0.23) 0.28(-0.18) 0.04£ (0.27) 0.80(0.08) dry mouth 20.46±22.84 0.63 (0.11) 0.90(-0.13) 0.67 (0.00) 0.13(-0.12) 0.31(0.07) bad taste 26.05±20.69 0.51 (0.06) 0.85(-0.22) 0.87 (0.01) 0.69(-0.26) 0.82(-0.02) recent loss of tooth# 0.00±0.46 0.60 (0.07) 0.86 (0.03) 0.61 (0.12) 0.17 (0.21) 0.24(0.25) loss of interproximal gingival tissue 17.24±22.74 0.81 (0.07) 0.96 (0.01) 0.53 (0.11) 0.42 (0.11) 0.80(0.01) sensitivity when drink cold drinks 32.19±20.13 0.59 (0.08) 0.50(0.05) 0.20(0.13) 0.25(-0.15) 0.90(0.03) correlation with* table 2. patients referred perception of aggressive periodontitis symptoms (mm) and its correlation with initial and reduction values of pi and gi and proportion of sites with probing depth (ppd) > 5 mm. * p (r value). # referred to modal value. £ statistical significance correlation (p<0.05) at spearman test. professional; this perception involving the psychosocial influence of disease to a greater extent than does its presence or severity. certain psychological models can be applied to explain levels of compliance with dental and medical treatment. the hbm proposes the following requirements for behavior change: (1) a perception of susceptibility to disease; (2) a belief that the impact of this disease will affect him/her biologically and/or psychosocially; (3) a belief that the potential benefits of the treatment outweigh the risks of the disease and its treatment; (4) an ability to surmount barriers to treatment23. the tm suggests that six stages of changes are involved in health behavior: pre-contemplation, contemplation, preparation, action, maintenance and termination24. in both models, the perception of the disease is an important variable that could determine the level of compliance with the treatment. thus, the analysis of self perception, as shown in the present study, could represent a key to achieving and improving patient compliance. moreover, the influence of the perception of symptoms could also be related to the leventhal’s theory. leventhal et al.25 postulated that people’s behavior in response to a disease is determined by their representation of that disease. disease representation has a cognitive and an emotional aspect and they are constructed through direct or vicarious experiences as well as information received from the social environment and health professionals. they propose that patients will only adhere to a treatment if they believe that this treatment will have a positive effect on their health and that they have the capacity to effectively act as required. the application of this model in periodontal treatment was tested by phillipot et al18. their results showed that the application of leventhal’s method produced a higher rate of compliance with oral hygiene instructions, producing a lower level of plaque accumulation within a month, than in patients who received traditional oral hygiene instructions. in this context, the self-perception of symptoms, and particularly the recognition of the changes after adherence to the oral hygiene instructions, could help patients believing their own capacity of acting on the instructions and adhere to the treatment. this could be seen in the results of present study, in which those patients who perceived their own symptoms of the disease showed better adherence to oral hygiene. the gi has been used as an indicator of compliance in previous studies12,17-18,26. this index is an important tool to evaluate significant adherence to the daily oral hygiene regime, since the reduction in bleeding represents efficient and continued plaque control. moreover, the use of the pi may represent efficient hygiene but it does not affirm that control is continuous or was only implemented near the revaluation day. therefore, the reduction in the gi observed in the present study represents an adherence to the oral hygiene instructions. the reduction in bleeding observed in the present study could be explained by improvement in the daily oral conditions after patients received prophylaxis and oral hygiene instruction (reinforced during the study) and represents a reduction in gingival inflammation, as seen in previous studies17-18. the role of the reduction of these aspects (bleeding and pi) during periodontal therapy, especially during the initial phase of the treatment, is well demonstrated in the literature. the supragingival plaque control due to the compliance with the oral hygiene instructions, sessions of prophylaxis and calculus/plaque retenders removal shown in recent studies be capable of produce favorable clinical and microbiological changes in periodontal disease27,28. however, in this pilot study, posttreatment follow up was done for only 1 month. long-term implication of initial adherence to the treatment is unknown, and has yet to be determined in future studies. therefore, a larger population of patients with aggressive periodontitis, specifically generalized periodontitis, should be evaluated to ratify the results of this study. in spite of study limits, the obtained results can be easily transported to daily practice, in which recognition by patients with greater levels of perception could be an indication of enhanced adherence to the initial treatment. nevertheless, the key idea of this study was to recognize patients with a lower perception, which provided an opportunity to plan a 391 self-perception of generalized aggressive periodontitis symptoms and its influence on the compliance with the oral hygiene instructions a pilot study braz j oral sci. 9(3):388-392 392 patient-specific approach to improve their knowledge and self-perception of symptoms and ultimately their compliance with the treatment. in conclusion, within the limitations of this study, patient adherence to oral hygiene instructions could be related to the self-perception of generalized aggressive periodontitis symptoms. references 1. morris aj, steele j, white da. the oral cleanliness and periodontal health of uk adults in 1998. br dent j. 2001; 191: 186-92. 2. ciancio s. improving oral health: current considerations. j clin periodontol. 2003; 30 suppl 5: 4-6. 3. wilson tg jr, glover me, schoen j, baus c, jacobs t. compliance with maintenance therapy in a private periodontal practice. j periodontol. 1984; 55: 468-73. 4. demetriou n, tsami-pandi a, parashis a. compliance with supportive periodontal treatment in private periodontal practice. a 14-year retrospective study. j periodontol. 1995; 66: 145-9. 5. novaes ab, novaes junior ab, moraes n, campos gm, grisi mf.compliance with supportive periodontal therapy. j periodontol. 1996; 67: 213-6. 6. ojima m, hanioka t, shizukuishi s.survival analysis for degree of compliance with supportive periodontal therapy. j clin periodontol. 2001; 28: 1091-5. 7. blinkhorn as. factors affecting the compliance of patients with preventive dental regimens. int dent j. 1993; 43: 294-8. 8. weinstein r, carrassi a, ciancaglini r. dento-periodontal pathology in a group of patients with down’s syndrome. mondo odontostomatol. 1983; 25: 7-9. 9. syrjala amh, knuuttila mle, syrjala lk. obstacles to regular dental care related to extrinsic and intrinsic motivation. community dent oral epidemiol. 1994; 22: 269–272. 10. kuhner mk, raetzke pb. the effect of health beliefs on the compliance of periodontal patients with oral hygiene instructions. j periodontol. 1989; 60: 51-6. 11. friedman hs, dimatteo mr. health psychology, pp. 68–100. englewood cliffs, nj: prentice-hall. 1989. 12. ojima m, kanagawa h, nishida n, nagata h, hanioka t, shizukuishi s. relationship between attitudes toward oral health at initial office visit and compliance with supportive periodontal treatment. j clin periodontol. 2005; 32: 364-8. 13. mendoza ar, newcomb gm, nixon kc. compliance with supportive periodontal therapy. j periodontol. 1991; 62: 731-6. 14. novaes ab jr, novaes ab. compliance with supportive periodontal therapy. part 1. risk of non-compliance in the first 5-year period. j periodontol. 1999; 70: 679-82 15. revere d, dunbar p. review of computer-generated outpatient health behaviour inteventions. j am med info assoc. 2001; 8: 62–79. 16. ainamo j, bay i. problems and proposals for recording gingivitis and plaque. int dent j. 1975; 25: 229-35. 17. gamboa ab, hughes fj, marcenes w. the relationship between emotional intelligence and initial response to a standardized periodontal treatment: a pilot study. j clin periodontol. 2005; 32: 702-7. 18. philippot p, lenoir n, d’hoore w, bercy p. improving patients’ compliance with the treatment of periodontitis: a controlled study of behavioural intervention. j clin periodontol. 2005; 32: 653-8. 19. monteiro da silva am, oakley da, newman hn, nohl fs, lloyd hm. psychosocial factors and adult onset rapidly progressive periodontitis. j clin periodontol. 1996; 23: 789-94. 20. da silva sr, castellanos fernandes ra. self-perception of oral health status by the elderly. rev saude publica. 2001; 35: 349-55. 21. vered y, sgan-cohen hd. self perceived and clinically diagnosed dental and periodontal health status among young adults and their implications for epidemiological surveys. bmc oral health. 2003; 13; 3(1): 3. 22. reisine st, bailit hl clinical oral health status and adult perceptions of oral health. soc sci med [med psychol med sociol] 1980; 14: 597-605. 23. ross dj, guggenheim fg. compliance and the health belief model: a challenge for the liaison psychiatrist. gen hosp psych. 1983; 5: 31-5. 24. prochaska jo, velicer wf. the transtheoretical model of health behavior change. am j health promotion. 1997; 12: 38-48. 25. leventhal h, diefenbach m, leventhal e. illness cognition: using common sense to understand treatment adherence and affect cognitive interaction. cognit psyc research. 1992; 16: 143–63. 26. walsh mm, heckman bh, moreau-diettinger r. use of gingival bleeding for reinforcement of oral home care behavior. community dent oral epidemiol. 1985; 13: 133-5. 27. gomes sc, piccinin fb, susin c, oppermann rv, marcantonio ra. effect of supragingival plaque control in smokers and never-smokers: 6-month evaluation of patients with periodontitis. j periodontol. 2007; 78: 1515-21. 28. ribeiro edp, bittencourt s, nociti jr fh, sallum ea, sallum aw, casati mz. the effect of one session of supragingival plaque control on clinical and biochemical parameters of chronic periodontitis. j appl oral sci. 2005; 13: 275-9. self-perception of generalized aggressive periodontitis symptoms and its influence on the compliance with the oral hygiene instructions a pilot study braz j oral sci. 9(3):388-392 404 not found oral sciences n3 braz j oral sci. 10(4):258-261 original article braz j oral sci. october | december 2011 volume 10, number 4 bone marrow mesenchymal cell adhesion to polished and nitrided titanium surfaces luciana bastos alves1, fernanda ginani2, josé sandro pereira da silva3, clodomiro alves junior4, carlos augusto galvão barboza5 1phd candidate, department of dentistry, federal university of rio grande do norte, brazil. 2msc candidate; department of morphology, federal university of rio grande do norte, brazil 3phd, department of dentistry, federal university of rio grande do norte, brazil 4phd, department of mechanical engineering, federal university of rio grande do norte, brazil 5phd, department of morphology, federal university of rio grande do norte, brazil correspondence to: carlos augusto galvão barboza universidade federal do rio grande do norte centro de biociências – departamento de morfologia av. salgado filho, 3000 – campus universitário natal/rn, brasil 59072-970 phone (fax): +55-84-3211-9207 e-mail: cbarboza@cb.ufrn.br abstract aim: to evaluate the adhesion of mouse bone marrow mesenchymal cells (mbmmc) on different titanium surfaces. methods: grade ii titanium discs (astm f86) received two different surface treatments: polished (s1) and cathodic cage plasma nitriding (s2). mbmmc were cultured on titanium discs in 24-well cell culture plates, at a density of 1 x 104 cells per well. after 24 h, the adhesion was evaluated using a hemocytometer. results: the mean adhesion was greater on s2, though without statistically significant difference from s2 (p>0.05). conclusions: it was demonstrated that titanium surface treatment with ionic nitriding in a cathodic cage is biocompatible since it preserved the integrity of the cultivated mbmmc for a period of 24 h, allowing their adhesion. keywords: mesenchymal stem cells, bone marrow, titanium. introduction titanium (ti) is currently considered the biomaterial of choice for the manufacture of intra-osseous implants because this metal has exceptional physical and chemical properties that allow its installation in living tissues with no incompatibility among them. ti is a very stable metal, although slight oxide formation occurs on its surface. this formation helps deposition and adhesion of the extracellular matrix on the bone-implant interface. these oxides form during the cutting, cleaning, and implant sterilization process and remain adherent to the surface. the foregoing properties allow the scarring and maintenance of tissue structure adhesion to the ti surface1. a wide range of approaches have been developed to thoroughly investigate cellular behavior on ti surfaces. maeda et al.2 (2007) observed that cell adhesion and proliferation, as well as the osteogenic differentiation of mouse mesenchymal stem cells (mscs) to ti discs were significantly similar to those on the plastic surface of the culture, indicating ti as an excellent material for repairing hard tissue in the field of bone tissue engineering. however, the interaction between cells and some biomaterials, or the received for publication: july 07, 2011 accepted: december 07, 2011 259 braz j oral sci. 10(4):258-261 biocompatibility, depends on the material surface properties, such as energy, texture, roughness, and chemical composition. these properties determine the adhesion and behavior of cells in contact with the surface. the term “adhesion” to the biomaterial refers to the most important phase, since the quality of it will influence morphology and the capacity of cell proliferation and differentiation3. the physicochemical properties of ti implant surfaces are fundamental to the success of osseointegration. to improve the biological responses for obtaining rapid osseointegration, the surfaces have been modified by a wide range of process involving mechanical, chemical, and physical surface treatment methods, such as: the ti plasma spray treatment, ti oxide blasting, laser deposition of ti carbide, and acid conditioning4. ionic nitriding is a surface treatment method which exhibits excellent mechanical properties, chemical stability, and biocompatibility when applied to ti. this process, also known as plasma nitriding, consists of an ionizing gas or a gaseous mixture containing nitrogen using glow-discharge generated by a difference in potential between the sample (cathode) and the anode in a low pressure atmosphere. the ions produced in the plasma are accelerated towards the sample (cathode), colliding with it, and supplying enough energy to heat it to the nitriding temperature5. the basic concept in using ionic nitriding to improve ti surface properties is based on the possibility of forming nitrides or carbides below the alloy surface. ti nitrides and carbides are brittle materials that improve tribologic surface properties; that is, they increase resistance to corrosion and surface roughness6. accordingly, the association of ti implants with bone tissue culture may contribute to bone tissue regeneration7. therefore, the present study aimed to evaluate the adhesion capacity of mouse bone marrow mscs to smooth and plasmanitrided ti surfaces in the cathodic cage configuration. material and methods the present study was approved by the research ethics committee of the federal university of rio grande do norte (ufrn; protocol 008/09) and was divided into two stages. first, sample preparation was carried out using the surface treatment of ti plates. a test was then performed in vitro with isolated mouse bone marrow cells cultivated on ti discs. sample preparation grade ii titanium discs (astm f86), 15 mm in diameter and 1.5 mm thick, were prepared according to the protocol established by alves jr et al.8 (2006). all the discs (n=12) were polished and then six discs were submitted to cathodic cage nitriding. a study of these surface characteristics have been previously published by da silva et al.9 (2011). the discs were subsequently sterilized by gamma radiation (25 kgy dose) released at a mean dose of 8.993 kgy/h (2h 46 min at a distance of 50 mm), in a gammacell 220 excel irradiator (mds nordion, ottawa, on, canada). bone marrow cell culture bone marrow was extracted from two male swiss albino mice in accordance with the protocol established by maniatopoulos et al.10 (1988). after anesthesia, the animals were dissected under aseptic conditions for femur and tibia removal. the marrow cavity was flushed out with α-mem medium containing 50 mg/l of gentamicin sulfate and 2 mg/l of amphotericin b (cultilab, campinas, brazil) and supplemented with 10% fetal bovine serum (fbs; gibco, carlsbad, ca, usa). the extracted marrow was cultivated in basic medium (α-mem 10% fbs) for 72 h in a humid atmosphere with 5% co 2 at 37ºc. after this stage, the medium was changed, thus making it possible to remove the non-adhered cells from the culture, and subsequent medium changes were performed every 3-4 days. in order to confirm the multi-lineage differentiation potential of periodontal ligament cells, aliquots of p1 cells were cultured in osteogenic, chondrogenic, or adipogenic differentiation media (stempro ® differentiation kits, invitrogen corp., carlsbad, ca, usa) for up to 21 days. by light microscopy, the cells showed typical osteoblast/ osteocyte, chondroblast, and adipocyte morphology and produced characteristic extracellular matrix components. bone marrow cell culture on ti discs bone marrow cells were cultivated in two 24-wells plates (ttp®), with a density of 1x104 cells per well. twelve ti discs were used, six from each group (polished and cathodic cage). the same cell density was cultivated in six wells without discs, as a positive control of cell proliferation. the disks are the same size of the well, so the growth area of disks and controls are the same. cell viability and adhesion data obtained by counting the cells that adhered to the ti surfaces (the polished group [s1], and the cathodic cage group [s2]), in the 24-h period after plating were used to analyze cell adhesions. the number of cells collected from each well was obtained from a viable cell count using a hemocytometer and the trypan blue dye exclusion method. all the titanium samples were also evaluated by scanning electron microscopy to check the reproducibility of the results, according to the protocol established by guerra neto et al.11 (2009). statistical analysis the data were subjected to non-parametric analysis. each counting value corresponds to the mean ± standard deviation of the mean (sd) of six samples per group. the differences between the groups were compared by the mann whitney statistical test. a statistical difference was considered when p<0.05. results results of mouse bone marrow cell adhesion to different titanium surfaces shows that the mean adhesion among mouse bone marrow mesenchymal cell adhesion to polished and nitrided titanium surfaces 260 braz j oral sci. 10(4):258-261 c s1 s2 mean± sd mean± sd mean ± sd p* value bone marrow cells 2.32 ± 0.78 1.03 ± 0.26 1.83 ± 0.83 0.16 table 2 cell adhesion between the three groups: control (c), polished (s1) and cathodic cage plasma nitrided (s2) ti surfaces. p* kruskal-wallis s1 s2 mean± sd mean ± sd p*value bone marrow cells 1.03 ± 0.26 1.83 ± 0.83 0.12 table 1 cell adhesion to polished (s1) and cathodic cage plasma nitrided (s2) ti surfaces. p* mann whitney bone marrow cells was greater in the cathodic cage group [s2] (1.83 ± 0.83) than in the polished group [s1] (1.03 ± 0.26). however, no statistically significant difference was observed (p=0.12) between the two groups (table 1). the control surface (plastic) showed the best result (2.32 ± 0.78), since it is the gold standard surface for cell adhesion and proliferation. however, no statistically significant difference was observed either (p=0.16) among the three groups (table 2). discussion mesenchymal cells were first isolated from a cell suspension of bone marrow by friedenstein and collaborators in the early 1970’s, and classified as adherent, fibroblastic and clonogenic cells, and it were initially called colony forming units – fibroblastic (cfu-f)12. this type cell is found in the bone marrow and it is called msc13-15. from the extraction and culture of bone marrow of mouse is possible to obtain a population of adherent cells with fibroblastic, elongated, spindle-shaped and pointed, called multipotent mesenchymal stromal cells, according to the nomenclature proposed by the international society for cellular therapy in 200516. in the present experiment, the adhesion capacity of mouse bone marrow mesenchymal cells to different ti surfaces was analyzed under conditions of cell cultivation. the plastic surface or polystyrene cell culture plate was used as a positive control, since it is the standard surface used in cell cultivation owing to its excellent hydrophilic characteristics2. material surface characteristics play an important role in cell adhesion because they provide the necessary adhesion conditions to adsorb and help in the cell adhesion process3. experimental studies7,17 comparing different surface types concluded that the best results were those obtained with textured surfaces, and that bone marrow cells and osteoblasts cultivated on different ti surfaces adhere and respond better on rougher surfaces18-23. however, other evaluations of in vitro biocompatibility of ti using cell culture have indicated that cell attachment was not affected by surface roughness24-26. in this study, ionic nitriding or the plasma nitriding technique was used. experiments11,22 with a cell line model (osteo-1 lineage and l929 mouse fibroblasts) demonstrated that cell adhesion to the ti surface was favored by the low energy ion irradiation surface treatment (plasma). in agreement with the related literature, it was possible to observe that adhesion and an initial interaction between cell and substrate occurred irrespective of the surface. the best cell adhesion results were obtained by the control surface (plastic) according to the results obtained by santiago et al.25 (2005) and resende et al.27 (2010), who showed no statistically significant difference among the different titanium surfaces and a larger number of cells on the polystyrene surface. even though more experiments are needed to explain the ti cell adhesion mechanism, the results suggest that ti surface characteristics are similar to those of a plastic surface, resulting in good cell adhesion capacity, in accordance with maeda et al.2 (2007). this result reinforces the argument that ionic nitriding treatment to the surface (s2) may contribute to better adhesion of bone marrow mesenchymal cells, corroborating a number of studies on roughness and wettability8,18-19,20. further studies, analyzing the capacity of proliferation and differentiation of these types of cells when in contact with the biomaterial may contribute to an understanding of the osseointegration process. furthermore, molecular studies that analyze the types of adhesion bonds involved might be important in explaining the mechanism by which each cell type adheres to different surfaces. references 1. hansson ha, albrektsson t, branemark pi. structural aspects of the interface between tissue and titanium implants. j prosthet dent. 1983; 50: 108-13. 2. maeda m, hirose m, ohgushi h, kirita t. in vitro mineralization by mesenchymal stem cells cultured on titanium scaffolds. j biochem. 2007; 141: 729-36. 3. stiehler m, lind m, mygind t, baatrup a, dolatshahi-pirouz a, li h, et al. morphology, proliferation, and osteogenic differentiation of mesenchymal stem cells cultured on titanium, tantalum, and chromium surfaces. j biomed mater res a. 2008; 86: 448-58. 4. silva mam, martinelli ae, alves jr c, nascimento rm, távora mp, vilar cd. surface modification of ti implants by plasma oxidation in hollow cathode discharge. surf coat technol. 2005; 200: 2612-26. 5. o’brien jm, goodman d. plasma (ion) nitriding. in: asm international handbook committee, editors. asm handbook: heat treating. utah: international library service; 1991. p.420-4. 6. yilbas bs, sahin az, al-garni az, said sam, ahmed z, abdulaleem bj, et al. plasma nitriding of ti-6al-4v alloy to improve some tribological properties. surf coat technol. 1996; 80: 287-92. 7. franzolin sob, francischone ce, bittencourt rec, felisbino sl, deffune e. diferenciação de célula-tronco hematopoética periférica humana em osteoblasto sobre diferentes superfícies de implantes de titânio. rev dent press periodont implantol. 2008; 2: 68-79. 8. alves jr c, de araújo fo, ribeiro kjb, da costa jap, souza rrm, de sousa rs. use of cathodic cage in plasma nitriding. surf coat technol. 2006; 201: 2450-4. bone marrow mesenchymal cell adhesion to polished and nitrided titanium surfaces braz j oral sci. 10(4):258-261 9. da silva js, amico sc, rodrigues ao, barboza ca, alves c jr, croci at. osteoblastlike cell adhesion on titanium surfaces modified by plasma nitriding. int j oral maxillofac implants. 2011; 26: 237-44. 10. maniatopoulos c, sodek j, melcher ah. bone formation in vitro by stromal cells obtained from bone marrow of young adult rats. cell tissue res. 1988; 254: 317-30. 11. guerra neto clb, da silva mam, alves jr c. in vitro study of cell behaviour on plasma surface modified titanium. surf eng. 2009; 25: 146-50. 12. friedenstein aj, piatetzky-shapiro ii, petrakova kv. osteogenesis in transplants of bone marrow cells. j embryol exp morphol. 1966; 16: 381-90. 13. pittenger mf, mackay am, beck sc, jaiswal rk, douglas r, mosca jd et al. multilineage potential of adult human mesenchymal stem cells. science. 1999; 284: 143-7. 14. caplan ai. the mesengenic process. clin plast surg. 1994; 21: 429-35. 15. donzelli e, salvadè a, mimo p, viganò m, morrone m, papagna r et al. mesenchymal stem cells cultured on a collagen scaffold: in vitro osteogenic differentiation. arch oral biol. 2007; 52: 64-73. 16. horwitz em, le blanc k, dominici m, mueller i, slaper-cortenbach i, marini fc et al. clarification of the nomenclature for msc: the international society for cellular therapy position statement. cytotherapy. 2005; 7: 393-5. 17. buser d, schenk rk, steinemann s, fiorellini jp, fox ch, stich h. influence of surface characteristics on bone integration of titanium implants. a histomorphometric study in miniature pigs. j biomed mater res. 1991; 25: 889-902. 18. anselme k, bigerelle m, noel b, iost a, hardouin pj. effect of grooved titanium substratum on human osteoblastic cell growth. j biomed mater res. 2002; 60: 529-40. 19. deligianni dd, katsala n, ladas s, sotiropoulou d, amedee j, missirlis yf. effect of surface roughness of the titanium alloy ti-6a1-4v on human bone marrow cell response and on protein adsorption. biomaterials. 2001; 22: 1241-51. 20. keller jc. tissue compatibility to different surfaces of dental implants: in vitro studies. implant dent. 1998; 7: 331-7. 21. perizzolo d, lacefield wr, brunette dm. interactions between topography and coating in the formation of bone nodules in culture for hydroxyapatiteand titaniumcoated micromachined surfaces. j biomed mater res. 2001; 56: 494-503. 22. abidzina v, deliloglu-gurhan i, ozdal-kurt f, sen bh, tereshko i, elkin i, et al. cell adhesion study of the titanium alloys exposed to glow discharge. nucl instr and meth phys res b. 2007; 261: 624-6. 23. vasconcellos lm, leite do, oliveira fn, carvalho yr, cairo ca. evaluation of bone ingrowth into porous titanium implant: histomorphometric analysis in rabbits. braz oral res. 2010; 24: 399-405. 24. rosa al, beloti mm. effect of cpti surface roughness on human bone marrow cell attachment, proliferation, and differentiation. braz dent j. 2003; 14: 16-21. 25. santiago as, santos ea, sader ms, santiago mf, soares gde a. response of osteoblastic cells to titanium submitted to three different surface treatments. braz oral res 2005; 19: 203-8. 26. silva ts, machado dc, viezzer c, silva júnior an, oliveira mg. effect of titanium surface roughness on human bone marrow cell proliferation and differentiation: an experimental study. acta cir bras. 2009; 24: 200-5. 27. resende cx, lima ir, gemelli e, granjeiro jm, soares ga. cell adhesion on different titanium-coated surfaces. materia 2010; 15: 386-91. 261bone marrow mesenchymal cell adhesion to polished and nitrided titanium surfaces oral sciences n3 braz j oral sci. 11(3):411-415 original article braz j oral sci. july | september 2012 volume 11, number 3 topographic relationship of impacted third molars and mandibular canal: correlation of panoramic radiograph signs and cbct images eduardo luiz delamare1, gabriela salatino liedke2, mariana boessio vizzotto3, heraldo luis dias da silveira4, tais weber furlanetto de azambuja4, heloisa emilia dias da silveira5 1bds, msc; private practice; porto alegre, rs, brazil 2bds, msc; phd student; department of surgery and orthopedics, school of dentistry, federal university of rio grande do sul, porto alegre, rs, brazil 3bds, msc, phd; department of surgery and orthopedics, school of dentistry, federal university of rio grande do sul, porto alegre, rs, brazil 4bds, msc, phd; associate professor; department of surgery and orthopedics, school of dentistry, federal university of rio grande do sul, porto alegre, rs, brazil 5bds, msc, phd; professor and department head; department of surgery and orthopedics, school of dentistry, federal university of rio grande do sul, porto alegre, rs, brazil correspondence to: mariana boessio vizzotto, department of surgery and orthopedics – ground floor, faculty of dentistry rua ramiro barcelos, 2492 – cep: 90035-003 porto alegre, rs brasil phone: +55 51 99596941 e-mail: mari_vizzotto@yahoo.com.br mari.vizzotto@gmail.com abstract aim: this study evaluated the proximity and relation of impacted lower third molars and mandibular canal on panoramic radiography. methods: radiographic signals associated with proximity of structures and pell & gregory and winter classifications of 78 impacted teeth were analyzed and compared with cbct images (gold standard). the associations between the findings were tested with pearson’s chi-square. results: direct contact between structures was observed in 85% of cases of radiolucent band over roots. conclusions: radiolucent band over roots is the image more associated with direct contact between structures and the one that indicates lingual positioning of the canal more consistently. some categories of pell & gregory and winter classifications suggested signs of the topographic location of the mandibular canal. keywords: radiography, tomography, third molar. introduction surgical removal of impacted lower third molars is common procedure in clinical dental practice and might be associated with post-operative complications related to inferior alveolar nerve (ian) injuries, and the risk of complications is sensibly increased whenever direct contact between the nerve and the impacted molar root is observed1-3. therefore, a precise topographic evaluation of the mandibular canal and the surrounding molars is deemed essential during preoperatory planning1,3-4. the panoramic radiograph is routinely used as an auxiliary examination for treatment planning of lower third molar removal, due to its wide availability, low cost and relatively low exposure dose5. for this kind of investigation, four signals have been more consistently associated with direct contact between mandibular canal and molar roots: interruption of the radiopaque line of the canal wall; received for publication: may 21, 2012 accepted: september 18, 2012 braz j oral sci. 11(3):411-415 412412412412412 type definition pg (horizontal) 3 over 50% of the crown’s largest mesiodistal dimension within the mandibular ramus. 2 over 50% of the crown’s largest mesiodistal dimension outside the mandibular ramus – or completely outside, but with no space for eruption. 1 crown’s largest mesiodistal dimension completely outside the mandibular ramus and with sufficient space between the distal surface of the second molar and the mandibular ramus anterior line. pg (vertical) c over 50% of the crown’s largest mesiodistal dimension below the cervical plane line. b over 50% of the crown’s largest mesiodistal dimension above the cervical plane line – or completely above, but without contact on the occlusal plane. a crown’s largest mesiodistal dimension completely above the cervical plane and in contact with the occlusal plane. winter* horizontal angle below 30º mesial angle between 30º and 60º vertical angle between 60º and 90º distal angle above 90º *angle formed posterior to the intersection of the occlusal plane and a perpendicular line to the crown’s largest mesiodistal dimension. fig. 1. criteria for pg and winter classification. radiolucent band over roots; deflection of the canal around the apices; and narrowing of the roots6-12. another traditionally accepted option for estimations of difficulty and risk during lower third molar removal is the evaluation of the impacted tooth according to the classification of pell & gregory (pg) and winter13. pg classification distributes impacted molars in three categories according to their vertical positioning (related to the occlusal and cervical plane of adjacent molars) and three horizontal categories (related to the mandibular ascending ramus). winter classification is divided in four categories based on tilting of the impacted molar in relation to its longitudinal axis. whenever these classifications and signals point towards categories correlated with higher difficulty levels, as well as with proximity between structures observed in panoramic radiographs, it should be noticed that the buccolingual aspect cannot be visualized. indeed, a further assessment regarding surgical removal of impacted lower third molars observed by ghaeminia et al.14 is that lingual positioning of the mandibular canal is significantly linked with injuries to the ian. due to the recent development and spread of cone beam computed tomography (cbct), three-dimensional images are becoming more easily available in dentistry, allowing extra investigation of the mandibular canal and surrounding molars. tantanapornkul et al.7 concluded that cbct was significantly superior to panoramic images in predicting neurovascular bundle exposure during extraction of impacted mandibular third molar. although cbct allows such evaluation, panoramic radiography is still often the first imaging method requested to the investigation of third molars5. moreover, alara principle should be a concern among professionals, seeking to extract maximum information from an imaging study, avoiding further exposure15. thus, this study aimed to evaluate more deeply the association between impacted lower third molars and mandibular canal based on radiographic signals suggesting proximity of structures and the pg and winter classifications, using cbct images as references, to increase the information that might be gather from panoramic radiographs. material and methods all patients who attended to oral surgery department for third molar removal during one year and had at least one impacted molar and one of the radiographic signals associated with proximity of structures – interruption of the radiopaque line of the canal wall; radiolucent band over roots; deflection of the canal around the apices; and narrowing of the roots were asked a tomographic examination. for all those cases in which the impacted molar exhibited two radiographic findings or more, the most striking feature has been selected. a total of 78 impacted teeth (from 41 patients 17 women; 24 men) comprised the sample. all of them read and signed informed consent forms and this study has been approved by the institution ethical and research boards. tomographic images were obtained with an i-cat tomography scanner (imaging sciences international, hatfield, pa, usa), and a 0.25 mm voxel has been used. the conventional panoramic radiographs were digitized with a transparency flatbed scanner (umax 2400s) and resolution of 300 dpi. all randomly coded images were analyzed with a 19" lcd screen by three calibrated examiners from the oral radiology department (kappa scores over 0.7 for intra and inter-observer reproducibility). image j version 1.42 (national institutes of health, bethesda, md, usa) software has been used to perform pg and winter classifications in the panoramic views (figure 1). i-cat vision v.7.2.4. implant screen function has been chosen to carry out the tomographic examination. identification of contact between structures was classified in: 1 – present; 2 – absent; and the topographic positioning of the mandibular canal as seen in the buccolingual aspect was divided in: 1 – inferior; 2 – lingual; 3 – inter-radicular; 4 – buccal. data was processed and analyzed with the statistical package for social sciences, version 15.0 (spss inc., chicago, il, usa). the association between the radiographic signals and the classifications of pg and winter with the tomographic images have been tested with pearson’s chi-square test. topographic relationship of impacted third molars and mandibular canal: correlation of panoramic radiograph signs and cbct images braz j oral sci. 11(3):411-415 413413413413413 dc ir n r r b present 30 58.1 61.5 85.7 absent 70 41.9 38.5 14.3 inferior 65 67.7 61.5 6.4 lingual 15 12.9 15.4 43.6 inter-radicular 5 0 15.4 21.4 buccal 15 19.4 7.7 28.6 mc evaluations ÷2 p radiograph signals 14.23 0.01 10.74 0.01 table 1. rates of direct contact between structures and the topographic relation of the mandibular canal (mc) regarding the radiographic signals. dc = deflection of the canal around the apices; ir = interruption of the radiopaque line of the canal wall; nr = narrowing of roots; rb = radiolucent band over roots mc evaluations ÷2 p horizontal mesial vertical/ distal present 57.1 56.7 55.6 0.01 0.99 absent 42.9 43.3 44.4 inferior 47.6 26.7 77.8 24.04 0.00 lingual 9.5 40.0 18.5 inter-radicular 0 20 0 buccal 42.9 13.3 3.7 winter classification table 2 – rates of direct contact between structures and the topographic relation of the mandibular canal (mc) regarding winter classification. mc evaluations ÷2 p ÷2 p 1 2 3 a b c present 45.5 61.1 46.2 1.58 0.45 60 64 50 1.30 0.52 absent 54.5 38.9 53.8 40 36 50 inferior 72.7 48.1 84.6 7.97 0.24 80 52 52.6 9.89 0.13 lingual 9.1 20.4 7.7 20 24 10.5 inter-radicular 0 11.1 0 0 4 13.2 buccal 18.2 20.4 7.7 0 20 23.7 horizontal component vertical component table 3. rates of direct contact between structures and the topographic relation of the mandibular canal (mc) regarding pell & gregory classification. results analyses of table 1 shows that direct contact between structures could be observed in 85% of cases of radiolucent band over roots (p<0.05); on the other hand, deflection of the canal demonstrated the smallest amount association with contact of molars and mandibular canal (30%). the connection between the radiographic signals and the topographic relation of the mandibular canal (visualized on cbct images) reveals that the lingual positioning of the canal has proven to be more closely associated with radiolucent band over roots. tables 2 and 3 present associations between winter and pg classifications and the mandibular canal (visualized on cbct images). it was observed that molars mesial impacted are consistently linked with lingual position of the canal. moreover, despite most pg categories were closely associated with inferior position of the canal, the lingual position was more observed with a, b and 2 components. table 3 explores the association between pg classification and the mandibular canal (visualized on cbct images). although no statistically significant data can be drawn, the observation of frequencies reveals that lingual position is more linked to type a and b. besides, when the horizontal component is evaluated, it can be reported that the lingual position of the mandibular canal is more linked to type 2. topographic relationship of impacted third molars and mandibular canal: correlation of panoramic radiograph signs and cbct images 414414414414414 braz j oral sci. 11(3):411-415 discussion several factors might be associated with higher risk of injury to the ian and increased difficulty during impacted third molar removal, and those can be divided in clinical and radiographic. within clinical factors, gender and body mass index appear to be consistently correlated 16. radiographic factors include panoramic signals like interruption of the radiopaque line of the canal wall, radiolucent band over roots, deflection of the canal around the apices and narrowing of the roots6-12, and positioning according to pg and winter classifications13. previous studies observed relationship between radiographic signals indicating proximity of structures and the mandibular canal: radiolucent band over roots and interruption of the radiopaque line of the canal wall were stronger associated with direct contact between the tooth roots and the mandibular canal17-19. the present study showed a statistically significant relation among three types of radiographic finding –interruption of the radiopaque line of the canal wall, narrowing of roots and radiolucent band over roots and the presence of contact with the mandibular canal. furthermore, some studies have evaluated the association between the lingual positioning of the mandibular canal and its relation to accidental exposure of the neurovascular bundle7,14. the results from this study detected that the lingual positioning of the canal is closely associated with radiolucent band over roots. this study has also aimed as well to evaluate pg and winter classifications and the topographic relationship of the mandibular canal as seen in cbct images. analysis of data relative to winter classification indicates that mesial positioning is associated with lingual positioning of the mandibular canal. regarding pg components, although no statistically significant associations could be established, table 3 reveals a higher frequency with direct contact between mandibular canal and positioning of mandibular canal in the lingual aspect for categories a, b and 2. almendro-marqués et al . 13 have warned for a higher probability of complications correlated with categories b2 and c2. therefore, whenever in face to mesially positioned impacted molars, it is advisable for clinicians to also look for the coexistence of signals associated with a higher probability of lingual positioning of the ian (such as radiolucent band over roots), since the association of this topographic relation and exposure of the neurovascular bundle has been observed7,14. some authors have stated that panoramic radiographs cannot be considered an ideal tool in terms of prediction of accidents related to ian20, and the requesting of cbct is supported in the presence of radiographic signals associated with proximity of structures7,17,21-23. however, facing that panoramic radiograph is the most frequently used image for the diagnosis and treatment recommendations for third molar surgery5, the information available with this technique is essential for dimensioning the need for further investigation. furthermore, cbct should be use consciously among professionals, especially because of the radiation dose received by patients. even if the radiation dose from cbct is significantly less than the conventional ct, it is several to many times higher than panoramic imaging 24, and, therefore, their use has to be fully justify over conventional techniques before they are carried out25-26. the present study evaluated the signals and classifications usually used to determine the injury risk to the ian by impacted lower third molars surgery using different diagnostic images methods. the results showed a tendency of critical positions and closeness between mandibular canal and third molars at the panoramic radiography based on its topographic position as seemed in cbct images. hence, the knowledge about the relationship between radiographic signals and the pg and winter classifications might improve the panoramic radiographic diagnosis, contributing to the clinical decision as well as to the teaching and learning process; moreover, the lower radiation dose received will benefit the patient. regarding radiographic signals indicating proximity between impacted lower third molars and mandibular canal in panoramic radiographs, radiolucent band over roots is the image more closely associated with direct contact between structures and the one that indicates lingual positioning of the canal more consistently. some categories of pg and winter classifications suggested signs of the topographic location of the mandibular canal encouraging further assessments exploring these aspects. references 1. cade ta. paresthesia of the inferior alveolar nerve following the extraction of the mandibular third molars: a literature review of its causes, treatment, and prognosis. mil med. 1992; 157: 389-92. 2. miloro m, dabell j. radiographic proximity of the mandibular third molar to the inferior alveolar canal. oral surg oral med oral pathol oral radiol endod. 2005; 100: 545-9. 3. savi a, manfredi m, pizzi s, vescovi p, ferrari s. inferior alveolar nerve injury related to surgery for an erupted third molar. oral surg oral med oral pathol oral radiol endod. 2007; 103: e7-9. 4. klinge b, petersson a, maly p. location of the mandibular canal: comparison of macroscopic findings, conventional radiography, and computed tomography. int j oral maxillofac implants. 1989; 4: 327-32. 5. marciani rd. third molar removal: an overview of indications, imaging, evaluation, and assessment of risk. oral maxillofac surg clin north am. 2007; 19: 1-13. 6. monaco g, montevecchi m, bonetti ga, gatto mr, checchi l. reliability of panoramic radiography in evaluating the topographic relationship between the mandibular canal and impacted third molars. j am dent assoc. 2004; 135: 312-8. 7. tantanapornkul w, okouchi k, fujiwara y, yamashiro m, maruoka y, ohbayashi n et al. a comparative study of cone-beam computed tomography and conventional panoramic radiography in assessing the topographic relationship between the mandibular canal and impacted third molars. oral surg oral med oral pathol oral radiol endod. 2007; 103: 253-9. 8. blaeser bf, august ma, donoff rb, kaban lb, dodson tb. panoramic radiographic risk factors for inferior alveolar nerve injury after third molar extraction. j oral maxillofac surg. 2003; 61: 417-21. topographic relationship of impacted third molars and mandibular canal: correlation of panoramic radiograph signs and cbct images braz j oral sci. 11(3):411-415 415415415415415 9. rood jp, shehab ba. the radiological prediction of inferior alveolar nerve injury during third molar surgery. br j oral maxillofac surg. 1990; 28: 20-5. 10. sedaghatfar m, august ma, dodson tb. panoramic radiographic findings as predictors of inferior alveolar nerve exposure following third molar extraction. j oral maxillofac surg. 2005; 63: 3-7. 11. de melo albert dg, gomes ac, do egito vasconcelos bc, de oliveira e silva ed, holanda gz. comparison of orthopantomographs and conventional tomography images for assessing the relationship between impacted lower third molars and the mandibular canal. j oral maxillofac surg. 2006; 64: 1030-7. 12. gallesio c, berrone m, ruga e, boffano p. surgical extraction of impacted inferior third molars at risk for inferior alveolar nerve injury. j craniofac surg. 2010; 21: 2003-7. 13. almendros-marques n, berini-aytes l, gay-escoda c. influence of lower third molar position on the incidence of preoperative complications. oral surg oral med oral pathol oral radiol endod. 2006; 102: 725-32. 14. ghaeminia h, meijer gj, soehardi a, borstlap wa, mulder j, berge sj. position of the impacted third molar in relation to the mandibular canal. diagnostic accuracy of cone beam computed tomography compared with panoramic radiography. int j oral maxillofac surg. 2009; 38: 964-71. 15. farman ag. alara still applies. oral surg oral med oral pathol oral radiol endod. 2005; 100: 395-7. 16. gbotolorun om, arotiba gt, ladeinde al. assessment of factors associated with surgical difficulty in impacted mandibular third molar extraction. j oral 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molars. j oral maxillofac surg. 2008; 66: 256-9. 21. flygare l, ohman a. preoperative imaging procedures for lower wisdom teeth removal. clin oral investig. 2008; 12: 291-302. 22. neugebauer j, shirani r, mischkowski ra, ritter l, scheer m, keeve e et al. comparison of cone-beam volumetric imaging and combined plain radiographs for localization of the mandibular canal before removal of impacted lower third molars. oral surg oral med oral pathol oral radiol endod. 2008; 105: 633-43. 23. susarla sm, dodson tb. preoperative computed tomography imaging in the management of impacted mandibular third molars. j oral maxillofac surg. 2007; 65: 83-8. 24. ludlow jb, davies-ludlow le, brooks sl, howerton wb. dosimetry of 3 cbct devices for oral and maxillofacial radiology: cb mercuray, newtom 3g and i-cat. dentomaxillofac radiol. 2006; 35: 219-26. 25. roberts ja, drage na, davies j, thomas dw. effective dose from cone beam ct examinations in dentistry. br j radiol. 2009; 82: 35-40. 26. scarfe wc. “all that glitters is not gold”: standards for cone-beam computerized tomographic imaging. oral surg oral med oral pathol oral radiol endod. 2011; 111: 402-8. topographic relationship of impacted third molars and mandibular canal: correlation of panoramic radiograph signs and cbct images oral sciences n3 braz j oral sci. 9(2):81-84 original article braz j oral sci. april/june 2010 volume 9, number 2 evaluation of enamel roughness after ceramic bracket debonding and clean-up with different methods geraldo de silveira albuquerque1, mário vedovello filho 2, adriana simoni lucato2 eloísa marcantonio boeck2, viviane degan2, mayury kuramae2 1dds, ms in orthodontics, department of orthodontics, university fundação hermínio ometto, uniararas, brazil 2dds, ms, phd, professor, department of orthodontics, university fundação hermínio ometto, uniararas, brazil correspondence to: mário vedovello filho a/c pós-graduação – mestrado em ortodontia av. maximiliano baruto, 500 jd. universitário cep.: 13607-339 araras –sp e-mail: vedovelloorto@terra.com.br received for publication: august 04, 2009 accepted: may 12, 2010 abstract aim: this study evaluated the surface roughness (ra) and the topography (scanning electron microscopy) of the dental enamel after use of different methods for removal of residual resin after debonding of orthodontic brackets. methods: nine roughness measurements in three directions were made on enamel surface of 60 human premolars before bracket bonding (ra initial). ceramic brackets were bonded with transbond xt and stored for 24 h/37°c before debonding with pliers. the specimens were divided in five groups according to the method used for removal of residual resin: control (c); carbide bur at slow-speed (cl); carbide bur at high speed (ch); shofu tip at low speed (sb); shofu tip at high speed (hb); debonding pliers (zp). nine final surface roughness measurements (ra final) were made and one specimen of each group was observed by sem. the data were analyzed statistically by anova, tukey’s test and paired t test (p<.05). results: ra final of sb was significantly higher than c, cl, ch, and zp groups. the ttest showed that the ra final was significantly higher than ra initial for sb and cl. conclusions: the method used for removal of residual resin influenced the roughness of the enamel. carbide bur at high speed presented the best results and shofu at low speed presented the worst results. keywords: orthodontics. dental debonding. orthodontic brackets. introduction direct bonding techniques using orthodontic brackets on enamel is possible due to mechanical interaction between restorative material and enamel surface. acid etching of enamel creates a micro-retentive surface and allows for the mechanical woven between enamel and resin materials1. in addition, the adhesive technique recommended by newman2 enabled the improvement of the orthodontic apparatus, resulting in a more effective bonding between enamel and brackets. bracket bonding has many benefits for the orthodontic treatment, such as reduction of the risk of caries and periodontal diseases, simplification of the technique, oral hygiene most adequate, and reduction of the esthetic discomfort3. due to the improvement of the bonding materials and techniques, bracket bonding is not a great operational obstacle currently. however, problems concerning to removal of the residual adhesive persist and damages on enamel surface can be created4. gwinnet and cens5 reported that the existence of small portions of unfilled resin remnant doe not predisposes to the accumulation of plaque and wear occurs over time. however, this does apply to filled resins because a good part of them has high resistance to wear and biofilm accumulates more easily, making it difficult the cleaning of these areas4. 82 braz j oral sci. 9(2):81-84 concern about the integrity of the dental enamel dates back to the development of enamel etching, and its use in bonding of orthodontic brackets6. however, there is little discussion among orthodontists about the amount of enamel lost during the bonding and debonding of brackets. the aims of debonding of brackets are to remove the accessories and all the resin attached to teeth, and restore the enamel surface as close as possible to pretreatment condition, without inducing iatrogenic damages. several methods and techniques have been proposed to eliminate resin remnants. ideally, the removal takes place so that the original surface remains smooth and the qualities of the enamel are preserved intact as close as possible to its original appearance. the fact that resins usually have similar color to that of enamel, especially when the tooth is wet, is an additional difficulty even when mechanical devices are employees7. there are several methods used for removal of residual resin after debonding of orthodontic brackets: sandblasting with aluminum oxide, carbide burs at high and low speed, debonding pliers, and abrasive wheels8. although orthodontic research has focused on the development of new techniques of brackets bonding, the problems arising from bracket debonding and removal of residual resin from enamel have not received the same attention. orthodontists do not have special care with possible changes in enamel during the removal of residual resin. however, damages on enamel are irreversible, and should be avoid in order to reduce the unnecessary removal of sound tissues. the aim of this study was to evaluate the surface roughness (ra) and the topography (scanning electron microscopy) of the dental enamel after use of different methods for removal residual resin after debonding of orthodontic brackets. material and methods sixty freshly extracted human maxillary premolars with intact buccal enamel were collected and stored in a solution of 0.2% (weight/volume) thymol. the criteria for tooth selection included intact buccal enamel not subjected to any bracket bonding procedures, no damage caused by the extraction forceps, and no caries. the crowns were separated from root using diamond disks (kg sorensen, rio de janeiro, rj, brazil) and embedded in acrylic resin (vipi flash, pirassununga, sp, brazil) with the buccal surface exposed. after mounting, the teeth were cleaned and polished with pumice and rubber prophylactic cups for 10 s. surface roughness (ra) was measured using a surface roughnessmeasuring device (surfcord model 170, japan). for each specimen, nine measurements in three different directions were made with a cutoff value of 0.8 mm. the measurements were made before (ra initial) and after (ra final) the adhesive remnant removal. roughness variation was obtained with the equation ra delta=ra final–ra initial. after the initial roughness measurements, 10 specimens were stored in distilled water until the final roughness measurements and the other specimens were subjected to bracket bonding procedure. the bonding area was delimitated using an adhesive tape (3m, nova odessa, sp, brazil) positioned at the center of the buccal surface. the teeth were acid etched with phosphoric acid 37% (scothcbond 3m/espe, st. paul, mn, usa) for 20 s, washed and dried for 10 s. the transbond xt primer (3m unitek, monrovia, ca, usa) was applied on the enamel surface and dried lightly with compressed air for 10 s. the resin composite transbond xt (3m unitek) was applied on the bottom of the ceramic brackets (henry schein inc., melville, ny, usa), positioned and fixed on teeth with manually pressure to extrude the excess material. the photoactivation was performed with halogen lamp (xl2500, 3m) for 40 s and the specimens were stored in distilled water for 24 h at 37°c before debonding. the brackets were removed using bracket-removing pliers (ice-mocar 346) and the remnant adhesive was checked under a stereomicroscope at 40x (carl zeiss, manaus, am, brazil) to certify that all adhesive was present on enamel. the specimens were divided in 5 groups (n=10) according to the method of removal of the remnant resin: 32-fluted tungsten carbide bur at slow-speed handpiece (cl); 32-fluted tungsten carbide bur at high-speed handpiece (ch); aluminum oxide tip shofu at low-speed hand-piece (sb); aluminum oxide tip shofu at high-speed handpiece (hb); zatty 934 debonding pliers (zp) (figure 1). the specimen surface was checked with a probe to certify that all remnant resin was removed. nine final surface roughness measurements in different directions were made for each specimen at the same sites of initial measurements. one representative specimen of each group was observed with a scanning electron microscope (model jeol jsm 5600 lv, tokyo, japan) to illustrate the effect of different methods of adhesive removal on enamel topography. the data were subjected to analysis of variance (anova) and tukey’s post-hoc test to compare the influence of different methods to remove the residual resin on enamel roughness. the paired t test was used to analyze the difference in surface roughness before (baseline) and after the adhesive removal. a level of significance of 5% was used for all analyses. fig. 1. materials used to remove the resin remnant. a – shofu tip; b – tungsten carbide bur; c – zatty 934 pliers. results the mean roughness values are presented on table 1. the anova showed that the method to remove the residual resin influenced significantly the roughness of the enamel evaluation of enamel roughness after ceramic bracket debonding and clean-up with different methods 83 braz j oral sci. 9(2):81-84 control 0.42 (0.06) a, a 0.33 (0.10) c, a -0.1 b debonding pliers 0.49 (0.12) a, a 0.72 (0.44) bc, a 0.24 b shofu at high speed 0.57 (0.10) a, a 0.72 (0.27) ab, a 0.16 b shofu at low speed 0.48 (0.14) a, b 1.19 (0.30) a, a 0.72 a carbide bur at high speed 0.47 (0.11) a, a 0.62 (0.29) bc, a 0.15 b carbide bur at low speed 0.48 (0.14) a, b 0.65 (0.19) bc, a 0.17 b roughness values (µm) (sd) methods ra initial ra final ra delta table 1. surface roughness (ra) (standard deviation) of enamel before bracket bonding and after removal the residual adhesive using different methods. lowercase letters in the columns and uppercase letters in the rows indicate statistically significant difference (p<0.05). (p<0.001). the shofu tip at low speed promoted the highest surface roughness on enamel compared to debonding pliers, carbide bur at high speed, carbide bur at low speed and control group (p<0.05). however, there was no significant difference between shofu at low and high speeds (p>0.05). carbide bur at low speed, carbide bur at high speed and debonding pliers showed no significantly difference compared to control group (p>0.05). the difference between the ra final and ra initial was calculated and described as ra delta. the results showed that shofu at low speed presented the highest ra delta, significantly higher (p<0.05) than the others methods, which showed no significant differences (p>0.05) among them (table 1). discussion the development of dental materials, mainly resin composite and adhesive systems, has led to a more effective adhesion between enamel and resin, reducing bracket bonding failure rate during orthodontic treatment. however, due to the increased adhesion to enamel, removing the residual resin after bracket debonding became more difficult. residual resin remains in enamel after debonding, or depending on the method of debonding used, cracks can be produced on enamel. therefore, the method of removal of the residual resin is very important to avoid damages on enamel, such as cracks, increased roughness of enamel, excessive enamel wear9, overheating of the tooth and pulp necrosis10. the results of this study showed that the enamel roughness after the clean-up with shofu at low speed was significantly higher than the other methods (p<.05). besides, t-test showed that the ra final was significantly higher than ra initial when the clean-up was performed with shofu and tungsten carbide bur at low speed (p<.05). shofu and tungsten carbide bur at low speed showed not be appropriate to remove the residual resin due to higher irregularity of the enamel after use of these methods. due to the scratches that the shofu tip generates on the enamel surface, it is indicated only for gross removal of the residual resin. retief and denys11 reported that tungsten carbide burs are most efficient for resin removal. however, carbide burs are harder than enamel12 and, when used at high speed, can damage the underlying enamel12-13. zachrisson and artun14 recommended using carbide burs but at low speeds. zarrinnia et al.15 recommended the removal of the bulk of the remaining resin with a 12fluted tungsten carbide finishing bur (tcb), operated at high speed (above 200,000 rpm) with adequate air cooling. all these investigations were carried out to evaluate the effects of different clean-up techniques on enamel surface. the literature is controversy about the most effective method of removal the residual resin. van waes et al.6 and zachrisson and artun14 concluded that a tcb at low speed produced the finest scratch pattern with the least enamel loss of 7.4 µm. retief and denys11 recommended the use of tcb at high speed with adequate air cooling, whereas rouleau et al.13 and campbell16 suggested water spray instead of air cooling for heating control. in clinical settings, cooling procedures that use air-water sprays are essential to ensure the prevention of pulpal damage. removal of resin remnants with a tungsten carbide bur using a high-speed handpiece without water cooling produced temperature increases exceeding the critical 5.58°c value for pulpal health. clean-up with water cooling never produced temperature changes exceeding the critical value10. it was related that the number of blades affects the wear of resin composite and the heating during the removal of resin remnants from enamel surface. temperature rise of 9.4°c was found using a 6-fluted bur, followed by the 12-fluted bur (6.5°c) and 1.2°c using a 40-fluted bur. the removal of residual adhesive after debonding is best performed with fine burs17. removal of unfilled resin remnants with hand instruments results in a mean enamel loss of 7.7 µm. rotary instruments, however, are required for cleaning up filled resin and the enamel lost for high-speed bur and low-speed bur was respectively 19.2 µm and 11.3 µm18. the enamel loss after the use of the high-speed tungsten carbide bur was greater compared to low speed. the least enamel loss occurred after the use of the slow-speed tungsten carbide bur or the debonding pliers. however, more residual adhesive are associated with the use of the debonding pliers9. the removal of residual resin with rotary instruments in low speed produces more vibration and generates discomfort for patients. although the enamel surfaces after treatment with the low-speed hand piece was irregular, the natural enamel also was slightly repetitive and spiky over the whole enamel surface area19. however, resin remnant removal with lowspeed instruments has disadvantages for pulpal health and patient comfort19. another disadvantage of removal of residual resin with rotary instruments is the generation of aerosols. it has been found that the potentially hazardous action of adhesive particulate aerosol produced by grinding, composite resin evaluation of enamel roughness after ceramic bracket debonding and clean-up with different methods 84 braz j oral sci. 9(2):81-84 particulates may act as endocrinological disruptors20. these results suggest the pliers as an appropriate method for removal of residual resin, since it produced little roughness of enamel. these results are in agreement with hossein et al.9, who found good results using the pliers, but are in disagreement with the findings of other studies13,21, which considered this method undesirable due to the higher occurrence of deep grooves in enamel produced by the removal of the residual resin. furthermore, some portions of the resin remains on the enamel after clean-up when pliers are used (figure 2). in this study, sem was used to give a better understanding of what happens to enamel with the different methods of fig. 2. representative sem micrographs of enamel surfaces at 50x magnification after cleaning clean-up of enamel with: a-tungsten carbide bur at high speed; b tungsten carbide bur at low speed; c shofu tip at high speed; d shofu tip at low speed; e zatty 934 pliers. resin removal tested. nonetheless, sem lacks a quantitative scale, cannot be used for the comparative assessment, and provides only subjective information22. tcb, shofu and pliers promoted the lowest roughness on the enamel in the present study. sem micrographs clearly demonstrate that enamel scarring was inevitable with both low and high-speed tcb and shofu (figure 2). using pliers, tcb and shofu tip with high speed seems to be very efficient ways to clean the surface and the least time consuming. the tcb and shofu with low speed were the most hazardous procedure to the dental enamel. however, the sem and surface roughness methods cannot measure the quantitative loss of enamel during clean-up procedures. therefore, when tcb or shofu are used at high speed, damages and excessive wear of enamel may occur due to difficulty of distinguishing the resin from enamel and controlling its wear13. the findings of this study showed that no method of removal the residual resin was able to leave the enamel surface with roughness similar to that of intact enamel. therefore, after removing the resin remnants from enamel surface following bracket debonding, polishing of tooth surface is recommended to minimize the grooves and irregularities left by the method used for resin removal and reduce the surface roughness. based on the methodology and analysis of results it was concluded that: 1. the method of removal the residual resin influenced the roughness of the enamel; 2. the best enamel roughness results were found with tungsten carbide bur at high speed and the worst results were obtained with shofu tip at low speed; 3. sem showed that no method eliminated all the irregularities left after the bonding/debonding of brackets. references 1. buonocore mg. a simple method of increasing the adhesion of acrylic filling materials to enamel surfaces. j dent res. 1955; 34: 849-53. 2. newman gv. epoxy adhesives for orthodontic attachments: progress report. am j orthod. 1965; 51: 901-12. 3. mandall na, millett dt, mattick cr, hickman j, macfarlane tv, worthington hv. adhesives for fixed orthodontic brackets. cochrane database syst rev. 2003: cd002282. 4. brown cr, way dc. enamel loss during orthodontic bonding and subsequent loss during removal of filled and unfilled adhesives. am j orthod. 1978; 74: 663-71. 5. gwinnett aj, ceen rf. an ultraviolet photographic technique for monitoring plaque during direct bonding procedures. am j orthod. 1978; 73: 178-86. 6. van waes h, matter t, krejci i. three-dimensional measurement of enamel loss caused by bonding and debonding of orthodontic brackets. am j orthod dentofacial orthop. 1997; 112: 666-9. 7. zachrisson b. bonding in orthodontics. in: vanarsdall rl, graber tm., editors. orthodontics: current principles and techniques. saint louis: mosby; 1994. p.542-626. 8. herion dt, ferracane jl, covell da jr. porcelain surface alterations and refinishing after use of two orthodontic bonding methods. angle orthod. 80: 167-74. 9. dragiff da. a new debonding procedure. j clin orthod. 1979; 13: 107-11. 10. almeida hc, vedovello filho m, vedovello sa, young aaa, ramirezyanez go. er: yag laser for composite removal after bracket debonding: a qualitative sem analysis. int j orthod. 2009; 20: 9-13. 11. mishima fd, valentim rg, araujo mt, ruellas ac, sant’anna ef. the effect of tooth bleaching on the enamel surface and the tensile force to debond orthodontic brackets. j orthod. 2009; 36: 236-42. 12. hosein i, sherriff m, ireland aj. enamel loss during bonding, debonding, and cleanup with use of a self-etching primer. am j orthod dentofacial orthop. 2004; 126: 717-24. 13. uysal t, eldeniz au, usumez s, usumez a. thermal changes in the pulp chamber during different adhesive clean-up procedures. angle orthod. 2005; 75: 220-5. 14. retief dh, denys fr. finishing of enamel surfaces after debonding of orthodontic attachments. angle orthod. 1979; 49: 1-10. 15. gwinnett aj, gorelick l. microscopic evaluation of enamel after debonding: clinical application. am j orthod. 1977; 71: 651-65. 16. rouleau bd jr, marshall gw, jr., cooley ro. enamel surface evaluations after clinical treatment and removal of orthodontic brackets. am j orthod. 1982; 81: 423-6. 17. zachrisson bu, arthun j. enamel surface appearance after various debonding techniques. am j orthod. 1979; 75: 121-7. 18. zarrinnia k, eid nm, kehoe mj. the effect of different debonding techniques on the enamel surface: an in vitro qualitative study. am j orthod dentofacial orthop. 1995; 108: 284-93. 19. campbell pm. enamel surfaces after orthodontic bracket debonding. angle orthod. 1995; 65: 103-10. 20. jonke e, weiland f, freudenthaler jw, bantleon hp. heat generated by residual adhesive removal after debonding of brackets. world j orthod. 2006; 7: 357-60. 21. pus md, way dc. enamel loss due to orthodontic bonding with filled and unfilled resins using various clean-up techniques. am j orthod. 1980; 77: 269-83. 22. kim ss, park wk, son ws, ahn hs, ro jh, kim yd. enamel surface evaluation after removal of orthodontic composite remnants by intraoral sandblasting: a 3-dimensional surface profilometry study. am j orthod dentofacial orthop. 2007; 132: 71-6. evaluation of enamel roughness after ceramic bracket debonding and clean-up with different methods oral sciences n3 braz j oral sci. 10(2):93-97 original article braz j oral sci. april | june 2011 volume 10, number 2 salivary characteristics of diabetic children gheena s.1, chandrasekhar t.2, pratibha ramani3 1mds, senior lecturer, department of oral and maxillofacial pathology, faculty of dental sciences, sri ramachandra university, chennai, india 2mds, professor and head of department, department of oral and maxillofacial pathology, saveetha dental college, saveetha university, chennai, india 3mds, professor, department of oral and maxillofacial pathology, saveetha dental college, saveetha university, chennai, india correspondence to: gheena.s dept of oral and maxillofacial pathology, faculty of dental sciences, sri ramachandra university, porur, chennai, india phone: +91 98840 33777 e-mail: gheena_ranjith@yahoo.co.in abstract aim: the objectives of this study were to evaluate the levels of glucose, cholesterol, protein and albumin in saliva, and to correlate the levels of glucose of the saliva to oral health and blood glucose of diabetic and non-diabetic children. methods: 32 children with type 1 diabetes mellitus formed the study group (dc) and 32 non-diabetic children formed the control group (nd). the patients had their saliva collected and evaluated for glucose, cholesterol, total protein and albumin. blood glucose analysis was also performed. the dental health status of the subjects was measured by dmft index and def index. independent student’s t-test was performed to compare metabolic status values in dc and nd groups. correlation test was applied between blood glucose and salivary glucose (spearman’s correlation), and salivary glucose and dmft/def (spearman’s test). results: a statistically significant difference was observed between dc and nd considering salivary glucose (p=0.000). elevated levels of cholesterol were evident in dc in correlation with nd. total protein and albumin had increased values in dc (nonsignificant p value). the dental health status was not statistically different. conclusions: salivary parameters can act as adjuncts in assessing the overall metabolic status of the patient. keywords: saliva, diabetes, pediatric, glucose, cholesterol. introduction diabetes mellitus (dm) comprises a group of common metabolic disorders that share the salient feature of hyperglycemia. several distinct types of dm exist and are caused by a complex interaction of genetics, environmental factors and lifestyle choices. the metabolic deregulation associated with dm causes secondary pathophysiological changes in multiple organ systems and imposes a tremendous burden on the individual with diabetes and the health care system. although all forms of dm are characterized by hyperglycemia, the pathogenic mechanisms by which hyperglycemia arises differ widely. the two broad categories of dm are designated as type1 (iddm) and type 2 (niddm). the incidence of childhood onset type 1 diabetes is increasing in many countries in the world, at least in the under 15-year-old age group. there are strong indications of geographic differences in trends but the overall annual increase is estimated to be around 3%.two international collaborative projects, the diabetes mondiale study (diamond)1 and the europe and diabetes study (eurodiab)2 have been instrumental in monitoring trends in incidence. by 2010, it is estimated that annually some 76,000 children aged less than 15 years develop type 1 dm worldwide. of the estimated 480,000 children with type 1 dm, 24% come from the south-east asian region, but the european region, where the most reliable and up-to-date estimates of incidence are available, comes a close second (23%)3.the incidence rate of type 1 dm in india is 4.2/100,000 population per year. received for publication: july 15, 2010 accepted: may 31, 2011 94 braz j oral sci. 10(2):93-97 a wide spectrum of oral manifestations of dm has been reported ranging from xerostomia, taste impairment, sialosis, dental caries and periodontal disease to fungal infections, oral lichen planus and fissured tongue4. however, in children, there is no agreement in results that relate alterations of salivary chemical composition and oral health5. an optimum metabolic control level correlates positively with the well being of the patient. assessing the metabolic control of the patient has traditionally been done through testing of glycosylated hemoglobin, fructosamine, and glycoalbumin along with other criteria such as body mass index, microalbuminuria and dyslipidemia6-7. fructosamine reflects the glycemic control over the previous 1-2 weeks and has been correlated positively with serum albumin and serum total protein5. saliva is a unique fluid and interest in it as a diagnostic medium has advanced exponentially in the last decade. advances in technology have helped to move saliva beyond evaluating oral health characteristics to where it now may be used to measure essential features of overall health8. thus, the objectives of this study were to evaluate the levels of glucose, cholesterol, protein and albumin in saliva, and to correlate the levels of glucose of the saliva to oral health and blood glucose of diabetic and non-diabetic children thereby analyzing whether saliva-based glucose testing can be an effective alternative to blood-based glucose testing in children. material and methods study population the study group comprised 32 diabetic children (dc) aged between 5 and 15 years with an already established diagnosis of type 1 dm. this group was selected from the patients attending the outpatient diabetes department, institute of child health and research centre, egmore, chennai. the control group comprised 32 non-diabetic children (nd) without any other pre-existing systemic diseases. the study was conducted in collaboration with the institute of child health and research centre and college of dental surgery, saveetha university, chennai with the ethical committee approval [(ihep no.) mds 34/su 63/06]. parental informed consent was obtained for all patients before they were examined and samples collected. saliva collection and processing sample collection was performed during the morning hours when the subjects are in the fasting mode. additionally, blood glucose values determined from venous blood samples drawn at the time of collection of saliva (fasting blood sugar) were also taken into account. standard ucla procedure was used to collect the saliva. the subjects were asked to refrain from eating, drinking or other oral hygiene procedures, for at least one hour prior to collection. drinking water was then given to the subjects, to rinse their mouth. five minutes after the oral rinse, unstimulated saliva was collected in 50 ml falcon tubes by the method of spitting the saliva. the patient was asked to swallow the saliva present in the mouth and then to remain still without moving the tongue or swallowing the saliva for one minute. the patient spat the saliva every 60 s for a total of 5 min into a falcon tube to which sodium fluoride9 has been added. the tubes were then placed on ice. about 1.5 ml of saliva was collected from each subject. the samples were then centrifuged at 2,500 rpm for 5 min. the centrifugation resulted in a saliva sample which is free of large particulate debris and reduced viscosity, thereby allowing a more accurate and reproducible analysis. determination of glucose glucose was estimated by using a glucose kit (crest biosystems, a division of coral clinical systems, goa, india) based on glucose oxidase-peroxidase method. it was standardized for saliva with 0.384mg/dl as the lower value. the absorbance of the standard and the test material (0.01 ml/10 µl) were measured against the blank without dilution within 60 min at a wavelength of 505 nm (hg 546 nm)/ green. total glucose in milligram/deciliter (mg/dl) is then derived as absorbance of test (abs.t)/ absorbance of standard (abs.s) x 100. determination of cholesterol cholesterol (ch) was estimated by using a commercially available cholesterol kit, which is based on cholesterol oxidase /pap method. the absorbance of the standard and the test material (0.01 ml/10µl) without dilution were measured against the blank within 60 min at a wavelength of505nm (hg 546nm)/ green. cholesterol in mg/dl = abs.t/ abs. s x 200. determination of total protein total protein (tp) was estimated by using a commercially available test kit based on the biuret method. the absorbance of the standard and the test material (0.02 ml/20 µl) are measured against the blank without dilution within 60 min at a wavelength of 550 nm (hg 546 nm)/ yellow – green. total proteins in gram (g)/dl = abs.t/abs.s x8 determination of albumin estimation of albumin (alb) was done by the colorimetric method based on the bcg method (commercially available test kits). the absorbance of the standard and the test material (0.01 ml/10 µl) are measured without dilution against the blank within 60 min at a wavelength of 630 nm (hg623nm)/ red. albumin in g/dl= abs.t/abs.s x 4. dental health status the dental health status of the subjects was measured by the dmf-t index and the def index. the dmf index pertains to the permanent teeth (d – decayed, mmissing and f – filled) and the def index (d – decayed, e – extracted, f – filled) to the deciduous teeth salivary characteristics of diabetic children braz j oral sci. 10(2):93-97 95 average dmf=total dmf total number of the subjects examined average def = total def total number: of the children examined statistical analysis independent students’t-test was performed to compare metabolic status values in dc and nd groups. correlation test was applied between blood glucose and salivary glucose (spearman’s correlation), and salivary glucose and dmft/ def (spearman’s test). results the mean value of salivary glucose (sg) of dc is 10.4 mg/dl and for nd is 17.6 mg/dl. the resulting p = 0.000 was 99.9% significant. a statistically significant difference (p = 0.000) was found between dc and nd with regard to cholesterol values. no statistically significant difference was found between dc and nd with regard to albumin (p = 0.22) and total protein (p = 0.47) (table 1). there was no significant correlation between salivary glucose (sg) and blood glucose (bg) within dc, but there was a significant correlation within nd (p=0.000) (table 2). the correlation between the dmft status and the salivary glucose values of the subjects yielded nonsignificant results. discussion whole saliva is a mixture of the secretions produced by the three pairs of large glands and the smaller glands of oral mucosa (labial, lingual, buccal and palatal). it may also contain fluid from the gingival pocket (gingival, or crevicular fluid)9. the serum constituents in saliva are derived from the local vasculature of the salivary glands as well as from gingival fluid. the increased salivary glucose evident in the whole saliva can be due to several contributing factors or it could be a simple reflection of the blood glucose levels since saliva is an ultra filtrate of plasma. the salivary analytes are derived from plasma generally by three mechanisms (passive diffusion, active transport and ultra filtration) and are thus found in saliva as mentioned by miller10. a chronic disease like dm, with its emphasis on patient involvement in the control of the disease, and its attendant problems, place a huge burden on the pediatric patients. our efforts should be directed at making it an easier burden to bear. if saliva could be used for diagnosis and monitoring, diabetic children would not require daily invasive tests, they can just collect their saliva in a sterilized collection tube, which could then be used in biosensors for the real-time, sensitive and specific detection of salivary diagnostic analytes to give an overall view of the child’s diabetic status. very little information is available on the potential of saliva in diabetic children in india. the salivary glucose values of the case subjects in our study were lower than the controls. this finding is similar to those of previous studies11-13. reuterving et al.11 determined whether salivary flow rate and salivary glucose concentration in patients with diabetes mellitus influence of severity of diabetes. salivary glucose concentration was lower during the period of better metabolic control. marchetti et al.12 found that the salivary glucose secretion rate was significantly lower (p less than 0.02) in diabetic patients with diabetic autonomic neuropathy than normal patients. kanji et al.13 measured the plasma and salivary concentrations of glucose and cortisol during insulin induced hypoglycemic stress in healthy nigerians. salivary glucose levels (fasting and after intravenous insulin) were unaffected by hypoglycemia and did not correlate with plasma glucose at any time point. we could not ascertain the reason for the low salivary glucose concentrations evident in dc in comparison to nd. one of the confounding variables may be that the cases in dc were under treatment as opposed to the cases in nd. more studies are needed on this facet. the salivary concentration of lipid soluble, unconjugated steroids such as cortisol, estriol, testosterone and progesterone, closely reflects the plasma concentration. the significantly high values of salivary cholesterol seen in the diabetic subjects could be an indication of the dyslipidemia, a group parameters correlation value p value diabetic children blood glucose 0.255 0.159(ns) salivary glucose non-diabetic children blood glucose 0.833 0.000** salivary glucose table 2 table 2 table 2 table 2 table 2 correlation between blood glucose and salivary glucose in diabetic children and non-diabetic children ** correlation is significant at the 0.01 level (2-tailed) salivary characteristics of diabetic children parameters diabetic children non-diabetic children significance mean(mg/dl) s d mean(mg/dl) s d blood glucose 326.34 104.33 84.5 5.94 0.000*** salivary glucose 10.48 8.26 17.60 4.34 0.000*** cholesterol 11.24 8.9 4.08 3.05 0.000*** total protein 0.47 0.60 0.34 0.16 0.22(ns) albumin 0.30 0.46 0.24 0.16 0.47(ns) table 1 -table 1 -table 1 -table 1 -table 1 metabolic status –blood glucose, salivary glucose, cholesterol, total protein and albumin in the groups *** p<0.001(99.9% significant) 96 braz j oral sci. 10(2):93-97 condition usually associated with diabetic subjects14-15. dyslipidemia is a common feature of dm and needs to be treated because of the potential complications especially atherosclerosis. cholesterol being a lipid moiety passes into saliva by passive diffusion and has the potential to closely reflect blood concentrations. the cholesterol of case subjects is statistically higher than that for the control subjects. karjailanen et al. 16 suggested that salivary cholesterol may be regarded as a transudate from serum, as suggested by slomiany et al. the positive correlation of serum and saliva cholesterol values further supported the concept that at least part of the salivary cholesterol originates from serum. they also concluded that, in healthy adults, salivary cholesterol concentrations reflect serum concentration to some extent and can be used to select individuals with high serum cholesterol levels. medical laboratory observer (2000) gives information about a salivabased cholesterol test that has demonstrated the potential to correlate a quantitative result with patient blood cholesterol level. ben aryeh et al.17 found no difference in salivary total protein, amylase, lactoferrin, or lysozyme among the three groups (iddm, niddm, controls) examined. streckfus et al. 18 recorded that all diabetic groups in their study demonstrated a significantly lower salivary total protein concentration when compared with controls, which is in contrast to our study values. there is a lack of literature on total protein and albumin estimations from the saliva of diabetic subjects, and the available data do not show a consensus on the results. these varied results indicate, as suggested by rantonen et al.19 that these proteins are subject to short-term variation. improved ways of evaluation of these elements in saliva with sophisticated techniques or another way of assessing the metabolic control in saliva needs to be considered. the diabetic group and the control group did not differ with regard to the dental status as opposed to the usual scenario where diabetic children are expected to have more carious lesions. this could be attributed to the reduction in the frequency of sticky food intake and food with high glycemic index and better oral hygiene practices by the diabetic children. swanljung et al.20 found that if the patients’ iddm is well controlled, their salivary and caries data does not differ from that of healthy controls. blanco et al.21 found no differences in the number of decayed, missing and filled teeth based on metabolic control, evolution time and existence of late complications of diabetes. the lack of correlation between the salivary glucose and blood glucose values in dc underscores the importance of more studies needed in this direction. both saliva and blood samples were taken during morning hours when the case subjects were in fasting mode. one of the confounding variables may be the fact that the cases in dc were under treatment as opposed to the cases in nd. the cholesterol in dc was significantly higher than that of nd. further research is needed, especially on the correlation of salivary and blood values of cholesterol, to have a better understanding of this relationship. total protein and albumin can be used to assess the metabolic status of diabetic subjects since both showed a slight increase as compared to controls. the dental caries experience correlation between the cases and controls was not significant and gives credence to the value of diet and oral hygiene measures in the subjects. salivary parameters can act as adjuncts in assessing the overall metabolic status of the patient. further studies should be performed to make these tests equivalent to a blood test. acknowledgements the authors express their sincere gratitude to dr. n. malathi, faculty of dental sciences, sru; director, institute of child health; dr. parivardhini, diabetes opd, institute of child health; mr. porchelvan and mr. ravi shanker, statisticians, sri ramachandra medical college and research institute for their help and motivation. references 1. diamond. project group. incidence and trends of childhood type 1 diabetes worldwide 1990-1999. diabet med. 2006; 23: 857-66. 2. patterson cc. dahlquist gg. gyürüs e. incidence trends for childhood type 1 diabetes in europe during 1989-2003 and predicted new cases 2005-20: a multicentre prospective registration study. lancet.2009; 373: 2027-33. 3. international diabetes federation. the diabetes atlas. 4th ed. montreal: international diabetes federation; 2009. 4. manfredi m, mc cullough mj, vescovi p, al-kaarawi zm, porter sr. update on diabetes mellitus and related oral diseases. oral dis. 2004; 10: 187-200. 5. lopez me, colloca me, paez rg, schallmach jn, koss ma, chervonagura a. salivary characteristics of diabetic children. braz dent j. 2003; 14: 26-31. 6. kaplan l, pesce a. clinical chemistry theory, analysis, and correlation. saint louis: mosby; 1984. 7. the clinical chemistry of diabetes, workshop, washington, dc, oct 1998. 8. streckfus cf, bigler lr. saliva as a diagnostic fluid. oral dis. 2002; 8: 69-76. 9. jenkins gn. the physiology and biochemistry of the mouth. oxford: blackwell; 1978. v.4. 10. miller sm. saliva: new interest in a non-traditional specimen. saliva as a multipurpose diagnostic fluid. med lab observ. 1993; 20: 31-5. 11. reuterving co, reuterving g, hagg e, ericson t. salivary flow rate and salivary glucose concentration in patients with diabetes mellitus influence of severity of diabetes. diabete metab. 1987; 13: 457-62. 12. marchetti p, tognarelli m, giannarelli r, grossi c, picaro l, di carlo a et al. decreased salivary glucose secretory rate: usefulness for detection of diabetic patients with autonomic neuropathy. diabetes res clin pract. 1989; 7: 181-6. 13. alkanji ao, ezenwaka c, adejuwon ca, osotimehin bo. plasma and salivary concentrations of glucose and cortisol during insulininduced hypoglycaemic stress in healthy nigerians. afr j med med sci. 1990; 19: 265-9. 14. ladeia am, adan l, couto-silva ac, hiltner a, guimaraes ac. lipid profile correlates with glycemic control in young patients with type 1 diabetes mellitus. prev cardiol. 2006; 9: 82-8. 15. wiltshire ej, hirte c, couper jj. dietary fats do not contribute to hyperlipidemia in children and adolescents with type 1 diabetes. diabetes care. 2003; 26: 1356-61. salivary characteristics of diabetic children 97 braz j oral sci. 10(2):93-97 16. karjalainen s, sewon l, soderling e, larsson b, johansson i, simell o et al. salivary cholesterol of healthy adults in relation to serum cholesterol concentration and oral health. j dent res. 1997; 76: 1637-43. 17. ben-aryeh h, serouya r, kanter y, szargel r, laufer d. oral health and salivary composition in diabetic patients. j diabetes complications. 1993; 7: 57-62. 18. streckfus cf, marcus s, welsh s, brown rs, peppers gc, brown rh. parotid function and composition of parotid saliva among elderly edentulous african-american diabetics. j oral pathol med. 1994; 23: 277-9. 19. rantonen pj, meurman jh. correlations between total protein, lysozyme, immunoglobulins, amylase, and albumin in stimulated whole saliva during daytime. acta odontol scand. 2000; 58: 160-5. 20. swanljung o, meurman jh, torkko h, sandholm l, kaprio e, maenpaa j. caries and saliva in 12-18-year-old diabetics and controls. scand j dent res. 1992; 100: 310-3. 21. arrieta blanco jj, villar bb, martinez ej,vallejo ps, arrieta blanco fj. bucco-dental problems in patients with diabetes mellitus (i) : index of plaque and dental caries. med oral. 2003; 8: 97-109. salivary characteristics of diabetic children oral sciences n3 braz j oral sci. 10(3):158-162 received for publication: december 07, 2010 accepted: june 21, 2011 original article braz j oral sci. july | september 2011 volume 10, number 3 use of dental dimensions estimated from personal portraits in human identification rachel lima ribeiro tinoco1, laíse nascimento correia lima1, mário marques fernandes2, luiz francesquini junior3, eduardo daruge junior4 dds, msc, department of forensic dentistry, piracicaba dental school, university of campinas, brazil 2dds, msc, biomedical service of prosecute council of rio grande do sul state, brazil 3dds, msc, phd, department of forensic dentistry, piracicaba dental school, university of campinas, brazil 4dds, msc, phd, assistant professor, department of forensic dentistry, piracicaba dental school, university of campinas, brazil correspondence to: rachel lima ribeiro tinoco piracicaba dental school university of campinas department of forensic dentistry av. limeira, 901 caixa postal 52 piracicaba sp cep 13414-903 phone: (55) 19 2106 5200 / (55) 21 9963 4751 e-mail: racheltinoco@live.com abstract many cases of human identification in which traditional methods are not applicable challenge the experts’ capability and versatility. in the absence of ante-mortem records, superimposition of skull images over photographs of a possible victim arises as a possible alternative. aim: the present study was a pilot work willing to validate a new method of sizing images of the face by the use of proportionality principles, taking as reference a few predetermined accessories: a pair of sunglasses, a hat and a necklace. methods: twenty-one volunteers were photographed using each one of the accessories mentioned above. pictures of the dental arches were also taken, with millimeter scale adjacent. the images with accessories were examined by a single operator, who estimated the mesiodistal width of the upper central incisor, for later comparison with the real measures. results: the accuracy of the method was evaluated by the student’s t-test, which showed that the estimated measures were statistically greater than the real ones. conclusions: the analysis of the data collected showed that the use of the accessories as a dimensional scale did not generate reliable results. keywords: forensic dentistry, victims’ identification, tooth. introduction forensic dentistry is an important science for human identification, especially when conventional methods cannot be applied, usually due to advanced decomposition, carbonization or fragmentation of the body. in such cases, and in the absence of antemortem dental data to be compared, craniofacial image superimposition can be an accurate and reliable technique for human identification1-3. however, before superimposing images of the skull over photographs of a possible victim, it is mandatory that they both are properly sized in the same scale4-5. authors that apply computer-assisted craniofacial superimposition size reach the scale through craniometric and cephalometric landmarks easily identifiable in the face and the skull6-7, even thought this assessment is influenced by the thickness of local soft tissue amount and disposition of fat which can vary among different populations8. considering that the teeth are naturally visible throughout life, not being influenced by soft tissue, and commonly shown in smiling pictures, they are reliable size reference for comparing images of skull and personal pictures9. although 159 braz j oral sci. 10(3):158-162 technical advances allow the three-dimensional facial reconstruction10-12, this is not an identification method, but rather a form to disseminate a facial approximation of that subject, willing to achieve someone who knew him/her, and can provide reliable antemortem records for human identification. human identification through craniofacial image superimposition has been tested and used by different researchers and experts. however, the proper scaling of the photographs to be compared is a precondition for an accurate evaluation. the aim of this pilot study was to test the feasibility of obtaining real size of teeth from personal photographs, using common accessories as size reference, in order to use them for the proper scaling required for image superimposition. material and methods sampling pictures of 21 volunteers (8 males and 13 females) were taken by a single photographer. all the subjects were aged between 21 and 60 years, and agreed to participate of the study by signing an appropriate document. subjects with absence or anomalies in the upper central incisors were excluded, as well as those with notorious systemic pathology. methods from each participant the following photographs were taken: • dental arch, in frontal view, with lip spacer, and scale number 2 from american board of forensic dentistry placed at the same visual plane of the upper central incisors; • bust photograph, wearing a pair of sun glasses, and smiling; • bust photograph, wearing a cap, and smiling; • bust photograph wearing a round pendant necklace, and smiling – only the female subjects. the images obtained were stored in a computer managed by windows vista™, and analyzed by the software adobe photoshop™. from the bust photographs, the mesiodistal dimensions of the upper central incisors were measured, which were called tmp (tooth measure at the picture). from the same picture, using the same zoom, the size of the adornment (sunglass, a rounded detail of the cap, or necklace) were measured, to obtain what were called amp (adornment measure at the picture), as shown in figure 1, with the cap. figure 2 shows the sunglasses and the necklace. all the measurements were stored in an excel™ file. then, the life-size of the adornments worn by the subjects were taken, with a digital caliper (digimess, são paulo, brazil), which was called adornment’s real measure (arm). from the measures taken, tooth’s real measure (trm) was estimated, according to algebra fundamental rules of proportion, translated by the following equation: fig. 1 example of the measurements taken from the bust photographs: tooth measure at the picture (tmp), and adornment measure at the picture (amp). fig. 2 sunglasses and necklace used as adornment. once the estimations were concluded and stored, the pictures of the dental arches with metric scale were analyzed, and the mesiodistal dimensions of the upper central incisor were taken with a manual caliper, according to the scale at the picture, as graphically represented in figure 3. the values obtained from this measuring procedure were then compared to the estimations. all measurements and estimations were made by a single examiner. statistical analysis all measures – real and estimated – were divided in three independent groups, according to the adornment used as reference to the estimation: sunglasses, cap or necklace. for each group, the results were analyzed by student’s t-test for paired samples, with the tooth’s real measure and the tooth measure at the picture (tmp) = tooth’s real measure (trm) adornment measure at the picture (amp) adornment’s real measure (arm) use of dental dimensions estimated from personal portraits in human identification 160 braz j oral sci. 10(3):158-162 subject sex tooth measures difference estimated measure real measure estimation – real measure 1 f 0.95 0.9 0.05 2 f 0.82 0.7 0.12 3 f 0.90 0.7 0.2 4 f 1.47 0.7 0.77 5 f 0.96 0.9 0.06 6 f 0.96 0.8 0.16 7 f 0.84 0.8 0.04 8 f 1.03 0.8 0.23 9 f 0.81 0.7 0.11 10 f 0.78 0.7 0.08 11 f 0.86 0.7 0.16 12 f 0.85 0.6 0.25 13 f 1.07 0.8 0.27 14 m 0.93 0.7 0.23 15 m 0.91 0.8 0.11 16 m 0.91 0.8 0.11 17 m 1.08 0.8 0.28 18 m 1.04 0.8 0.24 19 m 0.92 0.8 0.12 20 m 0.88 0.7 0.18 21 m 0.95 0.8 0.15 mean 0.9486 0.7619 0.1867 table 1 size estimations (in cm) calculated from the cap fig. 3 graphic presentation of tooth’s real measure (trm), according to the dental arch’s photograph, using a metric scale. tooth’s estimated measure as variables. results the dental dimensions estimated from the size of the cap were overestimated in 100% of the cases, with a mean difference of 0.1867 mm, as can be seen in table 1. according to the student’s t-test, the estimated dental width was statistically higher than the real width (p = 0.0000169), at a significance level of 5%. when estimating the dental width from sunglass dimensions, overestimated values were found again, except for two cases of underestimation (9.52% of the sample). the mean difference between estimated and real measurements was equal to 0.1190cm, as shown on table 2. the values estimated from measurements of the sunglasses were considered statistically higher than the actual measurements (p = 0.0000018), with a significance level of 5%. the estimated tooth measures from the dimensional relation with the necklace were overestimated in most cases (92.31%). it is noteworthy that this type of calculation, using the necklace as a reference point, was performed only in female participants, since they have virtual exclusivity in the use of this type of adornment. the mean difference between estimated and actual measurements was equivalent to 0.1262 cm, as shown in table 3. tooth measures estimated from the necklace were significantly higher (p = 0.0001307) than the actual measurements, with a significance level of 5%. discussion since the early twentieth century, researchers have analyzed craniofacial correlations and the relationship between the face and the underlying bone morphology. on this line, are noteworthy the studies about the shape of the nose and the nasal bone, as well as variations in nose shapes of different ethnic groups13-14. these studies underlie the current principles of facial reconstruction15, which became the main target for this type of research16. as occurs in the nose, eyes also show great relationship between soft tissue and bone structure. the position of ectocanthus was studied by whitnall in 2,000 skulls, and can be located in the so called tubercle of whitnall. this anatomical point constitutes the major reference for sizing use of dental dimensions estimated from personal portraits in human identification 161 braz j oral sci. 10(3):158-162 subject sex tooth measures difference estimated measure real measure estimation – real measure 1 f 0.96 0.9 0.06 2 f 0.87 0.7 0.17 3 f 0.89 0.7 0.19 4 f 0.84 0.7 0.14 5 f 0.81 0.9 -0.09 6 f 0.87 0.8 0.07 7 f 0.95 0.8 0.15 8 f 1.01 0.8 0.21 9 f 0.80 0.7 0.1 10 f 0.84 0.7 0.14 11 f 0.88 0.7 0.18 12 f 0.82 0.6 0.22 13 f 0.90 0.8 0.1 mean 0.88 0.7538 0.1262 table 3 size estimations (in cm) calculated from the necklace – only female subjects. subject sex tooth measures difference estimation – real measure estimated measure real measure 1 f 1.00 0.9 0.1 2 f 0.77 0.7 0.07 3 f 0.93 0.7 0.23 4 f 0.84 0.7 0.14 5 f 0.89 0.9 -0.01 6 f 0.91 0.8 0.11 7 f 0.91 0.8 0.11 8 f 0.92 0.8 0.12 9 f 0.84 0.7 0.14 10 f 0.91 0.7 0.21 11 f 0.63 0.7 -0.07 12 f 0.91 0.6 0.31 13 f 1.00 0.8 0.2 14 m 0.88 0.7 0.18 15 m 0.92 0.8 0.12 16 m 0.88 0.8 0.08 17 m 0.89 0.8 0.09 18 m 0.95 0.8 0.15 19 m 0.85 0.8 0.05 20 m 0.80 0.7 0.1 21 m 0.87 0.8 0.07 mean 0.8810 0.7619 0.1190 table 2 size estimations (in cm) calculated from the sunglasses and positioning the image of the skull in relation to the face in superimposition methods15. the objects used as a dimensional reference in this study were selected because they frequently appear in personal pictures, in the same spatial plane of the teeth. however, the facial sizing from the teeth dimensions naturally requires the recovery of these teeth, which does not always occur, especially considering the frequency of post-mortem loss of anterior teeth. the proposed method overestimated the mesiodistal width of the central incisor, in its best result – by measuring sunglasses 1.19 mm on average (15.62% of the real average). the worst performance was achieved by having the cap as size reference, overestimating the dental width in 1.867 mm on average. the less satisfactory result of the cap in relation to other items may be related to the position of the cap slightly oblique in relation to the plane of the central incisors. further studies are suggested. the analysis of a personal photograph for human identification purposes requires excellent image resolution for detailed observation and accurate superimposition with the cranial image. the analysis of teeth in photographs only becomes possible under optimal conditions, close and high resolution images. still, the use of tooth width as a size use of dental dimensions estimated from personal portraits in human identification braz j oral sci. 10(3):158-162 reference, given its small dimensions, creates a greater risk of failure, since fractions of a millimeter can represent important distances in the final result. thus, when sizing images using anatomical landmarks, the larger the reference, the greater the chance of success. although previous studies have been engaged in creating protocols and methods of sizing and positioning skull and face images for superimposition, the correct size and position are more easily achieved through successive attempts, and movement of the images3,6,1,17-19. the method evaluated in the present study is therefore inaccurate for sizing facial images for superimposition with human identification purposes. references 1. dorion rbj. photographic superimposition. j forensic sci. 1983; 28: 724-34. 2. chai ds, lan yw, tao c, gui rj, mu yc, feng jh et al. a study on the standard for forensic anthropologic identification of skull-image superimposition. j forensic sci. 1989; 34: 1343-56. 3. austin-smith d, maples w. the reliability of skull/photograph superimposition in individual identification. j forensic sci. 1994; 39: 446-55. 4. iscan my, helmer rp. forensic analysis of the skull: craniofacial analysis, reconstruction, and identification. new york: wiley-liss; 1993. 5. jayaprakash pt, srinivasan gj, amravaneswaran mg. cranio-facial morphhoanalysis: a new method for enhancing reliability while identifying skulls by photo superimposition. forensic sci int. 2001; 117: 121-43. 6. yoshino m, imaizumi k, miyasaka s, seta s. evaluation of anatomical consistency in cranio-facial superimposition images. forensic sci int. 1995; 74: 125-34. 7. yoshino m, matsuda h, kubota s, imaizumi k, miyasaka s, seta s. computer-assisted skull identification system using video superimposition. forensic sci int. 1997; 90: 231-44. 8. starbuck jm, ward re. the affect of tissue depth variation on craniofacial reconstructions. forensic sci int. 2007; 172: 130-6. 9. marks m, bennett jl, wilson l. digital video image capture in establishing positive identification. j forensic sci. 1997; 42: 492-5. 10. de greef s, willems g. three-dimensional cranio-facial reconstruction in forensic identification: latest progress and new tendencies in the 21st century. j forensic sci. 2005; 50: 12-7. 11. ricci a, marella gl, apostol ma. a new experimental approach to computer-aided face/skull identification in forensic anthropology. am j forensic med pathol. 2006; 27: 46-9. 12. quatrehomme g, balaguer t, staccini p, alunni-perret v. assessment of the accuracy of three-dimensional manual craniofacial reconstruction: a series of 25 controlled cases. int j legal med. 2007; 121: 469-75. 13. glanville ev. nasal shape, prognathism and adaptation in man. am j phys anthropol. 1969; 30: 29-37. 14. macho ga. an appraisal of plastic reconstruction of the external nose. j forensic sci. 1986; 31: 1391-403. 15. wilkinson c. forensic facial reconstruction. cambridge: cambridge university press; 2004. 16. prokopec m, ubelaker dh. reconstructing the shape of the nose according to the skull. forensic sci commun. 2002; 4: 1-4. 17. pesce delfino v, colonna m, vacca e, potente f, introna fj. computeraided skull/face superimposition. am j forensic med pathol. 1986; 7: 201-2. 18. gruner o. identification of skulls: a historical review and practical applications. in: iscan my, helmer rp. forensic analysis of the skull: craniofacial analysis, reconstruction, and identification. new york: wileyliss; 1993. 19. pesce delfino v, vacca e, potente f, lettini t, colonna m. shape analytical morphometry in computer-aided skull identification via video superimposition. in: iscan my, helmer rp. forensic analysis of the skull: craniofacial analysis, reconstruction, and identification. new york: wiley-liss; 1993. 162 use of dental dimensions estimated from personal portraits in human identification oral sciences n3 original article braz j oral sci. october|december 2010 volume 9, number 4 healing and cytotoxic effects of psidium guajava (myrtaceae) leaf extracts kristianne porta santos fernandes1, sandra kalil bussadori1, márcia martins marques2, nilsa sumie yamashita wadt3, erna bach3, manoela domingues martins4 1phd, full professor, department of rehabilitation sciences, nove de julho university, são paulo, sp, brazil 2phd, full professor, department of dentistry, dental school, university of são paulo, são paulo, sp, brazil 3phd, full professor, school of pharmacy, nove de julho university, são paulo, sp, brazil 4phd, full professor, department of oral pathology, dental school, federal university of rio grande do sul, porto alegre, rs, brazil correspondence to: kristianne porta santos fernandes curso de mestrado em ciências da reabilitação, universidade nove de julho 612, francisco matarazzo avenue são paulo sp – brazil 05001-100 phone/fax: +55-11-3665-9325 e-mail: kristianneporta@gmail.com received for publication: june 24, 2010 accepted: november 12, 2010 abstract aim: the aims of this study were to evaluate the wound healing potential in vivo and the cytotoxic effects in vitro of psidium guajava (myrtaceae) leaf extract and commonly used corticosteroids. methods: the healing effect was studied in vivo by the clinical and histological evaluation of traumatic lesions in the oral mucosa of rats treated with these substances. each rat received 2 daily applications of the medicine tested and the animals were sacrificed after 1, 3, 5, 7 and 14 days. tissue sections stained with hematoxylin & eosin were analyzed. the histological evaluation involved a 5-point scoring system based on the degree of healing, ranging from 1 (total repair of epithelium and connective tissue) to 5 (epithelial ulcer and acute inflammatory infiltrate). the kruskal-wallis test was used for statistical analysis of the histological scores. for the in vitro toxicity assay, each substance was applied to mucosa fibroblast cell cultures in conditioned media. the media were conditioned by placing the substances in contact with fresh culture medium for 24 h. the cytotoxicity analysis was performed using the mtt assay. data were analyzed statistically by anova and tukey’s test at 5% significance level. results: in vitro, the guava extract caused a decrease in the cell viability and growth when compared to the control and corticosteroids. in vivo, guava extract caused accelerated wound healing from the 3rd day, whereas the corticosteroids delayed tissue repair and were associated with bacterial surface colonization, the presence of micro-abscesses and intense inflammatory infiltrate in the submucosa. conclusions: although in a short-term cytotoxicity analysis, the guava extract reduced the cell population in vitro, while in vivo, the extract accelerated wound healing. keywords: psidium guajava, wound healing, cell culture, guava leaves, cytotoxicity. introduction medicinal plants contribute significantly to primary health care in many countries and serve as the starting point for several semi-synthetic analogs. numerous plants and plant components have demonstrated antiinflammatory and wound healing properties as well as cytotoxic activity, which illustrates the potential for novel agents to be identified from uncharacterized natural plant resources1-5. the guava tree, psidium guajava linnaeus (myrtaceae) (hereafter referred to as guava), is a tropical hardwood plant that can reach a height of 10 m. it is considered native of mexico and extends throughout south america, europe, braz j oral sci. 9(4):449-454 africa and asia6-8. guava is used medicinally in many parts of the world as an antiinflammatory and antiseptic as well as in the treatment of diabetes, hypertension, pain, fever, respiratory disorders, gastroenteritis, diarrhea and dysentery. the leaves are applied to wounds, ulcers and joints (for the relief of rheumatic pain) and are also chewed to relieve toothache8. the wound healing properties of a methanolic leaf extract of psidium guajava have been determined in only one paper using the excision wound model. more than 90% wound healing was observed after 14 days post-surgery, compared to 72% healing in the group treated with distilled water9. the wound healing process involves a variety of events (inflammation, cell proliferation and contraction of the collagen lattice) and may be hampered by the presence of oxygen free radicals or microbial infection. the aims of this study were to evaluate the wound healing potential in vivo and in vitro cytotoxic effects of a psidium guajava (myrtaceae) leaf extract and commonly used corticosteroids. material and methods plant collection and preparation guava leaves were collected from the city of valinhos in the state of são paulo, brazil and registered at the herbarium of the bioscience institute, são paulo, brazil. the leaves were dried for 96 h in an oven at 40° c and ground. the hydroalcoholic extract was prepared with 100 g of leaves through fractionated percolation (7 days by fraction), using 70% ethanol at room temperature, obtaining 100 ml of final extract. the extract was stored in a refrigerator at 5° c until use. leaves of psidium guajava l. were submitted a phytochemical analysis of the main groups of active compounds using the following methods: • tannins – dry leaves were boiled in water (decoction), filtered and tested with proteins (gelatin), heavy metals (iron, lead and copper) and alkaloids (pilocarpine hydrochloride) in precipitate reactions. • flavonoids – dry leaves were extracted with 70% alcohol, cooled and filtered. the extract was tested through colorimetric reactions with hydrochloric acid, magnesium metal, ferric chloride iii, sodium hydroxide and uv light (366 nm). • essential oil – the oil of the leaves was extracted through hydrodistillation (clevenger method). • saponnins – the dry leaves were boiled in water, cooled and filtered. ten ml were shaken for 15 s and set to rest for 15 min. the 1-cm layer of foam was analyzed. • free anthraquinones – the dry leaves were shaken with hexane. the supernatant was removed and tested with ammonium hydroxide. anthraquinones glycosides – the dry leaves were boiled in 10% hydrochloric acid and iron chloride, filtered and tested with ammonium hydroxide. • cardiac glycosides – a hydroalcoholic extract was purified by precipitation with lead acetate, filtered and extracted with chloroform. the chloroform fraction was evaporated. keller killiani, baljet, kedde, lieberman-burchard reactions were tested colorimetric methods. • alkaloids – dry leaves were boiled in 10% hydrochloric acid, cooled, filtered, basified, extracted with chloroform, dried and dissolved again with 10% hydrochloric acid. bertrand, mayer, bouchardat and dragendorff precipitation reactions were tested. the quantitative analysis of total polyphenols (taninns) was carried out by precipitation with dry skin powder. the results were detected by absorbance in a spectrophotometer (sp 22, biospectro, são paulo, brazil) with uv light l 715 nm. experimental groups for both the in vitro and in vivo studies, four groups were tested: group i (gi): control (no drugs); group ii (gii): guava tree leaf hydroalcoholic extract (10%) in orabase formulation; group iii (giii): topical triamcinolone acetonide (omcilon a, bristol myers squibb, sao paulo, sp, brazil); group iv (giv): 0.05% clobetasol propionate cream (psorex®, medley, campinas, sp, brazil) cytotoxicity analysis (in vitro study) the toxicity of the guava leaf formulation and corticosteroids used in the topical treatment of oral ulcers was determined in vitro. the response of human gingival fibroblasts to substances leached from these drugs was analysed. cell viability was determined using the mtt reduction test (short-term and long-term assays) following contact with the substances. cell culture was chosen, as this method enables the control of bias by eliminating factors related to the subject reaction5. cell culture the cells were cultured as described elsewhere5. briefly, the fmm1 cell line of human gingival fibroblasts was used. these cells were cultured in dulbecco’s modified eagle medium (dmem), supplemented with 10% fetal bovine serum (fbs, cultilab, campinas, sp, brazil) and 1% antimycotic-anti-biotic solution (10,000 units of penicillin, 10 mg of streptomycin and 25 ¼g of amphotericin b per ml in 0.9% sodium chloride; sigma chemical company, st. louis, mo, usa). the cells were kept in an incubator at 37° c and a humidified 5% co2 atmosphere. cultures were supplied with fresh medium every other day. cells between the 10th and 14th passages were used in all experimental procedures. culture medium conditioning in order to obtain the conditioned media (e.g., media containing the substances dissolved or leached from the test materials), 50-ml centrifuge tubes containing the materials were filled with dmem (sigma). conditioning was carried out for 24 h at 37° c, using 0.2 g of each substance per ml of fresh medium5. conditioned media were applied to the cell cultures and cell viability was determined after 1, 3 and 5 days through an analysis of mitochondrial activity. experiment for the determination of the effect of the substances on 450450450450450healing and cytotoxic effects of psidium guajava (myrtaceae) leaf extracts braz j oral sci. 9(4):449-454 cell viability, fmm1 cells were plated into 96-well microtitration plates (1.8 x 103 cells/well). the experimental cultures were grown under conditions of nutritional deficit (culture medium supplemented with 5% fbs). this in vitro situation produces an environmental situation that is potentially similar to in vivo stress conditions during wound healing5. mitochondrial activity analysis was performed to infer cell viability and plot cell growth curves. all experiments performed with eight replicates. analysis of mitochondrial activity mitochondrial activity was analyzed using the mttbased cytotoxicity assay. the mtt assay involves the conversion of the water soluble 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (mtt) into an insoluble formazan. the formazan is then solubilized and the concentration is determined by optical density at @570 nm. for such, an mtt reduction analysis kit (vybrant mtt, molecular probes, eugene, or, usa) was used. immediately after the end of the assay procedures, absorbance was read in a microplate reader (biotrak ii, biochrom ltd, eugendorf, austria) using a 562 nm filter. the absorbance data was used to plot the cell growth curves. statistical analysis the optical density data corresponding to cell viability obtained in eight replicates are presented as mean ± sem. the data were compared using analysis of variance (anova) complemented with tukey’s test. the level of significance was 5% (p<0.05). wound healing evaluation (in vivo study) wistar rats (rattus novegicus albinus, rodentia; mammalian) weighing 250 to 300 g were used in this study. the animals were fed before and during the experimental period with solid chow and water ad libitum. this study was conducted under the approval of the local ethics committee and is in accordance with national guidelines for the care and use of experimental animals that are in compliance with the national institutes of health guide for the care and use of laboratory animals. traumatic ulcers were made on the tongue using a circular scalpel with 3 mm of diameter. then each animal received two daily applications of the medicine tested with swab at 12-h intervals throughout the experiment. after 1, 3, 5, 7 and 14 days, 5 animals from each experimental group were sacrificed by anesthetic overdose. after ether inhalation, the animals were anesthetized with administration of 1 ml/kg of 1% ketamine hydrochloride (dopalen, vetbrands, são paulo, sp, brazil and 2% xylazine hydrochloride (anasedan, vetbrands). the tongue was removed and immersed in a 10% buffered formalin solution for 48 h. after washing with water, the specimens were dehydrated and embedded in paraffin. the tissue specimens were sectioned into 5-ìm-thick slices and stained with hematoxylin-eosin for routine histological analysis. the medial section of the ulcers was selected analysis under a light microscope at a magnification of x40. clinical evaluation the state of the ulcer repair process was clinically analyzed after sacrifice of the rats. the status of the repair process was then classified as ‘repaired’ when no clinical signs of the ulcer were observed or ‘not repaired’, when clinical signs of the ulcer were observed. histological evaluation the evaluation of tissue responses was performed considering the microscopic aspects of the epithelium and connective tissue. for the epithelial tissue analysis, the width of the wound covered by epithelial cells was taken into account. for the connective tissue, the status of the inflammatory process (determined by the type and amount of inflammatory cells) and the presence or absence of fibrosis were analyzed. the histological evaluation was based on the degree of tissue healing using a 5-point scoring system ranging from grade 1 (total repair of the epithelium and connective tissue) to grade 5 (epithelium ulcer and acute inflammatory infiltrate). the microscopic characteristics of each grade were as follows: grade1: total epithelium repair with underlying connective tissue with fibrosis and absence or presence of few inflammatory cells; grade2: total epithelium repair with underlying connective tissue with fibrosis and moderate number of inflammatory cells, represented by macrophages, plasma cells and lymphocytes; grade3: presence of ulcer with two-thirds of its width covered by epithelium and the connective tissue showing mild fibrosis and moderate number of inflammatory cells, represented by macrophages, plasma cells and lymphocytes; grade4: presence of ulcer with one-third of its width covered by epithelium and the underlying connective tissue with a moderate degree of fibrosis and number of inflammatory cells represented by macrophages, plasma cells and lymphocytes; grade 5: less than one-third of epithelium width repaired and connective tissue with moderate or large number of inflammatory cells, represented by neutrophil granulocytes, macrophages, plasma and lymphocytes; presence of microabscesses. statistical analysis the data on the scores obtained in 5 replicates per group at each experimental time are presented as mean ± sem. the data were compared using the kruskal-wallis test, complemented with dunn’s test. the level of significance was set at 5% (p<0.05). results phytochemical analysis the results from phytochemical analysis are displayed on table 1. total polyphenols (tannins) in the leaves and hydroalcoholic extract was 11.91% (±0.85) and 8.53% (± 451451451451451 healing and cytotoxic effects of psidium guajava (myrtaceae) leaf extracts braz j oral sci. 9(4):449-454 active compound presence taninns + flavonoids + essential oil + saponnins + free antraquinones antraquinones glycosides cardiac glycosides alkaloids table 1. phytochemical analysis of the main groups of active compounds. 0.64), respectively. the amount of tannins and the phytochemical screening of psidium guajava leafs are in agreement with previous studies7-8,10-12 showing that the extraction process was adequate. in vitro analysis cell viability of the four experimental groups is displayed in figure 1. all experimental groups exhibited positive cell viability at 24 h. the cultures treated with guava had the least cell viability among all groups (p<0.05) throughout the entire experiment. regardless of the drug formulation, the cultures treated with corticosteroids had similar cell viability to that of the control cultures at 24 h. on days 3 and 5 the viability of cells treated with corticosteroids was also significantly lesser than that in the control group. fig. 1. cell viability (mtt assay) in the four experimental groups. same letters indicate statistically significant difference among groups. in vivo analysis the clinical evaluation revealed no detectable repair for the first five days in any group. on day 7, the guava group exhibited the highest percentage of repair of all groups. total repair occurred only on day 14. the corticosteroid groups achieved the worst tissue repair results throughout the entire experiment (figure 2). the histological analysis (figure 3) revealed that the animals treated with guava exhibited faster wound healing than the control and corticosteroid groups. on days 5 and 7, the guava group exhibited significantly better histological findings than corticosteroid groups comparable with grade 1 (e.g., total epithelium repair with underlying connective tissue with fibrosis and either the absence or presence of few inflammatory cells). the experimental groups treated with corticosteroids reached this stage of tissue repair only after 14 days of treatment. moreover, the wound healing in the corticosteroid groups was significantly slower than that of the control group (p<0.05). fig. 3. histological analysis (scores) of the four experimental groups. same letters indicate statistically significant difference among groups. fig. 2. clinical evaluation (percentage of repair) of the four experimental groups. discussion a wide range of therapies for promoting wound healing have been sugested, such as topical corticosteroids, antimicrobial agents, cyanoacrylate adhesives, chemical 452452452452452healing and cytotoxic effects of psidium guajava (myrtaceae) leaf extracts braz j oral sci. 9(4):449-454 cautery, phototherapy with low-power lasers and other antiinflammatory, immunosuppressive, and immunomodulatory agents13,14. topical corticosteroids applied 4 times a day constitute the main armamentarium in controlling the symptoms of ulcerous and inflammatory diseases. however, the long-term and/or repeated application of corticosteroids could cause adrenal suppression15. the therapeutic use of medicinal plants would be of importance to avoid the undesirable side effects of corticosteroid therapy5. in the quest for drugs capable of replacing the most commonly used corticosteroids for improving wound healing, the present study analyzed guava, which has been proven to maintain cell survival (as shown by the continued metabolic activity, i.e., the reduction of the tetrazolium salt by cells) and was able to enhance the healing of experimental wounds in rats. the different phases of the wound healing process overlap and, ideally, an herbal medication should affect at least two different processes before it can be said to have some scientific support for a traditional use16. after the initial rally of neutrophils to a wound site, several other types of cells are recruited to carry forward the repair processes. these include monocytes and, more importantly, fibroblasts, which are attracted into the site to initiate the proliferative phase of repair. an extract that stimulates the growth of fibroblasts can therefore be considered useful in helping a wound to heal. fibroblasts can be grown in a culture and their proliferation is assessed by the metabolism of a colorful substance, such as mtt5,16. in the present study, although the viability of treated cells (cultured medium conditioned by the guava formulation) was significantly lesser than in all other groups, viable cells were still present in the culture plates after 5 days of contact with the drug. this result is in agreement with previous cytotoxic tests on guava leaf extracts2,17-18. wound infection is a frequent occurrence in ulcerative lesions and the postoperative period, which could delay the wound healing process and lead to complications culminating in chronic non-healing wounds. a number of plant products, including those from guava, are known to have antibacterial activity, which enhances their medicinal value1,4,6,8-9,12,19-23. based on the reduction in the cell population when in contact with the guava extract and the fact that the histological analysis revealed no formation of abscess on the ulcer surface, the results of the present study indicate that guava has antibacterial properties. in contrast, the corticosteroid groups exhibited no antibacterial activity, considering the extensive abscess formation and proliferation of cells in contact with the conditioned media, as observed in a previous study on chamomile5. the clinical and histological analyses revealed that rats treated with the guava leaf extract experienced faster healing than that in the groups treated with corticosteroids, thereby demonstrating the effectiveness of guava leaf extract in wound healing. this positive influence of guava leaf extract on wound healing has previously been documented6,9 and may be explained by the fact that this plant contains many different substances, including tannins and flavonoids. data suggest that these compounds have antiinflammatory, astringent, antibacterial and antifungal properties11. natural products have provided and will continue to provide a unique element of molecular diversity and biological functionality in drug research and development. based on the obtained results and within the limitations of the study (i.e., number of animals evaluated), we suggest that psidium guajava is a phytotherapy that may contribute to tissue healing. references 1. holetz fb, pessini gl, sanches nr, cortez da, nakamura cv, filho bp. screening of some plants used in the brazilian folk medicine for the treatment of infectious diseases. mem inst oswaldo cruz. 2002; 97: 1027-1031. 2. kaileh m, berghe wv, boone e, essawi t, haegeman g. screening of indigenous palestinian medicinal plants for potential anti-inflammatory and cytotoxic activity. j ethnopharmacol. 2007; 113: 510-16. 3. aridogan bc, baydar h, kaya s, demirci m, ozbasar d, mumcu e. antimicrobial activity and chemical composition of some essential oils. arch pharm res. 2002; 25: 860-64. 4. gonçalves fa, andrade neto m, bezerra jns, macrae a, sousa ov, fonteles-filho aa, et al. antibacterial activity of guava, psidium guajava linnaeus, leaf extracts on diarrhea-causing enteric bacteria isolated from seabob shrimp. xiphopenaeus kroyeri (heller). rev inst med trop sao paulo. 2008; 50: 11-15. 5. martins md, marques mm, bussadori sk, martins ma, pavesi vc, mesquita-ferrari ra, et al. comparative analysis between chamomilla recutita and corticosteroids on wound healing. an in vitro and in vivo study. phytother res. 2009; 23: 274-8. 6. jaiarj p, khoohaswan p, wongkrajang y, peungvicha p, suriyawong p, saraya mls, et al. anticough and antimicrobial activities of psidium guajava linn. leaf extract. j ethnopharmacol. 1999; 67: 203-12. 7. lutterodt gd, maleque a. effects on mice locomotor activity of a narcoticlike principle from psidium guajava leaves. j ethnopharmacol. 1988; 24: 219-31. 8. gutiérrez rmp, mitchell s, solis rv. psidium guajava: a review of its traditional uses, phytochemistry and pharmacology. j ethnopharmacol. 2008; 117: 1-27. 9. chah kf, eze ca, emuelosi ce, esimone co. antibacterial and wound healing properties of methanolic extracts of some nigerian medicinal plants. j ethnopharmacol. 2006; 104: 164-7. 10. belemtougri rg, constantin b, cognard c, raymond g, sawadogo l. effects of two medicinal plants psidium guajava l. (myrtaceae) and diospyros mespiliformis l. (ebenaceae) leaf extracts on rat skeletal muscle cells in primary culture. j zhejiang univ sci b. 2006; 7: 56-63. 11. lutterodt gd. inhibition of gastrointestinal release of acetylcholine by quercetin as a possible mode of action of psidium guajava leaf extracts in the treatment of acute diarrhoeal disease. j ethnopharmacol. 1989; 25: 235-47. 1. 12. arima h, danno g. isolation of antimicrobial compounds from guava (psidium guajava l.) and their structural elucidation. biosci biotechnol biochem. 2002; 66: 1727-30. 12. cavalcanti bn, rode sm, marques mm. cytotoxicity of substances leached or dissolved from pulp capping materials. int endod j. 2005; 38: 505-9. 13. femiano f, lanza a, buonaiuto c, gombos f, nunziata m, piccolo s, et al. guidelines for diagnosis and management of aphthous stomatitis. pediatr infect dis j. 2007; 26: 728-32. 14. vena ga, cassano n, d’argento v, milani m. clobetasol propionate 0.05% in a novel foam formulation is safe and effective in the short-term 453453453453453 healing and cytotoxic effects of psidium guajava (myrtaceae) leaf extracts braz j oral sci. 9(4):449-454 treatment of patients with delayed pressure urticaria: a randomized, doubleblind, placebo-controlled trial. br j dermatol. 2006; 154: 353-6. 15. houghton pj, hylands pj, mensah ay, hensel a, deters am. in vitro tests and ethnopharmacological investigations: wound healing as an example. j ethnopharmacol. 2005; 100:100-7. 16. chen kc, hsieh cl, peng cc, hsieh-li hm, chiang hs, huang kd, et al. brain derived prostate cancer du-145 cells are effectively inhibited in vitro by guava leaf extracts. nutr cancer. 2007; 58: 93-106. 17. manosroi j, dhumtanom p, manosroi a. anti-proliferative activity of essential oil extracted from thai medicinal plants on kb and p388 cell lines. cancer lett. 2006; 235: 114-20. 18. gnan so, demello mt. inhibition of staphylococcus aureus by aqueous goiaba extracts. j ethnopharmacol. 1999; 68: 103-8. 19. vieira rhsf, rodrigues dp, gonçalves fa, menezes fgr, aragão js, sousa ov. microbicidal effect of medicinal plant extracts (psidium guajava linn. and carica papaya linn.) upon bacteria isolated from fish muscle and known to induce diarrhea in children. rev inst med trop sao paulo. 2001; 43: 145-8. 20. cáceres a, fletes l, aguilar l, ramirez o, figueroa l, taracena am, et al. plants used in guatemala for the treatment of gastrointestinal disorders. confirmation of activity against enterobacteria of 16 plants. j ethnopharmacol. 1993; 38: 31-8. 21. chomnawang mt, surassmo s, nukoolkarn vs, gritsanapan w. antimicrobial effects of thai medicinal plants against acne-inducing bacteria. j ethnopharmacol. 2005; 101: 330-3. 22. abdelrahima si, almagboulb az, omerb mea, elegami a. antimicrobial activity of psidium guajava l. fitoterapia. 2002; 73: 713-5. 454454454454454healing and cytotoxic effects of psidium guajava (myrtaceae) leaf extracts braz j oral sci. 9(4):449-454 oral sciences n3 braz j oral sci. 10(3):193-198 original article braz j oral sci. july | september 2011 volume 10, number 3 comparative clinical evaluation of chemomechanical caries removal agent papacarie® with conventional method among rural population in india in vivo study sanjeet singh1, deepti jawa singh2, shipra jaidka3, rani somani4 1senior lecturer, department of oral and maxillofacial pathology, d j college of dental sciences and research, modinagar (u.p) india 2senior lecturer, department of paedodontics and preventive dentistry, d j college of dental sciences and research, modinagar (u.p) india 3reader, department of paedodontics and preventive dentistry, d j college of dental sciences and research, modinagar (u.p) india 4professor, department of paedodontics and preventive dentistry, d j college of dental sciences and research, modinagar (u.p) india correspondence to: deepti jawa singh a-28 ,defence colony, mawana road, meerut 250001, uttar pradesh , india phone: 09720038877, 0121-2622435 e-mail: jawadeepti@rediffmail.com abstract the use of minimally invasive procedures and attention to patient comfort are of great importance, especially for dental treatment in young children. this has led to the development of chemomechanical methods for caries removal. aim: to evaluate and compare the antimicrobial efficacy, efficacy in terms of time consumption and pain perception of chemomechanical caries removal agent papacarie® and conventional method of caries removal. methods: subjects for this study were chosen from children admitted to dental clinic for restorative procedures. forty children (age 4-8 years) with early childhood caries were included in this study. two primary teeth with comparable degrees of carious destruction were chosen in each child for caries removal with either papacarie® or rotary instruments. the time taken for caries removal was measured using stopwatch. pain response during caries removal was evaluated using the wong baker face pain scale. dentin samples of both groups were taken prior to, and after caries removal for microbiological analysis. results the time taken for caries removal in chemomechanical caries removal method was three times longer than the conventional method. pain score during chemomechanical method of caries removal was 1.525 as compared with 6.65 when conventional method was used. the antimicrobial efficacy of chemomechanical caries removal was significantly similar to conventional method. conclusions: papacarie® can be an effective clinical alternative treatment for the removal of occlusal dentinal caries in cavitated primary molars. keywords: chemomechanical caries removal, papacarie®, dental caries. introduction painless dentistry and minimal intervention providing comfort, relief, solace and instillation of positive attitude towards dental treatments are some of the factors justifying the specialty of pediatric dentistry. despite the decline of its prevalence, caries continues to affect a significant portion of world population and treatment of the decay is still a challenge for researchers. in children, especially received for publication: september 23, 2010 accepted: june 16, 2011 194 braz j oral sci. 10(3):193-198 those with dental anxiety, caries removal by means of conventional instruments is considered an unpleasant step of the restorative process mainly because of pain, drilling and noise1. furthermore, drilling results in rapid and excessive removal of tooth structure and may cause harmful thermal and pressure effects to the pulp2. these disadvantages of conventional method had led to a more gentle, comfortable and conservative caries excavation method aimed at providing minimal thermal changes, less vibration and less pain, and removal of infected dentine only. with an advancing era of science, much superior technique of removing dental caries by means of chemomechanical agents was first introduced in 1975 by habib et al.3 by using 5% sodium hypochlorite, which is a non specific proteolytic agent. as sodium hypochlorite was found eventually too corrosive to be used in healthy tissue, goldman et al.4 made an attempt to minimize the problem by introducing gk-101 for removal of dental caries in 19764. it was fda approved for use in usa in 1984 and was marketed in 1985 by the name of caridex system5. despite its effectiveness, caridex had certain limitations like long working time, short shelf life and requirement of large volume of solutions along with a special pump6. rolf bornstein et al in mid 1990’s introduced carisolv as a successor to caridex7-8.carisolv was quite a success in the field of dentistry but with its long use certain drawbacks of the system has been reported which includes requirement of customized instruments that increased the cost of solution. in 2003, a research project in brazil conducted by bassadori et al.9 led to the development of a new formula to universalize the use of the chemomechanical method for caries removal, which was launched for use in public health in 2005 under the brand name papacarie7. papain gel, the basic component of this product, is responsible for its bactericidal, bacteriostatic and inflammatory characteristics. various in vivo and in vitro studies have been done using different chemomechanical caries removal agents namely 5% sodium hypochlorite, gk101, caridex, carisolv, but literature on the efficacy of chemomechanical caries removal using papain gel is scanty3-8,10. thus the need to evaluate the recent material especially in young children arose. the aims of the present in vivo study were to compare the time taken in caries removal by papacarie ® as a chemomechanical caries removal agent with conventional method; to compare the pain response associated with the chemomechanical and conventional methods during caries removal; to evaluate and compare the antimicrobial efficacy of both methods. material and methods the present in vivo study was carried out at the department of pedodontics and preventive dentistry at d.j. college of dental sciences and research, modinagar (distt. gaziabad, state uttar pradesh, india), involving 40 healthy children aged 4-8 years. two contralateral cavitated primary molars, with occlusal caries having approximately equal-sized cavity openings (diameter 1.5-2 mm measured with a metallic caliper) with brown and softened dentine and having defects with comparable depths (less than 1.5-2 mm measured with a who periodontal probe) were chosen for the study. the patients selected were fully cooperative as judged by the frankle shiere and fogel four point behavior rating scale11. parents/guardians responsible for each child were fully informed of the details of the study, and asked to sign a consent form authorizing their child’s participation in the study in agreement with the ethical principles of the dj dental iec declaration with reference number djd/iec/05. thus, the 80 contralateral primary molars from the 40 children were divided equally for the conventional and chemomechanical methods of caries removal. in each child, one tooth was randomly selected to be treated with either papacarie® or the other conventional method. since both molars in each child were exposed to a similar oral environment, hence this study was more suitable to compare the two treatment modalities. both chemomechanical and conventional methods of caries removal were carried out under rubber dam isolation (figure 1) in order to obtain moisture control and avoid microbial contamination. the first sample from the superficial carious lesion from both cavities was removed with the help of a sterile spoon excavator and transferred to a sterile vial containing 20 ml of bhi broth for microbiological evaluation (figure 2). caries removal by chemomechanical method (figure 3) according to the manufacturer’s instructions, the syringe containing papacarie® was removed from the refrigerator 30 min before treatment. papacarie® was applied with the help of an applicator tip into the cavity and left for 30-40 s. the softened dentine tissue was removed using the excavator in a pendulum motion in a pressureless manner. the remaining gel was removed with cotton pellet soaked in saline. this procedure was repeated as many times as necessary, until the darkish color of the gel was revealed. the cavity was not washed or rinsed between the gel applications. the cavities were considered caries-free when there was no change in the color of papacarie® gel. fig. 1 rubber dam placement. comparative clinical evaluation of chemomechanical caries removal agent papacarie® with conventional method among rural population in india in vivo study braz j oral sci. 10(3):193-198 fig. 2 pretreatment collection of sample for microbiological evaluation fig. 3 caries removal using the chemomechanical caries removal agent papacarie® caries removal by the conventional method conventional caries removal was carried out using a sterile no. 16 straight bur on a contra-angle micromotor handpiece at slow and intermittent speed, without water spray (figure 4). after caries removal, dentine was considered cariesfree, using established ericksons clinical (optical and tactile) criteria7.the second sample from both cavities was then taken from the cavity floor with a sterile spoon excavator and transferred to another sterile vial containing 20 ml of bhi broth for microbiological eval-uation. the preparation time for each caries-removal technique fig. 4 caries removal using the conventional method was determined using a stopwatch. the time taken for the experimental group was calculated from the beginning of gel application until the end of the caries removal procedure after the removal of caries was completed, the wong baker faces pain scale12 was used to evaluate whether the child felt any pain during the procedure and accordingly the pain scores was given to them for both the methods used for caries removal separately. microbial cultivation and evaluation the dentin samples of both groups were processed in the microbiological laboratory within 1 hour of collection. each sample was vortexed for about 30 s in order to dislodge the bacteria from the dentin. a sterile loop full of sample was collected and was cultured with aseptic technique onto 10% blood agar plates by streaking method. the plates were incubated at 37ºc in 5% co 2 atmosphere in candle jar for 48 h. thereafter, the plates were observed for the growth of alpha hemolytic streptococci (figure 5). the alpha hemolytic green colored colonies from the primary plates were picked up with a sterile loop and were subcultured onto another blood agar plate for examination of colony characteristics and identification of viridans streptococci. during isolation, a disc of optochin was placed in the primary inoculum to exclude s. pneumoniae (figure 6). these plates were also incubated at 37ºc in 5%co 2 atmosphere in candle jar. after overnight incubation, the plates were observed for colony characteristics using magnifying lens. the plates were further evaluated using compound light microscope. the colonies showing convex appearance were identified as viridans streptococci. further gram staining was done to identify the streptococci in chains and the plates were divided into four quadrants and bacterial count was done using a magnifying lens. the total viable bacterial count was determined and expressed as number of bacteria per ml of medium. because of the wide range of total numbers, 5 classes were defined for the total viable count: 0: no growth; 1: < 103; 2: 1001-104; 3: 10001-105(discrete growth); 4: uncountable (confluent growth). fig. 5 alfa hemolytic colonies on blood agar plate (preoperatively) 195comparative clinical evaluation of chemomechanical caries removal agent papacarie® with conventional method among rural population in india in vivo study 196 braz j oral sci. 10(3):193-198 fig. 6 alpha hemolytic colonies on blood agar plate growing around optochin disc, confirming the presence of streptococcus viridians. data were analyzed statistically using the z-test for time and pain assessment at 1% level of significance and paired t test for microbiological evaluation at 1% level of significance. results in the present study, 80 primary molars obtained from 40 children aged 4 to 8 years were evaluated in terms of time spent for caries removal, pain response and microbiological assessment. caries removal method conventional (rotary instruments) chemomechanical (papacárie®) number of teeth 40 40 mean value of pain score 6,65 1,525 s.d. 1.888562 1.35847 s.e. 0.2986 0.2148 minimum value 4 0 maximum value 10 4 z value 13,92 13,92 p value p<0.01 table 2 mean value of pain scores for the chemomechanical and conventional methods of caries removal caries removal method conventional (rotary instruments) chemomechanical (papacárie®) number of teeth 40 40 mean time (in s) 124.6 328.5 s.d. 22.76502 45.2656 s.e. 3.5994 7.1571 minimum time (in s) 78 230 maximum time (in s) 169 430 z value 25.45 25.45 p value p<0.01 table 1 mean value of time taken for caries removal in both groups. number of teeth mean total viable bacterial count /ml s.d. t-cal p value pretreatment 80 3.575 0.635993 20.38 p<0.001 posttreatment (chemomechanical method) 40 0.675 0.693837 20.38 p<0.001 posttreatment (conventional method) 40 0.425 0.500641 26.34 p<0.001 table 3 total viable bacterial count after use of the chemomechanical and conventional methods of caries removal time consumption and pain perception table 1 shows that the mean time required for caries removal with the chemomechanical method (328.5 ± 45.26 s) was 193.9 s longer than the time spent with the conventional method (124.6 ± 22.76 s) (p<0.01). table 2 shows that the mean value of pain score using the conventional method was significantly higher (6.65 ± 1.888) compared with the chemomechanical method (1.525 ± 1.35847) (p<0.01). microbial count table 3 shows the total viable bacterial count before and after caries removal by both methods. the mean total viable count of s. viridians was 3.575 bacteria/ml before caries removal. the mean total viable streptococcus count was reduced to 0.675 after caries removal with the chemomechanical agent and 0.425 with conventional method (table 3). the difference between the pretreatment sample score and posttreatment sample score was found to be statistically significant in both groups (p<0.01). this corresponds to a mean reduction of total viable count of 87.94% and 81.12% for the conventional and chemomechanical methods, respectively (table 4). the results also indicated that both methods of caries removal had an almost similar percentage of reduction of bacterial count when analyzed statistically. discussion dental caries is still one of the most common diseases affecting the human population. it is one of the main etiological factors of dental pain, so its treatment is not only required, but also demanded since the inception of mankind. the development of caries removal techniques in mean % reduction total viable bacterial count /ml t-value p value conventional method 87.94% 1.082 p >0.001 chemomechanical method 81.12% 1.082 p >0.001 table 4 mean reduction in total viable bacterial count after use of the chemomechanical and conventional methods of caries removal. comparative clinical evaluation of chemomechanical caries removal agent papacarie® with conventional method among rural population in india in vivo study 197 braz j oral sci. 10(3):193-198 restorative dentistry is progressing towards a more biological and conservative direction. chemomechanical caries removal became an area of interest in dental research due to its concept of sound tissue preservation. as only carious dentin is removed, the painful removal of sound dentin is avoided and hence, the need for local anesthesia is minimized. the latest production of chemomechanical caries removal papacarie® has been developed in brazil in order to overcome the clinical limitations of other products. the present study was performed to assess the efficacy of this new product to remove carious lesion in primary teeth. as the microflora is one of the main etiological factors in caries occurrence, it is essential to reduce the microbial counts in carious lesions. with regard to oral microflora, evidence has shown that acidogenic species such as viridians streptococci (s. mutans and s. sobrinus) are strictly associated with the onset and presence of dental caries13. mutans streptococci are mainly implicated with the initiation of enamel caries and gradually increase with the completion of the primary dentition and presence of proximal contacts between primary molars. hence, in the present study, the efficacy of the chemomechanical method was assessed by evaluating its antimicrobial efficacy. papain, the main ingredient of papacarie®, is an enzyme similar to human pepsin, used in food technology and pharmaceutical and cosmetic industries. guzman and guzman14 performed clinical studies on patients with skin lesions caused by burns, observing that the enzymatic action of papain was considered excellent in areas with necrotic and purulent processes. udok and storojuk15 also verified that papain aided cleansing necrotic tissue and secretions, shortening the period of tissue repair. flindt16 demonstrated that papain acts only in infected tissues because infected tissues lack plasmatic anti protease called al anti trypsin, this is only present in sound tissues and it inhibit protein digestion16. the absence of al anti trypsin enzyme in infected tissues allows papain to break the partially degraded molecules. dawkins et al.17 showed that papain has bactericidal and bacteriostatic properties which inhibit the growth of gram positive and gram negative organism. in addition to papain, papacarie ® also contains chloramines, toluidine blue, salts and thickening vehicle. chloramine has bactericidal and disinfectant properties. the antiseptic properties of chloramine were recently documented in an in vitro study18. these are broadly used to chemically soften the carious dentine. according to maragakis et al.19, the partially degraded collagen in carious dentine was chlorinated by chemomechanical caries removal solutions. the chlorination affects the secondary or quaternary structure of collagen, by disrupting hydrogen bonding and thus facilitating the carious tissue removal. an in vitro evaluation of papacarie® cytotoxicity using different concentrations of papain (2, 4, 6, 8, and 10%) on fibroblast culture found not cytotoxic effects, suggesting that papacarie® is safe to use in pediatric patients9. in the present study, the efficacy of papacarie® in terms of time taken for caries and pain perception along with its comparison with the conventional method was evaluated. it was observed that chemomechanical caries removal was approximately 3.25 min longer in removing caries, which was statistically highly significant. this result is consistent with those of jawa d et al. and bassadori et al.9 it has been stated that papacarie® requires more than one application for its action to work20. carrillo et al. 21 reported that the chemomechanical removal of carious tissue using papacarie® had treatment duration of 8 min per tooth. the longer treatment time in that study was due to the evaluation done on special needs children. in the present study, the analysis of pain perception during caries removal according to the overall rating by patients revealed a higher comfort level with the chemomechanical method compared with the conventional method. these findings are in accordance with those of silva et al.15, who demonstrated that caries removal using papacarie® is significantly less painful in comparison with the conventional method. the wide difference in the pain scores between papacarie® and conventional method could be because papacarie® acts only on the dead infected cells and does not damage the healthy tissues. anusavice and kincheloe22 demonstrated that removing carious dentin generally elicits little or no painful sensation, while removing sound dentin often results in some level of pain. clinically, in general practice, soft and presumably irreversibly destroyed dentine is removed prior to restoring the cavity. the clinical criteria for complete caries removal differ around the world. over the years, several investigators have defined caries-free dentin and the number of microorganisms that can be left in the cavity that will not promote further disease progressl1. when excavating a lesion, the bulk of microorganisms are removed along with most of the necrotic dentin. this does not render the prepared cavity bacteria-free, and the rationale behind removal of carious dentin is still uncertain and based on rather blunt clinical criteria. banerjee et al.1 reviewed this problem and concluded that ‘it is not possible to remove all infected dentin. in the present study, the mean total viable count after caries removal was reduced to 0.675 score per ml from 3.525 using chemomechanical caries removal (papacarie®) and 0.425 score per ml with conventional drilling which mean less than 103 cfu /ml .these reductions were highly significant. kneist and heinrich-wetzien23, azrak et al. 24 and subramaniam et al.25showed that the mean total viable count was significantly reduced after caries removal using a chemomechanical caries removal agent (carisolv™) and conventional drilling, which is in accordance with the findings of the present study. although complete caries removal was achieved by both methods, jawa et al.20 observed under light microscopy that there was less marked destruction of dentinal tubules when caries was removed with papacarie®. in a scanning electron microscopic study, bassadori et al.9 observed conventional removal of carious tissue using diamond and/or stainless steel burs in permanent teeth with dentin caries left a residual smear layer, whereas the use of papacarie® resulted in more comparative clinical evaluation of chemomechanical caries removal agent papacarie® with conventional method among rural population in india in vivo study 198 braz j oral sci. 10(3):193-198 preservation of dentin structure and bacterial removal9,20. the results of this study indicated that papacarie® is efficient in caries removal from open and accessible occlusal lesions. this finding is in agreement with those of bussadori et al.9, kotb et al.26. papacarie®, a virtually painless, noninvasive technique of caries removal was proven efficient, easy to perform, inexpensive and comfortable to the patient. thus, its use can be recommended for caries removal in patients seeking an alternative to conventional treatment. references 1. banerjee a, watson tf, kidd eam. dentine caries excavation: a review of current clinical techniques. br dent j. 2000; 188: 476-82. 2. willershausen b, azrak b, wilms s. fear of dental treatment and its possible effects on oral health. eur j med res. 1999; 4: 72-7. 3. habib cm, kronman j, goldman m. a chemical evaluation of collagen and hydroxyproline after treatment with gk-101 (n-chloroglycine). pharmacol ther dent. 1975; 2: 209-15. 4. goldman m,kronam jh .a preliminary report on a chemomechanical means of caries removal. j am dent assoc. 1976; 93: 1149-53. 5. schutzbank sg, galaini j, kronman jh, goldman m, clarke rea. a comparative in vitro study of the effect of gk-101 and gk-101e in caries removal. j dent res. 1978; 57: 861-4. 6. beeley ja, yip hk, stevenson ag. chemomechanical caries removal: a review of techniques and latest developments. br dent j. 2000; 188: 427-30 7. ericson d, zimmerman m, raber h, götrick b, bornstein r, thorell j. clinical evaluation of efficacy and safety of a new method for chemo-mechanical removal of caries. a multicentre study. caries res. 1999; 33: 171-7. 8. bornstein r, wedenberg c. pulpal reactions in rat incisors to caridex. aust dent j. 1990; 35: 505-8. 9. bussadori sk, castro. lc, galvao ac. papain gel: a new chemo¬mechanical caries removal agent. j clin pediatr dent. 2005; 30: 115-19. 10. burke f m, lynch e. the effect of chemomechanical caries removal on the bond strength of glass polyalkenoate cement to dentine. j dent. 1994: 22: 283-91. 11. frankle sn, shiere fr, fogel hr. should the parent remain with the child in the dental operatory. j dent child. 1962; 29: 150-4. 12. wong dl, hockenberry-eaton m, wilson d, winkelstein ml, schwartz p. wong’s essentials of pediatric nursing. 6 ed. saint louis: mosby; 2001. 13. roeters fj, van der hoeven js, burgersdijk rc, schaeken mj. lactobacilli, mutans streptococci and dental caries: a longitudi¬nal study in 2 year old children up to the age of 5 years. caries res. 1995; 29: 272-9. 14. guzman ay, guzman mgs. the enzymatic debridement of suppura¬tions, necrotic lesions and burns with papain. j int coil surg, 1953, 20: 695-702. 15. . udok vm, storojuk vt. use of papain in trating suppurative postoper¬ative soft tissue complications and daises. khirurgiia.1981; 19: 99-101. 16. flindt m. health and safety aspects of working with enzymes. process biochem. 1979; 13: 3-7. 17. dawkins g ,hewitt h,wint y obiefuna p h,wint b. antimicrobial effects of carica papaya fruit on common wound organisms. west indian med j. 2003; 52: 290-2. 18. fuursted k, hjort a, knudsen l. evaluation of bactericidal activity and lag of regrowth (post antibiotic effect) of five antiseptics on nine bacterial pathogens antimicrob chemother. 1997; 40: 221-6. 19. margakis gm, hahn p, hellwig e. clinical evaluation by patients of chemomechanical varies removal in primary molars and its acceptance by patients. caries res. 2001; 35: 205-10. 20. jawa d, singh s, somani r, jaidka s, sirkar k, jaidka r comparative evaluation of efficacy of chemomechanical caries removal agent papacarie and conventional method of caries removal: an in vitro study. j ind soc pedod prev dent. 2010; 28: 73-7. 21. carrillo cm, tanaka mh, cesar mf, camargo ma, julianoy, novo nf. use of papain gel in disabled patients. j dent child. 2008; 75: 222-8. 22. anusavice kj, kincheloe je comparison of pain associated with mechanical and chemomechanical removal of caries j dent res. 1987; 66: 1680-3. 23. kneist s, heinrich-weltzien r: antibacterial action of carisolv. in: albrektsson t, bratthall d, glantz p, lindhe j, editors). tissue preservation in caries treatment. london: quintessence; 2001. p.205-19. 24. azrak b, callaway a, grundheber a, stender e comparison of the efficacy of chemomechanical caries removal (carisolv) with that of conventional excavation in reducing the cariogenic flora. int j paediatr dent. 2004; 14: 182-91. 25. subramaniam p, babu kl, neeraja g. comparison of the antimicrobial efficacy of chemomechanical caries removal (carisolv) with that of conventional drilling in reducing cariogenic flora. j clin pediatr dent. 2008; 32: 215-9. 26. kotb rm, abdella aa, el kateb ma, ahmed am. clinical evaluation of papacarie in primary teeth. j clin pediatr dent. 2009; 34: 117-23. comparative clinical evaluation of chemomechanical caries removal agent papacarie® with conventional method among rural population in india in vivo study oral sciences n3 braz j oral sci. 9(1):20-24 original article braz j oral sci. january/march 2010 volume 9, number 1 erosive effect of an antihistamine liquid formulation on bovine teeth: influence of exposure time viviane santos da silva pierro1, beatriz romeiro furtado2 , michele villardi3, lúcio mendes cabral4, eduardo moreira silva5, lucianne cople maia6 correspondence to: lucianne cople maia rua gastão gonçalves, 47/501 santa rosa 24.240-030 niterói, rj, brazil e-mail: rorefa@terra.com.br received for publication: october 13, 2009 accepted: march 25, 2010 abstract aim: to evaluate, in vitro, the effect of an oral antihistamine liquid formulation on roughness and topography of bovine enamel and the influence of exposure time on its erosive effect. methods: forty-one bovine enamel blocks were prepared leaving an exposed window of 0.8 mm2. thirtynine blocks were divided into three treatment groups according to media immersion: antihistamine formulation (histamin®), 0.6% citric acid (positive control), and distilled water (negative control). before immersion of the samples, ph, titratable acidity, calcium, phosphate and fluoride contents of all media were verified. enamel roughness was evaluated at baseline, and after 5, 15, and 30 min of immersion (9 samples per group). two specimens from each group and exposure time, and 2 additional specimens representing baseline, were analyzed by scanning electron microscopy (sem). data were analyzed by the kruskal-wallis test, and the mann-whitney test using the bonferroni correction (α=0.017). results: specimens immersed in citric acid showed the highest roughness (p<.001). sem images showed a progressive erosion pattern in samples immersed in citric acid and in antihistamine formulation. conclusions: the antihistamine liquid formulation did not promote significant alterations of enamel roughness. nevertheless, sem demonstrated that the antihistamine eroded bovine enamel, and the erosion pattern was influenced by exposure time. keywords: tooth erosion, pharmaceutical preparations, hydrogen-ion concentration, child. introduction dental erosion is the irreversible loss of dental hard tissue due to a chemical process of acid dissolution that does not involve bacterial plaque acid, and is not directly associated with mechanical or traumatic factors1. the etiology of erosion is related to different behavioral, biological and chemical factors and it can have extrinsic or intrinsic causes. extrinsic erosion is the result of exogenous acids, and dietary acids are, undoubtedly, its main causative factors. the most frequently consumed potentially damaging beverages are fruit juices and soft drinks2. however, several cases of tooth erosion have been attributed to oral administration of medicines3. medicines usually have low endogenous ph, high titratable acidity, and absence or low concentrations of ions including those of calcium, fluoride, and phosphate in their composition. the risk of dental erosion is increased when medications are used for treatment of chronic diseases with a high frequency of 1dds, ms, graduate student, department of pediatric dentistry and orthodontics, dental school, federal university of rio de janeiro, brazil 2dds, undergraduate student, department of pediatric dentistry and orthodontics, dental school, federal university of rio de janeiro, brazil 3dds, undergraduate student, department of medicaments, pharmacy school, federal university of rio de janeiro, brazil 4msc, phd, pharmacist, associate professor, department of medicaments, pharmacy school, federal university of rio de janeiro, brazil 5dds, ms, phd, associate professor, department of restorative dentistry, dental school, federal fluminense university, brazil 6dds, ms, phd, associate professor, department of pediatric dentistry and orthodontics, school of dentistry, federal university of rio de janeiro, brazil 21 ingestion (two or more times per day), at bedtime, or when they have side effects such as reduction of salivary flow rate, which happens with antihistamines4-6. although the contact of a solution with the teeth during intake is usually not as long as when the liquid is rinsed3, at least one of the daily medication doses are designated to be given at bedtime. additionally, liquid medicines usually take a longer time to clear from the mouth compared to tablets and capsules7. the ph of liquid oral medicines may be formulated to optimize efficacy and patient acceptability. the solubility of weak acids and bases is ph dependent, and acidic preparations are often necessary for drug dispersion. additionally, these acidic medicines often taste pleasanter, which may enhance patient compliance, especially children8. previous studies have already pointed out the erosive potential of liquid medicines or soluble tablets4-5,8-12, but few of them evaluated the effect of oral medication on tooth surface4,9-10,12, and only one study investigated the effect of exposure time on the development of dental erosion 9. therefore, this study aimed to evaluate, in vitro, the effect of an oral antihistamine liquid formulation on roughness and topography of bovine enamel and the influence of exposure time on its erosive effect, if any. material and methods medicine selection, ph analysis, titratable acidity and mineral content of the tested media the most widely distributed antihistamine liquid formulation (histamin, neo química, anápolis, go, brazil) by the pharmacies of public pediatric hospitals and other healthcare services in the city of rio de janeiro, brazil, was chosen for this study because of its possible impact on the oral health of children who usually seek for medical assistance at these facilities. negative (distilled water) and positive (0.6% citric acid) controls were also tested. all the 3 tested media were analyzed in triplicate with regard to ph, titratable acidity, calcium, phosphate and fluoride contents. the ph was measured with a calibrated ph meter (quimis q-400mt, diadema, sp, brazil), which was also used to determine titratable acidity by adding increasing quantities of 0.1 n sodium hydroxide (naoh) solution to 25 ml of the medicine and controls, followed by agitation and equilibration (2 min) for further ph measurements. a correction factor of 0.95 was obtained by factorizing 0.1 n naoh solution with potassium biphthalate standard. the total volume of 0.1 n naoh solution required to neutralize medicines and controls (raise their ph to 7.0) multiplied by the correction factor of 0.95 corresponded to the titratable acidity value. all the media were used in an undiluted form. phosphate and fluoride contents of the antihistamine formulation and controls were analyzed by ion chromatography13, while calcium content was determined by flame atomic absorption spectrophotometry14. preparation of bovine enamel specimens forty-one enamel blocks (3 x 3 x 3 mm) were prepared from bovine incisors stored in a 2% formaldehyde solution (ph 7.0) at room temperature. crowns were sectioned from the roots and enamel blocks were obtained with water-cooled diamond double-faced disk at low-speed. all enamel blocks were then embedded in acrylic resin using pvc rings as moulds with the labial surfaces facing toward the ring base. after resin acrylic polymerization, the labial enamel of samples were wet ground using 600, 800 and 1200-grit abrasive discs (3m, sumaré, brazil) for 10 min each and polished using felt discs (arotec ind.& com. ltda; são paulo, brazil) and 1 and 0.3 µm aluminum oxide suspension (south bay technology inc.; san clemente, usa) in a water-cooled grinding machine (panambra dpu-10, struers; copenhagen, denmark) to produce a flat surface. after the polishing procedure, samples were cleaned with an ultrasonic device and viewed under an optical microscope (aus jena, model 444181, with a 40 objective; astro optics division, montpelier, usa) in order to confirm that the surfaces were flat, polished, and free of irregularities that could interfere with roughness evaluation. red nail polish (two coats, 24 h drying) was then placed over the enamel blocks, except for a 0.8 mm2 window of exposed enamel. treatment groups thirty-nine specimens were randomly divided into three treatment groups, as follows: antihistamine liquid formulation (histamin®, neo química, anápolis, go, brazil), 0.6% citric acid (crystal pharm, niterói, rj, brazil) – positive control, and distilled water – negative control. the specimens were immersed in 100 ml of each solution for 5, 15, and 30 min at 37 °c and maintained upon agitation during the experiment. two additional specimens were set aside and kept in humid environment at 37°c to represent baseline on further sem analysis. roughness evaluation all measurements of enamel loss were made using a surface roughness tester (surftest sj 201, mitutoyo co., kawasaki, japan) to determine surface roughness (r a – µm) due to media exposure. before roughness measurements, all specimens were ultrasonicated both at baseline and after the experimental periods. three roughness measurements spaced at 60° were recorded for each specimen (cut-off length of 0.25 mm) at baseline and after each treatment period (5, 15 and 30 min). therefore, a mean roughness value was obtained for each sample according to treatment and exposure time (n=9), for both experimental and control groups. scanning electronic microscopy (sem) analysis sem analysis was performed to assess the topography of enamel surface at baseline and after each treatment period for the three tested media. two enamel specimens from each treatment group according to exposure time were prepared for sem analysis. the two specimens were mounted on aluminum stubs, fully-dried, sputter-coated with a thin layer braz j oral sci.9(1):20-24 erosive effect of an antihistamine liquid formulation on bovine teeth: influence of exposure time 22 fig. 1 – mean roughness values of bovine enamel specimens according to media and exposure time of gold-palladium and examined with a jeol jsm-35 scanning electronic microscope (tokyo, japan) with an acceleration voltage of 15kv. sem micrographs were made at 500x and 2000x magnification. statistical analysis statistical analysis was performed using spss software version 11.0 (spss inc., chicago, usa). after verifying that the roughness data presented a non-normal distribution for all treatment groups, kruskal-wallis test was used for comparison among the three treatment groups, and then the mann-whitney test using bonferroni correction (α=.017) was employed for group comparisons. results the chemical parameters of the media tested in the present study are shown in table 1. the results of surface roughness are shown in figure 1 and table 2. kruskal-wallis test detected significant difference for media (p<0.001). mann-whitney test with bonferroni correction showed that specimens immersed in citric acid presented the highest roughness values (p<.001) at all time periods, followed by the antihistamine liquid formulation and distilled water, without significance between them after the 5-min (p=.66), 15-min (p=.60) and 30-min (p=.66) exposures. qualitative analysis of sem micrographs showed that specimens immersed in citric acid presented the most severe erosion pattern with time (figure 2), followed by those immersed in antihistamine liquid formulation (figure 3). it seemed that specimens immersed in water did not suffer erosion irrespective of exposure time (figure 4). discussion erosive damage to the permanent teeth in early childhood may compromise the dentition for the child’s entire lifetime and this will certainly require expensive dental treatments in adult life15. however, individual susceptibility should be taken into account due to structural variations in enamel. bovine teeth were used in the present study based on a morphological investigation on progression of enamel erosion, and it has been shown that the surface ultrastructure of erosive lesions in prismatic human enamel did not differ from that observed in bovine tooth specimens16. previous in vitro studies have already shown that oral fig. 2 – representative sem micrographs of bovine enamel at baseline (a) and after immersion in 0.6% citric acid (positive control) for 5 min (b), 15 min (c) and 30 min (d). note extensive eroded areas at higher magnification (x2000) (e). fig. 3 – representative sem micrographs of bovine enamel at baseline (a) and after immersion in antihistamine liquid formulation (histamin®) for 5 min (b), 15 min (c) and 30 min (d). note the presence of eroded areas at higher magnification (x2000) (e). fig. 4 – representative sem micrographs of bovine enamel at baseline (a) and after immersion in distilled water (negative control) for 5 min (b), 15 min (c) and 30 min (d). note absence of eroded areas at higher magnification (x2000) (e). braz j oral sci. 9(1):20-24 erosive effect of an antihistamine liquid formulation on bovine teeth: influence of exposure time characteristics antihistamine formulation positive control negative control commercial name histamin® citric acid distilled water pharmaceutical name dexchlorpheniramine citric acid distilled water batch number 75974 ___ ___ expiration date 05/2009 02/12/2008 ___ ph 5.1 2.0 5.6 titratable acidity (volume of 0.1 n naoh – ml) 1.6 19 0.1 calcium (mg/l) 1.2 1.4 0.1 phosphate (mg/l) < 0.1 3.61 < 0.1 fluoride (mg/l) 0.99 0.23 < 0.1 table 1 – chemical parameters of the antihistamine formulation and controls tested in the present study 23 medicines could be related to dental erosion 4-5,8-12. additionally, johansson et al.17, in an in vivo investigation with saudi children, pointed out that high intake of acidic drinks and fruits, upper respiratory tract problems and frequent medications may constitute possible etiological and/or aggravating factors for severe dental erosion. the medicine selected for this study is an antihistamine liquid formulation extensively handled in public pediatric hospitals and healthcare services of rio de janeiro city and, therefore, consumed by a significant number of children that do not have access to private medical assistance. this antihistamine liquid formulation, like others, is most commonly used for treatment of respiratory allergies, which are usually present as a chronic health problem. as this medication is used chronically by allergic children, it is important to study its chemical properties to verify their role in children’s dental health. factors mostly related with the ingested substances per se could modify erosion patterns. ph and dissociation constants of the acids allied to the general chemical composition of the solutions may modify the degree of enamel dissolution16. in the present study, the specimens immersed in citric acid (positive control), presented remarkable changes in enamel surfaces than those immersed in antihistamine liquid formulation and distilled water (figures 2, 3 and 4). at a first glance, this finding could be related to the low ph of this acid (table 1). however, it is well-known that the ph gives only the initial concentration of h+ ions, and does not represent the presence of undissociated acid in the medium15,18-20. valinoti et al.21 using two acidic medicines with ph close to the same value (claritin/antihistamine – 2.57 and dimetapp/elixir – 2.51) showed, under sem, that claritin/antihistamine caused more surface alterations on some resin-based composites. according to these authors, the dissimilar results of these medicines were due to the differences in the titratable acidity between the medicines, 41.83 and 36.31 ml naoh, respectively. based on this, it is clear that the higher titratable acidity of citric acid in the present study have contributed more than ph to the changes produced in the enamel surfaces. in fact, titratable acidity can be claimed as a more accurate measure of the total acid content present in substances, and calcium-chelating properties may greatly enhance the erosive potential of beverages15,18-20. the studied antihistamine liquid formulation presented a ph slightly lower than distilled water, but it showed higher buffering capacity and ability to resist ph changes than distilled water because of its amount of neutralizing agent was tenfold greater than that of water. this probably explains the different patterns of erosion found in sem images, that is, eroded areas were present in antihistamine (ph 5.1; 1.6 ml naoh) treated enamel specimens, while no erosive pattern was verified in the samples treated with distilled water (ph 5.6; 0.1 ml naoh). ions like calcium, phosphate and fluoride have a protective effect against erosion15. however, despite the presence of fluoride and calcium in the antihistamine liquid formulation, and of fluoride, calcium and phosphate in citric acid (table 1), their ionic concentrations were not high enough to prevent the erosive effect viewed in the enamel surfaces (figures 2 and 3) on teeth treated with those substances. here, again, it seems obvious that the high titratable acidity overcame the probable positive effects of ions f3-, ca++ and po 4 3on preventing enamel erosion. the etiology of dental erosion can also be influenced by biological and behavioral factors, unusual eating, drinking and swallowing habits, for example holding an acid beverage in the mouth before swallowing, increase the contact time of an acid substance with the teeth and thus increase the risk of erosion15. furthermore, it should be emphasized that some oral medicines are usually given at bedtime without subsequent toothbrushing or water rinse, and oral clearance is usually also compromised during sleep. therefore, it seems reasonable to evaluate how exposure time could influence erosive dental patterns. the choice for 5-, 15and 30-min exposure times were intentionally proposed because they seem to be less aggressive than the time intervals proposed by babu et al.9. on this matter, the sem results of the present study differed from those of babu et al.9 because our images showed that severity of erosion’s increased with exposure time for both the positive control and the tested antihistamine liquid formulation, while those authors found more severe lesions after a 10-min than an 8-hour immersion period. this could probably be explained by the great difference between these time periods, which could have been enough to promote saturation of the liquid medium. quantitative methods are usually employed for determination of erosion severity along with sem evaluation15,22 and roughness analysis has already been used to evaluate the effect of acidic medicines, under ph-cycling conditions, on the surface degradation of composite resins21. however, in this study, roughness measurement was not sensitive enough to detect the alterations shown by sem braz j oral sci. 9(1):20-24 erosive effect of an antihistamine liquid formulation on bovine teeth: influence of exposure time mean 0.14 0.10 0.10 0.10 0.12 0.21 0.22 0.25 0.16 0.12 0.12 0.12 standard deviation 0.07 0.02 0.01 0.02 0.02 0.06 0.04 0.04 0.05 0.02 0.01 0.02 minimum 0.06 0.08 0.09 0.07 0.08 0.16 0.18 0.19 0.08 0.10 0.11 0.08 1st quartile 0.09 0.09 0.09 0.09 0.10 0.16 0.19 0.21 0.13 0.11 0.11 0.11 median 0.11 0.10 0.10 0.10 0.12 0.19 0.21 0.25 0.16 0.11 0.12 0.12 3rd quartile 0.18 0.12 0.12 0.12 0.13 0.26 0.26 0.28 0.19 0.14 0.14 0.14 maximum 0.30 0.13 0.12 0.12 0.14 0.32 0.30 0.30 0.23 0.14 0.14 0.15 0 5min 15min 30min 0 5min 15min 30min 0 5min 15min 30min antihistamine formulation(n=9) 0.6% citric acid (n=9)* distilled water (n=9) descriptive statistics table 2 – descriptive statistics of roughness values (ra – µm) by treatment groups and time intervals. *p<.001 (mann-whitney test with bonferroni correction). 24 analysis in enamel immersed in antihistamine liquid formulation. this was a limitation of the present study with regard to the quantification of mineral loss. further studies would benefit from combining sem evaluation with techniques that enable ultrastructural mineral loss quantification. the difficulty in reproducing the clinical situation in in vitro studies must be borne in mind. this is due to complexity of the oral environment. the absence of salivary pellicle and buffering by saliva may overestimate the in vivo occurrence of erosive demineralization4. however, antihistamine-containing medicines are known to reduce salivary secretion and it is important to alert for their erosive potential even, based mainly on in vitro studies5. in the present study, enamel specimens treated with the antihistamine liquid formulation showed erosion in sem images when compared to distilled water, and their erosive pattern increased with increasing exposure time. further studies testing low-ph antihistamine liquid formulations should be encouraged because allergic children make regular use of these medicines and could present dental erosion as an undesirable effect of oral medication. based on the experimental conditions of this study, the following conclusions can be made: among the tested media, only 0.6% citric acid produced a significant increase in enamel roughness. the sem analysis showed that antihistamine liquid formulation and 0.6% citric acid were capable of producing erosion in enamel and was influenced by exposure time. acknowledgements the authors are grateful to prof. paulo barrocas and letícia alves da silva for their support in the phosphate and fluoride content analysis at ensp/fiocruz (escola nacional de saúde pública/fundação oswaldo cruz). the authors also would like to thank cnpq (conselho nacional de desenvolvimento científico e tecnológico) for the research grant (302382/2009-7), and faperj (fundação carlos chagas de amparo à pesquisa do estado do rio e janeiro) (e-26/ 110.486/2010) for the financial support. references 1. o’sullivan e, milosevic a. uk national clinical guidelines in paediatric dentistry: diagnosis, prevention and management of dental erosion. int j paediatr dent. 2008; 18: 29-38. 2. magalhães ac, wiegand a, rios d, honório hm, buzalaf mar. insights into preventive measures for dental erosion. j appl oral sci. 2009; 17: 75-86. 3. moss sj. dental erosion. int dent j. 1998; 48: 529-39. 4. costa cc, almeida ics, costa filho lc. erosive effect of an antihistaminecontaining syrup on primary enamel and its reduction by fluoride dentifrice. int j paediatr dent. 2006; 16: 174-80. 5. pierro vss, abdelnur jp, maia lc, trugo lc. free sugar concentration and ph of paediatric medicines in brazil. community dent health. 2005; 22: 180-3. 6. pierro vss, barcelos r, maia lc, silva an. pediatricians´ perception about the use of antibiotics and dental caries – a preliminary study. j public health dent. 2004; 64: 244-8. 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jagger dc. an in vitro investigation of the effect of some analgesics on human enamel. j oral rehabil. 2006; 33: 529-32. 13. tanakaa m, margolis hc. release of mineral ions in dental plaque following acid production. arch oral biol. 1999; 44: 253-8. 14. udoh ap. atomic absorption spectrometric determination of calcium and other metallic elements in some animal protein sources. talanta. 2000; 52: 749-54. 15. lussi a, kohler n, zero d, schaffner m, megert b. a comparison of the erosive potential of different beverages in primary and permanent teeth using an in vitro model. eur j oral sci. 2000; 108: 110-4. 16. meurman jh, frank rm. progression and surface ultrastructure of in vitro caused erosive lesions in human and bovine enamel. caries res. 1991; 25: 81-7. 17. johansson a-k, sorvari r, birkhed d, meurman jh. dental erosion in deciduous teeth – an in vivo and in vitro study. j dent. 2001; 29: 333-40. 18. cairns am, watson m, creanor sl, fove rh. the ph and titratable acidity of a range of diluting drinks and their potential effect on dental erosion. j dent. 2002; 30: 313-7. 19. grenby th, phillips a, desai t, mistry m. laboratory studies of the dental properties of soft drinks. br j nutr. 1989; 62: 451-64. 20. larsen mj, nyvad b. enamel erosion by some soft drinks and orange juices relative to their ph, buffering effect and contents of calcium phosphate. caries res. 1999; 33: 81-7. 21. valinoti ac, neves bg, da silva em, maia lc. surface degradation of composite resins by acidic medicines and ph-cycling. j appl oral sci. 2008; 16: 257-65. 22. barbour me, rees js. the laboratory assessment of enamel erosion: a review. j dent. 2004; 32: 591-602. braz j oral sci. 9(1):20-24 erosive effect of an antihistamine liquid formulation on bovine teeth: influence of exposure time oral sciences n3 original article braz j oral sci. 8(4):193-196 esthetic perception towards different combinations of facial contours and upper incisor shape lidia yileng tay chu jon1; daniel rodrigo herrera morante1; eduardo bernabé2 manuel o. lagravere vich3; leyla antoinette delgado cotrina4 1undergraduate student, dental school, universidade estadual de ponta grossa, paraná, brasil. 2dds, ms, phd, department of epidemiology and public health, university college london, london, united kingdom. 3dds, ms, phd, research associate graduate orthodontic program, university of alberta, edmonton, canada. 4dds, ms, professor, depar tment of restorative dentistry, universidad peruana cayetano heredia, lima, peru. correspondence to: lidia yileng tay chu jon av carlos cavalcanti, 4748, uvaranas. ponta grossa, pr brasil cep 84030-900 e-mail: yilengt@gmail.com abstract aim: the aim of this study was to assess the esthetic perception of adults with primary, secondary and higher education and recent dental graduates towards different combinations of facial contours and upper central incisor shapes. methods: photographs of 6 individuals with square, tapered and ovoid facial contours (2 per type) were modified to have the 3 different types of tooth shapes (square, tapered and ovoid) in each facial contour (total of 18 photographs). the 195 participants rated each photograph using visual analogue scales. comparison between groups was performed using the chi-square t-test (a=0.05). results: no statistically significant differences (p>0.05) were found in the esthetic perception towards the frontal facial outline and upper incisor shape by participants’ educational level. in addition, the shapes of teeth perceived as the most esthetically pleasing were not always similar to the form of frontal facial outline evaluated. conclusions: no general agreement was found in the esthetic perception towards the frontal facial outline and upper incisor shape by educational level. dentists tend to prefer ovoid shape tooth for almost all frontal facial outline. keywords: esthetic perception, facial contour, dental shape introduction esthetics has great importance in restorative dentistry becoming synonymous of natural, healthy, and harmonious appearance1-2. an attractive smile increases an individual’s acceptability to society and improves interpersonal relationships. dentists and patients prefer equilibrated smiles with uniform teeth and a straight or mildly convex incisal plane3. this tooth relationship can be affected by several factors such as shape, size, color, texture, symmetry, and proportion4. knowledge of these details can satisfy the needs of every patient. beauty is not absolute but rather extremely subjective and perception is determined by the senses, knowledge, ethnic background, and preferences of each individual5-6. regarding teeth shape, several methods have been used to predict the shape of the missing anterior teeth to facilitate the restoration and maintenance of the anterior segment7. in 1914, williams8 established that to restore the upper central incisors they should be related to the facial contour. in this fact, he classified both teeth and facial contours into three categories: square, tapered and ovoid. studies have proven the existence of a relationship between upper central incisor shape and facial contour3 while other studies have proven the contrary4,7,9-10. however, the majority of these studies determined this relationship by using photographs of the facial contours and comparing them to the intraoral photographs of the upper central incisor. brisman3 has braz j oral sci. october/december 2009 volume 8, number 4 received for publication: august 13, 2009 accepted: january 18, 2010 194 braz j oral sci. 8(4):193-196 demonstrated that judgments on dental esthetics differed when the teeth shape is assessed jointly with the facial contours, indicating an influence of the facial presentation on the esthetic perception. on the other hand, several studies have reported that the level and type of education can influence people’s esthetic perception. anderson et al.11 evaluated the esthetic perception of tooth shapes when smiling and found discrepancies between the preferences of dental professionals and lay persons. brisman3 stated that the patients’ and dentists’ opinions differ when evaluating images and photographs of upper central incisor variations in shape, symmetry and proportion. however, no study has explored the influence that the level and type of education can have on people’s esthetic perception. to fill this gap in knowledge, a study was set out to compare the esthetic perception of adults with primary, secondary and higher education and recent dental graduates towards different combinations of facial contours and upper central incisor shapes. material and methods study sample one hundred and ninety five adults participated in the survey. the study sample included 47 subjects with primary, 50 with secondary and 50 with higher education as well as 48 recent dental graduates (>1 year) from the universidad peruana cayetano heredia. this was a convenience sample selected from the patients attending the dental clinic of the universidad peruana cayetano heredia (lima, peru). the institutional review board at the universidad peruana cayetano heredia approved the study protocol. furthermore, subjects signed a written consent agreeing their voluntarily participation in the survey. data collection the survey instrument consisted of 6 black-and-white photographs (3 of each gender). each photograph included an individual presenting a square, tapered and ovoid facial contour. subjects photographed did not present any severe facial or dental alterations. standardized frontal full dental smiling photographs (28 x 20 cm2) were taken of each subject such that the upper central incisors and up to 2 mm of gingiva were shown. teeth in each photograph were modified using adobe photoshop 7.0 (adobe systems incorporated, san jose, ca, usa) so as to obtain 3 different types of tooth shapes (square, tapered and ovoid) for each facial contour. a total of 18 photographs were included in the final version of the survey instrument, which were randomly arranged into a booklet. therefore, each page in the booklet showed the 3 photographs of the same individual presenting the 3 different incisal shapes (figures 16). during the survey, subjects were asked to select the most esthetically pleasing photograph from each set of 3 with the same facial contour. overall, each subject provided 6 answers. they had 30 s to view and rate each set of photographs. a pilot evaluation was undertaken to determine the reliability of the instrument before the main survey. ten individuals filled out the survey and, after 24 h, repeated it but with the photographs randomly rearranged to eliminate memory bias. the kappa value was 0.60, which is considered acceptable for studies on perception12. statistical analysis groups were initially compared with regard to their gender distribution, using the chi-square test, and their age, using the kruskal-wallis test. thereafter, the esthetic perception towards the different combinations of facial contours and tooth shape was compared between educational groups using the chi-square test (a=0.05). fig. 1 female square facial contour set. each photograph presents a different shape of upper central incisors. fig.2 male square facial contour set. each photograph presents a different shape of upper central incisors. fig. 3 female tapered facial contour set. each photograph presents a different shape of upper central incisors. fig.4 male tapered facial contour set. each photograph presents a different shape of upper central incisors. fig. 5 female ovoid facial contour set. each photograph presents a different shape of upper central incisors. esthetic perception towards different combinations of facial contours and upper incisor shape 195 braz j oral sci. 8(4):193-196 fig. 6 male ovoid facial contour set. each photograph presents a different shape of upper central incisors. results groups were not statistically different by their gender distribution (p=0.080), but were statistically different by age (p<0.001). the group with elementary education was the oldest group (46.60 + 11.5 years), table 1 aesthetic perception towards different combination of facial contours and upper incisors tooth shape: a – square female facial contour; b – square male facial contour; c – tapered female facial contour; d – tapered male facial contour; e – ovoid female facial contour; f – ovoid facial contour. followed by the groups with higher education (33.10 + 10.11 years), high school education (28.48 + 8.64 years) and dental graduates (23.79 + 2.21 years), respectively. table 1 illustrates the esthetic perception towards the different combinations of tooth shape and facial contour in the four groups compared. for the square female facial contour (a), subjects with elementary education and dental graduates preferred the ovoid tooth shape (46.8 and 64.6%, respectively) and subjects with high school and higher education preferred the tapered tooth shape (50% and 48%, respectively). this difference was not statistically significant (p=0.217). for the square male facial contour (b), subjects with elementary, high school and higher education (38.3, 50.0 and 54.0%, respectively) preferred the square tooth shape while dental graduates preferred the ovoid tooth shape (47.9%). however, this difference was not statistically significant (p=0.089). for the tapered female facial contour (c), subjects with primary a b c d e f esthetic perception towards different combinations of facial contours and upper incisor shape 196 braz j oral sci. 8(4):193-196 education preferred the square tooth shape (46.8%), those with secondary and higher education (48.0 and 54.0%, respectively) preferred the tapered tooth shape and dental graduates preferred the ovoid tooth shape (39.6%). this difference was statistically significant (p=0.003). on the other hand, all groups preferred the tapered tooth shape for the tapered male facial contour (d), with no significant difference (p=0.209). for the ovoid female facial contour (e), subjects with elementary, high school and higher education preferred a tapered tooth shape (48.9, 38.0 and 46.0%, respectively) whereas dental graduates preferred the ovoid tooth shape (45.8%). there was no significant difference between groups (p=0.453). for the ovoid male facial contour (f), subjects with elementary and higher education preferred the tapered tooth shape (48.9% and 54.0%, respectively) while those with high school education preferred the ovoid tooth shape (54.0%) and dental graduates the square tooth shape (39.6%). there was a significant difference between groups (p=0.003). discussion this study assessed the esthetic perception towards different combinations of facial contour and tooth shape by adults with different levels of education. from the 6 sets of photographs assessed, there were significant differences between groups only for the tapered female and ovoid male facial contours. in the former case, dental graduates preferred the ovoid tooth shape whereas the other groups preferred the ovoid or square tooth shapes. in the latter case, dental graduates preferred the square tooth shape while the other three groups preferred the tapered or ovoid tooth shape. there were two additional findings in this study that must be highlighted. the first relates to the homogenous preferences reported by groups with primary, secondary and higher education (i.e., the groups including lay people), which differed from those reported by dental graduates. whereas dental graduates preferred the ovoid tooth shape for 4 out of the 6 sets of photographs assessed, the other three groups preferred the tapered or ovoid tooth shape for 5 out of the 6 sets of photographs. as in the present study, anderson et al.11 concluded that dentists have a preference for ovoid teeth for the incisors. the second finding is that the shape of the selected teeth was not always similar to the form of frontal facial outline evaluated. contrary to expected, not even dental graduates preferred those photographs showing the tooth shape that corresponded to the facial contour (i.e., square-square, tapered-tapered or ovoid-ovoid). it occurred only in the case for the tapered male facial contour, for which all groups preferred the tapered tooth shape. williams6 related the facial contour with the shape of the anterior teeth. this theory has been proven and disproved by several studies2,4,7,9-10. however, the majority of these studies determined this relationship by using photographs of the facial contours and comparing them to the intraoral photographs of the upper central incisor. the analysis of the data was done individually with the objective to determine which tooth shape was the most prevalent for each specific facial contour. brisman3 stated that females should present more round and delicate teeth (tapered or ovoid) while males should have more angulated teeth (square). he also reported that when patients and dentists observed an incisor individually, they preferred it to be longer (3:5 proportion), but when the judgment was made jointly with the facial contours, shorter teeth were preferred (4:5 proportion) indicating an influence of the facial presentation on the esthetic perception. dentists have been searching for ways to standardize fixed characteristics to obtain the composition of each patient or group of patients. this has caused dentists to learn certain characteristics and concepts related to persons with respect to age, gender and personality1314. this can cause limitations in communication among dentists and patients since beauty concepts in society are influenced by other factors such as social, cultural, economic and psychological and each person forms their own perception, which is usually considered as the correct one. tjan5 corroborated this stating that beauty is not absolute but extremely subjective and perception is determined by the senses, knowledge, ethnic background, and preferences of each individual. mahshid et al.6 evaluated dental proportions in a harmonious smile and noted that cultural and individual characteristics, as well as esthetic perception, of each person played an important role in this area. some studies3,15 reported that the esthetic perception did not present statistical differences between genders as was found in this study. some limitations of this study need to be discussed. although groups were homogenous in size and gender distribution, they were statistically different with respect to their ages because it was difficult to find adults with primary education or less. future studies should try to match groups with similar ages in order to disentangle the potential influence of this factor on esthetic perception. no general agreement was found in the esthetic perception towards the frontal facial outline and upper incisor shape by educational level. dentists tend to prefer ovoid shape tooth for almost all frontal facial outline. references 1. newton jt, prabhu n, robinson pg. the impact of dental appearance on the appraisal of personal characteristics. int j prosthodont. 2003; 16: 429-34. 2. wolfart s, menzel h, kern m. inability to relate tooth forms to face shape and gender. eur j oral sci. 2004; 112: 471-6. 3. brisman as. esthetics: a comparison of dentists’ and patients’ concepts. j am dent assoc. 1980; 100: 345-52. 4. brodbelt rh, walker gf, nelson d, seluk lw. comparison of shape with tooth form. j prosthet dent. 1984; 52: 588-92. 5. tjan ah, miller gd, the jg. some esthetic factors in a smile. j prosthet dent. 1984; 51: 24-8. 6. mahshid m, khoshvaghti a, varshosaz m, vallaei n. evaluation of “golden proportion” in individuals with an esthetic smile. j esthet restor dent. 2004; 16: 185-2. 7. mavroskoufis f, ritchie gm. the face-form as a guide for the selection of maxillary central incisors. j prosthet dent. 1980; 43: 501-5. 8. williams jl. a new classification of human tooth forms, with special reference to a new system of artificial teeth. dent cosmos. 1914; 56: 627. 9. sellen pn, jagger dc, harrison a. computer-generated study of the correlation between tooth, face, arch forms and palatal contour. j prosthet dent. 1998; 80: 163-8. 10. bell ra. the geometric theory of selection of artificial teeth: is it valid? j am dent assoc. 1978; 97: 637-40. 11. anderson km, behrents rg, mckinney t, buschang ph. tooth shape preferences in an esthetic smile. am j orthod dentofacial orthop. 2005; 128: 458-65. 12. landis jr, and koch gg. the measurement of observer agreement for categorical data. biometrics. 1977; 33: 159-74. 13. rosenstiel sf, rashid rg. public preferences for anterior tooth variations: a web-based study. j esthet restor dent. 2002; 14: 97-106. 14. geron s, atalia w. influence of sex on the perception of oral and smile esthetics with different gingival display and incisal plane inclination. angle orthod. 2005; 75: 778-84. 15. moore t, southard ka, casko js, qian f, southard te. buccal corridors and smile esthetics. am j orthod dentofacial orthop. 2005; 127: 208-13. esthetic perception towards different combinations of facial contours and upper incisor shape oral sciences n3 braz j oral sci. 11(2):106-111 original article braz j oral sci. april | june 2012 volume 11, number 2 aging and thermocycling effects on adhesion of fiber posts to human radicular dentin abu-naba’a la1, abu-rujai m2 1bds, phd, mfdrcs, assistant professor of prosthodontics, department of substitutive oral sciences, taibah university, ksa, formerly: jordan university of science and technology, jordan 2master’s degree student, department of prosthodontics, jordan university of science and technology, irbid, jordan correspondence to: layla abu-naba’a assistant professor of prosthodontics, department of substitutive dental sciences faculty of dentistry, taibah university p.o.box 2898, madinah munawarah ksa phone: 00966545240240 fax: 0096648373231 e-mail: laylanabaa@hotmail.com abstract aim: this in vitro study evaluated the effect of different aging periods and thermocycling on the adhesion of fiber posts to human dentin at different depths. methods: twenty teeth were cleaned, decoronated, and endodontically treated. after one week, root filling was removed and a 10-mm post space was prepared. posts (relyx™ fiber size 2) were cemented with a self-adhesive resin cement (relyx™ unicem) and light cured. specimens (n=4, each) were stored in 100% humidity (0 day, 7 days, 1 month, 3 months) or thermo-cycled (at 7 days, 10,000 cycles, 5-55c°, 30 s). four 1-mm-thick sections were obtained from each tooth and a push-out test was performed and results compared. results: there was a significant difference (anova p<0.05) between the means of push-out test results (mpa ± se) at different storage periods: 24 hours: 15.2 ± 1.4; 7 days: 16.8 ± 1.5; 1 month: 20.3 ± 1.8; and 3 months: 12.5±2.0. shear strength was significantly different (chi-square, p<0.05) at the different slice positions. apical sections had an increase in strength at 7 days and 1 month, but without statistical significance. the coronal section increased significantly between 0 and 7 days. all sections had reduced strengths between 1 and 3 months, but only the apical sections decreased significantly (anova, p<0.05). there was no significant difference in shear strength between thermally cycled and non-cycled samples (mpa/se 16.7± 5.9, mpa/se 16.0 ± 9.3)(p>0.05). conclusions: long-term storage of test samples affected the bond strength of fiber posts to radicular dentin variably, and should be considered as part of invitro testing. keywords: adhesion, fiber post, push-out test, radicular dentin, storage, thermocycling. introduction damaged tooth structure get support and retention from various post materials and methods with emphasis on esthetic demands1. many in vitro tests of fiber post material analyze dentin-cement-post system behavior under proposed clinical situations. these tests were reviewed and reasons for the variability of strength results were indicated2-6: 1. tests were done on full-root samples (as pull-out and fracture compressive load), or root sections (as micro-push-out or hourglassand rectangular stick-shaped microtensile testing); 2. as the method of force application differed, values of adhesion reported varied; 3. pull-out of a whole post from full roots had highest values of bond strength, followed by push-out tests from root sections; 4. failures and high standard deviations of strengths reduce the reliability of test values. a large number of premature failures (during specimen preparation) occur with hourglass and stick tensile tests; 5. full root sample testing was dependent on the morphology of radicular dentin; 6. the push-out test of thin received for publication: december 04, 2011 accepted: april 18, 2012 braz j oral sci. 11(2):106-111 107107107107107 perpendicular root sections showed advantages over other testing methods: strength measurements were more consistent, stress distribution was more homogenous (finite element analysis), strength values were less variable, flaws were distributed uniformly and finely throughout the material (this is called a higher weibull modulus), fewer premature failures were found (confined mainly to root samples not embedded in resin blocks), and finally, there was differentiation of regional differences in bond strength; 7. moisture, heat and fatigue stresses interplay and reduce the longevity of fiber posts clinically, and were standardized conditions added to samples before testing. these were applied either to whole teeth (which best mimics the clinical situation), but other studies concluded from applying them to dentin powder, isolated dentin sections of coronal dentin, or unsealed samples with an enamel-bonded margin, or bovine teeth; 8. variability also resulted because tests were applied to different cements, adhesives, posts, dentin substrates and irrigant materials. the study reported here applies some of these clinical conditions to intact human roots that received fiber bonded posts and were tested by push-out tests with different storage periods and thermocycling. the null-hypothesis states that no difference will be detected after different aging periods and thermocycling of intact roots on the adhesion of fiber posts to radicular dentin at different root section locations. material and methods sample selection twenty, caries and crack-free human maxillary central incisors and canines with straight 14-mm-long roots and mature apices were selected. a single canal was verified by 2-angled radiographs. ethical approvals this study was supported by grant no. 149/2009, by jordan university of science and technology, jordan. the study was independently reviewed and approved by the research committee, (deanship of research) and the institutional research board (irb), specialized in humanresearch ethics, (faculty of medicine). sample preparation debris were cleaned and the intact teeth were disinfected (30 min, 3% sodium hypochlorite sonic bath; sultan chemists, 300 pro. sonic, branson ultrasonics) and stored in thymol solution until the time of testing. root canal treatment the crowns were removed 2 mm above the buccal cementoenamel junction (0.5 mm diamond wafering blade; isomet, buehler ltd, lake bluff, il, usa) under copious water cooling. a #15 k-type file was inserted until it could be seen at the apical foramen, and then 1 mm was subtracted from this length to determine the working length. crowndown technique prepared canals for root canal filling (s1, s2, s3, f1, f2 and f3 protaper files on x-smart handpiece; dentsply maillefer, switzerland). irrigation (1 ml of 1% naocl solution; pd, dentaires sa, switzerland,) and fileeze (18% edta, ultradent, south jordan, ut, usa,) were used between files. a final rinse (5 ml distilled water) and drying (protaper paper points, ref a 022w; dentsply maillefer, switzerland) were done, then ah26 resin sealer (dentsply deterey, konstanz, germany) was applied using a lentulo spiral onto the dry canal. cold lateral condensation technique was used to obturate the canals (gutta-percha cone and fine accessory points, protaper gutta percha points, dentsply maillefer, switzerland). the coronal part, 2 mm below the orifice, was cleaned and sealed (cavit-g; 3 m espe, seefeld, germany). the root tips were immersed in saline solution in a closed container to ensure high humidity. the container was stored in an incubator at a 37°c to allow setting of the sealer for at least 7 days. post space preparation root filling was removed (gates-glidden, gates, dentsply maillefer, switzerland) and a 10-mm post space was prepared, leaving at least 3 mm of the root filling apically (universal burr, size 2 drill, relyx™ fiber post kit; t, 3m espe, seefeld, germany). posts (relyx™ fiber size 2, 3m espe, seefeld, germany) were cleaned with alcohol, tried for the fit (binding was excluded) and protected from any further contamination. the post space was irrigated with normal saline and then 5 ml of distilled water. canals were dried with paper points (protaper paper points, dentsply maillefer, switzerland). cementation of the post the self adhesive resin cement (relyx™ unicem, 3m espe, seefeld, germany,) was activated, mixed (15 s capsule mixer, italy), and then applied into canal (elongatin tips, 3m espe, seefeld, germany) according to the manufacturer’s recommendation. the post was seated immediately, slightly twisted, and moderate pressure was applied to hold the post in its position while removing excess and during polymerization (600 mw/cm2, 40 s through the post, 20 s cervical, buccal and lingual surfaces). aging and storage specimens (n=4, each) were either stored in 100% humidity for different periods (0 days, 7 days, 1 month, 3 months) or thermocycled after 1 week of storage (5-55c°, 10,000 cycles, 30-s soaking, 12-s intervals). sectioning after the storage period, the teeth were mounted in molds of polyester resin (hm 190 unsaturated polyester resins, intermediate petrochemicals industries, jordan) for the ease of handling and sectioning. the specimens were then sectioned in a isomet sectioning machine (buehler ltd, lake aging and thermocycling effects on adhesion of fiber posts to human radicular dentin 108108108108108 braz j oral sci. 11(2):106-111 bluff, il, usa) with a 0.5 mm diamond wafering blade under copious water cooling, and loss between each slice was measured to be 0.6 mm. four 1-mm-thick sections were obtained from each tooth and identified as coronal (s1), sub-coronal (s2), epiapical (s3) and apical (s4) (figure 1). fig. 1: sectioning procedure followed at the cementoenamel junction and standardizing 4 sections: coronal s1, subcoronal s2, epiapical s3, and apical s4. the most apical portion of the post is not included. bond strength test a push-out test was performed in a (universal testing machine, jinan testing equipment corporation, prc). the sample mounted on the testing machine on a table with a 3mm-diameter central opening to allow the escape of the debonded post. an indenter with a diameter of 0.5 mm was used to apply the force on the post in an apico-coronal direction at a crosshead speed of 1 mm/min until the post was dislodged (figure 2). fig. 2: loading specimens for push-out test on the shear-strength universal testing machine. statistical analysis the retentive strength of the post segment was expressed in mpa, by dividing the load at failure (newton) by the interfacial area of the post fragment (sl). the latter, being the lateral surface of a truncated cone, was calculated by the formula: sl = ð (r+ r) .[(h2 (r r) 2] 0.5, where ð= 3. 14, r: coronal post radius, r: apical post radius and h: root slice thickness. the differences between groups were tested using anova to compare means of tested groups. chi square and tukey’s hsd test were used to test the results in terms of slice position (p<0.05). results effect of storage period there was a significant difference between the means of push-out test results at different storage periods (anova p<0.05) (figure 3). shear strength was significantly different at 1 and 3 months of storage (tukey hsd, p<0.05). immediate or baseline values were considered testing samples at baseline (24 hours of storage) and comparison with 1-week of storage showed that stored samples had a slight increase in test values (0 day: 15.2 ± 1.4; 7 day: 16.8 ± 1.5 mpa/se). then the strength increased reaching the maximum at 1 month (20.3 ± 1.8 mpa/se), then reduced to its lowest value at three months (12.5±2.0 mpa/se). fig. 3: total shear strength of all samples (mpa, mean se) per group, at different storage periods. effect of slice position shear strength was significantly different at different slice positions (chi-square, p<0.05). apical and epiapical sections had an increase in strength at 7 days and 1 month but without statistical significance. coronal sections had a significant increase in strength between 0 and 7 days. all sections had reduced strengths between 1 and 3 months, but apical section decreased significantly between 1 and 3 months (anova, p<0.05) (table 1). effect of thermal cycling at 1 week the number of cycles used here are 10,0000 and aging and thermocycling effects on adhesion of fiber posts to human radicular dentin braz j oral sci. 11(2):106-111 109109109109109 0 day 7 day 1 month 3 months total section mean sd mean sd mean sd mean sd mean sd coronal 9.2* 1.9 14.3 3.1 17.2 5.6 13.2 12.4 13.2* 6.8 subcoronal 13.9 6.5 12.4* 4.2 16.4 6.0 9.5 7.4 13.3* 5.9 epiapical 16.7 5.5 16.8 5.4 20.9 9.6 14.6 6.1 17.2* 6.5 apical 20.8** 5.7 23.6** 4.9 26.8 4.5 11.9 5.7 20.7** 7.3 anova** anova** anova— anova— anova** tukey hsd* tukey hsd* tukey hsd— tukey hsd— tukey hsd* table 1 mean shear strength (mpa ±sd) for each section at different storage periods. (anova**, p<0.05, anova—,p>0.05, tukey hsd*, p<0.05 between designated sections** with *, tukey hsd—,p>0.05). 7 days 7 days with thermocycling section mean sd mean sd coronal 14.3 3.1 11.1 7.6 subcoronal 12.4* 4.2 14.5 6.4 epiapical 16.8 5.4 13.3 6.2 apical 23.6* 4.9 25.0 11.9 anova** anova— tukey hsd* tukey hsd— table 2 mean shear strength (mpa ± sd) for each section at 1 week, comparing before and after thermocycling (anova**, p<0.05, anova—,p>0.05, tukey hsd*, p<0.05 between designated sections, tukey hsd-,p>0.05). performed over 10 days. there was no significant difference in shear strength between the two conditions for the whole samples (16.7± 5.9 and 16.0 ± 9.3 mpa/se)(p>0.05). subcoronal and apical sections had a non-significant increase, while coronal and epiapical sections had a non-significant decrease of stress values (table 2). discussion a five-year survival rates for fiber posts are considered satisfactory 7-8 and depend on the location of the tooth restored, type of final restoration, presence of proximal contacts from adjacent teeth, presence of a ferrule, remaining coronal structure and occlusal loads9-11. nevertheless, the failure mode is usually a restorable de-bonding of resin bonded fiber posts, and can be enhanced by using various pretreatment procedures12. in vitro studies have tried to test dentin bonding behavior from tests applied to dentin sections and not endodontically and post-restored intact teeth. testing the radicular dentin-bond-cement-post system could be proposed to have different results than testing sections or non-sealed dentin surfaces 2-6. even in this closed system, more standardization is needed. this study standardized the taper of endodontic preparation, endodontic filling, post space preparation, irrigation, cement, post placement, coronal seal, and light curing for all study samples. test variables storage and thermocycling have been subjected to intact and sealed roots. then, resin-block addition, around these root samples, aimed to only facilitate the sectioning procedure. standardizing the cement in this study, and using only relyx unicem, was essential. it had similar flexural strength to the conventional resin cements. even higher strength and modulus of elasticity were evident when light cured. its low initial ph is characterized by a more rapid rise to neutrality than other cements, regardless to the curing method11. although criticized, not being able to effectively remove the thick smear layer12, the cement formed sporadic resin tags and hybrid layers, and it had the highest bond strengths when compared to other conventional cements6,13. in addition, this higher chemical interaction between the cement and hydroxyapatite caused no displacement of the resin components across the dentin tubules12. its strength was not affected if present in thicker cement layers13. and when test samples were examined for failure modes, it was the only one to show an adhesive-cohesive mixed fracture14-16. the effect of storage storage of bonded restorations caused deterioration by infusion of fluid into its matrix, from periodontioum, coronal and apical ends of the restorations. if no peripheral failure is observed in restorations, this leakage is confined to fluids and not to macromolecules and microbial products. later, a progressive disintegration of the fibrilar network of the collagen occurs in hybrid layer. matrix metalloproteinase (mmps) bind to dentin matrix and become activated or come from pulp tissue, odontoblasts, saliva or microbial macromolecules2-6. further expression of their activity is produced by acidic challenges to dentin tissue during bonding procedures17. another effect of fluid ingression during storage is the plasticizing of resin bond and cement materials. however, a study, using multiple cements, concluded that 3-month storage of intact samples in deionized water had no influence on the hardness of 3 common post-cements, with the exception of relyx unicem. it had a significant increase in the hardness values after storage18. comparing strength values at different slice depth it could be proposed that the restored canal system behaves differently at different depths from the cementoenamel. one difference between layers is the adaptation of the post to canal walls, increasing adhesion forces, and friction, while reducing the low cohesive strength, aging and thermocycling effects on adhesion of fiber posts to human radicular dentin braz j oral sci. 11(2):106-111 fluid ingression and flaws relevant to the cement layer19. apical portions expected to be closer and could be matched in size with the post, by custom-post preparation burs. if a close fit is produced, by post relining for example, higher retention values were obtained than non-relined post, in all thirds, along with disappearance of differences among various depths19. when precise fitting was reached, relyx unicem had similar or higher strength values, compared with other cements, after water storage and thermocycling. in oversized post space, it was second after build-it, superbond and panavia13,20-21, in comparison with 3 other different cements with thermocycling only. fracture modes were shown by other cements to be mainly adhesive at the post surface or cohesive for precisely fitting posts and to occur between post and composite, except for relyx unicem with cohesive fractures, for the oversized spaces. finite element model found that indirect and direct restorations produced both strain and stress, off the scale in the middle third of the buccal aspect of the root surface. while minimum values were noticed at the level of both the apical portion of the post and the root apex22. in vitro studies not including the previous clinical conditions have shown the contrary. cervical (coronal sections) regions have higher strength values than middle and apical thirds, respectively2-6. the preference of light curing to easily accessible cervical regions could be one reason for this result. relyx unicem showed the same result in a previous study23. the reason could have been the amount of non-homogenous cement that was less in the cervical than in the middle and apical levels. the application tip used in this study significantly reduced these failures, but they were still more predominant in apical areas24. another problem was that the brush used to coat adhesives accessed cervical regions with more material, leading to better resin tag morphologies25. using the unique application tip, then inserting the post in it, excluded the formation of air inhibited layers in the cement. finally, contamination of apical regions with remnants of sealing material is more prominent in apical regions26. this final limitation was excluded from this study as the last 1.4 mm of the post was not included in the sections. this study was persistent with the results of the first group; apical sections were stronger than other sections that gradually decreased up to the coronal. the apical even benefitted to some extent from thermocycling, in the same way as observed in a previous study16, but was significantly affected by the storage, leading it to comply more with the second group27. studies that have shown a thermocycling effect used a larger number of cycles, 40,000, and were subjected to exposed sections, not intact teeth28. within the limitations of this study, it was observed that intact and sealed roots produced different fiber post bonding results in comparison with other studies applying these conditions to dentin sections, powder or un-sealed restorations. such variation should be taken into account when planning in vitro studies which test and conclude on the behavior of the radicular dentin-cement-post system on the long-term and at different regions within the root. acknowledgements this study was supported by grant no. 149/2009, by jordan university of science and technology, jordan. the study was independently reviewed and approved by the research committee, (deanship of research) and the institutional research board (irb), specialized in humanresearch ethics, (faculty of medicine). references 1. cagidiaco mc, garcía-godoy f, vichi a, grandini s, goracci c, ferrari m. placement of fiber prefabricated or custom made posts affects the 3-year survival of endodontically treated premolars. am j dent. 2008; 21: 179-84. 2. bitter k, kielbassa am. post-endodontic restorations with adhesively luted fiber-reinforced composite post systems: a review. am j dent. 2007; 20: 353-60. 3. saskalauskaite e, tam le, mccomb d. flexural strength, elastic modulus, and ph profile of self-etch resin luting cements. j prosthodont .2008; 17: 262-8. 4. goracci c, sadek ft, fabianelli a, tay fr, ferrari m. evaluation of the adhesion of fiber posts to intraradicular dentin. oper dent. 2005; 30: 627-35. 5. wrbas kt, kampe mt, schirrmeister jf, altenburger mj, hellwig e. .[retention of fiber posts dependent on different resin cements] schweiz monatsschr zahnmed. 2006; 116: 18-24. 6. bitter k, paris s, pfuertner c, neumann k, kielbassa am. morphological and bond strength evaluation of different resin cements to root dentin. eur j oral sci. 2009; 117: 326-33. 7. signore a, benedicenti s, kaitsas v, barone m, angiero f, ravera g. long-term survival of endodontically treated, maxillary anterior teeth restored with either tapered or parallel-sided glass-fiber posts and full-ceramic crown coverage. j dent. 2009; 37: 115-21. 8. piovesan em, demarco ff, cenci ms, pereira-cenci t. survival rates of endodontically treated teeth restored with fiber-reinforced custom posts and cores: a 97-month study. int j prosthodont .2007; 20: 633-9. 9. naumann m, blankenstein f, kiessling s, dietrich t. risk factors for failure of glass fiber-reinforced composite post restorations: a prospective observational clinical study. eur j oral sci. 2005; 113: 519-24. 10. naumann m, reich s, nothdurft fp, beuer f, schirrmeister jf, dietrich t. survival of glass fiber post restorations over 5 years. am j dent. 2008; 21: 267-72. 11. dorriz h, alikhasi m, mirfazaelian a, hooshm, t. effect of ferrule and bonding on the compressive fracture resistance of post and core restorations. j contemp dent pract. 2009; 10: 1-8. 12. de souza costa ca, hebling j, randall rc. human pulp response to resin cements used to bond inlay restorations. dent mater. 2006; 22: 954-62. 13. huber l, cattani-lorente m, shaw l, krejci i, bouillaguet s. push-out bond strengths of endodontic posts bonded with different resin-based luting cements. am j dent. 2007; 20: 167-72. 14. garcia ld, naves lz, correr-sobrinho l, consani s, pires-de-souza fd. bond strength of a self-adhesive resinous cement to root dentin irradiated with a 980-nm diode laser. acta odontol scand. 2010; 68: 171-9. 15. wrbas kt, kampe mt, schirrmeister jf, altenburger mj, hellwig e. .[retention of fiber posts dependent on different resin cements] schweiz monatsschr zahnmed. 2006; 116: 18-24. 16. dimitrouli m, günay h, geurtsen w, lührs ak. push-out strength of fiber posts depending on the type of root canal filling and resin cement. clin oral investig. 2011; 15: 273-81. 17. de munck j, van den steen pe, mine a, van landuyt kl, poitevin a, opdenakker g, et al. inhibition of enzymatic degradation of adhesivedentin interfaces. j dent res. 2009; 88: 1101-6. 110110110110110aging and thermocycling effects on adhesion of fiber posts to human radicular dentin braz j oral sci. 11(2):106-111 111111111111111 18. pedreira ap, pegoraro lf, de góes mf, pegoraro ta, carvalho rm. microhardness of resin cements in the intraradicular environment: effects of water storage and softening treament. dent mater. 2009; 25: 868-76. 19. faria-e-silva al, pedrosa-filho cde f, menezes mde s, silveira dm, martins lr. effect of relining on fiber post retention to root canal. j appl oral sci. 2009; 17: 600-4. 20. schmage p, pfeiffer p, pinto e, platzer u, nergiz i. influence of oversized dowel space preparation on the bond strengths of frc posts. oper dent. 2009; 34: 93-101. 21. naumann m, preuss a, frankenberger r. load capability of excessively flared teeth restored with fiber-reinforced composite posts and all-ceramic crowns. oper dent. 2006; 31: 699-704. 22. sorrentino r, salameh z, apicella d, auriemma t, zarone f, apicella a, et al. three-dimensional finite element analysis of stress and strain distributions in post-and-core treated maxillary central incisors. j adhes dent .2007; 9: 527-36. 23. boff ll, grossi ml, prates lh, burnett lh, shinkai rs. effect of the activation mode of post adhesive cementation on push-out bond strength to root canal dentin. quintessence int. 2007; 38: 387-94. 24. watzke r, blunck u, frankenberger r, naumann m. interface homogeneity of adhesively luted glass fiber posts. dent mater. 2008; 24: 1512-7. 25. vichi a, grandini s, ferrari m. comparison between two clinical procedures for bonding fiber posts into a root canal: a microscopic investigation. j endod. 2002; 28: 355-60. 26. serafino c, gallina g, cumbo e, ferrari m. surface debris of canal walls after post space preparation in endodontically treated teeth: a scanning electron microscopic study. oral surg oral med oral pathol oral radiol endod. 2004; 97: 381-7. 27. ma s, nakajima kf, nishiyama n. effects of storage temperature on the shelf life of one-step and two-step self-etch adhesives. oper dent. 2009; 34: 472-80. 28. mazzoni a, marchesi g, cadenaro m, mazzotti g, di lenarda r, ferrari m, et al. push-out stress for fibre posts luted using different adhesive strategies. eur j oral sci. 2009; 117: 447-53. aging and thermocycling effects on adhesion of fiber posts to human radicular dentin original article braz j oral sci. january/march 2009 volume 8, number 1 shear bond strength of a new composite for orthodontic use under different situations matheus melo pithon1, rogério lacerda dos santos1 1 specialist in orthodontics, faculdade de odontologia, universidade federal de alfenas (unifal), alfenas, mg, brazil; master and doctorate student, universidade federal do rio de janeiro (ufrj), rio de janeiro, rj, brazil received for publication: march 4, 2009 accepted: april 17, 2009 correspondence to: centro odontomédico dr. altamirando da costa lima matheus melo pithon avenida otávio santos, 395 – sala 705 – recreio cep 45020-750 – vitória da conquista (ba), brazil e-mail: matheuspithon@bol.com.br abstract aim: the aim of this study was to evaluate the shear bond strength of metallic orthodontic brackets bonded with eagle bond composite under different enamel surface conditions. methods: ninety bovine permanent lower incisors were divided into six groups (n = 15). in group 1 (control) and group 2 the brackets were bonded with xt primer/transbond xt composite (3m unitek) and eagle bond primer/eagle bond composite (american orthodontic), respectively, according to the manufacturers’ recommendations. in the other groups, the brackets were bonded with eagle bond composite as follows: group 3 had an enamel surface treated with transbond plus selfetching primer; group 4, bonding without application of eagle bond primer; group 5, use of homogenized eagle bond composite; and group 6, eagle bond primer/eagle bond composite applied on saliva/blood-contaminated dental surface. after bonding of the brackets, all specimens were tested in shear strength in an emic universal testing machine at a crosshead speed of 0.5 mm/min. results: there were no statistically significant differences among the groups (p > 0.05). a larger number of fractures were detected at the bracket/composite interface by the analyses of the adhesive remnant index scores. conclusions: it may be concluded that the modifications on the enamel surface did not compromise the shear bond strength of the composite eagle bond. keywords: shear strength, dental bonding, orthodontic brackets. introduction until the 1970s, orthodontic accessories were fixed by using bands in all teeth. according to zachrison1, such a bonding procedure had several disadvantages, which included, difficult cleaning, complexity, time-consuming clinical application and loss of esthetics. as a result, the technique for directly bonding orthodontic accessories to teeth was considered an essential advance for developing, simplifying and expanding orthodontics. this direct bonding technique was possible only after the advent of the acid etching2, which became a routine procedure for bonding fixed appliances. the first paper known to address the direct bonding of brackets to dental surface dates back to the late 1950’s3. according to nordenvall et al.4, such a technique brought several advantages to orthodontics, namely absence of proximal contact5, easy bonding and debonding of accessories, shorter chair-time, esthetics, improved oral hygiene and less incidence of gingival inflammation5,6. a wide array of materials have been developed in recent years for bonding orthodontic brackets and, thus the scientific knowledge of these materials is crucial for their clinical use. these composites usually have high bond strength, hardness and dimensional stability, and they also have some disadvantages regarding viscosity, preparation time7-11 and fluoride release12. 35shear bond strength of a new composite for orthodontic use under different situations braz j oral sci. 8(1): 34-37 eagle bond composite (american orthodontic, sheboygan, wi, usa) is one of the composites currently introduced to the dental market, and little research has been done with this material. according to the manufacturer, eagle bond is easily applied, has good bond strength and moderate viscosity. the actual need of testing newly introduced materials justifies the present study, whose objective was to evaluate both the shear bond strength and the adhesive remnant index (ari) of eagle bond composite, applied according to the manufacturer’s instructions and under different experimental conditions. material and methods ninety bovine permanent lower incisors were selected, properly cleaned, stored in 10% formaldehyde solution and kept refrigerated at 6 oc. the teeth were embedded in pvc cylinders (tigre, joinville, brazil) filled with acrylic resin (clássico, são paulo, brazil), in such a way that only their crowns were left exposed. the buccal surfaces of the crowns were positioned perpendicular to the shearing die’s base, using a tsquare to ensure that the mechanical test could be performed correctly. after resin polymerization, the specimens were stored in distilled water and maintained in refrigeration. prior to the bonding procedures, the buccal surfaces of all teeth were subjected to prophylaxis with a slurry of extra-fine pumice (s.s. white, juiz de fora, mg, brazil) and water in a rubber cup (viking, kg sorensen, barueri, sp, brazil) for 15 seconds. next, the specimens were washed with an air/water spray for 15 seconds and dried with oil/moisture-free air streams for 15 minutes. the rubber cup was replaced after every five consecutive applications in order to keep the experimental pattern. after prophylaxis, the specimens were randomly divided into six groups (n = 15), and upper central incisor brackets (abzil lancer, são josé do rio preto, sp, brazil) with a base area of 13.8 mm2 were used in the bonding procedures. the six groups are divided as follows: • group 1 (control): the enamel surfaces were etched with 37% phosphoric acid during 15 seconds, afterwards they were cleaned and dried in the same time. xt primer was applied and the brackets were bonded with transbond xt (3m unitek, monrovia, usa) composite. material in excess was removed with a sharp explorer (duf lex, juiz de fora, brazil); • group 2: the enamel surfaces were etched with 37% phosphoric acid during 15 seconds, then, cleaned and dried for the same period of time; eagle bond primer (american orthodontic) was applied on the etched enamel and light-cured for 15 seconds. eagle bond composite (american orthodontic) was applied on the base of the brackets, which were positioned, and the material in excess was removed; • group 3: transbond plus self etching primer tpsep (3m unitek) was applied by rubbing it onto the enamel surface during three seconds; air-thinning of the material; application of eagle bond composite (american orthodontic) on the base of the brackets, which were positioned, and the material in excess was removed; • group 4: the enamel surfaces were etched with 37% phosphoric acid during 15 seconds, then they were cleaned and dried for the same time; eagle bond composite (american orthodontic) was applied on the base of the brackets, which were positioned, and the material in excess was removed; • group 5: the enamel surfaces were etched with 37% phosphoric acid during 15 seconds, then they were cleaned and dried for the same time; eagle bond composite (american orthodontic, sheboygon, usa) was homogenized at a ratio of 1 g of composite to one drop of primer and applied to the base of the brackets, which were positioned, and the material in excess was removed; • group 6: the enamel surfaces were etched with 37% phosphoric acid during 15 seconds, then they were cleaned and dried for the same time; eagle bond primer (american orthodontic) was applied on the etched enamel and light-cured for 15 seconds; the enamel surfaces were contaminated with blood/saliva and dried; eagle bond composite was applied to the base of the brackets, which were positioned and the material in excess was removed. the composition of eagle bond is paste composed of sílica, bis-gm a, silane, n-dimethyl benzocaine, hexafluoride phosphate; primer with bis-gm a, silane, n-dimethyl benzocaine, hexafluoride phosphate. the brackets were light-cured during 40 seconds (ten seconds for each face –mesial, distal, incisal and gingival) at a distance of 1 mm, a halogen light-curing device (xl 1,500; 3m dental products, monrovia, usa) with light intensity of 450 mw/cm2 as measured with curing radiometer (demetron, danbury, ct, usa). after the bonding procedures, the specimens were stored in distilled water at 37 oc for 24 hours. a custom-made device was developed to hold the specimen completely stable during the mechanical test. the shear bond strength test was performed in a universal testing machine (emic dl 10.000; são josé dos pinhais, pr, brazil), at a crosshead speed of 0.5 mm/min through a chisel-shaped rod. the results were obtained in kgf, converted into n and, then divided by the base area of the bracket so that values in mpa could be obtained. after the mechanical test, the buccal surface of each specimen was evaluated with a stereoscopic magnifying glass (carl zeiss, goettingen, germany), at ×8 magnification in order to quantify the ari, according to the criteria established by artun and bergland7, that is, zero means no adhesive left on the enamel surface; one, less than half of the adhesive left on the enamel surface; two, more than half of the adhesive left on the enamel surface; and three, all the adhesive left on the enamel surface. shear bond strength mean values were analyzed statistically by analysis of variance and tukey’s test, in order to compare group 1 (control) to the experimental groups. kruskal-wallis test was used for assessing the ari scores. 36 pithon mm, santos rl braz j oral sci. 8(1): 34-37 results no statistically significant differences were found between group 1 (xt primer/transbond xt composite – control), group 2 (eagle bond primer/eagle bond composite), group 3 (transbond plus self-etching primer + eagle bond composite), group 4 (eagle bond composite without primer), group 5 (homogenized eagle bond composite), and group 6 (eagle bond composite applied to saliva/blood-contaminated enamel). however, as can be seen in table 1, group 2 presented the highest shear bond strength numerical mean values (p > 0.05), as can be seen in figure 1. the ari scores in each group are presented in table 2. regarding group 1, no statistically significant differences were found in relation to group 2 (p = 0.154), group 3 (p = 0.321), group 4 (p = 0.999), group 5 (p = 0.130), and group 6 (p =0.335). the same was observed for group 2 in relation to groups 4 (p = 0.154), 5 (p = 0.775), and 6 (p = 0.539), as well as between groups 4 and 5 (p = 0.130) and groups 4 and 6 (p = 0.335). however, statistically significant differences were observed between groups 2 and 3 (p = 0.002), groups 3 and 5 (p = 0.006), and groups 3 and 6 (p = 0.008). discussion transbond xt (group 1 – control), which has confirmed adhesive characteristics, was used in the present study according to the manufacturer’s instructions. no statistically significant differences were observed comparing the shear bond strengths of all groups, although group 2 had the lowest values. these results indicate that eagle bond is appropriate for bonding orthodontic accessories to enamel surface, with shear bond strength ranging from 5 to 20 mpa, which is considered by owens and miller13 to be sufficient to resist the orthodontic forces. as mentioned above, no statistically significant difference (p > 0.05) was found between group 2 (conventional eagle bond) and 3 (eagle bond + transbond plus self etching primer tpsep), which is consistent with the findings of previous studies8,14 using transbond xt under similar conditions. similar results were also reported by romano et al.15 using transbond xt and z 100, and by pithon et al.16, using orthobond composite. this finding is of clinical relevance, since tpsep has been shown to make the bonding procedure 65% faster, according to whyte17. aiming at simplifying the technique proposed by the manufacturer, eagle bond was used for bracket bonding without the priming step (group 4). the mean shear bond strength was higher than that obtained for group 2 (eagle bond with primer), though without statistical significance. this result is of great importance since a bonding step can be eliminated, shortening the clinical chairtime, and may be due to the lower resistance of the resin without load that was applied in group 2. however, it is important to have in mind that, although the elimination of this step reduces the clinical chairtime, the priming procedure protects the etched enamel that was not covered by the bracket after bonding. among the innumerous questions raised by other studies, regarding composites used for bonding orthodontic accessories, the behavior of these materials when previously homogenized should also be known. composite homogenization is justifiable for achieving a suitable distribution of the components, which would allow an improved flow during direct bonding of orthodontic brackets18,19. the bonding ability of homogenized eagle bond (group 5) was compared to the bonding ability of the conventional composite (group 2). no significant difference was found between the groups, suggesting that homogenized eagle bond would be a viable alternative when an improved flow is desirable. similar results were also found by patel et al.18, who tested the homogenization of the superbond composite. contamination of dental surface, with either blood or saliva during the bonding procedures, happens all the time. the interference of contamination after drying the contaminated area (group 6) was tested. no significant difference was found between the groups, which is also consistent with the findings of pithon et al.20, table 1. mean shear bond strength values and standard deviation groups mean values (mpa) 1 10.62 (3.64) 2 6.89 (4.62) 3 9.22 (2.38) 4 10.33 (4.69) 5 9.03 (2.58) 6 10.25 (2.98) table 2. adhesive remnant index (ari) scores and average rank for each group groups ari scores average rank 0 1 2 3 1 4 4 2 5 49.93 2 4 9 2 0 35.93 3 2 1 7 5 61.77 4 4 4 2 5 49.93 5 7 4 3 1 34.87 6 3 9 2 1 40.57 0: no adhesive left on the enamel surface; 1: less than half of the adhesive left on the enamel surface; 2: more than half of the adhesive left on the enamel surface; 3: all the adhesive left on the enamel surface. figure 1. box plot with shear bond strength mean values for all groups evaluated. 1 25.00 20.00 15.00 10.00 5.00 0.00 2 3 4 5 6 groups sh ea r b o n d s tr en g th * 3 * 22 37shear bond strength of a new composite for orthodontic use under different situations braz j oral sci. 8(1): 34-37 who used similar methodolog y with another composite. therefore, it was demonstrated that the whole clinical sequence did not need to be repeated, provided that the contaminated area was dried before bonding the brackets. once the enamel surface is contaminated with blood and saliva during the bonding procedure, it is necessary to dry the area to be bonded with eagle bond in order to obtain enough bond strength. regarding the ari, no statistically significant differences were found between the groups, except between groups 2 and 3, groups 3 and 5, and groups 3 and 6. such differences were due to the lower ari scores observed in groups 2, 5 and 6, compared to the higher ari scores observed in group 3, in which transbond plus self etching primer was used. the good adhesiveness to the teeth, promoted by associating eagle bond with tpsep, favored the achievement of higher shear bond strength mean values and, hence enamel protection during bracket debonding, that is, a greater amount of the adhesive material was left on the enamel surface. in groups 2 and 5, however, the majority of fractures occurred at the enamel/composite interface following the debonding process, with ari scores being predominantly zero or one, that is, no or lesser amount of composite adhered to enamel. such a result may be explained by the improved flow, provided by either primer application before bonding the brackets (group 2), or composite homogenization (group 5). these results are favorable as far as the maintenance of enamel integrity is concerned, since enamel micro or macrofragments can be removed together with bracket and composite during debonding. the values obtained in groups 2 and 5 are corroborated by most studies in the literature21,22. based on the results of the present study, the following conclusions may be drawn: conventional transbond xt and eagle bond systems showed good results in the shear bond strength testing, when bonded to enamel surface etched with 37% phosphoric acid; eagle bond composite presented good bond strength to enamel, treated with transbond plus self etching primer; the use of eagle bond primer was found to be facultative, that is, it was not necessary for achieving full adhesion; and homogenization of eagle bond composite did not reduce the shear bond strength values, being an alternative if improved flow is desirable. references 1. zachrisson bu. bonding in orthodontic. in: graber bf. (ed.) orthodontic current principle and technique. st. louis; 1985. p. 78. 2. buonocore mg. a simple method of increase the adhesion of acrylic filling materials to enamel surfaces. j dent res. 1955;34:849-53. 3. sadler j.f. a survey of some commercial adhesives: their possible application in clinical orthodontics. am j orthod dentofacial orthop. 1958;44:65-9. 4. nordenvall kj, branstron m, malmgren t. the effect of various pretreatment methods of the enamel in bonding procedures. am j orthod dentofacial orthop. 1978;74:52230. 5. boyd rl, baumrind s. periodontal considerations in the use of bonds or bands on molars in adolescents and adults. angle orthod. 1992;62:117-26. 6. ianni filho d, silva tbc, simplício ahm, loffredo lcm, ribeiro rp. avaliação in vitro da força de adesão de materiais de colagem em ortodontia: ensaios mecânicos de cisalhamento. r dental press ortodon ortop facial. 2004;9:39-48. 7. artun j, bergland s. clinical trials with crystal growth conditioning as an alternative to acid-etch enamel pretreatment. am j orthod. 1984;85:333-40. 8. bishara se, olsen me, damon p, jakobsen jr. evaluation of a new lightcured orthodontic bonding adhesive. am j orthod dentofacial orthop. 1998;114:80-7. 9. hobson rs, ledvinka j, meechan jg. the effect of moisture and blood contamination on bond strength of a new orthodontic bonding material. am j orthod dentofacial orthop. 2001;120:54-7. 10. littlewood sj, mitchell l, greenwood dc, bubb nl, wood dj. investigation of a hydrophilic primer for orthodontic bonding: an in vitro study. j orthod. 2000;27:181-6. 11. schaneveldt s, foley tf. bond strength comparison of moisture-insensitive primers. am j orthod dentofacial orthop. 2002;122:267-73. 12. pithon mm, dos santos rl, de oliveira mv, ruellas ac, romano fl. metallic brackets bonded with resin-reinforced glass ionomer cements under different enamel conditions. angle orthod. 2006;76:700-4. 13. owens se jr, miller bh. a comparison of shear bond strengths of three visible light-cured orthodontic adhesives. angle orthod. 2000;70:352-6. 14. cacciafesta v, sfondrini mf, gatti s, klersy c. effect of water and saliva contamination on the shear bond strength of a new light-cured cyanoacrylate adhesive. prog orthod. 2007;8:100-11. 15. romano fl, tavares sw, nouer df, consani s, borges de araújo magnani mb. shear bond strength of metallic orthodontic brackets bonded to enamel prepared with self-etching primer. angle orthod. 2005;75:849-53. 16. pithon mm, santos rl, oliveira mv, sant’anna ef, ruellas aco. avaliação da resistência ao cisalhamento do compósito orthobond em diferentes condições. rgo. 2008;56:405-10. 17. white lw. an expedited indirect bonding technique. j clin orthod. 2001;35: 36-41. 18. patel mp, pinzan a, francisconi pa, pinzan crm, ferreira kb. estudo da resistência ao cisalhamento na colagem de acessórios ortodônticos com e sem homogeinização da resina superbond. j bras ortodon ortop facial. 2004;9:242-7. 19. pinzan crm, pinzan a, franciscone pa, lauris jrp, freitas mr. estudo comparativo de resistencias às forças de cisalhmaneto, de colagem de bráquetes ortodônticos, testando dois tempos diferentes de condicionamento ácido, com e sem homogeinização prévia das pastas. rev dental press ortodon ortop facial. 2001;6:45-9. 20. pithon mm, oliveira mv, santos rl, ruellas aco. influência da contaminação de sangue misturado à saliva sobre a resistência ao cisalhamento da colagem e no índice de remanescente de adesivo. rev assoc paul esp orto ortop facial. 2003;1:24-8. 21. bishara se, vonwald l, zamtua j. effects of different types of light guides on shear bond strength. am j orthod dentofacial orthop. 1998;114:447-51. 22. cacciafesta v, sfondrini mf, de angelis m, scribante a, klersy c. effect of water and saliva contamination on shear bond strength of brackets bonded with conventional, hydrophilic, and self-etching primers. am j orthod dentofacial orthop. 2003;123:633-40. oral sciences n3 braz j oral sci. 10(2):146-151 original article braz j oral sci. april | june 2011 volume 10, number 2 temporomandibular disorder and generalized joint hypermobility: electromyographic analysis of the masticatory muscles fernanda pasinato1, juliana alves souza2, eliane castilhos rodrigues corrêa3, ana maria toniolo da silva4 1ms, assistant professor of physical therapy, federal university of pampa, brazil 2ms, physical therapist, university hospital of santa maria, brazil 3phd, adjunt professor, department of physical therapist, federal university of santa maria; graduate program in human communication disorders, federal university of santa maria, brazil 4phd, associate professor, department of speech pathology, federal university of santa maria; graduate program in human communication disorders, federal university of santa maria, brazil correspondence to: fernanda pasinato julio de castilhos street, number 2905, apt 102, downtown, uruguaiana city, rio grande do sul, brazil, cep: 97510-311 phone: (55) 81179089; (55) 81 34010225 e-mail: fepas.fisio@yahoo.com.br abstract aim: this study aimed to verify the presence of generalized joint hypermobility (ghj) in individuals with temporomandibular disorders (tmd) and asymptomatic individuals and to compare the activity of their masticatory muscles. methods: 61 female patients aged 18 to 35 years were evaluated: 34 with diagnosis of tmd by the research diagnostic criteria for temporomandibular disorders constituted the tmd group and 27 constituted the asymptomatic group. the subgroups were classified according to the presence of gjh by the beighton score. electromyographic recordings of the masseter and anterior temporal muscles were acquired bilaterally at mandibular rest and in maximal intercuspal position. results: gjh was present in 64.71% of the individuals with tmd and in 40.74% of the asymptomatic individuals. the electrical activity was significantly higher in the right masseter (p = 0.0111), left masseter (p = 0.0007) and right temporal (p = 0.0046) in the patients with tmd than in the asymptomatic individuals. the activity of the left masseter muscle was significantly higher (p=0.0072) in the volunteers with tmd and gjh compared with in the individuals with tmd but without hypermobility. also, the right temporal muscle showed higher activity in subjects with gjh and tmd compared with asymptomatic individuals without hypermobility (p=0.0248). conclusions: the electrical activity was higher at mandibular rest in tmd and tmd/ gjh patients. this result suggests that these muscles need to be recruited for the joint stabilization due to the low ligamentar resistance and a possible proprioceptive deficit. this recruitment appears to occur asymmetric and variedly among all muscles involved in this stabilization, which could compensate for the low ligamentar competence and a possible proprioceptive deficit in individuals with gjh. both tmd and gjh seem to have influenced the muscular activity. keywords: temporomandibular disorder, hypermobility, joint instability, electromyography, masticatory muscles. introduction the association between generalized joint hypermobility (gjh) and temporomandibular disorders (tmd) has been addressed by several studies1-8. it is received for publication: april 06, 2011 accepted: june 17, 2011 braz j oral sci. 10(2):146-151 believed that the temporomandibular joint is one of the hypermobile joints. on the other hand, the results of the studies regarding this relationship are conflicting generally due to discrepancies in the sampling and methodology used. gjh is characterized by the excessive range of motion of several joints due to ligamentous laxity, and may be associated with chronic and recurrent musculoskeletal symptoms in patients without any visible rheumatologic pathology9. the alteration of proprioceptive acuity may be the cause or the effect of hypermobility, encouraging the adoption of biomechanically inadequate postures and consequently joint trauma. moreover, loose ligaments produce down-regulation related to muscle stretch receptors, reducing the proprioception10. associated to these changes, joint instability in gjh patients can alter the modulation of muscle contraction. bird11 considers that gjh depends not only on ligament laxity, but also on skin, blood vessels and adjacent muscle tissue that allow the occurrence of this phenomenon. simmonds and keer12 reported that usually there is little muscle definition and the rest tone is low even when the individual is submitted to proper training. it is possible to consider that the proprioceptive changes in the modulation of muscle contraction in individuals with gjh may influence the pattern of electrical activity on masticatory muscles associated to a clinical state of tmd. in recent years, several studies involving electromyography of masticatory muscles have been performed13-16. these studies have demonstrated that there is higher electrical activity of the masticatory muscles at rest, especially the anterior temporal, in individuals with tmd. such condition can be explained by the higher activity required to keep the mandibular rest position in patients with tmd and myofascial pain14. although there are studies about gjh and tmd, there is neither consensus about their association nor studies that investigate the pattern of electrical activity of masticatory muscles in individuals with both conditions. understanding the musculoskeletal and ligament system as a complex formed by muscular and fascial chains linked, it should be investigated whether the presence of gjh influence the electrical activity of masticatory muscles. searching individuals with joint hypermobility is essential to identify patients with potential risk to develop certain injuries and prevent them. the pathogenetic mechanisms for the development of joint symptoms in the joint hypermobility are not well defined and may be related to excessive and/or inadequate use of the joint, which may not be a causative factor of tmd but would predispose to its occurrence17. therefore, this study aimed to verify the presence of gjh in individuals with tmd and asymptomatic individuals and to compare the electrical activity of their masticatory muscles. material and methods subjects sixty-one female volunteers aged between 18 and 35 participated in the study and were distributed in two groups. the study group (sg) was composed by 34 individuals presenting signs and chronic symptoms of tmd (for a period superior to 6 months) who sought the discipline of prosthodontics occlusion of the federal university of santa maria or the researchers in response to the research advertising in print and electronic media. the sg volunteers had one or more diagnoses according to the research diagnostic criteria for tmd (rdc/tmd). the control group (cg) was composed by 27 asymptomatic volunteers who did not present any signs and symptoms of tmd or bruxism, based on history and clinical signs according to the rdc/tmd. individuals were excluded from the study with neuropsychomotor impairment, history of orthopedic trauma or malformation of face; systemic or rheumatologic disease in physical, dental or speech therapy prior to the study, or using any medication. the study was approved by the university ethics committee under number 0281.0.243.000-08. all participants were informed about the nature and objectives of the study and signed an informed consent form before participating in the research. evaluation procedures the rdc/tmd classify tmd diagnoses into three groups: i) muscular (only myofascial pain or myofascial pain with limited opening); ii) disc displacement (with or without reduction with limited opening or without reduction without limited opening); iii) arthralgia and tmj osteoarthritis/ osteoarthrosis18. gjh has been evaluated by the criteria of carter and wilkinson modified by beighton19, which have been used in several studies1-2,4-9 on hypermobility. the beighton score examines 9 joints on 5 tests: apposition of thumb to the anterior forearm until they touch; passive dorsiflexion of the little finger until it is parallel to the forearm; trunk flexion with knees fully extended so palms touch the floor; and elbow and knee hyperextension beyond 10 degrees. each joint with hypermobility scores one point. gjh is diagnosed with a score equal or greater than 4. gjh is considered moderate (4-6 points) and severe (7-9 points)4. sg and cg groups were subdivided after this examination: sg with gjh (sgh), sg without gjh (sgn), cg with gjh (cgh) and cg without gjh (cgn). the electromyographic (emg) exam of masticatory muscles (masseter and anterior temporal) was carried out with an electromyography of eight channels with analog-digital conversion board of 16-bit model cad 10/26, sampling frequency of 2 khz, butterworth filter with high-pass cut-off frequency of 10hz and low-pass of 1000hz (lynx electronic technology ltda). the acquisition software bioinspector developed by lynx electronic technology ltda was used. before collecting the emg signal, the skin impedance was reduced by cleaning with isopropyl alcohol swab 70° (isek international society of electromyography and kinesiology). double-junction ag/agcl electrodes (hall indústria e comércio ltda) with circular shape, fixed distance of 20 mm, diameter of 10 mm, 2 mm contact surface, 20x 147 temporomandibular disorder and generalized joint hypermobility: electromyographic analysis of the masticatory muscles 148 braz j oral sci. 10(2):146-151 gain, input impedance of 10 gù and common mode rejection ratio >100 db were connected to active preamps with differential input (pa1020) from lynx electronic technology ltda. surface electrodes were fixed in the region of the muscle belly13 and a reference electrode was fixed on the region of the sternum bone20. for collecting and storing data a notebook dell latitude d520, intel (r) celeron (r) m cpu 430@1.73ghz, real speed of 1.69 ghz, 1536 mb of ram memory, microsoft windows xp professional operating system version 5.1.2600, was used disconnected from the electrical grid. the muscular electric activity was recorded bilaterally in two mandibular positions21: 1) mandibular physiologic rest position: emg signal collected for 10 s. 2) maximal intercuspal: oriented under the examiner’s verbal command “clench, clench, clench”. the signal was collected for 5 s, three times, with the material parafilm “m”® (chicago, il, usa) located between the premolars, the maxillary 1st and 2nd molars. a 2-min between each signal collecting was maintained in order to minimize the effects of muscle fatigue. the recordings was accomplished in the orthostatic posture, with the subject barefoot, feet parallel, arms along the body and stare at a target at eye level. the signal acquisition was carried out three times in each situation and the one with the best quality and lowest presence of noise was selected. emg amplitude values were quantified using the root mean square (rms) and expressed in microvolts (µv)14,21-23. this measure is recommended to represent emg amplitude of static contractions, such as isometric – maximal intercuspal24. the software aqdanalisys 7.0 (lynx electronic technology ltda) was used for the signal processing. statistical analysis statistical analysis was performed by the software statsoft statistica 7.1. the data showed normal distribution by shapiro wilk test (p <0.05). the difference between emg activity of masticatory muscles in sg and cg was analyzed by the student’s t-test for independent samples. comparison of emg activity among the subgroups with and without gjh was verified by one-way anova and tukey’s test was used for multiple comparisons when significant difference was detected. to verify the difference in the frequency of gjh between groups the chi-square and fisher’s tests were used. the level of significance was set at p< 0.05 for all analyses. results from the evaluation of tmd and hypermobility, participants were distributed in a sg (n=34) and a cg (n=27), as described before. these two groups were subdivided into four subgroups: sg with gjh (sgh), n = 22; sg without gjh (sgn), n = 12; cg with gjh (cgh), n = 11; and cg without gjh (cgn), n = 16. the mean age of the participants in the sg was 25.7 ± 5.0 years old and in the control group was 22.4 ± 2.3 years old. the sg was characterized by individuals who had signs and/or chronic symptoms of tmd (more than 6 months). among these symptoms, all participants reported complaints of bruxism concentric and/or eccentric. among subjects with tmd, 64.71% of participants had hypermobility and 35.29% had normal mobility. in the control group, 40.74% had hypermobility and 59.25% showed normal mobility. the chi-square test did not find statistically significant different between the groups (p = 0.0621). besides, the frequency of severe hypermobility (7-9 points on the beighton score) was higher in the sg (23.53%) than in the cg (7.41%). the percentage of severity degrees of gjh in both groups were evaluated using the chi-square and fisher’s tests. there were no statistically significant differences between groups for moderate (p = 0.5301) and for severe hypermobility (p = 0.0878). all tmd individuals were diagnosed with myofascial pain (group i), 41% had a diagnosis of disc disorders (group ii) and 91% had some type of joint involvement (group iii), especially arthralgia (79.41%). when the participants were subdivided according to gjh, there was a higher percentage of myofascial pain without limited mouth opening (ia) in individuals with gjh (81.82%) compared with those without gjh (58.33%). this difference was not significant in the chi-square test (p = 0.2468). disc displacement with reduction (iia) was diagnosed in 31.82% of hypermobility and 41.67% of normal joint mobility participants. the diagnosis of arthralgia (iiia) showed high percentages in both groups (81.82% and 83.33% in the groups with and without gjh, respectively) (table 1.) the results of emg signals of masticatory muscles (masseter and anterior temporal) of the sg and cg are shown in table 2. classification tmd (n=34) gn (n=12) gh (n=22) ia 70.59 58.33 81.82 group i ib 29.41 41.67 18.18 none 0.00 0.00 0.00 iia 38.23 41.67 31.82 group ii iib 0.00 0.00 0.00 iic 2.94 0.00 4.54 none 61.76 58.33 63.64 iiia 79.41 83.33 77.27 group iii iiib 11.76 8.33 13.64 iiic 2.94 0.00 4.54 none 8.82 8.33 9.09 table 1. table 1. table 1. table 1. table 1. results (%) of diagnostic classification of tmd by the rdc/tmd (dworkin & leresche, 199218) according to the presence of gjh legends: ia = myofascial pain; ib = myofascial pain with limited opening, iia = disk displacement with reduction; iib = disk displacement without reduction with limited opening; iic = disk displacement without reduction without limited opening; iiia= arthralgia; iiib = tmj osteoarthritis; iiic = tmj osteoarthrosis; gn = group without gjh; gh = group with gjh. temporomandibular disorder and generalized joint hypermobility: electromyographic analysis of the masticatory muscles 149 braz j oral sci. 10(2):146-151 r masseter l masseter r temporal l temporal rest sg (n =34) 3.92 ± 1.34 4.23 ± 1.62 5.67 ± 1.73 6.24 ± 3.4 cg (n= 27) 3.72 ± 0.97 4.03 ± 1.38 4.94 ± 1.44 4.61 ± 1.57 p 0.0111* 0.0007* 0.0046* 0.1191 maximal intercuspal sg (n=34) 271.03 ± 158.71 254.29 ± 156.51 258.91 ± 154.12 249.86±117.17 cg (n=27) 279.51±171.43 264.18±121.52 271.13±99.70 281.68 ± 61.58 p 0.9476 0.5272 0.3118 0.0513 table 2.table 2.table 2.table 2.table 2. mean and standard deviation of rms values (in µv) of masticatory muscles at mandibular rest and maximal intercuspal position of the study and control groups legends: sg = study group, cg = control group; significance p <0.05 table 3table 3table 3table 3table 3. mean and standard deviation of rms values (in µv) of masticatory muscles at mandibular rest and comparison between study and control groups, distributed on the presence of gjh muscles mean and sd sgh sgn c g h c g n p value r masseter 4.26±1.35 3.29±1.12 3.98±0.96 3.54±0.97 0.0667 l masseter 4.74±1.66 3.30±1.07 4.59±1.41 3.65±1.26 0.0072* r temporal 6.09±1.81 4.90±1.34 5.38±1.29 4.64±1.50 0.0248* l temporal 6.67±3.83 5.46±2.37 4.96±1.93 4.37±1.29 0.1173 legends: sg = study group, cg = control group; sgh = study group with gjh; sgn = study group without gjh; cgh = control group with gjh; cgn = control group without gjh; * statistical significance (p <0.05) when comparing sg and cg, it was observed a higher electrical activity at rest physiologic mandibular in the temporal compared with the masseter muscles, in individuals with tmd. a statistically significant difference (student’s t test) was found for right temporal and masseter muscles. there was no difference in emg activity between groups during the maximal intercuspal position. mean and standard deviations of rms values (in µv) of masticatory muscles at rest physiologic mandibular of the volunteers, classified according to the presence of gjh, are shown in table 3. in the comparison between groups by one-way anova, statistically significant differences were observed for the left masseter and right temporal muscles. the tukey’s test revealed the left masseter muscle was significantly more active in subjects with tmd and gjh (sgh) compared with patients with tmd and normal joint mobility (sgn). also the right temporalis muscle showed a value of rms significantly higher in sgh compared with cgn. yet, there was increased electrical activity in the temporalis muscles, with levels of hyperactivity, mainly in sg and sgh. there were no statistically significant differences in the emg analysis of masticatory muscles during maximal intercuspal among the individuals with normal mobility and with gjh. discussion the association between tmd and gjh has been investigated in several studies1-8 with inconclusive results. in the present study, gjh reached high percentages in both groups, even higher in the individuals with tmd (64.71%). however, there was no statistically significant difference in the incidence and severity of gjh between the study and control groups. this result agrees with conti et al.9 who did not observed differences in the incidence of gjh in symptomatic and asymptomatic groups for tmd, despite the high percentages in both groups. some authors1,7,25 found an association between tmd and gjh, with higher scores of hypermobility in symptomatic individuals. multiple diagnoses according to rdc/tmd were present in most individuals with tmd. however, disc disorders diagnoses were not more frequent in the gjh patients evaluated in this study than in the normal joint mobility, agreeing with conti et al9 and saez-yuguero et al.25. intraarticular dysfunctions have been correlated with gjh and tmj hypermobility1,6, since an excessive movement of the mandibular condyle beyond the articular eminence and the ligaments laxity would facilitate intra-articular joint disc displacements. however, in studies that considered older individuals2627, the diagnosis of disc disorders was more frequent and there are reports1 that the diagnosis of disc displacement increases with age. in the present study, it can be considered that the low mean age of the evaluated volunteers may have contributed for this result. furthermore, tmd individuals showed high incidence of arthralgia, regardless of the joint mobility condition, which may indicate an earlier stage of joint damage considering the young group studied. high percentage of myofascial pain without limited mouth opening was observed in individuals with gjh (81.82%) compared with those without gjh (58.33%), but there was no significant difference between them. hirsh et al.8 confirmed the lower risk of gjh subjects to develop limited mouth opening. the preservation of the mandibular motion range within physiological parameters, in these individuals, may lead to a low functional repercussion and late diagnosis. temporomandibular disorder and generalized joint hypermobility: electromyographic analysis of the masticatory muscles 150 braz j oral sci. 10(2):146-151 on the emg evaluation of masticatory muscles of tmd individuals and control group, there was significant higher electrical activity at rest physiologic mandibular in all assessed muscles, except the left temporal in the tmd group. several authors14-16,28 confirmed the higher electrical activity of masticatory muscles at rest, especially the anterior temporal, in patients with tmd. this behavior is explained by the need for greater muscle recruitment in patients with tmd and myofascial pain at mandibular rest14. in the present study, it was also observed a predominance of electrical activity of the temporal over the masseter muscles, with levels of hyperactivity in sg compared with the cg. rodrigues-bigaton et al. 15 also observed a higher electrical activity of masticatory muscles at rest in tmd patients compared with controls. however, this increase did not reach levels of muscular hyperactivity, but it was considered by the authors as a suggestive sign of tmd. the present study observed levels below of the hyperactivity 5 µv for all muscles studied only in cgn. it suggests that both tmd and hypermobility contribute to the muscle hyperactivity. multiple comparisons in the rest physiologic mandibular activity found significantly higher levels in the left masseter muscle when comparing individuals with tmd and gjh (sgh) and tmd without gjh (sgn). besides, the right temporal muscle was more active in individuals with tmd and gjh (sgh) compared with controls without gjh (cgn). the literature has been reporting higher activity at rest in the masticatory muscles in individuals with tmd14-15,28. the results of this present study indicate that the gjh may contribute to higher emg activity in these muscles. considering that in individuals with tmd associated with gjh the mandibular rest position, which should be maintained by the viscoelasticity of muscles, ligaments, articular capsule and the subatmospheric pressure of mouth13, is hampered by the reduction of ligament resistance10, it is assumed that the masticatory muscles may be recruited to participate in the tmj stabilization. this recruitment appears to occur asymmetric and variedly among all muscles involved in this stabilization, which could compensate the low ligamentar competence and a possible proprioceptive deficit in individuals with gjh. according to ferrell et al.29, besides the excessive motion range of some joints, the only recognized neurophysiologic abnormalities in individuals suffering from gjh were the proprioceptive deficit. on the emg evaluation of masseter and temporal muscles during maximal intercuspal it was not observed significant differences between groups. these results agree with those of rodrigues et al.30, who did not find differences between tmd and control groups during maximal intercuspal. on the other hand, other study14 observed lower emg activity in patients with tmd. in this study, the masticatory muscles of individuals with tmd and gjh presented a different and non-specific activation pattern. thus, it is assumed that tmd demands greater muscle recruitment and gjh determines difficulty in modulating the muscle contraction due to joint instability associated to a proprioceptive deficit. no studies were found in the literature associating emg variables to gjh. however, since gjh is a feature often found in individuals with tmd, it is important to study which effects this phenomenon can cause in the masticatory muscles. as this topic has not yet been explored, further studies are needed to generalize the obtained results. references 1. westling l, mattiasson a. general joint hypermobility and temporomandibular joint derangement in adolescents. ann rheum dis. 1992; 51: 87-90. 2. perrini f, tallents rh, katzberg rw, ribeiro rf, kyrkanides s, moss me. 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masticatory muscles 151 braz j oral sci. 10(2):146-151 gaithersburg, maryland: an aspen publication; 1998. 21. pedroni cr. diagnostic application of surface electromyography for temporomandibular disorders [thesis] piracicaba: fop-unicamp; 2007. 22. ribeiro ec, marchiori sc, da silva am. electromyographic muscle emg activity in mouth and nasal breathing children. cranio. 2004; 22: 145-50. 23. tecco s, epifania e, festa f. an electromyographic evaluation of bilateral symmetry of masticatory, neck and trunk muscles activity in patients wearing a positioned. j oral rehabil. 2008; 35: 433-9. 24. [seniam 7] the state of the art on signal processing methods for surface electromyography, deliverable of the seniam project. hermens hj, merletti r, freriks b, editors. freriks, roessingh research and development; 1999. 25. saéz-yuguero mdel r, linares-tovar e, calvo-guirado jl, bermejofenoll a, rodríguez-lozano fj. joint hypermobility and disk displacement confirmed by magnetic resonance imaging: a study of women with temporomandibular disorders. oral surg oral med oral pathol oral radiol endod. 2009; 107: 54-7. 26. manfredini gc, bosco m. research diagnostic criteria for temporomandibular disorders (rdc/tmd) axis i diagnosis in italian patient population. j oral rehabil. 2006; 33: 551-8. 27. tartaglia gm, moreira rodrigues da silva ma, bottini s, sforza c, ferrario vf. masticatory muscle activity during maximum voluntary clench in different research diagnostic criteria for temporomandibular disorders (rdc/tmd) groups. man ther. 2008; 13: 437-44. 28. bérzin f, sakai e. fundamentos da eletromiografia (emg) – da teoria à prática. in: sakai e., fiuza sc, martins ns, dominguez-rodrigues gc, grimberg j, pereira cb et al., organizators. nova visão em ortodontia e ortopedia funcional dos maxilares. são paulo: santos; 2004. p.311-30. 29. ferrel wr, tennant n, sturrock rd, ashton l, creed g, brydson g et al. amelioration of symptoms by enhancement of proprioception in patients with joint hypermobility syndrome. arthritis rheum. 2004; 20: 3323-8. 30. rodrigues d, siriani ao, bérzin f. effect of conventional tens on pain and electromyographic activity of masticatory muscles in tmd patients. braz oral res. 2004; 18: 290-5. temporomandibular disorder and generalized joint hypermobility: electromyographic analysis of the masticatory muscles oral sciences n3 case report braz j oral sci. january/march 2010 volume 9, number 1 prosthetic rehabilitation for a patient with hypohidrotic ectodermal dysplasia: a clinical case guna shekhar1, alluri ramaraju2, chandrasekhar rao3, sarada1 1associate professor, faculty of pediatric dentistry, vdc, ntr university of health sciences, ap, india 2professor, faculty of prosthetic dentistry, vdc, ntr university of health sciences, ap, india 3professor, faculty of pediatric dentistry, vdc, ntr university of health sciences, ap, india correspondence to: guna shekhar c/o m.prakash, 160, 5th cross, 28th main, sector-1, hsr layout, bangalore-102, india e-mail: drguna1234@yahoo.com received for publication: december 04, 2009 accepted: march 04, 2010 abstract hypohidrotic ectodermal dysplasia (hed) is a hereditary syndrome, characterized by a classic triad of hypotrichosis, hypodontia and hypohidrosis. the case of an 8-year-old girl with hed presenting oligodontia and marked resorption of maxillary and mandibular alveolar ridges is reported. a specially designed aluminum wire tray was used for taking impressions of both dental arches. prosthetic rehabilitation included a removable maxillary overdenture and a mandibular conventional acrylic resin denture made tissue-compatible by means of a soft denture liner attached to denture base. the proposed treatment plan provided improved esthetics, function, and quality of life. keywords: ectodermal dysplasia, oligodontia, soft liner, mucosa supported dentures. introduction ectodermal dysplasia (ed) is a group of hereditary diseases with abnormal development of two or more structures of ectodermal origin1. these disorders are relatively rare with an incidence of 1 in 10,000 to 1 in 100,000 live births2. ed can present numerous clinical alterations and up to 154 different types and 11 subgroups have been observed depending on the involvement of the hair, teeth, nails or sweat glands3. ed can be hypohidrotic or hidrotic depending on the sweat gland function2. hypohidrotic ectodermal dysplasia (hed) is characterized by dry and scaly skin due to absence of sweat and sebaceous glands and the affected child will develop intolerance to heat due to absence or reduced sweat glands causing elevated body temperature1. skeletal manifestations in individuals affected by hed include decreased lower facial height and depth, small cranial base width and calvarial height, prominent forehead and depressed nasal bridge4. oral findings include total or partial anodontia affecting both the primary and the permanent dentitions. the teeth are usually conical or peg shaped. alveolar ridges are underdeveloped resulting in reduced vertical dimension, thereby giving a senile facial appearance along with protuberant dry lips1,4-5. this paper describes the prosthetic rehabilitation of a child with hed presenting severely resorbed alveolar ridges by using specially designed aluminum trays and tissue-supported dentures. case report an 8 year-old girl was referred to the department of pediatric dentistry accompanied by her mother with the complaint of difficulty in eating due to braz j oral sci. 9(1):63-66 several missing teeth since infancy. she is the elder of the two siblings with a younger brother, and was born out of consanguineous marriage. past medical history revealed that the girl had several frequent episodes of fever throughout childhood along with frequent ear and nose infections. the pedigree of the patient could not be researched due to insufficient data from the parents. detailed dialogue history with mother revealed that the girl was intolerant to heat and takes frequent dips in summer to keep cool, and that she had difficulty in speech and social interaction. the girl appeared poorly nourished. extraoral features included frontal bossing, depressed nasal bridge, sunken cheeks, protuberant lips, periorbital pigmentation, dry skin, scanty scalp hair and decreased lower facial height, which suggested typical facial physiognomy of hypohidrotic ectodermal dysplasia (figure 1). intraoral examination revealed a relatively dry mucosa, bone atrophy of the alveolar ridges, on both mandible and the maxilla. mandibular arch was completely edentulous and the maxillary arch presented two widely spaced conoid deciduous incisors (figure 2). fig. 2: maxillary arch with two wide spaced conoid deciduous incisors and completely edentulous mandibular arch. radiographic investigations included digital panoramic radiograph, which displayed numerous agenesis of primary and permanent teeth, conical shaped maxillary deciduous incisors and unerupted malformed permanent central incisors. in the mandible, all of the anterior and posterior permanent tooth germs were missing (figure 3). the patient had a total of 26 congenitally missing permanent teeth excluding third molars. a clinical diagnosis of hed with oligodontia was made and the treatment plan included fabrication of a removable mucosa supported over denture and a conventional acrylic resin denture in the maxillary and mandibular arches respectively. fig. 3: digital panoramic radiograph showing multiple agenesis of permanent teeth and malformed unerupted conical maxillary incisors impression trays were specially designed and fabricated using an aluminum wire stabilized with green wax (figure 4) and impressions were made using putty and light viscosity additional silicone, simultaneous mix technique, for both maxillary and the mandibular arches (figure 5). master casts were obtained and temporary denture bases were prepared. jaw relations were recorded and acrylic artificial teeth were modified by trimming down their size to accommodate the jaw size and were then arranged in class i molar relation. fig. 4: maxillary and mandibular custom-made trays made of aluminum wire and tray check for mandibular arch. fig. 5: master impressions with aluminum trays. after wax try-in acrylic dentures were fabricated lined with a heat cure soft liner (molloplast b, detax gmbh & co, kg ettingen, germany). insertion and removal of the dentures was taught to the child and post-insertion instructions were given regarding mastication, hygiene and maintenance. patient was found to be extremely satisfied with her esthetics. she was instructed to speak and read daily for sometime in order to improve phonation. diet was restricted to semi-solid/liquid, till she was comfortable eating with dentures. during the follow-up visit at the end of 2 months she presented with marked improvement in articulation of speech by means of controlled mandibular movements. fig. 1: frontal and lateral initial views of the patients face. 64 braz j oral sci. 9(1):63-66 prosthetic rehabilitation for a patient with hypohidrotic ectodermal dysplasia: a clinical case the existing two conoid maxillary anterior teeth were preserved and served as retention for the prosthesis. after a 6and 12-month follow-up visit, the patient’s mother gave a positive feedback regarding peer-group interaction and significant improvement in her phonetics (figures 6 and 7). fig. 6: frontal and lateral intraoral views of the patient with the dentures at the 12month follow-up visit. fig. 7: frontal and lateral final extra oral views of the patient’s face at the 12-month follow-up visit. discussion hypohidrotic type, originally known as hed because of notable reduction of sweat gland function, is clinically characterized by triad of hypotrichosis, hypohidrosis and either hypodontia or anodontia6-7. the gene responsible for x-linked recessive trait has been mapped to xq12-q13.18. recent findings reported that mutations in the ectodysplasina (eda) gene are responsible for x-linked hed9 and isolated hypodontia10. van der hout et al.11 identified mutations in the ed1 gene in 24 (57%) of 42 unrelated european probands with hed. genetic counseling of affected patients or parents may be necessary so that they can be informed of the possible recurrent risks and the eventual dental problems that their offspring may encounter. majority of these children present multiple ocular, dental, infectious, and dermatologic problems and require multidisciplinary treatment 7. various rehabilitation possibilities in patients with ed may include removable partial/complete prostheses, fixed, and /or implant-supported prostheses12 or a combination of these options. treatment should be initiated as soon as possible so that further resorption or atrophy of the alveolar ridges due to partial or complete absence of teeth can be prevented and vertical dimension controlled. additionally, cosmetic and psychological issues become more important for the patient later in the childhood and adolescence. therefore early prosthodontic treatment of these patients has been strongly recommended13. the objective of treatment in the present case was not only to reestablish the masticatory function, but also to improve the emotional and social aspect of the child so that the child can lead a normal life-style with positive self esteem. the removal of the conoid teeth for subsequent placement of a prosthetic appliance would further reduce the alveolar ridge of the patient6. therefore the teeth were not extracted. in the present case, the conical teeth served as an aid in retention of the maxillary prosthesis. the child adapted herself well to the prosthesis with great personal satisfaction. severe resorption of the alveolar ridges in ectodermal dysplasia may contribute to instability and discomfort of the conventional acrylic resin dentures. these dentures may frequently irritate residual alveolar ridge tissues, which often are atrophic and minimally resistant to stress14. use of custommade aluminum wire trays will minimize unbalanced and excessive pressures on an already compromised alveolar ridges, and thereby reduce degenerative changes in bony and soft tissue architecture. as the aluminum wire used in the present case has a thin border, it does not impinge upon muscle attachments, which could produce dislodging forces. conventional acrylic resin dentures with a soft liner have proven to be advantageous in patients who has severely atrophic alveolar ridge. the soft liner accommodates ridge irregularities and changes such as excessive resorption, minimal keratinized ridge epithelium and decreased resistance to irritation due to nutritional and physiologic problems15. in the present case, the entire acrylic resin base was lined with a soft liner to prevent abuse to the soft tissues and accommodate the jaw growth. these prostheses need to be changed periodically according to the growth and the bone development. the choice for the treatment approach was based on the child’s physical condition and the need of esthetics. the masticatory and phonetic functions were markedly improved, which would otherwise have had a negative effect on the psychosocial development of the patient. franchi et al.16 in a serial cephalometric study of patients with ed found that the growth of both maxilla and mandible has been favored by the use of conventional prosthesis. the use of tissue-supported (soft liner) maxillary overdenture and conventional acrylic resin denture in the mandibular arch in the present case can be considered as a good practical alternative that provided a relatively quick, easy, acceptable and economical solution to the functional and esthetic oral rehabilitation. moreover, it helps stimulating the alveolar ridges for later treatment with implant-retained dentures, as a more stable alternative. references 1. neville bw oral and maxillofacial pathology. philadelphia: saunders; 1995. p.542-3. 2. della valle d, chevitarese ab, maia lc, farinhas ja. alternative rehabilitation treatment for a patient with ectodermal dysplasia. j clin pediatr dent. 2004; 28: 103-6. 3. pinheiro m, freire-maria n. ectodermal dysplasias: a clinical classification and a casual review. am j med genet. 1994; 53: 153-62. 65 braz j oral sci. 9(1):63-66 prosthetic rehabilitation for a patient with hypohidrotic ectodermal dysplasia: a clinical case 4. kargul b, alen tk, alabay u, atasu m. hypohidrotic ectodermal dysplasia: dental clinical genetic and dermatoglyphic findings of three cases. j clin pediatr dent. 2001; 26: 5-12. 5. ohno k, ohmori i. anodontia with hypohidrotic ectodermal dysplasia in young female: a case report. pediatr dent. 1999; 22: 49-52. 6. abadi b, herren c. clinical treatment of ectodermal dysplasia: a case report. quintessence int. 2001; 32: 743-45. 7. gorlin rj, cohen mm, hennekam rc. hypohidrotic ectodermal dysplasia. in: syndromes of the head and neck. 4th ed. oxford: oxford university press; 2001. p.540 5. 8. kere j, srivastava ak, montonen o, zonana j, thomas n, ferguson b, et al. x-linked anhidrotic (hypohidrotic) ectodermal dysplasia is caused by mutation in a novel transmembrane protein. nat genet. 1996; 13: 409-16. 9. huang c, yang q, ke t, wang h, wang x, shen j et al. a novel de novo frame-shift mutation of the eda gene in a chinese han family with hypohidrotic ectodermal dysplasia. j hum genet. 2006; 51: 1133-7. 10. fan huali, ye xiaogian, shi lisong, yin w, hua b, song g et al. mutations in the eda gene are responsible for x-linked hypohidrotic ectodermal dysplasia and hypodontia in chinese kindreds. eur j oral sci. 2008; 116: 412-7. 11. van der hout ah, oudesluijs gg, venema a, verheij jb, mol bg, rump p et al. mutation screening of the ectodysplasin-a receptor gene edar in hypohidrotic ectodermal dysplasia. eur j hum genet. 2008; 16: 673-9. 12. guckes ad, mc carthy gr, brahim j. use of endosseous implants in a 3year-old child with ectodermal dysplasia: case report and 5-year follow-up. pediatr dent. 1997; 19: 282-5. 13. hummel p, guddack s. psychosocial stress and adaptive functioning in children and adolescents suffering from hypohidrotic ectodermal dysplasia. pediatr dermatol. 1997; 14: 180-5. 14. granger er. practical procedures in oral rehabilitation. philadelphia: lippincott; 1962. p.33. 15. duncan jd, caswell cw, rommerdale eh. simplified technique for placement of a medical-grade adhesive silicone denture liner. j prosthet dent. 1985; 54: 599-602. 16. franchi l, branch r, tollaro i. craniofacial changes following early prosthetic treatment in a case of hypohidrotic ectodermal dysplasia with complete anodontia. j dent child. 1995; 65: 116-72. 66 braz j oral sci. 9(1):63-66 prosthetic rehabilitation for a patient with hypohidrotic ectodermal dysplasia: a clinical case oral sciences n3 original article braz j oral sci. july | september 2015 volume 14, number 3 canal transportation and centering ability of curved root canals prepared using rotary and reciprocating systems marlos barbosa-ribeiro1, silvio josé albergaria2, maria de fátima gesteira malvar2, iêda margarida crusoé-rebello2, brenda paula figueiredo de almeida gomes1, fabíola bastos de carvalho2 1universidade estadual de campinas unicamp, piracicaba dental school, department of restorative dentistry, area of endodontics, piracicaba, sp, brazil 2universidade federal da bahia ufba, dental school, department of dental clinic, area of endodontics, salvador, ba, brazil correspondence to: marlos barbosa-ribeiro faculdade de odontologia de piracicaba unicamp, área de endodontia avenida limeira, 901 13414-903 piracicaba, são paulo, brasil phone: +55 19 2106 5215. fax: +55 19 2106 0144 e-mail: ribeiro.marlos@yahoo.com.br abstract aim: to evaluate canal transportation and centering ability of protaper and waveone systems in curved root canals by cone-beam computed tomography. methods: twenty human molars were randomly divided into two groups according to the system used: group gp (protaper universal®) and group gw (waveone™). ct scans were performed before and after the chemo-mechanical preparation on three points: 2 mm, 3 mm and 4 mm from the tooth apex. the cuttings in dentin were measured and results were statistically analyzed. results: in gp, there was higher percentage in transport at 2 mm and 4 mm toward the mesial wall of the tooth root compared to gw (p<0.05). there was no difference between the systems regarding their centering ability. conclusions: none of the systems was able to touch all the dentin walls and stay centered during the chemical-mechanical preparation. keywords: root canal preparation; endodontics; cone-beam computed tomography. introduction development of nickel-titanium (niti) rotary instruments provided easier and faster root canal instrumentation1-8. many systems have different designs in their structure to minimize procedural errors and achieve a predictable canal preparation. wherefore, it is important to evaluate the mechanical action of these files in order to improve and optimize the endodontic instrumentation5,7-12. the protaper® rotary system has variable taper and some rigidity due to the considerable amount of metal in its structure. these factors may limit its use in curved and flattened root canals, since the instrument is subject to lock in the cervical third and does not touch properly all dentinal walls13. the waveone™ reciprocating system consists of biomechanical preparation with a single file that presents greater flexibility and resistance to cyclic fatigue compared with the conventional niti alloy, due to treatment of the m-wire alloy. in addition, their active part have less variation in taper, which could provide greater control of the instrument by the clinician2,4,9,11,14-19. cone-beam computed tomography (cbct) is a modern and noninvasive diagnostic feature with compact equipment, low dose radiation and allows evaluation of detailed images using different settings. it is useful in comparing anatomical structure of the root canal before and after biomechanical preparation, allowing to detect deviations and transportation3,20-23. it may also allow assessment received for publication: august 12, 2015 accepted: september 23, 2015 http://dx.doi.org/10.1590/1677-3225v14n3a08 braz j oral sci. 14(3):214-218 215215215215215 braz j oral sci. 14(3):214-218 of centering ability of endodontic instruments that indicate the ability of the instrument to remain centered in the root canal17,24. continuous evaluation of the mechanical behavior of endodontic files is important to understand their effect on the chemo-mechanical preparation. thus, the aim of this study was to evaluate canal transportation and centering ability of protaper and waveone systems in curved mandibular molar mesial canals by cbct. material and methods after approval by the ethics research committee of the school of dentistry of piracicaba, university of campinas (protocol number 094/2013), 20 human mandibular molars from the toth bank of the institute of health sciences, federal university of bahia were selected. the method described by estrela et al.22 was used to determine the degree of root curvature. only teeth with apical curvature between 20° and 40° in the mesial-distal direction were selected. the tooth crowns were cross-sectioned with a diamond disc (buehler, lake buff, il, usa) mounted in a metallographic cutter (isomet 1000, buehler) in low rotation (300 rpm) and under constant water irrigation to standardize the root length at 16 mm. pulp tissue removal from pulp chamber was performed with endo-z bur (dentsplymaillefer, ballaigues, switzerland). preparation of the specimens the teeth were randomly divided into 2 groups, n=10 per group (gp and gw) (table 1), identified and mounted in a jaw and attached with silicone impression material. these procedures allowed positioning for tomographic scanning and for standardizing the test for scans before and after root canal procedures. the mesial-buccal roots were marked in all their extension with a gutta-percha point in order to serve as a reference for the measurement on the images (figure 1). chemo-mechanical preparation (cmp) of root canals the working length (wl) was established at 15.0 mm. the cmp was performed according to the manufacturer’s recommendations (table 1). in gw were first used #013 and #016 pathfiles (dentsply-maillefer) to obtain the glide path of the root canal; in gp the glide path was obtained with #10 and #15 hand files. in both groups, irrigation/aspiration at each file exchange was performed with 5.0 ml of 1% naocl using a disposable syringe and needle. after completing cmp, a final flush was done with 3.0 ml of system motion cutting file tip angle protaper universal® continuous rotation slightly positive sx, s1, s2, f1, f2 no cutting waveone™ reciprocation reverse primary no cutting table 1.table 1.table 1.table 1.table 1. kinematics description and the files used in this study. (manufacturer: dentsplymaillefer, ballaigues, switzerland) fig. 1. specimens mounted in a jaw and attached with silicone impression material for positioning the scanner. 17% edta for 3 min followed by 3 ml of 1% naocl. finally, root canals were dried with absorbent paper points. the diameter d0 of the final file was standardized at 0.25 mm for both groups, whereas the time spent by each instrument in gp and the progressive insertion of the file in gw was 30 s. in gp the “shaping files” were introduced with brush movements on all root canal walls. the “finishing files” in gp and the “primary file” of gw were introduced with slight apical pressure in pecking motion up to the wl. the electric engine used for instrumentation in both groups was x-smart plus (dentsply-maillefer) with speed and torque pre-established by the handset. all groups were prepared by a single experienced clinician. analysis of images teeth were scanned before and after cmp perpendicular to the beam of the kodak 9000 3d unit (trophy, france) cbct device with the following settings: 72 kv, 12 ma and 0.1 mm voxel size. the specimen was allocated in a styrofoam box with water to attenuate the radiation beams and simulate the clinical conditions of the soft tissue. calculation and comparison of all scans were made by the cs 3d imaging software at 2 mm, 3 mm and 4 mm from the apex (figure 2). references were taken in axial, parasagittal and paracoronal dimensions and the distances mesial-canal, canal diameter, distal-canal and mesialdistal, as well as buccal-canal, canal diameter, lingual-canal and buccal-lingual were measured (figure 3). the parameters used to evaluate the centering ability followed the proportionality method proposed by gambill et al.17, using the following formula: d1=(x-x’)/(x1-x1') in the buccal-lingual direction and d2=(z-z’)/(z1-z1'), mesialcanal transportation and centering ability of curved root canals prepared using rotary and reciprocating systems 216216216216216 braz j oral sci. 14(3):214-218 fig. 2. computer screen displaying the multidimensional window of software cs 3d imaging recording the images according to the selected cuts. distal direction. if these numbers are not equal, the lower figure is considered as numerator of the ratio. result 1 indicates perfect centering. the direction of the canal transportation was observed using the same method by the following formula: t1=(x-x’)-(x1-x1') and t2=(z-z’)(z1, z1')4. result 0 (zero) means that there was no canal transport, positive (buccal/distal transportation), negative fig. 3. representative image of a cbct scan before (a) and after cmp (b). (lingual/mesial transportation). f o r p a i r e d s a m p l e s w a s u s e d r e p e a t e d m e a s u r e s anova and tukey’s multiple-comparison test or the friedman test for nonparametric data. the significance level was set at 5%. each tooth was evaluated at points 2, 3 a n d 4 m m i n t h e b u c c a l l i n g u a l a n d m e s i a l d i s t a l direction. canal transportation and centering ability of curved root canals prepared using rotary and reciprocating systems results the results showed a high rate of transportation at 2 mm level (p<0.05) for the mesial wall of the root canal in gp (table 2). comparing the groups, both presented a high rate of mesial transportation at the 4 mm level (p<0.05). in relation to the centering ability, both tested systems left untouched dentine areas (table 3) and created transport the root canal towards buccal and/or mesial directions. discussion the cbct is an effective tool for comparisons between mechanical performances of endodontic instruments and has been widely used to evaluate area, volume, centering ability of instruments and transportation of the root canal4-5,7. in this study we used the cs 3d imaging software (kodak dental systems), which allows performing measurements in detail and the analysis of the same point on different dimensions at the multidimensional reconstructions. the fidelity of this examination is the standard for this kind of assessment. during the cmp of curved root canals, files tend to be linear over the entire canal and have more wear of inner wall of curvature in the coronal third and toward the outer edge of the root 20,23. several studies have shown little decentralization and transportation of root canal during cleaning and shaping due to the flexibility of niti automated instruments3,5,9-10,14,19,24-26. the degree of curvature of the root canal, file taper and the canal flattening might influence negatively the cmp13. it could be the reasons why in both groups there was a higher rate of mesial transportation. in gp, there was a higher rate of transportation in the mesial canal wall, which reinforces the above-mentioned idea. the cross section and the surface treatment of the waveone system increase their mechanical efficiency and contribute to a balanced action in cmp. however, the time required for instrumentation seems to influence directly the appropriate modeling of the root canal system. this may have been the reason why waveone did not produce direction group 2 m m 3 m m 4 m m p value* buccal-lingual gp 0 ± 0.1 0 ± 0.3 0.1 ±0.3 0.875 gw -0.2 ± 0.3 -0.1 ± 0.3 0 ± 0.4 0.521 mesial-distal gp -0.04 ± 012** 0.13 ± 0.2 -0.1 ± 0.2 0.039* gw 0.06 ± 0.2 0.05 ± 0.3 0.07 ± 0.2 0.986 table 2.table 2.table 2.table 2.table 2. mean and standard deviation of transportation of root canal. * anova with repeated measures, ** tukey’s test (p<0.05). group distance point p-value 2 m m 3 m m 4 m m gp buccal-distal -0.22 ± 0.2 -0.22 ± 0.2 -0.16 ± 0.1 mesial-lingual -0.09 ± 0.1 -0.07 ± 0 -0.09 ± 0.1 p>0.05 gw buccal-distal -0.15 ± 0.1 -0.21 ± 0.1 -0.14 ± 0.1 mesial-lingual -0.16 ± 0.1 -0.14 ± 0.1 -0.13 ± 0.1 table 3.table 3.table 3.table 3.table 3. mean and standard deviation of centering ability. friedman test (p < 0.05). significant results in this study, corroborating with the results of kim et al.6 (2012). this indicates that the waveone singlefile system does not create excessive transportation compared with protaper, allowing inferring that the reciprocating systems are an effective alternative and tend to be routinely used in endodontic treatment, being a safe technique. according to berutti et al.2 (2012), the reciprocating movement allows a more centralized cmp compared to continuous rotary motion, especially in the apical third. the authors report that quality in debris removal is similar to the sequence of protaper instruments up to f2 file. in this study, despite of waveone having touched less mesial and lingual walls of the root canal than protaper, it remained more centered in the root canal, in the mesial-distal direction as well as in the buccal-lingual one. however, the fact that the instrument remained centered does not necessarily imply that a balanced motion and who managed to touch properly on the canal walls, possibly due to a clamping of the file in the coronal third. versiani et al.18 (2013), assessed by micro-computed tomography the instrumentation of oval canals by waveone, reciproc, self-ajusting (saf) and protaper systems. the authors observed areas with untouched dentin. protaper and waveone systems obtained similar results and the lowest untouched areas compared to saf. thus, it is possible to admit that frequently the number of untouched areas by the files is due either to a deficiency of the technique, but also to anatomical irregularities. this study corroborated this idea, since in both groups it was observed in certain points that the instrument was not able to touch the dentinal walls. this fact may result in clinical implications where portions of the root canal could remain contaminated, especially in the apical third, and this can maintain the infection. a differential of this study is the fact that it considered the centering ability and canal transportation in both root directions (buccal-lingual and mesial-distal) and not only in one direction (usually the mesial-distal) as most studies do4,17,24, allowing a wide approach to the mechanical action of the tested files. considering the methodological limitations of this 217217217217217 canal transportation and centering ability of curved root canals prepared using rotary and reciprocating systems braz j oral sci. 14(3):214-218 218218218218218 study, particularly the use of cbct instead of microcomputed tomography that has more accuracy, we conclude that there was a low percentage in the centering ability of the tested instruments with a considerable index of partially or completely untouched dentinal walls during cmp. the degree of transportation does not seem to compromise the preparation of root canals since both automated systems proved being able of providing good shaping. references 1. arias a, perez-higueras jj, macorra jc. differences in cyclic fatigue resistance at apical and coronal levels of reciproc and waveone new files. j endod. 2012; 38: 1244-8. 2. berutti e, paolino ds, chiandussi g, alovisi m, cantatore g, castellucci a, et al. root canal anatomy preservation of waveone reciprocating files with or without glide path. j endod. 2012; 38: 101-4. 3. gao y, cheung gs, shen y, zhou x. mechanical behavior of protaper universal f2 finishing file under various curvature conditions: a finite element analysis study. j endod. 2011; 37: 1446-50. 4. gergi r, rjeily ja, saber j, naaman a. comparison of canal transportation and centering ability of twisted files, pathfile-protaper system, and stainless steel hand k-files by using computed tomography. j endod. 2010; 36: 904-7. 5. hartmann msm, barletta fb, fontanella vrc, vanni jr. canal transportation after root canal instrumentation: a comparative study with computed tomography. j endod. 2007; 33: 962-5. 6. kim hc, kwak sw, cheung gs, ko dh, chung sm, lee w. cyclic fatigue and torsional resistance of two new nickel-titanium instruments used in reciprocation motion: reciproc versus waveone. j endod. 2012; 38: 541-4. 7. moraes sh, gonçalves m, tanomaru filho m, bonetti filho i. cutting ability of nickel-titanium rotary systems protaper, mtwo and k3. rsbo: rev sul-bras odontol. 2012; 9: 177-82. 8. ozer sy. comparison of root canal transportation induced by three rotary systems with noncutting tips using computed tomography. oral surg oral med oral pathol oral radiol endod. 2011; 111: 244-50. 9. basmaci f, oztan md, kiyan m. ex vivo evaluation of various instrumentation techniques and irrigants in reducing e. faecalis within root canals. int endod j. 2013; 46: 823-30. 10. elsherief sm, zayet mk, hamouda im. cone-beam computed tomography analysis of curved root canals after mechanical preparation with three nickel-titanium rotary instruments. j biomed res. 2013; 27: 326-35. 11. castelló-escrivá r, alegre-domingo t, faus-matoses v, román-richon s, faus-llácer vj. in vitro comparison of cyclic fatigue resistance of protaper, waveone, and twisted files. j endod. 2012; 38: 1521-4. 12. kim hc, kwak sw, cheung gs, ko dh, chung sm, lee w. cyclic fatigue and torsional resistance of two new nickel-titanium instruments used in reciprocation motion: reciproc versus waveone. j endod. 2012; 38: 541-4. 13. park sk, kim yj, shon wj, you sy, moon ym, kim hc, et al. clinical efficiency and reusability of the reciprocating nickel-titanium instruments according to the root canal anatomy. scanning. 2013; 36: 246-51. 14. aguiar cm, mendes da, câmara ac, figueiredo já. evaluation of the centering ability of the protaper universal rotary system in curved roots in comparison to nitiflex files. aust endod j. 2009; 35: 174-9. 15. berutti e, paolino ds, chiandussi g, alovisi m, cantatore g, castellucci a, et al. effect of canal length and curvature on working length alteration with waveone reciprocating files. j endod. 2011; 37: 1687-90. 16. burklein s, hinschitza k, dammaschke t, schafer e. shaping ability and cleaning effectiveness of two single-file systems in severely curved root canals of extracted teeth: reciproc and waveone versus mtwo and protaper. int endod j. 2012; 45: 449-61. 17. gambill jm, alder m, del rio ce. comparison of nickel-titanium and stainless steel hand-file instrumentation using computed tomography. j endod. 1996; 22: 369-75. 18. versiani ma, leoni gb, steier l, de-deus g, tassani s, pécora jd, et al. micro-computed tomography study of oval-shaped canals prepared with the self-adjusting file, reciproc, waveone, and protaper universal systems. j endod. 2013; 39: 1060-6. 19. webber j, machtou p, pertot w, kuttler s, ruddle c, west j. the waveone single-file reciprocating system. clin tech. 2011; 1: 28-33. 20. tepel j, schäfer e, hoppe w. properties of endodontic hand instruments used in rotary motion. part i. cutting efficiency. j endod. 1995; 21: 118-21. 21. torres mgg, campos psf, segundo npn, ribeiro r, navarro m, crusoé-rebello i. [evaluation of referential dosages obtained by conebeam computed tomography examinations acquired with different voxel sizes]. dent press j orthod. 2010; 15: 42-3. portuguese. 22. estrela c, bueno mr, sousa-neto md, pécora jd. method for determination of root curvature radius using cone-beam computed tomography images. braz dent j. 2008; 2: 114-8. 23. rödig t, hülsmann m, kahlmeieir c. comparison of roof canal preparation with two rotary niti instruments: profile .04 and gt rotary. int endod j. 2007; 40: 553-62. 24. gergi r, arbab-chirani r, osta n, naaman a; micro-computed tomographic evaluation of canal transportation instrumented by different kinematics rotary nickel-titanium instruments. j endod. 2014; 40: 1223-7. 25. junaid a, freire lg, da silveira bueno ce, mello i, cunha rs. influence of single-file endodontics on apical transportation in curved root canals: an ex vivo micro-computed tomographic study. j endod. 2014; 40: 717-20. 26. pasternak-júnior b, sousa-neto md, silva rg. canal transportation and centring ability of race rotary instruments. int endod j. 2009; 42: 499506. canal transportation and centering ability of curved root canals prepared using rotary and reciprocating systems braz j oral sci. 14(3):214-218 original articlebraz j oral sci. april/june 2009 volume 8, number 2 digital versus conventional radiography for determination of primary incisor length mariane emi sanabe1, maria daniela basso2, marcela almeida gonçalves3, rita de cássia loiola cordeiro4, lourdes santos-pinto4 1dds, md, phd, student in pediatric dentistry, faculdade de odontologia de araraquara, universidade estadual paulista “júlio de mesquita filho” (unesp), araraquara (sp), brazil 2dds, md, phd, associate professor, department of pediatric dentistry, cascavel dental school, universidade estadual do oeste do paraná (unioeste), cascavel (pr), brazil 3dds, md, phd, associate professor, departament of pediatric dentistry, universidade de franca (unifran), franca (sp), brazil 4dds, md, phd, associate professor, department of pediatric dentistry, faculdade de odontologia de araraquara, unesp, araraquara (sp), brazil received for publication: may 12, 2009 accepted: july 24, 2009 correspondence to: lourdes santos-pinto avenida queiroz filho, 972 – vila harmonia cep 14802-610 – araraquara (sp), brazil e-mail: lspinto@foar.unesp.br abstract aim: this in vitro study evaluated the accuracy of primary incisor lengths determined by digital and conventional radiography compared to the actual tooth length. methods: twenty extracted primary maxillary incisors were mounted in acrylic blocks. tooth length was estimated by using a straight-line measurement provided by the distance measurement tool of a digital dental imaging system (computed dental radiography, schick technologies inc.) and conventional e-speed film radiographs by using a digital caliper. two operators familiar with both radiographic methods performed the estimates. the estimated tooth lengths were compared to the actual tooth lengths measured with the digital caliper. data were statistically analyzed by dahlberg’s equation, paired t test, pearson’s correlation coefficient and anova at 5% significance level. results: there were no statistically significant differences (p = 0.85) between tooth length estimated on digital and conventional radiographs. admitting as clinically acceptable a 0.5-mm discrepancy between the actual tooth lengths and the radiographically estimated lengths, 60% of the radiographic measurements were considered as accurate. when the acceptable difference range was 1.0 mm, the accuracy of the radiographic measurements increased to 100%. conclusions: digital and conventional radiography provided similar tooth length measurements and were equivalent to the actual tooth lengths. keywords: dental radiography, dental digital radiography, primary tooth, odontometry. introduction radiographic determination of tooth length is one of the critical aspects of pulpectomy in primary teeth because minor degrees of resorption may not be obvious radiographically1 and an underling permanent tooth germ can cause image superimposition. consequently, the root apex is not clearly identified. in order to establish the correct working length (wl) for instrumentation of the root canal system, the tooth length should be estimated from a preoperative radiograph, an endodontic file should be inserted up to the established length and another radiograph should be taken to check whether the instrument is positioned at the right level. therefore, accurate tooth length measurements are extremely important to ensure that the file does not pass beyond the apical foramen and causes injuries to the periapical tissues2. overinstrumentation in primary teeth involves the additional risk of damage to the permanent tooth germ3,4. 102 sanabe me, basso md, gonçalves ma, cordeiro rcl, santos-pinto ld braz j oral sci. 8(2): 101-4 the wl is traditionally established based on the examination of conventional intraoral radiographs. the recent technological advances turned the digital radiography into a viable option for the determination of the wl. the reliability of wls established with use of digital radiography has been described as comparable to or even better than that of conventional radiography5-10. other studies11-14, however, reported that conventional radiography is more accurate in comparison to older digital radiographic systems. for most digital radiographic systems, image acquisition and manipulation may be performed using the proprietary softwares supplied by the manufacturers or other commercially available graphic softwares. among the options of digital radiographic systems are the linear measurements between two or more spots localized on the image using the mouse, which produce a numerical measurement, generally in millimeters. although this method has been used for wl determination in permanent teeth6,7,10, little is known about its reproducibility, consistence and accuracy for tooth length assessment in the primary dentition. the purpose of this study was to compare the accuracy of digital and conventional radiography as well as inter and intra-examiner agreement for determination of the length of primary incisors. material and methods after approval of the research protocol by the ethics committee of faculdade de odontologia de araraquara, children from the pediatric dentistry clinic with symptomatology and clinical findings of necrosis and radiographic diagnosis of periapical lesion in the primary incisors were invited to participate in this study. the informed consent was obtained from all parents/legal guardians. twenty primary maxillary incisors with less than 2/3 of root resorption were used in this study. the teeth were stored in 1% thymol solution at room temperature until use. each tooth was measured twice with a digital caliper (mitutoyo corp., tokyo, japan) and the average was recorded as the actual tooth length. the teeth were embedded in acrylic resin blocks and adapted to a custom-made radiograph positioning device that maintained the angle and distance (25 cm) constant between the radiation source and the tooth for both digital and conventional radiographs. a groove was made in the resin block to allow adaptation of the guiding shaft in order to adjust the cylinder of the x-ray equipment, thereby simulating the paralleling radiographic technique. the teeth were radiographed by a single calibrated operator. the exposure times were established in previous evaluations in which radiographs were obtained from extracted teeth with exposure setting ranging from 0.1 to 0.4 seconds. two experienced radiologists, after a careful comparison of the quality of the images provided by both types of radiographic techniques, chose the most appropriates radiographic density for tooth length determination. the digital radiographs were obtained using a computed dental radiography system and #2 sensor (cdr-schick technologies inc., long island city, ny, usa; version 2.6) and x-ray equipment (gnatus xr 6010; gnatus, ribeirão preto, sp, brazil), operating at 60 kvp, 7 ma. the digital images were stored in tiff format for further analysis. the conventional radiographs were taken with e-speed film (eastman kodak, rochester, ny, usa) using the same x-ray equipment. the films were developed by the time/temperature method and mounted on slide frames. on the conventional radiographs, tooth length was estimated using a digital caliper (mitutoyo corp.,). the radiographs were viewed on a light box with no magnification. on the digital radiographs, tooth length was measured directly on the screen of a high-resolution 17” monitor with 100% zoom magnification. the measurement method was the electronic ruler of the proprietary cdr system software (version 2.6; schick technologies inc.). using the left mouse button, a two-click measurement was performed for tooth length determination: one click at the visible edge of the crown and the other at the root apex. prior to the measurements, the electronic ruler was calibrated by measuring an object of known length, a #30 kerr file (les fils d’auguste maillefer s.a., switzerland). enhancement features, such as brightness and contrast, were not used for the on-screen measurements. two experienced pediatric dentists with expertise in both radiographic techniques were calibrated (kappa = 0.94) and assessed twice the length of the teeth with a two-month interval. intra and inter-observer variability was determined by calculating the error of the method from double measurements using dahlberg’s equation and systematic errors were detected through a paired t test15. the measurements obtained with conventional radiography and digital radiography were compared to the actual tooth lengths and analyzed by anova. a 5% significance level was set for all analyses. results the analysis of intra-examiner agreement showed a high reproducibility of tooth lengths for both examiners when digital and conventional radiographic measurements were performed (table 1). considering that both examiners presented excellent measurement reproducibility for both types of radiographic method and that there was small variation of tooth lengths and similar standard deviations, the means were used for results analysis. inter-examiner agreement was excellent for digital radiographic images; however, measurements in the conventional radiography presented random and systematic error (table 1). there was no statistically significant difference (p = 0.85) between the actual tooth lengths and the tooth lengths for both radiographic image types. however, overestimation of the actual tooth length occurred in 70% of the digital radiographs and in 75% of the conventional radiographs (table 2). admitting a 0.5-mm difference between the actual tooth lengths and lengths estimated on the radiographs as a clinically acceptable 103digital versus conventional radiography for determination of primary incisor length braz j oral sci. 8(2): 101-4 intra-examiners inter-examiners rx d rx c rx d rx c ex 1 ex 2 ex 1 ex 2 mean (sd) 11.16 (1.46) 11.26 (1.54) 11.06 (1.55) 11.36 (1.52) 11.21 (1.48) 11.21 (1.53) dalberg’s 0.06 0.04 0.00 0.01 -0.10 -0.29 p 0,469 0,494 0,959 0,695 0,319 .001* r 0.95 0.97 0.97 0.98 0.96 0.97 table 1. tooth length means and standard deviations (mm), random error (dalberg’s equation), systematic error (p) and pearson’s correlation coefficient (r) for intra and inter-examiners (ex) reliability for digital (rx d) and conventional (rx c) images sd: standard deviation; *diferença estatística. radiography overestimated (%) underestimated (%) maximun (mm) minimun (mm) mean (mm) digital 70 30 +0.72 0.90 0.40 conventional 75 25 +0.85 -0.90 0.44 table 2. percentage of overestimated and underestimated radiographic tooth lengths as well as maximum, minimum and mean discrepancies from the actual tooth lengths for each radiographic method discrepancy, 60% of the measurements were considered accurate for both digital and conventional radiography. when the acceptable difference range was 1.0 mm, the accuracy of the radiographic measurements increased to 100%. discussion the results of this study showed no significant difference between the primary tooth lengths obtained with digital and conventional radiographs, suggesting that digital radiography was effective in the assessment of primary anterior tooth length. these findings are compliant with those of similar studies that compared the accuracy of digital images and conventional radiographs for determination of the length primary teeth in vivo16, extracted primary teeth in vitro17 and extracted permanent teeth6,7,10 . the reproducibility of the measurements after a 2-month inter val was excellent for both examiners using either ty pes of radiographic image (table 1). an excellent correlation between digital and conventional radiography has also been found for estimation of canal leng th in permanent teeth w ith a two-week inter val between image acquisition18 as well as for assessment of endodontic w ls in permanent molars obtained from human cadaver ma xilla19,20. the f indings of the present study revealed excellent agreement between the examiners for digital radiographic images measured directly on the screen monitor w ith 100% zoom magnif ication. however, the performance of the examiners for conventional image presented random error (dalberg’s error = 0.29) and systematic error (p = 0.01), probably inf luenced by the radiographs v iew w ith no magnif ication and diff iculty to identif y the root apex when the root resorption was irreg ular. the results of the present study do not agree w ith those of ellingsen et al.11, who found high inter-examiner agreement in both radiov isiography and conventional radiography for determining the position of the tips of small-sized f iles relative to the radiographic apex in extracted permanent teeth. accepting clinically a 0.5-mm discrepancy between the actual tooth length and lengths estimated on the radiographs, 60% of the measurements obtained with either types of radiographic image were considered equivalent to the actual tooth lengths. these findings are consistent with those of martinez-martinez-lozano et al.6, who radiographically compared estimate and actual tooth lengths admitting as acceptable a 0.5-mm difference range, and found that conventional and digital radiological methods were satisfactory in 50.6 and 61.4% of cases, respectively. leddy et al.19 compared radiovisiography imaging and conventional film-based radiography in determining endodontic file length adjustment and reported a 50% increase in length adjustment estimates when a 0.5-mm difference between radiographic and actual measurements were considered as satisfactory. in the present study, when the acceptable difference range was 1.0 mm, the accuracy of the radiographic measurements increased to 100%. this result corroborates the findings of larheim and eggen21, who observed that, for conventional radiography, 95% of the sample presented wl estimates equivalent to the actual wls, admitting a 1.0-mm variation. most tooth length measurements were overestimated in less than 1 mm for both digital and conventional film-based radiographs. this discrepancy could be considered as clinically acceptable because, in order to determine the wl, a radiograph is taken with an endodontic file placed inside the canal 2 mm short of the tooth length. the 1 mm overestimated measurements observed in this study did not allow the file to extend beyond the actual tooth length and past the apical foramen. slightly overestimated root lengths have also been observed in digital radiographs of extracted permanent teeth7,18,22 and dry mandibular jaw sections23. this study controlled possible sources of error in radiographic images, such as the distance from the tooth to the radiation source and to the film or sensor, as well as the vertical and horizontal cone angulation. a custom-made positioning device allowed holding the specimen, the x-ray equipment cone and the film and maintaining the sensor in a fixed position. it was, therefore, possible to simulate the paralleling radiographic technique, in which the estimated tooth length is closer to the actual length21. 104 sanabe me, basso md, gonçalves ma, cordeiro rcl, santos-pinto ld braz j oral sci. 8(2): 101-4 digital image calibration was performed before each tooth length determination using the on-screen calibration tool to measure the image of an endodontic file of a known length. it was done because it has been shown that calibrated digital measurements are more accurate than uncalibrated measurements24. in conclusion, digital and conventional radiography prov ided similar tooth leng th measurements and were equivalent to the actual tooth leng ths, which validates both techniques for endodontic image acquisition in primar y teeth. the null hy pothesis is therefore accepted. it must be emphasized, however, that in this study the radiographic images were obtained under well controlled and standardized conditions, which is ver y diff icult to be reproduced in a clinical setting, especially w ith pediatric patients. notw ithstanding, some characteristics of the digital images, such as reduced patient exposure to radiation, possibility of producing instant images and elimination of the chemical solutions used for image processing, make them a valuable alternative for endodontic leng th measurements in primar y teeth. acknowledgements the authors wish to thank to fundação de amparo à pesquisa do estado de são paulo (fapesp) for the financial support. references 1. mente j, seidel j, buchalla w, koch mj. electronic determination of root canal length in primary teeth with and without root resorption. int endod j. 2002;35:447-52. 2. rodd hd, waterhouse pj, fuks ab, fayle sa, moffar ma. pulp therapy for primary molars. int j paed dent. 2006;16:15-23. 3. troutman kc, reisbick mh. pulp therapy. in: stewart re, barber tk, troutman kc, wei shy, editors. pediatric dentistry: scientific foundations and clinical practice. 1982; toronto: mo mosby co. p. 908-17. 4. katz a, mass e, kaufman ay. electronic apex locator: a useful tool for root canal treatment in the primary dentition. asdc j dent child. 1996;63:414-7. 5. cedeberg ra, tidwell e, frederiksen nl, benson bw. endodontic working length assessment: comparison of storage phosphor digital imaging and radiographic film. oral surg oral med oral pathol endod. 1998;85:325-8. 6. martinez-lozano ma, forner-navarro l, sanches-cortez jl, llena-puy c. methodological considerations in the determination of working length. int endod j. 2001;34:371-6. 7. mentes a, gencoglu n. canal length evaluation of curved canals by direct digital or conventional radiography. oral surg oral med oral pathol oral radiol endod. 2002;93:88-91. 8. radel rt, goodell gg, mcclanaban sb, cohen me. in vitro radiographic determination of distances from working length files to tooth ends comparing kodak rvg 6000, schick, and kodak insight film. j endod. 2006;32:566-8. 9. versteeg kh, sanderink gch, van ginkel fc, van der stelt pf. estimating distances on direct digital images and conventional radiographs. j am dent assoc. 1997;128:439-43. 10. woolhiser ga, brand jw, hoen mm, geist jr, pikula aa, pink fe. accuracy of filmbased, digital, and enhanced digital images for endodontic length determination. oral surg oral med oral pathol oral radiol endod. 2005;99:499-504. 11. ellingsen ma, harrington gw, hollender lg. radiovisiography versus conventional radiography for detection of small instruments in endodontic length determination part i: in vitro evaluation. j endod. 1995;21:326-31. 12. ellingsen ma, hollender lg, harrington gw. radiovisiography versus conventional radiography for detection of small instruments in endodontic length determination part ii: in vivo evaluation. j endod. 1995;21:516-20. 13. hedrick rt, dove sb, peters dd, mcdavid wd. radiographic determination of canal length: direct digital radiography versus conventional radiography. j endod. 1994;20:320-6. 14. sanderink gch, huiskens r, van der stelt pf, welander us, stheeman se. image quality of direct digital intraoral x-ray sensors in assessing root canal length. the radiovisiography, visualix/vixa, sens-a-ray, and flash dent systems compared with ektaspeed films. oral surg oral med oral pathol. 1994;78:125-32. 15. houston, wjb. the analysis of errors in orthodontic measurements. am j orthod.1983;83:383-90. 16. santos-pinto l, cordeiro r de c, zuanon ac, basso md, gonçalves ma. primary tooth length determination in direct digital radiography: an in vivo study. pediatr dent. 2007;29:470-4. 17. subramaniam p, konde s, mandanna dk. an in vitro comparison of root canal measurement in primary teeth. j indian soc pedod prev dent. 2005;23:124-5. 18. burger cl, mork to, hutter jw, nicoll b. direct digital radiography versus conventional radiography for estimation of canal length in curved canals. j endod. 1999;25:260-3. 19. leddy bj, miles da, newton cw, brown ce. interpretation of endodontic file lengths using radiovisiography. j endod. 1994;20:542-5. 20. sheaffer jc, eleazer pd, schetz jp, clark sj, farman ag. endodontic measurement accuracy and perceived radiograph quality: effects of film speed and density. oral surg oral med oral pathol oral radiol endod. 2003;96:441-8. 21. larheim ta, eggen s. determination of tooth length with a standardized paralleling technique and calibrated radiographic measuring film. oral surg oral med oral pathol oral radiol endod. 1979;48:374-8. 22. velders xl, sanderink gch, van der stelt pf. dose reduction of two digital sensor systems measuring file lengths. oral surg oral med oral pathol endod. 1996;81:607-12. 23. borg e, gröndahl hg. endodontic measurement in digital radiographs acquired by a photostimulable, storage phosphor system. endod dent traumatol. 1996;12:20-4. 24. loushine rj, weller n, kimbrough wf, potter bj. measurement of endodontic file lengths: calibrated versus uncalibrated digital images. j endod. 2001;27:779-81. oral sciences n3 original article braz j oral sci. july/september 2010 volume 9, number 3 received for publication: october 07, 2009 accepted: july 06, 2010 dental caries experience in children attending an infant oral health program leila maria cesário pereira pinto1, luiz reynaldo de figueiredo walter2, celio percinoto3, cássia cilene dezan4, murilo baena lopes5 1dds, ms, phd, professor, department of dentistry, university of north parana (unopar), londrina; and assistant professor, department of oral medicine and pediatric dentistry, state university of londrina (uel), brazil 2dds, phd, assistant professor, department of oral medicine and pediatric dentistry, state university of londrina (uel), brazil 3dds, ms, phd, professor, department of pediatric and social dentistry, são paulo state university (unesp), brazil 4dds, ms, phd, professor, department of oral medicine and pediatric dentistry, state university of londrina (uel), brazil 5dds, ms, phd, professor, department of dentistry, university of north parana (unopar), londrina, pr, brazil correspondence to: leila maria cesário pereira pinto av. rio de janeiro, 1630 apto 502 centro 86.010-150 londrina, pr, brazil e-mail: jolugui@sercomtel.com.br abstract aim: to investigate: a) caries experience in 5 year-old children and its relationship to the caries risk evaluation made before 1 year of age; b) compliance of parents to an infant oral health program as well as the abandonment reasons. methods: group a (ga) 242 children (60-71 months old) who stayed in the program and had been enrolled since their first year of life underwent a clinical examination according to the who’s criteria; their parents/caregivers were interviewed to ascertain their compliance to the program. group b (gb) – parents of 60 children, who had dropped out of the program, were interviewed to check the reasons of withdrawing. the caries risk classification was based on the file at the first appointment. chi-square test was used (α=0.05) for statistical analyses. results: most of the children were free of caries (71.1%). caries risk evaluation showed low sensibility (34.3%, 95% ci = 22.4–46.1) and high specificity (74.4%, 95% ci = 67.6–81.2) to caries experience. sixty-two mothers (25.6%) reported difficulty to follow the guidelines. the main reason for dropping out was changing to similar health services (40.0%). conclusions: caries risk in the first year of life was not efficient to predict dental caries experience at 5 years of age. educational practice should be improved to increase the compliance of the parents to the program. keywords: dental education, prevention, behavior. introduction prevalence, severity and level of dental caries progression have declined in the younger segment of the population of well developed countries over the last few decades1. in latin america and the caribbean, the decline in the severity of the disease has been less conspicuous in preschool age children (5-6 years old) than in schoolchildren (11-13 years)2. in the united states, the prevalence of caries has declined in 3 to 4-year-old children, but the severity of the disease has increased3. the role of fluoride in reducing caries is well documented, but little emphasis has been given to the educational practices in dental caries prevention and control. however, since dental caries is a disease than can develop as early as the first year of life4, parents and pediatricians should be made aware of this condition through education5. braz j oral sci. 9(3):345-350 346 in breast-fed infants, the guidance and motivation of parents to participate in caries prevention leads to their greater compliance in controlling the risk factors6. moreover, as a preventive strategy, caries risk evaluations involving the identification of children with a potential for developing the disease enables dental health services to target those most in need of such services, providing greater efficiency of the procedures, appropriate levels of assistance, and economic effectiveness7. according to twetman et al.8, the assessment of risk factors for the disease in the first year of life enhances the effectiveness of preventive procedures. the aim of this study was to investigate: a) caries experience in 5 year-old children and its relationship to the caries risk evaluation made before 1 year of age; b) compliance of parents to an infant oral health program as well as the abandonment reasons. material and methods this transversal study included retrospective data collection. the sample comprised all children (n = 484) between 60 to 71 months old enrolled in their first year of life at a public infant oral health program in a brazilian city with fluoride supplied water. the project was approved by the local ethics in research committee. the study began with an analysis of the patients’ file to classify the children participation in the program as follow: a) those whose time interval between the last appointment and the research clinical examination did not exceed 10 months, classified as group a (ga) “those that stayed in the program” b) those whose time interval between the last appointment and the research clinical examination exceed 10 months, classified as group b (gb) “those that dropped out the program”. the exclusion criteria were: a) refusal to participate in the survey; b) children with special needs9. among those that stayed in the program (ga), a caries risk classification based on the patient’s case history recorded on his/her file at the first appointment in the first year of life was made. patients classified as high risk were the ones presenting one or more caries risk indicatives, while those classified as low risk were infants presenting no indicative of caries risk or those who had no teeth, even when caries risk behaviors were encountered i.e. nocturnal feeding. figure 1 describes the criteria adopted for caries risk classification in the first year of life. an oral examination was then carried out to diagnose dental caries in ga. all exams were carried out by a trained and qualified examiner (kappa intra-rater index = 0.92). ten percent of the total sample was reexamined during the data collection (kappa = 0.98). caries diagnostic criteria were based on the methods recommended by the world health organization10. the teeth were dried with a gentle air stream and illuminated with artificial light. the examination was visual, aided by a flatsurfaced dental mirror. the dmft index was used to verify the caries experience of the participants in the study10. the parents/caregivers were interviewed to ascertain their compliance to the program, evaluating their difficulty to follow the guidelines of the program. the evaluated aspects were: nocturnal feeding, sugar intake, teeth brushing, use of fluoride and regular visits to the clinic. the data were recorded according to the frequency that the parents followed the guidelines of the program regularly (regular frequency), occasionally (irregular frequency), and hardly ever (rarely). the parents/caregivers enrolled in gb were interviewed by phone call, using a structured questionnaire to evaluate the possible cause for abandoning the program, as well as the continuity of their dental care in other locations. the data were analyzed using the statistical non-parametric chisquare test with 5% level of significance. results the evaluation of the patient’s files indicated that the total sample comprised 484 children of both genders aged 60 to 71 months, who were divided into: ga 300 (62.0%) who stayed in the program and had been enrolled since their first year of life; gb 184 (38.0%) children who had left the program before the case histories were evaluated for this survey. among children belonging to ga there were 58 losses. the reasons for losses were: 2 parents/caregivers refused to participate, 39 dropped out the program without explanation while the research was being conducted, 8 were transferred to similar health services and 9 could not be encountered. the final sample of those that stayed at the program was composed by 242 children. data about the age and number of erupted teeth registered on the patients records showed that at the first appointment: 23.8% were 6 months old or less and 76.2% were older than 6 months, 58.9% presented one or more erupted teeth and 41.1% did not. it was also observed that 85.3% had regular returns to the appointments, considered as children up to the age of 3, which time elapsed from one appointment to other did not exceeded 1 month of the scheduled appointment, and after this age, not exceeding 2 months. about children who had dropped out of the program (gb), 60 parents/caregivers could be contacted. a) children attended the infant oral health program (ga) dental caries prevalence in 5 years old at the time of the clinical examination, most of the children were free of caries (71.1%). the encountered dmft was 1.0 (sd = 2.1). no dental loss due to caries was recorded, and the number of filled teeth exceeded that of decayed (table 1). the dmft for the group of children presenting caries (n = 70) was 3.5 (sd = 2.5). relationship between the caries experience and the caries risk evaluation to evaluate if the caries risk indicators adopted in the program were efficient in predicting future caries lesions the sensibility and specificity of the method was calculated. the indicators adopted to determine the caries risk in the first year of life revealed low sensibility to caries experience at 5 years of age, once 34.3% (95% ci: 22.4 – 46.1) of the children with caries were identified as high risk at the dental caries experience in children attending an infant oral health program braz j oral sci. 9(3):345-350 347 number of lesions n dmft d m f mean s d mean s d mean s d mean s d 0 172 (71.1%) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1 to 3 43 (17.8%) 1.8 0.8 0.9 0.9 0.0 0.0 0.9 1.0 > 3 27 (11.1%) 6.1 2.1 2.5 2.0 0.0 0.0 3.6 2.2 total 242 (100.0%) 1.0 2.1 0.4 1.1 0.0 0.0 0.6 1.4 table 1. dmft index and its components according to the number of lesions in ga dmft > 0 dmft = 0 total n % n % n % high risk 24 35.3 44 64.7 68 100.0 low risk 46 26.4 128 73.6 174 100.0 total 70 28.9 172 71.1 242 100.0 caries experience at 5 years old caries risk before 12 months old n = number of children; chi-square test: χ2 = 1.46 with1 g.l. and value of p = 0.226 table 2. distribution of sample (n, %) according to the caries risk before 12 months of age and dental caries experience at the age of 5 in ga beginning of the program. however, the specificity was high, pointing out that among the children without caries at age 5, 74.4% (95% ci: 67.6 – 81.2) were identified as low risk in first year. the positive predictive value of the method was low, since 35.29% (95% ci: 23.2 – 47.4) of the children who entered the program with high risk developed the disease, while its negative predictive value is high, because it showed that 73.6% (95% ci: 66.7 – 80.4) of the children who entered the program with low risk did not develop the disease. no significant associations (p>0.05) was encountered between the risk evaluation at the beginning of the program and caries experience at age of 5 (table 2) compliance of parents/caregivers sixty-two mothers (25.6%) reported having encountered some difficulty to follow the guidelines of the program, while 180 (74.4%) did not have problem. the topics of the guidelines with more frequency of “occasionally” and “hardly ever” answers involved the control of nocturnal feeding (35.1%) followed by sugar intake (28.9%) (table 3). on the other hand, greater compliance (“regularly”) was recorded for the regular visits to the clinic (94.2%) followed by the use of fluoride (93.0%). b) children that dropped out the program (gb) abandonment reasons to the program the most frequent reason for abandoning the program regularly 157 64.9 172 71.1 192 79.3 225 93.0 228 94.2 occasionally 30 12.4 32 13.2 34 14.1 9 3.7 9 3.7 hardly ever 55 22.7 38 15.7 16 6.6 8 3.3 5 2.1 total 242 100.0 242 100.0 242 100.0 242 100.0 242 100.0 n % n % n % n % n % frequency of compliance to guidelines nocturnal feeding sugar intake brushing/cleaning use of fluoride regular visits to the clinic n = number of mothers/caregivers table 3. parents’ compliance (n, %) to guidelines in ga was changing to a similar health service (40.0%) in londrina or in another city (figure 2-a). according to the mothers’ report, 50.0% of the cases were due to the need of being attended in a place closer to their homes (figure 2-b). the current situation of dental care of those who dropped out of the program showed that 65.0% (n = 39) were still receiving dental care. among these, 79.9% (n = 30) were in preventive treatments, 15.4% (n = 6) in restorative treatment because of dental caries and 7.7% (n = 3) in orthodontic treatment. only 35.0% (n = 21) affirmed not to be under any kind of dental care. discussion the program described in this paper has been operating to promote oral health for children since 1985. inclusion criteria of the program are the age (the child must be underage of 12 months) and to be free of caries. the children are assisted from the first year of life up to the age of 71 months. the service is focused on parents’ education and preventive measures for the children, which are planned according to a periodic evaluation of caries risk. parents’ education is based on a collective educational approach and an individual follow-up in order to evaluate and guide parents about their children’s needs. the preventive measures applied to infants involve the removal of dental plaque and fluoride dental caries experience in children attending an infant oral health program braz j oral sci. 9(3):345-350 criteria* high risk low risk nocturnal cleaning or brushing absent or irregular diary done nocturnal feeding (breast, bottle or both) present absent nocturnal feeding followed by oral hygiene absent present sugar intake(including intake at meals and between meals on demand use of bottle/sippy cup containing liquid other than water; consumption of juice, carbonated beverages, or sports drinks; use of sweetened medications) >6 < 6 *a single risk indicator in any area of the “high risk” category classifies a child as being “high risk” fig. 1. criteria used for initial classification of risk fig. 2. reasons for abandoning the program (a) and justifications for changing service (b) in gb application. for the children enrolled in the program before dental eruption, the mothers behaviors and the children habits related to oral health are evaluated previously to the educative measures be performed. the parents’ education and preventive measures performed at every visit to the clinic generate feedback for their children oral health improvement11. the appointments were scheduled tri-monthly to the child up to 3 years old, and at a 6-month interval thereafter. there is some flexibility in the schedule of the dental appointments to assure parental compliance to the program. the nucleus of dentistry for babies is funded by the sus (brazilian public health system) and integrated to the municipal health system of londrina, state of parana, brazil. it provides health services to all socioeconomic levels of the local and regional community, with more than 17,000 infants enrolled in the program. the dmft found in 5-year-old children participating in the program (1.0, sd = 2.1) was lower than that reported for brazil (2.8, sd = 3.5), according to an epidemiological survey conducted by the sb brazil 2003 project12. the results of the dmft index by macro-region in brazil were also higher than those of the program (north – 3.2, sd = 3.6; northeast – 3.2, sd = 3.7; southeast – 2.5, sd = 3.3; south – 2.6, sd = 3.4; mid-west – 2.7, sd = 3.3)12. although the living conditions are different, the number of caries-free children (71.1%) was similar to that found in oral health programs in well-developed countries such as finland13, denmark14 and france15. these findings are also in agreement with those of ismail et al.16, who reported a significant correlation between regular visits to the dentist and low caries indices. in addition, the results confirm the importance of oral health promotion programs as an effective mean to facilitate the adoption of healthy behaviors and, hence, better health for children6,17-21. the successful promotion of health is associated with the development of different parental skills allied to the guidelines offered by the health services and the child’s regular visits to the clinic17. the method employed in the caries risk evaluation in the first year of life revealed a low positive predictive and a high negative predictive value for dental caries experience at the age of 5. in this way, the findings of this survey confirm the need for establishing specific protocols for risk evaluation in different age groups, since children’s habits are being formed and, if inappropriately established, they become resistant to changes. additionally, the importance of determining caries risk in preventive treatment planning are pointed and are in agreement with data reported in the literature, which identify risk evaluation as the key to a successful prediction of disease in all age groups13,22-23. the specificity of the method used in this study was good. the test demonstrated that among the children that were classified as low risk at the beginning of the program, 74.4% were free of caries. on the other hand, the sensibility was low, once from those children that were diagnosed as high risk at the beginning of the program only 34.3% developed caries lesions. the low sensibility may be related to the interposal of educative and preventive measures to reverse the caries risk from high to low since the first patients’ appointment to the clinical examination at 5 years old. legal and ethical aspects must be considered in this situation because the dentist is supposed to promptly advise the parents/caregivers when a patient is diagnosed as high caries risk. another element to be considered is the major dietary changes occurring at the period of transition from breast or 348dental caries experience in children attending an infant oral health program braz j oral sci. 9(3):345-350 349 bottle-feeding to familiarly diet in childhood. an obstacle to the clinical implementation of caries risk evaluation is the lack of studies investigating how the application of the risk evaluation methodology affects future oral health24. moreover, there is no single risk factor or combination of factors that has reached high positive and negative predictive values25, although previous caries experience is still the best indicator of the future development of caries. however, the caries experience is not commonly used with children because it is important to determine the risk of caries before the disease manifest24, especially in infants (1-23 months). the parents’ motivation through personalized guidelines shows a stronger effect in caries prevention compared to traditional health education6. however, this survey revealed that even with the personalized educational practices provided by the program, 25.6% of the parents reported finding some difficulty to follow the guidelines, especially those involving control of nocturnal feeding and sugar intake. thus, a more specific approach is required for individual assessment of each child. in addition to this, personalized alternatives could be indicated to control these factors, such as: a) feeding throughout the first year of life, but before eruption of the first teeth; b) establishing a maximum limit of 12 to 14 months for breast and bottle feeding26. these measures established in the first year of life could control the free demand for sugar intake from juices, soft drinks and other cariogenic liquids via bottles or cups. parents abandon oral health promotion programs for several reasons, e.g., choose to take their children to be treated by the family dentist, change their address, mothers may begin or go back to work when their children are old enough, lack encouragement or the may be even financial reason 17. in this study, the most frequent reason for abandoning the program was changing for similar health service (40.0%), which demonstrates the program’s ability in become parents conscious of their role in promoting their children oral health. the change from one clinic to another rather than giving up of oral health promotion means that the program has being reaching some of its goals. although the program offers daily services from mondays through fridays, morning and afternoon, difficulties were reported to appointment times, resulting in the child’s loosing the place in the program due to consecutive absences. the continuity of dental care by those who dropped out of the program can be considered an indicative of the education importance to a parents/caregivers preventive conscious. despite of the parents’ compliance difficulties, the educative-preventive measures lead to good oral health when adopted since the first year of life, independently of the caries risk in this age. the findings of this survey showed that caries risk in the first year of life was not efficient to predict dental caries experience at 5 years of age. the caries risks must be systematically checked between 1 and 5 years when hygiene and diet undergo drastic changes. the importance of oral health promotion should also be highlighted since the first year of the children’s life. although dental health promotion through educational and preventive practices since the child first year of life leads to a good oral health, educational practices should be improved to increase the compliance of the parents to the guidelines of the oral health program. the change from one clinic to another rather than giving up of oral health promotion program means that the program has reached some of its goals, showing that the parents become conscious of the importance of dental health promotion for their children. references 1. brambilla e, garcia-godoy f, strohmenger l. principles of diagnosis and treatment of high-caries-risk subjects. dent clin north am. 2000; 44: 507-40, vi. 2. bonecker m, cleaton-jones p. trends in dental caries in latin american and caribbean 5-6and 11-13-year-old children: a systematic review. community dent oral epidemiol. 2003; 31: 152-7. 3. douglass jm, montero mj, thibodeau ea, mathieu gm. dental caries experience in a connecticut head start program in 1991 and 1999. pediatr dent. 2002; 24: 309-14. 4. ferreira sh, beria ju, kramer pf, feldens eg, feldens ca. dental caries in 0to 5-year-old brazilian children: prevalence, severity, and associated factors. int j paediatr dent. 2007; 17: 289-96. 5. adewakun aa, beltran-aguilar ed. early childhood caries in anguilla, british west indies. gen dent. 2003; 51: 42-7. 6. weinstein p, harrison r, benton t. motivating parents to prevent caries in their young children: one-year findings. j am dent assoc. 2004; 135: 731-8. 7. stamm jw, disney ja, graves rc, bohannan hm, abernathy jr. the university of north carolina caries risk assessment study. i: rationale and content. j public health dent. 1988; 48: 225-32. 8. twetman s, garcia-godoy f, goepferd sj. infant oral health. dent clin north am. 2000; 44: 487-505. 9. american academy of pediatric dentistry. 2007-08 definitions, oral heath policies, and clinical guidelines. pediatr dent. 2007. 10. oral health surveys: basic methods. 4th ed. geneva: world health organization; 1997. 11. walter lrf, ferelle a, issao m. educación odontológica: necesidaes educativas. in: odontología para el bebé: odontopediatría desde el nacimiento hasta los 3 años. caracas: amolca; 2000. p.73-92. 12. brazil. bureau of health care secretary, department of basic care. project sb brazil 2003: oral health conditions of brazilian population: main results. brasília; 2004. p.1-68. 13. mattila ml, paunio p, rautava p, ojanlatva a, sillanpaa m. changes in dental health and dental health habits from 3 to 5 years of age. j public health dent. 1998; 58: 270-4. 14. wendt lk, carlsson e, hallonsten al, birkhed d. early dental caries risk assessment and prevention in pre-school children: evaluation of a new strategy for dental care in a field study. acta odontol scand. 2001; 59: 261-6. 15. adam c, eid a, riordan pj, wolikow m, cohen f. caries experience in the primary dentition among french 6-year-olds between 1991 and 2000. community dent oral epidemiol. 2005; 33: 333-40. 16. ismail ai, sohn w. the impact of universal access to dental care on disparities in caries experience in children. j am dent assoc. 2001; 132: 295-303. 17. harrison rl, wong t. an oral health promotion program for an urban minority population of preschool children. community dent oral epidemiol. 2003; 31: 392-9. 18. nurko c, skur p, brown jp. caries prevalence of children in an infant oral health educational program at a wic clinic. j dent child. 2003; 70: 231-4. 19. scavuzzi ai, de franca caldas junior a, couto gb, de vasconcelos mm, de freitas soares rp, valença pa. longitudinal study of dental caries in brazilian children aged from 12 to 30 months. int j paediatr dent. 2007; 17: 123-8. 20. sgan-cohen hd, mansbach ik, haver d, gofin r. community-oriented oral health promotion for infants in jerusalem: evaluation of a program trial. j public health dent. 2001; 61: 107-13. 21. kalyvas di, taylor cm, michas v, lygidakis na. dental health of 5-yearold children and parents’ perceptions for oral health in the prefectures of athens and piraeus in the attica county of greece. int j paediatr dent. 2006; 16: 352-7. dental caries experience in children attending an infant oral health program braz j oral sci. 9(3):345-350 350 22. ramos-gomez fj, tomar sl, ellison j, artiga n, sintes j, vicuna g. assessment of early childhood caries and dietary habits in a population of migrant hispanic children in stockton, california. asdc j dent child. 1999; 66: 395-403, 366. 23. tinanoff n. dental caries risk assessment and prevention. dent clin north am. 1995; 39: 709-19. 24. tinanoff n, douglass jm. clinical decision making for caries management in children. pediatr dent. 2002; 24: 386-92. 25. zero d, fontana m, lennon am. clinical applications and outcomes of using indicators of risk in caries management. j dent educ. 2001; 65: 1126-32. 26. nainar sm, mohummed s. diet counseling during the infant oral health visit. pediatr dent. 2004; 26: 459-62. dental caries experience in children attending an infant oral health program braz j oral sci. 9(3):345-350 oral sciences n3 original article braz j oral sci. july | september 2015 volume 14, number 3 degree of conversion of a flowable composite light-activated through ceramics of different shades and thicknesses adilson yoshio furuse1, daniel hatschbach glir2, fabio antonio piola rizzante1, rayane prochnow2, ana flávia sanches borges1, carla castiglia gonzaga2 1universidade de são paulo usp, school of dentistry of bauru, department of operative dentistry, endodontics and dental materials, bauru, sp, brazil 2universidade positivo up, school of dentistry, graduate program in dentistry, curitiba, pr, brazil correspondence to: adilson yoshio furuse departamento de dentística, endodontia e materiais odontológicos faculdade de odontologia de bauru, universidade de são paulo usp alameda dr. octávio pinheiro brisolla, 9-75 cep: 17012-901 bauru, sp, brasil phone: +55 14 3235-8253 e-mail: furuse@usp.br abstract the cementation of thin ceramic veneers employing fluid light-activated resin-based materials is a common procedure in the dental practice. aim: to evaluate the influence of ceramic thickness and shade on the degree of conversion (dc) of a flowable light-activated material. methods: flowable resin composite (tetric n-flow – shade a1) was light-activated through ceramic discs of two shades (ips classic a1 and a3) in three thicknesses (0.5, 0.7 and 1.0 mm). for control, the resin composite was light-activated without ceramic interposition. dc was evaluated by ftir (n=5). data were analyzed by one-way anova and dunnett’s t3 test (α=0.05). results: statistically significant differences between groups were observed (p<0.001). control group (without the interposition of ceramic) showed the highest mean for dc (71.9± 1.7). considering the interposed ceramic disc groups, the highest dc values were obtained when 0.5 and 0.7 mm a1 ceramics were used (64.6±1.2 and 64.4 5.0, respectively) and the lowest dc values were obtained for 0.7 and 1 mm a3 ceramics (61.74±0.9 and 62.0±1.9, respectively). conclusions: no flowable resin composite group with interposing ceramics reached a dc similar to the control group. keywords: composite resins; polymerization; dental materials; ceramics. introduction dental ceramics have become well known due their great esthetic quality, capacity of mimicking the dental structure, high wear and staining resistances, color stability and biocompatibility1. conversely, it is well established that ceramic veneers should be firmly fixed to dental structure in order to achieve better clinical longevity. thus, despite the superior physical-chemical properties of ceramic materials when compared to other dental esthetic restorative materials, such as resin composites, the clinical success of ceramic restorations depends on the properties of the cementation material as well as the cementation technique2. luting materials are considered clinically acceptable if they present appropriate resistance to solubilization, high adhesion to dental substrates, high strength under tension, good manipulative properties, and biocompatibility in the oral environment and to the dental tissues2-3. in order to improve these properties, the material should be properly polymerized. thus, one of the most important aspects related to the characteristics of resin-based materials is the braz j oral sci. 14(3):230-233 http://dx.doi.org/10.1590/1677-3225v14n3a11 received for publication: august 31, 2015 accepted: september 26, 2015 231231231231231 material type composition tetric n-flow flowable resin composite 36 wt.% dimethacrylate (including tegdma), 63 wt.% fillers (barium glass, ytterbium trifluoride, highly dispersed silica and mixed oxide) and 1 wt.% catalysts, stabilizers and pigments. the total content of inorganic fillers is 39 vol.%. the particle size of inorganic fillers is between 40 and 3000 nm ips classic feldspathic ceramic sio2, bao, al2o3, cao, ceo2, na2o, k2o, b2o3, mgo, zro2, p2o5, tio2, and pigments (composition according to the material safety data sheet) table 1table 1table 1table 1table 1 materials used in the study. polymerization level or degree of conversion (dc) of monomer chains into polymers4-5. dc is important because properties such as strength, color stability and biocompatibility are closely related to it6-8. the biocompatibility is related to the concept that unreacted monomers have cytotoxic potential in contact with biological tissues9. thus, since the physical/chemical properties of resin-based cements are related to how the polymerization occurs, it is important to study and understand the factors that may influence the dc, such as thickness and color of the ceramic material interposed between the light source and the resin cement. in the case of the cementation of ceramic veneers, the use of light-activated resin-based materials has increased due to the working time control 10. this clinical advantage, however, should not be the determinant factor for material choice in veneer cementation due to the possible attenuating effect provided by thickness, opacity and shades 11-13. additionally, the type and microstructure of the interposing ceramic material are significant factors in the properties of polymerized luting materials14. in general, the use of these light-activated cements should be limited to some clinical situations such as inlays, veneers and onlays, in which the thickness and shade of restoration do not affect the ability of light to reach and activate the cement15. considering the light-activated resin cements for veneers and onlays cementation, the polymerization quality is related to several factors such as photo-activation light source, irradiance, irradiation time, material composition and light attenuation caused by ceramic11,13-14,16-18. it was observed that the transmittance of light through ceramic decreases as function of the material interposed between the light source and the resin cement19. furthermore, for a single ceramic material with different shades and thickness, it can be hypothesized that the polymerization may vary due to differences in the transmission of photons through the ceramic. therefore, the clinical performance would be determined by the amount of light transmitted through the restoration that reaches the resinbased cement. thus, the aim of this study was to evaluate the influence of ceramic color and thickness on the dc of a flowable light-activated resin composite. the working hypothesis was that the shade and thickness of interposing ceramic material influence dc. material and methods the materials used in this research are in table 1. feldspathic ceramic discs (ips classic, ivoclar vivadent, schaan, liechtenstein) 12 mm diameter and 0.5, 0.7 and 1.0 mm thickness were prepared with the shades a1 and a3. all spacers were fabricated according to the manufacturer’s instructions. the thickness was measured by a digital caliper (starret, jiangsu, china) with 0.01 mm accuracy. for dc evaluation, a portion of approximately 0.05 g of the uncured shade a1 tetric n-flow (ivoclar vivadent, schaan, liechtenstein) flowable resin composite was dispensed onto a plastic film. another plastic film was placed over the material and the set was pressed with a pneumatic press (shimadzu, kyoto, japan) at 10 kn to obtain a thin film approximately 0.1 mm thick and 20.0 mm diameter (n=5). these composite films were light-activated through different interposed ceramic spacers with a 1200 mw/cm2 led curing light (radii-cal, sdi limited, bayswater, australia) for 45 s. the light intensity was determined before the beginning of the experiments by a radiometer (demetron, kerr, middleton, wi, usa) without the interposition of ceramic disks. the samples were subjected to fourier transformed infrared spectroscopy (ftir) with a resolution of 4 cm-1 and 32 scans ranging from 4,000 to 800 cm-1. the absorption peaks of aromatic double bonds at 1608 cm-1 (abs 1608) and aliphatic double bonds at 1636 cm-1 (abs 1636) were recorded before and after the light-activation. five measurements were performed for each condition. the ratio between abs 1636 and abs 1608 was calculated for both cured and uncured materials. the percentage of remaining double bonds (rdb) was determined according to the formula: %rdb = (abs 1636/abs 1608 for cured resin) x 100/ (1636 abs/abs 1608 for uncured resin) the percentage of the degree of conversion of double bonds (dc) was calculated by the following formula: %dc = 100 – %rdb data were submitted to one-way anova. multiple comparisons were performed using the dunnett’s t3 test. a global level of 0.05 significance was set. results the dc mean values and standard deviations for the flowable resin light-activated through ceramic of different shades and thicknesses are shown in table 2. statistically significant differences were observed between groups (p<0.001). the control group (without interposition of ceramic) showed the highest mean percentage of dc (71.9±1.7). the lowest dc values were obtained for the 0.7 degree of conversion of a flowable composite light-activated through ceramics of different shades and thicknesses braz j oral sci. 14(3):230-233 232232232232232 and 1 mm a3 ceramics (61.7±0.9 and 62.0±1.9, respectively). discussion this study confirmed the hypothesis that the shade and thickness of interposing ceramic material would influence the dc. although no flowable resin composite group with interposing ceramics reached dc similar to the control group, little difference in the dc was observed when ceramics of different thicknesses and shades were compared. possible explanations for this result are the high irradiance of the led curing light used in this study, the reduced thickness of the ceramic veneers (up to 1 mm) and the thin films of flowable resin composite used to determine the dc. thus, the possible attenuating effect of light by ceramic interposition was not sufficient to negatively affect dc. it should be noted that only one feldspathic ceramic was evaluated in the present study. ceramic systems with different levels of opacity are commercially available, and dc results could be different if ceramics presenting other opacities and microstructures were evaluated. more opaque ceramics tend to absorb more light, which would decrease the dc of light-cured resin-based materials20. it has been reported that the dc of resin-based materials may change considerably as function of the type and opacity of the interposing ceramics13-14. another aspect to be addressed is that one single shade of flowable resin composite was evaluated. as recently observed, the shade is an important factor for the development of mechanical properties of resin-based cements. darker shades may show inferior properties than the lighter ones21. thus, the results of the present study could be different if other shades of resin composite (i.e. darker or bleached teeth) were evaluated. additionally, the composition and irradiation time are important factors that influence dc of resin composites22-23. it should be noted that, according to the manufacturer, tetric n-flow is a radiopaque nano-hybrid light-activated flowable resin composite indicated for adhesive cementation of translucent ceramic and composite resin indirect restorations. although the resin composite evaluated in the present study is indicated for the cementation of ceramic restorations, other light-activated materials (i.e. the so called “veneer cements”) have been indicated for the cementation of thin veneers10 and also have shown good dc results when light-activated through ceramic materials13-14. as seen in table 2, the similar results for dc in most groups indicate that the shade and thickness of the evaluated interposed ceramic veneers did not influence the polymerization of the flowable resin composite. this result shows that a lightactivated flowable resin may be used to fix minimum thickness ceramic veneers without a great effect on the dc level. this result is in agreement with another study that used the same method of the present study, in which the dc was not affected when 1.5-mm-thick ceramics were interposed13. similar results were also observed by cho et al.11 (2015). other important factors to be considered are the exposure time and interposed ceramic opacity. archegas et al.20 (2011) observed that opaque ceramic resulted in lower degree of conversion, hardness and elasticity modulus of resin-based cements. an exposure time of 120 s produced higher degree of conversion values for all materials, regardless the opacity of the ceramic. moreover, the exposure of 120 s promoted higher hardness and modulus of elasticity values than those obtained when the cements were light-activated for 40 s. thus, the results of the present study could be different if longer light-activation times were used. it should be noted that, although the manufacturer of the resin composite evaluated in the present study recommends a 10-s lightactivation time when a curing device with irradiance higher than 1000 mw/cm2 is used, a 45-s light-activation time was used instead. this increased irradiance (1200 mw/cm2) and light-activation time (45 s) could have reduced the attenuation of light caused by the evaluated ceramics. besides the dc, it should be noted that the clinical longevity of all-ceramic restorations includes other factors, such as the used adhesive system, the mechanism of polymerization, the polymerization unit, and the microstructural characteristics and thickness of the ceramic material24. for this reason, other studies should be conducted addressing the long-term behavior of light-activated resin cements cured through ceramics when different protocols of adhesive system application are employed. there was no statistically significant difference between the evaluated thickness and shades, except when comparing 0.5 mm a1 and 1.0 mm a3 ceramics. these results indicate that this flowable resin may be used to cement minimum thickness laminate ceramic veneers without great impact on the conversion degree. nevertheless, no flowable resin composite group with interposing ceramics reached a dc similar to the control group. it may be concluded that the shade and thickness of interposing glass-ceramic did not influence the dc of a flowable resin composite when ceramic was interposed. acknowledgements this study was supported by the pibic/cnpq. the authors also acknowledge the manufacturer for providing the resin composite. degree of conversion of a flowable composite light-activated through ceramics of different shades and thicknesses braz j oral sci. 14(3):230-233 ceramic shade thickness (mm) dc* (in %) control (without ceramic interposition) 71.9±1.7 a a1 0.5 64.6±1.2 b 0.7 64.4±5.0 bc 1.0 62.7±4.7 bc a3 0.5 62.7±1.4 bc 0.7 61.7±0.9 c 1.0 62.0±1.9 c *dc degree of conversion table 2 table 2 table 2 table 2 table 2 mean values and standard deviations for dc. different superscript letters represent statistically significant differences (p<0.05). 233233233233233 references 1. lawson nc, burgess jo. dental ceramics: a current review. compend contin educ dent. 2014; 35: 161-6. 2. manso ap, silva nr, bonfante ea, pegoraro ta, dias ra, carvalho rm. cements and adhesives for all-ceramic restorations. dent clin north am. 2011; 55: 311-32. 3. rosenstiel sf, land mf, crispin bj. dental luting agents: a review of the current literature. j prosthet dent. 1998; 80: 280-301. 4. watts dc. reaction kinetics and mechanics in photo-polymerised networks. dent mater. 2005; 21: 27-35. 5. furuse ay, mondelli j, watts dc. network structures of bis-gma/ tegdma resins differ in dc, shrinkage-strain, hardness and optical properties as a function of reducing agent. dent mater. 2011; 27: 497-506. 6. atsumi t, iwakura i, fujisawa s, ueha t. the production of reactive oxygen species by irradiated camphorquinone-related photosensitizers and their effect on cytotoxicity. arch oral biol. 2001; 46: 391-401. 7. lapp ca, schuster gs. effects of dmaema and 4-methoxyphenol on gingival fibroblast growth, metabolism, and response to interleukin-1. j biomed mater res. 2002; 60: 30-5. 8. peutzfeldt a, asmussen e. the effect of postcuring on quantity of remaining double bonds, mechanical properties, and in vitro wear of two resin composites. j dent. 2000; 28: 447-52. 9. dos santos rl, de sampaio ga, de carvalho fg, pithon mm, guenes gm, alves pm. influence of degree of conversion on the biocompatibility of different composites in vivo. j adhes dent. 2014; 16: 15-20. 10. da cunha lf, pedroche lo, gonzaga cc, furuse ay. esthetic, occlusal, and periodontal rehabilitation of anterior teeth with minimum thickness porcelain laminate veneers. j prosthet dent. 2014; 112: 1315-8. 11. cho sh, lopez a, berzins dw, prasad s, ahn kw. effect of different thicknesses of pressable ceramic veneers on polymerization of light-cured and dual-cured resin cements. j contemp dent pract. 2015; 16: 347-52. 12. peixoto rt, paulinelli vm, sander hh, lanza md, cury la, 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dent. 2010; 35: 579-86. 18. ozturk b, cobanoglu n, cetin ar, gunduz b. conversion degrees of resin composites using different light sources. eur j dent. 2013; 7: 102-9. 19. pick b, gonzaga cc, junior ws, kawano y, braga rr, cardoso pe. influence of curing light attenuation caused by aesthetic indirect restorative materials on resin cement polymerization. eur j dent. 2010; 4: 314-23. 20. archegas lr, freire a, vieira s, caldas db, souza em. colour stability and opacity of resin cements and flowable composites for ceramic veneer luting after accelerated ageing. j dent. 2011; 39: 804-10. 21. ozturk e, bolay s, hickel r, ilie n. effects of ceramic shade and thickness on the micro-mechanical properties of a light-cured resin cement in different shades. acta odontol scand. 2015; 73: 503-7. 22. cornelio rb, kopperud hm, haasum j, gedde uw, örtengren u. influence of different mould materials on the degree of conversion of dental composite resins. braz j oral sci. 2012; 11: 469-74. 23. catelan a, kawano y, santos ph, ambrosano gmb, berdran-russo ak, aguiar fhb. radiant exposure effects on physical properties of methacrylate and silorane-composites. braz j oral sci. 2014; 13: 168-74. 24. rasetto fh, driscoll cf, prestipino v, masri r, von fraunhofer ja. light transmission through all-ceramic dental materials: a pilot study. j prosthet dent. 2004; 91: 441-6. degree of conversion of a flowable composite light-activated through ceramics of different shades and thicknesses braz j oral sci. 14(3):230-233 revista fop n 13 braz j oral sci. july/september 2008 vol. 7 number 26 1580 effect of chemical denture cleansers on flexural resistance and color changes of microwave-polymerized acrylic resins helena de freitas oliveira paranhos1; iara augusta orsi2; osvaldo zaniquelli2; maria cristina candelas zuccolotto3; fabrício magalhães3 1 phd, associate professor 2 phd, professor 3 ms, phd student faculty of odontology of ribeirão preto, department of dental materials and prosthodontics, university of são paulo, ribeirão preto, sp, brazil received for publication: april 14, 2008 accepted: september 09, 2008 correspondence to: helena de freitas oliveira paranhos faculdade de odontologia de ribeirão preto – usp, departamento de materiais dentários e prótese avenida do café s/nº monte alegre 14040-904 ribeirão preto, sp, brazil phone: +55-16-602-4006. fax: +55-16-16-633-0999 e-mail: helenpar@forp.usp.br a b s t r a c t aim: the aim of this study was to assess the flexural resistance and color alterations of microwave-polymerized acrylic resins immersed in denture cleansers for different periods of time. methods: forty-five rectangular specimens (65x10x3mm) of each commercial brand of the microwave-activated acrylic resins (vipi wave and onda cryl) were divided in three denture cleanser groups (bony plus, corega tabs and efferdent plus) and a control group (immersion in water). soaking trials of 15 min and 8 h simulated 30 days of use. the flexural strength test was carried out in a universal testing machine. color alterations were assessed by visual inspection of photographs. the results obtained in the flexural test, in kgf, were converted to mpa and these values were submitted to analysis of variance with a 5% significance level. results: there were no significant differences (p<0.05) between onda cryl (85.61±12.76) and vipi wave (89.8±19.95) after the soaking trials regarding the use of different denture cleansers. no differences were found in relation to the solutions [bony plus (88.52±9.89), corega tabs (88.75±12.71) and efferdent (85.86±12.11)], soaking periods [control (87.17±12.92), 15 min (88.05±11.74) and 8 h (87.91±10.30)], and interactions during the 30 days of simulated use. visual inspection did not detect any color alterations. conclusions: denture cleansers, when used according to the manufacturers’ instructions, did not cause any mechanical or visual alterations in the microwave-polymerized acrylic resins after a simulated period of 30 days of use. key words: complete dentures, acrylic resin, microwave, denture cleansers, flexion resistance, color. i n t r o d u c t i o n acrylic resins have been used to produce dentures for more than 60 years. microwave polymerization of acrylic resin was introduced by nishii1 in japan. polymerization by microwave irradiation has several advantages: a denture base can be fully polymerized in only 3 min, much faster when compared to the polymerization time of 9 h normally used for water-bath polymerization; a simpler equipment is required; only a fraction of the energy needed by conventional methods is required for microwave-activated polymerization; and less residual monomer remains in microwave-polymerized resins2. dentures can be cleaned mechanically, chemically or by the combination of both methods. mechanical methods are the most common way for biofilm removal from denture surfaces 3. the use of chemical cleansers is usually associated to its efficacy in removing stains and a reduction in biofilm formation on dentures’ irregularities has been reported 4. the most commonly used cleansers are represented by the group of alkaline peroxides5. they are effective to remove newly formed biofilm and also when used for extended immersion periods6. these products have been clinically tested, demonstrating an effective removal of biofilm on complete dentures7 and antimicrobial action against specific microorganisms8. factors like water temperature9-11 and immersion period1214 are considered critical when complete denture cleansers are used. sometimes, the prostheses need to be replaced due to the patients’ abuse of hygiene methods. the flexural resistance property has been tested in temporary soft liners15 and acrylic resins16-18 after the use of disinfecting solutions and specific products for cleaning total prostheses. 1581 the importance of following the manufacture’s instructions is emphasized because the transverse strength of acrylic resins depends on several factors, such as polymer molecular weight19, polymer bead size20, residual monomer levels19,21, plasticizer composition19,22, amounts of crosslinking agents23, internal porosity of the polymer matrix21,24, denture base thickness 24, patient factors 24, type of polishing25, and action of chemical agents. one of the problems frequently reported by chemical cleanser users is a whitening effect on the denture. denture base polymers are susceptible to color changes12 if the cleaning solutions are not used correctly. the whitening effect is related to the high temperature of the water used in the solution9,10,26. when peroxide-based cleansers are used in a warm water solution, as recommended by the manufacturer, no deleterious effects on correctly processed denture acrylic have been found18. considering that denture overall longevity also depends on the physical properties of the denture base resin22 and that the denture base polymers may fall clinically due to flexural fatigue, the assessment of the transverse strength of acrylic resins has been reported to be a reliable method to estimate resin behavior under different experimental conditions27. in the same way, it is of clinical importance to determine whether chemical solutions or denture cleansers alter the acrylic resins color14 when dentures are cleaned repeatedly and for various amounts of time. therefore, the aim of this study was to evaluate whether soaking of different microwave-polymerized acrylic resins in chemical solutions (alkaline peroxide-effervescent tablets) may affect the resin flexural strength and color when subjected to the recommended instructions of use for a simulated period of 30 days. material and methods dental stone casts were prepared in dental flasks (vipi vipi ind com ltda, pirassununga, sp, brazil), each flask containing three preformed teflon® (dupont™) dies (each 65x10x3mm). each die was coated with a thin layer of petroleum jelly before being invested. for the flask base, 120g of type iii dental stone (herodent; vigodent s/a, rio de janeiro, rj, brazil) was used. undercuts were placed in the stone for best retention of 80 g of type iv die stone (durone; dentsply, petrópolis, rj, brazil), where the dies were invested. a new coat of petroleum jelly was applied before pouring of 80g of die stone and final pouring of 300 g of dental stone. after complete final stone set, the 3 min 40% potency and 4 min 0% and 3 min at 90% manufacturer powder/liquid ratio (g/ml) curing cycle onda cryl clássico art. odontológicos ltd., são paulo, sp, brazil vipi-wave dental vipi ltd, são paulo, sp, brazil 14/6.5 20 min 20% potency and 5 min 60% 21/7 table 1 acrylic resins and denture cleansers employed flasks were opened and the dies were removed from the investing material. the mould cavities obtained were used for preparation of the acrylic resin test specimens. the resins (table 1) were mixed according to the manufacturers’ instructions. the monomer and polymer were mixed together until a doughy stage was reached, then kneaded and placed in the mould. after the end of the polymerization cycle, the flasks were allowed to slowly cool in a water bath at room temperature before deflasking. the acrylic specimens were trimmed with a tungsten bur (#9201 shape 84; edenta ag, hauptstrasse, switzerland) and ground wet to the final dimensions with 320-, 400-, 600-, 1000-grit silicon carbide papers (norton ind. e com. ltda, são paulo, sp, brazil). pumice and whiting were used for final polishing. after polishing, the specimens were marked individually with an identifying number. five specimens of each resin were assigned to each experimental group. the accuracy of the dimensions was verified with a digital caliper (cd-6" csx-b – mitutoyo, japan) and the dimensions were recorded. all specimens were stored in water at 50°c for 1 h to remove the excess of residual monomer, and then stored at room temperature until the time of the soaking trials. soaking trials five specimens of each resin were subject to soaking trials, according to the: • denture cleanser: a) bony plus (bonyf ag, liechtenstein, switzerland), b) corega tabs (block drug company, inc., usa) or c) efferdent plus (pfizer, morris plans, usa) dissolved in 250 ml of water at 45±2°c; • soaking time: a)15 min, three times a day for 10 days, simulating a 15-min soaking time once a day for 1 month and b) 8-h intervals for up to a total of 240 h, changing the solution every 8 h, to correspond to 30 overnight soaking periods. the control specimens were stored in water at room temperature, changing the water every 8 h. analysis of treated specimens flexural strength testing – three-point loading specimens were labeled on each end before testing so that fractured pieces could be reunited and examined subsequent to testing. a three-point loading test was carried out in a universal testing machine (emic, são josé dos pinhais – pr, brazil) running at a crosshead speed of 1 mm/min and 50-mm distance between the specimen braz j oral sci. 7(26):1580-1584 effect of chemical denture cleansers on flexural resistance and color changes of microwave-polymerized acrylic resins 1582 supports. a 50 kgf load cell was applied by a centrally located rod until fracture occurred (figure 1). the flexural strength was calculated with the following equation: 22 3 bd pl s = where s is flexural strength, p peak load applied, l span length, b sample width and d specimen thickness. color alteration the control specimens stored in water and the specimens immersed in the three denture cleansers, using daily soaking times of 15 min and 8 h for a simulated period of 30 days, were put side by side and photographed (camera: canon eos-100s; lens: canon 50mm f2.8 ef macro – canon, tokyo, japan) in a photographic studio. film was processed and visual inspection of photographs of the specimens was carried out independently by three investigators. each investigator received an initial photograph of the non-treated resin specimens (used as a control) and compared to the photograph of the treated specimens. yes or no answers were given depending on the presence or absence of color change. statistical analysis the results obtained in the flexural resistance test, in kgf, were converted to mpa. the preliminary statistical analysis showed that sample distribution was normal and homogeneous, thereby allowing the use of parametric tests. the analysis of variance was used to compare types of resins, solutions and immersion times. significance level was set at 5%. r e s u l t s the results of the anova (table 2) did not show significant differences (p<0.05) between the resins after the soaking trials regarding the use of different denture cleansers. no statistically significant differences were found regarding the resins [onda cryl (85.61±12.76) and vipi source of variation sum of squares df mean square (f) ratio prob. h 0 (%) solutions (s) 154.275 2 77.138 0.53 40.20 ns resins (r) 397.964 1 397.964 2.72 9.94 ns time (t) 13.608 2 6.804 0.05 5.53 ns interaction sxr 37.236 2 18.618 0.13 11.94 ns interaction txs 318.292 4 79.573 0.54 29.29 ns interaction txr 359.369 2 179.685 1.23 29.84 ns interaction txsxr 111.080 4 27.770 0.19 5.90 ns error 10530.362 72 146.255 total variation 11922.188 89 table 2 analysis of variance results ns= no significant difference (p>0.05) resins means (standard deviation) vipi wave 89.81 ± 9.95 onda cryl 85.61 ± 12.76 table 3 mean values (mpa) and standard deviations for resins p > 0 . 0 5 solutions means ± sd bony plus 88.52 ± 9.89 corega tabs 88.75 ± 12.71 efferdent 85.86 ± 12.11 table 4 mean values (mpa) and standard deviations (sd) for solutions p > 0 . 0 5 time (min) means ± sd 0 87.17 ± 12.92 15 88.05 ± 11.74 480 87.91 ± 10.30 p > 0 . 0 5 table 5 mean values (mpa) and standard deviations (sd) for immersion times wave (89.8±19.95)], solutions [bony plus (88.52±9.89), corega tabs (88.75±12.71) and efferdent (85.86±12.11)], soaking periods [control (87.17±12.92), 15 min (88.05±11.74) and eight h (87.91±10.30)] and interactions, during the 30 days of simulated use. tables 3-5 show the mean values and standard deviation obtained for the resins, solutions and immersion times. no statistically significant differences were found. visual examination of the photographs of the specimens did not show any clinically significant color alterations. d i s c u s s i o n denture immersion in chemical products aims to provide cleaning and decontamination. it is important to analyze braz j oral sci. 7(26):1580-1584 effect of chemical denture cleansers on flexural resistance and color changes of microwave-polymerized acrylic resins 1583 the efficacy of the cleaning product and how it acts on the denture materials28. it has been shown that immersion in certain cleansing solutions can affect the strength and the structure of denture base resins10. if denture cleansers lead to a reduction in strength, a higher incidence of denture fractures could occur. midline fracture of the denture base, for example, is one of the failures that may occur as a consequence of flexural fatigue, as a result of the cyclic deformation of the base during function. a supposed increased frequency of this last kind of failure due to the use of denture cleansers can be demonstrated by the flexural strength test. in the present study, flexural strength and color were not affected by the exposure to the tested cleaning products. using the same solutions and simulating the same usage period, sato et al.26 did not find any alterations in the flexural strength of conventional resins. one factor that contributed to this result was the use of products at the recommended temperatures. robinson et al.10 and arab et al.26 showed reduced flexural strength of acrylic resins when exposed to peroxides and hypochlorites at high temperatures, which is not recommended by the manufacturers. previous investigations have emphasized that the correct use of chemical cleansers is not associated to alterations in the mechanical properties of the materials for denture bases9-11,26. however, another factor to be taken into account is the immersion time, as extended immersions can damage certain materials used to manufacture the prostheses29. twenty-minute (short immersion) and 8-hour periods (extended or overnight immersion – during sleep period) were established to simulate the orientations patients received for the daily cleaning of total prostheses. the results showed that, even within an 8-hour period, no alterations occurred in the analyzed characteristics. factors that may contribute to the change in the color of materials include stain accumulation, dehydration and oxidation of the reacted carbon-carbon double bonds that produces colored peroxide compounds, and continuing formation of the colored degradation products30. color alterations can be objectively measured with a spectrophotometer. however, in this study, color alterations were evaluated only by visual examination because a clinically perceptible color change was considered more important than a measurement of color difference. within 30 days of simulated immersion, the tested acrylic resins did not show any noticeable color change with the use of the three cleansing agents. unlü et al.31 observed a whitening effect in acrylic resins after 30 days of simulated use of chemical cleansers, measuring the color alterations with a reflectometer. significant differences were dependent on the acrylic resin and the kind of cleansing agent used. these results do not agree with those of the present study, possibility due to methodological differences, which objectively measured the color values of the specimens with a reflectometer. this device detected color differences that the human eye could not perceive, which explains the color alterations reported by ünlu et al.31. jin et al.30 observed minimal color change in the tested heat polymerized materials, with no significant differences among the denture cleansers in which the materials were immersed (alkaline peroxide, neutral peroxide, neutral peroxide with enzyme and enzyme). the authors attributed this result to the fact that heat polymerized materials have a high polymerization rate and greater stability of physical properties. devlin and kaushik32 evaluated acrylic resin specimens placed in warm water (40°c) and boiling water (100°c) with an alkaline peroxide tablet (efferdent).one effect of the hot water was to cause a severe whitening of all acrylic specimens, whereas those treated with warm water were unaffected. they concluded that hot alkaline peroxide solution caused a water oversaturation of acrylic surface, resulting in surface whitening and softening. these findings are in agreement with those of the present study; when used according the manufacturer instructions, cleansers do not affect the flexural resistance and color of acrylic resins and the polymerization type can contribute to increase property stability. further research using longer immersion periods is needed. the effect of these solutions on other characteristics and properties of acrylic resins, such as superficial roughness, should also be investigated. it may be concluded that, when used according to the manufacturers’ instructions, denture cleansers did not cause alterations in flexural strength or color changes in microwavepolymerized acrylic resins after 30 days of simulated use. r e f e r e n c e s 1. nishii m. studies on the curing of denture base resins with microwave irradiation: with particular reference to heat curing resins. j osaka dent univ. 1968; 2: 23-40. 2. schneider rl, curtis er, clancyn jms. tensile bond strength of acrylic resin denture teeth to a microwaveor heat-processed denture base. j prosthet dent. 2002; 88: 145-50. 3. nikawa h,hamada t,yamashiro h,kumagai h. a review of in vitro and in vivo methods to evaluate the efficacy of denture cleansers. int j prosthodont. 1999; 12: 153-9. 4. paranhos hfo, silva-lovato ch, souza rf, cruz pc, freitas km, peracini a. effects of mechanical and chemical methods on denture biofilm accumulation. j oral rehabil. 2007; 34: 606-12. 5. shay k. denture hygiene: a review and update. j contemp dent pract. 2000; 1: 1-8. 6. shannon il, mccrary br, starcke en. removal of salivary deposits by commercial denture cleansers. gen dent. 1976; 24: 30-4. 7. gornitsky m, paradis i, landaverde g, mallo am, velly am. a clinical and microbiological evaluation of denture cleansers for geriatric patients in long term care institutions. j can dent assoc. 2002; 68: 39-45. 8. chan ec, iugovaz i, siboo r, bilyk m, barolet r, amsel r et al. comparison of two popular methods for removal and killing of bacteria from dentures. j can dent assoc. 1991; 57: 937-9. 9. crawford c-a, lloyd ch, newton jp, yemm r. denture bleaching: a laboratory simulation of patients’ cleaning procedures. j dent. 1986; 14: 258-61. braz j oral sci. 7(26):1580-1584 effect of chemical denture cleansers on flexural resistance and color changes of microwave-polymerized acrylic resins 1584 10. robinson jg, mccabe jf, storer r. denture bases: the effects of various treatments on clarity, strengh and structure. j dent. 1987; 15: 159-65. 11. arab j, newton jp, lloyd ch. the importance of water temperature in denture cleaning procedures. j dent. 1988; 16: 277-81. 12. mcneme sj, von gonten as, woolsey gd. effects of laboratory disinfecting agents on color stability of denture acrylic resins. j prosthet dent. 1991; 66: 132-6. 13. ma t, johnson gh, gordon ge. effects of chemical disinfectants on the surgace characteristics and color of denture resins. j prosthet dent 1997; 77: 197-204. 14. polyzois gl, yannikakis sa, zissis aj, demetriou pp. color changes of denture base materials after disinfection and sterilization immersion. int j prosthodont. 1997; 10: 83-9. 15. yilmaz h, aydin c, bal bt, ocak f. effects of different disinfectants on physical properties of four temporary soft denture-liner materials. quintessence int. 2004; 35: 826-34. 16. asad t, watkinson a, huggett r. the effect of disinfection procedures on flexural properties of denture base acrylic resins. j prosthet dent. 1992; 68: 191-5. 17. pavarina, ac, machado al, giampaolo et, vergani ce. effects of chemical didinfectants on the transverse strength of denture base acrylic resins. j oral rehabil. 2003; 30: 1085-9. 18. sato s, cavalcante mrs, orsi ia, paranhos hfo. zaniquelli o. assessment of flexural strenght and color alteration of heatpolymerized acrylic resins after simulated use of denture cleansers. braz dent j. 2005; 16: 124-8. 19. stafford gd, smith dc. some studies of the properties of denture base polymers. br dent j. 1968; 125: 337-42. 20. mutlu g, huggett r, harrison a. factors that affect the rheologic properties of acrylic resin denture base materials. j prosthet dent. 1994; 7: 186-91 21. dogan a, bec b, cevik nn, usanmaz a. the effect of preparation conditions of acrylic denture base materials on the level of residual monomer, mechanical properties and water absorption. j dent. 1995; 23: 313-8. 22. aldana l, marker va, kolsdat r, iacopino am effects of candida treatment regimens on the physical properties of denture resins. int j prosthodont. 1994; 7: 473-8. 23. arima t, murata h, hamada t. the effects of cross-linking agents on the water sorption and solubility characteristics of denture base resins. j oral rehabil. 1996; 23: 476-80. 24. honorez p, catalan a, angnes u, grimonster j. the effect of three processing cycles on some physical and chemical properties of a heat-cured acrylic resin. j prosthet dent. 1989; 61: 510-7. 25. orsi ia, andrade vg. effect of chemical disinfectants on the transverse strength of heat-polymerized acrylic resins submitted to mechanical and chemical polishing. j prosthet dent. 2004; 92: 382-8. 26. arab j, newton jp, lloyd ch. the effect of an elevated level of residual monomer on the whitening of a denture base and its physical properties. j dent. 1989; 17: 189-94. 27. chiotchumnong p, books cs, stafford gd. comparison of threeand four-point flexural strength testing of denture-base polymers. dent mat. 1989; 5: 2-5. 28. garcia rcmr, souza jr ja, rached rn, del bel cury a. a. effect of denture cleansers on the surface roughness and hardness of a microwave-cured acrylic resin and dental alloys. j prosthodont. 2004; 13: 173-8. 29. jagger dc, al-akkhazami l, harrison a, rees js. the effectiveness of seven denture cleansers on tea stain removal from pmma acrylic resin. int j prosthodont. 2002; 15: 549-52. 30. jin c, nikawa s, makihira s, hamada t,, furukawa m, murata h. changes in surface roughness and colour stability of soft denture lining materials caused by denture cleansers. j oral rehabil. 2003; 30: 125-130. 31. ünlü a, altay ot, sahmali s. the role of denture cleansers on the whitening of acrylic resins. int j prosthodont. 1996; 9: 266-70. 32. devlin h, kaushik p. the effect of water absorption on acrylic surface properties. j prosthodont 2005; 14: 233-8. braz j oral sci. 7(26):1580-1584 effect of chemical denture cleansers on flexural resistance and color changes of microwave-polymerized acrylic resins oral sciences n3 braz j oral sci. 11(1):36-41 original article braz j oral sci. january | march 2012 volume 11, number 1 atraumatic restorative treatment in permanent molars: a systematic review liana studart1, carolina da franca2, viviane colares3 1dds, msc, department of pediatric dentistry, university of pernambuco, brazil 2dds, msc, phd, department of pediatric dentistry, university of pernambuco, brazil 3dds, msc, phd, associate professor, department of pediatric dentistry, university of pernambuco, brazil correspondence to: liana peixoto carvalho studart av. generalnewton cavalcante 1650, tabatinga, 54753-220, camaragibe pe phone: + 55 81 31847659 e-mail: lianapcarvalho@hotmail.com received for publication: october 13, 2011 accepted: january 24, 2012 abstract aim: this paper presents a systematic review of studies published within the last 10 years on atraumatic restorative treatment (art) in permanent molars. methods: a systematic search was carried out in the medline and pubmed databases from april 1st 2001 to december 31st 2010 using the term “atraumatic restorative treatment” and in the lilacs database using the term “tratamento restaurador atraumático”. the selection of publications was based on the following criteria: texts written in english, spanish or portuguese; presence of an abstract; and investigation of clinical aspects related to art in permanent molars. results: the analysis of abstracts led to the selection of 26 studies. recent clinical studies on art in permanent molars of children, adolescents and young adults mainly address the survival of restorations through a comparison between caries removal methods and restorative materials. the publications report a survival success rate of art restorations ranging from 30 to 100% in permanent molars, which is similar to that reported for treatments employing conventional methods and materials. the success rate of art sealants ranged from 6.2 to 98.5%. the number of studies reporting on the retention and caries preventive effect of art sealants was small. conclusions: this systematic review revealed high survival rates for single-surface art restorations in permanent molars and lower success rates for multiple-surface restorations. clinical studies conducted with children, adolescents and young adults demonstrate a reliable clinical performance of single-surface and multi-surface art restorations for 3-year and 2-year periods, respectively. however, long-term clinical studies are scarce and most of them focus on one-surface cavities. the survival rates observed, especially for single-surface restorations, confirm the potential of the art approach for restoring and saving permanent molars. more clinical studies are required on the retention and caries preventive effect of art sealants. keywords: atraumatic restorative treatment, art, molar, glass ionomer, survival, restoration, sealants. introduction the atraumatic restorative treatment (art) approach was developed in the mid-1980s to provide dental preventive and restorative care to underserved populations in areas that are out of reach1-2. the method consists of the removal of infected (soft demineralized carious) tooth tissue with manual instruments only, followed by the filling of the cleaned cavity and associated pits and fissures with an adhesive restorative material1. the resulting sealant-restoration is thus both preventive and restorative. art is perfectly aligned with modern concepts in health care, which advocate maximal effort regarding preventive approaches and braz j oral sci. 11(1):36-41 main topic studies (n) survival of art restorations survival of art only restorations 6 comparison of restorative materials 7 comparison of restoration methods 7 retention and caries preventive effect of art sealants 6 total 26 table 1.table 1.table 1.table 1.table 1. distribution of studies on art in permanent molars of children, adolescents and young adults minimally invasive procedures 3. this approach requires neither electricity nor running water and can therefore be applied in almost any setting4. in addition, art has the other following advantages: the use of easily available and inexpensive hand instruments rather than more expensive electrically-driven dental equipment; conservation of sound t o o t h t i s s u e t h r o u g h t h e c h e m i c a l a d h e s i o n o f g l a s s ionomers; limitation of pain, minimizing the use of local anesthesia; and low cost. as a result, many people living in less developed areas can receive oral care by means of art. moreover, art is also suitable for patients with permanent or temporary physical disabilities. although initially developed to provide restorative dental treatment in areas of difficult access, art or modified art techniques are being increasingly introduced into dental clinics in industrialized countries4. since inception, different aspects of the technique have become the subject of numerous studies, which have served mainly to obtain information on technical aspects of the procedure, such as handling characteristics of the restorative material, and the survival of the restorations4. these studies have led to improvement of the technique5 and to new, more appropriate restorative glass ionomer cements developed specifically for art. the studies carried out before 1994 used a low-viscosity glass ionomer cement, whereas the later studies used a high-viscosity glass ionomer cement. art studies are currently underway using composite and compomer. because of its chemical adhesion to enamel and its improved physical properties, high-viscosity glass ionomer cement can also be used to seal pits and fissures in the absence of cavitation. these are called art sealants. after cleaning the surface, high-viscosity glass ionomer cement is pressed into the pits and fissures with an index finger. thus the art approach consists of two components; a sealant restoration and a preventive sealant1. the aim of the present study was to carry out a short systematic review of studies published in the last 10 years on clinical aspects related to the art approach in permanent molars of children, adolescents and young adults. material and methods a systematic search in the literature was carried out for publications indexed from april 1st 2000 to december 31st 2010 in medline and pubmed databases using the term “atraumatic restorative treatment”, and in lilacs database using the portuguese term “tratamento restaurador atraumático”. a total of 146 publications were found in medline, 161 in pubmed and 51 in lilacs. among the 358 publications, 149 were found in more than one database, resulting in a total of 209 publications for analysis. the selection of papers was based on the following criteria: texts written in english, spanish or portuguese; presence of an abstract; and investigation of clinical aspects related to atraumatic restorative treatment in permanent molars of children, adolescents and young adults. results a total of 186 of the 209 papers retrieved in the search were excluded for the following reasons: 20 for addressing a topic other than art; 26 for being literature reviews; 48 for investigating the primary dentition; 39 for being laboratory studies; nine for not having an abstract; eight for having been published in a language other than the three aforementioned languages; and 36 for being outside the scope of the aforementioned topic. thus, the critical analysis of abstracts resulted in 24 studies being eligible for inclusion in the systematic review. in general, recent studies on art in permanent molars mainly address the survival of art restorations, comparing different restorative materials and caries removal methods, and the retention and caries preventive effect of art sealants (table 1). art restorations most clinical studies focusing on the survival of art restorations in permanent molars referred to in this review used the same art criteria and involved a single surface4,618. evaluation time in these studies ranged from 4 months to 6 years. the survival rates for single-surface art restorations in the permanent molars ranged from 29.6 to 100%, regardless of the operator or material employed (table 2). regarding the survival rates of art restorations for multi-surface cavities, only a few clinical studies have been conducted18-20. evaluation time in these studies ranged from 6 months to 2 years. the survival rates for multi-surface art restorations in permanent molars ranged from 30.6 to 100%, regardless of the operator or material employed (table 3). art sealants a number of authors report on the retention and the caries-preventive effect of sealants using art approach in permanent molars9,21-25. the success rate of art sealants ranged from 6.2 to 98.5% (table 4). discussion although art may appear to be a simple technique, it embodies a philosophy of treatment based on evidence accumulated over 20 years of scientific research about health promotion. health promotion involves a holistic role in 3737373737 atraumatic restorative treatment in permanent molars: a systematic review braz j oral sci. 11(1):36-41 3838383838 author, year, country restorative materials evaluation time (months) success (%) lo et al.6, 2001, china glass ionomer (fuji ix) 24 96.0 glass ionomer (chem flex) 95.0 mandari et al.7, 2001, tanzania glass ionomer (fuji ii) 36 93.0 amalgam (non-gamma-2 amalgam) 89.0 yip et al.8, 2002, china glass ionomer (fuji ix) 12 100.0 glass ionomer (ketac molar) 93.8 ziraps and honkala9, 2002, kuwait glass ionomer (chem flex) 24 92.5 glass ionomer (fuji ix) 94.9 monse-schneider et al.10, 2003, germany encapsulated amalgam 24 94.4 souza et al.11, 2003, brazil glass ionomer (fuji ix) 8 86.2 glass ionomer(fuji plus) 88.4 taifour et al.12, 2003, syria glass ionomer (fuji ix and ketac molar) 36 82.1 amalgam tc 76.9 mandari et al.13, 2003, tanzania glass ionomer (fuji ii) 72 67.1 amalgam (non-gamma-2 amalgam) 74.0 lopez et al.14, 2005, mexico glass ionomer (fuji ix) 24 66.0 bresciani et al.15, 2005, brazil glass ionomer (ketac molar) 6 97.3 lo et al.16, 2007, china glass ionomer (ketac molar) 72 76.0 van gemert-sckirks4, 2007, suriname glass ionomer (ketac molar) 36 29.6 frencken et al.17, 2007, syria glass ionomer(fuji and ketac molar) 72 64.8 ercan et al.18, 2009, turkey glass ionomer (vitremer) 24 100.0 glass ionomer (ketac molar) 89.0 table 2.table 2.table 2.table 2.table 2. distribution of studies on longevity of single-surface art restorations author and year glass ionomer evaluation time (months) success (%) cefaly et al19, 2005, brazil fuji viii 6 100.0 ketac molar 96.6 cefaly et al.20, 2007, brazil fuji viii 12 100.0 ketac molar 93.0 ercan et al.18, 2009, turkey vitremer 24 100.0 ketac molar 41.2 table 3.table 3.table 3.table 3.table 3. distribution of studies on longevity of multi-surface art restorations author, year and country restorative material presence of caries (%) evaluation time (months) success(%) motsei et al.21, 2001, south africa fuji ix 1.1 12 10.4 abid et al.22, 2002, tunisia fuji ix 7.8 36 54.96 ziraps and honkala9, 2002, kuwait chem flex 0.0 24 70.0 fuji ix 0.0 77.8 beirute et al.23, 2006, syria fuji ix 14.4 60 12.0 visio-seal 48.6 14.0 vieira et al.24, 2006, brazil vidrion r 0.0 12 43.5 chem flex 0.0 41.8 oba et al.25, 2009, turkey ketac molar 6.0 36 44.7 fissurit f 6.48 6.2 table 4. table 4. table 4. table 4. table 4. distribution of studies on sealants used with art which dental practice emerges from its repetitive restorative cycle and returns to the prevention of caries. art was originally developed for use in underserved communities1-2. however, over the last 10 years, it has also been used in dental offices, providing quality treatment because it is in accordance with the concepts of modern dentistry of having minimally invasive techniques that preserve the maximum of dental tissues using hand instruments and adhesive materials. on the other hand, in some places it was mixed with the temporary restoration. however, art is a definitive treatment and should not be considered as part of the measures used to adequate the oral environment to receive restorations, which consists of a transition phase using temporary materials26. most studies that have investigated the art approach have used glass ionomer cement as the filling and sealant atraumatic restorative treatment in permanent molars: a systematic review 3939393939 braz j oral sci. 11(1):36-41 material. likewise the material most commonly used in the majority of studies published within the last 10 years on art in permanent molars has been the glass ionomer, in particular high-viscosity glass ionomer cement. the success rate obtained with the art approach has progressively decreased with time. considering only the studies with 1 or more years of follow-up, when using ketac molar, the success rate decreased from 93.8 to 65.2% in 1 to 10 years. when using fuji ix, the trend was the same as that observed in studies with ketac molar, the success rate decreased from 100% to 82.1% in 1 to 3 years. clinical studies conducted with children, adolescents and young adults have demonstrated a reliable clinical performance of single-surface and multi-surface art restorations for 3-year and 2-year periods, respectively. a number of studies have compared the clinical performance of different materials in art restorations in permanent molars6-7,9,11,13,18-20,27. lo et al.6 and ziraps and honkala9 compared the clinical performance of two different glass ionomer cements (high-viscosity glass ionomer cement chem flex and ketac molar) using the art approach in class i cavities. the authors noted that the clinical performance of both materials over a 24-month period was similar and there was no statistically significance between the two gic materials. souza et al.11 compared the clinical performance of single-surface restorations of two different glass ionomer cements (highviscosity glass ionomer cement – fuji ix and resin-modified glass ionomer cements – fuji plus) using the art approach and found no statistically significant differences between the materials. cefaly et al.19 and cefaly et al.20 evaluated the performance of multiplesurface restorations employing two different glass ionomer cements (highviscosity glass ionomer cement ketac molar and resin-modified glass ionomer fuji viii) and found no statistically significant differences between materials, after 6 and 12 months, respectively. in contrast, dulgergil et al.27 compared the clinical performance of the resin-modified glass ionomer (vitremer) material with the high-viscosity traditional glass ionomer cement (ketac molar) in permanent molar teeth with one or more carious cavities after 6 months. the achieved better results than those using the glass ionomer cement and the difference between the two groups was statistically significant. however, the duration of this study was too short for comparison with any other art studies27. ercan et al.18 compared the clinical performance of highviscosity glass ionomer cement (ketac molar) and resinmodified glass ionomer cement (vitremer) in singleand multiple-surface carious cavities. after 24 months, the authors observed that, irrespective of the number of surfaces, the resin-modified glass ionomer cement was significantly superior to the high-viscosity glass ionomer cement. their study demonstrates that the superior clinical performance of light-cured materials over those of chemical curing indicates that such materials are technically less sensitive than selfcure materials because, once irradiated, they do not require protection from moisture. two other studies have investigated the survival of single-surface art restorations using different glass ionomers compared to amalgam restorations7,13. mandari et al.7,13 reported no statistically significant differences between the success rates of both amalgam and glass ionomer restorations after two and six years. a meta-analysis is required to estimate the overall survival rates of amalgam and glass-ionomer restorations placed by the art approach, but the number of suitable studies to carry out such an analysis is too small. however, in the absence of such an analysis, single-surface art restorations using glass ionomer cements appear to survive as long as comparable amalgam art restorations in permanent molar teeth after 6 years13. based on the findings of this systematic review, it appears that in most studies, no statistically significant differences were found between the success rates for art restorations placed with different materials6-7,9,11,13,19-20. this comparison indicates that the survival rate of art restorations is acceptable, particularly if one considers that these restorations have generally been performed under field conditions28. the comparison further suggests that the survival rate of art restorations will become even higher if the procedure is performed in dental offices under more favorable conditions, as recently shown29. some studies have compared the effect of different cavity preparation methods in permanent molars7-8,12-13,17,30-31. yip et al.8 clinically evaluated two encapsulated more viscous esthetic conventional glass ionomer cements, placed using two cavity preparation methods (art and conventional cavity preparation method), and one encapsulated highcopper-content admixed non-gamma 2 amalgam alloy, placed using a conventional cavity preparation method, for the restoration of occlusal caries in permanent molar teeth. after 12 months, all the occlusal restorations were rated as satisfactory. taifour et al.12 compared the treatment of cavitated dentinal lesions in permanent molars through the art approach using high-viscosity glass ionomer with that using the traditional approach with amalgam. the authors observed no statistically significant differences between the survival of restorations placed through the two approaches after 3 years. gao et al.31 compared art and the conventional caries removal method using high-viscosity glass ionomer cement and amalgam, respectively. the authors concluded that art and hand instruments alone for relatively small occlusal cavity preparations in permanent molars took approximately twice as long as the use of conventional rotary instruments. after 30 months, only one restoration with glass ionomer cement failed. both glass ionomer cements had a substantial initial loss of material, but no caries were detected in the exposed fissures. mandari et al.7 compared the effectiveness of three caries removal methods: the conventional method with rotary instruments; a modified conventional method with portable dental equipment; and modified art using manual instruments and caridex for the chemomechanical removal of carious tissue. a total of 430 restorations were performed in contralateral pairs of permanent molars contralaterally. after six months, no statistically significant differences were found between groups with regard to the success rate: 91.0% for art, 96.0% for atraumatic restorative treatment in permanent molars: a systematic review 4040404040 braz j oral sci. 11(1):36-41 the conventional method and 96.0% for the modified conventional method. glass ionomer cement and amalgam were also compared and no significant differences were detected between the materials, despite a tendency toward better results with the glass ionomer cement. frencken et al.17 compared the atraumatic restorative treatment approach with the traditional amalgam approach. the authors reported that the longevity of small single-surface art restorations is comparable with conventional amalgam restorations, while the longevity of large art restorations is lower. it is important to note that the survival rate of small art restorations (90% and 85% after 3 and 6 years, respectively) is much higher than that of large ones (77% and 46% after 3 and 6 years, respectively). the importance of implementing early intervention rather than waiting till the dental caries reaches an advanced stage should be emphasized. in most of these studies, no statistically significant differences were found between the success rates of restorations performed by different cavity preparation methods7-8,12-13,31. some researchers have discussed on the retention and the caries-preventive effect of sealants using the art approach in permanent molars9,21-25. beirute et al.23 compared the preventive effect of glass ionomer cement and a composite sealant and found that 86% of composite resin and 88% of glass ionomer sealant did not survive after 5 years of evaluation, but there were statistically significantly more caries-free pits and fissures in the group sealed with glass ionomer than in the group sealed with composite resin material after 5 years. in a more recent study, oba et al.25 compared the preventive effect of glass ionomer cement and a composite sealant and found that the composite sealant had a significantly greater loss after three years (93.8% versus 55.3%). moreover, the incidence of caries following the loss of material was lower in the cases performed with glass ionomer cement, demonstrating the preventive effect of this material, even after its clinical failure. vieira et al. 2 4 investigated the use of two glass ionomer cements (vidrion r and chemflex) on recently erupted permanent first molars. the authors reported no statistically significant differences in retention rate or the incidence of caries between the conventional glass ionomer cement (vidrion r) and the highviscosity glass ionomer cement (chemflex) after a one-year follow-up period. unfortunately, studies of this kind are scarce. it is therefore not possible to draw conclusions from the findings of this systematic review other than to propose that more studies are required. the indications are, however, that art sealants are promising. in conclusion, the present systematic review revealed high survival rates for single-surface art restorations in permanent molars and lower success rates for multiple-surface restorations. clinical studies conducted with children, adolescents and young adults have demonstrated a reliable clinical performance of single-surface and multi-surface art restorations for 3-year and 2-year periods, respectively. however, long-term clinical studies are scarce and most of them focus on one-surface cavities. the survival rates observed, especially for single-surface restorations, confirm the potential of the art approach for restoring and saving permanent molar teeth. more clinical studies are required on the retention and caries preventive effect of art sealants. references 1. frencken je, pilot t, songpaisany y, phantumvanit p. atraumatic restorative treatment (art): rationale, technique, and development. j public health dent. 1996; 56: 135-40. 2. holmgren cj. the state of art (atraumatic restorative treatment) a scientific perspective. community dent oral epidemiol. 1999; 27: 419-60. 3. holmgren cj, pilot t. minimal intervention techniques for caries. j public health dent. 1996; 56: 161-3. 4. van gemert-schriks mc, van amerongen we, ten cate jm, aartman iha. three-year survival of singleand two-surface art restorations in a high-caries child population. clin oral invest. 2007; 11: 337-43. 5. phantumvanit p, songpaisan y, frencken je. atraumatic restorative treatment (art): a three-year community field trial in thailand-survival of one-surface restorations in the permanent dentition. j public health dent. 1996; 56: 141-5. 6. lo ec, luo y, fan mw, wei sh. clinical investigation of two glassionomer restoratives used with the atraumatic restorative treatment approach in china: two-years results. caries res. 2001; 35: 458-63. 7. mandari gj, truin gj, van‘t hof ma, frencken je. effectiveness of three minimal intervention approaches for managing dental caries: survival of restorations after 2 years. caries res. 2001; 35: 90-4. 8. yip kh, smales rj, gao w, peng d. the effects of two cavity preparation methods on the longevity of glass ionomer cement restorations: an evaluation after 12 months. j am dent assoc. 2002; 133: 744-51. 9. ziraps a, honkala e. clinical trial of a new glass ionomer for an atraumatic restorative treatment technique in class i restorations placed in latvian school children. med princ pract. 2002; 11(suppl 1): 44-7. 10. monse-schneider b, heinrich-weltz r, schug d, sheiham a, borutta a. assessment of manual restorative treatment (mrt) with amalgam in highcaries filipino children: results after 2 years. community dent oral epidemiol. 2003; 31: 129-35. 11. souza fm, cefaly df, terada rs, rodrigues cc, de lima navarro mf. clinical evaluation of the art technique using high density and resinmodified glass ionomer cements. oral health prev dent. 2003; 1: 201-7. 12. taifour d, frencken je, beiruti n, van´t hof ma, truin gj, van palenstein helderman wh. comparison between restorations in the permanent dentition produced by hand and rotary instrumentation: survival after 3 years. community dent oral epidemiol. 2003; 31: 122-8. 13. mandari gj, frencken je, van’t hof m.a. six-year success rates of occlusal amalgam and glass-ionomer restorations placed using three minimal intervention approaches. caries res. 2003; 37: 246-53. 14. lopez n, simpser-rafalin s, berthold p. atraumatic restorative treatment for prevention and treatment of caries in an underserved community. am j public health. 2005; 95: 1338-9. 15. bresciani e, carvalhowl, pereiralcg, baratatje, garcía-godoy f, navarro, mfl. six-month evaluation of art one-surface restorations in a community with high caries experience in brazil. j appl oral sci. 2005; 13: 180-6. 16. lo ecm, holmgren cj, hu d, van palenstein helderman w. six-year follow-up of atraumatic restorative treatment restorations placed in chinese school children. community dent oral epidemiol. 2007; 35: 387-92. 17. frencken je, van’t hof ma, taifour d, al-zaher i. effectiveness of art and traditional amalgam approach in restoring single-surface cavities in posterior teeth of permanent dentitions in school children after 6.3 years. community dent oral epidemiol. 2007; 35: 207-14. atraumatic restorative treatment in permanent molars: a systematic review 4141414141 braz j oral sci. 11(1):36-41 18. ercan e,dülgergil ct,soyman m, dalli m, yildirim i. a field-trial of two restorative materials used with atraumatic restorative treatment in rural turkey: 24-month results. j appl oral sci. 2009; 17: 307-14. 19. cefaly dfg, barata the, tapety cmc, bresciani e, navarro, mfl. clinical evaluation of multisurface art restorations. j appl oral sci. 2005; 13: 15-9. 20. cefaly df, barata tj, bresciani e, fagundes, tc, lauris jr, navarro mf. clinical evaluation of multiple-surface art restorations: 12 month follow-up. j dental child. 2007; 74: 203-8. 21. motsei sm, kroon j, holtshousen ws. evaluation of atraumatic restorative treatment restorations and sealants under field conditions. sadj. 2001; 56: 309-15. 22. abid a, chkir f, ben salem k, argoubi k, sfar-gandoura m. atraumatic restorative treatment and glass ionomer sealants in tunisian children: survival after 3 years. east mediterr health j. 2002; 8: 315-23. 23. beiruti n, frencken je, van´t hof ma, taifour d, van palenstein helderman wh. caries-preventive effect of a one time application of composite resin and glass ionomer sealants after 5 years. caries res. 2006; 40: 52-9. 24. vieira al, zanella nl, bresciani e, barata tde j, da silva sm, machado ma, navarro mf. evaluation of glass ionomer sealants placed according to the art approach in a community with high caries experience: 1-year follow-up. j appl oral sci. 2006; 14: 270-5. 25. oba aa, dülgergil t, sönmez is, dogan s. comparison of caries prevention with glass ionomer and composite resin fissure sealants. j formos med assoc. 2009; 108: 844-8. 26. lima dc, saliba na, moimaz sas. tratamento restaurador atraumático e sua utilização em saúde pública. rgo. 2008; 56: 75-9. 27. dulgergil ct, soyman m, civelek a. atraumatic restorative treatment with resin-modified glass ionomer material: short-term results of a pilot study. med princ pract. 2005; 14: 277-80. 28. van’t hof ma, frencken je, helderman vp holmgren cj. the atraumatic restorative treatment (art) approach for managing dental caries: a metaanalysis. int dent j. 2006; 56: 345-51. 29. scale ns, casamassimo ps. acess to dental care for children in the united states. a survey of general practitioners. j am dent assoc. 2003; 134: 1630-40. 30. frencken je, taifour d, van´t hof ma. survival of art and amalgam restorations in permanent teeth of children after 6.3 years. j dent res. 2006; 85: 622-6. 31. gao w, peng d, smales rj, yip kh. comparison of atraumatic restorative treatment and conventional restorative procedures in a hospital clinic: evaluation after 30 months. quintessence int. 2003; 34: 31-7. atraumatic restorative treatment in permanent molars: a systematic review oral sciences n3 braz j oral sci. 14(3):246-250 original article braz j oral sci. july | september 2015 volume 14, number 3 swelling of self-adhesive resin cement increases long-term push-out bond strength of fiber post to dentin fabrício mezzomo collares1, vicente castelo branco leitune1, carolina rocha augusto1, patrícia franken1, susana maria werner samuel1 1universidade federal do rio grande do sul – ufrs, school of dentistry, dental materials laboratory, porto alegre, rs, brazil correspondence to: fabrício mezzomo collares universidade federal do rio grande do sul dental materials laboratory, school of dentistry rua ramiro barcelos, 2492 rio branco cep:90035-003 porto alegre, rs, brasil phone: +55 51 3308-5198 e-mail: fabricio.collares@ufrgs.br abstract aim: to evaluate the long-term post push-out bond strength to dentin, water sorption, solubility and swelling of conventional and self-adhesive dual-cure resin cements. methods: forty-eight bovine roots were prepared for fiber post cementation with relyx arc and relyx u100. according to resin cement and storage time (24 h and 6 months), 4 groups were assessed using the push-out test. water sorption and solubility were performed according to iso 4049:2009. the swelling coefficient was obtained using cement disks of each material immersed in distilled water until the swelling equilibrium was reached. the mass of dry and swelled polymer and solvent density were used to calculate the coefficient. statistical data analysis was performed using student’s t-test for water sorption, solubility and swelling coefficient and the kruskal-wallis and dunn multiple comparison tests for push-out analysis with a significance level of 0.05. results: the immediate bond strength was not significantly different between relyx arc (3.09 mpa) and relyx u100 (3.78 mpa) (p>0.05). relyx u100 showed higher (p<0.05) bond strength after six months of storage (9.60 mpa) than relyx arc (6.65 mpa). the water sorption and solubility values were not significantly different (p>0.05) between groups. the swelling coefficient of the relyx u100 group was significantly higher than that of the relyx arc group (p<0.05). conclusions: relyx u100 resin cement showed a higher swelling coefficient than relyx arc, and the longitudinal push-out bond strength increased after six months. clinical significance: the clinical longevity of restorative treatment in root-filled teeth is dependent on the long-term properties and behavior of the cement used for post luting. results of this study suggest that the selfadhesive resin cement may be a reliable alternative. keywords: resin cements; dentin-bonding agents; solubility; water storage. introduction fiber posts are widely used to restore endodontically treated teeth as an alternative to metal posts and cores. the similarity in the elastic modulus among the fiber post, resin cement and dentin is advantageous for the improved performance of restorative procedures1. moreover, the chemical nature of the posts allows them to be bonded to canal walls with adhesive systems in combination with resin cements. the choice of a luting agent depends on the clinical situation and on the material’s physical, biologic and handling properties. in general, the resin cements are composed of a dimethacrylate-based polymeric matrix, filler particles, pigments received for publication: september 01, 2015 accepted: september 29, 2015 http://dx.doi.org/10.1590/1677-3225v14n3a14 and chemical substances to start the polymerization reaction. variation in the content of these components strongly influences the physicochemical properties of the material2. the use of conventional resin cements requires pretreatment of the root surface with an adhesive system3 toincrease the bond strength4. the adhesion strategy of etchand-rinse adhesive systems involves two or three steps with the successive application of an acid, followed by a primer and an adhesive resin. in self-etch adhesive systems, the etching and priming steps are combined, and in the most recent formulations, etching, priming, and bonding are combined into a single step5. recently introduced self-adhesive resin cements do not require the pretreatment of the tooth substrate. the adhesive properties of self-adhesive cements are attributed to acidic methacrylate monomers that simultaneously demineralize and infiltrate the tooth substrate, resulting in micromechanical retention 6. self-adhesive resin cements present reliable immediate bond strength to dentin1,7,8. however, the longevity of bond strength is still a concern for cementation procedures. therefore, the purpose of this study was to evaluate the long-term push-out bond strength of post to dentin with conventional and self-adhesive dual-cure resin cements. material and methods specimen preparation forty-eight bovine teeth with similar lengths and dimensions were used in this study. freshly extracted teeth were immediately immersed in distilled water and stored at 4 °c for no more than 6 months. to be included in this study, the following criteria had to be met: straight roots and root length of at least 15 mm. external debris were removed with a periodontal curette. the crown surfaces of each tooth were sectioned below the cementum-enamel junction, perpendicular to their long axis, using a low speed diamond disc with water coolant. after endodontic access, the working length was established by the direct method subtracting 1 mm from the real root length, determined by introducing a no 10 k-file (maillefer-dentsply, ballaigues, switzerland) until the file was visible through the apical foramen. the root canals were prepared with k-files using the step-back technique. the coronal portion of each canal was shaped with size 2 gatesglidden drills. the root canals were irrigated with 3 ml distilled water prior to each instrument. after final irrigation, the root canals were dried with absorbent paper points. cementation of fiber posts the post space of each specimen was enlarged with a no 2 drill from the exacto post system (angelus, londrina, pr, brazil), 4 mm before reaching the working length depth. the fiber post was 20 mm long, 1.4 mm cervical diameter, and 0.9 mm apical diameter. to standardize the method, the same operator performed all of the procedures. following post space preparations, the roots were randomly divided into 4 experimental groups of 12 teeth, according to material and storage time (n=12). the resin cements were applied according to the manufacturer’s instructions and are shown in table 1. the fiber posts were cleaned with 96% ethanol, and silane was applied with disposable microbrush tips9. in the relyx arc (3m espe) groups, the intracanal dentin was etched with 37% phosphoric acid for 15 s, rinsed with distilled water for 15 s, and then gently dried with absorbent paper points. the activator (3m espe) was applied to the canal with a paper point and gently air-dried followed by application of the primer (scotch bond multi-purpose plus, 3m espe). the catalyst (3m espe) was applied on the root dentin and the post. the cements were inserted only into the root canal10 with accudose (centix inc, shelton, ct, usa) needle tubes and a centrix syringe (centrix inc, shelton, ct, usa)11. the fiber post was inserted and excess cement was removed. light activation was performed through the cervical portion of the root for 30 s at the buccal and lingual surfaces for a total of 60 s of light exposure, with a 5 mm distance between the source and the root. the resin cement and adhesive were light activated with xl2500 (3m espe), with an output intensity of 600 mw/cm2. the power of the light curing unit was gauged with a radiometer (model 100, demetron research group, danbury, ct, usa). push-out test all the roots in all groups were stored in 37 °c distilled water for 7 days and then serially sectioned into 0.7 mmthick slices in a precision cutting machine (low speed saw, material composition relyx arc paste a bisgma, tegdma, silane treated ceramic, silane treated silica, functionalized dimethacrylates polymer, triphenylantimony. paste b bisgma, tegdma, silane treated silica, silane treated ceramic, functionalized dimethacrylates polymer, benzoyl peroxide. relyx u100 base glass powder, methacrylated phosphoric acid esters, triethylene glycol dimethacrylates, silane treated silica, sodium persulfate. catalyst glass powder, substituted dimethacrylate, silane treated silica, sodium p-toluenesulfinate, calcium hydroxide. scotch bond multi-purpose plus primer hema, copolymer of acrylic and itaconic acids, water. catalyst bisgma, hema, benzoyl peroxide, triphenylphosphine, triphenylantimony, hydroquinone. activator ethyl alcohol, sodium benzenesulfinate. table 1:table 1:table 1:table 1:table 1: chemical compositions of materials 247247247247247 swelling of self-adhesive resin cement increases long-term push-out bond strength of fiber post to dentin braz j oral sci. 14(3):246-250 248248248248248 buehler, lake bluff, il, usa) with constant water cooling. the slices of all roots from each group were stored in 37 °c distilled water for 24 hours or 6 months before the push-out tests. the cervical and apical diameters of the canal and the thickness of all of the slices were measured with a digital caliper. each section was marked on its apical side and positioned on a base with a central hole in a universal testing machine (dl2000, emic, são josé dos pinhais, pr, brazil). the push-out test was performed applying a compressive load to the apical side of each slice using a 0.7 mm-diameter cylindrical plunger attached to the upper portion of the testing machine. a crosshead speed of 0.5 mm/min was applied until bond failure occurred. to express the bond strength in mpa, the load upon failure was recorded in newton (n) and divided by the bond area (mm2)12. water sorption and solubility water sorption and solubility were determined based on the iso 4049:2009 standard specification, except for specimen size. cement disks (n=5) of each material were produced in a polytetrafluoroethylene matrix (6.0 mm diameter and 1-mm thick, in order to fit the light output guide of the qth curing unit). for specimen preparation, the cement was directly dispensed into the mold until it was filled. an acetate strip was placed on top of the cement and covered with a glass slide. the specimens were light activated for 20 s, removed from the mold and the opposite surface received additional light activation for 20 s. specimens were placed in a desiccator containing silica gel at 37 °c. the disks were repeatedly weighed after 24-h intervals on an analytical scale until a constant mass (m1) was obtained (i.e., until the mass loss of each specimen was not more than 0.1 mg in any 24-h period). the diameter and thickness of each specimen were measured with a digital caliper to calculate the volume (v) of each disk (in mm3). thereafter, the specimens were stored in sealed glass vials with 10 ml distilled water at 37 °c for 7 days. after seven days, the disks were weighed after being washed under running water and gently wiped with an absorbent paper to obtain the mass measure (m2) and then returned to the desiccator. next, the specimens were weighed until a constant mass (m3) was obtained (as described above). water sorption (ws) and solubility (sl) were calculated in micrograms per cubic millimeter13. swelling coefficient swelling coefficient measurements were performed gravimetrically. cement samples (n=3) of each material were produced in a polytetrafluoroethylene matrix (2 x 2 x 12 mm). next, the samples were immersed in distilled water at 37 °c until swelling equilibrium was reached. then, the samples were removed, the excess solution deposited on the film surface was quickly removed with blotting paper and the samples were weighed. the values of the swelling coefficients (α) of all materials were calculated using the following equation: α= ((meq – m0) / m0) x 1/ds fig. 1: bond strength values as a function of evaluation period and cement type. different letters represents statistically significant difference among groups (p<0.05). where meq is the mass of the sample (polymer+solvent) after equilibrium, m0 the mass of the polymer, and ds is the density of the solvent14. statistical analysis statistical analysis was performed using sigmastat (version 4, ashburn, ga, usa). the normality of the results was tested using the kolmogorov-smirnov test. the used statistical tests were student’s t-test for water sorption, solubility and swelling coefficient and the kruskal-wallis and dunn multiple comparison tests for push-out analysis, all at a significance level of 0.05. results the results of the bond strength analysis are presented in figure 1. the relyx arc group exhibited 3.09 (±1.72) mpa at 24 h and 5.65 (±5.77) mpa at 6 months (p>0.05). the relyx u100 group presented 3.78 (±1.84) mpa and 9.60 (±7.65) mpa at 24 h and 6 months (p<0.05), respectively. group ws (µg/cm3) sl (µg/cm3) ααααα (mlg) relyx arc 19.27 (±1.39) 2.19 (±0.45) 0.011 (±0.00006) relyx u100 19.94 (±2.98) 0.86 (±1.48) 0.027 (±0.00172) p 0.66 0.09 0.000 table 2:table 2:table 2:table 2:table 2: mean and standard deviation (±sd) of water sorption (ws), solubility (sl) and swelling coefficient (α) of the resin cement water sorption and solubility showed no statistical differences (p>0.05) between cements. the swelling coefficient of the relyx u100 group (0.027 mlg) was significantly higher than that of the relyx arc group (0.011 mlg) (p<0.05). the means and standard deviations are shown in table 2. swelling of self-adhesive resin cement increases long-term push-out bond strength of fiber post to dentin braz j oral sci. 14(3):246-250 249249249249249 discussion the clinical longevity of restorative treatment in rootfilled teeth relies on the properties of the used cement3. water movement from the intra-radicular dentin through the hybrid layer is one of the reasons for cement/dentin interface degradation over time15. in this study, the tested resin cements showed similar water sorption, solubility and immediate pushout bond strength. nevertheless, relyx u100 demonstrated higher swelling coefficients and longitudinal push-out bond strength values than relyx arc. water sorption of polymers could lead to degradation15. furthermore, the polymer could undergo hygroscopic expansion 2,16 due to the chemistry of monomers and polymerization linkages17. monomers like hema, bis-gma and tegdma are heteroatom structures composed of carbon and oxygen 15 revealing the presence of hydrolytically susceptible ester groups18. water sorption of resin composites depends on the degree of conversion of the polymer, the polar interaction, the particle size and morphology of the filler, and the surface area exposed to water17. in this study, all specimens presented water sorption after seven days of storage. the swelling coefficient was calculated based on the mass of the polymer before and after immersion, when the polymer shows a constant swelling mass. in this study, the resin cements only presented a stable mass after 29-day immersion. relyx u100 presented higher swelling coefficient than relyx arc. self-adhesive resin cement contains acidic monomers (e.g., carboxylated or phosphate-derivatized methacrylates) in its composition, which have polar structures, leading to increased water sorption19. retention of fiber posts in roots depends on the bond strength between post material and a resin luting agent, bond strength between post space dentin and resin luting agent1. despite the difference in the mechanism of adhesion of both tested materials, immediate push-out bond strength presented no difference in this study, corroborating other studies12,20-24. although resin cements present reliable immediate results, the longevity of the bond strength should be evaluated. adhesive interfaces are prone to degradation over time, leading to a decrease in bond strength. one could think that 6 months of water storage could decrease the push-out bond strength of resin cements. however, in this study, six months of storage significantly increased the bond strength values for the relyx u100 group. according to a systematic review there is a 3.81 mpa increase of resistance to dislodgement for bovine teeth when compared to human teeth 25. the increase in value is proportional to the used type of teeth. in the present study, bovine teeth were used in all groups. considering the water sorption value (19.94 µg/cm3) and low solubility (0.86 µg/cm3), relyx u100 presented hygroscopic expansion leading to material swelling. the hygroscopic behavior of materials is strongly influenced by filler composition17. the self-adhesive resin cement used in this study contains fluoraluminosilicate glass powder, which exhibits hygroscopic expansion26. glass-ionomer and resinmodified glass-ionomer luting cements presented delayed hygroscopic expansion increasing fiber post retention27. the hygroscopic expansion could explain the increased longterm bond strength values showed in this study. the relyx arc group exhibited lower push-out bond strength values after six months of storage, suggesting that a concurrent solubility (2.19 µg/cm3) process occurred during the water sorption stage28. the sorption and solubility features of resin cements have been studied29, and the structural characteristics of polymers are essential to determine the extent to which polymers are affected by an aqueous environment17. other parameters, such as the cross-linking density of the polymeric network 29, the amount of monomers 30 and polarity of functional groups may affect the cement behavior. the results presented by relyx arc are in agreement with the current literature31-32. despite the materials’ degradation over time, the selfadhesive resin cement showed higher bond strength after six months, presenting a high swelling coefficient and low solubility. considering that the leachability of dental polymers is a biological concern, polymers with low release of components are required. acknowledgements the authors would like to express their thanks to capes and fapeam for the financial support. references 1. das ak, muddugangadhar bc, amarnath gs, garg a, kumar u, rao tr. comparative evaluation of push out bond strength of a fiber post system using four different resin cements: an in vitro study. j int oral health. 2015; 7: 62-7. 2. braga rr, cesar pf, gonzaga cc. mechanical properties of resin cements with different activation modes. j oral rehabil. 2002; 29: 257-62. 3. silva ra, coutinho m, cardozo pi, silva la, zorzatto jr. conventional dual-cure versus self-adhesive resin cements in dentin bond integrity. j appl oral sci. 2011; 19: 355-62. 4. pashley dh, tay fr, breschi l, tjaderhane l, carvalho rm, carrilho m, et al. state of the art etch-and-rinse adhesives. dent mater. 2011; 27: 1-16. 5. vaidyanathan tk, vaidyanathan j. recent advances in the theory and mechanism of adhesive resin bonding to dentin: a critical review. j biomed mater res b appl biomater. 2009; 88: 558-78. 6. moraes rr, boscato n, jardim ps, schneider lf. dual and self-curing potential of self-adhesive resin cements as thin films. oper dent. 2011; 36: 635-42. 7. peumans m, voet m, de munck j, van landuyt k, van ende a, van meerbeek b. four-year clinical evaluation of a self-adhesive luting agent for ceramic inlays. clin oral investig. 2013; 17: 739-50. 8. frassetto a, navarra co, marchesi g, turco g, di lenarda r, breschi l, et al. kinetics of polymerization and contraction stress development in self-adhesive resin cements. dent mater. 2012; 28: 1032-9. 9. li r, zhou h, wei w, wang c, sun yc, gao p. effects of mechanical and chemical pretreatments of zirconia or fiber posts on resin cement bonding. plos one. 2015; 10: 1-12. 10. skupien ja, sarkis-onofre r, cenci ms, de moraes rr, pereira-cenci t. a systematic review of factors associated with the retention of glass fiber posts. braz oral res. 2015; 29: 1-8. swelling of self-adhesive resin cement increases long-term push-out bond strength of fiber post to dentin braz j oral sci. 14(3):246-250 250250250250250 11. souza ac, gonçalves f de c, anami lc, melo rm, bottino ma, valandro lf. influence of insertion techniques for resin cement and mechanical cycling on the bond strength between fiber posts and root dentin. j adhes dent. 2015; 17: 175-80. 12. kahnamouei ma, mohammadi n, navimipour ej, shakerifar m. pushout bond strength of quartz fibre posts to root canal dentin using total-etch and self-adhesive resin cements. med oral patol oral cir bucal. 2012; 17: e337-44. 13. collares fm, ogliari fa, zanchi ch, petzhold cl, piva e, samuel sm. influence of 2-hydroxyethyl methacrylate concentration on polymer network of adhesive resin. j adhes dent. 2011; 13: 125-9. 14. neumann mg, schmitt cc, catalina f, goi be. the relation between the polymerization rates and swelling coefficients for copolymers obtained by photoinitiation. polymer testing. 2007; 26: 189-94. 15. ferracane jl. hygroscopic and hydrolytic effects in dental polymer networks. dent mater. 2006; 22: 211-22. 16. zhu s, platt j. curing efficiency of three different curing modes at different distances for four composites. oper dent. 2011; 36: 362-71. 17. malacarne j, carvalho rm, de goes mf, svizero n, pashley dh, tay fr, et al. water sorption/solubility of dental adhesive resins. dent mater. 2006; 22: 973-80. 18. collares fm, leitune vcb, portella ff, ogliari fa, samuel smw. longterm bond strength, degree of conversion and resistance to degradation of a hema-free model adhesive. braz j oral sci. 2014; 13: 261-5. 19. nakaoki y, nikaido t, pereira pn, inokoshi s, tagami j. dimensional changes of demineralized dentin treated with hema primers. dent mater. 2000; 16: 441-6. 20. calixto lr, bandeca mc, clavijo v, andrade mf, vaz lg, campos ea. effect of resin cement system and root region on the push-out bond strength of a translucent fiber post. oper dent. 2012; 37: 80-6. 21. amaral m, rippe mp, bergoli cd, monaco c, valandro lf. multi-step adhesive cementation versus one-step adhesive cementation: push-out bond strength between fiber post and root dentin before and after mechanical cycling. gen dent. 2011; 59: e185-91. 22. bergoli cd, amaral m, druck cc, valandro lf. evaluation of four cementation strategies on the push-out bond strength between fiber post and root dentin. gen dent. 2011; 59: 498-502. 23. amaral m, rippe mp, konzen m, valandro lf. adhesion between fiber post and root dentin: evaluation of post surface conditioning for bond strength improvement. minerva stomatol. 2011; 60: 279-87. 24. gomes gm, gomes om, reis a, gomes jc, loguercio ad, calixto al. regional bond strengths to root canal dentin of fiber posts luted with three cementation systems. braz dent j. 2011; 22: 460-7. 25. collares fm, portella ff, rodrigues sb, celeste rk, leitune vc, samuel sm. the influence of methodological variables on the push-out resistance to dislodgement of root filling materials: a meta-regression analysis. int endod j. 2015; doi: 10.1111/iej.12539. 26. 26yiu ck, tay fr, king nm, pashley dh, carvalho rm, carrilho mr. interaction of resin-modified glass-ionomer cements with moist dentine. j dent. 2004; 32: 521-30. 27. cury ah, goracci c, de lima navarro mf, carvalho rm, sadek ft, tay fr, et al. effect of hygroscopic expansion on the push-out resistance of glass ionomer-based cements used for the luting of glass fiber posts. j endod. 2006; 32: 537-40. 28. wei yj, silikas n, zhang zt, watts dc. diffusion and concurrent solubility of self-adhering and new resin-matrix composites during water sorption/ desorption cycles. dent mater. 2011; 27: 197-205. 29. attar n, tam le, mccomb d. mechanical and physical properties of contemporary dental luting agents. j prosthet dent. 2003; 89: 127-34. 30. marghalani hy. sorption and solubility characteristics of self-adhesive resin cements. dent mater. 2012; 28: e187-98. 31. leme aa, coutinho m, insaurralde af, scaffa pm, da silva lm. the influence of time and cement type on push-out bond strength of fiber posts to root dentin. oper dent. 2011; 36: 643-8. 32. pereira jr, da rosa ra, só mv, afonso d, kuga mc, honorio hm et al. push out bond strength of fiber post to root dentin using glass ionomer and resin modified glass ionomer cements. j appl oral sci. 2014 ;22: 390-6. swelling of self-adhesive resin cement increases long-term push-out bond strength of fiber post to dentin braz j oral sci. 14(3):246-250 404 not found oral sciences n3 case report braz j oral sci. april/june 2010 volume 9, number 2 radiological study of three cases of loose bodies in the temporomandibular joint allan abuabara1 , dale a. miles2 , giuseppe v. cruz3, luis a. passeri4 1specialist in dental & maxillofacial radiology, health division of the city of joinville, brazil 2dds, ms, frcd(c), university of texas, san antonio; arizona school of dentistry and oral health, texas, usa. 3dds, ms, professor of dental & maxillofacial radiology, joinville university, brazil 4 dds, msc, phd, professor of oral and maxillofacial surgery, division of plastic surgery, department of surgery, school of medical sciences, state university of campinas, brazil correspondence to: allan abuabara rua fernando machado, 400, apto 201, cep: 89204-400 joinville-sc, brazil phone: +55-47-3801 2423 e-mail: allan.abuabara@gmail.com received for publication: november 24, 2009 accepted: june 9, 2010 abstract according to the literature, loose bodies in the temporomandibular joint (tmj) primarily prompt to synovial chondromatosis (sc). sc is a cartilaginous metaplasia of the mesenchymal remnants of the synovial tissue of the joints. its main characteristic is the formation of cartilaginous nodules in the synovium and inside the articular space, described as loose bodies. the main symptoms are pain, limitation of jaw movement, crepitation and inflammation. diagnosis is made by panoramic radiograph, computed tomography scan and mainly magnetic resonance imaging. sc is usually monoarticular. we report two cases of bilateral loose bodies in tmj and one monoarticular evaluated through plain radiograph and cone beam volumetric tomography (cbvt). clinical and radiologic findings are reviewed and discussed. references for diagnosis of sc affecting tmj are supported. patients presenting preauricular swelling, pain and restriction of tmj movements should be evaluated with plain radiography, cbvt and if necessary, magnetic resonance imaging. if loose bodies are found, synovial chondromatosis must be the first suspicion. the definitive diagnosis depends on histology. a differential diagnosis of chondrosarcoma should be considered because of the life-threatening features of chondrosarcoma. keywords: synovial chondromatosis, temporomandibular joint, temporomandibular joint disorders. introduction according to the literature, loose bodies in the temporomandibular joint (tmj) primarily prompt to synovial chondromatosis1. synovial chondromatosis (sc) is a cartilaginous metaplasia of the mesenchymal remnants of the synovial tissue of the joints. its main characteristic is the formation of cartilaginous nodules in the synovium and inside the articular space, the loose bodies2. other clinical signs and symptoms of sc are preauricular swelling, pain, crepitus and limitation of mandibular movement1-2. sc commonly involves large joints such as the knee, hip and elbow, but its presence in smaller joints has also been reported3. the incidence of sc is more common in the second and third decades of life. however, when it affects the tmj, it is more common in women (1,5:1) during their fourth and fifth decades of life and rare in children4-5. diagnosis is made by panoramic radiograph, computed tomography (ct) braz j oral sci. 9(2):133-136 braz j oral sci. 9(2):133-136 scan and mainly, magnetic resonance imaging2. sc is usually monoarticular. reports of bilateral sc of the tmj is scarce in literature6-7. this paper presents 3 cases of loose bodies in tmj, being two bilateral cases, evaluated by plain radiograph and cone beam volumetric tomography (cbvt). clinical and radiologic findings are reviewed and discussed. references for diagnosis of sc affecting tmj are supported. case 1 a 17-year-old white woman was referred to our radiological center due to pain in the tmjs on opening the mouth, and bilateral preauricular tenderness. physical examination revealed slight diffuse swelling of the preauricular region, normal open the mouth (40mm) without mandibular deviation. the patient had no history of trauma or rheumatoid arthrosis. an orthopantomograph of tmjs (instrumentarium imaging, tuusula, finland) (figures 1 and 2) revealed bilateral calcifications posterosuperior the mandible condyle (loose bodies). in positions of maximum intercuspation (mi) and open mouth, the loose bodies did not appear to accompany the condyle movement. the cortical bone of condyles and glenoid fossa were intact. clinical and radiologic findings prompted a diagnosis of bilateral sc of the tmj. no follow-up information was received from the referring clinician. fig. 1. case 1. orthopantomograph of right tmj: maximum intercuspation (mi) and open mouth. arrows point to the loose bodies. fig. 2. case 1. orthopantomograph of left tmj: maximum intercuspation (mi) and open mouth. arrows point to the loose bodies. case 2 a 68 year-old white female was referred to an imaging service for cbvt (i-cat, imaging sciences international, hatfield, pa, usa) evaluation of the tmj regions due to intermittent pain and “locking” problems. radiographic evaluation of the data volume revealed the bilateral presence of loose bodies consistent with sc as well as subchondral cyst formation and subchondral sclerosis (figures 3 and 4). diagnoses of osteoarthritis and sc were included in the “clinical findings” and “clinical impressions and recommendations” sections of the radiographic report. in addition, both stylohyoid ligaments were calcified and elongated, but did not relate to any of her symptomatology (figure 4). no follow-up information was received from the laboratory service or referring clinician. fig. 3. case 2. sagittal view (cbvt) of left and right condylar head. arrows point he loose bodies. fig. 4. case 2. panoramic view of cbvt. arrows point to the bilateral loose bodies and both stylohyoid ligaments calcified and elongated. case 3 a 78 year-old white female was referred to an imaging facility for the cbvt (i-cat, imaging sciences international) evaluation of potential implant sites in the maxilla and mandible. radiographic interpretation of the data set revealed multiple discrete calcifications in and around the “joint space” of the right tmj. in addition, there was a large lobular radiopaque mass located medially to the condylar head, originating from the temporal bone (figures 5 and 6). osteoarthritic changes were also seen. a clinical impression of sc as well as osteoarthritic changes was given in the radiology report. no follow-up information was received from the laboratory service or referring clinician. 134 radiological study of three cases of loose bodies in the temporomandibular joint braz j oral sci. 9(2):133-136 fig. 5. case 3. panoramic view of cbvt. arrow points to the loose bodies and a mass originating from the temporal bone. fig. 6. case 3. coronal image and 3-d reconstruction (cbvt) of right tmj. arrows point the loose bodies and a mass medially to the condylar head originating from the temporal bone. discussion the etiology of sc is still unknown. however, most researchers believe it to be associated with embryologic disorders, a cartilaginous metaplasia of synovial tissue remains. it can be associated with trauma or microtrauma, infection or articular disease, such as inflammatory and noninflammatory osteochondritis and arthroplasty4,8. conventional radiography is not particularly sensitive to the presence of calcified loose bodies, since they have been detected in less than 45% of reported cases9. oftentimes, loose bodies do not show bone formation, thus plain radiographs are not always suitable if sc is suspected5. an interesting finding is that loose bodies can be seen bilaterally in a plain radiograph (case 1). ct and magnetic resonance imaging (mri) had greatly improved radiological diagnosis of this condition1,10. conventional ct scan reformats series of parallel helical slices, which incorporate small errors in final scan. two important advantages of cbvt over conventional tomography are more accurate images and less radiation. cbvt captures volumes of data taken in one 194 to 360-degee rotation about patient’s head. each volume “touches” adjacent volume to avoid distortion and error in reformatted studies11. the radiation exposure to a patient from a conventional ct is approximately 100-300 microsieverts (ìsv) for the maxilla and 200-500 ìsv for the mandible12. the radiation exposure from cbvt is between 34-102 microsieverts (ìsv) depending on the time and resolution of the scan, the same magnitude of conventional dental radiographs13. according holmlund et al.5, mri is useful in demonstrating internal derangement of the tmj. intra-articular loose bodies, expansion of joint capsule and fluid accumulation within the joint space can be directly depicted by mri14. an advantage of mri over ct is in the detection of parotid tumor, which may clinically mimic sc15-16. a recent systematic review was carried out and reported that the prevalence of sc in the tmj is very low with less than 300 cases reported in the literature17. from january 1998 to july 2009, only 80 cases were retrieved. females are affected almost three times more than males. the right and left tmjs seem to be affected equally. only a single new case of bilateral involvement was reported17. because of the nonspecific symptom and signs, sc in tmj should be differentially diagnosed first with tmj disorder. leveling, erosion, destruction of articular bone surface (degenerative osteoarthritis), osteophyte formation and reduction of the joint space must be investigated. about 0.3% of the patients complaining of tmj pain and dysfunction were found with sc18. in addition, sc has been found to coexist with degenerative osteoarthritis as we can see in this study (cases 2 and 3), and sometimes with condylar hyperplasia19. in agreement with the gender predilection found in the literature, our patients were women. highlights of the cases presented hereby are the wide age difference among the patients (age range from 17 to 78 years) and the reporting of two cases of bilateral loose bodies, which is rare in literature. almost all cases of sc of the tmj that have been reported showed monoarticular occurrence. although our cases prompt to sc, differential diagnosis from other pathologies of the joint, such as osteoarthrosis, osteochondritis dissecans, intracapsular fractures and inflammatory arthritis must be considered and histopathologic study can be required20-21. rheumatologic conditions, dysfunction of the facial nerve and hearing disturbances also must be investigated. a differential diagnosis of chondrosarcoma should be considered because of the life-threatening features of chondrosarcoma, which is a rare primary malignancy of the tmj17. owing to the aggressive nature of sc cases for which a known etiology cannot be identified, it was suggested that primary cases may be neoplastic in origin and secondary cases metaplastic in origin, thus representing mild and benign tumoral variants. malignant transformation of sc is very rare, but some histopathological signs of sc, such as cellular atypia, may easily be misinterpreted as signs of malignancy17. histologically, sc often has atypical and multinucleated chondrocytes, so is essential to differentiate also from a chondrosarcoma21. perry et al.22 have reported malignant transformation of the cartilage with development of chondrosarcoma. yokota et al.16 presented a case of sc in the tmj with extension into the middle cranial fossa manifesting as swelling and exacerbation of pain. the preoperative diagnosis was chondrosarcoma or osteosarcoma, however the histological diagnosis was sc. treatment for sc is surgical, removing all the loose bodies through surgical exploration or arthroscopical 135radiological study of three cases of loose bodies in the temporomandibular joint 136 removal5,23-24. adachi et al..24 believe that when loose bodies alone are present, arthrocentesis could be an efficient method of treatment. the authors report completely resolution of the symptoms after arthrocentesis and no recurrence of the lesion after 18 months of follow up. monitoring is important in the light of possible malignant transformation of the cartilage with development of chondrosarcoma or recurrence25-26. surgery has always been recommended as the therapy of choice but some authors advocate less invasive techniques, such as arthroscopy and two-needle arthrocentesis, to remove the loose bodies from the joint space1. data from a systematic review do not support the hypothesis that minor surgery may be sufficient to treat sc17. the reported success rate for arthroscopy was no better than 55%, since in almost half the cases complete removal of loose bodies from the joint cavity was not achieved by arthroscopy and needle aspiration alone, an open surgery was needed to clear the synovia thoroughly. tmj arthroscopy has no advantage over arthrocentesis in terms of efficacy and none over open surgery in terms of post-surgical course. diagnosis of sc in tmj can be made by panoramic radiography, ct and mainly, mri. in particular, the utility of a cbvt in diagnosing a lesion involving the tmj is emphasized in this study. dentists must be aware of the availability of this imaging technology to diagnose tmj anomalies. patients presenting preauricular swelling, pain and restriction of tmj movements should be evaluated with plain radiography, cbvt and if necessary, mri. if loose bodies were found, synovial chondromatosis must be the first suspicion. the definitive diagnosis depends on the histological analysis. references 1. carls fr, von hochstetter a, engelke w, sailer hf. loose bodies in the temporomandibular joint. the advantages of arthroscopy. j craniomaxillofac surg. 1995; 23: 215-21. 2. mandrioli s, polito j, denes sa, clauser l. synovial chondromatosis of the temporomandibular joint. j craniofac surg. 2007; 18: 1486-8. 3. shearer h, stern p, brubacher a, pringle t. a case report of bilateral synovial chondromatosis of the ankle. chiropr osteopatv. 2007; 15: 18. 4. bonatti b de s, patrocinio lg, costa sa, costa jm, patrocinio ja. temporomandibular joint synovial chondromatosis. rev bras otorrinolaringol. 2008; 74: 480. 5. holmlund ab, eriksson l, reinholt fp. synovial chondromatosis of the temporomandibular joint: clinical, surgical and histological aspects. int j oral maxillofac surg. 2003; 32: 143-7. 6. keogh cf, torreggiani wc, munk pl. bilateral synovial chondromatosis of the temoromandibular joint. clin radiol. 2002; 57: 862. 7. peng lw, yan dm, wang yg, li yd. synovial chondromatosis of the temporomandibular joint: a case report with bilateral occurrence. j oral maxillofac surg. 2009; 67: 893-5. 8. lustmann j, zeltser r. synovial chondromatosis of the temporomandibular oint. review of the literature and case report. int j oral maxillofac surg. 1989; 18: 90-4. 9. koyama j, ito j, hayashi t, kobayashi f. synovial chondromatosis in the temporomandibular joint complicated by displacement and calcification of the articular disk: report of two cases. ajnr am j neuroradiol. 2001; 22: 1203-6. 10. wong wc, cheng pw, chan fl. mri appearance of synovial chondromatosis in the temporomandibular joint. clin radiol. 2001; 56: 773-4. 11. winter aa, pollack as, frommer hh, koenig l. cone beam volumetric tomography vs. medical ct scanners. n y state dent j. 2005; 71: 28-33. 12. dula k, mini r, van der stelt pf, lambrecht jt, schneeberger p, buser d. hypothetical mortality risk associated with spiral computed tomography of the maxilla and mandible. eur j oral sci. 1996; 104: 503-10. 13. brooks sl. effective dose of two cone-beam ct scanners: i-cat and newtom 3g. quarterly publication of the american association of dental maxillofacial radiographic technicians, winter 2005. available from: http:/ /www.aadmrt.org/static.aspx?content=currents/brooks_winter_05. 14. herzog s, mafee m. synovial chondromatosis of the tmj: mr and ct findings. ajnr am j neuroradiol. 1990; 11: 742-5. apud wong wc, cheng pw, chan fl. mri appearance of synovial chondromatosis in the temporomandibular joint. clin radiol. 2001; 56: 773-4. 15. thompson k, schwartz hc, miles jw. synovial chondromatosis of the temporomandibular joint presenting as a parotid mass: possibility of confusion with benign mixed tumor. oral surg oral med oral pathol. 1986; 62: 37780. 16. yokota n, inenaga c, tokuyama t, nishizawa s, miura k, namba h. synovial chondromatosis of the temporomandibular joint with intracranial extension. neurol med chir. 2008; 48: 266-70. 17. guarda-nardini l, piccotti f, ferronato g, manfredini d. synovial chondromatosis of the temporomandibular joint: a case description with systematic literature review. int j oral maxillofac surg. 2010 [article in press]. 18. ida m, yoshitake h, okoch k, et al. an investigation of magnetic resonance imaging features in 14 patients with synovial chondromatosis of the temporomandibular joint. dentomaxillofac radiol. 2008; 37: 213-9. 19. meng j, guo c, yi b, zhao y, luo h, ma x. clinical and radiologic findings of synovial chondromatosis affecting the temporomandibular joint. oral surg oral med oral pathol oral radiol endod. 2010; 109: 441-8. 20. balliu e, medina v, vilanova j, peláez i, puig j, trull jm, pedraza s. synovial chondromatosis of the temporomandibular joint: ct and mri findings. dentomaxillofac radiol. 2007; 36: 55-8. 21. forssell k, happonen rp, forssell h. synovial chondromatosis of the temporomandibular joint. report of a case and review of the literature. int j oral maxillofac surg. 1988; 17: 237-41. 22. perry be, mcqueen da, lin jj. synovial chondromatosis with malignant degeneration to chondrosarcoma. report of a case. j bone joint surg am. 1988; 70: 1259-61. 23. sembronio s, albiero am, toro c, robiony m, politi m. arthroscopy with open surgery for treatment of synovial chondromatosis of the temporomandibular joint. br j oral maxillofac surg. 2008; 46: 582-4. 24. adachi pl, kaba sp, martins mt, hueb ch, shinohara eh. arthrocentesis in the treatment of loose bodies of the temporomandibular joint associated with synovial chondromatosis. br j oral maxillofac surg. 2008; 46: 3201. 25. d’souza b, dimitroulis g. a case of recurrence of synovial chondromatosis of the temporomandibular joint. oral surg oral med oral pathol oral radiol endod. 2007; 104: e59-61. 26. wittkop b, davies am, mangham dc. primary synovial chondromatosis and synovial chondrosarcoma: a pictorial review. eur radiol. 2002; 12: 2112-9. braz j oral sci. 9(2):133-136 radiological study of three cases of loose bodies in the temporomandibular joint oral sciences n3 original article braz j oral sci. 10(1):74-78 original article braz j oral sci. january | march 2011 volume 10, number 1 metabolic activity of streptococcus mutans biofilms after treatment with different mouthwash formulations taciano r. cardoso1, alexandre s. carvalho1, marcelo e. beletti2, marcelo h. napimoga3, geraldo thedei jr1 1laboratory of biochemistry of microorganisms, university of uberaba, uberaba, brazil 2college of veterinary medicine, federal university of uberlandia, brazil 3laboratory of biopathology and molecular biology, university of uberaba, brazil correspondence to: geraldo thedei jr. pró-reitoria de pesquisa, pós-graduação e extensão universidade de uberaba. avenida nenê sabino, 1801 bloco r. bairro universitário uberaba mg cep 38055-500 phone: +55-34-3319-8959 fax: +55-34-3314-8910 e-mail: geraldo.thedei@uniube.br received for publication: november 16, 2010 accepted: march 22, 2011 abstract aim: the aim of this study was to investigate the metabolic activity of streptococcus mutans biofilms after treatment with mouthwashes with different compositions. methods: s. mutans biofilms were growth on polystyrene plates during 18 h, washed with sterile saline and treated with the following mouthwashes during 1 min: listerine®, oral b®, parodontax® and periogard® with and without alcohol. after the treatment, the biofilms were incubated with complete medium containing sucrose during 60, 120 or 180 min, and then samples were collected for ph measurements. in addition, biofilms were grown in microscope coverslips treated as described above, followed by staining with propidium iodide and fluoresceine for visualization with a confocal laser scanning microscopy. results: for all mouthwashes evaluated, treatment was deleterious to cell metabolism, since little or no acidification was observed at least 60 min after treatment. mouthwashes containing 0.2% chlorhexidine (parodontax®) or essential oils (listerine®) induced a significant reduction in the metabolic activity of biofilms during the tested time points (120 and 180 min after treatment), being thus more effective than the mouthwashes containing 0.12% chlorhexidine (periogard®) or cetylpyridinium plus fluoride (oral b®). the confocal analysis overall confirmed the results observed in the analysis of metabolic activity. conclusions: the treatment of biofilms with mouthwashes containing 0.2% chlorhexidine or essential oils induced significant reduction in s. mutans metabolism. keywords: streptococcus mutans, mouthwashes, chlorhexidine, biofilm. introduction dental caries is a chronic contagious disease caused by several interacting factors, which results in the irreversible destruction of the mineralized structures of teeth, compromising their vitality and fixation in the maxillomandibular complex1, 2. the gram positive bacteria streptococcus mutans are a substantial part of the oral microbiota and their importance in the dental caries etiology is unquestionable3. the carbohydrates present in the diet are the main energy source in an anaerobic process (mainly lactic fermentation) resulting in the production of organic acids. these acids decrease the ph to around 4.5 on the tooth surface, thus inducing its demineralization4. one important characteristic of s. mutans in promoting caries development is 75 braz j oral sci. 10(1):74-78 the ability to adhere firmly to the tooth surface in the presence of sucrose. this adherence is mediated mainly by the action of the gtf enzymes, which are considered fundamental to the virulence of s. mutans in the pathogenesis of dental caries 5-7. biofilm formation occurs as a result of a sequence of events: microbial surface attachment, cell proliferation, matrix production and detachment8. this process is partially controlled by quorum sensing, an interbacterial communication mechanism that is dependent on population density and is associated with radical changes in protein expression patterns8. mature biofilms demonstrate a complex threedimensional structure with numerous microenvironments differing with respect to osmolarity, nutritional supply and cell density. many antimicrobial agents that are effective against planktonic cells turn out to be ineffective against the same bacteria growing in a biofilm state9,10. planktonic and biofilm cells also exhibit different susceptibilities to a certain antimicrobial concentration. several studies focusing on the efficacy of mouthwashes with diverse chemical composition demonstrated that combination of sodium fluoride and sodium lauryl sulfate as well as essential oils is able to diminish the metabolic activity of microorganisms present in the dental biofilm11-13. foster, et al.14 (2004) studied the effects of mouthwashes containing essential oils, triclosan, cetylpyridinium chloride and chlorhexidine against streptococcus gordonii biofilms. the confocal laser scanning microscopy analysis demonstrated that all mouthwashes, except for cetylpyridinium chloride, were able to cause membrane damage after 60 s of incubation with s. gordonii biofilms. zhang, et al.15 (2004) evaluated the effect of a mouthwash with and without fluoride over metabolic activity of s. mutans biofilms and demonstrated that essential oil-containing mouthwashes, with or without 100 ppm of fluoride reduced the metabolic activity and the consequent acid production by approximately 36-44%. a significant reduction on total colony forming units (cfu) was observed in saliva of healthy volunteers after a single mouthwash with 0.2% or 0.12% chlorhexidine, but only the highest concentration showed bactericidal activity against salivary obligate anaerobes16. furthermore, an in vivo study showed that both essential oils and alcohol-free chlorhexidine mouthwashes were able to reduce plaque acidogenicity after a sucrose challenge, with no difference between both solutions17. although several studies have been undertaken, little data are available about the action of mouthwashes with different active principles on bacterial biofilm metabolism, especially s. mutans biofilms, and the effects of those mouthwashes on three-dimensional structure of biofilms. material and methods mouthwashes the following mouthwashes were evaluated in the present study: parodontax ® (composition: 0.2% chlorhexidine gluconate (w/v), batch: 168f, smithkline beecham consumer healthcare, united kingdom); listerine cool mint ® (composition: 0.092% eucalyptol (w/v), 0.042% menthol (w/v), 0.060% methyl salicylate (w/v), 0.064% thymol (w/ v), batch: 3558b01, johnson & johnson, sp, brazil); oralb® (composition: water, glycerin, polysorbate 20, flavor, methylparaben, 0.053% monohydrated cetylpyridinium chloride, 0.050% sodium fluoride (226 ppm fluoride), sodium saccharine, sodium benzoate, propylparaben, ci 42090, ci 47005 batch: 8114852516, rety laboratories, barranquilla, colombia) and periogard® with or without alcohol (composition: 0.12% chlorhexidine gluconate (w/v), batch br123a and br112a, respectively, colgate-palmolive, são bernardo do campo, sp, brazil). positive and negative controls were 70% ethanol (v/ v) and sterile 0.9% (w/v) saline, respectively. streptococcus mutans growth conditions the atcc 25175 strain of s. mutans was purchased from the andré tosello foundation, campinas, sp, brazil. the lineage was kept stored at -20ºc in 40% (v/v) glycerol (sigma, st. louis, mo, usa) medium and checked for purity before being grown in broth. the frozen s. mutans cultures were reactivated in 5 ml of triptic soy broth (tsb soybean-casein digest medium; difco, sparks, md, usa) and incubated at 37°c, under microaerophylic conditions for 18 h. the cultures were adjusted to a620 nm = 0.2 using a photocolorimeter (analyser com & ind. ltda. são paulo, sp, brazil) and 750 ml of this suspension was transferred to a tube containing 30 ml of previously autoclaved complete medium18 (10 g/l tryptone, 5 g/l yeast extract, 60 µmol/l mgso 4 , 1.3 µmol/l feso 4 , 1.5 µmol/l mncl 2 , 0.2 mmol/l kh 2 po 4 , 0.3 mmol/l k 2 hpo 4 , 0.7 mmol/l kcl, ph 7.0) supplemented with 50 mmol/l sucrose as carbon source. then, 600 ml of this suspension was inoculated in a 24-well cell culture plate (corning costar 3524, flat bottom) and incubated at 37°c, under microaerophilia, during 18 h. for biofilm formation as previously described7. effects of mouthwashes on s. mutans metabolism all procedures were carried out in as a blind experiment. after biofilm formation as described above, the culture medium of each well was removed and the ph was measured using a pg 1800 ph meter associated with a microelectrode (gehaka, são paulo, sp, brazil). the formed biofilms were washed 3 times with sterile 0.9% (w/v) saline and 1 ml of each the mouthwashes was added to each well. after 1 min of incubation, the mouthwashes were removed and the wells washed with abundant sterile 0.9% (w/v) saline. then, to each well was added 1 ml sterile complete medium supplied with 50 mmol/l sucrose as carbon source. the treated biofilm was incubated at 37ºc under microaerophilic conditions and samples were taken at 60, 120 and 180 min for further ph analysis. the positive control used was ethanol 70% (v/v) and the negative control was sterile 0.9% (w/v) saline. confocal laser scanning microscopy (clsm) for the clsm study, glass coverslips were inserted in previously autoclaved falcon tubes with 30 ml of complete metabolic activity of streptococcus mutans biofilms after treatment with different mouthwash formulations 76 braz j oral sci. 10(1):74-78 60 min 120 min 180 min saline 5.69 ± 0.31a 4.73 ± 0.22a 4.32 ± 0.10a ethanol 70% (v/v) 6.80 ± 0.02b 6.80 ± 0.01b 6.78 ± 0.01b listerine® 6.81 ± 0.01b 6.80 ± 0.01b 6.79 ± 0.01b parodontax® 6.81 ± 0.08b 6.75 ± 0.11b 6.59 ± 0.21b oral b® 6.75 ± 0.03b 6.58 ± 0.05b,c 6.00 ± 0.18c periogardò with alcohol 6.68 ± 0.01c 6.48 ± 0.01c 6.23 ± 0.12c periogardò without alcohol 6.54 ± 0.12c 6.14 ± 0.15c 5.64 ± 0.35c table 1: acidogenity of s. mutans biofilms after treatment with different mouthwashes. values are expressed as mean ± sd of three independent experiments. different letters indicate statistically significant difference among the groups (p,0.05). medium18 supplemented with 50 mmol/l sucrose as carbon source. suspension of 5 x 107 bacteria/ml of s. mutans were added and cultivated for 18h. the s. mutans biofilm formed in the coverslips were washed and treated with different mouthwashes during 1 min. after that, the coverslips were extensively washed with sterile saline and treated with 1 mm propidium iodide followed by 0.1% fluoresceine. the coverslips were mounted on individual slides and the images was captured for an emission wavelength at 500-530 nm or at 600-675 nm respectively at 63× magnification with a confocal laser scanning microscope (carl zeiss lsm 510 meta, jena, germany). the two color images obtained by a clsm, i.e. a green-filtered emission image and a red-filtered emission image, were converted to digital image and merged together using the zeiss lsm image browser. statistical analysis data are reported as the mean of triplicate measurement of three independent assays. one-way analysis of variance (anova) was used to determine the significance between treatments. to determine whether the means were statistically different from each other we used the bonferroni’s multiple comparison test, considered to be statistically significant at p<0.05 or less. results after 60 min of the mouthwashes treatment, all of the tested brands of mouthwashes differ significantly (p<0.05) from saline-treated biofilm (control), avoiding a more intense decrease in the ph. however, periogard® with or without alcohol demonstrated a more intense ph-drop, differing significantly (p<0.05) from the other mouthwashes evaluated (table 1, column 1), suggesting lower efficacy. in the second time analyzed (120 min after the treatments), the saline-treated biofilm showed an intense ph-drop statistically significant (p<0.05) when compared to all mouthwashes. moreover, ethanol 70, listerine®, parodontax® and oral b® demonstrated a slight ph-drop avoiding high variations and none of them differed statically among them (p>0.05). however, the biofilm treated with periogard® with or without alcohol demonstrated a higher ph-drop (acidification) statistically significant (table 1, column 2). finally, when analyzing the biofilm acidification 180 min after mouthwash treatment, saline-treated biofilm had a very low ph, characteristically demonstrating the metabolic activity and viability of the biofilm. furthermore, ethanol 70, listerine® and parodontax® showed the best effect avoiding an intense ph-drop, while oral b®, periogard® with or without alcohol showed the lowest metabolic activity of streptococcus mutans biofilms after treatment with different mouthwash formulations fig. 1: confocal laser scanning microscopy of biofilms treated with saline (a) after treatment with mouthwashes containing 70% ethanol (b) essential oils (c), 0.2% chlorhexidine (d), 0.12% chlorhexidine plus alcohol (e), 0.12% chlorhexidine without alcohol (f) and alcohol-free cetylpyridinium chloride plus fluoride (g). all images show a three-dimensional reconstruction rotated 90º in the y-z direction (above) and in the x-z direction (right side). 77 braz j oral sci. 10(1):74-78 ph, differing significantly (p<0.05) from all other mouthwashes (table 1, column 3). clsm was used to ascertain the viability of bacteria in the biofilm after mouthwash treatment. s. mutans biofilm without any treatment revealed great cell viability (fig. 1a), contrasting with a higher level of dead cells after 70% ethanol treatment (fig. 1b). furthermore, biofilm treated with mouthwashes containing essential oils (listerine®) or 0.2% chlorhexidine (parodontax®) caused extensive damage to biofilms (fig. 1c and d, respectively), comparable to or more extensive than lesions induced by ethanol. it was also possible to observe that both antimicrobial agents used effectively penetrated the biofilm. in a smaller extent, treatment of biofilms with 0.12% chlorhexidine plus alcohol (periogard® with alcohol, fig 1e) also was able to cause cell death, whereas alcohol-free 0.12% chlorhexidine (periogard® without alcohol, fig 1f) and alcohol-free cetylpyridinium chloride plus fluoride mouthrinse (oral b®, fig 1g) caused a low level of cell death, restricted to spots on biofilm and not throughout the biofilm. these results, in a greater extent, are corroborative with ph measurements after treatment of biofilms with mouthwashes (table 1). discussion the formation of dental biofilm is instantly initiated after tooth cleaning by the adsorption of salivary components to the enamel surface, followed by addition of initial colonizers, to which eventually, the climax community of matured dental biofilm will adhere11,19. biofilm bacteria are involved in a matrix of salivary proteins and microbial products20. this type of growth protects the bacteria from external agents, such as antibiotics 11, and mouthwash components21. in the present study, the mouthwashes with essential oils and 0.2% chlorhexidine showed similar efficacy to 70% (v/v) ethanol to reduce the acidogenicity from s. mutans biofilms (table 1). these results are in agreement with those of a recent study17, which demonstrated in vivo that using mouthwashes with essential oils or alcohol-free chlorhexidine during a 16-day period reduced plaque acidogenicity after a sucrose challenge. kocak, et al.22 (2009) showed that 0.12% chlorhexidine was effective against oral microorganisms. our results suggest that an alcohol-free mouthwash containing 0.12% chlorhexidine was able to reduce the bacterial metabolism as compared to the negative control, but failed, at any time evaluated, to reduce significantly the bacterial metabolism as compared to the positive control. the in vivo study of those authors 22 evaluated the efficacy of mouthwashes measuring the number of s. mutans cfu present in saliva after use of mouthwash, probably reflecting only cells that detached from biofilm and not the whole dental biofilm. in the present study, the whole biofilm was analyzed and the results clearly showed that 0.12% chlorhexidine failed to eliminate the metabolic activity and also to induce extensive membrane damage to biofilm growing s. mutans. thus, this result indicates that chlorhexidine concentration is determinant to its penetrability into the biofilm. tomás, et al.16 (2008) observed a reduction of total bacterial population after use of both 0.2% and 0.12% chlorhexidine mouthwashes. however, these authors16 also reported that only the higher concentration showed bactericidal activity, which agrees with our results for both acidogenicity and clsm assays. comparison between 0.12% chlorhexidine with and without alcohol showed a small advantage of the alcoholcontaining mouthwash, since it caused a 60 min delay in acidogenicity in comparison to the alcohol-free version (table 1). a similar result was found in a previous study23 that compared two chlorhexidine solutions against plaque re-growth and bacterial viability, showing that ethanol may contribute significantly to reduce bacterial vitality. interestingly, in the present study the worst results were obtained from alcohol-free mouthwashes, suggesting that the alcohol may contribute to a better penetrability of the active principle into the biofilm. witt, et al.24 (2005) observed no difference between an alcohol-free cetylpyridinium chloride mouthwash and a product containing essential oils, when using a modified quigley-hein plaque index. on the other hand, in the present study, the cetylpyridinium chloride plus fluoride mouthwash had the worst capacity to reduce s. mutans metabolism (figure 1), as shown in both acidogenity and clsm experiments. among the reasons to explain these results, we can arise that: (1) the penetrability of cetylpyridinium chloride might not have been sufficient to entirely permeate the biofilms; (2) the molecule could penetrate but the contact period between cetylpyridinium chloride and bacterial cells was insufficient to cause membrane damage; or (3) the cetylpyridinium chloride concentration present in the mouthwash used was below of the necessary to cause extensive membrane damage. our data from cslm strongly suggests that reduction of metabolic activity is due to cell damage as a result of mouthwash treatment. in the present study, among 5 mouthwashes tested, only 2 showed efficient penetration of the agents throughout the biofilm as observed in the positive control experiment, visualized by clsm. evidence of membrane damage extended from the bottom of coverslips to the surface of biofilms induced by 0.2% chlorhexidineand essential oil-containing mouthwashes suggests an effective penetration of these molecules through the biofilm. interestingly, 0.12% chlorhexidine showed poor efficacy when compared to 0.2% chlorhexidine, indicating that a small variation in concentration may compromise the penetrability and, consequently, bacterial inactivation. several previous studies 17,22,23 measured the efficacy of antimicrobials on dental plaque in vivo and some of these studies had high interindividual variations of the results17. the methodology employed in the present study is highly reproducible, low cost and easy to perform. furthermore, it was attempted to mimic exposure times often used in in vivo clinical studies (60 s)25-27. thus, it may be concluded that the mouthwashes containing essential oils or 0.2% metabolic activity of streptococcus mutans biofilms after treatment with different mouthwash formulations 78 braz j oral sci. 10(1):74-78 chlorhexidine showed higher efficacy than those containing cetylpyridinium chloride plus fluoride or 0.12% chlorhexidine. acknowledgements the authors would like to thank uniube and fapemig for the continuous support given to our laboratories, and hilara n. ruas for technical assistance. trc and asc were recipients, respectively, of master’s degree and undergraduate fellowships from fapemig. references 1. krasse b. caries risk: a pratical guide for a assessment and control. chicago: quintessence; 1985. p.113. 2. marsh pd. are dental diseases examples of ecological catastrophes? microbiology. 2003; 149: 279-94. 3. mikkelsen 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chapman and hall; 1992. p.98-132. 21. landa as, van der mei hc, busscher hj. detachment of linking film bacteria from enamel surfaces by oral rinses and penetration of sodium lauryl sulphate through an artificial oral biofilm. adv dent res. 1997; 11: 528-38. 22. kocak mm, ozcan s, kocak s, topuz o, erten h. comparison of the efficacy of three different mouthrinse solutions in decreasing the level of streptococcus mutans in saliva. eur j dent. 2009; 3: 57-61. 23. arweiler nb, boehnke n, sculean a, hellwig e, auschill tm. differences in efficacy of two commercial 0.2% chlorhexidine mouthrinse solutions: a 4-day plaque re-growth study. j clin periodontol. 2006; 33: 334-9. 24. witt jj, walters p, bsoul s, gibb r, dunavent j, putt m. comparative clinical trial of two antigingivitis mouthrinses. am j dent. 2005; 18:15a-17a. 25. moran j, addy m, newcombe r. a 4-day plaque regrowth study comparing an essential oil mouthrinse with a triclosan mouthrinse. j clin periodontol. 1997; 24: 636–9. 26. fine dh, furgang d, barnett ml, drew c, steinberg l, charles ch, et al. effect of an essential oil-containing antiseptic mouthrinse on plaque and salivary streptococcus mutans levels. j clin periodontol. 2000; 7: 157–61. 27. pan p, barnett ml, coelho j, brogdon c, finnegan mb. determination of the in situ bactericidal activity of an essential oil mouthrinse using a vital stain method. j clin periodontol. 2000; 27: 256–61. metabolic activity of streptococcus mutans biofilms after treatment with different mouthwash formulations oral sciences n3 original article braz j oral sci. january | march 2012 volume 11, number 1 received for publication: december 06, 2011 accepted: march 21, 2012 influence of viscosity and polymerization mode on bond strength of dual-cure resin luting agent to dentin marina di francescantonio1, thaiane rodrigues aguiar1, marcelo tavares de oliveira2, marcelo giannini3 1dds, ms, phd student, department of restorative dentistry, piracicaba school of dentistry, campinas state university, piracicaba, sp, brazil 2dds, ms, phd, assistant professor, department of operative dentistry, university nove de julho, são paulo, sp, brazil 3dds, ms, phd, associate professor, department of restorative dentistry, piracicaba school of dentistry, campinas state university, piracicaba, sp, brazil correspondence to: marcelo giannini department of restorative dentistry, operative dentistry section piracicaba school of dentistry state university of campinas av. limeira, 901, piracicaba, sp, brasil cep: 13414-903 phone: +55 19 21065340 fax: +55 19 21065218 e-mail: giannini@fop.unicamp.br abstract aim: to evaluate the influence of the viscosity and curing mode on the bond strength of two resin cements to dentin. methods: eight experimental groups were formed (n = 7) according to the dual-cure resin cements (nexus 2 kerr corp. and variolink ii ivoclar vivadent), curing modes (dual-cure or self-cure) and viscosities (low and high). resin cements were applied to pre-cured composite resin discs (2 mm thick, sinfony -3m espe), which were fixed to bonded dentin surfaces. the restored teeth were either light-activated (xl3000 3m espe) or allowed to self curing only. after 24 h, the teeth were both mesiodistally and buccolingually sectioned to obtain bonded beam specimens (0.8 mm2 cross-sectional area). each specimen was tested in microtensile strength at a crosshead speed of 0.5 mm/min until failure. results: data (in mpa) were analyzed statistically by three-way anova and tukey’s post-hoc test (pre-set á = 0.05). no significant difference was observed between resin cements (p=0.26) and viscosities (p=0.13), however, the curing mode affected the bs within the viscosities (p=0.01). statistically significant difference was observed for low viscosity: nexus 2: 23.8(10.6) (dual-cure) and 16.0(5.1) (self-cure); variolink ii: 28.7(8.7) (dual-cure) and 11.9(3.0) (self-cure). conclusions: light activation yielded higher bond strength for the low-viscosity versions of the resin cements. keywords: dentin bonding agents, resin cement, bond strength, dentin. introduction the adhesive cementation techniques for indirect metal-free restorations use dual-cure resin luting agent, and the clinical success of these restorations depends on the quality of the restorative material and its bonding to the mineralized dental tissues, among other factors. the luting agents are a combination of dual-cure resin cement and a bonding agent, which is responsible by the adhesion between the tooth and the resin cement1-5. resin cements can be dual-cure, only self-cure or only light-cure materials. dual-cure resin cements are indicated in clinical situations when no light is available to polymerize the material and the self-curing component should compensate for the absence of light. in other situations, there is a loss of light because of either the distance between the light-curing tip and the luting agent or light attenuation through the thickness of the indirect restoration6-10. the resin cements present different viscosities, which can produce cement braz j oral sci. 11(1):76-80 products (type) composition lot number variolink ii(resin cement) paste of dimethacrylates, bis-gma, tegdma, udma, inorganic fillers, ytterbium trifluoride, initiators, stabilizers, pigments, benzoyl peroxide. excite dsc(adhesive system) adhesive resin: alcohol, phosphonic acid acrylate, hema, sio2, initiators f54523 and stabilizers, dimethacrylates.activator: aromatic sodium sulfinate salt. nexus 2(resincement) monomers of methacrylic acid esters, ba–al–borosilicate glass, chemical and photoinitiators. optibond solo plus(adhesive system) adhesive resin: ethyl alcohol; bis-gma; hema; gpdm; photoinitiators; barium aluminoborosilicate glass; fumed silica (silicon dioxide); sodium hexafluorosilicate activator: ethyl alcohol; alkyl dimethacrylate resins; benzene sulfinic acid sodium salt. monobond s(silane coupling agent) ethanol, 3methacryloxy-propyl-trimetoxy-silane h24764 silane primer(silane coupling agent) ethylalcohol, organosilaneester. 32667 high: j24363 low: j19103 base: j19730 high: 438681 low: 452344 base: 452365 adhesive: 487047 activator: 100914 table 1.table 1.table 1.table 1.table 1. compositions of the resin cements used in this study. abbreviations: tegdma: triethylene glycol dimethacrylate; bis-gma, bisphenol a diglycidyl ether methacrylate; udma, urethane dimethacrylate; hema: 2-hydroxyethyl methacrylate; gpdm: glycerol phosphate dimethacrylate. layers with different thicknesses. these differences on thickness may affect some properties of the material, such as strength, modulus of elasticity and rheology, which can interfere in restoration longevity. resin cements provide a correct seating of the indirect restoration and are also responsible for the tooth-restoration interfacial sealing, which prevents the marginal leakage11-14. viscosity variation for resin cements is obtained by modifying the proportion between monomer composition and filler particle content15. nothing is known about the viscosities of resin cements and their influence when used with different polymerization modes (dual-cure or self-cure) on bond strength to dentin. thus, the purpose of this study was to measure the bond strength of prepolymerized composite discs to underlying tooth structure using dual-cure resin luting agents with two viscosities (high and low), which either were allowed to self curing in the absence of light or were exposed to light through the composite disc. in addition, the failure site morphology was analyzed and compared by material types and polymerization modes. the research hypothesis tested was that bond strength values would be significantly higher when the resin cement was subjected to light-activation (dual cure) than when they were only allowed to self curing. it was also hypothesized that the bond strength of low-viscosity dual-cure resin cement would be significantly lower than that of high-viscosity resin cement. material and methods specimen preparation and experimental groups the research protocol was approved by the ethics committee of the school of dentistry of piracicaba, university of campinas, sp, brazil (176/2006). fifty-six freshly extracted, erupted, human third molars were used. the teeth were stored in a saturated thymol solution at 5 °c for no longer than 3 months16. they were then sectioned transversally in the middle third of the crown, using a diamond blade saw (series 15hc diamond, buehler ltd, lake bluff, il, usa) on an automated sectioning device (isomet 2000, buehler ltd) under water cooling, exposing areas of midcrown dentin. the exposed dentin surfaces were wet polished (apl-4, arotec, cotia, sp, brazil) using 600-grit sic paper in order to create a flat surface with standardized smear layer before application of the bonding agents. the prepared teeth were then randomly divided into 8 groups (n = 7). two dual-cure resin cements that present commercial versions in high and low viscosity, nexus 2 (kerr corp., orange, ca, usa) and variolink ii (ivoclar vivadent, schaan, liechtenstein), and their respective adhesive systems and silane primers (optibond solo plus and silane primer kerr corp.; excite dsc and monobond s ivoclar vivadent) were used (table 1). fifty-six pre-polymerized, light-cure composite resin discs, 2 mm thick and 10 mm in diameter (b2d shade, sinfony, 3m espe, st. paul, mn, usa), were prepared to simulate overlying laboratory-processed composite resin restorations. the surface of each disc was sandblasted with 50 µm aluminum oxide (danville engineering inc, san ramon, ca, usa) for 10 s (air pressure = 0.552 mpa; distance from the tip = 1.5 cm) and silanated with coupling agents according to manufacturers’ instructions (monobond-s or silane primer). the adhesive systems and the resin cements were applied and used according to manufacturers’ instructions. the resin cements were mixed previously in the proportion of 1:1 (catalyst and base paste) and were applied to the sandblasted surface of the pre-polymerized composite resin disc, which was placed on the dentin surface. the resin cements were light-cured for 40 s (xl 3000; 3m espe) through the 7777777777 influence of viscosity and polymerization mode on bond strength of dual-cure resin luting agent to dentin braz j oral sci. 11(1):76-80 resin cement viscosity dual-cure self cure nexus 2 l o w 23.8 (10.6) aa 16.0 (5.1) ab nexus 2 high 20.2 (4.7) aa 18.9 (6.5) aa variolink ii l o w 28.7 (8.7) aa 11.9 (3.0) ab variolink ii high 16.5 (4.7) aa 13.1 (6.4) aa table 2.table 2.table 2.table 2.table 2. means (standard deviation) of bond strength to dentin for nexus 2 and variolink ii resin cements as a function of viscosity and curing mode (in mpa). similar letters indicate no statistically significant difference among values (uppercase letters compare viscosities within the same resin cement and lowercase letters compare curing modes). composite resin disc or were allowed to self curing only with a load of 0.5 kg applied horizontally for 5 min. in order to facilitate specimen gripping during bond testing, a 3-mmthick block of self-cure composite resin (concise, 3m of brazil, sumaré, sp, brazil) was added to the untreated, prepolymerized composite resin surface. microtensile bond strength restored teeth were stored in distilled and deionized water at 37° c for 24 h and were then vertically serially sectioned into several 1.0-mm-thick slabs. each slab was further sectioned perpendicularly to produce bonded beam specimens with 0,8mm2 in cross-section. each bonded beam was attached to the grips of a microtensile testing device with cyanoacrylate glue (super bonder; henkel/loctite, diadema, sp, brazil) and tensioned in an universal testing machine (4411; instron co., canton, ma, usa) at a crosshead speed of 0.5 mm/min until failure. after testing, the specimens were carefully removed and the cross-sectional area at the site of fracture was measured to the nearest 0.01 mm with a digital caliper (mod. 727-6/150, starret ind. e com. ltda., itu, sp, brazil). the specimen cross-sectional area was divided by the peak tensile load at failure to calculate stress at fracture (in mpa). a single failure stress value was then calculated for each tooth by averaging the values of 5 tested beams from that tooth. a three-way anova (two resin cements, two polymerization modes and two viscosities) was performed to determine the effect of these major factors on tensile strength. tukey’s post-hoc test was used to detect pair-wise differences among the experimental groups. all statistical testing was performed at a preset α of 0.05. failure pattern analysis fractured surfaces of tested specimens were sputter coated with gold (med 010, balzers, balzer, liechtenstein) and examined by a single individual using a scanning electron microscope (vp 435, leo, cambridge, england). failure patterns were classified as: (1) adhesive failure between adhesive resin and dentin; (2) cohesive within the adhesive resin, (3) adhesive failure between adhesive and resin cement (4) cohesive within the resin cement, (5) mixed failure involving different structures of dentin-resin disc bonded interface. representative areas of the failure patterns were photographed (85× to 1,900×). results summary statistics for the different experimental groups are shown in table 2. three-way anova indicated that the curing mode factor significantly influenced tensile strength results (p = 0.0005). the statistical analysis revealed difference only for curing mode (p= 0.0004), no significant differences for the triple interaction (resin cement × curing mode × viscosity, p = 0.4303) or for interaction between cement × viscosity (p = 0.1710) and cement × curing mode (p = 0.1312). the analysis only indicated an interaction between the viscosity x curing mode (p = 0.0127). when looking at data with respect to differences in curing mode, the bond strength of the low viscosity version of the resin cements was affected by curing mode (p< 0.05), while light-activation did not increase the bond strength of high viscosity versions (p> 0.05). the type of resin cement and the viscosity did not affect the bond strength (p> 0.05). figure 1 shows the proportional prevalence (%) of the failure patterns in all experimental groups, and representative images depicting failure modes are presented in figures 2 and 3. adhesive failures along the dentin surface were observed for all groups, except for nexus 2 in low-viscosity and self cure mode. for variolink ii in high viscosity and dual-cure mode, half of the specimens had adhesive failures (type 1) (figures 2a and 2b). highor low-viscosity dualcure nexus 2 exhibited high incidence of cohesive failures within the adhesive resin and adhesive failures between adhesive and resin cement (figures 3a and 3b, respectively). except for high-viscosity dual-cure nexus 2, cohesive failures within the resin cement was observed for all groups; however, it occurred mainly for self-cure groups (figure 3c). mixed failures involved two or more types of failures (adhesive and cohesive failures) in the same fractured end of specimens and all groups with variolink ii showed mixed failures (figures 2c, 2d, and 2e). 7878787878influence of viscosity and polymerization mode on bond strength of dual-cure resin luting agent to dentin braz j oral sci. 11(1):76-80 fig. 1. distribution (%) of failure modes among experimental groups. fig. 3. (a) fractured end of specimen cemented with nexus 2 (high viscosity and dual-cure) exhibiting cohesive failure within the adhesive resin (original magnification x95). (b) fractured end of specimen cemented with nexus 2 (high viscosity and dual-cure) exhibiting adhesive failure between adhesive and resin cement (original magnification x100). (c) fractured end of specimen cemented with nexus 2 (high viscosity and self-cure) exhibiting cohesive failure within the resin cement (original magnification x100). fig. 2. (a) fractured end of specimen cemented with variolink ii (high viscosity and dual-cure) exhibiting adhesive failure along dentin surface (original magnification x85). (b) higher magnification of figure 2a revealing fractures located predominantly within the hybrid layer and resin tags inside the dentinal tubules (original magnification x1900). (c) fractured end of specimen cemented with variolink ii (low viscosity and self-cure) exhibiting mixed failure mode, characterized by adhesive and cohesive failures within the resin cement (original magnification x100). ddentin; rcresin cement. (d) higher magnification (x500) of figure 2c demonstrating rc (resin cement) and d (dentin) within same fractured surface. (e) fractured end of specimen cemented with variolink ii (high viscosity and self-cure) exhibiting mixed failure mode, characterized by adhesive and cohesive failures within the resin cement and within indirect composite (original magnification x100). ddentin; rcresin cement; idindirect composite. discussion a more effective bonding to dentin can increase the strength of remaining dental structures and reduce microleakage between the tooth and restoration, which are important to tooth longevity17,18. for supporting use of these materials, we tested the research hypothesis that bond strength values would be significantly higher when the resin cements were light-activated, which was confirmed only for lowviscosity versions. the hypotheses that the bond strength of resin cements in low-viscosity versions would be significantly lower than those in high-viscosity cements was rejected, since no statistical difference was noted between lowand highviscosity materials. the low and high versions of resin cements are indicated for different purposes15,19, but the compositions that form both viscosities did not influence the bond strength. since the resin cements reach high monomer conversion after light curing, only the low-viscosity resin cements were able to produce higher bond strength. the high-viscosity version of both resin cements showed no significant difference on bond strength irrespectively of being light activated or not, since the increased amount of fillers improves the mechanical properties of the cement. the bond between the monomer components and filler particles by treatment of these particles, such as silanization, and the higher amount of fillers promote the increase of the cohesive strength of the resin cement19. however, the low-viscosity formulation of the resin cements did not maintain the bond strength mean value when the materials were allowed to self curing only. light activation of low-viscosity resin cements produced higher bond strength to dentin than self curing alone20. cohesive failures within the resin cement occurred predominantly in those specimens in which the resin cements were allowed to self curing only (figure 1). since the self curing reaction alone does not reach the monomer conversion promoted by light6,10, the resin cement layer may be the weak point at the resin-dentin interface. thus, in the present in vitro study, when the resin-dentin interface was under tension, the resin cement layer tended to fracture more easily than other parts of this interface. depending on the resin cement and its viscosity, light-curing caused different types of failure (figures 1 to 3). for the selfcure resin cements, the predominant failure pattern was the cohesive failure in resin cement, indicating that the lack of light activation reduced the cohesive strength of the cement (figures 2c, 2d, 2e and 3c). the dual-cure resin cements presented different failure patterns, depending on the type of adhesive systems used (figures 2a, 2b, 3a and 3b). the luting agents tested in this study showed no significant difference when compared by different viscosities and curing modes. these materials showed the same results in terms of bond strength to dentin, corroborating the findings of hikita et al.21 (2007). other studies showed that variolink ii presented higher bond strength than nexus 2, but they did not report viscosity of these resin cements used4,22. regarding the curing mode, few resin cement systems (nexus 2, kerr co.; variolink ii, ivoclar vivadent; panavia f, kuraray; relyx 7979797979 influence of viscosity and polymerization mode on bond strength of dual-cure resin luting agent to dentin braz j oral sci. 11(1):76-80 arc and relyx unicem, 3m espe) seem able to produce proper degree of conversion and high bond strength to dentin6,13-15,17. the bonding agents (optibond solo plus and excite dsc) used with resin cements are etch-and-rinse, two-step systems. they are simplified single-bottle adhesive systems that present a low ph value and might jeopardize resin cement conversion reducing the bond strength, if only autopolimerized23. as adhesive systems are spread into a thin layer, an incompletely polymerized resin monomer layer is formed on the adhesive surface. the oxygen inhibition layer formed by uncured acidic simplified etch-and-rinse systems impairs the adhesion between bonding agents and chemical-, lightor dual-cured resin-based restorative materials. the adverse reaction involves the uncured acidic adhesive layer and the tertiary amine catalytic component of the resin cement23,24. both adhesives contain an aromatic sodium sulfinate salt as a co-initiator to develop dual-cure reaction. excite dsc uses the co-initiator impregnated in the microbrush tip as a white salt powder, while optibond solo plus has an activator bottle that contains the benzene sulfinic acid sodium salt in an alcoholic solution4,20. in this study, the addition of these co-initiators to the bonding agents contributed to preserve the bond strength of the high-viscosity cements without light exposure (self curing), since the adhesive was not polymerized and the co-initiator could mix with cement, increasing the polymerization of cement. the same, however, did not occur with the low-viscosity version because the mixture of liquid solutions from the adhesive and activator with a hydrophobic flowable resin cement material may be poor and a larger amount of monomers requires a higher formation of free radicals for an efficient polymerization17. these mixtures involve an aqueous solution containing sodium sulfinate salt and a hydrophobic material, which resulted in lesser polymerization and lower bond strength for the self-cure cements. it may be concluded that light activation yielded higher bond strength for the low-viscosity versions of the resin cements. acknowledgements this study was supported by grants 06/57998-0 from fapesp and capes, brazil. the authors are indebted to the dental materials division (fop-unicamp) for providing use of the universal testing machine. references 1. arrais ca, giannini m, rueggeberg fa, pashley dh. microtensile bond strength of dual-polymerizing cementing systems to dentin using different polymerizing modes. j prosthet dent. 2007;97:99-106. 2. inokoshi s, willems g, van meerbeek b, lambrechts p, braem m, vanherle g. dual-cure luting composites: part i: ûller particle distribution. j oral rehabil. 1993;20:133-46. 3. milleding p, ortengren v, karlsson s. ceramic inlay systems: some clinical aspects. j oral rehabil. 1995;22:571-80. 4. piwowarczyk a, bender r, ottl p, lauer hc. long-term bond between dual-polymerizing cementing agents and human hard dental tissue. dent mater. 2007;23:211-7. 5. sjogren g, molin m, van dijken j, bergman m. ceramic inlays (cerec) cemented with either a dual-cured or a chemically cured composite resin luting agent. a 2-year clinical study. acta odontol scand. 1995;53:325-30. 6. arrais ca, rueggeberg fa, waller jl, de goes mf, giannini m. effect of curing mode on the polymerization characteristics of dual-cured resin cement systems. j dent. 2008;36:418-26. 7. dagostin a, ferrari m. in vivo bonding mechanism of an experimental dual-curing enamel-dentin bonding system. am j dent. 2001;14:105-8. 8. foxton rm, nakajima m, tagami j, miura h. bonding of photo and dual cure adhesives to root canal dentin. oper dent. 2003;28:543-51. 9. rasetto fh, driscoll cf, prestipino v, masri r, von fraunhofer ja. light transmission through all-ceramic dental materials: a pilot study. j prosthet dent. 2004;91:441-6. 10. rueggeberg fa, caughman wf. the influence of light exposure on polymerization of dual-cure resin cements. oper dent. 1993;18:48-55. 11. díaz-arnold am, vargas ma, haselton dr. current status of luting agents for fixed prosthodontics. j prosthet dent. 1999;81:135-41. 12. wilson pr. low force cementation. j dent. 1996;24:269-73. 13. de la macorra jc, pradíes g. conventional and adhesive 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gröfke m, hellwing e. influence of resin cement viscosity on microleakage of ceramic inlays. dent mater. 2001;17:191-6. 20. arrais ca, giannini m, rueggeberg fa, pashley dh. effect of curing mode on microtensile bond strength to dentin of two dual-cured adhesive systems in combination with resin luting cements for indirect restorations. oper dent. 2007;32:37-44. 21. hikita k, van meerbeek b, de munck j, ikeda t, van landuyt k, maida t et al. bonding effectiveness of adhesive luting agents to enamel and dentin. dent mater. 2007;23:71-80. 22. pekkan g, hekimoglu c. evaluation of shear and tensile bond strength between dentin and ceramics using dual-polymerizing resin cements. j prosthet dent. 2009;102:242-52. 23. sanares ame, itthagarun a, king nm, tay fr, pashley dh. adverse surface interactions between one-bottle light-cured adhesives and chemicalcured composites. dent mater. 2001;17:542-56. 24. giannini m, de goes mf, nikaido t, shimada y, tagami j. influence of activation mode of dual-cured resin composite cores and low-viscosity composite liners on bond strength to dentin treated with self-etching adhesives. j adhes dent. 2004;6:301-6. 8080808080influence of viscosity and polymerization mode on bond strength of dual-cure resin luting agent to dentin braz j oral sci. 11(1):76-80 revista fop n 13 1641 tobacco as risk factor for periodontal disease in green marble mine laborers of rajasthan, india santhosh kumar1; prabu d2; suhas kulkarni3; rushabh j dagli1 1post graduate student 2associate professor 3professor department of preventive and community dentistry, darshan dental college and hospital, udaipur, rajasthan, india received for publication : april 07, 2008 accepted: october 08, 2008 correspondence to: santhosh kumar tadakamadla department of preventive and community dentistry darshan dental college and hospital udaipur, rajasthan, india 313001 phone: +91-9928714533 fax : +91-2942452273. e-mail: santosh_dentist@yahoo.co.in a b s t r a c t aim: to determine the effect of tobacco usage on the severity of periodontal disease in green marble mine laborers. methods: marble mine laborers (n=585) aged 15–54 years were selected from four geographic zones in green marble mines area using a stratified random sampling procedure. a total of 517 (88%) laborers participated in the study. the sample were classified as tobacco users, non users, occasional users, ex users and the data regarding form, duration and frequency of tobacco consumption was also collected by personal interviews. periodontal status was assessed by community periodontal index. results: nearly three fourth (71.9%) of the population was tobacco users. among the smokers, bleeding and calculus accounted to 33% each whereas the prevalence of these periodontal indicators was 38% and 44% respectively among smokeless tobacco users. logistic regression analyses revealed that smokeless tobacco users were more liable for presence of periodontal pockets than smokers. duration had a significant influence with users since more than 20 years being liable for presence of pockets at least twice (or = 2.625, 95% ci 1.529 – 4.507) than the <5 years group. the odds ratio for presence of periodontal pockets increased by 2.143 (95 % ci 1.060 – 4.333) and 5.596 (95 % ci 2.901 – 10.639) for users of 6 – 10 units/ day and more than 10 units/day than the 1-2 times/day category. conclusions: tobacco usage had a significant impact on the severity of periodontal disease with users being more likely to present periodontal pockets than non users. moreover, the risk of periodontal pockets increased as the duration and frequency of tobacco consumption increased. key words: periodontal disease, risk factors, marble mine laborers, tobacco i n t r o d u c t i o n multiple cross sectional and longitudinal studies about the association between smoking and periodontal disease have demonstrated that pocket depth, attachment and alveolar bone loss are more prevalent and severe in smoker patients than non smokers1-3. nevertheless, sheiham4 concluded that smoking has no direct effect on periodontal disease and that differences could be due to higher plaque levels in smokers than non smokers. despite the controversial findings, it has been observed that smoking exerts a major effect on the protective elements of immune response, increasing the extent and severity of periodontal destruction. moreover, this habit has been associated with alterations in the neutrophil count and function in form of chemotaxis, phagocytosis and oxidative burst. significant alterations are present in the gingival microvasculature of smokers that can lead to decrease of blood flow and decrease in clinical signs of inflammation5. there is little information concerning tobacco use and periodontitis in developing countries like india6,7, where both oral hygiene levels and the severity of periodontitis may be worse than developed countries8. areca nut, often used with betel quid and chewable tobacco, is the fourth most commonly used psycho-active substance in the world, ranking after caffeine, alcohol and nicotine9. high prevalence of use of these items has been reported in south and south east asia10. having an ancient history, they are an integral part of the culture and sometimes erroneously believed to have medicinal benefits11. there are fewer studies stating the relationship between smokeless tobacco use and periodontitis than the ones that have examined the association between cigarette smoking and periodontitis6,12,13. the most of them are restricted to tobacco use in form of snuff dipping and braz j oral sci. october/december 2008 vol. 7 number 27 1642 tobacco chewing12. the habit of betel chewing with tobacco is a particular form of smokeless tobacco consumption that is predominantly practiced in the indian subcontinent13,14 either in the form of pan or guthka which is an industrially prepared mixture of areca nut, lime, catechu and tobacco9. the association between tobacco consumed in chewing form and oral cancer has been widely documented15,16. moreover, studies done on athletes13,17 and adults18,19, who have smokeless tobacco habit showed a strong relationship with oral white lesions. these lesions were found in regions where tobacco was placed for longer hours, with a prevalence more than 50%13,20,21. to the best of our knowledge, the effects of tobacco in smoke and smokeless forms, highly prevalent in india, on periodontal disease have not been documented. hence, the present study aimed to assess the effect of tobacco usage on the severity of periodontal disease in green marble mine laborers in india. materials and methods the study area is located in udaipur district of rajasthan and divided into four geographic zones. stratified cluster sampling procedure was executed to collect the representative population. the final sample consisted of 513 dentate adult male green marble mine laborers, aged from 15 to 54 years. there were no female subjects among the target group, since mining involves strenuous workload. intra-oral examinations were performed with adequate illumination by a single examiner (sk), using a mouth mirror and a who periodontal probe to record the community periodontal index22 (cpi), comprising the following scores: 0 (healthy); 1 (bleeding); 2 (calculus); 3 (pockets of 45 mm); 4 (pockets of 6 mm or more). calibration of the investigator for cpi was carried out against an experienced clinician and the kappa value23 was 0.91. ethical clearance for the study was obtained from the ethical committee for research of darshan dental college and hospital, udaipur, india. for assessment of tobacco habits, the sample was classified as non users, regular users, occasional users and ex-users24. the form of tobacco use (smoking, chewing and snuff), the number of years of consumption (duration) and the number of units used per day were recorded according to a pre formulated scale by subject interview. subjects were then classified based on duration of tobacco usage into less than 5 years, 610 years, 11-20 years and more than 20 years. number of units was measured under four categories, namely 1-2/day, 3-5/day, 6-10/day and more than 10/day. regular users constituted of individuals who are currently consuming tobacco at least once a day, non users are those who had never used tobacco. occasional users were those people who did not use tobacco at least for three consecutive days and ex users were subjects who had not used tobacco in any form since a year or more. smoking category included cigarettes, bidi and combination of both. bidi is the most popular smoking form of tobacco used in india because of the less cost, while chewable forms of tobacco comprised of pan, guthka, mawa, zarda and khaini. mawa is a mixture of areca nut, tobacco and slaked lime, while khaini comprises of powdered sun-dried tobacco mixed with slaked lime and is occasionally used with areca nut. zarda is prepared by boiling tobacco leaf in water along with lime and spices25. snuff dipping was recorded separately. there were many subjects in the study sample who used snuff orally though it can also be used through nasal route. to eliminate the bias, 25 subjects with systemic illness like diabetes and heart disease were excluded from study in addition to 47 subjects who were in the habit of using both smoke as well as smokeless forms of tobacco. therefore, a final sample of 513 individuals were included in the study statistical analysis the data collected was entered into the spreadsheets. spss (version 11.0) software package was used for statistical analysis. the pearson chi-square test was used to compare different percentages at the 1% level of significance26 in order to facilitate chisquare analysis, all the tobacco categories were dichotomized, users (regular, ex and occasional) and non users, smokers and smokeless users (chewers and snuff users), short (1 10 years) and long duration (more than 11 years) in addition to moderate (1 – 5 units/day) and heavy users (more than 5 units/day). logistic regression analysis was performed to determine the influence of tobacco on the severity of periodontal status. independent variables constituted age, use, form, duration and frequency of tobacco consumption. dependent variable for multiple logistic regression analysis constituted absence (scores 0, 1 and 2 of cpi) vs presence of periodontal pockets (scores 3 and 4 of cpi). odds ratio was calculated for the influence of age and the tobacco variables with 95% confidence intervals. the effect of each independent variable was assessed adjusting for all other variables in the model. r e s u l t s table 1 illustrates the general characteristics of the study population. the youngest and the oldest age groups constituted for a major and minor proportion of the final sample size with 33.3% and 8.8% respectively. the sample size in each age group was proportional to elementary units in each age group (optimal allocation). nearly three fourth (71.9%) of the population was tobacco users. chewers formed a major proportion of tobacco users and there were few subjects (5.3%) who were using tobacco since 20 years. for a majority of the sample, duration of braz j oral sci. 7(27):1641-1647 tobacco as risk factor for periodontal disease in green marble mine laborers of rajasthan, india 1643 tobacco consumption was in the range of 6 – 20 years whereas frequency ranged between 3 – 10 units per day. table 2 presents the sample distribution in accordance with cpi scores for users and non users of tobacco in different age groups. the majority of regular users belonged to the youngest age group. all the periodontal disease indicators were more predominant in tobacco users when compared to non users. there were no periodontal pockets observed in non users whereas they were evident among tobacco users. furthermore, there was a statistically significant proportion (χ2 = 13.965, p = 0.003) between the users and non users for all the indicators of periodontal disease. occurrence of periodontal disease among smokers and smokeless tobacco users in different age groups is presented in table 3. of the 432 tobacco users, 81 (18.7%) were smokers, whereas 351 (81.3%) were smokeless tobacco users. among the smokers, bleeding and calculus accounted to 33% each whereas the prevalence of these periodontal indicators was 38% and 44% respectively among smokeless tobacco users. chi square analysis revealed significant results (p=0.001) for presenting bleeding and calculus between smokers and smokeless tobacco users. table 4 illustrates the sample distribution according to cpi scores in each age group, considering the duration of tobacco use irrespective of the form consumed. long time users constituted 38% of the users and the respective proportion increased with the age, whereas among the short time users the trend was opposite. the proportions of subjects with bleeding, calculus and pockets for long time users were, respectively, 33%, 39% and 28%. the respective values for the short time users were 40%, 44% and 13%. there were significant differences (p = 0.001) for the presence of pockets between short time and long time users. frequency percentage (%) 15 – 24 171 33.3 25 – 34 162 31.6 35 – 44 135 26.3 age 45 – 54 45 8.8 healthy 9 1.7 bleeding on probing 234 45.6 calculus 198 38.6 shallow pockets 72 14.1 periodontal status deep pockets 9 1.7 non users 81 15.8 regular users 315 61.4 occasional users 54 10.5 tobacco usage ex users 63 12.3 smokers 81 15.8 chewers 315 61.4tobacco form snuff users 36 7.1 ? 5 years 81 15.8 6 – 10 years 189 36.8 11 – 20 years 135 26.3 duration of tobacco use > 20 years 27 5.3 1 – 2 units/day 90 17.5 3 – 5 units/day 126 24.6 6 – 10 units/day 135 26.3 frequency of tobacco consumption > 10 units/day 81 15.8 table 1background and general sample characteristics > braz j oral sci. 7(27):1641-1647 tobacco as risk factor for periodontal disease in green marble mine laborers of rajasthan, india 189 36.9 1644 age healthy bleeding calculus pockets 4 – 5 mm pockets ? 6mm total nu u nu u nu u nu u nu u nu u 15 – 24 9 9 18 72 9 36 0 18 0 0 36 135 25 – 34 0 0 18 36 18 72 0 18 0 0 36 126 35 – 44 0 0 9 27 0 63 0 27 0 9 9 126 45 – 54 0 0 0 27 0 9 0 9 0 0 0 45 total 9 9 45 162 27 180 0 72 0 9 81 432 ≥ table 2 distribution of tobacco users (u) and non – users (nu) in accordance with cpi scores in different age groups age healthy bleeding calculus pockets 4 – 5 mm pocket ? 6mm total s sl s sl s sl s sl s sl s sl 15 – 24 9 0 9 63 0 36 0 18 0 0 18 117 25 – 34 0 0 9 27 0 72 0 18 0 0 9 117 35 – 44 0 0 0 27 18 45 9 18 0 9 27 99 45 – 54 0 0 9 18 9 0 9 0 0 0 27 18 total 9 0 27 135 27 153 18 54 0 9 81 351 ≥ table 3 distribution of smokers (s) and smokeless (sl) tobacco users in accordance with cpi scores in different age groups sample distribution in accordance with periodontal disease levels in various age groups based on frequency of tobacco consumption is presented in table 5. the highest proportion of heavy users of tobacco was found in 15-24 age healthy bleeding calculus pockets 4 – 5 mm pocket ?6mm total st lt st lt st lt st lt st lt st lt 15 – 24 0 0 9 18 9 0 0 9 0 0 18 27 25 – 34 9 0 54 18 36 0 18 0 0 0 117 18 35 – 44 0 0 36 0 27 45 9 9 0 0 72 54 45 – 54 0 0 9 18 45 18 9 18 0 9 63 63 total 9 0 108 54 117 63 36 36 0 9 270 162 ≥ table 4 distribution of short time (st – 1 to 10 years) and long time (lt – greater than 10 years) tobacco users in accordance with cpi scores in different age groups years old age group (53%), followed by the oldest age group (40%). thirty-three percent of heavy users presented periodontal pockets where as only 4% among the moderate users had pockets (p = 0.001). χ2 = 18.173, p = 0.001 χ2 = 13.965, p = 0.003 χ2 = 66.851, p = 0.001 braz j oral sci. 7(27):1641-1647 tobacco as risk factor for periodontal disease in green marble mine laborers of rajasthan, india 1645 15-24 age 25-34 35-44 45-54 total ≥ table 5 sample distribution based on frequency of tobacco consumption in accordance with cpi scores in different age groups independent variables odds ration (95% ci) p value 15 – 24 1.00 <0.0001 25 – 34 1.059 (0.573 – 1.558) 35 -44 1.203 (1.070 – 1.353) age (years) 45 – 54 1.875 (1.277 – 4.523) non users 1.00 <0.0001 regular users 1.250 (1.183 – 1.321) occasional users 1.250 (1.080 – 1.447) tobacco usage ex users 1.200 (1.065 – 1.352) smokers 1.00 <0.0001 chewers 1.250 (1.191 – 1.974) tobacco form snuff users 1.333 (1.167 – 1.524) ? 5 years 1.00 <0.0001 6 – 10 years 1.167 (1.120 – 3.611) 11 – 20 years 1.175 (1.174 – 3.957) duration of tobacco use > 20 years 2.625 (1.529 – 4.507) 1 – 2 units/ day 1.00 <0.0001 3 – 5 units/ day 1.111 (1.037 – 1.190) 6 – 10 units/ day 2.143 (1.060 – 4.333) frequency of tobacco consumption > 10 units /day 5.596 (2.901 – 10.639) ≤ table 6 logistic regression analysis with cpi as dependent variable (absence vs presence periodontal pockets) and age, users of tobacco, form, duration and frequency of tobacco use as independent variables braz j oral sci. 7(27):1641-1647 tobacco as risk factor for periodontal disease in green marble mine laborers of rajasthan, india χ2 = 23.625, p = 0.001 mod – moderate (1 – 5 units/day) heavy (more than 5 units/day) 1646 logistic regression analysis was employed to determine the effect of the age and tobacco practices on periodontal status. the results showed that all the independent variables were statistically significantly related to periodontal disease. the relationship between age of the subjects and their periodontal status was evident (table 6) and the odds ratio of presenting shallow and deep pockets increased gradually as the age increased with oldest population being 1.875 times more likely to have pockets than the youngest age group. when form of tobacco consumed was taken into consideration, it was clear that chewers (or = 1.250; p = 0.0001) and snuff users (or = 1.333; p = 0.0001) were more liable to present periodontal pockets than smokers. duration had a significant influence on the periodontal disease, since more than 20 years tobacco users were liable to have periodontal pockets at least twice (or = 2.625; p = 0.0001) than the < 5 years group. the odds for presence of periodontal pockets increased by 2.143 and 5.596 times for users of 6 – 10 units/ day and more than 10 units /day than the 1-2 times/day category. d i s c u s s i o n in the most of the population studies the sample belongs to lower socio economic level. moreover, the population of the present study had government provided below poverty line (bpl) cards with them. they have poor access to formal health care services and particularly to oral health; about 90% of the subjects had stated that they have never been to a dentist and they completely rely on local quacks for their treatment. our study also revealed poor oral hygiene habits in the subjects, since none of them brushed their teeth twice or more a day, being the most common oral hygiene aids restricted to finger with tooth powder and neem sticks. the nature of this study was cross-sectional, thus precluding the ability to draw inferences about causal relationships. a limitation that can be considered is that periodontal status was assessed using cpi, which measures probing depth in selected index teeth without any measurement of clinical attachment loss. adjusted or of regular users of tobacco for the presence of periodontal pockets was 1.25 which is in accordance with a study among japanese adult population where the respective value was 1.38 among current smokers27. furthermore, a very small difference for the presence of periodontal pockets was noticed between regular users, ex-users and occasional users which lead to a conclusion that former and occasional use of tobacco play analogous role in the causation of pockets as that of regular use. the association between age of the subjects and their periodontal status was evident and the risk of presenting periodontal pockets increased gradually as the age increased, similarly to previous study28 with higher levels of periodontal disease in older age group compared to younger ones. several studies have shown a relationship between the smoking amount and the prevalence and severity of periodontitis. a relationship has been demonstrated between the prevalence of moderate to severe periodontal disease and the number of cigarettes smoked per day29-33 and number of years that the patient has smoked30-32,34,35. similar relationship was observed in the present study, since long time and heavy tobacco users were more liable for presenting periodontal pockets. we could not find any study that assessed the effect of quantity and duration of smokeless tobacco usage for the occurrence of periodontal pockets. smokeless forms of tobacco users presented pockets more frequently than smokers. chewers and snuff users presented, respectively, 1.250 and 1.333 more chance to present pockets in reference to smokers. nevertheless, previous studies have failed to demonstrate an association of these forms with periodontal disease36. it was difficult to compare the results of the present study with previous ones30,32 regarding the quantity, form of tobacco used and associated periodontitis because they have included only cigarette smokers, whereas the present study dealt with cigarette, bidi smokers, chewers of various forms of tobacco and snuff users. moreover, the quantity of tobacco used by the subjects32 was greater than the subjects of the present study. in this way it was observed that tobacco usage had a significant impact on the severity of periodontal disease, with users being more likely to present periodontal pockets than non users. moreover, the risk of periodontal pockets increased as the duration and frequency of tobacco consumption increased. extensive health education should be aimed for cessation of tobacco habit stating its effect on systemic and oral health. intervention in the form of curative services is desperately needed for subjects with shallow and deep periodontal pockets. there is a need to assess by longitudinal studies the relationship between tobacco use and periodontal disease in indian population, where the habits of smoke as well as smokeless tobacco use are more common. r e f e r e n c e s 1. clarke ng, hirsch rs. personal risk factors for generalized periodontitis. j clin periodontol. 1995; 22: 136-45. 2. john novak m, karen f novak. smoking and periodontal disease. in: newman, takei, klokkevold, carranza, editors. clinical periodontology. in: elsevier; 2007. p.251-8. 3. johnson gk, hill m. cigarette smoking and the periodontal patient. j periodontol. 2004; 75: 196-209. 4. sheiham a. periodontal disease and oral cleanliness in tobacco smokers. j periodontol. 1971; 42: 259-63. 5. newman, takei, klokkevold, carranza. clinical periodontology. in: john novak m, karen f novak. smoking and periodontal disease. elsevier; 2007. p.251-8. 6. bergström j, preber h. tobacco use as a risk factor. j periodontol. 1994; 65: 545-50. braz j oral sci. 7(27):1641-1647 tobacco as risk factor for periodontal disease in green marble mine laborers of rajasthan, india 1647 7. gelskey sc. cigarette smoking and periodontitis: methodology to assess the strength of evidence in support of a causal association. community dent oral epidemiol. 1999; 27: 16-24. 8. bali rk, mathur vb, talwar pp, chanana hb. national oral health survey and fluoride mapping 2002-2003, rajasthan. new delhi: dental council of india; 2004. p.104-6. 9. gupta pc, ray cs. epidemiology of betel quid usage. ann acad med singapore. 2004; 33: 31-6. 10. qidwai w, saleheen d, saleem s, andares m, azam si. are our people health conscious? results of a patients survey in karachi, pakistan. j ayub med coll abbottabad. 2003; 15: 10-3. 11. mack tm: the new pan-asian paan problem. lancet. 2001; 357: 1638-9. 12. mandel i. smoke signals: an alert for oral diseases. j am dent assoc. 1994; 125: 872-8. 13. robertson pb, walsh m, greene j, ernster v, grady d, hauck w. periodontal effects associated with the use of smokeless tobacco. j periodontol. 1990; 61: 438-43. 14. mendis s. tobacco use in a rural community in sri lanka. ceylon med j. 1988; 33: 23-6. 15. wray a, mcguirt wf. smokeless tobacco usage associated with oral carcinoma. incidence, treatment, outcome. arch otolaryngol head neck surg 1993; 119: 929-33. 16. tomar sl, winn dm, swango pa, giovino ga, kleinman dv. oral mucosal smokeless tobacco lesions among adolescents in the united states. j dent res. 1997; 76: 1277-86. 17. waerhaug j. prevalence of periodontal disease in ceylon. association with age, sex, oral hygiene, socio-economic factors, vitamin deficiencies, malnutrition, betel and tobacco consumption and ethnic group. final report. acta odontol scand. 1967; 25: 205-31. 18. creath cj, cutter g, bradley dh, wright jt. oral leuokoplakia and adolescent smokeless tobacco use. oral surg oral med oral pathol. 1991; 72: 35-41. 19. offenbacher s, weathers dr. effects of smokeless tobacco on the periodontal, mucosal and caries status of adolescent males. j oral pathol. 1985; 14: 169-81. 20. greer ro jr, poulson tc. oral tissue alterations associated with the use of smokeless tobacco by teen-agers, part i. clinical findings. oral surg oral med oral pathol. 1983; 56: 275-84. 21. poulson tc, lindenmuth je, greer ro jr. a comparison of the use of smokeless tobacco in rural and urban teenagers. ca cancer j clin. 1984; 34: 248-61. 22. oral health surveys: basic methods. world health organization. 4th ed. geneva: who; 1997. 23. bulman js, osborn jf. measuring diagnostic consistency. br dent j 1989; 166: 377-81. 24. harada s, akhter r, kurita k, mori m, hoshikoshi m, tamashiro h, et al. relationships between lifestyle and dental health behaviors in a rural population in japan. community dent oral epidemiol. 2005; 33: 17-24. 25. peter s. essentials of preventive and community dentistry. 1.ed. arya (medi). 2000: 370-415. 26. abramson jh, abramson zh. making sense of data: a selfinstruction manual on the interpretation of epidemiological data. new york: oxford university press; 2001. 27. ojima m, hanioka t, tanaka k, inoshita e, aoyama h. relationship between smoking status and periodontal conditions: findings from national databases in japan. j periodont res. 2006; 41: 573-9. 28. miyazaki h, pilot t, leclercq mh, barmes de. profiles of periodontal conditions in adults measured by cpitn. int dent j. 1991; 41: 74-80. 29. haber j, wattles j, crowley m, mandell r, joshipura k, kent rl. evidence for cigarette smoking as a major risk factor for periodontitis. j periodontol. 1993; 64: 16-23. 30. krall ea, dawson-hughes b, garvey aj, garcia ri. smoking, smoking cessation and tooth loss. j dent res. 1997; 76: 1653-9. 31. grossi sg, zambon jj, ho aw, koch g, dunford rg, machtei ee, et al. assessment of risk for periodontal disease. i. risk indicators for attachment loss. j periodontol. 1994; 65: 260-7. 32. grossi sg, genco rj, machtei ee, ho aw, koch g, dunford r, et al. assessment of risk for periodontal disease. ii. risk indicators for alveolar bone loss. j periodontol. 1995; 66: 23-9. 33. martinez-canut p, lorca a, magán r. smoking and periodontal disease severity. j clin periodontol. 1995; 22: 743-9. 34. haber j, kent rl. cigarette smoking in periodontal practice. j periodontol. 1992; 63: 100-6. 35. alpagot t, wolff lf, smith qt, tran sd. risk indicators for periodontal disease in a racially diverse population. j clin periodontol. 1996; 23: 982-8. 36. wouters fr, salonen lw, frithiof l, helldén lb. significance of some variables on interproximal alveolar bone height based on cross-sectional epidemiological data. j clin periodontol. 1993; 20: 199-206. braz j oral sci. 7(27):1641-1647 tobacco as risk factor for periodontal disease in green marble mine laborers of rajasthan, india oral sciences n3 original article braz j oral sci. january | march 2011 volume 10, number 1 quantitative light-induced fluorescence (qlf) in relation to other technologies and conventional methods for detecting occlusal caries in permanent teeth antônio carlos pereira1, hafsteinn eggertsson2, carlos gonzález-cabezas3, domenick t. zero2, george j. eckert4, fábio luiz mialhe1 1department of community dentistry, piracicaba dental school, university of campinas, sp, brazil 2indiana university school of dentistry, indianapolis, in, usa 3university of michigan school of dentistry, ann arbor, mi, usa 4indiana university school of medicine, indianapolis, in, usa correspondence to: antonio carlos pereira faculdade de odontologia de piracicaba fop/unicamp departamento de odontologia social av. limeira 901 areão 13414-903 piracicaba, sp, brazil phone: +55 10 2106 5209 fax: +55 19 2106 5218 e-mail: apereira@fop.unicamp.br received for publication: july 27, 2010 accepted: february 10, 2011 abstract aim: the aim of this in vitro study was to compare the reproducibility and accuracy of quantitative light-induced fluorescence (qlf) in relation to other technologies and conventional methods for detecting occlusal carious lesions. methods: ninety-six extracted permanent molar teeth were selected. three examiners carried out examinations using visual examination (vi), bitewing radiographs (rx), qlf, electrical conductance measurement (ecm), and diagnodent. twentyfive percent of the teeth were re-examined for repeatability. stereomicroscopic examination was used as the gold standard. results: intraand inter-examiner agreement ranged from 0.43 to 0.89. areas under roc curves were 0.82, 0.54, 0.84, 0.79 and 0.88, respectively, for vi, rx, qlf, ecm and diagnodent examinations, with rx significantly lower than the other methods. no significant statistical difference was found when comparing the areas under roc curve of visual inspection and qlf. conclusions: although qlf and other technologies for early caries detection may offer some advantages, this study did not find significant improvement in occlusal caries detection when compared to visual examination. keywords: dental caries, detection, diagnosis, pits and fissures, quantitative light-induced fluorescence, visual scoring of caries, radiograph, fluorescence, accuracy, ecm. introduction over the past 40 years there have been changes in the epidemiological pattern of dental caries around the world. these changes have been characterized by a reduction in prevalence and severity of carious lesions1-2. the reduction has been surface dependent, and the occlusal surfaces of teeth are the sites most frequently affected by dental caries1-2. the reduction in caries progression has resulted in changes in the morphology and pattern of lesions, making it more difficult for the clinician to detect and assess the extent of the lesion3. braz j oral sci. 10(1):27-32 28 detection of carious lesions in their earliest stages has important implications for preventing their progression before cavitation occurs. unfortunately, traditional diagnostic systems for detecting caries lesions, such as visual inspection and radiography, have limited accuracy and sensitivity when diagnosing occlusal caries at the pre-cavitated level3-4. to improve the accuracy of diagnosis, non-invasive instrumentbased techniques have been developed for detecting and quantifying demineralization5-7. these techniques include methods based on electrical conductance measurement and laseror light-induced fluorescence properties associated with demineralized dental tissues. at the same time, proposals have been put forward for improvement in the visual examination, which include the detection of caries lesions in pre-cavitated stages7-9. however, in relation to light-induced fluorescence (qlf), few studies have been published evaluating the diagnostic performance of the method on occlusal surfaces and comparing it with traditional and other techniques for caries detections89. therefore, the aim of this study was to evaluate in vitro the performance of qlf for detecting occlusal caries and compare the results with visual examination criteria adopted by ekstrand6-7, and with other technologies in permanent molars without cavitation. material and methods the study was approved by the research ethics committee of the piracicaba dental school, university of campinas, registration number 027/2004. ninety-six extracted permanent molar teeth exhibiting complete root formation were selected from an institutional teeth depository. the occlusal surfaces were visually diagnosed for caries and ranged from sound to varying degrees of fissure discoloration and possible microscopic breakdown of the surface structure (extractions especially for orthodontic reason). none of the teeth showed macroscopic signs of cavity formation. the teeth were stored in 0.1% thymol solution for less than 3 months from the time of extraction. teeth with dental fluorosis, tetracycline stain, hypoplasia or dental restorations were excluded. the teeth were cleaned with a prophylaxis brush using pumice slurry, rinsed with a 3-way syringe, and after the ecm examination, mounted in blocks in sets of 3 teeth. the occlusal surfaces were photographed (×4 magnification), and one site on each tooth was selected and marked on the photograph for identification during examinations. the teeth were stored under refrigeration in de-ionized water in individual plastic containers. three examiners participated in this study. all the examiners underwent a training session, which consisted of two-hours of theoretical training and four-hours of practice on extracted teeth. additionally, a pilot study was conducted, using 9 supplementary teeth. examinations visual examination visual examination was carried out using only a dental operating light and air-drying up to 5 s. no explorer was used during the examination. each surface was scored using the criteria described by ekstrand et al.7, as presented in table 1. radiographic examination the teeth were radiographed under standardized conditions and the exposures were made using a trophy general electric ge 1000 intraoral x-ray machine, operating at 70 kvp and 8 ma. the blocks of teeth were placed in a holder, specially designed to provide standardized projection geometry during exposure. the focus-film distance was 21 cm and a 15mm-thick soft tissue equivalent material was placed between the cone end and the blocks of teeth. the classification criteria for radiographic examination are shown in table 2. score criteria 0 no or slight change in enamel translucency after prolonged air drying (>5s) 1 opacity or discoloration hardly visible on the wet surface, but distinctly visible after air drying 2 opacity or discoloration distinctly visible without air drying 3 localized enamel breakdown in opaque or discolored enamel and/or grayish discolored from underlying dentin 4 cavitation in opaque or discolored enamel exposing the dentin beneath table 1: criteria for visual examination score criteria 0 no caries 1 radiolucency extending to the outer ½ of the enamel 2 radiolucency extending to the inner ½ of the enamel 3 radiolucency extending to the outer ½ of the dentin 4 radiolucency extending to the inner ½ of the dentin table 2: criteria for radiographic examination quantitative light-induced fluorescence (qlf) images of occlusal surfaces of tooth specimens were captured using a portable intra-oral camera device connected to a computer (qlf, inspektor research systems bv, amsterdam, the netherlands). each occlusal surface was illuminated with 13 mw/cm2 of the violet-blue light (wave length: 290-450 nm) and the images were captured through a yellow 520 nm high-pass filter using a custom-made software (qlf, inspektor research systems bv). the images were scored subjectively from the stored images displayed on a crt monitor. the scoring criteria are shown in table 3. electrical conductance measurement (ecm) electrical conductance method was performed using the electronic caries monitor iii (ecm iii, lode, groningen, quantitative light-induced fluorescence (qlf) in relation to other technologies and conventional methods for detecting occlusal caries in permanent teeth braz j oral sci. 10(1):27-32 the netherlands). the ecm examination had to be done before mounting to allow for a reference electrode to be attached to the root complex for measurement. before each measurement the occlusal surface of the tooth was moistened with de-ionized water then gently air-dried and a toothpaste gel was syringed into the fissure system as a conducting medium. the reference electrode was attached to the root and the measurement electrode probe placed in contact with the fissure enamel at the site identified in the photograph, activating the co-axial air flow (7.5 l/min) until stable readings were obtained. ecm readings ranged from 0.00 indicating low conductance, to 99.99 being the highest measurable conductance. a score of 15 or lower was considered to indicate the presence of caries. each site was examined three times, and the average of these readings was considered as a definitive score. laser fluorescent (lf) examination laser fluorescence measurements were made using the diagnodent device (kavo, biberach, germany). the device was calibrated before use on the standardized porcelain chip provided by the manufacturer. using probe tip a, the machine was then calibrated against a sound area on each tooth separately, by holding the tip against a sound smooth surface and pressing the ring button until calibration was completed. the conical probe tip was then positioned perpendicularly over each selected site and slightly rotated around its long axis. each site was measured 3 times using the abovementioned procedures, and the average of these readings (099 range) was considered as a definitive score. histological validation after all assessments were completed, the teeth were removed from the blocks, and approximately 150 to 200µm-thick buccolingual sections made using a silverstonetaylor microtome (silverstone-taylor, scientific fabrications co., lafayette, co, usa), by cutting through the pre-selected site in the occlusal surface. the histologic examination was done with a stereomicroscope at ×40 magnification. both sides of each tooth section and the more severe side scored for the specimen. three examiners underwent a training session, which consisted of 2 h of theoretical training and 4 h of practice on extracted teeth. additionally, a pilot study was conducted, using nine supplementary teeth. all examiners were university teachers who had graduated more than 10 yr previously, had experience in clinical teaching, and had up to 4 yr of experience in clinical practice10. caries was defined as being present when demineralization was observed, seen score criteria 0 no caries 1 demineralization extending to the outer ½ of the enamel 2 demineralization extending to the inner ½ of the enamel 3 demineralization extending to the outer ½ of the dentin 4 demineralization extending to the outer ½ of the dentin table 4: criteria for histological validation score criteria 0 no change in enamel fluorescence 1 slight change in enamel fluorescence 2 fluorescence loss distinctly visible without enamel broken, 3 fluorescence loss distinctly visible with enamel broken 4 fluorescence loss distinctly visible with cavitation table 3: criteria for qlf examination as white or discolored (yellow/brown) area. the histological criteria for caries lesion depth are presented in table 4. statistical analysis to assess the intra-examiner agreement, 25 teeth were re-examined. intraand inter-examiner reproducibility for the ordinal visual, radiographic and qlf scores were assessed using weighted kappa statistics11. intraand inter-examiner reproducibility for ecm and diagnodent were assessed using intraclass correlation coefficients (icc)12. in order to compare the sensitivity, specificity, accuracy, and area under the roc curve (auc) between methods, a bootstrap sampling procedure was used. a bootstrap sample was obtained by randomly selecting specimens with replacement, preserving the percentages of true positives and true negatives in the sample distribution. the estimates were obtained for each of the 1000 bootstrap samples for each method, and the difference between methods was calculated. bootstrap sampling allows p-values and confidence intervals to be generated for the differences between methods 10. the sensitivity, specificity, and accuracy were calculated for the d1 diagnostic threshold (histology score > 1) with the cutoff > 0 ratings for visual and cut-off ³ 2 for qlf measurements. for ecm site measurements, the cut-off for all lesions was < 15 and for diagnodent, the cut-off limits for all lesions were values ³ 5, adopted by lussi et al.13 area under the roc curve, an indicator of overall diagnostic performance which does not require cut-offs for the diagnostic methods, was computed using the c-statistic from logistic regression models for each examiner and for each method. spss and sas statistical software packages were used for statistical analysis. results the histological examination revealed that 41 sites (43%) were sound; 31 (32%) had demineralization in enamel and 24 (25%) had demineralization extending into dentin. table 5 shows weighted kappa values for intraand inter-examiner reproducibility for the visual examination, radiography, and qlf ranked scoring systems. weighted kappas for intra-examiner repeatability ranged from 0.58 to 0.89 and qlf presented the best results for examiner 2. in relation to inter-examiner reproducibility, qlf presented better results than visual and radiography methods. table 6 presents the values of intraclass correlation coefficients for intraand inter-examiner reproducibility for diagnodent and ecm diagnostic methods. intra-examiner 29 quantitative light-induced fluorescence (qlf) in relation to other technologies and conventional methods for detecting occlusal caries in permanent teeth braz j oral sci. 10(1):27-32 30 score method intra-examiner reproducibility inter-examiner reproducibility exam 1 exam 2 exam 3 exam 1-2 exam 1-3 exam 2-3 visual 0.78 0.84 0.89 0.64 0.49 0.43 radiography 0.58 0.80 0.75 0.58 0.57 0.54 qlf 0.79 0.87 0.77 0.86 0.54 0.50 table 5: weighted kappa values of intra-and inter-examiner reproducibility for visual, radiography and qlf for examiners. method intra-examiner reproducibility interexaminer reproducibility exam 1 exam 2 exam 3 exam 1, 2, 3 e c m 0.40 0.81 0.95 0.69 diagnodent 0.66 0.69 0.93 0.83 table 6: intraclass correlation coefficients for intra-and inter-examiner reproducibility for ecm and diagnodent diagnostic methods. detection method sensitivity specificity accuracy az visual inspection 0.81 b 0.72 b 0.77 a,b 0.82a,b radiography 0.20 d 0.88 a 0.49 c 0.54c qlf 0.96 a 0.38 c 0.72 b 0.84a,b e c m 0.62 c 0.80 ab 0.70 b 0.79b diagnodent 0.78 b 0.91 a 0.83 a 0.88a table 7: the performance of each diagnostic method at d1 diagnostic threshold assessed by all examiners expressed in terms of sensitivity, specificity, accuracy and the average area under the roc-curve (az). different superscript letters indicate statistically significant differences among detection methods (p<0.05). agreement for both ecm and diagnodent varied widely among examiners, with iccs ranging from 0.40 to 0.95, but it was clearly higher for one of the examiners (range: 0.930.95). inter-examiner agreement was higher for diagnodent (icc=0.83) than for ecm (icc=0.69). the performance of each diagnostic method in terms of sensitivity, specificity, accuracy and the area under the roccurve (az) are shown in table 7. lf presented the best sensitivity but the worst specificity values. diagnodent had a higher az, accuracy, and sensitivity than ecm; higher accuracy than qlf, and higher specificity than visual. qlf presented the best sensitivity but the worse specificity values. the radiographic method was significantly worse than any of the other methods for az, accuracy, and sensitivity while it had higher specificity than the visual and qlf methods. discussion in the present study, the traditional visual diagnosis of occlusal caries presented high sensitivity (0.81) and specificity (0.72) values. these findings are similar to those obtained by other studies3,6-7,14-16, that showed high sensitivity values (0.80-0.98) and moderate to high specificity values (0.38-0.98) at d1 and d2 diagnostic thresholds. the visual criteria used in the present and in these studies were those proposed by ekstrand et al.6 the better performance of the visual inspection compared with other methods assessed in this study, should also be compared with the traditional visual examination, as none of the teeth in the study had macroscopic cavitation, and therefore would have been called sound. it should also be born in mind that the majority of teeth had narrow and deep fissures typical of third molars. some authors have demonstrated that in teeth with narrow fissures, the overall sensitivity, specificity and the percentage of correctly diagnosed teeth decreased 30% on average, in comparison with those that had wide fissures14. studies comparing the areas under roc curve for qlf and visual inspection have shown conflicting results. some authors affirm that visual inspection is as good as qlf when the dentist is instructed to detect the early signs of caries, and have not found significant differences in the areas under roc curve between the methods17-18. on the other hand, qlf has been found to detect more non-cavitated caries lesions than visual inspection using the criteria of ekstrand8. however, due to its time-consuming image processing and analysis and higher cost, authors questioned the practicality of qlf for regular use in the dental office at the present stage. furthermore, it has been suggested that the use of qlf technology must be combined with visual clinical examination since qlf detects any hypocalcified area, including developmental defects and dental fluorosis17. the poor performance of bitewing radiography in the present study may be attributed to several reasons: the majority of the studies evaluating the performance of radiographic methods in detecting occlusal caries lesions serially sectioned the teeth in the buccolingual direction, the probability of a carious lesions being found is greater and the diagnostic performance of the method would potentially be better6-7,10,19. several in vivo and in vitro studies have demonstrated that qlf presented better repeatability and reproducibility values for the quantification of smooth-surface caries than in pit and fissures ones20-22. however, it is interesting to note that studies have found higher levels of intra/inter-examiner reliability for qlf when detection of occlusal caries lesions was done through quantitative analysis using the qlf analysis quantitative light-induced fluorescence (qlf) in relation to other technologies and conventional methods for detecting occlusal caries in permanent teeth braz j oral sci. 10(1):27-32 31 software than when using qualitative interpretation of the images, as was the case of the criteria utilized in the present study8-9,23. these differences could also be attributed to the threshold value chosen for a caries lesion to be considered absent or present. a review of the effectiveness of qlf to detect occlusal caries lesions showed that intra and interexaminer reproducibility values were above of 0.90 when the evaluations were based on d3 diagnostic threshold24. in the present study, the criteria used for qlf demonstrated a good performance in detecting occlusal caries lesions, represented by the area under roc curve (az) and no significant statistical difference in az was found with visual inspection and diagnodent. diagnodent showed the highest accuracy at d1 diagnostic threshold (0.83), which in agreement with the published literature13,19,25. strong positive correlation between the histological examination and diagnodent was observed, although it was lower than those obtained by other authors 16,19,26. the performance of this method could be dependent on the cutoff points used, as a probable source of variation, which could explain the different results in the performance of this method. here, the cut-off chosen was a value of 5 which is lower than the recommended value of 15 for clinically detected early lesions. this correction is to compensate for the fluorescence decrease that occurs in teeth stored in different media, including the solution used for tooth storage in this study27. an important difference between qlf and the other methods evaluated was its lower specificity, which could lead to more false-positive diagnosis and consequently overtreatments. low specificity values for qlf have been reported previously18,28. the sensitivity and specificity values for ecm found in the present study were lower than those found by other studies6,15-16,29. however, the estimated area under the roc curve for occlusal caries detection agreed with the data presented by other authors16,29-30. only a moderate correlation was found between depth of the lesions and the numerical ecm reading. these results were in agreement with those obtained by ricketts et al.30-31 but were lower than data presented in other studies6-7. this could be attributed to the differences in the histological scores used. although qlf, ecm and diagnodent provide an appealing high-tech approach in the dental office, the present results do not suggest they are a significant improvement over a well-trained eye using the new visual criteria. therefore, although the advantages of qlf, ecm and diagnodent provide quantitative information, allowing the progression or arrest of carious lesions to be monitored in the course of time, the effectiveness of these emerging technologies only for detecting occlusal enam el and dentinal lesions in comparison with traditional methods is questionable. however, the higher specificity values found by some of these technologies indicated that those diagnostic methods were more accurate than the visual method for detecting sound surfaces. considering the decrease in the prevalence of caries in many populations around the world, these methods could be used as adjuncts in sites where there is clinical uncertainty, thereby decreasing the probability of over-treatment. while qlf and ecm methods have several advantages, they are expensive, and a less expensive device, such as diagnodent, would probably find greater acceptance by clinicians. further studies should focus on the applicability and effectiveness of new visual methods, using different classification criteria, and the combination of these with emerging technologies. in conclusion, although qlf and other technologies for early caries detection present some advantages, this study did not find significant improvement in occlusal caries detection when compared to visual examination system. references 1. hugoson a, koch g, hallonsten al, norderyd j, aberg a. caries prevalence and distribution in 3-20-years-olds in jönköping, sweden, in 1973, 1978, 1983, 1993. community dent oral epidemiol. 2000; 28: 83-9. 2. hugoson a, koch g. thirty year trends in the prevalence and distribution of dental caries in swedish adults (1973-2003). swed dent j. 2008; 32: 57-67. 3. bader jd, shugars da, bonito aj. a systematic review of the performance of methods for identifying carious lesions. j public health dent. 2002; 62: 201-13. 4. pretty ia, maupomé g. a closer look at diagnosis in clinical dental practice: part 3. effectiveness of radiographic diagnostic procedures. j can dent assoc. 2004; 70: 388-94. 5. lussi a, imwinkelried s, pitts nb, longbottom c, reich e. performance and reproducibility of a laser fluorescence system for detection of occlusal caries in vitro. caries res. 1999; 33: 261-6. 6. shi xq, welander u, angmarmånsson b. occlusal caries detection with kavo diagnodent and radiography: an in vitro comparison. caries res. 2000; 34: 151-8. 7. zandona af, zero dt. diagnostic tools for early caries detection. j am dent assoc. 2006; 37: 1675-84. 8. ekstrand kr, ricketts dnj, kidd eam. reproducibility and accuracy of three methods for assessment of demineralization depth on the occlusal surface: an in vitro examination. caries res. 1997; 31: 224-31. 9. ekstrand kr, ricketts dnj, kidd eam, qvist v, schou s. detection, diagnosing, monitoring and logical treatment of occlusal caries in relation to lesion activity and severity: an in vivo examination with histological validation. caries res. 1998; 32: 247-54. 10. kühnisch j, ifland s, tranaeus s, hickel r, stösser l, heinrich-weltzien r. in vivo detection of non-cavitated caries lesions on occlusal surfaces by visual inspection and quantitative light-induced fluorescence. acta odontol scand. 2007; 65: 183-8. 11. kühnisch j, ifland s, tranaeus s, angmar-månsson b, hickel r, stösser l et al.. establishing quantitative light-induced fluorescence cut-offs for the detection of occlusal dentine lesions. eur j oral sci. 2006; 114: 483-8. 12. landis j r, koch gg. the measurement of observer agreement for categorical data. biometrics. 1977; 33: 159-74. 13. shrout pe, fleiss jl. intraclass correlations: uses in assessing rater reliability. psychol bull. 1979; 86: 420-8. 14. pereira ac, eggertsson h, martinez-mier ea, mialhe fl, eckert gj, zero dt. validity of caries detection on occlusal surfaces and treatment decisions based on results from multiple caries-detection methods. eur j oral sci. 2009; 117: 51-7. 15. el-housseiny aa, jamjoum h. evaluation of visual, explorer, and a laser device for detection of early occlusal caries. j clin pediatriat dent. 2001; 26: 41-8. 16. pereira ac, verdonschot eh, mcdnjm huysmans. caries detection methods: can they aid decision making of invasive sealant treatment? caries res. 2001; 35: 83-9. quantitative light-induced fluorescence (qlf) in relation to other technologies and conventional methods for detecting occlusal caries in permanent teeth braz j oral sci. 10(1):27-32 32 17. côrtes df, ellwood rp, ekstrand kr. an in vitro comparison of a combined foti/visual examination of occlusal caries with other caries diagnostic methods and the effect of stain on their diagnostic performance. caries res. 2003; 37: 8-16. 18. van der veen mh, de josselin de jong e. application of quantitative lightinduced fluorescence for assessing early caries lesions. monogr oral sci. 2000; 17: 144-62. 19. kano-wilson lh, ferreira zandona ag. comparing icdas, diagnodent, qlf, and neksduo in occlusal caries detection. j dent res. 2007: 86(spec issue a): abstract 2554. 20. tranaeus s, shi xq, lindgren le, trollsås k, angmar-månsson b. in vivo repeatability and reproducibility of the quantitative light-induced fluorescence method. caries res. 2002; 36: 3-9. 21. pretty ia, hall af, smith pw, edgar wm, higham sm. the intra-and inter-examiner reliability of quantitative light-induced fluorescence (qlf) analyses. br dent j. 2002; 193: 105-9. 22. heinrich-weltzien r, kühnisch j, ifland s, tranaeus s, angmar-månsson b, stösser l. detection of initial caries lesions on smooth surfaces by quantitative light-induced fluorescence and visual examination: an in vivo comparison. eur j oral sci. 2005; 113: 494-8. 23. pretty ia, ellwood rp. comparison of paired visual assessment and software analyses of changes in caries status over 6 months from fluorescence images. caries res. 2007; 41: 115-20. 24. pretty ia. a review of the effectiveness of qlf to detect early caries lesions. indianapolis, indiana: indiana university press; 2005. p.253-90. 25. baseren nm, gokalp s. validity of a laser fluorescence system (diagnodent) for detection of occlusal caries in third molars: an in vitro study. j oral rehabil. 2003; 30: 1190-4. 26. bamzahim m, shi xq, angmar-månsson b. occlusal caries detection and quantification by diagnodent and electronic caries monitor: in vitro comparison. acta odontol scand. 2002; 60: 360-4. 27. francescut p, zimmerli b, lussi a. influence of different storage methods on laser fluorescence values: a two-year study. caries res. 2006; 40: 181-5. 28. ferreira-zandoná ag, ando m, eggerston h. clinical validation of caries detection methodologies: preliminary results. j dent res. 2004; 83 (spec issue a): 2812a. 29. lussi a, firestone a, schoenberg v, hotz p, sitch h. in vivo diagnosis of fissure caries using a new electrical resistance monitor. caries res. 1995; 29: 81-7. 30. ricketts dnj, kidd eam, wilson rf. the effect of airflow on site-specific electrical conductance measurements used in the diagnosis of pit and fissure caries in vitro. caries res. 1997; 31: 111-8 . 31. ricketts dnj, kidd eam, wilson rf. the electronic diagnosis of caries in pits and fissures: site specific stable conductance readings or cumulative resistance readings? caries res. 1997; 31: 119-24. quantitative light-induced fluorescence (qlf) in relation to other technologies and conventional methods for detecting occlusal caries in permanent teeth braz j oral sci. 10(1):27-32 original articlebraz j oral sci. april/june 2009 volume 8, number 2 evaluation of three radiographic methods for detecting occlusal caries lesions antonio carlos pereira1, hafsteinn eggertsson2, analoui moustafa2, domenick t. zero3, george j. eckert4, fábio luiz mialhe5 1 phd, department of community dentistry, faculdade de odontologia de piracicaba, universidade estadual de campinas (unicamp), piracicaba (sp), brazil 2 phd, indiana university, indiana, united states of america 3 ms, associate dean, indiana university school of dentistry, indiana, united states of america 4 mas, indiana university, indiana, united states of america 5 phd, department of community dentistry, faculdade de odontologia de piracicaba, unicamp, piracicaba (sp), brazil received for publication: december 22, 2008 accepted: may 18, 2009 correspondence to: antonio carlos pereira departamento de odontologia social da faculdade de odontologia de piracicaba da unnicamp avenida limeira, 901 – areião cep 13414-903 – piracicaba (sp), brazil e-mail: apereira@fop.unicamp.br abstract aim: to compare, in vitro, the performance of three radiographic methods for the detection of occlusal caries in permanent teeth. methods: a total of 96 extracted molars with no apparent occlusal cavitation were selected, they were photographed and radiographed under standardized conditions using conventional e-plus films and two digital systems, cdr and sidexis. two examiners analyzed all films and images, recording the presence and lesion depth. one quarter of the teeth were re-examined for intraand interexaminer agreements. the teeth were subsequently bisected and examined under a stereomicroscope. the intra and interexaminer agreements and the diagnostic performance (sensitivity, specificity, accuracy and the area under receiver operating characteristic, roc curve) of each method were evaluated. results: out of 96 occlusal surfaces, 41 were sound, 31 had lesions in enamel, and 24 had dentin lesions. weighted kappa values for intraexaminer agreement varied widely, depending on both the observer and method. the interexaminer agreement was higher for the digital images than for the conventional films. the area under the roc curve for enamel and dentin caries (at d1 diagnostic threshold) was 0.55 for films, 0.60 for schick and 0.54 for sirona, which were not significantly different from each other. conclusions: digital images presented better results of interexaminer agreement; however, no additional effect in the diagnostic performance could be observed in comparison to conventional films. keywords: digital radiography, dental caries, roc curve, diagnostic test. introduction advances in radiographic detection methods include the development of several digital radiography systems for dental use1-5. these systems have shown a number of advantages over conventional radiography (film-based images), but their incorporation into dental offices has occurred slowly. several digital radiographic systems are available for the clinicians, who are replacing conventional radiography1,4. however, very little data is available on the diagnostic differences between intraoral digital systems and conventional radiographs to detect occlusal caries lesions4,6-8. in addition, there are few studies evaluating the diagnostic efficacy of charge-coupled device (ccd) based sensor systems for this purpose4,7. it is; therefore, necessary to continually evaluate these methods with regard to their clinical performances in order to obtain information that could help the dentists in selecting the best system for their clinical purposes. 68 pereira ac, eggertsson h, moustafa a, zero dt, eckert gj, mialhe fl braz j oral sci. 8(2): 67-70 the aim of this study was to compare the accuracy of conventional radiographs and two different digital systems – cdr and sidex – for detecting caries lesions in occlusal surfaces. material and methods the research protocol was reviewed and approved by the ethics committee in human research of faculdade de odontologia of universidade estadual de campinas (unicamp), under the protocol #028/2004. for this study, 96 permanent molars without fillings extracted, which had been stored in 10% buffered formalin, were selected. none of the teeth had any macroscopic occlusal cavity formation or clinically visible proximal caries, but presented several degrees of fissure discoloration. the teeth were first cleaned with prophylaxis brush and pumice slurry, rinsed with air/water spray and mounted in plaster blocks in sets of three teeth, simulating anatomic positions. the occlusal surfaces were photographed (4× magnification) and one site was chosen per tooth. then, they were radiographed under standardized conditions and the exposures were made using a trophy general electric ge 1,000 intra-oral x-ray unit (general electric company, crown point, in, usa), operating at 70 kvp and 8 ma and using exposure time of 0.25 second for conventional film and 0.08 for digital systems. the blocks of teeth were placed in a holder device specially designed to provide standardized projection geometry during exposure. the focus-film distance was 21 cm and a 15-mm thick acrylic material equivalent to soft tissue was placed, between the cone end and the blocks of teeth. the radiographs were taken using a conventional dental film (ektaspeed plus-eastman kodak co, rochester, ny, usa) and two different digital systems, sidexis (sirona, bensheim, germany), which uses a sensor with ccd technology, and cdr (schick technologies, ny, usa), which uses complementary metal-oxide semiconductor (cmos) technology. the films were processed in a dürr automatic unit (1330, ac 245 l, bietighein-bissingen, germany), mounted in frames and examined using a viewing box and a dental x-ray viewer (4× magnification). the digital images were displayed on a sva 17-inch monitor screen (dell computer corporation, austin, tx, usa). the conventional radiographs and the digital images were examined by two independent and previously calibrated observers, both experienced researchers in caries diagnosis. selected sites on each photograph were used to locate the precise investigation site on the radiograph in a mesiodistal plane. the occlusal caries depth was assessed using a rank scale with the following criteria5: 0 = no caries, 1 = radiolucency extending to the outer ½ of the enamel, 2 = radiolucency extending to the inner ½ of the enamel, 3 = radiolucency extending to the outer ½ of the dentin and 4 = radiolucency extending to the inner ½ of the dentin. one week elapsed between individual sets of recordings. after this interval, 25 teeth were re-examined to assess the intraand interexaminer agreement. the diagnostic performance of each observer with the three recording systems was compared to the histological diagnosis (gold standard). for validation of the true presence or absence of caries, the teeth were sectioned in a buccolingual direction through the selected investigation site using a silverstone taylor microtome (scifab, lafayete, colo, usa). the histologic examination was done with a stereomicroscope (model bh2, olympus optical co., ltd., tokyo, japan) at a 40× magnification and performed by the two observers (joint decisions). both sides of each tooth section were examined. caries was defined as being present when demineralization seen as a white or discolored (yellow/brown) area was observed. the following classification criteria were applied: 0 = no caries, 1 = demineralization extending to the outer ½ of the enamel, 2 = demineralization extending to the inner ½ of the enamel, 3 = demineralization extending to the outer ½ of the dentin and 4 = demineralization extending to the outer ½ of the dentin. statistical analysis intraand interexaminer agreements for the x-ray methods were assessed using weighted kappa statistics9. the diagnostic performance for occlusal enamel or deeper (cut off ≥ 1) was evaluated using the parameters sensitivity and specificity and area under receiver operating characteristic (roc) curve (az), having the histological ratings as a gold standard (at d1 threshold: caries in enamel in histological sections was considered “positive”). to compare the sensitivity, specificity, accuracy, and area under the roc curve (az) among the methods, a bootstrap sampling procedure was used. a bootstrap sample was obtained by randomly selecting specimens with replacement, preserving the percentages of true positives and negatives in the sample distribution. the estimates were obtained for each of the 1.000 bootstrap samples for each method, and the difference between the methods was calculated. bootstrap sampling allows p-values and confidence intervals to be generated for the differences between methods. area under the roc curve (az), an indicator of overall diagnostic performance which does not require cut offs for the diagnostic methods, was computed using the c-statistic from logistic regression models for each examiner and method. area under the roc curve was computed using the c-statistic from logistic regression models for each examiner and method at a 5% level of significance. results according to the histological examination, 41 teeth were sound, 31 had caries confined to the enamel, and 24 had caries in dentin. the intraexaminer agreement mean values for the two examiners, using weighted kappa, were respectively: 0.79 and 0.75 for conventional film (cf), 0.01 and 0.66 for cdr, and 0.40 and 0.67 for sidexis. interexaminer agreement was higher for the digital images (0.90 for cdr and 0.85 for sidexis) than for the cf (0.57). 69evaluation of three radiographic methods for detecting occlusal caries lesions braz j oral sci. 8(2): 67-70 the results of the diagnostic parameters are summarized in table 1. in general, cdr had the highest sensitivity, specificity, diagnostic accuracy, and area under the roc curve. cdr had significantly higher sensitivity than sidexis (p = 0.0397). no other differences among the three systems were statistically significant (p > 0.05). discussion the literature is still scarce in studies evaluating the accuracy of sidexis and cdr dental digital imaging systems, for detecting occlusal caries lesions4,7,10. studies in this field were mainly carried out using a storage phosphor digital system5-6,10-12 (digora; soredex, helsinki, finland). the majority of the studies comparing these two digital systems evaluated their performance for detecting proximal caries lesions3,8,13. although there is no other study in literature that compares these two systems for detecting occlusal caries lesions, data from other ones have demonstrated significant differences in the diagnostic accuracy among digital radiographic systems. the mean value of az for the sidexis system (0.54) in the present study was lower than that obtained by hintze et al.12 (0.8 to 0.92) and hintze7 (0.75 to 0.79). however, in these previous studies, the authors considered that the cut off for the presence of disease was caries in dentin, which naturally increases the diagnostic performance of the radiographic methods. data of some studies have demonstrated that sidexis presents poorer contrast in its images of dental structures, hindering the differentiation of relevant details8,14-15. to achieve optimal image clarity of the areas of interest, these authors modified the images by adjusting contrast and brightness, possibly influencing the performance of the method, but this was not done in the present study. in addition, data of another study14 show that the sidexis system underestimated the depth of proximal caries lesions by 51%, when compared with the gold standard examination. according to pfeiffer et al.15, this might have happened due to the automatic optimizing of the sidexis system, reducing the 12-bit digitized image into a 8-bit displayed image. regarding cdr, a new version of this equipment was introduced in 19982 which has a sensor with a smaller pixel size and uses the active pixel sensor (aps) and cmos technologies. this change improved the physical performance of this direct digital radiographic sensor when compared to the earlier generation2. analoui1 suggested that, based on the inherent characteristics of the new technologies used in the ccd sensors, they have the potential to surpass film in virtually every relevant aspect. studies on the performance of this new model of cdr for detecting carious lesions are needed, in order to assess whether they are better than conventional radiograph for this purpose. the poor performance of the radiographic methods evaluated in the present study may be attributed to several reasons. it is well known that a radiograph is unable to detect initially demineralized occlusal enamel and dentin lesions, resulting in low sensitivity10-11,16-23. in the present study, of all teeth presenting caries lesions, 56.3% had caries in enamel and 36.3% presented carious lesions extending to the outer half of the dentin, and only 7.4% of the lesions were located in the inner half of the dentin, which can lead to underestimate the performance of the methods. there are two aspects of the study design that limit the interpretation of the results. first, as no metal strip was fixed to the pre-selected site by tape before exposure to ensure its precise identification, the location of the clinical site in the radiographic examination was not precise. this could have allowed the observers to evaluate different sites from those selected in the individual photographs. second, the histological sections were obtained in a buccolingual direction through the selected investigation site only. although the study of ekstrand et al.16 used the same methodology, the majority of the studies evaluating the performance of radiographic methods in detecting occlusal caries lesions serially sectioned the teeth in various sections, in the buccolingual direction3,6,12,14,17,19,24-25. it is obvious that in these studies, the probability of detecting a carious lesion was higher, and the determination of the diagnostic performance of the method was probably more accurate. however, in daily clinical practice, the dentist frequently wants to know the radiographic extent of a clinically detectable lesion in a single site in the occlusal surface. therefore, further studies to evaluate the site-specific performance of radiographic methods are recommended. although in this study brightness and contrast of the images were not enhanced as in previous studies, it seems that there are no significant differences in the accuracy between enhanced and unenhanced images7,8. in addition, it appears that in clinical practice, dentists use the enhancement facilities of digital radiographic systems (brightness and contrast manipulation) very differently, and enhancements not properly used may actually reduce diagnostic accuracy26. however, further studies should be carried out to assess whether the management of these variables can influence the accuracy of the methods tested herein26-30. the present study demonstrates the diagnostic equivalence of two digital systems with conventional film using similar viewing conditions. these findings agree with the data presented by other studies, which found out that digital intraoral radiographic systems seem to be as accurate as current conventional dental films for detecting occlusal caries lesions5,8,13. in conclusion, digital images presented better results of interexaminer agreement; however, no additional effect in the diagnostic performance could be observed in comparison to conventional films. method sensitivity specificity accuracy az cf 0.20ab 0.89a 0.49a 0.55a cdr 0.27a 0.91a 0.55a 0.60a sidexis 0.15b 0.91a 0.48a 0.54a table 1. diagnostic performance for enamel and dentinal caries diagnosis of the three radiographic systems and two examiners, expressed as sensitivity, specificity and accuracy cf: conventional film. different superscript letters indicate statistically significant differences among the diagnostic methods (p < 0.05); az: area under the roc curve. 70 pereira ac, eggertsson h, moustafa a, zero dt, eckert gj, mialhe fl braz j oral sci. 8(2): 67-70 references 1. analoui m. digital diagnostic imaging: today and tomorrow. dentomaxillofac radiol. 1999;28:56-8. 2. attaelmanan ag, borg e, gröndahl hg. assessments of the physical performance of 2 generations of 2 direct digital intraoral sensors. oral surg oral med oral pathol oral radiol endod. 1999;88:517-23. 3. haiter-neto f, dos anjos pontual a, frydenberg m, wenzel a. a comparison of older and newer versions of intraoral digital radiography systems: diagnosing noncavitated proximal carious lesions. j am dent assoc. 2007;138:1353-9. 4. hintze h, wenzel a. influence of the validation method on diagnostic accuracy for caries. a comparison of six digital and two conventional radiographic systems. dentomaxillofac radiol. 2002;31:44-9. 5. wenzel a, borg e, hintze h, gröndahl hg. accuracy of caries diagnosis in digital images from charge-coupled device and storage phosphor systems: an in vitro study. dentomaxillofac radiol. 1995;24:250-4. 6. ashley pf, blinkhorn as, davies rm. occlusal caries diagnosis: an in vitro histological validation of the electronic caries monitor (ecm) and other methods. j dent. 1998;26:83-8. 7. hintze h. diagnostic accuracy of two software modalities for detection of caries lesions in digital radiographs from four dental systems. dentomaxillofac radiol. 2006;35:78-82. 8. tyndall da, ludlow jb, platin e, nair m. a comparison of kodak ektaspeed plus film and the siemens sidexis digital imaging system for caries detection using receiver operating characteristic analysis. oral surg oral med oral pathol oral radiol endod. 1998;85:113-8. 9. landis jr, koch gg. the measurement of observer agreement for categorical data. biometrics. 1977;33:159-74. 10. galcerá civera v, almerich silla jm, montiel company jm, forner navarro l. clinical and radiographic diagnosis of approximal and occlusal dental caries in a low risk population. med oral patol oral cir bucal. 2007;12:e252-7. 11. chong mj, seow wk, purdie dm, cheng e, wan v. visual-tactile examination compared with conventional radiography, digital radiography and diagnodent in the diagnosis of occlusal occult caries in extracted premolars. pediatr dent. 2003;25:341-9. 12. hintze h, wenzel a, frydenberg m. accuracy of caries detection with four storage phosphor systems and e-speed radiographs. dentomaxillofac radiol. 2002;31:170-5. 13. syriopoulos k, sanderink gc, velders xl, van der stelt pf. radiographic detection of approximal caries: a comparison of dental films and digital imaging systems. dentomaxillofac radiol. 2000;29:312-8 14. jacobsen jh, hansen b, wenzel a, hintze h. relationship between histological and radiographic caries lesion depth measured in images from four digital radiography systems. caries res. 2004;38:34-8. 15. pfeiffer p, schmage p, nergiz i, platzer u. effects of different exposure values on diagnostic accuracy of digital images. quintessence int. 2000;31:257-60. 16. ekstrand kr, ricketts dnj, kidd ea. reproducibility and accuracy of three methods for assessment of demineralization depth on the occlusal surface: an in vitro examination. caries res. 1997;31:224-31. 17. hintze h, wenze a. clinical and laboratory radiographic caries diagnosis. a study of the same teeth. dentomaxillofac radiol. 1995;25:115-8. 18. hugoson a, koch g, hallonsten al, norderyd j, aberg a. caries prevalence and distribution in 3-20-year-olds in jönköping, sweden, in 1973, 1978, 1983, and 1993. community dent oral epidemiol. 2000;28:83-9. 19. huysmans mc, longbottom c, pitts n. electrical methods in occlusal caries diagnosis: an in vitro comparison with visual inspection and bite-wing radiography. caries res. 1998;32:324-9. 20. machiulskiene v, nyvad b, baelum v. a comparison of clinical and radiographic caries diagnosis in posterior teeth of 12-year-old lithuanian children. caries res. 1999;33:340-8. 21. mejàre i, källestal c, stenlund h, johansson h. caries development from 11 to 22 years of age: a prospective radiographic study. prevalence and distribution. caries res. 1998;32:10-6. 22. richardson ps, mcintyre ig. the difference between clinical and bitewing detection of approximal and occlusal caries in royal air force recruits. community dent health. 1996;13:65-9. 23. weerheijm kl, groen hj, poorterman jhg. clinically undetected oclusal dentin caries in 1987 and 1993 in 17 years old dutch adolescents. abstract. caries res. 1999;33:288. 24. ricketts dn, kidd ea, smith bg, wilson rf. clinical and radiographic diagnosis of occlusal caries: a study in vitro. j oral rehabil. 1995;22:15-20. 25. van amerongen jp, penning c, kidd ea, ten cate jm. an in vitro assessment of the extent of caries under small occlusal cavities. caries res. 1992;26:89-93. 26. wenzel a. a review of dentists’ use of digital radiography and caries diagnosis with digital systems. dentomaxillofac radiol. 2006;35:307-14. 27. pereira ac, eggertsson h, martinez-mier ea, mialhe fl, eckert gj, zero dt. validity of caries detection on occlusal surfaces and treatment decisions based on results from multiple caries-detection methods. eur j oral sci. 2009;117:51-7. 28. swenson e, hennessy b. detection of occlusal carious lesions: an in vitro comparison of clinicians’ diagnostic abilities at varying levels of experience. gen dent. 2009;57:60-6. 29. novaes tf, matos r, braga mm, imparato jc, raggio dp, mendes fm. performance of a pen-type laser fluorescence device and conventional methods in detecting approximal caries lesions in primary teeth – in vivo study. caries res. 2009;43:36-42. 30. jablonski-momeni a, ricketts dnj, stachniss v, maschka r, heinzel-gutenbrunner m, pieper k. occlusal caries: evaluation of direct microscopy versus digital imaging used for two histological classification systems. j dent. 2009;37:204-11. oral sciences n3 original article braz j oral sci. july/september 2009 volume 8, number 3 assessment of flexural strength of two self-curing acrylic resins containing pigment vicente telles 1, raphael m. f. brito1, estela m. jurach 2, lincoln i. nojima 3 1undergraduate student, department of orthodontics, federal university of rio de janeiro, brazil 2 dds, ms, graduate student, department of orthodontics, federal university of rio de janeiro, brazil 3 dds, ms, associate professor, department of orthodontics, federal university of rio de janeiro, brazil received for publication: june 29, 2009 accepted: november 11, 2009 correspondence to: lincoln i. nojima universidade do brasil ufrj faculdade de odontologia programa de pós-graduação em odontologia av. professor rodolpho paulo rocco, 325 ilha do fundão rio de janeiro rj brasil cep: 21941-617 phone: 2590-2727 / fax: 21-2590-9771 e-mail: linojima@gmail.com abstract aim: to assess the flexural strength of orthodontic acrylic resins from two different manufacturers ( vipi, ortocril versus clássico, ortoclas) by comparing pigmented resins to colorless ones. methods: resins of blue, yellow and green colors were studied. a total of 120 specimens were made and then divided into groups of 15 elements each, all having the same dimensions. next, they were kept in aqueous medium until being subjected to mechanical testing. the flexural strength was tested in a universal test machine (emic dl 10000) in which the specimens were subjected to a gradual load until fracture occurred. results: pigmented resins had flexural strength values compatible with clinical use, being similar to those from colorless ones, except for ortoclas green-colored and yellow-colored resins, which showed greater flexural strength. the ortoclas green-colored resin was the most resistant to fracture (482.2 n), whereas the ortocril colorless resin was the least resistant (368.4 n). all ortoclas resins showed higher strength values compared to ortocril resins of same color, except for the ortocril’s blue-colored resin, which presented higher flexural strength than that of the other trademark. conclusions: the use of pigments seems to have no effect on decreasing the flexural strength of self-curing acrylic resins. therefore, pigmented resins are compatible with clinical use. keywords: acrylic resins, flexural strength, orthodontic appliances. introduction self-curing acrylic resins have been widely used in orthodontics for making plates for small tooth movements and space maintenance, palatal disjunction appliances, retention plates, and fixed inclined planes1. either orthodontists or even general practitioners can make some of these more simple appliances in order to prevent progression of malocclusions, which can potentially require a more complex and prolonged treatment in the future2. therefore, despite being inefficient in some orthodontic treatments, these appliances still play an important role in correcting malocclusions during deciduous and mixed dentition within each stage of the craniofacial development3-4. because of their limitations, these orthodontic appliances are used only in the treatment of children and teenagers as the practitioner can rely on both bone growth and eruptive tooth movement. esthetics is so valued today that the demand for orthodontic baseplates that are colored, decorated or inscribed with designs has increased. because these appliances can be fixed or removable, it is extremely important that the patient is satisfied and accepts them so that he or she can co-operate with the orthodontic treatment. in order to fulfill such a demand, pigmented acrylic resins has been increasingly accepted in the dental market as their preparation is the same as that regarding colorless resins and there is no need to add pigment during the laboratorial phase to obtain a colored resin. however, no study assessing the resistance of these materials after pigmentation is available in the literature. braz j oral sci. 8(3):137-140 although acrylic resin is widely used, this material can easily fracture due to its low resistance to impact, low flexural strength or low fatigue strength5-6. flexural strength is a physical property that determines the material’s resistance to bending. when a flexural force is applied, the material suffers an elastic deformation followed by plastic deformation and eventually fractures 7. another great disadvantage of acrylic resins is the rapid loss of esthetic, physical and mechanical properties within the oral medium because this material absorbs and releases water8. orthodontic appliances tend to fracture due to both occlusive forces and presence of metallic wires which promote dental movement because it suffers some deformation during placement and removal, thus leading to fatigue over the insertion areas of these wires9-10. because the pigments added to resins can bring impurities that might react with free radicals, the polymerization reaction could be compromised and consequently the physical properties of the material changed. therefore, the aim of the present study was to assess the flexural strength of pigmented resins compared to colorless resins. material and methods a total of 120 acrylic resin specimens (20.5 x 5.5 x 4.0 mm) distributed into 8 groups (n = 15) were fabricated using in a powder/liquid mixing ratio of 3:1, according to recommendations of the two manufacturers, namely, ortocril® (vipi, pirassununga, sp, brazil) and ortoclas® (artigos odontológicos clássico ltda., são paulo, sp, brazil). the specimens were made within a silicone condenzation mold (perfil, vigodent, rio de janeiro, rj, brazil) with internal dimensions of 21 x 6.2 x 4.0 mm that served as a negative control. polymerization occurred inside a vh softline pressure container (midas dental products ltda.) at constant pressure of 17 pounds/in2 or 87.93 cm/hg for 20 min. the excess material was removed from the specimens by using progressively 150, 400 600-grit sandpapers until obtaining the desired dimensions, which were measured with a starrett caliper (figure 1). after polishing the specimens were stored in containers with water for 24 h so that residual monomers could be released. after this period, water was replaced and the specimens remained immersed until being subjected to mechanical testing. ortoclas specimens were indicated by the abbreviation cl, whereas ortocril specimens were indicated by cr. these groups were also identified in terms of color as follows: colorless (l), yellow (y), blue (b) and green (g). the test consisted of gradually applying a force to each sepcimen by using a universal test machine (emic dl 1000; são josé dos pinhais, pr, brazil) at a crosshead speed of 5 mm/min until fracture occurred. the machine has three shafts in which the two inferior ones serves to hold the sample and the superior one serve to apply force to the centre of the sample. the three shafts have the same ray of 2.5 mm in order to avoid differences in the results. the center of the specimen was determined by using a millimeter rule and the resulting central point was marked with an ohp marker pen. the machine recorded the force that resulted in fracture (figure 2). statistical analysis was performed with the spss software for windows (v. 13.0) and using two-way anova and tukey’s test in order to determine the mean flexural strength of each group and to compare them regarding the trademarks. the control group consisted of colorless self-curing acrylic resins because of the absence of pigments. means and standard deviations were obtained for a significant level of 5% (table 1). results in the control group containing ortocril specimens, the fracture force was not significantly higher than that of other specimens of same trademark (table 1 and figure 3). with regard to ortoclas resins, containing yellow-colored and green-colored resin specimens had a significantly higher flexural strength than the blue-colored resin and colorless resin specimens (table 1 and figure 3). figure 2 – flexural strength test schematic‘s representation. figure 3 – box plot comparing trademarks and colours of acrylic resins. mean, sd, max, min. assessment of flexural strength of two self-curing acrylic resins containing pigment138 braz j oral sci. 8(3):137-140 figure 1 – samples after polishing. n mean s d m i n i m u m m a x i m u m statistics ortocril colorless 15 368.38 40.73 291.59 423.98 a ortocril yellow 15 401.37 32.03 348.01 448.56 a ortocril blue 15 406.17 46.29 334.60 494.92 a ortocril green 15 369.50 24.14 336.28 416.16 a ortoclas colorless 15 397.42 52.04 312.82 465.31 a ortoclas yellow 15 461.77 34.80 396.05 509.44 b ortoclas blue 15 402.49 39.97 332.37 452.47 a ortoclas green 15 482.17 20.81 448.00 512.79 b table 1 – descriptive statistical analysis of the mean flexural strength values (n). different letters mean statistically significant difference (p < 0.05). by comparing the trademarks, ortoclas yellow-colored and green-colored resins were significantly more resistant than the ortocril resins of same colors. with regard to blue-colored and colorless resins, no statistically significant differences were found between the trademarks (table 1 and figure 3). discussion acrylic resins are composed of polymeric chains of polymethylacrylate and monomers of methylmethacrylate with a small amount of ligant agents1,10. polymerization occurs by mixing polymer (powder) with monomer (liquid). a macromolecule is formed through a series of chemical reactions11-12. any impurity existing in the monomer may inhibit or delay polymerization if free radicals are involved, thus impeding the stage called propagation. chemically polymerized resins have 3-5% free monomers. a greater amount of these residual monomers implies a decrease in resistance and hardness of the material1,11-13. according to rocha filho et al.12, there is an amount of residual monomer following resin polymerization that is released mainly during the first 24 h. orthodontic resins have higher levels of residual monomers compared to the conventional ones. in order to avoid any influence from the residual monomer released, which might interfere with the final physical properties of the resin, the aqueous medium in which the specimens were stored was replaced following that period of time. residual monomers, if in contact with oral mucosa, can cause local or systemic tissue reactions due to their cytotoxic activity1415. pressure during polymerization raises the amount of residual monomer as well as compromise the powder-liquid mixing ratio, mainly in orthodontics as the salt-and-pepper technique is widely used to saturate the polymer with monomer1,15-16. according to rantala et al.10 and keyf and etikan8, acrylic resins undergo a water-absorbing process depending on the medium in which they are. the greater the amount of residual monomer, the greater is the water absorption. water absorption decreases the mechanical properties of the material in the oral cavity, since water acts as a plastifying agent penetrating into the spaces between the polymeric chains and decreasing the secondary chemical bonds such as the van der waals forces1,14. with the aim of meeting the reality, we decided to store the specimens in aqueous medium before submitting them to mechanical test. it has been reported that the addition of acrylic fibers or acrylic stain to the polymerized resin through microwaves did not affect the transverse strength, and that both methods were found to be esthetically and mechanically acceptable for clinical use17. the present study also shows that addition of pigments to acrylic resins does not reduce their physical properties. although the flexural strength values are compatible with masticatory forces, price18 emphasizes that the majority of fractures in acrylic resin are not the result of fatigue because such fractures generally occur outside the oral cavity due to accidental impacts (e.g.: falls). despite the fact that these accidental impacts are not classified as a functional stress, many manufactures have considered the impact strength as being relevant factor to classify a given resin as being “virtually unbreakable” under impact18. in order to increase the fracture strength of acrylic resin, several types of fibers have been used such as aramid fibers, glass fibers, nylon fibers, and carbon fibers, all showing favorable results9,19-20. nevertheless, because such techniques are not frequently used for making orthodontic baseplates, they were not assessed in the present study. the use of a pressure container is indicated because the resin cannot come in contact with oxygen during polymerization. therefore, the reaction speed and degree of polymerization are higher than those in polymerization under air atmosphere because oxygen reacts with free radicals, which results in less porosities12-13,20. the presence of porosity not only reduces the mechanical properties of the resin, but also interferes with the cleaning of orthodontic baseplates by allowing adhesion of substances and deposition of calculus, which may promote inflammatory processes in the surrounding mucosa11,16,21. due to this factor, finishing and polishing procedures using sandpapers as well as the use of a pressure device were shown to be extremely important. however, further studies are needed, as the pressure was used on an arbitrary basis. in addition, it was raised the question on why the ortoclas selfcuring acrylic resins of yellow and green colors had higher flexural strength. although no further investigation was carried out in the present study, it is possible that the polymer might be accounted because the specimens were made using the same monomer. therefore, the reasons why ortoclas acrylic resins of yellow and green colors have higher flexural strength compared to colorless resins could not be fully understood. the differences in the flexural strengths obtained in various studies may be explained by the different methods of polymerization and storage of the specimens before test. it may be concluded that the use of pigments seems to have no significant effect on decreasing the flexural strength of self-curing acrylic resins. although two pigmented resins had higher flexural strength, other colors had mean values compatible with clinical use and can be used according to the patient’s needs. in addition, the use of pigmented resins does not interfere with the construction of the orthodontic appliance. assessment of flexural strength of two self-curing acrylic resins containing pigment 139 braz j oral sci. 8(3):137-140 acknowledgments the authors acknowledge the financial support given by cnpq, capes and faperj the authors acknowledge the financial support given by cnpq, capes and faperj references 1. faltermeier a, rosentritt m, müssig d. acrylic removable appliances: comparative evaluation of different postpolymerization methods. am j orthod dentofacial orthop. 2002; 131: 301.e16-22. 2. melsen b. removable orthodontic appliances. dent clin north am. 1981; 25: 157-76. 3. pauw g, derweduwen k, dermaut l. are removable appliances obsolete? ned tijdschr tandheelkd. 2000; 107:151-4. 4. ward s, read mj. the contemporary use of removable orthodontic appliances. dent update. 2004; 31: 215-8. 5. kanie t, fujii k, arikawa h, inoue k. flexural properties and impact strength of denture base polymer reinforced with woven glass fibers. dental mater. 2000; 16: 150-8. 6. oliveira ag, panzeri h. flexural and fatigue strength of chemically activated resin in association with a hybrid fibre. biosci j. 2004; 20: 103-12 7. elias cn, lopes hp. materiais dentários – ensaios mecânicos. são paulo: santos; 2007. 8. keif f, etikan i. evaluation of gloss changes of two denture acrylic resin materials in four different beverages. dental mater. 2004; 20: 244-51. 9. mullarky rh. aramid fiber reinforcement of acrylic appliances. j clin orthod. 1985; 19: 655-8. 10. rantala li, lastumäki tm, peltomäki t, vallittu, pk. fatigue resistance of removable orthodontic appliance reinforced with glass fibre weave. j oral rehabil. 2003; 30: 501-6. 11. mantzikos t, epstein m. interior surface sealant for acrylic appliances. j clin orthod. 1998; 32: 152-3. 12. rocha filho r, paula lv, costa vc, seraidarian pi. evaluation of residual monomer in autopolymerizing acrylic resins: spectroscopy analysis rev dent press ortod ortop facial. 2007; 12: 96-104. 13. anusavice kj. materiais dentários. 10. ed. rio de janeiro: guanabara koogan; 1998. 14. urban vm, machado al, oliveira rv, vergani ce, pavarina ac, cass qb. residual monomer of reline acrylic resins. effect of water-bath and microwave postpolymerization treatments. dental mater. 2007; 23: 363-8. 15. gonçalves ts, schmitt vm, thomas m, souza mal, menezes lm. citotoxicity of two autopolymerized acrylic resins used in orthodontics. angle orthod. 2008; 78: 926-30. 16. gonçalves ts, spohr am, souza rm, menezes lm. surface roughness of auto polymerized acrylic resin according to different manipulation and polishing methods. angle orthod. 2008; 78: 931-4. 17. silva fap, silva tbp, rached rn, del bel cury aa. effect of intrinsic pigmentation on the flexural strength of a microwave-cured acrylic resin. braz dent j. 2002; 13: 205-7. 18. price ca. the effect of cross-linking agents on the impact resistance of a linear poly(methyl methacrylate) denture-base polymer. j dent res. 1986; 65: 987-92. 19. john j, gangadhar sa, shah i. flexural strength of heat-polymerized polymethyl methacrylate denture resin reinforced with glass, aramid, or nylon fibers. j prosthet dent. 2001; 86: 424-7. 20. chung k, lin t, wang f. flexural strength of a provisional resin material with fiber addition. j oral rehabil. 1998; 25: 214-7. 21. compagnoni ma, barbosa db, souza rf, pero ac. the effect of polymerization cycle on the surface roughness in microwave-processed denture base resin. rev odontol unesp. 2005; 34: 101-6. assessment of flexural strength of two self-curing acrylic resins containing pigment140 braz j oral sci. 8(3):137-140 oral sciences n3 original article braz j oral sci. april | june 2012 volume 11, number 2 musculoskeletal disorders in upper limbs in dental students: exposure level to risk factors patrícia petromilli nordi sasso garcia1, camila pinelli2, juliana dos reis derceli3, juliana álvares duarte bonini campos1 1dds, ms, adjunt professor, department of community dentistry, araraquara school of dentistry, unesp – univ estadual paulista, araraquara, sp, brazil 2dds, ms, assistant professor doctor, department of community dentistry, araraquara school of dentistry, unesp – univ estadual paulista, araraquara, sp, brazil 3dds, master student in restorative dentistry, department of restorative dentistry, ribeirão preto school of dentistry, usp – university of são paulo, ribeirão preto, sp, brazil correspondence to: patrícia petromilli nordi sasso garcia rua humaitá 1680, centro cep:14801-903 c.p.331 araraquara, sp brasil phone: +55 16 33016405 e-mail: psgarcia@foar.unesp.br abstract aim: this study assessed the risk factors of undergraduate students to develop musculoskeletal disorders (msd) in the upper limbs, regarding gender, type of dental clinical procedure, mouth region treated, and the four-handed dentistry practice. methods: dental students enrolled in the 8th semester in the araraquara school of dentistry, unesp, brazil, were photographed while practicing 283 dental procedures. the rapid upper limb assessment (rula) method was used to evaluate the working postures of each student. the photographs were evaluated and a final risk score was attributed to each analyzed procedure. the prevalence of risk factors of developing msd was estimated by point and by 95% confidence interval. the association between the risk factor of developing disorders and variables of interest were assessed by the chi-square test with a significance level of 5%. results: the risk factors of developing msd were high, regarding most dental procedures performed by the undergraduate students (score 5: 7.07%, ci 95% : 4.0810.06%; score 6: 62.54%, ci 95% : 56.90-68.18%). there was no significant association between the rula final score and gender (p=0.559), and type of dental procedure (p=0.205), and mouth regions by arch (p=0.110) or hemi-arch (p=0.560), and the use of four-handed dentistry (p=0.366). conclusions: it can be concluded that gender, type of dental clinical procedure, mouth region treated, and practice of four-handed dentistry did not influence the risk of developing msd in the upper limbs among the dental students evaluated; however, they are at a high risk of developing such disorders. keywords: human engineering, dentistry, occupational health, students, dental. introduction due to the restricted work area, the need for manual dexterity, the long time in a sitting position, without breaks, and the needed firmness and stability of the hands, dentistry is a profession that requires prolonged periods of static muscle activity, which can lead to musculoskeletal disorders (msd)1-4. the msd related to this type of work activity are mainly in the upper limbs and may result in sick leave, reduced productivity and/or having to abandon the professional career4-6. the most common msd in dentistry are chronic low back pain, neck tension syndrome, trapezius myalgia, shoulder joint injury e carpal tunnel syndrome and upper extremity tendonitis7. de carvalho et al.8 (2001) emphasize that dentists are among those most susceptible to develop msd, such as tendonitis, synovitis, tenosynovitis and bursitis. studies have indicated a wide variety of causative factors associated with braz j oral sci. 11(2):148-153 received for publication: may 03, 2012 accepted: june 15, 2012 braz j oral sci. 11(2):148-153 149149149149149 msd in dentistry. some of these result from the physical burden of clinical work and the psychosocial factors since the beginning of a dentists’ academic training, or during their undergraduate course3,5-6,9. within the psychosocial factors one can also highlight the lack of prospects for professional growth, pressures at work, work-related negative influence on private life, the need to deal with difficult people, work routine, problems with the dental staff and work-related stress10. however, there is little research investigating the prevalence of msd among dental students2-3,5,7-8,11. therefore, given the pressures of tertiary education and the physical burden of clinical training, this seems to be an area that lacks research, which needs to be explored, since the problems may actually begin during the training period. this study was carried out to assess the level of risk factors of msd in the upper limbs of undergraduate dental students by gender, regarding gender, type of dental clinical procedure, mouth region treated, and the four-handed dentistry practice. material and methods sample design this was an observational study. all students (n=75) of both genders, enrolled in the 8th semester of the undergraduate course at the school of dentistry araraquara – unesp were invited and agreed to participate. as sampling unit, all clinical procedures performed by students (n=283) were considered over a period of two months at the integrated clinic twice a week. on average, 3.8 procedures were performed per student. the study was approved by the local ethics committee (protocol # 40/08). recording the work postures the work postures during the various clinical procedures were photographed using a canon 5.0 g5 digital camera. in order to enable viewing the process of the body regions, three key points were defined in a pilot study, and then photographed in the experiment. the pilot study was based on the definition of points to take the photographs, training the operator and intra-examiner calibration. to define the points in a simulated procedure, several photographs were taken of different regions regarding the operator and the dental chair. in order to better visualize the regions to be evaluated by the rapid upper limb assessment (rula) method, during the training of the individual responsible for the photographs, it was stipulated that the photographs should be obtained sequentially, holding the camera at about 1.5 m. from the ground, at the height of the eyes. for intra-examiner calibration, 10 undergraduate students were photographed during 50 clinical procedures, which were not included in the definitive experiment. to estimate the reliability of the angular deviation measurement by the image-tool software (uthscsa, houston tx, usa), the photographs were analyzed in duplicate, with a one-week interval between assessments. the photographs were taken by a regular dental surgeon researcher, previously trained in the pilot study, who was different from the professor in ergonomics. such procedure avoided interferences with work postures, which means that the students were not behaving artificially while the photographs were taken. the photographs were taken at least 10 min after the surgical procedures started, hence allowing the students to become comfortable in their postures2. the sequential photographic records for different parts of the body were carried out with the photographer holding the camera close to the eyes at a distance of about 1.5 m from the ground, orthogonally. although the students had previously authorized being photographed for the study, the idea was that photographer would go unnoticed and without interfering in the dentistpatient procedure. all photographs were taken and then analyzed by the dental ergonomics professor, who was previously calibrated in the pilot study (k=0.91). the imagetool software12 was used for measuring the angles of the body regions of interest for further analysis with the rula method. posture assessment method the rula method was used, according to mcatamney and corlett13 (1993). it uses a posture assessment diagram and three score tables. when associated, such score tables allow assessing the exposure to risk factors for developing msd, by using the risk scores. this method analyzes only one side of the body and produces a final risk score for it. in the present study, the side evaluated corresponded to the dominant hand of the student. in accordance with rula, the individual’s body is divided into two groups: a and b. group a corresponds to the regions of the arm, forearm and wrist, and group b corresponds to the region of the neck, trunk and legs. for each of these six segments, a score is issued, depending on the observed posture. the lowest scores are related to the positions with the least risk factors. these scores (a and b) must be added to the scores related to the way the assessed muscle group is used (a static posture maintained for longer than a minute or repeated more than four times per minute) and the load/force the muscle is subjected to (endurance, intermittent force or load of the object being manipulated). it should be noted that score 1 was standardized for the muscle and score 0 for force/load, following the recommendations of gandavadi, ramsay and burke2 (2007), as the musculature used in dental work is static, and the external load used does not exceed 2 kg. according to the rula method, the final risk scores range from 1 to 7. scores 1 to 2 are considered low risk, in other words acceptable. scores 3 to 4 represent medium risk, pending further investigations for postural changes to be undertaken in the long term. scores 5 and 6 are of high risk, requiring a prompt investigation for changes to be made in the short term. a score 7 is considered a significantly high risk for msd, that is, investigations and changes in these work postures should be performed immediately to reduce the excessive load on the musculoskeletal system and the individual’s risk of injury. musculoskeletal disorders in upper limbs in dental students: exposure level to risk factors * significant statistical difference, α=0.05 gender procedure four-handed work arch quadrant scores male female 2 p preparatory restorative / rehabilitation 2 p yes no 2 p upper lower 2 p right left 2 p group a upper arm 1 43 86 26 103 79 50 89 39 78 50 2 24 85 22 87 76 33 54 53 51 56 3 14 25 8 31 24 15 18 17 15 20 4 1 5 5.104 0,164 3 3 3.159 0,368 6 5.316 0.150 2 4 11.557 0.009* 3 3 5.924 0.115 lower arm 1 77 194 59 212 177 94 158 106 140 124 2 5 7 0.981 0.322 12 3.301 0.069 8 4 0.009 0.923 5 7 1.569 0.210 7 5 0.130 0.719 wrist 1 2 3 76 148 43 181 142 82 124 93 114 103 4 6 53 12.809 0.001* 16 43 1.776 0.183 43 16 1.857 0.173 39 20 1.540 0.215 33 26 0.215 0.643 group b neck 1 2 3 21 38 14 45 34 25 26 33 24 35 4 61 163 1.586 0.208 45 179 0.375 0.540 151 73 1.975 0.160 137 80 6.974 0.008* 123 94 4.773 0.029* trunk 1 28 55 12 71 49 34 44 39 41 42 2 19 43 16 46 39 23 32 27 23 36 3 33 95 27 101 90 38 79 45 76 48 4 2 8 2.024 0.568 4 6 5.205 0.157 7 3 3.114 0.374 8 2 4.745 0.191 7 3 9.667 0.022* legs 1 49 105 41 113 105 49 92 57 80 69 2 33 96 1.327 0.249 18 111 6.829 0.009* 80 49 1.179 0.278 71 56 0.967 0.325 67 60 0.024 0.877 χ χ χ χ χ 150150150150150 braz j oral sci. 11(2):148-153 assessing the procedures performed a final risk score was obtained for each dental procedure by analyzing the photographs of students performing various clinical procedures. the dental procedures were categorized and divided into: “preparatory”, which are those that prepare the oral cavity to receive the restorative/rehabilitation treatment (clinical examination, dental scaling and polishing, endodontic treatments, extractions and periodontal surgery), and “restorative/rehabilitation”, which are those that restore the lost dental tissue (sealing with resin materials or glass ionomer cements, provisional restorations, glass ionomer cement, amalgam and composite resin restorations) or replace lost teeth (implant-supported dentures, fixed, removable and complete dentures). all procedures were categorized and divided according to guidelines adopted by the integrated clinic course at the school of dentistry of araraquara, unesp, brazil. statistical analysis the prevalence of the exposure level to risk factors for msd in the upper limbs was estimated by point and by 95% of confidence interval. the final score of the risk obtained by rula is the dependent variable, and gender, type of procedure performed, four-handed work practice and mouth region treated are the independent variables. the association between the variables of interest was analyzed using the chi-square test (χ2). a significance level of 5% was used for all analyses. results among the 283 clinical activities, female students performed 71.02%, and most activities were restorative/ rehabilitation procedures (60.78%), performed by the fourhanded method (65.37%) and carried out in the upper arch (57.60%). table 1 shows the results of the association between the scores assigned to the positions of body segments and the variables of interest (gender, type of procedure, four-handed work practice, and mouth region treated). the body parts with less risk score were the arm and forearm, while the wrist and neck showed the highest risk scores. a significant association was observed between wrist score and gender (χ2=12.809; p=0.001), with women showing higher wrist scores. there was only significant association between the scores ascribed to the surgical procedure performed and the legs of the students surveyed (χ2=6.829; p=0.009), with higher risk scores when performing restorative/rehabilitation procedures. no statistically significant association was observed among the scores ascribed to all body segments and to the four-handed procedure. regarding the associations with the region of the mouth (“upper”/“lower” and “right”/“left”), the procedures performed in the whole mouth were excluded (n=7) because they did not allow assessing the arches (upper/lower) independently. there were a larger number of procedures with high neck scores when working in the upper region (χ2=6.974; musculoskeletal disorders in upper limbs in dental students: exposure level to risk factors table 1. distribution of scores ascribed to the body segment postures assessed by rula method according to the variables of interest. 151151151151151 p=0.008) and on the right side (χ2=4.773; p=0.029) of the mouth. regarding the arm, there was a larger number of procedures in the upper region, when compared with the lower region of the mouth (χ2=11.557; p=0.009). the trunk showed a higher score, when working on the right quadrant (χ2=9.667; p=0.022), regardless of the low or top arch. considering the dental procedures performed, the risk of msd in the upper limbs, based on the final rula score, were classified as medium in 13.07% (ic 95% : 9.14-17.00%), high in 69.61% (ic 95% : 64.25-74.97%) and very high in 17.31% (ic 95% : 12.90-21.72%) of the individuals assessed. table 2 shows the distribution of the final risk classification of msd, according to the variables of interest. there was no significant association between the risk classification and the variables of interest (gender, type of procedure performed, four-handed work practice, and the mouth region treated). discussion the risk assessment of msd in the upper limbs among dentists is important due to the specificity of the work place and the activity performed. there are several risk assessment methods for such disorders13-19. in this study, the rula method was selected for assessing the risk, since it enables a prompt and appropriate study of the individuals’ workload, regardless of their professional area2,20-21. because the body is divided into segments, according to the rula method, it is possible not only to obtain a final risk score of the individual, but also to observe the body parts that contribute to a greater or lesser intensity, in order to obtain the individual’s final risk score. thus, with the accuracy of the methodology used in rula, as well as its reliability and validity, measured by the study of mcatamney and corlett13 (1993), the important contribution of the present study for the area of ergonomics applied to dentistry is endorsed, since there is a scarcity of studies, especially among undergraduate dental students5,7-8. it was found that the wrist and neck were the body parts that had higher risk scores. this result can be explained by the fact that the dentist works in an area that is small, dark and difficult to access, with the patient lying in the dental chair and the professional making movements to use several instruments to preform procedure that require force, extension, flexion, pronation, supination and rotation of the wrists. all this increases the risk of msd3,9,22-23. moreover, despite the patient’s proper positioning in the dental chair and the dentist striving to maintain a neutral and balanced posture according to the basic requirements of an ergonomic posture4, the continuous work performed in front of or below the dentist’s eyes leads to a slight head tilt forward and down to have the best viewing angle6, which is achieved when a distance of 30 cm to 40 cm is maintained between the operator’s eyes and the patient’s mouth4. if such distance is reduced, there is a need for a greater inclination of the neck forward. if this is done over a long period of time, the cervical spine is not able to support the whole spine and muscles of the neck. also, the upper chest will constantly constrict in order to bear the weight of the head forward, hence resulting in a painful symptom pattern called neck tension syndrome6. regarding the high risk score observed in the wrist of the individuals, it should be considered that despite all technological and scientific advances, a dental surgical procedure is essentially manual, which can cause an overload on the wrists. according to fish and morris-allen24 (1998), in professions such as dentistry, which require the use of braz j oral sci. 11(2):148-153 musculoskeletal disorders in upper limbs in dental students: exposure level to risk factors final rula score variable average h i g h very high χ χ χ χ χ2 p gender male 3 57 12 female 24 140 37 1.161 0.559 procedure preparatory 9 38 12 restorative/rehabilitative 28 159 37 0.955 0.620 four-handed work yes 21 129 35 n o 16 68 14 2.008 0.366 mouth region regarding arch whole mouth 3 4 upper 17 122 24 lower 20 72 21 4.417 0.110 mouth region regarding hemiarch whole mouth 3 4 right 17 107 23 left 20 87 22 1.158 0.5604 table 2. classification of the risk of musculoskeletal disorders using the rula method according to the variables of interest. small hand tools, the permanence of the wrist joint at rest seems impossible because certain medical procedures require that the wrist remains bent for a certain amount of time. this continuous flexing of the wrist may increase the risk of professionals to be affected by the carpal tunnel syndrome25. analyzing the risk of msd in relation to gender, it was observed that female subjects had higher wrist scores than males (table 1). according to coury et al.26 (2001) men and women may develop different movement strategies to perform the same task as they have different body size, muscle strength and aerobic capacity. this assertion can justify the results obtained in this study, which raises concern. the higher load on the wrists of female subjects, combined with a greater number of domestic tasks26-27 and the influence of sex hormones on the articulation26 may render this group to be more susceptible to developing msd in the wrist when actually performing the profession. regarding the type of procedure, there was a significant association only with the legs, with greater postural constraints when performing restorative/rehabilitation work (table 1). when observing the leg risk scores, it was found that body weight was inappropriately distributed between the legs and feet of the operators in this type of procedure, which can be explained by their higher mental and physical demands when treating patients. with regard to the four-handed work practice, there was no significant association with the risk score of different body parts. an interesting fact is that marshall et al.28 observed that professionals working four-handed reported having more pain than those who did not, suggesting that this type of work was unable to prevent msd and neurological symptoms. however, the professionals assessed reported that they had not been trained in four-handed work, which might have influenced or contributed to the results obtained. regarding the association between the scores of body parts and the oral cavity region to be treated, a significant association was observed for the arm and neck scores, when compared to the arch (upper/lower) and the neck and trunk, in relation to the quadrant (right/left) (table 1). the work in the lower region presented higher arm scores, because the students were possibly working with their shoulders and/or elbows raised, a posture usually adopted when they do not position the dental chair horizontally for oral cavity procedures. specifically in the lower arch, the students tend to keep the backrest of the seat relative to the seat at an angle greater than 30 degrees because they believe that the more it is raised, the more operative space they will be able to see. however, by raising the backrest, the patient’s mouth ultimately is much higher than the operator’s elbows, even with the backrest of the dental chair fully lowered. in this situation, to have access to the operative field, the student lifts his shoulders or elbows. in the case of the upper arch, the student does not position the patient with the backrest as high as he does for the lower arch, but then the patient is not in a position where the upper occlusal plane is perpendicular to the ground, which is recommended for a direct view when working on the arch4. he then needs to change his posture, and in this case, it is not the access to the operative field that is difficult, but rather his view. thus, to facilitate a direct view of the arch, he leans his neck too far out, with this inclination often associated to the trunk. regarding the work quadrant (right/left), the right side was related to higher scores in both the neck and trunk. while it is believed that the quadrant corresponding to the dentist’s working side, the right side, enables a more appropriate working posture, it is a mistake in some situations, because if the patient is not properly positioned in the dental chair, visualization of the operative field is in fact difficult, requiring the inclination and/or torsion of the neck and trunk. by analyzing the results of the arch and the work quadrant, the main factor assumed to have influenced getting the high risk scores in some parts of the body (arms, neck and trunk) was the patient’s inadequate positioning in the dental chair, which rendered access and viewing difficult to achieve. according to valachi and valachi4 (2003) placing the patient in an awkward position can result in a prolonged static muscle tension in the neck and shoulders. as a result, the authors recommended that the patient’s positioning must be according to the mouth region to be treated. the risk scores assessment for each segment of the body enabled observing the influence of specific body parts in the final rula score. however and additionally, getting the final rula score is vital, since it generally expresses the individual’s risk to develop msd in the upper limbs over time2,13,20. a high percentage of procedures performed by dental undergraduate students was observed and classified as high risk. there was no significant association between the final rula score and the variables of interest (table 2), indicating that the difficulties intrinsic to the dental practice can lead to possible msd3,9,22-23,28-29, irrespective of the nature of work performed. another fact worth mentioning is that the wrong posture adopted by students during the course may perpetuate and worsen over time, especially when they later work as dentists5. moreover, during their activities, while the undergraduate students might be subject to all kinds of discomforts, many health problems will only become apparent over time. unfortunately, during their undergraduate course, they are more concerned with the procedure they are performing than with how they are performing it. one should also consider that although these students, still in their pre-clinical life, are educated and instructed to work in the different regions of the oral cavity using ergonomic postures, with the patient and equipment properly placed, their postures may be influenced by harmful habits acquired from different experiences, not always based on the ergonomics requirements30. this way, studies that analyze early on the prevalence and progression of msd in dental students are truly needed, because the methods the students use to acquire manual dexterity in the first years of dental school can influence how these disorders will be developed and continued in the future11. 152152152152152 braz j oral sci. 11(2):148-153 musculoskeletal disorders in upper limbs in dental students: exposure level to risk factors 153153153153153 braz j oral sci. 11(2):148-153 in conclusion, in the analysis of the different body segments and the variables of interest, there was significant association only between gender and wrist, between type of procedure and legs, and between treated mouth region, arm, neck and trunk. for most procedures performed by the undergraduate students, the final risk score using the rula method was classified as high, with no significant association between them and the gender, type of procedure performed, four-handed work practice and treated mouth region. references 1. prasad v, pratheeth g, madhumietha a. repetitive strain injuries and its incidence in practicing dentists. int j comtemp dent. 2011; 2: 6-11. 2. gandavadi a, ramsay jre, burke fjt. assessment of dental student posture in two seating conditions using rula methodology a pilot study. br dent j. 2007; 203: 601-5. 3. melis l, abou-atme ys, cottogno l, pittau r. upper body musculoskeletal symptoms in sardinian dental students. j can dent assoc. 2004; 70: 306-10. 4. valachi b, valachi k. preventing musculoskeletal disorders in clinical dentistry: strategies to address the mechanisms leading to musculoskeletal disorders. j am dent assoc. 2003; 134: 1604-12. 5. hayes mj, cockrell d, smith dr. a systematic review of musculoskeletal disorders among dental professional. int j dent hyg. 2009; 7: 59-165. 6. valachi b, valachi k. mechanisms leading to musculoskeletal disorders in dentistry. j am dent assoc. 2003; 134: 1344-50. 7. werner ra, franzblau a, gell n, hamann c, rodgers pa, caruso tj, et al. prevalence of upper extremity symptoms and disorders among dental and dental hygiene students. j calif dent assoc. 2005; 33: 123-31. 8. carvalho mvd, soriano ep, caldas jr af, campello ric, miranda hf, cavalcanti fid. work-related musculoskeletal disorders among brazilian dental students. j dent educ. 2009; 73: 624-30. 9. lindfors p, thiele u, lundberg u. work characteristics and upper extremity disorders in female dental health workers. j occup health. 2006; 48: 192-7. 10. gambhir rs, singh g, sharma s, brar r, kakar h. occupational health hazards in current dental profession – a review. open occup health saf j. 2011; 3: 57-64. 11. rinsing dw, bennett bc, hursh k, plesh o. reports of body pain 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1987; 30: 273-9. 19. seth v, weston rl, freivalds a. development of a cumulative trauma disorder risk assessment model for the upper extremities. int j ind ergon. 1999; 23: 281-91. 20. fountain ljk. examining rula’s postural scoring system with selected physiological and psychophisiological measures. int j occup saf ergon. 2003; 9: 375-84. 21. massaccesi m, pagnotta a, soccetti a, masali m, masiero c, greco f. investigation of work-related disorders in truck drivers using rula method. appl ergon. 2003; 34: 303-7. 22. biswas r, sachdev v, jindal v, ralhab s. musculoskeletal disorders and ergonomic risk factors in dental practice. indian j dent pract. 2012; 4: 70-4. 23. sarkar pa, shigli al. ergonomics in general dental practice. people´s j scient res. 2012; 5: 56-60. 24. fish dr, morris-allen dm. musculoskeletal disorders in dentists. new york state dent j. 1996; 64: 44-8. 25. hamann c, werner ra, franzblau a, rodgers pa, siew c, gruninger s. prevalence of carpal tunnel syndrome and median mononeuropahty among dentists. j am dent assoc. 2001; 132: 163-70. 26. coury hjcg, porcatti ia, alem mer, oishi j. influence of gender on work-related musculoskeletal disorders in repetitive tasks. int j ind ergon. 2001; 29: 33-9. 27. nordander c, ohlsson k, balogh i, hansson ga, axmon a, persson r, et al. gender differences in workers with identical repetitive industrial tasks: exposure and musculoskeletal disorders. int arch occup environ health. 2008; 81: 939-47. 28. nutalapati r, gaddipati r, chitta h, pinninti m, boyapati r. ergonomics in dentistry and the prevention of musculoskeletal disorders in dentists. internet j occup health. 2010; 1: 1-9. 29. garbin ají, garbin cas, diniz dg, yarid sd. dental students’ knowledge of ergonomic postural requirements and their application during clinical care. eur j dent educ. 2011; 15: 31-5. 30. gupta s. ergonomic applications to dental practice. indian j dent res. 2011; 22: 816-22. musculoskeletal disorders in upper limbs in dental students: exposure level to risk factors oral sciences n3 original article braz j oral sci. 9(2):85-88 braz j oral sci. april/june 2010 volume 9, number 2 a comparative study of p53 expression in hyperplastic , dysplastic epithelium and oral squamous cell carcinoma sucheta bansal1, keya sircar2, sanjeev kumar joshi3, sanjeet singh4, varun rastogi4 1mds, post-graduate, department of oral and maxillofacial pathology, dj college of dental sciences and research, india 2mds, professor, department of oral and maxillofacial pathology, dj college of dental sciences and research, india 3mds, department of prosthodontics including crown and bridge and implantology, dj college of dental sciences and research, india 4mds, department of oral and maxillofacial pathology, dj college of dental sciences and research, india correspondence to: sucheta bansal department of oral and maxillofacial pathology, dj college of dental sciences and research, modinagar 201204, uttar pardesh, india e-mail: suchetabansal@gmail.com received for publication: october 14, 2009 accepted: may 12, 2010 abstract aim: to study oral hyperplastic epithelium, dysplastic epithelium and squamous cell carcinoma to determine (1) the prevalence of p53 protein immunoreactivity, (2) number of p53 positive cells, and (3) the area of localization of p53 protein immunoreactivity. methods: two contiguous sections from 30 tissue specimens (10 each from oral hyperplastic epithelium, dysplastic epithelium and squamous cell carcinoma) were subjected to hematoxylin and eosin (h/e) staining for histopathological diagnosis and immunohistochemical (ihc) staining for demonstration of p53. p53 positivity was looked for in each ihc stained slide and the number of positive cells amongst 1,000 epithelial cells were recorded. the localization of these p53 positive cells within the strata (i.e. basal/suprabasal, spinous and superficial layers) of epithelium between 3 groups, and also within each group according to histological grades was recorded. results: higher p53 positive cell counts were demonstrated in oral squamous cell carcinoma compared to hyperplastic and dysplastic tissues. the expression of p53 in epithelial hyperkeratosis was mainly localized to basal epithelial cells whereas in epithelial dysplasia, it was predominantly localized to spinous epithelial cells. conclusions: qualitatively p53 is not a specific marker for malignancy of oral epithelium. however the quantitative analysis of p53 positive cells and their localization in oral epithelium is of importance as a marker for oral squamous cell carcinoma. keywords: dysplasia, p53, oral squamous cell carcinoma, oral leukoplakia. introduction oral squamous cell carcinoma constitutes the sixth most common cancer worldwide and the third most common cancer in the developing countries1. oral squamous cell carcinoma is believed to develop through sequential stages of premalignant/pre-invasive lesions: hyperplasia, mild, moderate, severe dysplasia, carcinoma in situ, and finally invasive squamous cell carcinoma2. leukoplakia and erythroplakia are recognized oral precancerous lesions and may exhibit the histopathological features of epithelial dysplasia ranging from mild to severe. the proportion of epithelial dysplastic lesions that progress to squamous cell carcinoma varies between 6.6 and 36%, and the period over which this occurs may be >20 years. higher transformation rates are quoted when lesions exhibit epithelial dysplasia3. genetic changes leading to alterations in structure, function or expression 86 braz j oral sci. 9(2):85-88 level of proteins involved in cell cycle regulation are known to be one of the key events in the malignant transformation of the tissue4. mutations of the tumor suppressor gene, particularly p53, are the most commonly identified events in various human cancers5. the grading and staging of squamous cell carcinoma is indicative of the prognosis and the clinical course of the disease. the quantitative study of p53 suggested their aberrant expression in oral cancer. p53 over expression was also detected in 15-19% of oral pre-malignant lesions, including lesions with mild dysplasia in head and neck cancer patients6. the present study was conducted to correlate the p53 expression with histological diagnosis of oral hyperplastic, dysplastic lesions and oral squamous cell carcinoma. to the best of our knowledge, there are only few published studies on similar lines reported from india found on pubmed search7. material and methods the sample selected for this study consisted of 30 tissue specimens from 30 different patients, 10 each of oral hyperplastic epithelium, dysplastic epithelium and squamous cell carcinoma. the sections were sourced from the archives of the department of oral and maxillofacial pathology, dj college of dental sciences and research, modinagar, up, india. breast carcinoma tissue section was used as positive control whereas the negative control was the normal tissue from the palatal mucosa. each tissue specimen was fixed in 10% neutral buffered formalin and processed for histopathology. in each case, two contiguous 4-µm-thick sections were cut. of these, one section was stained with h/e staining and the second was subjected to immunostaining for p53 (fig. 1-7) using the protocol proposed by abbas nf8. p53 positivity was looked for in each ihc slide. one thousand epithelial cells were counted and the number of p53 positive cells was recorded. the localization of these p53 positive cells within the strata of epithelium between hyperplastic and dysplastic groups and also within each grade of dysplastic group was noted with respect to basal/suprabasal, spinous and superficial layers. results immunohistochemical staining for p53 was found exclusively in the nuclei of epithelial cells. breast carcinoma tissue section was used as positive control whereas the fig. 1 h/e (a,10x) and ihc staining with p53 expression (b,4x) showing hyperplasia. fig. 2 h/e (a) and ihc staining with p53 expression (b) showing mild dysplasia (10x). fig. 3 h/e (a) and ihc staining with p53 expression (b) showing moderate dysplasia (10x). fig. 4 h/e (a, 10x) and ihc staining with p53 expression (b, 4x) showing severe dysplasia. fig. 5 h/e (a) and ihc staining with p53 expression (b) showing well differentiated squamous cell carcinoma (10x). fig. 6 h/e (a) and ihc staining with p53 expression (b) showing moderately differentiated squamous cell carcinoma (10x). a comparative study of p53 expression in hyperplastic , dysplastic epithelium and oral squamous cell carcinoma 87 braz j oral sci. 9(2):85-88 type of tissue p53 positive cells per 1,000 epithelial cells 0-150 151-300 301-450 451-600 hyperplastic 8 (80%) 1 (10%) 1 (10%) 0 dysplastic 3 (30%) 5 (50%) 2 (20%) 0 oral squamous cell carcinoma 4 (40%) 1 (10%) 0 5 (50%) table 1. number and percentage of patients in each group in different class intervals of p53 positive cells. type of tissue layers of epithelium basal basal & spinous superficial hyperplastic 7 3 0 dysplastic mild 2 0 0 moderate 3 3 0 severe 0 2 0 table 2. distribution of cases according to predominant localization of p53 cells in the different study groups. fig. 7 h/e (a) and ihc staining with p53 expression (b) showing poorly differentiated squamous cell carcinoma (10x). negative control was the normal tissue from the palatal mucosa. negative control did not display brown staining in epithelial or any other cells. i.e., it was negative for p53. sections of breast cancer tissue showed consistent nuclear staining of neoplastic cells. p53 positivity was observed in each histological group. p53 positive cells were counted in each group for each patient. in different class intervals, the number and percentage of patients in each group were counted (table 1). the localization of p53 positive cells in the hyperplastic and dysplastic group and also within each grade of dysplastic group were recorded (table 2). p53 immunostaining was positive in all the tissues of hyperplastic oral epithelium, dysplastic oral epithelium and oral squamous cell carcinoma. hence, there was no significant qualitative difference in p53 positivity among three groups (fig. 1-7). higher p53 positive cell counts were demonstrated in oral squamous cell carcinoma with respect to hyperplastic tissue and dysplastic tissue (fig. 8). the number of p53 positive cells increased from hyperplastic and dysplastic lesions to oral squamous cell carcinoma. however, there were no significant differences in the expression of these proteins, within a group according to grading. in this study, p53 varied in location between the three groups, and also within different grades of dysplasia. the expression of p53 in epithelial hyperplasia was mostly limited to basal and parabasal epithelial cells, whereas in the epithelial dysplasia group it also extended to the spinous strata in some cases (fig. 9a and c). in the dysplasia group, as we move from mild to moderate and to severe dysplasia, it was observed that the p53 positive cells was predominantly localized to basal strata in mild dysplasia, localized to either basal alone or together in basal and spinous strata in moderate dysplasia and predominantly localized to spinous strata in severe dysplasia (fig. 9b and c). discussion the present study was carried out in oral hyperplastic and dysplastic epithelium and squamous cell carcinoma to determine p53 protein immunoreactivity (both presence and quantification) and to determine the area of its localization in tissue strata in different grades of dysplasia. this was achieved by performing h/e and p53 (ihc) staining on two contiguous sections from each tissue specimen. in the present study, all cases in all three groups showed p53 positivity. the reasons for this might be due to small sample size or the criteria applied for qualitative analysis for determining p53 positivity9. hence, there was no significant qualitative difference in p53 positivity among the three groups. this is similar to the findings of abbas et al.8 p53 regulates cell cycle proliferation, and so all tissues may be expected to exhibit some amount of cell proliferation. hence, fig. 9 ihc staining showing basal and suprabasal position of p53 positive cells (marked by arrows) in hyperplasia (a) and mild dysplasia (b), and spinous position in severe dysplasia (c) in different layers of oral epithelium. fig. 8 ihc staining with p53 (10x) showing well differentiated squamous cell carcinoma (a) with increased number of p53 positive cells as compared to dysplastic epithelium (b). a comparative study of p53 expression in hyperplastic , dysplastic epithelium and oral squamous cell carcinoma 88 braz j oral sci. 9(2):85-88 some p53 positive cells are expected in all cases including normal tissue8,10-11. in this study, the number of p53 positive cells per 1,000 epithelial cells was found to be in the range of 0-150 in the hyperplastic group, 150-300 in the dysplastic group and more than 450 in the squamous cell carcinoma group. thus higher p53 positive cell counts were demonstrated in oral squamous cell carcinoma compared to hyperplastic tissue and dysplastic tissue suggesting that the p53 expression peaked close to the time of transition from the precancer state to cancer. several studies have shown that the proportion of cases with positive p53 expression increases from hyperplasia to dysplasia to oral squamous cell carcinoma8-10,12. the number of immunopositive cells in mild, moderate and severe grades of dysplastic tissue and in different grades of oral squamous cell carcinoma was counted. there were no significant differences in the expression of these proteins, within a group according to grading. the results of the present study were in accordance with those of abbas et al.8 and regezi et al.13, who could not find a clear correlation between grades of dysplasia and the percentage of p53 positive cells in oral premalignant lesions. in contrast, wood et al. 14 reported a significant correlation between p53 expression and grades of dysplasia. in that study, a significantly higher number of p53 positive cells were found in the lesions showing moderate or severe dysplasia than in the lesions showing mild dysplasia. the distribution patterns or localization of p53 in different strata of epithelium were also assessed in the present study. the expression of p53 in epithelial hyperplasia was localized mainly in basal and parabasal epithelial cells whereas in epithelial dysplasia, p53 positive cells occupied lower two-third (basal, suprabasal and spinous cell layers) of whole thickness of epithelium. the results of this study are in accordance with those of piattelli et al. 12 who correlated the progression of preneoplastic lesions to neoplasms with the extension of expression of p53 from suprabasal to spinous layers. within different grades of dysplasia, as we move from mild dysplasia to moderate dysplasia and then to severe dysplasia, it was observed that the location of p53 positive cells extended from suprabasal cell layer to the spinous layer, indicating the association of suprabasal staining with the increasing severity of the grades of dysplasia. the results of the present study are similar to those of nylander et al.15, who reported that the suprabasal expression of p53 could indicate the proliferative activity of the suprabasal cells, rather than just the basal cells as expected in normal epithelium. these authors also suggested that there are more chances of malignant transformation in lesions with suprabasal staining. based on the obtained results, it may be concluded that presence of p53 is not a specific marker for malignancy of oral epithelium. however, the quantitative analysis of p53 positive cells and their localization in oral epithelium may be a significant biomarker for diagnosis of oral epithelial dysplasia and squamous cell carcinoma. references 1. carlos de vicente j, junqueira gutierrez lm, zapatero ah, fresno forcelledo mf, hernández-vallejo g, lópez arranz js. prognostic significance of p53 expression in oral squamous cell carcinoma without neck node metastases. head neck 2004; 26: 22-30. 2. zhang l, rosin mp. loss of heterozygosity: a potential tool in management of oral premalignant lesions? j oral pathol med. 2001; 30: 513-20. 3. oliver rj, mac donald g, felix dh. aspects of cell proliferation in oral epithelial dysplastic lesions. j oral pathol med. 2000; 29: 49-55. 4. haas s, hormann k and bosch fx. expression of cell cycle proteins in head and neck cancer correlates with tumour site rather than tobacco use. oral oncol. 2002; 38: 618-23. 5. xu l, gimenez-conti b, cunnigham e, collet am, luna ma, lanfranchi he et al. alterations of p53, cyclind1, rb, and h-ras in human oral carcinomas related to tobacco use. cancer 1998; 83: 204-12. 6. cruz ib, snijders pjf, meijer cj, braakhuis bj, snow gb, walboomers jm et al. p53 expression above the basal cell layer in oral mucosa is an early event of malignant transformation and has a predictive value for developing oral squamous cell carcinoma. j pathol. 1998; 184: 360-8. 7. pillay m, vasudevan dm, rao cp, vidya m. p53 expression in oral cancer: observations of a south indian study. j exp clin cancer res. 2003; 22: 447-51. 8. abbas nf, labib el-sharkawy s, abbas ea, abdel monem el-shaer m. immunohistochemical study of p53 and angiogenesis in the benign and preneoplastic oral lesion and oral squamous cell carcinoma. oral surg oral med oral pathol oral radiol endod 2007; 103: 385-90. 9. iamaroon a, khemaleelakul u, pongsiriwet s, pintong j. co-expression of p53 and ki67 and lack of ebv expression in oral squamous cell carcinoma. j oral pathol med. 2004; 33: 30-6. 10. montebugnoli l, felicetti l, gissi db, cervellati f, servidio d, marchetti c et al. predictive role of p53 protein as a single marker or associated to ki67 antigen in oral carcinogenesis. open dent j. 2008; 2: 24-9. 11. kannan s, chandran gj, pillai kr, mathew b, sujathan k, nalinakumary k r et al. expression of p53 in leukoplakia and squamous cell carcinoma of the oral mucosa: correlation with expression of ki67. clin mol pathol. 1996; 49: 170-5. 12. piattelli a, rubini g, fioroni m, iezzi g, santinelli a. prevalence of p53, bcl-2 and ki-67 immunoreactivity and of apoptosis in normal oral epithelium and in premalignant and malignant lesions of the oral cavity. j oral maxillofac surg 2002; 60: 532-40. 13. regezi ja, zarbo rj, regev e, pisanty s, silverman s, gazit d. p53 protein expression in sequential biopsies of oral dysplasias and in situ carcinomas. j oral pathol med. 1995; 24: 18-22. 14. wood mw, medina je, thompson gc, houck jr, min kw. accumulation of the p53 tumor-suppressor gene product in oral leukoplakia. otolaryngol head neck surg. 1994; 111: 758-63. 15. nylander k, dabelsteen e, hall pa. the p53 and its prognostic role in squamous cell carcinomas of the head and neck region. j oral pathol med. 2000; 29: 413-25. a comparative study of p53 expression in hyperplastic , dysplastic epithelium and oral squamous cell carcinoma oral sciences n3 received for publication: october 04, 2010 accepted: may 09, 2011 braz j oral sci. 10(2):105-108 original article braz j oral sci. april | june 2011 volume 10, number 2 effect of finishing and polishing techniques on the surface roughness of a nanoparticle composite resin vera lucia schmitt1, regina maria puppin-rontani2, fabiana scarparo naufel1, danielle ludwig3, julio katuhide ueda1, lorenço correr sobrinho2 1dds, msc, phd, department of restorative dentistry, paraná west state university, brazil 2dds, msc, phd, department of restorative dentistry, dental materials division, piracicaba dental school, state university of campinas, brazil 3undergraduate student, paraná west state university, brazil correspondence to: regina maria puppin-rontani department of restorative dentistry, dental materials division, piracicaba dental school, state university of campinas av. limeira, 901 caixa postal 52 piracicaba sp brazil 13414-903 phone: +55.19.2106-5200 fax.: +55.19.3421-0144 e-mail: rmpuppin@fop.unicamp.br abstract aim: to evaluate the surface roughness of the resin filtek z350 (3m espe) after different finishing and polishing techniques. methods: sixty specimens of 7x2 mm were made and distributed in 6 groups (n=10), according to the technique employed: g1 (control) – polyester strip – no finishing or polishing; g2multi-blade burs; g3diamond burs 3195f and 3195ff; g4diamond pro discs (fgm); g5sof-lex discs (3m espe); g6robinson bristle brushes with pumice paste for 20 s and felt disc with 2-4 µm diamond paste for 30 s. the specimens were stored in artificial saliva at 37°c for 7 days. after the finishing and polishing techniques, surface roughness (ra, µm) was measured using surf-corder profilometer se 1700. data were subjected to one-way anova and tukey’s test at 5% significance level. results: g3 presented the highest surface roughness mean value (0.61). g5 presented the lowest surface roughness mean value (0.15), but it was not significantly different from g1, g4 and g6. conclusions: according to the obtained results, z350 composite resin presented the lowest surface roughness when finishing and polishing systems were used (sof-lex and diamond pro discs and robinson bristle brush with pumice plus diamond® felt disc with diamond excel® paste). the use of diamond burs (g3) resulted in the highest composite surface roughness. there was no significantly different between g1, g4, g5 and g6. keywords: dental polishing, composite resin, nanotechnology. introduction due to their broad use in dentistry since their introduction, light-activated composite resins have been constantly improved. one of the most significant improvements regarding is related to the used of nanotechnology. the new composites, named nanocomposites, have advantages such as lower polymerization shrinkage, improved mechanical properties, favored optical behavior, better brightness, extended maintenance of surface smoothness, better color stability and lower wear1-3. filtek z350 composite resin (3m espe), one of those nanoparticle composites, presents zirconia and silica particles4, with approximate size between 5-20 nm and pre-polymerized nanoclusters ranging from 0.6 to 1.4 micrometers5. the organic matrix structure and the characteristics of fillers exert a direct influence on the surface roughness and staining susceptibility of composite resins. 106 braz j oral sci. 10(2):105-108 besides the effect of composition and conversion degree, finishing and polishing procedures can also influence the surface quality of composite resins and are related to roughness and staining6-7. clinical procedures including finishing and polishing of composite resins improve esthetic results and restoration longevity. rough surfaces predispose restorations to increase of bacterial biofilm accumulation, facilitating the development of secondary caries8, discoloration and staining9, and compromise final brightness and esthetics9-10. greater surface roughness also increases the absorption of chemical components from beverages and foods, which, once retained within the previously formed bacterial biofilm, diffuse into the composite possibly affecting the formed polymer, inducing degradation11. finishing and polishing procedures require sequential use of instruments with gradual decrease in particles abrasiveness, aiming to obtaining a brighter and smoother surface12. there is no consensus in the literature regarding the effectiveness of the different systems used for finishing and polishing of composite resins. while some reports state that the use of multi-blade burs prior to abrasive discs or rubbers is a key step to achieve adequate surface smoothness13, others advocate the effectiveness of “one-step” polishing systems14-16. moreover, heintze et al.17 observed a considerable decrease on the mean surface roughness after 20 s of polishing of practically every restorative materials tested in their study and stated that increasing the polishing time did not result in significant improvements on surface smoothness17. the aim of this study was to evaluate the surface roughness of the nanocomposite resin filtek z350 (3m espe) after different finishing and polishing techniques. the tested hypothesis was that different finishing and polishing techniques provide different surface roughness on the resin. material and methods sixty specimens of filtek z350 composite resin (3m/ espe) were used (n=10). the characteristics of filtek z350 are described on table 1. the procedures were performed in a special room according to the american dental association specification #27 for direct composite resin restorations. those specifications are related only to the temperature and humidity conditions to insert the composite resin into the matrix18. a metallic matrix (2 mm high and 7 mm diameter) was used to fabricate the specimens. composite resin was inserted into the matrix, covered with clear polyester matrix strips, manufacturer composition type shade amount of particles batch # 3m espe, st paul, mn, usa nanoparticle composite a3 78.5 wt.%59 vol.% 8nw matrix: bis-gma, tegdma, udmafiller particle: silica nanofillers (5-75 nm), zirconia/silica nanoclusters (0.6-1.4 µm) table 1table 1table 1table 1table 1 characteristics of filtek z350 composite resin. pressed with glass slides and light activated following the manufacturer’s instructions using a halogen light device (optilux 501, kerr corp., orange, ca, usa). the curing tip was positioned perpendicular to specimen surface and a power output density of 620 mw/cm2 was used, as frequently monitored with a curing radiometer. the specimens were stored at 37ºc and 100% relative humidity for 7 days prior to finishing and polishing procedures. finishing and polishing systems and their respective compositions are described in table 2. the specimens were divided into 6 groups (n= 10) according to the finishing and polishing systems as described on table 3. the roughness (ra, µm) readings were carried out using a profilometer (kosaka lab. se 1700) with 0.25 mm cutoff and 0.1 mm/s speed. three consecutive measurements in different areas on the polished surface were made and an average number was calculated. the specimens were polished by a single operator in order to reduce technique variability and surface roughness was measured again immediately after polishing. data were analyzed by one-way anova and tukey’s test for individual comparisons between groups. the significance level was set at 5%. results the mean values, standard deviation and statistical comparisons for surface roughness (µm) are shown on figure 1. a one-way anova test indicated significant effects of the finishing/polishing techniques. the use of fine and ultrafine diamond burs (g3) resulted in the roughest surfaces, followed by multi-blade burs (g2), although there was no statistically significant difference between them (p>0.05). robison bristle brush with pumice plus felt disc with diamond paste (g6) did not differ significantly from control (g1), diamond pro discs (g4), sof-lex discs (g5) and multi-blade burs (g2). g1, g4 and g5 presented the smoothest surfaces and differed significantly (p<0.05) from g2 and g3. fig. 1 roughness means according to experimental groups. black vertical lines means standard deviation. different small letters mean statistically significantly difference between means. effect of finishing and polishing techniques on the surface roughness of a nanoparticle composite resin 107 braz j oral sci. 10(2):105-108 product carbide multi-blade burs fine diamond burs ultrafine diamond burs diamond pro® sequential discs sof-lex® discs diamond® felt disc diamond excel® diamond polishing paste manufacturer kg sörensen, barueri, sp, brazil kg sörensen, barueri, sp, brazil kg sörensen, barueri, sp, brazil fgm, joinville, sc, brazil 3m espe, st. paul, mn, usa fgm, joinville, sc,brazil fgm, joinville, sc, brazil composition pressed carbide 46 µm diamonds 30 µm diamonds al2o3 discs 20 µm 10 µm 5 µm 03 µm al2o3 discs 29 µm 14 µm 5 µm natural or artificial felt diamond (2 to 4 ìm) batch # 2976511 39520308 061027 2011 081720027 2011 141207 table 2table 2table 2table 2table 2 finishing and polishing systems and their respective compositions. discussion the surface roughness of composite resin is dependent on the microstructure created by the sequence of physical procedures used to modify this surface. in this study the tested hypothesis was partially accepted. different finishing and polishing techniques provided different surface roughness values. the use of clear polyester strips over the last increment of material in composite resin restorations is a usual step to avoid the oxygen inhibition layer on the resin surface. however, the resulting surface is rich in organic matrix brought about from the material, leading to a relatively unstable surface. the use of finishing and polishing techniques is essential to favor the chemical stability and improve the mechanical properties of the composite resin surface13. however, these procedures can increase surface roughness at different degrees, depending on the polishing system and material used. in the present study, the smoothest surface was obtained using diamond pro (g4), sequential sof-lex system discs (g5), and robison bristle brush with pumice plus felt disc with diamond paste (g6), but they did not differ from the surfaces obtained with use of clear polyester strips (g1 control group). these results corroborate those of yap et al. 200419. the geometric structure of the filler particles content of filtek z350 3m espe might be a possible explanation for these results. furthermore, the micromorphology of composite resin surfaces after finishing and polishing is strongly influenced by the amount, geometry and size of fillers. as the tested material is a nanoparticle composite resin, the fillers are round, smaller and more homogeneously distributed, leading to less wear (which will also be more homogeneous if it occurs)13. composite resins with smaller fillers provide “protection” to the resin matrix and consequently a better clinical performance with less wear and improved polishing11. özgünaltay et al. (2003)20 stated that sof-lex discs provide smoother surfaces and can be indicated when necessary. other discs may also provide good polishing results. the diamond pro (fgm) sequential discs (g4) provided adequate polishing, group (n=10) group 1 group 2 group 3 group 4 group 5 group 6 material polyester strip matrix no finishing or polishing multi-blade carbide burs fine (46 µm) and ultrafine (30 µm) diamond burs diamond pro® sequential discs sof-lex® system robinson bristle brush with pumice and diamond® felt disc + diamond excel® diamond polishing paste technique direct contact with surface conventional rotation, mean time of 30 s. conventional rotation, mean time of 30 s (15 s each bur). intermittent use for 15 s for each grain at low speed. air/water spraying and air drying of composite surface at each change of disc intermittent use for 15 s for each grain at low speed. air/water spraying and air drying of composite surface at each change of disc robinson bristle brush with pumice for 15 s, air/water spraying and air drying of composite surface followed by 15 s application of diamond® felt disc with diamond excel® diamond polishing paste. final air/water spraying and air drying of composite surface. table 3table 3table 3table 3table 3 distribution of groups according to the finishing and polishing systems effect of finishing and polishing techniques on the surface roughness of a nanoparticle composite resin 108 braz j oral sci. 10(2):105-108 being similar to sof-lex system discs (g5), and similar results were found in this study. group 3 (diamond polishing burs) provided the highest roughness, differing significantly different from the other groups, except for g2 (multi-blade burs). this is possibly because the diamond bur is highly wear resistant6, but it makes difficult surface leveling for the final polishing. therefore, these bur should be only used for polishing in cases where extensive removal of composite resin is required1. the combination of polishing pastes after the use of abrasives (g6) did not show different mean surface roughness values from the the diamond pro (g4) sequential discs alone or the sof-lex polishing system (g5). polishing systems like diamond pro (g4) have smaller abrasive particles and, theoretically, they should have promoted the best composite polishing in association with felt discs, providing smooth and bright surfaces. however, this fact was not observed in the present study. according to costa et al. (2007)21, it could be explained by the quality of abrasive used in each system. differences in composition and the physical properties, such hardness, are expected to influence the surface polishing more than the dimensions of abrasive particles6. bollen et al. (1997)22 stated that surface roughness greater than 0.2 µm (ra) may lead to bacterial colonization onto the restoration and increase the risk of secondary caries. ra values lower than 0.2 µm were obtained in the present study for filtek z350 3m espe composite resin in the control, soflex, diamond pro sequential discs and robinson bristle brush/pumice + diamond paste/felt disc groups. according to the obtained results, filtek z350 composite resin presented the smoothest surface when no finishing and polishing was done and when these procedures were performed with sof-lex and diamond pro al2o3 flexible discs and robinson bristle brush with pumice followed by diamond® felt disc with diamond excel® diamond polishing paste. multi-blade and diamond polishing burs (bur/point) did not promote an adequate surface smoothness. references 1. jung m, eichelberger k, klimek j. surface geometry of four nanofiller and one hybrid composite after one-step and multiple-step polishing. oper dent. 2007; 32: 347-55. 2. marghalani hy. effect of finishing/polishing systems on the surface roughness of novel posterior composites. j esthet restor dent. 2010; 22: 127-38. 3. mitra sb, wu d, holmes bn. an application of nanotechnology in advanced dental materials. j am dent assoc. 2003; 134: 1382-90. 4. anusavice jk. phillips’ science of dental materials. 11th ed. saint louis: saunders; 2003. 5. 3m dental products filtek supreme plus universal restorative system. saint paul: technical product profile; 2005. 6. jefferies sr. abrasive finishing and polishing in restorative dentistry: a state-of-the-art review. dent clin north am. 2007; 51: 379-97. 7. venturini d, cenci ms, demarco ff, camacho gb, powers jm. effect of polishing techniques and time on surface roughness, hardness and microleakage of resin composite restorations. oper dent. 2006; 31: 11-7. 8. uno s, asmussen e. marginal adaptation of restorative resin polymerized at reduced rate. scand j dent res. 1991; 99: 440-4. 9. yap auj, tan kb, bhole s. comparison of aesthetic properties of toothcolores restorative materials. oper dent. 1997; 22: 167-72. 10. hoelscher dc, neme am, pink fe, hughes pj. the effect of three finishing systems on four esthetic restorative materials. oper dent. 1998; 23: 36-42. 11. ferracane jl. elution of leachable components from composites. j oral rehabil. 1994; 21: 441-52. 12. jones cs, billington rw, pearson gj. the in vivo perception of roughness of restorations. brit dent j. 2004; 196: 42-5 13. turssi cp, ferracane jl, serra mc. abrasive wear of resin composites as related to finishing and polishing procedures. dent mat. 2005; 21: 641-8 14. ergücü z, turkün ls, aladag a. color stability of nanocomposites polished with one-step systems. oper dent. 2008; 33: 413-20. 15. ergücü z, turkün ls. surface roughness of novel resin composites polished with one-step systems. oper dent. 2007; 32:185-92. 16. korkmaz y, ozel e, attar n, aksoy g. the influence of one-step polishing systems on the surface roughness and microhardness of nanocomposites. oper dent. 2008; 33: 44-50 17. heintze sd, forjanic m, rousson v. surface roughness and gloss of dental materials as a function of force and polishing time in vitro. dent mater. 2006; 22: 146-65. 18. nihei t, kurata s, kondo y, umemoto k, yoshino n, teranaka t. enhanced hydrolytic stability of dental composites by use of fluoroalkyltrimethoxysilanes. j dent res. 2002; 81: 482-6. 19. yap auj, yap sh, teo ck, ng jj. comparison of surface finish of new aesthetic restorative materials. oper dent. 2004; 29: 100-4. 20. özgünaltay g, yazici ar, görücü j. effect of finishing and polishing procedures on the surface roughness of new tooth coloured restoratives. j. oral rehabil. 2003; 30: 218-24. 21. da costa j, ferracane j, paravina rd, mazur rf, roeder l. the effect of different polishing systems on surface roughness and gloss of various resin composites. j esthet restor dent. 2007; 19: 214-24. 22. bollen cml, lambretchts p, quirynen m. comparison of surface roughness of oral hard material to the threshold surface roughness for bacterial plaque retention: a review of the literature. dent mater. 1997; 13: 258-69. effect of finishing and polishing techniques on the surface roughness of a nanoparticle composite resin oral sciences n3 braz j oral sci. 10(3):163-166 original article braz j oral sci. july | september 2011 volume 10, number 3 received for publication: january 31, 2011 accepted: april 29, 2011 ultrastructure of buffalo tooth enamel: a possible replacement for human teeth in laboratory research luana de nazaré silva santana¹, mayara sabrina luz², nayara cristina monteiro carneiro², aline marques dias², marcia cristina dos santos guerra³, rafael rodrigues lima³ 1master student in animal science, federal university of pará, belém, pará, brazil 2undergraduate student in dentistry, federal university of pará, belém, pará, brazil 3institute of biological sciences, federal university of pará, belém, pará, brazil correspondence to: rafael rodrigues lima instituto de ciências biológicas laboratório de neuroproteção e neurorregeneração experimental. universidade federal do pará rua augusto corrêa, 1. campus do guamá cep: 66075-900. belém pará brazil phone/fax:(55) 91 3201 7891 e-mail: rafalima@ufpa.br rafaelrodrigueslima@hotmail.com abstract buffalo production takes place in several areas worldwide. in brazil, buffalo are raised mainly in the northern region, specifically in the marajó archipelago, where most of the herd is slaughtered for meat. this makes possible the extraction of numerous healthy teeth from these animals as replacements for human teeth in laboratory tests. aim: to evaluate the morphology of enamel from species bubalus bubalis as a replacement for human enamel in laboratory research studies, considering its wider availability in the amazon region. methods: after removal, the teeth were prepared for scanning electron microscopy (sem). teeth were sectioned in different planes – some were subjected to abrasion and others were merely polished for observation of surface enamel. all samples were submitted to a cleaning process, dried, sputter-coated with a platinum alloy and set for observation under sem. results: the sem micrographs revealed an aprismatic surface enamel as well as prismatic enamel, the latter being similar to human enamel, in both arrangement and morphology. conclusions: buffalo enamel showed prismatic morphology, requiring further tests to corroborate its use as a substitute for human teeth. keywords: enamel, buffalo, sem. introduction buffalo production began in brazil during the late 19th century, particularly in the marajó archipelago, located in the northern region of the country. recent estimates show that brazil has the largest bubalus bubalis herd in the americas1. the large-scale buffalo production in that area is directed mainly towards meat production, resulting in high availability of biological material for other uses, including scientific studies. currently, laboratory dental research studies are limited by the small number of healthy extracted human teeth available, as well as by the ethical aspects in obtaining them. this has led to an increase in the illegal use of human teeth in research, through postmortem extraction and illegal trade in dental organs, which goes against law 9434, from february 4th, 19972. as an alternative to these limitations, studies have been proposed using different animals, including several 164 braz j oral sci. 10(3):163-166 mammal species, to be adopted as experimental models3-5. several investigations have been carried out using teeth from different animals, such as bovines4-8, swine4,7,9, equines1011 and others. among these, bovine teeth have been most commonly used, due to easy acquisition and to the fact of having several morphological aspects similar to human teeth1213. the dental arch of a nine-month old buffalo already has 8 erupted permanent incisors and 12 premolars; adult animals have 32, with more 12 molars in the dentition1,14. due to the need for an adequate substitute for human teeth in laboratory studies, buffalo teeth can be regarded as an interesting alternative animal model. given the difficulty in using human teeth in scientific research studies, due both to access factors and ethical issues, an animal substitute as similar as possible to human teeth becomes extremely important15. therefore, the objective of this study was to evaluate the morphology of tooth enamel from buffalo species bubalus bubalis as a replacement for human enamel in laboratory studies, using scanning electron microscopy (sem). material and methods this investigation began by submitting the project to the ethics committee for animal experiment research of the federal university pará (cepaeufpa) and granting its approval under the protocol #bio013/09. biological samples were obtained from 8 male adult buffaloes (bubalus bubalis) from marajó archipelago. all samples were obtained from animals slaughtered for commercial purposes. maxillary incisors were extracted, crowns removed from the roots, and organic residues adhered to the crown surfaces were mechanically removed using a soft-bristle toothbrush, preserving tissue integrity. next, specimens were sectioned in different planes using a double-sided diamond disk set in a low-speed motor, in order to obtain enamel samples of various depths and section planes. after sectioning, the samples used to visualize surface enamel were polished with 04-µm-grain diamond paste to obtain a smoother surface. the samples selected for enamel observation in deeper planes were submitted to progressive abrasion using 1200-, 1500and 2000-grit abrasive paper, after sectioning. sections were immersed in ultrasonic bath with distilled water for 30 s. next, the samples were kept for five min in a sodium hypochlorite solution at 1% in order to remove any remaining organic material, and returned to ultrasonic bath in distilled water for 30 s. they were immersed in hcl solution at 10% for 10 s in order to remove the smear layer resulting from the cutting process. for final detritus removal, the specimens were subjected to immersion in ultrasonic bath with distilled water for 60 s. the next process consisted of dehydrating the specimens in increasing concentrations of alcohol (70%, 90% and 100%), for 5 min in each concentration. the samples were then dried at room temperature, set and sputter-coated with a platinum alloy. sem micrographs of buffalo enamel were obtained for the different regions mentioned, using a scanning electron microscope (leo-1430; carl zeiss, oberkochen, germany) under different magnifications. results the ultrastructure pattern found in buffalo enamel revealed several morphological aspects similar to those found in human enamel. as seen in figure 1, obtained from deep enamel planes, prisms were observed arranged in different directions – perpendicular and parallel to the plane in which they are viewed. this aspect is similar to the prismatic pattern of human teeth, which follow an irregular course. in figure 2, under 700x magnification obtained from deep planes, the final portion of the long axis of the rods can be seen in a region where prisms are all arranged in the same direction. figure 3 shows a crossover of rods arranged in rows with alternate distributions. this complex organization gives greater resistance, durability and protection to teeth, and is also commonly found in human tooth enamel16-17. fig. 1sem micrograph showing the different prism trajectories. in the region “a” the enamel prisms with the upward trajectory and in the region “b” the enamel prisms with two different trajectories interspersed (zone mag = 300x). fig. 2 sem micrograph showing the final portion of the long axis of the rods in a region where prisms are all arranged in the same direction (zone mag = 700x). ultrastructure of buffalo tooth enamel: a possible replacement for human teeth in laboratory research 165 braz j oral sci. 10(3):163-166 fig. 4 sem micrograph showing an area of prismatic enamel with individualized rods. arrowheads indicate the enamel prisms with individualized rods (zone mag = 1.00 kx). fig. 3 sem micrograph showing a crossover of rods marked with asterisks, giving teeth greater resistance, durability and protection (zone mag = 300x). observing the surface structure (figure 4), prismatic enamel is evidenced in individualized rods, showing their diameters. in figure 5, also from the surface plane, this individual pattern is lost, suggesting the presence of aprismatic enamel. another relevant characteristic observed in figures 4 and 5 is the existence of a mineralized tissue that circles the enamel rods, an interrod enamel sheath. from the visualization of this histological finding, the existence of interprismatic enamel in buffalo dental tissue is proposed. these aspects approximate the ultrastructural pattern of both. discussion teeth featuring microscopic morphology similar to recent vertebrates appeared approximately 460 million years ago. some agnathan fish species developed surface structures named odontoids, which were initially located outside the oral cavity. odontoids consisted of tissues similar to those fig. 5 sem micrograph showing areas suggesting the presence of aprismatic enamel, as well as interprismatic enamel surrounding the prisms. asterisks indicate four areas with considerable concentration of aprismatic enamel (zone mag = 1.00 kx). found in current vertebrates – pulp chamber, dentin, covered by hypermineralized enameloid material. the evolution of these structures is their displacement towards the inner oral cavity led to the emergence of dental elements similar to those known today. feeding habits and ecological adaptations directly influenced the acquisition of different anatomic shapes, represented by incisors, canines, premolars and molars, as well as structural modifications in dental tissues15. enamel underwent evolution processes resulting in a prismatic pattern, currently found in higher mammals. it is known that human tooth enamel is a complexity arranged hypermineralized tissue, secreted by ameloblasts (cells of ectodermic origin). its extracellular matrix consists of approximately 96% mineral material and 4% organic matter and water; inorganic content is formed basically by hydroxyapatite crystals16. the basic structural units of enamel are prisms and interprismatic substance. prisms are rod-shaped structures formed basically by ordered and densely arranged hydroxyapatite crystals. these rods, involved in interrod enamel, represent most of the thickness of dental tissue – prismatic enamel. internally (the deepest layer, next to dentin) and externally (superficially) to it, there are thin layers of aprismatic enamel, without rods16,17. a microscopic morphology similar to that found in this investigation has been described in teeth from other mammals. according to lopes et al.9 (2006), teeth from monkeys, dogs and swine show an enamel mineralization pattern similar to humans. furthermore, other authors, such as fejerskov18 (1979), limeback et al.19 (1992) and popowics, rensberger and herring20 (2001) studied the dental tissues of these mammals and found several similarities to humans, such as size, macroand microscopic morphology, and development period. bovine teeth have shown results akin to human teeth in laboratory tests21-22. schilke et al.23 (1998) performed a study on enamel morphology and did not find differences in ultrastructure of buffalo tooth enamel: a possible replacement for human teeth in laboratory research 166 braz j oral sci. 10(3):163-166 hardness, which was similar to human enamel. moreover, the ratio of organic and inorganic components is similar in both tissues. oesterle et al.12 (1998) did not observe differences in the adhesion of materials to human or bovine enamel, and attributed these findings to the similar microstructure of both substrates. however, some authors reported small differences in the behavior of human and bovine enamel in certain laboratory tests24. this demonstrates the need to continue researching new animal models, as done in the present study. buffaloes emerge as a promising species in scientific studies. this study showed that the ultrastructural morphology of buffalo enamel was similar to that of human enamel, suggesting that it may be an alternative to human teeth in enamel studies. however, further studies are needed to evaluate the behavior of buffalo enamel in tests of adhesion to restorative materials, hardness evaluation, analysis of radiographic aspects, as well as more detailed investigations of its mineral composition. references 1. santos fcf, sousa al, machado júnior aan, lima fc, ribeiro f. análise morfológica dos dentes incisivos de búfalos e sua relação com a idade de abate. cienc animal bras. 2008; 9: 506-11. 2. brasil. lei n° 9.434. dispõe sobre a remoção de órgãos, tecidos e partes do corpo humano para fins de transplante e tratamento e dá outras providências. diário oficial da união. 1997. 3. weinberg ma, bral m. laboratory animal models in periodontology. j clin periodontol. 1999; 26: 335-40. 4. fonseca rb, haiter neto f, fernandes neto aj, barbosa gas, soares cj. radiodensity of enamel and dentin of human, bovine and swine teeth. arch oral biol. 2004; 49: 919-22. 5. camargo chr, sivieiro m, camargo sea, oliveira shg, carvalho cat, valera mc. topographical, diametral and quantitative analysis of dentin tubules in the root canals of human and bovine teeth. j endod. 2007; 33: 422-6. 6. resende amr, gonçalves sep. avaliação da infiltração marginal em dentes humanos e bovinos com dois diferentes sistemas adesivos. cienc odontol bras. 2002; 5: 38-45. 7. abuabara a, santos ajs, aguiar fhb, lovadino jr. evaluation of microleakage in human, bovine and swine enamels. braz oral res. 2004; 18: 312-6. 8. fonseca rb, haiter neto f, carlo hl, soares cj, sinhoreti mac, puppin-rotani rm et al. radiodensity and hardness of enamel and dentin of human and bovine teeth, varying bovine teeth age. arch oral biol. 2008; 53: 1023-9. 9. lopes fm, markarian ra, sendyk cl, duartz cp, arana-chavez ve. swine teeth as potential substitutes for in vitro studies in tooth adhesion: a sem observation. arch oral biol. 2006; 51: 548-51. 10. muylle s, simoens p, lauwers h. the dentinal structure of equine incisors: a light and scanning electron-microscopic study. cells tissues organs. 2000; 167: 273-84. 11. muylle s, simoens p, lauwers h. the distribution of intratubular dentine in equine incisors: a scanning electron microscopic study. equine vet j. 2001; 33: 65-9. 12. oesterle l, shellhart w, belanger g. the use of bovine enamel in bonding studies. am j orthod dentofacial orthop. 1998; 114: 514-9. 13. posada mc, sanches cf, gallego gj, vargas ap, restrepo lf, lópez jf. dientes de bovino como sustituto de dientes humanos para su uso em la odontologia. rev ces odontol. 2006; 19: 63-8. 14. seixas vnc, cardoso ec, araújo cv, pereira wla, viana rb. determinação da cronologia dentária de machos bubalinos (bubalus bubalis) criados no estado do pará. cienc animal bras. 2007; 8: 529-35. 15. koussoulakou ds, margaritis lh, koussoulakos sl. a curriculum vitae of teeth: evolution, generation, regeneration. int j biol sci. 2009; 5: 22643. 16. nanci a. ten cate, histologia oral: desenvolvimento, estrutura e função. rio de janeiro: elsevier; 2008. 17. durso g, abal a. variabilidad de la morfología de los prismas del esmalte dental humano. acta microscopica. 2008; 17: 1-8. 18. fejerskov o. human dentition and experimental animals. j dent res. 1979; 58: 725-34. 19. limeback h, schlumbohm c, sen a, nikiforuk g. the effects of hypocalcemia/hypophosphatemia on porcine bone and dental hard tissues in an inherited form of type 1 pseudo-vitamin d deficiency rickets. j dent res. 1992; 71: 346-52. 20. popowics te, rensberger jm, herring sw. the fracture behavior of human and pig molar cusps. arch oral biol. 2001; 46: 1-12. 21. muench a, da silva e.m, ballester ry. influence of different dentinal substrates on the tensile bond strength of three adhesive systems. j adhesive dent. 2000; 2: 209-12. 22. moreira dm, almeida jf, ferraz cc, gomes bp, line sr, zaia aa. structural analysis of bovine root dentin after use of different endodontics auxiliary chemical substances. j endod. 2009; 35: 1023-7. 23. schilke r, bauss o, lisson ja, schuckar m, geurtsen w. bovine dentin as a substitute for human dentin in shear bond strength measurements. am j orthod dentofacial orthop. 1998; 114: 514-9. 24. nakamichi i, iwaku m, fusayama t. bovine teeth as possible substitutes in the adhesion test. j dent res. 1983; 62: 1076-81. ultrastructure of buffalo tooth enamel: a possible replacement for human teeth in laboratory research oral sciences n3 original article braz j oral sci. july/september 2010 volume 9, number 3 effect of photochemical activation of hydrogen peroxide bleaching gel with and without tio 2 and different wavelengths taciana marco ferraz caneppele1, carlos rocha gomes torres2, adriana chung3, edson hidenobu goto4, soraya consolin lekevicius4 1phd student in restorative dentistry, department of restorative dentistry, são josé dos campos dental school, são paulo state university – unesp, brazil 2phd, department of restorative dentistry, são josé dos campos dental school, são paulo state university – unesp, brazil 3msc student in restorative dentistry, department of restorative dentistry, são josé dos campos dental school, são paulo state university – unesp, brazil 4dds, department of restorative dentistry, são josé dos campos dental school, são paulo state university – unesp, brazil received for publication: september 28, 2009 accepted: august 24, 2010 correspondence to: adriana chung muniz de souza, 1020 apt 181 cep 01534-001 são paulo – sp, brazil phone: 11-8778-2457/ 12-3308-4724 e-mail: adri_chung@yahoo.com.br abstract aim: to evaluate the effect of photochemical activation of hydrogen peroxide (h 2 o 2 ) bleaching gel with different wavelengths. methods: in the study, 80 bovine incisors were used, which were stained in 25% soluble coffee and divided in 4 groups. the initial color was measured with the easy shade spectrophotometer by cie lab. an experimental 35% h 2 o 2 bleaching gel was used, either with or without the presence of titanium dioxide (tio 2 ) pigment, associated with two light sources: g1 transparent gel (tg) and no activation; g2 gel with tio 2 and activation with blue led (l=470nm)\laser (easy bleach) appliance; g3 gel with tio 2 and activation with ultraviolet (l=345nm uv); g4 tg and activation with uv. three applications of the gels were made for 10 min, and in each, 3 activations of 3 min, with interval of 30 s between them. the coloration was evaluated again and the variation in color perception (de) was calculated. the data were submitted to one-way anova and tukey’s test at 5% significance level. results: there were significant differences between g1 and g4. the greatest “e value was observed in g4 (13.37). there was no statistically significant difference (p>0.05) between the groups 2, 3 and 4. conclusions: the presence of tio 2 particules in the bleaching gel did not interfere at the bleaching results. keywords: tooth bleaching, hydrogen peroxide, color. introduction dental bleaching was introduced in dentistry in 1877. however, due to modern standards of beauty, this treatment modality has been in great demand and has been the subject of various scientific researches. therefore, this procedure has undergone many changes. nevertheless, the action mechanism remains the same, consisting of an oxide reduction reaction with release of active oxygen, free radicals in a solvent (especially water) in which the substance to be bleached donates electrons to the bleaching agent, normally hydrogen peroxide1 . due to its low molecular weight, the bleaching agent penetrates through the porosities of the enamel prisms, reaches the dentin, comes into contact with a braz j oral sci. 9(3):393-397 394 large quantity of pigmented organic molecules and breaks them up into shorter and lighter chains that allow the absorption of light in shorter wavelengths2. to accelerate this process of oxidation, various sources of energy can be used, such as heat and light3-6. the use of heat consists of contact of a hot instrument with the bleaching agent, resulting in an excessive rise in temperature of the dental structure. this may cause crack formation in the enamel and pulp damage, in addition to the risk of contact with the soft tissues and burns. that is why this technique is no longer used7. heating can also be produced by electromagnetic radiation in the infrared region. this mechanism may also exceed the limits tolerated by dental tissues, contributing to an increase in postoperative sensitivity and pulp irritation8 . with the aim of increasing the efficacy and safety of bleaching treatment, other energy sources have been tested, such as, halogen lamps. although there are filters capable of minimizing the thermal waves reaching the tooth, heating will always occur9. as light is applied for long periods in bleaching procedures, and colorants are used to intensify light absorption, there is a greater risk for the occurrence of pulp damage. the most recent technology developed involves equipment provided with led (light emitted diode) and laser based energy sources, a highly concentrated and selective form of energy, whose radiation is emitted at a specific wavelength (peak of emission of 470nm)10. as this light per se is not capable of substantially heating a transparent hydrogen peroxide (h 2 o 2 ) gel, colorants or pigments are incorporated into the gels, capable of promoting maximum absorption of this light and converting it into heat3. titanium dioxide (tio 2 ) is a white pigment of inorganic constitution, chemically inert and thermally stable, and has high power of reflectance and opacity. when present in the composition of the bleaching agent it seems to have the same function as the colorants because when it is irradiated with blue light, its electrical charge undergoes alteration resulting in destabilization of hydrogen peroxide10. another property that tio 2 seems to present is its greater absorption of light by the gel. this phenomenon consists of the capacity of the pigment to scatter light in the adjacent areas due to refraction and diffraction. however, nothing has yet been proved. moreover, tio 2 is a photocatalyzer under ultraviolet rays11-13. this interaction between uv light and tio 2 has been extensively studied for the treatment of polluted water. however, there are no reports in the literature about this interaction for dental bleaching. therefore, the aim of the present study was to evaluate the effect of photochemical activation by different wavelengths in bleaching gels with tio 2 , with the purpose of accelerating the process of pigmented component oxidation. material and methods eighty bovine incisors were used, divided into 4 groups containing 20 teeth each (n=20). the teeth were cleaned with the aid of a carborundum disk (ssw, rio de janeiro, rj, brazil), driven by a micromotor at low speed (dabi atlante, ribeirão preto, sp, brazil). the teeth were sectioned transversally at the crown 11 mm from the cementoenamel junction (cej) and at the root 3 mm from the cej, as well as longitudinally to expose dentin, with the lingual half being removed and discarded, and the vestibular half used for the study. the buccal surfaces of the specimens were submitted to prophylaxis with sodium bicarbonate (gnatus, ribeirão preto, sp, brazil), while the lingual surfaces were etched with 37% phosphoric acid (dfl, rio de janeiro, rj, brazil) for 15 s, followed by washing with a jet of water/air for 30 s to expose the dentinal tubules. afterwards the specimens were immersed in an ultrasound bath (odontobrás, ribeirão preto, sp, brazil) for 20 min. in order to have better visualization and obtain quantification of the bleaching, the teeth were submitted to a staining process. this procedure was sufficient for the teeth to attain a color close to shade c4, according to the vita classical scale (vita zahnfabrick, bad säckingen, germany). the teeth were immersed in 400 ml of a recently prepared solution of instant coffee (pilão, barueri, sp, brazil), at a concentration of 25%, and kept in an oven at 37ºc for 14 days. after this, dentin exposed by the sections was made impermeable, in order to prevent distilled water from penetrating into the dentinal tubules during the long storage period to which they were submitted, and interfere in the color. they were made impermeable by the application of two coats of transparent nail varnish (impala, são paulo, sp, brazil). next, the enamel surfaces were polished with diamond paste (fgm, joinville, sc, brazil) for polishing associated with felt disks (figure 1). then the specimens were stored individually in test tubes, with a cap containing strands of cotton imbibed with distilled water, thus providing an environment with high relative humidity, and were kept in an oven at 37°c. after staining, the color of the teeth was measured, using a vita easyshade spectrophotometer (vita zahnfabrik, germany). for this purpose, the color of each specimen was evaluated in three regions of the buccal faces of the teeth, these being the gingival, middle and occlusal thirds, on an imaginary occlusal-cervical line that passes through the center of the tooth crown. para each specimen, means of the values of l*, a* and b* were found of the three regions analyzed, so that these means represent the color for this specimen, as described below: l* mean = (l* cervical + l* middle + l* occlusal )/3 a* mean = (a* cervical + a* middle + a* occlusal )/3 b* mean = (b* cervical + b* middle + b* occlusal )/3 after this, each of the groups containing 20 teeth received the following treatments (figure 2): g135% h 2 o 2 transparent gel (total bleach – clean line, taubaté, sp, brazil) without tio 2 and no activation; g235% h 2 o 2 gel (total bleach – clean line, taubaté, sp, brazil) with tio 2 (vetec quimica fina ltda; duque de caxias, rj, brazil) and activation by the appliance with blue leds (l=470 nm)\laser (easy bleach; clean line, taubaté, sp, brazil); g335% h 2 o 2 gel (total bleach;clean line) effect of photochemical activation of hydrogen peroxide bleaching gel with and without tio 2 and different wavelengths braz j oral sci. 9(3):393-397 395 fig. 2 – experimental design. fig. 1 – specimen preparation. with tio 2 (vetec quimica fina ltda) and activation with ultraviolet light (l=345nm uv), g435% h 2 o 2 transparent gel (total bleach; clean line) without tio 2 and activation by uv; for the irradiation with uv light, it was made a darkroom wood containing the ultraviolet lamp (revoluz equi. ilum., 60w – 4a – 230v). specimens were then placed inside the box, the lid closed and then the lamp was lit by a switch located outside the box. the 35% h 2 o 2 gel used in this study was the total bleach (clean line) whose composition is 50% h 2 o 2 + thickness agent and activator solution (alkalinization solution), the tio 2 particle being introduced in the groups 2 and 3 by the manufacturer. three applications of the gels were made for 10 min, and in each application, three activations were made for 3 min, with an interval of 30 s between them. after this, coloring was again evaluated. in sequence, the variation in color after the different treatments was calculated. to evaluate the change in color after each treatment the values of the variation in l* mean were calculated . (∆l*), of a* mean. (∆a*) and of b* mean. (∆b*). the values after each bleaching procedure were subtracted from those with the darkened tooth. the variation in the composition of color or total variation in color was also calculated, designated by the sign ∆e*ab. for this purpose the values obtained after darkening and after each bleaching treatment from those with the darkened tooth were considered. the following equations were used: ∆e*ab bleached x darkened . = [(∆l* bleached darkened. )2 + (∆a* bleached darkened. )2 + (∆b* bleached darkened )2]0.5 the data were subjected to one-way anova and tukey’s test at a significance of 5%. results the anova test showed a p value of 0.013, indicating the presence of significant differences among the groups. there was a greater degree of dental bleaching in the group whose transparent h 2 o 2 gel without tio 2 was irradiated with ultraviolet light (g4). however, there was no statistically significant difference (p>0.05) between groups 2, 3 and 4, suggesting that the presence of tio 2 particles in the bleaching gel do not interfere at the bleaching degree, since irradiated. the mean values, standard deviation and the results of the tukey’s test are shown in table 1. group mean s d homogeneous sets* g1 8.28 ± 5.73 a g2 9.93 ± 6.16 a b g3 12.83 ± 5.72 a b g4 13.37 ± 4.39 b table 1. the mean values, standard deviation (sd) and results of the tukey’s test. * sets accompanied by the same letters presented no statistically significant difference at 5%. discussion the present study evaluated the effect of photochemical activation by different wavelengths of light on bleaching gels with tio 2 , with the purpose of accelerating the process of pigmented component oxidation. the shade of the teeth was measured using vita easyshade spectrophotometer (vita zahnfabrik, germany), which automatically provides the values of l*, a* and b* for each area analyzed. several studies have successfully evaluated the effectiveness of dental effect of photochemical activation of hydrogen peroxide bleaching gel with and without tio 2 and different wavelengths braz j oral sci. 9(3):393-397 396 bleaching with the use of spectrophotometers and colorimeters14. this method has the advantage of eliminating subjectivity from the analysis and providing more precise results15. this system allows the numerical definition of color and the differentiation existent between two colors, by means of the mathematical calculation of ∆e (difference in color). the advantage of the cie l*a*b* system is that the differences in color are expressed in units that can be related to visual perception and clinical significance16-17. in the present study, bleaching gels containing or not the particle of tio 2 were evaluated. even the tio 2 particle has excellent pigmentation property18, the presence of the particle did not interfere at the calculation of the color variation (∆e) once the gel was removed from the tooth surface before color measurement and the cie l*a*b* system expresses the color in units, eliminating subjectivity. at present, h 2 o 2 is the most used bleaching agent is hydrogen peroxide. in spite of its action mechanism not being completely elucidated, the basic process involves an oxide reduction reaction with release of active oxygen and free radicals in a solvent (particularly water) in which the substance to be bleached donates electrons to the bleaching agent, normally h 2 o 2 1. this process can be accelerated by various sources of energy, such as heat and light3-6. torres et al.10 presented three theories that try to explain the action of light on activating the bleaching gel: controlled heating of the gel, whose radiant energy is converted into thermal energy, causes an increase in temperature; electronic excitation of the hydrogen peroxide molecules by the photons, provides rupture of the intra and inter molecular chemical bonds and the physical-chemical action on the colorant interferes in the h 2 o 2 stability. ishibashi et al.19 incorporated the particle of tio 2 into chemical bleaching agents and provided incidence of different wavelengths of light on the gel, thus obtaining the best results in the groups whose chemical agent was hydrogen peroxide and the wavelength of 385 nm. in the present study, the association of ultraviolet light with the gel containing tio 2 (g3) produced slightly more bleaching than was produced by the gel without tio 2 and no activation by uv (g1), though without significant differences. the tio 2 is a solid, has good dielectric properties, high ultraviolet absorption, high stability beyond being a semiconductor that in normal state, presents not continuous levels of energy 18. however, several authors 20-22 have explained that when the tio 2 particle is irradiated by uv light, a photocatalytic process is started through chemical reactions on the surface of the particle, which produce oxidant radicals (o 2 and oh) that cause the chemical degradation of any compound. furthermore, ishibashi et al.18 affirmed that these reactions generate h 2 o 2 in an aqueous solution and thickening agents. romero and robles13 and rigone18 explained that when tio 2 is irradiated an electronic excitement occurs resulting in the electron promotion of the layer of valence to the layer of conduction, leading to formation of pairs electrons/gap. these gaps located in the valence band have sufficiently positive potentials in the band measured from +2,0 to +3.5 against a saturated calomel electrode, depending on the semiconductor and ph. this gap is capable to oxidate water or the oh ion at the surface of the semiconductor, leading to the formation of highly oxidant hydroxyl radicals, capable of causing the chemical degradation of any compound. this means that from the thermodynamic point of view, practically any organic composition can be oxidated when displayed to this potential13,18 however, in the present study, the gel without incorporation of the tio 2 particle irradiated by uv light (g4) presented the highest degree of dental bleaching, demonstrating the uv light has action on hydrogen peroxide, irrespective of the presence of tio 2. another important factor that could explain the better performance of uv light on gel without tio 2 seen in this study is the size of the particle used. although the concentration of the particle in the gel was the same as that recommended by ishibashi et al.19, the size of the particle used may not have been optimum for the absorption of the light. if there is excess of white pigment in the system, almost all the light that reaches the teeth would be reflected, making light penetration difficult, and consequently, preventing photocatalysis in the bleaching gel. therefore, pigment excess as well as the size of tio 2 particle might have prevented the entrance of light in the bleaching gel, thus hindering the photocatalysis. as regards the result of the gel with the particle of tio 2 activated by the appliance with blue leds (l=470nm)\laser (easy bleach) (g2) not having been much efficient for bleaching the specimens in this study, this can be explained by the selectivity of the light source used. as the led units act by the absorption of the complementary color of the bleaching gel for greater effectiveness, the color orange being complementary to blue light10, the unsatisfactory result could be explained by the lack of interaction between the light and the white gel, leading to no statistically significant difference from the gel without activation (g1). as led/laser sources of radiation are the most recent developed technology and are widely used in clinical dentistry10, in this study was used this type of radiation. however, studies show that the ultraviolet light is indicated to generate photocatalysis when tio 2 is used11-13,18-19,23. uv radiation can be classified, for the effect to the human health and the environment, in uva (400 320 nm, also called “dark light” or long wave), uvb (320-280 nm, also called average wave) and uvc (280 100 nm, also short call of uv or “antimicrobial. the wavelength used in this study was 350 nm (uva) does not cause damage to the patient’s health if some precautions are taken. the discus dental company already sells a device including uv whitening (zoom!). since the patient is protected from this radiation by the use of suitable protective clothing and eyewear, avoiding contact with skin and gingival tissues, there is no risk to the patient health. thus, for the application of uv light, certain precautions are essential and necessary. moreover, the bleaching effect of h 2 o 2 generally depends effect of photochemical activation of hydrogen peroxide bleaching gel with and without tio 2 and different wavelengths braz j oral sci. 9(3):393-397 397 on the extent to which hydroxyl radicals are generated, which can be increased by ultraviolet light (uv) irradiation. this is because h 2 o 2 has a tendency to absorb uv, which then induces the molecular vibration and degradation of h 2 o 2 , subsequently leading to an increase in temperature24. this might explain the result of the present study in which the transparent h 2 o 2 gel without tio 2 particle was irradiated with uv light (g4) increased significantly the degree of dental bleaching compared to the non-irradiated group24. it could be concluded that the presence of tio 2 particules in the bleaching gel did not interfere at the bleaching results and the use of irradiation increased the degree of dental bleaching although with no statistical difference. references 1. goldstein gr, kiremidjian-schumacher l. bleaching: is it safe and effective? j prosthet dent. 1993 69: 325-8. 2. seghi rr, denry i. effects of external bleaching on indentation and abrasion characteristics of human enamel in vitro. j dent res. 1992; 71: 1340-4. 3. baik jw, rueggeberg fa, liewehr fr. effect of light-enhanced bleaching on in vitro surface and intrapulpal temperature rise. j esthet restor dent. 2001; 13: 370-8. 4. walsh lj. safety issues relating to the use of hydrogen peroxide in dentistry. aust dent j. 2000; 45: 257-69; quiz 89. 5. goldstein re. in-office bleaching: where we came from, where we are today. j am dent assoc. 1997; 128 suppl: 11s-5s. 6. chen jh, xu jw, shing cx. decomposition rate of hydrogen peroxide bleaching agents under various chemical and physical conditions. j prosthet dent. 1993; 69: 46-8. 7. greenwall l. bleaching techniques in restorative dentistry. london: taylor & francis; 2001. 8. sun g. the role of lasers in cosmetic dentistry. dent clin north am. 2000; 44: 831-50. 9. buchalla w, attin t. external bleaching therapy with activation by heat, light or laser—a systematic review. dent mater. 2007; 23: 586-96. 10. torres crg, borges ab, kubo ch, gonçalves sep, celaschi s, s gce et al. clareamento dental com fontes híbridas led/laser. são paulo: santos; 2007. p. 9-15. 11. freitas fs, machado aedh, velani v. avaliação da atividade fotocatalítica de compósitos de dióxido de titânio e ftalocianina de zinco. vi encontro interno de iniciação científica, x seminário de iniciação científica; 2006. uberlândia: universidade federal de uberlândia; 2006. 12. dias mg, azevedo eb. degradação fotocatalítica de corantes comerciais usando luz uv e solar. 14ª semana de iniciação científica. 2005. rio de janeiro: uerj; 2005. p. 218. 13. romero ih, robles gs. tratamiento fotocatalítico de águas residuales utilizando tio2 como catalizador. 2003. available from: http: // www.csva.gov.mx/foromundial/material/mision_ultimo.pdf. acessed 2010 apr. 14. auschill tm, hellwig e, schmidale s, sculean a, arweiler nb. efficacy, side-effects and patients’ acceptance of different bleaching techniques (otc, in-office, at-home). oper dent. 2005; 30: 156-63. 15. tung ff, goldstein gr, jang s, hittelman e. the repeatability of an intraoral dental colorimeter. j prosthet dent. 2002; 88: 585-90. 16. borges ab. the influence of bleaching agents on enamel bond strength of a composite resin according to storage time. j dent res. 2003; 82: b-191. 17. bowles wh, bokmeyer tj. staining of adult teeth by minocycline: binding of minocycline by specific proteins. j esthet dent. 1997; 9: 30-4. 18. rigoni re. degradação de corante alimentício amarelo crepúsculo utilizando fotocatálise [dissertation]. erechim: universidade regional integrada do alto do uruguai; 2006. 19. ishibashi t, higahiizumi e, sotoaka r, ishihara f, kakuda m, ogasawara m et al. tooth bleaching compositions and methods of bleaching discolored tooth. united states patent us 6843981. 2005 jan 18. 20. herrmann j-m, guillard c, disdier j, lehaut c, malato s, blanco j. new industrial photocatalysts for the solar detoxification of water containing various pollutants. appl catal b: environ. 2002; 35: 281-94. 21. grzechulska j, morawski aw. photocatalytic decomposition of azo-dye acid black in water over modified titanium dioxide. appl catal b: environ. 2001; 36: 45-51. 22. tseng j, huang cp, editors. international symposium on chemical oxidation technologies for the nineties. nashville, tn: vanderbilt university; 1991. 23. suyama y, otesuki m, ogisu s, kishikawa r, tagami j, ikeda m, kurata h, cho t. effects fo light sources and visible ligth-activated titanium dioxide phtotocatalyst on bleaching. dent mater j. 2009; 28: 693-9 24. joiner a, thakker g, cooper y. evaluation of a 6% hydrogen peroxide tooth whitening gel on enamel and dentin microhardness in vitro. j dent. 2004; 32: 27-34. effect of photochemical activation of hydrogen peroxide bleaching gel with and without tio 2 and different wavelengths braz j oral sci. 9(3):393-397 oral sciences n3 braz j oral sci. 11(3):416-421 original article braz j oral sci. july | september 2012 volume 11, number 3 craniocervical posture: cephalometric and biophotogrammetric analysis priscila weber1, eliane castilhos rodrigues corrêa2, jovana m. milanesi1, juliana corrêa soares1, maria elaine trevisan3 1physical therapist, msc in human communication disorders, federal university of santa maria ufsm, rs, brazil 2professor, program of human communication disorders, department of physical therapy, federal university of santa maria ufsm, rs, brazil 3professor, department of physical therapy, federal university of santa maria ufsm, rs, brazil correspondence to: priscila weber avenida presidente vargas 1855/1101, centro cep: 97015-513, santa maria, rs – brasil phone: +55 19 99678173 e-mail: prifisio07@yahoo.com.br abstract aim: to investigate the correlation between the craniocervical posture measurements obtained by the biophotogrammetric and cephalometric analysis. methods: 80 women aged between 19 and 35 years were evaluated by the cephalometric and biophotogrammetric methods. the cephalometric variables were: cva (position of flexion/extension of the head) cpl (forward head posture) cvt/evt ratio (cervical column curvature). for the biophotogrammetric analysis, photography were analyzed in right profile being two angles related to the head forward posture (a1 and a2) and one related to flexion-extension head position (fe). it was also measured the curvature of the cervical spine by the cervical distance. the correlation between the craniocervical posture variables, as measured by the two methods of assessment, was analyzed by the pearson’s correlation with a significance level of 5%. results: it was observed a moderate and significant agreement (p=0.00) between the postural craniocervical variables that analyzed the flexionextension head position (fe and cva) and the forward head (cpl and a1). the evaluation of the cervical curvature by the dc measure showed no correlation with the cephalometric variable cvt/evt. conclusions: the biophotogrammetric analysis can be preferably chosen for assessing the head posture. however, the cephalometric analysis appears to be the most indicated for the cervical curvature measurement, since it enables a more objective view of the bone structures without the influence of the soft tissues. keywords: evaluation, posture, cephalometry, photogrammetry. introduction the applicability and the methods of craniocervical postural analysis have been investigated in the literature in a multidisciplinary way, basically by experts in the field of physical therapy, dentistry and speech therapy. there are several procedures that serve as an aid to craniocervical postural analysis, being three of them considered more populars1. the classic method is based on visual analysis, using qualitative observations of postural asymmetries, having as a reference pattern the model proposed by kendall2 (1995). regardless of the examiner’s experience, this type of measure is considered of less credibility in relation to quantitative measures3. the second proposed method, the biophotogrammetry, stands out because it represents a simple, noninvasive and low-cost method. it consists in the analysis of angles and linear measurements of points marked on the skin in some of the received for publication: june 25, 2012 accepted: september 18, 2012 braz j oral sci. 11(3):416-421 417417417417417 individual body segments. such measures can be viewed and interpreted in digital photographic records. with the advancement of technology is considered the most requested method by professionals who perform postural assessment, both for investigative slant as to aid in the clinical diagnosis and in the therapeutic approach 4. moreover, the great advantage of biophotogrammetry is to allow changes in craniocervical posture to be quantified and small changes to be detected5. currently, the most accurate craniocervical postural analysis method is the cephalometry, in which it is used the lateral radiograph of the skull and of the cervical spine to perform cephalometric measurements. by means of the radiography, it is possible a more objective visualization of the craniocervicomandibular structures, without the influence of soft tissues, as occurs in the biophotogrammetry1. however, cephalometric analysis has little impact on clinical practice because the limitation of its applicability is related to high costs and technique complexity6. recent studies have sought to investigate the correlation between measurements of posture, obtained by biophotogrammetry and by visual analysis3,5. on the other hand, there are few studies investigating the correlation between the findings obtained by the biophotogrammetric and cephalometric analyses. considering the increasing number of studies using the biophotogrammetric analysis, because of the easy access to it in the clinical practice, it is necessary to develop studies to verify the agreement of the photographic method of the postural analysis with the radiographic method, which is more objective, but more expensive and with the individual’s exposure to radiation. in other words, are biophotogrammetric measures related to head posture and does the cervical spine reflect the postural changes observed in the cephalometric analysis? for its interdisciplinary approach, it is believed that this research is of interest to dentists, physical therapists, speech therapists and other professionals since it may assist them in the reflection of their clinical practice and in the context of scientific research. material and methods eighty women with mean age of 23.8 ± 3.65 years (1935 years) and bmi of 22.4 ± 3.83 kg/cm 2 recruited voluntarily through the print media participated in this study. this research project was approved by the ethics committee in health research under the number 0048.0.243.000-08, recognized by the national research ethics (conep). all individuals who participated in the study were included by accepting and signing a free informed consent form. the exclusion criteria of the study were: history of facial and/or craniocervical trauma, presence of acute and severe musculoskeletal pain, musculoskeletal deformities (scoliosis, congenital bone malformation), craniomandibular orthopedic surgical procedures and/or neck, and a previous diagnosis of cervical disc herniation. biophotogrammetric evaluation the posture of the head and of the cervical spine was evaluated by means of digital photograph, with biophotogrammetric analysis by the postural assessment software (sapo®). pictures were taken in standing position in right lateral view. anatomic points were marked in the subject’s body with white styrofoam balls wrapped in reflective tape and previously prepared with double-sided tape for attachment to the skin. during the image acquisition, the volunteers were oriented to keep the usual body posture with opened eyes glancing to the horizon line. the participants with suitable clothing, hair tied back and barefoot were photographed in a scenario constituted by a black background of 3x1.5 meters, a plumb line suspended in the roof beside the volunteer, a base with 10x40x20 centimeters of dimensions with the foot outline drawn in a rubber rug. a tripod (vanguard® vt 131) was positioned at a distance of, at least, 3 meters from the digital camera (sony® cybershot 7.2 megapixels) and in a 1.20 meter height from the floor. in order to reproduce the natural position of the head, the individual was instructed to look at the reflection of his eyes in a mirror positioned at one meter of distance7-10. four variables related to the craniocervical posture were evaluated (figure 1). three angles measured the position of the head, being two related to the forward head posture: a1 angle formed between the tragus, spinous process of the seventh cervical vertebra (vertex) with the horizontal11-12 and a2 angle formed between the external acoustic meatus (vertex), chin and sternal notch12. the third angle was related to the flexion-extension head position (fe) and it was formed by the points marked in the spinous process of the seventh cervical vertebra, tragus (vertex) and palpebral commissure11. the smaller the a1 angle and the greater the a2 angle, the more forward the head. regarding the fe, the greater the angle, the greater the hyperextension of the head on the upper cervical spine. the curvature of the cervical spine was measured by the horizontal distance from a vertical line tangent, by the apex of the thoracic kyphosis, called as thoracic plan, and by the point of the apex of the cervical concavity13. with a one-week interval, the images of 20 individuals were randomly assigned to a second analysis of the variables to verify the intra-examiner reliability of the measurements. cephalometric evaluation subjects underwent a right lateral radiograph of the skull and of the cervical spine in a normal standing position, thus preserving the true resting position of the head and of the cervical spine. the posture adopted by the individual in the cephalometric evaluation of the right profile was the same as required for the acquisition of the photographic record, so that the natural position of the head was also reproduced. craniocervical posture: cephalometric and biophotogrammetric analysis 418418418418418 fig. 2 – craniocervical cephalometric variables. a) craniovertebral angle (cvt); b) cvt/evt angle – cervical lordosis; c) cpl/hor angle– forward head posture. fig. 1 – craniocervical biophotogrammetric variables a) angle fe – head flexion/extension of b) angle a1 – forward head posture; c) angle a2 – forward head posture the cephalometric radiography to assess the craniocervical posture was performed with the orthophos plus equipment (siemens, erlangen, germany) with the chassis positioned parallel to the face and immediately adjacent to the shoulder of the individual to ensure the visualization of c7. it was used chassis with t mat g kodak film of de 18x24 cm, with a fixed focus distance of 1.52 m and in accordance with the protocol of radiation protection in accordance with ordinance 453 of 06/01/1998 (ministry of health, department of health surveillance). all angles were traced manually by a single, previously trained examiner. the cephalograms were drawn on acetate paper with the aid of a mechanical pencil (graphite 0.3 mm) using a light box in order to enable a better visualization of the structures. a protractor to perform the angular measurements and a millimeter ruler for the linear measurements were used. in the lateral radiographic record, three variables related to the posture of the head and of the cervical spine (figure 2) were evaluated. the position of flexion/extension of the head was measured using the cranio-vertebral angle of rocabado13 (1983), recently described in the literature3,14-15. this angle is formed by the mcgregor plane (a plane that touches the base of the occipital bone to reach the posterior nasal spine) and by the odontoid process (from the apex of the odontoid process of c2 to the most anterior and inferior point of the body of c2) . the smaller the cpl-hor angle, the greater the hyperextension of the head on the upper cervical spine. the forward head posture was measured by the angle formed by the intersection of the cpl line (craniocervical postural line) in relation to the horizontal8. the cpl line reaches the central points that are demarked in the body of the first six cervical vertebrae. the smaller the cpl-hor angle, the more forward the head. for the analysis of the cervical curve the cvt/evt ratio was used15-16. the cvt line intersects the apex of the tooth of the second cervical vertebra with the most posterior-inferior point of the body of the fourth cervical vertebra, while the evt line intersects the most posterior-inferior points of the body of the fourth and sixth cervical vertebrae. the smaller the cvt/evt angle, the more rectified the cervical curvature. the variables were measured by a single examiner through manual cephalometric tracing. with a one week interval, 20 radiographs were randomly assigned to a second analysis of the variables to verify the reliability of the performed tracing. statistical analysis the intraclass correlation coefficient (icc) verifies the reliability of the cephalometric measurements. the icc values should be greater than 0.75 to indicate good reliability and craniocervical posture: cephalometric and biophotogrammetric analysis braz j oral sci. 11(3):416-421 419419419419419 cephalometric variables i c c confidence interval p cvt/evt 0.979 0.947 – 0.992 *0.000 cva 0.995 0.987 – 0.998 cpl 0.901 0.754 – 0.960 biophotogrammetric variables f e 0.976 0.941 – 0.990 *0.000 a1 0.978 0.946 – 0.991 a2 0.997 0.993 – 0.999 dc 0.974 0.936 – 0.989 table 1 – intra-examiner reliability of cephalometric and biophotogrammetric variables cva: craniovertebral angle related to the flexion/extension head position; cvt/evt cervical curvature ratio; cpl: angle related to the position of forward head posture, fe: angle related to the flexion/extension head position, a1: angle related to the forward head posture with vertex at the seventh cervical vertebra, a2: angle related to the forward head posture with vertex in the external acoustic meatus, dc: cervical distance. correlated variables r p cva x fe 0.41 0.00* cpl x a1 0.68 0.00* cpl x a2 -0.11 0.14 cvt/evt x dc 0.07 0.52 table 3 – correlation between craniocervical variables evaluated by cephalometry and biophotogrammetry cephalometric variables: cva craniovertebral angle related to the flexion/extension head position; cvt/evt cervical curvature ratio; cpl: angle related to the forward head posture. biophotogrammetric variables: fe: angle related to the flexion/ extension head position, a1: angle related to the forward head posture with vertex at the seventh cervical vertebra, a2: angle related to the forward head posture with vertex in the external acoustic meatus, dc: cervical distance, measures the cervical spine curvature; level of significance: *p<0.05, **p<0.01, pearson’s correlation coefficient. (r). biophotogrammetric variables r p a1 x a2 -0.21 0.05 a1 x fe 0.62 0.00** a2 x fe 0.23 0.01** a1 x dc -0.68 0.00** a2 x dc 0.11 0.21 dc x fe 0.38 0.00** cephalometric variables cva x cpl 0.61 0.00* evt/cvt x cva 0.33 0.06 evt/cvt x cpl 0.15 0.11 table 2 correlation between the different craniocervical postures evaluated in each method a1: angle related to the forward head posture with vertex at the seventh cervical vertebra; a2: angle related to the forward head posture with vertex in the external acoustic meatus; fe: angle related to the flexion/extension head position; dc: cervical distance, measures the curvature of the cervical spine; cva: craniovertebral angle regarding the flexion/extension head position; cpl angle related to the position of forward head posture; cvt/evt: cervical curvature ratio; level of significance: *p<0.05, **p<0.01, pearson’s correlation coefficient (r). those below 0.75 indicate poor to moderate reliability17. a descriptive analysis of the demographic variables of age, weight, height and bmi was performed. additionally, we analyzed the correlation between variables related to craniocervical posture measured by cephalometry and by biophotogrammetry as well as the correlation between postural variables measured by the two methods of evaluation. for this purpose, the pearson´s coefficient was used ranging from -1 (negative correlation, variables vary in opposite direction) to 1 (positive correlation, variables vary in the same direction). the correlation was considered strong for values of correlation coefficient (r) greater or equal than 0.7, moderate when 0.3 <r <0.7, and weak when 0 < r < 0,318. analysis were performed using the statistica 7.1 (statsoft inc., tulsa, ok, usa) and spss 17.0 (spss inc., chicago, il, usa) softwares and a significance level of 5% was considered. results results of the icc showed excellent levels of reliability for all measurements in the biophotogrammetric and cephalometric analyses by the same examiner at two different times (table 1). correlations between biophotogrammetric and cephalometric variables tested separately for each method are presented in table 2. table 3 shows the values related to the correlation between the two assessment methods of craniocervical posture. it was observed a moderate and significant agreement (p=0.00) between the craniocervical postural variables that measured the head flexion-extension position (cva and fe) and the forward head posture (cpl and a1). discussion the computerized biophotogrammetry is considered a reliable and reproducible method being an important tool in the evaluation of body posture3,19. high values of icc for the craniocervical postural variables measured by means of the manual cephalometric tracing have been demonstrated20, corroborating the excellent levels of reliability for all drawing angles, in both biophotogrammetric and cephalometric analyses obtained in the present study. recent studies have used cephalometry as a tool for analyzing the alignment of the head and cervical spine in individuals with and without temporomandibular disorder, considering the hypothesis of a possible association between craniocervical posture: cephalometric and biophotogrammetric analysis braz j oral sci. 11(3):416-421 420420420420420 craniomandibular pain with the presence of postural changes in the craniocervical segment3,14,21. during radiographic and biophotogrammetric analysis, all volunteers were instructed to maintain their habitual posture. thus, it was important to adopt natural head position in this study for faithfully representing the position of the craniocervical structures. there are different biophotogrammetric measures capable of measuring the forward head as well as the cervical spine curvature and the head flexion/ extension. in a similar study, silva et al.22 (2010) considered the use of the c7-tragus horizontal angle to characterize the forward head and the tragus-horizontal-eye angle to characterize the extension of the head. regarding the angular measurements used to assess the forward head posture through the biophotogrammetry (a1 and a2), a moderate and negative correlation between a1 and the angle of cranial flexion-extension (fe) was found. thus, the forward head position required a head extension, possibly in order to maintain the horizontality of the optical plane23. a moderate and a negative correlation between a1 and the cervical distance was found, that is, the more forward head posture (lowest angle), the greater the cervical distance. neiva et al.24 (2009) reported that the forward head is characterized by the lower cervical flexion associated with the upper cervical extension, what in part, is in agreement with the photogrammetric findings of this study. the angles a1 and a2 showed to be consistent, but with a weak correlation. in the analysis of cephalometric measurements, there were positive and moderate correlations between cva and cpl. this finding strengthens the association between the forward head and the head extension. no significant correlation regarding the variable that measured the cervical curvature was observed (cvt/evt). comparing the two different methods of assessment, the negative and moderate correlation between fe (biophotogrammetry) and cva (cephalometry) reinforces the ability of the fe angle to evaluate head flexion/extension position. furthermore, it was observed a positive and moderate correlation between a1 (biophotogrammetry) and cpl (cephalometry), confirming the ability of the a1 angle to evaluate the presence of a forward head. a positive and significant correlation between these variables was also demonstrated by visscher et al.8 (2002), who also advocated the adoption of the natural head position during radiographic and photographic recordings. similar craniocervical angular measurements were used by van niekerk et al.25 (2008) in the correlation between the photography and radiography, but in the sitting position. the authors considered the photogrammetric measurements valid and reliable for postural assessment. gadotti et al.5 (2010) considered another angle formed between chin, external auditory meatus and sternal notch (in this paper named a2) for testing the sensitivity of photogrammetry in evaluating the head posture in the sagittal plane. in the present study, this same photogrammetric measure compared with equivalent purposes used in cephalometry did not present significant correlation. rocabado13 (1983) reported that the head position on the neck related to the cervical spine curvature can be assessed by a distance (in cm) of a horizontal line, which has as reference the tangent to the apex of the thoracic spine, and the deepest point of the cervical spine. however, in this study, the dc (biophotogrammetry) showed no correlation with the cephalometric variable cvt/evt expressing the cervical lordosis. thus, it is assumed that this photogrammetric measure is best used for assessment of the forward head posture and not for the cervical curvature. it is possible to study the craniocervical posture with reliable methods and without the exposure to radiation26. based on the association found between the two methods regarding the analysis of the measures that characterized the forward head and the head flexion/extension, it can be suggested the use of the biophotogrammetric method to assess the craniocervical posture. based on the correlations obtained between the postural assessment methods, it can be concluded that computerized biophotogrammetry, apart from being reliable, seemed to be adequate to evaluate the head position. however, for analysis of the cervical curvature, it is suggested the radiographic method as a more objective view of the bone structures which are not influenced by the soft tissues. references 1. armijo-olivo s, jara x, castillo n, alfonso l, schilling a, valenzuela e, et al. a comparison of the head and cervical posture between the self-balanced position and the frankfurt method. j oral rehabil. 2006; 33: 194-201. 2. kendall f p, mccreary ek, provance pg. muscles: testing and function, with posture and pain. são paulo: manole; 1995. 3. iunes dh, bevilaqua-grossi d, oliveira as, castro fa; salgado hs. comparative analysis between visual and computerized photogrammetry postural assessment. rev bras fisioter. 2009; 13: 308-15. 4. iunes dh, castro fa, salgado hs, moura ic, oliveira as, bevilaquagrossi d. intra and inter-examiner reliability and method repeatability of postural evaluation via photogrammetry. rev bras fisioter. 2005; 9: 327-34. 5. gadotti ic, biasotto-gonzalez da. sensitivity of clinical assessments of sagittal posture. j eval clin pract. 2010; 16: 141-4. 6. bryden l, fitzgerald d. the influence of posture and alteration of function upon the craniocervical and craniofacial region. craniofacial dysfunction and pain. in: craniofacial dysfunction and pain: manual therapy, assessment, and management. london: butterworth-heinemann; 2001. p.164-71. 7. solow b, sandham a. cranio-cervical posture: a factor in the development and function of the dentofacial structures. eur j orthod. 2002; 24: 447-56. 8. visscher cm, de boer w, lobbezoo f, habets llmh, naeije m. is there arelationship between head posture and craniomandibular pain? j oral rehabil. 2002; 29 : 1030-6. 9. ciancaglini r, colombo-bolla, g, gherlone ef, radaelli g. orientation of craniofacial planes and temporomandibular disorder in young adults with normal occlusion. j oral rehabil. 2003; 30: 878-86. 10. rosa lp, moraes lc. comparative study of the influence of the conventional method and natural head posture for cephalometric lateral x-rays obtainment using the craniocervical analysis . cienc odontol bras. 2009; 12: 56-62. 11. lee wi, okeson jp, lindroth j. the relationship between forward head posture and temporomandibular disorders. j orofac pain. 1995; 9: 161-7. craniocervical posture: cephalometric and biophotogrammetric 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head posture, scapula and thoracic spine in mouth-breathing children. int j pediatr otorhinolarygol. 2009; 73: 227-36. 25. van niekerk s, louw q, vaughan c, grimmer-somers k, schereve k. photographic measurement of upper-body sitting posture of high school students: a reliability and validity study. bmc musculoskelet disord. 2008; 9: 1-11. 26. cuccia am, caradonna c. the measurement of craniocervical posture: a simple method to evaluate head position, int j pediatric othorhinolaryngol. 2009; 73: 1732-6. craniocervical posture: cephalometric and biophotogrammetric analysis braz j oral sci. 11(3):416-421 oral sciences n3 braz j oral sci. 11(2):112-115 original article braz j oral sci. april | june 2012 volume 11, number 2 clinical reproducibility of a low cost electronic apex locator nathalia vilaça soares1, emmanuel joão nogueira leal da silva2, claudio malizzia alves ferreira1, renato liess krebs3, tauby de souza coutinho-filho3 1dds, private practice 2dds, msc, phd student, department of endodontics, university of campinas, brazil 3dds, msc, phd, department of endodontics, state university of rio de janeiro, brazil correspondence to: emmanuel joão nogueira leal da silva rua herotides de oliveira, 61/902 – icaraí cep: 24230-230 niterói rj brazil phone: +55 21 83575757 e-mail: emmanuel@fop.unicamp.br abstract aim: to evaluate the clinical reproducibility of three electronic apex locators (eals), joypex 5, romiapex a-15 and novapex. methods: twenty-five patients requiring endodontic treatment were selected. sixty root canals were measured by the three eals to determine their respective working length, which was defined as a zero reading on the eal. a new k-file of the same size was used for each measurement. the file length was fixed with a rubber stop and measured with a caliper to an accuracy of 0.1 mm. data were analyzed statistically using paired t-tests with the bonferroni correction and bland-altman plot to determine the reproducibility of clinical data among the eals. significance level was set at 5%. results: mean and standard deviation values measured by the three eals showed no statistically significant differences. identical readings by all three eals were found in 38% of root canals. fifty percent differed by less than ± 1.0 mm and only 1.3% exceeded a difference of 2.0 mm. conclusions: the clinical reproducibility of the three devices was confirmed with no significant differences among them, indicating that they are effective for clinical use. keywords: apex locator, working length, working length determination. introduction the determination and maintenance of the working length (wl) is an important step in root canal treatment, ensuring an efficient chemomechanical preparation and a hermetic seal of the root canal system, both of which are necessary to avoid damage to the periradicular tissues1. several methods have been proposed for determining the length of the root canal, such the patient’s response to pain caused by the passing of the instrument through the apical foramen and the use of conventional or digital radiographs2-3. the advent of electronic apex locators (eals) provides an additional feature to the endodontic arsenal for determining the correct wl determination. the latest generation of eals operates by measuring changes in impedance (alternating current). for this, two or more different frequencies are used and processed using different mathematical algorithms4. many studies report a high efficacy achieved by new generations of eals, even in adverse situations like the presence of irrigating solutions, blood, and the existence of large foramens2-5. received for publication: january 06, 2012 accepted: may 03, 2012 braz j oral sci. 11(2):112-115 113113113113113 with the necessity of effective techniques to locate the root apex, eals are being widely used and many models have been imported and commercialized in the international market. recently, various brands of inexpensive eals were launched in the dental market. although these eals have proven accurate in determining the correct wl6, there is little evidence regarding the clinical reproducibility of these devices. reproducibility indicates whether or not two or more techniques used to measure a given variable, in identical circumstances, produce the same result7. thus it is reasonable to expect that the wl measured by an eal should be reproducible by similar eals in the same root canal under the same clinical conditions. the aim of this study was to evaluate the reproducibility of the eals joypex 5 (denjoy®, china), romiapex a-15 (romidan, israel) and novapex (forum, israel) under clinical conditions. the null hypothesis is that there is no significant difference in clinical reproducibility between the low cost eal (joypex 5) and the other trademarks. material and methods twenty-five patients from the state university of rio de janeiro, including 13 men and 12 women, aged between 18 and 60 years, were selected. informed consent was obtained from all patients, and the study was conducted in compliance with the ethical principles of the helsinki declaration and good clinical practice. inclusion criteria were pulpal pathosis with or without radiographically confirmed periapical lesions in incisors, canines, premolars, and molars. teeth with previous endodontic treatment, internal or external root resorption, and/or intracanal calcification were excluded. patients with active systemic disease and physical or mental disability were also excluded. thirty-one teeth were treated including 11 incisors, 7 premolars and 13 molars, totaling 60 root canals. in each case, the diagnosis was based on the patient’s history, clinical examination, and radiographs. local anesthesia was applied in all cases. access cavities were prepared with a round diamond bur (dentsply maillefer, ballaigues, switzerland) and refined with a endo z bur (dentsply maillefer) using a high-speed handpiece. where necessary, all remaining occlusal cusps were flattened using a high-speed conical diamond bur (dentsply maillefer) to achieve a flat reference plane. after verifying the initial patency of the root canal with a size 10 k-file (dentsply maillefer,), pulp tissue was removed using 5.25% sodium hypochlorite as an irrigant. the eals were used in the following sequence: joypex 5, romiapex a-15, and novapex. in total, 180 measurements in 60 root canals were undertaken by the same experienced operator. the wl was defined as the most apical point of the root canal and was registered when the “0” or “ou” appeared on the eal display. the wl was determined by inserting a k-file into the root canal until any of the afor ementioned marks were reached and remained stable for at least 5s. the file was then removed and the measurement made with a digital caliper (mitutoyo, suzano, sp, brazil). data were analyzed statistically using paired t-tests with the bonferroni correction for multiple comparisons in order to control the overall type i error rate at 0.05. the blandaltman plots were used to determine inter-eal reproducibility. statistical analysis was performed using spss (spss inc., chicago, il, usa). results three measurements were performed for each canal, one with each eal. table 1 shows mean and standard deviation (sd) values of absolute differences between pairs of the tested eals. there were no significant differences between tested eals (p>0.05; paired t-tests with the bonferroni correction). pairs of eals mean absolute sd absolute difference (mm) difference (mm) joypex vs.romiapex 0.41a 0.48 joypex vs.novapex 0.40a 0.44 romiapex vs. novapex 0.45a 0.46 table 1. mean and standard deviation (sd) values of absolute differences among the three electronic apex locators (eals) compared in pairs. same letters indicate no statistically significant difference (p>0.05) table 2 shows the difference between the wl values measured by different pairs of eals, with a high reliability between the eals. in 38% of the canals full precision was achieved (difference of 0.0 mm) and, only in three cases, a difference greater than 2.0 mm was verified. pairs of eals 0 <0.5 <1.0 <1.5 <2.0 joypex x romiapex 23 (38%) 16 (26%) 15 (25%) 4 (6%) 2 (3%) joypex x novapex 22 (36%) 20 (33%) 13 (21%) 5 (8%) 0 (0%) romiapex x novapex 24 (40%) 10 (16%) 18 (30%) 7 (11%) 1 (1%) table 2. absolute difference distribution of working length measurements by different pairs of electronic apex locators (eals). figure 1 shows bland-altman plots of the differences in wl measurements in all patients with mean values and limits of agreement. limits of agreement indicated the range that contained 98% of the differences between pairs of eals and were between 0 and 2.0 for the three pairs of eals. blandaltman plots with differences randomly scattered around a mean value close to zero confirmed the reproducibility of the three eals. a random scatter of points along the x-axis on all three plots indicated that the inter-eal reproducibility was independent of the length of the root canals. a random scatter of points along the y-axis on all the three plots indicated that none of the three eals showed predominantly longer or shorter wl measurements compared to the other eals. clinical reproducibility of a low cost electronic apex locator 114114114114114 braz j oral sci. 11(2):112-115 discussion the apical foramen location is extremely important to endodontic treatment success8. in general, 0.5-1.0 mm short to the radiographic apex is used as a reference for the foramen location and the working length. apex locators have been developed to improve the accuracy of apical constriction determination and consequently increase the success rates of endodontic treatment2-5,9. several studies have reported the accuracy of eals in determining the wl, validating its clinical use as a simple and effective alternative and optimizing endodontic therapy25,9-11. although these studies have showed the clinical efficacy of eals, the cost of this technology hinders their wider dissemination and use. recently, new eals were released fig. 1. bland-altman plots of differences in wl measurements in all teeth. (a) joypex x romiapex (b) joypex x novapex (c) romiapex x novapex. x-axis shows the mean length (mm) of a certain root canal measured by each pair of electronic apex locators. with more accessible prices. a previous study has shown the efficacy of these eals in vitro6, but this is the first clinical study comparing the reproducibility of a less costly eal, joypex 5, in relation to other eals such as novapex and romiapex. the choice for novapex and romiapex was based on previous studies that have shown a good efficacy and accuracy of both eals12-13. all measurements were made by the same experienced and trained operator, thus eliminating the possibility of operator bias. although the issue of how the operator’s skill influences eal accuracy has not yet been described in the literature, extensive training and practice are required to ensure familiarity with these devices and consequently accurate measurements14-15. root canals with unstable readings or different initial and repeated readings were excluded from this study. only two root canals were excluded, indicating consistency of the tested eals. these results were observed in a previous study that demonstrated a high clinical reproducibility of eals12. a number of studies evaluated eals, but comparisons of results should take into account the adoption of the same parameters of apical limits and the use of similar methods. in the present study, standardization of the wl measurements involved the same file type and size, irrigant concentration, reference point, and teeth serving as their own controls. this approach is similar to that of other studies and allows certain variables to be controlled in the clinical setting12-18. the null hypothesis was upheld because no significant difference was observed in reproducibility among the tested eals. this result confirms the clinical reliability of these devices to determine the wl, as in 69 out of 180 measurements there was no difference (0.0mm) and in 92 measurements the differences were approximately ± 0.5mm and ± 1.0 mm. a previous study showed that even the same eal can have differences in the repeatability coefficient, showing different measurements values using the same sample13. also, during the measurement of files with the caliper, small differences could result is nonidentical results. in addition, only 3 cases showed a greater difference than 2.0 mm, demonstrating high reproducibility among the eals. there is no study in the literature comparing the reproducibility of joypex 5, romiapex a-15 and novapex either in vitro or in vivo, although several studies have shown the accuracy of novapex and romiapex a-1512-13,19-21. it is impossible to compare reproducibility and accuracy results as the former may be perfect even when the eals are inaccurate. furthermore, comparison of an accurate to inaccurate eal results in unacceptable reproducibility. the accuracy of each eal needs to be determined in order to differentiate which device is responsible for wide limits of agreement. the aim of study was not to determine which device was more or less accurate than others but to examine the reliability of various eals in reproducing “apex” readings under same clinical conditions. the results obtained in this study confirmed the clinical reproducibility of the eals joypex 5, romiapex a-15 and novapex. clinical reproducibility of a low cost electronic apex locator 115115115115115 braz j oral sci. 11(2):112-115 references 1. ricucci d. apical limit of root canal instrumentation and obturation. part 1. int endod j. 1998; 31: 384-93. 2. cianconi l, angotti v, felici r, conte g, mancini m. accuracy of three electronic apex locators compared with digital radiography: an ex vivo study. j endod. 2010; 36: 2003-7. 3. janner sf, jeger fb, lussi a, bornstein mm. precision of endodontic working length measurements: a pilot investigation comparing cone-beam computed tomography scanning with standard measurement techniques. j endod. 2011; 37: 1046-51. 4. nekoofar mh, ghandi mm, hayes sj, dummer pm. the fundamental operating principles of electronic root canal length measurement devices. int endod j. 2006; 39: 595-609. 5. ding j, gutmann jl, fan b, lu y, chen h. investigation of apex locators and related morphological factors. j endod. 2010; 36: 1399-403. 6. vardasca de oliveira pt, chita jj, silva pg, de vicente fs, pereira kf. accuracy of two apex locators made in china compared to root zx ii. pesq bras odontoped clin integr. 2010; 10: 83-8. 7. petrie a, sabin c. med stati at a glance. 3rd ed. oxford: wiley-brackwell; 2009. 8. kojima k, inamoto k, nagamatsu k, hara a, nakata k, morita i et al. success rate of endodontic treatment of teeth with vital and non-vital pulps. a meta-analysis. oral surg oral med oral pathol oral radiol endod. 2004; 97: 95-9. 9. grimberg f, banegas g, chiacchio l, zmener o. in vivo determination of root canal length: a preliminary report using the tri auto zx apex-locating handpiece. int endod j. 2002; 35: 590-3. 10. plotino g, grande nm, brigante l, lesti b, somma f. ex vivo accuracy of three electronic apex locators: root zx, elements diagnostic unit and apex locator and propex. int endod j. 2006; 39: 408-14. 11. ravanshad s, adl a, anvar j. effect of working length measurement by electronic apex locator or radiography on the adequacy of final working length: a randomized clinical trial. j endod. 2010; 36: 1753-6. 12. miletic v, beljic-ivanovic k, ivanovic v. clinical reproducibility of three electronic apex locators. int endod. j 2011; 44: 769-76. 13. d‘assunção fl, alburquerque ds, salazar-silva jr, dos santos vc, sousa jc. ex vivo evaluation of the accuracy and coefficient of repeatability of three electronic apex locators using a simple mounting model: a preliminary report. int endod j. 2010; 43: 269-74. 14. akisue e, gavini g, de figueiredo ja. influence of pulp vitality on length determination by using the elements diagnostic unit and apex locator. oral surg oral med oral pathol oral radiol endod. 2007; 104: 129-3. 15. renner d, grazziotin-soares r, gavini g, barletta f. influence of pulp condition on the accuracy of an electronic foramen locator in posterior teeth: an in vivo study. braz oral res. 2012; 26:106-11. 16. dunlap ca, remeikis na, begole ea, rauschenberger cr. an in vivo evaluation of an electronic apex locator that uses the ratio method in vital and necrotic canals. j endod. 1998; 24: 48-50. 17. venturi m, breschi la. a comparison between two electronic apex locators: an ex vivo investigation. int endod j. 2007; 40: 362-73. 18. elayouti a, dima e, ohmer j, sperl k, ohle c, lost c. consistency of apex locator function: a clinical study. j endod. 2009; 35: 179-81. 19. stein tj, corcoran jf, zillich rm. influence of the major and minor foramen diameters on apical electronic probe measurements. j endod. 1990; 16: 520-2. 20. olson dg, roberts s, joyce ap, collins de, mcpherson jc iii. unevenness of the apical constriction in human maxillary central incisors. j endod. 2008; 34: 157-9. 21. silveira lf, petry fv, martos j, neto jb. in vivo comparison of the accuracy of two electronic apex locators. aust endod j. 2011; 37: 70-2. clinical reproducibility of a low cost electronic apex locator oral sciences n3 case report braz j oral sci. january/march 2010 volume 9, number 1 acute myeloid leukemia: a case report with palatal and lingual gingival alterations mahesh chavan1, arun subramaniam2, hiral jhaveri3, shivaji khedkar1, sachin durkar4, ankush agrwal5 1 mds, lecturer, dept. of oral medicine and radiology dr. d. y. patil dental college and hospital, pune, india 2 mds, professor and head, dept. of oral medicine and radiology dr. d. y. patil dental college and hospital, pune, india 3 mds, lecturer, dept. of periodontics terna dental college and hospital, nerul, navi mumbai, india 4 mds, lecturer, dept. of orthodontics and dentofacial orthopedics dr. d. y. patil dental college and hospital, pune, india 5 mds, private practice in oral and maxillofacial surgery, pune, india correspondence to: mahesh chavan dept. of oral medicine and radiology, dr. d. y. patil dental college and hospital, pune 411018, india. e-mail: drmahesh_1000@yahoo.co.in received for publication: december 07, 2010 accepted: march 25, 2010 abstract acute myeloblastic leukemia (aml) is a malignant bone marrow disease. due to its high morbidity rate, early diagnosis and appropriate medical therapy are essential. dentists and physicians should be aware of the importance of recognizing oral manifestations of this systemic disease. here we report a case of gingival alterations aml. the interesting clinical findings about this case are the severe alterations of palatal and lingual gingiva with almost normal labial gingiva. the need for early diagnosis and referral of this fatal disease are also underlined. keywords: acute myeloid leukemia, gingival alterations. introduction leukemia results from the proliferation of a clone of abnormal hematopoietic (hp) cells with impaired differentiation, regulation, and programmed cell death (apoptosis). leukemic cell multiplication at the expense of normal hp cell lines causes marrow failure, depressed blood cell count (cytopenia), and death as a result of infection, bleeding, or both1. oral lesions may be the presenting feature of acute leukemias and are therefore important diagnostic indicators of the disease. such lesions may occur due to direct leukemic infiltration of tissues, or be secondary to immunodeficiency, anemia and thrombocytopenia. typical oral manifestations of acute leukemias include gingival swelling, oral ulceration, spontaneous gingival bleeding, petechiae, mucosal pallor, herpetic infections and candidosis2. aml is a clonal proliferation of immature myeloid cells. it presents with marrow failure and cytopenia. symptoms include fever, fatigue, pallor, mucosal bleeding, petechiae, and local infections1. the french-american-british (fab) classification system divides aml into 8 subtypes, m0 through to m7, based on the cell type from which the leukemia developed and its degree of maturity3. gingival infiltration represents a 5% frequency as the initial presenting complication of aml4-5. this report refers to a patient with aml who presented gingival hyperplasia, palatal ecchymoses and other related findings. case report a 35-year-old male patient was referred to the department of oral medicine after visiting two dentists with chief complaint of swollen, painful bleeding gingiva with 5 months of evolution. the patient had a habit of tobacco quid chewing and ganja smoking since the age of 15. braz j oral sci. 9(1):67-69 the patient had fever and pallor. the submandibular lymph nodes of both sides were enlarged, non-tender and freely movable. on the clinical examination, palatal and lingual gingiva (figures 1 and 2) of both the jaws appeared to be swollen, glazed, devoid of stippling and spongy in consistency. the color of the marginal and papillary gingiva was bluish black to deep purple which was indicative of necrosis. in addition, the patient had fetor oris and ulcerations, friability and bleeding of gingiva. hard and soft palatal mucosa (figure 3) showed large area of ecchymoses. interestingly, the labial gingiva (figure 4) had almost normal appearance with just a shiny and glazed surface. the local factors were not proportional to the severity. differential diagnoses of inflammatory gingival enlargement, acute leukemia, acute necrotizing ulcerative gingivitis (anug) and human immunodeficiency virus (hiv) infection were considered for this patient. the most probable clinical diagnosis of leukemia was considered for this case based on the severity and extent of gingival alterations without significant local factors like microbial dental plaque or calculus accumulation, history and duration of gingival overgrowth, gingival bleeding and the palatal ecchymoses. the patient was referred to hematological investigations. the peripheral blood smear showed elevated total white blood cells (wbc) count to 20,000/ mm3. platelet count was 40,000/ mm3 and red blood cells (rbc) were of microcytic and hypochromic type. the differential wbc count showed more than 30% of blast cells, mainly myeloblasts (figure 5), giving diagnosis of aml m2 variety, i.e. acute myeloblastic leukaemia with maturation. tests for hiv infection/aids were negative. the patient was referred to an oncologist and treatment started with chemotherapy to which he did not respond. within the following week, the patient developed high fever, diffused swelling of the neck and brownish black coating on tongue with possible necrotic ulcerations at the tip of the tongue, and died 4 weeks later. discussion the leukemias are subdivided into chronic and acute forms. chronic leukemias involve relatively well fig. 1. mandibular lingual gingiva showing enlargement and necrosis. fig. 2. maxillary palatal gingiva showing enlargement and necrosis. fig. 4. labial and buccal gingiva showing almost normal appearance. fig. 3. large area of ecchymoses on palatal mucosa. differentiated leukocytes, are slow in onset and run an indolent course. acute leukemias are characterized by an uncontrolled proliferation of poorly differentiated blast cells. they are abrupt in onset, and are aggressive and rapidly fatal 68 braz j oral sci. 9(1):67-69 acute myeloid leukemia: a case report with palatal and lingual gingival alterations if left untreated. oral manifestations are more common in acute leukaemias2. acute leukemia usually presents precipitously with bone marrow failure and associated anemia, infection, and bleeding. symptoms are generally flu-like with bone pain, joint pain, or both, caused by malignant marrow expansion. thrombocytopenia is manifested by petechial skin and posterior palatal hemorrhages and gingival bleeding. gingival infiltration by leukemic cells will also predispose the patient with leukemia to bleeding. gingival ulcerations may occur as a result of infection by normal oral flora in the setting of neutropenia1. more atypical oral findings that have been reported include cracked lips and the presence of hemorrhagic bullae on the anterior dorsum of the tongue, buccal and labial mucosa, toothache, tooth mobility and petechiae6. oral manifestations in patients with leukemia have been described in all subtypes of aml, chronic myeloid leukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia7. dreizen et al.8 reported that the patients with acute monocytic leukemia had the greatest incidence of gingival infiltrates (m5) (66.7%) followed by acute myelomonocytic leukemia (m4) (18.5%) and acute myeloblastic leukemia (m1, m2) (3.7%). the patient of this case was diagnosed as having acute myeloblastic leukemia with maturation (aml m2 variety). gingival hyperplasia is characterized by progressive enlargement of the interdental papillae, marginal gingiva and attached gingiva. it its most severe form, the tooth crowns may be covered. mucosal hemorrhages, ulcerative gingivitis, infectious gingivitis and odontalgia may be observed9. there are several etiologies for gingival overgrowth and each etiology usually has its own overgrowth characteristics. the inflammatory gingival enlargement is the most common form of gingival overgrowth and is associated with local factors, like plaque and calculus. anug typically presents with gingival necrosis and ‘punched-out’ ulceration involving the interdental papillae which are covered by a grayish-green pseudomembrane. it is also accompanied by excessive salivation, metallic taste and malodor10, but it is not associated with ecchymoses. hiv/ aids associated lesions like kaposi’s sarcoma (ks) (early lesions) can be confused with gingival enlargement and palatal ecchymoses in leukemia. ks can involve any oral site, but most commonly the palate, gingiva and tongue. ks lesions begin as blue purple or red purple flat discolorations that can progress to tissue masses that can ulcerate10. the clinical provisional diagnosis of this case was narrowed to acute leukemia considering the above-mentioned factors. leukemia cell gingival infiltrate is not observed in edentulous individuals, suggesting that local irritation and trauma associated with the presence of teeth may play a role in the pathogenesis of this abnormality8. there may be variation in presentation and severity of gingival overgrowth. this patient had gingival alterations on palatal and lingual gingiva, while the labial gingiva had an almost normal appearance with a shiny surface. the probable reason for this finding is the association of patient’s habit of ganja smoking, which is a source of constant irritation to the gingiva. petechiae, easy bruising, gingival bleeding and epistaxis are directly related to thrombocytopenia (decrease in the number of platelets). bleeding can also occur internally, with hemorrhage more likely when the platelet count is less than 20,000/ìl. in addition, these patients are prone to infections because of a decrease in circulating neutrophils. breakdown of mucosal barriers leads to the development of systemic infections from organisms colonizing the skin, throat, or gastrointestinal tract11. this patient also had a large area of ecchymoses on the palate suggestive of platelet depletion and also complained of gingival bleeding on slight provocation. infections and anemia are the major causes of death in leukemic patients10. untreated, acute leukemia has an aggressive course, with death occurring within 6 months or less1. the patient died in spite of our immediate referral because valuable time had been lost until the patient visited our department. in conclusion the fact that gingival alterations are sometimes the first manifestations of the disease implies that dental professionals must be sufficiently familiarized with the clinical manifestations of systemic diseases to ensure prompt detection and referral. considering the acuteness of this disease, early diagnosis and referral of leukemic patients should be done for better outcome of this fatal condition. references 1. mckenna sj. leukemia. oral surg oral med oral pathol oral radiol endod. 2000; 89: 137-9. 2. ak dean, jw ferguson, es marvan. acute leukaemia presenting as oral ulceration to a dental emergency service. aust dent j. 2003; 48: 195-7. 3. bennett j, catovsky d, daniel m, flandrin g, galton d, gralnick h, sultan c. proposals for the classification of the acute leukaemias. french-americanbritish (fab) co-operative group. br j haematol. 1976; 33: 451-8. 4. williams wj, beutler e, erslev aj, lichtman ma. hematology. 4th ad. new york: mcgraw hill; 1990. p.243-4. 5. wu j, fantasia je, kaplan r. oral manifestations of acute myelomonocytic leukemia: a case report and review of the classification of leukemias. j periodontol. 2002; 73: 664-8. 6. p gleeson. spontaneous gingival haemorrhage: case report. aust dent j. 2002; 47: 174-5. 7. bergmann oj, philipsen hp, ellegaard j. isolated gingival relapse in acute myeloid leukaemia. eur j hematol. 1988; 40: 473-6. 8. dreizen s, mccredie kb, keating mj, luna ma. malignant gingival and skin infiltrates in adult leukemia. oral surg oral med oral pathol. 1983; 55: 572 -9. 9. curtis l. cooper, ruth loewen, tsiporah shore. gingival hyperplasia complicating acute myelomonocytic leukemia. j can dent assoc. 2000; 66: 78-9. 10. greenburg m, glick m. burket’s oral medicine diagnosis and treatment. 10th edn. bc decker inc.; 2003. p.443-8. 11. parisi e, draznin j, stoopler e, schuster sj, porter d, sollecito tp. acute myelogenous leukemia: advances and limitations of treatment. oral surg oral med oral pathol oral radiol endod. 2002; 93: 257-63. 69 braz j oral sci. 9(1):67-69 acute myeloid leukemia: a case report with palatal and lingual gingival alterations fig. 5. peripheral blood smear showing myeloblasts. oral sciences n3 braz j oral sci. 10(4):262-267 original article braz j oral sci. october | december 2011 volume 10, number 4 effect of light-curing units on gap formation and microleakage of class ii composite restorations giovana mongruel gomes1 , bruna fortes bittencourt1 , gibson l. pilatti2 , joão carlos gomes3, osnara maria mongruel gomes3 , abraham lincoln calixto3 1dds, ms, phd student, department of restorative dentistry, school of dentistry, state university of ponta grossa, brazil 2dds, ms, phd, professor, department of periodontology, school of dentistry, state university of ponta grossa, brazil 3dds, ms, phd, professor, department of restorative dentistry, school of dentistry, state university of ponta grossa, brazil correspondence to: giovana mongruel gomes rua engenheiro schamber 452, ap 21 ponta grossa, pr, brasil 84010-340 phone: +55-42-32226560 fax: +55-42-32247351 e-mail: giomongruel@gmail.com abstract aim: this in vitro study evaluated gap width formation and marginal microleakage in class ii composite restorations light-cured with three different light-curing units. methods: standardized cavities in the proximal surfaces of 36 human third molars were made with margins located below the cementoenamel junction. cavities were restored with filtek p60 (3m espe), inserted with a photocondenser tip and light-cured with three different methods: gi optilux401 (halogen); gii – coltoluxled (led) and giii –ultralumeled5 (led). after finishing the restorations, teeth were subjected to a thermal cycling regimen of 500 cycles (5oc ± 2oc and 55oc ± 2oc), totalizing 500 cycles. thereafter, the teeth were sectioned in a buccolingual direction and in the center of the restorations. half of the specimens (18) were used to evaluate marginal microleakage, by measuring of dye penetration in cross-sectioned specimens, and the other half was used to analyzed the gap formation width by sem observations (1000x). data were submitted to kruskal-wallis (α=0.05). results: the mean values of gap width (µm) were: gi 3.28±3.34; gii 1.48±1.89 and giii 3.11±3.45, and microleakage was not affected by the light-curing units. conclusions: there were no differences between the light-curing methods in gap formation and marginal microleakage. keywords: composite resins, dental curing lights, polymerization. introduction light-cured composite remains in focus since its introduction in dentistry in the 1960’s1. its importance is due to several factors, among which aesthetics is considered essential. however, the adhesion between composite resin and dental tissues remains a challenge for dental practice after all these years2-5. resin-based materials have an inherent characteristic, which is due to the polymerization of monomers to polymers1. when this strength exceeds the adhesive bond strength to cavity walls, marginal gaps may appear between restoration and tooth6-7, predisposing the restoration to marginal infiltration8. a range of undesirable effects that include penetration of bacteria and oral fluids can result in staining, recurrent caries, postoperative sensitivity and ultimately irreversible pulp pathologies2, thereby decreasing the longevity of the restoration2,9. several techniques have been proposed to reverse the problems caused by polymerization shrinkage of resin, including the use of an adhesive layer with sufficient bond strength and elasticity modulus to withstand the stresses transmitted received for publication: august 01, 2011 accepted: november 10, 2011 braz j oral sci. 10(4):262-267 263 to this interface10-11, as well as the use of the so-called lowshrink composites currently available in the dental market12-16. the complexity of the factors that determine the stress generated by polymerization shrinkage is so great, that it could also be cited other contributing factors: cavity configuration1718, placement technique8,17, radiant exposure (dose)19, curing techniques 17-18,20 and inherent properties of resin-based materials, such as the amount of filler load12,17-21. it is known that resin composite restorations with enamel margins have higher bond strength values than those with margins located in dentin, because this substrate is more critic22. among different light-curing units (lcus) developed for photoactivation of resin-based materials, quartz-tungsten halogen (qth) lamps and light-emitting diodes (leds) are still the most common devices. these lcus emit blue light close to the absorption spectrum of camphorquinone (468 nm), the m ost common photoinitiator in resin-based materials23. despite their popularity, halogen lamps have some disadvantages, such as accelerated degradation of the internal components, due to overheat generated by the lamp, requiring frequent replacement. leds are smaller, cordless and do not require filters. there is no heat output, so a cooling fan is not needed. however, they cost more than conventional halogen lamps and their battery must be recharged24. studies have showed the lcus may interfere on staining susceptibility and conversion degree of composites25, as well as in the amount of residual monomers found in adhesives26. on the other hand, the literature has reported no significant differences on the color stability27, conversion degree28 and surface energy of various composite resins after curing by led or halogen devices29. several studies have compared microleakage and gap formation in class ii composite restorations and the influence of different lcus17,22,30-31, but with no consensus between the authors. gap formation and leakage studies have been used as in vitro indicators of both retention and marginal sealing abilities of composite restorations22. thus, the objective of this study was to evaluated gap formation and microleakage of class ii composite restorations light-cured with three different lcus (one halogen lamp and two led units). the null hypothesis tested was that lcus do not influence gap formation or microleakage of class ii composite restorations. material and methods specimen preparation and restorative procedures this study was approved by the ethics committee (coep) of the state university of ponta grossa – uepg, under the reports number 08/2005 (microleakage analysis) and 09/2005 (gap measurement) and protocols number 01211/ 05 and 01212/05, respectively. thirty-six sound human third molars extracted for orthodontic reasons, with no defects, were selected for this study. immediately after extraction they were hand scaled to remove tissue remnants and stored in distilled water at 4 ºc and used for no longer than 6 months after extraction32. two class ii cavities were made in each tooth – one on each proximal surface – totalizing 72 cavities. all cavities were prepared as vertical slots, with the cervical margin in cementum, using a diamond bur #4137 (kg sorensen, barueri, sp, brazil) mounted in a mechanical device (el quip, são carlos, sp, brazil) that allowed standardized preparation of cavities with the following dimensions: 4 mm oclusal-cervical length, 1.8 mm buccolingual width and 2.1 mm cervical-axial depth. a new bur was used after five preparations. teeth were randomly divided in three groups (12 teeth/group, totalized 24 restorations/group) according to the lcu used: group i: halogen optilux 401 (kerr/ demetron res. corp., orange, ca, usa); group ii: led – coltolux (coltène whaledent, altstatten, switzerland) and group iii: led – ultralume led 5 (ultradent, south jordan, ut, usa). table 1 shows more details concerning the lcus used in the study. all cavities were restored with the same adhesive system (adper single bond, 3m espe, st. paul, mn, usa) and composite resin (filtek p60, 3m espe). the composite resin was inserted according to the oblique technique, in increments of 2 mm, using a photocondenser tip (tdv dental ltda, pomerode, sc, brazil). each increment was light-cured for 40 s, using the tested lcus. the length of the photocondenser tip served as a guide to standardize the distance of the lcu from the restoration. after 24 h of water storage at 37ºc, the restorations were finished and polished with sof-lex discs (3m espe) according to the manufacturer’s instructions. specimens were then subjected to a thermal cycling regimen of 500 cycles (5ºc ± 2ºc and 55ºc ± 2ºc), with a 15-s dwell time in each bath. the 36 teeth were bisected in a buccolingual direction with a water-cooled diamond saw (isomet 1000, buehler, lake bluff, il, usa) to obtain mesial and distal halves, each one with a restoration, totalizing 72 restorations. then, each half was sectioned longitudinally in the middle of the restorations, in such a way that each half produced light curing unita type light intensity (mw/cm2) wavelength (nm) energy density per incrementb (j) optilux 401 qth 600 390 530 24 coltoluxled led 1000 450 480 40 ultralume 5 led led 800 370 – 500 32 table 1 – light-curing units used in the study a information supplied by the manufacturers. b calculated based on the curing time used for each increment, and the light intensity generated by each unit, as measured with the appropriate radiometer. effect of light-curing units on gap formation and microleakage of class ii composite restorations 264 braz j oral sci. 10(4):262-267 two hemi-sections: one buccal and one lingual. gap measurement eighteen teeth were taken to a vacuum desiccator and sputter coated with gold-palladium (polaron sc7620, quorum technologies ltd., east sussex, uk) for 5 min at 10 ma. each specimen was examined by a scanning electron microscope (jsm 6360lv, jeol ltd., tokyo, japan) at a 15kv accelerating voltage at ×1000 magnification and sem micrographs were taken for evaluation of gap width (µm) (figure 1). microleakage analysis the 18 teeth were used to evaluate dye penetration at the gingival wall of each specimen. specimens were coated with two layers of nail varnish, and then exposed to 50% silver nitrate solution (vetec química fina, xerém, rj, brazil) for 2 h, photodeveloped (kodak, eastman kodak company, rochester, ny, usa) for 16 h. afterwards, teeth were washed with tap water and nail varnish layers were removed with table 2 – mean, median, standard deviation, minimum and maximum values referent to gap width (µm) in all groups. gap width values (µm) group i optilux401 (halogen) group ii coltoluxled (led) group iii ultralumeled5 (led) mean 3.28 1.48 3.11 median 2.74 0.85 2.37 standard deviation 3.34 1.89 3.45 minimum 0 0 0 maximum 10.90 5.19 11.80 scores group i optilux401 (halogen) group ii coltoluxled (led) group iii ultralumeled5 (led) 1 7 2 5 2 5 8 6 3 1 1 4 1 total 12 12 12 table 3 – frequencies of marginal microleakage scores in all groups. the aid of manual cutting instruments. the teeth were sectioned in the same manner as previously described, and the microleakage was evaluated b y t w o e x a m i n e r s u s i n g o p t i c a l s t e r e o m i c r o s c o p e (olympus bx41, tokyo, japan) at ×40 magnification. leakage was scored using the following criteria: 1, no dye penetration; 2, dye penetration extending up to 1/2 of cervical wall; 3, dye penetration extending to cervical wall, but without reach the axial wall; 4, dye penetration e x t e n d i n g t o c e r v i c a l w a l l , r e a c h i n g a x i a l w a l l . i f examiners disagreed, a forced consensus was reached and the consensus score recorded. statistical analysis after measurements, as data did not confirm to the presuppositions of parametric analysis, the non-parametric kruskal-wallis test was used to compare the effect of each lcu on gap formation and marginal microleakage at a 5% significance level. results gap measurement table 2 summarizes the results (mean, median, standard deviation, minimum and maximum values) for gap formation in the experimental groups. the first null hypothesis was accepted because there were no statistically significant differences (p=0.25) among the groups with respect to gap width. marginal microleakage the frequencies of marginal microleakage scores are presented in table 3. the second null hypothesis was also accepted because, although group i showed lower microleakage scores follow by group iii and group ii, there were no statistically significant differences (p=0.072) among the experimental groups with respect to marginal microleakage. effect of light-curing units on gap formation and microleakage of class ii composite restorations fig. 1. scanning electron micrograph of a representative marginal gap. a gap can be seen at the interface between dentin and the composite restoration (original magnification ×1000). the asterisk indicates the gap width being measured. d dentin; g gap; cr – composite restoration. 265 braz j oral sci. 10(4):262-267 discussion the polymerization reaction of resin-based composites involves the conversion of c=c bonds in individual monomer molecules and the formation of c-c bonds to form polymer chains, causing volume reduction, as covalent bonds are created and molecular distances and free volume are reduced33. light-activated composite resins start the polymerization process through the absorption of light by the initiator (usually a diketone), which once activated, reacts with a reducing agent (aliphatic amine) to produce radicals that bind the monomer molecule, making it active. the unsaturated monomer link opens, and subsequently acts on the other molecules from the monomer, forming a crosslinked macromolecule. in this polymerization process, generally there are three stages: initiation, propagation and termination. in the first stage, photons of light energy at a wavelength around 450 nm to 500 nm, activate the initiator. the initiator depends on a co-initiator for the electron transfer and the formation of free radicals. the co-initiator usually is a tertiary amine that does not absorb light, but interacts with the camphorquinone when it is excited. free radical is a highly energetic molecule with an unpaired electron. it needs to form a covalent bond with another compound, which, in turn, may be the double bond (c=c) present in the monomers. propagation is the stage when the free radical reacts with the first monomer unit (c=c). the formation of a radical-monomer complex occurs, which aim to produce more connections with other monomers. in other words, it is a chain reaction with rapid growth of radical-monomer complex, forming a three-dimensional network, until the polymer formation. termination is when two macroradicals collide, or when the active extremities of two chains that propagate react with each other and bind in a bimolecular reaction to form a single molecule no more reactive, ending the chain growth34. the development of contraction stress of composites depends, among other factors, on the composition of the material (type of monomer, filler load, and their interaction), and factors related to resin composite polymerization (conversion degree, curing technique, placement technique, c-factor, and others)35. regarding the composition of the material, filtek p60 contains 61% of filler content (in volume) silica and zirconia fillers with mean size of 0.6 µm and a polymeric matrix consisting of bis-gma, bis-ema, udma and tegdma. as any resin-based composite, this material undergoes polymerization shrinkage. in the present study, it was used an only type of composite, since the objective was to verify the effect of different lcus on gap formation and marginal microleakage. variation between resin composites were excluded, as each material could have different compositions and, consequently, distinct degrees of polymerization shrinkage. the photoinitiator of filtek p60 composite resin is camphorquinone (maximum absorption spectrum at 468 nm), and all the lcus used in the present study work within this spectrum. it was also employed a device for standardization of the class ii cavities in order to avoid variations in their dimensions. each lcu has its own wavelength specifications, advantages, disadvantages, and curing efficiency. it has been observed that scattering is greater with the halogen units36. also, led units has more spectral purity than halogen lamps, as it has a narrow band of light emission with a wavelength between 450-490 nm, with peak emission at 470 nm, and this is the coincident blue light band with the absorption spectrum of most of the photoinitiators included in the composite resins, which allows full use of leds37. the type of photoinitiator in resin-based materials significantly influences the curing efficiency of the material across the width of a restoration38. it also determines the most appropriate lcu to cure a particular type of composite resin, as the wavelength emitted by a lcu should match the absorption spectrum or absorption peak of the photoinitiator in that composite. camphorquinone can be readily cured with halogen lamps and other units, but other photoinitiators (ppd, lucirin tpo) pose a great problem because most commercially available lcus do not match their spectrum24. another factor that affects light-curing efficiency is the filler particles of resin-based materials. these filler particles tend to scatter the light, and both filler content and size influence the light dispersion24. smaller filler particles (0.1 µm to 1.0 µm) have maximal scattering because these particle sizes correspond to the wavelength range of the photoinitiator. microfilled composites with smaller or greater particles scatter more light than microhybrid resins. if the refractive indices of the matrix and filler particles have an increased difference, light scattering is also increased. therefore, the size and concentration of filler particles should be controlled depending on the refractive indices of the filler and resin matrix, as it also influences the resin color39. the results of this study demonstrate that all groups showed gap formation. corroborating these results, other studies have previously reported that all groups, regardless of the composite material12,16,21 or the curing technique22,40 were free of marginal gaps at tooth/restoration interface. the results of this study are supported by the literature. researchers30-31,41 have found no differences in microleakage when leds or halogen lamps were used to photo-activate class ii composite restorations. small (2001)41 stated that although many improvements have been made, any material or method can ensure the effectiveness of the restoration if certain principles are respected, while another study30 found better overall results when 2nd generation leds and halogen lamps are used compared with 1st generation leds. on the other hand, differently from this study, fernandes et al. (2002)42 reported that the use of led units contributes to a better interface integrity, which is an extremely essential factor in procedures that utilize light-curing materials. studies have found that using a led20 or a halogen lamp18 in soft start mode reduces polymerization shrinkage and microleakage. all the discrepancies between these studies probably occur due to the different experimental designs of each study, especially the cavity size, polymerization effect of light-curing units on gap formation and microleakage of class ii composite restorations 266 braz j oral sci. 10(4):262-267 method22, light intensity of lcus and energy density per increment. accurate knowledge of the polymerization process and control techniques may also contribute to the clinical performance of the restorative procedure and better marginal adaptation, hence, decreasing marginal gap formation17. it is important to emphasize that this in vitro study used different lcus with the same curing time (according to the manufacturer’s instructions), with no influence of other factors, such as energy density or power intensity. it is also known that the stress caused by polymerization shrinkage of composites has a multifactorial nature7. therefore, select an appropriate restorative material, follow correct handling and placement techniques and use an appropriate lcu may allow for controlling polymerization shrinkage and having more aesthetic and durable class ii restorations due to reduction of marginal discoloration, recurrent caries and postoperative sensitivity17. within the limitations of this in vitro study, it may be concluded that there were no differences among the lcus tested with respect to gap formation and marginal microleakage of class ii composite restorations. further studies should be done to verify the interaction between the different factors that characterize gap formation and marginal microleakage, as well as ensure a better marginal adaptation at tooth/restoration 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geneve, switzerland: cd tr 11405; 2003. 33. ferracane jl. developing a more complete understanding of stresses produced in dental composites during polymerization. dent mater. 2005; 21: 36-42. effect of light-curing units on gap formation and microleakage of class ii composite restorations braz j oral sci. 10(4):262-267 34. rodrigues mr, neumann mg. fotopolimerização: princípios e métodos. polímeros: cien tecnol. 2003; 13: 276-86. 35. braga rr, ballester ry, ferracane jl. factors involved in the development of polymerization shrinkage stress in resin-composites: a systematic review. dent mater. 2005; 21: 962-70. 36. emami n, sjödahl m, söderholm kj. how filler properties, filler fraction, sample thickness and light source affect light attenuation in particulate filled resin composites. dent mater. 2005; 21: 721-30. 37. nomoto r. effect of light wavelength on polymerization of light-cured resins. dent mater j. 1997; 16: 60-73. 38. palin wm, senyilmaz dp, marquis pm, shortall ac. cure width potential for mod resin composite molar restorations. dent mater. 2008; 24: 1083-94. 39. lee yk. influence of scattering/absorption characteristics on the color of resin composites. dent mater. 2007; 23: 124-31. 40. ciucchi b, bouillaguet s, delaloye m, holz j. volume of the internal gap formed under composite restorations in vitro. j dent. 1997; 25: 305-12. 41. small bw. a review of devices used for photocuring resin-based composites. gen dent. 2001; 49: 457-60. 42. fernandes ds, corbellini c, derossi ca. leds diodo emissor de luz uma nova opção em fotopolimerização. anais do vi congresso catarinense de promoção de saúde bucal; 2002. p.40. abstract 069. 267effect of light-curing units on gap formation and microleakage of class ii composite restorations oral sciences n3 braz j oral sci. 10(3):199-203 received for publication: may 16, 2011 accepted: july 26, 2011 evaluation of a digital methodology for human identification using palatal rugoscopy karen christine dos santos1, clemente maia s. fernandes2, mônica da costa serra3 1dds, department of social dentistry, faculty of dentistry of araraquara, são paulo state university unesp, brazil 2dds, ll.b student, msc, phd, post-doctorate student, department of social dentistry, faculty of dentistry of araraquara, são paulo state university unesp, brazil 3dds, ll.b, msc, phd, post-doctor, department of social dentistry, faculty of dentistry of araraquara, são paulo state university unesp, brazil original article braz j oral sci. july | september 2011 volume 10, number 3 correspondence to: mônica da costa serra departamento de odontologia social, faculdade de odontologia de araraquara unesp rua humaitá 1680 araraquara sp brasil 14801-903 phone: (55) 16 33016353 / fax: (55) 16 33016343 e-mail: mcserra@foar.unesp.br abstract palatal rugoscopy, or palatoscopy, is the process by which human identification can be obtained by inspecting the transverse palatal rugae inside the mouth. aim: this study evaluated a digital method for human identification using palatoscopy, by comparing photographs of the palate against the images of cast models of the maxilla photographed with and without highlighting of the palatal rugae. methods: condensation silicone impressions were made from the upper arches of 30 adult subjects of both genders and their palates were then photographed. the first impression was made with heavy silicone, the second impression with light silicone, and then the models were cast in improved type iv dental stone. the casts were photographed, the palatal rugae of each one were highlighted with a pencil, and then the models were photographed again. using a free image-editing software, the digital photographs were overlapped over the images of the palatal rugae of the models with and without highlighting of the palatal rugae, in order to identify the pairs. results: the result of overlapping the digital photographs with the images of the models without highlighted palatal rugae resulted in 90% positive identification. for the overlapping of the digital photographs with the images of models with highlighted palatal rugae, there was 100% positive identification. conclusions: the digital method evaluated in this study was proven effective for human identification. keywords: forensic sciences, forensic anthropology, forensic dentistry, human identification, palatal rugae. introduction human identification corresponds to the set of different procedures to identify a person or object1. identification means demonstrating that a certain person or his/her relevant attribute, which appears in the examination at a given moment, is the same one that appeared on a previous occasion2-3. for an identification process to be applicable, five requirements need to be met: individuality, immutability, perenniality, practicality and classification4. in humans, unlike in other animals, the set formed by the rugae is asymmetric2,5. the palatal rugae are unique, unchanging, perennial and subject to classification6-10. it appears in the third month of the embryonic period, remaining for the entire life of the individual and for several days after his/her death1. when identification cannot be established by fingerprinting or by analysis of dental arches (dental records data), the palatal rugae can be considered as a 200 braz j oral sci. 10(3):199-203 source of comparative material8,11 because they are able to individualize a person, legitimizing an identification process12, even in extreme circumstances13. illnesses, chemical injury or trauma do not seem to modify the palatal rugae structure. muthusubramania et al.14 found that most victims of third-degree burns do not show changes in the palatal rugae, and when such changes do occur, they are less pronounced than those found on the body. the palatal rugae are better “preserved” due to the protection given by the bony scaffold, dental arches and soft structures. on the other hand, fingerprints can be easily destroyed in certain accidents or due to the action of the vast necrophagous fauna15. palatal rugoscopy can have a major role in human identification. the cast models and antemortem intraoral photographs can be found in dental records and serve as the first protocol to perform the identification process. furthermore, for at-risk populations (for example, aeronauts and firemen), this first record can be preventively arranged and archived for the possible need of identification. limson and julian11 compared the rugosities of university students by an impression with irreversible hydrocolloid and cast models in plaster type ii. the rugae were highlighted with well sharpened graphite pencils. the rugae were photographed and the models were scanned, achieving a 92-97% success rate. the authors claimed that this error rate of 3-8% can be reduced by using an intraoral scanner, with a direct transfer to the computer. some identification methods are available for edentulous victims, such as comparing the anatomy of the paranasal sinuses and comparing the bone patterns observed in radiographs. in addition to this, the victim’s own dentures can also be used, which are found inside the mouth or in their homes. among the evidence from an edentulous victim, the palatal rugae are considered to be one of the unique morphological features16. ohtani et al.17 studied the possibility of identifying edentulous individuals by comparing the rugae in denture molds against models obtained from impressions taken from the patients. impressions were taken from the mucosal surfaces of complete dentures with alginate impression materials and 146 maxillary casts were made from hard dental plaster. they then compared the rugae patterns to identify the pairs (dentures and plaster models). the median percentage of correct matches was 94%, and the error rate was attributed to 3 factors: poorly demarcated eminences of rugae, change of palatal height in some cases and non-complex rugae pattern. castellanos et al.16 reported the identification of the body of an adult woman in a city in colombia. the woman had been declared missing by her family 15 days before the skeletonized body was found. identification was made by comparing the palatal rugae obtained from the remainder of the body with the plaster model that was obtained from the denture that was with the family. it is often difficult to obtain dental arch impressions of corpses that arrive at the forensic medicine institutes to be compared with the antemortem data of the alleged victim. as the experts or technicians are not familiar with dental impression materials and there is no dental laboratory structure, photographs are more practical. antemortem models can be obtained from the victims’ dentists and then compared with the postmortem photographs using free image-editing software, which is more practical and economically feasible for the brazilian forensic medicine institutes. this study evaluated a digital method for human identification using palatal rugoscopy by comparing photographs of the palate with images of maxillary cast models photographed with and without the palatal rugae highlighted. material and methods thirty adult subjects of both genders participated in this study. impressions were made from their upper arches with condensation silicone and their palates were photographed. these subjects agreed to participate in the survey by signing an informed consent form and the study was approved by the ethics committee of araraquara dental school, unesp, brazil. casts were made of the upper dental arches of each of the 30 volunteers. in order to obtain a reliable copy of the palatal rugae positive, the first impression was prepared with heavy silicone, and the second impression with light silicone. the impressions were cast in plaster, and the corresponding models were assembled using type iv improved dental stone, in order to obtain greater strength and accuracy. the rugae of each model were classified according to carrea18 (table 1). the types of palatal rugae (i, ii, ii or iv) were determined according to their directions in relation to the raphae. the arch shapes were classified according to the method proposed by testut. digital photographs of the palate were taken with a 10.1 megapixel nikon digital camera. the plaster models were photographed with the same digital camera, and then the palatal rugae were highlighted. for the design and filling of the rugae in the plaster models, a 0.3 mm mechanical pencil was used; this procedure consisted of outlining the papilla, highlighting the rugae and determining the length of the median raphe. the models with the marked palatal rugae were also photographed. each photograph of the palate and each plaster model received a code. each model received a different code from the code given to the corresponding photograph of the same subject. the palate photographs received arabic numeric codes, and the models received roman numeric codes. the images of the highlighted rugae models received the same code, with the addition of a line, which meant that it was a model with highlighted rugae, for example: ii (model with no rugae highlighted) and ii’ (model with highlighted rugae). classification of palatal rugae characteristics type i posterior-anterior directed rugae type ii rugae perpendicular to the raphae type iii anterior-posterior directed rugae type iv rugae directed in several directions table 1types of palatal rugae, according to carrea’s classification. evaluation of a digital methodology for human identification using palatal rugoscopy 201 braz j oral sci. 10(3):199-203 the gimp (gnu image manipulation program), which is a free software raster graphics editor, was used for identification of the pairs (corresponding pictures and models)19. first, superimposition of the images from the digital photographs of the palate with the photographs of the palatal rugae of the models without highlighted rugae was performed; next, superimposition of the images from the digital photographs with the models that had the rugae highlighted by a pencil was carried out. before being overlapped and compared, the digital images had their size standardized. the identification of the 30 pairs was performed by an examiner who had no previous contact with the photographs of the palate nor with the specimens obtained. the examiner was not aware of the previously established codes, which assured the blind nature of this experiment. the percentages of positive identifications were analyzed on the images of the models with and without highlighted palatal rugae. results in the superimposition of the digital photographs with the photographs of the models without highlighted palatal rugae, the percentage of correct matches obtained by the examiner was 90%. of the 30 models evaluated, one model was not correctly matched with its corresponding photograph, and two other models were not identified by the examiner, after all of photographs had been evaluated. in the superimposition of the digital photographs with the models with highlighted palatal rugae, the percentage of correct answers obtained by the examiner was 100%. the classification of the dental arches and palatal rugae of the 30 individuals is presented in table 2. figures 1 to 5 show the correlation between the photographs of the palate, the images of a model without highlighted palatal rugae, a model with highlighted palatal rugae and their respective overlapped images. fig. 1 photograph of palatal rugae. fig. 2 cast with palatal rugae. fig. 3 cast with highlighted palatal rugae. fig. 4 superimposition – photograph and cast. evaluation of a digital methodology for human identification using palatal rugoscopy 202 braz j oral sci. 10(3):199-203 fig. 5 superimposition photograph and cast with highlighted palatal rugae. digital photograph code palatal rugae classification dental arch classification 1 type iii hyperbole 2 type iii parable 3 type iii hyperbole 4 type ii parable 5 type iii parable 6 type i parable 7 type iii hyperbole 8 type ii parable 9 type ii parable 10 type iii hyperbole 11 type i hyperbole 12 type iv parable 13 type iv hyperbole 14 type iii hyperbole 15 type iv parable 16 type iii parable 17 type i elliptic 18 type ii parable 19 type iv elliptic 20 type iv hyperbole 21 type iv parable 22 type iii elliptic 23 type iii parable 24 type i hyperbole 25 type iv parable 26 type iv hyperbole 27 type ii parable 28 type iii hyperbole 29 type iv parable 30 type i hyperbole table 2 classification of palatal rugae and dental arches. araraquara, 2010 discussion palatal rugoscopy is not a recent identification method. notwithstanding, there are few published research papers using such rugoscopy method in the international literature, and particularly using information technologies such as digital images, which does not enable to establish the comparative data found herein with several others. in this work, when the palatal rugae of the nonhighlighted models were superimposed with the digital photographs, there was a 90% rate of identification. in the overlapping of the palatal rugae of the highlighted models with the digital photographs, there was a 100% accuracy rate in the identification. these results are similar to those of martins filho20, who also obtained 100% identification in his study and limson and julian11, who achieved a success rate of 92-97% in the comparison. however, the first author compared the models with one another, not using the images from photographs of the palate. it is understood that the use of palate photographs, in a second record, represents the most practical form to be performed on a day-to-day basis at forensic medicine institutes for the palatal rugoscopy identification method. the 90% rate for the non-highlighted palatal rugae suggest that, in real situations, when there may be only a digital image of the antemortem plaster model (thus, without highlighted palatal rugae) of the alleged victim to be identified, this method can be used with a high success rate for palatal rugoscopy identification. having the plaster model physically documented, it can have the palatal rugae highlighted and can be photographed for later comparison with the digital photograph of the subject’s palate rugae, for whom an identity is sought. regarding the classification of the arches, 50% of the arches showed a parabolic form, 40% a hyperbolic form and 10% an elliptical form. martins filho20 found mostly parabolic (70%) and hyperbolic arches (21%) and a lower incidence of elliptical (8%) and epsilons arches (1%). adding the parabolic and hyperbolic arches (90%), the findings in this study are similar to those found by martins filho20 (91%), notwithstanding the individual results for the parabolic and hyperbolic forms. the location of the palatal rugae, protected by the anatomical structures that surround them, makes them more resistant to the action of external factors, which may be present when other structures that could serve as a basis for comparative analysis, characteristic of human identification methods, are destroyed or absent8,13,15. the findings in this study confirm the importance of using palatal rugoscopy as an identification method, not only by the traditional method, but also using information technology, digital imaging, which can facilitate performing the technique. in individual situations or in mass disasters, when the traditional methods of identification cannot be used, such as fingerprinting or identification by data comparison from dental records, the use of palatal rugoscopy, by digital imaging, can be very useful9,11. the fact that the palatal rugae are protected by the bony scaffold, dental arches and even by the soft parts, was also reported by castellanos et al.16, according to whom the procedure for body identifying depend on the state of preservation of the remains, that is, whether the body is complete or not, fresh, decomposed, charred, mutilated or skeletonized. these authors obtained a positive identification of an edentulous victim by using the palatal rugoscopy method. identification by dna analysis is also a reliable, but expensive and laborious method, in which in the absence of samples from the alleged victim, it is often necessary to obtain dna samples from relatives. the advent of new information technologies, and the opportunity afforded by evaluation of a digital methodology for human identification using palatal rugoscopy braz j oral sci. 10(3):199-203 203 using computerized tools, generates the convenience and ease of application of the method employed in this work. the digital method assessed herein was proven efficient for human identification. the method of overlapping the photographs of the palate with the images of the upper plaster models that had the palatal rugae highlighted is more accurate than overlapping the photographs of the models without the rugae highlighted. the human identification digital method by means of the analysis of palatal rugae investigated herein is viable and within range of being implemented. it may assist and facilitate the work of forensic human identification. references 1. vanrell jp. odontologia legal & antropologia forense. 2nd ed. rio de janeiro: guanabara koogan; 2009. 2. arbenz go. introdução à odontologia legal. são paulo: edition of the author; 1959. 3. 3 arbenz go. medicina legal e antropologia forense. são paulo: atheneu; 1998. 4. frança gv. medicina legal. 8th ed. rio de janeiro:guanabara koogan; 2007. 5. taylor p, wilson m, lyons t. forensic odontology lessons: multishooting incident at port arthur, tasmania. forensic sci. int. 2002; 136: 174-82. 6. caldas im, magalhães t, afonso a. establishing identity using cheiloscopy and palatoscopy. forensic sci int. 2007; 165: 1-9. 7. campos mlb. rugoscopia palatina. in: vanrell jp. odontologia legal & antropologia forense. rio de janeiro guanabara koogan; 2002. p.212-4. 8. ochoa ee. rugoscopia palatina y su importancia en la identification. rev dent. 1972; 19: 31-2. 9. mantecca mam, melani rfh. arcos dentários e rugoscopia palatina. in: silva m, organizator. compêndio de odontologia legal. rio de janeiro: medsi; 1997. p.237-42. 10. pretty ia, sweet d. a look at forensic dentistry – part 1: the role of teeth in the determination of human identity. br dent j. 2001; 190: 359-66. 11. limson ks, julian r. computerized recording of the palatal rugae pattern and an evaluation of its application in forensic identification. j forensic odontostomatol. 2004; 22: 1-4. 12. english wr, robison sf, summitt jb, oesterle lj, brannon rb, morlang wm. individuality of human palatal rugae. j forensic sci. 1988; 33: 718-26. 13. ermenc b, rener k. possibilities for dental identification in the cases of mass disaster in slovenia. forensic sci. 1999; 103: 867-75. 14. muthusubramania m, limson ks, julian r. analysis of rugae in burn victims and cadavers to simulate rugae identification in cases of incineration and decomposition. j forensic odontostomatol. 2005; 23: 26-9. 15. souza lima j. considerações sobre o estudo das rugosidades palatinas [doctorate’s thesis. belo horizonte: federal university of minas gerais; 1964. 16. castellanos dca, higuera lfh, avella amh, gutiérrez app, martínez jac. identification positiva por médio del uso de la rugoscopia en un município de cundinamarca (colombia): reporte de caso. acta odontol venez. 2007; 45: 446-9. 17. ohtani m, nishida n, chiba t, masayuki f, miyamoto y, naofumi y. indication and limitations of using palatal rugae for personal identification in edentulous cases. forensic sci int. 2008; 176: 178-82. 18. carrea ju. la identificacion de las rugosidades palatinas. rev ortod. 1937; 1: 3-23. 19. gimp (gnu software was used) image manipulation program. available from: http://www.gimp.org. 20. martins filho ie. simplificação de método para identificação humana por meio da rugoscopia palatina [master’s thesis]. bauru: university of são paulo; 2006. evaluation of a digital methodology for human identification using palatal rugoscopy oral sciences n3 braz j oral sci. 14(4):272-275 original article braz j oral sci. october | december 2015 volume 14, number 4 influence of shade, curing mode, and aging on the color stability of resin cements janes francio pissaia1, gisele maria correr1, carla castiglia gonzaga1, leonardo fernandes da cunha1 1 universidade positivo – up, dental school, department of operative dentistry, curitiba, pr, brazil correspondence to: janes francio pissaia graduate program in dentistry, universidade positivo, curitiba, pr, brazil rua professor pedro viriato parigot de souza 5300 curitiba pr cep: 81280-330 phone: 55 46 99301204 e-mail: janespissaia@hotmail.com abstract the color stability of resin cements is essential for aesthetic restorations. aim: to evaluate the influence of shade and aging time on the color stability of two light-cured and two dual-cured resin cements. methods: the cie-lab color parameters (n=6) were measured immediately after sample preparation and at 7, 30 and 90 days of aging in distilled water. the color difference (∆e) was calculated and then analyzed by three-way anova for repeated measures and tukey’s hsd test (α=0.05). results: ∆e was higher for transparent resin colors, followed by dark and light colors. the mean values of ∆e were lower for both light-cured resin cements compared to the dual-cured cements. as the aging time increased, ∆e values increased. conclusions: the light-cured resin cements showed greater color stability. the lighter shades of luting were more likely to display a greater color change. keywords: aging; color; dental cements; esthetics; dental materials. introduction resin-based cements have been extensively applied for aesthetic restorations with translucent material, like dental ceramic veneers1-2. it is essential that the color of the aesthetic materials remains stable over a long period in the oral environment3-4. previous studies have described color changes occurring in direct dental composites5-9. however, as koishi et al. have remarked4, the color stability of luting composites requires careful analysis. luting composites and direct composite resins have similar compositions (in terms of the polymer matrix and fillers), and therefore the color stability of luting composites may be affected by chemical components. inorganic oxide additives are typically used to create material shades that match the color of the tooth or other restorative materials used with resin-based cements10. depending on the manufacturer, one or multiple shades may be applied. uchida et al. reported that the discoloration of a direct composite resin is influenced by shade selection8: the lighter the shade, the higher the tendency for discoloration. as direct composite materials and luting cements have similar chemical components (organic matrix and fillers), the color stability of resin-based cements may also be affected by shade selection. resin-based cements may be classified based on whether they are chemically cured, light-cured or dual-cured materials11. light-cured resin cements present increased working time and the removal of excesses of material is easier than for chemically cured materials. on the other hand, dual-cured luting materials contain compounds for both chemical and light curing, and consequentially these materials possess beneficial traits from both polymerization systems. the combination of curing systems used with dual-cured resin cements is reported to reduce the number received for publication: november 18, 2015 accepted: december 11, 2015 http://dx.doi.org/10.1590/1677-3225v14n4a04 of remaining double bonds, which improves the degree of conversion12. as the color of resin-based materials is related to the degree of conversion13-14, dual-cured luting materials should show better color stability than the light-cured resin cements. in the case of aesthetic restoration materials, such as ceramic veneers, the color stability of the resin-based cement employed for the cementation procedure may be as important for the long-term clinical success of the restoration as the mechanical properties of veneering material. when ultrathin ceramic veneers are used, the color of the resin-based cement plays a major role. the aim of this study was to evaluate the color stability of four different luting cements with variations in curing mode, shade and aging time. the experimental design tested three null hypotheses: that there might be no difference in color stability between the evaluated shades, that the method of polymerization might not influence the color stability, and that aging in water might induce no changes in color. material and methods three shades (transparent, clear, and dark) of each four resin-based cements (rely x veneer 3m/espe, germany, light-cured; choice 2 bisco, usa, light-cured; variolink ii ivoclar-vivadent, germany, dual-cured; and allcem fgm, brazil, dual-cured) were evaluated in this study. the key attributes of each material are in table 1. all specimens were prepared in controlled relative humidity (55±5%) and temperature (23±1 ºc). each luting composite was mixed in accordance with the manufacturer’s instructions. six disk specimens, 5 mm diameter and 1 mm thick, were prepared for each shade and material. each specimen was made by inserting the resin-based cement into a teflon mold ring and pressed between two 1-mm-thick glass slides under finger pressure. all samples were continuously light-cured with a led light source (dental woodpecker led, 1200 mw/cm 2), according to the manufacturer’s instructions (rely x veneer 3m/espe 30 s,; choice 2 bisco – 40 s; variolink ii ivoclar-vivadent – 40 s; and allcem fgm – 40 s), by placing the light tip on the glass slide covering the entire area of each specimen. specimens were stored in dark canisters containing distilled water at 37 °c for 24 h. the color of each specimen was first determined after 24 h, and this was defined as the baseline color evaluation. the color measurements were performed with an easyshade spectrophotometer (vita zahnfabrik, bad säckingen, germany) according to the cielab (commision internationale de l’eclairage) l*, a*, b* coordinates. the cie l* parameter corresponds to the degree of light and darkness, whereas a* and b* coordinates correspond to red or green (+a*=red, -a*=green) and yellow or blue (+b*=yellow, -b*=blue), respectively. the specimens were then stored in dark canisters containing distilled water at 37 °c, and the color values were again measured after 7, 30 and 90 days. the specimens were placed on a white background during the measurement to prevent potential absorption effects on any of the measured color parameters. the mean of three measurements on each sample was used to represent the average values of the color parameters of each sample and these average values were used for the overall data analysis. the cie-lab coordinates were used to calculate the color difference (∆e) between the baseline color measurement and the aged specimens. before each color measurement, the immersed specimens were dried with absorbent paper. the ∆e for each experimental time was calculated using the following equation: ∆e = [(∆l*)2 + (∆a*)2 + (∆b*)2]1/2 where ∆l*, ∆a*, and ∆b* are the differences in the respective values before and after aging. the ∆e values were analyzed by three-way anova for repeated measures and tukey’s hsd test (α=0.05). results the mean values of ∆e for the three different variables in this study, along with standard deviations, are in tables 2-4. significant effects of curing mode, shade and aging time on the color stability were observed. as shown in the results of resin shade comparison in table 2, ∆e values were the highest for translucent resins (p=0.000009), and lowest for clear and dark resins. there were no significant differences 273273273273273 influence of shade, curing mode, and aging on the color stability of resin cements braz j oral sci. 14(4):272-275 material curing color color color mode transparent clear dark choice 2 light-cured translucent a1 a2 bisco rely x veneer light-cured translucent a1 a3 3m/ espe variolink ii dual-cured transparent light yellow-a1 opaque yellow-a3yellow/ ivoclar-vivadent white (110/a1) universal (210/a3) allcem dual-cured transparent a1 a3 f g m table 1.table 1.table 1.table 1.table 1. materials used in the present study. 274274274274274 material mean s d choice 2 2.79 a* 0.71 rely x veneer 3.49 a 0.77 variolink ii 3.60a 1.42 allcem 4.48b 0.99 table 3. table 3. table 3. table 3. table 3. mean ∆e values and corresponding sds for the tested four resin-based cements. *different letters indicate statistically significant difference between group ∆∆∆∆∆e (days) mean s d 7 2.79a* 0.51 30 3.73b 1.01 90 4.25c 1.40 table 4. table 4. table 4. table 4. table 4. mean ∆e values and corresponding sds for different aging times. *different letters indicate statistically significant difference between group influence of shade, curing mode, and aging on the color stability of resin cements braz j oral sci. 14(4):272-275 c o l o r mean s d clear 3.12a* 0.74 dark 3.25 a 1.13 transparent 4.41 b 1.04 table 2.table 2.table 2.table 2.table 2. mean ∆e values and corresponding standard deviations (sds) between different shades of resin-based cements. *different letters indicate statistically significant difference between group between clear and dark shades of the tested materials. comparing the four resins in terms of the type of curing, the dual-cured allcem cement had higher ∆e values when compared to light-cured materials (table 3) (p=0.000016). no significant difference was found between the dual-cured cement variolink ii and the light-cured materials. when aging time was considered, the mean values of ∆e increased with time from 7 to 30 to 90 days (table 4, p=0.000000). discussion all three evaluated null hypotheses in the present study were rejected: the experimental results revealed that the color stability varied among resin shades, curing methods and specimen aging times. storage in water is frequently used for in vitro aging of restorative composite materials. in this study, water exposure for longer periods of time caused increased changes in the cie color space coordinates of the composites (table 4). it is well known that water absorbed in the polymer matrix causes filler matrix debonding and hydrolytic degradation of the filler15-16, and it also could change the refractive index of the material4. therefore, the increase of the ∆e values might be affected by water sorption in the used luting composites. the ∆l values increased as the immersion period increased, suggesting that the color of a luting composite tends to darken over long-term clinical use4. other researchers have demonstrated that composite materials undergo discoloration by exposure to sources like uv light, visible light and/or heating6,17. in prosthetic composites, ruyer et al. reported that color changes with ∆e values lower than 3.3 were acceptable18; turgut et al. reported that ∆e values lower than 3.5 were also acceptable19. after aging, all specimens showed changes in äe values, but almost all the color differences had acceptable values. however, the mean ∆e values for transparent resin shades and for the dual-curable resin cement were above the acceptable. after 30 and 90 days of laboratory aging, ∆e values were also higher than acceptable. these results are related to the used method, because the uncovered resin disks used in this study, similar to those used in previous reported ageing tests 20-22, have increased exposure to potentially damaging environmental factors. different number of samples are used for this kind of research; for example, uchida et al. 1998 used 3 samples per group8. on the other hand, five specimens were prepared for each group by koishi et al. 20024, lu and powers 200423, furuse et al. 20086. in the present study, six samples were enough to provide statistically significant difference, thus no more samples were fabricated. the effect of shades on the color stability of luting composites was statistically significant. the relative values of de*ab were dark=clear<transparent. this suggests that higher color degradation occurred on the colorimetrically lighter and less chromatic shades in the studied composite systems. it was previously shown that lighter shades yield more visible color changes than dark shades in a semi quantitative comparison for direct composite resins 8. according to uchida et al. 8, the higher discoloration associated with lighter shades may result from environmental breakdown of the polymer. this breakdown leads to the release of monomers and causes the cured resin color to shift to the color of the monomers. alternatively, environmental damage can cause a color change by affecting the retention and/or stability of pigments and other additives in the polymer formulations. the aesthetic performance of porcelain laminates can be affected by the color of the resin cements used to bond them to teeth22. the current results indicate that lighter resin cements presented significantly more color change after aging. both the light-cured and the dual-cured cements presented color changes. degradation of residual amines and oxidation of residual unreacted carbon-carbon double bonds result in the formation of yellowing compounds in resinbased materials23. in the present study, the ∆e values for dual-cured specimens were significantly higher than those for respective light-cured specimens. these results are in agreement with brauer, who reported that veneer luting cement composites had better color stability when compared with the auto-cured materials containing tertiary aromatic amine accelerators17. aromatic tertiary amines used in selfcured resins are more likely to oxidize than the aliphatic amines used in light-cured materials; therefore, light-cured cements are expected to have more color stability23. factors such as monomer composition and filler content might also 275275275275275 influence of shade, curing mode, and aging on the color stability of resin cements braz j oral sci. 14(4):272-275 influence the color stability of luting composites4. this could explain the different color stability values between the two dual-cured resin cements tested in this study. many materials with different color and composition are commercially available. further studies are required to evaluate color changes of resin luting cements and factors related to them in different situations, in order to improve long-term aesthetic results and help the dentists to have a better understanding of this issue24-26. within the limitations of this in vitro study, the following conclusions were drawn: the translucent shade materials exhibited higher ∆e changes; the light-cured luting composite resin showed lower ∆e values than the dual cured materials; the ∆e values were influenced by the time of immersion period. references 1. oei jd, mishriky m, barghi n, rawls hr, cardenas hl, aguirre r, et al. development of a low-color, color stable, dual cure dental resin. dent mater. 2013; 29: 405-12. 2. salameh z, tehini g, ziadeh n, ragab ha, berberi a, aboushelib mn. influence of ceramic color and translucency on shade match of cad/ cam porcelain veneers. int j esthet dent. 2014; 9: 90-7. 3. turgut s, bagis b, turkaslan ss, bagis yh. effect of ultraviolet aging on translucency of resin-cemented ceramic veneers: an in vitro study. j prosthodont. 2014; 23: 39-44. 4. koishi y, tanoue n, atsuta m, matsumura h. influence of visible-light exposure on colour stability of current dual-curable luting composites. j oral rehabil. 2002; 29: 387-93. 5. berber a, cakir fy, baseren m, gurgan s. effect of different polishing systems and drinks on the color stability of resin composite. j contemp dent pract. 2013; 14: 662-7. 6. furuse ay, gordon k, rodrigues fp, silikas n, watts dc. colourstability and gloss-retention of silorane and dimethacrylate composites with accelerated aging. j dent. 2008; 36: 945-52. 7. reddy ps, tejaswi kl, shetty s, annapoorna bm, pujari sc, thippeswamy hm. effects of commonly consumed beverages on surface roughness and color stability of the nano, microhybrid and hybrid composite resins: an in vitro study. j contemp dent pract. 2013; 14: 718-23. 8. uchida h, vaidyanathan j, viswanadhan t, vaidyanathan tk. color stability of dental composites as a function of shade. j prosthet dent. 1998; 79: 372-7. 9. usumez a, ozturk n, ozturk b. two-year color changes of light-cured composites: influence of different light-curing units. oper dent. 2005; 30: 655-60. 10. anusavice k. phillips’ science of dental materials, 12th ed. saint louis: saunders; 2012. 11. krämer n, lohbauer u, frankenberger r. adhesive luting of indirect restorations. am j dent. 2000; 13(spec n.): 60d-76d. 12. peutzfeldt a. dual-cure resin cements: in vitro wear and effect of quantity of remaining double bonds, filler volume, and light curing. acta odontol scand. 1995; 53: 29-34. 13. furuse ay, mondelli j, watts dc. network structures of bis-gma/ tegdma resins differ in dc, shrinkage-strain, hardness and optical properties as a function of reducing agent. dent mater. 2011; 27: 497-506. 14. imazato s, tarumi h, kobayashi k, hiraguri h, oda k, tsuchitani y. relationship between the degree of conversion and internal discoloration of light-activated composite. dent mater. 1995; 14: 23-30. 15. ferracane jl, berge hx, condon jr. in vitro aging of dental composites in water—effect of degree of conversion, filler volume, and filler/matrix coupling. j biomed mater res. 1998; 42: 465-72. 16. söderholm kj, zigan m, ragan m, fischlschweiger w, bergman m. hydrolytic degradation of dental composites. j dent res. 1984; 63: 1248-54. 17. brauer gm. color changes of composites on exposure to various energy sources. dent mater. 1988; 4: 55-9. 18. ruyter ie, nilner k, moller b. color stability of dental composite resin materials for crown and bridge veneers. dent mater. 1987; 3: 246-51. 19. turgut s. bagis b. colour stability of laminate veneers: an in vitro study. j dent. 2011; 39 suppl 3: e57-64. 20. gürdal p, akdeniz bg, hakan sen b. the effects of mouthrinses on microhardness and colour stability of aesthetic restorative materials. j oral rehabil. 2002; 29: 895-901. 21. janda r, roulet jf, latta m, steffin g, rüttermann s. color stability of resin-based filling materials after aging when cured with plasma or halogen light. eur j oral sci. 2005; 113: 251-7. 22. alqahtani mq, aljurais rm, alshaafi mm. the effects of different shades of resin luting cement on the color of ceramic veneers. dent mater j. 2012; 31: 354-61. 23. lu h, powers jm. color stability of resin cements after accelerated aging. am j dent. 2004; 17: 354-8. 24. mendes ap, barceleiro mo, reis rs, bonato ll, dias kr. changes in surface roughness and color stability of two composites caused by different bleaching agents. braz dent j. 2012; 23: 659-66. 25. pires-de-souza fde c, garcia lda f, hamida hm, casemiro la. color stability of composites subjected to accelerated aging after curing using either a halogen or a light emitting diode source. braz dent j. 2007; 18: 119-23. 26. almeida jr, schmitt gu, kaizer mr, boscato n, moraes rr. resinbased luting agents and color stability of bonded ceramic veneers. j prosthet dent. 2015; 114: 272-7. oral sciences n3 original article braz j oral sci. october|december 2010 volume 9, number 4 received for publication: june 26, 2010 accepted: october 22, 2010 influence of selective acid etching on microtensile bond strength of a self-adhesive resin cement to enamel and dentin lúcia trazzi prieto1, cíntia tereza pimenta araújo2 , maria malerba colombi humel2 , eduardo josé souza-junior1, carlos tadeu dos santos dias3, luís alexandre maffei sartini paulillo4 1dds, ms student, department of restorative dentistry, piracicaba dental school, state university of campinas, piracicaba, são paulo, brazil 2dds, ms, phd student, department of restorative dentistry, piracicaba dental school, state university of campinas, piracicaba, são paulo, brazil 3agronomic engineer, ms, phd, associate professor, department of exact science, esalq university of são paulo, piracicaba, sp, brazil 4dds, ms, phd, full professor, department of restorative dentistry, piracicaba dental school, state university of campinas, são paulo, piracicaba, brazil correspondence to: eduardo josé souza-junior piracicaba dental school, university of campinas unicamp department of restorative dentistry, p.o. box 52 avenida limeira, 901, areião,13414-903 piracicaba sp brazil phone: 55 19 8841 5291 e-mail: edujcsj@gmail.com abstract aim: to evaluate the selective acid etching of enamel and dentin on microtensile bond strength (µtbs) of a self-adhesive resin cement. methods: forty-eight bovine teeth were selected and had the buccal surface ground to obtain a flat dentin (n=24) or enamel (n=24) surface. z250 composite resin blocks (4 x 3 x 8 mm) were prepared for cementation at enamel and dentin. each substrate received three experimental resin cementation strategies: 1) 37% phosphoric acid etching before the application of relyx unicem; 2) application of relyx unicem alone; 3) relyx arc as a control. samples were light-cured using a halogen light (optilux 501, 700mw/cm2), for 40 s. hourglass-shaped specimens were obtained and submitted to a tensile strength at a crosshead speed of 0.5 mm/min in a universal testing machine. for statistical analysis, data were submitted to anova and tukey’s test at a pre-set alpha (á=0.05). results: relyx unicem showed similar bond strength value (16.5 mpa) when compared to the pretreatment with acid etching (11.9 mpa) and to the conventional resin cement (18.1 mpa) for enamel. all luting strategies presented similar dentin bond strength, but significantly lower than enamel bond strength. conclusions: acid etching prior to relyx unicem application did not improve microtensile bond strength for enamel and dentin. keywords: resin cement, bond strength, dental enamel, dentin, dental acid etching. introduction with the development of adhesive dentistry, resin luting cements have been widely used due to their ability to bond indirect restorations to tooth structure1-2. these materials can minimize some adverse effects of direct composite restorations, like polymerization shrinkage stress magnitude3 and gap formation at tooth/ restoration interface4. conventional resin luting cements require prior application of bonding systems, which is more susceptible to procedure errors, due to unsatisfactory adhesive curing56, overdrying or overwetting of the dentin for hybridization7, cement incompatibility to some acidic monomers of the bonding system8-10. self-adhesive resin cements braz j oral sci. 9(4):455-458 were introduced in an attempt to simplify resin luting strategy, in which a single clinical step is achieved11. in this process, demineralization/infiltration of enamel and dentin is expected to occur simultaneously, not requiring acid etch and bond system application separately11. some studies state that bond strength values of self-adhesive cements are comparable to those of conventional ones12-13. therefore, bonding performance of these simplified cements on smear layer-covered tooth substrate still remains a concern14-17. also, a chemical reaction is suggested to occur between the self-adhesive resin cement and hydroxyapatite (3m espe relyx unicem product profile), similar to the reaction of glass ionomer cements18-19. notwithstanding that self-adhesive resin luting cements do not require pretreatment of the dental substrate, acid etching has been proposed for removal of the smear layer in an attempt to enhance the interaction between the cement and the enamel surfaces, thus increasing the bond strength14,20. it is not clear if pretreatment with phosphoric acid etching of enamel and dentin is really efficient, and this procedure is largely questioned11,21. since the real efficacy of selective enamel and dentin etching is not clear by the literature, the aim of this study was to evaluate the effect of prior acid etching of enamel and dentin on the microtensile bond strength of a selfadhesive resin luting cement, using a total-etch resin cement as the control. the hypothesis tested is pretreatment with phosphoric acid etching could increase microtensile bond strength of relyx unicem to both the dental substrates. material and methods forty-eighty bovine incisors were selected, cleaned and stored in a 0.5% chloramine t solution at 4°c for no more than 1 week. roots were sectioned 1 mm below the cementoenamel junction using a double-faced diamond saw (k.g. sorensen, são paulo, sp, brazil). then, the buccal surface of half of the specimens was ground on a water-cooled mechanical polisher (metaserv 2000, buehler, uk ltd, lake bluff, il, usa) using 320-, 600and 1200-grit silicon carbide paper (carbimet disc set, #305178180, buehler, uk ltd, lake bluff, il, usa) in order to obtain a flat dentin area of at least 8 mm2, and the other half, to obtain a flat enamel with the same area. specimens were randomly assigned into 3 experimental groups according to the luting protocol, per tooth substrate (n=8): 1) relyx unicem (3m/espe, st. paul. mn, usa); 2) 37% phosphoric acid etching (scotchbond, 3m/espe) followed by relyx unicem; 3) relyx arc, as control (3m/ espe). luting agents were mixed and placed according to manufacturer’s instructions. for relyx arc, the specimen was acid etched, scotchbond multi-purpose plus (3m/espe) was applied followed by resin cement application. the composition and manufacturer’s information of the resin luting cements are shown in table 1. composite resin blocks (3 mm thick, 8 mm long and 4 mm wide) were built with a microhybrid composite (z250, shade a3; 3m espe) into a silicon mold. composite was lightcured for 40 s with a halogen light (optilux 501, kerr corp, orange, ca, usa, at 700mw/cm2 of irradiance) and then photocured at an edg unit for 3 min. the composite surface that would be in contact to the luting cement was airboneparticle abraded with 50µm aluminum oxide particles (asfer indústria química ltda, são caetano do sul, brazil) for 10 s. then, a silane couple agent (relyx ceramic primer, 3m/ espe) was applied on the composite block bonding surface. resin luting cements were applied according to the tested groups on enamel or dentin using manufacturer’s instructions, and then the composite blocks were cemented. specimens were light-cured for 40 s from the buccal, lingual and occlusal directions. after storage in distilled water for 24 h, specimens were serially sectioned perpendicular to the bonding interface into slabs with a diamond saw (isomet 1000, buehler, uk ltd, lake bluff, il, usa), and then hourglass-shaped specimens were created with a crosssectional bonded area of approximately 1 mm2. the specimens were fixed to the grips of a universal testing machine (emic 500, são josé dos pinhais, pr, brazil) using a cyanoacrylate adhesive (loctite super bonder gel, henkel, d düsseldorf, germany) and tested in tension at a crosshead speed of 0.5 mm/min until failure. for statistical analysis, the obtained data were analyzed (sas 9.1, sas institute, inc, nc) and submitted to a two-way anova and tukey’s test at 5 % of significance results there was no statistically significant difference in the bond strength values for the tested conditions at each tooth substrate (p>0.05). however, significant differences were found between enamel and dentin, where enamel showed higher microtensile bond strength means compared to dentin, 456456456456456influence of selective acid etching on microtensile bond strength of a self-adhesive resin cement to enamel and dentin braz j oral sci. 9(4):455-458 resin cements relyx unicem(batch #270644) relyx arc(batch #gu9jg) composition powder: glass fillers, silica, calcium hydroxide, self-curing initiators, pigments, light-curing initiators, substituted pyrimidine, peroxy compound.liquid: methacrylated phosphoric esters, dimethacrylates, acetate, stabilizers, selfcuring initiators, light-curing initiators. paste a: silane treated ceramic, bis-gma, tegdma, photoinitiators, amine, silane treated silica, functionalized dimethacrylate polymer.paste b: silane treated ceramic, tegdma, bis-gma, silane treated silica, benzoil peroxide, functionalized dimethacrylate polymer. manufacturer 3m espe, st paul, mn, usa 3m espe, st paul, mn, usa table 1. materials used in the study with composition and manufacturer’s information relyx arc relyx unicem acid etch + relyx unicem enamel 18.1 (2.0)aa 16.5 (3.8)aa 11.9 (6.6)aa dentin 8.1 (3.6)ab 9.2 (7.0)ab 8.3 (3.7)ab table 2. means and standard deviations (in mpa) of microtensile bond strength for the tested groups. mean values followed by different lowercase letters in columns and uppercase letters in rows are significantly different (tukey’s test; p<0.05). regarding the resin luting protocol (p>0.05). these results are shown in table 2. discussion the resin luting cements used in this study (relyx unicem and relyx arc) are broadly used on clinical practice for cementation of indirect restorations. moreover, self-adhesive resin cements have been used because of its simplified resin luting strategy, reducing clinical steps for cementation procedure, although its bond strength it is not totally clarified56,11. some studies have shown that the acidity of nonrinsed acidic primers can affect enamel bond strength22-24. to improve enamel adhesion, selective phosphoric acid etching prior to the application of self-adhesives resin cements has been suggested14,20-21. in this work, the tested hypothesis was rejected, since pretreatment with phosphoric acid etching did not affect enamel bond strength of the self-adhesive resin cement tested. for enamel, similar microtensile bond strength values were found, regarding the fixing system. a possible reason for this fact is that relyx unicem would present a low ph at the initial stage of polymerization. immediately after mixing the self-adhesive cement is very acidic, lower than ph 2 at the first curing minute, reaching at about 5 after this point, according to the manufacturer. this cement only reaches a neutral ph (at about 7). the low ph after mixing promotes only a superficial etching, increasing slightly the surface free energy and improving mechanical bond mechanism. however, it is known that hybrid layer is not formed in this interaction and part of the adhesion of self-adhesive cements occur due to chemical bonding to tooth hydroxyapatite14,20. in this manner, enamel prisms would be affected by phosphoric acid deep etching, maybe interfering bond strength. it goes against the majority of studies14,20-21, which state that micromechanical interaction of resin penetration between the crystallites and enamel rods associated with chemical bonds to hydroxyapatite could explain the higher bond strength. these studies observed an increase on bond strength between enamel and selfadhesive cements after pretreatment with phosphoric acid etching . however, etching time, acid concentration, ph, pka, hydroxyapatite buffer potential, and orientation of enamel prisms may significantly affect the demineralization of enamel and consequently, the bond strength25-27. bonding to dentin has been referred to be a less reliable strategy, especially when compared to enamel bonding, because of the intrinsic characteristics of dentin, like higher organic content, variations in tubular structure and fluid flow28-30. in this work, pretreatment with phosphoric acid etching did not affected relyx unicem bond strength to dentin, rejecting the tested hypothesis, maybe because no further cement penetration would occur even with opened and plug-free dentinal tubules31-33. thereby, the viscosity of this cement may hamper the infiltration into dentin11. collagen exposure to phosphoric acid does not seem to improve the bonding values of the hydrophobic autoadhesive cement34. the exact bonding mechanism of these simplified materials remains not clarified. thus, the results are in agreement with the literature, showing higher bond strength for enamel compared to dentin either with relyx arc or relyx unicem regardless of pretreatment with phosphoric acid etching for the self-adhesive cement14,20. the mechanical test used in this work to evaluate the bond strength values was the microtensile bond test35. some advantages of this methodology are easier specimen collection and uniform loading stress distribution over a small bonded area36-39. sticks, dumbbell bars or hourglass shapes can be the presentation of the microtensile specimens40-41. also, tensile tests are shown to be the most common laboratory tests to evaluate adhesive strength of bonding systems to the tooth substrate38-39,41. however, there are some limitations to consider about microtensile bond strength test, as stress on the bonded interface due to the handling, cutting procedure and the way of attachment40. the use of bovine teeth could be considered a limitation of the present work. this substrate is not as accurate as human teeth when different interactions between bond system and enamel/dentin are investigated. thus, bovine teeth were selected because they are a suitable substrate for the tested methodology, easier to collect compared to human teeth and do not rely on ethical problems. bovine enamel and dentin are adequate alternatives for human teeth, in adhesion tests42. however, it has been observed that a good marginal quality is more difficult to ensure with these bovine substrates4. in this sense, a successful technique in bovine teeth also tends to be successful in human teeth42. additionally, the use of bovine substrate has been supported by several authors43-46. in conclusion, selective acid etching did not improve microtensile bond strength of relyx unicem to enamel and dentin. the self-adhesive resin luting cement presented higher bond strength to enamel compared to dentin and similar values when compared to a conventional dual-cured resin cement. the results of this work cannot be directly extrapolated to clinical in vivo conditions, since clinical trials are still required. references 1. bandeca mc, el-mowafy o, saade eg, rastelli ans, bagnato vs, porto-neto st. changes on degree of conversion of dual-cure luting lightcured with blue led. laser physics. 2009; 19: 1050-5. 2. faria-e-silva al, moraes rr, ogliari fa, piva e, martins lrm. panavia f. the role of the primer. j oral sci. 2009; 51: 255-9. 3. cunha lg, alonso rcb, pfeifer csc, correr-sobrinho l, ferracane jl, sinhoreti mac. contraction stress and physical properties development of a resin-based composite irradiated using modulated curing 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acidity of self-etching primers affect bond strength and surface morphology of enamel? j adhes dent. 2006; 37: 35-41. 23. perdigão j, gomes g, gondo r, fundingsland jw. in vitro bonding performance of all-in-one adhesives. part i—microtensile bond strengths. j adhes dent. 2006; 8: 367-73. 24. van landuyt kl, peumans m, de munk j, lambretchs p, van meerbeek b. extension of a one-step self-etch adhesive into a multi-step adhesive. dent mat. 2006; 22: 533-44. 25. pashley dh. the effects of acid etching on the pulpodentin complex. oper dent. 1992; 17: 229-42. 26. carvalho rm, santiago sl, fernandes ca, suh bi, pashley dh. effects of prism orientation on tensile strength of enamel. j adhes dent. 2000; 2: 251-7. 27. salz u, mücke a, zimmermann j, tay fr, pashley dh. pka value and buffering capacity of acidic monomers commonly used in self-etching primers. j adhes dent. 2006; 8: 143-50. 28. pashley dh, sano h, ciucchi b, yoshiyama m, carvalho rm. adhesion testing of dentin bonding agents: a 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dammaschke t, zuchner h, schafer e. chemical analysis and bonding reaction of relyx unicem and bifix composites – a comparative study. dent mat. 2006; 22: 934-41. 35. sano h, shono t, sonoda h, takatsu t, ciucchi b, carvalho r et al. relationship between surface área for adhesion and tensile bond strength – evaluation of a micro-tensile bond test. dent mater. 1994; 10: 236-40. 36. cardoso pe, braga rr, carrilho mr. evaluation of micro-tensile, shear and tensile tests determining the bond strength of three adhesive systems. dent mater. 1998; 14: 394-8. 37. shono y, ogawa t, terashita m, carvalho rm, pashley el, pashley dh. regional measurement of resin-dentin bonding as na array. j dent res. 1999; 78: 699-705. 38. schwartzer e, collares fm, ogliari fa, leitune vcb, samuel smw. influence of zinf oxide-eugenol temporary cement on bond strength of an allin-one adhesive system to bovine dentin. braz j oral sci. 2007; 6: 1423-7. 39. spazzin ao, galafassi d, gonçalves ls, moraes rr, carlini-júnior. bonding to wet or dry deproteinized dentin: microtensile bond strength and confocal laser micromorphology analysis. braz j oral sci. 2009; 8; 181-4. 40. el zohairy aa, de gee aj, de jager n, van ruijven lj, feilzer aj. the influence of specimen attachment and dimensiono n microtensile strength. j dent res. 2004; 83: 420-4. 41. el zohairy aa, saber mh, abdalla ai, feilzer aj. efficacy of microtensile versus microshear bond testing for evaluation of bond strength of dental adhesive systems to enamel. dent mater. 2010; 26: 848-54. 42. reis a,giannini m, kavaguchi a, soares cj, line srp. comparison of microtensile bond strength to enamel and dentin of human, bovine and porcine teeth. j adhes dent. 2004; 6: 117-21. 43. nakamichi i, iwaru m, fusayama t. bovine teeth as possible substitutes in the adhesion test. j dent res. 1983; 62: 1076-81. 44. peutzfeldt a, asmussen e. determinants of in vitro gap formation of resin composites. j dent. 2004; 32: 109-15. 45. correr gm, alonso rcb, puppin-rontani rm, correr-sobrinho l, sinhoreti mac. marginal and internal adaptation of composite restorations using a resin liner on deproteinized substrate. acta odontol scand. 2005; 63: 227-32. 46. fais lmg, marcelo cc, silva rhbt, guaglianoni dg, pinelli lap. human teeth versus bovine teeth: cutting effectiveness of diamond burs. braz j oral sci. 2010; 9: 39-42. 458458458458458influence of selective acid etching on microtensile bond strength of a self-adhesive resin cement to enamel and dentin braz j oral sci. 9(4):455-458 oral sciences n3 original article braz j oral sci. 8(4):197-200 dna damage in viscose factory workers occupationally exposed to carbon di-sulfide using buccal cell comet assay pappuswamy manikantan1, vellingiri balachandar2; keshavarao sasikala3; subramanium mohanadevi4 1m.sc, m.phil, ph.d, pgdmbt, junior research fellow (ugc), division of human genetics, school of life sciences, department of zoology, bharathiar university, coimbatore, tamilnadu, india 2m.sc, m.phil, ph.d, senior research fellow (csir), division of human genetics, school of life sciences, department of zoology, bharathiar university, coimbatore, tamilnadu, india 3m.sc, ph.d, faz, mafrc, professor and head, division of human genetics, school of life sciences, department of zoology, bharathiar university, coimbatore, tamilnadu, india 4m.sc, m.phil, b.ed, ph.d, senior research fellow (csir), division of human genetics, school of life sciences, department of zoology, bharathiar university, coimbatore, tamilnadu, india correspondence to: pappuswamy manikantan division of human molecular genetics department of zoology, school of life sciences bharathiar university, coimbatore, tamilnadu, india-641046 e-mail: humangentistmani@gmail.com abstract aim: the most important industrial use of carbon disulfide (cs 2 ) has been in the fabrication of regenerated cellulose rayon by the viscose process and cellophane. cs 2 leads to increased frequency of chromosomal aberrations in workers with occupational exposure to cs 2 . methods: in the present study, the dna damage was analyzed by using buccal cell comet assay for 30 viscose plant workers who are occupationally exposed to cs 2 and 30 healthy individuals. both groups were classified as smokers and non-smokers and only the experimental subjects were classified based on the exposure period. the data were analyzed statistically by the student’s t-test. results: the results of this study showed increased levels of dna damage among viscose plant workers. conclusion: the habit of cigarette smoking among the viscose workers had a synergistic effect on inducing dna damage. keywords: carbon di-sulphide, dna damage, smoking, buccal cell comet assay. introduction carbon disulfide (cs 2 ) is an important industrial liquid organic solvent, which is mainly used to treat alkali cellulose in the viscose process (a source of rayon and cellophane). cs 2 may react with chlorine in the presence of a catalyst to form carbon tetrachloride also toxicants. in past years, studies have shown different potential cytotoxic effects of cs 2 on mammals1-2. acute and subacute poisoning appear due to exposure to cs 2 concentrations of 500-3000 mg/m3 and are predominantly characterized by neurological and psychiatric symptoms, gastrointestinal disturbances and genderual disorders3-10, whereas exposure to cs 2 concentrations above 5000 mg/m3 may induce coma or even death11. the toxic effects of cs 2 on experimental animals12-15 have been extensively demonstrated and epidemiological studies on cs 2 exposure among workers in viscose rayon plants have been also reviewed, including studies of ischemic heart disease �ihd) mortality for workers in the viscose rayon industry16-18. comprehensive testing of the mutagenic potential of cs 2 has been performed on several types of bacteria (ames test) and drosophila, with no positive results19. further studies on salmonella typhimurium, drosophila, human fibroblasts cultures, human blood leucocytes and rats have been inconclusive20. numerous studies have shown elevated standard mortality ratios (smrs) for workers occupationally exposed to cs 2 . nonetheless, there are no reports available for cs 2 that provide strong evidence of genotoxic effects on dna. since the buccal epithelium provides an alternative source of tissue for monitoring human exposure to occupational and environmental genotoxins21. the present study was carried out to assess, using buccal cell comet assay, the genotoxicity among viscose plant workers who are occupationally exposed to cs 2 . braz j oral sci. october/december 2009 volume 8, number 4 received for publication: august 19, 2009 accepted: november 11, 2009 198 braz j oral sci. 8(4):197-200 materials and methods subject recruitment the study subjects were 30 viscose plant workers and 30 healthy individuals as controls who were selected from various cities of southern india between august 2008 and january 2009. prior to enrollment in the the study, all subjects gave written informed consent. a questionnaire was used to collect information on gender, age, duration of exposure, use of protective masks, general health status, smoking habits and exposure to drugs for each experimental and control subject. there were 12 smokers and 18 non-smokers in each group. the average cigarette consumption of smokers in both groups was nearly 13.4 ± 3.0 (mean±standard deviation) cigarettes/day. ethical approval for this study was granted by the ethics committee of bharathiar university. sample collection buccal cells were collected from subjects by oral brushing. prior to brushing, subjects washed their mouth with normal saline to avoid the interference of mucus. collected samples were taken in cold phosphate buffer saline (pbs) and cells were allowed to pellet down. the cells were then resuspended in 300 ìl pbs and 50 ìl of cell suspension were taken for comet assay. comet assay comet assay was performed under alkaline conditions by using a standard protocol22 with some modifications23. cells were embedded in low melting point agarose on glass slide precoated with 1% normal agarose. after solidification of gel, the slide was submerged into cool lysis solution [2.5m nacl, 100 mm edta, 10 mm tris (ph 10.0), 1% lss lauryl sarcosine sodium salt to which 10% dmso, 1% triton x100 were freshly added] and kept overnight at 4°c. the slides were then placed on the horizontal electrophoresis unit filled with freshly prepared alkaline electrophoresis buffer (300 mm naoh, 1 mm edta, ph 13) for 30 min and then subjected to electrophoresis at 25v/ 300ma for 40 min. after electrophoresis the slides were neutralized for ~60 min in 0.4 m tris/hcl, ph 7.5 on ice, followed by staining in ethidium bromide (stock concentration 25 ìg/ml in distilled water) and mounting on glycerol. all steps were performed on ice to prevent the removal of thin agarose gel layer from the slide. the stained slides were examined under nikon fluorescent microscope with a 580nm emission filter. statistical analysis results are expressed as mean ± standard deviation. student’s t-test was performed to compare the dna damage levels between the experimental and controls. statistically significant levels were considered at p < 0.05. results the subjects were selected from viscose plant workers who are occupationally exposed to cs2. the tail movement of comets observed in the buccal cells of experimental and controls are given in table 1. in the control group, the percentage of dna damage observed among smokers was higher than that observed among non-smokers, though without statistically significant data was observed except for the 3 subjects aged 26-35 years. an age-related increase in dna damage was observed in both control and experimental subjects. experimental subjects over 46 years of age showed maximum dna damage (25.3±0.3%). significant increase (p < 0.05) of dna damage percentage was identified in most individuals of the experimental groups when compared to the control subjects. an increased level of dna damage was observed in the viscose plant workers with smoking habits when compared to smoking controls and nonsmoking viscose plant workers. to determine the effect of duration of exposure to cs 2 on dna damage, the workers were divided into 2 groups depending on whether they had less than 10 years of exposure or more than 10 years of exposure (table 2). no statistically significant difference in dna damage was observed with increased duration of exposure to cs 2 (table 2). discussion mutagenesis is involved in the pathogenesis of many neoplasias. occupational exposure may contribute to the development of pernicious illnesses, many times through mechanisms that involve genotoxic changes. continuous efforts have been made to identify genotoxic agents, to determine conditions of harmful exposure and to monitor populations that are excessively exposed24-25. groups number of subjects percentage of dna damage <25 5 6.3 ± 0.1 26-35 years 3 8.1 ± 0.3* 36-45 years 2 14.0 ± 0.3* 46-55 years 2 16.6 ± 0.2 <25 7 7.2 ± 0.4 26-35 years 4 8.6 ± 0.2 36-45 years 3 11.8 ± 1.2 46-55 years 4 14.1 ± 0.4 <25 5 14.1 ± 0.6 26-35 years 3 16.1 ± 0.5** 36-45 years 2 21.2 ± 0.1 46-55 years 2 25.3 ± 0.3** <25 7 11.4 ± 0.7* 26-35 years 4 14.4 ± 0.4* 36-45 years 3 18.5 ± 1.4* 46-55 years 4 20.2 ± 0.3 subjects control smokers control non-smokers experimental smokers experimental non smokers * p < 0.05 compared with non-smoking control subjects; ** p < 0.05 compared with smoking control subjects. table 1 classes of comets and percentage of dna damage among the control and experimental subjects dna damage in viscose factory workers occupationally exposed to carbon di-sulfide using buccal cell comet assay 199 braz j oral sci. 8(4):197-200 exposure period number of subjects percentage of dna damage <10 years 12 12.9 ± 3.1 > 20 years 18 13.27 ± 4.7 table 2 percentage of dna damage according to duration of exposure in experimental subjects the present study was designed to assess the dna damage among viscose plant workers who are occupationally exposed to cs 2 . comet assay is a valuable method for detection of occupational and environmental exposures to genotoxicants, and it can be used as a tool in risk assessment for hazard characterization26-27, air pollution28, cigarette smoking29 and various in vitro and in vivo studies30. in the present investigation, a notable dna damage was observed among the healthy controls. it is due to the assay being widely used in studying dna damage in healthy individuals31 and day to day variation in buccal epithelial cell strand breaks32. there was significant difference between experimental and control subjects who are occupationally exposed to cs 2 . in past years, cs 2 concentrations in viscose rayon plants averaged about 250 mg/m3; they were subsequently reduced to 50-150 mg/m3 and more recently exposure levels of cs 2 are mostly below 31 mg/m3,33. a report on hypospermia, asthenospermia and teratospermia in young workers exposed to 40-80 mg/m3 of cs 2 confirmed gonadal injury34. le and fu (1996)35 showed that the cs 2 induce chromosome aberration in human sperm. numerous epidemiological reports concluded that the cs 2 is toxicant to viscose industry workers36-38. in this study, experimental subjects with smoking habits showed maximum levels of dna damage when compared to respective controls, which shows that the cs 2 exposure with cigarette smoking has synergistic effect on inducing dna damage. chromosomal aberrations were shown to be good indicators of future risk of cancer39. likewise, dna damages are the ultimate causes of cancer because dna base changes can be mutagenic40. the present findings highlight the importance of investigating the genotoxicity of cs 2 on viscose plant workers occupationally exposed to this organic solvent when the smoking habit is associated, since this information provides an increased degree of identification for the positive response. acknowledgements the authors express their sincere thanks to workers from various viscose industry in south india and authorities of bharathiar university for their kind technical support and providing facilities and encouragement during the study. references 1. national institute for occupational safety and health. criteria for a recommended standard occupational exposure to carbon disulfide. washington, dc: us department of health education and welfare; 1977. 2. carbon disulfide. geneva: world health organization; 1979. (environmental health criteria, no. 10). 3. izmerov nf, editor. carbon disulfide. moscow: centre of international projects (gknt ); 1983. (scientific reviews of soviet literature on toxicity and hazards of chemicals, no.41). 4. jun-yi l, xaio-min f. human sperm chromosome analysis study on human sperm chromosome mutagenesis induced by carbon disulfide. biomed environ sci. 1996; 9: 37-40. 5. reinhardt f, drexler h, bickel a, claus d, ulm k, angerer j, et al. electrophysiological investigation of central, peripheral and autonomic nerve function in workers with long-term low-level exposure to carbon disulphide in the viscose industry. int arch occup environ health. 1997; 70: 249-56. 6. chu cc, huang cc, chu ns, wu tn. carbon disulfide induced polyneuropathy: sural nerve pathology, electrophysiology and clinical correlation. acta neurol scand. 1999; 94: 258-63. 7. wronska-nofer t, chojnowska-jezierska j, nofer jr, halatek t, wisniewska-knypl j. increased oxidative stress in subjects exposed to carbon disulphide (cs2) e an occupational coronary risk factor. arch toxicol. 2002; 76: 152e7. 8. wang c, tan x, bi y. su y, yan j, ma s et al. cross-sectional study of the ophthalmological effects of carbon disulfide in chinese viscose workers. int j environ health. 2002; 205: 367-72. 9. krstev s, perunicic b, farkic b, banicevic r. neuropsychiatric effects in workers with occupational exposure to carbon disulfide. j occup health. 2003; 45: 81-7. 10. nishiwaki y, takebayashi t, o’uchi t., nomiyama t, uemura t, sakurai h et al. six-year observational cohort study of the effect of carbon disulphide on brain mri in rayon manufacturing workers. occup environ med. 2004; 61: 225-32. 11. benowitz n. cardiovascular toxicology. in: ladou j, editor. occupational medicine, norwalk: appleton & lange; 1990. p. 237-46. 12. hoffman p, klapperstuck m. effects of carbon disulfide on cardiovascular function after acute and subacute exposure of rats. biomed biochim acta. 1990; 49: 121-8. 13. hoffman p, muller s. subacute carbon disulfide exposure modifies adrenergic cardiovascular actions in rats. biomed biochim acta. 1990;49: 115-20. 14. mooreman mp, sills rc, collins bj, morgan dl. carbon disulfide neurotoxicity in rats: ii. toxicokinetics. neurotoxicology 1998; 19: 88-98. 15. sills rc, harry gj, valentine wm, morgan dl. interdisciplinary neurotoxicity inhalation studies: carbon disulfide and carbonyl sulfide research in f344 rats. toxicol appl pharmacol. 2005; 207 (2 suppl): 245-50. 16. swaen gm, braun c, slangen jj. mortality of dutch workers exposed to carbon disulfide. int arch occup environ health. 1994; 66:103-10. 17. liss g, finkelstein m. mortality among workers exposed to carbon disulfide. arch environ health. 1996; 51: 193-200. 18. sulsky si, hooven fh, burch mt, mundt ka. critical review of the epidemiological literature on the potential cardiovascular effects of occupational carbon disulfide exposure. int arch occup environ health. 2002; 75: 365-80. 19. beauchamp ro, jr a. critical review of the literature on carbon disulfide toxicity. crit rev toxicol. 1983; 11: 169-278. 20. commission of the european communities (cec). solvents in common use. 1988 21. stone jg, jones nj, mcgregor ad, waters r. development of a human biomonitoring assay using buccal mucosa: comparison of smoking-related dna adducts in mucosa versus biopsies. cancer res. 1995; 55: 1267-70. 22. maluf sm, erdtmann b. evaluation of occupational genotoxic risk in a brazilian hospital. gen mol biol. 2000; 23: 485-8. 23. maluf sm, erdtmann b. follow-up study of genetic damage in lymphocytes of pharmacists and nurses handling antineoplastics drugs evaluated by cytokinesis-block micronuclei analysis and single cell gel electrophoresis assay. mutat res. 2000; 471: 21-7. 24. collins a, dusinska m, franklin m, somorovská m, petrovská h, duthie s, et al. comet assay in human biomonitoring studies reliability, validation and applications. environ mol mutagen. 1997; 30: 139-46. 25. valverde m, del carmen lópez m, lópez i, sanchez i, fortoul ti, ostroskywegman p et al. dna damage in leukocytes and buccal and nasal epithelial cells of individuals exposed to air pollution in mexico city. environ mol mutagen. 1998; 30: 147-52. 26. moller p. the alkaline comet assay: towards validation in biomonitoring of dna damaging exposures. basic clin pharmacol toxicol. 2006; 98: 336-45. 27. dusinska m, collins ar. the comet assay in human biomonitoring: gene– environment interactions. mutagenesis. 2008; 23: 191-205. 28. valverde m, del carmen lópez m, lópez i, sanchez i, fortoul ti, ostroskywegman p et al. dna damage in leukocytes and buccal and nasal epithelial cells of individuals exposed to air pollution in mexico city. environ mol mutagen. 1998; 30: 147-52. 29. rojas e, valverde m, sordo m, ostrosky-wegman p. dna damage in exfoliated buccal cells of smokers assessed by the single cell gel electrophoresis assay. mutat res. 1996; 370: 115-20. 30. fairbairn dw, olive pl, o’neill kl. the comet assay: a comprehensive review. mutat res. 1995; 33: 37-59. 31. bajpayee m, pandey ak, parmar d, mathur n, seth pk, dhawan a. comet assay responses in human lymphocytes are not influenced by the menstrual cycle: a study in healthy indian females. mutat res. 2005; 565: 163-72. 32. szeto yt, benzie iff, collins ar, choi sw, cheng cy, yow cm et al. a buccal cell model comet assay: development and evaluation for human biomonitoring and nutritional studies. mutat res. 2005; 578: 371-81. 33. daemen e, van risseghem m, de bacquer d, bulat p, braeckman l, vanhoorne m. preliminary external quality assessment for the biological monitoring of carbon disulfide with urinary 2thiothiazolidine-4-carboxylic acid. ann occup hyg. 1999; 43: 125-30. 34. lancranjan i, popescu hi, klepsch i. changes of the gonadic function in chronic carbon disulfide poisoning. med lav. 1969; 60: 556-71. dna damage in viscose factory workers occupationally exposed to carbon di-sulfide using buccal cell comet assay 200 braz j oral sci. 8(4):197-200 35. le jy, fu xm. human sperm chromosome analysis – study on human sperm chromosome mutagens induced by carbon disulfide. biomed environ sci.1996; 9: 37-40. 36. guidotti tl, hoffman h. indicators of cardiovascular risk among workers exposed to high intermittent levels of carbon disulphide. occup med (lond). 1999; 49: 507-15. 37. wang q, fu k, wu q. effects on fertility and menstrual cycle of female workers exposed to carbon disulfide. chin publ health. 1999; 15: 215-7 [in chinese]. 38. wang yf, shiu yf. investigation on eye injury of workers exposed to cs2. j lab med. 2000; 17: 89 [in chinese]. 39. hagmar l, brøgger a, hansteen il, heim s, hogstedt b, knudsen l et al. cancer risk in humans predicted by increased levels of chromosomal aberrations in lymphocytes: nordic study group on the health risk of chromosome damage. cancer res. 1994; 54: 2919-22. 40. poirier mc. dna adducts as exposure biomarkers and indicators of cancer risk. environ health perspect. 1997; 105 (suppl. 4): 907-12. dna damage in viscose factory workers occupationally exposed to carbon di-sulfide using buccal cell comet assay revista fop n 13 1571 braz j oral sci. april/june 2008 vol. 7 number 25 effects of bonded rapid maxillary expansion applianceeffects of bonded rapid maxillary expansion applianceeffects of bonded rapid maxillary expansion applianceeffects of bonded rapid maxillary expansion applianceeffects of bonded rapid maxillary expansion appliance (brmea) in vertical and sagittal dimensions:(brmea) in vertical and sagittal dimensions:(brmea) in vertical and sagittal dimensions:(brmea) in vertical and sagittal dimensions:(brmea) in vertical and sagittal dimensions: a systematic reviewa systematic reviewa systematic reviewa systematic reviewa systematic review moara de rossi, dds, msc1; renata andréa salviti de sá rocha, dds, msc1; maria beatriz duarte gavião, md, phd2 1phd student in pediatric dentistry 2professor department of pediatric dentistry, dental school of piracicaba, university of campinas, brazil received for publication: march 07, 2008 accepted: june 12, 2008 correspondence to: profa. dra. maria beatriz duarte gavião faculdade de odontologia de piracicaba – unicamp av. limeira 901 cep 13414-903 piracicaba, são paulo, brasil phone: +55-19-2106-5368. e-mail: mbgaviao@fop.unicamp.br abstract aim: the aim of this systematic review was to evaluate the existing literature about the effects of bonded rapid maxillary expansion appliance (brmea) on vertical and sagittal dimensions, and the possible advantages of its use. methods: a comprehensive search was performed in medline, pubmed, and web of science full-text electronic journal databases to retrieve english-language articles referring to brma. the inclusion criteria to initially select abstracts were: human clinical trials involving the use of brmea, measurements made from lateral cephalometric radiographs, no surgical treatments or clinical reports. from a total of 24 abstracts dealing brmea, only 7 fulfilled all inclusion criteria. after reading the full text articles, 4 remained. results: critical review of these papers revealed a great heterogeneity in the methodologies regarding the evaluation periods, sample characteristics, linear and angular cephalometric measurements. the studies presented on this review showed that the vertical effects are only partially controlled with bonded devices. conclusion: there is not sufficient evidence to support the use of brmea to control the undesirable effects of rapid maxillary expansion (rme). there is a need to study rme with brmea considering the patient’s facial patterns in order to determine whether this appliance is actually efficient in controlling the undesirable effects of rme. key-words: palatal expansion technique, cephalometry, orthodontics. i n t r o d u c t i o n rapid maxillary expansion (rme) is a treatment modality for maxillary transverse discrepancy that was introduced by angell in 18601. since this time, different appliances have been developed to open the midpalatal suture, such as hass-type, hyrax-type and brmea (bonded rapid maxillary expansion appliance). while haas and hyrax appliances are banded to posterior teeth, the brmea is bonded to posterior teeth by a full acrylic surface coverage that encloses all occlusal surfaces. regarding the maxillary and mandibular responses to rme, several studies2-7 have shown downward and forward maxilla displacement, dental extrusion, lateral rotation of the maxillary segments and cuspal interferences. these events lead to posterior rotation of mandible, open bite and increased vertical face dimension, which can be undesirable for patients with pronounced vertical facial growth patterns. brmas have been reported to help eliminating some of the extrusive effects of palatal expansion, due to the additional surface coverage, which limits unwanted tipping and rotation of teeth by increased rigidity8-9. however, there are contradictions in the literature concerning vertical and sagittal changes following rme performed with brmea. therefore, the purpose of this systematic review is to evaluate the existing literature about the effects of brmea on vertical and sagittal dimensions, and the possible advantages of its use. material and methods this literature review consisted of a comprehensive search in the medline (from 1966 to week 1 of march 2008), pubmed (from 1966 to week 1 of march 2008) and web of science (from 1945 to week 1 of march 2008) electronic journal databases to retrieve english-language articles that referred to brmea. the inclusion criteria to initially select the abstracts were: human clinical trials involving the use of brmea, 1572 database results selected % of selected abstracts (7)* medline 15 4 57.14 pubmed 24 7 100 web of science 4 1 14.28 table 1 distribution of the number of abstracts referring to brmea, number of abstracts that met the inclusion criteria and percentage of abstracts selected for analysis of the full-text papers for each electronic journal database. *percentages do not add up to 100% because same references were found in different databases measurements made from lateral cephalometric radiographs (to determine the sagittal and vertical effects of rme with this appliance) and no surgical treatments or clinical reports. two researchers independently selected the articles to be reviewed by reading their titles and abstracts in each database. all articles that seemed to meet the inclusion criteria on the basis of their abstracts were retrieved. one hundred percent agreement was reached between the two researchers in this phase. the full-text papers were then examined in an independent manner by both researchers. their reference list was also searched manually for additional relevant publications that could have been missed in the electronic database search. a consensus was reached on which articles actually fulfilled all inclusion criteria and should be included in this systematic review. r e s u l t s the distribution of the number of abstracts referring to brmea, number of abstracts that met the inclusion criteria and percentage of abstracts selected for analysis of the full-text papers for each electronic journal database is presented on table 1. four articles found in web of science were also found in medline and pubmed. fifteen abstracts found in medline were also found in pubmed. pubmed provided the largest number of selected abstracts. three articles found in pubmed were not found in the other databases. only 7 out of 24 abstracts initially selected actually met the inclusion criteria. four of them referred to studies that used other appliances associated with brmea7,10-12. after reading of the full-text articles, 17 of those 4 articles was maintained because it had a group of patients who wore only brmea. the other 3 articles10-12 were discarded after analysis because the subjects wore brmea and other appliances at the same time, and thus the obtained results could be attributed to the combination of the brmea with other therapies, such as edgewise orthodontics10,12, incisor intrusion11 and vertical pull chincup therapy11-12. in the end, only 4 four articles remained 6-9. the methodologies and results of each selected paper are summarized in table 2. d i s c u s s i o n a critical review of the full-text articles a great heterogeneity in the methodologies regarding the evaluation periods, sample characteristics, such as age and gender, linear and angular cephalometric measurements. sarver and johnston8 reported the effects of brmea and compared their results to those of wertz4, whio used a banded appliance. the anterior movement of the maxilla in the bonded sample was lower than that observed in the banded sample. according to the authors8, limited anterior movement of the maxilla with the brmea would be an indication for use in class ii patients. this study showed that there was lesser extrusion of the maxilla with the brmea and postulated that the thickness of the acrylic acts as a deterrent for extrusion. asanza et al.9 also studied the difference between a banded appliance and the brmea and found anterior movement of the maxilla in group i (hyrax) and posterior displacement in group ii (brmea). in group i, the maxilla moved inferiorly with subsequent posterior and downward displacement of the mandible. the group treated with the brmea showed less inferior movement of the maxilla and a relative stability in the lower face height. these findings can be of importance in the treatment of patients with a long face, in which extrusion of the maxilla or the maxillary dentition would worsen the open bite situation and create more difficulty to treat vertical pattern89. it is important to note that these two studies presented similarities in their methodologies 8-9: the evaluation periods were “before treatment” and “after 3 months of retention period”, the appliances were similar, and 7 of the cephalometric measurements were the same in both studies. this could explain the similar results obtained in both articles. akkaya et al.6 determined the vertical and sagittal effects of bonded rapid and slow maxillary expansion procedures, and compared these effects between the groups. comparing these two treatment modalities is not the goal of the present review, but akkaya’s et al.6 study clearly demonstrates the effects of treatment with brmea (group i). the maxilla showed anterior displacement, and there was a posterior rotation of the mandible after use of brmea. bascifitci and karaman7 compared the effects of brme therapy alone to those of brme combined with vertical chin cap. in group i (brmea only), the mandible rotated downward and backward and the lower anterior facial height increased. in group ii (brmea with vertical chin cap), the mandibular plane decreased, and vertical displacement of the maxilla occurred only in group i, which is in agreement with the findings of akkaya et al.6 but differ from those of sarver and johnston8. the long lower facial height moved anteriorly in both groups. the authors affirm that the use of vertical chin cap during and after rme is important to control the vertical dimension, especially in subjects exhibiting long lower facial height. braz j oral sci. 7(25):1571-1574 effects of bonded rapid maxillary expansion appliance (brmea) in vertical and sagittal dimensions: a systematic review 1573 fi rst au th or year stu dy gr oups sample mean age (years ) evalua tion perio ds expansi on peri od (mean s ) retent io n peri od (mean s ) la te ral ceph alome tr ic meas u rements bascifitti 7 2002 i brmea ii brmea with vertica l chin cap i n=17 10 girls 7 boys ii n=17 15 girls 2 boys i 12.8 ii 12.6 t1before treatment t2after treatment t3aft er retent ion i 5.2 we eks ii 5.3 weeks i 12.1 weeks ii 12.9 weeks sna; snb; anb; sn-mp;sn-pp; pp-mp; sn? pns; sv? a; sv? b; n -ans; an s-me ; u1p-sn; l1 p-mp; sn? u1; sn? u6; sv? u1 ; sv? l1; mp? l1; mp? l 1; ul-e; ll-e akkaya 6 1999 i brmea ii bo nded slow mea i n=12 5 girls 7 bo ys ii n=12 5 girls 7 boys i 11.9 ii 12.3 t1-bef ore treatment t2after treatment t3aft er retent ion n ot cit ed 3 months n-s-ba; sna; sn/ans-pns; n-pg-a; sn b; sn/mp; anb; ans-pns/amp; sn/occlusal plane; 1/sn; 1´/1; overjet, ove rbite ; e plane asanza 9 1997 i hyra x ap plian ce ii brmea n=14 7 girls 7 boys assigned to 2 groups range: 8.5-16 yrs t1before treatment t2 after treatm ent n ot cit ed 3 months sna, snb, anb, sn-pp; sn-mp; sn-u1; sn-pns; sn-ans; ans-me;u6-pp; sapt. sarver 8 1989 i brmea ii band ed applia nce (wertz 4 ) i n=20 14 g irls 6 boys ii n =60 37 g irls 23 boys i 10.8 ii girls: 7-29 boys: 8 -14 t1before treatment t2after treatment varied 3 months sna; snb; anb; sn -pp; sn -mp; snpns; sn-ans; s-a, s-1; sn.1; sn-1 ii more downward movement than in group i i less downward movement than in group ii ii forward i backward ii downwar d i dow nwar d ii ba ck wa rd i backward sarver8 ii downward i less downward movement than in group ii ii backward i forward ii downwar d i dow nwar d ii ba ck wa rd i backward asanza9 not relevant for the review analysis i downward not relevant for the review analysis i forward not relevant for the review analysis i dow nwar d not rel ev ant for the rev iew an aly sis i backward akkaya6 ii no change i downward ii forward i forward ii no change i dow nwar d ii no ch an ge i backward bascifitti 7 maxilla vertical displacementaxillary sagittal displacement mandible vertical displacement mandibular sagittal displacement fisrt author ii more downward movement than in group i i less downward movement than in group ii ii forward i backward ii downwar d i dow nwar d ii ba ck wa rd i backward sarver8 ii downward i less downward movement than in group ii ii backward i forward ii downwar d i dow nwar d ii ba ck wa rd i backward asanza9 not relevant for the review analysis i downward not relevant for the review analysis i forward not relevant for the review analysis i dow nwar d not rel ev ant for the rev iew an aly sis i backward akkaya6 ii no change i downward ii forward i forward ii no change i dow nwar d ii no ch an ge i backward bascifitti 7 maxilla vertical displacementaxillary sagittal displacement mandible vertical displacement mandibular sagittal displacement fisrt author table 2 summary of the methodologies and results of each selected paper. table2 – extended although the brmea has been shown to cause less vertical alterations than the banded appliances, some alterations were still present8-9. downward movement of the maxilla, downward and backward rotation of the mandible using brmea alone were verified6,10. bascifitti and karaman9 showed a forward displacement of the maxilla. however, in asanza’s et al.7, and sarver and johnston’s8 samples, some subjects exhibited a forward displacement of the maxilla, which can be seen by the sna variation, that ranged from -5° to +1°, and from -3.6° to 1.7°, respectively. these differences may be attributed to changes in the sample characteristics to the distinct responses that each individual present to the treatments. the patient’s facial pattern is also an important issue to braz j oral sci. 7(25):1571-1574 effects of bonded rapid maxillary expansion appliance (brmea) in vertical and sagittal dimensions: a systematic review 1574 be considered and can interfere with the choice, response and prognosis of the whole orthopedic and orthodontic treatment. in all research articles retrieved for the present review, the subjects were enrolled without any consideration to their skeletal facial pattern. in this way, we believe that there is a need to study rme with brmea considering the patient’s facial patterns in order to determine whether this appliance is actually efficient to control the undesirable effects of rme in all situations. it has been reported that after rme therapy the maxilla will partially3 or completely4 return to its original position. haas2 has stated that active facial sutures and bones force the maxilla to return to its original position. the tendency of the maxillary skeletal segments to return to their origin can also be attributed to accumulated forces in the circummaxillary articulations13, occlusal forces, surrounding buccal musculature14, and stretched fibers of the palatal mucosa15. thus, the long-term changes can be of little, if any, clinical significance. the studies presented on this review6,7,9,12 showed that the vertical effects are only partially controlled with bonded devices. however, their observations were limited to the time of use of the brmea, including a 3-month stabilization period. this way, further studies should evaluate the dimensional changes occurring at longer periods after removable appliance retention. to the present, the orthodontist should be aware that brmea is an option for treatment of bilateral maxillary posterior deficiency, regardless of patient’s facial patterns. based on this systematic review, it maybe concluded that: the brmea caused less downward and backward displacement of the mandible than the banded appliances, but these alterations were not completely absent; there is no consensus in the literature regarding the maxillary sagittal displacement after rme; there is not sufficient evidence to support the use of brmea to control the undesirable effects of rme. a c k n o w l e d g e m e n t s this paper was prepared in the discipline “advanced pediatric dentistry issues” from the phd program in dentistry of the dental school of piracicaba, state university of campinas. the authors acknowledge fapesp ((process 05/03472-4)and cnpq for the phd scholarships granted to 1st and 2nd authors, respectively, and the professors of the department of pediatric dentistry, of the dental school of piracicaba, state university of campinas, for their incentive. r e f e r e n c e s 1. angell eh. treatment of irregularity of the permanent or adult teeth. dent cosmos. 1860; 1: 540-4. 2. haas aj. rapid expansion of the maxillary dental arch and nasal cavity by opening the midpalatal suture. angle orthod. 1961; 31: 73-9. 3. haas aj. the treatment of maxillary deficiency by opening the midpalatal suture. angle orthod. 1965; 35: 200-17. 4. wertz ra. skeletal and dental changes accompanying rapid midpalatal suture opening. am j orthod. 1970; 58: 41-66. 5. wertz ra, dreskin m. midpalatal suture opening: a normative study. am j orthod. 1977; 71: 367-81. 6. akkaya s, lorenzon s, ucem tt. a comparison of sagittal and vertical effects between bonded rapid and slow maxillary expansion procedures. eur j orthod. 1999; 21: 175-80. 7. basciftci fa, karaman ai. effects of a modified acrylic bonded rapid maxillary expansion appliance and vertical chin cap on dentofacial structures. angle orthod. 2002; 72: 61-71. 8. sarver dm, johnston mw. skeletal changes in vertical and anterior displacement of the maxilla with bonded rapid palatal expansion appliances. am j orthod dentofacial orthop. 1989; 95: 462-6. 9. asanza s, cisneros gj, nieberg lg. comparison of hirax and bonded expansion appliances. angle orthod. 1997; 67: 15-22. 10. reed n, ghosh j, nanda rs. comparison of treatment outcomes with banded and bonded rapid palatal expansion appliances. am j orthod dentofacial orthop. 1999: 116: 31-40. 11. pearson le, pearson bl. rapid maxillary expansion with incisor intrusion: a study of vertical control. am j orthod dentofacial orthop. 1999; 115: 576-82. 12. schulz so, mcnamara ja jr, baccetti t, franchi l. treatment effects of bonded rme and vertical-pull chincup followed by fixed appliance in patients with increased vertical dimension. am j orthod dentofacial orthop. 2005; 128: 326-36. 13. brossman re, bennet cg, merrow ww. facioskeletal remodeling resulting from a rapid expansion in the monkey (macaca cynomolglus). arch oral biol. 1973; 18: 987-94. 14. bishara se, staley rn. maxillary expansion: clinical implications. am j orthod dent orthop. 1989; 91: 3-14. 15. maguerza oe, shapiro pa. palatal mucoperiostomy: an attempt to reduce relapse after slow maxillary expansion. am j orthod. 1980; 78: 548-58. braz j oral sci. 7(25):1571-1574 effects of bonded rapid maxillary expansion appliance (brmea) in vertical and sagittal dimensions: a systematic review revista fop n 13 1585 clinical evaluation of packable resin class i restorations after 7 years joão batista novaes junior1; patrícia valente araújo2; fernanda damas3; alfonso gala-garcia2; maria esperanza cortés4 1dds, ms, phd, associated professor, department of clinic, pathology and surgery, cpc 2dds, ms, graduate student, department of restorative dentistry 3dds, private practitioner 4dds, ms, phd, associated professor, department of restorative dentistry federal university of minas gerais received for publication: may 06, 2008 accepted: september 09, 2008 correspondence to: patricia valente araujo av. antônio carlos, 6627 pampulha belo horizonte – mg cep 31270-901 email: patbhz@terra.com.br a b s t r a c t aims: compactable composite resins have been indicated as amalgam substitutes. however, longitudinal clinical trials are necessary to verify their wear resistance and integrity maintenance. the purpose of this study was to evaluate the clinical performance of a packable resin in class i restorations after 7 years. methods: forty restorations were placed in 15 young patients aged 13 to 30 years. the restorative material (prodigy/optibond-kerrâ) was used according to standard recommendations. the restorations were assessed at baseline at the year 2000 and after 2 and 7 years according to the usphs (united state public health service) criteria. results: after 7 years, 50% of the restorations were scored alpha and 37.5% scored bravo for color match, marginal discoloration and marginal adaptation. secondary caries were observed in only one restoration and none of them presented postoperative sensibility. conclusion: in accordance with the usphs method used, the packable resin presented appropriate clinical performance after a 7-year clinical evaluation. key words: packable resin, usphs criteria, clinical evaluation. i n t r o d u c t i o n in recent years, the placement of resin-based direct composite restorations has become a routine and wellestablished dental procedure. despite the excellent longterm results obtained with amalgam restorations, speculation about the possible healthy risks associated with mercury and the demand for esthetic restorative materials have contributed to the increase use of composite resins in posterior applications. in addition, bonded restorations provide a more conservative cavity preparation by preserving valuable tooth structure1. composite resins were introduced in the 1970s, but their clinical use revealed serious problems such as excessive wear, leakage, secondary caries and postoperative sensitivity2. some modifications were done and with the improvement of the physical and chemical properties, the composite resins started being indicated for posterior teeth restorations. at the present moment, they are substantially superior to those produced years ago, even though secondary caries and wear resistance still continue being reasons for investigation. in face of these difficulties, changes in the handling characteristics of composite resins have been made with the aim of improving the composite placement, and several new products, called condensable or packable resin composite, were introduced to the dental market3. the packable resins present a linear thermal contraction coefficient of 1.8% and 80% of load percentage in volume. the particles occupy their space more efficiently and due to a greater percentage of loads, the volume of matrix is reduced, resulting in a much smaller curing contraction and producing a superior marginal integrity than the conventional composite resins. the packable resins are indicated to recover areas of great masticatory efforts because of their capacity to resist to the deformation and because they have better viscoelasticity than the amalgam4. for some authors the wear resistance is similar to the one of the amalgam5-6. nevertheless, when these materials were compared with conventional resins, the chemical components were practically the same, only differing in proportions7, and the mechanical properties did not present great differences8-9. although analysis of mechanical and physical properties provides valuable information, no in vitro method can totally subject the materials to the comprehensive and hard test conditions of the oral cavity. ultimately, the clinical qualification can only be obtained in clinical studies3. a clinical evaluation of restorative materials, by clinical braz j oral sci. july/september 2008 vol. 7 number 26 1586 baseline (n=40) 2 year (n=40) 7 year (n=40) criteria a b c a b c a b c marginal staining 40 26 14 4 3 color match 40 34 6 4 3 secondary caries 40 40 6 1 superficial texture 40 34 6 4 3 table 1 clinical evaluation of packable resin after 2 and 7-year according to the usphs criteria observation, was described by cvar and ryge10 in 1971 and used in the method united state public health service (usphs). this method has been used in several studies in the evaluation of direct restorative materials, mainly for materials that would replace the amalgam restorations. the purposed evaluation made a longitudinal analysis of the restorative material, independent of other factors that could interfere with their clinical performance11. the aim of this in vivo study was to evaluate the clinical performance of classe i restorations restored with a packable resin, after 7 years, using the usphs method. material and methods this study was approved by research ethics committee of federal university of minas gerais. patients in treatment in the primary attention clinic 1 and 2 of the dental school of the federal university of minas gerais were selected. forty restorations were made in patients at ages ranging from 13 to 30 years. the clinical and radiographic diagnosis indicated the need of restoration of occlusal lesions. after a suitable disease control, including plaque control and sealing cavities to eliminate any microbial niche, the restorative treatment was executed. the packable resin used was prodigy/optibond-kerrâ. the restorations were done by following clinical steps: rubber dam isolation, removal of decay, preparation of the cavity and protection of the dentin-pulp complex. the specific operating steps of the material like acid conditioning, application of adhesive systems, material insertion and photopolymerization followed the manufacturer’s instructions. the restorations were made by the students supervised by the professors. the re-evaluations were made at baseline and after 2 and 7 years, by two calibrated operators. the criteria and codes used were those of method usphs, as described below: color match alfa (a): no mismatch of color, shade and translucency between restoration and adjacent tooth bravo (b): slight mismatch of color shade and translucency, but within normal clinical limits charlie (c): mismatch of color and non-esthetic appearance. unacceptable clinically. marginal staining alfa (a): no penetration of staining at the marginal interface bravo (b): penetration along the margin, but not in a pulpal direction charlie (c): penetration at the margin to the level of dentin or in a pulpal direction. unacceptable clinically. secondary caries: alpha (a): no caries lesions present in the restoration margins; bravo (b): caries lesions present in the restoration margins, indicating change of the restoration. superficial texture: alpha (a): smooth superficial texture similar to that of the enamel, as compared by the explorer; bravo (b): superficial texture slightly rougher than that of the enamel. acceptable clinically; charlie (c) superficial texture moderately rougher than that of the enamel. sandy surface texture, similar to that of chemically polymerized resins. acceptable clinically; delta (d): rough surface in depth and extension stopping the sliding of the explorer in the surface in a continuous way. when disagreements arose during evaluations, consensus was reached between examiners. because of the non-normal distribution observed by the shapiro-wilk test, the mc nemar test non parametric was performed at a significance level of á= 0.05. the analysis was performed with the data collected at baseline, 2-year and 7-year period. r e s u l t s the results are summarized in table1. at baseline, the 40 restorations evaluated received alpha score in all categories. all restorations were re-evaluated after 2 years, and an increase in marginal staining could be observed. no secondary caries was observed after 2 years (100% alpha). nevertheless, some restorations presented postoperative sensitivity and had to be replaced. after 7 years, only 8 restorations could be evaluated. one of them braz j oral sci. 7(26):1585-1590 clinical evaluation of packable resin class i restorations after 7 years 1587 had been replaced by amalgam in the period between the evaluation of 2 and 7 years. there was no postoperative sensitivity in any of the restorations evaluated after 7 years, and only 1 presented secondary caries. all restorations evaluated in this study demonstrated acceptable clinical performance within the evaluation period based on the alfa and bravo ratings for clinically satisfactory restorations. figure 1 shows the frequency distribution of the evaluated criteria. the figures 2 and 3 show the restorations evaluated after 2 years, presenting alpha and bravo criteria and the figure 4 shows restorations evaluated after 7 years. a statistically significant difference (p<0.05) was observed for marginal staining, surface texture and color match after 2 years follow-up and there was no significant difference (p>0.05) regarding secondary caries during this period. although a statistically significant difference (p<0.05) could be observed for the above-mentioned criteria, all restorations were considered clinically acceptable, since this difference was attributed to variations between alpha or bravo criteria, both considered satisfactory from a clinical standpoint. because of the low rate of patients evaluated after 7 years, statistical analysis was not possible. d i s c u s s i o n resin-based composites have been increasingly used as restorative materials and the new brands have been marketed in the same rate. thus, the greatest problem with clinical trials in the evaluation of posterior composite restorations is that the brands are constantly changed by manufacturers. fig. 3a and 3b packable composite resins restorations after 2 years of evaluation, showing bravo score for marginal staining, according to usphs criteria. fig. 2a and 2b packable composite resins restorations after 2 years of evaluation, showing alpha score according to usphs criteria. fig. 1 frequency distribution of usphs criteria braz j oral sci. 7(26):1585-1590 clinical evaluation of packable resin class i restorations after 7 years 1588 however, clinical investigations with composite resins are important for predicting the longevity of current and future ones2-3. interest in clinical studies focuses on the reasons for clinical failures. while collins et al.12 described the most common types of failures in composite resin restorations as bulk fractures and secondary caries, burke et al.13 reported secondary caries as the most prevalent reason for replacement of restorations. in the present clinical study, secondary caries were not observed in any of the restorations after 2 years and in only 1 restoration after 7 years. a possible explanation for this good clinical performance is the fact that all restorations were done in conservative cavities with all margins in enamel, which probably contributed for the more effective sealing, reducing marginal infiltration. in addition, all patients were instructed about oral hygiene and the restorations were done after a suitable disease control, including plaque control and sealing cavities. the marginal seal is another important parameter to be considered in a clinical evaluation. regarding marginal adaptation, a restoration could be considered clinically acceptable when no visible evidence of crevice along margin can be detected by explorer (alpha score) or when the crevice is detected, but without exposure of the dentin or base (bravo score). the charlie score presents dentin or base exposed and in the delta score the restoration is mobile or fractured. these last two scores are considered clinically unacceptable. although in this study we did not include this criterion in the clinical evaluation using usphs method, the lower percentage of secondary caries could indicate a good marginal adaptation. moreover, during the follow-up investigation fractures were not observed. other studies demonstrated a good marginal adaptation after clinical evaluation: in a 3-year evaluation, loguercio et al.3 observed that only 10% of the restorations showed evidence of a slight crevice along the marginal interface (bravo score). burke et al.13 evaluated the packable resin solitaireâ and found good results for marginal adaptation, when 88% of the restorations were considered alpha or bravo after a 2 years evaluation. although almost all restorations were considered clinically acceptable in the evaluated period, being rated alpha mostly and bravo in some cases, important findings were noted and must be mentioned. the most affected criterion was the marginal staining, where 35% of the restorations received bravo score after 2 years. this is consistent with findings of lopes et al.2, who reported that the performance of the prodigy condensable/optibond solo® (kerr) after 2 years was different from that at baseline. this composite resin showed a significant increase in marginal staining2. after 7 years, 37% of the evaluated restorations had bravo score for marginal staining. according to lopes et al.2, this might be due to the patients, especially their habits, oral hygiene, and the extent to which they are influenced by external factors such as drinks, food, cigarettes and other things that possess stain elements. the alterations in surface texture can be attributed to many variables related to the inorganic filler of resin composites like size, hardness and amount of inorganic loading. the color match is thought to be involved with the organic matrix of resin composites. however, the interaction of these factors, should not be overlooked, as materials with a very rough surface will retain plaque and stains more easily, which can certainly contribute to color mismatch3. this interaction could be noted in the present study, because in both evaluation periods, 2 and 7 years, all restorations that had bravo score for surface texture, showed the same score for color mismatch. changes in surface texture and color match could be used to indirectly measure the wear using the usphs criteria13. prodigyâ condensable resin (kerrâ), by the use of rheological control additive (rca) and a high filler loading (80% by figure 4a and 4b packable composite resins restorations after 7 years of evaluation, showing alpha and bravo score respectively. braz j oral sci. 7(26):1585-1590 clinical evaluation of packable resin class i restorations after 7 years 1589 weight), presents “packable” characteristics14. this material is characterized by a high filler load and a filler distribution giving them a stiffer consistency than hybrid composites. the preferential wear of the softer matrix over time may cause the protrusion of some high-sized particles. bayne et al.15 suggested that the presence of these large particles may theoretically cause greater wear of the restorative material and the antagonist enamel. according to this author, the stress concentration through the filler particle and into the resin matrix when the restoration is under masticatory function will lead to the easy removal of these particles in the surface, exposing the organic matrix and accelerating the wear process even more. the good clinical performance observed after 7 years, where 50% of the restorations were scored alpha and 37% bravo for surface texture, could be attributed to the cavity size. the conservative outline form, preserving tooth structure, allows less occlusal contact on the restoration and reduces roughness and wear. certainly, the more conservative cavity contributed for the maintenance of an adequate surface texture during the evaluated period. over the years, composite resins with different formulations such as macrofilled, microfilled, hybrid, and “packable” resins were introduced into the dental market14. the average annual wear of several recent-generation posterior resins has been shown in laboratory and clinical studies to be equivalent to that of silver amalgam14. according to lopes et al.14, the use of composite resins for the restoration of posterior teeth still presents other problems such as microleakage, postoperative sensitivity, secondary caries, and technical difficulties, although these problems have not been observed in the present study . it is important to observe that only 8 restorations could be evaluated after 7 years. the recall rate after 2 years was excellent, and all the restorations were re-evaluated. therefore, after 7 years, some difficulties were found as the recall of patients was done. firstly, it is important to mention that the dental school changed its address between the 2and 7-year evaluation and the new school is located very far from the old one. consequently, many patients did not update their records and then could not be reached. secondly, patients’ age was a problem. when they were first evaluated they were adolescents, with a more flexible schedule and after 7 years many had jobs and were therefore unable to attend the re-evaluations, since the class time at the university coincided with the working hour of the patients. the usphs method has been used for most the clinical studies1-3,14,16 and this method was selected for this study to facilitate the comparison with other studies. according to this method, after 3 years of use, no more than 10% of the total of restorations can be classified as “charlie” or unacceptable. such values must be maintained for 5 years. no stipulation is made for the minimum percentage of restorations classified as “bravo”. long term evaluations have demonstrated little variation of criteria usphs. a 7 year investigation on posterior composite restorations (n=70) using z100® (3m), clearfil ray-later® (kuraray) or prism tph® (dentsply) showed that 4 restorations failed due to the presence of secondary caries. no statistically significant difference was found between the materials when the color match, anatomic form and secondary caries were compared. clearfil ray-later® (kuraray) presented a rough surface texture different from the surrounding enamel, and it was statistically significantly different from the other materials evaluated. z100 (3m) showed more marginal staining compared to other resins after 5 years. all materials had marginal adaptation problems after 7 years, but their clinical performance was acceptable17. yip et al.18 evaluated 57 class i and 45 class ii composite resin restorations in permanent teeth, according to the usphs method. the packable resin used was surefil®, (dentsply) and the conventional hybrid resin was spectrum tph® (dentsply). three surefil® restorations failed before their initial evaluation. for both composite resins, the alpha score for marginal staining was given in 90% of the restorations. a small percentage of restorations was classified as alpha for color match, marginal integrity and gingival health. occasional postoperative sensitivity was verified in 4 surefil® and spectrum tph® restorations both restorative materials had a satisfactory clinical performance after 1 year and no difference in the evaluated parameters was perceived. regarding the evaluation period, few differences were detected and a failure rate of 6% was considered not significant in some studies19. these authors19 evaluated 47 restorations with surefil® (dentsply) after three years. in 31 restorations, no differences were observed regarding color match. two restorations of the same patient were lost after 1 month and were rated as charlie until the end of the study. after 3 years, only 5 restorations were classified as bravo and 1 as charlie for marginal staining. the score bravo was found in 5 restorations for marginal adaptation, and in 3 restorations for anatomic form (p<0.05). according to the authors, the clinical performance of these class ii restorations was considered excellent. based on the results obtained in this study, it may be concluded that the evaluated packable resins exhibited satisfactory clinical performance after 7 years with respect to color match, surface texture, marginal staining and secondary caries. a c k n o w l e d g m e n t s the authors thank to prpq (pró-reitoria de pesquisa da ufmg), which supported this project, and to kerrâ for supplying the material to be tested. r e f e r e n c e s 1. fagundes tc, barata tj, bresciani e, cefaly df, jorge mf, navarro mf. clinical evaluation of two packable posterior composites. clin oral invest. 2006; 10: 197-203. braz j oral sci. 7(26):1585-1590 clinical evaluation of packable resin class i restorations after 7 years 1590 2. lopes lg, cefaly df, franco eb, mondelli rf, lauris jr, navarro mf. clinical evaluation of two “packable” posterior composite resins: two year results. clin oral invest. 2003; 7: 123-8. 3. loguercio ad, reis a, hernandez pa, macedo rp, busato al. 3-year clinical evaluation of posterior packable composite resin restorations. j oral rehabil. 2006; 33: 144–51. 4. marghalani hy, al-jabab as. compressive creep and recovery of light-cured packable composite resins. dent mater. 2004; 20: 600-10. 5. yap au, teoh sh, chew cl. effects of cyclic loading on occlusal contact area wear of composite restoratives. dent mater. 2002; 18: 149-58. 6. suzuki s. does the wear resistance of packable composite equal that of dental amalgam? j esthet restor dent. 2004; 16: 355-65. 7. silva aab, veeck eb, oliveira jpp, souza phc. comparison of chemical composition of packable resin composites by scanning electron microscopy. j appl oral sci. 2005; 13: 65-3. 8. cobb ds, macgregor km, vargas ma, denehy ge.. the physical properties of packable and conventional posterior resinbased composites: a comparison. j am dent assoc. 2000; 131: 1610-5. 9. kelsey wp, latta ma, shaddy rs, stanislav cm. physical properties of three packable resin-composite restorative materials. oper dent. 2004; 25: 331-5. 10. cvar jf, ryge g. criteria for the clinical evaluation of dental restorative materials. san francisco: us public health service publication no 790-244, government office; 1971. 11. hawthorne ws, smales rj. factors influencing long-term restoration survival in three private dental practices in adelaide. aust dent j. 1997; 42: 59-63. 12. collins cj, bryant rw, hodge kl. a clinical evaluation of posterior composite resin restorations: 8-year findings. j dent. 1998; 26: 311-7. 13. burke fj, cheung sw, mjör ia, wilson nh. restoration longevity and analysis of reasons for the placement and replacement of restorations provided by vocational dental practitioners and their trainers in the united kingdom. quintessence int. 1999; 30: 234-42. 14. lopes lg, cefaly df, franco eb, mondelli rf, lauris jr, navarro mf. clinical evaluation of two “packable” posterior composite resins. clin oral invest. 2002; 6: 79-83. 15. bayne sc, taylor df, heymann ho. protection hypothesis for composite wear. dent mater. 1992; 8: 305-9. 16. oberländer h, hiller ka, thonemann b, schmalz g. clinical evaluation of packable composite resins in class-ii restorations clin oral invest. 2001; 5: 102-7. 17. turkun ls, aktener bo, ates m. clinical evaluation of different posterior resin composite materials: a 7-year report. quintessence int. 2003; 34: 418-26. 18. yip kh, poon bk, chu fc, poon ec, kong fy, smales rj. clinical evaluation of packable and conventional hybrid resinbased composites for posterior restorations in permanent teeth: results at 12 months. j am dent assoc. 2003; 134: 1581-9. 19. ernst cp, martin m, stuff s, willershausen b. clinical performance of a packable resin composite for posterior teeth after 3 years. clin. oral. investig. 2001; 5: 148-55. braz j oral sci. 7(26):1585-1590 clinical evaluation of packable resin class i restorations after 7 years original article braz j oral sci. january/march 2009 volume 8, number 1 cephalometric evaluation of effectiveness interland headgear on class ii, division 1, malocclusion ana zilda nazar bergamo de carvalho1, meire alves sousa2, vânia célia vieira de siqueira3 1 dds, ms, orthodontic division, faculdade de odontologia de piracicaba, universidade estadual de campinas (unicamp), piracicaba (sp), brazil 2 dds, ms, graduate student, department of radiology, orthodontic division, faculdade de odontologia de piracicaba, unicamp, piracicaba (sp), brazil 3 dds, ms, phd, professor, department of pediatric dentistry, orthodontic division, faculdade de odontologia de piracicaba, unicamp, piracicaba (sp), brazil received for publication: august 13, 2008 accepted: february 26, 2009 correspondence to: vânia célia vieira de siqueira rua josé corder, 87 – jardim modelo cep 13419-325 – piracicaba (sp), brazil e-mail: siqueira@fop.unicamp.br, anazildac@gmail.com abstract aim: the purpose of this study was to determine the effects of early treatment on the maxillary dental arches in children with mixed dentition. methods: it was evaluated 40 lateral cephalograms (20 pre and 20 posttreatment) from girls with mean age of 9.15 years, who had class ii, division 1 malocclusion and received occipital headgear treatment with 350 g of force applied bilaterally 14 hours per day, during 22.35 months. the values of: sn.gogn, fma, n-ans, ans-me, s-ptm, ptm-ans, sna, snb, anb, ptm-6, 1.na, 1.pp were evaluated. the dalhberg’s formula revealed a casual error smaller than 1.5° and 1.0 mm, indicating reliability of the obtained data. results: the data revealed that the growth pattern did not change with the treatment. the ans-me decreased. the jaw relationship changed positively, reducing the value of anb. anterior maxillary displacement was limited and the anteroposterior dimension increased. the maxillary molars were moved distally and incisors were retracted. conclusions: the early treatment with occipital headgear was effective in moving maxillary teeth distally and retracting incisors, improving the jaw relationship and favoring the second phase of the orthodontic treatment when necessary. keywords: extraoral traction appliances, interceptive orthodontics/cephalometry, class ii malocclusion introduction class ii, division 1 malocclusion occurs frequently in the brazilian population. in a previous study, on the prevalence of normal occlusion and malocclusion in the city of bauru, brazil, this type of malocclusion was found in 42% of a population of 7-to-11-year-old children1. most patients with moderate or severe class ii dental malocclusion present some skeletal imbalance and the required orthodontic treatment modifies the growth of the jaws2,3. early treatment in the developmental period allows tooth movements and dentoalveolar and skeletal changes to be obtained. the treatment can improve occlusion and facial esthetics, as well as promote the stability of occlusion2-4. the treatment is indicated in class ii and iii malocclusion with maxillary midface deficiency, anterior and posterior crossbite, midline discrepancies due to early loss of primary teeth with a midline shift, severe anterior open bite, severe deep bite, ectopic maxillary canines, thumb and finger-sucking habits, crowding resulting in ectopic positioning of permanent teeth, crowding resulting in periodontal compromise, congenitally missing teeth, and supernumerary teeth. the early treatment’s goal is to reduce the time of the second stage of the orthodontic treatment, when necessary, reduce the need for extractions in permanent dentition, root resorption, periodontal problems, impacted canines and orthognathic surgery4-10. the aim of the treatment in the prepubertal grow th spurt in class ii, division 1, hyperdivergent facial patterns is to rotate the maxilla in a clockwise direction, allowing 39cephalometric evaluation of effectiveness interland headgear on class ii, division 1, malocclusion braz j oral sci. 8(1): 38-43 material and methods the research protocol was reviewed and approved by the research ethics committee of dental school of piracicaba of universidade estadual de campinas (fop-unicamp), process #089/2005 the study sample consisted of 20 girls selected from schoolchildren attending public schools who met strict criteria for enrollment in the study. the initial mean age was 9.15 years old. the following clinical selection criteria were used: bilateral class ii molar relationship, and overbite and overjet greater than 3.0 mm (figures 1a, 1b, 1c). the cephalometric selection criteria were anb range 5º, sna range 80º, snb range 75º, sn.gogn range 35º, ptm-ans range 50 mm. the patients were treated with an interlandi occipital pull (figure 2), outer bow length was close to the first maxillary molar and 1 cm longer than the inner bow. the inner bow was attached to the tube placed on the first maxillary molar, and further than 4.5 mm from the maxillary incisors, parallel to the occlusal plane. the force was located in the resistance centre. in the first month, 250 g force was used on each side, and thereafter 350 g was used during 14 to 18 hours. after class i molar relationship was achieved, the force was reduced to 250 g during three months. then, the appliance was completely removed, and new documentation was requested (figures 3a, 3b, 3c). patients were asked to change the elastic everyday and there were visits scheduled once a month. the compliance and the dental alterations were assessed. the lateral cephalometric radiographs were obtained in the same position and tracing was done by the counterclockwise mandible rotate by inhibition of maxillary posterior dental eruption, guiding the mandibular grow th forwards rather than downwards11. several treatments may be instituted for the treatment of class ii, division 1 malocclusion. application of extraoral force is effective since it promotes arch expansion, superior distal molar moment and consequently premolar and alveolar remodeling12-14. the purpose of the present study was to verify the dentomaxillary alterations occurred after occipital headgear treatment in girls, in the mixed dentition stage during the prepubertal growth spurt. figure 2. interlandi occipital pull. figure 1. (a) intraoral frontal view before treatment; (b) intraoral left side before treatment; (c) intraoral right side before treatment. a b c 40 carvalho aznb, sousa ma, siqueira vcv braz j oral sci. 8(1): 38-43 same operator twice at a 30-day interval to obtain the dahlberg’s formula. the linear measurements selected were n-ans, ans-me, s-ptm, ptm-ans, ptm-6. the angular measurements were sn.gogn, fma, sna, snb, anb, 1.na, 1.pp (figures 4a, b, c). statistical analysis random error was calculated with dahlberg’s formula. method errors of the model measurements and cephalometric variables were less than 1.0 mm, for linear measurements, and 1.5° for angular measurements15,16. descriptive statistics was used to calculate the minimum, maximum, range, mean, standard deviation and median. in order to analyze the dentomaxillary alterations promoted by the extraoral force, paired sample t-tests were used to compare the pre and posttreatment cephalometric values for each patient at 5% significance level. results the dahlberg’s formula indicated the reliability of the initial (t1) and final (t2) data of the cephalograms (table 1). before the treatment, the skeletal pattern was predominantly dolicofacial, and did not change during the evaluated period (tables 3 and 4). the maxilla was close to a neutral skeletal position. the values of s-ptm in t1 were similar to normal average values (table 2). the maxilla was displaced backwards to some extent during the treatment (tables 3 and 4). the extraoral force remodeled the a point vifigure 3. (a) intraoral frontal view after treatment; (b) intraoral left side after treatment; (c) intraoral right side after treatment. a b c s-ptm ptm ptm-ans n-ans ans ans-me orptm-6 6 po s me nsn a n anb ans a 1.pp 1.na b me gn or sn b go fma po sn.gongn s pns po s ptm or ans a b ghme go pns 6 n figure 4. cephalometric tracing of the initial treatment. (a) points utilized – sella (s) is the center of the pituitary fossa; nasion (n) is the junction of the frontonasal suture; orbitale (or) is the lowest point in the inferior margin of the orbit; porion (po) is the most superior point of the external auditory meatus; pterygomaxillary fissure (ptm) is the center point of the pterygomaxillary fissure; anterior nasal spine (ans) is the tip of the bony anterior nasal spine; posterior nasal spine (pns) is the dorsal limit of the maxilla; a point (a) is the point at the deepest midline concavity on the maxilla; 6 is the middle molar point; b point (b) is the point at the deepest midline concavity on the mandibular symphysis; gonion (go) is the point of intersection of the ramus plane and the mandibular plane; menton (me); gnation (gn) this is the most anteroinferior point on the symphysis of the chin; (b) angular measurements – sna is the position of maxilla to cranial base; snb is the position of mandible to cranial base; anb is the skeletal anteroposterior jaw relationship, sn.gogn is the skeletal pattern; fma is the skeletal pattern; 1.na is the axial inclinations of the upper central incisors to na line; 1.pp is the axial inclinations of the upper central incisors to palatal plane. (c) linear measurements – s-ptm is the anteroposterior position of the maxillary jaw; ptmans is the maxillary length; n-ans is the anterosuperior facial height; ans-me is the anteroinferior facial height; ptm-6 is the anteroposterior position of the upper molar. a b c 41cephalometric evaluation of effectiveness interland headgear on class ii, division 1, malocclusion braz j oral sci. 8(1): 38-43 table 1. dahlberg’s formula cephalometric measurements dahlberg’s formula dahlberg’s formula t1 t2 sn.gogn° 0.48 0.48 fma° 0.63 1.42 s-ptm mm 0.38 0.37 sna° 0.31 0.00 ptm-ans mm 0.12 0.054 snb° 0.16 0.32 anb° 0.48 0.32 n-ans% 0.30 0.38 ans-me% 0.02 0.61 ptm-6 mm 0.19 0.81 1.na° 0.05 1.42 1.pp ° 0.95 1.22 dahlberg’s formula showed values less than 1.50 and 1.00 mm = no statistically significance error. table 3. comparison of starting forms at t2 cephalometric measurements mean sd minimum maximum t-test normal /t2 t2 t2 t2 t2 p s-ptm mm 17.36 1.59 14.18 20.71 0.33 ptm-ans mm 51.43 2.19 47.83 54.86 0.25 n-ans% 44.25 1.71 42.0 47.0 0.065 ans-me% 55.75 1.71 53.0 58.0 0.065 ptm-6 mm 15.61 2.71 9.26 20.32 0.53 fma° 28.40 4.97 21.0 40.0 0.006* sna° 79.45 3.58 75.0 89.0 0.005* snb° 74.95 3.38 70.0 82.0 0.012* anb° 4.50 1.50 2.0 9.0 0.15 sn.gogn° 35.25 5.24 28.0 45.0 0.012* 1.na° 21.40 5.66 9.0 32.0 0.641 1.pp° 71.10 5.57 62.0 83.0 0.39 * statistical significance. mean, sd, minimum, maximum and statistical significance for cephalometric values after orthodontic treatment. table 4. comparison of changes during treatment t1 to t2 cephalometric measurements md sd t p s-ptm 0.24 1.07 0.99 0.33 ptm-ans 0.52 2.06 1.12 0.28 n-ans 0.25 1.71 0.65 0.522 ans-me 0.25 1.71 0.65 0.522 ptm-6 1.23 1.62 3.38 0.003* fma 1.30 4.41 1.32 0.20 sna 0.40 1.39 1.28 0.21 snb 0.85 1.49 2.54 0.020* anb 1.25 1.16 4.48 0.0001* sn.gogn 0.70 3.76 0.83 0.42 1.na 1.55 4.67 1.48 0.154 1.pp 0.70 3.76 0.83 0.415 md: mean difference, sd: standard difference, *statistical significance. table 2. comparison of starting forms at t1. cephalometric measurements cephalometric measurements mean sd minimum maximum t-test normal/ t1 t1 t1 t1 t1 p s-ptm mm 17.59 1.40 15.22 21.14 0.074 ptm-ans mm 50.91 2.01 45.45 54.63 0.025* n-ans% 44.00 2.41 40.62 51.25 0.079 ans-me% 56.00 2.35 52.13 64.40 0.079 ptm-6 mm 16.84 2.01 12.80 20.35 0.077 fma° 27.10 3.24 22.00 33.00 0.009* sna° 79.85 3.68 75.0 89.0 0.017* snb° 74.10 2.95 70.0 80.0 0.0001* anb° 5.75 1.52 3.0 10.0 0.0001* sn.gogn° 35.95 4.94 28.0 44.0 0.002* 1.na° 22.95 5.57 12.0 34.0 0.455 1.pp° 70.00 6.13 60.0 80.0 1.0 * statistical significance. mean, sd, minimum, maximum and statistical significance for cephalometric values before orthodontic treatment. sualized for the sna values. the maxillary length was shorter than normal average indicated for the ptm-ans values, but increased during the treatment period (p = 0.28), as is seen in tables 2 and 4. the skeletal anteroposterior jaw relationship verified for the anb values was higher than the normal average ones (p = 0.0001) (table 2), but decreased significantly towards normal values during the treatment period (tables 3 and 4). in the pretreatment period, the relationship between n-ans and ans-me was balanced (p = 0.079) (table 2). in the course of the treatment period, the relationship remained balanced, but the ans-me decreased 0.25%. when t1 and t2 were compared, no statistically significant difference (p = 0.52) was verified (table 4). the maxillary first molar, indicated for the ptm-6, was forward positioned (table 2), and during orthodontic treatment using extraoral force, the maxillary first molar was displaced for backwards (table 3) with statistical significance (p = 0.003), as seen in table 4. the axial inclination of the maxillary incisors, shown for the 1.pp and 1.na values, decreased without statistical significance (tables 2, 3 and 4). discussion the orthodontic treatment alters the growth, improving the skeletal and dental balance, shortening treatment duration and complexity, and reducing the incidence of root resorption, periodontal problems and premolar extraction6. growth modification is often desirable and is more successful when undertaken during the prepubertal growth spurt17, at which stage more stable treatment outcomes can be obtained10. the effectiveness of early treatment is intimately related to a comprehensive diagnosis, an appropriate treatment plan based on the malocclusion characteristics, and the continuous surveillance until the permanent teeth erupt10. the headgear is an appliance that promotes movements in three planes of space. it is effective for treatment of discrepancy in the jaws and teeth, characteristics of class ii malocclusion18,19. with the occipital headgear, it is possible to restrain forward maxillary jaw displacement and its clockwise rotation, distal mo42 carvalho aznb, sousa ma, siqueira vcv braz j oral sci. 8(1): 38-43 lar movement and limit molar eruption, as well as to allow forward displacement of the mandibular jaw, and its counterclockwise rotation11,20,21. in the present study, the facial growth was not altered during therapy and no statistical significance was observed (p = 0.42) (table 4). only a minor reduction of the sn.gogn (tables 2 and 3) suggested the effectiveness of vertical control, which is consistent with the findings of other authors7,12, those which also observed an increase in sn.gogn. no significant changes were observed in the maxilla during the orthodontic treatment. a small restriction of forward displacement occurred, as demonstrated by the s-ptm values (tables 2 and 3). several authors2,3,7 verified modifications in the maxillary position with statistical significance and baumrind et al.22 reported a substantial retraction. the a point moved backwards visualize for the sna values (tables 2 and 3), in the same way as reported by o’reilly et al.23. the headgear redirected anteroposterior growth of the maxilla. ptm-ans increased, even though without statistical significance (p = 0.28). this result is in agreement with those of different authors8,14,19,24, who reported that, during the developmental period, the maxilla was displaced backwards and forwards, and the anteroposterior dimension increased. in the present study, the treatment promoted posterior displacement, and the anteroposterior dimension increased only 0.52 mm, demonstrating the effectiveness of the appliance. the jaw relationship improved, decreasing significantly (p = 0.0001), as seen in table 4. the appliance reduced the severity of skeletal discrepancy, which is likely to have an important impact on the improvement of malocclusion. several authors2,3,7,8 have verified an improvement in the anb and reported that the correction obtained in the growth period was stable in the post-contention period. the treatment did not have a significant effect on the facial height, but the ans-me decreased (table 2 and 3), though without statistical significance (p = 0.522) (table 4). this result suggests that the maxillary molar moved distally but was not extruded. similar results were observed in the study by baumrind et al.22. the maxillary first molar distal movement shown for the ptm-6 values was significant (p = 0.003) (table 4). the occipital headgear moved distally, but controlled the extrusion, which was verified by the vertical control visualized for sn.gogn and ans-me values (table 2 and 3). keeling et al.8, fioruz et al.20 and billet et al.25 found similar results. after the treatment, the axial inclination of the anterior teeth, shown for the 1.na and 1.pp angles, decreased without statistical significance (table 4). ghafari et al.7 and keeling et al.8, on the other hand, found an increase in the axial inclination of the anterior teeth. freman et al.11 reported that the success of early treatment in the prepubertal growth spurt of hyperdivergent facial patterns depended on three points: clockwise rotation of the maxilla, inhibition of posterior maxillary tooth eruption (allowing mandibular counterclockwise rotation), and its growth in a forward direction. according to the obtained results (tables 2, 3 and 4; figure 5), the present study showed that the treatment with occipital headgear reached these goals, which suggests its effectiveness. in conclusion, the facial grow th was not altered during therapy. vertical control was obtained. the jaw relationship improved, decreasing significantly. the maxillary first molar moved distally to a significant extent, and the class ii molar relation was corrected. after the treatment, the axial inclination of the anterior teeth decreased. references 1. da silva filho og, de freitas sf, cavassan ade o. prevalence of normal occlusion and malocclusion in bauru (sao paulo) students. 1. sagittal relation. rev odontol univ são paulo. 1990;4:130-7. 2. tulloch jf, phillips c, proffit wr. benefit of early class ii treatment: progress report of a two-phase randomized clinical trial. am j orthod dentofacial orthop. 1998;113:62-72. 3. proffit wr, tulloch jfc. preadolescent class ii problems: treat no or wait? am j orthod dentofacial orthop. 2002;121:560-2. 4. horn aj, thiers-jégou i. prévention et traitement aprés 10 ans dans une logique edgewise. orthod fr. 2006;77:285-301. 5. dugoni sa, lee js, varela j, dugoni aa. early mixed dentition treatment: postrentetion evaluation of stability and relapse. angle orthod. 1995;65: 311-20. 6. dugoni sa. comprehensive mixed dentition treatment. am j orthod dentofacial orthop. 1998;113:75-84. 7. ghafari j, shofer fs, jacobsson-hunt u, markowitz dl, laster ll. headgear versus function regulator in the early treatment of class ii, division 1 malocclusion: a randomized clinical trial. am j orthod dentofac orthop. 1998;113: 51-61.figure 5. composite tracings of treated t1 and t2. 43cephalometric evaluation of effectiveness interland headgear on class ii, division 1, malocclusion braz j oral sci. 8(1): 38-43 8. keeling sd, wheeler t t, king gj, garvan cw, cohen da, cabassa s, et al. anteroposterior skeletal and dental changes after early class ii treatment with bionators and headgear. am j orthod dentofacial orthop. 1998;113: 40-50. 9. fogle ll, southard ka, southard te, casko js. treatment outcomes of growing class ii division 1 patients with varying degrees of anteroposterior and vertical dysplasias, part 1. cephalometrics. am j orthod dentofacial orthop. 2004;125:450-6. 10. dugoni s, aubert m, baumrind s. differential diagnosis and treatment planning for early mixed dentition malocclusions. am j orthod dentofacial orthop. 2006;129(suppl):80-1. 11. freeman cs, mcnamara ja jr, baccetti t, franchi l, graff tw. treatment effects of the bionator and high-pull facebow combination followed by fixed appliances in patients with increased vertical dimensions. am j orthod dentofacial orthop. 2007;131:184-95. 12. burke m, jacobson a. vertical changes in high-angle class ii, division 1 patients treated with cervical or occipital pull headgear. am j orthod dentofac orthop. 1992;102:501-8. 13. baccetti t, franchi l, mcnamara ja jr, tollaro i. early dentofacial features of class ii malocclusion: a longitudinal study from the deciduous through the mixed dentition. am j orthod dentofacial orthop. 1997;111:502-9. 14. klocke a, nanda rs, kahl-nieke b. skeletal class ii patterns in the primary dentition. am j orthod dentofacial orthop. 2002;121:596-601. 15. houston wj. the analysis of errors in orthodontic measurements. am j orthod. 1983;83:382-90. 16. sandler pj. reproducibility of cephalometric measurements. br j orthod. 1988;15:105-10. 17. kopecky gr, fishman ls. timing of cervical headgear treatment based on skeletal maturation. am j orthod dentof orthop. 1993;104:162-9. 18. greenspan ra. reference charts for controlled extraoral force application to maxillary molars. am j orthod. 1970;58:486-91. 19. ochoa bk, nanda rs. comparison of maxillary and mandibular growth. am j orthod dentofacial orthop. 2004;125:148-59. 20. firouz m, zernik j, nanda r. dental and orthopedic effects of high-pull headgear in treatment of class ii, division 1 malocclusion. am j orthod dentofac orthop. 1992;102:197-205. 21. lahaye mb, buschang ph, alexander rg, boley jc. orthodontic treatment changes of chin position in class ii division 1 patients. am j orthod dentofacial orthop. 2006;130:732-41. 22. baumrind s, korn el, isaacson rj, west ee, molthen r. quantitative analysis of the orthodontic and orthopedic effects of maxillary traction. am j orthod. 1983;84:384-98. 23. o’reilly mt, nanda sk, close j. cervical and oblique headgear: a comparison of treatment effects. am j orthod dentofacial orthop. 1993;103:504-9. 24. enlow dh, bang s. growth and remodeling of the human maxilla. am j orthod. 1965;51:446-64. 25. billiet t, de pauw g, dermaut l. location of the centre of resistance of the upper dentition and the nasomaxillary complex. an experimental study. eur j orthod. 2001;23:263-73. oral sciences n3 190 original article braz j oral sci. january/march 2010 volume 9, number 1 selection of mandibular major connector based on a conventional impression technique adriana da fonte porto carreiro1, arcelino farias neto2, brunna moreira de farias pereira3, luana maria martins de aquino2 correspondence to: adriana da fonte porto carreiro av. alexandrino de alencar, 1384/14 tirol. cep 52015-350 natal-rn, brazil e-mail: adrianadafonte@hotmail.com received for publication: october 22, 2009 accepted: march 29, 2010 abstract the selection of the mandibular major connector of a removable partial prosthesis depends on the distance between the floor of the mouth and free gingival margin, height of the lingual frenum, presence of mandibular tuberosity, mobility of anterior teeth, major connector used in a previous denture and patient’s opinion, slope and retentivity of alveolar bone. however, the dental technician rather than the dentist often selects the major connector in the cast model. aim: to determine whether there is a difference between selecting the mandibular major connector clinically or in the cast model as determined by a conventional impression technique using alginate and a universal metallic tray. methods: the sample was composed of 64 patients under treatment at the department of dentistry of the federal university of rio grande do norte. the distance between the floor of the mouth and the free gingival margin of the remaining elements was measured with a millimeter periodontal probe in the oral cavity and in the cast models. results: the mean clinical distance between the free gingival margin and the floor of the mouth was 7.39 ± 2.13 mm, in contrast to the mean distance found in the models (9.03 ± 1.36). the mann-whitney test showed a significant difference (p < 0.001) between the two measures. conclusions: for the adequate selection of the mandibular major connector, the distance between the gingival margin and the floor of the mouth must be measured clinically when using the conventional impression technique. keywords: removable partial prosthesis, mandible, planning. introduction the fabrication of removable partial dentures (rpds) is one of the most affected areas of dentistry with the absence of sound scientific criteria, given that innumerable framework designs can be correctly performed for a same case. according to owall et al.1, current planning principles are not based on clinical studies and, consequently lack scientific evidence, although this does not mean that they are incorrect. one of the few components where there seems to be universal consensus in terms of its selection is the major bar lingual connector, which should be used whenever possible, owing to its advantages in hygiene and comfort2-5. it requires a minimum space of 8 mm between the free gingival margin of the anterior teeth and the mobile floor of the mouth (myohyoid and genioglossus muscles). four millimeters of this distance is needed for the occlusal-gingival diameter of the bar, a space that ensures rigidity and avoids flexion and fibromucosa trauma. the other 4 mm refer to the distance that the upper bar must remain from the gingival margin5. however, some authors reported that this distance can be even shorter, that is, only 3 mm2 or even 2 mm4,6 from the free gingival margin. braz j oral sci. 9(1):30-32 1dds, ms, phd, professor, department of dentistry, federal university of rio grande do norte, natal (rn), brazil 2dds, ms, graduate student, department of dentistry, federal university of rio grande do norte, natal (rn), brazil 3dds, graduate student, department of dentistry, federal university of rio grande do norte, natal (rn), brazil 31 measurement n clinical 64 7.39 2.13 cast model 64 9.03 1.36 table 1 distance (mm) between the floor of the mouth and the free gingival margin (mean and standard deviation). the distance between the gingival margin and the floor of the mouth can be measured clinically using a periodontal probe and instructing the patients to raise and protrude their tongue until the tip reaches the red part of the upper lip. thus, the floor of the mouth becomes active and raises the tissues to the maximum height that they attain during mastication. once the measure is obtained, it is transferred to the diagnostic cast model7. when there is not enough space for the lingual bar, because the mobile tissues of the floor of the mouth are very high or the ridge height is reduced due to advanced periodontal problems, the choice will be the lingual plate6. the lingual plate allows a higher placement of the lower border without compromising rigidity, in addition to not damaging the gum, owing to the greater relief permitted. however, its upper limit is established at the cingulum level of the anterior teeth, covering the entire area corresponding to the marginal gingival. thus, it is believed that this covering hinders the flow of saliva, oral self-cleaning and the physiological stimulus of gingival tissues promoted by the tongue in this area. studies have shown that patients with a lingual plate exhibited greater plaque accumulation on the lingual surface of the anterior teeth than those with a lingual bar, even with professional supervision8-9. thus, when the lingual plate is used, the patient no longer enjoys the benefits of the lingual bar. on the other hand, an improperly chosen lingual bar may cause a series of problems, such as trauma to the floor of the mouth tissues, displacement during mastication, undue covering of the free marginal gingival, difficult hygienization, greater plaque accumulation and periodontal problems. therefore, the correct measurement of the available space is crucial in selecting the mandibular major connector. however, given the large number of cast models that are sent to the prosthetic laboratory without any type of planning10-14, it is often observed that this measure is not obtained clinically, but rather in the cast model by the dental prosthetic technician, sometimes resulting in an incorrect selection. the aim of the present study was to determine whether there is a difference between the clinical selection of a major mandibular selector and the selection made in the cast model. material and methods the present study was conducted at the department of dentistry of the federal university of rio grande do norte, brazil after approval by the local ethics research committee (protocol nº 095/05) and all patients signed a written informed consent form. the sample consisted of 64 randomly selected patients who were in the final process of rpd installation in the partial removable and integrated clinic disciplines. all the metallic frameworks were made in cast models obtained from the alginate mold and type iv plaster (durone; dentsply, petrópolis, rj, brazil). clinical evaluation of the distance between the marginal gingival and the floor of the mouth was performed using a periodontal probe and instructing the patients to open mouth and to elevate and protrude their tongues until the tip reached the red part of the upper lip. the same tongue movement was performed during impression with metallic stock trays to obtain the cast model. one measure was carried out for each tooth (from 35 to 45), if they were present, measuring the free gingival margin at its most apical point, at the bottom of the floor of mouth. the smallest measure and its location were recorded, serving as a reference for taking a single measurement of the cast model and for comparing the values. descriptive data analysis was conducted using the mean and standard deviation and spss 16.0 software. the mann-whitney test was applied to evaluate the relationship between the study variables. the significant level was set at 5%. results the mean distance found clinically between the free gingival margin and the floor of the mouth was 7.39 mm, whereas in the models this distance was 9.03 mm. this difference was statistically significant (p < 0.0001) (table 1). discussion in addition to replacing lost structures, the main goal of rehabilitation with a rpd is to preserve and protect the remaining structures4. however, dentists are reluctant to plan rpd, possibly due to their lack of knowledge, and thus, many errors occur in the selection of the mandibular major connector15. on average, 90% of cast models are sent to the laboratory without any type of dental preparation or planning. rather, the responsibility falls on the dental prosthesis technician, who does not have the required knowledge to perform this task, a situation that leads to high failure rates and periodontal complications10-14. clinical studies show that the lingual bar is the most widely used type of connector in rpd16-19. the examination of 200 patients with rpd showed that this connector was used in 77% of inferior prostheses18. another study, with a sample of 25 patients with lower rpd found 96% with a lingual bar, and a lingual plate in only one patient, due to mobility in the pillar teeth. however, the mean distance between the superior border of the bar and the gingival margin found by the authors was only 2.82 mm19. according to carr et al.5, this distance should be at least 4 mm to maintain oral hygiene and gingival health. review of the literature shows no consensus regarding the ideal distance. most authors believe that 3 mm is sufficient, although even a distance of 2 mm would be acceptable2,4,6. considering a mean distance of 3 mm and adding to it the 4 mm corresponding to the diameter of the occlusal-gingival of the bar, a space of at least 7 mm would be necessary between the gingival margin and the floor of the mouth for a lingual bar to be indicated. in the present study, it was observed that only 59.3% of the patients met this clinical criterion. a similar result was found in a study with 80 partially braz j oral sci. 9(1):30-32 selection of mandibular major connector based on a conventional impression technique 32 edentulous patients free of periodontal disease, where this proportion was 60%20. if the 8 mm suggested by carr et al.5 were considered, this indication would be even more restricted, to only 48.4% of our sample. on the other hand, when the measures obtained in the models are used, 95.3% (7 mm) or 82.8% (8 mm) would be indicated to receive the lingual bar. thus, comparison of the data indicating a lingual bar obtained in the model and in the mouth between one another and with those of frequency of use observed in other studies1819 suggests that this type of connector is being improperly indicated, likely resulting from the laboratory selection made by the dental prosthesis technician. when the measures obtained clinically were compared to those of the cast models, a significant difference was observed between the two groups (p < 0.0001). a review of the literature did not find any study with a similar methodology. the difference between the measures of the two groups was 2.36 mm, which is a value high enough to cause errors in the selection of the inferior major connector. the highest mean was found for the measurements performed in the plaster model. although the patients were instructed to elevate and protrude their tongues until the tip reached the red part of the upper lip during impression, this result may be due to the displacement of the floor of the mouth caused by the alginate or by insufficient tongue movement during impression, given that the gingival margin was relatively adhered to the dental surface, and did not suffer displacement. also there is the possibility that the inner border of the tray touched the floor of the mouth avoiding its upward shift. thus, the error will likely occur in the determination of the lower limit of major connector positioning. perhaps, a custom-made tray would have been useful since it can be adjusted prior to impression. these findings suggest that those who perform the measurements in cast models are systematically incurring the risk of selecting a lingual bar that clinically invades the functional space of the soft tissues of the floor of the mouth, a situation that may provoke trauma or displacement of the rpd. furthermore, the need to adjust the rpd, due to patient complaints, may wear and weaken the bar. zavanelli et al.19 report that in case of incorrect selection, compression from the major connector on the tissues may result in local inflammation, gingival retraction, pain, bone loss and dental mobility, compromising the patient-professional relationship and discrediting the prosthetic rehabilitation treatment. thus, it is evident that for the proper selection of the mandibular major connector the distance between the marginal gingival and the floor of the mouth must be obtained clinically. dentists must show greater commitment during the fabrication of rpd in the sense of carefully planning their cases, thereby avoiding sending cast models to the dental prosthesis laboratory without any orientation or planning. the present results show that for the correct selection of the mandibular major connector it is essential that the distance between the marginal gingival and the floor of the mouth be measured in the oral cavity. the distance from the floor of the mouth to the free gingival margin of the remaining teeth is essential for the selection of mandibular major connector. considering the results of the present study, the reviewed literature and the issues discussed above, it seems reasonable to conclude that the impression technique used in this study to obtain a cast model does not offer an accurate measurement of space, leaving to the dentist and not to the dental prosthesis technician the decision for the type of mandibular major connector to be chosen. references 1. owall b, budtz-jörgensen e, davenport j, mushimoto e, palmqvist s, renner r et al. removable partial denture design: a need to focus on hygienic principles? int j prosthodont. 2002; 15: 371-8. 2. lavere am, krol aj. selection of a major connector for the extension-base removable partial denture. j prosthet dent. 1973; 30: 102-5. 3. mccraken wl. contemporary partial denture designs. j prosthet dent. 1958; 8: 71-84. 4. todescan r, silva eeb, silva oj. atlas de prótese parcial removível. são paulo: santos; 1998. 345p. 5. carr ab, mcgivney gp, brown dt. mccracken´s removable partial prosthodontics. saint louis: elsevier mosby; 2005. 458p. 6. klieman c, oliveira w de. manual de prótese parcial removível. são paulo: santos; 1998. 7. phoenix rd, cagna dr, defreest cf. prótese parcial removível clínica de stewart. são paulo: quintessence. 2007. 526p. 8. addy m, bates jf. plaque accumulation following the wearing of different types of removable partial dentures. j oral rehabil. 1979; 6: 111-7. 9. akaltan f, kaynak d. an evaluation of the effects of two distal extension removable partial denture designs on tooth stabilization and periodontal health. j oral rehabil. 2005; 32: 823–9. 10. todescan r, romanelli jh. por que fracassam os aparelhos parciais removíveis. rev assoc paul cirurg dent. 1971; 25: 13-22. 11. vieira df, todescan r. estarrecedora situação da prótese parcial removível. um “alerta!” à profissão odontológica. rev assoc paul odont. 1972; 26: 299-310. 12. bonachela wc, di creddo rc. insucessos das próteses parciais removíveis. rgo. 1990; 38: 262-4. 13. duarte arc, paiva hj. avaliação do nível de conhecimento e conscientização do cirurgião-dentista e do técnico em prótese dental, em relação ao planejamento e a execução de próteses parciais removíveis – estudo laboratorial. rev abo nac. 2000; 8: 232-7. 14. palomo e, teixeira ml, stegun rc. avaliação do comportamento dos cirurgiões-dentistas e protéticos na confecção de estruturas metálicas de próteses parciais removíveis nos laboratórios comerciais da cidade de são paulo. pcl. 2003, 5: 425-31. 15. mattos mgc, brun cl, matos rl, pagnano vo, ribeiro rf. perfil dos laboratórios de prótese dental e dos trabalhos de prótese parcial removível. pcl. 2001; 3: 505-10. 16. curtis da, curtis ta, wagnild gw, finzen fc. incidence of various classes of removable partial dentures. j prosthet dent. 1992; 67: 664-7. 17. owall g, bieniek kw, spiekermann h. removable partial denture production in western germany. quintessence int. 1995; 26: 621-7. 18. al-dwairi zn. partial edentulism and removable denture construction: a frequency study in jordanians. eur j prosthodont restor dent. 2006; 14: 13-7. 19. zavanelli ra, hartmann r, zavanelli ac, carvalho jrh. dimensions of major connector of removable partial denture and its relation with gingival tissue. rev odontol unesp. 2006; 35: 135-9. 20. cameron sm, torres gt, lefler tb, parker mh. the dimensions of mandibular lingual tissues relative to the placement of a lingual bar major connector. j prosthodont. 2002; 11: 74-80. braz j oral sci.9(1):30-32 selection of mandibular major connector based on a conventional impression technique braz j oral sci. 15(1):35-38 original article braz j oral sci. january | march 2016 volume 15, number 1 odontometric analysis of molars for sex determination larissa chaves cardoso fernandes1, carolina vieira lucena veloso2, julyana de araújo oliveira2, paloma rodrigues genu3, bianca marques santiago2, patrícia moreira rabello2 1universidade de pernambuco – upe, pernambuco dental school, area of forensic dentistry, recife, pe, brazil 2universidade federal da paraiba – ufpb, school of dentistry, department of clinical and social dentistry, area of forensic dentistry, joão pessoa, pb, brazil 3universidade federal de pernambuco – ufpe, school of dentistry, department of social medicine, area of forensic dentistry, recife, pe, brazil correspondence to: larissa chaves cardoso fernandes avenida epitácio pessoa, 4050, apartamento 1701 miramar – cep: 58032-000 joão pessoa, pb, brasil phone: +55 83 98887-6360 e-mail: larissaccfernandes@gmail.com abstract aim: to investigate the existence of sexual dimorphism between the first and second permanent molars. methods: a cross-sectional, observational, blind study using comparative and statistical descriptive procedures. the sample included 50 pairs of plaster casts from undergraduate dental students (25 men/25 women) from the federal university of paraíba, joão pessoa/pb, brazil, aged 20-26 years. odontometric measurements of first and second maxillary/mandibular, right/ left permanent molars were performed. mesiodistal (md) and buccolingual/palatal (bl/bp) widths and the distance between the lingual cusps of corresponding molars in opposite quadrants, were measured. the data were analyzed by student’s t test and anova with bonferroni (p≤0.05). results: the crowns of all first molars were statistically larger in men than in women (p<0.05). maxillary and mandibular left second molars (#27 and #37) did not differ in their md widths (p=0.66, p=0.75), whereas mandibular left and right second molars (#37 and #47) showed statistically different bl widths (p=0.007 and p=0.008). as to the distance between the lingual cusps, only the first left-to-right mandibular molars (#36-46) showed no sex dimorphism (p=0.107). conclusions: molars are larger in males than in females. individually, first molars demonstrated higher evidence of sex distinction than second molars. keywords: forensic dentistry. molar. odontometry. sex characteristics. introduction forensic dentistry is a branch of the forensic sciences that provides information to support the court of justice decisions, especially with regard to human identification and clarification of facts with legal interest1,2. approximately 70% of the identifications performed worldwide in cases of mass disasters are obtained by forensic dentistry3,4. human identification by the study of teeth has gained considerable importance, mainly in the absence of fingerprint data5. teeth are the most stable, durable and resistant organs of the human body6, able to stand up very high temperatures and the process of cadaveric decomposition7,8. dental features may assist the reconstruction of forensic biological profile of unknown individuals. as such, teeth could serve as important tools for estimating age and height at the death time as well as ethnic group and sex7,9-12. sex determination is of paramount importance in cases when visual identification of the sex is impossible, thus eliminating about 50% of possibilities in the examined population8. although some of the dental features may change in lifetime, anatomical peculiarities, and missing, decayed http://dx.doi.org/10.20396/bjos.v15i1.8647109 received for publication: april 20, 2016 accepted: may 24, 2016 36 and restored teeth do provide specific data for ante-mortem and post-mortem comparisons13. studies have shown statistically significant differences in the dental patterns of men and women by odontometric measures, that include the mesiodistal and buccolingual distances between the crowns of permanent teeth9,14. among all teeth, molars have greater potential for race differentiation15. herein, odontometric measures were used to evaluate sexual dimorphism in the first and second maxillary/mandibular permanent molars to assist the process of human identification. material and methods this study is in accordance with the brazilian resolution no. 466/12 of the national health council, ministry of health, which regulates research involving human beings, and had prior approval of the research ethics committee at the center for health sciences, federal university of paraíba (caae: 30967814.6.0000.5188). this was a cross-sectional, observational, blind study using comparative, statistical and descriptive procedures. the study approach was based on intensive direct observation of maxillary and mandibular plaster casts. the universe consisted of 191 pairs of maxillary and mandibular plaster casts belonging to students from the federal university of paraíba (ufpb), which were part of the occlusion discipline database in this institution. the casts were thoroughly analyzed and we excluded from the sample those with missing molars; poorly positioned teeth; restorations of free faces that could change dental dimensions; failures in the cast (e.g. bubbles and fractures) or those from individuals with previous history of orthodontic treatment. the final sample consisted of 50 pairs of plaster casts, where 25 were from females and 25 from males, aged 20 to 26 years. a pilot study including other 40 pairs of plaster casts from the same database was carried out prior to the full study for training the examiner. the time between the first and second analyses was eight days, yielding intraclass correlation coefficient values between 0.868 and 0.999. these casts were excluded from the final sample. the measurements were performed with a digital caliper (stainless-hardened® 150 mm, mauá, são paulo, brazil) and consisted in the following variables: mesiodistal width –the greatest distance between the proximal surfaces of the molar tooth (figure 1); buccolingual/palatal width – the distance between the outermost points of the molar crowns (figure 2) and the distance between the lingual cusps of the corresponding molar teeth in opposite quadrants (figure 3). the data collected were processed in the statistical package for social sciences v. 20.0 (spss inc., chicago, il, usa) software, then treated and analyzed using descriptive and inferential approaches, with a 5% significance level. the kolmogorov-smirnov test was used to check whether the odontometric measurement data had a normal distribution. the levene f test was used to verify the equal variances in the study variables. the other comparative analyses were performed using the student’s t test and repeated-measures analysis of variance (anova) with bonferroni. odontometric analysis of molars for sex determination braz j oral sci. 15(1):35-38 fig.2. buccolingual/palatal distance. source: current research. results table 1 describes the mean and standard deviation of the measurements according to tooth and sex. there was a significant difference between the md and bl/bp widths in all first molars, and male teeth were found to be larger than the female ones (p<0.05). as for second molars, only the maxillary and mandibular right second molars (#17 and #47, respectively) showed a statistically significant difference concerning their md width (p=0.034 and p=0.044, respectively). the mandibular left and right second molars (#37 and #47, respectively) showed a significant difference in their bl widths according to sex (p=0.007 and p=0.008, respectively). furthermore, there was a statistically significant sex differentiation in the segments from the maxillary right first molar (#16) to the maxillary left first molar (#26) (p=0.019); maxillary right second molar (#17) to the maxillary left second molar (#27) (p=0.001); and the mandibular right second molar (#37) to the mandibular left second molar (#47) (p=0.022). fig.1. mesiodistal distance. source: current research. fig.3. distance between the lingual cusps of the molars in opposite quadrants. source: current research. 37 as described in table 1, the mandibular left first molar (#36) showed the highest md width in males, statistically different from the other first molars. with regard to the other analyzed measurements, significant differences were observed both in first and second molars in opposite quadrants. for instance, mandibular teeth had higher md width than maxillary teeth, while maxillary teeth had greater bl width. similar findings were also observed in females, in which the measurements followed the same pattern of differences between maxillary and mandibular molars. discussion university students attending public high education institutions, such as ufpb, commonly come from different ancestries and social classes. this fact has been accentuated by social inclusion policies (university quotas) gradually implemented in these institutions16. unlike bones, teeth are not much affected by nutritional deficiencies17. hence, social factors and eating habits do not odontometric analysis of molars for sex determination braz j oral sci. 15(1):35-38 table 1 mean and standard deviation of the measurements according to tooth and sex. dental nomenclature follows the system proposed by the federacion international dental (fdi). joão pessoa, pb, brazil, 2015. sex male female measures tooth mean standard deviation mean standard deviation p-value1 mesiodistal 16 10.32a 0.57 9.84a 0.43 0.001* 26 10.26a 0.57 9.84a 0.50 0.008* 36 10.77b,c 0.67 10.25b 0.57 0.005* 46 10.66a,c 0.61 10.27b 0.55 0.020* p-value2 0.008* 0.002* buccolingual/ palatal 16 10.48a 0.65 10.05a 0.46 0.010* 26 10.49a 0.55 10.09a 0.52 0.010* 36 10.14b 0.57 9.72b 0.46 0.006* 46 10.08b 0.63 9.64b 0.53 0.010* p-value2 0.024* 0.002* mesiodistal 17 9.70 0.61 9.34a 0.55 0.034* 27 9.59a 0.62 9.28a 0.57 0.066 37 10.34b 0.81 9.99b 0.54 0.075 47 10.26b 0.77 9.85b 0.65 0.044* p-value2 <0.001* < 0.001* buccolingual/ palatal 17 10.40a 0.77 10.07a 0.50 0.074 27 10.43a 0.78 10.08a 0.46 0.061 37 9.96b 0.59 9.51 b 0.55 0.007* 47 9.82b 0.66 9.36b 0.51 0.008* p-value2 0.004 < 0.001* distances 16-26 37.81 2.51 36.13 2.42 0.019* 36-46 34.30 2.40 33.04 2.99 0.107 p-value2 <0.001* <0.001* distances 17-27 43.29 2.52 40.72 2.58 0.001* 37-47 40.68 2.62 39.02 2.33 0.022* p-value2 0.001* 0.018* affect significantly determination of sex by the odontometric measurements of teeth. this fact places teeth as important auxiliary tools in the human identification process. this study confirmed the existence of sex dimorphism based on odontometric measures of the maxillary/mandibular first and second permanent molars. male teeth were found to be larger than the female ones and the distances between the lingual cusps of the corresponding molars in opposite quadrants were also higher in men. these results corroborate those found by studies assessing other populations12,18-22. a limitation of this study lies in the inability to pinpoint a borderline numeric variation for the analyzed measures concerning the use of molar teeth for sex determination. this shortcoming may be explained by the small sample size, which may not have been sufficient to perform a discriminant statistical analysis, i.e., the comparison between sexes based on confidence intervals (95% ci)23. it is worth noting that one of the major difficulties in sample selection lied in the high frequency of ineligibility due to proximal restorations in the target teeth. a study12 carried out in greece investigated the degree of sex dimorphism in 344 permanent molars from 107 greek (53 men and 54 women). the authors used the diameters of the crowns and the diagonal length of the cervical region as parameters for determination. they concluded that men have larger molars than women. nevertheless, this greek population was found to have their maxillary second molars as the most dimorphic teeth, which is in contrast with the findings presented here, as we found that maxillary first molars are unanimously larger in males. in our study, the analysis of homologous teeth revealed that the mandibular first left and right molars (#36 and #46, respectively) in men showed a significant difference in their md widths. a study performed in peru24 investigated the relationship between the md widths of teeth and sex. the authors used 120 plaster casts belonging to students (60 men and 60 women) aged 12 to 18 years. they found a significant discrepancy between the md diameters of seven pairs of homologous teeth in men, including the mandibular first molars. they also observed an important relationship between the md width and sex dimorphism, as men were found to have greater md diameter than women. sex determination using dental parameters is employed in the process of forensic identification. another study25 aimed to verify sexual dimorphism by md and bl/bp measurements. two hundred plaster casts from brazilian adults of both sexes, aged 20 to 30 years, were analyzed. the author concluded that male teeth are larger than female teeth, which corroborates the findings of our study. however, no significant differences were observed between homologous teeth, except for the md width of the mandibular first right and left molars (#36 and #46, respectively). furthermore, measuring of canines and molars was the best dental indicator to identify sex in this population, with the maxillary first molars showing sex distinction. sexual dimorphism based on tooth size varies among populations and even within the same population due to historical and evolutionary reasons. accordingly, the criteria set down in one place may not apply to another, requiring determination of specific values for each population17. it is important to consider that the brazilian population is composed by miscegenation of different *statistically significant difference (p-value<0.05). 1student’s t test for equal variances. 2 repeated-measures anova. obs: different superscript letters indicate a statistically significant difference between the corresponding teeth according to bonferroni’s multiple (paired) comparisons test. 38 ethnic groups, which influences and hinders the identification of sex based on odontometric analysis. even so, when odontometry is employed with this purpose, the most commonly used dimensions are the md and bl distances1,7,20,26-29. participation of dentists in the forensic staff is beneficial to the victim and their family30. an expert analysis of the stomatognathic system, especially the teeth, is able to guide criminal investigations, in which the inclusion of forensic dentists is a unanimous requirement in accredited institutes. forensic dentistry is an area of great importance in the forensic practice and is a useful tool in the studies related to human identification methods, in particular the use of teeth. the results observed in this study led us to conclude that the maxillary first molars were found to have the highest percentage of sex dimorphism and that male molars are larger than those of the opposite sex. acknowledgements the authors are thankful to the national council for scientific and technological development for financial support. references 1. niquini btb, villalobos miob, manzi fr, bouchardet fch. 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[comparative study of the mesio-distal size between homologous teeth in permanent dentition]. odontol. sanmarquina. 2013;16(2):711. spanish. 25. martins filho ie. [relationship between gender and tooth measures: a brazilian study] [thesis]. são paulo: university of são paulo, the são paulo school of dentistry; 2013. portuguese. 26. ates m, karaman f, iscan my, erdem tl. sexual differences in turkish dentition. leg med (tokyo). 2006 oct;8(5):288-92. 27. angadi pv, hemani s, prabhu s, acharya ab. analyses of odontometric sexual dimorphism and sex assessment accuracy on a large sample. j forensic leg med. 2013 aug;20(6):673-7. doi: 10.1016/j. jflm.2013.03.040. 28. khan sh, hassan gs, rafique t, hasan n, russell sh. mesiodistal crown dimensions of permanent teeth in bangladeshi population. bsmmu j. 2011;4(2):81-7. 29. pereira c. [forensic dental medicine]. lisboa: lidel; 2012. 30. ribas-e-silva v, terada assd, silva rha. [the importance of the dentist´s specialized knowledge into brazilian forensic team]. rev bras odontol legal. 2015;2(1):68-90. portuguese. braz j oral sci. 15(1):35-38 odontometric analysis of molars for sex determination original articlebraz j oral sci. april/june 2009 volume 8, number 2 enhanced susceptibility of candida albicans to chlorhexidine under anoxia andressa marafon semprebom1, ana cláudia azevedo isidoro1, maria ângela naval machado1, patrícia maria stuelp campelo1, josé francisco höfling2, lakshman perera samaranayake3, edvaldo antonio ribeiro rosa1 1 laboratory of stomatology, dental school, pontifícia universidade católica do paraná (pucpr), curitiba (pr), brazil 2 laboratory of microbiology and immunology, faculdade de odontologia de piracicaba, universidade estadual de campinas (unicamp), piracicaba (sp), brazil 3 oral biosciences unity, dental school, university of hong kong, hong kong, hong kong sar. received for publication: may 26, 2009 accepted: july 13, 2009 correspondence to: edvaldo antonio ribeiro rosa faculdade de odontologia, pontifícia universidade católica do paraná. rua imaculada conceição, 1155 cep 80215901 – curitiba (pr), brazil e-mail: edvaldo.rosa@pucpr.br abstract aim: periodontal pockets can be colonized not only by bacteria, but also by candida albicans. however, its role in periodontitis is unknown. this study evaluated the inhibitory performance of chlorhexidine digluconate under normoxic and anoxic conditions against 16 strains of c. albicans from periodontal pockets and other 20 from the oral mucosa. methods: strains were grown in normoxia and anoxia to adapt themselves to the different atmospheric conditions. microdilution-based assays were carried out to determine the minimum concentrations of chlorhexidine that may restrain the conditioned candidal strains, in normoxia (normoxic mic) and anoxia (anoxic mic). the mann-whitney u test was used to evaluate the antimicrobial effect of chlorhexidine on c. albicans under normoxic and anoxic conditions (α = 0.05). results: the normoxic mic of chlorhexidine varied broadly from 150 to 1200 µg/ml, whereas its anoxic mic varied narrower from 2.34 to 37.5 µg/ml. regarding the origins of strains, no statistically significant differences (p > 0.05) were found. conclusions: these results indicate that anoxic environmental conditions, compatible with periodontal pockets, tend to enhance c. albicans susceptibility to chlorhexidine. keywords: candida albicans, chlorhexidine, anoxia. introduction periodontitis is a multifatorial inflammatory disease process that leads to the destruction of the periodontal tissues supporting the teeth1. the etiologic factor of periodontitis is the dental biofilm associated or not with calculus2. the progression of the disease is related to gingival crevice colonization by microorganisms such as aggregatibacter (actinobacillus) actinomycetemcomitans, porphyromonas gingivalis, prevotella intermedia, tannerella forsythia, and treponema denticola1,3,4. although bacteria has a major role in the pathogenesis of periodontal disease, the yeast candida albicans has also been isolated from periodontal pockets2, with prevalence ranging from 14 to 19%5. in a previous study, yeasts were found in 19.7% of individuals with periodontal pockets > 7 mm and in 15.6% of subjects with pockets ≤ 7 mm6. this organism has important virulence factors such as proteolytic activity and capacity to adhere and invade the epithelium7,8. although the presence of c. albicans in the periodontal pocket per se may not be 106 semprebom am, isidoro aca, machado mân, campelo pms, höfling jf, samaranayake lp, rosa ear braz j oral sci. 8(2): 105-10 directly associated with periodontitis, this yeast may take part in the pathogenic microbiota of some forms of periodontitis6. the chemotherapeutical eradication of periodontal yeasts does not follow the protocols indicated for bacteria once they are not affected by drugs commonly used in periodontics. as no antifungal therapy is routinely used, antiseptics may play an important adjuvant role. chlorhexidine {1,1’-hexamethylene-bis[5-(p-chlorophenyl) biguanide]}, a widely used antimicrobial agent, adversely affects the microbial eukaryotic plasma membrane by nonspecific electrostatic binding9 to negative protein and phospholipid moieties, causing alteration in the cellular membrane structure and in the cellular osmotic balance10,11. normal fungal cells have a negative internal charge12,13 that explains their susceptibility to chlorhexidine. based on the premise that periodontal sites are anoxic and no prior studies investigated the inhibitory effects of chlorhexidine on c. albicans in such environmental condition, the present study evaluated the performance of this biguanide on periodontal c. albicans strains under normoxic and anoxic conditions. material and methods sampling sixteen periodontium-related (so called “pp” strains) strains from periodontal pockets ≥ 4 mm were used. these strains were obtained from the culture collection of faculdade de odontologia de piracicaba, universidade estadual de campinas (unicamp), brazil. twenty mucosa-related c. albicans isolates (so-called “om” strains) were obtained from the culture collection of the dental school of the pontifícia universidade católica do paraná (pucpr), brazil. the research project was reviewed and approved by the ethics committee of the second institution. culture media the culture broth used throughout the study14 contains (per 900 ml of distilled water) 4 g of kh 2 po 4 , 3.2 g of nah 2 po 4 , 1.2 g of l-proline, and 0.7 g of mgso 4 .7h 2 o. l-proline was replaced by 0.5 g of l-lysine and 1 g of yeast extract was added. after autoclaving, the broth received 40 ml of 20% glucose, 0.5 ml of vitamin mixture, and 0.25 ml of mineral mixture. the vitamin mixture contains ( per 100 ml of 20% ethanol) 2 g of biotin, 20 mg of thiamine-hcl, and 20 mg of pyridoxine-hcl. the mineral mix contains ( per 100 ml of 100 mm hcl) 0.5 g of cuso 4 .5h 2 o, 0.5 g of znso 4 .7h 2 o, 0.8 g of mncl 2. 4h 2 o, and 0.5 g of feso 4 . the vitamin and mineral mixtures were filter-sterilized with 0.22 µm pore-sized cellulose nitrate membranes (whatman, maidstone, uk), and stored at 4 °c. for anaerobic growth of c. albicans, the broth was supplemented with 200 µl of 1 mm oleic acid in 100% methanol, 200 µl of 4 mm nicotinic acid, and 1 ml of 500 mm nh 4 cl. sterile l-cysteine was added up to 0.01%. the ph of the complete broth was 5.0. this modified broth was distributed in sterile disposable 96 wells polystyrene plates (difco laboratories, detroit, mi, usa) at 100 μl per well and stored at -20°c. aerobic inoculum preparation both sets of strains (pp and om strains) were inoculated in 3 ml of broth and incubated under normoxia at 37 °c for 24 h. after growth, the cells were harvested, washed three times in sterile deionized water, and suspended at 2 × 107 cells/ml. the suspensions were stored at 4°c for no more than four hours. anaerobic inoculum preparation one hundred microliters of aerobic inocula were inoculated in 3 ml of modified broth and incubated in hermetically sealed jars supplied with two disposable anaerobac® anoxia generator cartridges (probac co., são paulo, sp, brazil) at 37 °c for 48 h. one hundred milliliters of culture were transferred to 3 ml of modified broth and anaerobically incubated at 37 °c for 48 h. this procedure aimed turning the cells totally adapted to the anoxic condition. after growth, the cells were harvested, washed three times in sterile deionized water, suspended until obtaining a concentration of 2 × 107 cells/ml, and stored at 4 °c in vials whose headspaces were filled with sterile co 2 . susceptibility tests the technique of broth microdilution was used for determining the minimum inhibitory concentration (mic)15 for chlorhexidine digluconate (pharma nostra co., são paulo, brazil). chlorhexidine was diluted in modified broth and transferred to microdilution plates to obtain a range of 12 wells with doubling increased concentrations ranging from 0.122 to 500 μg/ml. each well received 10 μl of suspension of normoxic or anoxic c. albicans, obtaining final densities of 1 × 106 cells/ml. the microdilution plates were statically incubated at 37 °c in normoxic or anoxic atmospheres. the anoxic conditions were obtained using hermetically sealed jars supplied with two disposable anaerobac® anoxia generator cartridges (probac co.) at 37°c, as stated before. the normoxic growth was monitored up to 48 h and the anoxic growth was monitored up to 72 h. the cellular growth was visually compared to the growth in wells containing only culture broth. the mic determination assays were done in triplicate on three independent occasions. the mann-whitney u test was used to evaluate the antimicrobial effect on c. albicans under normoxic and anoxic conditions. a p value of 0.05 was assumed as threshold for differences. results during inoculum preparation, we observed that the normoxic cultures achieved an od 520nm of 0.450 ± 0.030 after 24 h of incubation at 37 °c, whereas anoxic-adapted cells achieved an od 520nm of 0.123 ± 107enhanced susceptibility of candida albicans to chlorhexidine under anoxia braz j oral sci. 8(2): 105-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 pp -f o p3 pp -f o p7 pp -p u c pr 3 pp -f o p6 pp -f o p5 pp -f o p9 pp -f o p4 pp -p u c pr 2 pp -f o p1 1 pp -f o p1 0 pp -f o p8 pp -f o p1 2 pp -p u c pr 4 pp -f o p2 pp -f o p1 pp -p u c pr 1 av er ag e m ic 90 (m ic ro g ra m s/ m ill ili te r) p < 0.0001 normoxia anoxia figure 1. minimum inhibitory concentration (mic) of chlorhexidine digluconate for periodontium-related (pp) candida albicans strains in normoxia and anoxia. 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 o m -f o p3 o m -f o p7 o m -p u c pr 3 o m -f o p6 o m -f o p5 o m -f o p9 o m -f o p4 o m -p u c pr 2 o m -f o p1 1 o m -f o p1 0 o m -f o p8 o m -f o p1 2 o m -p u c pr 4 o m -f o p2 o m -f o p1 o m -p u c pr 1 av er ag e p < 0.0001 normoxia anoxia m ic 90 (m ic ro g ra m s/ m ill ili te r) figure 2. minimum inhibitory concentration (mic) of chlorhexidine digluconate for mucosa-related (om) candida albicans strains in normoxia and anoxia. 0.018 after 24 h and an od 520nm of 0.420 ± 0.023 after 48 h. the predominant cell shape in normoxic cultures was budding yeast-like with some cells forming pseudo-hyphae; on the other hand, most part of cells grown in anoxia was true hyphae. by multiple gram staining, it was estimated that less than 5% of the fungal load grew as budding yeast-like cells in anoxia. figures 1 and 2 indicate the variation in the susceptibility performances for consensual values after nine repetitions of pp and om strains, respectively. for the pp strains, the results showed that chlorhexidine promoted growth restrain of all c. albicans strains with mic values varying from 15.62 to 125 µg/ml (mean = 70.31 ± 50.06 µg/ml) in normoxic conditions versus a decreased range from 0.97 to 15.62 µg/ml (mean = 8.45 ± 6.84 µg/ml) in anoxia. for the om strains, chlorhexidine promoted growth restrain of all c. albicans strains with mic values varying from 15.62 to 125 µg/ ml (mean = 70.31 ± 50.06 µg/ml) in normoxic conditions versus a decreased range from 0.97 to 7.81 µg/ml (mean = 5.09 ± 3.18 µg/ ml) in anoxia. 108 semprebom am, isidoro aca, machado mân, campelo pms, höfling jf, samaranayake lp, rosa ear braz j oral sci. 8(2): 105-10 in order to determine de minimum candidacidal concentration (mcc), after the mic determination, contents of all wells were independently recovered and transferred to tubes with 3 ml of broth without chlorhexidine. the growth in those tubes followed the same mic assay results. thus, it could be concluded that the mic and mcc were the same for these strains under the experimental conditions. in all cases, the nine values obtained (three repetitions in three independent situations) were exactly the same and no standard deviations could be noticed. the mann-whitney u test demonstrated a significant reduction in the resistance to chlorhexidine under anoxia. this test also showed no significant differences for mic in relation to the anatomic origin of strains (p = 0.9584). in order to evaluate the influence exerted by atmospheric oxygen on mic performance, the increment rate of effectiveness (ire) for each isolate was calculated through the equation: ire = mic normoxia / mic anoxia . figure 3 shows that the ire values for the pp strains varied from 2.0-fold to 128.8-fold, with higher frequencies of 2.0-fold (37.5%), 16-fold (18.75%), and 66.8-fold (18.75%). om strains had their susceptibility ranging from 2.0-fold to 66.8-fold, with higher frequencies of 4.0-fold (37.5%), 66.8-fold (32.25%), and 16-fold (18.75%). discussion studies addressing the action of chlorhexidine on yeasts living in anoxia are scarce16. until the present moment, no data referring to the susceptibility of pp c. albicans strains to antimicrobials under anoxia are available. it has been shown that c. albicans cells living in anoxic environments are protected against the action of most common antimycotics14. this anaerobic-related resistance to antifungal probably derives from a suppression of ergosterol biosynthesis at fungal cell membrane. as the ergosterol biosynthetic pathway is the main target of azoles and such via is not used when candidal cells are under anoxia, these antimycotics completely lose their efficacy. on the other hand, as ergosterol appears not to be synthesized under anoxic conditions, polyenes do not manifest their membrane interactive behavior either. furthermore, it has been shown that histatin-5, a potent salivary antifungal peptide, might have such effect abolished, once the mitochondrial energy level is very low when in anoxia17,18. in this study, the results showed that chlorhexidine had its candidacidal capacity increased in such environmental conditions. two plausible hypotheses to explain such phenomenon are proposed. firstly, this increased susceptibility to chlorhexidine in an oxygen-free environment may be better discussed taking into account that oxygen is also a positively charged element19 and has a cationic behavior. based on this premise, we herein postulate that, in normoxia, the existing atmospheric oxygen competes with chlorhexidine for binding sites, whereas it does not occur in an anoxic environment. plaut et al.20 calculated the sorption enthalpy for chlorhexidine and stated that it may be mediate by electrostatic-like bonding interactions. such interactions are weak and depend on the molecular size. akaho and fukumori21 stated that an area near to 548 å2 is required for the complete absorption of chlorhexidine molecule to an amphyphylic surface in order to accommodate its ionic and hydrophobic moieties responsible for the adsorption to solid surfaces. this value is much higher than the 1.2 å of molecular oxygen, commonly absorbed for the aerobic respiration. additionally, the fact that the deenergization caused by the dropdown in aerobic respiration rates affects significantly the plasmatic membrane of eukaryotes leading to a reduction in the negative potentials22-25. anoxic conditions cause a release of previously accumulated lipophilic cation tetraphenylphosphonium (tpp+) into saccharomyces cerevisiae26, which is compatible with the assumption that anoxia reduces de negative feature of fungal membranes. interestingly, the data hereby presented show that the inhibitory efficiency of chlorhexidine increases in such unfavorable environmental conditions. our assumption that the better action of chlorhexidine in the absence of oxygen must be derived from 35 30 25 20 15 10 5 0 2.5x 64x 10x 16.5x 1.3x 133x 2.5x 64x 10x 1.3x 133x 33.3x 16.5x 5x increment rates of e�ectiveness fr eq u en cy o f d is tr ib u ti o n a m o n g s tr ai n s (% ) periodontal-related strains mucosa-related strains figure 3. increment rate of effectiveness (ire) for minimal inhibitory concentrations (mic) of chlorhexidine digluconate for periodontium-related (pp) and mucosa-related (om) candida albicans strains in normoxia and anoxia. 109enhanced susceptibility of candida albicans to chlorhexidine under anoxia braz j oral sci. 8(2): 105-10 a disruption in the competition rates is reinforced by the fact that some cations may reduce its adsorption and antimicrobial effectiveness by competitive ways20,27,28. secondly, it has been previously reported that chlorhexidine substantially increases the cell permeability on apical and sub-apical segments of early-stage filamentous forms higher than for yeast forms29. this is compatible with the proposed mechanism of disruption of the membrane followed by rapid permeabilization. therefore, it is possible that fungal cells had their susceptibility have increased by the anaerobically-induced filamentation. interestingly, the origin of strains did not exert any influence on the susceptibility (p = 0.9584). however, as strains from culture collections were used in the present study, the long-term storage or the multiple re-inoculations might have decreased their susceptibility. although the sampling have been done six months before the study (data not shown), it was not possible to ensure whether or not it actually occurred. further studies enrolling freshly isolated strains may possibly clarify this issue. the extensive variation observed in the increase of effectiveness rates indicates a great heterogeneity for this characteristic. some strains presented only an increment of mere 1.3-fold susceptibility, whereas others had increased their effectiveness rates in 133 times. it may be suggested that it is variable according to the strains and no generalizations may be done. the results of the present study are applicable for growing planktonic cells. for c. albicans grown in biofilms, lamfon et al.30 previously reported that the resistance to chlorhexidine increases up to 8-fold the mic in relation to planktonic counterparts grown in normoxia. such result contrasts with those of the present study since it is widely accepted that the accessibility of oxygen to internal layers of biofilms is limited. there are two points that may clarify the differences between the results from lamfon’s et al.30 study and those expected after assuming that anoxia increases chlorhexidine effectiveness. firstly, those authors grew their biofilms under normoxic conditions; secondly, the extracellular matrix present in the biofilm may act as a barrier against the antiseptic diffusion. according to the results obtained in the present study, it may be concluded that chlorhexidine is effective to restrain c. albicans grown in the anoxic periodontal pocket with minor concentrations than those needed to kill cells living on surfaces under normoxic conditions. the origin of strains seems not to influence the growth of c. albicans under neither normoxia nor anoxia. however, despite these encouraging results, our opinion is that clinicians should not indicate lower chlorhexidine doses to their patients. the main advantage of lower mic is the maintenance of inhibition effects throughout the lixiviation of chlorhexidine. acknowledgements authors thank to professor sérgio a. ignácio for his assistance in the statistical analysis. this study was conducted using grants from araucaria foundation (fa63/07, protocol 9042) and was part of the master’s degree thesis of a.m.s. references 1. apatzidou da, riggio mp, kinane df. quadrant root planing versus sameday fullmouth root planing ii. microbiological findings. j clin periodontol 2004;31:141-8. 2. tozum tf, yildirim a, caglayan f, dincel a, bozkurt a. serum and gingival crevicular fluid levels of ciprofloxacin in patients with periodontitis. j am dent assoc. 2004;135:1728-32. 3. haffajee ad, socransky ss. microbial etiological agents of destructive periodontal diseases. periodontol 2000 1994,5:78-111. 4. song x, sun j, hansen bf, olsen i. oral distribution of genera, species and biotypes of yeasts in patients with marginal periodontitis. microb ecol health dis 2003;15:114-9. 5. hannula j, dogan b, slots j, ökte e, asikainen s. subgingival strains of candida albicans in relation to geographical origin and occurrence of periodontal pathogenic bacteria. oral microbiol immunol 2001;16:113-8. 6. reynaud ah, nygaard-ostby b, boygard gk, olsen i, gjermo p. yeasts in periodontal pockets. j clin periodontol 2001;28:860-4. 7. wiebe cb, putnins ee. the periodontal disease classification system of the american academy of periodontology – an update. j can dent assoc 2000;66:594-7. 8. oliveira lf, jorge aoc, santos ssf. in vitro minocycline activity on superinfecting microorganisms isolated from chronic periodontitis patients. braz oral res 2006;20:202-6. 9. freitas cs, diniz hf, gomes jb, sinisterra rd, cortes me. evaluation of the substantivity of chlorhexidine in association with sodium fluoride in vitro. pesq odontol bras 2003;17:78-81. 10. bonacorsi c, raddi ms, carlos iz. cytotoxicity of chlorhexidine digluconate to murine macrophages and its effect on hydrogen peroxide and nitric oxide induction. braz j med biol res 2004;37:207-12. 11. veerman ec, nazmi k, van’t hof w, bolscher jg, den hertog al, nieuw amerongen av. reactive oxygen species play no role in the candidacidal activity of the salivary antimicrobial peptide histatin 5. biochem j 2004;381:44752. 12. prasad r, hofer m. tetraphenylphosphonium is an indicator of negative membrane potential in candida albicans. biochim biophys acta 1986;861:37780. 13. liao rs, rennie rp, talbot ja. assessment of the effect of amphotericin b on the vitality of candida albicans. antimicrob agents chemother 1999;43:103441. 14. dumitru r, hornby jm, nickerson kw. defined anaerobic growth medium for studying candida albicans basic biology and resistance to eight antifungal drugs. antimicrob agents chemother 2004;48:2350-4. 15. national committee for clinical laboratory standards. reference method for broth dilution antifungal susceptibility testing of yeasts. approved standard m27-a2 – 2nd ed. wayne, pa.: national committee for clinical laboratory standards, 2002. 16. jensen je. the effect of chlorhexidine on the anaerobic fermentation of saccharomyces cerevisiae. biochemical pharmacology 1975;24:2163-6. 17. helmerhorst ej, breeuwer p, van’t hof w, walgreen-weterings e, oomen lc, veerman ec, amerongen av, et al. the cellular target of histatin 5 on candida albicans is the energized mitochondrion. j biol chem 1999;274:7286-91. 18. helmerhorst ej, troxler rf, oppenheim fg. the human salivary peptide histatin 5 exerts its antifungal activity through the formation of reactive oxygen species. proc natl acad sci usa 2001;98:14637-42. 19. goultschin j, levy h. inhibition of superoxide generation by human polymorphonuclear leukocytes with chlorhexidine. its possible relation to periodontal disease. j periodontol 1986;57:422-5. 20. plaut bs, davies djg, meakin bj, richardson ne. the mechanism of interaction between chlorhexidine digluconate and poly(2-hydroxyethylmethacrylate). j pharmacol 1981;33:82-8. 110 semprebom am, isidoro aca, machado mân, campelo pms, höfling jf, samaranayake lp, rosa ear braz j oral sci. 8(2): 105-10 21. akaho e, fukumori y. studies on adsorption characteristics and mechanism of adsorption of chlorhexidine mainly by carbon black. j pharm sci 2001;90:128897. 22. miller ag, budd k. evidence for a negative membrane potential and for movement of c1against its electrochemical gradient in the ascomycete neocosmospora vasinfecta. j bacteriol 1976;128:741-8. 23. komor e, tanner w. the determination of the membrane potential of chlorella vulgaris. evidence for electrogenic sugar transport. eur j biochem 1976;70:197204. 24. hofer m, kunemund a. tetraphenylphosphonium ion is a true indicator of negative plasma-membrane potential in the yeast rhodotorula glutinis. experiments under osmotic stress and at low external ph values. biochem j 1985;225:815-9. 25. gimmler h, weis u, weiss c, kugel h, treffny b. dunaliella acidophila (kalina) masyuk-an alga with a positive membrane potential. new phytol 1989;113:175-84. 26. boxman aw, barts pw, borst-pauwels gw. some characteristics of tetraphenylphosphonium uptake into saccharomyces cerevisiae. biochim biophys acta 1982;686:13-8. 27. waler sm, rolla g. plaque inhibiting effect of combinations of chlorhexidine and the metal ions zinc and tin. a preliminary report. acta odontol scand 1980;38:213-7. 28. ben-yaakov d, friedman m, hirschfeld z, gedalia i. fluoride enhancement of chlorhexidine uptake by hydroxyapatite and enamel powders. j oral rehabil 1984;11:65-70. 29. suci pa, tyler bj. action of chlorhexidine digluconate against yeast and filamentous forms in an early-stage candida albicans biofilm. antimicrob agents chemother. 2002;46:3522-31. 30. lamfon h, porter sr, mccullough m, pratten j. susceptibility of candida albicans biofilms grown in a constant depth film fermentor to chlorhexidine, fluconazole and miconazole: a longitudinal study. j antimicrob chemother 2004;53:383-5. oral sciences n3 braz j oral sci. 14(3):251-255 original article braz j oral sci. july | september 2015 volume 14, number 3 characterization of surface topography and chemical composition of mini-implants luegya amorim henriques knop1, ana prates soares2, ricardo lima shintcovsk1, lidia parsekian martins1, luiz gonzaga gandini jr. 1 1universidade estadual paulista – unesp, araraquara dental school, department of orthodontics and pediatric clinic, araraquara, são paulo, brazil 2universidade de são paulo – usp, hospital of rehabilitation of oralfacial anomalies, area of prosthodontics, bauru, são paulo, brazil correspondence to: luegya amorim henriques knop rua magno valente, 110, apartmento 1401 a, pituba cep: 41810-620 salvador, ba, brasil phone: +55 71 3358 3338 e-mail: luegya@gmail.com abstract aim: to assess the surface topography and chemical composition of three brands of as-received mini-implants (sin®, morelli®, and conexao®). methods: twelve mini-implants of each brand were analyzed by scanning electron microscopy and energy dispersive x-ray (edx). results: there was no significant differences among sin®, morelli®, and conexao® mini-implants comparing their surface topography by visualization of sem micrographs and analysis of scores. the edx analysis showed statistically significant difference among them for the amount of ti, al and v. miniimplants sin® presented also n and o in their composition. conclusions: in conclusion, the miniimplants morelli®, sin® and conexao® presented ti as main component of the alloy. remaining components, such as al and v, were also observed in all the analyzed brands, with differences among them. only sin® mini-implants presented n and o. as far as surface topography is concerned, there are no differences among the three brands of mini-implants. keywords: orthodontics; orthodontic anchorage procedures; titanium. introduction temporary anchorage devices (tads) such as mini-implants act as skeletal anchorage for orthodontic movements. tads are used when dental anchorage is insufficient or a large amount of dental movement is required1. these devices are widely used in orthodontics offering excellent results and solving anchorage problems that could not be addressed previously2 by overcoming the active versus reactive forces generated during tooth movement3. the optimal use of tad should have some requirements such as small size, placement without drilling, stability to withstand immediate and long term loading, easy removal and comfort for the patient4. all these features, especially the small dimensions of mini-implants, require a strong, high-grade titanium alloy. grade 5 ti, also known as ti-6al-4v, is composed of 6% aluminum, 4% nittro, 0.25% (maximum) iron, 0.2% (maximum) oxygen and ti (remaining percentage). the result is a combination of strength and fabricability 5. in biological terms, grade 5 machined ti promotes cell proliferation, good cytocompatibility and cell adhesion4 although it does not provide a good-quality osseointegration, which facilitates removal when needed6. the design of miniimplants should feature a head to engage elastic bands or sprains, a smooth transmucosal neck, an endossous self tapping body and a special groove in their tip to be used for cutting or tapping the bone during insertion, which is called lead angle1. http://dx.doi.org/10.1590/1677-3225v14n3a15 received for publication: august 26, 2015 accepted: september 30, 2015 manufacturers can create mini-implants in different shapes and sizes. while they usually supply information on outer diameter and length, chemical composition, depth, pitch, lead angle of the thread as well as surface characteristics are rarely provided7-8. the objective of this study was to assess the surface topography and chemical composition of three brands of mini-implants (morelli®, sin® and conexao®) by scanning electron microscopy (sem) and energy dispersive x-ray (edx). material and methods the sample was composed of 36 mini-implants of 3 different commercial brands: sin® (são paulo, sp, brazil; n= 12, 1.4 mm diameter, 8 mm length), morelli® (sorocaba, sp, brazil; n= 12, 1.5 mm diameter, 8 mm length), and conexao®; (aruja, sp, brazil; n= 12, 1.5 mm diameter, 8 mm length). all mini-implants were individually packaged and used as received from the manufacturers. packages were only opened at the beginning of each analysis and carefully handled in order to prevent contamination. analysis of surface topography by sem the specimens were fixed on sem-stub-holders and visualized through a fieldemission scanning electron microscope (fe-sem) type 6301f (jeol ltd., tokyo, japan) at 2kv with a working distance of 39mm and a small spot size. representative sem micrographs of the head, body and notch were taken from each sample of the different brands. a single experienced examiner viewed the samples at 30, 60 and 75x magnification to obtain the images after brightness, contrast and focus adjustments. surface texture was observed and described in a qualitative manner, comparing the groups. mini-implant topography was evaluated according to the following scoring system: (0) absence of defects and irregularities (like striations or protrusions); (1) presence of defects in up to 25% of the mini-implant threads; (2) presence of defects in up to 50% of the mini-implant threads; (3) presence of defects in up to 75% of the miniimplant threads; (4) presence of defects in all mini-implant threads. the mean scores were analyzed statically by anova and tukey’s test. significance level was t at 5%. analysis of chemical composition by edx the chemical composition of the same all miniimplants was analyzed by edx at the same sites of the topographical analysis. the edx generated graphics composed of the chemical compounds found in the device a n d t h e i r r e s p e c t i v e a m o u n t s . t h e i n f o r m a t i o n w a s g a t h e r e d i n a s i n g l e t a b l e , w h i c h w a s u s e d f o r t h e s t a t i s t i c a l a n a l y s i s . d a t a w e r e a n a l y z e d b y a n o v a f o l l o w e d b y t u k e y ’ s p o s t t e s t t o d e t e c t d i f f e r e n c e s regarding the amount of the studied chemical elements (c, al, ti, and v) in each mini-implant brand. significance level was t at 5%. results surface topography sin® mini-implants were characterized by a clearly wellpolished and visible head (figure 1), uniform unidirectional threads, likely to be the result of machining. the machined metal surface of sin® appeared to be more satisfactory defined with few structural defects among the 12 (figure 2). morelli® mini-implants revealed uniform threads, but with some surface defects, especially on the body. in addition, small irregularities, such as striations, were also visible on a typical machined metal surface (figure 3). conexao® mini-implants presented no equivalent distance among the threads, with larger distances among the threads on the body compared with the head. the surface was homogenous, well polished, and with few structural defects such as protrusion (figure 4). on the notch we observed a vertical design that is possibly very relevant to bone drilling (figure 5). the three mini-implant groups received score 1 (mean), with no statistically significant difference among the brands (p>0.05). fig. 1. sem micrograph of the head of a sin® mini-implant (x30) fig. 2.sem micrograph of the body of a sin® mini-implant showing a small defect (x60) characterization of surface topography and chemical composition of mini-implants 1 7 11 7 11 7 11 7 11 7 1252252252252252 braz j oral sci. 14(3):251-255 253253253253253 fig. 3. sem micrograph of the body of a morelli® mini-implant showing evident surface roughness (x60) fig. 4. sem micrograph of the body of conexao® mini-implant. a well-polished surface can be observed, with small defects (x60) fig. 5. sem micrograph of the notch of conexao® mini-implant (x75) chemical composition mini-implants from sin®, morelli® and conexao® had ti, o, al and v in their composition. carbon and nitrogen in smaller amounts were found as impurities in sin® miniimplants (figure 6). it was not possible to conduct statistical inference regarding elements n and o since their results for all the studied samples by morelli® and conexao® were zero (table 1). discussion translational research is an important component of orthodontic research since it can translate information from the laboratory to enhance the outcomes of patients’ treatments3. the present research analyzed mini-implants using sem and edx to verify their chemical composition and surface design, two aspects of major relevance when choosing the device to be used in a patient. titanium is a biocompatible metal with proper mechanical9 and corrosion resistance10, which makes its presence in the composition of mini-screws very important. mini-implants present a composition commercially known as ti-6al-4v with al and v in addition to ti. this type of alloy provides the mini-implant with greater resistance if compared with conventional implants with larger diameter. in contrast, such formulation generates lower biological compatibility decreasing the osseointegration phenomenon5. all the analyzed brands presented ti as main component, and lower amounts of al and v (score1). conexao® and morelli® mini-implants presented ti with quantities equivalent to the respective manufacturers’ commercial descriptions. mini-implant sin® presented lower ti concentration with statistically significant difference in relation to morelli® (p<0.0001) but no significance in relation to conexao®. alsamak et al.5 identified foreigner mini-implants brands with the presence of ti, v, al and o in quantities similar to the ones found in our research, which corroborates the composition of the ti 5 grade alloy. park et al.11 stated that the osseointegration phenomenon does not occur despite the presence of ti in mini-implants, which is a very important characteristic to facilitate their removal. sin® presented other components, as n (1.59%), and o (18.04%). chin et al.12 studied 5 different mini-implants associating their xps survey spectra with a constitution of primarily c, o and ti, but also detecting traces of n, ca, fe, cr, cu, pb, zn, and si. the authors reported it as an apparent problem of surface contamination. silverstein et al. 1 3 evaluated three different brands of mini-implants by xps and observed that the elements found in all them were mainly c, o, and ti. they also found were other metals in small amounts, and other trace elements. all three mini-screws showed very different characteristics in surface composition. jofré et al.14 evaluated the 2-year survival rate of miniimplants that came into contact with stainless steel prior to insertion. sem and edx analyses revealed c and o in all mini-implants. those that had contact with stainless steel, additional elements were identified, including si, ca, fe, and cr. the authors found that during the 2-year follow-up, characterization of surface topography and chemical composition of mini-implants braz j oral sci. 14(3):251-255 1 7 11 7 11 7 11 7 11 7 1254254254254254 c 2.33 (0.61) 2.32 (0.73) 2.40 (0.41) 0.9252 n 1.59 (0.77) 0.00 (0.00) 0.00 (0.00) —o 18.04 (2.96) 0.00 (0.00) 0.00 (0.00) —al 4.61 (0.17) a 5.75 (0.18) b 5.32 (0.72) b <0.0001 ti 72.88 (5.21) a 88.11 (0.64) b 77.98 (8.70) a <0.0001 v 3.15 (0.35) a 4.16 (0.23) b 4.01 (0.23) b <0.0001 element sin® morelli® conexao® p value % mean (s.d.) % mean (s.d.) % mean (s.d.) (anova and tukey’s test) table 1. table 1. table 1. table 1. table 1. chemical elements detected in each group (n=12) of mini-implants sin®, morelli® and conexao® results were calculated as percentage and are presented in the table mean and standard deviation. different letters in rows mean statistically significant difference among the mini-implants (p<0.05) fig. 6. graphic of the chemical composition of a sin® mini-implant showing ti, al, v, c, o and n amounts. one mini-implant failed (97.8% survival rate). so, the authors concluded that stainless steel surgical guides does not seem to generate contamination that compromises the survival of mini-implants. according to vezeau et al.15, the contamination appeared to occur during the manufacturing, packaging and handling processes, as well as to result from sterilization procedures involving undesirable located water condensation, and the heterogeneity of a mixed sterilizer load in an institutional setting. morra et al.16 affirm that airborne n contaminations in the implants are unavoidable and usual to a reasonable level of inadequate surface treatment and implant handling (during packaging, for example). this type of surface pollution is typically inhomogeneous surrounding the implant, and should not be mistaken with controlled chemical or biochemical modifications. generally, as-received mini-implants can present high amounts of c, indicating high level of particulate contamination on oxide surfaces12. the present study revealed all the groups with small quantity of c with non-statistically significant difference among the tested brands. the success of orthodontic mini-plants depends on the metallurgy applied in their production, which is especially associated with a great quality alloy surcharge and its proper handling. during the process of alloy turning to produce the mini-implants, metallurgical contamination must be prevented17. further studies should investigate whether the contamination of mini-implants surfaces interferes in their biocompatibility or in the stability of their clinical use. a systematic review by schatzle18 presented 363 or 15.3% failures out of 2374 mini-screws inserted in 1196 patients. the contamination of the mini-implant surface is a possible cause of clinical failure. the topography analysis revealed that the three brands presented some type of structural defect such as protrusion or striated surfaces, especially in the body, in addition to some roughness. in this study, it was given a score to quantify the surface topography homogeneity. all brands presented score 1, with no statistical difference among them. it is not certain how decisive the interference of such structural alterations are to the success of the mini-implant; although burmann et al.19 states that differences in mini-implant design and the presence of surface irregularities may influence the effectiveness of orthodontic anchorage. characterization of surface topography and chemical composition of mini-implants braz j oral sci. 14(3):251-255 255255255255255 according to melsen20 the head of mini-implants should be well polished to avoid the accumulation of biofilm on local tissues. this characteristic also decreases the possibility of causing injuries to the surrounding mucous membrane consequently increasing the possibility of success during the treatment. all the studied brands presented favorable characteristics in the area enabling their clinical use. further studies should be carried out in order to assess those mini-implants in vivo, which could enable their clinical use with improved safety. in conclusion, the mini-implants by sin®, morelli® and conexao® presented ti as main component of the alloy; the remaining components, such as al and v, were also observed in all the analyzed brands. sin® was the only brand presenting mini-implant with chemical elements o and n. all miniimplants presented structural defects in the sem analysis, with no differences among the groups. acknowledgements we would like to thank the osvaldo cruz foundation (fiocruz), especially the electron microscopy service, for supporting our research and providing a scanning electron microscope. references 1. prabhu j, cousley rr. current products and practice: bone anchorage devices in orthodontics. j orthod. 2006; 33: 288-307. 2. kalarickal b. group distal movement of teeth using micro-screw-implant anchorage-a case report. j clin diagn res. 2014; 8: 26-9. 3. rossouw e. translational mini-screw implant research. j orthod. 2014; 41: s8-s14. 4. galli c, piemontese m, ravanetti f, lumetti s, passeri g, gandolfini m, et al. effect of surface treatment on cell responses to grades 4 and 5 titanium for orthodontic mini-implants. am j orthod dentofacial orthop. 2012; 141: 705-14. 5. alsamak s, psomiadis s, gkantidis n. positional guidelines for orthodontic mini-implant placement in the anterior alveolar region: a systematic review. int j oral maxillofac implants. 2013; 28: 470-9. 6. mizrahi e, mizrahi b. mini-screw implants (temporary anchorage devices): orthodontic and pre-prosthetic applications. j orthod. 2007; 34: 80-94. 7. katiæ v, kamenar e, blaževiæ d, spalj s. geometrical design characteristics of orthodontic mini-implants predicting maximum insertion torque. korean j orthod. 2014; 44: 177-83. 8. walter a, winsauer h, marcé-nogué j, mojal s, puigdollers a. design characteristics, primary stability and risk of fracture of orthodontic miniimplants: pilot scan electron microscope and mechanical studies. med oral patol oral cir bucal. 2013; 18: e804-10. 9. gonçalves jp, shaikh aq, reitzig m, kovalenko da, michael j, beutner r, et al. detonation nanodiamonds biofunctionalization and immobilization to titanium alloy surfaces as first steps towards medical application. beilstein j org chem. 2014; 26: 2765-73. 10. carlsson l, rostlund t, albretsson b, albretsson t, branemark pi. osseointegration of titanium implants. acta orthop scand. 1986; 57: 385-9. 11. park hs, kwon tg, sung jh. nonextraction treatment with microscrew implants. angle orthod. 2004; 74: 539-49. 12. chin myh, sandham a, de vries j, van der mei hc, busscher hj. biofilm formation on surface characterized micro-implants for skeletal anchorage in orthodontics. biomaterials. 2007; 28: 2032-40. 13. silverstein j, barreto o, frança r. miniscrews for orthodontic anchorage: nanoscale chemical surface analyses. eur j orthod. 2015; 13. pii: cjv007. 14. jofré j, conrady y, carrasco c. survival of splinted mini-implants after contamination with stainless steel. int j oral maxillofac implants. 2010; 25: 351-6. 15. vezeau pj, koorbusch gf, draughn ra, keller jc. effects of multiple sterilization on surface characteristics and in vitro biologic responses to titanium. j oral maxillofac surg 1996; 54: 738-46. 16. morra m, cassinelli c, bruzzone g, carpi a, di santi g, giardino r, et al. surface chemistry effects of topographic modification of titanium dental implant surfaces: 1. surface analysis. int j oral maxillofac implants. 2003; 18: 40-5. 17. carano a, velo s, leone p, siciliani g. clinical applications of the miniscrew anchorage system. j clin orthod. 2005; 39: 9-24. 18. schätzle m, männchen r, zwahlen m, lang np. survival and failure rates of orthodontic temporary anchorage devices: a systematic review. clin oral implants res. 2009; 20: 1351-9. 19. burmann pf, ruschel hc, vargas ia, de verney jc, kramer pf. titanium alloy orthodontic mini-implants: scanning electron microscopic and metallographic analyses. acta odontol latinoam. 2015; 28: 42-7. 20. melsen b. mini-implants, where are we? j clin orthod. 2005; 39: 539-47. characterization of surface topography and chemical composition of mini-implants braz j oral sci. 14(3):251-255 oral sciences n3 original article braz j oral sci. october | december 2015 volume 14, number 4 influence of implant-abutment angulations and crown material on stress distribution on central incisor: a 3d fea mohamed i. el-anwar1, khairy e. al-azrag2, mohamed h. ghazy3, lamia e. dawood3 1national research centre, department of mechanical engineering, giza, egypt 2university of almergib, faculty of dentistry, libya 3university of mansoura, faculty of dentistry, department of conservative dentistry, mansoura, egypt correspondence to: mohamed i. el-anwar national research centre mechanical engineering dept., 33 el bohouth st., dokki, giza p.o. 12622, egypt. phone: +2 0122 2431297 / fax: +2 02 33370931 e-mail: anwar_eg@yahoo.com abstract aim: to investigate the effect of implant-abutment angulation and crown material on stress distribution of central incisors. finite element method was used to simulate the clinical situation of a maxillary right central incisor restored by two different implant-abutment angulations, 15° and 25°, using two different crown materials (ips e-max cad and zirconia). methods: two 3d finite element models were specially prepared for this research simulating the abutment angulations. commercial engineering cad/cam package was used to model crown, implant abutment complex and bone (cortical and spongy) in 3d. linear static analysis was performed by applying a 178 n oblique load. the obtained results were compared with former experimental results. results: implant von mises stress level was negligibly changed with increasing abutment angulation. the abutment with higher angulation is mechanically weaker and expected to fail at lower loading in comparison with the steeper one. similarly, screw used with abutment angulation of 25° will fail at lower (about one-third) load value the failure load of similar screw used with abutment angulated by 15°. conclusions: bone (cortical and spongy) is insensitive to crown material. increasing abutment angulation from 15° to 25°, increases stress on cortical bone by about 20% and reduces it by about 12% on spongy bone. crown fracture resistance is dramatically reduced by increasing abutment angulation. zirconia crown showed better performance than e-max one. keywords: finite element analysis; dental implant-abutment design; incisor; materials. introduction dental implant restoration has been widely accepted as one of the treatment modalities to replace missing teeth and restore human masticatory function. the biomechanical properties of the bone–implant interface determine the implant stability. the bone–implant interface properties depend on amount of implant surface in contact with mineralized bone tissue and bone tissue quality around the interface11111. the interface has a complex biomechanical nature due to (i) its roughness, (ii) the fact that bone is in partial contact with the implant, (iii) adhesion phenomena between bone and the implant and (iv) the time-evolving nature of the interface properties. therefore, remodeling phenomena of bone tissue around the interface are difficult and highly complicated. a single tooth implant with crown has greater survival rate than a fixed partial denture (fpd)22222. the abutment angulation is a mechanical variable in implantology33333 http://dx.doi.org/10.1590/1677-3225v14n4a13 received for publication: november 15, 2015 accepted: december 13, 2015 braz j oral sci. 14(4):323-329 that may influence the internal and external structure of bone tissue33333. thus, the bone behavior is related to the stress and deformation induced on it. the influence of angled abutments on stress is a matter of debate44444. it has been widely accepted that increased stress on implants and bone has been associated with the use of angled abutments55555. the dental implants design is driven by an imitator marketing approach rather than by scientific advances11111. clinicians used implants in new applications before research was carried out based on their basic science. empirical approaches may have some advantages but remain limited when it comes to understand the interaction of the various mechanisms, playing a role in bone healing around an implant11111. ips e-max lithium disilicate glass ceramic, a material that provides optimum esthetics, yet has the strength to enable conventional or adhesive cementation. it has a needle-like crystal structure that offers excellent high flexural strength, roughly 360 to 400 mpa and durability, as well as outstanding optical properties. it can be traditionally pressed or contemporarily processed via cad/cam technology. due to its strength and versatility, the material can be utilized for anterior/posterior crowns, inlays/onlays, veneers, thin veneers, telescopic crowns, implant restorations and anterior three units bridgework up to the second premolar (press only)66666. yttrium-stabilized tetragonal zirconia (y-tzp) is having increased use in dentistry due to its good mechanical properties. it is currently used as a core material in all-ceramic dental restorations and implant superstructures77777. compared to other dental ceramics, its superior mechanical properties, such as higher strength and fracture toughness, are due to the transformation toughening mechanism, similar to that observed in quenched steel88888. finite element analysis (fea) is an accepted and accurate numerical technique used for solving complicated stress analysis problems. it has proven to be a reliable method in dentistry as it provides reliable evaluation of stresses in complex geometries99999. in this study, the influence of implant-abutment angulation (15° and 25°) supporting different central incisor crown material on stress distribution was estimated. material and methods implant fixture (hexacone hc2 3.7 13 mm) and two abutments (tla15 hc1, and tla25 hc1; dr. ihde dental gmbh, eching, germany), were modeled using commercial cad/cam “autodesk inventor” software version 8.0 (autodesk inc., san rafael, ca, usa). bone geometry was simplified and simulated as two co-axial cylinders. the inner one represents the spongy bone (14 mm diameter x 22 mm high) filling the internal space of the outer cylinder (1 mm thick shell) that represents cortical bone (16 mm diameter x 24 mm high)10-1110-1110-1110-1110-11. the crown dimensions were obtained from the anatomical data1212121212 of the maxillary right central incisor. the cement layer was designed with a 50 µm thickness. the geometric models were exported from the cad/ cam software as several components (sat and iges files) to be assembled together in ansys version 14.5 environment model 1 model 2 implant abutment 15° implant abutment 25° nodes elements nodes elements implant 28463 25131 28017 24918 abutment 86354 78355 7819 7624 screw 10605 9227 9881 8597 crown 84959 73388 23643 20816 cortical bone 2708 3676 2966 3940 spongy bone 44070 36174 43492 35801 gutta-percha 18948 15835 1598 1658 cement layer 23036 35108 1858 2825 table 2:table 2:table 2:table 2:table 2: number of nodes and elements after meshing models’ components..... material modulus of elasticity [mpa] poisson’s ratio cortical bone 13600 0.26 cancellous bone 1360 0.31 titanium 110000 0.25 ips e-max cad 96000 0.23 zirconia 205000 0.22 rely x unicem 4900 0.30 aplicap cement gutta-percha 0.00069 0.45 table 1:table 1:table 1:table 1:table 1: material’s properties used in the fe models1313131313..... (ansys inc., canonsburg, pa, usa). a set of boolean operations was performed to obtain two fe models, for 15° and 25° abutment angulations. element type “solid 186” (higher order 20 node) was utilized for meshing the model’s components, as it has three degrees of freedom (translations in the global directions x, y and z). complete osseointegration was assumed. in addition isotropic, homogenous, and linearly elastic materials’ properties were fed into the finite element (fe) software based on previous studies9-11,139-11,139-11,139-11,139-11,13 and manufacturer’s information (table 1). meshing density was then evaluated and adequate mesh of the models’ components was used in the analysis. the number of nodes and elements in each component are in table 2. figures 1 and 2a illustrate models’ components on ansys screen. load of 178 n1414141414 was applied on each model on the palatal surface of the maxillary right central incisor at oblique directions 45° to the long axis of the implant fixture (figure 2b)1515151515. the boundary conditions were defined by fixing the lower surface of the cylinder representing cortical bone. additionally, the implant fixture, abutment, screw, cementlayer, gutta-percha, crown, cortical and spongy bone were assumed to be perfectly bonded together1616161616. the finite element models were verified against previous experimental studies 13,1513,1513,1513,1513,15, where two groups each of 14 implant-abutment complexes (angled 15° and 25°) were gradually loaded up to failure in a universal testing machine. the fea results showed very good agreement with experiments’ results. influence of implant-abutment angulations and crown material on stress distribution on central incisor: a 3d fea324324324324324 braz j oral sci. 14(4):323-329 fig. 1: modeled and meshed components (implant, screw, gp, abutment, cement layer, crown) screen shots from inventor / ansys screens fig. 2: (a) complete meshed model (b) schematic for load direction results the von mises stress distributions and their maximum values were discussed in details. figure 3 illustrates the increase of maximum value of von mises stress with increasing abutment angulation from 15° to 25° on cortical bone, and the stress distribution did not change. on the other hand, spongy bone von mises stress distribution with different abutment angulation is in figure 4, where the spongy bone showed lower values with increasing abutment angulation. contrarily, crown material change did not affect bone stress. from mechanical point of view, the lower-angulated abutment was expected to survive against more loading than the higher-angulated ones. increasing abutment angulation increases the abutment von mises stress and may change its distribution. as indicated in figure 5, the 15° angulated abutment stress level is about 25% less than the one of the 25 angulated one. similarly, screw behavior with different abutment angulations indicated higher stress values under the screw head for 25° angulated abutment (figure 6), which may fail by its head removal, and/or screw bent with load (in the same direction). figure 7, illustrates that the cement layer will suffer influence of implant-abutment angulations and crown material on stress distribution on central incisor: a 3d fea 325325325325325 braz j oral sci. 14(4):323-329 fig. 3: cortical bone von mises stress distribution comparison between (a) 15°, (b) 25° angulated abutment under zirconia crown fig. 4: spongy bone von mises stress distribution comparison between (a) 15°, (b) 25° angulated abutment under ips e-max cad crown. fig. 5: abutment von mises stress distribution comparison between (a) 15°, (b) 25° angulated abutment under zirconia crown. more with 15° abutment angulation, about double the von mises stress, in comparison with 25° abutment angulation. the location of maximum von mises stress was expected to be at the finish line with 15° abutment angulation, and at abutment step with 25° abutment angulation (as crown will tend to slide inward from its original position). comparing the different crown materials investigated in this study, as shown in figure 8, the place of maximum influence of implant-abutment angulations and crown material on stress distribution on central incisor: a 3d fea326326326326326 braz j oral sci. 14(4):323-329 fig. 6: screw von mises stress distribution comparison between (a) 15°, (b) 25° angulated abutment under zirconia crown. fig. 7: cement layer von mises stress distribution comparison between (a) 15°, (b) 25° angulated abutment under ips e-max cad crown. fig.8: von mises stress distribution on different crown materials (a) ips emax cad, (b) zirconia supported by 15° abutment. von mises stress of indicated expected crown failure as two similar parts with using vertical cutting plane. finally, table 3 compares maximum values of von mises stress exerted on all components on the studied models. discussion nowadays the advantages of monolithic zirconia restorations with an increased mechanical stability made them influence of implant-abutment angulations and crown material on stress distribution on central incisor: a 3d fea 327327327327327 braz j oral sci. 14(4):323-329 model 1: e-max model 1: zirconia model 2: e-max model 2: zirconia cortical bone 111.67 111.61 133.14 133.15 spongy bone 9.637 9.636 8.181 8.181 implant 218.30 218.22 224.50 244.51 abutment 1193.52 1225.24 1566.55 1563.47 screw 82.29 89.19 269.66 269.36 gutta-percha 53.09 49.29 30.79 30.02 cement layer 332.34 342.11 174.05 175.74 crown 456.80 607.62 822.91 824.76 table 3:table 3:table 3:table 3:table 3: values of maximum von mises stresses [in mpa] induced in the side of load application under oblique loading condition in all models..... possible to expand their clinical indications1717171717. many dentists and patients choose zirconia for its advantages, like high strength similar to metals, high biocompatibility, similar color and translucency to natural teeth and low risk of inflammation due to an unlikely dental plaque in accumulation. in a recent clinical report 1818181818, elimination of veneered porcelain on posterior zirconia crowns and fixed dental prostheses was performed for a clinical trial and presented an acceptable esthetic result. in this study, zirconia crowns showed better performance than the e-max due to their high rigidity. thus, better load transfer pattern was expected on the following parts, in comparison with less rigid material (ips e-max cad). the obtained results in this research matched previous studies’ findings, that using low rigidity crown material reduces the stresses generated on the jaw bone (cortical and spongy), that it absorbs more energy from the applied load and transfers less energy to implant-abutment complex and bone1010101010. in addition, this finding was proven experimentally1313131313, that all zirconia crowns did not fail under 178 n oblique load. failure occurred in screws supporting angulated abutments whatever the abutment angulation (15° or 25°). about 50% of e-max crowns failed under load and the other failures occurred in screw. in other words, regardless the crown material, the increased abutment angulation resulted in increasing the lateral stresses exerted on the whole assembly rather than the apical stresses. lateral stress increases may affect the screw of the abutment, as it represents the weakest component of the whole assembly. these results were in full agreement with those found by ellakwa et al.1111199999 as their results assessed the effect of three implant abutment angulations and three core thicknesses on the fracture resistance of overlaying cam milled zirconia, and found that the 30° implant abutment angulation significantly reduced the fracture resistance of the overlaying cam milled zirconia single crowns. in addition, the cervical areas are the most critical on the abutments due to the force concentration that may be a reason for failures, i.e. increasing the abutment angulation had a negative influence on the fracture load. former experimental studies13,1513,1513,1513,1513,15 showed different modes of failure for the 15° and 25° implant abutment angulations with ips e-max cad crowns. about half the specimens had screw fracture and the other half had crown fracture. this was assigned to the fact that the flexural strength of ips emax cad crown (460 mpa) is near to that of titanium screw (500 mpa). on the other hand, zirconia crowns have flexural strength of 900-1400 mpa, which is superior to the titanium screw. the fractures in the ceramic crowns typically occurred at the cervical portion of the abutment and at the screw. according to previous studies20-2220-2220-2220-2220-22, these abutment areas have the highest stress concentrations due to levering effects. using angulated abutments with different types of restorative materials to construct the overlaying crowns are significant factors in determining the amount and distribution of stresses loaded onto the superstructure and implant under functional forces2323232323. most fe models in dental researches 9-10,249-10,249-10,249-10,249-10,24 assumed perfect bond between assembled model components to simulate natural condition, in addition to assuming linear, static and isotropic material properties. the film thickness of the resin cement might significantly affect the shortand long-term bond strengths. it was reported that greater resin cement film thickness (100 ìm vs. 50 ìm) resulted in lower bond strength of resin materials to lithium disilicate ceramics 1313131313. another study 2525252525 showed that the zirconia bond strengths were significantly reduced with thicker (100 ìm) resin cement layer. thus, in this study the film thickness was considered to be 50 µm. finally, the results of this study were in agreement with literature33333 when abutments with 0, 15°, and 25° angulations were evaluated in the maxilla by 3d fem. that concluded to the superiority of abutments with less-angulation than 25°, which increased stresses on the peri-implant region and demonstrated higher stress concentration on the opposite side of loading with angulated abutments. within the limitations of this study, the following conclusions can be drawn: 1implant von mises stress level was negligibly changed with increased abutment angulation, which indicated good implant-abutment complex design. 2abutment with higher angulation is mechanically weaker and is expected to fail at lower load level in comparison with steeper one. 3screw used with abutment angulation of 25° will fail at lower (about one-third) of the failure load of similar screw used with abutment angulated by 15°. 4cement layer placed above the 15° angulatedabutment will fail at lower load than that one placed on the influence of implant-abutment angulations and crown material on stress distribution on central incisor: a 3d fea328328328328328 braz j oral sci. 14(4):323-329 25° angulated-abutment, as it rests on smaller area of abutment lowest surface. 5bone (cortical and spongy) is insensitive to crown material. increasing abutment angulation from 15° to 25°, increased stress on cortical bone by about 20%, and reduced it by about 12% on spongy bone. 6more rigid crown material (zirconia), showed better distribution of load on the following parts, in comparison with less rigid material (ips e-max cad). references 1. mathieu v, vayron r, richard g, lambert g, naili s, meningaud j, et al. biomechanical determinants of the stability of dental implants: influence of the bone–implant interface properties. j biomech. 2014; 47: 3-13. 2. misch ce. contemporary implant dentistry. 3rd ed. saint louis: mosby elsevier; 2008. 3. bidez mw, misch ce. force transfer in implant dentistry: basic concepts and principles. j oral implantol. 1992; 18: 264-74. 4. hasan i, roger b, heinemann f, keilig l, bourauel c. influence of abutment design on the success of immediately loaded dental implants: 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fracture resistance of yttria-stabilized zirconia dental implant abutments. j prosthodont. 2009; 18: 17-22. 22. nothdurft fp, doppler ke, erdelt kj, knauber aw, pospiech pr. fracture behavior of straight or angulated zirconia implant abutments supporting anterior single crowns. clin oral invest. 2011; 15: 157-63. 23. brunski jb. biomechanics of oral implants: future research directions. j dent educ. 1988; 52: 775-87. 24. deepa rh, surendra kumar gp, satish babu cl, shetty s, jnandev kr, rohit p, et al. influence of occlusal forces on stress distribution on preloaded dental implant abutment screw: a finite element analysis. int j oral implant clin res. 2013; 4: 16-23. 25. gehrke p, dhom g, brunner j, wolf d, degidi m, piattelli a. zirconium implant abutments: fracture strength and influence of cyclic loading on retaining-screw loosening. quintessence int. 2006; 37: 19-26. influence of implant-abutment angulations and crown material on stress distribution on central incisor: a 3d fea 329329329329329 braz j oral sci. 14(4):323-329 oral sciences n3 braz j oral sci. 11(1):42-46 original article braz j oral sci. january | march 2012 volume 11, number 1 effect of crude extract and essential oil of cordia verbenacea in experimental periodontitis in rats fernanda vieira ribeiro1, guilherme emerson barrella2 , renato corrêa viana casarin1, fabiano ribeiro cirano1, mary ann foglio3, suzana peres pimentel1 1dds, ms, phd, professor, dental research division, department of periodontology, school of dentistry, paulista university, brazil 2dds, ms student, dental research division, department of periodontology, school of dentistry, paulista university, brazil 3dds, ms, phd, professor, multidisciplinary center for chemical, biological and agricultural research (cpqba), university of campinas, brazil correspondence to: fernanda vieira ribeiro departamento de odontologia universidade paulista unip av. dr. bacelar, 1212, 4o andar, cep: 04026-002 vila clementino, são paulo, sp brazil, phone/fax: +55 11 5586-4000 e-mail: fernanda@ribbeiro.com received for publication: november 08, 2011 accepted: january 30, 2012 abstract aim: to evaluate the effect of crude extract and essential oil of cordia verbenacea (c.v.), systemically administered, on ligature-induced periodontitis in rats. methods: periodontitis was induced in 54 wistar rats: one of the first mandibular molars was randomly assigned to receive a ligature, whereas the contralateral molar was left unligated. then, animals were randomly assigned to one of the following groups: non-treatment group (n=18): animals that received 10 ml/day of vehicle; c.v. extract group (n=18): animals that received 100 mg/kg/day of crude extract of c. verbenacea; and c.v. essential oil group (n=18): animals that received 100 mg/kg/day of essential oils free of c. verbenacea. all therapies were administered orally 3 times daily, for 11 days. next, the animals were sacrificed, and the specimens were processed for morphometric analysis. bone loss was determined on the buccal surface of the lower first molars by the distance of the cementoenamel junction from the alveolar bone. results: both extract and essential oil of c. verbenacea orally administered decreased alveolar bone loss in the ligated teeth when compared with the non-treated group (p<0.05). conclusions: the present study demonstrated that systemic administration of both formulations of cordia verbenacea may attenuate the progression of ligatureinduced periodontitis. keywords: alveolar bone loss, cordia, inflammation, periodontitis. introduction periodontitis is characterized by an infectious condition leading to inflammation of the periodontal supporting tissues, attachment loss, and alveolar bone destruction. although the etiological role of microorganisms in the pathogenesis of this disease is clear, the host’s immune-inflammatory response can lead to protective and/or destructive effects on periodontal tissues1. thus, an unbalanced host response to periodontopathogens is an essential determinant in the outcome of the disease1. innumerous biological systems have been suggested as alternatives to modulate the host’s immune-inflammatory response involved in periodontal disease2. among them, the nonsteroidal anti-inflammatory drugs (nsaids) are probably the most studied medications, showing effective outcomes in controlling braz j oral sci. 11(1):42-46 periodontal breakdown in pre-clinical experiments when systemically administered3-5. nevertheless, clinical trials have not observed consistent benefits when nsaids are associated with conventional mechanical therapy5. additionally, these drugs, both non-selective inhibitors and selective cyclooxygenase-2 inhibitors, are frequently associated with side effects, impairing patients’ compliance to their use6. this way, the search for new drugs that effectively interfere with the immune-inflammatory process is currently important. in this context, evidence has shown that the use of medicinal plants is greatly relevant in folk medicine to treat different inflammatory conditions, including periodontitis7-11. the species cordia verbenacea, formally classified as c. curassavica, is a native brazilian medicinal plant belonging to the boraginaceae family, distributed along the brazilian coastal regions and popularly known as erva baleeira. studies have demonstrated that, in the form of alcoholic extracts, decoctions, and infusions, c. verbenacea exhibits important anti-rheumatic, anti-inflammatory, analgesic, and healing properties which are related to a protective effect on the gastric mucosa, as well as very low toxicity when orally or topically administered12-17. however, no information has previously been available regarding the effects of c. verbenacea on periodontal disease. taking into account the extensive presence of c. verbenacea in brazilian folk medicine, and considering the anti-inflammatory activity previously reported12-17 our group was prompted to assess the impact of c. verbenacea in modulating periodontal disease progression. therefore, the present study investigated the role of systemically administered c. verbenacea in induced-ligature periodontitis. moreover, two formulations of the drug (crude extract and essential oil) were evaluated to determine whether they produce different therapeutic effects when used in a pathological situation, such as periodontitis. the hypothesis of this investigation was that systemic therapy with both formulations of c. verbenacea could positively modify the progression of experimentally induced periodontitis in rats, representing a promising new approach for the management of periodontal diseases. material and methods animals fifty-four male wistar rats were obtained from the butantan institute (são paulo, sp, brazil). the rats were 90day-old and weighed 304 ± 23 g at the beginning of the study. during the acclimatization (5 days) and experimental period (11 days), each animal was housed in a plastic cage with access to food (labina, purina, paulínia, sp, brazil) and drinking water ad libitum in the animal care facility of paulista university. the protocol was approved by the paulista university institutional animal care and use committee (036/10 cep/ics/unip). plant material and extraction of crude extract and essential oil fresh leaves and stems of c. verbenacea were collected from multidisciplinary center for chemical, biological and agricultural research (cpqba) of the university of campinas (unicamp). a voucher specimen (uec 112744) is deposited at the unicamp’s biological institute. to obtain the plant crude extract, the material was allowed to dry under circulating air (40 ºc) and ground prior to use. the powder was submitted to dynamic maceration with ethanol for 4-h periods. this procedure was repeated 3 times. concentration of the extract under reduced pressure yielded extracts which were denoted as ethanol crude extracts. the essential oil was extracted from fresh chopped leaves by hydrodistillation for 4 h, using a clevenger-type apparatus. under these conditions, the yield of essential oil was 0.37% (considering 80% humidity). both the crude ethanol extract and volatile oil were analyzed by gc/ms (hp 6890/mass detector hp 5975 / automatic injector 7673 agilent technologies, palo alto, ca) using a hp-5 fused silica capillary column (30 m x 0.25 mm x 0.25 µm / stationary phase 5% methyl silicone). helium was used as the carrier gas (1.0ml.min-1). the detector was acquired by electron impact (scan mode) using an ionization energy of 70 ev. one microliter of sample was injected in the splitless mode. the column was initially heated at 60oc and then heated at 3oc min-1 to 240oc. injector and detector temperature were 220oc and 250 oc, respectively. the compounds were identified by comparing their mass spectra with the system data bank nist-2005. a homologous series of n-hydrocarbons c-9-c18 and c-20 was co-injected with the ample in order to calculate the retention index and co-injection of authentic standards to provide additional criteria for identification. the essential oil components were therefore identified crossing their retention index, with comparison of their mass spectrums compared with those of authentic samples. for the quantitative determination of α-humulene calibration was carried out using a standard solution of αhumulene in acetone (25–127 µg/ ml) containing dibutylphthalate (200 µg/ml) as the internal standard. the correlation between the peak area ratio and the concentrations of the compound was linear over the range tested. in order to determine the contents of α-humulene, oil samples (100.00 ± 0.1 mg) were dissolved in acetone (10 ml) containing the internal standard (200 µg/ml) and aliquots (1.0 µl) injected into the gc/ms. all chemical analyses were performed in triplicate. purity of α-humulene was 99%. ligature placement general anesthesia was obtained by intramuscular administration of ketamine hydrochloride (10 mg/kg) (dopalen®, agribrands brasil ltda., paulínia, sp, brazil) and xylazine hydrochloride (10 mg/kg) (rompun®, bayer s.a., são paulo, sp, brazil). one of the mandibular first molars of each animal was randomly assigned to receive a cotton ligature (corrente algodão no 10; coats corrente, são paulo, sp, brazil) in a cervical position. briefly, the thread was introduced in the proximal space between the first and second molars and the ligatures were kept in position in order to 4343434343 effect of crude extract and essential oil of cordia verbenacea in experimental periodontitis in rats 4444444444 braz j oral sci. 11(1):42-46 allow biofilm accumulation over 11 days5,9-10. the contralateral tooth was left unligated to be used as a control. treatment after ligature placement, animals were randomly assigned to one of the following groups, according to a computergenerated code: non-treatment group (n=18): animals received orally 10 ml/day of vehicle; c.v. extract group (n=18): animals received orally 100 mg/kg/day of crude extract of c. verbenacea; and c.v. essential oil group (n=18): animals received orally 100 mg/kg/day of essential oils isolated from c. verbenacea of flow rate). treatments were administered by oral gavage with a 1 ml syringe, using 0.3 ml of the respective substances, 3 times daily (7 a.m., 1 p.m., and 8 p.m.) for 11 days. the animals were evaluated at each of these moments (7 a.m., 1 p.m., and 8 p.m.) throughout the experiment, to assess possible clinical or toxicological symptoms. at the conclusion of the experiment, the animals’ weights were monitored and compared with the baseline weight. morphometric analysis the animals were sacrificed by co2 inhalation on the 12th d of periodontitis induction. subsequently, the mandibles were excised and defleshed after immersion in 8% sodium hypochlorite for 4 h. the specimens were washed in running water and immediately dried with compressed air. to outline the cementum enamel junction (cej), 1% methylene blue (sigma-aldrich®, saint louis, mo, usa) was applied to the specimens for 1 min and then washed in running water. photographs were obtained with a 6.1-megapixel digital camera (canon® 40d) on a tripod to keep the camera parallel to the ground at the minimal focal distance. the specimens were fixed in wax with their occlusal planes parallel to the ground and long axes perpendicular to the camera. photographs of the buccal aspects were made both in test and control sides. to validate measurement conversions, a millimeter ruler was photographed with all specimens18. alveolar bone loss was determined on the buccal surface of the lower first molars, by the distance of the cej from the alveolar bone crest (abc), measured at 3 equally distant sites. measurements were made along the axis of each root in 3 regions of the first molar (3 roots) (figure 1). the total alveolar bone loss was obtained by taking the sum of the linear recordings from the buccal tooth surface of the end of the root and dividing by 3. the measurements were performed by the same calibrated masked examiner after intraexaminer calibration, by evaluating 10 non-study images presenting alveolar bone loss similar to the present study. the examiner measured the linear measurements of all photographs twice within 24 h. the intraclass correlation showed 93% reproducibility. statistical analyses to test the null hypothesis that both crude extract and essential oil of c. verbenacea had no influence on alveolar bone loss, an intergroup analysis was performed by anova. fig. 1 morphometric parameter evaluated: red lines represent the distance to the cementoenamel junction from the alveolar bone crest when statistical difference was found, analysis of the difference was determined using the tukey’s test. in addition, the paired student’s t-test was used for intragroup comparisons between ligated and unligated teeth. the significance level set for all analyses was 5% (p<0.05). results the essential oil was obtained in 1% yield from freshly collected plants. the main component identified in the essential oil was α-humulene, which represented 80% of the total oil’s content, whereas the ethanol crude extract was obtained by reflux in a soxhlet system. the α-humulene yield content of this extract represented 0.047%. the animals did not lose weight throughout the experimental period. indeed, the tested therapies did not promote side effects or alterations in the animals’ behavior and in their general activity related to the toxicity. deaths were not observed. morphometric results a significant difference in the alveolar bone loss between unligated and ligated teeth was observed for all experimental groups (p<0.05), showing that the cotton ligatures around the teeth were able to promote bone loss (table 1). both groups treated with c. verbenacea crude extract and essential oil presented a significant reduction of the alveolar bone loss in ligated teeth when compared with the non-treated groups ligated teeth unligated teeth non-treatment 1.71 ± 0.11 aa 1.21 ± 0.08 ab c.v. extract 1.53 ± 0.15 ba 1.16 ± 0.11 ab c.v. essential oil 1.59 ± 0.10 ba 1.12 ± 0.15 ab table 1. table 1. table 1. table 1. table 1. means and standard deviation of alveolar bone loss [mm] for ligated and unligated teeth in all experimental groups. means followed by different capital letters in a column represent significant intergroup differences by anova/tukey test, p<0.05. means followed by different noncapital letters in a line represent significant inter-group differences by student’s ttest, p<0.05. effect of crude extract and essential oil of cordia verbenacea in experimental periodontitis in rats 4545454545 braz j oral sci. 11(1):42-46 group (p<0.05), and no difference was observed between crude extract and essential oil formulations (p>0.05) (table 1). figure 2 illustrates the morphometric findings. fig. 2 representative photographs illustrating the morphologic findings of nontreatment [a], c.v. extract [b], and c.v. essential oil [c] groups. discussion studies have demonstrated that c. verbenacea extracts or essential oil display marked anti-inflammatory effects in several models of inflammation and currently this substance has been used in the therapy of inflammatory conditions, such as tendinitis and muscular or articular pain12-16. however, to date, no study has investigated the impact of c. verbenacea in periodontitis. in this investigation, the effect of c. verbenacea orally administered on ligature-induced periodontitis in rats was assessed. indeed, two formulations of c. verbenacea [crude extract and essential oil] were evaluated, in order to determine whether they possess different effects. the outcomes indicated that the c. verbenacea therapy was effective for reducing alveolar bone loss in experimentally induced periodontitis, independent of the plant formulation. to the best of our knowledge, there have been no clinical or pre-clinical studies that have previously evaluated the impact of c. verbenacea in periodontitis, hampering a more direct comparison with the outcomes of the present investigation. however, the results of this study corroborate some previously published data on other inflammatory conditions16, supporting the view that this medicinal plant can control chronic inflammation. the possible mechanisms underlying the pronounced effect of c. verbenacea in attenuating inflammatory disorders seems to have a relationship, at least in part, with the downregulation of pro-inflammatory mediators, such as tnf-α and il-1β16,18. in fact, alternative therapeutic approaches based on inhibiting tnf-α production have been successfully used in clinical treatment of chronic inflammatory diseases, particularly rheumatoid arthritis19-20. on the other hand, passos et al.16 (2007) demonstrated that the anti-inflammatory action of c. verbenacea was not associated with the reduction of prostaglandin (pg) e2 levels, indicating that the mechanism of action of this medicinal plant seems to be distinct from that of non-steroidal anti-inflammatory drugs 21-22. contradictorily, other data have suggested that essential oil from c. verbenacea, given orally, greatly reduced the generation of pge2 in the rat paw18. indeed, this study evidenced that the anti-inflammatory effects of these compounds seem to be closely associated with their ability to inhibit the up-regulation of important inflammatory proteins, such as cyclooxygenase-2 (cox-2) and inducible nitric oxide synthase (inos) enzymes18. in fact, previous data have indicated that both ethanolic extract and essential oil from c. verbenacea leaves have presented a wide variety of constituents, such as flavonoids and sesquiterpenes áhumulene and trans-caryophyllene, recognized agents responsible for the anti-inflammatory actions of c. verbenacea13,16,18,23-24. this supports the promising outcomes obtained in the current investigation by using both formulations of the studied plant. studies have shown that this medicinal plant may also present antibacterial activity against some gram-positive and gram-negative bacteria24-25. michielin et al.25 (2009) showed that c. verbenacea contains high relative amounts of oxygenated monoterpenes and sesquiterpenes, the main components responsible for its antibacterial activity. indeed, the antibacterial potential related to c. verbenacea could be attributed to the presence of the aromatic compounds in this natural substance25. nevertheless, the antibacterial role of systemic therapy with c. verbenacea in periodontitis remains unclear and further investigations are needed to clarify this issue. altogether, the data obtained in the current investigation showed for the first time a significant effect of c. verbenacea systemically administered, both as crude extract well as essential oil, in controlling bone loss in experimental periodontitis. the results of the present study confirm and extend those of earlier reports, which have demonstrated the potent anti-inflammatory in vivo activity of c. verbenacea12-14 and support the systemic use of this natural product as an attractive alternative to prevent further periodontal disease development. recently, besides the better understanding of the paradigm of periodontal disease and new information with regard to the role of host immune response in modulating periodontal breakdown, researchers have focused on the development of novel therapeutic strategies and directions of host-modulatory agents for the management of periodontal diseases. in this context, the nsaids represent an essential pharmacologic class of agents that has been well studied as modulators of the host response in periodontitis. pre-clinical evidence has indicated that therapies using either selective (cox-2) or traditional nsaids can positively modify the progression of periodontal disease3-5,26. clinical studies have also pointed out that the systemic administration of nsaids may provide additional benefits in the periodontal condition when associated with non-surgical periodontal therapy (scaling and root planning) by modulating the host’s immunoinflammatory response27-29, although these findings remain controversial2. nevertheless, chronic treatments using both non-selective and selective inhibitors of cox-2 are related to innumerous adverse effects, such as gastroduodenal problems and renal toxicity, inhibiting patient compliance to their use, especially when prolonged periods of administration are required6,30. therefore, the use of natural plants such as c. verbenacea to modulate periodontal breakdown could present advantages when taking into account the well-known systemic side effects attributed to nsaids. in this context, previous studies indicated that the extracts of c. verbenacea leaves exhibited an anti-inflammatory activity linked to an important protective effect on the gastric effect of crude extract and essential oil of cordia verbenacea in experimental periodontitis in rats braz j oral sci. 11(1):42-46 4646464646 mucosa, and very low toxicity in acute models of experimentation in rats when orally administered12-14,17. indeed, roldão et al.17 (2008) found that c. verbenacea leaf extract produced an important antiulcer effect, contributing to the maintenance of mucosal integrity. thus, this therapeutic strategy with a medicinal plant is safe and would be a more practical and viable approach, allowing the possibility of a longer period of drug administration in periodontal disease treatment. in conclusion, the systemic use of both crude extract and essential oil of c. verbenacea could represent a new therapeutic option for the treatment of inflammatory diseases, especially those presenting a chronic profile. the low cost and easy access to c. verbenacea justify additional studies on the efficacy of this compound as an adjunct in periodontal therapy in clinical practice, providing new insight for the modulation of periodontal disease progression in individuals suffering from this inflammatory condition. however, additional studies are required to evaluate the molecular mechanism by which these active compounds exert their effects. acknowledgements the authors thank professor daclé juliani macrini for making the laboratory available in order to carry out this project. references 1. de souza ap, da silva ra, da silva mad, catanzaro-guimarães sa, line srp. matrix metalloproteinases: the most important pathway involved with periodontal destruction. braz j oral sci. 2005; 4: 884-90. 2. reddy ms, geurs nc, gunsolley jc. periodontal host modulation with antiproteinase, anti-inflammatory, and bone-sparing agents. a systematic review. ann periodontol. 2003; 8: 12-37. 3. gurgel bc, duarte pm, nociti fh jr, sallum ea, casati mz, sallum aw, et al. impact of an anti-inflammatory therapy and its withdrawal on the progression of experimental periodontitis in rats. j periodontol. 2004; 75: 1613-8. 4. holzhausen m, spolidorio dm, muscará mn, hebling j, spolidorio lc. protective effects of etoricoxib, a selective inhibitor of cyclooxygenase-2, in experimental periodontitis in rats. j periodontal res. 2005; 40: 208-11. 5. queiroz-junior cm, pacheco cm, maltos kl, caliari mv, duarte id, francischi jn. role of systemic and local administration of selective inhibitors of cyclo oxygenase 1 and 2 in an experimental model of periodontal disease in rats. j periodontal res. 2009; 44: 153-60. 6. fitzgerald ga, patrono c. the coxibs, selective inhibitors of cyclooxygenase-2. n engl j med. 2001; 345: 433-42. 7. calixto jb, campos mm, otuki mf, santos ar. anti-inflammatory compounds of plant origin. part ii. modulation of pro-inflammatory cytokines, chemokines and adhesion molecules. planta med. 2004; 70: 93-103. 8. cai x, li c, du g, cao z. protective effects of baicalin on ligature-induced periodontitis in rats. j periodontal res. 2008; 43: 14-21. 9. toker h, ozan f, ozer h, ozdemir h, eren k, yeler h. a morphometric and histopathologic evaluation of the effects of propolis on alveolar bone loss in experimental periodontitis in rats. j periodontol. 2008; 79: 1089-94. 10. botelho ma, martins jg, ruela rs, i r, santos ja, soares jb, et al. protective effect of locally applied carvacrol gel on ligature-induced periodontitis in rats: a tapping mode afm study. phytother res. 2009; 23: 1439-48. 11. napimoga mh, benatti bb, lima fo, alves pm, campos ac, penados-santos dr, et al. cannabidiol decreases bone resorption by inhibiting rank/rankl expression and pro-inflammatory cytokines during experimental periodontitis in rats. int immunopharmacol. 2009; 9: 216-22. 12. sertié ja, woisky rg, wiezel g, rodrigues m. pharmacological assay of cordia verbenacea; part 1. anti-inflammatory activity and toxicity of the crude extract of the leaves. planta med. 1988; 54: 7-10. 13. sertié ja, basile ac, panizza s, oshiro tt, azzolini cp, penna sc. pharmacological assay of cordia verbenacea. iii: oral and topical antiinflammatory activity and gastrotoxicity of a crude leaf extract. j ethnopharmacol. 1991; 31: 239-47. 14. sertié ja, woisky rg, wiezel g, rodrigues m. pharmacological assay of cordia verbenacea v: oral and topical anti inflammatory activity, analgesic effect and fetus toxicity of a crude leaf extract. phytomedicine. 2005; 12: 338-44. 15. ticli fk, hage li, cambraia rs, pereira ps, magro aj, fontes mr, et al. rosmarinic acid, a new snake venom phospholipase a2 inhibitor from cordia verbenacea (boraginaceae): antiserum action potentiation and molecular interaction. toxicon. 2005; 46: 318-27. 16. passos gf, fernandes es, da cunha fm, ferreira j, pianowski lf, campos mm, et al. anti-inflammatory and anti-allergic properties of the essential oil and active compounds from cordia verbenacea. j ethnopharmacol. 2007; 110: 323-33. 17. roldão ede f, witaicenis a, seito ln, hiruma-lima ca, di stasi lc. evaluation of the antiulcerogenic and analgesic activities of cordia verbenacea dc. (boraginaceae). j ethnopharmacol. 2008; 119: 94-8. 18. fernandes mi, gaio ej, oppermann rv, rados pv, rosing ck. comparison of histometric and morphometric analyses of bone height in ligature-induced periodontitis in rats. braz oral res. 2007; 21: 216-21. 19. van assche g, rutgeerts p. anti-tnf agents in crohn’s disease. expert opin investig drugs. 2000; 9: 103-11. 20. feldmann m. development of anti-tnf therapy for rheumatoid arthritis. nat rev immunol. 2002; 2: 364-71. 21. tilley sl, coffman tm, koller bh. mixed messages: modulation of inflammation and immune responses by prostaglandins and thromboxanes. j clin invest. 2001; 108: 15-23. 22. murakami m, kudo i. recent advances in molecular biology and physiology of the prostaglandin e2-biosynthetic pathway. prog lipid res. 2004; 43: 33-5. 23. sertié ja, basile ac, panizza s, matida ak, zelnik r. anti inflammatory activity and sub-acute toxicity of artemetin. planta med. 1990; 56: 36-40. 24. de carvalho pm jr, rodrigues rf, sawaya ac, marques mo, shimizu mt. chemical composition and antimicrobial activity of the essential oil of cordiaverbenacea d.c. j ethnopharmacol. 2004; 95: 297-301. 25. michielin em, salvador aa, riehl ca, smânia a jr, smânia ef, ferreira sr. chemical composition and antibacterial activity of cordia verbenacea extracts obtained by different methods. bioresour technol. 2009; 100: 6615-23. 26. bezerra mm, de lima v, alencar vb, vieira ib, brito ga, ribeiro ra, et al. selective cyclooxygenase-2 inhibition prevents alveolar bone loss in experimental periodontitis in rats. j periodontol. 2000; 71: 1009-14. 27. ng vw, bissada nf. clinical evaluation of systemic doxycycline and ibuprofen administration as an adjunctive treatment for adult periodontitis. j periodontol. 1998; 69: 772-6. 28. aras h, caðlayan f, güncü gn, berberoðlu a, kilinç k. effect of systemically administered naproxen sodium on clinical parameters and myeloperoxidase and elastase-like activity levels in gingival crevicular fluid. j periodontol. 2007; 78: 868-73. 29. azoubel mc, sarmento va, cangussú v, azoubel e, bittencourt s, cunha fq, et al. adjunctive benefits of systemic etoricoxib in non-surgical treatment of aggressive periodontitis: short-term evaluation. j periodontol. 2008; 79: 1719-25. 30. emery p. clinical implications of selective cyclooxygenase-2 inhibition. scand j rheumatol suppl. 1996; 102: 23-88. effect of crude extract and essential oil of cordia verbenacea in experimental periodontitis in rats oral sciences n3 braz j oral sci. 11(2):154-157 original article braz j oral sci. april | june 2012 volume 11, number 2 role of periodontics in the treatment to traumatized teeth: a 4-year retrospective evaluation adriana de jesus soares1, mariana amade mendes brazão2, renato correa viana casarin3, juliana yuri nagata4, márcio zaffalon casati3, francisco josé de souza filho5 1collaborator professor, department of restorative dentistry, endodontics area, unicamp university of campinas, piracicaba, sp, brazil 2master’s degree student, são leopoldo mandic institute and research center, campinas, sp, brazil 3full professor, unip paulista university, são paulo, sp, brazil 4phd student, department of restorative dentistry, endodontics area, unicamp university of campinas, piracicaba, sp, brazil 5full professor, department of restorative dentistry, endodontics area, unicamp university of campinas, piracicaba, sp, brazil correspondence to : adriana de jesus soares av. doutor nelson noronha gustavo filho, 150, cep:13092-526, apto 112b, edifício alpha park, bairro vila brandina, campinas, sp, brasil e-mail: ajsoares.endo@uol.com.br abstract treatment of dental trauma should be planned based on a multidisciplinary approach. it is known that the maintenance of oral health in these situations depends not only on the viability of dental tissues, but also on the periodontal damage occurred and the emergency therapeutic procedures performed. aim: the purpose of this epidemiological retrospective study was to assess the periodontal involvement in traumatized teeth treated at the piracicaba dental school/,unicamp dental trauma attendance service (dtas), during a period of 4 years. methods: clinical charts of patients (n=203) treated between 2006 and 2009 at dtas were retrospectively analyzed. results: a total of 295 traumatized teeth were assessed about history, type of dental trauma, and periodontal tissue damage detected during clinical and radiographic examination. moreover, the treatment associated with periodontal damage resolution was also determined. of the 295 traumatized teeth, 184 had periodontal damage (62.3%) and from this total, crown-root fractures with invasion of biological width were found in 42% of the cases, followed by bone injury (30%). regarding the periodontal treatment performed, periodontal surgery to re-establish the biological width prevailed between procedures with 39% of the cases. conclusions: it could be concluded that periodontics has an essential role in the multidisciplinary approach for the treatment of traumatized patients and that the conjunct work is important to re-establish the overall oral health. keywords: prevalence diagnosis, tooth fractures. introduction the incidence of dental trauma in children and school-age young people has increased considerably over the years1-2. epidemiological studies show a strong relationship between trauma occurrence and anatomic characteristics such as accentuated overjet and inadequate lip seal3-4. in addition to anatomic factors, the causes of the growing incidence of dental trauma in younger people may be associated with the practice of contact and radical sports, although automobile accidents and urban violence also figure in the prevalent level of dental trauma5. dental trauma usually involves damage to cementum, periodontal ligament, received for publication: may 08, 2012 accepted: june 15, 2012 155155155155155 braz j oral sci. 11(2):154-157 dentin, and pulp. its severity may be classified according to the level of damage to the supporting tissues as well as to the type of fracture3. in this context, it is important to consider that the traumatic sequel may involve not only the dental structures but also their surrounding tissues6. it brings a necessity to adopt a multidisciplinary vision for the treatment planning with the goal of maintenance and re-establishment of oral health7. it is known that dental trauma, even in its mildest form, invariably injuries the periodontal ligament fibers2-3,8. thus, correct diagnosis and treatment protocols focused on the re-establishment of the health of periodontal tissues become essential to the treatment of traumatized patients. however, there are few published studies that evaluate the role of periodontics during diagnosis and attendance of traumatized teeth. the multidisciplinary involvement aims to correlate the clinical variables for each specialty, ensuring comprehensive care to the trauma patient. in this context, the purpose of this epidemiological retrospective study was to assess the periodontal involvement in traumatized teeth, assisted by a dental trauma service for a period of 4 years. material and methods the study design was approved by the ethics committee of piracicaba dental school, university of campinas (protocol #106/2007). patient charts, containing the clinical and radiographic files of patients treated at the piracicaba dental school/,unicamp dental trauma attendance service (dtas), between 2006 and 2009, were reviewed by an expert examiner. data collection patient personal data including age, gender and medical history were collected. in addition, trauma history related to tooth affected, etiology and type of injury were analyzed. related to clinical examination, tests of sensibility, palpation, mobility, vertical and horizontal percussion tests were performed. regarding periodontal involvement, signals and symptoms of the teeth such as gingival bleeding, disruption of periodontal ligament fibers, and presence of subgingival fractures were assessed. according to these signals, periodontal injury was categorized in: biologic width invasion, bone injury, gingival inflammation, and aesthetic impairment. signals of periodontal disease occurred before the moment of the trauma was excluded, according to wholemouth periodontal examination. moreover, the entire periodontal treatment to these traumatized teeth was determined and performed by a periodontist. data were subjected to statistical analysis for fisher’s nonparametric samples (p<0.05). results two hundred and three patients (295 teeth) were treated at dtas between 2006 and 2009. of this total, 135 (66%) patients presented at least one tooth with periodontal involvement, which represents 184 (62.3%) teeth with association between dental trauma and periodontal injuries (figure 1). patient age varied from 8 to 70 years old, and the majority of them were males (68%). the most common type of trauma was crown-root fracture (n=61 teeth, 33%); followed by avulsion (n=38 teeth, 20%), and lateral luxation (n=29 teeth, 15.8%) (table 1). periodontal involvement fig. 1. number of teeth with periodontal involvement (red) in the total of traumatized teeth attended in dental trauma attendance service (dtas) per year. periodontal injury n % biologic width invasion 77 42 bone injury 55 30 gingival inflammation 32 17 aesthetic impairment 20 11 total 184 100 table 2. prevalence of periodontal injuries observed in traumatized teeth of patients treated at dtas between 2006 and 2009. dental trauma type n % enamel-dentin fracture 10 5.4 enamel-dentin-pulp fracture 23 12.5 crown-root fracture 61 33.2 avulsion 38 20.7 lateral dislocation 29 15.8 intrusion 23 12.5 total 184 100 table 1. prevalence of trauma types in teeth with periodontal injuries. regarding the periodontal involvement, biologic width invasion was the most common condition, which was observed in 77 teeth (42%). bone injury was the second type of periodontal involvement, affecting 55 teeth (30%) (table 2). the treatment adopted in the majority of the cases was a periodontal surgery to re-establish the biologic width with adjunct of gingivectomy and osteotomy, performed in 39% of the cases. although gingivectomy was a frequent procedure (52 cases), alternatives to treat subgingival crown root fracture such as orthodontic extrusion and fracture re-anatomization were also performed in 6% of the cases (table 3). role of periodontics in the treatment to traumatized teeth: a 4-year retrospective evaluation braz j oral sci. 11(2):154-157 periodontal treatment n % biological width reestablishment 72 39.1 gingivectomy 58 31.5 root fracture re-anatomization 5 2.7 orthodontic extrusion 6 3.3 basic therapy 15 8.2 gingival graft 7 3.8 exploratory surgery 21 11.4 total 184 100.0 table 3. periodontal treatment in traumatized teeth. discussion retrospective studies allow a clear view of the subject matter and demonstrate its most important aspects 9-10. however, there is no study focusing on the role of periodontics in attendance of a traumatized patient. considering that most of the trauma promotes periodontal and pulpal damage, the information provided by epidemiological studies could be important in planning future approaches to the treatment of traumatized patients. in this retrospective study, most of the traumatized teeth (62.3%) were associated with a periodontal injury. the number of traumatized teeth follows an ascending order each year, which can be related to the disclosure of dental trauma services in schools of in piracicaba and region2. the most common trauma related to periodontal injuries was complex crown root fractures involving enamel, dentin, pulp and cement, with 61 teeth involved (33.2%). periodontal injury was more related to biologic width invasion (n=77 teeth, 42%). this is probably due to the angle and direction of the impact in which the trauma is related that may cause a deeper destruction. when the biologic width is invaded, an inflammatory reaction occurs on the periodontium, which causes alveolar bone resorption in order to generate more space for new connective tissue. consequently, a deepening gingival groove is opened which can lead to chronic inflammation11. the average of biologic width is established, as follows12: gingival sulcus: 0.69 mm; junctional epithelium: 0.97 mm; and connective tissue attachment: 1.07 mm. so, there is 2 to 3 mm of sound tooth structure coronal to the alveolar bone crest to the junctional epithelium and connective tissue attachment that can properly join the tooth. in these situations, the re-establishment of the biologic width with increased clinical crown may be performed through surgical crown lengthening procedures, surgical or orthodontic extrusion, each one when properly indicated6-7. surgical crown lengthening procedures are the most common technique that allows rehabilitation of the biologic width, especially in cases of caries, prosthetic preparations and invoices13. they are important for the rehabilitation of periodontal health and removal of irritation that might damage the periodontium. the millimeter that is needed from the bottom of the junctional epithelium to the tip of the alveolar bone is held responsible for the lack of inflammation and bone resorption, and as such the development of periodontitis14. in cases of dental trauma, these procedures are indicated when there is inadequate clinical crown for retention due to tooth fracture and root resorption within the cervical third of the root in teeth with adequate periodontal attachment; placement of sub gingival restorative margins; unequal, excessive or unaesthetic gingival levels for aesthetics; teeth with inadequate interocclusal space for proper restorative procedures due to supraeruption; and restorations which violate the biologic width15. meantime, there are some contraindications of these techniques namely deep fracture requiring excessive bone removal; post-surgery unaesthetic outcomes; tooth with inadequate crownroot ratio (ideally 2:1 ratio is preferred); non-restorable teeth; teeth with increased risk of furcation involvement; unreasonable impairment of aesthetics; and unreasonable impairment of adjacent alveolar bone support14. considering dental trauma frequently involves anterior teeth in young individuals, who are highly associated with a severe overjet and an inadequate coverage lip16, aesthetic involvement should be taken into account. in these cases, as the aesthetics of the patient may be affected, surgical crown lengthening procedures are not indicated because of the risks of gingival recession13. heithersay17 1973, said that to minimize gingival recession caused by gingivectomy associated with removal of bone, orthodontic extrusion (forced eruption) should be performed prior to surgery, mainly in fractures. it may be explained because this extrusive moving acts not only on the tooth but also on its oral support, gingival ligament fibers and alveolar bone, allowing a posterior osteotomy18. an example occurs when lateral luxation promotes bone break-up, needing a periodontal surgery in the location of the fracture for the re-establishment of biologic width. periodontal grafts with bone or soft tissue may also be performed in order to have better aesthetic post-trauma, matching with the periodontal plastic surgery, which aims to improve the environment periodontal facilitation of hygiene and ensure appropriate aesthetics. plastic surgery is used to reduce periodontal gingival recession, preservation and reconstruction of the alveolar, interdental papilla gain and to improve aesthetics, especially after avulsion and replantation, as well as intrusions 5,10,19. some of the contraindications to forced eruption are inadequate crownto-root ratio, lack of occlusal clearance for the required amount of eruption and any possible periodontal complications14. another periodontal treatment is the exploratory surgery, performed in cases where the fracture line is not radiographically visible. this is frequently held, especially in cases of fractures and oblique fractures of the crown and root. these conditions require periodontal access not only to establish the patient’s oral health but also to allow the optimization of the placement of rubber sheet for absolute isolation, a fundamental procedure in endodontics. in some cases, such as oblique fractures of the teeth prior to biologic width invasion, a re-anatomization using diamond drill bits that return the correct spacing after an exploratory periodontal surgery helps exposing the fracture line. in these conditions, 156156156156156role of periodontics in the treatment to traumatized teeth: a 4-year retrospective evaluation 157157157157157 braz j oral sci. 11(2):154-157 there is no need for gingivectomy or orthodontic extrusion20. since 2003, dtas has treated traumatized patients with multidisciplinary care in the areas of periodontics, restorative dentistry and oral-maxillofacial surgery with a high-quality professional staff. this conjunct work favors integrated patient treatment, considering that support tissues are frequently affected when a dental trauma occurs, as can be seen in the present study. thus, the improvement of health and full reestablishment of these patients may be achieved beginning with interdisciplinarity between endodontics and periodontology in treating dental trauma. in view of the present results, it could be concluded that periodontal damage occurs frequently in dental trauma, and its treatment has an essential role in the multidisciplinary approach to re-establish overall oral health. references 1. kumar a, bansal v, veeresha kl, sogi gm. prevalence of traumatic dental injuries among 12to 15-year-old schoolchildren in ambala district, haryana, india. oral health prev dent. 2011; 9: 301-5. 2. souza-filho fj, soares aj, gomes bpfa, zaia aa, ferraz, ccr, almeida jfa. evaluation of the traumatic dental injuries attended at the traumatic dental center of dental school of piracicaba, brazil. rgo. 2009; 14: 111-6. 3. bonini gc, bönecker m, braga mm, mendes fm. combined effect of anterior malocclusion and inadequate lip coverage on dental trauma in primary teeth. dent traumatol. 2012 feb 27. doi: 10.1111/j.16009657.2012.01117.x. [epub ahead of print]. 4. borzabadi-farahani a, borzabadi-farahani a, eslamipour f. an investigation into the association between facial profile and maxillary incisor trauma, a clinical non-radiografic study. dent traumatol. 2010; 26: 311-6. 5. stewart gb, shields bj, fields s, comstock rd, smith ga. consumer products and activities associated with dental injuries to children treated in united states emergency departments, 1990-2003. dent traumatol. 2009; 25: 399-405. 6. eichelsbacher f, denner w, klaiber b, schlagenhauf u. periodontal status of teeth with crown-root fractures: results two years after adhesive fragment reattachment. j clin periodontol. 2009; 36: 905-11. 7. poi wr, cardoso lc, de castro jc, cintra lt, gulinelli jl, de lazari ja. multidisciplinary treatment approach for crown fracture and crownroot fracture a case report. dent traumatol. 2007; 23: 51-5. 8. sharma d, garg s, sheoran n, swami s, singh g. multidisciplinary approach to the rehabilitation of a tooth with two trauma episodes: systematic review and report of a case. dent traumatol. 2011; 27: 321-6. 9. guedes oa, de alencar ah, lopes lg, pécora jd, estrela c. a retrospective study of traumatic dental injuries in a brazilian dental urgency service. braz dent j. 2010; 21: 153-7. 10. santos se, marchiori ec, soares aj, asprino l, de souza filho fj, de moraes m, et al. a 9-year retrospective study of dental trauma in piracicaba and neighboring regions in the state of são paulo, brazil. j oral maxillofac surg. 2010; 68: 1826-32. 11. eichelsbacher f, denner w, klaiber b, schlagenhauf u. periodontal status of teeth with crown-root fractures: results two years after adhesive fragment reattachment. j clin periodontol. 2009; 36: 905-11. 12. shobha ks, mahantesha, seshan h, mani r, kranti k. clinical evaluation of the biological width following surgical crown-lengthening procedure: a prospective study. j indian soc periodontol. 2010; 14: 160-7. 13. kina jr, dos santos ph, kina ef, suzuki ty, dos santos pl. periodontal and prosthetic biologic considerations to restore biological width in posterior teeth. j craniofac surg. 2011; 22: 1913-6. 14. nugala b, kumar bs, sahitya s, krishna pm. biologic width and its importance in periodontal and restorative dentistry. j conserv dent. 2012; 15: 12-7. 15. jorgic-srdjak k, dragoo mr, bosnjak a, plancak d, filipovic i, lazic d. periodontal and prosthetic aspect of biological width part ii: reconstruction of anatomy and function. acta stomatol croat. 2000; 34: 441-4. 16. gupta s, kumar-jindal s, bansal m, singla a. prevalence of traumatic dental injuries and role of incisal overjet and inadequate lip coverage as risk factors among 4-15 years old government school children in baddibarotiwala area, himachal pradesh, india. med oral patol oral cir bucal. 2011; 16: e960-5. 17. rotundo r, bassarelli t, pace e, iachetti g, mervelt j, pini prato g. orthodontic treatment of periodontal defects. part ii: a systematic review on human and animal studies. prog orthod. 2011; 12: 45-52. 18. barboza ep, monte-alto rf, ferreira vf, carvalho wr. supracrestal gingival tissue measurements in healthy human periodontium. int j periodontics restorative dent. 2008; 28: 55-61. 19. soares aj, gomes bpfa, zaia aa, ferraz ccr, souza-filho fj. relationship between clinical–radiographic evaluation and outcome of teeth replantation. dental traumatol. 2008; 24: 183-8. 20. bate al, lerda f. multidisciplinary approach to the treatment of an oblique crown-root fracture. dent traumatol. 2010; 26: 98-104. role of periodontics in the treatment to traumatized teeth: a 4-year retrospective evaluation oral sciences n3 original article braz j oral sci. january | march 2011 volume 10, number 1 antioxidant effect on the shear bond strength of composite to bleached bovine dentin masoomeh hasani tabatabaei1, sakineh arami1, atefeh nojoumian1, mansooreh mirzaei1 1 department of operative dentistry, school of dentistry, tehran university/medical science, tehran, iran correspondence to: sakineh arami department of operative dentistry, school of dentistry, tehran university/medical science, tehran, iran phone: +98 21 44657647 e-mail: nasrin.arami@gmail.com received for publication: august 13, 2010 accepted: february 3, 2011 abstract several studies have shown that compromised bonding to bleached enamel can be reversed with antioxidants. aim: the aim of this study was investigate the effect of the antioxidant treatment on the micro-shear bond strength of a composite resin with a clinically acceptable antioxidant usage time taken into account. methods: using in vitro techniques, the effect of the antioxidant sodium ascorbate (sa) was evaluated on the micro-shear bond strength of a hybrid composite resin (tetric® a 2 ivoclar vivadent) to dentin, which was bleached with 35% carbamide peroxide (opalescence quick, ultradent products inc). thirty-five intact flat buccal dentin surfaces from bovine incisors were randomly assigned to five groups which were subjected to the following treatment protocols: group 1, bleached for 45 min and bonded immediately afterwards; groups 2 and 3, bleached and then treated with 10% sa for 10 and 5 min before bonding, respectively; group 4, stored in distilled water for seven days after bleaching and before bonding; group 5, received no bleaching or antioxidant treatment. after the bonding procedure, specimens were subjected to a micro-shear bonding test. data were analyzed by anova and a post-hoc tukey’s test. results: one-way anova revealed significant differences in bond strength among the five groups. conclusions: it was found that the shear bond strength was reduced by carbamide peroxide bleaching, and that the antioxidant sa was ineffective at reversing the composite strength at the concentrations and treatment times examined. keywords: antioxidant, shear bond strength, composite, bleaching. introduction hydrogen peroxide bleaching is effective at lightening discolored teeth1. when bonding is performed immediately after bleaching, hydrogen peroxide and carbamide peroxide (cp) bleaching agents alter the bonding strength of composites to acid-etched enamel2-3. delays in bonding of 1 to 3 weeks are recommended following bleaching, to avoid the clinical problems related to bleaching-mediated compromised bond strength4. one mechanism that may account for the lower bond strengths of bleached teeth is the presence of residual oxygen, which inhibits the polymerization of adhesive monomers5. recent studies have revealed that the use of the antioxidant sodium ascorbate (sa) before the bonding process reverses the bleaching-induced reduction in bonding strength6-9. antioxidants can neutralize free hydroxyl radicals and prevent their adverse biological effect10. previous studies have different opinions about the duration of applying the antioxidant. the aim of this study was investigate the effect of this antioxidant treatment on the microshear bond strength of a composite resin with a clinically acceptable antioxidant usage time. braz j oral sci. 10(1):33-36 34 material and methods after slaughtering, 35 two-year-old intact bovine incisors were immediately extracted. the teeth were cleaned and sectioned at the cementoenamel level, pulp was removed using endodontic instruments, and the pulp chamber was rinsed with saline solution. the teeth were mounted with the buccal surface upwards in a self-curing acrylic resin using a heavy-body silicon mold. the teeth were stored in distilled water at 4oc for less than 3 months. teeth were sectioned buccolingually parallel to the line axes at 2-mm slices, using a low speed machine (isomet, buehler ltd., lake bluff, il, usa) to expose the dentin. the dentin surface was ground (polished) with wet 600and 1000-grit silicon carbide abrasive paper to create a flat dentin surface. specimens were randomly assigned to five groups (1 control and 4 experimental) of 7 teeth each (table 1). two or three composite samples were placed over each tooth, for a total of 17 specimens per group. the lingual sides of the teeth were placed in distilled water to simulate the humid condition of the oral cavity. for the bleaching treatment, a 35% cp bleaching gel (opalesence quick, ultradent products inc., usa) was placed on the dentin surfaces of the whole buccal aspect, and the teeth were placed in an incubator at 37oc and 100% humidity for 45 min. one group of teeth received no bleaching, as a positive control group (group 5). for the antioxidant treatment, a 10% sa solution was prepared by solving 10 g of sa powder (merck kgaa, darmstadt, germany) in distilled water. sa solution was prepared immediately before its application on the teeth. specimens from groups 2 and 3 were treated with 10% sa for 10 min and 5 min, respectively, followed by agitation with sa solution and thorough rinsing with tap water for 30 s. specimens in group 4 were immersed in distilled water in an incubator at 37°c during 1 week after bleaching. for the bonding treatment, two or three composite cylinders were applied to each sample. application of the self-etching non-rinsing primer dentin adhesive system (clearfil se bond, kuraray medical inc., japan) was according to the manufacturer’s instructions. the above adhesive was used in this study because it is a current and clinically acceptable adhesive. the bonded surfaces were light-activated for 10 s using a visible light-curing unit (optilux 501; demetron kerr, danbury, ca, usa) with an output intensity level of 410 mw/cm2. silicon tubes (0.7 mm internal diameter and 2 mm length) were placed on the dentin surface, and dental composite (tetric ceram, ivoclar-vivadent, group treatment agent sodium ascorbate treatment delayed bonding g1 35% carbamide peroxide g2 35% carbamide peroxide 10 min g3 35% carbamide peroxide 5 min g4 35% carbamide peroxide 1 week g5 no bleaching table 1: groups, treatment agent used in the experiment. liechtenstein) was inserted into the tubes in one step. each specimen was totally cured for 80 s. after curing, the silicon was removed and the specimens were subjected to 1000 thermal cycles between water baths of 5°c and 55oc, with a dwelling time of 15 s each. specimens were then stored in distilled water at 4oc for one week. the micro-shear bond strength was measured with a micro-shear testing machine (bisco, schaumburg, il, usa) using a wire and loop method. the loop was prepared with orthodontic ligature wire, with one side placed around the sample and the other side around the testing machine rod. specimens were loaded at a speed of 0.5 mm/min. bond strengths (mpa) were calculated using the load required to debond the specimen. results were subjected to one-way anova followed by a post-hoc tukey’s test at a 0.05% significance level. statistical analysis was processed with spss for windows xp. differences with p < 0.05 were considered significantly different. results the results of the micro-shear bond strength tests are summarized in table 2. one-way anova revealed significant differences in bond strength among the five groups. the posthoc test indicated differences between groups 1 (cp + bonding), 2 (cp + sa for 10 min), 3 (cp + sa for 5 min) and groups 4, 5. the cp and two antioxidant groups (groups 2 and 3) showed significantly lower bond strength averages than the positive control (group 5) and the group where specimens were immersed in water for 1 week (group 4). no differences were observed when groups 2, 3, and 1 were compared with each other, and group 4 was not significantly different from group 5 (table 2). discussion in the present study, it was found that bleaching treatments of 35% cp decreased the micro-shear bonding strength of a hybrid composite resin to dentin. the use of 10% sa for 10 or 5 min before bonding was unable to neutralize the oxidizing effects of the bleaching agent. bond strength reduction by cp may be caused by the presence of residual peroxide, which can interfere with resin attachment and inhibit resin polymerization5,7-8. previous scanning electron microscopy studies have shown that antioxidant effect on the shear bond strength of composite to bleached bovine dentin braz j oral sci. 10(1):33-36 bleached resin interfaces display a few short, fragmented resin tags compared to interfaces with unbleached enamel. the bleaching of human tooth alters the enamel surface characteristics, causing surface loss and increased surface porosity2,9. these alterations may affect the bonding of composite resin, and contribute to microleakage if it is restored with a bonded composite11. bleaching can significantly reduce the relative concentrations of calcium and phosphorous and cause morphological alterations in the most superficial enamel crystallites12. peroxide-containing bleaching agents affect the organic phase of enamel, affecting both the outer and inner enamel surfaces13. theoretically, enamel pores, dentin and dentin fluid can all act as peroxide/oxygen reservoirs12,14. dentin may be the most important reservoir of the three ones15, and is more affected by hydrogen or carbamide peroxide due to its lower mineral content and more organic matrix. thus, dentin proteins may be denatured by hydrogen-based materials that produce morphological changes, which could negatively impact resin performances12,16. in particular, since the surface calcium levels affect dentin bonding13, it is predicted low shear bond strength values for dentin samples after bleaching. several methods have been proposed to avoid the clinical problems related to bleaching-associated reduced bond strength. the most common recommendation is delayed bonding for composite resin restorations16-18. studies have identified 2 weeks as a satisfactory waiting period between bleaching and composite restoration for enamel and dentin17,19. some in vitro studies indicate that the immersion of specimens in distilled water, saliva, or saline reverses the reduced enamel or dentin bond strength2-3,16. it is observed that the immersion of specimens in distilled water for 7 days after bleaching effectively reversed the reduced dentin bonding strength. enamel removal can also restore bonding strengths to their normal levels20. barghi et al.1 reported that the adverse effects of enamel bleaching on bonding can be reduced or eliminated by treating the bleached surface with a water displacement solution, such as a dentin-bonding agent that contains acetone1. this has led others to recommend the use of alcohol-based bonding agents, particularly when restorative work is performed immediately after bleaching21. the inorganic and organic components of both dentin and cementum are reportedly affected by 3% hydrogen peroxide22, which remains active in the pulp chamber or dentin tubules after bleaching due to its interaction with certain dentin components. catalase has been suggested as an effective adjunct following bleaching to prevent the adverse effects of hydrogen peroxide10. turkun et al.6 found that the bond strength of composite resin to bovine enamel increased after treating bleached teeth with 10% sa for 10 min6. another report showed that antioxidant treatment for 10 min immediately after bleaching reversed the tensile bond strength of brackets23. zhao et al.24 speculated that peroxide ions may be temporarily substituted for the hydroxyl radicals in the apatite lattice, producing a patite. it may be that these lattice substitutions are thermodynamically unfavorable, and may be reversed by an antioxidant9. on the other hand the use of sa to reverse bleaching-related oxidation required a substantial treatment time that may not be clinically acceptable9. another study found that the use of a 10% sa hydrogel for 3 h before bonding neutralized the oxidizing effects of bleaching, increasing the enamel bond strength25. also, similar studies reported that compromised bonding can be reversed with 3 h application of hydrogel or solution from of sodium ascorbate26-27. results of another study showed that the antioxidant gel should be applied to enamel for at least 60 min for maximum effectiveness28. nevertheless, it is sought to reverse the bleaching-associated reduction in bond strength by neutralizing the residual oxygen with 10% sa, it is found that sa treatment before composite bonding was ineffective. also, in an in vitro study to investigate the neutralizing effect of antioxidant agents on the bond strength of bleached enamel, gomes torres et al.15 found that only cataluse application increased the bond strength relative to the pc (positive control) group, and none of the tested treatments could completely neutralize the deleterious effects of bleaching. although it could have been effective if the application time were increased to at least one-third of the bleaching time, this may not be clinically acceptable. therefore, it is recommended that treatment be postponed for 1 week after bleaching. the results of this study showed significant differences in bond strength among the five groups. the cp and two antioxidant group showed significant lower bond strength means than the control and distilled water-immersed (delayed bonding) groups. the effects of sa at different forms could be studied. references 1. barghi n, godwin jm. reducing the adverse effect of bleaching on composite-enamel bond. j esthet dent. 1994; 6: 157-61. 35 antioxidant effect on the shear bond strength of composite to bleached bovine dentin braz j oral sci. 10(1):33-36 group treatment mean(sd) range g1 bleach 15.46(8.48)b* 11.09-19.82 g2 bleach + antioxidant (10 min) 15.69(6.73)b 12.22-19.15 g3 bleach + antioxidant (5 min) 15.18(5.097)b 12.56-17.80 g4 bleach + distilled water (1 week) 24.04(8.99)a* 19.41-28.66 g5 no bleach 23.79(8.52)a 19.41-28.17 table 2: mean micro-shear bond strength values, standard deviation and range of each group. different letters indicate statistically significant difference at p<0.05. 36 2. nour el-din ak, miller bh, griggs ja, wakefield c. immediate bonding to bleached enamel. oper dent. 2006; 31(1): 106-14. 3. stokes an, hood ja, dhariwal d, patel k. effect of peroxide bleaches on resin enamel bonds. quintessence int. 1992; 23: 769-71. 4. cavalli v, reis af, giannini m, ambrosano gmb. the effect of elapsed time following bleaching on enamel bond strength of resin composite. oper dent. 2001; 26: 597-603. 5. dishman mv, covey da, baughan lw. the effects of peroxide bleaching on composite to enamel bond strength. dent mater. 1994; 9: 33-6. 6. turkun m, kaya ad. effect of 10% sodium ascorbate on the shear bond strength of composite resin to bleached bovine enamel. j oral rehabil. 2004; 31: 1184-91. 7. titely kc, torneck cd, ruse nd. the effect of carbamid-peroxide gel on the shear bond strength of a microfil resin to bovine enamel. j dent res. 1992; 71(1): 20-24. 8. titely kc, torneck cd, ruse nd, krmec d. adhesion of a resin composite to bleached and unbleached human enamel. j endod. 1993; 19: 112-5. 9. lai scn, tay fr, cheung gsp, mak yf, carvalho rm, wei sh et al. reversal of compromised bonding in bleached enamel. j dent res. 2002; 81: 477-81. 10. rostein l. role of catalase in elimination of residual hydrogen peroxide following tooth bleaching. j endod. 1993; 19: 567-70. 11. josey al, meyers ia, romaniuk k, symons al. the effect of a vital bleaching technique on enamel surface morphology and the bonding of composite resin to enamel. j oral rehabil. 1996; 23: 244-50. 12. perdigao j, francci c. ultra-morphological study of the interaction of dental adhesives with carbamid peroxide-bleached enamel. am j dent. 1998; 11: 291-301. 13. hegedus c, bistey t, flora-nagy e. an atomic force microscopy study on the effect of bleaching agents on enamel surface. j dent. 1999; 7: 509-15. 14. edward j, swift jr, perdigao j. effects of bleaching on teeth and restorations. compendium. 1998; 19: 815-20. 15. gomes torres cr, fuzuko koga a, borges ab. the effects of antioxidant agents as neutralizers of bleaching agents on enamel bond strength. braz j oral sci. 2006; 5: 971-6. 16. basting rt, de freitas pm, pimenta laf, serra mc. shear bond strength after dentin bleaching with 10% carbamide peroxide agents. braz oral res. 2004; 18: 162-7. 17. van der vyver pj, lewis sb, marais jt. the effect of bleaching agent on composite enamel bonding. j dent assoc s afr. 1997; 52: 601-3. 18. kayo ad, tukun m. reversal of dentin bonding to bleached teeth. oper dent. 2003; 6: 825-9. 19. shinahara ms, peris ar, pimenta la, ambrosano gm. shear bond strength evaluation of composite resin on enamel and dentin after non vital bleaching. j esthet restor dent. 2005; 1: 22-29. 20. cvitko e, denehy ge, swift ej, pires ja. bond strength of composite resin to enamel bleached with carbamide peroxide. j esthet dent. 1991; 3: 100-2. 21. sung ec, chan sm, mito r, caputo aa. effect of carbamide peroxide bleaching on the shear bond strength of composite to dental bonding agent enhanced enamel. j prosthet dent. 1999; 82: 595-9. 22. rostein i, lehr z, gedalia i. effect of bleaching agents on organic components of human dentin and cementum. j endod. 1992; 18: 290-3. 23. bulut h, kaya ad, turkun m. tensile bond strength of brackets after anti oxidant treatment on bleached teeth. eur j orthod. 2005; 27: 466-71. 24. zhao h, li x, wang j, qu s, weng j, zhang x. characterization of peroxide ions in hydroxyapatitic lattice. j biomed mater rest. 2000; 52: 157-63. 25. kimyai s, vafizadeh. the effect of hydrogel and solution of sodium ascorbate on bond strength in bleached enamel. oper dent. 2006; 4: 496-9. 26. paul p, rosaline h, balagopel s. the effect of hydrogel and solution of sodium ascorbate on the bond strength of bleached enamel. j conserv dent. 2007; 10: 43-7. 27. kimyai s, savadi oskoee s, rafighi a, valizadeh h. comparison of the effect of hydrogel and solution forms of sodium ascorbate on orthodontic bracket-enamel shear bond strength immediately after bleaching: an in vitro study. indian j dent res. 2010; 21; 54-8. 28. kaya ad, turkun m, arici m. reversal of compromised bonding in bleached enamel using antioxidant gel. oper dent. 2008; 33: 441-7. antioxidant effect on the shear bond strength of composite to bleached bovine dentin braz j oral sci. 10(1):33-36 oral sciences n3 original article braz j oral sci. july/september 2010 volume 9, number 3 received for publication: december 03, 2009 accepted: july 06, 2010 cariogenic and erosive potential of industrialized fruit juices available in brazil leopoldina de fátima dantas de almeida1, gisely maria freire abílio2, mônica tejo cavalcante3, ricardo dias castro4, alessandro leite cavalcanti5 1undergraduate student, school of dentistry, federal university of paraiba, joao pessoa, pb, brazil 2bs, pharmacist, assistant professor, federal university of paraiba, joão pessoa, pb, brazil 3phd student, department of process engineering, federal university of paraiba, joão pessoa, pb, brazil 4dds, msc, assistant professor, department of community dentistry, school of dentistry, federal university of paraiba, joão pessoa, pb, brazil 5dds, msc, phd, professor, department of pediatric dentistry, school of dentistry, state university of paraiba, campina grande, pb, brazil correspondence to: alessandro leite cavalcanti avenida manoel moraes, 471/802 manaíra 58038-230 joão pessoa, pb, brasil phone: +55 83 3315-3326 e-mail: dralessandro@ibest.com.br abstract aim: this in vitro study evaluated the cariogenic and erosive potential of different industrialized fruit juices available in the brazilian market. methods: twenty-five samples of fruit juices were analyzed physically and chemically by means of the following parameters: ph, titratable acidity (ta) and total soluble solid content (tssc), reducing sugars (e.g.: glucose), non-reducing sugars (e.g.: sucrose) and total sugars. the analyses were made in triplicate. data were collected by a single examiner and were recorded in study-specific charts. data were statistically analyzed by anova and tukey’s post-test (p<0.05). results: all fruit juices showed ph below the critical value of 5.5, with significant differences among the samples (p<0.0001). mango juice (jandaia®) presented the lowest tssc (10.25 obrix), while orange juice (del valle®) presented the highest tssc (12.75 obrix), with no significant differences among the samples. the lowest and the highest ta values were recorded for cashew juice (jandaia®) (0.13%) and passion fruit (del valle®) (0.52%), respectively (p<0.0001). for reducing sugars (glucose), the highest value was recorded for purple fruit juice (skinka®) (10.85 g/100ml) and the lowest was recorded for strawberry juice (kapo®) (1.84 g/100ml). regarding non-reducing sugars (sucrose), the values ranged from 0.45 g/100ml (passion fruit/del valle®) to 9.07 g/100ml (orange/del valle®). purple fruit juice (skinka®) presented the highest total sugars content (12.09 g/100ml), while guava juice (jandaia®) presented the lowest content (7.25 g/100ml). there were significant differences among the samples for reducing, non-reducing and total sugars (p<0.0001). conclusions: the industrialized fruit juices evaluated in this study presented low ph and a high total sugar content, differing in their erosive and cariogenic potential, respectively. keywords: beverages, hydrogen-ion concentration, dietary sucrose, dental caries, tooth erosion. introduction important innovations in the fruit juice industry in the last 100 years have included the use of pasteurization and the introduction of juice concentrates1. fruit juices are products defined as liquids obtained by expression or extraction of ripe fruit, by means of adequate technological processes and with characteristic color, aroma and flavor2. in general, juice drinks contain between 10 and 99% juice and added sweeteners, flavors and sometimes fortifiers, such as vitamin c or braz j oral sci. 9(3):351-357 calcium. these ingredients must be listed on the label, according to the us fda regulations 3. the brazilian legislation for foods is ruled by the ministry of health’s national health surveillance agency (anvisa) and by the ministry of agriculture, cattle raising and food supply (mapa). ready-to-use fruit juices should fulfill the mapa’s requirements with respect to the definition, classification, registration, standardization, labeling and quality demands, as well as the anvisa’s requirements with respect to labeling of packed food, namely main nutritional information, portions, complementary nutritional information and information on the presence of gluten in the product4-6. the desirability of a healthful lifestyle has led to an increased consumption of juices. fruit juice intake by children aged 1 to 6 years should be limited to 118-177 ml per day7. however, over 10 percent of american preschoolers consume at least 350 ml of fruit juices daily7. a combination of infant/ child feeding practices and repeated sequential consumption of fermentable carbohydrates, such as sweetened beverages, highly processed starchy/sugary foods or acidic beverages, increases the mineral loss of dental tissues8-9. the titratable acidity and a low ph are related to erosive lesions; and all these factors plus sugar content are involved in the development of carious lesions. a ph of 5.5 is traditionally considered to be the ‘critical ph’ for enamel dissolution, although mineral loss may begin at higher phs10. the most common cause of dental erosion in young people is the consumption of acidic beverages such as carbonated sports drinks and fruit juices. these have been reported to be associated with severe loss of dental enamel, particularly when consumed during periods when there is low salivary flow, such as immediately after heavy sports activities11. recently, there has been increased interest in determining some physical and chemical properties of fruit juices, such as endogenous ph12-14, titratable acidity (ta)15-16, total soluble solid content (tssc) or degrees brix (obx)17 as well as their effects on dental biofilm18. the acidity of a composition may be expressed in terms of ta, which is a measure of the percent weight of acid present in a solution as calculated from the volume of sodium hydroxide (naoh) or potassium hydroxide (koh) required neutralizing the acidic species. in practice, ta is measured potentiometrically with a standardized naoh or koh solution of a known concentration at 20oc19. soluble solids are compounds that are mixed or dissolved in the fruits, and are mainly formed by sugars, which give a sweet flavor, and acids, which give a sour taste. the brix scale or obx is numerically equal to the percentage of sugar and other solids dissolved in the solution. the food industry uses this scale for measuring the approximate amount of sugars in fruit juices and other beverages. thus, a solution that is 25 degrees brix has 25 g of sugar per 100 ml of solution20. sugars may be classified as reducing or non-reducing based on their reactivity with fehling’s reagents. sugars that contain aldehyde groups that are oxidized to carboxylic acids are classified as reducing sugars (e.g. glucose, fructose, maltose, lactose). those that are unable to reduce the above oxidizing agents are called non-reducing sugars (e.g.: sucrose). fruit juices are examples of foods that contain a mixture of these three soluble sugars (fructose, sucrose and glucose), with the concentration varying according to the type and maturation status of the fruit. such sugariness, coupled with an acidic nature, has caused fruit juice to be cited as a risk factor dental decay. in view of the high consumption of fruit juices among brazilian children13 and the lack of international studies addressing this subject, the purpose of this study was to evaluate in vitro the cariogenic and erosive potential of different industrialized fruit juices available in the brazilian market by assessing some physical and chemical parameters of these products. material and methods determination of endogenous ph, tssc, ta and sugar levels was undertaken in samples of 25 industrialized fruit juices commercialized in the city of joao pessoa, pb, brazil. the products were selected according to their availability in the market (table 1). each analysis was made in triplicate. data were collected by a single calibrated examiner, recorded on study-specific charts. ph measurement the ph of each juice was determined using a ph meter (tec-2 ph meter; tecnal, são paulo, sp, brazil) placed directly into each solution. the ph meter accurate to 0.1 was first calibrated according to the manufacturer’s instructions, employing buffer standards of ph 7 and ph 4. twenty milliliters of each pure beverage was placed in a beaker, the ph meter electrode was immersed in the juice and the reading was recorded12. between readings, the electrode was rinsed in distilled water to ensure that no crosscontamination occurred. degrees brix (obx) the obx readings were made by refractometry using an abbe refractometer (pzo-rl1, warsaw, poland). as the refractive index of a sugar-containing solution is also temperature-dependent, refractometers are typically calibrated at 20oc12. the equipment was calibrated with deionized water (refraction index = 1.3330 and 0º brix at 20ºc) and the readings of the samples were performed (obrix or g/100ml)12. titratable acidity (ta) ta was measured according to the method adopted by the association of official analytical chemists21, that is, the amount of 0.1 n koh solution needed for the product to reach a neutral ph or a ph value above it. a 10 ml aliquot of the diluted product was titrated (10% solution of the sample) with the 0.1 n koh solution until the substance reached a ph value between 8.2-8.4, corresponding to the endpoint of the phenolphthalein. readings were done with 352cariogenic and erosive potential of industrialized fruit juices available in brazil braz j oral sci. 9(3):351-357 353 fruit juice commercial name manufacturer pineapple jandaia jandaia sucos do brasil sa guava jandaia jandaia sucos do brasil sa cashew jandaia jandaia sucos do brasil sa acerola jandaia jandaia sucos do brasil sa mango jandaia jandaia sucos do brasil sa hog plum jandaia jandaia sucos do brasil sa passion fruit jandaia jandaia sucos do brasil sa citric fruits (orange, tangerine and lemon) and peach tampico ultrapan ind. com. ltda. wild fruits (apple, strawberry, raspberry and black mulberry) tampico ultrapan ind. com. ltda. grape tampico ultrapan ind. com. ltda. grape kapo coca cola ind. ltda. strawberry kapo coca cola ind. ltda. passion fruit kapo coca cola ind. ltda. pineapple kapo coca cola ind. ltda. acerola and orange isis isis ind. ltda. citric fruits (orange, tangerine and lemon) citrus indaiá brasil águas minerais ltda. acerola and orange citrus indaiá brasil águas minerais ltda. grape citrus indaiá brasil águas minerais ltda. purple fruits (grape, jabuticaba and raspberry) skinka schincariol ind. cerveja e refrigerantes ltda. red fruits (apple, strawberry and black mulberry) skinka schincariol ind. cerveja e refrigerantes ltda. citric fruits (orange, tangerine and lemon) skinka schincariol ind. cerveja e refrigerantes ltda. green fruits (apple and kiwi fruit) skinka schincariol ind. cerveja e refrigerantes ltda. passion fruit del valle del valle do brasil ltda. orange del valle del valle do brasil ltda. guava del valle del valle do brasil ltda. table 1. fruit juices, commercial name and manufacturers. a ph meter (tec-2r; tecnal, são paulo, sp, brazil). when this value was reached, the spent koh volume was recorded and the acidic percentage of the substance was calculated using the following equation, with the result being expressed as percentage of citric acid. acidity (%citric acid) = v x nap x f x meq-g(citric acid) x 100 sample where: v = koh volume; nap = normal concentration of the koh base; f = normality correction factor; meq-g = miliequivalent per gram of citric acid; sample= volume of the medicine. reducing sugars, non-reducing sugars and total sugars reducing sugars (e.g.: glucose), non-reducing sugars (e.g.: sucrose) and total sugars were measured according to the method adopted by the association of official analytical chemists21 and the results were expressed in g/ml. reducing sugars the measuring unit for reducing sugars is expressed as gram of glucose per 100 ml of the sample. for determination of this type of sugar, 5 ml of each sample were diluted in 50 ml of distilled water. the resulting solution was heated in water bath for 5 min and, after cooling and filtering, its volume was completed to 100 ml with distilled water. next, 10 ml of the fehling solution were mixed with 3 drops of 1% blue methylene (vetec®, rio de janeiro, rj, brazil). this mixture was titrated under warming blanket with the previously prepared sample solutions until the reducing sugars present in the sample reduced completely the fehling solution, as demonstrated by color change from blue to colorless, and by the formation of a brick red precipitated. the percentage of reducing sugars was calculated using the following equation: % of reducing sugars in glucose = 100 x v x cf sv x tv v = volume (ml) of the sample solution cf = calibration factor of the fehling solution sv = volume of the sample used for preparation of the solution tv = volume of the sample solution used in the titration total sugars in this analysis, the non-reducing sugars were subjected to acid hydrolysis with hydrochloride acid (vetec®, rio de janeiro, rj, brazil). these sugars were converted into reducing sugars, which were further subjected to titration and reduced the fehling solution, according to the same method used for determination of reducing sugars. the results were expressed as grams of sugars per 100 ml of sample. non-reducing sugars the non-reducing sugars were estimated by subtracting the reducing sugars from the total sugars and multiplying this value by the conversion factor of glucose in sucrose (0.95). the results were expressed as grams of sucrose per 100 ml of sample. cariogenic and erosive potential of industrialized fruit juices available in brazil braz j oral sci. 9(3):351-357 354 commercial name fruit juice p h ta (% citric acid) pineapple 3.92±0.0a 0.18±0.01 guava 3.73±0.0b 0.17±0.0 cashew 3.87±0.0ª 0.13±0.0 jandaia acerola 3.57±0.0c 0.25±0.01a mango 3.73±0.0b 0.23±0.0b hog plum 3.75±0.0b 0.20±0.0 passion fruit 3.53±0.0d 0.31±0.01c citric fruits 3.46±0.0de 0.41±0.01d tampico wild fruits 3.37±0.0df 0.30±0.0ce grape 3.50±0.0eg 0.37±0.0 grape 4.32±.0h 0.24±0.0abfg kapo strawberry 4.29±0.0hij 0.24±0.0abfhi passion fruit 4.28±0.0hij 0.27±0.01b pineapple 4.28±0.0hi 0.29±0.0e isis acerola and orange 3.53±0.0cgk 0.43±0.0j citric fruits 3.60±.0cl 0.39±0.01d citrus acerola and orange 3.58±0.0ckml 0.44±0.01 grape 3.53±0.0cgkmn 0.43±0.0j purple fruits 3.46±0.0deg 0.22±0.0bikl skinka red fruits 3.36±0.0df 0.27±0.0m citric fruits 3.60±0.0clm 0.27±0.01m green fruits 3.51±0.0cegk 0.23±0.01bkn passion fruit 3.36±0.0df 0.52±0.0 del valle orange 4.44±0.0 0.23±0.0bghln guava 3.94±0.0a 0.25±0.0agh table 2. distribution of the fruit juices according to the mean values for endogenous ph and titratable acidity (ta). same letter in columns for the same parameter indicate no statistically significant difference (anova and tukey’s post-hoc test; p>0.05). statistical analysis all recorded data were analyzed with the software graphpad prism version 5.0. data were statistically analyzed by anova and tukey’s post-test. the significance level adopted was 5% for all statistical analyses. results the results of the physical and chemical parameters varied among the evaluated brands of juices. table 2 displays the distribution of ph and ta mean values for the tested fruit juices. all fruit juices showed ph below the critical value of 5.5, with significant differences among the samples (p<0.0001). the ph values ranged from 3.36 (passion fruit/ del valle ®) to 4.44 (orange/del valle®). mango juice (jandaia®) presented the lowest tssc (10.25 obrix), while orange juice (del valle®) presented the highest tssc (12.75 obrix), with no significant differences among the groups (p>0.05). the lowest ta value was recorded for cashew juice (jandaia®) (0.13%) and the highest ta value was recorded for passion fruit (del valle®) (0.52%), with significant differences among the samples (p<0.0001). table 3 displays the distribution of reducing sugars, non-reducing sugars and total sugars mean values for the tested fruit juices. for the reducing sugars, the highest value was recorded for purple fruit juice (skinka®) (10.85 g/100ml) and the lowest value was recorded for strawberry juice (kapo®) (1.84 g/100ml). with regards to non-reducing sugars, the values ranged from 0.45 g/100ml (passion fruit/del valle®) to 9.07 g/100ml (orange/del valle ®). fourteen samples presented total sugar content over 10 g per 100 ml. purple fruit juice (skinka®) and guava juice (jandaia®) presented the highest (12.09 g/100ml) and the lowest (7.25 g/100ml) total sugar, respectively. there were significant differences among the samples for reducing, non-reducing ad total sugars (p<0.0001). discussion fruit drinks have been increasingly consumed by young infants in the form of diluted squashes and juices 7. historically, fruit juice has been recommended by pediatricians as a source of vitamin c and an extra source of water for healthy infants and young children as their diets expanded to include solid foods with higher renal solute3. because juices taste good, children readily accept them. however, though juice consumption has some benefits, it also has potential detrimental effects3, including shortness of stature and obesity22, development of dental caries23 and dental erosion9,14. therefore, fruit juices should be used as part of a meal or snack, and should not be sipped throughout the day or used as a means to pacify an unhappy infant or child3. the measurement of the ph is a practical method to assess the erosive potential of acidic drinks. in agreement with previous in vitro investigations12-13,17,24-25, the present study showed that any of the tested fruit juices could cause dental erosion because all of them had ph values below the critical value assumed for dental demineralization (5.5). although the values were numerically very similar, it must be kept in mind that ph is a logarithmic scale. small changes in ph values therefore equate with larger changes in the cariogenic and erosive potential of industrialized fruit juices available in brazil braz j oral sci. 9(3):351-357 355 commercial name fruit juice rs(g/100ml) nrs(g/100ml) ts (g/100ml) tssc(obrix,g/100ml) jandaia pineapple 5.04±0.04 4.36±0.04 9.40±0.04 11.50±0.0* guava 5.73±0.04ª 1.44±0.02 7.17±0.1 11.50±0.1* cashew 7.38±0.1bc 2.09±0.02 9.47±0.12 11.50±0.0* acerola 4.65±0.05 3.55±0.06 8.20±0.04 10.75±0.1* mango 3.48±1.02df 4.21±0.08ª 7.69±0.98 10.25±0.1* hog plum 3.33±0.02gh 4.16±0.06ª 7.49±0.08 10.50±0.0* passion fruit 2.00±0.06ijk 5.40±0.02 7.40±0.26 10.75±0.1* citric fruits 10.09±0.02 1.07±0.08b 11.16±0.1 11.75±0.1* tampico wild fruits 7.03±0.04m 2.96±0.06cd 9,99±0.02 12.00±0.0* grape 10.68±0.06n 0.81±0.03e 11,49±0.09 12.50±0.0* grape 3.00±0.02go 8.13±0.04 11.13±0.06 12.50±0.0* kapo strawberry 1.84±0.04ipq 8.77±0.03 11.61±0.07 12.50±0.1* passion fruit 1.94±0.08jpr 8.45±0.02 10.39±0.06 11.50±0.1* pineapple 3.07±0.02ho 6.68±0.04 9.75±0.02 11.00±0.0* isis acerola and orange 7.22±0.0bms 2.93±0.12cf 10.15±0.12 12.00±0.0* citric fruits 5.99±0.04 3.76±0.12 9.75±0.16 11.50±0.0* citrus acerola and orange 7.83±0.04t 1.84±0.03 9.67±0.06 11.75±0.1* grape 3.69±0.06du 7.12±0.02 10.81±0.08 12.00±0.0* skinka purple fruits 10.85±0.02n 1.18±0.1b 12.03±0.12 12.25±0.1* red fruits 10.29±0.08l 0.85±0.04e 11.14±0.04 11.75±0.0* citric fruits 7.53±0.02cst 3.03±0.06df 10.56±0.08 11.75±0.1* green fruits 8.60±0.04 0.46±0.04g 9.06±0.08 11.75±0.0* del valle passion fruit 9.63±0.02 0.45±0.07g 10.08±0.09 11.25±0.0* orange 1.88±0.02kqr 9.07±0.06 10.95±0.34 12.75±0.1* guava 4.00±0.06fu 4.80±0.04 8.80±0.02 10.50±0.0* table 3. distribution of the fruit juices according to the mean values for reducing sugars (rs), non-reducing sugars (nrs), total sugars (ts) and tssc. same letter in columns for the same parameter indicate no statistically significant difference (anova and tukey’s post-hoc test; pe”0.05). *no statistically significant difference among the groups (p=4790; anova and tukey’s post-hoc test). there were significant differences among the samples for the parameter total sugar (p<0.0001 –anova). hydrogen ion concentration14. regarding ta (i.e., amount of base required to bring a solution to neutral ph), the values obtained for the tested industrialized juice brands were lower than those reported in the literature26. the type of acid present in the beverages seems to influence the demineralizing capacity of the product; citric acid, for example, has a greater erosive potential than maleic, phosphoric and hydrochloric acids27-30. the high erosive potential of the citric acid is associated with its capacity of forming complexes with the calcium ions present in the hydroxiapatite30. therefore beverages with low ph and containing citric acid are considered as being potentially erosive31. baseline ph values give only a glimpse of the initial hydrogen ion concentration and therefore provide no indication as to the presence of undissociated acids. it is currently thought that ta is a more accurate measure of the total acid content of a drink and may, therefore, be a more realistic means of predicting erosive potential32. another evaluated parameter was °brix, which is a measure of total content of soluble solids (proteins, lipids, glucides, mineral salts, vitamins, organic acids, pigments and other substances) in a sample33, which has a direct relationship with the viscosity of the ingested foods, possibly facilitating the retention of diet components on the dental surfaces. in this study, the tssc (°brix values) ranged from 10.25 °brix (mango juice, jandaia®) to 12.75 °brix (orange juice, del valle®), which is in agreement with the findings of previous brazilian studies13,26,34. regarding this parameter, all fruit juices evaluated in the present study seem to have similar viscosity, as they did not significantly differ in °brix analysis. the cariogenic potential of foods is linked to the content of a variety of sugars, monosaccharides and disaccharides. water is the predominant component of fruit juice. carbohydrates, including sucrose, fructose, glucose and sorbitol, are the next most prevalent nutrients in juice3. all three sugars, sucrose, glucose and fructose, are fermentable to acid by a variety of oral microorganisms15. sucrose can be split into its two component sugars (glucose and fructose). this process is called inversion, and the product is called invert sugar. invert sugar is used mainly by food manufacturers to retard the crystallization of sugar and to retain moisture in the packaged food. glucose and fructose can reduce the content of cations, such as copper ions, which are present in the fehling solution, transforming the copper into an oxidized product. sucrose, however, does not have the same characteristic and it has to be acidulated in order to have its content measured35. this study evaluated the content of reducing sugars (e.g.: glucose), non-reducing sugars (e.g.: sucrose) and total sugars. fourteen samples presented total sugar content over 10 g per 100 ml, which is an important value as far as liquid intake by children is concerned. some studies have analyzed sugar content of commercial brands of juices of fruits and found values ranging from 2.833 to 7.3 g per 100 g26. therefore, the total sugar content values obtained in the present study may be considered high, which leads to the conclusion that frequent and excessive ingestion of such products combined with poor oral hygiene, may contribute to the initiation of carious lesions. cariogenic and erosive potential of industrialized fruit juices available in brazil braz j oral sci. 9(3):351-357 both reducing (glucose) and non-reducing sugars (sucrose) can be metabolized by s. mutans. however, these microorganisms have greater capacity of forming glucans from sucrose. this sugar is a substrate for glycosyltransferase (gtf), which can be of three types: gtf-b, gtf-c and gtfd. the glucans mediate the adherence of microbial cells to dental surface, favoring biofilm formation36-37. the implication is that an important concern with the consumption of these beverages by infants should be their erosiveness rather than their cariogenicity. however, it obviously must be borne in mind that since all these products contain sugars, no matter whether natural or added, if they are allowed to remain in the mouth over long periods as part of a frequent, protracted sugar intake pattern, it is likely that they will be able to contribute to the caries process, in which sugars serve as substrate for acid formation15. it is important to emphasize some limitations of this study, among which the fact that it was not possible to have an equal distribution of flavors among the commercial brands because this variability is not available in the market. another important point to be considered is the need of evaluating different lots of products, as the three measurements were done in different samples of the same lot in the present study. the national and international literature is scarce in studies investigating the presence of reducing, non-reducing and total sugars in industrialized fruit juices. in addition, to the best of our knowledge, there is no standardized value in the literature to establish the cariogenic potential of the sugars present in beverages. it should also be considered that dental caries and erosion have a multifactorial etiology and that an in vitro study, such as the present one, does not meet all requirements for extrapolating the obtained results to the clinical conditions. thus, further studies about this issue are required. public health advocates widely believe that poor infant feeding practices, particularly feeding with juice in a bottle at bedtime, are associated with the development of caries in primary teeth23. parents and caregivers should limit young children’s consumption of fruit juice to less than 350 ml per day7. moreover, instructing parents/caregivers on the negative impacts of an excessive intake of fruit juices by their children is an important aspect to be considered in the prevention of dental erosive lesions. in conclusion, the industrialized fruit juices evaluated in this study presented low ph and a high total sugar content, differing in their erosive and cariogenic potential, respectively. therefore, parents/caregivers should be instructed on the potential deleterious effects of such beverages on the dental hard tissues when often consumed by children. furthermore, information on healthy dietary habits and instructions on adequate oral hygiene measures must be an integral part of health education actions. references 1. . wolf a, bray ga, popkin bm. a short history of beverages and how our body treats them. obes rev. 2008; 9: 151-64. 2. tojo sierra r, dalmau serra j, alonso franch m, crespo ps, esteban mm, ferri nl et al. consumo de zumos de frutas y de bebidas refrescantes por niños y adolescentes en españa. implicaciones para la salud de su mal uso y abuso an pediatr 2003; 58: 584-93. 3. committee on nutrition. american academy of pediatrics: the use and misuse of fruit juice in pediatrics. pediatrics. 2001; 107: 1210-3. 4. brasil. agência nacional de vigilância sanitária. regulamento técnico sobre rotulagem nutricional de alimentos embalados. rdc 360, 23 de dezembro de 2003. diário oficial da união. 2003 26 dez; (251): 33; seção 1. 5. brasil. ministério da agricultura, pecuária e abastecimento. altera dispositivos do regulamento aprovado pelo decreto 2.314, 4 de setembro de 1997, que dispõe sobre a padronização, a classificação, o registro, a inspeção, a produção e a fiscalização de bebidas. decreto 3.510, 16 de junho de 2000. 6. ferrareze ac. interpretation of the consumer, evaluation of the intention to purchase and the physicochemical characteristics of the nectar and readyto-use orange juice [master’s dissertation]. araraquara: faculdade de ciências farmacêuticas de araraquara, universidade estadual paulista “júlio de mesquita filho; 2008. 104p. 7. rampersaud g, bailey l, kauwell g. national survey beverage consumption data for children and adolescents indicate the need to encourage a shift toward more nutritive beverages. j am diet assoc. 2003; 103: 97-100. 8. touger-decker r, mobley cc. american dietetic association. position of the american dietetic association: oral health and nutrition. j am diet assoc. 2003; 103: 615-25. 9. clena c, forner l. dietary habits in a child population in relation to caries experience. caries res. 2008; 42: 387-93. 10. cairns am, watson m, creanor sl, foye rh. the ph and titratable acidity of a range of diluting drinks and their potential effect on dental erosion. j dent. 2002; 30: 313-17. 11. khan f, young wg, law v, priest j. cupped lesions of early onset dental erosion in young southeast queensland adults. aust dent j. 2001; 46: 100-7. 12. cavalcanti al, oliveira os, evangelista apa, vieira ff, granville-garcia af, cavalcanti cl. ph and total soluble solid content in concentrated and diluted in natura tropical fruit juices. acta stomatol croat. 2008; 42: 229-34. 13. cavalcanti al, oliveira kf, paiva ps, rabelo mvd, costa skp, vieira ff. determination of total soluble solids (obrix) and ph in milk drinks and industrialized fruit juices. pesq bras odontoped clin integr. 2006; 6: 57-64. 14. hunter ml, patel r, loyn t, morgan mz, fairchild r, rees js. the effect of dilution on the in vitro erosive potential of a range of dilutable fruit drinks. int j paediatr dent. 2008; 18: 251-5. 15. grenby th, mistry m, desai t. potential dental effects of infants’ fruit drinks studied in vitro. br j nutr. 1990; 64: 273-83. 16. cairns am, watson m, creanor sl, foye rh. the ph and titratable acidity of a range of diluting drinks and their potential effect on dental erosion. j dent. 2002; 30: 313-7. 17. argentieri a, pistochini a, doño r. analysis of intrinsic ph, soluble solids and buffer effect of non-alcoholic drinks. acta odontol latinoam. 2003; 16: 35-44. 18. banan lk, hegde am. plaque and salivary ph changes after consumption of fresh fruit juices. j clin pediatr dent. 2005; 30: 9-13. 19. cavalcanti al, fernandes lv, barbosa as, vieira ff. ph, titratable acidity acidity and total soluble solid content of pediatric antitussive medicines. acta stomatol croat. 2008; 42: 164-70. 20. ball dw. concentration scales for sugar solutions. j chem educ. 2006; 83: 1489-91. 21. association of official analytical chemists. official methods of analysis. 16th ed. arlington; 1995. 1141p. 22. dennison ba, rockwell hl, baker sh. excess fruit juice consumption by preschool aged children is associated with short stature and obesity. pediatrics 1997; 99: 15-22. 356cariogenic and erosive potential of industrialized fruit juices available in brazil braz j oral sci. 9(3):351-357 357 23. dye ba, shenkin jd, ogden cl, marshall ta, levy sm, kanellis mj. the relationship between healthful eating practices and dental caries in children aged 2-5 years in the united states, 1988-1994. j am dent assoc. 2004; 135: 55-66. 24. seow wk, thong km. erosive effects of common beverages on extracted premolar teeth. aust dent j. 2005; 50: 173-8. 25. touyz lzg. the acidity (ph) and buffering capacity of canadian fruit juice and dental implications. j can dent assoc. 1994; 60: 454-8. 26. pinheiro am, fernandes ag, fai aec, prado gm, sousa phm, mais ga. chemical, physicochemical and microbiological evaluation of single strength fruit juices: pineapple, cashew apple and passion. cienc tecnol aliment. 2006: 26: 98-103. 27. hannig c, hamkens a, becker k, attin r, attin t. erosive effects of different acids on bovine enamel: release of calcium and phosphate in vitro. arch oral biol. 2005; 50(6): 541-52. 28. west nx, hughes ja, addy m. erosion of dentine and enamel in vitro by dietary acids: the effect of temperature, acid character, concentration and exposure time. j oral rehabil. 2000; 27: 875-80. 29. magalhães ac, wiegand a, rios d, honório hm, buzalaf mar. insights into preventive measure for dental erosion. j appl oral sci. 2009; 17: 7586. 30. wiegand a, stock a, attin r, werner c, attin t. impact of the acid flow rate on dentin erosion. j dent. 2007; 35: 21-7. 31. burato em, andrade l, rath ibs, tames dr. evaluation of the erosive potential of the brazilian sport drinks to the dental hard tissues. rev abo nac. 2002; 10: 109-12. 32. tahmassebi jf, duggal ms, malik-kotru g, curzon me. soft drinks and dental health: a review of the current literature. j dent. 2006; 34: 2-11. 33. chaves mcv, gouveia jpg, almeida fac, leite jca, silva flh. physicochemical characterization of the acerola juice. rev biol ciênc terra. 2004; 4: 1-10. 34. caleguer, vf, toffoli, c, benassi, m t. acceptance of commercial powdered orange flavored refreshment and their correlation physicochemical parameters. semina: ciênc agrár. 2006; 27: 587-98. 35. demiate im, wosiacki g, czelusniak c, nogueira a. analysis of total and reducing sugar in foods. a comparative study between colorimetric and titration techniques. exact and soil sciences, agrariam s and engineering. 2002; 8: 65-78. 36. ooshima t, matsumura m, hoshino t, kawabata s, sobue s, fujiwara t. contributions of three glucosyltransferases to sucrose-dependent adherence of stretococcus mutas. j dent res. 2001; 80: 1672-7. 37. borges mf, castilho arf, pereira cv. influence of sucrose, lactose and glucose + fructose on the cariogenic potential of s. mutans: an in situ and in vitro study. rev odonto ciênc. 2008; 23: 360-4. cariogenic and erosive potential of industrialized fruit juices available in brazil braz j oral sci. 9(3):351-357 revista fop n 13 1648 the effect of repeated bracket recycling on the shear bond strength of different orthodontic adhesives nádia lunardi1; gustavo hauber gameiro2*; maria beatriz borges de araújo magnani3; darcy flávio nouer4*; vânia célia vieira de siqueira3*; simonides consani5*; joão sarmento pereira-neto3* 1dds, ms, private practice 2dds, ms, phd student, department of pediatric dentistry, orthodontics division 3dds, ms, phd, assistant professor, department of pediatric dentistry, orthodontics division 4dds, ms, phd, professor, department of pediatric dentistry, orthodontics division 5dds, ms, phd professor, department of restorative dentistry, dental materials division *piracicaba dental school, university of campinas, piracicaba, sp, brazil received for publication: april 08, 2008 accepted: november 24, 2008 correspondence to: joão sarmento pereira-neto faculdade de odontologia de piracicaba – unicamp av. limeira 901 c.p. 52, cep 13414-903, piracicaba, sp, brasil phone.: +55-19-2106-5288 fax.: +55-19-21065218 e-mail: sarmento@fop.unicamp.br a b s t r a c t aim: the aim of this study was to evaluate the effect of two consecutive recycling procedures on the shear bond strength of different orthodontic adhesives. methods: edgewise brackets were bonded to the buccal surfaces of 40 bovine incisors using the following bonding materials: concise (group i), transbond xt (group ii), smart bond (group iii) and fuji ortho (group iv). the teeth were stored in water at 37°c for 24 h, thermocycled between 5 and 55°c, and debonded using an instron machine at a crosshead speed of 0.5 mm/min. in all groups, the bonded brackets were detached and rebonded after recycling by 50-µm particle aluminum oxide blasting. after the second recording of retentive strengths, the recycling procedure, the rebonding and the shear bond strength test were repeated. data were analyzed statistically by anova and tukey’s test at 5% significance level. results: the results showed that repeated recycling did not interfere in retention of brackets, regardless of the adhesive used. the highest shear bond strength values were obtained after bonding with transbond xt, independent of the recycling procedure. conclusion: repeated bracket recycling using 50-µm aluminum oxide particle air abrasion did not affect the shear bond strength of metallic brackets bonded with different orthodontic adhesives. key words: orthodontic brackets, bonding, recycling, aluminum oxide air abrasion. i n t r o d u c t i o n one problem that clinicians face during treatment is bracket failure. this is usually the consequence of either a patient’s accidentally applying inappropriate force to the bracket or a poor bonding technique. thus, a significant number of teeth must be rebonded in a busy orthodontic practice. one solution is to recycle the brackets1. the recycling process basically consists in removing bonding agent remnants from the bracket base, thus allowing the brackets to be reused without causing damage to the retention mesh and preserving its retentive characteristics2. although the clinical use may produce small distortions on the brackets, removal phase is responsible for most distortions and damages observed3. bracket recycling can be performed either in the dental office (immediate method) or by specialized companies without altering the slot positions4. two methods are commonly used for industrial bracket recycling: 1) heat application to burn the bonding agent followed by electrolytic polishing for oxide removal; 2) use of chemical solvents to dissolve the bonding agent in combination with high frequency vibrations and electrochemical polishing5-7. however, these are impractical to perform at the chairside. thus, several in-office bracket reconditioning methods have been introduced 1,8. immediate recycling of debonded brackets can be performed using silicon carbide stone grinding or aluminum-oxide blasting, which enhances bracket bonding to tooth structure by producing micromechanical retention on base surface. this process increases the area of composite bonding, which is essentially mechanical due to the micro-asperity of the bracket mesh. in spite of its increasingly widespread use for recycling purposes, aluminum-oxide blasting technique was originally intended braz j oral sci. october/december 2008 vol. 7 number 27 1649 to enhance the mechanical retention of new brackets and improve bracket bonding to restored teeth as well as to prepare the enamel surface8-9. several in vitro studies evaluating the effect of recycling on bracket bond strength have shown that reconditioning produces a reduction in bond strength, which is statistically significant compared to new brackets10-14. however, one must take in consideration that the effects of recycling depend on the type of reconditioning process used, the type of steel from which the bracket is constructed, and whether the bracket has a mesh pad or a non-mesh undercut integral pad15. tavarez et al.16 have shown that brackets recycled by aluminum oxide blasting had similar shear bond strength when compared with new ones. moreover, the bond strength values obtained after this recycling procedure were consistently higher than those obtained by an industrial process at a specialized company and those obtained by silicon carbide stone grinding. the reduced cost of using recycled brackets represents a significant financial advantage when bonding orthodontic brackets. to date, however, the bonding performance of brackets submitted to repeated recycling has not been investigated. thus, the purpose of the present study was to evaluate in vitro the effect of two consecutive recycling procedures on the shear bond strength of different orthodontic adhesives. material and methods forty freshly extracted bovine permanent mandibular incisors were collected, cleaned of soft tissue and stored in a saline, which was renewed every 7 days, and maintained under refrigeration at 4ºc. the criteria for tooth selection included intact buccal enamel, no pretreatment with chemical agents (eg, hydrogen peroxide), no cracks caused by the extraction forceps and no caries. the teeth were embedded in chemically activated acrylic resin (vipi flash; dentalvipi, pirassununga, sp, brazil) using pvc rings (tigre, cotia, sp, brazil) as moulds (20 mm in internal diameter; 20 mm in height), leaving only the crowns exposed. the buccal surfaces were cleaned with water/pumice slurry in robinson bristle brushes (kg sorensen, rio de janeiro, rj, brazil) at slow speed for 15 s, rinsed and dried with an air stream for 10 s each. the robinson brushes were replaced for new ones every five teeth to maintain the same mechanical cleaning action for all specimens. forty edgewise metallic brackets for central incisors (ultraminitrim-dentaurum, ispringen, germany) without angulation or torque were used. the teeth were randomly divided into 4 groups. the brackets were bonded to the teeth according to protocols following the manufacturer’s instructions, except for group iv, as described in the discussion section: group i bonding protocol with concise orthodontic (3m dental products, st. paul, mn, usa): 10 teeth were etched with 37% phosphoric acid gel for 30 s, washed with running water for 30 s, and dried for 20 s with oil free light compressed air jets. before bonding, a thin layer of adhesive was applied to the etched area. the bracket was applied to the tooth with a constant force, and the excessive material was carefully removed. a 5-min period was allowed for self-polymerization group ii bonding protocol with transbond xt (3m unitek, monrovia, ca, usa):10 teeth were etched with 37% phosphoric acid gel for 30 s. the teeth were thoroughly washed (30 s) and air dried (20 s). the sealant was applied, and the brackets were then bonded and lightcured with a halogen light-curing unit (3m unitek, sumaré, sp, brazil) for 20 s on the mesial side and 20 s on the distal side (total curing time, 40 s). group iii bonding protocol with smartbond adhesive system (gestenco international, göthenburg, sweden): 10 teeth were etched with 35% phosphoric acid for 10 s followed by thorough washing (30 s) and drying (20 s). a moist cotton roll was used to wet the enamel surface before the adhesive was applied. once the smartbond adhesive came into contact with the wet enamel surface, the clinician had 3 to 5 s to adjust the placement of the bracket before the adhesive starts to set within 3 to 5 min. group iv bonding protocol with fuji ortho lc (gc corporation, tokyo, japan): 10 teeth were etched with 37% phosphoric acid for 30 s, washed with running water for 30 s, and dried for 20 s. each tooth was then wiped with a moist cotton roll to ensure that the bonding surface was not desiccated, and excess water was removed. fuji ortho lc rmgi capsule was triturated for 10 s and then applied to the bracket without bubbles or voids. the bracket was applied to the tooth with a constant force, and the surrounding flash was carefully removed. the adhesive was light-cured with a halogen light-curing unit (3m unitek, sumaré, brazil) for 20 s on the mesial side and 20 s on the distal side (total curing time, 40 s). after bonding, the mounted teeth were thermocycled between 5°c and 55°c for 500 cycles. the exposure to each bath was 20 s, and the transfer time between the two baths was 5-10 s. debonding was performed at room temperature. the brackets were debonded using a universal testing machine (instron corp., canton, mass) at a crosshead speed of 0.5 mm/min. the samples were fixed to the testing machine by wire rings (0.019× 0.025-inch) attached to the bracket slot and to the machine’s clamps (figure 1). in this test, however, the resulting stress in the tooth-bracket bonding zone represented the shear bond strength. the rings were replaced with new ones for every 10 shear tests. the shear bond strength values were obtained in kgf and were divided by the bracket’s area to convert them into mpa. the bases of the brackets were sandblasted with 90µm diameter particle aluminum oxide air-abrasion (bio-art, são carlos, sp, brazil) for 15 s. a 10-mm distance was kept between the device tip and the bracket base17. after braz j oral sci. 7(27):1648-1652 the effect of repeated bracket recycling on the shear bond strength of different orthodontic adhesives 1650 each debonding, all visible residual adhesive was removed with a multiblade carbide burs (9114f; kg sorensen) at low speed, which were replaced by new ones every 5 teeth. after the second recording of retentive strengths, the recycling procedure, the rebonding and the shear bond strength test were repeated. since the data had a normal distribution (shapiro-wilk test), the results of the shear bond strength were submitted to analysis of variance and tukey test in order to compare the 4 adhesives at each bonding/debonding sequence (1st and 2nd recycling) and to determine whether significant differences existed in the shear bond strength of the 2 recycling procedures s within each adhesive. the level of significance was set at 5%. the brackets were examined with a scanning electron microscope (leo 435 vp; leo electron microscopy ltd., cambridge, england) to observe the base meshes before and after recycling procedures. r e s u l t s the descriptive statistics for the shear bond strength at the 2 recycling procedures for the 4 adhesives are presented in the table 1. within each adhesive type, no statistically significant differences (p<0.05) were found between new brackets and recycled ones for both 1st and 2nd recycling procedures. the comparisons among the 4 adhesives at each recycling procedure indicated significant differences. transbond had a significantly higher shear bond strength than that that of the other adhesives at each bonding/debonding sequence, except for transbond xt versus smartbond in new brackets, and transbond xt versus concise in recycled brackets -2nd recycling (table 1). the sem micrographs of each studied group are shown in figures 2 to 5. fig. 1. shear strength testing in the instron machine. fig. 2. scanning electron micrographs of a bracket base (a) after first (b) and second recycling (c) with aluminum oxide blasting. roughness of recycled bracket provided significant mechanical retention during bonding process (in this example, brackets were bonded with concise original magnification x500) fig. 3 scanning electron micrographs of a bracket base (a) after first (b) and second recycling (c) with aluminum oxide blasting. roughness of recycled bracket provided significant mechanical retention during bonding process (in this example, brackets were bonded with transbond original magnification x500) fig. 4 scanning electron micrographs of a bracket base (a) after first (b) and second recycling (c) with aluminum oxide blasting. roughness of recycled bracket provided significant mechanical retention during bonding process (in this example, brackets were bonded with smartbond original magnification x500) fig. 5 scanning electron micrographs of a bracket base (a) after first (b) and second recycling (c) with aluminum oxide blasting. roughness of recycled bracket provided significant mechanical retention during bonding process (in this example, brackets were bonded with fuji ortho lc original magnification x500) d i s c u s s i o n bond failure during orthodontic treatment is relatively frequent and undesirable. as a result, the shear bond strength of new and recycled brackets has been a subject of great interest in orthodontic research. the present investigation demonstrated that, when using four different orthodontic adhesives, the shear bond strength of recycled brackets was not significantly different from that new ones. also, the repeated recycling did not affect the shear bond strength braz j oral sci. 7(27):1648-1652 the effect of repeated bracket recycling on the shear bond strength of different orthodontic adhesives 1651 adhesive system new brackets 1st recycling 2nd recycling concise 4.168 (2.397) b 4.821 (2.328) b 5.769 (3.21) ab transbond 8.177 (4.612) a 8.460 (3.632) a 8.101 (2.055) a smartbond 4.808 (2.546) ab 4.468 (1.857) b 3.434 (1.989) b fuji ortho 3.013 (1.352) b 4.559 (2.688) b 3.740 (1.608) b table 1 descriptive statistics and results of anova/tukey tests comparing shear bond strength (mean in mpa and sd) within and among the 4 adhesive systems after repeated recycling procedures different letters indicate statistically significant difference within and among materials at each debonding sequence (p<0.05) when two recycling procedures were compared with the first bonding strength. although some studies have shown that recycling of bonded and rebonded orthodontic attachments adversely affect the shear bond strengths10-14, it is important to note that the effects of recycling depend fundamentally on the type of reconditioning process used. this fact can be observed in the study of tavarez et al.16 in which brackets recycled by aluminum oxide blasting had similar shear bond strength when compared with new ones, and the bond strength values obtained after 90-µm particle aluminum oxide blasting were consistently higher than those obtained by an industrial process at a specialized company or by silicon carbide stone grinding. several studies have reported that sandblasting bracket bases greatly increases their retentive surface which produces a significant reduction in the probability of failure relative to the non-sandblasted samples8,18. in a previous study, it was reported that sandblasting the meshbase of the stainless steel bracket for 3 s increased the bond strength of the conventional glass-ionomer cements to a level that may be clinically acceptable8. in this study, the time spent for bracket recycling with aluminum oxide air-abrasion was of 15 s and a10-mm distance was kept between the device tip and the bracket base. the time and distance settings were used to provide data directly comparable to those of other studies 16,17. the good mechanical retention between the enamel surface and the air-abraded recycled brackets is probably due to the fact that this method creates an effective microroughened surface on the bracket base, which increases the area available for composite bonding in comparison to the control brackets19 (figures 2 to 5). because orthodontic adhesives are routinely subjected to thermal changes in the oral cavity, it is important to determine whether such temperature variations introduce stresses in the adhesive that might influence bond strength. some investigators found that the shear bond strength of resin-modified glass ionomers is clinically acceptable following thermocycling20-22. whereas others concluded that bond strengths were acceptable only when phosphoric acid is used as an etchant23. in the present study, the tooth substrates were submitted to thermocycling and the phosphoric acid was used before bonding with fuji ortho lc to simulate the clinical condition when this protocol is commonly used. examining the base of the bracket with the scanning electron microscope (sem) suggested that the amount of mechanical retention created by the 50µm particle aluminum oxide blasting could be responsible for the similar shear bond strengths observed in recycled and new brackets, even after two recycling procedures. bishara et al.24 showed that rebonded teeth have significantly lower bond strength because of the residual adhesive on the enamel surface. the residual adhesive was present even after the surface was cleaned with the finishing bur and the enamel surface regained its gloss. although the residual adhesive was removed with a multiblade carbide bur in the present study, some residual material might be present. therefore, the current findings suggest that this problem could be compensated by mechanical retention created by the recycling procedures. among the tested adhesives, transbond xt presented significantly higher shear bond strength compared to the other adhesives at each bonding/debonding sequence, excep for transbond xt versus smartbond in new brackets, and transbond xt versus concise in brackets recycled twice. higher shear bond strength for transbond xt than for the other adhesives has already been observed21,25. in summary, aluminum oxide air-abrasion has been proved a good option for bracket recycling by offering a simple and easy-of-handle technique that can be performed in the dental office, which reduces the costs and working time. therefore, recycled brackets can be of benefit to the profession, as long as the orthodontist is aware of the various aspects of the recycling methods, and that patients are informed about the type of bracket that will be used for their treatment. concluding, repeated bracket recycling using 50µm aluminum oxide particle air abrasion did not affect the shear bond strength of metallic brackets bonded with different orthodontic adhesives. r e f e r e n c e s 1. basudan am, al-emran se. the effects of in-office reconditioning on the morphology of slots and bases of stainless steel brackets and on the shear/peel bond strength. j orthod. 2001; 28: 231-6. 2. maccoll ga, rossouw pe, titley kc, yamin c. the relationship between bond strength and base surface area using conventional braz j oral sci. 7(27):1648-1652 the effect of repeated bracket recycling on the shear bond strength of different orthodontic adhesives 1652 and micro-etched foil-mesh bases. am j orthod dentofacial orthop. 1996; 109: 338-9. 3. oliver rg, pal ad. distortion of edgewise orthodontic brackets associated with methods of debonding. am j orthod. 1989; 71: 65-71. 4. hixson me, brantley wa, pincsak jj, conover jp. changes in brackets slot tolerance following recycling of direct-bond metallic orthodontic appliances. am j orthod. 1982; 81: 447-54. 5. buchman djl. effects of recycling on metallic direct-bond orthodontics brackets. am j orthod. 1980; 77: 654-68. 6. mascia ve, chen sr. shearing strengths of recycled directbonding brackets. am j orthod. 1982; 82: 211-6. 7. buchwald a. a three-cycle in vivo evaluation of reconditioned direct-bonding brackets. am j orthod. 1989; 95: 352-4. 8. millet d, mccabe jf, gordon ph. the role of sandblasting on the retention of metallic brackets applied with glass ionomer cement. br j orthod. 1993; 201: 117-22. 9. senay c, iiken k, ela a. the effect of enamel air abrasion on the retention of bonded metallic orthodontic brackets. am j orthod dentofacial orthop. 2000; 117: 15-9. 10. jassem ha, retief dh, jamison hc. tensile and shear strengths of bonded and rebonded orthodontic attachments. am j orthod. 1981; 79: 661-8. 11. mascia ve, chen sr. shearing strengths of recycled directbonding brackets. am j orthod. 1982; 82: 211-6. 12. wright wl, powers jm. in vitro tensile bond strength of reconditioned brackets. am j orthod. 1985; 87: 247-52. 13. buchwald a. a three-cycle in vivo evaluateon of reconditioned direct-bonding brackets. am j orthod dentofacial orthop. 1989; 95: 352-4. 14. regan d, van noort r, o’keeffe c. the effects of recycling on the tensile bond strength of new and clinically used stainless steel orthodontic brackets: an in vitro study. br j orthod. 1990; 17: 137-45. 15. postlethwaite km. recycling bands and brackets. br j orthod. 1992; 19: 157-63. 16. tavares sw, consani s, nouer df, magnani mb, nouer pr, martins lm. shear bond strength of new and recycled brackets to enamel. braz dent j. 2006; 17: 44-8. 17. tavares sw, consani s, nouer df, magnani mbba, pereira neto js, romano fl. evaluation in vitro of the shear bond strength of aluminum oxide recycled brackets. braz j oral sci. 2003; 7: 378-81. 18. maccoll ga, rossouw pe, titley kc, yamin c. the relationship between bond strength and orthodontic bracket base surface area with conventional and microetched foil-mesh bases. am j orthod dentofacial orthop. 1998; 113: 276-81. 19. sonis al. air abrasion of bonded metal brackets: a study of shear bond strength and surface characteristics as determined by scanning electron microscopy. am j orthod dentofacial orthop. 1996; 110: 96-8. 20. meehan mp, foley tf, mamandras ah. a comparison of the shear bond strengths of two glass ionomer cements. am j orthod dentofacial orthop. 1999; 115: 125-32. 21. rix d, foley tf, mamandras a. comparison of bond strength of three adhesives: composite resin, hybrid gic, and glass-filled gic. am j orthod dentofacial orthop. 2001; 119: 36-42. 22. bishara se, ostby aw, laffoon jf, warren j. shear bond strength comparison of two adhesive systems following thermocycling. a new self-etch primer and a resin-modified glass ionomer. angle orthod. 2007; 77: 337-41. 23. toledano m, osorio r, osorio e, romeo a, de la higuera b, garcia-godoy f. bond strength of orthodontic brackets using different light and self-curing cements. angle orthod. 2003; 73: 56-63. 24. bishara se, laffoon jf, vonwald l, warren jj. the effect of repeated bonding on the shear bond strength of different orthodontic adhesives. am j orthod dentofacial orthop. 2002; 121: 521-5. 25. romano fl, tavares sw, nouer df, consani s, borges de araujo magnani mb. shear bond strength of metallic orthodontic brackets bonded to enamel prepared with self-etching primer. angle orthod. 2005; 75: 849-53. braz j oral sci. 7(27):1648-1652 the effect of repeated bracket recycling on the shear bond strength of different orthodontic adhesives oral sciences n3 original article braz j oral sci. october|december 2010 volume 9, number 4 received for publication: january 10, 2010 accepted: november 12, 2010 pain behavior to electroacupuncture in rabbit tooth pulp delane viana gondim1, krishnamurti de morais carvalho2, mariana lima vale3 1msc, department of clinical medicine, medical school, federal university of ceará, fortaleza, ce, brazil 2 dds, department of physiology, state university of ceará, fortaleza, ce, brazil 3 dds, department of physiology and pharmacology, medical school, federal university of ceará, fortaleza, ce, brazil correspondence to: delane viana gondim departamento de medicina clínica, faculdade de medicina, universidade federal do ceará ufc. r. cel. nunes de melo, 1127, rodolfo teófilo 60430-270 fortaleza, ce, brasil. phone: +55-85-3366-8588 fax: +55-85-3366-8333 e-mail: delanegondim@yahoo.com.br abstract aim: the aim of this study was to verify the pain behavior to electroacupuncture (eacp) in rabbit tooth-pulp assay. methods: albino rabbits weighing 1.5-2.0 kg) were pretreated with saline or morphine (5mg/kg, e.v.) 10 min before the nociceptive test (nt). in another group, eacp (rectangular pulses, f1=2 hz, f2=0.1 s, 3 ma) was applied in acupoints and sham points, before and during the nt. after 120 min, eacp was withdrawn and the nociceptive threshold was measured every 10 min until the initial nociceptive threshold was achieved. results: eacp, using the yintang, st4 and st5 acupoints, induced an increased in the nociceptive threshold and this effect persisted for up to 2 h, even after the removal of electric stimulation. application of eacp at sham points did not show significant analgesic activity. the present results demonstrated that males presented a higher initial level of analgesia, but a poorer maintenance of analgesic effect after the eacp procedure, while females demonstrated a long lasting analgesic effect even after discontinuation of eacp. conclusions: eacp presented an analgesic effect in a rabbit tooth pulp assay that was probably due to the release of endogenous opioids. the duration of this analgesic effect seems to be different for males and females. keywords: electroacupuncture, tooth pulp, analgesic effect, endogenous opioids, pain. introduction acupuncture is part of chinese traditional medicine, one of the most ancient and well known human health systems, which is approximately 5,000 years old. acupuncture is a technique for correcting reversible physiological malfunction of several parts of the body by physiological mechanisms, acting mostly through the application of skin stimuli by inserting needles in specific points called acupuncture points or acupoints1-2. acupoints are skin spots with a rich concentration of sensory nerve terminations and they have been determined over thousands of years of medical practice3. these regions are also rich in blood vessels, muscles, and articular capsules4. stimulation of acupoints enables direct access to the central nervous system (cns)5. results from some animal and clinical studies provide evidence of nervous and endocrine system involvement in the action of acupuncture 6-7. acupuncture is also a reflex therapy, which means that a stimulus in one area acts on another(other) one(s), with this effect mainly occurring through the nociceptive system8. it has been suggested that the analgesic effect of acupuncture is the result from the saturation phenomenon that melzack and wall9 have called “gate control braz j oral sci. 9(4):415-420 theory”. the discovery of the central endorphin system was a prominent step towards understanding the analgesic effects of acupuncture and many studies have been performed to investigate the mechanisms of acupuncture analgesia8,10-14. electroacupuncture (eacp) is based on acupuncture and is used as a therapeutic method for general purposes. eacp stimulates acupuncture points with an electric current that enters into the body through needles rather than with manual stimulation, and it is used for disease treatment and as an analgesic method for acute and chronic pain12. studies have demonstrated that eacp, at different frequencies, can induce the release of different neuropeptides in the cns. low-frequency eacp accelerates the release of encephalin and b-endorphin at supra-spinal levels, whereas high frequency eacp increases the release of dynorphin at spinal level, causing extensive physiological effects8. in this case, the antinociceptive action consists of afferent peripheral nervous stimulations through the anterolateral tract of the spinal cord, raphe nucleus, and reticular formation, thus reaching different cns areas including various sections of the hypothalamus, thalamus, hippocampus, and hypophysis5. the evidence found in the literature about the analgesic activity of acupuncture, acting through the release of endogenous opioid peptides, has motivated the development of this study. thus, using the classic rabbit tooth pulp stimulation model15, the antinociceptive effects of lowfrequency eacp were investigated, assessing its duration, the significance of needle localization, and differences between males and females. material and methods animals experiments were carried out with 54 albino rabbits (48 males and 6 females) weighing 1.5-2.0 kg that were housed in a temperature-controlled room with access to water and food ad libitum and 12 h of dark-light cycles. all experiments were approved by animal care and use committee of state university of ceará (protocol number 156/06) and carried out in agreement with its guide for the care and use of laboratory animals. tooth cavity procedure a cavity was prepared on the distal surfaces of anterior-superior teeth close to the gingival region using a 0.5 mm round bur in a low-speed dental handpiece immediately after anesthesia16-17. two shallow cavities were drilled until exposure of dentin18-19. sterile saline and air spray were used to minimize thermal damage. for these procedures, animals were anaesthetized with a mixture of k e t a m i n e x y l a z i n e h y d r o c h l o r i d e s ( 9 0 1 5 m g / k g ) administered by the intramuscular route, and were kept in the anesthetic condition by checking a blink reflex and tail flick by pinch. animals were also shaved at the face or at the dorsal side. rabbit tooth pulp assay the nociceptive test used in this study was the rabbit tooth pulp assay, originally described by piercey and schroeder (1980) 15. animals were acclimated to the immobilization procedure during 5 days before the experiment for 20 min once a day. on the 5th day, the animals were shaved (face and back) and the tooth cavity procedure was performed as described above. on the 6th day, animals were placed in boxes, where they remained for 10 min. after this acclimation period, an electric current (voltage of 0 to 24 v from a direct-current source with an electrical resistance of 1 kù)20 was applied to the exposed dentin cavities via finewire steel electrodes held in each of the cavities18-19. before the experiment, the electric stimulator equipment was tested with a voltmeter to measure and confirm the output current. the nociceptive threshold was measured by a trained observer through the observation of patterned lick-chew response associated with reflex head-jerk withdrawal movement or simple reflex muscle twitches15,21. another observer registered the reached voltage in the nociceptive test. mean threshold voltages were established for controls using an average of three determinations. rabbits having control values greater than 6v were excluded from the study. generally, non-treated animals reached a threshold of 3 to 6v. eacp and drug treatment for this experimental study, rabbits were subjected to eacp using stainless steel needles (0.25 x 30 mm) in predetermined acupuncture points (st4, st5, and yintang)22 or in randomly chosen sham points on the face and on the back. yintang point is located on the forehead, at the midpoint between the eyebrows (glabella). st4 and st5 points are located on the lateral corner of the mouth and anterior to the angle of the mandible, respectively. the facial and dorsal sham points were located near the ears and on the cervical column, respectively, and are not related to dental analgesia. sham and acupuncture points were bilaterally stimulated with low-frequency rectangular pulses (f1=2 hz, f2=0, recurrence time=1 sec, intensity= 3 ma) from the nklel530 eacp equipment. eacp was performed 20 min before the nociceptive test14,23-24 and was applied for 120 min. in another group, the opioid antagonist naloxone (2 mg/kg) was injected via intraperitoneal route 10 min before eacp. morphine (5mg/kg; e.v.) was administrated 10 min before the nociceptive test in a group of rabbits not submitted to eacp procedure. the nociceptive threshold was measured every 10 min up to 120 min. after this period, eacp procedure was finished and the needles removed. after needle removal, the nociceptive threshold was still measured every 10 min until the recovery of the initial nociceptive threshold. drugs and equipment the following drugs were used: morphine (cristália, itapira, sp, brazil), naloxone (rhodia farma; são paulo, sp, brazil), ketamine (francotar; virbac do brasil ind. e com. 416416416416416pain behavior to electroacupuncture in rabbit tooth pulp braz j oral sci. 9(4):415-420 ltda, roseira, sp, brazil), xilasine (rompun; bayer s.a. saúde animal, são paulo, sp, brazil). sterile acupuncture needles were obtained from wujiang shenli medical and health material co. ltd, são paulo, brazil, and electroacupuncture was applied using the nkl el530 instrument (bioaacus, são paulo, sp, brazil). neiko ac/dc deluxe digital multimeter tol13451 (surplus computers, fremont, ca, usa).the electric stimulator equipment was kindly made and donated by dr. roberto brasil from the state university of ceará uece, brazil. statistical analysis results are presented as means ± s.e.m. of measurements made on at least 6 animals for each group. differences between responses were evaluated by anova followed by tukey’s test. statistical differences were considered to be significant at p<0.05. the student’s t-test was used to compare two means. results comparing the analgesic effect of low-frequency eacp when applied at acupuncture points versus facial and dorsal sham points, no significant differences (p>0.05) were observed between the non-treated group and the eacp group in the first 20 min after the eacp procedure. eacp at facial sham points initially showed significant nociceptive activity (148%; p<0.05), which was not maintained after 20 min of experiment. at 60, 90, and 120 min after the low-frequency eacp procedure, the eacp group showed a significant difference in nociceptive threshold (p<0.001) when compared to the untreated group (165.17%, 209.3% and 223.68%, respectively), facial sham group (111.44%, 108.43% and 170.0% respectively), and dorsal sham group (114.19%, 163.63% and 141.66%, respectively). at these times, no significant differences were observed between the untreated group and the dorsal sham group and facial sham group (figure 1). during the evaluation of the differences between lowfrequency eacp and morphine’s antinociceptive effect, it was observed a significant difference (138%, p<0.05) between the morphine group and the untreated group that lasted 50 min, while no statistically differences was observed with eacp group. after 30 min, eacp induced a significant increase in nociceptive threshold in comparison to untreated animals (104%; p<0.01), but it remained lower than the analgesic effect observed in the morphine group (159%; p<0.001). at first there was no statistical difference between the eacp and morphine groups, but at 50 min the morphine analgesic effect was significantly different from the eacp effect (75.18%; p<0.001; figure 2). after 50 min the morphine analgesic effect decreased and at 90 min no antinociceptive activity was observed, whereas the eacp antinociceptive activity remained throughout the whole test, up to a 180.23% difference from the non-treated group (p<0.001; figure 2). the effect of pretreatment with naloxone on the antinociceptive activity of low-frequency eacp in the rabbit fig. 1. evaluation of differences among acupoints and facial and dorsal sham points on the analgesic effect of low-frequency electroacupuncture (eacp) in rabbit tooth pulp assay. the nociceptive threshold was measured before and after eacp procedure, every 10 min up to 120 min. animals were treated with eacp at acupoints (acupoints; yintang, st4, st5; 2 hz, 30 ma; 20 min before test up to 120 min), facial sham points (facial sham; 2hz, 30 ma; 20 min before test up to 120 min) and at back sham points (dorsal sham; 2hz, 30 ma; 20 min before test up to 120 min). nt represents non-treated animals. the points represent the means ± sem of 6 animals per group. asterisks indicate statistically significant differences between the nt group and the others (*p<0.05; anova followed by turkey’s test). fig. 2. evaluation of analgesic effect differences between low-frequency electroacupuncture (eacp) and morphine in rabbit tooth pulp assay. the nociceptive threshold was measured before and after eacp procedure or morphine injection every 10 min up to 120 min. animals were treated with eacp (yintang, st4, st5 acupoints; 2 hz, 30 ma; 20 min before test up to 120 min), morphine (5mg/kg; 1 ml; e.v.; 20 min before test) or saline (nt; 1 ml; e.v.; 20 min before test). the points represent the means ± sem of 6-12 animals per group. asterisks indicate statistically significant differences between the nt group and the others (*p<0.05; anova followed by turkey’s test). tooth pulp assay was also evaluated. animals were pretreated with saline (1 ml; i.p.) or naloxone (2 mg/kg; i.p.) 10 min before applying eacp to acupuncture points. naloxone pretreatment significantly inhibited the eacp antinociceptive effect up to 298.31% (p<0.001) when compared to the eacp plus saline treated group (figure 3). when gender was assessed to evaluate differences in the antinociceptive effect of low-frequency eacp in the rabbit tooth pulp assay, statistically significant differences (p<0.05) 417417417417417 pain behavior to electroacupuncture in rabbit tooth pulp braz j oral sci. 9(4):415-420 fig. 3. effects of pretreatment with naloxone on the analgesic activity of lowfrequency electroacupuncture (eacp) in rabbit tooth pulp assay. the nociceptive threshold was measured before and after 30 min of eacp procedure. animals were pretreated with saline (1 ml; i.p.) or naloxone (2 mg/kg; i.p.) 10 min before eacp treatment (yintang, st4, st5 acupoints; 2 hz, 30 ma; 20 min before test up to 120 min). nt represents non-treated animals. bars represent the means ± sem of 12 animals per group after 30 min of test. asterisks indicate statistically significant differences between eacp-saline group and eacp-naloxone group (*p<0.05; student’s t-test). were observed between males and females after 30 (91.56%), 60 (82%), 80 (89.62%), 90 (80%) and 100 min (70.47%) (figure 4). when the eacp procedure was discontinued, males showed a shorter duration of the antinociceptive effect than females. the antinociceptive effects on female rabbits remained until, at least, 2 h after discontinuation of the eacp procedure (figure 5). discussion acupuncture is part of traditional chinese medicine and is used in several countries all over the world. in recent fig. 4. male-female differences in the analgesic effect of low-frequency electroacupuncture (eacp) in rabbit tooth pulp assay. the nociceptive threshold was measured before and after eacp procedure every 10 min up to 120 min. males and females were treated with eacp (yintang, st4, st5 acupoints; 2 hz, 30 ma; 20 min before test up to 120 min). the points represent the means ± sem of 6 animals per group. asterisks indicate statistically significant differences between males and females (*p<0.05; student’s t-test). years, the academic and scientific communities have had a growing interest in this area in order to find answers to nervous-anatomic-physiological problems. the effectiveness of acupuncture has always been a controversial topic for traditional medicine. acupuncture treatment uses thermal and electrical stimulation, pressure, laser radiation, and needle insertion at specific points called acupuncture points. results from animal and human studies provide evidence of cns, autonomic nervous system, and endocrine system involvement in the mechanism of action of acupuncture11,14,25-26. pain has different causes and is characterized by distinct somatosensory, visceral, affective, cultural, and cognitive qualities 27. following these patterns, the tooth pulp stimulation method can be used to test the central antinociceptive activity in rabbits15-17, and has been employed in different species18-20,28. the tooth pulp stimulation test has been successfully used to evaluate the antinociceptive effect of opiates drugs, non-opiate antiinflammatory and antipyretic drugs after intravenous and intramuscular injections15-17,21. electric stimulation of the tooth pulp in rabbits induces typical reactions such as licking, biting, chewing and agitation of the head, which can be easily observed15,21. we have modified the original model described by piercey and schroeder15, since these authors used isolated pulses and pulp exposition. other authors have also performed pulp exposure during experiments with this model11-12,16-17,2829, but we preferred not to expose the tooth pulp in order to avoid possible contamination and necrosis as done by iwata et al. (1998)19. instead of isolated pulses, a direct-current source with a voltage from 0 to 24 volts was used. this methodological variation was chosen to analyze eacp nociceptive activity in the presence of more aggressive stimuli and to control its effects through time. physical and psychological stressors are known to cause a variety of behavioral and biochemical alterations, including fig. 5. male-female differences in analgesic effect after low-frequency electroacupuncture (eacp) treatment in rabbit tooth pulp assay. the nociceptive threshold was measured after 120 min eacp (yintang, st4, st5 acupoints; 2 hz, 30 ma) procedure in males and females. the points represent the means ± sem of 6 animals per group. asterisks indicate statistically significant differences between the nt group and the others (*p<0.05, student’s t-test). 418418418418418pain behavior to electroacupuncture in rabbit tooth pulp braz j oral sci. 9(4):415-420 blood pressure and norepinephrine levels. studies have demonstrated that animals subjected to eacp with forced immobilization show a reduction in blood pressure and in plasma catecholamines24,28. based on these facts, we have observed the eacp antinociceptive effect by tooth pulp electrical stimulation over 120 min. higher antinociceptive activity was registered at 60, 90, 100, and 110 min. the nociceptive behavior of untreated animals and sham group did not show an increase in the nociceptive threshold, during the same period of time (120 min), when compared to the eacp group. this reinforces the theory that eacp antinociceptive activity is not related to stress in this experimental protocol. several studies have utilized 20-40 min of eacp treatment10,12-14. however, we standardized a 120 min treatment period, with 10 min intervals, to evaluate the eacp antinociceptive effect kinetics and observe its maximum effect during all the eacp treatment period. our results suggest that eacp, on the points yintang, st4, and st5, produced an antinociceptive response at the all observed times as well as after needle removal. it was measured by the increase in the nociceptive threshold. in the chinese traditional medicine, li4 and st44 points, which are located on the dorsal side of the forward paw (between the first and second fingers) and on the hind paw (between the second and third fingers), are essential to tooth pain22,30; however, they were not used in this study due the impossibility of paw immobilization. these points, in rabbits, could only be used with previous sedation, which could change the eacp antinociceptive response. for this reason, we used only local points, which distinguish this work from previous studies10,12. the neuronal pathway stimulated by acupuncture differs based on the frequency of the applied stimulus. we chose a low frequency (2 hz) stimulus, which is the most common modality of eacp therapy and has been associated with less irritability. low-frequency eacp facilitates animal manipulation, which results in a lower stress effect. lowfrequency eacp promotes the release of encephalin and âendorphin in the brain and spinal cord, which interact with ì and ä receptors in the cns31. this mechanism of action was demonstrated in the present work through the reversion of eacp analgesic activity by pretreatment with the nonselective opioid antagonist naloxone. in this experimental model, eacp produced a prolonged antinociceptive effect that lasted longer than the morphine antinociceptive effect. however, the early antinociceptive effect of morphine was more potent than eacp but with a shorter duration. this difference is probably related to the metabolic degradation of morphine, but more data are needed to confirm this suggestion. after the eacp procedure was finished, the antinociceptive effect was maintained for at least 2 h. this prolonged effect gives us a perspective of use of acupuncture in relieving acute pain in dentistry patients. the traditional chinese acupuncture literature emphasizes the precise localization and correct combination of acupuncture points to elicit an adequate therapeutic response. our findings agree with these data, since sham points did not produce a significant antinociceptive response when compared to acupuncture points. regarding gender differences, female hormones have been speculated to play an important role in the development and maintenance of chronic and acute pain. estrogen has been considered a modulator of the nociceptive afferent primary trigeminal fibers. some authors32,33 have demonstrated that a glutamate injection into the temporomandibular joint (tmj) can induce the jaw muscle reflex, which is more intense in female rats than in males. gonadectomy significantly reduced the magnitude of glutamate-evoked digastric muscle activity in female rats. treatment of gonadectomized female rats with estrogen increased the magnitude of glutamateevoked digastric muscle activity32. the results of the present study suggest male-female differences for this experimental model, since males presented a higher initial level of analgesia, but a lower capacity of maintaining the analgesic effect after termination of eacp, while females demonstrated a weaker, but longer lasting analgesic effect, even persisting after eacp was discontinued. these data need further investigation considering that female hormone cycles have effects on nociception. the findings of this study indicate that analgesia induced by eacp through the combination of stimulation at the yintang, st4, and st5 sites probably occurs through endogenous opioid peptide release and the analgesic response to eacp depends on gender. our results also suggest that precise acupuncture point localization is necessary to obtain the desired analgesic effect. however, further research is required to increase the understanding of the neuroendocrine effects of acupuncture point combination. this analgesic response could represent a promising technique for 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myofascial pain by the reproductive hormones: a preliminary report. j prosthet dent. 1998; 79: 663-70. 420420420420420pain behavior to electroacupuncture in rabbit tooth pulp braz j oral sci. 9(4):415-420 oral sciences n3 braz j oral sci. 10(4):226-232 received for publication: march 09, 2011 accepted: july 20, 2011 original article braz j oral sci. october | december 2011 volume 10, number 4 quality of life and self-perceived oral health among workers from a furniture industry cristina gomes de macedo1, dagmar de paula queluz2 1dds, master student, piracicaba dental school, university of campinas (unicamp), brazil 2dds, msph, phd, professor, department of community dentistry, piracicaba dental school, university of campinas (unicamp), brazil correspondence to: dagmar de paula queluz departamento de odontologia social faculdade de odontologia de piracicaba unicamp avenida limeira, 901 cep 13414-900 piracicaba sp brasil e-mail: dagmar@fop.unicamp.br abstract oral health problems have more and more been recognized as important causers of negative impact to workers in the daily activities and quality of life. aim: to identify the profile and selfperceived oral health among workers from a furniture industry. methods: data were obtained from a cross-sectional study in which the oral health impact profile questionnaire (ohip 14 – short form) was applied to the workers focusing on their demographic profile and self-perceived oral health results: 111 workers out of a total of 170 agreed to participate in the study; 84.68% (n=94) were men and the majority was under 35 years of age (68.81%, n=75). the following results were obtained for the different parameters evaluated: level of education, occupation, mean frequency of toothbrushing (number of times per day) (3.19), general health, dry mouth (7.2%, n=8), smoking (15.32 %, n=17), gingival bleeding (51.35%, n=57) and use of medications for toothache relief (38.74%, n=43). the mean ohip value found was 0.51. significant association was found between the mean ohip value and general health and gingival bleeding. no significant association was found between the use of medications for toothache relief and gingival bleeding (p=0.729) or dry mouth (p=0.704). significant association was found between use of medications for toothache relief and smoking (p=0.007). conclusions: knowing the habits and the profile of workers is a relevant factor to promote an oral health attention in the context of the occupational health; gingival bleeding was a strong indicator in self-perceived oral health and workers’ quality of life. keywords: oral health, quality of life, self assessment (psychology), occupational health. introduction oral health problems have more and more been recognized as important causers of negative impact to workers in their daily activities and quality of life1-2. the orofacial region is usually an area of significant concern because it draws the most attention from other people in interpersonal interactions and is the primary source of vocal, physical, and emotional communication3. the oral health organization has recognized that oral diseases cause pain, psychological suffering, social constraints and privations, leading to damage in an individual and collective level4. few studies have so far investigated the relationship between the oral conditions and their impact on people’s life, but in recent years, the use of clinical indicators (e.g: gingival bleeding, caries, tooth loss) in epidemiological studies has been widely recommended. however, these clinical indicators have been frequently 227 braz j oral sci. 10(4):226-232 used only to determine the presence or absence of the disease/ condition, but their impact on patients and populations have not been investigated 5. measuring the impacts of oral conditions on quality of life is an important part of oral health needs assessment. there are some instruments described in literature for measuring the impact of oral health on the quality of life. however, these instruments differ with respect to size, content, structure of the response form scale and methods for obtaining the scores. a common problem in assessing the strength of the measures obtained with these instruments is that researchers usually do not specify why it is important to evaluate a specific clinical indicator in the studied population and they often only reproduce existing methodologies and compare their data with those of previous studies6. validated and reliable instruments for measuring subjective oral diseases were introduced in the 1990s to supplement the clinical indicators used routinely. they are used for collecting oral health data at both individual and population levels, and can be applied in education, preventive and curative programs as well as by other health care professionals7. subjective indicators can be either onedimensional, when they cover only one aspect (e.g.: chewing difficulty or pain intensity) or multidimensional, when they encompass different dimensions (e.g.: pain, constraints and psychological well-being of individuals). one of the most widely used indicators in different cultures and sociodemographic profile is the oral health impact profile (ohip). developed by slade and spencer8, the proposed questionnaire measures dysfunction, discomfort and disability attributed to oral condition. originally composed of 49 items, the conceptual index involves seven dimensions: functional limitation (e.g., chewing difficulty), physical pain (e.g., toothache), psychological discomfort (e.g., self-confidence), physical disability (e.g., food restriction), psychological incapacity (e.g., affected concentration), social disability (e.g., is less lenient with others) and functional incapacity (e.g., become completely incapable functionally). the questionnaire is answered on a five-point lickerttype, scale (always, often, sometimes, rarely and never). a short ohip version has emerged as a powerful instrument in the subjective assessment of oral health related to the quality of life. containing 14 items, this version is preferred over ohip-49 by many researchers because of its convenience and validation. the short version of this instrument, the ohip14, was developed by slade in 19979, and validated for use in adult population in england10 and scotland11. it was translated and trans-culturally adapted in 10 languages12-17 including portuguese14. studies9,18 have shown that ohip-14 presents good psychometric properties when employed in more distinct populations. interest in the oral health of workers has increased due to need of knowing the oral health profile of this population group as little information is available about this subject. there is still the need to understand how employees perceive the influence of oral health on their quality of life. this information is relevant for the development of oral health strategies that meet the specific needs of this population, benefiting their well-being and general health. thus, the aim of this study was to identify the profile and self-perceived oral health among workers from a furniture industry. material and methods the research protocol was reviewed and approved by the ethics committee of the university of campinas, under the protocol number 009/2009. all 170 workers from a furniture industry located in the municipality of itatiba, sp, brazil, were invited to participate in the study. the investigation was designed as a crosssectional study and the data collection was held through questionnaires focusing questions on profile and selfperceived oral health. the demographic characteristics comprised questions about age, gender, school level (incomplete fundamental school, complete fundamental school, complete high school, complete college education), occupation (administration, production, project, reception). habits and health problems comprised questions about smoking (no, yes), gingival bleeding (never, sometimes, always), use of medications for toothache relief (no, yes), dry mouth (no, yes), general health (medium, good, excellent), mean frequency of toothbrushing (one, two, three, four, more than four times/day, never). self-perceived oral health was evaluated using ohip 14 short form9. the ohip is composed of 14 items divided into 7 conceptual dimensions: functional limitation (items 1 and 2), physical pain (items 3 and 4), psychological discomfort (items 5 and 6), physical disability (items 7 and 8), psychological disability (items 9 and 10), social disability (items 11 and 12) and physical limitation (items 13 and 14). the answers of the workers to the ohip questionnaire were analyzed using a lickert-type scale in which answers are encoded into five categories. each category received a score between 0 and 4, which was multiplied by the weight of each question: 0 = never 1 = almost never, 2 = sometimes 3 = with reasonable frequency and 4 = very frequent9. the application of the questionnaire was accompanied by a psychologist of the department staff in different shifts, after instructing the workers on how to fill out the questionnaire. during conduction of the study, the identities of workers were shielded in the questionnaires. all procedures were carried out with treatment ensuring reliability and credibility to the worker. descriptive statistics (frequency distribution, average, standard deviation) were used to characterize the workers. epidemiological studies using the ohip have found that oral diseases are associated with increasing levels of impact on well being. based on the answers to the ohip, the workers were divided into two major groups: no impact (0 = never, 1 = almost never and 2 = sometimes) and impact (3 = with reasonable frequency and 4 = very frequent). if at least one dimension was scored 3 or 4 the worker was considering as belonging to the impact group. data were tabulated using excel19 software (2007) and quality of life and self-perceived oral health among workers from a furniture industry 228 braz j oral sci. 10(4):226-232 were then analyzed using the sas20 (2002) system. the mantel and haenszel chi square tests, fisher’s exact test, and the coefficient v cramer were used to analyze the association between ohip and demographic characteristics (age, gender and school level) and the related variables measures health. a significance level of 0.05 was adopted for all tests. results from the 170 individuals working at the furniture industry, 111 accepted to take part in the study. the industry presents a variation of 10-15 workers per year and many outsourced workers in other distant occupations of the search local. the age range was from 17 to 64 (mean age 31.38 ± 9.34 years). the majority of workers (68.81%, n = 75) were under the age of 35 years and 31.19% (n=34) were over this age (table 1). the majority of workers were male (n= 94, 84.68%). with regard to school level most workers (64.48%, n = 69) had at least graduated from high school and 23 workers (21.50%) had completed the fundamental school (table 1). regarding the occupation in the industry, most were production workers (n=72, 66.67%) covering sectors like polishing, maintenance, glass factory, carpentry, sorting, warehouse and management (table 1). the majority of the workers (n=94; 84.68%) were nonsmokers. the mean frequency of tooth brushing (number of times per day) was 3.19 (table 1). fifty-four workers (48.65%) had never had gingival bleeding and only 3 workers (2.70 %) said to have gingival bleeding frequently. the most serious instances with frequencies are only three cases being that, among those who had bleeding, the majority (n = 54) said to be a low frequency event (table 1).about medications for toothache relief, 68 workers (61.26%) had never used while 43 workers (38.74%) had already used some kind of medications for toothache (table 1). there was no significant association between the use of medications for toothache relief and gingival bleeding (p=0.729) or dry mouth (p=0.704); but it was positively associated with smoking (p=0.007). in relation to dry mouth, the frequency of workers with dry mouth was low (n=8, 7.21%) against a total of 103 workers (92.79%) who had never experienced this condition (table 1). about the general health, 15 workers (13.51%) considered their general health as excellent, 77 workers (69.37%) considered as good and 19 workers (17.12%) considered as medium (table 1). as much as 92 workers (82.88%) of the workers considered their general health as excellent or good. however, regarding the impact of oral health on quality of life, the mean ohip value of this group was 1.74, which is much higher than the mean value of 0.51 recorded for the total population (figures 1 and 2). the fisher’s exact test did not find significant association between the demographic characteristics and the ohip value; such a difference was only detected using chi square mantel and haenszel test, which are more specific to test the hypothesis of absence of linear association between the measures assessed. there was significant linear (p<0.01) association between ohip values and self-assessed general health. this association can be illustrated by the fact that people who perceived their general health as medium (73.68%) recorded ohip values above the mean value for the total population while those who perceived their general health as good or excellent (26.32%) recorded ohip value below the mean value for the total population (figure 1). significant association was found between the ohip value and gingival bleeding (figure 2), as higher incidence of gingival bleeding coincided with higher ohip value. we could observe the same inversion that we observed in the association of ohip and general health. lower ohip values were most often associated with workers who had never had gingival bleeding (73.68%), while higher ohip values were most frequently associated with workers who always have gingival bleeding (73.33%) (figure 2). quality of life and self-perceived oral health among workers from a furniture industry demographic characteristics age frequency percentage 17 |— 24 31 28.44 24 |— 35 44 40.37 35 |— 64 34 31.19 gender male 17 15.32 female 94 84.68 school level incomplete fundamental school 15 14.02 complete fundamental school 23 21.50 complete high school 51 47.66 complete college education 18 16.82 occupation in the industry administration 22 20.37 factory 72 66.67 projects 9 8.33 reception 5 4.63 habits and health problems smokers frequency percentage n o 94 84.68 yes 17 15.32 gingival bleeding never 54 48.65 sometimes 54 48.65 always 3 2.70 medicine for toothache n o 68 61.26 yes 43 38.74 dry mouth frequency percentage n o 103 92.79 yes 8 7.21 general health frequency percentage medium 19 17.12 good 77 69.37 excellent 15 13.51 table 1. frequencies and percentages the study population of demographic characteristics; habit and health characteristics. 229 braz j oral sci. 10(4):226-232 fig.1. percentages of workers in the categories of ohip and general health fig. 2. percentages of workers in the categories of ohip and gingival bleeding. discussion the analysis of the demographic characteristics of the studied population revealed that the majority of the workers were young (<35 years), male and had at least graduated from high school. the brazilian formal worker profile prepared by the ministry of labor and employment’s social service for industry traced the full picture of the reality of brazilian formal workers in 5,500 municipalities 21. in 2003, the evolution of employment by gender did not present major differences. however, despite the participation of women in the workforce in recent years, the number of formal male employees remains largely superior to female workers. in 2003, for example, the number of formal work spots occupied by men was 50.3% higher than those occupied by women. regarding the behavior of formal workers according to the level of education, employment opportunity is lower in the lower levels of education21. this dynamics can be explained by two factors: firstly, we could be facing a supply of qualified workers. the second factor is that workers with the highest level of qualification would be required due to technological modernization21. there was a prevalence of non-smokers in the studied population. prevalence of tobacco use has declined in some high-income countries, but it continues to increase in lowincome and middle-income countries, especially among young people and women. undoubtedly, the increasing number of smokers and smokeless tobacco users among young people in some parts of the world will considerably affect the general and oral health of future generations. the prevalence of tobacco use in most countries is higher among people of low educational background and among poor and marginalized people22. the mean frequency of toothbrushing (3.19 times per day) found in this study is considered as an indicator of good oral hygiene habits and concern about health and appearance. hugoson et al. 23 in 2005 compared data on dental care habits and knowledge of oral health in four cross-sectional epidemiological studies carried out in 1973, 1983, 1993, and 2003. a questionnaire about dental care habits and knowledge of oral health was used in connection with a clinical and radiographic examination. the same questions were used in all four studies. the knowledge of the etiology of dental diseases did not change much between 1973 and 2003. the frequency of tooth brushing increased from 1973 and in 2003 more than 90% of all investigated individuals brushed their teeth twice or once a day. the use of dental floss and toothpicks decreased in 2003 compared with 1983 and 1993. in 2003, almost all individuals used fluoride toothpaste. it was obvious that the dental team constituted the main source of oral health information. for the 20-30year-old age group, information from friends and relatives was also important. in the 3-20-year-old age group, up to 45% of the individuals consumed soft drinks every day or several times a week. we did not find evidence of association between demographic variables with the workers’ self-perceived general health, i.e. the different strata of age, gender and school level did not have an important influence on the general health. as the workers did not present so distinct demographic characteristics, as expected, these characteristics did not exert an effect on the concept of general health. contributed considerably to the absence of effect, the uniformity of the population, which was predominantly composed of men aged between 17 and 64 years, with a predominantly high school level for the brazilian standards, since more than 85% of the workers had completed fundamental education and 64.48% had at least graduated from high school. good health is a major resource for social, economic and personal development. political, economic, social, cultural, environmental, behavioral and biological factors can enhance or impair health. almost forty percent of employees had already used medications for toothache relief, which is not negligible a percentage, and 57 workers said to always or sometimes suffer from gingival bleeding. there are few reports in the literature about oral health conditions in adults as well as a shortage of structured collective health programs for this economically active population. however, there is a consensus that adults constitute the vast majority of the population that demand for dental care and influence the behavior of their quality of life and self-perceived oral health among workers from a furniture industry braz j oral sci. 10(4):226-232 230 dependants24. oral health-related quality of life can be assessed positively, by measuring satisfaction with mouth, or negatively, by measuring oral impact on the performance of daily activities. slade et al.25 compared subjective oral health of adults in the uk and australian populations. cross-sectional studies were conducted with people aged 18+ years in the 1998 uk adult dental health survey and the 1999 australian national dental telephone interview survey. subjective oral health was measured using the 14-item oral health impact profile questionnaire (ohip-14). while the percentage of adults reporting adverse impacts of oral health was similar, australians reported a larger number of impacts and more severe impacts than dentate people in the uk. differences in the number and severity of impacts between the two populations may be an artifact of different data collection methods or may reflect relatively subtle socio-cultural differences in subjective oral health between these populations. although the effect of disorders on psychological wellbeing is well-known, there are few studies focusing on oral conditions at the worksite. another study examined the association between psychological well-being and oral conditions of japanese workers. a cross-sectional study was performed using data from 1,381 japanese civil service officers aged 20 to 59 year. results did not show that psychological well-being was associated with oral conditions measured by clinical indicators. however, an association was found between some oral symptoms and psychological wellbeing. absence of oral symptoms seems to be related to better psychological well-being26. using a questionnaire containing a spontaneous smiling photograph of the participant, geld et al.27 investigated selfperception of smile attractiveness to determine the role of smile line and other aspects correlated with smile attractiveness and their influence on personality traits, concluding on the psychosocial importance and the dental significance of an attractive smile. brennan et al.28, in a study with middle-aged and older adults, investigated tooth loss and chewing ability and their association with oraland general-health-related quality of life and life satisfaction. the authors concluded that chewing ability was related to oral-health-related quality of life and general health, possibly reflecting the impact of chewing on food choice and enjoyment of meals and diet, and also indicating the importance of oral health to general well-being. oral health deficits can have a significant effect on workers’ general health and their ability to carry out normal activities. significant differences were found between the mean ohip value and general health in this study. lower ohip values were most often associated with excellent selfperceived health and higher ohip values were most frequently associated with medium self-perceived health. this difference between the average and median is quite representative in light of the magnitude of value and possibly be unduly influenced in the case of average by highest values. the standard deviation of 2.69 indicates that the variability is bigger than the average, which characterizes a high variability in data, although a large proportion of people recorded a very low ohip value. general health indicator was incurred and in conjunction with ohip want to allowed assessment on the quality of life. in another study, quandt et al.29 observed that although farm workers have been found to lack access to dental care, few studies have documented their oral health status or its impact on quality of life. this research describes the oral health problems experienced and oral health care received by latino farm workers in north carolina, and explores the association between oral health and quality of life. data were collected using face-to-face interviews from a representative sample of 151 farm workers; data included oral health-related quality of life (ohip-14) and general health-related quality of life (sf-12 health survey). workers reported a high number of unmet needs: 52% reported caries, and 33% reported missing teeth. these findings indicate that the high rate of unmet oral health needs is associated with poorer farm worker quality of life. the consequences of suffering on-going dental pain for work performance, sleep, and nutritional status are unknown. kieffer and hoogstraten30 studied the association among oral health, general health, and quality of life. the oral health impact profile (ohip-49) and the rand-36 were distributed amongst 118 psychology freshmen. additionally, two single items self-rated general health (srgh) and self-rated oral health (sroh) were administered. kruskal-wallis and mannwhitney u-tests were used to evaluate differences between srgh and sroh categories, regarding ohip subscale scores and rand subscale scores. more than 75% of the subjects rated their oral and general health as good. mean ohip scores and rand scores indicated a relatively good oraland general health-related quality of life, respectively. the correlation between oral and general health was weak. the findings suggest that oral health, general health, and quality of life have different determinants. furthermore, oral health and general health appear to be mostly unrelated in this seemingly healthy population. it is proposed that if no apparent disease is present, oral and general health must be regarded as separate constructs. the use of subjective indicators in dentistry has been increasing. a population-based cross-sectional study in the city of chapecó, santa catarina state, brazil, analyzed the relationship between oral health conditions and quality of life in 35-44-year-old adults (n = 622). oral health status was found to interfere in the daily routine of 20.7% of participants and was more severe for 11.4%. household crowding, low-income neighborhoods, female gender, and orofacial pain in the previous six months were associated with higher interference in quality of life. the use of subjective indicators in public health services helps planning the delivery of oral care to priority groups31. significant differences were found between the mean ohip value and gingival bleeding in this study. higher ohip values were most often associated with workers who always quality of life and self-perceived oral health among workers from a furniture industry braz j oral sci. 10(4):226-232 had gingival bleeding. several studies have linked periodontitis and consequently the gingival bleeding with numerous diseases, such as: premature birth and low weight of newborn children, lung disease, osteoporosis, stroke and diabetes. thus, gingival bleeding affects the workers’ selfesteem and consequently their quality of life, interfering in their social and labor coexistence32-33. drumond et al.32 evaluated the potential impact of periodontal disease on quality of life in diabetics. a total of 159 dentate diabetic individuals registered at the municipal hospital in itaúna, mg, brazil, were examined and interviewed. the clinical periodontal parameters recorded were: gingival bleeding, probing depth, and clinical attachment level. the ohip-14 form was used to evaluate the impact of periodontal disease on quality of life. association between diagnosis of periodontal disease and impact on quality of life was significant in individuals with periodontitis (p < 0.001). gingival bleeding, probing d e p t h , a n d c l i n i c a l a t t a c h m e n t l e v e l > 4 m m w e r e associated with intensely negative impact on quality of life (p = 0.013, p < 0.001, and p = 0.012 respectively). diabetics with mild-to-moderate and advanced periodontitis had more negative impact on quality of life than those who were periodontally healthy or with gingivitis. another study33 assessed the periodontal health status in the danish adult population, analyzed how the level of periodontal health is associated with age, gender, urbanization, socioeconomic factors, and dental visiting habits, and compared the periodontal health status of danish adults with that of adults in other industrialized countries. using a cross-sectional model, a random sample of 1,115 danish adults aged 35-44 years and 65-74 years. data were collected by means of personal interviews and by clinical examinations in accordance with the world health organization basic methods criteria. the clinical examination revealed a low prevalence of healthy periodontal conditions in both age groups: at age 35-44 years 7.7% and at age 65-74 years 2.4% had healthy periodontal conditions. a high proportion of the elderly had scores of severe periodontal health; more than 82% of older participants had pockets of 4-5 mm or deeper against 42% in younger adults. in both age groups, the mean number of teeth with periodontal pockets deeper than 4-5 mm was high in individuals with low education. the multivariate analysis showed that participants with low or medium levels of education had significantly more teeth with shallow and deep pockets than those with high education. people with regular dental visiting habits had fewer teeth with gingival bleeding, shallow and deep pockets than individuals with irregular dental visiting habits. the authors concluded that reorientation of the danish dental health-care services is needed with further emphasis on preventive care, and public health programs should focus on risk factors shared by chronic diseases in order to improve the periodontal health of danish adults. developing more effective approaches for prevention has been a permanent challenge for occupational health. a major obstacle to this goal has been the lack of communication and knowledge of problems affecting workers. until recently, few studies had investigated the relationship between oral conditions and their impact on people’s lives; but in the last decade, there has been increased interest in quantifying the diseases. various instruments were developed in an attempt to understand and assess how the oral problems have affected the daily lives of people. open or structured interviews provide greater understanding of human behavior and people’s beliefs. the absence of predetermined interview responses offers the possibility of new perspective for social and cultural variables often judged by the researcher. in conclusion, taken together and considering the applied methodology, knowing the habits and the profile of the workers is a relevant factor to promote an oral health attention in the context of the occupational health. gingival bleeding was found to be a strong indicator in workers’ selfperceived oral health and quality of life. references 1. queluz dp. labour dentistry: a new specialty in dentistry. braz j oral sci. 2005; 4: 766-72. 2. queluz dp. odontologia do trabalho. in: pereira ac, organizador. tratado de saúde coletiva em odontologia. nova odessa: napoleão; 2009. p.217-38 3. kiyak ha. does orthodontic treatment affect patient’s quality of life? j dent educ. 2008; 72: 886-94. 4. world health organization. who technical report series 916. geneva; 2003. 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original article braz j oral sci. july/september 2009 volume 8, number 3 mixture of betel leaf, areca nut and tobacco chewing is a risk factor for cytogenetic damage in construction workers from south india sellappa sudha1, balakrishnan mythili2, vellingiri balachandar3 1associate professor, department of biotechnology, school of life sciences, karpagam university coimbatore, tamil nadu, india 2m.sc., m.phil., research scholar, department of biotechnology, school of life sciences, karpagam university, coimbatore, tamil nadu, india 3m.sc., m.phil., phd., research scholar, human genetics laboratory, school of life sciences, bharathiar university, coimbatore. tamilnadu, india received for publication: july 23, 2009 accepted: october 16, 2009 correspondence to: sellappa sudha department of biotechnology, school of life sciences, karpagam university coimbatore-641021, tamil nadu, india phone: +91-422-2611146. fax: +91-422-2611043 e.mail: sudhasellappa@yahoo.co.in abstract aim: to determine the cytogenetic effect of betel leaf, areca nut and tobacco mixture usage among female construction workers in tamilnadu, southern india. methods: totally 236 buccal cells and blood samples were collected from 80 betel quid users and 76 users with tobacco snuffing habit which were compared with 80 healthy subjects. peripheral blood leukocyte cultures were analyzed for chromosomal aberrations (ca) and exfoliated cells from the buccal mucosa were examined for micronucleus (mn). results: statistically significant (p<0.01) increase in ca and mn were observed in users with snuffing habit when compared to users without snuffing habit and controls as confirmed by chi-square test. therefore, specific biomarkers on cytogenetic endpoints might help in planning precautionary measures to reduce oral cancer risks. conclusions: the present study can be concluded that a mixture of betel quid, areca nut and tobacco chewing/snuffing is unsafe for oral health. the genotoxic effect of smokeless tobacco should be considered in addition to other known hazards for assessing health risks. keywords: betel leaf, areca nut, tobacco snuff, chromosomal aberration, micronucleus. introduction worldwide, betel quid is among the most common addictions subsequent to tobacco, alcohol and caffeine. its use is very popular in india. many indian women in rural areas regularly chew betel quid, a combination of areca nut, betel leaf ( from piper betle), lime paste, and leaf tobacco. users are easily identified because the quid causes the teeth to turn black brown and stain the tongue and oral mucosa. the habit of betel quid chewing is quite common throughout southeast asia. it is estimated that between 10% and 20% of the world’s population chews betel quid1-2. the international agency for research on cancer (iarc) regards the chewing of betel leaf and areca nut to be a known human carcinogen3, which have role in multistage progression of oral cancer4. smokeless tobacco contains nitrosonornicotine and 4-(methylnitrosamino)-1-(3pyridyl) 1-butanone; areca nut contains arecoline and 3-(methylnitrosamino) propionitrile, while lime provides reactive oxygen radicals, each of which has a role in oral carcinogenesis5. chewing betel quid without tobacco is an independent risk factor for developing oral cancer6. when betel quid with tobacco is consumed with alcohol and smoking the relative risk increases 11-fold7. betel leaf contains large amounts of carcinogens called safrole, which is readily metabolized and excreted in urine. betel quid and areca nut chewing leads to oral sub-mucous fibrosis, a painful disabling and potentially precancerous condition of the oral mucosa. betel quid chewing is a major risk factor for cancer in mouth, pharyngeal cavity and upper digestive tract8. regular braz j oral sci. 8(3):145-148 use of betel quid have several adverse effect on oral cavity and upper digestive tract, including inflammation, development of white or gray patches on the tongue and buccal mucosa and oral cancer 9. chewing a mixture of betel leaf, areca nut and tobacco is a complex behavior and is poorly studied. betel and areca nut chewing has been extensively studied in populations in many part of the world. however genotoxic effect of combinational use of betel quid with snuff has received less attention. the purpose of this study was to evaluate the mn and ca of individuals regularly using a mixture of betel leaf, areca nut and tobacco with snuff tobacco. chromosomal aberrations (ca) in peripheral blood lymphocytes and micronucleus (mn) in buccal mucosa are considered as reliable biomarkers of genotoxic exposure to both physical and chemical agents10, and an increase in ca/ mn frequency indicates the risk of exposure to clastogenic and/or aneugenic agents. in addition, cytogenetic end points in peripheral blood lymphocytes have been used as biomarkers which allow a reasonable epidemiological evaluation of cancer predictability11. material and methods the subjects were selected by random sampling. the study group consisted of 156 healthy female construction laborers and 80 healthy subjects who did not use any form of tobacco or alcohol and working in the same environment were selected as controls. study area is coimbatore city, south india. the exposed group includes 80 individuals (group i) who regularly chewed a mixture of betel leaf, areca nut and tobacco (users) and 76 users with snuffing habit (group ii). for assessment of smokeless tobacco habits among construction workers, the sample was classified as users and users with snuffing. the form of smokeless tobacco use (chewing and/or snuff ), the number of years of consumption (duration) and the number of units used per day were recorded by subject interview. subjects were then classified based on duration of tobacco usage into less than 10 years and more than 10 years. before collecting the sample, all subjects were interviewed to evaluate their habits, according to the protocol published by the international commission for protection against environmental mutagens and carcinogens12. venous blood samples (5 ml) were drawn in heparinized syringes from each subject for the chromosomal analysis. the work was carried out in accordance with the ethical standards laid down in the 1964 declaration of helsinki13. chromosomal aberration assay blood samples were used to establish leukocyte cultures by following standard procedures6. 0.5 ml blood was added to 4.5 ml rpmi 1640 medium supplemented with 10% calf fetal serum, 2 mm l-glutamine, 1% streptomycin-penicillin, 0.2 ml reagent grade phytohemagglutinin, and was incubated at 37 ºc. after 50 h, cultures were treated with 0.1 g/ml colcemid to arrest the cells at metaphase in mitosis. lymphocytes were harvested after 52 h by centrifuging cell suspension to remove culture medium (800-1000 rpm), addition of hypotonic solution (kcl 0.075 m) at 37 ºc for 20 min to swell the cells, and treated twice with carnoy’s fixative (3:1 ratio of methanol: acetic acid). slides were carefully dried on a hot plate (56ºc, 2 min). later, the slides were stained using the giemsa technique. for the ca analysis, 100 well spread complete metaphase cells in first cell cycle were evaluated per subject under a microscope at ×100 magnification to identify numerical and structural ca. chromatid-type cas: (chromatid gaps; chromatid breaks) chromosome-type cas: (break; gap; exchange) were observed. micronucleus assay buccal cells were sampled with a tooth brush from the inside of both cheeks and placed in 50ml tubes containing 25 ml of buffer solution (0.1m edta. 0.01m tris hcl 0.02 m nacl) ph 7.0, the cells were washed thrice in the buffer solution by centrifugation and slides for microscopic analysis were prepared14. cell suspension was dropped onto clean slides and cell density was checked using a microscope. the slides were allowed to dry and then fixed in 80% cold (0°c) methanol. the samples were then applied to clean microscope slides. smears were air dried and fixed in methanol: acetic acid (3:1). slides were stained with may-grunewald giemsa method (sigma st louis mo). the mn analysis was done with a light microscope, at x 100 magnification, using coded slides. 1000 cells from each individual were examined. only unfragmented cells that were not smeared, clumped or overlapped and that contained intact nuclei were included in the analysis. criteria used for identification of micronuclei were according to the method of countryman and heddle15. results table 1 shows the total number of subjects, age range, mean age and mean duration of exposure. the study subjects were categorized into two groups based on type of exposure (users and users with snuff ). table 2 shows frequencies of chromosome aberrations and frequency distribution of micronuclei. the mean percentages of mn cells in group i was 1.55 ± 0.64 for less than 10 years of habitual exposure and 2.76 ± 0.90 for more than 10 years of exposure. in the controls, the mean percentage of mn cells was 0.52 ± 0.26. statistically significant differences were observed between the experimental subjects compared to controls (p < 0.01). the mean values ± sd of ca in experimental and control subjects group exposed group i group ii control total number of subjects n=236 156 80 76 80 age range(yrs) 32-51 30-59 31-54 mean age(yrs) 46 49 47 mean period of exposure(yrs) 7.59 16.75 s.no 1. 2. table 1 characterization of the participants group i – users (betel leaf, areca nut and tobacco chewing). group ii users with snuff. control – non users/non snuffers. mixture of betel leaf, areca nut and tobacco chewing is a risk factor for cytogenetic damage in construction workers from south india146 braz j oral sci. 8(3):145-148 in group i was 8.25 ± 3.27 for more than 10 years exposure group and 4.68 ± 2.64 for less 10 years exposure group respectively; in group ii it was 9.92 ± 4.08 (> 10 years exposure) and 7.19 ± 2.73 (< 10 years exposure), respectively. statistically significant results were obtained in experimental subjects compared to control groups (p < 0.01), confirmed by chi-square test. users with snuff habit having increased percentage of ca and mn cells when compared to users with out snuffing habit. in group ii the mean percentages of mn cells were 2.97 ± 0.97 for more than 10 years habitual users with snuffing and 1.93 ± 0.75 for less than 10 years of exposure. discussion india has the largest betel quid consuming population in the world. a large-scale survey reported an overall 33.0% of betel quid chewing in mumbai, india. the prevalence of oral cancer was also noted to be high in india16. similarly, in karachi of pakistan, there is a high prevalence of betel quid chewing and also a high prevalence of oral cancer17. the habit of chewing tobacco is increasing because of its free availability, cheaper cost and increasing education about w ell established hazards of smoking. studies have confirmed that use of tobacco leaf along with betel quid is as harmful as smoked tobacco18. gajalakshmi et al. 19 conducted a large case-control study in chennai and reported that tobacco is a major risk factor for mortality. a mixture of betel leaf, areca nut and tobacco chewing addiction is frequent in southern parts of india. smokeless tobacco products including chewing and/or snuffing are believed to face less cancer risk than smokers, but are still at greater risk than people who do not use tobacco products20. in order to elicit the above issues, the present study has been carried out to determine the cytogenetic damage in betel leaf, areca nut and tobacco users in coimbatore city. mn and ca have for many years been applied as biomarkers of genotoxic exposure and early effects of genotoxic carcinogens21-22. the mn test has received increasing attention as a simple and sensitive short-term assay for the detection of environmental genotoxicants23. the evaluation of cytogenetic damage performed in the present study helps understanding the health hazards as well as the cancer risk involved in using them. the present data shows an increased number of ca and mn in the users with snuffing compared to users and controls. chromosomal instability has been described in many human dysplastic lesions and is considered a primary event in neoplastic transformation as well as a marker of progression to cancer24-25. a significant increase in the mortality ratio for all types of cancer in subjects with increased levels of ca in their lymphocytes has been found26-27. the present study confirmed that duration of exposure to smokeless tobacco plays an important role in genetic damage. while our previous study on micronucleus was based on the buccal cells among a mixture of betel leaf, areca nut and tobacco chewing population with smoking gave a significant increase in genetic damage28. the present control group shows a minimum number of mn and ca when compared to the smoke less tobacco exposed groups. the ca in controls might have been due to factors like their age, working environment and lifestyle. several studies have found a significant influence of age on ca frequency, whereas others have found no association at all. recently, age and lifestyle factors have been found to be strongly associated with the frequency of ca measured by the chromosome painting technique29. ca and mn assay is a cost-effective procedure, accurate and easy to carry out for population-based studies. furthermore, in vivo evaluations allow for considering the influence of the individual susceptibility in screened humans. our previous reports have established that buccal cells are useful not only for characterizing the molecular mechanisms underlying tobacco-associated oral cancers, but also as exfoliative cells that express diverse changes that appear promising as candidate biomarkers for the early detection of oral cancer30. in the present study rather than directly assessing the mn present in the buccal cells, by analyzing peripheral blood leucocytes indirectly confirm the genomic instability. in conclusion, betel quid chewing in southern parts of india is the most prevalent among construction and agricultural workers over many years. betel quid usage is strongly associated with tobacco snuffing in most of the construction workers. efforts to reduce habitual betel quid consumption and snuffing might be of benefit in reduction of oral cancer incidence. a strong and intriguing relation between the use of betel quid chewing and tobacco snuffing was found to be a public health hazard. acknowledgement the authors are grateful to the authorities of karpagam university, coimbatore, tamil nadu, south india for the use of facilities and encouragement and we also extend our thanks to all the participants of this study. group i n=80 group ii n=76 control n=80 < 10 years (n=42) > 10 years (n=38) < 10 years (n=38) > 10 years (n=38) 1.55 ± 0.64 2.76 ± 0.90 1.93 ± 0.75 2.97 ± 0.97 0.52 ± 0.26 3.42 ± 1.54 5.01 ± 2.01 4.01 ± 1.67 5.21 ± 2.51 0.73 ± 0.66 1.26 ± 1.10 3.24 ± 1.26 3.18 ± 1.06 4.21 ± 1.57 0.09 ± 0.29 4.68 ± 2.64 8.25 ± 3.27 7.19 ± 2.73 9.92 ± 4.08 0.82 ± 0.95 chromatid type aberration chromosomal aberration total cells with micronuclei (mn)mean ± sdyear of exposuresubjects chromosomal aberration (ca) mean ± sd table 2 chromosomal aberration (ca) and micronucleus (mn) frequency in exposed and control groups mixture of betel leaf, areca nut and tobacco chewing is a risk factor for cytogenetic damage in construction workers from south india 147 braz j oral sci. 8(3):145-148 references 1. r aghavan v, baruah hk . areca nut: india’s popular masticator yhistory,chemistry and utilization. econ botany. 1958; 12:315-45. 2. marshall m. an overview of drugs in oceania. in: lindstrom l, editor. drugs in western pacific societies: relations of substance. lanham, md: university press of america; 1987. p. 13-50. 3. chang mj, ko cy, lin rf hsieh li. biological monitoring of environmental exposure to safrole and the 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tobacco chewing is a risk factor for cytogenetic damage in construction workers from south india148 braz j oral sci. 8(3):145-148 original article braz j oral sci. april/june 2009 volume 8, number 2 shear bond strength of metallic brackets bonded with a new orthodontic composite fábio lourenço romano1, américo bortolazzo correr2, lourenço correr sobrinho3, maria beatriz borges de araújo magnani4, vânia célia vieira de siqueira4 1 dds, ms, phd, graduate student, department of pediatric dentistry, faculdade de odontologia de piracicaba, universidade estadual de campinas (unicamp), campinas (sp), brazil 2 dds, ms, phd, graduate student, department of restorative dentistry, faculdade de odontologia de piracicaba, unicamp, campinas (sp), brazil 3 dds, ms, phd, professor, department of restorative dentistry, faculdade de odontologia de piracicaba, unicamp, campinas (sp), brazil 4 dds, ms, phd, assistant professor, department of pediatric dentistry, faculdade de odontologia de piracicaba, unicamp, campinas (sp), brazil received for publication: march 11, 2009 accepted: june 30, 2009 correspondence to: fábio lourenço romano avenida engenheiro josé herbert faleiros, 600, casa 78 – núcleo são luis cep 14098-780 – ribeirão preto (sp), brasil e-mail: flromano@terra.com.br abstract aim: the aim of this study was to assess the shear bond strength of orthodontic brackets in different enamel surfaces using the transbond plus color change composite (tpcc-3m unitek), and to analyze the adhesive remnant index (ari). methods: seventy-two human premolars were divided into six groups (n = 12), as follows: group 1(control) transbond xt conventional; in groups 2 to 6, tpcc was used under the following enamel treatment conditions: phosphoric acid and xt-primer; transbond plus self-etching primer (tpsep); phosphoric acid only; phosphoric acid, xt-primer and saliva; and tpsep and saliva, respectively. twenty-four hours after bonding, the brackets were debonded with an instron machine at a crosshead speed of 0.5 mm/min, and ari was evaluated by using a stereoscopic magnifying glass. results: the mean shear strength values (mpa) for groups 1 to 6 were 24.6, 18.7, 17.5, 19.7, 17.5 and 14.8, respectively. data were submitted to anova and tukey’s test (α = 0.05). group 1 had significantly higher shear bond strength values than groups 3, 5, and 6 (p < 0.05), but did not differ significantly from groups 2 and 4 (p > 0.05). no statistically significant differences (p > 0.05) were found between groups 2, 3, 4, 5 and 6. conclusions: bracket bonding using tpcc showed adequate adhesion for clinical use, and the type of enamel preparation had no influence. keywords: composite resins, shear strength, orthodontic brackets, orthodontics. introduction composites are the most common materials used for bonding dental accessories to enamel directly because of the adequate adhesive values obtained in laboratory and clinical experiments1-4. in order to bond brackets using composites conventionally, the enamel surface must be properly prepared by prophylaxis and acid etching before application of the bonding agent. all these procedures are time-consuming, increase the clinical chairtime, make it more difficult to keep the operative field dry and increase the risks of bracket debonding due to salivary or moisture contamination5,6. in order to simplify the bonding procedures, new bonding systems combining etchant and primer in one solution have emerged – the self-etching primers (seps). one of these systems is transbond plus self-etching primer (tpsep, 3m unitek, orthodontic products, monrovia, ca, usa), an orthodontic bonding agent tested in several laboratory and clinical 77shear bond strength of metallic brackets bonded with a new orthodontic composite braz j oral sci. 8(2): 76-80 experiments as an enamel-etching agent to be used before bracket bonding procedure, with promising adhesive results2,5,7-16. a new adhesive composite, transbond plus color change (tpcc, 3m unitek), has been recently developed. it is characterized as having an initial pink color, that facilitates the removal of excess material, and becoming transparent after photo-activation. according to the manufacturer, this material releases fluoride and has hydrophilic characteristics that allow its use under conditions of contamination and presence of moisture without decreasing its adhesiveness. enamel surface preparation for use of this material should be carried out with 37% phosphoric acid and bonding agent or tpsep only. the aim of the present study was to assess in vitro the shear bond strength of metallic brackets bonded with tpcc under different enamel conditions, that is, in a conventional way or using tpsep only, no xt primer, and saliva-contaminated enamel surfaces. the adhesive remnant index (ari) was also assessed after bracket debonding. material and methods teeth seventy-two healthy human maxillary and mandibular right and left premolars were used, all presenting intact buccal surface with no restoration, caries, fissure or cracks. teeth that had been submitted to previous application of chemical agents or orthodontic/endodontic treatment were excluded. the teeth were cleaned with periodontal curettes, placed in 0.1% thymol solution for one week and then stored in distilled water at 6oc until its use. the research project was reviewed and approved by the ethics committee of faculdade de odontologia de piracicaba, universidade estadual de campinas, são paulo, brazil (process number 128/2008). specimen preparation the roots were centrally inserted into pvc cylinders (20 mm height x 20 mm internal diameter; tigre, joinville, sc, brazil) containing selfcuring acrylic resin ( jet; clássico artigos odontológicos ltda., são paulo, sp, brazil) in such a way that the buccal surface of each tooth was positioned perpendicularly to the base. resin excesses were removed by using a le cron spatula (duflex, juiz de fora, mg, brazil), so that no resin was left in contact with the crown. in order to assure the correct positioning of the tooth, a glass angle square was placed onto the buccal surface and the cylinder. bonding procedures prior to bracket bonding, the buccal surface of all teeth was cleaned by prophylaxis with slurry of fluoride-free pumice paste (s.s. white, petropolis, rj, brazil) and water in rubber cups at low rotation for ten seconds, flowed by air drying for the same time. the rubber cups were replaced every five uses in order to keep standard procedures. the specimens were randomly assigned to six groups (n = 12). in group 1, the brackets were bonded to enamel surface with transbond xt (control) according to the manufacturer’s recommendations (3m unitek). in groups 2, 3, 4, 5 and 6, the brackets were bonded to enamel surface submitted to different treatments and using tpcc (3m unitek), as described in table 1. the enamel surfaces from groups 1, 2, 4 and 5 were etched with 37% phosphoric acid gel for 15 seconds, washed and air-dried for equal amounts of time. in groups 3 and 6, tpsep was rubbed on enamel for three seconds and gently air-dried. the xt primer used in groups 1, 2 and 5 was applied to the acid-etched enamel with a microbrush and spread over with a gentle air stream. the saliva used in group 5 and 6, collected from a donor one hour before the procedure, was applied onto the enamel surface with a dropper and the excess was removed with air stream, keeping the surface contaminated. brackets seventy-two orthodontic brackets (code 10.30.208, morelli, sorocaba, spbrazil) with base area of 15.78 mm2 were centrally positioned onto and pressed against the buccal surface of the teeth by using a pair of pliers (ortopli corp, philadelphia, pa, united states). composite excesses were removed with a sharp explorer. composite photoactivation a xl 2500 quartz-tungsten-halogen light-curing unit (3m/espe, st paul, mn, usa) was used in all bonding procedures during 40 seconds (10 seconds for mesial, distal, occlusal, and gingival margins) at 500 mw/cm2, as maintaining a distance of 1 mm from the bracket base. light intensity for each photoactivation cycle was measured with curing radiometer (demetron, danbury, ct, usa). shear bond strength testing after a 24-hour storage in distilled water at 37°c to simulate the oral conditions, the brackets were tested in shear strength in an instron testing machine (model 4.11, canton, ma, usa) at crosshead speed of 0.5 mm/min, with its chisel tip placed onto the enamel/composite interface. the results in kgf were converted into n and divided by the bracket area, as providing values in mpa. adhesive remnant index (ari) after bracket debonding procedures, each enamel surface was evaluated with a stereoscopic magnifying glass (carl zeiss, gottingen, germany) at ×8 magnification and characterized according to the adhesive remnant index (ari) scores established by artun and bergland17, as follows: 0: no composite remaining on the tooth; 1: less 78 romano fl, correr ab, correr sobrinho l, magnani mbba, siqueira vcv braz j oral sci. 8(2): 76-80 than half of the composite remaining on the tooth; 2: more than half of the composite remaining on the tooth; 3: all composite remaining on the tooth. statistical analysis enamel surface preparation was the factor taken into account for statistical analysis. the shear strength bond mean values were subjected to one-way analysis of variance and tukey’s test. kruskal0 1 2 3 4 5 6 7 8 9 score 0 score 1 score 2 score 3 group 1 group 3 group 6group 5group 4group 2 figure 1. ari scores. groups enamel surface preparation composite 1 37% phosphoric acid + xt primer* transbond xt 2 37% phosphoric acid + xt primer* transbond plus color change 3 transbond plus self etching primer** transbond plus color change 4 37% phosphoric acid transbond plus color change 5 37% phosphoric acid + xt primer* + human saliva transbond plus color change 6 transbond plus self etching primer** + human saliva transbond plus color change table 1. experimental groups * bonding agent; **self-etching pimer from 3m unitek groups mean (standard deviation) tukey’s test (5%) 1transbond xt (conventional) 24.6 (5.2) a 2transbond plus color change (conventional) 18.7 (5.5) ab 3tpsep + transbond plus color change 17.5 (4.1) b 4transbond plus color change without primer 19.7 (4.7) ab 5transbond plus color change (conventional) + saliva 17.5 (4.0) b 6tpsep + transbond plus color change + saliva 14.8 (5.3) b mean values expressed in mpa. different letters indicate statistically significant difference at 5%. table 2. shear bond strength results groups mean rank mean statistics 1 28.20 1.41 a 2 37.54 1.75 abc 3 46.08 2.16 bc 4 49.91 2.33 c 5 24.79 1.25 a 6 32.45 1.58 ab table 3. adhesive remnant index (ari) scores and statistical comparison different letters indicate statistically significant difference at 5%. wallis and student-newman-keuls tests were used for comparing the ari scores. a significance level of 5% was set for all analyses. results table 2 shows the shear bond strength mean values obtained in the six groups and data statistical analysis. group 1 had shear bond strength values significantly higher than groups 3, 5, and (p < 0.05), but did not differ significantly from groups 2 and 4 (p > 0.05). no statistically significant differences (p > 0.05) were found between groups 2, 3, 4, 5 and 6. the mean ari rank for each group and statistical analysis are presented in table 3. there were statistically significant differences (p = 0.009) between groups. the following pairs of groups differed significantly: groups 1 and 3 (p = 0.036); groups 3 and 5 (p = 0.021); groups 4 and 5 (p = 0.003), and groups 4 and 6 (p = 0.041). most fractures (94.4%) after bracket debonding occurred at the bracket/composite interface, where some amount of remaining composite could be seen on the enamel (figure 1). ari score 1 (less than half of the composite on the tooth) was predominantly seen in groups 1, 5 and 6, whereas ari score 2 (more than half of the composite on the tooth) was more common in groups 2, 3 and 4. ari score 0 (no composite remaining on the tooth) was found only in four specimens (figure 1). discussion transbond xt composite was specifically developed for bonding orthodontic accessories to the enamel. the main advantages offered by this material are: reduced working time, no need of mixing, and good adhesion to enamel1,8, thus being largely used in clinical orthodontics and experimental studies as controls3,6,11. in the present investigation, this composite was used in the control group and yielded a mean shear strength value of 24.6 mpa, which confirms its high adhesiveness to dental enamel8,10,12. transbond plus color change (tpcc), which is characterized by its color change form pink to transparent following photoactivation was the composite evaluated in the present in vitro study. though not being the objective of this study, it was observed that its pink color changed even when exposed to room light during the bonding procedures. this fact makes color change a relative advantage as the orthodontist needs time to handle the material, 79shear bond strength of metallic brackets bonded with a new orthodontic composite braz j oral sci. 8(2): 76-80 place the accessory correctly, and remove excess material, and all these clinical steps are performed under both natural and artificial light. tpcc’s manufacturer provides this information on early color change in lightened environment and such fact was observed in the present study. transbond xt and tpcc composites are very similar, but there are small differences in their formulation and the proportion of their compounds18. while transbond xt has 14% bis-gma, 9% bis-ema and 77% load particles, tpcc has 12, 8 and 80%, respectively. however, these differences in the proportions of their components did not seem to influence the shear strength values, since no statistically significant differences were found between groups 1 and 2, which used the same composite conventionally. the hypothesis that changes in the enamel surface preparation interfere with the shear strength values was also tested in this study. in group 3, tpsep was applied to dry enamel before using tpcc for bonding the brackets. since its introduction in 2000, this self-etching agent has been tested in several bonding experiments, mostly yielding adhesive results similar to those of conventional systems3,6,10,14. in the present study, the combination between tpsep and tpcc for dry enamel resulted in a mean shear strength value of 17.5 mpa. although this value was statistically inferior to that of group 1 (conventional transbond xt), such statistical difference was not observed when the same composite was used conventionally (group 2). this finding indicates that the etching pattern that uses either phosphoric acid or tpsep did not interfere with the shear strength values6,10,14,19. despite the different types of enamel surface preparations, no statistically significant differences were found between the groups (2 to 6) in which tpcc was used. the conventional use of adhesive composites requires well-defined steps in order to assure adequate adhesion to enamel. elimination of one of these steps without compromising the adhesiveness would facilitate the bonding procedure and prevent brackets from debonding. in group 4, the tpcc composite was used with no previous application of xt primer despite the manufacturer’s instructions, yielding a shear strength value of 19.7 mpa. no statistically significant differences were found between groups 4 and 2, which used the same composite conventionally. the other groups did not show statistically significant differences either. the results obtained in the present study corroborate those of other authors20,21, who found no significant differences in shear bond strength values regardless of the use of bonding agent. on the other hand, some authors have reported that the bonding agents penetrate more deeply into the enamel, thus forming deeper and wider resin tags in addition to protecting the conditioned dental surface not occupied by the bracket base20,22,23. saliva contamination decreases the adhesion of composites to enamel when they are applied conventionally5,7,24, resulting, in many cases, in bracket debonding during the orthodontic treatment. in order to reduce the number of cases involving loosen brackets, the manufacturers have developed composite resins, etching agents, and hydrophilic primers that allow adhesion to occur even under conditions of salivary or moisture contamination. in group 5, tpcc, which is a hydrophilic composite, was used conventionally, but the enamel surface was contaminated by human saliva following application of xt primer. the value of 17.5 mpa was statistically inferior to that of group 1 (control), although no significant differences had been found between group 5 and other groups the used tpcc. this similarity between values, including those referring to conventional bonding procedures, is possibly due to the hydrophilic characteristics of tpcc. tpsep is another hydrophilic material that was used in groups 3 and 6 as etching agent; in group 6, however, the brackets were bonded with tpcc to saliva-contaminated enamel. group 6 presented the lowest mean shear bond strength value (14.8 mpa) of all groups, but differed significantly only from the control group. no statistically significant difference was found when group 6 was compared to group 3, in which tpsep was applied to dry enamel. these findings confirm that moisture can reduce the adhesiveness, but an adequate adhesion may be achieved by means of hydrophilic materials. in the present study, all groups showed higher shear strength values than those reported by reynolds25 despite some statistical differences, which indicates that tpcc can be used for bracket bonding under different enamel conditions as tested here. in laboratory experiments involving materials for bonding orthodontic accessories to enamel, both differences and similarities regarding shear bond strength values usually do not correspond to the ari results4,10. this fact was also observed in the present study, since statistically significant differences in shear bond strength (table 2) did not correspond to the ari rank (table 3). it is important to evaluate the ari scores following debonding in order to verify the amount of composite left on enamel surface, that is, the more adhered the material is, the better (ari = 3). however, one can be sure that no enamel fracture has occurred at all. in this study, most fractures occurred at the bracket/composite interface with some material left on enamel (ari scores = 1, 2, and 3), whereas only four specimens had no amount of composite adhered to enamel (ari score = 0). these results are commonly found in studies using composites as bonding material for orthodontic accessories4,14,16. the following conclusions may be drawn: tpcc composite yielded adequate adhesive results for all enamel surface preparations; the type of enamel preparation did not influence the shear strength values obtained with tpcc; when tpcc was used in enamel conditioned with tpsep and/or contaminated by saliva, the adhesive results were inferior to those obtained with transbond xt; finally, in all groups, most fractures involved the bracket/composite interface. references 1. bishara se, vonwald ba, laffoon jf, warren jj. the effect of repeated bonding on the shear bond strength of a composite resin orthodontic adhesive. angle orthod. 2000;70:435-41. 2. cal-neto jp, miguel jam. an in vivo evaluation of bond failure rates with hydrophilic and self-etching primer systems. j clin orthod. 2005;39:701-2. 80 romano fl, correr ab, correr sobrinho l, magnani mbba, siqueira vcv braz j oral sci. 8(2): 76-80 3. pandis n, polychronopoulou a, eliades t. failure rate of self-ligating and edgewise brackets bonded with convenvional acid etching and a self-etching primer. angle orthod. 2006;76:119-22. 4. romano fl, correr-sobrinho l, magnani mbba, nouer df, sinhoretti mac, correr ab. shear bond strength of metallic brackets bonded under various enamel conditions. braz oral res. 2006;20(spec iss):28-33. 5. grandhi k, combe ec, speidel tm. shear bond strength of stainless steel orthodontic brackets with a moisture-insensitive primer. am j orthod dentofacial orthop. 2001;119:251-5. 6. sfondrini mf, cacciafesta v, scribante a, de angelis m, klersy c. effect of blood contamination on shear bond strength of brackets bonded with conventional and self-etching primers. am j orthod dentofacial orthop. 2004;125:357-60. 7. cacciafesta v, sfondrini mf, de angelis m, scribante a, klersy c. effect of water and saliva contamination on shear bond strength of brackets bonded with conventional, hydrophilic, and self-etching primers. am j orthod dentofacial orthop. 2003;123:633-40. 8. romano fl, tavares sw, consani s, magnani mbba, nouer df. shear bond strength of metallic orthodontic brackets bonded to enamel prepared with self-etching primer. angle orthod. 2005;75:849-53. 9. miller ra. laboratory and clinical evaluation of a self-etching primer. j clin orthod. 2001;35:42-5. 10. dorminey jc, dunn wj, taloumis lj. shear bond strength of orthodontics brackets bonded with a modified 1-step etchant and primer technique. am j orthod dentofacial orthop. 2003;124:410-3. 11. grubisa his, heo g, raboud d, glover ke, major pw. an evaluation and comparison of orthodontic bracket bond strengths achieved with self-etching primer. am j orthod dentofacial orthop. 2004;126:213-9. 12. vicente a, bravo la, romero m, ortiz aj, canteras m. shear bond strength of orthodontic brackets bonded with self-etching primers. am j dent. 2005;18:256-60. 13. noble rr, salas-lopez a, english jd, powers jm. clinical evaluation of orthodontic self-etching primers. texas dent j. 2006;123:274-8. 14. turk t, elekdag-turk s, isci d. effects of self-etching primer on shear bond strength of orthodontic brackets at different debond times. angle orthod. 2007;77:108-12. 15. vicente a, bravo la. shear bond strength of precoated and uncoated brackets using a self-etching primer. angle orthod. 2007;77:524-7. 16. uysal t, sisman a. can previously bleached teeth be bonded safely using selfetching primer systems? angle orthod. 2008;78:711-5. 17. artun j, bergland s. clinical trials with crystal growth conditioning as an alternative to acid-etch enamel pretreatment. am j orthod. 1984; 85:333-40. 18. bishara se, olsen m, von wald l. comparisons of shear bond strength of precoated and uncoated brackets. am j orthod dentofacial orthop. 1997;112:617-21. 19. buyukyilmaz t, usumez s, karaman ai. effect of self-etching primers on bond strengthare they reliable? angle orthod. 2003;73:64-70. 20. moin k, dogon il. indirect bonding of orthodontic attachments. am j orthod. 1977;72:261-75. 21. jassem ha, retief dh, jamison hc. tensile and shear strengths of bonded and rebounded orthodontic attachments. am j orthod. 1981;79:661-8. 22. prevost ap, fuller jl, peterson lc. use of an intermediate resin in the acid etched procedure: retentive strength, microleakage and failure mod analysis. j dent res. 1982;61:412-8. 23. menezes lf s, chevitarese o. sealant and resin viscosity and their influence on the formation of resin tags. angle orthod. 1994;64:383-8. 24. kula ks, nash td, purk jh. shear-peel bond strength of orthodontic primer in wet conditions. orthod craniofacial res. 2003;6:96-100. 25. reynolds ir. a review of direct orthodontic bonding. br j orthod. 1975; 2:171-8. oral sciences n3 original article braz j oral sci. april/june 2010 volume 9, number 2 influence of preparation height and luting agent type on crown retention in molars marcelo marchiori1, cezar augusto garbin2, lilian rigo3, daniel becker nunes1, genaro marcial mamani gilapa4 1specialist in dental prosthesis, postgraduate program, dental school, uningá, brazil 2phd, dental school, university of pernambuco; coordinator of the postgraduate program, dental school, uningá, brazil 3phd, dental school, university of pernambuco; professor of scientific methodology, postgraduate program, dental school, uningá, brazil 4phd, professor, electromechanical production engineering course, federal technological university of paraná, brazil correspondence to: lilian rigo av. major joão schell, 1121 vila santa terezinha passo fundo, rs – cep 99020-020 phone: +55 (54) 33131081 e-mail: lilianrigo@via-rs.net received for publication: october 27, 2009 accepted: june 16, 2010 abstract aim: mechanical characteristics of the preparation along with luting agent are significant elements on the maintenance of fixed prostheses. this study aimed at assessing the retention of metal complete crowns luted with two different luting agents under different preparation height. methods: forty human third molars were selected and prepared to receive total crowns, and were randomly divided in 4 groups: (1) 5-mm preparation height (ph) and relyx u100 self-adhesive resin cement (sa); (2) 5-mm ph and zinc phosphate (zp); (3) 3-mm ph and sa; and (4) 3-mm ph and zp. crowns were cast in nickel-chromium alloy. the tensile strength was tested in a universal testing machine. results: mean tensile strength values to crown displacement (kgf) and standard deviation were 39.6 (13.0) for group 1; 16.9 (8.1) for group 2; 32.2 (7.9) for group 3; and 10.6 (3.2) for group 4. overall, the crowns cemented with sa presented significantly higher mean tensile strength values than zp, and the 5-mm ph presented significantly higher mean tensile strength values than 3-mm ph. conclusions: the self-adhesive resin cement and higher preparation height improved crown retention. keywords: crown retention, luting agents, preparation design. introduction the stabilizing properties of abutments used in cemented complete coverage restorations have been ascribed to retention and resistance form1-3. geometric configuration of the prepared tooth is essential to promote retention between axial walls of preparation and the prosthetic restoration4. the retention shape obtained by tooth preparation must avoid dislocation of the restoration along its insertion axis, acting against tensile forces5. reduction of convergence angle, higher axial surface and minimal occlusal reduction contribute to increase the retentive capability of the prepared tooth6-8. several types of luting agents are available and the choice for a material depends of various factors. the retention of most luting agents is based on mechanical imbrications in the irregularities of tooth-restoration interface9-10. zinc phosphate cement is still the most used for crown luting due to its low cost, easy of handling and good mechanical properties11-13. however, it has some negative properties namely solubility in oral environment and absence of adhesive bonding14. the resin cements has significant role in increasing crown retention by promoting an adhesive bonding between tooth and restoration, which has increased the use of these luting agents15. nevertheless, the use of these materials requires braz j oral sci. 9(2):89-93 several steps, mainly for treatment of dental substrate, which make them technically sensitive16. self-adhesive resin cements have been recently introduced on the market. the goal of these luting agents is to combine the easiness of use offered by zinc phosphate cements (they do not demand pretreatment steps) with the favorable mechanical properties, esthetics and adequate adhesion to dental structure of conventional resin cements17,18. the aim of this study was evaluate the influence of preparation height (3or 5-mm) and type of luting agent (zinc phosphate or self-adhesive resin cement) on the retention of metal complete crowns on molars. material and methods the study was approved by the research ethics committee of uningá (protocol 0089/08). forty sound human third molars with similar shapes and dimensions, which had been freshly extracted due to orthodontic or periodontal reasons, were used in this study. the selected teeth were scaled, cleaned for debris and stored in saline, which was constantly renewed. specimen preparation the roots were centrally inserted into pvc cylinders (25-mm height x 20-mm internal diameter; tigre, joinville, sc, brazil) containing self-curing acrylic resin (jet; clássico artigos odontológicos ltda., são paulo, sp, brazil). the acrylic resin was maintained 2 mm below to the cementoenamel junction12,19. one horizontal perforation was made in the root equidistant from the cementoenamel junction and tooth apex, enabling the passing of a rectangular orthodontic wire (dental morelli ltda., sorocaba, sp, brazil) to endure that teeth were not removed from the acrylic resin during the tensile test. preparation was standardized by using a mechanical lathe, according to previous studies20-21. tooth reduction was initiated by positioning the cutting instrument at the cementoenamel junction and then moving it along the axial wall in a cervical-occlusal direction, under constant cooling. following axial reduction, the external portion of the root was reduced to standardize the cervical area (figure 1). tapered-shaped preparations had the following characteristics: 6.5-mm upper diameter, 16 degree of convergence and the cervical finish line was in straight-shoulder shape. finishing was made at 45 degree angle on the edge formed by the axial and occlusal walls, where an approximately 1-mm-wide groove was made all around the preparation using a cylindrical diamond bur (#1090; kg sorensen, barueri, sp, brazil) in a handpiece under water cooling.. the diamond bur was used in almost the whole area of preparation, simulating the clinical condition. the teeth were randomly divided into four groups (n = 10), according to the preparation height and luting agent: group 1 – tooth prepared with 5 mm of height and crowns fixed using self-adhesive resin cement (relyx™ u100, lot #318258; 3m espe, seefeld, germany), according manufacturer’s directions; group 2 – tooth prepared with 5 fig. 1 preparation on mechanical lathe. mm of height and crowns fixed using zinc phosphate cement (lot #06809007 powder and lot #0040807 liquid, ss white, são paulo, sp, brazil), according manufacturer’s directions; group 3 – tooth prepared with 3 mm of height and crowns fixed using self-adhesive resin cement as group 1; and group 4 – tooth prepared with 3 mm of height and crowns fixed using zinc phosphate cement as group 2. the preparation heights are presented in figure 2. standardization of height was done using a cylindrical diamond bur (#1090; kg sorensen) in a handpiece under water cooling. the last cut was made in a groove shape, with 0.5 mm depth in the proximal walls along the tooth long axis to guide the insertion pathway of the cast crown. areas of pulp chamber exposure were closed with a light-cured resin (z350; 3m espe, st. paul, mn, usa). specimens were stored in constantly renewed saline until the luting procedure. crown cast crowns were waxed directly on the prepared teeth6. a handle was done on each crown for future joining to the testing machine, using wax wires (bego, bremer, germany) with 1.5 mm of diameter. the wax crowns were invested with proportion of 100 g for 22.5 ml of liquid (begosol; bego) and 2.5 ml of water, according to the manufacturer’s directions. the crowns were cast in a nicr alloy (wironia; 90 braz j oral sci. 9(2):89-93 influence of preparation height and luting agent type on crown retention in molars fig. 2 – preparation heights: 3 mm (left) and 5 mm (right). bego). after divesting, fit was checked, the internal surfaces of the cast crowns were airborne-particle abraded with 110ìm aluminum oxide (korox 110; bego) under 2 bar of pressure, and cast crowns prepared for luting as presented in figure 3. crown luting the preparation was cleaned using rotary brush and pumice in low-speed hand piece, washed and then dried using sterile cotton10,22. metallic crown were sandblasted (microjet; bio art equipamentos odontologicos ltda, sao carlos, sp, brazil) with a 50-µm particle stream, cleaned with alcohol and water, and dried with an air stream free of water or oil. luting procedure was performed according to each manufacturer’s instructions. the crowns were finger pressed after insertion on the preparation, and were then subjecte dto constant axial pressure of 5 kg for 10 min using a custom-made press that allowed cement flow and correct crown fit on the preparation12,19,23 (figure 4). excess material was removed with a sharp explorer. fig. 3 cast crowns prepared for luting. fig. 4 standardization of luting load. excesses of relyx u100 were first light-cured using led curing unit (radii cal; sdi, australia; 1.200 mw/cm2) for 2 s to facilitate the removal. the margins of the preparation were then light-cured for 30 s at each face. specimens were stored in distilled water at room temperature for 10 days before of the tensile strength test. tensile strength test crown retention was measured by applying a tensile force to the loop attached to the cast crown in a universal testing machine (emic dl 20000, são josé dos pinhais, pr, brazil) at a cross-head speed of 0.5 mm/min13,24. each specimen was positioned on the lower part of the machine. the upper part of the testing machine had a 2.5-mm diameter s-shaped steel hook to which the handles of crowns were connected (figure 5). tensile force values required to separate the crowns (kgf) was recorded by machine’s software. lavenes’s test was applied to verify the homogeneity of variances, as well as the kolmogorov-smirnov’s test to verify data distribution. as normal distribution was observed, statistical analysis was performed by two-way anova and tukey’s post-hoc test at 5% significance level. visual examination of the debonded surfaces of the teeth and crowns was performed. fig. 5 – specimen positioned in the testing machine. results the results are presented in the table 1. both factors, height preparation and luting cement had significant influence on crown retention. crowns cemented with relyx u100 (groups 1 and 3) presented significantly higher mean tensile strength values (p<0.0001) than crowns cemented with zinc phosphate cement (groups 2 and 4). the 5-mm preparation height promoted significantly higher mean tensile strength values than 3-mm preparation height (p=0.018). there was no significant interaction between the factors (p=0.847). 91 braz j oral sci. 9(2):89-93 influence of preparation height and luting agent type on crown retention in molars luting agent preparation height 5 mm 3 mm self-adhesive resin cement 39.6 (13.0) aa 32.2 (7.9) ba zinc phosphate cement 16.9 (8.1) ab 10.6 (3.2) bb table 1. means (standard deviations) tensile strength values (kgf) for the different experimental conditions. means followed by different uppercase letters in the same line and different lowercase letters in the same column are significantly different (p<0.05). debonded adhesive failure types were predominantly found. for groups 1 and 3, the failures were adhesive between cement and preparation on the axial surfaces; and between cement and crown on the occlusal surface. for groups 2 and 4, on the other hand, the failures were adhesive between cement and crown on the axial surfaces; and between cement and preparation on the occlusal surface. discussion retention is considered an important requirement in the fixation of prosthetic crowns, and its achievement is dependent on some factors, namely favorable relation between surface geometry of the prepared tooth and the definitive restoration4,9,25-28. clinically, a crown would hardly undergo such great tensile efforts as those applied in this study, but the tested experimental conditions serve as parameters to evaluate different properties and behaviors of the materials used. according to the obtained results, minimum occlusal reduction during preparation contributes significantly to increase the retention6-8, regardless of the type of luting agent used. the results of the present study may be explained by the fact that higher preparation height promotes greater superficial area with the crown5,26,28-29. although other factors may influence on crown retention, the preparations were standardized (cervical diameter, taper, roughness, piece fit), thus eliminating or minimizing the interference of these variables on the results. rubo et al. 5 concluded that preparation 2 mm higher contributes significantly for a better retention of crowns. other studies have shown greater crown retention for preparation with greater height24,29-32. concerning of the luting agents, the results showed greater retention (table 1) for the relyx™ u100 selfadhesive resin cement when compared to the zinc phosphate cement, probably due to the adhesive capacity of the resin cement to dentin by hybrid layer formation, improving the retention compared to conventional cements13,17-18. moreover, better mechanical properties of resin cement in relation to zinc phosphate cement also influence their tensile, compressive and shearing strengths. the lower tensile strength of the zinc phosphate cement may be related to its ceramic composition, which makes this material friable and less resistant to tensile forces33. zinc phosphate cement does not have chemical adhesion to any dental substrate, acting only as luting agent by mechanical or frictional retention. thus the height, taper and area of the preparation important aspects for its success as a luting material 32,34. therefore, in situations where preparation retention is deficient, for example, a short clinical crown and accentuated taper of the preparation, the choice for a luting agent lies on resin cement, leading to a more favorable clinical prognosis15. however, the difference in tensile strength between the luting agents found in this study does not contraindicates use of the zinc phosphate cement because the retention values obtained were higher that the forces expected clinically, which is around of 4 kgf23. tjan and li35 compared the retention of cast crowns luted with adhesive resin cement (panavia ex®) and zinc phosphate cement (flecks®), concluding that the resin cement panavia ex® provided almost twice the retention values given by zinc phosphate. browning et al.12 compared the retentive capacity of three cements (resin, glass ionomer and zinc phosphate) on crown preparations that presented adverse conditions for retention, and found that the resin cement showed significantly higher retention values than the conventional cements. the findings of other studies13,36 are in agreement with ours. piwowarczyk et al.16 compared the shear bond strength of luting agents used in fixed prostheses, stating that the self-adhesive resin cement relyx® unicem did not differ significantly from other conventional resin cements. however, it presented significantly higher bond strength than glass ionomer, zinc phosphate and resin-modified glass ionomer cements. martins pinto31 verified the tensile strength of metal crowns luted with relyx™ unicem, relyx™ arc and hy bond® (zinc phosphate). the author observed higher retention for self-adhesive resin cement relyx™ unicem than for the other cements. under the tested conditions, it may be concluded that self-adhesive resin cements should be preferred for luting of metal crowns and minimum occlusal reduction should be done on preparation. further studies using other variables such as thermal cycling, cyclic loading and long-term storage should be performed to confirm the results and hypotheses addressed in the present study. references 1. shillingburg ht, hobo s, whitsett ld, jacobi r, brackett se. fundamentals of fixed prosthodontics. 3rd ed. chicago: quintessence; 1997. p.139-42, 151-2. 2. potts rg, shillingburg ht jr, duncanson mg jr. retention and 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oral sci. 15(1):75-78 original article braz j oral sci. january | march 2016 volume 15, number 1 erosive potential of sugar-free hard candies dissolved in water and artificial saliva maria mercês aquino gouveia farias1, magatha marquetti lazzaris de oliveira1, beatriz helena eger schmitt1, eliane garcia da silveira1, silvana marchiori de araújo1 1universidade do vale do itajaí, school of dentistry, area of pediatric dentistry, itajaí, sc, brazil correspondence to: maria mercês aquino gouveia farias rua bartolomeu de gusmão 209, carianos cep: 88047-520 florianópolis, sc e-mail: mercesfarias@gmail.com abstract aim: to compare the acidity of sugar-free hard candies dissolved in water and artificial saliva. methods: sugar-free flopi florestal hard candies (grape, strawberry, cherry, orange, ginger, lemon balm, fennel) were selected and grouped in 2 groups: g-1 (candies dissolved in distilled water) and g-2 (candies dissolved in artificial saliva). candies were triturated with a porcelain pestle, yielding two samples of 20 g. samples were dissolved in 120 ml distilled water (g-1) and 120 ml artificial saliva (20 mm nahco3, 3 mm nah2po4.h2o and 1 mm cacl2.2h2o) (g-2), obtaining three samples of 30 ml for each of the flavors and groups. ph was measured using potentiometer and combined glass electrode. titratable acidity was evaluated by adding 100 μl 1m naoh aliquots until reaching ph 5.5. for statistical analysis, analysis of variance (anova) was used. means were compared by the tukey test at 5% significance level (p<0.05) results: all flavors of g-1 showed ph values below 5.5. comparison of groups in the same flavor showed a significant increase in ph in flavors of g-2. comparison of the titratable acidity between g-1 and g-2, showed that fruit flavors were significantly different from each other, with reduced acidity in g-2. conclusions: all evaluated candies are acid, and dilution in artificial saliva raised their ph and lowered their titratable acidity, reducing their erosive potential. keywords: tooth erosion. candies. saliva, artificial. hydrogen-ion concentration. acidity. introduction dental erosion is a multifactorial disorder in which factors such as structural characteristics of dental tissues, physiological properties of saliva, acidic sources from intrinsic and extrinsic origin, as well as behavioral, educational, socio-economic factors should be considered1,2. its prevalence is increasing and its clinical management requires diagnosis and assessment of risk factors in order to understand the various factors associated with its etiology3. the most important exposure to extrinsic acids comes from diet1. some physicochemical characteristics influence the erosivity of food like the type of acid, ph, titratable acidity, chelating potential, calcium and phosphate concentration, temperature and adhesion4. especially, ph, titratable acidity and calcium content are the most important features for determining the erosive potential of a product4. in addition, several biological factors related to salivary protection mechanisms have an important role in the erosive wear, especially dilution of the erosive agent, salivary cleaning, film formation and buffer capacity, which are able to modulate the erosive wear2. recently, along with the economic development in many countries, there has been a wide range of industrial acidic products accompanied by intense advertisement, increasing the exposure of children and adolescents to these products at increasingly http://dx.doi.org/10.20396/bjos.v15i1.8647129 received for publication: august 10, 2016 accepted: august 25, 2016 76 early ages5. acidic candies are among these products and the literature about their erosivity is relatively scarce, compared to studies addressing the erosive potential of industrialized beverages6. recent studies have shown the consumption of acidic sweets as a potential risk factor for dental erosion7,8. sugar-free products are accepted as safe for teeth for being noncariogenic. however, the presence of acidic components (citric, phosphoric, ascorbic, malic, tartaric, oxalic, carbonic and fumaric acids) may turn them an acid source, which is a risk factor for dental erosion, making them able to cause damage to the tooth structure9,10. a previous study compared the erosive potential of original-flavor candies and sour candies dissolved in artificial saliva and water. the authors observed that saliva was able to reduce the erosive potential of both flavors, but reduction was more significant for original-flavor candies than on sour candies11. seeking to contribute to the research of the erosive potential of candies available in brazil, the aim of this study was to measure and compare the erosive potential of sugar-free hard candies dissolved in water and artificial saliva. material and methods this was a two-factor experiment. factor a: 2 dilution media: group 1 (g1) distilled water and group 2 (g2), artificial saliva. factor b: seven flavors of sugar-free hard candies flopi florestal® (florestal alimentos s.a.; lajeado, rs, brazil) available at supermarkets, all in a single package with opening and closing system as a common feature (chart 1). erosive potential of sugar-free hard candies dissolved in water and artificial saliva braz j oral sci. 15(1):75-78 the ph of all solutions was measured (g-1 and g-2) at room temperature and constant stirring (magnetic stirrer; fisaton equipamentos científicos, são paulo, sp, brazil), using potentiometer and combined glass electrode (tec-2; tecnal equipamentos para laboratórios, piracicaba, sp, brazil) previously calibrated with ph 7.0 and ph 4.0 standard solutions, before each reading. only in solutions with ph values below 5.5 (g-1 and g-2), titratable acidity (buffer capacity) was measured by adding aliquots of 100 μl of 1 m naoh under constant stirring (fisaton) to reach ph 5.5. the results were statistically analyzed by twoway anova, followed by tukey’s multiple-comparison test at p<0.05. results all candies dissolved in water showed ph values below 5.5. when dissolved in artificial saliva only, fruit flavors maintained their acidity. the anova f-test was significant (p<0.05) in the comparison among the seven flavors and between groups (g1 and g2) (table 1). comparing groups for titratable acidity, fruit flavors had significantly lower values when dissolved in artificial saliva (p<0.05) (table 2). the flavors ginger, lemon and fennel showed low titratable acidity when dissolved in water. flavors g1water g2 -artificial saliva grape 2.55 ± 0.02 d b 4.42 ± 0.01 f a strawberry 2.82 ± 0.06 c b 4.80 ± 0.01 c a cherry 2.86 ± 0.06 c b 4.54 ± 0.02 e a orange 3.05 ± 0.03 b b 4.67 ± 0.01 d a ginger 4.33 ± 0.06 a b 7.44 ± 0.02 ab a lemon balm 4.40 ± 0.05 a b 7.37 ± 0.02 b a fennel 4.44 ± 0.03 a b 7.50 ± 0.05 a a table 1 mean ± standard deviation values of ph of the different flavors of candies, after dilution in water (g1) or artificial saliva (g2). means followed by the same capital letter in columns and small letter in rows do not differ significantly from each other by the tukey’s test (p<0.05). flavors composition sugar-free flopi (florestal®) flavors: cherry, orange, grape, strawberry, lemon balm, fennel vitamin c, sweetener sorbitol, arabic gum thickener, citric acid, flavor, artificial color. chart 1 selected flavors and product composition according to the manufacturer. flavors g1water g2 -artificial saliva grape 733.3 ± 57.74 a a 266.7 ± 57.74 b b strawberry 566.7 ± 57.74 b a 200.0 ± 0.0 b b cherry 566.7 ± 57.74 b a 166.7 ± 57.74 b b orange 600.0 ± 0.0 b a 200.0 ± 0.0 b b ginger 100 ± 0.0 0 lemon balm 100 ± 0.0 0 fennel 100 ± 0.0 0 table 2 mean ± standard deviation values of titratable acidity* of the different flavors of candies, after dilution in water (g1) or artificial saliva (g2). means followed by the same capital letter in columns and small letter in rows do not differ significantly from each other by the tukey test (p <0.05). *volume (µl) of 1m naoh required to reach ph 5.5. analysis of ph and titratable acidity two packs of 40 g of sugar-free flopi florestal® candies were employed (cherry, strawberry, orange, grape, lemon, fennel and ginger). candies from each package were ground by a 305 ml porcelain pestle (nalgon equipamentos científicos; itupeva, sp, brazil). from the resulting powder, two samples of 20 g were weighed for this purpose, on an analytical and precision electronic scale (ae200s mettler-toledo ind. e com. ltda., barueri, sp, brazil). the powder samples were dissolved in 120 ml of distilled water (ph=6.2) (g-1) and in 120 ml artificial saliva (20 mm nahco3, 3 mm nah2po4.h2o and 1 mm cacl2.2h2o) (ph=7.39) (g-2) with a glass rod until reaching a homogeneous solution. from these solutions were obtained three samples of 30 ml for each flavor in each group. this process allowed for reading ph and titratable acidity in triplicate11. 77erosive potential of sugar-free hard candies dissolved in water and artificial saliva braz j oral sci. 15(1):75-78 discussion in recent years, the production and supply of new types of candies/sweets increased significantly. the relationship between the consumption of candies/sweets and tooth decay is widely reported in literature5. however, in addition to their cariogenic potential, many candies contain organic acids (citric, malic, fumaric, ascorbic, acetic, lactic, tartaric acids) in their composition, isolated or combined, in different concentrations5,6. the excessive consumption of these products was pointed out as a potential risk factor for dental erosion7,8. the analyzed products had the same components, but their concentrations were not described by manufacturers. probably the variation in concentration of components is responsible for variations in ph and titratable acidity, as found in this study. there are various artificial saliva formulations. the formulation used in this study was the same used in a previous study that analyzed the erosive potential of candies11. in this study, candies diluted in water had ph values below 5.5, corroborating several studies11-16. this ph value refers to the critical value for dissolution of hydroxyapatite used as a reference for determining the erosive potential of these products3. the ph values increased upon dissolution in artificial saliva, reaching neutrality in non-fruit flavors, but remaining acid in fruit flavors. this ph rise after dilution in saliva was also found in a previous study11. this may be due to the dilution and buffering effect of saliva. the chemical erosion of hard dental tissues occurs by the action of the hydrogen ion, h+ and/or other anions that act as acid-derived chelating substances. the h+ ion dissociates in water attacking the crystal surface, dissolving it by combining with carbonate or phosphate ions, which are components of the mineral structure of dental tissues, causing the demineralization of dental tissues17. in all analyzed products in this study, ascorbic and citric acids were present in their composition, according to the manufacturer. but observing the changes in ph and titratable acidity, it suggests that there are variations in their concentrations among the different flavors. citric acid has a more complex interaction with the tooth structure. in an aqueous solution, it is a mixture of h+ ions, acid anions (citrate) and non-dissociated acid molecules. the amounts of each component vary depending on ph and the acid dissociation constant17. the capacity of citrate to link with complex calcium ions, removes the crystal surface and/or saliva, and so increases the erosive wear17. in addition to ph, titratable acidity also strongly influences the erosive potential of a product18. analyzing the titratable acidity, it was observed that only fruit candies in both groups showed significant acidity. it was also demonstrated that after dissolution in artificial saliva, fruit flavors reduced acidity significantly. this reduction was also observed in a previous study with single-flavor11 candies. a recent study demonstrated that the titratable acidity of a beverage influences more the salivary ph after consumption of an acidic beverage than the ph of the beverage19. the literature suggests that during consumption of acid candies, there is a drop in ph and increased salivary flow. only after consumption, with times ranging from 2 to 5 min, salivary ph returns to neutral14,20,21. studies on the consumption of hard candies, as analyzed in the present study, have shown that the average consumption time may reach 15 min14, a period in which there is change in salivary ph. thus, the frequent consumption of acidic hard candies may contribute to the development of dental erosion, as they are slowly dissolved by saliva in the oral cavity20,21. thus, while saliva plays a protective role, its effect is partial11,21. whereas the normal salivary flow of stimulated saliva ranges from 1 to 3ml/min, in low flow from 0.7 to 1.0 ml/min and under hypo salivation conditions, it may reach values below 0.7ml/ min22 the salivary protection undergoes significant variations among individuals. this is especially important in patients with compromised protective saliva capacity due to the presence of diseases and use of drugs that affect the salivary flow. in these patients, even products with low erosivity may represent a risk to dental erosion. in this research was used a ratio of 1 g of candy powder to 6 ml of artificial saliva, which would amount to a condition of salivary flow of 2-6 min (3 ml-1) for each gram of candy powder. the formulation of the artificial saliva of this study contains phosphate and bicarbonate buffer, as in natural saliva. the concentration of these compounds in natural saliva presents variations both among individuals and in stimulated and nonstimulated salivation. in stimulated salivation, the phosphate system has low concentration, but in non-stimulated saliva, it can reach peaks of 10 mm, showing its maximum buffer capacity in ph range of 6.8-7.2. the concentration of bicarbonate ions ranges from less than 1 mm in non-stimulated saliva to 60 mm in high flow rates22. in view of the above, the complexity of the salivary dynamics cannot be fully described by in vitro studies, which is a limitation of this study. in addition, the erosive acid products comprise the interaction among physical, chemical and biological and behavioral factors2. thus, it may be inferred that the analyzed candies have erosive potential, but their erosivity must be investigated by in situ and in vivo studies that include all the dynamics of the oral environment and quantify the mineral loss on exposure to potentially erosive agents. according to the present results, all the evaluated candies are acid and fruit flavors presented the lowest ph values. dilution in artificial saliva raised their ph and lowered their titratable acidity, reducing their erosive potential. aiming to prevent and control dental erosion, the most significant strategy against extrinsic factors is the reduction or elimination of the erosive agent. in the case of candies, one should avoid their daily consumption and for extended periods. additional measures, such as chewing sugar-free candies, rinsing teeth with water after candy consumption and using fluoridated toothpaste are also recommended6. acknowledgements to the scientific initiation program probic/ ice-dean of research, graduate program, extension and culture at the 78 braz j oral sci. 15(1):75-78 university of vale do itajaí univali, which funded the research. references 1. lussi a, schlueter n, rakhmatullina e, ganss c. dental erosion-an overview with emphasis on chemical and hispopatholigicol aspects. caries res. 2011;45 suppl 1:2-12. doi: 10.1159/000325915. 2. magalhães ac, rios d, honorio hm, buzalaf mar. insights into preventive measures for dental erosion. j appl oral sci. 2009 marapr;17(2):75-86. 3. west nx, joiner a. enamel mineral loss. j dent. 2014 jun;42 suppl 1:s2-11. doi: 10.1016/s0300-5712(14)50002-4. 4. furtado jr, freire vc, messias dcf, turssi cp. [physicolchemical aspects related to the erosive potential of acid beverages]. rfo upf. 2010;15(3): 325-30. portuguese. 5. gambon dl, brand hs, veerman eci, dental erosion in the 21st century: what is happening to nutritional habits and lifestyle in our society? br dent j. 2012 jul 27;213(2):55-7. doi: 10.1038/ sj.bdj.2012.613. 6. feltham eb. the power of sour candies: a dental hygienist’s battle against dietary dental erosion. cdha j. 2010;25(1):16-8. 7. correa msnp, corrêa fnp, correa jpnp, murakami c, mendes fm. prevalence and associated factors of dental erosion in children and adolescents of a private dental practice. 2011 nov;21(6):451-8. doi: 10.1111/j.1365-263x.2011.01150.x. 8. sovik jb, skudutyte-rysstad r, tveit a b, sandvik l, mulic a. sour sweets and acidic beverage consumption are risk indicators for dental erosion. 2015;49(3):243-50. doi: 10.1159/000371896. 9. moynihan pj. the role of diet and nutrition in the etiology and prevention of oral diseases. bull world health organ. 2005 sep;83(9):694-9. 10. nadimi h, wesamaa h, janket sj, bollu p, meurman jh. are sugarfree confections really beneficial for dental health? br dent j. 2011 oct 7;211(7):e15. doi: 10.1038/sj.bdj.2011.823. 11. wagoner sn, marshall ta, quian f, wefel js. in vitro enamel erosion associated with commercially available original and sour candies. j am dent assoc. 2009 jul;140(7):906-13. 12. davies r, hunter l, loyn t, rees j. sour sweets: a new type of erosive challenge? br dent j. 2008 jan 26;204(2):e3; discussion 84-5. 13. brand hs, gambon dl, paap a, bulthuis ms, veerman ec, amerongen av. the erosive potential of lollipops. int dent j. 2009 dec;59(6):358-62. 14. brand hs, gambon dl, van dop lf, van liere le, veerman ec. the erosive potential of jawbreakers. int j dent hyg. 2010 nov;8(4):308-12. doi: 10.1111/j.1601-5037.2010.00450.x. 15. silva jg, farias mmag, silveira eg, araújo sm, schmitt bhe. evaluation of the erosive potential of acidic candies consumed by children and teenagers. j pharm nutr sci. 2013;3(4):262-5. 16. lazzaris m, farias mmag, araújo sm, schmitt beh, silveira eg. erosive potential of commercially available candies. braz res pediatr dent integr clin. 2015;15(1):7-12. doi: 0.4034/pboci.2015.151.01. 17. featherstone jdb, lussi a. understanding the chemistry of dental erosion. monogr oral sci. 2006;20:66-76. 18. lussi a, jaeggi t. chemical factors. monogr oral sci. 2006;20:77-87. 19. tenuta lma, fernández ce, brandão acs, cury ja. titratable acidity of beverages influences salivary ph recovery. braz oral res. 2015;29. pii: s1806-83242015000100234. doi: 10.1590/1807-3107bor-2015. vol29.0032. 20. gambon dl, brand hs, amerongen avn. [acidic candies affect saliva secretion rates and oral fluid acidity]. ned tijdschr tandheelkd 2007 aug;114(8): 330-4. dutch. 21. jensdottir t, nauntofte b, buchwald c, bardow a. effects of sucking acidic candy on whole mouth saliva composition. caries res 2005 nov-dec;39(6):468-74. 22. tenevuo j, lagerloj f. [saliva]. in: thylstrup a, fejerskov o. [clinical cariology]. são paulo: santos; 1995. p.17-43. portuguese. erosive potential of sugar-free hard candies dissolved in water and artificial saliva braz j oral sci. 15(3):201-204 combining the effects of undersized drilling and bone density on implant insertion torque bernardo alievi camargo, dds, ms1; daniel becker nunes, dds2; aloísio oro spazzin, dds, ms, phd3; leonardo federizzi, dds, ms4; chirstian schuh, dds, ms5; érica alves gomes dds, ms, phd6 1faculdade meridional imed, school of dentistry, department of prosthodontics and oral implantology, passo fundo, rs, brazil 2faculdade meridional imed, school of dentistry, department of prosthodontics and oral implantology, passo fundo, rs, brazil 3faculdade meridional imed, school of dentistry, department of prosthodontics and oral implantology, passo fundo, rs, brazil 4universidade de ribeirão preto unaerp, school of dentistry, department of prosthodontics, ribeirão preto, sp, brazil 5faculdade meridional imed, school of dentistry, department of prosthodontics and oral implantology, passo fundo, rs, brazil 6universidade de ribeirão preto unaerp, school of dentistry, department of prosthodontics, ribeirão preto, sp, brazil correspondence to: prof. dr. érica alves gomes department of prosthodontics, school of dentistry university of ribeirão preto, unaerp av. costábile romano, 2.201, cep 14096-900, ribeirão preto, sp, brazil tel.: + 55 (16) 3603-7000 e-mail: ericaagomes@yahoo.com.br abstract aim: this study evaluated the influence of surgical undersized drilling on insertion torque of an implant system at low bone density. methods: implant site preparations were made in two polyurethane foam blocks with different densities, where two preparation techniques were considered: control group (c): conventional drilling, following the manufacturer’s instructions; and group (e): undersized drilling, experimental technique using a final surgical drill with reduced diameter. the artificial bone blocks were selected based on density: d1 (0.64 g/cm 3) and d2 (0.32 g/cm 3). three groups were considered according to the preparation technique and bone density used (n=10): cd1 – conventional drilling technique in artificial bone with higher density; cd2 – conventional drilling technique in artificial bone with lower density; and ed2 – undersized drilling in artificial bone with lower density. externalhexagon implants (11 × 4 mm) were inserted and the insertion torque values were measured using a digital torque-meter. data were submitted to one-way anova followed by tukey’s test (α=0.05). results: the results showed significant statistical differences between groups (p<0.001), where the highest insertion torque was found for the group cd1 (48.9 ncm) followed by group ed2 (22.6 ncm) and group cd2 (11.7 ncm). conclusions: the undersized drilling technique for implant site preparation leads to increased insertion torque on low bone density. keywords: dental implantation. torque. bone density. drilling techniques. introduction the insertion torque obtained during the surgical moment of implant placement is essential to improve the primary stability, and consequently, to generate a successful osseointegration, especially during the immediate loading procedure1,2. primary stability is defined as the absence of implant movement achieved after implant placement, and it depends of the implant design, bone density and surgical preparation technique3,4. additionally, a decreased insertion torque presents a higher risk of early implant failure considering an immediate loading, whereas high stability allows smaller micro-motions of the implant inside the bone providing good conditions for osseointegration5. the bone density is one of the most important factors related to a higher insertion torque. higher bone density creates greater bone-implant contact and consequently, it received for publication: december 19, 2016 accepted: june 12, 2017 original article braz j oral sci. july | september 2016 volume 15, number 3 http://dx.doi.org/10.20396/bjos.v15i3.8649981 202 increases the primary stability. on the other hand, a low bone density is associated to a higher implant failure rate6,7. clinical studies shows that implants placed in the jaw present higher survival rates compared to the ones placed on the maxilla8,9, especially when considered the maxillary posterior region witch usually presents a thinner cortical bone combined with thicker trabecular bone10. clinically, the quality and quantity of bone are local factors that cannot be controlled; whereas, surgical technique and implant design may be changed to adapt specific bone situations and improve insertion torque1,11. several strategies have been proposed to improve implant insertion torque in low-density bone. therefore, the undersized drilling technique recommends a site preparation smaller than the diameter of the implant, providing a press-fit situation in which strain propagates into the supporting bone. this so-called undersized drilling technique was introduced with the aim to locally optimize bone density and consequently improve the insertion torque and the primary stability of the implant2,12-14. this possibility becomes interesting mainly in situations of low bone density, where the direct contact with the implant surface will be smaller. however, literature suggests that the use of undersized drilling technique may not fully compensate the effect of low bone density, once the factors related to implant diameter/length and bone density may also affect the level of implant insertion torque6. therefore, the purpose of this study was to evaluate the influence of surgical undersized drilling on the insertion torque of an externalhexagon dental implant system at a lower bone density. the null hypothesis was that there would be no significant differences in the insertion torque regardless of the preparation technique used. material and methods three groups (n=10) were considered according to the preparation technique (conventional drilling x undersized drilling) and two different bone densities (high density x low density) used, as described on table 1. table 1 the combination of the drilling technique and bone density group bone density (g/cm3) drilling technique cd1 0.64 #2.0 mm, #2/3 pilot, #3.0 mm, #3.3 mm cd2 0.32 #2.0 mm, #2/3 pilot, #3.0 mm, #3.3 mm ed2 0.32 #2.0 mm, #2/3 pilot, #3.0 mm all implant preparations and insertions were made using a surgical hand-piece (sg20; nsk, kanumashi, japan) coupled to a surgical motor unit (neosurg xt plus; nsk) under plentiful and constant saline irrigation and at a constant speed of 800 rpm. a single calibrated and trained operator performed all the implant site preparations and implant insertions. it highlights that the artificial bone blocks were attached to a support jig to avoid movement during all procedures. implant site preparations were made in two rigid polyurethane foam blocks (nacional ossos, jaú, sp, brazil), with dimensions of 18 × 13 × 4 cm; which were produced in conformity with astm f1839-97. the artificial bone blocks used in the present study were selected based on different densities, as following: d1 (0.64 g/cm3) and d2 (0.32 g/cm 3). a total of thirty implant site preparations were performed using surgical drills (neodent, curitiba, pr, brazil), where two preparation techniques were evaluated: one following the fabricant instructions (conventional drilling/control groups, c); and the other, using an experimental technique, with a reduced final surgical drill size (undersized drilling, e). first, the pilot holes were performed using a lance drill (2.0 mm of diameter, neodent, curitiba, pr, brazil) in 11 mm of depth for all groups; and then, a final #4.1 mm countersink drill (neodent, curitiba, pr, brazil) was used in all groups following fabricant instructions. for the conventional drilling (control groups – c), the implant site preparations were performed using the drill sequence (neodent, curitiba, pr, brazil), #2.0 mm, #2/3 pilot, #3.0 mm, #3.3 mm drills. while, for the undersized preparation (experimental group – e) it was used the same sequence of drills until the #3.0mm (i.e. the last one #3.3 mm was not used). after that, externalhexagon implants (cylindrical shape and dimensions of 4.0 mm in diameter and 11.0 mm length; titamax ti cortical; neodent, curitiba, pr, brazil) were inserted and the final insertion torque, (ncm) was measured with a digital torque-meter that presents a 0.1-n.cm precision (tq8800; lutron, taipei, taiwan). the results were statistically analyzed by kolmogorov-smirnov test (data distribution test), and as they presented a parametric (normal) distribution the one-way anova (analysis of variance) and the post-hoc tukey’s test were employed (α=0.05). results statistical data from insertion torque values are summarized in figure 1. one-way anova showed a statistically significant difference between groups (p<0.001), where all of them were different from each other. the highest insertion torque was achieved on the group submitted to the conventional drilling technique at the higher density bone (cd1, 48.9 ± 9.3 ncm) followed by the undersized drilling technique at the low density bone (ed2, 22.6 ± 3.2 ncm); and the worst scenario was observed on the conventional drilling and low density bone (cd2, 11.7 ± 2.8 ncm). figure 1 mean insertion torque values (ncm) for different experimental groups: control technique and higher bone density (cd1); control technique and lower bone density (cd2); and undersized drilling technique and lower bone density (ed2). different capital letters above the bars represent significant statistical difference between the groups (p<0.001). combining the effects of undersized drilling and bone density on implant insertion torque braz j oral sci. 15(3):201-204 203 discussion today, it lacks consensus regarding a standard experimental design to investigate the effect of implant site preparations. different bone models based on cadaveric bone (acquired from tissue banks), synthetic blocks, and resin models have been described15. the american society for testing materials (astm f-1839-08) has been considering the use of solid rigid polyurethane foam blocks as the gold standard materials for simulate artificial bone on laboratory tests, as they present similar mechanical properties to the human bone. besides, they present the advantage of high structural homogeneity in comparison to the other alternatives (human cadaveric bone or animal bones). thus, these artificial bone blocks have already been used successfully in previous studies4,15-20. concerning the evaluation method to assess the implant primary stability, several noninvasive techniques have been tested in scientific literature. among all existing options the most reported in laboratory and clinical studies are the insertion torque and resonance frequency analysis4,21,22. the resonance frequency analysis use a particular device (ostell), while the insertion torque is evaluated in the moment of implant placement using a surgical torque-meter, surgical motor, or digital torque meter. however, there is no consensus concerning which would be the more efficient method to evaluate these outcomes. therefore, in the current study we opted to use the digital torque meter. the null hypothesis was rejected, since the insertion torque is dependent of the technique for implant site preparation in low density bone. our data support that the highest insertion torque mean (48.9 n.cm) was achieved with the group submitted to conventional drilling in the artificial bone with higher density (cd1); while, the group submitted to conventional drilling with lower bone density (cd2) presented the lowest insertion torque mean (11.7 n.cm). similar results were found in other studies4,19,23-25 where a positive relation between implant stability and artificial bone block density was noticed. besides, magno filho et al.23 evaluating the insertion torque and resonance frequency analysis of implants placed in maxilla and mandible with different bone densities, observed higher implant insertion torque and resonance frequency analysis on most dense bone, elucidating a positive correlation between these factors. when considering the undersized drilling, the results of this current study support that this technique leads to a higher insertion torque for low density bone (ed2), in comparison to the conventional drilling technique (technique recommended by the manufacturer) at the same scenario (cd2). these results are in agreement with a previous study26. however, it is important to emphasize that this technique (undersized drilling ed2) still leads to lower insertion torque in comparison to the one obtained by conventional drilling technique at higher bone density (cd1). therefore despite it increases the insertion torque; it still does not fully compensate the lower density of the foam bone block. thus, undersized drilling of the implant site is shown as an efficient technique for lower density bone to ensure primary stability27. however, a potential risk of bone necrosis has been assumed due to over compression inserted in bone tissue around of the implant28. on this sense, literature shows that this technique should be not the first choice in regions of higher density bones, which was the main reason why the current study did not simulated this scenario. another factor that could influence the final insertion torque is the shape of the implant, where a previous study29 showed that tapered shape implants with surface treatments, leads to higher insertion torque in comparison to cylindrical ones with machined surface. despite the methodological differences presented between clinical and laboratorial studies, it becomes evident a strong correlation between surgical technique, bone density, insertion torque and primary stability of the implant. however, as other factors (different physical and biological features) may be involved in obtaining an adequate insertion torque and primary stability on a clinical scenario, it is still strongly recommended the execution of clinical studies evaluating technological innovations on regards of shape and surface treatments of dental implants aiming to achieve higher insertion torque especially in regions with lower density bone. thus, within the limitations of this in vitro study and according to the obtained results, it may be concluded that the undersized drilling of the implant site preparation increased the insertion torque in lower bone density. references 1. heinemann f, hasan i, bourauel c, biffar r, mundt t. bone stability around dental implants: treatment related factors. ann anat. 2015 may;199:3-8. doi: 10.1016/j.aanat.2015.02.004. 2. javed f, ahmed hb, crespi r, romanos ge. role of primary stability for successful osseointegration of dental implants: factors of influence and evaluation. interv med appl sci. 2013 dec;5(4):162-7. doi: 10.1556/imas.5.2013.4.3. 3. chong l, khocht a, suzuki jb, gaughan j. effect of implant design on initial stability of tapered implants. j oral implantol. 2009;35(3):130-5. doi: 10.1563/1548-1336-35.3.130. doi: 10.1563/1548-1336-35.3.130. 4. mohlhenrich sc, heussen n, elvers d, steiner t, holzle f, modabber a. compensating for poor primary implant stability in different bone densities by varying implant geometry: a laboratory study. int j oral maxillofac surg. 2015 dec;44(12):1514-20. doi: 10.1016/j. ijom.2015.08.985. 5. trisi p, berardi d, paolantonio m, spoto g, d’addona a, perfetti g. primary stability, insertion torque, and bone density of conical implants with internal hexagon: is there a relationship. j craniofac surg. 2013 may;24(3):841-4. doi: 10.1097/scs.0b013e31827c9e01. 6. ostman po, hellman m, wendelhag i, sennerby l. resonance frequency analysis measurements of implants at placement surgery. int j prosthodont. 2006 jan-feb;19(1):77-83. 7. akkocaoglu m, uysal s, tekdemir i, akca k, cehreli mc. implant design and intraosseous stability of immediately placed implants: a human cadaver study. clin oral implants res. 2005 apr;16(2):202-9. 8. jemt t, stenport v. implant treatment with fixed prostheses in the edentulous maxilla. part 2: prosthetic technique and clinical maintenance in two patient cohorts restored between 1986 and 1987 and 15 years later. int j prosthodont. 2011 jul-aug;24(4):356-62. 9. jemt t, stenport v, friberg b. implant treatment with fixed prostheses in the edentulous maxilla. part 1: implants and biologic response in two patient cohorts restored between 1986 and 1987 and 15 years later. int j prosthodont. 2011 jul-aug;24(4):345-55. 10. shibli ja, mangano c, mangano f, rodrigues ja, cassoni a, bechara k, et al. bone-to-implant contact around immediately loaded direct laser metal-forming transitional implants in human posterior maxilla. j combining the effects of undersized drilling and bone density on implant insertion torque braz j oral sci. 15(3):201-204 204 combining the effects of undersized drilling and bone density on implant insertion torque periodontol. 2013 jun;84(6):732-7. doi: 10.1902/jop.2012.120126. 11. friberg b, ekestubbe a, sennerby l. clinical outcome of branemark system implants of various diameters: a retrospective study. int j oral maxillofac implants. 2002 sep-oct;17(5):671-7. 12. irinakis t, wiebe c. initial torque stability of a new bone condensing dental implant. a cohort study of 140 consecutively placed implants. j oral implantol. 2009;35(6):277-82. doi: 10.1563/aaidjoi-d-09-00020.1. 13. tabassum a, meijer gj, wolke jg, jansen ja. influence of surgical technique and surface roughness on the primary stability of an implant in artificial bone with different cortical thickness: a laboratory study. clin oral implants res. 2010 feb;21(2):213-20. doi: 10.1111/j.16000501.2009.01823.x. 14. bilhan h, geckili o, mumcu e, bozdag e, sunbuloglu e, kutay o. influence of surgical technique, implant shape and diameter on the primary stability in cancellous bone. j oral rehabil. 2010 dec;37(12):900-7. doi: 10.1111/j.1365-2842.2010.02117.x. 15. mohlhenrich sc, modabber a, steiner t, mitchell da, holzle f. heat generation and drill wear during dental implant site preparation: systematic review. br j oral maxillofac surg. 2015 oct;53(8):679-89. doi: 10.1016/j.bjoms.2015.05.004. 16. mohlhenrich sc, heussen n, loberg c, goloborodko e, holzle f, modabber a. three-dimensional evaluation of implant bed preparation and the influence on primary implant stability after using 2 different surgical techniques. j oral maxillofac surg. 2015 sep;73(9):1723-32. doi: 10.1016/j.joms.2015.03.071. 17. wu sw, lee cc, fu py, lin sc. the effects of flute shape and thread profile on the insertion torque and primary stability of dental implants. med eng phys. 2012 sep;34(7):797-805. doi: 10.1016/j. medengphy.2011.09.021. 18. hsu jt, huang hl, chang ch, tsai mt, hung wc, fuh lj. relationship of three-dimensional bone-to-implant contact to primary implant stability and peri-implant bone strain in immediate loading: microcomputed tomographic and in vitro analyses. int j oral maxillofac implants. 2013 mar-apr;28(2):367-74. doi: 10.11607/ jomi.2407. 19. wang tm, lee ms, wang js, lin ld. the effect of implant design and bone quality on insertion torque, resonance frequency analysis, and insertion energy during implant placement in low or lowto mediumdensity bone. int j prosthodont. 2015 jan-feb;28(1):40-7. doi: 10.11607/ijp.4063. 20. hsu jt, fuh lj, tu mg, li yf, chen kt, huang hl. the effects of cortical bone thickness and trabecular bone strength on noninvasive measures of the implant primary stability using synthetic bone models. clin implant dent relat res. 2013 apr;15(2):251-61. doi: 10.1111/j.1708-8208.2011.00349.x. 21. suer bt, yaman z, buyuksarac b. correlation of fractal dimension values with implant insertion torque and resonance frequency values at implant recipient sites. int j oral maxillofac implants. 2016 jan-feb;31(1):55-62. doi: 10.11607/jomi.3965. 22. wang r, eppell sj, nguyen c, morris n. relative contribution of trabecular and cortical bone to primary implant stability: an in vitro model study. j oral implantol. 2015 apr;42(2):145-52. doi: 10.1563/ aaid-joi-d-14-00322. 23. magno filho lc, cirano fr, hayashi f, feng hs, conte a, dib ll, et al. assessment of the correlation between insertion torque and resonance frequency analysis of implants placed in bone tissue of different densities. j oral implantol. 2014 jun;40(3):259-62. doi: 10.1563/aaid-joi-d-11-00183. 24. herekar m, sethi m, ahmad t, fernandes as, patil v, kulkarni h. a correlation between bone (b), insertion torque (it), and implant stability (s): bits score. j prosthet dent. 2014 oct;112(4):805-10. doi: 10.1016/j.prosdent.2014.02.011. 25. degidi m, daprile g, piattelli a. primary stability determination by means of insertion torque and rfa in a sample of 4,135 implants. clin implant dent relat res. 2012 aug;14(4):501-7. doi: 10.1111/j.17088208.2010.00302.x. 26. coelho pg, marin c, teixeira hs, campos fe, gomes jb, guastaldi f, et al. biomechanical evaluation of undersized drilling on implant biomechanical stability at early implantation times. j oral maxillofac surg. 2013 feb;71(2):e69-75. doi: 10.1016/j.joms.2012.10.008. 27. al-marshood mm, junker r, al-rasheed a, al farraj aldosari a, jansen ja, anil s. study of the osseointegration of dental implants placed with an adapted surgical technique. clin oral implants res. 2011 jul;22(7):753-9. doi: 10.1111/j.1600-0501.2010.02055.x. 28. fanuscu mi, chang tl, akça k. effect of surgical techniques on primary implant stability and peri-implant bone. j oral maxillofac surg. 2007;65 (12):2487–91. 29. dos santos mv, elias cn, cavalcanti lima jh. the effects of superficial roughness and design on the primary stability of dental implants. clin implant dent relat res. 2011 sep;13(3):215-23. doi: 10.1111/j.1708-8208.2009.00202.x. braz j oral sci. 15(3):201-204 braz j oral sci. 15(2):119-123 original article braz j oral sci. april | june 2016 volume 15, number 2 prevalence of endodontic diseases: an epidemiological evaluation in a brazilian subpopulation bruna paloma de oliveira1, andréa cruz câmara1, carlos menezes aguiar1 1universidade federal de pernambuco – ufpe, dental school, department of prosthodontics and oral and maxillofacial surgery, recife, pe, brazil correspondence to: bruna paloma de oliveira universidade federal de pernambuco, centro de ciências da saúde, departamento de prótese e cirurgia bucofacial av. prof. moraes rego s/n, cidade universitária, 50670-901 recife, pe brazil phone: +55 81 2126-8817 e-mail: bruna_paloma@msn.com abstract aim: to determine the prevalence of pulp and periradicular diseases in a brazilian subpopulation, correlating the prevalence with sex, age and most affected teeth. methods: data collected from medical records of patients treated at the clinic of specialization in endodontics of the federal university of pernambuco between 2003 and 2010. the following variables were recorded for each patient: sex, age, affected teeth and diagnosed endodontic disease. using pearson’s chi-square and fisher’s exact tests, the collected data analysis was set at a 5% significance level. results: from all the treated teeth, 57% were diagnosed with pulp diseases, with the symptomatic irreversible pulpitis being the most prevalent (46.3%), while among the diagnosed periradicular diseases (43%), chronic apical periodontitis was the most prevalent (81%). pulp diseases were detected in men and women in an unequal mode (p=0.008). subjects under 40 years old had higher prevalence of pulp disease (p=0.286), and patients over 50 years were most affected by periradicular diseases (p=0.439). maxillary incisors and mandibular first molars were the most affected teeth by endodontic diseases. conclusions: in the evaluated subpopulation, the endodontic diseases were more prevalent in the maxillary incisive, affected indiscriminately individuals of different age groups and chronic apical periodontitis was the most prevalent diagnosed disease. keywords: endodontics. dental pulp. periapical diseases. epidemiology. introduction in normal physiological conditions, the pulp tissue of a healthy tooth and its surrounding dentine is protected from the external environment by enamel and cementum. nevertheless, aggressive agents like caries, dental trauma and restorative procedures may produce disruption of the tissue integrity that protects the pulp, allowing occurrence of infections in the dentin-pulp complex, eventually leading to a pulpal disease that may spread to the periradicular tissue1-2. in brazil, dental caries is one of the main damaging agents to dental pulp. a 2010 report on the oral health status of the brazilian population,, issued by the brazilian ministry of health3, showed that 76.1% of adolescents between 15-19 years old had at least one decayed tooth. among the individuals aged between 35-44 and 65-74 years, only 0.9% and 0.2% respectively, were caries-free. oral diseases are the fourth most expensive disease to treat in most industrialized countries4, affecting not only the teeth but also the surrounding tissues and, in later stages, compromising the phonetic functions, nutrition, aesthetics5 and even the overall http://dx.doi.org/10.20396/bjos.v15i2.8648762 received for publication: june 27, 2016 accepted: september 08, 2016 120 health status of the individual6. this situation calls for investment in prevention and programs to promote oral health by the decisionmakers to implement programs that should, ideally, rely on the real needs of a population. therefore, data from endodontic epidemiological surveys may contribute to the improvement of this knowledge7. investigations on the prevalence of pulp and periradicular diseases in the brazilian population produced few studies8-11 and most of them were conducted exclusively based on radiographic assessments for the diagnosis of such infections. considering the importance of epidemiological investigations, the objective of this study was to determine the prevalence of pulp and periradicular diseases diagnosed in a brazilian subpopulation, correlating this information to gender, age group and most affected teeth. material and methods after approval by the research ethics committee of the federal university of pernambuco-ufpe, information was collected from 1346 medical records of patients treated at the specialization course in endodontics of ufpe, a reference institution for endodontic treatment in the brazilian northeastern region, between april 2003 and march 2010. only medical records containing complete information on the medical and dental history were included. from each medical record, the following variables were collected: sex, age, affected teeth and diagnosed endodontic diseases. the diagnosis of pulp and periradicular diseases was made by evaluating the following information: main complaint and pain symptomatology; examination of extra and intraoral tissues; tests of pulp vitality, including cold and heat sensitivity; clinical assessments of periapical health, including percussion and palpation; and interpretation of periapical radiographs. the classification for the diagnosis of endodontic diseases was the one used by the american association of endodontists12, as follows: reversible pulpitis, symptomatic irreversible pulpitis, asymptomatic irreversible pulpitis, pulp necrosis, internal and/or external root resorption, acute apical periodontitis, chronic apical periodontitis, acute apical abscess, chronic apical abscess, phoenix abscess and flare up. the collected data were analyzed using descriptive statistics, after which pearson’s chi-square and fisher’s exact tests were applied with the significance level set at 5%. the statistical package for social sciences, version 21 (spss, chicago, il, usa) was used. results one thousand and six hundred teeth from 1346 patients were examined. most individuals (n=908, 67.5%) were female. more than half (57.0%) of all treated teeth were diagnosed with pulp diseases. among the diagnosed pulp diseases, symptomatic irreversible pulpitis was the most prevalent (46.3% pulp diseases, 26.38% endodontic diseases), while root resorption was the least prevalent (0.8% pulp diseases, 0.5% endodontic diseases). chronic apical periodontitis was the most prevalent periradicular disease (81% periradicular diseases, 34.81% endodontic diseases) while flare up was not diagnosed (0%). regarding gender, pulp diseases were detected in men and women in an unequal mode (p=0.008), as statistically significant differences were verified for symptomatic irreversible pulpitis, which affected more females (p=0.005), and for asymptomatic irreversible pulpitis, which affected more males (p<0.001). the periradicular diseases were diagnosed in men and women similarly (p=0.895) (table 1). prevalence of endodontic diseases: an epidemiological evaluation in a brazilian subpopulation braz j oral sci. 15(2):119-123 table 1 prevalence of treated teeth due to pulp and periradicular pathologies according to gender. gender pathologies male female total p value n % n % n % pulp p(1) = 0.008* reversible pulpitis 8 2.4 10 1.7 18 2.0 symptomatic irreversible pulpitis 132 40.1 290 49.7 422 46.3 asymptomatic irreversible pulpitis 66 20.1 67 11.5 133 14.6 pulp necrosis 109 33.1 194 33.3 303 33.2 internal root resorption 0 0 external root resorption 1 0.3 2 0.3 3 0.3 internal and external root resorption 1 0.3 4 0.7 5 0.5 normal pulp (endodontic treatment with prosthetic purpose) 12 3.6 16 2.7 28 3.1 total 329 100.0 583 100.0 912 100.0 periradicular p(1) = 0.953 acute apical periodontitis 7 3.0 18 4.0 25 3.6 chronic apical periodontitis 192 82.4 365 80.2 557 81.0 acute apical abscess 7 3.0 15 3.3 22 3.2 chronic apical abscess 27 11.6 56 12.3 83 12.1 phoenix abscess 1 0.2 1 0.1 flare up 0 0 total 233 100.0 455 100.0 688 100.0 (*): significant association at the level of 5.0%. (1): using fisher’s exact test. 121 there were no statistically significant differences between different age groups and the prevalence of endodontic diseases (p=0.388) (table 2). the prevalence of pulp diseases was higher in patients under 50 years old, whereas the age group 50 to 59 years showed the lowest prevalence (p=0.286). the periradicular diseases affected a greater proportion of patients over 50 years of age, while individuals under 18 years were less affected (p=0.439). pulp and periradicular diseases affected the maxillary and mandibular teeth with the same frequency (p=0.581). in the maxilla, the second molars (75.6%) were the most affected teeth by pulp diseases, while the central and lateral incisors (57.5%) were the most affected by periradicular diseases (p<0.001). in the mandible, the teeth most affected by the pulp diseases were the third molars (82.6%), while the first premolars (49.2%) were the most affected by periradicular diseases (p=0.107) (table 3). the dental elements most affected by endodontic diseases in the maxilla were the incisors (31.87%), while in the mandible, the first molars were the most affected (41.28%). discussion this cross-sectional study had as its primary objective describing the prevalence of pulp and periradicular diseases in a brazilian urban subpopulation by analysis of medical records of patients treated at the clinic of the postgraduate course in endodontics in recife (pe), in the brazilian northeastern region. since the studied patients do not represent a random sample of the population, but individuals who sought dental treatment, the extrapolation of results for the general brazilian population should be made with caution. however, we emphasize that this dental service is a reference in the region for endodontic care, which may portray the reality of the geographical region reached by our clinic. the results of this study demonstrate that the most prevalent endodontic disease in both sexes was chronic apical periodontitis. the prevalence of chronic apical periodontitis (34.81%) agreed with studies conducted in other populations in buenos aires in argentina13, bucaramanga and floridablanca in colômbia14, and barcelona in spain15, where it ranged 27%to 49%. however, these results were higher than those reported in other surveys conducted in english16 (4.1%), scottish17 (5.8%) and kosovan18 (12.3%) populations. the discrepancies observed among these studies may be justified by the different degrees of human development among these populations. the prevalence of chronic apical periodontitis in brazilian subpopulations was target of previous studies11,19, which found a prevalence of chronic apical periodontitis of 3.4%11 and 5.9%19, respectively, whereas in our study, which evaluated a larger population sample than those studies, we detected a higher prevalence, i.e., 34.81%. the discrepancies observed between the results of the mentioned studies may be justified by lack of homogeneity in the analyzed population, lack of standardized evaluation methods, and the populations’ general level of oral health, factors that hamper comparing results from different studies. the results of this study showed that the pulp diseases were detected in men and women in an unequal mode (p=0.008), where the symptomatic irreversible pulpitis affected more females (p=0.005), while asymptomatic irreversible pulpitis affected more males (p<0.001). this difference may be explained by the fact that women take more care of their health and appearance than men8, which may lead them to seek dental care immediately in symptomatic cases, while men choose to postpone treatment20. on the other hand, men and women presented a similar prevalence of periradicular diseases (p=0.953), confirming what has been previously reported in other studies11,19,21. the results presented here indicated that the prevalence of pulp diseases was higher in people under 50 years of age, whereas periradicular diseases affected more patients over 50 years of age. there were no statistically significant differences between the age groups and the prevalence of endodontic diseases (p=0.388). these findings agree with those of kamberi et al.18 (2011), paes da silva ramos fernandes et al.11 (2013), peters et al.21 (2011), terças et al.19 (2006), who observed an increase in the prevalence of periradicular lesions with increasing patient age. according to terças et al.19 (2006), this result is expected since, with increased age, the tooth is exposed to caries, periodontal disease, friction and various operative procedures that increase the risk of pulpal inflammation, which if not treated may progress to periapex. in this study, the central and lateral incisors and the first molars were the teeth most affected by endodontic diseases. as reported by al-negrish22 and berlinck et al.8 (2015), the reason for the incisors to be most the affected may be associated with dental traumas, which usually affect the teeth that are in anterior position and more prominent in the dental arch. on the other hand, the higher prevalence of the first molars may be related to their morphology, with multiple grooves that facilitate the largest plaque retention, and the fact that they are the first permanent teeth to erupt in the oral cavity23. prevalence of endodontic diseases: an epidemiological evaluation in a brazilian subpopulation pathologies age groups all patients pulp periradicular both p value n % n % n % p(1) = 0.388 ≤18 226 125 55.3 82 36.3 19 8.4 19-29 300 155 51.7 116 38.7 29 9.7 30-39 334 180 53.9 123 36.8 31 9.3 40-49 266 139 52.3 105 39.5 22 8.3 50-59 165 75 45.5 78 47.3 12 7.3 ≥60 55 26 47.3 25 45.5 4 7.3 total 1346 700 52.0 529 39.3 117 8.7 (1): using pearson’s chi-square test. table 2 prevalence of treated patients due to pulp and periradicular pathologies according to age groups. braz j oral sci. 15(2):119-123 122 prevalence of endodontic diseases: an epidemiological evaluation in a brazilian subpopulation although the percentage of assessed maxillary teeth was higher than the mandibular ones, there was no significant difference between the maxilla and mandible in the incidence of teeth affected by endodontic diseases, which is consistent with the results of other authors18,24. on the other hand, the studies of al-negrish23 and scavo et al.13 (2011) reported that in their studies the maxillary teeth were predominantly more affected in relation to the mandibular. although our results do not describe a random sample of the brazilian population, but accounting that brazil is a continentwide country, these results provide relevant information on the prevalence of pulp and periradicular diseases that most affect a subpopulation this may help to define new treatment strategies and prevention of endodontic diseases. based on the results obtained in this study, we conclude that in the evaluated subpopulation: chronic apical periodontitis and symptomatic irreversible pulpitis were the most prevalent diagnosed endodontic diseases; symptomatic irreversible pulpitis affected more females, whereas asymptomatic irreversible pulpitis was more prevalent in males; endodontic disease affected indiscriminately individuals of different age groups and the maxillary and mandibular teeth; incisors and first molars were the most affected teeth by endodontic diseases. references 1. love rm. intraradicular space: what happens within roots of infected teeth? ann r australas coll dent surg. 2000 oct;15:235-9. 2. nair pn, pajarola g, schroeder he. types and incidence of human periapical lesions obtained with extracted teeth. oral surg oral med oral pathol oral radiol endod. 1996 jan;81(1):93-102. 3. brazil. brazilian ministry of health. [sb brazil 2010: national research on oral health: main results]. brasília: ministry of health; 2010 [cited 2015 jun 17]. 92p. available from: http://dab.saude.gov.br/cnsb/ sbbrasil/arquivos/projeto_sb2010_relatorio_final.pdf. portuguese. 4. petersen pe, bourgeois d, ogawa h, estupinan-day s, ndiaye c. the global burden of oral diseases and risks to oral health. bull world health organ. 2005 sep;83(9):661-9. 5. corrêa-faria p, paixão-gonçalves s, paiva sm, martins-júnior pa, vieiraandrade rg, marques ls, et al. dental caries, but not malocclusion or developmental defects, negatively impacts preschoolers’ quality of life. int j paediatr dent. 2016 may;26(3):211-9. doi: 10.1111/ipd.12190. 6. cotti e, mercuro g. apical periodontitis and cardiovascular diseases: previous findings and ongoing research. int endod j. 2015 oct;48(10):92632. doi:10.1111/iej.12506. 7. kirkevang ll. root canal treatment and apical periodontitis: what can be learned from observational studies? endod topics. 2011 mar;18(1):51-61. 8. berlinck t, tinoco jm, carvalho fl, sassone lm, tinoco em. epidemiological evaluation of apical periodontitis prevalence in an urban brazilian population. braz oral res. 2015;29:51. doi: 10.1590/18073107bor-2015. 9. costa th, de figueiredo neto ja, de oliveira ae, lopes e maia m de f, de almeida al. association between chronic apical periodontitis and coronary artery disease. j endod. 2014 feb;40(2):164-7. doi: 10.1016/j. joen.2013.10.026. 10. hebling e, coutinho la, ferraz cc, cunha fl, queluz dp. periapical status and prevalence of endodontic treatment in institutionalized elderly. braz dent j. 2014;25(2):123-8. 11. paes da silva ramos fernandes lm, ordinola-zapata r, húngaro duarte ma, alvares capelozza al. prevalence of apical periodontitis detected in cone beam ct images of a brazilian subpopulation. dentomaxillofac radiol. 2013;42(1):80179163. doi: 10.1259/dmfr/80179163. 12. aae consensus conference recommended diagnostic terminology. j endod. 2009 dec;35(12):1634. 13. scavo r, martinez lalis r, zmener o, dipietro s, grana d, pameijer ch. frequency and distribution of teeth requiring endodontic therapy in an argentine population attending a specialty clinic in endodontics. int dent j. 2011 oct;61(5):257-60. doi: 10.1111/j.1875-595x.2011.00069.x. 14. moreno jo, alves fr, gonçalves ls, martinez am, rôças in, siqueira jf jr. periradicular status and quality of root canal fillings and coronal restorations in an urban colombian population. j endod. 2013 may;39(5):600-4. doi: 10.1016/j.joen.2012.12.020. pathologies dental groups all pulp periradicular p value teeth n % n % maxillary p (1) < 0.001* incisors 320 136 42.5 184 57.5 canines 62 33 53.2 29 46.8 1st premolars 160 90 56.3 70 43.8 2nd premolars 164 105 64.0 59 36.0 1st molars 212 139 65.6 73 34.4 2nd molars 78 59 75.6 19 24.4 3rd molars 8 5 62.5 3 37.5 subtotal 1004 567 56.5 437 43.5 mandibular p (1) = 0.107 incisors 38 22 57.9 16 42.1 canines 19 14 73.7 5 26.3 1st premolars 61 31 50.8 30 49.2 2nd premolars 93 50 53.8 43 46.2 1st molars 246 138 56.1 108 43.9 2nd molars 116 71 61.2 45 38.8 3rd molars 23 19 82.6 4 17.4 subtotal 596 345 57.9 251 42.1 total 1600 912 57.0 688 43.0 (*): significant association at the level of 5.0%. (1): using pearson’s chi-square test. table 3 prevalence of treated teeth due to pulp and periradicular pathologies according to dental groups. braz j oral sci. 15(2):119-123 123prevalence of endodontic diseases: an epidemiological evaluation in a brazilian subpopulation 15. abella f, patel s, durán-sindreu f, mercadé m, bueno r, roig m. an evaluation of the periapical status of teeth with necrotic pulps using periapical radiography and cone-beam computed tomography. int endod j. 2014 apr;47(4):387-96. doi: 10.1111/iej.12159. 16. di filippo g, sidhu sk, chong bs. apical periodontitis and the technical quality of root canal treatment in an adult sub-population in london. br dent j. 2014 may;216(10):e22. doi: 10.1038/sj.bdj.2014.404. 17. dutta a, smith-jack f, saunders wp. prevalence of periradicular periodontitis in a scottish subpopulation found on cbct images. int endod j. 2014 sep;47(9):854-63. doi: 10.1111/iej.12228. 18. kamberi b, hoxha v, stavileci m, dragusha e, kuçi a, kqiku l. prevalence of apical periodontitis and endodontic treatment in a kosovar adult population. bmc oral health. 2011 nov 29;11:32. doi: 10.1186/14726831-11-32. 19. terças ag, de oliveira ae, lopes ff, maia filho em. radiographic study of the prevalence of apical periodontitis and endodontic treatment in the adult population of são luís, ma, brazil. j appl oral sci. 2006 jun;14(3):183-7. 20. courtenay wh. constructions of masculinity and their influence on men’s well-being: a theory of gender and health. soc sci med. 2000 may;50(10):1385-401. 21. peters lb, lindeboom ja, elst me, wesselink pr. prevalence of apical periodontitis relative to endodontic treatment in an adult dutch population: a repeated cross-sectional study. oral surg oral med oral pathol oral radiol endod. 2011 apr;111(4):523-8. doi: 10.1016/j.tripleo.2010.10.035. 22. al-negrish ar. incidence and distribution of root canal treatment in the dentition among a jordanian sub population. int dent j. 2002 jun;52(3):125-9. 23. mukhaimer r, hussein e, orafi i. prevalence of apical periodontitis and quality of root canal treatment in an adult palestinian sub-population. saudi dent j. 2012 jul;24(3-4):149-55. doi: 10.1016/j.sdentj.2012.02.001. 24. wayman be, patten ja, dazey se. relative frequency of teeth needing endodontic treatment in 3350 consecutive endodontic patients. j endod. 1994 aug;20(8):399-401. braz j oral sci. 15(2):119-123 braz j oral sci. 15(2):180-184 incisor proclination and gingival recessions: is there a relationship? márlio vinícius de oliveira1, matheus melo pithon2, márcia luzia lacerda xavier1, rodrigo villamarim soares1, martinho campolina rebello horta1, dauro douglas oliveira1 1pontifícia universidade católica de minas gerais – puc minas, school of dentistry, department of orthodontics, belo horizonte, mg, brazil 2universidade do estado da bahia – uneb, school of dentistry, area of orthodontics, jequié, ba, brazil correspondence to: dauro douglas oliveira pontifícia universidade católica de minas gerais av. dom josé gaspar 500, prédio 46, sala 101, cep 30535-901 belo horizonte mg brazil e-mail: dauro.bhe@gmail.com abstract aim: to test the hypothesis that there is no relationship between the amount of vestibular inclination of mandibular incisors and the appearance of gingival recession in this region. methods: this study included 20 patients selected in accordance with the following inclusion criteria: 1. adult patients without gingival recession in the mandibular incisors before treatment (t0), 2. no previous orthodontic treatment, 3. treated without tooth extraction in the mandibular arch, 4. bonded mandibular splinting from one canine to another after the active stage of orthodontic treatment (t1), 5. no visible wear of the incisal edge of the mandibular incisors, 6. preand post-treatment teleradiography and plaster casts, and plaster casts 3 years post-treatment (t2). depending on the amount of inclination of the mandibular incisors after treatment, the sample was divided into two groups: group 110 individuals (impa t1-t0 ≤ 5˚) and group 210 individuals (impa t1-t0 > 5˚). the measurement of length of the clinical crown (lcc) of the four mandibular incisors, distance between the incisal edge and vestibular marginal gingiva were made in plaster casts at t0, t1 and t2. results: in spite of group 1 presenting a reduction in lcc at t2, there was no statistically significant difference in lcc in the 3 time intervals evaluated in the two groups. conclusions: the null hypothesis was accepted. the variation in the amount of vestibular inclination of the mandibular incisors during orthodontic treatment and 3 years after conclusion of treatment did not promote the appearance of gingival recessions in this group of patients. keywords: gingival recession. orthodontics, corrective. cephalometry. introduction a gingival recession is defined as the displacement of the marginal tissue apical to the cementoenamel junction1-3. although its etiology is unclear, periodontal disease and mechanical trauma are considered the primary factors in the pathogenesis of gingival recessions4. orthodontic treatment might promote the development of recessions5, with a possible mechanism being that orthodontic tooth movement can result in root positions close to or outside alveolar cortical plates; this can lead to bone dehiscences6,7. as a result, a marginal gingiva without proper alveolar bone support can migrate apically, leading to root exposure8. furthermore, a fixed orthodontic appliance creates retention areas for dental plaque. in case of inadequate plaque removal, gingival inflammation could lead to periodontal breakdown9. gingival recessions are more frequently observed in mandibular than in maxillary teeth. with increasing age, they are more frequent on buccal than on lingual surfaces10. received for publication: december 06, 2016 accepted: march 08, 2017 original article braz j oral sci. april | june 2016 volume 15, number 2 http://dx.doi.org/10.20396/bjos.v15i2.8648780 181 canines, first premolars, and first molars in the maxilla, and central incisors and first premolars in the mandible are at the highest risk for labial gingival recessions11. the data suggest that gingival recession is universal and a common manifestation in most populations. prevalence varies from 3% to 100% depending on the population and the methods of analysis12, and appears to be lower in younger groups, in which the incidence increases over time13. it has been estimated that over half of the adults in the united states have gingival recession, and on average, it affects about one fourth of the dentition14. several studies have demonstrated that labial movement of incisors in humans may be a risk factor for gingival recessions, but their conclusions were contradictory15,16. some publications have shown association between incisor proclination and development of recessions and others have demonstrated the lack of such correlation17,18. to date, studies that have focused on the development of gingival recessions in orthodontic patients have evaluated samples either immediately17,18 or several years after treatment19. the latter types were, however, limited to only 1 long-term observation. consequently, the dynamics of the development of gingival recessions could not have been evaluated. moreover, the studies did not establish safe limits with regard to the amount of vestibular inclination of mandibular incisors allowed, so that clinicians may make their decisions at the time of leveling the curve of spee5,12. the aim of this retrospective study was to test the following null hypothesis: there is no relationship between the amount of buccal inclination of mandibular incisors and the appearance of gingival recession in this region. in addition, an evaluation was made of whether an increase in the inclination of these teeth promoted increase in the length of their clinical crown (lcc). material and methods this study was approved by the research ethics committee (44066415000005137). the sample was selected from the files of patients treated at the orthodontic clinic. the patients were selected in accordance with the following inclusion criteria: 1. adult patients without gingival recession in the mandibular incisors before treatment (t0), 2. no previous orthodontic treatment, 3. treated without tooth extraction in the mandibular arch, 4. bonded mandibular splinting from one canine to another after the active stage of orthodontic treatment (t1), 5. no visible wear of the incisal edge of the mandibular incisors, 6. preand post-treatment teleradiography and plaster casts, and plaster casts 3 years post-treatment (t2). this retrospective study included 20 patients who met the inclusion criteria (13 men and 7 women). to evaluate the alteration that occurred in the inclination of the mandibular incisors after treatment, the measurement of the angle formed between the long axis of the mandibular incisor and the mandibular plane was used (impa). depending on the amount of inclination of the mandibular incisors after treatment, the sample was divided into two groups: group 110 individuals (impa t1t0 ≤ 5˚) and group 210 individuals (impa t1-t0 > 5˚) (table 1). after obtaining the cephalometric tracing, the following points were marked: mandibular incisor edge and root apex, mentum (most inferior point of the mandibular symphysis), and gonium (the most inferior and posterior point of the mandibular angle). to identify the alterations in the inclination of the mandibular incisors after treatment, tracings were superimposed according to the mandibular superimposition technique, with stable natural structures used as reference16. the radiographic tracings were oriented, based on: 1anterior contour of the pogonion region; 2-internal cortical contour at the level of the inferior edge of the mandibular symphysis; 3-trabecular structure of the mandibular symphysis; 4mandibular canal contour; and 5contour of the inferior edge of the mandible. references in stable anterior and posterior structures of the mandible were used in the pretreatment tracing (t0) and were transferred to the post-treatment tracing (t1) by means of superimposition. all the cephalograms were traced and digitized by a single investigator. the teleradiographs were digitized in the resolution of 9.600 x 4.800 dpi in a microtek scanmaker i800 scanner (microtek international, inc., carson, usa), coupled to a pentium microcomputer. the images were transferred to the dolphin imaging premium 10.5 program (dolphin imaging & management solutions, chatsworth, usa), by means of which the cephalometric points of interest were marked and the superimpositions were made (figure 1). incisor proclination and gingival recessions: is there a relationship? braz j oral sci. 15(2):180-184 fig.1. agroup 1(impa t1-t0 ≤ 5˚). bgroup 2 (impa t1-t0 > 5˚). a b 182 the measurement of lcc of the four mandibular incisors, distance between the incisal edge and vestibular marginal gingiva were made in plaster casts at t0, t1 and t2 (figure 2). the measurements were taken by a single researcher, by using a digital pachymeter (model 100.174b, digimess, são paulo, brazil) with a precision of 0.01mm. renkema et al.20 validated this method of evaluating the clinical crown by means of plaster casts. to determine the intra-examiner agreement, all the measurements were made twice, in an interval of one month. the intraclass correlation coefficient (icc) showed an excellent intra-examiner agreement. greater vestibular inclination that occurred in group 2. none of the groups evaluated presented gingival recession after the active stage of treatment (t1) or during the post-splinting period (t2), in spite of the greater vestibular inclination of the mandibular incisors in group 2 after treatment. incisor proclination and gingival recessions: is there a relationship? fig.2. measurement of lcc of the four mandibular incisors. the data were initially submitted to the kolmogorov-smirnov test of normality, which demonstrated their normal distribution. the groups were divided by using two criteria: time (t0, t1 and t2) and variation in impa between t1 and t0 (≤5˚ and >5˚). the repeated measurements two-way anova test, followed by the bonferroni post hoc test for comparison between pairs was used to evaluate whether there were differences in lcc between the times (t0, t1 and t2). this evaluation was performed separately for each variation of the impa (≤5˚ and >5˚). the two-way anova test, followed by the bonferroni post hoc test for comparison between pairs was used to evaluate whether there were differences in lcc between each impa (≤5˚ and >5˚). this evaluation was made separately for each of the times (t0, t1 and t2). the student t test was used to assess if the two evaluated groups were well paired. the level of significance was established at 5%. the analyses were performed with the use of graphpad prism 6.05 software (graphpad software, san diego, california, usa). results the two groups evaluated were well paired and their characteristics are presented in table 1. the impa at t0 was greater in group 1 (101.6˚) than in group 2 (90,6˚), whereas, at the end of treatment (t1), their values were similar, due to the p value obtained by the student t test; ns = not significant (p>0.05) table 1 description of subjects. group 1(n=10) group 2(n=10) mean sd mean sd p value age, years 25.3 7.7 30.3 7.8 ns treatment time, months 32.5 6.3 33.3 4.5 ns sna t0, degrees 81.5 3.3 80.7 2.5 ns snb t0, degrees 78.1 3.3 77.4 3.7 ns anb t0, degrees 3.4 2.1 3.3 2.9 ns sn-gome, degrees 28.9 5.4 34.5 8.1 ns impa to, degrees 101.7 6.4 90.6 6.4 <0.05 impa t1, degrees 103.9 6.0 102.4 10.1 ns a in the lines, means followed by the same capital letters do not show statistically significant differences (p> 0.05). p values were obtained by repeated measurements two-way anova followed by bonferroni post hoc test to compare pairs. a in the columns, means followed by the same lowercase letters do not show statistically significant differences (p> 0.05). p values were obtained by two-way anova followed by bonferroni post hoc test to compare pairs. table 2 the mean and standard deviation of lcc and its comparison between the different times (t0, t1 and t2) and between the different variations of impa (group 1 ≤5˚ and group 2 >5˚). impa (t1-t0) t0 t1 t2 ≤5˚ 8.06 ± 0.69 a,a 8.03 ± 0.81 a,a 7.95 ± 0.96 a,a >5˚ 8.26 ± 1.27 a,a 8.10 ± 1.23 a,a 8.24 ± 1.09 a,a the mean and standard deviation of lcc and its comparison between the different times (t0, t1 and t2) and between the different variations of impa (group 1 ≤5˚ and group 2 >5˚) are presented in table 2. in spite of group 1 presenting a reduction in lcc at t2, there was no statistically significant difference in lcc in the 3 time intervals evaluated in the two groups. discussion wedrychowska-szulc and syrynska21 verified that the majority of patients seek orthodontic treatment for esthetic reasons. therefore, gingival recessions may compromise the esthetic results in addition to causing tooth hypersensitivity. although its etiology has not been completely elucidated, gingival recession may be associated with orthodontic treatment20,22. therefore, knowing that gingival recession may be a side effect of orthodontic therapy, identifying the factors that may contribute to the development of recessions is of great importance5. in this study, we investigated the relationship between the amount of change in inclination of the mandibular incisors during braz j oral sci. 15(2):180-184 183 orthodontic treatment, and the changes that occurred in the lcc of these teeth, immediately after removal of the appliance, and three years later. our results showed that in spite of the difference in the amount of vestibular inclination of the mandibular incisors during treatment, the lcc remained unaltered in the two groups. even in group 2, in which there was greater vestibular inclination of the incisors, no appearance of gingival recessions had occurred in this region 3 years after orthodontic treatment. thus, our results are in agreement with the findings of yared et al.19, ruf et al.23 and djeu et al.24. yared et al19 evaluated the periodontal condition of mandibular incisors after orthodontic treatment and concluded that there was no correlation between gingival recession and the amount of vestibular inclination of these teeth. ruf et al.23, in a sample of adolescents treated with the herbst appliance, analyzed the alterations in the inclination of mandibular incisors and the development of gingival recessions 6 months after treatment. they verified that a mean vestibular inclination of 8.9 degrees of the mandibular incisors did not increase the risk of recession. djeu et al.24 concluded that an inclination of 5 degrees in the mandibular incisors after orthodontic treatment in adolescents and adults, had no correlation with gingival recession. after a vestibular inclination of 5 degrees in the mandibular incisors, allais and melsen25 found no association between the amount of vestibular inclination of the mandibular incisors during orthodontic treatment in adults, and the prevalence and severity of gingival recession. furthermore, they reported that around 5% of the patients had a reduction in gingival recession after treatment. other studies, however, found association between the alteration in inclination of the mandibular incisors and increase in the risk of gingival recession. in the study of slutzkey and levin26 the prevalence of recession was correlated with previous orthodontic treatment and the use of dental piercing. they examined 303 young adults (18-22 years) and found strong correlation between severity and extension of recession, and orthodontic treatment. choi et al.22, in class iii patients decompensated before orthognathic surgery, evaluated whether the periodontal alterations in the mandibular incisors that underwent minimal vestibular inclination were similar to those that were highly tipped bucally. they observed that the mandibular incisors that had been very inclined towards the vestibular region during dental decompensation presented greater retraction of the vestibular cortical bone as well as a reduction in the strip of keratinized gingiva. nevertheless, the amount of gingival recession appears to be clinically insignificant. the difference between our results and those found by the cited studies may be explained by the fact that none of our patients had gingival recession before treatment, which would show a more favorable periodontal biotype. previous studies have used intraoral photographs to evaluate periodontal alterations8,14. however, in some patients this method of evaluation was not ideal, because the retractors generally covered some part of the gingiva. consequently, we opted to use the method of measuring the lcc directly in plaster casts, as described and validated by renkema et al.20. other factors, such as inflammation and gingival biotype, a narrow strip of keratinized gingiva are considered predisposing factors for gingival recession. a systematic review conducted by jossvassalli et al.27 evaluated the effects of orthodontic treatment on the inclination of mandibular incisors and the occurrence of gingival recession. the authors concluded that further randomized clinical studies that included an examination of oral hygiene and the gingival condition before, during and after treatment are necessary in order to demonstrate the axial changes of the incisors, and the occurrence of gingival recession. the limitation of the present study was that some of the above-mentioned periodontal parameters were not evaluated. however, as regards oral hygiene care, all the patients were monitored and were able to perform adequate plaque control during orthodontic treatment, and in the post-splinting period. our results indicate that further prospective clinical studies that control the primary etiological factors of gingival recession, before, during and after treatment should be conducted. these studies must also classify patients according to their periodontal biotype. the null hypothesis was accepted. the variation in the amount of vestibular inclination of the mandibular incisors during orthodontic treatment and 3 years after conclusion of treatment did not promote the appearance of gingival recessions in this group of patients. even in group 2, in which there was greater vestibular inclination of the incisors, there was no development of gingival recession in this region. references 1. garcia-de-la-fuente am, aguirre-zorzano la, estefania-fresco r, roig-odena l, aguirre-urizar jm. histologic and clinical study of gingival recession treated with subepithelial connective tissue graft (sctg): a case report. int j periodontics restorative dent. 2017 jan/feb;37(1):89-97. doi: 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central incisors after orthodontic proclination in adults. am j orthod dentofacial orthop. 2006 jul;130(1):6 e1-8. 20. renkema am, fudalej ps, renkema a, bronkhorst e, katsaros c. gingival recessions and the change of inclination of mandibular incisors during orthodontic treatment. eur j orthod. 2013 apr;35(2):249-55. 21. wedrychowska-szulc b, syrynska m. patient and parent motivation for orthodontic treatment--a questionnaire study. eur j orthod. 2010 aug;32(4):447-52. 22. choi yj, chung cj, kim kh. periodontal consequences of mandibular incisor proclination during presurgical orthodontic treatment in class iii malocclusion patients. angle orthod. 2015 may;85(3):427-33. 23. ruf s, hansen k, pancherz h. does orthodontic proclination of lower incisors in children and adolescents cause gingival recession? am j orthod dentofacial orthop. 1998 jul;114(1):100-6. 24. djeu g, hayes c, zawaideh s. correlation between mandibular central incisor proclination and gingival recession during fixed appliance therapy. angle orthod. 2002 jun;72(3):238-45. 25. allais d, melsen b. does labial movement of lower incisors influence the level of the gingival margin? a case-control study of adult orthodontic patients. eur j orthod. 2003 aug;25(4):343-52. 26. slutzkey s, levin l. gingival recession in young adults: occurrence, severity, and relationship to past orthodontic treatment and oral piercing. am j orthod dentofacial orthop. 2008 nov;134(5):652-6. 27. joss-vassalli i, grebenstein c, topouzelis n, sculean a, katsaros c. orthodontic therapy and gingival recession: a systematic review. orthod craniofac res. 2010 aug;13(3):127-41. braz j oral sci. 15(2):180-184 braz j oral sci. 15(4):293-297 influence of aesthetic coating on the loaddeflection ratio of nickel–titanium archwires marcus vinicius neiva nunes do rego, msc, phd,1 gislayne maria mesquita de araújo,2 elizabeth ferreira martinez, scd,3 kássio rafael de sousa lima,4 pio thiago feitosa fortes,5 leanne matias portela leal, msc6 1professor of orthodontics, centro universitário uninovafapi; professor of orthodontics, universidade federal do piauí (ufpi), teresina, pi, brazil. phone: +55-86-99482-2248. e-mail: marcus_rego@yahoo.com.br 2dentist, centro universitário uninovafapi, teresina, pi, brazil. phone: +55-86-99976-1706. e-mail: gislaynemesquita21@hotmail.com 3scd in cell and tissue biology, institute of biomedical sciences, universidade de são paulo (usp); postdoctorate researcher in dentistry, instituto e centro de pesquisas são leopoldo mandic, campinas, sp, brazil. phone: +55-11-99625-2801. e-mail: dr.efmartinez@gmail.com 4student, specialization program in orthodontics, centro universitário uninovafapi; dentist, ufpi, teresina, pi, brazil. phone: +55-86-99972-7335. e-mail: kassiorafaelsl@gmail.com 5student, specialization program in orthodontics, centro universitário uninovafapi; dentist, ufpi, teresina, pi, brazil. phone: +55-86-99972-7335. e-mail: thi-fortes@hotmail.com 6professor of orthodontics, centro universitário uninovafapi, teresina, pi, brazil. phone: +55-86-99424-2303. e-mail: leannempleal@yahoo.com.br correspondence to: marcus vinicius neiva nunes do rego rua vitorino ortiges fernandes, 6123, bairro uruguai 64073-505 teresina, pi, brazil marcus_rego@yahoo.com.br phone: +55-86-2106.0700 abstract aim: to assess the influence of aesthetic surface coating on load-deflection ratios in nickel-titanium (niti) orthodontic wires compared with uncoated wires. methods: niti wires (0.016") from four different manufacturers (morelli, sorocaba, sp, brazil; tp, la porte, in, usa; eurodonto, curitiba, pr, brazil; ortho organizers, san marcos, ca, usa) were divided into eight groups, according to presence or absence of coating: group 1, morelli coated wire; group 2, morelli uncoated; group 3, tp coated; group 4, tp uncoated; group 5, eurodonto coated; group 6, eurodonto uncoated; group 7, ortho organizers coated; group 8, ortho organizers uncoated. to determine the load-deflection ratio, a three-point bending test was performed in a ags-x 250 kn (shimadzu) universal testing machine. results: the results showed that aesthetic coatings did not influence load-deflection ratio in niti orthodontic wires at 1-mm and 2-mm activation. however, comparison across the four tested brands revealed that eurodonto coated wires exhibited the greatest force levels at 1-mm, 2-mm, and 3-mm deflection. at 3-mm deflection, ortho organizers coated wires exhibited lower force levels than all other tested brands, except for tp wires. conclusions: we conclude that the load-deflection ratio of niti wires was not influenced significantly by aesthetic coatings, especially at lower activations. keywords: orthodontic wires. aesthetics. alloys. received for publication: january 19, 2017 accepted: june 28, 2017 original article braz j oral sci. october | december 2016 volume 15, number 4 http://dx.doi.org/10.20396/bjos.v15i4.8650043 introduction individual variations considered, orthodontic treatment usually extends over months or years. therefore, the appearance of orthodontic appliances has become a significant factor in orthodontic treatment decisions, particularly due to increasing demands from adult patients1-3. these demands mean that aesthetic considerations now extend beyond ceramic or composite brackets and ligatures and are now a concern for archwires as the three-point bending (flexural) test was used to assess load-deflection ratio, as it is regarded as the assay that best simulates the conditions of real-life orthodontic practice24,25. all assays were performed in accordance with the iso 15841:2006 standard (figure 1). as suggested in the iso standard, the wire span between supports was 10 mm, the crosshead rate was set at 6.0 mm/min, and the radius of both fulcrum and indenter was 0.1 mm. assays were carried out in a servo elétrica ags-x 250 kn (shimadzu) universal testing machine coupled to a workstation running trapezium x software (shimadzu). the span between support and fulcrum was 5 mm, and deflection was carried out with a centrally placed indenter. specimens measured 30 mm and were obtained from the straightest section of each archwire with distal end cutters. each of the eight groups comprised six specimens. thus, a total of 48 specimens were tested at a temperature of 37 °c, obtained by using heating lamps in a thermal chamber coupled to the universal testing machine (figure 1). all wires were isolated from the outside environment to ensure temperature stability during testing. deflection was quantified with the ags-x 250 kn electronic deflectometer (shimadzu), with the universal testing machine programmed to impart a maximum deflection of 3.0 mm. these assays yielded force (gf) vs. deflection (mm) curves, which were analyzed and compared. data were described as means and standard deviations and entered into tables. the shapiro-wilk test was applied to evaluate the normality of data distribution. for the 1-mm and 3-mm activations, values fell outside the normal distribution curve (p = 0.009 and p = 0.029 respectively), and thus required nonparametric tests for comparison. for the 2-mm activation, all values fell within the normal distribution curve (p = 0.331), and groups were thus compared by parametric tests. to assess the null hypothesis that the eight tested groups of independent samples would be homogeneous in terms of loaddeflection ratio distribution, the kruskal-wallis test was applied to 1-mm and 3-mm activation values. when the null hypothesis was rejected, dunn's multiple comparisons test was used to ascertain which groups differed. for the 2-mm activation, analysis of variance (anova) was used to test the null hypothesis of absence of difference across groups, with tukey's multiple comparisons used to ascertain which groups differed. the level of significance was set at 5% (p < 0.05) for all tests. all statistical procedures were performed in the statistical package for the social sciences (spss) 22.0 software environment. 294 well, which has led to the advent of aesthetic orthodontic wires4,5. aesthetic orthodontic wires may be divided into three categories: a) stainless-steel or nickel-titanium (niti) wires coated with epoxy resin, which are manufactured by depositing or embedding an approximately 0.002"-thick layer of epoxy resin onto the wire; b) stainless-steel or niti wires coated with teflon (polytetrafluoroethylene, ptfe), which mimics tooth color. ptfe coatings are applied by an atomic process that yields a mean layer thickness of 20-25 µm; and c) fiber-reinforced composite resin wires6,7. unsolved questions remain as to whether the mechanical properties of metal archwires, such as the load-deflection ratio, could be affected by such coatings and by modifications in wire dimension made to compensate for the added thickness of the coating layer8. the load-deflection ratio has been used as a marker of how much force is released with each millimeter of wire activation, as well as to determine the elastic limit of the wire9. several advantages of aesthetically coated niti archwires in their original, as-received condition have been described, including reduced surface roughness, increased corrosion resistance, and reduced friction10-15. however, some disadvantages of ptfe and epoxy coatings have been reported, including poor durability of the coating, discoloration, cracking and pitting, increased surface roughness, and predisposition to a buildup of amorphous organic matter, both through the mechanical action of masticatory forces and tooth brushing and due to the effects of oral enzymes. these changes or losses of coating material have been observed after clinical use and even after exposure to simulated oral environments2,11,16-23. within this context, given the limited scientific evidence regarding the potential benefits of aesthetically coated niti archwires in orthodontic practice, the present investigation sought to assess the influence of aesthetic coatings on the load-deflection ratio of niti wires. methods sample size calculation was based on an alpha level significance of 1% (α=0.01) and a beta level of 20% (β=0.20), with a power of 80% to detect a mean difference between the four groups of 125.04 g/mm, with a standard error deviation of 5.58, which is in agreement with the study of silva et al.20. therefore, a sample size of 6 wire segments per group was required. aesthetically coated niti orthodontic wires from four different brands and their respective control (uncoated) wires were evaluated in this study (table 1). to assess the influence of aesthetic coating on load-deflection ratio, the sample was divided into eight groups: group 1, morelli coated wire; group 2, morelli uncoated; group 3, tp coated; group 4, tp uncoated; group 5, eurodonto coated; group 6, eurodonto uncoated; group 7, ortho organizers coated; group 8, ortho organizers uncoated. all wires in each group were obtained from the same batch. these brands were selected because we aimed to test different coatings types (epoxy resin or teflon), coated surfaces (buccal or total surface), and wires from different origins (nationally manufactured or imported). influence of aesthetic coating on the load-deflection ratio of nickel–titanium archwires braz j oral sci. 15(4):293-297 table 1 characteristics of the nickel–titanium aesthetic archwires and respective control wires. manufacturer dimension coating type coated surface tp orthodontics, la porte, in, usa 0.016" teflon buccal ortho organizers, san marcos, ca, usa 0.016" teflon total eurodonto, curitiba, pr, brazil 0.016" teflon total morelli, sorocaba, sp, brazil 0.016" epoxy resin total 295 results table 2 lists the means and standard deviations obtained in each group after bending tests, as well as the differences influence of aesthetic coating on the load-deflection ratio of nickel–titanium archwires braz j oral sci. 15(4):293-297 table 1 mean and standard deviation loads (gf) in three-point bending tests, stratified by brand and presence of aesthetic coating. groups 1 mm activation 2 mm activation 3 mm activation mean sd sig mean sd sig mean sd sig 1 116.22 11.78 a,b,c,d,g,h 192.64 46.35 a,b,c 276.41 65.18 a,b,c,d,f,h 2 131.67 46.76 b,c,d,f,g,h 225.11 46.76 a,b,c 321.57 29.79 b,e,f,h 3 142.94 25.52 c,d,f,g,h 204.21 14.53 a,b,c 219.96 29.81 c,d,g 4 114.78 26.67 d,g 161.56 44.21 a 238.04 11.40 d,g 5 195.38 26.32 e 263.23 76.86 b 373.45 77.99 e 6 165.65 35.13 e,f 251.26 37.64 c 305.70 32.29 f 7 122.71 27.73 f,g,h 175.15 49.46 a,c 204.34 26.47 g 8 148.71 25.01 f,h 233.29 34.23 a,b,c 288.17 41.80 d,e,f,h fig. 1 specimen positioned for three-point bending test at 37 °c. across groups, as determined by dunn's test (1-mm and 3-mm activations) and tukey's test (2-mm activation). at 1 mm and 2 mm of activation, aesthetic coating did not have a significant influence on the load-deflection ratio when comparing coated and uncoated wires from the same brand. at the 3-mm activation, load/deflection ratios were significantly increased in eurodonto coated wires and were reduced in ortho organizers wires. comparison between different brands revealed that, at 1 mm, 2 mm and 3 mm of activation, most groups were not significantly different, except for the eurodonto coated wires, which exhibited the highest load/deflection levels. at 3-mm activation, it was also observed that ortho organizers coated wires had lower load/deflection levels than those of morelli and eurodonto coated wires. discussion in the present study, the load-deflection ratio of orthodontic archwires made of nickel-titanium—the alloy of choice for the initial leveling and aligning phases of orthodontic treatment—was tested by means of a three-point bending test. as recommended in the literature, tests were performed without brackets, and ambient temperature was kept at 37 °c throughout the experiment21,24-27. the results showed that, at 1 and 2 mm of activation, aesthetic coatings did not have a significant influence on the load-deflection ratio of wires from the same brand. this is consistent with the findings of silva et al.20, neves et al.13, and washington et al.3. at 3-mm activation, however, coating did have an influence on the behavior of wires from two brands. in the present study, eurodonto coated wires exhibited increased load-deflection ratios compared with their uncoated counterparts. this result is in agreement with that obtained by bradley et al.21, who found increased load-deflection ratios in coated wires at greater activations and after clinical use. conversely, ortho organizers coated wires demonstrated decreased load-deflection force levels compared with uncoated wires from the same brand. this finding corroborates those of prior studies by elayyan et al.17, elayyan et al.23, alavi and hosseini2, kaphoor and sundareswaran28, and ryu et al.29, which found a reduction in load-deflection ratio in aesthetically coated wires. same lowercase letters in the same column denote absence of statistical significance (p > 0.05). different lowercase letters in the same column denote statistically significant differences (p < 0.05). 296 influence of aesthetic coating on the load-deflection ratio of nickel–titanium archwires on comparison across the four tested brands, eurodonto coated wires were found to exhibit the greatest force levels at all activations, whereas ortho organizers coated wires exhibited lower force levels than all other tested brands at 3-mm deflection, except for tp wires. interestingly, while coating tended to increase force levels in eurodonto, it had the opposite effect (reduction of force levels) in ortho organizers wires. similar results were reported by silva et al.20, who found different force levels in coated wires from several brands. according to previous studies28,30, this finding may be explained by the fact the archwires of some brands do not undergo a reduction in cross-section to compensate for the thickness of the aesthetic coating. conversely, ortho organizers wires have reduced cross-sections20,30. since the present study compared wires made of the same metallic alloy (niti), our results may not be explained by the type of alloy used for manufacturing the wires. thus, the diameter of the wire, along with the thickness of coating, were found to be the factors that have the greatest influence on load-deflection ratios, which became more evident at the greatest activation (3 mm). this aspect is relevant when choosing the wire to be used in the initial stages of alignment and leveling, especially in patients with moderate to severe crowding, in which wires with a low load-deflection ratio are needed to reduce the biological burden of orthodontic movement. the cross-sections of morelli and eurodonto coated archwires have not been analyzed in other studies. thus, by extrapolating the results of the present study to clinical practice, the presence of aesthetic coating on the archwire (in as-received condition) should not have a significant influence on load-deflection ratio, especially at lower activations (1 mm and 2 mm). however, other studies2,11,16-22 have reported substantial loss of coating in the oral milieu, with consequent increase in wire roughness. in cases of moderate to severe crowding, which require massive deflection, the increased friction caused by loss of the aesthetic coating could hinder achievement of proper alignment and leveling. conclusion in assays performed under the conditions of the present study, aesthetic coatings did not have a significant influence on the load-deflection ratio of niti orthodontic wires, especially at lower activations. however, comparison across the four tested brands revealed that eurodonto coated wires exhibited the greatest force levels at all activations. references 1. zhang m, mcgrath c, hagg u. patients' expectations and experiences of fixed orthodontic appliance therapy. impact on quality of life. angle orthod. 2007 mar;77(2):318-22. 2. alavi s, hosseini n. load-deflection and surface properties of coated and conventional superelastic orthodontic archwires in conventional and metal-insert ceramic brackets. dent res j (isfahan). 2012 mar;9(2):1338. 3. washington b, evans ca, viana g, bedran-russo a, megremis s. contemporary esthetic nickel-titanium wires: do they deliver the same forces? angle orthod. 2015 jan;85(1):95-101. doi: 10.2319/092513701.1. 4. russell js. aesthetic orthodontic brackets. j orthod. 2005 jun;32(2):14663. 5. aksakalli s, malkoc s. esthetic orthodontic archwires: literature review. j orthod res. 2013;1(1):2-4. 6. husmann p, bourauel c, wessinger m, jager a. the frictional behavior of coated guiding archwires. j orofac orthop. 2002 may;63(3):199-211. 7. brunharo ihvp, quintão kca. fios ortodônticos: indicação e aplicação clínica. ln: ortodontia em um contexto multidisciplinar. maringá: dental press; 2013. p.184-203. 8. ramadan aa. removing 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degradation on the frictional force of an experimental coated niti wire. angle orthod. 2011 may;81(3):484-9. doi: 10.2319/042810-232.1. 19. zegan g, sodor a, munteanu c. surface characteristics of retrieved coated and nickel-titanium orthodontic archwires. rom j morphol embryol. 2012;53(4):935-9. 20. silva dl, mattos ct, sant' anna ef, ruellas ac, elias cn. crosssection dimensions and mechanical properties of esthetic orthodontic coated archwires. am j orthod dentofacial orthop. 2013 apr;143(4 suppl):s85-91. doi: 10.1016/j.ajodo.2012.09.009. 21. bradley tg, berzins dw, valeri n, pruszynski j, eliades t, katsaros c. an investigation into the mechanical and aesthetic properties of new generation coated nickel-titanium wires in the as-received state and after clinical use. eur j orthod. 2014 jun;36(3):290-6. doi: 10.1093/ejo/cjt048. 22. rongo r, ametrano g, gloria a, spagnuolo g, galeotti a, paduano s, et al. effects of intraoral aging on surface properties of coated nickel-titanium archwires. angle orthod. 2014 jul;84(4):665-72. doi: 10.2319/081213-593.1 23. elayyan f, silikas n, bearn d. mechanical properties of coated braz j oral sci. 15(4):293-297 297influence of aesthetic coating on the load-deflection ratio of nickel–titanium archwires superelastic archwires in conventional and self-ligating orthodontic brackets. am j orthod dentofacial orthop. 2010 feb;137(2):213-7. doi: 10.1016/j.ajodo.2008.01.026. 24. kapila s, sachdeva r. mechanical properties and clinical applications of orthodontic wires. am j orthod dentofacial orthop. 1989 aug;96(2):1009. 25. krishnan v, kumar kj. mechanical properties and surface characteristics of three archwire alloys. angle orthod. 2004 dec;74(6):825-31. 26. yoneyama t, doi h, hamanaka h, okamoto y, mogi m, miura f. superelasticity and thermal behavior of ni-ti alloy orthodontic arch wires. dent mater j. 1992 jun;11(1):1-10. 27. parvizi f, rock wp. the load/deflection characteristics of thermally activated orthodontic archwires. eur j orthod. 2003 aug;25(4):417-21. 28. kaphoor aa, sundareswaran s. aesthetic nickel titanium wires--how much do they deliver? eur j orthod. 2012 oct;34(5):603-9. 29. ryu sh, lim bs, kwak ej, lee gj, choi s, park kh. surface ultrastructure and mechanical properties of three different white-coated niti archwires. scanning. 2015 nov-dec;37(6):414-21. doi: 10.1002/ sca.21230. 30. silva dl, mattos ct, de araujo mv, de oliveira ruellas ac. color stability and fluorescence of different orthodontic esthetic archwires. angle orthod. 2013 jan;83(1):127-32. doi: 10.2319/121311-764.1. braz j oral sci. 15(4):293-297 braz j oral sci. 15(4):242-247 comparative dimensional tomographic study of the mandibular condyle of individuals with transverse maxillary deficiency andréa guedes barreto gonçales1, victor tieghi neto2, eduardo sanches gonçales3, ana lúcia álvares capelozza4 1dds, msc. stomatology department. são paulo university. bauru dental school 2dds, phd. stomatology department. são paulo university. bauru dental school 3dds, phd. associated professor of oral and maxillofacial surgery. stomatology department. são paulo university. bauru dental school 4dds, phd. full professor of radiology. stomatology department. são paulo university. bauru dental school correspondence to: andréa guedes barreto gonçales fob/usp. departamento de estomatologia al. otávio pinheiro brizolla, 9-75. cep: 17017901 tel: +55 14 32358241 e-mail: andreagoncales72@gmail.com abstract transverse maxillary deficiency is characterized by posterior uni or bilateral crossbite, crowded and rotated teeth, as well as high palate. its treatment in adult individuals is surgically assisted rapid palatal expansion. the aim of this study was to verify the occurrence of dimensional alterations in the mandibular condyles of patients with tmd submitted to surgically assisted maxillary expansion. measurements of the mandibular condyles using the distance tool in cone beam computed tomography icat software were performed. the values obtained were submitted to statistical analysis by the paired t-test and the results showed statistically significant dimensional reduction in the axial posterior-anterior lateral (-0.74mm), axial posterior-anterior lateral left (-0.90mm) and coronal medium right (-1.24mm) dimensions. the coronal inferior (1.13mm), coronal inferior left (1.78mm) and coronal superior-inferior right (0.76mm) measurements showed statistically significant dimensional increase. the results allowed us to conclude that dimensional alterations occurred in the mandibular condyles in individuals with maxillary transversal deficiency that underwent surgically assisted rapid palatal expansion (sape), which can be understood by remodeling, since they are characterized by dimensional increase or reduction, depending on the location where the measurement was performed. keywords: computed tomography. malocclusion. mandibular condyle. introduction the dento-facial skeletal deformities can be defined as a defect of conformation of an organ or body part and result from changes in the growth and development of the bones of the face, especially maxilla and mandible. however other structures, organs and systems related to bones involved may be affected, causing problems in positioning of teeth, dental arches, masticatory function, phonation, swallowing, temporomandibular joints, breathing and facial aesthetics1. transverse maxillary deficiency (tmd) is characterized by posterior uni or bilateral crossbite, crowded and rotated teeth, as well as high palate2,3. there are three basic alternatives for the treatment of tmd: orthopedic maxillary expansion, surgically assisted palatal expansion (sape) and le fort i received for publication: july 06, 2016 accepted: august 25, 2016 original article braz j oral sci. october | december 2016 volume 15, number 4 http://dx.doi.org/10.20396/bjos.v15i4.8650028 243 segmental osteotomy. sape is indicated for the treatment of subjects with tmd larger than 5 mm, orthodontic/orthopedic expansion failure, need of expansion greater than 7 mm and for individuals with tmd and skeletal age of 15 years or more2,. the treatment of tmd in individuals with skeletal maturity is surgically assisted palatal expansion (sape) and this treatment has shown good results4, even with the description of different surgical techniques2. the effects of sape are observed not only in the dental arch5, but also in the adjacent structures6-10 and mandibular dental arch11. the procedure is not free of complications, such as the occurrence of pain in the temporomandibular joint (tmj) in 3% of the cases, mobility (3%) and loss (3%) of the upper central incisors, sinusitis (6%), paresthesia (3%), bleeding (3%), external resorption of teeth (3%) and displacement and tilt of the teeth (3%)12. cone beam computed tomography (cbct) has been used in the planning and follow up of oral and maxillofacial surgeries, allowing the comparison of bone changes13. since sape exerts effects on the adjacent maxillary structures, like changes in occlusion during this procedure, possible pain and changes in the tmj, and that cbct images allow the visualization and measurements of bony structures, the aim of the present study was to verify the occurrence of dimensional changes in mandibular condyles of individuals with tmd that underwent sape. material and methods in the present study, measurements were done by the use of cbct of the mandibular condyles. for this, preoperative and postoperative measurements (mm) of mandibular condyle of 14 individuals that underwent sape were done. it was approved by the research ethics committee of the bauru school of dentistry of são paulo university (protocol no. 1702011/11-30-2011). in axial view, measurements of the lateral-medial, posterior-anterior lateral, posterior-anterior central and posterior-anterior medial sizes of the articular surface were performed. in the coronal reformats, the superior lateral-medial, inferior lateral-medial, medium lateral-medial and superior-inferior sizes were measured. all scans were performed on an i-cat classic (imaging science international, hatfield, pennsylvania, usa) using the following protocol: 0.3mm voxel and extended height 20/20sec. the cbcts were filed on cd and the linear measurement tool (distance) of the i-cat vision program was used for obtaining the measurements as described below. on the i-cat vision homepage, we choose the name of the individual and open the cbct by double clicking on the individual name. after the cbct loads, the screen mpr option was selected, obtaining the sagittal, axial and coronal views (figures 1, 2 and 3). the standardization of the condyle positions was obtained as follows: the mandible image was positioned on sagittal reformatting showing the articular cavity, articular eminence, mandibular angle, ramus and condyle. in this image, we positioned the red reference line (corresponding to axial reformatting) at the lower point of the articular eminence surface. the blue reference line (corresponding to the coronal reformatting) was positioned in the center of the condyle, perpendicular to the red reference line. the distance tool was used to determine the center of the condyle by the measurement of the distance from the posterior to the anterior portion of the condyle surface on the red reference line. the condyle center corresponded to half the distance measured on the red reference line (figure 1) and all the measures done in the axial and coronal views (figures 2 and 3) were made with this standardization. the measures done on axial reformatting are shown in figure 2: a) the lateral-medial distance of the articular surface measured between the lateral and medial condyle poles (axial lateral-medial), b) posterior-anterior lateral size of the condyle articular surface measured in the lateral portion of the condyle, c) posterior-anterior central size of the condyle articular surface measured in the central portion of the condyle, d) posterior-anterior medial size of the condyle articular surface measured in the medial portion of the condyle. for the determination of the sites to be measured, the distance between the lateral and medial poles of the condyles was initially measured (axial lateral-medial measurement) on axial reformatting. the center of this measure determined the position of the axial posterior-anterior central measurement, which was measured perpendicular to the external surfaces. similarly, the axial posterior-anterior lateral and medial distances were measured, since its position corresponded to half the distance between the condyle center (axial posterior-anterior central measurement) and the lateral and medial poles, respectively (figure 2). comparative dimensional tomographic study of the mandibular condyle of individuals with transverse maxillary deficiency braz j oral sci. 15(4):242-247 fig.1. standardization of the positioning in sagittal reformatting. 244 the following measurements were performed on coronal reformatting: a) the lateral-medial superior size, measured from the lateral to medial surface at the bottom of the condyle joint surface (coronal superior measurement), b) medium medial-lateral size, measured from lateral to the medial, in the intermediate portion of the condyle, equidistant from the upper and lower measures, c) inferior medial-lateral size, measured from the lateral to medial in the lower portion of the condyle, and d) superior-inferior measure, taken from the articular surface of the condyle center until the lower cortical bone (figure 3). in a similar way implemented in axial reformatting, on coronal reformatting the locations to be measured were standardized as follows: the distance between the lateral and medial poles of the condyle were measured at the lower bound of the articular surface and this was the superior coronal measurement. at the center of this measurement, perpendicular to the outer surface of the articular surface, from upper to lower, the coronal superior-inferior measurement was done. the lower bound of this measure was the intersection of the central measurement line with the external cortical of the condyle. the distance between the coronal superior measurement and the lower limit of the condyle was then measured. it was then divided into three and the coronal medium measurement was measured in the lateral-medial sense in the upper third of the measure, while the coronal inferior measurement was performed in the lower third, also in the medial-lateral direction (figure 3). all measurements were obtained in preoperative and postoperative cbcts, tabulated and submitted to statistical analysis by paired t test. after 15 days of the completion of the first data collection14, all measurements were redone to determine the random and systematic error by the intraclass correlation coefficient (icc)15. results we used 28 cbcts from fourteen individuals with tmd, submitted to sape. five of the individuals were men and nine were women, with an average age of 24.28 years (dp 4.39). six individuals (42.86%) used the hass expander device while 8 (57.15%) used the hyrax expander device. the average postoperative period was 192 days (sd 16.03). table 1 shows the average (a), standard deviation (sd), difference between preoperative and postoperative averages (dif), paired t-test result (p), when the right and left condyles were considered together, while table 2 represents the average (a), standard deviation (sd), difference between preoperative and postoperative averages (dif), paired t-test result (p), when the right and left condyles were considered separately. in general, it was observed that there were changes in dimensional mandibular condyles, characterized by increase or decrease, depending on the site of the measure. the analysis of the condyles together (per individual) was shown to be similar to the analysis of the isolated condyles. thus, the results of the measures showed an increase in the axial lateral-medial (0.19mm), axial posterior-anterior central (0.15 mm), coronal superior (0.27 mm), coronal inferior (1.13 mm) and coronal comparative dimensional tomographic study of the mandibular condyle of individuals with transverse maxillary deficiency braz j oral sci. 15(4):242-247 fig.2. axial measurements: lateral-medial (red), posterior-anterior central (green), posterior-anterior lateral (pink) and posterior-anterior medial (light pink), carried out in the condyles. the other measures that appear (yellow, light blue and salmon) were performed to determine the locations of the axial measurements. fig.3. coronal superior measurements (red), coronal superior-inferior measurement (green), coronal medium measurement (salmon) and coronal inferior measurement (orange), carried out in the condyles on coronal reformatting. the other measures that appear (yellow, light pink, pink and light blue) were performed to determine the locations of the coronal measures. 245 comparative dimensional tomographic study of the mandibular condyle of individuals with transverse maxillary deficiency superior-inferior (0.48 mm) measures. the reduction of the measures was observed at the axial posterior-anterior lateral (-0.74 mm), axial posterior-anterior medial (-0.35 mm) and coronal medium (-0.28 mm) measures. only the axial posterioranterior lateral and coronal inferior measures were statistically significant (table 1). axial posterior-anterior central right (0.62 mm), axial posterioranterior central right (0.42 mm), coronal superior left (0.67 mm), coronal inferior right (0.47 mm) and left (1.78 mm) and coronal superior-inferior right (0.76 mm) and left (0.20 mm) measures. the isolated analysis of each condyle allowed us to also see a reduction in axial dimensions of the posterior-anterior lateral right (-0.58 mm) and left (-0.90 mm), posterior-anterior central left (-0.11 mm), posterior-anterior medial right (-0.05 mm) and left (-0.65 mm), coronal superior right (-0.15 mm) and coronal medium right (-1.24 mm). when we analyzed the condyles separately, apart from the axial posterior-anterior lateral left, coronal medium right, coronal superior left and coronal superior-inferior right measures (table 2), these values were not statistically significant. for error analysis, the icc was used; all measures were carried out by the same examiner and respecting the time of no less than 15 days. the icc was excellent, since all their values were greater than 0.75. discussion in individuals who have reached skeletal maturity due to the consolidation of craniofacial sutures16, the tmd treatment is orthodontic and surgical and the surgical step is known as surgically assisted maxillary expansion (sape)2,3. after the surgical procedure, maxillary expansion itself is accomplished by gradual, periodic and successive activation of the expander device that results in distribution of forces through the maxillas and adjacencies, which lead to the separation and enlargement of them17,18. the occurrence of tmj pain was evidenced in 3% of the cases of individuals subjected to sape12, which may be considered a low incidence, and the incidence of other complications was also low, even though those that occurred more often reached a maximum of 6% (sinusitis)12. the incidence of tmj pain allow us to believe that the anatomical configuration of the human face, especially the relationship between the maxilla and mandible, tmjs, muscles of mastication and the dental occlusion may lead to the occurrence of sape effects in the mandible. it should be noted that albuquerque et al.12 (2013) considered tmj pain in a generic form, perhaps not only including isolated pain in the tmj, but also muscle aches. since the mandibular condyle is one of the components of tmj, by the factors described above, it is subjected to the effects of sape. this study was a retrospective study that evaluated only the preoperative and postoperative cbct of the individuals submitted to sape. thus there are no conditions to discuss about signs and symptoms or even about clinical features as albuquerque et al.12 (2013) did. our results corroborate the aforementioned because there were changes in the dimensional mandibular condyles, characterized by increase or decrease, based in the preoperative and postoperative measurements carried out, depending on the measure. such changes could have taken place, for example, by the occlusal overload generated by premature dental contacts, leading to changes of mandibular position and eventually in the condyle remodeling. braz j oral sci. 15(4):242-247 table 1 average (a), standard deviation (sd), difference between preoperative (preop) and postoperative (postop) averages (dif), paired t-test result (p), when the right and left condyles were considered together (same individual) (*p<0.05). measure preop postop a sd a sd dif. p axial lateral-medial 18.32 2.51 18.51 2.53 0.19 0.372 axial posterior-anterior lateral 7.05 1.15 6.31 1.02 -0.74 0.001* axial posterior-anterior central 7.32 1.36 7.47 1.49 0.15 0.487 axial posterior-anterior medial 6.86 1.15 6.51 1.33 -0.35 0.147 coronal superior 14.87 2.48 15.14 2.76 0.27 0.320 coronal medium 13.62 2.53 13.33 2.85 -0.28 0.410 coronal inferior 7.25 1.21 8.37 1.78 1.13 0.000* coronal superior inferior 18.58 3.25 19.06 3.66 0.48 0.090 table 2 average (a), standard deviation (sd), difference between preoperative (preop) and postoperative (postop) averages (dif), paired t-test result (p), when the right and left condyles were considered separate (*p<0.05). r = right. l = left. measure preop postop a sd a sd dif. p axial lateral-medial r 18.61 2.51 18.65 2.64 0.05 0.860 axial lateral-medial l 18.03 2.56 18.37 2.51 0.34 0.344 axial posterior-anterior lateral r 6.61 1.18 6.03 0.97 -0.58 0.094 axial posterior-anterior lateral l 7.49 0.98 6.59 1.02 -0.90 0.005* axial posterior-anterior central r 6.89 1.36 7.31 1.67 0.42 0.303 axial posterior-anterior central l 7.74 1.27 7.63 1.33 -0.11 0.513 axial posterior-anterior medial r 6.69 1.51 6.65 1.43 -0.05 0.889 axial posterior-anterior medial l 7.02 0.63 6.37 1.26 -0.65 0.061 coronal superior r 14.54 2.72 14.39 2.70 -0.15 0.621 coronal superior l 15.21 2.27 15.89 2.70 0.68 0.121 coronal medium r 13.54 2.91 12.30 3.00 -1.24 0.000* coronal medium l 13.69 2.19 14.36 2.36 0.67 0.234 coronal inferior r 7.07 1.38 7.54 1.70 0.47 0.123 coronal superior l 7.42 1.04 9.20 1.48 1.78 0.000* coronal superior-inferior r 18.16 3.69 18.92 3.88 0.76 0.007* coronal superior-inferior l 19.00 2.81 19.20 3.58 0.20 0.694 when analyzed separately, each condyle showed an increase in axial lateral-medial right (0.05 mm) and left (0.34 mm), however, even when an increase in dimensions of axial measures occurs, when separately analyzing each condyle, only the axial posterior-anterior lateral left, coronal medium right, coronal superior left and coronal superior-inferior right measurements were statistically significant (*p<0.05). such fact might demonstrate that the remodeling occurs preferentially in certain areas and these areas may vary according to the occlusal interference and because of this, they are asymmetrical. it is expected that where there was a statistically significant decrease of the measures, the condyle was reabsorbed and a statistically significant increase occurred because of bone formation. the tmj is a bilateral joint in which both sides work simultaneously, and therefore we also evaluated the condyles in sets. similarly, only the axial posterior-anterior lateral and coronal inferior measures were statistically significant. it is believed that, even when the individual is considered, the occlusal interference might be responsible for possible asymmetric condyle remodeling. although studies19-21 showed that occlusal forces can affect the condyle morphology and may lead to condyle remodeling and cbct was a useful tool for monitoring condylar bone changes, it should be noted that the statistical result found can be due the number of studied subjects or the methodology employed. this study did not verify the occurrence of occlusal changes and premature contacts, as well as the presence or absence of symptoms related to tmjs or masticatory muscles. thus, one can only infer the occurrences just described. with regard to the method employed, the applicability of cbct in dentistry appears to be questionable because some studies19,22-26 claimed that the cbct was a reliable and accurate method for verification of changes and carry out measures in tmj as a whole and specifically in the mandibular condyles. this is especially true when the sagittal and coronal views are considered, being efficient in the determination of changes related to the positioning and shape of the condyle and joint surfaces27,28. it is believed that cited studies , corroborated the methodology used, since they correlated cbct with dimensional and positioning and shape changes of condyles. despite having difficulties in detection by the cbct of the condylar defect smaller than 2mm29,30, this difficulty would not be expected to occur in the present study, since the distances representing the condylar dimensions were measured, but not bone defects. t. in addition, the icc15 was excellent, confirming the accuracy of the measurements performed. thus, based on the methodology used and the sample studied, it seems justified to conclude that in individuals with tmd submitted to sape, dimensional changes occur in the mandibular condyles, perhaps by remodeling, since they were characterized by an increase and reduction of the condylar dimensions. references 1. assis dsfr, duarte ma, gonçales es. clinical evaluation of the alar base width of patients submitted to surgically assisted maxillary expansion. oral maxillofac surg. 2010 sep;14(3):149-54. doi: 10.1007/s10006-0100211-3. 2. betts nj, vanarsdall rl, barber hd, higgins-barber k, fonseca rj. diagnosis and 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gp. assessment of linear and angular measurements on three dimensional cone-beam computed tomographic images. oral surg oral med oral pathol oral radiol endod. 2009 sep;108(3):430-6. doi: 10.1016/j.tripleo.2009.01.032. 26. zain-alabdeen eh, alsadhan ri. a comparative study of accuracy of detection of surface osseous changes in the temporomandibular joint using multidetector ct and cone beam ct. dentomaxillofac radiol. 2012 mar;41(3):185-91. doi: 10.1259/dmfr/24985981. 27. accorsi m, velasco l. 3d diagnosis in orthodontics: the cone-beam tomography applied. nova odessa, são paulo: napoleão; 2011. portuguese. 28. bayrama m, kayipmazb s, sezginb os, küc m. volumetric analysis of the mandibular condyle using cone beam computed tomography. eur j radiol. 2012 aug;81(8):1812-6. doi: 10.1016/j.ejrad.2011.04.070. 29. bastos lc, campos psf, ramos-perez fmm, pontual aa, almeida sm. evaluation of condyle defects using different reconstruction protocols of cone-beam computed tomography. braz oral res. 2013 nov-dec;27(6):503-9. doi: 10.1590/s1806-83242013000600010. 30. patel a, tee bc, fields h, jones e, chaudhry j, sun z. evaluation of cone-beam computed tomography in the diagnosis of simulated small osseous defects in the mandibular condyle. am j orthod dentofacial orthop. 2014 feb;145(2):143-56. doi: 10.1016/j.ajodo.2013.10.014. 247 comparative dimensional tomographic study of the mandibular condyle of individuals with transverse maxillary deficiency braz j oral sci. 15(4):242-247 oral sciences n3 original article braz j oral sci. october|december 2010 volume 9, number 4 association between temporomandibular disorder and body mass index in institutionalized children tatiana o. de santis1, manoela d. martins2, lara j. motta3, olga m. s. amancio4, kristianne p. s. fernandes5, sandra k. bussadori6 1dds, msc student, rehabilitation sciences program, university nove de julho, uninove, são paulo, sp, brazil 2ph.d. in oral pathology, professor of the master’s degree program in rehabilitation sciences, university nove de julho – uninove, são paulo, sp,brazil 3msc in rehabilitation sciences. professor of pediatric dentistry, university nove de julho – uninove, são paulo, sp, brazil 4ph.d. associate professor, department of pediatrics, federal university of são paulo, são paulo, sp brazil 5ph.d. in immunology, professor of the master’s degree program in rehabilitation sciences, university nove de julho – uninove, são paulo, sp, brazil 6dds, msc, ph.d., professor of the master’s degree program in rehabilitation sciences, university nove de julho – uninove, são paulo, sp, brazil received for publication: june 30, 2010 accepted: september 17, 2010 correspondence to: tatiana oliveira de santis rua bom jesus 1151, apto. 62ª cep 03344-000 tatuapé são paulo sp brasil phone: (11) 77370483 e-mail: tasamara@bol.com.br abstract temporomandibular disorders (tmd) is a term applied to functional changes of the temporomandibular joint (tmj) and associated structures of mastication. aim: to investigate the correlation between body mass index (bmi) and tmd in children. methods: 70 children between 6 and 14 years enrolled in the rogationist benevolent institute of charity in the city of são paulo (brazil) underwent clinical examination by one calibrated examiner for the use of research diagnostic criteria for tmd. weight and height were determined by anthropometric assessment for the diagnosis of nutritional status, and bmi was calculated by dividing weight in kilograms by height in meters squared (kg/m2). descriptive analysis (mean and standard deviation) was used to characterize the sample. anova complemented by the least significant difference test was used to compare the mean anthropometric measurements between the genders and the groups with and without tmd. the significance level was set at 0.05. results: we found a significantly high prevalence of tmd, as well as a significant association between tmd light and low body weight. conclusions: the results show a high prevalence of tmd and a slight association between tmd and children with low body weight. keywords: temporomandibular disorders, stomatognathic system, body mass index, orofacial structures, masticatory muscles. introduction temporomandibular disorders (tmd) is a blanket term applied to functional abnormalities of the temporomandibular joint (tmj) and associated masticatory structures1. the main signs of tmd are joint noises and limited range of motion or deviation during mandible function, while the main symptom is pain in the preauricular region, tmj and/or masticatory muscles2-4. tmd has a multi-factor etiology, including occlusal disharmony, parafunctional oral habits and posture imbalance as well as psychosocial and behavioral alterations5. braz j oral sci. 9(4):459-463 mastication is the action of biting, shredding and pasteurizing food. it is considered the most important function of the stomatognathic system, as it is the initial phase of the digestive process, in which foods are mechanically broken down to an adequate size for swallowing. the force, movements, duration and type of mastication are influenced by morphology, health status of orofacial structures, functional capacity of muscles and joints and characteristics of the food. a number of studies on the association between the prevalence/severity of tmd in children and types of food ingested have demonstrated a significant impact of tmd on the quality of life of affected individuals when compared to those with no complaints of pain. functional problems, such as difficulty chewing certain foods, are reported to be fourfold higher among patients with tmd6-11. however, no studies were found in the literature correlating tmd with nutritional status in children. monitoring nutritional status is important in all age groups and it is at the core of healthcare actions directed at childhood and adolescence. the importance of nutritional status in these phases of life is related to the growth and development process. poor nutrition can lead to early health problems and the risk of disease, especially considering the growing prevalence of overweight/obesity in the world12-15. the world health organization recommends assessing the nutritional state of children using weight/height and height/age indicators16. in recent decades, different criteria for this assessment have emerged and the body mass index (bmi) had been proposed as an indicator of nutritional status 17-21. the bmi is used to identify possible weight problems. the centers for disease control and prevention and american academy of pediatrics recommend the use of the bmi to determine overweight beginning at two years of age. however, it is not a diagnostic tool. for example, the bmi may indicate whether a child is overweight; however, in order to determine whether the excess body fat is a problem, the physician needs to perform tests such as the measurement of skinfolds and carry out assessments of diet, degree of physical activity, family history, etc.22. in children, along with the weight variation, the bmi also changes with height and age and its direct application is therefore unsatisfactory. the calculation of bmi percentile is based on weight and height percentile tables and is more adequate for use on children. another simple method is the use of graphs that correlate weight and height by age and provide a clear, immediate visualization of the magnitude of the disproportion between weight and height. if the height of a child is between the 50th and 75th percentiles, ideal weight is located between these same percentiles. using a curve, the estimate of the amount of weight that should be lost (when the degree of excess weight is considerably large) can be determined or the amount of time in which the current weight should be maintained until it is adequate for the height (when the excess weight is not accentuated) can be estimated. the bmi percentile actually only numerically expresses the extent to which a child’s weight deviates from normal values on the weight versus height-for-age curve23. considering the close relationship eating habits have with bmi and the development of tmd, the aim of the present study was to investigate the association between bmi and tmd in brazilian children. material and methods a cross-sectional study was carried out for the assessment of the association between the body mass index and temporomandibular disorder among 70 children between 6 and 14 years of age enrolled at the rogationist benevolent institute of charity in the city of são paulo (brazil). the following were the inclusion criteria: age between 6 and 14 years; presence of primary 2nd molar and permanent 1st molar among children from 6 to 10 years of age; and presence of permanent 1st molar among children from 11 to 14 years of age. the following were the exclusion criteria: current medical, psychological or dental treatment; and dental/facial deformities. all individuals had the same eating pattern – 4 daily meals under the supervision of a nutritionist on staff at the institution. the study was carried out in compliance with the norms that regulate studies involving human subjects in resolutions 196/96 and 251/97 of the brazilian national health council and received approval under process nº 249781. the parents/guardians of the children signed informed consent forms. all participants responded to a screening questionnaire for orofacial pain and temporomandibular disorder recommended by the american academy of orofacial pain, made up of 10 self-explanatory questions with yes/no answers on the most common signs and symptoms of tmd2. the participants were submitted to a clinical exam by single examiner calibrated for use of the research diagnostic criteria for tmd2 (axis i)2. the exam constituted an extraoral and intraoral inspection of the teeth and occlusion, palpation of the trapezium, sternocleidomastoid, temporal, masseter, digastric and medial pterygoid muscles, palpation of the tmjs, determination of joint noises and analysis of mandible movement using a digital pachymeter (mytutoio®) for the measurement of maximal mouth opening and lateral movement. information on frequent headaches, facial pain, facial muscle fatigue, difficulty chewing, teeth grinding, psychological aspects, non-nutritive sucking habits and nail biting was also collected. weight and height were determined for the anthropometric evaluation. weight was determined on a digital scale (filizola) with a capacity of 150 kg and a precision of 100 g, with the individual barefoot and wearing shorts and a tshirt. height was determined with the individual barefoot, standing erect against a flat vertical surface, arms down, palms of the hands on the thighs, heels together and feet apart, knees in contact and head adjusted to the frankfurt plane. the measurement was taken using a stadiometer with a capacity of two meters and precision of 0.1 cm. for the diagnosis of nutritional state, the body mass index (bmi) was calculated dividing weight in kilograms by height in meters squared (kg/m2). the children were classified 460460460460460association between temporomandibular disorder and body mass index in institutionalized children braz j oral sci. 9(4):459-463 age without tmdn (%) with tmdn(%) total 6 years 1 (20%) 4 (80%) 5 (100%) 7 years 2 (25%) 6 (74%) 8 (100%) 8 years 2 (16.7%)* 10 (83.3%)* 12 (100%) 9 years 0 6 (100%) 6 (100%) 10 years 5 (35.7%)* 9 (64.3%)* 14(100%) 11 years 4 (50%) 4 (50%) 8 (100%) 12 years 3 (37.5%) 5 (62.5%) 8 (100%) 13 years 3 (33.3%) 6 (66.7%) 9 (100%) total 20 (28.6%) 50 (71.4%) 70 (100%) table 2. number and percentage of participants with and without tmd according to age * statistically significant association; p<0.05 n(%) mean bmi(standarddeviation) underweight healthy weight risk of overweight overweight 6 years 5 (7.1%) 16.81 (2.08) 3 (60%) 2 (40%) 0 0 7 years 8 (11.4%) 16.36 (2.26) 6 (75%) 1 (12,5%) 0 1 (12.5%) 8 years 12 (17.1%) 17.04 (1.62) 10 (83.3%) 1 (8.3%) 0 1 (8.3%) 9 years 6 (8.6%) 17.69 (2.82) 0 6 (100%) 0 0 10 years 14 (20.0%) 18.73 (3.97) 0 11 (78.6%) 2 (14.3%) 1 (7.1%) 11 years 8 (11.4%) 20.17 (3.18) 2 (25%) 4 (50%) 1 (12.5%) 1 (12.5%) 12 years 8 (11.4%) 20.83 (2.45) 0 5 (62.5%) 3 (37.50%) 0%) 13 years 9 (12.9%) 16.81 (2.08) 1 (11,1) 5 (55.6%) 3 (33.3%) 0 table 1. mean bmi and weight classification according to age considering bmi values according to gender and age using the percentile distribution proposed by must et al.13, which was designed for the classification of adults and children beginning at 6 years of age according to gender, age and race23. the calculated bmi was then plotted on the bmi diagram for age and gender in order to determine the percentile classification. the data were organized in tables and graphs and submitted to statistical tests. descriptive analysis (mean and standard deviation) was used for the characterization of the sample. anova complemented by the least significant difference test, was used to compare the mean anthropometric measurements between genders and groups with and without tmd. the level of significance was set at 0.05. results among the 70 children evaluated, 39 (54.92%) were female and 31 (45.07%) were male; mean age was 9.67 years (standard deviation = 2.152); 50 (71.4%) exhibited some degree of tmd, 27 (54%) of whom were female. there was no significant association between gender and tmd (p=0.648). among the individuals with tmd, 29 (58%) had mild tmd, 16 (32%) had moderate tmd and 5 (10%) had severe tmd. table 1 displays the mean and standard deviation values for bmi in relation to age and the percentile classification of the age category. thirty-five children (50.0%) had a healthy weight; 9 (12.9%) were at risk of overweight; 22 (31.4%) were underweight; and 4 (5.7%) were overweight. table 2 displays the distribution of individuals according to age and presence/absence of tmd. there was a statistically significant association (p<0.05) between the presence of tmd and age among the participants between eight and 10 years of age. the distribution of individuals according to degree of tmd and bmi per percentile reveals that, among the 50 children with tmd, 4 (8%) were underweight, 40 (80%) had a healthy weight, 5 (10%) were at risk of overweight; and 1 (2%) was overweight. among the 20 children without tmd, one (5%) was underweight, 12 (60%) had a healthy weight, four (20%) were at risk of overweight and 3 (15%) were overweight. analyzing the degree of tmd in relation to bmi per percentile, there was a statistically significant association (p<0.05) between underweight children and mild tmd (table 3). discussion the present study found a high prevalence of tmd among institutionalized children as well as a significant association between mild tmd and low body weight. these data corroborate the theory that tmd has a multifactor origin caused by the association of psychological, structural and postural factors that disharmonize the functional balance between the three fundamental elements of the stomatognathic system: dental occlusion, masticatory muscles and tmj16,14,17,21,24,25 the etiological factors of tmd in children are similar to those in adults. it is widely accepted that there are predisposing, initiating and perpetuating factors. predisposing factors increase the risk of tmd and are divided into systemic, psychological and structural. initiating factors cause the onset of tmd. perpetuating factors affect the progression and cure of this condition14-24. the multifactor etiology of tmd in young populations involves parafunctional habits, trauma and occlusal, systemic and psychological factors. the prevalence of tmd in children varies considerably with patient age and the criteria used during the examination. comparing different studies on the prevalence of tmd in children, pahkala and laine (1991)24 found a prevalence of signs and symptoms of tmd of 40% among 5-to-8-yearolds, 46% among 9-to-12-year-olds and 31% among 13-to461461461461461 association between temporomandibular disorder and body mass index in institutionalized children braz j oral sci. 9(4):459-463 underweight healthy weight risk of overweight overweight total n % n % n % n % n % without tmd 1 5.0 12 60.0 4 20.0 3 15.0 2 100.0 mild tmd 21* 72.4 4 13.8 4 13.8 0 0 2 100.0 moderate tmd 0 0 14 87.5 1 6.3 1 6.3 1 100.0 severe tmd 0 0 5 100.0 0 0 0 0 5 100.0 0 9 6 table 3. distribution of individuals according to the severity of tmd and bmi per percentile * statistically significant association; p<0.05 15-year-olds; the authors report that the signs and symptoms of tmd in children range from mild to moderate and may even be unconscious, thereby reflecting physiological and psychological changes, rather than a pathology24. there was no association between an increase in age and the presence of tmd in the present study, but previous studies have reported such an association. a stronger association between age and the presence of tmd was found among the participants between eight and 10 years of age. according to bertoli et al.25 (2009), during the transition from the primary to the permanent dentition and the growth and development of the craniofacial complex, children may exhibit disorders, as a series of adaptive changes in the tmj occurs in this period25. mastication is one of the most important functions of the stomatognathic system and is influenced by a number of general and local factors. general factors include changes in eating habits influenced by culture as well as the socioeconomic and psychological profiles of the population. local factors that affect mastication include occlusal equilibrium, the presence/absence of teeth, dental health and status of the tmj. besides directly influencing the function of the stomatognathic system, eating habits have been associated to nutritional status, as measured by the bmi26-28. the present investigation is the first study to demonstrate an association between mild tmd and low body weight in children, which may indicate that eating habits not only influence the bmi, but may also contribute toward the development of tmd. the children analyzed in the present study had the same eating pattern, as they were all offered the same types of food. thus, it may be inferred that the variations in bmi per percentile are associated to the amount rather than the quality of the food ingested. murray et al. 29 (1996) analyzed 121 adults in order to determine whether there is an association between tmd, bmi, eating habits and quality of life. the authors found that the bmi among individuals with tmd deviated from normal values toward both overweight and underweight and such individuals exhibited orofacial pain, psychological disorders and a lower quality of life. individuals with mild to moderate tmd had a lower bmi than those without tmd. the present study corroborates these findings and lends support to the statement by murray et al. 29 (1996) that one of the explanations for this association is that the difficulty in chewing foods may be governed by the characteristic pain during mastication in cases of tmd29. it should be stressed that many of the individuals analyzed in the present study were in the mixed dentition phase, which is a transition stage of the occlusion. as tmd has a multifactor etiology and may be the result and/or cause of psychological and eating alterations, multidisciplinary treatment involving a physician, dentist, nutritionist, psychologist and physical therapist is needed while the patient is still in childhood. moreover, the signs and symptoms of tmd tend to exacerbate with age 28. therefore, preventive and therapeutic measures need to be initiated early. further studies need to be carried out for a detailed assessment of other aspects involved in the physiopathology of tmd and its correlation with nutritional status and psychological state. in conclusion, the results of the present study reveal a high prevalence of tmd and an association between mild tmd and children with low body weight. references 1. american society of temporomandibular joint surgeons (astjs). guidelines for diagnostic and manegement of desordens involving the temporomandibular joint and related musculoskeletal atructures. cranio. 2003; 21: 68-76. 2. 02. chaves tc, oliveira as, grossi db. principais instrumentos para avaliação da disfunção temporomandibular, parte i: índices e questionários; uma contribuição para a parte clínica e de pesquisa. fisioter pesq. 2008; 15: 92-100. 3. 03. dworkin sf, huggins kh, leresche l, von korff m, howard j, truelove e, et al. epidemiology of signs and symptoms in temporomandibular disordens: clinical signs in case and controls. j am dent assoc. 1990; 120: 273-81. 4. bertoli fmp, antoniuk sa, bruck i, xavier grp, rodrigues dbc, losso em. evaluation of the sings and symptoms of temporomandibular disorders in children with headaches. arq. 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http://www.abeso.org.br/revista/revista11/metodos.htm. access. 22. dworkin sf, leresche l. research diagnostic criteria for temporomandibular disorders: review, criteria, examinations and specifications, critique. j craniomandib disord. 1992; 6: 301-55. 23. biasotto-gonzalez da, bérzin f. electromyographic study of patients with masticatory muscles disorders, physiotherapeutic treatment (massage) braz j oral sci. 2004; 3: 516-21. 24. munõz gc, silva c,misaki jk,gomes igd, carvalho arrc. analise dos potenciais elétricos do músculo masseter durante a mastigação de alimentos com rigidez variada. rev cefac. 2004; 6: 127-34. 25. moreno bgd, maluf sa, marques ap, crivello-júnior o. avaliação clínica e da qualidade de vida de indivíduos com disfunção temporomandibular. rev bras fisioter. 2009; 3: 210-4. 26. bertoli fmp, losso em, noresca rc. disfunção da articulação temporomandibular em criançasrevisão de literatura. rsbo ver sulbras odontol. 2009; 6: 83-7. 27. amantéa dv, novaes ap, 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parameters in the mining employees and the general population a comparative study tarulatha r shyagali1*, mohamed helmy salama2, deepak p bhayya1 *corresponding author: college of dentistry, majmaah university, al zulfi e mail id:drtarulatha@gmail.com, t.shyagali@mu.edu.sa mobile no:0504793029 received: february 6, 2017 accepted: august 10, 2017 1 associate professor, college of dentistry, majmaah university, al zulfi ksa 2 assistant professor, college of dentistry, majmaah university, al zulfi, ksa. associate professor, college of dentistry, al-azhar university, egypt. objective: comparison of the prevalence of usage of tobacco products and its effect on the periodontal health parameters of the mining laborers and the general population of udaipur city, india. materials and methods: the cross sectional study was performed on the sample of 980 adults including 500 mining employees and 480 subjects from the general population. the presence or absence of the tobacco products usage was noted. the ada type iii examination using the community periodontal probe was done. the data obtained was subjected to chi-square and cramer’ v statistical analysis. results: 79.40% of the mining-employees and 61.67% of the general population used different tobacco products, the difference noted was statistically significant (p=0.000). the most common periodontal condition amongst the mining employees and the general population was the presence of calculus (39% and 35.63% respectively) followed by the 4-5mm periodontal probing depth (29.20% and 26.04% respectively). the difference spotted was significant (p=0.018). there was statistically significant difference between the tobacco users and nonusers for the different periodontal conditions (p=0.000). conclusion: the tobacco product usage was high in mining employees group. there was a positive correlation between the usage of tobacco products and the periodontal health parameters. keywords: mining, tobacco use, periodontium, community periodontal index of treatment needs 2 shyagali et al. introduction mining is one of the ancient occupations in the world. it’s viability to cause the disease and injury are well known1. there are thousands of people who are working in the mining sector2 and the literature is explicit with the ill effects of mining on the general health of the mining employees3,4. however, the studies related to oral health condition on the mining employees are very scanty and whatever the few, have explored the areas of oral health conditions like dental caries, the periodontal status, dental wear, cancerous and precancerous lesions5-10. the dust swirling from the mining areas, the populated air and water can be hazardous to human health and it can affect the oral health as well. in addition, the strenuous working condition drives the miners to indulge in adverse habits which can aggravate the ill health condition caused due to the polluted environment. these habits are known to cause the detoriation of oral health. among the various adverse habits in which the mining employees indulge, oral smokeless tobacco (slt) and the smoking remain the most prominent. india being a vast country has many states and each state uses its own variant of tobacco product. bidi and cigarette are widely consumed in india11.the smokeless tobacco is consumed either through oral or nasal route. various slt used across in india are gutkha, zarda, mawa and khaini. but, the most popular product among the smokeless tobacco is khaini12. these are either consumed alone or in various combinations with the other tobacco products13. accordingly, it’s been reported that one in four adults and one in ten school students (13-15 years) in india use slt11. the studies have also shown that the oral tobacco products available commercially in india and other asian countries are highly toxic owing to the presence of more than 4000 chemicals and are known to be highly carcinogenic and mutagenic14,15. the prevalence of periodontal disease in india varies from one state to other state, but the 100% prevalence of periodontal disease is reported in the state of rajasthan and the orissa16. the association between the tobacco consumption and the periodontal disease is a well-established fact. both the forms of tobacco are known to cause poor periodontal status, however the smokeless tobacco caused greater attachment loss17. about 95% of the indian population suffers from periodontal diseases18. according to world health organization (who) report (1997) in india prominent tobacco products used were bidis (34%), cigarettes (31%), chewing tobacco (19%), hookah (9%), cigars-cheroots (5%), and snuff (2%)19. however, the change in the trend of usage of tobacco product was noticed in the recent survey by cancer patient association of india, their report suggests that the prevalence of the tobacco usage was in the form of cigarettes (20%), bidis (40%) and the rest 40% was consumed in the form of chewable tobacco, pan masala, snuff, gutkha, masheri and tobacco toothpaste20. with the noticed trend of tobacco consumption popularity in this part, this study was taken up with the aim to evaluate the periodontal status of the mining employees of the udaipur city, india and its association with the prevalence of usage of tobacco products. the study also compared the periodontal status of the mining employees with that of the general population. 3 shyagali et al. materials and methods the cross sectional study was done on 500 mining employees and 480 general populations of age group 18-50 years (table 1). the comparative group was selected from the population which visited the dental hospital for the routine dental visits. initially a list of all the mining companies situated in and around the udaipur city was made. the lottery system was employed to select the different regions for the collection of the data. the post graduate research board of mohanlal sukhadia university granted the ethical clearance. the prior permission was obtained from the mining owners for examining the mining employees. the purpose of the study was explained to the subjects and the signed informed consent was obtained before the examination. a questionnaire was prepared to procure the demographic details of the individuals and to record the presence and absence of the tobacco usage. the subjects who consumed any of the tobacco products less than four times a week, who had a systemic disease and who were on medication for such diseases were excluded from the study. further, the subjects who were indulged in the usage of tobacco products for more than four times a week and for not less than a year were considered for the study. the questionnaire was filled by the single examiner and the same examiner examined the patient. american dental association (ada) type iii examination was carried out with the minimal equipment’s which included mouth mirror, community periodontal probe and the chair. the examination of the periodontium was done as per the recommendation of the who21. the natural light was used during the examination if the natural light was insufficient than the torch light was used. the cold sterilization procedure was employed to sterilize the instruments. the collected data was tabulated and the data was subject for the statistical analysis. the chi-square test and the cramer’s v test were performed to evaluate the difference between the mining and nonmining population and between the tobacco users and the non-users. results table 1 show the sample distribution of the study population. there were 980 subjects belonging to the age group of 18-50 years. there were 500 mining employees and the control group comprised of 480 subjects from the general population who were not exposed to the mining environment and who belonged to the udaipur city proper. the main aim of including the control population was to know whether mining environment apart from the tobacco usage has any influence on the periodontal status of the employees. the demographic data obtained showed that the employees belong to different strata of the working hierarchy, depending upon this the study population was divided into administrative group, maintenance group, transportation group. cutting and polishing groups. table 2 shows the sample distribution pertaining to the mining employees. table 1. distribution of study population based on groups study population no % marble mining employees (study group) 500 51.02 general population (comparative group) 480 48.98 total 980 100 4 shyagali et al. the table 3 depicts the data in relation to the usage of the tobacco products by the mining employees and the general population. amongst the mining employees, 79.40% indulged in tobacco products, whereas, 61.67% of the general population used the tobacco products. the difference noted was statistically significant. the results pertaining to the commonly used tobacco products among the study population is illustrated in table 4. cigarette, bidi, tobacco leaf, pan, and gutkha were most commonly consumed tobacco products amongst both the study and comparative population. among mining employees, a majority were using gutkha (38.60%) followed by tobacco chewing (19.20%) and bidi smoking (16.0%). similar trend was noted for the general population in terms of usage of different tobacco products with gutkha being the most popular product (22.08%), followed by tobacco chewing (15.42%) and bidi smoking (13.75%). comparison of prevalence of various tobacco habits between mining employees and general population yielded statistically significant differences in the prevalence of pan chewing (p=0.004), and gutkha chewing (p=0.000). within mining employees’ highest prevalence of gutkha (52.14%) was seen among of transportation unit workers. while highest prevalence of bidi smoking was seen among polishing unit workers (28.79%) compared to other units. whereas prevalence of cigarette smoking and pan chewing was highest among administrative unit. the differences in use of tobacco products among mining employees were statistically significant for all the tobacco products (p<0.001) except for the tobacco leaf. table 2. distribution of marble mining labourers based on work units mining employees (m.e) no % administrative unit (a) 43 8.60 maintenance unit (m) 45 9.00 transportation unit (t) 140 28.00 cutting unit (c) 140 28.00 polishing unit (p) 132 26.40 total 500 100 table 3. distribution of study population according to use of tobacco products tobacco use factory employees (f.e) f.e total g.p total a m t c p user no 33 37 120 105 102 397 296 693 % 76.74 82.22 85.71 75.00 77.27 79.40 61.67 70.71 non-user no 10 8 20 35 30 103 184 287 % 23.26 17.78 14.29 25.00 22.73 20.60 38.33 29.29 total no 43 45 140 140 132 500 480 980 % 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 chi-square test χ2 = 5.839, df = 4, p= 0.211 (>0.05), not sig. χ2 = 37.188, df = 1, p = 0.000 (<0.001) very high sig. cramer’s v 0.108 0.195 5 shyagali et al. the data pertaining to the periodontal health parameters is shown in the table 5. the most common periodontal condition spotted amongst the mining employees and the general population was the presence of calculus (39% and 35.63% respectively) followed by the 4-5mm periodontal probing depth (29.20% and 26.04% respectively). the difference spotted between the two groups was significant. table 6 represents the data for the periodontal health status of the mining and the general population in the tobacco and non-tobacco users. the healthy periodontal parameters were appreciated in only 2.02% of the tobacco user mining population and in 29.13% of the non-tobacco users. the difference noted was statistically significant. there was statistically significant difference between the tobacco users and nonusers for the different periodontal conditions in both the mining employees group (p<0.001) and the general population (p<0.001). discussion the indulgence in tobacco has multitude of oral health problems which can range from developing oral cancerous and precancerous lesions to the detoriation of periodontal health. it can also cause delay in oral wound healing, can contribute significantly for the halitosis, can cause staining of teeth, can trigger negative immune table 4. distribution of study population according to type of tobacco products tobacco use mining employees m.e total g.p total a m t c p cigarette no 08 04 10 00 00 22 30 52 % 18.60 8.89 7.14 0.00 0.00 4.40 6.25 5.31 chi-square test χ2 = 37.805, df = 4, p = 0.000 (<0.001) very high sig. χ2 = 1.668, df = 1, p = 0.197 (>0.05) not sig. cramer’s v 0.275 0.041 bidi no 01 08 08 25 38 80 66 146 % 2.33 17.78 5.71 17.86 28.79 16.00 13.75 14.90 chi-square test χ2 = 33.529, df = 4, p = 0.000 (<0.001) very high sig. χ2 = 0.978, df = 1, p = 0.323 (>0.05) not sig. cramer’s v 0.259 0.032 tobacco leaf no 08 10 28 25 25 96 74 170 % 18.60 22.22 20.00 17.86 18.94 19.20 15.42 17.35 chi-square test χ2 = 0.501, df = 4, p = 0.973 (>0.05) not sig. χ2 = 2.445, df = 1, p = 0.118 (>0.05) not sig. cramer’s v 0.032 0.050 pan no 04 00 01 00 01 06 20 26 % 9.30 0.00 0.71 0.00 0.76 1.20 4.17 2.65 chi-square test χ2 = 26.553, df = 4, p = 0.000 (<0.001) very high sig. χ2 = 8.346, df = 1, p = 0.004 (<0.01) highly sig. cramer’s v 0.230 0.092 gutkha no 12 15 73 55 38 193 106 299 % 27.91 33.33 52.14 39.29 28.79 38.60 22.08 30.51 chi-square test χ2 = 18.825, df = 4, p = 0.001 (<0.01) highly sig. χ2 = 31.511, df = 1, p = 0.000 (<0.001) very high sig. cramer’s v 0.194 0.179 6 shyagali et al. response, can alter the ph of the oral cavity22,23.the current article explored the association of usage of tobacco products and the periodontal health parameters in the mining employees and the general population. around 79.40% and 61.67% of the mining employees and the general population indulged in one or the other form of tobacco usage. the difference found was very highly significant indicating that the tobacco usage was more common in the mining employees and the reason for this might be the tobacco can act as a relaxer after the strenuous table 5. distribution of study population according to community periodontal index (cpi) scores. cpi factory employees f.e total g.p. total a m t c p healthy no 10 8 5 8 7 38 50 88 % 23.26 17.78 3.57 5.71 5.30 7.60 10.42 8.98 bleeding no 5 10 20 26 28 89 116 205 % 11.63 22.22 14.29 18.57 21.21 17.80 24.17 20.92 calculus no 15 16 60 44 60 195 171 366 % 34.88 35.56 42.86 31.43 45.45 39.00 35.63 37.35 4 -5 mm no 11 8 46 51 30 146 125 271 % 25.58 17.78 32.86 36.43 22.73 29.20 26.04 27.65 6mm or more no 2 3 9 11 7 32 18 50 % 4.65 6.67 6.43 7.86 5.30 6.40 3.75 5.10 total no 43 45 140 140 132 500 480 980 % 100 100 100 100 100 100 100 100 chi-square test χ2 = 40.351, df = 16, p = 0.001 (<0.01) highly sig. χ2 = 11.910, df = 4, p = 0.018 (<0.05) sig. cramer’s v 0.142 0.110 *a administrative unit; m-maintenance unit; t-transportation unit; c-cutting unit; p-polishing unit; f.e –factory employees; g.p-general population table 6. distribution of cpi scores among user and non-user of tobacco products among study population cpi scores factory employees general population nonusers users non-users users healthy no 30 8 44 6 % 29.13 2.02 23.91 2.03 bleeding no 15 74 44 72 % 14.56 18.64 23.91 24.32 calculus no 40 155 78 93 % 38.83 39.04 42.39 31.42 4 – 5 mm no 15 131 12 113 % 14.56 33.00 6.52 38.18 6mm or more no 3 29 6 12 % 2.91 7.30 3.26 4.05 total no 103 397 184 296 % 100 100 100 100 chi-square test χ2 = 91.840, df = 4, p = 0.000 (<0.001) very high sig. χ2 = 99.866, df = 4, p= 0.000 (<0.001) very high sig. cramer’s v 0.429 0.456 7 shyagali et al. laboring involved in the mining occupation. the earlier reports also highlight this finding that the tobacco usage is most commonly seen amongst the middle and low income countries and that too in population belonging to low socioeconomic strata and low literacy rate24,25. there was no difference amongst the different categories of the mining employees for the usage of tobacco products. however, the transportation unit and the maintenance unit used tobacco more than the other units. the type of tobacco which was used by most of the mining employees were gutkha and the tobacco leaves, which were both the chewable forms of the tobacco. the oral hygiene habit showed that all the mining employees had the habit of brushing their teeth once daily. presence of the calculus (39%, 35.63%) was the most prevalent of all the periodontal conditions in the mining employees and the general population respectively, this was followed by the presence of periodontal pocket of 4-5mm depth which was seen in 29.20% of mining employees and 26.04%in the general population. the periodontal conditions were more in tobacco using group than the rest and the same finding is proved in the earlier studies as well26-33. the healthy periodontium was seen more in case of general population (10.42%) than the mining employees (7.60%). the difference noted between the mining employees and the general population was statistically significant (p<0.05). the periodontal health parameters noticed for the non-mining population in the current study are in agreement with the results of the previous studies conducted on the general population18. the difference in the periodontal condition for the different units of mining employees was statistically highly significant (p<0.01) with greater incidence of calculus in polishing unit employees. higher incidence of pocket depth of 4-5mm was seen in the cutting unit employees. this difference noted amongst the different group of mining employees is probably associated with the type of tobacco product they consumed. for example, the polishing unit and the cutting unit employees consumed bidi (a type of tobacco used for smoking) more than the other unit employees. apart from this the duration and the frequency of indulgence in the habit is directly proportional to the adversity of the periodontal condition. the mining employees and the general population both indulged in the usage of tobacco products not less than four times a day. the same fact has been emphasized in the earlier studies as well28,31. around 29.13% of the tobacco non-user group of the mining employees had healthy periodontium. however, the users both in the mining population and the general population had around 2% healthy periodontium. most common periodontal conditions seen in the tobacco users in the mining population was presence of calculus (39.04%) and the pocket depth of 4-5 mm (33%). similar findings have been reported in the literature33,34. however, in the non-tobacco users the presence of the calculus (38.83%) was most commonly spotted. the difference noted for the periodontal parameters in both the mining employees and the general population for the tobacco users and non-users was statistically very highly significant (p<0.001). this again proves the significant contribution of tobacco usage in increasing the oral health burden globally. the results of the current study are in agreement with previous study of the similar nature29,35-37. the scope of the current manuscript was to find the prevalence rate of periodontal 8 shyagali et al. disease status in tobacco usage sample, nevertheless the study carries the further scope to explore the relationship between the periodontal disease and the frequency of usage of tobacco products. conclusion the periodontal status of the current group of mining employees is very poor in comparison to general population and its association with the usage of tobacco product is well proven. this emphasizes the need to conduct regular dental health education and checkup camps to improve the awareness of the effects of the tobacco product usage. references 1. donoghue am. occupational health hazards in mining: an overview. occup med (lond). 2004 aug;54(5):283-9. 2. semple s, green da, mcalpine g, cowie h, seaton a. exposure to particulate matter on an indian stone-crushing site. occup environ med. 2008 may;65(5):300-5. 3. oliveira a, cacodcar j, motghare dd. morbidity among iron ore mine workers in goa. indian j public health. 2014 jan-mar;58(1):57-60. doi: 10.4103/0019-557x.128171. 4. nandi ss, dhatrak sv, chaterjee dm, dhumne um. health survey in gypsum mines in india. indian j community med. 2009 oct;34(4):343-5. doi: 10.4103/0970-0218.58396. 5. petersen pe, gormsen c. oral conditions among german battery factory workers. community dent oral epidemiol. 1991 apr;19(2):104-6. 6. petersen pe, henmer p. oral conditions among workers in the danish granite factory industry. scand j work environ health. 1988 oct;14(5):328-31. 7. petersen pe, tanase m. oral health status of an industrial population in romania. int dent j. 1997 aug;47(4):194-8. 8. sanadhya s, nagarajappa r, sharda aj, asawa k, tak m, batra m, et al. the oral health status and the treatment needs of salt workers at sambhar lake, jaipur, india. j clin diagn res. 2013 aug;7(8):1782-6. doi: 10.7860/jcdr/2013/5887.3275. 9. sharma a, thomas s, dagli rj, solanki j, arora g, singh a. oral health status of cement factory workers, sirohi, rajasthan, india. j health res rev. 2014;1(1):15-9. 10. sood m, blaggana a, blaggana v, sharma v. occupational dental wear among ceramic factory workers an observational study. j all india dent assoc. 2011;5(4):472-3. 11. gupta pc, arora m, sinha d, asma s, parascondola m. smokeless tobacco and public health in india. government of india, ministry of health and family welfare; 2012. p. 1-14. 12. singh a, ladusingh l. prevalence and determinants of tobacco use in india: evidence from recent global adult tobacco survey data. plos one. 2014 dec 4;9(12):e114073. doi: 10.1371/journal.pone.0114073. 13. mohan p, lando ha. oral tobacco and mortality in india. indian j clin med. 2016;7:5-12. 14. nair u, bartsch h, nair j. alert for an epidemic of oral cancer due to use of the betel quid substitutes gutkha and pan masala: a review of agents and causative mechanisms. mutagenesis. 2004 jul;19(4):251-62. 9 shyagali et al. 15. bhisey r a. chemistry and toxicology of smokeless tobacco. indian j cancer. 2012 oct-dec;49(4):364-72. doi: 10.4103/0019-509x.107735. 16. shah n, pandey rm, duggal r, mathur vp, rajan k. oral health in india: a report of the multi centric study. directorate general of health services, ministry of health and family welfare, government of india and world health organisation collaborative program; 2007 dec. 17. katuri kk, alluri jk, chintagunta c, tadiboina n, borugadda r, loya m et al. assessment of periodontal health status in smokers and smokeless tobacco users: a cross-sectional study. j clin diagn res. 2016 oct;10(10):zc143-zc146. 18. chandra a, yadav op, narula s, dutta a. epidemiology of periodontal diseases in indian population since last decade. j int soc prev community dent. 2016 mar-apr;6(2):91-6. doi: 10.4103/2231-0762.178741. 19. who. tobacco or health: a global status report. who publication: geneva: who; 1997 20. chaly pe. tobacco control in india. indian j dent res. 2007 jan-mar;18(1):2-5. 21. oral health survey-basic methods. 5th ed. geneva, switzerland: who; 2013. 22. mubeen k, chandrashekhar h, kavitha m, nagarathna s. effect of tobacco on oral-health an overview. j evol med dental sci. 2013 may;2(20):3523-34. 23. malhotra r, kapoor a, grover v, kaushal s. nicotine and periodontal tissues. j indian soc periodontol. 2010 jan;14(1):72-9. doi: 10.4103/0972-124x.65442. 24. johnson, nw, bain c. tobacco and oral disease. eu-working group on tobacco and oral health. br dent j. 2000 aug;189(4):200-6. 25. mackay j, eriksen m. the tobacco atlas. geneva: world health organization; 2002. p. 1-128. 26. grossi sg, zambon jj, ho aw, koch g, dunford rg, machtei ee, et al. assessment of risk for periodontal disease. i. risk indicators for attachment loss. j periodontol. 1994 mar;65(3):260-7. 27. grossi sg, genco rj, machtei ee, ho aw, koch g, dunford r, et al. assessment of risk for periodontal disease. ii. risk indicators for alveolar bone loss. j periodontol. 1995 jan;66(1):23-9. 28. martinez-canut p, lorca a, magán r. smoking and periodontal disease severity. j clin periodontol. 1995 oct;22(10):743-9. 29. haber j, kent rl. cigarette smoking in periodontal practice. j periodontol. 1992 feb;63(2):100-6. 30. alpagot t, wolff lf, smith qt, tran sd. risk indicators for periodontal disease in a racially diverse population. j clin periodontol. 1996 nov;23(11):982-8. 31. wouters fr, salonen lw, frithiof l, helldén lb. significance of some variables on interproximal alveolar bone height based on cross-sectional epidemiological data. j clin periodontol. 1993 mar;20(3):199-206. 32. machuca g, rosales i, lacalle jr, machuca c, bullon p. effect of cigarette smoking on periodontal status of healthy young adults. j periodontol. 2000 jan;71(1):73-8. 33. bergstrom j. tobacco smoking and supragingival dental calculus. j clin periodontol. 1999 aug;26(8):541-7. 34. muller hp, stadermann s, heinecke a. longitudinal association between plaque and gingival bleeding in smokers and non-smokers. j clin periodontol. 2002 apr;29(4):287-94. 35. bergstrom j. cigarette smoking as risk factor in chronic periodontal disease. community dent oral epidemiol. 1989 oct;17(5):245-7. 36. amarasena n, ekanayaka an, herath l, miyazaki h. tobacco use and oral hygiene as risk indicators for periodontitis. community dent oral epidemiol. 2002 apr;30(2):115-23. 37. tomar sl, asma s. smoking-attributable periodontitis in the united states: findings from nhanes iii. national health and nutrition examination survey. j periodontol. 2000 may;71(5):743-51. oral sciences n3 original article braz j oral sci. 8(4):201-205 chlorhexidine chip and tetracycline fibers as adjunct to scaling and root planing – a clinical study ruchi srivastava1; pushpendra kumar verma2; pradeep tandon3; ramesh kumar m4 ; krishna kumar gupta5 ; amitabh srivastava6 1 bds, postgraduate student, department of periodontology & implantology, sardar patel postgraduate institute of dental & medical sciences, lucknow, uttar pradesh, india 2bds, postgraduate student, department of conservative dentistry and endodontics, government dental college, calicut, kerala673008, india 3mds, professor and head of the department, department of periodontology & implantology, sardar patel postgraduate institute of dental & medical sciences, lucknow, uttar pradesh, india 4mds, professor and head of the department, department of conservative dentistry and endodontics, government dental college, calicut, kerala673008, india 5mds, professor, department of periodontology & implantology, sardar patel postgraduate institute of dental & medical sciences, lucknow, uttar pradesh, india 6 mds, reader,department of periodontology & implantology, sardar patel postgraduate institute of dental & medical sciences, lucknow, uttar pradesh, india correspondence to: ruchi srivastava postgraduate student, department of periodontology & implantology,sardar patel postgraduate institute of dental & medical sciences, lucknow, uttar pradesh, india e-mail: lichi_ruchi@yahoo.com abstract aim: prevention of periodontal disease progression is the primary goal of periodontal therapy. when conventional therapy is found inadequate to attain periodontal health in chronic periodontitis, local antimicrobial agents have been used as adjunct to scaling and root planing, producing encouraging results. hence, a study was undertaken to evaluate clinically, the newly released sustained drugs, periocoltmcg (chlorhexidine chxchip) with periodontal plus abtm ( tetracycline fibers). methods: patients were allocated in 3 experimental treatment groups, group asrp + chx chip, group bsrp + tetracycline fibers, and group csrp alone (control group). forty-five sites in 14 patients (9 females and 5 males) with chronic periodontitis (5-8mm probing depth), were evaluated clinically for probing depth (pd) and relative attachment level (ral). results: all the treatment groups were found to be efficacious in the treatment of periodontal disease as demonstrated by improvement in pd and ral. conclusion: combination of srp + chx chip (group a) resulted in added benefits compared to the other two treatment groups. keywords: chlorhexidine chip, chronic periodontitis, tetracycline fibers. introduction chronic periodontitis results in a progressive loss of attachment and formation of periodontal pocket. the process of periodontal pocket formation represents the pathologic sequela of microbial and inflammatory mediated degradation of collagenous connective tissue and alveolar bone1. mechanical therapy may however fail to eliminate the pathogenic bacteria because of their location within gingival tissues or in other areas inaccessible to periodontal instruments2. hence the use of several antimicrobial agents started gaining prominence as chemical aids would compensate for technical limitations and prevent early microbial recolonization, to ultimately ensure, the best chance for clinical improvements. these chemical agents may gain access into the periodontal pocket through both a systemic and local route of delivery. since systemic use of antibiotics may cause several side effects (sensitivity, resistant strains and superinfections), contemporary research is now focused on the role of topical antimicrobial agents in the treatment of periodontitis. various agents have been used to prevent further progression of periodontal disease braz j oral sci. october/december 2009 volume 8, number 4 received for publication: november 2, 2009 accepted: january 15, 2010 202 braz j oral sci. 8(4):201-205 either as monotherapy or as an adjunct to scaling and root planing (srp) procedure. these include tetracycline, doxycycline, minocycline, chlorhexidine3, metronidazole4, enzymes and quaternary ammonium compounds, which have been administered topically in pure forms by their incorporation in chewing gums, dentifrices, acrylic strips, hollow fibers, films, ointments, gels etc. it is clear that for local antimicrobial therapy to be clinically effective, successful mechanisms to deliver sustained and adequate concentration of the active agent to the periodontal pocket are required5. topical antiseptics have been successfully used for treating plaque-related gingivitis, among which chlorhexidine (chx) remains one of the most effective antimicrobials reported till now and is not known for any appreciable resistance to oral microorganisms. however, subgingival irrigation using chx solution or chx gels turned out to be poorly effective in the treatment of periodontitis, due to the inability to retain biologically significant concentrations of the drug for sufficient lengths of time within the confines of the periodontal pocket. however, it is difficult to maintain the effective antibacterial concentrations, for a sufficient period in periodontal pockets for a variety of reasons like poor penetration by mouth rinses, rapid dissipation of irrigation solutions, relatively low localized concentrations achievable with high systemic dose of antibiotics. hence, slow-release devices have been developed. there are two subtypes: “sustained release devices”, delivering the drug for less than 24 h, and “controlled delivery devices” (cdds), releasing the agent over an extended period of time. goodson (1989) 6 pointed out that; successful control of periodontal microflora requires a delivery of an intrinsically effective antimicrobial agent, according to the fundamental pharmacokinetic principles. these agents reach the site of action, i.e. the periodontal pocket, and maintain minimum effective concentration for a sufficient duration to produce the desired specific therapeutic effect. among the tested antibiotics, tetracyclines were the first and had their efficacy evaluated in a number of periodontal clinical studies. tetracyclines have been incorporated into a variety of delivery systems for insertion into periodontal pockets. these include hollow fibers7, ethylene vinyl acetate copolymer fibers8, ethyl cellulose fibers9, acrylic strips5, collagen preparations10. recently, a new local drug delivery system, periocoltm-cg, which contains fibrilar collagen of fish origin with 2.5 mg (approximately) of chx (eucare pharmaceuticals, chennai, india), and another local drug, periodontal plus abtm, which contains 25 mg pure fibrilar collagen with approximately 2 mg of evenly impregnated tetracycline hydrochloride (advanced biotech products, chennai, india) have been introduced. since research with drug delivery systems is limited, the present clinical study evaluated comparatively the efficacy of two commercially available new controlled-release drugs chx chip (periocoltm -cg) and tetracycline fibers (periodontal plus ab)tm as adjunct to srp in the treatment of chronic periodontitis. material and methods forty-five bleeding sites, with a probing depth 5-8mm, were selected in 14 patients of both genders (9 females and 5 males) aged between 20 to 50 years from the outpatient department of periodontics at sardar patel postgraduate institute of dental and medical sciences, lucknow, uttar pradesh, india. the ethical committee of sardar patel postgraduate institute of dental and medical sciences, lucknow, uttar pradesh, india, approved the study and written informed consent was obtained from all patients. patients with good systemic health, patients who had not received any surgical or non-surgical periodontal therapy in the past 6 months, who had not received antibiotic therapy in the past 6 months, who were diagnosed as suffering from chronic generalized periodontitis, and patients who had periodontal pocket measuring 5-8mm in different quadrants were enrolled. individuals with history of using anti-microbial mouthrinses within 2 months of the baseline visit or on routine basis or patients having a history of allergy to tetracycline; chx or lidocaine, were excluded from the study. the selected sites were randomly divided into 3 groups: test group a (srp + chx chip) – included 15 sites treated by srp with chlorhexidine chip. test group b (srp + tetracycline fibers) included 15 sites treated by srp with tetracycline fibers. test group c (srp alone) included 15 sites treated with srp alone. the clinical parameters recorded are the probing depth (pd) using unc-15 periodontal probe and relative attachment level (ral) using customized acrylic stent (figure 1). after recording clinical parameters from each site at baseline, a thorough srp was done, in all the 3 groups. the clinical parameters were assessed at baseline, after 1 month and 3 months after receiving all the 3 treatments in a same patient; as it is a split-mouth study. fig.1. measurement of probing depth and relative attachment level. chlorhexidine chip (periocoltmcg) (figure 2) periocol – cg is a small, orange-brown in a rectangular chip form (rounded at one end) for easy insertion into periodontal pockets. size of the chip is 4 x 5mm and thickness is 0.25-0.32mm and 10 mg weight. each chip contains approximately 2.5 mg of chx in a biodegradable matrix of fibrilar collagen of fish origin (eucare pharmaceuticals, chennai, india). tetracycline fibers (periodontal plus abtm) (figure 3) the product contains 25 mg pure fibrilar collagen, containing approximately 2 mg of evenly impregnated tetracycline hcl. periodontal plus ab fibers are available in strips containing four individually packed and separable sterile product packs. chlorhexidine chip and tetracycline fibers as adjunct to scaling and root planing – a clinical study 203 braz j oral sci. 8(4):201-205 fig. 2. chlorhexidine chip (periocoltmcg). fig. 3. tetracycline fibers (periodontal ab plustm). administration (figures 4 and 5) subgingival administration of chlorhexidine chip was accomplished by inserting the round end of the chip directly into the base of the pocket. chip was pressed apically so that it rest subgingivally at the base of the pocket. whereas, soaked tetracycline fibers were inserted into the base of the periodontal pocket. gentle force was used with straight probe, so that the material fills the depths and curves of the pocket. the gingiva was subsequently and carefully adapted to close the entrance of the gingival margin and hand pressure was applied for just a few minutes to encourage hemostasis and initial setting of the material inside the pocket. the gingival margin was sealed with coepak to prevent the dislodgement of the drug and to prevent the ingress of oral fluids. both these drugs degrade within 7-10 days after insertion. patients were recalled after 7 days for coe-pak removal and were evaluated for any inflammatory response. patients were instructed not to chew any hard, crunchy or sticky food for at least 1 week, postpone brushing and flossing on the treated site for 1weeek, not to disturb the area with tongue, finger or tooth pick, and to report immediately if the material is dislodged before the scheduled recall visit or if pain, swelling or any other problem occurs. the results were averaged out for each parameter. values are depicted as mean ± sd. for comparisons between different time intervals paired t-test were used. t test for independent samples were used for intergroup comparisons at the different time intervals, the confidence level of the study was kept at 95% and hence a ‘p’ value fig. 5. placement of tetracycline fibers <0.05 was considered as significant. paired t-test and student’s t-test were used to evaluate and establish differences between baseline, 1 month and 3 months values. fig. 4. placement of chlorhexidine chip results results are presented in tables 1 to 3. the recording of all the clinical parameters was done at baseline and after 1 month and 3 months. finally, the complete data were statistically analyzed. none of the subjects reported any oral symptoms at any time during the trial such as toothache (including dental or gingival), painful symptomatology (including oral pain, tenderness, soreness, discomfort or sensitivity), inflammation, allergy, abscess, altered taste or increased salivation, etc. probing depth from baseline to 3 months, group a (srp +chx chip) had significantly higher mean percentage reduction as compared to group b (p=0.007) as well as group c (p<0.001). these findings were similar to that of previous studies11-15. relative attachment level from baseline to 3 months group a (srp +chx chip) had significantly higher mean percentage gain as compared to group b (p=0.046) as well as group c (p=0.001). these findings were similar to that of other authors16-20, who studied the changes in probing depth following 2 years of periodontal maintenance therapy including adjunctive controlled release of biodegradable chx chip. chlorhexidine chip and tetracycline fibers as adjunct to scaling and root planing – a clinical study 204 braz j oral sci. 8(4):201-205 baseline 1 month 3 months baseline 1 month 3 months baseline 1 month 3 months 8.00 6.00 3.00 7.00 5.00 4.00 7.00 6.00 5.00 7.00 5.00 3.00 8.00 6.00 4.00 6.00 5.00 5.00 7.00 6.00 4.00 7.00 6.00 6.00 5.00 4.00 4.00 6.00 6.00 5.00 8.00 7.00 5.00 6.00 5.00 5.00 6.00 4.00 3.00 8.00 5.00 4.00 6.00 4.00 4.00 7.00 4.00 3.00 7.00 4.00 3.00 7.00 6.00 5.00 8.00 5.00 3.00 8.00 6.00 4.00 7.00 5.00 5.00 8.00 5.00 4.00 8.00 5.00 4.00 7.00 5.00 4.00 8.00 6.00 5.00 6.00 5.00 4.00 5.00 4.00 4.00 6.00 4.00 3.00 7.00 5.00 5.00 5.00 4.00 5.00 8.00 6.00 4.00 5.00 4.00 4.00 6.00 4.00 5.00 5.00 4.00 3.00 6.00 5.00 5.00 6.00 5.00 5.00 6.00 4.00 3.00 7.00 6.00 6.00 5.00 4.00 3.00 8.00 6.00 5.00 8.00 6.00 4.00 6.00 5.00 5.00 8.00 5.00 4.00 7.00 5.00 3.00 6.00 4.00 5.00 group a (chx chip + srp) group b (ttc fibers + srp) group c (srp alone) table 1. probing depth values (in mm). baseline 1 month 3 months baseline 1 month 3 months baseline 1 month 3 months 12.00 8.00 6.00 11.00 8.00 8.00 10.00 7.00 7.00 11.00 7.00 6.00 12.00 8.00 7.00 9.00 7.00 6.00 11.00 7.00 7.00 10.00 8.00 7.00 8.00 6.00 7.00 10.00 7.00 5.00 11.00 8.00 8.00 9.00 7.00 6.00 10.00 9.00 8.00 11.00 9.00 8.00 10.00 8.00 8.00 11.00 8.00 8.00 12.00 9.00 8.00 11.00 9.00 9.00 11.00 6.00 6.00 11.00 7.00 7.00 10.00 7.00 7.00 11.00 7.00 6.00 11.00 7.00 6.00 9.00 6.00 6.00 12.00 7.00 7.00 8.00 6.00 6.00 7.00 6.00 5.00 10.00 8.00 7.00 9.00 7.00 7.00 8.00 6.00 6.00 12.00 9.00 8.00 9.00 7.00 6.00 8.00 6.00 6.00 9.00 7.00 6.00 10.00 7.00 7.00 9.00 8.00 8.00 10.00 8.00 6.00 10.00 8.00 8.00 8.00 7.00 7.00 11.00 8.00 7.00 11.00 7.00 7.00 9.00 7.00 6.00 11.00 8.00 6.00 11.00 8.00 7.00 10.00 8.00 6.00 group a (chx chip + srp) group b (ttc fibers +srp) group c (srp alone) table 2. relative attachment level values (in mmm). measurement groups 0-1 month 0-3 months probing depth(in mm) significance relative attachmentlevel (gain) (in mm) significance group a group b group c group a group b group c 2.000±0.795 1.803±0.716 1.340±0.489 p < 0.01 sig. 3.200±1.099 2.866±0.788 2.000±0.605 p < 0.01 sig. 3.400±0.995 2.803±1.231 1.400±0.754 p < 0.01 sig. 4.200±1.214 3.333±0.821 2.334±1.000 p < 0.01 sig. table 3. post treatment clinical changes and comparison between the 3 groups discussion a periodontal disease essentially comprises of a group of oral infections, whose primary etiological factor is dental plaque, which results in an inflammatory lesion in the supporting tissues. removal of the cause (and its effects) is the primary aim of both non-surgical and surgical treatment regimens. the major non-surgical therapeutic approach involves mechanical srp. the infective nature of the disease has lead to the widespread use of antimicrobials, as an adjunct to srp. local delivery of controlled release antimicrobials has some advantages over the systemic route, including the high local drug levels in the periodontal pockets and avoidance of drug compliance issues. in the present study, an attempt was made to e valuate effectiveness of chx chip (periocoltmcg) and tetracycline fibers (periodontal ab plustm), in the treatment of chronic periodontitis, as an adjunct to srp. chx and tetracycline were chosen in the present study, because of their proven efficacy in the management of periodontal diseases. tetracycline is known for its antibacterial actions and also due to number of additional properties that have been recently identified. these include colagenase inhibition, anti-inflammatory actions and inhibition of bone resorption. chx is a widely used broad-spectrum antimicrobial agent, encompassing gram-positive and gram-negative bacteria, yeasts, chlorhexidine chip and tetracycline fibers as adjunct to scaling and root planing – a clinical study 205 braz j oral sci. 8(4):201-205 dermatophytes and some lipophilic viruses. the antibacterial mode of action is explained by the fact that the cationic chx molecule is rapidly attracted by the negatively charged bacterial cell surface. after adsorption, the integrity of the bacterial cell membrane is altered, which results in a reversible leakage of bacterial low molecular-weight components at low dosage or more severe membrane damage at higher doses (bactericidal). tetracycline-hcl is a bacteriostatic agent that inhibits bacterial protein synthesis and, as such, requires a significantly longer exposure time for bacterial damage than, for example, metronidazole or chx. it however, has the ability to bind to the hard tissue walls of pockets to establish a drug reservoir. the crux of the present study clearly shows that, the locally delivered chx chip (periocoltm-cg) along with mechanical debridement resulted in a clinically meaningful improvement of all clinical parameters, which was maintained significant throughout the study duration. in order to be effective, a pharmaceutical agent should reach the entire periodontal pocket up to the bottom and should be maintained long enough at a sufficient concentration for the intended pharmaceutical effect to occur. periodontal pockets, however, possess complicating anatomic characteristics. furthermore, periodontal pathogens in the subgingival environment reside in a biofilm adhering to the exposed root cementum or to the soft tissue, or even invading the pocket epithelium, the underlying connective tissue or the root dentin. the aggregation of bacteria in a biofilm impairs the diffusion or may even inactivate antimicrobial agents. thus, high concentrations of antimicrobial agents are needed before a beneficial effect can be expected. various biofilm experiments indicate that the necessary minimum inhibitory concentrations (mic) of antimicrobial agents, are at least 50 times higher (or even 210,000 times), than for bacteria growing under planktonic conditions21-23. moreover, the minimum contact time for an antimicrobial agent to be active depends on the mechanism by which the agent inhibits or destroys target bacteria. chx (which kills microorganisms by compromising the integrity of the cell membrane) and povidone-iodine (which kills bacteria on contact) require a shorter exposure time than, for example, a bacteriostatic agent, such as tetracycline, which inhibits protein synthesis23. however, the substantivity of a topically applied agent is increased spontaneously, if it binds to the soft and/or hard tissue surfaces within the pocket. chx has the advantage of prolonged supragingival substantivity, because it can bind to the intraoral soft and hard tissues24. ciancio et al.25, reported that tetracycline applied via ttc-fibers does not penetrate into the gingiva a significant distance to kill or suppress tissue invasive organisms, such as a. actinomycetemcomitans. besides the pharmacokinetics, the patient’s comfort, ease of placement of the drug into the periodontal pocket and the costbenefit ratio are key elements for determining the selection and efficacy of a product. the results from the present study suggest that the application of chx chip combined with srp is beneficial in the treatment of chronic periodontitis and improving periodontal parameters for 3 months duration. in spite of the proven additive benefits, the availability and costs associated with controlled delivery devices have so far limited their application. however, as this material i.e. periocoltmcg (chx chip) is of indian origin, easy to place in periodontal pocket, less time consuming and is relatively cost effective, its use can be expanded easily in indian population. references 1. drisko cl, cobb cm, killoy wj, michalowicz bs, pihlstrom bl, lowenguth ra et al. evaluation of periodontal treatment using controlled release tetracycline fibers: clinical response. j periodontol. 1995; 66: 692-9. 2. slots j, rams te. antibiotics in periodontal therapy: advantages and disadvantages. j clin periodontol. 1990; 17: 479-95 3. cetin eo, buduneli n, atlihan e, kirilmaz l. in vitro studies on controlled-release cellulose acetate films for local delivery of chlorhexidine, indomethacin, and meloxicam. j clin periodontol. 2004; 31: 1117-21. 4. kaner d, bernimoulin j-p, hopfenmuller w, kleber b-m, friedmann a. controlled-delivery chlorhexidine chip versus amoxicillin/ metronidazole as adjunctive antimicrobial therapy for generalized aggressive periodontitis: a randomized controlled clinical trial. j clin periodontol. 2007; 34: 880-91. 5. addy m, rawle l, handley r, newman hn, coventry jf. the development and in-vitro evaluation of acrylic strips and dialysis tubing for local drug delivery. j periodontol. 1982; 53: 693-9. 6. goodson jm. pharmacokinetic principles controlling efficacy of oral therapy. j dent res.. 1989; 68: 1625-32. 7. goodson jm, haffajee a, socransky ss. periodontal therapy by local delivery of tetracycline. j clin periodontol. 1979; 6: 83-92. 8. goodson jm, holborow d, dunn rl, hogan p, dunham s. monolithic tetracycline containing fibers for controlled delivery to periodontal pockets. j periodontol. 1983; 54: 575-9. 9. friedman m, golomb g. new sustained release dosage form of chlorhexidine for dental use. 1. development and kinetics of release. j periodont res. 1982; 17: 323-8. 10. minabe m, takeuchi k, tamura t, hori t, umemoto t. subgingival administration of tetracycline on a collagen film. j periodontol. 1989; 60: 552-6. 11. jeffcoat mk, bray ks, ciancio sg, dentino ar, fine dh, gordon jm et al. adjunctive use of a subgingival controlled-release chlorhexidine chip reduces probing depth and improves attachment level compared with scaling and root planing alone. j periodontol. 1998; 69: 989-97. 12. killoy wj. assessing the effectiveness of locally delivered chlorhexidine in the treatment of periodontits. j am dent assoc. 1999; 130: 567-90. 13. heasman pa, heasman l, stacey f, mccracken gi. local delivery of chlorhexidine gluconate (periochiptm) in periodontal maintenance patients. j clini periodontol. 2001; 28: 90-5. 14. r. mythili, t. ramakrishnan, biju mammen. effect of controlled-release device containing chlorhexidine gluconate in the treatment of adult periodontitis – a clinical and microbiological study. j ind soc periodontol. 2006; 10: 321-4. 15. ronald hw cheng, w. keung leung, esmonde f. corbet. non-surgical periodontal therapy with adjunctive chlorhexidine use in adults with down syndrome. a prospective case series. j periodontol. 2008; 79: 379-85. 16. soskolne wa, proskin hm, stabholz a. probing depth changes following 2 years of periodontal maintenance therapy including adjunctive controlled release of chlorhexidine. j periodontol. 2003; 74: 420-7. 17. chen yt, hung sl, lin lw, chi ly, ling lj. attachment of periodontal ligament cells to chlorhexidine loaded guided tissue regeneration membranes. j periodontol. 2003; 74: 1652-59. 18. juliana carvalho, m. john novak, lf mota. evaluation of the effect of subgingival placement of chlorhexidine chips as an adjunct to scaling and root planing. j periodontol. 2007; 78: 997-1001. 19. paolantonio m, d’angelo m, grassi rf, perinetti g, piccolomini r, pizzo g et al. clinical and microbiologic effects of subgingival controlled release delivery of chlorhexidine chip in the treatment of periodontitis : a multicenter study. j periodontol. 2008; 79: 271-82. 20. mizrak t, guncu gn, caglayan f, balci ta, aktar gs, ipek f. effect of a controlled release chlorhexidine chip on clinical and microbiological parameters and prostaglandin e2 levels in gingival crevicular fluid. j periodontol. 2006; 77: 437-43. 21. rodrigues if, machion l, casati mz, nociti fh jr, de toledo s, sallum aw, sallum ea. clinical evaluation of the use of locally delivered chlorhexidine in periodontal maintenance therapy. j periodontol. 2007; 78: 624-8. 22. iyad hussein, meena ranka, angela gilbert and kevin davey. locally delivered antimicrobials in the management of periodontitis: a critical review of the evidence for their use in practice. dent update. 2007; 34: 494-506. 23. quirynen m, teughels w, de soete m, van steenberghe d. topical antiseptics and antibiotics in the initial therapy of chronic adult periodontitis: microbiological aspects. periodontol 2000. 2002; 28: 72-90. 24. bonesvoll p, gjermo p. a comparison between chlorhexidine & some quaternary ammonium compounds with regard to retention, salivary concentration & plaque inhibiting effect in human mouth after mouth rinses. arch oral biol. 1978; 23: 289-94. 25. ciancio sg, cobb cm, leung m. tissue concentration and localization of tetracycline following site specific tetracycline fiber therapy. j periodontol. 1992; 63: 849-53. chlorhexidine chip and tetracycline fibers as adjunct to scaling and root planing – a clinical study revista fop n 13 1575 forensic anthropology and molecular biology: independent orforensic anthropology and molecular biology: independent orforensic anthropology and molecular biology: independent orforensic anthropology and molecular biology: independent orforensic anthropology and molecular biology: independent or complementary sciences in forensic dentistry? an overviewcomplementary sciences in forensic dentistry? an overviewcomplementary sciences in forensic dentistry? an overviewcomplementary sciences in forensic dentistry? an overviewcomplementary sciences in forensic dentistry? an overview ricardo henrique alves da silva1; rogério nogueira de oliveira2 1dds, msc, phd, dental school of ribeirão preto, university of são paulo, ribeirão preto,, sp, brazil 2dds, msc, phd, dental school, university of são paulo, são paulo, sp, brazil received for publication: august 8, 2007 accepted: december 5, 2007 a b s t r a c t human identification is currently one of the most outstanding areas of forensic sciences. forensic anthropology is a branch of the forensic sciences concerned with the application of general anthropological knowledge and methods to the process of law. postmortem human identification is one of the major areas of study and research in legal medicine and forensic dentistry and both sciences work with the same type material and the human body in several conditions. every human being has an identity in life, which should be recognized after death both for family consolation and for juridical purposes. therefore, the aim of this literature review was to discuss and elaborate on the relationship between forensic anthropology and molecular biology in the expert practice of forensic dentistry. the use of dna profile tests in forensic dentistry offers a new perspective in human identification. molecular biology techniques should definitely be added to the constellation of investigative resources of the forensic sciences, providing a variety of tools to forensic anthropology practice in human identity research, especially when the usual identification methods fail due to the deleterious effects of heat, trauma or autolytic processes on the body or body remains. key words: forensic dentistry; forensic anthropology; molecular biology correspondence to: ricardo henrique alves da silva rua minas gerais, 12-67, parque paulistano, bauru – sp brazil cep: 17030-511 e-mail: ricardohenrique@usp.br introduction forensic anthropology is a branch of the forensic sciences concerned with the application of general anthropological knowledge and methods to the process of law. its main goal is related to the medical-legal identity, defined as the aggregate of unique characteristics of persons, animals and objects. taken together, positive or negative signs, marks and physical characteristics individualize the human beings, making possible to distinguish one person from another1. molecular biology has been recently applied in forensic anthropology. until the 1980’s, the science of identification of criminal cases was based only on serological analyses of protein polymorphism, blood groups and some genetic markers. forensic examination of biological samples started in the beginning of the 20th century by application of the abo blood group system in evidences related to crimes or human identification2. the discovery of the double-helix structure of dna, in 1953, which is responsible for the genetic inheritance in human beings, caused a major impact and led to remarkable changes in nearly all fields of science. this knowledge has been the basis for development of techniques that allow characterizing each person’s individuality based on the dna sequence. three decades later, jeffreys et al.3 created radioactive molecular probes that could recognize certain highly sensitive regions of dna and determine specific patterns of each individual, which were named dna “fingerprints”. the currently performed dna profile tests are very reliable, being accepted as legal proofs in courts for investigation of paternity and human identification. there are many acceptable methods for human identification, each one with its limitations. fingerprints have historically been used to determine people’s identity. however, in some situations (e.g. in fires, decomposing bodies or skeletonized bodies), fingerprints are easily destroyed. identification of human remains using genetic tests has thus been recognized as an important and reliable resource of forensic anthropology. hairbrush, toothbrush and other objects of personal use may contain material that can be used for comparison. nevertheless, only the presence of hair or oral cells does not provide all elements necessary for identification. in the same way as for dactyloscopy, for dna fingerprint molecular typing techniques to be of value in human identification or crime solving, it is also necessary to have a previous registration or available offspring, in addition to a sophisticated sample braz j oral sci. april/june 2008 vol. 7 number 25 1576 processing. furthermore, although the anthropologic analysis can provide useful information about stature, race and gender, it does not determine someone’s identity 4. in this way, postmortem human identification is one of the most important areas of study in forensic dentistry, as it can work with the human body in several conditions (quartered, lacerated, carbonized, macerated, putrefied, in skeletonization process and skeletonizated), pursuing to establish human identity5. the advent of molecular biology has introduced new methods, equipments and perspectives to the contemporary forensic dentistry, though it still have some limitations. according to sweet6, because every human being has an identity in life, there is a basic societal need for this identity to be recognized after death, both for family consolation and for juridical purposes. human identification has a legal implication and is part of the process of investigation of death causes, since a responsible person should be indicated, even in a suicide case7. in this sense, the technical requirements of human identification techniques for any recommendation could be listed as follows8: uniqueness (identification elements should allow an accurate and clear distinction between the suspected and the others); immutability (the analyzed features should not suffer alteration with the course of time); perenniality (the analyzed features should be present throughout the life); practicability (safe and rapid procedure for collection of identification data in order to avoid creating constraint for the identified p e r s o n a n d a l l o w i n g a g o o d s a f e t y a n d r e l i a b i l i t y degree); classification (it should allow comparison among collected data, in a synthetic and accurate way, quickly distinguishing a subject in a population); and r e p r o d u c i b i l i t y ( c a p a c i t y t o r e p r o d u c e t h e s a m e technique under the same conditions, anywhere and at any time). a critical analysis of these ideal conditions for human identity research shows that only few methods fulfill all these requisites, for example dactyloscopy, dental arch identification, genetic identification tests (except for identical twins who have the same genetic load). other tests can be used, such as analysis of the palatal rugae pattern (a less used method that is simple to perform, but do not provide a clear classification); and biometric tests, which include patterns and comparison of iris, voice, retina, and signature. as far as human identity is concerned, it is important to emphasize the difference between recognition and identification. while recognition can be understood as an empiric identification (by family, friends and/or colleagues) without scientific criteria, identification is established using well-established scientific knowledge and techniques5,7-9. there are two types of human identification: comparative and reconstructive. the first is based on antemortem evidence, allowing individual identification by medical and dental records. in the reconstructive process, it is not possible to use these data and it is necessary to make general identification by defining gender, age and race10. the purpose of this literature review is to discuss and elaborate on the role of forensic anthropology and molecular biology in human identification, as well their application in forensic dentistry. the legal role of forensic dentistry in human identification the legal competency of the dentist for procedure in cases of forensic expert identification is based on the brazilian federal law no. 5081/6611, which rules: “art. 6. it is the dentist competency: i – the practice of all the acts pertinent to the dentistry, resulting from the knowledge acquired in a regular undergraduate or postgraduate course; (…) iv – to proceed with forensic dentistry expert examination in civil, criminal, and labor court in administrative headquarters”. furthermore, the brazilian federal council of dentistry by its resolution no 63/200512 determines in art. 64, the expert ability of forensic dentistry. “art. 64. the competency areas to the practice of the forensic dentistry expert include: a) human identification; b) expert analysis in civil, criminal, and labor court; c) expert analysis in administrative area; d) expert analysis, evaluation and planning in labor hazard; e) forensic thanatology; f) elaboration of: 1) records, reports, opinions; 2) records and certificates; g) legal dentistry traumatology; h) forensic ballistic; i) logistics expert analysis in alive, dead, complete or fragmented persons; j) expert analysis in correlated vestiges, including blot or liquids from oral cavity or present in it; l) image examination with expert analysis purposes; m) deontology n) forensic dentistry orientation to the professional practice; o) image examination with legal dental purposes.” according to sweet6, the dentist has a key role in the search for human identity. this author has emphasized that oral records are currently used in three kinds of identification, two of which have been used for many decades and are characterized as the main responsibility of the forensic dentist. the first is dental identification and involves comparison between antemortem and postmortem records. the second refers to the reconstruction braz j oral sci. 7(25):1575-1579 forensic anthropology and molecular biology: independent or complementary sciences in forensic dentistry? an overview 1577 of the postmortem dental profile, which is used in cases when the subject’s or his/her offspring’s identity are unknown. the third refers to the application of modern dna profile techniques. in this way, the collaboration of forensic dentistry with forensic anthropology can be verified by human identification using body remains, such as skull, working by comparison with dental records, photographs and clinical records7,13. moreover, forensic dentistry can act in the identification of victims of mass events such as natural disasters14,15, bus accident involving body carbonization16,17, plane crashes18-20, conflagration21, train accidents22, military accidents and wars23, in addition to the study of allelic frequency to be used in forensic investigation24,25. odontological identification examination of a decedent is based on a systematic comparison of the antemortem and postmortem dental traits of the individual according to the analysis of dental records and supported by radiographs (periapical films, panoramic radiographs and cranial radiographs)26. molecular biology applied to human identification: activities in forensic dentistry. dna is responsible for storing all genetic information. it is found in the cell nucleus chromosomes (genomic dna) and in the mitochondria (mitochondrial dna). the currently performed dna profile tests are totally reliable, being accepted as legal proofs in courts for investigation of paternity and human identification27. it is possible to acquire dna from practically any human tissue, like blood, oral smear, saliva, bone, tooth, tissue, organs, hair, semen, urine, among other biological materials28. the amount of extracted dna, however, varies depending on the tissue12. in forensic investigations, dna studies are usually done by the analysis of tandem regions of a determined base sequence. strs (short tandem repeat) are the most used technique in forensic samples because they indicate a repetition sequence (from 2 to 9 base pairs), which make loci smaller than 300 base pairs. these repeated sequences are spread throughout the human genome and present a sufficient variety to be used in human identification tests, and allows a variability of choice in forensic identification29. in forensic investigations, the characterization of the biologic sample aims to limit or to reduce the number of subjects that could be the material origin in analysis, since the application of dna technology provides a valid discriminatory capacity to reach the required limit for human identification30. this demonstrates the applicability of dental samples in several conditions. the environmental influence on the concentration, integrity and recovery of dna extracted from dental pulps has been previously measured by schwartz et al.31. the authors varied the ph (3.7 and 10.0), temperature (4°c/39.2°f, 25°c/77°f, 37°c/ 98.6°f and tooth incineration), humidity (20, 66 and 98%), type of the soil in which the teeth were buried (sand, potting soil, garden soil, submersion in water and burying outdoors) and periods of inhumation (one week to six months). it was determined that the environmental conditions examined did not affect the ability to obtain high-molecular-weight human dna from dental pulp. in order to verify heat resistance, remualdo32 evaluated the pcr amplification of dna retrieved from teeth subjected to heat (200°c/392°f, 400°c/752°f, 500°c/932°f, and 600°c/1,112°f) during 60 minutes, testing 3 different dna extraction methods (organic; ammonia acetate/ isopropanol and silica). the authors concluded that amplification of all samples at all temperatures was possible using the ammonium/isopropanol acetate method, which gives a high credibility to the use of teeth in dna-based forensic investigations, regardless of tooth conditions. in order to evaluate the different dental tissues as dna sources in forensic analyses, malaver and yunis33 conducted a study in which 20 teeth were obtained from unidentified bodies buried in 1995 and exhumed in 2000, providing 45 dna samples (5 from the pulp, 20 from dentin and 20 from cementum). the pulp produced the strongest pcr amplification signals, while dentin and cementum signals were very similar to each other. there is a good possibility of using teeth as sources of dna material, even in cases where dna extraction and retrieval seem impossible. if there is a structure in the human body that can resist the most different environment conditions and still provide material for analysis, this structure is the tooth. in addition to the teeth, molecular biology applied to forensic dentistry also permits the analysis of cells from oral smear and human saliva, which can be obtained in cases of physical violence, such as sexual crimes, murders, child abuse, where bite marks can be found on the skin34. koh et al.35 has reported that saliva is a very useful dna source because it can be collected in a painless and noninvasive way and can be used even when stored in many different ways. the usefulness of saliva for bimolecular techniques is due to the fact that approximately 99% of its composition is water, having also leucocytes (25 to 650,000) and desquamated epithelial cells (6 to 600,000)36. the amount of saliva deposited on the skin is often very small in cases of bite impression, being necessary to use specific collection methods, which can recover the maximum amount of saliva and minimize any potential contaminations. anzai-kanto et al.37 studied saliva obtained from volunteers was deposited on skin and recovered for dna extraction and typing in order to evaluate its usefulness for practical case investigation and discuss the contribution of forensic dentistry to saliva dna typing. it was verified that the double-swab technique was sensible and efficient for collection of saliva deposited on the skin and use for salivary dna analysis in forensic cases. braz j oral sci. 7(25):1575-1579 forensic anthropology and molecular biology: independent or complementary sciences in forensic dentistry? an overview 1578 furthermore, sweet et al.38 used simulated situations of bite impressions in two experimental series. for such purpose, three 40 ìl saliva samples were deposited over 27 skin corpses (on 33 different sites) and three 100 ìl saliva samples were deposited over 5 skin corpses (on 12 different sites). saliva was then collected by the doubleswab techniques in periods of 5 minutes, 24 hours and 48 hours. it was observed a decrease in concentration within the first 24 hours and stability between 24 and 48 hours. amplification was successful regardless of the time elapsed after saliva deposition. no cases of contamination were detected. in a case of expert analysis, sweet and shutler39 used dna analysis by pcr of a saliva sample recovered from a bite mark found in a corpse that was recovered after approximately 5.5 hours submerged in a lake. enough dna from the bite impression was recovered to allow a genotypic contribution to identify the aggressor. the respective efficiency is attested by the fact that the saliva in contact with the intact skin is kept in stable conditions and can be recovered even 60 hours after its deposition40. however, it is not always possible to recover dna from a bite impression because the sample may be subject to a series of modifications, such as contamination, degradation, and putrefaction, depending on the circumstances to which the body and/or object had been submited41. in this way, either or not associated to molecular biology techniques, forensic dentistry provides an effective cooperation to human identification with analysis of dental structures and/or saliva. forensic anthropology and molecular biology: independent or complementary sciences? molecular biology is a helpful tool in forensic cases. discussions involving the areas pertinent to forensic investigation are important to elucidate some issues. legal medicine has benefited from the unquestionable advance deriving from the study of human genome42. therefore, a marked evolution has been observed in both civil and criminal identification, widening the scopes in this area and making it an important branch of legal medicine haemogenetics. in the expert analysis, the cartesian method should be considered, i.e., from the simplest exams to the most complex ones, starting performance in forensic anthropology with the use of personal impressions, measurements, dental arch analysis, and, if necessary, application of modern biomolecular techniques, with the purpose of establishing identity. nonetheless, the cost of genetic tests varies from region to region, as well as the need for formation of qualified human resources in this area. the main restrictive exogenous factors that may hinder the retrieval of evidence from body remains, even with the use of biomolecular technology, and hence compromise human identification processes, are the burning elements, such as fire, heat and explosions. furthermore, tissue deterioration, incorrect preservation and other external factors may also influence sample quality. from all issues discussed hereby, what is clear in the relationship between forensic anthropology and molecular biology is that, even with the inestimable contribution of the anthropometric resources, their application does not allow a precise nomination of the individual because the sole analysis of bones gives an estimative of the specie, gender, race, age and stature, but not in an accurate manner. according to campos43, in the forensic expert analysis, when the skeleton is complete, the legal physician works, mainly, with the bones from the axial, limb and cingulum skeleton, while the legal dentist examines the teeth, bone from the neurocranium and viscerocranium or their fragments. as emphasized by silva et al.2, forensic dentists should incorporate these new technologies in their work because forensic expert analysis will never have totally predictable in results. further studies in the fields of forensic anthropology and molecular biology are necessary in the search for new approaches in forensic dentistry. it may be concluded that molecular biology techniques should definitely be added to the constellation of investigative resources of the forensic sciences, providing a variety of tools to forensic anthropology practice in human identity research. references 1. croce d, croce-júnior d. manual de medicina legal. 5th ed. são paulo: saraiva; 2004. cap.2, p.264-397. 2. silva rha, oliveira, ft, sales-peres a, oliveira rn, salesperes shc. use of dna technology in forensic dentistry. j appl oral sci. 2007; 15: 156-61. 3. 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sci. 1997; 42: 320-2. 39. sweet d, shutler gg. analysis of salivary dna evidence from a bite mark on a body submerged in water. j forensic sci. 1999; 44: 1069-72. 40. smith bc, holland mm, sweet d, dizinno ja. dna and the forensic odontologist. in: bowers cm, bell gl. manual of forensic odontology. 3rd ed. colorado springs: american society of forensic odontology; 1997. cap.12, p.283-98. 41. silva rha, musse jo, melani rfh, oliveira rn. human bite mark identification and dna technology in forensic dentistry. braz j oral sci. 2006; 5: 1193-7. 42. frança gv. fundamentos de medicina legal. rio de janeiro: guanabara-koogan; 2005. 43. campos mlb. identificação craniométrica. in: vanrell jp. odontologia legal & antropologia forense. rio de janeiro: guanabara koogan; 2002. p.224-31. braz j oral sci. 7(25):1575-1579 forensic anthropology and molecular biology: independent or complementary sciences in forensic dentistry? an overview revista fop n 13 1539 braz j oral sci. april/june 2008 vol. 7 number 25 two-year clinical wear performance of two polyacid-modified resin composites (compomers) in posterior permanent teeth rafael guerra lund1 ; evandro piva2; flávio renato reis de moura3; flávio fernando demarco1; janaína de oliveira lima4; paulo eduardo capel cardoso4 1dds, phd student, department of restorative dentistry, dental school, federal university of pelotas, brazil 2dds, ms, phd, department of restorative dentistry, dental school, federal university of pelotas, brazil 3dds, ms, department of public health dentistry, lutheran university of brazil, cachoeira do sul campus, brazil 4dds, ms, department of dental materials, dental school, university of são paulo, brazil received for publication: march 31, 2008 accepted: july 1, 2008 correspondence to: rafael lund departamento de odontologia restauradora faculdade de odontologia de pelotas, universidade federal de pelotas rua gonçalves chaves, 457 / 504, cep: 96015-000 pelotas, rs, brasil phone/fax: +55-53-3222-6690. e-mail:rafael.lund@gmail.com a b s t r a c t aim: the aim was to compare the clinical wear of two compomers (f2000; 3m/espe and dyract ap; dentsply) placed in occlusal cavities in permanent molars within a two-year follow-up period using an indirect method of evaluation. methods: twenty-one patients, whose treatment plans included class i restorations, were selected. each patient received two occlusal class i restorations in permanent molars. one and 2 years after placement of the restorations, the 21 teeth restored with each material were submitted to wear evaluation. polyvinylsiloxane impressions were taken and casts were made (baseline, 1 year and 2 years). the casts were classified by comparative evaluation using sets of 18 calibrated standard models (0 to 900 ìm), according to leinfelder’s indirect method. paired and unpaired t-tests were used for comparisons between the evaluations for the same material and between materials for each evaluation period, respectively. results: the occlusal wear was higher after 2 years than after 1 year (p<0.001). the t-test demonstrated that the wear values, after 1 year, were similar for both compomers (f2000=17.6 ìm and dyract ap=12.8 ìm). however, after 2 years, f2000 restorations (40.6 ìm) suffered significantly more wear (p<0.05) than dyract ap restorations (29.8 ìm). although compomers performed similarly after 1 year, dyract showed less occlusal wear after 2 years. all occlusal class i compomer restorations presented more occlusal wear after 2 years, but were considered as clinically acceptable within the evaluation period. conclusions: the mean wear values found for both compomers do not indicate the need for either repair or replacement of the restorations. key words: dental restoration wear; compomers; clinical trials; dyract ap; f2000. i n t r o d u c t i o n polyacid-modified resin composites, also called compomers, have been developed to improve the physical and mechanical properties of conventional glass ionomer cements (gics)1, and have been classified as intermediate materials between gics and composite resins2. when compared to resin-modified glass ionomers (rmgics), compomers contain larger amounts of monomers in their formulation and release low fluoride levels with a recharge capacity. the better clinical performance of compomers when compared to gics has been demonstrated3. moreover, compomers offer excellent esthetics and are ease to handle, which are some of the reasons for their popularity4, especially in pediatric dentistry5 6. compomers are basically composed of dimethacrylates, modified monomers and fluoride-releasing charges, and contain a proportion of hydrophilic monomer in their organic matrix. the monomer has carboxylic acid groups and polymerizable methacrylate groups, which enables a free-radical polymerization by light curing and an acidbase reaction if water is present. due to continuous improvements in their physical properties and clinical performance, compomers have been used to restore primary teeth and cavities in stress-bearing areas on permanent teeth7-8. the wear resistance is an important property to be evaluated in materials indicated for posterior teeth. the restoration should not only be satisfactory at the time of placement, but also remain this way over time. toothbrushing, 1540 interdental contacts and masticatory movements are some of the wear-inducing factors. the abrasive agents abrade the resin matrix and exposes fillers, which may then be exfoliated from surface. the increase of surface roughness causes accumulation of bacterial biofilm, pigments and food debris, which impairs the longevity of the restoration9. taylor et al.10 evaluated the wear of posterior composite restorations using a series of methodologies. two of them were widely used measuring techniques: a direct evaluation, based on the clinical observation of the restoration, and an indirect method that measures the wear in models obtained from restorations in the mouth. while the direct method provides a subjective evaluation, the indirect method provides a numeric estimative, using standard models of worn restorations. this method is more objective and eliminates many variables. compomers have shown a good performance in primary molars6,11-13. when used in ultraconservative cavities in permanent posterior teeth, the clinical performance of these materials was considered as satisfactory14-15. other studies investigating the behavior of compomers in stress-bearing areas7,16-17 have also observed a good clinical performance. the purpose of this study was to compare the clinical wear of two compomers placed in occlusal cavities in permanent molars within a two-year follow-up period using an indirect method of evaluation. material and methods patient selection patients attending the operative dentistry clinic at the dental school of the federal university of pelotas for routine restorative care were examined as eligible patients for the trial. subjects were enrolled if they needed at least one pair of restorations in the posterior permanent teeth and if the cavities were no larger than 1/3 of the intercuspal distance. patients with tooth loss, parafunctional habits or prostheses were not included in this study. twenty-five patients aged 20 to 32 years (mean age = 25 years) were selected. before any restorative procedure, patients were informed about the study purposes and signed a written informed consent. the research protocol was approved by the research ethics committee of the federal university of pelotas (medical school). restorative procedures right after medical history review, clinical and radiographic examinations were performed. local anesthesia was administrated and the procedures were carried out under rubber dam isolation. cavity preparation was restricted to caries removal with dentin excavators and round carbide burs under air/water cooling. cariesdisclosing agents were used to help distinguishing the outer and the inner carious dentin layers and perform minimal dentin removal without affecting adhesion18. in deep cavities, indirect pulp capping was performed with a calcium hydroxide-based cement (hidro c; dentsply ind. e com. ltda., petrópolis, rj, brazil) prior to the adhesive protocol. fifty cavities were prepared in molars (24 maxillary and 26 mandibular). both materials were applied in the same patient and they were equally distributed between maxillary and mandibular teeth. for the adhesive restorative protocol, the cavities were etched with a 35% phosphoric acid gel (dentsply, milford, de, usa) for 20 s, rinsed and gently dried with a mild air stream for 5 seconds at a distance of approximately 10 cm from tooth surface in order to keep the dentin moist. two coats of prime & bond 2.1 (dentsply) or single bond (3m/ espe, st. paul, mn, usa) were applied and then the cavities were incrementally filled with either dyract ap (dentsply) or f2000 (3m/espe), respectively. each increment of less than 2 mm was light cured for 40 seconds using a halogen light-curing unit with irradiance higher than 450 mw/cm2 (xl3000; 3m/espe) as measured with a curing radiometer. the occlusion was checked after removal of rubber dam. the restorations were finished and polished one week later using multi-blade carbide burs (kg sorensen, alphaville, sp, brazil), enhance polishing system (dentsply) and diamond polishing pastes (diamond; fgm, pomerode, sc, brazil). only one operator performed all the procedures. wear evaluation polished restorations were considered the baseline and impressions were taken with polyvinylsiloxane (express; 3m/espe). to be included in the clinical evaluation, restorations should be classified as alfa for all the items of the criteria of modified usphs system19. a single examiner blinded to the materials used in each case and not the researcher who placed the restorations, performed all evaluations. after 1 and 2 years, the same examiner revaluated the restorations clinically and took new impressions. models for indirect evaluation of wear were obtained with type iv die (durone; dentsply). twenty-one patients with 42 restorations (84% recall rate within 2 years) returned in all clinical appointments. models of the leinfelder scale were used to perform the visual readings of wear in comparison to the models obtained from the compomer restorations. the scale was composed by models of different simulated wear in occlusal cavities: 0, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 500, 600, 700, 800 and 900 ìm. an experienced examiner made the comparisons at the laboratory of dental materials of the university of são paulo, brazil. the occlusal surface of each tooth was uniformly divided in four parts (quadrants). the mean of maximum wear in each quadrant (deeper area at restorations margins) was recorded and a mean wear value was obtained for each tooth. statistical analysis the values at baseline, 1 year and 2 years were used for braz j oral sci. 7(25):1539-1542 two-year clinical wear performance of two polyacid-modified resin composites (compomers) in posterior permanent teeth 1541 dyract ap f2000 1 year 12.8c ± 11.4 17.6c ± 11.1 2 years 29.8b ± 18.1 40.6a ± 20.5 table 1mean wear values and standard deviations (±sd) for both compomers at 1 and 2-year evaluations. * different superscript letters indicate statistically significant differences between means (p<0.05). the statistical analysis. data had a normal distribution and equal variances, allowing parametric tests to be performed. a paired t-test was used to compare each material in both follow-up periods and a t-test was used to identify differences between materials for each evaluation period. a 95% confidence level (p<0.05) was set for all tests and sigmastat 3.0 for windows statistical package (systat software inc., chicago, il, usa) was used. r e s u l t s table 1 presents the mean wear values and standard deviations (±sd) for both compomers after 1 and 2-year evaluations. the paired t-test, applied for comparisons at the different periods for each material showed an increase in the wear of the restorations after 2 years compared to the results obtained at 1 year (p<0.001). f2000 had a significantly higher wear mean value at 2 years compared to dyract ap (p<0.05). the materials had similar behaviors at 1-year evaluation. for both materials, the wear of the restorations at 2 years was approximately 2.3 times greater than that recorded at the 1-year evaluation. d i s c u s s i o n in vitro studies are less expensive and faster than clinical trials, and play a key role as an initial characterization of the mechanical properties of restorative materials. on the other hand, only long-term clinical evaluations can determine the real performance of the materials in the oral environment20. a clinical evaluation period of at least 2 years has been recommended before any dental restorative material can be launched to the market21. in the present study, compomer restorations were placed used after a total-etch adhesive technique. according to dyract ap’s manufacturer, tcb (tetracarboxylic butane acid), a hydrophilic monomer with two methacrylate and two carboxylic groups was added to this product. therefore, 50% of the reactive groups of each molecule are composed by carboxylic acid. these groups are responsible for the bonding of the material to the dental structure. in the present study, the total-etch technique was used to improve bonding ability20 because there are more mechanical requirements in class i cavities in permanent posterior teeth than in cavities prepared in primary teeth. the size of the inorganic fillers can influence the clinical performance and may account for the differences observed in this study. while dyract ap has 2.5 µm strontiumaluminum-fluoride-silicate glass filler, with a mean filler size of 0.8 µm, f2000 contains a glass filler of fluoridealuminum-silicate, with 3-10 µm size range and mean particle size of 6.5 µm. in addition, colloidal silica is added to increase the packability of the inorganic matrix. it is well known that smaller fillers provide better resistance to wear89,22. data from the 1-year evaluation, first stage of the present clinical trial16 showed greater surface roughness for f2000 restorations compared to dyract ap restorations. a rougher surface can accelerate the abrasion caused by foods, leading to an increase in the restoration wear9. the interaction between the matrix and water is probably the reason for the higher wear of the compomers when it is compared to the composites22. the desired fluoride release from compomers can cause material dissolution23, leading to superficial degradation of the restoration. this finding could also influence the increase in clinical wear observed at the 2-year follow-up. clinical studies in primary molars6 and permanent molars8,16 have showed satisfactory performance for dyract ap. in the present study, dyract ap exhibited better performance than f2000 after 2 years, which may be related to the improvements undertaken in dyract ap formulation. dyract ap received an additional crosslinker, and the size of the glass particles was reduced to improve the resistance to wear6. in the present study, occlusal class i cavities with a maximum intercuspal distance of 1/3 were prepared in permanent teeth. manhart et al.24 demonstrated that class i restorations are more likely to have a good clinical performance than class ii restorations. in a 17-year clinical evaluation of posterior composite restorations, rodolpho et al.25 verified that the survival rates decreased significantly for 2, 3 and 4-surface restorations compared to 1-surface occlusal restorations. comparing the follow-up periods of the present investigation, the restorations were submitted to clinical conditions responsible for material aging and higher wear values was recorded in the second moment. there is a positive correlation between the length of the evaluation period and the failure rate. thus, short-time studies have the tendency to produce more favorable results for the tested materials24-26. two experimental groups were evaluated in this study and a control group was not employed, which an important limitation of the study. the use of a control group with a well established composite could provide important parameters for comparison. however, if another group were used in this study, we would probably have some difficulties to fulfill all requirements for inclusion criteria, i.e., the number of class i cavities needed per patient with similar characteristics. the indirect method used in the present study (leinfelder scale) has been considered a good resource to evaluate the wear of dental materials in clinical studies27. however, the leinfeder’s method assess only marginal wear and not the wear on contact-free area/occlusal contact area. it should braz j oral sci. 7(25):1539-1542 two-year clinical wear performance of two polyacid-modified resin composites (compomers) in posterior permanent teeth 1542 be highlighted that small cohesive failures at restoration margins and degradation events other than tooth and food attrition may also affect wear, which means that clinical wear is a complex process. three-dimensional laser digitizing technique has been advocated and could be significantly more effective than subjective evaluations, like cast models evaluations (leinfelder scale) in establishing restoration wear rates28. additionally, the cast model-set employed for wear measurements is critical because significant differences can be found with different scales (m-l scale and leinfelder scale)10. manhart et al.24 performed a review reporting the longevity of restorations in stress-bearing posterior cavities in permanent teeth. the mean (sd) annual failure rates in posterior cavities for some materials were: 3.0% (1.9) for amalgam restorations, 2.2% (2.0) for direct composites, 1.1% (1.2) for compomer restorations, 7.2% (5.6) for regular glass ionomer restorations, 6.0% (4.6) for art glass ionomers, 2.9% (2.6) for composite inlays, 1.9% (1.8) for ceramic restorations, and 1.4% (1.4) for cast gold inlays and onlays. however, most cavities prepared to receive compomer restorations were more conservative than those prepared for direct composites. the adhesive technique used in the present study provided an adequate retention level along 2 years of clinical wear evaluation. the wear observed for both materials (approximately 30 and 40 µm for dyract ap and f2000, respectively) is within the range required for full acceptance of materials intended for use in posterior restorations, according to ada acceptance guidelines requirements29. the mean wear values found for both compomers do not indicate the need for either repair or replacement of the restorations. r e f e r e n c e s 1. mclean jw, nicholson jw, wilson ad. proposed nomenclature for glass-ionomer dental cements and related materials. quintessence int. 1994; 25: 587-9. 2. blackwell g, kase r. technical characteristics of light curing glass-ionomers and compomers. academy of dental materials proceedings of conference on clinically appropriate alternatives to amalgam: biophysical factors in restorative decisionmaking. acad dent mater proc.1996; 9: 77-88. 3. welbury rr, shaw aj, murray jj, gordon ph, mccabe jf. clinical evaluation of paired compomer and glass ionomer restorations in primary molars: final results after 42 months. br dent j. 2000; 189: 93-7. 4. burgess jo, walker r, davidson jm. posterior resin-based composite: review of the literature. pediatr dent. 2002; 24: 465-79. 5. garcia-godoy f. resin-based composites and compomers in primary molars. dent clin north am. 2000; 44: 541-70. 6. pascon fm, kantovitz kr, caldo-teixeira as, borges af, silva tn, puppin-rontani rm et al. clinical evaluation of composite and compomer restorations in primary teeth: 24month results. j dent. 2006; 34: 381-8. 7. huth kc, manhart j, selbertinger a, paschos e, kaaden c, kunzelmann kh et al. 4-year clinical performance and survival analysis of class i and ii compomer restorations in permanent teeth. am j dent. 2004; 17: 51-5. 8. kramer n, garcia-godoy f, reinelt c, frankenberger r. clinical performance of posterior compomer restorations over 4 years. am j dent. 2006; 19: 61-6. 9. bayne sc, taylor df, heymann ho. protection hypothesis for composite wear. dent mater. 1992; 8: 305-9. 10. taylor df, bayne sc, sturdevant jr, wilder ad. correlation of m-l, leinfelder, and usphs clinical evaluation techniques for wear. dent mater. 1990; 6: 151-3. 11. marks la, weerheijm kl, van amerongen we, groen hj, martens lc. dyract versus tytin class ii restorations in primary molars: 36 months evaluation. caries res. 1999; 33: 387-92. 12. peters tc, roeters jj, frankenmolen fw. clinical evaluation of dyract in primary molars: 1-year results. am j dent. 1996; 9: 83-8. 13. roeters jj, frankenmolen f, burgersdijk rc, peters tc. clinical evaluation of dyract in primary molars: 3-year results. am j dent. 1998; 11: 143-8. 14. cehreli zc, altay n. three-year clinical evaluation of a polyacid-modified resin composite in minimally invasive occlusal cavities. j dent. 2000; 28: 117-22. 15. demirci m, sancakli hs. five-year clinical evaluation of dyract in small class i cavities. am j dent. 2006; 19: 41-6. 16. de moura fr, piva e, lund rg, palha b, demarco ff. oneyear clinical evaluation of two polyacid-modified resin composites (compomers) in posterior permanent teeth. j adhes dent. 2004; 6: 157-62. 17. luo y, lo ec, fang dt, smales rj, wei sh. clinical evaluation of dyract ap restorative in permanent molars: 2-year results. am j dent. 2002; 15: 403-6. 18. piva e, meinhardt l, demarco ff, powers jm. dyes for caries detection: influence on composite and compomer microleakage. clin oral investig. 2002; 6: 244-8. 19. barnes dm, blank lw, gingell jc, gilner pp. a clinical evaluation of a resin modified. glass ionomer restorative material. j am dent assoc. 1995; 126: 1245-53. 20. jokstad a, bayne s, blunck u, tyas m, wilson n. quality of dental restorations. fdi commission project 2-95. int dent j. 2001; 51: 117-58. 21. kramer n, garcia-godoy f, frankenberger r. evaluation of resin composite materials. part ii: in vivo investigations. am j dent. 2005; 18: 75-81. 22. zantner c, kielbassa am, martus p, kunzelmann kh. sliding wear of 19 commercially available composites and compomers. dent mater. 2004; 20: 277-85. 23. hicks j, garcia-godoy f, donly k, flaitz c. fluoride-releasing restorative materials and secondary caries. dent clin north am. 2002; 46: 247-76, vi. 24. manhart j, chen h, hamm g, hickel r. buonocore memorial lecture. review of the clinical survival of direct and indirect restorations in posterior teeth of the permanent dentition. oper dent. 2004; 29: 481-508. 25. da rosa rodolpho pa, cenci ms, donassollo ta, loguercio ad, demarco ff. a clinical evaluation of posterior composite restorations: 17-year findings. jdent. 2006; 34: 427-35. 26. brunthaler a, konig f, lucas t, sperr w, schedle a. longevity of direct resin composite restorations in posterior teeth. clin oral investig. 2003; 7: 63-70. 27. bayne sc, taylor df, rekow ed, wilder ad, heymann ho. confirmation of leinfelder clinical wear standards. dent mater. 1994; 10: 11-8. 28. perry r, kugel g, kunzelmann kh, flessa hp, estafan d. composite restoration wear analysis: conventional methods vs. three-dimensional laser digitizer. j am dent assoc. 2000; 131: 1472-7. 29. ada. american dental association. council on scientific affairs. acceptance program guidelines restorative materials. chicago: ada; 1996. braz j oral sci. 7(25):1539-1542 two-year clinical wear performance of two polyacid-modified resin composites (compomers) in posterior permanent teeth 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1506/1159 1/7 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1506/1159 2/7 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1506/1159 3/7 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1506/1159 4/7 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1506/1159 5/7 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1506/1159 6/7 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1506/1159 7/7 oral sciences n3 original article braz j oral sci. october | december 2015 volume 14, number 4 evaluation of hemosponge in promoting dental socket healing after 3rd mandibular premolar extraction in a feline model azin tavakoli1, alireza sagart1 1islamic azad university, faculty of veterinary medicine, garmsar branch, gamsar, iran correspondence to: azin tavakoli po box: 3581631167 garmsar, iran phone: 0098 9127693797/fax:0098 2334552121 e-mail: azin.tavakoli@gmail.com abstract aim: to investigate the healing process following use of collagen sponges in the dental socket after extraction. wound complications during the study were also evaluated. methods: 32 cats were included in this study. iv administration of the combination of diazepam (0.22 mg/kg) and ketamine (10 mg/kg) was used to induce general anesthesia. surgical extraction of both 3rd mandibular premolars was performed. the open dental sockets were divided in two groups. in group a, the open dental socket on the left side was closed using 4-0 monocryl in simple interrupted pattern. in group b, the right dental socket was filled with lyophilized hydrolyzed collagen and the buccal and lingual flaps were sutured using 4-0 monocryl and simple interrupted pattern. meloxicam (0.2 mg/ kg) was used to manage the post-extraction pain in all cats. ampicilline 20 mg/kg was used as prophylaxis. the wounds were observed during the study to evaluate any signs of inflammation or dehiscence. radiographs were taken to compare healing of the socket 3 weeks after the procedure. a 1 mm biopsy punch sample was taken from sockets in all cats for comparison of the healing in both groups. results: hemorrhage occurred only in the sockets of group a. remission of radiolucent area occurred in both groups. mean score of inflammation was lower and mean scores of fibrotic reaction and fibroplasia were higher in group b (p<0.05). conclusions: use of hemosponge in alveolar socket may accelerate fibroplasia and formation of the connective tissue and reduce inflammation after tooth extraction. therefore, post-extraction use of the hemostatic agent in the dental socket is recommended. keywords: tooth socket; tooth extraction; wound healing; cats. introduction dental extraction is a very frequent procedure performed both in human and veterinary dentistry. following dental extraction complications may occur leading to discomfort for the patient. the most frequent complications occurring after dental extraction include hemorrhage from the socket, swelling and pain. more serious complications are infection of the socket, alveolar osteitis and osteomyelitis13. both anatomical and physiological changes in the socket after exodontia, which are generally defined as alveolar bone resorption, affect adversely the following implant therapy as well4-6. different techniques are used to manage and preserve the alveolar socket after extraction. simple closure with sutures and soft tissue coverage of the wound prevents the accumulation of debris and fluid in the socket. moreover, topical applications of antibiotics or anti-inflammatory medications and different absorbable or non-absorbable biomaterials have been suggested to http://dx.doi.org/10.1590/1677-3225v14n4a14 received for publication: november 16, 2015 accepted: december 22, 2015 braz j oral sci. 14(4):330-333 be used in the dental socket7-8. sponges made of collagen, the oldest known protein, are widely used for preservation of dental socket. several studies have been performed to assess the effect of collagen sponges in the healing of dental sockets9-11. reports indicated that the collagen sponges are advantageous and accelerate healing of the wound by stabilizing the blood clot and protecting the alveolar lining of the socket 8. there is no report available about the histopathological changes in the socket when collagen sponges are used. therefore, the aim of this study was to compare dental socket healing with and without topical application of collagen sponges after 3rd mandibular premolar extraction in a feline model. material and methods the study started after the ethical approval was received from the research and ethical committee of the university (# 5238), according to the national regulations in researches on indigenous animals. thirty-two adult domesticated short hair cats were included in the study. food was restricted 4 to 6 h prior to surgery in the animals. im acepromazine (0.05 mg/kg, neurotranq®, alfasan, woerden, the netherlands) was used as premedication and general anesthesia was induced by iv administration of diazepam (0.22 mg/kg zepadic®; caspian tamin pharmaceutical co., rasht, iran) and ketamine (10 mg/kg ketalar®; alfasan) combination. cleansing of the mouth with 0.2% chlorhexidine solution was performed to reduce oral contamination prior to the procedure. the incision was made with a scalpel blade #11 on the buccal and lingual gingival margin of the right and left 3rd mandibular premolar teeth in all cats. a dental elevator was introduced into the periodontal space to stretch the periodontal ligament. finally, dental forceps was used and the tooth extracted. following extraction, the sockets were divided into two groups. in group a, the dental socket on the left side was closed using 4-0 monocryl in simple interrupted pattern. the right dental socket was filled with lyophilized hydrolyzed collagen (hemospon; technew, rio de janeiro, rj, brazil) and in group b, the buccal and lingual flaps were sutured using 4-0 monocryl with simple interrupted suture pattern. intravenous injection of meloxicam (0.2 mg/kg metacam®; boehringer-ingelheim, ingelheim, germany) was used to manage the post-extraction pain in all cats. intramuscular administration of ampicilline 20 mg/kg was used as prophylaxis. the wounds were observed daily in the first week and then weekly during the study to evaluate any signs of inflammation or dehiscence. radiographs were taken to compare healing of the dental socket 3 weeks after the procedure in both groups. in all cats, a biopsy sample was taken from the dental socket using a 1 mm biopsy punch for histopathological evaluation 3 weeks following the procedure. sixty-four dental sockets were evaluated in the present study. the tissue was fixed in 10% buffered formalin and processed in a tissue processor. paraffin-embedded tissue sections were stained by harris hematoxylin and eosin (h&e) method. the stained sections were viewed under light microscope at 4x and 10x magnifications for examination. a single veterinary pathologist viewed the slides and graded them according to epithelialization, fibrotic reaction, fibroplasia, inflammation and edema. epithelialization was graded as non-existent (1), start of migration (2), covering less than half the wound surface (3), covering more than half the wound surface (4), irregular covering of the wound (5) and normal covering of the wound (6). inflammation was graded as infiltration of acute inflammatory cells in wound clot and perivascular regions more than 1.2 of high power field (hpf) (1), between 1.2-1.4 of hpf (2), less than 1.4 of hpf (3), infiltration of acute inflammatory cells in the clot, perivascular regions and in connective tissue (4), infiltration of acute inflammatory cells in wound clot and perivascular regions (5), infiltration of acute inflammatory cells just in wound clot (6), infiltration of acute inflammatory cells in wound margin (7) and no inflammation(8). fibrotic reaction graded as no deposition of collagen (1), new collagen bundle deposition (2), lamina propria fibrosis (3), dermal fibrosis (4) and fibrosis in all layers (5). fibroplasias graded as no granulation tissue (1), loose granulation tissue (2), cell-rich granulation tissue (3), a few collagen fiber depositions (4), more collagen deposition (5) and remodeling of collagen bundles (6). means of the measured variables were compared between the groups using t-test. p values less than 0.05 were considered statistically significant. results hemorrhage was reported only in the sockets of group a. wound dehiscence occurred in 9 sockets and infection was observed in 2 sockets in the same subject, mostly in group a following the first week after the procedure. radiographs revealed remission of radiolucent areas in both groups. however, soft tissue swelling was noted in radiographs of 9 patients in group a and 4 patients in group b, respectively. histopathological studies indicated that the mean scores of fibroplasia and fibrotic reaction in group b were statistically higher in comparison with group a (p 0.05) (figures 1 and 2). the inflammation scores of wound edges were also significantly lower in group b than in group a (p<0.05). epithelialization scores were not statistically different between the groups (p>0.05) (table 1). clinical evaluation of the wounds was performed during the study. the observed complications are listed in table 2. discussion the purpose of the present study was to investigate the healing process following the use of collagen sponges in the dental socket after extraction. it was also evaluate the efficacy of collagen sponges by assessing the rate of wound complications during the study. the healing of 64 dental sockets after 3rd mandibular premolar extraction was evaluated evaluation of hemosponge in promoting dental socket healing after 3rd mandibular premolar extraction in a feline model331331331331331 braz j oral sci. 14(4):330-333 group epithelialization fibroplasia fibrotic reaction inflammation group a 4.4±0.55 3±0.36* 2.8±0.13* 6.9±0.9* group b 4.8±0.45 4.1±0.55 4.1±0.66 4.1±0.33 table 1table 1table 1table 1table 1mean±sd histopathological scores following 3rd mandibular premolar extraction in the groups of the study. group hemorrhage swelling during the week dehiscence infection group a 11 9 8 1 group b 0 4 1 1 table 2table 2table 2table 2table 2the frequency of wound complications after dental extraction in the groups of the study. fig. 1. granulation tissue formation in group a. arrows point to edema and granulation tissue formation. inflammation and edema are evident. (x 400h & e staining) fig. 2. fibrotic reaction and fibroplasia in group b. arrows point to angiogenesis of the tissue. (x 400h & e staining) in the present study. wound complication rate was higher in dental sockets that were routinely closed without the hemosponge. hemorrhage after closure was evident in 11 patients and occurred only in the sockets of group a, which highlights the nature of hemosponge as a hemostatic device. wound dehiscence occurred in 8 and 1 of the subjects in group a and b, respectively, probably due to the inflammation that caused discomfort on the left side. bleeding occurs immediately after dental extraction and inflammation, which is the first phase of wound repair that takes place within a few minutes after exodontia. clot fills the remaining socket. the clot acts as a lining to the socket, which preserves the alveolar bone and exposed nerve ends1213. fibroplasia continues and the clot is replaced by granulation tissue usually a week after the procedure. then the epithelial cells recognize the granulation tissue as a connective tissue and start to migrate and cover the newly formed granulation tissue. remodeling begins in the underneath granulation tissue to form the provisional matrix. finally gradual mineralization of the matrix occurs to form the lamellar bone14-15. therefore, clot formation works as a beginning stage of the socket-healing cascade. then it works as a matrix for angiogenesis and fibroplasia. if the clot is washed away or fibrinolysis happens, early swelling and sever pain will occur. thus, the more stable the clot in the dental socket, the less inflammation, alveolar osteitis and pain will be expected16-17. hemosponge is a soft, white, sterile and hemostatic spongelike structure made of lyophilized, hydrolyzed collagen of porcine origin, which is capable of absorbing and holding blood many times its weight. the sponge is completely absorbed usually within 4 to 6 weeks18. the risk of postextraction bleeding was reported to decrease when the collagen hemostatic sponge is used in the extraction socket19. elimination of the radiolucent area of the dental socket was observed in both groups, which underscores that the healing process continued without complication in most sockets. histopathological evaluation of the wounds 3 weeks after the extraction showed that the score of inflammation was significantly less when the hemosponge was used as in group b in comparison to simple closure, as in group a. considering the score of fibroplasia and fibrotic reaction, the best results were obtained in group b. as mentioned above, the maintenance of blood clot acts as an inflammation preventive factor during socket healing. therefore, efforts evaluation of hemosponge in promoting dental socket healing after 3rd mandibular premolar extraction in a feline model * significant difference (p<0.05) 332332332332332 braz j oral sci. 14(4):330-333 were made to stabilize the clot at the early stages of the healing. hemostatic sponges not only preserve the clot and work as hemostatic agent, but also prevent the collapse of newly formed soft tissue into the socket. this extracellular matrix establishes a scaffold so that fibroplasia and formation of the granulation tissue is accelerated. thus, bone healing in the socket is stimulated. jenkins20 suggested that the spongy structure of the gelatin sponge is initially responsible for the stimulation of clot formation, and that the structure causes damage to the platelets in bleeding. this means that releasing of prothrombin and calcium in the clot could be sufficient to start the clotting mechanism. in the present study, fewer complications were observed in the sockets treated with hemosponge, which is consistent with previous studies. choo et al.8 reported less rates of complication in patients with 3rd molar extraction by the use of absorbable collagen sponges. the scores of epithelialization were not significantly different between the groups. margo21studied the healing process of the dental socket in a rat model using microfibril collagen hemostat. he reported that the device interferes with the early stage of healing of the socket, but it does not affect the final result of the healing. this finding is in agreement with our findings. although the hemosponge did not facilitate the epithelialization, occurrence of wound dehiscence and inflammation was not increased. we have limited access to information about the animals after discharges, so that a longterm follow up of the subjects was not undertaken. therefore, clinical trials and long-term prospective studies of the use of hemosponge are advised. post-operative computed tomography scans of the dental sockets were not available. if available they may provide more detailed and precise information about the healing and bone formation of the dental socket. infection of the socket occurred in both sockets of the same patient. oral hygiene of the patient seems important for reduction of the infection risk in the socket after extraction. therefore, postextraction wound care is highly recommended specially during the first week after dental removing. hemosponge is a non-toxic and inexpensive structure that provides hemostasis, stabilizes clot, promotes healing and ridge preservation of the socket22-23. in addition, less inflammation of the related soft tissues causes less pain and discomfort for the patient following extraction. based on clinical, radiological and histological results of the present study, the use of hemosponge in the alveolar sockets may accelerate fibroplasia and formation of the connective tissue and reduces inflammation of the socket after extraction of the 3 rd mandibular premolar teeth. therefore, post-extraction use of the hemosponge in the dental socket is recommended. references 1. bouloux gf, steed mb, perciaccante vj. complications of third molar surgery. oral maxillofac surg clin north am. 2007; 19: 117-28. 2. bui ch, seldin eb, dodson tb. types, frequencies, and risk factors for complications after third molar extraction. j oral maxillofac surg. 2003; 61: 1379-89. 3. hwang jk, kim kw. complications of impacted third molar extraction: retrospective study. j korean assoc oral maxillofac surg. 2010; 36:119-24. 4. pietrokovski j, massler m. ridge remodeling after tooth extraction in rats. j dent res. 1967; 46: 222-31. 5. boyne pj. osseous repair of the postextraction alveolus in man. oral surg oral med oral pathol. 1966; 21: 805-13. 6. devlin h, sloan p. early bone healing events in the human extraction socket. int j oral maxillofac surg. 2002, 31: 641-5. 7. alexander re. dental extraction wound management: a case against medicating postextraction sockets. j oral maxillofac surg. 2000; 58: 538-55. 8. choo h, jung hd, kim bj, kim ch, jung ys. complication rates in patients using absorbable collagen sponges in third molar extraction sockets: a retrospective study. j korean assoc oral maxillofac surg. 2015; 41: 26-9. 9. 9oliveira mr, martins eg, mariano rc, sonoda ck, garcia r, morais w. tissue engineering: using collagen type i matrix for bone healing of bone defects. 10. j craniofac surg. 2013; 24:e394-6. 11. 10lee ch, singla a, lee y. biomedical applications of collagen. int j pharm. 2001; 221:1-22. 12. 11rao kp. recent developments of collagen-based materials for medical applications and drug delivery systems. j biomater sci polym ed. 1995; 7: 623-45. 13. 12amler mh. the time sequence of tissue regeneration in human extraction wounds. oral surg oral med oral pathol.1969; 3: 309-18. 14. clafin rs. healing of disturbed and undisturbed extraction wounds. j am dent assoc. 1996; 23: 945-59. 15. pagni g, pellegrini g, giannobile wv, rasperini g. postextraction alveolar ridge preservation: biological basis and treatments. int j dent. 2012; 2012: 1-13 16. liu j. mechanism of guided bone regeneration: a review. open dent j. 2014; 8: 56-65. 17. nilsson bg. fibrinolytic activity in alveoli after tooth extraction. odontology, 1968, 19: 197-204. 18. vezeau pj. dental extraction wound management: medicating postextraction sockets. j oral maxillofac surg. 2000; 58: 531-7. 19. ogle oe. perioperative hemorrhage. in: dym h, ogle oe. atlas of minor oral surgery. philadelphia, pa: saunders; 2000. p.62-3. 20. svensson r, hallmer f, englesson cs, svensson pj, becktor jp. treatment with local hemostatic agents and primary closure after tooth extraction in warfarin treated patients. swed dent j. 2013; 37: 71-7. 21. jenkins hp, janda r. studies on the use of gelatin sponge or foam as a hemostatic agent in experimental liver resections and injuries to large veins. ann surg. 1946; 124: 952-61. 22. magro fo, garcia r, magro fn. histologic study of use of microfibrillar collagen hemostat in rat dental sockets. braz dent j. 2003; 14: 12-7. 23. mahesh l, kurtzman g, shukia s. regeneration in periodontics: collagen a review of its properties and applications in dentistry. compend contin educ dent. 2015; 36: 358-63. 24. park bj. ridge preservation following tooth extraction using an absorbable gelatin sponge. ohdm. 2015; 14: 271-3. evaluation of hemosponge in promoting dental socket healing after 3rd mandibular premolar extraction in a feline model333333333333333 braz j oral sci. 14(4):330-333 oral sciences n3 original article braz j oral sci. october | december 2015 volume 14, number 4 in vitro effects of erosive challenge on the surface properties of sealants isabella cavalcante medeiros1, bruna palmeira costa1, brenna louise cavalcanti gondim1, hugo lemes carlo1, rogério lacerda dos santos1, fabíola galbiatti de carvalho1 1universidade federal da paraíba ufpb, health science center, department of clinical and social dentistry, joão pessoa, pb, brazil correspondence to: fabíola galbiatti de carvalho universidade federal da paraíba centro de ciências da saúde – programa de pós-graduação em odontologia campus i cep: 58051-900 joão pessoa, paraíba, brasil phone: +55 83 32167251 fax:+55 83 32167797 e-mail: fabigalbi@yahoo.com.br abstract aim: to assess in vitro the surface roughness (ra), vickers hardness (vhn) and surface morphology of resin and glass ionomer materials used for sealants after dynamic erosive challenge. methods: twenty specimens of each material were prepared and divided into experimental (erosive challenge) and control groups (n=10): protect riva (sdi), opallis flow (3m espe), fluroshield (dentsply), filtek z350 xt flow (3m espe). the erosive challenge was performed 4 times per day (90 s) in cola drink and for 2 h in artificial saliva for 7 days. the control specimens were maintained in artificial saliva. ra and vhn readings were performed before and after erosion. the percentage of hardness loss (%vhn) was obtained after erosion. the surface morphology was evaluated by scanning electron microscopy (sem). the data were analyzed by anova, tukey and paired t tests (α=0.05). results: after erosion and saliva immersion, there was an increase in ra values for all groups and riva group showed the highest ra values. after erosive challenge, riva and filtek groups showed significant decrease in vhn values, but filtek group showed the greatest %vhn. for all groups there was inorganic particle protrusion and matrix degradation after erosion visualized by sem images. conclusions: erosive challenge affected the surface properties of all materials used as sealants, particularly in the riva and filtek groups. keywords: tooth erosion; composite resins; glass ionomer cements; hardness tests. introduction dental erosion is defined as the loss of tooth substance by the chemical process of acid exposure and dissolution, involving no bacterial plaque acid1. the acids in food and drink are considered the major etiological factors responsible for erosive lesions in enamel1-2. there is evidence that the prevalence of erosion is increasing because of the high consumption of soft drinks3. the most important aspects in the treatment of patients with dental erosion are diagnosis and prevention of lesion progression to limit additional destruction of tooth tissue. elimination of the acid source and preventive approaches using fluoride compounds are indicated to control the tooth erosion or the dental tissue softening caused by acidic solutions4-7. the effects of dentin-bonding agents and restorative materials used to seal dental structures have also been advocated to control or prevent the development of erosion lesions8-10. it was shown that resin-based bonding agents could protect against erosion and abrasion in situ8,10. wegehaupt et al.11 (2012) demonstrated that resin-based materials were also able to reduce the erosive demineralization of bovine enamel after immersion in hydrochloric and citric acids over consecutive days. however, to simulate what occurs in the oral cavity, in vitro studies used braz j oral sci. 14(4):276-281 received for publication: november 18, 2015 accepted: december 12, 2015 http://dx.doi.org/10.1590/1677-3225v14n4a05 277277277277277 dynamic erosive ph-cycling challenge, with daily cycles of immersion in acid solution and artificial saliva, to mimic the daily ingestion of acidic beverages6,12. it would be interesting to investigate the surface properties of materials used as sealants after erosive challenge because the surface properties of sealant materials can be used to evaluate the surface degradation of the material and to predict its resistance in the oral environment. however, few studies have evaluated the surface properties of materials used as sealants after dynamic erosive challenge12. thus, the aim of this study was to evaluate the surface microhardness and roughness of ionomeric and resin-based materials used as sealants and their morphological surface characteristics using scanning electron microscopy (sem), after erosive challenge with a cola beverage. the tested hypothesis was that there would be differences in microhardness, roughness and morphological surface characteristics among the sealants after erosive challenge. material and methods specimen preparation four materials used as sealants were investigated in this study: three resin-based sealants – filtek z350 xt flow (3m/ espe, st. paul, mn, usa), fluroshield (dentsply, rio de janeiro, rj, brazil) and opallis flow (fgm, joinville, sc, brazil); and one glass ionomer sealant – riva protect (sdi, bayswater, victoria, australia). the compositions of the evaluated materials are in table 1. twenty specimens of each material were fabricated using silicone molds (4 mm diameter x 2 mm high), according to the manufacturers’ instructions. the capsules of riva protect were triturated for 10 s in an amalgamator (ultramat 2, sdi, bayswater, victoria, australia). then, the material was inserted into the matrix with the riva applicator. the specimens were sealant/batch number riva protect (sdi, bayswater, victoria, australia, f1111031) fluroshield (dentsply, rio de janeiro, rj, brazil, 946229g) filtek z350 xt (3m/espe, st. paul, mn, usa, n385003) opallis flow (fgm, joinville, sc, brazil, 040612) type glass ionomer resin resin resin composition* capsule: compartment 1 (powder): fluoro aluminosilicate glass (90% load size not specified) and polyacrylic acid (10%); compartment 2 (liquid): polyacrylic acid (25%), tartaric acid (10%) and balanced ingredient (non-hazardous) (65%) ued-bis-gma (<40%), barium aluminoborosilicate glass (30%), polymerizable dimethacrylate resin (<10%), bis-gma (<5%), sodium fluoride (<5%), dipentaerythritol pentaacrylate phosphate (<5%), titanium dioxide (<3%), amorphous silica (<2% load size not specified) silane-treated ceramic (52-60%), bis-gma (10-15%), tegdma (10-15%), bisema (1-5%), silane-treated silica (3-11%), silane-treated zirconium oxide (3-11% mean load size 0.01-6 µm), functionalized dimethacrylate polymer (1-5%) udma (5-10%), tegdma (5-10%), bis-ema (5-10%), silanized inorganic filler, ba-al-si micro-particles and sio2 in nanoparticles (0.05 and 5.0 µm) (~72%) *bis-gma: bisphenol-a-glycidyl methacrylate; tegdma: triethylene glycol dimethacrylate; ued-bis-gma: urethane-modified bis-gma dimethacrylate; bis-ema: etoxylated bisphenol-a-diglycidyl methacrylate table 1table 1table 1table 1table 1 compositions of materials used as sealants in the study. covered with acetate strips (probem ltda, catanduva, são paulo, brazil) and were pressed flat with a glass slide for 5 min to obtain a smooth surface. the resin-based materials were inserted into the matrix and were covered with acetate strips and polymerized for 20 s with a led curing light (1200 mw/cm² – radii cal; sdi). the specimens were maintained in relative humidity for 24 h, before the baseline roughness and microhardness measurements were obtained as described above. specimens of each material were divided in two groups (n=10): erosion (erosive challenge) and control (artificial saliva immersion) groups. dynamic erosive ph-cycling challenge the specimens were immersed in cola drink (coca-cola®, sp, brazil ph 2.3) at room temperature in individual containers (10 ml/specimen) for 90 s four times/day4-5. subsequently, the specimens were rinsed thoroughly with deionized water and were immersed in artificial saliva with a 7.0 ph (10 ml/block) at room temperature for 2 h, both between erosive challenges and overnight4-5. the artificial saliva was fabricated according to carvalho et al.13 (2013). this erosive challenge was repeated for 7 days. the cola drink and artificial saliva were changed after every cycle. during the acidic cycles, the samples were kept in hermetically sealed containers to prevent the loss of carbonation from the cola drink. the specimens in the control group were immersed in artificial saliva for 7 days. the artificial saliva was changed every day. surface roughness measurements at the end of the erosive challenge, the specimens were ultrasonically washed for 10 min and dried with absorbent paper. they were then fitted to a surface roughness-measuring instrument (tr200, digimess, são paulo, sp, brazil). in each specimen, three successive measurements in the central area in vitro effects of erosive challenge on the surface properties of sealants braz j oral sci. 14(4):276-281 roughness values (ra) sealants erosion group artificial saliva group baseline after erosion baseline after saliva riva protect 0.29 ± 0.07a,a* 0.45 ± 0.06b,a** 0.29 ± 0.03a,a 0.35 ± 0.02b,a opallis flow 0.04 ± 0.01a,b 0.07 ± 0.01b,b 0.04 ± 0.01a,b 0.06 ± 0.01b,b filtek z350 xt 0.06 ± 0.02a,b 0.10 ± 0.02b,b 0.07 ± 0.03a,b 0.06 ± 0.01a,b fluroshield 0.07 ± 0.01a,b 0.13 ± 0.02b,b 0.08 ± 0.01a,b 0.08 ± 0.01a,b * the same uppercase letters indicate that there was no significant difference between the initial and post-treatment values of each sealant material (paired t test, p > 0.05). ** the same lowercase letters indicate that there were no significant differences among the sealants materials at baseline or after treatment (two-way anova and tukey’s test, p>0.05). table 2table 2table 2table 2table 2 surface roughness (ra) of the sealant materials after erosive challenge and immersion in artificial saliva (control). values are expressed as means ± standard deviations (µm). microhardness values (vhn) sealants erosion group artificial saliva group baseline after erosion %vhn loss baseline after saliva %vhn loss riva protect 64.2 ± 1.8a* 59.4 ± 3.3b -7.3b** 64.3 ± 1.5a 64.0 ± 2.4a -2.7a** opallis flow 43.5 ± 2.6a 40.2 ± 1.9a -7.6b 41.5 ± 3.1a 39.6 ± 2.1a -7.9a filtek z350 xt 44.1 ± 0.7a 24.1 ± 0.9b -44.6a 43.3 ± 0.9a 43.1 ± 1.2a -2.7a fluroshield 21.4 ± 2.6a 20.4 ± 2.4a -5.6b 20.3 ± 1.6a 19.9 ± 1.9a -6.0a table 3table 3table 3table 3table 3 surface microhardness (vhn) and percentage of vhn loss of sealant materials after erosive challenge and immersion in artificial saliva (control). values expressed as means ± standard deviations *the same uppercase letters indicate that there was no significant difference between the initial and post-treatment values of each sealant material (paired t test, p > 0.05). **the same lowercase letters indicate that there were no significant differences among sealant materials at baseline or after treatment (two-way anova and tukey’s test, p>0.05). in different directions were performed by the same examiner, and the mean surface roughness values (ra) were obtained and expressed in micrometers. the roughness test was performed at baseline and 24 h after erosive challenge. surface vickers microhardness the microhardness measurements were obtained with a hardness tester (hmv ii; shimadzu corporation, kyoto, japan), using a vickers indenter (vhn) and a 200 g load, with a 15 s dwell time14. five indentations were made in each specimen, at least 50 µm apart, and the mean vhn value was obtained. in addition, the percentage of microhardness loss (%vhn) was calculated using the following formula12: %vhn = 100 (vhn(f) – vhn(i))/vhn(i), where vhn(i) is the average of the initial (baseline) microhardness measurements, and vhn(f) is the average of the ûnal (after erosive challenge) microhardness values. scanning electron microscopy (sem) three representative specimens from each group were mounted on aluminum stubs and sputter-coated with gold in vacuum (balzers-scd 050 sputter coater, balzers, liechtenstein). three extra specimens were prepared for baseline (without saliva or coca immersion) evaluation. a leo 1430 scanning electron microscope (zeiss inc., thornwood, ny, usa) was used for sem analyses. analyses were performed at 1500 x and 2000 x magnification before and after the erosive challenge and immersion in artificial saliva. statistical analysis the data were analyzed using graphpad instat software, version 2.0 (graphpad software, la jolla, ca, usa) at a significance level α=0.05. the sample size was calculated considering the minimum difference between the average of treatments (mean ± standard deviation) of 0.5 ± 0.05 µm for roughness testing and 13.0 ± 3.0 vhn for microhardness testing. with a significance level of 0.05 and a power of 95%, a minimum of four specimens per group was required. all tested variables satisfied the assumptions of normal distribution, therefore two-way anova and tukey’s test were performed for statistical comparisons of ra and vhn measurements among the sealants. student’s paired t-test was used compare ra and vhn measurements before and after erosive challenge for the same sealant. unpaired t-test was used to compare ra and vhn values after erosive challenge and artificial saliva immersion for each sealant. results the results of the roughness and microhardness tests are in tables 2 and 3, respectively. when the ra values between baseline and post-treatment (after erosion or after saliva) were compared, there was a statistically significant increase in ra values in all the groups (p=0.001), except for the fluroshield and filtek groups, which did not show significant differences in ra values after artificial saliva immersion (p=0.06 and p=0.08, respectively) (table 2). when the comparison was made among sealants after erosion 278278278278278in vitro effects of erosive challenge on the surface properties of sealants braz j oral sci. 14(4):276-281 279279279279279 sealants microhardness values (vhn) after erosion after saliva riva protect 59.4 ± 3.3b* 64.0 ± 2.4a opallis flow 40.2 ± 1.9a 39.6 ± 2.1a filtek z350 xt 24.1 ± 0.9b 43.1 ± 1.2a fluroshield 20.4 ± 2.4a 19.9 ± 1.9a * the same uppercase letters indicate that there was no significant difference in vhn values between the erosion and saliva treatments of each sealant material (unpaired t test, p > 0.05). table 5table 5table 5table 5table 5 microhardness (vhn) measurements of sealant materials after erosive challenge and artificial saliva immersion. values are expressed as mean ± standard deviation. sealants roughness values (ra) (µm) after erosion after saliva riva protect 0.45 ± 0.06 a* 0.35 ± 0.02b opallis flow 0.07 ± 0.01a 0.06 ± 0.01a filtek z350 xt 0.10 ± 0.02a 0.06 ± 0.01b fluroshield 0.13 ± 0.02a 0.08 ± 0.01b * the same uppercase letters indicate that there was no significant difference in ra values between the erosion and saliva treatments of each sealant material (unpaired t test, p > 0.05). table 4table 4table 4table 4table 4 surface roughness (ra) measurements of sealant materials after erosive challenge and artificial saliva immersion. values are expressed as mean ± standard deviation. and after saliva immersion, the riva group showed the highest ra values (p=0.001) and there were no significant differences among other groups after both treatments (p=0.07) (table 2). after erosive challenge, the riva and filtek groups showed significant decrease in vhn values (p=0.03 and p=0.001, respectively), but after saliva immersion, there were no significant vhn alterations in any group (table 3). after erosion, when the %vhn values were compared , the filtek group had the greatest value (p=0.001) and riva, fluroshield and opallis groups did not show significant differences among them (p=0.08). however, after artificial saliva immersion, there were no significant differences in %vhn among the groups (p=0.10) (table 3). tables 4 and 5 show the comparison of ra and vhn values of each material between “after erosion” and “after saliva immersion” treatments, respectively. for all the materials, the erosion treatment showed significantly higher ra values when compared to saliva immersion treatment, except for the opallis group (p=0.08) (table 4). for vhn measurements, riva and filtek groups showed higher vhn values after saliva immersion compared to after erosion treatment (p=0.03 and p=0.001, respectively) (table 5). figures 1, 2, 3 and 4 show the sem images at baseline, after erosive challenge and after saliva immersion. for all the groups, after the erosive challenge, a protrusion of the inorganic particles and degradation of the ionomeric or resin matrix (figures 1b, 2b, 3b and 4b) was observed. the riva group had greater surface degradation than the other groups, showing a porous surface and matrix deterioration (figure 1b). the cracks visualized on ionomeric sealant images fig.1. sem images of the surface morphology for the riva protect group (1500 x): (a) baseline; (b) after erosive challenge and (c) after saliva immersion; ( ): filler particles. fig. 2. sem images of the surface morphology for the fluroshield group (1500 x): (a) baseline; (b) after erosive challenge and (c) after saliva immersion; ( ): filler particles. fig. 3. sem images of the surface morphology for the filtek z350xt flow group (1500 x): (a) baseline; (b) after erosive challenge and (c) after saliva immersion; ( ): filler particles. fig. 4. sem images of the surface morphology for the opallis flow group: (a) baseline (2000x); (b) after erosive challenge (1500 x) and (c) after saliva immersion (1500 x). (figure 1) were artifacts caused by vacuum during sample preparation. after artificial saliva immersion, slight surface degradation was observed in the riva and fluroshield groups (figures 1c and 2c), but the other groups did not show morphological differences between baseline and the treatments. furthermore, the opallis group showed the smallest surface morphological changes among baseline, erosive challenge and artificial saliva immersion (figure 4). discussion resin and ionomeric materials can be used in clinical practice to seal tooth structures and to prevent dental erosion8,11. it is known that, during consumption, beverages contact only with the tooth surface and restorative materials for a short time before they are washed away by saliva15. in previous studies, the sealant surfaces have usually contacted acidic beverages for prolonged periods of time, or the studies did not include the saliva in their methodologies11,16. the in vitro effects of erosive challenge on the surface properties of sealants braz j oral sci. 14(4):276-281 280280280280280 present study was designed to overcome these limitations of in vitro erosion studies, using a dynamic erosive ph-cycling model. this dynamic erosive model simulated the typical consumption of individuals considered to be at risk for dental erosion4-6, using a beverage (coca-cola, ph 2.3) that is widely consumed by the population and that has high erosive potential due to its low ph and fluoride/calcium concentrations2. the results showed an increase in ra values for all of the sealants after the erosive challenge (table 2). francisconi et al.12 (2008) found wear of approximately 0.3 µm for resin and glass ionomer restorative materials after an erosive challenge similar to that used in the present study. according to these authors, roughness values of materials after erosive challenge of approximately 0.1-0.4 µm could be considered low, like the ra values in the present study. for resin-based sealant materials, the phosphoric acid found in cola beverage could induce softening of the bisphenol-a-glycidyl methacrylate (bis-gma) polymers in resin sealants, which could result from the leaching of diluent agents, such as triethylene glycol dimethacrylate (tegdma)12. additionally, softening of the resin matrix could favor the displacement of inorganic fillers, contributing to the formation of a rough surface. the ionomeric sealant (riva group) showed the highest ra values before and after erosion and artificial saliva immersion (table 2). sem images also showed greater surface degradation of in the riva group than in other groups, with a porous surface and higher matrix deterioration (figure 1b). because the size of the glass filler particles in ionomeric cements is larger than in resin materials17 (figure 1), there is likely less homogeneity between the filler and matrix, thus increasing its surface roughness in both immersion media. furthermore, in glass ionomer cements, there is microcrack formation in a ionic-crosslinked polyalkenoate matrix, leading to subsequent loss of particle adherence, which could also cause increases in roughness and wear17. similar to resinbase sealants, acidic beverages could dissolve the siliceous hydrogel layer and the matrix peripheral to the glass particles in ionomeric sealant, causing a rougher surface. the dissolution of siliceous hydrogel matrix could also explain the significant decrease in vhn values in the riva group compared to other groups after the erosive challenge (table 3). the study by francisconi et al.12 (2008) also showed that glass ionomer cements had greater microhardness losses compared to resin composites, because the acid attack on the resin matrix occurred in a lesser extent than in the siliceous hydrogel matrix. after artificial saliva immersion, there were no significant differences among the sealants in %vhn loss; it seems that the acidic ph of beverages could interfere to a greater extent in the organic matrix degradation of the tested materials. in the present study, the filtek group showed the greatest %vhn loss, compared to other resin-based sealants after erosive challenge (table 3). it is known that inorganic filler particles reduce polymerization shrinkage at the same time as they enhance the mechanical properties of the resin material18. the higher percentage of inorganic filler (72%) and the lower organic content (30%) of opallis sealant, compared with filtek z350 xt (52-60% and 3-11%, respectively) (table 1) probably resulted in a smaller %vhn loss in the opallis group19. the higher percentage of inorganic filler and its possible displacement due to matrix degradation could also explain the higher ra values after saliva immersion for opallis group. furthermore, the percentage of ethoxylated bisphenol-a dimethacrylate (bis-ema) in the organic matrix of opallis was higher than in the filtek group. the decreased flexibility and elimination of the hydroxyl groups from the bis-gma monomer to bis-ema increased the hydrophobicity of bisema monomer20. this characteristic reduced the water uptake by the matrix and its plasticization after contact with oral liquids. thus, the percentage of bis-ema monomer may be partially responsible for the biochemical stability of opallis sealant in aqueous environments, regardless the ph of the solution., the opallis group was the only one that showed no significant differences in ra or vhn values after erosive challenge and immersion in artificial saliva (tables 4 and 5). after erosion, sem images also showed a surface with matrix degradation and the protrusion of inorganic filler in the fluroshield and filtek groups (figures 2b and 3b, respectively), whereas the opallis group showed a homogeneous surface similar to that at baseline (figures 4a and b). the concentration and particle size of the glass ionomer cement is greater than that of the resin sealants. thus, it was possible to visualize more easily the matrix degradation and the protrusion of the particles after erosive challenge. a limitation of this study was not showing a higher magnification of sem images of resin sealants, but even in 1500x magnification it was still possible to verify the same degradation characteristics (protrusion of the particles) after erosion in figures 2b and 3b related to fluroshield and filtek z350 materials. for opallis group (figure 4) it was not possible to verify the degradation on the surface because this material suffered less degradation, as explained before. hardness is a physical property possibly related to the degree of conversion and to the amount of filler particles in resin-based materials21. thus, it is likely that the relatively high degree of conversion of the bis-gma/ued-bis-gma resin matrix for fluroshield might have compensated for the small percentage of filler particles, resulting in a smaller %vhn loss than in the filtek group (table 3). it is generally accepted that crosslinked polymers are more resistant to degradation and solvent uptake in aqueous environments, whereas linear polymers present more spaces and pathways for molecules to diffuse within their structures and to degrade the material21. these facts may also be the reasons why the fluroshield group did not show significant differences between vhn values before and after erosive challenge and saliva immersion (tables 3 and 5). for the other groups, except for opallis, the erosive challenge caused greater degradation of the surface properties than artificial saliva immersion (tables 4 and 5). sem images also showed similarities between the surface morphology after erosion and after saliva immersion for the fluroshield and opallis in vitro effects of erosive challenge on the surface properties of sealants braz j oral sci. 14(4):276-281 281281281281281 groups (figures 2b, 2c and 4b, 4c). the oral cavity is the adequate environment for predicting the behavior of dental materials, but in vitro models are very important for providing insight into the fundamental mechanisms of biodegradation12. clinically, a thin layer of sealant is applied on tooth structure to prevent against erosion. this study used specimens 2 mm thick, but the analysis was restricted to surface properties and the thickness of specimens did not interfere in the results. although sealants materials may degrade with time9, it may have a role in prevention of tooth erosion and may be a less patient-dependent approach compared to fluoride application, because it does not depend on the patient compliance. in general, the present study showed that that erosive challenge with cola beverage caused changes in the surface properties of sealant materials, and the opallis group had a better in vitro performance related to surface properties. the hypothesis tested in the present study was accepted because there were differences among the sealants in microhardness, roughness or morphological surface characteristics after erosive challenge. longer periods of erosive challenge should be used, and other important properties of the sealants, such as adhesion and microleakage, should be studied under erosive challenge. acknowledgements the authors are grateful to laisa daniel gondim for contributions to the roughness surface analysis and to professor severino jackson guedes de lima and isaque jerônimo porto of rapid solidification laboratory/federal university of paraíba (lsr/ufpb) for use of sem. references 1. lussi a, carvalho ts. erosive tooth wear: a multifactorial condition of growing concern and increasing knowledge. monogr oral sci. 2014; 25: 1-15. 2. salas mms, dantas rvf, sarmento hr, vargas-ferreira f, torriani d, demarco ff. tooth erosion and dental caries in schoolchildren: is there a relationship between them? braz j oral sci. 2014; 13: 12-6. 3. gurgel cv, rios d, buzalaf ma, da silva sm, araújo jj, pauletto ar, et al. dental erosion in a group of 12and 16-year-old brazilian schoolchildren. pediatr dent. 2011; 33: 23-8. 4. levy fm, magalhães ac, gomes mf, comar lp, rios d, buzalaf ma. the erosion and abrasion-inhibiting effect of tif4 and naf varnishes and solutions on enamel in vitro. int j paediatr dent. 2012; 22: 11-6. 5. magalhães ac, levy fm, rios d, buzalaf ma. effect of a single application of tif4 and naf varnishes and solutions on dentin erosion in vitro. j dent. 2010; 38: 153-7. 6. medeiros ic, brasil vl, carlo hl, santos rl, de lima ba, carvalho fg. in vitro effect of calcium nanophosphate and high-concentrated fluoride agents on enamel erosion: an afm study. int j paediatr dent. 2014; 24: 168-74. 7. huysmans mc, young a, ganss c. the role of fluoride in erosion therapy. monogr oral sci. 2014; 25: 230-43. 8. azzopardi a, bartlett dw, watson tf, sherriff m. the surface effects of erosion and abrasion on dentine with and without a protective layer. br dent j. 2004; 196: 351-4. 9. bartlett d, sundaram g, moazzez r. trial of protective effect of fissure sealants, in vivo, on the palatal surfaces of anterior teeth, in patients suffering from erosion. j dent. 2011; 39: 26-9. 10. sundaram g, watson t, bartlett d. clinical measurement of palatal tooth wear following coating by a resin sealing system. oper dent. 2007; 32: 539-43. 11. wegehaupt fj, tauböck tt, sener b, attin t. long-term protective effect of surface sealants against erosive wear by intrinsic and extrinsic acids. j dent. 2012; 40: 416-22. 12. francisconi lf, honório hm, rios d, magalhães ac, machado ma, buzalaf ma. effect of erosive ph cycling on different restorative materials and on enamel restored with these materials. oper dent. 2008; 33: 203-8. 13. carvalho fg, brasil vl, silva filho tj, carlo hl, santos rl, lima ba. protective effect of calcium nanophosphate and cpp-acp agents on enamel erosion. braz oral res. 2013; 27:463-70. 14. fúcio sb, carvalho fg, sobrinho lc, sinhoreti ma, puppin-rontani rm. the influence of 30-day-old streptococcus mutans biofilm on the surface of esthetic restorative materials: an in vitro study. j dent. 2008; 36:833-9. 15. honório hm, rios d, francisconi lf, magalhães ac, machado ma, buzalaf ma. effect of prolonged erosive ph cycling on different restorative materials. j oral rehabil. 2008; 35: 947-53. 16. fatima n, abidi sya, qazi f, jat as. effect of different tetra pack juices on microhardness of direct tooth colored-restorative materials. saudi dent j. 2013; 25: 29-32. 17. de fucio sb, de paula ab, de carvalho fg, feitosa vp, ambrosano gm, puppin-rontani rm. biomechanical degradation of the nano-filled resinmodified glass-ionomer surface. am j dent. 2012; 25: 315-20. 18. furuse ay, mondelli j, watts dc. network structures of bis-gma/tegdma resins differ in dc, shrinkage-strain, hardness and optical properties as a function of reducing agent. dent mater. 2011; 27: 497-506. 19. borges bc, barreto as, gomes cl, silva tr, alves-júnior c, pinheiro iv, et al. preheating of resin-based flowable materials in a microwave device: a promising approach to increasing hardness and softening resistance under cariogenic challenge. eur j esthet dent. 2013; 8: 558-68. 20. cornelio rb, wikant a, mjøsund h, kopperud hm, haasum j, gedde uw, et al. the influence of bis-ema vs bis-gma on the degree of conversion and water susceptibility of experimental composite materials. acta odontol scand. 2014; 72: 440-7. 21. anagnostou m, mountouris g, silikas n, kletsas d, eliades g. chemical, mechanical and biological properties of contemporary composite surface sealers. dent mater. 2015; pii: s0109-5641(15)00411-x (epub ahead of print). in vitro effects of erosive challenge on the surface properties of sealants braz j oral sci. 14(4):276-281 revista fop n 13 1596 effect of a denture cleanser on hardness, roughness and tensile bond strength of denture liners antônio de luna malheiros-segundo1; marina xavier pisani2; helena de freitas oliveira paranhos3; raphael freitas de souza4; cláudia helena silva-lovato4 1dds, ms, phd student 2dds, graduate student 3dds, ms, phd, associated professor 4dds, ms, phd, assistent professor department of dental materials and prosthodontics, dental school of ribeirão preto, university of são paulo, brazil received for publication: april 14, 2008 accepted: september 09, 2008 correspondence to: cláudia helena lovato da silva departamento de materiais dentários faculdade de odontologia de ribeirão preto, usp av. do café, s/n, 14040-904, ribeirão preto, sp, brasil tel: +55-16-3602-4006 fax: +55-16-3633-0999. e-mail: chl@forp.usp.br a b s t r a c t aim: this study aim to investigate the effect of a denture cleanser on hardness, roughness and tensile bond strength of a rigid (kooliner) and a soft denture liner (elite soft) after 7, 60 and 120 days of immersion. methods: thirty circular and twenty rectangular specimens of each material were randomly distributed in two groups: control immersion in artificial saliva at 37°c; and experimental immersion in artificial saliva at 37ºc combined with immersion in the cleanser for 5 min. hardness was measured using either a vickers or shore a hardness tester, and a surface roughness tester was used to measure the surface roughness. tensile bond strength was carried out on a universal testing machine. data were analyzed statistically by anova or kruskal-wallis test (α=0.05). results: no significant difference was found between the groups for the tested properties (p>0.05). there was an increase in the hardness of both materials after 60 days (p>0.05). no difference between the immersion periods was found for the roughness of kooliner (p>0.05), although the roughness of elite soft decreased after 120 days in both groups (p>0.05). kooliner presented higher tensile bond strength than elite soft (p>0.05). conclusions: both materials showed alterations on the tested properties during the experimental period, but these changes were not promoted by the denture cleanser. key words: acrylic resins, denture liners, denture cleanser, properties. i n t r o d u c t i o n the search for increased quality for patients who use removable dentures means improving adaptation of the bases after residual ridge resorption. because of their resilience, soft denture liners provide a better distribution of functional loads on the denture foundation area, having a key role in modern removable prosthodontics because of their capacity to restore health to inflamed and injured mucosa1-2. dentures made from two different materials can only be successful if there is an adequate bond between the materials1. however, the most common reason for failure of soft-lined dentures is the basic structural differences between the materials. hardness is one of the most important properties of liners, with a direct impact on ductibility, malleability and resistance to abrasion. surface roughness is also an important clinical property. a rough denture surface can lead to biofilm accumulation and colonization by candida albicans, which the major etiologic factor for denture-induced stomatitis 2. nevertheless, these properties can be affected when the material is submitted to daily immersions in denture cleansers3. when immersed in soaking solutions or placed in the oral cavity, soft denture liners undergo two processes: leaching out of plasticizers and other soluble materials, and sorption of water or salivary components. the fluctuation between these two processes affects the properties of the denture liner material4. the use of denture liners has become popular in the fabrication of complete dentures. therefore, in clinical practice, the choice for an appropriate denture liner, especially long-term materials as well efficient chemical cleanser that does not interfere with the liner’s properties is of paramount importance. the purpose of this study was to evaluate hardness, roughness and tensile bond strength between a heat-processed acrylic resin and two braz j oral sci. july/september 2008 vol. 7 number 26 1597 different types of denture liners (a soft and a rigid one), following daily immersion in a sodium perborate effervescent cleansing solution. material and methods a self-curing reline acrylic resin (kooliner; gc america, inc., alsip, il, usa), and a elastomeric liner, (elite soft; zhermack s.p.a., badia polesine, italy) were selected for this study. the main components of kooliner are poly(ethyl) methacrylate (powder) and isobutyl methacrylate (monomer), whereas elite soft is a twocomponent paste system composed by polyvinyl siloxane. hardness and roughness thirty specimens of each material were obtained for analysis of each property, using a rectangular aluminum matrix containing circular moulds (20x4mm) fixed to a glass plate. the liners were processed according to the manufacturer’s instructions, inserted in the moulds and manually compressed by another glass plate. after setting, the specimens were removed, and excess was trimmed with burs (kooliner) or a sharp penknife (elite soft). next, the specimens were randomly divided into two groups: g1 (control; n=15) immersion in artificial saliva at 37°c; and g2 (experimental; n=15) immersion in artificial saliva at 37°c combined with daily immersion for 5 min in a sodium perborate effervescent cleansing solution (corega tabs; block drug company, inc. jersey city, nj, usa). the artificial saliva was prepared at the school of pharmaceutical sciences of ribeirão preto, university of são paulo, sp, brazil, and had the following composition: potassium diacid phosphate, potassium dibasic phosphate, kcl, nacl, mgcl 2 (6 h 2 o), cacl 2 (2 h 2 o), naf, 70% sorbitol, aromatizer and pigment, preservative (10 ml; nipagin/nipasol), inspissator, water q.s.p (1.0 l). the hardness of elite soft was evaluated using a shore a durometer (instrument and manufacturing co. inc, freeport, ny, usa) and the hardness of kooliner was evaluated using a vickers hardness tester (hmv-2, shimadzu, kyoto, japan) immediately after finishing the specimens (t0) and after 60 days (t60) of immersion in the cleansing solution. three indentations were made in each sample. data were analyzed statistically by two-way anova for each material. the significance level for all comparisons was set at α=0.05. roughness (ra) was evaluated in three areas of each specimen using a surface roughness tester (sj 201-p, mitutoyo, kanagawa, japan) with a 0.8 mm cutoff for an evaluated length of 4.0 mm. roughness readings were made at t0 and after 120 days of immersion (t120) in the cleansing solution. data were analyzed statistically by kruskal-wallis test at 5% significance level. tensile bond strength eighty blocks measuring 40 mm in length, 10 mm in height, and 10 mm thickness were fabricated from an pmma specimens (vipi, dental vipi ltd. ind., pirassununga, sp, brazil). pairs of blocks with a 3-mm-thick layer of liner were then bonded in a sandwich configuration, providing 40 specimens measuring 83 mm in total length and with a cross-sectional area of 10 x 10 mm (figure 1). twenty specimens were prepared for each material (2 materials x 2 times x 2 groups x 5 repetitions = 40) (figure 1). fig. 1. specimen of acrylic resin and elite soft the pmma specimens were prepared by investing polyurethane patterns measuring 40 mm x 10 mm and 3mm-thick brass spacer in a denture flask. the patterns and the spacer were invested in a hard but flexible silicone rubber (zetalabor; zhermack s.p.a., zhermack s.p.a., badia polesine, italy) to allow easy removal of the processed specimens from the flask. after removal of the polyurethane dies, the pmma specimens was mixed, packed into a mold with a brass spacer, and processed as recommended by the manufacturer in an automatic polymerization machine (termocycler t-100, ribeirão preto, são paulo, sp, brazil). after heat polymerization, the brass spacer and the pmma specimens were removed from the mold, trimmed, and the surfaces to be bonded with liners were prepared and treated according to the manufacturer’s instructions for each denture liner. for elite soft, one coat of bonding agent was applied to the pmma specimens. the interface of the acrylic resin specimens that was joined to kooliner was prepared with burs (tungsten point) for acrylic resin finishing. the pmma specimens were then returned to the molds, the denture liners were packed into the space created by the brass spacer and auto-cured, according to the manufacturers’ directions. after curing, the specimens were removed from the flask and the denture liner was finished. the 20 specimens of each material were randomly divided into two groups according to the immersion protocol (n=10; g1control and g2 experimental). for the tensile bond strength test, 5 specimens of each group were loaded until failure in a universal testing machine (mem 2000, emic, são josé dos pinhais, pr, brazil) at a crosshead speed of 5 mm/min after 7 days (t7) and other 5 specimens after 60 (t60) days of immersion. bond strength (mpa) was calculated as stress at failure divided by the crosssectional area of the specimen. the results were tested by anova test (material, time and groups). all data were analyzed at a 0.05 level of significance. r e s u l t s hardness and roughness braz j oral sci. 7(26):1596-1601 effect of a denture cleanser on hardness, roughness and tensile bond strength of denture liners 1598 the analysis of variance of the data relative to the material’s hardness indicated a statistically significant difference between the immersion periods (table 1). there was no statistically significant difference between the groups. hardness means and standard deviations of the tested materials in both evaluation periods are present in figure 2. the kruskal-wallis test did not show significant difference (p>0.05) between immediate and 120-day immersion periods or with the use of the sodium perborate on kooliner roughness. the roughness of elite soft reliner decreased significantly during the 120 days in both groups (p<0.05). the use of the sodium perborate had no influence on the roughness of this material. comparing both materials, elite soft presented a significantly lower roughness means (p<0.05) than kooliner. roughness (mm) means and standard deviations of the tested materials in both evaluation periods are present in table 2. tensile bond strength the results of anova indicate significant differences kooliner df sum of squares mean square f p. val ue groups (g) error i immersion period (ip) g x ip error ii 1 28 2 2 56 9.2095 169.0958 147.9866 0.8889 49.0389 9.20 6.03 73.99 0.44 0.87 1.52 84.50 0.51 0.22 0.00 0.39 elite soft df sum of squares mean square f p. value groups (g) error i immersion period (ip) g x ip error ii 1 28 1 1 28 0.63 14.65 272.95 0.46 29.26 0.63 0.52 272.95 0.46 1.04 1.22 261.16 0.88 0.278 0.00 0.357 table 1 two-way anova for hardness of the kooliner and elite soft specimens. table 2 roughness means and standard deviations (sd) of the tested materials. different small letters mean statistical difference between times in each evaluation period different capital letters mean statistical difference between materials fig. 2 hardness means and standard deviations of the tested materials. between the tensile bond strength (p<0 .05) of the materials. kooliner presented significantly higher bond strength means than elite soft in both groups. regarding the immersion period, the mean bond strength of kooliner ranged from 2.97±0.47 (t7) to 2.62±1.51 (t60) mpa in braz j oral sci. 7(26):1596-1601 effect of a denture cleanser on hardness, roughness and tensile bond strength of denture liners 1599 gi, and from 2.67±0.42 (t7) to 3.45±1.10 (t60) mpa in gii. elite soft presented an increase in the mean bond strength throughout the 60 days in gi (t7=0.32±0.20, t60=1.19±0.29) and in gii (t7=0.43±0.32, t60=1.16±0.30). however, no statistical significance was any of the materials. there was no significant difference (p>0.05) between the groups, which indicates that the denture cleanser solution did not affect the materials’ bond strength. d i s c u s s i o n one of the most serious problems found in the use of denture liners is the adhesion failure between the liner and the denture base4. some factors are expected to affect the bond between lining materials and denture bases, including aging in water, use of a primer with the lining material and the nature of the material base4. hardness is one of the most challenging factors in the use of complete denture liners, since the most of them are not stable in a moist environment such as the mouth. the literature does not establish an ideal hardness value for resilient liners. craig and gibbons (1961)5 suggested that the greater the softness, the greater the extension in absorbing the impact effect. thus, less hardness is a desirable characteristic for soft liners. according to the literature, the denture cleansing method is one of the factors that can modify the superficial characteristics of the liners6. the prosthesis should be immersed cleansing solutions in order to remove the biofilm from the liner’s surface, which might increase the possibility of increasing the instability of the resilient materials. two structurally different materials were selected for comparison of the tensile bond strength: kooliner (hard acrylic or polymethyl methacrylate resin) and elite soft relining (a silicone-based material). there are different methods for obtaining adhesion with acrylic resins. kooliner has chemical affinity and elite soft relining requires the use of an adhesive to obtain this union. as acrylic resin-based materials present similar components when compared to the conventional acrylic resin, they form a molecular network that simultaneously penetrates in both union surfaces of similar compositions7. these findings can explain the results found in this work. kooliner presented the highest tensile bond strength means. this material does not have an adhesive in its kit, but a surface sealant is supplied. sealant’s application produces a pack that makes water absorption difficult and reduces the deterioration of the base, thus extending the material’s useful life. elite soft relining is supplied with the adhesive and the sealant. the role of the adhesive is to increase the bond strength between the silicone-based soft liner and the acrylic resin. these results are similar to those of aydin et al.8. in the present study, the significant difference found between the materials was apparently due to their individual characteristics, such as the capacity to humidify the surface of the thermopolymerized acrylic resin, properties and chemical composition7. the literature recommends that the tensile bond strength should not be less than 0.45 mpa in order to these materials be clinically used9. considering this criterion, kooliner seems to be the most indicated material. although elite soft relining presented a lower mean initial bond strength than this value in both groups, at the end of 60 days the mean bond strength was higher than 0.45 mpa. regarding the immersion solutions, artificial saliva at 37ºc and artificial saliva with daily 5-min immersions in a denture cleanser, neither of the materials presented statistically significant difference when compared to immersion in the artificial saliva. similar results were found by rodrigues garcia et al.10, who stated that the bond strength increased throughout the 60 days in both evaluated groups. such increase in the bond strength over time was also found by craig and gibbons5 and aydin et al.8, and was attributed to the leaching out of the plasticizer, resulting in an increase of stiffness. the favorable results found in this study about the use of the denture cleanser solution can be attributed to the fact that the solution does not contain any chemical component that affects the dissolution of the tested materials. moreover, the thermopolymerized acrylic resin used contains “cross-linking” agents, whose function is to increase the resistance of solvents and surface stresses. however, other authors2,4 have observed a weakness of the adhesion at the acrylic resin/liner interface in the presence of water. on the other hand, the authors found that diffusion of the water in the area of union between the acrylic resin and the liner did not have a deleterious effect in the adhesion capacity between the materials. since different materials were evaluated, an acrylic resinbased (kooliner hard) and a silicon-based (elite soft) material, we could not compare their hardness, but it was possible to compare their behavior regarding the use of the denture cleansing product (control and experimental) and the period of immersion (60 days). for both materials, it was observed that immersion in sodium perborate did not influence hardness significantly. these results are in agreement with those of davenport et al.11 and haywood et al.12. materials that present cross-linking agents in their composition demonstrate a greater hardness stability when stored in water. unlike elite soft, kooliner does not present this component. however, both materials presented the same behavior with the cleansing solution. contradictory results were found by pavan et al.13. these authors observed an increase in the hardness of resilient materials immersed in cleaning products. tan et al.14 and botega et al.15, on the other hand, demonstrated a decrease in the hardness of the tested silicon based liners after the treatment with different denture hygiene solutions. according to davenport et al.11 , silicon-based materials do not possess plasticizers, but rather contain load in their composition, braz j oral sci. 7(26):1596-1601 effect of a denture cleanser on hardness, roughness and tensile bond strength of denture liners 1600 and the water absorption caused by the presence of this component reduces the hardness. regarding the immersion time, a significant increase in the hardness could be noticed throughout the 60-day period in both groups for both materials. polyzois and frangou16 verified an increase in hardness in all tested materials during the first month of immersion; after that period, the materials presented a stabilization of hardness values. hardness is a important property for resilient material and should remain constant for a long period so that the materials can efficiently fulfill their function. however, the findings in literature and the results of the present study show that time is a limiting factor. studies must be conducted with the intention to increase hardness stability of resilient materials in such a way that its clinical indication is carried through with certainty. although kooliner is a hard liner, its hardness is well below that of acrylic resin, therefore offering a greater capacity to absorb impact. the ideal hardness or softness for providing a greater comfort to the patient can be obtained with the use of soft materials, but they still have many properties along with unstable use. surface roughness was determined based on the value of ra, which represents the average of peaks and depressions on the surface, enabling us to evaluate the possibility of bacteria colonizing the area. this parameter was selected for being the most used in literature, allowing comparisons with the results of this study. previous studies have already mentioned alterations on the surface of liners when immersed in effervescent alkaline solutions. however, nikawa et al.17 and jin et al.18 affirmed that not only the active component, but also other components of the cleansing chemical agent as well as the ph can cause damage to the material. according to jagger and harrison19, the effervescent hygiene agents have a chemical and a mechanical cleaning action, resulting in the production of bubbles created by oxygen release during the reaction, which could increase the material’s surface roughness. the materials evaluated in this study did not undergo a significant alteration in the surface roughness caused by immersion in sodium perborate throughout the 120 days of evaluation when compared to the immersion in artificial saliva for the same time. these results are in agreement with those found by tan et al.14. the immersion time did not modify the surface roughness of kooliner, but promoted a significant reduction in the surface roughness means of elite soft over the 120 days. regarding the roughness of soft liners, rodrigues garcia et al.10 stated that apparently when these materials were immersed in cleansing products, a loss of soluble components occurred leaving empty voids or bubbles, which are responsible for surface roughness. these voids or bubbles underwent an increase in size that resulted in craters. the limits of the craters are probably smaller when compared to the bubbles, leaving the specimens smoother. comparing the materials, kooliner presented significantly higher means than elite soft. pavan et al.13 evaluated siliconand acrylic resin-based liners and observed that the silicon presented surfaces that were smoother than the acrylic resin ones. these results were contrary to those of zissis et al.20. the smoothness of the specimens produced by the glass plate used in the methodology of this study does not correspond to the clinical reality, as the glass provides a more polished surface compared to the denture processed using plaster models and the finishing that is provided by bur, sandpapers or polishing products. the roughness difference of the silicon compared to the acrylic resin-based materials is probably related to their consistencies. kooliner presents a more fluid consistency than elite soft and, although its application is easier, until the moment of flasking with the glass plate, the material is already more consistent, raising the hypothesis that kooliner is less capable of reproducing the details of the extremely smooth glass surface on which the specimens were processed, thus providing higher roughness values. clinical factors as the oral environment and the conformation of the denture base were not considered. the results of in vitro investigations should be applied to the clinical conditions with caution. the final evaluation of the material’s performance should be determined by means of clinical tests in vivo, in addition to performing the physical tests. in conclusion, the use of sodium perborate did not modify the hardness, roughness and the tensile bond strength of the evaluated materials. kooliner showed a greater increase in the tensile bond strength throughout the 60 days of immersion when compared to elite soft. kooliner and elite soft presented a significant increase in the hardness values at the end of the 60 days in both groups. only elite soft presented a reduction in the roughness values after 120 days of immersion. both materials showed alterations on the properties tested during the experiment, but these changes were not promoted by the denture cleanser. a c k n o w l e d g e m e n t s this investigation was supported by the são paulo state research foundation (process # 2004/09878-0). r e f e r e n c e s 1. wright ps. characterization of the adhesion of soft lining materials to poli (methyl methacrylate). j dent res. 1982; 61: 1002-5. 2. wright ps. the success and failure of denture soft-lining materials in clinical use. j dent. 1984; 12: 319-27. 3. kazanji mnm, watkinson acm. soft lining materials: their absorption of, and solubility in artificial saliva. br dent j. 1988; 165: 91-4. 4. sinobad d, murphy wm, hugget r, brooks s. bond strength and rupture properties of some soft denture liners. j oral rehabil. 1992; 19: 151-60. 5. craig rg, gibbons p. properties of resilient denture liners. j am dent assoc. 1961; 63: 382-90. braz j oral sci. 7(26):1596-1601 effect of a denture cleanser on hardness, roughness and tensile bond strength of denture liners 1601 6. budtz-jørgensen e. the significance of candida albicans in denture stomatits. scand j dent res. 1974; 82: 151-90. 7. amin wm, fletcher am, ritchie gm. the nature of the interface between polymethylmethacrylate denture base materials and soft lining materials. j dent. 1981; 9: 336-46. 8. aydin ak, terzioðlu h, akinay ae, ulubayram k, hasirci n. bond strength and failure analysis of lining materials to denture resin. dent mater. 1999; 15: 211-8. 9. kawano f, dootz er, koran a 3rd, craig rg. comparison of bond strength of six soft denture liners to denture base resin. j prosthet dent. 1992; 68: 368-71. 10. garcia rm, léon bt, oliveira vb, del bel cury aa. effect of a denture cleanser on weight, surface roughness, and tensile bond strength of two resilient denture liners. j prosthet dent. 2003; 89: 489-94. 11. davenport jc, wilson hj, spence d. the compatibility of soft lining materials and denture cleansers. br dent j. 1986; 161: 13-7. 12. haywood vb. color measurement symposium 2003. j esthet restor dent. 2003; 15: s3-4. 13. pavan s, arioli filho jn, dos santos ph, nogueira ss, batista au. effect of disinfection treatments on the hardness of soft denture liner materials. 14. j prosthodont. 2007; 16: 101-6. 15. tan h, woo a, kim s, lamoureux m, grace m. effect of denture cleansers, surface finishing, and temperature on molloplast b resilient liner color, hardness, and texture. j prosthodont. 2000; 9: 148-55. 16. botega dm, carmo filho jl, mesquita mf, nóbilo maa, henriques gep. influence of toothbrushing on surface roughness of soft dednture liners: na in vitro study. rev pós grad. 2004; 11: 125-9. 17. polyzois gl, frangou mj. influence of curing method, sealer, and water storage on the hardness of a soft lining material over time. j prosthodont. 2001; 10: 42-5. 18. nikawa h, jin c, makihira s, egusa h, hamada t, kumagai h. biofilm formation of candida albicans on the surfaces of deteriorated soft denture lining materials caused by denture cleansers in vitro. j oral rehabil. 2003; 30: 243-50. 19. jin c, nikawa h, makihira s, hamada t, furukawa m, murata h. changes in surface roughness and color stability of soft denture lining materials caused by denture cleansers. j oral rehabil. 2003; 30: 125-30. 20. jagger dc, harrison a. denture cleansing—the best approach. br dent j. 1995; 178: 413-7. 21. zissis aj, polyzois gl, yannikakis sa, harrison a. roughness of denture materials: a comparative study. int j prosthodont. 2000; 13: 136-40. braz j oral sci. 7(26):1596-1601 effect of a denture cleanser on hardness, roughness and tensile bond strength of denture liners oral sciences n3 received for publication: december 07, 2009 accepted: july 06, 2010 original article braz j oral sci. july/september 2010 volume 9, number 3 relation between oral stereognosis and masticatory efficiency in complete denture wearers: an in vivo study 1 postgraduate student, department of prosthodontics, a.b. shetty memoral institute of dental sciences (absmids) , rajiv gandhi university of health sciences, deralakatte, mangalore, karnataka, india 2 bds, mds, professor, department of prosthodontics, a.b. shetty memoral institute of dental sciences (absmids) , rajiv gandhi university of health sciences, deralakatte, mangalore, karnataka, india 3 bds, mds, head of the department of prosthodontics, a.b. shetty memoral institute of dental sciences (absmids), rajiv gandhi university of health sciences, deralakatte, mangalore, karnataka, india atul bhandari1, chethan hegde2, krishna prasad d3 correspondence to: atul bhandari department of prosthodontics a.b. shetty memoral institute of dental sciences (absmids) deralakatte – mangalore – karnataka (575018) india e-mail: dr.atulbhandari@gmail.com abstract aim: the purpose of the present study was to evaluate the possible association between the oral stereognostic ability and masticatory efficiency at the time of denture insertion and after 6 months in complete denture wearers. methods: sixty edentulous patients were selected. the study was conducted in two parts. first, on the day of denture fit-in oral stereognostic ability test was conducted, followed by masticatory efficiency was carried out. the patients were recalled for check up after 6 months and the same test was performed again. data were tabulated and analyzed statistically by paired t-test and pearson’s correlation coefficient. results: there was weak correlation between oral stereognosis and masticatory efficiency. many opinions have been presented in the literature in the past regarding oral stereognosis. the issue whether oral stereognosis also helps in patients’ adaptation towards complete denture prosthesis is still ambiguous. no consensus has been reached. this study is further an attempt to understand the basic physiology of oral stereognosis and whether there is any possible relation between oral stereognosis and masticatory efficiency. conclusions: within the limitation of this study, it can be concluded that oral stereognostic ability improves with time, which might be due to adaptation to the denture. as adaptation towards denture improves masticatory efficiency improves as well. this study showed that there might be a weak association between oral stereognosis and masticatory efficiency. keywords: oral stereognosis, oral perception, oral sensorimotor function, masticatory efficiency, masticatory performance. introduction the efficiency and success of complete dentures are difficult to measure objectively. it is important to be able to do so because patients sometimes complain with no clinical evidence. most patients expect dentures to be comparable, both functionally and esthetically, to natural dentition1. several investigators have reported that the patient’s adaptation to complete dentures may be predicted by oral stereognostic tests. stereognosis has been employed to evaluate the integrity of sensory feedback and is used in neurological braz j oral sci. 9(3):358-361 359 evaluation. it involves identification of forms of objects without the aid of vision by hand or oral manipulation. the oral stereognosis test consists of placing objects into the mouth without being seen by the patient and having the patient identify the form. a correct identification of the form of the object is recorded as a score2-3. the chief purpose of fitting oral prostheses is to enable the patients to recover their oral function. therefore, to diagnose the effectiveness of the prosthesis, it is important to evaluate the oral function4. masticatory performance and efficiency are defined as the capacity to reduce food during mastication, and by counting the number of masticatory strokes required to reduce food to a certain particle size reduction, respectively4. the purpose of this study was to evaluate any possible association between the oral stereognostic ability and masticatory efficiency at the time of denture insertion and after 6 months in complete denture wearers. material and methods the present in-vivo study was conducted on sixty edentulous subjects reported to the department of prosthodontics a.b.s.m.i.d.s mangalore. the inclusion criteria were: completely edentulous patients with age ranging from 50 to 60 years, and with no past denture history. the exclusion criteria were: patients with mucosal lesions, under the influence of neurological drugs, under intoxications, and having any psychological problems or perverted habits. the various forms used in this study were: square shape form, rectangle shape form, triangle shape form, star-shaped form, circle shape form, semicircle shape form. the test forms were fabricated from heat cured acrylic resin by conventional technique. the test forms used were 2-3mm in length. six forms were used to prevent the fatigue. for identification purpose, similar but 5-6 times oversized test forms were fabricated with plaster of paris. for evaluating masticatory efficiency, wrigley’s orbit sugar free pellet form chewing gum was used. measurement of the weight loss was carried out by electronic weigh balance (axpert enterprises iso 9001) having max weigh limit of 2 kg and minimum 0.2 g standard error of 0.01 g. sixty edentulous subjects were selected. the aim of the study and the procedures were fully explained to the subjects and due written consent was obtained. this study was conducted in two parts. first, on the day of denture fit-in oral stereognostic ability test was conducted, followed by masticatory efficiency was carried out. oral stereognostic ability test on day 1, oral stereognosis test was carried out after denture fit and insertion. day 1 means the day of final delivery of the prosthesis as this is the first day the patient will experience the new prosthesis. thus on day 1 always slight amount of discomfort patient will experience. to correct this discomfort and to make them adjusted to this test day n mean standard deviation day 1 60 7.133 ±1.39 after 6 months 30 8.9000 ±1.295 table 1. mean and standard deviation for oral stereognostic ability of subjects on the day of denture fit and insertion and after 6 months of denture usage new prosthesis, the term fit and insertion was used. initially, patients were made familiarized with plaster forms with digital manipulation and then acrylic test forms were placed in the mouth and movements like sucking, pressing against the palate and lips were advised to help them in identification. after identification, subjects were advised to point towards one of the plaster forms. sterilization was carried out by autoclaving these test forms. the readings hence recorded were grouped in three categories (2 point if the answer was correct, 1 point if the answer was partly correct, 0 point if the answer was wrong). patients’ oral stereognostic scores were collected in the following way at denture insertion and after 6 months of denture usage2. measuring masticatory efficiency of the patient masticatory efficiency of the subject was carried out with the help of chewing gums, which were pre-weighed by using electronic (axpert) weighing balance. for each subject four orbit chewing gums were used for 5, 10, 20 and 30 strokes. the chewing gums were then desiccated and the total weight loss of the sweetener chewed out was recorded. the same procedure was followed after a period of 6 months. thus the weight loss of the chewing gum was recorded at the time of insertion and 6 months after denture usage4. data were tabulated and analyzed statistically by paired t-test and pearson’s correlation coefficient. results from the on-going study the results obtained shows that: the mean value for oral stereognostic ability testing for 60 edentulous subjects was 7.133±1.39, and for 30 subjects after 6 months of denture usage it was 8.9000±1.295. of the 60 subjects only 30 subjects returned after 6 months of denture usage, and so the study was conducted with 30 patients only (table 1). comparative analysis of these subjects was conducted by paired sample t-test. the results suggested that the mean value for oral stereognostic ability on the day of denture fit and insertion was 6.6667±1.51, and the mean value after 6 month of denture usage was 8.9000±1.295. the mean difference of oral stereognosis between day 1 and after 6 months of denture usage was 2.233, which is highly significant (p=0.001) (table 2). masticatory efficiency of the thirty denture subjects who returned after 6 months of denture usage. the mean value for weight loss of the sugar content on the day of denture fit and insertion from the chewing gum was 4.806±0.766 and after 6 months of denture usage was 4.623±0.669.there was a decrease in value after 6 months of denture usage with a mean difference of 0.183 (p=.003), which shows high significance (table 3 relation between oral stereognosis and masticatory efficiency in complete denture wearers: an in vivo study braz j oral sci. 9(3):358-361 360 pair 1 n mean mean difference standard deviation significance day 1 30 6.6667 ±1.516 2.233 0.001 after 6 months 30 .9000 ±1.295 table 2. a comparative analysis of oral stereognostic ability test on the day of denture fit and insertion and after 6 months of denture usage by using paired sample t-test n mean standard deviation day 1 60 4.78 ±0.651 after 6 months 30 .623 ±0.669 table 3. mean and standard deviation for masticatory efficiency of subjects on the day of denture fit and insertion and 6 months of denture usage pair 1 n mean mean difference standard deviation significance day 1 30 4.806 -0.183 ±0.766 0.003 after 6 months 30 4.623 ±0.669 table 4. a comparative analysis of masticatory efficiency on the day of denture fit and insertion and after 6 months of denture usage by using paired sample t-test and 4). the pearson’s correlation coefficient obtained (-0.066) suggests weak correlation that exists between oral stereognosis and masticatory efficiency. discussion oral stereognosis is the neurosensorial ability of the oral mucosa to recognize and discriminate the forms of objects in the oral cavity3,5-7. oral stereognostic ability has been employed in many studies to evaluate oral perception. altered masticatory efficiency with age again is a multifactorial problem which may or may not be associated with loss of teeth8-10. the relationship between oral stereognosis and masticatory efficiency has not been clearly identified. this study is further an attempt to understand any possible relation that exists between these two conditions. in the present study, 6 forms were used. the study was conducted with dentures on the day of denture fit-in and 6 months after denture usage11-12. to measure masticatory efficiency, the technique as proposed by heath et al. has been followed, which uses chewing gum as a test material. the possible advantages of using chewing gum over other materials are that kneading and folding are easily done with chewing gum and no fracture of particles occurs on chewing, which causes no discomfort for the patient4. the mean oral stereognostic ability scores obtained on the day 1 of denture fit and insertion was 7.133±1.39 (table 1). this when compared with the maximum value the subject could have scored [12 points (2 points each for 6 forms)] suggested that the mean oral stereognosis score was 58.3% for 60 edentulous subjects. this decreased oral stereognostic scores may be explained due to loss of natural teeth as proprioception from the periodontal ligament was lacking, or increased age, or patient’s satisfaction13. the 30 subjects out of 60, who returned after 6 months of denture usage showed a mean oral stereognostic ability score of 8.9000±1.295. the mean oral stereognostic ability score of these 30 subjects on the day of denture fit and insertion was 6.666±1.516. by comparing the mean of oral stereognostic ability for day 1 and 6 months after denture usage a highly significant improvement has been noticed (p=0.001) (table 2) (figure 1). the above results demonstrate that as the time elapsed there was an highly significant improvement in oral stereognostic ability. this improvement may be due to improved adaptability with the dentures with due course of time7,14-16. regarding the neurological mechanism involved in recognizing various forms, the oral phenomenon known as stereognosis involves elaborate functions of the parietal cortex. received sensations are synthesized in the cortex and compared with previous sensorial memories. part of the somatosensorial cortex (2) is composed of broadmann areas (3a, 3b. 1. 2). sensorial input of muscle and articulations are fig. 1. comparison between oral stereognostic ability on day 1 and after 6 months of denture usage osa day-1 (oral stereognostic ability on day 1) **osa -2after 6 months( oral stereognostic ability after 6 months) relation between oral stereognosis and masticatory efficiency in complete denture wearers: an in vivo study braz j oral sci. 9(3):358-361 conducted to 3a, whereas skin sensorial input is conducted to 3b and processed in area 1, combined with other information in area 2. the s-i area projects the signal to other locations of the parietal lobe, where the somatosensorial impulses are used for the learning of new discriminatory sensations. the sensorial area for the tongue, lips, thumbs and index lingers is greater than other sensorial parts for the rest of the body. testing one’s oral stereognostic level can involve some motor activity and manipulation of test pieces inserted into the oral cavity and their interactions with lips, tongue, and teeth. oral stereognostic testing can also measure recognition times, surface texture of objects, and sensibility thresholds17. the mean score for masticatory efficiency after 6 months of denture usage was 4.62±0.66. when this value is compared to the initial score of masticatory efficiency on the day 1 of denture fit and insertion (4.80±0.766), a significant improvement in masticatory efficiency is noticed (p=0.003) (table 3 and 4) (figure 2). the results obtained were in accordance with muller et al.14, who stated that as the adaptation towards the prosthesis improves, masticatory efficiency also improves with time. those authors suggested that good oral motor ability supports the adaptation to new dentures7,14-16. when the results were analyzed altogether, it could be noted that over a period of 6 months there was an improvement in masticatory efficiency with improved oral stereognostic ability. however, pearson’s correlation coefficient denotes that there was a weak association exists between oral stereognosis and masticatory efficiency (-0.066). a possible explanation for this co-relation can be based on multiple factors (age, neurosensory ability, patients’ psychological status, salivary flow rate) which exists and are subjective in nature12,18-19. the results obtained in this study are suggestive but not conclusive because of the relatively small sample size, the infrequent follow-ups, and the lack of standardization of the sweetener lost to determine good, intermediate or poor masticatory efficiency. the initial dissolution of the sweetener in the saliva was also a limitation of the study. following conclusions were drawn from this study: by comparing the mean of oral stereognostic ability for day 1 and 6 months after denture usage a highly significant improvement has been noticed. by comparing the mean of masticatory efficiency for day 1 and 6 months after denture usage, a highly significant improvement has been noticed. there was a weak association between oral stereognosis and masticatory efficiency, but further studies are still required to evaluate this association. references 1. ahmad sf. an insight into the masticatory performance of complete denture wearer. ann dent. 2006; 13: 24-33. 2. al-rifaiy mq, sherfudhin h, aleem ma. oral stereognosis in predicting denture success. saudi dent j. 1996; 8: 126-30. 3. smith pw, mccord jf. oral stereognostic ability in edentulous and dentate individuals. eur j prosthodont restor dent. 2002; 10: 53-6. 4. anastassiadou v, heath mr. the development of a simple objective test of mastication suitable for older people, using chewing gums. gerodontology. 2001; 18: 79-86. 5. litvak h, silverman si, garfinkel l. oral stereognosis in dentulous and edentulous subjects. j prosthet dent. 1971; 25: 139-50. 6. mantecchini g, bassi f, pera p, preti g. oral stereognosis in edentulous subjects rehabilitated with complete removable dentures. j oral rehabil. 1998; 25: 185-9. 7. zarb ga. oral motor patterns and their relation to oral prostheses. j prosthet. dent. 1982; 47: 472-8. 8. gambareli fr, serra md, pereira lj, gavião mb. influence of measurement technique, test food, teeth and muscle force interactions in masticatory performance. j texture stud. 2007; 38: 2-20. 9. ikebe k, amemiya m, morii k, matsuda k, furuya-yoshinaka m, yoshinaka m et al. association between oral stereognostic ability and masticatory performance in aged complete denture wearers. int j prosthodont. 2007; 20: 245-50. 10. demers m, bourdages j, brodeur jm, benigeri m. indicators of masticatory performance among elederly complete denture wearer. j prosthet dent. 1996; 75: 188-93. 11. grasso je, catalanatto fa. the effects of age and full palatal coverage on oral stereognostic ability. j prosthet dent. 1979; 41: 215-9. 12. langer a, michman j. occlusal perception after placement of complete dentures. j prosthet dent; 1968; 19: 246-51. 13. jacobs r, van steenberghe d. role of periodontal ligament receptors in the tactile function of teeth: a review. j periodontal res. 1994; 29: 153-8. 14. muller f, link i, fuhr k, utzkh studies on adaptation to complete dentures, oral stereognosis and tactile sensibility. j oral rehabil. 1995; 22: 759-67. 15. engelen l, van der bilt a, bosman f. relationship between oral sensitivity and masticatory performance. j dent res. 2004; 83: 388-92. 16. hirano k, hirano s, hayakawa i. the role of oral sensorimotor function in masticatory ability. j oral rehabil. 2004; 31: 199-205. 17. rossetti pho, bonachela wc, nunes lmo. oral stereognosis related to the use of complete dentures: a literature review. int j oral med sci. 2004; 2: 57-60. 18. van aken aa, van waas ma, kalk w, van rossum gm. differences in oral stereognosis between complete denture wearers. int j prosthodont. 1991; 4: 75-9. 19. grasso je, catalanatto fa. the effects of age and full palatal coverage on oral stereognostic ability. j prosthet dent. 1979; 41: 215-9. 361 fig. 2. comparison between weight loss on the day 1 of denture insertion and after 6 months of denture usage. chew_ini: initial weight loss of chewing gum on the day 1 of denture fit and insertion chew_fin: final weight loss of chewing gum after 6 months of denture usage relation between oral stereognosis and masticatory efficiency in complete denture wearers: an in vivo study braz j oral sci. 9(3):358-361 original article braz j oral sci. january/march 2009 volume 8, number 1 study on the training of brazilian dentists and physicians who treat patients with chronic pain maria da graça rodrigues bérzin1, josé tadeu tesseroli de siqueira2 1 phd, department of morphology, faculdade de odontologia de piracicaba, universidade estadual de campinas (unicamp), piracicaba (sp), brazil 2 phd, orofacial pain team, dentistry and neurology divisions, hospital das clínicas da faculdade de medicina, universidade de são paulo (usp), são paulo (sp), brazil received for publication: september 22, 2008 accepted: april 7, 2009 correspondence to: maria da graça rodrigues bérzin rua dom pedro i, 818 – apto. 91 cep 13400-410 – piracicaba (sp), brazil e-mail: graberzin@yahoo.com.br abstract aim: to describe the characteristics of the professional training of dentists and physicians who treat patients with pain. methods: a sample of 87 dentists and 63 physicians, selected at random and based on a stratified strategy, responded to a questionnaire with questions about training in pain. the statistical analysis of the data was done by the chi-square test and the student’s t-test at a 5% significance level. results: the mean age was 45 years. there was no gender prevalence among the dentists and a male predominance among physicians; 80.20% had worked professionally for more than ten years; 81.61% of the dentists and 79.37% of the physicians were specialists. residence training was reported by 55.56% of the physicians and 12% of the dentists; 48.27% of the dentists and 34.92% of the physicians had a master’s and/or doctorate degree; 69.33% declared sufficient knowledge in pain; both physicians and dentists prioritized their own areas regarding the study of pain. the doctor/patient relationship was more valued by physicians, while the technical training was more valued by dentists. dentists reported more difficulties in prescribing medications and physicians had more difficulties with the patient’s behavior. conclusions: dentists and physicians had different professional experience and had valued specific aspects of their specialty. there is a need for a uniform curriculum designed for training in pain. keywords: pain, orofacial pain, chronic pain, health education, professional-patient relations. introduction there is currently a progressive increase in the prevalence of chronic pain. it is considered an important problem in public health and produces a substantial negative impact on people’s lives1,2. among the types of pain prevalent in the general population, chronic orofacial pain stands out3. although it involves a small segment of the human body, chronic orofacial pain has multiple origins and its diagnosis is a real challenge for healthcare professionals4-6. the practice of differential nosological diagnosis of headaches and craniofacial pain frequently requires efforts on the part of physicians and dentists7-8. despite this and the extensive scientific literature on the subject, there still are some aspects that need more studies, as in the case of the training of the healthcare professional who treat patients with chronic pain4,9-10. in this aspect, the scientific literature is lacking with respect to the training of dentists and physicians who practice in this complex area. when the quality of care rendered to patients with chronic orofacial pain is examined, signs of theoretical-technical deficiencies are noted, which question the professional training in dealing with pain, among other aspects2,11-14. from an academic point of view, literature emphasizes the curriculum deficiencies in medical and dental training, which do not address adequately the subject in all its breadth and complexity. the same can be said of the technical training of future professionals in the diagnostics and therapeutics of many clinical pictures that comprise orofacial pain, which 45study on the training of brazilian dentists and physicians who treat patients with chronic pain braz j oral sci. 8(1): 44-9 should not dispense the humanistic view that helps understanding patient suffering15-16, even though there is a concern of international associations with respect to the need for an appropriate curriculum aimed at professional training in pain, including the distinction between acute and chronic pain4,11,17. the signs of deficiency in academic and professional training are evidenced by the fragmented view of pain, with excessive focus on medical and dental specialization, difficulties in the practice of differential diagnosis, in the perception of chronic pain as a complex phenomenon and the lack of information with respect to methods and appropriate care for the patient with pain that considers important concepts such as prevention and interdisciplinary management17. moreover, the lack of information by the clinicians with respect to the differences between acute and chronic pain, methods of assessing pain, and insistence on the utilization of empirical and inadequate medications, also contributes to the difficulties in the diagnosis and treatment of orofacial pain12-13, in addition to the difficulties and barriers in the relationship between professionals and patients with pain18. it is known that the number of dentists and physicians has grown significantly in the brazilian work force, mainly in the large urban centers of the south and southeast regions19-20. a study published in 2004 by the brazilian federal council of medicine points out that, among the 12 universities that train most of the doctors in the country, six are in the southeast region21. specialization among physicians has also increased over the years. a survey conducted in 1996 by the federal council of medicine pointed out that 40.7% of physicians were specialists. in 2004, this number increased to 66.5% of the medical specialists8. the same tendency in the increased pursuit of master’s and doctorate degrees was observed among dentists and physicians. in 1996, 11% of dentists were registered with official master’s or doctorate degrees. the number increased, in 2002, to 20.8% but in 2003, a survey made by the brazilian institute for socioeconomic research revealed only 14.4% of dentists with such titles8. the task of treating patients with pain, especially chronic, with a humanistic focus, presumes a special condition by the clinicians. they need to work with all the personal concerns of their patients, by establishing a true process of interpersonal relationship that values empathy. thus, it is possible to establish verbal and non-verbal communication with the patients and their family members and to offer availability and time to dedicate themselves to this relationship. such conditions stimulate the formation of an important affective link between these two, which contribute positively to the clinical work22. however, medical and dental education shows a tendency of techniques and has not prioritized questions relative to the relationship between doctor and patient23-24. despite the difficulties observed in the work routine of physicians and dentists who treat persons with pain, there is a lack of studies on this population of professionals. given the progressive increase in the prevalence of chronic pain in brazil and its adverse impact on people’s lives, the deficiencies in academic training of this kind of professionals, the difficulties in their clinical practice and the lack of studies on this population of professionals, the aim of this study was to describe the characteristics of the theoretical-technical training and clinical practice of brazilian dentists and physicians who treat patients with chronic pain. it is expected that the findings of this study may contribute to a better understanding of the difficulties inherent in the clinical practice of professionals of different specialties who treat patients with pain. material and methods the study followed the ethics principles recommended in resolution 196/96 of the brazilian national health council and was approved by the research ethics committee of faculdade de odontologia de piracicaba da universidade estadual de campinas (fop/unicamp). a total of 150 healthcare professionals (87 dentists and 63 physicians) who treat patients with orofacial pain were investigated. the following three categories of specialists were included. 1) dentists and physicians who are active members of at least one of the following associations involved in the study of pain: sociedade brasileira para o estudo da dor – sbed (brazilian chapter of international association for study of pain – iasp); sociedade brasileira de dor orofacial (sobrad); sociedade brasileira de cefaleia (sbce) and academia brasileira de fisiopatologia crânio-oro-cervical (abfcoc); 2) dentists with the title of specialist in temporomandibular dysfunction and orofacial pain granted by the brazilian federal council of dentistry; 3) physicians with clinical experience in the area of orofacial pain. those with no current clinical practice in orofacial pain, despite being a member of the above-mentioned professional associations were excluded as participants. the volunteers were investigated by means of a self-administered, research questionnaire composed by 46 questions about sociodemographic data of dentists and physicians related to the various aspects of the theoretical-technical training in the area of pain, which had been previously validated in a pilot-study of ten volunteers with a profile compatible to the sample to be studied. the questionnaires were sent by mail or delivered personally to the volunteers in an envelope identified as “for research purposes”. regarding the composition of the sample, the sample size was carried out based on the lists of active members provided by the offices of the associations involved in the study of pain mentioned above, in the first quarter of 2006. the level of significance was 5% and the confidence interval, 12%. initially, the official number of members of each association was obtained. then, the names were listed and numbered for random drawing. the final list of volunteers was composed from the statistical analysis system’s (sas) function of generating pseudorandom numbers with a uniform distribution. thus, it was a universe sample that allowed the composition of a random sample, rather than a convenience. the power analysis was 0.818 for comparison of the two groups, with mean difference of four and standard deviation of eight. the distribution of the volunteers, 46 bérzin mgr, siqueira jtt braz j oral sci. 8(1): 44-9 based on the criteria for inclusion in the sample, corresponded to 14 states, five regions and 52 cities in brazil. the data were entered by a person trained in the use of software specially developed for research in visual basic, version 6.0, called dof data, analyzed by a specialist in systems analysis and a technician in informatics who incorporated routines of data storage and applied consistency during digitization. the chi-square test was used for comparison of the proportions and the association, for rows and columns of tables. the student’s t-test was used for comparison of the characteristics, the specialties of the two professionals evaluated in the study. in all the analyses, a level of significance of 5% was adopted. results the study involved dentists and physicians who practice in the area of pain in various regions of brazil. the distribution of the volunteers showed a greater concentration of professionals in the capitals of seven states in the south and southeast regions of the country (91.36%), particularly in the state of são paulo (64.67%). the distribution of the dentists by gender was statistically similar, but the group of physicians was predominantly male. with respect to civil status, there was a predominance of married dentists and physicians in the sample. the sociodemographic characteristics of the sample are described in table 1. the types of institution in which the volunteers had their academic training, as well the time of professional training and practice in the area of pain, are presented in table 2. the majority of professionals in the sample were trained more than ten years ago. however, the proportion of professionals with more than ten years of practice in the area of pain is significantly higher among dentists (86.20%), than physicians (35.48%). the proportion of physicians and dentists with specialist titles reached 79.37% and 81.61%, respectively. these findings can be seen as one of the indicators of changes in the professional profile of dentists and physicians, observed in brazil. in the present study, 34.92% of physicians had a master’s or doctorate degree. among the dentists, 48.27% had such degrees and some post-doctoral training. only 16.42% of the volunteers did not have a graduate degree beyond medical or dental degree. thus, it appears that the sample of this study was composed of a significant number of professionals dedicated to professional advancement. the self-assessment of the volunteers on their theoretical-technical knowledge in the field of pain, and on their need to improve this knowledge showed that 64.37% of the dentists and 78.69% of the physicians declared having sufficient understanding in the area of pain. it even showed that 37.93% of the dentists and 43.54% of the physicians did not recognize the necessity to enhance their knowledge in pain. the study also investigated how the topic of pain was studied during medical/dental school courses of the volunteers. the reduced number of citations presented by the volunteers suggests that this topic was not covered substantially in the professional education of both the dentists and physicians surveyed. while the most frequently cited disciplines by dentists were endodontics (13 citations), surgery (12), pharmacolog y (7) and occlusion (8), physicians cited more often anesthesiolog y (11), neurolog y (10), physiolog y (5) and clinical medicine (4). the disciplines most cited by the physicians appeared to be linked to their specialties. it is noted that the discipline regarding pain treatment was referred to by only two dentists and two physicians. in addition, the main topics linked to pain studied by the volunteers over the course of their professional career were inquired in this study. a substantially varied field of interests was noted. dentists referred to a greater number of topics of interest, than did the physicians (321 citations from the dentists and 187 from the physicians). the topics most cited by the two groups of volunteers were neuroanatomy, neurophysiolog y, physiopatholog y, categories of pain, treatments in various areas, semiolog y, diagnoses, clinitable 1. sociodemographic data of dentists and physicians who responded to the questionnaire sociodemographic data dentists (%) physicians (%) profession (n = 150 – p value = 0.0500) 87 (58.00)a 63 (42.00)b gender male 37 (42.53)a 48 (76.19)a female 50 (57.47)a 15 (23.81)b p-value 0.1634 < 0.0001 mean age (p = 0.8325) 45.63 (10.60) a 45.24 (12.09) a civil status (married – p value = 0.5050) 66 (75.86)a 43 (68.26)a profession, gender and civil status were analyzed by the chi-square test and mean age by the student’s t-test. the lowercase letters indicate the comparison of data from the same professional group. the uppercase letters indicate the comparison of data between dentists and physicians. table 2. characterization of the academic training of the sample by profession, type of institution, time of professional activity and of practice in the area of pain dentists physicians graduation study public 52 (59.77%)a 46 (73.02%)a private 35 (40.23%)a 17 (26.98%)b p value 0.0684 0.0003 post-graduation study no post-graduate course 06 (6.90%) c 06 (9.52%) c lato sensu 71 (81.61%)a 50 (79.37%)a stricto sensu 42(48.27%)b 22 (34.92%)b p value – no post. vs lato sensu < 0.0001 < 0.0001 p value – no vs stricto sensu < 0.0001 0.0025 p value – lato sensu vs stricto sensu 0.0064 0.0010 time of professional activity less than 10 years 12 (13.79%)b 16 (25.80%)b more than 10 years 74 (86.20%)a 47 (74.19%)a p value < 0.0001 < 0.0001 time of practice in the area of pain less than 10 years 12 (13.79%)b 40 (64.51%)a more than 10 years 74 (86.20%)a 22 (35.48%)b p value < 0.0001 0.0223 often treats chronic pain (p value < 0.0001) 68 (77%) 63 (100%) p value calculated based on the chi-squared test. the lowercase letters indicate the comparison of data from the same professional group. the uppercase letters indicate the comparison of data between dentists and physicians. 47study on the training of brazilian dentists and physicians who treat patients with chronic pain braz j oral sci. 8(1): 44-9 cal practice, headache, neuropathies, pharmacolog y, specific diseases and musculoskeletal pain. none of the physicians referred to temporomandibular disorder, trigeminal neuralgia and bruxism. dentists made only 13 citations of the themes neck and musculoskeletal pain, neuropathies and fibromyalgia topics. among the less cited subjects by the general sample were psychological aspects, pain behavior, depression, interdisciplinary relationship and physician-patient relationship. the greatest majority of volunteers (88.50%), being all physicians and 77% of the dentists, reported treating patients with chronic pain. also in large numbers, 92.66% of the volunteers declared adopting an interdisciplinary approach in their work. figures 1 and 2 illustrate the aspects considered most important for working in the area of pain and the principal difficulties faced by the volunteers in their daily work routine. discussion although in the last decades the number of dentists and physicians has increased in the brazilian work force, no changes in the distribution of these professionals have been observed in the country. the concentration of professionals is mainly in the large urban centers, that is, in the south and southeast regions of the country19-20. this scenario was also seen in the present study, since the absolute majority of professionals was from the southeast region, particularly from the state of são paulo. the present study confirms the increase in the prevalence of men among physicians (76.19%); the mean percentage of male physicians in brazil was 60.40% in 2000 and 69.80%, in 200419,21. with respect to dentists, women prevailed, which confirms the slight prevalence of this gender among brazilian dentists20. married professionals predominated in the present study, which is a higher prevalence than that found in the brazilian work force20. the sample from the present study showed that 2/3 of the physicians evaluated received their education in public schools, which is in accordance w ith the f indings of the federal council of medicine21. on the other hand, 59.77% of the dentists who treated patients w ith chronic pain also received their education in public schools. this points out a higher percentage than that found in the brazilian work force, in which the majority of dentists are educated in private schools20. another important aspect is that the majority of the sample for both professions reported hav ing some post-graduate title, in contrast to other studies that showed that less than half the dentists20 and only 21.90% of the physicians had some post-graduate title21. the international scientif ic literature shows that the interest of dentists in getting a specialization is increasing. taken altogether, the f indings of this sur vey suggest that the majority of professionals involved w ith the treatment of pain, in the two professions, maintain a continuing interest in study and specialization. a lthough this f inding is not conclusive, one possible reason is the diff iculty that this f ield presents due to its complexity and lack ing in the formal education system. it is possible that the grow ing search for technical advancement is intimately linked to the worsening standard of dental and medical schools in brazil, obser ved in the last years, and by the need of entering a work force increasingly more competitive and demanding 20 -21. interestingly, with regard to professional specialization, our data show that at least a quarter of the dentists consider the physicianpatient relationship important in caring for patients with pain versus half of the physicians. these findings suggest that theoretical-tech39.08% 17.24% 31.03%31.75% 22.22% 52.38% choice of treatment prescribing medications behavior of the patient dentists physicians figure 1. more important aspects for working in the pain field. 40.98% 22.95% 24.24% 42.42% professional/patient relationship theoretical-technical training dentists physicians figure 2. main difficulties found in work routine in the pain field. 48 bérzin mgr, siqueira jtt braz j oral sci. 8(1): 44-9 nical training continues to be priority for the majority of dentists and some of physicians. although these professionals demonstrated an interest in continuing education, the results of this study did not clearly showed the adoption of a clinical approach of chronic pain, as recommended by the modern literature and the institutions involved in the study and treatment of pain17,25-26. on the contrary, medical residency which is considered an excellent form of interdisciplinary learning and experience, is decreasing among physicians21 and is rare among dentists, especially in the area of pain16. another explanation for the greater interest in post-graduate courses could be the fact that the majority of professionals in this sample, who treat chronic pain, is composed by those who were trained more than ten years ago. however, the period they work with patients with pain is less than ten years, especially among physicians (64.51%), which suggests that the topic of pain, as a specific area of interest and professional practice, is very recent in the practice of the volunteers. the time of practice in the area of pain was significantly higher among dentists (86.20%). however, the proportion of physicians (100%) that treat patients with chronic pain was significantly higher than dentists (77%). topics that approach pain were not much cited as part of the professional training of both professions, and dentists evaluated demonstrated a greater interest in disciplines concerning basic studies such as physiopathology of pain, although both groups considered themselves capable of treating patients with chronic pain. however, the low rate of citations that were considered important with regard to psychological aspects, pain behavior and interdisciplinary relationship, revealed a technique tendency in the training of the volunteers, and also questions the declarations of the great majority of the sample who stated having sufficient knowledge to work in the area of pain, particularly chronic pain. pain is generally not much emphasized in medical/dental school courses. moreover, it is not presented in a way that enables an integrated view of the subject25. therefore, dentists and physicians are not much aware of this subject and conclude their schooling overlooking the importance and consequences of the lack of preparation to work in the area of pain, especially chronic pain27. similarly, pharmacology was considered as deficient in professional training, which is in accordance with the experience of professors in the area28. this study shows the little interest of the volunteers in issues related to other areas of health. issues such as temporomandibular disorders and toothache were of little interest to physicians, although they affirmed that they treat chronic orofacial pain, while dentists had little interest in issues such as neck and musculoskeletal pain, neuropathies and fibromyalgia. different forms of orofacial pain occur in the same body region, innervated by a complex system, namely the trigeminal29, which has multiple etiologies4 and frequently cross borders of medicine and dentistry8, requiring an interdisciplinary management, including professionals such as psychologists and physical therapists. it is evident that professional training, as for dentists or physicians, in the area of orofacial pain, requires a curriculum appropriate for training and practice, respecting the respective areas of professional practice3,14. certainly, professional training in pain that enables a better care of patients cannot be viewed only as the result of theoreticaltechnical knowledge and many years of professional experience. it is much more than that. it has also to be considered factors of psychological and social nature, in addition to the personal skills of these professionals, which determine their pattern of behavior in the daily dealing with the suffering of patients with pain. it is the combination of these technical, humane and social factors that enable the training of persons adapted to their profession, satisfied with their work and eager to improve their knowledge and competence. this subject, however, needs to be better studied. in conclusion, taken together and considering the applied methodolog y, the findings of this survey show that the majority of professionals in the studied population who treat patients with chronic pain: have more than ten years of training and have more post-graduate education, when compared to professionals in the work force; reveal a technique tendency in the professional training; consider the professional/patient relationship, emotional aspects of pain and pain behavior of little importance, based on the low rate of citation of these as important themes; and consider themselves competent in treating patients with chronic pain. the analysis of the theoretical-technical difficulties pointed out by professionals of this study confirms the deficiencies in quality of care to patients with pain in our country, and the need of reflection by clinicians, professors, scholars and public health associations linked to the study of pain. references 1. carlson cr, reid ki, curran sl, studts j, okeson jp, falace d, et al. psychological and physiological parameters of masticatory muscle pain. pain. 1998;76: 297-307. 2. breivik h, collett b, ventafridda v, cohen r, gallacher d. survey of chronic pain in europe: prevalence, impact on daily life, and treatment. eur j pain. 2006;10(4):287-333. 3. lipton ja, ship ja, larach-robinson d. estimated prevalence and distribution of reported orofacial pain in the united states. j am dent assoc. 1993;124:115-21. 4. okeson jp. bell’s orofacial pain. the clinical management of orofacial pain. chicago: quintessence; 2006. 5. okeson jp. orofacial pain: guidelines for assessment, diagnosis and management. chicago: quintessence; 1996. 6. siqueira jt, lin hc, nasri c, siqueira sr, teixeira mj, heir g, et al. clinical study of patients with persistent orofacial pain. arq neuropsiquiatr. 2004;62:988-96. 7. headache classification committee of the international headache society. classification and diagnostic criteria for headache disorders, cranial neuralgias and facial pain. cephalalgia 1988; 8(suppl 7):1-96. 8. blasberg b, greenberg ms. orofacial pain. in: greenberg ms, glick m (eds). burket’s oral medicine: diagnosis and treatment. 10th ed. new york: bc decker inc. 2003. p. 307-40. 9. [no authors listed]. classification of chronic pain. descriptions of chronic pain syndromes and definitions of pain terms. prepared by the international association for the study of pain, subcommittee on taxonomy. pain suppl. 1986;3:s1-226. 10. bonica jj. the management of pain. 2. ed. philadelphia: febiger publisher; 1990. 11. wilson, jf, brockopp gw, kryst s, steger h, witt wo. medical students’ attitudes toward pain before and after a brief course on pain. pain. 1992;50:251-6. 49study on the training of brazilian dentists and physicians who treat patients with chronic pain braz j oral sci. 8(1): 44-9 12. turk dc, melzack r. the measurement of pain and the assessment of people experiencing pain. in: turk dc, melzack, r. handbook of pain assessment. new york: guilford publisher; 1992. p. 3-12. 13. leila nm, pirkko h, eeva p, eija k, reino p. training medical students to manage a chronic pain patient: both knowledge and communication skills are needed. eur j pain. 2006;10:167-70. 14. siqueira jtt. the past, present and future of temporomandibular disorders and orofacial pain in brazil. in: s-c chung, j fricton (eds). the past, present and future of temporomandibular disorders and orofacial pain. seoul: shinhung international; 2006. p. 315-48. 15. morris db. success stories: narrative, pain, and the limits of storylessness. in: narrative, pain and suffering. daniel b carr, hohn d loeser, david b morris (eds). progress in pain research and management. seattle: iasp press; 2005. p. 269285. v. 34. 16. siqueira jtt . dores mudas. as estranhas dores de boca. são paulo: artes médicas; 2007. 17. fields h. core curriculum for professional education in pain. 2. ed. seattle: iasp press; 1995. 18. greene, cs. science transfer in orofacial pain. in: lund jp, lavigne gj, dubner r, sessle b. orofacial pain – from basic science to clinical management. quintessence publishing co, inc.; 2002. p. 281-86. 19. brasil. ministério da saúde. dinâmica das graduações em saúde no brasil: subsídios para uma política de recursos humanos. brasília: ministério da saúde; 2006. disponível em: http://portal.saude.gov.br/portal/arquivos/pdf/ dinamica.pdf [2006 mar 9]. 20. instituto brasileiro de estudos e pesquisas sócio-econômicos. perfil do cirurgião-dentista no brasil. cfo 2003 apr. disponível em: http://www.cfo.org. br/download/pdf/perfil_cd.pdf [2006 feb 15]. 21. federal council of medicine. the doctor and his work: methodological aspects and results of brazil. brasilia: federal council of medicine; 2004. 22. bérzin mgr. chronic pain: a psychological approach. br j oral sciences. 2004;10:480-3. 23. kulich kr, rydén o, bengtsson h. a descriptive study of how dentists view their profession and the doctor-patient relationship. acta odontol scand. 1998;56:206-9. 24. maynard dw, heritage j. conversation analysis, doctor-patient interaction and medical communication. med educ. 2005;39:428-35. 25. merskey h, bogduk n. classification of chronic pain. 2. ed. seattle: iasp press; 1994. 26. loeser j. pain, suffering and the brain: a narrative of meanings. in: loeser jd, carr db, morris d (eds). narrative, pain and suffering. seattle, iasp press, 2005. 27. pimenta cam, figueiró jab, teixeira mj, siqueira jtt, perissinotti dmn, castro ces, et al. proposta de conteúdo mínimo sobre dor e cuidados paliativos nos cursos de graduação da área da saúde. rev simbidor. 2001;2:23-35. 28. andrade ed, ranali j, volpato mc. uso de medicamentos na prevenção e controle da dor. in: andrade ed. terapêutica medicamentosa em odontologia: procedimentos clínicos e uso de medicamentos nas principais situações da prática odontológica. são paulo: artes médicas; 2002. p. 45-64. 29. sessle bj. peripheral and central mechanisms of orofacial pain and their clinical correlates. minerva anestesiol. 2005;71:117-36. 1 volume 16 2017 e17057 original article 1 dds, department of restorative dentistry, school of dentistry, universidade de são paulo (usp), sp, brazil. 2 dds, msc, phd, department of restorative dentistry, university of uberaba, uberaba, mg, brazil. 3 dds, msc, department of restorative dentistry, school of dentistry, universidade de são paulo (usp), sp, brazil. 4 dds, msc, phd, department of dental materials, piracicaba dental school, faculdade de odontologia de piracicaba (unicamp), piracicaba, sp, brazil. 5 dds, msc, phd, special laboratory of lasers in dentistry, department of restorative dentistry, school of dentistry, universidade de são paulo (usp), sp, brazil. corresponding author: patricia moreira de freitas laboratório especial de laser em odontologia (lelo) faculdade de odontologia – universidade de são paulo av. professor lineu prestes, 2226 – cidade universitária, butantã, são paulo, brazil +55 11 3091-7645 pfreitas@usp.br received: may 31, 2017 accepted: september 14, 2017 bond strength of composite resin containing biomaterial s-prg to eroded dentin alessandra sanchez coelho lourenço1, ana paula almeida ayres2, taís fonseca mantilla3, marcelo giannini4, patricia moreira de freitas5 aim: to evaluate the bond strength of composite resin containing or not biomaterial (s-prg) to sound/eroded dentine. methods: occlusal dentin of 30 human molars (n=15) had half of its surface kept uneroded, while on the other half an erosive lesion was produced by cycling in citric acid (ph 2.3) and supersaturated solution (ph 7.0). on both eroded (ed) and non-eroded (sd) substrates, two restorative systems (containing or not s-prg) were tested. composite resin cylinders were built and, after storage in water (24h), were submitted to bond strength test. the analysis of the fracture pattern was performed under an optical microscope (40x). the obtained values of bond strength (mpa) were submitted to anova (two factors) and tukey multiple comparisons tests (p<0.05). results: according to the results, there was difference between substrates (<0.001) and restorative materials (p=0.002) evaluated. for the microtensile bond strength, the values obtained were: sdnb (47.6±12.2 mpa), sdwb (34.1±15.8 mpa), ednb (31.1±8.3 mpa) and edwb (15.5±13.6 mpa), revealing a statistically significant difference in the evaluated substrates and restorative materials. conclusion: bond strength of eroded substrate is inferior to the sound substrate and the restorative system containing s-prg biomaterial influences negatively the results of bonding to sound/eroded dentin. keywords: biomaterial s-prg. erosion. union resistance. http://dx.doi.org/10.20396/bjos.v16i0.8651050 2 lourenço et al. introduction dental erosion is a chemical process characterized by the dissolution of hard dental tissues resulting from exposure to a variety of acids of non-bacterial origin1. intrinsic factors such as gastroesophageal reflux and regurgitations resulting from eating disorders (anorexia and/or bulimia) are associated with this pathology, as well as extrinsic factors, which are more common nowadays due to acidic diets resulting from healthy foods or conditions related to profession2,3. the factors that generate tooth erosion may be of multiple origins and its diagnosis should be followed by measures to control its progression or even to restore the function and aesthetics of the compromised teeth4. in this context, direct restorative techniques are indicated on the affected surfaces, being performed on the enamel or, in more advanced cases (severe lesions), on dentin4. although in the last decade the technological evolution has been huge with respect to adhesive restorative materials, it is still a great challenge to achieve an equally effective adhesion in different dental tissues, with dentin being the most critical substrate for adhesion5. among the adhesive systems available in the market and of interest in the context of the control of the dental substrate demineralization process, fluoride adhesive systems, which can release fluoride ions6, are distinguished from other restorative materials by their capability to penetrate into the dentin and provide a more effective source of fluoride7,8. however, it is not known if the fluoride concentration released would be able to offer some benefit to the dentin, either for the inhibition of demineralization or the activation of dentin remineralization. also, the actual role of fluoride release in restorative materials in erosion prevention is not yet fully understood9. a composite resin containing the s-prg particle (surface pre-reacted glass/pre-activated surface ionomer, patented and exclusive technology giomer-shofu inc., kyoto, japan) was developed with properties much like that of a glass ionomer cement in many aspects and at the same time with physical properties of a composite. the advantage of the material seems to be the release of six different ions (fluoride, sodium, strontium, aluminum, silicate and borate), known to have bioactive properties10. the s-prg filler particles are formed by an acid-base reaction between fluoroaluminosilicate glass and polyacrylic acid1 and are capable of fluoride releasing and recharging. therefore, the material is able to provide significant effects on remineralization throughout cariogenic challenges, inhibiting carbohydrate metabolism in the biofilm, promoting acid neutralization in the oral cavity, and being able to control the demineralization process of the dental substrate10-12. up to the moment, the literature reports studies on the adhesion of this material only to sound or demineralized (secondary caries lesions) dental surfaces13. considering that fluoride containing products can be of great interest for the restoration of erosive lesions, controlling future tooth mineral loss, this in vitro study evaluated the bond strength of composite resin restorations, containing or not s-prg biomaterial, to the sound and eroded dentin surfaces. the working hypotheses tested in this study were: (a) there were no differences in bonding effectiveness to eroded and non-eroded dentin surfaces irrespective of which restorative system was applied and (b) a restorative 3 lourenço et al. system with s-prg particles in the composition would not influence the bond strength to dentin (eroded or not) when compared to the a conventional restorative system. materials and methods ethical aspects this study protocol was approved by the committee of ethics on research of the school of dentistry of the university of sao paulo (protocol n. 1.730.958). study design the sample units of the present study consisted on 60 human dentin discs, obtained from 30 human third molar teeth. the factors under study were the substrate type in 02 levels (sound dentin/sd and eroded dentine/ed) and restorative systems in 02 levels (containing/wb or not/nb the biomaterial s-prg). samples were randomly divided in 4 groups (n = 15): sdnb (sound dentin/no biomaterial in the restorative system), sdwb (sound dentin/ with biomaterial in the restorative system), ednb (eroded dentin/ no biomaterial in the restorative system) and edwb (eroded dentin/ with biomaterial in the restorative system). the response variable obtained was the microtensile bond strength (mpa) between the sound/eroded dentin surfaces and the restorative system. the evaluations were performed after 24h storage in deionized water. the analysis of the fracture pattern was also performed in a descriptive way. fluxogram of samples’ distribution is shown in figure 1. sample preparation thirty recently extracted third molars were collected from the human teeth bank of the school of dentistry of the university of são paulo. the teeth were stored in 0.5% chloramine t solution under refrigeration (4oc) for a maximum period of three months after the extraction date. the teeth were cleaned using periodontal curettes (duflex, ss white, rio de janeiro, rj, brazil) and polishing with pumice (ss white, rio de janeiro, rj, brazil) and water with the aid of robinson’s brush (kg sorensen, barueri, sp, brasil), followed by washing with distilled water. figure 1. fluxogram of samples’ distribution. 60 human dentin discs eroded dentin samples (ed) non-eroded dentin samples (sd) sdwb (n=15) (sound dentin/with biomaterial in the restorative system) sdnb (n=15) (sound dentin/no biomaterial in the restorative system) ednb (n=15) (eroded dentin/no biomaterial in the restorative system) edwb (n=15) (eroded dentin/with biomaterial in the restorative system) (30 human third molar teeth) 4 lourenço et al. after cleaning, the teeth were fixed with sticky wax (asfer indústria química ltda, são paulo, brazil) on a metal plate (gibraltar’s jig), with the dentin-enamel junction perpendicular to the plate. initially, the teeth had the occlusal enamel removed with a double-sided diamond disc (buehler, uk ltd, lake bluff, il, usa), in a metallographic slicer for exposing the dentin surface at low speed (100 rpm), coupled to a cutting machine (isomet 1000, buehler ltd, lake buff, il, usa) and under water cooling. then, the specimens were polished and planned manually in a low speed (100 rpm) polishing machine (ecomet 3 buehler ltd, lake buff, il, usa) using silicon carbide discs (buehler ltd, lake buff, il, usa) of decreasing granulations #120, #240, #400, under constant irrigation. between the use of each polishing sandpaper disc, the specimens were ultrasonically washed with deionized water for 5 minutes. all specimens were included in polyvinyl chloride (pvc) tubes containing acrylic resin (jet clássico, são paulo, sp, brazil) to enable specimen fixation. after inclusion, the specimens were polished with #600 granulation sandpaper to standardize the smear layer. the dentin occlusal surface (dentin hemi-disc) was protected with adhesive tape, except in a delimited area that corresponded to the sound surface. the artificially eroded dentin was produced on the exposed dentin surface. erosive cycling the demineralization was done by immersing the specimens in 10 ml of 0.05 m citric acid solution (e. merck, d-6100 darmstadt, f.r., germany), ph 2.3, 6 immersions of 10 minutes each, during 5 days14. between each immersion in acidic solution, with an interval of 1 hour each, and during the remaining time (other periods of the day in which the samples were not immersed in the acid solution), the specimens were stored in 10 ml of remineralizing solution, consisting of h3po4 4.08 mm, 20.10 mm kcl, 11.90 mm na2co3, and 1.98 mm cacl2, ph 6.7, under gentle stirring (30 rpm) (polymax 1040, incubator 1000, heidolph instruments gmbh & co. kg, schwabach, germany). after each immersion, the samples were carefully washed with distilled water for 1 minute and gently dried with absorbent paper and then placed in the container of the next solution. the solutions were renewed at the beginning of each day of the experiment and the ph value of all the solutions was controlled at the beginning and at the end of each experimental day. after the last erosive cycling, the samples were immersed for 24 hours in supersaturated solution and then stored in a humid environment with distilled-deionized water until the surface treatments and adhesive procedures were performed. surface treatments after obtaining the hard and artificially eroded dentin hemidiscs, 15 samples were treated with the restorative system not containg the s-prg particles and 15 samples with the restorative system containg the s-prg particles, both used according to the manufacturer’s instructions (table 1). after the application of the adhesive systems, composite resin blocks (a3 color) were built on the dentin surfaces that were previously delimited with a pen to differentiate between sound and eroded dentin. each 2-mm-thick increment was photoactivated for 20 seconds using an led light curing device (radii sdi, victória, au) with a power density of approximately 1,200 mw/cm2. the intensity of the light-curing source was monitored by means of a radiometer (curing light meter 105, demetron research corporation, usa). 5 lourenço et al. microtensile bond strength test after the adhesive procedures and the composite resin blocks were obtained, the samples were stored in distilled-deionized water (37°c) in a greenhouse for a period of 24 hours and then sectioned to obtain sticks15. the samples were fixed in wax (asfer indústria química ltda, são paulo, brazil) on a metal plate (isomet 1000, buehler ltd., lake bluff, illinois, usa), the most perpendicular to the double-sided diamond disk (buehler ltd lake bluff, il, usa). each sample was sectioned in the buccal-lingual direction and in the mesio-distal direction perpendicular to the dentin-composite union interface, with a rotation of 100 rpm, under water cooling, obtaining stick-shaped specimens with dimensions of approximately 1.0 x 1.0 mm. sticks obtained from the margins of the resin block were discarded. after being examined in a stereoscopic magnifying glass (olympus, tokyo, japan) in a 30x magnification with the objective to exclude those with defects in the adhesive interface, bubbles, cracks or remnants of enamel, approximately 2 to 6 sticks per sample were selected and stored for another 24 hours to be submitted to the microtensile bond strength test. the device containing the stick sample was coupled to the universal test machine (instron 5942, canton, ma, usa), and loaded with 50 n (speed of 0.5 mm/min) until fracture of the sample. microtensile bond strength was recorded in newton (n) and values were calculated and expressed in mpa, using the formula: bond strength (mpa) = strength (n)/sample cross-sectional area (mm2). table 1. description of the adhesive materials used in the present study (information given by manufacturers). material (manufacturer) composition (# bath) application’s mode adper single bond 2 (3m espe, st. paul, usa), ph 4.7 acid conditioner: 35% phosphoric acid ph = 0.6 adhesive: bis-gma, hema, dimethacrylates, photoinitiator, functional methacrylate, polyacrylic acid copolymer and polyacrylic acid, 10% by weight of 5 nm diameter spheroidal silica particles, water, ethanol (1703000272) acid conditioning apply for 15 s, wash for 15s application of the adhesive for 20 s. gently dry (air jet) for 5 s. photopolymerization for 10 s. filtek z350 xt (3m espe, st. paul, usa) bis-gma, bisema, udma, tegdma, combination of silica and zirconia aggregates (5-20 nm), and nanoparticles of silica (20 nm) / 78.5% by weight (1636100289) application of increment with a maximum of 2 mm of thickness. photopolymerization for 20 s. fl-bond il adesive system (shofu inc., kyoto, japan) ph 2.2 primer: ethanol, methacrylate adhesive monomer bonding resin: hema, udma, tegdma and sprg glass particles (041501) passive application of the primer on the surface, leaving it for 10 s. gently dry (air jet) for 5 sec. apply the adhesive over the entire surface. photopolymerization for 10 s. beautifil ii/shofu inc., kyoto, japan bis-gma, tegdma, multi-functional glass particles, s-prg particles (based on fluoroboroaluminosilicate) (081470) application of increment with a maximum of 2 mm of thickness. photopolymerization for 20 s. 6 lourenço et al. failure mode after the microtensile bond strength test was performed, all specimens were mounted on glass slides and the fractured surfaces were analyzed under a stereoscopic magnifier (olympus sz60, olympus corporation, tokyo, japan), with a magnification of 60x. the failure mode was classified as follows: type i—adhesive; type ii—cohesive in dentin; type iii—cohesive in composite resin; type iv—mixed. data analysis each tooth was considered a sample unit, so all sticks of a single tooth gave a mean value of bond strength. the data were submitted to statistical analysis by means of anova (two factors, repeated-measures) and tukey’s multiple comparisons tests, using the statistical software sigmaplot (systat software, inc), version 12.5, considering the level of significance of 5 %. results microtensile bond strength test the obtained values of microtensile bond strength (mpa) were submitted to anova (two factors, repeated-measures) and tukey’s multiple comparisons tests (p<0.05) and consisted on: sdnb (47.6 ± 12.2 mpa), sdwb (34.1 ± 15.8 mpa), ednb (31.1 ± 8.3 mpa) and edwb (15.5 ± 13.6 mpa). according to the results, there was no statistically significant difference on the interaction between substrate and restorative materials (0.711). however, as shown in table 2, there was difference between the substrates and the restorative materials evaluated. failure mode in groups sdnb and sdwb (sound dentin), the predominant fracture mode for both adhesive systems was adhesive failure, corresponding to 65.9% in sdnb and 88.67% in sdwb. the percentages of mixed failure were 27.7% for sdnb and 13.2% for sdwb, followed by cohesive failure in dentin of 6.87% in the sdnb group and 3.77% in the sdwb group (fig. 2). in dentin submitted to erosive cycling, the ednb obtained 70.88% of adhesive fractures, 26.58% of mixed fracture and 8.86% of cohesive fractures in resin, and in edwb there were 100% of adhesive failures (fig. 2). table 2. microtensile bond strength (mpa) values and standard deviation of each experimental group. restorative system substrate 3m/espe (pc) shofu (se) p value sound dentin (sd) sdpc 47.6 ± 12.2 sdse 34.13 ± 15.80 0.008 eroded dentin(ed) edpc 31.07 ± 8.31 edse 15.50 ± 13.60 0.003 p value <0.001 <0.001 7 lourenço et al. discussion compared to sound dentin, eroded dentin was consistently related to lower microtensile bond strength, irrespective of the adhesive system. therefore, the two hypotheses might be rejected, since eroded dentin did not present the same bonding effectiveness than non-eroded dentin and the restorative system containing biomaterial s-prg had a negative influence on dentin bond strength. different modifications on the surface of restorative materials and dental surfaces have been reported after an erosive challenge. erosion jeopardizes properties as morphology16, surface roughness17, hardness18 and wear depth19. the reduction of dentin bond strength is widely reported20,21 and it is probably a consequence of the combination of these deleterious effects. only one study reported that eroded dentin does not jeopardize the immediate microtensile bond strength of the adhesive restorative materials evaluated22. this result might be related to the different ph cycling mode, products and test utilized in the contradictory study. eroded dentin exhibit structural alteration of inorganic content23,24 causing unprotected collagen exposure24. the acidic nature of erosion agents might also activate metalloproteinases, contributing to enzymatic degradation of the substrate over time25. these differences between sound and eroded dentin might have interfered with the bonding properties of the restoration materials evaluated, irrespective to the presence of the biomaterial s-prg. apparently, the collagen fibrils’ disorganization of the softened and demineralized dentin is not modified by acid etching pretreatment and impairs monomers infiltration, thus hindering the formation of an adequate hybrid layer26. other possible explanation is that the altered morphological structure of the eroded dentin presents more porosities that jeopardize the evaporation of hydrophilic components, resulting on inferior bond strength26. in dental literature, etch-and-rinse bonding systems tend to provide higher bond strength than self-etching ones27. ultra-morphologically, there is a marked difference between the hybrid layers of these adhesive techniques bonded to dentin28. the adhesive system fl bond ii is considered “mild” regarding the acidity as it has a relatively high ph, around 2.2. this category of self-etching adhesives promotes incomplete smear layer dissolution and/or incorporation, while the phosphoric acid used in etchand-rinse adhesives removes the smear debris and demineralize the surface of the dentin29. after the rinsing phase, the adhesive is applied to dentin and resin monomers figure 2. percentage of failure mode for each experimental group. g4 g3 g2 g1 0% 20% 40% 60% 80% 100% i adhesive ii cohesive in detin iv mixed 8 lourenço et al. infiltrate into the dentinal tubules and into the collagen fibrils network, forming long resin tags and a thick hybrid layer, respectively. conversely, a very thin hybrid layer and short resin tags are formed with mild-acidity self-etching adhesive systems applied to dentin29. the intense micromechanical interlocking produced by phosphoric acid etching might explain the higher bond strength observed to the adper single bond 2. contradictory results30 found better results for self-etch approach when compared to etch-and-rinse bond strength results, which infers that the bonding effectiveness to eroded dentin might be product-dependent. forgerini et al.21 (2017) reported that the use of an universal adhesive did not provide the same bonding efficacy on eroded dentin as on sound dentin, irrespective of the etching mode. in all groups the most frequent failure mode was adhesive. it infers a good reliability of the present µtbs test conditions and correlates well with the literature for eroded dentin bonded to adhesive systems20,30-32. on the other hand, eroded dentin restored with ionomer based materials usually shows higher incidence of cohesive and mixed failure22. this fact might be related to the intrinsic cohesive material properties and also associated to the very common presence of air bubbles in glass ionomer cements that can act as stress points, increasing the likelihood of cohesive fracture within the cement33. in the present study, the eroded dentin restored with the self-etch adhesive presented 100% of adhesive failure, which is compatible with the worst bond strength result and indicates that the weakest zone is concentrated in the hybrid layer. although preventive effects of different fluoride formulations on erosive tooth wear34 and on dentin mechanical properties35 have shown positive results, our findings suggest that the fluoride-releasing restorative material used in this study showed no evidence of bond strength improvement to eroded dentin. according to the respective manufacturer, the s-prg particles of fl-bond ii adhesive system establish chemical bonding to the inorganic content of the dentin and releases fluoride. since the erosion demineralizes superficial dentin, less hydroxyapatites are available to interact with the ions present in the s-prg molecules, which might partially explain the lower bond strength found to this adhesive system. bollu et al. (2016) reported high microleakage for giomer and speculate that it was caused by polymerization shrinkage stress of this material36, while some authors suggest that the main cause the for the margin deterioration observed might be the intrinsic hygroscopic expansion of this restorative material37. other possible explanation for the lower performance of fl-bond ii is that due to its mild acidity, the adhesive components may not penetrate the whole depth of the demineralized layer produced by erosion, resulting in incomplete resin infiltration. the hypothesis that a prolonged primer application time would results in better penetration was tested by deari et al.32, applying optibond fl prime (kerr, scafati, italy) for a prolonged duration (60 s instead of 15 s). however, this technique did not improve the bond strength of eroded dentin. despite the lower immediate bond strength results using composite resin containing biomaterial s-prg, further studies should be conducted to investigate the influence of the chemical interaction between s-prg and inorganic content of eroded dentin in long-term storage evaluation. 9 lourenço et al. in conclusion, eroded dentin compromises the bonding quality of adhesive systems. the adhesive system not containing the biomaterial showed higher bond strength values to non-eroded and eroded dentin than the s-prg based restorative system. references 1. lussi a, schlueter n, rakhmatullina e, ganss c. dental erosion an overview with emphasis on chemical and histopathological aspects. caries res. 2011;45 suppl 1:2-12. 2. zero dt. etiology of dental erosion extrinsic factors. eur j oral sei. 1996 apr; 104(2):16277. 3. salas mm, nascimento gg, vargas-ferreira f, tarquinio sb, huysmans mc, demarco ff. diet influenced tooth erosion prevalence in children and adolescents: results of a meta-analysis and meta-regression. j dent. 2015 aug;43(8):865-75. 4. carvalho ts, colon p, ganss c, huysmans mc, lussi a, schlueter n, et al. consensus report of the european federation of conservative dentistry: erosive tooth wear-diagnosis and management. clin oral lnvestig. 2015 sep;19(7):1557-61. 5. van meerbeek b, yoshihara k, yoshida y, mine a, de munck j, et al. state of the art of self-etch adhesives. dent mater. 2011 jan;27(1):17-28. 6. mccabe jf, carrick te, kamohara h. adhesive bond strength and compliance for denture soft lining materials. biomaterials. 2002 mar;23(5):1347-52. 7. featherstone jdb, o’relly mm,shariati m, brugler s. enhacement of remineralization in vitro and in vivo. in: leach sa. factors affecting deand remineralization of the teeth. oxford: irl press; 1986. p. 23-34. 8. han l, edward c, okamoto a, iwaku m. a comparative study of fluoridereleasing adhesive resin materials. dent mat j. 2002; 21(1): 9-19. 9. soares le, lima lr, vieira lde s, do espírito santo am, martin aa. erosion effects on chemical composition and morphology of dental materials and root dentin. microsc res tech. 2012 jun;75(6):703-10. 10. gordan vv, mondragon e, watson re, garvan c, mjor ia. a clinical evaluation of a self-etching primer and a giomer restorative material: results at eight years. j am dent assoc. 2007;138(5):621-7. 11. moretto sg, azambuja n jr, arana-chavez ve, reis af, giannini m, eduardo cde p,et al.. effects of ultramorphological changes on adhesion to lased dentin-scanning electron microscopy and transmission electron microscopy analysis. microsc res tech. 2011 aug;74(8):720-6. 12. shiozawa m, takahashi h, iwasaki n. fluoride release and mechanical properties after 1-year water storage of recent restorative glass ionomer cements. clin oral investig. 2014 may;18(4):1053-60. 13. ayres apa, tabchoury cp, bittencourt berger s, yamauti m, bovi ambrosano gm, giannini m. effect of fluoride-containing restorative materials on dentin adhesion and demineralization of hard tissues adjacent to restorations. j adhes dent. 2015 aug;17(4):337-45. 14. ganss c, klimek j, schãffer u, spall t. effectiveness of two fluoridation measures on erosion progression in human enamel and dentin in vitro. caries res. 2001 sep oct;35(5):325-30. 15. sano h, shono t, sonoda h, takatsu t, ciucchi b, carvalho r, et al. relationship between surface area for adhesion and tensile bond strength-eval uation of a micro-tensile bond test. dent mater. 1994 jul;10(4):236-40. 16. honório hm, rios d, francisconi lf, magalhães ac, machado maam, buzalaf mar. effect of prolonged erosive ph cycling on different restorative materials. j oral rehabil. 2008;35(12):947-53. https://www.ncbi.nlm.nih.gov/pubmed/11804290 https://www.ncbi.nlm.nih.gov/pubmed/11804290 https://www.ncbi.nlm.nih.gov/pubmed/22131274 https://www.ncbi.nlm.nih.gov/pubmed/22131274 https://www.ncbi.nlm.nih.gov/pubmed/20945461 https://www.ncbi.nlm.nih.gov/pubmed/20945461 https://www.ncbi.nlm.nih.gov/pubmed/20945461 https://www.ncbi.nlm.nih.gov/pubmed/26258176 https://www.ncbi.nlm.nih.gov/pubmed/26258176 https://www.ncbi.nlm.nih.gov/pubmed/26258176 10 lourenço et al. 17. badra vv, faraoni jj, ramos rp, palma-dibb rg. influence of different beverages on the microhardness and surface roughness of resin composites. oper dent. 2005;30(2):213-9. 18. wongkhantee s, patanapiradej v, maneenut c, tantbirojn d. effect of acidic food and drinks on surface hardness of enamel , dentine , and tooth-coloured filling materials. j dent. 2006 mar;34(3):214-20. 19. pedroso c, anderson at, hara t, jorge s, mo dæ. study on the potential inhibition of root dentine wear adjacent to fluoride-containing restorations. j mater sci mater med. 2008 jan;19(1):47-51. 20. amsler f, lussi a. long-term bond strength of self-etch adhesives to normal and artificially eroded dentin : effect of relative. 2017;19(2):169-77. 21. forgerini tvrocha r de oliveira, soares fzm, lenzi tl rjf. role of etching mode on bonding longevity of a universal adhesive to eroded dentin. j adhes dent. 2017;19(1):69-76. 22. cruz jb, lenzi tl, tedesco tk, guglielmi c de ab, raggio dp. eroded dentin does not jeopardize the bond strength of adhesive restorative materials. braz oral res. 2012;26(4):306-12. 23. wang x, lussi a. assessment and management of dental erosion. dent clin north am. 2010;54(3):565-78. 24. prati c, montebugnoli l, suppa p, valdrè g, mongiorgi r. permeability and morphology of dentin after erosion induced by acidic drinks. j periodontol. 2003;74(4):428-36. 25. buzalaf mar, kato mt, hannas ar. the role of matrix metalloproteinases in dental erosion. adv dent res. 2012;24(2):72-6. 26. wang y, spencer p. effect of acid etching time and technique on interfacial characteristics of the adhesive-dentin bond using differential staining. eur j oral sci. 2004;112(3):293-9. 27. masarwa n, mohamed a, abou-rabii i, abu zaghlan r, steier l. longevity of self-etch dentin bonding adhesives compared to etch-and-rinse dentin bonding adhesives: a systematic review. j evid based dent pract. 2016 jun;16(2):96-106. 28. giannini m, makishi p, ayres apa, vermelho pm, fronza bm, nikaido t, et al. self-etch adhesive systems : a literature review. braz dent j. 2015 jan-feb;26(1):3-10. 29. wang y, spencer p. quantifying adhesive penetration in adhesive/dentin interface using confocal raman microspectroscopy. j biomed mater res. 2002;59(1):46-55. 30. ramos tm, ramos-oliveira tm, de freitas pm, azambuja n, esteves-oliveira m, gutknecht n, et al. effects of er:yag and er,cr:ysgg laser irradiation on the adhesion to eroded dentin. lasers med sci. 2015;30(1):17-26. 31. frattes fc. bond strength to eroded enamel and dentin using a. universal adhesive system. j adhes dent. 2017;19(2):121-7. 32. deari s, wegehaupt j, tauböck tt. influence of different pretreatments on the microtensile bond strength to eroded dentin. j ades dent. 2017;19(2):147-55. 33. burrow m., nopnakeepong u, phrukkanon s. a comparison of microtensile bond strengths of several dentin bonding systems to primary and permanent dentin. dent mater. 2002;18(3):239-45. 34. lussi a, carvalho ts. the future of fluorides and other protective agents in erosion prevention. caries res. 2015;49(suppl 1):18-29. 35. paula a, guedes a, moda md, umeda ty, gustavo a, godas dl, et al. effect of fluoride-releasing adhesive systems on the mechanical properties of eroded dentin. braz dent j. 2016 mar-apr;27(2):153-9. 36. bollu ip, hari a, thumu j, velagula ld, bolla n, varri s, et al. comparative evaluation of microleakage between nano-ionomer, giomer and resin modified glass ionomer cement in class v cavitiesclsm study. j clin diagnostic res. 2016;10(5):zc66-zc70. 37. sunico mc, shinkai k, katoh y. two-year clinical performance of occlusal and cervical giomer restorations. oper dent. 2005;30(3):282-9. 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1519/1172 1/11 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1519/1172 2/11 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1519/1172 3/11 28/01/2019 pdf.js 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dos santos1, luciane zanin2, flávia martão flório1 1são leopoldo mandic slmandic, school of dentistry and research center, area of public health, campinas, sp, brazil 2faculdade de medicina de jundiaí – fmj, medical school, area of public health, jundiaí, sp, brazil and fundação hermínio ometto uniararas, school of dentistry, area of public health, araras, sp, brazil correspondence to: roberta blanco dos santos praça general portinho nº6 apt 203 cep: 20271-010 maracanã, rio de janeiro, rj, brasil phone: +55 21 999633963 e-mail: robertablanco@gmail.com abstract aim: to demonstrate the feasibility of enabling trained acupuncturist as professionals able to detect oral lesions, with a role in the screening of oral cancer. methods: this study was conducted in two phases. in the first, 11 acupuncturists were trained for visual detection of oral lesions and support content available on : <www.kankou.com.br>, especially developed for this study. they were evaluated at the end of training by a written exam. the inclusion criteria were to sign the consent form and to obtain a minimum of 90% of correct answers in the assessment. four acupuncturists fulfilled both criteria and were included as study subjects. in the second phase, the subjects and the researcher (gold standard) examined 43 volunteer patients. the inclusion criterion for volunteers was just to sign the consent form. the volunteers’ sample was established by saturation. volunteers with changes deemed suspicious by the subjects were reexamined 15 days after the first examination. results: first exam: mouth alterations were detected in 56% (n=24) of the volunteers. among the alterations, 21% (n=5) were deemed suspicious by the subjects and 8% (n=2) by gold standard. second exam: two mouth alterations remained suspicious and these volunteers were referred to the dental specialty center for diagnosis and treatment. compared to the gold standard, no injury remained undetected by the subjects. conclusions: this study concluded for the feasibility of enabling trained acupuncturist to visually detect oral lesions and have a role in oral cancer screening. keywords: acupuncture; mouth neoplasms; pathology, oral diagnosis; training. introduction oral cancer is considered the 12th most common malignant tumor worldwide1. the incidence in 2008, for both sexes, was 263,020 cases for cancer in the lip and oral cavity, with a mortality rate of 127,6542. in brazil, mortality rates due to oral cancer in 1994 were 2,899 and 707 per 100,000 for men and women, respectively. in 2004, they increased to 4,418 and 1,045. in 2008, the number of deaths due to oral cancer reached 6,265 combining men and women3. most tumors arise in the oral cavity preceded by potentially malignant lesions or even as a carcinoma in situ, with intraoral manifestation as a white or red patch. therefore, early diagnosis is consensually paramount4. statistically, almost half of all cases are detected at advanced stages, therefore, braz j oral sci. 14(3):234-239 http://dx.doi.org/10.1590/1677-3225v14n3a12 received for publication: august 30, 2015 accepted: september 28, 2015 50% of patients have a probable average survival of five years only5-6. early detection of oral lesions is best achieved by dentists, who perform oral examinations routinely in their professional practice7-8. the incorporation of professionals other than dentists and physicians, such as community health care workers, dental nurses and dental hygienists to identify oral lesions has been suggested in literature to improve early diagnosis and treatment of these lesions4,9-10. acupuncture is a therapy advocated by chinese medicine and is regarded as traditional medicine11, based on its own medical rationality. according to dr. madel luz, the term “medical rationality” must be understood as an operational concept to the style of a weberian model, which establishes that any medical rationality denotes a complex, symbolic and empirically structured system in six fundamental dimensions: cosmology, medical doctrine, morphology, physiology or human vital dynamics, a diagnostic system and a therapeutic intervention system12. in brazil, acupuncturists are included in the unified health system (sus), in accordance with the national policy for complementary and integrating practices (pnpic – política nacional de práticas integrativas e complementares)13 and similar to dentists, acupuncturists have regular access to the oral cavity to perform tongue examination, analyzing the shape, color, coating and changes of the tongue, to gather information about the patient’s health. this is an important stage of the anamnesis, according to traditional chinese medicine/acupuncture semiology14. this may be an opportunity for early detection of oral precursor lesions and, therefore, prevent oral cancer, provided they are properly trained. patients seeking acupuncture are not after an oral examination primarily, but a well-trained acupuncturist may by occasion visually detect the presence of oral lesions that can be suggestive of oral cancer and refer the patient for diagnosis, thus acting within the principles of referral and counter-referral13. in view of the aforementioned arguments, the purpose of this study was to evaluate the feasibility of including adequately trained acupuncturists in the team of professionals able to detect oral lesions, thus widening the spectrum of professionals that may help oral cancer prevention. material and methods this study was performed according to principles established by resolution 196/96 of the national health council of the ministry of health, approved by committee of ethics for research of são leopoldo mandic school of dentistry and research center (nº 2012/0363). the subjects and volunteers signed an informed consent form. phase 1 – capacitation twelve acupuncturists were invited to participate in the capacitation phase, and 11 accepted the invitation. the training was carried out by a dentist also acupuncturist aiming to prepare the acupuncturists for detects oral lesions visually. the training process consisted of six-hour theory lessons, for which the candidates were present. the lessons comprised the below-described subjects, based on the literature2-3,9,15-17 and adapted to this study: anatomy of the oral cavity, variations of normality, epidemiology of oral cancer, types of lesions found in the oral cavity, risk factors for oral cancer, visual examination of the mouth for lesions, using a especially designed map for this study. the acupuncturists enrolled in the capacitation course received all lecture materials in printed form as well as supporting references on the website www.kankou.com.br, specifically designed for this study17. in standard mandarin, official language of the people’s republic of china, birthplace of acupuncture, the name kankou ( 看口 kànkǒu ) of the site may be translated as “look in the mouth”. the site was developed in five languages. at the end of the theory lessons, the enrolled acupuncturists were subjected to a written exam to demonstrate the knowledge they acquired on routine examination of the mouth, risk factors for oral cancer and visual detection of oral lesions or absence thereof. photographs of the oral cavity exhibiting some alterations were used. the acupuncturists were expected not only to identify them but also to classify them into categories based on the atlas issued by the international agency for research on cancer (iarc) 15, “a digital manual for the early diagnosis of oral neoplasia”, adapted for this study: a) anatomical variations: anatomical structures that may suggest pathology but are normal; b) common lesions: idiopathic changes or alterations related to non-neoplastic pathology; c) benign lesions: alterations compatible with benign tumors or similar to benign tumors; d) potentially malignant lesions: changes that suggest a potential for cancer development; e) malignant lesions: changes highly suspicious of oral cancer. in order to qualify for the second phase of the study, the acupuncturists were expected to achieve 90% of correct answers in the written exam and to sign the consent form. four acupuncturists were successful in fulfilling the inclusion criteria. phase 2 – exams in the second phase of the study, the subjects chose patients from the acupuncture outpatient clinic at pedro de alcântara hospital rio de janeiro rj, who hereafter will be referred to as ‘the volunteers’. the inclusion criterion for the volunteers was only their acceptance to take part by signing a consent form, as proposed by the acupuncturists to the volunteers. the final sample of volunteers was defined by saturation18, i.e., inclusion was suspended when the obtained data started to present, according to the researcher, 235235235235235 feasibility of including trained acupuncturists in the detection of oral lesions and oral cancer screening braz j oral sci. 14(3):234-239 236236236236236 redundancy and repetition. therefore, during the data collection period, from december 2012 to april 2013, 43 volunteers were included. an oral examination form was specifically designed for this study and after the examination a copy was attached to the patient’s records. it should be stressed that, in this phase of the study, the subjects were instructed to classify the lesions with a potentially malignant or malignant aspect into a single group, namely suspicious lesions, since diagnosis is no part of the acupuncturist’s scope. the volunteers were randomly examined by the subjects, following the outpatient routine of the institution, e.g., at the time of the consultation, the volunteer would be seen by any of the subjects available at the time. the volunteers that wore removable prosthetic appliances were instructed to remove them for examination purposes. as acupuncture contraindicates the often necessary tongue traction maneuvers to examine the posterior third of the tongue because of possible pressure-induced changes to color or coating status, as it could compromise the data deemed necessary for acupuncture, a set of specific guidelines was designed and made available for acupuncturists at the website: www.kankou.com.br17. the examination performed by the head researcher was considered as gold standard and, therefore, she re-examined the volunteers on the same day. according to the national institute of cancer (inca) directions16, volunteers that presented suspicious oral lesions were re-examined 15 days after the first examination by the same subject and then by the gold standard. the volunteers presenting oral changes compatible with common lesions and/or benign lesions were advised to have regular follow-ups with a dentist. at the end of the study, the volunteers with suspicious oral lesions were referred to the oral medicine service of the dental specialty center (centros de especialidades odontológicas ceo) for diagnosis and treatment whenever applicable. education and health promotion all volunteers received instructions regarding the risks involved with tobacco use and other risk factors for oral cancer as well as treatment options to help quitting their tobacco habits should they be smokers3,19. data analysis the data were recorded on microsoft excel 2010 and analyzed descriptively using figures containing data on distribution as well as absolute and relative frequencies. results phase 1 – capacitation from the 11 acupuncturists that took part in the capacitation, 64% (n=7) had 90% or more correct answers, whilst 36% (n=4) scored between 80 to 90% in the written exam. the results showed that among the subjects, 18% (n=2) were male and 82% (n=9) female. the mean age was 52.6 years (sd=6.87). those with a college degree were 82% (n=9) against 18% (n=2) with a high school degree. subjects with a health-related background were 55% (n=6), humanities background were 36% (n=4) and technology background 9% (n=1). regarding the acupuncture training, 46% (n=5) were qualified by a postgraduate course in acupuncture, 36% (n=4) by a technical course and 18% (n=2) by an open course. the average time in practice as professional acupuncturists was 9 years. four acupuncturists were included as subjects, all of them females aged between 50 to 60 years. regarding education level, 75% (n=3) had a college degree (physical education, social sciences and psychology) and 25% (n=1) had a high school degree. regarding their acupuncture training, 75% (n=3) were qualified by a technical course acknowledged by the ministry of education (mec) and the state education secretariat (see) – rj, whereas 25% (n=1) was qualified by an open course. their average acupuncture practice time was 9 years. phase 2 – examination among the volunteers, 35% (n=15) were male and 65% (n=28) were female. their age ranged from 12 to 79 years, with a mean age of 55 years (sd=15.42). regarding tobacco use, 11% (n=5) were current smokers, 5% (n=2) were former smokers and 84% (n=36) were non-smokers. once the oral examination was performed both by the subjects and the gold standard, visible alterations were identified in 56% (n=24) of the volunteers, whilst no changes were observed in 44% (n=19). figure 1 shows the total results in relation to the gold standard. amongst the detected alterations, 16% (n=4) were considered as variations of normal anatomy, 46% (n=11) as common changes, 17% (n=4) as benign lesions and 21% (n=5) as suspicious lesions. fig. 1: total results of the first examination. subjects and gold standard. feasibility of including trained acupuncturists in the detection of oral lesions and oral cancer screening braz j oral sci. 14(3):234-239 237237237237237 according to the gold standard of this study, among the lesions found in the first examination, 17% (n=4) were classified as anatomical variations, 58% (n=14) as common lesions, 17% (n=4) as benign lesions and 8% (n=2) as suspicious lesions. the gold standard of this study identified 60% (n=3) of the lesions identified as suspicious by the subjects as low severity on visual examination. the results from the second examination of the oral cavity performed 15 days after the first, as advised for the volunteers detected by the subjects as suspicious lesions and therefore needing confirmation, are described in figure 2. among the five subjects re-examined, 40% (n=2) presented persisting lesions and were referred to the ceo for diagnosis and treatment, according to the criteria established by the dentist in charge of the local specialty service. fig. 2: total results of the second examination. subjects and gold standard. discussion in brazil, the demand for acupuncture in the public sector increased 128% in two years, according to data from the ministry of health, increasing from 97,274 consultations in 2007 to 221,862 in 200920. the increased supply of acupuncture treatment in the unified health system (sus) was one of the basic aspects in this study. the aim was to evaluate the feasibility of including appropriately trained acupuncturists to visually detect oral lesions, on account that these professionals have regular access and familiarity with the oral cavity to examine the tongue in their routine practice, as well as being part of sus, according to the national policy for complementary and integrating practices (pnpic – política nacional de práticas integrativas e complementares)13. the scientific literature suggests the inclusion of other professional categories for the task of detecting oral lesions suggestive of cancer, thus favoring early diagnosis of the disease, as reported by lehew et al.9, where training was provided to physicians, nurses and a hospital administrator for that purpose. melo et al.10 and morse et al.4 encourage the capacitation of community health workers and other professionals involved in primary care, such as dental hygienists and nurses, in order to increase the chances of early detection, which therapeutically would result in less complex and more effective approaches, thus improving prognosis and reducing treatment costs4,9-10. there are no studies in the literature regarding the feasibility of training acupuncturists for visual detection of oral lesions. this study is the first to demonstrate a successful outcome from training acupuncturists and, differently from lehew et al.9, they were trained for visual detection of oral lesions, supervised during examination and assessed both at the capacitation and clinical examination phases. the availability of an electronic source for support (<www.kankou.com.br>)17 made it easier for the subjects to access the teaching material anytime and anywhere, which was also used during the examination phase to clarity questions that could relative to the detected oral changes. expanding knowledge through digital media has been used by the ministry of health in an attempt to overcome the territorial and sociocultural distances of the brazilian population and health professionals. at the same time, it contributes to ongoing education in health, diagnosis and information on general health. advantages such as fast and easy access, as well as interaction and research incentives make the digital media an important permanent tool that may encourage the professionals to search for information in order to complement their practice in the health services21-23. regarding screening programs, the canadian task force on preventive health care (ctfphc) and the united states preventive service task force (uspstf), do not advise the inclusion of organized oral cancer screening in routine practice, according to a systematic review performed by the american dental association council on scientific affair. instead, they recommend that screening be directed only at those at higher risk of developing oral cancer24. opportunistic screening has yet no firm evidence on effectiveness to be included or excluded from routine practice16,25. this study suggests that opportunistic screening may occur during a consultation with a trained acupuncturist, especially at the primary care level established by sus, where the acupuncturist is included according to the pnpic13 and considering that 221,862 acupuncture consultations were recorded in 200920. during the clinical examination phase, initial resistance by the patients (volunteers) to allow a complete oral examination was noticed, e.g., embarrassment upon denture removal. in order to solve this issue, the posture and explanations offered by the acupuncturist, in terms of objectives and possible benefits, the ease, speed, lack of risk and cost of the examination during the history were essential. care and a competent patient approach are narrowed down to the concept of humanization, which is based on respect and dignity, thus constituting a process that aims at cultural institutional change, via the construction of ethical commitments and methods for health care26. in this study, following explanation to the volunteers, full compliance was obtained and the screening process was performed satisfactorily. currently, acupuncture has different levels of academic qualifications in brazil, especially those as a specialist degree feasibility of including trained acupuncturists in the detection of oral lesions and oral cancer screening braz j oral sci. 14(3):234-239 238238238238238 (latu sensu) offered by universities. there are still some technical courses running in some states of the federation regulated by mec and see, as well as open courses. although acupuncture is not a registered profession per se, in 1982 the ministry of work and employment – mte designed the brazilian classification of occupations, which included the occupation of acupuncturist. a newer version of the classification was made based on entry nº397/2002, describing the profession of acupuncturist as independent from any other professional class, including medicine, with its own registration nº 3221-0527. according to the gold standard, at the first examination, a higher severity was attributed to some oral alterations. such conduct did not imply risks to the patient. on the second examination, both subjects and gold standard showed agreement. taking into account the fears and expectations experienced by the volunteers facing suspicious lesions, despite the advice to wait 15 days to follow them up, acupuncturists should promptly refer patients with suspicious lesions to a dentist, a dental school or other health services in their region. acupuncture is mostly known in the west for the analgesic activity, acting by the release of endogenous opioid peptides28 but its semiology and diagnosis methods deserve our attention. based on the results presented in this study, the inclusion of acupuncturists in the detection of oral lesions that suggest oral cancer may increase the number of patients undergoing oral examination secondary to acupuncture sessions. such opportunistic screening may also be widened to include the private sector, where most acupuncturists are placed29, thus contributing even further for early referral, diagnosis and treatment of oral cancer. the afore mentioned data and the results presented in this study suggest that if training were to be offered in regular acupuncture courses, the dentist should be administering its content, since they are the professionals with suitable experience to perform opportunistic oral cancer screening and they also have the knowledge to train professionals to assist them in doing so7,10,30. this study established an interdisciplinary team approach focusing on the visual detection of oral lesions, capacitating and including acupuncturists in a field primarily dominated by dentists and eventually by physicians. decuyper et al.31 highlighted that cooperation, communication and the opportunity to confront and integrate knowledge and individual competencies are fundamental actions and, moreover, experiencing a constructive conflict that reveals the diversity of opinions may guide interdisciplinary actions, thus integrating and shaping cooperative active professionals within their working environment. therefore, although this study may present some limitations such as a small sample, it verified the feasibility of including appropriately trained acupuncturists into the practice of visual detection of oral lesions, thus contributing to early detection of oral cancer in the public and private health services, via visual examination of the entire oral cavity as part of their clinical practice. references 1. eadie d, mackintosh am, macaskill s, brown a. development and evaluation of an early detection intervention for mouth cancer using a mass media approach. br j cancer. 2009; 101 (suppl 2): s73-9. 2. iarc. globocan 2008 fast stats. section of cancer information 2010 [cited: 2013 aug 27]. available form: http://globocan.iarc.fr/factsheets/ populations/factsheet.asp?uno=900#both. 3. who health statistics and health information systems 2013 [cited 2013 aug 26]. available fromt: http://www.who.int/healthinfo/mortality_data/en/ index.html. 4. morse de, vélez-vega cm, psoter wj, vélez h, buxó cj, baek ls, et al. perspectives of san juan healthcare practitioners on the detection deficit in oral premalignant and early cancers in puerto rico: a qualitative research study. bmc public health. 2011; 11: 391. 5. kujan o, glenny am, duxbury j. evaluation of screening strategies for improving oral cancer mortality: a cochrane systematic review. j dent educ. 2005; 69: 255-65. 6. gomez is, warnakulasuriya s, varela-centelles pi, lopez-jornet p, suarezcunqueiro m, diz-dios p, et al. is early diagnosis of oral cancer a feasible objective? who is to blame for diagnostic delay? oral diseases. 2010; 16: 333-4. 7. applebaum e, ruhlen tn, kronenberg fr, hayes c, peters es. oral cancer knowledge, attitudes and practices: a survey of dentists and primary care physicians in massachusetts. j am dent assoc. 2009; 140: 461-7. 8. epstein jb, sciubba jj, banasek te, hay lj. failure to diagnose and delayed diagnosis of cancer: medicolegal issues. j am dent assoc. 2009; 140: 1494-503. 9. lehew cw, epstein jb, koerber a, kaste l. training in the primary prevention and early detection of oral cancer: pilot study of its impact on clinicians’ perceptions and intentions. ear nose throat j. 2009; 88: 748-53. 10. melo ns, figueiredo pts, leile af, souza tac, lucena, ehg, zanetti chg. early oral diagnosis in the uniûed national health system: individual analysis, health surveillance and teamwork as a (re)structuring possibility. tempus actas saude colet. 2011 [cited 2013 sep 21]; 5: 89-103. available from: http://www.tempusactas.unb.br/index.php/tempus/article/view/1045. 11. choi sh. who traditional medicine strategy and activities “standardization with evidence-based approaches”. j acupunct meridian stud. 2008; 1: 153-4. 12. tesser cd, luz mt. [medical rationalities and integrality]. cienc. saude colet. 2008; 13: 195-206. available from: http://www.scielo.br/pdf/csc/v13n1/ 23.pdf. portuguese. 13. brazil. ministry of health. national policy on integrative and complementary practices in unified health system extension attitude access. brasília: ministry of health, 2006. 14. world health organization (who). consultation on acupuncture guidelines on basic training and safety in acupuncture. who: cervia; 1996. 15. iarc. iarc screening group. a digital manual for the early diagnosis of oral neoplasia [cited 2013 aug 27]. available from: http://screening.iarc.fr/ atlasoral.php. 16. brazil inca. national cancer institute. mouth cancer. 2012 [cited 2013 jul 27]. available from: http://www2.inca.gov.br/wps/wcm/connect/ tiposdecancer/site/home/boca/evidencias_cientificas_para_rastreamento. 17. kankou the acupuncturist and the inspection of the tongue. learn to optimize your routine clinical examination and participate in the prevention of mouth cancer. 2013 [cited 2014 mar 18]. available from: http://www.kankou.com.br. 18. denzin nk, lincoln ys, editors. handbook of qualitative research. thousand oaks: sage publications; 1994. 19. santos jdp, duncan bb, sirena sa, vigo a, abreu msn. [indicators of effectiveness of the unified health system smoking cessation program in minas gerais, brazil, 2008]. epidemiol serv saude. 2012; 21: 579-88. portuguese. feasibility of including trained acupuncturists in the detection of oral lesions and oral cancer screening braz j oral sci. 14(3):234-239 239239239239239 20. aranda f. ig homepage. [health and wellbeing]; 2010 aug 24 [cited 2014 mar 3]. available from: http://saude.ig.com.br/bemestar/ e m + d o i s + a n o s + a c u p u n t u r a + g r a t u i t a + c r e s c e + 1 2 8 + n o + p a i s / n1237750946329.html. portuguese. 21. cardoso bap, pacheco cma, souza rf. [website development on the internet. permanent improvement in nursing: contribution for continuing education]. enfermeria global. 2010 [cited 2013 aug 27]; (19). available from: http://scielo.isciii.es/pdf/eg/n19/miscelanea2.pdf. spanish. 22. rezende ejc, melo mcb, tavares ec, santos af; souza c. [ethics and ehealth: reflections for a safe practice]. rev panam salud publica. 2010; 28: 58-65.portuguese 23. rendeiro mmp, vieira p, figueiredo dltm. [infoculture: a barrier of access to permanent learning?] rev hosp univ pedro ernesto. 2012 [cited 2014 jan 1];11(suppl 1). available at: http://revista.hupe.uerj.br/ detalhe_artigo.asp?id=303. 24. rethman mp, carpenter w, cohen ee, epstein j, evans ca, flaitz cm et al. evidence-based clinical recommendations regarding screening for oral squamous cell carcinomas. j am dent assoc. 2010; 141: 509-20. 25. brazil. ministry of health. [screening: series a standards and technical manuals. primary care guide, n. 29]. brasília: ministry of health; 2010. portuguese. 26. rios ic. [health humanization pathways practice and reflection]. são paulo: áurea; 2009 [cited 2015 nov 3]. available from: http:// p e s s o a c o m d e f i c i e n c i a . s p . g o v. b r / u s r / s h a r e / d o c u m e n t s / caminhos_da_humanizacao_na_saude.pdf. portuguese. 27. kurebayash lsf, oguisso t, freitas gf. acupuncture in brazilian nursing practice: ethical and legal dimensions. acta paul enferm. 2009; 22: 210-2. 28. gondim dv, carvalho km, vale ml. pain behavior to electroacupuncture in rabbit tooth pulp. braz j oral sci. 2010; 9: 415-20. 29. sousa im, bodstein rc, tesser cd, santos f de a, hortale va. [integrative and complementary health practices: the supply and production of care in the unified national health system and in selected municipalities in brazil]. cad saude publica. 2012; 28: 2143-54. portuguese. 30. wade j, smith h, hankins m, llewellyn c. conducting oral examinations for cancer in general practice: what are the barriers? fam pract. 2010; 27: 77-84. 31. decuyper s, dochy f, van den bossche p. grasping the dynamic complexity of team learning: an integrative model for effective team learning in organisations, educ res rev. 2010; 5: 111-33. feasibility of including trained acupuncturists in the detection of oral lesions and oral cancer screening braz j oral sci. 14(3):234-239 revista fop n 13 1653 the effect of luting techniques on the push-out bond strength of fiber posts andré luis faria-e-silva1;andré figueiredo reis2; luis roberto marcondes martins3 1dds, ms, graduate student, department of restorative dentistry, piracicaba dental school, university of campinas, piracicaba, sp, brazil 2dds, ms, phd, professor, department of restorative dentistry, guarulhos university, guarulhos, sp, brazil 3dds, ms, phd. professor, department of restorative dentistry, piracicaba dental school, university of campinas, piracicaba, sp, brazil received for publication: may 27, 2008 accepted: september 22, 2008 correspondence andré luis faria-e-silva department of restorative dentistry, piracicaba dental school, university of campinas, av. limeira, 901, vila areião, 13414-903 piracicaba, sp, brasil. tel: +55-19-2106-5340 fax: +55-19-2106-5218. e-mail: andrelfs@fop.unicamp.br abstract aim: this study aimed to evaluate the effect of using a chemical co-initiator and/or an additional coat of a hydrophobic resin on the bond strength of fiber posts luted with a dual-cured resin cement. in addition, the use of the resin cement only was also evaluated. methods: fiber posts were luted with dual-cured resin cement (enforce) and using one of five adhesive procedures: g1 prime&bond 2.1 (pb), g2 pb + self-cure activator (sc), g3 pb + bonding agent of scotchbond multi-purpose (smp), g4 – pb + sc + smp and g5 – no adhesive. the root samples were sectioned transversally into three slabs (coronal, middle and apical third) that were submitted to push-out testing. data were submitted to split-plot anova and tukey’s test (α = 0.05). results: when the adhesives were used, the hydrophobic resin application (g3 and g4) increased fiber post retention in comparison to g2 only in the coronal third. for the other thirds, there was no difference between the adhesive methods. g5 presented the lowest values in the coronal and middle thirds, but it was similar to all other groups in the apical third. conclusion: post retention seemed not to benefit from the adhesive application in the apical region. i n t r o d u c t i o n some single-bottle etch-and-rinse adhesives have been demonstrated to be incompatible with chemically cured composites due to an adverse chemical interaction between unpolymerized acidic adhesive resin monomers and the basic tertiary amine catalyst in the composite1. this fact prevents composite polymerization and can create an area susceptible to fracture propagation. thus, different types of chemical co-initiators have been introduced to overcome this shortcoming2. however, this adverse interaction is only partially responsible for such an incompatibility. the other factor responsible for impairing bonding is the fact that some adhesives can behave as permeable membranes that permit the passage of fluids after polymerization3-5. in the presence of a slow-setting composite, water diffusion from dentin tends to be exacerbated. the water that migrates to the composite-adhesive interface can be trapped as water blisters, which might act as stress raisers that may result in debonding of the resin-dentin interface6. clinically, this incompatibility can occur during luting of endodontic posts. the use of dual-cure resin cements is recommended in order to provide a controlled working time, which cannot be controlled in self-cure cements. however, pfeifer et al.7 demonstrated that incompatibility between single-bottle adhesives and dual-cured cements can occur when the resin-based luting cements are not photoactivated. the most apical areas of the root canal rely mostly on the chemical cure rather than the physical cure8. during fiber post cementation, the low compliance of the canal space renders it impossible to accommodate resin cement polymerization shrinkage. thus, in addition to the low bond strength that can occur due to the high complexity of the adhesive procedures in the root canal, debonding can occur even in the absence of incompatibility. goracci et al.9 observed no difference in post retention with or without the application of a dentin bonding agent. for the authors, the main factor contributing to displacement resistance of the bonded and unbonded fiber posts was the sliding friction. this study examined the effect of using a chemical coinitiator and/or an additional coat of a more hydrophobic bonding resin used with a single-bottle etch-and-rinse adhesive on the push-out bond strength of a fiber post luted with a dual-cure resin cement. in addition, the importance of application of a dentin adhesive on post retention was evaluated. the tested null hypotheses were: braz j oral sci. october/december 2008 vol. 7 number 27 1654 coronal middle apical pb2.1 + sbmp (g3) 21.05(1.90)aa 14.36(5.39)ba 10.27(3.44)ba pb2.1 + sc + sbmp (g4) 19.52(2.56)aab 13.49(4.51)ba 11.12(3.38)ba prime&bond 2.1 (g1) 16.22(4.52)aab 10.20(2.72)bab 8.67(4.50)ba pb2.1 + sc activator (g2) 13.70(3.41)ab 8.87(2.86)bab 8.61(3.66)ba no adhesive (g5) 4.21(1.94)ac 4.39(1.98)ab 5.62(2.23)aa table 1 push-out bond strength values in mpa (sd) for the different luting procedures at the three root canal thirds pb = prime&bond 2.1; sbmp = scotchbond multi-purpose; sc = self-cure activator. means followed by different uppercase letters in the rows and different lowercase letters in columns differ significantly by tukey’s test at the 95 % confidence level. (1) the different luting procedures do not affect the pushout bond strength and (2) the push-out bond strength is not different at the different regions tested. material and methods twenty-five bovine incisors with mature apices and roots with no curves were selected for this study. the crowns were removed in order to obtain a remaining 17-mm long root segment. for the endodontic treatment, a step-back preparation technique was used with stainless-steel k-files and gates-glidden drills #2 to #4. all enlargement procedures were followed by irrigation with 2.5% sodium hypochlorite. the prepared root canals were obturated with gutta-percha cones using the lateral condensation technique and sealer-26 resin sealer (dentsply indústria e comércio ltda., petrópolis, rj, brazil). the filled roots were stored in 100% relative humidity for at least 72 h to allow the resin sealer to set. post-holes were prepared by standardization of the length at 9 mm and the preparation was performed with a size 5 largo drill. a 1.5-mm diameter glass fiber-reinforced composite post system reforpost® (angelus, londrina, pr, brazil) was used in this study. roots were randomly divided into three groups (n=5) according to the adhesive procedure to be used. the bonding procedures were carried out as follows: group 1: the canal walls were etched with 35% phosphoric acid (dentsply indústria e comércio ltda.) for 15 s, rinsed for 15 s and gently air-dried. excess water was removed from the post space with absorbent paper points. two coats of prime & bond 2.1 (dentsply indústria e comércio ltda.) adhesive system were applied, gently air dried and lightcured for 40 s. group 2: the bonding procedure was performed as described for group 1. however, before adhesive application, prime & bond 2.1 was mixed with the selfcure activator (dentsply indústria e comércio ltda.) at a 1:1 ratio. group 3: the bonding procedure was performed as described for group 1. afterwards, one coat of a hydrophobic adhesive resin (bonding agent, scotchbond multi-purpose: 3m/espe, st. paul, mn, usa) was applied, the excess was removed with paper points and the adhesive was light-cured for 40 s. group 4: the bonding procedure was performed as describe for group 2. after adhesive light-curing, the bonding agent of sbmp was applied and light-cured for 40s group 5: the resin cement was used with no adhesive. in all groups, the fiber posts were treated with a silane coupling agent and then the respective bonding agent of each group was applied onto the posts (the hydrophobic resin was used for group 3 and 4, while no adhesive was applied onto the posts of group 5) and light cured for 20 s. afterwards, the dual-cured resin cement enforce (dentsply indústria e comércio ltda.) was inserted into the root canal with a #40 lentulo spiral. the post was cemented into the root canal with light pressure, and excess luting material was removed. light activation was performed at the coronal portion of the root for 60 s. the specimens were stored in distilled water for 1 week at 37º c. after the storage period, the specimens were sectioned transversally. three 1.5-mm-thick slabs were obtained per root and identified as coronal, middle and apical specimens. each slab was positioned on the push-out jig, which was placed in a universal testing machine (model 4411, instron corp., canton, ma, usa) with a cell load of 500 n. load was applied at a crosshead speed of 0.5 mm/min until post dislodgement occurred. statistical analysis was performed using split-plot two-way anova followed by tukey’s post-hoc test at 95% confidence level. r e s u l t s split-plot anova showed that there were statistically significant differences for the factors “bonding procedure” (p<0.0001), “root region” (p<0.0001) and for interaction between factors (p = 0.0059). tukey’s test was used for the interactions between factors and the results are shown in table 1. in the coronal third, g3 presented higher mean push-out bond strengths than g2. the no-adhesive group (g5) presented the lowest values in this third. when adhesive was used, there was no statistically significant difference between the other groups. in the middle third, g3 and g4 (hydrophobic resin) presented higher values than g5 (no adhesive). however, there were no differences between adhesive procedures in this third. in the apical third, all groups presented similar push-out bond strengths. in all groups, except for g5, the coronal third presented higher braz j oral sci. 7(27):1653-1656 the effect of luting techniques on the push-out bond strength of fiber posts 1655 bond strength values than the middle and apical thirds, which were not significantly different from each other. in g5, there were no significant differences between the bond strengths recorded in the different regions evaluated. d i s c u s s i o n the first null hypothesis was rejected based on the results of the push-out bond strength test. it was demonstrated that the push-out bond strengths differed depending on the luting procedure used. the only difference between adhesive methods occurred in the coronal third, where hydrophobic resin adhesive application produced higher values of fiber post retention than the use of self-cure activator only. in this third, the resin cement benefits largely from both light and self-curing as they are readily accessible to the curing light10. thus, incompatibility is not expected to occur. one possible reason for this is a difference in adhesive degree of conversion and in adhesive layer quality. when the adhesive is applied, it can flow to the apical region and reduce the adhesive layer thickness in the coronal region. the solvent content in prime & bond 2.1 is approximately 80 wt%11. solvent evaporation might result in additional reduction of the adhesive layer thickness. the self-cure activator contains only 2% of chemical co-initiator and the remainder is solvent (ethanol and acetone). the use of self-cure activator increases the solvent content and can result in a thinner adhesive layer12. as the adhesive layer is thin, blisters can be formed even when the resin cement is light-cured due to rapid water movement across the adhesive13, reducing the bond strength. even though positive pulpal pressure is not present in endodontically treated teeth, dentin is still hydrated, which might compromise adhesion. rinsing the etchant with water during bonding procedures probably result in the retention of substantial amounts of water within the widened tubular entrances created by acid-etching14. this water is not completely removed with the use of paper points and may be responsible for the occurrence of fluid droplets in the adhesive layer. these droplets may act as stress raisers and contribute to crack propagation during the push-out testing, reducing post retention15. in addition, zheng et al.16 reported that thin adhesive layers are not adequately polymerized due to the inhibition caused by oxygen. thus, the additional application of a more hydrophobic bonding resin might have improved the polymerization degree of the adhesive layer and eliminated or reduced the permeability17-18. however, this effect did not occur when the hydrophobic resin and selfcure activator were used in the same procedure. this might be attributed to the low degree of conversion of the first adhesive layer formed by prime&bond 2.1 and the selfcure activator (unpublished data). the increased solvent content by use of self-cure activator hinders its evaporation from the adhesive layer19. residual solvents might impair monomer conversion, forming pores that can act as flawinitiating sites during push-out testing, and increase adhesive permeability. an interesting observation in the present investigation was that the highest values of fiber post retention occurred in the coronal third for all adhesives used. the apical and middle thirds presented the lowest values and no difference was observed among them. thus, the second null hypothesis was also rejected. the direct light curing of both adhesive and resin cement promoted higher bond strengths in the coronal third, independently of the adhesive method. moisture control and adhesive light activation are even more critical procedures in the apical region, which can contribute to the lower bond strengths to dentin. in addition, resin cement photoactivation is also compromised, and the self-cure component of the polymerization system is responsible for most or all polymerization reaction. in the absence of photoactivation, a chemical incompatibility between single-bottle adhesives and dual-cured cements may occur7. the slower curing rate of resin cement in these thirds can result in water flow to the cement/adhesive interface. the morphology of root dentin at different areas (coronal, middle and apical) and the hybridization ability of these areas may also help to explain the results. ferrari et al.20 demonstrated a reduction of bonding ability toward apical third of root canal dentin. this is related mainly to the density of dentin tubules in the coronal and middle thirds compared to the apical third. all these factors can be responsible for the reduction in fiber post retention. it is important to emphasize that incompatibility is not expected to occur when a hydrophobic adhesive resin is used, but low bond strength is still likely to occur in the more apical regions. in the coronal third, all adhesive procedures produced higher fiber post retention compared to the use of resin cement alone. however, in the middle third, adhesive application produced higher bond strengths than the nonadhesive treatment only when a hydrophobic resin was used. lack of differences among the groups was found only in the apical third. the highest push-out bond strength obtained with the use of hydrophobic adhesive in the middle third probably occurred because this last adhesive coat eliminated the incompatibility between the singlebottle adhesives and the dual-cured resin cement. however, the same effect was not observed in the apical third. in the groups that used the hydrophobic resin adhesive, the first adhesive layer was formed by prime & bond 2.1 or by its combination with the self-cure activator. due to adhesive flow, the adhesive layer is thicker in the apical third. as vapor pressure is reduced in this area, an increase in residual solvent is also expected to occur5. this fact can result in a reduced degree of conversion and in the presence of flaws in the adhesive layer. these two events probably reduced the bond strength to dentin. both bonding and sliding friction contribute to the resistance to dislocation of fiber posts during the push-out testing. as the bond strength is braz j oral sci. 7(27):1653-1656 the effect of luting techniques on the push-out bond strength of fiber posts 1656 very low in the apical region, it is likely that the sliding friction has the main contribution to fiber post dislocation resistance9. this friction results from the contact between the resin cement and the root canal walls. however, it is important to observe that the degree of conversion of the resin cement did not influence the sliding friction, since there was no difference between the three regions when only the resin cement was used. sigemori et al.8 demonstrated that the degree of conversion of enforce resin cement decreases remarkably as cavity depth increases. a high degree of conversion often results in improved physical and mechanical properties of resin cements. despite the improvement in the mechanical properties, higher degree of conversion increases the polymerization shrinkage. since friction occurs by contact, it is reasonable to assume that closer contact between resin cement and root dentin improves fiber post retention. the highest bond strengths were observed in the coronal third of the root. in this region, both sliding friction and adhesive bonding opposes fiber post dislocation. a decrease in bond strength was observed towards the apical regions and the post retention did not benefit from the adhesive application in the apical area. the clinical implications of the reduced bond strengths observed in the middle and apical regions have not yet been reported, but the development of materials with improved adhesive properties to the root canals would certainly improve the quality and reliability of restorative procedures in endodontically treated teeth. in conclusion, the highest bond strengths were observed in the coronal third. the application of a hydrophobic resin produced increased fiber post retention, except in the apical third. on the other hand, application of the self-cure activator did not provide any additional benefit to the interface. r e f e r e n c e s 1. sanares am, itthagarun a, king nm, tay fr, pashley dh. adverse surface interactions between one-bottle light-cured adhesives and chemical-cured composites. dent mater. 2001; 17: 542-56 2. ikemura k, endo t. effect on adhesion of new polymerization initiator systems comprising 5-monosubstituted barbituric acids, aromatic sulfinate amides, and tert-butyl peroxymaleic acid in dental adhesive resin. j appl polymer sci. 1999; 72: 1655-68. 3. tay fr, frankenberger r, krejci i, bouillaguet s, pashley dh, carvalho rm, et al. single-bottle adhesives behave as permeable membranes after polymerization. i. in vivo evidence. j dent. 2004; 32: 611-21. 4. van landuyt kl, snauwaert j, de munck j, coutinho e, poitevin a, yoshida y, et al. origin of interfacial droplets with one-step adhesives. j dent res. 2007; 86: 739-44. 5. yiu ck, hiraishi n, chersoni s, breschi l, ferrari m, prati c, et al. single-bottle adhesives behave as permeable membranes after polymerisation. ii. differential permeability reduction with an oxalate desensitiser j dent. 2006; 34: 106-16. 6. cheong c, king nm, pashley dh, ferrari m, toledano m, tay fr. incompatibility of self-etch adhesives with chemical/ dual-cured composites: two-step vs. one-step systems. oper dent. 2003; 28: 747-55. 7. pfeifer c, shih d, braga rr. compatibility of dental adhesives and dual-cure cements. am j dent. 2003; 16: 235-8. 8. sigemori rm, reis af, giannini m, paulillo la. curing depth of a resin-modified glass ionomer and two resin-based luting agents. oper dent. 2005; 30: 185-9. 9. goracci c, fabianelli a, sadek ft, papacchini f, tay fr, ferrari m. the contribution of friction to the dislocation resistance of bonded fiber posts. j endod. 2005; 31: 608-12. 10. faria e silva al, arias vg, soares le, martin aa, martins lr. influence of fiber-post translucency on the degree of conversion of a dual-cured resin cement. j endod. 2007; 33: 303-5. 11. reis af, oliveira mt, giannini m, de goes mf, rueggeberg fa. the effect of organic solvents on one-bottle adhesives’ bond strength to enamel and dentin. oper dent. 2003; 28: 700-6. 12. de silva al, lima da, dias de souza gm, dias ct, paulillo la. influence of additional adhesive application on the microtensile bond strength of adhesive systems. oper dent. 2006; 31: 562-8. 13. tay fr, pashley dh, suh bi, hiraishi n, yiu ck. water treeing in simplified dentin adhesives deja vu? oper dent. 2005; 30: 561-79. 14. chersoni s, acquaviva gl, prati c, ferrari m, grandini s, pashley dh, et al. in vivo fluid movement trough dentin adhesives in endodontically treated teeth. j dent res. 2005; 84: 223-7. 15. faria e silva al, casselli ds, ambrosano gm, martins lr. effect of the adhesive application mode and fiber post translucency on the push-out bond strength to dentin. j endod. 2007; 33: 1078-81. 16. zheng l, pereira pn, nakajima m, sano h, tagami j. relationship between adhesive thickness and microtensile bond strength. oper dent. 2001; 26: 97-104. 17. carvalho rm, pegoraro ta, tay fr, pegoraro lf, silva nr, pashley dh. adhesive permeability affects coupling of resin cements that utilize self-etching primers to dentine. j dent. 2004; 32: 55-6. 18. king nm, tay fr, pashley dh, hashimoto m, ito s, brackett ww, et al. conversion of one-step to two-step self-etch adhesives for improved efficacy and extended application. am j dent. 2005; 18: 126-34. 19. cho b, dickens sh. effect of the acetone content of single solution dentin bonding agents on the adhesive layer thickness and the microtensile bond strength. dent mater. 2004; 20: 107-15. 20. ferrari m, mannocci f, vichi a, cagidiaco mc, mjör ia. bonding to root canal: structural characteristics of the substrate. am j dent. 2000; 13: 255-60. braz j oral sci. 7(27):1653-1656 the effect of luting techniques on the push-out bond strength of fiber posts untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8652904 volume 17 2018 e18139 original article 1 department of oral and maxillofacial surgery, erasto gaertner hospital, curitiba, paraná, brazil. 2 universidade federal do paraná, curitiba, paraná, brazil. 3 department of oral and maxillofacial surgery of the municipal hospital and maternity são josé dos pinhais, paraná, brazil. corresponding author: bruna da fonseca wastner rua guilherme tragante, 377, tarumã, curitiba, paraná, brazil, 82800-090. email: bru.wastner@hotmail.com received: december 06 2017 accepted: may 11, 2018 odontogenic abscess with severe and fast evolution in a young and healthy patient bruna da fonseca wastner1, salmo cortiglio 1, joana letícia vendruscolo 2, josé luis dissenha3 deep cervical abscesses are infections of the different spaces on the the neck that may have a mild or severe outcome and requires fast and assertive approach. the most usual cases of severe infections are associates with comorbidities and older patients, but sometimes, in rare cases, it may affect the younger and healthier. aim: the purpose of this is to communicate shortly a case of a severe cervical abscess with fast evolution in a 28 years old patient, to highlight the possibility of this outcome even in unexpected cases. key-words: abscess. infection. neck. 2 wastner et al. introduction deep cervical abscesses are defined by the presence of pus in regions of the head and neck, and can reach separately or together the spaces: retropharyngeal, peritonsillar, masseteric, submandibular, parapharyngeal, pterygopalatine and parotid, among others. in some cases, especially when the treatment is not adequate, they can progress both superior and inferior, increasing the risk of death. a rapid and accurate approach is required in order to avoid complications such as airway obstruction, necrotizing fasciitis, jugular vein thrombosis, aspiration pneumonia and carotid artery thrombosis (1). it is observed that even with the use of broad-spectrum antimicrobials and surgical drainage, severe complications may occur, mainly, but not exclusively, in patients with comorbidities (2). the literature shows that airway obstruction forced emergency tracheostomy in 7.9% of the cases and in those individuals with retropharyngeal spaces and mouth floor, 75% presented this need (3). the trismus due to the involvement of the masticatory muscles may also be a predisposing factor, even if there is no respiratory distress. mortality rates in these cases are described as close to 2% (2,3). case report our patient was treated in the emergency room of the municipal hospital and maternity são josé dos pinhais, paraná, brazil, with an odontogenic abscess associated with the left lower molars. intraoral exam showed pericoronitis in the third left molar and a carious lesion in the second left molar. despite this, patient had a good oral hygiene and absence of periodontal disease. the patient presented hyperthermia and edema in the left submandibular region without a fluctuation point, with dysphagia and dyspnea. he referred that started to feel pain in the region in the day before and, when he woke up on the next day, the swelling was present. he was classified as asa i, have no comorbidities and all serologies came negative. initially, empiric intravenous antibiotic therapy was used with cefazolin and metronidazole, but the patient evolved with increased edema and worsened general condition. it was surgically approached through extraoral drainage of two localized colletions, one submandibular and one sublingual. after a few hours, the patient got worse, presenting cervical erythema extending to the clavicle. at this time, the triple antimicrobial regimen was instituted, with the addition of gentamicin, thinking of a more severe infection with gram negative aerobes. however, a few hours later, the erythema reached the lower sternal region and the patient complained of dyspnea and chest pressure. computed tomography of the neck and thorax showed a purulent collection medial to the left sternocleidomastoid muscle, without compromising the mediastinum and a purulent collection in the retropharyngeal space and on the deep neck space, compromising the airway (figures 1 and 2). the patient underwent a new surgical drainage. in conjunction with the thoracic surgeon, cervicotomy was performed to access the collection medial to the left sternocleidomastoid muscle and abundant irrigation with a 0.9% sodium chloride heated solution, maintaining two penrose drains. the wound was left open for healing by second intention (figure 3). in the same surgical procedure, the infected tooth was extracted. culture of the secretion collected in the trans-operative revealed 3 wastner et al. figure 1. computed tomography of the neck and thorax in sagittal view showing a purulent collection in the retropharyngeal space and on the deep neck space, compromising the airway. figure 2. computed tomography of the neck and thorax in coronal view showing a purulent collection medial do the sternocleidomastoid muscle. figure 3. transoperatory view showing the surgical wound and the poor aspect of the fascia, that appears with a pale color. 4 wastner et al. an abundance of gram-positive cocci, which lead us to withdraw gentamicin from the scheme because it was not effective in this situation. them, the antibiotics were changed to ceftriaxone and clindamycin. after this approach, the patient had a significant and progressive improvement of the condition, with regression of erythema and cervical edema, improvement of dyspnea and dysphagia. pre-discharge laboratory tests showed normalization of laboratory tests. the patient did not show impairment of renal function or hydroelectrolytic imbalance. he remained hospitalized for 14 days, feeding through a nasoenteral tube, breathing in ambient air without effort. the widest cervical wound was covered daily with special dressings for 14 days, with complete closure. the patient remains well and had no other similar episodes. acknowledgements the authors deny any conflict of interests. references 1. ogle oe. odontogenic infections. dent clin north am. 2017 apr;61(2):235-252. doi: 10.1016/j.cden.2016.11.004. 2. brito tp, hazboun im, fernandes fl, bento lr, zappelini cem, chone ct, et al. deep neck abscesso: study of 101 cases. braz j otorhinolaryngol. 2017 may jun;83(3):341-348. doi: 10.1016/j.bjorl.2016.04.004. 3. huang tt, liu tc, chen pr, tseng fy, yeh th, chen ys. deep neck infection: analysis of 185 cases. head neck. 2004 oct;26(10):854-60. 1http://dx.doi.org/10.20396/bjos.v16i0.8650497 volume 16, number 3 2017 e17052 original articlebjos isoenzyme genotyping and phylogenetic analysis of oxacillin-resistance staphylococcus aureus isolates marcelo fabiano gomes boriollo1,2,*, manoel francisco rodrigues netto1,2, jeferson júnior da silva1, thaísla andrielle da silva2, maysa eduarda de castro2, júlio césar elias2 and josé francisco höfling1 *corresponding author: marcelo f. g. boriollo, universidade estadual de campinas, faculdade de odontologia de piracicaba, departamento de diagnóstico oral, laboratório de microbiologia e imunologia, avenida limeira, 901 – cep 13414-903, piracicaba, são paulo, brasil. telephone / fax: +55 019 21065250 / 2106-5251 / 2106-5355. e-mail marcelofgb@yahoo.com.br received: may 25, 2017 accepted: august 09, 2017 1 universidade estadual de campinas, faculdade de odontologia de piracicaba, departamento de diagnóstico oral, laboratório de microbiologia e imunologia, piracicaba, são paulo, brasil. 2 universidade josé do rosário vellano, faculdade de medicina, laboratório de farmacogenética e biologia molecular & centro de pesquisa em ciência animal, departamento de patologia e farmacologia animal, alfenas, minas gerais, brasil. aim: the propagation of s. aureus in hospital and dental environments is considered an important public health problem since resistant strains can cause serious infections in humans. the genetic variability of 99 oxacillin-resistant s. aureus isolates (orsa) from the dental patients (oral cavity) and environments (air) was studied by isoenzyme genotyping. methods: s. aureus isolates were studied using isoenzyme markers (alcohol dehydrogenase, sorbitol dehydrogenase, mannitol-1-phosphate dehydrogenase, malate dehydrogenase, glucose dehydrogenase, d-galactose dehydrogenase, glucose-6-phosphate dehydrogenase, catalase and α/β-esterase) and genetic (nei’s statistics) and cluster analysis (upgma algorithm). results: a highly frequent polyclonal pattern was observed in this population of orsa isolates, suggesting various sources of contamination or microbial dispersion. genetic relationship analysis showed a high degree of polymorphism between the strains, and it revealed three taxa (a, b and c) distantly genetically related (0.653≤dij≤1.432) and fifteen clusters (i to xv) moderately related (0.282≤dij<0.653). these clusters harbored two or more highly related strains (0≤dij<0.282), and the existence of microevolutionary processes in the population of orsa. conclusion: this research reinforces the hypothesis of the existence of several sources of contamination and/or dispersal of orsa of clinical and epidemiologically importance, which could be associated with carriers (patients) and dental environmental (air). keywords: oxacillin-resistant staphylococcus aureus. genetic variability. propagation dynamics. mlee. clustering analysis. mailto:marcelofgb@yahoo.com.br 2 boriollo et al. introduction the dissemination of s. aureus is considered an important public health problem because resistant strains can cause serious infections, especially in children and hospital patients1–3. dentists treat a wide variety of patients, a fact that exposes these health professionals to people colonized or infected with resistant microorganisms2,4. high rates of resistance to antibiotics used during odontological prophylaxis have been detected in pathogens associated with bacterial endocarditis, for example, s. aureus5–8. strains of s. aureus can be disseminated during dental treatment and occasionally lead to the contamination and infection of patients and dentists. certain aspects of odontological practice can contribute to the dissemination of microorganisms9,10. the skin, environment and instruments can be contaminated with saliva, blood or debris during routine odontological treatment10,11. several researchers have noted an increase in the amount of microorganisms present during clinical procedures in odontological environments, suggesting contamination from aerosols, especially when high-speed devices or ultrasonic scalers are used12,13. among the species identified in microbiological studies, streptococci of the group viridans and staphylococcus spp. are the most prevalent microorganisms found on surfaces of odontological equipment12–15, including methicillin-resistant s. aureus, which has been detected on odontological operatory surfaces, air-water syringes and recliner chairs16. additionally, bacteria and fungi were significantly more frequent in dentist’s hand with rings than those without rings, being staphylococcus aureus, escherichia coli and candida albicans highly prevalent among the isolated potentially pathogenic microorganisms10. phenotypic methods (biotyping, serotyping, bacteriophage or bacteriocin typing and antimicrobial susceptibility profiles) and genotypic [pulsed-field gel electrophoresis (pfge) and other methods based on the restriction of genomes, analysis of plasmids, typing methods based on polymerase chain reaction (pcr)] of microbiological characterization have elucidated the relationship and the distribution of human pathogens, which is considered essentially important for the epidemiology and control of hospital infections17. isoenzymatic typing [multilocus enzyme electrophoresis (mlee)] has been used for several decades as a “gold standard” in population genetics studies of eukaryotes18–20 and systematic studies21, as well as in large-scale studies for determining the genetic diversity and structure of natural populations of a variety of bacteria species22–24 and fungi25–27. this method represents an invaluable complement to the more recently developed molecular typing methods, particularly for largescale epidemiological studies28. in addition, mlee possesses excellent typability (i.e., the percentage of different strains obtained) and reproducibility (i.e., the percentage of strains that display the same results in repetitive tests) and is associated with great discriminatory power (i.e., the ability to differentiate unrelated strains)23–33. epidemiological studies are necessary for the implementation of effective prevention measures. genotyping of strains from patients in odontological clinical treatment and their environments can provide information that can potentially help control and prevent the spread of s. aureus involved in the processes of colonization and human infection. this scientific research evaluated the genetic diversity of natural populations of oxacillin-resistance s. aureus dental isolates (dental patients and environments). the 3 boriollo et al. frequency of strains and operational taxonomic groups (taxon and cluster) and possible epidemiological correlations were investigated by using isoenzymatic markers (mlee) and genetic and grouping analysis. material and methods microbiological sampling a total of ninety-nine bacterial samples of oxacillin-resistant s. aureus (orsa), from the bacteria collection of the laboratório de farmacogenética e biologia molecular, faculdade de ciências médicas and centro de pesquisa e pós-graduação (unifenas), alfenas, mg, brazil, were kindly provided and used for the present research. these samples were previously isolated from dental patients and clinical environment (air) (faculdade de odontologia, unifenas) (approved by committee of ethics in human research, protocol no. 174/2009) and characterized using microbiological methods of identification [i.e., stain of gram, growth in chromogenic medium chromagar staphylococcus aureus®, catalase test, coagulase test (coagu-plasma, laborclin produtos para laboratórios ltda.), clumping factor a test (staphy test, probac do brasil produtos bacteriológicos ltda.), fermentation of mannitol test and dnase test]34 and antimicrobial susceptibility testing (i.e., diffusion disk and confirmatory triage for resistance to oxacillin)35. multilocus enzyme electrophoresis (mlee) preparation of cell extracts, electrophoresis procedures, enzyme staining and genetic interpretation of mlee patterns were performed according to methods previously reported23,25,26,31. to ensure reproducibility of the results, the cellular enzymes of the s. aureus atcc® 25.923tm reference strain were systematically used. a total of nine metabolic enzymes (table 1) was investigated using systems and solutions previously established for the mlee analyses23,25,26,31. the discriminatory power of the mlee method was determined using the numeric index of discrimination (d), in accordance with the probability that two unrelated isolates sampled from a test population are classified into different types (i.e., strains or ets)25,26. grouping analysis the statistic of nei (1972)36 was used to estimate the genetic distance (dij) among the isolates/strains (ets) of oxacillin-resistant s. aureus. the interpretation in terms of enzyme loci infers that, on average, from zero to an infinite number of allele substitutions are detected (for electrophoresis) for every 100 existing loci from a common ancestral strain. a tree with two-dimensional classification (dendrogram), based on the matrix dij, was generated by the grouping sahn method (sequential, agglomerative, hierarchic, nonoverlapping clustering methods) and the upgma algorithm (unweighted pair-group method using an arithmetic average). once mlee provided all levels of relationship that must be solved by dna fingerprinting methods (i.e., identification of the same strain between independent isolates, identification of microevolutionary changes in the same strain, identification of clusters of moderately related isolates and identification of completely unrelated isolates), a threshold (average value: dij ) in the dendrogram was established to identify identical isolates and highly related isolates, clusters 4 boriollo et al. table 1. systems and solutions utilized for the mlee analyses of the s. aureus metabolic enzymes. enzyme compound for staining ec number name symbol substrate buffer salt coenzyme dye and catalyser 1.1.1.1. alcohol dehydrogenase adh ethanol (3ml) isopropanol (2ml) 200mm tris-hcl ph 8.0 (q.s.p. 50ml) a nad 1% (2ml) pms 1% (500µl) mtt 1.25% (1ml) 1.1.1.17 mannitol-1phosphate dehydrogenase m1p mannitol 1-phosphate (5mg) 200mm tris-hcl ph 8.0 (q.s.p. 50ml) a nad 1% (2ml) pms 1% (500µl) mtt 1.25% (1ml) 1.1.1.37. malate dehydrogenase mdh 2m malic acid (6 ml) b 200mm tris-hcl ph 8.0 (q.s.p. 50ml) a nad 1% (2ml) pms 1% (500µl) mtt 1.25% (1ml) 1.1.1.47 glucose dehydrogenase gdh d-glucose (500 mg) 200mm tris-hcl ph 8.0 (q.s.p. 50ml) a nad 1% (2ml) pms 1% (500µl) mtt 1.25% (1ml) 1.1.1.48 d-galactose dehydrogenase gldh galactose (450ml) tris-hcl 100mm ph 8.4 (q.s.p. 50ml) c nad 1% (1ml) pms 1% (500µl) mtt 1.25% (1ml) 1.1.1.49 glucose-6phosphate dehydrogenase g6pdh glicose-6phosphate disodium salt (100 mg) 200mm tris-hcl ph 8.0 (q.s.p. 50ml) a 100mm mgcl2 (1 ml) d nadp 1% (1ml) pms 1% (500µl) mtt 1.25% (1ml) 1.11.1.6 catalase cat e 3.1.1.1. αand β esterase est αand βnaphthyl acetate (1% solution in acetone) (1.5ml) 50mm sodium phosphate ph 7.0 (q.s.p. 50ml)f fast blue rr salt (25 mg) electrode buffer: tris–citrate ph 8.0 [83.2 g of c4h11no3 (tris), 33.09 g of c6h8o7.h2o (citric acid), 1 liter of h2o]; gel buffer: electrode buffer diluted 1:29. a 24.2 g of c4h11no3 (tris), 1 liter of h2o (ph adjusted with hcl); b 26.8 g of c4h6o5 (dl-malic acid) and 16g of naoh in 100 ml of h2o (caution: potentially explosive reaction); c 12.1 g of c4h11no3 (tris), 1 liter of h2o (ph adjusted with hcl); d 2.03 g of mgcl2.6hcl (magnesium chloride) in 100 ml of h2o; e incubate gel slice for 30 min at 0 °c in 50 ml of 0.1 m sodium phosphate ph 7.0 buffer, then pour off solution, and immerse it in 50 ml of 1.5% potassium iodide solution (ki) for 2 min. therefore, rinse gel slice with water, and immerse it in 50 ml of 0.03% hydrogen peroxide (h2o2) solution. mix gently and remove stain solution when white zones appear on dark-blue background; f sodium phosphate buffer ph 7.0: mix equal parts of 27.6 g of nah2po4.h2o (monobasic) in 1 liter of water and 53.6 g of na2hpo4.7h2o in 1 liter of water, then dilute the mixture 1:25 with water. 5 boriollo et al. (0 ≤ dij < dij ) and taxa (singular taxon, i.e., taxonomic group of any nature or rank) (dij ≥ dij ). correlation coefficients based on the pearson product-moment was used as a measure of agreement between the genetic distance values implicit in the upgma dendrogram and the original explicit values in the matrix of genetic distance dij. all these analyses were performed using the ntsys-pc program version 2.125,26,32. results the electrophoretic isoenzyme patterns of oxacillin-resistant s. aureus isolates were reproducible in different gels after three repetitions of each electrophoretic run. according to haploid nature of s. aureus, these patterns displayed the following characteristics (table 2): all 30 enzymatic loci were polymorphic for one, two, three, four, five and six alleles (one allele: cat-2, β-est-3, gdh-2, g6pdh-3, sdh-1; two alleles: adh-1, cat-1, α-est-1, β-est-2, m1p-2, m1p-3, mdh-3 and sdh-2; three alleles: gdh-3, mdh-1, sdh-3 and mdh-4; four alleles: adh-2, adh-3, α-est-3, β-est-1, g6pdh-2 and mdh-2; five alleles: m1p-1, gdh-1, gldh-2 and g6pdh-4; six alleles: α-est-2, g6pdh-1 and gldh-1). the average number of alleles per polymorphic locus was equal to 3.16 ±1.62. the existing combination in 30 enzymatic loci revealed 79 strains (ets) [79% of the isolates, including the reference strain of s. aureus atcc® 25.923, that is, identical isolates that match the same strain et (dij = 0.000)]. based on the genetic interpretation of electrophoretic patterns, the discriminatory power of the mlee genotyping method was equal to 0.99051, that is, there was a 99% probability that two non-related isolates of s. aureus, from the test population, would be classified as distinct strains ets. the genetic diversity among the strains of oxacillin-resistant s. aureus was evaluated using the matrix dij and the upgma dendrogram (figure 1). considering the threshold obtained (0 ≤ dij < 0.282: isolates identical or highly related; 0.282 ≤ dij < 0.653: isolates moderately related; dij ≥ 0.653: isolates genetically distantly related), the results indicated three main groups or taxa, designated a, b and c. taxon a comprised nine isolates/strains (et2 g22.5, et6 g11.66, et8 g11.86, et9 g11.129, et22 g18.66, et24 g18.8, et32 g18.46, et68 g11.36 and et76 g18.137) and eight moderately related clusters (from i to viii; a total of 60 isolates 60% or 43 ets 54.4%): • cluster i: thirteen identical and/or highly related isolates, including the reference strain; 11 highly related strains (et1 atcc 25.923 and g13.172, et41 g18.100 and g20.44, et43 g20.12, et44 g18.155, et45 g22.55, et46 g22.22, et47 g18.51, et48 g15.100, et49 g16.140, et50 g13.165 and et51 g15.40). • cluster ii: five identical and/or highly related isolates; two highly related strains (et36 g18.20 and et39 g18.110, g18.111, g18.166 and g18.156). • cluster iii: three highly related isolates; two highly related strains (et13 g11.135, et15 g13.47 and et21 g18.95). • cluster iv: three highly related isolates; three highly related strains (et3 g6.15, et5 g5.38 and et4 g6.12). • cluster v: four identical and/or highly related isolates; three highly related strains (et10 g11.139, et11 g11.58 andg11.19 and et12 g11.39). 6 boriollo et al. table 2. allele profiles of oxacillin-resistant s. aureus isolates (79 strains/ets), sourced from odontological clinical and environment samples, and obtained for the genetic interpretation of mlee patterns. discriminatory power (d) = 0.99051. (-) allele null. et alleles in 30 enzyme loci adh sdh m1p mdh gdh gldh g6pdh cat α-est β-est 1 2 3 1 2 3 1 2 3 1 2 3 4 1 2 3 1 2 1 2 3 4 1 2 1 2 3 1 2 3 et1 2 2 2 1 1 1 1 1 2 5 1 et2 2 2 2 1 6 1 2 5 2 et3 2 2 1 1 2 1 2 3 2 2 5 1 et4 2 2 1 1 2 1 2 3 2 2 6 4 2 et5 2 1 1 4 1 2 3 2 2 5 1 et6 2 1 2 1 2 1 1 2 6 2 4 2 et7 2 1 2 1 4 2 4 et8 2 1 2 1 1 1 2 1 et9 2 1 2 1 4 1 2 1 et10 4 2 2 5 2 4 1 5 1 5 1 1 2 5 1 et11 4 2 4 4 5 5 3 2 3 1 et12 4 2 4 4 3 5 1 3 2 3 1 et13 1 2 3 2 2 5 et14 3 5 1 4 2 5 et15 1 3 2 2 5 1 1 et16 1 5 3 1 2 2 et17 2 1 4 2 et18 1 4 2 2 et19 2 3 1 3 6 2 et20 2 2 1 3 2 5 1 2 et21 2 3 1 3 1 2 5 et22 3 3 4 1 2 5 et23 1 3 2 2 1 2 et24 1 3 2 2 2 5 et25 2 3 1 4 5 6 2 1 et26 1 4 1 1 2 2 2 et27 2 2 2 3 1 1 1 2 1 1 et28 2 2 5 2 3 1 1 1 2 1 1 et29 2 2 2 1 3 1 1 1 2 2 1 et30 2 2 2 1 1 1 2 2 1 et31 2 2 2 1 1 1 2 2 1 et32 3 2 2 1 2 1 1 2 2 1 et33 2 2 2 1 1 1 1 2 2 1 et34 2 2 2 1 1 1 1 2 1 et35 2 2 2 1 3 1 1 1 2 3 et36 2 2 5 2 1 1 1 1 2 2 6 3 et37 2 2 2 1 3 1 1 2 2 3 et38 2 2 5 2 1 1 1 1 2 2 3 et39 2 2 5 2 3 1 1 1 2 6 3 et40 2 2 5 2 1 1 1 1 2 1 3 et41 2 2 2 1 1 1 1 2 6 3 et and [-] correspond to electrophoretic type (bacterial strain) and allele null, respectively. continue 7 boriollo et al. table 2. allele profiles of oxacillin-resistant s. aureus isolates (79 strains/ets), sourced from odontological clinical and environment samples, and obtained for the genetic interpretation of mlee patterns. discriminatory power (d) = 0.99051. (-) allele null. continuation et alleles in 30 enzyme loci adh sdh m1p mdh gdh gldh g6pdh cat α-est β-est 1 2 3 1 2 3 1 2 3 1 2 3 4 1 2 3 1 2 1 2 3 4 1 2 1 2 3 1 2 3 et42 2 2 2 1 1 1 2 1 3 et43 2 2 2 3 1 1 1 2 5 1 et44 2 2 2 1 1 3 2 5 1 et45 2 2 2 1 1 1 2 5 1 et46 2 2 2 1 1 1 1 2 5 1 et47 2 2 2 1 1 3 1 2 5 1 et48 2 2 2 1 1 1 2 5 3 1 et49 2 2 2 2 1 1 1 2 4 3 et50 2 2 2 2 1 1 1 2 5 1 et51 2 2 2 2 1 1 1 2 4 1 et52 2 1 1 2 et53 4 2 3 2 2 3 1 5 5 5 2 1 et54 2 1 1 2 2 et55 2 3 2 et56 1 1 2 2 et57 1 3 1 2 2 et58 1 2 et59 1 3 2 et60 3 1 1 5 5 2 et61 1 1 2 4 1 1 2 et62 2 et63 1 3 2 1 2 4 et64 2 2 et65 1 5 2 et66 4 1 2 et67 2 1 et68 4 5 1 2 2 et69 2 2 et70 1 4 2 et71 1 2 1 2 et72 4 5 2 et73 2 1 2 et74 1 1 1 5 2 et75 1 1 3 1 et76 1 1 3 1 5 3 et77 2 2 1 1 1 et78 2 3 1 et79 2 1 1 et and [-] correspond to electrophoretic type (bacterial strain) and allele null, respectively. 8 boriollo et al. • cluster vi: eight identical and/or highly related isolates; six highly related strains (et27 g18.33, g18.104 and g20.45, et29 g18.14, et30 g19.43, et31 g22.48, et33 g18.135 and et34 g20.14). • cluster vii: three highly related isolates; three highly related strains (et35 g18.45, et37 g19.10 and et42 g18.126). figure 1. genetic diversity of 99 oxacillin-resistant s. aureus isolates sourced from a population of odontological clinical and environment samples. the upgma dendrogram (rjk = 0.79908) was generated from a matrix of genetic distance dij (nei, 1972). et1 et1 et45 et46 et50 et51 et41 et41 et48 et49 et43 et44 et47 et36 et39 et39 et39 et39 et13 et15 et21 et3 et5 et4 et24 et76 et2 et22 et10 et11 et11 et12 et6 et68 et8 et27 et27 et27 et29 et30 et33 et31 et34 et35 et37 et42 et32 et9 et28 et28 et28 et38 et40 et40 et40 et40 et40 et40 et40 et40 et14 et60 et16 et20 et61 et66 et70 et70 et18 et55 et59 et65 et74 et58 et64 et71 et73 et19 et72 et25 et53 et52 et69 et54 et56 et62 et62 et67 et67 et23 et26 et57 et63 et7 et17 et75 et75 et78 et77 et79 cluster i clusters and/or isolates/ets moderately related dij = 0.282 ± 0.371 dij = 0.653 isolates/ets identical and/or highly related clusters and/or isolates/ets distantly genetically related a b c cluster ii cluster iii cluster iv cluster v cluster vi cluster vii cluster viii cluster x cluster ix cluster xi cluster xii cluster xiii cluster xiv cluster xv 0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700 0.800 0.900 0.100 1.100 1.200 1.300 1.400 1.500 1.600 1.700 1.800 9 boriollo et al. • cluster viii: twelve identical and/or highly related isolates; three highly related strains (et28 g18.94, g18.9 and g21.1, et38 g18.55, et40 g15.52, g16.40, g18.142, g18.26, g18.50, g18.74, g22.63 and g19.21). taxon b comprised eight isolates/strains (et14 g13.112, et60 g14.71, et16 g12.14, et25 g18.89, et53 g14.126, et56 g15.159, et57 g13.41 and et63 g16.258) and six moderately related clusters (from ix to xiv; a total of 33 isolates 33% or 30 ets 37.9%): • cluster ix: thirteen identical and/or highly related isolates; four highly related strains (et20 g18.15, et61 g13.120, et66 g5.31, et70 g13.174 and g14.199). • cluster x: nine highly related isolates; nine highly related strains (et18 g13.51, et55 g17.68, et58 g17.86, et59 g16.269, et64 g15.131, et65 g17.13, et71 g15.64, et73 g17.128 and et74 g17.62). • cluster xi: two highly related isolates; two highly related strains (et19 g11.13 and et72 g17.63). • cluster xii: three highly related isolates; three highly related strains (et52 g16.88, et54 g16.49 and et69 g13.142). • cluster xiii: four identical and/or highly related isolates; two highly related strains (et62 g17.115 and g16.167 and et67 g11.131 and g11.32). • cluster xiv: two highly related isolates; two highly related strains (et23 g18.91 and et26 g18.10). taxon c comprised three isolates/strains (et7 g11.96, et17 g12.13 and et79 g22.64) and only one moderately related cluster (xv; a total of six isolates 6% or five ets 6.3%): • cluster xv: four identical and/or highly related isolates; three highly related strains (et75 g17.42 andg18.124, et78 g20.48 and et77 g19.44). discussion in this study, the enzyme electrophoretic profiles of oxacillin-resistant s. aureus isolates on different gels were reproducible after three repetitions of each electrophoretic run. the discriminatory capacity (i.e., 99% probability that two unrelated isolates sampled from a population test are classified in different strains ets) of the mlee method, based on genetic interpretation of electrophoretic enzyme patterns, was also observed (i.e., the combination of existing alleles on 30 enzyme loci revealed 79 ets). once again, mlee proved to be a powerful tool for the typing of s. aureus in epidemiological studies. these results are in agreement with previously reported data on the discriminatory power and reproducibility of the mlee method as applied to bacteria and yeasts of medical importance23–27,31, but the discriminatory power was higher than the values reported for s. aureus by other groups of researchers29,30. genetic polymorphism has been found in almost all natural populations and at all levels of genetic organization, from genotype characteristics to phenotypic traces. the possible reasons of its existence have been the subject of a long debate in the population genetics and molecular evolution fields37,38. s. aureus is a heterogeneous species (polymorphic)39 that has been observed to have a clonal population structure40. therefore, it is believed that s. aureus does not suffer extensive recombination, diversifies extensively by nucleotide mutations and displays a high degree of linkage disequilibrium (non-random asso10 boriollo et al. ciations between gene loci). a particular structural gene locus is defined as polymorphic when the frequency of its more common allele presents a value below 0.99 (99%). some of the measures used to quantify this variability in populations of organisms are the allele and gene frequencies, the percentage of polymorphic loci, the average number of alleles per locus and heterozygosity41. in this study, quantitative and qualitative variations of polymorphic loci (30 100% polymorphic enzyme loci to one, two, three, four, five and six alleles) and the average number of alleles per polymorphic locus (3.16 ±1.62) were observed in the population of oxacillin-resistant s. aureus. these variations have been observed in several studies of genetic diversity of populations of s. aureus obtained from human and bovine sources29,30,42,43. in addition, the genetic polymorphism observed in the population of oxacillin-resistant s. aureus isolates revealed a highly frequent polyclonal pattern and infrequent monoclonal pattern, suggesting various sources of contamination or microbial dispersion from an epidemiological point of view. the genetic relationship between the oxacillin-resistant s. aureus strains was determined by using the statistic dij of nei (1972) and the upgma dendrogram 25,26,32,36, which displayed a value rjk acceptable (rjk ~ 0.8) based on the correlation coefficient of pearson’s product-moment [i.e., good agreement between the elements dij (matrix of genetic distance) and cjk (correlation matrix derived from upgma dendrogram)]. a high degree of genetic polymorphism (0.000 ≤ dij ≤ 1.705) was observed between the orsa isolates (i.e., on average, from zero to 170.5 allele substitutions were detected in each 100 loci from a common ancestor strain). these isolates were allotted to three taxa (a, b and c), which were distantly genetically related (0.653 ≤ dij ≤ 1.705). taxon a presented a larger number of isolates, strains or clusters of bacteria (60 isolates 60%, 43 ets 54.4% and eight clusters i-viii), followed by taxon b (33 isolates 33%, 30 ets 37.9% and six clusters ix-xiv) and taxon c (six isolates 6%, five ets 6.3% and one cluster xv). each taxon presented one or more clusters and/or moderately related isolates (0.282 ≤ dij < 0.653). in turn, these clusters harbored two or more identical and/or highly related isolates (0 ≤ dij < 0.282). considering that highly related isolates/strains highly come from a common ancestor [i.e., descendants have suffered microevolutions and adaptations as a result of recombination (not extensive), nucleotide mutations and non-random association between gene loci (linkage disequilibrium)39,40, these results suggest the existence of microevolutionary processes in the population of oxacillin-resistant s. aureus, as demonstrated in each cluster (i.e., on average, from zero to < 28.2 allele substitutions were detected in each 100 loci from a common ancestor strain). however, these data reinforce the hypothesis of the existence of several sources of contamination and/or dispersal of oxacillin-resistant s. aureus of clinical and epidemiologically importance, which could be associated with carriers (patients) and dental environmental (air). these epidemiological investigations have also been a goal of our research group and contribute to (i) knowledge about the dynamics of the spread and retention of s. aureus strains resistant to antibiotics in hospital and odontological environments (i.e., surgical devices, dental instrumentation, various surfaces, air and other) and (ii) the implementation or restructuring of containment barriers, use of personal protective equipment, means of identification and periodic treatment from professionals carriers of microorganisms (nasal cavities, oral and oropharyngeal, perineum and armpits), techniques and devices for air purification, hygiene and more efficient prophylaxis. 11 boriollo et al. certain aspects of practicing dentistry may contribute to the transmission of microorganisms9. the skin, environment, and instruments can be contaminated with saliva, blood or organic debris during routine dental treatment11. in the dental environment, investigators have observed an increase in the amount of microorganisms during clinical procedures, suggesting contamination by aerosols, especially when high-speed handpieces or ultrasonic scalers are used12,13,15. among the species identified in microbiological studies, streptococcus viridans and staphylococcus spp. are the most prevalent microorganisms found on the surfaces of dental equipment12–15. in addition, the high-speed drills and cavitrons used in dental offices generate aerosols and droplets that are contaminated with blood and bacteria and may be a route for the transmission of diseases such as sars (severe acute respiratory syndrome), tuberculosis, and legionnaires’ disease44–46. methicillin-resistant s. aureus (mrsa) has frequently been detected on surfaces in dental operatories, including the air-water syringe and reclining chair16. nosocomial infections or the colonization of mrsa occurred in eight out of 140 patients who displayed no evidence of mrsa upon admission to a clinic. antibiogram tests revealed that the isolates from the eight patients were of the same strain as those from the surfaces of the dental operatory, suggesting s. aureus transmission between the patients and the dentist via the clinical environment16. the frequency of s. aureus isolated from the noses, hands, and tongues of students and patients and from the clinical environment of a pediatric dentistry clinic before and after dental treatment was determined47. the highest concentration of s. aureus was found in the noses and on the tongues of children and among the dental students, and the highest level of contamination was observed on gloved hands, which was followed by the tongue and hands without gloves before clinical care. at the end of dental treatment, s. aureus colonies isolated from the gloved hands of students decreased significantly. considering the clinical environment, s. aureus dissemination increased at the end of dental procedures, and the most contaminated areas were the auxiliary table and the storeroom, which was located at the center of the clinic. such results can be explained by the intense circulation of people in the clinic and the use of high-speed dental handpieces. however, it is still speculated that much of the s. aureus contamination detected in the clinical environment came from other sources, such as direct contact, skin exfoliation or the improper handling of plates, and it is concluded that the dental clinic is an appropriate environment for s. aureus cross-transmission. because molecular-based epidemiological studies are useful in identifying possible sources of the spread of microorganisms in hospitals and dental clinical settings, this study contributes to our knowledge on the dynamics of the spread of s. aureus strains resistant to antibiotics and points to the need for containment barriers, use of personal protective equipment, periodic identification and treatment of carriers among clinical staff, and installation of air purifiers. thus, infection control guidelines and published research pertinent to dental infection control principles and practices must be applied by the dentist as a matter of routine in academic dental offices. this research showed a genetic polymorphism in the population of oxacillin-resistant s. aureus isolates (dental patients and air of the clinical environment) and a highly frequent polyclonal pattern of these bacterial strains, supporting the hypothesis of various sources of contamination or microbial dispersion in the dental clinic environment. the isoenzyme typing and genetic relationship analysis revealed also some taxa and 12 boriollo et al. clusters exhibiting different frequencies of strains and possibly microevolutionary changes. in addition to the genetic information of s. aureus, the present methodology potentially collaborates with measures of prevention, management, and tracking of s. aureus, especially in dental clinics with great workflow. acknowledgements this study was supported by the fundação de amparo à pesquisa do estado de minas gerais (fapemig process. apq-3897-4.03/07) and the 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is staphylococcus aureus? j bacteriol. 2003 jun;185(11):3307-16. 41. hartl dl, clark ag. principles of population genetics. suderland: sinauer associates; 2007. 42. musser jm, kapur v. clonal analysis of methicillin-resistant staphylococcus aureus strains from intercontinental sources: association of the mec gene with divergent phylogenetic lineages implies dissemination by horizontal transfer and recombination. j clin microbiol. 1992 aug;30(8):2058-63. 43. kapur v, sischo wm, greer rs, whittam ts, musser jm. molecular population genetic analysis of staphylococcus aureus recovered from cows. j clin microbiol. 1995 feb;33(2):376-80. 44. harrel sk, molinasi j. aerosols and splatter in dentistry: a brief review of the literature and infection control implications. j am dent assoc. 2004 apr;135(4):429-37. 45. hubar js, pelon w. low-cost screening for microbial contaminants in aerosols generated in a dental office. gen dent. 2005 jul-aug;53(4):270-2. 46. kohn wg, collins am, cleveland jl, harte ja, eklund kj, malvitz dm, et al. guidelines for infection control in dental health-care settings--2003. mmwr recomm rep. 2003 dec 19;52(rr-17):1-61. 47. negrini tc, duque c, oliveira acm, hebling j, spolidorio lc, spolidorio dm. staphylococcus aureus contamination in a pediatric dental clinic. j clin pediatr dent. 2009 fall;34(1):13-8. _goback 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1523/1176 1/14 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1523/1176 2/14 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1523/1176 3/14 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1523/1176 4/14 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1523/1176 5/14 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1523/1176 6/14 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1523/1176 7/14 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1523/1176 8/14 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1523/1176 9/14 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1523/1176 10/14 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1523/1176 11/14 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1523/1176 12/14 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1523/1176 13/14 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1523/1176 14/14 untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8652649 volume 17 2018 e18134 original article 1 d.d.s., m.s., ph.d., postgraduate program in dentistry, federal university of maranhão, são luís, maranhão, brazil. 2 d.d.s., m.s., ph.d., department of dentistry i, federal university of maranhão, são luís, maranhão, brazil. 3 d.d.s., m.s., ph.d., department of dentistry ii, federal university of maranhão, são luís, maranhão, brazil. 4 school of medicine, federal university of maranhão, são luís, maranhão, brazil. corresponding author: maria carmen fontoura nogueira da cruz department of dentistry ii, av. dos portugueses, 1966, bacanga, são luís – ma, brazil. cep 65080-805 e-mail address: ma.carmen@uol. com.br phone number: +55 98 3272-8572 received: november 20, 2017 accepted: march 28, 2018 histopathological evaluation of dental follicles associated with impacted third molars rogério vera cruz ferro marques1, daniele meira conde marques2, fernanda ferreira lopes3, leonardo victor galvão-moreira4, maria carmen fontoura nogueira da cruz3 aim: to histologically evaluate dental follicles of impacted third molars with no radiographic evidence of pathology. methods: we carried out both a quantitative and qualitative analysis of pericoronal follicles removed from impacted third molars and investigated the association with clinical data. the sample included 36 extracted dental follicles of impacted third molars, obtained from 28 patients, which presented with no radiographic evidence of pathologies. results: none of the follicles analyzed showed any pathological entity. the epithelial lining was observed in 61.1% of samples, being identified as reduced enamel epithelium. we found a significant relation between the the presence of inflammatory infiltrate and the group aged over 21 years (64.3%; p<0.05). conclusions: considering the absence of pathological lesions, we suggest that the removal of impacted third molars, particularly in young-aged individuals, should be carefully indicated. keywords: impacted tooth, third molar, dental follicle, pathology 2 marques et al. introduction the therapeutic removal of third molars is a common maxillofacial surgery that takes into account clinical diagnostic criteria, including the presence of symptoms or pathologies, preventive measures or orthodontic indications in asymptomatic patients1,2. several issues have been linked to the maintenance of these teeth, such as root resorption or carious lesions on the second molar, trismus, pericoronaritis, localized pain and development of cysts or tumors. in the presence of such complications, the surgical removal has been widely indicated; however, this decision becomes more difficult when there is no symptom or associated pathology3,4. even though prophylactic surgery has been justified on the basis that third molars might have a greater potential to develop associated diseases, this practice remains largely controversial5,6. some authors have argued against surgery in asymptomatic impacted teeth based on the low incidence of pathologies related to third molars. most studies have used data obtained only through radiographic analysis; nevertheless, the prevalence of pathological conditions may be higher than usually is expected on radiographies7,8. thus, only radiographic aspects are inconclusive for the proper diagnosis of lesions associated with impacted third molars9. moreover, although histological changes may exist in the surrounding tissues of impacted teeth, there is no accurate information regarding the prevalence of diseases in the pericoronal tissue, which is thought to range from 10% to 58.5%2,10. hence, further research is necessary to identify a prevalence of alterations that can justify the prophylactic removal of impacted teeth. in this way, we aimed to histologically evaluate dental follicles of impacted third molars with no radiographic evidence of associated pathologies. material and methods the present study was approved by the research ethics committee of the federal university of maranhão (protocol #23115.016756/2011-40). we conducted clinical, morphological and radiographic evaluations of dental follicles of impacted third molars with no radiographic evidence of associated pathologies. the sample included 36 dental follicles obtained from 28 patients seeking orthodontic treatment in a private clinic in são luís, brazil. inclusion criteria were the presence of impacted third molars with an indication for extraction and a dental follicle radiographic space of up to 2.5mm10. patients with any local or systemic inflammatory and/or infectious disease and those who did not agree to participate in the study were not included. data obtained from each patient included age, gender, dental unit extracted, and indication for surgery. the radiographic evaluation was carried out by a specialist in a blind fashion, using scanned panoramic radiographies at the ratio of 1:1 that were processed by the radiocef studio 2 software (radio memory, belo horizonte, mg, brazil). the width 3 marques et al. of the pericoronal space was used as the largest distance from the crown to the surrounding bone. the winter’s classification of impaction was also registered. for morphological analysis, dental follicles obtained during the extraction of third molars were fixed in 10% formaldehyde solution and embedded in paraffin. histological cuts (5µm) were performed, stained using the hematoxylin-eosin technique and observed on a microscope. the histopathological analysis of dental follicles was carried out by two independent oral pathologists that were blind to clinical and radiographic data. the cohen’s kappa coefficient (κ) was calculated using spss 18.0 (ibm corp, armonk, ny, usa). the following characteristics were taken into account for the analysis of dental follicle specimens. connective tissue: dense, loose, or myxoid, which may present occasional mineralization; epithelial odontogenic residues; and unspecific inflammatory infiltrate. limiting epithelium: reduced enamel epithelium or absence of the epithelial lining. the presence of a stratified squamous epithelium was considered normal, with up to 3 cell layers, fragmented or separated from the surrounding connective tissue11 (figure 1). data were analyzed using spss 18.0. to verify statistically significant associations among variables, the pearson’s chi-squared test (χ2) was utilized at a 5% significance level. results a total of 36 pericoronal follicles of impacted third molars were collected from 28 patients, aged 14-37 years (mean age: 21.1 years). a total of 55.6% of follicles were obtained from females (n = 20) and 44.4% were from males (n = 16). mean age for figure 1. histological analysis of a dental follicle from a patient under 21 years old: a) limiting enamel epithelium exhibiting discontinuity. b) connective tissue fragment. (original magnification, ×40; he stain). 4 marques et al. females was 19.75 years (95% ci: 18.58–20.92) and 22.88 (95% ci: 19.48–26.27) for males. the sample was then divided into two age groups: under 21 years old and over 21 years old. most teeth included in the present study were lower left third molars (38.9%), followed by lower right third molars (36.1%). in relation to the third molars impaction type, according to the winter’s classification, 47.2% were mesioangular, 30.6% vertical, 11.1% distoangular, and 11.1% horizontal. in regard to their location, 75% of all impacted teeth were in the mandible (table 1). we found no association between classification and location with histological features. regarding the histopathological analysis, inter-rater agreement was satisfactory (κ = 0.84). microscopically, 38.9% of the dental follicles did not present epithelial lining, while 61.1% presented with a fragmented reduced enamel epithelium or separated from the surrounding connective tissue. the connective tissue exhibited altered myxomatosis in 72.2% of cases, while epithelial odontogenic residues and calcification were found in 88.9% and 36.1%, respectively, but there was no association between these variables and the age group (p>0.05; table 2). we observed the presence of nonspecific chronic inflammation in 41.7% of samples to be significantly associated with the age range of patients. the presence of inflammatory infiltrate was positive in both groups, but was higher in patients aged over 21 years old (p=0.028) as shown in table 2. table 1. distribution of groups according to the winter’s classification and anatomical location. winter’s classification n % mesial 17 47.2 vertical 11 30.6 horizontal 4 11.1 distal 4 11.1 location n % mandible 27 75 maxilla 9 25 table 2. association between the histological characteristics of samples and the age group. age (years) myxomatous áreas p epitelial odontogenic residues p calcifications p inflammatory infiltrate p yes no yes no yes no yes no n (%) n (%) n (%) n (%) n (%) n (%) n (%) n (%) < 21 14 (53.8) 8 (80.0) 0.14 19 (59.4) 3 (75.0) 0.54 8 (61.5) 14 (60.9) 0.96 6 (27.3) 16 (72.7) 0.02* > 21 12 (46.2) 2 (20.0) 13 (40.6) 1 (25.0) 5 (38.5) 9 (39.1) 9 (64.3) 5 (35.7) *p<0.05, according to the pearson’s chi-squared test. 5 marques et al. discussion from data obtained, we observed an increased frequency of surgical removal of impacted third molars in female patients aged over 21 years old, corroborating with previous findings10,11. still, there was a higher incidence in the mandible as 75% of impacted teeth were lower third molars, similarly to the findings of stathopoulos et al.5 (2011). regarding the radiographic evaluation, we considered as a normal pericoronal space a width of up to 2.5mm. according to the literature, the normal space varies from 2mm to 4mm; however, it is important to correlate the radiographic findings with clinical and histopathological aspects3. small cystic histological alterations may be present in dental follicles tissue and not be featured on radiographies, and the reverse may also occur if the enlargement of the pericoronal space represents a normal tissue on the histopathological exam10. regarding the dental follicle, stathopoulos et al.5 (2011) described a high transformation potential in cysts or neoplasms. on the other hand, khorasani and samiezadeh12 (2008) reported that this potential may be overrated as dental follicles can be misdiagnosed as pathological lesions, considering that the distinction between a small dentigerous cyst and a large dental follicle may be difficult, requiring thereby histological, clinical and radiographic correlation. immunohistochemical and molecular tests may be useful tools for differential diagnosis among these follicular alterations13. in this study, all dental follicles analyzed presented without any pathological entity. histologically, the dental follicles analyzed were identified as elements of fibrous connective tissue with different amounts of altered myxomatosis, epithelial calcification, epithelial odontogenic residues and epithelial lining. the incidence of epithelial lining on dental follicles has been reported in 69-87.8% of cases12,13. here, we observed the presence of epithelium (reduced enamel epithelium) in 61.1% of samples, most probably due to the average age of patients (21.1 years). the reduced enamel epithelium is more common in young individuals, whereas the incidence of squamous epithelium is regularly found in older subjects. a significant relation between the increasing age of patients and the presence of inflammatory infiltrate in the connective tissue was observed. prior studies that investigated the connection between the inflammatory infiltrate and oral tissues proliferation found inflammation to cause chronic irritation, stimulate epithelial cells proliferation and modify a normal epithelium into a squamous epithelium, which is more resistant to external factors14. metaplasia can be considered an adaptive reaction in cells vulnerable to stress, improving their tolerance to inadequate environmental conditions15, but this epithelial type was not found in the present study. furthermore, 88.9% of samples presented odontogenic epithelium inside the connective tissue, more than the 79% reported by kim and ellis16 (1993). a similar reduction of epithelial islands in relation to increasing age was also reported in this study; however, this relation was not significant. our data shows that only 27.8% of dental follicles presented with myxomatous areas as compared to 40% reported by khorasani and samiezadeh12 (2008). a significant relationship between altered myxomatosis, age and gender was not found. in conclusion, although our histopathological evaluation of dental follicles did not show pathological alterations, the participants aged over 21 years presented with unspecific chronic inflammation. therefore, we suggest that impacted third molars should be 6 marques et al. extracted only due to pathological processes that may influence the patient’s well-being or by specific indications, especially in young-aged patients. we also encourage further studies that include immunohistochemical and molecular analysis. references 1. fardi a, kondylidou-sidira a, bachour z, parisis n, tsirlis a. incidence of impacted and supernumerary teetha radiographic study in a north greek population. med oral patol oral cir bucal. 2011;16:e56-61. 2. kotrashetti vs, kale ad, bhalaerao ss, hallikeremath sr. histopathologic changes in soft tissue associated with radiographically normal impacted third molars. indian j dent res. 2010 jul-sep;21(3):385-90. doi: 10.4103/0970-9290.70809. 3. adelsperger j, campbell jh, coates db, summerlin dj, tomich ce. early soft tissue pathosis associated with impacted third molars without pericoronal radiolucency. oral surg oral med oral pathol oral radiol endod. 2000 apr;89(4):402-6. 4. marciani rd. is there pathology associated with asymptomatic third molars? j oral maxillofac surg. 2012 sep;70(9 suppl 1):s15-9. doi: 10.1016/j.joms.2012.04.025. 5. stathopoulos p, mezitis m, kappatos c, titsinides s, stylogianni e. cysts and tumors associated with impacted third molars: is prophylactic removal justified? j oral maxillofac surg. 2011 feb;69(2):405-8. doi: 10.1016/j.joms.2010.05.025. 6. brkić a, mutlu s, koçak-berberoğlu h, olgaç v. pathological changes and immunoexpression of p63 gene in dental follicles of asymptomatic impacted lower third molars: an immunohistochemical study. j craniofac surg. 2010 may;21(3):854-7. doi: 10.1097/scs.0b013e3181d809ab. 7. curran ae, damm dd, drummond jf. pathologically significant pericoronal lesions in adults: histopathologic evaluation. j oral maxillofac surg. 2002 jun;60(6):613-7; discussion 618. 8. van der linden w, cleaton-jones p, lownie m. diseases and lesions associated with third molars. review of 1001 cases. oral surg oral med oral pathol oral radiol endod. 1995 feb;79(2):142-5. 9. dudhia r, monsour pa, savage nw, wilson rj. accuracy of angular measurements and assessment of distortion in the mandibular third molar region on panoramic radiographs. oral surg oral med oral pathol oral radiol endod. 2011 apr;111(4):508-16. doi: 10.1016/j.tripleo.2010.12.005. 10. simşek-kaya g, ozbek e, kalkan y, yapici g, dayi e, demirci t. soft tissue pathosis associated with asymptomatic impacted lower third molars. med oral patol oral cir bucal. 2011 nov;16:e929-36. 11. costa fw, viana ts, cavalcante gm, de barros silva pg, cavalcante rb, nogueira as et al. a clinicoradiographic and pathological study of pericoronal follicles associated to mandibular third molars. j craniofac surg. 2014 may;25(3):e283-7. doi: 10.1097/scs.0000000000000712. 12. khorasani m, samiezadeh f. histopathologic evaluation of follicular tissues associated with impacted third molars. j dent med tehran univ med sci. 2008;5:65-70. 13. villalba l, stolbizer f, blasco f, mauriño nr, piloni mj, keszler a. pericoronal follicles of asymptomatic impacted teeth: a radiographic, histomorphologic, and immunohistochemical study. int j dent. 2012;2012:935310. doi: 10.1155/2012/935310. 14. de paula am, carvalhais jn, domingues mg, barreto dc, mesquita ra. cell proliferation markers in the odontogenic keratocysts: effect of inflammation. j oral pathol med. 2000 nov;29:477-82. 15. kumar v, abbas ak, fausto n. robbins and cotran. pathologic basis of disease. 7th ed. philadelphia: saunders; 2005. 16. kim j, ellis gl. dental folliculartissue: misinterpretation as odontogenictumors. j oral maxillofac surg. 1993 jul;51(7):762-7. 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1517/1170 1/2 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1517/1170 2/2 untitled 1 volume 16 2017 e17068 original article 1 dds, ms, phd student, universidade estadual do oeste do paraná – unioeste, school of dentistry, department of restorative dentistry, cascavel, paraná, brazil 2 dds, ms, phd, adjunctive professor, universidade estadual do oeste do paraná – unioeste, school of dentistry, department of restorative dentistry, cascavel, paraná, brazil 3 dds, ms, phd, universidade estadual de campinas, faculdade de odontologia de piracicaba – fop/unicamp, school of dentistry, department of dental materials, piracicaba, são paulo, brazil 4 dds, ms, phd, adjunctive professor, universidade estadual de campinas, faculdade de odontologia de piracicaba – fop/ unicamp, school of dentistry, piracicaba, são paulo, brazil corresponding author: bianca medeiros maran, engenharia 464 – universitário – cascavel, pr, zip code: 85819-190 – brazil. phone: +55 45 99968-3042 email: medeiros.bianca@hotmail.com received: june 25, 2017 accepted: september 20, 2017 biological and mechanical degradation affecting the surface properties of aesthetic restorative bianca medeiros maran1, fabiana scarparo naufel2, andréia bolzan de paula3, giovana spagnolo albamonte araújo3, regina maria puppin-rontani4 aim: to evaluate the roughness (ra), knoop hardness (khn) and change of color (∆e) of esthetic restorative materials (filtek z350-composite nanoparticle; empress direct-composite nanohybrid and ips e.max-ceramic) subjected to contact with the streptococcus mutans biofilm (biological degradation) associated with abrasion generated by tooth brushing (mechanical degradation). methods: ten specimens of each material were prepared, and the surface properties initial were evaluated. all specimens were exposed to streptococcus mutans inoculum; after 7 days, surface properties were evaluated. the specimens were submitted to a 30,000 toothbrushing cycles, using a toothpaste slurry, then, surface properties were evaluated again. data were analyzed by proc-mixed, one-way anova, tukey-kramer and tukey’s tests (α = 0.05). results: at the baseline, ceramic showed the highest ra and khn values; after the biological degradation the composites showed increased ra, but khn did not change; after the mechanical degradation, empress showed decreased ra and z350 showed similar ra, the khn increased to both composites, and all materials had increased lightness after the mechanical degradation. conclusions: the results suggest that, when exposed to streptococcus mutans biofilm and toothbrush abrasion, the ceramics undergoes minimal degradation and the composites exhibited variable degradation, depending on the composition of the material. keywords: biofilms. surface properties. dental materials. http://dx.doi.org/10.20396/bjos.v16i0.8651058 2 maran et al. introduction all restorative materials are susceptible to degradation. the degradation of restorative materials can be caused by low ph because of the cariogenic biofilm, consumption of acidic drinks or foodstuffs, and toothbrushes and the water present on saliva as well other components of the saliva1. tooth brushing is the most used and efficient mechanical method to remove dental biofilm from all accessible tooth surfaces2. published studies have shown that this method may cause tooth and composite abrasion. this degradation process may lead to several drawbacks, such as an increase in wear and surface roughness, softening and a decrease in the hardness of dental materials3-5. over time, intraoral degradation also interferes with the fracture strength of the material, culminating in a lower durability of the restoration in the long term6. surface texture, gloss and color are also included among the important characteristics that determine the aesthetic effect of these composite restorations, and they are also influenced by the intraoral surroundings7,8. it is important to know about this process, because the search for dental esthetics has been one of the main reasons why patients seek a dentist. thus, the need for tooth-colored fillings has increased, decreasing the use of metal restorations and dental amalgam fillings or cast metal, unlike the use of aesthetic materials, such as composite and ceramics, which have been increasingly used9. ceramics are considered the most inert of all dental materials used for restorations, composed of metal elements (aluminum, calcium, lithium, magnesium, potassium, sodium, lanthanum, tin, titanium and zirconium) and non-metal substances (silicon, boron, fluorine and oxygen), and characterized by two phases: a crystalline phase surrounded by a vitreous layer10. so far, little information about surface degradation by biofilm is available in the literature. some studies have evaluated the interaction between biofilm and ceramics, but they verified only the biofilm characteristics instead of the biodegradation produced on the material surfaces11,12. composites are currently the most used material in the field of restorative dentistry. basically, these materials are composed of three chemically different components: a polymeric matrix of dimethacrylate monomers, filler particles (dispersed phase) and an organosilane, which is a coupling agent that bonds the fillers to the polymeric matrix13. in this context, nanotechnology, consisting of nanofillers, has emerged in the dental market14. this technology came with the intention of improving the electrical, chemical, mechanical and optical properties of restorative materials with advantages such as less toothbrush abrasion, greater hardness and better translucency, polish, gloss and opacity options being used for restorations of anterior and posterior teeth; as a result, studies have been done to prove these characteristics15,16. the nanohybrid composite ips empress direct promises similar aesthetics to those made of ceramics in addition to the advantages of easy handling of the composite (ivoclar vivadent). therefore, it becomes interesting to compare the ips empress direct composite with a 100% nanofiller resin such as z350 (3m espe st. paul, mn, usa), as well as with the 3 maran et al. ceramics ips e.max (ips empress; ivoclar-vivadent, schaan, liechtenstein), considering the material that undergoes minimal degradation. thus, the aim of this study was to test the hypothesis that aesthetic restorative materials submitted to the streptococcus mutans biofilm associated with brushing abrasion would differ in surface stability to degradation, depending on their composition. materials and methods specimen preparation 10 specimens of each material tested (described in table 1) were fabricated using silicon molds (express 3m espe, st. paul, minn, usa) of 8 mm in diameter and 2 mm deep, with the exception of the ceramics. composite materials were inserted in a mold using incremental technique and covered with polyester strips and a glass slide to obtain a smooth flat surface. all specimens light cured using a led light unit (elipar freelight, 3m espe, st. paul, mn, usa) for 40 s on the top surface. the light intensity of the curing (1000 mw/cm2) device was checked with a curing light meter (hilux dental curing light meter, benlioglu dental inc., demetron, ankara, turkey). then, specimens were storage for 24 h in 100% relative humidity at 37°c, and then, they were polished with sequential abrasive discs (soflex pop-on, 3m espe, st. paul, mn, usa). table 1. material, composition, color and batch of the tested materials. materials composition mean filler size color batch # composite filtek z350 xt (3m espe st. paul, mn, usa) bis-gma (1-10 wt%); udma (1-10 wt%); tegdma (< 5 wt%); bis-ema (1-10 wt%); pegma (< 5 wt%) silica, zirconia, zirconia/ silica (78.5 wt%) 0.6-1.4 µm (cluster) 5–20 nm (nanofiller) a3e 1124300109 ips empress direct (ivoclarvivadent, schaan, liechtenstein) udma (10-<20 wt%); tegdma (3-<5 wt%); bis-gma (2.5-<3 wt%) barium glass, ytterbium trifluoride, mixed oxide, silicon dioxide and copolymer (77.5-79 wt%) additives, catalysts, stabilizers and pigments (<1.0 wt%) 0.4 to 0.7 µm a3e n32078 ceramic ips e.max (ivoclarvivadent, schaan, liechtenstein) sio 2 , li 2 o, k 2 o, mgo, zno, al 2 o 3 , p 2 o 5 and others oxides -a3e p82207 4 maran et al. ceramic specimens were fabricated with the same dimensions of the composite, in a prosthetic laboratory using the pressing process in an oven (programat p500–ivoclar vivadent, schaan, liechtenstein), and they were glazed. then, all specimens were stored in water at 37° c for 24 h for the evaluation of the baseline properties. measurements of surface roughness the surface roughness (ra) was measured in a rugosimeter (surfcorder se 1700, kosaka, tokyo, japan) at a constant speed of 0.5 mm/s with a load of 0.7 mn. the cut-off value was set at 0.25 mm to maximize the filtration of the surface waviness. the ra values for each specimen were taken across the diameter over a standard length of 1.25 mm. the mean surface roughness values (μm) of the specimens were obtained from three successive measurements of the center of each disk, in different directions (45°). a calibration was done periodically to check the performance of the surface roughness-measuring instrument. measurements of hardness three knoop hardness (khn) indentations were made on the surface of the specimen under a load of 50 g for 10 s (hmv-2, shimadzu, tokyo, japan). the khn for each specimen was recorded as the average of the three readings distant 100 µm each other. measurement of color the readings were performed using a spectrophotometer (cm-700d, konica minolta, osaka, japan). initially, the ambient light was calibrated in a light cabin, (gti mini matcher mm1e, gti graphic technology inc., newburgh, ny, usa), and the specimens were positioned in a sample carrier for the baseline readings. the parameters l*, a* and b* from the color space, referred to as cielab (l*, a*, b*), were recorded. the l* indicates lightness (l* + = lightness and l* = darkness), the a* coordinate represents the red/green range (a* + = redness and a* = greenness) and the b* coordinate represents the yellow/blue range (b* + = yellowness and b* = blueness). the l*a*b* system allows the numeric definition of a color, as well as the difference between two colors using the following formula: δe= [(δl)2 + (δa)2 + (δb)2]1/2. the data acquisition was performed by a microcomputer using on color qc lite software (konica minolta, osaka, japan). biofilm growth – biological degradation after the measurements of surface roughness, hardness and color, the all the specimens were sterilized for 4 h in an ethylene oxide chamber (ferlex, são paulo, sp, brazil). a streptococcus mutans (ua 159) strain was obtained from the culture of the department of microbiology and immunology, piracicaba dental school, university state of campinas. to prepare the inoculums, the streptococcus mutans was first grown on mitis salivarius agar plates (difco laboratories, sparks, mi, usa) at 37°c for 24 h in an environment supplemented with 5% co2. subsequently, single colonies were inoculated into 5 ml of brain-heart infusion (bhi) broth (difco laboratories, detroit, mi, usa) and incubated at 37º c for 24 h. the spec5 maran et al. imens were exposed under static conditions to 25 μl of streptococcus mutans inoculums adjusted to an optical density of 0.6 at 550 nm (approximately 8x1011 cfu/ml), and after 2 hours at room temperature the non-adhering cells were removed by washing twice with 0.9% nacl solution (saline). a single material disk was inserted in each well of 48-well polystyrene plates (nunc multidish, sigma, st. louis, mo, usa) with 2 ml of sterile, fresh bhi broth with the addition of 1% sucrose (wt/vol). the bacterial accumulation occurred at 37ºc in an environment supplemented with 5% co2, developing 7-day-old biofilms. the medium was renewed every 48 h. at the end of the experimental period, the specimens were ultrasonically (unique 1400, indaiatuba, sp, brazil) washed for 10 minutes, and soon after the measurements were repeated. three-body abrasion test – mechanical degradation. three-body abrasion test – mechanical degradation after biological degradation, the tooth brushing test was conducted at 250 cycles/min for 30,000 cycles with a 200 gf load. the oral b pró saúde toothpaste (procter & gamble, são paulo, sp, brazil) was diluted in distilled water (1:2) and used as an abrasive third body. specimens were washed in an ultrasonic bath for 10 min and gently dried with absorbent paper. then, three final surface roughness readings were taken from each specimen in the opposite direction to that of the tooth brushing movement; knoop hardness and color were also evaluated as previously reported after mechanical degradation using the same pattern described above. statistical analysis for ra, knh and l*, after the exploratory data analysis and selection of the best covariance structure, data were analyzed by means of mixed models (proc-mixed) and tukey-kramer test (α = 0.05). the data of hardness suffered logarithmic transformation to meet the assumptions of a parametric analysis. for δe, after the exploratory analysis the data were analyzed by one-way anova and tukey’s test (α = 0.05). results there was a significant difference among the materials studied (p < 0.0001 – for all the variables analyzed) and between the degradation methods (baseline/biological biodegradation/mechanical degradation) (for ra and khn: p < 0.0001; for l*: p = 001; for initial δe: p = 0005); in addition, there was significant difference for the interaction between the three studied factors (for ra: p < 0.0001; for khn: p = 0.0327; for l*: p = 0.05). table 2 shows the ra averages found by different materials and different degradation processes. at baseline, e.max showed the highest roughness and the empress and z350 composite showed very low and similar roughness. after biological degradation, the ra values of e.max increased still remaining statistical similarity to the baseline, but it was showed a significant increase for z350 and empress composite, being that z350 showed the lowest roughness. however, after mechanical degradation, the roughness of z350 remained similar to the biological degradation and higher than baseline values; for empress and e.max, ra was smaller than biological values; being statistically similar to the baseline values for e.max, but for empress it was lower than those obtained after biological degradation but higher than baseline values (table 2). 6 maran et al. table 3 shows the khn values obtained for different materials and degradation models. at baseline, empress and z350 composite showed the lowest and similar khn values. after the s mutans degradation, it can be observed that all materials experienced a decreasing on khn values. however, in a descending significant order it can be observed that e.max showed the highest khn values followed by empress and z350. after the mechanical degradation, the hardness increased for z350 and remained similar to the biological degradation values for empress. in all conditions, baseline, biodegradation and mechanical degradation exposition, e.max showed the highest khn values (table 3). table 4 shows the δe values for different materials submitted to different degradation models. after the biological biodegradation, the empress composite showed the lowest δe, similar to e.max values. there was no statistical difference between all materials studied after the mechanical degradation (table 4). table 5 shows the values obtained for lightness of the materials submitted to different degradation models. all materials studied showed the same performance when submitted to different degradation methods. at baseline, all materials showed similar lightness pattern. however, the exposition to s mutans biofilm provided the lowest lightness for empress table 2. means (standard deviations) of surface roughness (ra) (µm) for the different experimental conditions. materials baseline biological biodegradation mechanical degradation z350 0.26 (0.09) bb 1.51 (1.08) ab 1.48 (0.70) aab empress 0.24 (0.07) cb 2.71 (0.43) aa 0.86 (0.34) bb e.max 2.60 (0.71) aba 3.26 (0.98) aa 2.20 (0.79) ba means followed by different capital letters in the same line and small letters in the same column were significantly different (p < 0.05). table 3. means (standard deviations) of the knoop hardness (khn) for the different experimental conditions. materials baseline biological biodegradation mechanical degradation z350 62.1 (24.0) bb 51.6 (15.38) bc 82.38 (17.8) ab empress 82.2 (15.8) ab 80.4 (13.5) ab 106.2 (16.7) ab e.max 811.7 (139.9) aa 656.8 (105.6) aa 757.8 (151.1) aa means followed by different capital letters in the same line and small letters in the same column were significantly different (p < 0.05). table 4. means (standard deviations) of color change (δe) for the different experimental conditions. materials biological biodegradation mechanical degradation z350 2.8 (1.0) a 1.9 (0.5) a empress 2.1 (0.5) b 1.2 (0.4) a e.max 3.0 (0.6) a 1.7 (1.1) a groups denoted by a different letter represent significant difference (p<0.05). 7 maran et al. when compared with e.max and z350. a significant increase on lightness can be observed for all materials studied after mechanical degradation, although the highest values were observed for z350 and the lowest for empress. e.max showed intermediary lightness (table 5). discussion aesthetic restorative materials are prone to a gradual degradation process in the oral cavity because of ph changes (chemical or bacterial action), temperature, chewing and brushing, depending on the composition of the restorative material15,17,18. this study revealed that the composites showed similar average roughness after polishing. after biological degradation, the composites show different variations of roughness, which may depend on the hydrolytic stability of the polymer matrix15,16. according to sarkar19 (2000), these changes are due to the absorption and diffusion of water and organic acids from the bacterial metabolism, internal resin matrix, interfaces between the inorganic particles, pores and other defects. the greater increase in the roughness of the composite empress compared to z350 can be attributed to the fact that the second one is a composite which has only nanoparticles particles as fillers, with less interstitial spacing of the matrix, which decreases its hydrolysis; in addition, we should mention the presence of bis-ema, a hydrophobic monomer, which favors the hydrolytic stability13,16,20,21. at baseline, the ceramics showed higher roughness than the polished composites, which is due to the irregularities in the surface from the resulting glazing process. after the biological biodegradation, there was no significant variation in the surface roughness of the ceramics, which may be due to the stability of the material, as it is considered the most inert dental material10. these results are in agreement with the study of padovani et al.1. the final roughness of the ceramics was comparable to the original, which is in agreement with studies evaluating resistance to toothbrush abrasion8,22. the surface hardness of z350 remained statistically similar after biological degradation; the presence of the tegdma monomer is justified in both composites, which increases the degree of conversion, reducing leaching and softening10,21.hardness becomes an important parameter to measure the performance of materials in the oral environment, being correlated with the resistance to compression and abrasion, and it indirectly reflects the rate of polymerization of the material5. materials with decreased hardness have reduced longevity and may require early replacement of the restoration5,23. table 5. means (standard deviation) of lightness (l*) for the different experimental conditions. materials baseline biological biodegradation mechanical degradation z350 72.96 (0.23) ba 72.78 (0.69) ba 73.50 (0.29) aa empress 68.59 (0.53) ba 71.81 (0.71) bb 72.50 (0.51) ab e.max 72.60 (0.53) ba 73.18 (0.55) ba 73.30 (0.51) aab means followed by different capital letters in the same line and small letters in the same column were significantly different (p < 0.05). 8 maran et al. the hardness of the z350 composite was lower than the empress after the biological biodegradation, and this can be attributed to differences in size and distribution of the fillers on these materials4,24; beyond the aforementioned factor, it can be speculated that this is the association of the consequent hydrolysis of the polymeric matrix with the inorganic framework differences of the studied composites24. the ceramic, showed higher hardness, in all degradation methods than the composites studied, which is due to their glass character, as there is the coalescence of the particles and higher solid density in the sintering process1,10. the nanofilled composite may be prone to absorbing liquids because of the greater contact area-load matrix, and this interface is more susceptible to fluid accumulation in the bacterial biofilm, or alternatively, the spaces resulting from the presence of the imperfect engagement of charged particles in the polymeric matrix. spaces or “microvoids” in the polymeric matrix can increase the retention of acids and thereby increase the degradation of z35024. in the oral cavity, the deleterious effects of the biodegradation are generally associated with toothbrush abrasion, as the abrasiveness of the toothpaste along with the toothbrush may promote the displacement of charged particles, which is directly proportional to the size of these effects25. the hardness of the composite z350 increased, this can be attributed to the process of maturation or late polymerization of the composite26, and that the nano-sized loads have greater contact surface with the organic phase, improving the hardness of the material27. the empress composite showed decreased roughness after the mechanical degradation, but not returning to equivalent values to the baseline; this may be due to the losses of larger particles, which weakens the softened matrix and enhances the abraded mass of the polymer, removing the softened layer13,20. the roughness of z350 remained similar to that observed after the biological degradation, and greater than at the baseline, which can be attribute to the effect of the bristles of the toothbrush atop the smaller interstitial space in the polymeric matrix, which could result in a higher abrasion resistance16. the lightness and stability of the color, important properties of aesthetic restorative materials, are influenced by various factors such as the composition of the inorganic portion, diet, habits or even the organic matrix. the sensitivity of the human eye to detect color variation translates to δe> 3.3; thus, the color changes were imperceptible to human sensitivity22,28,29. however, analysis of the cielab color scale coordinates (l *, a *, and b *) showed significant changes in the values of l*. the lightness is the ability of the material to reflect direct light and is closely related to the surface characteristics of the material, ranging from light (100) to dark (0)8,30. after the mechanical degradation, there were increases in the lightness for all materials studied, probably the optical changes that occurred reflect physical and chemical reactions: i) internal – such as hydrolysis – or ii) in the surface – such as increased roughness –, as these affect the lightness through changes in the refractive index and reflection, respectively28, since the specimens were not exposed to any coloring agent and there was standardization of the thickness of the specimens. 9 maran et al. the biodegradation provided on composite materials as ra, was recovered after mechanical degradation for all materials. however, for hardness, only empress direct has recovered that after bio and mechanical degradation. color was significantly affect after mechanical degradation for all materials studied. based on the experimental conditions described, the results showed that the degradation process associated with toothbrush abrasion promoted increased roughness of the nanoparticulate composite z350; the nanohybrid composite empress exhibited less variations in roughness and hardness than that of the nanoparticle; finally, the ceramic e.max was more stable and resistant to degradation in the oral environment. acknowledgements this investigation was supported by the romanini dental prosthesis laboratory. address: avenida rio de janeiro, 1306, 86010150 londrina – pr e-mail: contato@romanini.com.br espaço da escrita/coordenadoria geral da unicamp references 1. padovani g, fucio s, ambrosano g, sinhoreti m, puppin-rontani r. in situ surface biodegradation of restorative materials. oper dent. 2014;39(4):349-60. doi: 10.2341/13-089-c. pubmed pmid: 24555699. 2. srivastava n. a comparative evaluation of efficacy of different teaching methods of tooth brushing in children contributors. j oral hyg health. 2013;01(03). doi: 10.4172/2332-0702.1000118. 3. da silva ma, 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artificial accelerated aging on the optical properties and monomeric conversion of composites used after expiration date. gen dent. 2013;61:1-5. 24. da silva em, goncalves l, guimaraes jg, poskus lt, fellows ce. the diffusion kinetics of a nanofilled and a midifilled resin composite immersed in distilled water, artificial saliva, and lactic acid. clin oral investig. 2011;15(3):393-401. doi: 10.1007/s00784-010-0392-z. 25. da silva em, dória j, da silva jdjr, santos gv, guimarães jga, poskus lt. longitudinal evaluation of simulated toothbrushing on the roughness and optical stability of microfilled, microhybrid and nanofilled resin-based composites. j dent. 2013;41(11):1081-90. 26. alrahlah a, silikas n, watts dc. post-cure depth of cure of bulk fill dental resin-composites. dent mater. 2014;30(2):149-54. doi: 10.1016/j.dental.2013.10.011. 27. kango s, kalia s, celli a, njuguna j, habibi y, kumar r. surface modification of inorganic nanoparticles for development of organic–inorganic nanocomposites—a review. prog polymer sci. 2013;38(8):1232-61. doi: 10.1016/j.progpolymsci.2013.02.003. 28. oliveira dc, souza-junior ej, prieto lt, coppini ek, maia rr, paulillo la. color stability and polymerization behavior of direct esthetic restorations. j esthet restorative dent. 2014;26(4):288-95. doi: 10.1111/jerd.12113. 29. prodan da, gasparik c, mada dc, miclăuş v, băciuţ m, dudea d. influence of opacity on the color stability of a nanocomposite. clin oral investig. 2015;19(4):867-75. 30. tan b, yap a, ma h, chew j, tan w. effect of beverages on color and translucency of new tooth-colored restoratives. oper dent. 2015;40(2):e56-e65. original article braz j oral sci. january/march 2009 volume 8, number 1 genetic polymorphism of streptococcus mutans strains associated with incomplete caries removal cristiane duque1, thais de cássia negrini2, nancy tomoko sacono3, marcelo fabiano gomes boriollo4, josé francisco hofling5, josimeri hebling6, denise madalena palomari spolidorio7 1 dds, phd, post-doctorate student, department of microbiology and immunology, faculdade de odontologia de piracicaba, universidade estadual de campinas (unicamp), piracicaba (sp), brazil; department of orthodontics and pediatric dentistry, faculdade de odontologia de araraquara, universidade estadual paulista “júlio de mesquita filho” (unesp), araraquara (sp), brazil 2 dds, ms, doctorate student; department of clinical analysis, faculdade de ciências farmacêuticas, unesp, araraquara (sp), brazil 3 dds, ms, doctorate student, department of orthodontics and pediatric dentistry, faculdade de odontologia de araraquara, unesp, araraquara (sp), brazil 4 dds, ms, phd, professor, department of microbiology, universidade josé do rosário vellano, alfenas (mg), brazil 5 bsc, phd, professor, department of microbiology and immunology, faculdade de odontologia de piracicaba, unicamp, piracicaba (sp), brazil 6 dds, phd, professor, department of orthodontics and pediatric dentistry, faculdade de odontologia de araraquara, unesp, araraquara (sp), brazil 7 bsc, phd, professor, department of physiology and pathology, faculdade de odontologia de araraquara, unesp, araraquara (sp), brazil received for publication: march 15, 2009 accepted: april 22, 2009 correspondence to: cristiane duque department of microbiology and immunology faculdade de odontologia de piracicaba universidade estadual de campinas av. limeira, 901 cep 13414-903 – piracicaba (sp), brazil e-mail: cristianeduque@yahoo.com.br abstract aim: despite the antibacterial properties of dental materials, the survival of residual bacteria under restorations has been demonstrated after incomplete caries removal. the aim of this study was to evaluate the genetic polymorphism of streptococcus mutans strains isolated from deep dentinal lesions before and three months after incomplete caries removal. methods: samples of carious dentin were collected from 33 primary and/or permanent molars before and after indirect pulp treatment and processed for microbiological isolation of mutans streptococci (ms). after three months of the dental treatment, positive cultures for ms were detected in only ten of these teeth. dna of ms isolates were obtained and subjected to polymerase chain reaction (pcr) for identification of s mutans. the arbitrary primed-pcr method (primer opa-13) was used to detect the genetic polymorphism of s. mutans strains. results: identical or highly related s. mutans genotypes were observed in each tooth, regardless of the collect. considering each tooth separately, a maximum of nine genotypic patterns were found in each tooth from all the collects. in addition, at least one genotypic pattern was repeated in the three collects. genetic diversity was observed among the s. mutans isolates, obtained from different teeth after three months of the dental treatment. conclusions: the persistence of identical genotypic patterns and the genetic similarity among the isolates, from the same tooth in distinct collects, showed the resistance of some s. mutans strains after incomplete caries removal treatment. keywords: streptococcus mutans, polymerase chain reaction, polymorphism, genetic, dental caries. introduction the wide distribution and variety of oral bacteria demonstrate their ability to survive among their human hosts, as a result of the efficient transmission of strains and their persistence in the oral cavity1. there are several microenvironments within the mouth that harbor communities of pathogenic or non-pathogenic bacteria, called biofilms, which are mainly found in the hard surfaces of the teeth2. the biochemical instability between tooth substance and the overlying biofilm, determined by intense production of acids derived from bacterial metabolism, and may lead to dissolution of the dental hard tissues and consequently to the development of a carious lesion3. among the oral pathogens, mutans streptococci (ms) comprise a group of seven bacterial species of which only streptococcus mutans, s. sobrinus, s. rattus and s. cricetus can be found on the human oral cavity4. the highest prevalence of s. mutans (74 to 94%) among oral streptococci, 3genetic polymorphism of streptococcus mutans strains associated with incomplete caries removal braz j oral sci. 8(1): 2-8 isolated from carious lesions demonstrates their association with caries development5. s. mutans has the ability of tolerating continuous cycles of acid shock, produced in the oral environment by mechanisms of proton extrusion from the cell via membrane-associated processes and by acid end product efflux, protecting important cellular components, especially dna, from aggressive effects of acidification6. in addition, in adverse environments with nutrient restriction, some bacteria, such as s. mutans, are able to obtain carbohydrates from host-derived saliva or serum glycoproteins7-8. genetic diversity among strains of the same species may reflect their ability to survive extreme environmental changes. therefore, selection of strains best fitted to a given environment can be related to the generation of genetic variants, representing clonal populations, which conserve dna into single cell lines, or non-clonal populations, which incorporate dna from other cells9. several studies have shown genetic heterogeneity, among s. mutans strains, using modern molecular typing techniques, such as arbitrary primed polymerase chain reaction (ap-pcr), multilocus enzyme electrophoresis (mlee), restriction fragment length polymorphisms (rflp) and repetitive extragenic palindromic pcr (rep-pcr)9-15. ap-pcr is a valuable dna analysis tool that have been used mainly in streptococcal epidemiology and transmission studies, by virtue of its rapidity, efficiency and reproducibility in generating genetic fingerprints of bacterial isolates10,16. several studies using ap-pcr have demonstrated that caries-active children have greater genotypic diversity of s. mutans, compared to caries-free children14,17. in addition, a site-specific colonization pattern of s. mutans genotypes in coronal and root caries lesions has been reported12,18. the survival of oral streptococci has been observed even months or years after incomplete caries removal and cavity sealing19-21, suggesting the generation of treatment-resistant strains. paddick et al.15 have detected different genotypes of streptococcus oralis and actinomyces naeslundii, five months after incomplete carious dentin removal, with reduced phenotypic and genotypic diversity compared to strains isolated from the initial samples before treatment. therefore, the aim of this study was to evaluate the genetic polymorphism of s. mutans strains isolated from deep dentinal lesions before and three months after incomplete caries removal. materials and methods thirty-three molars (27 primary and 6 young permanent) with deep occlusal caries lesions were obtained from 20 children of both genders, aged four to ten years and subjected to indirect pulp treatment. the absence of irreversible pulpal or periapical diseases was determined through clinical and radiographic examinations of all selected teeth. this study was conducted under a protocol approved by the research ethics committee of faculdade de odontologia de araraquara of universidade estadual paulista, in brazil, “júlio de mesquita filho” (unesp) and a signed informed consent was obtained from the legal tutors for the the children’s participation. clinical procedures and microbiological sampling indirect pulp treatment was divided in two sessions. at the first session, anesthesia was delivered and a rubber dam applied to isolate the tooth and prevent contamination by saliva. rubber cup pumice prophylaxis and antisepsis of operative area, using 0.2% chlorhexidine digluconate, were performed. if necessary, access to infected dentin was gained using a sterile # 245 carbide bur (kg sorensen, barueri, sp, brazil) at high speed. after removing the superficial necrotic dentin, an initial collect (a) of carious dentin was sampled with a sterile excavator, immediately immersed in 1 ml of saline and maintained in refrigerated boxes (4 ºc). a sterile round steel bur at low speed was used to clean all carious tissue from the dentinoenamel junction, leaving a layer of soft dentin on the cavity floor to avoid pulp exposure. after washing and air-drying the cavities to remove debris, a second collect (b) of carious dentin was obtained from the mesial portion of the cavity floor for microbiological analysis, as previously described. in order to standardize the amount of collected dentin, a small cavity was created at the flat end of an amalgam plugger, which was completely filled with dentin. the remaining carious dentin was covered with resin-modified glass ionomer cements (vitrebond; 3m/espe, st. paul, mn, usa or fuji lining lc; gc corp., tokyo, japan) or a calcium hydroxide-based cement (dycal; dentsply, milford, de, usa). the cavities were restored with temporary zinc oxide-eugenol cement (irm; dentsply). after three months, the second session of the indirect pulp treatment was undertaken. under the same initial conditions of anesthesia and isolation, teeth were reopened and the liner materials, carefully and completely removed. a third collect (c) of carious dentin was sampled from the distal portion of the cavity floor, as already described. later, the teeth were lined with glass ionomer cement and restored definitively with silver amalgam (dispersalloy, dentsply international, usa). microbiological procedures the tubes containing the carious dentin samples were vortexed for two minutes and the suspension was serially diluted with saline. for each dilution, 25 μl of the samples were placed duplicated in agar sacarose bacitracin (sb) – sb-2022 and incubated at 37 oc for 24 hours. after incubation, six to eight representative colonies of streptococci were collected and inoculated individually in 5 ml of brain heart infusion (bhi) (brain heart infusion, difco laboratories, bd, sparks, md, usa) broth for 24 hours. the purity of the cultures were confirmed using gram technique and aliquots of the subculture frozen at -20 oc, in 10% glycerol bhi for posterior molecular analysis of bacterial isolates. polymerase chain reaction the extraction of chromosomal dna was described by nociti et al.23 and modified by nascimento et al.18. briefly, overnight cultures in 4 duque c, negrini tc, sacono nt, boriollo mfg, hofling jf, hebling j, spolidorio dmp braz j oral sci. 8(1): 2-8 bhi broth were centrifuged, followed by washing twice with trisedta buffer (10 mm tris-hcl, 1 mm edta, ph = 8.0) and boiled for ten minutes. after centrifugation, 60 μl of supernatant was collected and used as templates for the pcr. in order to confirm s. mutans molecular identity, dna from ms isolates obtained from carious dentin collects was submitted to pcr method, using specific primers for portions of the glucosyltransferase b gene (gtf b) following the bases sequences’: 5’ – act aca ctt tcg ggt ggc ttg g – 3’ e 5’ – cag tat a ag cgc cag ttt cat c – 3’, to amplify a517 bp dna fragment24. each pcr mixture contained 5 μl of the dna template, 5 µl of 10x pcr amplification buffer (100 mm tris-hcl, 500 mm kcl, ph = 8.3), 0.2 mm of dntps (dna polymerization mix), 3.0 mm mgcl 2 , 1 μm of each primer, 2.5 u of taq dna polymerase and sterile distilled water, in order to make a final volume of 25 μl. all pcr reagents were obtained from invitrogen, life technologies, são paulo, brazil. positive and negative controls of pcr were purified in genomic dna of s. mutans (atcc 25175) and sterile water, respectively. the amplification of dna was performed in a thermocycler (geneamp pcr system 2400, perkin-elmer’s applied biosystems division, foster city, ca, usa) with initial denaturation at 95 oc for five minutes, followed by 30 cycles of denaturation at 95o c for 30 seconds, annealing at 59 oc for 30 seconds, extension at 72 oc for one minute, ending with final extension at 72 oc for seven minutes24. the pcr amplification products were separated by electrophoresis in 1% agarose gels in tris-borato-edta (tbe), running buffer (ph = 8.0) at 75 v for two hours. gels were stained with 0.5 μg of ethidium bromide/ml and visualized under ultraviolet light illumination (ultralum; labtrade do brasil, são paulo, brazil). a 100 bp dna ladder was included as a molecular-size marker in each gel. ap-pcr strains identified as s. mutans by pcr method were used for genotyping. ap-pcr amplification was performed with primer opa-13 (5’cagcacccac – 3’)10,14,18. all reactions were processed in a volume of 25 μl, containing 2.5 μl of 10x pcr amplification buffer (100 mm tris-hcl, 500 mm kcl, ph = 8.3), 7 mm mgcl 2 , 0.2 μm of dntp, 1 μm of primer, 2.5 u of taq dna polymerase, 50 ηg of dna and distilled water18. the amplification was performed in the same thermocycler with an initial denaturation at 94 oc for five minutes and 45 cycles of denaturation at 94 oc for 30 seconds, annealing at 36 oc for 30 seconds, extension at 72 oc for one minute, ending with final extension at 72 oc for three minutes. amplicons generated by ap-pcr were analyzed electrophoretically in 1.4% agarose gel, in tbe running buffer and stained in 0.5 μg/ml ethidium bromide. a 1 kb dna ladder was used as molecular-size marker. the gels were photographed and their images, captured with a digital imaging system (kodak digital science 1d; eastman kodak company, rochester, n y, usa). the molecular weights for each band or amplicon were computed and analyzed using the sigma gel software ( jandel scientific, san rafael, ca, usa). the amplicons were converted in binary data and submitted to ntsys-pc software (applied biostatistics, inc., setauket, n y, usa), using the simple matching coefficient (s sm ), and the unweighted pair group method with mathematic average (upgma) cluster analysis to generate similarity dendrograms. results only ten deciduous teeth were included in the study that showed positive culture in all collects (a, b and c). from the 429 selected ms colonies, 377 (87.9%) were identified as s. mutans. table 1 shows the number of ms isolates and s. mutans strains, according to the collected period and material group. one hundred and seventy-three s. mutans isolates were chosen for ap-pcr analysis considering the three collects (a, b and c). because the distribution of the teeth was not similar among the material groups, data analysis was performed for all teeth, independent of the lining material. all of them were subjected to ap-pcr. each isolate received a specific code according to the following sequence: number of the tooth (from 1 to 10), collect (a, b and c) and number of the bacterial isolate (from one to eight), for example: 5a2. the amplification of genomic dna of this species resulted in fragments (amplicons or eletrophoretic bands), ranging from 0.3 to 2.2 kb in size. some of them were species-specific, whereas others were found in only one or few s. mutans strains. the ap-pcr fingerprinting profile analysis with primer opa-13 showed distinct genotypes patterns of s. mutans obtained from caries samples. in this study, the same genotypic pattern was considered for identical or highly related samples with genetic similarity (s sm ) ≥ 0.869 (threshold). considering each tooth separately, a maximum of nine genotypic patterns were found in each tooth from all the collects (a, b and c). in addition, at least one genotypic pattern was repeated in the three collects. table 2 shows the total number of genotypic patterns detected in each tooth, without repetitions among the collects (genetic similarity s sm ≥ 0.869). lining material ms colonies total s. mutans strains total (%)baseline reentry baseline reentry a b c a (%) b (%) c (%) vitrebond 70 51 14 136 57 (81.5) 44 (86.3) 14 (100) 115 (84.6) fuji lining lc 57 55 47 154 55 (96.5) 55 (100) 37 (78.7) 142 (92.2) dycal 59 51 25 135 49 (83.1) 47 (92.3) 19 (76) 115 (85.2) total 186 157 86 429 161 (86.6) 146 (93) 70 (81.4) 377 (87.9) table 1. ms colonies isolated from sb-20 medium and s. mutans strains identified by pcr 5genetic polymorphism of streptococcus mutans strains associated with incomplete caries removal braz j oral sci. 8(1): 2-8 based in the matrices generated by the upgma analysis using coefficient s sm , the genetic similarity levels obtained among the s. mutans strains are illustrated in figure 1, which shows representative dendrograms of s. mutans isolates obtained for each tooth (1 to 10). the genetic similarity values ranged from 0.555 ≤ s sm ≤ 1. two to four s. mutans groups (clusters) containing identical or highly related isolates (s sm ≥ 0.869) were found in each tooth. these groups can be identified by tonalities of gray in the dendrograms. identical or highly related isolates were found in all collects in the same tooth. comparing the genot y pic patterns in col lect c, which were obta ined three months af ter denta l treatment , a dendrog ram w ith the sm isolates is show n in fig ure 2 and the genetic similarit y index obser ved was 0.621 ≤ s sm ≤ 1. u pgm a ana lysis revea led 11 g roups conta ining identica l or hig h ly related isolates (0.869 ≤ s sm ≤ 1). seven of these g roups had isolates corresponding to a specif ic tooth (groups 1, 3, 7, 8, 9, 10 and 11). other three g roups had isolates f rom t wo d istinct teeth (groups 4, 5 and 6) and one g roup had isolates f rom four teeth (group 2), demonstrating genetic similarit y among s . mutans stra ins, obta ined f rom the same tooth and g reater poly mor phism among isolates f rom d if ferent teeth. discussion s. mutans is the major pathogen associated with dental caries in humans. several studies have demonstrated its capacity to tolerate extreme ph changes, varying from alkalinity 25 to high levels of acidity6. furthermore, in situations of environmental nutrient stress, such as after cavity sealing, s. mutans is capable of producing glycosidic enzymes that release carbohydrates from serum glycoproteins7-8 present in the dentinal tubules15. therefore, it is plausible to suggest that specific phenotypic characteristics could be expressed by genotypes best fitted to survive and/or grow in adverse environments1. in the present study, three months after incomplete caries removal, genetic diversity of s. mutans strains was observed in samples of deep caries lesions even after the contact with antibacterial materials, such as glass-ionomer cements26. analyzing each tooth individually, a maximum of nine different genotypic patterns was observed from all the collects. several studies have demonstrated genetic heterogeneity among s. mutans strains, obtained from saliva or dental biofilm samples10,13, especially in caries-active individuals14,17. some investigators have suggested that the genotype frequency can even vary among the oral sites, but a site-specific colonization of genotypes seems to exist in dental caries lesions12,18. some identical or highly genotypic patterns were isolated in more than one collect, in a same tooth. although few clones (s sm = 1) have been detected, the great genetic similarity could indicate resistance of s. mutans strains. genetic groups containing highly related s. mutans isolates are thought to have derived from a single ancestral cell9, which could have originated new strains with genetic variations. it is commonly accepted that genetic polymorphism between close species is determined by modifications in base pairing, by deletion or insertion of new genetic sequences27, in addition to clone transmission to external sources28. however, the frequency of these events in vivo is not known yet8. as a part of the evolution process, bacteria have the ability to gain genetic material from other cells using the mechanism of transformation. for recombination to occur, the foreign dna must share between 70 and 100% identity with the sequence in the recipient strain’s chromosome29. some s. mutans strains may acquire several cariogenic properties30, fluoride31 and antibiotic resistance32 by transformation. it is possible that s. mutans could act as a donor of dna to another species, such as s. sanguis and s. milleri33. cvitkovitch2 suggested that bacterial transformations can occur in environments which experience extreme changes and fluctuations in population dynamics, such as the oral cavity. li et al.34 have demonstrated that s. mutans cells are hypertransformable when grown in biofilms in vitro. bacteria in these environments are frequently exposed to various stress conditions, such as nutrient excess or shortage, low ph, high osmolarity and the consumption of antimicrobial agents by the host9,34. therefore, natural genetic transformation could be considered an important mechanism of cell’s adaptation to environmental changes, providing microbial resistance, genetic variation and rapid evolution of the virulence factors2,29,34. comparing s. mutans genotypic patterns isolated from carious samples after three months of the dental treatment, genetic diversity was observed among the teeth and high intra-tooth similarity was detected. these results may suggest that different resistant s. mutans strains have tooth-specific colonization, because it was not possible to verify a common genetic pattern within the sample subjected to the treatment proposed in this study. some investigators have shown genetic similarity among s. mutans genotypes obtained from members of the same family11,13,35-36 or individuals that cohabit in environments, such as nursery schools, denoting horizontal transmission37. generally, non-related subjects rarely share identical s. mutans genotypes10. nascimento et al.18 have compared 40 genetic types determined by ap-pcr, isolated from nine different patients and it was observed a great diversity among them, demonstrating that the maximum value of the similarity indices (s sm = 0.960) was observed only in isolates from the same individual. despite some deficiencies, such as the difficulty to visualize low-intensity bands and the need for more than one primer to increase the technique accuracy14, studies have shown the efficacy of ap-pcr in the detection of genetic polymorphism of various bacteteeth 1 2 3 4 5 6 7 8 9 10 isolates 15 12 15 16 21 15 18 20 21 20 genotypic patternsa 2 3 4 3 7 5 2 9 6 3 table 2 total of s. mutans strains and genotypic patterns found in each tooth from all collects (a, b and c) a genotypic patterns (genetic similarity s sm ≥ 0.869) among the collects were considered only once. 6 duque c, negrini tc, sacono nt, boriollo mfg, hofling jf, hebling j, spolidorio dmp braz j oral sci. 8(1): 2-8 0.00 0.25 0.50 0.75 1.0 tooth 1 tooth 2 tooth 3 tooth 4 tooth 5 0. 968 pc 1b2 1b3 1b5 1b6* 1c2 1c1 1c3 1c7 1c5 1a2 1b1 1a3 1c9 1a5 1c8 pc 2c3 2c2 2c8 2c5 2c6* 2c7 2c1* 2a1 2a2 2a8 2a7 2a5 pc 3a2 3a5 3c5 3c4* 3b7 3b1* 3a6 3b2 3b5 3c3 3a1 3a4 3a3 3b6 3c2 pc 4c3 4c4 4c5* 4b4 4b2 4b3 4a4 4a6 4c1 4a3 4c2 4c6* 4b1 4a1 4b5 4a2 pc 5b6 5b7 5a2 5a3 5a4 5a5 5b1 5b3 5c6* 5c7 5c3 5c4 5c5 5c2 5c1 5a1 5c8* 5b4* 5a6* 5b5* 5a7*  0.584 s strainssm 1.000 0.941 1.000 0.837 0.941 1.000 1.000 1.000 0.918 1.000 0.941 1.000 1.000 1.000 -------0.662 0.941 1.000 0.922 1.000 0.812 0.941 0.790 1.000 1.000 1.000 1.000 ------ 0.576 0.941 0.912 0.824 0.814 0.941 0.840 0.941 1.000 1.000 1.000 1.000 1.000 0.912 0.941 -------0.804 1.000 0.941 0.760 0.941 1.000 0.892 1.000 1.000 0.941 1.000 1.000 0.856 0.882 1.000 1.000 -------0.429 1.000 0.941 1.000 1.000 1.000 1.000 1.000 1.000 0.800 1.000 0.941 1.000 1.000 0.878 0.912 0.941 0.846 0.768 0.709 0.682 ------0.00 0.25 0.50 0.75 1.0 tooth 6 tooth 7 tooth 8 tooth 9 tooth 10 0. 968 pc 6c4* 6b4 6b1 6b2 6a4 6a6* 6b6* 6b3* 6c3 6a1 6a3 6b5 6a2 6a5 pc 7c1 7a2 7b6 7a4 7b1 7b2 7c5 7a3 7b5 7a1 7c7 7c2 7c3 7c6 7b3 7b4* 7a6 7a5 pc 8a6 8a5 8c4 8c2 8b5 8b7 8b8* 8b1 8b2 8a4 8b3* 8a8 8b6* 8a7 8c3 8a3* 8c5* 8a1* 8c1* 8b4* pc 9b1 9b6 9b7* 9a3 9c2 9c3 9a4 9a6 9a5 9c4 9c7 9a1 9c5 9c8 9c6 9c1* 9b2* 9b3 9b4 9b5* 9a2* pc 10a1 10a3 10c4 10a2 10a5 10a6 10b1 10b6 10b5 10b4 10b3 10b2 10c5 10c1 10c2 10c3* 10c8* 10c6 10a4 10c7 0.941 0.865 0.941 1.000 0.922 1.000 0.778 0.790 0.863 0.912 0.941 0.882 0.941 1.000 -- 0.702 0.941 0.912 0.941 1.000 1.000 1.000 0.888 1.000 0.941 0.880 0.941 1.000 1.000 0.926 0.918 0.555 0.941 ----- 0.693 0.941 0.876 1.000 0.941 1.000 1.000 0.839 1.000 0.941 0.922 0.868 0.941 0.827 0.882 0.882 0.812 0.737 0.670 0.706 ----- 0.774 1.000 0.941 0.787 0.941 1.000 0.922 1.000 0.882 0.941 0.912 0.874 0.941 1.000 1.000 0.926 0.817 0.843 1.000 0.941 0.739 ----- 0.747 0.941 1.000 0.876 0.941 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.935 0.929 1.000 1.000 0.809 0.843 0.941 1.000 ------strains ssm figure 1. genetic similarity indices (ap-pcr method, primer opa-13) verified among s. mutans strains sampled from caries lesions of each teeth, submitted to indirect pulp treatment. a: dendrograms obtained to isolates of teeth 1 to 5. b: dendrograms obtained to isolates of teeth 6 to 10. individual bands were analyzed by matrices generated by upgma analysis using coefficient ssm (simple matching). tonalities of gray in the dendrograms illustrate identical or highly related isolates (s sm ≥ 0,869) found in each tooth. * different genotypic patterns obtained in each tooth (s sm < 0,869). a b 7genetic polymorphism of streptococcus mutans strains associated with incomplete caries removal braz j oral sci. 8(1): 2-8 figure 2. genetic similarity indices (ap-pcr method, primer opa-13) verified among s. mutans strains sampled from caries lesions three months after dental treatment – collect c. dendrogram generated from upgma analysis, using coefficient s sm (simple matching). different tonalities of gray in the dendrograms illustrate identical or highly related isolates (s sm ≥ 0,869). pc 6c4 4c3 4c4 4c5 7c1 3c4 9c1 7c5 9c5 9c8 9c6 8c5 9c7 9c4 9c2 9c3 3c5 2c7 2c1 4c1 4c2 4c6 6c3 10c4 10c1 10c3 10c2 10c5 3c3 3c2 7c2 7c3 7c6 7c7 10c6 10c7 10c8 8c3 8c4 8c2 1c2 1c1 1c3 1c5 1c7 1c8 1c9 2c3 2c2 2c8 2c5 2c6 8c1 5c6 5c7 5c3 5c4 5c5 5c1 5c2 5c8 5b4 0.00 0.25 0.50 0.75 1.0 0. 968 0.941 0.838 1.000 0.941 0.863 0.809 1.000 0.926 0.941 1.000 1.000 0.873 1.000 0.941 0.856 1.000 0.789 0.762 0.941 0.892 0.941 1.000 0.833 0.941 0.912 1.000 1.000 0.871 0.802 0.882 0.941 1.000 1.000 0.926 0.853 0.941 0.846 0.733 0.882 1.000 0.710 0.941 1.000 1.000 1.000 0.929 1.000 0.621 0.941 1.000 0.922 1.000 0.776 0.731 1.000 0.926 1.000 1.000 0.941 0.873 0.849 0.750 -- ssm group 1 group 2 group 3 group 4 group 5 group 6 group 7 group 8 group 9 group 10 group 11 0.869  ssm  1 strains rial species10,16, obtaining similar results to more sophisticated techniques like mlee14. in this study, genetic heterogeneity was verified among s. mutans strains isolated form caries lesions, even three months after incomplete caries removal. the persistence of some identical genotypes and high genetic similarity among isolates of the same tooth, in distinct collects, denoted resistance of some s. mutans strains to dental treatment. the polymorphism observed in different teeth may suggest that resistant strains are specific to each tooth, since a common genetic pattern among individuals was not found. however, further studies are necessary to evaluate the phenotypic characteristics of different s. mutans genotypes resistant to indirect pulp treatment or another incomplete caries removal technique, observing possible similarities in the production of enzymes that participate of several virulence mechanisms of this species. acknowledgements the authors thank andréia cristina celi and carina bento luis macera for laboratorial assistance. this work was supported by the brazilian agencies: fundação de amparo à pesquisa do estado de são paulo (fapesp) by grant 04/00677-1, and coordenação de aperfeioçoamento de pessoal de nível superior (capes). references 1. bowden ghw, hamilton ir. survival of oral bacteria. crit rev oral 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transformation of fluoride resistance genes in streptococcus mutans. infect immun. 1989;57:1968-70. 32. murchison hh, barrett jf, cardineau ga, curtiss r 3rd. transformation of streptococcus mutans with chromosomal and shuttle plasmid (pya629) dnas. infect immun. 1986;54:273-82. 33. kuramitsu hk, trapa v. genetic exchange between oral streptococci during mixed growth. j gen microbiol. 1984;130:2497-500. 34. li yh, lau pc, lee jh, ellen rp, cvitkovitch dg. natural genetic transformation of streptococcus mutans growing in biofilms. j bacteriol. 2001;183:897-908. 35. klein mi, flório fm, pereira ac, höfling jf, gonçalves rb. longitudinal study of transmission, diversity and stability of streptococcus mutans and streptococcus sobrinus genotypes in brazilian nursery children. j clin microbiol. 2004;42:4620-6. 36. lindquist b, emilson cg. colonization of streptococcus mutans and streptococcus sobrinus genotypes and caries development in children to mothers harboring both species. caries res. 2004;38:95-103. 37. mattos-graner ro, li y, caufield pw, duncan m, smith dj. genotypic diversity of mutans streptococci in brazilian nursery children suggests horizontal transmission. j clin microbiol. 2001;39:2313-6. braz j oral sci. 15(1):39-44 original article braz j oral sci. january | march 2016 volume 15, number 1 epidemiological features of patients with nonsyndromic cleft lip and/or palate in western parana helenara salvati bertolossi moreira1,2, renato assis machado1, sibele nascimento de aquino3, ana lúcia carrinho ayrosa rangel2, hercílio martelli júnior1,4, ricardo della coletta1 1universidade estadual de campinas – unicamp, piracicaba dental school, department of oral diagnosis, piracicaba, sp, brazil 2universidade estadual do oeste do paraná – unioeste, department of physiotherapy, cascavel, pr, brazil 3universidade federal de juiz de fora ufjf, school of dentistry, area of stomatology clinic, governador valadares, mg, brazil 4universidade estadual de montes claros – unimontes, dental school, area of stomatology clinic, montes claros, mg, brazil correspondence to: renato assis machado department of oral diagnosis school of dentistry, unicamp cep 13414-018, piracicaba, são paulo, brazil phone: +55 19 21065315 e-mail: renatoassismachado@yahoo.com.br abstract aim: : to describe the clinical, demographic and environmental features associated with nscl/p (nonsyndromic cleft lip and/or palate) patients born in western parana state, brazil. methods: this cross-sectional, observational, retrospective study included 188 patients attended at the association of carriers of cleft lip and palate apofilab, cascavel-parana, between 2012 and 2014. information on demographic characteristics, medical and dental histories and life style factors were obtained from records and personal interviews. results: among the 188 patients, cleft lip and palate (clp) was the most frequent subtype (55.8%), followed by cleft lip only (clo, 25.0%) and cleft palate only (cpo, 19.2%). caucasian males were the most affected, although no differences among types of cleft were observed. the otorhinolaryngologic and respiratory alterations were the most frequent systemic alterations in nscl/p patients, and more than 80% of the nscl/p mothers reported no vitamin supplements during the first trimester of pregnancy. conclusions: this study revealed that the prevalence of nonsyndromic oral cleft types in this cohort was quite similar to previously reported prevalence rates. systemic alterations were identified among 23.4% of the patients and patients with clp were the most affected. history of maternal exposition to environmental factors related to nonsyndromic oral clefts was frequent and most mothers reported no vitamin supplements during the pregnancy. this study highlights the importance of identifying systemic alterations and risk factors associated with nscl/p in the brazilian population for planning comprehensive strategies and integrated actions for the development of preventive programs and treatment. keywords: cleft lip. epidemiology. risk factors. introduction nonsyndromic cleft lip and/or palate (nscl/p) represents the most frequent head and neck congenital malformations in the world1,2. they are traditionally divided in cleft lip only (clo), cleft lip and palate (clp) and cleft palate only (cpo). however, as there are similarities in both epidemiologic features and embryologic timing for both clo and clp, they are considered variants of the same defect and grouped together to form the group cleft lip with or without cleft palate (cl±p). the incidence of nscl/p is approximately 1 in 500-2500 live births and influenced by ethnic and environmental http://dx.doi.org/10.20396/bjos.v15i1.8647121 received for publication: april 11, 2016 accepted: june 15, 2016 40 factors3,4. in brazil, epidemiological studies demonstrated that the incidence of nscl/p varies from 1 per 685 to 2800 births5,6. approximately 70% of cl±p and 50% of cpo are isolated defects and the remaining are related to syndromes including clefts in the clinical spectrum7,8. the etiology of nscl/p, which involves both genetic and environmental factors, is highly complex and the molecular basis remains largely unknown3,9,10. epidemiological and experimental data suggest the exposure to tobacco smoke, agrotoxics, alcohol, poor nutrition and drugs as the main environmental factors to the development of oral clefts2,11,12. on the other hand, maternal ingestion of folic acid or multivitamins during early pregnancy can reduce the risk of nscl/p13-15. nscl/p results in morphological and functional morbidities associated with nutrition, speech, hearing, psychological and social aspects and imposes a substantial financial burden3,16. several of the nscl/p effects may extend into adulthood like an increased risk of cancer17-19. previous studies have also shown a high risk of cancer in relatives of patients with nscl/p18,20. this association has been explained, because cancer and nscl/p may share similar genetic defects, which may be segregated within family21,22. the aim of the present study was to report the epidemiological and clinical features of 188 patients with nscl/p from western parana state, brazil, and to evaluate the family background relevant to the disease. material and methods this observational cross-sectional study comprised a population of 188 patients with nscl/p treated at a reference association in cascavel-parana, brazil. this reference service has a multidisciplinary team of health care specialists, including plastic surgeons, dentists, psychologists, physiotherapists, pediatricians, nutritionists and speech therapist. patients were enrolled between 2012 and 2014 and all subjects were born in the study area. experienced professionals evaluated the patients and only those identified with nonsyndromic oral cleft were included in this study. mothers of nscl/p patients were interviewed and physical examination of the patients was made to establish the subtype of cleft and involved anatomical structures. the incisive foramen was the reference structure to classify nonsyndromic oral clefts. clefts were also classified by laterality and extent23. information on demographic characteristics, medical and dental histories and life style factors were obtained from medical records and personal interviews by a trained staff. the variables included gender, age, race/ethnicity, oral cleft type, systemic alterations, mother’s exposure to environmental factors, family history and parental consanguinity. race/ethnicity was established by a multivariate evolution based on skin color in the medial part of the arm, and hair color and texture as previously described24. all mothers of nscl/p patients were asked about their habits during pregnancy. they were considered smokers or to consume alcohol during the first trimester of pregnancy if any smoking or alcohol consumption was reported. maternal passive epidemiological features of patients with nonsyndromic cleft lip and/or palate in western parana braz j oral sci. 15(1):39-44 smoking was defined as being exposed to the smoke from more than one cigarette per day at home or at the workplace during the first trimester. the same is true for indirect contact with agrotoxics. positive exposure was considered when the person had environmental contact with agrotoxics or living near places with constant pesticide spraying25. direct contact with paints, solvents, fuel or laboratory substances were considered as other substances. mothers who used drugs like antibiotics, anticonvulsants or corticosteroids during the first trimester of pregnancy were classified as positive contact. data on vitamin supplement intake were also derived from the interview. women were asked whether they used multivitamins or folic acid supplements during the onemonth preconceptional period or first trimester. written informed consents were obtained from all participants, and the study was carried out with approval of the institutional human research ethics committee of the university (071/2012). statistical analysis was performed using the graphpad prism software version 6.01 for windows with a 5% significance level. chi-square tests assessed the frequency distributions of clinical, demographic and environmental characteristics with the subtypes of cleft. results a description of study participants is in table 1. among the 188 patients, 121 (64.4%) were male, resulting in a male to female 1.8:1 ratio. all types of oral clefts were more frequent in males. the median age of the patients at the first visit was 13 years. except for a few patients all were classified as caucasian. bilateral clefts were significantly more frequent in clp patients (33.3%) than in clo patients (4.3%, p=0.0001) and clefts with complete extent were significantly more common in clp compared to clo and cpo (p=0.0001). forty-four patients presented diagnosed systemic alterations associated with nscl/p. thirty-five patients had only 1 systemic alteration, whilst 9 of the nonsyndromic cleft patients had 2 or more alterations. otorhinolaryngologic and respiratory alterations were most frequent among nscl/p patients (table 2). palate involvement was essential for otorhinolaryngologic alterations, since patients with clo did not exhibit any associated alteration in the ears, nose or throat. allergic bronchitis was the most common associated alteration, more frequent in patients with clo and clp (table 2). table 3 depicts the rates of maternal exposure to environmental factors during the first trimester of pregnancy. in general, no significant differences were detected among the nscl/p groups regarding environmental factors, but the frequency of mothers that did not take vitamin supplements was very high. more than 80% of the mothers reported no vitamin supplements during the first trimester of pregnancy. no mother reported preconceptional use of vitamins or folic acid. according to table 4, consanguinity and history of miscarriages and stillbirths were not significantly different among the three types of cleft. similarly, no significant differences were observed regarding presence of orofacial cleft and cancer in the first degree relatives. 41epidemiological features of patients with nonsyndromic cleft lip and/or palate in western parana braz j oral sci. 15(1):39-44 cleft lip n (%) cleft lip and palate n (%) cleft palate n (%) otorhinolaryngology otitis 0 5 (16.7) 5 (22.7) tonsillitis 0 4 (13.3) 4 (18.2) allergic rhinitis 0 3 (10.0) 4 (18.2) sinusitis 0 0 2 (9.1) pharyngitis 0 1 (3.3) 0 respiratory allergic bronchitis 3 (27.3) 7 (23.3) 3 (13.6) recurrent pneumonia 1 (9.1) 1 (3.3) 1 (4.5) cardiovascular benign heart murmur 2 (18.2) 2 (6.7) 2 (9.1) dermatologic allergic dermatitis 1 (9.1) 3 (10.0) 0 gastrointestinal reflux 2 (18.2) 1 (3.3) 0 neurological seizure 0 2 (6.7) 0 ophthalmic visual deficiency 0 1 (3.3) 1 (4.5) musculoskeletal scoliosis 1 (9.1) 0 0 facial facial asymmetry 1 (9.1) 0 0 total 11 (17.5) 30 (47.6) 22 (34.9) cleft lip n (%) cleft lip and palate n (%) cleft palate n (%) p value gender male 30 (63.8) 69 (65.7) 22 (61.1) female 17 (36.2) 36 (34.3) 14 (38.9) 0.88 age 0-2 years 2 (4.3) 7 (6.7) 0 2-12 years 27 (57.5) 49 (46.7) 25 (69.4) 13--20 years 8 (17.0) 33 (31.4) 9 (25.0) >20 years 10 (21.2) 16 (15.2) 2 (5.6) 0.08 race/ethnicity caucasian 42 (89.4) 93 (88.6) 32 (88.8) non-caucasian 5 (10.6) 12 (11.4) 4 (11.2) 0.98 cleft side unilateral 45 (95.7) 70 (66.3) 0 bilateral 2 (4.3) 35 (33.3) 0 0.0001 cleft extent incomplete 25 (53.2) 18 (17.2) 18 (50.0) complete 22 (46.8) 87 (82.8) 18 (50.0) 0.0001 table 1 distribution by gender and race and clinical extent of the clefts. table 2 distribution of systemic alterations in patients with nonsyndromic cleft lip and/or palate. discussion different studies were conducted worldwide to evaluate nscl/p distribution, often resulting in varying prevalence rates26,27. however, in most studies, the percentage of patients with clp is higher compared to that of clo or cpo28-30. similar results were described in previous studies with brazilian nscl/p patients29,31. in the present study, the findings revealed 105 (55.8%) patients with clp, 47 (25%) with clo and 36 (19.2%) with cpo. moreover, in some studies were observed differences in the distribution of nscl/p between males and females28,29. this study showed that clo, clp and cpo prevailed in males. investigating the epidemiological features of nscl/p patients treated at the center for rehabilitation of craniofacial anomalies in minas gerais, brazil, martelli-junior et al. (2008) revealed prevalence of cpo in females, whereas males were more affected by other types of cleft32. unilateral involvement was more common than bilateral in clo and clp patients. however, according to the literature, frequency of bilateral clp is significantly higher than bilateral clo28,33,34. in addition, patients with bilateral clp presented more frequently nasal deformities and nasopharyngeal depths than unilateral clp35,36. involvement of the palate in patients with oral cleft is related to facial and airway structures disruption and in an increased risk of sleep and breathing disorders37. in this study, complete clp was significantly more frequent than complete clo or cpo. according to the literature, alterations in nscl/p patients are complex and beyond the facial structure. feeding difficulties, speech alterations, recurrent middle ear infections and other difficulties are frequently observed3,28. in this study, 23.4% of oral cleft patients had at least one associated systemic alteration. the highest incidence of systemic alterations was observed in clp patients. generally, the most anomalies occur in cases involving the palate rather than the lip and higher rates of hospitalization before age two in children with cp or clp are reported38. in a study of 5,449 cases from 23 european birth registries, anomalies were found in 20.8% of clo patients and in 34.0% of clp patients39, and another study found anomalies in 12.2% of clo patients, 35.1% with clp and 36.7% with cpo38. this study confirmed that associated otorhinolaryngologic, respiratory and cardiovascular defects are frequent38,40,41. children born with cleft palate may need more attention and closer monitoring for a long time. many studies have identified a relationship between environmental risk factors and nscl/p2,11,12. although cigarette smoking, alcohol consumption (drinking) and exposure to agrotoxics among the mothers were the most common environmental factors, the present results showed no differences among types of cleft. with respect to lack of association between environmental factors of parents and risk for orofacial cleft in this study, in contrast to other studies11,42,43. the current data support the possibility that exposure to risk factors has a different effect on cleft risk among parents, which may reflect a role for genetic susceptibility factors in cleft development9,44. in this study, over two-thirds of mothers did not take vitamin supplements in early pregnancy. low concentrations of micronutrients increase significantly the risk of orofacial cleft45. recent reports suggest that 42 epidemiological features of patients with nonsyndromic cleft lip and/or palate in western parana table 3 frequency of maternal exposure to environment factors during the first trimester of pregnancy, according to patients with nonsyndromic cleft lip and/or palate. cleft lip n (%) cleft lip and palate n (%) cleft palate n (%) p value cigarette smoking no 40 (85.1) 93 (88.6) 30 (83.3) yes 7 (14.9) 12 (11.4) 6 (16.7) 0.67 passive smoking no 44 (93.6) 92 (87.6) 34 (94.4) yes 3 (6.4) 13 (12.4) 2 (5.6) 0.33 alcohol consumption no 43 (91.5) 99 (94.3) 33 (91.7) yes 4 (8.5) 6 (5.7) 3 (8.3) 0.76 illicit drugs no 46 (97.9) 105 (100) 36 (100) yes 1 (2.1) 0 0 0.22 drugs (medications) no 39 (83.0) 97 (92.4) 30 (83.3) yes 8 (17.0) 8 (7.6) 6 (16.7) 0.14 chemicals non-exposure 39 (83.0) 74 (70.5) 28 (77.8) agrotoxics 1 (2.1) 12 (11.4) 4 (11.1) environment contact with agrotoxics 4 (8.5) 14 (13.3) 4 (11.1) other 3 (6.4) 5 (4.8) 0 0.34 vitamin supplementation no 37 (78.7) 89 (84.8) 25 (69.4) yes 10 (21.3) 16 (15.2) 11 (30.6) 0.13 table 4 characteristics of parents and relatives of the patients with nonsyndromic cleft lip and/or palate. cleft lip n (%) cleft lip and palate n (%) cleft palate n (%) p value consanguinity no 45 (95.7) 100 (95.2) 34 (94.4) yes 2 (4.3) 5 (4.8) 2 (5.6) 0.96 miscarriage no 43 (91.5) 98 (93.3) 35 (97.2) yes 4 (8.5) 7 (6.7) 1 (2.8) 0.56 stillbirth no 45 (95.7) 102 (97.1) 36 (100) yes 2 (4.3) 3 (2.9) 0 0.48 orofacial cleft in 1st degree relative no 37 (78.7) 80 (76.2) 28 (77.8) yes 10 (21.3) 25 (23.8) 8 (22.2) 0.93 cancer in 1st degree relative no 30 (63.8) 74 (70.5) 20 (55.6) yes 17 (36.2) 31 (29.5) 16 (44.4) 0.24 use of vitamins containing folic acid during the early pregnancy may decrease risk of nscl/p46,47. additionally, variations on genes related to absorption, transport and metabolism of vitamins may have key roles in nscl/p predisposition48. regarding family recurrence, 43 (29%) patients had a family history of nscl/p among first-degree relatives with little variation among cleft types. this is similar to other studies where a prevalence of oral clefts in other family members was found to range between 18% and 30.5%34,49. furthermore, the finding that clo patients were less likely to have a positive family history of oral clefts than patients with clp and cpo was consistent with grosen et al. (2010)50. akin marriages are an important factor in the development of a host of genetic anomalies as well as increased risk of oral cleft34,51. the akin relationships observed in the present study (4.9%) were slightly more common than what was observed by leite and koifman (2009)52. in addition to the congenital anomalies, the consanguinity is also associated with increased risks of low birth weight, preeclampsia, which in turn are risk factors for stillbirth, especially preterm stillbirth51. in this study, the frequency of miscarriage and stillbirth was of 6% and 2.4% respectively, which are quite similar to average population. this study also revealed that cancer frequency in relatives of patients born with oral clefts ranged from 29.5 to 44.4%. these findings agree with previous studies which suggested that individuals born with nscl/p have a higher risk for cancer18,19,53. recent evidences from genetic studies have supported the hypothesis that some genes are simultaneously 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np, elkodary hm, little j, mossey pa. passive smoking in the etiology of non-syndromic orofacial clefts: a systematic review and meta-analysis. plos one. 2015 mar 11;10(3):e0116963. doi: 10.1371/journal.pone.0116963. 44. wu t, schwender h, ruczinski i, murray jc, marazita ml, munger rg, et al. evidence of gene-environment interaction for two genes on chromosome 4 and environmental tobacco smoke in controlling the risk of nonsyndromic cleft palate. plos one. 2014 feb 6;9(2):e88088. doi: 10.1371/journal.pone.0088088. 45. mckinney cm, chowchuen b, pitiphat w, derouen t, pisek a, godfrey k. micronutrients and oral clefts: a case-control study. j dent res. 2013 dec;92(12):1089-94. doi: 10.1177/0022034513507452. 46. figueiredo rf, figueiredo n, feguri a, bieski i, mello r, espinosa m, et al. the role of the folic acid to the prevention of orofacial cleft: an epidemiological study. oral dis. 2015 mar;21(2):240-7. doi: 10.1111/ odi.12256. 47. molina-solana r, yanez-vico rm, iglesias-linares a, mendoza-mendoza a, solano-reina e. current concepts on the effect of environmental factors on cleft lip and palate. int j oral maxillofac surg. 2013 feb;42(2):177-84. doi: 10.1016/j.ijom.2012.10.008. 48. de aguiar pk, coletta rd, de oliveira am, machado ra, furtado pg, de oliveira la, et al. rs1801133c>t polymorphism in mthfr is a risk factor for nonsyndromic cleft lip with or without cleft palate in the brazilian population. birth defects res a clin mol teratol. 2015 apr;103(4):292-8. doi: 10.1002/bdra.23365. 49. ravichandran k, shoukri m, aljohar a, shazia ns, al-twaijri y, al jarba i. consanguinity and occurrence of cleft lip/palate: a hospital-based registry study in riyadh. am j med genet a. 2012 mar;158a(3):541-6. doi: 10.1002/ajmg.a.34432. 50. grosen d, chevrier c, skytthe a, bille c, molsted k, sivertsen a, et al. a cohort study of recurrence patterns among more than 54,000 relatives of oral cleft cases in denmark: support for the multifactorial threshold model of inheritance. j med genet. 2010 mar;47(3):162-8. doi: 10.1136/ jmg.2009.069385. 51. maghsoudlou s, cnattingius s, aarabi m, montgomery sm, semnani s, stephansson o, et al. consanguineous marriage, prepregnancy maternal characteristics and stillbirth risk: a population-based case-control study. acta obstet gynecol scand. 2015 oct;94(10):1095-101. doi: 10.1111/ aogs.12699. 52. leite ic, koifman s. oral clefts, consanguinity, parental tobacco and alcohol use: a case-control study in rio de janeiro, brazil. braz oral res. 2009 jan-mar;23(1):31-7. 53. menezes r, marazita ml, goldstein mchenry t, cooper me, bardi k, brandon c, et al. axis inhibition protein 2, orofacial clefts and a family history of cancer. j am dent assoc. 2009 jan;140(1):80-4. original articlebraz j oral sci. april/june 2009 volume 8, number 2 freshwater and salt-water influence in human identification by analysis of dna: an epidemiologic and laboratory study jamilly de oliveira musse1, amanda da costa nardis2, evelyn k. anzai3, mário h. hirata4, regina maria barreto cicarelli5, rogério nogueira de oliveira6 1 dds, msc, phd student in community dentistry, faculdade de odontologia, universidade de são paulo (usp), são paulo (sp), brazil 2 undergraduate dental student, faculdade de odontologia, usp, são paulo (sp), brazil 3 phd in clinical analysis, faculdade de ciências farmacêuticas, usp, são paulo (sp), brazil 4 dds, msc, phd, associate professor, faculdade de ciências farmacêuticas, usp, são paulo (sp), brazil 5 dds, msc, phd, full professor, faculdade de ciências farmacêuticas, usp, são paulo (sp), brazil 6 dds, msc, phd, professor, faculdade de odontologia, usp, são paulo (sp), brazil received for publication: december 22, 2008 accepted: may 18, 2009 correspondence to: jamilly de oliveira musse avenida prof. mello de moraes, 1.235 – bloco c – apto. 205 – cidade universitária – butantã cep 05508000 – são paulo (sp), brazil e-mail: jamillymusse@usp.br; musse_jo@hotmail.com abstract aim: to investigate the casuistry of drowning cases by reviewing the records from the forensic medicine institute nina rodrigues in the city of salvador, ba, brazil, and to verify the potential of dna recovery in human teeth immersed in water. methods: an epidemiological survey was conducted followed by a laboratorial phase, in which 40 teeth were immersed in fresh and salt-water, the dna was extracted by the organic method and amplified by polymerase chain reaction, using the amelogenin as initiator. the electrophoresis initially occurred in agarose gel and later in polyacrylamide gel. results: in the present survey, 346 deaths from drowning were observed, most of them in salt-water (51.73%), with a predominance of male victims (86.13%) aged from 18 to 35 years-old (37.94%). dentists identified 14.74% of the victims. dna was recovered in 37.5% from the samples, most from teeth immersed in freshwater. polyacrylamide gel analysis in samples that were amplified in agarose gel allowed correct gender identification in 83.3% of the cases. however, allele loss was observed in samples of two victims, jeopardizing gender determination. conclusions: dental exposure to water interfered in dna recovery. the gender investigation using the amelogenin as initiator was effective. keywords: human identification, forensic dentistry, teeth, dna, drowning. introduction forensic dentistry has contributed to human identification for a long time. dentists, enforced by law #5.081/66, are able to perform reports involving biological materials derived from human body under several conditions (quartered, dilacerated, carbonized, macerated, decomposed, in skeletonization and skeletonized), with the goal of establishing human identity1. historically, fingerprints have been used in identification. however, in certain situations, in which the body is found in advanced stage of putrefaction, fingerprints are easily destroyed. moreover, the technical experts frequently need to use comparative antemortem elements, such as dental records, in human body identification, but they are not always available. before the development of molecular biology techniques, these situations would make the experts establish the victim’s identification without the comparative elements. whenever the documen72 musse jo, nardis ac, anzai ek, hirata mh, cicarelli rmb, oliveira rn braz j oral sci. 8(2): 71-5 tation was available, but the corpses were degraded, the exams would try to recover the individual’s profile by establishing the species, gender, built, age, phenotype, skin color, among other characteristics2. nowadays, biomolecular resources have been employed in human identification whenever the use of traditional methods is not viable. applying these resources allows identifying victims even without antemortem information or with deteriorated biological material in insignificant amounts, which are relatively frequent conditions in forensic analysis, especially in mass accidents3. in this context, some authors have emphasized the importance of gender investigation in victims’ identification and the need of the joint action among several healthcare workers (physicians, dentists, psychologists, geneticists, radiologists) and professionals from related sciences (anthropologists, technical examiners, among others) in this process4,5. recently reported in the media, events such as the tsunami, in thailand, in 2004 and in indonesia, in 2006, the hurricanes “katrina” in new orleans and “rita” in texas, both in 20056, reinforce the need of mastering dna extraction techniques in the process of identifying skeletonized bodies or bodies in advanced state of putrefaction, under the influence of aqueous environment7. the practical applicability of these methods can be observed in several studies that used dna-based techniques in the identification of victims, whose bodies had undergone the action of water8,3. the influence of environmental factors on dna stability has been emphasized, since corpses or their pieces are frequently found carbonized, submersed or buried. according to schwartz et al.9, those factors can interfere in the amount of recovered dna and influence in the identification process9. according to bender et al.10, this is due to the fact that the rate of dna degradation varies according to light conditions, water content and temperature, and bacterial and fungal contamination might occur followed by microbial growth, and resulting in the physical, chemical and biological degradation of the genomic dna10. although mass accidents, including natural disasters, have been poorly documented in brazil, records from the ministry of health report a significant increase in mortality rate due to external causes, among which drowning is mentioned. in this sense, the official records report the occurrence of 57,595 deaths by drowning and accidental submersion between 1996 and 2004, which represents 0.67% from the total number of deaths that occurred in the country in this period11. concern is expressed when the brazilian coastal line extension and its population concentration are considered, especially in bahia, where, according to the brazilian institute of geography and statistics12, there is a prevalence of coastal cities. the present study aimed at understanding the casuistry in situations involving drowning cases, and verifying the potential of dna recovery from teeth immersed in water. material and methods an epidemiological survey was conducted with records from the forensic medicine institute nina rodrigues in salvador, ba, brazil, between 2003 and 2005. initially, information such as number of deaths by drowning, gender, age, type of water in which the body was submerged (freshwater or salt-water) and the contribution or not of forensic dentistry in the victims’ identification was collected. later, a research laboratorial phase was carried out. the sample was composed by 40 teeth from 20 individuals (two from each victim). saliva in mgm® cards was collected from oral mucosa swabs. the teeth were submitted to a physicochemical process for tooth-cleaning, through scaling with curettes and 70% alcohol-wash and then immersed in fresh and salt-water for one month, according to schwartz et al.9. after the immersion, the tooth crown was sectioned13 and pulverization was done according to the method proposed by sweet and hildebrand14, adapted to the use of a mortar and pestle, and manual storage by immersion in liquid nitrogen. dna extraction from the saliva stored on cards was performed with those fragments using a fta kit. the protocol to extract dna from the teeth was based on the organic method described by hochmeister et al.15. the region for amplification by polymerase chain reaction (pcr) was the amelogenin. the sequences of initiators were sense: acctcatcctgggcaccctgg (21bp) and anti-sense: aggcttgaggcca accatcag (21bp). the following cycling protocol was used: denaturation at 96° c for two minutes, followed by ten denaturation cycles at 94 °c for one minute, hybridization at 64 °c for one minute, elongation at 72 °c for one minute and 30 seconds and final elongation at 72 oc for ten minutes. the pcr products were initially analyzed in agarose gel at a 1.5% concentration. electrophoretic separation occurred in 100 v and 60 ma for 40 minutes. later, the samples that amplified in the agarose gel were submitted to electrophoresis in silver-stained 8% polyacrylamide gel. results the review of the records referring to the total of deaths occurred between 2003 and 2005 showed a prevalence of only 2.7% deaths resulting from drowning. the records also revealed a higher prevalence of death in male individuals and young adults (37.94%), as seen in figures 1 and 2. regarding the type of water in which the bodies were submerged, salt-water prevailed, followed by freshwater and swimming pool water, except for 2003, when the first two categories were inversed (figure 3). when evaluating forensic dentistry contribution to victim identification, little participation of the forensic dentist was observed (figure 4). during the laboratorial phase, the dna was initially by electrophoresis in 1.5% agarose gel (figure 5). in this phase, genetic material recovery was observed in 37.5% of the dental samples, with a predominance of teeth immersed in freshwater (45%) over teeth immersed in salt-water (30%). all saliva samples used as controls presented positive amplification. the analysis of polyacr ylamide gel in 27 samples (15 teeth and 12 saliva samples), from 12 v ictims w ith positive amplif ica73freshwater and salt-water influence in human identification by analysis of dna: an epidemiologic and laboratory study braz j oral sci. 8(2): 71-5 87.50% 86.90% 88.89% 12.50% 13.10% 11.11% 0.00% 10.00% 20.00% 30.00% 40.00% 50.00% 60.00% 70.00% 80.00% 90.00% 100.00% 2003 2004 2005 male female figure 1. percent distribution of drowning victims according to gender, salvador, 2003 to 2005. 15% 17.64% 37.94% 21.77% 1.76% 5.89% 0% 5% 10% 15% 20% 25% 30% 35% 40% 0 to 11 years 12 to 17 years 18 to 35 years 36 to 59 years 60 or more not informed figure 2. percent distribution of drowning victims according to age, salvador, 2003 to 2005. 46% 28,70% 36.68% 43.00% 59.00% 57.77% 3.90% 2.46% 1.11% 7% 9.84% 4.44% 0% 10% 20% 30% 40% 50% 60% 70% 2003 2004 2005 freshwater salt-water pool not informed figure 3. percent distribution of drowning victims according to the place where the body was found, salvador, 2003 to 2005. 33% 4.00% 4.44% 67.20% 96.00% 95.56% 0% 20% 40% 60% 80% 100% 120% 2003 2004 2005 yes no figure 4. percent distribution of drowning cases according to the contribution of forensic dentistry in victim identification, salvador, 2003 to 2005. 1 2 3 4 5 6 figure 5. photograph in 1.5% agarose gel stained with ethidium bromide, after amel locus amplification. samples: 1 = 100 pb marker; 2 = saliva; 3 = tooth submerged in salt-water; 4 = tooth submerged in freshwater; 5 = pcr negative control and 6 = pcr positive control. 1 2 3 4 5 6 7 8 9 10 a b c figure 6. photograph in 8% polyacrylamide gel stained with silver. samples: 1, 5 and 8 = tooth submerged in salt-water; 2 and 6 = tooth submerged in freshwater; 3, 7 and 9 = saliva; 4 and 10 = 10 pb standard. a: male; b: female; c: no gender identification. tion in agarose gel, allowed the v isualization of the amelogenin different alleles, enabling the correct identif ication of gender in 83.3% of the cases (ten indiv iduals). moreover, an adequate correlation was obser ved between band intensit y and the t y pe of material used in the exam (tooth or saliva) (fig ure 6). however, loss of alleles in the polyacr ylamide gel was obser ved in the samples of two indiv iduals, jeopardizing gender identif ication in these cases. 74 musse jo, nardis ac, anzai ek, hirata mh, cicarelli rmb, oliveira rn braz j oral sci. 8(2): 71-5 discussion the mortality rates due to external causes in brazil, among which drowning, have increased significantly in the last few years, reaching the second place in brazilian mortality statistics16. thus, it is necessary to know the population profile that have mostly contributed to this statistics, in such a way that planners and executors of government policies are able to define, in concrete basis, actions that should be a priority, in order to contemplate prevention and attention to those victims. in this sense, the findings of the present epidemiological survey showed prevalence of 2.7% of deaths by drowning in the city of salvador. baptista et al.17 and martins and andrade18 have found 26 and 19.5% of deaths by drowning in portugal and in paraná/brazil, respectively. these discrepant percent values can be attributed to the methodological differences in the studies and their duration. regarding the distribution of deaths by drowning, the findings of the present study agree with the literature19,20 regarding the most prevalent gender (male) and age group (adults). the findings of a 12-year survey of deaths by drowning stated that 63% of the cases occurred in freshwater18, while another study19 found a predominance of death cases in salt-water. in the present research, most cases occurred in salt-water, which is probably due to the fact that 95% of the water in the brazilian territory is formed by salt-water20. there is a low prevalence of forensic dentistry contribution in the identification of victims by drowning in the present study. forensic dentists participated in only 14.74% of the cases within three years. this result is probably due to the lack of forensic experts in bahia during the period of this study, since the first public selection for these positions occurred in 200621. the results of the review of the records referring to the total of deaths in the forensic medicine institute served as basis for the next phase of the present study: the laboratorial. considering the recent world casuistry of mass accidents involving the aquatic environment, and the consequent need of mastering the dna-based technique in the process of identifying victims that were under the influence of water, teeth were immersed in fresh and salt-water in order to simulate cases of forensic investigation. in this sense, one of the first steps to proceed with the victim identification is gender investigation4, usually performed according to anatomical characteristics from male and female genitals. however, when forensic examination has to be done in skeletonized corpses or bodies in advanced state of putrefaction, bones and teeth are, many times, the only materials available and identification can be performed by molecular biology techniques, using amelogenin analysis22. in the present paper, the analysis by electrophoresis in agarose gel showed only one dna region for gender investigation (amelogenin). genetic material recovery was possible in only 37.5% of the teeth, demonstrating that the water interfered directly in dna preservation. on the other hand, dna from all saliva samples used as controls was successfully amplified. there was greater dna degradation in salt-water (70%) compared to freshwater (55%), probably due to the chemical composition of both aqueous conditions. both types of water present ions such as: calcium, magnesium, sodium, potassium, bicarbonate, chloride, sulfate, nitrate, among others. traces of lead, copper, arsenic, manganese and a large spectrum of organic compounds from decomposition of organic matter of animal and vegetal origin can also be found. in addition, in some cases, residues from agricultural fields and disposal of effluents from domestic and industrial origin are present, varying from humic acids to synthetic organic compounds such as detergents, pesticides and solvents20. it was not possible to determine the mechanism by which the water affects dna recovery. however, some components present in aquatic ecosystems have already been reported as being able to degrade genetic material (microbial growth, humidity)10 or inhibit the taq dna polymerase enzyme (humic acid). this enzyme is responsible for the incorporation of free nucleotides, which will form a new fragment of the dna molecule. thus, its inhibition is a hindrance to pcr 23. the analysis of the agarose gel does not allow gender identification due to a slight difference in base pairs6 present in men and women, when the amelogenin is used as a region. in this sense, mukherjee and biwas24 have suggested the use of polyacrylamide gel in those cases because it promotes a better band separation and, consequently, allele visualization. running polyacrylamide gel was performed in samples that amplified in agarose gel, enabling the accurate visualization of the amelogenin alleles, and identifying correctly the gender of ten victims. however, although gender determination using amelogenin is a recognized and scientifically accredited methodology, loss of alleles in the polyacrylamide gel was observed in the samples of two individuals. pinheiro1 comments on this possibility when he states that sometimes dna degradation, instead of impeding the results, can cause the visualization of one allele, instead of two, and the disappearing of the allele with larger dimension is more frequent. hence, when old vestiges are analyzed and homozygous profile is obtained by some systems, one should be careful using the results because they might be from a heterozygous profile. an alternative to solve this problem was proposed by steinlechner et al.25, while studying the genetic profile of 29,432 men stored in an australian database, they verified the absence of pcr product specific of the amelogenin related to chromosome y in six individuals. after changing the methodology and analyzing eight short tander repeat (str) from the same chromosome, the complete genetic profile of five victims was obtained, confirming their male genotype. the error rate in gender investigation using the amelogenin test in the present work was 0.018%. a factor that may lead to dna non-amplification is the quality of the forensic biological sample. the insignificant amount of biological material to dna extraction may result in absence of the target sequence in the fraction used for the reaction or the same can be degraded, not allowing dna amplification by pcr24. in an attempt to minimize the effects of this degradation and even as legal endorse75freshwater and salt-water influence in human identification by analysis of dna: an epidemiologic and laboratory study braz j oral sci. 8(2): 71-5 ment to avoid the exam result to be questioned, it is necessary that forensic laboratories keep the chain-of-custody of the samples. the records must be readily accessible and followed since data collection until the final disposition, in such a way to warrant that every precaution has been taken to avoid falsification, break, loss or contamination of the samples. in addition, inadequate procedures that may lead to sample contamination, by the lab investigators and forensic professionals, may result in erroneous interpretation of the genetic profile26. therefore, the maintenance of good laboratorial practices is essential and specific training on dna-based identification techniques is mandatory. the following conclusions may be draw from the obtained results: the epidemiological survey allowed outlining the population profile that most contributed to the statistics surveyed, during a three-year period in the forensic medicine institute of salvador; there was a predominance of adult male victims exposed to the action of salt-water; tooth exposure to water interfered directly in dna recovery; despite its well-established efficacy, recognition and credibility, gender investigation by amelogenin, as every procedure that uses biological molecular resources, requires a careful interpretation of the results by the researcher, therefore, the observation of all the criteria related to the maintenance of the chain-of-custody and 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[2006 jan. 12]. disponível em: url: http://www. egba.ba.gov.br/diario/_dodia/do_frm0.html. 22. murakami h, yamamoto y, yoshitome k, ono t, okamoto o, shigeta y, et al. forensic study of sex determination using pcr on teeth simples. acta med okayama. 2000;54:21-32. 23. burger j, hummel s, hermann b, henke w. dna preservation: a microsatellitedna study on ancient skeletal remains. electrophoresis. 1999;20:1722-8. 24. mukherjee kk, biswas r. short tandem repeat (strs) and sex specific amelogenin analysis of blood samples from neurosurgical female transfused patients. j clin forensic med. 2005;12:10-3. 25. steinlechner m, berger m, niederstätter h, parson w. rare failures in the amelogenin sex test. int j legal med. 2002;116:117-20. 26. bonaccorso ns. aplicação do exame de dna na elucidação de crimes. são paulo: faculdade de direito da usp; 2005. 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1534/1187 1/9 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1534/1187 2/9 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1534/1187 3/9 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1534/1187 4/9 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1534/1187 5/9 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1534/1187 6/9 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1534/1187 7/9 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1534/1187 8/9 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1534/1187 9/9 revista fop n 13 1543 braz j oral sci. april/june 2008 vol. 7 number 25 susceptibility of susceptibility of susceptibility of susceptibility of susceptibility of candidacandidacandidacandidacandida spp. oral isolates for azolic spp. oral isolates for azolic spp. oral isolates for azolic spp. oral isolates for azolic spp. oral isolates for azolic antifungals and amphotericin bantifungals and amphotericin bantifungals and amphotericin bantifungals and amphotericin bantifungals and amphotericin b vivian fernandes furlletti1,2; rita de cássia mardegan1,2; gustavo alberto obando-pereda1; paula cristina aníbal1,2; marta cristina texeira duarte2; reginaldo bruno gonçalves1; josé francisco höfling1 1department of microbiology and immunology, dental school of piracicaba, university of campinas unicamp, 13414-903 piracicaba, sp, brazil. 2research center for chemistry, biology and agriculture, p.o.box 6171 university of campinas – unicamp, 13083-970 campinas, sp, brazil. received for publication: may 8, 2008 accepted: july 31, 2008 correspondence to: josé francisco höfling piracicaba dental school, university of campinas, piracicaba, são paulo, brazil cep: 13414-903 phone: +55 19 21065321/ 21065322 email address: hofling@fop.unicamp.br a b s t r a c t aims: among the oral infections, candidosis may be considered the most frequent, and c. albicans the most prevalent species. meanwhile, the non-albicans species may also be related to other infections processes and be able to affect the oral cavity, including periodontal disease. in this sense, understanding the relationship between candida spp. and host, it is necessary and justified the search of mechanisms modulators of infections and treatments against diseases associated with these yeasts. methods: nineteen patients with periodontal disease were involved in this study. the aim was evaluate the susceptibility to azoles antifungals fluconozole, itraconazole, ketoconazole and the polienic anfotericin b against candida spp isolated from three different sites of the oral cavity from these patients (periodontal disease, being periodontal pocket, oral mucosa and ridge gingival), by the minimum inhibitory concentration method – mic. results: among the samples of c. albicans, 88% showed susceptibility depending on the concentration (scd) and 3.6 % were resistant to at least one antifungal azole studied. among the others species, 57% presented sdc and 42.8% showed resistance to at least one of the antifungal azole tested. regarding to anfotericin b, 90% of the c. albicans isolates and 3% of the nonalbicans showed resistance. there was no occurrence of resistance to the fluconazole and only 3.6% of c. albicans and 40% of the non-albicans were sdc to this antifungal. conclusions: patients with periondontal disease showed relevant levels of colonization by candida spp, mainly at the oral mucosa and periodontal pocket showing important occurrence of sdc and resistance to the antifungals drugs tested. key words periodontal disease, candida spp., antifungical, minimal inhibitory concentration. i n t r o d u c t i o n fungal infections are more common today than ever before. there are a number of reasons for this. people are living longer, and older people are more likely than younger people to have compromised immune systems, a major risk factor for fungal infection. similarly, the widespread use of antibiotics has contributed to the growing infection rate (fungal infections are known to occur after antibiotic therapy, which has the effect of killing the beneficial bacteria that normally suppress fungi). finally, the success in treating diseases like hiv/aids has created a subgroup of the population susceptible to fungal infections1. the most common organism implicated in fungal infections is the ubiquitous candida, which is found in the human digestive tract, mouth, and genital region2. under normal circumstances, levels of candida are controlled by commensal bacteria. however, if the bacteria-fungi balance is upset by the use of antibiotics, for example, or if the immune system is compromised, an overgrowth of candida could occur, resulting in infection3. more than 20 different species of candida have been reported as etiologic agents of invasive candidiasis in humans4-5 though more than 90% of invasive infections due to candida spp. can be attributed to species c. albicans, c. glabrata, c. parapsilosis, c. tropicalis, and c. krusei. the roll of reported species continues increasing as laboratories are pushed to provide an identification to the species level as an aid in optimizing therapy of candidal infections6-10. likewise, the diverse array of opportunistic yeasts and yeast-like fungi and their variable susceptibilities to both 1544 new and established antifungal agents has made the need for prompt identification of non-candida yeasts from clinical material much more compelling 6,11-12. our understanding of the frequency of occurrence and the antifungal susceptibility of both candida and non-candida yeasts has been enhanced in recent years through the efforts of several large surveillance programs conducted throughout the world8,13-20. fungal overgrowth is encouraged by certain ph levels and the availability of sugar (glucose) 21-23. people with the right conditions for fungal infection, such as a high sugar diet, are at higher risk. also, candida infections can be spread to vulnerable people with depressed immune systems who are in the hospital, where the fungus is commonly found on the hands of caregivers and where indwelling catheters can allow an infection to take hold. the number of available drugs for the treatment of systemic fungal infections is limited. the antifungals known nowadays can be classified into azolic and polienics. the azolics are elected in the first instance for treating these diseases and are generally fungistatics, while the latter are fungicides. among azolic are the fluconazole, ketoconazole and itraconazole. among polienics we found the amphotericin b and nystatin. in recent years, the amphotericin b and azoles mainly ketoconazole, fluconazole and itraconazole have been the drugs of choice in therapy24. the mechanism of action of the azoles drugs is based on the inhibition of sterol-14-to-desmetilase, an enzyme system dependent microsomal cytochrome p450, hindering the synthesis of ergosterol in cytoplasmic membrane and leading to accumulation of 14-to-methylsterols. these methyl-sterols not have the same form and physical properties that ergosterol and lead to the formation of the membrane with properties changed, that does not perform the basic functions necessary for the development of the fungus. the polienics connecting to a portion sterol, basically ergosterol present in the membrane of fungus is forming pores or channels. the result is an increase in permeability of the membrane that allows the escape of several small molecules, leading to cell death. the amphotericin b is a broad-spectrum antibiotic fungicide and powerful, but its use is associated with significant adverse effects such as nephrotoxicity with chills and fever, and acute reaction to the intravenous infusion, since the pharmacokinetics of this drug does not allow oral administration24. new formulations of amphotericin b, in the form of liposomes and colloidal dispersion, produce fewer side effects, as a result of redistribution of the drug in tissue and the selectivity of release, but the price of these formulations is often greater than that of old 25-27. the azoles cause less adverse reactions that the amphotericin b, but are less powerful than the same. they may have action fungistatic or fungicide. excessive use of azoles led to the emergence of resistance in species susceptible. moreover, the azoles still have the disadvantage of cross-resistance24, 28. the availability of information microbiological and epidemiological helps the doctors to choose the most appropriate antimicrobial agent for the treatment of infections. these susceptibility tests measure the ability of a microbial agent inhibit the growth of microorganisms in vitro. the microorganisms can be classified in categories according to drugs susceptibility, which may be sensitive (s), concentration dependent susceptibility (sdc) or resistance (r). sensitive is the microorganism whose infection caused by it are prone to respond to treatment with the drug; sensitivity concentration dependent is the one whose infection depends on adequacy of dose to be controlled and resistance is the body that does not respond to a particular drug regardless of adequacy of the dose29. the aim of this study was to determine the pattern of susceptibility of yeast for some azoles antifungals and amphotericin b. material and methods samples. c. albicans, c. tropicales, c. parapsilosis, c. krusei, c. famata, c. norvegensis, c. dubliensis and c. lusitaniae. the yeasts were selected from microbiology and immunology laboratory collection – piracicaba dental school, university of campinas (fop-unicamp), piracicaba, sp, brazil, including 140 isolates from three different sites of the oral cavity from 19 patients with periodontal disease, known as: periodontal pocket (a), oral mucosa (m) and ridge gingival (b). after activation, the identification of isolates was confirmed in chromogenic chromagar candida® (difco), api 20 system® kit (aux system biomérieux, france) and microculture. the samples were maintained in sabouraud dextrose agar (merck) covered with glycerol at 4 oc and yeast peptone dextrose with 15% glycerol at – 70 oc. the roll and samples code of candida spp isolates are presented in table 1. the codes of samples correspond to number of voluntary and precedence, being: a: periodontal pocket; b: healthy ridge gingival and m: oral mucosa. antifungal drugs. the following antifungal drugs were used for susceptibility tests: fluconazol, ketoconazol, itraconazol (azolics) and amphotericin b (polienic) neon®. susceptibility assay minimal inhibitory concentration (mic) test. the yeasts was grown overnight at 36 °c in sabouraud dextrose agar (merck) plates. inocula for the assays were prepared by diluting scraped cell mass in 0.85% nacl solution, adjusted to mcfarland scale 0.5 and confirmed by spectrophotometric reading at 580 nm. cell suspensions were finally diluted to 104 ufc ml”1 in rpmi-1640 medium (difco) for use in the assays. mic tests were carried out according to clsi (2002)30, using tissue culture testplate (96 wells), containing 100 µl rpmi-1640 medium.the stock solutions of the antifungals were diluted and transferred into the first well, braz j oral sci. 7(25):1543-1549 susceptibility of candida spp. oral isolates for azolic antifungals and amphotericin b 1545 sample code candida specie sample code candida specie sample code candida specie sample code candida specie 1-b1 c. albicans 15-a2 c. albicans 41-m4 c. albicans 55-a3 c. albicans 1-b2 c. albicans 15-a3 c. albicans 41-m5 c. albicans 55-a4 c. albicans 1-b3 c. albicans 15-a4 c. albicans 42-b1 c. albicans 55-a5 c. albicans 1-b4 c. albicans 15-a5 c. albicans 42-b2 c. albicans 55-b1 c. albicans 1-b5 c. albicans 15-m1 c. albicans 42-b3 c. albicans 55-b2 c. albicans 1-m1 c. albicans 26-a2 c. dubliniensis 42-b4 c. albicans 55-b3 c. albicans 1-m2 c. albicans 26-a3 c. dubliniensis 42-b5 c.albicans 55-b4 c. albicans 1-m3 c. albicans 26-a4 c. dubliniensis 42-m1 c. albicans 55-b5 c. albicans 1-m4 c. albicans 26-a6 c. tropicalis 42-m2 c. albicans 55-m1 c. albicans 1-m5 c. albicans 26-a7 c. tropicalis 45-m1 c. parapsilosis 55-m4 c. albicans 3-a1 c. albicans 26-a8 c. tropicalis 45-m2 c. parapsilosis 56-m1 c. famata 3-a2 c. albicans 30-a6 c. tropicalis 45-m4 c. parapsilosis 56-m3 c. krusei 3-a3 c. albicans 30-a7 c. tropicalis 45-m5 c. parapsilosis 56-m4 c. krusei 3-a4 c. albicans 30-a8 c. tropicalis 46-m1 c. lusitaniae 56-m5 c. krusei 3-a5 c. albicans 31-a1 c. albicans 47-a1 c. albicans 56-m6 c. krusei 3-m1 c. albicans 31-a2 c. albicans 47-a2 c. albicans 56-m7 c. famata 3-m2 c. albicans 31-a3 c. albicans 47-a3 c. albicans 56-m8 c. famata 3-m3 c. albicans 31-a4 c. albicans 47-a4 c. albicans 56-m9 c. norvegensis 3-m4 c. albicans 31-m1 c. albicans 47-a5 c. albicans 56-m10 c. norvegensis 3-m5 c. albicans 31-m2 c. albicans 47-m1 c. albicans 56-m11 c. norvegensis 13-a1 c. albicans 34-a6 c. tropicalis 47-m2 c. albicans 58-a1 c. albicans 13-a2 c. albicans 34-a7 c. tropicalis 50-a1 c. albicans 58-a2 c. albicans 13-a3 c. albicans 34-a8 c. tropicalis 50-a2 c. albicans 58-a3 c. albicans 13-a4 c. albicans 34-a9 c. tropicalis 50-a3 c. albicans 58-a4 c. albicans 13-a5 c. albicans 34-a10 c. tropicalis 50-a4 c.albicans 58-a5 c. albicans 14-a1 c. albicans 34-b2 c. tropicalis 50-m1 c. albicans 58-b1 c. albicans 14-a2 c. albicans 34-m2 c. tropicalis 50-m2 c. albicans 58-b2 c. albicans 14-a3 c. albicans 41-a1 c. albicans 50-m3 c. albicans 58-b3 c. albicans 14-a4 c. albicans 41-a2 c. albicans 50-m4 c. albicans 58-b4 c. albicans 14-a5 c. albicans 41-a3 c. albicans 50-m6 c. parapsilosis 58-b5 c. albicans 14-b1 c. albicans 41-a4 c. albicans 53-m6 c. tropicalis 58-m1 c. albicans 14-m1 c. albicans 41-a5 c. albicans 53-m7 c. tropicalis 58-m2 c. albicans 14-m2 c. albicans 41-m1 c. albicans 53-m8 c. tropicalis 58-m3 c. albicans 14-m4 c. albicans 41-m2 c. albicans 55-a1 c. albicans 58-m4 c. albicans 15-a1 c. albicans 41-m3 c. albicans 55-a2 c. albicans 58-m5 c. albicans table 1 roll and samples code of candida spp. isolated from oral cavity of patients undergo periodontal disease. and serial dilutions were performed so that concentrations in the following range were applied in the wells: fluconazol, 640 1.25 µg ml+1; ketoconazole, 80-0.15 µg ml+1, itraconazole, 40-0.078 µg ml+1 and amphotericin b, 160-0.3 µg ml+1. the yeast inocula were added to all wells and the plates were incubated at 36 °c for 24 h. antimicrobial activity was detected by change in the rpmi-1640 medium color (the pink color of the medium change to yellow after yeast growth by ph alteration). r e s u l t s a total of 140 yeasts belonging to microbiology and immunology laboratory collection (fop-unicamp) were tested for drugs susceptibility, as c. albicans (105 isolates), c. tropicales (16 isolates), c. parapsilosis (5 isolates), c. krusei (4 isolates), c. famata (3 isolates), c. norvegensis (3 isolates), c. dubliensis (3 isolates) and c. lusitaniae (1 isolate). the isolates of candida spp. were studied for the susceptibility to antifungals fluconazole, ketoconazole and itraconazole, and were classified as sensitive (s), susceptibility dependent of concentration (sdc) and resistant (r), according to clsi30 presented in table 2. for amphotericin b, the mic has been identified as the braz j oral sci. 7(25):1543-1549 susceptibility of candida spp. oral isolates for azolic antifungals and amphotericin b 1546 susceptibility fluconazole itraconazole ketoconazole amphotericin b s ≤ 8 ≤ 0,125 ≤ 0,125 ≤1 sdc 16 e 32 0,25 e 0,5 0,25 e 0,5 ---r ≥ 64 ≥ 1 ≥ 1 ≥ 2 table 2 criteria for ranking for susceptibility to fluconazole, itraconazole, ketoconazole and amphotericin b (ìg.ml”1). values of reference of the clsi m27-a2 (2002)30. 47 18 0 0 0 3 1 0 36 0 5 10 15 20 25 30 35 40 45 50 p e ri o d o n ta l p o c k e t (a ) o ra l m u c o s a (m ) r id g e g in g iv a l (b ) o ra l m u c o s a (m ) p e ri o d o n a ta l p o c k e t (a ) r id g e g in g iv a l (b ) r id g e g in g iv a l (b ) p e ri o d o n ta l p o c k e t (a ) o ra l m u c o s a (m ) susceptibles resistances sdc n u m b e r o f i s o l a t e s s u s c e p t i b i l i t y fig. 1a susceptibility of c. albicans isolates to fluconazole for sites collected. 12 8 1 0 0 0 8 6 0 0 2 4 6 8 10 12 14 o ra l m u c o s a (m ) p e ri o d o n ta l p o c k e t (a ) r id g e g in g iv a l (b ) o ra l m u c o s a (m ) p e ri o d o n ta l p o c k e t (a ) r id g e g in g iv a l (b ) o ra l m u c o s a (m ) p e ri o d o n ta l p o c k e t (a ) r id g e g in g iv a l (b ) susceptibles resistances sdc n u m b e r o f i s o l a t e s s u s c e p t i b i l i t y fig.1b susceptibility of candida nonalbicans isolates to fluconazole for sites collected. 7 6 0 3 1 0 41 26 21 0 5 10 15 20 25 30 35 40 45 p e ri o d o n ta l p o c k e t (a ) o ra l m u c o s a (m ) r id g e g in g iv a l (b ) o ra l m u c o s a (m ) p e ri o d o n ta l p o c k e t (a ) r id g e g in g iv a l (b ) p e ri o d o n ta l p o c k e t (a ) o ra l m u c o s a (m ) r id g e g in g iv a l (b ) susceptibles resistances sdc n u m b e r o f i s o l a t e s s u s c e p t i b i l i t y fig. 2a susceptibility of c. albicans isolates to ketoconazole for sites collected. 12 6 1 4 0 0 8 4 0 0 2 4 6 8 10 12 14 o ra l m u c o s a (m ) p e ri o d o n ta l p o c k e t (a ) r id g e g in g iv a l (b ) o ra l m u c o s a (m ) p e ri o d o n ta l p o c k e t (a ) r id g e g in g iv a l (b ) p e ri o d o n ta l p o c k e t (a ) o ra l m u c o s a (m ) r id g e g in g iv a l (b ) susceptibles resistances sdc s u s c e p t i b i l i t y n u m b e r o f i s o l a t e s fig. 2b susceptibility of candida nonalbicans isolates to ketoconazole for sites collected. 10 8 4 1 0 0 17 38 27 0 5 10 15 20 25 30 35 40 p e ri o d o n ta l p o c k e t (a ) o ra l m u c o s a (m ) r id g e g in g iv a l (b ) o ra l m u c o s a (m ) p e ri o d o n ta l p o c k e t (a ) r id g e g in g iv a l (b ) r id g e g in g iv a l (b ) p e ri o d o n ta l p o c k e t (a ) o ra l m u c o s a (m ) susceptible resistances sdc n u m b e r o f i s o l a t e s s u s c e p t i b i l i t y fig.3a susceptibility of c. albicans isolates to itraconazole for sites collected. 8 4 0 9 5 1 5 3 0 0 1 2 3 4 5 6 7 8 9 10 o ra l m u c o s a (m ) p e ri o d o n a tl p o c k e t (a ) r id g e g in g iv a l (b ) o ra l m u c o s a (m ) p e ri o d o n ta l p o c k e t (a ) r id g e g in g iv a l (b ) p e ri o d o n ta l p o c k e t (a ) o ra l m u c o s a (m ) r id g e g in g iv a l (b ) susceptible resistances sdc s u s c e p t i b i l i t y n u m b e r o f i s o l a t e s fig. 3b susceptibility of candida non albicans isolates to itraconazole for sites collected. braz j oral sci. 7(25):1543-1549 susceptibility of candida spp. oral isolates for azolic antifungals and amphotericin b 1547 6 4 0 14 10 1 0 2 4 6 8 10 12 14 16 o ra l m u c o s a (m ) p e ri o d o n ta l p o c k e t (a ) r id g e g in g iv a l (b ) o ra l m u c o s a (m ) p e ri o d o n ta l p o c k e t (a ) r id g e g in g iv a l (b ) susceptible resistances s u s c e p t i b i l i t y n u m b e r o f i s o l a t e s fig. 4b susceptibility of candida non albicans isolates to amphotericin b for sites collected. 8 13 1 35 28 20 0 5 10 15 20 25 30 35 40 o ra l m u c o s a (m ) p e ri o d o n ta l p o c k e t (a ) r id g e g in g iv a l (b ) p e ri o d o n ta l p o c k e t (a ) o ra l m u c o s a (m ) r id g e g in g iv a l (b ) susceptible resistances s u s c e p t i b i l i t y n u m b e r o f i s o l a t e s fig. 4a susceptibility of c. albicans isolates to amphotericin b for sites collected. lowest concentration of drugs in middle-free growth of yeast, and classified as sensitive (s) or resistant (r). the results are shown in figure 1a until 4b. d i s c u s s i o n among the 53 patients investigated, 19 undergo oral colonization by candida spp, and 14 of them (26.4%) had colonization in the periodontal pocket. the presence of yeast in periodontal pockets has been reported in about 20% of patients with severe periodontal disease 31-32. although not fully defined the participation of these organisms in the pathogenesis of this disease, the results are suggestive, considering the fact that all patients had not analyzed systemic diseases or factors that may contribute to the development of yeast infections, such as immunosuppression by use of antibiotics or steroids. the occurrence of resistance to azoles antifungal was greater for the group of candida non-albicans (42.8%) compared to that seen in c. albicans (3.6%). it was possible to observe the occurrence of cross resistance between the antifungal ketoconazole and itraconazole, for five samples of three patients. although the occurrence of resistance was low for azolic, there is a decrease in susceptibility to these antifungals, demonstrated by the high level of sdc, especially for ketoconazole and itraconazole. the fluconazole show to be the most effective for most samples from all tested species of candida. studies have shown that the susceptibility to antifungals azolic among isolates of candida spp. of periodontal pockets can be variable; occurring cross resistance33-34 also highlighted the occurrence of cross resistance between the antifungal azolic, based in the presence of similar mechanisms of action. this shows different patterns of susceptibility of these strains to drugs tested1.this fact is corroborated recent data, which indicate a growing resistance among species of candida to azoles antifungals, suggesting that the oral cavity could be a reservoir of resistant yeast35-36. a correct diagnosis should be considered in microbiological periodontal lesions refractory to conventional treatment. the joint application of antifungal treatment can be useful in cases of opportunistic infections by candida spp. in periodontology, especially in patients at high risk of developing systemic candidiasis37. the high incidence of resistance to amphotericin b found between the species analyzed indicates the need to make more accurate studies for this antifungal. actually, considering the potential of azoles antifungal fungistatics and fungicide potential of amphotericin b (polienics) would be expected to find greater number of isolates sensitive to the latter and not the other drugs. considering the relevant literature, it was found that there is a pattern of behavior of unusual clinical isolates regarding susceptibility to antifungals tested, since different authors found different results in this respect38 concluded in their work, that the effectiveness of two types of drug amphotericin b and fluconazole was equivalent. thus, the results of this research may be suggestive of the occurrence of of strains resistant selection to conventional amphotericin b. the same has not happened with another form of presentation of amphotericin b, lipossomal, whose formulation allows the release gradual, but constant, the principle active38-40. this formulation has been regarded as a better therapeutic option in cases of resistance. thus, the preliminary identification of microorganisms causing the disease and the appropriate concentration of the drug are important factors to avoid the selection of resistant strains. ruhnke41 described the development of resistance to fluconazole in a patient with aids infected by c. albicans and c. dubliniensis simultaneously. the patient had recurrent oral candidosis because of the two species and under use of fluconazole for four years. molecular analyses showed persistence of isolates during the same period, affecting the development of resistance in the two species. researchs involving studies on the growth and susceptibility of candida spp strains to a wide variety of antifungal drugs has expressed cross-resistance to braz j oral sci. 7(25):1543-1549 susceptibility of candida spp. oral isolates for azolic antifungals and amphotericin b 1548 antifungal amphotericin b and azoles in individuals who had or not contact with them, suggesting that it is not an effect on the metabolism of sterol, but that new mechanisms of resistance are involved in clinical isolates30,42-43. these data, added to those obtained in this study, suggest that to achieve effective therapy are necessary comparative studies between the various azolics and polienics antifungal normally used, confronting their mechanisms of action and factors of local origin as bad oral hygiene, presence of caries and prostheses, which caused the microbial accumulation. in addition, must still be considered factors of a systemic disorder related to the metabolism of the host, immunosuppressive therapies, malnutrition and infection by the hiv virus since the union of these conditions to the opportunistic microorganism contributes to the worsening of the disease 33,44. the development of a program that includes routine tests for sensitivity to antifungal for tracking oral samples is necessary to determine the drug and the efficient concentration for the treatment. this also would avoid the selection of resistant strains whose susceptibility depends on the concentration of the drug33,44. studies that will contribute to a greater understanding of the relationships between organisms and antimicrobials should be carried out in order to elucidate the complex mechanisms involving microbial resistance to drugs. concluding, patients with periodontal disease showed relevant levels of colonization by candida spp., mainly in the oral mucosa and periodontal pocket. species of c. albicans was the most prevalent yeast in the oral microbiota considering the various sites analyzed, which confirm the previous findings. there is also an increase in the incidence of non-albicans species in these sites, together with a greater resistance to the latest drugs tested. moreover, the patterns of susceptibility from c. albicans and non-albicans isolates to different antifungal studied differ considerably. there was no occurrence of resistance to fluconazole and small part of candida spp. isolates showed susceptibility dependent on concentration (sdc) in relation to this drug, experiencing the opposite for ketoconazole and itraconazole. most isolates are resistant to amphotericin b. a c k n o w l e d g m e n t s this research was supported by a grant from cnpq (brazil). r e f e r e n c e s 1. nola i, kostovic k, oremovic l, soldo-belic a, lugovic l. candida infections today how big is the problem? acta dermatovenerol croat. 2003; 11: 171-7. 2. eggimann p, garbino j, pittet d. epidemiology of candida species infections in critically ill non-immunosuppressed patients. lancet inf dis. 2003; 3: 685-702. 3. braunwald e, fauci as, kasper dl, hauser sl, longo dl, jameson jl, editors. harrison’s principles of internal medicine. 15 ed. new york: 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witherly sa. dietary supplement of neosugar alters the fecal flora and decreases activities of some reductive enzymes in human subjects. am j clin nutr. 1996; 63: 709-16. 22. howard md, gordon dt, garleb ka, kerley ms. dietary fructooligosaccharide, xylooligosaccharide and gum arabic have variable effects on cecal and colonic microbiota and epithelial cell proliferation in mice and rats. j nutr. 1995; 125: 2604-9. 23. mcginnis mr, molina tc, pierson dl, mishra sk. evaluation of the biolog microstation system for yeast identification. j med vet mycol. 1996; 34: 349-52. 24. goodman ls, gilman a, editors. as bases farmacológicas da terapêutica. 9.ed. rio de janeiro: mcgraw-hill; 1996. 25. graybill jr. lipid formulations for amphotericin b: does the emperor need new clothes? ann int med. 1996; 124: 9213. 26. harste s, bolard j. amphotericin b: new life for an old drug. trends pharmac sci. 1996; 17: 445-9. 27. hoppe-tichy t. systemische pilzerkrankungen: klinik und antimykotische therapie. pharm ztg. 1997; 142: 2161-8. 28. williams da, lemke tl. foye’s principles of medicinal chemistry. 5.ed. philadelphia: lippincot williams & wilkins; 2002. 29. molina p, puchades mj, aparicio m, garcía ramón r, miguel a. experience in fungal peritonitis in a dialysis unit for 10 years .nefrologia. 2005; 25: 393-8. 30. clsi. método de referência para testes de diluição em caldo para determinação da sensibilidade de leveduras à terapia antifúngica: norma aprovada. 2 ed. clinical and laboratorial standards institute, clsi 2002; norma m27-a2, v.22, n.15. 44p. 31. dahlen g, wikstrom m. occurrence of enteric rods, staphylococci and candida in subgingival samples. oral microbiol immunol. 1995; 10: 42-6. 32. slots j, rams te, listgarten ma. yeasts, enteric rods and pseudomonads in the subgingival flora of severe adult periodontitis. oral microbiol immunol. 1988; 3: 47-52. 33. carrillo-muñoz aj, tur c, montsant l, carceller a, hernandezmolina jm, torres-rodrigues jm. resistencia in vitro al fluconazol e itraconazol en aislamientos clinicos de candida spp e cryptococcus neoformans. rev iberoam micol. 1997; 14: 50-4. 34. arikan a, kulak y, kadir t. comparison of different treatment methods for localizad and generalized simple denture stomatitis. j oral rehalbil. 1995; 22: 365-9. 35. ito cyk, martins cap, loberto jcs, santos ssf, cardoso jao. in vitro antifungal susceptibility of candida spp. isolates from patients with chronic periodontitis and control patients. braz oral sci. 2004; 18: 80-4. 36. kuriyama t, williams dw, bagg j, coulter wa, ready d, lewis ma.in vitro susceptibility of oral candida to seven antifungal agents. oral microbiol immunol. 2005; 20: 349-53. 37. pizzo g, giuliana g, milici me. effect of dietary carbohydrates on the in vitro epithelial adhesion of candida albicans, candida tropicalis, and candida krusei. new microbiol. 2000; 2: 63-71. 38. marra a, camargo lf a. fluconazol ou anfotericina b no tratamento de candidemias em pacientes internados na uti. rev assoc med bras. 2002; 48: 93-117. 39. magaldi s. in vitro susceptibility of 137 candida spp. isolates from, hiv patients to several antifungal drugs. micopathologia. 2001; 149: 63-8. 40. paniagua lg, monroy w, negrete e, vaca s. susceptibility to 5fluorocytosine, miconazole and amphotericin b of candida braz j oral sci. 7(25):1543-1549 susceptibility of candida spp. oral isolates for azolic antifungals and amphotericin b albicans strains isolated from the throat of nonaids patients. rev latinoam microbiol. 2002; 44: 65-8. 41. ruhnke m, schmidt-westhausen a, morschhäuser j. development of simultaneous resistance to fluconazole in candida albicans and candida dubliniensis in a patient with aids. j antimicrob chemother. 2000; 46: 291-5. 42. perea s. azole resistance in candida albicans rev esp quimioter. 2000; 13: 314-17. 43. swinne d, watelle m, van der flaes m. in vitro activities of variconazole(uk-109, 496), fluconazole, itraconaole and amphotericin b against 132 nonalbicans bloodstream yeast isolates(canari study). mycoses. 2004; 47; 177-83. 44. lindhe j. tratado de periodontia clinica e implantologia oral. rio de janeiro: guanabara koogan ; 1999. braz j oral sci. 15(3):205-208 evaluation of the maternal perception of the oral health of the inpatient infant ana de lourdes sá de lira1, joyce de moura crisóstomo1, sylvana thereza de castro pires rebelo1 1universidade estadual do piauí – uespi, school of dentistry, department of clinical dentistry, area of integrated clinic, parnaíba, pi, brazil correspondence to: ana de lourdes sá de lira universidade estadual do piauí faculdade de odontologia rua senador joaquim pires 2076 ininga fone (86) 999595004 cep: 64049-590 teresina-pi-brasil email: anadelourdessl@hotmail.com abstract aim: to evaluate the mother’s perception of the oral health of their inpatient infants in maternity or infirmary units of a public hospital. methods: questionnaire applications were scheduled and educational lectures were carried out on how to sanitize the infant's mouth after breastfeeding, even in the absence of primary teeth, emphasizing the importance of breastfeeding. results: a significant number of mothers reported that they received no guidance regarding the oral health care of their infants. they had never attended lectures by dental practitioners, as well as they did not know that the use of pacifier, baby bottle and digital sucking habit could interfere with their infant’s oral heath over time. conclusions: the majority declared that they had no care with the oral health of their infants. only five mothers of newborns reported that they performed the oral hygiene of them once a day after the first breastfeeding. the mothers showed lack of knowledge on the diseases which can affect their children during early infancy as they had no information on how to prevent them. they did not know that early caries lesions could affect the infant and that harmful oral habits can predispose to the development of malocclusions. keywords: odontopaediatrics. prevention. infants. introduction the parents assume a caretaker role which is crucial for promoting and maintaining the health of their children, and for this reason they should be the primary source of information on oral health. mothers have more influence on the child because they are more present as a caretaker, and when they are well instructed by healthcare practitioners, can form attitudes and behaviours and transform them into generation of healthy habits1,2. dental caries is the most prevalent chronic disease in the infancy, being classified even as a “pandemic” condition characterised by a high rate of untreated cavities, thus causing pain, distress and functional restrictions among the children3. a poor oral health can increase the risk of caries in infants, since the mother’s saliva is the main source of cariogenic bacteria for the infant’s oral microbiota. mothers who have untreated decayed teeth are more risky of transmitting bacteria to their children4,5. in order to reduce the risk of dental caries and promote the children’s oral health, hygiene measures should be implemented even before the eruption of the first deciduous tooth. the teeth should be cleaned at least twice a day under supervision of the caretaker, who can use a diaper or soft toothbrush to decrease the bacterial colonisation by disorganising dental plaques6-8. healthy or harmful habits are behavioral patterns which are learnt by the child. the importance of breastfeeding and infant oral hygiene should be taught to the received for publication: december 15, 2016 accepted: may 3, 2017 original article braz j oral sci. july | september 2016 volume 15, number 3 http://dx.doi.org/10.20396/bjos.v15i3.8649982 206 parents during medical and dental healthcare services throughout gestation and during routine medical visits9. non-nutritive sucking habits can lead to undesirable effects on dental arches. the continuous presence of the thumb or finger inside the oral cavity can exert pressure enough to deform the upper arch, palate or both. if this digital sucking habit persists while the permanent teeth are erupting, then harmful effects on dentofacial development can occur10. the parents should be informed about the benefits to the infant from breastfeeding during the first 6 months of life in terms of normal development of the dentofacial skeleton. the non-nutritive sucking habit should be diagnosed in time so that the development of posterior cross-bite, anterior open-bite and class ii malocclusion can be reduced9. it is believed that the pregnant women during the prenatal and after delivery are not oriented on the preventive measures of caries and malocclusion of infants, although healthcare professionals know that the infant oral health care depends on the parent’s awareness. it is further believed that even in the territories covered by the family health strategy, with or without the participation of the dentist even if the dentist does not participate in the home visit, the community health agents usually fails do it, and does not refer them for healthcare in the basic health unit whenever necessary. it occurs especially in cities of poor states or in rural areas where socioeconomic conditions hamper monthly consultations during prenatal care. in this context, we sought to know whether the mothers were being instructed on infant oral hygiene, so that guidance programs on the oral health of infant could be improved, in basic health units, where prenatal care is performed. therefore, the objective of this work was to evaluate the mother’s perception of the oral health of their inpatient infants in maternity or infirmary units of a public hospital. material and methods the researcher obtained a signed informed consent form from the public hospital of parnaíba (piauí) authorising the development of the study, which was also approved by the human research ethics committee of the state university of piauí (cep/uespi) according to protocol number caae 16604613.2.0000.5209. the researchers carried out the study according to an individual schedule of activities as detailed in the project. informed consent forms were distributed to the mothers, including those younger 18 years old, explaining the objective of the study. in this way, it was possible to obtain their consent on a free-will basis after they agreed and signed the forms. only mothers with infants aged up to 18 months old (age limit for infants) in the maternity or infirmary units during the second semester of 2014 participated in the study, totalizing 42 mothers, being 34 residents in the rural area, who were with their infants hospitalized for medical treatment. we opted to carry out the study in the maternity unit of the dirceu arcoverde state hospital, since we believed that these parturients were less instructed by the hospital staff about infant oral health compared to those attended in private hospitals. it should also be emphasised that we already expected that the population attended in this public hospital had low income and low education level, which would contribute to their lack of knowledge on the importance of breastfeeding and keeping healthy habits for the oral health of their infants. the present research not only respected the determinations of the resolution number 466/12 of the national health council, but also guaranteed confidentiality, anonymity and non-use of information in detriment of the individuals, thus reducing the risk to the subjects (the results only contribute to the present study by means of statistical assessment of the data obtained with the questionnaire). a pilot survey was initially undertaken by one examiner for calibration purposes, and the same questionnaire was applied to five mothers who were with their infants in the infirmary of the same hospital on the first sunday of the first week of july in 2014. the questionnaire was applied again to the same mothers on the friday of the same week, prior to the discharge of their infants, thus totalizing 10 questionnaires. no discrepancies in the answers were observed between the questionnaire applications in both periods of time. the visits for questionnaire applications were scheduled in the mornings on tuesdays and thursdays of each week during the second semester of 2014. educational lectures were carried out on how to sanitize the infant's mouth after breastfeeding, even in the absence of primary teeth, emphasizing the importance of breastfeeding, prevention of digital sucking habits or pacifiers. before the first teeth erupted it was emphasized that oral hygiene should be performed using clean diaper / gauze moistened with filtered or boiled water at the time of body hygiene5,7. for the child who was being breastfed exclusively at the breast, it was advised that mouth cleaning should be restricted to the tongue once a day because breast milk contains immunoglobulins that protect the child's mouth from infections. if the child is using artificial breast-feeding, cleaning should be performed throughout the oral cavity twice a day6,7. this is a cross-sectional, quantitative, qualitative study whose resulting data were analysed by using the pasw statistical package, version 21. descriptive statistical analyses (measurements of central tendency and dispersion) were also performed, including the spearman’s rank correlation (one-tailed test). results the study used a convenience sample (non-probabilistic) of 42 mothers with inpatient infants in a public hospital in the city of parnaiba (piauí). the subjects had a mean age of 23.04 years old (sd = 6.01, range between 17 and 39 years). table 1 lists the social-economic data of our sample. with regard to the infant age group, 88.1% (n = 37) were newborns and five were between 12 and 18 months old. in table 2, one can see the mothers’ answers on whether they received some guidance on infant oral hygiene. the majority of the mothers interviewed during their stay in the hospital reported that they received no visit of a dentist, corresponding to 90.5% evaluation of the maternal perception of the oral health of the inpatient infant braz j oral sci. 15(3):205-208 207evaluation of the maternal perception of the oral health of the inpatient infant braz j oral sci. 15(3):205-208 (n = 38) of the subjects. only one mother reported that she had received such a visit, whereas other three mothers did not answer. table 1 social-economic data of the mothers participating in the study. variables frequency n % education level elementary education 9 21.4 incomplete elementary education 20 47.6 intermediate education 5 11.9 incomplete intermediate education 4 9.5 higher education 2 4.8 incomplete higher education 1 2.4 illiterate 1 2.4 marital status single 19 45.2 married 12 28.6 stable union 11 26.2 income 1 to 2 minimum wages 41 97.6 3 to 4 minimum wages 1 2.4 source: direct research parnaíba 2015. table 2 questionnaire on the oral health of infants aged between 0 to 18 months old. variables frequency n % have you ever received some guidance on infant oral hygiene? no 38 90.5 yes 4 9.5 by whom? where? nurse 4 9.5 basic health unit 1 2.4 physician 1 2.4 hospital 1 2.4 no answer 35 83.3 source: the respondents could answer more than one choice. direct research parnaíba 2015. table 3 question on caries lesions. variables frequency n % which is the best measure to prevent caries from occurring in the future? hygiene of the mouth 15 35.7 food control 7 16.7 visit to the dentist 16 38.1 food control and visit to the dentist 1 2.4 hygiene of mouth and food control 1 2.4 no answer 2 4.8 source: the respondents could answer more than one choice. care. three mothers refused to answer this question. three of the subjects knew to define the so-called baby bottle tooth decay, but 92.9% (n = 39) reported having no knowledge about it. table 3 lists the data from the question on the best measures to be taken to prevent caries. in the time of the study, it was also asked whether caries lesion could be transmitted from the mother to the infant, with 63.4% (n = 27) answering negatively while 36.6% (n = 15) believing it was true. however, only one mother reported that this might occur during a very close conversation with the infant. among the 42 mothers, 76.2% (n = 32) reported that their infants were breastfed only, whereas four were fed with artificial milk and six fed with both. in the present study, 92.9% (n = 39) of the mothers reported they did not know that the use of pacifier, baby bottle and digital sucking habit could interfere with their infant’s oral heath over time. all the mothers were unanimous in stating that their infants had the habit of putting the finger into the mouth. the mothers were asked how many times a day they performed the oral hygiene of their infants. the majority (73.8%; n = 31), declared that they had no care with the oral health of their infants. only five mothers of newborns reported that they performed the oral hygiene of them once a day after the first breastfeeding. with regard to the mothers of infants between 12 and 18 months, one did it twice a day, in the morning and after the lunch; three did it three times a day, in the morning, after the lunch and before sleeping; and only one mother reported that she did it more than 3 times a day, always after any meal. the mothers were also asked on whether they had ever participated in some lecture by a dentist. the majority answered negatively (81%; n = 34), whereas only eight reported that they had already participated. only seven mothers reported that they knew that caries and candidiasis could be the result of poor oral hygiene, whereas 76.2% (n = 32) answered that they did not know which diseases might affect the infant in the absence of such discussion a social-economic questionnaire was used in the present study to determine the profile of the population sample, which can contribute to the development of further contextualised policies so that people’s needs can be met according to the characteristics observed in the population studied. the subjects had a mean age of 23.4 years old, with 47.6% (n = 20) not completing the elementary school, 45.2% (n = 19) being single, 97.6% (n = 41) having monthly income of 1 minimum wage, and 88.1% (n = 37) having newborn infants (table 1). in the present study, after performing the spearman’s rank correlation (one-tailed test) between the variables (i.e. education level versus mothers’ knowledge on diseases which might affect the infant due to poor oral hygiene), it was possible to observe a significantly positive relationship between both (r = -0.45* p< 0.01). it was observed that the higher the education level of the mother, the greater of knowledge on diseases which might affect the infant as a result of poor oral hygiene. campos et al.11 (2010), by analysing the social-economic aspect of the mothers, found that the lower the social-economic class, the higher the percentage of lack of knowledge. this finding was also observed in the present study. the majority of the mothers (90.5%) reported that they had never received any information on infant oral health (table 208 evaluation of the maternal perception of the oral health of the inpatient infant 2), a finding not corroborated by guarienti et al12 (2009), who concluded that 83% of the mothers interviewed reported they had such information, but out of the dental office, hospital or healthcare unit environment. also drawing attention was the finding that 73.8% of the mothers declared that they took no care of the oral health of their infants. in another study, 96% of the mothers stated that infant oral health is necessary, but 63% did not know to explain how it should be done13. in a study of pregnants, the future mothers were asked on whether they knew about caries in the early childhood, the so-called baby bottle tooth decay, and 96.25% of them had no knowledge about it14. such finding was also reported in our study. according to the literature, early dental caries or baby bottle tooth decay should be prevented as this condition progresses rapidly, resulting in destruction of tooth crowns in a short space of time, or even early loss of teeth, which can lead to malocclusion10-14. guarienti et al.12 (2009) conducted a research in which half of the mothers responded that they did not know whether caries could be transmitted from the mother to the child. similarly, the subjects of our study (61.9%) responded they did not know about such a possibility. serpa and freire15 (2012) found that 85% of the mothers interviewed were aware that one of the best ways to prevent caries from occurring in the future would be an adequate oral hygiene. this finding is also corroborated by our study, since only 38.1% of the mothers know this correlation (table 3). by means of questionnaires, charchut et al.16 (2003) and massoni et al.17 (2010) found that prolonged breastfeeding beyond the first two years of life can favour the establishment of non-nutritive sucking habits, such as the use of pacifiers. the interviewees considered the prolonged use of pacifiers harmful to the child. however, a few ones had knowledge about the age limit for stopping this habit in order to avoid compromising the dentofacial development, since self-correction occurs up the age of 3 years approximately. differently from our study, 92.9% of the mothers did not believe that the use of pacifier, baby bottle and digital sucking habit could interfere with the infant oral health over time. during the study, the mothers were instructed by the researchers about the need to perform the infant oral hygiene, including different ways of doing it, as well as the harms affecting the infants as a result of non-nutritive sucking habits. also, educational folders were handed out to the mothers. it is necessary to provide more information for mothers, especially those not attending public hospitals, so that they can take care of the oral health of their infants accordingly, regardless of being in healthcare units, hospitals or lectures by dentists or other healthcare practitioners in the community. on conclusion, the majority declared that they had no care with the oral health of their infants. only five mothers of newborns reported that they performed the oral hygiene of them once a day after the first breastfeeding. those participating in the study showed lack of knowledge on the diseases which can affect their children braz j oral sci. 15(3):205-208 in early infancy, since they received no information on how to prevent them. particularly, the mothers did not know that early tooth decay can affect the infant and that harmful oral habits can predispose to the development of malocclusion. references 1. sisson kl. theoretical explanations for social inequalities in oral health. community dent oral epidemiol. 2001 apr;35(2):81-8. 2. wandera m. factors 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[pacifier sucking habit and associated dental changes. importance of early diagnosis]. an pediatr (barc). 2012 dec;77(6):374-80. doi: 10.1016/j.anpedi.2012.02.020. epub 2012 may 18. spanish. 11. campos l, bottan er, birolo jb, silveira eg, schmitt bhe. [knowledge of mothers from different social classes regarding oral health in the city of cocal do sul (sc)]. rev sul-bras odontol. 2010 oct-dec;7(4):287-95. portuguese. 12. guarienti ca, barreto vc, figueiredo mc. [parents' and caregivers' knowledge of oral health in the early childhood]. pesq bras odontoped clin integr. 2009;9(3):321-5. portuguese. 13. praetzel jr, ferreira fv, lenzi tl, melo gp, alves ls. [maternal perceptions of dental, speech and hearing care during pregnancy]. rgo. 2010 aprjun;58(2):155-60. portuguese. 14. marín c, pereira cc, koneski k, andrades kmr, miguel lcm, ávila lfc. evaluation of pregnant adolescent women’s knowledge about their baby’s oral health. arq odontol. 2013;49(3):133-9. portuguese. 15. serpa em, freire pll. [perception of pregnants from in joão pessoa-pb on the oral health of its babies]. odontol clin-clent. 2012;11:121-5. portuguese. 16. charchut sw, alfred en, needleman hl. the effects of infant feeding on the oclusion of the primary dentition. j dent child (chic). 2003;70(3):197-203. 17. massoni aclt, paulo sf, forte fds, freitas chsm, sampaio fc. [children's oral health: knowledge and interest of parents/caregivers]. pesq bras odontop clin integr. 2010;10:257-64. portuguese. untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8651902 volume 17 2018 e18026 original article 1 dds, msc, phd, professor, department of restorative dentistry, são paulo state university (unesp), school of dentistry, araçatuba, são paulo, brazil. 2 msc, master student, department of child and social dentistry, são paulo state university (unesp), school of dentistry, araçatuba, são paulo, brazil. 3 dds, msc, phd, department of child and social dentistry, são paulo state university (unesp), school of dentistry, araçatuba, são paulo, brazil. 4 dds, msc, phd, full professor, department of restorative dentistry, são paulo state university (unesp), school of dentistry, araçatuba, são paulo, brazil. 5 dds, msc, phd, full professor, department of child and social dentistry, são paulo state university (unesp), school of dentistry, araçatuba, são paulo, brazil. 6 dds, msc, phd, full professor, department of child and social dentistry, são paulo state university (unesp), school of dentistry, araçatuba, são paulo, brazil. corresponding author: full professor suzely adas saliba moimaz department of child and social dentistry araçatuba dental school, unesp univ estadual paulista address: r. josé bonifácio, 1193, cep: 16015-050, araçatuba-sp, brazil tel: (+55) 18 3636 3260 fax: (+55) 18 3636 3233 e-mail: sasaliba@foa.unesp.br received: october 01, 2017 accepted: january 25, 2018 microscopic evaluation of rotatory and handle caries removal on glass ionomer cement/dentin interface silvio josé mauro1, adriana beatriz silveira pinto fernandes2, fernando yamamoto chiba3, renato herman sundfeld4, suzely adas saliba moimaz5, cléa adas saliba garbin6 aim: this study evaluated the interface between glass ionomer cement (gic) and dentin using the conventional and atraumatic restorative treatment (art) techniques for removal of decayed tissue. materials and methods: after preparation of the cavities by the conventional (group i – gi) and art (group ii – gii) techniques, the specimens were divided into 3 subgroups according to the gic used (n=10). the conventional gic ketac fill (3m/espe) was used in the groups gi-1 and gii-1, the conventional gic vidrion r (ss white) in the groups gi-2 and gii-2, and the modified gic fuji ii lc (gc corporation) in the groups gi-3 and gii-3. for the microscopic analysis, the teeth were decalcified in a solution containing equal portions of 50% formic acid and 20% sodium citrate, dehydrated and submitted to paraffin baths. the samples were sectioned (6mm in thickness), stained by the brown and brenn method, and evaluated in a light microscope. results: the microscopic analysis revealed interaction between the material and dentin structure only with the fuji ii lc. in addition, the behavior was superior for the conventional technique in comparison to the art technique. conclusion: it was concluded that the technique used for carious dentin removal does not seem to be determinant for the bonding of ionomeric materials to the dental structure, except for the resin-modified gic in which the formed hybrid layer was higher using the conventional technique. keywords: dental atraumatic restorative treatment. dental caries. glass ionomer cements. 2 mauro et al. introduction the atraumatic restorative treatment (art) technique is an alternative method recognized by the world health organization, which intends to provide dental treatment for destitute communities. this technique is indicated for treatment of carious lesions preserving the healthy dental structure. so, the art can be applied in social programs with low technical financial funds1-5. the art technique consists on coronal removal of the decayed tissue using only hand instruments and cavity restoration with glass ionomer cement. among the properties of the glass ionomer cements, the adhesion to the dental structure (enamel and dentin) through ionic exchanges allows the formation of an acid-resistant adhesive layer between the ionomeric material and the tooth6,7. the majority of the studies concerning the art technique performed longitudinal analysis of the restorations obtained by this method in comparison to the conventional techniques1. however, few studies evaluated the interaction between the ionomeric materials and the dental structure with different caries removal techniques. this evaluation is important to indicate a material and elucidate the reasons for the supremacy of this material in comparison to the others. the mechanical properties of the glass ionomer bond interface to the tooth structure might be the main concern among researchers8. the conventional method for carious dentin removal using burs may generate better results since it eliminates a great quantity of infected and affected carious tissue9. when compared to the art, the use of bur can easily eliminate the affected carious dentin, which provides better conditions for bonding procedures with glass ionomer9. on the other hand, some studies demonstrated a pause in the carious lesion progress and also a significant reduction in the number of bacteria when using the art technique4,10. hence, it has been observed the development of reparative dentin and preservation of pulp vitality4. considering that the adhesion of glass ionomer to dentin is more critical than to enamel, this factor should be further evaluated by the researchers6. the electron microscopy is a common procedure to investigate the adhesive interface11. however, the polarized light microscopy may also represent an additional method for evaluation. the light microscopy is advantageous since several slices with 6µm in thickness can be obtained and, therefore, the total adhesive area can be evaluated as opposed to a small portion of the interface. the development of materials with better mechanical properties increases the longevity of restorations in both deciduous and permanent teeth. these investigations may contribute to the use of the art as a definitive restorative procedure not only in social programs but also in private practices1,9. the aim of this study was to evaluate the hypothesis that the interaction between glass ionomer materials and dentin is not affected by the mechanism of caries removal – burs or hand instruments. 3 mauro et al. material and methods sixty human carious molars with active shallow deep cavitated dentinal lesions (lesions radiographically extending less than the pulpal third of dentine12) were used in this study after approval by the research ethics committee of foa – unesp (protocol foa 1538/2003). after the verification of extension of the carious lesion with radiographic examinations the teeth were randomly distributed among the groups to keep similar conditions for the tests. from the 60 teeth, 30 were prepared by the conventional technique (group i gi) and the remaining by the art technique (group ii gii). the cavities in group gi were prepared with spherical diamond burs (1016 kg sorensen, barueri, sp, brazil) in high speed under abundant water-air spray cooling. selective removal to firm dentine was performed by using spherical burs compatible with the cavity size in low speed and without cooling. selective removal to firm dentine was confirmed with the aid of an explorer to assess the consistency of the remaining dentin13,14. the cavities in group gii were prepared with hand cutting instruments and the selective removal to soft dentine was done with dentin excavators (number 5, 11½ duflex ss white artigos dentários, rio de janeiro, rj, brazil) with circular movements until resistance for selective removal of carious tissue. it is worthwhile to note that hard dark or pigmented dentin remained in the cavity4,5. a sharp hand excavator was used to check the hardness of the remaining dentine13,14. considering the adhesive properties of the restorative materials used in this study, no cavity contour or convenience forms were established, maintaining the enamel without dentin support. after cavity preparation, the teeth were randomly divided into the subgroups according to the materials used (n=10): conventional glass ionomer cement ketac-fill (3m espe dental products, st paul, mn, usa); conventional glass ionomer cement vidrion r (ss white, rio de janeiro, rj, brazil); and resin-modified glass ionomer cement fuji ii lc (gc corporation, tokyo, japan) (tables 1 and 2). table 1. materials used and respective manufacturers and batches material manufacturer batch ketac fill – plus – powder (conventional glass ionomer cement) 3m espe 150374 ketac fill – plus liquid (conventional glass ionomer cement) 3m espe 114819 fuji ii lc (resin-modified glass ionomer cement) gc corporation 0206201 vidrion powder (conventional glass ionomer cement) s.s.white 00c vidrion liquid (conventional glass ionomer cement) s.s.white 00b polyacrylic acid s.s.white 00r 4 mauro et al. all cavities were conditioned with 11.5% polyacrylic acid (dentin conditioner – ss white, rio de janeiro, rj, brazil) for 20 seconds, rinsed for 15 seconds and completely dried6. the ionomeric materials were proportioned and manipulated according to the manufacturer’s instructions and inserted in the cavities with the aid of a centrix syringe (c-r®.syringe centrix™ speed slot). in the subgroups using the glass ionomers ketac-fill and vidrion r, a clear nail polish layer was applied to protect the surface 5 minutes after filling the cavity. then, 15 minutes later, the teeth were immersed in distilled water for 24 hours. in the subgroups using the glass ionomer fuji ii lc, the material was polymerized for 40 seconds with the light curing unit ultralux el (dabi atlante, ribeirão preto sp, brazil) at 450 mw/cm² potency and, subsequently, immersed in distilled water for 24 hours. afterwards, the teeth were decalcified in a solution containing equal portions of 50% formic acid and 20% sodium citrate changed every 5 days. the complete decalcification of each specimen was radiographically checked. this process completely removes the dental enamel and just maintains the demineralized dentin tissue, which was the subject of this study. after decalcification, the restorations were carefully removed for paraffin embedment. the specimens were serially sectioned in longitudinal direction through the crown with 6μm in thickness and sequentially mounted in glass slides. fifteen slides of each specimen containing approximately six sections were selected by systematic sampling with an interval proportional to the number of sections obtained for each specimen. these sections were stained with brown and brenn15 staining technique. then, the better histological section of each slide was analyzed on a light microscope axiophot (zeiss dsm-940 a, oberkochen, germany) under 400x of magnification with a micrometric ocular 40/075. the interface of each section was carefully analyzed in the entire extension of the histological section by a single calibrated examiner. when an interaction layer between the ionomeric restorative material and dentin was detected, it was measured using a micrometric ocular 40/075 under 400x of magnification. representative samples of each group were photographed with a film speed asa 100. table 2. group distribution according to technique and restorative material used groups n sub groups n conditioning restorative material group g i 30 g i 1 10 polyacrylic acid ketac fill g i 2 10 polyacrylic acid vidrion r g i 3 10 polyacrylic acid fuji ii lc group g ii 30 g ii 1 10 polyacrylic acid ketac fill g ii 2 10 polyacrylic acid vidrion r g ii 3 10 polyacrylic acid fuji ii lc 5 mauro et al. figure 1. (a) preparation accomplished with the convention technique in group gi – regular dentin surface obtained by the spherical drill (microscopy 200x). (e) preparation accomplished with the art technique in group gi – irregular dentin surface obtained by the instruments used in the art technique (microscopy 200x). dentin adhesive surface using the glass ionomer ketac fill, vidrion r and fuji ii lc after carious tissue removal by the conventional technique in b, c and d, respectively (microscopy 400x). dentin adhesive surface using the glass ionomer ketac fill, vidrion r and fuji ii lc after carious tissue removal by the art technique in f, g and h (microscopy 400x). a b c d e f g h 6 mauro et al. results regarding the preparation, the uniformity obtained with the spherical drill used in low rotation was observed for the group i (figure 1a). however, the cavity floor surface presented irregular shape in the group ii (figure 1e). an absence of ionomeric restorative material on the dentin adhesive surface was noted in the specimens of the groups gi-1(figure 1b), gi-2 (figure 1c), gii-1 (figure 1f) and gii-2 (figure 1g). the glass ionomer cement and a stained area were observed on the remaining dentin surface in the groups gi-3 (figure 1d) and gii-3 (figure 1h). this indicates the infiltration of the product into the dentin surface, which means a very significant glass ionomer and dentin interaction. the mean thickness was 7.5μm for the group gi-3 (figure 1d) and 2.5μm for the group gii-3 (figure 8). all sections exhibited the same pattern (table 3). considering that no variability was observed for the means between the groups gi-3 and gii-3, no statistical analysis was applied. however, it was evident that 7.5µm in thickness represents a better hybrid layer than 2.5µm. discussion in this study, the groups submitted to the conventional technique presented smoother and more uniform dentin surfaces than those submitted to the art technique, which presented very irregular dentin surfaces. the art could be an important treatment method to children, especially those that have high treatment needs, hard access to dental care and limited financial resources. moreover, this technique can be performed in faster clinical sessions and with reduced cost of treatment being recommended by the international dental federation and world health organization in worldwide16. researches showed that the longevity of art restorations in primary teeth are similar from those produced using traditional methods using either amalgam or resin composite17,18. frencken et al.19 related that an effective method to remove decayed dentine is by using a sharp metal hand excavator, because the rotating metal dental drill may promote exacerbated removal of dental tissue in the prepare the cavity. indeed, there are studies that reinforce the effectiveness of art technique in treatment of carious lesion20. according to this study, the quality of cavity preparation with the conventional technique for removal of carious dentin tissue could result in a better condition for glass ionomer table 3. average measurements of the interaction layer between the glass ionomer cement and dentine, according to each treatment group groups gi 1 gii 1 gi 2 gii 2 gi 3 gii 3 n 10 10 10 10 10 10 averages 0 μm 0 μm 0 μm 0 μm 7.5 μm 2.5 μm 7 mauro et al. and dentin interaction10. the regular and uniform preparation favors the material flowage and improves the intimate contact between the material and dentin tissue, which is very important for ionic changes between the glass ionomer and dentin5,7. dentin quality improves glass ionomer adhesion since it is more complete and with greater amount of calcium. this characteristic results in greater amount of carboxyl groups interacting with hydroxyapatite calcium and, thus, a greater adhesive resistance7. although these considerations, it was not possible to maintain the bonding of the conventional glass ionomers ketac fill and vidrion r to dentin submitted to the demineralization procedure. destruction of the acid-resistant layer during the preparation of the specimens is recommended by some authors which allowed the restoration dislodgment in the cavities without any additional force. however, the use of the resin-modified glass ionomer cement fuji ii lc revealed the interaction layer with dentin. this layer was thicker (7.5μm) when the conventional technique was used for removal of carious dentin in comparison to the art technique (2.5μm). although the interaction layer obtained with the art technique was thinner, it could characterize an appropriate strong interaction for a long-term adhesive maintenance11. therefore, it will not affect the maintenance of infected dentin when the glass ionomer fuji ii lc is used in the restorative procedure. bond strength testing should be performed to test this hypothesis. these results demonstrate that the technique for carious dentin removal does not affect the interaction between the conventional glass ionomers and dentin since the acid-resistant layer was eliminated for both techniques. nevertheless, a different result was observed when the glass ionomer cement fuji ii lc was used, revealing that dentin quality was important for its interaction with the material. so, it seems that the ionomeric material properties are decisive for the interaction with a sound or demineralized dentin. however, independent of the technique used for caries removal, the use of cariostatic restorative materials allows the arrest of the carious lesion progress and remineralization of the remaining affected dentin4,10. for maltz et al.10, the success of this technique certainly depends on the complete cavity sealing, which could be obtained using a glass ionomer due to its adhesiveness and great potential for fluoride release. glass ionomer cements present a great remineralizing potential generally associated with mineral deposition in dentin tubules even for a carious dentin4,21,22. this property could be a factor for increasing tissue resistance and limiting the action of acidic biofilms4,10. this study demonstrated that dentin and glass ionomer cements exchanged minerals and organic elements, forming an intermediate layer. this layer was characterized as an acid-resistant adhesive layer that was not dissolved even under critical demineralization conditions23,24. although studies report the layer formation9, these observations were partially confirmed in the present study, since the dentin layer incorporated by the ionomeric material was not observed when the conventional glass ionomers ketac fill and vidrion r were used with both techniques. however, when the resin-modified glass ionomer cement fuji ii lc was used, a highly evident interaction layer between the material and dentin was demonstrated. this condition may result from the presence of the hydrophilic resinous components on the 8 mauro et al. materials composition24. such components generate the acid-resistant adhesive layer and also a micro-mechanical union more resistant to demineralization conditions than the adhesive layer23. this established greater retention of the material to dentin, independent of the method for carious tissue removal. according to the methodology used, an interaction between the conventional glass ionomers ketac fill and vidrion r and the dental structure more vulnerable to demineralization conditions was demonstrated in comparison to the resin-modified glass ionomer cement fuji ii lc. vidrion r and ketac fill spontaneously debonded from the cavities with both techniques, especially during decalcification. however, fuji ii lc did not debond from the cavity after decalcification, requiring additional force for its removal. so, this study demonstrated advantage on using resin-modified ionomer cement in comparison to conventional cements considering the cariogenic challenges frequently observed in dental elements. studies showed that art using high-viscosity glass-ionomer can be safely and reliably performed in single-surface cavities in both primary and permanent posterior teeth25. therefore, it is important to evaluate the cost-benefit of the ionomeric restorative material used in dental programs for destitute communities. when the art technique is used, it would be ideal to use resinous ionomer cements for restorations. it was concluded that the technique used for carious dentin removal does not seem to be determinant for the bonding of ionomeric materials to the dental structure, except for the resin-modified gic in which the formed hybrid layer was higher using the conventional technique. acknowledgements financial support provided by fundunesp – são paulo – brazil (process # 874/03-dfp). references 1. studart l, franca c, colares v. atraumatic restorative treatment in permanent molars: a systematic review. braz j oral sci. 2012 jan-mar;11(1):36-41. 2. schmalz g. art – a method on its way into dentistry. clin oral invest. 2012;16(5):1335-6. 3. gibilini c, de paula js, marques r, sousa mlr. atraumatic restorative treatment used for caries control at public schools in piracicaba, sp, brazil. braz j oral sci. 2012 jan-mar;11(1):14-8. 4. massara ml, alves jb, brandao pr. atraumatic restorative treatment: clinical, ultrastructural and chemical analysis. caries res 2002;36(6):430-6. 5. schriks mc, van amerongen we. atraumatic perspectives of art: psychological and physiological aspects of treatment with and without rotary instruments. community dent oral epidemiol 2003;31(1):15-20. 6. chen x, du m, fan m, mulder j, huysmans mc, frencken je. effectiveness of two new types of sealents: relation after 2 years. clin oral invest 2012;16:1443-50. 7. yiu ck, tay fr, king nm, pashley dh, sidhu sk, neo jc, et al. interaction of glass-ionomer cements with moist dentin. j dent res 2004;83(4):283-9. 8. yiu ck, tay fr, king nm, pashley dh, carvalho rm, carrilho mr. interaction of resin-modified glass-ionomer cements with moist dentine. j dent 2004;32(7):521-30. 9 mauro et al. 9. frencken je, wolke j. clinical and sem assessment of art high-viscosity glass-ionomer sealants after 8-13 years in 4 teeth. j dent 2010;38(1):59-64. 10. maltz m, de oliveira ef, fontanella v, bianchi r. a clinical, microbiologic, and radiographic study of deep caries lesions after incomplete caries removal. quintessence int 2002;33(2):151-9. 11. wadenya ro, yego c, mante fk. marginal microleakage of alternative restorative treatment and conventional glass ionomer restorations in extracted primary molars. j dent child (chic) 2010;77(1):32-5. 12. schwendicke f, frencken je, bjørndal l, maltz m, manton dj, ricketts d, et al. managing carious lesions: consensus recommendations on carious tissue removal. adv dent res 2016;28(2):58-67. 13. innes np, frencken je, bjørndal l, maltz m, manton dj, ricketts d, et al. managing carious lesions: consensus recommendations on terminology. adv dent res 2016;28(2):49-57. 14. frencken je, innes np, schwendicke f. managing carious lesions: why do we need consensus on terminology and clinical recommendations on carious tissue removal? adv dent res 2016;28(2):46-8. 15. brown jh, brenn l. a method for differential staining of gram positive and gram negative bacteria in tissue reactions. bull johns hopkins hosp. 1931;48;69-73. 16. kateeb et, warren jj, damiano p, momany e, kanellis m, weber-gasparoni k, et al. teaching atraumatic restorative treatment in u.s. dental schools: a survey of predoctoral pediatric dentistry program directors. j dent educ 2013;77(10):1306-14. 17. mickenautsch s, yengopal v, banerjee a. atraumatic restorative treatment versus amalgam restoration longevity: a systematic review. clin oral investig 2010;14(3):233-40. 18. mickenautsch s, yengopal v. failure rate of atraumatic restorative treatment using high-viscosity glass-ionomer cement compared to that of conventional amalgam restorative treatment in primary and permanent teeth: a systematic review update. j minim interv dent. 2012;5:63-124. 19. frencken je, peters mc, manton dj, leal sc, gordan vv, eden e. minimal intervention dentistry for managing dental caries a review: report of a fdi task group. int dent j 2012;62(5):223-43. 20. kateeb et, warren j, gaeth g, damiano p, momany e, kanellis mj, et al. the willingness of us pediatric dentists to use atraumatic restorative treatment (art) with their patients: a conjoint analysis. j public health dent 2014;74(3):234-40. 21. bolzan pa, bruschi arc, spagnolo aga, puppin rj, lourenço cs, maria prr. influence of chemical degradation and abrasion on surface properties of nanorestorative materials. braz. j oral sci 2015 apr-jun;14(2):100-5. 22. prado gk, ribeiro mm, carlos rj, camila ap, aquino mc, joana rj. one-year clinical evaluation of the retention of resin and glass ionomer sealants on permanent first molars in children. braz. j oral sci. 2015 jul-sep;14(3):190-4. 23. shintome lk, nagayassu mp, di nicoló r, myaki si. microhardness of glass ionomer cements indicated for the art technique according to surface protection treatment and storage time. braz oral res. 2009;23(4):439-45. 24. wang l, cefaly df, dos santos jl, dos santos jr, lauris jr, mondelli rf, etal. in vitro interactions between lactic acid solution and art glass-ionomer cements. j appl oral sci 2009;17(4):274-9. 25. de amorim rg, leal sc, frencken je. survival of atraumatic restorative treatment (art) sealants and restorations: a meta-analysis. clin oral investig 2012;16(2):429-41. 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1509/1162 1/8 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1509/1162 2/8 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1509/1162 3/8 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1509/1162 4/8 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1509/1162 5/8 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1509/1162 6/8 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1509/1162 7/8 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1509/1162 8/8 braz j oral sci. 15(4):248-251 microbial contamination of a university dental clinic in brazil victor hugo marques coelho1, gisely naura venâncio*2, thiago fontanella cestari3, maxine ennata alves de almeida4, carolinie batista nobre da cruz5 1dentistry department, university of nilton lins, school of dentistry, manaus, amazonas, brazil. victor_marques_@hotmail.com 2msc, health secretary of manaus city, manaus, amazonas, brazil. ginaura@gmail.com 3dentistry department, university of nilton lins, school of dentistry, manaus, amazonas, brazil. thiagofc10@gmail.com 4dentistry department, university of nilton lins, school of dentistry, manaus, amazonas, brazil. maxinealmeida@hotmail.com 5biology department, university of nilton lins, school of biological sciences, manaus, amazonas, brazil. carol_nobre24@yahoo.com.br correspondence to: gisely naura venâncio mário ypiranga monteiro avenue, 1695 – adrianópolis zip code: 69057-001 manaus-amazonas, brazil 92-991764966 abstract pathogens of the oral cavity of a patient can be transferred to the dental office surfaces by direct contact, aerosol instruments and blood or saliva. the objective of this study was to investigate the microbiological contamination presents in the stands, chairs and spittoons in the university nilton lins dental clinics, in manaus, amazonas. samples were collected with sterile swabs and seeded in different microbiological culture media for the isolation of microorganisms collected from each room. then, assays were carried out for identification of strains isolated from each environment, such as: gram stain, dna purification, amplification of 16s rrna genes and sequencing. all these experiments were performed in the lbs / ilmd / fiocruz. it was found 40 cfu / ml in the stands, 43 on the chairs and 47 in the spittoons and it was also possible to identify microorganisms like klebsiella pneumoniae, shigella sonnei and staphylococcus aureus. the greatest number of cfus was found in clinic 3 and it was observed that the spittoon was the dental surface with the highest number of cfus. some of the bacterial species isolated are opportunists, suggesting that more severe biosecurity measures must be taken in order to prevent cross-infection. keywords: students, dental; biosecurity; exposure to biological agents. introduction oral microbiota are a potentially significant source of contamination and crossinfection in the dental clinic1. the oral cavity is a natural habitat for a large number of microorganisms which, during dental practice, can be transferred to the equipment and instruments used in routine clinical activities of the dentist, posing a risk of cross contamination and infection and may even cause systemic infections2. dental health care students and professionals are at a risk of diseases including hbv, hcv, herpes simplex virus type 1, hiv, influenza, rubella, and besides that, fixed dental units, water lines and handpieces can become vehicles of cross-infection in dental offices3-5. the main bacteria that can cause infection hazards linked to dental practice are streptococcus pneumoniae, mycobacterium tuberculosis, klebsiella pneumoniae, escherichia coli, legionella pneumophila and pseudomonas aeruginosa6,7. this received for publication: november 4, 2016 accepted: june 15, 2017 original article braz j oral sci. october | december 2016 volume 15, number 4 http://dx.doi.org/10.20396/bjos.v15i4.8650030 249 aspect is critical in the dental field, where there is constant daily exposure to a wide variety of microorganisms of the oral microflora of patients8. even in non-invasive procedures4, such as x-rays, potentially pathogenic fungal species are found in x-ray apparatus, which can serve as reservoirs or fungal vectors representing a risk of acquisition of cross-infection for the patient, as well as for the dental team5. during a dental appointment, there is dispersion of splashes and aerosols containing pathogenic microorganisms that can be transmitted through saliva, blood and oral secretions onto countertops and materials, furniture and the dental unit itself6. aerosols can be a source of infection for dentists and an indirect cause of occupational hazards at work7. to avoid infection in the oral cavity, cleaning methods or disinfection and sterilization of the dental material to be used in the patient are recommended8. the professional must follow standard procedures such as risk evaluation and patient protection, personal protection, sterilization and chemical disinfection, sterilization of equipment, appropriate waste disposal, among others9. the aim of this study was to investigate the microbiological contamination presents in the stands, chairs and spittoons in the university nilton lins dental clinics, in manaus, amazonas, through the use of pcr 16s rrna. materials and methods study model samples (a total of 9 chairs, 9 benches and 9 spitters) were collected in dental clinics of the university nilton lins and the experiments were performed in the biodiversity laboratory of the health institute leonidas and maria deane of the oswaldo cruz foundation (lbs / ilmd / fiocruz). study population three chairs, 3 benches and 3 spitters were collected from each of the three dental clinics. inclusion criteria: chairs, benches and spitters in perfect condition, used daily by the students. exclusion criterion: chairs with some defect, making it impossible for students to use it. sampling the study was conducted in dental clinics of the nilton lins university, where samples were collected in triplicate from the stands, chairs and spittoons with sterile swabs. the sample collection was carried out in different areas in order to cover the entire clinic. prior to this, the surfaces were disinfected with 70% alcohol. all handling was performed using sterile gloves. bacteriological assays samples were immediately inoculated in assay tubes containing 3 ml of brain heart infusion (bhi difco) and incubated at 37ºc for 24 hours. after incubation (24 hours / 37°c), the tubes that showed turbidity of the culture medium had the material placed in petri dishes containing macconkey agar, pia agar, bhi agar, muellerhinton agar, sabouraud agar culture media. the plates were incubated (24 hours / 37°c)8. plates were then analyzed by counting the number of grown colonies, their size, hemolysis and staining. following this, gram staining of each selected strain was performed. dna purification each isolated strain was grown in luria bertani liquid (24 hours / 37°c) with stirring. the culture was transferred to microtubes and centrifuged (16000 g / 10 min / 4°c). the supernatant was discarded and the pellet resuspended in 300 ul (microliter) of buffer and homogenized by vortexing. after adding 30 ul of lysozyme the samples were incubated (30 min / room temperature). they were then added to samples of 50 ul of triton x-100 10% 20 ul of 3 m nacl. following this they were heated in a dry bath (5 min / 60°c) and 2 ul rna were added, and then incubated in an oven (15 min / 37°c). 25 ul of 10% sds were added to the samples and homogenized by vortexing, adding 3 ul of proteinase and then incubated (15 min / 37°c). thereafter, 500 ul of phenol was added and the mixture was stirred manually for 5 minutes. the sample was centrifuged (16000 g / 10 min) at room temperature. the supernatant was removed, recovering the aqueous phase and replacing the microtube by adding 400 ul of chloroform, followed by manual shaking for 5 minutes. once again the samples were centrifuged (16000 g / 10 min) at room temperature and the supernatant was discarded. then the microtube was replaced and 25 ul of 3m nacl was added. finally, there was the careful addition of 1 ml of 100% ethanol at 20°c. the samples were centrifuged (16000 g / 10 min / 4°c), resuspended in 70% ethanol and homogenized by vortexing, centrifuging again under the same conditions, discarding the supernatant carefully. the dna microtube was dried under flow and resuspended in 200ul of buffer (10mm tris-hcl, ph 7.5 + 1 mm edta), and maintained at -20°c. the extraction protocol was performed as described in literature, however with modifications10,11. amplification of 16s rrna genes and sequencing the reaction was performed under the following conditions: 40 ng of dna, 10x0.25 mm buffer of dntps, 2.5 mm of mgcl, 5 mm of each primer, 2.5 u taq polymerase and sterile deionised water to a total volume of 25 ul. the primers used were: 530f and 1492r of the 16s rrna gene. the polymerase chain reaction (pcr) program used in a thermocycler (eppendorf) comprised the following steps: initial denaturation (5 min / 95°c); followed by a 45 times cycle with a denaturing step (15 sec / 95°c), annealing of primers (20 sec / 65°c) and an extension step of the tapes (2 min / 72°c); in addition to the final extension (2 min / 72°c). it was used agarose gel electrophoresis to visualize the pcr products and observe the effects the different annealing temperatures had on the pcr reaction12. the samples were then sequenced by the fiocruz / ilmd platform, using the automatic sequencer abi prism 3100 genetic analyzer ™ (applied biosystems) (figure 1). microbial contamination of a university dental clinic in brazil braz j oral sci. 15(4):248-251 250 results the sequences obtained were processed for removal of poor quality sequences using the "phred / phrap" program. each sequence was compared to sequences deposited in the "genebank" of national center for biotechnology information and with sequences deposited in the ribosomal database project. a total of 27 samples distributed in 3 bench samples, 3 chair samples and 3 spittoon samples for each clinic were analyzed. bacteriological analysis of samples taken showed that 27 (100%) samples had microbial growth. the results obtained in this study showed variations in total cfu / ml between clinics and dental units (figures 2 and 3). figure 2 shows the total number of cfus found in each university dental clinic. thus, the greatest number of cfus was found in clinic 3, indicating that the decontamination process was less efficient in relation to the others. the microorganisms found on the surfaces of the dental clinics of the university are listed in table 1 and it was possible to identify 16 bacterial species and 3 genera. microbial contamination of a university dental clinic in brazil braz j oral sci. 15(4):248-251 table 1 correlation between clinics, places and species / bacterial genus found. clinic 1 chair bacillus subtilis, exiguobacterium sp stand rhizobium sp spittoon bacillus pumilus, bacillus cereus clinic 2 chair klebsiella pneumoniae, stand klebsiella pneumoniae, staphylococcus aureus, bacillus cereus spittoon staphylococcus warneri, enterobacter cancerogenus, clinic 3 chair staphylococcus saprophyticus, bacillus megaterium, bacillus safensis, acinetobacter sp stand shigella sonnei spittoon staphylococcus pasteuri, enterobacter amnigenus, citrobacter freundii, escherichia coli fig. 1 electrophoretic profile of genes in multiplex pcr found in the samples in 1.5% agarose gel. fig. 2 correlation cfu / ml between the clinics. figure 3 shows the total number of cfus found in each type of surface of the dental equipment, and it was observed that the spittoon was the dental surface with the highest number of cfus, due to the surface having direct contact with the secretions produced by the patient. fig. 3 correlation cfu / ml between the sites collected. discussion the highest concentration of microorganisms in the dental office is found in the patient's mouth13. the hands of dental professionals, once contaminated with saliva, sulcular fluid and / or blood, are the major vehicles for contamination of surfaces14. in this context, the microbial contamination of dental clinics, such as dental unit water systems it was already found p. aeruginosa and legionella spp. 7* so, the use of barriers, such as cellophane paper or plastics, are recommended and do not interfere in clinical procedures15. in multidisciplinary clinics, as seen in dentistry courses, biosecurity measures for infection control play a major role in daily care. staphylococcus aureus is found in human skin, especially nares and perineum and it has been linked to different types of infection, including pneumonia, meningitis, osteomyelitis, infections of the skin and soft tissue16. the bacillus genus comprises about 50 species of chain forming gram-positive bacilli which are also capable of forming spores. bacteria which are usually found in the bacillus subtilis environment are used as biological indicators for testing the 251 microbial contamination of a university dental clinic in brazil sterilization effectiveness of autoclaves17. escherichia coli is a gram-negative bacillus mobile belonging to the enterobacteriaceae family. all enterobacteriaceae are potentially pathogenic and responsible for various types of infections such as diarrheal diseases, urinary tract infections and sepsis18. acinetobacter spp. are non-fermentative bacteria, and potentially pathogenic environmental contaminants19. shigella sonnei is a bacterium that is an important agent of diarrheal infectious diseases that usually affects children, the elderly and immunocompromised patients. shigella ssp. has the ability to invade human intestinal mucosa and cause dysentery, spreading efficiently through a low dose of fecal-oral transmission20. klebsiella pneumoniae is a gram-negative bacterium commonly found in the skin and saliva that plays an important role as a resistance mechanism in the general hospital setting and confers resistance to carbapenem antibiotics, and inactivates penicillins, cephalosporins and monobactams21. professionals have to be aware of the inherent risk in their work, and be trained and encouraged to apply biosecurity procedures in order to reduce or eliminate these microorganisms5. conclusions given the results of this study and published reports, it is clear that the contamination of surfaces of dental units exists, and thus it is a matter of great importance that should be discussed among professionals and students of dentistry to search for more effective ways to prevent cross-infection. in order to decrease the risks it is necessary to establish a biosafety protocol in dental clinics which includes the correct preparation procedures of dental units, use of ipe (individual protection equipment) and adequate disinfection of surfaces. references 1. ghosh s, mallick sk. microbial bofilm: contamination in dental chair unit. ind med gaz. 2012 oct;145(10):383-7. 2. laheij am, kistler jo, belibasakis gn, välimaa h, de soet jj. healthcare-associated viral and bacterial infections in dentistry. j oral microbiol. 2012;4:1-10. doi: 10.3402/jom.v4i0.17659. 3. artini m, scoarughi gl, papa r, dolci g, de luca m, orsini g et al. specific anti cross-infection measures may help to prevent viral contamination of dental unit waterlines: a pilot study. infection. 2008 oct;36(5):467-71. doi: 10.1007/s15010-008-7246-5. 4. alavian sm. hepatitis c virus infection: epidemiology, risk factors and prevention strategies in public health in i.r.iran. gastroenterol hepatol bed brench. 2010;3(1):5-14. 5. dahiya p, kamal r, sharma v, kaur s. “hepatitis” – prevention and management in dental practice. j educ health promot. 2015 may 19;4:33. doi:10.4103/2277-9531.157188. 6. castiglia p, liguori g, montagna mt, napoli c, pasquarella c, bergomi m, et al. italian multicenter study on infection hazards during dental practice: control of environmental microbial contamination in public dental surgeries. bmc public health. 2008 may 29;8:187. doi: 10.1186/14712458-8-187. 7. pasquarella c, veronesi l, castiglia p, liguori g, montagna mt, napoli c, et al. italian multicentre study on microbial environmental contamination in dental clinics: a pilot study. sci total environ 2010 sep 1;408(19):404551. doi: 10.1016/j.scitotenv.2010.05.010 8. umar d, basheer b, husain a, baroudi k, ahamed f, kumar a. evaluation of bacterial contamination in a clinical environment. j int oral health 2015 jan;7(1):53-5. 9. cardoso ct, pinto júnior jr, pereira ea, barros lm, freitas abda. [contamination of composite resin tubes handled without a protective barrier]. robrac. 2010;18(48):71-5. portuguese. 10. santos ko, mobin m, borba cm, noleto ims. [isolation of fungi from dental radiographic equipment]. rgo. 2011;59(3):411-6. portuguese. 11. barreto acb, vasconcelos cpp, girão cms, rocha mmnp, mota oml, pereira s ls. [environmental contamination by aerosols during treatment using ultrasonic devices]. periodontia. 2011;21(2):79-84. portuguese. 12. kumar s, atray d, paiwal d, balasubramanyam g, duraiswamy p, kulkarni s. dental unit waterlines: source of contamination and cross infection. j hosp infect. 2010 feb;74(2):99-111. doi: 10.1016/j. jhin.2009.03.027. 13. spolidorio p, duque c. [microbiology and general and dental immunology]. são paulo: artes médicas; 2013. portuguese. 14. engelmann ai, dal aa, miura csn, bremm ll, boleta-cerantio dc. [evaluation of procedures performed by suregen-dentists from cascavel state of parana and surroundings for biossecurity control]. odontol clin cient. 2010 apr-jun;9(2):161-5. portuguese. 15. zadoks rnw, van leeuwen b, kreft d, fox lk, barkema hw, schukken yh, et al. comparison of staphylococcus aureus isolates from bovine and human skin, milking equipment, and bovine milk by phage typing, pulsed-field gel electrophoresis, and binary typing. j clin microbiol. 2002 nov;40(11):3894-902. 16. casamayor eo, schäfer h, bañeras l, pedrós-alió c, muyzer g. identification of and spatio-temporal differences between microbial assemblages from two neighboring sulfurous lakes: comparison by microscopy and denaturing gradient gel electrophoresis. appl environ microbiol. 2000 feb;66 (2):499-508. 17. matsuki t, watanabe k, fujimoto j, kado y, takada t, matsumoto k, et al. quantitative pcr with 16s rrna gene targeted species-specific primers for analysis of human intestinal bifidobacteria. appl environ microbiol. 2004 jan;70(1):167-73. 18. tenaillon o, skurnik d, picard b, denamur e. the population genetics of commensal escherichia coli. nat rev microbiol. 2010;8:207-17. 19. peleg ay, hooper dc. hospital-acquired infections due to gramnegative bacteria. n engl j med. 2010. 362:1804-13. doi:10.1056/ nejmra0904124. 20. molinari ja. infection control its evolution to the current standard precautions. j am dent assoc. 2003 may;134(5):569-74. 21. chong sl, lam yk, lee fkf, ramalingam l, yeo acp, lim cc. effect of various infection-control methods for light-cure units on the cure of composite resins. oper dent. 1998 mar-apr;23(3):150-4. braz j oral sci. 15(4):248-251 1http://dx.doi.org/10.20396/bjos.v16i0.8650490 volume 16 2017 e17016 original articlebjos 1 phd student, dental materials laboratory, department of conservative dentistry, school of dentistry, federal university of rio grande do sul, porto alegre, rs, brazil 2 professor, dental materials laboratory, department of conservative dentistry, school of dentistry, federal university of rio grande do sul, porto alegre, rs, brazil 3 undergraduate student, dental materials laboratory, department of conservative dentistry, school of dentistry, federal university of rio grande do sul, porto alegre, rs, brazil corresponding author: fabrício mezzomo collares. dental materials laboratory, school of dentistry, federal university of rio grande do sul, ramiro barcelos street 2492, porto alegre, rs, brazil. telephone: +5551 33085198. fabricio.collares@ufrgs.br received: february 6 2017 accepted: july 26, 2017 effect of beverages on surface properties of resin-based sealants bruna genari1, vicente castelo branco leitune2, joão henrique macedo saucedo3, susana maria werner samuel2, fabrício mezzomo collares2 aim: the aim of this study was to determine degree of conversion of resin-based sealants and the effect of beverages on surface roughness and color change of materials. methods: two commercial resin-based sealants were evaluated (defense chroma® and bioseal®). degree of conversion (dc) was initially measured using fourier-transformed infrared (ftir). specimens of each sealant were maintained in distilled water and immersed one hour daily in grape juice and cola drink for 30 days. one group was maintained only in distilled water, as a control. surface roughness and color change were measured before immersion, after seven days of immersion and after 30 days of immersion. results were analyzed using t-test, paired t-test, one-way repeated measures anova, one-way anova and post-hoc tests (0.05 level of significance). results: there was no significant difference regarding degree of conversion and initial surface roughness comparing both commercial sealants. surface roughness increased for bioseal® immersed in cola drink for 30 days. after 30 days, all groups presented significant color change. conclusions: the effect of beverages on color stability and surface roughness of resin-based sealants depended on exposure time and kind of beverage. keywords: methacrylates. aging. discoloration. surface properties. mailto:fabricio.collares@ufrgs.br 2 genari et al. introduction the majority of caries occurs in fissures and pits of molars of children and adolescents1,2. although glass ionomer cements are known by their fluoride release property, resin-based sealants are also commonly used to protect fissures and pits due to its higher retention rate3. its fluoride release through a slow diffusive of filler particles contributes to caries inhibition4. resin materials are submitted to physical stresses and chemical process. one of these factors is the exogenous substances taken in the diet, which contain a variety of acids such as tartaric acid present in grape juice and phosphoric acid in cola beverages5. chemistry and duration of exposure are important to determine the interaction between molecule and material polymeric network6. chemical degradation of materials also depends on composition of material. resin-based materials are susceptible to degradation, mainly those with no or low inorganic filler content7,8, such as sealants. the early effect of degradation is an increased surface roughness, which leads to raise biofilm accumulation and color change due stain penetration into the polymeric matrix8. sealants have been the subject of numerous studies evaluating effectiveness, adaptation and penetration into fissures and fluoride release4,9. in the oral environment, dental material is also subjected to chemical and physical insults. hence, the purpose of this study was to determine degree of conversion of resin-based sealants and the effect of beverages on surface roughness and color change of materials. methods two resin-based sealants were evaluated: defense chroma® (angelus, londrina, brazil) and bioseal® (biodinâmica, ibiporã, brazil), whose composition is in table 1. degree of conversion (dc) the dc was evaluated by ftir spectrometer. a drop (5 μl) of each sealant (n=3) was photoactivated for 20 s by a light-emitting diode (led) with irradiance value of 1200 mw/cm2 (radii cal, sdi, bayswater, australia). absorbance spectra were obtained before and immediately after light polymerization. the dc was calculated considering the intensity (peak height) of aliphatic carbon-carbon double bond stretching vibration at 1635 cm-1 and the symmetric ring stretching at 1610 cm-1 from the polymerized and unpolymerized samples. table 1. composition of resin-based sealants used in the present study. sealant manufacturer composition lot photoactivation time bioseal® biodinâmica, ibiporã, brazil tegdma, bis-gma, bht, silicon dioxide, sodium fluoride, calcium fluoride and catalyst 27025 20 s defense chroma® angelus, londrina, brazil bis-gma, modified urethane, tegdma, barium aluminum borosilicate, tetra-acrylic ester, phosphoric acid, sodium fluoride, n-methyl diethanolamine and camphorquinone 20412 20 s 3 genari et al. immersion of specimens in beverages fifteen disc-shaped specimens (10 mm x 2 mm) were prepared for each resin-based sealant. materials was photoactivated for 20 s, according to the manufacturer’s recommendations. after 24 h, the specimens were polished using a manual polisher (aropol 2v, arotec, cotia, brazil) through a series of silicon carbide (sic) papers (400-, 600-, 800and 1200-grit) for 2 minutes each and with a felt disc saturated with an alumina suspension. to evaluate surface roughness and color stability of materials in beverages, specimens of each sealant were randomly distributed in three subgroups (n=5) of beverages (distilled water, dellvalle® grape juice, and coke® cola drink). beverages were maintained at 4 oc during the period of study (30 days) and at room temperature before placing specimens for acid challenge. each specimen was individually immersed in a vial containing 5 ml of beverage for one hour daily at room temperature. specimens were washed with distilled water and stored again in distilled water at 37 oc. as a control group, specimens were individually immersed in vials containing distilled water for 24 h per day, spending one hour at room temperature. vials were sealed during immersion to prevent the evaporation of beverages. all solutions were renewed daily. ph measurement determination of beverages’ ph was performed at room temperature (22 ± 2 oc) using a ph meter (dm-22, digimed, campo grande, brazil). results of ph were obtained from an average of three measurements. it was measured before using beverages. surface roughness surface roughness (ra) was measured using a roughness tester (sj-201, mitotoyo, kawasaki, japan) on surface discs of resin-based sealants. results were obtained by averaging three equidistant lines of 2 mm. surface roughness was measured at baseline, after seven days of immersion and after 30 days of immersion of specimens. color change color measurements of specimens were performed according to the cie l*a*b* color scale, relative to standard illuminant d65, over a black and white backgrounds by a reflection spectrophotometer (cm-2500d, konika-minolka, tokyo, japan), with inclusion of ultraviolet (uv) component and exclusion of specular component (sce) geometry. the aperture size was 3 mm, illuminating and viewing configurations were cie diffuse/10o geometry. before measurement, the spectrophotometer was calibrated using the supplied white calibration standard. the reflectance spectra were obtained from 400 to 700 nm. due to the use of black and white backgrounds, kubelka-munk’s theory was used to calculate reflectance spectra of infinite thickness. chromaticity coordinates (x, y, z) of cie (the comission internationale de l’eclairage) were calculated using reflectance spectra of infinite thickness. the coordinates were converted to cie l*a*b* parameters. the color measurements were performed at baseline, after seven days of immersion and after 30 days of immersion. color change (δe) was calculated from differences between l*a*b* parameters between baseline and different times. 4 genari et al. statistical analysis statistical analysis was performed using sigma plot 11.0. the results were initially analyzed using the kolmogorov–smirnov test. t-test was used to analyze initial surface roughness and dc, comparing both sealants. one-way repeated measures anova was performed to compare roughness among different times. paired t-test was used to compare color change among times. in addition, one-way anova and tukey post-hoc tests were performed to analyze color changes among groups of different sealants and immersion beverages. the statistically significant level was 0.05. results degree of conversion for defense chroma® was 49.23 ± 2.46% and for bioseal® was 52.28 ± 2.86%, presenting no statistically significant difference between sealants. values of ph for distilled water, grape juice and cola drink were 5.53, 3.09 and 2.72, respectively. results of surface roughness are presented in table 2. there is no difference in initial surface roughness comparing sealants, presenting a roughness of 0.17 (±0.05) for defense chroma® and 0.18 (±0.09) for bioseal®. surface roughness did not alter, except for bioseal® immersed in cola drink for 30 days (p < 0.05). defense chroma® presented higher δe than bioseal® after seven days of immersion (p < 0.05) independently of beverage (table 2). after immersion for 30 days, all groups presented higher δe than after seven days (p < 0.05). although color change occurred for all specimens, higher difference was between bioseal® immersed in cola drink and defense chroma® in distilled water. table 2. mean and standard deviation of surface roughness (μm) and color change (δe) for bioseal® and defense chroma® sealants immersed in distilled water, grape juice and coca-cola® for 7 and 30 days. initial surface roughness color change (δe) 7 days 30 days 7 days 30 days bioseal® distilled water 0.11 (± 0.03)a 0.17 (± 0.04)a 0.15 (± 0.06)a 1.92 (± 0.49)aa 15.38 (± 0.64)abb grape juice 0.24 (± 0.18)a 0.24 (± 0.11)a 0.24 (± 0.12)a 1.28 (± 0.91)aa 15.32 (± 0.41)abb cola 0.20 (± 0.06)a 0.26 (± 0.06)a 0.49 (± 0.13)b 1.33 (± 0.41)aa 15.89 (± 0.69)ab defense chroma® distilled water 0.15 (± 0.05)a 0.17 (± 0.06)a 0.16 (± 0.06)a 5.11 (± 2.44) ba 13.70 (± 0.77)bb grape juice 0.19 (± 0.06)a 0.20 (± 0.12)a 0.21 (± 0.07)a 4.53 (± 0.48)ba 14.27 (± 1.02)abb cola 0.19 (± 0.08)a 0.24 (± 0.14)a 0.25 (± 0.06)a 5.34 (± 1.47)ba 14.31 (± 1.33)abb values followed by identical lower case letters denote no significant difference (p > 0.05) in the same row. values followed by identical capital letters denote no significant difference (p > 0.05) in the same column. 5 genari et al. discussion sealants protect high-risk children and adolescents from caries2,10. low amount or no fillers of these resin-based materials, which promote enough flow to allow adequate penetration of material into pits and fissures influence properties of material3,11. in the present study, sealants presented similar degree of conversion and susceptibility to chemical degradation in challenge conditions, except for bioseal® immersed in cola drink after 30 days. regarding performance of application of sealants, previous studies extensively discussed about retention, which is higher for resin-based materials than for glass ionomer cements12. in addition, the use (or not) of adhesive systems with sealants was also explored3. here is the first step toward the goal of elucidating surface properties of resin-based sealants in challenge conditions. high degree of conversion represents low amounts of released unreacted monomers and high resistance to degradation6. degree of conversion of bioseal® presented no significant difference compared to defense chroma®. their results are comparable to other experimental and commercial adhesives13, sealants14 and composite resins15. although sealants had different filler particles, they showed no difference in polymerization behavior, such as previously results, also evaluating other resin-based materials16. color stability and surface roughness are mostly tested for composite resin by immersion into red wine, tea and coffee10,17. there is a lack of studies related to pediatric situations, when sealants are often used as a preventive method for caries. sealants are used for children and adolescents at high risk for caries, who generally consume sugary beverages10. most of these have strong pigments and erosive potential due to their acidity. in the present study, simulating high daily frequency, it was possible to notice color change in the medium term and increased roughness for bioseal® immersed in cola drink for 30 days. increased time of exposure to beverages results in higher color change and roughness alteration because it allows more chemical interaction between beverage and organic matrix. besides hydrolytic degradation, the interaction with organic acids of beverages induces faster leaching of monomers by catalysis of ester groups from monomers18. phosphoric acid, presented in cola drinks, results more erosive effect in present sealants and also in tooth tissue19. degradation effects on polymeric matrix are initially perceived by increased surface roughness8,20, which facilitates the retention of bacterial plaque and extrinsic stains8,21. bioseal® presented increased roughness, due to higher hydrophilic monomers and different filler particles that lead to chemical degradation8,22,14. extrinsic compounds interact with the polymeric network in free volume spaces between polymer chains, resulting in colored oxidation products8,23,24. urethane methacrylate, due to its low water sorption and solubility6, presents more resistance to staining than hydroxyl groups25. defense chroma® contains urethane groups in its composition, which explains its resistance to discoloration. defense chroma® and bioseal® presented higher color change than acceptable difference (δ e > 3.3)26 in 30 days, even immersed in water. 6 genari et al. measurement of color parameters is based on reflectance spectra. evaluating a non-opaque material, there is a tendency to result in a gray effect, as occurs using a black background24 and in class iii and iv restorations27. in this study, black and white backgrounds were used to avoid a gray effect in non-opaque sealant evaluation. in this study, kubelka-munk was required to simulate an infinite thickness to calculate color parameters. the reflectance at infinite thickness is also known as reflectivity and describes an inherent reflectance and color of material28. although consumption was simulated in the present study, there are also the dilution effects of saliva and other fluids, intermittent exposure to beverages and effect of brushing, which could change exposure time necessary to alter surface properties. conclusions sealants present discoloration after 30 days. bioseal® presented increased surface roughness after 30 days of immersion into cola drink. the effect of 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optical and surface properties of model composites. j dent. 2013 nov;41 suppl 5:e45-53. doi: 10.1016/j.jdent.2013.05.011. 25. martim gc, detomini tr, schuquel it, radovanovic e, pfeifer cs, girotto em. a urethane-based multimethacrylate mixture and its use in dental composites with combined high-performance properties. dent mater. 2014 feb;30(2):155-63. doi: 10.1016/j.dental.2013.11.002. 26. ruyter ie, nilner k, moller b. color stability of dental composite resin materials for crown and bridge veneers. dent mater. 1987 oct;3(5):246-51. 27. ikeda t, sidhu sk, omata y, fujita m, sano h. colour and translucency of opaque-shades and body-shades of resin composites. eur j oral sci. 2005 apr;113(2):170-3. 28. mikhail ss, azer ss, johnston wm. accuracy of kubelka-munk reflectance theory for dental resin composite material. dent mater. 2012 jul;28(7):729-35. doi: 10.1016/j.dental.2012.03.006. untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8652939 volume 17 2018 e18371 original article 1 msc, phd, department of prosthodontics and periodontology, piracicaba dental school, state university of campinas, piracicaba, sp, brazil 2 msc, graduate program in dentistry, school of dentistry, federal university of pelotas, rs, brazil 3 phd, graduate program in dentistry, school of dentistry, federal university of pelotas, rs, brazil 4 phd, professor, department of restorative dentistry, school of dentistry, federal university of pelotas, rs, brazil. corresponding author: prof. dra. f. faot department of restorative dentistry, school of dentistry, federal university of pelotas, gonçalves chaves st., 457, center, pelotas, rs, brazil, 96015560 phone/fax: +55 53 32256741 e-mail: fernanda.faot@gmail.com received: march 07, 2018 accepted: june 01, 2018 simplified micrometric surface characterization of different implant surfaces available on the brazilian market luiz carlos do carmo filho1, ana paula pinto martins2, amália machado bielemann3, anna paula da rosa possebon3, fernanda faot4 aim: this study characterized the implant surfaces available on the brazilian market in terms of topography, chemical composition, and roughness. methods: the following brands were selected according to their surfaces: kopp (ko), signo vinces (sv), neodent (ne), osseotite (os), nanotite (nt), sin (si), titanium fix (tf), conventional straumann (str), slactive (sla). the morphological analysis and the alloy impurities and implant surface contaminants were analyzed by sem-eds. surface roughness parameters and 3-d reconstructions were obtained by laser microscopy (20x). two distinct areas were evaluated: i) the cervical portion (no surface treatment), and ii) the middle third (treated surface). results: the characterization of the implant surfaces by sem showed morphological differences between the thread geometries and surface morphology at 800x and 2000x magnification. the eds elemental analysis showed a predominance of titanium (ti) for all implants. the sla surface showed only peaks of ti while other implants brands showed traces of impurities and contaminants including al, c, pr, f, mg, na, ni, o, p, and sr. the implant surface roughness in the cervical portion did not exceed ra 0.5–1.0 μm, constituting a minimally rough surface and obtaining acceptable standards for this region. only nt, str, and sla presented ra above 2 μm in the middle third area showing a rough surface favorable for osseointegration. conclusion: this study concluded that there is no established standard for morphology, chemical composition and implant surface roughness that allows a safe comparison between the available dental implant surfaces. national implant brands generally contain more impurities and surface contaminants than their international counterparts and were consequently more sensitive to the surface treatment techniques. keywords: dental implants, surface properties, energy dispersive x-ray spectroscopy. mailto:fernanda.faot@gmail.com 2 do carmo filho et al. introduction the discovery of osseointegration enabled treatment of totally and partially edentulous patients with dental implants 1. in a previous study, branemark (1977) 2 stated that direct contact between vital bone tissue and implant is totally predictable, safe and long-lasting, and thus constitutes an important parameter for clinical success 3. insights in healing and repair processes contributed to the modification of the implant surface treatments and designs. surface roughness, macro, and microgeometry influence the cell proliferation and differentiation, extracellular matrix synthesis, local cell production factors, and even cell shape 4. therefore, the treatment used on the implant surface directly affects the implant’s survival rate and has been the focus of intense study 3. when the osseointegration is triggered, the osteoblast adhesion to the implant surface is required for the cell to receive signals that induce osteoblastic proliferation 5. adopting rough implant surfaces can facilitate the retention of the osteogenic cells and can accelerate their migration through osseoconduction 6. consequently, a high quality bone-implant interface guarantees faster and stronger bone formation that in turn promotes greater stability during the repair process 7–9. the primary stability is a predictor for successful osseointegration 10. geometry can also be factor, as spherical materials induce less fibrous encapsulation than cylindrical or sharp angles 11. the quality of the bone-implant interface also depends on the implant’s ability to support loading, mainly because overload biomechanics increase the bone density in the long-term and overloading might influence peri-implant tissue breakdown when plaque accumulation is present 12. the systematic review of in vivo data evidenced a differential peri-implant bone tissue response to overloading, depending on the mucosal health: supra-occlusal contacts in a non-inflamed peri-implant environment did not negatively affect osseointegration and are even anabolic 12. a combination of all the aforementioned factors can influence the clinical success of dental implants. in recent years, some studies 5,7,13,14 have measured the dental implants surfaces at macro, micro, and nanometric scales, to investigate how the different surfaces influence the bone repair process. although there are studies available that analyze the influence of surface roughness, little is known about the physico-chemical properties of the implant surfaces. this information is generally restricted to the implant package specifications and informational catalogs provided by the manufacturers 15. furthermore, the gold standard test to characterize the physical properties of implant surface treatments is interferometry, an expensive and time-consuming technique. this study aims to characterize the surface roughness of different commercial implant brands available in brazil using scanning electron microscopy images in conjunction with laser microscopy, a simplified interferometrical technique that has been sucessfully applied in the material engineering sciences 16. materials and methods seven commercial brands available on the brazilian market with nine different surfaces were randomly selected for this study: kopp (ko, hex ø3.75 x 11.0mm), neodent (ne, titamax ti cortical ø3.5 x 11.0mm), signo vinces (sv, duo ø3.8 x 10mm), 3 do carmo filho et al. sin (si, tryon ø3.75 x 10mm), straumann (str, sla and sla, slactive with dimensions ø4.1x8.0mm), titanium fix (tf, self-tapping implant ø4.0 x 13.0mm), biomet 3i (nt, nanotitetm ø4.0 x 8.5 and, os, osseotite® ø3.75 x 8.5mm). the description of the surface treatment of each implant is listed in table 1. surface analysis was performed using a laser microscope (lext ols 4000 olympus), with 20x objective lenses (mplapon), 1x zoom, with a magnification of 432x 3.456x, and field of view of 640-80 μm. the in-house software provided with the equipment allowed to describe several parameters of roughness (r) in micrometers in a single surface reading. in this study, we used the following parameters: rp (maximum profile peak height / peak height of the highest roughness); ry (maximum distance between peak and valley); rz ( maximum height of profile / represents the arithmetic mean of the 5 values); rt (total height of profile); ra (arithmetic mean deviation of profile / represents the average profile roughness amplitude parameter defined as integral and absolute value of the height of peaks and valleys, along the evaluated profile) 17. in addition, the surfaces studied were reconstructed in a 3-d format using the ols 4000 2.1 software to process and obtain 20x magnified images (figure 1). table 1. description of the type of surface treatment for the studied commercial brands. brand surface teatment description kopp sandblasting and double acid subtraction25 signo vinces sandblasting with aluminum oxide particles and chemical treatment by double acid etching26 neodent sandblasting and acid subtraction (neoporos surface)27,28 sin double acid-etching29 titanium fix sandblasting and acid-etching30 osseotite® double acid-etching31,32 nanotite® double acid-etching with cap crystals deposition29,31,32 straumann sla sandblasting with large grits of 0.25 to 0.50 mm and acid-etching with hcl/h2so423 straumann slactive sandblasting, large-griting, acid-etching and rinsed under nitrogen submerge in nacl solution33 a b c d e f g h i figure 1. 3d reconstructions showing the surface morphology of the implants: a: kopp; b: signo vinces; c: neodent; d: sin; e: titanium fix; f: biomet 3i osseotite; g: biomet 3i nanotite; h: straumann sla; i: straumann slactive. 4 do carmo filho et al. the implants surface morphologies and the elemental analysis were performed using a scanning electron microscopy (sem) and energy dispersive x-ray spectrometry (eds) (ssx-550; shimadzu, tokyo, japan) to identify the elements present on the implant surface. two regions of the implant were selected to perform the analyses: i) the cervical region (without surface treatment), and ii) the medium third (treated surface). results the 3-d reconstructions showing the surface morphology of the 7 implant types are shown in figure 1, while figure 2 presents the sem images showing the implant surface morphologies. table 2 lists the results of the implant surface roughness analysis for the different commercial brands in their cervical and middle regions. the cervical portion of all implants can be considered minimally rough, while the middle region may range from minimally rough to rough (ra> 2.0 μm). a 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 b c d e f g h i a 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 b c d e f g h i figure 2. scanning electron microscopy images showing (1) thread geometry (original magnification 30x 500µm), (2) surface morphology (magnification 800x 20µm) and (3) surface morphology (magnification 2000x 5µm): a: kopp; b: signo vinces; c: neodent; d: sin; e: titanium fix; f: biomet 3i osseotite; g: biomet 3i nanotite; h: straumann sla; i: straumann slactive. table 2. roughness parameters of the studied implant surfaces in the cervical and middle third regions of the different commercial brands at the micrometric level (μm). implant rp ry rz rt ra cervical middle cervical middle cervical middle cervical middle cervical middle kopp 2.696 5.256 1.037 4.228 3.733 9.485 7.659 14.651 0.455 1.557 signo vinces 2.26 5.494 0.876 8.419 3.136 13.913 9.187 32.53 0.492 1.814 neodent 1.738 4.638 0.487 2.973 2.225 7.611 8.256 9.681 0.291 1.034 sin 2.419 2.862 0.71 2.848 3.129 5.71 8.647 7.779 0.323 0.7 titanium fix 1.553 10.332 0.921 4.156 2.474 14.488 5.622 86.219 0.337 1.404 osseotite 5.883 5.037 0.686 4.474 6.568 9.511 11.444 27.264 0.706 1.086 nanotite 1.68 20.548 1.796 9.461 3.477 30.01 9.704 44.742 0.469 3.436 straumann sla 0.699 9.95 0.873 8.279 1.572 18.229 3.96 24.917 0.243 3.091 straumann slactive 0.831 11.304 1.723 8.691 2.554 19.955 10.821 46.28 0.305 2.997 5 do carmo filho et al. the eds elemental analysis shows that only the sla implant surface is composed of pure titanium; all other implant surfaces contained significant amounts of different elements, as described in table 3 and figure 3. discussion the characteristics of the bone-implant interface and the methods to improve this relationship such as implant surface modifications have been intensely studied18. the average surface roughness of the implants, represented by ra, is a widely investigated parameter by interferometry. our study was the first that described a simplified laser methodology for implants surface roughness characterization. according to albrektsson and wennerberg13 (2004), the surfaces can be classified according to the ra value as: i) minimally coarse: ra 0.5 – 1.0 μm, present in machine turned implants; ii) moderately rough: ra 1.0 – 2.0 μm, found in implants with acid-conditioned, blasting or anodized surfaces; and iii) rough: ra > 2.0 μm, as found in implants treated with plasma spray. however, the precise characterization of the surface morphologies is still a topic of discussion. most implant surface studies ignore the chemical aspects 18; chemical characterization of commercially available products is extremely scarce in the literature 19,20. in contrast, surface morphology and micrometer-scale topography are commonly used, but the optimal method to quantify microstructures remains a source of debate 21. the implant surface characteristics can influence the initial biofilm formation, because the adhesion of the microorganisms is directly proportional to the surface roughness. therefore, the cervical portion of the implants ideally should have a smoother surface, because this region of the implant is most exposed to the buccal environment. a smoother surface reduces bacterial adhesion and consequently reduces the incidence of peri-implant pathologies that can lead to implant failure 22. the ra values in the cervical portion of (table 2) did not exceed 1.0 μm in the national implant brands, characterizing a minimally rough surface 13. the latter implies that all brands presented acceptable micrometric patterns, capable of safely avoid biofilm accumulation in the peri-implant region. however, when other roughness parameters are included, a great disparity of values is observed, and all implants surfaces evaluated. the nt surface table 3. elemental composition of the studed implant surfaces (in weight %) measured with energy dispersive spectrometry (eds). implant ti al mg c f na sr pr ni o p kopp 95.289 0.580 0.605 3.526 x x x x x x x signo vinces 98.559 1.441 x x x x x x x x neodent 81.807 x x 1.463 0.637 1.011 15.082 x x x sin 73.092 0.582 x 5.571 x x 20.755 x x x titanium fix 18.849 5.880 0.231 2.252 1.317 x x x 29.487 10.856 x osseotite 91.967 x x 8.033 x x x x x x x nanotite 69.997 6.688 x 6.643 x x x 16.199 x x 0.473 straumann sla 99.905 0.095 x x x x x x x x straumann slactive 100.000 x x x x x x x x x x 6 do carmo filho et al. figure 3. spectra obtained by eds analysis of the middle third of the surface topography of the evaluated implants: a: kopp; b: signo vinces; c: neodent; d: sin; e: titanium fix; f: biomet 3i osseotite; g: biomet 3i nanotite; h: straumann sla; i: straumann slactive a 4000 [c ou nt s] [kev] 3000 2000 1000 0 2 4 6 8 10 12 14 4000 [c ou nt s] [kev] 3000 2000 1000 0 2 4 6 8 10 12 14 [kev] 4000 [c ou nt s] 3000 2000 1000 0 2 4 6 8 10 12 14 [kev] 4000 [c ou nt s] 3000 2000 1000 0 2 4 6 8 10 12 14 4000 [c ou nt s] [kev] 3000 2000 1000 0 2 4 6 8 10 12 14 [kev] 4000 [c ou nt s] 3000 2000 1000 0 2 4 6 8 10 12 14 4000 [c ou nt s] [kev] 3000 2000 1000 0 2 4 6 8 10 12 14 4000 [c ou nt s] [kev] 2000 0 2 4 6 8 10 12 14 [kev] [c ou nt s] 15000 10000 5000 0 2 4 6 8 10 12 14 b c d e f g h i 7 do carmo filho et al. generally presented the highest roughness values in the middle portion of the implant (4 of 5 parameters evaluated) while the sla surface presented the lowest roughness parameters in the cervical part, in accordance with the available literature13,23. in our study, str and sla surfaces presented similar roughness as described in previous studies, yet sla surfaces presented greater bone-to-implant contact areas during the early stages of bone healing (2 and 4 weeks)7,23. the surfaces nt, str and sla presented mean ra values above 2 μm in the middle third region. while high roughness promotes the retention of the osteogenic cells on the surface 5,6, ra values exceeding 2 μm can lead to an impaired and unenhanced bone response 5. the sin implant surface was characterized as minimally rough (ra 0.7 μm) and presented the lowest values of roughness in the middle region compared to other commercial brands, mainly in terms of the ry and rz parameters. the nt surface presented the highest rp value (maximum height of the highest peak of the roughness) and the tf surface presented a high value of rt (the total height of the profile). these rough spots probably represent the locations where the first osteoblastic cells will attach. the eds analysis showed that only the implant with sla surface is free of contaminations and impurities (figure 1 and table 3). this implant is immersed in water when sold, thus minimizing previous contamination. all other implants can be considered contaminated, and these impurities can determine the biological performance of the implant, and may be responsible for future osseointegration failures 24. these results corroborate the study of dohan ehrenfest et al.18 (2011) that detected inorganic contaminations, such as na, p, ca, f, and s in the evaluated implants. therefore, even if dental implants are carefully manufactured, the results are not homogeneous; similar implant surface treatments do not necessarily yield identical results 7. in addition, when the macrometric topography of an implant surface changes, its micrometric and chemical characteristics can also undergo changes, sometimes accidentally. therefore, it is essential that each implant design has a suitable surface treatment to achieve an acceptable roughness 15. a large number of experimental investigations have clearly demonstrated that the bone response is influenced by the implant surface. our study adopted a simplified methodology and showed that there is no pre-established roughness pattern that allows a safe comparison of different commercial brands available in brazil. therefore, it is necessary to perform more laboratory and clinical studies to investigate the ideal roughness characteristics that accelerate and maintain osseointegration. a limitation of this study is that only a qualitative analysis with one implant per brand was performed, precluding a statistical analysis. in conclusion, dental implants are currently marketed without clearly defined surface characteristics. our study was the first that described a simplified laser methodology for implants surface 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https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1508/1161 4/9 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1508/1161 5/9 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1508/1161 6/9 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1508/1161 7/9 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1508/1161 8/9 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1508/1161 9/9 diagnostic performance of fractal dimension and radiomorphometric indices from digital panoramic radiographs for screening low bone mineral density angela jordão camargo1, arthur rodriguez gonzalez cortes1, eduardo massaharu aoki1, marina gazzano baladi1, emiko saito arita1, plauto christopher aranha watanabe2 1universidade de são paulo – usp, school of dentistry, department of stomatology, university of são paulo, são paulo, sp, brazil 2universidade de são paulo – usp, ribeirão preto dental school, department of stomatology, area of public oral health and forensic dentistry. ribeirão preto, sp, brazil correspondence to: emiko saito arita departmento de estomatologia, fo-usp av. lineu prestes, 2227 cep: 05508-000 são paulo, sp, brazil phone and fax number: +55 11 3091-7831 e-mail address: esarita@usp.br abstract a number of panoramic radiographic measurements have been associated with osteoporotic alterations. however, little is known about the differences in sensitivity and specificity among these measurements for screening low bone mineral density (bmd). aim: to correlate and compare precision, sensitivity and specificity of panoramic radiomorphometric indices and fractal dimension (fd) for screening low bmd (i.e. osteopenia and osteoporosis). methods: sixty-eight female patients (42.78±15.59 years) were included in this study. body mass index (bmi), mandibular cortical index (mci), mandibular cortical width (mcw), fd and connectivity (c) were assessed. low bmd was diagnosed by peripheral dual-energy x-ray absorptiometry (p-dxa). non-parametric correlations were assessed among all variables. in addition, sensitivity and specificity of mci, mcw and fd were estimated for screening low bmd. results: significant correlation was found between fd and bmi (p=0.013; r=0.269). in addition, fd was the most sensitive method for screening low bmd (70.8%, p=0.001). fd and mci presented a significant and relatively high sensitivity, whereas mcw presented a high specificity for screening low systemic bmd conclusions: among the analyzed methods, fd and mci offer a significant and relatively high sensitivity, whereas mcw offers a high specificity for screening low bmd. keywords: radiography, panoramic. osteoporosis. mandible. introduction osteoporosis is a major health problem and affects a significant number of people. this skeletal disorder is characterized by bone fragility due to deterioration of the bone microarchitecture, which is related to bone strength and quality1,2. screening for osteoporosis is currently recommended for all women over 65 years old, since the complications of this disease may be prevented by early detection1. diagnosis is currently based mainly on bone mineral density (bmd) measurements using dual-energy x-ray absorptiometry (dxa), considered the “gold standard” method. however, bone densitometry is expensive and has limited availability for use in population screening3,4. furthermore, bmd assessment alone does not allow for predicting osteoporotic fractures, since bmd values may overlap between cases with and without fractures. accordingly, cases with osteopenia also present high rates of bone fractures5. other clinical parameters such as body mass index (bmi)6 received for publication: june 21, 2016 accepted: october 17, 2016 original article braz j oral sci. april | june 2016 volume 15, number 2 http://dx.doi.org/10.20396/bjos.v15i2.8648764 braz j oral sci. 15(2):131-136 132 and trabecular micro-architecture7 have also been associated with bmd. such parameters may also provide useful information for the diagnosis of osteoporotic alterations and prediction of bone fractures6,7. several studies have demonstrated that individuals with osteoporotic alterations present altered mandibular morphology. furthermore, there is evidence that intraoral and panoramic radiographic findings may be indicators of both osteopenia and osteoporosis1,6-12. the bmd of the mandible was correlated with the bmd of the lumbar spine and femoral neck, in osteopenia and osteoporosis cases8. panoramic radiography (pr) stands out in this context because it is commonly used as the initial dental examination. furthermore, it is fast, low-cost and uses low-dose x-radiation. pr is useful to diagnose systemic13 and alveolar14 bone quality by assessing the shape (i.e. mandibular cortical index – mci) and width of the inferior mandibular cortex at the mental foramen (i.e. mandibular cortical width – mcw). these measurements have been used as indices to predict osteopenia and osteoporosis since they are correlated with systemic bone mineral density (bmd) values measured with dxa15. it is also possible to assess bone morphometric parameters such as trabecular area, connectivity and fractal dimension (fd) on pr images. moreover, fd of trabecular bone has been associated with bone strength16,17. however, little is known about the difference in diagnostic performance among the above-mentioned methods. thus, the aim of this study was to assess the diagnostic performance of panoramic radiomorphometric indices and parameters in detecting low bone density. material and methods sixty-eight pr images of female patients referred to dental treatment (42.78±15.59 years) were included in this study. patients had a minimum age of 30 years and were indicated to bmd assessment. other metabolic disorders were considered as exclusion criteria. demographic data including age and body mass index (bmi) were recorded for all patients. all digital pr images (veraviewepocs 2d, morita, tokyo, japan) were performed with the same exposure conditions (60 kv, 4 ma, 0.5-mm copper filter). all patients willing to participate in this study signed an informed consent form. approval for conducting retrospective analyses was obtained from the ethics committee of the university of são paulo. the standards for the reporting of diagnostic accuracy studies (stard) and the guidelines of the helsinki declaration were followed in this investigation. trabecular pattern analysis morphometric analyses of the trabecular bone pattern was performed using the methodology proposed by a previous study18, using an imaging processing software (imagej, national institutes of health, bethesda, md). the morphological factors assessed were fd, using a box-counting method and connectivity. first it was selected the region of interest (roi). the choice was a rectangular selection tool, in a fixed manner, measuring 230 x 130 pixels located in the region of the mandibular body below the right mandibular canine tooth apex. this selection avoids overlapping anatomical structures such as the mental foramen, genis apophyses and regions of masticatory stress (premolars and molars)19. thus, the same size rectangular pictures were cut in all pr20. the chosen roi in the images of digital pr included the region of the mandibular body, anterior to the mental foramen and posterior to the mandibular symphysis. the trabecular pattern was analyzed in the roi using the imagej software. the following steps were followed18: 1) roi image duplication; 2) image blurring (from gaussian image) with 33 radius (pixels) this step removes all the fine and medium scale structures and retains only large variations in density (low-pass filter); 3) image subtraction the blurred image was subtracted from the original of the same patient; 4) adding this step adds a constant to each pixel of the resulting subtracted 8-bit image (256 pixel values), according to the image histogram. this generates an image by thresholding on a mean pixel value of 128, thus segmenting the image into components that radiographically approximate the trabeculae and marrow (fig. 1a), according to a previous methodology18; 5) the image is then converted to binary image (fig. 1b). 6) the resulting image is then eroded and 7) dilated to reduce noise. 8) next, the image of the trabeculae is inverted to make the trabecular pixels visible and 9) skeletonized, that is, automatically eroded by using a specific software tool so that only the central line of pixels remains visible (fig. 1c). superimposition of the skeletonized trabecular image on the original image of the bone demonstrates that the studied skeletal structure corresponds to the trabeculae of the original image (fig. 1d). the skeletonized images from each patient were saved in tiff format. using the resulting images, the imagej software did the following analyses: measurement of the average particular size (aps), and trabecular number (tb.n). the values of the aforementioned analyses are associated with the connectivity of the trabecular meshwork. therefore, a ratio of those parameters was proposed to facilitate the analysis, according to the following equation: mandibular cortical width (mcw) the mcw was measured at both mental foramen regions according to a previous methodology15. briefly, images were corrected using a magnification factor of 1.3. spatial calibration was set at a scale of 1 pixel per 96 mm. then, a line tangential to the inferior border of the mandible was drawn. a line perpendicular to this tangent intersecting the inferior border of the mental foramen was drawn, along which the mcw was measured. mandibular cortical index (mci) mci was assessed by evaluating the appearance of the cortical bone below the mandibular foramen, using a previously described classification21. briefly, the inferior mandibular cortex was classified as follows: c1 = normal, when it presented an even and distinct endosteal margin; c2 = moderately eroded, presenting evidence of lacunar resorption or endosteal cortical residues; and c3 = severely eroded, when unequivocal porosity was observed12. diagnostic performance of fractal dimension and radiomorphometric indices from digital panoramic radiographs for screening low bone mineral density braz j oral sci. 15(2):131-136 connectivity (c) = aps tb.n fig.1. methodology used to assess fd on panoramic radiographic images. a) optimized panoramic radiographic image b) conversion to binary image c) skeletonized image d) skeletonized image projected over the regular optimized panoramic radiographic image. 133 bone mineral density (bmd) bone densitometry measurements were carried out in peripheral dual energy x-ray absorptiometry (pdexa, norland; norland medical systems, inc., white plains, ny, usa). the scanning resolution was 1.00 x 1.00 mm, prior to scanning. the radiation dose was less than 0.03 msv for each examination. the region of choice for scanning was the distal forearm22,23. the chosen region of interest was defined as a rectangle with a fixed longitudinal size of 20 mm and a lateral extension large enough to cover both radius and ulna. its distal margin was defined to be coincidental with the location where the ulna and the radius start to superimpose. patients were diagnosed based on bmd values of the forearm, measured according to world health organization (who) criteria, as normal (t score > -1.0), osteopenic (t score, -1.0 to -2.5) and osteoporotic (t-score ≤ -2.5 sd)24. statistical analysis sample size was determined using the uncorrected chisquare test, to detect a minimum diagnostic odds ratio of 5 and to give the study a power of 80%, at a 5%significance level. all panoramic radiomorphometric measurements were performed in random order by two trained observers (i.e. dentists with expertise in oral radiology). intra-observer reliability was assessed by measurements performed 2 weeks apart to eliminate memory bias. intra and inter-observer agreement was assessed using the intraclass correlations coefficient (icc) and the kappa test for mcw and mci, respectively. normality for continuous variables was assessed using the shapiro-wilk test. correlation analyses were performed among all demographic (age and bmi) and clinical variables (dxa, mci, mcw, fd and c) analyzed in the study, using the spearman’s test. a diagnostic performance analysis was conducted to address the sensitivity and specificity of each imaging examination for screening low bmd (i.e. cases with either osteopenia or osteoporosis that have been included in the same category, following a previously described methodology)21. fisher’s exact test was calculated to address the significance of each diagnostic test. all variables were stratified using cutoff points. for continuous variables, mean cut-off points were chosen. for mci, “c1” category was chosen as the cut-off point, since this is the only category representing the lack of alterations in the inferior mandibular cortex21. all statistical analyses were performed at a 5% significance level , using ibm spss statistics 17 (spss®, inc, chicago, il, usa). results sixty-eight pr images were analyzed. mean bmi for participants was 25.04±4.61 kg/m2. ten patients were diagnosed with osteoporosis (t-score≤-2.5 sd), while 41 patients were diagnosed with osteopenia (t score, -1.0 to -2.5). as a result, 51 patients were included in the category of low bmd, considered in the diagnostic performance analyses (i.e. sensitivity and specificity). the remaining 17 patients presented normal bmd (t score>-1.0). all cases were considered in the correlation analyses. normality was rejected for all variables, according to the shapiro-wilk test (p<0.05). intra-observer reproducibility and inter-observer reliability were confirmed for the mcw (icc=0.87, p=0.001), as well as for mci categorical measurements (kappa=0.83, p=0.01). t-score from dxa was significantly correlated with mcw (r=0.215; p=0.039) and mci (r=0.238; p=0.026). in addition, diagnostic performance of fractal dimension and radiomorphometric indices from digital panoramic radiographs for screening low bone mineral density braz j oral sci. 15(2):131-136 134 bmi was significantly correlated with trabecular connectivity (r=0.269; p=0.013). no other correlations were significant (p>0.05). diagnostic performance analysis (table 1 and fig. 2) revealed a higher sensitivity and a lower specificity of fd (70.8% and 37.1%, respectively) as compared with mci (68.9% and 42.9%, respectively) and mcw (51.3% and 75%, respectively) for screening low bmd. fisher’s exact test analysis revealed a significant association between the aforementioned three variables and the t-score outcome (p=0.001). parameters from the panoramic radiographic device. variations in parameters such as kvp, exposure time, ma and different image receptors, as well as the presence of soft tissue may affect the radiographic assessment of the trabecular pattern18. on the other hand, radiomorphometric methods such as mci and mcw are less susceptible to methodological variations25 and are well described by studies on bone mineral density10,18,26-34. this finding is supported by the present study, according to the significant correlations and satisfactory diagnostic performance results using the above-mentioned radiomorphometric indices. a previous study found sufficient evidence that the trabecular pattern affects individuals with either medium or high risk of osteoporosis35, using periapical radiographs. accordingly, it was found a significant correlation between bmi and connectivity using pr images, which are useful as initial examination at patient’s first attendance36. this result suggests that, in addition to weight-bearing bones, low bmi could also affect the trabecular structure of the mandible. despite these results, no significant non-parametric correlation between fd and dxa results could be found. a previous study11 concluded that t-scores below or equal to -1 classify the patient with low bone mineral density and at risk for osteoporosis. who defines osteoporosis as "a disease characterized by low bone mass and deterioration of bone microarchitecture, leading to increased bone fragility and a consequent increase in fracture risk”24. thus, evaluating the conditions of trabecular bone may have an effect on the analysis of maxillofacial bone quality. according to the present diagnostic performance results, fd and mci presented a high sensitivity and average specificity, whereas mcw presented an average sensitivity and high specificity for screening osteoporotic alterations. therefore, none of these methods may be considered accurate and reliable enough to be used solely in the final diagnosis of osteoporosis, which agrees with a previous study37. however, our significant results (p<0.001) also suggest that a combination of the analyzed methods could yield better and more accurate results. further diagnostic studies with larger sample sizes could be recommended to test the diagnostic performance of a combination of the above-mentioned tests, in comparison with laboratorial morphometric measurements and dxa. a limitation of this study is that the present diagnostic performance analyses were conducted to screen low bmd, which includes diagnoses of both osteopenia and osteoporosis. this is in accordance with a previous methodology for relatively small sample sizes21. another limitation of this study is that only posterior mandibular rois were included in the analyses, which could influence the results since bmd may vary from site to site38. further studies would also be recommended to test and compare different rois from different regions, in association with systemic bone density. the morphological filter used in this study was similar to the one used by kumasaka (1997) for extracting skeletal pattern of trabecular bone on images from non-digital panoramic radiographic devices, which were transformed into binary images with enhanced geometric components10,18,26. accordingly, our results support that digital panoramic radiographs, which use lower radiation doses, are also useful for bone density assessments. variations of the present methodology have been described by table 1 diagnostic performance of fd, mci and mcw for screening low bmd. test fd mci mcw sensitivity 70.8% 68.9% 51.3% (95% ci) (53.3-93.5) (43.8-84.5) (26.2-75.6) specificity 37.1% 42.9% 75% (95% ci) (18.4-56.7) (23-61.2) (49.8-90.2) p* 0.001 0.001 0.001 abbreviations: ci, confidence interval; fd, fractal dimension; mci, mandibular cortical index; mcw, mandibular cortical width. * significance according to the fisher’s exact test (p<0.05 indicates statistical significance). fig.2. diagnostic performance of the analyzed methods for screening low bmd. abbreviations: fd, fractal dimension; mci, mandibular cortical index; mcw, mandibular cortical width. discussion despite the lack of contrast and image resolution, as compared with morphometric laboratorial methods, the literature has suggested that panoramic radiographic images could be useful to assess trabecular bone characteristics12,14,18,21. this is the first study proposing a formula to address trabecular bone connectivity from digital panoramic radiographic images, using specific software tools. however, trabecular bone density may be underestimated on panoramic radiographs due to a modified window level from the imaging software, as well as a number of diagnostic performance of fractal dimension and radiomorphometric indices from digital panoramic radiographs for screening low bone mineral density braz j oral sci. 15(2):131-136 studies on periapical radiographs, commonly used during dental treatments18,39-45. in addition, the chosen roi is free of artifacts resulting in images with higher details to assess trabecular skeletal pattern. on the other hand, further studies are required to compare and define the most accurate binary processing threshold in order to enhance the technique and obtain adequate results. this technique has also been described as useful for the computer-assisted analysis of bone structure, which assesses, for instance, the prognosis following surgery, or even to follow-up certain medical treatment outcomes26,39. in conclusion, among the analyzed tests, fd and mci have a significant and relatively high sensitivity, whereas mcw has a high specificity for screening low systemic bone density. furthermore, the diagnostic performance outcomes and significant correlations found in this study suggest that it is possible to obtain evidences of the trabecular bone pattern by assessing panoramic radiographic images. references 1. o'connor km. evaluation and treatment of osteoporosis. med clin north am. 2016 jul;100(4):807-26. doi: 10.1016/j.mcna.2016.03.016.. 2. paolucci t, saraceni vm, 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trans biomed eng. 2013 sep;60(9):2384-92. doi: 10.1109/tbme.2013.2256908. 42. arifin az, asano a, taguchi a, nakamoto t, ohtsuka m, tsuda m, et al. computer-aided system for measuring the mandibular cortical width on dental panoramic radiographs in identifying postmenopausal women with low bone mineral density. osteoporos int. 2006;17(5):753-9. 43. devlin h, allen pd, graham j, jacobs r, karayianni k, lindh c, et al. automated osteoporosis risk assessment by dentist: a new pathway to diagnosis. bone 2007 apr;40(4):835-42. 44. devlin h, karayianni k., mitsea a, jacobs r, lindh c, van der stelt p, et al. diagnosing osteoporosis by using dental panoramic radiographs the osteodent project. oral sur oral med oral pathol oral radiol endod 2007 dec;104(6):821-8. 45. ruttimann ue, webber rl, hazelrig jb. fractal dimension from radiographs of peridental alveolar bone. a possible diagnostic indicator of osteoporosis. oral surg oral med oral pathol 1992 jul;74(1):98-100. 136 diagnostic performance of fractal dimension and radiomorphometric indices from digital panoramic radiographs for screening low bone mineral density braz j oral sci. 15(2):131-136 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1541/1194 1/12 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1541/1194 2/12 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1541/1194 3/12 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1541/1194 4/12 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1541/1194 5/12 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1541/1194 6/12 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1541/1194 7/12 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1541/1194 8/12 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1541/1194 9/12 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1541/1194 10/12 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1541/1194 11/12 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1541/1194 12/12 braz j oral sci. 15(4):298-303 treatment of dento-alveolar trauma: knowledge evaluation from southern brazilian dentists renato azevedo de azevedo1, marcos britto corrêa1, marcos antonio torriani2, flávio fernando demarco1, rafael guerra lund1 1dds, ms, phd, post-graduate program in dentistry, pelotas dental school, universidade federal de pelotas, pelotas, rs, brazil 2dds, ms, phd, department of surgery and bucco-maxilo-facial traumatology, pelotas dental school, universidade federal de pelotas, pelotas, rs, brazil correspondence to: rafael guerra lund faculdade de odontologia da universidade federal de pelotas rua gonçalves chaves, 457, sala 503 – centro cep: 96015568 pelotas, rio grande do sul, brazil phone: +55 53 9125 7668 fax: +55 53 3222 6690 e-mail: rafael.lund@gmail.com abstract aim: this study aimed to evaluate through a specific survey the knowledge of dentists on dental trauma in southern brazil regarding their conduct facing some dental trauma injuries. methods: a survey with five personal and five specific questions on knowledge about dental trauma was carried out with all dentists regularly registered in pelotas, brazil (n=276). the data was submitted to descriptive statistical analysis and associations were tested by chi-square test (p≥0.05). results: there were a higher number of dentists with up to 10 years since graduation (45.4%) who worked in private dental office (66.1%) and with some specialization (63.7%). dentists with more years since graduation were associated to less knowledge on dental trauma management (p<0.001). conclusions: the knowledge of the dentists related to dento-alveolar trauma is lowered with higher time in clinical practice. continuing education courses should be offered to the dentists by educational institutions. keywords: tooth injuries, knowledge, cross sectional survey, dentists. received for publication: january 23, 2017 accepted: july 23, 2017 original article braz j oral sci. october | december 2016 volume 15, number 4 http://dx.doi.org/10.20396/bjos.v15i4.8650044 introduction it is estimated that in the near future the prevalence of dental alveolar injuries will exceed caries and periodontal diseases, and as a consequence it is considered a major public health problem among children and adolescents1,2. this is justified not only because of its relatively high prevalence reported in population studies, but also because it affects aesthetics, psychological and physical conditions of the individual, leading to a reduction in quality of life3,4. furthermore, treatment of dento-alveolar trauma frequently requires complex treatment and long term follow-ups, causing significant economic consequences5. due to the importance of injuries caused by dental trauma, the international association of dental traumatology (iadt), which is a professional organization responsible for promoting optimal prevention and treatment services regarding dental trauma lesions through interaction with doctors and dentists, makes available in its website three guidelines: the first dealing with the management of injuries with fracture dislocations of permanent teeth with closed or opened apex; the second dealing the total number of registered dentists (target population of dentists from pelotas) was 276 professionals. dentists who did not work anymore (retired or working in other area) were excluded of the study. this study was submitted to the ethics committee in research of a faculty of dentistry of an university, with approval number 116/2009. previously to the interview, the participants signed a consensual agreement. the identity of the professionals who accepted to take part in the survey was protected. a questionnaire was developed containing structured questions concerning dentist professional background and five specific questions about their knowledge about the procedures to be adopted facing several kinds of dento-alveolar trauma, such as fractures with pulp involvement, root fractures and avulsions. these questions were taken from a questionnaire described in a study by hu et al.9 undergraduate dental students visited the dentists participants in the second semester of 2010. the questionnaires were handed in person and the importance and aim of the study was explained. approximately one week after the delivery of the questionnaires, they were collected together with the consensual agreement form signed. this way, the dentists were not interrupted in their workplace at working hours and did not feel uncomfortable with the interviewers. the answers to the questions related to dento-alveolar trauma were analyzed independently and grouped. for analysis of associations, the number of questions answered correctly was quantified and further dichotomized in: 0 to 2 correct answers or 3 or more correct answers. the independent variables were gender, skin color, time since graduation, workplace, level and area of specialization. the variable skin color was self-referred by the professional according to the classification of brazilian institute of geography and statistics (ibge) in white, black, brown, yellow and indian. time since graduation was recorded in a continuous way and was categorized in up to 10 years, 11 to 20 years, 21 to 30 years and more than 30 years since graduation. the workplace was recorded by asking in which place the dentist worked most of the time, and categorized as public work, private dental office, and teaching (university). the degree of specialization was recorded by asking if the dentist had any kind of post-graduation. it was categorized in yes or no. data obtained from the questionnaires were processed in excel spreadsheet and submitted to descriptive analysis to obtain the relative and absolute frequencies of the answers with their respective 95% confidence intervals. the answers considered correct were based on iadt guidelines. the analysis of associations between independent variables and knowledge on dento-alveolar trauma was performed by chi-square test. the software used for statistical analysis was stata11. results out of 276 dentists (target population), 187 (sample obtained, 68%) answered the questionnaire. table 1 shows the 299 with management of injuries such as avulsion of permanent teeth with open or closed apex; and the third dealing with management of dental injuries in deciduous teeth6. in all guidelines it can be easily found the immediate and most adequate treatment for each case, as well as the immediate recommendations for post-treatment and follow up of each case. these guidelines were designed by a group of experts who came to a consensus after a literature review and group discussions on each subject7. facing a dento-alveolar trauma, a diagnosis based on an adequate clinical examination with a written, radiographic and in many cases photographic record, can eliminate many doubts, which the dentist may have, leading to a correct choice of treatment. considering that in patients who suffered dental trauma many of the late complications are consequence of failure in the first assessment8, it is of great importance that the dentist has the necessary knowledge to identify the various types of dental trauma and their possible treatments. in this case, the correct choice of emergency procedure will result in a more favorable prognosis for the affected tooth9. the organization of services and primary care teams for oral health present, in general, a low level of knowledge on management of dento-alveolar trauma10,11. even though there are available guidelines for dental professionals, some studies in different countries have also demonstrated that the knowledge of dental professionals on dento-alveolar trauma is insufficient11-13. in brazil some studies were carried out on the subject9,14-16, with results similar to those observed in other countries, showing lack of dentist’s knowledge on management of dental trauma emergency. besides the low level of knowledge, it was verified that dentists with a longer time since graduation had an even worse performance14, highlighting the need for constant updates in this area of expertise. thus, it is clear that the knowledge of dental professionals should be evaluated regarding their conduct facing dental trauma emergency injuries, aiming at identifying the main weaknesses related to this issue and allowing the development of strategies in order to improve this knowledge. this study aims to assess the knowledge of dentists from the city of pelotas, brazil, regarding their conduct towards dento-alveolar trauma and to test its association to the time since graduation, expertise area and gender. material and methods this cross-sectional study was carried out in pelotas, a city in an affluent area of south of brazil. this city is the third most populated in rio grande do sul state (327,778 inhabitants) with an area of 1,609 km2, and 95% of its population lives in urban areas. according to cro – conselho regional de odontologia (regional council of dentistry), 4.3% of all dentists from rio grande do sul state are working in pelotas. all dentists from pelotas regularly registered in the regional council and with updated address information were contacted and asked to respond the questionnaire. treatment of dento-alveolar trauma: knowledge evaluation from southern brazilian dentists braz j oral sci. 15(4):298-303 300treatment of dento-alveolar trauma: knowledge evaluation from southern brazilian dentists table 1 descriptive analyses of the relative variables related to dentists in pelotas, brazil (n=187). table 2 absolute (n) and relative (%) frequencies of dentist’s answers to questions about dental trauma. pelotas, brazil. (n=187). variables/categories absolute frequency (n) relative frequency [% (ci, 95%)] gender male 89 47.6 (40.3-55.0) female 98 52.4 (45.0-59.7) color white 180 96.8 (93.1-98.8) black 2 1.1 (0.1-3.8) brown 4 2.2 (0.1-5.3) years since graduation up to 10 84 45.4 (38.2-58.9) 11 to 20 43 23.2 (17.2-29.7) 21 to 30 28 15.1 (10.2-20.9) more than 30 30 16.2 (11.1-22.1) workplace private 121 66.1 (59.1-73.0) public 39 21.3 (15.7-28.0) teaching 23 12.6 (8.4-18.5) specialization without post-graduation 66 36.3 (29.5-43.7) with post-graduation 116 63.7 (56.3-70.5) question n [% (ci, 95%)] 1. what kind of splint and for how long should it be used for? (0) rigid, during 2 weeks or until the mobility of the tooth is reduced (1) semi-rigid or rigid, for 1 month *(2) semi-rigid, during 2 weeks or until the mobility of the tooth is reduced 40 [24.4(18.6-31.4)] 26 [15.9(11.1-22.1)] (3) no splint should be used 93 [56.7(49.3 63.9)] 5 [3.0(1.2-6.8)] 2. in this case, would you prescribe any medicine? (0) no (1) yes, antibiotics of narrow spectrum, anti-inflammatory, analgesic (2) yes, anti-inflammatory, analgesic *(3)yes, broad spectrum antibiotics, anti-inflammatory and analgesic 11 [6.4(3.4-10.9)] 36 [21.1(15.3-27.4)] 29 [17.0(12.0-23.3)] 95 [55.6(48.2-62.9)] a patient who suffered an accident the day before arrives at the dental office. after radiographic examination it is observed that tooth no. 21 showed root fracture. 3. the immediate treatment is: (0) extraction of the tooth (1) endodontic treatment *(2) thermal test, rigid splint (3) thermal test, semi-rigid splint 27 [18.4(12.9-24.5)] 27 [18.4(12.9-24.5)] 57 [38.8(32.0-46.4)] 36 [24.5(18.6-31.4)] a patient came to the dental office with an avulsed tooth, kept dry for 7 h. 4. the immediate treatment is: (0) cleaning the root and the alveolar socket with saline solution, tooth replant, splint and antibiotics therapy. *(1) place the tooth in a fluoride solution (2.4% sodium fluoride), cleaning of the alveolar socket with saline solution, replant, endodontic treatment, splint and antibiotic therapy. *(2) place the tooth in a fluoride solution (2.4% sodium fluoride), cleaning of the alveolar socket with saline solution, endodontic treatment, replant, splint and antibiotic therapy. (3) replacement of the missing tooth by prosthesis. 36 [21.7(16.2-28.5)] 30 [18.1(12.9-24.5)] 66 [39.8(32.5-47.0)] 34 [20.5(14.8-26.8)] a 7 year-old boy who was hit in the face with a football, came to the dental office showing a fracture involving enamel and dentin with pulp exposure. after radiographic examination it was verified that the stage of root formation was incomplete (open apex). 5. the immediate treatment is: (0) pulpectomy *(1) pulpotomy (2) endodontic treatment in one session 20 [12.1(8.0-17.9)] 141 [85.5(79.7-90.3)] 4 [2.4(0.1-5.4)] description of the sample that took part in the study according to the variables surveyed. it can be observed that the distribution of dentists regarding gender was proportional between men and women, while 96.8% of them were of white color. in relation to the variables relating to profession, there were a higher number of dentists with up to 10 years since graduation (45.4%) who worked in private dental office (66.1%) and with some specialization (63.7%). in table 2, relative and absolute frequency values are shown for each alternative to the questions answered by the dentists. the correct alternatives to each question in the table are in bold and italics. it is possible to observe that the majority of the dentists interviewed answered correctly the questions. in question number 3 (table 2) that addresses a case of root fracture, the majority of dentists interviewed (38.8%) indicated the treatment with rigid splint, which contradicts the iadt guidelines. this was the only question with a relative frequency of correct answer lower than 50%. table 3 shows the number of right answers, where it can be observed that, from 187 dentists interviewed, 13 (7%) did not answer any question correctly and 14 (7.5%) answered five questions correctly. the majority of the interviewed dentists answered two (24.6%) and three (28.9%) questions correctly. in the association analysis between the number of correct answers and the independent variables studied (table 4), only the variable related to years since graduation was associated to the outcome (p<0.001), and a downward trend in the number of correct answers was observed as the years after graduation increased. braz j oral sci. 15(4):298-303 301 discussion the present study showed a low level of knowledge of dentists regarding management of dental injuries related to dento-alveolar trauma. dentists were considered with a low level knowledge when these professionals scored 0 to 2 correct answers, which included 45.5% of the dentists interviewed. this low level of knowledge increased in a linear way with the increase in the time since graduation. our findings are similar to the studies reported by hu et al.9, krasti et al.12, and de frança et al.14 who also showed that recently graduated dentists presented a better knowledge of the techniques for treatment of dental trauma. on the other hand, granville-garcia et al.17 found a different result. in their study dentists presented a good knowledge for treatment of dento-alveolar trauma regardless the length of professional experience. if we observe the descriptive analysis of variables related to dentists (table 1), we could see that there is no predominance of gender among them, demonstrating an equality, which was not seen decades ago when the predominance of males in the profession reached 90%18. another variable that calls attention do to its great disproportion is skin color, where 96.8% of the dentists interviewed were white. the competition for admission in courses of high social prestige, e.g. dentistry, usually after expensive preparatory training, makes these courses real monopolies of higher social classes19. thus, it is expected that black population, still economically disadvantaged in our country20, would be underrepresented in the profession. although the majority of dentists have some kind of specialization, in our study this variable did not contribute to the improvement of the knowledge in the management of dentoalveolar trauma. in the studies by hu et al.9 and de frança et al.14, dentists who worked in post-graduate courses or who had experience in dento-alveolar trauma presented a higher knowledge score than other professionals. hu et al.9 also reported a poor correlation between time since graduation and knowledge score. in our study it was evident a clear correlation between years since graduation and decrease of knowledge (p=0.004) which confirms the results obtained in a study by de frança et al.14 the place where the dentist worked did not interfere in the knowledge; however a similar study in china compared the knowledge on dento-alveolar trauma among chinese dentists from rural and urban areas, and observed a better knowledge in the dentists from the urban area. however, they concluded that in both cases it was found a low level of knowledge on management of dento-alveolar trauma21. yet, a study by zadik et al.11 evaluated the data diffusion of iadt guidelines one year after publication in 2007, and showed that there was an increase in dentists knowledge, concluding that knowledge has to be reinforced. in the first question (table 2), more than half of the dentists interviewed chose semi-rigid splint, and this kind of splinting allows small physiological movements, which is desirable for periodontal healing and appears to reduce the risk of ankylosis and external root resorption when used for short periods22. in question 3, almost one quarter of the respondents (24.5%), chose semi-rigid splint. this treatment is contrary to the one proposed by sanabe et al.23 and by iadt guidelines6 that recommend rigid splint, which would have better outcome in the treatment. in this case, in order to have the healing of the dentinal-cement complex, it is required the immobilization of the tooth. in question 4 (table 2) we considered two correct answers, based on a recent work by kumar24, who reported that there is no difference in performing extra-oral or intra-oral endondontics. this is a discussion not yet well defined, as pohl et al.25 argues that endodontic should be performed immediately after avulsion preferably extra-oral. in opposite, other studies argue that endodontic should be performed seven to ten days after the tooth replantation26-28, while other researchers suggest that the treatment should be performed for a period of two to three weeks29. although there is disagreement regarding the time in which the endodontic treatment should be performed, all the researchers agree that ankylosis and root resorption substitution is present in all cases but it is the desirable outcome. the last question was the one that the respondents obtained the highest relative frequency of correct answers (85.5%) following iadt guidelines, that is, teeth with open apexes should be kept with their pulp vitality to ensure the root development and apex closure. the lack of standardized techniques for management treatment of dento-alveolar trauma: knowledge evaluation from southern brazilian dentists table 3 absolute and relative frequencies of the number of correct answers to the questions asked to dentists on dental trauma. pelotas, brazil (n=187). number of correct answers absolute frequency (n) relative frequency [% (ci, 95%)] 0 13 7.0 (3.8-11.6) 1 26 13.9 (9.3-19.7) 2 46 24.6 (18.6-31.4) 3 54 28.9 (22.5-35.9) 4 34 18.2 (12.9-24.5) 5 14 7.5 (4.2-12.2) table 4 absolute and relative frequencies of the number of correct answers to the questions asked to dentists on dental trauma. pelotas, brazil (n=187). variables/categories number of correct answers (%) p value 0 to 2 3 to 5 total 85 (45.5) 102 (54.5) gender 0.669* male 39 (43.8) 50 (56.2) female 46 (46.9) 52 (53.1) post-graduation 0,556* without post-graduation 32 (48.5) 34 (51.5) with post-graduation 51 (44.0) 65 (56.0) workplace 0,550* private 59 (48.8) 62 (51.2) teaching 16 (41.0) 23 (59.0) public 9 (39.1) 14 (60.9) years since graduation 0.004** up to 10 30 (35.7) 54 (64.3) 11 to 20 20 (46.5) 23 (53.5) 21 to 30 14 (50.0) 14 (50.0) more than 30 20 (66.7) 10 (33.3) * chi square test (χ²) ** χ² test for linear trend braz j oral sci. 15(4):298-303 of dental trauma, despite iadt guidelines has possibly been a hindrance to the acquisition of full knowledge. our findings demonstrate the need to implement new strategies in order to improve dentist’s knowledge regarding the management of injuries of dento-alveolar trauma. the traditional dental curriculum presents limited opportunities for students to evaluate and treat patients with dental trauma. as a result of this lack of exposure, most graduating dentists are undertrained and ill-prepared to manage dental trauma30. there is a need for courses in dental traumatology in the society to increase the knowledge level among the professionals. to improve the quality of education in dental trauma, continuing education through courses and seminars to dentists and the general public are important tools for improving this knowledge. guidelines also contribute to a higher standard of care. additionally, changings in the curriculum of dental institutions are necessary, aiming to improve the willingness of dentists to treat patients with traumatic dental injuries, the sustained knowledge of management of dental trauma, as well as the community involvement stimulated by the universities to support prevention31. moreover, courses of the emergency management of traumatic injuries is an important topic to be covered30. it is important to highlight that the use of questionnaires is a useful tool for data collection, allowing obtaining of data of good quantity in large geographic areas and in a short period of time. the enclosed and auto-applicable questionnaire had as an advantage to allow the dentists to answer the questions without interruption to their daily activities in the dental office. the disadvantage of this kind of auto-applicable questionnaire is that the dentist may not answer all the questions or may search for information when faced with questions that the answer is unknown. in this study, the response rate was 68%, with losses and refusals reduced by visiting the dental clinics at least twice to retrieve the questionnaires. this response rate is acceptable, since response rates of about 50% have been reported in similar surveys12,32-34. if the dentists were interviewed personally, instead of the auto-applicable version of the questionnaire, the presence of missing data in the questionnaire could be reduced, but a higher number of refusals could be expected, because the normal routine of the dental office would be disturbed or by the fear of not knowing the correct answer. within the limitations of the present study, we can conclude that: 1) the knowledge of the dentists regarding the management of dental injuries related to dento-alveolar trauma is less since the passing years after graduation pass because it seems there are no familiarity with current treatment guidelines; 2) other independent variables such as gender, workplace and post-graduation were not associated to the number of correct answers; 3) moreover, the findings of this study points out a lack of experience from professionals to perform some attendance without the appropriate knowledge to perform this. changings int the dental curricula and continuing education courses should be offered to the dentists by educational institutions, in order to keep these professionals updated regarding management of dentoalveolar trauma injuries. references 1. lam r. epidemiology and outcomes of traumatic dental injuries: a review of the literature. aust dent j. 2016 mar;61 suppl 1:4-20. doi: 10.1111/ adj.12395. 2. damé-teixeira n, alves ls, susin c, maltz m. traumatic dental injury among 12-year-old south brazilian schoolchildren: prevalence, severity, and risk indicators. dent traumatol. 2013 feb;29(1):52-8. doi: 10.1111/j.1600-9657.2012.01124.x. 3. freire-maia fb, auad 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[level of knowledge of nursing, physical education and dentistry professionals on dentoalveolar trauma and avulsion]. rev cir traumatol buco-maxilo-fac. 2010 jan-mar;10(1):95-102. portuguese. 16. menezes mc, carvalho rg, accorsi-mendonça t, de-deus g, moreira ej, silva ej. knowledge of dentists on the management of tooth avulsion injuries in rio de janeiro, brazil. oral health prev dent. 2015;13(5):457-60. doi: 10.3290/j.ohpd.a33923. 17. granville-garcia af, balduíno junior jb, ferreira jms, menezes va, fotes lbc, cavalcanti al. [knowledge of the dentist about tooth avulsion at the program family health in campina grande, pb, brazil]. odonto (sao bernardo campo). 2009 jan-jun;17(33):35-41. portuguese. 302treatment of dento-alveolar trauma: knowledge evaluation from southern brazilian dentists braz j oral sci. 15(4):298-303 303 18. ward p. the changing shape of the dental profession. br dent j. 2008 jan 12;204(1):1. doi: 10.1038/bdj.2007.1207. 19. almeida-filho n. higher education and health care in brazil. lancet. 2011 jun 4;377(9781):1898-900. doi: 10.1016/s0140-6736(11)60326-7. 20. boakari fm. black university students’ perceptions of the future in brazil’s multi-racial society. world stud educ. 2005;6(2):53-83. doi: 10.7459/ wse/06.2.04. 21. zhao y, gong y. knowledge of emergency management of avulsed teeth: a survey of dentists in beijing, china. dent traumatol. 2010 jun;26(3):281-4. doi: 10.1111/j.1600-9657.2010.00877.x. 22. von arx t, filippi a, buser d. splinting of traumatized teeth with a new devise: tts (titanium trauma splint). dent traumatol. 2001 aug;17(4):180-4. 23. sanabe me, bezerra l, cavalcante b, coldebella cr, abreu-elima fcb. [dental traumatism urgencies: classification, signs and procedures]. rev paul pediatr. 2009 dec;27(4):47-51. doi: 10.1590/ s0103-05822009000400015. 24. kumar sks. extraoral endodontic treatment is not detrimental for avulsed permanent teeth replanted after more than 60 minutes´ dry time. j am dent assoc. 2010 dec;141(12):1467-9. 25. pohl y, fillipi a, kirschner h. results after replantation of avulsed permanent teeth. i. endodontic considerations. dent traumatol. 2005 apr;21(2):80-92. 26. trope m. clinical management of the avulsed tooth: present strategies and future directions. dent traumatol. 2002 feb;18(1):1-11. 27. flores mt, andersson l, andreasen jo, bakland lk, malmgren b, barnett f, et al. guidelines for the management of traumatic dental injuries. ii. avulsion of permanent teeth. dent traumatol. 2007jun;23(3):130-6. 28. heithersay gs. life cycles of traumatized teeth: long‐term observations from a cohort of dental trauma victims. aust dent j. 2016 mar;61 suppl 1:120-7. doi: 10.1111/adj.12403.. 29. vasconcelos bce, filho jrl, fernandes bc, aguiar erb. [dental reimplantation]. rev cir traumat buco-maxilo-facial. 2001 juldec;1(2):45-51. 30. townsend j, king b, ballard r, armbruster p, sabey k. interdisciplinary approach to education: preparing general dentists to manage dental trauma. dent traumatol. 2017 apr;33(2):143-148. doi: 10.1111/ edt.12309. 31. abu‐dawoud m, al‐enezi b, andersson l. knowledge of emergency management of avulsed teeth among young physicians and dentists. dent traumatol. 2007 dec;23(6):348-55. 32. owen-smith v, burgess-allen j, lavelle k, wilding e. can lifestyle surveys survive a low response rate? j public health. 2008 dec;122(12):1382-3. doi: 10.1016/j.puhe.2008.05.008. 33. yeng t, parashos p. an investigation into dentists’ management methods of dental trauma to maxillary permanent incisors in victoria, australia. dent traumatol. 2008 aug;24(4):443-8. doi: 10.1111/j.16009657.2008.00609.x. 34. skaare ab, pawlowski aa, maseng aas al, espelid i. dentists’ self‐ estimation of their competence to treat avulsion and root fracture injuries. dent traumatol. 2015 oct;31(5):368-73. doi: 10.1111/edt.12186. treatment of dento-alveolar trauma: knowledge evaluation from southern brazilian dentists braz j oral sci. 15(4):298-303 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1535/1188 1/8 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1535/1188 2/8 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1535/1188 3/8 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1535/1188 4/8 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1535/1188 5/8 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1535/1188 6/8 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1535/1188 7/8 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1535/1188 8/8 revista fop n 13 braz j oral sci. january/march 2008 vol. 7 number 24 1470 drugs and nonsyndromic orofacial cleft: an update annalisa palmieri1 anna avantaggiato2 giorgio brunelli marzia arlotti1, luca scapoli1 marcella martinelli1 furio pezzetti1 francesco carinci2 1center of molecular genetics, carisbo foundation, and institute of histology and general embryology, school of medicine, university of bologna, italy 2chair of maxillofacial surgery, school of medicine, university of ferrara, italy received for publication: september 25, 2007 accepted: march 27, 2008 correspondence to: francesco carinci, md chair of maxillofacial surgery university of ferrara arcispedale s. anna corso giovecca, 203 44100 ferrara italy phone/fax: +39.0532.455582 web: www.carinci.org e-mail: crc@.unife.it abstract nonsyndromic orofacial cleft (ofc) derives from an embryopathy with failure of the nasal processes and/or fusion of the palatal shelves. this severe birth defect is one of the most common malformations among live births. human cleft is composed of two separate entities: cleft of the lip with or without palate (cl±p) and cleft palate only (cpo). both have a genetic origin, whereas environmental factors contribute to these congenital malformations. in this review we analyze the role of drugs related to the onset of cleft. the data were obtained from (i) epidemiologic studies (ii) animal models and (iii) human genetic investigations. these studies have demonstrated a relation between certain drugs (steroids and anticonvulsants) taken during pregnancy and a higher risk of generating offspring with ofc whereas no clear relation has been demonstrated between aspirin and ofc. key words: cleft; steroid, anticonvulsivant; cortisone; phenytoin 1471 braz j oral sci. 7(24):1470-1475 drugs and nonsyndromic orofacial cleft: an update introduction nonsyndromic cleft or orofacial cleft (ofc) is due to an embryopathy, which causes failure of the fusion of the nasal processes and/or palatal shelves. this severe birth defect is one of the most common malformations among live births. indeed, incidence is in the range of 1/700-1/1,000 among caucasians1,2. clefts of the human face can be classified anatomically as those including the secondary palate alone (the posterior and/or soft palate) or cleft palate only (cpo), and those that involve the primary palate, encompassing cleft of the lip with or without the palate (i.e. cl±p) 3. this distinction is biologically important and supported by embryological grounds: indeed, the primary and secondary palates are formed independently. furthermore, it is unusual to find a familial cpo if the index case has cl±p, and vice versa1. since one-fifth of patients in different populations have a positive family history, genetic factors are thought to play an important role in the etiology of this congenital defect. fogh-andersen 4 provided the first population-based evidence that ofc has a strong genetic component. fraser1 divided cpo and cl±p. the elevated concordance rate observed in monozygotic twins (36%) with respect to dizygotic twins (4.7%) provides, further evidence of genetic predisposition5-8. population-based studies have shown that non-genetic factors play an important role in clefting: teratogens like phenitoin and valproic acid are known to cause ofc. murine models were developed to investigate drug-induced embryopathy and, more recently, to obtain information regarding genes and biochemical pathways. comprehension of the results is complicated by the fact that nonsyndromic cleft lip in mice, as well as in humans, is genetically complex, and is distinct from isolated cleft palate9. in this review we focus on drugs related to the onset of cleft. the data here presented have been obtained from epidemiologic studies (ii) animal models and (iii) human genetic investigations. steroids corticosteroids are first-line drugs used for the treatment of a variety of conditions in women of childbearing age; in animal models the clefting role of corticosteroids is well documented. some studies have examined the association between corticosteroid use by women during the periconceptional period (one month before to 3 months after conception) and delivery of newborns with selected congenital anomalies. carmichael and shaw 10 found an increased risk for nonsyndromic cl±p and cpo. park-wyllie et al.11 have also demonstrated that, although prednisone does not represent a major teratogenic risk in humans at therapeutic doses, it does increase by an order of 3.4-fold the risk of oral cleft, which is consistent with the existing animal studies. in a case-control survey of children with nonsyndromic cleft lip or palate, edwards et al.12 have shown a significant increase in the prevalence of maternal use of topical corticosteroid preparations in the first trimester of pregnancy (odd ratio 13.154). in a case-control analysis, pradat et al.13 demonstrated the possible association between oral cleft in the newborn and maternal exposure to corticoids during pregnancy. the study includes data on 11,150 malformed infants with a cleft palate or cleft lip and a history of maternal first-trimester drug exposure. they observed a slight association between exposure to corticoids for systemic use and the occurrence of cleft lip with or without cleft palate (or, 2.59; 95% ci, 1.18-5.67). another study conducted on 1142 sweden infants with ofc and maternal exposure to drug in early pregnancy found association between glucocorticoid use and infant cleft. in particular this risk seemed to be strongest for median cleft palate14. additional strength was added by the same author in a recent report where anti-asthmatic drugs were analysed. an increased risk ofc, specifically for median cleft palate, was detected for inhaled corticosteroids15. cleft lip (cl) and cleft palate (cp) are induced in the mouse progeny when glucocorticoids were administered to pregnant mice. the incidence varies among inbred mouse strains and with the dosage and stage of gestation when the drug is given. the inbred a/j strain, which has spontaneous cleft lip, displays lateral median processes that are smaller than in other resistant strains; these diverge slowly to impede the fusion process. diewert et al.16 found not only that cortisone affected the content of the extracellular matrix and the number of palatal shelf cells in a/j mice, but also shelf elevation was delayed; besides, only half of the cortisone-treated palates achieved complete horizontal positioning of the shelves in all regions of the palate. melnick et al.17 studied the teratologic effects on lip morphogenesis following administration of triamcinolone hexacetonide at the eighth day of gestation. the frequency of cleft lip in treated a/j mice was found to be more than three times higher than spontaneous frequency in untreated controls. affected a/j embryos showed a severe reduction in the size of the lateral nasal processes. gasser et al.18 examined strains of mice for susceptibility to cortisoneinduced cleft palate, and confirmed the role of genes linked to h-2 complex (homologous to human hla system) on chromosome 17. later, the same group19 refined the map of the chromosome region carrying the cps-1 gene (cleft palate susceptibility-1). juriloff et al.9,20 mapped a major cl±pcausing gene on mouse chromosome 11, in a region having linkage homology with human 17q21-24. by setting up an in vitro organ culture system with developing mouse palates, shimizu et al.21 were able to demonstrate that exposure to hydrocortisone (hc) in a concentration-dependent manner inhibits the palatal fusion process by preventing apoptosis 1472 in the epithelial cells at the tip of palatal shelves. jaskoll et al.22 analyzed the developmental expression of four glucocorticoid-responsive genes (tgf-beta 1, tgf-beta 2, tgf-beta 3, and egf-r) in developing mouse palates in the presence or absence of exogenous glucocorticoids. these molecules delay the down-regulation of palatal tgf-beta 2 transcript, which is known to inhibit cell proliferation. the authors thus hypothesized that tgf-beta 2 is the mediator of the glucocorticoid effect. in the same year, fawcett et al.23 demonstrated that endogenous corticosterone did not contribute significantly to the incidence of cleft palate induced by the exogenous corticosteroid. these studies support the concept of a threshold in the dose-response relationship for corticosteroid-induced cleft palate in mice. anticonvulsants anticonvulsants (phenytoin/hydantoin, oxazolidinediones and valproic acid) are associated with a clearly demonstrated increased risk of congenital defects24. all three therapeutic classes induce cleft lip and/or cleft palate, as a part of severe embryopathies. it is worth noting that a significant increase of benzodiazepine usage was detected in mothers of infants with cpo, while no such significant increase was found in mothers of cl±p infants25. safra and oakley26 instead reported association of cl±p with first-trimester exposure to diazepam. these evidences were confirmed in 1990 by laegreid 27 for benzodiazepine in general. in a further study, eros et al.28 addressed the question of benzodiazepine teratogenicity as a whole, verifying undetectable teratogenic risk due to treatment with this kind of anticonvulsants. although diazepam is a weak teratogen in susceptible mice at very high doses, its interference with the forming human face is probably very modest or inexistent. in a more recent study wikner et al.29 studied the effects of benzodiazepines during foetal life by means data derived from the swedish medical birth register and they do confirm the earlier proposed increased risk for ofcs. it is well known that women with epilepsy have an increased risk of offspring with cl±p. this risk has been attributed mostly to the teratogenic effects of antiepileptic drugs, though other risk factors have been suggested, such as epilepsy itself, or some underlying genetic defects associated with epilepsy 30. however, it has been noted that a monotherapy of antiepileptic drugs reduces the risk of developing fetal anticonvulsant syndrome31. in 1987 tocco et al.32 demonstrated that diazepam produced a significant increase in cleft palate frequency in mice. subsequently, it was confirmed that the fusion of palatal shelves was inhibited dose-dependently by treatment with diazepam 33. moreover, knockout mice for the gamma aminobutyric acid (gaba)-producing enzyme glutamic acid decarboxylase (gad1) gene or the beta3 subunit of the gabaa receptor (gabrb3) gene results in neurological alteration and clefting of the secondary palate34-35. the striking similarity in phenotype between the receptor and ligand mutations clearly demonstrated a role for gaba signaling in normal palate development. a well-devised experiment showed that neural expression of gabrb3 in knockout mice can rescue the neurological phenotype, but not avoid cleft palate36. this indicates that non-neuronal gaba signaling is implicated in palate development. additional evidences of the pivotal role of gaba came from a microarray-based experiment37. in a recent study, our group 38 observed a significant relationship between the beta 3 subunit of the gammaaminobutyric acid receptor (gabrb3) and cl±p, suggesting that the gabrb3 gene is involved in this congenital disease. although gabr is the target of benzodiazepine, none of our patients presented neurologic diseases. in the same study, it was also demonstrated that the gad1 gene, which encodes the gaba-producing enzyme, is not involved in cl±p pathogenesis. kanno et al.39 studied the possible association between glutamic acid decarboxylase 67 (gad67) and development of nonsyndromic ofc in japanese patients. they screened 99 parent-offspring trios using 5 snps at the gad67 gene. the frequency distribution of the haplotype differed between ofc patients and controls. in transmission disequilibrium test (tdt) they found haplotypes preferentially transmitted to the patients with cl±p suggesting that gad67 is involved in the pathogenesis of ofc in the japanese population. some studies show that neuroprotective peptides prevented fetal death and learning deficits caused by prenatal alcohol exposure 40. the gamma-aminobutyric acid a (gaba) receptor subunit gababeta3 plays a critical role for nervous system and palate development. toso et al.41 demonstrated, with a study in vivo, that treatment with neuropeptides prevents the alcohol-induced decline in gababeta3 expression 10 days after alcohol exposure. because palate formation continues through e18, neuropeptides may be beneficial for the prevention of cleft lip and palate. as well as gabaergic signaling systems, other molecules like transforming growth factor-beta (tgf-beta) and retinoic acid (ra), are potentially involved in palatogenesis. baroni et al.42 studied the phenotypic differences between primary cultures of fibroblasts from subjects with familial nonsyndromic cl±p and age-matched normal fibroblasts. they found that cl±p fibroblasts exhibit an abnormal phenotype in vitro and respond differently to ra treatment. they suggest that altered crosstalk between ra, gabaergic, and tgf-beta signaling systems could be involved in human cl±p fibroblast phenotype. moreover they demonstrated that gaba receptor (gabrb3) mrna expression was up regulated in human cl±p fibroblasts. braz j oral sci. 7(24):1470-1475 drugs and nonsyndromic orofacial cleft: an update 1473 by using an animal model, hansen et al.43,44 analyzed the mechanism of phenytoin (pht) teratogenicity. in a first report, they found an increased incidence of cleft palate and a fall in maternal plasma folate levels in mice treated with pht during gestation43. since methylenetetrahydrofolate reductase (mthfr) activity was decreased in the hepatic tissue of pregnant mice, but it was unaltered in embryos, they inferred that pht affects maternal folate metabolism. in a second report the same authors found a relationship between the plasma levels of pht and corticosterone44. they suggest that the lengthy increase in plasma corticosterone during organogenesis may represent a factor in the increased incidence of cleft lip and palate observed after administration of pht to a/j mice. by exposing explants of mouse palates to diphenylhydantoin (dph), shimizu and colleagues21 obtained results indicating that the teratogen causes cleft palate by inhibiting mesenchymal and epithelial cell proliferation. recent epidemiologic studies to evaluate the possible association between some drug treatments during pregnancy and ofc confirmed the inducing effect of phenytoin45. experimental evidence suggests that the fetal adverse effects of pht’s are associated with potential embryonic bradycardia/arrhythmia and hypoxia-related damage during a restricted developmental period46-48. this hypoxia, through an undefined downstream mechanism, leads to the development of cl. an hypoxia-related teratogenic mechanism by pht is supported by indirect evidence from teratology studies46,49,50. longer periods of hypoxia result in embryonic death while shorter periods of severe hypoxia result in growth retardation and the same type of stage-specific malformations (distal digital reductions, orofacial clefts, and cardiovascular defects). nonsteroidal anti-inflammatory drugs it has been controversial whether aspirin use during pregnancy increases the risk of congenital abnormalities. saxen51 suggested that aspirin consumption during the first trimester of pregnancy was involved in the etiology of oral cleft. however, a review of a large body of published experimental animal and human epidemiological data provided no direct conclusive evidence of adverse effects in the pregnant woman and her developing fetus52. a meta-analysis, based on 22 studies published between 1971 and 2002, has suggested an increased risk of neural tube defects (odds ratio [or] = 2.2; 95 %ci: 0.93-5.17), gastroschisis (or = 2.37; 95% ci: 1.44-3.88), and cleft lip and palate (or = 2.87; 95% ci 2.04-4.02) after aspirin use in early pregnancy53. nørgård et al.54 examined the association between maternal aspirin use in the first weeks of gestation and the most frequent congenital abnormalities: neural tube defects, gastroschisis, cl±p and cpo. by using a large case-control dataset from hungary they revealed no increased risk of congenital abnormalities finally, ericson and källén55 performed a study on congenital malformations in infants whose mothers used nonsteroidal anti-inflammatory drugs in early pregnancy. an increased risk was demonstrated for ofc and it correlated with the use of naproxen. concluding, it is well-established that nonsyndromic ofc is composed of two separate entities: cl±p and cpo. both have a genetic origin, and environmental factors play a role in the onset of these malformations.epidemiologic studies have demonstrated a relationship between certain drugs (steroids and anticonvulsants) during pregnancy and a higher risk of having a child with ofc. in all cases, the molecular mechanisms whereby these environmental factors produce their effects are still unknown. further investigations will be necessary before a complete picture can be obtained of the main factors involved in lip and palate formation. these elements will enable to better understand this complex disease and to provide improved treatments. acknowledgements this study was supported, in part, by grants from telethon n. ggp05147 (p.c.), far (f.c.), rfo (l.s., m.m. and f.p.) and prin 2005 (f.c.). references 1. fraser fc. the genetics of cleft lip and palate. am. j. hum. genet. 1970; 22: 336-52. 2. bonaiti-pellie c, briand ml, feingold j, pavy b, psaume j, migne-tuffer et al. an epidemiological and genetic study of facial clefting in france. i. epidemiological and frequency in relatives. j. med. genet. 1982;11: 374-7. 3. ferguson mwj. palate development. 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z, siracusa ld, brilliant mh. nonneuronal expression of the gaba(a) beta3 subunit gene is required for normal palate development in mice. dev. biol. 2003; 254: 93101. 37. brown nl, knott l, halligan e, yarram sj, mansell jp, sandy jr. microarray analysis of murine palatogenesis: temporal expression of genes during normal palate development. dev. growth differ. 2003; 45: 153-65. 38. scapoli l, martinelli m, pezzetti f, carinci f, bodo m, tognon m, et al. linkage disequilibrium between gabrb3 gene and nonsyndromic familial cleft lip with or wiyhout cleft palate. hum. gent. 2002; 110: 15-20. 39. kanno k, suzuki y, yamada a, aoki y, kure s, matsubara y. association between nonsyndromic cleft lip with or without cleft palate and the glutamic acid decarboxylase 67 gene in the japanese population. am j med genet a. 2004; 27: 11-6. 40. spong cy, abebe dt, gozes i, brenneman de, hill jm. prevention of fetal demise and growth restriction in a mouse model of fetal alcohol syndrome. j pharmacol exp ther. 2001; 297: 774-9. 41. toso l, roberson r, abebe d, spong cy. neuroprotective peptides prevent some alcohol-induced alteration in gammaaminobutyric acid a-beta3, which plays a role in cleft lip and palate and learning in fetal alcohol syndrome. am j obstet gynecol. 2007; 196: 259-65. 42. baroni t, bellucci c, lilli c, pezzetti f, carinci f, becchetti e, et al. retinoic acid, gaba-ergic, and tgf-beta signaling systems are involved in human cleft palate fibroblast phenotype. mol med. 2006; 12: 237-45. 43. hansen dk, billings re. phenytoin teratogenicity and effects on embryonic and maternal folate metabolism. teratology. 1985; 31: 363-71. 44. hansen dk, holson rr, sullivan pa, grafton tf. alterations in maternal plasma corticosterone levels following treatment with phenytoin. toxicol. appl. pharmacol. 1988; 96: 24-32. 45. puhó eh, szunyogh m, métneki j, czeizel ae. drug treatment during pregnancy and isolated orofacial clefts in hungary. cleft palate craniofac j. 2007; 44: 194-202. 46. danielsson br, azarbayjani f, skold ac, webster ws. initiation of phenytoin teratogenesis: pharmacologically induced embryonic bradycardia and arrhythmia resulting in hypoxia and possible free radical damage at reoxygenation. teratology. 1997; 56: 271-81. 47. azarbayjani f, danielsson br. embryonic arrhythmia by inhibition of herg channels: a common hypoxia-related teratogenic mechanism for antiepileptic drugs? epilepsia. 2002; 43: 457-68. braz j oral sci. 7(24):1470-1475 drugs and nonsyndromic orofacial cleft: an update 1475 48. azarbayjani f, danielsson br. phenytoin-induced cleft palate: evidence for embryonic cardiac bradyarrhythmia due to inhibition of delayed rectifier k+ channels resulting in hypoxiareoxygenation damage. teratology. 2001; 63: 152-60. 49. brent rl, franklin jb. uterine vascular clamping: new procedure for the study of congenital malformations. science. 1960; 132: 89-91. 50. leist kh, grauwiler j. fetal pathology in rats following uterinevessel clamping on day 14 of gestation. teratology. 1974; 10: 55-67. 51. saxen, i. prolonged in vitro closure of the mouse secondary palate by salicylates. scand j dent res. 1975; 83, 202-8. 52. corby dg. aspirin in pregnancy: maternal and fetal effects. pediatrics. 1978; 62(suppl): 930-7. 53. kozer e, nikfar s, costei a, boskivic r, nulman i, koren g. aspirin consumption during the first trimester of pregnancy and congenital anomalies: a meta-analysis. am j obstet gynecol. 2002; 187: 1623-30. 54. nørgård b, puhó e, czeizel ae, skriver mv, sørensen ht. aspirin use during early pregnancy and the risk of congenital abnormalities: a population-based case-control study. am j obstet gynecol. 2005; 192: 922-3. 55. ericson a, källén ba. nonsteroidal anti-inflammatory drugs in early pregnancy. reprod toxicol 2001; 15: 371-5. braz j oral sci. 7(24):1470-1475 drugs and nonsyndromic orofacial cleft: an update braz j oral sci. 15(3):166-172 comparison of two different methods for detecting periodontal pathogenic bacteria telma blanca lombardo bedran*; guilherme josé pimentel lopes de oliveira**; luís carlos spolidorio, phd§; joni augusto cirelli, phd**; denise palomari spolidorio, phd§ *department of dentistry, nove de julho university, são paulo, sp, brazil **department of oral diagnosis and surgery, araraquara dental school, state university of são paulo, araraquara, sp, brazil §department of physiology and pathology, araraquara dental school, state university of são paulo, araraquara, sp, brazil correspondence to: telma blanca lombardo bedran department of dentistry, nove de julho university rua vergueiro, 235 são paulo sp brazil 19-99504-7008 telmabedran@hotmail.com abstract aim: to perform a comparative analysis between two methods for detecting porphyromonas gingivalis, tannerella forsythia and porphyromonas endodontalis in periodontal plaque samples. methods: the study sample consisted of twenty systemically healthy patients showing generalized chronic periodontitis. the subgingival samples for microbiological analysis were collected before (baseline) and 60 days after a basic periodontal therapy from 30 non-adjacent affected sites (probing depth (pd): 5-7 mm, clinical attachment loss (cal) ≥ 5 mm, positive for bleeding on probing (bop)). microbiological analysis was performed by pcr and qpcr. to allow a comparative analysis between both methods, qpcr was divided in three different scores (score 2: presence of more than 100 bacteria; score 1: presence of 10-100 bacteria, and score 0: absence of bacteria), in accordance to dna quantity, while for pcr two scores were assigned: presence or absence of bacteria. results: qpcr demonstrated higher sensitivity in the detection of these pathogens compared with pcr when scores 1 and 2 were considered positive. however, when only score 2 was considered positive, pcr and qpcr showed better agreement. conclusions: qpcr demonstrated higher sensitivity than conventional pcr for detection of low numbers of microorganisms and can be useful for the quantification of periodontopathogens. keywords: periodontal diseases. polymerase chain reaction. bacteria. introduction periodontitis is an inflammatory oral disease caused by specific microorganisms that colonize the periodontal pocket and leads to destruction of the tooth-supporting tissues, including gingival connective tissue and alveolar bone1. bacteria species that have been strongly associated with periodontitis include members of the red complex2: porphyromonas gingivalis (p. gingivalis), tannerella forsythia (t. forsythia) and treponema denticola (t. denticola). in addition, some studies have reported the presence of additional subgingival bacterial species in diseased periodontal sites1,2. among these pathogens, porphyromonas endodontalis, an asaccharolytic, black-pigmented, gram-negative anaerobic bacteria, is noteworthy3. mechanical therapy is considered as the gold standard treatment of periodontitis. this therapy consists in combining scaling and root planning performed by a professional and plaque removal achieved by the patient. in some cases, this treatment received for publication: june 22, 2016 accepted: april 19, 2017 original article braz j oral sci. july | september 2016 volume 15, number 3 http://dx.doi.org/10.20396/bjos.v15i3.8649599 167 may not be sufficient to prevent the progression of periodontitis4. for these patients, an adjunctive antimicrobial therapy associated with mechanical therapy is often used4. microbiological analysis is an important procedure to i) identify and quantify pathogens in periodontal pockets from patients with periodontitis, ii) clarify the diagnosis and etiology of severe forms of periodontitis, and iii) help to select the correct adjunctive antimicrobial therapy to treat patient4,5. bacterial culture is considered the gold standard method for the identification of periodontopathogens, since it is the only technique capable of isolating viable pathogens and to test their susceptibility to antimicrobial agents. however, there are some limitations associated with this procedure. indeed, cultivation cannot detect non-viable bacteria, as well as, hardly cultivated bacteria important in the etiology of periodontitis, such as t. denticola6,7. nowadays, alternative nucleic acid-based methods, more sensitive than bacterial culture, have been used to detect and quantify pathogens present in oral cavity5,6,8. more specifically, polymerase chain reaction is a technique based on the detection of nucleic acids and has been used by many laboratories to quantify periodontopathogens5-7. conventional pcr is a technique based on replication of dna that is used to detect pathogens in periodontal pocket with a greater sensitivity and specificity than cultivation9-11. however, this technique has some limitations, such as the difficulties to detect pathogens in sample containing small quantities of dna, since more than 100 bacteria in periodontal pockets is required for pcr9,10. in addition, the exact amount of bacteria cannot be determined since it is a qualitative method of diagnostic, and therefore cannot be used to differentiate health and disease samples6,8,9,11,12. in order to overcome some of these drawbacks, different strategies of pcr have been introduced in many laboratories. more specifically, quantitative pcr (qpcr) allows the quantification of low number of bacterial loads in subgingival plaque samples12-14. qpcr is able to detect approximately 10 bacteria and is able to quantify the exact amount of bacteria present in subgingival crevicular fluid6,12,15. a large number of studies in the literature compared qpcr and anaerobic culture for the detection and quantification of pathogens in periodontal samples6,16. however, only few studies compared conventional pcr and qpcr for the detection of periodontopathogens. therefore, the purpose of this study was to compare pcr and qpcr for the detection of p. gingivalis, t. forsythia and p. endodontalis in subgingival samples from patients with chronic periodontitis before and after periodontal treatment. this comparison of the two procedures should demonstrate benefits and limitations of each method. material and methods subject population this study was designed as a double-blind, controlled trial, which all patients were prospectively assigned for two months after a basic periodontal treatment of scaling and root planning, to evaluate the effect of periodontal treatment on the reduction of periodontopathogens on the periodontal pocket and to compare pcr and qpcr for the detection of p. gingivalis, t. forsythia and p. endodontalis in plaque samples from patients with chronic periodontitis, before and after basic periodontal treatment. the study was approved by the ethics committee in human research (protocol # 26/08), and all study participants signed a free and informed consent form. the sample size calculation was based on previous studies using qpcr for the detection of periodontal pathogens12,13,15. briefly, first we selected similar studies to find the approximate sample size that would be necessary (we found sample size between 20-30). based on this number, we performed the sample calculation to find the smallest number that could be used in this study to get statistically significant results. the sample size comprised twenty patients randomly selected (8 men and 12 women 35-55 years of age) with moderate to severe chronic periodontitis and without history of systemic diseases. inclusion criteria were as follows: presence of at least 20 teeth; minimum of three non-adjacent teeth with bleeding on probing (bop), clinical attachment level (cal) > 5 mm and probing depth (pd) between 5 and 7 mm. no patients used antibiotic therapy in the 6 months prior to the study. forty-five days before beginning the periodontal treatment, all the patients selected for this study received oral hygiene instructions every week. after 30 days of oral hygiene instruction, periodontal examination was performed for patients with a visible plaque index of less than 30%. study design after providing the oral hygiene instructions, we selected 30 non-adjacent posterior sites from 20 patients with a pd of 5-7 mm, positivity for bop and a cal ≥ 5 mm. the selected teeth had no dysfunctions in relation to occlusion and had no prostheses. in order to standardize the position of the manual probe (williams®, são paulo, brazil) and the position of paper point to collect plaque samples in sites selected for microbiological analysis, alginate molds of the dental arches were made to prepare acetate stents. the periodontal clinical measurements were performed by a single trained examiner, while another oral health professional performed the basic periodontal treatment followed by oral hygiene instruction and microbiological assessment. the clinical measurements and the plaque samples collections were performed at baseline and at 60 days after the periodontal treatment. clinical parameters clinical examination was performed by a trained and calibrated examiner, whose intra-exam repeatability was determined at baseline (kappa score = 0.91). the clinical parameters measured included cal, bop, 0/1 (negative/positive) and pd, determined at six different sites per tooth (mesiobuccal, buccal, distobuccal, distolingual, lingual and mesiolingual) using a periodontal probe (williams®, são paulo, brazil). subgingival sample collection plaque samples for microbiological analyses were collected seven days after the initial clinical examination and comparison of two different methods for detecting periodontal pathogenic bacteria braz j oral sci. 15(3):166-172 168 then 60 days after completion of the basic periodontal treatment and storage at -20oc. briefly, supragingival biofilm was removed and the selected sites were isolated with cotton rolls and gently air-dried. two sterile paper points (no. 30; dentsply, maillefer, petrópolis, rj, brazil) were inserted in the gingival pocket up to the apical portion for 30 seconds and the subgingival fluid was collected. the paper points were immediately placed in sterile eppendorf vials containing 500 µl of te buffer (10 mm trishcl, ph 8.0, 1 mm edta, ph 8.0) and stored at -20° c until dna was extracted for microbial analysis by pcr and qpcr. basic periodontal treatment the basic periodontal treatment was performed under local anesthesia and with the aid of manual instruments (mccall and gracey curettes and hirschfeld file scaler hu friday®). patients received a non-surgical periodontal treatment including scaling and root planning (srp) followed by oral hygiene instructions seven days after completing the microbiological sample collection. dental polishing was performed immediately after each session of srp with rubber cups and paste. after srp, supragingival biofilm control (maintenance phase) was performed via prophylaxis and oral hygiene instruction weekly for 60 days, at which time the second sample plaque collection and periodontal examination was performed. bacterial strains and growth conditions the bacterial species p. gingivalis (atcc 33277), t. forsythia (atcc 43037) and p. endodontalis (e203) were used to determine the specificity and detection levels of the pcr method. briefly, bacteria were grown anaerobically on tryptone soy blood agar plates supplemented with hemin (0.5 µg/ml) and menadione (1 µg/ml) at 37° c in 85% n2, 5% co2 and 10% h2 in an anaerobic chamber17. dna extraction the extraction of dna from subgingival fluid samples and from bacterial reference cultures was performed using standard methods. to determine the concentration of dna, the relationships between the absorbance at 260 and 280 nm were determined with a uv spectrophotometry. as a quality parameter, a260/a280 values between 1.8 and 2.0 were considered appropriate. thereafter, dna suspensions were divided and used for conventional pcr and qpcr. conventional pcr the presence of bacteria was confirmed using a non-specific oligonucleotide18. the positive samples for non-specific reaction were then processed in pcr amplification with a specific primer: p. gingivalis (forward: 5’-aat cgt aac ggg cga cac ac-3’, reverse: 5’-ggg ttg ctc ctt cat cac ac3’ – 593pb)18, t. forsythia (forward: 5’-gcg tat gta acc tgc ccg ca-3’, reverse: 5’-tgc ttc agt gtc agt tat acc t-3’ – 641pb)18 and p. endodontalis (forward: 5’-gct gca gct caa ctg tag tc-3’, reverse: 5’-ccg ctt cat gtc acc atg tc-3’– 672pb)18 (invitrogen tech-linesm). this conventional pcr, which is an older method, was used as a gold standard. pcr amplification was performed as described previously18. briefly, the reaction was carried out in a volume of 25 µl with template dna (50 ng/µl) and 1 mm oligonucleotide of specific bacteria (invitrogen tech-linesm). the positive control consisted of genomic dna (50 ng/µl) from tested bacteria. the reactions were carried out in a thermocycler (mycyclertmthermal cycler biorad). pcr products were analyzed by 1.5% agarose gel electrophoresis at 100 v for 90 min. a 1 kb dna ladder digest (invitrogen tech-linesm) served as molecular weight marker. quantitative pcr reactions for the quantitative analysis, plasmids containing the target genes were used as standard. pcr amplification was initially performed for the 16s rrna of p. endodontalis, p. gingivalis and t. forsythia. the amplicons were cloned using the topo ta cloning kit (invitrogen, carlsbad, ca, usa) according to the manufacturer’s instructions and plasmids were transformed into e. coli. the dilutions were used as template dna in the qpcr reactions. in each reaction, data obtained from the standard curve were used to convert the ct scores (cycle threshold) obtained with patient samples into the exact numbers of dna copies19,20. the detection and quantification by qpcr was performed using universal (applied biosystems®)21 and specific primers for p. gingivalis (forward: 5’-acc tta ccc ggg att gaa atg-3’, reverse: 5’-caa cca tgc agc acct ac ata gaa-3’83pb), t. forsythia (forward: 5’-agc gat ggt agc aat acc tgt c-3’, reverse: 5’-ttc gcc ggg tta tcc ctc-3’88pb) and p. endodontalis (forward: 5’-gct gca gct caa ctg tag tct tg-3’, reverse: 5’-tca gtg tca gac gga gcc tag tac-3’-110pb) (applied biosystems®)22 .the species-specific primer sets were designed based on the variable regions of each target gene. the specificity of the primers was confirmed by multiple alignments of relevant sequences from closely related species and by a basic local alignment search tool (blast) homology search16. the qpcr reactions were performed with the use of a step onetm qpcr system (applied biosystems®). all reactions were performed in duplicate, and average values were used to calculate the bacterial load. the total volume of each reaction was 10 µl containing 5 µl of sybr green er qpcr supermix universal (invitrogen tech-linesm), 0.1 µm of each primer pair (applied biosystems®) and 50 ng/µl template dna. the thermocycling program included incubation at 95°c for 10 minutes, 40 cycles of 95° c for 15 seconds and 60°c for 1 minute. after the pcr reactions, the dissociation curve (melting curve) was obtained using temperatures between 60° c and 95° c to determine primer specificity. melting curve analysis revealed only one peak of amplification. all reactions were performed in 48-well microamp optical plates covered with optical adhesive (applied biosystems). data were analyzed by step onetm software (applied biosystems). analysis of data the detection levels of conventional pcr and qpcr are 102 and 10 bacteria/subgingival sample, respectively. in the comparison of two different methods for detecting periodontal pathogenic bacteria braz j oral sci. 15(3):166-172 169 comparison of two different methods for detecting periodontal pathogenic bacteria present study, to allow the comparison between both techniques, the qpcr had the results adjusted (threshold level 102 bacteria/ plaque sample). the results were presented as a score system in the following ranges for conventional pcr: presence (score 1) and absence (score 0), and for qpcr: >102 bacteria (score 2), 10-100 bacteria (score 1), and no detection (score 0). statistical analysis the data were analyzed using the graphpad prism r 3.1 (graphpad software, inc. la jolla, ca, usa). interand intra-group differences were analyzed for the various periods. the prevalence of bacteria determined by both techniques was analyzed using the wilcoxon test. the sensitivities of the two techniques were determined by comparing the qpcr results with the conventional pcr results. agreement between the conventional pcr and qpcr results was determined using the kappa test (k). to determine agreement, the percentages of total samples that were positive or negative for both techniques were used, as described previously5,6. results the average age of the study group was 46 ± 7.49, with 8 men (40%) and 12 women (60%). the average number of teeth was 24 ± 3. since no statistically significant differences in age and number of teeth were observed in the sample set, it was considered as homogeneous. the collection sites for the microbiological analysis had a prevalence of bop of 100% at baseline that decreased to 13.33% after treatment (p < 0.0001). in addition, pd (5.33 ± 0.54 mm, baseline) and cal (5.4 ± 0.62 mm, baseline) showed statistically significant reductions (p <0.0001) after periodontal therapy (3.63 ± 0.76 mm and 3.83 ± 0.95 mm for pd and cal, respectively). detection of microorganisms the standard curve for each bacterial species by qpcr was obtained with the use of specific primers and four serial dilutions (101-104 bacteria) of the genomic dna of p. gingivalis, t. forsythia and p. endodontalis. the reaction efficiency (provided by software) for each organism was 96.3 (p. gingivalis), 97.8 (t. forsythia) and 97.1 (p. endodontalis). the correlation coefficient for the mean ct values was r2 >0.99. all 3 sybr green assays were highly specific and amplified only dna extracted from the periodontopathogens. the detection of all periodontopathogens targeted was significantly higher using the qpcr technique. a marked difference was noted for all the bacteria (p. gingivalis, t. forsythia and p. endodontalis) between both methods. the detection of microorganisms through conventional pcr technique at baseline was 46.6% for p. gingivalis, 53.3% for t. forsythia, and 56.6% for p. endodontalis. a significant statistical reduction at day 60 was observed: 10% for p. gingivalis, 3.3% for t. forsythia, and 20% for p. endodontalis (figure 1). on the other hand, the qpcr technique allowed the identification of more positive samples in regard to the presence of periodontopathogens. p. gingivalis was at 96.6% at baseline and at 93.3% at day 60 t. forsythia was detected at 90% at baseline with a statistically significant reduction to 66.6% at day 60. for p. endodontalis, the detection was 83.3% at baseline with a reduction to 43.3% at day 60 (figure 1). fig.1. bacteria detection (%) through qpcr and conventional pcr. comparison between conventional pcr and qpcr without adjusting the threshold table 1, 2 and 3 summarizes the comparison between both techniques to detect pathogens. the detection of pathogens through qpcr without adjusting the results was significant higher when compared to conventional pcr detection. at baseline, conventional pcr detected only 14 samples positive for p. gingivalis in opposite to 29 samples positive through qpcr, resulting a sensitivity of 100% and specificity only 6% for qpcr, and the kappa values showed poor agreement (k=0.06). at 60 days post-treatment, only 3 of 28 samples positive for p. gingivalis by qpcr were also positive by conventional pcr, resulting in a sensitivity of 100% and specificity of only 7% and poor agreement (k= 0.02) (table 1). the comparison between techniques to detect t. forsythia at baseline showed a sensitivity of 94% and specificity of 14% for the qpcr. same results were found at 60 days post-treatment with an increased sensitivity to 100% and specificity to 35%. the kappa values showed poor agreement between techniques, at baseline (k=0.01) and at 60 days post-treatment (k=0.03) (table 2). at baseline, only 15 of 25 samples positive for p. endodontalis by qpcr were also positive for this bacteria by conventional pcr, resulting in a sensitivity of 88%, a specificity of 23% and fair agreement between techniques (k=0.25). sixty days after periodontal treatment, the sensitivity was 83% and an increased specificity to 67% and agreement to k=0.35 (table 3). braz j oral sci. 15(3):166-172 comparison between conventional pcr and qpcr adjusting the threshold when the detection threshold of qpcr to 102 bacteria (score 2) was adjusted, the agreement between both methods increased. at baseline, the comparison between techniques for the detection of p. gingivalis showed a good agreement (k=0.67) that corresponded to sensitivity of 100% and specificity of 69%. similar results were obtained at 60 days post-treatment with a sensitivity of 67% and a specificity of 85%, although the kappa value showed a fair agreement between techniques (k=0.36) (table 1). after adjusting the threshold, 13 samples were positive for t. forsythia for both techniques at baseline. three samples were positive only for conventional pcr and 5 samples were positive only through qpcr, resulting in a sensitivity of 81%, specificity of 64%, and kappa value showed a moderate agreement (k=0.46). after periodontal treatment, the agreement between both methods increased (k=0.53), with corresponding a sensitivity of 100% and specificity of 86% (table 2). the comparison between both techniques for the detection of p. endodontalis at baseline showed a moderate agreement between techniques (k=0.51), each one corresponding to a sensitivity of 88% and a specificity of 61%. at 60 days post-treatment, the kappa value increased and showed a good agreement (k=0.63), corresponding to a high sensitivity (83%) and specificity (87%) (table 3). discussion the detection of periodontopathogens in samples from patients with periodontitis has become important in order to monitor the effect of periodontal treatments, especially patients that need complementary therapy4,5. as described by socransky et al.2 (1998), all cases of periodontitis are associated with the presence of periodontopathogens, although, the disease will not necessarily develop as long as the microorganisms does not exceed the threshold for the host. periodontal therapy is not exclusively based on microbiological diagnostic. indeed, for some cases, the clinical analysis can be associated with microbiological diagnostic, for example, metronidazole is recommended for patients that have the presence of the red complex pathogens (p. gingivalis, t. forsythia and t. denticola) but not for patients showing the presence of a. actinomycetemcomitans5,23. this statement supports the importance of quantifying pathogens in periodontal pockets6. bacterial cultures, considered a gold standard microbiological test, is routinely used in many microbiological laboratories, but hassome limitations, such as the need of at least 103 bacteria samples to allow the detection by culture. nowadays, other detection methods that might be more sensitive than microbiological culture, including methods based on nucleic acid detection, are routinely used, but still need evaluation and validation24. conventional pcr is 10-100 times more sensitive than anaerobic culture to detect periodontopathogens, although the intrinsic limitations of pcr cause a loss of qualitative information, because at least 102 pathogens are required for positive detection5,13,23. in some cases, conventional pcr shows false negative results and, therefore, can hide the real presence of pathogens and lead to a misdiagnosis. the qpcr is a more sensitive technique than conventional pcr, because only 10 bacteria are required for positive detection 170 comparison of two different methods for detecting periodontal pathogenic bacteria table 1 comparison between pcr and qpcr for the detection of p. gingivalis. pcr result n. (%) of samples with the qpcr result more than 10 bacteria/subgingival sample* n. (%) of samples with the qpcr result more than 102 bacteria/subgingival sample# positive negative total positive negative total ba se li nn e positive 14 (46.6) 0 14 14 (46.6) 0 14 negative 15 (50) 1 (3.3) 16 5 (16.6) 11 (36.6) 16 total 29 (96.6) 1 30 19 (63.3) 11 30 positive negative positive negative 60 d ay s positive 3 (10) 0 3 2 (6.7) 1 (3.3) 3 negative 25 (83.3) 2 (6,7) 27 4 (13.3) 23 (76.7) 27 total 28 (93.3) 2 30 6 (20) 24 (80) 30 baseline: * sensitivity 100%; specificity 6%; k 0,06; # sensitivity 100%; specificity 69%; k 0,67 60 days: *sensitivity 100%; specificity 7%; k 0,02; #sensitivity 67%; specificity 85%; k 0,36 table 2 comparison between pcr and qpcr for the detection of t. forsythia. pcr result n. (%) of samples with the qpcr result more than 10 bacteria/subgingival sample* n. (%) of samples with the qpcr result more than 102 bacteria/subgingival sample# positive negative total positive negative total ba se li nn e positive 15 (50) 1 (3.3) 16 13 (43.3) 3 (10) 16 negative 12 (40) 2 (6.7) 14 5 (16.7) 9 (30) 14 total 27 (90) 3 (10) 30 18 (60) 12 (40) 30 positive negative positive negative 60 d ay s positive 1 (3.3) 0 1 1 (3.3) 0 1 negative 19 (63.3) 10 (33.3) 29 4 (13.3) 25 (83.4) 29 total 20 (66.6) 10 30 5 (16.6) 25 30 baseline: * sensitivity 94%; specificity 14%; k 0,01; # sensitivity 81%; specificity 64%; k 0,46 60 days: *sensitivity 100%; specificity 35%; k 0,03; #sensitivity 100%; specificity 86%; k 0,53 table 3 comparison between pcr and qpcr for the detection of p. endodontalis. pcr result n. (%) of samples with the qpcr result more than 10 bacteria/subgingival sample* n. (%) of samples with the qpcr result more than 102 bacteria/subgingival sample# positive negative total positive negative total ba se li nn e positive 15 (50) 2 (6,7) 17 15 (50) 2 (6.7) 17 negative 10 (33.3) 3 (10) 13 5 (16.7) 8 (26.7) 13 total 25 (83.3) 5 (16.7) 30 20 (66.7) 10 (33.4) 30 positive negative positive negative 60 d ay s positive 5 (16.6) 1 (3.3) 6 5 (16.7) 1 (3.3) 6 negative 8 (26.7) 16 (53.3) 24 3 (10) 21 (70) 24 total 13 (43.3) 17 (56.6) 30 8 (26.7) 22 (73.3) 30 baseline: * sensitivity 88%; specificity 23%; k 0,25; # sensitivity 88%; specificity 61%; k 0,51 60 days: *sensitivity 83%; specificity 67%; k 0,35; #sensitivity 83%; specificity 87%; k 0,63 braz j oral sci. 15(3):166-172 and allows a real quantification of periodontopathogens in periodontitis sites, turning it an important criteria to distinguish among healthy and diseased sites. qpcr is able to detect and quantify small changes in the number of pathogens following periodontal treatments5,16. on the other hand, the most important disadvantage of qpcr is related to its cost and the fact that it requires special equipment6. in this study, we compared qpcr with conventional pcr for the detection of p. gingivalis, t. forsythia and p. endodontalis in patients with chronic periodontitis before and after periodontal treatment. conventional pcr, the oldest method, was used as a gold standard4. as reported by mullis10 (1990) and higuchi et al.25 (1992) the detection limit by qpcr is 10 bacteria while 102 bacteria is required by conventional pcr. for this reason, in this study, the agreement between both techniques was analyzed at two different threshold levels. therefore, the threshold level of qpcr was adjusted to 102 bacteria to allow an unbiased comparison between pcr and qpcr6. our results are in accordance with other microbiological studies that reported the presence of most periodontopathogens even in low quantity and proportions following a periodontal treatment5. through conventional qualitative pcr, small differences before and after periodontal treatment can be masked5,13. these results showed the importance of qpcr to quantify low proportion of periodontophatogens, that cannot be detected though conventional pcr21. since microbiological analysis are often used for a therapeutic strategy for patients who do not initially respond to conventional mechanical treatment, qpcr may be an interesting tool for the selection of antibiotics or for monitoring the efficacy of the antibiotic treatment16. the comparative analysis of both techniques showed a higher sensitivity of qpcr, resulting in a low chance of false negative results, when compared with conventional pcr. even though both pcr and qpcr techniques are more sensitive in the detection of pathogens when compared with anaerobic culture, a detection without quantification may be irrelevant in terms of disease activity, since the presence of microorganisms will not necessarily lead to the development of disease12. our results showed a poor agreement between both techniques with a high sensitivity and low specificity associated with qpcr when was compared with conventional pcr without adjusting the threshold. however, when the threshold of qpcr was adjusted, the agreement between techniques increased. this results, confirms the requirement of a good microbiological test diagnostic with high sensitivity that allow the detection of low number of periodontopathogens, because sometimes the number of bacteria present in periodontal pockets can be very low due to the difficulty to collect samples from subgingival pockets. this may be related to the insertion of paper points in the subgingival pocket and the capacity of paper points to absorb enough amount of gingival fluid. nowadays, the qpcr technique becomes an important tool to quantify the exact number of microorganisms and differentiate the stages of health and periodontal disease in combination with the clinical data. this technique allows identifying the presence of periodontopathogens in subgingival pockets and helps to choose the best treatment for some patients that need additional periodontal therapy12. the qpcr in comparison with conventional pcr has the advantages that it does not require the post-pcr processing of amplicons, eliminating some contaminations and the assay can be completed within 2,5 h. considering the limitation of this study and the small number of samples, qpcr showed to be a fast, efficient and a sensitive technique to detect and quantify p. gingivalis, t. forsythis, and p. endodontalis in subgingival samples from patients with chronic periodontitis. the qpcr approache can contribute to recognize new putative pathogens present in subgingival pocket from patients with periodontitis, and allow the development of new periodontal therapy. additional studies with larger sample size are required to validate this difference between conventional pcr and qpcr. acknowledgements the authors thank the financial support from fapesp (fundação de amparo à pesquisa do estado de são paulo, são paulo, brazil). conflicts of interest this manuscript does not have any kind of conflict of interest. references 1. 1. krishnan k, chen t, paster bj. a practical guide to the oral microbiome and its relation to health and disease. oral dis. 2017 apr;23(3):276-286. doi: 10.1111/odi.12509. 2. socransky ss, haffajee ad, cugini ma, smith c, kent rl jr. microbial complexes in subgingival plaque. j clin periodontol. 1998 feb;25(2):13444. 3. lombardo bedran tb, marcantonio ra, spin neto r, alves mayer mp, grenier d, spolidorio lc, et al. porphyromonas endodontalis in chronic periodontitis: a clinical and microbiological cross-sectional study. j oral microbiol. 2012;4. doi: 10.3402/jom.v4i0.10123. 4. cosgarea r, bäumer a, pretzl b, zehaczek s, kim ts. comparison of two different microbiological test kits for detection of periodontal pathogens. acta odontol scand. 2010 mar;68(2):115-21. doi: 10.3109/00016350903514848. 5. eick s, straube a, guentsch a, pfister w, jentsch h. comparison of real-time polymerase chain reaction and dna-strip technology in microbiological evaluation of periodontitis treatment. diag microbiol infections dis. 2011 jan;69(1):12-20. doi: 10.1016/j. diagmicrobio.2010.08.017. 6. jervøe-storm pm, koltzscher m, falk w, dörfler a, jepsen s. comparison of culture and real-time pcr for detection and quantification of five putative periodontopathogenic bacteria in subgingival plaque samples. j clin periodontol. 2005 jul;32(7):778-83. 7. bolerázska b, mareková m, markovská n. trends in laboratory diagnostic methods in periodontology. acta medica (hradec kralove). 2016;59(1):3-9. doi: 10.14712/18059694.2016.47. 8. savitt ed, strzempko mn, vaccaro kk, peros wj, french ck. comparison of cultural methods and dna probe analyses for the detection of actinobacillus actinomycetemcomitans, bacteroides gingivalis, and bacteroides intermedius in subgingival plaque samples. j periodontol. 1988 jul;59(7):431-8. 9. feng x, zhang l, xu l, meng h, lu r, chen z, et al. detection of eight 171 comparison of two different methods for detecting periodontal pathogenic bacteria braz j oral sci. 15(3):166-172 172 comparison of two different methods for detecting periodontal pathogenic bacteria periodontal microorganisms and distribution of porphyromonas gingivalis fima genotypes in chinese patients with aggressive periodontitis. j periodontol. 2014 jan;85(1):150-9. doi: 10.1902/jop.2013.120677. 10. mullis k. the unusual origin of the polymerase chain reaction. sci am. 1990 apr;262(4):56-61, 64-5. 11. decat e, cosyn j, de bruyn h, miremadi r, saerens b, van mechelen e, et al. optimization of quantitative polymerase chain reactions for detection and quantification of eight periodontal bacterial pathogens. bmc res notes. 2012 dec 2;5:664. doi: 10.1186/1756-0500-5-664. 12. marín mj, figuero e, gonzález i, o'connor a, diz p, álvarez m, et al. comparison of the detection of periodontal pathogens in bacteraemia after tooth brushing by culture and molecular techniques. med oral patol oral cir bucal. 2016 may 1;21(3):e276-84. 13. guglielmetti mr, rosa ef, lourenção ds, inoue g, gomes ef, de micheli g, et al. detection and quantification of periodontal pathogens in smokers and never-smokers with chronic periodontitis by real-time polymerase chain reaction. j periodontol. 2014 oct;85(10):1450-7. doi: 10.1902/jop.2014.140048. 14. rudney jd, chen r, pan y. endpoint quantitative pcr assays for bacteroides forsythus, porphyromonas gingivalis and actinobacillus actinomycetemcomitans. j periodontal res. 2003 oct;38(5):465-70. 15. tellapragada c, eshwara vk, acharya s, bhat p, kamath a, vishwanath s, et al. prevalence of clinical periodontitis and putative periodontal pathogens among south indian pregnant women. int j microbiol. 2014; 2014:420149. doi: 10.1155/2014/420149. 16. boutaga k, van winkelhoff aj, vandenbroucke-grauls cm, savelkoul ph. periodontal pathogens: a quantitative comparison of anaerobic culture and real-time pcr. fems immunol med microbiol. 2005 aug;45(2):1919. 17. umeda je, missailidis c, longo pl, anzai d, wikström m, mayer mp. adhesion and invasion to epithelial cells by fima genotypes of porphyromonas gingivalis. oral microbiol immunol. 2006 dec;21(6):4159. 18. ashimoto a, chen c, bakker i, slots j. polymerase chain reaction detection of 8 putative periodontal pathogens in subgingival plaque of gingivitis and advanced periodontitis lesions. oral microbiol immunol. 1996 aug;11(4):266-73. 19. persson gr, hitti j, paul k, hirschi r, weibel m, rothen m, et al. tannerella forsythia and pseudomonas aeruginosa in subgingival bacterial samples from parous women. j periodontol. 2008 mar;79(3):508-16. doi: 10.1902/ jop.2008.070350. 20. lages ej, costa fo, cortelli sc, cortelli jr, cota lo, cyrino rm, et al. alcohol consumption and periodontitis: quantification of periodontal pathogens and cytokines. j periodontol. 2015 sep;86(9):1058-68. doi: 10.1902/jop.2015.150087. 21. nonnenmacher c, dalpke a, rochon j, flores-de-jacoby l, mutters r, heeg k. real-time polymerase chain reaction for detection and quantification of bacteria in periodontal patients. j periodontol. 2005 sep;76(9):1542-9. 22. martin ef, nadkarni ma, jacques na, hunter n. quantitative microbiological study of human carious dentine by culture and real-time pcr: association of anaerobes with histopathological changes in chronic pulpitis. j clin microbiol. 2002 may;40(5):1698-704. 23. shaddox lm, walker c. microbial testing in periodontics: value, limitations and future directions. periodontol 2000. 2009;50:25-38. doi: 10.1111/j.1600-0757.2008.00285.x. 24. döring g, unertl k, heininger a. validation criteria for nucleic acid amplification techniques for bacterial infections. clin chem labor med. 2008;46(7):909-18. doi: 10.1515/cclm.2008.152. 25. higuchi r, dollinger g, walsh ps, griffith r. simultaneous amplification and detection of specific dna sequences. biotechnology (n y). 1992 apr;10(4):413-7. braz j oral sci. 15(3):166-172 original article braz j oral sci. january/march 2009 volume 8, number 1 relationship between orofacial pain and absenteeism among workers in southern brazil anderson nardi1, edgard michel-crosato2, maria gabriela haye biazevic3, edgard crosato4, eduardo pizzatto5, dagmar de paula queluz6 1ms, universidade do oeste de santa catarina (unoesc), joaçaba (sc), brazil 2 dds, ms, phd, assistant professor, department of community dentistry, faculdade de odontologia, universidade de são paulo (usp), são paulo (sp), brazil 3dds, ms, phd, post-doctored student, department of community dentistry, faculdade de odontologia, usp, são paulo (sp), brazil 4 dds, ms, phd, professor, department of community dentistry, faculdade de odontologia, usp, são paulo (sp), brazil 5dds, msph, phd, professor, faculdade de odontologia, universidade positivo, curitiba (pr), brazil 6 dds, msph, phd, professor, department of community dentistry, faculdade de odontologia de piracicaba, universidade estadual de campinas (unicamp), campinas (sp), brazil. received for publication: july, 17, 2008 accepted: february 04, 2009 correspondence to: edgard michel crosato departamento de odontologia social da faculdade de odontologia da usp avenida professor lineu prestes, 2.227 – cidade universitária cep 05508-000 – são paulo (sp), brazil e-mail: michelcrosato@usp.br abstract aim: to verify the relationship between orofacial pain and absenteeism in workers of slaughter and meat processing industries in the southern region of brazil. methods: a cross-sectional study, with the random sample of 401 workers of slaughter and meat processing industries in the southern region of brazil, was carried out. a questionnaire referred to the situation of absenteeism caused by nine different types of orofacial pain and also the amount of time the employee was kept from work. results: only 60 workers (15%) reported having missed work due to orofacial pain in the six months prior to the study. the prevalence of absenteeism resulting from orofacial pain was of 15%. the types of orofacial pain that resulted in absenteeism were: spontaneous toothache (9.7 %); toothache caused by cold or hot liquids or by sweet foodstuff (6.5%) and pain around and behind the eyes (3.2%). there was a predominance of absenteeism in half and full work shifts for the types of orofacial pain experienced. associations between absenteeism from induced toothache and gender (p < 0.05), absenteeism and spontaneous toothache and family income (p = 0.011), and between absenteeism and the self awareness of their oral health condition, as well as the nine types of orofacial pain (p < 0.001) were observed. conclusions: the prevalence of absenteeism as a result of orofacial pain was low. keywords: absenteeism, facial pain, facial pain/epidemiology, oral health. introduction absenteeism is an issue of growing interest as a result of the economic importance of competitiveness, driving companies to seek means to reduce its occurrence and, consequently, to increase the profitability and achieve sustained growth1,2. various epidemiological studies have demonstrated that the prevalence of absenteeism resulting from dental reasons varies from 10 to 35%, and the average number of working hours lost varies from 1.24 to 6.20 working hours/workers/years3-9. the pain is a private percept that arises in a conscious brain, typically in response to a noxious provoking stimulus, but, sometimes, in the absence of a stimulus. the relation of the percept to the stimulus is variable, and depends on the individual’s prior expectations and beliefs, and on his/her cognitive and emotional state – not just on the nature of the stimulus itself. while acute pain is, by definition, a brief and self-limiting process, chronic pain comes to dominate the life and concerns of the patient, and often also family, friends and other caregivers. in addition to the severe erosion in quality of life of the pain sufferer and those around 51relationship between orofacial pain and absenteeism among workers in southern brazil braz j oral sci. 8(1): 50-4 him/her, chronic pain imposes severe financial burdens on many levels. these include: costs of healthcare services and medication, job absenteeism and disruption in the workplace, loss of income, non-productivity in the economy and in the home, financial burden on family, friends and employers, worker compensation costs and welfare payments. the workers can reduce absenteeism and healthcare utilization from the risks from dental disease by incorporating dental education into workplace wellness. the aim of this study was to verify the relationship between orofacial pain and absenteeism in workers of slaughter and meat processing industries in the southern region of brazil. material and methods the study population was composed of a sample of 401 workers of slaughter and meat processing industries in the southern region of brazil. the company workforce was composed of 1,187 employees. sample size calculation was done using the following criteria: confidence level 95%, sample mistake 4% and unknown prevalence, and estimated prevalence 50%. the selection process of the samples was realized through simple random drawings. an observational and cross-sectional study design was used. data collected referred to absenteeism resulting from orofacial pain, as well as social and economic characteristics of the study population (gender, age, marital status, area of work, address, educational level and family income). a questionnaire developed by locker & grushka6-7 was used as an instrument for the survey, which was validated in brazil in the bambuí health and ageing study (bhsa)10. the questions in the survey instrument referred to the situation of absenteeism caused by nine different types of orofacial pain and also the amount of time the employee was kept from work. the questionnaire was prepared to gather information about orofacial pain and absenteeism at present or in the recent past. all questions referred to the period encompassing the six months prior to the survey. this relatively short period of time is normally used to minimize bias due to possible lapse of memory of the surveyed study population11,12. about the family income, low means until two minimum wage, and high means two or more minimum wages. the oral health, it was self related good or poor. the questionnaire was first applied to 15 employees. thereafter, it was applied by duly trained surveyors to the employees that agreed to be enrolled as volunteers after granting authorization from the company management and after the participants had signed an informed consent form to take part in the study. the research project was approved by the research ethics committee of the universidade de passo fundo (upf) under the protocol number 194/03. data were analyzed by the chi-square test using the stata 8.0 software and presented in tables, according to the frequency distribution. a 5% significance level was adopted for all analyses. results the social and economic status of the studied population is shown on table 1. among the participants in the study, 60 employees declared having lost working hours due to orofacial pain in the six months prior to the survey. consequently, the prevalence of absenteeism due to orofacial pain in this study was of 15%. the types of orofacial pain that cause more absenteeism were: spontaneous toothache (9.7%); toothache caused by cold or hot liquids or sweet foodstuff (6.5%) and pain around and behind the eyes (3.2%). table 2 shows the percentage of loss of working hours for each of the nine types of orofacial pain described. the predominance of loss of half and full work shifts was observed for all types of orofacial pain experienced. a half work shift was considered as a period of four hours, in other words, half a day’s work. the types of orofacial pain that caused two and three days or more of absence from work were the spontaneous toothache, toothache caused by hot or cold liquids or sweet foodstuff, pain in front of the ears and pain around and behind the eyes. considering the prevalence of absenteeism according to the number of types of pain experienced by workers, 26 employees informed having been absent from work due to one type of orofacial pain (6.5%), 18 employees informed having been absent as a result of two types of orofacial pain (4.5%), nine employees informed having missed work as a result of feeling more than three types of orofacial pain and only one worker informed having missed working days as a result of feeling the nine types of orofacial pain (0.2%) in the last six months (table 2). the association between absenteeism and gender indicated that the prevalence of absenteeism resulting from toothache caused by hot or cold liquids or sweet foodstuff (p = 0.043) was higher for male workers (table 3). variable category n % gender male 285 71.1 female 116 28.9 age 18 to 21 91 22.7 22 to 31 191 47.6 32 to 41 99 24.7 42 to 51 20 5.0 marital status single 178 44.4 married 223 55.6 education level cannot read or write 1 0.2 incomplete elementary school 75 18.7 complete elementary school 78 19.5 incomplete high school 73 18.2 complete high school 142 35.4 college education 32 8.0 family income from 1 to 2 minimum wages 180 44.9 from 3 to 5 minimum wages 182 45.4 from 6 to 10 minimum wages 33 8.2 from 11 to 20 minimum wages 6 1.5 table 1. percentage of gender, age, marital status, education level, family income among workers of a meat processing industry. joaçaba (sc), brazil, 2003 52 nardi a, michel-crosato e, biazevic mgh, crosato e, pizzatto e, queluz dp braz j oral sci. 8(1): 50-4 table 4 shows that the prevalence of absenteeism resulting from spontaneous toothache was higher among workers with the worst level of family income (p = 0.011). an association was also observed between absenteeism resulting from orofacial pain and self awareness of oral health (p < 0.001). employees who declared having poor oral health conditions presented a higher prevalence of absenteeism resulting from orofacial pain, in the nine types of orofacial pain studied, than those who declared having a good oral health status (table 5). discussion the prevalence of absenteeism due to orofacial pain for the workers of the meat processing industry was of 15%, and the average number of lost working hours as a result of dental causes was of 0.88 working hours/employee/year. when comparing the results of this study to those of various epidemiological studies3-9, it was noted that the prevalence of absenteeism for dental reasons in the study population matched the previously found values. however, the average number of working hours lost by the employees of the meat processing industry was below those results previously presented. the orofacial pains that caused the highest levels of absenteeism were: spontaneous toothache (9.7%); toothache caused by cold or hot liquids or sweet foodstuff (6.5%) and pain around and behind the eyes (3.2%). these pains have the characteristic of acute cases with a relatively short duration and that can be rapidly treated, reestablishing the workers’ capabilities to perform their functions and enabling their prompt return to their jobs. considering the amount of time absent from work as a result of orofacial pain, there was a predominance for the loss of half or a full work shift as shown in table 2. the average number of leave-of-absence days due to dentist certificates was of 1.5 days/employee/year. these results are consistent with those presented in previous studies11-14. absenteeism caused by dental reasons in this meat processing industry was smaller than absenteeism resulting from medical causes in both average length and total number of leaves of absence, being these data similar to the survey by reisine15. employees who declared having poor oral health status presented a higher prevalence of absenteeism due to orofacial pain than those who declared having a good oral health status. in this sense, type of orofacial pain no absence (%) absent on half a shift (%) absent for a full shift (%) absent for 1 day (%) absent for 2 days (%) total (%) spontaneous toothache 90.7 5.2 1.4 2.2 0.5 100.0 induced toothache 93.5 3.7 1.4 1.2 0.2 100.0 burning sensation on the tongue 98.8 0.5 0.2 0.5 0.0 100.0 tmj pain 97.5 1.2 0.8 0.5 0.0 100.0 pain on chewing 98.0 1.0 0.5 0.5 0.0 100.0 pain on opening the mouth 99.0 0.5 0.3 0.2 0.0 100.0 pain in front of the ears 97.5 1.5 0.0 0.5 0.5 100.0 pain in the face or cheeks 97.3 1.5 1.0 0.2 0.0 100.0 pain around or behind the eyes 97.0 1.3 1.4 0.3 0.0 100.0 table 2. percentage of working hours lost for each type of orofacial pain among workers of a meat processing industry. joaçaba (sc), brazil, 2003 causes of absenteeism gender odds ratio confidence interval (95%) p male female spontaneous toothache no 253 109 1.970 0.843 4.599 0.111 yes 32 7 induced toothache no 262 113 3.307 0.973 11.236 0.043 yes 23 3 burning sensation on the tongue no 281 115 1.637 0.181 14.804 0.658 yes 4 1 tmj pain no 278 113 0.948 0.241 3.733 0.940 yes 7 3 pain on chewing no 279 114 1.226 0.244 6.164 0.805 yes 6 2 pain on opening the mouth no 283 114 0.403 0.056 2.894 0.350 yes 2 2 pain in front of the ears no 280 111 0.396 0.113 1.396 0.137 yes 5 5 pain in the face or cheeks no 277 113 1.088 0.283 4.175 0.902 yes 8 3 pain around or behind the eyes no 275 113 1.370 0.370 5.070 0.636 yes 10 3 table 3. association between absenteeism resulting from facial pain and gender among the workers of a meat processing industry. joaçaba (sc), brazil, 2003 53relationship between orofacial pain and absenteeism among workers in southern brazil braz j oral sci. 8(1): 50-4 causes of absenteeism income odds ratio confidence interval (95%) p low high spontaneous toothache no 155 207 2.385 1.200 4.738 0.011 yes 25 14 induced toothache no 166 209 1.469 0.662 3.261 0.342 yes 14 12 burning sensation on the tongue no 177 219 1.856 0.307 11.229 0.494 yes 3 2 tmj pain no 174 217 1.871 0.520 6.733 0.331 yes 6 4 pain on chewing no 176 217 1.233 0.304 5.000 0.769 yes 4 4 pain on opening the mouth no 178 219 1.230 0.172 8.822 0.836 yes 2 2 pain in front of the ears no 174 217 1.871 0.520 6.733 0.331 yes 6 4 pain in the face or cheeks no 174 216 1.490 0.447 4.963 0.514 yes 6 5 pain around or behind the eyes no 175 213 0.761 0.244 2.367 0.636 yes 5 8 table 4. association between absenteeism from orofacial pain and family income among the workers of a meat processing industry. joaçaba (sc), brazil , 2003 causes of absenteeism oral health odds ratio confidence interval (95%) p good poor spontaneous toothache no 37 325 6.787 3.309 13.923 0.000 yes 17 22 induced toothache no 39 336 11.748 5.043 27.370 0.000 yes 15 11 burning sensation on the tongue no 50 346 27.680 3.033 252.646 0.000 yes 4 1 tmj pain no 47 344 17.078 4.269 68.321 0.000 yes 7 3 pain on chewing no 48 345 21.563 4.231 109.892 0.000 yes 6 2 pain on opening the mouth no 50 347 7.940 6.127 10.289 0.000 yes 4 0 pain in front of the ears no 49 342 6.980 1.950 24.984 0.001 yes 5 5 pain in the face or cheeks no 49 341 5.799 1.705 19.722 0.002 yes 5 6 pain around or behind the eyes no 45 343 17.150 5.073 57.980 0.000 yes 9 4 tabela 5. association between absenteeism from orofacial pain and self-awareness of oral health among workers of a meat processing industry. joaçaba (sc), brazil, 2003 self-awareness of the oral health status coincided with the impact observed through the application of the instruments: work-related activities are more severely affected among employees who presented the perception of their poor oral health condition. this study did not demonstrate any associations between prevalence of absenteeism due to orofacial pain and age, marital status, schooling, geographical area (rural or urban), company sector or work shift. there are two types of absenteeism: absenteeism through the absence of work and physically present absenteeism. the first type can be measured and its costs can be calculated through the absence. the second type cannot be measured, since it represents the worker who cannot perform his/her normal working activities due to pain, despite being physically present to the workspace13. labor is increasingly becoming effective and instrumental in the social-economical advancements in our society14. the attention of the authorities responsible for the implementation of the directives of a country that has its economy based on labor should be oriented towards the health and welfare. consequently, workers should be the objective of measures and policies to preserve their physical, mental and social well-being. therefore, maximum productive capacity is achieved when the worker is satisfied in his basic health needs11-16. 54 nardi a, michel-crosato e, biazevic mgh, crosato e, pizzatto e, queluz dp braz j oral sci. 8(1): 50-4 studies have shown that oral problems caused difficulties or incapacity to perform normal working activities, study or sleep in a percentage that ranges from 8 to 60%16-18. in the present study, associations between absenteeism from induced toothache and gender (p = 0.05); absenteeism, spontaneous toothache and family income (p = 0.011); and absenteeism, selfawareness of the oral health condition and the nine types of orofacial pain (p = 0.00) were observed. the prevalence of absenteeism as a result of orofacial pain was low. it is important to point out that the methodological aspects of future epidemiological studies about orofacial pain and absenteeism must be standardized, in such a way that the results of the various studies could be compared with greater reliability. since absenteeism for dental reasons was not pronounced, the company did not present losses in productivity because their work force contemplates a surplus percentage of employees to compensate for absences. references 1. gift hc, reisine st, larach dc. the social impact of dental problems and visits. am j public health. 1992;82:1663-8. 2. berndt er, bailit hl, keller mb, verner jc, finkelstein sn. health care use and at-work productivity among employees with mental disorders. health aff. 2000;19:244-56. 3. midorikawa et. odontology in worker’s health as a new professional specialty: definition of the activity field and functions of the surgeon dentist in worker’s health team. [doctoral tesis]. são paulo: faculdade de odontologia of universidade de são paulo; 2000. 337p. 4. hollister mc, weintraub ja. the association of oral status with systemic health, quality of life, and economic productivity. j dent educ. 1993;57:901-12. 5. schou l. oral health promotion at worksites. int dent j. 1989;39:122-8. 6. locker d, grushka m. the impact of dental and facial pain. j dent res. 1987;66:1414-7. 7. locker d, grushka m. prevalence of oral and facial pain and discomfort: preliminary results of a mail survey. community dent oral epidemiol. 1987;15:169-72. 8. hooper ha. dental services in industry: observations on their effects in the reduction on absenteeism. industrial medicine. 1942;11:157-62. 9. bailit h, beazoglou t, hoffman w. work loss and dental disease. report to the robert wood johnson foundation. university of connecticut health center; 1982. 10. matos dl, lima costa mf, guerra hl, marcenes w. projeto bambuí: avaliação de serviços odontológicos privados, públicos e de sindicato. rev saúde pública. 2002;36:237-43. 11. macfarlane tv, blinkhorn as, davies rm, kincey j, worthington hv. orofacial pain in the community: prevalence and associated impact. community dent oral epidemiol. 2002;30:52-60. 12. reisine st, miller j. a longitudinal study of work loss related to dental diseases. soc sci med. 1995;21(12):1309-14. 13. jaafar n, razak ia, zain rb. the social impact of oral and facial pain in an industrial population. ann acad med. 1989;18:553-5. 14. naito m, yuasa h, nomura y, nakayama t, hamajima n, hanada n. oral health status and health-related quality of life: a systematic review. j oral sci. 2006;48:1-7. 15. reisine st. dental disease and work loss. j dent res. 1984;63:1158-61. 16. reisine st. dental health and public policy: the social impact of dental disease. am j public health. 1985;75:27-30. 17. reisine st. the impact of dental conditions on social functioning and the quality of life. annu rev public health. 1988;9:1-19. 18. johnson nw, glick m, mbuguye tn. oral health and general health. adv dent res. 2006;19:118-21. original article braz j oral sci. january | march 2016 volume 15, number 1 does social vulnerability for caries predict caries status of children in sub-urban nigeria? morenike oluwatoyin folayan1, olujide olusesan arije2 1obafemi awolowo university, department of child dental health, ile-ife, nigeria 2obafemi awolowo university teaching hospitals complex, department of community health, ile-ife, nigeria correspondence to: morenike oluwatoyin folayan department of child dental health, obafemi awolowo university, ile-ife, nigeria phone: +234 706 2920 394 e-mail: toyinukpong@yahoo.co.uk abstract it is important to identify groups of people vulnerable to a disease condition. aim: to determine the association between social vulnerability to caries and caries status of children in ile-ife, nigeria. methods: a composite vulnerability index for caries was developed using data generated for 992 children. wilks’ lambda test to verify relationship between vulnerability and its variables. logistic regression analysis was conducted to determine if the social vulnerability for caries index was a good predictor for caries status. results: the social vulnerability to caries index could not predict caries status. the study found that sex, age and number of siblings were the significant predictors of caries status in the study population. females (aor: 1.63; 95%ci: 1.08 – 2.46; p=0.02) and children with more than two siblings had higher odds of having caries (aor: 2.61; 95%ci: 1.61 – 4.24; p<0.001) while children below 5 years had lower odds of having caries (aor: 0.62; 95%ci: 0.39 – 1.00; p=0.05) conclusions: the social vulnerability index for caries could not predict the caries status of children in the study population. sensitive tools to identify children with caries in the study population should be developed. keywords: social vulnerability. indexes. dental caries. child. nigeria. introduction vulnerability is by definition a measure of possible future harm1. it is the ability to anticipate, resist, cope with and respond to a hazard2 and the intrinsic predisposition of an individual or a collective to be susceptible to damage when exposed to a hazard3. it has been studied in the context of physical and social vulnerabilities. physical vulnerability takes into consideration the risk of exposure to natural disasters while social vulnerability studies the impact of social inequalities4,5. social vulnerability is a multifaceted concept often defined as the entirety of the social deficits faced by patients, including social and environmental inequalities and deprivation, which affect their social cohesion and capacity to respond to situations of social risk6. it is associated with the health/disease process and encompasses various individual dimensions linked to exposure to risk factors and threats6,7. it includes consideration of access to food, public health services and the ability to actively respond to risks8. understanding social vulnerability helps to identify and understand which group of people may be more sensitive and susceptible to a disease condition and why9. understanding this will help governments respond appropriately to the needs of vulnerable populations by promoting targeted interventions and strategies, and increase future social capacity and resilience9. vulnerability increases risk for diseases and other stressors for individuals who live especially in conditions of poverty. poverty reduces people’s ability to cope http://dx.doi.org/10.20396/bjos.v15i1.8647130 received for publication: may 23, 2016 accepted: august 22, 2016 braz j oral sci. 15(1):79-85 80 with, recover from, or adapt to external stresses that affect their livelihood and well-being10. data on social vulnerability assessments are the guide for government and international agencies in efforts to reduce individual and community vulnerability to disease and other stressors, and to strengthen the coping mechanisms10. methods and indicators for assessing vulnerability vary depending on the research or policy context8. several indices to characterize social vulnerability to various issues at different spatial scales were developed over the past decade11-13. some of the variables in indices used for measuring social vulnerability include residential area14 socioeconomic status5, educational status15 and financial status16. these same measures had been used over the years to assess the vulnerability of individuals and communities to diseases, as they reflect how the material reality of life increases the predisposition to poor nutrition17 and serves as barrier to access health services, due to the cost of medical care and distance to facilities13,18, among other things. numerous studies that examined the associations between socio-demographic, economic and individual factors such as race19, ethnicity20, socioeconomic status21, oral health literacy22 and compliance23, and oral health outcomes. the outcomes of these measurements are complex with many of the studies conducted in developed countries. the studies on vulnerability to health has a relatively brief history in lowand middle-income countries. publication of the findings of such research just started to appear in the late 20th century3. studies on social vulnerability to oral health are less common, especially from developing nations like nigeria. the few direct studies on social vulnerability to oral diseases explore social vulnerability of adults to oral diseases14,24 and fewer studies examined social vulnerability of children to oral diseases25-28. no studies were identified measuring the social vulnerability of children from an african country to oral diseases, when a pubmed and google scholar search was made for this study. in view of the need to determine the vulnerability of individuals and populations to diseases as an important public health response, this study aimed to develop an index for social vulnerability to caries for children in ile-ife, nigeria; and to determine the association between the social vulnerability to caries and the actual caries status of children in the study location. material and methods this was an analysis of secondary data generated by a household survey. part of the study was reported by kolawole et al.29 and folayan et al.30. the study recruited 992 children aged 1-12 years for assessing the association between digit-sucking and caries. data were obtained by a questionnaire administered by experienced field workers who had been engaged in past national surveys and trained on the study protocol. the interviewers collected all information from respondents and submitted to survey supervisors who reviewed the questionnaires. mothers were requested to respond on behalf of children below eight years, based on evidence that responses of mothers on questionnaires have a higher correlation with children’s response31. where the does social vulnerability for caries predict caries status of children in sub-urban nigeria? mother was unavailable, fathers completed the questionnaires. the socio-demographic characteristics were obtained for each child. information from the interview included number of siblings, age at last birthday, gender, socioeconomic status, maternal level of knowledge on caries prevention practices and number of meals eaten per day as proxy measure for wealth status32. study location: the study was conducted in ife central local government of ile-ife, a sub-urban town in southwestern nigeria. according to the 2004 national population census, the population of the local government area (lga) was 138,818, with about 14,000 (10%) children among them. the study site also hosted oral health clinics, thereby making it possible to refer for oral health care pupils screened with lesions. sample size determination: the sample size was calculated using leslie fischer’s formula32 for study population >10,000. based on a prevalence of 34.1% of oral habits in children aged four to 15 years old, determined by quashie-williams et al.33, a sample size of 1,011 children was required to identify 345 children with oral habits, with a non-response rate of about 10%. sampling technique: the sampling procedure was a threelevel multi-stage cluster sampling aimed at selecting eligible persons with known probability. stage 1 involved the random selection of enumeration areas within the lga; at the sites, every third household on each street was chosen. stage 2 involved listing eligible individuals within households. stage 3 involved selection of actual respondents for interview. only children present in the house at the time of the study were eligible to participate in the study. in the chosen houses only one child was selected. details of this sampling technique were reported by kolawole et al.29 and folayan et al.30 in an earlier publication from this same database. clinical examination: oral examination was conducted at the homes of all participants to determine the presence of caries and oral hygiene status. the children were examined seated, under natural light, using sterile dental mirrors and probes by trained dentists; radiographs were not used in the study. the teeth were examined wet and debris removed with gauze where present. diagnosis of caries: caries was diagnosed using the world health oral health survey recommendations34. each tooth was examined for dental caries using a plane mouth mirror, using natural light while the child was seated on a chair. caries status was assessed using the decayed missing and filled (dmft/dmft) index. a decayed (d/d) tooth was defined as any which crown had an unmistakable cavitation on pits or fissures, or on a tooth surface or a filled crown with decay, when it had one or more permanent restorations that were decayed. the f/f was defined as a filled crown with no decay, when it had one or more permanent restorations, and there were no caries anywhere on the crown. the m/m was defined as a tooth missing due to caries; when a tooth had been extracted due to caries. to arrive at a dmft/dmft score for an individual patient’s mouth, three values had to be determined: the number of teeth with carious lesions, the number of extracted teeth due to caries and the number of teeth with fillings or crowns35. the number of teeth was then summed to give the dmft/dmft score for the permanent dentition. for the purpose of analysis, caries status was further divided into present or absent caries. social vulnerability to caries scale: this study adapted the method by tirapanildos et al.36 to develop a scale that assessed braz j oral sci. 15(1):79-85 81does social vulnerability for caries predict caries status of children in sub-urban nigeria? the social vulnerability of study participants to caries. social vulnerability to caries was defined according to the results of factor analysis, cluster analysis, and discriminant analysis conducted using individual and social caries-predictive variables for the study participants in the study area from six variables. the six candidate variables for the vulnerability scale were gender of the child, maternal knowledge of caries prevention, socioeconomic category of the child, child’s age, number of siblings the child had and the number of meals the child had per day. these variables were the social factors associated with the risk of caries in children in the study by prior studies. they include child’s age, sex, socioeconomic status, number of siblings and maternal knowledge of oral health: younger children28,29, women28-30, those with lower socioeconomic status30, children with mothers who had poor knowledge of oral health30, and children with more siblings37 had higher risk for caries. we also included number of meals per day as a proxy measure for poverty38 in view of evidences that showed a strong association between poverty and health outcomes39,40. a score for maternal knowledge of oral health was created by summing up 8 items that assessed maternal knowledge. the questions asked if the respondents agreed or disagreed with the following statements: “fluoridation of drinking water is an effective, safe and efficient way to prevent holes from forming on the teeth’; ‘use of fluoride-containing toothpaste is an effective, safe and efficient way to prevent holes from forming on the teeth’; ‘the number of times you eat sugar containing food has a great role in producing holes in the teeth’; ‘fissure sealant is effective in the prevention of holes developing in newly erupted molars’; ‘rinsing teeth with a lower amount of water after tooth-brushing reduces the risk of caries’, ‘using fluoride toothpaste is more important than the brushing per se for preventing holes from forming on the teeth’, ‘brushing twice daily with fluoride containing toothpaste is effective for preventing holes from developing in the teeth’; and ‘it is important to visit the dental clinic regularly as a measure for preventing holes from forming in the teeth’. the ensuing score was dichotomized into poor and good knowledge using k means cluster analysis. a correlation matrix of the six candidate variables for the vulnerability scale showed that the highest correlation coefficient between any pair of variables was 0.317, between number of siblings and child’s age group; this correlation was statistically significant (p<0.001). therefore, the variable for number of siblings was dropped from the scale because the age of the child was considered a more important variable for assessing social vulnerability to caries. the five remaining items for the social vulnerability to caries scale were subjected to factor analysis. assessment of suitability of the data for factor analysis showed that the kaiser-meyer-oklin value was 0.51, exceeding the recommended value of 0.5 and the barlett’s test of sphericity41 reached statistical significance, supporting the factorability of the correlation matrix. factor analysis using principal components showed the five items could be grouped into two factors with eigen-value exceeding 1 and explaining 23.1% and 22.1% of the variance respectively. it was decided to retain both factors (table 1). the five items were joined to give the social vulnerability to caries score and cluster analysis. the k means was used to create a binary variable for caries vulnerability dividing the score into vulnerable and non-vulnerable categories. discriminant analysis was used to validate the grouping created by k means cluster analysis. a model that allowed for classification of all patients into vulnerable and non-vulnerable groups was developed and validated using the wilks’ lambda test. the test indicated that the model was appropriate for the study population: among 992 children, 100% were classified correctly by the proposed model, indicating that the proposed allocation process would ensure correct classification (tables 2 and 3). validation of factor analysis % of variability kmo 0.51 23.1 bartlett test 28.85 22.1 p-value <0.001 factor loadings (rotated)* factor 1 factor 2 individual factor household factor socio economic status 0.61 child’s age group 0.61 child’s gender -0.59 average number of meals per day 0.73 maternal knowledge of caries 0.69 table 1 results of factor analysis. *considered values >+0.40 or <-0.40 for better understanding group number percentage vulnerable 684 70.7% non-vulnerable 283 29.3% total 967 100.0% table 3 results of discriminant analysis using data of 992 study participants in ile-ife. group number percentage percentage valid vulnerable 684 69.0% 70.7% non-vulnerable 283 28.5% 29.3% omitted from the study 25 2.5% total 992 100.0 100.0 table 2 results of cluster analysis using data of 992 study participants in ile-ife. standardization of clinical examiners: the clinical investigators were qualified dentists undergoing postgraduate residency training as paedodontists or orthodontists who were calibrated on the study protocol and the who criteria for caries diagnosis, including the dmft/dmft index and ohi-s. training was followed by practice on patients; each investigator examined and scored children for oral lesions as prescribed in the study protocol. results were subjected to a cohen’s weighted kappa score analysis to determine intraand inter-examiner variability. the intra-examiner variability ranged between 0.89 0.94, while inter-examiner variability ranged between 0.82 – 0.90 for caries detection. data analysis: the profile of study participants was reported by vulnerability classification. bivariate analysis was conducted to braz j oral sci. 15(1):79-85 82 test the association between dependent variables (presence of caries and severity of caries measured by the dmft/dmft score) and the independent variables (socially vulnerable and non-vulnerable groups for caries). where appropriate, chi square tests were conducted. multivariate logistic regression was used for inferential analysis. the logistic regression model included only variables whose p values were <0.4 and entered into the subsequent models. the estimated coefficients were expressed as odds ratios (ors) and their 95% confidence intervals were also calculated. statistical analysis was conducted with stata software (version 11) for the logistic regression. statistical significance was fixed at p<0.05. ethical considerations: ethical approval was obtained from the ethics and research committee of the obafemi awolowo university teaching hospital complex ile-ife (erc/2013/07/14). approval to conduct the study was obtained from the local government authority. the study was conducted in full compliance with the study protocol. written informed consent was obtained from the parents of study participants after duly explaining study objectives, risks and benefits, voluntary nature of participation and freedom to withdraw at any time. all children aged eight to 12 years also provided written assent. efforts to minimize risks such as loss of confidentiality and discomfort to participants were made. all data were collected without the identifier (names and addresses) of participants. participants experienced no direct benefit and no compensation was paid, however they were given token gifts of stationery or a small tube of toothpaste. none of the gifts exceeded $0.50. results table 4 shows the outcome of the association between social vulnerability, the dmft/dmft and presence of caries. there was no significant association found between the social vulnerability for caries and the dmft (p=0.64), the dmft (p=0.78), a combination of dmft and dmft (p=0.28) and the dental caries status (p=0.53). does social vulnerability for caries predict caries status of children in sub-urban nigeria? variables social vulnerability chi square test fisher’s exact non-vulnerable (n=684) vulnerable (n=283) freq % freq % dmft p = 0.64 0 611 89.3 258 91.2 1 31 4.5 8 2.8 2 27 3.9 8 2.8 3 6 0.9 4 1.4 4 3 0.4 2 0.7 5 2 0.3 2 0.7 6 1 0.1 1 0.4 8 2 0.3 0 0.0 9 1 0.1 0 0.0 dmft p = 0.78 0 666 97.4 276 97.5 1 11 1.6 4 1.4 2 4 0.6 1 0.4 3 0 0.0 1 0.4 4 2 0.3 1 0.4 9 1 0.1 0 0.0 dmft + dmft p = 0.28 0 602 88.0 253 89.4 1 33 4.8 11 3.9 2 30 4.4 7 2.5 3 8 1.2 4 1.4 4 4 0.6 5 1.8 5 2 0.3 2 0.7 6 0 0.0 1 0.4 7 1 0.1 0 0.0 8 3 0.4 0 0.0 18 1 0.1 0 0.0 dental caries p = 0.53 no caries 602 88.0 253 89.4 in primary dentition 64 9.4 23 8.1 in permanent dentition 10 1.5 6 2.1 in primary and permanent dentition 8 1.2 1 0.4 table 4 social vulnerability, dmft, dmft and presence of caries. braz j oral sci. 15(1):79-85 table 5 shows the predictors of caries status. with simple logistic regression analysis, the social vulnerability to caries status of the child was not a significant predictor of caries status. rather, children classified as socially vulnerable to caries had insignificantly lower odds of having caries than children classified as socially vulnerable to caries. (or: 0.87; 95% ci: 0.55 – 1.36; p=0.54). the significant predictors of caries status were sex, number of siblings and age: women had significantly higher odds of having caries than men (or: 1.70; 95% ci: 1.14 – 2.52; p=0.009); children with more than two siblings had higher odds of having caries than children with 2 or less siblings (or: 3.05; 95% ci: 1.93 – 4.83; p<0.001); and children under 5 years of age had significantly higher odds of having caries than children 5 years and older (or: 0.43; 95% ci: 0.27 – 0.67; p<0.001). following adjustment for confounders, the three significant predictors of presence of caries were sex, age and number of siblings. women had significantly higher odds of having caries than men (aor: 1.63; 95% ci: 1.08 – 2.46; p=0.02). children under the age of 5 were less vulnerable to caries than children five years and above (aor: 0.62; 95% ci: 0.39 – 1.00; p=0.05); and children with more than two sibling children had higher odds of having caries than children with two or less siblings (aor: 2.61; 95% ci: 1.61 – 4.24; p<0.001). discussion the study showed that the developed social vulnerability index for caries was not a sensitive determinant of caries status for this study population. rather, the study found that gender, age of the child and the number of siblings were the three social variables predictive of caries status of children in the study population. these findings have a few implications. first, our findings may imply that the social vulnerability of each child is not a determinant of its caries status in the study population. rufai et al.42 also noted that exclusion of social vulnerability dimensions pertinent to specific hazards or the overrepresentation of weakly influential dimensions may lead to misleading conclusions about social vulnerability. conscientious effort was made to develop a social vulnerability index for caries that included social vulnerability dimensions identified earlier by multiple studies as social risk factors for caries for individuals in the study population. the use of location-specific variables to construct a contextually specific social vulnerability index for caries in this study was based on the understanding that geographical and time-varying characteristics are important and essential for deconstructing vulnerability42-44. the developed index was found appropriate for the population, and the wilks’ lambda test indicated that the proposed allocation process would ensure correct classification. the development of a quantitative indicator to measure caries risk for this study population with low caries prevalence45 was important because a sensitive and specific index can facilitate the identification of populations most vulnerable to caries. this in-turn, can enhance political decision making processes about resource allocation and project prioritization especially in a limited resource setting like nigeria. this study found that the developed social vulnerability for caries index developed was not a sensitive and specific tool for determining caries status in the study population. 83does social vulnerability for caries predict caries status of children in sub-urban nigeria? variables caries absent caries present or p-value aor p-value frequency (%) frequency (%) gender male 462 (52.7) 46 (39.7) 1 1 female 414 (47.3) 70 (60.3) 1.70 (1.14 – 2.52) 0.009 1.63 (1.08 – 2.46) 0.02 maternal knowledge of caries prevention good 506 (57.8) 68 (58.6) 1 poor 370 (42.2) 48 (41.4) 0.96 (0.65 – 1.43) 0.86 socioeconomic status high 281 (32.1) 41 (35.7) 1 low/moderate 595 (67.9) 74 (64.3) 0.85 (0,57 – 1.28) 0.44 age ≥5 years 512 (58.4) 89 (76.7) 1 1 under 5 years 364 (41.6) 27 (23.3) 0.43 (0.27 – 0.67) <0.001 0.62 (0.39 – 1.00) 0.05 number of siblings 2 or less 414 (47.5) 26 (22.8) 1 1 more than 2 458 (52.5) 88 (77.2) 3.05 (1.93 – 4.83) <0.001 2.61 (1.61 – 4.24) <0.001 number of meals per day 2 or less 844 (98.7) 110 (97.3) 1 1 more than 2 11 (1.3) 3 (2.7) 2.09 (0.57 – 7.62) 0.26 2.49 (0.66 – 9.53) 0.18 social vulnerability to caries non vulnerable 602 (70.4) 82 (73.2) 1 vulnerable 253 (29.6) 30 (26.8) 0.87 (0.55 – 1.36) 0.54 table 5 frequency distribution and results of logistic regression analysis for predictors of caries status in a sample of 992 children. braz j oral sci. 15(1):79-85 the tool development process had a limitation: the variables in the index were allotted equal weights in the model. weights usually have an important impact on the composite indicator value and are based on statistical adequacy, social and economic significance among others46. however, weights are based on value judgment increasing the propensity to introduce bias and possibly affecting the comparison of data across countries46. unfortunately, while we had evidence to justify the variables derived for the construction of the composite index, we did not have the evidence required to make the needed value judgement to place weights on any of the components of the composite index. we intend to conduct further studies to identify how weights can be allotted to the composite variables in the developed index and use this information to refine the current index. we will then evaluate if the refined index for social vulnerability to caries can be more sensitive for assessing caries status in the study population. divaris47 also highlighted the limitations of attempting to transfer and apply population-derived risk estimates to individual risk assessment. he called this the privatization of risk and it can be misleading, as the risk factors may be insufficient to cause diseases at the individual level. rather, individual level caries risk assessment should be made at the tooth level. the findings of this study may be a validation of this postulation. the study outcomes may also be an indication that social conditions may be reflective of vulnerability in populations with high risk for caries or in highly heterogenic societies where race and ethnicity are factors for social inequity and vulnerability for diseases and health outcomes. the social environment in ile-ife is highly homogenous, with little disparity in race and ethnicity. this made it difficult to include ethnicity as a variable in the composite index developed to measure social vulnerability to caries. the study outcome may also reflect the limitations of using social vulnerability for assessing individual caries status in the study population. there are multiple efforts made to help increase the precision of dentists in taking decisions about the caries risk of individuals at their care. however, the use of a social vulnerability to caries index goes beyond being clinically applicable; the focus is to develop a quantitative assessment tool that can enhance the translation of oral health care research into policies and programs that benefit the general population. vulnerability assessment may help governments develop effective responses to the needs of vulnerable populations. the development of a social vulnerability to caries index can serve this purpose though we do acknowledge that translating social vulnerability processes into composite indicators can indeed be a complex endeavor. further research efforts are therefore required to help develop a social vulnerability index for children that can help facilitate government’s effort to prioritize its limited resources to respond to the children most vulnerable to caries. the three significant risk factors identified as independent predictors of caries status in this study were female children, children older than 5 years and children with more than two siblings. although these variables were included in the developed social vulnerability for caries index, the index was still not predictive of caries status although these factors were. further qualitative and quantitative studies are required to understand the role these three variables play in increasing the risk of children to caries in the 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int j adolesc med health. 2015 nov 10. pii: /j/ijamh. ahead-of-print/ijamh-2015-0059/ijamh-2015-0059.xml. doi: 10.1515/ ijamh-2015-0059. 39. raphael d. poverty in childhood and adverse health outcomes in adulthood. maturitas. 2011 may;69(1):22-6. doi: 10.1016/j. maturitas.2011.02.011. 40. wagstaff a. poverty and health sector inequalities. bull world health organ. 2002;80(2):97-105. 41. bartlett ms. a note on the multiplying factors for various chi square approximations. j r stat soc series b stat methodol. 1954;16:296-8. 42. rufai s, tate e, burton cg, maroof as. social vulnerability to floods: review of case studies and implications for measurement. int j disaster risk reduct. 2015;14(4):470-86. doi: 10.1016/j.ijdrr.2015.09.013. 43. kasperson re, dow k, golding d, kasperson jx, editors. understanding global environmental change: the contributions of risk analysis and management. worcester, ma: clark university; 1990. p.27-44. 44. mitchell jk, devine n, jagger k. a contextual model of natural hazard. am geogr soc. 1989 oct;79(4):391-409. 45. petersen p. sociobehavioural risk factors in dental caries international perspectives. community dent oral epidemiol. 2005 aug;33(4):274-9. 46. composite indicators research group. weighting. 2016 [cited 2016 may 15]. available from: https://composite-indicators.jrc.ec.europa. eu/?q=content/step-6-weighting. 47. divaris k. predicting dental caries outcomes in children: a ‘risky’ concept. j dent res. 2016 mar;95(3):248-54. doi: 10.1177/0022034515620779. 85does social vulnerability for caries predict caries status of children in sub-urban nigeria? braz j oral sci. 15(1):79-85 revista fop n 13 1662 quantitative role of mast cells in odontogenic cystic enlargement shailja chatterjee1; sumita mahajan2; karen boaz3; thomas george4 1mds, senior lecturer, 2mds, former professor and head 3mds, associate professor 4mds, reader department of oral pathology, manipal college of dental sciences, mangalore (karnataka), india received for publication: june 06, 2008 accepted: september 16, 2008 correspondence to: shailja chatterjee its centre for dental studies & research delhi-meerut road, ghaziabad (u.p.), india email: shailjadoc@indiatimes.com a b s t r a c t aim: mast cells have been hypothesized to play a significant role in pathogenesis of odontogenic cysts. the aim of this study was to evaluate mast cell distribution in cystic lining and the capsule to formulate a mechanism of cystic expansion. methods: ten formalin-fixed paraffin embedded tissue blocks each of okc, dentigerous and radicular cysts were selected. toluidine blue staining (1% in 1% nacl solution) was done in 5µm thick sections and counting performed in 10 areas using an ocular grid. areas counted were divided into 4 zones: intraepithelial, subepithelial, intermediate and deep zones (group i, ii, iii and iv respectively). statistical analysis: mean ±s.d. was calculated in each group followed by paired ‘t’ test. results: mast cells had greatest concentration in subepithelial zone. ‘t’ test showed no significant differences between group i and ii zones in okc but a highly significant difference between groups i and ii in dentigerous cyst. radicular cysts showed a significant difference between groups ii and iii. conclusion: mast cell degranulation releases numerous hydrolytic enzymes that facilitate breakdown of capsular matrix increasing the hydrostatic pressure due to raised osmolality. influx of tissue fluids results in their enlargement coupled with resorption at the bone-cyst interface. key words: mast cells, degranulation, toluidine blue, odontogenic cysts i n t r o d u c t i o n odontogenic cysts are possibly the most common benign destructive lesions in the human maxillofacial skeleton. three most common jaw cystsradicular cysts, odontogenic keratocysts and dentigerous cysts (of developmental odontogenic origin) are characterized by an expansile non – infiltrative growth, resulting in a smooth and usually unilocular cavity containing fluid or semi fluid material, lined by an epithelium and supported by a fibrous connective tissue capsule1. the expansion of the jaw cyst involves destruction of the extra-cellular matrix due to proteolysis of collagen fibers, osteoid – derived gelatin and protein components of basement membrane 1. mast cells contain numerous cytoplasmic granules, which are degranulated into the extra-cellular space upon activation. in addition to preformed granule contents, activated mast cells can synthesize de novo vasoactive mediators, for example, platelet – activating factor, chemotactic mediators, and several proinflammatory cytokines such as il-1α, il-3, il-6 and tnf – α. furthermore, mast cells are a rich source of heparin and proteolytic enzymes, such as tryptase, chymase and hyaluronic acid, which participate in connective tissue breakdown in the capsule during normal metabolic turnover, as well as in inflammation1. products released by mast cell activation and subsequent breakdown products of connective tissue elements are released into the cyst lumen increasing the hydrostatic pressure with subsequent enlargement. the aim of this study was to evaluate mast cell distribution in cystic lining and the capsule to formulate a mechanism of cystic expansion using morphometric analysis. material and methods paraffin – embedded formalin fixed tissue blocks of odontogenic keratocysts, dentigerous cysts and radicular cysts (10 each) were retrieved from the archives of the department of oral pathology, manipal college of dental sciences, mangalore. sections of 5µm were cut and stained with freshly prepared toluidine blue solution (1% tolonium blue in 1% sodium chloride), mounted with dpx, and followed by mast cells counting under 40x magnification. braz j oral sci. october/december 2008 vol. 7 number 27 1663 counting of mast cells: mast cells were counted in ten areas under 40x magnification using an ocular grid with a total area of 0.30625mm2, divided in four zones: 1. intraepithelial 2. sub-epithelial 3. intermediate 4. deep for intraepithelial counting, the graticule was oriented along the basement membrane and along the connective capsular tissue at the epithelial-capsular junction for counting in subepithelial zone. every alternate microscopic field was counted. the graticule was then moved further down into two microscopic fields into the capsule and the procedure repeated for intermediate zone. it was then moved further down two microscopic fields into the capsule to a third level (deep zone) and the counting was performed in similar manner. statistical analysis means and standard deviation of the mast cell counting were calculated in each layer zone. for comparisons between zones, the t test was applied. r e s u l t s statistical analysis revealed an increase in mast cell count in all the three cysts at the sub-epithelial zone. paired ‘t’ test showed no statistically significant difference (table 1) between intraepithelial (i) and sub-epithelial zones (ii) in odontogenic keratocysts (p=0.08), whereas a highly significant difference (table 2) was noted between the intraepithelial (group i) and sub-epithelial zones (ii) of tissue blocks intraepithelial(i) sub-epithelial(ii) intermediate(iii) deep(iv) 1 11 96 50 11 2 0 10 7 0 3 5 11 0 0 4 13 14 10 9 5 10 18 13 4 6 0 1 0 0 7 0 22 17 6 8 0 10 7 0 9 2 12 17 21 10 4 11 3 2 mean±sd 4.5±5.1 20.5±21.6 12.4±14.6 5.3±6.1 t test (p values) (i) (ii) (iii) (ii) 0.083 (iii) 0.123 0.416 (iv) 0.769 0.102 0.180 table 1 mast cell distribution in odontogenic keratocysts (mean ± standard deviation and p values of t test between zones) braz j oral sci. 7(27):1662-1665 quantitative role of mast cells in odontogenic cystic enlargement table 2 mast cell distribution in dentigerous cysts (mean ± standard deviation and p values of t test between zones) tissue blocks intraepithelial(i) sub-epithelial(ii) intermediate(iii) deep(iv) 1 1 6 5 0 2 0 3 0 0 3 4 29 28 36 4 3 15 1 0 5 1 1 0 0 6 5 7 0 0 7 2 28 26 0 8 2 9 3 0 9 8 23 10 0 10 0 41 30 24 mean±sd 2.6±2.5 16.2±13.4 10.3±12.6 6±12.1 t test (p values) (i) (ii) (iii) (ii) 0.005 (iii) 0.074 0.324 (iv) 0.426 0.101 0.462 1664 tissue blocks intraepithelial(i) sub-epithelial(ii) intermediate(iii) deep(iv) 1 0 11 2 34 2 0 6 0 0 3 9 4 3 3 4 2 26 6 1 5 1 9 1 0 6 2 11 3 0 7 0 6 2 4 8 1 2 0 0 9 12 16 7 0 10 1 5 1 0 mean±sd 2.8±4.2 9.6±7.1 2.5±2.4 4.2±10.6 t test (p values) (i) (ii) (iii) (ii) 0.017 (iii) 0.846 0.008 (iv) 0.702 0.196 0.626 table 3 mast cell distribution in radicular cysts (mean ± standard deviation and p values of t test between zones) dentigerous cysts (p=0.005). on the other hand, radicular cysts showed a highly significant difference (table 3) between sub-epithelial (ii) and intermediate (iii) zones (p=0.007). a significant elevation in mean±sd values was noted in mast cell population in odontogenic keratocysts in the subepithelial zone as compared to dentigerous and radicular cysts. 1.intraepithelial sub-epithelial intermediate deep d i s c u s s i o n mast cells are found widespread throughout the connective tissue wall of all the cysts particularly in the subepithelial zone and are source of a variety of proteolytic enzymes found in the cystic fluid. the results of the present study showed a great concentration of mast cell in subepithelial zone in all cyst walls. this concentration was higher in okcs than dentigerous and radicular cysts, suggesting an increased breakdown of capsular matrix in okcs. okc epithelium has been shown to be nonkeratinized at places, which causes a transport of breakdown matrix products into the cystic lumen2, and consequently can determine an elevated osmolality of the cystic fluid, which partly explains the greater aggressiveness of okc comparing to other odontogenic cysts. smith et al. 1 found a considerable amount of mast cells in the walls of odontogenic keratocysts, dentigerous and radicular cysts with the highest concentration seen in sub-epithelial zone. mast cells were also observed in the epithelial linings, which the authors suggested to be due to a chemotactic stimulus attracting them to the epithelial lining or luminal fluid contents. smith et al. 2 concluded that the major source of glycosaminoglycans and proteoglycans in cystic fluid was from the ground substance of the connective tissue capsule, released because of normal metabolic turnover and inflammatory degradation. degranulating mast cells release heparin and other hydrolytic enzymes, which facilitate breakdown of glycosaminoglycans and proteoglycans2,3. histochemical investigations of the connective tissue capsule in odontogenic cysts have demonstrated that hyaluronic acid, a product of mast cell degranulation, is the predominant glycosaminoglycan present along with less amounts of sulphated glycosaminoglycans2,3. the release of glycosaminoglycans and proteoglycans into the luminal fluid contributes significantly to osmotic and hydrostatic pressure by increasing the osmolality of the cyst fluid, thereby raising the internal hydrostatic pressure2,3. cyst expansion is also affected by the rate in which the surrounding bone is destroyed particularly at the cyst-bone interface4. teronen et al. 4 stated that activated mast cells can synthesize vasoactive and chemotactic mediators (e.g., platelet – activating factor) as well as several pro inflammatory cytokines such as il-3, il-6 and tnf-α de novo. these chemical mediators increase vascular permeability thereby facilitating influx of highly osmolar substances in cystic lumen. the highest concentration of mast cells in okcs explains a greater expansion as compared to other odontogenic cysts. the authors also found a high number of extensively degranulated mast cells in the area of cyst expansion at the border with the bony wall suggestive of high activity of mast cells in this area. mast cell degranulation also releases tryptase and prostaglandins which aid in bone resorption which is a feature in cyst enlargement at cyst-bone interface. in addition, interleukin-1α in okc cyst wall has been found to have an enhancing effect on matrix metalloproteinases secreted by fibroblasts5. braz j oral sci. 7(27):1662-1665 quantitative role of mast cells in odontogenic cystic enlargement 1665 several other studies have also substantiated the effect of mmps along with tissue inhibitor of metalloproteinases and collagenases in cyst walls6,7. these cell products have been found to be stimulated by mast cell derivatives79. based upon the literature review analysis, it can be proposed that the degranulating mast cells release products that contribute to cystic enlargement in four ways: 1. by direct release of heparin in luminal fluid 2. by release of hydrolytic enzymes which degrade capsular extracellular matrix components thereby facilitating their passage into the fluid 3. by the action of histamine on smooth muscle contraction and vascular permeability encouraging translation of serum proteins 4. by stimulating the production of prostaglandins, interleukin-1á, timp and other collagenases, which are said to be important in bone resorption and thus, cyst growth10 based upon the present study and similar investigations, it can be concluded that mast cells play a vital role in the pathogenesis of odontogenic cysts as an elevated number of mast cells was found in the connective tissue capsule of all three odontogenic cysts. the luminal fluid which accumulates as a result of osmolar concentration of mast cell by-products plays an important in cyst enlargement. r e f e r e n c e s 1. smith g, smith aj, basu mk. mast cells in human odontogenic cysts. j oral pathol med. 1989; 18: 274 – 8. 2. smith g, smith aj, browne rm. histochemical studies on glycosaminoglycans of odontogenic cysts. j oral pathol. 1988; 17: 55-9. 3. smith g, smith aj, browne rm. glycosaminoglycans in fluid aspirates from odontogenic cysts. j oral pathol. 1984; 13: 614-21. 4. teronen o, hietanen j, lindqvist c, salo t, sorsa t, eklund kk, et al. mast cell–derived tryptase in odontogenic cysts. j oral pathol med. 1996; 25: 376 – 8. 5. kubota y, oka s, nakagawa s, shirasuna k. interleukin-1alpha enhances type i collagen-induced activation of matrix metalloproteinase-2 in odontogenic keratocyst fibroblasts. j dent res. 2002; 81: 23-7. 6. docherty aj, murphy g. the tissue metalloproteinase family and the inhibitor timp: a study using cdnas and recombinant proteins. ann rheum disease. 1990; 49: 469-79. 7. teronen o, salo t, laitinen j, tornwall j, ylipaavalniemi p, konttinen yt et al. characterization of interstitial collagenases in jaw cyst wall. eur j oral sci. 1995; 103: 141-7. 8. taylor ac. collagenolysis in culture tissue ii. role of mast cells. j dent res 1971; 50: 1301-6. 9. suzuki k, lees m, newlands gfj, nagase h, woolly de. activation of precursors for matrix metalloproteinases 1 (interstitial collagenases) and 3 (stromelysin) by rat mast cell proteinases i and ii. biochem j 1995; 305: 301-6. 10. mundy gr. cytokines and local factors which affect osteoclast function. int j cell cloning 1992; 10: 215-22. braz j oral sci. 7(27):1662-1665 quantitative role of mast cells in odontogenic cystic enlargement oral sciences n3 original article braz j oral sci. october | december 2015 volume 14, number 4 perception of hiv among pregnant women in the public health system in two municipalities of the state of são paulo cléa adas saliba garbin¹, karina tonini dos santos pacheco2, thaís fonseca santiago3, simone miyada1, artênio josé ísper garbin1, suzely adas saliba moimaz1 1universidade estadual paulista – unesp, araçatuba dental school, department of pediatric and social dentistry, araçatuba, sp, brazil 2universidade federal do espírito santo – ufes, centro de ciências da saúde, departament of dentistry, area of dental clinic, vitória, es, brazil 3dental surgeon – caraguatatuba, sp, brasil correspondence to: cléa adas saliba garbin departmento de odontologia infantil e social faculdade de odontologia unesp rua josé bonifácio, 1193 cep: 16015-050 araçatuba, sp, brasil phone: +55 18 3636 3249 fax: +55 18 3636 3332 e-mail: cgarbin@foa.unesp.br abstract aim: to verify the knowledge of pregnant women on mother-to-child transmission (mtct) of hiv, the availability of hiv tests in the public health system and counseling on the disease in two cities, birigui and piacatu, são paulo state, brazil. methods: this is a descriptive and exploratory research using as samples, the files of 141 pregnant women attending the basic health unit. data were collected by survey, followed by a semi-structured questionnaire with open and closedend questions. data were analyzed on epi info™ 7.1.4, by the chi-square and exact fisher tests. results: from all the 141 pregnant women, 119 were interviewed and 92.4% reported to have been informed about the need of taking the hiv test during prenatal exams. however, only 5.9% were counseled and 20.2% reported to be aware of how to prevent mtct of hiv, usually mentioning lactation suppression and prescribed medication. the association between the knowledge about how to prevent mtct of hiv and some social, demographic and economic variables like ethnics, educational level, home location, occupation, age and parenting was not verified. conclusions: it is necessary to advise pregnant women on the importance of taking the hiv test regardless of the examination outcome, which was not observed in the cities where the research was conducted. keywords: maternal and child health; hiv; public health. introduction the prevalence of hiv among parturient women in brazil is approximately 12,000 cases a year, and the detection rate has presented a significant statistical raise in the past 10 years1. this result has reflected the adaptation of prenatal care health policy with the aid of hiv vaccines2. the hiv tests made during pregnancy guarantee the care and treatment for women and their children3. the health ministry advises both the hiv tests and counseling during pregnancy at the first prenatal visit and, whenever possible, the reapplication of vaccines at the beginning of the third trimester. several guidelines have been established by the health ministry to direct how counseling should be conducted before and after the hiv test2. counseling is a practice that allows both reflection and decision making. it is based on active listening, by which the health professionals seek to establish trust bonds, providing strategies braz j oral sci. 14(4):282-286 http://dx.doi.org/10.1590/1677-3225v14n4a06 received for publication: october 30, 2015 accepted: december 12, 2015 that facilitate the recognition of users as participants in their own health condition4. knowledge of possible ways of hiv transmission from the mother to the infant is the most successful means of prevention5. however, researches report that social and demographical variables, such as educational level, age, urban life and the fact that these women are housewives or not, affect the mothers’ knowledge about transmission6-9. the purpose of this study was to verify the knowledge of pregnant women about mtct transmission of hiv, availability of hiv tests in the public health system and counseling on the disease. material and methods the present study is a descriptive exploratory research, approved by ethics committee of araçatuba dental school, unesp, brazil (process number foa 2007-01422). two cities in the state of são paulo participated in the study, birigui and piacatu. the records on all pregnant women from the basic health units were used to determine the sample, according to their medical records and appointments, accounting to 141 pregnant women.the data were collected from patients scheduled to a gynecological visit at the health unit. the sample group was interviewed at the basic health unit, whereas the patients who were not programmed timely were called and an interview with the pregnant women was scheduled at their homes. the subjects were given explanations on the study objectives, and those who agreed to participate had their identities protected for ethical reasons. a pilot study was carried out in order to validate the research instrument and standardize the investigators. data were collected through a series of interviews, following semi-structured directions with open and closedend questions, designed especially for research purposes. the proposed questions comprised social, economical and demographic features, as well as knowledge about hiv prevention, mtct of hiv, hiv testing and also the subsequent counseling. the collected data were analyzed on epi info™ 7.1.4. the chi-square using the yates correction and exact fisher tests were used in order to verify the association between the patients’ knowledge about mtct of hiv and some social and economic variables, such as: ethnics, educational level, home location, occupation, age and parenting. results among the 141 pregnant women, 119 (84.4%) agreed to take part in the study. the age of the population under study varied from 14 to 41 years old (mean age of 24.7 years old) and 48.7% were brown-skinned. concerning the educational level, the majority (60.5%) completed high school and only 8 (6.7%) completed higher education. regarding the home location, the majority (96.6%) reported to live in urban areas. as for their occupations, 52.29% were housewives and 48.7% were primigravidae (table 1). the pregnant women who reported to have information on how to prevent hiv (89.1%) indicated the television, school and health units as main sources of information; the relatives were less frequently indicated as sources of information. c o l o r caucasian black brown level of education illiterate elementary school high school superior area of residence rural urban occupation employed unemployed age* young adult number of children primigravidae non-primigravidae characteristics n (%) 47 (39.5) 14 (11.8) 58 (48.7) 1 (0.8) 38 (32.0) 72 (60.5) 8 (6.7) 4 (3.4) 115 (96.6) 56 (47.1) 63 (52.9) 91 (76.5) 28 (23.5) 58 (48.7) 61 (51.3) table 1table 1table 1table 1table 1 – numeric distribution and percentage of social, economical and demographical variables. *young: 14-29 years old; adult: 30-49 years old the majority of pregnant women (92.4%) were informed about the need of taking the anti-hiv test during prenatal and 94.1% took the test. however, during the prenatal, 69.6% took the test only once and when questioned about counseling, only 7 women (5.9%) answered positively. only 20.2% out of the sample reported knowing how to prevent mtct of hiv and they mentioned only one method to avoid it. the most frequent answers were “not to breastfeed the baby” and “take medication” (table 2). the statistics showed a 0.0123 significance in the association between “hiv/aids awareness” and “level of education”, as well as “age” (p<0.0001). no association was verified among “realization of the tests”, “counseling” and “prevention of mtct” to the social, demographical and economical variables, such as ethnicity, level of education, home location, occupation and number of children” (table 3). discussion the profiles of the interviewed women match the ones found in the literature references10-13. it exposes a set of perception of hiv among pregnant women in the public health system in two municipalities of the state of são paulo283283283283283 braz j oral sci. 14(4):282-286 variables 284284284284284 characteristics which fit in the pattern of favorable situations to the infection by hiv/aids, such as low level of instruction, customs, imposed sexual guidelines to women, lack of economical opportunities, lack of control over the relationships as well as gender vulnerabilities14. propagation of the knowledge on hiv/aids has arisen a conscious and preventive attitude in the population, especially regarding the conduction of the hiv tests, which cooperates to control dissemination of the disease and to the improvement of the population’s health conditions. it was also verified all the pregnant women were doing prenatal visits, since their recruiting was through the basic health unit, according to veloso et al.15 (2010). moreover, most of them took the hiv test, which strengthens other literature references11, setting its availability in the public health service. in a previous study 16, although the majority of interviewed pregnant women (89.1%) reported to have received information on how to prevent hiv, there was little specific knowledge of transmission from mother to child, since 79.8% did not know how to prevent transmission. these results differ from those of byamugisha et al.10 (2010), who reported that the majority of the interviewees were aware of the risks of hiv transmission and the ways to prevent contamination, accomplishing a successful outcome of a strong policy of counseling, monitoring, treatment and conduction of the hiv test. the low levels of knowledge about mtct of hiv may be explained by lack of necessary information and counseling. it is acknowledged that the pregnant women’s serological status, availability of information and counseling are contributing factors to reduce the mtct of hiv17. it must be stressed that the availability of information and advice is essential, so that there will be knowledge of the risks and means of prevention. filippi et al.18 (2006) showed that education is strongly associated with health. the perception of health problems and the ability to understand information on health problems, adoption of healthy lifestyles, use of health services, as well perception of hiv among pregnant women in the public health system in two municipalities of the state of são paulo braz j oral sci. 14(4):282-286 categories frequency n % lactation suppression– “the mother must not breastfeed”. 14 58.3 medication– “the doctor prescribes a medication to avoid transmitting hiv to the baby” 6 25.0 labor – “health cares to the umbilical cord”. 3 12.5 total 24 100 table 2 –table 2 –table 2 –table 2 –table 2 – percentage and absolute frequency distribution of the categories referring to the positive answers about the awareness of prevention of mtct of hiv. variables c o l o r caucasian black brown educational level illiterate elementary school high school superior area of residence rural urban occupation employed unemployed age* young adult number of children primigravidae non-primigravidae information on hiv/aids n(%) 44 (93.6) 12 (85.7) 50 (86.2) 0 (0.0) 32 (84.2) 66 (91.7) 8 (100.0) 4 (100.0) 102 (88.7) 51 (91.1) 55 (87.3) 83 (91.2) 23 (82.1) 51 (83.6) 55 (94.8) p 0.4384 0.0144 1.0000 * * 0.7661 * * 0.2959 * * 0.9233 conduction of test n (%) 45 (95.7) 12 (85.7) 55 (94.8) 1 (100.0) 37 (94.7) 67 (93.1) 8 (100.0) 4 (100.0) 108 (93.9) 53 (94.6) 59 (93.7) 85 (93.4) 27 (96.4) 53 (91.4) 59 (96.7) p 0.3564 0.6861 1.0000 * * 1.0000 * * 0.6862 * * 0.2644 * * conduction of counseling n (%) 2 (4.3) 0 (0.0) 5 (8.9) 0 (0.0) 2 (5.4) 4 (5.8) 1 (12.5) 0 (0.0) 7 (6.3) 3 (5.5) 4 (6.7) 6 (6.9) 1 (3.6) 4 (6.7) 3 (5.5) p 0.3896 0.8648 1.0000 * * 1.000 * * 0.6862 * * 0.7125 * * prevention of mtct n (%) 13 (27.7) 1 (7.1) 10 (17.2) 0 (0.0) 8 (21.1) 13 (18.1) 3 (37.5) 0 (0.0) 24 (20.9) 10 (17.9) 14 (22.2) 20 (22.0) 4 (14.3) 8 (13.1) 16 (27.6) p 0.1807 0.5801 0.5817** 0.7163 0.4339 * * 0.1439 p – probability with significance level of 5%. *young: 14-29 years old; adult: 30-49 years old. ** exact fisher test utilization table 3table 3table 3table 3table 3 – numeric and percentage distribution of positive answers given by pregnant women about information on hiv/aids, conduction of tests and counseling and prevention of mtct, according to some social, economical and demographic variables. 285285285285285 as submission to therapeutic procedures are all consequences of the individual’s educational level19. it was found that the educational level is associated with the fact that pregnant women receive information on aids prevention of, that is, pregnant women who have a higher educational level have shown a greater ability to assimilate information on how to prevent disease. it was also verified that, besides the lack of awareness about mtct of hiv, most of the women in this research were not counseled, as only 5.9% answered positively when asked whether they had been counseled or not. thus, even if there is availability of medication for all the pregnant women and their vaccine records are acknowledged, it was noted that, in the surveyed cities, the great barrier to hiv prevention is the lack of counseling and knowledge about the subject. in such cases, the prenatal care providers have an important role in the prevention of mtct of hiv, for the risk of transmission to the baby is minimum when the physicians routinely offer the hiv test, conduct counseling regardless of the risk, guide those who refuse to take the test and still have proficiency to treat the ones whose outcome is positive. therefore the importance of training the prenatal care providers is fundamental, so they may acquire more skills on prevention and treatment of mtct of hiv6. the pre-test counseling can be done in groups with the intent of sharing similar situations and risks among the pregnant women, creating an exchange of knowledge which can motivate them to take the hiv test and even to accomplish the prenatal care, in case the patient is hiv-positive. in this last case, the counseling must be conducted individually, because the health condition of each patient is unique and it requires specific treatment and counseling from the health care providers20. it must be stressed that the availability of information and counseling is indispensable, so the patients can be aware of the risks and means of prevention21. in order to have positive outcome of national control and prevention of mtct of hiv, it is necessary to implement a solid policy in the public health system, in which parents are encouraged to take hiv testing, receive prenatal routine counseling, have the availability of arv medications, have skilled health care providers and humanized care for hivpositive patients22-24. education is the basis for the prevention of hiv/aids contamination, and therefore, information and awareness campaigns for the entire population can collaborate with increased knowledge of the disease, and consequently, its prevention methods. however, when it comes to keeping the new educational precepts, the cultural aspect can negatively impact the expected outcome, that is, knowledge can reach the pregnant woman, but the use of preventive methods of mtct during pregnancy relies solely on the mother25. access to hiv testing is critical to reduce mtct rates, but, based on the study findings, it appears that it is not enough. adequate prenatal care with counseling, monitoring, treatment and testing for hiv, should be implemented in early pregnancy. thus, research aimed to evaluate the public health system should be carried out in the prenatal visits, which improve and strengthen the struggle against mtct of hiv. although most pregnant women have taken the hiv test, many of them did it only once during the prenatal care and very few received counseling. this factor probably reflected the remarkable lack of knowledge concerning the prevention mtct of hiv, even by those who reported knowing how to avoid it. in this scenario, it is suggested that the state improve management skills and train health professionals and service providers, aiming for excellence in promoting public health. dissemination of information on this subject by more effective media campaigns is also recommendable. given this situation, the health care models in these municipalities have to be revised in accordance with the institutional dynamics and a more humane treatment for pregnant women with hiv. acknowledgements all authors acknowledge the financial support by capes and fapesp in granting scientific initiative and doctoral grants. references 1. brazil. ministry of health. [epidemiological bulletin hiv – aids]. brasília: ministry of health; 2014 [cited 2015 jan 9]. avalilable from: http:// www.aids.gov.br/sites/default/files/anexos/publicacao/2014/56677/ boletim_2014_final_pdf_15565.pdf. portuguese 2. brazil. ministry of health. [recommendations for the prophylaxis of vertical transmission of hiv and antiretroviral therapy in pregnant women: pocket handbook]. brasília: ministry of health; 2010 [cited 2015 jan 9]. available from: http://www.aids.gov.br/sites/default/files/ consenso_gestantes_2010_vf.pdf. portuguese 3. ononge s, karamagi c, nakabiito c, wandabwa j, mirembe f, rukundo gz, et al. predictors of unknown hiv serostatus at the time of labor and delivery in kampala, uganda. int j gynaecol obstet. 2014;124: 235-9. 4. passos sc, oliveira mic, gomes júnior sc, silva ks. hiv rapid test counseling among parturient. rev bras epidemiol. 2013; 16: 278-87. 5. birhane t, tessema ga, alene ka, dadi af. knowledge of pregnant women on mother-to-child transmission of hiv in meket district, northeast ethiopia. j pregnancy. 2015; 2015: 960830. 6. soeiro cm, miranda ae, saraceni v, lucena no, talhari s, ferreira lc. mother-to-child transmission of hiv infection in manaus, state of amazonas, brazil. rev soc bras med trop. 2011; 44: 537-41. 7. amoran oe, salami of, oluwole fa. a comparative analysis of teenagers and older pregnant women in the utilization of prevention of mother to child transmission [pmtct] services in western nigeria. bmc int health hum rights. 2012; 12: 13. 8. malaju mt, alene gd. determinant factors of pregnant mothers’ knowledge on mother to child transmission of hiv and its prevention in gondar town, north west ethiopia. bmc pregnancy childbirth. 2012; 12: 73. 9. asefa a, beyene h. awareness and knowledge on timing of mother-tochild transmission of hiv among antenatal care attending women in southern ethiopia: a cross sectional study. reprod health. 2013; 10: 66. 10. byamugisha r, tumwine jk, ndeezi g, karamagi cas, tylleskär t. attitudes to routine hiv counselling and testing, and knowledge about prevention of mother to child transmission of hiv in eastern uganda: a cross-sectional survey among antenatal attendees. j int aids soc. 2010; 13: 52. perception of hiv among pregnant women in the public health system in two municipalities of the state of são paulo braz j oral sci. 14(4):282-286 286286286286286 11. falnes ef, tylleskär t, paoli mm, manongi r, engebretsen ims. mothers’ knowledge and utilization of prevention of mother to child transmission services in northerntanzania. j int aids soc. 2010; 13: 36. 12. soares ml, oliveira mi, fonseca vm, brito as, silva ks. predictors of unawareness of hiv serostatus among women submitted to the rapid hiv test at admittance for delivery. cienc saude coletiva. 2013; 18: 1313-20. 13. holla r, maroli s, wettasinghe d, unnikrishnan b, kamath s, de r, et al. perception of hiv testing and counseling among integrated counseling and testing center clients of tertiary level hospitals. j int assoc provid aids care. 2015 jan 14. pii: 2325957414567683. 14. garbin cas, garbin aji, moimaz sas, rocha nb. [the hiv vertical transmission through the perception of brazilian women]. rev odontol araçatuba. 2012; 33: 41-5. portuguese 15. veloso vg, bastos fi, portela mc, grinsztejn b, joão ec, pilotto jh, et al. hiv rapid testing as a key strategy for prevention of mother-to-child transmission in brazil. rev saude publica. 2010; 44: 803-11. 16. kominami m, kawata k, ali m, meena h, ushijima h. factors determing prenatal hiv testing for prevention of mother to child transmission in dar es salaam, tanzania. pediatr int. 2007; 49: 286-92. 17. unaids. countdown to zero: global plan towards the elimination of new infections among children by 2015 and keeping their mothers alive: 20112015. 2011 [cited 2015 jan 9]. available from: http: //www.unaids.org/ sites/default/files/media_asset/20110609_jc2137_global-plan-eliminationhiv-children_en_1.pdf. 18. filippi v, ronsmans c, campbell om, graham wj, mills a, borghi j, et al. maternal health in poor countries: the broader context and a call for action. lancet. 2006; 368: 1535-41. 19. araujo esp, friedman rk, camacho lab, derrico m, moreira ri, calvet ga, et al. cascade of access to interventions to prevent hiv mother to child transmission in the metropolitan area of rio de janeiro, brazil. braz j infect dis. 2014; 18: 252-60. 20. goldani mz, giugliani er, scanlon t, rosa h, castilhos k, feldens l, et al. voluntary hiv counseling and testing during prenatal care in brazil. rev saude publica. 2003; 37: 552-8. 21. united nations children’s found. children orphaned by aids: front-line responses from eastern and southern africa. new york: unicef; 1999 [cited 2015 jan 9]. available from: http: //www.unicef.org/publications/ files/pub_aids_en.pdf. 22. pottie k, medu o, welch v, dahal gp, tyndall m, rader t, et al. effect of rapid hiv testing on hiv incidence and services in populations at high risk for hiv exposure: an equity-focused systematic review. bmj open. 2014; 4: e006859. 23. passos aa, moura erf. process indicators in the program for humanization of prenatal care and childbirth in ceará state, brazil: analysis of a historical series (2001-2006). cad saúde pública. 2008; 24: 1572-80. 24. paintsil e, andiman wa. update on successes and challenges regarding mother-to-child transmission of hiv. curr opin pediatr. 2009; 21: 94-101. 25. von linstow ml, rosenfeldt v, lebech am, storgaard m, hornstrup t, katzenstein tl, et al. prevention of mother-to-child transmission of hiv in denmark,1994–2008. hiv med. 2010; 11: 448-56. perception of hiv among pregnant women in the public health system in two municipalities of the state of são paulo braz j oral sci. 14(4):282-286 revista fop n 13 dental caries and associated factors among brazilian adolescents: a longitudinal study leila grando amorim mendes1; maria gabriela haye biazevic2; edgard michel-crosato3; maria odete amorim mendes4 1dds, ms, assistant professor, department of dentristry, university of oeste de santa catarina, brazil 2dds, ms, phd post-doctoral student, department of social dentistry, university of são paulo, brazil 3dds, ms, phd assistant professor, department of social dentistry, university of são paulo, brazil 4dds, private practice, água doce, sc, brazil received for publication: june 06, 2008 accepted: september 30, 2008 correspondence to: edgard michel crosato faculdade de odontologia da usp fousp departamento de odontologia social avenida professor lineu prestes, 2227 cidade universitária 05508-000 são paulo, sp, brasil phone: +55-11-3091-7875. fax: +55-11-3091-7874. e-mail: michelcrosato@usp.br a b s t r a c t a decrease in caries experience among children has been observed in all countries. however, this subject has yet to be further investigated in adolescents. the aim of this study was to describe the prevalence and incidence of caries disease and determine possible factors associated to this condition among adolescents. a 2-phase longitudinal study was performed. the first phase comprised a prevalence study of caries carried out with children aged 11 to 13 years (n=247). the second phase was performed with the same participants at the ages of 15 to 17 years. dmf-t and significant caries index (sic) were used to assess dental caries experience. a logistic regression model analysis of data was performed. on the first phase, 69.23% of the subjects presented caries. the dmft was 2.52 ± 2.54 and the sic was 4.23 ±2.72. in the second phase, 88.26% of the subjects presented caries. the dmft and the sic were 5.27 ±4.10 and 10.58 ±3.13, respectively. caries incidence was 2.94 ±3.05. the multivariate analysis identified that the individuals belonging to the most deprived social classes presented a 2.51-fold (1.22-5.19; p=0.012) higher chance of having experience caries (1st phase). the chance was 2.51-fold (1.21-2.55; p=0.013) higher in the 2nd phase. in conclusion, high caries prevalence and incidence were observed in the studied population and social factors were associated with these findings. socioeconomic factors showed a direct association with caries experience. key words: adolescent; dental caries; epidemiology; risk. i n t r o d u c t i o n although dental caries experience among children and adolescents in brazil is still a great concern to the dental public health services, a decrease in its occurrence has been observed in the last decades1. a similar reduction has been observed in developed and developing countries alike2-3. the last nationwide study performed in 2003 noted that 68.92% of the children at the age of 12 had caries experience4. this situation was observed to be above the world health organization (who) suggested levels by the year 2000. on the other hand, the dmft for the same age was of 2.78, below the target suggested by who5. between the ages of 15 and 18, 88.94% of the individuals presented caries experience, and the dmft was 6.172, indicating a considerable decrease in oral health conditions among the brazilian adolescents. epidemiological studies on dental caries have explored the association of aggravating factors of various natures, such as socioeconomic conditions1, food intake habits, oral hygiene characteristics, access to fluoridated water, access to health services, among others6. several of these studies were cross-sectional investigations and the caries disease measurements were taken at one moment in time. incidence measurements to verify aggravating factors associated with health for a same cohort during the course of time have not yet been performed in adolescents. the aim of this study was to describe the prevalence and incidence of dental caries and to verify the possible influence of socioeconomic, behavioral and clinical factors among brazilian adolescents. material and methods the research project was reviewed and approved by the university of west of santa catarina’s ethics research committee braz j oral sci. july/september 2008 vol. 7 number 26 1614 a census was performed. the study population was composed of all children aged between 11 and 13 years living in a rural city of southern brazil (água doce, sc) in the year 2001 (n=247). all children had access to systemic fluoride since the city had uninterrupted public water supply fluoridation since 1989. this is a 2-phase longitudinal study. on the first phase a study of prevalence of dental caries was carried out and information regarding socioeconomic conditions and oral health behavior was collected by means of a structured questionnaire. the second phase was performed in 2005 (48 months after the first phase), in which a new collection of data on caries prevalence was done. the brazilian association of market survey institutes (anep – abipeme) questionnaire was used to evaluate the subjects’ socioeconomic conditions and a structured questionnaire was used to collect oral health behavior data. during the study period, the participants did not have access to any oral health preventive measures. access was limited to curative procedures, such as dental fillings. information concerning caries was obtained through dmft index. all examinations were performed according to who criteria7. the adolescents were examined by two calibrated dentists after obtaining an appropriate kappa (>0.80). the significant caries index (sic) was used to measure the polarization of caries experience in one-third of the participants who presented more disease8. socioeconomic, behavioral and clinical-epidemiological characteristics were classified using dichotomic variables. the socioeconomic characteristics were: parental schooling; family income, in minimal wages (one minimum wage corresponds to approximately 150 u.s. dollars). the social level was divided according to methodology from the brazilian association of market survey institutes (anep – abipeme), as published elsewhere9; classes a and b represent the categories with a higher socioeconomic level and classes c, d and e represent those with the lowest socioeconomic conditions; and the number of people per household. regarding oral health, the individuals were asked whether they had seen a dentist in the previous 12 months, and the frequency of the brushing, flossing and weekly use of mouthwash with fluoride. concerning the clinical-epidemiological characteristics, were observed: the prevalence of caries at baseline, caries prevalence in the same participants 48 months after baseline and the incidence of carious lesions during the study period, according to the dmft. data were analyzed by stata 8.0 software. the continuous variables were presented as means and standard deviation. the dichotomic variables were represented in the form of absolute (n) and relative (%) frequency. the nonparametric wilcoxon matched-pair signed-rank test was used to determine the difference in dmft means between the two exams. the nonparametric mann-whitney test was used to verify the differences between the mean dmft variables baseline 48 months incidence median ± sd median ± sd median ± sd d 0 ± 1.24 1 ± 2.06* 0 ± 2.11 m 0 ± 0.54 0 ± 0.71* 0 ± 0.64 f 1 ± 1.90 3 ± 3.14* 1 ± 2.31 dmft 2 ± 2.54 5 ± 4.10* 2 ± 3.05 sic 4 ± 2.72 10 ± 3.13* 6 ± 2.96 table 1 median and standard deviation (sd) of the dmft and the significant index for caries (sic) in adolescents (água doce-sc, 2006) * statistically significant differences when comparing the baseline examination to the 48-month examination (p<0.05; wilcoxon test). and sic in the dichotomic variable groups. single and multivariate logistic regressions were used to verify factors associated with caries prevalence and incidence. for the multivariate analysis, the statistically significant variables from the single variate regression were introduced in the model. r e s u l t s the study was carried out with 247 adolescents, of which 131 were male (53.04%). one-hundred and sixty-six mothers (65.99%) and 157 (63.56%) fathers reported having less than 8 years of schooling. of the total cohort, 154 (62.60%) participants belonged to families with a monthly income of up to 3 minimum wages; 188 (76.11%) belonged to the least favored social groups (c, d and e) and 194 (78.54%) lived in households with up to 5 people. at baseline, 171 (69.23%) participants had caries experience. the mean dmft was 2.52 (± 2.54) and the mean sic was 4.23 (±2.72) (table 1). on phase 2 of the study, 218 (88.26%) participants had caries experience. the mean dmft was 5.27 (±4.10) and the mean sic was 10.58 (±3.13) (table 1). the mean incidence of caries disease (dmft) was 2.94 (±2.96) and the median was 2 (table 2). a higher and statistically significant mean was observed between the d, m and f components and the dmft in the second phase of the study, compared to the baseline (table 1). the results of the analysis of the differences between the mean dmft and the dichotomic variable groups are shown in table 2. in the first phase of the study, a higher mean dmft was observed among the participants whose mothers had less years of schooling (p = 0.001), whose fathers had less years of schooling (p = 0.001), who belonged to families with a smaller monthly income (p = 0.05), who flossed less than once a day (p = 0.019). on the multivariate analysis, the greater risk of caries was associated with social class and frequency of brushing. participants who belonged to the least favored social classes presented a 2.51-fold (1.22-5.19) higher chance of having caries experience, when compared to those individuals of more favored social classes (p = 0.012); those individuals 1615 braz j oral sci. 7(26):1614-1619 dental caries and associated factors among brazilian adolescents: a longitudinal study that reported smaller a frequency of brushing had a 2.03fold (0.99-4.15) higher chance of having caries when compared to individuals who reported a higher frequency of brushing (p=0.052) (table 3). on the second phase, a higher mean dmft was observed among participants whose mothers had less years of schooling (p = 0.001), whose fathers had less years of schooling (p = 0.001), among adolescents who belonged to families with smaller income (p = 0.048), who flossed less than once a day (p = 0.034), who had caries experience in the first phase of the study (p = 0.001) and who were in the one-third of the participants with higher dmft on the first phase of the study (p = 0.001) (table 2). participants belonging to the least favored social classes presented a 2.51-fold (1.21-2.55) higher chance of presenting caries experience when compared to the most favored social classes (p=0.013) (table 3). concerning the incidence of caries, a higher mean dmft was observed among participants whose mothers had less years of schooling (p = 0.019), whose fathers had less years of schooling (p = 0.003), who lived in households with more than 5 people (p = 0.035), who had experience of caries in the first phase of the study (p = 0.001), and were in the one-third of the participants with higher dmft variables baseline 48 months incidence % dmft >=l; median ± sd % dmft >=l; median ± sd % dmft >=3; median ± sd g e n d e r male 70.63; 2 ± 2.35 89.23; 5 ± 4.33 46.15; 2 ± 3.44* female 70.43; 2 ± 2.74 87.07; 5 ± 3.83 46.55; 2 ± 2.54 schooling of the mother complete elementary 56.79; 1 ± 2.47a 78.57; 3 ± 3.83 h 38.10; 2 ± 2.83n incomplete elementary 77.64; 32.86 ± 2.51a 93.25; 66.02 ± 4.10h 50.31; 33.19 ± 3.13n schooling of the father complete elementary 57.14; 1 ± 2.11b 78.16; 3 ± 3.65i 34.48; 2 ± 2.70o incomplete elementary 78.06; 3 ± 2.66b 93.63; 6 ± 4.14i 52.23; 3 ± 3.20o family income/month 3 or more minimum wages 60.92; 2 ± 2.68c 81.52; 4 ± 3.89 41.30; 2 ± 2.96 up to 3 minimum wages 75.97; 2 ± 2.44c 92.21; 5 ± 4.14 48.70; 2 ± 3.07 social class (anep) class a and b 50.82; 1 ± 2.02d 75.00; 3 ± 3.65j 32.81; 1 ± 2.74p class c, d and e 77.35; 3 ± 2.63d 92.90; 6 ± 4.09j 50.82; 3 ± 3.12p number of people in the household up to 5 69.31; 2 ± 2.68 87.11; 5 ± 4.18 44.33; 2 ± 3.16q 5 or more 75.47; 3 ± 1.97 92.45; 6 ± 3.73 52.83; 3 ± 2.57q visited the dentist in the last 12 months yes 71.88; 2 ± 2.38 85.94; 5 ± 4.13 43.75; 2 ± 3.18 no 70.22; 2 ± 2.60 89.07; 4 ± 4.00 46.99; 2 ± 2.63 brushes teeth at least 3 times a day yes 66.28; 2 ± 2.56 86.44; 5 ± 4.25 43.50; 2 ± 3.26 no 81.43; 2 ± 2.46 92.86; 6 ± 3.64 52.86; 3 ± 2.42 flosses at least once a day yes 74.34; 3 ± 2.71 f 88.79; 6 ± 4.04k 50.00; 3 ± 2.81 no 67.44; 2 ± 2.33 f 87.79; 4 ± 4.10k 42.75; 2 ± 3.26 uses fluoridated mouthwashes at least once a week yes 68.47; 2 ± 2.56 90.35; 5 ± 4.01 41.23; 2 ± 2.61 no 72.52; 2 ± 2.52 86.47; 5 ± 4.16 50.38; 3 ± 3.37 caries prevalence (baseline) yes na** 100.00; 6 ± 3.44l 54.97; 1 ± 2.63 r no na** 59.15; 1 ± 3.59l 21.13; 3 ± 3.58r sic (dmft – baseline) 1st and 2nd tertile na** 82.94; 4 ± 3.94m 58.33; 2 ± 3.03s 3rd tertile na** 100.00; 7 ± 3.67m 34.41; 3 ± 2.85s table 2 median and standard deviation (sd) of the dmft according to risk variables, in adolescents (água doce-sc, 2006) *equal letters means that the groups analyzed were different (p<0.05), using the mann-whitney test; ** na= not applicable; * 1 minimum wage corresponds to approximately usd 150 1616 braz j oral sci. 7(26):1614-1619 dental caries and associated factors among brazilian adolescents: a longitudinal study variables aor* ic 95% p risk model for the baseline social class (anep) class a and b 1a class c, d and e 2.51 1.22-5.19 0.012 schooling of the father complete elementary 1a incomplete elementary1.51 0.71-3.22 0.279 schooling of the mother complete elementary 1a incomplete elementary 1.55 0.75-3.22 0.237 family income/month* 3 or more minimum wages 1a up to 3 minimum wages 0.91 0.44-1.87 0.788 brushes teeth at least 3 times a day yes 1a no 2.03 0.99-4.15 0.052 risk model for the examination after 48 months social class (anep) class a and b 1a class c, d and e 2.51 1.21-5.22 0.013 schooling of the father complete elementary 1a incomplete elementary 1.45 0.69-3.18 0.316 schooling of the mother complete elementary 1a incomplete elementary 1.69 0.81-3.57 0.164 family income/month* 3 or more minimum wages 1a up to 3 minimum wages 0.99 0.78-2.04 0.980 risk model for the incidence caries prevalence (baseline) yes 1a no 1.19 0.98-1.21 0.127 social class (anep) class a and b 1a class c, d and e 1.72 0.86-3.43 0.127 schooling of the father complete elementary 1a incomplete elementary 1.67 0.84-3.32 0.142 schooling of the mother complete elementary 1a incomplete elementary 1.03 0.53-2.00 0.938 table 3 multivariate logistic regression analysis according to caries prevalence (dmft=0 vs dmft>0) and incidence (inc d”3 vs inc >3) in the adolescents. água doce-sc, 2006 a reference value; *statistically significant (p<0.005); *aor= or adjusted; b 1 minimum wage corresponds to approximately usd 150. in the first phase of the study (p = 0.001) (table 2). on the multivariate analysis, none of the variables studied were associated with the incidence of caries (table 3). d i s c u s s i o n during the first phase of the study, at the age of 12, 69.23% of the children presented caries experience; on the other hand, the dmft for the same age group was 2.52, lower than the who recommendations of dmft d” 35. countries such as england, sweden and denmark have dmft that ranges between 0.9 and 1.1 for this age group10. there is a very large variation among experts with regard to the impact of various possible factors in explaining the caries experience decrease among the countries, and the use of fluoride toothpaste has been a consensus of a definite positive effect in this reduction11. 1617 braz j oral sci. 7(26):1614-1619 dental caries and associated factors among brazilian adolescents: a longitudinal study among countries that have caries prevalence below 3, such as those mentioned above, the sic index is recommended in order to monitor caries experience in one-third of the participants that presented higher indices of the disease12. on the first phase of the study, the sic was 4.23, which is higher than the target set for the year 2015 (<313). sweden and senegal are some of the few countries that have already achieved this goal13-14. in 1997, england presented a sic of 3.5 for children at the age of 12, the united states 3.6, germany 4.1, france 4.7, mexico 5.0 and costa rica 13.713. even though this new index is being proposed, the who goal defined for the year 2000 should still be considered, especially for those countries with a mean dmft above this value, or even below but close to it, such as the case of this study12. on the second phase of the study (after 48 months), 88.26% of the participants presented caries experience, and the dmft was 5.27, indicating a considerable decrease of the oral health status of the adolescents. the incidence of the dmft was 2.94, in which the filled (f) component presented the highest incidence (1.64), indicating the curative characteristics of the brazilian dental services. nevertheless, these values were inferior to the nationwide study that found a dmft of 6.17 in the 15-to-18-year-old age group2. during this phase of the study, the sic was 10.58, which is considered elevated, if compared to the results of marthaler, et al.15(2005), who found a sic of 4.31 at the age of 15. however, it was below the values found among adolescents in the 15 to 19 year age group in the state of são paulo, brazil2. marthaler, et al.15 (2005) suggest the adoption of the sic target of 5.0 at the age of 15. in the present study, the mean dmft was higher in those groups whose parents presented lower schooling levels, lower family income and no flossing at least once a day. these findings reinforce the results found by other authors6, who noted that socioeconomic inequalities were associated with higher prevalence of caries disease. the second moment of the survey (48 months after the first examination) and the incidence presented similar standards. on the multivariate analysis, it was noted that participants belonging to the least favored social classes presented a 2.51-fold higher risk for caries experience in both phases of the study, thus reinforcing the influence of living conditions on oral health16. in the first phase of the study, it was observed that participants who brushed their teeth less than three times a day had 2.03-fold higher chances of having caries, which is in agreement with the findings of previous studies17-18. the ideal brushing frequency depends on the efficiency of the cleaning18. from a public health standpoint, this should not be the main objective for intervention in high-risk populations19. our investigation confirmed the results of studies by antunes, et al.1 (2004) and marthaler, et al.15 (2005) regarding the polarization of caries disease, in which a small group of people is normally responsible for a higher proportion of the disease within a population. nevertheless, it has been discussed that new cases of the disease have not appeared in the group with the highest caries experience20. although this is a longitudinal study to determine factors associated with the risk of caries, the dmft index was used. for longitudinal studies, the most indicated index would be the dmfs21. the reason why de dmft index was adopted was because the study was carried out in 2 phases, and in the first phase, the use of dmft had the purpose of serving as a parameter for the local planning of health services. possible factors that contribute to the high caries incidence for age groups older than 12, should be further investigated. in conclusion, high caries prevalence and incidence were observed in the studied population and social factors were associated with these findings. socioeconomic factors showed a direct association with caries experience. r e f e r e n c e s 1. antunes jlf, narvai pc, nugent zl. measuring inequalities in the distribution of dental caries. community dent oral epidemiol. 2004; 32: 41-8. 2. gushi ll, soares mc, forni tib, vieira v, wada rs, sousa mlr. dental caries in 15-to-19-year-old adolescents in são paulo state, brazil, 2002. cad saúde públ. 2005; 21: 1383-91. 3. petersen pe, bourgeois d, ogawa h, estupinan-day s, ndiaye c. the global burden of oral diseases and risks to oral health. bull world health organ. 2005; 83: 661-9. 4. brasil. ministério da saúde. projeto sb brasil 2003. condições de saúde bucal da população brasileira 2002-2003. resultados principais. brasília; 2004. 5. fdi. federation dentaire internacionale. global goals for oral health in the year 2000. int dent j. 1982; 32: 74-7. 6. thomson wm, poulton r, milne bj, caspi a, broughton jr, ayers kms. socioeconomic inequalities in oral health in childhood and adulthood in a birth cohort. community dent oral epidemiol. 2004; 32: 345-53. 7. world health organization. oral health surveys. basic methods. 4th ed. geneva: orh/epid; 1997. 8. nishi m, bratthall d, stjernswärd j. how to calculate the significant caries index (sic index). who collaborating centre / faculty of odontology, university of malmö, sweden; 2001. 9. michel-crosato e, biazevic mgh, crosato e. relationship between dental fluorosis and quality of life: a population based study. braz oral res. 2005; 19: 150-5. 10. world health organization. global oral databank. genebra, who; 2003. disponível em: www.whocollab.od.mah.se/ euro.html. 11. bratthall d, hänsel-petersson g, sundberg h. reasons for the caries decline: what do the experts believe? eur j oral sci. 1996; 104: 416-22. 12. bratthall d. introducing the significant caries index together with a proposal for a new global oral health goal for 12-yearolds. int dent j. 2000; 50: 378-84. 13. nishi m, stjernswärd j, carlsson p, bratthall d. caries experience of some countries and areas expressed by the significant caries index. community dent oral epidemiol. 2002; 30: 296-301. 14. sembene m, kane sw, bourgeois d. caries prevalence in 12year-old schoolchildren in senegal in 1989 and 1994. int dent j. 1999; 49: 73-5. 15. marthaler t, menghini g, steiner m. use of ths significant caries index in quantifying changes in caries in switzerland from 1964 to 2000. community dent oral epidemiol. 2005; 33: 159-66. 1618 braz j oral sci. 7(26):1614-1619 dental caries and associated factors among brazilian adolescents: a longitudinal study 16. locker d. deprivation and oral health: a review. community dent oral epidemiol. 2000; 28: 161-9. 17. angelillo if, torre i, nobile cg, villari p. caries and fluorosis prevalence in communities with different concentrations of fluoride in the water. caries res. 1999; 33: 114-22. 18. campus g, lumbau a, lai s, solinas g, castiglia p. sócioeconomic and behavioral factors related to caries in twelveyear-old sardinian children. caries res. 2001; 35: 427-34. 19. koerber a, burns jl, berbaum m, punwani i, levy sr, cowell j, et al. toothbrushing patterns over time in at-risk metropolitan african-american 5th-8th graders: a brief communication. j public health dent. 2005; 65: 240-3. 20. rose g. sick individuals and sick populations. int j epidemiol. 1985; 14: 32-8. 21. broadbent jm, thomson wm. for debate: problems with the dmf index pertinent to dental caries data analysis. community dent oral epidemiol. 2005; 33: 400-9. 1619 braz j oral sci. 7(26):1614-1619 dental caries and associated factors among brazilian adolescents: a longitudinal study 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1511/1164 1/11 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1511/1164 2/11 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1511/1164 3/11 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1511/1164 4/11 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1511/1164 5/11 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1511/1164 6/11 28/01/2019 pdf.js viewer 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hypochlorite effects on dentin bond strength and acid-base resistant zone formation by adhesive systems marina di francescantonio1, hamid nurrohman2, tomohiro takagaki2, toru nikaido2, junji tagami2, marcelo giannini1 1 universidade estadual de campinas unicamp, piracicaba dental school, department of restorative dentistry, piracicaba, sp, brazil 2 tokyo medical and dental university, department of cariology and operative dentistry, tokyo, japan correspondence to: marcelo giannini departamento de odontologia restauradora faculdade de odontologia de piracicaba – unicamp av. limeira, 901, piracicaba, sp, brasil cep: 13414-903 phone: + 55 19 21065340 fax: + 55 19 21065218 e-mail: giannini@fop.unicamp.br abstract aim: to evaluate the effects of 10% naocl gel application on the dentin bond strengths and morphology of resin-dentin interfaces formed by three adhesives. methods: two etch-and-rinse adhesives (one-step plus, bisco inc. and clearfil photo bond, kuraray noritake dental) and one self-etch adhesive (clearfil se bond, kuraray noritake dental) were applied on dentin according to the manufacturers’ instructions or after the treatment with 10% naocl (ed-gel, kuraray noritake dental) for 60 s. for interfacial analysis, specimens were subjected to acid-base challenge and observed by sem to identify the formation of the acid-base resistant zone (abrz). for microtensile bond strength, the same groups were investigated and the restored teeth were thermocycled (5,000 cycles) or not before testing. bond strength data were subjected to two-way anova and tukey’s test (p<0.05). results: naocl application affected the bond strengths for one-step plus and clearfil photo bond. thermocycling reduced the bond strengths for clearfil photo bond and clearfil se bond when used after naocl application and one-step plus when used as recommended by manufacturer. abrz was observed adjacent to the hybrid layer for self-etch primer. the etch-and-rinse systems showed external lesions after acid-base challenge and no abrz formation when applied according to manufacturer’s instructions. conclusions:10% naocl changed the morphology of the bonding interfaces and its use with etch-&-rinse adhesives reduced the dentin bond strength. formation of abrz was material-dependent and the interface morphologies were different among the tested materials. keywords: dental bonding; dentin; dental caries susceptibility; dentin-bonding agents; sodium hypochlorite; scanning electron microscopy. introduction the success and durability of restorations depends on the type of adhesive technique and materials used. the advancement of adhesive dentistry produces restorations with excellent aesthetic and mechanical properties; even so, the restorative procedures still present some clinical problems related to microleakage and degradation. such problems could occur from gap formations between tooth and adhesive restoration, which can lead to secondary caries and fractures in adjacent dental structures, affecting the longevity of restorations1. many studies have evaluated the prevention and control of secondary caries around composite restorations, mainly in dentin. the focus of these investigations has been on e resin-adhesive interfaces after cariogenic challenge. different methods received for publication: november 17, 2015 accepted: december 22, 2015 braz j oral sci. 14(4):334-340 http://dx.doi.org/10.1590/1677-3225v14n4a15 335335335335335 material one-step plus clearfil photo bond clearfil se bond composition biphenyldimethacrylate, hema, acetone, dental glass catalyst liquid: bis-gma, mdp, hema, hydrophobic dimethacrylate, benzoyl peroxide, camphorquinoneuniversal liquid: n,n’-diethanol p-toluidine, sodium benzene sulûnate, ethyl alcohol primer: hema, mdp, hydrophilic aliphatic dimethacrylate, dl-camphorquinone, water, accelerators, dyes and othersbond: hema, mdp, bis-gma, hydrophobic dimethacrylate, dl-camphorquinone, n,n-diethanol-p-toluidine, silanated colloidal silica batch number 0800005538 catalyst: 00453a universal:00549a primer:01633a adhesive: 01088b table 1. table 1. table 1. table 1. table 1. composition and batch number of the adhesive systems used in this study (information supplied by the msds of each manufacturers). abbreviations: bis-gmabis-phenol a diglycidyl, hema2-hydroxyethyl methacrylate, mdp10-methacryloyloxydecyl dihydrogen phosphate. can be used to analyze the effects of cariogenic challenge on dentin-resin interfaces, such as scanning electron microscopy (sem), polarized light microscopy, microhardness analysis, microradiography, confocal laser scanning microscopy and x-ray analytical microscopy2-5. tsuchiya et al.6 (2004) using sem identified the formation of an acid-base resistant zone (abrz) beneath the hybrid layer, which was characterized by argon-ion etching technique. however, they reported that abrz was observed only for some categories of adhesive systems7. sodium hypochlorite (naocl) is a well-known nonspecific proteolytic agent able to dissolve organic material and has been used in restorative dentistry as a deproteinizing agent for dentin8-10. the rationale for using naocl is that it facilitates the infiltration of adhesive resins into an etched dentin substrate, creating the bond without collagen fibrils and hybrid layer11-13. depending on each test methodology and/or specific composition of dentin bonding agents, the application of naocl on acid etching may increase or decrease bond strengths14-19. this in vitro study was conducted to determine by sem the effects of thermocycling and 10% naocl gel application after acid etching on the dentin bond strengths and resindentin interface morphology. the first tested hypothesis was that 10% naocl and thermocycling would not reduce the dentin bond strength, regardless the type of adhesive system used. the second was that 10% naocl application would not provide the abrz formation. material and methods eighty-four caries-free human third molars stored in an aqueous solution containing 0.2% thymol were used after approval by the ethics research committee (process # 90/ 2009). this study tested three adhesive systems : two etchand-rinse adhesive systems: one-step plus (bisco inc., schaumburg, il, usa) and clearfil photo bond (kuraray noritake dental, tokyo, japan); and one self-etch adhesive system: clearfil se bond (kuraray noritake dental). table 1 displays the composition and lot number of these adhesive systems. bond strength test sixty teeth were used in this part of study (n=5). the occlusal enamel of teeth was removed using a low speed diamond saw (buehler, lake bluff, il, usa) to expose a flat middle-depth coronal dentin surface. the exposed dentin surface was ground using 600-grit silicon carbide paper (fuji star, sankyo rikagaku, saitama, japan) under water-cooling. adhesive systems were applied to the dentin surfaces according to the manufacturers’ instructions or following 10% naocl application (ad-gel, kuraray noritake dental). the experimental groups are described in table 2. when 10% naocl was used with two acid-etch adhesives (one-step plus and clearfil photo bond), it was applied for 1 min after phosphoric acid etching, rinsed for 20 s and gently air-dried for 10 s, followed by adhesive application. for clearfil se bond, 10% naocl was used after se-primer. afterwards, acetone (for 30 s) and water rinsing (for 30 s) were used to remove functional monomers that did not react with calcium ions from the dentin surface. next, the clearfil se bond bonding resin was applied to dentin and lightactivated for 10 s (optilux 501, demetron-kerr corp., danbury, ct, usa). a 6 mm-high composite block (clearfil majesty, kuraray noritake dental, tokyo, japan and aelite ls, bisco inc) was built-up incrementally over the bonded dentin. three increments of 2.0 mm were placed and each layer was light activated for 20 s. after 24 h in distilled water at 37 °c, half of the stored specimens and the other halves were thermocycled 5,000 times in a water bath between 5 °c and 55 °c with a 30 s dwell time in each bath and a transfer time of 10 s. afterwards, the teeth were serially sectioned perpendicular to the adhesive-dentin interface in mesiodistal and buccal-lingual directions to obtain bonded beams of approximately 1.0 mm2 cross-sectional area using a slow-speed diamond saw. the specimens were tested individually by attaching them to a microtensile jig with cyanoacrylate glue (model repair ii, dentsply-sankin, tochiji, japan). the bonded beams were tested in tension using a universal testing machine (ez test, shimadzu corp., kyoto, japan) at a 1.0 mm/min crosshead speed. the load in kgf and the bonding surface area of the specimen were recorded and microtensile bond strength data sodium hypochlorite effects on dentin bond strength and acid-base resistant zone formation by adhesive systems braz j oral sci. 14(4):334-340 336336336336336 restorative system treatment (adhesive + composite) one-step plus + aelite ls one-step plus + aelite ls clearfil photo bond + majesty clearfil photo bond + majesty clearfil se bond + majesty clearfil se bond + majesty treatment manufacturer’s instructions 10% naocl application manufacturer’s instructions 10% naocl application manufacturer’s instructions primer removal with acetone and water+ 10% naocl application table 2.table 2.table 2.table 2.table 2. experimental groups and application technique for adhesive systems. were expressed in mpa. statistical analysis was performed with statistical software (minitab 15, minitab, state college, pa, usa). two-way analysis of variance (anova) for the “restorative system” and “thermocycling” factors was performed, followed by a tukey’s post hoc test (p<0.05). the fractured surfaces of the tested specimens were sputter-coated with gold (med 010, balzers union, balzers, liechtenstein) and examined using a scanning electron microscope (vp 435, leo electron microscopy ltd., cambridge, uk). failure patterns were classified as: (1) failure within adhesive resin layer; (2) failure within hybrid layer; (3) cohesive failure in dentin; and (4) mixed failure involving the adhesive resin and hybrid layer. acid-base resistant zone evaluation the methodology for specimen preparation (figure 1) followed a previous report20. twenty-four caries-free human third molars were used in this part of study (n=4). teeth were prepared according to the bond strength methodology and the same application technique for adhesives and composites was used. after bonding and composite placement, teeth were stored in distilled water at 37 °c for 24 h and then were vertically sectioned through the dentin-adhesive interface and embedded in an epoxy resin (epoxicure resin, buehler). the embedded teeth were placed in 100 ml of a buffered demineralizing solution, containing 2.2 mmol/l cacl2, 2.2 mmol/l nah2po4 and 50 mmol/l acetic acid (ph 4.5) for 90 min to create artificial recurrent caries20. after this, the teeth were immersed in 5% naclo for 20 min to remove any demineralized dentin collagen fibrils, followed by rinsing fig. 1. schematic illustration of the specimen preparation for sem. with water for 30 s. the 4-meta/mma-tbb resin (super bond c&b, sun medical, moriyama, japan) was used as infiltrant agent to protect the demineralized surface during polishing procedure. after curing of infiltrant resin, the samples were vertically sectioned through the dentin-adhesive interface to obtain 1-mm thick specimens and then polished with diamond pastes (struers a/s, copenhagen, denmark). the polished surfaces were conditioned with a beam of argonions (eis-ie, elionix inc., tokyo, japan) for 6 min. the operating conditions for argon ion beam etching were 1 kv accelerating voltage and 0.2 ma/cm2 current density with the ion beam positioned directly perpendicular to the polished surface. the specimens were gold-sputter coated and the morphological changes at the dentin-adhesive interfaces produced by acid-base challenge were observed by sem (jsm-5310lv, jeol, tokyo, japan). results bond strength the dentin bond strength means of the experimental groups are shown in table 3. two-way anova indicated that the “restorative system” and “thermocycling” factors influenced the bond strength results (p<0.0001 and p<0.0001, respectively), and the interaction factor was also significant (p=0.0004). dentin bond strengths of one-step plus and clearfil photo bond decreased following 10% naocl application (p<0.05), while clearfil se bond was not affected (p>0.05). thermocycling decreased the bond strength of clearfil se bond after naocl application. thermocycling affected also the bond strength of clearfil photo bond (with or without naocl application) and one-step plus when following the manufacturer’s instructions (p<0.05). table 4 shows de percentage distribution of failure modes. failure within adhesive resin layer, within hybrid layer and mixed were observed for all experimental groups. in general, higher incidence of cohesive failure in dentin occurred when teeth bonded with clearfil photo bond and clearfil se bond were thermocycled. acid-base resistant zone the sem images of the dentinresin interfaces after acid-base challenge are shown in figure 2. the outer lesion created by the acid challenge was observed in all groups; it was approximately 10 to 20 ìm thick. one-step plus and clearfil photo bond used following the manufacturer’s instructions formed thick hybrid layers of approximately 3 to 4 µm (figures 2a and 2c). however, the hybrid layer formed by one-step plus was destroyed by acid-base challenge (figure 2c) and wall lesions were observed for it and clearfil photo bond adhesives. the etchand-rinse adhesives applied after 10% naocl did not form hybrid layer. the adhesive resins were bonded to irregular dentin wall created by the acid etching and no wall lesion was found along the adhesive interface (figures 2b and 2d). for clearfil se bond, 2-ìm-thick abrz formation was observed (figure 2e). a very thin abrz, approximately 0.5 ìm thick, was found with 10% naocl treatment after sodium hypochlorite effects on dentin bond strength and acid-base resistant zone formation by adhesive systems braz j oral sci. 14(4):334-340 337337337337337 restorative system one-step plus and aelite ls clearfil photo bondand majesty clearfil se bond and majesty naocl treatment no y e s no y e s no y e s without tc 50.5 (3.3) a a 38.0 (3.4) c a 50.4 (2.6) aa 42.4 (5.1) bc a 49.9 (6.5) ab a 44.1 (6.0) abc a with tc 35.2 (4.5) bcd b 32.4 (2.7) d a 41.1 (4.1) abc b 34.5 (3.2) cd b 44.2 (6.0) a a 32.5 (3.5) d b table 3. table 3. table 3. table 3. table 3. mean dentin bond strengths (standard deviation) of restorative systems (composite and adhesive) after thermocycling (tc) or not (in mpa). capital letters compare “restorative system/naocl treatment” within the same column. lower case letters compare “thermocycling or not” (row) for the same “restorative system/naocl treatment”. demineralization with the self-etch primer for clearfil se bond (figure 2f). discussion the first tested hypothesis, that 10% naocl and thermocycling would not reduce dentin bond strengths regardless the type of adhesive system used, was rejected because the dentin bond strengths of adhesives were reduced with 10% naocl application and thermocycling. the second hypothesis stating that 10% naocl application would not provide the abrz formation was also rejected, because the self-etch adhesive system formed abrz even with naocl application after etching. secondary caries and restorative material fractures have been considered as major causes for the failure of composite restorations1 and the same adhesive procedures seemed not providing restoration margins free of gaps and microleakage21. regarding caries progression in the dentin margins around adhesive restorations, the presence of abrz below the hybrid layer could protect dentin against recurrent caries attack. the abrz formation is related to penetration of adhesive monomers into the mineralized dentin, in which chemical interaction occurs between the 10-mdp functional monomer and hydroxyapatite22. in this study, etch-and-rinse adhesive systems used according to the manufacturers’ instructions showed external lesions adjacent to hybrid layer and no abrz formation was detected at dentin-resin interface after the acid-base challenge, which corroborates previous studies11-13. however, the hybrid layer disappeared and no erosion lesion was found at the interface after 10% naocl. the hybridization produced by etch-and-rinse adhesives is not a uniform layer, especially with respect to the existence of nanospaces within the hybrid layer, which correspond to dentin etched without monomer infiltration and filled with water or organic solvents23. when 10% naocl was applied to etched dentin, the exposed collagen fibrils were removed and the adhesives were unable to penetrate into underlying dentin, which resulted in no hybrid layer formation11-13. however, it was possible to note that there was a good contact without gaps between adhesives and dentin. for self-etch adhesive, a very thin hybrid layer is formed and after acid-base challenge, clearfil se bond presented abrz approximately 2 mm-thick. when the se primer is applied on the dentin surface, this acidic primer demineralizes the smear layer on the surface and next the underlying dentin. mdp-containing self-etch primer is able to create two layers of demineralized dentin, one totally demineralized and another partially demineralized. thus, when it is applied on mineralized dentin surface, a superficial demineralization adhesive / treatment/ thermocycling 1 2 3 4 one-step plus / no treatment / none 36 30 18 16 / no treatment / thermocycled 26 35 10 20 / 10% sodium hypochlorite / none 13 51 10 39 / 10% sodium hypochlorite / thermocycled 12 40 10 38 clearfil photo bond / no treatment / none 23 25 0 52 / no treatment / thermocycled 12 24 12 51 / 10% sodium hypochlorite / none 14 37 12 37 / 10% sodium hypochlorite / thermocycled 11 36 17 36 clearfil se bond / no treatment / none 34 24 0 62 / no treatment / thermocycled 24 28 6 62 / 10% sodium hypochlorite / none 19 36 10 35 / 10% sodium hypochlorite / thermocycled 13 30 18 39 table 4. table 4. table 4. table 4. table 4. failure modes distribution (%) according to the experimental groups. 1failure within adhesive resin layer; 2failure within hybrid layer; 3cohesive failure in dentin; 4mixed failure involving the adhesive resin and hybrid layer. sodium hypochlorite effects on dentin bond strength and acid-base resistant zone formation by adhesive systems braz j oral sci. 14(4):334-340 338338338338338 fig. 2. sem observations of the adhesive-dentin interfaces after acid-base challenge (x2.000). aclearfil photo bond, bclearfil photo bond applied after 10% naocl, cone-step plus, done-step plus applied after 10% naocl, eclearfil se bond and fclearfil se bond/10% naocl (c: composite; b: adhesive layer; hl: hybrid layer; d: dentin; *: hybrid layer destroyed; arrows: acid-base resistant zone). around 1 mm deep is formed and adjacent dentin at the bottom is also affected, but it is partially demineralized and contains hydroxyapatite, where the mdp chemical interaction occurs24-25. when dentin surface was treated with se primer, rinsed with acetone/water to remove the excess primer on the surface and treated with 10% naocl to deplete the exposed collagen, a very thin, approximately 0.5 mm-thick. abrz was observed. this very thin abrz formation at bottom (sub-surface region) was due to mdp-hydroxyapatite interaction with the remaining hydroxyapatite after collagen removal 8-10. nurrohman, et al.26 (2012) using transmission electron microscopy detected 5 mm hybrid layer formation on demineralized dentin for clearfil photo bond etch-and-rinse adhesive. they also evaluated the interaction between specific functional groups and apatite crystals and showed chemical bonding potential of 10-mdp with the remaining crystals, which produced the abrz zone. another study detected strong affinity of 10-mdp-based self-etch system to deproteinized dentin by formation of 10-mdp-ca salt19. the acid-base challenge destroyed the hybrid layer sodium hypochlorite effects on dentin bond strength and acid-base resistant zone formation by adhesive systems braz j oral sci. 14(4):334-340 formed by one-step plus, while the hybrid layer remained intact for clearfil photo bond. these results suggested that different qualities of hybridization promoted by etch-andrinse adhesive systems maybe caused by difference in the monomer compositions. one-step plus contains bpdm and hema monomers, while clearfil photo bond presents hema and mdp, which contribute to monomer penetration into etched dentin and chemical interaction with hydroxyapatite at the bottom of the hybrid layer, respectively. the application of 10% naocl decreased the dentin bond strength for the tested etch-and-rinse adhesives. however, the results are rather controversial on this matter. while the findings of this study agree with the outcomes from some authors16-18, other studies reported no changes in dentin bond strength when 10% naocl was used 11,19. conversely, some reports showed that the treatment of etched dentin with 10% naocl is beneficial, depending on the adhesive system evaluated12-15. these inconsistent outcomes in the literature hindered a widespread use of this technique. besides, deproteinization with 5% naocl removes collagen from demineralized dentin and also exposes lateral secondary tubules at the intertubular region and peritubular area12. according to prati et al.13 (1999), treatment of etched dentin with naocl produced an unusual type of resin infiltration of mineralized dentin, which may explain the mechanism of resin bonding to naocl-treated dentin. a previous study also reported significant reduction in bond strength to dentin after thermocycling for some adhesives, independent whether they were applied on deproteinized dentin or not19. because 10% naocl removes collagen fibrils8-10, the bonding is formed in absence of collagen fibrils and only by the contact between adhesive monomer and dentin11-13. thus, it was suggested that the only artificial material at the interface, the polymer formed after light activation of adhesives, could undergo degradation depending on monomeric composition, resulting in decrease of bond strength. some hydrophilic monomer resins, such as those in the current adhesives are highly prone to absorb water27. since adhesives used in the present study contain high concentration of hydrophilic monomers such as hema, water sorption of these monomers could contribute to bond strength reduction. in addition, residual water entrapped at the deepest regions of demineralized and/or deproteinized dentin forms poorly polymerized polymer chains28, which are weaker and less stable over time than those formed in absence of moisture. the failure patterns related for each group depended on the adhesive system used and naocl treatment. after thermocycling, a slightly higher percentage of cohesive dentin failure was reported, suggesting degradation of the collagen matrix of dentin or monomeric components of the adhesive systems infiltrated in dentin. secondary caries can initiate at defects, like gaps in the marginal areas of restorations. the self-etch primer system used in this study demonstrated good dentin bonding performance and sealing ability, and also resistance against the acid-base challenge6,20,22,29-30. therefore, formation of an abrz is important for the control of secondary caries around restoration, but this effect depends on the type of adhesive system7,22. the application of 10% naocl changed the demineralization pattern around composite restoration, because there was no hybrid layer formation. the bond strength of one-step plus etch-and–rinse adhesive to sodium hypochlorite-treated dentin did not reduce with thermocycling even without the hybrid layer. however, the use of 10% naocl represents an extra step and may not assure a superior bonding performance when dentin deproteinization is performed after acid etching. within the limitations of this study, the following conclusions were drawn: 1the application of 10% naocl after acid etching did not improve the bond strength to dentin neither after thermocycling; 2abrz formation is materialdependent. references 1. demarco ff, corrêa mb, cenci ms, moraes rr, opdam nj. longevity of posterior composite restorations: not only a matter of materials. dent mater. 2012; 28: 87-101. 2. carvalho fg, puppin-rontani rm, soares le, santo am, martin aa, nociti-junior fh. mineral distribution and clsm analysis of secondary caries inhibition by fluoride/ mdpb-containing adhesive system after cariogenic challenges. j dent. 2009; 37: 307-14. 3. pinto cf, vermelho pm, aguiar tr, paes leme af, oliveira mt, souza em et al. enamel and dentin bond strength, interfacial ultramorphology and fluoride ion release of self-etching adhesives during a ph-cycling regime. j adhes dent. 2015; 17: 27-34. 4. kuper nk, van de sande fh, opdam nj, bronkhorst em, de soet jj, cenci ms et al. restoration materials and secondary caries using an in vitro biofilm model. j dent res. 2015; 94: 62-8. 5. almeida ayres ap, tabchoury cp, bittencourt berger s, yamauti m, bovi ambrosano gm, giannini m. effect of fluoride-containing restorative materials on dentin adhesion and demineralization of hard tissues adjacent to restorations. j adhes dent. 2015; 17: 337-45. 6. tsuchiya s, nikaido t, sonoda h, foxton rm, tagami j. ultrastructure of the dentin-adhesive interface after acid-base challenge. j adhes dent. 2004; 6: 183-90. 7. nikaido t, nurrohman h, takagaki t, sadr a, ichinose s, tagami j. nanoleakage in hybrid layer and acid-base resistant zone at the adhesive/ dentin interface. microsc microanal. 2015; 21: 1271-7. 8. tartari t, de almeida rodrigues silva e souza p, vila nova de almeida b, carrera silva júnior jo, facíola pessoa o, silva e souza junior mh. a new weak chelator in endodontics: effects of different irrigation regimens with etidronate on root dentin microhardness. int j dent. 2013; 2013: 743018. 9. gowda l, das um. effect of various concentrations of sodium hypochlorite on primary dentin: an in vitro scanning electron microscopic study. j clin pediatr dent. 2012; 37: 37-43. 10. sakae t, mishima h, kozawa y. changes in bovine dentin mineral with sodium hypochlorite treatment. j dent res. 1988; 67: 1229-34. 11. vargas ma, cobb ds, armstrong sr. resin–dentin shear bond strength and interfacial ultrastructure with and without a hybrid layer. oper dent. 1997; 22: 159-66. 12. perdigao j, thompson jy, toledano m, osorio r. an ultra-morphological characterization of collagen-depleted etched dentin. am j dent. 1999; 12: 250-5. 13. prati c, chersoni s, pashley dh. effect of removal of surface collagen fibrils on resin-dentin bonding. dent mater. 1999; 15: 323-31. 14. inai n, kanemura n, tagami j, watanabe lg, marshall sj, marshall gw. 339339339339339 sodium hypochlorite effects on dentin bond strength and acid-base resistant zone formation by adhesive systems braz j oral sci. 14(4):334-340 1 7 11 7 11 7 11 7 11 7 1340340340340340 adhesion between collagen depleted dentin and dentin adhesive. am j dent. 1998; 11: 123-7. 15. kanca j, sandrik j. bonding to dentin. clues to the mechanism of adhesion. am j dent. 1998; 11: 154-9. 16. de castro ak, hara at, pimenta la. influence of collagen removal on shear bond strength of one-bottle adhesive systems in dentin. j adhes dent. 2000; 2: 271-7. 17. perdigao j, lopes m, geraldeli s, lopes gc, garcia-godoy f. effect of a sodium hypochlorite gel on dentin bonding. dent mater. 2000; 16: 311-23. 18. uceda-gomez n, reis a, carrilho mro, loguercio ad, rodrigues filho le. effect of sodium hypochlorite on the bond strength of an adhesive system to superficial and deep dentin. j appl oral sci. 2003; 11: 223-8. 19. zhou l, wang y, yang h, guo j, tay fr, huang c. effect of chemical interaction on the bonding strengths of self-etching adhesives to deproteinized dentine. j dent. 2015; 43: 973-80. 20. inoue g, tsuchiya s, nikaido t, foxton rm, tagami j. morphological and mechanical characterization of the acid-base resistant zone at the adhesivedentin interface of intact and caries-affected dentin. oper dent. 2006; 31: 466-72. 21. makishi, p, shimada, y, sadr, a, tagami, j, and sumi, y. non-destructive 3d imaging of composite restorations using optical coherence tomography: marginal adaptation of self-etch adhesives. j dent. 2011; 39: 316-25. 22. nikaido t, inoue g, takagaki t, waidyasekera k, iida y, shinohara ms et al. new strategy to create “super dentin” using adhesive technology: reinforcement of adhesive–dentin interface and protection of tooth structures. japan dent sci rev. 2011; 47: 31-42. 23. ferreira jc, pires pt, azevedo af, oliveira sa, melo pr, silva mj. influence of solvents and composition of etch-and-rinse and self-etch adhesive systems on the nanoleakage within the hybrid layer. j contemp dent pract. 2013; 14: 691-9. 24. yoshihara k, yoshida y, hayakawa s, nagaoka n, irie m, ogawa t et al. nanolayering of phosphoric acid ester monomer on enamel and dentin. acta biomater. 2011; 7: 3187-95. 25. fujita k, ma s, aida m, maeda t, ikemi t, hirata m et al. effect of reacted acidic monomer with calcium on bonding performance. j dent res. 2011; 90: 607-12. 26. nurrohman h, nikaido t, takagaki t, sadr a, ichinose s, tagami j. apatite crystal protection against acid-attack beneath resin-dentin interface with four adhesives: tem and crystallography evidence. dent mater. 2012; 28: 89-98. 27. feitosa vp, sauro s, ogliari fa, yoshihara k, zanchi ch, correr-sobrinho l et al. impact of hydrophilicity and length of spacer chains on the bonding of functional monomers. dent mater. 2014; 30: e317-23. 28. toledano m, cabello i, yamauti m, giannini m, aguilera fs, osorio e et al. resistance to degradation of resin-dentin bonds produced by one-step self-etch adhesives. microsc microanal. 2012; 18: 1480-93. 29. inoue g, nikaido t, foxton rm, tagami j. the acid-base resistant zone in three dentin bonding systems. dent mater j. 2009; 28: 717-21. 30. takagaki t, nikaido t, tsuchiya s, ikeda m, foxton rm, tagami j. effect of hybridization on bond strength and adhesive interface after acid-base challenge using 4-meta/mma-tbb resin. dent mater j. 2009; 28: 185-93. sodium hypochlorite effects on dentin bond strength and acid-base resistant zone formation by adhesive systems braz j oral sci. 14(4):334-340 untitled 1 volume 16 2017 e17079 original article 1 graduate student, graduate program in dentistry, universidade positivo, curitiba, pr, brazil. 2 professor, graduate program in dentistry, universidade positivo, curitiba, pr, brazil. corresponding author: carla castiglia gonzaga universidade positivo rua prof. pedro viriato parigot de souza, 5300 – 81280-330 – curitiba – pr – brasil phone: (5541) 3317-3180 fax: (5541) 3317-3082 e-mail: carlacgonzaga2@gmail.com received: july 25, 2017 accepted: november 23, 2017 effect of hydrogen peroxide and universal adhesive application on the bond strength of glass ƽfiv�tswxw fernando dalitz, dds, msc1; orides ferrari de oliveira junior, dds1; raisa carolina hintz, dds, msc1; gisele maria correr, dds, msc, phd2; leonardo fernandes da cunha, dds, msc, phd2; carla castiglia gonzaga dds, phd2 aim: this study aims to evaluate the effect of different wyvjegi�xviexqirxw�sr�xli�fsrh�wxvirkxl�sj�kpeww�ƽfiv�tswxw�� methods: 2mrix]� yrmvvehmgypev� tviqspevw� leh� xlimv� gvs[rw� viqszih� erh� [ivi� irhshsrxmgepp]� xviexih�� %jxiv� ��l�� tswx� wtegiw�[ivi�tvitevih�erh�xli�vssxw��r!�� �[ivi�hmzmhih�mrxs� wm\�kvsytw�eggsvhmrk�xs�xli�tswx�wyvjegi�xviexqirx��28�ɓ�rs� xviexqirx�� 9%� ɓ� yrmzivwep� ehliwmzi� �7mrkpi� &srh� 9rmzivwep �� 79%�ɓ�wmperi��4vswmp ��jspps[ih�f]�yrmzivwep�ehliwmzi��,�ɓ�, 2 o 2 �;lmxriww�,4�1e\\���� ��kip����qmr ��,9%�ɓ�, 2 o 2 �� jspps[ih� f]� yrmzivwep� ehliwmzi�� erh� ,79%� ɓ� , 2 o 2 �� jspps[ih� f]� wmperi� erh�yrmzivwep�ehliwmzi��8li�tswxw�[ivi�giqirxih�[mxl�hyep� gyvih� viwmr� giqirx� �6ip]<� 9pxmqexi �� &srh� wxvirkxl� [ew� izepyexih�[mxl�typp�syx�xiwx������qq�qmq �ejxiv���l�wxsveki� mr�hmwxmppih�[exiv�ex���o'��(exe�[ivi�wyfqmxxih�xs�%23:%�erh� tukey’s test (d!� ��results: ,79%�kvsyt�wls[ih�xli�lmkliwx� fsrh�wxvirkxl�zepyi��������2 �jspps[ih�f]�,9%��������2 ��28� �����2 �erh�79%������2 �kvsytw��9%�kvsyt�wls[ih�xli�ps[iwx� bond strength value (60 n). conclusion:�8li�ywi�sj�, 2 o 2 , the wmperi�ettpmgexmsr�sv�xli�gsqfmrexmsr�sj�xliwi�x[s�xviexqirxw� wmkrmƽgerxp]�mrgviewih�fsrh�wxvirkxl�zepyiw�mr�kvsytw�mr�[lmgl� xli�yrmzivwep�ehliwmzi�[ew�ywih��%�wmqtpmƽih�tvsgihyvi��wygl� ew� gpiermrk� [mxl� epgslsp�� ger� fi� vigsqqirhih� ew� wyvjegi� xviexqirx�jsv�xli�giqirxexmsr�sj�kpeww�ƽfiv�tswxw� keywords:� 4swx� erh� gsvi� xiglrmuyi�� ,]hvskir� tivs\mhi�� dentin-bonding agents. http://dx.doi.org/10.20396/bjos.v16i0.8651187 mailto:carlacgonzaga2@gmail.com 2 dalitz et al. introduction the dental restoration of endodontically treated teeth is considered one of the most gvmxmgep�viwxsvexmzi�tvsgihyviw��hyi�xs�mxw�gsqtpi\�rexyvi�erh�mrzspziqirx�sj�hmjjivirx� wtigmepxmiw��wygl�ew�)rhshsrxmgw�erh�3tivexmzi�(irxmwxv]��-r�wsqi�wmxyexmsrw��tevxmgypevp]�[lir�xlivi�mw�wmkrmƽgerx�psww�sj�gsvsrep�xssxl�wxvygxyvi��xli�ywi�sj�tswx�erh�gsvi� w]wxiqw�mw�vigsqqirhih��+peww�ƽfiv�tswxw�kemrih�tstypevmx]� mr�xli�pewx�higehiw� figeywi�evi�qsvi�ƽi\mfpi�xli�qixep�tswx�gsviw�erh�lezi�ipewxmg�qshypyw�wmqmpev�xs� the dentin1��1svisziv��wmrgi�xli]�ger�fi�ehliwmzip]�giqirxih��qewxmgexsv]�wxviwwiw� xirh�xs�fi�qsvi�izirp]�hmwxvmfyxih�mr�xli�vssx��viwypxmrk�mr�ji[iv�jvegxyviw�sv�qsvi� jezsvefpi�sriw��rsx�gexewxvstlmg ��[lmgl�ger�fi�wyfnigxih�xs�vitemv1-3. -r�svhiv�xs�tvszmhi�fixxiv�vixirxmsr�jsv�kpeww�ƽfiv�tswxw��pmxivexyvi�vigsqqirhw�ywmrk� zevmsyw�wyvjegi�xviexqirx�xiglrmuyiw��iwtigmepp]�gpiermrk�tswx�wyvjegi�[mxl�epgslsp�� gsrhmxmsrmrk�[mxl�tlswtlsvmg�egmh�sv�l]hvsƽysvmg�egmh��werhfpewx�[mxl�epyqmryq�s\mhi�� wmpmgexmpm^exmsr�� wmperi� erh�sv� l]hvstlsfmg� ehliwmzi� ettpmgexmsr�� erh�� qsvi� vigirxp]�� ettpmgexmsr�sj�l]hvskir�tivs\mhi��, 2 o 2 ) at different concentrations and times4-10. 0mxivexyvi�tviwirxw�wmkrmƽgerx�viwypxw�vikevhmrk�fsrh�wxvirkxl�zepyiw�xs�hirxmr�[lir� kpeww� ƽfiv� tswxw� [ivi� ixglih� [mxl� l]hvskir� tivs\mhi5,9,11-14�� ,]hvskir� tivs\mhi� ex� gsrgirxvexmsrw�sj��� �xs��� �mw�ors[r�xs�ijjigxmzip]�viqszi�e�wyvjegi�pe]iv�sj�its\]� viwmr�sj�kpeww�ƽfiv�tswxw9��,s[iziv��xli�ettpmgexmsr�xmqi�sj���xs����qmryxiw�ywih�mr� earlier studies can be considered long9,15��*i[�wxyhmiw�izepyexih�xli�ywi�sj�, 2 o 2 at hmjjivirx�gsrgirxvexmsrw�erh�ps[iv�ettpmgexmsr�xmqiw5,8��-x�mw�epws�mqtsvxerx�xs�rsxi�xlex� , 2 o 2 �mw�gsqqsrp]�ywih�mr�hirxep�tvegxmgi�jsv�xssxl�[lmxirmrk�erh��xlivijsvi��iewmp]� eggiwwmfpi�jsv�ywi�ew�tswx�wyvjegi�xviexqirx5��1svisziv��mx�mw�mqtsvxerx�xs�iqtlewm^i� xlex�xlivi�mw�rsx�]ix�irsykl�mrjsvqexmsr�efsyx�xli�fiwx�gsrgirxvexmsr�erh�ettpmgexmsr� xmqi�sj�l]hvskir�tivs\mhi�mr�xli�wyvjegi�xviexqirx�sj�kpeww�ƽfiv�tswxw5,9,11,13-15. 9rmzivwep� ehliwmziw� evi� wmqtpmƽih� ehliwmzi� w]wxiqw� xlex� ger� fi� ywih� [mxl� fsxl� ettvsegliw��xsxep��erh�w�wipj�ixglmrk �erh��figeywi�sj�xlmw��evi�gsrwmhivih�qsvi�zivsatile16,17. these universal adhesives differ from current self-etching systems by incortsvexmrk�qsrsqivw�getefpi�sj�tvshygmrk�e�gliqmgep�fsrh�[mxl�xli�hirxep�xmwwyiw17,18. ,s[iziv��xlivi�mw�pmxxpi�mrjsvqexmsr�sr�xli�tivjsvqergi�sj�xliwi�ri[�yrmzivwep�ehlisives. chen et al.19������ �vitsvxih�xlex�xli�fsrh�wxvirkxl�sj�e�yrmzivwep�ehliwmzi�xs� dentin is not different from that of totaland self-etching adhesives. this study also 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adhesive (hua) 206.5 r 64.6 ab hydrogen peroxide, silane and universal adhesive (hsua) 236.5 r 70.1 a different superscript letters indicate statistically significant differences among the groups (p < 0.05). 5 dalitz et al. discussion 8li�l]tsxliwmw�wxyhmih�mr�xli�tviwirx�[svo�[ew�vinigxih�figeywi�xli�hmjjivirx�wyvjegi�xviexqirxw�sj�kpeww�ƽfiv�tswxw�mrgviewih�xli�fsrh�wxvirkxl�zepyiw�xs�vssx�hirxmr�� *syv�kvsytw�wls[ih�wxexmwxmgepp]�wmqmpev�fsrh�wxvirkxl�zepyiw�xs�xli�gsrxvsp�kvsyt�� 3rp]�xli�yrmzivwep�ehliwmzi�kvsyt�wls[ih�ps[iv�fsrh�wxvirkxl�zepyiw�xlex�[ivi�wxexmwxmgepp]�hmjjivirx�jvsq�xli�sxliv�kvsytw�erh�epws�jvsq�gsrxvsp�kvsyt� 8li�ywi�sj�hmjjivirx�wyvjegi�xviexqirxw�mr�kpeww�ƽfiv�tswxw�emqw�xs�tvsqsxi�e�fixxiv� fsrh�fix[iir�xli�tswx�erh�xli�viwmr�giqirxw�ywih�jsv�pyxmrk��+peww�ƽfiv�tswxw�evi� gszivih�f]�er�its\]�viwmr�pe]iv�xlex�lew�lmkl�hikvii�sj�gsrzivwmsr�erh�ji[�viegxmzi� sites for chemical adhesion to other resin materials22,23��8lmw�lmklp]�vixmgypexih�erh�ps[� 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viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1539/1192 9/9 1 volume 16 2017 e17053 original articlebjos 1 dds, msc, phd, professor of the graduate program in dentistry, operative dentistry area, universidade positivo, curitiba, pr, brazil. 2 dds, msc, graduate student of the graduate program in dentistry, operative dentistry area, universidade positivo, curitiba, pr, brazil. corresponding author: leonardo fernandes da cunha universidade positivo 5300 professor pedro viriato parigot de souza street curitiba, pr zip code: 81280-330 e-mail: cunha_leo@me.com; tel: +55 41 3317-3403; fax: +55 41 3317-3082 received: may 27, 2017 accepted: july 26, 2017 physical properties of two bis-acryl interim materials: color stability, flexural strength and shear bond strength to flowable composite resin as add-on material leonardo fernandes da cunha1*, ana beatriz franco fernandes2, amanda mahammad mushashe2, gisele maria correr1, carla castiglia gonzaga1 aims: the objective of this study was to evaluate the mechanical properties of two bis-acryl interim resin materials, such as color stability, flexural strength and shear bond strength to flowable composite resin, simulating clinical situations when this material has to be used for repair as add-on. two shades of two bis-acryl interim resin materials [structur 2 sc (shades bleach and a2); protemp 4 (shades a1 and a2)] were evaluated. discs (5 x 1 mm) were fabricated and baseline color was determined after 1 h. ten specimens were immersed at 37oc in solutions of distilled water (control) and cola-based soft drink (coca-cola). color measurements were performed with a spectrophotometer using cielab parameters. color readings were again measured after 2 hours, 4 hours, 24 hours and 7 days. flexural strength was determined using the three-point bending test (10 x 1 x 2 mm) on a universal testing machine (0.5 mm/min) (n = 10). discs of bis-acryl resin were embedded in acrylic resin, planned and distributed in 2 groups: g1 filtek z350 flow/protemp4 and g2 grandio so flow/structure 3 (n = 15). cylinders (3.5 x 2 mm) were made with the flowable composite resins and polymerized for 20 s. the specimens were stored in distilled water at 37oc for 24 h and subjected to shear bond strength test. data were analyzed using one-way anova and tukey’s test (α = 0.05). δe values were higher for structur bleach (3.08)a compared with protemp 4 (shade a1, 2.22)b (shade a2, 2.25)b. there were no significant differences between structur bleach and structur a2 (2.62)ab. coca-cola presented higher δe values (3.08)a than (2.00)b. regarding time, http://dx.doi.org/10.20396/bjos.v16i0.8650498 2 cunha et al. introduction bis-acryl interim resin materials have been extensively applied for prosthetic treatments. properties such as fastness of set process, ease of handling and aesthetic quality of these materials allow its use in several clinical situations during prosthetic rehabilitation, such as temporary restorations and construction of dental mock up for aesthetic and functional evaluations. as interim material, it is essential that they remain stable over the period of rehabilitation. in order to achieve these purposes, they should present good mechanical properties and good color stability1-4. in the case of aesthetic rehabilitations, such as anterior interim restorations, the color stability of bis-acryl interim resin may be considered one of the most important factors for the clinical success. different composite resins or different luting cements present distinctive degree of the color change5,6. thus, different bis-acryl interim resin materials may also present distinctive color stability. color changes over time due to the use of beverages with staining potential (e.g., water, wine, and cola-based soft drink) have been previously described to resin-based materials5,6. in addition, inherent properties of different bis-acryl materials can interfere in their color stability. materials with smoother surfaces and less hydrophilicity tend to present less sorption of staining solutions, therefore, present better color stability7. in this way, the effect of different solutions with staining potential on bis-acryl interim resin must be evaluated. in some clinical situations, interim restorations need to stay in function for a longer period than originally planned. besides color stability, another important aspect in these cases of long-span interim restoration is their flexural strength4. low flexural strength can lead to fractures, resulting in functional and esthetic problems. however, there is a lack of sufficient information about the mechanical properties of bis-acryl interim resin materials4. additionally, bis-acryl interim resins may need add-on material for repair, modification of form or reduce discrepancies at the margin8. according to the manufacturer recommendation and lee and lee8 (2015), bis-acryl-based interim material has favorable add-on properties when used with a flowable composite resin. thus, the aim of this study is to evaluate the mechanical properties of two bis-acryl interim resin materials, such as color stability, flexural strength and shear bond strength δe values increased from 1.84a after 2 h to 2.31b after 4 h. the higher values were observed after 24 h and 7 days (2.93c and 3.09d, respectively). no significant differences were observed for the flexural strength of structur (22.05 mpa)a and protemp 4 (19.01 mpa)a. the repairs executed with structur/grandio flow (9.21 mpa)a were similar to those performed with protemp 4/z350xt flow (10.71 mpa)a. it can be concluded that the two bis-acyl resins evaluated showed similar physical and mechanical properties. keywords: mechanical properties, provisional materials, shear strength, flexural strength, color stability. 3 cunha et al. to flowable composite resin, simulating clinical situations when this material has to be used for repair as add-on. the hypotheses evaluated were: i) there would be differences between the two bis-acryl materials evaluated regarding color stability, flexural strength and shear bond strength to flowable composite resin; and ii) immersion in different staining solution would induce color changes in the two bis-aryl resins. material and methods the materials used in this study and their composition are described in table 1. optical and mechanical properties of two bis-acryl materials were evaluated by three steps: evaluation of color stability after immersed in different storage solutions, by means of a spectrophotometer; three bending point assay to test flexural strength and shear bond strength between the bis-acryl materials and two flowable composites. color stability two shades of two bis-acryl interim resin materials [structur 2 sc (shades bleach and a2) voco, germany; protemp 4 (shades a1 and a2) 3m espe, st. paul, mn, usa] were evaluated in this study. all specimens were prepared at the same laboratory with controlled humidity (55 ± 5%), temperature (23 ± 1ºc) and illumination conditions. twenty disk specimens (5 mm in diameter and 1 mm in thickness) were prepared for each shade and material. each specimen was made by inserting the bis-acryl resin in a teflon mold ring and pressed between two 1-mm-thick glass slides (separated by mylar strips) under finger pressure. all samples were prepared according manufacturers’ instructions (4 minutes). the samples were subjected to polishing procedures as described by the manufacturers. the color measurements were performed with a spectrophotometer (easyshade advance, vita zahnfabrik, bad sackingen, germany) according to the cielab coordinates, under a standardized white background. afterwards, ten specimens were immersed at 37oc in solutions of distilled water (control) and cola-based soft drink (coca-cola). the specimens were then stored in dark canisters containing water or coca-cola at 37oc, and the color parameters were again measured after 2 hours, 4 hours, 24 hours and 7 days. the cielab coordinates were used to calculate the color difference (δe) between the “before” (baseline) and “after” periods. table 1. compositions of materials used in this study material composition protemp 4 (3m espe, usa) bis-gma, dimethacrylate polymer, zirconia silica, fumed silica, silane structur 2 sc (voco, germany) bis-gma, bht, amines, benzoyl peroxide, dimethacrylates, glass particles filtek z350 (3m espe, usa) bisgma, bisema, udma.tegdma, sílica, zirconia,clusters, zirconia/sílica aggregated particles grandio so flow (voco, germay) bis-gma, bis-ema, tegdma, glass ceramic, functionalized sio2 nano-particles 4 cunha et al. before each color measurement, the disks were ultrasonic cleaned for 60 seconds and then specimens were dried with absorbent paper. the δe for each experimental time was calculated using the equation: δe = [(δl)2 + (δa)2 + (δb)2]1/2 where δl, δa and δb are the differences in the respective values before and after aging. the results of δe were analyzed by three-way anova with repeated measures (material, solution and time) and tukey’s hsd test (α= 0.05). flexural strength ten specimens were made for each interim restorative material for the evaluation of the flexural strength. a split stainless steel was used to produce the specimens with 10 x 2 x 1 mm. bis-acryl resins were injected into the mold according, as described previously for disks. excess material was removed, the specimens were polished and then stored in distilled water at 37oc for 24 hours. the flexural strength was determined using the three-point bending test on a universal testing machine (emic dl 2000, são josé dos pinhais, pr, brazil) at a crosshead speed 0.5 mm/min. the flexural strength (s) in mpa was calculated by the following formula: s = 3fl/2bh2 where f is fracture load (n), l is the span length (6 mm), b and h are, respectively, the width and height of the specimen (mm). the results of the flexural strength were analyzed by student’s t test (α= 0.05). shear bond strength thirty bis-acryl specimens of 6 mm of diameter and 2 mm of thickness were fabricated, as described previously (n = 15 for structur and protemp4). after polishing, the specimens were embedded in pvc cylinders with acrylic resin (jet, artigos odontológicos clássico, são paulo, brazil). a teflon matrix (3.5 mm in diameter and 1 mm in thickness) were placed onto the bis-acryl resin surface and filled with two flowable composite resins (shade a2, grandio flow, for structur samples and filtek z350 xt flow for protemp samples, 3m espe, st. paul, mn, usa). the specimens were light-cured through for 20 s with a led curing unit (poly wireless, kavo, joinville, sc, brazil) at 1100 mw/cm2. before adding the flowable resins on bis-acryl specimens, no surface treatment was performed. the specimens were then stored in distilled water at 37oc for 24 h. after the storage period, the specimens were submitted to shear bond strength test in a universal testing machine at a crosshead speed of 0.5 mm/min. data were analyzed using student’s t test (α= 0.05). results color stability means and standard deviations for δe values are presented in table 2. significant differences were observed for material (p = 0.008822), solution (p = 0.000001) and time (p = 0.000001). all double (p < 0.0001) and triple (p = 0.040033) interactions were also significant. δe values were higher for structur bleach (δe = 3.08 ± 2,27 )a compared with 5 cunha et al. protemp 4 (shade a1, δe = 2.23 ± 1,04)b (shade a2, δe = 2.26 ± 0.86)b. there were no significant differences between structur bleach and structur a2 (δe = 2.62 ± 1.48)ab. it was observed that coca-cola presented higher δe values (δe = 3.08 ± 1.85)a when compared to water (δe = 2.00 ± 1.09 )b. when the storage times were considered, the mean δe values increased from 1.84 ± 1.03 a after 2 hours to 2.32 ± 1.30b after 4 hours. the higher values were observed after 24 hours and 7 days (δe = 2.93 ± 1.78c) and 3.10 ± 1.65 d, respectively. flexural strength student’s t-test showed no significant difference between the flexural strength of structur and protemp4 (p = 0.115). the structur flexural strength (22.05 ± 5.71 mpa) was similar to the one presented by protemp 4 (19.01 ± 3.06 mpa). shear bond strength there were no statistically significant differences between the materials tested (p = 0.228). the repairs executed with structur/grandio flow (9.21 ± 3.72 mpa)a were similar to those performed with protemp4/z350xt flow (10.71 ± 2.86 mpa)a. all the failures were adhesive. discussion the hypotheses tested about color stability in the present study were accepted. there were significant differences between the shades evaluated, the immersion solutions used and different periods of time. the staining of dental materials is the result of both extrinsic and intrinsic factors9. cola-based soft drinks, wine, tea and coffee, for example, are commonly consumed beverages and other studies have demonstrated the discoloration of composite materials upon exposure to these solutions9-11. although the immersion for longer periods of time does not represent a clinical situation, this method provides a better understanding of the staining potencial of several solutions and color stability of materials subjected to immersion. other studies used this method to verify color stability of materials10-13. in the present study, immersion in water and cola-based soft drink caused changes in color of both materials studied, however, the color table 2. means and standard deviations for ∆e values. material solution ∆e 2 h 4 h 24 h 7 days structur bleach water 1.18±0.46 de 0.97±0.72e 2.32±1,26 bcde 1.61±0.93 cde coca-cola 2.25±0.53 bcde 3.92±1.42 b 6.51±1.66 a 5.91±1.29a structur a2 water 2.60±1.77bcde 2.91±1.80bcde 1.77±0.73bcde 3.15±1.12bcd coca-cola 1.70±0.84cde 2.21±0.63bcde 3.15±1.56bcd 3.46±2.19bc protemp a1 water 1.34±0.75cde 1.65±0.54cde 1.75±0.6bcde 1.80±0.4bcde coca-cola 1.99±1.20bcde 2.78±1.0bcde 3.10±0.78bcde 3.41±0.9bc protemp a2 water 2.04±0.65bcde 1.84±0.79bcde 2.37±0.93bcde 2.77±0.94bcde coca-cola 1.65±1.05cde 2.25±0.94bcde 2.47±0.79bcde 2.65±0.84bcde *means follow by different superscript letter are significantly different (p < 0.05). 6 cunha et al. changed was significantly higher for the cola-based drink. the results of this study indicated that the dietary habits of the patient could present a potential discoloration risk to provisional restorations. also, the water absorbed by the resin matrix can cause filler matrix debonding and hydrolytic degradation of the material14,15. however, in the present study, the two bis-acryl materials tested demonstrated no significant differences for color change. the effect of shades on the color stability of composite resins and luting cements was previously described6,15-18. in the present study, the color change for the materials with lighter and less chromatic shades was higher. these results corroborate with those found by uchida et al.17 (1998). these authors, by means of a quantitative analysis of color stability, verified a higher δe for the lighter shades of two composites. this study suggests that this fact may result from two factors: (a) discoloration through environmental breakdown of the polymer leading to release of monomers and the shift of color from the cured resin to that of the monomers and (b) the environmental effect on the retention and/or stability of pigments and other additives in the polymer formulations. the environmental effect on the pigments and other additives need clarification through additional research. δe values showed a tendency to increase as immersion period increased, suggesting that the color of the material would tend to change over long-term clinical use6,15. however, all the values reported color changes with δe values lower than the 3.3 threshold, being clinically acceptable19. the flexural strength of an interim restorative material is considerably tested during mastication. acceptable flexural strength is crucial to avoid repair procedures that can be time consuming or the fracture of these restorations can lead to functional and aesthetic problems. this factor is especially important in multiple-unit or long-span prosthesis, whose pontics are constantly submitted to flexural tensions during function4. in the present study, the materials tested presented similar acceptable flexural strength. flexural strength test usually follow the iso 4049 standard that state specimen with 25 x 2 x 2 mm. the dimensions used in the present study are different from the iso, however also observed in prior studies20-22. other dimensions are also used in the literature, such at vieira et al.22 (2012) and firoozmand and pagani23 (2009). distinctive methods can evaluate the bond strength to determine the adhesion between different materials, such as microtensile, microshear or shear bond strength24. besides its irregular distribuition of stress within the surface, shear bond strength tests are easier to perform when compared to microtensile and micro shear assays. similarly to many studies8,25-28, here, the shear bond strength method was used to determine the bond strength between bis-acryl interim resin materials and flowable composite resin, simulating an add-onrepair or modification of form. this method has the advantage to mimic the clinical condition very closely because it results in stress on the interface between the materials8,24-28. this study showed no differences between the two materials tested, regarding bond strength. also, additional treatments have been proposed to improve the bond strength of these materials, such as, applying bonding agent or additional light polymerization8. lee and lee8 (2015), verified that the use of those methods increased the shear bond strength of add-on materials to bis-acryl resins when compared to the untreated specimens. 7 cunha et al. however, other studies are still necessary to corroborate the clinical use of these strategies, particularly in cases of interim restorations used in oral rehabilitation that remain in function for long periods of time. this study has a number of limitations, mostly related to the conditions represented. in order to promote a better clinical correlation of the outcomes, more variables should be assessed, such as thermocycling of optical and mechanical properties specimens and immersion in solutions with different ph. in addition, different polishing and finishing protocols can be evaluated to identify the changes in color stability of bis-acryl resins. thus, future studies must be performed to evaluate the clinical performance of these materials. therefore, based on the results of the present study, it can be concluded that the color stability of the bis-acryl resin tests decreased with storage time and storing in cola-based soft drink. there was no difference between materials regarding fleruxal strength and shear bond strength. references 1. haselton dr, diaz-arnold am, dawson dv. color stability of interim crown and fixed partial denture resins. j prosthet dent. 2005 jan;93(1):70-5. 2. stansbury jw, trujillo-lemon m, lu h, ding x, lin y, ge j. conversion-dependent shrinkage stress and strain in dental resins and composites. dent mater. 2005 jan; 21(1):56-67. 3. balkenhol m, knapp m, ferger p, heun u, wostmann b. correlation between polymerization shrinkage and marginal fit of temporary crowns. dent mater. 2008 nov; 24(11): 1575-84. 4. mehrpour h, farjood e, giti r, barfi ghasrdashti a, heidari h. evaluation of the flexural strength of interim restorative materials in fixed prosthodontics. j dent (shiraz). 2016 sep;17(3):201-6. 5. spina dr, grossi jr, cunali rs, baratto filho f, da cunha lf, gonzaga cc, et al. evaluation of discoloration removal by polishing resin composites submitted to staining in different drink solutions. int sch res notices. 2015 aug; 20(1):2015:853975. doi: 10.1155/2015/853975. 6. pissaia jf, correr gm, gonzaga cc, cunha lf. influence of shade, curing mode, and aging on the color stability of resin cements. braz j oral sci. 2015 oct;14(4): 272-5. 7. berber a, cakir fy, baseren m, gurgan s. effect of different polishing systems and drinks on the color stability of resin composite. j contemp dent pract. 2013 jul;14(4): 662-7. 8. lee j, lee s. evaluation of add-on methods for bis-acryl composite resin interim restorations. j prosthet dent. 2015 oct;114(4):594-601. doi: 10.1016/j.prosdent.2015.02.020.  9. reddy ps, tejaswi kls, shetty s, annapoorna bm, pujari sc, thippeswamy hm. effects of commonly consumed beverages on surface roughness and color stability of the nano, microhybrid and hybrid composite resins: an in vitro study. j contemp dent pract. 2013 jul;14(4):718-23. 10. rutkunas v, sabaliauskas v, mizutani h. effects of different food colorants and polishing techniques on color stability of provisional prosthetic materials. dent mater j. 2010 mar;29(2):167-76. 11. ferracane jl, 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composite resin materials for crown and bridge veneers. dent mater. 1987 oct;3(5):246-51. 17. uchida h, vaidyanathan j, viswanadhan t, vaidyanathan tk. color stability of dental composites as a function of shade. j prosthet dent. 1998 apr;79(4):372-7. 18. lee yk, powers jm. color and optical properties of resin-based composites for bleached teeth after polymerization and accelerated aging. am j dent. 2001 dec;14(6): 349-54. 19. kadiyala kk, badisa mk, anne g, anche sc, chiramana s, muvva sb, et al. evaluation of flexural strength of thermocycled interim resin materials used in prosthetic rehabilitationan in-vitro study. j clin diagn res. 2016 sep;10(9):zc91-zc95. 20. fonseca rb, marques as, bernades k de o, carlo hl, naves lz. effect of glass fiber incorporation on flexural properties of experimental composites. biomed res int. 2014;2014:542678. doi:10.1155/2014/542678. 21. fonseca rb, de paula ms, favarão in, kasuya av, de almeida ln, mendes ga, et al. reinforcement of dental methacrylate with glass fiber after heated silane application. biomed res int. 2014;2014:364398. doi: 10.1155/2014/364398. 22. vieira c, silva-sousa yt, pessarello nm, rached-junior fa, souza-gabriel ae. effect of highconcentrated bleaching agents on the bond strength at dentin/resin interface and flexural strength of dentin. braz dent j. 2012;23(1):28-35 23. firoozmand lm, pagani c. influence of bleaching treatment on flexural resistance of hybrid materials. acta odontol latinoam. 2009;22(2):75-80. 24. da cunha lf, nascimento bm, baratto ssp, gonzaga cc, furuse ay, mondelli j, et al. influence of different surface treatments on the shear bond strength of a methacrylate resin composite repaired with silorane-based resin. rsbo. 2013 jul-sep;10(3):240-4. 25. hagge ms1, lindemuth js, jones ag. shear bond strength of bis-acryl composite provisional material repaired with flowable composite. j esthet restor dent. 2002;14(1):47-52. 26. chen hl, lai yl, chou ic, hu cj, lee sy. shear bond strength of provisional restoration materials repaired with light-cured resins. oper dent. 2008 sep-oct;33(5):508-15. doi: 10.2341/07-130. 27. shim js, park yj, manaloto a, shin s, lee j, choi y, ryu j. shear bond strength of four different repair materials applied to bis-acryl resin provisional materials measured 10 minutes, one hour, and two days after bonding. oper dent. 2014 jul-aug;39(4): e147-53. doi: 10.2341/13-196-l. 28. shim js, lee jy,choi yj, shin sw, ryu jj. effect of light-curing, pressure, oxygen inhibition, and heat on shear bond strength between bis-acryl provisional restoration and bis-acryl repair materials. j adv prosthodont. 2015 feb;7(1):47-50. doi: 10.4047/jap.2015.7.1.47. untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8652906 volume 17 2018 e18150 original article 1 universidade estadual do piauí – uespi, school of dentistry, department of clinical dentistry, area of integrated clinic, parnaíba, pi, brazil. corresponding author: ana de lourdes sá de lira universidade estadual do piauí, faculdade de odontologia rua senador joaquim pires 2076 ininga. fone (86) 999595004 cep: 64049-590 teresina-pi-brasil email: anadelourdessl@hotmail.com received: december 25, 2017 accepted: june 24, 2018 relationship between oral candidiasis and users of dental prostheses ana de lourdes sá de lira1, aline cardoso torres1 aim: to investigate the relationship between oral candidiasis and users of dental prostheses. methods: a cross-sectional study was carried out which the inclusion of adults and elderly people of both genders using dental prostheses. lectures were given with guidelines on oral hygiene techniques and dental prostheses, preventive measures of candidiasis, highlighting their influence on the oral and general health of the individual for the 240 users of dental prostheses. of this number, 153 did not want to participate in the clinical examination. signals and symptoms were analyzed in 87 patients, and lesions were present in only 21, and a cytological complement of the lesions was performed. data were analyzed using spss version 15.0 with chi-square (χ2) test at p < 0.05. results: the 21 patients presented oral candidiasis, with erythematous (prosthetic) stomatitis being the most prevalent. there was a correlation between the presence of candida and the use of prostheses (pvalue 0.00*). candidiasis was associated with poorly adapted prostheses, poor oral hygiene, inadequate prostheses and prolonged use of them. conclusion: there was a correlation between oral candidiasis and users of dental prostheses. the most prevalent type of candidiasis was erythematous stomatitis (prosthetic), and nystatin was used to treat all cases. the preventive activities carried out were extremely important, since patients were not aware of oral candidiasis and the factors that trigger it, such as poor oral hygiene and dental prostheses. keywords: candidiasis, oral. dental prosthesis. candida albicans. mailto:anadelourdessl@hotmail.com 2 lira et al. introduction the higher prevalence of different candida species in denture weares in contrast to no denture weares demonstrated that denture insertion induces plaque formation favoring the increased population of potentially pathogenic bacteria and candida spp. the most of the denture plaque occurs on the denture-base of the upper denture, and hence the tissue contact surface of the upper denture was considered for isolation of candida1. the most prevalent fungal infection in the oral cavity of humans is oral candidiasis or candidosis. it mainly affects the jugal mucosa, palate and back of the tongue. the fungus responsible for this disease is of the genus candida, and there are several types of the same species, for example: c. tropicalis, c. glabrata, c. krusei and c. albicans. however, the species c. albicans is the most present and pathogenic of all species of the genus candida1-4. among the main etiological factors for the development of candidiasis infection are immunosuppressive diseases such as hiv and aids, cancer treatments, organ transplants, diabetes, use of corticosteroids and antibiotic therapy. it is an opportunistic disease and depending on the state of the host can manifest as bacterial sepsis and spread through several organs causing morbidity or even death of the individual5. oral candidiasis can be divided into pseudomembranous which is characterized by the appearance of removable white plaque, erythematous by the presence of erythema, occurring more frequently on the palate and dorsum of the tongue, and may or may not have small petechiae or granules scattered throughout the affected region. this is the typical form found in elderly patients with total or partial dentures, known as stomatitis prosthetic. hyperplastic manifestation is less common with white lesions due to hyperkeratosis, and are unstable by scraping. the mucocutaneous is characterized by the presence of white plaques and reddish areas³, 4. the use of dental prosthesis associated with poor hygiene, both buccal and prosthesis, are also important local etiological factors4. people who use removable prostheses are more likely to have candidiasis compared to individuals who do not use them because of the ease of adhesion of microorganisms to acrylic prostheses, leading to the development of infection6. mechanical factors are also related to oral candidiasis, such as: loss of vertical dimension of occlusion with flaccidity of the upper lip over the lower one at the angle of the mouth, absence of teeth and use of maladaptive prostheses, the elderly population being more likely to get this infection. generally they tend to develop successive infections in short intervals, therefore, it is necessary to take therapeutic measures to avoid recurrence of the disease7-9. the importance of prevention, early diagnosis and immediate treatment of the present oral lesions is emphasized with dissemination of knowledge of good oral hygiene habits, promoting educational actions, emphasizing the importance of hygiene of dental prostheses, through materials and appropriate techniques. the alkalinization of oral ph and use of antifungal drugs are effective behaviors adopted in the treatment and control of the condition, with nystatin being the most commonly used drug10. 3 lira et al. the aim of this research was to investigate the relationship between oral candidiasis and users of dental prostheses. it is believed that oral candidiasis is directly related to maladaptive dentures or in poor conditions due to the long time of use, or precarious oral hygiene. material and methods a cross-sectional study was carried out which the inclusion of adults and elderly people of both genders using dental prostheses. the sample totaled 240 people, after the sample calculation, who were attended monthly by the family health strategy doctor in the basic health unit (bhu) of the district planalto and samaritana in the city of parnaíba-pi. these patients had already had dental consultations. the descriptors used were: oral candidiasis, dental prosthesis and candida albicans. for the review of literature and discussion, searches were indexed in the databases lilacs, medline, scielo, bireme, pubmed. the researchers were provided with the letter of consent of the nurses heads of the basic health units (bhu) in the city of parnaíba, piauí, after authorization to carry out the research development and approval of the research ethics committee of the state university of piauí, on the basis of opinion 1665.758. oral lectures with emphasis on preventive measures and early diagnosis (figure 1) on oral candidiasis, its etiological factors and consequences were conducted biweekly during the year of the research, coinciding with the day of routine medical care with the oral health strategy (bhu). then, the free and informed consent form (ficf) was given to each patient, and the identification, health and oral hygiene and the prosthesis questionnaire were applied to be drawn the profile of each research member. the questionnaire was applied with fifteen objective questions contained demographic information such as age, sex, time of wearing, and other questions to know the attitude of denture hygiene habits, frequency of cleaning, and nocturnal denture wearing habits. two researchers were trained at the clinical school of dentistry (csd) of the state university of piauí to identify possible lesions of oral candidiasis. subsequently, calibration exercises were performed with 11 persons not participating in the sample plan, in a bhu of the municipality, according to the methodology described in another publication10. the pilot study involving 30 elderly people was carried out with the objective of testing the proposed methodology. as a result, its viability was observed, without adjustments. figure 1. scraping with swab(1), application of the material(2) and fixation with cytological fixative(3) and storage of the blade in blade holder(4). 4 lira et al. in order to assess intraand inter-examiner diagnostic reproducibility, 10% of the total sample was double-examined by each of the examiners, with the kappa coefficient obtained for intra and inter-examiner agreement 0.97 and 0.91, respectively. of the 240 patients who participated in the lectures, only 87 patients (55 elderly ≥ 60 years and 32 adults) users of dental prostheses were performed oral examinations in the bhu. among the reasons that justify the non-participation of the others stand out shame when removing the prosthesis, lack of interest and availability of time. the objective of the examination was to investigate the possible presence of candidiasis, prosthetic conditions and the need for treatment, using the clinical examination method (signs and symptoms) using natural light and a led flashlight for better visualization, gloves for procedures and wooden spatula and complementary examination (exfoliative cytology)9,11. white lesions, when easily removed, are pathognomonic signs of pseudomembranous candidiasis. cytological examination was performed in these 21 patients, regardless of the type of lesion present, although some authors9,10 affirm that in the red lesions the yeast and filamentous forms of candidiasis are generally not present, and cytology is not necessary. the sites where these lesions were detected were previously dried with sterile gauze and scraped with sterile swab for collection (dalian goodwood wooden ltd, youjiacun xinzhaizi town, dalian, china). after swab scraping, the smear was deposited on the blade (cral disposable labware products ltd, cotia, são paulo, brazil), in a single movement from left to right so as not to damage the harvested material, which was then fixed with cytological fixative (figure 1). the slides were stored in the slide holder (deskarplas disposable products, mogi das cruzes, são paulo, brazil) and sent to the laboratory to be stained using the papanicolaou papanicolaou stain or harris hematoxylin (specialized chemistry erich ltda, são paulo, brazil) and visualized in binocular biologic electronic microscope (m50a, nikon instruments inc, melville, new york, united states of america)¹. after the staining process, the clinical analyzes were performed for laboratory diagnosis, in the laboratory of the federal university of piauí (campus ministro reis veloso). the diagnosis of oral candidiasis was based on clinical signs and symptoms, aided by microscopic examination of the smear. patients who presented lesions, independently of the laboratory report, were treated with nystatin, under the supervision of dental surgeons of the ubh, for having exclusive effect on candida and absence of systemic effects11-13. based on ferreira et al12. protocol the patients were oriented to use disinfectant solutions to clean dental prosthesis and found that naocl at 0, 5% was the most effective in the adhesion control of candida yeasts and they also were oriented to remove the dental prosthesis during sleep, because of the relaxation and rest of the tissues, including the cleaning action executed by the saliva, tongue and cheeks are fundamental to maintain the oral mucosa healthy. the measures adopted were as follows: prescription nystatin oral suspension (micostatin® 100,000 iu / ml) -blood for 1 minute and swallow 5 ml, 4 times daily for 5 lira et al. 7 to 15 days until lesion remission; oral hygiene instruction; guidance on the chemical disinfection of prostheses with water solution and sodium hypochlorite (0.25%), in the proportion of 1 ml to 10 ml of drinking water, for half an hour, twice a week, at least; suggestion for nocturnal resting of the prostheses and making new prostheses if necessary. in the removable partial dentures, the hygiene was performed with neutral soap, dental brush, and placed in a glass with water during sleep14. a descriptive analysis of the data was performed using frequency and number tables using microsoft windows excel 2013 software. the results were compared using the nonparametric chi-square (x2) test at a significance level of 5% (p< 0.05) in the statistical package spss version 15.0 (social package statistical science). results regarding the sociodemographic situation of the research, the 240 members of the research participated in the lectures and answered the questionnaire. their mean age was 59.20 years (sd = 13.02). of these, 86.6% (n = 208) were of the female gender and 13.33% (n = 32) of the masculine gender who declared having a monthly income of r$1.093 (sd = 67.20). of the 87 people who were willing to perform the oral exam, 70 were female and 17 male. of these, 21.25% (n = 51) reported frequent use of antibiotics, but only 13.33% (n = 32) did so only by prescription. in addition, 10% (n = 24) reported consuming alcoholic beverages, 7.08% (n = 17) smoked and 6.25% (n = 15) had any sexually transmitted diseases (stds). regarding chronic diseases, it was observed that 44.58% (n = 107) reported having hypertension, followed by allergic problems respectively 15.83% (n = 38), diabetes and anemia 12.91% (n = 31), respiratory problems 11.25 % (n = 27) and heart problems 7.91% (n = 19). about the oral hygiene conditions of the total sample, the majority reported adequate hygiene of 96.25% (n = 231), but it was reported that they did not use dental floss and only removed their dentures to sanitize them once daily, being the majority with toothpaste and water (n = 165) and the others with neutral soap and water (n = 75). in addition, a large number of participants do not remove the prosthetic devices to sleep (n = 210), and those who do so place them in a glass of water. regarding the type of dental prosthesis used, it was observed that the prosthetic combinations were: removable partial prosthesis (rpp) in both arches in 75% (n = 180), superior total prosthesis (tp) associated with the lower rpp in 17.08% (n = 41) and tp in both arches in 7.92% (n = 19). however, 17.5% (n = 42) reported feeling some discomfort due to their poor adaptation. cracks were observed in 8.33% (n = 20) and spots in 4.58% (n = 11) of the prostheses. when asked which professional had made the prosthesis, most did not know how to inform. however 30% (n = 72) reported that it was the dental surgeon and 15% (n = 36) the prosthetic. in descending order, the oral clinical examinations made possible to find the following pathologies: caries, periodontal disease, tasty tongue, residual roots, tongue lost and ulcers. of the 87 examined 21 (24.14%) had lesions of candidiasis, 14 of them female, with ages between 47 and 65 years and 11 of the male, with age between 60 and 72. 6 lira et al. among the women 12 they used rpp superior and inferior. all males and two females used upper tp and lower rpp. there was a correlation between the presence of candida and the use of prostheses(pvalue 0.00*). prosthetic stomatitis was found only on female gender. oral lesions were found on the cheeks, tongue, palate and labial commissure. the distribution of the types of candidiasis found was demonstrated in table 1 and figure 2, after the diagnosis, evaluating the signs and symptoms of the disease, since the hyphae and spores were found in only 3 smears, suggestive of pseudomembranous candidiasis (figure 3). figure 2. horizontal sequence of images of the types of oral candidiasis found: hiperplastic(1); acute atrophic erythematous(2); chronic multifocal erythematous(3); mucocutaneous(4); erythematous (prosthetic stomatitis)(5); pseudomenbranous(6). 1 2 3 4 5 6 table 1. types of candidiasis9 diagnosed in patients with lesions (n=21). types of candidiasis women men total % of 87 examined p-value pseudomenbranous 2 1 3 3.45 0.02* hiperplastic 1 1 2 2.3 0.05 mucocutaneous -1 1 1.15 0.01* acute atrophic erythematous 1 -1 1.15 0.01* erythematous (prosthetic stomatitis) 10 1 11 12.64 0.00* chronic multifocal erythematous 3 -3 3.45 0.02* figure 3. blade smear with hyphae(1) and spores(2). 1 2 7 lira et al. discussion it is believed that oral candidiasis can affect quality of life and make feeding difficult due to the presence of bleeding, exudation, burning sensation, pain, halitosis, unpleasant taste and dry mouth, corroborating with other authors15-18. according to the questionnaire, the exposure of the target public to the various predisposing factors of candidiasis, such as: antibiotic therapy, smoking, stds, diabetes, anemia, respiratory problems, which depending on the individual’s condition may lead to the development of the disease4. maladaptive prosthetics with cracks or spots are considered irritating factors for the buccal mucosa and may cause several lesions such as prosthetic stomatitis, inflammatory fibrous hyperplasia, inflammatory papillary hyperplasia, angular cheilitis and traumatic ulceration and cadidiasis14. in the clinical examination, it was also observed that all the patients with lesions had caries, periodontal disease, tasty tongue and residual roots. according to salerno et al.16, bacteria, such as streptococcus spps, induce the formation of proteases that degrade the epithelium and cause inflammation, facilitating bacterial proliferation and colonization by candida spp. periodontal disease is also directly associated with candidiasis because it allows the colonization of fungi and fungi, increasing the chances of infection². all 240 participants in the study used dental prostheses in the period between 11 and 20 years, extrapolating the average life span of 5 years15. some people said they felt discomfort from the use of the prosthesis, others reported maladaptation, besides showing cracks and blemishes. such facts were observed by other authors 7-9,14 who recommended a periodic visit to the dental surgeon to evaluate the adaptation, hygiene of the prostheses and the need to replace them. the prerequisites for prosthetic assessment are retention, stability, reciprocity, fixation and aesthetics. if one of these requirements is not obeyed, the prosthesis should be replaced. usually patients, in self-assessment, prioritize aesthetics, chewing and speech. when one of these factors is compromised, they are interested in looking for the dental surgeon16,17. it was observed in this research the lack of knowledge of the participants on the importance of the observance of prosthetic quality for oral health such as on others studies17-19. poor oral hygiene associated with the use of dental prosthesis predisposes to the development of buccal candidiasis6,7,13. regarding the hygiene of the prostheses, most of them performed with a toothbrush and toothpaste, but it can cause roughness on the surface of the prosthesis, facilitating the creation of biofilms. thus, the recommended method for sanitizing tp is the 0.25% sodium hypochlorite solution because it reduces biofilm with the remission of candidiasis18. most reported not removing the prosthesis to sleep. those who remove it put it in a glass with water, which is the most correct for conditioning, because when it is in a dry environment it suffers contraction, making it difficult to adapt it to the mouth19. prosthetic erythematous stomatitis was the most prevalent (12.64%) characterized by extensive reddish areas on the palate, followed by chronic multifocal erythematous (3.45%) with reddish atrophic spots and several subclinical types6. the lack of oral hygiene and prostheses promote biofilm formation and the colonization of candida 8 lira et al. albicans9, which suggests being responsible for the high prevalence of prosthetic stomatitis. similar results were found by other authors7,19,23. according to the literature, in the suspected hyperplastic candidiasis biopsy is recommended, but for the mucocutaneous and erythematous types, the indicated test is the culture medium, and the material harvested from the prosthesis. however, the most commonly used diagnostic method when any type of candidiasis is suspected is the clinical examination, the removal of predisposing factors, the use of antifungal agents until remission of the disease, and the need for complementary tests9. pseudomembranous candidiasis was found in 3.45% of the subjects, characterized by removable plaques with gauze or spatula, burning sensation and bad breath. accompanying mainly immunosuppressed people. the clinical diagnosis is what differs this type from the others found. however, through the smear on the lamina, filamentous (hyphae) and spore forms were observed in three smears, typical of pseudomembranous candidiasis, corroborating with other authors6,11. this examination is recommended according to schiboch et al.10 (2005) when pseudomembranous candidiasis is suspected and it is possible to visualize under the microscope, after smearing and staining of the slides, fungi in yeast and filamentous forms with epithelial cells. hyperplastic candidiasis, found in 2.3% of the patients examined, is characterized by an asymptomatic non-removable thick white plaque, mainly found in the anterior region of the jugal mucosa and dorsum of the tongue, whose main etiological factor is smoking6,20. its diagnosis was obtained by clinical examination associated with administration of antifungal for 15 days for both patients and if it had not regressed, the biopsy would be performed. this is because this type of candidiasis may be associated with epithelial dysplasia called leukoplakia. in these cases, biopsy is recommended and should be considered a true dysplasia, with the possibility of becoming a malignant lesion12. mucocutaneous candidiasis was present in one of the participants with candidiasis, characterized by white plaques that stood out during scaling, exposing reddish areas. his diagnosis was confirmed by clinical examination and remission of the lesions after the use of antifungal for 20 days. the main predisposing factors are immune dysfunctions and the use of antibiotics. it can reach several organs and become systemic, leading to death. as noted in the questionnaire, there is a lack of knowledge about candidiasis. however, it is notorious that there is a need to pass on guidelines on preventive measures and their consequences21,22. chronic multifocal erythematous (2.45%) and acute atrophic erythematosa (1.15%) are subdivisions of erythematous candidiasis that were diagnosed in the study. the chronic multifocal type was presented by red macules on the palate and burning sensation. this type mainly affects the individuals with immunosuppression and who do antibiotic therapy, which was verified in the 3 treated patients. the acute atrophy presented as red atrophic mucosal areas due to atrophy of the papillae. the main etiologic factor of this type is immunosuppression23, which was reported by the patient with atrophic candidiasis. conventional treatment, which implies the application of topical antifungal drugs, provides an effective response, but there is a frequent occurrence of recurrence of the disease due to its multifactorial etiology, which is caused mainly by lack of oral hygiene, in addition to low immunity and person-to-person contamination. another 9 lira et al. important factor in the recurrence of candida albicans is deficiency in the hygiene of the prosthesis. most microorganisms adhere to the acrylic surfaces, demonstrating a great mechanical resistance, especially in poorly adapted prostheses12,14,24,25. nystatin is one of the reference antifungal agents for the treatment of candidiasis, because it has no side effects or interactions with other drugs, with specific action against fungi of the genus candida¹². therefore, it was the drug of choice for the treatment of patients, and it was obtained from ubs pharmacies as oral suspension (micostatin® 100,000 iu / ml), 5ml, 4 times a day, and was chewed for 1 minute and then swallowed for 15 days, except those with hyperplasia, who used the drug for 20 days, as suggested by some authors14,16,21,22. patients were supervised during treatment and up to 90 days after lesion remission. the users of badly adapted or compromised prostheses were referred to make new prostheses after treatment. new research on the pathologies caused by the use of dental prosthesis as a factor predisposing to the alteration of oral microflora are important for diagnosis and reduction of complications. it was concluded that there was a correlation between oral candidiasis and users of dental prostheses, the most prevalent type being erythematous (prosthetic stomatitis). the preventive activities carried out were extremely important, since patients were not aware of oral candidiasis and the triggering factors, such as poor oral hygiene and dental prostheses. treatment was performed with nystatin and all participants in the sample were guided on local and systemic predisposing factors (baseline disease), so that relapses were avoided. individuals with lesions caused by removableprosthesis must be offered correct treatment which includes new prosthesis or adjustments of the old one. periodical visits to the dentist are mandatory to prevent oral health problems. research on the pathologies caused by the use of dental prosthesis as predisposing factor for oral microflora alteration are important for diagnosis and reduction of complications. references 1. andreola ad, galafass ebe, elsemann. [comparative study between the production of extracellular phospholipases and proteinases of the genus candida isolated from oral cavity infections]. rev odontol unesp. 2016 jul/aug;45(4):219-26. portuguese. 2. lourenço ag, ribeiro aera, nakao c, motta acf, antonio lgl, machado aa, et al. oral candida spp carriage and periodontal diseases in hiv infected patients in ribeirão preto, brazil. rev. inst med trop. 2017 mar;59(29):1-7. doi: 10.1111/odi.12669. 3. ezenwa bn, oladele ro, akintan pe, fajolu ib, oshun po, oduyebo oo, et al. invasive candidiasis in a neonatal intensive care unit in lagos, nigeria. niger postgrad med j. 2017 jul-sep;24(3):150-4. doi: 10.4103/npmj.npmj_104_17. 4. miyazima ty, ishikawa kh, mayer mpa, saad smi, nakamae aem. cheese supplemented with probiotics reduced the candida levels in denture wearersrct. oral dis. 2017 oct;23(7):919-25. doi:10.1111/myc.12680. 5. chanda w, joseph tp, wang w, padhiar aa, zhong m. the potential management of oral candidiasis using anti-biofilm therapies. med hypotheses. 2017 sep;106:15-18. doi: 10.1016/j.mehy.2017.06.029. 10 lira et al. 6. bianchi cm, bianchi ha, tadano t, paula cr, hoffmann-santos hd, leite jr dp, hahn rc. factors related to oral candidiasis in elderly users and non-users of removable dental prostheses. rev inst med trop sao paulo. 2016 mar;58(17):1-5. doi: 10.1590/s1678-9946201658017. 7. przybyłowska d, rubinsztajn r, chazan r, swoboda-kopeć e, kostrzewa-janicka j, mierzwińska-nastalska e. the prevalence of oral inflammation among denture wearing patients with chronic obstructive pulmonary disease. adv exp med biol. 2015 mar; 858:87-91. doi: 10.1007/5584_2015_128. 8. carli jp, giaretta bm, vieira rr, linden mss, ghizoni js, pereira jr. 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[use and need for dental prosthesis in brazil: progress, prospects and challenges]. saude transf social. 2016 apr;7(1):41-8. portuguese. 18. costa rs, filho hblf, chaves fn, vasconcellos aa. [lesions associated with the use of partial or complete removable dentures – features and treatment]. prosth labor sci. 2015;5(17):62-8. portuguese. 19. celakil t, baca e, topcuoglu en, röhling bg, evlioglu g, özcan m. prevalence of candida albicans and streptococcus aureus on maxillary obturators, maxillary defects and in saliva. a cross-sectional study. 2017 jul/sep;20(3):45-51. doi: 10.14295/bds.2017.v20i3.1366. 20. lause m, kamboj a, fernandez faith e. dermatologic manifestations of endocrine disorders. transl pediatr. 2017 oct;6(4):300-12. doi: 10.21037/tp.2017.09.08. 21. veverka kk, feldman sr. chronic mucocutaneous candidiasis: what can we conclude about il-17 antagonism? j dermatolog treat. 2017 nov;21:1-6. doi: 10.1080/09546634.2017.1398396. 22. li j, vinh dc, casanova jl, puel a. inborn errors of immunity underlying fungal diseases in otherwise healthy individuals. curr opin microbiol. 2017 nov;9(40):46-57. doi: 10.1016/j.mib.2017.10.016. 23. paraguassú gm, pimentel pa, santos ar, gurgel cas, sarmento va. prevalence of oral lesions associated with use of removable dental prostheses in a stomatology service. rev cubana estomatol. 2011 jul/set;48(3):268-76. 11 lira et al. 24. simões rj, fonseca p, figueira mh. [oral infections by candida spp]. odontol clín-cient. 2013 jan/ mar;12(1):19-22. portuguese. 25. prakash b, shekar m, maiti b, karunasagar i, padiyath s. prevalence of candida ssp. among healthy denture and nondeture wearers with respect to hygiene and age. j indian prosthodont soc. 2015 jan-mar;15(1):29-32. doi: 10.4103/0972-4052.155041. braz j oral sci. 15(1):45-50 original article braz j oral sci. january | march 2016 volume 15, number 1 factors associated with the technical quality of root canal fillings performed by undergraduate dental students in a malaysian dental school choon yoong wong1, yan xia liaw1, jhiew zhan wong1, lee chian chen1, abhishek parolia1, allan pau1 1international medical university, school of dentistry, division of clinical dentistry, kuala lumpur, malaysia correspondence to: abhishek parolia division of clinical dentistry, school of dentistry, international medical university, 126, jalan jalil perkasa 19, bukit jalil 57000 kuala lumpur, malaysia e-mail: abhishek_parolia@imu.edu.my abstract aim: a retrospective clinical audit was carried out on records of endodontic treatment performed by dental undergraduates. the audit was performed to evaluate the technical quality of root canal fillings performed by dental undergraduates and determine the associated factors. methods: 140 records of patients who had received root canal treatment by dental undergraduates were evaluated through periapical radiographs by two examiners (κ =0.74). the root canal fillings had their quality evaluated according to extent, condensation and presence of procedural mishap. possible factors associated with technical quality such as tooth type, canal curvature, student level and quality of record keeping were evaluated. data were statistically analyzed using chi-square test (p<0.05). results: among the 140 root-filled teeth, acceptable extent, condensation and no-mishap were observed in 72.1%, 66.4% and 77.9% cases respectively. overall, the technical quality of 68 (48.6%) root-filled teeth was considered acceptable. overall, non-acceptable root canal fillings were significantly more likely to be observed in molars (69.2%), moderately and severely curved canals (71.4%) and junior students (61.5%). there was no association between acceptable root canal fillings and quality of record keeping. conclusions: the technical quality of root canal fillings was acceptable in 48.6% cases and it was associated with tooth type, degree of canal curvature and student seniority. keywords: endodontics. root canal therapy. education. introduction endodontic treatment encompasses procedures designed to maintain the health of all or part of the dental pulp. when the dental pulp is diseased, endodontic treatment aims at preserving healthy periradicular tissues. however, if apical periodontitis occurs, the treatment aims restoring the periradicular tissue health. the purpose of endodontic treatment is to disinfect the entire root canal system and maintain asepsis1. the european society of endodontology stated that an appropriate endodontic treatment involves the following parameters: exposure of a good quality preoperative radiograph, administration of local anesthesia, isolation of tooth, preparation of access cavity, determination of working length, shaping, cleaning and obturation of the root canal system1. there is substantial evidence that the technical quality of root canal fillings has a significant impact on the outcome of the treatment and long-term retention http://dx.doi.org/10.20396/bjos.v15i1.8647122 received for publication: april 06, 2016 accepted: june 27, 2016 46 of endodontically treated teeth1. many studies reported that root canal fillings placed within 0-2 mm of the radiographic apex are associated with lesser chances of radiographic and clinical failures than those that are under-filled or over-filled2-6. clinical audit is defined as a quality improvement process that seeks to improve patient care and outcomes by systematic review of care against explicit criteria and the implementation of change. the use of clinical audit in endodontic treatment has been widely reported5,7-14. this study was a clinical audit on technical quality of root canal fillings performed by undergraduates. the aims of this study were to evaluate the quality of root canal fillings performed by dental undergraduates in a malaysian dental school and to determine factors associated with the technical quality of root canal fillings. material and methods the present study was restricted to patients who attended the school of dentistry, international medical university, kuala lumpur, malaysia, for root canal treatment. a retrospective clinical audit was carried out on the records of endodontic treatment performed only by undergraduate students. all 142 electronic records of endodontic treatment performed by dental undergraduates from january 2011 to april 2013 were retrieved and printed. inclusion criterion of this study was endodontic treatment completed by dental undergraduates. incomplete endodontic treatment and records without post-operative radiographs were excluded. two records were excluded due to incomplete treatment and missing postoperative radiograph respectively. the remaining 140 records fulfilled the inclusion criteria. two fourth year dental undergraduates carried out the audit after undergoing training in clinical audit, which consisted of: • a 2-h seminar on the principles and practice of clinical audit. • desktop research on criteria for assessing the technical quality of root canal fillings and quality of record keeping. • development of audit checklist form and protocol for auditing the records. • training on radiographic evaluation of root canal fillings by an endodontist. root canal treatment procedure the dentist first examined patients who attended imu oral health centre seeking for treatment and then referred these cases to undergraduate students or endodontic specialists based on the complexity of the tooth/root canal and economic concern of the patients. the subjects were patients who had a specific endodontic disease needing emergency treatment. after thorough clinical examination and considering the technical difficulties related to root canal therapy, the endodontic staff allotted these patients to different cohorts of undergraduates based on their seniority and competency level. the chief concern of the patient was obtained together with patient’s medical, dental, pain history. appropriate investigations were carried out to come to a diagnosis. all root canal procedures were performed using a rubber dam and aseptic technique. access cavities were prepared using endodontic access burs (dentsply factors associated with the technical quality of root canal fillings performed by undergraduate dental students in a malaysian dental school braz j oral sci. 15(1):45-50 maillefer, ballaigues, switzerland). the working lengths (1 mm short of the radiographic apex) were established using electronic apex locators and reconfirmed radiographically. the hybrid technique was used for shaping and cleaning procedures. the coronal two-thirds of the canals were prepared sequentially using gates gliden burs, (dentsply maillefer) and k-flex file (dentsply maillefer). the apical third was instrumented to the master apical file 1 mm short from the radiographic apex. root canals were further instrumented with step-back enlargement to three sizes larger than the master apical file. finally, circumferential filing was done to complete the shaping of the root canal. 2% sodium hypochlorite (clorox company, broadway, oakland, ca, usa), saline (to flush sodium hypochlorite and prevent any interaction in between sodium hypochlorite and edta) and 18% ethylenediaminetetraacetic acid (edta, ultradent products inc., south jordan, ut, usa) were used as irrigants. all patients were treated in multiple visits and non-setting calcium hydroxide (calcicur, voco gmbh, cuxhaven, germany) as an intracanal dressing and reinforced zinc oxide temporary cement (irm, dentsply caulk, milford, de, usa) or cavit (3m espe, st. paul, mn, usa) as a temporary restorative materials were used between appointments. in the subsequent visits all root canals were filled using guttapercha points with ah plus (dentsply maillefer) root canal sealer using lateral compaction technique. following, all root canals were sealed with glass ionomer cement (gc corporation, tokyo, japan) and thereafter the most suitable postendodontic coronal restorations (amalgam/composite/post and core) were placed after assessing the amount of remaining tooth structure. evaluation of the technical quality of root canal fillings three periapical radiographs were examined for each root canal filling: pre-operative, working length determination and post-operative. two examiners evaluated independently all the radiographs. radiographs were examined in the form of full screen images that could be enhanced and zoomed in on the computers. if disagreement in interpretation occurred between the examiners, the radiographs were re-evaluated until an agreement was reached. kappa value for inter-examiner agreement was 0.74. the technical quality of root canal fillings was evaluated according to condensation of root canal fillings, extent of root canal fillings and presence of procedural mishaps (table 1)8. for a multi-rooted tooth, all canals were evaluated simultaneously and an overall score was given to the tooth (e.g. technical quality of a multi-rooted tooth was considered acceptable only when the technical quality of all the root canals was acceptable). possible factors associated with the technical quality of root canal fillings anatomical characteristics of tooth: for each root-treated tooth, the tooth type and complexity (root canal curvature) were assessed. the complexity (root canal curvature) was determined based on the pre-operative radiographs according to the schneider’s classification of curvature15. tooth with moderate (10-20°) and severe curvature (25-70°) canals were combined and compared against tooth with straight canal (5° or less). 47factors associated with the technical quality of root canal fillings performed by undergraduate dental students in a malaysian dental school braz j oral sci. 15(1):45-50 variables criteria definition condensation acceptable no void identified in root canal filling or between root canal filling and root canal walls non-acceptable presence of void in root canal filling or between root canal filling and root canal walls extent acceptable root canal filling material is within the root canal system and within 2 mm of the radiographic apex under-filled root canal filling material is >2 mm short of the radiographic apex over-filled root canal filling material is extruded beyond the radiographic apex mishap no mishap no mishap identified ledge root canal filling is at least 1 mm shorter than the working length and is deviated from the original canal curvature perforation apical termination of filled canal is different from the original canal terminus or root canal filling material is extruded through the apical foramen separation of instruments separated instrument identified others other mishaps identified overall acceptable acceptable condensation and extent of root canal filling with no mishap non-acceptable non-acceptable condensation and/or nonacceptable extent of root canal filling with/without mishap table 1 criteria used to assess technical quality of root canal fillings. student seniority: third and fourth year undergraduates were combined and compared to fifth year undergraduates. quality of the record keeping of endodontic treatment: evaluation of quality of record keeping was based on the records of each endodontic treatment performed by undergraduate students. all the records were audited independently by two examiners against the set of criteria that had been previously developed accordingly to the european society of endodontology guideline1. the presence or absence of recording of each criterion (total=17) was observed: use of an electric pulp test, use of local anesthesia, name of local anesthesia, dosage of local anesthesia, use of rubber dam isolation, working length, reference point of working length, size of initial apical file, size of master apical file, applied intracanal dressing, medication prescribed including analgesics and antibiotics, size of master cone, pre-operative radiograph, working length radiograph, master cone radiograph, post-operative radiograph and advice on final restoration in the follow-up visit. statistical analysis data were entered into spss 18.0 (spss, inc., chicago, il, usa) for analysis. the frequency distributions of the technical quality of root canal fillings were calculated. chi-square tests were performed to determine associations between the technical quality of root canal fillings and possible associated factors (e.g. tooth type, root canal curvature, student seniority and quality of record keeping). the significance level was set at p<0.05. results technical quality of root canal fillings the present study reviewed the records 140 root-filled teeth. the distribution of this sample according to the tooth type and student seniority is in table 2. sixty-five (46.4%) teeth were treated by third and fourth year undergraduates and the remaining by the fifth year undergraduates. the most commonly treated tooth types were molars (37.1%), followed by premolars (32.9%), incisors (23.6%) and canines (6.4%). the condensation of root filling was acceptable in 93 (66.4%) cases, extent was acceptable in 101 (72.1%) cases and no mishaps were observed in 109 (77.9%) cases (table 3). overall, the technical quality of 68 (48.6%) of all 140 root-filled teeth was considered acceptable (table 3). possible factors associated with the technical quality of root canal fillings among the different tooth types, mishaps were significantly more likely to be observed in molars (32.7%, table 4) compared to premolars (8.7%, p<0.05). overall non-acceptable root canal fillings were significantly more likely to be noted in molars (69.2%) compared to other tooth types (p<0.01). underand over-extended of root canal fillings were significantly more likely to be observed in moderately and severely curved canals (47.6%) compared to straight canals (24.4%, p<0.05). overall non-acceptable root canal fillings were significantly more likely to be observed in moderately and severely curved canals (71.4%) compared to straight canals (47.9%, p<0.05). according to student seniority, non-acceptable condensation was more common among third and fourth year students (49.2%) compared to fifth year students (20.0%, p<0.01). similarly, non-acceptable extent was more common among junior students (36.9%) compared to senior students (20.0%, p<0.05). overall non-acceptable root canal fillings were significantly associated with junior students (61.5%) compared to senior students (42.7%, p<0.05). curved canals18. under-filled and over-filled root canal fillings occurred more frequently in teeth with curved canals and this may be attributed to the failure to realize canal curvature before preparation of the root canal system, leading to internal and external transportation, over-preparation, and straightening of the curved canals. in consequence, the loss of apical stop may result in over-filling of root canals. to improve the technical quality of root canal fillings in the molars and teeth with curved canals, endodontic training must emphasize the difficulties of treating molars and teeth with curved canals. increase in pre-clinical laboratory training and addition of seminars would be beneficial for students to familiarize with necessary procedures and precautions required for managing molars and teeth with curved canals. the use of dental magnification such as magnifying loupes and operating microscopes while performing molar endodontics would enhance visualization of the treatment field and increase accuracy of the endodontic procedures22. in addition; the use of rotary instruments may be considered, especially for molar endodontics20,23. however, it is not used by our undergraduate students. 48 student seniority incisors canines premolars molars total (%) 3rd and 4th 19 3 24 19 65 (46.4%) 5th 14 6 22 33 75 (53.6%) total (%) 33 (23.6%) 9 (6.4%) 46 (32.9%) 52 (37.1%) 140 (100.0%) table 2 distribution of teeth in the sample. discussion the key findings of this retrospective study were that the technical quality of root canal fillings performed by the fifth year dental undergraduates were superior to the third and fourth year undergraduates, and the overall technical quality was better in anterior teeth and teeth with straight canals irrespective of student seniority. overall, the technical quality of root canal fillings performed by dental undergraduates in this study was considered acceptable in nearly half the sample. this result is lower than those reported for scottish13, lithuanian9, irish11, greek10, iranian16, turkish17 and serbian students18, but better when compared to turkish8, french12, jordanian19, saudi arabian7 and spanish students20. wide variations in proportions of acceptable root canal fillings have been reported in the literature. they may be related to the different criteria used in evaluation, size and types of selected sample and the training the students received. for example, the lithuanian study9 did not take procedural mishap into account for evaluation of the technical quality and the findings were only based on condensation and extent of root canal fillings. lynch & burke (2006) reported that 63.0% of root canal llings were technically acceptable11. however, only single-rooted teeth were included in their study as compared to the present study, which evaluated the technical quality performed on both single-rooted and multi-rooted teeth. in the present study, non-acceptable root canal fillings were more common in the molars compared to the incisors, canines and premolars. the reason for this was the high incidence of procedural mishaps in the molars. this is consistent with results reported by balto et al.7, moussa-badran et al.12, and moradi et al.21. the high incidence of procedural mishaps may be attributed to the anatomy of molars (e.g. multiple root canals and greater canal curvatures) and difficulty to have adequate coronal access during the procedure21. the results also highlighted that nonacceptable root canal fillings were more common in teeth with variables criteria definition condensation acceptable 93 (66.4%) non-acceptable 47 (33.6%) extent acceptable 101 (72.1%) under-filled 19 (13.6%) over-filled 20 (14.3%) mishap no mishap 109 (77.9%) ledge 12 (9.3%) perforation 16 (11.4%) separation of instruments 1 (0.7%) others 1 (0.7%) overall acceptable 68 (48.6%) non-acceptable 72 (51.4%) table 3 acceptable and non-acceptable root canal fillings performed by undergraduate students. total total of overall non-acceptable non-acceptable condensation under-filled and over-filled mishap tooth type incisors 33 16 (48.5%) * 9 (27.3%) 8 (24.2%) 8 (24.2%) ** canines 9 2 (22.2%) 0 (0.0%) 1 (11.1%) 2 (22.2%) premolars 46 18 (39.1%) 16 (34.8%) 10 (21.7%) 4 (8.7%) molars 52 36 (69.2%) 22 (42.3%) 20 (38.5%) 17 (32.7%) complexity straight 119 57 (47.9%)** 39 (32.8%) 29(24.4%)** 25 (21.0%) moderate and severe 21 15 (71.4%) 8 (38.1%) 10 (47.6%) 6 (28.6%) student seniority 3rd and 4th 65 40 (61.5%)** 32 (49.2%)* 24 (36.9%) ** 14 (21.5%) 5th 75 32 (42.7%) 15 (20.0%) 15 (20.0%) 17 (22.7%) quality of record keeping good † 69 33 (47.8%) 20 (29.0%) 19 (27.5%) 14 (20.3%) poor † 71 39(54.9%) 27 (38.0%) 20(28.2 %) 17 (23.9%) table 4 non-acceptable root canal fillings according to tooth type, complexity, student seniority, and quality of record keeping. *p <0.01. ** p <0.05. † good quality of record keeping (11-17 criteria recorded in record); poor quality of record keeping (<11 criteria recorded in record). factors associated with the technical quality of root canal fillings performed by undergraduate dental students in a malaysian dental school braz j oral sci. 15(1):45-50 49factors associated with the technical quality of root canal fillings performed by undergraduate dental students in a malaysian dental school the technical quality of root canal fillings performed by fifth year dental undergraduates was significantly better than by the third and fourth year undergraduates. on one hand, this is to be expected as the fifth year dental undergraduates are more experienced; however, all patients treated by undergraduates should expect the same high quality endodontic treatment regardless the student’s seniority. to minimize the percentage of non-acceptable root canal fillings performed by third and fourth year dental undergraduates, closer supervision is recommended as well as case selection according to the degree of difficulty24. simple cases of endodontic treatment (e.g. anterior teeth and teeth with straight canals) should be treated by third and fourth year dental undergraduates whilst a little bit more complex cases (including molar teeth with a slight root curvature) should be treated by the fifth year dental undergraduates. moreover, teeth with moderate or high level of difficulty should be referred to specialists. this will allow the third and fourth year dental undergraduates to gain adequate competency in managing simple cases prior to performing more complex cases of endodontic treatment. in this retrospective study, only half of the clinical records kept by dental undergraduates met the criteria of good record keeping, despite implementation of general guidelines on record keeping for endodontic treatment in the dental clinic. this finding emphasizes the need to reinforce the guidelines on record keeping. although the present results showed that the quality of record keeping had no significant association with the technical quality of root canal fillings, inadequate record keeping may increase the opportunities for potential litigation by the patients25. therefore it is mandatory for the dental undergraduates to record details of the procedure performed to avoid medicolegal issues. in all cases performed by dental undergraduates, the working length measurement (i.e 1 mm before the apex) was established using electronic apex locators and reconfirmed radiographically, which is routinely used in most of the dental schools26. the instruments used for canal preparation were stainless steel hand k flex files and gates glidden burs (dentsply maillefer). canal preparation was carried out using a combination of crown down and step back technique. this technique has been shown to be useful for teaching dental undergraduates11. 2% sodium hypochlorite (clorox) and 18% edta (ultradent products inc.) were used as irrigants. alternate use of sodium hypochlorite and edta hwas found to be very effective in removing the smear layer from the root canals27,28. calcium hydroxide (calcicur, nordiska dental ab, angelholm, sweden) was used as an intra-canal medicament that provides antimicrobial activity and prevents recontamination of the root canal system29,30. lateral compaction technique with gutta-percha points and ah plus (dentsply maillefer, usa) root canal sealer was used to obturate all the canals, a technique used in other countries2,8,31. there are a few limitations in this retrospective study. the sample size is relatively small and the radiographical analysis does not entirely reflect the technical quality of root canal fillings because it is not a direct indication of the disinfection quality. besides that, a single periapical radiograph only provides a twodimensional image. therefore it is sometimes difficult to separate superimposed anatomical structures (e.g. multiple root canals) while evaluating the radiographs16. in conclusion, the technical quality of root canal fillings performed by dental undergraduates was acceptable in nearly half the cases. the results have highlighted points that should be considered during the case selection for undergraduate students and also in the teaching of endodontics. factors found to be associated with the technical quality of root canal fillings were tooth type, root canal curvature and student seniority. references 1. european society of endodontology. quality guidelines for endodontic treatment: consensus report of the european society of endodontology. int endod j. 2006 dec;39(12):921-30. 2. hayes sj, gibson m, hammond m, bryant st, dummer pm. an audit of root canal treatment performed by undergraduate students. int endod j. 2001 oct;34(7):501-5. 3. kerekes k, tronstad l. long-term results of endodontic treatment performed with a standardized technique. j endod. 1979 mar;5(3):83-90. 4. marques md, moreira b, eriksen hm. prevalence of apical periodontitis and 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critical review. int endod j. 1999 sep;32(5):361-9. 31. elsayed ro, abu-bakr nh, ibrahim ye. quality of root canal treatment performed by undergraduate dental students at the university of khartoum, sudan. aust endod j. 2011 aug;37(2):56-60. doi: 10.1111/j.17474477.2010.00273.x. factors associated with the technical quality of root canal fillings performed by undergraduate dental students in a malaysian dental school braz j oral sci. 15(1):45-50 untitled 1http://dx.doi.org/10.20396/bjos.v16i0.8651051 volume 16 2017 e17048 original article 1 dds, ms, professor, department of restorative dentistry,dental school, shahid beheshti university of medical sciences,tehran,iran email address; sh_kasraie@yahoo.com. 2 dds, ms, assistant professor, department of restorative dentistry, hamedan university of medical science, hamedan,iran. email address; yarmohammadi1380@yahoo.com. 3 phd, assistant professor, department of biostatistics,school of public health and research center for health sciences, hamadan university of medical sciences,hamadan,iran. email address; maryam_ farhadian80@yahoo.com. 4 dds, ms ,department of restorative dentistry,hamedan university of medical science, hamedan,iran. corresponding author: mona malek hamadan university of medical sciences, hamadan, iran e-mail: mona_mlk@yahoo.com received: may 13, 2017 accepted: september 14, 2017 effect of proteolytic agents on microleakage of etch and rinse adhesive systems shahin kasraei1, ebrahim yarmohammadi2, maryam farhadian3, mona malek4* aim: this study aimed to assess the effect of treatment of phosphoric acid etched dentin surface with 5% bromelain enzyme and nd:yag laser prior to the use of etch and rinse adhesive systems on microleakage margins of class v composite restorations. materials and methods: sixty sound premolar teeth were selected. standard class v cavities were prepared in the buccal and lingual surfaces of each teeth. preparation in that way 1 mm of the cavity was above and 1 mm of it was below the cementoenamel junction. the teeth were then randomly divided into three groups including 20 teeth (40cavities in each gorup). two ethanol base etch and rinse system (adper single bond and optibond solo) was applied in each group . in the control group, cavities were etched with 37% phosphoric acid. adper single bond was applied to 20 and optibond solo was applied to the remaining 20 cavities and they were restored with z250 composite (n=40). the same procedures were performed in bromelain and nd:yag laser groups with the difference that prior to the application of adhesive, aqueous 5% bromelain was applied in bromelain group while nd:yag laser was irradiated in laser group. all teeth were thermocycled and their apices were sealed with sticky wax. the tooth surfaces were coated with nail varnish except for 1 mm around the restoration margins, and the teeth were then immersed in fuchsine and sectioned by a diamond disc into mesial and distal halves. marginal microleakage at both sides was determined and scored under a stereomicroscope at x40 magnification. then data were analyzed using the kruskal wallis and mann whitney tests (α=0.05). results: according 2 kasraei et al. the result of mann whitney test, cervical margins showed significantly higher microleakage than the occlusal margins (p=0.000) but no significant difference was noted in microleakage of occlusal (p=0.362) or gingival (p=0.147) margins among the three groups by kruskal wallis test. conclusion: in conclusion, application of proteolytic agents(5% bromelain solution and nd:yag laser) on acid-etched dentin surface prior to the application of adhesive has no significant effect on marginal microleakage of class v composite restorations. keywords: microleakage. bromelain. laser. etch and rinse bonding agents. introduction the integrity of adhesive resin bond to dentin plays an important role in success of composite restorations1-4. the hybrid layer is the most important structure affecting micromechanical retention of resin restorations and sealing of dentin. however, the hybrid layer and the adhesive interface is the most common site of failure of these restorations5,6. proper bond of adhesive resin to dentin depends on many factors such as removal of minerals from dentin substrate, wetting of the substrate with adhesive and penetration of adhesive into demineralized dentin1. acid etching completely demineralizes a few micrometers of dentin surface and exposes the collagen fibers3,7. in etch and rinse adhesives, a reduction in concentration gradient of monomer is noted by an increase in penetration depth of resin into demineralized dentin. the highest concentration of resin is found at the surface. the hybrid layer has a lower concentration of resin and the least concentration is found at the deepest part of the demineralized dentin8,9. the discrepancy between the depth of dentin demineralization and penetration depth of resin results in nano-leakage in deep, water-rich parts of the hybrid layer8-11. moreover, outward movement of fluid in dentinal tubules of vital teeth decreases resin penetration into collagen matrix, causing weak points in the hybrid layer8,9. nano-leakage in this area results in enzymatic degradation and hydrolysis of resin or collagen and undermining of hybrid layer and derangement of collagen fibers12. type one collagen is the most abundant organic component in extracellular matrix of dentin. it plays an important role in formation of hybrid layer and adhesion to dentin structure. aside from type one collagen, non-collagen proteins such as proteoglycans and glycoproteins are also present in spaces between collagen fibers13-16. covering the etched dentin surface with these compounds can decrease the penetration of large molecules (bis-gma) into dentin, and only smaller molecules such as hema can penetrate deep into dentin8. hema-rich areas in the hybrid layer have high strain, causing fatigue of collagen fibrils and making them susceptible to degradation8. therefore, recent studies have focused on improving the quality of the hybrid layer by removing the demineralized collagen network and enhancing further penetration of resin into acid-etched areas1,2,8,17. materials used for this purpose often have shortcomings such as toxicity and instability18 or require long-term use, which limits their application in the clinical setting19-22. materials used for this purpose must save time in the clinical setting, have low cytotoxicity and protect the resin-dentin interface23. 3 kasraei et al. some previous studies discussed that removal of collagen network by use of proteolytic enzymes such as collagenase or bromelain may increase the bond strength and decrease marginal microleakage of composite restorations3,17. it appears that decreased dentin microleakage following the use of bromelain is due to the ability of this enzyme to remove the residual proteins and collagen network after etching3. since the etching of dentin surface with phosphoric acid lead to dissolving inorganic component of smear layer and remaining amorphous protein layer. this surface collagen smear decrease the rate of resin monomer penetration and form weak hybrid layer that affect the composite bond8 .bromelain solution can eliminate collagen smear layer and organic component from surface of dentin substrate and increase resin penetration into dentin , improve the quality of hybrid layer and decrease microleakage3. nd:yag laser is an electromagnetic infrared radiation with 1065 nm wavelength. it can selectively ablate the collagen network3 and deproteinize dentin without affecting its mineral content1,3. opening of dentinal tubules and absence of smear layer on the surface increase the bond strength of composite to laser-treated dentin surface1. this study aimed to assess dentin surface treatment methods to enhance further resin penetration into the etched dentin surface and facilitate the bonding process in the clinical setting. the null hypothesis was that dentin preparation with 5% bromelain and nd:yag laser irradiation prior to the application of etch and rinse (adper single bond and optibond solo) adhesive systems would have no effect on occlusal and marginal microleakage in class v composite restorations. materials and methods this study was performed on 60 sound human premolars with no carious lesions, occlusal wear or previous restorations. the teeth had been extracted within the past three months for orthodontic reasons and had stored in 0.2% thymol solution. one week before the experiment, the teeth were transferred to distilled water. standard class v cavities measuring 3 mm mesiodistally and 2 mm occluso-gingivally with 1.5 mm depth were prepared on buccal and lingual surfaces of each tooth according to iso/ts11405 using 008/835 diamond bur (microdont, new york, usa) and high speed hand piece under water and air spray by a single operator such that 1 mm of the cavity was above and 1 mm of it was below the cementoenamel junction. occlusal margin was beveled by 0.5 mm. the teeth were then randomly divided into three groups (n=20). each tooth had two class v cavities (one in the buccal and one in the lingual surface; a total of 40 cavities). groups were prepared as follows: control group: the enamel margin of the cavities was etched for 30 seconds. dentin was etched for 15 seconds using 37% phosphoric acid (gel etchant, kerr spa, salerno, italy). the cavities were rinsed for 60 seconds and excess water was removed by a cotton pellet. in 20 cavities, adper single bond (3m espe, st. paul, mn, usa) was applied on cavity walls according to the manufacturer’s instructions in two layers and gently air sprayed for 5 seconds in order for the solvent to evaporate; 10 seconds of light curing was then performed using a light curin unit bluephase; ivoclar vivadent, schaan, lichtenstein) with a light intensity of 1200 mw/cm2 . in the remaining 20 cavities, optibond solo (kerr spa, salerno, italy) was applied by an applicator on cavity walls according to the manufactur4 kasraei et al. er’s instructions and gently air sprayed for 3 seconds followed by 20 seconds of light curing. a3 shade of z250 microhybrid methacrylate composite (3m espe, st. paul, mn, usa) was applied by a spatula in three increments in the gingival, occlusal and middle areas. each segment was separately cured for 20 seconds using a light curing unit (bluephase; ivoclar vivadent, schaan, lichtenstein) with a light intensity of 1200 mw/cm2. after completion of restoration, post-curing was performed for 40 seconds. bromelain group: in this group, 5% bromelain solution was applied after acid etching of the cavity. to prepare 5% bromelain, 5g of bromelain powder was dissolved in 100 cc of distilled water. the solution was applied by an applicator on cavity surfaces, allowed 30 seconds, rinsed with water for 15 seconds and excess water was removed by cotton pellet. bonding agent and composite were applied as in the control group. nd:yag laser group: after acid etching (similar to the control group), the teeth were subjected to nd:yag laser irradiation with 1064 nm wavelength, 1.5 w output power, 100 mj pulse energy, short pulse width and 15 hz frequency. laser was irradiated manually using a hand piece with fiber optic tip with 300 µm diameter from 1 mm distance for 10 seconds using overlapping sweeping motion with a speed of 2 mm/second. the bonding agent and composite were applied as in the control group24. restored teeth were immersed in water and incubated at 37°c for 24 hours and were then polished using coarse to fine-grit polishing discs (opti disc, kerr, hawe sa, bioggio, switzerland). the teeth were then subjected to 5000 thermal cycles between 5-55°c with a dwell time of 30 seconds and transfer time of 30 seconds. next, the apices were sealed with wax. all parts of the teeth and cavity walls were coated with two layers of nail varnish except for 1 mm around restoration margins. the teeth were then immersed in 2% fuchsine (merck, darmstadt, germany) at room temperature for 72 hours. next, the teeth were sectioned into mesial and distal halves using a cutting machine and a diamond disc with 0.3 mm thickness. microleakage at both sides was determined under a stereomicroscope at x40 magnification by the same operator twice and the greater value was reported as the amount of microleakage. microleakage was scored in occlusal and gingival margins as follows (figure 1): 0 occlusal margin enamel dentin dentin axial wall composite filling 3 2 1 3 2 1 0 cervival margin figure 1. schematic view of microleakage scores in the occlusal and gingival margins 5 kasraei et al. 0: no dye penetration 1: dye penetration to half the gingival or occlusal wall 2: dye penetration to more than half the gingival or occlusal wall but not reaching the axial wall 3: dye penetration at the tooth-restoration interface reaching the axial wall to assess the micromorphology of the resin-dentin interface under a scanning electron microscope (sem), the slabs were wet-polished with 600, 800 and 1000 grit silicon carbide abrasive papers. after etching, the slabs were immersed in 5.25% sodium hypochlorite for 20 minutes and immersed in ascending concentrations of ethanol (33, 50, 70, 85, 95 and 100%) and were then dried in a critical point dryer. dehydrated samples were mounted in aluminum stubs (3m, sao paulo, sp, brazil) using conductive tape and were sputter-coated with gold-palladium alloy for 120 seconds (bal-tec, scd, 005; zurich, switzerland). the samples were then evaluated under a sem (jeol, jxa840, tokyo, japan) and some selected areas at the resin-dentin interface were photographed at x1000 and x2500 magnifications (two photographs). data were analyzed using spss version 19 via descriptive statistics and kruskal wallis and mann whitney tests (α=0.05). results the results of kruskal wallis test showed no significant difference in microleakage at the occlusal and cervical margins among the three groups (p>0.05, table 1). the mann whitney test showed that in all groups, microleakage at the cervical margins was significantly greater than that at the occlusal margins (p=0.000). figures 2 to 4 show selected sem micrographs of the micromorphology of the resin-dentin interface in different groups. table 1. composition and instruction of adhesive system used in the study. adhesive agents composition instructions for use adper single bond 2 (3m espe, st paul, mn, usa) ethyl alcohol, bis-gma, silanetreated silica, 2-hydroxyethyl methacrylate (hema), glycerol 1,3-dimethacrylate, diurethane dimethacrylate, copolymer of acrylic and itaconic acids apply two consecutive coats of adhesive to the tooth surface with gentle agitation for 15 seconds; gently air thin; light cure for 10 seconds optibond solo (kerr spa, salerno, italy) bis-gma, gpdm, gdma, hema, ethyl alcohol, mono and difunctional methacrylate monomers, cq, fumed silica, barium aluminum borosilicate glass, sodium hexafluorosilicate apply the adhesive and rub for 15s and dry for 3s. light cure for 20s 6 kasraei et al. a b c d t t p p figure 2. selected sem micrographs of resin-dentin interface in the control group; (a) single bond at x1000 magnification; (b) single bond at x2500 magnification; (c) optibond at x1000 magnification; (d) optibond at x2500 magnification. conical resin tags with a rough surface (t) and a few accessory canals filled with resin (p) are also seen. a b c d t t p p p t figure 3. selected sem micrographs of resin-dentin interface after the application of bromelain; (a) single bond at x1000 magnification; (b) single bond at x2500 magnification; (c) optibond at x1000 magnification; (d) optibond at x2500 magnification. hybrid layer in this group was thicker than that in the other two groups. the resin tags were thicker and conical in shape (t). spherical residues were seen on resin tags, which are probably formed by slight penetration of resin into accessory canals in dentin (p). 7 kasraei et al. discussion pulpal response to restorative material is related to marginal leakage. if a dentin adhesive system does not adhere intimately to the dentin substrate,an interfacial gap eventually develops,and bacteria are able to penetrate through this gap3. type of solvent present in the bonding system is an important factor directly affecting the penetration ability of etch and rinse adhesive systems into dentin substrate. by an increase in penetration depth of solvent into etched dentin, hydrophilic properties decrease and chemical integration between hydrophilic dentin and hydrophobic resin improves, resulting in a stronger bond1. thus, in this study, adhesive systems with the same solvent (ethanol) were selected. however, it should be noted that since both a b c d t figure 4. selected sem micrographs of resin-dentin interface after nd:yag laser irradiation; (a) single bond at x1000 magnification; (b) single bond at x2500 magnification; (c) optibond at x1000 magnification; (d) optibond at x2500 magnification. hybrid layer was formed in limited areas. resin tags were scarce and irregularly distributed (t). table 2. score of microleakage in the study groups pvalue microleakage score groupsmargin 3210 0.362 002(5%)38(95%)controla occlusal margin 001(2.5%)39(97.5%)bra 00040(100%)nda 0.147 2(5%)2(5%)10(25%)26(65%)controlb cervical margin 03(7.5%)3(7.5%)34(85%)brb 2(5%)3(7.5%)6(15%)29(72.5%)nd b comparisons between margins are coded by a and b. comparisons between assessment points in each group (kruskal wallis) are coded by uppercase letters. similar uppercase letters indicate statistically similar means. 8 kasraei et al. adhesive systems are commercially available, the amount of solvent and other resin components in chemical composition of the two systems are different. in this study, application of 5% bromelain did not cause a significant reduction in microleakage, which is in contrast to the results of previous studies3,17. dayem et al. reported that application of bromelain on etched dentin significantly decreased leakage; they attributed this finding to the ability of bromelain to remove collagen network of etched dentin1,3. chauhan et al. reported similar results. they stated that bromelain increases the permeability of dentin substrate by depletion of collagen from the acid-etched surface and causes widening of dentinal tubules in the outer surface. it also increases the surface energy and penetration and infiltration of monomers into dentin17. since hydroxyapatite has high and collagen has low surface energy, collagen removal from etched dentin decreases the organic content, increases the surface energy and changes the hydrophilic properties of dentin, causing better penetration of adhesive monomers into dentin3. dayem and tameesh3 in sem assessment of bromelain group, represent that the orifices of the dentinal tubules look wider compared to that in nd:yag laser group3. in our study, in bromelain group, hybrid layer was thicker and resin tags were thicker and conical in shape. also, slight penetration of resin into accessory canals in dentin was also noted (figure 3). however, no statistically significant difference was found in marginal microleakage of bromelain and control groups, which may be due to the effect of other influential factors such as percentage and duration of use of bromelain. in this study, 5% bromelain was used for 30 seconds in order to simulate short clinical application and have the lowest possible toxicity while chauhan et al, and dayem et al. applied pure bromelain for one minute on dentin surfaces3,17. on the other hand, by activation of matrix metalloproteinases in the collagen matrix, insoluble components may be broken down into small peptides and amino acids, eliminated from the hybrid layer and replaced with water. however, since the tested protocol does not change the hydrophilic nature of adhesive and solvent evaporation, it does not decrease the permeability of interface either. thus, it cannot be expected that no leakage occurs in use of bromelain. our results showed that proteolytic agents did not prevent adhesive monomer penetration, solvent evaporation or adhesive polymerization; otherwise, we would have noticed greater microleakage compared to that in the control group23. based on the results of this study, no significant difference was noted in microleakage of control and nd:yag laser groups. but studies have reported controversial results regarding the effect of nd:yag laser for dentin surface preparation prior to restoration of cavity. some previous studies reported an increase in bond strength25 and a reduction in microleakage25. according to dayem research, laser irradiation caused higher number of resin tags and better penetration of adhesive1 while ribeiro et al.26 reported the greatest microleakage with the same laser irradiation parameters26. castro et al.27 stated that due to the absence of significant structural changes in the hybrid layer, laser irradiation cannot increase the bond strength to etched dentin27. some authors reported that application of laser prior to the use of adhesive system decreases the bond strength due to the obstruction of tubules and melting of dentin28. laser irradiation of dentin surface can increase the calcium and phosphorus content and decrease the concentration of oxygen in dentin and create a glossy surface. it can also cause thermal fusion, re-freezing and fusion of the smear layer 9 kasraei et al. and result in partial obstruction of dentinal tubules. moreover, it can decrease the microbial count. elimination of smear layer and microorganisms is favorable for the bonding process24. it appears that nd:yag laser irradiation results in formation of stronger bond by the adhesive system via fusion and re-crystallization of dentin apatites5, opening of tubules and elimination of smear layer1. in agreement with these findings, gan et al.29, in 2016 used the same bonding agent and pulse energy of laser as in our study and reported removal of collagen fibrils from dentin surface and lower microleakage in assessment by attenuated total reflectance-fourier transform infrared spectroscopy29. moreover, in addition to polymerization shrinkage and marginal gap, presence of higher water content, greater moisture and higher organic content in the gingival margin impair the bonding process and cause higher microleakage at this margin24. based on all the above, microleakage is expected to be lower in nd:yag laser group. but it should be noted that the interaction of laser with tooth structure is determined by laser irradiation parameters such as wavelength, laser energy, repetition rate, distance from the surface and optical properties of tissue. different results in different studies may be due to variability in irradiation parameters and time of laser irradiation (before or after the bonding process)28. since in this study microleakage after surface preparation with 5% bromelain and nd:yag laser was not significantly different from that in the control group, the null hypothesis was accepted. further studies using higher percentage of bromelain and nd:yag laser with other irradiation parameters are required to confirm or refute the results of this study. due to the limitation of technique that used to evaluate the microleakage, additional work is also needed to determine the effect of this protocols on dentin bonding. within the limitations of this study, application of 5% bromelain and nd:yag laser for deproteinization of etched dentin surface had no significant effect on microleakage of etch and rinse adhesive systems. references 1. dayem rn. assessment of the penetration depth of dental adhesives through deproteinized acidetched dentin using neodymium: yttrium–aluminum–garnet laser and sodium hypochlorite. lasers med sci. 2010 jan;25(1):17-24. doi: 10.1007/s10103-008-0589-4. 2. dayem rn. a novel method for removing the collagen network from acid-etched dentin by neodymium: yttrium-aluminum-garnet laser. lasers med sci. 2009 jan;24(1):93-9. 3. dayem rn, tameesh ma. a new concept in hybridization: bromelain enzyme for deproteinizing dentin before application of adhesive system. contemp clin dent. 2013 oct;4(4):421-6. doi: 10.4103/0976-237x.123015. 4. xu c, wang y. cross‐linked demineralized dentin maintains its mechanical stability when challenged by bacterial collagenase. j biomed mater res b appl biomater. 2011 feb;96(2):242-8. doi: 10.1002/jbm.b.31759. 5. scheffel dl, hebling j, scheffel rh, agee ka, cadenaro m, turco g, et al. stabilization of dentin matrix after cross-linking treatments, in vitro. dent mater. 2014 feb;30(2):227-33. doi: 10.1016/j.dental.2013.11.007. 10 kasraei et al. 6. spencer p, ye q, park j, topp em, misra a, marangos o, et al. adhesive/dentin interface: the weak link in the composite restoration. ann biomed eng. 2010 jun;38(6):1989-2003. doi: 10.1007/s10439-010-9969-6. 7. scheffel dls, hebling j, scheffel rh, agee k, turco g, de souza costa ca, et al. inactivation of matrix-bound matrix metalloproteinases by cross-linking agents in acid-etched dentin. oper dent. 2014 mar-apr;39(2):152-8. doi: 10.2341/12-425-l. 8. liu y, tjäderhane l, breschi l, mazzoni a, li n, mao j, pashley dh, et al. limitations in bonding to dentin and experimental strategies to prevent bond degradation. j dent res. 2011 aug;90(8):953-68. doi: 10.1177/0022034510391799. 9. pashley dh, tay fr, breschi l, tjäderhane l, carvalho rm, carrilho m, et al. state of the art etch-and-rinse adhesives. dent mater. 2011 jan;27(1):1-16. doi: 10.1016/j.dental.2010.10.016. 10. carrilho m, geraldeli s, tay f, de goes mf, carvalho rm, tjäderhane l, et al. in vivo preservation of the hybrid layer by chlorhexidine. j dent res. 2007 jun;86(6):529-33. 11. zhang s-c, kern m. the role of host-derived dentinal matrix metalloproteinases in reducing dentin bonding of resin adhesives. int j oral sci. 2009 dec;1(4):163-76. doi: 10.4248/ijos.09044.. 12. summitt jb, robbins jw, hilton tj, schwartz rs. fundamentals of operative dentistry: a contemporary approach. quintessence pub.; 2006. 13. lu s, zhao s-j, gao y, sun y, li x, chen j-h. proteoglycans affect monomer infiltration in the etch-and-rinse bonding technique. dent mater. 2014 nov;30(11):e289-99. doi: 10.1016/j.dental.2014.05.015. 14. breschi l, lopes m, gobbi p, mazzotti g, falconi m, perdigão j. dentin proteoglycans: an immunocytochemical feisem study. j biomed mater res. 2002 jul;61(1):40-6. 15. breschi l, gobbi p, lopes m, prati c, falconi m, teti g, et al. immunocytochemical analysis of dentin: a double‐labeling technique. j biomed mater res a. 2003 oct 1;67(1):11-7. 16. shin tp, yao x, huenergardt r, walker mp, wang y. morphological and chemical characterization of bonding hydrophobic adhesive to dentin using ethanol wet bonding technique. dent mater. 2009 aug;25(8):1050-7. doi: 10.1016/j.dental.2009.03.006 17. chauhan k, basavanna rs, shivanna v. effect of bromelain enzyme for dentin deproteinization on bond strength of adhesive system. j conserv dent. 2015 sep-oct;18(5):360-3. doi: 10.4103/0972-0707.164029. 18. han b, jaurequi j, tang bw, nimni me. proanthocyanidin: a natural crosslinking reagent for stabilizing collagen matrices. journal of biomedical materials research part a. 2003;65(1):118-124. 19. al‐ammar a, drummond jl, bedran‐russo ak. the use of collagen cross‐linking agents to enhance dentin bond strength. j biomed mater res a. 2003 apr 1;65(1):118-24. 20. bedran‐russo akb, pashley dh, agee k, drummond jl, miescke kj. changes in stiffness of demineralized dentin following application of collagen crosslinkers. j biomed mater res b appl biomater. 2008 aug;86(2):330-4. 21. castellan cs, bedran-russo ak, karol s, pereira pnr. long-term stability of dentin matrix following treatment with various natural collagen cross-linkers. j mech behav biomed mater. 2011 oct;4(7):1343-50. doi: 10.1016/j.jmbbm.2011.05.003. 22. macedo g, yamauchi m, bedran-russo a. effects of chemical cross-linkers on caries-affected dentin bonding. j dent res. 2009 dec;88(12):1096-100. doi: 10.1177/0022034509351001. 23. hass v, luque-martinez iv, gutierrez mf, moreira cg, gotti vb, feitosa vp, et al. collagen cross-linkers on dentin bonding: stability of the adhesive interfaces, degree of conversion of the adhesive, cytotoxicity and in situ mmp inhibition. dent mater. 2016 jun;32(6):732-41. doi: 10.1016/j.dental.2016.03.008. 24. savadi oskoee s1, alizadeh oskoee p, jafari navimipour e, ahmad ajami a, pournaghi azar f, rikhtegaran s, et al. comparison of the effect of nd: yag and diode lasers and photodynamic therapy on microleakage of class v composite resin restorations. j dent res dent clin dent prospects. 2013;7(2):74-80. doi: 10.5681/joddd.2013.013.. 11 kasraei et al. 25. wen x, liu l, nie x, zhang l, deng m, chen y. effect of pulse nd: yag laser on bond strength and microleakage of resin to human dentine. photomed laser surg. 2010 dec;28(6):741-6. doi: 10.1089/pho.2009.2579. 26. ribeiro cf, anido aa, rauscher fc, yui kck, gonçalves sedp. marginal leakage in class v cavities pretreated with different laser energy densities. photomed laser surg. 2005 jun;23(3):313-6. 27. castro fl, andrade mf, hebling j, lizarelli rf. nd: yag laser irradiation of etched/unetched dentin through an uncured two-step etch-and-rinse adhesive and its effect on microtensile bond strength. j adhes dent. 2012 apr;14(2):137-45. doi: 10.3290/j.jad.a21854. 28. marimoto a, cunha l, yui k, huhtala mf, barcellos dc, prakki a, et al. influence of nd: yag laser on the bond strength of self-etching and conventional adhesive systems to dental hard tissues. oper dent. 2013 jul-aug;38(4):447-55. doi: 10.2341/11-383-l 29. gan j, liu s, zhou l, wang y, guo j, huang c. effect of nd: yag laser irradiation pretreatment on the long-term bond strength of etch-and-rinse adhesive to dentin. oper dent. 2017 jan/feb;42(1):62-72. doi: 10.2341/15-268-l. untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8652940 volume 17 2018 e18887 original article ¹ school of dentistry, são paulo state university “júlio de mesquita filho”/brazil ² school of engineering, federal technological university of paraná/ brazil 3 faculty of science and technology, são paulo state university “júlio de mesquita filho”/brazil 4 school of dentistry, guarulhos university/brazil correpondence to: gabriel pereira nunes school of dentistry, são paulo state university “júlio de mesquita filho”/ brazil. gabriel.pereira.nunes@hotmail.com received: april 11, 2018 accepted: may 30, 2018 the importance of student monitoring in academic learning: a two-year follow-up gabriel pereira nunes¹, beatriz ommati pirovani¹, hiago guimarães silva², ana victória butarelo¹, juliana da silva rossini¹, jefferson martins costa³, larissa pereira nunes4, karina vieira martins³ abstract: monitory exercises must provide conditions that support academic and personal development for students during graduation, once it allows a leveling possibility whereas the monitor meets the demands from the students in earlier years. aim: this study has as objective to analyze the importance of monitory in advantage of the best academic achievement in students. methods: in the years of 2015 and 2016, sophomore odontology students were monitored on pharmacology, originated from the need of remedial studies due to its high incidence of failure. for such, weekly monitoring on theoretical classes, study groups and content reviews were done. after two years of monitoring the number of failures and respective percentages were determined. the data was submitted to variance analysis and student-newman-keuls test (p< 0,001). results: the monitory had expressive results, since it reduced significantly failure numbers, statistically much inferior than previous years. conclusion: hence, it was possible to verify that monitory had positive effects on students learning, allowing access to knowledge and being imperative to the covered content systematization on the subject, since advising and monitoring students made their learning more natural. keywords: student monitoring. learning. pharmacology. 2 nunes et al. introduction academic monitoring is characterized as a program to foster education, arising from the need and importance of contributing to the advance of the monitor’s academic knowledge, as well as providing a leveling possibility whereas the monitor meets the demands of the students in earlier years. it is a necessary opportunity for the enrolled student to prepare itself for learning activities. even as a serious experience to break down opinions of limited mind about the global universe implicating learning and teaching, for through it some doubts are clarified, both in the students learning scope and on the educational scope, serving as base to build the educator’s identity of the student-monitor. besides the monitor’s academic and personal development, these activities have served as support to curricular subjects study and, thereafter advise the student to acquire greater knowledge, seeking information and success while fulfilling the program content of such subjects. given that some subjects have a continuously high level of failure, monitory also has the function to help students to acquire study skill, support with regard to their difficulties. this situation is reinforced by ferreira by mentioning that “uniting knowledge makes us wiser and more productive. it is necessary to discover new perspectives, dividing and adding up to each other”3. variables of many dimensions can hinder learning during childhood and adolescence. however such issue is also customary in higher education. learning difficulties may be due to cognitive shortfalls that harms knowledge acquiring, as also, in most cases, results only from educational or environmental problems that are not associated to cognitive shortfalls. this training program aims to provide interdisciplinarity and unite theoryand practice during the developed activities, aiding the teacher, facilitate and maximize students learning, arousing their interest into the importance of the academic subject. monitoring is an activity that puts the student in interaction with didactic experiences. the teaching routine, class preparation as well as stance training facing the most diverse situations found in teaching, serve as solid bases to those who covet academic carriers. to avoid difficulties and even to enhance the monitor’s efficiency and an ideal performance in the role demanded by this activity, the monitor must be always searching for information and updating itself, to avoid setbacks and inconveniences such as disbelief from the aided students. if the monitor, while student-advisor, loses his identity, the learning process gets misguided, flipping the scales solely to the professor side, causing the monitor to lose its meaning against the class and its leader. this way academic monitoring will contribute very little or, in worst cases scenario, nothing at all to its growth in the teaching approach. the initiative and relevance to study the theme of this paper came from the need to verify if academic monitory contributes in a positive way to the students learning, favoring and supporting the studies, here restricted to the educational scope of bachelor degree in odontology, as well as the experience lived by the monitor in its first steps towards teaching, if monitoring can assign relevant considerations as, for example, to evaluate the monitor’s profile and if the developed activities guarantee conclusive bases to determine a possible conduction and motivation to a sequence in teaching carrier. 3 nunes et al. materials and methods the monitory activities in pharmacology (sophomore year) in the odontology course at the state university “júlio de mesquitafilho” araçatuba’s campus were developed on class days and in extra class times. the monitor attended the subject’s classes weekly, aiding the students when needed. furthermore, the monitor would schedule program content reviews to classes of both terms, in a way that they had an explanatory feature in on class and answering doubts and questions on the next. they were all executed in opposite time to the scheduled classes, at night for day time classes and vice-versa. outlined study groups with fewer students for better exploitation of the meeting were made, even with the prerogative of answering all doubts and difficulties they had. these study groups took place on the campus library or in the students’ home (when these had enough room to accommodate them in an organized and plausible fashion). according to perpétuo and gonçalvez7. the group dynamic composes a valuable educational instrument that can be used to work the teaching-learning when it chooses an educational conception that values as much as theory as practice and considers all involved in this process as assets. another accessory idealized and accomplished was the auxiliary system assistance to home exercise regimen, therefore, the students that went through some kind of scholar withdraw, for example, illness or surgical procedure, were accounted for and aided in their homes to be aware of the contents taught in class and updated as to assignments. likewise in the year of 2015, the monitor (first author) created, by his own hand, a study tutorial, which covered all subjects and contents from the politic-pedagogical program of the subject, with the goal to guide and direct the students during the time they would be studying on their own, just as it is imperative to every student the individual dedication to acquire knowledge. in addition, throughout the school year, the students supported by the monitor, held a virtual group to solve exercises or work doubts out on a faster and safest way that involved everyone, so they could share their inquiries, suggestions and deliberations. parrenoud8 highlights that the teacher’s figure on formative evaluation demands disposition, and a stance that won’t end in the instrumentality, but an active positioning and a teaching methodology planning filled with intentionality to face learning difficulties. after the development of the monitory in the discipline, a data collection was executed on the institution data base, having as register the information obtained about the number of enrolled students and failures and, afterwards, such data was processed and segmented (related to day-time and night-time courses) to subserve the making of charts, aiming for a better visualization and comprehension of the reached results. to statistical analysis, it was considered as variable the number of failures and as variation factor the presence or not of the monitory in the corresponding school year. the data presented normal distribution and was subjected to variance analysis, followed by student-newman-keuls test. 4 nunes et al. results and discussion table 1. percentage of students’ disapprovals of the integral period in the discipline of pharmacology of foa / unesp, according to the school year analyzed school year analyzed number of enrolled students number of failures failures percentage 2011 78 12 15,3% a 2012 81 09 11,2% b 2013 79 13 16,4% a 2014 76 12 15,8% a 2015 74 03 4,1% c 2016 78 04 5,1% c different superscript letters indicate a statistical difference between the percentage of disapprovals in the school years described (student-newman-keuls, p <0.001). table 2.percentages of failures o nigh-time course students in the pharmacology subject at foa/unesp, according to the school year analyzed school year analyzed number of enrolled students number of failures failures percentage 2011 25 05 20,0% b 2012 28 06 21,4% b 2013 29 08 27,5% a 2014 24 05 20,8% b 2015 24 02 8,3% c 2016 18 00 0,0% d different superscript letters indicate a statistical difference between the percentage of disapprovals in the school years described (student-newman-keuls, p <0.001). according to the information contained in the previous charts, it is noted that the pharmacology subject in the years prior to the start of the monitoring had high levels of failure as can be seen through the percentage (%) of failures, as much in the day-time course as in the night-time course. after the monitory’s annexation to the subject, it is observed on the first year (2015) a reduction in the percentage of failure in both courses (average o ~12% for day-time and ~19% for night-time courses) when compared to the year of 2014, when there wasn’t a monitor performing its role in this activity. as there is a prospect showing high levels of failure in the four prior to the monitory’s introduction, it is reasonable to imply that it was ruling for a better performance of the students in the subject and, hence a significant reduction in the number of failures, being categorical in this action, for these are future professionals that will render aid to health and lives of others. in addition to the mitigation of the failures number, monitory also achieved impressive and lush deeds, since some students that had failed the subject several times in a dependency system were approved. specifically, one student, that had failed the subject for five years, was given special attention and approach, so through solid accompaniment and motivational assistance the student, that laid unmotivated by the adverse situation, adopted the task of studying as primordial and necessary for his life, being able to overcome the obstacles and difficulties imposed by the content 5 nunes et al. and achieved the approval he so longed for, becoming an example for persistency and effort in the pursuit for admission. indeed, educating an individual presupposes to transform it, help it develop its potential, trying to discover others. it is need to take under consideration the genetic and environmental factors and the interaction between both that education will then modify9. justifiably, the peculiarity that the monitor when studying the subject himself went through the same difficulties and requisitions made it easy the way he performed his duties, the way he should transmit the information to the monitored students, as well as the understanding related to the problems the students faced and the applicable measure to answer their questionings10. according coelho, from the moment that education starts to understand the principles of the learning process, the problems that may occur in this area will be treated and solved without taboos and without traumas. once that, considering the intrinsic role of the educator in providing support to the process of learning so the student is able to develop itself safely, in the constant quest for its independence and autonomy11. the evaluation of learning is comprehended as a rigorous practice of accompaniment and reorientation of the student, facing the difficulties of learning and thus, ensure students development. in relation to the total number of failures in the night-time course in the year of 2016, being of 0%, is mainly justifiable for the smaller number of students in class and in a way this made possible to have greater control and perception over the students individual difficulties, as a result, knowledge and information transmission were done in a holistic manner, since the students difficulties were clear, everyone could expose their its setback with less shyness, even because collective experience allowed greater exposition and the immediate feedback cooperated in the students critical analysis development10. another explanation is based on the average class size making it able to combine a diversity of students profile with the monitor’s attention. kokkelenberg13 studied the fact that the number of students (n) in a classroom interfere in the study-learning e as a consequence in the college students grade. just as, bedard and kuhn14, analyzed how the size of the classrooms interfere in the evaluation made by students about teachers’ performances, whose smaller classrooms were the ones with the best mentor’s efficiency. a crucial goal of the monitory program is to intensify the cooperation between teachers and students in the academic activities, being processed as something of extreme revolution to contribute to enhance graduation teaching, for in this context, the teacher works in the condition of understanding learning construction process and interfering to lead the group into a redetermination of learning. the interaction between the actors involved around knowledge acquisition becomes a central element in evaluation. esteban15 considers that, to evaluate doesn’t mean to judge its learning, it must be a moment that reveals the process of knowledge construction, when the student is not yet aware of its possibilities for advance and overcoming. the monitoring is a learning space for the student, favoring its personal, academic and professional growth. it tends to refine its abilities as a teacher and motivate it to follow this path, or helps it realize the hardships faced by the professionals in this area, for its own development16. in this context, being a monitor propelled the student into an orientation about its professional future, directing it to pursue a carrier in 6 nunes et al. teaching and it was a landmark for an evaluation and personal conclusion to define its prosecution area: its ingress in post-graduation. thus, it was possible to verify that the monitory had positive effects on the students learning, allowing also verifying that the formative assistance has positive effects in the students formation, ensuring the access to knowledge and it was indispensable to the subjects covered content systematization, since advising and monitoring the students made them acquire greater ease in studying. furthermore, the presence of the monitor contributes deeply to the right performance of the students, for it enhances academic formation for both aided students and the monitor itself. therefore it would be viable to institute the monitory in all subjects, when possible; because this is also developed as a positive instrument in the process of facing learning difficulties. references 1. foundation federal university of rondônia. resolution nº 129/consea, 2006 jul 13. [establishes norms for the monitoring program and gives other measures]. amended by resolution nº 291/ consea, 2012 oct 23. portuguese. 2. cordeiro as, oliveira bp. [academic monitoring: the importance for undergraduate chemistry students]. in: 2nd meeting of forensic science and expertise of the rn. natal: annq; 2011 [cited 2018 jan 25]. available from: disponível em http://annq.org/eventos/upload/1325330899.pdf. portuguese. 3. ferreira as, pacheco ab. [psychopedagogical intervention in a multidisciplinary perspective: working to develop the potential of adolescent students]. in: federal council of psychology. [professional experiences in the construction of educational processes in the school]. brasília: cfp; 2010. p. 53-76. available from: https://site.cfp.org.br/wp-content/uploads/2010/09/construcao_de_processos_ educativos_publicacao.pdf. portuguese. 4. araujo rb, tavares lb. [family and learning difficulties.pedagogia faip]. 2011 jan 24 [cited 2018 mar 2]. available from: http://pedagogiafaip.blogspot.com/2011/01/familia-e-dificuldades-deaprendizagem.html. portuguese. 5. soares maa, santos kf. [monitoring as a subsidy to the teaching-learning process: the case of financial administration in cchsa-ufpb]. in: xi teaching initiation meeting. joão pessoa: federal university of paraíba; 2008 [cited 2018 mar 5]. available from: www.prac.ufpb.br/anais/xenex_xienid/ xi_enid/monitoriapet/anais/area4/4cchsadcsamt04.pdf. portuguese. 6. sousa júnior ja, silva al, magno a, santos mbh, barbosa ja. [importance of the monitor in the teaching of organic chemistry in the search of the professional training of the agricultural sciences]. in: xi teaching initiation meeting. joão pessoa: federal university of paraíba; 2008 [cited 2018 mar 5]. available from: ww.prac.ufpb.br/anais/xenex_xienid/xi_enid/monitoriapet/anais/ area4/4ccadcfsmt03.pdf. portuguese. 7. perpétuo sc, gonçalvez am. [dynamics of groups in leadership formation]. rio de janeiro: dp&a; 2005. portuguese. 8. perrenoud p. [evaluation: from excellence to regulation of learning between two logics]. porto alegre: artes médicas; 1999. portuguese. 9. novaes mh. [school psychology]. petrópolis: vozes; 1972. portuguese. 10. turner am, prihoda tj, english dk, chismark a, jacks me. millennial dental hygiene students’ learning preferences compared to non-millennial faculty members’ teaching methods: a national study. j dent educ. 2016 sep;80(9):1082-90. http://annq.org/eventos/upload/1325330899.pdf https://www.ncbi.nlm.nih.gov/pubmed/27587575 7 nunes et al. 11. coelho mt. [learning problems]. são paulo: ática; 1990. portuguese. 12. luckesi cc. [learning evaluation: component of the pedagogical act]. são paulo: cortez; 2011. portuguese. 13. kokklenberg ec, dillon m, christy sm. the effects of class size on student grades at a public university. econ educ rev. 2008 apr;27(2):221-33. 14. bedard k, kuhn p. where class size really matters: class size and student ratings of instructor effectiveness. econ educ rev. 2008 jun;27(3):253-65. 15. eeteban mt, organizator. [assessment: a practice in search of new meanings]. rio de janeiro: dp&a; 2001. portuguese. 16. oliveira aa, maia filho af, siqueira lbo. [monitoring of: the first steps in academic life]. in: xi teaching initiation meeting. joão pessoa: federal university of paraíba; 2008 [cited 2018 mar 5]. available from: www.prac.ufpb.br/anais/xenex_xienid/xi_enid/monitoriapet/anais/ area4/4ccsademt04.pdf. portuguese. 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1513/1166 1/6 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1513/1166 2/6 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1513/1166 3/6 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1513/1166 4/6 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1513/1166 5/6 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1513/1166 6/6 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1520/1173 1/13 28/01/2019 pdf.js viewer 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https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1527/1180 9/9 braz j oral sci. 15(3):173-175 odontogenic infections in a dental emergency care unit: eleven-year epidemiological analysis antônio luis neto custódio1, raphael márcio egídio cota2, leandro junqueira de oliveira3 1maxillo-facial surgeon associate professor, department of morphology of the biological sciences institute, federal university of minas gerais, belo horizonte, minas gerais, brazil professor, department of oral and maxillofacial surgery, pontifical catholic university of minas gerais, belo horizonte, minas gerais, brazil 2graduate student of pontifical catholic university of minas gerais 3maxillo-facial surgeon substitute professor of the department of oral and maxillofacial surgery, pontifical catholic university of minas gerais, belo horizonte, minas gerais, brazil correspondence to: leandro junqueira de oliveira av. brasil, 1438, sala 708, bairro funcionários. belo horizonte – mg brazil cep: 30140-003 tel: (31) 8471-2440 fax: (31) 2127-1816 email: leojunq@hotmail.com abstract aim: to evaluate medical records from patients who underwent abscess drainage due to odontogenic infections in a public hospital in the city of belo horizonte (minas gerais, brazil) during the period of 2003 and 2013. methods: a retrospective analysis of cases which required drainage of abscesses due to dental infections. the types of therapeutic procedures analyzed were endodontic drainage, intraoral mucosa drainage, periodontal drainage, and extraoral drainage. results: 162,902 cases required dental assistance, and 32,352 cases required drainage of abscesses due to dental infections. the most frequent approach was endodontic drainage (21,313 procedures); the least frequent procedure was extraoral drainage (922 procedures). conclusions: odontogenic infection is a common clinical condition in dental clinics. it should be diagnosed and treated as quickly as possible to avoid or minimize progression to more severe cases. keywords: focal infection; dental; epidemiology; retrospective studies. introduction initially well-localized dental infections of low complexity are usually resolved without complications when appropriate therapy is conducted in cases where patient does not have predisposing systemic conditions. however, inefficient therapy or lack of treatment may result in progression to severe conditions, such as osteomyelitis, ludwig's angina, airway compromise, necrotizing fasciitis, intracranial structure involvement and mediastinitis, which can be life-threatening complications1,2. rapid and aggressive treatment of odontogenic abscesses is necessary to avoid these complications2. dental abscesses usually include secondary caries, trauma, or periodontal or endodontic infections3. therapy can include the elimination of the causative focus, extraction, endodontic treatment, surgical drainage, and/or administration of antibiotics1. received for publication: august 15, 2016 accepted: april 19, 2017 original article braz j oral sci. july | september 2016 volume 15, number 3 http://dx.doi.org/10.20396/bjos.v15i3.8649600 174 the objective of this study was to review and analyze the treatment of odontogenic abscesses at the emergency department of the municipal hospital odilon behrens (hmob) from 2003 to 2013. material and methods this retrospective study was conducted in the dental department of hmob of belo horizonte city, mg, brazil, and was approved by the ethics committee on human of the hospital (research number: 56442116.7.0000.5129). records dating from 2003 to 2013 were analyzed to determine the total number of drainage procedures performed for abscesses in the maxillofacial region. the factors evaluated were the types of drainage procedures performed: endodontic, intraoral or extraoral mucosa extraoral and sulcular (periodontal abscesses). the number of cases of ludwig's angina, which may develop in simpler cases, was recorded for comparison between cases of different complexities. results analyses of 11 years of data from the dental dpartment showed that 162,902 patients were treated (average, 14,809/year). abscess drainage was performed 32,352 times or an average of 2,941 times per year (19.85%). the most frequent surgical procedure was endodontic abscess drainage with an average of 1,937 cases per year (65.86%), followed by intraoral tissue drainage (average of 683 cases 23.23%) and periodontal abscess drainage (average of 224 cases 7.61%). the least frequent procedure was extraoral drainage, with an annual average of 84 cases (2.84%). these results are shown in table 1. ludwig's angina is a complication still less frequent, with 135 cases in 11 years (0.41%). the hospitalization for at least one day was necessary in 388 patients (average of 35 cases per year 1.19%). the mortality occurred in 17 cases (average 1.5 cases per year 0.05%). these results are shown in table 2. odontogenic infections in a dental emergency care unit: eleven-year epidemiological analysis braz j oral sci. 15(3):173-175 table 1 total number of patients from 2003 to 2013 in hmob’s dental department and the frequency of infections and procedures. year total patients total infections extraoral drainage intraoral mucosal drainage periodontal drainage endodontic drainage ludwig's angina n n % n % n % n % n % n % 2003 15,126 3,211 21.23 80 2.49 602 18.74 252 7.84 2,257 70.28 20 0.62 2004 14,908 2,970 19.92 70 2.35 533 17.94 231 7.77 2,122 71.44 14 0.47 2005 14,681 2,835 19.31 74 2.61 584 20.59 288 10.15 1,876 66.17 13 0.45 2006 14,496 2,975 20.52 80 2.68 528 17.74 254 8.53 2,100 70.58 13 0.43 2007 14,568 3,349 22.98 61 1.82 679 20.27 256 7.64 2,340 69.87 13 0.38 2008 15,119 3,693 24.42 72 1.94 697 18.87 325 8.80 2,585 69.99 14 0.37 2009 16,671 3,527 21.15 65 1.84 921 26.11 296 8.39 2,231 63.25 14 0.39 2010 15,787 3,207 20.31 76 2.36 784 24.44 263 8.20 2,069 64.51 15 0.46 2011 15,329 2,500 16.30 143 5.72 637 25.48 138 5.53 1,566 62.64 16 0.64 2012 13,803 1,990 14.41 96 4.82 759 38.14 128 6.43 1,004 50.45 3 0.15 2013 12,414 2,095 16.86 105 5.01 793 37.85 33 1.57 1,163 55.51 0 0 n = number; % = percentage table 2 the frequency of hospitalization and mortality of patients from 2003 to 2013 in hmob’s dental edpartment. year hospitalized patients mortality n n 2003 42 2 2004 37 1 2005 41 3 2006 39 1 2007 29 2 2008 33 1 2009 29 1 2010 42 1 2011 46 3 2012 34 2 2013 16 0 discussion suppurative odontogenic infections represent an important challenge in the dental clinic because of the risk of propagation and serious potential complications4. the assessment of infection involves the extent, etiology, and systemic conditions. clinical data, images, and laboratory tests can determine if there is a localized infection or suggest the propagation of infection1, and, based on these findings, therapeutic modalities are chosen. however, the most important procedures for orofacial suppurative infections are surgical drainage and removal of the primary source of infection5. in this study, treatment procedures were divided as follows: extraoral drainage, intraoral mucosal drainage, periodontal drainage, and endodontic drainage. it is important to emphasize that the hmob did not use tooth extraction as a protocol for pain relief. hmob has adopted drainage for treating dental infections 175 and prefers the continuation of appropriate treatment. the number of cases of ludwig's angina was cited to evaluate and compare the number of low complexity infections with that of highly complex infections without the intention to discuss local and systemic treatments for this type of infection. well-localized infections may result in severe conditions and can be life-threatening; in 11 years, 388 patients (1.19%) required internment of at least one day and 17 patients (0.57%) died due to complications of these infections. after analyzing the data presented, infections involving the maxillofacial region represented 19.85% of all dental emergencies in this hospital. of these patients, 96.71% underwent some kind of intraoral drainage. extraoral drainages, which require greater surgical experience, anatomical knowledge and result in an external scar, represented only 2.84% of cases. furthermore, the cases diagnosed as ludwig's angina amounted to only 0.41% of all cases observed during this period. comparison of the number of procedures performed revealed that treatments of low complexity infections are more common daily for a dental surgeon than the high complexity ones. this confirms the need for professional knowledge to make a diagnosis and treat low complexity infections to avoid progression to serious conditions. many studies have been published on the epidemiology of dental infections1,6-11. however, studies on the anatomical drainage locations (local treatment) and their prevalence are few. this study was conducted in the dental department of hmob of belo horizonte city, mg, brazil, which is a reference center for dental emergencies. it has full-time dentists and oral maxillofacial surgeons. it aims to serve an estimated of 4 million people in the belo horizonte metropolitan area. studies like the one conducted at metro health medical center (cleveland, ohio, usa), which serves 3.4 million people in northern ohio, showed that 86 cases of odontogenic infection were treated from 1983 to 19896. another study conducted in well taub general hospital in houston (texas, usa) analyzed 50 cases of oral maxillofacial infection from 1987 to 1990; of these, 43 cases were of odontogenic origin1. from 1972 to 1990, 561 patients were admitted to the maxillofacial surgery division at the university of turin, italy, with acute infections. in the present study, we found that 32,352 patients had dental infections, an average of 2,941 cases annually requiring some abscess drainage procedure. factors such as geographical location, population density, socio-economic status, and the registration period should be considered in any comparison of statistical results12. additionally, the oral health of the population must be considered, taking into account hygiene and access to preventive and curative dental treatment. this study, conducted at hmob, on the incidence and odontogenic infections in a dental emergency care unit: eleven-year epidemiological analysis braz j oral sci. 15(3):173-175 evolution of these infections improves current understanding of dental care emergencies in a representative sample of the population of belo horizonte and provides data that could be helpful for developing strategies for preventing odontogenic infections. acknowledgements assistance from colleagues: luiz augusto lima former manager of the emergency department of municipal hospital odilon behrens fernando sartori rocha campos er manager of the municipal hospital odilon behrens references 1. krishnan v, johnson jv, helfrick jf. management of maxillofacial infections: a review of 50 cases. j oral maxillofac surg. 1993 aug;51(8):868-73. 2. baqain zh, newman l, hyde n. how serious are oral infections? j laryngol otol. 2004 jul;118(7):561-5. 3. chavez de paz le. redefining the persistent infection in root canals: possible role of biofilm communities. j endod. 2007 jun;33(11):652-62. 4. opitz d, camerer c, camerer dm, raguse jd, menneking h, hoffmeister b, et al. incidence and management of severe odontogenic infections-a retrospective analysis from 2004 to 2011. j craniomaxillofac surg. 2015 mar;43(2):285-9. doi: 10.1016/j.jcms.2014.12.002. 5. bahl r, sandhu s, singh k, sahai n, gupta m. odontogenic infections: microbiology and management. contemp clin dent. 2014 jul;5(3):30711. doi: 10.4103/0976-237x.137921. 6. haug rh, hoffman mj, indresano at. an epidemiologic and anatomic survey of odontogenic infections. j oral maxillofac surg. 1991 spe;49(9):976-80. 7. saito ct, gulinelli jl, marão hf, garcia ir jr, filho om, sonoda ck, et al. occurrence of odontogenic infections in patients treated in a postgraduation program on maxillofacial surgery and traumatology. j craniofac surg. 2011 sep;22(5):1689-94. doi: 10.1097/scs.0b013e31822e5c8d. 8. ottaviani g, costantinides f, perinetti g, luzzati r, contardo l, visintini e, et al. epidemiology and variables involved in dental abscess: survey of dental emergency unit in trieste. oral dis. 2014 jul;20(5):499-504. doi: 10.1111/odi.12164. 9. sánchez r, mirada e, arias j, paño jr, burgueño m. severe odontogenic infections: epidemiological, microbiological and therapeutic factors. med oral patol oral cir bucal. 2011 aug;16(5):e670-6 10. bierderman gr, dodson tb. epidemiologic review of facial infections in hospitalized pediatric patients. j oral maxillofac surg. 1994 oct;52(10):1042-5. 11. igoumenakis d, gkinis g, kostakis g, mezitis m, rallis g. severe odontogenic infections: causes of spread and their management. surg infect (larchmt). 2014 feb;15(1):64-8. doi: 10.1089/sur.2012.178. 12. peters es, fong b, wormuth dw, sonis st. risk factors affecting hospital length of stay in patients with odontogenic maxillofacial infections. j oral maxillofac surg. 1996 dec;54(12):1386-91. revista fop n 13 1550 braz j oral sci. april/june 2008 vol. 7 number 25 using overdenture on implants and complete dentures:using overdenture on implants and complete dentures:using overdenture on implants and complete dentures:using overdenture on implants and complete dentures:using overdenture on implants and complete dentures: effects on postural maintenance of masticatoryeffects on postural maintenance of masticatoryeffects on postural maintenance of masticatoryeffects on postural maintenance of masticatoryeffects on postural maintenance of masticatory musculaturemusculaturemusculaturemusculaturemusculature carla moreto santos1; mathias vitti2; wilson matsumoto3; renato josé berro4; marisa semprini2; jaime eduardo cecílio hallak5; rodrigo galo6; simone cecílio hallak regalo2 1dds, phd student, department of morphology, stomatology and physiology, dental school of ribeirão preto, university of são paulo, brazil 2dds, msc, phd, professor, department of morphology, stomatology and physiology, dental school of ribeirão preto, university of são paulo, brazil 3dds, msc, phd, assistant professor, department of prosthodontics, dental school of ribeirão preto, university of são paulo, brazil 4dds, msc, phd, assistant professor, dental association of ribeirão preto (aorp), brazil 5dds, msc, phd assistant professor, department of neuropsychiatry and psychological medicine, medical school of ribeirão preto, university of são paulo, brazil 6dds, msc, phd student, department of prosthodontics, dental school of ribeirão preto, university of são paulo, brazil received for publication: march 06, 2008-08-18 accepted: june 12, 2008 correspondence to: simone cecilio hallak regalo depto. de morfologia, estomatologia e fisiologia / forp-usp avenida do café, s/n, bairro monte alegre, 14040-904 ribeirão preto, sp, brasil phone: +55-16-3602 4015 fax: 55-16-3633-0999 e-mail: schregalo@forp.usp.br a b s t r a c t aims: thanks to advances in osseointegration, oral rehabilitation specialists have had the option of using implants to improve retention and stability in treatments with complete dentures. this study compared the masticatory muscle electromyographic activity in implant-supported overdenture wearers, complete denture wearers and dentate individuals. the electromyographic activity of the right and left masseter muscles, and the right and left anterior temporalis muscles was analyzed in 10 implant-supported overdenture wearers (group 1), 10 conventional complete denture wearers (group 2), and 10 dentate individuals (group 3), with mean age of 65 years, at rest and during postural position maintenance. the analysis was performed using the myosystem-br1 electromyographer with differential active electrodes. analysis of variance tests were carried out to compare the groups and muscles and revealed different electromyographic values that were statistically significant at 1% significance level. duncan’s pos-hoc test showed that group 3 presented the smallest values (pd”0.01). the electromyographic contraction pattern was similar between groups 1 and 3 (p>0.01), and hyperactivity of anterior temporalis muscles was observed in group 2 (pd”0.01). conclusions: dentate individuals had smaller electromyographic values of masticatory muscles and the overdenture use caused electromyographic contraction patterns similar to those of dentate individuals in both positions. key words masticatory muscle, overdenture, complete denture, electromyography. i n t r o d u c t i o n geriatric dentistry developed as a consequence of world population aging and has contributed to improve the quality of life of elderly individuals by both preventive and curative measures. elderly people who are edentulous or use dentures that are worn out or inappropriately adjusted may have functional limitations, such as chewing difficulties. due to such limitations, these individuals may limit their food choices1. in addition to reducing their eating pleasure, this change in feeding may compromise their overall health, since they often choose foods that have less fiber and are of a low nutritional value. aging has different effects on the organs and systems of each human being. in the oral cavity, it appears to have a great influence and may reduce chewing efficiency due to either tooth loss and/or bad conditions of remaining teeth, which results in difficulties in eating appropriately1-2. the aging process produces a typical structural deterioration of the stomatognathic system as well as muscles and nerves throughout the entire body. in case of tooth loss, parts of the mandibular bone are reabsorbed and the oral mucosa loses its morphological characteristics, the muscular fibers become atrophic, a great part of motor neurons and their receptors are lost, and there is also a reduction of neurotransmitters3. as age advances, sensory functions, including taste, smell, and touch (for texture and temperature) become less precise. studies of mandibular masticatory movements have shown there is a reduction in vertical 1551 development during the chewing cycle in elderly individuals, complete denture wearers, when compared to dentate young individuals 4-6. karlsson and carlsson7 attributed this difference to several factors such as volume reduction in masticatory muscles, poor neuromotor coordination, and energy reduction in muscle cells. oral rehabilitation treatment may have a direct influence on various structures, including muscle activity. this may occur because rehabilitating intervention is performed in the oral cavity, which is part of the stomatognathic system, in which all structures function harmoniously8-9. good denture fitting is essential for complete and partial denture wearers to achieve proper speech articulation and to preserve chewing and swallowing functions. thus, it is of utmost importance to study muscular activity of the masticatory system in cases of oral rehabilitation. only with complete understanding of such functions one can obtain results with the highest level of efficiency. many authors consider elderly edentulous individuals as oral invalids and, like complete denture wearers, they have a reduced capacity in many masticatory system functions compared to individuals with natural dentition10-11. complete dentures supported by osseointegrated implants in the edentulous jaw have provided occlusion stabilization that shows considerable improvement in muscular activity and mandible movements. this type of treatment has been increasingly used by oral rehabilitation specialists to improve the satisfaction and functional comfort of edentulous individuals. this study compared the masticatory muscle electromyographic activity in implant-supported overdenture wearers, complete denture wearers and dentate individuals (control group). the results may provide valuable data to be considered when choosing one of these total oral rehabilitation treatments for elderly individuals and allow for detailed diagnosis and prognosis, which will improve their quality of life. material and methods volunteers this study was approved by the research ethics committee of the dental school of ribeirão preto, university of são paulo (forp-usp) in compliance with the resolution 196/ 96 of the brazilian national health council. the volunteers were informed about the experiment and agreed to take part in the study by signing an informed consent form (process number 2003.1.752.58.9). thirty subjects with mean age of 65 years were enrolled in this study. as inclusion criteria, the volunteers should have overall good health conditions, present similar clinical buccofacial conditions, no complaints of orofacial pain, no history of previous orthodontic treatment and no evidence for other pathologies. the selected subjects were allocated into 3 groups, as follows. group 1: 10 individuals (6 women and 4 men), aged between 46-75 years (mean age = 64.4 years), wearers of upper complete dentures and lower implant-supported overdentures for at 6 six months. the prostheses were evaluated considering the main factors that promote acceptable support, stability, and retention for appropriate functioning of the conventional upper complete dentures. the lower overdentures were supported by 4 implants located in the foramen mental region. a bar was attached to these implants, connected to the prostheses by a plastic clip located in the midline. one ball was placed on either end of the bar and connected to capsules using an o-ring system. the disocclusion guides were grouped, bilaterally balanced. factors such as occlusal vertical dimension, resting vertical dimension and centric relation were evaluated by methods proposed by misch12 and were considered as appropriate. group 2: 10 volunteers (5 men and 5 women), aged between 51-82 years (mean age = 68 years), wearers of upper and lower conventional complete dentures for at least 6 months. these participants were clinically evaluated in the same way as in group 1 and were selected according to the satisfactory conditions of their dentures. the disocclusion guides were grouped, bilaterally balanced. factors such as occlusal vertical dimension, resting vertical dimension and centric relation were evaluated in the same way as in group 1 and were considered as appropriate. group 3: 10 dentate volunteers with natural dentition (4 men and 6 women), aged between 60-75 years (mean age = 67.9 years). the inclusion criteria for this group were to present complete permanent dentition, angle class i occlusion and no symptoms of temporomandibular joint dysfunction. an examination of the oral cavity was performed, including a periodontal evaluation. electromyography this analysis was performed using a myosystem-br1 electromyographer with differential active electrodes (silver bars 10 mm apart, 10 mm long, 2 mm wide, x20 gain, input impedance 10 g&! and 130 db cmrr). surface differential active electrodes were placed on the skin over the belly of the left and right masseter, and on the skin over the anterior portion of the left and right temporalis muscles. electrode positions were determined by palpation and the electrodes were fixed using adhesive tape, with the silver bars perpendicular to muscle fibers. a circular stainless-steel electrode (3 cm in diameter) was also used as a reference electrode (ground electrode), fixed on the skin over the sternum. the skin region where electrodes were to be positioned was previously cleaned with alcohol and shaved, if needed. the electromyographic (emg) signals were analogically amplified with a gain of 1000·, filtered by a pass-band of 0.01-1.5 khz and sampled by a 12-bit a/d converter with a 2 khz sampling rate. the signals were digitally filtered by a pass-band filter of 10–500 hz for data processing. emg signals were captured with volunteers comfortably seated in an office-type chair with their arms next to their body and hands on their thighs. muscle activity was recorded while subjects maintained the following postures for ten seconds: rest position (rp), left laterality (ll), right laterality (rl), and protrusion (pr). at the end of the exam, activity during braz j oral sci. 7(25):1550-1554 using overdenture on implants and complete dentures: effects on postural maintenance of masticatory musculature 1552 dental clenching at maximum habitual intercuspation (mhi) was recorded and these values were used to normalize the subjects’ activities of posture maintenance. the data comprised the gross values’ root mean square (rms) of the emg signal collected during the exam. statistical analyses analysis of variance (anova) was used to compare the emg activity of the masseter and temporalis muscles in overdenture wearers, complete denture wearers and dentate individuals (controls) at rest and during different postural positions. two anova tests were used: one to compare the groups (overdenture wearers, complete denture wearers and dentate individuals), and another to compare the muscles (right and left masseter, and right and left anterior temporalis muscles). statistical analysis was performed using the spss software (spss inc., chicago, il, usa) with significance level set at p d”0.01. r e s u l t s complete denture and overdenture wearers presented higher emg activity than dentate individuals (table 1) during all positions tested in this study, including rest. no statistically significant difference (p>0.01) was observed for mandibular rest when the groups (overdenture wearers, complete denture wearers and dentate individuals) were compared. however, comparing the masticatory muscles, statistically significant difference (pd”0.01) was observed in the group of complete denture wearers. duncan’s posthoc test revealed higher emg activity for the right temporalis. dentate individuals were the only group that showed a balance of muscle activity for the right and left masseter, and for the right and left temporalis (figures 1 and 2). there was a statistically significant difference (pd”0.01) for the right temporalis muscle while maintaining the posture 0.19 ±0.18 0.38 ±0.22 0.32 ±0.22 0.2 ±0.17 0.48 ±0.40 0.31 ±0.27 0.32 ±0.22 0.65 ±0.55 0.49 ±0.48 0.16 ±0.15 0.35 ±0.18 0.25 ±0.20 lt 0.16 ±0.09 0.47 ±0.23 0.19 ±0.14 0.37 ±0.29 0.62 ±0.24 0.35 ±0.30 0.18 ±0.11 0.5 ±0.23 0.16 ±0.11 0.17 ±0.25 0.33 ±0.13 0.18 ±0.12 rt 0.29 ±0.17 0.39 ±0.21 0.62 ±0.48 0.23 ±0.19 0.31 ±0.13 0.41 ±0.40 0.22 ±0.16 0.29 ±0.13 0.63 ±1.08 0.15 ±0.16 0.2 ±0.09 0.23 ±0.29 lm 0.32 ±0.23 0.38 ±0.18 0.84 ±1.31 0.22 ±0.16 0.24 ±0.10 0.35 ±0.35 0.25 ±0.17 0.34 ±0.18 0.36 ±0.24 0.13 ±0.08 0.15 ±0.06 0.18 ±0.20 rm dentcdoverdentcdoverdentcdoverdentcdovermuscle protrusionright lateralityleft lateralityrestactivities 0.19 ±0.18 0.38 ±0.22 0.32 ±0.22 0.2 ±0.17 0.48 ±0.40 0.31 ±0.27 0.32 ±0.22 0.65 ±0.55 0.49 ±0.48 0.16 ±0.15 0.35 ±0.18 0.25 ±0.20 lt 0.16 ±0.09 0.47 ±0.23 0.19 ±0.14 0.37 ±0.29 0.62 ±0.24 0.35 ±0.30 0.18 ±0.11 0.5 ±0.23 0.16 ±0.11 0.17 ±0.25 0.33 ±0.13 0.18 ±0.12 rt 0.29 ±0.17 0.39 ±0.21 0.62 ±0.48 0.23 ±0.19 0.31 ±0.13 0.41 ±0.40 0.22 ±0.16 0.29 ±0.13 0.63 ±1.08 0.15 ±0.16 0.2 ±0.09 0.23 ±0.29 lm 0.32 ±0.23 0.38 ±0.18 0.84 ±1.31 0.22 ±0.16 0.24 ±0.10 0.35 ±0.35 0.25 ±0.17 0.34 ±0.18 0.36 ±0.24 0.13 ±0.08 0.15 ±0.06 0.18 ±0.20 rm dentcdoverdentcdoverdentcdoverdentcdovermuscle protrusionright lateralityleft lateralityrestactivities table 1. normalized electromyographic data (rms). averages and standard deviation (sd) of masseter muscle activity (rm and lm) and temporalis muscle activity (rd and lt) while at rest (rp), left laterality (ll), right laterality (rl) and protrusion (pro) of overdenture wearers (over), complete denture wearers (cd) and dentate individuals (dent). of left laterality (figure 1). the group of complete denture wearers had the highest emg values. for right laterality, the right temporalis muscle presented significantly higher emg activity in the group of complete denture wearers (figure 2) by analysis of the duncan’s complementary test results. in protrusion, the right and left masseter muscles showed slightly higher emg activity than the right and left temporalis muscles for overdenture wearers and dentate individuals. on the other hand, complete denture wearers did not show this correlation. when the three groups were compared, a statistical significance (pd”0.01) was found for the right temporalis muscle activities in all subjects (figure 1). duncan’s post-hoc test showed higher emg activity for the group of complete denture wearers when the right temporalis was analyzed. d i s c u s s i o n it is known that complete denture wearers have lower masticatory efficacy in comparison to that of individuals with natural dentition13, 14. allen et al.15 performed a study in which the satisfaction of complete denture wearers with the treatment was assessed by validated questionnaires. the results revealed that the individuals who received new complete dentures had a lower level of satisfaction than those who received overdentures. however, those who received implant-supported overdentures showed the highest level of satisfaction. the best way to ensure masticatory efficacy, and consequently health on postural muscular function, with aging is to maintain the highest possible number of healthy teeth7-8. in cases of tooth loss, patients turn to rehabilitation seeking for a function similar to that of the natural teeth. however, it cannot be provided by dentures supported by edentulous ridges. this is why the use of implant techniques in dentistry has increased continuously15. braz j oral sci. 7(25):1550-1554 using overdenture on implants and complete dentures: effects on postural maintenance of masticatory musculature 1553 the main topics concerning the treatment of edentulous patients researched by rehabilitation specialists include the relations of masticatory muscle functions, dental occlusion, craniofacial relations, and temporomandibular joint dysfunctions. this highlights the importance of the evolution of emg methods in order to make possible performing electromyographic exams routinely in a near future. analysis of masseter and temporalis muscles permits determining muscle activity during fucntion. ferrario et al.16 analyzed emg activity of the masseter and temporalis muscles of edentulous overdenture wearers during unilateral gum chewing and during maximum tooth clenching. these fig. 1 normalized electromyographic averages (rms) of the groups: overdenture wearers, complete denture wearers, and dentate individuals. recordings of right masseter (rm) and left masseter muscles (lm), and right (rt) and left temporalis (lt); at rest, left laterality, right laterality and protrusion. ** statistically significant if pd”0.01. fig. 2 normalized electromyographic averages (rms) of right masseter (rm) and left masseter muscles (lm), and right (rt) and left (lt) temporalis in the groups: overdenture wearers (over), complete denture wearers (cd), and dentate individuals (dent); recordings at rest, left laterality, right laterality and protrusion. ** statistically significant if pd”0.01. authors found that when compared to complete denture wearers, dentate individuals and overdenture wearers showed higher emg contraction. furthermore, only the temporalis muscle showed different values between groups during maximum tooth clenching. tallgren et al.17 observed the emg activity of the masseter and temporalis muscles of 21 complete denture wearers and observed that emg activity of the temporalis increased soon after fixing the complete dentures, whereas activity of the masseter remained constant throughout the tests. it was concluded that the temporalis muscle was sensitive to changes in intermaxillary relation and to the stability of braz j oral sci. 7(25):1550-1554 using overdenture on implants and complete dentures: effects on postural maintenance of masticatory musculature 1554 complete dentures during deglutition. this may explain the hyperactivity found in the present study for the temporalis muscle compared to the activity of the masseter muscle in complete denture wearers during all postural positions. it is likely that such a factor did not occur in overdenture wearers due to the greater stability provided by this type of prosthesis during function. therefore, in this case, the temporalis muscle is not hyperactivated, permitting the musculature to remain balanced and maintain posture and rest positions. chen et al.18 performed emg tests on 40 denture wearers, of which 14 were complete denture wearers, 12 wore overdentures supported by natural teeth, and 14 wore overdentures supported by osseointegrated implants, during fixed masticatory sequence with standardized portions of 2 food staples. the authors observed that temporalis muscle activity prevailed when compared to the activity of the masseter muscles in all groups. thirty-two of the 40 subjects showed predominant activity of the temporalis muscles over the masseter muscles. masticatory efficiency was also tested and the results revealed that the group of subjects with implant-supported overdentures had the highest masticatory efficiency, followed by subjects with overdentures supported by natural teeth. the group of complete denture wearers showed the lowest masticatory efficiency. in the present study, there was also a prevalence of activity for the temporalis muscles in the studied groups to stay at rest and all studied postural positions. this means that because complete denture wearers have reduced masticatory efficiency, the temporalis muscle becomes hyperactive during mastication in an attempt to overcome this deficit. rather than producing strong movements, the temporalis are muscles specialized in producing fine movements, and therefore become fatigued and hyperactive even at rest and during posture maintenance. the elevator musculature may become hyperactive due to fatigue and stress, which may also cause parafunction of the musculature during posture maintenance19,20. advances of oral implantology brought better conditions to support complete dentures in edentulous individuals with limitations to receive fixed treatments. this research showed that overdentures wearers have masticatory conditions more similar to that of dentate individuals than complete dentures wearers. this advantage is so important to provide ideal nutritional conditions and better quality of life to the increasing elderly population. in conclusion, the instability inherent to the biomechanical function of complete dentures sensitizes and unbalances the emg activity of temporalis muscles. the temporalis musculature is fatigued by complete dentures due to factors such as instability and masticatory ineffectiveness inherent to this type of prosthesis. hence, the musculature becomes hyperactive at rest and during posture maintenance. the use of overdentures supported by 4 implants with a barclip and o’ring retention system cause muscular activity, at rest and during posture maintenance, that is more similar to that of dentate individuals than complete denture wearers. a c k n o w l e d g e m e n t s this work received financial support from fapesp (process number 04/05324-0). r e f e r e n c e s 1. semba rd, blaum cs, bartali b, xue ql, ricks mo, guralnik jm et al. denture use, malnutrition, frailty, and mortality among older women living in the community. j nutr health aging. 2006; 10:161-7. 2. galo r, vitti m, santos cm, hallak je, regalo sc. the effect of age on the function of the masticatory system-an electromyographical analysis. gerodontology. 2006; 23: 177-82. 3. gallagher ma, cuomo f, polonsky l, berliner k, zuckerman jd. effects of age, testing speed, and arm dominance on isokinetic strength of the elbow. j shoulder elbow surg. 1997; 6: 340-6. 4. feine js, grandmont p, boudrias p, brien n, lamarche c, tache r et al. within-subject comparisons of implant-supported mandibular prostheses: choice of prosthesis. j dent res. 1994; 73: 1105-11. 5. fontiin-tekamp fa, slagter ap, van der bilt a, van ‘t hof a, witter dj, kalk w et al. biting and chewing in overdentures, full dentures, and natural dentitions. j dent res. 2000; 79: 1519-24. 6. van kampen fmc, van der bilt a, cune1 ms, bosman f. the influence of various attachment types in mandibular implantretained overdentures on maximum bite force and emg. j dent res. 2002; 81: 170-3. 7. karlsson s, carlsson ge. characteristics of mandibular masticatory movement in young and elderly dentate subjects. j dent res. 1990; 69: 473-6. 8. herring sw. masticatory muscles and the skull: a comparative perspective. arch oral biol. 2007; 52: 296-9. 9. karkazis hc. emg activity of the masseter muscle in implant supported overdenture wearers during chewing of hard and soft food. j oral rehabil. 2002; 29: 986-91. 10. carlsson ge. masticatory efficiency: the effect of age, the lost of teeth and prostetic rehabilitation. int dent j. 1984; 34: 93-7. 11. walls awg, steele jg. the relationship between oral health and nutrition in older people. mech ageing dev. 2004; 125: 853-7. 12. misch, ce. implantes dentários contemporâneos. são paulo: santos; 2000. p.637-9. 13. carr ab. postural contractile activities of human jaw muscles following use of an occlusal splint. j oral rehabil. 1991; 18: 185-91. 14. wastell dg, barker gp, devlin hd. differences in the emg lower spectrum of the masseter muscle in dentate and edentulous subjects. med sci res. 1987; 15: 1159–60. 15. allen pf, mcmillan as, walshaw d. a patient-based assessment of implant-stabilized and conventional complete dentures. j prosthet dent. 2001; 85: 141-7. 16. ferrario vf, tartaglia gm, maglione m, simion m, sforza c. neuromuscular coordination of masticatory muscles in subjects with two types of implant-supported prostheses. clin oral implants res. 2004; 15: 219-25. 17. tallgren a, lang br, holden s, miller rl. longitudinal electromyographic study of swallowing patterns in complete denture wearers. int j prosthodont 1995; 8: 467-78. 18. chen l, xie q, feng h, lin y, li j. the masticatory efficiency of mandibular implant-supported overdentures as compared with tooth-supported overdentures and complete dentures. j oral implantol. 2002; 28: 238-43. 19. yemm r. a neurophysiological approach to the pathology and aetiology of temporomandibular dysfunction. j oral rehabil. 1985; 12: 343-53. 20. mao j, stein rb, osborn j. fatigue in human jaw muscles: a review. j orofac pain. 1993; 7: 135-42. braz j oral sci. 7(25):1550-1554 using overdenture on implants and complete dentures: effects on postural maintenance of masticatory musculature 26/06/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1545/1198 1/11 26/06/2019 pdf.js viewer 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2017 e17080 original article 1 msc in public health, dds, universidade federal do rio grande do norte. natal/rn, brazil. 2 dds, universidade federal do rio grande do norte. natal/rn, brazil. 3 dds, universidade federal do rio grande do norte. natal/rn, brazil. 4 msc in public health, dds, universidade federal do rio grande do norte. natal/rn, brazil. 5 associate professor, phd, dds, department of dentistry. universidade federal do rio grande do norte. natal/rn, brazil. 6 associate professor, phd, dds, department of dentistry. universidade federal do rio grande do norte. natal/rn, brazil. corresponding author: msc anderson nicolly fernandes-costa senador salgado filho ave., 1787, lagoa nova, natal/rn, brazil. zip code: 59056-000. phone: +55 84 98716-6865. e-mail: anson.fernandes@hotmail.com received: july 26, 2017 accepted: september 14, 2017 quality of life in patients rehabilitated with implant-supported prostheses anderson nicolly fernandes-costa1, micaella pollyana silva do nascimento costa2, tâmara cabral rodrigues3, karyna de melo menezes4, patrícia dos santos calderon5, bruno césar de vasconcelos gurgel6 aim: to evaluate the impact of rehabilitation with implantsupported prostheses on the quality of life (qol) of patients. methods: the ohip-14 questionnaire was applied to 114 patients and information regarding gender, age, type of prosthesis and time of use were obtained. to analyze whether xlivi� [ivi� er]� wxexmwxmgepp]� wmkrmƽgerx� hmjjivirgiw� fix[iir� the mean scores of the seven parameters of the ohip-14, the mann-whitney and kruskal-wallis test were used. all data were izepyexih�ywmrk�e�wmkrmƽgergi�pizip�sj�� ��results: patients [ivi�tvihsqmrerxp]�jiqepi������ �erh�wmrkpi�gvs[rw�ywivw� ����� ��4exmirxw�vitsvxih�e�kssh�5s0������ ��4w]glspskmgep� discomfort and physical pain were the worst dimensions izepyexih� f]� xli� wyfnigxw�� 6iwypxw� hmjjivih� wmkrmƽgerxp]� (p<0.05) only for functional limitations and psychological discomfort in the different genders. conclusions: patients presented a high level of quality of life, regardless of age, duration of use and the type of prosthesis used. however, women presented more psychological distress and functional limitations than men. keywords: quality of life. dental prostheses, implantsupported. patient satisfaction. mouth rehabilitation. http://dx.doi.org/10.20396/bjos.v16i0.8651059 2 fernandes-costa et al. introduction therapy with implant-supported prostheses implants has been largely studied for the oral rehabilitation of edentulous patients1. due to the limitations of conventional prosthesis treatments, implant-supported prostheses (single crowns; bridges; complete hirxyviw��ƽ\ih�sv�viqszefpi �ger�fi�tperrih�xs�mqtvszi�wxefmpmx]��vixirxmsr��qewxmgetory function and phonetics in patients. furthermore, these approaches also improve the physical, psychological and social well-being of the patient2 and are considered the gold standard for the treatment of edentulism3. ,s[iziv��epp�xliwi�firiƽxw�ger�figsqi�qiermrkpiww�mj�[lex�xli�texmirx�gsrwmhivw� important for his/her satisfaction with treatment is different to that of the dentist. (s�rsx�gsrwmhivmrk�texmirxwƅ�i\tigxexmsrw�sr�xli�tevx�sj�tvsjiwwmsrepw�ger�gsrxvmfute to oral rehabilitation failure and produce typical psychosocial responses, such ew�er\mix]��mrwigyvmx]��ps[�wipj�iwxiiq�erh�mrxvszivwmsr4. during the initial planning of xviexqirx��mx�mw�rigiwwev]�xs�yrhivwxerh�xli�texmirxƅw�i\tigxexmsrw��wmrgi�xli�wyfnigxƅw� perception of his/her oral health is related to his/her quality of life (qol)5. (yi�xs�mxw�wyfnigxmzmx]��gsqtpi\mx]�erh�mrhmzmhyep�tivgitxmsr��mx�mw�hmƾgypx�xs�izepyexi�5s0�� according to the world health organization (who), qol is the perception, on the part of individuals or groups, regarding the satisfaction of their own needs and what is not denied in propitious for their happiness6. besides that, the who emphasizes the importance of oral health-related qol to use in its campaigns not only images portraying pain-free life but also aesthetic images with beautiful smiles as an image of complete well-being7. 8vehmxmsrepp]�� xli� wyvzi]w� evi� gsrgivrih� [mxl� xviexqirx� iƾgeg]� mrzspzmrk� gpmrmgep� parameters rather than individual patient perspective. in contrast, as a result of this difference, recent research is refocused to consider how oral health affects far reaching aspects of life such as psychosocial interaction, self-esteem, intimacy, overall health, and performance at work8. ;lir�5s0�mw�izepyexih�mr�vipexmsr�xs�svep�liepxl��qshmƽgexmsrw�mr�svep�liepxl�lezi�fiir� found to negatively interfere in an individual’s qol and this can also be affected by the satisfaction or dissatisfaction with an oral condition9. mcgrath and bedi10 have shown xlex�tvsfpiqw�i\tivmirgih�f]�texmirxw�[ls�lezi�pswx�xiixl�[ivi�fsxl�jyrgxmsrep�erh� psychosocial and were mainly associated with negative feelings. therefore, rehabilitation with implant-supported prostheses provides a positive effect on the qol associated with oral health and has a strong social, psychological and emotional impact on the daily life of each patient11. several instruments have been developed with the aim of evaluating and quantifying 5s0��8li�3vep�,iepxl�-qtegx�4vsƽpi��3,-4 �mw�sri�sj�xliwi�mrwxvyqirxw��mxw�wmqtpmƽih� form (ohip-14) was developed by slade and spencer12, derived from the original version (ohip-49) also developed by slade and spencer12. even in its abbreviated form, this questionnaire is reliable and valid in several languages13 and is considered a good mrhi\�jsv�izepyexmrk�5s0��figsqmrk�e�qixlshspsk]�sj�glsmgi�jsv�wygl�wxyhmiw5,11. as ohip-14 is easy and quick to apply, it is possible to verify the effectiveness of oral rehabilitation with implant-supported prostheses, evaluating the physical and emo3 fernandes-costa et al. tional state of the patient, their future limitations, level of performance in their daily life, degree of satisfaction of their needs and the impact on their qol. therefore, the aim of this study was to evaluate the impact of oral rehabilitation with prostheses supported or retained by implants on patients’ qol. materials and methods sample this cross-sectional study was performed in patients with implant-supported prostheses treated at the department of dentistry of the federal university of rio grande do norte (ufrn) during the period from 2000 to 2010. this search was approved by the ethics committee of the ufrn (protocol: 349.152/2013) and it has been conducted in full accordance with the world medical association declaration of helsinki. all patients who agreed to participate of this search assigned a written consent. patients [ls�lezi�tvswxliwmw�piww�xler�wm\�qsrxlw�mr�jyrgxmsr�[ivi�i\gpyhih�jvsq�xli�weqtpi�� the calculation of the sample size was based on the mean observed in our study for kirivep�3,-4�����%�jsvqype�jsv�ƽrmxi�weqtpi�tstypexmsrw�[ew�ywih�xs�gepgypexi�xli� weqtpi�sj�xlmw�wxyh]�[mxl�e�wmkrmƽgergi�sj�� �erh�ts[iv�xiwx�sj��� ��;mxl�er�ƽrmxi� population of 155 individuals, the sample of patients required for ohip-14 application was 109 individuals. data collect after the selection of patients, the ohip-14 was applied by previously trained researchers and information about gender, age, type of prosthesis and time of use of prosthesis were obtained from the patient’s medical records. in cases of patients that used more than one type of prosthesis, the type of prostheses with the highest number of implants was considered. all patients were informed about the ohip-14 methodology and the interviewer was always available for providing help. the ohip-14 consists of 14 items subdivided into seven parameters (functional limitation, physical pain, psychological discomfort, physical incapacity, psychological incapacity, social disability and handicap). for these items, participants were asked xs�izepyexi�ls[�sjxir�xli]�jipx�erh�i\tivmirgih�er�mqtegx�sr�svep�liepxl�ejxiv�vilefmpmxexmsr�xviexqirx�fi�gsrgpyhih�ywmrk�e�0moivx�wgepi�sj�ƽzi�tsmrxw�gshih����ƈrizivɖ ���� �ƈvevip]ɖ �����ƈwsqixmqiwɖ �����ƈjviuyirxp]ɖ ��erh����ƈep[e]wɖ �� the calculation of the impact of the prosthesis on the patient’s qol was performed f]�e�wxerhevh�qixlsh�sj�gepgypexmsr�sj�3,-4�����ywmrk�xli�wtigmƽg�[imklx�jsv�iegl� question2��*mrepp]��[lir�epp�tyrgxyexmsr�jvsq�iegl�uyiwxmsr�[ew�ehhih��e�ƽrep�wgsvi� was obtained, and the higher submitted score, the greater the negative impact on qol for the individual14. statistical analysis for the statistical analysis of parameters in relation to gender, the mann-whitney test was used. the kruskal-wallis test was utilized for the variables; age, type of prostheses and time of use of prosthesis. for statistical purposes, all variables were cate4 fernandes-costa et al. ksvm^ih��8li�3,-4����zevmefpi�erh�mxw�hmqirwmsrw�[ivi�gexiksvm^ih�ew�ƈmqtegxɖ�sr� 5s0��"� �erh�ƈrs�mqtegxɖ�sr�5s0��!� ��8li�mqtegx�sj�e�teveqixiv�sr�5s0�mrhmgexiw�e� negative effect of the rehabilitation the individual’s age was categorized according to the age, as adult (<60 years) or elderly (≥60 years). the type of prosthesis was categorized as single crowns, overdentures and multiple partial prostheses or total prosxliwiw��ƽ\ih ��*sv�xli�xmqi�sj�ywi�sj�xli�tvswxliwiw��gexiksvm^exmsr�[ew�tivjsvqih�mr� mrhmzmhyepw�[ls�leh�ywih�xliq�jsv�yt�xs�wm\�]ievw�sv�jsv�qsvi�xler�wm\�]ievw� results the ohip-14 was applied in 114 patients during the period of may/2013 to 1e]�������%�lmkliv�tvstsvxmsr�sj�jiqepiw������ �erh�mrhmzmhyepw�[ls�ywih�wmrkpi�tvswxliwiw������ �[ivi�sfwivzih��%ggsvhmrk�xs�kirhiv��fix[iir�qir�xlivi� [ew�lmkliv�tvizepirgi�sj�qypxmtpi�tevxmep�tvswxliwiw�sv�xsxep�tvswxliwiw���� �erh� fix[iir�[sqir�xli�qswx�tvizepirx�[ew�wmrkpi�tvswxliwiw������ ��8li�weqtpi� had a mean age of 55.46 years (±12.91) with a variation from 18 to 84 years. furxlivqsvi��xli�qensvmx]�sj�xli�texmirxw�tviwirxih�tvswxliwiw�[mxl�qsvi�xler�wm\� years of mean time of use. results demonstrate that the patients evaluated have a high qol, as the value of the general ohip-14 was close to zero (3.07), with a variation from 0 to 56. however, it can be seen that the dimensions of psychological discomfort and physical pain presented the highest means, even though these parameters demonstrated low values (fig. 1). the distribution of the analysis of the ohip-14 or the parameters of gender, age, type sj�tvswxliwmw�erh�xmqi�sj�ywi�sj�tvswxliwmw�hiqsrwxvexih�wmkrmƽgerxp]�wxexmwxmgep�hmjferences (p<0.05) just for gender, with regard to functional limitations and psychological discomfort, which presented the worst qol scores (tables 1 and 2). caption: circles and stars are respectively outliers and extreme outliers. figure 1. box-plot distribution of ohip-14 and its dimensions. general ohip -14 functions limitations physical pain psychological discomfort physical incapacity psychological incapacity social incapacity invalidity 25 20 15 10 5 0 5 fernandes-costa et al. table 2. distribution of the impact of oral health on qol, as evaluated by ohip-14, with regard to the type of prosthesis used and time of use of prosthesis. type of prosthesis p time of use of prosthesis psingle prostheses n (%) overdenture n (%) multiple partial or total prostheses n (%) ≤6 years n (%) >6 years n (%) general ohip-14 no impact 14 (29.8) 3 (13.6) 16 (35.6) 0.176 9 (24.3) 24 (31.2) 0.593 impact 33 (70.2) 19 (86.4) 29 (64.4) 28 (75.7) 53 (68.8) functional limitations no impact 40 (85.1) 20 (90.9) 40 (88.9) -a 33 (89.2) 67 (87.0) 1.000 impact 7 (14.9) 2 (9.1) 5 (11.1) 4 (10.8) 10 (13.0) physical pain no impact 28 (59.6) 8 (36.4) 28 (62.2) 0.111 21 (56.8) 43 (55.8) 1.000 impact 19 (40.4) 14 (63.6) 17 (37.8) 16 (43.2) 34 (44.2) continue table 1. distribution of oral health impact on qol, as evaluated by the ohip-14, and parameters according to gender and age. gender p age pmale n (%) female n (%) adult n (%) elderly n (%) general ohip-14 no impact 10 (40.0) 23 (25.8) 0.259 21 (32.3) 12 (24.5) 0.482 impact 15 (60.0) 66 (74.2) 44 (67.7) 37 (75.5) functional limitations no impact 25 (100.0) 75 (84.3) 0.037 56 (86.2) 44 (89.8) 0.765 impact 14 (15.7) 9 (13.8) 5 (10.2) physical pain no impact 12 (48.0) 52 (58.4) 0.484 41 (63.1) 23 (46.9) 0.126 impact 13 (52.0) 37 (41.6) 24 (36.9) 26 (53.1) psychological discomfort no impact 18 (72.0) 38 (42.7) 0.018 33 (50.8) 23 (46.9) 0.829 impact 7 (28.0) 51 (57.3) 32 (49.2) 26 (53.1) physical incapacity no impact 23 (92.0) 75 (84.3) 0.516 58 (89.2) 40 (81.6) 0.377 impact 2 (8.0) 14 (15.7) 7 (10.8) 9 (18.4) psychological incapacity no impact 24 (96.0) 76 (85.4) 0.298 55 (84.6) 45 (91.8) 0.382 impact 1 (4.0) 13 (14.6) 10 (15.4) 4 (8.2) social disability no impact 24 (96.0) 81 (91.0) 0.681 61 (93.8) 44 (89.8) 0.495 impact 1 (4.0) 8 (9.0) 4 (6.2) 5 (10.2) handicap no impact 25 (100.0) 87 (97.8) 1.000 64 (98.5) 48 (98.0) 1.000 impact 2 (2.2) 1 (1.5) 1 (2.0) 6 fernandes-costa et al. discussion the application of the ohip-14 questionaire in this study showed that patients using prostheses present a satisfactory qol. according to slade14, this instrument presents �� �sj�gsrgsvhergi�[mxl�3,-4�����kssh�vipmefmpmx]��zepmhmx]�erh�tvigmwmsr�erh�viuymviw� less time for application, favoring its use during the evaluation of oral health and qol. additional and recent studies also used the ohip-14 version to evaluate qol in individuals who used implant-supported prostheses1-2,5,11, as this test is proven to be a valid instrument that is reproducible, reliable and simple for use. other studies have also reported the ohip-14 to represent a good instrument for evaluation of qol1,5,15-16. despite these considerations, the cross-sectional evaluation used herein presents wsqi�pmqmxexmsrw��*sv�i\eqtpi��mx�mw�rsx�tswwmfpi�xs�jsvq�er]�gsrgpywmsrw�vikevhmrk� the whole population with regard to improvements in qol or evaluate cause-effect relationships between qol and implant-supported prostheses use17. however, wsqi�eyxlsvw�lezi�hiqsrwxvexih�e�wmkrmƽgerx� mqtvsziqirx� mr�5s0�[lir�gsqparing before and after treatment with implant-supported prostheses1-2,5,11,15. this fact corroborates the low values found in this study. other studies have reported xli�ywi�sj�mqtperxw�jsv�tvswxliwmw�vixirxmsr�tvsqsxiw�e�wmkrmƽgerx�mqtvsziqirx�mr� 5s0�erh��mj�[ipp�tperrih��xli�xviexqirx�mw�efpi�xs�higviewi�qewxmgexsv]�hiƽgmirg]� and psychological discomfort, in addition to providing aesthetic, retention and satisfactory stability18-19. in this study, gender had an impact on the two parameters where women complained more about functional limitations and psychological discomfort. it is commonly considered that women are more interested in their appearance than men. this the higher dissatisfaction level for females could be related to the idea that the self-esteems of females could be affected from physical injuries more than males20-21. continuation psychological discomfort no impact 23 (48.9) 8 (36.4) 25 (55.6) 0.336 16 (43.2) 40 (51.9) 0.503 impact 24 (51.1) 14 (63.6) 20 (44.4) 21 (56.8) 37 (48.1) physical incapacity no impact 40 (85.1) 17 (77.3) 41 (91.1) -a 31 (83.8) 67 (87.0) 0.860 impact 7 (14.9) 5 (22.7) 4 (8.9) 6 (16.2) 10 (13.0) psychological incapacity no impact 44 (93.6) 19 (86.4) 37 (82.2) -a 33 (89.2) 67 (87.0) 1.000 impact 3 (6.4) 3 (13.6) 8 (17.8) 4 (10.8) 10 (13.0) social disability no impact 43 (91.5) 22 (100.0) 40 (88.9) -a 35 (94.6) 70 (90.9) 0.755 impact 4 (8.5) 5 (11.1) 2 (5.4) 7 (9.1) handicap no impact 47 (100.0) 22 (100.0) 43 (95.6) -a 37 (100.0) 75 (97.4) 1.000 impact 2 (4.4) 2 (2.6) a could not perform the test due to presenting cells with lower than the expected counts of 5. 7 fernandes-costa et al. the perception of most negative may show women are more worried with their oral health when they evaluate their qol13. according to cohen-carneiro et al.13, these differences in gender could vary according to sociodemographic characteristics. due to xli�pmqmxih�ryqfiv�sj�wxyhmiw�mr�xli�pmxivexyvi�xs�i\tpemr�xliwi�hmjjivirgiw��mx�mw�hmƾgypx� to justify or discuss these discrepancies in relation to gender. 7mqmpevp]�xs�xli�viwypxw�sj�xli�tviwirx�wxyh]��sxliv�wxyhmiw�lezi�rsx�sfwivzih�wmkrmƽgerx�hmjferences in relation to the time of use of prostheses, according to ohip-145-6,22. kouppala et al.2 also compared groups of patients for 3 to 12 years and 13 to 22 years of follow-up erh�hmh�rsx�ƽrh�er]�wxexmwxmgep�hmjjivirgiw�mr�xli�viwypxw�jsv�3,-4�����0srkmxyhmrep�izepyexmsrw�sj�xliwi�texmirxw�evi�rigiwwev]�xs�hixivqmri�sv�gsrƽvq�xli�lmkl�5s0�sziv�xmqi� &ewih�sr�x]ti�sj�tvswxliwmw��xli�tviwirx�wxyh]�hmh�rsx�wls[�wmkrmƽgerx�hmjjivirgiw�[mxl� regard to the type of prosthesis. however, cakir et al.1 demonstrated that qol values were ps[iv�mr�ƽ\ih�tevxmep�tvswxliwmw�ywivw�erh�lmkliv�mr�szivhirxyvi�vixemrih�mqtperx�ywivw�� mainly due to functional limitations, physical incapacity and psychological discomfort hmqirwmsrw��8li�eyxlsvw�nywxmƽih�er�szivhirxyvi�gsyph�vitviwirx�e�fixxiv�mqtvsziqirx� jsv�er�ihirxypsyw�texmirx�xler�e�ƽ\ih�tevxmep�tvswxliwiw�vitviwirxw�jsv�e�tevxmepp]�ihirxylous patient. these types of prostheses present advantages and disadvantages, although mx�viqemrw�hmƾgypx�xs�hiƽri�xli�fiwx�x]ti�sj�tvswxliwmw�gexiksvmgepp]�erh�mxw�mrƽyirgi�sr� 5s0��8liwi�uyiwxmsrw�evi�epws�mrƽyirgih�f]�xli�xiglrmgep�ors[pihki�sj�xli�tvsjiwwmsrep�� ew�[ipp�ew�wsgmep��w]wxiqmg��ƽrergmep�erh�tl]wmgep�ewtigxw�sj�texmirxw22. 3zivhirxyvi�sjjivw�xli�texmirx�er�i\gippirx�efmpmx]�xs�wtieo�erh�gli[23 and less mobility in the mouth than to conventional prostheses, and the cost over multiple prostheses or total tvswxliwiw��ƽ\ih �mw�er�ehzerxeki�jsv�ps[�mrgsqi�mrhmzmhyepw24-26. the advantages and the important factors for the choice of treatment with multiple prostheses or total prostheses �ƽ\ih �evi�xli�gsqjsvx�erh�wxefmpmx]�geywih�f]�xli�ryqfiv�sj�mqtperxw�tpegih��wygl�ew� xlex�i\tivmirgih�[mxl�rexyvep�xiixl23,26,27. therefore, more research relating qol and costs, considering the number of implants and the type of prosthesis, are necessary. 7sqi�gexiksvmiw�tviwirxih�mqtegx�vipexih�xs�eki�erep]^ih��epxlsykl�rs�wmkrmƽgergi�� 8li�wgmirxmƽg�pmxivexyvi�wls[ih�xlex�texmirxw�sziv����]ievw�[ivi�qsvi�wexmwƽih�[mxl� xlimv� mqtperx�wyttsvxih� tvswxliwiw��8lmw� qe]� fi� i\tpemrih� f]� xli� jegx� xli� iphivp]� sjxir�i\tivmirgi�ihirxypmwq�erh�evi�wexmwƽih�[mxl�er�svep�liepxl�fips[�xlex�gsrwmhivih�xs�fi�i\gippirx��[lmpi�]syrkiv�texmirxw�[mxl�tvsjiwwmsrep�gsqqmxqirxw�lezi�xs� hiep�[mxl�hmjjivirx�gsrxi\xw�erh�wsgmep�wmxyexmsrw2. the results of this cross-sectional study indicate prosthetic rehabilitation with implants tswmxmzip]�mrƽyirgiw�xli�5s0�sj�texmirxw��[lmgl�gsyph�lezi�fsxl�wsgmep�mqtegxw�erh�firiƽxw�jsv�hemp]�egxmzmxmiw��%hhmxmsrep�wxyhmiw�xlex�izepyexi�sxliv�wsgmshiqskvetlmg�mrjsvmation and patients’ perception, beyond those commonly and routinely observed in rate success (biological and mechanical factors of rehabilitations), should be performed. it can be concluded patients with implant-supported prostheses present a satisfactory qol. however, increased attention should be given by professionals in relation xs� [sqirƅw� i\tigxexmsrw�� hyi� xs� lmkliv� tw]glspskmgep� hmwgsqjsvx� erh� jyrgxmsrep� limitations. therefore, the ohip-14 used to evaluate the impact of rehabilitation with 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in partially edentulous patients before and after implant therapy: a 2-year longitudinal study. oral implantol (rome). 2013 oct;6(2):37-42. 11. 7pehi�+(��7tirgiv�%.��(izipstqirx�erh�izepyexmsr�sj�xli�3vep�,iepxl�-qtegx�4vsƽpi��'sqqyrmx]� dent health 1994;11(1):3-11. 12. cohen-carneiro f, souza-santos r, rebelo mab. quality of life related to oral health: contribution from social factors. cienc saude colet. 2011;16 supl 1:1007-15. 13. 7pehi�+(��(ivmzexmsr�erh�zepmhexmsr�sj�e�wlsvx�jsvq�svep�liepxl�mqtegx�tvsƽpi��'sqqyrmx]�(irx�3vep� epidemiol. 1997 aug;25(4):284-90. 14. &ivvixmr�*ipm\�+��2ev]�*mpls�,��4ehszerm�'6��1eglehs�;1��%�psrkmxyhmrep�wxyh]�sj�uyepmx]�sj�pmji�sj� iphivp]�[mxl�qerhmfypev�mqtperxwyttsvxih�ƽ\ih�tvswxliwiw��'pmr�3vep�-qtp�6iw�������.yp����� �������� hsm����������n����������������������\� 15. preciado a, del río j, lynch cd, castillo-oyagüe r. impact of various screwed implant prostheses on oral health-related quality of life as measured with the qolip-10 and ohip-14 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groups of patients. j dent 1996 sep;24(5):335-8. 20. 7xvenrmʽ�0��&ypexszmʽ�(��7xer˃mʽ�-��̩mzoszmʽ�6��7ipj�tivgitxmsr�erh�wexmwjegxmsr�[mxl�hirxep�ettievergi� and aesthetics with respect to patients’ age, gender, and level of education. srp arh celok lek. 2016;144(11-12):580-9. 21. gates wd 3rd, cooper lf, sanders ae, reside gj, de kok ij. the effect of implant-supported removable partial dentures on oral health quality of life. clin oral implants res. 2014 feb;25(2):207-13. doi: 10.1111/clr.12085. 22. ,i]higoi�+�ɸ&syhvmew�4�ɸ%[eh�1%�ɸ(i�%pfyuyivuyi�6*�ɸ0yrh�.4�ɸ*imri�.7��;mxlmr�wyfnigx� gsqtevmwsrw�sj�qe\mppev]�ƽ\ih�erh�viqszefpi�mqtperx�tvswxliwiw��4exmirx�wexmwjegxmsr�erh�glsmgi�sj� prosthesis. clin oral impl res 2003 feb;14(1):125-30. 23. sadowsky sj. the implant-supported prosthesis for the edentulous arch: design considerations. j prost dent 1997 jul;78(1):28-33. 24. kim y, park sy, park jy, jeong yj, kim j, oh sh et al. economic evaluation of dental implants in korea. seoul, korea: national evidence-based healthcare collaboration agency; 2011. 25. emami e, de souza rf, bernier j, rompre p, feine js. patient perceptions of the mandibular three-implant overdenture: a practice-based study. clin oral implants res. 2015 jun;26(6):639-43. doi: 10.1111/clr.12351. 26. 4vigmehs�%��(ip�6ms�.��0]rgl�'(��'ewxmpps�3]ekyi�6��%�ri[��wlsvx��wtigmƽg�uyiwxmsrremvi��5s0-4��� �jsv� evaluating the oral health-related quality of life of implant-retained overdenture and hybrid prosthesis wearers. j dent 2013;41:753-63. doi: 10.1016/j.jdent.2013.06.014. braz j oral sci. 15(4):304-307 analysis of mmp-3 polymorphism in osseointegrated implant failure francielle boçon de araujo munhoz1, paula regina bach nogara2, francisco rafael da costa junior3, filipe polese branco4, maria cristina leme godoy dos santos5 1, 2, 3phd student department of cell biology, university federal of paraná, curitiba, pr, brazil 4phd professor faculty avantis of balneário camboriú, balneário de camboriú, sc, brazil 5phd professor department of cell biology, university federal of paraná, curitiba, pr, brazil correspondence to: francielle boçon de araujo munhoz av. cel francisco h dos santos, s/n, jardim das américas, 81531-990, curitiba pr, brazil. phone: +55-041-33611598 fax: + 55-041-3266-2042 e-mail: francielle.bocon@hotmail.com abstract polymorphisms in matrix metalloproteinases (mmps) genes have been associated with several pathologies, including dental implant loss. mmp-3 is crucial to the connective tissue remodeling process. the objective of this study was to investigate the possible relationship between -1612 mmp-3 polymorphism and the early implant failure. a sample of 240 non-smokers was divided: test group 120 patients with one or more early failed implants and control group 120 patients with one or more healthy implants. genomic dna from oral mucosa was analyzed by pcr-rflp. no association of early implant loss with genotypes and alleles of the -1612 polymorphism in mmp-3 were found by the chi-squared test. only the presence of the -1612 polymorphism of mmp-3 is not a genetic risk factor for early loss of implants. keywords: polymorphism. metalloproteinase. implant loss. risk factor. received for publication: february 15, 2017 accepted: june 20, 2017 original article braz j oral sci. october | december 2016 volume 15, number 4 http://dx.doi.org/10.20396/bjos.v15i4.8650045 introduction dental implants have become important therapeutic option and are now the most chosen option for oral rehabilitation in edentulous and partially dentate patients because of its high predictability and success rate. however, the literature reported global failure rates of 1.9-3.6%1, despite adequate surgical and medical treatment. several risk factors, like osteolysis, medical preconditions, poor bone quality, smoking, one-or-two-step surgery, have been proposed in literature2. in addition studies have demonstrated that implant material is an important determinant of treatment outcome3. the fact that only in a minority of titanium particles induce inflammation and osseo-disintegration, suggests an important role of host factors, in particular the immune response to titanium. in addition this, multiple implant failures in the same patient, the cluster phenomenon, indicate that individual's host response play a significant role in the implant loss4. gene polymorphisms are a biologically normal condition which individuals may exhibit genetic variations, which may increase their susceptibility to a certain disease. polymorphisms in matrix metalloproteinases (mmps) genes have been associated with a number of pathologies, including dental implant loss5-7. matrix metalloproteinases (mmps) are the major class of enzymes capable of cleaving all extracellular matrix substrates, including collagens, fibronectin, laminin, vitronectin and proteoglycans8, and are involved in physiological and pathological control group, 120 patients with one or more healthy implants for a minimal period of 1 year, and test group, 120 patients that had suffered one or more early implant failures, considered when presented mobility and/or pain before or during the abutment connection and needed to be removed. genotyping dna from epithelial buccal cells was extracted using the procedure described by aidar and line15. dna concentration (ng/ μl) was estimated by measurements of optical density 260/280 nm ratio greater than 1.9. the mmp-3 genotype was determined by the pcrrflp assay. the pcr primers used for amplifying the m m p 3 p o l y m o r p h i s m w e r e : f o r w a r d p r i m e r 5'-ggttctccattcctttgatggggggaaaga-3' and reverse primer 5'-cttcctggaattcacatcactgccaccact-3'. the forward primer for amplifying the mmp-3 fragment was mutated from a to g at the second nucleotide close to the 3’ end to create a tth111i recognition site in the case of a 5a allele. pcr were carried out in a total volume of 10 μl, containing 400ng genomic dna, 5 μl of taq green jumpstar taq readymix (amersham pharmacia-biotech, uppsala, sweden) and 200 nmol of each primer. a 6 μl aliquot of pcr products were then digested with 1 unit of tth111i enzyme at 37oc overnight. the total amount aliquot of the digest was electrophoresed on a 10% vertical non-denaturing polyacrylamide gel at 20 ma. the gel was stained by ethidium bromide. statistical analysis mann–whitney u test was used to determine any significant differences between ages and gender. the significance of the differences in the observed frequencies of polymorphism between both groups was assessed using the chi-squared test with p<0.05 indicating statistical significance. the program arlequin (v. 2.0 — schneider et al., 2000)16 was used to verify the hardy–weinberg equilibrium in the studied sample. to verify statistical power of our sample, we used g*power software. results the primers used were efficient to amplify the fragment and the tth111i enzyme digestion cleaves the pcr products in two fragments when the polymorphism site contained the 5a allele. electrophoresis produced dna bands of 97 and 32 bp for 5a alleles and a band of 129 bp for 6a alleles, whereas the heterozygote displayed a combination of both alleles (129, 97 and 32 bp). genotype distribution was in hardy–weinberg equilibrium. statistical power estimation for our sample showed 99% for association detection. the statistical analysis did not show significant differences in the alleles and genotypes (p=0.2) of the -1612 5a/6a in mmp-3 between the two sample groups. the result shows that in both groups was the higher frequency of allele 6a and the 5a/6a genotype (table i). 305 processes. mmps regulate a variety of cell behaviors such as cell proliferation and motility, apoptosis, angiogenesis, effects on the immune system and host defense, and modulation of the bioactivity of chemokines8. in fact, mmps are expressed by the major inflammatory and connective tissue cells in response to specific stimuli of remodeling, including implant osseointegration. besides, the literature demonstrated that mmp levels in peri-implant sulcular fluid are high, including mmp-139. mmp-3, or stromelysin-1, have broad substrate specificity and have an important role in remodeling of connective tissue. it participated of turnover of diverse extracellular matrix components, including non-fibrillar collagens, laminin, proteoglycan and fibronectin. it also activates other mmps, such as mmp-1, mmp-2 and mmp-9; as well as its own proenzyme10,11. mmp-3 can be produced by fibroblasts, macrophages, neutrophils, chondrocytes, synovial cells, and smooth muscle cells and can be induced in reaction to local stimulation such as mechanical loading12 and inflammation13. the mmp-3 gene, located on chromosome 11, has functional polymorphisms in the promoter region -1612 characterized by containing either five or six consecutive adenines (5a/6a) and has been associated with various pathologies including periodontal disease14. in present study the purpose was to investigate the relationship between -1612 polymorphism in the mmp-3 gene and early failure of osseointegrated oral implants. material and methods study population a sample of 240 non-smoking subjects, > 18 years of age, were recruited for study from the patient pool at the dental clinics of the faculty of dentistry of piracicaba – university of campinas (unicamp), piracicaba, são paulo, brazil, latin american institute for dental research, curitiba, paraná, brazil and private implantology clinical in são paulo, bahia, paraná brazil. the rate of implant loss of these centers was less than 3%. all patients were advised previously about the nature of the study and signed a consent form within a protocol approved by an institutional review board (ethical committee in research at fop-unicamp, protocol 006/2002). this study has followed the guidelines of helsinki declaration. all subjects were in good general and oral health and did not have any of the following exclusion criteria: a history of diabetes or osteoporosis, hepatitis or hiv infection, immunosuppressive chemotherapy, history of any disease known to severely compromise immune function. it also excluded patients that submitted a precocious prosthesis load or regenerative surgery, such bone grafting, and have had postsurgical complications, such as infection. all patients have a transgingival healing concept performed.. the groups were matched by gender and age; with 66% female and mean age 49 (range 18-80). the groups were matched by implant position; with 61% mandibular region and 67% posterior region. subjects were divided into two groups: analysis of mmp-3 polymorphism in osseointegrated implant failure braz j oral sci. 15(4):304-307 306 discussion biological, microbiological and biomechanical factors can be accredited implant loss, however the exact cause and mechanism of early implant failure are still uncertain. an abnormal immune-inflammatory response, involving fibroblasts, keratinocytes, macrophages, neutrophils, lymphocytes, endothelial cells, osteoclasts and osteoblasts, can destroy the peri-implant and periodontal tissues17. moreover, an intense inflammatory process is mediated by cytokines which point to different cell types and stimulating the production of prostaglandins and matrix metalloproteinases, which are associated with bone and connective tissue breakdown18. the cluster phenomenon supports the evidence that individual characteristics play an important role in transcription of these inflammatory mediators and may influence the osseointegration success. some studies show the influence of genetic polymorphisms in inflammatory mediators in implant loss. cosyn et al.19 (2016) demonstrated that the il-1b (+3954) gene polymorphism affect osseointegration, beside previous studies not found evidence20. the genotype 2/2 of il1rn polymorphism and the c allele of il-4 polymorphism was associated with susceptibility to dental implant loss20,21. in nonsmokers, have been show that a polymorphism in the promoter region of mmp-1 and mmp-8 gene is strongly associated with the early implant loss5-7. our group suggested that haplotype g-1607gg and a-519g of mmp-1 may be associated with the osseointegration process6. however, many others studies found no significant association between dental implant loss and polymorphism in inflammatory mediators such as mmp-9, il-1, il-2, il-6, il10, tnf-a, tgf-b, vntr, and others5,20-23. in this study, the -1612 5a/6a polymorphism in the promoter region of mmp3 gene was also not associated with early implant failure in non-smokers. in meta-analysis studies24,25 point that is important methodological and study design restricted to validate the associations, even though it has been raised as one of the potential risk indicators. early implant failure in non-smokers have a reduced frequency. nevertheless, smoking is a strong risk factor for early implant failure smokers have a 3% greater chance of losing an implant compared to non-smokers26, so studies that analysis of mmp-3 polymorphism in osseointegrated implant failure includes smokers would possibly mask the genetic influence. in this study, we observed a number of patients in the test group with reliable estimated statistical power, despite put out smokers. others risk factors with age, status periodontal, and medically compromised were exclude or matched. in osseointegration, as with any complex process seems to be a combination of several polymorphisms act synergistically significant risk that increases the susceptibility to failure. so, it is important to consider that this mmp-3 polymorphism may have their effects masked by polymorphisms in different regions of a gene or other genes involved with periodontal inflammatory mediators. however, to identify the influence of each allele, it is essential to analyze the relative contribution of each polymorphism. since mmp-3 activates the mmp-1 and polymorphism in mmp-1 was associated with implant loss, it seems important to assess the linkage disequilibrium between the polymorphisms of mmp-3 and mmp-1 which are located on chromosome 11q22.3 adjacent to each other. it seems to be of great value to understand osseointegration process and the mechanisms of functional compensation of the individual. in future studies of the investigation of polymorphisms in the mmp-3 gene, mainly in haplotype combination, remains to be considered regarding implant loss due to the importance of this gene in osseointegration. to understand the complex osseointegration failure is important analyze haplotype frequencies and imbalances between various polymorphisms. it could be help in clinical investigation of individuals at high risk to implant loss and, in future, guide the development of individual therapeutics to increasing the implants success rates. conclusion in conclusion, no associations were found between -1612 5a/6a polymorphisms of the mmp-3 gene promoter and early implant failure, suggesting that the presence of this polymorphism alone are not a genetic risk factor for predisposition to early implant loss. therefore, the investigation of polymorphisms in the mmp-3 gene, mainly in haplotype combination, remains to be considered regarding implant loss due to the importance of this gene in osseointegration. acknowledgement the authors wish to thank professor dr. silvio sanches veiga for his assistances. this study was supported by the fundação araucária and capes references 1. holm-pedersen p, lang np, muller f. what are the longevities of teeth and oral implants? clin oral implants res. 2007 jun;18 suppl 3:15-9. 2. chrcanovic br, albrektsson t, wennerberg a. reasons for failures of oral implants. j oral rehabil. 2014 jun;41(6):443-76. doi: 10.1111/joor.12157. table 1 distribution of the mmp-3 allele and genotype in the control and test group. mmp-3 (-1612) control group test group chisquared or (95% ic) n % n % allele n = 240 n = 240 5a 102 42.5 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(i). success criteria and epidemiology. eur j oral sci. 1998 feb;106(1):527-51. braz j oral sci. 15(4):304-307 untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8652650 volume 17 2018 e18135 original article ¹ dds, msc, graduate student of the graduate program in dentistry, operative dentistry area, universidade positivo, curitiba, pr, brazil. 2 dds, undergraduate student in dentistry, universidade positivo, curitiba, pr, brazil. 3 dds, msc, phd, professor of the graduate program in dentistry, operative dentistry area, universidade positivo, curitiba, pr, brazil. *corresponding author: leonardo fernandes da cunha : cunha_leo@me.com universidade positivo 5300 professor pedro viriato parigot de souza street curitiba pr zip code: 81280-330 tel: +55 41 3317-3403; fax: +55 41 3317-3082 received: november 20, 2017 accepted: april 23, 2018 surface topography and bacterial adhesion of cad/ cam resin based materials after application of different surface finishing techniques raphael meneghetti hamerschmitt1*, paulo henrique tomazinho1, kaíke lessa camporês2, carla castiglia gonzaga 3, leonardo fernandes da cunha 3, gisele maria correr 3 aim: this study evaluated the surface topography and bacterial adhesion of a hybrid ceramic and a nano ceramic resin composite after different surface finishes. methods: hybrid ceramic (vita enamic, vita en) and nano ceramic resin composite (lava ultimate, 3m/espe lv) blocks of 12 x 14 x 18 mm were cut into 1 mm slices. each slice was divided into four specimens (6 x 7 mm) that were randomly allocated into 4 groups (n=8) according to the surface finishing: ctl without surface finish (control); db wear with a diamond bur; vt polishing system for hybrid ceramic (vita); and dd polishing system for ceramics (dedeco). the specimens were analyzed regarding surface roughness parameters (ra, rz, rq), sterilized and subjected to bacterial adhesion. representative specimens from each group were observed by sem and confocal laser scanning microscopy. data were submitted to two-way anova and tukey’s test (α=0.05). results: en had lower surface roughness and bacterial adhesion than lv (p<0.05), regardless of the surface finish. the highest values for all roughness parameters was observed in lvdb group, differing from the other groups, which were not significantly different. smaller bacterial adhesion values (cfu/ml) were observed for endd and envt, which differed significantly from the other groups, except enctl. for lv groups there was no significant difference between the different surface finishes (p>0.05). the type of material and surface finish system significantly interfered with surface roughness parameters and bacterial adhesion. the hybrid ceramic performed better after polishing than the nano-ceramic resin. conclusion: an adequate finishing/polishing technique should always be performed after any kind of adjustment to indirect restorations made with these materials tested. keywords: bacterial adhesion, ceramics, composite resins, dental polishing. 2 hamerschmitt et al. introduction all exposed surfaces in the oral cavity are coated with a salivary pellicle that enables microbial adhesion and may cause damage to teeth and restorative materials1. failures of all-ceramic restorations are due to several factors, such as fracture of the restoration, marginal discoloration, and secondary caries2. particularly, discoloration and secondary caries involve cariogenic microorganisms3. ceramic restorations with rough surface finishes may result in increased wear of the antagonist teeth and bacterial adhesion; they can also lead to tooth decay and periodontal disease4. in contrast, well-polished restorations show less wear on the opposing tooth, lower bacterial adhesion, improved color stability, and suitable optical properties5. thus, the final surface polishing in ceramic restorations should ideally remain intact. however, in some cases, it is necessary to make adjustments to ceramics; this requires new polishing procedures. some studies have found that manual final finishing has better clinical performance than glaze in regards to surface roughness and shade matching6,7. while a wide variety of restorative materials are available, new materials compatible with dental structure and function are still necessary. ceramics have several advantages, such as a high flexural strength and excellent color stability; however, they also have disadvantages, such as opposing tooth wear (when ceramics are not properly polished) and requiring a minimum thickness to prevent fracture8. hybrid materials, such as those based on a ceramic network infiltrated with polymers and resins with ceramic nanoparticles, have been developed to minimize the disadvantages of ceramics. these materials exhibit promising characteristics, such as mechanical properties similar to teeth, such as elastic module similar to dentin9. they can be applied in thin layers but are still strong enough to prevent material cracks. these new materials are applicable for minimally invasive restorations, the treatment of young patients, patients suffering from hereditary diseases (i.e., imperfect amelogenesis), and patients suffering from bruxism and dental erosion10. vita enamic® is a hybrid material with a combination of ceramic and polymeric properties. it is a ceramic network infiltrated with resin. the filler particles (silica based) confer optical characteristics, while the monomers (i.e., bis-gma, udma, utma, and bis-ema) determine the organic contents. this material is manufactured in two steps: first, a network of pre-sintered ceramics is produced and conditioned by a binding agent, and then, the network is infiltrated with a polymer by capillary action11. lava ultimate® is a nano-ceramic resin based material composed of about 80% nano-ceramic particles that form a nano-cluster of silica and zirconia, which are wrapped in a highly polymerized polymer matrix12. there is no standard protocol for finishing ceramic restorations and there have been limited studies on the effects of different finishing techniques on the surface topography and bacterial adhesion of these new hybrid materials13. thus, the objectives of this study were to evaluate the surface topography and bacterial adhesion of a hybrid ceramic and a nano-ceramic resin after different surface finishing techniques. 3 hamerschmitt et al. the null hypotheses of this study are: there is no difference in surface roughness or bacterial adhesion between materials, and there is no difference in surface roughness or bacterial adhesion among finishing techniques. material and methods specimen preparation blocks (12 × 14 × 18 mm) of hybrid ceramic vita enamic® (vita zahnfabrik, bad säckingen, germany) and nano-ceramic resin lava ultimate® (3m espe, st. paul, mn, usa) were purchased. the blocks were sectioned (isomet 1000, buehler, lake buff, usa) into eight 1-mm slices, and each slice was divided into 4 specimens (6 x 7 mm). the specimens were randomly assigned into 4 surface finishing technique groups (n = 8): ctl no finish (control); db worn with diamond bur (#3203, jota, ruthi, switzerland); vt polished using hybrid ceramic system (vita zahnfabrik, bad säckingen, germany); and dd polished using ceramic system (dedeco international inc, long eddy, ny, usa). the specimens were placed on a glass slide fixed with wax, and the finishing techniques were performed using a high-speed turbine with diamond bur (#3203, jota) for the db group, or a straight hand piece for the vt and dd groups. all instruments were intermittently placed on the specimen surface in one direction for 10 s. the same operator performed all procedures. surfaces were gently washed and dried after completing the surface finishing procedures. surface roughness eight specimens from each group were gently dried with absorbent paper and fixed to a glass slide with wax. surface roughness was analyzed with a surface roughness-measuring instrument (surftest sj-210p, mitutoyo, tokyo, japan) equipped with a 2-µm radius diamond needle. the needle moved at a constant speed (0.5 mm/s) under a 0.7 mn load. to maximize filtration of surface waviness, three readings were recorded for each specimen at different parallel positions (2 mm apart) with a length of 2.5 mm and a cut-off of 0.25 mm. the average of the three readings was used as the roughness value for each specimen. in this study, it was assessed three roughness parameters: average roughness (ra), the arithmetical average value of all absolute distances of the roughness profile from the centerline within the measuring length the average of peaks and valleys recorded in each sampling length (rz), and the effect of the profile values that deviate from the average (rq). means and standard deviations of ra, rz, and rq were calculated. after the analysis of surface roughness, specimens were sterilized in ethylene oxide under the following conditions: eto, 500-750 mg/l; temperature, 50-60°c, humidity 40-90%, 180 minutes exposure time over 4 hours of incubation. bacterial adhesion a 3 ml suspension of streptococcus mutans (atcc 35688), adjusted to the macfarland scale #2 (~6 × 108 cells/ml), was sequentially diluted by the addition of sterile physiological solution (0.9% sodium chloride). the number of cells in suspension was measured 4 hamerschmitt et al. via spectrophotometry (uv-vis double beam with scanning, quimis, curitiba, brazil); specifically, optical density (0.135 nm) and wavelength (0.303 nm) were quantified. colony forming units (cfu) were calculated against reference standards (r2 = 0.9214). bacterial adhesion was performed in an aseptic environment in a laminar airflow chamber. specimens from each group received 20 µl of standard s. mutans suspension on the center of their surface and incubated at 37°c for 1 hour. the specimens were removed, rinsed twice with sterile distilled water to remove materials with low adhesion, placed in individual tubes with 1.5 ml of sterile saline (0.9% nacl), and sonicated in a vortex mixer (vortex mixer, quimis, diadema, sp, brazil) for 30 seconds to disperse the adhered bacteria. samples (1 ml) were measured on the spectrophotometer to obtain average cfu/ml values. scanning electron microscopy (sem) analysis one specimen from each group was subjected to bacterial adhesion analysis by scanning electron microscopy. the specimens were fixed in 10% formaldehyde for 1 h, dehydrated in successive baths of increasing concentrations of ethanol, and dried in a bacteriological incubator at 37oc for 24 hours. the specimens were then mounted on aluminum stubs using a copper tape and coated with gold. surface topography was observed and photographed using a scanning electron microscope (feg quanta 450, fei, oregon, usa) operating at 15 kv with 1000 and 5000 magnification. confocal laser scanning microcopy (clsm) analysis three samples from each group were removed from the growth medium and stained for live/dead baclight bacterial viability and cell counting using two fluorescent nucleic acid dyes: syto 9, which penetrates bacterial membranes and stains cells green; and isopropidium iodide, which penetrates cells with damaged membranes and stain cells red. the dyes were each diluted in sterile saline (0.9% nacl) in an opaque container at a ratio of 10 ml of saline solution per 4 µl of dye. they were then placed on each specimen and incubated for 15 minutes under light protection. finally, the specimens were washed with sterile saline solution to remove excess dye and non-adherent bacteria. the surface of each sample was analyzed by clsm (nikon eclipse ti, curitiba, brazil) using excitation wavelengths of 488 nm and 535 nm for syto 9 and isopropidium iodide, respectively, and light emission between 500 and 560 nm. samples were observed via optical lenses at 20x and 63x magnification. hr nis-elements software was used to check for viable bacteria on the surface. three images in different areas were obtained via clsm for each group. since it was not possible to quantify the biofilm volume or thickness in this study, a score was established for each image according to the presence of viable bacteria (stained green): no viable bacteria in the entire image (score of 0); presence of viable bacteria in up to 25% of the image area (score of 1); presence of viable bacteria in 50% of the image area (score of 2); presence of viable bacteria by more than 50% of the image area (score of 3), and; presence of viable bacteria covering the entire image area (score of 4). 5 hamerschmitt et al. all images were evaluated by a qualified examiner twice (7-days apart). the kappa test score was 0.864 (p<0.001), indicating excellent agreement between duplicate examinations. statistical analysis data were analyzed using the statistica software version 10.0. the homogeneity of variances and normal distribution of the data was checked by kolmogorov-smirnov and levene tests. the dependent variables of the study were as follows: surface roughness (continuously quantitative), bacterial adhesion (continuously quantitative), and viable bacteria (categorically qualitative). the independent variables in the study were the different materials (i.e., hybrid ceramic (vita enamic en) and nano ceramic resin (lava ultimate lv)) and the different surface finishing techniques (i.e., ctl, db, vt, and dd). roughness and bacterial adhesion data were analyzed using analysis of variance (two-way anova) considering the factors “material” and “surface finish”, as well as the tukey test (α = 0.05). the scores for viable bacteria were compared among groups using the chi-square test (5% significance level). results surface roughness the mean surface roughness values ranged from 0.13 to 1.45 µm, 0.83 to 7.59 µm, and 0.17 to 1.84 µm for ra, rz, and rq, respectively (table 1). analysis of variance (two-way anova) showed a statistically significant difference for the factors “material” (p = .021), “finish” (p = .000000), and interaction “material*finish” (p = .0000001) for all roughness parameters (ra, rz and rq). table 1. mean and standard deviation of roughness (µm) and bacterial adhesion (cfu/ml) for the different groups (n=8). material finish ra (µm) rz (µm) rq (µm) bacterial adhesion (ufc/ml) vita enamic (en) ctl 0.32 (±0.12) a 2.21 (±0.79) a 0.42 (±0.16) a 2.40 x 108 (±2.5 x 107) ab db 0.48 (±0.16) a 2.85 (±0.68) a 0.61 (±0.18) a 2.58 x 108 (±2.9 x 107) b vt 0.31 (±0.15) a 1.75 (±0.71) a 0.39 (±0.18) a 2.22 x 108 (±2.0 x 106) a dd 0.16 (±0.03) a 1.12 (±0.16) a 0.22 (±0.03) a 2.20 x 108 (±2.0 x 106) a lava ultimate (lv) ctl 0.23 (±0.03) a 1.68 ± (0.24) a 0.31 (±0.04) a 2.60 x 108 (±1.7 x 107) b db 1.45 (±0.78) b 7.59 (±3.97) b 1.84 (±0.95) b 2.73 x 108 (±4.0 x 106) b vt 0.13 (±0.03) a 0.83 (±0.21) a 0.17 (±0.04) a 2.70 x 108 (±1.0 x 106) b dd 0.15 (±0.02) a 0.91 (±0.12) a 0.18 (±0.03) a 2.70 x 108 (±1.0 x 106) b * values followed by the same letters are statistically similar (p>0,05). 6 hamerschmitt et al. according to tukey’s test, the greatest roughness parameter value was observed for the lvdb group, where the nano-ceramic resin (lv) was ground with a diamond bur (db); this value was different from the other groups, which were not significantly different from each other. a significant difference was observed for “material” in regards to ra (p = .021). the hybrid ceramic (en) had a lower average surface roughness (ra) than the nano-ceramic resin (lv) regardless of surface finishing. there was significant difference in “finish” between groups (p = .0000001). the mean values of surface roughness (ra) for the different treatments were as follows: dd = vt = ctl < db. the groups treated with diamond bur (db) showed higher average surface roughness (ra) compared with other groups, which did not differ regardless of the material (i.e., hybrid ceramic (en) or nano ceramic resin (lv)). bacterial adhesion analysis of variance (two-way anova) showed statistically significant differences for the factors “material” (p = .000001), “finish” (p = .034), and the interaction “material*finish” (p = .043) (table 2). lower bacterial adhesion (cfu/ml) was observed for the polished hybrid ceramic (endd and envt) than the other experimental groups; these two groups had levels similar to the control group (enctl). there was no significant difference between surface finish for the nano-ceramic resin groups. when the factors were considered individually, there was significant difference for the factor “material” (p = .00001). the hybrid ceramic (en) showed less bacterial adhesion than the nano-ceramic resin (lv) regardless of surface finish. there was significant difference among groups for the factor “finish” (p = .034). the mean values of bacterial adhesion to the groups polished with the dedeco system (dd) were lower than the groups treated with the diamond bur (db). the group polished with the vita system (vt) and the control group had intermediate values that were not different from the other groups regardless of the material used (i.e., hybrid ceramic or ceramic nano-resin). sem analysis the sem images of the materials’ surfaces before bacterial adhesion show that the polishing methods produced a regular and smoother surface for both materials. the sem images also show that all surfaces of the hybrid ceramic (en) showed bacterial adhesion with higher cellular accumulation (s. mutans) in the regions of higher surface irregularity (fig. 1). it is also evident that all surfaces of the nano-ceramic resin show similar bacterial adhesion (fig. 2). clsm analysis there was a statistically significant difference in bacterial viability between the experimental groups (fig. 3, chi-square = 66.69, p < 0.001). there was a similar distribution found for the two materials. all of the group finished with the diamond bur (db) scored 4 (i.e., the entire image area had viable bacteria). the other groups (i.e., polishing and control) were mostly scored 1 (i.e., less than 25% of viable bacteria in the image). figure 4 shows representative images of scores found for both materials (endb and lvdb – score 4, and endd and lvdd – score 1). 7 hamerschmitt et al. a b c d figure 1. photomicrographs of the surfaces of hybrid ceramic (en) after different finishes and bacterial adhesion. control (a), diamond bur (b), vita polishing system (c), and dedeco polishing system (d) (5000 x). a b c d figure 2. photomicrographs of surfaces of nano ceramic resin (lv) after different finishes and bacterial adhesion. control (a), diamond bur (b), vita polishing system (c), and dedeco polishing system (d) (5000 x). pe rc en ta ge (% ) hybrid ceramic (en) nano ceramic resin composite (lv) 100 90 80 70 60 50 40 30 20 10 0 do score 4 vtdbctldovtdbctl score 3 score 2 score 1 score 0 figure 3. scores attributed in different groups. 8 hamerschmitt et al. discussion according to the results, the first null hypothesis should be rejected. there was a significant difference between the materials regarding the surface roughness and bacterial adhesion,,the hybrid ceramic (en) had a lower roughness and lower bacterial adhesion than the nano-ceramic resin (lv). these differences could be related to materials composition and microstructure. vita enamic is a hybrid material (ceramic/resin) with a porous three-dimensional structure of feldspathic ceramic infiltrated with resin. lava ultimate is a resin composite with nano-zirconia particles, cost effective, easy to repair and is also for machining in cad/cam systems14. although both materials include resin, their microstructures differ significantly; this is evident in scanning electron microscopy images (fig. 1, 2). the flexural strength and modulus of elasticity of these materials are similar to dentin and lower than ceramics; this makes them more suitable for indirect restorations15. other studies have also shown that ceramic has lower roughness and bacterial adhesion than resin composites16-18. awad et al.19 defined an ascending order of surface roughness for restorative materials: ceramic, feldspathic ceramic, hybrid ceramics, resin-based composites, and polymethylmethacrylate (pmma). however, fasbinder and neiva20 observed a lower surface roughness in a nano-ceramic resin material than in a hybrid ceramic. a b c d 100µm100µm 100µm100µm figure 4. representative images of scores found for both materials after staining using live/dead baclight bacterial viability and cell counting method. viable/live bacteria stained in green and dead bacteria stained in red. (a) hybrid ceramic treated with diamond bur (endb); (b) hybrid ceramic polished with dedeco system (endd); (c) nano ceramic resin composite treated with diamond bur (lvdb); and (d) nano ceramic resin composite polished with dedeco system (lvdd). 9 hamerschmitt et al. regarding the finishing techniques, the second null hypothesis should also be rejected. there was also a difference in surface roughness and bacterial adhesion among surface finishing techniques (db groups presented higher roughness and bacterial adhesion). the superficial characteristics of the materials interfere in the bacterial adhesion, while treatment/polishing also influences the surface roughness and subsequently affects bacterial adhesion21. different polishing materials are available; in this study, two polishing systems were selected: the vita polishing kit, which was specially developed for vita enamic, and; the dedeco kit, which is used to polish ceramics worldwide. both kits were used according to the manufacturer’s recommendations. the surfaces of the polished specimens had values similar to the control group for both materials, and there were no differences between the different polishing kits (i.e., dd or vt). the sem images show that polishing produces a regular and smooth surface on both materials. for the hybrid ceramic (en), the ceramic polishing system (dd) produced a more regular and smooth surface compared to the other system (vt); this conforms to recommendations by hybrid ceramic manufacturers. both polishing systems produced regular and smooth surfaces on the nano-ceramic resin (lv). sem images revealed that bacteria adhered to the all hybrid ceramic (en) specimens with the highest biofilm accumulations. s. mutans were observed in areas with higher surface irregularities, such as those present in the db group; in these cases the bacteria were deposited in cracks and depressions present in the material. the group finished with the dedeco system (dd) also had biofilm accumulation; however, it was at a lower intensity than the other groups. clsm images showed that, independent of the material, the greatest concentration of viable bacteria was present in the groups finished with the diamond bur (db). the other groups (i.e., control and polished) had comparable distributions of viable bacteria on the surface. the clsm images confirmed the distribution of bacteria observed in the sem images and also enabled analysis of bacterial viability2,22. images from the hybrid ceramic (en) have higher amounts of green coloring; this indicates that there may be an interaction between the dye (syto 9) and the material. thus, the visualization of bacterial adhesion may be more complex. according to bollen et al.23, the roughness values (ra) of the intraoral hard tissue should be approximately 0.2 µm or lower to limit bacterial adhesion. it was observed surface roughness values (ra) close to or smaller than 0.2 µm for both materials except for the samples finished with the diamond bur (db). thus, the surfaces subject to polishing techniques are considered clinically acceptable. the highest surface roughness values (ra, rz, and rq) were observed in the nano-ceramic resin (lv) finished with the diamond bur (lvdb); these values differed significantly from the other groups. the polishing technique, independent of the selected system (i.e., vt or dd), resulted in a more regular and smooth surface with less accumulation of biofilm for the hybrid ceramic (en) (fig. 1). similar data were recently found by vo et al.21, who assumed that the surface roughness of a lithium disilicate-based material was a critical factor for s. mutans adherence. thus, an additional polishing of this surface leading to lower surface roughness is expected to reduce bacterial adherence. 10 hamerschmitt et al. clinically, the superficial characteristics of restorative materials and biofilm adhesion on these surfaces are related to the degradation of these restorative materials, the development of recurrent caries lesions, and gingival inflammation24. thus, bacterial adhesion to restorative materials interferes with clinical performance23. here, we show that the surface characteristics of restorative materials should be taken into consideration during the selection of these materials. for the cad/cam resin-based materials tested, if mouth adjustments are necessary using diamond burs or after cad/cam procedures, the use of specific systems for finishing and polishing of indirect restorations is required7. in conclusion, the type of the material and the finishing technique significantly influence surface roughness and bacterial adhesion. the hybrid ceramic performed better after polishing than the nano-ceramic resin. treatment with a diamond bur increased surface roughness and bacterial adhesion. an adequate finishing/polishing technique should always be performed after any kind of adjustment to indirect restorations made with these materials (hybrid ceramic or nano-ceramic resin). acknowledgements the authors thank the conventional fluorescence and confocal microscopy center of federal university of parana, for help obtaining the confocal laser scanning microscopy (clsm) images. references 1. meier r, hauser-gerspach i, lüthy h, meyer j. adhesion of oral streptococci to all-ceramics dental restorative materials in vitro. j mater sci mater med. 2008 oct;19(10):3249-53. doi: 10.1007/s10856-008-3457-7. 2. anami lc, pereira ca, guerra e, souza roa, jorge aoc, bottino ma. morphology and bacterial colonization of tooth/ceramic restoration interface after different cement excess removal techniques. j dent. 2012 sep;40(9):742-9. doi: 10.1016/j.jdent.2012.05.005. epub 2012 may 19. 3. lassila lvj, garoushi s, tanner j, vallittu pk, söderling e. adherence of streptococcus mutans to fiber-reinforced filling composite and conventional restorative materials. open dent j. 2009 dec 4;3:227-32. doi: 10.2174/1874210600903010227. 4. kawai k, urano m, ebisu s. effect of surface roughness of porcelain on adhesion of bacteria and their synthesizing glucans. j prosthet dent. 2000 jun;83(6):664-7. 5. boaventura jmc, nishida r, elossais aa, lima dm, reis jmsn, campos ea, et al. effect finishing and polishing procedures on the surface roughness of ips empress 2 ceramic. acta odontol scand. 2013 may-jul;71(3-4):438-43. doi: 10.3109/00016357.2012.690570. 6. akar gck, pekkan g, çal e, eskitaşçioğlu g, özcan m. effects of surface-finishing protocols on the roughness, color change, and translucency of different ceramic systems. j prosthet dent. 2014 aug;112(2):314-21. doi: 10.1016/j.prosdent.2013.09.033. 7. özarslan mm, büyükkaplan uş, barutcigil ç, arslan m, türker n, barutcigil k. effects of different surface finishing procedures on the change in surface roughness and color of a polymer infiltrated ceramic network material. j adv prosthodont. 2016 feb;8(1):16-20. doi: 10.4047/jap.2016.8.1.16. 8. mörmann wh, stawarczyk b, ender a, sener b, attin t, mehl a. wear characteristics of current aesthetic dental restorative cad/cam materials: two-body wear, gloss retention, roughness and martens hardness. j mech behav biomed mater. 2013 apr;20:113-25. doi: 10.1016/j.jmbbm.2013.01.003. 11 hamerschmitt et al. 9. coldea a, swain mv, thiel n. mechanical properties of polymerinfiltrated-ceramic-network materials: official publication of the academy of dental materials. dent mater. 2013 apr;29(4):419-26. doi: 10.1016/j.dental.2013.01.002. 10. dirxen c, blunck u, preissner s. clinical performance of a new biomimetic double network material. open dent j. 2013 sep 6;7:118-22. doi: 10.2174/1874210620130904003. 11. della bona a, corazza ph, zhang y. characterization of a polymer-infiltrated ceramic-network material. dent mater. 2014 may;30(5):564-9. doi: 10.1016/j.dental.2014.02.019. 12. koller m, arnetzl gv, holly l, arnetzl g. lava ultimate resin nano ceramic for cad/ cam: customization case study. inter j computer dent 2012;15(2):159-64. 13. da silva tm, salvia acrd, carvalho rf, pagani c, rocha dm, da silva eg. polishing for glass ceramics: which protocol? j prosthodont res. 2014 jul;58(3):160-70. doi: 10.1016/j.jpor.2014.02.001. 14. elsaka se. repair bond strength of resin composite to a novel cad/cam hybrid ceramic using different repair systems. dent mater j. 2015;34(2):161-7. doi: 10.4012/dmj.2014-159. 15. albero a, pascual a, camps i, grau-benitez m. comparative characterization of a novel cad-cam polymer-infiltrated-ceramic-network. j clin exp dent. 2015 oct 1;7(4):e495-500. doi: 10.4317/jced.52521. 16. koizumi h, saiki o, nogawa h, hiraba h, okazaki t, matsumura h. surface roughness and gloss of current cad/cam resin composites before and after toothbrush abrasion. dent mater j. 2015;34(6):881-7. doi: 10.4012/dmj.2015-177. 17. rosentritt m, hahnel s, gröger g, mühlfriedel b, bürgers r, handel g. adhesion of streptococcus mutans to various dental materials in a laminar flow chamber system. j biomed mater res b appl biomater. 2008 jul;86(1):36-44. 18. aykent f, yondem i, ozyesil ag, gunal sk, avunduk mc, ozkan s. effect of different finishing techniques for restorative materials on surface roughness and bacterial adhesion. j prosthet dent. 2010 apr;103(4):221-7. doi: 10.1016/s0022-3913(10)60034-0. 19. awad d, stawarczyk b, liebermann a, ilie n. translucency of esthetic dental restorative cad/cam materials and composite resins with respect to thickness and surface roughness. j prosthet dent. 2015 jun;113(6):534-40. doi: 10.1016/j.prosdent.2014.12.003. 20. fasbinder dj, neiva gf. surface evaluation of polishing techniques for new resilient cad/cam restorative materials. j esthet restor dent. 2016 jan-feb;28(1):56-66. doi: 10.1111/jerd.12174. 21. vo dt, arola d, romberg e, driscoll cf, jabra-rizk ma, masri r. adherence of streptococcus mutans on lithium disilicate porcelain specimens. j prosthet dent. 2015 nov;114(5):696-701. doi: 10.1016/j.prosdent.2015.06.017. 22. guilbaud m, piveteau p, desvaux m, brisse s, briandet r. exploring the diversity of listeria monocytogenes biofilm architecture by high-throughput confocal laser scanning microscopy and the predominance of the honeycomb-like morphotype. appl environ microbiol. 2015 mar;81(5):1813-9. doi: 10.1128/aem.03173-14. 23. bollen cm, lambrechts p, quirynen m. comparison of surface roughness of oral hard materials to the threshold surface for bacterial plaque retention: a review of the literature. dent mater. 1997 jul;13(4):258-69. 24. rashid h. the effect of surface roughness on ceramics used in dentistry: a review of literature. eur j dent. 2014 oct;8(4):571-9. doi: 10.4103/1305-7456.143646. braz j oral sci. 15(3):209-214 periapical injection of betamethasone to control postoperative pain in emergency endodontic care a randomized double blind clinical trial marcos lp pinheiro1, ricardo lopes-rocha2, eduardo d. de andrade3 1phd, department of basic sciences, biologic and health sciences faculty, universidade federal dos vales do jequitinhonha e mucuri, diamantina, minas gerais state, brazil 2msc, department of dentistry, biologic and health sciences faculty, , universidade federal dos vales do jequitinhonha e mucuri, diamantina, minas gerais state, brazil 3phd, department of pharmacology, anesthesiology and therapeutics, piracicaba dental school, universidade estadual de campinas, piracicaba, são paulo state, brazil correspondence to: dr. marcos luciano pimenta pinheiro rua da glória, 187, centro diamantina city minas gerais state, brazil email: marcospimenta@ufvjm.edu.br fone number: +55 038 3532 1200 – ramal abstract aim: the objective of this study was to evaluate the effect of betamethasone in the control of postoperative pain in patients undergoing endodontic treatment. methods: patients of both genders (n = 120), after being submitted to emergency endodontic treatment, received a single dose of betamethasone solution (0.05 mg / body weight) or sterile saline solution intramucosally, in the periapical region of the treated tooth. the study evaluated the intensity of pain experienced by the patient and the number of analgesics consumed during periods of 4, 24 and 48 hours after endodontic treatment. to compare the level of pain among the groups and the use of analgesics the fisher’s exact test was used, adopting a significance level of 95%. results: patients who received betamethasone felt less pain in 4 hours (p = 0.0177) and 24 hours (p = 0.0012) compared to those who received the placebo. conclusions: betamethasone at a dose of 0.05 mg / body weight administered in the periapical region is a advantageous protocol due to its effectiveness, and also because of the comfort it provides to patients in the prevention or control of inflammatory pain in endodontics. keywords: betamethasone, endodontics, pain. introduction the presence of pain, swelling or both after endodontic treatment is a condition defined as flare-up, with prevalence of 16%1 and a multifactorial etiology of mechanical, chemical and microbial processes that occur during the procedure1,2. also influential are the location of the tooth in question, the number of clinic visits and the condition of tooth and periapex before treatment2. patients seeking endodontic treatment in teeth that already present periapical radiolucency are up to nine times more likely to experience flare-up than those who did received for publication: december 6, 2016 accepted: may 5, 2017 original article braz j oral sci. july | september 2016 volume 15, number 3 http://dx.doi.org/10.20396/bjos.v15i3.8649983 210 not have this condition3; and those who undergoing multiple visits have a higher risk; up to three times more chance of developing flare-ups4. as endodontic treatment does not immediately or totally eliminate periapical inflammatory reaction during the procedure itself, harmful substances such as bacterial toxins can escape from the root canal system into the periapex, causing the persistence of pain in the postoperative period5,6. corticosteroids are potent anti-inflammatory drugs that suppress inflammatory phenomena as pain and edema7. thus, the use of this drug is an effective supplement to reduce of these symptons after endodontic treatment8, and its use in the treatment and prevention of pain and endodontic post-treatment swelling has been shown to be effective and safe8-16. among the corticosteroids, betamethasone has fast, potent and long-lasting action, a dose of 0.5 mg being necessary for an equivalent 20 mg of endogenous hydrocortisone17, without causing the undesirable mineralocorticoid activity of sodium retention11 and it is even more effective than dexamethasone, equivalent dose of which is 0.75 mg17. the hypothesis is that the use of betamethasone is as good or better than the use of dexamethasone, which is already well documented in clinical studies8-14,16. nobuhara et al.16 (1993) developed an experimental model to study the effects of dexamethasone on the periapical tissues of rats after endodontic overinstrumentation. these authors demonstrated by means of histological techniques that the submucosal infiltration of dexamethasone in the periapical region of the mandibular molars produces a significant antiinflammatory effect characterized by the inhibition of neutrophil migration to the periapical tissues. extrapolating to the clinic, the administration of a corticosteroid through intrabuccal infiltrative injection would be a technique that would offer great familiarity to the dentist without the need for specialized equipment. furthermore, it is assumed that optimum levels of the medication would be obtained in the inflamed region, with the further advantage of the application being painless, since at the time of infiltration the region would be anesthetized. the objective of this randomized double-blind clinical trial was to evaluate the effect of the systemic administration of betamethasone – through infiltration in the oral mucosa – to control pain and edemas in patients undergoing emergency endodontic treatment. material and methods the design of this randomized, double-blind clinical trial was conducted in full compliance with the ethical principles of the helsinki declaration, was approved by the research ethics committee of the piracicaba dental school, state university of campinas, under the protocol 007/2004, and was conducted in accordance with the consolidated standards of reporting trials statement. it is registered at clinicaltrials. gov with number nct02865746. (https://clinicaltrials.gov/ct2/ results?term=nct+02865746&search=search). participants were informed about the purpose of the study, agreed to participate and signed the term of free and informed consent. sample to calculate the sample, data obtained by krasner and jackson13 (1986) was used. by adopting a significance level of 95% and power of test of 90%, the study reached the minimum sample size of 54 subjects per group. this number was increased by 10% to compensate for occasional losses, totaling 60 subjects per group. eligibility criteria inclusion criteria: to be included in the sample individuals had to have a tooth with pain of endodontic origin, diagnosed clinically and radiographically as irreversible pulpitis or necrosis associated to pericementitis and presence of pain, justifying emergency care. they had to provide a general health history without alterations, and have no condition, local or systemic, that counterindicates the use of the drugs applied in this research study. exclusion criteria: individuals who presented any of the following conditions were not included in the study – pregnancy or lactation; use of corticosteroids; history of hypersensitivity to the drugs used in this study; pain associated with abscesses of endodontic origin; or anyone having any of the following diseases: tuberculosis, systemic fungal infections, simple ocular herpes, glaucoma, acute psychosis or psychotic tendencies. operative procedures emergency endodontic care was carried out in all patients, in a single session, by students majoring in dentistry, who were previously trained and assisted by a teacher for this study. local anesthesia a local anesthesia technique adequate to the tooth in question was applied, the solution used being lidocaine based at 2%, with epinephrine at proportion 1:100,000 (alphacaine ® dfl industry and trade ltd.), limiting the use to a maximum of 3.6 ml, equivalent to two vials of this anesthetic. two anesthetic tubes were used because it is the procedure recommended in the endodontics clinic of the school of dentistry of piracicaba unicamp. individuals who required more tubes were removed from the study. endodontic procedures after a diagnostic x-ray, the procedure to remove the carious dentin began, as well as the reconstruction of any lost wall that could compromise the isolation with the use of a rubber dam. the emergency endodontic procedure was carried out through the implementation of the first phase of the chemical-mechanical preparation by using the hybrid technique described by valdrighi et al.18 (1991). a cotton ball was placed at the entrance of the canals, and these were then sealed with a temporary restorative material (cotosol® vigodent rio de janeiro, brazil). after removal of rubber dam isolation, the occlusion of the patient was checked. quently, the patients received the drug prescription to be used in case of pain, they were given the following steps to carry out the remainder of the endodontic treatment and were discharged. quantitative transportation assessment in simulated curved canals after large apical preparations braz j oral sci. 15(3):209-214 211quantitative transportation assessment in simulated curved canals after large apical preparations braz j oral sci. 15(3):209-214 formation of study groups and randomization the random allocation of patients was carried out by means of a draw. 120 opaque equal envelopes were prepared. 60 of them, contained a piece of paper with the number "1" written on it, and the other 60 contained the number "2". all envelopes were sealed, mixed and stored. after endodontic procedures, one of the envelopes was randomly selected for each patient in order to allocate them to group "1" or group "2", by a person who was not directly involved in the patients evaluation. group 1 – received the injection of solution "1", of betamethasone disodium phosphate at a concentration of 4 mg / ml (celestone injetável® ampoules 4mg / ml chemical and schering plough pharmaceuticals a / s). group 2 received the injection solution "2", of sterile saline solution (sodium chloride 0.9% 1 ml ampoules) to calculate the volume of the solution to be injected, the concentration of betamethasone solution was used in the proportion of 0.05 mg / kg. thus, for an 80 kg patient, the maximum volume of solution administered was 1 ml. patients weighing over 80 kg received the same volume. the injections were performed with the aid of syringes fitted with ultrafine needles (bd ultrafine u-100, 0.3 x 8 mm), submucosally near the tooth’s periapex of the periapical region of the tooth involved, by using the submucosal infiltration technique. blinding of information the solutions were placed in similar ampoules containing 1 ml of solution and labeled merely with numbers "1" or "2". only the responsible pharmacist knew the content of the ampoules and this professional was not directly involved in the procedures. this information was kept in a sealed envelope and was made available only after the end of statistical analysis. thus, the patient and the investigator remained blinded to the type of drug used until the end of statistical analysis. postoperative care at the end of the session, three pills of dipirona (sodium metamizol) (tablets of 500 mg, generic medicine) were provided to each patient, to take after the effects of the local anesthesia ceased if they felt pain or discomfort. in case of persistent pain for longer periods, subjects also received a prescription for analgesics to be taken at intervals of four hours. evaluation method of postoperative pain and analgesic use in the periods of 4, 24 and 48 hours after treatment, each patient was contacted by phone to answer two questions, and they were instructed to write down the answers on a card (point verbal rating scale modified vrs4 jensen et al., 1986)19. the questions were: 1 how's your toothache? a) it passed and i do not feel any more pain b) i still feel a certain discomfort c) only hurts when i touch or chew (above 3 until 6); d) it got worse, the pain is severe (above 6 until 9). 2 how many analgesic pills have you taken so far? a) none; b) 1; c) 2; d) 3 or more. another question was whether they had experienced any of the possible side effects of the medication. statistical analysis after a descriptive analysis of the data, the presence of pain or discomfort among the groups was compared, considering the age and gender by using the fisher’s exact test. to compare the consumption of analgesics among the groups the fisher's exact test was also used, considering a significance level of 95%. results the sample consisted of individuals who came to the emergency service of the dentistry college of piracicaba, unicamp, with endodontic pain symptoms. from a total of 120 individuals, 44 were men (36.7%) and 76 women (63 %); age ranged from 10 to 72 years, constituting an average of 30.89 years (sd ± 3.7). pulpitis was diagnosed in 73 patients (60.8%) and necrosis in 47 (39.2%). there was no waiver of any patient. the list of individuals who reported different pain intensities (no pain, mild pain, moderate pain and severe pain) at the intervals of 4, 24 and 48 hours is itemized in table 1. table 2 discriminates the distribution of subjects in relation to the quantity of ingested analgesics, when comparing the groups. when comparing gender, there were no statistically significant differences in postoperative pain reports. fig. 1 flow diagram according to consort statement. 212 quantitative transportation assessment in simulated curved canals after large apical preparations discussion the sample consisted of individuals who came to the emergency service of the dentistry college of piracicaba, unicamp, with endodontic pain symptoms. from a total of 120 individuals, 44 were men (36.7%) and 76 women (63 %); age ranged from 10 to 72 years, constituting an average of 30.89 years (sd ± 3.7). pulpitis was diagnosed in 73 patients (60.8%) and necrosis in 47 (39.2%). there was no waiver of any patient. the list of individuals who reported different pain intensities (no pain, mild pain, moderate pain and severe pain) at the intervals of 4, 24 and 48 hours is itemized in table 1. table 2 discriminates the distribution of subjects in relation to the quantity of ingested analgesics, when comparing the groups. when comparing gender, there were no statistically significant differences in postoperative pain reports. given the notion that endodontic pain after treatment is the result of periapical inflammation, treatment should be based on the administration of anti-inflammatory medication, such as corticosteroids14. this study showed a good tolerability of the use of betamethasone, in the absence of infection reports or other adverse reactions, confirming the results of similar studies – although in these the corticosteroid used was dexamethasone, a similar corticosteroid to betamethasone5,8,10,13-15,20. corticosteroids were used in this study due to their anti-inflammatory power to control pain in endodontics8. corticosteroids have an inhibitory effect on the transcription of the cox-2 enzyme and, consequently, on the synthesis of pro-inflammatory prostaglandins, which may explain the potent anti-inflammatory effect of these agents. on the other hand, they do not exert any effect on the cox-1 expression, which may explain their noninterference in certain biological processes21. prolonged administration of corticosteroids, such as during the postoperative period may result in suppression of the hypothalamic-pituitary-adrenal axis (hha), leading to bone mineral loss, causing osteoporosis and may increase the risk of cataracts, glaucoma, cutaneous effects and vascular changes22,23, effects on the central nervous system, changes in body fat distribution, increased susceptibility to infection, increased appetite and emotional disturbances24-26. the dose of betamethasone used in this study (0.05 mg / kg) is 40 times smaller than that of 2 mg / kg of dexamethasone (which is equipotent to betamethasone), employed by czerwinsnky et al. (1972)27 which, according to these authors, did not produce significant adverse side effects. this also corroborates the results of mehrvarzfar et al.15 (2008) who used a dose of dexamethasone of 4 mg in the periapical region15. in fact, there were no reports of adverse effects in relation to the use of betamethasone by patients. this can be explained by the fact that corticosteroids, when used as a single dose or in restricted time periods, are virtually devoid of clinically significant side effects28. in this study, the significant reduction of pain at 4 and 24 hours with the administration of betamethasone corroborates with the results reported by shantiaee et al.8 (2012), although these researchers used dexamethasone. not having carried out the control of pain after 8 and 12 hours was a limitation of this betamethasone (n = 60) placebo (n = 60) none n (%) mild n (%) mod n (%) sev n (%) none n (%) mild n (%) mod n (%) sev n (%) p 4 hours 46 (76.7) 9 (15.0) 1 (1.67) 4 (6.67) 30 (50.0) 14 (23.3) 7 (11.67) 9 (15.0) 0.012* 24 hours 53 (88.33) 6 (10.0) 1 (1.67) 37 (61.67) 12 (20.0) 6 (10.0) 5 (8.33) 0.004* 48 hours 57 (95.0) 3 (5.0) 53 (88.33) 3 (5.0) 4 (6.67) 0.13 table 1 distribution of patients in relation to the degree of reported pain intensity according to postoperative time and treatment groups. none = no pain; mild = small discomfort; mod (moderate) = pain when touched or chew; sev (severe) = intense pain (vrs4). * significant values for fisher’s exact test betamethasone (n = 60) placebo (n = 60) 0 n (%) 1 n (%) 2 n (%) 3 or more n (%) 0 n (%) 1 n (%) 2 n (%) 3 or more n (%) p 4 hours 49(81.67) 11 (18.33) 31 (51.67) 27 (45.0) 2 (3.33) 0.002* 24 hours 55(91.67) 5 (8.33) 41 (68.33) 17 (28.33) 2 (3.33) 0.005* 48 hours 57(95.0) 3 (5.0) 55 (91.67) 4 (6.67) 1 (1.67) 0.55 table 2 distribution of patients in relation to the number of ingested analgesics according to postoperative time and treatment groups. * significant values for fisher’s exact test intensity of pain postoperative time number of analgesics postoperative time braz j oral sci. 15(3):209-214 study; the comparison of the results with other studies was only possible in the periods of 4, 24 and 48 hours8,10,13,15. betamethasone or dexamethasone at low doses, such as 1 to 2 mg, do not produce anti-inflammatory effects that outweigh the suppression of the ß-endorphins involved with the modulation of the pain threshold; consequently they do not decrease pain intensity compared to a placebo29. however, doses of 5 to 6.5 mg of dexamethasone (corresponding to 0.07 to 0.09 mg / kg) are considered optimal for pain control in endodontic procedures5. in this study used a dose of 0.05 mg / kg and a maximum volume of 1 ml, because the place planned for the injection does not support a large volume without exerting discomfort to the patient. however, the dose of 4 mg of betamethasone may be standardized, regardless of body weight, because it does not present risks, and at the same time it avoids unnecessary calculations of dosing and volume of the solution. through the data obtained and the comparisons carried out, it is possible to infer that betamethasone was infiltrated through the periapical tissues in sufficient quantities to exert its pharmacological action, which can prove the effectiveness of this corticosteroid by submucosal administration these results are supported by wayman et al.30 (1994), who reported a good distribution of dexamethasone, administered in the mucosa of the vestibule of rat jaws, indicating an affinity of this corticosteroid with bone tissue; and corroborates with the results of mehrvarzfar et al.15 (2008), who tested dexamethasone injected into the periapical region of the treated tooth, with similar results to those of the present study. the main advantages of the submucosal route in relation to the others are the drug concentration near the surgical site and the low systemic absorption of the drug, which consequently results in fewer side effects31. the limitation was the application of a maximum volume of 1 ml, equivalent to a dose of 4mg, which has already been demonstrated in previous studies as effective in endodontics32. the lower consumption of analgesics by patients treated with betamethasone confirms the findings by liesinger et al.5 (1993), which showed a consumption of analgesic 2.5 times lower in the group of patients previously treated with corticosteroids compared to those treated with the placebo. consumption of analgesics indicates the presence of postoperative pain, although there are other possible reasons for this intake, such as pain not associated with endodontic treatment or for prevention of potential pain33 facts that were not reported in this study. there is general agreement that pain is a difficult variable to quantify due to its subjective and multifactorial characteristics34,35, which may lead to the occurrence of failures in the evaluations. in this study, the measurement accuracy of the pain experienced by patients also had this limitation, as patients noted their experience of pain intensity subjectively. in addition, another limitation that we must report is that the pain was not measured in the initial period of treatment. in cases of already installed pain, the use of drugs which directly reduce the activity of nociceptors may be desirable because they can reduce the hyperalgesia – the standard drug in this group being dipyrone36. dipyrone is routinely used in brazil, russia, india and other south american, caribbean, african and asian countries37-40. 213quantitative transportation assessment in simulated curved canals after large apical preparations it is important to highlight that all patients who reported pain became asymptomatic after 24 hours, result that is very close to that found by krasner and jackson13 (1986) and glassman et al.10 (1989). furthermore, no case of flare-up was observed, confirming the results of imura and zuolo41 and being very close to the results found by iqbal et al.3. the results found by akbar et al.42 (2013) do not corroborate with those reported above, however these authors did not use drugs as postoperative medication. another aspect that should be considered is related to the expectation of post-instrumentation pain. when planning an endodontic therapy in previously asymptomatic teeth, the endodontist usually does not prescribe an anti-inflammatory. however, after the procedure and depending on the outcome the professional may choose to prescribe one. in these cases, a simple local infiltration of the corticosteroid in the apical region of the tooth involved may help minimize patient discomfort after the cessation of the effects of local anesthesia. to control pain and inflammation, the administration of drugs directly on the site of injury may be a more effective action than waiting for absorption through the gastrointestinal tract and distribution by the organism43. thus, administering the drugs submucosally near the tooth’s periapex, will presumably reach optimum corticosteroid levels in the inflamed region16. this administration may be painless because at the time of application the effect of the anesthetic used in the endodontic procedure will still be present. in conclusion, betamethasone showed satisfactory effects in the dosage used, as well as in the proposed route of administration. considering the limitations of this study, this protocol is an advantageous practice due to its effectiveness, and also because of the comfort it provides to patients in the prevention or control of inflammatory pain in endodontics. further studies should be performed to elucidate possible variations not considered in this study. acknowledgments thanks to prof. francisco josé de souza filho (in memoriam), who contributed and revolutionized on the field of endodontics. thanks to prof. joana ramos jorge for her contribution in statistics. the authors have no any conflicts of interest. 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geometry assessed by micro-computed tomography. int endod j. 2015 feb;48(2):162-70. doi: 10.1111/iej.12296. 11. van der sluis lw, shemesh h, wu mk, wesselink pr. an evaluation of the influence of passive ultrasonic irrigation on the seal of root canal fillings. int endod j. 2007 may;40(5):356-61. 12. elayouti a, kiefner p, hecker h, chu a, löst c, weiger r. homogeneity and adaptation of endodontic fillings in root canals with enlarged apical preparation. oral surg oral med oral pathol oral radiol endod. 2009 sep;108(3):e141-6. doi: 10.1016/j.tripleo.2009.04.022. 13. brunson m, heilborn c, johnson dj, cohenca n. effect of apical preparation size and preparation taper on irrigant volume delivered by using negative pressure irrigation system. j endod. 2010 apr;36(4):721-4. doi: 10.1016/j.joen.2009.11.028. 14. silva ej, tameirão md, belladonna fg, neves aa, souza em, dedeus g. quantitative transportation assessment in simulated curved canals prepared with an adaptive movement system. j 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reciprocating niti instruments in simulated curved canals. restor dent endod. 2012 nov;37(4):220-7. doi: 10.5395/rde.2012.37.4.220. 25. freire lg, gavini g, cunha rs, santos md. assessing apical transportation in curved canals: comparison between cross-sections and micro-computed tomography. braz oral res. 2012 may-jun;26(3):222-7. 214 quantitative transportation assessment in simulated curved canals after large apical preparations braz j oral sci. 15(3):209-214 1http://dx.doi.org/10.20396/bjos.v18i0.8656585 volume 18 2019 e191436 original article 1 social dentistry department, community dentistry division, school of dentistry, universidade de são paulo – usp, são paulo, brazil. corresponding author: prof. dr. e. michel-crosato. social dentistry department, community dentistry division, school of dentistry, universidade de são paulo – usp, av. prof. lineu prestes, 2227 cidade universitária butantã, são paulo – sp, 05508-000, brazil. phone: +55113091-7891 e-mail: michelcrosato@usp.br https://orcid.org/0000-0001-8559-9769 conflicts of interest: none. received: october 29, 2018 accepted: june 02, 2019 functional dentition and prosthodontic status in an indigenous population from the south of brazil gustavo hermes soares¹, nayara fernanda pereira¹, adrielly garcia ortiz¹, maria gabriela haye biazevic¹, edgard michel-crosato1,* aim: to analyze the prevalence of different definitions of functional dentition, prosthodontic status and associated factors, in an indigenous population from brazil. methods: a cross-sectional oral health survey was conducted with indigenous adults aged 35-44 years. a single examiner collected clinical data through oral examinations and sociodemographic data using a structured questionnaire. dentitions were classified according to four classification systems of functional dentition: fdwho (> 20 teeth), fdgroup2 (> 10 teeth in each arch), fdgroup3 (all anterior teeth), and fdgroup4 (> 10 teeth in each arch, all anterior teeth, and sufficient posterior region). use and need of prosthodontics was also evaluated. uni and multivariate analysis were conducted at the level of significance of 5%. results: indigenous adults presented considerably low frequencies of prosthodontic use and functional dentition, independently of the definition analyzed. substantial differences of prevalence rates were observed among the four definitions of functional dentition, ranging from 48.62% to 11.93%. age and municipality were associated with use of dental prosthesis and prosthodontic need, respectively. significant discrepancies in functional dentition rates were observed regarding sex and time of the last dental appointment. conclusions: indigenous adults are severely affected by tooth loss and, consequently, by low frequencies of functional dentition. the scenario is worsened by the elevated need of the population for prosthodontics. the phenomenon was associated with age, sex, access to specialized dental care and time of the last dental visit. keywords: oral health. health services, indigenous. population groups. health equity. https://orcid.org/0000-0001-8559-9769 2 soares et al. introduction oral health inequalities are particularly striking among indigenous peoples from different nations1. native populations constitute culturally different societies, with highly heterogeneous epidemiological profiles and treatment needs. although it is not normally explored in studies with indigenous, tooth loss might be an important parameter for assessing oral health status in different populations2. tooth loss represents one of the most severe injuries to oral health due to its mutilating effects and the potential repercussions to the quality of life3. the resulting sequels reflect on a complex combination of epidemiological, socioeconomic, cultural, racial, and health-related factors4-6. vulnerable groups and individuals belonging to lower social strata from different societies are most strongly affected by the problem, which implicates in identifying these injuries as a manifestation of oral health inequalities6-8. the world health organization (who) defines the retention of a natural dentition of at least 20 teeth throughout life not requiring prosthetic replacement as the goal for oral health in adult populations9. this quantitative concept of functional dentition (fd) has been largely employed in dental research, although its appropriateness in assessing functionality has been challenged8,10-12. by questioning whether simply the number of teeth present is adequate to describe the functional status of dentitions, more comprehensive definitions of fd have emerged. according to gotfredsen and walls13 (2007), oral functionality must encompass aspects of masticatory efficiency and ability, appearance, psychological and social comfort, tactile perception, speech ability, and taste. thus, in accordance with more demanding notions of fd, new classificatory systems that consider teeth distribution, aesthetics, and occlusal units have been tested in different populations14-17. furthermore, the absence of a functional dentition is directly associated with the individual’s prosthodontic status, since it constitutes a therapeutic approach to compensate functional and aesthetic implications of tooth loss. we hypothesize that more complex and qualitative concepts of functional dentition present greater implications to public health and oral rehabilitation of indigenous populations than concepts that are essentially quantitative. studies assessing the oral health of indigenous populations have essentially focused on the epidemiological analysis of dental caries. this context of epidemiological invisibility undermines the construction of scientific evidence needed to expose and reduce situations of health inequalities18. thus, investigating functional dentition and prosthodontic status offers an original perspective to evaluate the extent and severity of edentulousness in vulnerable ethnic groups. the guarani e kaingang ethnic groups represent the second and third largest indigenous peoples from brazil, respectively. yet, data regarding their health status remains scant. the guarani people belong to the tupi linguistic family and is present in eight states, from pará to rio grande do sul. the total guarani population living in brazil is estimated at 85,255 individuals. the kaingang people belong to the jê linguistic family and occupy approximately 30 territories distributed over são 3 soares et al. paulo, paraná, santa catarina e rio grande do sul. the total kaingang population is estimated at 45,620 individuals. although guarani and kaingang peoples present different linguistic, cultural and historical trajectories, it is not unusual that these two groups co-inhabit the same territory19. this study aimed to analyze the prevalence of different definitions of functional dentition, prosthodontic status and associated factors in an indigenous population from brazil. materials and methods this exploratory, cross-sectional, population-based study was conducted from february and to august 2017, among the kaingang and guarani indigenous peoples living on the guarita indigenous reservation, rio grande do sul state. the research project was previously presented to the local indigenous leaders, who agreed to its realization. human ethics research approval was granted by the research ethics committee of the university of são paulo dentistry school, and the national research ethics committee (process n. 1.756.066). this paper was prepared in accordance with the strobe checklist for cross-sectional studies. the guarita indigenous reservation is located in the northwest region of the state of rio grande do sul, southern region of brazil, and is legally recognized as a land traditionally occupied by the kaingang and guarani peoples. the indigenous population is distributed in 12 villages belonging to three different municipalities (tenente portela, redentora, and erval seco). the infrastructure of all villages includes a public health care facility, electric energy network, and a public elementary school. drinking water is extracted from the local drilled wells and does not receive fluoride addition. access to all villages is carried out by land, and the distances to the indigenous health office vary between 2 and 40 kilometers. the total population of the guarita reservation is estimated by the special secretariat for indigenous health (sesai) at 5867 individuals. of those, the guarani correspond to a small group of approximately 200 individuals concentrated in the most remote village. the number of inhabitants in each community varies from 181 to 744 people. in order to obtain a representative sample, a census-based strategy was employed. thus, all indigenous adults from the guarita reservation who met the inclusion criteria were potentially eligible to be participants of the study. inclusion criteria were individuals self-identified as kaingang or guarani and aged 35-44 years (age group specified by who to assess the oral health status of adults). all households from all villages were visited and individuals within the selected age group were invited to take part in the study by signing a written consent form. in addition to their native languages, all participants were portuguese speakers. individuals unable to provide informed consent due to cognitive or physical impairment were excluded. the total number of adults aged 35-44 years is estimated by the sesai at 300 individuals. only 12 individuals actively refused to be included in the study. number of losses was expressively affected by a seasonal outflow of 4 soares et al. indigenous workers, mainly men, to another region of the state during the time of the study. oral examinations were performed by a single examiner (ghs) previously trained and calibrated, in open areas near the houses of the participants, under natural light, with the aid of sterilized clinical instruments (flat mouth mirror and classic-round periodontal probe) and individual protection equipment. conditions of dental crowns, periodontal status, and use and need of prosthodontics were evaluated following the methodology recommended by the who to oral health surveys20. dental crowns were classified according to the decayed, missing and filled (dfmt) index. periodontal status was assessed by the community periodontal index (cpi). use of prosthodontics was assessed based on the presence of any type of removable dental prosthesis at the time of examination. prosthodontic need was measured based on the extension, location, and number of edentulous areas in both dental arches. in case of prosthodontic users, dental prostheses were visually inspected, and their general condition was considered in the classification of prosthodontic need. therefore, participants might be prosthodontic users and still present a prosthodontic need. intra-examiner agreement was determined by re-examining 10 participants with an interval of 5 to 7 days between the two assessments. the cohen’s kappa coefficient showed substantial level of agreement (kappa=0.817). for the statistical analysis, only data regarding number of teeth present, and use and need of dental prosthesis were considered. the total number of teeth was determined by the sum of dental elements present in the mouth, except third molars. all missing teeth due to caries or other reasons were considered as dental losses. posterior occluding pairs (pops) were defined as the presence of two opposing teeth. sociodemographic data were collected using a structured questionnaire. participants were classified dichotomously in relation to the use of upper and lower dentures at the moment of the examination and need of some kind of upper and lower prosthesis. the outcomes related to the presence of functional dentition were classified according to four different concepts: 1. outcome 1 – who definition of functional dentition (fdwho): presence of 20 or more teeth9; 2. outcome 2 well distributed teeth (fdgroup2): presence of 10 or more teeth in each arch13; 3. outcome 3 aesthetic functional dentition (fdgroup3): presence of 12 anterior teeth; 4. outcome 4 functional dentition classified by aesthetics and occlusion (fdgroup4): presence of at least 10 teeth in each arch, all anterior teeth, three or four pops between premolars, and at least one pop between molars bilaterally14; this set of concepts was adapted from a classification system previously tested in the brazilian population by chalub et al.21. participants were classified according to sex, age, years of study, monthly household income, time of the last dental appointment, presence of periodontal pocket, 5 soares et al. and municipality. age was the only continuous variable analyzed. education was classified as up to 4 years of formal education or more than 4 years of education. regarding monthly household income, the amount corresponding to half of the brazilian minimum wage in 2017 (us$147.00) was adopted as a cutoff point. it was primarily determined in the brazilian currency and converted to the us dollars (mean exchange rate in august 2017: r$3.15 = us$1.00). time of last dental appointment was categorized either as within the previous 2 years, or in 3 or more years. considering the cultural, linguistic, and geographic barriers that kaingang and guarani peoples face to access regular dental treatment, we find reasonable to use a 2-year period to classify time of the last dental appointment, rather than adopting the normative recommendation of annual dental check-ups. periodontal status was classified as the presence of at least one periodontal pocket (probing depth ≥4mm), or its absence. municipality was classified as tenente portela or others. this division is due to the fact that the municipality of tenente portela provides a dental specialties center (ceo – centro de especialidades odontológicas) in the public health care network. on the other hand, individuals living in the villages located in redentora and erval seco are referred for specialized dental treatment in a municipality located 116 km away from the indigenous reservation. initially, descriptive analyses were carried out in order to describe the sample. prevalence of fd and respective 95% confidence interval were calculated for each of the four definitions used. association between the dependent and independent variables was verified through poisson regression with robust variance and presented through prevalence ratio (pr) and confidence interval (95% ci). for the multivariate analysis, fd definitions were incorporated separately into the poisson regression models created. associations were considered statistically significant at the 5% probability level (p≤0.05). analysis was performed using stata 12.0 software (statacorp, college station, texas, usa). results of the 109 individuals examined, only 2 participants were from the ethnic group guarani. mean age was 39.4 (sd 3.3). the majority of the participants was female, with a monthly household income of less than us$147.00, and living in the villages situated in the municipalities of redentora and erval seco (table 1). there were no participants fully edentulous, and only 3.7% presented complete dentition. mean number of present teeth was 18.6 (sd 6.1; min 2; max 28). nearly all dentures were inadequate due to cracks, fractures, or ill-fitting, requiring to be replaced. about 30% of the participants presented complete anterior dentition. prevalence rates of the different definitions of fd ranged from 48.62% to 11.93% (table 2). table 3 displays the univariate and multivariate analyses for the associations between prosthodontic status and independent variables. age presented a statistically significant association with the use of upper (pr = 1.09, ci = 1.03-1.16) and lower dentures (pr = 1.27, ci = 1.06-1.53). indigenous adults living in the villages located in tenente portela presented a significant difference regarding the need of upper dental prosthesis when compared to individuals from the other municipalities (rp = 0.74, 6 soares et al. ci = 0.55-0.99). need for a lower dental prosthesis was not associated with any independent variable. associations between the four fd outcomes and independent variables are shown in table 4. after adjustments for the other variables, the prevalence of fdwho in the final model was 67% (95% ci 1.04-2.69) greater among men in comparison to women. when considering fdgroup2, prevalence of fd was twofold greater among men (pr 2.06; 95% ci 1.16-3.65). indigenous adults who visited a dentist in the previous 2 years presented a significantly lower prevalence of fdgroup4 (pr 0.27; 95% ci 0.08-0.91). there was no statistically significant association between the presence of a complete anterior dentition (fdgroup3) and independent variables. table 1 – sample distribution according to sociodemographic and clinical characteristics. factor category n. % sex male 26 23.85 female 83 76.15 municipality tenente portela 34 31.19 others 75 68.81 education up to 4 years 52 47.71 more than 4 years 57 52.29 household monthly income up to us$147.00 46 52.27 more than us$147.00 42 47.73 last dental visit within last 2 years 80 73.39 3 or more years 29 26.61 periodontal pocket yes 35 32.11 no 74 67.89 table 2. indigenous distribution regarding the use and need of prosthodontics and prevalence of functional dentition according to four different definitions. outcome n. % 95% ci prothodontics upper denture user 50 45.87 36.63-55.41 lower denture user 5 4.59 1.89-10.68 upper denture need 82 75.23 66.13-82.53 lower denture need 100 91.74 84.75-95.69 functional dentition dfwho 53 48.62 39.25-58.09 dfgroup2 43 39.45 30.60-49.05 dfgroup3 33 30.28 22.28-39.67 dfgroup4 13 11.93 6.99-19.60 dfwho: at least 20 teeth; dfgroup2: at least 10 teeth in each arch; dfgroup3: all anterior teeth; dfgroup4: at least 10 teeth in each arch, all anterior teeth, 3 or 4 pop between premolars, and at least one pop between molars bilaterally. 7 soares et al. table 3. unadjusted and adjusted analysis for prosthodontic use and need and associated factors according to multiple poisson regression model with robust variance. outcome category univariate multivariate prun 95% ci p prad 95% ci p upper denture use sex male 0.43 0.20-0.90 0.026 0.45 0.20-1.01 0.052 municipality tenente portela 0.93 0.88-0.98 0.012 1.30 0.91-1.86 0.145 education up to 4 years 0.91 0.60-1.37 0.660 1.38 0.95-2.03 0.088 household monthly income up to us$147.00 1.14 0.75-1.72 0.518 1.07 0.74-1.56 0.712 last dental visit within last 2 years 1.15 0.70-1.88 0.583 1.11 0.71-1.75 0.648 periodontal pocket yes 1.29 0.86-1.95 0.212 1.31 0.91-1.90 0.145 age in years 1.07 1.01-1.14 0.023 1.09 1.03-1.16 0.002 lower denture use sex male 0.79 0.09-6.89 0.838 0.78 0.09-6.99 0.823 municipality tenente portela 1.02 0.82-1.29 0.803 0.62 0.07-5.49 0.665 education up to 4 years 0.22 0.02-1.99 0.182 4.41 0.52-37.02 0.172 household monthly income up to us$147.00 0.73 0.12-4.20 0.725 0.71 0.17-2.99 0.646 last dental visit within last 2 years 0.54 0.09-3.11 0.494 0.46 0.05-3.91 0.474 periodontal pocket yes 1.41 0.24-8.12 0.701 1.24 0.13-12.24 0.851 age in years 1.30 1.04-1.62 0.021 1.27 1.06-1.53 0.010 upper denture need sex male 0.96 0.73-1.25 0.778 0.92 0.71-1.21 0.566 municipality tenente portela 1.02 0.99-1.05 0.084 0.74 0.55-0.99 0.048 education up to 4 years 0.91 0.73-1.13 0.404 1.02 0.82-1.28 0.843 household monthly income up to us$147.00 0.81 0.64-1.03 0.091 0.79 0.62-1.00 0.056 last dental visit within last 2 years 1.12 0.85-1.47 0.401 1.17 0.86-1.58 0.316 periodontal pocket yes 0.93 0.72-1.18 0.546 1.07 0.85-1.36 0.556 age in years 1.01 0.97-1.04 0.489 1.01 0.97-1.04 0.595 lower denture need sex male 1.00 0.88-1.14 0.903 1.06 0.92-1.22 0.395 municipality tenente portela 0.99 0.97-1.00 0.414 1.03 0.91-1.17 0.630 education up to 4 years 1.02 0.91-1.15 0.627 0.98 0.86-1.13 0.813 household monthly income up to us$147.00 1.04 0.91-1.18 0.543 1.05 0.91-1.20 0.507 last dental visit within last 2 years 1.09 0.92-1.27 0.295 1.07 0.88-1.29 0.482 periodontal pocket yes 1.04 0.93-1.16 0.472 1.02 0.89-1.18 0.748 age in years 0.99 0.97-1.00 0.371 0.99 0.55-2.92 0.567 prun – unadjusted prevalence ratio prad – adjusted prevalence ratio ci – confidence interval 8 soares et al. table 4. unadjusted and adjusted analysis for functional dentition and associated factors according to multiple poisson regression model with robust variance. outcome category univariate multivariate prun 95% ci p prad 95% ci p fdwho sex male 1.64 1.14-2.35 0.007 1.67 1.04-2.69 0.035 municipality tenente portela 0.99 0.94-1.04 0.736 1.44 0.91-2.30 0.117 education up to 4 years 1.18 0.80-1.76 0.387 0.65 0.40-1.05 0.80 household monthly income up to us$147.00 0.89 0.56-1.42 0.643 0.98 0.62-1.56 0.939 last dental visit within last 2 years 0.92 0.60-1.40 0.693 1.02 0.60-1.74 0.926 periodontal pocket yes 0.76 0.48-1.20 0.242 0.79 0.48-1.31 0.365 age in years 0.95 0.89-1.01 0.128 0.95 0.88-1.02 0.134 fdgroup2 sex male 2.08 1.36-3.19 0.001 2.06 1.16-3.65 0.013 municipality tenente portela 0.97 0.92-1.04 0.511 1.60 0.90-2.85 0.109 education up to 4 years 1.26 0.78-2.04 0.331 0.62 0.34-1.12 0.116 household monthly income up to us$147.00 0.90 0.50-1.60 0.724 1.00 0.57-1.75 0.990 last dental visit within last 2 years 0.68 0.42-1.07 0.099 0.72 0.40-1.29 0.272 periodontal pocket yes 0.82 0.48-1.39 0.462 0.81 0.43-1.53 0.525 age in years 0.97 0.90-1.04 0.432 0.96 0.87-1.05 0.346 fdgroup3 sex male 1.59 0.89-2.84 0.112 1.56 0.70-3.47 0.275 municipality tenente portela 1.06 0.98-1.15 0.114 0.75 0.33-1.70 0.487 education up to 4 years 0.85 0.48-1.52 0.602 1.21 0.60-2.42 0.594 household monthly income up to us$147.00 0.73 0.36-1.44 0.369 0.80 0.40-1.61 0.538 last dental visit within last 2 years 0.83 0.45-1.54 0.560 0.82 0.39-1.70 0.588 periodontal pocket yes 0.68 0.34-1.35 0.267 0.58 0.25-1.34 0.204 age in years 0.96 0.88-1.06 0.497 0.98 0.89-1.07 0.747 fdgroup4 sex male 2.73 1.00-7.45 0.049 0.95 0.22-4.02 0.947 municipality tenente portela 1.11 0.97-1.26 0.107 0.78 0.16-3.77 0.761 education up to 4 years 1.06 0.38-2.97 0.905 1.18 0.31-4.43 0.806 household monthly income up to us$147.00 0.31 0.06-1.43 0.135 0.38 0.07-1.89 0.236 last dental visit within last 2 years 0.42 0.15-1.16 0.095 0.27 0.08-0.91 0.034 periodontal pocket yes 0.63 0.18-2.17 0.469 0.71 0.80-2.70 0.613 age in years 0.89 0.76-1.05 0.195 0.91 0.74-1.13 0.409 prun – unadjusted prevalence ratio prad – adjusted prevalence ratio ci – confidence interval 9 soares et al. discussion this study presents the first evaluation of fd in an indigenous population. the employment of such measures represents an important shift in the paradigm of dental research from a disease-focused approach to positive outcomes of health. for instance, it was presented herein the mean number of teeth present rather than number of losses, which is a modest yet significant modification. this change is particularly relevant for the scientific literature covering the oral health of indigenous peoples, which is typically restricted to the epidemiological analysis of dental caries findings of this study might be useful for clinicians when deciding which criteria of functionality are suitable to guide prosthodontic rehabilitation. for vulnerable populations with high prosthodontic need, this information could lead to more appropriate therapeutic decisions and reduction of oral health inequalities. for instance, employing purely quantitative criteria (e.g., number of missing teeth) for prosthodontic treatment in an indigenous population may reify existing inequalities by not identifying a significant proportion of individuals in need of oral rehabilitation. understanding the burden of tooth loss for the aesthetics and functionality in a population is essential in order to plan effective and adequate oral health programs. the employment of traditional sampling strategies to conduct research among hard-to-reach populations such as indigenous peoples imposes important methodological challenges. it is likely that the high dispersion of the studied population throughout the territory and the difficulty to locate eligible participants prevented the accomplishment of a greater sample size. missing data for household monthly income was substantial due to the number of participants who did not know or declined to provide this information (19,3%). the comparison of results between kaingang and guarani individuals was not possible due to the low number of participants of the latter. this study also presented limitations inherent to the cross-sectional design regarding the impossibility of establishing causal conclusions. cultural and social characteristics of indigenous peoples prevent the generalizability to other native communities and might be explored in further studies. subjective perception of functionality was not assessed. in this study, prevalence rates of fd ranged considerably according to the definition assessed. nevertheless, our findings contrast with national data, suggesting that disparities between indigenous and non-indigenous adults persist regardless of the definition investigated. data from the 2010 national oral health survey (nohs) for brazilians in the age group of 35-44 years present prevalence rates of fdwho, fdgroup2 and fdgroup4 of 77.9%; 72.9%; and 42,6%, respectively 21. frequencies of fd among vietnamese and chinese non-indigenous populations are similarly high and contrast with the findings reported in this study14,22. such differences are most likely resulting from health inequalities related to ethnicity and structural determinants of health. a study with indigenous peoples from the xingu park found that tooth losses accounted for 80% of the dmft score of mother in the 35-44 age bracket23. lack of access to fluoridated water seems to be an important explanatory factor to the fd prevalence rates observed in this study as it has been associated with lower frequencies of fdwho, fdgroup2, and fdgroup4 10,21. 10 soares et al. in the final model, age was statistically associated with use of upper and lower prosthodontics. greater prevalence of dental replacements among individuals of higher age was also described in a study with chinese adults22. although it is likely to be a result of the cumulative effect of dental caries and periodontal disease over time, such association in a well-delimited age group might indicate a trend of rapid and premature deterioration of the oral status. the lower need of upper dental prosthesis among indigenous adults from the villages of tenente portela (pr: 0.74; ci: 0.55-0.99) seems to be an effect of the dental specialties offered in the local secondary care system. indigenous residents of the other municipalities might experience financial restraints related to the costs of travelling to a distant location in order to receive the same level of health care. substantial differences in relation to sex were observed for fdwho and fdgroup2, with higher prevalence rates among men. similar results were previously described for fdgroup4 in brazilian adults 21. a study with sudanese tribes found that women were twice as likely to present less than 20 teeth when compared to men24. greater risks of dental caries, tooth loss, and oral health impairment have also been reported for indigenous women of brazil and australia25-27. this phenomenon may possibly be explained by the social positions attributed to gender as well as biological markers. researchers have incorporated periodontal status as an additional level in the classificatory system of functional dentition, even though it did not seem to significantly affect the results14,15. in this study, periodontal status was included as an independent variable and was not statistically associated with functional dentition nor prosthodontic status. additionally, income and years of education have been reported as associated factors for fd in brazilian adults10,21. it is likely that these associations have not been observed in this study due to the similar socioeconomic characteristics of the participants and the modest sample size. time of the last dental visit was the only factor associated with fdgroup4. indigenous adults who had not seen a dentist for an interval higher than 2 years presented better outcomes of fd in comparison to those who had a recent dental appointment. sudanese adults who visited a dentist more frequently presented significantly fewer teeth than those who did not visit a dentist at all24. on the other hand, higher prevalence rates of fdwho and fdgroup2 have been reported to brazilian individuals who attended a dental appointment in the previous 12 months21. dental care may affect tooth loss in opposing directions according to the type of service offered. restrictive and mutilating dental practices have historically promoted edentulousness as a health practice in the field of oral health in brazil7. dental health teams in both developed and developing countries seem to operate mainly by reinforcing the curative praxis of care, and often perform clinical practices that are biased by characteristics such as ethnicity and social class4,28. health inequities resulting from this complex interplay of social determinants tend to be expressed as extremely high demands for health treatments among indigenous groups. nearly all individuals observed in this study presented need for prosthodontic rehabilitation. the scant frequency of denture users and the general unsuitable conditions of the prostheses worn seem to aggravate the situation. meanwhile, approxi11 soares et al. mately 69% of brazilian adults in the same age group present need for some type of dental prosthesis29. a strategic goal set by the who stipulates that 96% of all adults aged 35-44 years should present a fd comprising at least 20 teeth30. while brazilian and vietnamese adults have not reached so far the goal for fd set by the who12,14, the situation of indigenous from the guarita reservation is far worse. in fact, the prevalence of fdwho observed in this study is similar to the frequency reported to the brazilian adult population in 2003 (54%)31. improvements in the oral health status of brazilian adults, with a significant reduction in the number of missing teeth, resulted in a 44% increase in the prevalence rate of fdwho in the 2010 nohs 15,32. longitudinal data regarding indigenous oral health is needed in order to assess whether the gap between indigenous and non-indigenous is narrowing or widening. nevertheless, the who’s classification of fd might not guarantee a sufficient number of occlusal contacts for adequate functionality due to the multiple possible configurations of the teeth14. chalub et al.21 (2016) questions whether aiming at a fd based purely on quantitative criteria without considering teeth distribution and condition might lead to increasing oral health inequities. conversely, the classification proposed by nguyen et al.14 (2011) (here referred as fdgroup4) offers a qualitative system that is more compatible with functional and perceived demands21,33. the low prevalence of fdgroup3 indicates the magnitude of the aesthetic impairment affecting this population. exploring subjective perceptions related to the low frequency of intact anterior dentition might provide a better understanding of its implications to social life. despite limitations of the fdwho definition, it may still represent an important instrument for comparison across populations and for the establishment of health goals. on the other hand, more demanding concepts such as fdgroup4 might be appropriate to both planning of individualized treatments and formulation of public policies34. the findings of this study suggest that tooth loss is an eloquent marker of social and 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[sb brazil 2010: national research on oral health: main results]. brasília: ministry of health; 2012. 116p. portuguese. 30. world health organization iafdr. 4th world congress on preventive dentistry, “trends in prevention — promotion of oral health within general health care. possibilities and limitations in preventive dentistry”. sweden: university of umed; 1993. 31. ministry of health of brazil. [sb brasil 2003 project: oral health conditions of brazilian population 2002–2003: principal results]. brasília: ministry of health; 2004. portuguese. 32. nascimento s, frazão p, bousquat a, antunes jl. [dental health in brazilian adults between 1986 and 2010]. rev saude publica. 2013 dec;47(suppl 3):69-77. portuguese. 33. nguyen tc, witter dj, bronkhorst em, gerritsen ae, creugers nh. chewing ability and dental functional status. int j prosthodont. 2011 sep-oct;24(5):428-36. 34. chalub llfh, ferreira rc, vargas amd. influence of functional dentition on satisfaction with oral health and impacts on daily performance among brazilian adults: a population-based cross-sectional study. bmc oral health. 2017 jul 11;17(1):112. doi: 10.1186/s12903-017-0402-5. 1http://dx.doi.org/10.20396/bjos.v16i0.8650491 volume 16 2017 e17026 original article 1 school of medicine, universidade federal do maranhão – ufma, são luís, brazil; 2 d.d.s., department of oral and maxillofacial surgery, university hospital, universidade federal do maranhão – ufma, são luís, brazil; 3 d.d.s., school of dentistry, universidade federal do maranhão – ufma, são luís, brazil; 4 d.d.s., m.s., ph.d., department of dentistry ii, universidade federal do maranhão – ufma, são luís, brazil. corresponding author: maria carmen fontoura nogueira da cruz, d.d.s., m.s., ph..d., associate professor department of dentistry ii, universidade federal do maranhão – ufma. av. dos portugueses, 1966, bacanga, zip code: 65085-580, são luís, maranhão, brazil. phone number: +5598 32728575 e-mail: ma.carmen@uol.com.br received: march 13, 2017 accepted: august 25, 2017 factors affecting hospital discharge in maxillofacial trauma patients: a retrospective study leonardo victor galvão-moreira1, andre luis costa cantanhede2, aluisio cruz de sousa neto3, maria carmen fontoura nogueira da cruz4 aim: to determine factors that may affect the time of discharge from hospital in patients who underwent maxillofacial trauma. methods: the sample included 115 patients seen at a public hospital in brazil, to whom surgical maxillofacial treatment was delivered. data were obtained from patients’ medical records and then followed by a statistical analysis using a 5% significance level. results: the location of fractures and other clinical features such as the presence of edema and ecchymosis were found to be significantly associated with increased time of discharge from hospital (p < 0.05). when data were modeled using a gml approach, male gender was associated with a lower likelihood of prolonged hospital discharge (or = 0.84; 95% ci: 0.72–0.98; p < 0.05), while the presence of edema was associated with greater time of hospital discharge (or = 1.30; 95% ci: 1.14–1.49; p < 0.001). no significant association with age, etiology of trauma, and number of fractures was observed (p > 0.05). conclusion: female gender was associated with greater time of hospital discharge, and further concerns should be addressed to the management of lesions following maxillofacial trauma surgical interventions. keywords: facial injuries. medical records. outcome assessment (health care). 2 galvão-moreira et al. introduction maxillofacial fractures remain a major component of all traumas, representing a challenge for public health services worldwide due to the high incidence and significant financial cost1-4. considering that these fractures may result in functional or cosmetic deformities, maxillofacial surgery aims at consistently restoring patients back to their pre-injury form and function5-7. in both developed and developing countries, despite seat belt and alcohol legislation, maxillofacial injuries are likely associated with traffic accidents, being the main cause of facial trauma. other etiological factors often described include physical agressions, falls, interpersonal violence, sports or work-related activities, and animal-related accidents3,7-16. although maxillofacial trauma is thought to be more prevalent in younger age groups, its incidence in the elderly has increased, likely due to an increase in life expectancy and active lifestyle among this population12. factors such as geographic region, population density, socioeconomic status, education and culture affect the results of epidemiological investigations regarding the incidence, etiology, clinical presentation and length of stay in hospital17. importantly, maxillofacial trauma exhibit extremely variable resulting injuries, often promoting severe morbidities, deformities and functional limitations, whose treatments involve long periods of removal of patients from their professional activities6-7. nevertheless, data regarding the time of discharge from hospital of patients who have undergone maxillofacial trauma remains poorly investigated. the significance of epidemiological research addressing maxillofacial trauma is on the search for strategies to improve the quality of care, prevention and treatment protocols, and identification of injuries’ patterns1,3,14. therefore, since there is a shortage of studies in this regard, we aimed at investigating factors that could affect the time of hospital discharge of maxillofacial trauma patients at a public hospital in northeast brazil. methods sample selection clinical records of 100 male and 15 female patients (n = 115), aged 11-59 years (mean: 30 years), were obtained from the department of medical archives at the university hospital of the federal university of maranhão (são luís, brazil). a retrospective study was carried out by collecting data from all available medical records of maxillofacial trauma patients that underwent surgical treatment in the period 2009-2013. the present study was approved by the local research ethics committee (protocol #721873/14; brazil). data collection the following data collected were included and transcribed to a clinical record: gender, age, time when surgery was performed, etiology of trauma; location of the fracture (upper third, middle third and lower third), fractured bone(s) involved, signs and symptoms reported, and time of hospital discharge. the etiology of trauma comprised the following causes: car, motorcycle or road accidents; physical aggression; projectile injury; others. 3 galvão-moreira et al. statistical analyses data regarding gender, age, etiology of trauma, location/number of fractures, clinical features, and time of hospital discharge were expressed as frequencies, and the pearson’s chi-squared test was then applied to investigate any associations between these variables. the shapiro-wilk test was used to compare numerical variables between the groups and was followed by the non-parametric mann-whitney test. when comparing more than one group, the kruskal-wallis test was utilized. in addition, spearman’s correlation was used to investigate correlations among numerical variables. the level of significance considered for all statistical analyses was 5%. finally, we developed a generalized linear model (glm) using a gamma with log link function to test the effects of several variables on the time of hospital discharge. data obtained in this study were analyzed using the statistical package for social sciences – ibm spss statistics 23.0 (spss inc., il, usa). results in the present investigation, a predominance of male patients (86.95%) was observed. motorcycle accidents, followed by car accidents were found to be the most prevalent causes of maxillofacial trauma. in regard to the fractured sites, the lower facial third was the most affected one, presenting with 100 cases of mandibular fractures, 54 of which linked to more than one injury in the same bone. it was followed by the middle third, where 58 fractures were observed, whereas the upper third was the less affected site (data not shown). in terms of the treatment delivered, all patients underwent surgical procedures, 93.92% of whom waited up to one week for the surgery, while 6.08% waited up to one month for the definitive surgical procedure. most of patients (79.13%) who underwent general anesthesia and surgery procedures were found to have a 1-2 day time of discharge. nevertheless, there were patients who stayed longer in the hospital. the longest post-surgical stay period in hospital observed was 8 days, but there was no association between the number of fractures and time of hospital discharge (fig. 1, p > 0.05). four 7-8 days 5-6 days 3-4 days 1-2 days 0 10 20 40 6030 50 70 three two one figure 1. distribution of fractures according to the number of fractures and the time of discharge from hospital (p > 0.05, according to the pearson’s chi-squared test). 4 galvão-moreira et al. the associations between the age, gender and clinical features of maxillofacial trauma patients are available in table 1. none of the clinical features evaluated was found to be associated with either the gender or the age group (p > 0.05). additionally, there was no statistically significant association between the etiology of trauma and either the gender or the age group (p > 0.05; data not shown). interestingly, several variables evaluated related to the clinical features of patients and location of fractures had some level of association between each other (table 2, p < 0.05; p < 0.001). yet, the gender and number of fractures or time of discharge from hospital were not found to be associated (table 3, p > 0.05). furthermore, both the age group (table 4) and the etiology of trauma (table 5) did not demonstrated significant association with either number of fractures or time of discharge (p > 0.05). table 6 illustrates associations between several signs and symptoms demonstrated by the patients with both number of fractures and time of hospital discharge. a potential correlation between age, number of fractures and time of discharge was tested, but no statistically significant correlation was shown (p > 0.05; data not shown). lastly, in table 7, a glm approach showed that male gender was associated with a lower likelihood of prolonged hospital discharge (p < 0.05), and the presence of edema was associated with greater time of hospital discharge (p < 0.001). discussion in the present study, there was higher prevalence of men affected by maxillofacial trauma. this is in agreement with prior research that shows male patients within second and third decades of life to be the most affected by maxillofacial injuries, mainly table 1. association between gender, age group and clinical features in maxillofacial trauma patients. variable gender (n) or (95% ci) p value age group (n) p valuemale female 10-19 20-29 30-39 40-49 50-59 facial asymmetry 72 11 0.935 (0.27–3.18) 0.91 7 34 24 12 6 0.53 crepitation 10 2 0.722 (0.14–3.67) 0.69 1 7 3 1 0.63 paresthesia 21 2 1.728 (0.36–8.26) 0.49 1 10 7 2 3 0.37 malocclusion 71 11 0.89 (0.26–3.02) 0.85 11 35 24 10 2 0.06 limited movement 79 11 1.368 (0.39–4.73) 0.62 9 35 30 11 5 0.45 ecchymosis 14 2 1.058 (0.21–5.20) 0.94 1 7 4 3 1 0.91 edema 26 3 1.405 (0.36–5.37) 0.62 1 13 10 3 2 0.53 upper third fractures 4 (––) 0.43 2 1 1 0.45 middle third fractures 38 9 0.409 (0.13–1.23) 0.10 4 18 14 8 3 0.89 lower third fractures 66 8 1.699 (0.56–5.08) 0.34 9 34 20 8 3 0.20 ci: confidence interval; or: odds ratio; pearson’s chi-squared test. 5 galvão-moreira et al. ta bl e 2. a ss oc ia tio n an d ris k am on g cl in ic al fe at ur es in m ax ill of ac ia l t ra um a pa tie nt s, p re se nt ed w ith o r (9 5% c i) . fa ci al as ym m et ry c re pi ta tio n p ar es th es ia m al oc cl us io n li m ite d m ov em en t ec ch ym os is ed em a u pp er th ird m id dl e th ir d c re pi ta tio n 0. 74 (0 .2 0– 2. 67 ) p ar es th es ia 1. 49 (0 .5 0– 4. 43 ) 5. 05 * (1 .4 5– 17 .5 7) m al oc cl us io n 0. 36 (0 .1 2– 1. 04 ) 4. 95 (0 .6 1– 40 .0 5) 0. 90 (0 .3 3– 2. 44 ) li m ite d m ov em en t 2. 58 * (1 .0 2– 6. 53 ) 0. 81 (0 .2 0– 3. 26 ) 0. 55 (0 .1 9– 1. 55 ) 1. 22 (0 .4 6– 3. 19 ) ec ch ym os is 0. 82 (0 .2 6– 2. 59 ) 2. 30 (0 .5 5– 9. 64 ) 2. 89 (0 .9 2– 9. 04 ) 0. 45 (0 .1 5– 1. 35 ) 2. 11 (0 .4 4– 10 .0 1) ed em a 1. 28 (0 .4 8– 3. 39 ) 1. 56 (0 .4 3– 5. 62 ) 0. 78 (0 .2 6– 2. 35 ) 0. 20 ** (0 .0 8– 0. 49 ) 2. 97 (0 .8 2– 10 .8 1) 5. 07 * (1 .6 8– 15 .3 0) u pp er th ird 1. 16 (0 .1 1– 11 .6 0) (– – ) 4. 28 (0 .5 7– 32 .2 0) 0. 38 (0 .5 2– 2. 87 ) 0. 26 (0 .0 3– 1. 95 ) 2. 13 (0 .2 0– 21 .8 7) 3. 11 (0 .4 1– 23 .1 5) m id dl e th ird 1. 21 (0 .5 2– 2. 80 ) 0. 11 * (0 .0 10. 90 ) 1. 42 (0 .5 6– 3. 57 ) 0. 38 * (0 .1 6– 0. 87 ) 0. 36 * (0 .1 4– 0. 91 ) 2. 79 (0 .9 3– 8. 31 ) 1. 02 (0 .4 3– 2. 41 ) 4. 56 (0 .4 6– 45 .3 3) lo w er th ird 0. 92 (0 .3 9– 2. 17 ) 6. 98 * (0 .8 6– 56 .1 9) 0. 83 (0 .3 2– 2. 12 ) 3. 07 * (1 .5 8– 8. 62 ) 2. 97 * (1 .1 9– 7. 37 ) 0. 50 (0 .1 7– 1. 45 ) 0. 87 (0 .3 6– 2. 09 ) (– – ) 0. 00 3* * (0 – 0. 02 ) c i: co nfi de nc e in te rv al ; o r : o dd s ra tio ; * p < 0 .0 5, * * p < 0 .0 01 , a cc or di ng to th e p ea rs on ’s c hi -s qu ar ed te st . 6 galvão-moreira et al. due to higher risk-taking behavior1,3,8,14,17. our findings also showed motorcycles accidents to be the most prevalent etiology of maxillofacial injuries. motorcycle is the mean of transportation used by the majority of population living in northeast of brazil, and these statistics corroborated with other studies conducted in brazilian cities3,16. in a cross-sectional study, leles et al.3 (2010) reported that the commonest etiology of facial fractures was related to motorcycle accidents, in which 76% of victims were using helmets; however, 60.5% of them were not full-face helmets. it is important to stress out that most of motorcycles users in low-income communities in northeast brazil are not used to wear helmets, thereby leading them to experience severe maxillofacial injuries. table 3. difference of medians of the number of fractures and time of hospital discharge between male and female maxillofacial trauma patients. variable gender p value male female n mean median sd n mean median sd number of fractures 100 1.4 1 0.58 15 1.4 1 0.5 0.83 time of discharge 2.37 2 0.9 2.8 2 1.93 0.15 sd: standard deviation; mann-whitney test. table 5. difference of medians of the number of fractures and time of hospital discharge according to the etiology of trauma in maxillofacial trauma patients. etiology of trauma n number of fractures time of discharge median (95% ci) p value median (95% ci) p value accidental fall 14 1 (1.07–2.06) 0.81 2 (1.91– .22) 0.23 car accident 24 1 (1.2–1.7) 2 (2.13–3.61) motorcycle accident 48 1 (1.16–1.46) 2 (2.11–2.68) road accident 7 1 (0.72–2.08) 2 (---) physical aggression 14 1 (1.07–1.64) 2 (1.83–2.45) projectile injury 3 1 (-0.1–2.76) 2 (-0.2–5.53) other 5 2 (1.07–2.06) (2 1.84–3.29) ci: confidence interval; or: odds ratio; kruskal-wallis test. table 4. difference of medians of the number of fractures and time of hospital discharge according to the age group in maxillofacial trauma patients. variable age group p value 10-19 20-29 30-39 40-49 50-59 n median (95% ci) n median (95% ci) n median (95% ci) n median (95% ci) n median (95% ci) number of fractures 13 1 (1.14 – 1.77) 45 1 (1.26 – 1.66) 34 1 (1.13 – 1.51) 16 1 (1.1 – 1.64) 7 1 (0.83 – 1.73) 0.80 time of discharge 2 (1.53 – 3.54) 2 (2.04 – 2.44) 2 (2.09 – 2.84) 2 (1.91 – 3.08) 2 (1.22 – 4.77) 0.83 ci: confidence interval; or: odds ratio; kruskal-wallis test. 7 galvão-moreira et al. table 6. difference of medians of the number of fractures and time of hospital discharge according to the clinical features in maxillofacial trauma patients. clinical feature status n number of fractures p value time of discharge p value median (95% ci) median (95% ci) facial asymmetry yes 83 1 (1.28–1.53) 0.79 2 (2.23–2.77) 0.59 no 32 1 (1.17–1.57) 2 (2.04–2.39) crepitation yes 12 1 (0.99–1.84) 0.93 2 (1.79–2.53) 0.28 no 103 1 (1.28–1.5) 2 (2.23–2.67) paresthesia yes 23 1 (1.1–1.59) 0.55 2 (2.13–2.91) 0.2 no 92 1 (1.29–1.53) 2 (2.16–2.63) malocclusion yes 82 1 (1.26–1.53) 0.79 2 (2.14–2.51) 0.45 no 33 1 (1.21–1.56) 2 (2.12–3.2) limited movement yes 90 1 (1.3–1.55) 0.28 2 (2.22–2.72) 0.78 no 25 1 (1.09–1.46) 2 (2.02–2.45) ecchymosis yes 16 1 (1.1–1.64) 1.0 2 (2.10–4.02) 0.04* no 99 1 (1.28–1.52) 2 (2.14–2.5) edema yes 29 2 (1.36–1.87) 0.01* 2 (2.34–3.65) 0.01* no 86 1 (1.21–1.43) 2 (2.08–2.37) upper third fractures yes 4 2 (0.95–2.54) 0.12 3.5 (2.22–5.27) <0.001** no 111 1 (1.27–1.49) 2 (2.17–2.57) middle third fractures yes 47 1 (1.19–1.48) 0.53 2 (2.28–3.11) 0.01* no 68 1 (1.28–1.59) 2 (2.05–2.41) lower third fractures yes 74 1 (1.31–1.60) 0.18 2 (2.04–2.36) 0.02* no 41 1 (1.14–1.43) 2 (2.35–3.30) ci: confidence interval; or: odds ratio; * p < 0.05, ** p < 0.001, according to mann-whitney test. table 7. generalized linear model showing predictors for the time of hospital discharge, which was used as a dependent variable. variable or (95% ci) p value male 0.84 (0.72–0.98) 0.02* female 1 asymmetry 1.06 (0.95–1.20) 0.25 crepitation 0.88 (0.73–1.05) 0.18 paresthesia 1.06 (0.92–1.21) 0.38 malocclusion 1.07 (0.94–1.22) 0.25 ecchymosis 1.11 (0.94–1.30) 0.20 limitation 1.08 (0.95–1.23) 0.23 edema 1.30 (1.14–1.49) <0.001* upper third fractures 1.34 (0.99–1.81) 0.05 middle third fractures 0.95 (0.76–1.18) 0.65 lower third fractures 0.82 (0.65–1.04) 0.11 age 1 (0.99–1.00) 0.12 number of fractures 0.93 (0.85–1.02) 0.17 ci: confidence interval; or: odds ratio; *p < 0.05; *p < 0.001. 8 galvão-moreira et al. regarding fractures’ patterns, the lower third (mandible) accounted for most frequent location of fractures in our study, specifically with high incidence of angle and body sites isolated or associated. the major clinical features related to these injuries were crepitation, malocclusion and limited movement. in a greek population study, it has been found mandible fractures to be more prevalent (56%), mostly related to motorcycles vehicles crashes, though condylar and symphysis/parasymphysis were the most prevalent sites of injuries8. importantly, the fact that most patients surgically treated for maxillofacial trauma underwent general anesthesia might directly affect the time of treatment and hospital length of stay. in a 5-year study with 394 patients, van hout et al.14 (2013) found a mean of discharge time within one to four days; however, when other injuries were present it was nearer 22 days. additionally, al-dajani et al.1 (2015) reported a mean length of hospital stay lasting 2 to 7 days, addressing longer stay periods to older patients (>7 days) and shorter to children (< 2 days). kostakis et al.8 (2012) accounted longer mean periods of hospital stay of 12.1 days (work-related accidents), 11.7 days (motorcycles accidents), and 7.3 days for assaults. these authors mention that besides serious concomitant injuries associated with maxillofacial trauma, patients experienced prolonged waiting between hospital admission and definitive treatment due to lack of infrastructure8. in the present study, the length of hospitalization was similar for all age groups (mean: 2 days). this relatively short period that patients victims of maxillofacial fractures stayed in hospital might reflect the implementation and availability of rigid internal devices and trained residency programs. in fact, adequate use of plates, miniplates and screws can greatly benefit patients with a proper maintenance of reduced bones segments, eliminating longer maxilo-mandibular blockage periods, providing thus better esthetics outcomes and early functional return16,18. other comorbidities aspects that could increase the length of stay in hospital, such as leg fractures or systemic complications were not analyzed in this study. these aspects could be correlated with maxillofacial fractures by increasing the length of patients’ hospital discharge. in conclusion, we found gender and some clinical features such as the location of trauma and the presence of edema and ecchymosis to be associated with increased time of discharge from hospital. conversely, no association was observed between age, etiology of trauma, number of fractures, and time of hospital discharge in maxillofacial trauma patients. hence, we emphasize that female patients that underwent maxillofacial trauma should receive special care as well as further attention should be given to the management of lesions towards reducing the time of hospital discharge in the population. acknowledgements the authors would like to thank the state government agency for support to research and scientific and technological development, maranhão, brazil (fapema) for the research grant provided to a.c.s. neto (bic-02691/13). 9 galvão-moreira et al. references 1. al-dajani m, quinonez c, macpherson ak, clokie c, azarpazhooh a. epidemiology of maxillofacial injuries in ontario, canada. j oral maxillofac surg. 2015 apr;73(4):693.e1-9. doi: 10.1016/j.joms.2014.12.001. 2. salentijn eg, collin jd, boffano p, forouzanfar t. a ten year analysis of the traumatic maxillofacial and brain injury patient in amsterdam: complications and treatment. j craniomaxillofac surg. 2014 dec;42(8):1717-22. doi: 10.1016/j.jcms.2014.06.005. 3. leles jlr, santos ej, jorge fd, silva et, leles cr. risk factors for maxillofacial injuries in a 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elderly patients with maxillofacial trauma: study of mandibular condyle fractures. dent traumatol. 2015 feb;31(1):73-6. doi: 10.1111/edt.12129. 13. lin s, levin l, goldman s, peled m. dento-alveolar and maxillofacial injuries: a 5-year multi-center study. part 1: general vs facial and trauma dental. dent traumatol. 2008 feb;24(1):53-5. doi: 10.1111/j.1600-9657.2006.00510.x. 14. van hout wm, van cann em, abbink jh, koole r. an epidemiological study of maxillofacial fractures requiring surgical treatment at a tertiary trauma centre between 2005 and 2010. br j oral maxillofac surg. 2013 jul;51(5):416-20. doi: 10.1016/j.bjoms.2012.11.002. 15. yamamoto k, matsusue y, murakami k, horita s, sugiura t, kirita t. maxillofacial fractures in older patients. j oral maxillofac surg. 2011 aug;69(8):2204-10. doi: 10.1016/j.joms.2011.02.115. 16. brasileiro bf, passeri la. epidemiological analysis of maxillofacial fractures in brazil: a 5-year prospective study. oral surg oral med oral pathol oral radiol endod. 2006 jul;102(1):28-34. http://www.ncbi.nlm.nih.gov/pubmed?term=nogami%20s%5bauthor%5d&cauthor=true&cauthor_uid=25233910 http://www.ncbi.nlm.nih.gov/pubmed?term=yamauchi%20k%5bauthor%5d&cauthor=true&cauthor_uid=25233910 http://www.ncbi.nlm.nih.gov/pubmed?term=yamashita%20t%5bauthor%5d&cauthor=true&cauthor_uid=25233910 http://www.ncbi.nlm.nih.gov/pubmed?term=kataoka%20y%5bauthor%5d&cauthor=true&cauthor_uid=25233910 http://www.ncbi.nlm.nih.gov/pubmed?term=hirayama%20b%5bauthor%5d&cauthor=true&cauthor_uid=25233910 http://www.ncbi.nlm.nih.gov/pubmed?term=tanaka%20k%5bauthor%5d&cauthor=true&cauthor_uid=25233910 http://www.ncbi.nlm.nih.gov/pubmed?term=van%20hout%20wm%5bauthor%5d&cauthor=true&cauthor_uid=23218202 http://www.ncbi.nlm.nih.gov/pubmed?term=van%20cann%20em%5bauthor%5d&cauthor=true&cauthor_uid=23218202 http://www.ncbi.nlm.nih.gov/pubmed?term=abbink%20jh%5bauthor%5d&cauthor=true&cauthor_uid=23218202 http://www.ncbi.nlm.nih.gov/pubmed?term=koole%20r%5bauthor%5d&cauthor=true&cauthor_uid=23218202 10 galvão-moreira et al. 17. mijiti a, ling w, tuerdi m, maimaiti a, tuerxun j, tao yz, et al. epidemiological analysis of maxillofacial fractures treated at a university hospital, xinjian, china: a 5-year retrospective study. j craniomaxillofac surg. 2014 apr;42(3):227-33. doi: 10.1016/j.jcms.2013.05.005 18. jensen j, pedersen-sindet s, christensen l. rigid fixation in reconstruction of craniofacial fractures. j oral maxillofac surg. 1992 jun;50(6):550-4. braz j oral sci. 15(4):252-257 accuracy of partially edentulous arch impressions obtained from different alginates and storage times lucas de oliveira tomaselli1, rafael pino vitti2, marcus vinicius loureiro bertolo3, gabriel abuna3, william cunha brandt4, mário alexandre coelho sinhoreti5 1dds, msc student, university of campinas, piracicaba dental school, department of restorative dentistry, piracicaba, sp, brazil 2dds, msc, phd, associate professor, university of taubaté, school of dentistry, department of prosthodontics, taubaté, sp, brazil 3dds, msc, phd student, university of campinas, piracicaba dental school, department of restorative dentistry, piracicaba, sp, brazil 4dds, msc, phd, associate professor, university of santo amaro, school of dentistry, department of implantology, são paulo, sp, brazil 5dds, msc, phd, full professor, university of campinas, piracicaba dental school, department of restorative dentistry, piracicaba, sp, brazil correspondence to: marcus vinicius loureiro bertolo university of campinas avenida limeira, 901 piracicaba – sp cep: 13414-903 phone/fax: +55 21 99856-9898 e-mail: marcusbertolo@hotmail.com abstract purposes: this study aimed to assess the dimensional accuracy of five commercial alginates verified in stone casts. methods: each alginate impression (cavex colorchange, cavex holland bv; jeltrate plus, dentsply; hydrogum, zhermack; kromopan 100, lascod; ezact kromm, vigodent) was performed on partially edentulous standard stainless steel mandibular arch cast with reference points on teeth 33, 43, 37 and 47. on the stainless steel cast, the anteroposterior (33-37 and 43-47) and transverse (33-43 and 37-47) distances were measured in a stereomicroscope at 30x magnification and 0.5 μm accuracy. the distances between these points were measured three times, obtaining an average, which was analyzed statistically and compared with the distances obtained from the stone casts. for each alginate the casts were poured gypsum (n = 5) immediately and after a period of 1, 2, 3 and 5 days of the impression procedure. results: the dimensional accuracy values of stone and stainless steel casts were analyzed statistically by two-way anova and tukey's test (α=0.05). the results showed significant differences between the alginates; however, no differences in dimensional accuracy were found among the different storage times of alginate impression. conclusions: it can be concluded that the alginate impressions can be stored for up to 5 days. keywords: dimensional measurement accuracy; dental impression material; mandibular prosthesis. introduction impression materials are used in dentistry to create accurate casts of buccal tissues for pre-treatment planning, fabrication of prostheses, and post-treatment records. they can be classified in inelastic or elastic materials, which return to their initial form after be removed from mouth due to elastic recovery, and also according to chemical reaction (irreversible or reversible). the alginate (irreversible hydrocolloid) is an impression material that appeared in dentistry on ‘40s, when the agar supplies (reversible hydrocolloid) became insufficient due to the world war ii. the impression materials must copy the anatomy of buccal tissues and remain dimensionally stable. alginate is an impression material widely used in in dentistry and its popularity is due to the easy manipulation, low cost, and its hydrophilicity1. satisfactory results are obtained with stone casts made by alginate impression and used for the preparation of orthodontic studies, making plackers mouth guard, and dental received for publication: november 23, 2016 accepted: may 16, 2017 original article braz j oral sci. october | december 2016 volume 15, number 4 http://dx.doi.org/10.20396/bjos.v15i4.8650031 253 prosthesis2,3. alginates are commonly used as powder and water. the powder contains, in weight, diatomaceous earth (60%) and oxide zinc (4%) as filler particles, calcium sulfate (16%) as reactor, sodium or potassium alginates (15%) as soluble alginate, potassium titanium fluoride (3%) as accelerator (gypsum hardener), and sodium phosphate (2%) as retarder1. the good dimensional accuracy is required for a reliable copy of the molded anatomy. alginates are commercially available at pre-dosed or customized packages, bulk, bags, pots, sachets, daypacks or private label4. the gelation process is a sol-gel reaction, where at the contact with water, the sodium alginate (or potassium alginate) reacts with calcium sulfate, resulting in a molecular network of crosslinking (alginate gel)5. the hydrocolloids are 85% water approximately, then they are submitted to distortion due to the expansion, that occurs with humidity absorption (imbibition) or shrinkage due to water loss by syneresis or evaporation6,7. these processes (expansion/shrinkage) are mainly related to the storage medium of the alginate impression. another factors like the kind of the tray used at the impression, the material handling, disinfection process, temperature variance, quantity and quality of the chemical components present at alginate powder, also could interfere at the stability and dimensional accuracy1. some studies8-12 recommend that alginate impressions must be poured as fastest as possible, avoiding the excessive air exposure (syneresis and evaporation). if the pouring procedure is not possible, the most recommended step is to keep the impression at 100% humidity environment in order to preserve the hydric balance within the material13. following the manufacturer's recommendations, the casts could be poured until 12h after the alginate gelation. shaba et al.10 (2007) suggested that this time must be reduced until 30 minutes contrary to other authors. sedda et al.9 (2008) concluded that alginate impressions remains dimensionally stables after 24h, one of this was hydrogum (zhermack) which was the only one that remains dimensionally acceptable to be poured after 72h. some alginates commercially available may be stored up to five days without jeopardizing the stability and dimensional accuracy according the manufacturer's recommendations, but this information is questionable, since there is no consensus about it8-12. moreover, there is no study with a cast representing a clinical situation to evaluate dimensional stability and/or accuracy. due to these properties, the storage recommendations of this material, it appears the question if it is possible to retain the original dimensions after 5 days without interfere with the dimensional accuracy. the aim of this study was to evaluate the dimensional accuracy of five commercial alginates on casts poured at different times of a partially edentulous mandibular arch. the hypothesis is that the stone casts obtained immediately after the impression presents less dimensional accuracy alteration. materials and methods five commercial alginates impression materials were used: cavex colorchange-ch (cavex holland bv, haarlem, north holland, netherlands), jeltrate plus-jt (dentsply, petrópolis, rj, brazil), hydrogum-hy (zhermack, badia polesine, rovigo, ltaly), kromopan 100-kr (lascod, florence, tuscany, italy) and ezact kromm-ez (vigodent, rio de janeiro, rj, brazil). in order to simulate a clinical condition to obtain the impressions, all procedures were realized on a standard stainless steel cast representing a partially edentulous mandibular arch with reference points14 on the left and right canines (33 and 43), and left and right second molars (37 and 47). the anteroposterior (3337; 35.22 mm and 43-47; 35.06 mm) and transverse (33-43; 25.06 mm, and 37-47; 50.01 mm) distances were measured by olympus measuring microscope stm (olympus optical co., tokyo, kanto, japan) at 30x magnification and 0.5 μm accuracy. the ocular lens were adjusted to the inner vertical and horizontal border of the reference point and compared to the opposite reference point, at a linear trajectory15. every distance was measured three times by a single calibrated operator, obtaining an average, which was compared to the stone casts. metal stock trays i-3 (tecnodent, bologna, emilia-romagna, italy) were used for the impression procedure. the alginates were mixed following the manufacturer's instructions at a temperature and humidity controlled environment (23°c ± 2°c and 50% ± 10%)16, controlling the factors that lead to dimensional alteration. after the impression materials handling, they were placed into tray to impression of the stainless steel cast. the set tray/impression material was positioned on the stainless steel cast from posterior to anterior direction. after the gelation, the tray was carefully removed with a single and vertical movement. metallic gadgets were adapted to the trays, which were joined to pneumatic equipment, to standardize the detachment movement of the tray from the stainless steel cast in order to avoid distortions in the mold16. dental stone type iv (durone, dentsply, petrópolis, rj, brazil) was used in a water/powder ratio of 28.5 ml/150 g for stone cast pouring. the stone casts were poured immediately and 1, 3 or 5 days after impression. the stored groups were maintained in a humidifier with 100% relative humidity and room temperature (23°c ± 2°c). five models were obtained (n = 5) for each commercial alginate and storage condition13,16-20. after dental stone set, the stone casts were evaluated for the presence of pores or other defects, being discarded those who present any defect in one of the reference points. as in the stainless steel cast, three readings were performed by a single calibrated operator at each of the four distances between the dental elements (33-43, 33-37, 43-47 and 37-47), obtaining the average of each distance. all readings occurred in the same environment with the same temperature and humidity. the original values of dimensional change were converted into percentage, considering as original the measures stainless steel cast. if negative, indicate that there was a decrease of distances (shrinkage) and, if positive, indicate an increase (expansion). all data were submitted to normality test (kolmogorov-smirnov) and analyzed statistically by 2-way anova (material x storage time) for each distance and the means were compared by tukey's test (α=0.05). results table 1 shows that stone casts poured for distance 37-47 presented no statistical difference in dimensional accuracy for 2-5 accuracy of partially edentulous arch impressions obtained from different alginates and storage times braz j oral sci. 15(4):252-257 254 days of storage times (p>0.05), except for hy (2 days) group (p<0.05), which was statistically similar to hy (1 day) group (p>0.05). the lowest values of dimensional alteration were found for impressions poured with dental stone immediately or after 1 day of storage times. for the other transverse distance (33-43) similar values of dimensional accuracy were found for all storage times (table 2) (p>0.05). in general, for both transverse distances all alginates impressions showed stone casts with expansion (positive values) and similar dimensional alteration, except for 37-47 distance poured immediately and after 1 day, where hy presented the highest means of dimensional alteration and for 33-43 distance poured all periods of time, where jt showed the lowest means of dimensional alteration (p<0.05). still, table 2 shows also that jt did not differ statistically from ez, ch, and hy for 1 day (p>0.05). accuracy of partially edentulous arch impressions obtained from different alginates and storage times table 1 mean of dimensional alteration and sd (%) of stone casts obtained with different alginates and poured in different periods of time (distance 37-47). data with different lowercase letters in column and capital letters in row are statistically different (p<0.05). immediately 1 day 2 days 3 days 5 days hy 0.108±0.013 a,c 0.239±0.003 a,bc 0.348±0.018 b,b 0.418±0.008 a,a 0.371±0.018 a,a kr -0.036±0.017 bc,b 0.166±0.011 b,b 0.421±0.024 a,a 0.391±0.025 ab,a 0.367±0.021 a,a ch -0.081±0.018 bc,b 0.115±0.014 bc,b 0.371±0.018 ab,a 0.347±0.020 ab,a 0.392±0.030 a,a ez -0.005±0.009 b,b 0.017±0.008 cd,b 0.308±0.009 b,a 0.300±0.024 b,a 0.301±0.019 a,a jt -0.127±0.014 c,b -0.013±0.005 d,b 0.374±0.028 ab,a 0.331±0.015 ab,a 0.305±0.14 a,a table 2 mean of dimensional alteration and sd (%) of stone casts obtained with different alginates and poured in different periods of time (distance 33-43). data with different lowercase letters in column and capital letters in row are statistically different (p<0.05). immediately 1 day 2 days 3 days 5 days hy 0.122±0.014 a,a 0.138±0.018 b,a 0.240±0.022 a,a 0.154±0.016 a,a 0.253±0.034 a,a kr 0.173±0.020 a,a 0.325±0.014 a,a 0.266±0.028 a,a 0.197±0.033 a,a 0.241±0.012 a,a ch 0.124±0.005 a,a 0.107±0.021 b,a 0.253±0.028 a,a 0.246±0.037 a,a 0.216±0.044 a,a ez 0.095±0.012 a,a -0.056±0.019 c,a 0.160±0.024 a,a 0.266±0.033 a,a 0.130±0.013 a,a jt -0.094±0.036 b,a 0.010±0.016 bc,a -0.029±0.036 b,a -0.056±0.012 b,a -0.050±0.010 b,a table 3 mean of dimensional alteration and sd (%) of stone casts obtained with different alginates and poured in different periods of time (distance 33-37). data with different lowercase letters in column and capital letters in row are statistically different (p<0.05). immediately 1 day 2 days 3 days 5 days hy -0.256±0.035 ab,a -0.298±0.044 a,a -0.215±0.033 b,a -0.223±0.037 b,a -0.201±0.017 b,a kr -0.044±0.009 b,b -0.209±0.025 a,a -0.228±0.027 b,a -0.229±0.020 b,a -0.297±0.019 b,a ch -0.203±0.019 b,a -0.211±0.008 a,a -0.233±0.006 b,a -0.151±0.017 b,a -0.181±0.011 b,a ez -0.281±0.024 ab,a -0.339±0.005 a,a -0.238±0.033 b,a -0.289±0.021 b,a -0.242±0.033 b,a jt -0.332±0.032 a,b -0.323±0.030 a,b -0.446±0.027 a,b -0.508±0.009 a,a -0.619±0.017 a,a table 4 mean of dimensional alteration and sd (%) of stone casts obtained with different alginates and poured in different periods of time (distance 43-47). data with different lowercase letters in column and capital letters in row are statistically different (p<0.05). immediately 1 day 2 days 3 days 5 days hy -0.391±0.008 a,a -0.068±0.004 bc,b -0.019±0.031 c,b -0.023±0.008 b,b -0.046±0.012 b,b kr -0.081±0.033 b,a -0.188±0.025 b,a -0.120±0.008 bc,a -0.082±0.013 b,a -0.093±0.018 ab,a ch -0.033±0.016 b,a -0.048±0.022 c,a -0.184±0.006 b,a -0.091±0.016 b,a -0.060±0.021 b,a ez -0.042±0.022 b,a -0.102±0.019 bc,a -0.086±0.012 bc,a -0.073±0.023 b,a -0.121±0.044 ab,a jt -0.488±0.046 a,a -0.392±0.023 a,ab -0.375±0.040 a,ab -0.291±0.035 a,bc -0.216±0.023 a,c in general, the results were similar in the 33-37 anteroposterior distance (table 3). no statistical difference were found in dimensional accuracy for all storage times (p>0.05), except for kr (immediately) and jt (immediately, 1, and 2 days) groups which presented lower dimensional alteration than others different pouring times for each alginate group (p<0.05). on other hand, for 43-47 anteroposterior distance (table 4), no statistical difference were found in dimensional accuracy for all storage times for kr, ch, and ez groups (p>0.05), but hy alginate showed highest dimensional alteration for immediate pouring time and jt lowest dimensional alteration mean values for 3 and 5 days of pouring times (p<0.05). braz j oral sci. 15(4):252-257 255 accuracy of partially edentulous arch impressions obtained from different alginates and storage times comparing the alginates, jt showed the worst dimensional stability for all pouring times (p<0.05) and did not differ from hy for immediate time in 43-47 distance (p>0.05). however, for 33-37 distance jt presented the highest mean of dimensional alteration only for 2, 3, and 5 days of pouring times (p<0.05). in contrast to transverse distances, both anteroposterior distances for all alginates and storage times showed stone casts with shrinkage (negative numbers) as compared to those of the stainless steel cast. descriptive data summarization presents the behavior of the alginates in all distances along the time (figure 1). elastomers, except for the polyether and modified addition silicone, are hydrophobic1. faria et al.17 (2008) suggest that alginates may replace the condensation (polydimethylsiloxanes) or addition (polyvinyl siloxanes) silicones in some clinical situations. this study corroborate with findings of the present study in terms of dimensional accuracy. both specifications for alginate impression materials used in dentistry to make impressions of the teeth and buccal tissues (iso 1563:1990)21 contains no requirement for dimensional stability neither has any limits on dimensional changes. it is known in the literature for elastomeric impression materials that their dimensional change should be no more than ±0.5% upon setting and after storage time16. thus, the dental stone models made from alginate impressions used in this study poured up to 5 days showed dimensional changes within the limits recommended for elastomeric impression materials. to obtain a good dimensional accuracy is required a rigid control of the factors that may compromise the dimensional stability of alginates, decreasing the effects of expansion and shrinkage11. in this study, besides the standardized ratio and handling of the impression material in an environment with temperature and humidity controlled, the molds were placed in humidifiers with rigid humidity control (100%), decreasing the effects of syneresis and evaporation, responsible for alginate shrinkage, and imbibition, responsible for the expansion of the material. at first, the alginate has expansion due the excess of water added to handle the impression material. this initial expansion is compensate by alginate syneresis as a function of time due to the continued gelation process, which compresses the water onto the surface of gel structures. the syneresis may be highly accelerated in an environment with low water saturation8. in this study, the humidity was maintained in 100%. thus, the syneresis speed decreased and the alginate remained dimensionally stable up to 5 days. it was not possible to claim that the stone cast had expansion or shrinkage, because some areas (transverse distances) showed expansion due to tension release after the mold removal while others (anteroposterior distances) presented shrinkage due the syneresis towards the center of the alginate impression, besides of the posterior border of the mandibular tray is open, then does not occur adhesion between alginate and tray. moreover, the setting expansion of stone casts might have compensated the shrinkage caused by alginate syneresis18. for this occurred is necessary that the stone cast expand uniformly in three dimensions, but using an open tray the stone does not uniformly expand toward the impression. the stone cast increases only in width and does not in height. thus, stone cast does not become geometrically similar to the master model19. the tilting movement during removal of the impression from the buccal cavity may also jeopardize dimensionally the alginate15. in the present study, the detachment movement was standardized in a vertical direction. another responsible for the dimensional instability of the impression materials is the thermal contraction inherent the differences between buccal cavity and environment temperatures. the impression procedures and the mold storage were realized in an environment with standard temperature, which prevented or decreased the thermal contraction. this explains the excellent results found in this study to the alginate. however, clinically is not always possible to work in suitable environments discussion anatomical dental stone casts, which accurately reproduce the shape of the teeth and adjacent buccal tissues, are used for many diagnostic and treatment purposes in dentistry. a dimensionally accurate impression (negative mold) is essential for fabricating an accurate anatomical stone casts1. based on results of the current study, the hypothesis was rejected, since the stone casts poured immediately after impression procedure showed similar dimensional accuracy values than stone casts produced after different storage times in an environment with 100% relative humidity and room temperature (23°c ± 2°c) for 1, 2, 3 or 5 days. to verify any properties of the impression materials there are specifications as international organization for standardization (iso) 1563:199021. these specifications use models, casts and matrices presenting regions which may be easily measured. although the specifications provide the possibility to compare the dimensional stability of different alginates, the tests used in these statements differ from clinical reality17. the use of a standard stainless steel cast representing a partially edentulous mandibular arch in this study represents a situation routinely observed in clinical practice. according to faria et al.17 (2008) the molds made by alginate or elastomers impression material have similar dimensional accuracy. thus, alginate may be considered highly accurate impression material. one advantage is that alginates are hydrophilic, while fig. 1 frequency of periodontal sites. braz j oral sci. 15(4):252-257 and to control every these factors. even some studies8,9,11 have no a rigid control of the factors that may compromise the dimensional changes in impression materials, which explains some studies where the alginate does not present good results of dimensional accuracy or the storage of alginate molds is not suitable for more than one day9,10. on the other hand, studies18,20 controlling the sample preparation procedures corroborate with results of this study. these studies did not show difference in the dimensional accuracy of stone casts made from different alginate impression materials and stored for different times up to 5 days18. the differences in dimensional accuracy between alginates tested is related mainly with the different chemical compositions of materials such as, for instance, the amount and type of filler present in each material, the proportion of matrix/filler and the quality of the matrix components22,23. these factors should be considered when the properties of the alginate are evaluated, such as dimensional stability22. the largest dimensional change occurred in the anteroposterior distance, observed mainly in the jt alginate. it can be explained by bilateral adhesion of the impression material to tray when considering the transversal direction of the mold and only unilateral anteroposterior direction. the free end of the mold may offer less restrictive strength to shrinkage, allowing dimensional changes in anteroposterior distance16. furthermore, the transverse distance 37-47 showed greater dimensional changes at 2, 3, and 5 days than stone casts poured immediately and 1 day (table 1), but these dimensional changes were smaller than ±0.5% which is recommended for high accuracy impression material as elastomeric impression materials16. the optical measuring microscope provides greater accuracy than digital models (0.5 μm accuracy) to evaluate dimensional stability. it is a methodology supported by iso21 specification. however, this method does not represent a common clinical application and dimensional instability of small distances (< 1.0 μm) are clinically insignificant in partially edentulous arch impressions, since the dental stone has a crystalline structure that could not reproduce such amount of detail5. furthermore, the stone casts made from alginate tend to lose details in angle areas used as reference points in optical measuring microscope for measurement of the distances7,24. a limitation of in vitro studies is that we could not evaluate the effect of any factors such as blood, saliva, and/or temperature difference between buccal cavity and room where the mold will be stored (thermal shrinkage) on the dimensional accuracy/stability of impression materials9,16. in this study, the accuracy of alginates was measured indirectly on dental stone casts. this situation avoid inaccuracies promoted by time of measurement in measuring microscope, since temperature, humidity, and storage time may compromise dimensional stability and accuracy of impression materials1,9,16,20. this methodology is valid in terms of clinical application since the conditions would not be highly controlled in a clinical situation and the impression material is always used together dental stone in prosthodontic treatment of partially edentulous patients. thus, the prosthesis misfit is due to loss of accuracy and dimensional instability and these conditions are time-dependent distortion of the mold storage and poured stone cast24, since alginate polymerization shrinkage occurs toward the center of the mold and it is speculated that, under clinical conditions, expansion of the dental stone could compensate partly this shrinkage25. some distances showed shrinkage (negative values) due to alginate gelation while others present expansion (positive values) due to expansion of the dental stone. the differences between stone casts and stainless steel cast presented positive and negative values, but in order to avoid false results (positive and negative values canceling each other out), the data were converted to absolute values11. an accurate stone cast is important for the manufacturing of fixed or removable and total or partial prosthesis. thus, the choice of impression material and storage time is relevant. the differences in dimensional accuracy between the alginates seemed to be due the differences in the chemical composition of each material. alginate impressions can be stored up to 5 days before pouring with dental stone, since the factors responsible for dimensional instability of the material (humidity, temperature and correct powder/liquid ratio) be controlled. references 1. craig rg. review of dental impression materials. adv dent res. 1988 aug;2(1):51-64. 2. nassar u, aziz t, flores-mir c. dimensional stability of irreversible hydrocolloid impression materials as a function of pouring time: a systematic review. j prosthet dent. 2011 aug;106(2):126-33. doi: 10.1016/s0022-3913(11)60108-x. 3. wadhwa ss, mehta r, duggal n, vasudeva k. the effect of pouring time on the dimensional accuracy of casts made from different irreversible hydrocolloid impression materials. contemp clin dent. 2013 jul;4(3):313-8. doi: 10.4103/0976-237x.118368. 4. amalan a, ginjupalli k, upadhya n. evaluation of properties of irreversible hydrocolloid impression materials mixed with disinfectant liquids. dent res j (isfahan). 2013 jan;10(1):65-73. doi: 10.4103/17353327.111795. 5. rueggeberg fa, beall fe, kelly mt, schuster gs. sodium hypochlorite disinfection of irreversible hydrocolloid impression material. j prosthet dent. 1992 may;67(5):628-31. 6. rubel sb. impression materials: a comparative review of impression materials most commonly used in restorative dentistry. dent clin north am. 2007 jul;51(3):629-42, vi. 7. walker mp, burckhard j, mittis da, williams kb. dimensional change over time of extended storage alginate impression materials. angle orthod. 2010 nov;80(6):1110-5. doi: 10.2319/031510-150.1. 8. miller mw. syneresis in alginate impression materials. br dent j. 1975 dec 2;139(11):425-30. 9. sedda m, casarotto a, raustia a, borracchini a. effect of storage time on the accuracy of casts made from different irreversible hydrocolloids. j contemp dent pract. 2008 may 1;9(4):59-66. 10. shaba op, adegbulugbe ic, oderinu oh. dimensional stability of alginate impression material over a four hours time frame. nig q j hosp med. 2007 jan-mar;17(1):1-4. 11. chen sy, liang wm, chen fn. factors affecting the accuracy of elastometric impression materials. j dent. 2004; 32, 603-9. 12. kulkarni mm, thombare ru. dimensional changes of alginate dental impression materials-an in vitro study. j clin diagn res. 2015; 9, zc98102. 13. imbery ta, nehring j, janus c, moon pc. accuracy and dimensional stability of extended-pour and conventional alginate impression materials. j am dent assoc. 2010 jan;141(1):32-9. braz j oral sci. 15(4):252-257 256 accuracy of partially edentulous arch impressions obtained from different alginates and storage times 257 accuracy of partially edentulous arch impressions obtained from different alginates and storage times 14. consani rlx, domitti ss, mesquita mf, consani s. influence of flask closure and flask cooling methods on tooth movement in maxillary dentures. j prosthodont. 2006 jul-aug;15(4):229-34. 15. boscato n, consani rlx, consani s, del bel cury aa. effect of investment material and water immersion time on tooth movement in complete denture. eur j prosthodont restor dent. 2005 dec;13(4):164-9. 16. vitti rp, da silva ma, consani rl, sinhoreti ma. dimensional accuracy of stone casts made from silicone-based impression materials and three impression techniques. braz dent j. 2013 sep-oct;24(5):498-502. doi: 10.1590/0103-6440201302334. 17. faria ac, rodrigues rc, macedo ap, mattos mda g, ribeiro rf. accuracy of stone casts obtained by different impression materials. braz oral res. 2008 oct-dec;22(4):293-8. 18. guiraldo rd, moreti af, martinelli j, berger sb, meneghel ll, caixeta rv, et al. influence of alginate impression materials and storage time on surface detail reproduction and dimensional accuracy of stone models. acta odontol latinoam. 2015;28(2):156-61. doi: 10.1590/ s1852-48342015000200010. 19. teraoka f, takahashi j. dimensional changes and pressure of dental stones set in silicone rubber impressions. dent mater. 2000 mar;16(2):145-9. 20. guiraldo rd, borsato tt, berger sb, lopes mb, gonini-jr a, sinhoreti ma. surface detail reproduction and dimensional accuracy of stone models: influence of disinfectant solutions and alginate impression materials. braz dent j. 2012;23(4):417-21. 21. international organization for standardization. dental alginate impression material. iso 1563:1990(e). 2. ed.; 1990. 22. kawara m, iwasaki m, iwata y, komoda y, inoue s, komiyama o, et al. rheological properties of elastomeric impression materials for selective pressure impression technique. j prosthodont res. 2015 oct;59(4):25461. doi: 10.1016/j.jpor.2015.07.002. 23. carlo hl, fonseca rb, gonçalves ls, correr-sobrinho l, soares cj, sinhoreti ma. analysis of filler particle levels and sizes in dental alginates. mater res. 2010 apr/jun;13(2):261-4. 24. tan hk, hooper pm, buttar ia, wolfaardt jf. effects of disinfecting irreversible hydrocolloid impressions on the resultant gypsum casts: part iii–dimensional changes. j prosthet dent. 1993 dec;70(6):532-7. 25. kambhampati s, subhash v, vijay c, das a. effect of temperature changes on the dimensional stability of elastomeric impression materials. j int oral health. 2014 feb;6(1):12-9. braz j oral sci. 15(4):252-257 signs and symptoms of temporomandibular disorder and patients’ satisfaction before and after orthognathic surgery edmar ricardo pozzobon christovam1, eloisa marcantonio boeck2, silvia amélia scudeler vedovello1, heloisa cristina valdrighi1, viviane veroni degan1, mário vedovello filho1 1fundação hermínio ometo – fho/uniararas, school of dentistry, department of orthodontics, araras, sp, brazil 2universidade de araraquarauniara, centro universitário de araraquara, school of dentistry, department of orthodontics, araraquara, sp, brazil correspondence to: edmar ricardo pozzobon christovam fundação hermínio ometo – fho/uniararas, curso de odontologia av. maximiliano baruto, 500 jd. universitário, cep: 13607-339 araras –sp, brasil phone: +55-19-3861-0472. fax: +55-19-3861-0178 e-mail: edmarchristovam@ortodontista.com.br abstract aim: to evaluate signs and symptoms of temporomandibular joint disorder and satisfaction in patients before and after orthognathic surgery. methods: the sample consisted of 15 patients aged between 19 and 47 years old, indicated for orthodontic-surgical treatment. all patients answered an anamnesis questionnaire based on helkimo anamnestic index to evaluate subjective symptoms and underwent a clinical evaluation based on helkimo disfunction index, applied at three time points: before (t0), three (t1) and six months (t2) after surgery. statistical models used were χ2 test (chi-square), tukey test, confidence interval and analysis of variance (anova). results: statistical analysis revealed no significant difference in the incidence of joint sounds, maximum mouth opening, deviation of mouth opening and pain in the tmj region (p>0.05). no patient presented worsening of the symptomatology. as regards muscular pain, there was a statistically significant improvement with time (p<0.05) and 86.7% of patients reported that they were satisfied with the obtained results. conclusions: improvement of tmd after orthognathic surgery may not be the result of correcting malocclusion and satisfaction with the results can be a factor of tmd improvement. keywords: orthodontics. temporomandibular joint. malocclusion. self-concept. introduction surgical correction of dentofacial deformities is recommended to improve facial esthetics and social relationships, to establish static and functional occlusion health, stable results1,2 and adaptation of temporomandibular joint (tmj)3. the psychosocial impact of a dentofacial deformity may alter an entire lifestyle and be critical for self-esteem due to the psychological effects caused by facial and dental appearance1. the prime importance of stable results in orthognathic surgery is tmj health. if the tmj is not in good condition, the surgical outcome procedure may be unsatisfactory in terms of function, aesthetics, stability and pain; and any type of pain or dysfunction must be assessed before performing an orthognathic surgery4. some patients with open bite or large incisors overlap should be viewed as a risk factor before starting orthodontic, prosthodontic, routine oral surgery (third molar extraction) or orthognathic surgery, because these skeletal features may suggest an increase in mandibular pain and dysfunction after treatment5. anyway, a previous evaluation is important to properly identify these signs and symptoms while planning an orthognathic surgery6. there are controversies concerning the adequate procedure in patients with preexistent temporomandibular disorder (tmd) that need orthodontic-surgical treatment received for publication: june 26, 2016 accepted: october 27, 2016 original article braz j oral sci. april | june 2016 volume 15, number 2 http://dx.doi.org/10.20396/bjos.v15i2.8648765 braz j oral sci. 15(2):137-143 138 to correct dentofacial deformities. there are two distinct theories: one states that surgical procedures can reduce tmj dysfunction’s symptoms7, while other asserts that orthognathic surgery causes future deleterious effects on tmj and worsens the dysfunction’s symptoms8. signs and symptoms, consisting of tmj pain, pain in masticatory muscles, headache, limited mouth opening and joint noise may define tmd5,9. helkimo10 (1974), with the aim of facilitating both scientific investigations and patients routine exams with different types of symptoms and severity degrees, suggested a method comparing signs and symptoms of tmd. the patients were submitted to a clinical masticatory system dysfunction evaluation, an anamnesis interview and occlusion evaluation. data were collected and a numerical classification of the prevailing type was established, determining three indexes: clinical dysfunction index, anamnestic dysfunction index and index for occlusal state. from then on, several authors11-15 have studied the subject based on helkimo’s work. onizawa et al.8 (1995) investigated tmj alterations at three and six months after orthognathic surgery, comparing patients who underwent orthognatic surgery with patients who did not. they concluded that the joint symptoms alterations after surgery are not always the result of correcting malocclusion, but in practice due to factors like the influence of the orthognathic surgery on the masticatory muscles. gaggl et al.16 (1999) reported that the major problem faced by oral and maxillofacial surgeons is not only placing the bone bases into esthetic and ideal functional positions, but also repositioning bone bases in a way that tmj is restablished in a physiological manner. using magnetic resonance preoperatively and 3 months after orthognathic surgery and clinical data like maximum mouth opening, presence of joint sounds and palpation of masticatory muscles, the authors concluded that clinical findings combined with the magnetic resonance images supported that, in many cases, clinical improvement of signs and symptoms of tmd are acceptable after correcting joint complex position in orthodontic-surgical treatment. there are patient variations regarding psychological and physiological surgery effects6, and the risk of developing tmd in orthognathic patients has been attributed to various factors including psychological distress17,18. orthognathic surgery presents an interesting model to study pain and function of the masticatory system19. investigating tmd signs and symptom characteristics and post-operative changes in orthognathic patients leads to a better understanding of orthognathic surgery influence on tmd7. this study aimed to evaluate signs and symptoms of tmd in patients before and after orthognathic surgery and their satisfaction after orthodontic surgical treatment. material and methods the research was developed after approval of the research project by the research ethics and merit committee (protocol number 763/2007). the sample was selected at center of research and treatment of orofacial deformities (cedeface) according to the following inclusion criteria: patients with dentofacial deformities referred to orthodontic-surgical treatment, irrespective of facial pattern, with signs and symptoms of tmd, assessed by a questionnaire. the exclusion criteria were: patients who underwent previous treatment of tmj, or suffered facial traumatism, fissured lip and with ankylosis of the tmj. thus, among 20 assessed patients, 15 were selected; 12 were women and 3 were men, aged between 19 and 47 years. all patients filled out a questionnaire based on anamnestic disfunction index10, to evaluate subjective symptoms, and were also subjected to a clinical examination based on helkimo’s dysfunction index10, applied pre-surgical and three and six months post-surgical procedure. the anamnesis questionnaire approached matters concerning sounds in tmj (clicking and crepitation), confirmed with a stethoscope as suggested by gaggl et al.16 (1999); face pain, by asking the patients to indicate the most sensitive region on a face front view drawing; pain at tmj region at mouth opening, including mandibular movements and asking the patients to indicate pain intensity using a numerical visual scale suggested by wolford et al.7 (2003), ranging from 1 (pain absence) to 10 (intense pain). at last, the patients were asked if the general pain symptoms after surgery improved, turned worse or had no change. the clinical examination evaluated mandibular movements as maximal mouth opening, protrusive movement, group function and canine guidance, and muscular pain on palpation. to obtain an average, maximal mouth opening and the range of protrusive movement were measured three times, at the same moment, with a dry tip compass with a locking device, by the interincisal distance and then measured with a millimetric ruler16. group function or canine guidance movements were determined visually. masticatory muscles were evaluated bilaterally by palpation, by the same examiner, which was performed as follows: the masseter muscle was palpated extraorally from its upper insertion point to the area of the mandibular angle; the temporal muscle was palpated extraorally in the temporal region of the head; the lateral pterygoid muscle was palpated intraorally near the distal, lateral and upper region of the maxillary tuberosity; the medial pterygoid muscle was palpated extraorally behind the mandibular angle and intraorally in the inferior and posterior portion of the lingual groove of the mandible20. patients were subjected to a mandible or a maxilla surgery and/or a combination of both. for mandible, the used technique was bilateral sagittal osteotomy of mandibular ramus21,22, and for maxilla it was the le fort i type osteotomy. the mandible proximal segments were positioned manually, without using holding devices for the mandible head. at surgery, acrylic resin interocclusal plates (surgical guides) were used to establish the correct occlusion position until rigid internal fixation procedure with mini-plates and titanium screws was performed. for better stability, the first fixation was on the larger bone contact area, avoiding torsion of the mandibular head. after fixation, new occlusion passivity was checked. during the post-surgical phase, no patient had physical therapy follow-up. three and six months after surgery, all data were collected again, and in the last evaluation, patients answered if they were signs and symptoms of temporomandibular disorder and patients’ satisfaction before and after orthognathic surgery braz j oral sci. 15(2):137-143 139 satisfied with the results of treatment. the hypothesis refers to equality comparison between proportions or averages and the statistical models used to verify such hypothesis was the χ2 test (square chi), analysis of variance (anova), confidence interval and tukey test. results it was possible to verify if the results of subjective evaluation, which the study analyzed and compared, showed subsidies to reject the hypothesis that the occurrence of clicking versus time after surgery was independent events (table 1). applying the χ2 test there was a significant reduction (p<0.05) in clicking. similarly, in spite of the reduction of crepitation at the evaluated timepoints , the results were not significant compared with the evaluated total percentage (p=0.18). regarding face pain (table 1), most patients pointed on temporal region of the drawing. for statistical analysis, data were divided and the χ2 test was applied, so that on temporal region the presence or absence of pain regardless the time point, presented a significant decrease (p=0.001). the absence of pain showed a smaller number of patients in a given time compared with the consecutive time point, the opposite that occured with the pain. the relevance to assess pain intensity on tmj region referred to the question: does the degree of pain at initial time, reported by patients based on a visual scale, decrease with time after surgery? this question was assessed using statistical analysis of confidence intervals to 95% for true average, using only the patients who had pain at the initial time. thus, the sample was reduced to 9 patients and the statistical analysis showed that the true averages (middle grade), estimated at the three moments, were statistically equal to each other. this is because the confidence intervals of 95% presented common points, which made us deduce that the degree of pain showed no decrease over time (figure 1). for general pain symptoms with time, the results showed that no patient experienced worsening of pain he/she felt before surgery, and that despite the pain improvement, this alteration in the time intervals (from t0 to t1 and from t1 to t2) were independent events (p=0.361), calculated by distribution χ2 (square chi) with one (1) degree of freedom (figure 2). signs and symptoms of temporomandibular disorder and patients’ satisfaction before and after orthognathic surgery table 1 frequency of tmd symptomatology according to time. symptom initial (t0) after 3 months (t1) after 6 months (t2) total clicking presence 9 (60.0) 8 (53.3) 2 (13.3) 19 (42.2) (p<0.05) absence 6 (40.0) 7 (46.7) 13 (86.7) 26 (57.8) total 15 (100.0) 15 (100.0) 15 (100.0) 45 (100.0) crepitation presence 5 (33.3) 4 (26.7) 1 (6.7) 10 (22.2) (p>0.05) absence 10 (66.7) 11 (73.3) 14 (93.3) 35 (77.8) total 15 (100.0) 15 (100.0) 15 (100.0) 45 (100.0) face pain presence 12 (80.0) 3 (20.0) 1 (6,7) 16 (35.6) (temporal region) absence 3 (20.0) 12 (80.0) 14 (93.3) 29 (64.4) (p<0.05) total 15 (100.0) 15 (100.0) 15 (100.0) 45 (100.0) absolute frequency: outside of parenthesis/relative frequency: inside parenthesis fig.1. middle grade of pain intensity on tmj region according to time. t0 (before surgery); t1 (3 months after surgery); t2 (6 months after surgery). fig.2. general pain symptoms after surgery. braz j oral sci. 15(2):137-143 signs and symptoms of temporomandibular disorder and patients’ satisfaction before and after orthognathic surgery clinical assessment indicated the means of three measurements of mouth opening and protrusive movement that the patient could make when opening his/her mouth as far as possible. they were statistically analyzed using the statistical model of anova at a fixed criterion, which was time, thus verifying that the hypothesis of maximum mouth opening was the same at each time point of surgery. this model provided a meaningful probability (p=0.0001); but additional tukey test revealed that at three and six month points after surgery, these means were statistically equal and lower than the mean of maximum mouth opening in the initial instant (figure 3). for the protrusive movement, the application of the anova (figure 4) model derived a non-meaningful probability (p=0.891). ignoring the datum “unable to do” due to the value under 1 and working only with the data of the canine guidance and group function, it was observed that the canine guidance movement was easier to make along time (p<0.05), while group function movement was more difficult (table 2). fig.3. means of mouth opening according to time. 0.00= t0 (before surgery); 1.00= t1 (3 months after surgery); 2.00 = t2 (6 months after surgery). fig.4. means of protrusive movement according to time. 0.00= t0 (before surgery); 1.00= t1 (3 months after surgery); 2.00 = t2 (6 months after surgery). regarding pain on palpation of masseter muscle, the sample showed subsidies to not reject the hypothesis that the occurrence or not of pain versus time after surgery were independent events (p=0.36) and the meaning of this fact was that the percentage of absent pain in each post-surgery time were statistically equal to the total percentage of the absence of this pain (table 3), which in this case was 73.3% implying that those percentages were statistically equal to each other, and the percentage of the pain in each post-surgery time were statistically equal to the total percentage of the presence of this pain, which in this case was 26.7%, implying that those percentages were statistically equal to each other. for temporal muscle (table 3), the sample showed subsidies to reject the hypothesis that the occurrence or not of pain versus time after surgery were independent events (p=0.08), and the results indicated that with post-surgery time, pain on palpation of this muscle decreased. for lateral pterygoid and medial pterygoid muscles (table 3), the sample showed subsidies to reject the hypothesis that the occurrence or not of pain versus time after surgery were independent events (p=0.001 and p=0.0004 respectively).; it was noted that for lateral pterigoyd muscle from time point to three months after surgery, all patients showed absence of pain. for medial pterigoyd muscle the percentage of pain at each time post-surgery was statistically different from the percentage of presence of this pain, which in this case was 20.0% and at six month time point, all patients had absence of pain on palpation in this muscle. six months after surgery (t2) the patients were asked if they were satisfied with the results obtained from orthodonticsurgical treatment. most of them (86.7%) declared they were satisfied with the performed surgical procedures, which was statistically significant. discussion several studies have been conducted to elucidate tmd relationship with orthognathic surgery11-15,17-20. in addition to improve the appearance, an important goal of orthodontic surgical treatment is to improve functional occlusion with masticatory function1. defined as a series of clinical problems that affect masticatory muscles, tmj and its related structures generally, the etiology of tmd is considered multifactorial and malocclusion does not seem to be a significant etiological factor, but only one of the factors in a complex etiological context8,23. maximum mouth opening is an indicator of mandibular function24 and in the present study there was a significant reduction, which may have been influenced by the post-operative edema16. also protrusive movement means showed no significance over time, maybe because the sample presented negative and positive overjet. this requires further studies with homogenous patient samples of dentofacial deformities and the same tmd24. lateral excursive movements were visually assessed regarding canine guidance or group function and the results showed an improvement on canine guidance after six months anyway, it was not possible to associate it to reduction of mandibular mobility and clicking. the clicking in the assessments at three and six months after orthognathic surgery had a significant reduction, while the presence 140 braz j oral sci. 15(2):137-143 of crepitation remained unaltered with time. these results may be associated with the significant reduction in maximum mouth opening after surgery, presented by the patients12,13 or due to changes in condylar position caused by surgery25. reduction of painful sensitivity of the masticatory muscles after surgery was an important result of this study, both in the subjective assessment and clinical evaluation by means of muscular palpation13,25. on the other hand, some studies did not observe any change in muscular pain symptomatology with time, leading to conclusion that orthognathic surgery did not influence the relief of muscular pain and that improvement in tmd was unpredictable11,12,19, requiring follow up of the symptoms for several years8 and independent treatment11. tmd must be carefully assessed before orthognathic surgery in patients who have clear dysfunction signs and symptoms as well in those who are completely asymptomatic5,8. patients with tmd before the orthognathic surgery tended to have significant increase in dysfunction signs and symptoms7,24,26. contrarily, evaluating before and after orthognatic surgery, the results of this study showed that patients with previous tmd signs and symptoms, regardless of time, had significant improvement of subjective and clinical tmd signs and symptoms7,13,15,17,26-29 and no patient presented worsening symptoms after orthognathic surgery. therefore, the improvement of the signs or symptoms of tmd after orthodontic-surgical treatment seems to happen due to alteration of mandible posturing25, morphofunctional balance of soft tissue, muscles and occlusion29 established by the treatment and by monitoring patients with an interdisciplinary team28. nevertheless, tmd must not only be the object of systematic investigation, but must also indicate adequate therapeutic treatment for all the individuals who underwent combined orthodontic-surgical treatment for correction of dentofacial deformities30. the correction of malocclusion by orthognatic surgery does not cause significant increase or decrease in signs and symptoms of tmd25,31 and the relationship among the type of malocclusion, pattern, type of orthognatic surgery and tmd is complex and not clearly established13. in fact, there are more questions than answers32. it is important to point out that the aim of this study was not to compare the type of malocclusion, pattern or orthognathic surgery performed with the alterations in signs and symptoms of tmd, but to evaluate if these alterations remained constant or underwent significant changes over three and six months after surgery. therefore, it was not possible to determine what type of malocclusion showed significant increase or decrease signs and symptoms of tmd. similarly, the data of this study were collected using helkimo index without using auxiliary methods of diagnosis, like radiographs and computed tomography, magnetic resonance or electromyography, and therefore it was not possible to explain whether the alterations in the signs and symptoms of tmd were related to anatomical changes that occurred in the patients. the helkimo index, despite being widely used, is a very limited tool to assess tmd or muscular pain15. some authors used rdc/ tmd and noted that this research criterion may help establishing postoperative treatment plans by evaluating the patient’s psychological and psychosocial state11,17,33. signs and symptoms of temporomandibular disorder and patients’ satisfaction before and after orthognathic surgery table 2 clinical assessment of mandibular movements according to time. movement initial (t0) after 3 months (t1) after 6 months (t2) total right laterality canine guidance 0 (0.0) 3 (20.0) 3 (20.0) 6 (13.3) group function 15 (100) 12 (80.0) 11 (73.3) 38 (84.4) n. c. ex. 0 (0.0) 0 (0.0) 1 (6.7) 1 (2.3) left laterality canine guidance 0 (0.0) 5 (33.3) 6 (40.0) 11 (24.4) group function 14 (93.3) 8 (53.4) 9 (60.0) 31 (68.9) n. c. ex. 1 (6.7) 2 (13.3) 0 (0.0) 3 (6.7) absolute frequency: outside of parenthesis/relative frequency: inside parenthesis. table 3 frequency of muscular pain on palpation according to time. muscle initial (t0) after 3 months (t1) after 6 months (t2) total masseter (p>0.05) presence 6 (40.0) 3 (20.0) 3 (20.0) 12 (26.7) absence 9 (60.0) 12 (80.0) 12 (80.0) 33 (73.3) total 15 (100.0) 15 (100.0) 15 (100.0) 45 (100.0) temporal (p>0.05) presence 6 (40.0) 3 (20.0) 1 (6.7) 10 (22.2) absence 9 (60.0) 12 (80.0) 14 (93.3) 35 (77.8) total 15 (100.0) 15 (100.0) 15 (100.0) 45 (100.0) lateral pterygoid (p<0.05) presence 6 (40.0) 0 (0.0) 0 (0.0) 6 (13.3) absence 9 (60.0) 15 (100.0) 15 (100.0) 39 (86.7) total 15 (100.0) 15 (100.0) 15 (100.0) 45 (100.0) medial pterygoid (p<0.05) presence 8 (53.3) 1 (6.7) 0 (0.0) 9 (20.0) absence 7 (46.7) 14 (93.3) 15 (100.0) 36 (80.0) total 15 (100.0) 15 (100.0) 15 (100.0) 45 (100.0) absolute frequency: outside of parenthesis/relative frequency: inside parenthesis. 141 braz j oral sci. 15(2):137-143 signs and symptoms of temporomandibular disorder and patients’ satisfaction before and after orthognathic surgery several orthognathic surgery procedures involve functional changes and a considerable impact on the patients’ esthetic appearance. these changes provide the patients physical and psychological benefits. however, their expectations for improved esthetics may possibly be higher than the expectations of functional improvement. patients will only be satisfied with surgical results if their expectations have been exceded. in this study, most patients subjected to orthognathic surgery reported they were satisfied with the obtained results and had self esteem improvement after surgery34, while some of them showed to be dissatisfied1,15. dantas et al.1 (2015), observed that 97,6% of the patients were satisfied with the obtained results, presenting a psychosocial and functional improvement. some authors related that appearance improvement is a psychological factor that leads to functional improvement11,17,35, as observed in this study. early rehabilitative physiotherapy could facilitate early recovery in mandibular range of motion, so it is important to emphasize the need for interaction between specialists in cases treated with orthognathic surgery, including orthodontics, oral maxillofacial surgery, physical therapy, especially in the first six months36. a jointly prepared diagnosis and treatment plan guarantee better results, but long-term follow up also contributes to psychological improvement and a better quality of life in these patients32. however, in this study, no patient had physical therapy and it was not possible to conclude that this kind of therapy could improve tmd signs and symptoms. it was concluded that all patients showed alterations of signs and symptoms of tmd after orthognathic surgery. however, no patient presented worse symptoms. it was not possible to correlate such alterations with malocclusion correction and with the evaluated time intervals , despite the reduction in clicking, face pain and pain in masticatory muscles at the first moment (t1) after surgery, which may have occurred due to other factors, like surgical influence or psychological factors. most patients were highly satisfied with the results obtained after orthodontic-surgical treatment. in fact, orthognathic surgery can bring beneficial effects to tmd and quality of life, but a longer follow-up study could improve the understanding of tmd in orthognathic patients. references 1. dantas jfc, neto jnn, carvalho shg, martins imclb, souza rf, sarmento va. satisfaction of skeletal class iii patients treated with different types of orthognatic surgery. int j oral maxillofac surg. 2015;44(2):195-202. 2. baek sh, kim tk, kim mj. is there any difference in the condylar position and angulation after asymmetric 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temporomandibular disorders in patients undergoing orthognathic surgery compared with a control group. oral surg oral med oral pathol oral radiol endod. 2002;94(5):554-60. 14. landes ca, sterz m. proximal segment positioning in bilateral sagittal split osteotomy: intraoperative controlled positioning by a positioning splint. j oral maxillofac surg. 2003;61(12):1423-31. 15. martin md. orthognatic surgical treatment may produce improved temporomandibular functional status in adults with pretreatment dysfunction. j evidbased dent pract. 2011;11(4):191-3. 16. gaggl a, schultes g, santler g, karcher h, simbrunner j. clinical and magnetic resonance findings in the temporomandibular joints of patients before and after orthognathic surgery. br j oral maxillofac surg. 1999;37(1):41-5. 17. mladenovic i, dodic s , stosic s , petrovic d, čutovic t , kozomara r. tmd in class iii patients reffered for orthognatic surgery: psychological and dentition-related aspects. j cranio-maxillofac surg. 2014;42(8):16049. 18. kim yk, kim sg, kim jh, yun py, oh js. temporomandibular joint and psychosocial evaluation of patients after orthognatic surgery: a preliminary study. j cranio-maxillofac surg. 2013;41(5):e83-6. 19. farella m, michelotti a, bocchino t, cimino r, laino a, steenks mh. effects of orthognathic surgery for class ii malocclusion on signs and symptoms of temporomandibular disorders and on pressure pain thresholds of the jaw muscles. int j oral maxillofac surg. 2007;36(7):5837. 20. flynn b, brown dt, lapp th, bussard da, roberts we. a comparative study of temporomandibular symptoms following mandibular advancement by bilateral sagittal split osteotomies: rigid versus nonrigid fixation. oral surg oral med oral pathol. 1990;70(3):372-80. 21. trauner r, obwegwser hl. the surgical correction of mandibular prognathism and retrognathia with consideration of genioplasty. part i. oral surg. 1957;10(7):677-89. 22. epker bn. modification in the sagittal osteotomy of the mandible. j oral surg. 1977;35:157-9. 23. sartoretto sc, bello yd, bona ad. [evidências científicas para o diagnostico e tratamento da dtm e a relação da oclusão e a orthodontia]. rfo 2012;17(3):352-9. portuguese. 24. al-belasy f, tosoglu s, dolwick mf. mandibular hipomobility after orthognatic surgery: a review article. j oral maxillofac surg. 2013;71(11):1967.e1-1967.e11. 142 braz j oral sci. 15(2):137-143 25. rodrigues-garcia rcm, sakai s, rugh jd, hatch jp, tiner bd, sickels je, et al. effects of major class ii occlusal corrections on temporomandibular signs and symptoms. j orofac pain. 1998;12(3):18592. 26. abrahamsson c, henrikson t, nilner m, sunzel b, bondmark l, eckberg ec. tmd before and after correction of dentofacial deformities by orthodontic and orthognatic treatment. int j oral maxillofac surg. 2013;42(6):752-8. 27. al-ryiami s, cunningham s, moles dr. orthognatic treatment and temporomandibular disorders: a systematic review. part 2. signs and symptoms and meta-analyses. am j orthod dentofacial orthop. 2009;136(5):626.e1-626.e16. 28. silva mma, ferreira at, migliorucci rr, nari filho h, berretin-felix g. influence of orthodontic-surgical treatment on signs and symptoms of temporomandibular dysfunction in subjects with dentofacial deformities. rev soc bras fonoaudiol. 2011;16(1):80-4. 29. togashi m, kobayashi t, hasebe d, funayama a, mikami t, saito i, et al. effects of surgical orthodontic treatment for dentofacial deformities on signs and symptoms of temporomandibular joint. j oral maxillofac surg med pathol. 2013;25(1):18-23. 30. cascone p, paolo c, leonardi r, pedullà e. temporomandibular disorders and orthognathic surgery. j craniofac surg. 2008;19(3):687-92. 31. lindenmeyer a, eghtessad m, goulden r, speculand b, harrys m. oral and maxillofacial surgery and chronic painful temporomandibular disorders a systematic review. j oral maxillofac surg. 2010;68(11):275564. 32. nadeshah m, mehra p. orthognatic surgery in presence of temporomandibular dysfunction. what happens next? oral maxillofac surg clin north am. 2015;27(1):11-26. 33. kim yk, kim sg, kim jh, yun py. clinical survey of the patients with temporomandibular joint desorders using research diagnostic criteria (axis ii) for tmd: preliminary study. j cranio-maxillofac surg. 2012;40(4):366-72. 34. guimarães fo. r, oliveira jr. ec, gomes trm, souza tda. [quality of life in patients submitted to orthognatic surgery: oral health and selfsteem]. psicol cienc prof. 2014;34(1):242-51. portuguese. 35. silvola a s, tolvanen m, rusanen j, sipila k, lahti s, pirttiniemi p. do changes in oral health-related quality-of-life, facial pain and temporomandibular disorders correlate after treatment of severe malocclusion. acta odontol scand. 2016;74(1):44-50. 36. teng tt, ko ew, huang cs, chen yr. the effect of early physiotherapy on the recovery of mandibular function after orthognatic surgery for class iii correction: part i – jaw motion analysis. j cranio-maxillofac surg. 2015;43(1):131-7. signs and symptoms of temporomandibular disorder and patients’ satisfaction before and after orthognathic surgery 143 braz j oral sci. 15(2):137-143 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1531/1184 1/8 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1531/1184 2/8 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1531/1184 3/8 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1531/1184 4/8 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1531/1184 5/8 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1531/1184 6/8 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1531/1184 7/8 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1531/1184 8/8 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1515/1168 1/11 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1515/1168 2/11 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1515/1168 3/11 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1515/1168 4/11 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1515/1168 5/11 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1515/1168 6/11 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1515/1168 7/11 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1515/1168 8/11 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1515/1168 9/11 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1515/1168 10/11 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1515/1168 11/11 revista fop n 13 1320 effect of blood contamination during adhesive restorative procedures on dentin-resin cement shear bond strength carlos josé soares1 carolina assaf branco2 priscilla barbosa ferreira soares1 rodrigo borges fonseca1 hugo lemes carlo1 alfredo júlio fernandes neto2 1department of operative dentistry and dental materials, dental school federal university of uberlândia. 2department of occlusion and fixed prosthodontics, dental school federal university of uberlândia. received for publication: november 14, 2006 accepted: may 01, 2007 correspondence to: carlos josé soares universidade federal de uberlândia – faculdade de odontologia departamento de dentística e materiais odontológicos av. pará, nº 1720, bloco 2b, sala 2b-24, cep 38405-902 uberlândia – minas gerais – brazil phone: +55-34-32182255 fax: +55-34-32182279 e-mail: carlosjsoares@umuarama.ufu.br abstract the aim of this study was to evaluate the effect of dentin blood contamination during different steps of the restorative procedure on resin cement shear bond strength to dentin. crows of 120 bovine incisors were prepared to obtain flat superficial dentin surfaces. dentin was etched with phosphoric acid and contaminated with fresh blood for 10 seconds, before or after adhesive system application. different treatments were tested in contaminated dentin, resulting on eight groups (n=15). composite resin restorations (tph spectrum, dentsply) were adhesively fixed (excite, ivoclar-vivadent) with resin cement (variolink 2, ivoclar-vivadent) and shear bond strength test (0.5 mm/min) was performed. morphologic observations were carried out with scanning electron microscopy (sem). data (mpa) were submitted to one-way anova following tukey’s test (p<0.05), showing that blood contamination during adhesive procedure negatively affects bond strength, and decontamination methods do not recover original bond strength. the negative effects of blood contamination on shear bond strength to dentin and resin cement were significant in all contaminated groups; none of the tested dentin treatment procedures resulted in higher bond strength irrespective of the moment on which blood contamination took place. key words: blood contamination, adhesive system, dentin bonding, indirect restoration, shear bond strength braz j oral sci. april-june 2007 vol. 6 number 21 1321 introduction stable adhesion between resin composite and tooth structure is essential for the clinical success of restorations1. the creation of stable resin-dentin bonds relies on the formation of a hybrid layer that consists of partially or completely demineralized dentin and well-infiltrated, well-polymerized resins2. achieving a good moisture control is essential to avoid any contamination during the adhesive procedure, but this is a common problem in restorative dentistry, especially when rubber dam isolation is not feasible3. many carious lesions, which require the use of dentin bonding agents, are located on areas that are difficult to isolate and, therefore, dentin contamination with blood may occur. blood contamination of the adhesive surfaces may affect the bond strengths of adhesive resins3-7. recent studies have evaluated the effect of blood contamination of adhesives to dentin using different testing methods6-9, showing a significant reduction in bond strength values. shear and microtensile tests are the most common methods used for determining the bond strengths of adhesive systems, revealing valuable clinical information10. the negative effect of blood on adhesion can be attributed to its high protein content (6-7%)8. the high protein content together with macromolecules as fibrinogen and platelets, can form a thin film on the dentin surface and prevent adhesive infiltration8. on the other hand, if contamination occurs on previous infiltrated dentin, the interaction between adhesive system and resin cement will be affected6. regardless the time of blood contamination (before or after the adhesive application), different decontamination procedures have been used on contaminated dentin to recovery the original bond strength values6,8, but literature is still contradictory. some studies show that decontamination procedures can recover bond strength6,9, but others show the contrary7. then, it seems possible that contaminant-removing treatments after blood contamination could increase the bond strength. a re-etching, water rinsing or adhesive reapplication are some of these techniques3,6,8. the purpose of this study was to evaluate the effect of blood contamination at different steps during the restorative procedure on the shear bond strength between dentin and resin cement, and to determine the best decontamination method to re-establish the original resin-dentin bond strength. the null hypothesis tested was that different treatments of the blood contaminated dentin do not affect the bond strength. material and methods one hundred and twenty freshly bovine mandibular incisors extracted from two-to-three year old cattle were stored in thymol 0.2% at 4oc for up to two weeks and randomly assigned to eight groups (n=15). the roots of the teeth were cut-off with a double faced diamond disk (kg sorensen, barueri, brazil) and any tissue remnant and debris were removed. the crowns were then mounted in a cylinder with self-curing polystyrene resin (cromex, piracicaba, brazil) and their labial surface was ground with wet 180-, 320and 600-grit sic abrasive paper (norton abrasivos, campinas, brazil) in order to obtain flat superficial dentin surfaces and to produce a standardized smear layer. an adhesive tape with a 3mm central perforation was positioned on the flat dentin surface to demarcate the bonding area. this methodology ensured a standardized measurement of the bonding area, facilitating the obtainment of accurate bond strength values. a condensation silicone (zeta plus and oral wash, zhermack, italy) was used to take an impression of a metallic cylinder (3x3mm), and after polymerization the cylinder was removed and the impression used to build indirect composite restorations. the composite resin (tph spectrum, dentsply, milford, usa) was inserted in three increments and light-polymerized for 40 seconds with a conventional halogen light curing unit xl 2500 600mw/cm2 (3m-espe, st. paul, usa); then, samples were post-polymerized in a autoclave at 110ºc for 15 minutes. dentin was etched with phosphoric acid (35%; ivoclarvivadent, schaan, liechtenstein) for 15 seconds, copiously washed for 30 seconds and excess of water removed with an tissue paper (snack, melhoramentos papéis ltda, caieiras, brazil). in accordance with the ethics committee of federal university of uberlândia, brazil, human blood was collected from one volunteer and immediately used. blood was dropped to contaminate the dentin surface for 10 seconds, before or after adhesive system application. different protocols were tested due to blood contamination before or after adhesive application, resulting in eight groups (figure 1): g1 (control): after acid etching (ae), an one-bottle adhesive system (excite, ivoclar-vivadent) (ex) was applied with a disposable brush, left untouched for 20s, re-applied and light-cured for 20s, and the indirect composite restoration (ic) was fixed with a resinous cement (variolink 2, ivoclarvivadent); in g2, g3 and g4 contamination occurred before adhesive application: g2: after ae, blood contamination (bc) was carried out by drooping blood from a capillary tube on all exposed dentin surface, followed by air drying for 5 seconds (ad), ex, and ic; g3: ae, bc, water spray washing for 10 seconds (ws), ex and ic; g4: ae, bc, ws, ae, ex and ic; in groups g5, g6, g7 and g8 contamination occurred after adhesive application: g5: ae, ex, bc, ad and ic; g6: ae, ex, bc, ws and ic; g7: ae, ex, bc, ws, ex and ic; g8: ae, ex, bc, ws, ae, ex and ic. restorations were fixed with a dual-cure resinous cement (variolink 2, ivoclar-vivadent) under 500g of load and lightcured in two opposite directions for 40s. samples were stored in distilled water at 37ºc immediately after bonding, for 24hs, and then a shear bond test was conducted in a testing machine (emic dl 2000, são josé dos pinhais, brazil) at a crosshead speed of 0.5mm/minute. the knife-edge chisel was applied parallel to the dentin surface with a source-to-sample braz j oral sci. 6(21):1320-1325 effect of blood contamination during adhesive restorative procedures on dentin-resin cement shear bond strength 1322 fig. 1 experimental groups and surface treatments. footnotes for figure 1: ae: acid etching; ex: one-bottle adhesive system application (excite, ivoclar-vivadent); ic: indirect composite restoration cementation; bc: blood contamination; ad: 5 seconds air drying; ws: 10 seconds washing with water spray. distance of approximately 0.2mm, and perpendicular to the composite restoration. shear bond strength was calculated by dividing the ultimate load (n) by the bonded surface area (mm²). two samples of each group were prepared for sem evaluation. specimens were soaked in 6 mol/l hcl for 5s, followed by immersion in 5% sodium hypochlorite for 5 min, dehydrated in ascending concentrations of ethanol (50% for 10 min; 70% for 10 min; 95% for 10 min; and 100% for 30 min) and fixed in 2.5% glutaraldehyde. thereafter, the specimens were gold sputter-coated and observed at the sem (zeiss dsm 940 a, germany). data was analyzed by one-way anova, followed by tukey’s multiple comparisons test with a 5% level of significance. results mean shear bond strength values were expressed in mpa. data presented a normal and homogeneous distribution, which enabled parametric analysis. one-way anova revealed that there was a significant difference on the bond strength of the different groups (table 1). there was a significant decrease in bond strength due to blood contamination, regardless the cementation protocol used for each group. there was no statistical difference control contamination before bond system application contamination after bond system application g1 g2 g3 g4 g5 g6 g7 g8 11.6±3.8a 2.9±1.2c 7.3±3.2b 8.4±2.1b 7.6±2.0b 7.1±2.0b 8.3±1.9b 6.7±1.1b groups means±sd table 1 means and standard deviations (in mpa), and results of statistical analysis of the shear bond strength of experimental groups by anova following tukey’s test (a=0.05). sd: standard deviation values. different letters mean statistical significant differences (p<0.05) braz j oral sci. 6(21):1320-1325 effect of blood contamination during adhesive restorative procedures on dentin-resin cement shear bond strength 1323 between contaminated groups, except when the blood contamination was followed by air spray and adhesive application (g2), resulting in the lowest bond strength mean. sem evaluation showed the presence of erythrocytes within partially infiltrated dentin (figure 2) and adhesive failures between dentin and adhesive system (figure 3) or between adhesive system and resin cement (figure 4). fig.4 specimen from group g5 – contamination with blood occurred after adhesive application (ex), similar to the specimens from groups g6, g7 and g8. ir: indirect restoration; rc: resin cement; hl: hybrid layer; d: dentin. (original magnification x1000). fig. 2 specimen from group g2 contamination with blood occurred before adhesive application (ex), similar to the specimens from groups g3 and g4. the black arrow shows clusters of erythrocytes right under the hybrid layer (hl), jeopardizing bond strength. rc: resin cement; hl: hybrid layer; d: dentin. (original magnification x3000). fig. 3 specimen from group g3, showing fracture within hybrid layer (hl) that was caused by blood contamination before adhesive application. ir: indirect restoration; rc: resin cement; hl: hybrid layer; d: dentin. (original magnification x1000). discussion blood contamination is a major clinical problem during restorative dental treatment8. kaneshima et al.6 stated that the effects of blood contamination on bond strength of adhesive resin to dentin may vary greatly depending on the adherent surface conditions. according to basic concepts of adhesion, the closer the contact between the adhesive and the adherent, the stronger is their junction11. therefore, the contaminated layer may become a strong mechanical inhibitor of adhesion, preventing both adhesive system infiltration and polymerization, thus adversely affecting its bonding with the resin cement6. dental surface contamination can occur at two critical times during the bonding procedure: after the tooth surface had been etched (figures 2 and 3) and/or after the adhesive system had been applied (figure 4). as a result, bonding can be compromised at both times. in this study, the negative effects of blood contamination on shear bond strength to dentin and resin cement were significant in all contaminated groups. previous studies showed a decrease on shear bond strength when blood contamination occurred before the adhesive system application6,8. blood residues or reactants obstructing the dentin tubules can inhibit primer infiltration into dentin6,8, thus interfering with hybrid layer formation. figure 2 shows the presence of erythrocytes right below the hybrid layer, which seems to be responsible for failures within the hybrid layer, as seen in figure 3. in this study, blood air drying after contamination (g2) caused a catastrophic decrease in bond strength between the adhesive and dentin, which is in braz j oral sci. 6(21):1320-1325 effect of blood contamination during adhesive restorative procedures on dentin-resin cement shear bond strength 1324 agreement with previous published results6,8. similarly to kaneshima et al.6 when the blood was applied and just dried on the surface, a great number of blood residues were observed on dentin surface (figure 2). this situation might have left more blood contaminants on dentin surface and also caused collagen fibrils to collapse, preventing adhesive monomers to infiltrate12. the blood protein components trapped on the dentin surface interferes with the bonding agent ability to form a uniform surface coating3,8. according to abdalla et al.8 resin tags formed on air-dried dentin surfaces were found to be fewer and much shorter than that in control conditions and blood contamination prevents adhesive infiltration into dentin tubules. in spite of the fact that most of dentin bond strength is due to resin infiltration in intertubular dentin12, the absence of tag formation can be interpreted as a direct result of the presence of contaminants which would also jeopardize infiltration into collagen fibrils reducing bond strength, as seen in this study. then, the use of air drying on contaminated dentin, before adhesive application, should be avoided. water spray (ws) after dentin contamination increased bond strength (g3) to the same level that when ws was followed by re-etching (g4). in a study by xie et al.9 plasma contamination lowered bond strengths by 33 – 70% for both enamel and dentin, but re-etching restored bond strength. in this study the use of blood instead of just plasma contamination showed that dentin re-etching did not improve bond strength. then, in this situation re-etching should be avoided because a severe dentin decalcification can denature collagen fibrils1, and there is not a significant increase in bond strength. the use of an organic solvent on the contaminated surface to re-establish the capacity of formation of the hybrid layer seems to be a good alternative in cases of blood contamination. kaneshima et al.6 found reduced bond strength unless the most superficial layer (exposed collagen fibrils) was removed from the surface, before or after blood contamination, with hypochlorite solution6,13. they concluded that large blood corpuscle elements could have been completely rinsed away; then the adverse interaction between the exposed collagen meshwork and the blood protein components that could inhibit primer infiltration into dentin was eliminated. the removal of surface collagen fibrils eliminates the creation of a hybrid layer and can produce high bond strength by the closer contact between adhesives and dentin14 but collagen impregnation and the formation of high quality hybrid layers are still regarded as the main mechanism of dentin adhesion15. when blood contamination occurs after adhesive application the interaction between adhesive system and resin can be jeopardized6. eiriksson et al.3 evaluated the effects of blood contamination on resin-resin interfaces showing that blood protein components were not able to be completely rinsed away by water spray, lowering the surface energy of the cured composite. they concluded that the application of a more fluid adhesive layer significantly increased the resin-resin bond strength. in the present study, irrespective of the cementation procedure, when blood contamination occurred after adhesive system application, a new adhesive layer (g7 and g8) did not result on higher bond strength, probably because blood protein components still impaired an effective adhesive interaction. figure 4 shows failure right above the hybrid layer as a signal of blood contamination after adhesive application. in spite of the fact that no significant differences were found between g8 and the other contaminated groups, except g2, the mean shear bond strength of g8 was relatively lower when compared to the other treatments. in this group, blood contamination occurred after adhesive system application, and it was followed by water spray, re-etching and application of a new adhesive layer. re-etching either seemed not able to clean the surface or it could also have made this surface more acidic causing an adverse surface interaction16. the new etching over previous polymerized adhesive layer was rinsed off by water spray for 10s, but if any remnant of the phosphoric acid was still present it could react with the adhesive system tertiary amine initiator thus interfering with the polymerization of the new adhesive layer16. this occurrence may be the cause for g8 having lower, but not significant, bond strength than g7, where a new adhesive layer was applied but it was not preceded by re-etching. further studies are necessary to confirm this hypothesis. the bond strength reported in this study when blood contamination occurred after adhesive system application was substantially lower than those reported in previous investigations3,6,8,13. however, published researches that deal with the effects of blood contamination on adhesive restorations are limited and comparisons are difficult. these differences in results between studies may especially be due to variations in interpretation of what constitutes a contaminated dentin surface8, and the bond strength test used17-18. in addition, several variables have been identified to influence the results of bond strength measurements, including bonding systems, type of substrate, type of blood fresh or anticoagulated19, the origin and condition of the dentin substrate, the etching, priming and bonding procedures and storage of the specimens. few researchers had used freshly drawn blood3,6,8,20, as in this study. dietrich et al.19 found significantly higher percentages of marginal openings after contamination with fresh capillary blood compared to anticoagulated blood. another important point is the bond strength test used on each research. each test keeps its advantages and limitations in determining the significance of the results obtained. hence, comparisons among studies are difficult because a variety of tests, and are not always feasible. if different tests are used, it seems difficult to compare and draw conclusions with data gathered from different studies10. the shear bond strength test used in this study is an extensively used test4-5,8,21-22 and it has been one of the most common laboratory techniques for evaluating braz j oral sci. 6(21):1320-1325 effect of blood contamination during adhesive restorative procedures on dentin-resin cement shear bond strength 1325 adhesives on resin-bonded restorations17. for sure, testing adhesion with shear bond strength may not be the most specific method17 but shear represents a destructive force which occurs in almost all restorations in clinical function, due to a complex stress distribution of bite forces23. in spite of the limitations imposed by this method, its direct association with what happens in real situations may be the reason for many researchers still use it. the results from the present study clearly indicated that, when the dentin surface was contaminated either before or after application of the adhesive system, any of the cementation protocols recovered the bond strength to the level of the control group. it is likely that contaminants may have remained on the dentin surface, thus interfering with the formation of a hybrid layer or inhibiting the bonding of adhesive system to resin cement. therefore, the most important factor for ensuring optimal bonding is to avoid blood contamination. however, furthers studies varying the conditions, specially the laboratory technique and the adhesive system, are necessary for definitive conclusions. acknowledgements authors are grateful to ivoclar-vivadent and dentsply for partial donation of the materials used in this study. the authors have no financial interest in any of the products mentioned in this article. references 1. torii y, itou k, nishitani y, ishikawa k, suzuki k. effect of phosphoric acid etching prior to self-etching primer application on adhesion of resin composite to enamel and dentin. am j dent. 2002; 15: 305-8. 2. van meerbeek b, de munck j, yoshida y, inoue s, vargas m, vijay p, et al. buonocore memorial lecture. adhesion to enamel and dentin: current status and future challenges. oper dent. 2003; 28: 215-35. 3. eiriksson so, pereira pn, swift ej, heymann ho, sigurdsson a. effects of blood contamination on resin-resin bond strength. dent mater. 2004; 20: 184-90. 4. cacciafesta v, sfondrini mf, scribante a, de angelis m, klersy c. effects of blood contamination on the shear bond strengths of conventional and hydrophilic primers. am j orthod dentofacial orthop. 2004; 126: 207-12. 5. hobson rs, ledvinka j, meechan jg. the effect of moisture and blood contamination on bond strength of a new orthodontic bonding material. am j orthod dentofacial orthop. 2001; 120: 54-7. 6. kaneshima t, yatani h, kasai t, watanabe ek, yamashita a. the influence of blood contamination on bond strengths between dentin and an adhesive resin cement. oper dent. 2000; 25: 195-201. 7. yoo hm, pereira pn. effect of blood contamination with 1step self-etching adhesives on microtensile bond strength to dentin. oper dent. 2006; 31: 660-5. 8. abdalla ai, davidson cl. bonding efficiency and interfacial morphology of one-bottle adhesives to contaminated dentin surfaces. am j dent. 1998; 11: 281-5. 9. xie j, powers jm, mcguckin rs. in vitro bond strength of two adhesives to enamel and dentin under normal and contaminated conditions. dent mater. 1993; 9: 295-9. 10. de munck j, van landuyt k, peumans m, poitevin a, lambrechts p, braem m, et al. a critical review of the durability of adhesion to tooth tissue: methods and results. j dent res. 2005; 84: 118-32. 11. baier re, shafrin eg, zisman wa. adhesion: mechanisms that assist or impede it. science. 1968; 162: 1360-8. 12. pashley dh, carvalho rm. dentine permeability and dentine adhesion. j dent. 1997; 25: 355-72. 13. wakabayashi y, kondou y, suzuki k, yatani h, yamashita a. effect of dissolution of collagen on adhesion to dentin. int j prosthodont. 1994; 7: 302-6. 14. prati c, chersoni s, pashley dh. effect of removal of surface collagen fibrils on resin-dentin bonding. dent mater. 1999; 15: 323-31. 15. wang y, spencer p. hybridization efficiency of the adhesive/ dentin interface with wet bonding. j dent res. 2003; 82: 141-5. 16. tay fr, pashley dh, yiu ck, sanares am, wei sh. factors contributing to the incompatibility between simplified-step adhesives and chemically-cured or dual-cured composites. part i. single-step self-etching adhesive. j adhes dent. 2003; 5: 2740. 17. della bona a, van noort r. shear vs. tensile bond strength of resin composite bonded to ceramic. j dent res. 1995; 74: 15916. 18. pashley dh, sano h, ciucchi b, yoshiyama m, carvalho rm. adhesion testing of dentin bonding agents: a review. dent mater. 1995; 11: 117-25. 19. dietrich t, kraemer ml, roulet jf. blood contamination and dentin bonding—effect of anticoagulant in laboratory studies. dent mater. 2002; 18: 159-62. 20. garey dj, tjan ah, james ra, caputo aa. effects of thermocycling, load-cycling, and blood contamination on cemented implant abutments. j prosthet dent. 1994; 71: 12432. 21. cacciafesta v, sfondrini mf, de angelis m, scribante a, klersy c. effect of water and saliva contamination on shear bond strength of brackets bonded with conventional, hydrophilic, and self-etching primers. am j orthod dentofacial orthop. 2003; 123: 633-40. 22. park jw, lee kc. the influence of salivary contamination on shear bond strength of dentin adhesive systems. oper dent. 2004; 29: 437-42. 23. anusavice kj. phillips’ science of dental materials. 11th ed. st. louis: wb saunders; 2003. braz j oral sci. 6(21):1320-1325 effect of blood contamination during adhesive restorative procedures on dentin-resin cement shear bond strength untitled 1 volume 16 2017 e17090 original article 1 dds. school of dentistry, heminio ometto university center, uniararas. dr. maximiliano baruto, 500. araras-sp. 13607-339, brazil.taozaniboni@hotmail.com 2 phd. school of dentistry, heminio ometto university center, uniararas. dr. maximiliano baruto, 500 ararassp 13607-339, brazil mario@ vedovelloeassociados.com.br 3 phd. division of periodontics, school of dentistry, state university of são paulo, unesp. av. eng. francisco jose longo, 777. são josé dos campos – sp. 12245-000, brazil mauro.santamaria@fosjc.unesp.br 4 phd. division of periodontics, school of dentistry, state university of são paulo, unesp.av. eng. francisco jose longo, 777. são josé dos campos – sp. 12245-000, brazil mauro.santamaria@fosjc.unesp.br 5 phd. department of oral pathology, bauru dental school, university of são paulo, usp. alameda otávio pinheiro brisolla, 9-75. vila universistária. bauru-sp. 17012-901, brazil. mfmartinsortiz@gmail.com 6 phd. department of oral pathology, bauru dental school, university of são paulo, usp. alameda otávio pinheiro brisolla, 9-75. vila universistária. bauru-sp. 17012-901, brazil.consolaro@uol.com.br 7 phd. school of dentistry, heminio ometto university center, uniararas. dr. maximiliano baruto, 500 araras-sp 13607-339, brazil santamariajr@ig.com.br corresponding author: milton santamaria jr heminio ometto university center, uniararas. dr. maximiliano baruto, 500 araras-sp 13607-339, brazil received: january 14, 2017 accepted: november 23, 2017 root morphology can be a risk factor for periodontal damage and root resorption in orthodontic movement ewerton zaniboni1, mário vedovello filho2, mauro pedrine santamaria3, maria aparecida neves jardini4, maria fernanda martins-ortiz5, alberto consolaro6, milton santamaria jr7*. aim: the study evaluated, using histomorphometry, the percentage of hyaline area in periodontal ligament (pdl) and root resorption in orthodontic tooth movement (otm). methods: ten rats were divided into two groups. g3 group (n=5), with 3 days of otm and g7 group (n=5), with 7 days of otm. a control group (n=5) consisted of contralateral teeth of each animal, which were not moved. maxillary left first molar was moved, using stainless steel spring connected to the incisors with 40g force. microscopic analysis was done in transversal sections of the mesiovestibular (mv) and distovestibular (dv) roots in the cervical level. results: there was a pdl hyaline area in the dv root of 6.2% in g3 and 1.8% in g7. the root resorption area in g7 was 0.9%. on mv root and control group were not found occurrences of hyaline areas in pdl and no root resorption. conclusions: based on the results obtained, it might be concluded that smaller roots showed higher frequency of hyaline areas and root resorption. keywords: tooth movement. periodontal ligament. root resorption. orthodontics. http://dx.doi.org/10.20396/bjos.v16i0.8651188 2 zaniboni et al. introduction the complexity of events in orthodontic tooth movement (otm) involves forces of compression and traction on the periodontal ligament (pdl) and alveolar bone, inducing morphological and microscopic reactions controlled by cytokines and growth factors, promoting tooth displacement1-4. in orthodontics, a mechanical stimulus induces tooth movement5-6. the orthodontic force causes an imbalance in pdl, an inflammatory process7-8 where cytokines produced by osteoclasts, osteoblasts and osteocytes control bone remodeling around pdl9-10. experimental studies allow clinical and microscopic evaluation of the tooth movement biology and root resorption. different aspects may interfere in the effectiveness of otm, such as the intensity and type of force applied, local and systemic diseases, bone alterations and the root morphology11. when the pdl is overly compressed, it can cause cell death, creating a hyaline area, cementoblast layer damage and root resorption12. hyaline areas in pdl will delay tooth movement and will facilitate the presence of root resorptions13-15. root resorption during otm is a frequent phenomenon, but must not be considered normal or physiological. in some orthodontic treatments, apical root resorption is inevitable, but predictive factors can be defined12-14. it will depend on the magnitude, duration and type of force, which may cause many degrees of root resorption16-18 in different root morphologies17,19,20. this study proposes to demonstrate the microscopic aspects of tooth movement in rats, by analyzing the hyaline areas in pdl and root resorptions in otm, on roots with different types of morphology. methods sampling the procedures of this research were performed in compliance with the ethical and legal recommendations specified by the animals ethics commission (ceua) of fundação hermínio ometto uniararas (report no 030/2013). the research used wistar rats (rattus norvegicus, albinos), male, three month old. they were kept at temperature of 25oc, in plastic cages and were provided food (nuvilab, quimtia s.a, colombo/pr, brazil) and water ad libitum. ten animals were divided into three groups. group g3 (n=5): three days of orthodontic tooth movement (otm), group g7 (n=5): seven days of orthodontic tooth movement (otm) and control group (n=5): contralateral teeth not moved of each animal. orthodontic movement the animals were anesthetized with three parts of ketamine hydrochloride, 100mg/ml (cetamin, syntec do brasil ltda, cotia/sp, brazil) one part of the xylazine hydrochloride, 20mg/ml (xilasin, syntec do brasil, cotia/sp, brazil) at the dose of 1ml/kg, applied intramuscularly. 3 zaniboni et al. a closed stainless steel spring was placed between the maxillary left first molar (point of force application) and the maxillary incisors (point of anchorage), tipping forward the first molar, applying 40gf (figure 1)21. quantitative histomorphometric analysis the animals were euthanized with an overdose of the anesthetic ketamine and xylazine mixture. the maxillae were placed in a 10% buffered formol solution for fixation during 2 days. they were demineralized with edta (monobasic sodium phosphate 4.4g; dibasic sodium phosphate 45g; edta 70g; deionized h2o, 1000 ml; formaldehyde pa 50 ml) in a period of eight weeks. the maxillae were embedded in paraffin and cut in transversal cross sections (5µm), stained with hematoxylin-eosin and the roots were analyzed in the cervical level22. the mesiovestibular (mv) and distovestibular (dv) roots were analyzed in optical microscope with an objective with 10x magnification (zeiss ks 300, version 3.0). five sections per animal were used for histomorphometrical quantification (figure 2). figure 1. device used in the movement of the first molar. spring installed between the first molar and the incisors. figure 2. a microscopic cross section of the jaw (staining he, magnification-4x). b microscopic cross section of the upper first molar, occlusal view. mv mesiobuccal root. dv distobuccal root (staining he, magnification-10x). a b mv dv 4 zaniboni et al. division of hyaline area by the total pdl area gives the percentage of periodontal hyaline area (figure 3). the percentage of root resorption was calculated at the same way, with the division of resorption area by the total root area (figure 4). the frequency of root resorption events was also verified in each animal by total in group (n=5) in the different periods of otm and roots. the mean of hyaline area percentage of pdl and percentage of root resorption were compared by anova and the tukey post-test (p<.05). figure 3. cross section of distobuccal root (dv) with 3 days of orthodontic movement. a section area of the pdl. b * periodontal hyaline area (staining he, magnification-40x). a b figure 4. cross section of distobuccal root (dv) with 7 days of orthodontic movement. a section area of the root, including cementum and dentin (staining he, lens 4x). b * root resorption area (staining he, magnification-40x). a b * * 5 zaniboni et al. results hyaline area percentage in pdl and root resorption percentage, according to the otm period and analyzed root are presented in table 1. the mv root presented no segmental hyaline areas in pdl. there were hyaline areas in otm groups of the dv root, and were statistically significant in comparison of the control and mv groups (p≤0.05). the hyaline areas were greater in 3rd day (mean of 6.2%) than in 7th day (mean of 1.8%). on mv root, there was no root resorption. on the dv root resorption appeared in the 7th period of otm (mean of 0.9%). the frequency of root resorption occurred in the period of 7 days of otm. this frequency of root resorption was 3:5 (60% of the animals) in the dv root (table 2). discussion animals as experimental models allows clinical reproducibility22. this in vivo research provides relevant data on physiological and pathological conditions that may be useful for establishing more effective clinical interventions23. orthodontic tooth movement (otm) causes resorption of alveolar bone on the compression side and osteogenesis on the tension side. studies investigating the mechanisms involved in this process are important to improve orthodontic treatment24. this otm experimental design21, tips forward the first molar, without interfering on the rat craniofacial structure. it is efficient to study bone remodeling and root resorptions in orthodontics. this model allowed studies such as auxiliary therapies25 and drug interference26 on bone remodeling in orthodontic movement. transversal sections give a direct view of the alveolar bone between the roots, and also allow evaluation of the cortical bone in the same section. thus, all the structable 1. mean of hyaline percentage area of pdl and percentage of root resorption, found in each group, according to the otm period and root analyzed. root hyaline area (%) root resorptions area (%) control 3 days 7 days control 3 days 7 days mv 0 0 0 0 0 0 dv 0 6.2* 1.8* 0 0 0.9* * statistically significant compared to the control group on each root analyzed (p≤0.05). mv mesiovestibular root. dv distovestibular root. table 2. frequency of root resorption (animals by total in group), according to the otm period and root analyzed. root control 3 days 7 days mv dv (0:5) 0% (0:5) 0% (0:5) 0% (0:5) 0% (0:5) 0% (3:5) 60% 6 zaniboni et al. tures could be analyzed simultaneously, and compared with the same structures on the contralateral control side, without otm, being an important control of the biologic reactions found. in the longitudinal sections it is not possible to visualize all the roots at the same time and neither to simultaneously analyze the different anatomic regions, such as cortical and medullary bone27. mesiovestibular (mv) and distovestibular (dv) roots are exposed to moderate forces and intense forces, respectively, during otm. the root morphology has an influence on the orthodontic force intensity and pdl tissue reaction, and in the production of root resorptions. in this research, two roots of the rat first molar were compared and evaluated simultaneously. the mv root, bigger, presented no root resorption. bigger roots with larger dimensions present better distribution of the applied forces. this fact was confirmed given that no hyalinization of the pdl was found in the mv root in the otm groups. the presence of hyalinized tissue within the pdl is a microscopic sign of excessive compression, resulting from the intense application of forces. when these forces are intense and prolonged, the cells that line the tooth roots, the cementoblasts are injured. the cementoblasts protect the root surface, because they do not have receptors for the mediators that participate in bone remodeling12-14. the majority of external root resorptions, as an initial phenomenon, present large destruction of the layer of cementoblasts, denuding the mineralized dentin surface and exposing it to the action of the bone remodeling cells12-14. therefore, excessive orthodontic forces may result in undesired root resorptions. root resorption could be seen with abundance in the dv root, in the period of 7 days of movement, in which the percentage and the frequency were greater than in the mv root. this result is explained because its morphology is smaller in comparison with those of the mv root. on the 3th day of movement, there was clear evidence of the high incidence of forces on the mesial surface of this dv root, with the presence of large segmental pdl hyaline areas. the results demonstrated the anatomic influence on root resorptions. the dv root dimension is smaller, with its conical shape, as in a single-rooted tooth, promoting greater pressure and less distribution of forces on pdl walls. thus, smaller roots may present evident effects, such as root resorption and hyaline area, because they have poor ability to dissipate compressive force during tooth movement.20 references 1. krishnan v, davidovitch z. cellular, molecular, and tissue-level reactions to orthodontic force. am j orthod dentofacial orthop. 2006 apr;129(4):469.e1-32. 2. cattaneo pm, dalstra m, melsen b. strains in periodontal ligament and alveolar bone associated with orthodontic tooth movement analyzed by finite element. orthod craniofac res. 2009 may;12(2):120-8. doi: 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[experimental tooth movement in murines: study period and direction of microscopic sections]. rev dent press ortod ortop facial. 2009 jan-feb;14 (1):143-57. doi: 10.1590/s1415-54192009000100014. portuguese. 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1542/1195 1/9 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1542/1195 2/9 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1542/1195 3/9 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1542/1195 4/9 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1542/1195 5/9 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1542/1195 6/9 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1542/1195 7/9 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1542/1195 8/9 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1542/1195 9/9 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1521/1174 1/9 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1521/1174 2/9 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1521/1174 3/9 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1521/1174 4/9 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1521/1174 5/9 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1521/1174 6/9 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1521/1174 7/9 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1521/1174 8/9 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1521/1174 9/9 revista fop n 13 1631 influence of sex on temporomandibular disorder pain: a review of occurrence and development ana paula botelho1; maria cecília ferraz de arruda veiga2 1ms, graduate student 2phd, professor, department of physiology piracicaba dental school, university of campinas, piracicaba, sp, brazil received for publication: july 21, 2008 accepted: september 09, 2008 correspondence to: maria cecília ferraz de arruda veiga departmento de ciências fisiológicas faculdade de odontologia de piracicaba, unicamp cx postal 52, 13414-903 piracicaba, sp, brasil phone: +55-19-2106-5306 fax: +55-19-2106-5218 e-mail: cveiga@fop.unicamp.br abstract aim: the aim of this study was to develop a narrative literature review using international research to present the influence of sex on occurrence and development of temporomandibular disorder (tmd) pain. methods: the data sources were computer-based searches in pubmed between 1987 and feb 2008 using appropriate keywords. for inclusion in this review, articles had to meet the following criteria: be written in english; include human and nonhuman subjects; be published a full-text paper in a peer-reviewed medical journal. results: the studies considered eligible for this narrative review presented results in agreement with the difference in sex and orofacial pain. patients were almost always adults, with particular focus on patients’ sex. clinical conditions were predominantly tdm pain. since sexual dimorphism was detected in tmd pain, the results are focused on women. conclusion: the findings of this review suggest that there is difference in the occurrence and development of pain according to the individual’s sex, women being more susceptible to tmd pain. keywords: temporomandibular disorder; pain; sex difference; review. i n t r o d u c t i o n being male or female is one of the most important predictors of an individual’s health. compared to women of similar age, women outnumber men for stress-related bodily complaints such as chronic pain1. the most common cause of chronic facial pain conditions involves temporomandibular disorder (tmd)2. tmd pain is the most common symptom that compels patients to seek therapy; its management, however, mostly involves a multidisciplinary approach2. dentists, orthodontists, psychologists, physical therapists, and physicians work together to address the condition of the patient with tmd3. for a very long period, the sex of subjects used to study pain was rarely taken into account in either basic or clinical studies4. epidemiological studies on nonpatient populations in the early 1970s reported that the prevalence of tmd signs and symptoms was similar for men and women. studies of tmd signs and symptoms in nonpatients revealed either no gender difference or a somewhat greater prevalence among women. in the 1990s, a longitudinal study5, however, showed that the course of tmd symptoms differed significantly with respect to gender: women who had reported symptoms during adolescence consistently reported symptoms 1 decade later, whereas only 60% of men reported symptoms later. in view of the need for dealing with pain during tmd treatment, the objective of this review was to present the influence of sex on the occurrence and development of tmd pain. identification and review of studies computer-based searches in pubmed full-text paper electronic database were conducted using combinations of the following keywords: pain, temporomandibular disorder, orofacial pain, sex-related difference, sexual dimorphism and gender difference pain. reference sections from published articles in the field were also used as sources. no attempt was made to contact study authors. to be included in this review, articles had to meet the following criteria: (1) be written in english; (2) include human and nonhuman subjects; (3) be published as a fulltext paper in a peer-reviewed medical journal between 1987 and feb 2008. occurrence of temporomandibular disorder pain chronic orofacial pain affects approximately 10% of adults braz j oral sci. july/september 2008 vol. 7 number 26 1632 and up to 50% of the elderly. there is evidence that sex differences in masticatory muscle pain and tenderness emerge as early as 19 years of age1. childbearing-age women, mainly those in their 40s, seek treatment for orofacial pain more frequently in comparison with men by a 2:1 ratio. although women are more likely to seek medical care for pain, they also report more pain for which they do not seek treatment6. moreover, the difference between the two sexes is multifaceted, involving the occurrence of chronic pain, the type of pain syndromes experienced, the characteristics of the complications that develop, etc. there could be several reasons for the higher reactivity of women compared to men to a similar painful stimulation, ranging from genes to hormonal and cultural influences4. converging lines of evidence suggest that there are important sex-related influences on the experience of pain. women report more pain than men and are at greater risk for developing many forms of chronic pain7. laboratory studies consistently report lower pain threshold and tolerance among women, and these effects are moderate in magnitude. in addition to these human data, abundant nonhuman animal research indicates sex differences in nociceptive responses. while the clinical implications of these sex differences in pain responses are not yet precisely defined, it is becoming increasingly clear that future improvements in the effectiveness of pain management will require taking the patient’s sex into account8. tmd refers to a group of conditions, whose principal symptom is pain in the masticatory muscles and/or temporomandibular joints on palpation and during function (e.g., chewing, mouth opening, speech)9-10. the classification and epidemiology of orofacial pain presents challenges because of the many anatomic structures involved, diverse causes, unpredictable pain referral patterns and symptoms, and a lack of consensus with regard to differential diagnostic criteria6,11. a number of aspects of the prevalence pattern of tmd suggest that reproductive hormones may play a role in these pain conditions12: the prevalence of tmd pain prior to adolescence is low (2-4%), and does not seem to differ for boys and girls. however, prevalence rates are higher in adult women than in adult men, and the prevalence is lower for women in the postmenopausal years than for those of reproductive age13. the existence of sex differences in pain and analgesia, and the fact that the developmental profile of some types of pain clearly parallels reproductive function strongly suggest that gonadal steroid hormones significantly influence pain14. tmd are 1.5-2 times more prevalent in women than in men in the community, and 80% of treated cases of tmd are women15-16. moreover, women are at significantly greater risk than men of experiencing tmd-related disability, which is associated with significant use of health services and increased use of opioid and sedative hypnotic medications. in addition, treatment of tmd can be associated with severe iatrogenic consequences17. furthermore, chronic tmd has been found to interfere with normal social activity and interpersonal relationships and to negatively affect the ability to maintain employment3. individuals react to stressful events in different ways, and differences in the physiological stress response are important determinants of health. a stressful stimulus results in the activation of several physiological pathways including the hypothalamic-pituitary-adrenal axis (hpaa) and the autonomic nervous system. a considerable body of research during recent years has linked the function of both of these systems with the pathogenesis of several common disorders, including coronary arterial disease, type 2 diabetes, metabolic syndrome, depression and stressrelated bodily complaints. importantly, both systems show a clear sex-specific pattern of response. therefore, stress reactivity is a major candidate for a mechanism explaining why some diseases are more common in men and others in women1. tmd is usually manifested by one or more of the following signs or symptoms: pain, joint sounds, limitation in jaw movement, muscle tenderness, and joint tenderness. it also is commonly associated with other symptoms affecting the head and neck region such as headache, ear-related symptoms, and cervical spine disorders. patients with chronic tmd frequently report symptoms of depression, stress, anxiety, poor sleep quality, and low energy3,18. knowing what biological mechanisms underlie such profound differences may be extremely helpful in elucidating the pathogenesis of various common disorders, a crucial step in developing their prevention and treatment1. the prevalence of several pain conditions located in the craniofacial region and the mechanisms that underlie sexrelated differences remain obscure and probably involve both physiological and psychosocial factors9,19. development of orofacial pain nociception results from the activation of primary afferent nociceptors and the transmission of the nociceptive information to the spinal cord from where it is relayed to supra spinal levels. following tissue injury and inflammation, primary afferent nociceptors are sensitized by mediators released from diseased or damaged tissue or from the immune system in such a way that previously slight or ineffective stimulation becomes effective in inducing nociception. this primary sensory nociceptor sensitization is referred to as hyperalgesia20. inflammatory pain is a pervasive problem and usually results in both spontaneous pain and hyperalgesia. although the hyperalgesic state does not necessarily involve ongoing pain, the nociceptive threshold is lowered in this state, and the application of a nonnoxious mechanical, thermal, or chemical stimulus induces a nociceptive behavior response. however, spontaneous braz j oral sci. 7(26):1631-1635 influence of sex on temporomandibular disorder pain: a review of occurrence and development 1633 inflammatory pain is characterized by a continuous endogenous stimulation of nociceptors caused by the release of inflammatory mediators that directly stimulate them. postsurgical or traumatic pain is usually referred to as spontaneous pain in a hyperalgesic state21. inflammatory temporomandibular joint (tmj) conditions can result in tmj hyperalgesia produced by peripheral sensitization of tmj nociceptors and by central sensitization of the nociceptive neurons of the trigeminal brainstem sensory nuclear complex. peripheral sensitization, as well as central sensitization is characterized by an increase in the neuronal membrane excitability by inflammatory mediators released at the site of injury and by neuropeptide and excitatory amino acid released at the trigeminal brainstem sensory nuclear complex, respectively. some of the inflammatory mediators released at the site of injury including pge2 are present at high levels in the synovial fluid of patients with tmd. during hyperalgesic states, the nociceptive threshold is lowered and a nonnoxious stimulus such as jaw movement can induce pain, and noxious stimulus can also induce increased pain. the inflammatory mediators released at the site of tissue injury, such as prostaglandins, sensitize nociceptors. nonsteroidal anti-inflammatory drugs (nsaids) are frequently used to manage inflammatory pain. the analgesic action of these drugs results from the blockade of prostaglandins synthesis, thus preventing the peripheral sensitization of nociceptors21-22. the spinal cord has been shown to be a cns region in which components of opioid analgesic pathways and their regulation manifest sexual dimorphism. for example, the density of the kappa-opioid receptor (kor) and its distribution within axon terminals differs between the spinal cord of male and female rodents23. functional kor are located within the tmj of rats; peripherally acting kor agonists could be of benefit in the treatment of tmj pain, especially in women24, because the analgesic effect of a class of drugs, nalbuphine, pentazocine and butorphanol, which are thought to induce analgesia predominantly by action on kor, produce greater analgesia in women25. the receptors of gonadal steroids are referred to as the hormones produced by the ovaries and testes (gonads). they are present in many brain areas including some involved in pain transmission and modulation4. the man products of the tests are the androgens: testosterone and dihydrotestosterone. the ovaries primarily produce two types of steroid hormones: estrogens (e.g., estradiol, estriol, estrone) and progestins (e.g., progesterone; so-called because it promotes gestation and pregnancy). testosterone is a precursor to estradiol, so the ovaries also make testosterone. conversely, estradiol is a metabolite of testosterone, so the testes also produce some estrogens. the aromatization of testosterone to estradiol is greatly facilitated by the enzyme aromatase. this means that tissues containing aromatase can convert testosterone to estrogen and thereby make use of estrogen through estrogen receptors. in women, testosterone is produced in the adrenal cortex (25%) and ovaries (25%) and by transformation (50%) in the liver, kidneys, bowel, lungs, adipose tissue, and cns26. furthermore, since tmj pain in women is highest at times of lowest estrogen, the effects of peripherally acting kappa opioid receptor agonists on the treatment of tmj pain in women across the menstrual cycle should be better evaluated24. the physiological basis for the sex-related difference in analgesic response to a kor agonist is not completely known. it is possible that a male related hormone, such as testosterone, interacts negatively with kor agonists; or that female-related hormones, such as progesterone or estrogen, potentiate the action of kor27. thus, the treatment of choice for tmd is conservative because the symptomatology of the condition is often improved by the use of medication, occlusal splints, physical therapy, and orthodontic treatment3. influence of female gonadal hormones on orofacial p a i n during the menstrual cycle, serum levels of estrogen and progesterone fluctuate. in women, estrogen and progesterone levels are both relatively low at the beginning of the cycle. during the follicular phase, estrogen levels gradually increase, peaking prior to ovulation, and then moderately decrease during the luteal phase. progesterone levels rapidly increase after ovulation, peaking during the middle of the luteal phase. at the end of the luteal phase, both estrogen and progesterone levels drastically decrease28. however, menopause induces changes in the endogenous hormone balance: ovarian production of estrogens dramatically decreases. thereafter, the adrenal cortex is responsible for estrogen production via aromatization of androgens to estradiol in peripheral tissue (e.g., fat), which is significant in obese postmenopausal women. nevertheless, few researchers determine testosterone and estradiol blood concentrations in their experimental subjects at the time of testing14. several mechanisms by which hormones could influence tmd pain can be postulate29. peripherally, hormones could act directly on the temporomandibular joint and associated soft tissues. for example, estrogen is known to increase joint laxity, at least during pregnancy, and laxity of the temporomandibular joint is thought to play a role in the development of some of these disorders30. another possibility is that estrogen enhances a number of specific inflammatory responses in the tmj, and estrogen receptors have been found only in the tmj tissues of female primates, but not in males31. the classic animal experiment testing the relationship between hormones and nociception involves ovarectomizing female animals to examine the effects of braz j oral sci. 7(26):1631-1635 influence of sex on temporomandibular disorder pain: a review of occurrence and development 1634 hormone deficit, and then replacing hormones exogenously and observing the effects of hormone replacement. such studies are obviously not feasible in humans. however, the natural experiment of postmenopausal hormone replacement therapy presents an interesting parallel to the animal model: hormones are depleted (either slowly with the natural aging process, or more abruptly by surgery) and then replaced from exogenous sources. if female reproductive hormones increase the risk of a particular pain condition, those post-menopausal women who replace their depleted endogenous hormones from exogenous sources would be hormonally more similar to younger women than those postmenopausal women, who chose not to use hormone replacement therapy, and the users of hormone replacement therapy would be expected to be at higher risk of the specific pain condition31. few studies have investigated the role of hormonal fluctuations in the frequency or intensity of musculoskeletal pains, such as tmd, where episodes tend to be longer than for headache. there was a report on the variability of myofascial pain of tmd over three menstrual cycles in 12 female subjects. users of oral contraceptives tended to show less variable pain intensity levels, and fewer pain-free days than women experiencing hormonal fluctuations related to their naturally occurring menstrual cycles. however, the differences were not statistically significant and a predominant temporal pattern could not be discerned in this small sample13. tmd pain, abdominal pain, migraine and tension-type headache are more prevalent in adult women than in men. epidemiological studies have also found higher prevalence of these conditions, and sometimes back pain, among adolescent girls when compared to boys. recently, use of hormone replacement therapy in postmenopausal women has been identified as a risk factor for back pain and tmd pain32. concluding, the findings of this narrative literature review suggest that there is difference in the occurrence and development of pain according to sex, women being more susceptibly to orofacial pain. however, the conclusions drawn from these studies have considerable methodological limitations and this area requires further assessment using stricter randomized controlled trials to assess the difference between sexes. it is hoped that this review will highlight the fact that the patient’s sex should be taken into account during tmd therapy. r e f e r e n c e s 1. kajantie e, philips diw. the effects of sex and hormonal status on the physiological response to acute phychosocial stress. psychoneuroendocrinology. 2006; 31: 151-78. 2. gameiro gh, andrade as, nouer df, veiga mcfa. how many stressful experiences contribute to the development of têmporomandibular disorders? clin oral invest. 2006; 10: 261-8. 3. mcneely ml, armijo olivo s, magee dj. a systematic review of the effectiveness of physical therapy interventions for temporomandibular disorders. phys ther. 2006; 86: 710-25. 4. aloisi am, bonifazi m. sex hormones, central nervous system and pain. horm behav. 2006; 50: 1-7. 5. isberg a, hagglund m, paesini d. the effect of age and gender on the onset of symptomatic temporomandibular joint disk displacement. oral surg oral med pathol oral radiol endod. 1998; 85: 252-7. 6. shinal rm, fillingim rb.overview of orofacial pain: epidemiology and gender differences in orofacial pain. den clin n am. 2007; 51: 1-18. 7. sherman jj, leresche l. does experimental pain response vary across the menstrual cycle? a methodological review. am j physiol regul integr comp physiol. 2006; 291: r245-6. 8. fillingim rb, gear rw. sex differences in opioid analgesia: clinical and experimental findings. eur j pain. 2004; 8: 413-25. 9. cairns be. the influence of gender and sex steroids on craniofacial nociception. headache. 2007; 47: 319-24. 10. fischer l, clemente jt, tambeli ch. the protective role of testosterone in the development of temporomandibular joint pain. j pain. 2007; 8: 437-42. 11. song pc, schwartz j, blitzer a. the emerging role of botulinum toxin in the treatment of temporomandibular disorders. oral dis. 2007; 13: 253-60. 12. butkevich ip, barr ga, vershinina ea. sex differences in formalin-induced pain in prenatally stressed infant rats. eur j pain. 2007; 11: 888-94. 13. leresche l, mancl l, sherman jj, gandara b, dworkin sf. changes temporomandibular pain and other symptoms across the menstrual cycle. pain. 2003; 106: 253-61. 14. craft rm, mogil js, aloisi am. sex differences in pain and analgesia: the role of gonadal hormones. eur j pain. 2004; 8: 397-411. 15. von korff m, dworkin sf, leresche l, kruger a. an epidemiologic comparison of pain complaints. pain. 1988; 32: 173-83. 16. dworkin sf, huggins kh, leresche l, von korff m, howard j,truelove e, sommers e. epidemiology of signs and symptoms in temporomandibular disorders: clinical signs in cases and controls. j am dent assoc. 1990: 120: 273-81. 17. von korff m, dworkin sf, leresche l.graded chronic pain status: an epidemiologic evaluation. pain. 1990; 40: 279-91. 18. gameiro gh, gameiro ph, andrade as, pereira lf, arthuri mt, marcondes fk et al. nociception and anxiety-like behavior in rats submitted to different periods of restraint stress. physiol behav. 2006; 87: 643-9. 19. wiesenfeld-hallin z. sex differences in pain perception. gend med. 2005; 2: 137-45. 20. oliveira mcg, pelegrini-da-silva a, parada ca, tambeli ch. 5ht acts on nociceptive primary afferents through an indirect mechanism to induce hyperalgesia in the subcutaneous tissue. neuroscience. 2007; 145: 708-14. 21. jorge s, parada ca, ferreira sh, tambeli ch. interferential therapy produces antinociception during application in various models of inflammatory pain. phys ther. 2006; 86: 800-8. 22. rodrigues llfr, oliveira mcg, pelegrini-da-silva a, veiga mcfa, parada ca, tambeli ch. peripheral sympathetic component of the temporomandibular joint inflammatory pain in rats. j pain. 2006; 7: 929-36. 23. harris ja, chang pc, drake ct. kappa opioid receptors in rat spinal cord: sex-linked distribution differences. neuroscience. 2004; 124: 879-90. 24. clemente jt, parada ca, veiga mcfa, gear rw, tambeli ch. sexual dimorphism in the antinociception mediated by kappa opioid receptors in the rat temporomandibular joint. neuroscience lett. 2004; 372: 250-5. 25. gear rw, gordona nc, miaskowski c, paul sm, heller ph, levine jd. sexual dimorphism in very low dose nalbuphine postoperative analgesia. neuroscience lett. 2003; 339: 1–4. braz j oral sci. 7(26):1631-1635 influence of sex on temporomandibular disorder pain: a review of occurrence and development 1635 26. brody dj, bracken mb. short interpregnancy interval: a risk factor for low birth weight. am j perinatol. 1987; 4: 50-4. 27. liu nj, von gizycki h, gintzler ar. sexually dimorphic recruitment of spinal opioid analgesic pathways by the spinal application of morphine. j pharmacol exp ther. 2007; 322: 654-60. 28. kuba t, quinones-jenab v. the role of female gonadal hormones in behavioral sex differences in persistent and chronic pain: clinical versus preclinical studies. brain res bull. 2005; 66: 179-88. 29. arthuri mt, gameiro gh, tambeli ch, veiga mcfa. peripheral effect of a kappa opioid receptor antagonist on nociception evoked by formalin injected in tmj of pregnant rats. life sci. 2005; 76: 1177-88. 30. westling l. temporomandibular joint dysfunction and systemic joint laxity. swed dent j suppl. 1992; 81: 1-79. 31. leresche l, saunders k, von korff mr, barlow w, dworkin sf. use of exogenous hormones and risk of temporomandibular disorder pain. pain. 1997; 69: 153-60. 32. le resche l, mancl la, drangsholt mt, saunders k, von korff m. relationship of pain and symptoms to pubertal development in adolescents. pain. 2005; 118: 201-9. braz j oral sci. 7(26):1631-1635 influence of sex on temporomandibular disorder pain: a review of occurrence and development erratum braz j oral sci. 2008; 7: 1559-62 cytogenetic damage in khaini users of tamilnadu, southern india. raman sangeetha keshavarao sasikala. braz j oral sci. 2008; 7: 1535-8 ethical aspects concerning endodontic instrument fracture. received for publication: march 23, 2008 accepted: june 30, 2008 1http://dx.doi.org/10.20396/bjos.v19i0.8656779 volume 19 2020 e206779 original article 1 private practice, chapecó, sc, brazil. 2 school of dentistry, community university of chapecó region (unochapecó), chapecó, sc, brazil. 3 health sciences post-graduate program, community university of chapecó region (unochapecó), chapecó, sc, brazil. corresponding author: prof. dr. sinval adalberto rodrigues-junior community university of chapecó region (unochapecó), health sciences area servidão anjo da guarda 295-d – efapi, zip code 89809-000, chapecó, sc, brazil phone number: +55 (49) 3321-8069 e-mail: rodriguesjunior.sa@ unochapeco.edu.br received: september 23, 2019 accepted: march 28, 2020 in vitro tooth whitening effectiveness of whitening mouth rinses ana paula morona rodrigues1, mariele cristina klein1, mauro antonio dall agnol2, sinval adalberto rodrigues-junior3,* regardless of the extensive availability of mouth rinses that claim to whiten teeth, evidence of achievement of such effect is still missing. aim: therefore, this study assessed in vitro the whitening effectiveness of whitening mouth rinses. methods: sixty intact bovine incisors were embedded in acrylic resin and had their buccal surface flattened and polished. then, the specimens were randomly allocated to three conventional (colgate plax, cepacol and listerine cool mint) and three whitening mouth rinse groups (colgate luminous white, cepacol whitening and listerine whitening extreme) (n=10). following, the specimens were immersed twice a day in the mouth rinses for one minute for 28 days. in between each immersion period, the specimens remained in artificial saliva at 37oc. color was measured at baseline, 7, 14, 21, and 28 days using a portable spectrophotometer (easyshade, vita zahnfabrik, germany) with a 6 mm of diameter probe. color change was analyzed considering the parameters of ∆l*, ∆a*, ∆b* and, ultimately, ∆e*. the whitening efficacy of the mouth rinses was analyzed using the whiteness index for dentistry (wid). data of ∆s was analyzed using two-way anova and tukey’s test (α=0.05). results: the type of mouth rinse affected significantly all the ∆ parameters (p<0.05). a non-whitening (conventional) mouth rinse produced the highest ∆e*, followed by the three whitening mouth rinses. the application time also affected ∆e* (p<0.05), with emphasis on the third week of treatment. only the hydrogen peroxide-containing mouth rinse (listerine whitening extreme) presented a whitening effect, with an increasing trend over time. conclusion: although the overall color change was not different when comparing conventional and whitening mouth rinses, the hydrogen peroxide-containing whitening mouth rinse produces an increasing whitening trend over time. not every mouth rinse that claims to whiten teeth produces the desired effect. keywords: color. mouthwashes. nonprescription drugs. tooth bleaching. tooth bleaching agents. 2 rodrigues et al. introduction over-the-counter (otc) whitening products have been available in the dental market since the 2000s, increasing the number of alternatives to meet the tooth whitening demand. different from in-office tooth whitening and tray-based home whitening, professional prescription and orientation are not mandatory for applying otc products1. dentifrices, mouth rinses, whitening strips, dental flosses, and paint-on gels fall into the category of otc whitening products and eventually contain low concentrations of hydrogen peroxide1,2. chemically-induced tooth whitening derives from the interaction of the whitening agent, which is usually hydrogen peroxide, with the dental structure. when applied, hydrogen peroxide diffuses into the dental structure and releases reactive oxygen molecules that, through oxidation, break the double bonds of organic and inorganic coloring molecules3. this process relies on both the concentration of the whitening agent and the contact time with the tooth4. we may apply hydrogen peroxide directly to the tooth or it may result from a chemical reaction from sodium perborate or carbamide peroxide. the concentration of the former presentation may vary from 5% to 35%, and the latter, from 10% to 35%3, but a 10% carbamide peroxide solution produces only 3.35% hydrogen peroxide5. whitening mouth rinses may contain sodium hexametaphosphate, which protects the tooth surface from pigments2. they may also contain hydrogen peroxide at low concentrations, usually around 1.5% to 2%2,6. nonetheless, this concentration is close to the hydrogen peroxide concentration of the ada-recommended at-home whitening technique based on 10% carbamide peroxide gel3, suggesting a whitening potential for this oral hygiene technology. however, the contact time of mouth rinses with the tooth surface is low, lasting for only a few minutes and leading to questioning about the whitening capacity of these products under the application circunstances. while a long-term randomized controlled trial7 confirmed the whitening efficacy and safety of professionally supervised tooth whitening procedures, studies attesting the efficacy of whitening mouth rinses are scarce. from the best of our knowledge, there are no clinical trials attempting to verify the whitening efficacy and the possible production of adverse effects by whitening mouth rinses. also, the few existing in vitro studies that compare whitening mouth rinses between themselves and with other whitening alternatives vary a lot methodologically. based on that, discrepancies related to the whitening potential, either in favor of the whitening mouth rinses6,8,9 or towards no difference10 may have been caused by different methodological decisions. this scenario impairs comparisons between products and a sound decision-making process by either consumers or dental professionals responsible for indicating tooth whitening and oral hygiene products. therefore, the present study aimed to inform about the in vitro whitening efficacy of whitening mouth rinses, testing the hypothesis that there is no difference in color change results from the type of mouth rinse. 3 rodrigues et al. materials and methods specimen preparation for this in vitro study, 60 extracted intact bovine incisors were stored in 0.1% thymol solution for seven days and cleaned with pumice and robinson brush using a low-speed handpiece (kavo, joinville, sc, brazil). next, the roots were removed using a high-speed cutting saw (figure 1-a) and the teeth were embedded in chemically cured acrylic resin (vipiflash, vipi, pirassununga, sp, brazil) (figure 1-b). then, the buccal surfaces of the specimens were ground and polished using #400, 600, and 1200 grit sandpapers under water cooling, leaving a flat surface area of at least 6 mm2 in the center and cervical regions of the tooth (figure 1-c). teeth that had their enamel removed during grinding and polishing were excluded. the included specimens were stored in artificial saliva prior to the experiment. immersion protocol an online random sequence generator (www.random.org) randomized the specimens to any of the six mouth rinse groups (n=10) (table 1). considering that most manufacturers recommend a one-minute mouthwash, the specimens were immersed in the mouth rinses for one minute, twice a day. the trial period lasted for 28 days. after immersion, the mouth rinses were discarded, and the specimens were returned to the artificial saliva and maintained at 37oc. the artificial saliva was replaced daily. color assessment a single operator measured color with the help of an easyshade portable spectrophotometer (vita zahnfabrik, bad säckingen, germany), at baseline and after 7, figure 1. specimen preparation: a – root removal; b – tooth embedding in chemically cured acrylic resin; c – grinding and polishing of the specimen´s buccal surface a b c 4 rodrigues et al. 14, 21, and 28 days of immersion. color was read after a minimum interval of two hours after the last immersion in the mouth rinses, by positioning the 6-mm tip of the spectrophotometer between the middle and cervical thirds of the specimen, perpendicular to the specimen’s flat surface. a standard white background was used for all measurements, under the same lightning. color expression was based on the three-dimensional color space of the ciel*a*b* system consisting of three distinct axes. the l* axis varies from 0 to 100 and represents the degree of lightness in color, considering 0 totally black and 100 totally white; a* represents the variation between green (a-) and red (a+); and b* represents the variation between blue (b-) and yellow (b+). the l*, a*, and b* values in each assessment interval allowed determining a ∆ for each parameter and ultimately calculating ∆e*, which is a non-directional summary of color change based on the ∆s of each directional parameter, as follows: δe*ab = (l2* – l1*) 2 + (a2* – a1*) 2 + (b2* – b1*) 2 analysis of whitening efficacy the whitening efficacy of the mouth rinses was assessed using the whitening index for dentistry (wid) 12, which is calculated as follows: table 1. substances used in the study product manufacturer components artificial saliva (santos11 2008) unochapecó pharmacology laboratory potassium chloride, sodium chloride, magnesium chloride, potassium phosphate, calcium chloride, nipagin, nipasol, carboxymethylcellulose, sorbitol, distilled water colgate plax colgate-palmolive, são bernardo do campo, sp, brazil water, glycerin, propylene glycol, sorbitol, poloxamer 338, poloxamer 407, aroma, peg-40 hydrogenated castor oil, cetylpyridinium chloride, potassium sorbate, sodium fluoride, sodium saccharin, citric acid, sucralose, ci 42053 cepacol sanofi-aventis farmacêutica ltda., suzano, sp, brazil alcohol, water, cetylpyridinium chloride, disodium edta, sodium saccharin, polysorbate 80, glycerin, sodium phosphate, disodium phosphate, eucalyptol, menthol, methyl salicylate, aroma, parfum (benzyl alcohol, cinnamal), ci 19140 listerine cool mint johnson & johnson industrial ltda., yumbo, valle, colombia water, sorbitol, alcohol, poloxamer 407, benzoic acid, sodium saccharin, eucalyptol, aroma (d-limonene), thymol, methyl salicylate, sodium benzoate, menthol, ci 42053 colgate luminous white colgate-palmolive, são bernardo do campo, sp, brazil water, glycerin, propylene glycol, sorbitol, tetrapotassium pyrophosphate, polysorbate 20, tetrasodium pyrophosphate, zinc citrate, pvm/ma copolymer, aroma, benzyl alcohol, sodium fluoride, sodium saccharin, ci 42051 cepacol whitening sanofi-aventis farmacêutica ltda., suzano, sp, brazil water, sorbitol, glycerin, sodium benzoate, sodium saccharin, sodium cyclamate, poloxamer 407, peg-40 hydrogenated castor oil, pvp, methylparaben, aroma (eugenol, d-limonene and linalool), citric acid, propylene glycol, cetylpyridinium chloride and sodium fluoride listerine whitening extreme johnson & johnson industrial ltda., yumbo, valle, colombia water, alcohol, 2.5% hydrogen peroxide, aroma, poloxamer 407, sodium saccharin, menthol, phosphoric acid, disodium phosphate, sodium fluoride, sucralose 5 rodrigues et al. wid = 0.511l* – 2.324a* – 1.100b* according to the authors12, high positive values of the index indicate high whiteness in the specimen; low values, and even negative values indicate low values of whiteness. data analysis for statistical data analysis of the ∆s, the adherence of data to the normal distribution was verified using the anderson-darling test. levene’s test was used to check for equal variances and grubb’s test for the presence of outliers. when identified, outliers were removed from the dataset. two-way anova and tukey’s test verified the influence of time and the type of mouthwash on color and color change (∆), at a 5% significance level (minitab 17.0, minitab llc, state college, pa, usa). data of wid were analyzed descriptively considering each mouth rinse and the application time period, based on the parameters set by perez et al.12 (2016). results figure 2 shows the changes in l*, a*, and b* by immersion in the mouth rinses. table 2 presents the results of ∆l*, ∆a*, ∆b*, and ∆e*. the mouth rinse affected ∆l* significantly (p=0.017), while time (p=0.107) and the mouth rinse x time interaction (p=0.971) were not significant. listerine whitening extreme produced the lowest reduction in l* (-3.60), while listerine cool mint and cepacol whitening generated the greatest reduction (-10.29 and -9.91, respectively). similarly, the mouth rinse significantly affected ∆b* (p<0.0001), while time (p=0.844) and the mouth rinse x time interaction (p=0.229) were not significant. listerine whitening extreme produced the greatest reduction in b* values (-5.78), followed by colgate luminous white (-2.94). the mouth rinse affected ∆a* significantly (p=0.02), which did not occur for either time (p=0.142) or the mouth rinse x time interaction (p=0.791). listerine whitening extreme provided the greatest reduction in a* values (-0.88), while cepacol whitening increased a* (0.91). mouth rinse (p<0.0001) and time (p=0.002) affected ∆e* significantly, which did not occur for their interaction (p=0.739). listerine cool mint produced the highest ∆e* (13.72), while cepacol showed the lowest ∆e* (7.99). the three whitening mouth rinses produced intermediate ∆e* values. the period of 21 days of mouth rinse application resulted in the highest ∆e* (14.26). as to wid results, all groups presented negative mean values, except listerine whitening extreme. also, the index values per application time were always negative in all groups. again, the exception was the hydrogen peroxide-containing listerine whitening extreme, which raised the index values from baseline and became positive only after 14 days-application time (figure 3). the mean, minimum and maximum wid values of the groups are as follows: cepacol (mean=-10.9; minimum=-28.5; maximum=5.31); cepacol whitening (mean=-15.4; minimum=-27.6; maximum=0.03); colgate plax (mean=-8.3; minimum=-23.6; maximum=8.9); colgate luminous white (mean=-10.7; minimum=-25.1; maximum=5.2); listerine cool mint (mean=-13.4; minimum=-33.4; maximum=8.4); listerine whitening extreme (mean=0.11; minimum=-21.5; maximum=25.8). 6 rodrigues et al. figure 2. behavior of l* (a), a* (b) and b* (c) parameters, respectively, throughout the experiment colgate plax cepacol listerine cool mint colgate luminous white cepacol whitening listerine whitening extreme colgate plax cepacol listerine cool mint colgate luminous white cepacol whitening listerine whitening extreme colgate plax cepacol listerine cool mint colgate luminous white cepacol whitening listerine whitening extreme baseline 7 days 14 days 21 days 28 days baseline 7 days 14 days 21 days 28 days baseline 7 days 14 days 21 days 28 days 10 20 30 40 50 0 2 4 6 8 100 90 80 70 60 a b c 7 rodrigues et al. table 2. results of δl*, δa*, δb* and δe* (mean and sd) for each assessment interval time colgate plax cepacol listerine cool mint colgate luminous white cepacol whitening listerine whitening extreme δl* b-7 -6.11 (5.68)a -4.12 (6.40)a -8.26 (6.99)a -3.21 (14.14)a -9.30 (7.33)a -5.10 (9.16)a b-14 -7.14 (9.69)a -7.65 (5.43)a -11.83 (12.38)a -5.90 (11.78)a -9.17 (9.51)a -3.00 (10.41)a b-21 -10.45 (8.58)a -9.06 (5.47)a -14.05 (13.62)a -9.27 (9.99)a -10.45 (8.14)a -3.92 (10.63)a b-28 -2.78 (7.84)a -2.93 (4.30)a -7.02 (9.25)a -8.07 (14.47)a -10.71 (7.57)a -2.39 (9.92)a δa* b-7 1.31 (2.60)a 0.58 (2.63)a 0.42 (2.17)a -1.02 (3.77)a 1.68 (3.02)a -0.55 (2.01)a b-14 -0.45 (1.97)a 0.60 (0.83)a 0.10 (3.59)a -0.07 (2.62)a -0.08 (1.39)a -0.67 (2.01)a b-21 1.23 (2.16)a 1.36 (2.67)a 0.72 (4.60)a -1.36 (3.70)a 1.92 (3.32)a -1.31 (2.92)a b-28 -0.64 (1.97)a -0.55 (1.64)a -0.99 (3.60)a -0.34 (2.18)a 0.13 (1.53)a -0.98 (2.04)a δb* b-7 0.23 (6.23)ab 2.37 (2.82)ab -1.31 (2.38)ab -1.28 (5.20)ab 1.90 (2.55)ab -5.29 (3.60)bc b-14 -0.45 (3.54)ab 1.61 (2.92)ab 0.47 (6.51)ab -2.44 (2.63)abc -0.74 (2.28)ab -3.29 (5.89)bc b-21 1.01 (4.80)ab 4.68 (5.12)a -0.38 (7.29)ab -4.45 (6.67)bc 1.61 (3.84)ab -9.38 (8.15)c b-28 0.50 (3.05)ab 0.26 (2.93)ab 1.00 (7.21)ab -3.59 (3.88)bc -0.69 (2.39)ab -5.16 (5.73)bc δe* b-7 9.08 (5.46)ab 7.17 (4.91)b 9.04 (6.81)ab 12.41 (9.19)ab 11.15 (5.87)ab 11.17 (4.76)ab b-14 9.62 (8.04)ab 8.18 (5.70)ab 15.38 (10.07)ab 10.10 (4.11)ab 12.09 (5.41)ab 11.06 (5.79)ab b-21 11.69 (8.58)ab 11.50 (5.82)ab 18.47 (10.05)a 15.46 (3.44)ab 12.95 (5.84)ab 15.49 (6.18)ab b-28 7.35 (4.88)b 5.12 (3.25)b 11.99 (6.90)ab 15.07 (7.97)ab 11.89 (6.03)ab 11.15 (5.86)ab * different letters indicate statistically significant differences between groups within each δ ** b means baseline – each value represents the δ between the baseline and each mouth rinse application time figure 3. wid results of each mouth rinse over time 5 0 -5 -10 -15 -20 w i d baseline 7 days 14 days 21 days 28 days mouthrinse application time colgate plax cepacol listerine cool mint colgate luminous white cepacol whitening listerine whitening extreme 8 rodrigues et al. discussion the limited evidence on the whitening effect of whitening mouth rinses associated with the extensive availability of these over-the-counter (otc) products motivated this study. the influence of the type of mouth rinse on the overall color change (∆e*) led to the rejection of the study hypothesis. interestingly, a conventional (non-whitening) mouth rinse (listerine cool mint) produced the highest ∆e*, followed by whitening mouth rinses, which did not show statistical differences. on the other hand, wid results showed a growing whitening effect over time from the hydrogen peroxide-containing mouth rinse listerine whitening extreme (figure 3), suggesting that the presence of this active ingredient is key for a mouth rinse that claims to whiten teeth to, in fact, provide this effect. the whitening potential of whitening substances has been assessed, traditionally, by changes in ciel*a*b* coordinates, expressed as ∆s (∆l*, ∆a*, ∆b* and ∆e*)6-8. still, most recently, the whiteness index for dentistry (wid) was developed aiding at determining more precisely the amount of whiteness produced by tooth whitening technologies12. according to perez et al.12 (2016), the index has a very straightforward interpretation: the higher the positive index value, the whiter the tooth. lower values, and even negative values are considered as poorly associated to whiteness12. in this study, all wid values at baseline were negative. the only ascending tendency by immersion in mouth rinse over time was observed with the whitening mouth rinse containing hydrogen-peroxide, which only achieved positive values after 14 days of immersion. as to ciel*a*b* measures, the increase of lightness (increasing l*) and the reduction of yellowness (decreasing b*) are the main inducers of tooth whitening. the reduction of redness (decreasing a*) affects whitening to a lesser extent8,13. our results showed a reduction of l* (figure 2 and table 2), irrespective of the treatment group or application time, characterizing darkening of the tooth structure throughout the experiment. figure 2 also shows a tendency of lightness recovery by some mouth rinses from day 21 to day 28, although not fully restoring it. the presence of organic substances in the artificial saliva10 and the demineralizing effect of some low ph mouth rinses8 supposedly explain the lowering effect of lightness for in vitro settings. a significant yellowness (∆b*) reduction was observed only for the 2.5% hydrogen peroxide listerine whitening extreme mouth rinse. this mouth rinse also presented the lowest reduction of l*, meaning a reduced darkening of the tooth, although not statistically significant. finally, this mouth rinse, along with colgate luminous white, produced negative a* (again, not statistically significant), which means a reduced redness that tends to manifest after longer periods of whitening and to a lesser extent, representing improved tooth whitening8. some substances with whitening potential are common to whitening toothpastes and whitening mouth rinses. phosphate-derived substances such as pyrophosphate, tripolyphosphate, and hexametaphosphate have prevented superficial stains14. tetrapotassium pyrophosphate and tetrasodium pyrophosphate are present in colgate luminous white, which also tended to reduce a* and b* values. moreover, considering the general results, it seems that effective tooth whitening relates somewhat to the 9 rodrigues et al. presence of hydrogen peroxide15. among the three whitening mouth rinses, listerine whitening extreme was the only one to contain hydrogen peroxide, to reduce b* substantially (figure 2) and to present a true whitening effect (figure 3). torres et al.6 (2013) observed a similar in vitro whitening effect for a mouth rinse containing 2% hydrogen peroxide and 10% carbamide peroxide gel. considering that the 10% carbamide peroxide decomposes into 3.35% hydrogen peroxide3, the concentrations of the active whitening substance are similar. methodological disparities hindered the comparison of results of previous and the present in vitro study. they involve the type of tooth (human8 or bovine6,9,10), pre-staining (yes6,9,15/no8,10), varying application protocols (number of immersions – 1x6, 2x8,9, 3x10; and immersion times – 1 minute6,8,9 or 2 minutes10), assessment times (30 days10, 45 days8, up to 8 weeks9, and up to 12 weeks6,15), and color assessment procedures (standard digitized photographs8 or spectrophotometers6,9,10). regarding the choices made in this study, bovine teeth have long been a reasonable alternative to mimic the characteristics of human teeth10. we did not pre-stain the teeth in this study, because artificial staining creates conditions to confirm a whitening effect in the presence of chromogenic molecules that may not be very intense in the teeth of a person demanding tooth whitening. most mouth rinse manufacturers recommend a one-minute immersion. although there is no specific recommendation on the frequency of daily applications, twice a day8,9 would be a clinically feasible frequency and it would improve the whitening effect as compared with a single daily application. the total application time of 28 days almost doubled some enhanced protocol times7,16 for tooth whitening with carbamide or hydrogen peroxide, which rarely takes one month in present days. the authors understand that this application time was sufficient to confirm any whitening effect from whitening mouth rinses. finally, the digital spectrophotometer has long been reported as a reliable method for measuring the color change in whitening studies17 and, together with the wid 12, it presents a good scenario of the whitening effect of whitening substances. we assessed color weekly within a 28-day application period, generating four assessment intervals. the application time only affected ∆e*, with emphasis on the third week of whitening, which presented the highest ∆e* values from the baseline (table 2). nonetheless, the yellowing reduction with listerine whitening extreme was evident from day 7 (figure 2). chemically-induced peroxide tooth whitening products showed accumulation over time, enhancing the total contact time with the tooth structure4. therefore, we may speculate that a longer experiment period would show clearer results from the hydrogen peroxide mouth rinse and that it could even affect the overall color change. the color of the mouth rinses varied. products from cepacol had a yellow color, while those from colgate were blue and those from listerine presented a blueish/greenish color. regardless of that, one believes that the product color may not have influenced the color results of teeth, since they remained in contact with the substances for a short period. also, following the immersion protocols, the artificial saliva with some residual pigment was exchanged daily. one limitation of the study was that the ph of the mouth rinses was not assessed. 10 rodrigues et al. whitening mouth rinses are easy to acquire and represent low-cost alternatives for tooth whitening10. however, in vitro studies using different methods showed controversial results6,8,10, which are difficult to pool and compare. this study, for instance, used the conventional non-whitening counterparts as controls and revealed that some whitening mouth rinses do not achieve the expected whitening results. dental researchers and dental clinicians could incorporate this information to design comprehensive independent clinical studies and advise potential consumers of whitening products. as for therapeutic technology, a sound decision-making process and the indication of whitening mouth rinses for tooth whitening depend on the scrutiny of proven effects resulting from randomized controlled trials attesting clinical efficacy and safety. the overall color change produced by whitening mouth rinses in one month is not different from that produced by conventional mouth rinses. the whitening mouth rinse containing hydrogen peroxide reduced yellowing significantly during such application period and was the only whitening mouth rinse to present a true whitening effect. acknowledgements this study was funded by fapesc, grant no. 06/2017. references 1. american dental association. council on scientific affairs. tooth whitening/bleaching: treatment considerations for dentists and their patients. chicago: ada. 2009 sept [cited 2019 nov 25]. 12p. available from: https://www.ada.org/~/media/ada/about%20the%20ada/files/ada_house_of_ delegates_whitening_report.ashx. 2. demarco ff, meireles ss, masotti as. over-the-counter whitening agents : critical assessment of the otc products for home-use bleaching. braz oral res. 2009;23(spec iss 1):64-70. doi: 10.1590/s1806-83242009000500010. 3. kwon sr, wertz pw. review of the mechanism of tooth whitening. j esthet restor dent. 2015 sep-oct;27(5):240-57. doi: 10.1111/jerd.12152. 4. serraglio cr, zanella l, dalla-vecchia kb, rodrigues-junior sa. efficacy and safety of overthe-counter whitening strips as compared to home-whitening with 10 % carbamide peroxide gel—systematic review of rcts and metanalysis. clin oral investig. 2016 jan;20(1):1-14. doi: 10.1007/s00784-015-1547-8. 5. fasanaro ts. bleaching teeth: history, chemicals, and methods used for common tooth discolorations. j esthet dent. 1992 may-jun;4(3):71-8. 6. torres c, perote l, gutierrez n, pucci c, borges a. efficacy of mouth rinses and toothpaste on tooth whitening. oper dent. 2013 jan-feb;38(1):57-62. doi: 10.2341/11-360-l. 7. meireles ss, santos is, bona a della, demarco ff. a double-blind randomized clinical trial of two carbamide peroxide tooth bleaching agents: 2-year follow-up. j dent. 2010 dec;38(12):956-63. doi: 10.1016/j.jdent.2010.08.003. 8. lima fg, rotta ta, penso s, meireles ss, demarco ff. in vitro evaluation of the whitening effect of mouth rinses containing hydrogen peroxide. braz oral res. 2012 may-jun;26(3):269-74. 9. karadas m, duymus zy. in vitro evaluation of the efficacy of different over-the-counter products on tooth whitening. braz dent j. 2015 jul-aug;26(4):373-7. doi: 10.1590/0103-64402013x0111. 11 rodrigues et al. 10. nahsan fps, reis mjo, francisconi-dos-rios lf, leão l, paranhos lr. effectiveness of whitening mouthwashes on tooth color: an in vitro study. gen dent. 2018 mar-apr;66(2):e7-e10. 11. santos pa. [infuence of the light source on color stability of a compoiste resin. effect of storage medium and storage times] [thesis]. araraquara: são paulo state university “júlio de mesquita filho”; 2008. portuguese. 12. pérez mdm, ghinea r, rivas mj, yebra a, ionescu am, paravina rd, et al. development of a customized whiteness index for dentistry based on cielab color space. dent mater. 2016 mar;32(3):461-7. doi: 10.1016/j.dental.2015.12.008. 13. cie. commission internationale de i’eclairage. recommendations on uniform color spaces-color difference equations, psyhometric color terms. supplement no. 2 to cie publication no. 15 (e.-1.3. 1) 1971/(tc-1.3.). 1978. 14. joiner a. whitening toothpastes: a review of the literature. j dent. 2010;38 suppl 2:e17-24. doi: 10.1016/j.jdent.2010.05.017. 15. oliveira j, sarlo r, bresciani e, caneppele t. whitening efficacy of whitening mouth rinses used alone or in conjunction with carbamide peroxide home whitening. oper dent. 2017 may/jun;42(3):319-26. doi: 10.2341/15-361-l. 16. chemin k, rezende m, loguercio a, reis a, kossatz s. effectiveness of and dental sensitivity to at-home bleaching with 4% and 10% hydrogen peroxide: a randomized, triple-blind clinical trial. oper dent. 2018 may/jun;43(3):232-40. doi: 10.2341/16-260-c. 17. meireles ss, demarco ff, santos is, dumith sc, bona a della. validation and reliability of visual assessment with a shade guide for tooth-color classification. oper dent. 2008 mar-apr;33(2):121-6. doi: 10.2341/07-71. 1 volume 16 2017 e17062 original article 1 phd. professor at oral and maxillofacial surgery residency training program at university hospital of campos gerais (hucg), school of dentistry and health sciences post-graduate program, state university of ponta grossa (uepg). 2 oral and maxillofacial surgeon. preceptor at oral and maxillofacial surgery residency training program at university hospital of campos gerais (hucg), state university of ponta grossa (uepg). 3 m.d. in pathology. private practice. 4 phd. professor at school of dentistry são leopoldo mandic (slmandic). 5 phd student. professor at oral and maxillofacial surgery residency training program at university hospital of campos gerais (hucg), school of dentistry, state university of ponta grossa (uepg). corresponding author: marcelo carlos bortoluzzi universidade estadual de ponta grossa (uepg)campus uvaranas, bloco m, faculdade de odontologia. av. general carlos cavalcanti, 4748 bairro uvaranas ponta grossa paraná, brasil. zip(cep) 84030-900 tel:+55 42 3220 3104 email: mbortoluzzi@gmail.com received: june 09, 2017 accepted: november 04, 2017 classic kaposi’s sarcoma (non-hiv-associated) of oral cavity: a case report marcelo carlos bortoluzzi1, ramon cesar godoy gonçalves2, cristina maria de freitas zanellato3, juliana cama ramacciato4, roberto de oliveira jabur5 kaposi’s sarcoma (ks) is a locally aggressive multicentric mucocutaneous malignant neoplasm. the aim of this article is to report and discuss the immunohistochemical profile of a rare case of classic primary oral kaposi’s sarcoma presenting on the hard palate of a female patient which was non-hiv and was not immunocompromised. keywords: kaposi’s sarcoma. oral cavity. classic kaposi’s sarcoma. doi: http://dx.doi.org/10.20396/bjos.v16i0.8651052 2 bortoluzzi et al. introduction kaposi’s sarcoma (ks) is a locally aggressive multicentric mucocutaneous malignant neoplasm which belongs to the group of intermediate type of vascular/ endothelial origin and may also involve mucosal sites, lymph nodes and visceral organs. ks may appear in four main clinical forms as, respectively, (a) classic or mediterranean which occurs among elderly men of ashkenazic jewish and east european origin, (b) epidemic or acquired immunodeficiency syndrome (aids)-associated, (c) iatrogenic or post-transplant seen in patients undergoing immunosuppressive therapy, (d) and endemic or african which involves children, adolescents, and adults with a high frequency of extracutaneous manifestations1-4. currently, there is strong evidence that all types of ks are caused by the human herpesvirus (hhv-8) infection, known as kaposi’s sarcoma-associated herpesvirus (kshv)1,2,4-8. the epidemic ks is the variant which most commonly affect the oral cavity, however, the classic form of ks may also but rarely occur in the oral cavity (oks) and, the initial oral involvement existing as the sole presentation of the condition, is an even rarer occurrence with only a few previously reported cases3,8-13. clinically, oks most often affects the hard and soft palate, gingiva and dorsal tongue with plaques or tumors of coloration ranging from non-pigmented to brownish-red or violaceous. early lesions may appear as erythematous or ecchymotic patches which progress to papular, nodular, and exophytic forms. it could invade bone and create tooth mobility and morbidity may be associated with pain, bleeding, and functional interferences3,8,9,14. the aim of this article is to report and briefly discuss the immunohistochemical profile of a rare case of classic primary oks presenting on the hard palate of a female patient which was hiv-negative and wasn’t immunosuppressed. clinical case a brazilian female patient with 63 years old, with distant polish descent, searched treatment due to an asymptomatic increase of volume on had palate with two months of evolution. the main complain was the increase of volume itself and occasional bleeding. clinical evaluation evidenced an ulcerated soft nodular lesion red-purplish (figure 1). blood cells count test showed to be within the normal parameters. an incisional biopsy was recommended. the hematoxylin and eosin stain (h&e) showed a hypercellular tumoral mass being composed of bland-appearing spindle cells, ill defined or atypical vascular channels, and extravasated red blood cells (figures 2). due to its unspecific h&e pattern a immunohistochemical study was required and included: hhv-8 (clone ln53), positive (figure 3); cd31 (clone (jc/70a), positive; cd34 (clone qbend10), positive; cytokeratin 40,48, 50 and 50.6 kda (clone ae1/ae3), negative; desmin (clone d33), negative; s-100 protein (policlonal), negative; erg ets family (clone ep111), positive. the lesion immunohistochemistry profile is summarized in table 1. based on the clinical, histopathological and immunohistochemical findings it was possible to reach the diagnosis of oks. due to a low suspicion for oks, the anti-hiv test was performed just after the final diagnosis and showed to be negative. the patient underwent to careful medical screening searching any undiagnosed additional disease or condition which may induce to an immunological deficit and none has been found. 3 bortoluzzi et al. the intra-oral lesion was treated through a conservative oral surgery including a five millimeters free margin from visible lesion and the area was left to second intention healing. the patient was also referred for additional radiotherapy; however, since intra-oral cone radiation therapy was not available, the patient refuted this additional treatment. the patient is clinically disease free for seventeen months and maintains periodic monitoring (figure 4). figure 1. oral kaposi’s sarcoma at hard palate. table 1. oral cavity kaposis’s sarcoma immunohistochemistry profile. antibody / clone reactivity hhv-8 (clone ln53) positive cd31 (clone (jc/70a) positive cd34 (clone qbend10) positive erg ets family (clone ep111) positive cytokeratin 40,48, 50 and 50.6 kda (clone ae1/ae3) negative desmin (clone d33) negative negative; s-100 protein (policlonal) negative figure 2. low (a-100x) and high (b-400x) power microscopic view of oral kaposi’s sarcoma, showing a hypercellular tumoral mass with atypical vascular channels and extravasated red blood cells (hematoxylin & eosin). a b 4 bortoluzzi et al. discussion while oks is a common finding in patients with hiv infection and is infrequently described in the immunocompromised population, this manuscript aimed to report a rare case of classic oks in a non-hiv and non-immunocompromised patient. kshv has been frequently identified all types of ks lesions, suggesting a causative role and, according to duus et al.6 (2004), kshv is harbored in the oral cavity of healthy individuals and it is capable of infection in oral epithelial cells, even in absence of severe immunosuppression and the infection may persist at a low level for the lifetime of the host, being controlled by the immune system. the diagnosis of oks may be challenging in non-hiv individuals or for patients undergoing through immunosuppressive therapy due to its rarity and clinical similarities with common oral lesions such as pyogenic granuloma and haemangioma and this may lead to misdiagnosis. histopathologic evaluation is necessary to achieve a definitive diagnosis and hematoxylin/eosin (h&e) is the standard staining method for it. microscopically, in the early stages of the tumor growth, the diagnostic spindle cell proliferation is not always evident and later it may resemble benign vascular lesion such as pyogenic granulomas. atypical vascular channels, extravasated red blood cells, hemosiderin, and inflammatory cells are characteristic of advanced ks, figure 3. high power view (400x) of oral kaposi’s sarcoma showing a positive immunohistochemistry stain for hhv-8. figure 4. patient remains disease free after conservative surgery in the 17 months follow-up. 5 bortoluzzi et al. however, typical ks lesions do not exhibit marked cellular pleomorphism, necrosis or many mitotic figures, though, mildly atypical endothelial cells and monomorphic spindle cells may be observed8,9,11,15. therefore, it is also possible that a biopsy can be initially misdiagnosed and for that reason ks should be diagnosed with histology and immunohistochemistry due to a large range of similarities within diseases2,8,16. the differential diagnosis of ks includes benign and malignant tumors and for early lesions can be included lesions such as benign vascular proliferation like as hemangioma, acroangiodermatitis, benign lymphangioendothelioma, bacillaryangiomatosis and pyogenic granuloma. more advanced and cellular lesions must be differentiated from angiosarcoma, lymphoma, haemangioendothelioma, aneurysmal fibrous histiocytoma, spindle cell hemangioma, dermatofibrosarcoma protuberans, vascular or pilar leiomyomas and even undifferentiated scamous cells carcinoma2,8,10,11. the immunohistochemistry panel suggested for this particular case included the detection of kshv/hhv8 which, when present in a lesion, it may strongly indicate the diagnostic for ks and because all kshv-infected cells express lana-1, both lana-1 and kshv/hhv8 immunohistochemistry are useful for diagnosis of ks. those markers also act as differential diagnosis since angiosarcoma and benign vascular tumors are negative for those antigens2,8,10. by performing immunohistochemical staining for endothelial cell markers such as cd31 and cd34 which are endothelial markers expressed in vascular tumors, ks can be differentiated from nonvascular neoplasms10,16,17. the sample also showed immuno-positivity for erg (erythroblast transformation-specific family or ets) which is a highly specific marker for benign and malignant endothelial/vascular tumors and ks, while carcinomas and epithelial tumors usually were erg negative16,18 the negative stain for s-100, cytokeratins (40, 48, 50 and 50.6 kda) and desmin antibodies exclude respectively other tumors of neural, epithelial and muscle origins17. on the basis of the above immohistochemistry observations, the h&e and clinical features it was possible to conclude the oks final diagnosis. in conclusion, this manuscript described a rare case of classic primary oks presenting on a female patient who was hiv-negative and wasn’t immunosuppressed alerting clinicians and pathologists to be aware of the clinical and histopathologic features of ks so as not to misdiagnosis it, leading to a profound impact on patient therapeutic and prognostic. ks must also therefore be diagnosed including clinical, histopathology and immunohistochemistry due to a large range of similarities within diseases including, mainly, antibodies against kshv/hhv8 and lana-1. competing interests the authors of this manuscript declare no conflict of interest. funding this case report manuscript received no direct funding. ethical approval this case report manuscript is in compliance with declaration of helsinki ethical principles for medical research involving human subjects. 6 bortoluzzi et al. references 1. barnes l, eveson jw, reichart p, sidransky r, editors. world health organization classification of tumors. pathology and genetics head and neck tumors. lyon: iarc press; 2005 [cited 2017 jun 5]. available from: https://www.iarc.fr/en/publications/pdfs-online/pat-gen/bb9/bb9.pdf. 2. fukumoto h, kanno t, hasegawa h, katano h. pathology of kaposi›s sarcoma-associated herpesvirus infection. front microbiol. 2011 aug 25;2:175. doi: 10.3389/fmicb.2011.00175. 3. fatahzadeh m, schwartz ra. oral kaposi’s sarcoma: a review and update. int j dermatol. 2013;52(6):666-72. doi: 10.1111/j.1365-4632.2012.05758.x. 4. dal maso l, polesel j, ascoli v, zambon p, budroni m, ferretti s, et al. classic kaposi’s sarcoma in italy, 1985-1998. br j cancer. 2005;92(1):188-93. 5. chang y, cesarman e, pessin ms, lee f, culpepper j, knowles dm, et al. identification of herpesvirus-like dna sequences in aids-associated kaposi’s sarcoma. science. 1994;266(5192):1865-9. 6. duus km, lentchitsky v, wagenaar t, grose c, webster-cyriaque j. wild-type kaposi’s sarcoma-associated herpesvirus isolated from the oropharynx of immune-competent individuals has tropism for cultured oral epithelial cells. j virol. 2004;78(8):4074-84. 7. seleit i, attia a, maraee a, samaka r, bakry o, eid e. isolated kaposi sarcoma in two hiv negative patients. j dermatol case rep. 2011;5(2):24-6. doi: 10.3315/jdcr.2011.1067. 8. pantanowitz l, khammissa ra, lemmer j, feller l. oral hiv-associated kaposi sarcoma. j oral pathol med. 2013;42(3):201-7. doi: 10.1111/j.1600-0714.2012.01180.x. 9. mohanna s, bravo f, ferrufino jc, sanchez j, gotuzzo e. classic kaposi’s sarcoma presenting in the oral cavity of two hiv-negative quechua patients. med oral patol oral cir bucal. 2007;12(5):e365-8. 10. bottler t, kuttenberger j, hardt n, oehen hp, baltensperger m. non-hiv-associated kaposi’s sarcoma of the tongue. case report and review of the literature. int j oral maxillofac surg. 2007;36(12):1218-20. 11. reis-filho js, souto-moura c, lopes jm. classic kaposi’s sarcoma of the tongue: case report with emphasis on the differential diagnosis. j oral maxillofac surg. 2002;60(8):951-4. 12. markopoulos ak, papanayotou p, trigonidis g. kaposi’s sarcoma of the oral cavity: case reports. quintessence int. 1994;25(6):415-8. 13. gordon e, searles ge, markman s, yazdi hm. primary oral kaposi’s sarcoma of the hard palate. j am acad dermatol. 1990;23(3 pt 1):518-9. 14. wild r, balmer mc. have we forgotten? oral manifestations of kaposi’s sarcoma. sex transm infect. 2015;91(5):345. doi: 10.1136/sextrans-2015-052116 15. regezi ja, sciubba jj, jordanrck. oral pathology: clinical pathologic correlations. 4th ed. saunders; 2003. cap. 4: red-blue lesions. p.204. 16. ordóñez ng. immunohistochemical endothelial markers: a review. adv anat pathol. 2012;19(5):281-95. doi: 10.1097/pap.0b013e3182691c2a. 17. bishop pw. an immunohistochemical vade mecum. 2007 [cited 2017 jun 5]. available fom: www.e-immunohistochemistry.info. 18. miettinen m, wang zf, paetau a, tan sh, dobi a, srivastava s, et al. erg transcription factor as an immunohistochemical marker for vascular endothelial tumors and prostatic carcinoma. am j surg pathol. 2011;35(3):432-41. doi: 10.1097/pas.0b013e318206b67b. http://www.e-immunohistochemistry.info oral sciences n3 original article braz j oral sci. october | december 2015 volume 14, number 4 clinical evaluation of two materials in the restoration of abfraction lesions fabianna da conceição dantas de medeiros¹, marquiony marques santos1, isaac jordão de souza araújo2, isabela pinheiro cavalcanti lima2 ¹universidade estadual do rio grande do norte – uern, department of dentistry, area of dental materials, caicó, rn, brazil 2universidade estadual de campinas unicamp, piracicaba dental school, area of dental materials, piracicaba, sp, brazil correspondence to: fabianna da conceição dantas de medeiros departament of dentistry caicó campus, uern rua andré sales, 667, paulo vi, cep 59300-000 caicó, rio grande do norte, brasil phone: +55 84 3421 6513 e-mail: fabianna.89@hotmail.com abstract aim: to evaluate the clinical performance of a composite resin (cr) and a resin-modified glassionomer cement (rmgic) for the treatment of abfraction lesions. methods: thirty patients with abfraction lesions in at least two premolar teeth were selected and invited to participate in this study. all restorations were made within the same clinical time frame. one tooth was restored with cr z100tm (3m, st. paul, mn, usa), and the other was restored with rmgic vitremertm (3m). the restorations were assessed immediately and 1, 6 and 12 months after the restoration, using modified us public health service (usphs) criteria: marginal integrity, marginal discoloration, wear, retention, secondary caries and hypersensitivity. the statistical analysis was based on friedman anova test and mann-whitney test, considering p<0.05 for statistical significance. results: both materials demonstrated satisfactory clinical performance after one year. in the individual analysis of each material, there was a significant difference (p<0.05) in the criteria marginal integrity and wear, for both cr and rmgic. rmgic exhibited more damage one year after the restoration. comparing both materials, it was found a significant difference only for marginal discoloration, while the rmgic restorations showed the worst prognosis after a year of evaluation. there was no significant difference in the number of retentions, caries or hypersensitivity between cr and rmgic. conclusions: it was concluded that cr exhibited the best clinical performance according to the cost-effectiveness and evaluation criteria used in this study. keywords: composite resins; glass ionomer cements; tooth wear. introduction abfraction is a concept that explains the pathological loss of cervical tooth structure caused by eccentric occlusal forces on the teeth1-2. problems such as dentinal hypersensitivity, increased bacterial biofilm retention and caries incidence are commonly related to abfraction lesions and require the replacement of the lost tooth tissue with restorative materials3. the treatment of abfraction lesions depends on the elimination of the etiological factors, and it is done by a combination of preventive and therapeutic measures. in addition, performing occlusal adjustment prior to restoration is of paramount importance for a successful treatment4. commonly used restorative materials are composite resin (crs), glass-ionomer cement or a combination of both materials and their appropriate adhesive systems. the dentist’s choice of the restorative material is usually determined by factors such as micromechanical adhesive retention, tooth structure preservation, and good aesthetic and functional outcomes5. however, several studies report failure of cervical restorations of abfraction lesions and an incessant search for the most suitable restorative material4,6-8. braz j oral sci. 14(4):287-293 http://dx.doi.org/10.1590/1677-3225v14n4a07 received for publication: november 04, 2015 accepted: december 12, 2015 therefore, this study aimed to evaluate the clinical performance of a cr compared with a resin-modified glass-ionomer cement (rmgic) in the treatment of abfraction lesions. material and methods study design and sample this study involved the clinical follow up of patients treated at the integrated dentistry clinic at universidade estadual do rio grande do norte (uern), caicó, rn, brazil. the volunteers (n=30) who participated in this clinical trial were 18–50 years old. all participants were informed about the conditions and goals of this study and signed a written informed consent. the study project has been previously reviewed and approved by the local research ethics committee (process no. 785.023 / 2014). the sample size was determined considering a retention rate of 98% for rmgic after one year9 and a unilateral test with an alpha level of 5% and a power of 80%. thus, in order to detect a difference of 20%, the minimum sample size was set at 30 volunteers. each volunteer received two restorations of abfraction lesions, totaling 60 dental restorations. all procedures were performed in accordance with the recommendations of the american dental association (ada) for clinical trials with restorative materials10. the criteria for participation were good general health, current treatment at the dentistry clinic at uern, satisfactory oral hygiene and acceptable oral conditions. the required occlusal restorations were already placed on all posterior teeth except those with abfraction lesions. additionally, in order to participate in the study, volunteers were required to have at least 20 teeth and one abfraction lesion in two different premolar teeth, in contact with antagonist teeth. all premolars presented wedge-shaped cervical lesions with e”1 mm depth on the vestibular sides. volunteers were excluded from the study if they required dental prostheses in the spaces contiguous to target teeth, a diagnosis of caries coincident with the cervical lesion, or an indication for irreversible endodontic treatment of the target teeth. other exclusion criteria were the existence of moderate-to-severe periodontal damage and/or parafunctional habits. patients selected for the study were already under treatment in the integrated dental clinics, including those forwarded and screened in the temporomandibular disorders extension project, which excluded patients with severe occlusal problems and more severe parafunctional habits, avoiding bias in the study. the methodology was based on the split-mouth system. for standardization purposes, the tooth pairs chosen for restoration had to be similar in size and depth. a premolar from each dental arch, located in different hemi-arches was selected. clinical protocol of the restoration for standardization purposes, the same researcher and his/her assistant performed all restorative procedures. each patient received two restorations: one with cr z100tm (3m, st. paul, mn, usa) and the other with rmgic vitremertm (3m, st. paul, mn, usa). the restorative materials were randomly assigned to patients, through a simple lottery. prior to restoration, all patients underwent complete occlusal adjustment via selective grinding. all restorations were carried out with rubber dam isolation without cavity preparation, apart from prophylaxis and cleaning of the walls to be restored with pumice paste and water. the materials were handled and inserted according to the manufacturers’ recommendations. for cr restorations the acid etching was made with 37% phosphoric acid (dentscare, joinville, sc, brazil) for 30 s, followed by rinsing for 30 s and drying with an air syringe for 15 s. the adhesive adper single bondtm (3m espe, st. paul, mn, usa) was applied using a microbrush compatible with the abfraction size, light cured with a conventional lightcuring unit (elipar free light, 3m espe) emitting at 1200 mw/cm2 for 20 s. z100 cr (3m espe) was inserted according to an incremental technique in increments not thicker than 2.0 mm light cured with the same light-curing unit (elipar free light) for 40 s at each increment. in rmgic restorations, the primer from kit vitremertm (3m espe) was applied with a microbush on the surface of the abfraction, left for 30 s and light cured with elipar free light 1200 mw/cm 2 for 20 s. rmgic was prepared considering a powder/liquid ratio 1:1 by agglutination for approximately 45 s until a moist and shiny mass with creamy consistency was obtained. the material was inserted into the abfraction in 2.0-mm-thick increments light cured with the same light-curing unit (elipar free light) for 40 s at each increment. after the restorations with both materials, the gross excesses were removed and initial finishing was performed. in the following session, finishing and polishing were completed using composite polishing burs and rubber tips. clinical evaluation data collection two calibrated and trained dentists blinded to the materials and methodology of the study evaluated the restorations. the study’s coordinator conducted calibration of the evaluators. the clinical parameters were explained and a trial data collection with the equivalent of 10% of the sample was performed. kappa cohen test was applied to confirm the calibration of the evaluators and satisfactory results were obtained (kappa=0.85). the examiners evaluated all restorations by filling out an individual record sheet for each patient. these records were made using the following modified usphs criteria11: marginal integrity, marginal discoloration, wear, retention, secondary caries and hypersensitivity (table 1). the restorations were assessed immediately, one, six and twelve months after placement. statistical analysis in order to investigate the relationship between the dependent (marginal integrity, marginal discoloration, wear, retention, secondary caries, and hypersensitivity) and the independent variables (time gap of evaluations, materials – 288288288288288clinical evaluation of two materials in the restoration of abfraction lesions braz j oral sci. 14(4):287-293 289289289289289 criteria marginal integrity wear marginal discoloration retention recurrent caries post operative sensitivity score alfa (a) bravo (b) charlie (c) alfa (a) bravo (b) charlie (c) alfa (a) bravo (b) charlie (c) alfa (a) charlie (c) alfa (a) charlie (c) alfa (a) bravo (b) charlie (c) description of the criteria there is no visual evidence of marginal fracture in the interface tooth/restoration. there are visible and tactile evidence of the presence of cleft, but the dentin is not exposed. the explorer penetrates the tooth/restoration interface, with exposed dentin. the restoration has continuity with the anatomical shape of the existing tooth. the restoration has continuity with the anatomical, but no has dentin exposure. there is loss of restorative material leading to dentin exposure there is no visual evidence of marginal discoloration on the tooth/restoration interface. there is visual evidence of marginal discoloration on the tooth interface/restoration. there is visual evidence of marginal discoloration, with extent of penetration in the pulp. presence of restoration. partial absence or total restoration. there is no visual evidence of decay in the tooth/restoration interface. there is visual evidence of decay in the tooth/restoration interface. painful symptoms of absence to thermal stimuli and / or percussion. presence of painful symptoms when the performance of the different stimuli. presence of spontaneous symptoms reported by the patient. table 1 –table 1 –table 1 –table 1 –table 1 – description of the criteria used for the clinical evaluation of restorations. reference: santiago et al (2010)12. cr or rmgic) the following non-parametric tests were used: friedman’s anova, the wilcoxon post-hoc test and the mann-whitney test. all tests were performed using spss version 20.0 (spss inc., chicago, usa). the level of significance was set to 5% (p<0.05). results four patients were lost at the last follow-up interval 1 year. this loss is equivalent to eight restorations, which did not affect the comparison of results. table 2 summarizes the average results of the criteria used for assessing cr restorations immediately, one month, six months and one year after placement. among the analyzed criteria, there were significant changes (p<0.05) in marginal integrity and wear after 6 months and one year post-placement, respectively. although there were significant changes in marginal discoloration (p<0.05), no significant difference (p>0.05) was found between the evaluation periods times. table 3 summarizes the average results of the criteria used for assessing rmgic restorations immediately, one, six and twelve months after placement. according to table 3, there were statistically significant changes (p<0.05) in marginal integrity and wear of rmgic restorations. there were statistically significant changes (p<0.05) in marginal integrity of rmgic restorations after six months and one year. there were also significant changes (p<0.05) in the wear of restorations at one year post-placement. rmgic restorations showed marginal discoloration at six months post-placement. for both materials, in the individual evaluation, no significant differences (p>0.05) were observed over time criteria for secondary caries, hypersensitivity and retention, as shown in tables 2 and 3. table 4 shows the comparison of the mean for cr and rmgic over the evaluated intervals. except for marginal discoloration after one year of evaluation, there were no significant differences (p>0.05) between both materials for all evaluated criteria. discussion in general, the multifactorial etiology, pathogenesis, diagnosis and selection of procedures for the restoration of abfraction lesions have challenged modern dentistry and generated a good deal of discussion. the findings of this study are in line with the study by santiago et al. (2010)12, who found no significant difference in marginal integrity and wear for cr at one year postplacement. the flaws identified in the marginal integrity of the cr restorations after six months may be due to failures in the interface between the tooth and the restorative material, especially near the cavosurface angle of the abfraction lesions. moreover, the tooth is subjected to occlusal loads with time3,13. the poor marginal fit of cr restorations denotes adhesive bond degradation and polymerization shrinkage, which is a difference from the thermal expansion coefficient between the material/tooth and the occlusal loads 14-15. the high modulus of elasticity of micro-hybrid crs (e.g. z100) characterizes them as rigid materials. this means that if subjected to occlusal load, they are unable to flex along with the tooth15-16. in their in vitro study, pereira et al. (2012)8 clinical evaluation of two materials in the restoration of abfraction lesions braz j oral sci. 14(4):287-293 290290290290290 variable times average dp± p marginal integrity immediately a 2.88 0.44 0.001 1 month a,c 2.80 0.58 6 months b 2.56 0.65 1 year b,c 2.44 0.71 wear immediately a 2.88 0.33 0.001 1 month a 2.88 0.33 6 months a,b 2.68 0.56 1 year b 2.40 0.76 marginal discoloration immediately a 2.88 0.33 0.018 1 month a,b 2.84 0.37 6 months b 2.72 0.46 1 year b 2.64 0.49 retention immediately a 2.96 0.20 0.194 1 month a 2.92 0.40 6 months a 2.84 0.47 1 year a 2.64 0.76 recurrent caries immediately a 3.00 0.00 1.000 1 month a 3.00 0.00 6 months a 3.00 0.00 1 year a 3.00 0.00 sensitivity immediately a 2.92 0.28 0.274 1 month a 2.88 0.33 6 months a 2.80 0.41 1 year a 2.96 0.20 table 3 table 3 table 3 table 3 table 3 comparison of the average results obtained for the usphs criteria used for assessing rmgic restorations immediately, one month, six months and one year after placement variable times average dp± p marginal integrity immediately a 2.84 0.37 0.001 1 month a,b 2.72 0.46 6 months b 2.60 0.50 1 year b 2.52 0.59 wear immediately a 2.84 0.37 0.021 1 month a,b 2.80 0.41 6 months a,b 2.72 0.46 1 year b 2.60 0.50 marginal discoloration immediately a 2.92 0.28 0.083 1 month a 2.80 0.41 6 months a 2.76 0.44 1 year a 2.88 0.33 retention immediately a 2.96 0.20 0.234 1 month a 2.92 0.28 6 months a 2.84 0.37 1 year a 2.76 0.60 recurrent caries immediately a 3.00 0.00 1.000 1 month a 3.00 0.00 6 months a 3.00 0.00 1 year a 3.00 0.00 sensitivity immediately a 2.88 0.33 0.954 1 month a 2.88 0.33 6 months a 2.84 0.37 1 year a 2.88 0.33 table 2 table 2 table 2 table 2 table 2 comparison of the average results obtained for the usphs criteria used for assessing cr restorations immediately, one month, six months and one year after placement clinical evaluation of two materials in the restoration of abfraction lesions braz j oral sci. 14(4):287-293 291291291291291 variable materials average dp± p marginal integrity immediately c r 2.84 0.37 0.490 rmgic 2.88 0.44 marginal integrity 1 month c r 2.72 0.46 0.269 rmgic 2.80 0.58 marginal integrity 6 months c r 2.60 0.50 0.964 rmgic 2.56 0.65 marginal integrity 1 year c r 2.52 0.59 0.809 rmgic 2.44 0.71 wear immediately c r 2.84 0.37 0.720 rmgic 2.88 0.33 wear 1 month c r 2.80 0.41 0.492 rmgic 2.88 0.33 wear 6 months c r 2.72 0.46 0.931 rmgic 2.68 0.56 wear 1 year c r 2.60 0.50 0.471 rmgic 2.40 0.76 marginal discoloration immediately c r 2.92 0.28 0.393 rmgic 2.88 0.33 marginal discoloration 1 month c r 2.80 0.41 0.741 rmgic 2.84 0.37 marginal discoloration 6 months c r 2.76 0.44 0.372 rmgic 2.72 0.46 marginal discoloration 1 year c r 2.88 0.33 0.049 rmgic 2.64 0.49 retention immediately c r 2.96 0.20 1.000 rmgic 2.96 0.20 retention 1 month c r 2.92 0.28 0.584 rmgic 2.92 0.40 retention 6 months c r 2.84 0.37 0.953 rmgic 2.84 0.47 retention 1 year c r 2.76 0.60 0.653 rmgic 2.64 0.76 caries immediately c r 3.00 0.33 1.000 rmgic 3.00 0.28 caries 1 month c r 3.00 0.00 1.000 rmgic 3.00 0.00 caries 6 months c r 3.00 0.00 1.000 rmgic 3.00 0.00 caries 1 year c r 3.00 0.00 1.000 rmgic 3.00 0.00 hypersensitivity immediately c r 2.88 0.33 0.393 rmgic 2.92 0.28 hypersensitivity 1 month c r 2.88 0.33 0.451 rmgic 2.88 0.33 hypersensitivity 6 months c r 2.84 0.37 0.740 rmgic 2.80 0.41 hypersensitivity 1 year c r 2.88 0.33 0.302 rmgic 2.96 0.20 table 4 table 4 table 4 table 4 table 4 comparison of the average results obtained for rmgic e cr restorations immediately, one month, six months and one year after placement. found changes in the marginal integrity of teeth restored with z100 resin. the high modulus of elasticity of this resin is insufficient to dissipate the stress of polymerization shrinkage. this is a persistent feature crs, which ultimately leads to adhesion failure and seal collapse. awliya and elsahn (2008)17 showed that the class-v lesions restored with high-elasticity resins exhibited a satisfactory distribution of stresses, without marginal deterioration. clinical evaluation of two materials in the restoration of abfraction lesions braz j oral sci. 14(4):287-293 292292292292292 in line with the findings of other studies9,12,18, we found unsatisfactory marginal integrity results for rmgic restorations. in contrast, other studies found that a rmgic (vitremer) was the best material for keeping the integrity at the dentin edge8,18. despite the favorable biomechanical characteristics of rmgic, the striking marginal deterioration of rmgic restorations may result from the hygroscopic expansion of glass ionomer components and fractures due to excessive contouring of the edges by materials subjected to occlusal loads19. although finishing or beveling of the enamel walls has not yet been performed, the very configuration of lesion may lead to the occurrence of abfracted, friable, and unsupported enamel edges that surpass the cavosurface angle20. thus, minor marginal fractures are associated with the small volume of rmgic in the interface between tooth and restoration. when there is a satisfactory bond between the restorative material and the tooth, the forces generated by the compression of the restoration will act more upon the body than on the adhesive interface. as a consequence, the adhesive bond will be preserved. this will allow favorable retention of the restoration even if the marginal integrity is affected8,18, as shown in this study. according to van landuyt et al. (2011)21, although the retention rates for current adhesive systems have improved, marginal sealing remains problematic. analogous to this research, burrow (2011) 22 also found that marginal deterioration was more outstanding than discoloration for both materials. however, this marginal deterioration did not compromise the overall performance of the restorations23, because both restorations were considered clinically acceptable and did not have to be replaced. marginal discoloration is expected following damage to the edges of the restoration. in the initial assessments in this study, the statistical analysis revealed some change in the color of the edges for both study materials. this may relate to difficulties with the finishing and polishing of the cervical restorations. it may also relate to the excess of restorative material (not removed after polishing) ) ) ) ) in nonconditioned areas, as this facilitates the impregnation of pigments24. the initial color stability of rmgic is generally satisfactory. it has ben demonstrated, however, that this material tends to change its color over time9. this may be related to surface changes within the material, as indicated by loss of anatomic contour and wear. this was also evident in the results of the present study. in agreement with other studies 6,9,12,22, the present investigatiin found a significant difference in wear of both materials after one year of placement, mainly in cr. santiago et al. (2010)12 observed significant differences over time in the anatomical shape of the materials, especially cr, as compared to glass-ionomer cement. a possible explanation for the wear of cr restorations may be that the tensile stress during tooth bending would favor the formation of cracks in the body of the restoration, facilitating the detachment of inorganic resin fillers when they are subjected to conditions of mechanical friction and chemical abrasion, as during toothbrushing14. the long-term anatomical wear of glass-ionomer restorations may be attributed to inferior physical properties25 relative to cr. glass-ionomer has low resistance to the wear26 associated with excessive brushing force. van dijken and pallesen (2008)27, however, found that vitremer was able to satisfactorily maintain its anatomical shape, which may be due to the improved physical properties of hybrid ionomer restorative materials. glass-ionomer cements tend to incorporate air bubbles during handling. the air bubbles and filler particles (which become exposed following brushing) result in porosities, increased roughness and wear8. these effects can be observed clinically. the secondary caries criterion remained unchanged for both materials throughout the evaluation period. similarly, no statistically significant difference was observed for postoperative hypersensitivity. in agreement with these findings, pollington and van noort (2008)28 found that out of the 60 cr restorations placed during their study, none showed signs of caries or postoperative hypersensitivity. in this study, however, some patients exhibited hypersensitivity immediately and one month after restoration. the hypersensitivity experienced immediately after restoration may result from mechanical damage to the gums. this may occur during the finishing and polishing of the restorative materials or when excess material remains in contact with the soft tissue29. gingival recession and root exposure immediately after placement or following finishing and polishing have been associated with tooth hypersensitivity in one-month postrestoration30. this was also observed by stojanac et al. (2013)14. in their study, the hypersensitivity measured in the initial assessment decreased over time and disappeared completely after two years. several similar studies found no secondary caries associated with cervical restorations9,12,14,29-30. the absence of caries in the assessed restorations may be due because the participants were undergoing oral treatment (in the phase after conditioning of the oral environment) and maintained satisfactory oral hygiene. moreover, the evolution in gic has contributed to the reduction of dental caries because of its characteristics31-32. based on the findings of this study, both materials provided satisfactory clinical performance in the restoration of cervical abfraction lesions, with peculiarities inherent to the physicochemical properties of each one of them. nevertheless, cost-effectiveness should be considered when choosing a restorative material. rmgic is more expensive than cr. the authors believe that understanding the biomechanics of cervical restorations makes the selection of restorative materials easier and enables a better prognosis. this is due to a broader understanding of all associated factors. in conclusion, the composite can be a good option to restore abfraction lesions. the use of this material or rmgic should be inexorably bound to the balance of the occlusal clinical evaluation of two materials in the restoration of abfraction lesions braz j oral sci. 14(4):287-293 load, in order to increase the longevity of the restoration and preserve the remaining tooth structure. given the limitations of this study, further research into the treatment of abfraction lesions is recommended. future studies should employ indirect review methods and functional analysis of occlusion. references 1. grippo jo, simring m, coleman ta. abfraction, abrasion, biocorrosion, and the enigma of noncarious cervical lesions: a 20-year perspective. j esthet restor dent. 2012; 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21: 229-34. 13. brandini da, trevisan cl, barioni srp, pedrini d. clinical evaluation of the association between noncarious cervical lesions and occlusal forces. j prosthet dent. 2012; 108: 298-303. 14. stojanac il, premovic mt, ramic bd, drobac mr, stojsin im, petrovic lm. noncarious cervical lesions restored with three different tooth-colored materials: two-year results. oper dent. 2013; 38: 12-20. 15. petrovic lm, drobac mr, stojanac il. a method of improving marginal adaptation by elimination of singular stress point in composite restorations during resin photo-polymerization. dent mater. 2010, 26: 449-55. 16. kubo s, yokota h, hayashi y. three-year clinical evaluation of a flowable and a hybrid resin composite in non-carious cervical lesions. j dent. 2010; 38: 191-200. 17. awliya wy, el-sahn am. leakage pathway of class v cavities restored with different flowable resin composite restorations. oper dent. 2008; 33: 31-6. 18. francisconi lf, scaffa pmc, barros vr, coutinho m, francisconi pa. glass ionomer cements and their role in the restoration of non-carious cervical lesions. j appl oral sci. 2009; 17: 364-9. 19. santamaria mp, da silva feitosa d, nociti jr fh, casati mz, sallum aw, sallum ea. connective tissue graft plus resin-modified glass ionomer restoration for the treatment of gingival recession associated with noncarious cervical lesion: a randomized-controlled clinical trial. j clin periodontol. 2009; 36: 791-8. 20. baig mm, mustafa m, jeaidi zaa, al-muhaiza m. microleakage evaluation in restorations using different resin composite insertion techniques and liners in preparations with high c-factor – an in vitro study. king saud univ j dent sci. 2013; 4: 57-64. 21. van landuyt kl, peumans m, de munck j, cardoso mv, ermis b, van meerbeek b. three-year clinical performance of a hema-free one-step self-etch adhesive in non-carious cervical lesions. eur j oral sci. 2011; 119: 511-6. 22. burrow mf. clinical evaluation of non-carious cervical lesion restorations using a hema-free adhesive: three-year results. aust dent j. 2011; 56: 401-5. 23. chee b, rickman lj, satterthwaite jd. adhesives for the restoration of non-carious cervical lesions: a systematic review. j dent. 2012; 40: 44352. 24. heintze sd, thunpithayakul c, armstrong sr, rousson v. correlation between microtensile bond strength data and clinical outcome of class v restorations. dent mater. 2011; 27: 114-25. 25. lund rg, ogliari f, lima gs, del-pino fab, petzhold cl, piva e. diametral tensile strength of two brazilian resin-modified glass ionomers cements: influence of the powder/liquid ratio. braz j oral sci. 2007; 6: 1353-6. 26. torabzadeh h, ghasemi a, shakeri s, baghban aa, razmavar s. effect of powder/liquid ratio of glass ionomer cements on flexural and shear bond strengths to dentin. braz j oral sci. 2011; 10: 204-7. 27. van dijken jw, pallesen u. long-term dentin retention of etch-and-rinse and self-etch adhesives and a resin-modified glass ionomer cement in noncarious cervical lesions. dent mater. 2008; 24: 915-22. 28. pollington s, van noort r. a clinical evaluation of a resin composite and a compomer in non-carious class v lesions. a 3-year follow-up. am j dent. 2008; 21: 49-52. 29. veitz-keenan a, barna ja, strober b, matthews ag, collie d, vena d, et al. treatments for hypersensitive noncarious cervical lesions: a practitioners engaged in applied research and learning (pearl) network randomized clinical effectiveness study. j am dent assoc. 2013; 144: 495-506. 30. loguercio ad, raffo j, bassani f, balestrini h, santo d, do amaral rc, et al. 24-month clinical evaluation in non-carious cervical lesions of a twostep etch-and-rinse adhesive applied using a rubbing motion. clin oral investig. 2011; 15: 589-96. 31. pithon mm, ruellas aco, sant’anna ef. effect of bleaching with hydrogen peroxide into different concentrations on shear strength of brackets bonded with a resin-modified glass ionomer. braz j oral sci. 2008; 7: 1483-8. 32. graciano kp, moysés mr, ribeiro jc, pazzini ca, melgaço ca, ramos-jorge j. one-year clinical evaluation of the retention of resin and glass ionomer sealants on permanent first molars in children. braz j oral sci. 2015; 14: 190-4. 293293293293293 clinical evaluation of two materials in the restoration of abfraction lesions braz j oral sci. 14(4):287-293 absenteeism study in a steel industry of são josé dos campos, sp, brazil isa azevedo de almeida marote1, dagmar de paula queluz2 1uniodonto, private dental clinic, são josé dos campos, sp, brazil 2universidade de campinas – unicamp, piracicaba dental school, department of community dentistry, piracicaba, sp, brazil correspondence to: dagmar de paula queluz faculdade de odontologia de piracicaba – unicamp departamento de odontologia social avenida limeira, 901, cep: 13414-900 piracicaba, sp, brasil phone: +55 19 21065277 e-mail: dagmar@fop.unicamp.br abstract aim: to identify the factors involved in absenteeism in a steel industry in the city of são jose dos campos, são paulo, brazil. methods: a cross-sectional study was carried out after obtaining permission from institutional ethics committee in a steel industry. worker's detailed information regarding absenteeism was obtained from the health department files, from january 2005 to december 2008. specifical data were: sex, function, certificate type (medical or dental), the working sector, according to the large groups (lg) of brazilian classification of occupations 2002, working periods, duration of absenteeism (lost days), the main causes of absenteeism (international classification of diseases icd-10). results: a total of 570 workers were recorded. the majority of workers were males (97%), with medical certificate (97%), lg-7 workers in the production of industrial goods and services (62.09%), and operators of industrial machinery (68.3%) and the working period was shift 4 monday to friday from 8 am to 12 am and 1 pm to 5 pm (44.43 %). the total duration of absenteeism was 3,187 days, the main causes of diseases being problemas in the musculoskeletal system and connective tissue (18.48%), respiratory diseases (17.11%), injuries, poisoning and other external causes (11.24%), and diseases of the digestive tract (11.24%). conclusions: it was concluded that there was under-reporting of absenteeism due to dental causes (41 events) during the study period. more studies in this field are required. keywords: absenteeism. workers. iron and steel industry. introduction in the early 20th century, the brazilian steel-making sector had very little economic meaning. however, when the national steel company was created in 1941, as an answer to the national steel problem, the domestic steel-making industry was much more significant1. the search for total quality in the 1980s and 1990s has stimulated the competitivity among companies, forcing them to adapt to the market2,3. with the globalization, the industry is under international competition, with stress on important things like technology, efficiency, maximum productivity, zero waste, sustainability, lower cost, better quality and flexibility to pursue the business activity. thus, constant changes in production and marketing, the workers’ needs to improve their daily commitment and new updates, stress and diseases caused by the specific work activity, like exposure to heavy metals, acids, high temperatures and toxic products. in addition to stress, it may trigger systemic diseases such as increased blood pressure, heart attacks and other heart problems; failing immune system; depression and gynecological infections in women4. health is the word for one of the fundamental pillars for the development of a nation. the employee is a key component in the production process of the industry, playing an important role in the economy, so that all results are achieved successfully; therefore, received for publication: june 17, 2016 accepted: september 09, 2016 braz j oral sci. 15(2):124-130 original article braz j oral sci. april | june 2016 volume 15, number 2 http://dx.doi.org/10.20396/bjos.v15i2.8648754 125 providing general health assistance to the individual is necessary5-10. the work "de morbis artificium datriba"11 points out that there is a relationship between health and disease in the egyptian papyri, which is considered the mark in the history of work diseases, since they describe diseases related to more than 50 occupations11. for rouquayrol and almeida filho12: "the health of the worker can be considered a social practice that is established in the scope of public health, seeking to contribute to the transformation of the workers health and, by extension, of the general population, the way to understand particular work processes, in an articulated way with the consumption of goods and services and the set of values, beliefs, ideas and social representations characteristic of a moment in human history". according to höfelmann and blank13, health self-evaluation is an important predictor of morbidity and mortality and most of its effects are influenced by the chronic diseases and/or symptoms. absenteeism is a complex phenomenon with a multifactorial etiology including psychosocial, economic and work environment factors, so that it may provide important information about the health status of the population. however, its prevalence and its main causes are still poorly documented. for this reason, it is difficult to develop prevention and rehabilitation programs for this population2,5-7,14,15. in the work environment, oral health is a differential. with a specialist in occupational dentistry, some enterprises stand out by their social commitment, increased quality of life for their employees, also aiming to decrease absenteeism, because in the oral cavity may be located the so-called "occupational diseases" with manifestations in the mucosa and some alterations of enamel. therefore, it is possible to consider that certain diseases have their initial manifestation in the oral tissues, allowing an early and preventive diagnosis5,16-18. studies have demonstrated that absenteeism resulting from dental reasons varies from 10 to 35% of the total amount, and the average number of lost working hours varies from 1.24 to 6.20 working hours/worker/year19. therefore, it is up to the specialist in occupational dentistry to work with professionals involved in the area of occupational health and safety in the company, according to #4 regulatory norm9. he/she should be concerned with the social and political analysis of the issue, interacting with the working class and management. compatibility between the work activity and the preservation of the worker's oral health should be the permanent issue. to identify, evaluate and follow up the environmental factors that may constitute a risk to oral health at work, at any stage of the productive process. technical advice and attention on safety health, ergonomics and hygiene at work, as well as on personal protective equipment, are included in the multidisciplinary health team of the working persons. planning and implementation of campaigns and permanent programs is important for the education of workers, regarding work accidents, occupational diseases and health education. to organize morbidity and mortality statistics with oral cause and investigate their possible relationships with the work activities; dental examinations for labor purposes20. the purpose of this study was to identify the factors involved in absenteeism in a steel industry in the city of são josé dos campos, sp, brazil. material and methods study area the study was carried out in a steel industry in the city of são josé dos campos, são paulo state. occupying over 1,200 square meters, the unit has an installed production capacity of 7,200 tons per year. this industry operates in the manufacture of screens, trusses and columns, among others, used in construction, from buildings and roadways to small hydroelectric power stations. in 2010 the industry had 570 employees, with a working period of twenty-two days per month. study design this is a cross-sectional study, descriptive and quantitative, addressing absenteeism and prevalence of causes of absenteeism in the steel industry, between january 2005 and december 2008 data collection the research was carried out from search in the digital file of the medical sector of the industry, data on medical and dental certificates issued in the study period. medical records are filed at the health department of the steel industry. data were collected on the following variables: • with respect to the worker: sex (male and female), function • with regard to absenteeism: * duration of absenteeism or lost days (days) * causes of absenteeism (international classification of diseases – 10th revision, icd-1021) * certificate type: medical and dental * work sector, according to the large groups (lg) of brazilian classification of occupations – 200222 (chart 1) * working periods: shift 1: monday to friday 6 am – 2 pm and saturday 6 am – 1 pm; shift 2: monday to friday 2 pm – 10 pm and saturday 1 pm – 8 pm, shift 3: monday to friday 10 pm – 6 am and sunday 11 pm – 6 am; shift 4: monday to friday 8 am to noon and 1 pm to 5 pm, shift 5 (workers of 3-shift changes), shift 6 (workers of 2-shift changes), ½ period, trainees. absenteeism study in a steel industry of são josé dos campos, sp, brazil chart 1 brazilian classification of occupations in large groups / titles according to the level of competence22. brazilian classification of occupations (bco-2002) in large groups (lg) level of competence 0 armed forces, police and military firefighters undefined 1 members of public authorities, managers of public interest organizations and companies and managers undefined 2 professionals of the sciences and the arts 4 3 middle level technicians 3 4 administrative service workers 2 5 service workers, sellers of shops in shops and markets 2 6 agricultural, forestry, hunting and fishing workers 2 7 workers in the production of industrial goods and services (crafts) 2 8 workers in the production of industrial goods and services (machines) 2 9 maintenance and repair workers 2 braz j oral sci. 15(2):124-130 126 data analysis the study adopted the concept of medical cause of absenteeism from the international labor organization23, i.e., “the period of absence from work which is accepted as attributable to an inability of the individual, except for the one derived from normal pregnancy or prison.” it is understood by the uninterrupted period of absence from work, told from the very beginning, regardless of its duration. we used the recommendations of the subcommittee on absenteeism of the international society for occupational health, addressing the indices of frequency, severity, percentage rate of absenteeism, average length of absences, as indicators of absenteeism, represented by the formulas below:23. frequency index = total number of cases/number of employees severity index = total lost days/number of workers percentage rate of absenteeism = total lost days x 100/ total days worked x number of workers average length of absences = total lost days/total number of cases the prevalence of abdsences was addressed by the international classification of diseases – 10th revision (icd-10) and the specific highlights were the greatest highlights in terms of lost days and number of occurrences, also distributed by sex and function. the dependent variables were the number of lost days and the number of events. short-term offsets were those of up to five days, medium duration between six and fifteen days and large deviations of more than sixteen days. as independent variables, sex, function and medical nature (icd-10) of the condition were used. for the statistical analysis, the statistical package ssp was used. anova and the student's t-test were used to verify the differences between the means of the number of lost days and the number of events, adopting a significance level of 5% (p<0.05). the results were expressed by tables, in frequency and proportions. ethical considerations this research was approved by the ethics committee of the dental school of piracicaba, unicamp, under approval protocol number 051/2009. results a total of 570 workers were included in the final analysis. among the 570 workers in the steel industry, 63% (n=354) presented absenteeism and 37% (n=216) did not present absenteeism, from january 2005 to december 2008, in a total of 1245 certificates. it was observed that 97% (n=1209) of the presented certificates were for male and 3% (n=36) were for female workers. there was a higher percentage of medical certificates (97%; n=1204), in relation to the dental certificates (3%; n=41). in the steel industry, since there are several functions, it was necessary to group them according to the brazilian classification of occupations 2002, in large groups (lg)22. the large groups lg-6 and lg-8 were not part of this research, since they do not apply to the studied field (table 1). absenteeism study in a steel industry of são josé dos campos, sp, brazil table 1 distribution of cases of absenteeism in relation to the large groups of the brazilian classification of occupations (bco-2002). large groups cases of absenteeism lg-1 0.41% (n=5) lg-2 3.05% (n=38) lg-3 12.85% (n=160) lg-4 8.51% (n=106) lg-5 0.88% (n=11) lg-7 62.09% (n=773) lg-9 10.28% (n=128) others 1.93% (n=24) total 100% (n=1245) table 2 distribution of cases of absenteeism in relation to the large group (lg) 7 of the brazilian classification of occupations (bco-2002). functions of the lg-7 cases of absenteeism production auxiliaries 21.35% (n=165) production manager 0.13% (n=1) thermal treatment forge 0.52% (n=4) forklift operator 2.07% (n=16) industrial machine operators 68.3% (n=528) machine operator 0.39% (n=3) bridge operators 5.82% (n=45) financial accountant 1.03% (n=8) welder 0.39% (n=3) total 100% (n=773) table 3 distribution of absenteeism cases related with working periods. working periods cases of absenteeism shift 1 5.62% (n=70) shift 2 3.21% (n=40) shift 3 3.05% (n=38) shift 4 44.42% (n=553) shift 5 27.07% (n=337) shift 6 15.18% (n=189) ½ period 0.25% (n=3) trainees 1.2% (n=15) total 100% (n=1245) braz j oral sci. 15(2):124-130 among the 773 workers' certificates classified in lg-7, the industrial machine operators presented the highest percentage (68.3%), followed by production auxiliaries (21.35%) and bridge operators 5.82% (table 2). the workers were divided into 8 different working periods, separated according to the denominations used by the forgery. higher absenteeism prevalence as found in the labor shift 4 with 44.42% (n=553), shift 5 with 27.07% (n=337) and shift 6 with 15.18% (n=189) (table 3). 127 table 4 distribution of the number of cases of absenteeism according to the international classification of diseases (icd-10), sex (male and female) and lg (bco-2002). international classification of diseases (icd-10) (code description and diagnosis code) cases of absenteeism lg(bco-2002) sex 1 2 3 4 5 7 9 others m f chapter i some infectious and parasitic diseases (a00-b99) 102 0 0 3 9 5 0 71 9 5 chapter ii neoplasms [tumors] (c00-d48) 4 2 0 1 1 2 0 1 0 1 chapter iv endocrine, nutritional and metabolic diseases (e00-e90) 5 1 0 1 2 0 0 3 0 0 chapter v mental and behavioral disorders (f00-f99) 23 0 0 0 5 3 0 11 2 0 chapter vi diseases of the nervous system (g00-g99) 10 0 0 0 1 0 0 7 1 1 chapter vii diseases of the eye and attachments (h00-h59) 80 2 1 3 21 5 0 34 17 1 chapter viii diseases of ear and mastoid process (h60-h95) 26 2 0 0 2 3 0 19 4 0 chapter ix diseases of the circulatory system (i00-i99) 50 1 1 1 8 3 1 29 8 0 chapter x diseases of the respiratory system (j00-j99) 210 3 1 3 21 13 1 149 18 4 chapter xi diseases of the digestive system (k00-k93) 126 4 0 4 19 12 1 71 17 6 chapter xii diseases of the skin and subcutaneous tissue (l00-l99) 18 0 0 2 3 3 0 7 2 1 chapter xiii diseases of the musculoskeletal system and connective tissue (m00-m99) 227 3 0 4 17 16 3 175 14 1 chapter xiv diseases of the genitourinary system (n00-n99) 33 4 0 4 3 13 0 14 2 1 chapter xv pregnancy, childbirth and the puerperium (o00-o99) 1 0 0 0 0 0 0 1 0 0 chapter xviii symptoms, signs, and abnormal findings of clinical and laboratory exams not elsewhere classified (r00-r99) 70 4 0 3 13 7 1 43 7 0 chapter xix lesions, poisoning and some other consequences of external causes (s00-t98) 138 2 0 1 17 13 0 91 16 2 chapter xx external causes of morbidity and mortality (v01-y98) 1 0 0 0 0 0 0 1 0 0 chapter xxi factors influencing health status and contact with health services (z00-z99) 88 5 1 6 16 8 4 46 11 1 in the study of 1245 absences in relation to icd-10, the highest incidence was of musculoskeletal and connective tissue diseases (m00-m99) (n=230); diseases of the respiratory system (j00-j99) (n=213); injuries, poisoning and some other consequences of external causes (s00-t98) (n=140); diseases of the digestive tract (k00-k93) (n=130), infectious and parasitic diseases (n=102) (table 4). among the 130 occurrences of the digestive system tract diseases (k00-k93), 31% (n=40) represented diseases of the oral cavity, salivary glands and jaws (k00-k14). out of the 140 occurrences of lesions, poisoning and some other consequences of external causes, 4% (n=6) were by head trauma (s00-s09) and 1% (n=1) were by dental fracture (s02.5). the total number of lost days in the four study years was 3,187. the highest percentages were: with lg-7 accounting for 72.95% (n=2,325), lg-3 for 10.98% (n=350), lg-9 for 7.37% (n=235). lg-1 and lg-5 had low percentage of lost days (table 5). absenteeism study in a steel industry of são josé dos campos, sp, brazil braz j oral sci. 15(2):124-130 128 discussion prevalence of certificates of male workers (97%) aree with the results found by biswas et al. (2014)2, höflmann and blank (2008)13, manjunatha et al. (2011)24, lacerda et al. (2008)25, teles (2005)26. however, perez et al. (2006)15 and danatro (1997)27 found a larger number of absences. there was a higher percentage of medical certificates (97%) compared with höfelmann and blank (2008)13, danatro (1997)27, paulo (2007)28, ito (2007)29, martins et al. (2005)30, panzer (2004)31, mesa and kaempffer (2004)32, tomasini (2001)33, rocha (1981)34. of the 773 attestations of workers classified as lg-7, the industrial machine operators presented the highest percentage (68.3%), similar to the ones found in the studies of biswas et al. (2014)2, manjunatha et al. (2011)24; panzer (2004)31, mesa and kaempffer (2004)32, tomasini (2001)33. in relation to icd-10, among 1245 absences, the highest incidence was of musculoskeletal and connective tissue diseases (m00-m99), which corroborates the studies by biswas et al. (2014)2, manjunatha et al. (2011)24; panzer (2004)31, mesa and kaempffer (2004)32, danatro (1997)27. for gillies (1994)35, it were respiratory diseases, digestive disorders, circulatory problems, gynecological disorders and pathological neuroses. studies by guo et al. (2015)36, wada et al. (2013)37 point out that the highest amount is of mental and behavioral disorders. over the years, interest in the subject has been expanding and new research has been carried out, but there are still few nationally published studies related to the buccal health of the worker in the industries13,15,25,28-30,34,38-40. it is expected that the specialists in occupational dentistry will carry out new studies demonstrating to the employers the benefit of having a worker with good oral health, regardless of being or not a a company’s obligation. the results of our study were similar to rocha's34 study regarding the lack of odontological reasons for the one that contributed least to absenteeism, and medical causes were the most common. rocha34 developed a study in a metallurgic industry in canoas, rio grande do sul, brazil, whose objective was to determine the level of absenteeism from work for medical reasons by analyzing medical service data and questionnaires applied to workers, the level of absenteeism due to work accident, the degree of influence of dental causes in absenteeism (all in relation to the variables age, sex, employee's role in the company and length of service) and the level of perception of the workers regarding oral health and the dental service of the industry. he concluded that the lack of dental care was the one which contributed the least to absenteeism, which was overcome by work-related accidents and medical causes, which were the most common. the results of our study were similar to martins et al.30, where absences due to dental reasons had little weight on the total number of absences due to illness. martins et al.30 studied absenteeism for dental and medical reasons, in the public (in the municipality of araçatuba, sp, brazil) and private (acrylic manufacture) services, from january to june 2002. they found that: absence due to dental reasons had little weight on the total number of absences due to illness, in addition to causing a shorter period of absence. the variables age, sex, function and employment regime influenced the absenteeism at work. table 5 distribution of lost days in relation to the large groups, bco-2002 in the four study years. lg (bco-2002) lost days lg-1 0.29% (n=9) lg-2 1.91% (n=61) lg-3 10.98% (n=350) lg-4 5.27% (n=168) lg-5 0.29% (n=9) lg-7 7.37% (n=235) lg-9 72.95% (n=2325) others 0.94% (n=30) total 100% (n=3187) following the recommendations of the subcommittee on absenteeism of the international society of occupational health23, addressing the indices of frequency, severity, percentage rate of absenteeism, average length of absences, as indicators of absenteeism, were as follows: frequency index = 2.1842 severity index = 5.5912 percentage rate of absenteeism = 2.5598 average duration of absences = 0.58% the prevalence of withdrawals was addressed by the international classification of diseases (icd-10) grouping, and by specific morbidities of the major highlights in terms of lost days (groups v and xiii) and number of occurrences (groups xiii, x, xix and xi), also distributed by sex and function (tables 4 and 6) the differences between the means of the number of days of withdrawal and the number of events were performed by analysis of variance (anova) obtaining f=36.41 (p=0.00000003) and the student t test equal to 1.479 with an error margin of 0.5%. figure 1 shows the distribution of lost days in relation to number of cases in the studied four years. fig.1. distribution of lost days in relation to number of cases in the four study years. absenteeism study in a steel industry of são josé dos campos, sp, brazil table 6 distribution of the number of absentee days in relation to the number of cases. number of days number of cases average (%) 1-5 (*) 203 1.9556 6-15 86 9.7906 ≥16 65 29.969 (*) we included the 32 cases that an error in the system recorded as 0 days, but they were ½ period absences. braz j oral sci. 15(2):124-130 129 absenteeism study in a steel industry of são josé dos campos, sp, brazil paulo28 studied dental absenteeism in a metallurgical industry in the region of contagem, mg, brazil, in which he performed a survey of absences for health leave registered during four months. he collected data from all medical and dental certificates from june to september 2006. he found that medical certificates were 99%, while dental certificates presented 1%. our study found that medical certificates were 97%. höfelmann and blank13 studied 482 workers from a metalwork industry in santa catarina, brazil, in order to identify the factors that confounded the association between chronic diseases and/or symptoms and self-assessment of health among workers. using self-administered questionnaires and anthropometric measures, hierarchical models of multiple logistic regression with a response rate of 98.6% (n=475) were adjusted. there were 84.8% in the male sex, 79.4% in the productive sector. the most common complaint was back pain and association between chronic diseases and health self-evaluation presented an odds ratio of 7.3 (95% ci: 3.7; 14.5). with statistical modeling, psychosocial (-25.59%), socioeconomic (-9.29%) and occupational (10.54%) variables were identified as confounding factors between the outcome and chronic diseases and/ or symptoms. they concluded that the way in which the diseases and/ or symptoms act in the health self-evaluation among workers goes beyond the physical aspects. our study found the highest percentage (68.3%) in the industrial machine operators. danatro27 conducted a survey of data from medical certificates issued between july 1, 1994 and june 30, 1995 in a public institution with 1,474 employees (594 in the administrative sector), in the city of montevideo, uruguay. 1,604 certificates had medical causes and 40 non-medical causes. the 1,604 certificates of medical cause generated 10,085 days of absence; the majority of the certificates were 1 day; the most representative diseases of the study were respiratory causes, musculoskeletal and connective tissue disorders, presence of risk factors, contact with health services and digestive diseases. our study found the distribution of lost days in relation to number of cases in the four years study, the majority was between 0 and 10 lost days. it was concluded that: • most certificates were for males (97%), medical (97%), lg-7 (62%), industrial machinery operators (68%) and shift 04 (44.43%); • total duration of the absences was 3,187 days. the main causes were osteomuscular and connective tissue diseases (18.48%), respiratory diseases (17.11%), lesions, poisoning and some other consequences of external causes (11.24%), diseases of the digestive system (11.24%); • the greatest amount of lost days was in groups v and xiii and the greatest number of occurrences was in groups xiii, x, xix and xi; • the differences between the means of the number of lost days and the number of events were f = 36.41 (p = 0.00000003) and the student t test was equal to 1.479 with a margin of error of 0.5%; • there is a suggestion of under-reporting regarding the origin of dental absenteeism (41 events) in the study period; • further studies in this field are required. references 1. barros g. [the development of the brazilian steel industry between 1900 and 1940: growth and import substitution]. estud econ. 2015 janmar;45(1):153-83. available from: http://www.scielo.br/pdf/ee/v45n1/01014161-ee-45-01-0153.pdf. portuguese. 2. biswas mj, koparkar ar, joshi mp, hajare st, kasturwar nb. a study of morbidity pattern among iron and steel workers from an industry in central india. indian j occup environ med. 2014 sep-dec;18(3):122-8. doi: 10.4103/0019-5278.146909. 3. baroni g. [the influence of toothache]. rev cipa. 1996;17(202):40-51. portuguese. 4. schour i, sarnat bg. oral manifestation of occupational origin. jama. 1942;120:1197-207. 5. macedo cg, queluz dp. quality of life and self-perceived oral health among workers from a furniture industry. braz j oral sci. 2011 octdec;10(4):226-32. 6. macedo cg, queluz dp. medical and dental absenteeism in workers from a furniture industry in itatiba, sp, brazil. braz j oral sci. 2010 octdec;9(4):443-8. 7. nardi a, michel-crosato e, biazevic mgh, crosato e, pizzatto e, queluz dp. relationship between orofacial pain and absenteeism among workers in southern brazil. braz j oral sci. 2009 jan-mar;8(1):50-4. 8. lima jr. [absenteeism for dental reasons: comparative analysis between employees of the municipality of são josé dos campos and insured persons of the national institute of social security] 2001 [cited 2016 mar]. available from: http:www.odontologia.com.br/artigos.asp. portuguese. 9. queluz dp. labour dentistry: a new specialty in dentistry. braz j oral sci. 2005 jul-sep;4(14):766-72. 10. probst tm. safety and insecurity: exploring the moderating effect of organizational safety climate. j occup health psychol. 2004 jan;9(1):3-10. 11. ramazzini b. de morbis artificum diatriba [diseases of workers]. 1713. am j public health. 2001 sep;91(9):1380-2. 12. rouquayrol mz, almeida nf. [epidemiology and health]. 5. ed. rio de janeiro: medsi; 1999. portuguese. 13. höfelmann da, blank n. association between self-reported diseases and symptoms and self-rated health. cad saude publica. 2008;24(5):983-92. 14. andrade tb, souza mgc, simões mpc, andrade fb. [prevalence of absenteeism among public service workers]. sci med. 2008;18(4):166-71. portuguese. 15. peres shcs, peres as, oliveira ft, adachi a, silva al, morandini acf, et al. [absenteeism: a review of the literature on absenteeism related to dentistry]. rev odontol araçatuba, 2006;27(2):96-100. portuguese. 16. yaedú ryf. [epidemiological survey of oral lesions and their relationship to the profession, gender, age, biotype and origin of the patients attended at the stomatology clinic of the faculty of bauru – usp]. [dissertation]. bauru: usp/ fo; 2005. portuguese. 17. rushton l, betts b. collection of data for occupational epidemiologic research – results from a survey of european industry. scand j work environ health. 2000 aug;26(4):327-31. 18. almeida tf, vianna mip. [the role of epidemiology in planning the worker's oral health actions]. saude soc. 2005;14(3):144-54. portuguese. 19. santos ej, queluz dp. factors involved in dentistry absenteeism since the foundation of the labor court from 1986 to 2008. braz j oral sci. 2012 oct-dec;11(4):492-504. 20. federal dentistry council. [cited 2016 mar]. available from: http://www. cfo.org.br. 21. world hearth organization. international classification of diseases. [cited 2016 mar]. available from: www.who.int/whosis/icd10. 22. ministry of labor and employment. [cited 2016 mar]. available from: http:// www.mtecbo.gov.br/cbosite/pages/informacoesgerais.jsf. 23. oit. oficina internacional del trabajo. enciclopedia de salud. seguridad e higiene en el trabajo. madrid: centro de publicaciones del ministerio braz j oral sci. 15(2):124-130 de trabajo y seguridad social; 1991. 24. manjunatha r, kiran d, thankappan kr. sickness absenteeism, morbidity and workplace injuries among iron and steel workers a cross sectional study from karnataka, southern india. australas med j. 2011;4(3):144-7. doi: 10.4066/amj.2011.576 25. lacerda, jt, traebert j, zambenedetti ml. [orofacial pain and absenteeism in workers of the metallurgical and mechanical industry]. saude soc. 2008;17(4):182-91. portuguese. 26. teles mp. [oral health conditions in workers of a metallurgical industry in the metropolitan region of salvador ] [dissertation]. salvador; ufb/ fos; 2005. portuguese. 27. danatro d. ausentismo laboral de causa medica en una instituición pública de montevideo: 1994-1995. rev med uruguay. 1997;13(2):101-9. 28. paulo vc. [absenteeism due to odontological reasons withing a metallurgical industry in the region of contagem-mg]. abo-mg. 2007;59 p. portuguese. 29. ito sc. [working conditions and dental absenteeism in a brazilian cold store: a contribution to occupational health management] [dissertation]; são paulo; centro universitário senac; 2007. portuguese. 30. martins rj, garbin cas, garbin ají, moimaz sas. [absenteeism for dental and medical reasons in public and private services]. rev bras saude ocup. 2005;30(111):9-15. portuguese. 31. panzer ra. [correlation between overtime and work accidents, complaints of musculoskeletal pain, absenteeism and productivity in a metallurgical company in porto alegre, in the years 2002 and 2003] [dissertation] porto alegre: ufrs; 2004. portuguese. 32. mesa frm, kaempffer amr. 30 años de estudio sobre ausentismo laboral en chile: una perspectiva por tipos de empresas. rev med chile, 2004;132(9):1100-8. 33. tomasini a. [development and application of ergonomic management model for a metallurgical industry company] [dissertation] porto alegre: ufrs; 2001. portuguese. 34. rocha jad. [absenteeism to work by disease and the implication of oral health as one of its factors in a metallurgical industry in the city of canoas] [dissertation]. natal: universidade federal do rio grande do norte; 1981. portuguese. 35. gillies da. gestión en enfermaria. barcelona: masson-salvat; 1994. p.321-7. 36. guo h, guo h, yang y, sun b. internal and external factors related to burnout among iron and steel workers: a cross-sectional study in anshan, china. plos one. 2015;10(11): e0143159. doi: 10.1371/journal. pone.0143159 37. wada k, arakida m, watanabe r, negishi m, sato j, tsutsumi a. the economic impact of loss of performance due to absenteeism and presenteeism caused by depressive symptoms and comorbid health conditions among japanese workers. ind health. 2013 sep;51(5):482-9. 38. pinto vg, lima mop. [epidemiological study of oral health in industrial workers. brazil; 2002-2003] [cited 2016 mar]. available from: http://www. sesi.org.br/programas/saude_bucal/saude_bucal/pdf/estudo_saude_ bucal_completo.pdf. portuguese. 39. lido yjvm. [analysis of dental care provided by companies in the metropolitan region of campinas] [dissertation]. piracicaba: fop/ unicamp; 2008. portuguese. 40. teles mp, almeida tf, cangussu mct, vianna mip. [occupational exposure and oral health of the worker]. rev cienc med biol. 2006;5(1):4854. portuguese. braz j oral sci. 15(2):124-130 absenteeism study in a steel industry of são josé dos campos, sp, brazil 130 untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8651903 volume 17 2018 e18028 original article 1 college of veterinary medicine/ al muthanna university / formerly senior lecturer/ school of dental sciences/ universiti sains malaysian 2 college of dentistry /al muthanna university 3 school of dental sciences/ universiti sains malaysia corresponding author: e-mail: mrvsa59@gmail.com received: october 06, 2017 accepted: january 25, 2018 clinico-radiologic features of four cases of ameloblastoma al-salihi k a 1*, ihsan abdullah2, ling yoke li 3 ameloblastomas are jaw tumors that are locally aggressive with a high capacity for recurrence. it arises from the epithelium of the dental lamina. radiograph remains the first and an essential investigation, despite the development of various cross-sectional imaging modalities. aim: this article intends to describe the clinical findings and orthopantomograph (opg) supported by computed tomography scan (ct scan) and mri of four cases of ameloblastomas in the hospital university sains malaysia. materials and methods: data concerning the patients’ ages, sex, tumor locations, and surgical treatment history, as well as the radiographic findings, were analysed. results: the patients’ ages ranged from 23 to 41 years (mean, 30.5 years). the gender of patients were two male (50%) and 2 (50%) females. three cases (75%) of ameloblastomas located in the mandible and only one case found in the maxilla. the swelling was the most common symptom and experienced by all patients. radiographically, two cases were unilocular with a well-demarcated border, and the remaining 2 cases were multilocular. typical features of unicystic ameloblastoma appeared in case 1. this case previously presented with a lesion in the same area diagnosed as a dentigerous cyst. the other 3 cases showed variant types of ameloblastoma. these were plexiform, acanthomatous and granular cell type; follicular and plexiform type; and follicular and plexiform type in case 2, 3 and 4 respectively. conclusion: in conclusions, determination the location and density of jaws lesion and its margin relation to the tooth using radiological tool accompanied with clinical data, aids in the narrowing of the differential diagnosis and it is necessary for early diagnosis of ameloblastoma. a long-term follow-up at regular intervals after surgery is recommended for all ameloblastomas cases. keywords: radiograph. universiti sains malaysia. unicystic ameloblastoma. jaws mailto:mrvsa59@gmail.com 2 al-salihi et al. introduction ameloblastoma is the most common epithelial benign odontogenic tumor that can be locally aggressive and invasive. it represents only 1% of tumors arising in the jaws1,2,3. it develops from various sources of odontogenic epithelium, including residual epithelium from tooth germ, the stratified squamous epithelium, the epithelium of odontogenic cysts and the enamel organ epithelium4. ameloblastoma appears most commonly in the third to fifth decades. over 80% occur in the mandible, with 70% of these arising in the molar-ramus area, and they are occasionally associated with unerupted third molar teeth. they are usually benign in growth pattern but frequently invade locally and seldom metastasize. because of their neoplastic nature, surgical treatments differ from those of other cystic lesions. ameloblastoma is usually resected en bloc and sometimes with hemimandibulectomy or partial maxillectomy, if the lesion is highly infiltrative and extensive. therefore, a precise preoperative diagnosis has important therapeutic implications. several studies regarding clinicopathological characteristics of ameloblastoma reported in thailand5, singapore6, and japan7. most cases of ameloblastoma have a characteristic radiographic appearance although it is not diagnostic. radiographically, ameloblastomas described as a multilocular or unilocular cyst-like lesion. they are typically radiolucent and rounded areas, with moderately well-defined margins. lingual expansion may sometimes be seen, but it is not pathognomonic of ameloblastoma. other variants are a honeycomb pattern, a single well-defined cavity indistinguishable from a radicular, or rarely, a dentigerous cyst8. four radiographic features are significant in ameloblastoma these include the expansion of the cortical plate, the presence of corticated scalloped margin, the multilocular appearance of the lesion, and the resorption of the adjacent roots of teeth9. in malaysia, the distribution of this tumor reported in children10 moreover, separated case studies have also been published11. siar and ng (1993)12 mentioned that radiological features of the multilocular type were the most common, followed by unspecified type, unilocular and mixed radiolucent-radiopaque lesions. in their review, reichart et al. (1995)13 indicated that the frequent radiographic findings were embedded tooth, root resorption, and undefined borderline. approach to treatment of ameloblastoma is controversial and can be changed with the clinical-radiologic variant, anatomic location and clinical behaviour of a tumour. in addition, the age and the general state of health of the patient are important factors. this article intends to describe the clinical and opg features supported by ct scan and mri of four cases of ameloblastomas in the hospital university sains malaysia. material and methods patients the study comprised of 4 patients of ameloblastomas (1 unicystic type and 3 variant types). these cases were referred to the school of dental sciences/ universiti sains malaysia (hospital of universiti sains malaysia / health campus, kelantan) with a painless swelling in the jaws. the opg was done for all patients as one of the diagnostic tools. moreover, the patients were also admitted to ct scan and mri. 3 al-salihi et al. ethical approval this research has been approved by the ethics and research committee, with reference usmkk/ppsp®/ tindakan minit 149.6. results clinical findings and radiological presentation case 1 /unicystic ameloblastoma this case was a unicystic ameloblastoma in 27 years aged female affecting the left maxilla. the patient had swelling over the palatal region for six years. the swelling size was increased gradually over the past two years. it was non-tender, painless, no bleeding and ulceration. however, there was teeth mobility adjacent to the area of swelling. she also had a nasal blockage, rhinorrhea, and epiphora. patient’s nose deformed due to the massive growth of the tumor. she had no palpable cervical lymph node and no history of malignancy. however, she previously diagnosed with a dentigerous cyst in the same area of the current growth. the radiological findings showed unilocular tumor with a smooth margin and associated with a resorbed root of the 1st premolar and the expansion of the left palatal arch. displaced tooth of the upper left quadrant also was present. tumour was enlarged causing severe facial deformity. it extended into the nasal septum and paranasal sinuses, causing marked nasal obstruction. extension into the orbital floor of maxilla bone also seen. coincidently, ameloblastoma in this patient originated from a previous dentigerous cyst (figure 1) (table 1). figure 1. shows the unicystic ameloblastoma. (a) opg showed a large area of unilocular radiolucency, showing teeth displacement and root resorption on 24. (b) coronal ct scan shows a large unilocular mass compressing the nasal septum, nasal passage and left maxillary sinus. (c) coronal ct scan showed a large mass on the left cheek extending into the oral cavity, nasal airway, maxillary sinus, left orbital floor and surrounding soft tissue. a b c r l 4 al-salihi et al. case 2/ variant ameloblastoma this case was presenting a variant type of ameloblastoma in the left mandible of 31 years female. the patient claimed to have a swelling of chin and halitosis for many years. initially, it was small, but its sized increased gradually. she went for a traditional massage that worsened the swelling. she had loose teeth at the posterior arch of the left quadrants surrounding area of the swelling, which subsequently extracted. however, her socket never healed. there were palpable small lymph nodes with multiple mucosal ulceration on the buccal cheek. the swelling was firm, not fixed to the skin, no redness and pulsation. the opg findings showed tumor extended into surrounding tissues and caused bony destruction. it was a multiloculated mass on the left jaw extending into the left maxillary sinus and expanding the buccal cortex of the lower left mandible. (figure 2). table 1. shows the clinical and radiological findings and diagnosis of ameloblastomas case no age/ years and sex location clinical findings radiological findings diagnosis 1 27 / female left maxilla previous history of dentigerous cyst, swelling of the palatal region nontender, painless, no bleeding and no ulceration, teeth mobility adjacent to the area of swelling, nasal blockage, rhinnorrhea epiphora, previously diagnosed with a dentigerous cyst of the same area with the current growth. unilocular , smooth margin, resorbed root of the 1st premolar , expansion of the left palatal arch, severe facial deformity, extension into the nasal septum and paranasal sinuses, marked nasal obstruction and extension into the orbital floor of maxilla bone unicystic ameloblastoma 2 31/ female left mandible swelling of chin, mobile teeth at the surrounding area, subsequently the teeth were extracted, socket never healed, palpable small lymph nodes with multiple mucosa ulceration on the buccal cheek. tumour extended into surrounding tissues and caused bony destruction. it was a multiloculated mass on the left jaw extending into the left maxillary sinus and expanding the buccal cortex of the lower left mandible. variant ameloblastoma 3 41/ male mandible swelling over anterior lower jaw, numbness of the lower lip, mobile teeth grade i. ,painless and non-tender, non-compressible multiloculated radiolucency extending from 33 to 44. no well-defined margins, presence of roots resorption adjacent to the tumour, expanded the lingual cortex. variant type ameloblastoma 4 23/ male mandible a painless, swelling,bony hard, non-tender, fixed to underlying structure overlying the skin. well defined uniloculated radiolucency, smooth borders, displacement of teeth 45 and 46, slight expansion of cortex. variant type ameloblastoma summary age’s range: 23-41 years & age average: 30.5 sex: 2 female (50%) & 2 male (50%) location: 1 maxilla (25%) & 3 mandible (75%) type: 1 unicystic ameloblastoma & 3 variant type ameloblastoma 5 al-salihi et al. case 3/ variant ameloblastoma this case was presenting a variant type of ameloblastoma in the mandible anterior part of 41 years aged male. the patient had swelling over his anterior lower jaw. he complained of numbness of the lower lip. he had loose teeth grade i. the swelling was painless and non-tender, non-compressible and it was firm. this patient also suffered from a recurrent lesion of ameloblastoma. the radiographs in figure 3 showed the multiloculated radiolucency extending from 33 to 44. the well-defined margins were absence with roots resorption adjacent to the lesion. a tumour had also expanded the lingual cortex. (figure 3). case 4/ variant ameloblastoma this case was presenting a variant type of ameloblastoma in the right body of the mandible of 23 years aged male. the patient suffered from a painless swelling on the right mandible region. it was bony hard, non-tender, and fixed to underlying structure overlying the skin. he did not complain of loosening teeth, toothache or gum bleeding. radiographs investigation showed a very well defined uniloculated radiolucency with smooth borders. the displacement of teeth 45 and 46 and slight expansion of cortex were also observed (figure 4). figure 2. shows the variant type ameloblastoma (a) 3d radiograph showed extension of tumour and bony destruction. (b) mri showed a multiloculated mass of the left jaw causing compression of left maxillary sinus and extension into surrounding soft tissue. (c) mri scan showed an enlarging mass expanding the buccal cortex of the lower left mandible. a b c figure 3. shows the variant type ameloblastoma. (a) opg showed a multiloculated radiolucency extending from 33 to 44 with absence of the well-defined margins accompanied by roots resorptions. (b) skull x-ray showed an area of radiolucency at the anterior labial segment of the lower mandible. (c) coronal ct scan showed a large mass arising posterior to the mandible causing expansion of lingual cortex. a b c r l 6 al-salihi et al. discussion ameloblastoma, compounds of two words the english word “amel” which means enamel and the greek word “blastos” which means germ12 and the term was coined by lucas (1984) 14 . it is a rare structure of benign odontogenic tumor and is manifest by its aggressive local behavior and the high recurrence rate15,16 that may influence by certain factors. these factors include the clinicopathological variant of the tumor17,18 the anatomic site19,20 adequacy of surgery and the histological variant of the ameloblastoma that acts to be of prognostic significance in regards of recurrence. in the maxilla, ameloblastomas occurred at 22%, where the canine and antral regions of the upper jaw are the most localized area. moreover, ameloblastomas occurred in the mandible at 80%, where 70%, 20% and 10% found in the ascending ramus or the molars region and the premolar region, and the anterior region respectively21,22. however, it seldom affects the soft tissue (peripheral ameloblastoma)23. in addition, about 10-15% of ameloblastomas associated with a non-erupted tooth22. ameloblastomas occur with higher frequency in the third or fourth decade of life. besides, the average age at diagnosis is 34 years with a range of five to 74 years, except in the case of the unicystic variety, which was diagnosed amidst the ages of 20 and 30 years, albeit these case were reported in children21,24,25. the routine radiographic examination acts as an important tool in the discovering of ameloblastoma coinciding with a clinically observed developing mass. however, in most cases, a unilocular or multilocular radiolucency with a honeycomb or soap bubble appearance, is the characteristic features of ameloblastomas26. a b c r l figure 4. shows variant type ameloblastoma. (a) and (c) opg and mri showed a very well defined uniloculated radiolucency with smooth borders. there were displacement of teeth 45 and 46 seen in the opg. slight expansion of cortex was noted. (b) skull x-ray showed a radiolucent area at the right angle of mandible with some root resorption of the adjacent teeth. 7 al-salihi et al. clinical and radiological features of four cases of ameloblastomas were described in this study. these cases presented with variant types of ameloblastoma except one case with unicystic ameloblastoma. the results of the current study are in accord with those reported previously21,24,25 with the age range 23-41 years and average 30.5 years. also, the previous study revealed that ameloblastoma occurred in equal frequency among men and women27. even though, a lower frequency in male than females has been described22,28,29. in the current study, and according to gender, the percentage of patients was equally distributed, 2 female (50%) and 2 male (50%) and these percentages are compatible with previous records elsewhere in the world22,26. in this study, the unicystic ameloblastoma lesion occurred at left maxilla, which is compatible with its clinical features with typical clinical findings reported previously in japan 200930. ito et al. (2009)30 reported a case of unicystic ameloblastoma in a 26-year-old male. the patient was suffering from a history of a left maxillary third molar extraction, three years previously. this patient also revealed a well-defined 3.5 cm diameter radiolucency of the left maxilla. moreover, the case was confirmed histologically as unicystic ameloblastoma, plexiform type. the unicystic ameloblastoma patient that reported in this study revealed unilocular, smooth margin, resorbed root of the 1st premolar, expansion of the left palatal arch, severe facial deformity, extension into the nasal septum and paranasal sinuses, marked nasal obstruction and extension into the orbital floor of maxilla bone. the previous report mentioned that the radiographic features of unicystic ameloblastoma are typically unilocular accompanied with the area of radiolucency29. therefore, this lesion is often misdiagnosed as an odontogenic keratocyst or a dentigerous cyst. konouchi et al. (2006)31 carried out contrast-enhanced (ce)-mri to investigate 13 cases of unilocular, round radiolucent lesions observed by panoramic radiography and/or ct. konouchi et al. (2006)31 investigation approved that unicystic ameloblastoma revealed low signal intensity (si) on the t1weighted images and evidently high si on t2wis; and relatively thick rim-enhancement with/ without small intraluminal nodules on ce-t1wis. they considered that ce-mri to be useful in the diagnosis of unicystic ameloblastoma. the patient (case 1) with unicystic ameloblastoma reported in this study showed symptomatic swelling of the left posterior maxilla of a 27-year-old female. however, the most frequent primary site of this lesion is the angle of the mandible and commonly occurs at a younger age group of 1st and 2nd decade of life with a prominent preponderance in the male. so, it is not possible to conclude age and location of the lesion due to the limited number of this study. unicystic ameloblastoma classically contains a tooth and appears as a dentigerous cyst in radiology. these radiological and histological criteria’s presented in (case 1) patient. this patient was suffering from the dentigerous cyst, which characterized by non-tender swelling of the palatal region, no bleeding, and ulceration, teeth mobility adjacent to the area of swelling, nasal blockage, and rhinorrhea epiphora. it is often difficult to differentiate between some lesions that occur in the jaw because they have a similar radiographical appearance. in this patient, the radiograph technique approved its ability to investigate the unicystic ameloblastoma as well as the previous dentigerous cyst, which appeared at the same site. 8 al-salihi et al. in this study, the variant type of ameloblastoma presented in the mandible of the three others patients. the radiograph investigation revealed multiloculated and uninoculated radiolucency in the patients g15/04 & g9/05 and g25/03 respectively. the clinical and radiographical features of these cases are compatible to that mentioned in previously published cases1,8,9. the radiological features of ameloblastoma’s lesions revealed thin cortex of the buccal-lingual plane and enormous mass. characteristically, the lesions showed a “soap bubble” or “honeycomb” appearance multilocular cystic32,33. the prominent clinical signs appeared on case 2, case 3, case 4 patients were a swelling in the jaws and the first step in diagnosis was panoramic radiography. accordingly, pmg considered as a preferred technique for early diagnosis even though it only reveals the re-interfaces between a tumor and healthy bone. however, mri used in this study and it was a necessity in the confirmation of the diagnosis and identifies the interfaces between tumor and normal soft tissue such as the lesion contours, contents, and extension into the soft tissues that plain radiographs cannot be catch it. the mri can show both types of interface the coronal and axial views, while only the axial view is seen in contrast-enhanced ct images34. both mri and ct have ability for detecting the cystic component of the tumor and imaging papillary projections inside the cystic cavity. the exact content of maxillary ameloblastomas can identify by mri and thus determine the prognosis for surgery13,15. the posterior mandible is the most common site of ameloblastoma that associated with impacted teeth and follicular cysts. in advanced cases, expansion of the cortical plates accompanied with scalloped margins, perforations, and resorption of the affected tooth occurs. radiographically an ameloblastoma may be mistaken for an odontogenic keratocyst, aneurysmal bone cyst, fibrosarcoma, or a giant cell tumor. the invasiveness nature and a rare chance of metastases of ameloblastomas; the tumor have been subject to the definition from a benign odontogenic epithelial tumor to a slow-growing malignant tumor35. if ever metastases were delivered by an ameloblastoma, the most common sites are the lung (76.7 %), followed by regional lymph nodes (37.8 %), pleura (16.2 %), vertebrae (13.5 %), skull (10.8 %), diaphragm (8.1 %), liver and parotid (5.4 %) and, even more rarely, the spleen and the kidney36,37. the metastasis is probably a result of tumor cells associated with surgery, particularly in cases having undergone multiple operations due to recurrences. this aspect is being discussed in this study as one of the patients showed recurrence. the three variants of the benign ameloblastoma, are designated as solid or multicystic, unicystic and peripheral9,38. the solid variety has the highest propensity for local infiltration and therefore the highest potential for recurrence9. the anatomic site should be considered as one of the important factor in the recurrence of ameloblastoma39. up to 95% of ameloblastomas occur in the mandible. the dense cortical bone of the mandible prevents the tumor from spreading extensively for several years, however, its spreading in the central cancellous bone is beyond the radiographic margins of the tumor40. complete surgical removal of ameloblastomas lesions needs a careful assessment of the anatomic extent of the tumor. the lesions that are completely intraosseous can be adequately assessed with standard radiography. radiologically, the ameloblastoma lesions are expansile, with thinning of cortex of the buccal-lingual plane. the variant type of ameloblastoma examined in the present study by 3d radiograph, mri and mri scan showed extension 9 al-salihi et al. of tumour and bony destruction; a multiloculated mass of the left jaw causing compression of left maxillary sinus and extension into surrounding soft tissue and an enlarging mass expanding the buccal cortex of the lower left mandible respectively. finally, the histological variant of the ameloblastoma has been suggested to be of prognostic significance in terms of recurrence. the multilocular ameloblastomas have higher recurrence rates than unilocular ones. since ameloblastomas infiltrate within the cancellous spaces more, the tumor margin goes beyond the apparent clinical and radiographic margin. the attempts to remove the tumor by curettage may leave small tumor islands in bone, which may later occur as recurrences41. in conclusions, radiologic examination recognized the features of ameloblastomas lesion’s including location, density, relation to the tooth, and margin accompanied with clinical data, generally aids in the narrowing of the differential diagnosis and is necessary for early diagnosis of ameloblastoma. however, in young people, the diagnosis remains in doubt after clinical signs and a biopsy is needed. a long-term follow-up at regular intervals after surgery is recommended. references 1. becelli r, carboni a, cerulli g, perugini m, iannetti g. mandibular ameloblastoma: analysis of surgical treatment carried out in 60 patients between 1977 and 1998. j craniofac surg. 2002 may;13(3):395-400; discussion 400. 2. iordanidis s, makos c, dimitrakopoulos j, kariki h. ameloblastoma of the maxilla — case report. aust dent j. 1999 mar;44(1):51-5. 3. nakamura n, mitsuyasu t, higuchi y, sandra f, ohishi m. growth 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oct;51(10):1064-70. 18. gardner dg. controversies in the nomenclature, diagnosis and treatment of ameloblastoma. in: worthington p, evans jr, editors. controversies in oral and maxillofac surgery. philadelphia, pa: saunders; 1994. p.362. 19. gardner dg, pecak am. the treatment of ameloblastoma based on pathologic and anatomic principle. cancer. 1980 dec 1;46(11):2514-9. 20. collings sj, harrison a. recurrent ameloblastoma? an historic case report and a review of literature. br dent j. 1993 mar 20;174(6):202-6. 21. torres-lagares d, infante-cossío p, hernández-guisado jm, gutiérrez-pérez jl. mandibular ameloblastoma. a review of the literature and presentation of six cases. med oral patol oral cir bucal. 2005 may-jul;10(3):231-8. 22. hollows p, fasanmade a, hayter jp. ameloblastoma -a diagnostic problem. br dent j. 2000 mar 11;188(5):243-4. 23. ogunsalu c, daisley h, henry k, bedayse s, white k, jagdeo b, et al. a new radiological classification for ameloblastoma based on analysis of 19 cases. west indian med j. 2006 dec;55(6):434-9. 24. tozaki m, hayashi k, fukuda k. dynamic multislice helical ct of maxillomandibular lesions: distinction of ameloblastomas from other cystic lesions. radiat med. 2001 sep-oct;19(5):225-30. 25. artés-martínez mj, prieto-rodríguez m, navarro-hervás m, peñas-pardo l, camañas-sanz a, vaquero de la hermosa mc, et al. ameloblastoma. diagnosis by means of fnab. report of two cases. med oral patol oral cir bucal. 2005 may-jul;10(3):205-9. 26. small ia, waldron ca. ameloblastoma of the jaws. oral surg oral med oral pathol. 1955 mar;8(3):281-97. 27. small ia. recurrent ameloblastoma, 25 years after hemimandibulectomy. oral surg oral med oral pathol. 1956 jul;9(7):699-706. 28. adekeye eo. ameloblastoma of the jaws: a survey of 109 nigerian patients. j oral surg. 1980 jan;38(1):36-41. 29. gardner af, apter mb, axelrod jh. a study of twenty-one instances of ameloblastoma, a tumor of odontogenic origin. j oral surg anesth hosp dent serv. 1963 may;21:230-7. 30. ito s, mandai t, ishida k, kitamura n, deguchi h, hata t, et al. unicystic ameloblastoma of the maxilla : a case report. kawasaki med j. 2009;35(1):95-8. 31. konouchi h, asaumi j, yanagi y, hisatomi m, kawai n, matsuzaki h, et al. usefulness of contrast enhanced-mri in the diagnosis of unicystic ameloblastoma. oral oncol. 2006 may;42(5):481-6. 32. rampton p. teeth and jaws. in: sutton d, editor. textbook of radiology and imaging. philadelphia: churchill-livingstone; 1998. p.1388-9. 33. gümgüm s, hoşgören (2005). clinical and radiologic behaviour of ameloblastoma in 4 cases. j can dent assoc. 2005 jul-aug;71(7):481-4. 11 al-salihi et al. 34. cihangiroglu m, akfirat m, yildirim h. ct and mri findings of ameloblastoma in two cases. neuroradiology. 2002 may;44(5):434-7. 35. moe h, clausen f, philipsen hp. the ultrastructure of the simple ameloblastoma. acta pathol microbiol scand. 1961;52:140-54. 36. shatkin s, hoffmeister fs. ameloblastoma: a rational approach to therapy. oral surg oral med oral pathol. 1965 oct;20(4):421-35. 37. ueno s, mushimoto k, shirasu r. prognostic evaluation of ameloblastoma based on histologic and radiographic typing. j oral maxillofac surg. 1989 jan;47(1):11-5. 38. shafer wg, hine mf, levy bm. a textbook of oral pathology. 4th ed. saunders; 1983. chapter 4: cysts and tumours of odontogenic origin. 39. el-sissy na. immunohistochemical detection of p53 protein in ameloblastomas types. east mediterr health j. 1999 may;5(3):478-89. 40. vered m, shohat i, buchner a. epidermal growth factor receptor expression in ameloblastoma. oral oncol. 2003 feb;39(2):138-43. 41. vogelstein b, kinzler kw. the genetic basis of human cancer. 2nd ed. new york: mcgraw hill; 2002. chapter 23: syndrome by david malkin. p.387-401. 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1536/1189 1/8 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1536/1189 2/8 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1536/1189 3/8 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1536/1189 4/8 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1536/1189 5/8 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1536/1189 6/8 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1536/1189 7/8 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1536/1189 8/8 untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8652908 volume 17 2018 e18162 original article 1 dds, ph.d. professor. department of oral surgery and pathology. school of dentistry. pontifical catholic university of minas gerais, brazil. 2 dds, ph.d. professor. department of oral surgery and pathology. school of dentistry. university of vale do rio verde. belo horizonte, brazil 3 undergraduate student. department of oral surgery and pathology. school of dentistry. federal university of minas gerais. belo horizonte, brazil. 4 dds, ph.d. professor. school of dentistry. department of pediatric dentistry. federal university of minas gerais. belo horizonte, brazil. 5 dds, ph.d. professor. department of oral surgery and pathology. school of dentistry. federal university of minas gerais. belo horizonte, brazil. corresponding author: soraya de mattos camargo grossmann faculdade de odontologia da ufmg disciplina de patologia bucal, sala 3202-d av. antônio carlos, 6627 pampulha 31270-901 belo horizonte-mg, brazil e-mail: sorayagrossmann@gmail.com voice: 55-31-84372310; 55-31-3499-2479; 55-31-3499-2499 fax: 55-31-3499-2430 received: january 29, 2018 accepted: june 06, 2018 the use of social networks in scientific research with questionnaires soraya de mattos camargo grossmann1, mariela dutra gontijo moura2, michelle danielle porto matias3, saul martins paiva4, ricardo alves mesquita5 aim: the aim of this study was to determine the response rate of the sf-36 quality of life questionnaire sent and received by mail and over a social network to caregivers of individuals with epidermolysis bullosa (eb) in brazil. methods: all volunteers were first-degree relatives of patients with eb that directly spent time helping them with their basic activities of daily living. a maximum of two caregivers per patient could answer the questionnaire. volunteers were divided into two groups: for group 1, questionnaires were sent to 53 members of a support association for the disease by mail, and for group 2, 798 members of the facebook™ page of friends and relatives of patients with eb were invited to participate using an access link to google drive™ to gain access to the questionnaire. the data from both groups were analyzed 150 days after the start the study. descriptive analysis was performed by epiinfo8 and the return of questionnaires was evaluated according to age, sex, and time of return using the chi-squared and fisher’s exact test. results: after 150 days, 30 questionnaires were returned, 17 (56.7%) of which from group 1 and 13 (43.3%) from group 2. approximately 12 questionnaires were returned by mail and 11 over the social network. conclusions: the data collection of the sf-36 quality of life questionnaire from caregivers of individuals with eb over a social network seems to be efficient mainly when it is necessary to collect results within a short timeframe, highlighting the importance of social networks as a means for conducting this type of research. however, in our study, the most efficient method was distributing the questionnaires by mail. keywords: epidermolysis bullosa. electronic mail. quality of life. social support. surveys and questionnaires. 2 grossmann et al. introduction questionnaires are an important tool in clinical research for outlining a situation or the way of life of a population1-6. usually, the forms are distributed to the public through the mail or via censuses3,5. however, with the rise of the internet and the advent of electronic social networks, such as facebook™ in 2004, distances to the target population were shortened, especially in brazil1,5,7-8. studies have assessed the acceptability and representability of social networks in information science in brazil1, which led researchers to apply electronic surveys more often3,5,8. in addition, the scielo organization (online library) held a seminar in 2012 on the use of social networks in scientific communication, emphasizing the importance of this communication vehicle1,2,7-8. the distribution of questionnaires through social networks might be an effective approach to study epidermolysis bullosa (eb), which is a serious and rare dermatological genetic disorder with a prevalence of 5:100,000 live births and no racial or gender difference9. eb is a group of inherited connective tissue disorders characterized by the absence of a cohesion protein that results in the defective connection between epidermis and dermis, making the skin fragile10-11. based on the skin layer that is affected, eb can be classified into four main types: eb simplex, junctional eb, dystrophic eb, and the recently established kindler syndrome9,12. depending on the severity of the disease, which can vary from benign to life threatening, symptoms can include skin fragility, blistering of the skin following mild friction or trauma, and blistering of mucous membranes or internal organs11-13. currently, the disease has no cure and clinical management is focused on relieving symptoms11,13. topical agents and dressings are typically used for the treatment of skin lesions, and appropriate follow-up is essential to monitor the patient and his caregivers for psychosocial problems and psychiatric symptoms11-14. in addition, the occurrence of problems related to mobility, self-care, and usual activities, pain, discomfort, anxiety, and depression also impact the quality of life of patients and caregivers12,13. non-professional caregivers, who are often relatives, provide informal care spending time helping the individuals with eb with their basic activities of daily living15. patients with eb have direct medical costs with hospital admissions, emergency visits, and outpatient care (rehabilitation, medical tests and examinations, visits to health professionals and home medical care) and non-medical costs with transportation, social care services (formal care), and caregiver’s time. the time dedicated to the patient and the high informal care costs account for more than half of eb’s socio-economic burden. the hidden social costs of eb are relevant and have consistent impact on the quality of life of patients and their caregivers, especially when considering the impact on family income levels15. thus, the aim of this study was to determine the response rate of the sf-36 quality of life questionnaire delivered by mail and through a social network to first-degree relatives of individuals with eb in brazil. we discuss the use of surveys and questionnaires in eb caregivers, emphasizing the acceptability of social networks as an important tool for scientific research. 3 grossmann et al. materials and methods sample characteristics this study was approved by the human research ethics committee from the federal university of minas gerais (protocol number etic 285 204). each participant was informed about the research, and answered questions on their relationship with the individual with eb. all agreed by signing the informed consent that was sent with the sf-36 questionnaire. the inclusion criteria for the participants were first-degree relatives that spent time directly helping patients with their basic activities of daily living. the maximum of two caregivers could answer the questionnaire and they were divided into two groups. group 1 was selected from 83 files from the minas gerais association of relatives, friends and patients with epidermolysis bullosa (ampapeb), and the questionnaire was sent to 53 members by mail. group 2 was selected from the facebook™ page of the association, and the questionnaire was administered over this social network to 798 members, including parents, health professionals, family members, and friends of eb patients. study design as recommended for cross-sectional studies2-16, the addresses of participants from group 1 were confirmed by telephone and 53 updated records were obtained. an envelope was sent to everyone containing the following items: 1) a letter explaining the research, 2) two informed consent forms, 3) two sf-36 quality of life questionnaires, and 4) a prepaid and labelled envelope to increase the likelihood of the questionnaire being returned. after being accepted as a friend in the association’s facebook™ page, the researcher invited all first-degree relatives from group 2 members to participate in the study, to a maximum of two caregivers per individual with eb. the inform consent form and the questionnaire were sent through google drive™ software, which were filled out by an access link. previously, a pilot study with a virtual questionnaire was performed among people not involved in this research. the pilot test was done with 5 persons (10% of the expected total sample) and included a professor and students of dentistry. data collection sf-36 quality of life questionnaire data was stored in a database. the feedback for both groups was expected to be received no more than 150 days from the start date of the study. after 30 days, the same material (letter, 2 consent forms, and 2 questionnaires) was resent to non-responders in the same way as the first delivery: by mail for group 1 and through facebook™ and google drive for group 2. additionally, the researchers had access to the date and time of the response through the google drive™ software in group 2. statistical analysis descriptive statistical analysis was performed with epi info 8™ (center for disease control and prevention-cdc, usa). the return of questionnaires was evaluated for 4 grossmann et al. groups 1 and 2 according to age, sex, and time of return. data were analyzed using the chi-squared and fisher’s exact test. the level of significance of the analyses was set at 5%3. results after 30 days, five envelopes with seven questionnaires were returned from group 1 and eight were filled out by group 2. after 150 days, 10 more questionnaires were returned by mail, and five filled out over the social network. thus, after 150 days from the study start date, 30 questionnaires were returned, 17 (60.0%) from group 1 (from 53 members, 32.1% return rate) and 12 (40.0%) from group 2 (from 798 members, 1.50% return rate). during the first 30 days, group 2 had a rapid return, although it did not persist until the end of the evaluation (figure 1). females were the majority among volunteers (23/76.7%), returning 13 (56.5%) questionnaires by mail and 10 (43.5%) through the social network. for males, questionnaire returns were more common by mail (5 questionnaires, 71.4%) than though the social network (2 questionnaires, 28.6%). no association was observed between gender and type of return (p=0.40) (figure 2). figure 1. return rate of questionnaires during the initial 30 days (a) and after 150 days of the start of the research (b). 0 1 2 3 4 5 6 w ee k 1 w ee k 2 w ee k 3 w ee k 4 w ee k 5 w ee k 6 w ee k 7 w ee k 8 w ee k 9 w ee k 10 w ee k 11 w ee k 12 w ee k 13 w ee k 14 w ee k 15 w ee k 16 w ee k 17 w ee k 18 w ee k 19 w ee k 20 mail social networking 5 grossmann et al. participant age ranged from 22 to 92 years (mean 39.2 years). persons of 40 years of age or older (19–63.3%) asked questions about the questionnaire more frequently by mail (13–43.3%), and persons of 39 years of age or younger asked with almost the same frequency in both methods (5 persons by mail and 6 over the social network). no association was observed between age and type of return (p=0.19) (figure 2). the general health score of the questionnaire for the total sample was 60.23. among the seven domains of quality of life evaluated, the average scores obtained (ranging from 0 to 100) were: 63.66 for social functioning, 62.83 for physical functioning, 59.23 for pain, 53.33 physical role functioning, 53.33 for emotional role functioning, 52.0 for vitality, and 51.86 for mental health. discussion the internet presents an opportunity for innovative recruitment modalities1-2,7-8,17. social networks were developed to bring people together and increase the sharing of information. today, facebook™ is one of the most used social networks and has been considered a viable recruitment option for health behavior assessment in young adults18. the use of a social network in human research has been reported; however, dental research has not yet been conducted using this recruitment method1,3,5. a systematic review found that using mailed questionnaires for health research can improve the quality of the data19. according to leece et al.20 and kongsved et al.21, the prevalence of returned questionnaires via internet is significantly lower than via mail. however, the study of frandsen et al.22 and skonnord et al.8 demonstrated the opposite. the study conducted by mazzon et al.16 showed a return of 15.1% of questionnaires by mail, while our study observed a return of 32.1%, a high rate compared to the return by social network of only 1.50%. it is important to emphasize, however, that figure 2. relative distribution of questionnaires according to sex and age during the initial 30 days and after 150 days from the start date of the research. 0 5 10 15 20 25 30 female male 30 days 150 days 3rd decade 4rd decade 5rd decade 7rd decade 8rd decade 10rd decade 6 grossmann et al. many persons in the social network were friends, aunts, uncles or health professionals that did not meet the inclusion criteria. thus, we considered that the evaluation of the method using return rate of data collected through a social network is questionable. the social network as a means of data collection allows easy access to the caregivers of eb individuals because most brazilians have internet access. however, the researcher needs to constantly monitor data collection since many people might not be familiar with this type of approach. thus, new strategies should be employed in future studies to increase response rates and quality of research such as reminder system by telephone and softwares2,8. the literature demonstrates that most of the questionnaires sent by mail are returned within the first two weeks of research2,3,19-25, which is corroborated by the current study. moreover, a higher prevalence of return was observed through the social network in the first 30 days; after 150 days, however, the mailed questionnaires had a higher return rate, resulting in an overall higher rate (18 – 60.0%). this finding suggests that social networks are important tools for questionnaire-based research when data can be collected within a short timeframe23-25; for research in general, the most efficient way to send questionnaire and receive responses is by mail. women were more prevalent (76.7%) in both groups, and more men in the mail group returned the questionnaire (71.4%). these findings may be because women use social networks more frequently. the average age of participants who returned the questionnaire was 39.2 years; those who responded over the social network were younger (43.75 years) than those who responded by mail (52.85 years). these findings show that older people tend to be more interested in participating in independent research through forms sent by mail. research using questionnaires is necessary and practiced widely2-8,23-25. it is worth emphasizing that the internet, in addition to its technological benefits, has been asserted as a powerful research mechanism2,22. therefore, it is worthwhile to consider the expansion of social networks and their rapidly increasing number of users as a research method through sending and receiving questionnaires via the internet. these approaches may also be understood as important tools for conducting questionnaire-based-research, especially when a quick data collection is needed, to assess large samples, investigate rare cases, or decrease problems related to long distances from research centers and target population22-25. according to fenner et al.17, the success of this method has worldwide implications for future research. it is worth emphasizing that our goal was to discuss the use of a social network to determine the response rate of a quality of life questionnaire sent to caregivers of individuals with eb; in this respect, social networks have become an important means of gaining access to these individuals. the recruitment of young people for health research by traditional methods has become more expensive and challenging over recent decades17 and social networks have the potential to overcome this disadvantage, especially in terms of having a fast turnover, lower operating costs, and broad coverage. however, over a long term, mailed material still shows greater effectiveness of return. 7 grossmann et al. although we found that data collection by mail was more effective, we believe that social networks can help researchers to recruit more people and become an important tool for conducting research using questionnaires, mainly when data need to be collected within a short timeframe, people living far from the research need to be reached, and especially for investigations involving rare diseases. references 1. bufrem ls, gabriel junior rf, sorribas tv. 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[marketing: applications of quantitative methods]. são paulo: atlas; 1983. portuguese. 17. fenner y, garland sm, moore ee, jayasinghe y, fletcher a, tabrizi sn, et al. web-based recruiting for health research using a social networking site: an exploratory study. j med internet res. 2012 feb 1;14(1):e20. doi: 10.2196/jmir.1978. 18. ramo de, prochaska jj. broad reach and targeted recruitment using facebook for an online survey of young adult substance use. j med internet res. 2012 feb 23;14(1):e28. doi: 10.2196/jmir.1878. 19. edward p, robert i, clarke m, diguiseppi c, pratap s, wentz r, et al. increasing response rates to postal questionnaires: systematic review. bmj. 2002 may 18;324(7347):1183. 20. sprague s, swiontkowski mf, schemitsch eh, tornetta p, et al. internet versus mailed questionnaires: a randomized comparison (2). j med internet res. 2004 sep 24;6(3):e30. 21. kongsved sm, basnov m, holm-christensen k, hjollund nh. response rate and completeness of questionnaires: a randomized study of internet versus paper-and-pencil versions. j med internet res. 2007 sep 30;9(3):e25. 22. frandsen m, walters j, ferguson sg. exploring the viability of using online social media advertising as a recruitment method for smoking cessation clinical trials. nicotine tob res. 2014 feb;16(2):247-51. doi: 10.1093/ntr/ntt157. 23. dainesi sm, goldbaum m. e-survey with researchers, members of ethics committees and sponsors of clinical research in brazil: an emerging methodology for scientific research. rev bras epidemiol. 2012 dec;15(4):705-13. 24. globo. g1. [brazil overtakes us and bric countries in use of social networks, says research]. 2013 jul 2[access 2017 dec 28]. available from: http://g1.globo.com/tecnologia/noticia/2013/07/brasilsupera-eua-e-paises-do-bric-em-uso-de-redes-sociais-diz-pesquisa.html. portuguese. 25. close s, smaldone a, fennoy i, reame n, grey m. using information technology and social networking for recruitment of research participants: experience from an exploratory study of pediatric klinefelter syndrome. j med internet res. 2013 mar 19;15(3):e48. doi: 10.2196/jmir.2286. https://www.ncbi.nlm.nih.gov/pubmed/23512442 https://www.ncbi.nlm.nih.gov/pubmed/23512442 https://www.ncbi.nlm.nih.gov/pubmed/23512442 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1518/1171 1/10 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1518/1171 2/10 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1518/1171 3/10 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1518/1171 4/10 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1518/1171 5/10 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1518/1171 6/10 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1518/1171 7/10 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1518/1171 8/10 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1518/1171 9/10 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1518/1171 10/10 1 volume 16 2017 e17056 original article relationship between masseter muscle thickness and overbite values in a nigerian population olabimpe abigail soyoye1,otuyemi donald olayinka2, kolawole kikelomo adebanke2, ayoola oluwagbemiga oluwole3 corresponding author: dr olabimpe abigail soyoye department of child dental health, obafemi awolowo university teaching hospitals complex, ile-ife, nigeria. phone +2347031010372, e-mail: bimpe211@yahoo.com received: may 31, 2017 accepted: august 14, 2017 1 bds, fmcds. department of child dental health, obafemi awolowo university teaching hospitals complex, ile-ife, nigeria. 2 bchd, fwacs. department of child dental health, obafemi awolowo university, ile-ife, nigeria. 3 mb.chb, fmcr. . department of radiology, obafemi awolowo university, ile-ife, nigeria. objective: this study determined the relationship between masseter muscle thickness and overbite values among nigerians. methods: the subjects included 66 consecutive patients (21 males and 45 females) who presented for fixed orthodontic appliance treatment. overbite values were measured from standard lateral cephalometric radiographs taken for all patients,who were thereafter divided into three groups of reduced overbite (n=22, mean -1.11+ 2.18mm), normal overbite (n=22, mean 2.59+0.50mm) and increased overbite (n=22, mean 5.21+1.39mm). the masseter muscle thickness of each patient was measured bilaterally using ultrasonography. associations between masseter muscle thickness and different overbite values were analyzed using unpaired t-test, anova and tukey’s multiple comparison analysis. results: mean masseter muscle thickness was 11.23 ± 2.40 mm during relaxation and 12.81 ± 2.64 mm during contraction for study participants. the masseter muscle on the habitual side of mastication of participants was generally thicker but the difference was not statistically significant (p>0.05). there was a progressive increase in masseter muscle thickness from reduced overbite through normal overbite to increased overbite groups and the differences were statistically significant (p<0.01). tukey’s multiple comparisons showed significant differences between all the three overbite groups (p<0.05). conclusion: there was a direct relationship between the muscle thickness and overbite variations. keywords: masseter muscle thickness. overbite values. nigerian population. http://dx.doi.org/10.20396/bjos.v16i0.8650499 mailto:bimpe211@yahoo.com 2 soyoye et al. introduction the practice of contemporary orthodontics involves the understanding and application of many biomechanical principles that enable the orthodontist achieve a desired outcome. orthodontists have developed many techniques based on these principles to effect favourable changes in the dento-facial complex. treatment planning in orthodontics is, however not based entirely on biomechanical considerations, but also requires adequate knowledge of the craniofacial and muscular environment of each patient1,2. muscles of mastication that derive their origin or insertion from the maxilla and mandible, especially the masseter muscle contribute to craniofacial growth, and are of paramount importance in the aetiology of malocclusion as well as stability of orthodontic treatment3-5. the masticatory muscles, including the masseter muscle, affect craniofacial growth in two major ways; first, the formation of bone at the point of muscle attachment depends on the activity of the muscles, secondly the musculature is an important part of the total soft tissue mass whose growth normally carries the jaws downward and forward. loss of part of the musculature from any cause often results in underdevelopment of that part of the face. excessive muscle contraction also causes growth restriction on the affected part of the face, thus producing facial asymmetry6. on the other hand, a major decrease in the muscle tonicity as seen in muscular dystrophy, cerebral palsy and muscle weakness syndromes, allows the mandible to drop downward away from the rest of the facial skeleton, resulting in increased anterior facial height, distortion of facial proportion and mandibular form, excessive eruption of the posterior teeth, narrowing of the maxillary arch and an anterior open bite7. for normal vertical growth and development, growth in the anterior part of the face must be proportional to that occurring in the posterior segment. in the absence of this, a relative growth rotation of the mandible can develop. should growth in the posterior part of the face exceed that occurring anteriorly, then the net effect is an anterior, forward closing rotation of the mandible producing the typical short face deformity and deep overbite associated with the short face syndrome8-10. conversely, reduction in growth in the posterior part of the face in comparison with what occurs in the anterior segment results in an opening or clockwise rotation of the mandible with the net effect being an excessive anterior facial height. this frequently leads to an anterior open bite with associated long face deformity. the masseter muscle thickness can be assessed by various imaging techniques including ultrasound scanning, computed tomography (ct) and magnetic resonance imaging (mri). ct was used by weijs and hillen11 to measure masticatory muscle thickness in adults and they reported that the masseter and medial pterygoid muscles were thicker in persons with short faces and a small jaw angle. mri was used by hannam and wood12 who found a correlation between the cross section of masseter and bizygomatic width in 22 adult males. ultrasonography [us] has been used by a number of investigators to assess masseter muscle thickness. us is a non invasive imaging technique of assessing the parameters of the masticatory muscle and has the advantage over ct and mri of being relatively 3 soyoye et al. inexpensive, reproducible, less complicated to use and with no known cumulative biological effect13. ultrasonography is an indicated technique for evaluating muscles in vivo, for longitudinal studies and for evaluation in children since its advantages outweigh the disadvantages13. moreover, it adds more valuable information to that found by conventional methods for the study of muscles of mastication and other muscles and organs of the human body. in addition, it is readily accepted by patients and their parents. raadsheer et al.14, compared results of measurement of mid-belly masseter muscle thickness using us and mri and showed us to be an accurate and reproducible imaging technique. however, us is not without its attendant limitations, it has lagged behind the other imaging techniques i.e. ct and mri in establishing practical three dimensional (3d) visualisation due to problems associated with acquiring and displaying 3d sets data15. abnormalities in the vertical dimensions, whether in children or adults present the greatest difficulties during active treatment and maintenance of treatment outcome due to the strong influence of the masticatory muscles8. therefore, the aims of this study were to determine the masseter muscle thickness among a group of nigerians using ultrasonography, as well as to relate masseter muscle thickness to their different overbite values. materials and method this research was approved by the research ethics committee of the hospital; protocol number erc/2013/07/16. the sample size was determined by utilizing the formula for calculating sample size for comparative research16 study. sixty-six participants (45 females and 21 males) who presented for fixed orthodontic appliance therapy participated in the study. the participants were informed about the objectives, risks and benefits, the voluntary nature of participation and freedom to withdraw from the study. written informed consent was thereafter obtained from each willing participants. for those younger than 18years, parental consent was obtained including the child’s assent. participants in this study experienced no direct benefit and no compensation was paid to them. the selection criteria used were participants aged 12 to 30 years who presented at the orthodontic clinic of the hospital with malocclusion, no previous history of orthodontic treatment or orthognathic surgery and participants who gave their consent. exclusion criteria applied were participants with marked jaw asymmetry or craniofacial disorders and those with congenital developmental anomalies of the lips, mouth or face. the participants, having met the inclusion criteria were consecutively allotted into three groups of equal number based on their overbite values : reduced overbite (overbite less than 2mm), normal overbite (overbite between 2-3mm) and increased overbite (overbite greater than 3mm)17. the overbite was measured as the vertical overlap of the upper and lower incisors. the masseter muscle thickness was measured using ultrasonography at the department of radiology of the hospital by one of the authors (aoo). the thickness of the masseter muscle was measured using a real time mindray dc-7 ultrasound machine with a 7.5mhz linear probe. bilateral measurements were made with the subjects in supine position and their heads turned side-ways as described by kiliaridis and kalebo18, that is, the angle of the probe during scanning was adjusted to produce the strongest echo from the mandibular 4 soyoye et al. ramus, achieved when the scan plane is perpendicular to its surface. imaging and measurements were performed three times, with an interval of five minutes between each measurement. the intra-observer reliability coefficient was calculated for the three repeated measurements and was found to be 0.94. all measurements were made by the same operator (aoo) to eliminate inter-observer error. in order to register the scan plane at right angle to the long axis of the muscle, the probe was oriented at an estimated angle of 30 degrees to the frankfort plane. the orientation of the probe was maintained manually, while the full length of the muscle was scanned from origin to insertion. the site of measurement was in the thickest part of the masseter muscle close to the level of the occlusal plane, halfway between the zygomatic arch and the gonial angle, approximately in the middle of the mediolateral distance of the ramus. imaging and measurements for each individual were performed twice for the masseter muscle; during relaxation and during maximal clenching. in the relaxed state, the participants were asked to maintain slight inter-occlusal contacts in order to avoid muscle stretching as a result of the mouth opening. in the contracted state, the participants were asked to clench maximally in the intercuspal position . the measurements were made directly from the image at the time of scanning and all the distances were read to the nearest 0.1mm. in addition, information was obtained from the participants with regards to the habitual side of mastication after measurements. statistical method spss version 20 was used for data entry and analysis. analysis of variance (anova) test was used to determine the relationship between the masseter muscle thickness and overbite values. tukey’ multiple comparison analysis was used to assess the relationship between the masseter muscle thickness and the different overbite groups. the level of significance was set at p < 0.05. results a total of 66 subjects who fulfilled the inclusion criteria were recruited into the study. this consisted of 21 males (31.82%) and 45 (68.18%) females. the participants’ ages ranged from 12 to 30 years with a mean age and standard deviation of 19.15 ± 4.73 years. mean age and standard deviation of male participants was 19.90 ± 5.35 years and that of the females was 18.80 ± 4.43 years, with no statistically significant gender difference (p = 0.416) (table 1). table 1. characteristics of participants according to gender characteristics of participants male female distribution of sample according to number (%) 21( 31.8%) 45(68.2%) age mean ±sd (years) 19.90 ± 5.35 18.80 ± 4.43 median age (years) 20 18 age range (years) 13 – 29 12 – 30 occlusal vertical dimension (overbite) reduced (-1.11+2.18mm) 8(36.4%) 14(63.6%) normal overbite (2.59+0.50mm) 9(40.9%) 13(59.1%) increased overbite (5.21+1.39mm) 4(18.2%) 18(81.8%) 5 soyoye et al. the masseter muscle thickness was greater during contraction ( mean – 12.81 ± 2.64) than the relaxation phase ( mean – 11.23 ± 2.40), although the difference was not statistically significant. the muscle thickness was greater on the right side both during relaxation and contraction phases among the study participants. there was no statistically significant difference in the muscle thickness among both gender. ( p > 0.05) (table 2). a large number of the participants (91%) were found to masticate on the right. the masseter muscle was generally thicker on the habitual side of mastication but the differences was not statistically significant (p> 0.05) (table 3). table 4 shows two-way anova of muscle thickness in relation to different occlusal vertical dimensions (overbite). in the entire study population, there was a progressive increase in the muscle thickness across the groups from reduced overbite through normal overbite to increased overbite during both relaxation and contraction. these differences were statistically significant. table 2. gender distribution of masseter muscle thickness during relaxation and contraction phases characteristics of participants male (n =21) female (n =45) total (n =66) p value right masseter muscle relaxation 11.35 ± 2.30 11.58 ± 2.65 11.51 ± 2.52 0.726 left masseter muscle relaxation 10.85 ± 2.42 11.00 ± 2.74 10.95 ± 2.63 0.826 right masseter muscle contraction 13.47 ± 2.74 13.22 ± 2.94 13.30 ± 2.86 0.744 left masseter muscle contraction 12.28 ± 3.02 12.32 ± 2.75 12.31 ± 2.82 0.954 mean masseter muscle relaxation 11.10 ± 2.17 11.29 ± 2.53 11.23 ± 2.40 0.761 mean masseter muscle contraction 12.88 ± 2.70 12.77 ± 2.64 12.81 ± 2.64 0.884 table 4. two-way anova of muscle thickness in relation to occlusal vertical dimensions (overbite) masseter muscle status sex normal overbite (n=22) increased overbite (n=22) f2 p value relaxation male 9.81 ± 1.54 10.90 ± 1.70 10.37 0.001* female 10.00 ± 2.34 11.60 ± 1.86 3.05 0.058 total 9.93 ± 2.05 11.32 ± 1.79 11.52 0.002* contraction male 11.26 ± 1.62 16.71 ± 2.65 11.01 0.001* female 11.15 ± 2.23 13.91 ± 2.58 5.22 0.009* total 11.19 ± 1.99 14.42 ± 2.76 10.76 0.001* *statiscally significant table 3. distribution of masseter muscle thickness according to the habitual side of mastication side of mastication right (n = 59) mean ± sd left (n = 6) mean ± sd p value right masseter muscle relaxation 11.64 ± 2.49 10.03 ± 2.79 0.141 left masseter muscle relaxation 10.86 ± 2.63 11.20 ± 2.58 0.767 right masseter muscle contraction 13.45 ± 2.82 11.27 ± 2.56 0.073 left masseter muscle contraction 12.26 ± 2.83 12.07 ± 2.49 0.871 6 soyoye et al. comparison of masseter muscle thickness in the different occlusal vertical dimensions using tukey’s multiple comparison analysis showed a statistically significant difference in the muscle thickness between the reduced and normal overbite groups both during relaxation and contraction (p < 0.05). similarly, there was a highly statistically significant difference in the muscle thickness between the reduced and increased overbite group both under relaxation and contraction (p < 0.01). a statistically significant difference was attained only during contraction between the normal and increase overbite groups (table 5). discussion the active functional and resting forces of the masticatory muscles particularly the masseter muscle, are responsible for the integrity of the dental arches and relation of the teeth to each other. any aberration of these muscles either in terms of size, thickness or function will affect the integrity of the dental and skeletal structures or cause malocclusion. the mean masseter muscle thickness during relaxation and contraction phases in this study are lower than those reported from previous studies from the nigerian, turkish and japanese populations. the previous nigerian study by egwu et al.19 reported a higher mean thickness. this may be due to ethnic and/or environmental differences as the sample population of the earlier work was amongst the igbo extraction (south-eastern nigeria), well known for their built and physique unlike this present study carried out amongst the population in the south western nigeria (yoruba ethnic group) with less built. satiroglu et al.20 and kubota et al.21 reported higher mean thickness for turkish and japanese population respectively. however, this study recorded higher values than the values reported by benington et al.15 for a british population. these variations in thickness of the masseter muscle across the different populations may be associated with racial differences and the relative indulgence in masticatory activities, possibly from different diet that may have led to the attendant adaptive variation in size. it may also be associated with the orientation and size of the muscle fibres which may have genetic and environmental backdrop19. the higher values obtained during contraction of the muscle compared to relaxation in this study is consistent with those of previous studies by egwu et al.19, satiroglu et al.20 and kubota et al.21 this disparity between the values in masseter muscle thickness during relaxation and during maximal clenching table 5. comparison of masseter muscle thickness in different occlusal vertical dimensions using tukey’s multiple comparison analysis comparison of overbirte values masseter muscle state relaxation contraction p-value p-value reduced overbite/normal overbite 0.021* 0.012* increased overbite/normal overbite 0.107 0.035* reduced overbite/increased overbite 0.001* 0.001* *statiscally significant 7 soyoye et al. (i.e contraction) can be explained by the fact that during the contraction phase, as the mandible is elevated, there is enlargement and thickening of the muscle fibres which may account for the observed higher thickness in the clenched state. the right masseter muscle was thicker than that of the left both during relaxation and contraction in all the participants. this finding is consistent with the findings of chan et al.4, satiroglu et al.20 and rani and ravi22 who reported that the right massetter muscle was much thicker than the left side. a possible explanation may be due to the fact that most of the participants in this study masticated more on the right side of their mouth which may amount to exercising the right masseter muscle more than the left. exercising the muscle has been known to increase its thickness and the bite force, and a significant positive correlation has been found between bite force magnitude and the ultrasound thickness of the masseter muscle5,23,24. this is also supported by previous studies of he et al.25 and kiliaridis et al.26 who reported that reduced activity of the masseter muscle resulted in thin muscle fibres. however, a previous study by raadsheer et al.24 in 1999 reported greater thickness on the left side, whereas raadsheer et al.5 and marquezin et al.27 found no side differences in the thickness of the muscle in subjects with normal occlusion. of the three study groups considered in this study, participants in the increased overbite group had the highest masseter muscle thickness, followed by the normal overbite group with the reduced overbite group demonstrating the least thickness, both during the relaxation and contraction phases of the muscle. this is in agreement with the studies of by satiroglu et al.20, pereira et al.28 and van spronsen et al.29 who reported that individuals with increase (deep) overbite tend to have thicker masseter muscle. rasheed et al.30 in their study of electromyographic and ultrasonographic evaluation of the circum-oral musculature also found that deep-bite subjects demonstrated greater thickness of masseter muscle compared with normal and open bite subjects. a possible explanation for the higher value of masseter muscle thickness recorded in subjects with increased or deep overbite and a thinner result recorded in the reduced overbite group in this study may be due to the fact that individuals with deep overbite tend to have short face while those with reduced overbite or an anterior open bite tend to demonstrate a long face morphology. this is consistent with the results of previous studies by weijs and hillen11, benington et al.15, kiliaridis and kalebo18, and satiroglu et al20 which showed that the masseter muscle is thicker in individuals with short face who tend to have deep overbite, and thinner in those with long face who tend to have reduced overbite or an anterior open bite. another possible reason for the greater thickness recorded for the deep bite and the smaller thickness for the reduced overbite subjects may be because the type ii muscle fibers are present in greater numbers in these patients than those seen in normal and reduced overbite subjects. in contrast, patients with reduced overbite demonstrate not only a reduced number of type ii fibers, but these fibers are also smaller in size31. also, reduced overbite subjects tend to have thinner masseter muscle because the superficial masseter muscle is anteriorly inclined and obliquely oriented relative to the occlusal plane and has a superior positioning of its insertion on the mandible compared to deep overbite individuals who have vertically oriented masseter muscle3. 8 soyoye et al. in conclusion, the masseter muscle was thicker during contraction than during the relaxation phase. in addition, the muscle was thicker on the habitual side of mastication. additionally, there was a direct relationship between masseter muscle thickness and overbite variations, that is, as the overbite 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as, gameiro gh, gaviao mb,benington pmc. evaluation of sexual dimorphismand the relationship between craniofacial, dental arch and masseter muscle characteristics in mixed dentition stage. rev cefac. 2014 jul-aug; 16(4):1231-8. doi: 10.1590/1982-021620149613. 10 soyoye et al. 28. pereira lj, gaviao mbd, bonjardim lr, castelo pm, van der bilt a . muscle thickness, bite force, and cranio-facial dimensions in adolescents with signs and symptoms of temporomandibular dysfunction. eur j orthod. 2007 feb;29(1):72-8. 29. van spronsen ph, weijs wa, valk j, prahl-andersen b, van ginkel fc. a comparison of jaw muscle cross-sections of long-face and normal adults. j dent res. 1992 jun;71(6):1279-85. 30. rasheed sa, munshi ak. electromyographic and ultrasonographic evaluation of the circum-oral musculature in children. j clin pediatr dent. 1996 summer;20(4):305-11. 31. hannam ag, mcmillan as. internal organization in the human jaw muscles. crit rev oral biol med. 1994;5(1):55-89. _enref_3 _enref_5 _enref_20 _enref_34 _goback revista fop n 13 1666 antiinflammatory activity of ricinus communis derived polymer adriana cristina valderramas1*; sérgio henrique pereira moura2*; maira couto3, silvana pasetto5* ; gilberto orivaldo chierice4; sérgio augusto catanzaro guimarães5*; ana claudia bensuaski de paula zurron5* 1master degree in oral biology, prorector for research and postgraduate affairs, 2undergraduate student, department of biological sciences 3biologist, department of biological sciences, sacred heart university, bauru, sp, brazil 4professor, institute of chemistry of são carlos, school of engineering of são carlos, university of são paulo, são carlos, sp, brazil. 5professor, prorector for research and postgraduate affairs, sacred heart *university of sagrado coração, bauru, sp, brazil. received for publication: august 01, 2008 accepted: november 11, 2008 correspondence to: ana claudia bensuaski de paula zurron universidade do sagrado coração (usc), rua irmã arminda, 10-50, jardim brasil, 17011-160 bauru, sp, brasil phone +55-14-2107-7260 fax +55-14-2107-7254 e-mail: anabensuaski@yahoo.com.br a b s t r a c t aim: the present study aimed to evaluate the antiinflammatory activity of the polymer derived from ricinus communis and its mechanism of action. methods: the antiinflammatory activity was investigated in chronic and acute animal models and the mechanism of action involved in the antiinflammatory activity was determined by the in vitro phospholipase a 2 (pla 2 ) enzyme assay. results: in mouse ear edema (10.0 mg/ear) and granulomatous tissue formation (500 mg/kg) models, the polymer inhibited the inflammatory response in 75.08 ± 1.80% and 61.70 ± 1.80% of the cases, respectively (p<0.001). oral administration of the ricinus communis polymer (500 mg/kg) inhibited 72.00 ± 1.20% of formalin-induced inflammation. topical administration of the polymer on oral lesions of mice showed that the oral mucosa was recovered in 60.00 ± 1.40% (p<0.05) of the cases. in in vitro assay, the phospholipase a 2 enzyme was inhibited by the ricinus communis polymer (5.0 mg/ml) in a dose-dependent manner (84.60 ± 1.41%). conclusion: the polymer derived from ricinus communis showed a significant antiinflammatory activity, confirming that the pharmacological mechanism involved in this antiinflammatory action was related to the inhibition of the pla 2 enzyme. key words: polymer, ricinus communis, phospholipase a2, antiinflammatory activity. i n t r o d u c t i o n reconstruction of local bone defects resulting from trauma or bone tumors is a major problem in orthopedics and dental surgery. currently, large amounts of autogenous bone grafts are used to manage these clinical situations. although autogenous bone grafts, which combine osteoconductive, osteogenic and osteoinductive properties, are the gold standard for treating bone defects, only a few donor sites in the skeletal system are appropriate for supplying autogenous bone1. polymers are the most versatile class of biomaterials, being extensively applied in medicine and biotechnology2. a natural polyurethane resin obtained by polymerization of the polyester polyol is derived from the castor beam plant (ricinus communis), which native of tropical regions3. according to kojima et al. 3, this polymer has been developed for bone repair because of its capacity to stimulate new fibroblast formation and its progressively replacement by bone around and inside the material’s porosities material with no late inflammatory response and no signs of systemic toxic effects. the methanolic extract obtained from ricinus communis root showed a significant antiinflammatory and free radical scavenging activity4. the pharmacological activity may be due to the presence of phytochemicals in the plant extract, like flavonoids, alkaloids and tannins, which have various biological actions4. in endodontics, a polymer detergent derived from ricinus communis has been developed for use as a root canal irrigant5-8. the polymer detergent has similar antimicrobial activity to that of 0.5% sodium hypochlorite when used for irrigation of necrotic root canals5-8. although sodium hypochlorite is still the most widely used root canal irrigant, studies have searched for alternative solutions and therapeutic resources that might improve the success rate of endodontic treatment8. barros et al.1 reported that polymers offer the advantage braz j oral sci. october/december 2008 vol. 7 number 27 1667 that has biocompatibility with dental tissue, is osteoconductive and osteogenic, and provide space for the formation of new bone. the ricinus communis polymer showed fibroblastic neoformation progressively replaced by bone around and inside the porosities of material with absence of late inflammation reaction1. specific inhibitors of phospholipase a 2 (pla 2 ) have been sought for a variety of purposes. since this enzyme is believed to control a number of processes ranging from mobilization of eicosanoids to metabolism of phospholipids, these inhibitors could be useful for controlling inflammatory processes such as rheumatoid arthritis, asthma and psoriasis9. enzyme inhibition assays are important tools in the search for new drugs, and so the pla 2 assay could determine the mechanism of action of ricinus communis polymer in the antiinflammatory study. the present study investigated the acute and chronic antiinflammatory activity of the polymer obtained from ricinus communis in mouse ear edema, formation of granulomatous tissue in rats, formalin-induced paw edema in mice and in oral mucosa lesions in mice. the antiinflammatory mechanism of action of ricinus communis was assessed using the pla 2 enzyme assay. morphological analyses were also performed by means of topical antiinflammatory activity tests. material and methods animals fasted male wistar rats (200-250 g) and male swiss mice (20-25 g) obtained from the central animal house of sacred heart university (usc) were used. the animals had free access to tap water. experimental protocols were approved by the institutional ethics committee and were conducted according to recommendations of the canadian council on animal care10. all experiments were performed in the morning, according to current guidelines for laboratory animal care and ethical guidelines for the investigation of experimental inflammation in conscious animals11. chemical agents and reagents a natural detergent obtained from ricinus communis was used in this research. this substance was produced by the group of analytic chemistry and technology of polymers from university of são paulo, são carlos, sp, brazil. the technology created at the institute of chemistry of são carlos produced a polymer that had characteristics of a bone substitute in brazil12. other substances were also used in the experiments: arachidonic acid, hydrocortisone, indomethacin, dexamethasone, formalin, sodium taurocholate, calcium chloride, phosphatidylcholine dipalmitate, phospholipase a 2 and sodium hydroxide (sigma-aldrich corp., st louis, mo, usa), acetone, propolix (herbarium laboratório botânico ltda, colombo, pr, brazil.), tween 80, ethyl alcohol and xylene (merck kgaa, darmstadt, germany), xylazine hydrochloride (bayer s/a, são paulo, sp, brazil), ketamine (laboratórios pfizer ltda, são paulo, sp, brazil). acute toxicity in animals the acute toxicity of the ricinus communis polymer was investigated by intraperitoneal and oral administration in male mice (n=50) weighing 20-25 g. in this assay, increasing doses of the test substance were orally and intraperitoneally administered to groups of 10 animals per dose (100, 300, 500, 700, 1,000 mg/kg). the animals were observed for 14 days and the mortality rate was recorded13. mouse ear edema induced by arachidonic acid the in vivo antiinflammatory activity of ricinus communis polymer was assessed in the mouse ear edema model using arachidonic acid to induce inflammation14. control mice received only the irritant agents, whereas experimental mice also received the ricinus communis polymer (5 and 10.0 mg/ear) applied topically together with the irritant agent. arachidonic acid was dissolved in acetone at concentrations of 10 mg/ml and each mouse received 0.5 µg/ear of arachidonic acid on the left ear. the drugs were applied topically to the inner surface of the ear with an automatic pipette in a volume of 5 µl of arachidonic acid. the right ear (control) received 20 µl of acetone (vehicle). the mice were killed by cervical dislocation 1h after treatment with arachidonic acid. each ear was removed with a metal punch (6 mm diameter disc) and the edema was calculated by subtracting the weight of the right ear (control) from the left treated ear. hydrocortisone (217 µg/ ear) was used as a positive control. drug effects were expressed as percentage of inhibition, according to the following equation: [weight of left minus right control ears] [weight of left minus right treated ears] x 100¸ weight of left control ear phospholipase activity the inhibition of pla 2 activity (purified from apis mellifera bee venom) by the polymer obtained from ricinus communis was assayed by measuring the decrease in the ph of the incubation mixture using a ph electrode in a closed stirring chamber. the test substance was incubated for 30 min with the pla 2 enzyme and added to the assay medium containing 4 mm sodium taurocholate, 12 mm calcium chloride and 7 mm phosphatidylcholine dipalmitate (sigma-aldrich corp.). this technique is reliable under ph 5.015. in the present study, the mean initial ph of the phospholipids mixture was 8.0. positive controls were set up using purified pla 2 (0.33 µg/ml) from bee venom. the total incubation volume was 2.5 ml. the four different concentrations of the polymer used in these in vitro experiments were 2.5, 3.0, 3.5 and 5.0 mg/ml. the pla 2 inhibition was calculated according to the following equation: [d ph (treated/min)¸ d ph (control/60 min)] x100 = % enzymatic reaction braz j oral sci. 7(27):1666-1672 antiinflammatory activity of ricinus communis derived polymer 1668 granulomatous tissue formation cotton rolls (johnson and johnson, new brunswick, nj, usa) were cut into 5-mm pieces and sterilized in groups of four pellets (160 mg). rats were anesthetized with xylazine hydrochloride (50 mg/kg) and ketamine hydrochloride (180 mg/kg) and then the pellets were implanted subcutaneously into four symmetrically distributed positions in the abdomen16-17. two hours after implanting the cotton pellets, the animal groups (n=10 each) were treated orally by gastric gavage with tween (10 ml/kg), dexamethasone (0.2 mg/kg), and the ricinus communis polymer at doses of 250 and 500 mg/kg. daily application of these substances continued for 6 days. on the 7th day, the animals were killed by cervical dislocation, the cotton pellets removed, dried (60oc) and weighed. the difference between the initial and final dry weight corresponded to the weight of the granulomatous tissue formed. formalin-induced paw edema in mice the method applied was similar to that described by henriques et al.18. groups of male animals were treated orally by gastric gavage with tween (10 ml/kg), indomethacin (30 mg/kg) used as positive controls or with the ricinus communis polymer (250 and 500 mg/kg, p.o. respectively). the polymer was administered 30 min before the injection of 2% formalin in pbs (30 µl/paw) into the sub plantar area of the left hind paw. paw volume was measured 4 h after formalin injection. edema was calculated as the difference (µl) between the injected and control paw. the area under the curve (auc) versus ä paw volume was calculated for each animal and edema was expressed as the mean ± sem of auc. effect of ricinus communis in oral mucosa lesions of mice male swiss mice were used in this study. an ulcerated lesion was produced with topical application of naoh (40%) in the oral mucosa of anesthetized animals19-20. lesions of the experimental group were treated with a daily topical administration of the polymer derived from ricinus communis at dose of 250 and 500 mg/kg. lesions of negative and positive control animal groups were treated with a solution of tween 80 and a propolis solution (propolix 11%), respectively. fourteen days later, the animals were sacrificed. the ulcerated lesions were counted and classified according to level of severity presented21. histological analysis tissues obtained from the ears and oral mucosa lesions of mice were fixed in bouin for 24 h, then dehydrated through ascending concentrations of ethyl alcohol, cleared with xylene, embedded in histosec (merck; 11609), and prepared for microtomy. eight-micrometer-thick sections were then deparaffinized and rehydrated through descending concentrations of ethyl alcohol. these samples were stained with hematoxylin and eosin for morphological analysis, using hematoxylin for 15 min. the samples were washed in running water for 10 min and sections turned from red to blue. sections were stained with eosin for 10 min and washed in water, dehydrated, placed in 95% alcohol, cleared and mounted in resin22. photomicrographs were obtained with an axiophot in photomicroscope (d7082; carl zeiss, jena, germany). the lesions were counted by histomorphometry analysis. statistical analysis results were reported as means ± sem and were analyzed statistically by analysis of variance followed by tukey’s test. p values of less than 0.05 were considered significant. r e s u l t s acute toxicity in animals ricinus communis polymer at doses of up to 1,000 mg/kg did not cause any mortality in male albino mice (n=30) during 14 days after intraperitoneal injection and oral administration. mouse ear edema induced by arachidonic acid the mouse ear edema reached a maximum at 1h after arachidonic acid application. the ricinus communis polymer significantly inhibited swelling, considerably reducing the vascular permeability response to arachidonic acid application. the polymer (5.0 and 10.0 mg/ear) inhibited the inflammation induced by arachidonic acid in a concentration-dependent manner in 54.40 ± 1.60% and 75.08 ± 1.80% (p<0.001, respectively) (table 1; figure 1). the positive control, hydrocortisone (217 µg/ ear) inhibited 77.00 ± 1.20% of the topical inflammation (p<0.001). histologically, the topical application of arachidonic acid caused neutrophil accumulation (figure 1a). in this experiment, a rapid, albeit transient edema, was induced accompanied by erythema. ricinus communis polymer application immediately after the irritant agent inhibited acute inflammation caused by arachidonic acid. in this treatment, neutrophils were absent (figure 1b), confirming the antiinflammatory effect mentioned by ricinus communis polymer. treatment dose inhibition (%) control (acetone) 10.0 ml/ear hydrocortisone 217.0 µg/ear 77.00 ± 1.20(**) ricinus communis polymer 5.0 mg/ear 54.40 ± 1.60 (**) ricinus communis polymer 10.0 mg/ear 75.08 ± 1.80 (**) table 1 effect of topical application of ricinus communis on arachidonic acid (0.5 mg/ear)-induced mouse ear edema each value is the mean ± sem for 7 animals. each value differed significantly from the respective control value. anova f (3, 24) = 35.0. **p<0.001, tukey’s test braz j oral sci. 7(27):1666-1672 antiinflammatory activity of ricinus communis derived polymer 1669 a b fig. 1 arachidonic acid (aa)-induced mouse ear edema. a: animals treated with aa and acetone show a typical acute inflammation with neutrophils (arrow) inside blood vessels. bar = 100 mm, original magnification: 40x. b: acute inflammation was inhibited (no neutrophils inside blood vessels) in animals treated with aa and 10 mg/ear of ricinus communis. bar = 500 µm, original magnification 40x. ricinus communis polymer 2.5 35.70 ± 1.20 ricinus communis polymer 3.0 67.70 ± 0.98 (*) ricinus communis polymer 3.5 80.00 ± 1.34 (*) ricinus communis polymer 5.0 84.60 ± 1.41 (*) concentration (mg/ml) drug inhibition (%) table 2 inhibitory action of ricinus communis on phospholipase a 2 activity. data are reported as the mean % inhibition ± sem for 5 experiments (n=5). the concentrations (2.5; 3.0; 3.5; 5.0 mg/ml) of the ricinus communis polymer are significantly different (p<0.05) (3.0, 3.5 and 5.0 ‘“ 2.5 mg/ml). * p<0.05, tukey’s test phospholipase activity at concentrations up to 5.0 mg/ml, the polymer obtained from ricinus communis had a significant inhibitory activity against pla 2 enzyme (0.33 µg/ml bee venom) (table 2). the polymer inhibited the pla 2 activity around 84.60 ± 1.41% (p<0.05). these results showed that the possible mechanism of action of ricinus communis involved in its antiinflammatory activity would occur by pla 2 inactivation, that releases arachidonic acid during the beginning of the inflammatory cascade. granulomatous tissue formation treatment for 6 days with dexamethasone and the polymer of r. commus inhibited the formation of granulomatous tissue induced by implantation of subcutaneous cotton pellets into the abdominal region (table 3). the polymer at the dose of 250 and 500 mg/kg inhibited the inflammatory process by 55.70 ± 1.20 and 61.70 ± 1.80%, respectively (p<0.001). dexamethasone was effective in inhibiting inflammation by 72.30 ± 1.60% (p<0.001). formalin-induced paw edema in mice intradermal injection of formalin (1%, 20 µl), into one of the hind paw of normal rats (control group-treated orally with tween) caused a local inflammatory response, which reached a maximum intensity of edema at hour 4 after application of the phlogistic agent. ricinus communis polymer (250 and 500 mg/kg, p.o.) significantly decreased paw swelling by 61.80 ± 1.30% and 72.00 ± 1.20%, respectively (table 4, p<0.001). treatment of the animals with indomethacin (30 mg/kg), the reference antiinflammatory, significantly reduced the intensity of edema induced by formalin around 80.00 ± 2.00% (p<0.001). tween 10ml/kg 258.0 ±2.8 491.3 ± 1.8 dexamethasone 0.2 mg/kg 245.02 ±2.1 172.72 ±1.2 72.30 ± 1.60 (**) ricinus communis polymer 250 mg/kg 243.7 ± 1.6 188.0 ± 1.4 55.70 ± 1.20 (**) ricinus communis polymer 500 mg/kg 238.0 ± 1.2 176.3 ± 1.3 61.70 ± 1.80 (**) initial final treatment dose dry weight of granuloma (mg) inhibition (%) table 3 effect of oral administration of ricinus communis on rat granuloma tissue formation each value is the mean ± sem for 10 animals. the ricinus communis polymer at the dose of 250, 500 mg/kg and the positive control (dexamethasone) were significantly different compared to the respective control value. anova f (3,36) = 35.0. **p<0.001, tukey’s test braz j oral sci. 7(27):1666-1672 antiinflammatory activity of ricinus communis derived polymer 1670 effect of ricinus communis in oral mucosa lesions of mice in experiments of oral mucosa lesions induced by sodium hydroxyl 40%, the polymer obtained of ricinus communis (250 and 500 mg/kg) healed respectively 35.00 ± 1.20% and 60.00 ± 1.40% of the oral mucosa lesion (p<0.05) (table 5; figure 2). the positive control (propolix) healed 78.00 ± 1.60 (p<0.05). when only naoh was applied topically, it was observed that the surface epithelium was destroyed (figure 2a). in figure 2b, the oral lesion was healed and the surface epithelium was recovered in mice treated with the ricinus communis polymer. treatment dose inhibition (%) tween 10 ml/kg indomethacin 30 mg/kg 80.00 ± 2.00 (**) ricinus communis polymer 250 mg/kg 61.80 ± 1.30 (**) ricinus communis polymer 500 mg/kg 72.00 ± 1.20 (**) table 4 effect of ricinus communis on formalininduced mouse paw edema each value is the mean ± sem for 7 animals. each value differed significantly from the respective control value, anova f (3, 24)= 34.6 (p<0.001). **p<0.001, tukey’s test treatment dose inhibition (%) tween 10 ml/kg propolix 10 µl 78.00 ± 1.60 (*) ricinus communis polymer 250 mg/kg 35.00 ± 1.20(*) ricinus communis polymer 500 mg/kg 60.00 ± 1.40 (*) data are reported as the mean % inhibition ± sem for 9 animals. *p<0.05, tukey’s test table 5 topical effect of ricinus communis on oral mucosa lesions of mice a b fig. 2. effect of ricinus communis on oral mucosa lesions of mice. a: oral lesion caused by sodium hydroxide (40%) applied topically and treated with tween. damage to epithelial tissue is noted and indicated by arrow. bar = 100 mm, original magnification = 10x. b: the photomicrography show oral mucosa epithelium intact (regenerate in 14 days) by the ricinus communis polymer (500 mg/ kg) after induction of lesion by sodium hydroxyl 40%. bar = 500µm, original magnification = 10x.d i s c u s s i o n the development of polyurethane resins derived from ricinus communis widened the scopes in different fields of medical and dental research, as they were shown to have biocompatibility and potential applicability in several areas8. in endodontics, a detergent derived from ricinus communis has been proven to have similar antimicrobial activity as that of 0.5% sodium hypochlorite when used for irrigation of necrotic root canals5-6. in the present study, we examined the antiinflammatory activity of the polymer obtained from seeds of ricinus communis. this substance is a detergent derived from castor bean oil, has similar antimicrobial activity to that of 0.5% sodium hypochlorite when used for irrigation of necrotic root canals, is biocompatible with the periapical tissues, increases dentinal permeability and has similar ability to remove smear layer from the root canals as that of 17% edta8. studying the extract of leaves and root of ricinus communis, ilavarasan et al. 4 observed a significant antiinflammatory activity in rats, possibly due to the presence of flavonoids, alkaloids and tannins present in the plant extract. it was also reported that flavonoids obtained from ricinus communis root extract would explain its free radical scavenging activity. the excessive generation of reactive oxygen species (ros) leads to a variety of pathological processes, such as inflammation, diabetes, hepatic damage and cancer4. inflammation is generally defined as the response of living tissue to an injurious stimulus23. the usual features of inflammation include the activation of epithelial cells and resident macrophages, and the recruitment and activation of neutrophils, eosinophils, monocytes and lymphocytes24. leukotriene and prostaglandin synthesis is involved in arachidonic acid-induced ear edema25. chemically induced edema represents an acute local inflammation eliciting a complex series of physiological events involving many braz j oral sci. 7(27):1666-1672 antiinflammatory activity of ricinus communis derived polymer 1671 processes in which components of a plant extract may interact, inhibiting kinins and prostaglandins on vascular permeability that appear to be involved with inflammatory process26-27. acute and chronic inflammatory processes can be induced by several means, and antiinflammatory agents exert their effects through different manners28-29. for screening of new antiinflammatory compounds, the croton oil-induced mouse ear edema assay is widely used in conjunction with the in vitro phospholipase a 2 assay30-31. enzyme inhibition assays are important tools in the search for new drugs. is has been established that the inflammation induced by arachidonic acid involves an increase in pla 2 activity, which, in turn, leads to the release of arachidonic acid and subsequent biosynthesis of leukotrienes and prostaglandins, thus also involving the lipooxygenase pathway32. bresnick et al. 33 stated that pla 2 catalyzes the sn-2 hydrolysis of phospholipids releasing free fatty acids, predominantly arachidonic acid and lysophospholipids. these products can have biological actions or be further metabolized to form a variety of proinflammatory lipid mediators including prostaglandins, leukotrienes plateletactivating factor and thus the inhibition of pla 2 by pharmacological agents should have led to an antiinflammatory effect. glycosides obtained from ipomoea imperati also showed activity in both tests mentioned above, reducing mouse ear edema induced and inhibiting bee-venom phospholipase a 2 (pla 2 ) activity32. the cotton pellet-induced granuloma was used as a chronic model to evaluate the antiinflammatory effects of natural products and have good predictive value for screening antiinflammatory agents34. this method is a suitable test for assessing the antiinflammatory activity drugs and widely used to evaluate the transudative and proliferative components of chronic inflammation 35. in chronic inflammatory states, the efficacy of antiinflammatory agents can be indicated by inhibiting the increase in fibroblasts and the infiltration of neutrophils and exudation36-37. when macrophages, epithelioid cells and multinucleate giant cells predominate at the site of inflammation, the lesion is named nodular chronic inflammation or granuloma. the fluid absorbed by the pellet greatly influences the wet weight of the granuloma. dry weight correlates well with the amount of granulomatous tissue formed38. catanzaro guimarães et al.38 reported that the inflammatory process is represented by edema, which is formed in the early stages of granuloma development, or as a result of immune reactions participating in the pathogenesis of these lesions. these reactions originate immune complexes that activate the complement system to generate vasodilatator mediators. the latter mechanism is represented by cells, collagen fiber and newly formed bloods vessels38. the formalin’s test is a well-known model of chemically induced inflammation and nociception39. the intra-plantar injection of diluted formaldehyde causes inflammation that release mediators such as bradykinin, histamine, sympathomimetic amines, tumor necrosis factor-á and interleukins40-43. in addition to these mediators, local prostaglandin levels are responsible for the progress of nociception and are targeted by most non-steroidal antiinflammatory drugs43. in the formalin assay, edema that is maintained during the plateau phase occurs due to kinin-like substances. later, the swelling phase prevails due to the release of prostaglandin-like substance44. thereby, the formalin assay is well suited for the comparative bioassay of antiinflammatory agents since the relative potency estimates obtained for most drugs tend to reflect clinical experience45. in experiments of oral mucosa lesions induced by sodium hydroxyl 40%, the ricinus communis polymer was tested. ulcers are defined as local defects on the surface of tissues or organs. these defects are produced by loss of surface epithelium with exposure of connective tissue. contact of the mucous membrane with physical or chemical agents represents the main source of mouth ulcers46. oral ulcers are one of the most common complaints in the dental practice and are usually caused by mechanical trauma induced by ill-fitted dentures, orthodontic appliances and fractured crowns and restorations47. with major loss of cells involved in oral lesion formation, the inflammatory and repair processes occur by secondary union and involve a series of vascular, cellular, neurological and humoral events. ulcer healing is a dynamic process of filling mucosal defects with proliferating and migrating of epithelial cells as well as connective tissue, resulting in the reconstruction of the mucosal architecture48. in conclusion, the findings of the present study showed a potent antiinflammatory activity of ricinus communis polymer, this antiinflammatory effect being related to the phospholipase a 2 enzyme inhibition. this study establishes the therapeutic rationale of using ricinus communis polymer in various inflammatory events, which has antimicrobial activity and has been used for root canal irrigation. r e f e r e n c e s 1. barros vmr, rosa al, beloti mm, chierice, g. in vivo biocompatibility of three different chemical compositions of ricinus communis polyurethane. j biomed mater res. 2003; 67a: 235-9. 2. willians, df. definitions in biomaterials (progress in biomaterials) new york: elsevier press; 1987. p 4. 3. kojima ke, ohara gh, rossi jc, telles ml, soares tvc, sanda m. estudo experimental da biocompatibilidade do polímero da mamona implantado intra-ósseo e intra articular em coelhos. acta ortop bras. 1995; 3: 62-8. 4. ilavarasan r, mallika m, venkataraman s. antiinflammatory and free radical scavenging activity of ricinus communis root extract. j ethnopharmacol. 2006; 103: 478-80. 5. ferreira cm, bonifácio kc, froner ic, ito i.y. evaluation of the antimicrobial activity of three irrigating solutions in teeth with pulpal necrosis. braz dental j. 1999; 10: 115-21. 6. ferreira cm, rosa ops, torres as, ferreira fba, bernardinelli n. activity of endodontic antibacterial agents against selected anaerobic bacteria. braz dental j. 2002; 13: 118-22. 7. mantesso a. in vitro evaluation of a new intracanal irrigating braz j oral sci. 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modified formalin test: characteristic biphasic pain response. pain.1989; 38: 347-52. 41. le bars d, gozariu m, cadden sw. animal models of nociception. pharmacol. rev. 2001; 53: 597-652. 42. parada ca, tambeli ch, cunha fq, ferreira sh. the major role of peripheral release of histamine and 5-hydroxytryptamine in formalin-induced nociception. neuroscience. 2001; 102: 937-44. 43. ferreira sh. peripheral analgesic sites of action of antiinflammatory drugs. int j clin pract suppl. 2002; 128: 2-10. 44. ismail ts, gopalakrishnan s, hazeena begum v, elango v. antiinflammatory activity of salacia oblonga wall and azima tetracantha lam. j ethnopharmacol. 1997; 56: 145-52. 45. winter ca, risley ea, nuss cw. carrageenan-induced edema in hind paw of the rats as an assay for antiinflammatory drugs. proc soc exp biol med. 1962; 111: 544-7. 46. scully c, shotts r. mouth ulcers and others causes of orofacial soreness and pain. bmj. 2000; 321: 162-5. 47. soares de lima aa, trindade gregorio am, tanaka o, machado man, frança bhs. treatment of the mouth traumatic ulcers caused by orthodontic appliances. rev dent press ortodon ortop facial. 2005; 10: 30-6. 48. sasaki e, pai r, halter f, komurasaki t, arakawa t, kobayashi k, et al. induction of cyclooxygenase-2 in a rat gastric epithelial cell line by epiregulin and basic fibroblast growth factor. j clin gastroenterol.1998; 27 (suppl. 1): s21-7. braz j oral sci. 7(27):1666-1672 antiinflammatory activity of ricinus communis derived polymer braz j oral sci. 15(1):1-7 original article braz j oral sci. january | march 2016 volume 15, number 1 impact of dental caries on quality of life of adolescents according to access to oral health services: a cross sectional study angela xavier1, érica silva de carvalho1, roosevelt da silva bastos2, magali de lourdes caldana2, patrícia ribeiro mattar damiance2, josé roberto de magalhães bastos2 1universidade do estado do amazonas – uea, school of dentistry, area of community health, manaus, am, brazil 2universidade de são paulo usp, bauru dental school, department of orthodontics and community health, bauru, sp, brazil correspondence to: angela xavier avenida carvalho leal n° 1777 bairro: cachoeirinha cep: 69079-030 manaus, am, brazil phone: +55 14 997356040 e-mail: angelaxmonteiro@yahoo.com.br abstract aim: this study presents the prevalence of dental caries and its relation to the quality of life of adolescents according to the access to dental health services. methods: two hundred and fifty-six adolescents between 15 and 19 years of age participated in the study; they were all enrolled in public schools in a countryside municipality of the são paulo state. data related to dental caries were evaluated by the dmft index, and ohip-14 was used for evaluating the quality of life. mann whitney and spearmann correlation tests were also used (p<0.05). results: a dmft of 3.09 (±3.30) was found with a higher prevalence among the adolescents who used public dental services (3.43±3.34) compared with those who used private services (2.94±3.28). a statistically significant relationship between the decay component of dmft with physical pain (0.020), physical disability (0.002) and quality of life (0.017) was verified. conclusions: a low prevalence of dental caries was observed, and it was higher in adolescents who used public oral health services rather than private ones, evidencing the low influence of oral health on the quality of life of the participants. keywords: dental caries. adolescent. quality of life. health services accessibility. introduction adolescents are recognized as important part of the global public health. an approach associated to the quality of life can increase the understanding and knowledge of the adolescents’ health and help to establish policies that promote their health and well being1. the discussion about the relationship between health and quality of life demonstrates that this is a social representation based on subjective parameters, such as well being, happiness and objectives based on the needs of a certain population2. in this sense, oral health-related quality of life describes how an individual’s day-to-day living is disrupted by oral disorders. it is a multidimensional concept that involves different health domains, and it is increasingly recognized as an integral part of general health, with an important role for understanding subjective patient evaluations and the experience with oral health care and determining the assessment of needs3,4. the impact of oral health problems on society is defined as the outcomes related to the limitations of functional capabilities and performance of expected roles. oral health problems such as dental caries have been associated with absenteeism and decrease in children’s and adolescent’s school performance5. dental caries still remains one of the most prevalent oral diseases in our country. in a nationwide survey conducted in 2010 in brazil, a mean decayed, http://dx.doi.org/10.20396/bjos.v15i1.8647090 received for publication: january 26, 2016 accepted: april 25, 2016 2 missing and filled teeth (dmft) index of 4.25 was established for adolescents aged 15 to 19 years, with the highest mean found in the midwest region (5.94) and the lowest mean found in the southeastern region (3.83)6. in addition, a higher percentage of dmft in this age group remains untreated and it is associated with a negative impact on general health, development, productivity, school performance and oral health-related quality of life7. in order to improve oral health indicators, one of the required factors is the access to dental health services; however, there are difficulties concerning such access for a substantial part of the population. this can be explained by several factors, like the socioeconomic and educational levels, the high cost of private services and the deficiencies in the availability of oral health services in primary health care. the demand for public dental services is still high and the private sector accounts for a significant coverage of these services8. results from the national epidemiologic survey conducted in brazil in 2010 showed an overview of access to oral health services of adolescents between 15 and 19 years old. it is still unsatisfactory: 13.60% have never been to the dentist, with the lowest prevalence of teenagers that have never been to the dentist in the southern region of the country (5.00%) and the highest values in the midwest region (19.40%)6. based on these considerations, this study aimed to evaluate the prevalence of dental caries and its relation to the quality of life of adolescents aged between 15 and 19 years, according to the access to oral health services in a countryside municipality of são paulo state, brazil. material and methods this study was approved by the ethics committee of bauru dental school, university of são paulo (process number 174 ⁄ 2011), in accordance with the resolution 196/96 of the brazilian national health council. this research was conducted in full accordance with the world medical association declaration of helsinki. all participants or their legal guardians signed the informed consent form before participating in any part of the research. this cross-sectional study was conducted in the city of agudos, located in the midwest region of the são paulo state and according to the latest census it has 35,000 inhabitants (ibge, 2010). the city had 6 basic health units and 3 family health units and a number of 3,091 adolescents between 15 to 19 years old. the municipality has 5 public schools with high school in the urban region, and 3 of them were randomly selected for the study, with a total of 716 adolescents aged 15 to 19 years old. all participants provided an informed consent form signed by them or their legal representative, for those under 18 years of age, as required by the brazilian law9. the sample size was calculated using the correlation coefficient, based on the total adolescent population of the city (n=3,091) with a 0.05 error level and a correlation coefficient (r) of 0.20, resulting in study population of 256 adolescents to be examined. the examinations were conducted in 2012, between march and june. the adolescents were examined by a single calibrated examiner (kappa = 0.95) in order to ensure uniform interpretation, understanding, reproducibility and application of the who criteria. examinations were performed in an outdoor setting under natural light, with the examiner and the adolescent sitting in chairs. the examiner used a dental mirror and a community periodontal index (cpi) ballpoint probe. the who criteria for decayed, missing and filled teeth (dmft) were used to evaluate dental caries. these data provide the information to calculate the significant caries (sic) index10 and the care index10. percentages of dmft and caries-free children were used to describe the dental caries distribution among the teenagers. significant caries index (sic index) and care index were employed to assess the unequal distribution of dental caries and oral health care. sic index was calculated by the mean dmft of the one third of the individuals with the highest dmft values in a given population, and was used to measure the polarization of the dental caries occurrence among schoolchildren. the care index was calculated using the dmft means without the cariesfree children. the component “f” (filled teeth) was divided by dmft and multiplied by 10011. the oral health services and oral health-related quality of life questionnaires were also used in the study. the questionnaire from the national survey by household sampling12, was used to evaluate access to oral health services. it contains 8 questions about access to oral health services, time since the last dental visit, reason for consultation and if the treatment was by public or private service. the ohip-14 was used to access the impact of oral health in the adolescent’s quality of life. this instrument evaluates the experiences of the subject in the 12 months prior to the dental caries epidemiological examination13. the dimensions assessed by this instrument were functional limitations, physical pain, psychological discomfort, physical disability, psychological disability, social disability and handicap. two questions apply to each dimension. possible responses were: 0 = never; 1 = rarely; 2 = sometimes; 3 = often, and 4 = always. the total maximum score ranged from 0 to 28. data were analyzed descriptively by absolute and relative frequencies. they were stratified according to the access to dental healthcare (access to private or public dental healthcare services) and according to ethnic groups. in this case, the ethnic groups were stratified into whites and non-whites and since there was no oriental or indigenous individuals, it was a sample composed by white, black or brown participants. the mann-whitney test was used to compare findings in relation to ethnic and assistance groups on the dmft index, its components and quality of life and its domains. spearman’s correlation was used to estimate the correlations between the dmft, its components and the access to oral health service with ohip 14 and its domains. all statistical procedures adopted a significance level of 0.05 and tests were performed using statistica version 9.1. results the response rate was 35.75%, while the losses were especially due to parental refusal, incomplete or unanswered questionnaires, and adolescents who were not in school at the examination times. impact of dental caries on quality of life of adolescents according to access to oral health services: a cross sectional study braz j oral sci. 15(1):1-7 3 regarding sample distribution, 40.87% of the adolescents used public dental services, 59.13% used private dental services and 4.93% of them had never been to the dentist. concerning sex, 35.16% were male and 64.84% were female (table 1). a dmft mean of 3.09 was found, with a higher expressivity of the filled component and a statistically significant difference between the white and non-white ethnic groups in the decayed component (p=0.03). a higher dmft mean was observed in the public assistance group compared with the private assistance group, but without statistically significant difference between them (table 2). in addition, a sic index twice as high the value of dmft was found for the overall sample, ethnic groups and type of assistance, showing a polarization of dental caries in the group. when the influence of oral health on adolescents’ quality of life was assessed, a mean of 6.62 (+4.41) was observed, showing a low impact of oral health conditions on quality of life, with a minimum score of 0.00 and a maximum score of 18.40. according to the different responses to the domains questionnaire, a higher mean in the psychological discomfort and a lower mean in the domain deficiency was verified in the overall sample, the ethnic group and the type of assistance. no difference was found in the oral health related to the quality of life between white and non-white ethnic groups and between the private and the public access to oral health service groups, due to the higher expression of the filled component in both groups, as shown in table 3. the spearman correlation test assessed the relationship among the independent variables (dental caries, its components and access to oral health services) under the influence of oral health on quality of life and its domains. however, this study did not verify the causal relationship among the assessed variables due to its cross-sectional design. the results of the correlation test are in table 4, in which a statistically significant correlation can be observed among the decay component, the physical pain, physical disability and the final score of quality of life. a relationship was verified between the missing component with psychological discomfort, psychological disability, deficiency and final score of the quality of life. in relation to the access to oral health services, a statistically significant correlation was verified with the physical pain and physical disability domains and this relation was weak. impact of dental caries on quality of life of adolescents according to access to oral health services: a cross sectional study table 1 sample distribution according to the access to oral health services, agudos, sp, 2012. sealant/batch number public health service private health service have never been to the dentist total age n (%) 15 37 (14.68) 68 (26.98) 2 (0.79) 107 (41.79) 16 31 (12.30) 47 (18.65) 1 (0.40) 79 (30.86) 17 27 (10.71) 28 (11.11) 1 (0.40) 56 (21.88) 18 8 (3.17) 6 (2.38) 0 (0.00) 14 (5.47) sex n (%) male 35 (13.89) 53 (21.03) 2 (0.79) 90 (35.16) female 68 (26.98) 96 (38.10) 2 (0.79) 166 (64.84) ethnic groups n (%) white 75 (29.76) 112 (44.44) 3 (1.19) 190 (74.22) non-white 28 (11.11) 37 (14.68) 1 (0.00) 66 (25.78) total 103 (40.23) 149 (59. 13) 4 (1.59) 256 (100.00) *mann-whitney test. table 2 dental caries and components according to gender, ethnic groups and type of assistance, agudos, sp, 2012. decay (±sd) missing (±sd) filled (±sd) dmft (±sd) sic index (±sd) caries free (%) care index (%) ethnic groups white 0.45 (0.90) 0.07 (0.38) 2.53 (3.20) 3.05 (3.50) 7.17 (2.85) 35.79 82.95 non-white 0.80 (1.09) 0.18 (0.46) 2.24 (2.42) 3.23 (2.68) 5.68 (2.59) 22.73 69.35 p 0.03* 0.36 1.00 0.41 access to oral health service public health service 0.68 (+1.13) 0.07 (+0.29) 2.68 (+3.09) 3.43 (+3.34) 7.21 (+2.82) 25.24 78.13 private health service 0.46 (1.10) 0.12 (0.48) 2.36 (2.99) 2.94 (3.28) 6.68 (2.72) 36.24 80.27 have never been to the dentist 0.25 (0.50) 0.00 (0.00) 0.00 (0.00) 0.25 (0.50) 0,50 (0.71) 75.00 0.00 p* 0.38 0.73 0.68 0.49 total 0.54 (+1.11) 0.10 (+0.41) 2.45 (+3.02) 3.09 (+3.30) 6.86 (+2.76) 32.42 79.29 braz j oral sci. 15(1):1-7 4 impact of dental caries on quality of life of adolescents according to access to oral health services: a cross sectional study *mann-whitney test. table 3 oral health-related quality of life and domains according to gender and ethnic groups, agudos, sp, 2012. functional limitations (±sd) physical pain (±sd) psychological discomfort (±sd) physical] disability (±sd) psychological disability (±sd) social disability (±sd) deficiency (±sd) quality of life (±sd) ethnic groups white 0.74 (0.71) 1.13 (0.81) 1.56 (1.12) 0.63 (0.78) 0.83 (0.79) 1.12 (0.89) 0.51 (0.80) 6.53 (4.15) non-white 0.77 (0.70) 1.39 (0.77) 1.51 (0.96) 0.73 (0.73) 0.89 (0.92) 1.07 (0.89) 0.55 (0.85) 6.90 (4.14) p 0.79 0.49 0.75 0.87 0.71 0.96 0.58 0.48 access to oral health service public health service 0.75 (±0.68) 1.13 (±0.80) 1.59 (±1.09) 0.53 (±0.69) 0.83 (±0.78) 1.03 (±0.91) 0.48 (±0.86) 6.35 (±3.98) private health service 0.74 (±0.71) 1.23 (±0.81) 1.49 (±1.06) 0.73 (±0.81) 0.85 (±0.85) 1.15 (±0.86) 0.54 (±0.77) 6.73 (±4.21) never 0.99 (0.91) 1.59 (0.99) 2.45 (1.29) 1.23 (0.87) 1.15 (1.01) 1.35 (1.26) 0.69 (0.82) 9.44 (5.28) p* 1.00 0.79 0.30 0.75 0.70 0.62 1.00 1.00 total 0.75 (±0.70) 1.19 (±0.81) 1.55 (±1.08) 0.66 (±0.77) 0.85 (±0.83) 1.11 (±0.89) 0.52 (±0.81) 6.62 (±4.41) dental caries access to oral dmft decay filled missing health services r(p) r(p) r(p) r(p) r(p) functional limitations 0.021 (0.733) 0.050 (0,427) -0.014 (0.814) 0.074 (0.239) 0.013 (0.837) physical pain 0.102 (0.102) 0.145 (0.020)* 0.039 (0.529) 0.112 (0.073) 0.167 (0.008)* psychological discomfort 0.021 (0.737) 0.116 (0.065) -0.035 (0.572) 0.159 (0,011)* 0.026 (0.676) physical disability 0.114 (0.069) 0.185 (0.002)* 0.020 (0.747) 0.110 (0.079) 0.159 (0.011)* psychological disability 0.028 (0.650) 0.077 (0.216) -0.014 (0.821) 0.183 (0,003)* 0.020 (0.753) social disability -.005 (0.941) 0.019 (0.760) -0.028 (0.652) 0.113 (0.070) 0.012 (0.851) deficiency 0.085 (0,177) 0.066 (0.294) 0.046 (0.459) 0.183 (0.003)* 0.026 (0.677) quality of life 0.066 (0,291) 0.149(0.017)* -0.010(0.870) 0.192(0.002)* 0.069(0.274) table 4 spearman correlation (r) between dental caries, its components and access to oral health services with quality of life and its domains, agudos, sp, 2012. *statistically significant correlation (p<0.05). discussion adolescence is a period of growth and development, with strong internal and external changes in the intellectual and emotional area and in the sexual maturation; therefore, it is a period of great changes14. during this period young people develop behavioral patterns and lifestyle that may influence their morbidity pattern and health care. the pattern of health services use is considered an important factor in the health conditions study, since this pattern is related to the treatment needs, concerns and self-care. the present study found that 59.13% of the adolescents sought private oral health care. on the other hand, gomes et al.15 (2014) in a study carried out in the state of maranhão, found a very lower dental visit rate in the population: among children, only 9.0% used oral health services being that 61.2% used public oral health service; among adults, 28% used dental services being that 55.6% used private dental services15. these results show that a large part of the population sought treatment in the private clinics; therefore, this sector still represents a significant part of the provision of oral health services to the studied group. the reasons for this fact is the population disbelief’ concerning the public oral health services and the difficulty to access them. such results were similar to other studies carried out in the country16-18. the unified health system provides universal access to health services to all individuals and there are advances in the public policies regarding the oral health, by the inclusion of oral health teams in the family health program and implementation of specialized dental clinics. even so, wider public policies are still necessary to increase the access of the whole population to the oral health care, as the private sector still represents a significant part of the service coverage in this country19,20. there are few studies regarding this specific period of the human development relative to the oral health conditions. concerning dental caries, this study found a dmft mean of 3.09 (±3.30), lower than the ones found in other studies21-24, and higher than the research by cangussu et al.25 (2001). compared with a national survey carried out in 2010, the dmft found in this study braz j oral sci. 15(1):1-7 5 impact of dental caries on quality of life of adolescents according to access to oral health services: a cross sectional study is lower than the national average (4.25) and lower than the mean of the southeast region (3.83)6. moreover, a higher expressivity in the filled component was verified, according to several studies6,24-26, except for the research by rebelo et al.22 (2009), who found a higher expression in the decayed component of dmft. the low values of dmft found in this study may be explained by the high access to private dental services by the studied population (59.13%). furthermore, the city had six basic health units and three family health units. in this study, the main expression in the filled component was the care index, which showed that the adolescents had regular access to oral health care23,25,26. a difference was found in the decay component between white and non-white ethnic groups (p=0.03). this difference could be observed in other studies in the country associated to lower socioeconomic conditions, in which non-white ethnic groups (black and brown) have similar socioeconomic status compared to the white ones and it was not due to biological differences26,27. a higher mean dmft was found in the public oral health services group (3.43±3.34) compared to the private oral health services group (2.94±3.28), but there was no statistically significant difference between private and public groups. according to narvai et al.28 (2006) there is an agreement on the existence of a polarization when, in one pole, there is absence of the disease in a large number of people and there is another large proportion of cases concentrated in a small group of individuals. according to the author, the polarization is a phenomenon that possibly reflects the effectiveness of preventive measures and disease control, based on population strategy. it evolves from a high prevalence of the disease to a panorama of a large percentage of caries-free individuals28. in this study, it was recorded a sic index of 6.86, a value twice as high as the dmft, with a higher concentration of the disease in a lower percentage of the population; this was also observed in other studies29. the identification of polarized groups is important to guide oral health practices in the public health service and reduce inequalities in oral health conditions. the assessment of the oral health conditions by strictly clinical criteria does not consider socio-behavioral characteristics, that is, how changes in the oral health affect people’s daily lives. the incorporation of perception measurements to the clinical indicators could help making decisions regarding the best type of treatment for individuals, considering the social and psychological factors previously ignored by the normative systems that determine such needs30,31. instruments of oral health-related quality of life were developed in order to quantify the extent of oral health problems, which interferes in the well being of people’s daily lives and assesses the impact of oral health on the physical and psychosocial development. the ohip questionnaire was developed by slade and spencer in 1994, and subsequently, a simplified version was developed in 1997, the ohip 14, which assesses the impact of oral health in different dimensions30. a study using the ohip 14 showed good psychometric properties when administered to adolescents and could be a promising tool for the selection of the group care priority32. in this study, the psychological discomfort (1.55) and physical pain domains (1.19) were observed to have a higher influence on oral health status in adolescents’ lives, both in the overall sample and in relation to ethnicity and access to oral health services. the physical pain domain aims to show how changes in oral health conditions may cause pain or discomfort when eating, and the psychological discomfort domain refers to concerns or nervousness regarding oral health conditions; the most expressive of these parameters shows that there is an evident concern of the adolescents regarding the oral health status and its possible consequences. these results are similar to the ones found in the study by paredes et al.33 (2015), who found more expression of the physical pain domain. they are also similar to the ones found in the study by silveira et al.34 (2014), who found higher scores for the psychological discomfort dimension and also noted that the greater the need for treatment, the greater the perception of the severity of the physical and psychosocial dimensions impact. according to the authors, this association is due to the understanding that the dental caries can cause pain, functional limitations, disappointment or concerns regarding the oral health34. in the same way, these results are similar to the study by bastos et al.35 (2012) with adolescents in the municipality of bauru, sp, which found a correlation between the dmft index and an ohip-14 score in suburban area subjects in the physical pain and psychological disability dimensions. in addition, there was no difference in the oral health related quality of life (ohrqol) in the young people who accessed public health services and the ones who accessed the private sector. in relation to the ethnic groups, difference was observed in the decay component between white and non-whites groups. this result may be related to the size of the municipality as, in cities with less than 100,000 inhabitants, health policies can be more available and better controlled. an assessment of the correlations between the dimensions of the ohip-14 and the dependent variables of the dmft index showed a significant relation between the decay component of dmft with the physical pain and physical disability domains on quality of life and among the missing component and the psychological discomfort, psychological disability and deficiency, but the relation was weak. this weak relation may be explained by the low prevalence of untreated dental caries in the studied group and by the low expression of the missing component in the dmft observed in this research. despite these facts, the present study demonstrates that both untreated dental caries and its clinical consequences have impact on the ohrqol and require immediate treatment. the evaluation of oral health-related quality of life consists in the psychosocial perception in a non-normative evaluation of the oral health condition. this suggests that there are difficulties in this population about the full knowledge of the problems they face, despite the importance of personal impressions of individuals; it also reinforces the importance of the professional examination. an important aspect of this study results refers to the psychological issues related to tooth loss and the consequences on the quality of life, evident in the psychological discomfort domain and its relation to the missing component of dmft, which despite the low expressivity, showed dental mutilation at an early age. the dmft index represents the intensity of the dental caries attack and its relation to the care needs of the population. the correlation braz j oral sci. 15(1):1-7 6 between the index and quality of life (qol) indicator can help policymakers to better understand how to develop dental policy plans specifically designed to meet the needs of the people rather than fulfil the normative criteria of dentists. a positive relation was found between the access to oral health services and the physical pain and physical disability domains; the more distant the period of the last dental visit, the greater the influence of the oral health conditions on physical pain and physical disability. the identification of the groups most affected by the psychosocial impacts caused by diseases may provide support for the selection of treatment priorities in regions or municipalities with limited financial resources and suppressed demand; therefore, self-reported measures can express the experiences of illness complementing clinical assessments28. this study has some limitations, as the sample is not representative of the entire population and the correlation coefficient does not represent a cause and effect relationship because it is a cross-sectional study. despite these considerations, low prevalence of dental caries and low impact of oral health conditions were found on adolescents’ quality of life, which may evidence that most subjects consider their oral health in a positive way. the adolescents showed regular access to dental services, with a higher use of private oral services than the public ones, but the results of this study showed that untreated dental caries and its consequences still cause impact on oral health and quality of life of the adolescents, demonstrating the need for greater attention to oral health of this group by health managers and professionals. the results of this study were significant for re-directing the oral health attention towards the adolescents, based on the impact of oral health conditions in this population, seeking an articulation of the scientific knowledge and the practices with the implication of the oral health-disease process for this specific age group. acknowledgements we thank all principals, teachers, young people and their parents for their valuable contribution to the development of this study. references 1. matos mg, gaspar t, simões c. health-related quality of life in portuguese children and adolescents. psicol reflex crit. 2012;25(2):2307. 2. costa sm, vasconcelos m, abreu mhng. [impact of dental caries on quality of life among adults resident in greater belo horizonte, state of minas gerais, brazil]. cien saude colet. 2013 jul;18(7):1971-80. portuguese. 3. wong mcm, lau awh, lam kf, mcgrath c, lu h-x. assessing consistency in oral health-related quality of life (ohrqol) across gender and stability of ohrqol over time for adolescents using structural equation modeling. community dent oral epidemiol. 2011 aug;39(4):325-35. doi: 10.1111/j.1600-0528.2010.00600.x 4. soares ahr, martins aj, lopes mcb, britto jaa, oliveira cq, moreira mcn. [quality of life in children and adolescents: a literature review]. cien saude colet. 2011 jul;16(7):3197-206. portuguese. 5. piovesan c, antunes jlf, mendes fm, guedes, rs, ardenghi tm. influence of children’s oral health-related quality of life on school performance and school absenteeism. j public health dentistry. 2012 spring;72(2):156-63. doi: 10.1111/j.1752-7325.2011.00301.x. 6. brazil. ministry of health of brazil. [sb brazil 2010: national research on oral health: main results]. 2012 [cited 2016 jan 21]. 116p. available from: http://bvsms.saude.gov.br/bvs/publicacoes/pesquisa_nacional_ saude_bucal.pdf. portuguese. 7. mota-veloso i, soares mec, alencar bm, marques ls, ramos-jorge ml, ramos-jorge j. impact of untreated dental caries and its clinical consequences on the oral health-related quality of life of schoolchildren aged 8–10 years. qual life res. 2016 jan;25(1):193-9. doi: 10.1007/ s11136-015-1059-7. 8. rocha racp, goes psa. [comparison of access to oral health services in areas covered and not covered by family health program in campina grande, paraíba, brazil]. cad saude publica. 2008 dec;24(12):2871-80. portuguese. 9. brazilian institute of geography and statistics (ibge). ibge cidades@. rio de janeiro: ibge; 2013 [cited 2016 jan 21]. available from: http:// cidades.ibge.gov.br/painel/painel.php? 10. world health organization (who). basic epidemiological survey of oral health: instruction manual. 4. ed. genebra: who; 1997. 11. xavier a, carvalho fs, bastos rs, caldana ml, bastos jrm. dental caries-related quality of life and socioeconomic status of preschool children, bauru, sp. braz j oral sci. 2012 oct-dec;11(4):463-8. 12. brazilian institute of geography and statistics (ibge). national research by household sampling. rio de janeiro: igbe; 2012 [cited 2016 jan 21]. available from: http://www.sidra.ibge.gov.br/bda/tabela/listabl. asp?z=pnad&o=10&i=p&c=2526. 13. montero-martín j, bravo-pérez m, albaladejo-martínez a, hernándezmartín la, rosel-gallardo em. validation the oral health impact profile (ohip-14sp) for adults in spain. med oral patol oral cir bucal. 2009 jan;14(1):e44-50. 14. soares af, marques me. grow inside: the contact barrier in the adolescent process through rorschach. anal psicol. 2009;27(3):259-67. 15. gomes amm, thomaz ebaf, britto e alves mtss, silva aam, silva ra. [factors associated with the use of oral health services: population-based study in municipalities of maranhão, brazil]. cien saude colet. 2014 feb;19(2):629-40. portuguese. 16. lisbôa ic, abegg c. [oral hygiene habits and use of dental services by adolescents and adults of canoas, state of rio grande do sul, brazil]. epidemiol serv saude. 2006;15(4):29-39. portuguese. 17. baldani mh, mendes ybe, lawder jac, lara api, rodrigues mmas, antunes jlf. inequalities in dental services utilization among brazilian low-income children: the role of individual determinants. j public health dent. 2011 winter;71(1):46-53. 18. davoglio rs, aerts drgc, abegg c, freddo sl, monteiro l. [factors associated with oral health habits and use of dental services by adolescents]. cad saude publica. 2009 mar;25(3):655-67. portuguese. 19. rocha racp, goes psa. comparison of access to oral health services in areas covered and not covered by family health program in campina grande, paraíba, brazil. cad saude publica. 2008 dec; 24(12):2871-80. portuguese. 20. baldani mh, pupo ym, lawder jac, silva ffm, antunes jlf. [individual determinants of recent use of dental services for adolescents and young adults low income]. pesq bras odontopediat clin integ. 2011 jan-mar;11(1):91-8. portuguese. 21. gushi ll, soares mc, forni tib, vieira v, wada rs, sousa mlr. relationship between dental caries and socio-economic factors in adolescents. j appl oral sci. 2005 sep;13(3):305-11. 22. rebelo mab, lopes mc, vieira jmr, parente rcp. dental caries and gingivitis among 15 to 19 year-old students in manaus, am, brazil. braz oral res. 2009 jul-sep;23(3):248-54. 23. moreira pvl, rosenblatt a, passos ia. [prevalence of cavities among adolescents in public and private schools in joão pessoa, paraíba, impact of dental caries on quality of life of adolescents according to access to oral health services: a cross sectional study braz j oral sci. 15(1):1-7 brazil]. cien saude colet. 2007 sep-oct;12(5):1229-36. portuguese. 24. figueira acg, machado fca, amaral ba, lima kc, assunção iv. [oral health of school´s teenager]. holos. 2016; 32(1):161-72. portuguese. 25. cangussu mct, castellanos ra, pinheiro mf, albuquerque sr, pinho c. [dental caries in 12and 15-year-old schoolchildren from public and private schools in salvador, bahia, brazil, in 2001]. pesqui odontol bras. 2002;16(4):379-84. portuguese. 26. frias ac, antunes jlf, junqueira sr, narvai pc. [individual and contextual determinants of the prevalence of untreated caries in brazil]. rev panam salud publica. 2007 oct;22(4):279-85. portuguese. 27. bastos jl, antunes jlf, frias ac, souza mlr, peres kg, peres ma. color/race inequalities in oral health among brazilian adolescents. rev bras epidemiol. 2009;12(3):313-24. 28. narvai pc, frazão p, roncalli ag, antunes jlf. [dental caries in brazil: decline, polarization, inequality and social exclusion]. rev panam salud publica. 2006 jun;19(6):385-93. portuguese. 29. gushi ll, rihs lb, soares mc, forni tib, vieira v, wada rs, et al. [dental caries and treatment needs in adolescents from the state of são paulo, 1998 e 2002]. rev saude publica. 2008 jun;42(3):480-6. portuguese. 30. miotto mhmb, barcellos la, velten db. [evaluation of the impact on 7 impact of dental caries on quality of life of adolescents according to access to oral health services: a cross sectional study quality of life caused by oral health problems in adults and the elderly in a southeastern brazilian city]. cien saude colet. 2012 feb;17(2):397406. portuguese. 31. ravaghi v, ardakan mmm, shahriari s, mokhtari n, underwood m. comparison of the cohip and ohip14 as measures of the oral health-related quality of life of adolescents. community dent health. 2011 mar;28(1):82-8. 32. ferreira ca, loureiro ca, araújo ve. psycometrics properties of subjetive indicator in children. rev saude publica. 2004;38(3):1-7. 33. paredes so, leal júnior os, paredes ao, fernandes jmfa, menezes va. oral health influence on the quality of life of school adolescents. rev bras prom saude. 2015 apr-jun;28(2):266-72. 34. silveira mf, marôco jp, freire rs, martins amebl, marcopito lf. impact of oral health on physical and psychosocial dimensions: an analysis throught structural equation modeling. cad saude publica. 2014 jun;30(6):1169-82. 35. bastos jrm, carvalho es, xavier a, caldana ml, bastos jrm, lauris jrp. dental caries related to quality of life in two brazilian adolescent groups: a cross-sectional randomised study. int dent j. 2012 jun; 62(3):137-43. doi: 10.1111/j.1875-595x.2011.00105.x braz j oral sci. 15(1):1-7 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1537/1190 1/12 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1537/1190 2/12 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1537/1190 3/12 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1537/1190 4/12 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1537/1190 5/12 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1537/1190 6/12 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1537/1190 7/12 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1537/1190 8/12 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1537/1190 9/12 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1537/1190 10/12 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1537/1190 11/12 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1537/1190 12/12 untitled 1http://dx.doi.org/10.20396/bjos.v16i0.8651060 volume 16 2017 e17050 original article 1 orthodontic department, dental faculty, international islamic. university malaysia, pahang, malaysia 2 periodontics department, dental faculty, international islamic. university malaysia, pahang, malaysia 3 periodontics department, dental faculty, international islamic university malaysia, pahang, malaysia 4 basic medical science department, dental faculty, international islamic university malaysia, pahang, malaysia corresponding authors: asst prof dr noraini abu bakar dds(usm), msc orthodontics(london), morth rcs (edinburgh) head of paediatric dentistry, orthodontics and dental public health department dental faculty, international islamic university malaysia kuantan campus, 25200 kuantan, pahang, malaysia email: nor_aini@iium.edu.my phone:600127140094 received: may 22, 2017 accepted: september 20, 2017 saliva leptin levels in tooth movement during initial stage of orthodontic alignment: a pilot study noraini abu bakar1*, wisam kamil2, lina al bayati3, basma ezzat mustafa4 abstract: during orthodontic tooth movement, the early response of periodontal tissues to mechanical stress is an acute inflammatory reaction. mechanical stress from orthodontic appliances is believed to induce cells in the periodontal ligament (pdl) to form biologically active substances, such as enzymes and cytokines, responsible for connective tissue remodeling (nishijima y et al 2006). leptin, a polypeptide hormone has been classified as a cytokine (zhang et al 1994). earlier findings concluded that leptin at high local concentrations protects the host from inflammation and infection as well as maintaining bone levels. it has been also suggested that leptin plays a significant role in bone formation by its direct effect on osteoblasts (alparslan et al 2010). this pilot study aimed to study leptin in saliva and its association with tooth movement during initial orthodontic alignment. aim: to determine if there are any differences in saliva leptin level before and after orthodontic alignment. methods: ten orthodontic patients (7 girls and 3 boys; mean age, 16.76 ± 1.1 years) with crowding (up to 5mm) that required orthodontic fixed appliances, on a non-extraction basis as part of the treatment plan, were recruited in this longitudinal study. orthodontic study models were constructed at baseline and at 6weeks after orthodontic treatment commenced. full fixed orthodontic appliances with initial 0.014” nickel titanium archwire placed. the amount of crowding was measured, before and after initial alignment with an electronic digital caliper (max-cal, japan micrometer ltd, tokyo, japan) with an accuracy of up to 0.01mm. unstimulated morning saliva sample were collected at all visits, after at least an 8-hour period of fasting and no-toothbrushing. after centrifugation (4000x g;10min), the samples were stored at -25c and tested using leptin abnova lep human elisa kit (ka3080) which was subsequently mailto:nor_aini@iium.edu.my 2 abu bakar et al. analyzed. subjects’ periodontal health status was also monitored throughout the study. ethical approval (id irec 262) was received on 7th april 2014 from international islamic university malaysia research ethics committee (irec). results: leptin concentration in saliva was significantly decreased in a time-dependant manner (t(9)=8.60, p<0.001), from before orthodontic treatment (7016.45± 425.15 pg/ml) and 6 weeks after bond-up (4901.92± 238.64 pg/ml). conclusion: leptin concentration in saliva is decreased during orthodontic tooth movement in initial alignment stage. keywords: leptin, saliva, tooth movement introduction leptin is a highly hydrophilic protein that circulates in plasma as a 16-kda protein. it is produced in adipose tissue and also recently described to be synthesized by placental tissue. plasma concentration of leptin is positively correlated to body fat mass, and administration of recombinant leptin to mice indicates that leptin participates in the regulation of food intake and energy expenditure. leptin is released primarily by adipose tissue, and it is strongly correlated with body weight and body fat mass1. leptin has been reported to influence various biological mechanisms, including the immune and inflammatory response, haematopoiesis, angiogenesis, bone formation, and wound healing2; it also has an anti-inflammatory action3. it has been reported that serum leptin levels were increased by surgical stress and acute sepsis. in these states, increased stress-induced hormones and cytokines, such as cortisol, tnf-α, il-1, and il-6 have been thought to cause the increment of serum leptin level4. since the discovery of this relatively new hormone, many studies have been conducted to know more about its role in various fields. this includes leptin’s role in regulating bone metabolism that was first described in year 20005. leptin was also associated with inflammatory response including periodontitis. it has been suggested that the salivary leptin concentration significantly changed in chronic periodontitis patients and may reflects the disease activity6. leptin was classified as a cytokine7 that plays a role in the host defense immune system where stimulates the immune system by enhancing pro-inflammatory cytokine production and phagocytosis by macrophages1. this was further supported by the study of bozkurt 2006, who suggested that an elevated level of leptin in gingival crevicular fluid of healthy periodontium prolonging the life span of human primary osteoblasts by inhibiting apoptosis8. it is assumed that leptin has a role in protecting gingival tissues9, leptin stimulates the immune system and enhances bone formation by acting directly on osteoblasts10. as periodontal disease progresses, the protective role of leptin on the gingiva is lost owing to a decrease in the leptin level. during orthodontic tooth movement, the early response of periodontal tissues to mechanical stress is an acute inflammatory reaction11. remodeling process (resorption and apposition) takes place in periodontal tissues induced by the changes in the stress-strain distribution in the periodontium after the 3 abu bakar et al. application of orthodontic forces12. furthermore, a local damage-repair process with inflammation-like reactions, including high vascular activity with many leukocytes and macrophages and involvement with the immune system may occur during orthodontic tooth movement5. changes in the stress-strain distribution in the periodontium after the application of orthodontic forces trigger remodelling processes. these forces compress the pdl fibers and reduce the pdl space in the pressure area. at the tension site, pdl fibers are stretched, and orthodontic force results in widening of the periodontal membrane13. study of leptin therefore is a useful guide to determine its relationship with tooth movement in both tension and pressure sites and the role of this cytokine in controlling the local inflammation around the tooth. detection of the leptin level in gcf at sites under orthodontic movement had been tested and it was found that the concentration of leptin in gcf is decreased by orthodontic tooth movement12. this pilot study aimed to venture leptin in saliva and its association with tooth movement during initial orthodontic alignment. the specific aim is to determine if there is any differences in saliva leptin level before and after orthodontic alignment material and methods: a convenient sampling of ten orthodontic patients (7 girls and 3 boys; mean age, 16.76 ± 1.1 years) were selected according to the inclusion criteria. ethical approval with id no irec 262 received from international islamic university malaysia research ethics committee (irec). inclusion criteria were: • patients with mild to moderate crowding (up to 5mm) malocclusion, requiring orthodontic treatment with fixed appliances on a non-extraction basis • good health • normal body mass index, according to the who chart (bmi of 18.5-22.9) • no history of the use of anti-inflammatory drugs within the month preceding the sample collection • no history of the use of antimicrobial therapy within the previous 6 months • healthy periodontal tissues with generalized probing depths of ≤ 2 mm, with minimal bleeding and no sign of attachment loss • no radiographic evidence of periodontal bone loss patients were identified in orthodontic specialist clinic, international islamic university malaysia. patients who met the criteria were given ample information about the study in addition to the research information sheet. informed consent obtained from patients who agreed to participate and patients’ rights were protected according to good clinical practice guideline. subjects that met the inclusion criteria were seen in 3 appointments as shown in table 1. 4 abu bakar et al. morning, unstimulated whole saliva (5ml) sample were collected by a modified draining method14 as a diagnostic fluid, at all 3 visits. patients were required to fast from midnight till the time the saliva samples were taken at 8am (after at least 8 hours of fasting). patients also were not allowed to brush the morning of the appointment as to avoid risks of gingival trauma/bleeding during sample collection. participants were asked to expectorate into disposable tubes every 30 sec over a period of 5 min. after centrifugation(4000x g;10min), the samples were stored at -25c and tested using leptin abnova lep human elisa kit (ka3080). at baseline visit, all the clinical periodontal parameters were measured with a goldman/fox williams probe calibrated in millimetres by one trained dentist. these parameters include, bleeding on probing (bop) and the plaque control index (ps), while probing pocket depth (ppd) was calculated as the measurement from gingival margin to the base of probing crevice. this was to ensure that subjects were free from periodontal diseases and as a tool to monitor periodontium status throughout the study. as part of our routine pre orthodontic protocol, we implement professional plaque control, and all patients received full mouth supragingival supra and subgingival scaling using piezoelectric scaler with oral hygiene instruction including brushing twice a day using modified bass brushing technique. all the clinical periodontal parameters have been re-evaluated 6 weeks after the scaling session and re-assessed 6 weeks after the orthodontics treatment. dental impressions for study models were taken before and after orthodontic treatment commenced. full fixed orthodontic appliances(with mbt prescriptions) bonded with 0.014” nickel titanium wires ligated on both upper and lower arches, 6 weeks after scaling was done. amount of crowding was evaluated by using an electronic digital caliper (max-cal, japan micrometer ltd, tokyo, japan) with an accuracy of 0.01mm over the occlusal surface of study models to measure the mesio-distal width of misaligned teeth table 1. procedures done and time frame of the three visits. visit time frame actions 1 0 week • basic periodontal examination • first unstimulated saliva sample taken (baseline) • scaling and polishing • oral hygiene instructions given • impressions for baseline orthodontic study model taken 2 6 weeks after visit 1 • basic periodontal examination • second unstimulated saliva sample taken (using felcon sterile tube50ml) • upper and lower orthodontic fixed appliances bonded with 0.014” nickel titanium wires ligated on both upper and lower arch. 3 6 weeks after visit 2 • basic periodontal examination • third unstimulated saliva sample taken • impressions for second orthodontic study model taken 5 abu bakar et al. and available space in the archform selected15. the same technique was applied to measure the amount of tooth movement 6 weeks after braces placement. the total amount of tooth movement was recorded in mm. data was analyzed using ibm spss statistics for windows, version 20.0 and a significance level was set at 95% (p ≤ 0.05).data was presented using mean and standard deviation (sd). repeated measures anova test was used to test mean differences between the three visits, while the paired t-test was used to detect mean differences between two visits. p <0.05 was considered statistically significant. results changes in periodontal parameters between the three visits the clinical periodontal parameters of 10 recruited patients were scored at the baseline visit and were 89.58(8.63), 61.36(12.20) and 1.91(0.18) for ps, bop and ppd respectively. all patients’ periodontal scores were slightly decreased at 2nd visit after receiving nonsurgical periodontal treatment that includes supra and subgingival scaling using piezoelectric scaler with oral hygiene instruction including brushing twice a day using modified bass brushing technique, but didn’t reach the statistical significance as shown in table 2. on the other hand the ps 90.30(8.78) and bop 65.82(9.56) were increased after the orthodontics treatment compared to baseline and 2nd visit, but p-value was statistically not significant. this result showed that all the patients’ periodontium remains healthy throughout the study thus eliminating the possibility of leptin changes due to periodontitis. changes in leptin level between the three visits figure 1 shows the saliva leptin level between the three visits. as shown in table 3, there was a statistically significant decrease in leptin level between visit 3 (m = 4901.923, sd = 754.657) and visit 1 (m = 7016.457, sd = 1344.468); t(9) = 8.601, p = 0.000. there was also a statistically significant decrease in the number of leptin level between visit 1 (m = 7016.457, sd = 1344.468) and visit 2 (m = 5018.528, sd = 901.327); t(9) = 8.312, p = 0.000. however, no significant difference was found between visit 2 and visit 3 (t(9) = 1.081, p > 0.05). table 2. changes in periodontal parameters from baseline to 6 weeks after bond-up variables baseline (visit 1) 2nd visit (visit 2) p-value¶ 3rd visit (visit 3) p-value§ p-value¥ ps 89.58(8.63) 86.244(4.10) 0.320 90.30(8.78) 0.271 0.394 bop 61.36(12.20) 60.085(13.63) 0.759 65.82(9.56) 0.170 0.317 pdv 1.91(0.18) 1.792(0.22) 0.101 1.83(0.20) 0.475 0.174 data are given as mean (standard deviation) unless stated otherwise. ps: percentage of sites with plaque scores bop: percentage of sites with bleeding on probing ppd: probing pocket depth ¥: repeated measures anova test among three visits ¶: paired t-test (between baseline and 2nd visit) §: paired t-test (between 2nd and 3nd visits) 6 abu bakar et al. leptin concentrations decreased in a time-dependent manner during the study period. when compared with baseline, the decrease was statistically significantly 6 weeks after orthodontic alignment. changes in tooth movement after initial alignment there was significant tooth movement between 1st visit and 3nd visit (p <0.0001) as shown in table 4. on average, the tooth movement were 2.20 mm (95% ci: 1.6357, 2.7643) 6 weeks after patients in initial alignment stage. table 4. changes in tooth movement after initial alignment one-sample statistics 95% confidence interval of the differencemean std. deviation t df sig. (2-tailed) tooth movement 2.2000 .78881 8.820 9 .000 (1.6357,2.7643) table 3. saliva leptin level differences between the three visits mean std. deviation std. error mean t df sig. (2-tailed) pair 1 leptine 1 leptine 2 1997.92900 734.59400 232.29902 8.601 9 .000 pair 2 leptine 2 leptine 3 116.60500 341.15040 107.88123 1.081 9 .308 pair 3 leptine 1 leptine 3 2114.53400 804.44456 254.38771 8.312 9 .000 data was analysed using paired t-test by the statistical package for the social sciences (spss 20). figure 1. saliva leptin level between the three visits leptin level pg/ml leptin level pg/ml visit 1 visit 2 visit 3 8000 7000 6000 5000 4000 3000 2000 1000 0 7 abu bakar et al. discussion the result shows that, similar to leptin in gingival crevicular fluid, leptin in saliva also decreased in tooth movement. it does in some way potentially relate leptin as one of the mediators associated with tooth movement. this study opens a path to a bigger study with a larger sample size to further understand the role of leptin in orthodontics. to certify that this leptin changes happen only due to tooth movement and not periodontal issues, in methodology, we emphasized on professional plaque control regime throughout the sample taking, as orthodontic treatment may negatively affect the periodontal health status16-19. as leptin was observed among patients with irreversible periodontal disease (periodontitis)20 , patients with periodontitis were excluded from the study. constant monitoring of the periodontium health was also done to ensure no patients develop periodontitis at the duration of study. for saliva collection, the use of unstimulated saliva was implemented over the stimulated one to overcome the modulation of the fluid ph since the later provide less suitable saliva for diagnostic applications due to dilution in the concentration of the salivary protein of interest21 two conclusions can be drawn: • the concentration of leptin in the saliva is significantly decreased in time dependent manner in orthodontic tooth movement in alignment stage. • leptin may be one of the mediators associated with orthodontic tooth movement. the knowledge gain from this study will enable us to have a better idea of the relationship between leptin and tooth movement and the role of this cytokine in controlling the local inflammation around the tooth. acknowledgement: we would like to acknowledge international islamic university malaysia for grants given to carry out and publish this study: edw-b-14-103-0988 and rigs16-139-0303. references: 1. ahima rs, flier js. leptin. annu rev physiol. 2000;62:413-37. 2. włodarski k, włodarski p. leptin as a modulator of osteogenesis. ortop traumatol rehabil. 2009 jan-feb;11(1):1-6. 3. fantuzzi g, faggioni r. leptin in the regulation of immunity, inflammation, and hematopoiesis. j leukoc biol. 2000 oct;68(4):437-46. 4. wallace am, sattar n, mcmillan d. the co-ordinated cytokine/hormone response to acute injury incorporates leptin. cytokine. 2000 jul;12(7):1042-5. 5. ducy p, amling m, takeda s, priemel m, schilling af, beil ft, et al. leptin inhibits bone formation through a hypothalamic relay: a central control of bone mass. cell. 2000 jan 21;100(2):197-207. 6. purwar p1, khan ma, mahdi aa, pandey s, singh b, dixit j, sareen s. salivary and serum leptin concentrations in patients with chronic periodontitis. j periodontol. 2015 apr;86(4):588-94.doi: 10.1902/jop.2014.140581. 8 abu bakar et al. 7. zhang y, proenca r, maffei m, barone m, leopold l, friedman jm. positional cloning of the mouse obese gene and its human homologue. nature. 1994 dec 1;372(6505):425-32. 8. bozkurt fy, yetkin ay z, sutçu r, delibas¸ n, demirel r. gingival crevicular fluid leptin levels in periodontitis patients with long-term and heavy smoking. j periodontol. 2006 apr;77(4):634-40. 9. karthikeyan bv, pradeep ar. gingival crevicular fluid and serum leptin: their relationship to periodontal health and disease. j clin periodontol. 2007 jun;34(6):467-72. 10. reseland je, syversen u, bakke i, qvigstad g, eide lg, hjertner o, et al. leptin is expressed in and secreted from primary cultures of human osteoblasts and promotes bone mineralization. j bone miner res. 2001 aug;16(8):1426-33. 11. nishijima y, yamaguchi m, kojima t, aihara n, nakajima r, kasai k. levels of rankl and opg in gingival crevicular fluid during orthodontic tooth movement and effect of compression force on releases from periodontal ligament cells in vitro. orthod craniofac res. 2006 may;9(2):63-70. 12. alparslan d, nihat k,tugba a, f nesibe, meltem z, caglar b. leptin levels in gingival crevicular fluid during orthodontics tooth movement angle orthod. 2010 may;80(3):504-8. doi: 10.2319/072109-402.1. 13. thilander b, rygh p, reitan k. tissue reactions in orthodontics. in graber 14. tm. orthodontics: current principles and techniques. saint louis: mosby; 2000. p. 117-56. 15. malamud d. saliva as a diagnostic fluid. bmj. 1992 jul 25;305(6847):207-8. 16. o’higgins ea, lee rt. how much space is created from expansion or premolar extraction? j orthod. 2000 mar;27(1):11-3. 17. zachriss bu, alnaes l. periodontal condition in orthodontically treated and untreated individuals. i. loss of attachment, gingival pocket depth and clinical crown height. angle orthod. 1973 oct;43(4):402-11. 18. alfuriji s, alhazmi n, alhamlan n, al-ehaideb a, alruwaithi m, alkatheeri n, et al. the effect of orthodontic therapy on periodontal health: a review of the literature. int j dent. 2014;2014:585048. doi: 10.1155/2014/585048. 19. kouraki e, bissada nf, palomo jm, ficara aj. orthodontic attachments on the gingival health of permanent second molars. am j orthod dentofacial orthop. 1991 oct;100(4):337-40. 20. kaufman e, lamster ib. gingival enlargement and resolution during and after orthodontic treatment. n y state dent j. 2005 jun-jul;71(4):34-7. 21. purwar p1, khan ma, mahdi aa, pandey s, singh b, dixit j, sareen s. salivary and serum leptin concentrations in patients with chronic periodontitis. j periodontol. 2015 apr;86(4):588-94. doi: 10.1902/jop.2014.140581. 22. rudneyjd, kajander kc, smith qt. correlations between human salivary levels of lysozyme, lactoferrin, salivary peroxidase and secretory immunoglobulin a with different stimulatory states and over time. arch oral biol. 1985;30(11-12):765-71. https://www.ncbi.nlm.nih.gov/pubmed/?term=nishijima%20y%5bauthor%5d&cauthor=true&cauthor_uid=16764680 https://www.ncbi.nlm.nih.gov/pubmed/?term=yamaguchi%20m%5bauthor%5d&cauthor=true&cauthor_uid=16764680 https://www.ncbi.nlm.nih.gov/pubmed/?term=kojima%20t%5bauthor%5d&cauthor=true&cauthor_uid=16764680 https://www.ncbi.nlm.nih.gov/pubmed/?term=aihara%20n%5bauthor%5d&cauthor=true&cauthor_uid=16764680 https://www.ncbi.nlm.nih.gov/pubmed/?term=nakajima%20r%5bauthor%5d&cauthor=true&cauthor_uid=16764680 https://www.ncbi.nlm.nih.gov/pubmed/?term=kasai%20k%5bauthor%5d&cauthor=true&cauthor_uid=16764680 oral sciences n3 original article braz j oral sci. july | september 2015 volume 14, number 3 enterococcus spp. isolated from root canals with persistent chronic apical periodontitis in a chilean population gabriela sánchez-sanhueza1, gerardo gonzález-rocha2, mariana dominguez2, helia bello-toledo2 1universidad de concepción, faculty of dentistry, department of restorative dentistry, concepción, chile 2universidad de concepción, faculty of biological sciences, department of microbiology, research laboratory on antibacterial agents, concepción, chile correspondence to: helia bello-toledo laboratorio de investigación en agentes antibacterianos departamento de microbiología facultad de ciencias biológicas universidad de concepción, concepción, chile phone: +56 41 2661201 fax: +56 41 2245975 e-mail: hbello@udec.cl abstract aim: to isolate and identify in a chilean population, enterococcus spp. from root canals with persistent chronic apical periodontitis (cap) and to investigate the potential correlation between the bacteria and the observed clinical features. methods: twenty patients with indication for endodontic retreatment due to persistent cap were selected. data from patient general health and dental clinical history were recorded. during retreatment, a microbial sample was obtained from the root canal and inoculated in a selective enterococcus medium. using bacterial cultivation methods, bacterial isolates belonging to the genus enterococcus were identified. the relationship between the number of colony-forming units of enterococcus spp. and patient clinical data was assessed statistically by the pearson chi square and fisher exact tests. finally, a polymerase chain reaction (pcr) assay to determine the most prevalent species of enterococcus spp. was conducted in the clinical samples, and the results were analyzed by a proportion comparison test. results: enterococcus spp. strains were isolated in 70% of the patients. most of them (98.8%) accounted for enterococcus faecalis and only 1.2% for enterococcus faecium. a high frequency of e. faecalis was found in teeth with inadequate endodontic treatment or dental crown restorations. conclusions: this study concluded that e. faecalis is prevalent in root canals with persistent cap in a chilean population. e. faecium as found in a single case with the poorest root canal filling. further studies are still required to investigate the presence of other species, which may be linked to persistent chronic apical periodontitis. keywords: periapical granuloma; enterococcus faecalis; enterococcus. introduction one of the prevalent causes of lesions that appear or remain after endodontic treatment is the presence of microorganisms or contamination in the root canal system1. several authors reported that bacteria could survive within the root canal even after careful mechanical-chemical preparation2. such bacterial presence may lead to clinical symptoms of persistent chronic apical periodontitis (cap), which has high prevalence in some countries, ranging from 40% to 61%3-4. the treatment of persistent cap aims the removal of root canal filling material, disinfection of canals with irrigants and use of intra canal pastes such as calcium hydroxide. even so, sometimes the microorganisms persist even after treatment5. in cases of primary endodontic infections, there is a higher bacterial load than in secondary infections6-7. after biomechanical preparation of root canals, braz j oral sci. 14(3):240-245 http://dx.doi.org/10.1590/1677-3225v14n3a13 received for publication: may 31, 2015 accepted: september 29, 2015 241241241241241 the bacterial load is reduced by at least 95% and the remaining viable bacteria may be the cause of refractory lesions8. teeth with endodontic failure have been associated with the presence of enterococcus species9-10. these bacteria probably reach the root canal system after treatment, but the source is still unclear. enterococci do not seem to be colonizers of the oral cavity, unless there is a preferable site, such as a necrotic or obturated root canal without sealing. probably, the transient microorganisms in the oral cavity and changes in the microenvironment create favorable conditions for secondary or refractory endodontic infection11. enterococcus faecalis is a commensal organism, part of the normal microbiota of the digestive system, but it can also be an opportunistic human pathogen. moreover, e. faecalis has the ability to maintain its viability for twelve months without additional nutrients. this bacteria is most frequently isolated in cap cases after endodontic tooth treatment, but it may also be found in cases of necrotic pulps in very few cases12. viable e. faecalis strains could be trapped inside the root canal in the filling material and provide a niche for subsequent infection in the long term13. in chile, there are yet no studies about the presence of this bacterial genus in cap. the aim of this study was to identify enterococcus spp. from the root canals of teeth that had previous endodontic treatment with diagnosis of cap in a chilean population and to correlate its presence with the patient’s clinical features. material and methods patient selection twenty adult patients, aged eighteen and older, were chosen. all were treated at the faculty of dentistry, university of concepción, chile, in november 2013, with endodontic retreatment indicated in the mandibular and maxillary canine, premolar, first or second molar. teeth that had endodontic treatment two or more years before and showed radiographic evidence of periapical disease were considered to require retreatment. the radiolucent area size was recorded, and classified as widened periodontal ligament space (<1 mm) or periapical lesion (≥1 mm). pre-treatment quality was recorded after radiographic examination. root canal fillings with clearly visible voids in the filling material were classified as inadequate. patients who had antibiotic treatment during the preceding three months and whose teeth could not be fully isolated with a rubber dam and/or with periodontal disease in the selected or adjacent teeth were excluded. the following characteristics for each patient and correlated with the microbial findings were recorded: age, sex, tooth type, and time when the previous treatment was performed. clinical signs and symptoms included the quality of the dental crown restoration, spontaneous pain, pain history, sensitivity to percussion, pain on palpation, mobility, history of antibiotic therapy or any other relevant medication9. the ethics committee of the faculty of dentistry at the university of concepción approved the study protocol (c.i.y.b. nº08/14). before sampling, patients understood and gave written informed consent, according to the declaration of helsinki. sampling procedure a single trained operator collected samples under strict asepsis, as described below. the tooth surface was cleaned with pumice stone powder and isolated with a rubber dam. the tooth and the operation field were disinfected with 3 ml of 5.25% sodium hypochlorite solution, which was then absorbed with sterile gauze. the solution was inactivated with 5% sodium thiosulphate in order to avoid interference with the bacteriological sampling14. coronal restorations were removed using high-speed sterile carbide drills. to expose the root filling, the access cavity was prepared with sterile drills (denstply, maillefer, switzerland) without water spray but under manual irrigation with sterile saline solution (b. braun, barcelona, spain). then, the operating field, including the pulp chamber, rubber dam and clamp, was cleaned with 2.5% sodium hypochlorite solution (clorox regular bleach, oakland, ca, usa), and inactivated with 5% sodium thiosulphate (hash, loveland, co, usa). to control the effectiveness of disinfection, samples from the operating field were obtained and the tooth crown was disinfected with two f1 paper points (denstply, maillefer, switzerland). next, they were transferred to a tube containing 3ml of trypticase soy broth (tsb) (difco, detroit, mi, usa) and incubated for 72 h at 35 °c. the root canal filling was removed with hedström files, k and/or h type, and gatesglidden drills (denstply, maillefer, switzerland). protaper nickel-titanium retreatment instruments d1 d3 (denstply, maillefer, switzerland) were also used. solvent was not used in order to avoid harm to bacterial cells. the working length was set at 1 mm from the radicular apex. radiographs were taken to ensure that all the filling material had been removed. subsequently, 5 tsb drops were placed in the root canal and a sterile endodontic file was introduced at a level of approximately 1 mm from the apex of the tooth. the content from the root canal was absorbed with 4 sterile paper points. each paper point was maintained in position inside the duct at the established working length for one minute and then transferred into a tube with 1 ml tsb15. from this sample, an aliquot was taken to perform bacterial counts and the remainder was incubated for 24-48 h. if the plate count was negative, the remainder of the sample was allowed to seed on a kf streptococcus agar plate (merck, d-61 darmstadt, germany) to definitively rule out the presence of enterococcus spp.5,16. bacterial count the bacterial count was carried out in triplicate by seeding onto the surface of kf streptococcus agar plates (merck, d-61 darmstadt, germany). plates were incubated for 72 h at 37 °c. subsequently, six presumptive enterococcus spp. colonies were chosen from each patient sample. metabolic and biochemical strain characterization to identify the presumptive enterococcus spp. colonies, braz j oral sci. 14(3):240-245 enterococcus spp. isolated from root canals with persistent chronic apical periodontitis in a chilean population conventional tests, such as gram staining, growth in 6.5% nacl broth, esculin hydrolysis and the arabinose fermentation were carried out17. each enterococcus spp. strain was stored at -80 ºc in a tsb and 50% glycerol mixture at a ratio of 2:1. molecular identification by pcr identification at the species level was performed by pcr using the specific primers 5'-atc aag tac agt tag tct t-3' and 5'-acg att caa agc taa ctg-3' targeted against the ddl e. faecalis gene, 5'-gca agg ctt ctt aga ga-3' and 5'-cat cgt gta agc taa ctt c-3' targeted against ddl e. faecium gene, and 5'-ggt atc aag gaa acc tc3' and 5'-ctt ccg cca tca tag ct-3' targeted against the vanc-1 e. gallinarum gene18-19. the adn template was obtained according to sepulveda et al. (2002)18. briefly, four enterococcus spp. colonies after 24 h culture were suspended in 200 µl sterile distilled water (sdw) and centrifuged for 5-10 s at 14,000 rpm. then, 2 µl of the supernatant from the suspension were added to a reaction mixture containing 1 µl 50 mm mgcl2 (invitrogen, carlsbad, ca, usa), 2.5 µl 1.25 mm dntps (invitrogen, carlsbad, ca, usa), 2.0 µl of each primer (20 pmol/µl) (integrated dna technologies, coralville, ia, usa), 2.5 µl of 10x pcr buffer (invitrogen, carlsbad, ca, usa), 0.15 µl of recombinant taq dna polymerase (5 u/µl) (invitrogen, carlsbad, ca, usa) and adequate sdw to complete a 25 µl reaction volume. escherichia coli atcc 25922, e. faecalis atcc 29212, e. faecium vre 11, and e. gallinarum 132674 strains, available at the culture collection department of microbiology at the university of concepción, were used as controls in pcr. the amplification was performed in a veriti 96-well thermal cycler (applied biosystems, foster city, ca, usa) under the following program: 2 min at 94 ºc and 30 cycles at 94 °c, 1 min at 54 °c and 1 min at 72 ºc, with a final step of 10 min at 72 ºc. amplicons were separated by electrophoresis in 1.5% agarose gel at 100 v for 30-45 min in 0.5x tae buffer and stained by ethidium bromide (0.5 µg/ml) for 15 min, followed by washing in sdw for 15 min. dna bands were visualized with a uv white light transilluminator (vilber lourmat, torcy, z.i. sud, france)15,18. later, gels were photographed with a kodak id lpu 120 camera (rochester, ny, usa) and analysed by the id 3.02 kodak digital science software (rochester, ny, usa). data analysis prevalence of enterococcus spp. was recorded as percentage of positive cases. the relationship between the presence of e. faecalis and the clinical variables was analysed with the pearson, chi-square and fisher’s exact tests using spss version 15 (spss inc., chicago, il, usa). the results of pcr assay to detect e. faecalis in clinical samples were analysed with the proportion comparison z-test, adopting a 0.05 significance level. results presumptive colonies of enterococci were found on kf streptococcus agar plate in 14 out of 20 samples obtained from the root canals. the colonies were circular with convex elevation and smooth margins, deep red and 1 mm diameter (figure 1). the bacterial counts ranged from zero cfu/ml to 2.1x105 cfu/ml. they represent the distribution of e. faecalis countings over the samples (table 1). after regrowth time, aliquots seeded again in kf agar, reporting some colonies (few) not characteristic to enterococcus, presumably positive for other microorganism. sample median minimum maximum (cfu/ml) (cfu/ml) (cfu/ml) 1 0 0 0 2 2 x 101 0 9 3 9 0 9 4 1.3 x103 1.3 x 102 1.5 x103 5 1.4 x 103 1.5 x 102 1.4 x 103 6 9 0 9 7 1.1 x 103 9 x 101 1.3 x 103 8 3.4 x 103 3.5 x 102 7 x 103 9 8.5 x 104 7.9 x 104 9.1 x 104 10 4 x 101 0 4 x 101 11 0 0 0 12 2.1 x 105 2 x 105 2.1 x 105 13 0 0 0 14 0 0 0 15 9 0 9 16 9 0 9 17 0 0 0 18 8 x 101 5 x 101 10 x 101 19 0 0 0 20 9 0 9 table 1: table 1: table 1: table 1: table 1: counts of e. faecalis from root canals with persistent chronic apical periodontitis cfu= colony forming unit fig. 1: enterococcus spp. colony forming units (cfus) on kf agar plate (10-3 dilution). 242242242242242 braz j oral sci. 14(3):240-245 enterococcus spp. isolated from root canals with persistent chronic apical periodontitis in a chilean population 243243243243243 phenotypic identification indicated that 83/84 (98%) selected strains corresponded to e. faecalis, which was confirmed by pcr, as amplified bands of 941 bp were obtained with the specific primers for this species in all assays (figure 2). e. faecium (2%) was the only strain with different characteristics identified through phenotypic and molecular tests. in this case, a 550 bp amplification product was obtained using specific primers for this species (figure 3). regarding the clinical aspects, no significant differences were found between gender, age and medical history variables. most teeth with presence of enterococcus spp. were maxillary and posterior. for the variable related to time after previous endodontic treatment, increased presence of enterococcus spp. was observed in the range of two to five years. for the variable regarding quality of root canal filling and dental crown restorations, a high microorganism frequency was found in treatments labelled as “inadequate”. in relation to the clinical evaluation, there was a direct association between the absence of signs and symptoms and fig. 3: identification by pcr of e. faecium isolated from patient 6. lane 1: patient 6 strain 3 with primers for e. faecalis. lane 2: patient 6 strain 3 with primers for e. faecium. lane 3: patient 6 strain 3 with primers for e. gallinarum. lane 5: positive control (e. faecalis atcc 29212). lane 6: positive control (e. faecium vre 11). lane 7: positive control (e. gallinarum 132674). fig. 2: identification by pcr of enterococcus spp. isolated from patient 6. lane 1 to 6: six strains obtained from patient 6. lane 7: positive control (e. faecalis atcc 29212). lane 8: negative control: e. coli atcc 25922. lane 9: positive control (e. gallinarum 132674). lane 10: positive control (e. faecium vre 11). the presence of enterococcus spp. for the periapical variable, there was no association between the lesion size and the count. however, no statistically significant relationship was found between the clinical variables and bacterial count. discussion due to the physical limitations of the root canal system, obtaining a representative sample from this site is not an easy task, and it is even more difficult in patients with an indication for retreatment, in which the number of microorganisms may be low and a significant number of microbial cells can be lost during root canal desobturation procedures. as a result, the number of bacterial cells in the sample can fall below the detection rate of the isolation method and a particular species’ prevalence may be underestimated20. this was one limitation of the present study, in which the impossibility of using liquid solvents made the surgical procedure last for three hours in order to completely braz j oral sci. 14(3):240-245 enterococcus spp. isolated from root canals with persistent chronic apical periodontitis in a chilean population 244244244244244 remove all root canal filling material. this could have influenced those cases that were negative for enterococci. the appearance of some colonies (few) not characteristic to enterococcus can be explained because in the kf streptococcus agar may grow other species like listeria monocytogenes, streptococcus bovis group, pediococcus and staphylococci. other organisms (e.g., micrococci, candida, corynebacteria and gram-negative bacteria) may appear as clear small colonies or produce growth traces. a study carried out to compare the detection rate of e. faecalis from cultivation and pcr test from the same sample found that 46.6% of the sampled canals had e. faecalis14. in a lithuanian population study, 56% of the cases were reported to be positive for e. faecalis, using only culture techniques for identification21. in this study, 45% of the samples were positive for enterococcus spp. using culture techniques. however, performing sample regrowth and subsequent seeding again on kf streptococcus agar plates, the detection percentage yielded up to 70%, depending on the total number of samples, which was confirmed by the pcr test; which is consistent with previous studies. it should be underscored that the sample size in the present case is smaller than in other studies and it may have influenced the higher percentage obtained. the authors know that sample size calculation may determine the exact number of samples is required to strictly determine the prevalence of enterococcus spp. the authors developed a descriptive, cross-sectional study. relative frequency enterococcus spp isolated from root canals was evaluated in a limited time. in this case, the sample was not probabilistic. it was taken depending on availability of filed cases in a given time. this limited the number of sampled patients. the prevalence of e. faecalis and its association with clinical findings among patients in beijing, china, showed that root canal treatments displaying unsatisfactory obturation presented a greater diversity of cultivable species than those with satisfactory obturation 22. those canals probably provided more space and nutrition than wellobturated canals. in fact, in that study, e. faecalis was more frequent in the canals than in saliva, but this was not the aim of our study. on the contrary, the well-obturated canals maintained an obliged anaerobic environment, which does not favor the survival and growth of e. faecalis. in that chinese study, as well as in the present study, no association was found between these clinical features and the prevalence of e. faecalis in radicular canals, which is also attributed to the low sensitivity of fisher’s exact test in small samples22. e. faecalis has been commonly found in filled canals with radiographic evidence of peri-radicular periodontitis or without injury than in those with a radiolucent area, suggesting that the entry of bacteria may occur after filling, which is consistent with the findings of this study23. the observation of similar percentages of e. faecalis leakage, regardless of the obturation technique, reinforces this idea24. confirmation of the identity of e. faecalis by pcr has proven to be more sensitive than conventional e. faecalis culture methods. in this study, the use of a selective medium (kf streptococcus agar) and some biochemical tests enabled us to focus on the identification of this bacterium in relation to other species. in this case, there was no presence of e. faecalis in 30% of the samples in comparison with other reports of 6.8%25 to 20%21 of teeth. combining molecular and culture techniques, including biochemical profile is most likely the best available method to provide a comprehensive information on the bacterial populations associated with endodontic infections. ultimately, the observation that 98.8% of the strains were e. faecalis versus the 1.2% that turned out to be e. faecium was a probable outcome, considering the results of a study on non-dental clinical isolates from a hospital environment in a chilean population18. it should be stressed that the most relevant clinical characteristic of one of the patients, from whom the strain of e. faecium was isolated, was that he presented the worst quality of obturation, which was evident in the root canal filling material, the absence of dental crown restorations and sensitivity to palpation. however, his initial count was <10 cfu/ml as an estimated value. clearly, further studies are required to analyse other enterococci populations that may be linked to the presence and establishment of refractory chronic apical periodontitis during or after endodontic treatment, and which may form a multi-species biofilm. e. faecalis has been regarded for more than 30 years as a leading cause of endodontic failures. however, this assertion was placed in doubt based on recent publications reporting that other species, such as streptococci, may be predominant pathogens associated with persistent endodontic infections. the difference may be due to different methodological approaches for sampling and detection, different clinical conditions or socio-geographical differences in the studied subjects6,18,26. finally, it is necessary to advocate for a comprehensive approach to find new antibacterial substances that completely eradicate the microorganisms from the root canal system, which in its genesis is formed to maintain sterility27. this study has shown that there is a high prevalence of e. faecalis in root canal-treated teeth in a chilean population. further studies are required to analyse the presence and role of other species, which may be linked to refractory chronic apical periodontitis. acknowledgements the authors wish to acknowledge the support of the faculty of dentistry of the university of concepción, the public health institute of chile for supplying the strain of e. faecium vre 11 (vanb) and the research laboratory on antibacterial agents of the faculty of biological sciences at the university of concepción. this study was supported by the scholarship “conicyt pcha/national doctorate/ 2013-folio 21130022”, for phd in sciences, mention in microbiology, university of concepción, chile. references 1. ricucci d, siqueira jf jr. anatomic and microbiologic challenges to achieving success with endodontic treatment: a case report. j endod. 2008; 34: 1249-54. braz j oral sci. 14(3):240-245 enterococcus spp. isolated from root canals with persistent chronic apical periodontitis in a chilean population 245245245245245 2. rasimick b, shah r, musikant b, deutsch a. bacterial colonization of root canal 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731-4. 18. sepulveda m, bello h, ruiz m, hormazábal f, dominguez m, gonzález g et al. classic and molecular methodologies for the identification of enterococcus species. rev med chile. 2002; 130: 45-9. 19. dutka-malen s, evers s, courvalin p. detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by pcr. j clin microbiol. 1995; 33: 24-7. 20. rocas in, siqueira jfj. characterization of microbiota of root canal-treated teeth with posttreatment disease. j clin microbiol. 2012; 50: 1721-4. 21. peciuliene v, balciuniene i, eriksen hm, haapasalo m. isolation of enterococcus faecalis in previously root-filled canals in a lithuanian population. j endod. 2000; 26: 593–5. 22. wang qq, zhang cf, chu ch, zhu xf. prevalence of enterococcus faecalis in saliva and filled root canals of teeth associated with apical periodontitis. int j oral sci. 2012; 4: 19-23. 23. kaufman b, spångberg l, barry j, fouad af. enterococcus spp. in endodontically treated teeth with and without periradicular lesions. j. endod. 2005; 31: 851–6. 24. nabeshima ck, rosa martins gh, pasquali leonardo mf, furukava shin, silvana cai rc, lima machado me. comparison of three obturation techniques with regard to bacterial leakage. braz j oral sci. 2013; 12: 212-5. 25. zhu x, wang q, zhang c, cheung gs, shen y. prevalence, phenotype, and genotype of enterococcus faecalis isolated from saliva and root canals in patients with persistent apical periodontitis. j endod. 2010; 36: 1950–5. 26. tennert c, fuhrmann m, wittmer a. karygianni l, altenburger m, pelz k et al. new bacterial composition in primary and persistent/ secondary endodontic infections with respect( to clinical and radiographic findings j endod. 2014; 40: 670-7. 27. paloma b, cabral dos santos c, alves f, lima l, machado c. in vitro antimicrobial photoinactivation with methylene blue in different microorganisms. braz j oral sci. 2014; 13: 53-7. braz j oral sci. 14(3):240-245 enterococcus spp. isolated from root canals with persistent chronic apical periodontitis in a chilean population 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1522/1209 1/8 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1522/1209 2/8 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1522/1209 3/8 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1522/1209 4/8 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1522/1209 5/8 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1522/1209 6/8 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1522/1209 7/8 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1522/1209 8/8 revista fop n 13 1555 braz j oral sci. april/june 2008 vol. 7 number 25 influence of light-curing units on the flexural strengthinfluence of light-curing units on the flexural strengthinfluence of light-curing units on the flexural strengthinfluence of light-curing units on the flexural strengthinfluence of light-curing units on the flexural strength and flexural modulus of different resin compositesand flexural modulus of different resin compositesand flexural modulus of different resin compositesand flexural modulus of different resin compositesand flexural modulus of different resin composites william cunha brandt1; leandro cardoso2; rafael ratto de moraes1; lourenço correr-sobrinho3; mário alexandre coelho sinhoreti3 1 dds, ms, graduate student department of restorative dentistry, dental materials division, piracicaba dental school, university of campinas, brazil 2 ms, graduate student, department of prosthodontics, piracicaba dental school, university of campinas, brazil 3 dds, ms, phd, professor, department of restorative dentistry, dental materials division, piracicaba dental school, university of campinas, brazil received for publication: june 25, 2008 accepted: july 18, 2008 correspondence to: william cunha brandt depto. de dentística restauradora, área de materiais dentários avenida limeira 901, cep: 13414-903, piracicaba sp, brasil telephone: +55 19 2106.5345. fax: +55 19 2106.5218. e-mail: wcbrandt@fop.unicamp.br a b s t r a c t aim: this study aimed to evaluate the flexural strength and flexural modulus of different resin composites (masterfill, opallis, z250, supreme) after photoactivation with quartz-tungsten-halogen (qth xl2500) or light-emitting diode (led radii and ultrablue is) light-curing units (lcus). methods: the irradiance and spectral emission of each unit were evaluated using a power meter and a spectrometer. flexural strength (mpa) was determined in a three-point bending test in accordance with the iso4049 standard specifications. flexural modulus (gpa) was measured from the linear-elastic range on the stress-deformation profile. data were submitted to two-way anova and tukey’s test (p<0.05). results: different values for irradiance (850, 780 and 590 mw/cm2) and peak of emission (484, 456, 467 nm) were detected for xl2500, radii and ultrablue is, respectively. flexural strength and modulus were dependent on both material and lcu. among the resins, z250 and supreme showed significantly higher strength and modulus than masterfill and opallis. comparing the lcus, ultrablue is showed significantly lower flexural strength and modulus than the others. conclusion: flexural strength and modulus were dependent on the irradiance and the spectral emission of the curing units, as well on the resin composite tested. key words composite resin, light, physical properties, polymers, photoactivation. i n t r o d u c t i o n the ultimate goal of dental restorative materials is to replace the biological and functional properties of healthy tooth structures, with physical and esthetic properties matching those of natural teeth. since their introduction about 40 years ago, restorative resin composites have proven to be successful1, and it is expected that their use for restoring both anterior and posterior teeth will continue to increase. although considerable improvements have been made in the properties of these materials over the last decade2, brazilian formulations are increasingly available, but little is know about their performance. for this reason, studies evaluating their properties are warranted. likewise composites, several light-curing units (lcus) are available, each one having specific characteristics of light emission, such as irradiance level and spectral emission range and peak. although quartz-tungsten-halogen (qth) lcus are the most traditional ones, blue light-emitting diodes (leds) are increasingly popular among clinicians. these lcus emit a narrow spectrum of wavelengths that is better correlated with the spectral absorbance of camphorquinone (cq), the most commonly used photoinitiator in dental composites3. in comparison to leds, qth lcus also present some drawbacks that may interfere with their long-term exposure efficiency, such as high heat generation within the quartz bulb, degradation of filters over the course of time, and relatively overall short efficient working life span4-5. however, although some studies describe similar curing efficiency for qth and led units, the actual efficiency of leds still needs further evaluation. therefore, the aims of this study were to evaluate the light characteristics (irradiance level and spectral emission) of led and qth lcus, and to investigate their influence on flexural strength and flexural modulus of different resin composites. the tested null-hypothesis was that no significant differences in strength and modulus would be observed regardless of the lcu or resin composite. 1556 material manufacturer composition* filtek z250 3m espe bisgma, bisema, udma, tegdma, zirconia/silica (75 wt%) filtek supreme 3m espe bisgma, bisema, udma, tegdma, zirconia/silica (78.5 wt%) opallis fgm bisgma, bisema, tegdma. ba-al-si glass particles/silica (79 wt%) masterfill biodinâmica bisgma, udma, ba-al-si glass particles (79 wt%) table 1 materials used in the study *as informed by the manufacturers unit manufacturer irradiance* peak of emission** xl2500 qth 3m espe 850 mw/cm2 484 nm radii led sdi 780 mw/cm2 467 nm ultrablue is led dmc 590 mw/cm2 456 nm table 2 characteristics of the light-curing units *values confirmed with a digital power meter. **data obtained with a computer-controlled spectrometer. fig. 1 spectral distribution of each light-curing unit material and methods four commercial hybrid resin composites, shade a3, were evaluated: masterfill (biodinâmica, ibiporã, pr, brazil), opallis (fgm, joinville, sc, brazil), filtek z250 and filtek supreme (3m espe, st. paul, mn, usa). materials’ compositions are shown in table 1. light characteristics three lcus were investigated: xl2500 qth (xl 3m espe), ultrablue is led (ub dmc, são carlos, sp, brazil) and radii led (rd sdi, victoria, australia). the lcus were connected to a voltage stabilizer and the output power (mw) of each unit was measured with a digital power meter (ophir optronics inc., danvers, ma, usa). the diameter of each light guide tip (cm) was measured with a digital caliper (mitutoyo, tokyo, japan) accurate to 0.01 mm. irradiance (mw/cm2) was computed as the ratio of the output power by the area of the light guide. additionally, the spectral distribution of each lcu was obtained using a computer-controlled spectrometer (usb2000; ocean optics, dunedin, fl, usa). flexural strength flexural strength test was conducted in accordance with the iso4049 standard specifications6. five rectangular barshaped specimens (25 x 2 x 2 mm) were prepared for each material-unit combination by placing the composites into a stainless steel mold held between two glass microscopic slides. after light-activation procedures, the specimens were removed from the mold and stored in distilled water at 37°c in the dark. after 24 h, the height and width of the specimens were measured with a digital caliper (mitutoyo) and the samples were subjected to a three-point bending test in a mechanical testing machine (dl500; emic, são josé dos pinhais, pr, brazil), at a crosshead speed of 0.5 mm/min until failure. flexural strength (fs) was determined as follows: fs = 3p f l / 2wh2 where p f is the measured maximum load (n) at the time of specimen fracture, l is the distance between the supports on the tension surface (20 mm), w is the mean specimen width, and h is the mean height of the specimen between the tension and compression surfaces. data were submitted to two-way anova and tukey’s test (p<0.05). flexural modulus a chart plotter recorded the stress-deformation profile during the flexural test. flexural modulus (fm) was calculated from the linear-elastic range, between bending force and specimen displacement before fracture, as follows: fm = (df / dy) x (l3 / 4wh3) where df / dy is the change in force (df) per unit change in deflection of the center of the specimen (dy). data were submitted to two-way anova and tukey’s test (p<0.05). r e s u l t s light characteristics table 2 presents the light characteristics for each lcu. figure 1 shows a comparison for the spectrum of wavelengths. for xl, a spectrum concentrated in the 400braz j oral sci. 7(25):1555-1558 influence of light-curing units on the flexural strength and flexural modulus of different resin composites 1557 520 nm wavelength range was observed. for rd and ub, the spectrum was concentrated in the 430-530 and 420520 nm range, respectively. flexural strength results for flexural strength are shown in table 3. the factor ‘lcu’ was not significant (p=0.179), while the factor ‘material’ (p<0.001) and the interaction unitmaterial (p=0.018) were significant. for xl, opallis showed significantly lower flexural strength than the other composites (p<0.001), which were similar to each other (pe”0.947). correspondingly, for rd, z250 and supreme were similar between them (p=0.554), while masterfill was similar only to supreme (p=0.119); opallis showed significantly lower strength than z250 and supreme (pd”0.004), but similar to masterfill (p=0.565). on the other hand, for ub, all composites presented similar results (pe”0.349). when comparing the different lcus for each material, no significant differences were observed for masterfill (pe”0.269) and opallis (pe”0.091). for z250 and supreme, rd showed the highest flexural strength values, which were similar to xl (pe”0.31), but significantly higher than ub (pd”0.041); xl and ub presented similar results (pe”0.176). masterfill opallis z250 supreme xl2500 124.2 (14.9)a,a 74.4 (14.1)b,a 130.4 (30.9)a,ab 124.4 (16.8)a,ab radii 106.4 (13.3)bc,a 91.6 (14.5)c,a 147.2 (3.7)a,a 132.2 (9.8)ab,a ultrablue is 114.2 (7.4)a,a 98.8 (21.8)a,a 117.8 (29.0)a,b 103.8 (18.4)a,b table 3 means (standard deviations) for flexural strength (mpa) means followed by different uppercase letters in the same row and lowercase letters in the same column, are significantly different at p<0.05. masterfill opallis z250 supreme xl2500 9.0 (0.4)c,a 8.1 (0.3)c,a 12.2 (0.8)a,a 10.7 (0.7)b,a radii 8.2 (0.6)b,ab 8.7 (0.4)b,a 11.8 (0.7)a,ab 10.8 (0.3)a,a ultrablue is 7.6 (1.1)c,b 7.9 (0.6)c,a 11.2 (0.9)a,b 9.3 (0.8)b,b table 4 means (standard deviations) for flexural modulus (gpa). means followed by different uppercase letters in the same row and lowercase letters in the same column, are significantly different at p<0.05. flexural modulus results for flexural modulus are shown in table 4. the factors ‘material’ and ‘lcu’ were significant (p<0.001), but not their interaction (p=0.165). for xl and ub, z250 and supreme showed significantly higher values than both masterfill and opallis (pd”0.012), which performed similar (pe”0.147). for rd, z250 and supreme presented similar results (p=0.072), and both presented significantly higher modulus than masterfill and opallis (p<0.001), which were similar (p=0.677). when comparing the different lcus for each material, no significant differences were detected for opallis (pe”0.194). for masterfill and z250, xl and rd were similar (pe”0.155), and so were rd and ub (pe”0.295), but xl yielded significantly higher modulus than ub (pd”0.04). for supreme, xl and rd were similar (p=0.992), generating significantly higher modulus than ub (pd”0.004). d i s c u s s i o n flexural strength is a measure of composite strength: the higher the value, the stronger the material. the iso4049 standard6 classifies dental polymer-based restorative materials into 2 types: type i materials are those claimed by the manufacturers to be suitable for restorations involving occlusal surfaces, while type ii are all other polymer-based restorative materials. the minimum flexural strength requirement for type i materials is 80 mpa6-7. the results of this study showed that all composites presented flexural strength higher than 80 mpa, except for opallis photoactivated with qth. indeed, opallis showed the lowest strength values among all tested composites, regardless of the lcu. therefore, the nullhypothesis is rejected. the probable explanation for this result may rely on the fact that the composites tested in this study present distinct compositions, and differences in filler load and type, organic matrix components, and even surface treatments of the particles, may affect the mechanical behavior of the materials. on the other hand, flexural modulus describes stiffness, a measure of material’s resistance to deformation under load. there are debates on how much modulus resin composites should possess8. class v cervical cavities, for example, demand a low modulus restorative material to flex with the tooth. a relatively high modulus, on the other hand, is expected for posterior restorations to withstand the occlusal forces and preserve the adhesive interface. theoretically, the ideal value would be similar to that of dental structures, so the restorative would have similar deformation to the surrounding tooth under loading. however, when compared to the modulus of human enamel (84 gpa)9, resin composites have much lower values; in comparison to human dentin (14 gpa)9-10, some composites may present similar values. in the present study, z250 and supreme generally presented higher modulus than the other tested composites. this result might be related to differences in resin formulation or inorganic load among the materials, which strongly affect the properties of the dental composites. opallis and masterfill possess irregular glass particles, while the other composites possess spherical ceramic particles that present higher strength and improve the packing of the inorganic fillers, enhancing the properties of the composite. composites made with irregular particles could have lower flexural strength and modulus than those made with braz j oral sci. 7(25):1555-1558 influence of light-curing units on the flexural strength and flexural modulus of different resin composites 1558 spherical fillers due to the fact that the stress concentration around the fillers would be expected to be greater for materials loaded with irregular-shaped particles11. the lcus produced different results among the resin composites. in general, rd and xl generated higher values for strength and modulus. this is probably explained by the fact that these lcus presented higher irradiance than ub. the amount of light energy delivered to the specimens might affect the conversion of double bonds which, in turn, might interfere with the development of mechanical properties. moreover, a photo-polymerization initiated at low light level is generally associated with relatively few centers of polymer growth, possibly resulting in a more linear final polymer structure, with lower network strength. on the other hand, irradiance at high levels produces a multitude of growth centers and leads to the formation of densely cross-linked polymers12-13, which might also explain the results observed for rd and xl. in addition to irradiance, the polymerization potential by photoactivation is also dependent on the correlation between the light spectrum emitted by the lcu and spectrum of absorption of the photoinitiator14-15. therefore, one could expect better results for rd, since the peak of emission for this lcu was centered on the absorption peak of cq, as shown in figure 1. nonetheless, similar results for flexural strength and modulus were detected for both rd and xl, irrespective of the resin composite. this result might be attributed to the fact that both lcus presented high and similar irradiance values, which might have accounted for their similar results16. the present results have clinical implications, since both flexural strength and flexural modulus were found to be dependent on the material and lcu tested. the brazilian resin composites generally presented poorer properties as compared with the other composites tested, while highintensity lcus generated polymers with enhanced strength and modulus. notwithstanding, the results of the present study should be restricted to the conditions tested here. further studies evaluating different resin composites and curing devices are necessary. a c k n o w l e d g e m e n t s the authors are grateful to the dental school of the federal university of pelotas, rs, brazil, for the access to its facilities, and to biodinâmica and fgm for donating the materials used in the study. r e f e r e n c e s 1. leonard dl, charlton dg, roberts hw, cohen me. polymerization efficiency of led curing lights. j esthet restor dent. 2002; 14: 286-95. 2. ferracane jl. developing a more complete understanding of stresses produced in dental composites during polymerization. dent mater. 2005; 21: 36-42. 3. taira m, urabe h, hirose t, wakasa k, yamaki m. analysis of photo-initiators in visible-light-cured dental composite resins. j dent res. 1988; 67: 24-8. 4. bennett aw, watts dc. performance of two blue lightemitting-diode dental light curing units with distance and irradiation-time. dent mater. 2004; 20: 72-9. 5. segreto d, brandt wc, correr-sobrinho l, sinhoreti ma, consani s. influence of irradiance on the push-out bond strength of composite restorations photoactivated by led. j contemp dent pract. 2008; 9: 89-96. 6. international standard organization. iso 4049. dentistry — polymer-based filling, restorative and luting materials. 3rd ed.; 2000. 7. leonard dl, charlton dg, roberts hw, cohen me. polymerization efficiency of led curing lights. j esthet restor dent. 2002; 14: 286-95. 8. choi kk, ferracane jl, hilton tj, charlton d. properties of packable dental composites. j esthet dent. 2000; 12: 216-26. 9. meredith n, sherriff m, setchell dj, swanson sa. measurement of the microhardness and young’s modulus of human enamel and dentine using an indentation technique. arch oral biol. 1996; 41: 539-45. 10. sano h, ciucchi b, matthews wg, pashley dh. tensile properties of mineralized and demineralized human and bovine dentin. j dent res. 1994; 73: 1205-11. 11. turssi cp, ferracane jl, vogel k. filler features and their effects on wear and degree of conversion of particulate dental resin composites. biomaterials. 2005; 26: 4932-7. 12. brandt wc, de moraes rr, correr-sobrinho l, sinhoreti ma, consani s. effect of different photo-activation methods on push out force, hardness and cross-link density of resin composite restorations. dent mater. 2008; 24: 846-50. 13. moraes rr, schneider lf, correr-sobrinho l, consani s, sinhoreti ma. influence of ethanol concentration on softening tests for cross-link density evaluation of dental composites. mat res. 2007; 10: 79-81. 14. stahl f, ashworth sh, jandt kd, mills rw. light-emitting diode (led) olymerization of dental composites: flexural properties and polymerization potential. biomaterials. 2000; 21: 1379-85. 15. neumann mg, miranda wg, jr., schmitt cc, rueggeberg fa, correa ic. molar extinction coefficients and the photon absorption efficiency of dental photoinitiators and light curing units. j dent. 2005; 33: 525-32. 16. luiz bk, prates lh, bertolino jr, pires at. influence of light intensity on mechanical properties of the resin composite. braz j oral sci. 2006; 5: 1048-53. braz j oral sci. 7(25):1555-1558 influence of light-curing units on the flexural strength and flexural modulus of different resin composites untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8652651 volume 17 2018 e18148 original article 1 proclin department, school of dentistry, state university of rio de janeiro (uerj), rio de janeiro, rj, brazil 2 department of endodontics, school of dentistry, grande rio university (unigranrio), duque de caxias, rj, brazil 3 deparment of microbiology, school of dentistry, state university of rio de janeiro (uerj), rio de janeiro, rj, brazil corresponding author: marina carvalho prado estrada do campinho 298a. campo grande, rio de janeiro, rj, brazil. 23080-420. tel/fax: (55) 21 24131442. e-mail: marinaprado@dentistas.com.br received: december 22, 2017 accepted: april 08, 2018 improved sealing ability promoted by calcium silicate-based root canal sealers tatiana vieira de melo, dds, msc1; marina carvalho prado, dds, msc1,2; raphael hirata jr, dds, phd3; sandra rivera fidel, dds, phd1; emmanuel joão nogueira leal da silva, dds, phd1,2; luciana moura sassone, dds, phd1 aim: this study investigated the sealing ability produced by ah plus (dentsply detrey, konstaz, germany), endosequence bc sealer (brasseler usa, savannah, ga, usa), guttaflow (coltène/ whaledent, altstätten, france) and mta fillapex (angelus, londrina, brazil). methdos: a total of forty-six single-root human canines were prepared and randomly divided into four experimental groups (n=10): mta fillapex, endosequence bc sealer, ah plus or guttaflow. teeth with intact crowns served as negative controls (n=3) and teeth filled with only gutta-percha served as positive controls (n=3). teeth were mounted in a two-chamber apparatus and exposed to enterococcus faecalis. the number of days over a 60-days period was recorded for the appearance of turbidity in the lower chamber. kaplanmeier method was used to estimate the survival curves. the nonparametric log-rank test was used to compare the survival curves using a significance level of 0.05. results: the results at the end of the observation time were further analyzed by chi-square testing. all positive controls leaked within 24 h, whereas none of the negative controls leaked after 60 days. endosequence bc sealer and mta fillapex had significant less bacterial leakage (p<0.05) than the other tested sealers. no significant difference between ah plus and guttaflow was observed (p>0.05). conclusion: in conclusion, calcium-silicate based root canal sealers promoted improved sealing ability when compared to other endodontic sealers. key words: dental materials. endodontics. root canal sealers. 2 de melo et al. introduction the aim of the root canal filling is to create a fluid-tight apical, lateral and coronal seal, thus perpetuating the state of disinfection obtained after chemomechanical preparation and intracanal medication1. this procedure minimizes the risks of infection or reinfection of the root canal system1. the most widely accepted technique for root canal filling is the association of gutta-percha with an endodontic sealer. the main goals of the root canal sealer is to fill the interface between gutta-percha and the dentin walls, the voids inside gutta-percha, to fill root canal imperfections and increase adaption of the root filling, obtaining a hermetic seal1. over the last years, a new class of root canal sealers, named calcium-silicate-based sealers, has been commercially available. these sealers, such as mta fillapex (angelus, londrina, pr, brazil) and endosequence bc sealer (brasseler usa, savannah, ga, usa), can be regarded as an outgrowth of the mineral trioxide aggregate (mta)’s clinical and scientific success. this means that the intention is to extrapolate the mta’s remarkable balance between biological and physical-chemical properties creating a close-to-ideal sealer, but showing proper flow rate to be used with gutta-percha cones and conventional and warm vertical filling techniques. moreover, theoretically, handling, retreatability and bioactivity are present; thus, the so-called biomineralization process would be able to take place in the critical sealer-dentine interface2. mta fillapex is a paste-paste sealer basically composed of mta, salicylate resin, natural resin, bismuth and silica. according to the manufacturer, mta fillapex has adequate working time, high radiopacity and low solubility, providing sealing through expansion during setting. it was shown that mta fillapex has suitable physicochemical properties, however several studies demonstrate some concerns regarding its biological properties3,4. endosequence bc sealer is a premixed and injectable calcium silicate root canal sealer and, according to the manufacturer’s description, it is composed of zirconium oxide, calcium silicates, calcium phosphate monobasic, calcium hydroxide and thickening agents5-7. it is a hydrophilic, insoluble, radiopaque and aluminum-free material based on calcium silicate composition, which utilizes the moisture naturally present in the dentinal tubules to initiate and complete its setting reaction. it has been demonstrated that endosequence has good bond strength5 and adequate physicochemical properties6,7. although some physicochemical properties of calcium silicate-based sealers have been extensively evaluated, there are only little inconsistent data about the sealing ability of this group of sealers using bacterial penetration methodology. therefore, the present study was designed to investigate the sealing ability, a relevant aspect, produced by endosequence bc sealer and mta fillapex. ah plus (dentisply detrey, konstaz, germany), a resin-based sealer, and guttaflow (coltène/whaledent, altstätten, france), a silicon-based sealer, were also evaluated in the present study. the bacterial penetration assessment was used to test the hypothesis that the calcium silicate-based sealers (endosequence bc sealer and mta fillapex) produced higher sealing ability than the ah plus and guttaflow. 3 de melo et al. material and methods sample selection forty-six human mandibular canines were selected and autoclaved. then, each tooth were submitted to buccolingual and mesiodistal planes of periapical radiographs for analysis. the presence of lateral and accessory canals, caries, isthmus, cracks or fracture lines excluded specimens from this study. following this, the total sample were stored in 10% neutral formalin. chemomechanical preparation from the total sample, three teeth with intact crowns did not receive any preparation, serving as a negative control group. therefore, only forty-three teeth were prepared by the same operator, using the same technique. access cavity was conventionally made using diamond burs and endoz (dentsply maillefer, ballaigues, switzerland). before and after the completion of preparation procedures, the patency of each canal was checked by exceeding a size #15 k-file through the apical foramen. after that, the working length was stablish up to 1 mm short of the foramen and root canals were instrumented using a full sequence of protaper universal files (dentsply maillefer, ballaigues, switzerland) as follows: (1) s1 file, (2) sx file, (3) s2 file, (4) f1, (5) f2, (6) f3, (7) f4 and (8) f5 files. during each change of files, canals were irrigated with 2 ml of freshly prepared 5.25% naocl. following preparation, root canals received a final flush of 2 ml of 17% edta for 3 min and were dried with f5 paper points (dentsply maillefer). thereafter, prepared teeth were randomly divided into one positive control group (n=3) and four experimental groups (n=10). root canal filling all forty-three prepared teeth were root filled by the same operator using the cold lateral compaction technique. the three specimens selected for the positive control group were filled with gutta-percha and no root canal sealer. the forty teeth from the 4 experimental groups were filled with gutta-percha and one of the following root canal sealers: mta fillapex, endosequence bc sealer, ah plus or guttaflow. the sealers were prepared following the manufactures instructions. a f5 master gutta-percha cone was placed into the canals to the full working length. lateral compaction technique was undertaken by inserting ten accessory gutta-percha cones (mf, odous de-deus, belo horizonte, mg, brazil) with the use of a size b endodontic finger spreader (dentsply maillefer). the excess of coronal gutta-percha was removed by a heated instrument. except from the apical and coronal extremities, two coats of nail varnish were applied on the external surface of all specimens. then, filled teeth were stored at 37°c and 100% humidity for 14 days. bacterial penetration model afterwards, the bacterial penetration test was performed in a two chamber set-up as previously described8. ten ml glass assay tubes (bd vacutainer, juiz de fora, mg, brazil) with rubber stops were adjusted for application. then, a hole was prepared in the 4 de melo et al. central point of each rubber stopper using heated instrument, in order to insert a cylinder that was prepared from insulin syringes. specimens tooth crown was tightly adjusted into the rubber tube and sealed by cyanoacrylate (loctite 496, henkel ltda, são paulo, sp, brazil). then, on the opposite side of this rubber tube, syringe cylinders were fitted to produce a reservoir for the medium and cyanoacrylate was inserted in all junctions of this structure. the apparatus external surface was sealed with cyanoacrylate and parafilm m (laboratory film, american national can, chicago illinois, usa) (figure 1). after that, the apparatus was overnight sterilized with ethylene oxide gas (bioxxi esterilization services ltda, rio de janeiro, rj, brazil). the setup was accomplished in a laminar airflow hood, in which 3 ml sterile brain heart infusion (bhi, oxoid ltda, basingstoke, uk) was inserted in the glass assay tubes in such a way as to immerse approximately 2 mm of the resected root in the broth. to assure sterilization of this bacterial penetration model, the entire system was incubated during 4 days at 37°c. the upper chamber was filled with medium without bacteria on day 1 and checked for penetration until day 0. enterococcus faecalis (atcc-29212) was added to the upper chamber and the time for e. faecalis to eventually penetrate into the lower chamber was noted. the apparatus was incubated at 37°c and daily checked. bacteria penetrating along the root filling were detected by turbidity observed in the lower chamber. maximum observation time was 60 days. statistical analysis the kaplan–meier method was used to estimate the survival curves. specimens that did not leak until the end of the observation time were computed with an event time figure 1. setup of the model design. bhi teeth rubber cover glass tube teeth sealing glass/eppendorf with parafilm “m” eppendorf 5 de melo et al. of 60 days as censored variables. the nonparametric log-rank test was used to compare the survival curves using a significance level of 0.05. the results at the end of the observation time (60 days) were further analyzed by chi-square testing. results all specimens of the positive control group leaked within 24 h, whereas no bacterial penetration was observed in the negative control group after the observation time of 60 days. overall, 60% of the samples of ah plus and 40% of guttaflow group were fully contaminated after 60 days, whereas 10% and 20% of the endosequence bc sealer and mta fillapex groups were fully contaminated, respectively. endosequence bc sealer and mta fillapex demonstrated significant lower specimens to become contaminated in comparison with ah plus and guttaflow (p<0.05) (figure 2). no significant difference between ah plus and guttaflow was observed (p>0.05). discussion according to the current results, bacterial penetration occurred in all tested groups regardless of the endodontic sealer used, thus, we currently cannot achieve the ideal outcome with the existent materials. however, the use of calcium silicate-based root canal sealers resulted in significantly fewer samples being contaminated at the end of the experimental period when compared to the other tested sealers (p<0.05). therefore, the tested hypothesis was sustained. previous studies demonstrated good sealing ability of calcium-silicate based root repair materials9,10 and indicated the clinical use of mta-based sealers as apical barrier in wide-open apices as it may stop/reduce the fluid flow rate through the apex11. moreover, other studies demonstrated suitable sealing ability of calcium-silicate based sealers using fluid-filtration method12,13; however, there are only little inconsistent data about the sealing ability of this group of sealers using bacterial penetration methodology. endosequence bc sealer presented a superior sealing ability than ah plus in a dye penetration evaluation14, consisting with figure 2. kaplan-meier survival curves for the experimental root canal sealers in the bacterial leakage test. c um ul at iv e su rv iv al observation time in days 1,2 1 0,8 0,4 0,2 0 0 15 30 45 55 0,6 ah plus endosequence guttaflow mta fillapex 10 25 40 505 20 35 60 6 de melo et al. the present results. in contrast, mta fillapex has demonstrated controversial sealing performance in endodontic literature13,15-18, what may result from its physicochemical properties19 and differences in experimental designs such as the evaluation technique. the results obtained by calcium silicate-based sealers compared to the other sealers may be caused, in part, by its extremely small particle size and excellent level of viscosity, which enhances flow of the sealer into dentinal tubules, anatomic irregularities, and gutta-percha3,7. additionally, endosequence bc sealer and mta fillapex exhibits minimal or no shrinkage during the setting phase6, which may have contributed to the higher sealing ability values. moreover, the performance of calcium silicate-based sealers may be attributable to its bioactivity20, that is, the capacity to produce spontaneously an apatite layer when in contact with phosphate-containing physiological fluids. during set, the calcium silicate that is present in these sealers generates calcium hydroxide and calcium silicate hydrogel. following this, part of the produced calcium hydroxide reacts with phosphate, providing hydroxyapatite and water. this spontaneous precipitation may promote a biomineralization process creating a chemical adhesion, improving sealing ability of the sealer21. epoxy resin-based ah plus is well known for its long-term dimensional stability, expansive properties and ability to bond to dry dentine9,10,12. moreover, ah plus is thought to be able to react with any exposed amino groups in collagen to form covalent bonds between the resin and collagen. despite these good properties, in the present study ah plus allowed more samples to become contaminated over the experimental period, in agreement with several previously published studies8,22,23. sealing ability is related to different phenomenon such as porosity, marginal adaptation and hydrophilicity, explaining why ah plus did not have the best sealing ability amongst the tested materials24,25. another possible explanation is the inadequate bonding between ah plus sealer and gutta-percha, allowing bacteria penetration at this interface. many laboratory studies comparing the sealing ability of guttaflow has also been published8,22,23. however, there is a lack of consensus in regard to the results obtained by this sealer. the discrepancies among studies could be explained on the basis of differences in experimental designs (eg. obturation technique and/or methodological differences). in the present study, using the same experimental design, guttaflow showed inferior sealing ability when compared to endosequence bc sealer and mta fillapex (p<0.05). it is important to emphasize that this is one of the first’s studies to compare the sealing ability promoted by calcium silicate-based endodontic sealers in comparison to silicone-based endodontic sealers. in the past, leakage was assessed using dye penetration methodologies. however, the reliability, reproducibility, and clinical relevance of these methods are questionable26,27. moreover such dyes have low molecular weights and can penetrate into sites where protein and pathogens cannot penetrate28. studies using bacterial cultures have been used widely to test the penetration resistance of endodontic sealers as it might be more meaningful and provides more precise and reproducible data29,30. such tests may be considered to have more biological significance than dye leakage tests as they reflect more closely the clinical situation, and also allows the evaluation of the samples at specific periods29,30. however, it still uses a static model, thus it requires long periods of observation and it does not allow the quantification of the penetrat7 de melo et al. ing bacteria29. e. faecalis was chosen as the test bacteria, as they are part of normal microbiota in humans and are frequently isolated in secondary infections. using human saliva is advantageous to some degree because it closely approximates to the real clinical situation; on the other hand, it does not simulate temperature changes, the influence of diet, and salivary flow29. in conclusion, calcium-silicate based root canal sealers promoted improved sealing ability when compared to other endodontic sealers. acknowledgment: the authors would like to thank faperj (proc. e-26/202.839/2015) for scholarship support. references 1. peters lb, wesselink pr, moorer wr. the fate and role of bacteria left in root dentinal tubules. int endod j. 1995 mar;28(2):95-9. 2. gandolfi mg, taddei p, tinti a, de stefano dorigo e, rossi pl, prati c. kinetics of apatite formation on a calcium-silicate cement for root-end filling during ageing in physiological-like phosphate solutions. clin oral investig. 2010 dec;14(6):659-68. doi: 10.1007/s00784-009-0356-3. 3. silva ej, rosa tp, herrera dr, jacinto rc, gomes bp, zaia aa. evaluation of cytotoxicity and physicochemical properties of calcium silicate-based endodontic sealer mta fillapex. j endod. 2013 feb;39(2):274-7. doi: 10.1016/j.joen.2012.06.030. 4. silva ej, santos cc, zaia aa. long-term cytotoxic effects of contemporary root canal sealers. j appl oral sci. 2013 jan-feb;21(1):43-7. 5. shokouhinejad n, gorjestani h, nasseh aa, hoseini a, mohammadi m, shamshiri ar. push-out bond strength of gutta-percha with a new bioceramic sealer in the presence or absence of smear layer. aust endod j. 2013 dec;39(3):102-6. doi: 10.1111/j.1747-4477.2011.00310.x. 6. zhou hm, shen y, zheng w, li l, zheng yf, haapasalo m. physical properties of 5 root canal sealers. j endod. 2013 oct;39(10):1281-6. doi: 10.1016/j.joen.2013.06.012. 7. candeiro gt, correia fc, duarte ma, ribeiro-siqueira dc, gavini g. evaluation of radiopacitiy, ph, release of calcium ions, and flow of a bioceramic root canal sealer. j endod. 2012 jun;38(6):842-5. doi: 10.1016/j.joen.2012.02.029. 8. de-deus g, brandão mc, fidel ra, fidel sr. the sealing ability of guttaflow in oval-shaped canals: an ex vivo study using a polymicrobial leakage model. int endod j. 2007 oct;40(10):794-9. 9. jeevani e, jayaprakash t, bolla n, vemuri s, sunil cr, kalluru rs. evaluation of sealing ability of mm-mta, endosequence, and biodentine as furcation repair materials: uv spectrophotometric analysis. j conserv dent. 2014 jul;17(4):340-3. doi: 10.4103/0972-0707.136449. 10. nair u, ghattas s, saber m, natera m, walker c, pileggi r. a comparative evaluation of the sealing ability of 2 root-end filling materials: an in vitro leakage study using enterococcus faecalis. oral surg oral med oral pathol oral radiol endod. 2011 aug;112(2):e74-7. doi: 10.1016/j.tripleo.2011.01.030. 11. prati c, siboni f, polimeni a, bossu m, gandolfi mg. use of calcium-containing endodontic sealers as apical barrier in fluid-contaminated wide-open apices. j appl biomater funct mater. 2014 dec 30;12(3):263-70.doi: 10.5301/jabfm.5000162. 12. ersahan s, aydin c. solubility and apical sealing characteristics of new calcium silicate-based root canal sealer in comparison to calcium hydroxide-, methacrylate resinand epoxy resin-based sealers. acta odontol scand. 2013 may-jul;71(3-4):857-62. doi: 10.3109/00016357.2012.734410. 8 de melo et al. 13. asawaworarit w, yachor p, kijsamanmith k, vongsavan n. comparison of the the apical sealing ability of calcium silicate-based sealer and resin-based sealer using the fluid-filtration technique. med princ prac. 2016;25(6):561-5. 14. pawar ss, pujar ma, makandar sd. evaluation of the apical sealing ability of bioceramic sealer, ah plus & epiphany: an in vitro study. j conserv dent. 2014 nov;17(6):579-82. doi: 10.4103/0972-0707.144609. 15. jafari f, sobhani e, samadi-kafil h, pirzadeh a, jafari s. in vitro evaluation of the sealing ability of three newly developed root canal sealers: a bacterial microleakage study. j clin exp dent. 2016 dec;8(5):e561-5. 16. razavian h, barekatain b, shadmehr e, khatami m, bagheri f, heidari f. bacterial leakage in root canals filled with resin-based and mineral trioxide aggregate-based sealers. dent res j (isfahan). 2014 sep;11(5):599-603. 17. ahuja l, jasuja p, verma kg, juneja s, mathur a, walia r, et al. a comparative evaluation of sealing ability of new mta based sealers with conventional resin based sealer: an in-vitro study. j clin diagn res. 2016 jul;10(7):zc76-9. doi: 10.7860/jcdr/2016/18909.8194. 18. singh r, pushpa s, arunagiri d, sawhny a, misra a, sujatha r. the effect of irrigating solutions on the apical sealing ability of mta fillapex and adseal root canal sealers. j dent res dent clin dent prospects. 2016 fall;10(4):251-256. doi: 10.15171/joddd.2016.040. 19. vitti rp, prati c, sinhoreti ma, zanchi ch, souza e silva mg, ogliari fa, et al. chemical-physical properties of experimental root canal sealers based on butyl ethylene glycol disalicylate and mta. dent mater. 2013 dec;29(12):1287-94. doi: 10.1016/j.dental.2013.10.002. 20. han l, okiji t. bioactivity evaluation of three calcium silicate-based endodontic materials. int endod j. 2013 sep;46(9):808-14. doi: 10.1111/iej.12062. 21. reyes-carmona jf, felippe ms, felippe wt. biomineralization ability and interaction of mineral trioxide aggregate and white portland cement with dentin in a phosphate-containing fluid. j endod. 2009 may;35(5):731-6. doi: 10.1016/j.joen.2009.02.011. 22. bouillaguet s, shaw l, barthelemy j, krejci i, wataha jc. long-term sealing ability of pulp canal sealer, ah-plus, guttaflow and epiphany. int endod j. 2008 mar;41(3):219-26. 23. brackett mg, martin r, sword j, oxford c, rueggeberg fa, tay fr, et al. comparison of seal after obturation techniques using a polydimethylsiloxane-based root canal sealer. j endod. 2006 dec;32(12):1188-90. 24. resende lm, rached-junior fja, versiani ma, souza-gabriel ae, miranda ce, silva-sousa yt, et al. a comparative study of physicochemical properties of ah plus, epiphany and epiphany se root canal seralers. int endod j. 2009 sep;42(9):785-93. doi: 10.1111/j.1365-2591.2009.01584.x. 25. schwartz rs. adhesive dentistry and endodontics. part 2: bonding in the root canal system-the promise and the problems: a review. j endod. 2006 dec;32(12):1125-34. 26. de bruyne ma, verhelst pc, de moor rj. critical analysis of leakage studies in endodontics. rev belge med dent. 2005;60(2):92-106. 27. wu ml, van b, wesselink pr. diminished leakage along root canals filled with gutta-percha without sealer over time: a laboratory study. int endod j. 2000 mar;33(2):121-5. 28. barthel cr, moshonov j, shuping g, orstavik d. bacterial leakage versus dye leakage in obturated root canals. int endod j. 1999 sep;32(5):370-5. 29. siqueira jf jr, rôças in, favieri a, abad ec, castro aj, gahyva sm. bacterial leakage in coronally unsealed root canals obturated with 3 different techniques. oral surg oral med oral pathol oral radiol endod. 2000 nov;90(5):647-50. 30. siqueira jf jr, rôças in, lopes hp, de uzeda m. coronal leakage of two root canal sealers containing calcium hydroxide after exposure to human saliva. j endod. 1999 jan;25(1):14-6. revista fop n 13 1467 clear cell myoepithelioma of the hard palate a.k. agarwal1 ashwani sethi2 shamit chopra3 deepika sareen3 1ms, director, professor 2ms, senior resident 3mbbs, junior resident department of ent & head and neck surgery, maulana azad medical college and associated l. n. hospital received for publication: august 7, 2007 accepted: november 11, 2007 correspondence to: ashwani sethi e-80, naraina vihar new delhi-110028, india phone no: + 91 11 9811338978 e-mail: dr_sethi@rediffmail.com abstract salivary gland myoepitheliomas are rare tumours. we report the case of a 40-year-old female who presented to us with a hard palatal mass. the tumour was excised with a healthy rim of surrounding tissue. histological and immunohistochemical analyses revealed the tumour to be a myoepithelioma of the clear cell variety. key words: myoepithelioma; hard palate; salivary gland tumours; minor salivary gland braz j oral sci. october-december 2007 vol. 6 number 23 1468 introduction myoepithelial cells are contractile cells derived from ectoderm and are seen in major and minor salivary glands, lacrimal glands, breast, sweat glands and prostate1. these cells are thought to be responsible for expulsion and propagation of secretions from the acini and through the ductal network of these tissues. these cells may assume several distinct formsas a spindled, plasmacytoid, clear, stellate or a basket-like cell2. the tumours arising exclusively from these cells are rare and account for less than 1% of all salivary gland tumours3. most of these tumours have been reported in the parotid gland and minor salivary glands on the hard palate4. although, clear cell myoepitheliomas have been reported at other sites5-8, most of the cases of palatal myoepitheliomas have been plasmacytoid or spindle cell type9. we did not find any report of a clear cell myoepithelioma of the hard palate in the review of medical literature. the extreme rarity of such an occurrence prompted us to report this case. case report a 40-year-old female presented to us with a palatal mass of 6 months duration, which has been gradually increasing in size and was painless. the patient had no other significant complaints. the patient had history of chewing tobacco for the past 15 years. examination of the oral cavity revealed a firm, non-tender, well-circumscribed mass located on the hard palate on the right side, measuring 2 cm x 2 cm with a partly ulcerated overlying mucosa (figure 1). nicotine stains could be seen on the teeth. the patient had no trismus or cervical lymhadenopathy. routine hematological and urine analyses and chest x-ray were normal. elisa for hiv was non-reactive. a fine needle aspiration cytology (fnac) of the tumour revealed a cellular minor salivary gland tumour suggestive of a pleomorphic adenoma. an enbloc excision of the tumour with a rim of surrounding normal mucosa was performed and subjected to histopathological evaluation. the postoperative period was insignificant and the patient is totally asymptomatic for the disease without any signs of recurrence one year postoperatively. the specimen consisted of a firm, circumscribed tumour with a smooth external and a solid tan cut surface. on light microscopy, it was found to be surrounded by a thick fibrous capsule. the tumour was uniformly composed of large polyhedral cells with glassy, clear eosinophilic cytoplasm arranged in closely packed interlacing bundles (figure 2). the nuclei were round to oval, eccentrically placed and were vesicular with small nucleoli. immunohistochemically, the clear cells were immunoreactive for cytokeratin, s-100 and muscle specific actin (msa) and non-reactive for epithelial membrane antigen (ema), vimentin and carcinoembryonic antigen (cea). a final diagnosis of clear cell myoepithelioma of the hard palate was made. discussion myoepitheliomas are rare, generally benign tumours with the parotid gland and palate accounting for three-fourths of all cases2. the age range is wide, and there is no significant gender predisposition of this tumour2,4. the plasmacytoid variant appears to have a predilection for the palate in slightly younger individuals, while the spindle cell type tends to occur in the parotid gland of older individuals5-6. on the other hand, clear cell tumours of the salivary glands are almost invariably malignant in nature, with rare exceptions in the form of myoepitheliomas and oncocytomas6. when benign, the tumour most often presents as an asymptomatic mass that slowly enlarges over a course of several months to years. parotid lesions never produce facial dysfunction and those of the palate rarely ulcerate2. our patient had a history of 6 months with a partly ulcerated, slowly growing mass. pathologically, myoepitheliomas vary from 1 to 5 cm in fig. 2 large polyhedral cells with clear eosinophilic cytoplasm and round to ovoid nucleus (h & e staining, 400x). fig.1mass located on the hard palate on the right side with a partly ulcerated overlying mucosa, measuring 2 cm x 2 cm braz j oral sci. 6(23):14671469 clear cell myoepithelioma of the hard palate 1469 greatest dimensions and are well demarcated with a smooth, sometimes bosselated external appearance and a uniform white, tan or gray cut surface. they are generally encapsulated except for palatal myoepitheliomas, which may or may not have a capsule2. in or patient, the tumour measured 2 cm x 2 cm and was encapsulated. microscopically, primarily spindle-shaped cells, occasionally plasmacytoid cells, or a combination of these two cell types constitute these tumours, being the stellate and clear cell variants rare2. immunohistochemically, a large number of markers have been used for establishing the diagnosis of myoepitheliomas. the myoepithelial cells are usually found to be immunoreactive to s-100, actin and prekeartin, and are non-reactive to desmin and ema. other markers that have been used in some reports are cea, secretory piece, high molecular weight keratin, cytokeratin and factor viii antigen9. the reactivity to msa and vimentin has been reported variably in different reports2,4,10. in our patient, the tumour cells were found to be immunoreactive to msa, s-100 and cytokeratin and negative for vimentin and ema. there is a close correlation between histological appearance and prognosis. tumours showing a more aggressive behaviour are mitotically active, more anaplastic and contain fewer myofilaments. cellular pleomorphism, increased mitotic activity, necrosis and invasion indicate towards possibility of malignancy2. these features were absent in our case. wide local excision with a rim of surrounding normal tissue is considered the treatment for myoepitheliomas. our patient also underwent similar treatment without any evidence of recurrence one year following the surgery. myoepitheliomas of the salivary glands are uncommon tumours with clear cell variants still rarer. in our review of medical literature, we did not come across any report of a palatal clear cell myoepithelioma. the diagnosis of these tumours is based on a combination of routine microscopy as well as immunocytochemistry. the treatment is surgical with wide local excision. in conclusion, myoepitheliomas are rare benign tumours arising from myoepithelial contractile cells present in minor and major salivary glands. these tumours are difficult to diagnose on routine histochemical staining and require immunohistochemical staining for diagnosis. the clear cell variants of myoepitheliomas are extremely rare on the palate with no case reported in the pertinent medical literature in the past. wide local excision is usually curative. references 1. hamperl h. the myoepithelia (myoepithelial cells). normal state; regressive changes; hyperplasia; tumors. curr topics pathol. 1970; 53: 161-220. 2. barnes l, appel bn, perez h, el-attar am. myoepitheliomas of the head and neck: case report and review. j surg oncol. 1985; 28: 21-8. 3. scuibba jj, brannon rb. myoepithelioma od salivary glands. report of 23 cases. cancer. 1982; 49: 562-72. 4. ellyn jew, gnepp dr. myoepithelioma of the palate in a child. int j pediatr otolaryngol 1986; 11: 5-13. 5. de steffani a, lerda w, bussi m, valente g, cortesina g. tumours of the parapharyngeal space: case report of clear cell myoepithelioma of the parotid gland and review of literature. acta otolaryngol ital. 1999; 19: 276-82. 6. maiorano e, altini m, favia g. clear cell tumors of the salivary glands, jaws and oral mucosa. semin diagn pathol. 1997; 14: 203-12. 7. son hj, jung sh, lee sy, moon ws. glycogen-rich clear cell mammary malignant myoepithelioma. breast. 2004; 13: 50609. 8. boniuk m, halpert b. clear cell hidradenoma or myoepithelioma of the eyelid. arch ophthalmol. 1964; 72: 59-63. 9. kanazawa h, furuya t, watanabe t, kato j. plasmacytoid myoepithelioma of the palate. j oral maxillofac surg. 1999; 57: 857-60. 10. das dk, haji be, ahmed ms, hossain mnr. myoepithelioma of the parotid glan initially diagnosed by fine needle aspiration cytology and immunocytochemistry. a case report. acta cytol. 2005; 49: 65-70. braz j oral sci. 6(23):14671469 clear cell myoepithelioma of the hard palate braz j oral sci. 15(1):51-56 original article braz j oral sci. january | march 2016 volume 15, number 1 expression of timp-2 in hpv-16 infected oral squamous cell carcinoma in patients in iraq habib a manal1, habib a huda2, hussain a israa1, karima al-salihi3 1university of baghdad, baghdad college of medicine, department of pathology, iraq 2university of baghdad, al-kindy college of medicine, department of community, iraq 3the university of nottingham, school of veterinary medicine and science, loughborough, leicestershire, uk correspondence to: karima al-salihi. the university of nottingham, school of veterinary medicine and science, college road, sutton bonington, loughborough, leicestershire le12 5rd uk e-mail: kareema.nasrullah@nottingham.ac.uk; kama_akool18@yahoo.co.uk; mrvsa59@gmail.com abstract aim: to determine the expression of tissue inhibitors of metalloproteinases (timp-2) in oral squamous cell carcinoma (oscc) and the difference in its expression level between positive and negative hpv-16 (human papilloma virus16) oscc patients. methods: this study was conducted on 33 biopsies obtained from patients with oscc and 10 normal oral mucosa as controls. in situ hybridization (ish) was used to investigate the presence of hpv-16, while immunohistochemistry (ihc) was used to estimate the expression level of timp-2. results: the timp-2 was expressed in 27 (81.8%) of oscc sections with no significant difference between its expression level in hpv-16 positive and hpv-16 negative oscc cases (p=0.058). timp-2 was found to be highly expressed in oscc sections, and the presence of hpv was not related to its overexpression. conclusions: the percentage of samples that appeared to accommodate detectable hpv-16 was high, but no significant difference was observed in relation to timp-2 expression level. future studies with a larger number of patients are highly recommended to address the possible association between timp-2 and oscc positive hpv-16. keywords: timp-2. hpv. iraq. oral cancer. introduction the major neoplasm worldwide is oral cancer. it is accompanied with high death rate, which is associated with a routine late discovery in its development. the recurrence is a serious prognostic influence in patients with oral squamous cell carcinoma (oscc)1-3. according to data from the american oral cancer foundation published in 2014, over 640,000 new cases are found every year4, whereas there were 128,000 deaths due to oral cancer worldwide in 2008 according to the international agency for research on cancer (iarc)5,6. in iraq, cancer made up 10% of total deaths of all ages. oral cancer constituted approximately 4.5% of all cancer cases and oscc represents about 91.5% of all oral cancer and 37% of head and neck cancer according to iraqi cancer registry7. the prevalence of oral cancer during 2003-2006 in iraqi governorates in relation to sex, age and site has been estimated, and the study found that the highest prevalence (55.81%) was observed in tongue, while the lowest (3.87%) was found in the floor of the mouth (al-kawaz, 2010)8. moreover, baghdad governorate was found to have the highest prevalence of oscc (41.08%)8. mohammed et al. (2015)9, detected human herpesvirus-1 antigen in oscc and apparently healthy control in iraq. the detection of hhv-1 antigen, chemicals and radiation, found to play important role in the development of oral cancers in iraq. http://dx.doi.org/10.20396/bjos.v15i1.8647123 received for publication: april 13, 2016 accepted: july 07, 2016 52 epidemiological and molecular studies have determined that the persistence of infection with high-risk human papillomaviruses (hr-hpvs) types is the most common risk factor for the development of head and neck malignancies10-12. hpvs are small non-enveloped, double-stranded, circular dna viruses that encode two viral oncoproteins, e6 and e7. the best-characteristic properties of e6 and e7 proteins from hr-hpvs are their ability to mediate the degradation of p53 and prb, respectively. these viral proteins also interact with other several cellular factors and lead to revoke normal cell cycle checkpoint stand cell death mechanisms13. a possible malignant disorder is the proliferative verrucous leukoplakia (pvl), which is often undergone malignant transformation to oscc14,15. akrish et al., (2014)16 reported and compared the histologic, immunohistochemical and clinical features as well as the survival rates of carcinoma arising in patients with pvl (p-scca) and conventional squamous cell carcinoma (c-scca). the authors found that p-scca revealed considerably better prognostic factors and limited survival rates and longer duration of disease in compare to c-scca. they also suggested that p-scca may show a distinct entity, which may have clinical implications on treatment16. development of most solid tumors is characterized by an increase in secretion and activation of matrix metalloproteinases (mmps) produced by either the tumor cells or tumor-associated fibroblasts. these proteins play a master role in diverse physiological processes and diseases such as the homeostatic tissue remodeling and cancer17. proteolytic activity of mmps can be regulated at different levels including gene expression, protein compartmentalization zymogen to active enzyme conversion and the presence of specific inhibitors18. tissue or extracellular mmps are regulated by endogenous inhibitors named tissue inhibitor metalloproteinase family 1 to 4 (timp-1-4). among them, timp-2 is unique because it may function both as an mmp activator and inhibitor17. although timps are known to act as inhibitors of mmps, they exhibit other biological functions. indeed, timps play complex divergent roles in metastasis, functioning both as anti-invasive agents and as prognostic indicators. another study suggested that the overexpression of timps is correlated with poor outcome in colorectal and breast carcinomas18. in oscc, mmp-9 and timp-2 expressions have shown a predictive value for tumor metastases and cause-specific survival. as in other tissue carcinomas, high expression of timp-2 is the most independent factor for worse prognosis in early-stage oral scc10,12,17. however, expression of timp-2 (mmp) in hpv-16 infected oscc patients is still incomplete and needs further investigations14, whereas, the reliable markers for progression of high-risk hpvinfected epithelium to malignancy are not yet available19. chandolia et al., (2016)20 also approved that oscc shows higher mmp-9 expression as compared to oral epithelial dysplasia followed by epithelium from normal oral mucosa. however, no correlation was found between the histological grades of oscc. a previous immunohistochemical study reported the presence of hpv type 16 infected patients with oscc in iraq21. overexpression of p53 tumor suppressor gene showed significant prevalence of high oncogenic hpv genotypes in patients with oscc in iraq. they found herald marks for the spread of hpv among iraqi general population, which played an important role in oral carcinogenesis21. the expressions of p53 and its related gene expression of timp-2 in hpv-16 infected oral squamous cell carcinoma in patients in iraq braz j oral sci. 15(1):51-56 mdm4 in oral, laryngeal and cutaneous squamous cell carcinoma have also been investigated using tissue microarray in iraq22. it was found that p53 and mdm4 were frequently overexpressed in scc cases with significant correlation between these markers, which was considered as an indicator for prognostic factor in regard to tumor grading22. several molecular markers have been suggested as reliable prognostic cancer biomarkers for the classification of oscc19,21-26. considering all the previous facts regarding ossc in iraq, this study intended to investigate the expression of timp-2, and the difference in its expression level between positive and negative hpv-16 oscc cases. material and methods sample collection the study was conducted on 33 retrospective cases of ossc taken from the maxillofacial center in surgical specialty hospital in baghdad between 2007 and 2009. the age of patients ranged between 30 and 95 years. none of the cases had received radiotherapy or chemotherapy. as negative controls, ten normal oral tissues, were obtained from a buccal mucosa of individuals underwent plastic surgery at the same hospital. this study was approved by the ethical committee / college of the medicine/ university of baghdad (no.2012, 11-12 ar). the expression of hpv-16 and timp-2 were investigated using in situ hybridization (ish) and immunohistochemistry (ihc) respectively. immunohistochemistry fixed paraffin tumor and control tissues were sectioned into 5 µm thickness and stained according to the timp-2 kit manufacture’s protocol (chemicon international, usa). the slides were placed in 10mm citric acid buffer at ph 6.0 and underwent antigen retrieval for 10 min at 680 w in a microwave oven. mouse monoclonal antibody against timp-2 in 5ug/ml (chemicon international, usa) was then added and kept overnight at room temperature. after application of peroxidase labeled secondary antibody and the dab, the sections were finally counterstained and mounted. about 10 representative areas containing 100 scc cells were analyzed. immunostaining was scored using combined quantitative (percentage of tumor cells with immunoreactivity) and qualitative criteria (intensity of staining: none, weak, or strong). these parameters were then combined as: 0= no staining; 1= weak (<10% of tumor cells); 2= weak (≥10% of tumor cells), and 3=strong (≥10% of tumor cells)27. paraffin-embedded sections of colon cancer were immunostained as positive control. in situ hybridization (ish) ish procedure was done using dna probe hybridization/ detection system in situ kit (maxim biotech, usa), according to manufacturer’s protocol. the biotinylated cdna probe for hpv16 (maxim biotech, usa) was diluted to 7% (0.7 µl of the probe diluted in 9.3 µl of hybridization solution). positive control was made with housekeeping gene probe, while the negative control made with hybridization solution without probe. determination of positive reaction was made with nuclear and/or cytoplasmic blue staining of cells28. 53expression of timp-2 in hpv-16 infected oral squamous cell carcinoma in patients in iraq braz j oral sci. 15(1):51-56 statistical analysis statistical analysis of observed data was performed by utilizing spss 22.0 (spss inc., chicago, il, usa) with the application of chi-square and fisher exact tests. results the median age of the patients was 55 years with a mean ± s. d. of 54.5 ± 12.9 years. twenty-five of patients were males (75.8%) and eight (24.2%) were females. the majority of cases (27, 81.8%) were of stage 4 (according to tnm staging system); 4 cases (12.1%) were stage 2 and 2 cases were stage 1 (6.1%). the grades (using border’s system) of the tumor were as follows: low, 4 (12.1%); intermediate, 26 (78.8%) and high, 3 (9.1%). lymph nodes involvements were positive in 23 (85.2%) cases. nuclear hybridization signals for hpv-16 were observed in 24 (72.7%) of oscc paraffin-embedded tissues, while 6 (18.2%) cases appeared negative (table 1). no detectable signals were seen in the sections from the control group. the immunoreactivity of timp-2 observed in 24 (72.7%) and 3 (9.1%) of positive and negative hpv-16 oscc cases, respectively (table 1). immunoreactivity to timp-2 was localized mainly on the cell membrane and the cytoplasm of tumor cells (figure 1. a, b). it was also detected on some stromal cells surrounding the tumor cells. no detectable signals were observed in the sections from the control group (figure 2). the total expression of timp-2 was detected in 27 (81.8%) of oscc cases (table 1) and its expression scores are illustrated in table 2. there was no significant difference in timp2 expression between hpv-16 infected and non-infected cases (p=0.058). in addition, the expression score of timp-2 revealed no significant difference between cases with and without lymph node metastases, p = 0.176 (table 3), whereas, its expression showed no significant difference considering the tumor grade and stage, p = 0.838 and p = 0.169, respectively (table 4). discussion according to a recent review article6, there are shortage in the studies regarding the correlation between oscc, detection of hpv and expression of strictly related tumor markers. however, published articles have shown that high oncogenic hpv genotypes have an essential etiologic role in the development of oscc worldwide17,28-36. the correlation between hpv and oscc was first suggested by syrjänen et al., (1983)14, who detected koilocytotic atypias in malignant oral lesions by optical microscopy and the presence of viral dna by means of ish14. the current study revealed positive nuclear hybridization signals for hpv-16 in 24 (72.7%) of oscc cases. this result is in accordance with those of previous studies, which reported a similar detection rate among hnscc tumors with 90% of the hpv types identified as hpv-1634,35. a previous study also confirmed that oral infection with hpv increased the risk of tumorigenesis of oropharyngeal cancer independent of tobacco use35. the result of the present study is also in agreement with a previous study in iraq, which reported the significant prevalence of high oncogenic hpv genotypes in oscc patients21. the high-risk hpv adopts mechanism to generate the malignant progression of previously benign lesions. earlier investigations have proved that high-risk hpvs and the two hpv-related oncoproteins e6 and e7 can immortalize and transform oral keratinocytes in vitro34,35. pérez-sayáns garcía et al. (2012)32 stated that “the expression of timps in oscc is higher in tumors than in normal tissue, which correlates with an increase of metastatic risk and regional lymph node affectation”. however, the role of timp-2 in cervical carcinoma has been approved to be down-regulated by acute expression of hpv oncoproteins that may favor deregulated mmp activity in the context of hpv infection15,30. fig.1. immunohistochemical expression of timp-2 in a: colon cancer (positive control), b: oscc brown cytoplasmic granular staining in poor differentiated oral squamous cell cancer, strong immunostaining (score 3), (x 200). in this study, immunoreactivity to timp-2 was observed in 27 (81.8%) of oscc cases. this result is in accordance with previous studies, and reflect a pathological role of timp-2 in oscc17,18,3639. the finding of increased timp-2 expression in oral cancer might be explained by the growth-promoting activity of timps on a variety of cell types, or the induction of timps by secreted mmps from tumor–host interaction in the extracellular milieu39. in addition, many immunohistochemical studies addressed the positive role for timp-2 in tumor progression and metastasis3,16,18,21-23,26. it is believed that timp-2 suppresses the tumor invasion and metastases by inhibiting mmp-2. high activation of mmp-2 in oscc has been reported to be associated with high expression of timp-2 in tumor cells26,39,40 timp-2 over-expression has been linked with local tumor invasion, nodal status and clinical stage, as well as with disease-free survival in oral cancer, especially in tongue scc41. in this study, 72.7% of oscc appeared to harbor detectable hpv-16, whereas, no significant difference was observed in relation between positive hpv-16 and timp-2 expression level. however, the p value was borderline (p = 0.058). the results of this study also revealed no significant association between level of timp-2 expression and tumor stage, tumor grade or lymph node metastases. however, many other researchers found significant relationships, and such studies include the following correlations between: a) the expression of timp-2 and tumor size or lymphatic metastasis9,41. b) the expression of timp-1 and lymphatic metastasis9,41,42; c) the expression of mmp-2 and histologic grade or lymphatic metastasis4,14,39,42-45. in conclusion, this study investigated the expression of timp2 in hpv-16 infected tumor tissues of oscc in iraq, and found that the percentage of samples that appeared to accommodate detectable hpv-16 reached up to 72.7%. however, no significant difference was observed in relation to timp-2 expression level. future studies with a larger number of patients are highly recommended to address the possible association between timp-2 and oscc positive hpv-16. references 1. wang b, zhang s, yue k, wang xd. the recurrence and survival of oral squamous cell carcinoma: a report of 275 cases. chin j cancer. 2013 nov;32(11):614-8. doi: 10.5732/cjc.012.10219. 2. scully c, 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10.1016/j.virusres.2009.06.008. expression of timp-2 in hpv-16 infected oral squamous cell carcinoma in patients in iraq braz j oral sci. 15(1):51-56 56 braz j oral sci. 15(1):51-56 37. ikebe t, shinohara m, takeuchi h, beppu m, kurahara s, nakamura s, et al. gelatinolytic activity of matrix metalloproteinase in tumor tissues correlates with the invasiveness of oral cancer. clin exp metastasis. 1999 jun;17(4):315-23. 38. o-charoenrat p, rhys-evans p, eccles sa. expression of matrix metalloproteinases and their inhibitors correlates with invasion and metastasis in squamous cell carcinoma of the head and neck. arch otolaryngol head neck surg. 2001 jul;127(7):813-20. 39. grignon dj, sakr w, toth m, ravery v, angulo j, shamsa f, et al. high levels of tissue inhibitor of metalloproteinase-2 (timp-2) expression are associated with poor outcome in invasive bladder cancer. cancer res. 1996 apr 1;56(7):1654-9. 40. ondruschka c, buhtz p, motsch c, freigang b, schneider-stock r, roessner a, et al. prognostic value of mmp-2, -9 and timp1, -2 immunoreactive protein at the invasive front in advanced head and neck squamous cell carcinomas. pathol res pract. 2002;198(8):509-15. 41. sternlicht md, bissell mj, werb z. the matrix metalloproteinase stromelysin-1 acts as a natural mammary tumor promoter. oncogene. 2000 feb 21;19(8):1102-13. 42. lee ji, jin bh, kim ma, yoon hj, hong sp, hong sd. prognostic significance of cxcr-4 expression in oral squamous cell carcinoma. o r a l s u r g o r a l m e d o r a l p a t h o l o r a l r a d i o l e n d o d . 2 0 0 9 may;107(5):678-84. doi: 10.1016/j.tripleo.2008.12.047. 43. qu h, xin ze, jing x. expression and correlation of mmp-2. timp-2 in oral squamous cell cancer. j hard tissue biol. 2005;14(2):311-2. 44. singh rd, haridas n, patel jb, shah fd, shukla sn, shah pm, et al. matrix metalloproteinases and their inhibitors: correlation with invasion and metastasis in oral cancer. 2010 jul;25(3):250-9. doi: 10.1007/ s12291-010-0060-8. 45. blancato j, singh b, liu a, liao dj, dickson rb. correlation of amplification and overexpression of the c-myc oncogene in high–grade breast cancer: fish, ish and immunohistochemical analysis. br j cancer 2004 apr 19;90(8):1612-9. expression of timp-2 in hpv-16 infected oral squamous cell carcinoma in patients in iraq braz j oral sci. 15(3):176-180 prevalence of porphyromonas gingivalis fima ii genotype in generalized aggressive periodontitis patients richelle soares rodrigues1, catarina martins tahim1, virgínia regia silveira2, nadia accioly pinto nogueira3, rodrigo otavio rego2* 1dds, ms, universidade federal do ceará(ufc), graduate program in dentistry, school of pharmacy, dentistry and nursing, fortaleza, ce, brazil 2 dds, ms, phd, universidade federal do ceará(ufc), department of dentistry, school of dentistry at sobral, sobral, ce, brazil 3msc, phd, universidade federal do ceará(ufc), department of clinical and toxicological analyses, school of pharmacy, dentistry and nursing, fortaleza, ce, brazil correspondence to: rodrigo otavio rêgo federal university of ceara school of dentistry at sobral st. estanislau frota, s/n, sobral-ce 62.011-000, brazil e-mail: rodrigorego@yahoo.com tel/fax: +55 85 33668232 abstract purpose: the objective of this study was to evaluate the prevalence of porphyromonas gingivalis (pg) and its fima ii genotype in a sample of brazilian patients with generalized aggressive periodontitis (gagp) and to correlate the presence of each pathogen/genotype with clinical parameters. methods: we used polymerase chain reaction (pcr) to evaluate the presence of pg and fima ii genotype in subgingival plaque samples collected from the deepest site of 45 brazilian patients aged 15-40 years with gagp and correlated findings with age and clinical parameters (plaque index, gingival bleeding index, probing depth and clinical attachment loss). results: pg was identified in 64.4% patients. fima ii genotype was present in 82.6% of pg-positive patients. the presence of pg and fima ii genotype was significantly associated with greater clinical attachment loss at the sampled periodontal site. pg-positive patients were slightly older than pg-negative patients. conclusions: pg and fima ii genotype were highly prevalent in brazilian patients with gagp. pg was more commonly observed in slightly older individuals and in sites with more clinical attachment loss. keywords: aggressive periodontitis. bacterial fimbriae. porphyromonas gingivalis. introduction aggressive periodontitis (agp) is a rapidly progressive form of periodontitis affecting systemically healthy patients1. less common and more severe than chronic periodontitis, agp tends to run in families, with a preference for younger individuals2. such characteristics suggest the presence of highly virulent pathogens and/or a high level of susceptibility to the disease1,2. over the past few years, much research has been conducted on the etiology and pathogenesis of agp3,4. strong evidence exists for the role of aggregatibacter actinomycetemcomitans (aa), especially the jp2 clone, as an etiological factor in the pathogenesis of agp5. however, in some populations pg can also be associated to agp3,4. this organism displays great genotypical and phenotypical diversity resulting in variations in virulence and ability to induce periodontal destruction6. the virulence of pg is consistently associated with the presence of fimbriae, structures related to cell adhesion7. the fimbriae play an important role in the invasion and colonization of periodontal tissues8. fimbrillin (fima), a subunit protein of the fimbriae in this organism, is encoded received for publication: december 2, 2016 accepted: april 19, 2017 original article braz j oral sci. july | september 2016 volume 15, number 3 http://dx.doi.org/10.20396/bjos.v15i3.8649601 177 by the fima gene6,9. based on nucleotide sequence, six fima genotypes have been identified: fima i, ib, ii, iii, iv and v7,9,10. fima ii is the most prevalent in patients with periodontitis, usually followed by fima iv and ib6,9. the genotypical diversity of fima genotype has been evaluated for a range of periodontal conditions, but to our knowledge only two studies have focused on agp3,11. in these studies, agp was strongly associated with genotype fima ii in japanese11 and chinese subjects3. microbiological variations in agp patients related to ethnicity and socio-demographic conditions justify conducting studies on different populations12,13. in addition, by correlating the presence of periodontal pathogens with clinical parameters, individuals at risk may be identified. thus, we evaluated the prevalence of pg/fima ii genotype in generalized aggressive periodontitis (gagp) brazilian patients and correlated the presence of each pathogen/genotype with clinical parameters. material and methods selection of subjects this study was conducted in 45 gagp patients selected at the periodontology clinic of the school of pharmacy, dentistry and nursing, at the federal university of ceara, brazil. gagp patients were classified according to the clinical criteria suggested by the american academy of periodontology2: generalized interproximal attachment loss affecting at least three permanent teeth other than the first molars and incisors in systemically healthy individuals with rapid attachment loss and bone destruction. to be eligible for the study, patients had to be aged 12-40 years and have at least 20 teeth other than third molars, of which at least three first molars and five incisors. the clinical diagnosis was confirmed by evidence of inter-proximal bone loss on full-mouth periapical radiographs. the exclusion criteria were as follows: periodontal treatment within the previous 6 months, antibiotic therapy within the previous 3 months, smoking habits and systemic changes capable of interfering with periodontal health. the research protocol was approved by the ethics committee of the federal university of ceara, brazil (no 20/08). after being informed about the purpose of the study, all participants or guardians gave their written consent. clinical measurements clinical measurements were made on all the completely erupted permanent teeth, except the third molars, using a periodontal probe (pcp-unc 15, trinity, são paulo, sp, brazil). the following parameters were evaluated: plaque index (pi)14, gingival index (gi)14, probing depth (pd) and clinical attachment loss (cal). pd and cal were taken at six sites per tooth (mesiobuccal, buccal, distobuccal, mesiolingual, lingual, and distolingual). a single examiner evaluated all the clinical periodontal parameters, and measurement reproducibility was calculated by using the intraclass correlation coefficient (icc) for pd and cal. the agreement between replicate measurements was high (icc>0.80). microbiological sampling and evaluation the supragingival plaque was removed with curettes and sterile cotton pellets, and the area was isolated with sterile cotton rolls. subgingival plaque samples were collected by means of two sterile paper points (dentsply maillefer 35, dentsply, rio de janeiro, rj, brazil) at the selected site for 20 seconds. the selected site was the proximal site with the greatest pd and cal of molars or incisors of each patient. the samples were separately immersed in microtubes containing 1 ml ringer’s sterile solution15 and stored at -80°c until they were processed. each sample was processed separately. the microtubes containing the samples were thawed on ice. the bacterial cells or suspension were dispersed by vortexing at the maximum setting for 1 min and centrifuged at 12,000x g for 10 min. genomic dna was extracted from the pellet (instagene matrix, bio-rad laboratories, hercules, ca, usa) and a 20 μl aliquot of the resulting supernatant was added to 30 μl reaction mixture containing 25 μm pcr buffer (promega corporation, madison, wi, usa), 25 μm mgcl2 (promega corporation, madison, wi, usa), 0.2 μm dntp mix (promega corporation, madison, wi, usa), 1.25 u taq polymerase (promega corporation, madison, wi, usa), and 100 ng of each primer (invitrogen, são paulo, sp, brazil), resulting in a final volume of 50 μl16. negative and positive controls were included in each reaction. f i r s t , p c r w a s p e r f o r m e d w i t h u n i v e r s a l p r i m e r s ( 5 ′ g g a c t a y a g g g t a t c t a a t 3 ′ ; 5′-agagtttgatcmtgg-3′)17 for the 16s ribosomal dna (16s rdna) to confirm the presence of bacterial dna. subsequently, the samples were evaluated by pcr with specific primers for the presence of pg (5′-tgtagatgactgatggtgaaaacc-3′; 5 ′ a c g t c a t c c c c a c c t t c c t c 3 ′ ) 1 8 a n d f i m a i i g e n o t y p e ( 5 ´ a c a a c t a t a c t t a t g a c a a t g g 3 ´ ; 5´-aaccccgctccctgtattccga-3´)7. the primers resulted in amplicons of 197 and 257 bp from strains of pg (#hw24d-2) and genotype fima ii (#hw24d-2), respectively, which were used as controls. amplification (biocycler, biosystems, curitiba, pr, brazil) of the 16s rdna was performed with an initial cycle at 94°c for 10 min, followed by 30 cycles at 96°c for 30 s, 55°c for 30 s and 72°c for 30 s, with a final extension at 72°c for 10 min17. for pg, amplification was performed with an initial cycle at 95°c for 5 min, followed by 30 cycles at 94°c for 30 s, 58°c for 30 s and 72°c for 30 s, followed by a final extension at 72°c for 7 min7. for fima ii genotype, amplification was performed with an initial cycle at 95°c for 5 min, followed by 35 cycles at 94°c for 30 s, 58°c for 30 s and 72°c for 30 s, followed by a final extension at 72°c for 7 min9. the amplification products were analyzed by electrophoresis on agarose gel. a 1.5% gel was used to assess the 16s rdna and the presence of pg, while a 2.0% gel was used to assess the fima ii genotype. the gels were stained with sybr® safe (invitrogen, são paulo, sp, brazil) and photographed (canon powershot a640, canon, usa) under ultraviolet light (lta/ltb ge, loccus biotecnologia, são paulo, sp, brazil). pcr was repeated three times for each sample and for each microorganism. statistical analysis the normality of the data distribution was verified with the kolmogorov-smirnov test. comparisons between clinical prevalence of porphyromonas gingivalis fima ii genotype in generalized aggressive periodontitis patients braz j oral sci. 15(3):176-180 178 data and bacterial findings were made with the non-paired t test (for normally distributed data) and the mann-whitney test (for non-normally distributed data). the level of significance was determined to be 5%. all data analyses were performed with the software graphpad instat (graphpad software inc., san diego, ca, usa). results the sample included 45 subjects with gagp aged 15-40 years (mean: 28.8). the clinical findings of the sample are presented in table 1. pg was present in 29 patients (64.4%) and fima ii genotype in 25 (86.2%) of the 29 subjects testing positive for pg. table 2 shows mean periodontal findings according to the presence/absence of pg and fima ii genotype. statistically significant difference was found for age between pg+ and pgpatients. mean clinical attachment loss was significantly greater at the sampled site for microbial analysis in pg+ and also in fima+ patients. prevalence of porphyromonas gingivalis fima ii genotype in generalized aggressive periodontitis patients discussion this study present results to support the idea that pg play an important role in the etiology of agp. it has been associate to agp in certain populations [3,4]. pg was one of the most frequently detected species in agp chinese patients, harboring almost a hundred percent of the patients3. in the current study pg was detected in 64.4% of the subjects. in addition, fima ii genotype was detected in over 85% of patients colonized by pg. among the different pg fimbriae genotypes, fima ii is the most virulent and has been related to periodontal disease progression7,19 and agp3,11. agp is often associated with aa2,20, however, it has been showed that the virulence of aa decreases over time21. there is evidence that the aa leukotoxicity decreased in patients with localized aggressive periodontitis (lagp) originally colonized by highly leukotoxic clones21. another study found an increased prevalence of aa in shallow and intermediate pockets of lagp patients only, whereas the prevalence of pg was high in both lagp and gagp, suggesting aa is predominantly associated with lagp while pg is associated with the progression of lagp and with gagp19. once the patients of the current study presenting pg are older and presented sites with greater periodontal destruction as well as the ones presenting the fima ii genotype, it can be suggested that over time p. gingivalis can play an important role in agp progression. the prevalence of pg observed in this study (64.4%) was lower than the prevalence generally reported in the literature. in three brazilian studies, pg was found in 80%22, 86.7%6 and 73.3%23 of agp patients. in chile24 and in japan11, the prevalence were 88.8% and 79.8%, respectively. in china, it was even higher, colonizing nearly 100% of a sample of chinese agp patients. the smaller prevalence of pg found in the present study also may be related by the fact that our samples were collected from the site of greatest probing depth in each patient, whereas the above-cited studies pooled samples from multiple sites. however, a study comparing samples from one against three deepest periodontal sites, evaluated by molecular biology technique, concluded that there was no difference for the frequency of pg25. to our knowledge, only two studies reported prevalences for pg fima genotypes in patients with agp3,11. the prevalence of fima ii genotype observed in this study may be considered high (86.2%) compared to another results, that evaluated the prevalence of six genotypes in agp patients and found fima ii to be the most common type: 40.5%3 and 47.3%11. these two studies analyzed patients with characteristics similar to ours, including mean age and periodontal destruction. however, while the number of agp patients recruited was greater to feng et al.3, who studied 81 chinese patients, miura et al.11 analysed only 18 japanese patients. fima ii genotype is considered particularly virulent due to its association with periodontal destruction26. fima ii adheres to and invades human epithelial cells more efficiently than other genotypes8. studies also demonstrated that fima ii genotype can induce a stronger inflammatory reaction27,28. in addition, when comparing pg genotypes in periodontitis patients with diabetes after periodontal treatment, fima ii was detected only in subjects with increased levels of glycated hemoglobin (hba1c), while improvements in hba1c values were observed in subjects without table 1 clinical data of the gagp patients. number of patients 45 mean age; age range (years) 28.8 ± 5.9; 15-40 mean number of teeth 26 ± 2 mean pd fm (mm) 3.4 ± 0.7 mean cal fm (mm) 3.8 ± 0.9 mean pd at sampled site (mm) 9.0 ± 1.9 mean cal at sampled site (mm) 9.9 ± 2.2 mean gi (%) 12.7 ± 9.8 mean pi (%) 32.1 ± 17.4 pd ≥5 mm (% of sites) 26.5 ± 14.5 pd ≥5 mm (% of teeth) 59.0 ± 20.6 cal ≥5 mm (% of sites) 31.7 ± 17.0 cal ≥5 mm (% of teeth) 65.2 ± 19.1 values are presented as mean±standard deviation. gagp: generalized aggressive periodontitis; pd: probing depth; cal: clinical attachment loss; gi: gingival index; pi: plaque index; fm: full mouth. table 2 characteristics of the patients according to the presence/ absence of the microorganism/genotype studied. pg+ pgfima ii+ fima iin 29 16 25 4 mean age (years) 30.1 ± 5.7* 26.3 ± 5.5 30.9 ± 5.0 25.5 ± 8.2 mean pd fm (mm) 3.4 + 0.5 3.3 ± 0.9 3.4 ± 0.5 3.5 ± 0.6 mean cal fm (mm) 3.9 ± 0.8 3.7 ± 1.1 3.9 ± 0.8 3.7 ± 0.7 mean pd at sampled site(mm) 9.4 ± 1.8 8.2 ± 1.8 9.6 ± 1.9 8.3 ± 1.3 mean cal at sampled site(mm) 10.5 ± 2.2* 8.8 ± 1.9 10.8 ± 2.1** 8.5 ± 1.3 pi (%) 37.3 ± 16.6 25 ± 16.5 38.2 ± 13.3 29.7 ± 9.7 gi (%) 13.5 ± 11 11.3 ± 8.3 14.7 ± 11.3 7.3 ± 6.5 values are presented as mean±standard deviation. *statistically significant difference (p<0.05) between pg+ and pg-. **statistically significant difference (p<0.05) between fima ii+ and fima ii-. pg+: presence of porphyromonas gingivalis; pg-: absence of porphyromonas gingivalis; fima ii+: presence of fima ii genotype; fima ii-: absence of fima ii genotype; pd: probing depth; cal: clinical attachment loss; pi: plaque index; gi: gingival index fm: full mouth. braz j oral sci. 15(3):176-180 179 prevalence of porphyromonas gingivalis fima ii genotype in generalized aggressive periodontitis patients type ii clones, suggesting that glycemic levels in diabetes are affected by the persistence of pg fima ii genotype29. these studies suggest that fima ii genotype is an important factor of virulence in pg and may play a role in inflammatory response not only in periodontitis, but also in systemic diseases28,29. in our study, the presence of pg and genotype fima ii was associated with greater clinical attachment loss at the site sampled for microbial analysis. these findings are supported by studies that observed a higher prevalence of pg at sites with greater attachment loss in patients with chronic periodontitis4,13, and that founded the prevalence of fima ii genotype to be greater in deeper periodontal pockets7,26. therefore, the presence of this genotype may be used to identify individuals at risk, establish plan treatments and prognosis. these findings can also be useful in the development of novel treatment strategies, such as passive immunotherapy30. in conclusion, in the present sample of brazilians with gagp, the prevalence of pg and fima ii genotype was high. additionally, pg was more prevalent among slightly older patients and at sites with greater periodontal destruction, and fima ii genotype was more present in sites with greater clinical attachment loss. conflict of interest no potential conflict of interest relevant to this article was reported. acknowledgements this study was supported by grants from cnpq (478161/20077) and capes (procad nf 2313/2008). the authors would like to thank the oral microbiology group from academic centre for dentistry amsterdam (acta) for providing porphyromonas gingivalis strains. references 1. albandar jm. aggressive periodontitis : case definition and diagnostic criteria. periodontol 2000. 2014 jun;65(1):13-26. doi: 10.1111/prd.12014. 2. american academy of periodontology. parameter on aggressive periodontitis. j periodontol. 2000 may;71(5 suppl):867-9. 3. feng x, zhang l, xu l, meng h, lu r, chen z, et al. detection of eight periodontal microorganisms and distribution of porphyromonas gingivalis fima genotypes in chinese patients with aggressive periodontitis. j periodontol. 2014 jan;85(1):150-9. doi: 10.1902/ jop.2013.120677. 4. yano-higuchi k, takamatsu n, he t, umeda m, ishikawa i. prevalence of bacteroides 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krieger jk, eickholz p. two subgingival plaque-sampling strategies used with rna probes. j periodontol. 2007 jan;78(1):72-8. 26. zhao l, wu yf, meng s, yang h, ouyang yl, zhou xd. prevalence of fima genotypes of porphyromonas gingivalis and periodontal health status in chinese adults. j periodontal res. 2007 dec;42(6):511-7. 27. kato t, kawai s, nakano k, inaba h, kuboniwa m, nakagawa i, et al. virulence of porphyromonas gingivalis is altered by substitution of fimbria gene with different genotype. cell microbiol. 2007 mar;9(3):753-65. 28. sugano n, ikeda k, oshikawa m, sawamoto y, tanaka h, ito k. differential cytokine induction by two types of porphyromonas gingivalis. oral microbiol immunol. 2004 apr;19(2):121-3. 29. makiura n, ojima m, kou y, furuta n, okahashi n, shizukuishi s, et al. relationship of porphyromonas gingivalis with glycemic level in patients with type 2 diabetes following periodontal treatment. oral microbiol immunol. 2008 aug;23(4):348-51. doi: 10.1111/j.1399302x.2007.00426.x. 30. hijiya t, shibata y, hayakawa m, abiko y. a monoclonal antibody against fima type ii porphyromonas gingivalis inhibits il-8 production in human gingival fibroblasts. hybridoma (larchmt). 2010 jun;29(3):201-4. doi: 10.1089/hyb.2009.0109. braz j oral sci. 15(3):176-180 1http://dx.doi.org/10.20396/bjos.v18i0.8655466 volume 18 2019 e191431 original article 1 faculty of dentistry. state university of paraiba, campina grande – pb, brazil 2 postgraduate program in dentistry, federal university of pernambuco ufpe, recife pe, brazil; 3 postgraduate program in dentistry, federal university of minas gerais ufmg, belo horizonte mg, brazil. corresponding author: renata cimões federal university of pernambuco ufpe, av. prof. moraes rego, 1235 cidade universitária, 50670-901, recife-pe, brazil email: renata.cimoes@globo.com received: october 22, 2018 accepted: may 06, 2019 evaluation of oral health-related quality of life in individuals with type 2 diabetes mellitus raulison vieira de sousa¹, roberto carlos mourão pinho2, bruna de carvalho farias vajgel2, saul martins paiva3, renata cimões2,* aim: the aim of the present study was to evaluate the impact of oral problems on the quality of life of individuals with type 2 diabetes mellitus (dm2). methods: a population-based, cross-sectional study was conducted with a random sample of 302 individuals with dm2 who answered the oral health impact profile 14 (ohip-14) questionnaire as well as a questionnaire addressing socioeconomic and oral health characteristics. after filling out the questionnaires, the participants were submitted to a clinical dental examination periodontal diseases, dental caries and edentulism. data analysis involved descriptive statistics, bivariate analysis and logistic regression. results: the prevalence of impact on oral health-related quality of life (ohrqol) was 47%. in the multivariate analysis, the variables that remained significantly associated with a negative impact on quality of life were xerostomia (or= 2.15; 95% ci: 1.07-4.30), denture need (or= 3.71; 95% ci: 1.17-11.73) and periodontitis (or= 5.02; 95% ci: 2.19-11.52). conclusion: the prevalence rate of impact on ohrqol was high in the sample studied. xerostomia, denture need and periodontitis posed a risk of negative impact on the quality of life of individuals with dm2, independently of socioeconomic status. keywords: oral health. quality of life. diabetes mellitus. 2 de sousa et al. introduction type 2 diabetes mellitus (dm2) is a metabolic disorder characterized by high levels of glucose in the blood due to defects in the action and secretion of insulin1. dm2 is the most common form of diabetes, accounting for 90 to 95% of all cases and generally occurs in obese adults over 40 years of age. however, there has been an increase in cases diagnosed in younger people due to the association between dm2 and obesity, the incidence of which is also high among younger people2. from the epidemiological standpoint, diabetes is considered a public health problem in both developed and developing countries2. the number of individuals with diabetes was 171 million throughout the world in 2000 and is expected to reach 366 million by 20303. in brazil, the most recent study published on this issue reports approximately 10.3 million individuals with diabetes4. dm2 is associated with systemic complications, such as microvascular diseases (retinopathy, nephropathy and neuropathy) as well as cerebrovascular and cardiovascular diseases5. dm2 accounts for 5.2% of deaths in brazil and is an important risk factor for cardiovascular disease, which accounts for 31.3% of deaths in the country6. among the oral problems found in patients with dm2, high prevalence rates of periodontitis, dental caries, edentulism and xerostomia have been described5,7-11. there are no characteristic phenotypic features that are unique to periodontitis in patients with diabetes mellitus. on this basis diabetes-associated periodontitis is not a distinct disease. nevertheless, diabetes is an important modifying factor of periodontitis, and should be included in a clinical diagnosis of periodontitis as a descriptor. according to the new classification of periodontitis, the level of glycemic control in diabetes influences the grading of periodontitis12. studies reveal that individuals with dm2 are at greater risk for the development of periodontal disease due to the diminished defense mechanisms against the action of biofilm (bacterial plaque)9,11,13. although not pathognomonic of dm2, these oral problems are highly prevalent in this population and can exert a negative influence on quality of life due to the functional, psychological and social impacts8,14. the few studies have evaluated the impact of oral problems on the quality of life of individuals with diabetes have methodological limitations, such as the absence of a population-based sample, the studies in the review vary in quality and have several common methodological limitations. these include: lack of reported response rates, varying questionnaires used to measure study outcomes; limited validated questionnaires and inadequate discussion of confounding factors that may have affected the findings (age, education, income level). studies included were from both high and low income countries and therefore it is not known whether the different health care systems and cultural beliefs across these countries could have affected the knowledge, attitudes and practices of people with diabetes in relation to oral health care. self-reported data from the studies also limit the generalization of the findings. the systematic review undertaken also has limitations. there is also the possibility of outcome reporting bias15. thus, the aim of the present study was to investigate the impact of oral problems on the quality of life of individuals 3 de sousa et al. with type 2 diabetes mellitus in a population-based study conducted in northeastern brazil. materials and methods characterization of sample a population-based sample was conducted involving a randomly selected sample by simple lottery of 302 male and female individuals with dm2 (mean age: 63.1 years) registered with primary care units of the family health program in the municipality of pombal, state of paraíba, brazil. the participants were selected from a total population of 778 individuals with dm2 according to data furnished by the municipal secretary of health. the municipality of pombal is located in northeastern brazil and has an estimated population of 32,766 inhabitants as well as a human development index of 0.63416. the sample size was calculated using a proportion estimate for a finite population and considering a 5% margin of error, 95% confidence interval and 50% prevalence rate of the oral problems investigated. the minimum sample was determined to be 258 individuals, to which 20% was added to compensate for possible dropouts. thus, the sample was composed of 310 individuals. ethical aspects this study received approval from the human research ethics committee of the universidade federal de pernambuco (certificate number: 47981015.8.0000.5208) and was conducted in compliance with the precepts stipulated in resolution nº 466 of december 12, 2012 of the brazilian national board of health. all participants received clarifications regarding the objectives and procedures of the study and agreed to participate by signing a statement of informed consent. eligibility criteria the inclusion criteria were registration with a primary care unit of the family health program in the municipality of pombal, diagnosis of dm2 at least one year earlier based on the criteria recommended by the brazilian society of diabetes (fasting blood glucose ≥ 126 or glycated hemoglobin > 6.5%),2 age 18 years or older and signed statement of informed consent. the exclusion criteria were neuropsychomotor disorder, pregnancy and systemic complications of dm2 that could lead to an underestimation of the impact of oral problems on quality of life, such as amputations and blindness. training and calibration exercises the training and calibration exercises were conducted by a researcher with ample experience in the use of the epidemiological indices employed in this study. the first step consisted of theoretical explanations of the indices and the data collection routine. in the practical phase, the experienced researcher and the examiner being trained performed clinical examinations of 30 patients using the indices employed in the study. the level of agreement between the examiner and experi4 de sousa et al. enced researcher regarding the diagnoses was determined. the 30 patients were examined again after a seven-day interval for the determination of intra-examiner agreement. cohen’s kappa statistic was used for this purpose, which furnished the following minimum coefficients for the variables collected: inter-examiner k = 0.85 and intra-examiner k = 0.8717. pilot study a pilot study was conducted with 30 individuals prior to the main study to test the methods as well as the use of the questionnaire and clinical charts. the sample in the pilot study was composed of individuals with dm2 from the same municipality. these individuals were not included in the main study. data collection non-clinical data the participants answered a questionnaire administered in interview form addressing socioeconomic characteristics and aspects related to oral health, such as oral hygiene frequency and visits to a dentist. the brazilian version of the oral health impact profile 14 (ohip-14) was used for the assessment of oral health-related quality of life (ohrqol)18. this scale has 14 items distributed among seven domains (functional limitation, pain, psychological discomfort, physical disability, psychological disability, social disability and handicap). each item has five response options on a rating scale: never = 0; rarely = 1; sometimes = 2; often = 3; and very often = 4. as recommended by the authors of the questionnaire, impact on ohrqol was considered when at least one item was scored ≥ 2 (response options “sometimes”, “often” and “very often”) and the absence of impact was considered when all items were scored ≤ 1 (response options “never” and “rarely”). clinical data after filling out the questionnaires, the participants were submitted to a clinical dental examination by the dentist who had undergone the training and calibration exercises. the clinical examinations were conducted at the dental offices of the primary care units in the municipality of pombal. the examiner used individual protective equipment (white coat, mask, gloves and protective eyewear). all instruments and materials used during the examination, such as a mouth mirror (prisma, são paulo, sp, brazil), university of north carolina periodontal probe (pcpunc 15® hu-friedy, chicago, il, usa), ball point probe (golgran, são paulo, sp, brazil) and dental gauze, were sterilized and packed into individual kits for each patient. after the examination, individuals with oral problems were sent for treatment. the conditions investigated during the examination were periodontitis, dental caries, xerostomia and edentulism. for the analysis of peridontitis, all teeth were examined, except third molars and teeth indicated for extraction. each tooth was probed at six sites (mesio-vestibular, mid-vestibular, disto-vestibular, disto-lingual, mid-lingual and mesio-lingual). peri5 de sousa et al. odontitis was diagnosed based on gingival recession, probing depth, clinical attachment loss, bleeding on probing and tooth mobility, regarding dental mobility, the following classification was used: grade 1 (mobility of the tooth crown 0.2 1.0mm horizontally); grade 2 (mobility of the dental crown exceeding 1.0mm horizontally); and grade 3 (mobility of the tooth crown in the vertical and horizontal directions)12,19,20. the criteria established by the american academy of periodontology21 were used for the classification of severity based on the occurrence of at least one site with the following combinations of periodontal findings (table 1). also using the criteria of the american academy of periodontology21, the extent of periodontitis was classified as localized (≤ 30% of teeth affected) or generalized (> 30% of teeth affected). dental caries was assessed using the decayed, missing and filled teeth (dmft) index recommended by the world health organization22. edentulism was classified based on the number of missing teeth (edentulous arch, short arch or complete arch) as well as the location of the missing teeth (anterior loss, posterior loss or anterior and posterior loss)23. denture need was evaluated using an adaptation of the criteria used in the 2010 brazil smiling program24 :absence of need (all teeth present, some missing teeth with dentures in adequate condition for use and complete edentulism with dentures in adequate condition for use) and presence of need (missing teeth with no dentures, complete edentulism with no dentures or dentures present but inadequate for use). xerostomia was evaluated based on the study conducted by busato et al. (2012). the following question was posed: “have you had a sensation of dry mouth every day for the last six months?” xerostomia was considered present when the respondent answered “yes”25. statistical analysis descriptive statistics were performed for the characterization of the sample with regard to socioeconomic, oral health and clinical data as well as the ohip-14 items. in the bivariate analyses, the chi-square test and likelihood ratio test were used to determine associations between the independent variables and negative impact on quality of life (p < 0.05). multivariate logistic regression analysis was then performed using the forward stepwise procedure, in which each variable with a p-value < 0.20 in bivariage analysis was incorporated into the model one by one. the data were entered an excel spreadsheet and subsequently analyzed using the spss for windows, version 20.0 (spss, chicago, il, usa). table 1. classification of severity based on the occurrence of at least one site with the following combinations of periodontal findings mild moderate severe probing depth > 3 and < 5 mm ≥ 5 and < 7 mm ≥ 7 mm bleeding on probing present present present clinical attachment loss 1-2 mm 3-4 mm ≥ 5 mm 6 de sousa et al. results three hundred two individuals with dm2 participated in the present study, corresponding to 97.4% of the total number of individuals selected based on the sample calculation. the eight dropouts (2.6%) were individuals who declined to participate during the data collection. however, the final number of participants was higher than the minimum number determined during the calculation of the sample size. the analysis of the distribution of the sample according to the socio-demographic data revealed that the female sex accounted for 71.2% of the sample. mean age was 63.1 years and 58.9% of the participants were between 51 and 70 years of age. the majority was married (58.9%). monthly household income ranged from r$ 80 to r$ 10.000 and 60.3% earned up to the brazilian monthly minimum wage. a total of 77.8% of the participants had an incomplete primary school education, 50.3% reported being retired and 22.5% reported having paid employment. moreover, 48.7% reported going to the dentist due to pain and 37.4% reported brushing their teeth three times a day. with regard to the clinical diagnoses, 49.3% of the individuals examined had a short arch, 47.7% had an edentulous arch; 85.4% had anterior and posterior tooth loss and 72.2% had denture needs. the prevalence of xerostomia was 52.6% and 29.5% had at least one tooth with caries experience. bleeding on probing occurred in 47.7% of the patients and periodontitis was diagnosed in 38.4%, among whom 49.1% had severe periodontitis, 25% had moderate periodontitis and 25.9% had mild periodontitis. with regard to extent, 68.1% of these individuals with had generalized periodontitis and 31.9% had localized periodonditis. tooth mobility was diagnosed in 30.2% of the sample, 37.1% of whom had grade 1, 31.4% had grade 2 and 31.4% had grade 3 (table 2). the prevalence of impact on ohrqol in the sample was 47%. the ohip-14 items with the greatest frequency of impact were items 3 (“have you had painful aching in your mouth?”) and 4 (“have you found it uncomfortable to eat any foods because of your teeth, mouth or dentures?”), with rates of 53% and 57.9%, respectively (table 3). these items belong to the pain domain, which was the most prevalent (74.5%), followed by the physical disability (56.3%) and psychological discomfort (51.0%) domains (table 4). in the bivariate analysis, the independent variables significantly associated with the impact on ohrqol were edentulism (p < 0.001), denture need (p = 0.002), bleeding on probing (p = 0.007), periodontitis (p = 0.000) and degree of mobility (p = 0.017) (table 5). in the multivariate analysis, xerostomia (or = 2.15; 95% ci: 1.07 to 4.30), denture need (or = 3.71; 95% ci: 1.17 to 11.73) and periodontitis (or = 5.02; 95% ci: 2.19 to 11.52) remained significantly associated with a negative impact on ohrqol (table 6). in the analysis per ohip-14 domain, xerostomia was significantly associated with all domains (p < 0.05), except physical disability (p = 0.082) and social disability (p = 0.132). denture need was significantly associated with the pain, psychological discomfort and physical disability domains. periodontitis was associated with all domains (p < 0.05) except social disability (p = 0.062). 7 de sousa et al. table 2. distribution of the sample according to clinical diagnosis data variable n % edentulism full arch 9 3,0 short arch 149 49,3 toothless arch 144 47,7 location of dental loss loss only anterior 1 0,3 loss only posterior 34 11,3 loss anterior and posterior 258 85,4 no information ( full arch) 9 3,0 denture need no 84 27,8 yes 218 72,2 xerostomia no 143 47,4 yes 159 52,6 number of carious teeth (nc) nc=0 69 22,8 nc>0 89 29,5 no information (tothless arch) 144 47,7 bleeding after probing absent 14 4,6 present 144 47,7 no information (tothless arch) 144 47,7 periodontitis ausent 42 13,9 present 116 38,4 no information (tothless arch) 144 47,7 severity of periodontitis light 30 25,9 moderate 29 25,0 severe 57 49,1 total 116 100,0 periodontitis extension localized 37 31,9 generalized 79 68,1 total 116 100,0 dental mobility no 81 69,8 continue 8 de sousa et al. continuation yes 35 30,2 total 116 100,0 grade of dental mobility grade 1 13 37,1 grade 2 11 31,4 grade 3 11 31,4 total 35 100,0 total 302 100,0 table 3. prevalence of impact of oral alterations on ohip-14 quality of life among subjects with dm2 ohip (questions) whithout impact (never; rarely) with impact (sometimes, repeatedly, always) total n % n % n % q01 speech 240 79,5 62 20,5 302 100,0 q02 palate 199 65,9 103 34,1 302 100,0 q03 pain 142 47 160 53 302 100,0 q04 chewing 127 42,1 175 57,9 302 100,0 q05 worried 169 56 133 44 302 100,0 q06 tense 216 71,5 86 28,5 302 100,0 q07 alimentation 155 51,3 147 48,7 302 100,0 q08 meal 172 56,9 130 43,1 302 100,0 q09 relax 223 73,9 79 26,1 302 100,0 q10 shame 194 64,2 108 35,8 302 100,0 q11 irritation 251 83,1 51 16,9 302 100,0 q12 – daily activities 238 78,8 64 21,2 302 100,0 q13 life 220 72,9 82 27,1 302 100,0 q14 work 239 79,2 63 20,8 302 100,0 table 4. prevalence of impact of oral changes in quality of life per ohip-14 domain among individuals with dm2 ohip (dimension) without impact with impact total n % n % n % functional limitation 176 58,3 126 41,7 302 100,0 pain 77 25,5 225 74,5 302 100,0 psychological discomfort 148 49,0 154 51,0 302 100,0 physical inability 132 43,7 170 56,3 302 100,0 psychological inability 162 53,6 140 46,4 302 100,0 social inability 209 69,2 93 30,8 302 100,0 disability 190 62,9 112 37,1 302 100,0 9 de sousa et al. table 5. impact on quality of life (qol) according to the variables independentes variable impact qol total valor p or (ic 95%) without impact with impacto n % n % n % sex male 44 27,5 43 30,3 87 28,8 0,5941 0,87 (0,53-1,44) female 116 72,5 99 69,7 215 71,2 total 160 100,0 142 100,0 302 100,0 age up to 50 years 21 13,1 23 16,2 44 14,6 0,1331 (-) from 51 to 70 years 89 55,6 89 62,7 178 58,9 over to 70 years 50 31,3 30 21,1 80 26,5 total 160 100,0 142 100,0 302 100,0 civil status single 18 11,3 15 10,6 33 10,9 0,4791 (-) married 90 56,3 88 62,0 178 58,9 divorced 7 4,4 9 6,3 16 5,3 widower 45 28,1 30 21,1 75 24,8 total 160 100,0 142 100,0 302 100,0 income up to r$937,00 95 59,4 87 61,3 182 60,3 0,6642 (-) from r$937,00 to r$2811,00 56 35,0 51 35,9 107 35,4 from r$2811,00 to r$4685,00 6 3,8 3 2,1 9 3,0 over r$4685,00 to r$ 14055,005 3 1,9 1 0,7 4 1,3 total 160 100,0 142 100,0 302 100,0 scholarity up to grade 1 incomplete 129 80,6 106 74,6 235 77,8 0,3891 (-) 1st to 2nd grade 15 9,4 21 14,8 36 11,9 2nd grade until univers incomplete 12 7,5 9 6,3 21 7,0 univ comp to postgrad / graduate 4 2,5 6 4,2 10 3,3 total 160 100,0 142 100,0 302 100,0 work activity work 32 20,0 36 25,4 68 22,5 0,0921 (-) housewife 38 23,8 44 31,0 82 27,2 retired 90 56,3 62 43,7 152 50,3 total 160 100,0 142 100,0 302 100,0 visit to the dentist never 16 10,0 9 6,3 25 8,3 0,1601 (-) because of the pain 75 46,9 72 50,7 147 48,7 once a year 48 30,0 31 21,8 79 26,2 twice a year 13 8,1 16 11,3 29 9,6 more than twice a year 8 5,0 14 9,9 22 7,3 total 160 100,0 142 100,0 302 100,0 continue 10 de sousa et al. continuation brush the teeth less than once a day 5 3,1 5 3,5 10 3,3 0,9491 (-) once a day 23 14,4 22 15,5 45 14,9 twice a day 57 35,6 52 36,6 109 36,1 three times a day 63 39,4 50 35,2 113 37,4 more than three times a day 12 7,5 13 9,2 25 8,3 total 160 100,0 142 100,0 302 100,0 edentulism full arch 9 5,6 9 3,0 <0,0012 (-) short arch 67 41,9 82 57,7 149 49,3 toothless arch 84 52,5 60 42,3 144 47,7 total 160 100,0 142 100,0 302 100,0 location of dental losses loss anterior 1 0,7 1 0,3 0,3272 (-) loss posterior 20 13,2 14 9,9 34 11,6 loss anterior e posterior 131 86,8 127 89,4 258 88,1 total 151 100,0 142 100,0 293 100,0 denture need no 57 35,6 27 19,0 84 27,8 0,0021 2,36 (1,39-4) yes 103 64,4 115 81,0 218 72,2 total 160 100,0 142 100,0 302 100,0 xerostomia no 84 52,5 59 41,5 143 47,4 0,0741 1,55 (0,99-2,45) yes 76 47,5 83 58,5 159 52,6 total 160 100,0 142 100,0 302 100,0 number of carious teeth (nc) nc=0 37 48,7 32 39,0 69 43,7 0,2881 1,48 (0,79-2,79) nc>0 39 51,3 50 61,0 89 56,3 total 76 100,0 82 100,0 158 100,0 bleeding after probing no 12 16,0 2 2,4 14 8,9 0,0073 7,62 (1,6435,29) yes 63 84,0 80 97,6 143 91,1 total 75 100,0 82 100,0 157 100,0 periodontitis no 32 42,1 10 12,2 42 26,6 0,0001 5,24 (2,3511,69) yes 44 57,9 72 87,8 116 73,4 total 76 100,0 82 100,0 158 100,0 severity periodontitis light 13 29,5 17 23,6 30 25,9 0,1941 (-) moderate 14 31,8 15 20,8 29 25,0 severe 17 38,6 40 55,6 57 49,1 total 44 100,0 72 100,0 116 100,0 periodontitis extension localized 18 40,9 19 26,4 37 31,9 0,1551 1,93 (0,87-4,29) generalized 26 59,1 53 73,6 79 68,1 total 44 100,0 72 100,0 116 100,0 dental mobility no 35 79,5 46 63,9 81 69,8 0,1151 2,2 (0,92-5,28) yes 9 20,5 26 36,1 35 30,2 total 44 100,0 72 100,0 116 100,0 continue 11 de sousa et al. discussion studies on ohrqol are more complete that those restricted to measuring clinical data due to the ability to express the extent of the negative impact of oral problems on the lives of populations and therefore constitute an important collective health tool that can contribute to the planning of public health policies26-28. studies have evaluated quality of life in patients with dm2, but few have investigated ohrqol in this population27-29. in the present study, oral problems exerted a negative impact on quality of life among nearly half of the population with dm2. a similar result is reported in a study conducted in the united states, in which the prevalence of impact on ohrqol was 47.7%28. other studies, however, report lower prevalence rates ranging from 22.5 to 34.4%10,25,29. such divergences may be explained by cultural differences among the populations surveyed as well as differences in the methods employed in the studies. a strong point of the present investigation is the fact that it was the population-based study with a randomized, representative sample. the ohip-14 items related to pain were the most prevalent. the population studied reported greater impact on items 3 (“have you had painful aching in your mouth?”) and 4 (“have you found it uncomfortable to eat any foods because of problems with your teeth, mouth or dentures?”), with rates of 53% and 57.9%, respectively. these findings are similar to those reported in studies conducted in iran14, the united kingdom30 and brazil31, suggesting that oral problems with the potential to cause physical pain and discomfort have the greatest negative impact on quality of life. continuation grade of dental mobility grade 1 5 55,6 8 30,8 13 37,1 0,0172 (-) grade 2 4 44,4 7 26,9 11 31,4 grade 3 11 42,3 11 31,4 total 9 100,0 26 100,0 35 100,0 1pearson’s chi-square test; 2 likelihood ratio test; 3 chi-square with continuity correction; r$ real. table 6. multivariate analysis of the association between independent variables and impact on ohrqol variable coef. e.p. χ2 valor p or1 ic 95%  minimum maximum denture need 1,31 0,59 4,98 0,026 3,71 1,17 11,73 xerostomia 0,76 0,35 4,63 0,031 2,15 1,07 4,30 periodontitis 1,61 0,42 14,53 0,000 5,02 2,19 11,52 constante -2,70 0,70 14,72 0,000 0,07 hosmer-lemeshow test p-valor   test omnibus p-valor r2 of nagelkerke 2,91 0,573   27,912 0,000 0,216 legend: : c2 chi-square; 1-or-odds ratio; ci confidence interval; coefcoefficient of the variable; e.p standard error; r2 coefficient of determination 12 de sousa et al. the socio-demographic data were not significantly associated with ohrqol. this finding is in agreement with data described in a study conducted in iran14, but is in disagreement with findings described in other studies8,26,32. in the present investigation, the sample was quite homogeneous with regard to socio-demographic variables, especially sex, income and schooling, which may have influenced the results, as reported in study of mohamed et al ( 2013)10, where educational level was originally measured as (0 = illiterate, 1 = literate, 2 = primary school, 3 = middle school, 4 = high school, 5 = college, 6 = post-graduation studies) and was recoded into illiterate = 1 (including the original category 0) and literate = 2 (including the categories 1–6). employment status was measured as (0 = unemployed, 1 = student, 2 = housewife, 3 = retired, 4 = employed), then recoded into unemployed = 1 (including the original categories 0–3) and employed = 2 (including the original category 4). the literature reports that individuals with inadequate oral hygiene habits and infrequent visits to a dentist have a greater chance of having an unfavorable oral health status, which can exert a negative impact on quality of life26,33. such findings suggest that the effects of self-care and dental treatment can improve ohrqol. however, this association was not found in the present study, which may be explained by the profile of the sample. the fact that nearly half of the sample was composed of completely endentulous individuals may have led to an underestimation of the role of oral problems, such as dental caries and periodontal disease, which are dependent on the control of biofilm and are therefore related to hygiene habits. there is a consensus in the literature regarding the role of dental caries as a factor associated with a negative impact on quality of life in different populations, including individuals with dm214,34. caries is an oral problem that can cause pain and, in some situations, have a negative impact on esthetics, with functional, psychological and social repercussions35. in the present study, however, no such association was found in the individuals with dm2. one should bear in mind that the sample was composed mainly of older adults and other oral problems, such as periodontal disease, are more prevalent than dental caries in this age group35 and therefore have a greater impact on quality of life. xerostomia is of the most prevalent oral manifestations in diabetic patients and exerts a negative impact on quality of life due to the fact that it affects speaking, the use of dentures and food intake14,23,36,37. this condition was highly prevalent in the present investigation, which is similar to data described in a study conducted in sweden involving adults with dm28. however, a difference observed between the two studies regarding the impact on quality of life. unlike the study conducted in sweden, xerostomia was associated with a negative impact on quality of life in nearly all the domains of the ohip-14 in the present investigation. in a brazilian study involving patients with type 1 diabetes mellitus, the authors also report the impact of xerostomia on quality of life, demonstrating the importance of the prevention and treatment of this condition for improving the quality of life of diabetic patients17. it should be pointed out that xerostomia is a condition that may or may not be associated with hyposalivation. the evaluation of xerostomia is limited to self-reported information and is considered to be an important aspect of ohrqol in patients with dm217. however, further studies 13 de sousa et al. should be conducted involving the analysis of saliva flow to determine the impact of hyposalivation of ohrqol in this population. a strong association was found between periodontitis and the negative impact on quality of life, which is in agreement with data described in previous studies10,14,38. the multivariate analysis revealed that individuals with dm2 and a diagnosis of periodontitis had a fivefold greater risk of a negative impact on quality of life. moreover, all domains of the ohip-14, except social disability, were significantly associated with periodontitis. these findings confirm the fact that periodontitis is the most important oral complication of diabetes due not only to its high prevalence, but also its impact on quality of life, underscoring the need for specific strategies aimed at minimizing the negative effects of periodontal disease on the quality of life of individuals with dm2. in many cases, tooth loss is a consequence of periodontal disease. edentulism has functional and esthetic repercussions that compromise quality of life8. differently from the findings of previous studies8,10,39, however, no significant association was found between edentulism and quality of life in the present investigation when considering either the number or location of missing teeth. the divergence in comparison to other studies may reflect differences in the meaning attributed to tooth loss in different social and cultural contexts. for some populations, tooth loss is understood as a natural circumstance of the ageing process40, which may make this condition not have a negative impact on quality of life. as reported in other studies41,42, individuals with denture needs in the present investigation had a greater chance of experiencing a negative impact on quality of life. in the analysis per domain, denture need was associated with the domains related to pain, psychological discomfort and physical disability. the absence of the impact of tooth loss and the associations between denture need and the domains cited reveal that tooth loss is not important to the population studied provided that prosthetic rehabilitation is adequate. moreover, this finding supports the inference that the experience of pain, stress and functional loss stemming from edentulism prevails over the impact on esthetics and its psychological and social repercussions. the present study has limitations that should be addressed. the cross-sectional design places limits on causal inferences between the independent variables and the occurrence of impact on quality of life. therefore, longitudinal studies should be performed to confirm the inferences revealed in the present investigation. moreover, there is the possibility of memory bias with regard to questions related to the past. however, the present investigation was a population-based study randomized, and the results can be extrapolated to the population, making the findings useful for the definition of priorities that need to be considered in the planning of public health policies directed at the population with dm2. the predictive factors of a negative impact on ohrqol differ among different populations and therefore the needs of each population should be analyzed in an individualized manner. oral healthcare policies for individuals with type 2 diabetes mellitus should encompass specific strategies based on studies addressing ohrqol 14 de sousa et al. in conclusion, for patients with this systemic condition, the present findings reveal that xerostomia, denture need and periodontitis constitute risks for the negative impact oral 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[impact of periodontal disease on quality of life for dentate diabetics]. cad saude publica. 2007 mar;23(3):637-44. portuguese. 32. ahola aj, saraheimo m, forsblom c, hietala k, sintonen h, groop ph, et al. health-related quality of life in patients with type 1 diabetes-association with diabetic complications (the finn diane study). nephrol dial transplant. 2010 jun;25(6):1903-8. doi: 10.1093/ndt/gfp709. 16 de sousa et al. 33. niesten d, witter dj, bronkhorst em, creugers nhj. oral health care behavior and frailty-related factors in a care-dependent older population. j dent. 2017 jun;61:39-47. doi: 10.1016/j.jdent.2017.04.002. 34. ahmad ms, bhayat a, zafar ms, al-samadani kh. the impact of hyposalivation on quality of life (qol) and oral health in the aging population of al madinah al munawarrah. int j environ res public health. 2017 apr 20;14(4). pii: e445. doi: 10.3390/ijerph14040445. 35. ragghianti ms, greghi sl, lauris jr, sant’ana ac, passanezi e. influence of age, sex, plaque and smoking on periodontal conditions in a population from bauru, brazil. j appl oral sci. 2004 dec;12(4):273-9. 36. arrieta-blanco jj, bartolomé-villar b, jiménez-martinez e, saavedra-vallejo p, arrieta-blanco fj. buccodental problems in patients with diabetes mellitus (i): index of plaque and dental caries. med oral. 2003 mar-apr;8(2):97-109. 37. carda c, mosquera-lloreda n, salom l, de ferraris meg, peydró a. structural and functional salivary disorders in type 2 diabetic patients. med oral patol oral cir bucal. 2006 jul 1;11(4):e309-14. 38. lopes mwf, gusmão es, alves rv, cimões r. the impact of chronic periodontitis on quality of life in brazilian subjects. acta stomatol croat. 2009 jan;43(2):89-98. 39. silva mes, villaça el, magalhães es, ferreira ef. [impact of tooth loss in quality of life]. cien saude colet. 2010;15(3):841-50. doi: 10.1590/s1413-81232010000300027. portuguese. 40. ferreira aaa, piuvezam g, werner cwa, alves mscf. [the toothache and toothloss: social representation of oral care]. cien saude colet. 2006;11(1):211-8. doi: 10.1590/s1413-81232006000100030. portuguese. 41. araújo ac, gusmão es, batista je, cimões r. impact of periodontal disease on quality of life. quintessence int. 2010 jun;41(6):e111-8. 42. jones ja, orner mb, spiro a, kressin nr. tooth loss and dentures: patient’s perspectives. int dent j. 2003;53(5 suppl):327-34. 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1528/1181 1/9 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1528/1181 2/9 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1528/1181 3/9 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1528/1181 4/9 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1528/1181 5/9 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1528/1181 6/9 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1528/1181 7/9 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1528/1181 8/9 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1528/1181 9/9 braz j oral sci. 15(4):264-268 impact of the use of ethanolic extract of propolis, flavonoid and non-flavonoid propolis for direct pulp capping in collagen type i density ardo sabir1, latief mooduto2, cahyono kaelan3, sherly horax4 1department of conservative dentistry, faculty of dentistry, hasanuddin university, makassar 90245, indonesia 2department of conservative dentistry, faculty of dentistry, airlangga university, surabaya 60132, indonesia 3department of pathological anatomy, faculty of medicine, hasanuddin university, makassar 90245, indonesia 4department of pediatric dentistry, faculty of dentistry, hasanuddin university, makassar 90245, indonesia correspondence to: dr. ardo sabir, dds, m.kes department of conservative dentistry faculty of dentistry hasanuddin university, makassar 90245, indonesia phone : +62-81524301953, +62-411-586012; fax : +62-411-584641 e.mail : ardo.sabir@yahoo.com abstract aim: to analysis collagen type i density on inflamed rat dental pulp after capping with propolis. methods: flavonoid and non-flavonoid substances were purified from propolis. eighty male rats were divided into five groups, each group consisting of 16 rats. as a negative control (group i), rats were not conducted any treatment. a class i cavity was prepared on the occlusal surface of right maxillary first molar. dental pulp was exposed and allowed in oral environment for 60 minutes, then dental pulp capping with ethanolic extract of propolis (group ii), flavonoid propolis (group iii), non-flavonoid propolis (group iv), or calcium hydroxide as positive control (group v). rats were sacrificed at 6 hours, 2, 4 or 7 days, biopsy samples were obtained, stained and viewed by light microscope. data was statistically analysis using friedman and kruskal-wallis tests. results: except in group i, collagen type i density was increased in group ii, iii, and v with the longer of observation time periods. however, in group iv, collagen type i density increased only on day 7. no statistically significant differences of collagen type i density among the groups for each time period were found. conclusions: propolis and flavonoid propolis may increase collagen density on inflamed rat dental pulp. keywords: propolis. collagen type i. inflammation. dental pulp. rat. received for publication: december 12, 2016 accepted: july 23, 2017 original article braz j oral sci. october | december 2016 volume 15, number 4 http://dx.doi.org/10.20396/bjos.v15i4.8650037 introduction propolis, or bee glue is a natural wax-like resinous substance collected by bees from buds and exudates of the plants1. honey bees used propolis as antibiotic, seal hole or cracks of its combs, and also protect it from insects2. propolis has been used since long time ago as traditional medicine due to its several biological properties, such as antibacterial3, anti-inflammatory4, and immunomodulatory5. among of its constituents, both flavonoid and non-flavonoid substances of propolis also showed anti-bacterial and anti-inflammatory activities. the chemical composition of propolis is very complex, depends on the collecting location, time, and plant source6. commonly, the composition of propolis primarily consists of resinous (50%), wax (30%), essential and aromatics oils (10%), bee pollen (5%), and other substances (5%)7. flavonoid and non-flavonoid fraction, which were then subjected to thin layer chromatography using silica gel gf254 precoated plates with n-butanol: acetic acid: water (3:1:1 v/v) as mobile phase. examination under ultraviolet light at λ 253 and λ 366 nm and treatment with ammonia showed that the flavonoids group from propolis contains flavones, flavonoles, flavanols, and chalcones19. experimental groups and treatment rats were divided into five groups randomly, each consisting of 16 animals. group i as a negative control was not conducted any treatment. in group ii, iii, iv and v, rats were anesthetized intramuscularly with ketamine (ketalar®, warner lambert, ireland) (65 mg kg-1 body weight) and xylazine-hcl (xyla®, interchemie, netherlands) (7 mg kg-1 body weight), and then class i (black’s classification) cavities were prepared on the occlusal surface of right maxillary first molar using a low-speed tapered round diamond bur (intensiv®, switzerland) (0.84 mm in diameter). the dental pulp was then exposed at the cavity floor using a dental explorer (martin®, germany) (0.35 mm in tip diameter) and allowed in the oral environment for 60 minutes, after that, dental pulp was immediately capping with ethanolic extract of propolis (eep) (group ii) (0.5 mg), extract of flavonoid propolis (group iii) (0.5 mg), extract of non-flavonoid propolis (group iv) (0.5 mg), or calcium hydroxide (ca(oh)2) (hydcal®, technew, rio de janeiro, brazil) as positive control (group v) (0.5 mg), respectively. each cavity was then air-dried and filled with glass ionomer cement (hs posterior extra®, gc, tokyo, japan) as permanent filling. histological examination four rats were sacrificed at 6 hours, 2 days, 4 days and 7 days respectively. the teeth and the surrounding bone were resected, fixed in bouin’s fixative solution for 24 hours, decalcified with acetic acid/ formal saline for 7 days, embedded in paraffin wax blocks and sliced into 6 μm-thick sections serially at bucco-palatal direction. the slides were stained with mallory and examined under a light microscope (leica®, olympus bx41-u-ca, tokyo, japan) with 400x magnification. the scoring of collagen type i density on rat dental pulp was blindly evaluated by two separate independent pathologists. the density of collagen type i was scored according to the absent or presence of collagen type i, using a 0 to 2 score system : score 0 no collagen type i was detected; score 1 thin collagen type i was detected; and score 2 thick collagen type i was detected. if there was any disagreement between the evaluators, the sample under discussion was jointly analyzed until a consensus was reached. statistical analysis statistical anaylsis was performed by using the spss-pc package for windows (version 8.0, spss inc, chicago, usa). the friedman test was used to analysis the significant difference of collagen type i density between observation time periods for each group. meanwhile, for analysis the significant difference between the groups for each observation time period were evaluated by kruskal-wallis test. a value of p<0.05 was considered statistically significant. 265 in recent years, a new trend of using complementary and alternative medicines including apitherapy has increased worldwide. using propolis in conservative dentistry and endodontic treatment to treat tooth and pulp diseases is a popular practice such as cariostatic agent in suppressing cariogenic bacteria8, desensitizing agent to treat hypersensitivity dentin9, intracanal irrigant10, cavity disinfecting agent in atraumatic restorative treatment11, medicament during root canal treatment12, and also as direct pulp capping agent13. dental pulp is a loose connective tissue uniquely situated within the rigid encasement of mineralized dentin. however, dental pulp may become exposed due to caries, accidental mechanical during cavity preparation, tooth fracture or attrition14. collagen is a major organic component of macromolecular structures in the dental pulp, designated as collagen fibers. collagen type i play pivotal role during wound healing process, especially in hemostatis, inflammatory response, cell growth, differentation and migration15,16. previous studies have demonstrated that propolis is toxic to dental pulp fibroblasts at 2 mg or above17 and not reduced the viability of dental pulp fibroblasts at 1 mg/ml18. one of honeybee species that breeding by many beekeepers in south sulawesi province, indonesia is trigona sp. this honeybee species is stingless and can produce a lot of propolis. therefore, the aim of the present study was to evaluate the impact of the use of ethanolic extract of propolis, flavonoid propolis and non-flavonoid propolis for direct pulp capping in collagen type i density. material and methods the experimental protocol was approved by the ethical committee of faculty of medicine, hasanuddin university number 0032/h04.8.4.5.31/pp36-kometik/2015. animals eighty sprague dawley rats (male, 8-16 weeks old, weight 200-300 g), obtained from the animal research development center, gadjah mada university, yogyakarta indonesia with standard food and water ad libitum throughout the experiment. the room was maintain on a 12 h light-dark schedule at a temperature of 26 ± 2°c and a relative humidity of 60-70%. propolis raw propolis was purchased from honey bee development center, hasanuddin university, makassar which collected from honeycombs in luwu regency, south sulawesi province, indonesia in the early monsoon season. extraction of propolis dried propolis (250 g) was sliced and squashed with a mortar and pestle, and the extract was cultured for five days in the dark at 45°c with continous shaking (100 rpm) in a flask containing ethyl alcohol (95%) at a ratio of 1:5. it was then filtered through whatman paper, and the crude extract of propolis was dried at 60°c using a vacuum rotary evaporator to get ethanolic extract of propolis. the residue was separated using toluene solution to yield impact of the use of ethanolic extract of propolis, flavonoid and non-flavonoid propolis for direct pulp capping in collagen type i density braz j oral sci. 15(4):264-268 266 results histogram of collagen type i density on rats dental pulp tissue of all groups after 6 hours, 2 days, 4 days and 7 days of application can be seen at figure 1. it was showed that collagen type i density was increased at group ii, iii, and v along with the increase of the observation time periods. meanwhile, at impact of the use of ethanolic extract of propolis, flavonoid and non-flavonoid propolis for direct pulp capping in collagen type i density group iv collagen type i density slight increased only on day 7. however, the result of friedman and kruskal-wallis tests showed no significant difference (p>0.05) of the collagen type i density among 4 time periods of each group (table 1) and among 5 groups of each time period (table 2). no necrotic pulp tissues were found in all animals of treatment groups throughout the study. table 1 the difference of collagen type i density among time periods for each group. table 2 the difference of collagen type i density among groups for each time period. groups mean rank freidman test p6 hours 2 days 4 days 7 days no treatment 2.50 2.50 2.50 2.50 0.00 1.00 eep 2.00 2.50 2.50 3.00 2.400 0.494 flavonoid 1.88 2.38 2.88 2.88 2.538 0.468 non-flavonoid 2.38 2.38 2.38 2.88 3.000 0.392 ca(oh)2 2.00 2.50 2.50 3.00 3.000 0.392 time periods mean rank kruskal wallis test pno treatment eep flavonoid non-flavonoid ca(oh)2 6 hours 10.50 10.50 10.50 10,50 10.50 0.000 1.000 2 days 9.00 11.50 11.50 9.00 11.50 2.235 0.693 4 days 8.50 11.00 13.50 8.50 11.00 4.156 0.385 7 days 7.00 12.00 12.00 9.50 12.00 3.341 0.503 note: *significant at p<0.05 there was no statistically significant difference among the groups in each period (p>0.05). braz j oral sci. 15(4):264-268 fig. 1 histogram of percentage of collagen type i density on rats dental pulp tissue of all groups after 6 hours, 2 days, 4 days and 7 days of materials test application. fig. 2 collagen type i density on rats dental pulp tissue after materials test application for 6 hours and 7 days in groups i (a-b), ii (c-d), iii (e-f), iv (g-h) and v (i-j) with no treatment (negative control), ethanolic extract of propolis (eep), extract of flavonoid-propolis, extract of non-flavonoid propolis and calcium hydroxide (ca(oh)2) (positive control), respectively. white arrows show collagen type i. mallory stain, original magnification 400 x. 267 the photomicrograph of collagen type i density evaluation is presented here in only by the section from all groups at 6 hours and 7 days (figure 2). impact of the use of ethanolic extract of propolis, flavonoid and non-flavonoid propolis for direct pulp capping in collagen type i density and angiogenesis process will followed with connective tissue regeneration by collagen as results fibroblast cell synthesis20. the present study showed that except in negative control group (without treatment), all treatment groups showed collagen type i in different density and tends to increased with the longer of observation time periods. however in group iv (non flavonoid propolis), collagen type i density increased only on day 7. (figure 1). the present results are not surprising, since propolis is known to have antibacterial, anti-inflammatory, antioxidant and immunomodulatory properties that permits cells regeneration and the healing process in dental pulp with collagen formation21,22. previously study by bretz et al.23 (1998) reported that propolis was effective to maintain the presence of fibroblast cells, low inflammatory and microbial cell population as well as in stimulating the formation of reparative dentin. these effects are mediated by the presence some of propolis constituents -such as active flavonoids, caffeic acid phenethyl ester, terpenoid, steroid, vitamin and mineralsthat have a significant role in healing process24. the results of present study also showed that the collagen type i density was increased on inflamed rat dental pulp after capping with flavonid propolis. study by kandhare et al.25 (2014) in ulcer rat found that naringin (flavanone glycoside) was significant upregulation of mrna expression of growth factor and collagen type i whereas mrna expression of pro-inflammatory mediators was down-regulated. further results suggest that angiogenesis was improved via naringin-mediated inhibition of oxidative stress, down-regulation of inflammatory mediator expression and up-regulation of growth factor expression, leading to improved wound healing of ulcer. moreover, when inflammation was occurs, damage of blood vessels increases capillary permeability. flavonoid substances have a significant role in maintaining permeability and increasing resistance of capillary blood vessels26. it will also increase biosynthesis mucopolysaccharide acid process of ground substances that finally caused both the number of new capillary blood vessel and collagen density were increased27. our previous study in rats showed that flavonoid propolis could delay inflammation process and stimulated reparative dentin in direct pulp capping treatment28. in contrast, collagen type i density on inflamed rat dental pulp increased only on day 7 after capping with non-flavonoid propolis. (figure 1). this result may due to that the antibacterial and anti-inflammatory properties of non-flavonoid substances weaker than other material test. havsteen26 (2002) stated that flavonoids are considered as the most biologically active substance in propolis. our previous study found that antibacterial activity of non-flavonoid propolis weaker than flavonoid propolis against streptococcus mutans29. calcium hydroxide was used in this study as material of positive control group because until now it was known as the most promising capping agent for direct pulp capping treatment to preserve tooth vitality in an exposed pulp cavity. nelson-filho et al.30 (1999) reported that ca(oh)2 initially induce the formation of a necrotic zone when contact with dental pulp tissue due to its high ph (11-12). following infiltration of inflammatory cells, fibroblast-like cells proliferate and migrate to the injury site. this action is followed by the formation of new collagen that is arranged in contact with the superficial necrotic zone31. discussion wound healing represents an interactive process which requires highly organized activity of many cells, synthesizing cytokines, growth factors, chemical mediators, extracellular matrix and collagen16. healing mechanisms are a dynamic and continuous process that occurs when no inflammation is present. proliferation braz j oral sci. 15(4):264-268 268 the results of this present study showed that the application of all material test on inflamed rats dental pulp tissue increased collagen type i density along with the increase of the observation time period. however, ethanolic extract of propolis, flavonoid propolis, and ca(oh)2 have better effect than non-flavonoid propolis in stimulated collagen type i on inflamed rats dental pulp tissue. therefore, the present results suggest that both extract ethanolic of propolis and flavonoid propolis increase collagen type i density when used for direct pulp capping, presenting a comparable effect to ca(oh)2. acknowledgements the authors would like to thanks to prof. s pramono, ph.d (faculty of pharmacy, gadjah mada university, yogyakarta, indonesia) for his technical assistance in propolis preparation. the authors state that they have no conflicts of interest. references 1. kuropatnicki ak, szliszka e, krol w. historical aspects of propolis research in modern times. evid based complement alternat med. 2013;2013:964149. doi: 10.1155/2013/964149. 2. bogdanov s. propolis: composition, health, medicine: a review. bee prod sci. 2017; 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2015. p.457. 20. velnar t, bailey t, smrkolj v. the wound healing process: an overview of the cellular and molecular mechanisms. j int med res. 2009 sepoct;37(5):1528-42. 21. ramos afn, miranda jl. propolis: a review of its anti-inflammatory and healing actions. j venom anim toxins incl trop dis. 2007;13(4):697-710. doi: 10.1590/s1678-91992007000400002 22. abdel-wahed re, el-kammar mh, korittum as, edrees ir. surgical and histological evaluation of the effectiveness of propolis on wound healing. alexandria j vet sci. 2013;39(1):52-63. 23. bretz wa, chiego dj jr, marcucci mc, cunha i, custódio, schneider lg. preliminary report on the effects of propolis on wound healing in the dental pulp. z naturfosch c. 1998 nov-dec;53(11-12):1045-8. 24. jain s, rai r, sharma v, batra m. propolis in oral health: a natural remedy. world j pharm sci. 2014 jan;2(1):90-4. 25. kandhare ad, ghosh p, bodhankar sl. naringin, a flavanone glycoside, promotes angiogenesis and inhibits endothelial apoptosis through modulation of inflammatory and growth factor expression in diabetic foot ulcer in rats. chem biol interact. 2014 aug 5;219:101-12. doi: 10.1016/j. cbi.2014.05.012. 26. havsteen bh. the biochemistry and medical significance of flavonoids. pharmacol ther. 2002 nov-dec;96(2-3):67-202. 27. di carlo g, mascolo n, izzo aa, capasso f. flavonoids: old and new aspects of a class of natural therapeutic drugs. life sci. 1999;65(4):33753. 28. sabir a, tabbu cr, agustiono p, sosroseno w. histological analysis of rat dental pulp tissue capped with propolis. j oral sci. 2005 sep;47(3):135-8. 29. sabir a. [antibacterial activity of flavonoid and non-flavonoid of propolis trigona sp. toward the growth of streptococcus mutans (in vitro)]. jitekgi (sci and tech dent j). 2010;7(1):37-42. indonesian. 30. nelson filho p, silva la, leonardo mr, utrilla ls, figueiredo f. connective tissue responses to calcium hydroxide-based root canal medicaments. int endod j. 1999 aug;32(4):303-11. 31. vesna d, elena k, vesna l. histological evaluation of odontoblast-like cells response after capping application of calcium hydroxide and hydroxilapatite in dog’s pulp. acta vet. 2007 jan;57(5-6):573-84. braz j oral sci. 15(4):264-268 untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8652941 volume 17 2018 e181097 original article 1 department of prosthodontics and periodontology, piracicaba dental school, university of campinas (unicamp), piracicaba, sao paulo, brazil 2 department of restorative dentistry, school of dentistry, federal university of minas gerais (ufmg), minas gerais, brazil corresponding author: anna gabriella camacho presotto department of prosthodontics and periodontology university of campinas (unicamp), piracicaba dental school av limeira, 901, piracicaba, são paulo, 13414-903, brazil. tel.: +55-19 2106 5719 e-mail: annapresotto@gmail.com received: may 22, 2018 accepted: july 09, 2018 photoelastic and finite element stress analysis reliability for implant-supported system stress investigation anna gabriella camacho presotto1, cláudia lopes brilhante bhering2, ricardo armini caldas1, rafael leonardo xediek consani1, valentim adelino ricardo barão1, marcelo ferraz mesquita1 aim: to compare the reliability between photoelastic and finite element (fe) analyses by evaluating the effect of different marginal misfit levels on the stresses generated on two different implant-supported systems using conventional and short implants. methods: two photoelastic models were obtained: model c with two conventional implants (4.1×11 mm); and model s with a conventional and a short implant (5×6 mm). three-unit cocr frameworks were fabricated simulating a superior first pre-molar (p) to first molar (m) fixed dental prosthesis. different levels of misfit (µm) were selected based on the misfit average of 10 frameworks obtained by the single-screw test protocol: low (<20), medium (>20 and <40) and high (>40). stress levels and distribution were measured by photoelastic analysis. a similar situation of the in vitro assay was designed and simulated by the in silico analysis. maximum and minimum principal strain were recorded numerically and color-coded for the models. von mises stress was obtained for the metallic components. results: photoelasticity and fe analyses showed similar tendency where the increase of misfit generates higher stress levels despite of the implant design. the short implant showed lower von mises stress values; however, it presented stresses around its full length for the in vitro and in silico analysis. also, model s showed higher µstrain values for all simulated misfit levels. the type of implant did not affect the stresses around pillar p. conclusions: photoelasticity and fea are reliable methodologies presenting similarity for the investigation of the biomechanical behavior of implant-supported rehabilitations. keywords: biomechanical phenomena. dental implants. optical phenomena. finite element analysis. 2 presotto et al. introduction despite the high success rates of implant-supported rehabilitation demonstrated over the years,1 this issue continues to be an ongoing research goal in dentistry. the attention in this field persists as clinical problems may occur with implants due to the considerable difference in the biomechanical behavior of implants and natural teeth.2 differently from natural teeth, bone tissue and implants are rigidly connected and do not allow stress absorption as promoted by the presence of periodontal ligament.3 as a consequence, misfit can generate stresses in the implant system even without load application.3,4 since the presence of misfit is a clinical reality,4 obtaining acceptable fit levels is very important to the longevity of the implant treatment;5,6 otherwise biological and mechanical complications can potentially occur.7 however, not only the presence of misfit affects the stresses in the implant-supported system, but also the selected components such as implant types and prosthetic abutments.8 in this sense, short and wide implants reduce the stress and strain in the periimplant bone in the posterior maxilla in comparison to conventional implants placed in the grafted sinus.9,10 also, short implants (<10 mm) can avoid surgical procedures such as ridge augmentation and sinus lift, which involves greater morbidity for the patient, longer treatments and higher costs.8–10 short implants can be a predictable alternative when used under rigorous clinical protocols,11 such as the optimization of occlusion of the prostheses,8 where its deficiency is a common cause of implant-supported rehabilitation failure.12 in addition, the increase in diameter of short implants shows benefit for stress distribution.13 a previous review article evaluating the biomechanical behavior of short implants showed a similar survival rate and bone resorption when compared to the conventional implants.8 however, despite the decrease of the stress values around the implants,9,10 the influence of marginal misfit in this type of implants was not evaluated. in vitro investigations of stress levels can be considered a challenge in prosthodontics researches as each methodology has its own singularities. among the main methods used for in vitro stress investigation are photoelasticity2,4 and finite element analysis (fea).2,14,15 photoelasticity provides a qualitative result of the stress distribution and mimics the clinical scenario by using real components (i.e. implants, abutments and frameworks).2,4,16 fea allows for detailed information of the stress data16 and also provides the stress in the components of the implant-supported system,2 which is not possible through photoelasticity. based on these particularities of the methodologies, previous studies recommended their combined use.2,14,16 despite this recommendation, the reliability between photoelasticity and fea methodologies are not yet well established for the investigation of the biomechanical behavior of the implant-supported system. thus, a comparison between these two methodologies under the same simulation is warranted. therefore, this study aimed to compare the reliability between photoelasticity and fea methodologies by evaluating the stresses patterns in conventional and short implants with three different marginal misfit levels. the research hypotheses were as follows: (1) the photoelasticity and fea methodologies generate similar stress pattern under the same simulation; (2) the increase of marginal misfit is less critical for short and wide implants than conventional implants. 3 presotto et al. materials and methods study design two conditions were evaluated: 1) model c: two standard branemark implants (external hexagon (eh), 4.1 × 11 mm; sin – sistema de implante, sao paulo, sp, brazil); and 2) model s: one conventional implant (eh, 4.1 × 11 mm) and one short and wide implant (eh, 5 × 6 mm; sin – sistema de implante, sao paulo, sp, brazil), simulating the placement of a short implant in posterior maxilla. frameworks simulating 3-unit fixed denture prosthesis (fdps) were obtained by the overcasting method (n=10). three different levels of marginal misfit were selected based on their misfit average, obtaining three groups: low (< 20 µm), medium (> 20 µm and < 40 µm) and high (> 40 µm) misfit. qualitative photoelastic analysis was used to evaluate the stress level and distribution in periimplant region under two situations: non-loaded (after tightening of frameworks to the models); and 100-n loaded on the molar. also, fea was conducted similarly to the photoelasticity design. maximum and minimum principal strain were recorded numerically and color-coded for the models (non-ductile material) while von mises stress was obtained for the metallic components (ductile materials) (figure 1). master model frameworks fabrication (n=10) photoelastic models fabrication photoelastic analysis stress level and distribution in periimplant region von mises stress for the metallic components maximum and minimum principal strain for the models finite element analysis (conducted similarly to experimental analysis) selection of three misfit levels: low (< 20 µm) medium (> 20 µm and < 40 µm) high (> 40 µm) model c misfit analysis model s figure 1. flowchart of study methodology design. 4 presotto et al. frameworks fabrication a steel master model with dimensions of 30 × 20 × 15 mm was fabricated with two drill holes 18 mm from each other (center to center) and two mini abutment analogs screwed on the model. overcasted mini abutment cylinders (sin – sistema de implante; sao paulo, sp, brazil) were tightened on the master model. the frameworks simulating fdps for superior first pre-molar (pillar p) to first molar (pillar m) were waxed with a low-shrinkage acrylic resin (duralay ii; reliance dental mfg. co., chicago, usa). all waxed patterns were sectioned and splinted with a low-shrinkage acrylic resin. the frameworks (n=10) were overcasted in cocr alloy (starloy c; degudent, dentsply, hanau-wolfgang, hesse, germany) after including in investment material (gilvest hs; bk giulini, ludwigshafen, rheinland-pfalz, germany). the frameworks were processed by airborne-particle abrasion (110-µm al2o3 particles under 0.55 mpa air pressure), followed by finishing and polishing with tungsten carbide drills at a low speed, excepted on the metallic strap region. photoelastic model fabrication a silicone impression (silibor; silibor industria e comercio ltd., sao paulo, sp, brazil) was obtained from the master model/transfer set. the implants with mini pillars (mini abutment eh 4.1 × 2 mm; sin – sistema de implante, sao paulo, sp, brazil) were tightened on transfers of silicone impression. the photoelastic resin (araldite gy 279 br and catalyst aradur hy 2963; araltec chemicals ltd., guarulhos, sp, brazil) was manipulated with the proportion of 2 parts of resin to 1 part of catalyst for 1 minute, leaving a homogeneous mixture. the resin was placed for 20 minutes in a pressure chamber coupled to an air injection tube and at a pressure of 60 kgf/cm2 to prevent bubbles in the material. the photoelastic resin was slowly poured over the impression. the same procedure was performed with short and wide implant and conventional implants to obtain the second photoelastic model. after 72 hours, the transfers were removed from the silicone mold and photoelastic models were obtained for evaluation. thus, a photoelastic model for each situation was obtained and identified as model c (conventional) that has two standard branemark implants, and model s (short) that has a short and wide implant and a standard branemark implant. marginal misfit evaluation the marginal misfit was performed at 120× magnification using a 1.0-μm precision microscope (vmm-100-bt; walter uhl, asslar, germany) equipped with a digital camera (kc-512nt; kodo br eletronics ltd, sao paulo, sao paulo, brazil) and analyzer unit (qc 220-hh quadra-check 200; metronics inc., bedford, massachusetts, usa). the procedures also involved a calibrated examiner with an intraclass correlation coefficient of 0.995 (p<0.0001), according to the single-screw test protocol,3 which shows the marginal misfit reading of the loop while the screw of the opposite pillar is tightened. the frameworks were positioned on the model and tightened with 10 ncm using a 0.1-ncm precision digital torque meter (torque meter tq-8800; – lutron, taipei, taiwan). the readings for pillar m were performed on the buccal and lingual sides in diametrically opposite positions after the pillar p screw was tightened and vice versa. the measurements were performed on both pillars, and an average value of mis5 presotto et al. fit was obtained for each framework. three average values of marginal misfit were selected obtaining three groups: low (misfit average < 20 µm); medium (misfit average > 20 µm and < 40 µm); and high (misfit average > 40 µm). photoelastic analysis a horizontal transmission polariscope developed in the mechanical design laboratory henner alberto gomide, school of mechanical engineering of federal university of uberlandia was used and consisted of two ¼-retardant wave filters and two polarizing filters, called polarizer and analyzer. a standard position for the photoelastic models was obtained by markings on the polariscope platform. each framework was tightened to the photoelastic model with 10-ncm standardized torque, always following the tightening sequence p-m. a layer of mineral oil was applied on the photoelastic models to improve the view of the fringes. the photoelastic models were positioned on the polariscope, and the images were obtained using a digital camera (canon sx50hs; miyazaki daishin canon inc., miyazaki, japan) in two different conditions: after tightening the frameworks to the photoelastic models, and after applying a load of 100-n on the first molar. in the interval among the analyses, the models were kept under 37°c for 10 minutes until no stress was observed using the polariscope, avoiding the presence of residual stress from the previous analysis. the analysis of each image was performed with a graphic software (adobe photoshop cs5®; adobe systems, san jose, ca, usa) according to the visualization of isochromatic fringe order where fringe order of 0 = black; 1 = violet/blue transition; and 2, 3, 4 = red/green transition.18 all images were evaluated by the same operator. the analysis was separated according to the intensity (fringe order) and distribution of stress for different misfit levels and implant designs. finite element analysis a similar situation of the in vitro assay was modeled and simulated. the framework was scanned by a 3d contact scanner (modela mdx-20; roland, japan), and the images were imported into the autodesk meshmixer 3.0 software (san diego, ca, usa) to generate the final post-scan image. all metallic components were created based on the real components measurements (vmm-100-bt; walter uhl, asslar, germany; and qc 220hh quadra-check 200; metronics inc., bedford, ma, usa) in solidworks 2010 (solidworks, concord, ma, usa). then, six models were assembled based on the misfit level (low, medium and high) and implant design (conventional and short). the ansys workbench 14 software (ansys inc.; canonsburg, pa, usa) was used to perform the pre-processing, processing and post-processing analyses. all materials were considered as homogeneous, isotropic and elastically linear. the material properties used are shown in table 1. all contacts were set to frictional (μ = 0.3),17 with exception of thread interfaces (screws and implant/araldite), which were set as bonded condition. the meshes were refined in contact areas and checked by element quality (figure 2), varying from 931.651 to 1,005.189 nodes and 633.436 to 685.492 elements. then, the analysis was divided in two simulations, mechanical loading and molar screw torque. each simulation was divided in 3 steps, being step 1 and 2 the same for both simulations, which: step 1: bolt pre-tension (200-n) at the abutment screws; step 2: 6 presotto et al. bolt pre-tension (100-n) at the prosthetic p screw.19 step 3a (m screw torque): the m prosthetic mini screw neck was set to fixed (zero degrees of freedom) and an axial dislodgement (high, medium and low) was applied at molar screw site to simulate misfit sealing by pre-loading. these results were used in comparison to the screw torque test due to simulation limitations with no negatives consequences at obtained results. step 3b (mechanical loading): an axial 100-n force was applied at molar to simulate the masticatory force, and the force opposition was set to araldite basis (zero degress of freedom). contact prediction was programed between prosthesis and abutment. the results were obtained after the third step. maximum and minimum principal strains were obtained for the models, and von mises stress was obtained for the metallic components. results in vitro data from photoelastic analysis table 2 and figure 3 show the stress levels and distribution, respectively, in the models c and s for different levels of misfit under non-loaded and loaded conditions. the stress intensity is presented according to the higher fringe order observed for each implant. greater stress level was noted with the increase in the misfit level for both models under nonloaded and loaded conditions (table 2). after tightening the frameworks, models c and s showed similar stress distribution for all situations of misfit. however, peri-implant stress was observed in the full length of the short implant and the same condition is observed after load application (figure 3). despite of the load application, both models showed a higher concentration of stress around implant m (table 2). however, the stresses were distable 1. materials properties used in the finite element models. material young’s modulus (gpa) poisson’s ratio (v) reference araldite 2.07 0.41 anami et al.27 titanium 110 0.28 spazzin et al.31 cocr alloy 185 0.35 archangelo et.45 a b c figure 2. finite element mesh refined at contact interfaces (a, vestibular view of model; b, model c sectioned; c, model s sectioned). 7 presotto et al. tributed to implant p after load application in model c, whereas for model s, the stresses were located almost entirely around the short implant (implant m) (figure 3). in silico data from finite element analysis figure 4 and table 3 show the stress maps distribution and von mises stress (mpa) levels, respectively, for the metallic components of models c and s for the different levels of misfit. a similar stress maps distribution is observed for all simulated conditions (figure 4). however, in both models a slight increase of stress is observed in the distocervical region of implant/abutment p as higher is the misfit level. overall, higher von mises stress levels in the components is observed for higher misfit levels conditions despite of the implant design (table 3). the von mises stress levels of components of pillar p are not influenced by the presence of short implant (table 3). comparing implant m data, lower von mises stress values were found for model s despite of the misfit level (table 3). table 2. stress intensity (fringe order) according to the model and implant without and with load application for different misfit levels. misfit level model c model s no load with load no load with load implant m implant p implant m implant p implant m implant p implant m implant p low (< 20 µm) 0 0 5 1 0 0 4 1 medium (> 20 µm and < 40 µm) 1 0 5 2 1 0 5 0 high (> 40 µm) 3 1 9 4 3 2 7 2 * for model s, the implant m represents the short implant. m p m p m p m p model c no load low misfit level <20 µm medium misfit level >20 µm and <40 µm high misfit level >40 µm with load model s no load with load figure 3. stress distribution in models c and s without load and with load for different misfit levels. 8 presotto et al. table 3. maximum von mises stress (mpa) levels of the metallic components according to the model for the different misfit levels. component misfit level low (< 20 µm) medium (> 20 µm and < 40 µm) high (> 40 µm) model c model s model c model s model c model s framework 155 138 158 148 158 159 abutment p 78 83 98 100 117 114 abutment m 135 128 125 121 134 146 abutment screw p 340 334 339 343 298 306 abutment screw m 354 322 276 279 278 325 prosthetic screw p 248 241 238 241 282 279 prosthetic screw m* ------------implant p 130 130 134 131 144 147 implant m 130 105 134 114 139 118 maximum value 354 334 339 343 298 325 * the site of load application eliminates the need of prosthetic screw m component creation for fea analysis. model c low misfit level <20 µm medium misfit level >20 µm and <40 µm high misfit level >40 µm model s 355 100 87.5 75 62.5 50 37.5 25 12.5 0 figure 4. von mises stress (mpa) distribution in the metallic components according to the model (model c and s) and the misfit levels (low, medium and high). 9 presotto et al. the µstrain distribution and levels for models c and s for the different misfit levels are shown in figures 5 and 6. maximum and minimum principal elastic strain data were selected to illustrate the stress distribution in the resin blocks due to the viewing similarity with the photoelasticity patterns. the µstrain data was selected to represent the stress levels in the resin block by being the same unity generated by the photoelastic analysis. the higher the misfit level, the greater is the strain concentration (figure 5). model s showed higher µstrain values under all misfit levels simulation when compared to model c (figure 6). model s showed higher percentage variation (%) of von mises stress values between low and high misfit level (figure 7). maximum principal elastic strain model c low misfit level <20 µm medium misfit level >20 µm and <40 µm high misfit level >40 µm model s minimum principal elastic strain model c model s 10 x10-6 0 -142 -285 -428 -571 -714 -857 -1000 -6100 10 x10-6 428 500 357 285 214 142 71 0 -20 figure 5. maximum and minimum principal µstrain distribution of the resin block according the model (model c and s) and misfit level (low, medium and high). µ st ra in 6000 4000 2000 0 -2000 -4000 -6000 -8000 model c 2653 -3062 -5365 -3460 -5909 -4674 -6049 4017 2496 4367 2835 4464 model s low misfit medium misfit high misfit model c model s model c max min model s figure 6. maximum and minimum principal µstrain levels of the resin block according the model (model c and s) and misfit level (low, medium and high). 10 presotto et al. discussion photoelasticity and fe analyses showed reliable and similar results, accepting the first research hypothesis. both methodologies detected that the increase of misfit levels generated higher stress levels and produced similar stresses pattern around the implants. such similar results between photoelasticity and fea were also reported by a previous study that investigated the stresses generated around implants with different internal-cone abutments.14 photoelasticity is an experimental analysis and allows the use of real components; therefore, the 3d misfit generated by the casting procedure can be accurately reproduced.4 in contrast, the reproduction of misfits by fea is usually simplified by a gap between two components with parallel contact surfaces.2,14 such situation can underestimate the resultant stress levels as noted in the present study. the photoelastic analysis was more sensible to detect the increase of stress with the increase of marginal misfit. however, fea presents an important advantage providing the stress levels on the components of the system. for these results, a slight difference in the stress concentration is observed among the different misfit levels (figure 4). such slight difference can be also justified by the limited simulation of misfit levels by fea. thus, the combined use of photoelasticity and fea is suggested for the investigation of the biomechanical behavior of implant-supported rehabilitations. in this study, two conditions for the rehabilitation of the posterior maxilla region were simulated. the first condition involved available bone tissue for the placement of two conventional implants. however, in the posterior region the bone tissue is often severely resorbed, requiring surgical procedures such as sinus lift prior to implant placement. the placement of a short implant would avoid these procedures, with promising results in the literature.10,20 according to the photoelasticity and fea results, higher misfit increased the stress levels in the implant-supported system independent of the supporting condition. for figure 7. variation (%) of von mises stress values of model c and s between low and high misfit level. the reference of total value was the low misfit data. *the prosthetic screw m was not created for fe analysis. pe rc en ta ge v ar ia tio n (% ) 0.6 0.5 0.4 0.3 0.2 0.1 0 -0.1 -0.2 -0.3 framework abutment p abutment m abutmentscrew p abutment screw m prosthetic screw p prosthetic screw m implant p implant p 0.15 0.5 0.37 -0.007 0.14 -0.12 -0.08 -0.21 0.009 0.14 0.16 0.11 0.13 0.17 0.12 * component model c model s 0.02 11 presotto et al. this study, the condition of total passivity (no misfit) was not evaluated since the absence of a totally passive fit framework is expected.4 although some authors suggest empirical values of misfit between 10 and 150 μm to be clinically acceptable,7 the findings of this study are that small increments of misfit resulted in an increase of stress intensity and distribution independent of the stress analysis methodology. these findings corroborate that small misfits for screwed implant-supported prosthesis could create a high degree of stress around the implant bodies due to the limited movement (i.e., the absence of periodontal ligament).6 the different designs of implants promote differences between the models when compared under the same level of misfit. in contrast with the conventional implant m of model c, the full length of the short implant in the model s was involved by stresses areas, observed in both stress analysis. this could be explained by the location of the load; however, this stress concentration pattern is maintained even when the load is not applied in the photoelastic analysis. although fea shows stresses in the coronal region of loaded conventional implant (model c) as well (figure 5), its length allows for a better distribution of stresses levels when compared to the short implant. the presented µstrain data (figure 6) confirm the higher concentration of stresses in the full model s. this condition can be potentially damaging for the rehabilitation with short implants. corroborating these findings, a previous study21 concluded that stresses around short and wide implants is increased when compared to conventional implants. also, the presence of stresses around the coronal third of short implants emphasizes the concern about an increase of bone resorption and risk of failure.22 thus, because bone reabsorption is more critical for short implants due to their length, minimum misfit values should be emphasized for the production of implant fixed prosthesis,5 obtaining a condition close to passivity and reducing concentration of stress around coronal region of this implant design. concerning the metallic components analysis by fea, higher von mises stress values were noted with the increase of misfit level. however, the misfit values simulated in the present study were not sufficient to predict a failure in the components. all the von mises stress values were lower than the tensile strength for all materials evaluated: cocr (552 to 1034 mpa)17 and cpti (480 to 552 mpa)23 indicating that no failure or mechanical complications would occur under such misfit levels. independently of the “safe” von mises stress values found in components, there are important differences between models c and s. the presence of the short implant did not affect the von mises stresses of all components of pillar p (table 3). however, for the pillar m, lower von mises stress data was found for the short implant when compared to conventional implant. this can be justified by the higher diameter of the short implant that allows for a better stresses dissipation in the region of the implant/ abutment connection. however, despite of the lower stress values observed in the component, the presence of short implants induced higher µstrain values on the surrounding system. in addition, higher stress values were detected for almost all components of the model with short implant. this means that the components of model s showed higher difference on von mises stress values between the low and high misfit levels. this can suggest that the model s is more influenced by the misfit increasing. higher levels of misfit could potentially promote failures for all biomechanical system, 12 presotto et al. including in the metallic components.7,24 thus, as the increase of marginal misfit is more critical for short and wide implants for all implant-supported system, the second research hypothesis was denied. other types of implant connection and different lengths of short implant were not investigated. therefore, future studies with different implant connections and lengths are warranted to verify the improvement of biomechanical behavior of short and wide implants. concerning the stress analysis methodologies, even though photoelasticity and finite element analysis have particularities, there are similarities and important complementary capacity between then. thus, their combined use may be encouraged for the investigation of the biomechanical behavior of implant-supported rehabilitations. from this study it can be concluded that the results of photoelasticity and fea are complementary and present similarities for periimplant evaluation. also, the increase in marginal misfit produces higher stress concentration in a 3-unit fdp implant-supported rehabilitation. however, it is more critical when a posterior short implant is used. acknowledgements this study was supported by the sao paulo research foundation (fapesp) (grant #2014/19264-0) and coordination for the improvement of higher education personnel (capes) (grant #1778/2014 proex). the authors are grateful to professor mauro a. a. nobilo for the polariscope facility. references 1. lindquist lw, carlsson ge, jemt t. a prospective 15-year follow-up study of mandibular fixed prostheses supported by osseointegrated implants. 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fora federal university 2 master student, juiz de fora federal university 3 undergraduate student, juiz de fora federal university 4 undergraduate student, juiz de fora federal university corresponding author: paula liparini caetano ivon josé cury 2, juiz de fora mg, 36037-467, brazil 55 32 3231-1444; 55 32 98853-3166 paulaliparini@gmail.com and xpaulinha1@hotmail.com received: april 7, 2017 accepted: august 09, 2017 transverse dimension of the alveolar bone in different masticatory patterns marcio josé da silva campos1, paula liparini caetano2, fernanda farage da costa felipe3, gabriella moreira de carvalho4, marcelo reis fraga1, robert willer farinazzo vitral1 aim: the aim of this study was to evaluate, through cone beam computed tomography (cbct), the transverse dimension of the alveolar bone in the posterior region of the maxilla and mandible in subjects with different patterns of mastication, comparing both sides of the arches according to the performance of the masticatory function. methods: 39 subjects not orthodontically treated, with normal occlusion or symmetrical malocclusion, and normal periodontal condition were selected. twenty-one subjects (54%) were identified as having preferential unilateral mastication, 11 subjects (28%) had bilateral mastication and 7 (18%) had exclusive unilateral mastication. all participants were submitted to cbct and the buccolingual dimension of the posterior regions was evaluated at a height of 2, 4, 6, 8, and 10mm from the alveolar crest. results and conclusion: subjects with bilateral mastication showed statistically significant difference between the right and left sides at the heights of 6 (p=0.030) and 8mm (p=0.023) between the first and second maxillary premolars. there was no difference in the transverse dimension of the alveolar bone in the posterior regions of maxilla and mandible between preferred and non-preferred sides in subjects with preferred unilateral mastication and between right and left sides in subjects with bilateral mastication. keywords: alveolar process. bone. cone-beam computed tomography. mastication. 2 campos et al. introduction mastication is one of the most important functions of the stomatognathic system because it is related to nutrition, maturation of the orofacial musculature, occlusal stability, temporomandibular joints and growth and development of the craniofacial complex, as well as a necessary factor for a perfect homeostasis of the human being1. when considering the health of the stomatognathic system, a desirable pattern of mastication is required, which is characterized by unilateral cycles with periodic alternation of food between the right and left sides, distributing the force of mastication on the teeth and structures of support on both sides of the dental arches2,3, what results in greater masticatory efficiency4-6. however, this bilateral pattern of distribution of food is not present in most of the population, where a preferred chewing side is more common7,8. the choice for a preferred chewing side is a voluntary decision that becomes involuntary with function performance9 and may be associated with the control of the central nervous system10 or related to peripheral factors9. since teeth are supported by the alveolar process, this bone structure is directly exposed to mechanical loads created by the functional performance of the maxilla and mandible11. bone tissue is in a constant process of functional adaptation through modeling and remodeling mechanisms, which are significantly influenced by mechanical stimuli12. thus, the masticatory function provides mechanical stimuli capable of influencing the formation, maintenance and remodeling of the craniofacial skeleton, exerting an important function in the regulation of the mass and bone architecture of this region13. changes in intensity and/or frequency of functional load may be accompanied by changes in the alveolar bone12,14-16. in studies with rats, implementation of soft diet14,17 or placement of anterior18 and posterior12 occlusal build-ups produced changes in the occlusal force applied to the teeth and distributed in the alveolar process, resulting in significant qualitative and quantitative alterations in the alveolar bone tissue. however, the hypothesis that the dimension of the alveolar bone can be influenced by functional loading was not tested. the purpose of this study was to evaluate, through cone beam computed tomography (cbct), the transverse dimension of the alveolar bone in the posterior regions of the maxilla and mandible in subjects with different patterns of mastication, comparing both sides of the arches according to the performance of the masticatory function. the hypothesis of this study is that the transverse dimension of the alveolar bone in the posterior regions of the maxilla and mandible is greater in the preferred side than in the non-preferred side in subjects with preferred and exclusive unilateral mastication. such difference does not exist between the right and left sides in subjects with bilateral mastication. material and methods the sample consisted of 39 subjects, not orthodontically treated, between 19.2 and 44.6 years (mean: 24,3 years) with complete permanent dentition (except 3 campos et al. third molars), normal occlusion or symmetrical malocclusion, and normal periodontal condition, as assessed by visual evaluation. this study was approved by the research ethics committee of the of the juiz de fora federal university, and all participants signed an informed consent form. determination of the preferred chewing side the first phase of this research consisted in determining the masticatory pattern of the subject by means of a visual method19,20 where the participant was seated in an upright position in a chair with their backs towards a white background, their hands resting on their legs, and looking fixedly at the digital video camera (sony mhs-pm5), which was placed on a fixed tripod one meter away from the chair back at the height of their mandible. each subject was filmed chewing on a piece of french bread in their habitual manner for approximately 90 seconds. the videos were analyzed by 3 speech therapists who counted the masticatory cycles in each hemi-arch and determined, unanimously, the masticatory pattern of each participant, classifying it as bilateral (occurrence of up to 60% of the cycles in one side), preferred unilateral (occurrence from 61% to 77% of the cycles in one side) or exclusive unilateral (occurrence from 78% to 94% of the cycles in one side)21. from the total of 39 subjects, 21 (54%) were identified as having preferred unilateral chewing (13 right and 8 left), 11 (28%) bilateral chewing, and 7 (18%) exclusive unilateral chewing (2 right and 5 left). evaluation of the transverse dimension of the alveolar bone all participants were submitted to cone-beam computed tomography (cbct) (i-cat-imaging sciences international, hatfield, pa – usa), operated at 120kv and 3-8 mma, voxel of 0.25 mm, rotation time of 26.9s, and field of view with a diameter of 160 mm and height of 100 mm. for image acquisition, each subject was seated with the chin on the chin rest, with the frankfort plane parallel to the floor, the midsagittal plane perpendicular to the floor and with the participant in maximum intercuspation position. the field of view was positioned in a way that the occlusal plane occupied its vertical center and the anterior nasal spine was at 35 mm from its anterior border. images were analyzed with the i-cat vision (imaging sciences international inc., hatfield, usa) software, on mpr visualization mode (multiplanar reconstruction), with 0.5 mm-thick slices. initially, for the definition of the images of the posterior interdental regions, the line corresponding to the coronal slice was centrically positioned in the posterior interdental areas (vertical line – figure 1b) and perpendicular to the alveolar process buccolingually (horizontal line – figure 1a). the line corresponding to the sagittal slice was positioned in the long axis of the alveolar process (vertical line – figure 1c). the posterior interdental regions between maxillary and mandibular canine and first premolar, first and second premolar, second premolar and first molar and first and second molars of both sides were evaluated (figure 1b). 4 campos et al. buccolingual dimensions of the posterior interdental regions were determined at the heights of 2, 4, 6, 8 and 10 mm from the alveolar bone crest (figure 1c), including the bone height where the roots of the posterior teeth were located. measurements were performed by two examiners in a blind manner, where examiner 1 measured teeth quadrants 1 and 3 and examiner 2 measured teeth quadrants 2 and 4. statistics intraand inter-examiner agreement was determined by intraclass correlation coefficient, which was calculated on the basis of the values of the buccolingual dimensions of 3 (240 measurements) randomly chosen participants and measured twice by the examiners with a 20-day interval. the distribution pattern of the values of the buccolingual dimensions was evaluated by the shapiro-wilk test, where the variable showed normal distribution. the comparison between the preferred chewing side and the opposite side for the participants with preferred and exclusive unilateral mastication and between the right and left sides for the subjects with bilateral mastication was performed with the student t test for paired samples. statistical analysis used a level of significance of a = 0.05 and the data were processed with the spss statistics 17.0.0 software (spss, chicago, il, usa). results intraand inter-examiner agreement for the buccolingual dimensions were 0.959 (p<0.001) and 0.979 (p<0.001) respectively, demonstrating excellent agreement. the mean values for the maxillary and mandibular buccolingual dimensions of the alveolar process in subjects with preferred unilateral, bilateral and exclusive unilateral masticatory patterns, as well as the comparison between comparable sides (the preferred and non-preferred sides, right and left sides, and mastication and balancing sides) are shown in tables 1 to 3. subjects with preferred unilateral and exclusive unilateral mastication did not show statistically significant difference between the preferred and non-preferred sides and the chewing and balancing sides, respectively, for both the maxilla and mandible. subjects with bilateral mastication showed statistically significant difference between the right and left sides at the heights of 6 mm (p=0.030) and 8 mm (p=0.023) between the first and second maxillary premolars. 1 2 3 4 5 a b c figure 1. definition of the interdental image and determination of the transverse dimension of the alveolar process on axial (a), sagittal (b), and coronal (c) cuts. 5 campos et al. table 1. mean values of the buccolingual dimensions of the alveolar bone in subjects with preferred unilateral mastication. interdental region height  maxilla mandible preferred side  non-preferred side p value  preferred side  non-preferred side p value mean  sd  mean  sd mean  sd  mean  sd canine  x  1rst premolar  2mm  7.89  0.75  7.92  1.00  0.892  7.47  1.18  7.40  1.07  0.758  4mm  8.67  0.94  8.83  0.98  0.332  8.58  1.39  8.55  1.55  0.929  6mm  9.15  1.22  9.11  1.33  0.841  8.90  1.37  8.76  1.41  0.432  8mm  9.45  1.62  9.28  1.52  0.509  9.14  1.47  9.05  1.53  0.685  10mm  9.88  1.96  9.77  2.03  0.765  9.47  1.66  9.53  1.80  0.843  1rst premolar  x  2nd premolar  2mm  9.38  0.96  9.19  0.86  0.239  8.16  1.83  8.02  1.27  0.681  4mm  9.83  1.09  9.89  1.17  0.680  9.35  1.79  8.98  1.34  0.201  6mm  9.95  1.34  10.02  1.20  0.722  9.67  1.70  9.53  1.51  0.528  8mm  10.05  1.38  10.02  1.40  0.852  9.94  1.59  9.76  1.59  0.380  10mm  10.31  1.69  10.20  1.78  0.535  10.08  1.55  10.15  1.55  0.702  2nd premolar  x 1rst molar  2mm  10.34  1.16  10.34  1.22  1.000  9.65  1.29  9.50  1.04  0.539  4mm  11.15  1.11  11.22  1.07  0.562  10.80  1.34  10.58  1.22  0.303  6mm  11.38  1.62  11.30  1.59  0.577  11.08  1.40  11.15  1.53  0.705  8mm  11.38  1.88  11.40  1.81  0.942  11.20  1.64  11.41  1.65  0.314  10mm  12.13  2.00  12.13  1.98  1.000  11.25  1.60  11.52  1.88  0.237  1rst molar x 2nd molar  2mm  13.01  1.09  13.11  0.90  0.597  11.19  1.19  11.04  1.33  0.219  4mm  13.67  1.02  13.92  0.81  0.165  12.40  1.43  12.34  1.38  0.717  6mm  14.33  1.14  14.36  1.14  0.900  13.32  1.65  13.21  1.58  0.623  8mm  14.18  0.85  14.45  1.13  0.355  13.43  1.62  13.33  1.46  0.565  10mm  15.80  2.01  16.45  2.98  0.552  13.22  1.81  13.34  1.58  0.521  table 2. mean values of the buccolingual dimensions of the alveolar bone in subjects with bilateral mastication. interdental region  height  maxilla mandible right side left side  p value  right side  left side  p value mean  sd  mean  sd  mean  sd  mean  sd  canine  x  1rst premolar  2mm  8.00  1.31  8.02  1.11  0.952  7.09  0.91  7.40  1.31  0.358  4mm  8.93  1.28  9.15  1.10  0.447  8.50  1.30  8.75  1.27  0.345  6mm  9.22  1.30  9.27  1.19  0.896  8.97  1.55  8.93  1.41  0.829  8mm  9.36  1.50  9.31  1.33  0.899  9.04  1.77  9.13  1.65  0.777  10mm  9.56  1.46  9.34  1.03  0.615  9.18  1.83  9.43  2.00  0.448  1rst premolar  x  2nd premolar  2mm  9.15  1.08  9.09  0.11  0.732  7.88  1.48  8.20  1.71  0.322  4mm  9.90  1.63  9.38  1.02  0.118  9.15  1.91  9.29  1.81  0.523  6mm  9.97  1.45  9.45  1.16  0.030*  9.72  1.74  9.61  1.45  0.598  8mm  9.79  1.53  9.27  1.32  0.023*  10.13  1.49  10.13  1.26  1.000  10mm  9.77  0.80  9.50  1.31  0.323  10.50  1.46  10.38  1.46  0.450  2nd premolar  x  1rst molar  2mm  10.31  1.37  10.77  1.10  0.064  9.61  1.24  9.50  1.58  0.742  4mm  11.40  1.24  11.20  0.07  0.455  10.75  1.46  10.72  1.57  0.921  6mm  11.56  0.61  11.22  1.43  0.151  11.36  1.69  11.40  1.51  0.835  8mm  11.57  2.09  11.25  1.64  0.231  11.61  1.81  11.47  1.55  0.628  10mm  11.17  3.04  11.28  2.22  0.807  11.65  1.91  11.63  1.76  0.918  1rst molar  x  2nd molar  2mm  12.88  1.01  13.13  1.37  0.490  11.18  1.30  11.15  1.28  0.884  4mm  14.22  1.00  14.34  1.16  0.713  12.34  1.46  12.18  1.40  0.396  6mm  14.38  0.71  14.11  1.23  0.263  13.25  1.72  13.09  1.60  0.494  8mm  14.33  1.19  14.02  1.29  0.179  13.77  1.88  13.54  1.97  0.331  10mm  13.50  1.62  13.81  1.14  0.504  13.77  2.03  13.43  2.28  0.180  * statistically significant difference according to student t test for paired samples. 6 campos et al. statistical power was calculated based on the group of subjects with exclusive unilateral mastication where, hypothetically, a greater difference between both sides was expected. besides, this group represents the weakest statistical condition, because it has a fewer amount of patients. alveolar dimensions of 1mm were considered as of clinical relevance. the power of statistical significance was found to be 0.78, which corresponds to a 78% chance of having a real effect. discussion the ideal masticatory pattern presents a similar number of masticatory cycles on both sides of the arches2. however, most people show preference for one chewing side during the masticatory function7-9, which may be due to an adaptive process to problems such as tooth loss, occlusal interferences, morphology of the craniofacial bones and muscular and temporomandibular joint problems9. in the present study, the occlusal and dental characteristics that might interfere asymmetrically in the alveolar bone structure were controlled, and from the total of 39 participants, 72% had unilateral mastication (54% preferred and 18% exclusive) and 28% had bilateral mastication. this distribution reflects what can be found in the general population where 73% to 77% have a preferred chewing side22. table 3. mean values of the buccolingual dimensions of the alveolar bone in subjects with exclusive unilateral mastication. interdental region  height  maxilla mandible chewing side  balancing side  p value  chewing side  balancing side  p value mean  sd  mean  sd  mean  sd  mean  sd  canine  x  1rst premolar  2mm  7.67  0.64  7.92  0.97  0.533  7.67  1.54  7.92  1.51  0.356  4mm  8.39  0.92  8.46  1.36  0.875  9.17  2.03  9.07  2.12  0.573  6mm  8.57  1.37  8.85  1.67  0.493  9.67  2.17  9.64  2.14  0.818  8mm  8.25  1.58  8.85  2.16  0.222  9.35  2.78  9.92  2.50  0.388  10mm  8.57  1.61  9.17  3.06  0.422  10.17  2.88  10.07  2.79  0.695  1rst premolar  x  2nd premolar  2mm  9.28  1.43  9.07  1.24  0.585  8.67  1.68  8.25  1.43  0.111  4mm  9.82  1.59  9.71  1.12  0.815  9.67  2.26  9.60  1.88  0.778  6mm  9.92  1.71  9.92  1.79  1.000  10.28  2.33  10.03  1.91  0.403  8mm  10.39  1.95  10.35  1.74  0.945  10.42  2.46  10.60  2.11  0.499  10mm  10.60  2.51  10.60  2.27  1.000  10.46  2.95  10.92  2.35  0.308  2nd premolar  x  1rst molar  2mm  10.25  1.02  10.39  0.80  0.558  9.39  1.48  9.96  2.37  0.403  4mm  11.16  0.58  11.75  0.93  0.052  10.39  2.02  10.89  1.36  0.197  6mm  11.79  1.02  11.91  1.47  0.832  10.96  2.00  11.53  1.53  0.084  8mm  12.10  1.52  12.40  1.85  0.485  11.17  2.12  12.00  1.53  0.054  10mm  13.00  2.88  13.18  3.27  0.547  11.60  2.56  12.07  1.85  0.239  1rst molar  x  2nd molar  2mm  12.25  0.76  12.82  1.37  0.306  10.96  1.26  11.00  1.02  0.864  4mm  13.85  1.00  13.75  1.10  0.448  12.28  1.04  12.28  1.30  1.000  6mm  14.25  0.88  14.54  1.27  0.384  13.60  1.12  13.10  1.26  0.221  8mm  14.50  0.90  15.12  1.49  0.098  14.14  1.42  14.03  1.29  0.796  10mm  15.37  1.45  16.12  2.21  0.223  14.32  1.95  14.28  1.79  0.890  7 campos et al. besides being an adaptive process to the occlusal pattern, the choice for a preferred chewing side may be associated with the type of food and its texture3. in this study, the type of food was standardized for all analyses. each participant received a portion of french bread sufficient to perform the filming of approximately 90 seconds. french bread was used in other research concerning the masticatory system with good acceptance from the population studied19. the filming technique and subsequent speech therapy evaluation are recommended strategies for the analysis of the masticatory pattern8,20,22. the video and the evaluations made by 3 speech therapists permitted a careful analysis by means of repeated visualizations and discussion of each case. cone beam computed tomography was used to evaluate the buccolingual dimension of the alveolar bone because it provides images without superimposition of structures with resolution and reliability sufficient to analyze the amount of bone and allow tridimensional manipulation of the structures under study23. research have evidenced the relationship between the preference for a chewing side and facial anthropometric measurements, demonstrating that unilateral masticatory function results in asymmetric changes of the maxilla and mandible24. studies with rats have correlated masticatory hypofunction with reduction of the alveolar bone11,12,17. in the present study, however, subjects with preferred or exclusive unilateral mastication pattern did not show statistically significant differences in the buccolingual dimension of the alveolar bone between the preferred and non-preferred sides in the interdental regions evaluated in the maxilla and mandible.  statistically significant differences were found at the heights of 6 mm and 8 mm between the first and second premolars on the right and left sides of the maxilla in patients with bilateral chewing. since from a total of 40 comparisons made for these subjects (5 heights x 4 interdental regions in maxilla and mandible), only 2 showed such variation, this result seems to reflect an isolated difference in the sample and it does not allow to infer that the masticatory pattern was responsible for the alterations found. conclusions there was no difference in the transverse dimension of the alveolar bone 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formation in tooth extraction sites. an experimental study in dogs. j clin periodontol. 2003 sep;30(9):809-18. 16. araujo mg, lindhe j. dimensional ridge alterations following tooth extraction. an experimental study in the dog. j clin periodontol. 2005 feb;32(2):212-8. 17. denes bj, mavropoulos a, bresin a, kiliaridis s. influence of masticatory hypofunction on the alveolar bone and the molar periodontal ligament space in the rat maxilla. eur j oral sci. 2013 dec;121(6):532-7. doi: 10.1111/eos.12092. 18. suhr es, warita h, iida j, soma k. the effect of occlusal hypofunction and its recovery on the periodontal tissues of the rat molar: ed1 immunohistochemical study. orthod waves. 2002;61:165-72. 19. hitos sf, solé d, periotto mc, fernandes ml, weckx ll, guedes zc. standardization of the registration and analysis of mastication: proposal for clinical application. int j orofacial myology. 2011 nov;37:47-56. 20. barcelos dc, da silva ma, batista gr, pleffken pr, pucci cr, borges ab, et al. absence or weak correlation between chewing side preference and lateralities in primary, mixed and permanent dentition. arch oral biol. 2012 aug;57(8):1086-92. doi: 10.1016/j.archoralbio.2012.02.022. 21. felício cm, folha ga, ferreira cl, medeiros ap. expanded protocol of orofacial myofunctional evaluation with scores: validity and reliability. int j pediatr otorhinolaryngol. 2010 nov;74(11):1230-9. doi: 10.1016/j.ijporl.2010.07.021. 9 campos et al. 22. macdonnell st, hector mp, hannigan a. chewing side preferences in children. j oral rehabil. 2004 sep;31(9):855-60. 23. garib dg, calil lr, leal cr, janson g. is there a consensus for cbct use in orthodontics? dental press j orthod. 2014 sep-oct;19(5):136-49. doi: 10.1590/2176-9451.19.5.136-149.sar. 24. nascimento gkbo, lima lm, freitas mcr, silva egf, balata pmm, cunha da, et al. preference side masticatory and facial symmetry in total laryngectomy: clinical and electromyographic study. rev cefac. 2013 nov-dec;15(6):1525-32. doi: 10.1590/s1516-18462013000600015. braz j oral sci. 15(3):215-220 oral health impact profile (ohip-14) and its association with dental treatment needs of adolescents in a rural nigerian community lawal folake barakat, bds, mds,fmcds, fwacs1; ifesanya joy ucheonye bds, mph, fmcds2 1 department of periodontology and community dentistry, university of ibadan, ibadan and university college hospital, ibadan 2 department of child oral health, university of ibadan, ibadan and university college hospital, ibadan correspondence: dr joy u ifesanya, department of child oral health, faculty of dentistry, college of medicine, university of ibadan, pmb 5017, ibadan, nigeria 200212 e-mailjoyifesanya@yahoo.co.uk telephone+2348055623129 abstract aim: to validate and determine the applicability of ohip-14 in assessing the impact of unmet dental treatment needs on the quality of life of adolescents in a rural community. methods: the ohip-14 questionnaire and the aesthetic component (ac) of index of orthodontic treatment need (iotn) were data collection instruments in a cross sectional survey among students in a rural community. the reliability and validity of the ohip-14 as well as the association between it and dental treatment needs including malocclusion was assessed. data obtained was analyzed using mann whitney u test. results: the mean age of participants was 14.9 (±1.6) years. the ohip-14 had acceptable cronbach alpha value of 0.8. it could discriminate between respondents with or without dental treatment needs due to caries and dental trauma (p <0.001). the ohip-14 did not differentiate between respondents with or without orthodontic treatment need (p= 0.808). however, significant association existed between being irritable with people and unmet orthodontic treatment needs (p= 0.032). conclusion: the ohip-14 is a valid and reliable quality of life assessment tool in young adolescents in this rural community. however, only the social disability domain component discriminated significantly between those with or without orthodontic treatment needs. keywords: quality of life. ohip-14. malocclusion. dental treatment needs. adolescents. introduction the concept of oral health related quality of life(ohrqol), involves the use of multidimensional constructs that assess the absence or presence of negative impacts of oral health conditions and diseases on the day to day wellbeing of an individual1,2. ohrqol was borne out of the paradigm shift from the assessment of oral health merely on the basis of clinical presence or absence of disease3. ohrqol does not assess health solely from the standpoint of the managing physician who assesses clinical signs and symptoms as the major yardsticks, but primarily incorporates the subjective (selfperceived) opinion of the patient affected by the oral condition being assessed. the subjective assessment of the impact of oral conditions on quality of life has been found useful in the planning and evaluation of oral health programmes, dental care services and instituted treatments4,5. these tools are especially valuable in rural communities of developing countries where appropriate allocation, monitoring and evaluation of sparse resources are very important. the additional merit of providing insights into individuals’ perception of the effect of oral diseases and conditions on their daily performances is of received for publication: november 11, 2016 accepted: june 12, 2017 original article braz j oral sci. july | september 2016 volume 15, number 3 http://dx.doi.org/10.20396/bjos.v15i3.8649984 216 great value6. this is especially true as it has been established that the absence of perceived needs is a major factor for not utilizing dental services3. rural communities in many parts of africa are faced with inaccessibility to and poor utilization of dental care services and high unmet dental needs both in children and adults7-10. children in these communities are noted to have poor oral hygiene and high prevalence of untreated dental caries8,11,12. unmet dental needs due to dental caries have negative impact on the daily activities of children with functional domain mostly affected13. oral conditions such as malocclusion have also been associated with impacts on the quality of life of affected individuals14 and affecting the psychological and social domains most15,16. this is especially important among adolescents who are undergoing physical, psychological, emotional and social life changes, and are under various forms of pressure to be accepted by their peers, while maintaining their ‘status’ in the home front17. the effect of treating the aforementioned oral conditions have shown significant reduction or elimination of the self-perceived negative impact on the quality of life of those affected13,18. this is one of the major advantages of using quality of life measures. numerous ohrqol measures exist which have not been utilized in nigerian rural communities. one of these is the oral health impact profile (ohip-14), which is easy to use, and has been validated and found acceptable in many regions19-21 as well as among adult nigerians in an urban region22. the evaluation of this instrument for comprehensive use both in the adult and adolescent nigerian population will require its validation, as well as the determination of its applicability in the young adolescent in rural communities with diverse cultural norms and values. the ohip14 has however been evaluated and documented among brazilian adolescents where it was found to be valid and acceptable for assessing ohrqol23. its discriminative properties to determine adolescents’ response to orthodontic treatment has also been previously assessed23. this study aimed to validate and determine the applicability of ohip-14 in assessing the impact of unmet dental treatment needs on the quality of life of adolescents in a rural community in nigeria. materials and methods this is a cross sectional study conducted among secondary school students in randomly selected schools in a rural community in southwestern nigeria over a period of six weeks. a minimum sample size of 384 was calculated at 5% α-level (zα=1.96) and difference margin of 5% using maximum prevalence of 50% with the formula for cross sectional studies24. following ethical approval from the state ethics review committee, approval and permission to conduct the study in selected schools was obtained from the local inspector of education and the local government schools’ board. schools from which students were recruited for the study were selected by simple random sampling technique through balloting from the list of schools within the community that was provided by the local government central schools’ board. the head teachers of the selected schools and class teachers of junior secondary school one to three (grade 7-9) were approached, the purpose of the study was explained and permission to conduct the study was obtained. all grade 7-9 students who were eligible for the study were approached and only those who assented and were available at the time of the study were recruited. illness and negative parental consent led to exclusion from the study. instrument of data collection was ohip-14 questionnaire, which comprises of 14 questions with two questions each under seven domains. the domains include; functional limitation, physical pain, psychological discomfort, physical disability, psychological disability, social disability and handicap19. responses to the questions are based on a likert scale which ranged from 0“never” to 4“very often”. variables also included in the questionnaire were: demographic variables and satisfaction ratings of teeth appearance and selfrating of tooth condition. the satisfaction ratings of tooth appearance assessed how satisfied the participants were with the appearance of their teeth with a response of “satisfied or dissatisfied”. the selfrating of tooth condition assessed oral condition as being “good or poor”. the satisfaction ratings and self-ratings of oral health were variables used to assess the construct validity of ohip-14 in the respondents. the questionnaire was translated to yoruba language by language professionals and back-translated by two independent research assistants with minimal modification required. the backtranslated questionnaire showed that the true meaning of the domains of the ohip-14 was preserved in the yoruba version. due to the significant language barrier in the rural community of interest, the yoruba language translation of the questionnaire was pretested among 30 students from other schools different from those selected for the study to ascertain the feasibility of self-administration as well as its comprehensiveness. the yoruba translation of the ohip-14 was subsequently administered to the students assessed in this study. in addition to administration of the ohip-14 questionnaire, oral examination was conducted by a trained examiner to determine the treatment needs of the participants. the orthodontic treatment need was assessed using the aesthetic component (ac) of the index of orthodontic treatment need (iotn). the iotn-ac scores were coded as: • no orthodontic treatment need ac grade 1-4 • borderline need ac grade 5-7 • definite treatment need ac grade 8-10 need for orthodontic treatment comprised of ac scores (5-10); borderline grade (5-7) and definite treatment need grade (8-10) were coded together in this category. need for other dental treatments due to presence of dental caries and trauma were documented as “present or absent” and considered as “other unmet dental treatment needs”. the reliability of ohip-14 for internal consistency was determined by cronbach’s alpha and its stability assessed by the intra-class correlation coefficient. the cohen kappa statistics for the test re-test reliability was conducted among 20 students who had duplicate interviews at an interval of one week. the total ohip score for the respondents was calculated by adding the responses score for each item together to give a minimum score of 0 and maximum score of 56. an impact on the quality of life was considered at a response level of hardly braz j oral sci. 15(3):215-220 oral health impact profile (ohip-14) and its association with dental treatment needs of adolescents in a rural nigerian community 217 ever. the face validity of the ohip-14 was determined by interviewing dentists, while the construct validity was assessed by the association between self-ratings of oral health and satisfaction of the participant with their ohip-14 scores. the discriminant validity was determined by comparing ohip-14 scores in those with or without treatment needs for malocclusion with ohip-14 scores of subjects with or without treatment need as a result of dental caries and trauma. data collected were processed and analyzed with spss version 21. test of association was done using mann whitney u statistics since the data was skewed. level of significance was set at <5%. kappa statistics was used to determine test-retest reliability of ohip-14 and cut off level for significance set at p<5%. results overall, 395 students participated in the study. the mean age of the study participants was 14.9 (±1.6) years. the male gender accounted for 222 (53.6%) of the participants, the rest were females. reliability of ohip-14 the cronbach alpha value for internal consistency of the ohip-14 was 0.84. the inter item coefficient ranged from 0.1 to 0.6 with no negative values and intra class correlation coefficient was 0.83. deletion of any of the items of the ohip-14 resulted in lower cronbach alpha values compared with the standardized alpha value (table1). table1 internal consistency of ohip-14 ohip item scale mean if item is deleted cronbach alpha if item is deleted trouble pronouncing words 3.06 0.825 worsened sense of taste 3.10 0.826 painful aching 2.90 0.836 uncomfortable eating 3.03 0.824 self-consciousness 3.14 0.827 felt nervous 3.14 0.829 diet been unsatisfactory 3.01 0.822 meals interrupted 3.04 0.826 difficulty to relax 3.16 0.825 embarrassment 3.10 0.825 irritable with other people 3.21 0.833 difficulty doing school work 3.25 0.831 life less satisfying 3.26 0.829 unable to perform usual function 3.23 0.835 scale mean 3.35 cronbach alpha 0.84 validity of ohip-14 the face and content validity was assessed by a team of dentists and the participants, and it was made known that the instrument assessed how oral health affected the daily activities of individuals, all questions were simple to understand and straightforward. the construct validity as assessed by comparison of ohip14 scores with a proxy since there is no overall gold standard to evaluate the criterion validity. the ohip-14 scores when compared with pain, self-perceived treatment need, global self-rating of oral health, satisfaction rating of oral health condition and tooth appearance were all statistically significant (table 2). the mean and median ohip-14 score was higher in participants who were dissatisfied with their oral health condition and tooth appearance, who rated their oral health poorly, perceived a need for dental treatment or experienced pain compared with those who did not report any of the aforementioned variables (p<0.05). table 2 the discriminant and construct validity of the ohip-14 questionnaire variable ohip-14 p value mean (sd) median self-rating of oral health good 2.5 (± 4.6) 0.0 <0.001* poor 6.8 (± 8.0) 5.0 satisfaction ratings of oral condition satisfied 2.7 (± 4.7) 0.0 <0.001* dissatisfied 6.4 (± 8.4) 3.0 satisfaction ratings of tooth appearance satisfied 2.7 (± 5.0) 0.0 <0.001* dissatisfied 7.6 (± 7.5) 6.0 perceived need for treatment no 1.9 (± 4.1) 0.0 <0.001* yes 5.4 (± 6.8) 3.0 pain no 1.7 (± 3.4) 0.0 <0.001* yes 8.2 (± 7.8) 6.0 normative orthodontic treatment need no 3.1 (± 5.3) 1.0 0.808 yes 3.7 (± 6.2) 1.0 other normative dental treatment needs no 3.0 (± 5.2) 0.0 <0.001* yes 6.5 (± 7.4) 6.0 categories of orthodontic treatment needs no need 3.1 (± 5.3) 1.0 0.920 borderline 3.3 (± 5.2) 1.0 definite need 5.8 (± 9.6) 0.0 *statistically significant with mann whitney u test ohip-14 and dental treatment needs there were 157 (39.7%) respondents with “orthodontic treatment needs” of which 28 (7.1%) had definite orthodontic treatment needs. thirty-nine respondents (9.9%) had “other dental treatment needs” due to dental trauma and caries. the ohip-scores ranged from 0-37 with a mean score of 3.4± 5.7. the mean ohip-14 score for those with “other dental treatment needs” was 6.5± 7.4, and was generally higher than mean score for those with “orthodontic treatment needs” (table 2). two hundred and two (51.5%) respondents perceived an impact on their quality of life due to oral diseases and conditions, while 155 (39.0%) had some form of need for orthodontic treatment as assessed by the aesthetic component of the iotn. mean ohip scores increased with increased need for orthodontic treatment; however, this was not statistically significant. (table 2) a significantly higher proportion of respondents with “other dental needs” reported impacts on their quality of life from; braz j oral sci. 15(3):215-220 oral health impact profile (ohip-14) and its association with dental treatment needs of adolescents in a rural nigerian community 218 worsened sense of taste, painful aching, discomfort with eating, self-consciousness, nervousness, unsatisfactory diet, meals being interrupted, embarrassment, irritability, difficulty doing school work and inability to function (table 3). however, the only significant impact effect as a result of unmet orthodontic treatment need was being irritable with people (table 3). table 3 relationship between the ohip-14 questionnaire items and the various unmet dental treatment needs ohip item orthodontic treatment need pvalue other dental treatment needs p value yes n (%) no n (%) yes n (%) no n (%) functional limitation trouble pronouncing words impact 21 (13.4) 20 (8.4) 0.742 5 (12.8) 36 (10.1) 0.330 no impact 136 (86.6) 203 (81.6) 34 (87.2) 320 (89.9) worsened sense of taste impact 25 (42.4) 34 (57.6) 0.655 10 (25.6) 20 (5.6) 0.001* no impact 132 (39.3) 204 (60.7) 29 (74.4) 336 (92.3) physical pain painful aching impact 27 (17.2) 41 (17.2) 0.468 9 (23.1) 59 (16.6) <0.001* no impact 130 (82.8) 197 (82.8) 30 (76.9) 297 (83.4) uncomfortable eating impact 15 (9.6) 31 (13.0) 0.580 9 (23.1) 37 (10.4) 0.002* no impact 142 (90.4) 207 (87.0) 30 (76.9) 319 (89.6) psychological discomfort self-consciousness impact 12 (7.6) 12 (5.0) 0.122 6 (15.4) 18 (5.1) 0.001* no impact 145 (92.4) 226 (95.0) 33 (84.6) 338 (94.9) felt nervous impact 15 (9.6%) 11 (4.6) 0.079 6 (15.4) 20 (5.6) 0.013* no impact 142 (90.4) 227 (95.4) 33 (84.6) 336 (94.4) physical disability diet been unsatisfactory impact 16 (10.2) 26 (10.9) 0.641 11 (28.2) 31 (8.7) <0.001* no impact 141 (89.8) 212 (89.1) 28 (71.8) 325 (91.3) interrupted meals impact 22 (14.0) 17 (7.1) 0.073 4 (10.3) 35 (9.8) 0.024* no impact 135 (86.0) 221 (92.9) 35 (89.7) 321 (90.2) psychological disability difficulty to relax impact 7 (4.5) 17 (7.1) 0.606 3 (7.7) 21 (5.9) 0.516 no impact 150 (95.5) 221 (92.9) 36 (92.3) 335 (94.1) embarrassment impact 14 (8.9) 15 (6.3) 0.676 7 (17.9) 22 (6.2) 0.023* no impact 143 (91.1) 223 (93.7) 32 (82.1) 334 (93.8) social disability irritable with other people 0.032* 6 (15.4) 8 (2.2) <0.001* impact 9 (5.7) 5 (2.1) 33 (84.5) 348 (97.8) no impact 148 (94.3) 233 (97.9) difficulty doing school work impact 4 (2.5) 10 (4.2) 0.581 3 (7.7) 11 (3.1) 0.020* no impact 153 (97.5) 228 (95.8) 36 (92.3) 345 (96.9) handicap life less satisfying impact 3 (1.9) 10 (4.2) 0.051 4 (10.3) 9 (2.5) 0.064 no impact 154 (98.1) 228 (95.8) 35 (89.7) 347 (97.5) unable to function impact 5 (3.5) 9 (3.8) 0.606 1 (2.6) 13 (3.7) <0.001* no impact 152 (96.5) 229 (96.2) 38 (97.4) 343 (96.3) *statistically significant with mann whitney u test discussion this study has observed that the ohip-14 is a valid tool for assessing oral health related quality of life among this group of rural dwelling adolescents as it demonstrated a value for internal consistency higher than the recommended 0.7 value25. the construct validity is also of great value among these children as the ohip-14 was able to detect significant difference in quality of life experiences among participants who required oral health intervention when compared to those that did not. in this study, the mean ohip-14 scores were highest among people who felt pain and those who were dissatisfied with the appearance of their teeth followed by those who rated their oral health as being poor or were dissatisfied with their oral conditions. this is similar to finding in previous studies where higher impacts were observed when oral health impact profile (ohip-14) and its association with dental treatment needs of adolescents in a rural nigerian community braz j oral sci. 15(3):215-220 219 self-perceived oral health was poor26,27. more participants in this rural community based study reported an impact of their general dental wellbeing on their quality of life than in a previous study among urban nigerian children28. this is probably a reflection of the lower oral health utilization among rural dwellers in this environment with attendant higher disease burden10,29. in the present study, when ‘other oral health needs’ were compared to orthodontic treatment need however, the ohip-14 had better discriminant value for assessing oral health related quality of life associated to caries and trauma than quality of life associated with malocclusion. previous studies have reported weak relationship between orthodontic indices and ohrqol tools30,31. this is partly because malocclusion itself is not necessarily a disease, but a series of deviations from the dental norm1. as a result, perception of need for treating malocclusion may be overlooked as long as functionality and aesthetics are not affected32,33. on the other hand, orthodontists view the occlusion more intensively than lay individuals do; and unless the malocclusion is critically severe, the patients’ views may never match that of the orthodontist. the insignificant relation between the ohip-14 and ohrqol due to malocclusion in this study may also be attributed to the fact that rural children are less knowledgeable about malocclusion than urban children. anosike et al., had reported more social impact on ohrqol due to malocclusion among urban dwelling children than observed in this rural based study28. in addition, studies have reported that social class is a major factor among people seeking treatment of malocclusion34,35. since most of these rural children are not from the high socioeconomic class, the low social status may also be a contributory factor. as a result of the aforementioned, these rural children cannot claim to be affected by a concept or condition they are not fully aware of. this argument poses a limitation on this study and needs to be verified by a study comparing ohrqol experiences and malocclusion between urban and rural children. another limitation is that only the aesthetic component of the iotn was assessed in this study which does not necessarily impact on the objective functional capabilities of the adolescents assessed. the social disability domain of the ohip-14 detected that children who felt a need for orthodontic treatment were more irritable than those who did not and this agrees with a previous study of urban children where the psychosocial domain was most noted to impact on quality of life of the children as far as self-perceived orthodontic need was concerned28. this further emphasizes the social impact of malocclusion on ohrqol of individuals. in conclusion, the ohip-14 is a valid tool for assessing ohrqol of dental treatment needs as a result of caries and trauma among rural nigerian children. however, it appears to be valid only in detecting the social impact of normative orthodontic treatment needs. validating 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m, albaladejo-martinez a, hernandez-martin la, rosel-gallardo em. validation the oral health impact profile (ohip-14sp) for adults in spain. med oral patol oral cir bucal. 2009 jan 1;14(1):e44-50. available from: http://www.medicinaoral. com/pubmed/medoralv14_i1_p44.pdf. 27. hongxing l, list t, nilsson i-m, johansson a, astrøm an. validity and reliability of oidp and ohip-14: a survey of chinese high school students. bmc oral health. 2014 dec 19;14:158. doi: 10.1186/14726831-14-158. available from: http://bmcoralhealth.biomedcentral.com/ articles/10.1186/1472-6831-14-158. 28. anosike an, sanu oo, da costa oo. malocclusion and its impact on quality of life of school children in nigeria. west afr j med. 2010 nov-dec;29(6):417-24. 29. sofola oo. implications of low oral health awareness in nigeria. niger med j. 2010;51(3):131-3. 30. de oliveira cm, sheiham a. orthodontic treatment and its impact on oral health-related quality of life in brazilian adolescents. j orthod. 2004 mar;31(1):20-7. 31. ashari a, mohamed am. relationship of the dental aesthetic index to the oral health-related quality of life. angle orthod. 2016 mar;86(2):337-42. doi: 10.2319/121014-896.1. 32. roberts-harry d, sandy j. orthodontics. part 11: orthodontic tooth movement. br dent j. 2004 apr;196(7):391-4. 33. agarwal ss, jayan b, chopra ss. an overview of malocclusion in india. j dent health oral disord ther. 2015;3(3):1-4. 34. adeyemi at, aderiokun ga, denloye oo. socio-economic status and utilization of orthodontic services in a nigerian hospital. odontostomatol trop. 2008 jun;31(122):27-33. 35. badran sa, al-khateeb s. factors influencing the uptake of orthodontic treatment. j public health dent. 2013 fall;73(4):339-44. doi: 10.1111/jphd.12034. oral health impact profile (ohip-14) and its association with dental treatment needs of adolescents in a rural nigerian community braz j oral sci. 15(3):215-220 oral sciences n3 original article braz j oral sci. 8(4):189-192 comparative analysis of the electronic and radiographic determination of root canal length of primary molars– an ex vivo study lucineide de melo santos1; josé ivo limeira dos reis1 1 dds, msc, phd, adjunct professor, dental school, federal university of alagoas, brazil correspondence to: lucineide de melo santos faculdade de odontologia-foufal av. lourival melo mota, s/n, tabuleiro do martins maceió al, cep: 57072-970 e-mail: jilreis@uol.com.br abstract aim: ́ to evaluate comparatively the radiographic and electronic root canal length determination in primary molars. methods: 128 canals from 66 primary molars were selected. the root of each tooth was cut open on the occlusal face, and the real tooth length (rtl) was measured and the gold standard working length ( wl) was determined by subtracting 1 mm from this measurement. the teeth were then placed in a plastic receptacle holding alginate and saline for the electronic determination of the root length using a root zx apex locator. the radiographic determination of the wl was obtained by subtracting 1 mm from the measurement highest cusp to the root apex appearing in the radiography. the data were analyzed statistically by the chi-square test at a 5% significance level. results: there was statistically significant difference (p<0.05) between each tested method and the gold standard. the root zx apex locator and the radiographic method presented satisfactory results in 75.78% and 54.68% of the cases, respectively. conclusions: the root zx apex locator is a reliable method to determine the wl of primary teeth, since it showed greater accuracy than the radiographic method. keywords: root zx, radiography, root canal length measurement, primary molars. introduction although dental caries is well known disease, primary teeth with deep carious lesions reaching the root canals is still a reality in pediatric dentistry, especially in economically deprived regions, which makes root canal therapy on these teeth necessary1. in this context, pulpectomy is still an important treatment in primary molars when the root pulp, infected or not, is not vital or is irreversibly inflamed2-3. the determination of the working length (wl) is one of the earliest stages and is a crucial point of endodontic therapy4-5, especially in primary teeth6. estimating the exact length of the root canal during endodontic therapy is fundamental to avoid injuring the permanent successor tooth6-7. while the factors related to pulpal damage in primary and permanent teeth are similar, the clinical management of these teeth can be different, based mainly on the differences between the two types of teeth8. several techniques have been proposed to determine root canal length, but the ideal procedure has yet to be identified. radiography has been widely used in the determination of root length1-2,7,9. however, radiography may generate inaccurate results, since x-rays provide a two-dimensional image of a three-dimensional streucture10. it is impossible to see, for example, the buccolingual aspect due to the superposition of the dentin, cementum, cortical bone and alveolus11. moreover, radiographic images may suggest the presence of instruments inside the canal when, in fact, they are outside it. small degrees of resorption are n ot clearly visible radiog raphically, resulting in an increased ri sk of overinstrumentation and/or overobturation2. braz j oral sci. october/december 2009 volume 8, number 4 received for publication: july 27, 2009 accepted: november 9, 2009 190 braz j oral sci. 8(4):189-192 table 1 – working length scoring system. score situation 0 working length (wl) equal to gold standard 1 working length (wl) from 0.5 to 1mm shorter than gold standard 2 working length (wl) > 1mm shorter than gold standard 3 working length (wl) exceeds gold standard table 2 – means and standard deviations of the differences between the gold standard wl and the wl obtained with the root zx and radiographic methods root zx radiography mean a0.14mm b0.68 mm standard deviation 0.45 0.88 number of canals 128 128 different letters indicate statistically significant differences (p<0.05). groups no. of cases total canals percentage (%) root zx 97a 128 75.78 radiography 70b 128 54.68 tab l e 3 – numb er of cases i n w hich th e e l ectronical ly and radiographically measured wl fell between scores 0 and 1. different letters indicate statistically significant differences (p<0.05). electronic apex locators were developed in 196212, in an attempt to obtain more precise measurements for the determination of root length and to establish the apical limit of instrumentation, and have been widely used on permanent teeth since then5,13-16. the root zx apex locator (morita®, são paulo, brazil) is a third-generation electronic device which can detect the smallest diameter of the root canal in both moist and dry conditions, and performs well on permanent teeth17, even when these teeth present root resorption18. the literature does not indicate differences when using the locator in permanent and primary teeth. however, there are some limitations in evaluating the accuracy of apex locators in primary teeth, including millimeter measurements and visualizing the exact location of the file tip, particularly in resorbed primary teeth19. moreover, the shape, dimension, and position of the root apex are often continuously altered2. several studies have focused on evaluating the use of these devices in primary dentition2,6-7,20-22, achieving accuracy rates of 64-96.7%. in view of the above considerations, this ex vivo study evaluated the electronic and radiographic determination of root canal length in primary molars comparatively with the measurement obtained by direct viewing of the endodontic file. materials and methods this work was approved by the research ethics committee of the federal university of alagoas (ufal), under the protocol number 011436/2005-98. sixty-six primary molars with up to one third of root resorption were obtained from the tooth bank of the dental school of federal university of alagoas, totalizing 128 canals. molar roots with more than 1/3 apical resorption were excluded from this study. all the teeth showed some degree of root resorption. the root of each tooth was cut open on the occlusal face with carbide ( jet brand; wheeling, il, usa) and endo-z burs (dentsply-maillefer, ballaigues, switzerland) mounted in a high-speed handpiece. the real tooth length (rtl) was then measured using a magnifying glass (x2). a 21-mm-long size 10 k-file (dentsply-maillefer) was introduced into the canal until its tip appeared at the apical foramen, or at the point of root resorption, and this measurement was marked with a rubber stop at the height of the reference cusp. the file was then placed in a digital caliper (mitutoyo, tokyo, japan) and the rtl was measured. this measure minus 1mm was recorded as the wl gold standard. the measurements were taken by a calibrated examiner and recorded on specific charts. each tooth was measured three times, and the mean value was calculated. the root canal length was determined electronically using a root zx (morita, são paulo, sp, brazil) apex locator. for such purpose, the teeth were numbered 1 to 66 and embedded in alginate ( jeltrate; dentsply ind. e com. ltda., rio de janeiro, rj, brazil) with saline, which acts as a conductor simulating the periodontium23. the canals were then flushed with 2.5% sodium hypochlorite and dried with a paper point. t he wl was m easured w ith a size 10 k-f il e. al l measurements were taken within a 2-h interval, when the gel was still sufficiently damp. the device was used according to the manufacturer’s instructions. the lip electrode was attached to the alginate. the file was inserted slowly into the canal until the signal on the lcd screen display bar reached the apex signal. at this point, the instrument was removed gradually until it displayed a measurement of 1 2 1. measurements were considered valid if the instrument remained stable for at least 5s20. each tooth was measured three times with the device and the mean value was calculated. all measurements were made by the same operator, who was blinded to the resorption stage of the teeth. the wl was determined radiographically according to the paralleling technique) using an x-ray equipment operating at 8ma and 70kvp (dabi atlante®, ribeirão preto, sp, brazil). a conventional dental x-ray film (ektaspeed e-speed group; kodak, rochester, ny, usa) was placed perpendicular to the teeth, maintaining a 20 cm focus-film object and 0.4 exposure time. the wl was determined as follows: the length of the tooth was measured on the diagnostic radiographic image, from the highest cusp to the root apex, and 1 mm was subtracted from this length. the same procedure was employed in the case of curved roots. all measurements were taken by the same operator. the electronically and radiographically measured wls were compared with the gold standard wl and scores were attributed to the resulting values24 (table 1). data were analyzed statistically by the chi-square test at 5% significance level. results there was statistically significant difference (p<0.05) between the gold standard wl and the wls obtained with the root zx and radiographic methods (table2). a comparison of the number and proportion of cases in which the wl measurements were scored 0 and 1 indicated a significant difference (p<0.05) between the two techniques, with the root zx method b eing statistical ly superior to radiog raphy (table 3). comparison of the data referring to the number and proportion of cases in which the wl measurements fell between scores 2 and 3 also showed statistically significant difference (p<0.05) between the two techniques (table 4). comparative analysis of the electronic and radiographic determination of root canal length of primary molars– an ex vivo study 191 braz j oral sci. 8(4):189-192 groups no. of cases total canals percentage (%) root zx 31a 128 24.21 radiography 58b 128 45.31 different letters indicate statistically significant differences (p<0.05). tabl e 4 – numbe r of cas es in w hich th e e l ectronical ly and radiographically measured wl fell between scores 0 and 1. discussion the correct determination of the length of root canals is essential to avoid injuring the permanent successor tooth during root canal therapy6-7. most root canal length determination methods are based on radiographic examination1-2,7. however, radiographic images may lead to misinterpretations, since slight degrees of resorption may not be visible and the superposition of adjacent anatomical structures may impair the clarity of the image9. moreover, the exposure of children to x-rays should be reduced25. hence, the electronic method has been proposed as an alternative2,7. in the present study, the root zx device provided wl measures that were closer to the gold standard than those obtained with the radiographic technique. these data were confirmed in the comparison with the mean scores attributed to the differences of the measurements, since it was found that most measurements obtained radiographically were 1 mm shorter than the gold standard wl, while those obtained with the root zx device were closer with the gold standard. these findings are similar to several studies reported in the literature that evaluate the accuracy of the root zx method in primary teeth, which have demonstrated that electronic measurements are closer to the wl than those obtained radiographically6-7, 9. when the apical constriction is destroyed by apical root resorption, it is very difficult to determine the wl based only on an xray image, and the root zx method can be used in these cases18. it should be emphasized, at this point, that physiological26 or pathological root resorption frequently occurring in primary teeth should not be seen as an obstacle preventing the use of electronic apex locators. root resorption does not involve more than two thirds of the root; therefore, endodontic therapy is counter-indicated in these cases. the electronic apex locator can work accurately in primary teeth with root resorption because the root canal typically has a decreasing taper towards the defect23. in the present study, all teeth presented root resorption, though without affecting the performance of the root zx device, which confirms the findings of previous studies2,6-7,19-20. with regard to the number and proportion of cases when the wl measurements were the same or 0.5 to 1 mm shorter than the gold standard wl, it was observed that the root zx technique presented a higher percentage (75.78%) than the radiographic technique (54.68%), a statistically significant difference. these results are partially consistent with those obtained in previous studies6-7. by comparing radiographic and electronic measurements to determine the root canal length, katz et al.7 and subramaniam et al.9 found no significant difference between the methods, although the x-ray based measurements were higher than those obtained with the root zx device. a probable explanation for this difference in results lies in the methodology used in the present research, which established scores to evaluate the data obtained, unlike the cited studies. some studies investigating the accuracy of electronic apex locators in permanent teeth have shown that the accuracy of the root -zx device varied from 75 to 82.3%5, 27. the proportion of cases in which the wl measurements were more than 1 mm shorter or longer than the gold standard wl was different, although the root zx method presented superior results. this means that the use of the electronic apex locator enabled us to obtain wl measurements that are closer to the gold standard wl and shorter than the apical foramen or the earliest stages of resorption. considering that the objective of the endodontic therapy in primary teeth is to eliminate infection and keep the tooth functional until its physiological exfoliation, without compromising the permanent successor28, it can be inferred that the radiographic technique is less accurate than the root zx technique, since it presented a higher proportion of cases in which the wl was more than 1 mm shorter than the gold standard wl. it may be concluded that the root zx locator is a reliable method to determine the wl of primary teeth, since the root zx method showed s smaller difference from the gold standard in determining the acceptable wl than the radiographic method. references 1. garcia–godoy f. evaluation of an iodoform paste in root canal therapy for infected primary teeth. j dent child. 1987; 54: 30-4. 2. mente j, seidel j, buchalla w, koch mj. eletronic determination of root canal length in primary teeth with and without root resorption. int endod j. 2002; 35: 447-52. 3. rodd hd, waterhouse pj, fuks ab, fayle sa, moffat, ma. pulp therapy for primary molars. int j paediatr dent. 16 (suppl.1): 2006; 15-23. 4. ingle ji, beveridge ee. endodontics. 2a ed. philadelphia: lea & febiger; 1976. 5. wrbas kt, ziegler aa, altenburger m.j., schirrmeister j.f. in vivo comparison of working length determination with two electronic apex locators. int endod j. 2007; 40: 133-8. 6. kielbassa am, muller u, munz i, monting js. clinical evaluation of the measuring accuracy of root zx in primary teeth. oral surg oral med oral pathol oral radiol endod. 2003; 95: 94-100. 7. katz a, mass e, kaufman ay. eletronic apex locator: a useful tool for root canal treatment in the primary dentition. j dent child. 1996; 63: 414-7. 8. koshy s, love rm. endodontic treatment in the primary dentition. aust endod j. 2004; 30: 59-68. 9. subramaniam p, konde s, mandanna dk. an in vitro comparison of root canal measurement in primary teeth. j indian soc pedod prev dent. 2005; 23: 124-5. 10. pineda f, kuttler y. mesiodistal and buccolingual roentgenographic investigation of 7.275 root canals. oral surg med oral pathol. 1972; 33: 101-10. 11. gupta d, grewal n. root canal configuration of deciduous mandibular first molars an in vitro study. j indian soc pedod prev dent. 2005; 23: 134-7. 12. sunada i. new method for measuring the length of the root canal. j dent res. 1962; 41: 375-87. 13. kaufman ay, fuss z, keila s, waxenberb s. realibility of different electronic apex locators to detect root perforations in vitro. int endod j. 1997; 30: 403-7. 14. ebrahim ak, wadachi r, suda h. in vitro evaluation of the accuracy of five different electronic apex locators for determining the working length of endodontically retreated teeth. aust endod j. 2007; 33: 7-12. 15. 1williams cb, joyce ap, roberts s. a comparison between in vivo radiographic working length determination and measurement after extraction. j endod. 2006; 32: 624-7. 16. d’assunção fl, albuquerque ds, de queiroz ferreira lc. the ability of two apex locators to locate the apical foramen: an in vitro study. j endod. 2006; 32: 560-2. 17. pagavino g, pace r, baccetti t. a sem study of in vivo accuracy of the root zx electronic apex locator. j endod. 1998; 24: 438-41. 18. goldberg f, de silvio ac, manfré s, nastri n. in vitro measurement accuracy of an eletronic apex locator in teeth with simulated apical root resorption. j endod. 2002; 28: 461-3. 19. leonardo mr, silva lab, nelson-filho p, silva rab, lucisano mp. ex vivo accuracy of an apex locator using digital signal processing in primary teeth. pediatr dent. 2009: 31: 320-2. 20. tosun g, erdemir a, eldeniz au, sermet u, sener y. accuracy of two electronic apex locators in primary teeth with and without apical resorption: a laboratory study. int endod j. 2008; 41: 436-41. 21. leonardo mr, silva lab, nelson-filho p, silva rab, raffaini msgg. ex vivo evaluation of the accuracy of two eletronic apex locators during root canal length determination in primary teeth. int endod j. 2008; 41: 317-21. comparative analysis of the electronic and radiographic determination of root canal length of primary molars– an ex vivo study 192 braz j oral sci. 8(4):189-192 22. angwaravong o, panitvisai p. accuracy of electronic apex locator in primary teeth with root resorption. int endod j. 2007; 42: 115-21. 23. ounsi hf, haddad g. in vitro evaluation of the reliability of the endex electronic apex locator. j endod. 1998; 24: 120-1. 24. menezes jvnb. estudo in vitro do grau de fidelidade de técnicas de odontometria em molares decíduos [master ’s thesis]. florianópolis: universidade federal de santa catarina; 1999. 25. bagett fj, mackie ic, worthington hv. an investigation into the measurement of the working length of immature incisor teeth requiring endodontic treatment in children. br dent j. 1996; 181: 96-8. 26. harokopakis-hajishengallis e. physiologic root resorption in primary teeth: molecular and histological events. j oral sci. 2007; 49: 1-12. 27. tselnik m, baumgartner jc, marshall jg. an evaluation of root zx and elements diagnostic apex locators. j endod. 2005; 31: 507-9. 28. reddy s, ramakrishna y. evaluation of antimicrobial efficacy of various root canal filling materials used in primary teeth: a microbiological study. j clin pediatr dent. 2007; 31: 193-8. comparative analysis of the electronic and radiographic determination of root canal length of primary molars– an ex vivo study braz j oral sci. 15(4):308-314 systematic review of the measurement properties of instruments utilized to diagnose temporomandibular disorders according to the cosmin checklist marília barbosa santos garcia,1 ana paula amaral,1 cid andre fidelis de paula gomes,2 fabiano politti,1 daniela aparecida biasotto-gonzalez,1 1postgraduate program in rehabilitation sciences, núcleo de apoio a pesquisa em análise do movimento, universidade nove de julho (uninove), rua profa maria jose barone fernandes, 300, são paulo, sp 02117-020, brazil. mariliabsg@gmail.com; ap.fisioterapeuta@gmail.com; politti@uninove.br; dani_atm@uninove.br 2postgraduate program in biophotonics applied to health sciences, universidade nove de julho (uninove), rua vergueiro, 235, são paulo, sp, 01504-001, brazil. cid.andre@gmail.com correspondence to: marília barbosa santos garcia rua andrea del sarto, 205, jardim martineli, itatiaia cep: 27498000, rj, brazil telephone: +55 24988332338 email: mariliabsg@gmail.com abstract introduction: most of the instruments used in brazil to diagnose temporomandibular disorders (tmd) were developed in another language. to effectively use instruments that were created in another language, it must be translated into the relevant target language before cross-cultural adaptation. clinimetric tests should also be performed. measurement properties consist of quality criteria related to evaluation instruments. these criteria are necessary to determine the quality of the instruments used in brazil. objectives: the aim of the present systematic review was to assess the quality of the measurement properties of instruments utilized to diagnose tmd. methods: systematic searches were performed of the pubmed, scielo, lilacs and science direct databases. studies addressing questionnaires translated and cross-culturally adapted for use in brazil were retrieved and the quality of the measurement properties was analyzed using the cosmin checklist. results: in the 11 eligible articles, 10 instruments were identified. the studies were evaluated based on their analysis of structural validity, internal consistency, reproducibility (concordance and reliability), responsiveness, ceiling effect and floor effect. none the assessment tools had all its measurement properties tested. conclusion: the measurement properties of the instruments were not completely tested. thus, care must be taken when interpreting the scores of these questionnaires. keywords: temporomandibular disorder, measurement properties, questionnaire. received for publication: april 19, 2017 accepted: june 14, 2017 original article braz j oral sci. october | december 2016 volume 15, number 4 http://dx.doi.org/10.20396/bjos.v15i4.8650046 introduction temporomandibular disorder (tmd) regards a set of clinical problems involving articular noises, limited range of motion and/or deviations in mandibular function, pre auricular pain, as well as pain in the temporomandibular joint (tmj) and/or pain” and “questionnaire” or “index” or “scale” or “score” or “assessment” or “evaluation” or “self-report” or “inventory” and “brasil” or “brazil” or “portuguese” or “brazilian-portuguese” or “brazilian”. no restrictions were imposed regarding language or publication date. the last search was performed on dez 17th, 2015. inclusion criteria the study included instruments used in brazil to diagnose tmd that had been submitted to a procedure for testing the measurement properties. this study included instruments with variable characteristics of the sample (with tmd, without tmd, or both), adult or pediatric population and different statistics analyses. only studies that were published in complete texts were included. exclusion criteria texts that were part of theses and dissertation, congress summaries or books were excluded. data extraction and assessment of the methodological quality of the eligible studies data referring to measurement properties were extracted from each study and analyzed according to the cosmin checklist10-14. when assessing the quality of an instrument, one can distinguish three domains of quality: reliability; validity and responsiveness. each domain contains one or more measurement properties. the domain reliability contains three measurement properties: internal consistency; reliability and measurement error. the domain validity also contains three measurement properties: content validity; structural validity and criterion validity. the domain responsiveness contains only one measurement property, also known as responsiveness. a number of measurement properties contain one or more aspects, which were defined separately: validity of the content included face validity; structural validity includes structural validity, tests of hypotheses and crosscultural validity10-14. according to cosmin, the main measurement properties are the following: internal consistency; validity; reliability; the measurement error and ceiling and floor effects. internal consistency is a measurement of the homogeneity of an instrument and indicates the degree to which the items of a determined instrument are correlated, thereby measuring the same construct. validity indicates whether the instrument is assessing the construct it proposes to measure and can be used to measure the criterion validity (in the case of a “gold standard”) or the structural validity (when there is no “gold standard” for comparison). reliability refers to the capacity of a certain test to obtain similar results for stable individuals. the measurement error confirms the errors in patients scores that did not attribute real changes to the construct that was measured. ceiling and floor effects refer to the number of individuals interviewed that reached the maximum and minimum score possible, respectively10-14. the cosmin checklist has 12 boxes, ten of which can be used to evaluate whether a study meets the requirements of adequate methodological quality. nine of these boxes have norms for measurement properties: box a (internal consistency); box b (reliability); box c (measurement error); box d (content validity, including face validity); box e (structural 309 masticatory muscles1. the literature offers different instruments to diagnose tmd, with distinct categorizations: questionnaires, anamnestic indices, clinical indices and diagnostic criteria2-7. most of the instruments used in brazil to diagnose temporomandibular disorders (tmd) were developed in another language. to effectively use instruments that were created in another language, it must be translated into the relevant target language before cultural adaptation. clinimetric tests should also be performed to evaluate the measurement properties. this procedure is fundamental due to the different customs, cultures, languages and perceptions of health found in different countries. the culturally adapted questionnaire overcomes linguistic and cultural barriers8. that assessment instruments are only useful and capable of providing scientifically robust results when they demonstrate satisfactory measurement properties, that is, that all the measurement properties have been tested with adequate sample and also that they present values statistically indicated by the criterion of quality followed when performing the clinimetric tests8. despite the significant increase in the quantity of assessment scales and/or questionnaires, many of them have not been developed and/or validated appropriately9. studies that evaluate the measurement properties of assessment tools should have a high degree of methodological quality. to evaluate the quality of such studies, criteria are needed to classify the study design and statistical analyses. the consensusbased standards for the selection of health status measurement instruments (cosmin) checklist provides these criteria10-14. this list was developed in an international, multidisciplinary study involving the participation of 43 specialists in measurement properties in the field of health10-14. according to the cosmin group, studies assessing measurement properties should exhibit a high methodological quality in order to ensure appropriate conclusions concerning the validity of the instrument10-14. the aim of the present systematic review was to employ the cosmin checklist to analyze the methodological quality of measurement properties of tmd assessment tools for use in brazil. methods the present study was a systematic review, which followed the recommendations of the prisma checklist. it was registered under number 2014 crd42014014286 in prospero (international prospec tive register of systematic reviews) and can be accessed at http://www.crd.york.ac.uk/prospero/display_ record.asp?id=crd42014014286. the details of the protocol of this systematic review can be accessed using the following link: http://www.crd.york.ac.uk/prosperofiles/14286_ protocol_20140920.pdf. study selection systematic searches were performed of the pubmed, scielo, lilacs and science direct databases. the search terms and operators (and, or or not) used in the electronic databases were as follows: “temporomandibular disorder” or “temporomandibular dysfunction” or “temporomandibular systematic review of the measurement properties of instruments utilized to diagnose temporomandibular disorders according to the cosmin checklist braz j oral sci. 15(4):308-314 310 validity), box f (hypotheses testing); box g (cross-cultural), box h (criterion validity) and box i (responsiveness). box j is used for the interpretability of the results of a given study. box irt is used for item response theory and the generalizability box is used for the possibility of the generalization of a study regarding one or more measurement properties13. part of the cosmin group developed an evaluation scale to classify each measurement property as excellent, good, reasonable or weak based on the scores of the items in the corresponding box. methodological quality using this scale is defined by the worst score of a given box. thus, a box with some items classified as excellent or good, but one item classified as poor is classified as having poor methodological quality (“worse score counts”)14. the extraction of the data and evaluations were performed by a single rater and verified by an independent reviewer, who then met to discuss the findings. no divergences of opinion were found between the rater and independent reviewer. results in total, 513 studies were found in the searches, although only 11 were considered eligible for the data analysis (figure 1). systematic review of the measurement properties of instruments utilized to diagnose temporomandibular disorders according to the cosmin checklist reduced version of the qaado25. a number of the measurement properties from all of the instruments found in the searches were tested. the table 1 displays the sample size, sample description and statistical values of each measurement property assessed in the studies included in this systematic review. the table 2 displays the assessments of measurement properties, according to the cosmin checklist10-14. in summary, the measurement properties reliability, internal consistency and content validity were tested for the rdc/tmd15,16. reliability and internal consistency were tested for the multimedia version of the rdc/tmd17. reliability, internal consistency and the content validity were tested for the mandibular function impairment questionnaire (mfiq)18. internal consistency and reliability were analyzed for the fai19. only the content validity was analyzed in the cr-10 for tmd20. internal consistency, reliability and validity were tested for the reduced version of the fai21. internal consistency, reliability and the criterion validity were analyzed for the self-report of oral conditions22.internal consistency, reliability and the structural validity were tested for the brasil-mopds23. only reliability was tested for the qaado24, whereas reliability, internal consistency and the content validity were tested for the reduced version of the qaado25. lucena et al.15 analyzed the internal consistency (box a), reliability (box b) and structural validity (box e) of the rdc/ tmd, for which the respective classifications were good, fair (based on the moderate sample size [n = 45], although other items were classified as good and excellent) and poor. the poor classification for structural validity was due to an error regarding the formulation of hypotheses, which were not described prior to testing the validity. campos et al.16 tested the internal consistency (box a) and reliability (box b) of the rdc/tmd, for which the respective classifications were good and fair (due to the sample size [n = 36]). campos et al.19 analyzed the internal consistency (box a) and reliability (box b) of the fonseca index, for which the respective classifications good (due to the failure to perform factor analysis) and fair (due to the moderate sample size [n = 40]). ferreirabacci et al.20 analyzed the content validity (box d) of the cr-10 questionnaire used to measure pain associated with tmd, for which the classification was fair. cavalcanti et al.17 analyzed the internal consistency (box a) and reliability (box b) of the multimedia version of the rdc/tmd, for which both classifications were fair due to the failure to perform factor analysis and the moderate sample size (n = 30). campos et al.18 tested the measurement properties of the mfiq. internal consistency (box a) was classified as good, with some items were classified as excellent, such as the use of factor analysis. reliability (box b) was classified as good due to the adequate sample size (n = 62). content validity (box d, including face validity) was classified as excellent. this was the only study to employ cronbach’s alpha coefficient in combination with factor analysis, which is an important analysis, as it allows the identification of subscales on a questionnaire and cronbach’s alpha coefficient should be calculated separately for each subscale26. the studies that evaluated the rdc/tmd and the multimedia version of the rdc/tmd calculated cronbach’s alpha coefficients, but did not employ factor analysis. fig. 1 flowchart of systematic review. in the 11 eligible articles, ten instruments were identified: the research diagnostic criteria for temporomandibular disorders(rdc/tmd)15,16; the multimedia version of the rdc/ tmd17; the mandibular function impairment questionnaire (mfiq)18; the fonseca anamnestic index (fai)19; the category-ratio scale (cr-10)20; the reduced version of the fai21; the self-report for oral conditions22; the brazilian version of the manchester orofacial pain disability scale (brasil-mopds)23; a screening questionnaire for orofacial pain and temporomandibular disorders, recommended by the american academy of orofacial pain (qaado)24; and the braz j oral sci. 15(4):308-314 311 systematic review of the measurement properties of instruments utilized to diagnose temporomandibular disorders according to the cosmin checklist legend: k =kappa; icc = intraclass correlation coefficient; ci = confidence interval; kr-20 =kuder-richardson reliability coefficient; cvr= content validity ratio; d1= functional capacity dimension; d2= nourishment dimension; es= effect size; ave= average variance extracted; cr = composite reliability; who= world health organization; ohip14= oral health impact profile-14; ohip= oral health impact profile. vas= visual analogue scale; mifq= mandibular function impairment questionnaire. table 1 classification of the measurement properties of the articles included in the review according to the cosmin checklist. study sample size sample description reliability internal consistency responsiveness validity lucena et al., 2006 (rdc/tmd) n=155 n=45 (retest) volunteers with tmd kappa between 0,73 and 0,91 (p<0,01) cronbach alpha value= 0,72 _ spearman correlation between the ohip14 and ohip 0.598 0.349 0.336 0.261 campos et al., 2007 (rdc/tmd) n=109 n=36 (retest) volunteers with tmd intra-examiner: iccquestions: 4a = 0, 9869 4b = 0, 9856 7 = 0, 8302 8 = 0, 9869 9 = 0, 9661 11 = 0, 9850 12 = 0, 9785 13 = 0, 9265 cronbach alpha value= 0, 8479 (intensity of pain and disability) 0, 8971 (limitation of mandibular function) 0, 8673 (non-specific physical symptoms including pain) 0, 8080 (non-specific physical symptoms excluding pain) 0, 9270 (depression) _ _ ferreira-bacci et al., 2009 (cr-10) n=121 volunteers with tmd _ _ _ pearson correlation between the vas (p=0,76) campos et al., 2009 (fai) n=1230 n=40 (retest) volunteers without tmd kappa question1=0.787 question 2=0.725 question 3=0.771 question 6=0.805 question 7=0.838 kr-20= 0.5594 _ _ cavalcanti et al., 2010 rdc/tmd multimedia version) n=30 (15 responded in writing and 15 responded to the multimedia version) volunteers with tmd _ cronbach alpha value= 0.94 _ spearman correlation with the rdc/ tmd written version 0.670 to 0, 913 p < 0.01 campos et al., 2012 (mfiq) n=62(responded twice – reproducibility) n=219 (validation) volunteers with tmd iccd1= 0,895, 95% ic d1= 0,832 to 0,935 iccd2= 0,825, 95% ic d2= 0, 726 to 0, 891 cronbach alpha valued1= 0, 874 cronbach alpha value d2= 0, 918 _ content validity (face validity) cvr minimum of 0.43 manfredri et al., 2001 (qaado) n=46 volunteers with tmd _ k= 0, 454 (muscle disorders) k= 0, 043 (intra-joint disorders _ _ kallás et al., 2012 (brasil-mopds) n=50 volunteers with orofacial pain and tmd icc (inter-examiner)= 0.92 icc (intra-examiner)= 0.98 cronbach alpha value= 0.9 _ structural validity (pearson) with ohip14 (r= 0.85) and with vas (r= 0.75) franco-micheloni et al., 2014 (qaado reduced version) n=1307 volunteers with and without tmd k=0.840 kr-20= 0.673 _ content validity cvr= 0.42 campos et al., 2014 (fai – reduced version) n=700 volunteers without tmd _ cronbach alpha value= 0.745 _ structural validity ave = 0.513, cr = 0.878 concurrent validity with the mifq r=0.66, p<0.01 pinelli and loffredo, 2007 (self-report of oral conditions) n=200 volunteers with orofacial pain (k = 0.85 for the condition of the tmj) (k = 0.81 for the periodontal condition)and (k = 0.69 for the dental condition) _ _ criterion validity with clinical examinations of the who manual braz j oral sci. 15(4):308-314 312 construct validity presented a good methodological quality. the self-report of oral conditions had the properties of measure reliability and internal consistency tested through the study of pinelli and loffredo22.the reability was classified as reasonable because it did not use icc in statistical analysis. the internal consistency obtained a weak degree because cronbach's alpha was not calculated. the criterion validity (box e) of the self-report of oral condition was also tested. a reasonable classification was reached because it did not use a gold standard questionnaire in the statistical comparison. discussion the aim of the present study was to assess the quality of the measurement properties of instruments to diagnose temporomandibular disorder that had been translated to portuguese. none of the questionnaires completely tested all measurement properties. reliability was tested in 81.8% of the questionnaires, with 66.7% classified as reasonable. this classification was mainly due to the fact that none of the articles mentioned which type of intraclass correlation coefficient (icc) was used to measure reliability. it is extremely important to specify which type of icc was used in different tests, given that different iccs can lead to completely different results, which would underestimate or overestimate the reliability, depending on the icc used27. internal consistency was also assessed in 81.8% of the instruments, with 77.8% receiving a classification of good. questionnaires did not receive a classification of excellent if they did not use the cronbach alpha test in combination with factorial analysis. this analysis is important since it can identify how many scales are present in a questionnaire. if there is more than one scale, the cronbach alpha value should be calculated for each sub-scale separately26. only the reduced version of the fai and the mifq used the cronbach alpha test in combination with factorial analysis. measurement errors were not tested in any of the questionnaires. the measurement error confirms errors in the scores of patients that did not attribute real changes in the construct that was measured14. the criterion validity was tested in 9.09% of the instruments and was classified as reasonable. the content validity was analyzed in 18.9% of the studies and was classified as good. the structural validity was tested in 46.1% of the instruments. in these studies, there were no classifications of excellent. the studies used pearson’s correlation test to correlate a specific questionnaire with other similar measurements. however, prior to testing the structural validity, it is important to formulate hypotheses that should specify the range and the direction of the expected correlation. hypotheses were not formulated in any of the studies included in this review. without specific hypotheses, the risk of bias can be high since it is easier to develop an alternative explanation for low correlations, rather than concluding that the questionnaire may not exhibit high indices for the validity of the construct26. systematic review of the measurement properties of instruments utilized to diagnose temporomandibular disorders according to the cosmin checklist table 2 classification of questionnaires related to tmd according to the cosmin checklist.. studies measurement property analyzed classification according to the cosmin checklist lucena et al., 2006 (rdc/ tmd) internal consistency reliability structural validity good reasonable weak cavalcanti et al., 2010 (rdc/ tmd) internal consistency reliability reasonable weak campos et al., 2007 (rdc/tmd) internal consistency reliability good reasonable ferreirabacci et al., 2009 (cr-10) structural validity weak campos et al., 2012 (mifq) internal consistency reliability content validity good reasonable good manfredi et al., 2001 (qaado) reliability weak campos et al., 2009 (fonseca) internal consistency reliability good reasonable kallás et al., 2012 (brazil-mopds) internal consistency reliability structural validity good reasonable good franco-micheloni et al., 2014 (qaado reduced version) internal consistency reliability content validity good reasonable good campos et al., 2014 (fai, reduced version) internal consistency structural validity good good pinelli & loffredo, 2007 (self-report of oral conditions) internal consistency reliability criterion validity weak reasonable reasonable manfredi et al.24 analyzed the reliability (box b of the cosmin checklist) of the qaado. although the sample size (n = 46) was classified as fair and other items were classified as good, the study was classified as poor with regard to reliability due to the failure to calculate the intraclass correlation coefficient (icc), since the “worst score counts” on the cosmin checklist14. only the studies analyzing the rdc/tmd15 and mifq18 mentioned the type of intraclass correlation coefficient (icc) employed to measure reliability. both studies used 95%. it is extremely important to state the type of icc employed, as different iccs can demonstrate completely different results, which can either underestimate or overestimate reliability27. the study that analyzed the brazil-mopds23 questionnaire tested the following measurement properties: internal consistency (box a), reliability (box b) and structural validity (box e). the internal consistency obtained a good classification. the reliability reached a reasonable classification because it did not present the type of icc used in the analysis. and the structural validity was classified as good. the study developed by franco-micheloni et al.25 tested the following measurement properties of qaado instruments reduced version: internal consistency (box a), reliability (box b) and construct validity (box e). the internal consistency has been classified as good due to the factorial analysis. the reliability obtained a reasonable classification because it did not present the type of icc used in the analysis. finally, the braz j oral sci. 15(4):308-314 313 systematic review of the measurement properties of instruments utilized to diagnose temporomandibular disorders according to the cosmin checklist responsiveness was not assessed in any of the studies included in this systematic review. responsiveness represents the ability of a questionnaire to detect clinical changes over time14. in addition, none of the studies tested ceiling/floor effects and consequently, it is not clear if the instruments assessed would fail to detect an improvement or a worsening in certain patients. costa et al.28 found the same problems in a systematic review on cross-cultural adaptations and measurement property tests of a questionnaire designed to assess pain intensity (mcgill pain questionnaire). among the 44 different versions of the questionnaire for 26 different languages/cultures, most measurement properties were either not tested or were tested inadequately. the same was found in a systematic review of assessment tools designed for the evaluation of low back pain29, for which most studies evaluated reliability and structural validity, but failed to test internal consistency, responsiveness, the ceiling effect and the floor effect. bot et al.30, conducted a systematic review to analyze the measurement properties of questionnaires that assessed shoulder disorders and found different assessment methods for measurement properties, as well as flaws in assessments of the structural validity, internal consistency and reliability. in most of the instruments assessed, hypotheses related to the range and direction of the expected correlations with other instruments were not formulated. factorial analysis was also not conducted. when it was used, it did not always confirm the dimensions that the questionnaire proposed to measure. responsiveness was usually tested in samples of inadequate sizes. most of the studies did not adequately describe the study method and/or data analysis. it should be pointed out that other guidelines can be used for the evaluation of procedures for testing measurement properties that do not require all the criteria found on the cosmin checklist. however, the decision was made to employ this checklist based on the fact that its quality criteria are the most updated and widely accepted in the literature10-14,26. every effort was made in the systematic search of the electronic databases to identify studies on tmd assessment tools in brazilian portuguese. however, it is possible that unpublished data on the measurement properties of the assessment tools analyzed could be found in dissertations and theses. such texts were not considered in the selection process, which may be interpreted as a possible limitation of the present review. the measurement properties of the instruments included in this systematic review ranged in classification from good to weak, according to the criteria of the cosmin checklist. these questionnaires are used in many brazilian scientific epidemiological or clinical researches. thus, care must be taken when interpreting the scores of questionnaires that have not had their measurement properties completely tested or were not tested in accordance with quality criteria. finally, we recommend that instrument 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j. self-report outcome measures for low back pain: searching for international cross-cultural adaptations. spine (phila pa 1976). 2007 apr 20;32(9):1028-37. 30. bot sd, terwee cb, van der windt da, bouter lm, dekker j, de vet hc. clinimetric evaluation of shoulder disability questionnaires: a systematic review of the literature. ann rheum dis. 2004 apr;63(4):335-41. braz j oral sci. 15(4):308-314 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1512/1165 1/10 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1512/1165 2/10 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1512/1165 3/10 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1512/1165 4/10 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1512/1165 5/10 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1512/1165 6/10 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1512/1165 7/10 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1512/1165 8/10 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1512/1165 9/10 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1512/1165 10/10 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1538/1191 1/15 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1538/1191 2/15 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1538/1191 3/15 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1538/1191 4/15 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1538/1191 5/15 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1538/1191 6/15 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1538/1191 7/15 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1538/1191 8/15 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1538/1191 9/15 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1538/1191 10/15 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1538/1191 11/15 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1538/1191 12/15 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1538/1191 13/15 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1538/1191 14/15 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1538/1191 15/15 oral sciences n3 original article braz j oral sci. october | december 2015 volume 14, number 4 braz j oral sci. 14(4):294-298 incidence and classification of bifid mandibular canals using cone beam computed tomography george borja de freitas1, alessandra de freitas e silva1, lucas alexandre morais2, milena bortolotto felippe silva1, thiago coelho gomes da silva2, luiz roberto coutinho manhães júnior1 1 são leopoldo mandic slmandic, dental school, department of oral and maxillofacial radiology, são paulo, sp, brazil 2 universidade de pernambuco – upe, pernambuco dental school, department of oral and maxillofacial surgery, recife, pe, brazil correspondence to: george borja de freitas rua dr. josé rocha junqueira, 13 bairro ponte preta cep:13045-755 campinas sp phone: +55 19 3518 3600 +55 87 99657 0044 e-mail: george_borja@hotmail.com abstract aim: to determine the prevalence and classification of bifid mandibular canals using cone beam computed tomography (cbct). methods: the sample comprised 300 cbct scans obtained from the radiology and imaging department database at são leopoldo mandic dental school, campinas, sp, brazil. all images were performed on classic i-cat® cbct scanner, with standardized voxel at 0.25 mm and 13 cm fov (field of view). from an axial slice (0.25 mm) a guiding plane was drawn along the alveolar ridge in order to obtain a cross-section. results: among 300 patients, 188 (62.7%) were female and 112 (37.3%) were male, aged between 13 to 87 years. changes in the mandibular canal were observed in 90 patients, 30.0% of the sample, 51 women (56.7%) and 39 men (43.3%). regarding affected sides, 32.2% were on the right and 24.5% on the left, with 43.3% bilateral cases. conclusions: according to the results obtained in this study, a prevalence of 30% of bifid mandibular canals was found, with the most prevalent types classified as b (mesial direction) and bilateral. keywords: anatomy; mandible; classification. introduction surgical procedures such as exodontia of mandibular third molars, osteotomy, mandibular anesthetic technique, bone remodeling and implant placement involve the risk of injuring the mandibular alveolar nerve, buccal nerve and lingual nerve, with consequent transient or permanent paresthesia. the mandibular nerve has a complex pathway, originating from the trigeminal ganglion and branching down to the inferior alveolar nerve in the infratemporal fossa. the mandibular canal is located within the ramus and body of the mandible, creating a pathway that begins at the mandibular foramen, externalizing in the mental foramen. this pathway may or may not follow an intraosseous route towards the mental region as a single canal. it is also characterized by curvatures along a posterior anterior direction, crossing obliquely the entire body of the mandible. topographically, it is located closer to the inner bone wall up to the mesial aspect of the first molar, moving across to the outer bone wall until it reaches the mental foramen1. the radiographic appearance of the mandibular canal is characterized by a radiolucent strip between two radiopaque lines2 generally as a single structure, presenting different positions inside the body of the mandible in both the upperlower and the medial-lateral directions3 with occasional duplicate pathways and bifurcations4, and even trifurcations5 in some cases. knowledge of the jaw anatomy and the path of the mandibular alveolar http://dx.doi.org/10.1590/1677-3225v14n4a08 received for publication: november 09, 2015 accepted: december 13, 2015 295295295295295 nerve in the mandibular canal is of great importance to dentists, especially for those planning orthognathic surgery, mandibular reconstruction, extraction of third molars or installation of osseointegrated implants6-7. it is therefore paramount to investigate the frequency and classification of bifid mandibular canals using cbct to aid surgical planning at posterior regions of the mandible, minimizing the risk of accidents and operative complications. material and methods this study was approved by the research ethics committee of the são leopoldo mandic dental school, campinas, sp, brazil (process number 811.741) in agreement with resolution no. 466/12 by the brazilian national health council. the sample was established retrospectively within a three-year interval, using the department of radiology image database at são leopoldo mandic dental school, campinas, sp, brazil. cbct images from 500 patients who had undergone cbct imaging for diagnostic or treatment planning purposes were examined, from which 300 images were selected according to the inclusion and exclusion criteria described below. the sample comprised cbct scans from both male and female patients aged between 13 to 87 years. image selection was performed at random, i.e., regardless of ethnicity, gender, age or presence/absence of teeth. only images with satisfactory tomographic quality were considered. images were excluded if the patient had a history of trauma and bone lesions in the mandible as well as orthognathic surgery or restorative bone procedures in the posterior mandible. images were selected using the classic i-cat® (imaging sciences international, hatfield, pa, usa) with standardized voxel at 0.25 mm, fov (field of view) of 13 cm and acquisition time of 40 s according to the manufacturer’s fig. 1 illustration of the methods used to evaluate the ct images. a – axial reconstruction tracing the jaw line to obtain the cross-sections; b – panoramic reconstruction; c – cross-sectional reconstruction. standards and useful radiation time of 6.6 s. the settings used for the acquisitions were those pre-established by default, i.e. 120 kv fixed and 5 to 7 ma variation, according to the used resolution. all images were processed and adjusted using xorancat® software (xoran technologies, ann arbor, mi. usa). ct image analysis was performed on the tomography workstation, correcting the anatomical planes using the multiplane reconstruction page (mpr). only bifid canals with a diameter larger than 1 mm were included in this study, aiming to achieve clear clinical relevance and standardization of the results. images were selected by chronological order in which they were taken on xorancat scanner software to highlight those with alterations in the mandibular canal (figure 1). all images were evaluated in a quiet environment with dim lighting by a single operator, a specialist in oral radiology with experience in cbct scans. the analysis was performed on cross sections of three planes (axial, sagittal and coronal), always following the path of the mandibular canal. all analyses and evaluations were performed using the xoran 3.0.34 program (xoran) in multiplanar reconstructions of three slicing planes: axial, coronal and sagittal. a 3x3 filter was used to sharpen the 0.25-mm thick images. identification of the mandibular canal was optimized by applying small changes to the slicing plane along the bone ridge, as well as corrections in brightness, contrast and image filter, since the mandibular canal is not linear and must be individualized for each side. whenever a bifid canal was detected, oblique slices were applied to obtain images in the buccolingual direction. the frequency of bifid mandibular canals was evaluated in the scans against gender, age and affected side and described in terms of percentage and subsequent descriptive analysis. braz j oral sci. 14(4):294-298 incidence and classification of bifid mandibular canals using cone beam computed tomography 296296296296296 an excel spreadsheet (microsoft, seattle, wa, usa) was used for data collection to identify the image number, presence of bifid mandibular canals, type of bifid canal, affected side, age and gender. mandibular canal bifurcations were classified into six distinct categories: classes a, b, c, d, e and f, according to the classification by naitoh et al.2 (table 1), which is based on the pathway of the bifid canal, starting from the mandibular foramen. view of a tomographic and clinical changes of the mandibular canal are illustrated in figures 2 and 3, respectively. the statistical analysis of cbct images from 300 participants was descriptive of gender and age in both absolute numbers (n) and percentages (%). the student t test for independent samples was applied to check for age differences between genders. the findings relating to bifid mandibular canals on cbct were described as absolute and relative frequencies, according to gender and location (right unilateral, left unilateral and bilateral). additionally, associations between bifid mandibular canals, gender and location were investigated using fisher’s exact and chi-square tests. the classification of bifid mandibular canals was also analyzed descriptively by frequencies. statistical calculations were performed on spss 20 (spss inc., chicago, il, usa) and bioestat 5.0 (fundação mamirauá, belém, pa, brazil) at a significance level of 5% (0.05). results descriptive data revealed that 112 (37.3%) images were from males whilst 188 (62.7%) were from females and the 300 patients were aged between 13 and 87 years with a mean age of 48.4 years (sd ±15.0 years). male individuals were aged between 13 and 77 years (mean 46.4 years, sd ±16.1 years), whereas the females were aged between 14 and 87 years (mean was 49.5 years, sd ±14.2 years). the student t test for independent samples revealed no difference in age between males and females in this sample (p=0.077). regarding the number of canals, 210 (70.0%) images revealed a single mandibular canal, whereas 90 images revealed the presence of bifid mandibular canals, indicating that the prevalence of this event in the study sample was 30.0%. among the females, 27.1% (51 out of 188 participants) had bifid mandibular canals against 34.8% in males (39 of 112). the fisher’s exact test revealed no significant difference between genders (table 2). table 3 shows the absolute and relative frequencies of mandibular canals according to location, indicating that 29 of 90 cases (32.2%) of bifid mandibular canals occurred exclusively on the right side, while 22 cases (24, 5%) were on the left side. thirty-nine participants (43.3%) had bilateral bifid mandibular canals. the chi-square test revealed that the proportion of patients with unilateral bifid canals located either on the right or on the left sides were significantly lower than the proportion of subjects with bilateral bifid mandibular canals (p=0.026). among the 39 cbct from male patients with bifid mandibular canals, 41.0% were located exclusively on the right side, 23.1% on the left and only 35.9% were bilateral. fig. 3 clinical photograph showing a retromolar accessory canal (class d) in close proximity with the unerupted third molar. fig. 2 ct image showing a retromolar accessory canal (class d) in close proximity with the unerupted third molar. braz j oral sci. 14(4):294-298 incidence and classification of bifid mandibular canals using cone beam computed tomography table 1 -table 1 -table 1 -table 1 -table 1 classification of bifid mandibular canals: class direction class a buccal direction – bifid mandibular canal towards the buccal surface of the mandible class b mesial direction bifid mandibular canal towards the mesial or anterior aspect of the mandible class c alveolar direction bifid mandibular canal towards the alveolar ridge class d retromolar direction bifid mandibular canal towards retromolar aspect class e lingual direction bifid mandibular canal towards the lingual surface of the mandible class f base of the mandible bifid mandibular canal towards the lower aspect of the mandible or the base of the mandible 297297297297297 location* bifid mandibular canals p-value** present absent unilateral left 29 (32.2%) 61 (67.8%) unilateral right 22 (24.5%) 68 (75.5%) 0.026 bilateral 39 (43.3%) 51 (56.7%) location male bifid mandibular canals p-value present absent unilateral left 16 (41.0%) 23 (59.0%) unilateral right 9 (23.1%) 30 (76.9%) 0.223 bilateral 14 (35.9%) 25 (64.1%) location female bifid mandibular canals p-value present absent unilateral left 13 (25.5%) 38 (74.5%) unilateral right 13 (25.5%) 38 (74.5%) 0.015 bilateral 25 (49.0%) 26 (51.0%) * no distinction between genders (male, female) ** p-values refer to the chi-square test table 3 -table 3 -table 3 -table 3 -table 3 absolute (n) and relative (%) frequencies of bifid mandibular canals in cbct images according to location gender bifid mandibular canals total p-value* present absent male 39 (34.8%) 73 (65.2%) 112 (37.3%) female 51 (27.1%) 137 (72.9%) 188 (62.7%) 0.193 total 90 (30.0%) 210 (70.0%) 300 (100.0%) * the p-value refers to the fisher’s exact test. table 2 table 2 table 2 table 2 table 2 absolute (n) and relative (%) frequencies of bifid mandibular canals in cbct images according to gender the chi-square test revealed no difference between the proportions of bifid mandibular canals in different locations (left, right and bilateral, p=0.223) (table 3). in the 90 patients with bifid mandibular canals, 39 males and 51 females, there were 129 accessory canals. as shown in figure 4, 86 of them (66.7%) were mesially oriented (class b), against 25 (19.4%) in the retromolar direction (class d). the e (lingual) and f (base of the mandible) categories were observed in 6 (4.6%) and 12 (9.3%) cases, respectively. no cases were found of bifid mandibular canals classified as class a (buccal direction) or c (alveolar direction), as illustrated in figure 4. fig. 4 pie chart of the relative frequency (%) of bifid mandibular canals in cone beam computed tomography, according to pathway classification. class b: mesial or anterior direction; class d: retromolar direction; class e: lingual direction; class f: base of the mandible direction. discussion the mandibular canal is most often found as a single one, but canal duplication may occur, and they are also known as bifid canals, which are among the most reported anatomical variations of the mandibular canal in the literature. they can be grouped into different classifications, as proposed by various authors8-9. chávez-lomeli et al.10 questioned the definition of the term “bifid” assigned to the mandibular canal with variation in number, claiming there is no such division of the mandibular canal into two or more canals, but the persistence or non-fusion of embryologically defined branches. neves et al.11 reported that the mandibular canal has been extensively investigated with regards to its location and pathway, as well as the possible variations in its normal anatomy, which varies considerably, displaying accessory canals with different configurations. naitoh et al.2 reported that these accessory canals may occasionally be observed in panoramic radiographs and may contain a neurovascular bundle. according to fukami et al.12, the vast majority of dentists have no knowledge of the anatomical variations of the mandibular canal and these variations may have a number of clinical implications if not identified prior to invasive interventions. according to neves et al.11 it is up to the dentist to recognize the possible anatomical variations of the mandibular canal in order to reduce the risk of failure during surgical or anesthetic approaches. for orhan et al.13 knowledge of the anatomy of this canal is essential for successful dental interventions and regional mandibular anesthesia, as well as dental implant placement and jaw surgery. for kuribayashi et al.14 anatomical variations of the mandibular canal can be identified on panoramic radiographs. however, when more precise information on the path of this canal is required, the most appropriate imaging techniques are the ct scans. naitoh et al. 2 compared panoramic radiographs with cbct and were able to visualize bifid mandibular canals and accessory foramens in 48.6% of cases where they were not noted on the panoramic radiographs. in the present study, bifid mandibular canals were observed in 30.0% of cases. previous studies with panoramic radiographs reported incidences of less than 1%6,9. studies with cbct images have shown a much higher incidence, with prevalence ranging from 15.6% to 65% 2,13-15, thus reiterating that conventional radiographs are not suitable to detect anatomical variations of the mandibular canal. the differences in incidence may be related to ethnic, geographical as well as methodological differences. according to ohran et al.13 there are several methods to braz j oral sci. 14(4):294-298 incidence and classification of bifid mandibular canals using cone beam computed tomography classify anatomical alterations of the mandibular canal, which take into account the characteristics of bifid canals, associations with additional foramens, width and length. naitoh et al.2 suggested the classification of bifid mandibular canals into four types: buccolingual (type i), mesial direction (type ii), alveolar ridge direction (type iii) and retromolar direction (type iv). in our study, a more comprehensive classification was used, taking into account directions not included in previous classifications, class a (buccal direction), class b (mesial direction), class c (alveolar direction), class d (retromolar direction), class e (lingual direction) and class f (mandible base direction). in this study, we found a 19.4% prevalence of retromolar canals and 7.33% of additional foramens. sawyer & kiely apud16 found a prevalence of retromolar foramens in the order of 7.7% of cases with significant occurrence of accessory mandibular foramens. bilecenoglu & tuncer16 reported a prevalence of 25% for the retromolar foramen and demonstrated histologically that these canals contained myelin fibers, an artery and numerous venules, which provided innervation to part of the third molar and also the mucosa of the retromolar area. regarding the affected side, the right side was most affected in the present study, 32.2% against 24.5% on the left side. these findings are corroborated by ohran et al.13 and carvalho17, who also reported a higher prevalence on the right side of about 57.2 to 62.5%. some auhors6,9,13,17 reported a higher prevalence of bifid mandibular canals among women. no significant difference in the prevalence of bifid mandibular canals was however observed between genders in the used dataset. in the present study, the mean age of the patients affected by changes in the mandibular canal was 48.4 years. this was consistent with the study by running et al.18 who reported a mean age of 48.2 years. other studies also reported the use of cbct to investigate the prevalence of bifid mandibular canals13-14,17. in the present study, as well as in the cited literature, cbct is regarded as appropriate for assessing anatomical variations of the mandibular canal, in view of the difficulties when using conventional radiographs. according to the results obtained in this study, a prevalence of 30% of bifid mandibular canals was reported, with the most prevalent types being class b (mesial direction) and bilateral. references 1. kawai t, asaumi r, kumazawa y, sato i, yosue t. observation of the temporal crest canal in the mandibular ramus by cone beam computed tomography and macroscopic study. int j cars. 2014 9: 295-299. 2. naitoh m, hiraiwa y, aimiya h, gotoh k, ariji e. observation of bifid mandibular canal using cone beam computed tomography. int j oral maxillofac implants. 2009, 24: 155-9. 3. neves sn, nascimento cc, oliveira ml, almeida sm, bóscolo fn. comparative analysis of mandibular anatomical variations between panoramic radiography and cone beam computed tomography. oral maxillofac surg. 2014; 18: 419-24. 4. niek l, gerlach md, gert j, thomas jj, frits a. reproducibility of 3 different tracing methods based on cone beam computed tomography in determining the anatomical position of the mandibular canal. j oral maxillofac surg. 2010; 68: 811. 5. oliveira-santos c, souza ph, de azambuja berti-couto s, stinkens l, moyaert k, van assche n, et al. characterization of additional mental foramina through cone beam computed tomography. j oral rehabil. 2011; 38: 595-600. 6. imada ts, fernandes lm, centurion bs, de oliveira-santos c, honório hm, rubira-bullen ir. accessory mental foramina: prevalence, position and diameter assessed by cone-beam computed tomography and digital panoramic radiographs. clin oral implants res. 2014; 25: e94-e99. 7. mizbah k, gerlach n, maal tj, bergé sj, meijergj. bifid and trifid mandibular canal. a coincidental finding. ned tijdschr tandheelkd. 2010; 117: 616-8. 8. langlais rp, broadus r, glass bj. bifid mandibular canals in panoramic radiographs. j am dent assoc. 1985; 110: 923-6. 9. nortjé c j, farman a g, joubert j j v. the radiographic appearance of the inferior dental canal: an additional variation. br j oral surg. 1977; 15: 171-2. 10. chávez-lomeli me, mansilla-lory j, pompa ja, kjaer i. the human mandibular canal arises from three separate canals innervating different tooth groups. j dent res. 1996; 75: 1540-4. 11. neves fs, almeida sm, bóscolo fn, haiter-neto f, alves mc, rebello ic, et al. risk assessment of inferior alveolar neurovascular bundle by multidetector computed tomography in extractions of third molars. surg radiol anat. 2012; 34: 619-24. 12. fukami k, shiozaki k, mishima a, kuribayashi a, hamada y, kobayashi k. bifid mandibular canal: confirmation of limited cone beam ct findings by gross anatomical and histological investigations. dentomaxillofac radiol. 2012; 41: 460-5. 13. orhan k, aksoy s, bicenoglu b, sakul bu, paksoy cs. evaluation of bifid mandibular canals with cone beam computed tomography in a turkish adult population: a retrospective study. surg radiol anat. 2011; 33: 501-7. 14. kuribayashi a, watanabe h, imaizumi a, tantanapornkul w, katakami k, kurabayashi t. bifid mandibular canals: cone beam computed tomography evaluation. dentomaxillofac radiol. 2010; 39: 235-9. 15. oliveira-santos c, souza p h c, berti-couto s a, stinkens l, moyaert k, rubira-bullen irf, et al. assessment of variations of the mandibular canal through cone beam computed tomography. clin oral invest. 2012; 16: 387-93. doi: 10.1007/s00784-011-0544-9. 16. bilecenoglu b, tuncer n. clinical and anatomical study of retromolar foramen and canal. j oral maxillofac surg. 2006; 64: 1493-7. 17. mizbah k, gerlak n, maal tj, bergé sj, meijer gj. the clinical relevance of bifid and trifid mandibulars canals. oral maxillofac surg. 2012; 16: 147-51. 18. correr gm, iwanko d, leonardi dp, ulbrich lm, araújo mr, deliberador tm. classification of bifid mandibular canals using cone beam computed tomography. braz oral res. 2013; 27: 510-6. 298298298298298 braz j oral sci. 14(4):294-298 incidence and classification of bifid mandibular canals using cone beam computed tomography 1http://dx.doi.org/10.20396/bjos.v19i0.8656950 volume 19 2020 e206950 original article 1 growth and development department, ajman university, united arab emirates. corresponding author: dr. afraa salah hussain ajman university department of growth and development united arab emirates/ajman pox: 364 /uae phone: 009716-747-2222 email: a.salah@ajman.ac.ae received: october 04, 2019 accepted: may 02, 2020 knowledge of tooth avulsion first aid management among parents residing in uae afraa hussain1,*, raghad hashim1 , aisha khamees1 aim: this study aimed to evaluate the knowledge of parents visiting a specialized dental center in ajman on permanent tooth avulsion and the required first aid procedure to achieve better prognoses. methods: a cross-sectional study was conducted in ajman emirate, uae. three hundred eighty-eight parents from three nationalities (emirati, egyptian and indian) residing in uae answered a constructed questionnaire that included demographic information related to the participants and questions related to permanent tooth avulsion and the required first steps for its management. results: study findings reflect deficiencies in the following areas. of the 388 parents, (236, 60.8%) had poor knowledge about permanent tooth avulsion. parents who had previous knowledge obtained information from unsupported sources, such as friends (145, 37.4%). out of 388 parents, (324, and 83.5%) would not replant the tooth. finally, the majority of the participants did not know the proper media to store the avulsed permanent tooth with one-third of parents choosing cotton or water (142, 36.6%). conclusion: this study showed a lack of knowledge regarding permanent tooth avulsion among parents from the major nationalities residing in uae. however, the respondents represent major differences regarding related to knowledge, replant, and storage of avulsed permanent teeth. keywords: ethnic groups. knowledge. parents. tooth. tooth avulsion. https://orcid.org/0000-0002-5895-6131 2 hussain et al. introduction dental trauma is one of the common types of accidents that children may experience during their primary and mixed dentitions stages1. dental trauma may range from crown fracture to complete removal of the tooth out of its socket2. permanent tooth avulsion is the complete removal of a primary or permanent anterior or posterior tooth out of its socket because of trauma or accident2,3. the major scenes of dental trauma include school and home; the most common reasons for permanent tooth dental trauma are falling, accidents, fights, and sports involving physical activities3. implantation of an avulsed tooth is positively related to the immediate and accurate management of the avulsed permanent tooth4. epidemiological studies showed that parents and school nurses are generally responsible for the primary management of an avulsed permanent tooth; however, their knowledge that the avulsed tooth should be maintained, cleaned and stored in proper media was inappropriate and insufficient4,5. an avulsed permanent tooth requires certain first aid steps to keep the tooth in good condition until a specialist in a dental center replants the tooth. these steps include tooth cleaning with running water without surface scratching and maintaining the tooth in proper storage media, such as saline, milk or saliva to prevent dehydration and surface cell death6. tap water was unacceptable storage media for avulsed tooth since it will cause pdl cell damage due to the low osmolarity of tap water7. the necessary steps to save the avulsed permanent tooth are commonly known among dental team members; however, epidemiological studies showed that nonprofessionals (parents, caregivers, school teachers) are not aware of information regarding the immediate steps needed to save an avulsed permanent tooth4,5. immediate management of an avulsed permanent tooth requires important steps to keep the tooth in a proper condition until replanted into the socket by dental professionals. many studies represented the poor or absence of parents’ knowledge about the option of keeping the tooth and bringing it with the child to the dental center for replantation. some studies presented different failure percentages in saving the avulsed permanent tooth due to the caregiver’s failure to take action to save the tooth8,9. caregivers did not know that the permanent avulsed tooth should not be out of the socket for more than 30 minutes for a good prognosis5,10. other studies found that caregivers were unaware of the transport media for avulsed permanent tooth storage until the dental center was reached9. in addition to being unaware of information regarding cleaning and holding, information on how to properly treat the avulsed permanent tooth was also lacking3,6. the united arab emirates has a wide diversity. however, the three major nationalities residing in uae are indian (37%), emirati (20%), and egyptian (12%)8. the purpose of this study is to evaluate parents’ knowledge from three nationalities residing in the uae regarding emergency management of permanent avulsed teeth. 3 hussain et al. materials and methods study design and population this cross-sectional study was conducted for a period of 4 months (august 2018 to november 2018). the participants were recruited from parents who accompanied their children attending ajman university dental specialized center for dental treatment. the institutional ethical committee approved the study (ref. gd 2018/6 11). g*power version 3.1.9.2 software was used to calculate the sample size. the effect size was calculated based on 80% power and a significance level of α = 0.05in which 30% of parents could not demonstrate any knowledge of the emergency management of dental avulsion. the minimum sample size was 350: however, an additional 20% was added to compensate for the expected drop out of the study. thus, a total sample size of 420 was selected. a total of 388 randomly selected parents participated in this study. parents from (emirati, egypt, and india) were recruited randomly and parents from other nationalities were not included in this study. questionnaire evaluation was pretested on 30 parents attending the medical center where certain words were changed for more clarification of the questions. participants from this pilot study were excluded from the main study. study questionnaire the study questionnaire was constructed based on a literature review of previously used questionnaires related to the topic of permanent teeth avulsion. the questionnaire was designed to include two parts. the first part represents demographic information of the parents, including gender, age, level of education, and nationality. the second part includes questions related to parents’ assessment of their knowledge and attitude as well as emergency management of the avulsed permanent tooth. the researcher provided the questionnaire in english and arabic (the local language in uae) for arab and non-arab parents. a pilot study was conducted as a preliminary study to identify any grammar or technical problems. questions the first part of the questionnaire represented parents’ demographic information in the following order: sex as male and female; age categorized into five categories (20-30), (30<40), (40<50), (50<60), and (60<); and parents’ nationality categorized into three scales: (emirates), (egyptian), (indian). the second part of the questionnaire includes the following questions: do you have any information regarding dental trauma do you have any information about tooth avulsion? what are the sources of your information? what action did you take when your child experienced trauma that resulted in permanent tooth avulsion? what action would you take if your child had an avulsed primary tooth? what action would you take if your child had an avulsed permanent tooth? what would you do with the avulsed permanent tooth? what action would you take with a dirty avulsed tooth? what would you do to clean an avulsed tooth? did you replant the avulsed tooth? how would you handle the avulsed tooth? what storage media would you use for an avulsed permanent tooth? 4 hussain et al. statistical analysis data analysis performed using statistical package for social science software version 22.0. recorded data tabulated and expressed by using a descriptive analysis test to get the required frequencies and percentages of participant’s respondents. results demographic information study participants consisted of 272 (70.1%) women and 116 (29.9%) men. the majority of the participants were in the (30-40) age range (44.3%). out of 388 participants, 215 (55.4%) had a university degree. in total, 182 (46.9%) of the participants were of egyptian nationality followed by indian nationality 110 (28.4) and emirati nationality 96(24.7). details on the demographic information presented in table 1. dental knowledge the percentage of the parents who had previous information about tooth avulsion was 152 (39.2%). however, the majority of parents (236, 60.8%) felt they had inadequate information about traumatic dental injuries.100 (26%) egyptian parents, 70 (18%) emirati parents, 66(17%) indian parents (figure 1). table 1. sociodemographic characteristics of the parents (n= 388). characterestics variables frequency percentages n % sex male 116 29.9 female 272 70.1 age 20-30 105 27.1 31-40 172 44.3 41-50 85 21.9 51-60 26 6.7 level of education non educated 2 0.5 primary 20 5.2 mid-school 28 7.2 high –school 99 25.5 college 215 55.4 master 23 5.9 phd 1 0.3 nationalities egyptian 182 28.4 indian 110 46.9 emirates 96 24.7 5 hussain et al. knowledge sources regarding the source of the information (if they had any previous information), more than one-third of the parents reported that they received information from friends (145, 37.4%). in total, 90(23.2%) of indian parents received information from a physician. 80 (20.6%) of emirati parents obtained information from a dentist. finally, 73(18.8%) of egyptian parents received information from the internet. (figure 2) figure 1. dental knowledge among parents residing in uae 39% 26% 18% 17% dental knowledge parents with dental knowledge emirati parents egyption parents indian parents figure 2. sources of dental knowledge 37.40% 23.20% 20.60% 18.80% sources of dental knowledge friends indian parents (physician) emarati parents (dentist) egyption parents (social media) 6 hussain et al. tooth replantation out of 388, only 64 (16.5%) of the respondents reported that they would replant an avulsed permanent tooth in its socket. however, the majority 83.5% (n=324) of the respondents said they would not replant it. 170 (41.3%) egyptian parents, 80 (19.4%) emirati parents, 94(22.8%) indian parents (figure 3). types of media for avulsed tooth storage regarding the media for storing an avulsed permanent tooth until reaching the dentist, indian parents 101(26%) chose no physiological media (paper tissue). egyptian parents 142 (36.7%) chose cotton or water. emirati parents chose milk 85(21.9%). 60 (15.4%) of the three nationalities chose more than two options (figure 4). 41.30% 22.80% 19.40% tooth replant reluctance egyption parents indian parents emarati parents figure 3. tooth replant reluctance 36.70% 26% 21.90% 15.40% types of prefered storage media cotton or water (egyption parents) paper tissue (indian parents) milk (emarati parents) more than two options figure 4. types of storage media for avulsed tooth 7 hussain et al. discussion children play activities that are highly susceptible to dental trauma7. permanent tooth avulsion is the only critical dental condition where unprofessional individuals (parents, caregivers, and schoolteachers) who were present at the accident area performed immediate action9. in this study, the majority of the respondents (60.8) from the three nationalities revealed that they lack adequate information regarding permanent tooth avulsion conditions and proper management. however, egyptian parents showed higher percentages. this finding was similar to other studies in different countries, such as kuwait, jordan, brazil, tehran, and hong kong1-5. this unfortunate finding indicates the lack of dental health knowledge among people, which should be managed by lectures and brochures through different channels, such as schools, universities, media, and meetings6. this study showed that sources of required management of permanent tooth avulsion received from different channels; however, this information could be incorrect or missing important issues necessary for tooth prognosis. most indian and emirati parents of the study respondents received their knowledge on permanent tooth avulsion from physicians and dentists (23.2% and 20.6%, respectively). however, they realized that the information was improper or adequate to help provide action at the accident scene. this finding similar to the finding of another study in india that suggested that physicians and dentists should provide an adequate and serious explanation of such knowledge to people through preventive measures or workshops8. one-third of parents get their knowledge from friends (37.4%), which represents a social source for this issue. a study in egypt showed a similar finding that necessitates the presence of leaflets and posters to increase individual awareness through community channels11. the final source of information in this study was the internet, which was cited by egyptian parents (18.8%) higher than other respondents. this finding was similar to previous studies where the internet represents a powerful source for different issues; however, a high percentage of incorrect knowledge could be found on the internet especially for nonprofessionals1,4. high expenses of dental treatment and lack of dental insurance are the major causes of the absence of dental visits of the residents in uae which affect their meeting with the dentists to had the correct information from the correct source. the majority of our respondents in this study would not replant a primary or permanent avulsed tooth (83.5%). the higher percentages were among egyptian parents (41.3%). this major finding was consistent with the results of previous studies in india12,13, turkey9, egypt, and kuwait1,10,11. this finding could be attributed to the fact that the majority of people who do not have any information about the required first aid to replant an avulsed permanent tooth could not perform any procedure at the time of the accident. such a finding acts as an alarm to the responsible specialty to provide necessary information through numerous channels. regarding storage media selection for an avulsed permanent tooth, the respondents reported different opinions. indian parents chose paper tissue as a temporary stor8 hussain et al. age media for the avulsed tooth (26.0%). this finding was similar to previous studies in india14,15. emirates respondents chose milk (21.9%). this finding is consistent with many previous articles; however, the responses exhibited different percentages11,16,17. egyptian respondents replied with tap water or cotton as storage media for the avulsed permanent tooth (36.7%). this finding is similar to egyptian parents’ responses regarding avulsed tooth storage media11. the general findings of this study revealed a lack of information regarding permanent tooth avulsion and the required necessary steps and precautions to be considered when the parents were responsible for the required action. different responses could be related to different cultures, which affect people’s attitudes towards health conditions and their management. important steps required to increase community awareness regarding immediate management of tooth avulsion through many channels like social media, schools, medical centers, and hospitals18. in conclusion, this study showed that egyptian parents need more dental awareness regarding dental conditions. indian and egyptian parents did not receive dental knowledge from the dentist due to the higher cost of dental treatment. egyptian nationality represents the higher percentage of parents’ refusal to replant avulsed teeth. emirati parents had the best knowledge in storing the avulsed tooth in the best media. highlights of the study dental trauma is an important clinical condition in dentistry. children may experience dental trauma during their first years of life and mixed dentition stages. most dental trauma conditions receive treatment in a dental clinic. however, permanent tooth avulsion requires first aid procedures at the trauma location before final treatment in a dental clinic. the background knowledge of nonprofessional individuals on the required first aid steps to store an avulsed permanent tooth is critical. acknowledgement this study accomplished by the help of the dental general practitioners dr. fatima abbas, dr. hanan albannaai and dr. latifa alsaeed in questionnaire distribution and collection. statement of ethics:this study was approved by the ethical committee of ajman university. disclosure statement: the authors have no conflicts of interest to declare. funding sources: this research is self-funded. references 1. alyahya l, alkandari sa, alajmi s, alyahya a. knowledge and sociodemographic determinants of emergency management of dental avulsion among parents in kuwait: a cross-sectional study. med princ pract. 2018;27(1):55-60. doi: 10.1159/000486095. 2. al-jundi sh. knowledge of jordanian mothers with regards to emergency management of dental trauma. dent traumatol. 2006 dec;22(6):291-5. doi: 10.1111/j.1600-9657.2005.00371.x. 9 hussain et al. 3. ahlawat b, sharma a, kaur a, chaudhary n, sharma s, kumar a. tooth avulsion: its perspective among athletically active children of chandigarh, india. j oral res rev. 2016 jan;8(1):12-5. doi: 10.4103/2249-4987.182494. 4. hashim r. investigation of mothers’ knowledge of dental trauma management in united arab emirates. eur arch paediatr dent. 2012 apr;13(2):83-6. doi: 10.1007/bf03262849. 5. chan aw, wong tk, cheung gs. lay knowledge of physical education teachers about the emergency management of dental trauma in hong kong. dent traumatol. 2001 apr;17(2):77-85. doi: 10.1034/j.1600-9657.2001.017002077.x. 6. al-asfour a, andersson l. the effect of a leaflet given to parents for first aid measures after tooth avulsion. dent traumatol. 2008 oct;24(5):515-21. doi: 10.1111/j.1600-9657.2008.00651.x. 7. bazmi ba, singh ak, kar s, mubtasum h. storage media for avulsed tooth – a review. indian j multidiscip dent. 2013 may;3(3):741-9. 8. population of uae.global review of the implementation of the fundamental principles of official statistics. 2018. 9. qazi sr, nasir ks. first-aid knowledge about tooth avulsion among dentists, doctors and lay people. 2009 jun;25(3):295-9. doi: 10.1111/j.1600-9657.2009.00782.x. 10. shashikiran nd, reddy vv, nagaveni nb. knowledge and attitude of 2,000 parents (urban and rural 1,000 each) with regard to avulsed permanent incisors and their emergency management, in and around davangere. j indian soc pedod prev dent. 2006 sep;24(3):116-21. doi: 10.4103/0970-4388.27891. 11. loo tj, gurunathan d, somasundaram s. knowledge and attitude of parents with regard to avulsed permanent tooth of their children and their emergency management--chennai. j indian soc pedod prev dent. 2014 apr-jun;32(2):97-107. doi: 10.4103/0970-4388.130781. 12. namdev r, jindal a, bhargava s, bakshi l, verma r, beniwal d. awareness of emergency management of dental trauma. contemp clin dent. 2014 oct;5(4):507-13. doi: 10.4103/0976237x.142820. 13. ozer s, yilmaz ei, bayrak s, tunc es. parental knowledge and attitudes regarding the emergency treatment of avulsed permanent teeth. eur j dent. 2012 oct;6(4):370-5. 14. nikam ap, kathariya md, chopra k, gupta a, kathariya r. knowledge and attitude of parents/ caretakers toward management of avulsed tooth in maharashtrian population: a questionnaire method. j int oral health. 2014 sep-oct;6(5):1-4. 15. marino tg, west la, liewehr fr, mailhot jm, buxton tb, runner rr, et al. determination of periodontal ligament cell viability in long shelf-life milk. j endod. 2000 dec;26(12):699-702. doi: 10.1097/00004770-200012000-00005. 16. holan g, shmueli y. knowledge of physicians in hospital emergency rooms in israel on their role in cases of avulsion of permanent incisors. int j paediatr dent. 2003 jan;13(1):13-9. doi: 10.1046/j.1365-263x.2003.00414.x. 17. young c, wong ky, cheung lk. the effectiveness of educational poster on knowledge of emergency management of dental trauma part 2: cluster randomized controlled trial for secondary school students. plos one. 2014 aug 5;9(8):e101972. doi: 10.1371/journal.pone.0101972. 18. american academy of pediatric dentistry. guidelines on management of acute dental trauma. pediatric dentistry reference manual 2012-13;34:230-8. 10 hussain et al. (appendix-1) patients attending aust (from august 2018 – november2018) gender: male female age: 1. 20 30 years 2. 31 40 years 3. 4150 years 4. 51 60 years 5. > 60 years level of education: 1. not educated 2. primary 3. middle school 4. high school 5. college 6. master 7. phd nationality: 1. emirates 2. egypt 3. india questionnaire: *avulsed tooth = fallen by an accident or trauma choose the appropriate answer: 1. have any of your children had dental trauma? • yes, had trauma other than tooth avulsion. • yes, had avulsed primary tooth • yes, had avulsed permanent tooth • no, had not 11 hussain et al. 2. do you have any previous information about tooth avulsion? • yes • no 3. if you had such information, what is the source of that information: • dentist • physician • media • friends 4. if your child has an avulsed primary tooth, what will you do? • searching to find the tooth • not searching for it 5. if you would find the primary tooth, what will you do? • replant the tooth • not replanting it 6. if your child have an avulsed permanent tooth, you will: • search to find the tooth. • not searching for the tooth. 7. if you would find the permanent tooth, what will you do? • replant the tooth • not replanting it if you would search for the tooth, please complete the following questions: 8. if you found the tooth covered with dirt, what will you do? • clean the tooth then save it • save the tooth only • replant the tooth • i don’t know 9. if you would clean the tooth, you will use: • tooth brush • tap water • antiseptics • i don’t know 12 hussain et al. 10. if you would replant the tooth, you will do that: • immediately • within half an hour • at any time 11. you will handle the avulsed tooth from: • crown • root • either 12. if you will transport the avulsed tooth to the dentist, you will use: • paper tissue • cotton • tap water • milk • i don’t know untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8651895 volume 17 2018 e18013 original article 1 department of periodontology and community dentistry, university of ibadan and university college hospital, ibadan, nigeria corresponding author: dr. folake b. lawal, department of periodontology and community dentistry, college of medicine, university of ibadan, pmb 5017 ibadan 200212, nigeria +2348023658988 folakemilawal@yahoo.com received: august 20, 2017 accepted: november 27, 2017 non-clinical factors and predictors of self-rating of oral health among young adolescents in a rural nigerian population folake b. lawal, bds, mds, fwacs, fmcds; mumini a. dauda, bds aim: the aim of the study was to assess the non-clinical predictors of self-rating of oral health among young adolescents in a nigerian rural population. methods: a cross-sectional survey was conducted among adolescents of 11-13 years old in igboora, nigeria. information on self-rating of oral health, self-assessed satisfaction with oral health condition and tooth appearance, pain history, consultation with the dentist and oral hygiene measures were obtained using structured questionnaires translated to the local language. data were analysed using spss version 23; chi square and logistic regression were used to establish associations between variables and predictors with p value < 0.05 statistically significant. results: a total of 400 respondents participated in the study. most 346 (86.5%) rated their oral health positively. those who expressed dissatisfaction with the appearance of their teeth, 17 (44.7%) dissatisfaction with their oral health condition, 25 (45.5%) had toothache in the preceding six months, 44 (19.7%) perceived a need for dental treatment, 43 (16.7%) or cleaned their teeth once daily or less frequently, 37 (20.9%), rated their oral health poorly (p < 0.001, p < 0.001, p = 0.001, p = 0.012, p < 0.001, respectively). the significant predictors of self-rating of oral health were self-assessed satisfaction with oral health condition, toothache in the preceding six months and frequency of tooth cleaning. conclusion: satisfaction with oral health condition, toothache in the preceding six months and frequency of tooth cleaning are factors that predict self-rating of oral health in young adolescents in the rural community studied. keywords: adolescent health. global self-rating. non-clinical factors. self-perception. predictors. mailto:folakemilawal@yahoo.com 2 lawal and dauda introduction self-rating of oral health (sroh) subjectively evaluates oral health and has been used in clinical, epidemiological and public health settings1-7. it is a useful tool for oral disease screening, assessment of oral health needs and disease surveillance2,4. it also complements clinical evaluation of oral health in planning, monitoring and evaluation of oral health intervention programmes2,4. sroh has been found valid in distinguishing between individuals with or without oral health problems; poor self-rating of oral health has been associated with oral diseases such as dental caries7-11. in addition, simplicity of sroh as a single item tool and its ability to evaluate the overall oral health of an individual amongst others makes it a valuable tool in underserved regions like rural communities. sroh is however influenced by and associated with non-clinical factors1,8,10,12,13. in spite of the advantages of the sroh especially in rural communities in developing countries where dentists are rarely found, very little is known regarding the utility of this tool among adolescents. this is pertinent in view of the need to get preventive oral health across to adolescents at an age when habits are formed or cemented. this becomes important as contributory effects of cultural norms, more prevalent in rural communities, to validity of self-rating of oral health has been documented in a previous study5. furthermore, identification of predictors of self-rating of oral health could also help in stratifying target groups for oral health intervention, especially in rural settings in developing countries where the disproportionately poorer allocation of resources to oral health is more obvious. the aim of the study was to assess the non-clinical predictors of self-rating of oral health of young adolescents in a rural nigerian population. materials and methods this was a descriptive cross-sectional survey conducted among adolescents aged 11 to 13 years in igboora, a rural agrarian town in south-western nigeria. following ethical approval from the state’s ethical review committee (ad/13/479/649), 400 consenting adolescents were recruited from schools selected through simple random sampling technique from the town between january and june 2015. three primary schools were randomly selected from the list of 23 primary schools obtained from ibarapa central local government schools’ board. all the pupils in the sixth grade of the selected schools aged 11 to 13 years were then approached in the three schools and those who gave consent, and whose parents did not give a negative consent were approached consecutively until 400 students were recruited. a sample size of at least 384 was arrived at based on a probabilistic prevalence of 50% in the absence of prevalence values from the literature14, an allowable error (d) of 5% and z statistic of 1.96 (confidence interval of 95%) all inputted into the formula to calculate sample size in cross-sectional studies15. structured interviewer administered questionnaires were used to obtain information from the students. the biodata of the respondents and basic information about the parents’ tribes, religious beliefs and occupational status were recorded. information 3 lawal and dauda was also obtained on self-rating of oral health, self-assessed satisfaction with oral health condition and tooth appearance, history of dental pain, perception of need for dental treatment, prior consultation with the dentists and oral hygiene measures. the self-rating7 of oral health was recorded using a likert scale with responses scored from 1 “very poor”, 2 “poor”, 3 “neither good nor poor”, 4 “good” to 5 “very good”. the responses were subsequently recoded on the computer as “poor” (very bad, bad and neither good nor bad) or “good” (good and very good). self-assessed satisfaction with appearance of the teeth and satisfaction with oral health condition were also graded using a likert scale with responses from 1 “dissatisfied”, 2 “dissatisfied”, 3 “neither dissatisfied nor satisfied”, 4 “satisfied” to 5 “very satisfied”. the responses were recoded in each case for the two variables as “dissatisfied” (very dissatisfied, dissatisfied and neither dissatisfied nor satisfied) or “satisfied” (satisfied and very satisfied). the history of dental pain was asked with a single question: “have you experienced toothache in the last six months that was serious enough for you to mention to your parents?” the response was either “yes” or “no”. an independent translator translated the questionnaire to the local language, with a back translation having confirmed retention of test questions in spite of linguistic differences by another independent translator. the questionnaire was pre-tested and transculturally adapted among 30 school going pupils in another town. only pupils who understood the local language and who consented were included in the study. those with special needs were excluded from the study. the questionnaire was self-administered under supervision of a trained dentist. data were analyzed using the statistical package for the social sciences version 23 (armonk, ny: ibm corp). univariate analysis was presented using proportions, percentages and means (with standard deviations) as appropriate. chi square statistics was used to test for associations between variables with non-clinical factors considered as independent variables and self-rating of oral health as the dependent variable with the reference category being poor rating. row percentages were presented for clarity. logistic regression was done by considering independent variables that were significant during bivariate analysis in order to identify predictors of self-rating of oral health among the respondents. wald test was used to test for the statistical significance of the predictors. a p value < 0.05 was considered to be statistically significant for this study. results four hundred adolescents with a mean age of 12.4 (sd = 0.7) years were recruited into the study of which 205 (51.3%) were females. the predominant occupations of the fathers were: commercial motorcycling (81, 20.3%), trading (63, 15.8%) and farming (60, 15.0%) and those of the mothers were: trading (261, 65.3%) and teaching (43, 10.8%). they were mostly of the yoruba tribe (379, 94.8%), the dominant tribal group in southwest nigeria. most of the respondents (346, 86.5%) rated their oral health positively, 362 (90.5%) were satisfied with the appearance of their teeth and 345 (86.3%) were satisfied with their oral health condition. a total of 223 (54.8%) respondents have had significant toothache in the preceding six months. only 81 (20.3%) had been to a dentist before. the majority (258, 64.5%) perceived a need for dental treatment and the main 4 lawal and dauda treatment thought to be required were scaling and polishing (74, 18.5%) and relief of tooth/gum ache (51, 12.8%). more than half of the respondents (223, 55.8%) cleaned their teeth twice each day, the rest did so once a day or on most days of the week. a higher proportion of the respondents who expressed dissatisfaction with the appearance of their teeth self-rated their oral health as poor compared to those who were satisfied with the appearance of their teeth and rated their oral health similarly (44.7% vs. 10.2%, p < 0.001). the proportion of those who rated their oral health poorly and were dissatisfied with their oral condition (45.5%) was higher than that of the respondents who rated their oral health poorly and were satisfied with their oral health condition (8.4%), p < 0.001. similar relationships were observed between reporting toothache in the preceding six months, perception of need for dental treatment and frequency of tooth cleaning on one hand and self-rating of oral health on the other hand (table 1). the respondents who were dissatisfied with their oral condition were nearly eight times more likely to self-rate their oral health status as poor (or = 7.69, 95% ci: 3.16, 18.75, p < 0.001). poor rating of oral health status was nearly three times higher in those who had reported toothache in the preceding six months than in those who had not (or = 2.54, 95% ci: 1.10, 5.82, p = 0.028). respondents who cleaned their teeth once daily or less frequently were three times more likely to rate their oral health negatively than those who cleaned their teeth at least twice daily (or = 3.20, 95% ci: 1.74, 5.91, p < 0.001). satisfaction with appearance of the teeth (p = 0.124) and perception of dental treatment need (p = 0.050) could not predict the self-rating of oral health status (table 2). table 1. relationship between non-clinical factors and self-rating of oral health status of the respondents non-clinical factors self-rating of oral health poor no (%) good no (%) total no (%) χ2 p value appearance of teeth dissatisfied 17 (44.7) 21 (55.3) 38 (100.0) 35.085 < 0.001* satisfied 37 (10.2) 325 (89.8) 362 (100.0) oral condition dissatisfied 25 (45.5) 30 (54.5) 55 (100.0) 55.760 < 0.001* satisfied 29 (8.4) 316 (91.6) 345 (100.0) toothache in the past 6 months yes 44 (19.7) 179 (80.3) 223 (100.0) 10.376 0.001* no 10 (5.6) 167 (94.4) 177 (100.0) perceived need for treatment yes 43 (16.7) 215 (83.3) 258 (100.0) 6.241 0.012* no 11 (7.7) 131 (92.3) 142 (100.0) cleaning of teeth once daily or less 37 (20.9) 140 (79.1) 177 (100.0) 14.904 < 0.001* twice daily 17 (7.6) 206 (92.4) 223 (100.0) total 54 (13.5) 346 (86.5) 400 (100.0) * statistically significant (p < 0.05) 5 lawal and dauda discussion the present study conducted amongst adolescents in a rural town in a developing country showed a very favourable rating of oral health by individuals. there was significant relationship between non-clinical factors and self-rating of oral health condition. these findings have impact on the utility of self-rating as a subjective measure of oral health status in underserved communities where access to dentists and clinical tools to diagnose oral conditions may be inadequate. the self-rating of oral health is a single item summary tool that has been validated for evaluation of oral health7. it is easy to administer and does not have the limitation imposed by inadequate oral health professionals. it is a subjective assessment of oral health by individuals and is comparable to quality of life measures like ohip-14, which has been evaluated and found appropriate in determining the unmet dental treatment needs of adolescents in similar settings16. this study found that the majority of the participants rated their oral health positively as very good and good, similar to previous studies5,17-19. contrasting findings to this, was however noted by jiang et al. 8, and yamane-taukechi et al.20, where 39% and 36.8% of the study participants rated their oral health as good or very good respectively. the differences in self-rating of oral health as reported by the studies may be attributed to varying perception of oral health that may occur among individuals. bivariate analysis showed that positive rating of oral health was associated with self-perceived satisfaction with appearance of teeth and oral condition, this is in line with previous findings that subjective assessment of oral health correlate strongly with each other as reported by authors of previous studies21-24. however, on multivariate analysis, only the relationship of satisfaction with oral condition was statistically significant. this finding may due to the fact that satisfaction with teeth appearance may vary extensively among individuals more so that it ultimately does not result in dysfunction of the dentition. moreover, perception of tooth appearance is highly subjective and influenced by what an individual considers as ideal25. in addition, the strong association of satisfaction rating of oral health condition and sroh may be contributory to their relevance as validation tools for other instrument of subjective assessment of oral health26,27. table 2. logistic regression analysis of relationship between non-clinical factors and self-rating of oral health status of the respondents variable categories of variable or 95% ci p value satisfaction with tooth appearance dissatisfied 2.26 0.80 – 6.41 0.124 satisfied satisfaction with oral condition dissatisfied 7.69 3.16 – 18.75 <0.001* satisfied toothache in preceding 6 months yes 2.54 1.10 – 5.82 0.028* no perceived treatment need yes 2.40 1.00 – 5.75 0.050 no tooth cleaning once daily or less 3.20 1.74 – 5.91 <0.001* twice daily *statistically significant; reference category on logistic regression = poor self-rating of oral health 6 lawal and dauda many of the respondents perceived a need for dental treatment and scaling and polishing (oral prophylaxis) was the main treatment mentioned. this is a probable reflection of self-awareness of the significance of poor oral hygiene among this study group, which is commendable. perceived need for treatment by the adolescents was significantly associated with rating of their oral health as poor in this study on bivariate analysis, similar to reports by other authors19,28,29. the presence of oral disease and conditions, which has been associated with poor rating of oral health, hence, perceiving a need for treatment, may be an explanation for this. perceived need for treatment was however, not a significant factor on multivariate analysis, thus not a determinant of sroh in this study. participants with history of toothache rated their oral health poorly more often than those without toothache in the last six months. this finding has been corroborated by others29 who reported the impact pain has on subjective assessment of oral health. pain is a significant factor that impacts negatively on the quality of life of individuals30. in addition, adolescents’ perception of oral health has been defined as presence or absence of disease or pain31. more than half of the students cleaned their teeth twice or more often daily, a reflection of good oral health practices among the study participants, which may be partly attributable to the school outreach programs previously conducted in the community. multivariate analysis, furthermore, confirmed that twice or more daily tooth cleaning was significantly associated with sroh as good or very good in this study. significant relationships between good oral health behaviour and self-perceived oral health have been documented in previous studies8,9. in addition, tooth cleaning described as one of the action-based definition of oral health concept in a qualitative study among adolescents in a rural county in sweden31 is a strong supportive evidence for this finding. the importance of good oral hygiene behaviour in achieving good oral health is therefore to be promoted among the studied group in view of it being a determinant of positive rating of oral health among adolescents in a rural setting. a major limitation of this study was the inability to establish a cause and effect relationship between the self-rating of oral health and oral health status of the adolescents, which is inherent in the study design. in conclusion, satisfaction with oral health condition, twice daily tooth cleaning and pain are factors that predict self-rating of oral health in young adolescents in rural communities. acknowledgement the authors are grateful to the students who participated in the study as well as to the teachers and principals who supported the study and gave the necessary approvals. references 1. atchison k, gift h. perceived oral health in a diverse 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2002;60(3):167-73. 1http://dx.doi.org/10.20396/bjos.v18i0.8656589 volume 18 2019 e191504 original article 1 science and technology institute, são paulo state university (unesp), são jose dos campos, sp, brazil. 2 department of dentistry, prosthodontics unit, federal university of rio grande do norte (ufrn), natal, rio grande do norte, brazil 3 department of restorative dentistry, federal university of juiz de fora, juiz de fora, mg, brazil. corresponding author: jean soares miranda https://orcid.org/0000-0001-5379-0155 address: av engenheiro francisco josé longo, 777, jardim são dimas, são josé dos campos, são paulo, brazil. zip code: 12245-000. phone: +55 32 988239151 e-mail: jeansoares@msn.com received: january 20, 2019 accepted: june 02, 2019 different surface treatment protocols of a y-tzp ceramic with a superficial glaze layer jean soares miranda1,*, ronaldo luís almeida de carvalho1, aline serrado de pinho barcellos1, rodrigo othávio assunção e souza2, estevão tomomitsu kimpara1, fabíola pessôa pereira leite3 aim: evaluate the influence of etching time with hydrofluoric acid on the bond strength of a yttrium-stabilized polycrystalline tetragonal zirconia (y-tzp) ceramic with a superficial glaze layer and a resin cement. methods: y-tzp blocks were cut to obtain 40 samples. they were distributed into four groups (n = 10): control treated by sandblasting with silica-coated alumina (rs) and three glazed experimental groups with different etching times: gs20s, gs60s and gs100s. cementation was done with a universal adhesive and a resin cement. two cement cylinders were made in each block. after thermocycling, the shear bond test was performed. two extra samples of each group were made to obtain profilometry, scanning electron microscopy, mapping and backscattered electron detector images. energy dispersive spectrometry and goniometry were also performed. results: kruskal-wallis and dunn tests demonstrated bond strength differences only between the rs (22.10mpa) and the gs groups (gs20s: 8,10mpa; gs60s: 10.49mpa; gs100s: 7.53mpa) (p = 0.001), but there was no difference among the experimental groups (p > 0.05). the contact angles were 55.33º (rs); 70.78° (gs100s); 48.20º (gs60s) and 28.73º (gs20s). anova and tukey test demonstrated similar wettability of rs to gs60s and gs100s (p > 0.05), but all the experimental groups were statistically different between them (p < 0.001). qualitative image analysis revealed an irregular glaze distribution after etching. the thickness of the remaining glaze layer measured by profilometry was 5±1μm (gs20s), 4±1μm (gs60s) and 3±1μm (gs100s). conclusion: the etching time of glazed zirconia did not influence the adhesive strength of the ceramic to the resin cement. keywords: zirconium. glass. dental cements. https://orcid.org/0000-0001-5379-0155 2 miranda et al. introduction yttrium-stabilized polycrystalline tetragonal zirconia (y-tzp) has high chemical stability, high flexural strength, radiopacity, biocompatibility and low thermal conductivity, what makes it a good material for oral rehabilitation1-6. however, the clinical success of dental ceramic prostheses also depends on a luting protocol1-7. however, this is not yet establish for zirconia8. in order to achieve a durable bond between zirconia and resin cement, surface treatments are required to this material, creating micro mechanical -retentions2-5,9-11. they are: sandblasting with aluminum oxide particles12, sandblasting with silica-coated alumina1,12,13; er:yag laser irradiation1 and plasma spraying14. the sandblasting with silica-coated alumina presents good bond results1,12,13. it can chemically modify the ceramic making it reactive with the resin, creates roughness and irregularities, what increases the surface area and the wettability, allowing the cement to flow3,5,6,9,10,15. nevertheless, there are concerns about this treatment. the impact of this abrasion may lead to long-term surface changes, to local silica network distortions that are not sustained, and/ or due to the emergence of a new zirconia phase, create stress and form lateral cracks14. thus, use of alternative methods to abrasion have been suggested, such as application of a thin glaze layer and/or the use of universal primers on the zirconia surface6,10,16. the surface treatment referred as vitrification involves applying a thin glass layer under the surface of the y-tzp ceramic. this aims to enrich the surface with a vitreous material and allow the hydrofluoric acid (hf) etch of this surface, changing the topography and providing area of mechanical retention3,5,7,17-20. in addition, this etching would increase the ceramic surface energy and its adhesive potential, a prerequisite for a stable and durable bonding of the resin cement to the substrate3,5,7,17-20. however, the ideal hf etching time on this surface is not yet defined. following a simplified strategy, some new universal adhesives have been developed to be used with various restorative materials. they allow the bond to zirconia without the use of primers because they have silane, which promotes adherence to silica-based surfaces, and mdp (10-methacryloyloxydecyl dihydrogen phosphate), which is designed to create a chemical bond to metal oxides such as zirconium1,3,10,21,22. it has been suggested that these monomers can interact with the y-tzp and the resin cement, enabling chemical adhesion through van der waals forces or hydrogen bonds21,22. studies are being published with the intention of achieving better adhesion to dental zirconia2,3,5,6,9,10,15,21-22. they involve a variety of surface treatment methods, adhesion promoters or cements, but an efficient and long-lasting protocol for zirconia luting has not yet been established2. y-tzp vitrification followed by hf etching is a method already reported in the literature11,19. however, in a temptive to establish a luting protocol, some problems should be better studied, such as the influence of the hf etching time on this new vitreous layer. thus, this research aimed to evaluate the influence of different etching times with 10% hydrofluoric acid on the bond strength between a vitrified y-tzp ceramic and a resin cement. these times were chosen from the conventional 60s etching to vitreous ceramic. the intention was also to verify if some higher or lower etching time would modify the bond strength. the null hypotheses were that the surface treatment type would not influence the bond strength and the etching time of the experimental groups would not alter this result. 3 miranda et al. materials and methods sample preparation: the materials used in this study, their trademarks, manufacturers, composition and lots are presented in table 1. y-tzp blocks (ips e.max® zircad ivoclar-vivadent, schaan, liechtenstein) were cut to a standard size of 15x15x2mm with a diamond cutting disc (extec high concentration, enfield ct, usa) in a precision cutting machine (isomet® 1000 precision saw, buehler, lake buff-il, usa) to obtain 48 samples. both sides of the samples were regularized with # 180, # 600 and # 1200 granule sandpaper (norton saintgobain, são paulo, brazil). prior to sintering, the samples were washed in an ultrasonic bath (cristófoli ultrasonic washer, campo mourão, paraná, brazil) in isopropyl alcohol for eight minutes. the sintering was carried out in a zyrcomat t oven (vita, zahnfabrick, germany) up to the temperature of 1530°c. after this process, the final samples dimensions were 12x12x1.5 mm. the ceramic blocks were then randomly distributed into four groups (n = 10) by radom alocater (mads haahr, dublin, irish). one was the control group, in which sandblasting with silica-coated alumina was performed by rocatec soft (3m espe, st. paul, minnesota, usa) (rs), and three experimental groups were vitrified with glaze spray vita akzent plus (vita zanhfabrik, bad sachingen, germany) on the adhesion surface and etched with 10% hf for 20s (gs20s), 60s (gs60s) or 100s (gs100s). the rs group had sandblasting done at a distance of 10mm between the zirconia surface and the tip of the apparatus (dento-preptm, rønvig a / s) with a 45º slope, at 2.8 bars of pressure for 15s. in the experimental groups, the glaze spray vita table 1. commercial brand, use, manufacturer, composition and lot of materials used in the research. brand material type manufacturer composition lot ips e.max® zircad y-tzp ceramic ivoclar-vivadent, schaan, liechtenstein zro2 + hfo2 (94.4 wt%), y2o3 (5.2 wt%), al2o3 (0.2–0.5 wt%) m24091 rocatec® soft silica-coated alumina 3m espe, st. paul, minnesota, usa 30µm silica-coated alumina 424975 vita akzent® plus glaze spray vita zanhfabrik, bad sachingen, germany 111-29-5 pentano-1,5-diol a0764 single bond universal universal adhesive 3m espe, sumaré, sp, brazil mdp, vitrebond copolymer, hema, silane, dimethacrylate resins, fillers, initiators and ethanol 1511900505 relyx ultimate resin cement 3m espe, sumaré, sp, brazil base: methacrylate monomers/ radiopaque, silanated fillers, initiators, stabilizers, rheological additives. catalyst: methacrylate monomers, radiopaque, alkaline fillers, initiators, stabilizers, pigments, rheological additives, fluorescence dye, dark cure activator for scotchbond universal adhesive. 1509800356 condac porcelana hydrofluoric acid fgm, pinheiros, sp, brazil 10% hf, water, thickener, surfactant and colorant 250215 4 miranda et al. akzent plus (vita zahnfabrik) was applied at a standard of 3cm distance from the ceramic surface, taking the time required for the ceramic surface to be completely covered. then, the samples were taken to a vita vacumat 6000 mp oven (vita, zahnfabrik, germany) for glaze firing process19. sample luting: the experimental group samples were etched with 10% hf (condac porcelana fgm, pinheiros, sp, brazil) at different time intervals according to the group, and washed by air-water spray for twice the hf etching time. the blocks were then cleaned again in sonic bath (cristófoli ultrasonic washer) for 5 min in distilled water to remove the acid precipitate. these blocks were fixed in a cylinder of acrylic resin and single bond universal adhesive (3m espe®, st. paul, minnesota, usa) was applied with a microbrush (vigodent, rio de janeiro, rj, brazil) for 60s, then left to act for 20s and light air was applied for 5s, without light curing. soon after, two silicon transparent cylindrical matrices (tygon tubing, tyg-030, saint-gobain performance plastic, miami lakes, florida, usa) of 3mm internal diameter by 3mm height were placed under the samples, totaling twenty adhesive interfaces per group to be tested. then, relyx ultimate dual resin cement (3m espe, st. paul, minnesota, usa) was manipulated following the manufacturer’s recommendations and immediately inserted into the matrices with the aid of a centrix syringe (polidental ind. e com. ltda, são paulo, sp, brazil). light curing was performed with the radii-cal led (sdi, pinheiros, sp, brazil) with an intensity of 1200mw/cm and an application time of 40s on each side20. after luting, all samples were stored in distilled water (olidef, ribeirão preto, são paulo, brazil) at 37ºc for 24 hours. next, the silicon matrices were removed with number 12 blades (becton dickinson, new jersey, usa), obtaining the final specimens for the research. shear bond strength test and failure mode analysis: all the specimens were subjected to aging by thermocycling for 6,000 cycles (nova ética, são paulo, sp, brazil) between two water baths of 5oc and 55oc, with a time of 30s each3,20. after this, they were subjected to shear bond strength testing using a universal testing machine (emic, dl 2000, são josé dos pinhais, paraná, brazil) at a crosshead speed of 1.0 mm/min. shear bond strength (in mpa) was calculated by dividing the load at fracture (in newtons), with the bonding interface area (28.26 mm2). for failure analysis, a stemi 2000-c stereomicroscope (karl zeiss) with 16x magnification was used coupled with a digital camera. all samples were analyzed for failure type classification: adhesive (at the adhesive-ceramic interface), cohesive (involving only one of the substrates) or mixed (involves the adhesive interface and also one of the substrates). contact angle analysis: for contact angle analysis, two extra non-cemented specimens of each group were made. the contact angle was measured by a goniometer (ramé hart-inc, 100-00-115, mountain lakes, nova jersey, eua) in a controlled-temperature environment. the 5 miranda et al. goniometer was connected to a computer equipped with specific software (rhi 2001 imaging software), and the sessile drop technique was implemented. a drop of distilled water was placed on the ceramic surface using a syringe, and the contact angle was measured for 10 seconds (30 frames per second). five measurements were performed for each sample, totaling 10 measurements per group. surface analysis: one of the extra samples was also examined in a digital optical profilometer (wyko, modelo nt 1100, veeco, tucson, usa) connected to a computer with image software (vision 32, veeco, tucson, usa) to perform surface micrographs (qualitative analysis of three-dimensional geometry). the glaze layer after 10% hf etching was also measured using the profilometer, performed by four micrographs per sample. then, the glaze layer was calculated by averaging the obtained values. the same samples were then cleaned with 70% alcohol (alves santa cruz ltda. guarulhos, são paulo, brazil), dried and metallized (emitech sc7620), receiving a thin layer (12nm) of gold alloy. they were examined using a scanning electron microscope (sem; inspect s50, fei, czech republic) to obtain mapping, backscattered electron detector (bse) and conventional sem images. energy dispersive spectrometry (eds) was then used for chemical element analysis. the readings were performed at a distance of 12mm and 20kv accelerating voltage. the main elements were analyzed in 100s real time for each measured area (1mm2). statistical analysis: to evaluate the surface treatment influence on bond strength, the data were submitted to kruskal-wallis and dunn statistical tests. in order to evaluate the influence of these different treatments on the zirconia wettability, one-way anova and tukey tests were applied. the significance level for all tests was 95%. results shear bond strength test and failure mode: the kruskal-wallis test revealed a significant interaction of the surface treatment (p = 0.001). using dunn’s test (p ≤ 0.05), it was possible to verify that bond strength mean values of the rs group were statistically higher than the gs20s, gs60s and gs100s groups. however, the etching time did not influence the bond strength of these experimental groups (table 2). pre-test failures occurred in gs20s (9), gs60s (14) and gs100s (16) groups. they were characterized by cement detachment during thermocycling. stereomicroscopic analysis revealed complete adhesive failures (100%). contact angle: the control group was similar to gs60s and gs100s. gs20s group had lower contact angle and therefore better wettability. it was observed that the lower the glaze layer etching time, the better the surface wettability. in addition, all experimental groups were statistically different from each other (p<0.001) (table 2). 6 miranda et al. surface analysis: three-dimensional geometry analysis by profilometry revealed a decrease in the glaze layer thickness; it was inversely proportional to the 10% hf etching time. mean of the residual glaze layer thickness was: 5 ± 1μm for gs20s, 4 ± 1μm for gs60s, and 3 ± 1μm for gs100s (figure 1). the sem of the rs group showed a homogeneous surface composed by zirconia modified by the silica oxide 30μm sandblasting. the gs20s sample presented table 2. dunn test results for bond strength values (mpa) and contact angles results (º) analyzed by oneway anova and tukey test (p<0.001). different letters indicate a significant difference between groups. n bond strength mean (mpa) n contact angle (º) rs 20 22.10 (2.78) a 10 55.33 (10.70) a b gs20s 11 8.10 (5.59) b 10 70.78 (6.69) c gs60s 6 10.49 (5.38) b 10 48.20 (10.57) b gs100s 4 7.53 (4.62) b 10 28.73 (19.75) a figure 1. profilometry 3d image of the of gs20s (a), gs60s (c) and gs100s (e) showing irregularity in the arrangement of applied glaze spray and their remaining glaze thickness measurements, indicating the average layer thickness 5 ± 1μm (b), 4 ± 1μm (d) and 3 ± 1μm (f). a c e f d b um um x: 128.3817 um z : 5.6274 um 0 20 40 60 80 100 120 140 160 180 200 4 2 0 -2 -4 um um x: 121.8445 um z : 4.3724 um 0 20 40 60 80 100 120 140 160 180 200 4 2 0 -2 um um x: 112.0332 um z : 3.4175 um 0 20 40 60 80 100 120 140 160 4 2 0 -1 -2 220 3 -1 1 5 3 1 7 miranda et al. a surface predominantly composed of zirconia with few residual glaze layer areas dispersed on the ceramics (“glaze islands”). the other vitrified groups (gs60s and gs100s) also demonstrated a similar morphology, but with an even smaller residual glaze remaining on the surface. the same pattern could be observed in the mapping and bsd images (figure 2). therefore, as the 10% hf etching time increases, the amount of remaining glaze becomes scarcer. figure 2. sem, mapping and bse (1000x) of the control group rs (a,b,c), showing homogenous surface; and the experimental gs20s (d,e,f), gs60s (g,h,i) and gs100s (j,k,l), which reinforce the irregular distribution of the glass layer on the y-tzp surface, forming the “glaze islands”. map data 114 se mag 1000x hv. 25.0kv wd: 11.9mm 100 µmict unesp hv 25.00 kv mag 1000 x wd 11.9 mm det bsed spot 5.0 se o zr au al se o zr au alsic cse o zr au alsi kna se o zr au alsi k na 40 µm 40 µm 40 µm 40 µm map data 115 se mag 1000x hv. 25.0kv wd: 11.9mm 100 µmict unesp hv 25.00 kv mag 1000 x wd 11.9 mm det bsed spot 5.0 map data 118 se mag 1000x hv. 25.0kv wd: 12.0mm 100 µmict unesp hv 25.00 kv mag 1000 x wd 12.0 mm det bsed spot 5.0 map data 117 se mag 1000x hv. 25.0kv wd: 12.1mm 100 µm ict unesp hv 25.00 kv mag 1000 x wd 12.1 mm det bsed spot 5.0map data 117 map data 118 map data 115 map data 114 a b c d e f g h i j k l 40 µm 40 µm 40 µm 40 µm 8 miranda et al. eds analysis showed the presence of the following elements in its composition: aluminum (al), calcium (ca), potassium (k), sodium (na), oxygen (o), silica (si) and zirconia (zr). the weight (%) for each chemical element in the rs group was: al (1.9%), o (30.0%), si (1.8%) and zr (66.3%). for gs20s: al (1.3%), k (1.7%), na (1.7%) o (38.7%), si (6.6%) and zr (60.1%). for gs60s: al (0.4%), o (24.5%), si (0.8%) and zr (74.3%). and finally for gs100s: al (0.9%), k (0.4%), na (0.5%) o (25.3%), si (1.9%) and zr (71.4%). in the extra gs60s sample, one more measurement of the chemical composition was performed specifically on an “glaze island” area (figure 3). in this area, the weight (%) for each chemical element was: al (5.5%), ca (2.6%), k (5.9%), na (6.2%), o (42%), and si (37.9%). discussion previous studies showed that chemical and/or mechanical modification by the application of a thin glass layer in the y-tzp ceramic surface positively influences bonding strength to resin cements3,19-20. as such, this research was carried out in order to evaluate the effect of three different hf etching times on the glaze surface of a y-tzp ceramic associated with the use of a universal adhesive and resinous dual-cure cement. the control group of this research was constituted by sandblasting with silica-coated alumina y-tzp, with subsequent application of the universal adhesive containing a silane coupling agent3,15,23. this procedure promotes chemical adhesion between the ceramic surface and the resin cement organic matrix3. this happens by the attachment of silane monomers that react with the silica-coated surface within the silanol groups, thus forming hydrogen bonds and finally a covalently bonded very thin silane film6,12. then, silane film with its free carbon–carbon double bonds reacts with the double bonds of resin composite luting cement12. thus, good bond strength is obtained. an acceptable range of bond strength is 10 to 13mpa24. in the present study, even after aging, the control group bond strength was 22 mpa. this high result may figure 3. sem (20,000x) of the gs60s group indicating the area analyzed by eds (a) and the graphical representation of the eds analysis indicating a considerable presence of silica (37.9%) in one of the “glaze islands” (b). imagem de elétrons 6 a b espectro 8 espectro 8 o si na k al ca wt% 42.0 37.9 6.2 5.9 5.5 2.6 σ 0.2 0.1 0.1 0.1 0.1 0.1 cp s/ ev 10 µm 60 40 20 0 0 0.5 1 1.5 2 2.5 kev ca o na al si 9 miranda et al. be related to the use of this surface treatment associated to an adhesive system with mdp1,6,10,21. the chemical interaction of this monomer can improve bond strength of crystalline ceramics such as zirconia, since this monomer has two bonding ends: one end has vinyl groups that react with the monomers of the resin cement when cured, and the other has phosphate ester groups which have strong hydrophilic bonding to metal oxides2,21. despite the promising results of bond strength using sandblasting with silica-coated alumina treatment, previous studies have demonstrated that this method can create a critical damage zone involving grooves and defects that can generate clinical failures3,14. because of this, an alternative approach was introduced to improve bond strength to y-tzp ceramics and resin cement3,6,7. this other treatment involves a thin glass layer applied to the zirconia surface3,7. this enriches the surface with silicon oxides, which facilitates chemical bonding through the silane application. in turn, this produces a siloxane bond between the silica contained in this new vitrified layer and the resin cement organic matrix3,7. in addition, the vitrification allows hf etching of the glass layer, which modifies the surface topography and creates micromechanical retentions, similar to the vitreous ceramics mechanisms3,5,11,17. however, denying the first null hypothesis, the bond strength values in the vitrified groups were significantly lower than the control group. the gs60s had bond strength values closer to those considered acceptable in the literature24, while gs20s and gs100s presented lower values. nonetheless, as in a previous study on a naturally vitreous ceramic18, the hf etching time on the glass layer revealed no statistical difference in the bond strength results of the ceramic with a resinous cement, leading to the acceptance of the second null hypothesis. however, from the statistical point of view, the small number of samples per group in this research may be responsible for the high variation coefficient and caused a low power of the test, increasing the probability of a false negative result8. martins et al.5 stated that the amount of silica on the zirconia surface is higher when sandblasting with silica-coated alumina compared to vitrification, which could justify the obtained result; however, our eds results do not corroborate this information, because the silica percentage on the different groups was similar. the most acceptable justification for this is that sandblasting was able to create surface irregularities by which the adhesive system and cement penetration occurred on the y-tzp (20). despite this, other authors claim that the vitrification technique is an advantageous surface treatment for zirconia as a whole25; it is easy to apply, has satisfactory cost-benefit, and does not induce damage to the ceramic5,11,14,25. the glaze layer remaining after hf etching was not uniform, which does not favor the chemical and mechanical union desired from vitrification, and can justify the results. through these tests, it was found that 10% hf etching of the glazed surface irregularly removes a considerable part of the glaze applied to the surface of the y-tzp, leaving only “glaze islands” and large regions without vitreous content on the zirconia. this removal was proportional to the application time of hf. therefore, it is suggested that adhesion in these specimens are more related to the mdp presence in the adhesive system22. there is a statement that in order to produce better bond strength results, this monomer must be present in both the cement and the 10 miranda et al. adhesive8,21,22; however, the present adhesive system only had mdp in the adhesive composition, but not in the cement. six thousand thermal cycles was adopted in this research, which is a quantity also used by other researchers3,12,16,23. some experimental group samples were lost during aging. previous studies have reported reduced bond strength or premature failure due to thermocycling, even in specimens treated witch silica-coated alumina15. this is due to the combination of hydrolytic degradation, water diffusion into the interfacial layer and thermal irradiation during cycles3. therefore, it is observed that y-tzp adhesive interfaces are susceptible to aging23. however, it is known that even though zirconia cemented with mdp-containing adhesive systems reduces adhesion after thermocycling8, the presence of this phosphate monomer generates better conditions to support this aging26. this is due to the monomers’ chemical bonding to the metal oxides by van der waals forces or hydrogen bonds at the resin cement/zirconium interface10. failure analysis indicated that these were always adhesive, independently of the groups, and the zirconia blocks were adhesive and cement free. this has also been observed in other studies1,8,12,21,26. these failures may be associated with several factors: thermal expansion difference between the materials8,25, processing techniques, phase transformation and factors related to the adhesive system25. in the universal adhesive chemical composition there are the mdp, dimethacrylate, 2-hydroxyethyl methacrylate, vitrebond copolymer, ethanol, water, initiators and silane. however, mixing these constituents in the same flask containing a greater amount of solvents can hinder the adhesion between resin cement and ceramic, because they react differently in each substrate13. in addition, kim et al.27 claimed that silane incorporation into the universal adhesive appears to be ineffective, and that the mdp may prevent optimal chemical interaction between silane and ceramic, which is due to the tendency for premature hydrolysis in an acidic environment. some authors argue that systems containing metal primer and silane in separate flasks promote better chemical bonding5,13,26. lastly, through eds of a “glaze island” area it was possible to observe the high presence of silica content (37.9%) (figure 3). in addition, the contact angle results showed that lower etching time results in a higher amount of glaze being maintained on the surface, higher wettability and better adhesion. therefore, the application and more importantly the maintenance of this glass layer on the y-tzp surface seems to be a promising path for zirconia adhesive luting. new protocols have yet to be evaluated, such as application of a double or triple glaze layer on a zirconia surface or the use of powder/liquid glaze by brush technique, in order to obtain better standardization of the glaze application, which does not seem to be guaranteed with the spray application. in conclusion, the shear bond results demonstrated that the bond strength between y-tzp glazed and an adhesive system with mdp was not influenced by different conditioning times with hydrofluoric acid. however, the image tests and goniometry indicate that a shorter hf etching time is more favorable for the adhesive surface of this zirconia. 11 miranda et al. references 1. cavalcanti an, foxton rm, watson tf, oliveira mt, giannini m, marchi gm. bond strength of resin cements to a zirconia ceramic with different surface treatments. oper dent. 2009 may-jun;34(3):280-7. doi: 10.2341/08-80. 2. özcan m, bernasconi m. adhesion to zirconia used for dental restorations: a systematic review and meta-analysis. j adhes dent. 2015 feb;17(1):7-26. doi: 10.3290/j.jad.a33525. 3. amaral m, belli r, cesar pf, valandro lf, petschelt a, lohbauer u. the potential of novel primers and universal adhesives to bond to zirconia. j dent. 2014 jan;42(1):90-8. doi: 10.1016/j.jdent.2013.11.004. 4. gargava s, ram sm. evaluation of surface conditioning of zirconia and its effect on bonding to resin-luting agent. j contemp dent. 2013 jan-apr;3(1):7-10. doi: 10.5005/jp-jourals-10031-1026. 5. martins arm, gotti vb, shimano mm, borges ga, gonçalves ls. improving adhesion between luting cement and zirconia-based ceramic with an alternative surface treatment. braz oral res. 2015;29:54. doi: 10.1590/1807-3107bor-2015.vol29.0054. 6. pereira ll, campos f, dal piva am, gondim ld, souza ro, özcan m. can application of universal primers alone be a substitute for airborne-particle abrasion to improve adhesion of resin cement to zirconia? j adhes dent. 2015 apr;17(2):169-74. doi: 10.3290/j.jad.a33974. 7. ntala p, chen x, niggli j, cattell m. development and testing of multi-phase glazes for adhesive bonding to zirconia substrates. j dent. 2010 oct;38(10):773-81. doi: 10.1016/j.jdent.2010.06.008. 8. zhao l, jian yt, wang xd, zhao k. bond strength of primer/cement systems to zirconia subjected to artificial aging. j prosthet dent. 2016 nov;116(5):790-6. doi: 10.1016/j.prosdent.2016.03.020. 9. attia a. bond strength of three luting agents to zirconia ceramic influence of surface treatment and thermocycling. j appl oral sci. 2011 aug;19(4):388-95. 10. tanıs mt, akay c, karakıs d. resin cementation of zirconia ceramics with different bonding agentes. biotechnol biotechnol equip. 2015 mar 4;29(2):363-7. 11. vanderlei a, bottino ma, valandro lf. evaluation of resin bond strength to yttria-stabilized tetragonal zirconia and framework marginal fit: comparison of different surface conditionings. oper dent. 2014 jan-feb;39(1):50-63. doi: 10.2341/12-269-l. 12. özcan m, yetkiner e. could readily silanized silica particles substitute silica coating and silanization in conditioning zirconium dioxide for resin adhesion? j adhes sci technol. 2016;30(2):186-93. doi: 10.1080/01694243.2015.1095628. 13. alves m, campos f, bergoli cd, bottino ma, özcan m, souza r. effect of adhesive cementation strategies on the bonding of y-tzp to human dentin. oper dent. 2016 may-jun;41(3):276-83. doi: 10.2341/15-052-l. 14. melo rm, souza r, dursun e, monteiro e, valandro lf, bottino ma. surface treatments of zirconia to enhance bonding durability. oper dent. 2015 nov-dec;40(6):636-43. doi: 10.2341/14-144-l. 15. usumez a, hamdemirci n, koroglu by, simsek i, parlar o, sari t. bond strength of resin cement to zirconia ceramic with different surface treatments. lasers med sci. 2013 jan;28(1):259-66. doi: 10.1007/s10103-012-1136-x. 16. rippe mp, amaral r, oliveira fs, cesar pf, scotti r, valandro lf, et al. evaluation of tensile retention of y-tzp crowns cemented on resin composite cores: effect of the cement and y-tzp surface conditioning. oper dent. 2015 jan-feb;40(1):e1-e10. doi: 10.2341/13-310-l. 17. bottino ma, snellaert a, bergoli cd, özcan m, bottino mc, valandro lf. effect of ceramic etching protocols on resin bond strength to a feldspar ceramic. oper dent. 2015 mar-apr;40(2):e40-6. doi: 10.2341/13-344-l. 12 miranda et al. 18. leite fp, ozcan m, valandro lf, moreira chc, amaral r, botino ma, et al. effect of the etching duration and ultrasonic cleaning on microtensile bond strength between feldspathic ceramic and resin cement. j adhes. 2013;89:159-73. doi: 10.1080/00218464.2013.739024. 19. miranda js, malta nv, carvalho rla, souza roa, machado jpb, leite fpp. which low-fusing porcelain glaze treatment technique is better to promote a vitreous surface on y-tzp ceramic? rev odonto cienc. 2017;32(4):174-9. doi: 10.15448/1980-6523.2017.4.28749. 20. simões ac, miranda js, souza roa, kimpara et, leite fpp. bond and topography evaluation of a y-tzp ceramic with a superficial low-fusing porcelain glass layer after different hydrofluoric acid etching protocols. rev odontol unesp. 2018 nov-dec;47(6):1-6. doi: 10.1590/1807-2577.10118. 21. gotti vb, calabrez filho s, shimano mm, borges ga, borges lg, gonçalves ls. influence of ceramic primers on microshear bond strength between resin cements and zirconia-based ceramic. braz j oral sci. 2011 apr-jun;10(2):124-9. 22. ahn j, yi y, lee y, seo d. shear bond strength of mdp-containing self-adhesive resin cement and y-tzp ceramics: effect of phosphate monomer-containing primers. biomed res int. 2015;2015:389234. doi: 10.1155/2015/389234. 23. sarmento hr, campos f, sousa rs, machado jp, souza ro, bottino ma, et al. influence of air-particle deposition protocols on the surface topography and adhesion of resin cement to zirconia. acta odontol scand. 2014 jul;72(5):346-53. doi: 10.3109/00016357.2013.837958. 24. lüthy h, loeffel o, hammerle ch. effect of thermocycling on bond strength of luting cements to zirconia ceramic. dent mater. 2006 feb;22(2):195-200. 25. anami lc, lima jm, corazza ph, yamamoto et, bottino ma, borges al. finite element analysis of the influence of geometry and design of zirconia crowns on stress distribution. j prosthodont. 2015 feb;24(2):146-51. doi: 10.1111/jopr.12175. 26. kim mj, kim yk, kim kh, kwon ty. shear bond strengths of various luting cements to zirconia ceramic: surface chemical aspects. j dent. 2011 nov;39(11):795-803. doi: 10.1016/j.jdent.2011.08.012. 27. kim rjy, woo js, lee ib, yi ya, hwang jy, seo dg. performance of universal adhesives on bonding to leucite-reinforced ceramic. biomater res. 2015 may 22;19:11. doi: 10.1186/s40824-015-0035-1. http://www.ncbi.nlm.nih.gov/pubmed/?term=kim rj%5bauth%5d http://www.ncbi.nlm.nih.gov/pubmed/?term=woo js%5bauth%5d http://www.ncbi.nlm.nih.gov/pubmed/?term=lee ib%5bauth%5d http://www.ncbi.nlm.nih.gov/pubmed/?term=yi ya%5bauth%5d influence of curing protocols on water sorption and solubility of a self-adhesive resin-cement dilcele silva moreira dziedzic1, joão paulo stanislovicz prohny1, gledson luiz picharski2, adilson yoshio furuse3 1universidade positivo – up, school of dentistry, area of dental material, curitiba, pr, brazil 2faculdades pequeno príncipe, department of biostatistics, curitiba, pr, brazil 3universidade de são paulo – usp, bauru dental school, department of operative dentistry, endodontics and dental materials, bauru, sp, brazil correspondence to: adilson yoshio furuse departamento de dentística, endodontia e materiais odontológicos faculdade de odontologia de bauru universidade de são paulo al. octávio pinheiro brisolla, 9-75, bauru são paulo, brasil cep: 17012-901 phone: +55 14 3235-8253 e-mail: furuse@usp.br abstract aim: to evaluate the effect of different activation protocols on the polymerization of a self-adhesive dual cured resin-based cement. methods: thirty disc-shaped specimens were prepared with the resin cement relyx u200 (3m espe) and divided according to three protocols: immediate light-activation for 40 s, delayed light-activation (10 min after manipulation, for 40 s) or self-curing without lightactivation. the specimens were desiccated, kept in water at 37 °c for 7 days and desiccated again to calculate water sorption, solubility and mass variation. data were analyzed by shapiro-wilk test and wilcoxon tests (α=0.05). images after the specimens’ final desiccation were also made. results: the wilcoxon test revealed a significant difference for sorption and mass variation (p<0.05) and the highest value was observed in self-curing or chemical activation group (ca), followed by delayed light-activation (dl) and immediate light-activation (il). besides the water sorption parameters, there were also microvoids on the discs from the delayed and no light-activation groups. conclusions: the light-activation immediately after manipulation is recommended for the evaluated resin cement. keywords: resin cements. solubility. polymerization. introduction the degree of conversion and polymerization rate of dual-curing resin-based cements can determine the longevity of the indirect restoration, because it is directly related to the materials’ properties. besides the bonding mechanisms to both dental tissues and restorations, the success of luting cements depends on the mechanical properties developed during polymerization to be preserved during clinical life. therefore, all involved procedures and the used materials for cementation of indirect restorations and intracanal posts are critical to achieving adequate performance of indirect restorations. dual-curing resin cements present two activation systems (chemical and light activation) for a thorough and uniform conversion of monomers into polymers, even when the composition, thickness and color of the ceramic restoration could hamper the transmission of the curing light used to polymerize the cement1. thus, different light-activation protocols should be investigated, as the most significant effects on the properties of resin cements are related to the type of cement, exposure time, followed by translucency, ceramic type and thickness of the ceramic restoration1-3. the effect of light activation on dual-cured resin cements should not be neglected for some cements4-7. however, the immediate light-activation of a dual-cured resinreceived for publication: august 08, 2016 accepted: november 01, 2016 original article braz j oral sci. april | june 2016 volume 15, number 2 http://dx.doi.org/10.20396/bjos.v15i2.8648766 braz j oral sci. 15(2):144-150 145 cement, as recommended by the manufacturer, may have a deleterious effect on polymerization if it stiffens the polymer, limiting the process of chemical activation. this prevents the cement from achieving its maximum degree of conversion and mechanical properties8. the light-activation delay to allow more time for the chemical activation to occur before light-activation seems to affect the dual-cured resin cement properties depending on the material’s composition and the time interval between the mixture until light-activation. light-activation delay of 5 min, was suggested as being advantageous to reduce shrinkage stress during polymerization, without impairing the degree of conversion, the extent of polymerization and hardness of some dual-cured cements9-11. it was also demonstrated that a 6-minute delay after the manipulation of resin cements may result in increased color stability2, but a 10-minute delay may be harmful12. during polymerization, individual polymer chains are formed by covalent bonds, which are crosslinked by secondary bonds. the degree of conversion of the material, which is the average number of monomers that form the polymer chains, is directly related to the length and crosslinking of the network and indirectly to water sorption. the polymer network of a resin-based material is composed by crosslinked molecules and unreacted monomers not linked to the chains. inadequate polymerization, in addition to reducing the mechanical properties10,13, may have biological consequences14,15 related to the unreacted free monomers.16 water sorption and hydrogen bonds with the polar groups of polymers produce separation of the molecules and reduction of hydrogen bonds between them17. the expansion resulting from water sorption favours the release of the monomers not linked into chains, reduction of mechanical properties18 and the cement plasticisation can generate unsupported areas, causing fracture to indirect restorations19. sorption and solubility tests are used for in vitro evaluation of resin-based materials20, including dual-cured resin cements12, to evaluate and recommend the materials with lower sorption and solubility for indirect restorations21. the water absorption measured by sorption, influences negatively the material, causing expansion, weakening of matrix and filler bonding, discoloration and reduction of mechanical properties20. the solubility of the material reflects the amount of released residual monomers, revealed by its mass reduction20. the high water sorption and solubility influence negatively the resinous materials, indicating lower chemical stability, lower biocompatibility, higher expansion and lower mechanical properties. sorption, solubility and mass variation are considered indicators of the chemical, physical and mechanical properties observed in the structure of the polymeric network12,21. alterations of these properties can lead to deleterious clinical effects. therefore, parameters of sorption, solubility and mass variation can be used to demonstrate the incompatibility of the material for certain clinical applications. dual-curing resin cements of different brands showed different behaviours as to the degree of conversion and mechanical properties when cured with different light-activation protocols22-24. while some cements are more suitable for situations where immediate light-activation is possible, others show a better behaviour when the light-activation is limited23. the mechanical properties of cements are mostly affected by their composition rather than the used activation mode and the delay of 5 min for the polymerization did not interfere with the assessed properties22. the period of 5 min for chemical conversion can even improve the clinical performance of restorations by reducing shrinkage stress without interfering with the degree of conversion9. the aim of the present study was to evaluate sorption (s), solubility (sl) and mass variation (m%) of the dual-cured selfcuring cement relyx u200 (3m espe) when three different polymerization protocols were used. the protocols were: immediate light-activation, 10 min delayed light-activation and self-activation. the tested null hypothesis was that there was no difference between the polymerization protocols for the values of each parameter: s, sl and m%. material and methods thirty disc-shaped specimens were prepared with the dual-cured self-adhesive resin cement (relyx u200; 3m espe deutschland gmbh, neuss, germany, colour a3 opaque, lot: 506385). the main components of the cement, presented by weight in the material safety data sheet, are silane treated glass powder (65%) and substituted dimethacrylate (30%) in the catalyst paste; silane treated glass powder (45–55%), methylesterdimethacrylate acid (20–30%), triethylene glycol dimethacrylate (10–20%), and silane treated silica (10%) in the base paste. both pastes also contain initiator components, stabilizers and rheological additives. the two pastes (1:1 ratio) were hand mixed on a mixing pad for 10 s and placed into a circular nylon mould (1 mm thick with a central orifice with 7 mm diameter), between two mylar strips and two 1-mm-thick glass slides. in order to obtain parallel surfaces, light finger pressure was applied on the top glass slide. the cement was light-activated on the top exposed surface in continuous mode for 40 s with a led curing device (wireless poly; kavo, joinville, sc, brazil) at 1,100 mw/cm2. three groups were evaluated according to the light-activation protocol: immediate light-activation, delayed light-activation or chemical activation. the immediate light-activation (il) was done approximately 1 min after specimen preparation. in the delayed light-activation group (dl), after manipulation of the cement, the samples were protected from light for 10 min before light-activation. the light-activation of these groups was initially conducted through a 1 mm-thick glass plate, which was then removed and the process continued through the mylar strip. chemical activation (ca) or self-curing specimens were prepared, covered with black plastic and away from light exposure for 24 h, with no light-activation. each resin cement disc was visually inspected to exclude those with porosities. excess material at the edges was removed with a #15 scalpel blade and the borders were smoothed with 600-grit sandpaper. discs were stored in amber glass bottles and retained from any light for 24 h at room temperature. the discs were washed with distilled water in an ultrasonic cleaner (cristófoli, campo mourão, pr, brazil) for 2 min and dried with absorbent paper before desiccation. dark plastic canisters with calcium chloride and the amber glass vials containing the specimens were placed in a desiccator with silica and under influence of curing protocols on water sorption and solubility of a self-adhesive resin-cement braz j oral sci. 15(2):144-150 146 vacuum suction (400 mmhg) for 2 h at room temperature. the vials, kept in desiccation and dark conditions, were transferred to an incubator (labstore equipment, curitiba, pr, brazil) at 37 °c for 24 h. the specimens were weighed every 24 h on an analytical scale (boeco, hamburg, germany) with a ±0.01 mg accuracy to obtain a constant mass (m1), with a variation lower than 0.2 mg. the thickness and the diameter of the discs were measured using a digital calliper (starret 799 a-6/150, itu, sp, brazil), at four places around the circumference, in order to calculate the volume of the specimen (v, in mm3) with the following equation: v=π r2h where r is the mean radius of the specimen, and h is the average thickness. the specimens were kept in amber glass bottles containing 10 ml distilled water (ph 7.2) for 7 days, sealed at 37 c. the water was changed every 48 h during this period. the samples were dried with absorbent paper and weighed again (m2) before a second desiccation cycle, as described previously, to obtain the final mass (m3). the water sorption (s) and solubility (sl) were calculated by the following equations, respectively: s=(m2-m3)/v sl=(m1-m3)/v where m1 is the initial mass of the desiccated specimen before immersion in distilled water, m2 is the mass of saturated sample after immersion in water for 7 days, m3 is the final mass after desiccation, and v is the volume of sample. the percent change in mass (m%) was calculated by the following equation: m%= (m2-m1)/m1 x100 the data for water sorption (s), solubility (sl) and percentage change in mass (m%) were analyzed by shapiro-wilk test and wilcoxon nonparametric test (r core team software; r foundation for statistical computing, vienna, austria), with a 5% significance level. if the p value obtained from the test was statistically significant, the group differed from the others. for the purpose of evaluating the surface characteristics like voids, the discs were examined with an optical microscope (olympus bx41, tokyo, japan) at 10x magnification and the images were captured with the image pro plus program. results significant differences in water sorption between the protocols used for light-activation were observed and are presented in tables 1 and 2, and figures 1 and 2. the wilcoxon tests revealed highly significant differences among the three activation protocols for mass variation with no exception (table 2). after storage in water, there was a significant difference between chemical activation and the other two groups, (p<0.05). a significant difference was also observed between immediate and delayed light-activations, the two lowest values for this parameter (tables 1 and 2, figures 1 and 2). influence of curing protocols on water sorption and solubility of a self-adhesive resin-cement fig.1. water sorption characteristics (µg/mm3) of the cement under three different activation protocols after seven days of water immersion (mean and standard error). table 1 mean values and standard deviations for water sorption, solubility, and percentage of mass variation of the groups after 7 days of storage in water (37 °c). sorption (μg/ mm³) solubility (μg/mm³) mass variation (%) immediate lightactivation 28.09 (3.77) a 1.28 (3.10)a 1.49 (1.10)a delayed light-activation 32.07 (3.36) b 0.04 (1.46)a 1.74 (0.12)b chemical activation 37.58 (3.55)c -0.53 (0.75) a 2.06 (0.17) c water sorption and solubility presented in absolute values (μg/mm³), mass variation presented in percent (%). different capital letters in columns indicate significant difference (p<0.05). table 2 p values of wilcoxon tests of water sorption, solubility and percent of mass variation of the groups after 7 days of storage in water (37 °c). evidence of difference between groups when p<0.05. sorption solubility mass variation immediate chemical immediate chemical immediate chemical chemical <0.001 0.32 <0.001 delayed 0.009 0.008 0.79 0.79 <0.001 <0.001 braz j oral sci. 15(2):144-150 147 days of storage in water the major components removed from the resinous materials are the residual monomers that have not reacted to form polymeric chains18. the highest concentration of eluted monomers in 7 days was also observed by other authors5. fig.2. mass variation percent (%) of the cement under different activation protocols after seven days of water immersion (mean and standard error). fig.3. images of representative discs after the water sorption process (desiccation, water immersion for 7 days and desiccation). (a) the immediate light-activation group showed a more homogenous mixture, and fewer microvoids than the other groups. (b) the delayed light-activated group exhibited microvoids. (c) the chemical activation group showed more and larger microvoids than the others. field width of each image= 600 µm. the solubility, a measure of the amount of unreacted monomer eliminated in the water during 7 days storage at 37 °c, exhibited no evident differences between the activation protocols (tables 1 and 2). statistical analysis also expressed significant differences among the three protocols for mass variation (p<0.001). the highest mass variation was observed in ca followed by dl and il (figure 2). images showed a more homogenous mixture in the immediate light-activation group, and microvoids were observed in the delayed light-activation group and throughout the chemical activation group (figure 3). there were no cracks in any disc. discussion in the present study the dual-cured self-adhesive resin cement relyx u200 was activated according three different curing protocols: il, dl and ca. under the experimental conditions, the results demonstrated that these protocols influenced significantly the investigated properties of water sorption and mass variation. thus, the null hypothesis was rejected. the immediate light-activation significantly decreased water sorption and mass variation. this result may indicate that a higher degree of conversion of this dual-cured self-adhesive cement was attained with immediate light-activation. this means that in clinical practice during cementation procedure the removal of excesses and lightactivation should be performed immediately, since according to the present study a long delay may generate a polymer with inferior properties. the water storage period of seven days, chosen for the present study, has been extensively studied. svizero et al. verified water sorption daily for 2 weeks and found that the greatest increase of mass was observed in the first day in water for the cements analysed after three curing protocols12. during seven influence of curing protocols on water sorption and solubility of a self-adhesive resin-cement (a) (b) (c) braz j oral sci. 15(2):144-150 besides the polymerization protocol, sorption and solubility are also explained by the affinity of the organic and inorganic components with water. hydrophilic monomers and monomers with polar characteristic may account for greater water sorption than hydrophobic monomers5,25. the greater density of inorganic filler reduces sorption18, but the reaction between filler particles and water can also promote water retention5. the sorption and release of residual monomers are related to the degree of polymerization, the composition of the material: organic matrix and filler particles; and also the solvents used for in vitro studies5,21,25-27. among the factors that affect the hygroscopic and hydrolytic properties of the cements, the degree of polymerization is linked to the sorption, solubility, hydrolytic degradation and the formed polymer, because the quality of the network formed by polymerization regulates the solvent sorption and expansion of the material18. polymer networks with higher density of covalent cross-links have lower absorption and expansion due to the proximity of the polymer chains, reducing the space to be filled by solvent diffusion, as the degree of conversion of the polymer and the pending molecules within the network also influence the absorption of water18. therefore, the polymerization protocols of 10 min-delay and no light-activation may have disturbed the conversion of the dual-cured resin cement producing shorter chains, resulting in significantly higher water sorption. it should be noted that a 10-min delay was proven to be harmful in another study12 and it is a time too long for a clinical procedure. the expansion promoted by the solvent inside the resin matrix, separating the polymer chains, promotes plastification of the material and diffusion of the unreacted monomer, called elution16,18. this elution of unreacted monomers during the immersion in water period, results in mass loss and is calculated as solubility. in this study, solubility of the cement under three activation protocols was not significantly different and presented low levels (≤0.1%), even lower than some reported in the literature12,21. the average values of water sorption and solubility of the resin cement relyx u200 for the three tested polymerization protocols were lower than 40 μg/mm3 and 7.5 μg/mm3, respectively20. the estimated mean value of solubility for the chemical activation was negative, suggesting that this group of cement, instead of presenting only the expected decrease in mass due to elution of unreacted monomers, showed an increase in mass. authors observed mass increase in sorption and solubility studies, despite the simultaneous release of free monomers5,25. possible explanations for this increase in mass (m3>m1) have been suggested: instead of elution of the unreacted monomers, the material retained some of the solvent which was absorbed25 and/or the reaction between filler particles and water, with the formation of hydroxides, can result in increased mass5. studies pointed out that the delay of the light-activation to the maximum tolerable time clinically, from 5 to 10 min, offers an interval for conversion only by chemical activation, which is followed by light-activation to ensure the highest possible degree of conversion8,19,23. this study investigated the possibility that lightactivation shortly after mixing the pastes could limit the chemical conversion, preventing a thorough and uniform polymerization, with the different curing protocols. however, the tested 10 min delay, with the purpose of extending the self-curing reaction, prevented the achievement of the best properties in sorption, solubility and mass variation. maybe the results could be different if a 5-min-delay was also evaluated. inadequate light-activation of dual-cured resin cements may occur under more opaque or thicker ceramic restorations1. therefore, the characteristics in the group without light-activation may be considered clinically relevant. the highest evidence of difference in the values of sorption and mass variation were found in the group with chemical activation compared to the immediate light-activation (p<0.001), where only the polymerization rate of the cement was altered. therefore, the lowest degree of conversion of the material and/or crosslinking, directly related to water sorption5, may explain the higher water sorption of specimens without light-activation. a possible hypothesis for the higher values of sorption and mass variation in delayed light-activation and no light-activation groups, is that these protocols resulted in polymers with shorter chain length28, more sensitive in water sorption probably due to lesser crosslinking, but still with a sufficient degree of conversion to have solubility values as low as the immediate light-activation. according to marghalani (2012), the percentage of the mass variation or the weight increase of the specimen by absorption of water does not include the free monomers, which are not bonded to the polymer chain and are eliminated simultaneously in water absorption, resulting in reduced mass of the specimen21. it was suggested that the structural parameters of the polymers that affect sorption and solubility are crosslinking density and porosity of the polymer chains25. microscope images showing the density of microvoids in the different groups indicated a good agreement with the numerical data. more microvoids were observed in delayed and no light-activation groups, the ones with higher sorption. microvoids may be produced by trapped unreacted monomers21, enhancing solubility and forming sites for solvent accumulation18,25. however, the present study did not use any method to evaluate the degree of conversion. studies are recommended to confirm the possibility that delay and no lightactivation result in lower molecular weight and crosslink density, causing higher mobility and relaxation between the chains. dual-curing resin cements of different brands showed different results as regards the degree of conversion23 and mechanical properties22, when different polymerization protocols were employed. a comparative study concluded that certain cements are suitable for situations where immediate lightactivation is possible but not in other circumstances where this type of activation could be limited, so that the conversion should not be compromised23. two polymerization protocols employed in this study affected the conversion of the dual-cured self-adhesive cement relyx u200. however, the immediate light-activation resulted in the best characteristics of significantly lower sorption and mass variation. further studies should assess the ideal time frame between mixing and light-activation (inferior to 10 min) to verify any interference of light-activation with the self-curing mechanism, in order to determine the longest light-activation delay recommended for this cement. the parameters evaluated in the present study provided the basis for a recommendation on how dentists should use the cement in their daily clinical routine. the lower the sorption, solubility 148influence of curing protocols on water sorption and solubility of a self-adhesive resin-cement braz j oral sci. 15(2):144-150 and microvoid formation, the better the material in clinical use. furthermore, the effect of the assembly stability during the delayed light-activation and the chemical activation periods must be known. two studies describe that finger pressure and 0.5 kg weight were applied to permit extrusion of excess material13,21, while another9 used clamps during light-activation delay periods. most studies do not use any device for stability, with constant support. even though constant pressure during these periods was not employed by other authors4-6,10,12,13,22-24,29, and it is not clinically appropriate, this parameter may have influenced the results, because any loss of stability may favour the formation of microvoids and consequently increase water sorption and solubility. more research examining other properties may elucidate the effect of light exposure conditions on hardness, degree of conversion and mechanical properties4,30. yan et al. observed that resin cement materials with three different activation modes were cured within 24 h after mixing or after light activation. the light exposure for light or dual-cured materials produced significantly higher degree of conversion values than the chemically activated ones, compared with the material’s initial conversion promoted by light activation30. the light activation of dual materials is recommended to produce higher rates of conversion30 and hardness4, because the relative contributions of the light-cure and chemical-cure mechanisms may differ depending on the material brand. in conclusion, the alteration of the polymerization protocol from the recommended immediate light-activation to 10-min delay and no light-activation, disturbed the conversion of dual-cured self-adhesive resin cement, resulting in higher water sorption, higher mass variation and presence of microvoids. acknowledgements this study was carried out during the undergraduate research program at positivo university, curitiba, pr, brazil. the cement employed was kindly supplied by 3m espe. references 1. calgaro pa, furuse ay, correr gm, ornaghi bp, gonzaga cc. influence of the interposition of ceramic spacers on the degree of conversion and the hardness of resin cements. braz oral res. 2013;27(5):403-9. 2. furuse ay, santana lo, rizzante fa, ishikiriama sk, bombonatti jf, correr gm, et al. delayed light activation improves color stability of dual-cured resin cements. j prosthodont. 2016 jul 25. doi: 10.1111/ jopr.12509. [epub ahead of print] 3. runnacles p, correr gm, baratto filho f, gonzaga cc, furuse ay. degree of conversion of a resin cement light-cured through ceramic veneers of different thicknesses and types. braz dent j. 2014;25(1):3842. 4. braga rr, cesar pf, gonzaga cc. mechanical properties of resin cements with different activation modes. j oral rehabil. 2002;29(3):25762. 5. ortengren u, wellendorf h, karlsson s, ruyter ie. water sorption and solubility of dental composites and identification of monomers released in an aqueous environment. j oral rehabil. 2001;28(12):1106-15. 6. da silva fonseca as, mizrahi j, menezes lr, valente ll, de moraes rr, schneider lf. the effect of time between handling and photoactivation on self-adhesive resin cement properties. j prosthodont. 2014;23(4):302-7. 7. kim ar, jeon yc, jeong cm, yun mj, choi jw, kwon yh, et al. effect of activation modes on the compressive strength, diametral tensile strength and microhardness of dual-cured self-adhesive resin cements. dent mater j. 2016;35(2):298-308. 8. manso ap, silva nr, bonfante ea, pegoraro ta, dias ra, carvalho rm. cements and adhesives for all-ceramic restorations. dent clin north am. 2011;55(2):311-32, ix. 9. faria-e-silva a, boaro l, braga r, piva e, arias v, martins l. effect of immediate or delayed light activation on curing kinetics and shrinkage stress of dual-cure resin cements. oper dent. 2011;36(2):196-204. 10. ramos mb, pegoraro ta, pegoraro lf, carvalho rm. effects of curing protocol and storage time on the micro-hardness of resin cements used to lute fiber-reinforced resin posts. j appl oral sci. 2012;20(5):556-62. 11. soares cj, bicalho aa, verissimo c, soares p, tantbirojn d, versluis a. delayed photo-activation effects on mechanical properties of dual cured resin cements and finite element analysis of shrinkage stresses in teeth restored with ceramic inlays. oper dent. 2016 sep-oct;41(5):491-500 . doi: 10.2341/15-090-l. 12. svizero nda r, silva ms, alonso rc, rodrigues fp, hipolito vd, carvalho rm, et al. effects of curing protocols on fluid kinetics and hardness of resin cements. dent mater j. 2013;32(1):32-41. 13. fonseca rg, santos jg, adabo gl. influence of activation modes on diametral tensile strength of dual-curing resin cements. braz oral res. 2005;19(4):267-71. 14. kingman a, hyman j, masten sa, jayaram b, smith c, eichmiller f, et al. bisphenol a and other compounds in human saliva and urine associated with the placement of composite restorations. j am dent assoc. 2012;143(12):1292-302. 15. lodiene g, kopperud hm, orstavik d, bruzell em. detection of leachables and cytotoxicity after exposure to methacrylateand epoxybased root canal sealers in vitro. eur j oral sci. 2013;121(5):488-96. 16. kwon hj, oh yj, jang jh, park je, hwang ks, park yd. the effect of polymerization conditions on the amounts of unreacted monomer and bisphenol a in dental composite resins. dent mater j. 2015;34(3):32735. 17. park j, eslick j, ye q, misra a, spencer p. the influence of chemical structure on the properties in methacrylate-based dentin adhesives. dent mater. 2011;27(11):1086-93. 18. ferracane jl. hygroscopic and hydrolytic effects in dental polymer networks. dent mater. 2006;22(3):211-22. 19. pegoraro ta, da silva nr, carvalho rm. cements for use in esthetic dentistry. dent clin north am. 2007;51(2):453-71,x. 20. heintze sd, zimmerli b. relevance of in vitro tests of adhesive and composite dental materials, a review in 3 parts. part 1: approval requirements and standardized testing of composite materials according to iso specifications. schweiz monatsschr zahnmed. 2011;121(9):80416. 21. marghalani hy. sorption and solubility characteristics of self-adhesive resin cements. dent mater. 2012;28(10):e187-98. 22. faria-e-silva al, piva e, lima gs, boaro lc, braga rr, martins lr. effect of immediate and delayed light activation on the mechanical properties and degree of conversion in dual-cured resin cements. j oral sci. 2012;54(3):261-6. 23. pereira sg, fulgencio r, nunes tg, toledano m, osorio r, carvalho rm. effect of curing protocol on the polymerization of dual-cured resin cements. dent mater. 2010;26(7):710-8. 24. ozcan m, zamboniota s, valandro f, bottino m, bagis b. microhardness of dual-polymerized resin cement around a translucent fiber post in the intraradicular environment. j conserv dent. 2011;14(4):370-3. 25. sideridou id, achilias ds, karabela mm. sorption kinetics of ethanol/ 149 influence of curing protocols on water sorption and solubility of a self-adhesive resin-cement braz j oral sci. 15(2):144-150 water solution by dimethacrylate-based dental resins and resin composites. j biomed mater res b appl biomater. 2007;81(1):207-18. 26. malacarne j, carvalho rm, de goes mf, svizero n, pashley dh, tay fr, et al. water sorption/solubility of dental adhesive resins. dent mater. 2006;22(10):973-80. 27. malacarne-zanon j, pashley dh, agee ka, foulger s, alves mc, breschi l, et al. effects of ethanol addition on the water sorption/ solubility and percent conversion of comonomers in model dental adhesives. dent mater. 2009;25(10):1275-84. 28. cekic-nagas i, ergun g. effect of different light curing methods on mechanical and physical properties of resin-cements polymerized through ceramic discs. j appl oral sci. 2011;19(4):403-12. 29. giraldez i, ceballos l, garrido ma, rodriguez j. early hardness of self-adhesive resin cements cured under indirect resin composite restorations. j esthet restor dent. 2011;23(2):116-24. 30. yan yl, kim yk, kim kh, kwon ty. changes in degree of conversion and microhardness of dental resin cements. oper dent. 2010;35(2):203-10. 150influence of curing protocols on water sorption and solubility of a self-adhesive resin-cement braz j oral sci. 15(2):144-150 tooth whitening recovers the color of pre-stained composites camila bruscato farinon1, giovanna signori pasqualotto1, kauani carraro1, sinval adalberto rodrigues-junior1 1universidade comunitária da região de chapecó – unochapecó, school of dentistry, health sciences area, chapecó, sc, brazil correspondence to: sinval adalberto rodrigues-junior universidade comunitária da região de chapecó área de ciências da saúde – caixa postal 1141 av. senador atílio fontana, n. 591-e – efapi cep 89809-000 – chapecó – sc – brasil phone: +55 49 3321-8069 e-mail: rodriguesjunior.sa@unochapeco.edu.br abstract aim: to assess the effect of home-whitening on aged and stained composite. methods: fifteen discshaped specimens (10 mm diameter x 2 mm thick) of filtek z350xt, shade a2e were fabricated, polished and embedded in wax, leaving exposed the top surface. the specimens were allocated to three groups (n=5): a – the specimens remained dry; b – conditioning in distilled water and; c – conditioning in distilled water and coffee. they were next subjected to whitening with 10% carbamide peroxide (whiteness perfect) for 4 h daily for 1, 2 and 4 weeks. shade was measured with a spectrophotometer easyshade and expressed based on the cie l*a*b* system (δe*). surface roughness was measured with a roughness meter (ra-µm). data were analyzed with two-way anova and tukey’s test (α=0.05). results: conditioning, grouping and the interaction between both, influenced the δe* (p<0.0001), which exceeded the 3.3 threshold for visible color change after conditioning of group c in black coffee. whitening for 1 week significantly reduced δe* in this group. there were no significant changes in surface roughness. conclusions: home-whitening did not alter significantly the color of the nanocomposite, except when it was previously stained. one-week whitening was sufficient to recover color change to an acceptable level. keywords: tooth bleaching. tooth bleaching agents. dental restoration, permanent. introduction tooth whitening has become a major elective demand for dentists in restorative dentistry nowadays1. usually, the protocol involves advising patients that restored anterior teeth to be whitened would probably have the restoration replaced after tooth whitening, due to discrepancies of color between the recently whitened tooth structure and the restoration2,3. however, it is not uncommon the report of dentists that replacing the restoration was not necessary due to color match with the bleached tooth. based on this observation, it is hypothesized that the resin composite also undergoes color change during tooth whitening. color change in resin composites is well known, either due to intrinsic or extrinsic factors4,5. intrinsic factors involve the hydrolytic degradation and release of constituents, while extrinsic factors involve the incorporation of pigments by staining substances from the diet4. hydrolytic degradation is inherent to dimethacrylate-based composites in contact with a wet environment, leading ultimately to a more porous restoration6. although the increase of porosity of the restoration makes it more prone to staining, water diffusion alone revealed being insufficient to lead to significant, visible color change of a composite restoration4. color change in dentistry has been expressed quantitatively, based on the cie l*a*b* system. this system provides a summarizing measure (δe*) that represents received for publication: july 20, 2016 accepted: november 16, 2016 braz j oral sci. 15(2):163-166 original article braz j oral sci. april | june 2016 volume 15, number 2 http://dx.doi.org/10.20396/bjos.v15i2.8648755 164 the overall color change, and may be classified into three distinct intervals, considering the ability of the human eye to recognize differences in colors: a δe*<1 is considered imperceptible by the eye, while 1 <δe*< 3.3 is considered visible only to a skilled person and δe* > 3.3 is easily detected by anyone, characterizing the color change of a restoration as clinically unacceptable7,8. other studies have confirmed the color changing effect of several extrinsic pigments from dietary sources, including grape juice, red wine and coffee4,8-10. considering the capillarity of the restorative composite to staining molecules, it seems fair to assume that the hydrogen peroxide (h2o2) resulting from the tooth whiteners would also percolate the material’s structure, due to its low molecular weight1. prior studies have shown some evidence of dental composites whitening3,9, but, the extent to which it is whitened and whether this is sufficient to maintain the restoration following tooth whitening still remains uncertain3. on that basis, this study was designed to verify whether tooth whitening alters the color of a universal nanocomposite and to test the hypothesis that significant color alteration after whitening only occurs when the composite contains acquired extrinsic pigments. material and methods fifteen disc-shaped specimens with 10 mm diameter and 2 mm thick were built up in a single increment with the universal composite filtek z350xt (3m/espe, st. paul, mn, usa), shade a2e. the sample size was calculated by g*power 3.1.9.2 (universität düsseldorf, düsseldorf, germany) considering a type i error probability of 0.05, a test power of 95%, one tail and a 3.3 effect size, based on the clinically visible δe*7. the specimens were light cured for 20 s using an ultraled lightcuring unit (dabi atlante, joinville, sc, brazil) with a minimum irradiance of 600 mw/cm2. next, the specimens were polished with diamond pro (fgm, joinville, sc, brazil) sequential discs of decreasing particle sizes at low speed and embedded in white wax, except for the irradiated/polished surface. the specimens were randomly allocated to three groups (n=5), according to figure 1. group a: a total 16-day conditioning period prior to whitening involved water degradation and staining of the specimens. specimens of group a remained dry during this conditioning period for control purposes. group b: the specimens were stored for 9 days in distilled water as a means of imposing hydrolytic degradation. the ageing period was previously determined as required for total water sorption and solubilization of components of a 2-mm thick specimen11. the specimens were kept in distilled water during the next 7-day period. group c: these specimens were conditioned for 9 days in distilled water, similar to group b and then for 7 days in black coffee. after this, dry, degraded and pigmented specimens were submitted to whitening. conditioning occurred at 37oc. following the 16-day conditioning period, the specimens of the three groups were submitted to whitening with 10% carbamide peroxide gel (whiteness perfect, fgm) for 4 h daily during 1, 2 and 4 weeks. between each conditioning and whitening period the specimen’s shade was measured with a spectrophotometer easyshade (vita zahnfabrik, bad säckingen, germany) and expressed based on the cie l*a*b* system. color was measured with the spectrophotometer’s tip in contact and perpendicular to the specimen’s surface. a standard white background was used for all measurements. the cie l*a*b* is a three-dimensional color space. l* stands for lightness and varies from 0 (black) to 100 (white). a* varies between green (negative a*) and red (positive a*) and b* varies between blue (negative b*) and yellow (positive b*). variations between measures throughout different periods generate the deltas (δl*, δa* and δb*) that, ultimately, yield the δe*, which represents the overall color change by the following equation. additionally, the surface roughness was measured with a rp-200 roughness meter (instrutherm, são paulo, sp, brazil), by three parallel readings along 4 mm length with a cut-off setting of 0.8 mm. data were analyzed with two-way analysis of variance and post-hoc tukey test (α=0.05). results variance analysis depicted significant differences in δe* due to conditioning (p<0.0001), to grouping (p<0.0001) and to the interaction between both (p<0.0001). color change was highly significant following conditioning of group c in black coffee, exceeding the 3.3 threshold for visible color change. such color change returned to acceptable levels after whitening for 1 week and kept decreasing during the two-week and four-week whitening (table 1 and figure 1). this reflected the significant decrease of l* (darkening) and increase of b* tooth whitening recovers the color of pre-stained composites braz j oral sci. 15(2):163-166 δeab = √(l2‒l1) 2 + (a2‒a1) 2 + (b2‒b1) 2* * * ** * * fig.1. study flow diagram involving conditioning and whitening procedures and times. 165 (yellowing) following storage of specimens of group c in black coffee (figure 1-a and c). recovery of lightness was observed following one-week whitening, while significant reduction of yellowing occurred only after four-week whitening. to what occurs in the clinical situation13. the susceptibility of the composite to percolation of staining substances is related to its chemical composition, which includes the type of monomer, the type of inorganic filler, the used photoinitiator and the quality of the matrix/filler interface14. it is also modulated by its degree of conversion and network characteristics, since a low degree of conversion leads to degradation of the polymer network and release of byproducts6,12,15,16. the nanocomposite used in this study is recommended to both anterior and posterior restorations and has been characterized elsewhere17, revealing a degree of conversion of 46% on the top surface and of 38% on the bottom surface of a 2 –mm thick specimen. according to the authors, light-scattering effects of the nanoclusters could possibly explain the reduction of degree of conversion in depth in this composite17. conditioning in distilled water for nine days generated a δe* of 1.2 for all groups. interestingly, specimens from group a, which remained dry during this conditioning period, also had the same color change as the ones stored wet. also, color change was below the 3.3 threshold and within the interval of detection only by a skilled person, meaning that an insignificant color change to the patient’s eyes occurs due to hydrolytic degradation4,9. according to mendes et al.14 (2012), degradation of the composite is a modulating factor for the uptake of extrinsic pigments. water sorption and swelling affect the total volume of the restoration and its porosity12,15, enabling the uptake of staining solutions. conditioning of the specimens from group c in coffee following degradation, significantly affected δe* as a reflection of yellowing (significant increase of b* color coordinate) and darkening (significant reduction of l* color coordinate) (table 1). staining would probably be higher if the other surfaces of the specimen were not protected with wax. even so, an easily perceptible 6.3 δe* was observed in group c as a consequence of conditioning in coffee. as to the specimens from groups a and b, stored dry and wet, respectively, no significant color change was observed. the staining potential of coffee has been attributed to the presence of yellow colorants4, which could be confirmed by the significant alteration in the b* axis. garoushi et al.18 (2013) observed that staining solutions not containing yellow colorants presented a lower staining potential. bagheri et al.19 (2005) noticed that color changes higher than 3.3 are often associated to darkening and yellowing, the l* component being the most significant for color change. in our study, lightness was recovered with one-week whitening, while yellowing took four weeks to reduce significantly. this may be explained by the whitening mechanism of hydrogen peroxide that breaks dark complex pigment molecules into simpler structures with lighter optical characteristics. therefore, lightness is more quickly achieved than the recovery of yellowing. whitening with 10% carbamide peroxide for one week significantly reduced color change from 6.3 to 2.8, and kept reducing it during the subsequent weeks to levels similar to the groups that were not stained (table 1). the diffusion of h2o2 across enamel and dentin is a well-recognized phenomenon1,20 that occurs based on the fick’s second law of diffusion, considering enamel and dentin as semipermeable membranes and several other variables, such as the surface area, the diffusion coefficient, the concentration and the diffusion distance1. water sorption by composites is also known to respect the same laws of diffusion12; nevertheless, little is known tooth whitening recovers the color of pre-stained composites table 1 results of l*, a*, b*, δe* and average surface roughness (ra µm) (mean and sd) after conditioning and whitening. groups conditioning group a group b group c baseline l* 79.3 (1.6)a 79.0 (0.5)a 78.9 (0.9)a a* 0.3 (0.2) 0.1 (0.3) -0.1 (0.2) b* 19.9 (1.3)b 19.4 (0.3)b 19.0 (0.8)b ra 0.204 (0.064) 0.298 (0.164) 0.244 (0.102) conditioning step 1† l* 78.8 (1.8)a 79.7 (0.7)a 79.8 (0.8)a a* -0.1 (0.2) 0.5 (0.3) 0.2 (0.2) b* 18.9 (1.3)b 20.2 (0.4)b 19.7 (0.9)b δe* 1.2 (0.5)bcd 1.2 (0.3)bcd 1.2 (0.5)bcd ra 0.214 (0.075) 0.301 (0.149) 0.236 (0.090) conditioning step 2‡ l* 78.9 (2.0)a 78.7 (0.6)ab 76.1 (1.3)c a* 0.1 (0.4) 0.7 (0.2) 0.6 (0.6) b* 19.8 (1.6)b 19.4 (0.5)b 24.6 (2.0)a δe* 1.1 (0.6)cd 0.7 (0.2)d 6.3 (2.2)a ra 0.198 (0.068) 0.220 (0.047) 0.261 (0.109) whitening 1 week l* 78.2 (1.6)ab 78.2 (1.2)ab 77.7 (0.9)ab a* 0.6 (0.2) 1.2 (0.2) 0.8 (0.4) b* 19.5 (1.5)b 19.5 (0.4)b 21.3 (1.2)ab δe* 1.3 (0.4)bcd 1.4 (0.3)bcd 2.8 (0.9)b ra 0.218 (0.069) 0.222 (0.050) 0.235 (0.074) whitening 2 weeks l* 78.7 (1.5)ab 78.4 (0.3)ab 78.6 (0.7)ab a* 1.2 (0.4) 1.3 (0.3) 1.0 (0.3) b* 19.9 (1.6)b 19.6 (0.3)b 21.1 (1.0)ab δe* 1.3 (0.4)bcd 1.4 (0.2)bcd 2.5 (0.6)bc ra 0.195 (0.073) 0.222 (0.034) 0.254 (0.058) whitening 4 weeks l* 78.4 (1.7)ab 78.8 (0.6)a 78.0 (0.6)ab a* 1.3 (0.3) 1.6 (0.2) 0.9 (0.3) b* 19.2 (1.7)b 19.2 (0.4)b 20.2 (0.5)b δe* 1.7 (0.4)bcd 1.6 (0.2)bcd 2.0 (0.4)bcd ra 0.204 (0.073) 0.205 (0.040) 0.261 (0.092) † immersion of specimens of groups b and c in water for degradation means. specimens of group a were kept dry. ‡ immersion of specimens of group c in coffee for staining. specimens of group a were kept dry and of group b were kept in water. different letters in the row represent statistically significant difference between groups. no significant changes in surface roughness were observed with either conditioning or whitening procedures (p>0.05) (table 1). discussion this study was designed to test the hypothesis that whitening of the composites occurs following a degradation period and the incorporation of extrinsic staining molecules. the degradation period of 9 days was based on prior calculation that considered the time for water sorption in a 2-mm thick specimen11. according to ferracane12 (2006), clinically placed restorations take up to two months to fully saturate with solvent and leach unconverted components. other surfaces than the irradiated/polished surface were protected in wax, exposing a single way of percolation through the specimen, similar braz j oral sci. 15(2):163-166 166 about the diffusion of h2o2 into previously degraded composite restorations, an issue that demands further research. still, one could observe significant whitening of the stained specimens. the whitening mechanism, as it is understood up to now, involves the release of reactive oxygen and its interaction with high molecular weight organic chromophore molecules breaking them into simpler structures with final optical properties that reduce the appearance of stain1,9,21. this was clearly observed in specimens of group c. however, the lack of significant color change in the specimens from groups a and b reinforces the hypothesis that whitening of composites occurs by removal of extrinsic stain, not to intrinsic color change9,22. no significant change in surface roughness was observed throughout the treatment conditions, in either group. expectations were that degradation would lead to solubilization of non-converted monomers, oligomers, catalysts, silane and other by-products, which would expose and dislodge filler particles, increasing surface roughness6,12. this would lead to a more porous surface rising the percolation way for either the staining solution or the whitener. however, neither conditioning in water, nor in the staining solution, nor the whitening procedure were sufficient to increase surface roughness, similar to other studies2,14. prior studies have shown that the effect of whiteners on restorative materials are material-dependent3,9, and vary according to the material’s composition and microstructure. the constituents of the composite generate a tridimensional microstructure that involves the polymer matrix, the inorganic filler and the matrix/ filler interface17. hydrolytic degradation of the matrix or the matrix/ filler interface generates percolation paths to both staining molecules and whitener. according to fontes et al.8 (2009), the presence of specific monomers, like tegdma, make the composite more prone to degradation. li and wang3 (2009) also claim that if the composite is highly cross-linked by high-molecular weight polymer molecules, the whitener may require more time to diffuse through the material. in our study we focused on a single, extensively studied and well-known nanofill universal composite, which may produce unique interactions with water, the staining solution and specially with the tooth whitener. therefore, our results should be regarded with caution, not extrapolated to other tooth-coloring materials. this study also attempted to simulate clinical whitening by a home-whitening protocol based on 10% carbamide peroxide and revealed that a 3.35% hydrogen peroxide release1 during a 4-hour/day period was sufficient to recover color change of the composite following staining with coffee. based on that, one could infer that a clinical in-office whitening protocol based on 35% hydrogen peroxide would easily reach the same results. the 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ecv, gacia lfr, hebling j, souza costa ca. effective tooth-bleaching protocols capable of reducing h2o2 diffusion through enamel and dentin. j dent. 2014 mar;42(3):351-8. doi: 10.1016/j. jdent.2013.09.001. 21. durner j, obermaier j, ilie n. investigation of different bleaching conditions on the amount of elutable substances from nano-hybrid composites. dent mater. 2014 feb;30(2):192-9. doi: 10.1016/j.dental.2013.11.003. 22. el-murr j, ruel d, st-georges aj. effects of external bleaching on restorative materials: a review. j can dent assoc. 2011; 71:b59. tooth whitening recovers the color of pre-stained composites braz j oral sci. 15(2):163-166 untitled 1 volume 16 2017 e170082 original article 1 msc. graduate program in clinical dentistry, positivo university, curitiba, pr, brazil. 2 phd. professor of the graduate program in clinical dentistry, positivo university, curitiba, pr, brazil. 3 phd student in graduate program in clinical dentistry, positivo university, curitiba, pr, brazil. 4 master´s student in graduate program in clinical dentistry, positivo university, curitiba, pr, brazil corresponding author: tatiana miranda deliberador rua professor pedro viriato parigot de sousa, 5300 campo comprido, cep: 81280-330, curitiba/pr, brasil e-mail: tdeliberador@gmail.com phone: + 55 41 99764948 fax: + 55 41 33173403 received: july 26, 2017 accepted: october 20, 2017 the influence of the protein wnt10b as a marker of bone repair of critical size defects fille with autogenous adipose tissue graft: a study in rabbit calvaria luiz gustavo klug 1 , carmen lucia mueller storrer 2 , aline monise sebastiani 3 , bruna lorena barbaresco, allan fernando giovanini 2 , tatiana miranda deliberador 2 the proteins wnts are considered a key regulator of the early development of the skeleton. aim: the aim of this study was to evaluate the presence of the protein wnt10b as a marker of bone repair in critical size defects surgically created in the calvaria of rabbits treated with fragmented autogenous adipose tissue graft. methods: a total of 28 rabbits were divided into two groups: the control group (c) and adipose tissue graft group (atg). a csd measuring 15 mm in diameter was created in the calvaria of each animal. in rabbits of the c group, the defect was filled only with blood clot, and in atg group, the defect was filled with fragmented adipose tissue graft. the two groups were divided into two subgroups (n = 7) for euthanasia 15 and 40 days after surgery. histological and immunohistochemically analyses were performed to evaluate the neoformed bone and the presence/concentration of wnt10b protein. the kruskal-wallis test was performed to compare the means and standard deviations of the number of wnt10b + cells/mm2 in both groups in each postoperative period. it was assumed a significance level of 5%. results: after 40 days, the mean concentration of the protein wnt10b in atg group was 26.26 (+-6.97) significant higher (p<0,001) than the mean in c group that was 305 (37.41). conclusion: the protein wnt10b would play a crucial role in the signaling of bone formation in bone defects treated with fragmented autogenous adipose tissue graft. keywords: adipose tissue. bone regeneration. rabbits. wnt proteins. http://dx.doi.org/10.20396/bjos.v16i0.8651053 mailto:tdeliberador@gmail.com 2 klug et al. introduction the growing occurrence of bone disorders and the increase in aging population have resulted in the need for more effective therapies to meet this request. bone tissue engineering strategies, by combining biomaterials, cells, and signaling factors, are seen as alternatives to conventional bone grafts for repairing or rebuilding bone defects1. the successful repair of bone deficiencies still remains a great challenge to surgeons worldwide2. recently, the adipose-derived mesenchymal stem cells have raised a lot of attentions in bone regeneration owing to their easy accessibility and availability in relatively large quantities3. adult stem cells derived from adipose tissue are multipotent cells that can differentiate into osteoblasts in vitro4. when these cells are combined with appropriate scaffolds, they can contribute towards bone neoformation in critical size bone defects. in 2011, zou et al.5 have demonstrated that cells derived from adipose tissue have the potential to differentiate into an osteogenic lineage both in vitro and in vivo. the authors stated that this new type of cell-based therapy can be useful for the treatment of bone defects in the clinical field. to understand bone signaling when adipose tissue graft is used, it is important to know that a signaling mechanism occurs by means of cell markers, such as the wnt/β catenin. this marker signals the control pathway for the differentiation of various cells because it regulates the expression of target genes. a study in vitro has shown that the wnt/β catenin signaling pathway inhibited the adipogenic differentiation potential and altered the cell fate from adipocytes to osteoblasts6. an in vivo prior study demonstrated that the expression of wnt 10b in the bone marrow of mice resulted in an increase in the metaphyseal bone volume, which made the bone mechanically stronger7. wnt signaling pathways are a key regulator of the early development of the skeleton. the activation of the canonical pathway results in the stabilization of β-catenin. the suppression of wnt target genes occurs in the absence of β-catenin. therefore, β-catenin can be considered an important molecule that regulates the canonical wnt signaling pathway to embryogenesis8. in a genetic study, the inactivation of β-catenin, in mesenchymal lineage cells, resulted in severe loss of bone mass due to reduced maturation of osteoblasts as well as increased differentiation of osteoclasts9. in addition, an increased wnt signaling pathway, through a stabilized form of β-catenin, can produce strengthened ossification as well as the suppression of chondrogenesis9,10. although the wnts may inhibit adipocyte differentiation through β-catenin dependent or independent mechanisms, there is some evidence that the wnt/β-catenin signaling pathway can be considered an important regulator in the adipocyte differentiation11. there are more than 19 proteins and more than 19 receptors and co-receptors, which combine to activate pathways. the proteins wnt 1, 2, 3, 3a, 8a, 8b, 10a e 10b are proven by the canonical wnt/β-catenin; wnt 4, 5b, 6, 7, 7b and 11 are responsible for activation of the wnt/ca2+ pathway which elevates the cytoplasmic levels of ca2+ and as wnt 5a and 11 proteins via planar cell polarity (pcp) responsible for the cytoskeletal reorganization12,13. the aim of this study was to evaluate the presence of the protein wnt10b as a marker of bone repair in critical size defects surgically created in the calvaria of rabbits treated with fragmented autogenous adipose tissue graft. 3 klug et al. materials and methods this study was approved by the ethics and research committee from positivo university, curitiba-pr, brazil (protocol number: 009/2009). a total of 28 male rabbits oryctolagus cuniculus (new zealand strain), aged approximately 6 months and weighing from 2.0 to 3.5 kg were used. the animals were healthy and had a period of 15 days to adapt themselves to the environment. the rabbits were kept in individual cages at the positivo university biotery. they were given commercial feed and mineral water ad libitum. the animals were randomly divided into two groups of 14 animals: c group (control) and atg group (fragmented autogenous adipose tissue graft). one hour before surgery, rabbits received intramuscular injection of enrofloxacin (0.1 ml / kg) (baytril, bayer s.a., são paulo, sp, brazil). for the surgical procedure, all the animals received as preanesthetic medication: 1 mg/kg of midazolam (união química, são paulo, sp, brazil) administered intramuscularly. after sedation, the animals received an anesthetic medication containing a combination of 35 mg/kg of 10% ketamine hydrochloride (syntec, coria, sp, brazil) with 5 mg/kg of 2% xylazine hydrochloride (vetbrands, brazil) intramuscularly. after induction of the anesthetic plan, the venous access was established by the marginal vein of the ear for anesthetic maintenance during the surgical procedure. the rabbit was then positioned on the surgical table in dorsal decubitus position. the anesthesia was maintained by the vaporization of isoflurane (cristália, itapira, sp, brazil) by facial mask. critical-size defects (csd) measuring 15 mm were created in the calvaria of each animal according to a research methodology described by oliveira et al.14. in c group, the defect was filled only with blood clot, and in atg group, the defect was filled with fragmented autogenous adipose tissue graft. the method used to obtain autogenous adipose tissue was described by torres et al. [28]. an incision measuring between 2 and 3 cm was performed with a scalpel blade 15, about 5 cm posterior to the skull base, along the dorsomedial line, towards the craniocaudal direction. the adipose tissue was carefully removed from the exposed area with sterile scissors and tweezers (figure 1). then, it was macerated with a scalpel blade 15, through random cuts in different directions, and immediately placed in the surgical defect. figure 1. incision and exposure for the removal of adipose tissue from animal’s dorsal region. 4 klug et al. the soft tissues were then repositioned and sutured for a primary closure of the wound. the suture was performed in two layers. in the first layer, the periosteum was sutured with absorbable suture thread (4-0 medcryl, medevolution, tamil nadu, india). in the second layer, dermis and epidermis were sutured with 4-0 silk suture thread (ethicon, johnson & johnson, são josé dos campos, sp, brazil). each animal was given intramuscular injections of enrofloxacin (5 ml per kg of body weight) once a day for five days. to control postoperative pain, the animals were given morphine (3mg/kg intramuscular) at the end of the surgery and 4 hours after the first injection. analgesia was maintained with paracetamol dissolved in water (200 mg/kg) 3 times a day for 5 days. the c and atg groups were divided into two subgroups with 7 animals for euthanasia in 15 and 40 days’ time after surgery. the euthanasia was performed through a fast intravenous injection of thiopental sodium 2.5% (10 ml/kg). tissue processing the original surgical defect area and its surrounding tissue were removed in blocks. the specimens were fixed in 10% neutral formalin, washed in tap water, and decalcified in 20% formic acid. after decalcification, each specimen was hemi-sectioned in its core, parallel to the sagittal suture. the specimens were processed and embedded in paraffin. longitudinal serial sections, 3 µm thick each, were performed, starting from the core of the original surgical defect. the sections were stained with the technique of hematoxylin and eosin (he) for posterior analyses with light microscopy. histological analysis two histological sections were selected from each animal, representing the core of the original surgical defect. these analyses were performed by a single operator previously calibrated. for histological analysis, the images were analyzed in 12x and 40x magnification by means of an optical microscope (021/3 quimis, diadema, sp, brazil) and the following parameters were assessed: the closure of the bone defect, the amount of the newly bone matrix deposition, a quantity of presence of dense connective tissue, the presence of acute (neutrophylic) or chronic (mononuclear) inflammatory infiltrate. the closure of the defect was considered complete when all its extensiveness was filled with newly formed bone. each parameter was considered such as negative (-), scarce (+), moderated (++) and intense (+++). immunohistochemical analysis the immunohistochemical antiperoxidase was performed with the use of the protein wnt 10b. the immunoexpression of the protein was obtained through the method streptavidin-biotin immunoperoxidase. three-micrometer-thick cuts of each specimen were deparaffinised and submitted to antigen retrieval in 1% trypsin solution (ph 6.8) for 60 minutes at 37-c for the anti-wnt 10b and 10 mm citrate solution (ph 6.0) for 45 minutes in a double-boiler at 95-c for the anti-cd34 antibody. the slides containing the histologic specimens were immersed in 3% hydrogen peroxide for 30 minutes 5 klug et al. to extinguish endogenous peroxidase activity, followed by incubation with 1% phosphate-buffered saline (ph 7.4). the sections were incubated with the primary antibody anti-wnt 10b (0.5 mg/ml, ab 6285; abcam, cambridge, uk) with a dilution factor of 1:100. a labelled streptavidin biotin antibodybinding detection system (universal hrp immunostaining kit; diagnostic biosystem, foster city, ca) was used to detect primary antibodies. the immune reaction was revealed with diaminobenzidine tetrachloride chromogen solution (diagnostic biosystem), which produced a brown precipitate at the antigen site. the specimens were counterstained with mayer hematoxylin. a negative control was performed for all samples using rabbit polyclonal isotype igg (2 kg/ml, ab 27472, abcam) for 10 minutes at room temperature as a primary antibody. for each specimen, 3 slides were used for incubation with each antibody. the images of each field were obtained with a light microscope with a magnification of x 400 through bright-field fixed focus. then, the images displayed on a tv monitor connected to a computerized system were scanned by a system of cameras (samsung sdc-310, korea) attached to a light microscope (021/3 quimis, diadema, sp, brazil). after that, a manual counting of the nuclei that expressed immunostaining of the protein was performed. the immunopositive cells for the antibody wnt 10b were counted twice in every surgical defect by a single observer. for the statistical analysis of the immunohistochemical expression of the antigen investigated, the results were expressed as a percentage, taking into account the relation between the number of positive cells and the number of total cells showing mesenchymal morphology. statistical analysis the data were submitted to descriptive and inferential statistical analysis. the non-parametric statistical test of kruskal-wallis was performed to compare the mean and standard deviations of the number of wnt10b + cells/mm2 between groups. was assumed a significance level of 5%. all histological parameters were analyzed by moda. results histological analysis the histometric aspects found in the present study is revealed in the table1, the qualitative results is summarized bellow. no amount of newly formed bone was observed. table 1. histometric aspects found in histological analysis parameter analyzed 15 days 40 days c atg c atg closure of the bone defect + acute (neutrophylic) inflammatory infiltrate chronic (mononuclear) inflammatory infiltrate + presence of connective tissue +++ +++ ++ + bone matrix + +++ 6 klug et al. group control (c) 15 days after surgery: there was the presence of chronic inflammatory infiltrate amidst thin dense connective tissue whose fibers were arranged parallel in the anteroposterior direction (figure 2-a). no amount of newly formed bone was observed. group control (c) 40 days after surgery: only scarce osteoid matrix were identified mainly on surgical defect, but there was one case in which the bone formation occurred in the core areas of the defect as well (figure 2-c). the defect was composed by intense dense connective tissue. group fragmented adipose tissue graft (atg) 15 days after surgery: a complete bone closure of the surgical defect was not observed in this period. the reparative histological analysis showed the presence of dense connective tissue with figure 2. a) microscopic view of c group c in the postoperative period of 15 days. this view shows an intense deposition of collagen fibers (cf) parallel to the defect. (he staining, original magnification × 40). b) microscopic aspect of atg group in the postoperative period of 15 days. microscopy reveals remaining adipose tissue (at) surrounded by osteoid tissue and intense deposition of newly formed compact bone (cb) tissue encircling the defect area. (he staining, original magnification × 40 in). c) microscopic view of group c in the postoperative period of 40 days. there was neoformation of compact bone (cb) mimicking areas of intramembranous formation during this period. a large area of the defect was predominantly filled with dense connective tissue. (he staining, original magnification × 40). d) microscopic aspect of group atg in the postoperative period of 40 days. the microscopic view shows intense deposition of mature compact bone (cb) tissue filling a large part of the artificial defect. most adipose tissue (at) was replaced by bone tissue or loose connective tissue (lct), mimicking the bone marrow extracellular matrix. (he staining, original magnification × 40). a b c d cf cb cb control group 15 days 40 days adipose tissue graft group cb cb lct at at cb 7 klug et al. bundles of collagen fibers amidst the remaining adipose tissue, sometimes encircling the tissue. these fibers also had a pale coloring that characterizes the formation of osteoid structure, in which isolated fragments of mature newly formed tissue were found. newly formed bone tissue was evident in the margins of the defect. the tissue thickness was similar to the original bone volume of the skullcap (figure 2-b). group fragmented adipose tissue graft (atg) 40 days after surgery: the results were heterogeneous among the specimens. some specimens had a larger amount of osteoid tissue while others had areas filled with newly formed compact haversian bone tissue, not only surrounding the defects but also amidst the bundles of collagen fibers. discrete adipose tissue remaining in the center of the defect could be seen as well (figure 2-c). immunohistochemical analysis of the protein wnt 10b all specimens showed the presence of wnt10b + cells. the atg group showed intense positivity to cells/mm2 in areas with dense connective tissue, osteoid tissue, and configure 3. a) immunohistochemical aspects of control group in the postoperative period of 15 days. verify the presence of wnt10b localized on specific stromal area (arrows) b) immunohistochemical aspects of atg group in the postoperative of 15 days. there are important presence of wnt10b on nucleus of cells spread on fibrous tissue (head of arrow) and immunopresence of wnt10b in mineralization areas surrounding the adipose tissue (arrows) e) immunohistochemical aspects of control group in the postoperative of 40 days. it shows intense presence of dense connective tissue surrounding scarce area of osteoid wnt10b + (arrows) f) immunohistochemical aspects of atg group in the postoperative of 40 days (original magnification × 40). verify the intense presence of wnt10b + cells peripherally to bone matrix deposition on deffects (arrows) and also in the conective tissue (head of arrows). a b c d control group 15 days 40 days adipose tissue graft group 8 klug et al. nective tissue septa. in c group, a greater amount of positive cells was found in the edges of the defect. in c group, the amount of positive cells/mm2 remained regular in both periods, reaching an immunostaining peak in the postoperative period of 15 days (figure 3-a). in the period of 40 days, these values decreased coinciding with the deposition of bone neoformation (3-c). the atg group showed in postoperative of 15 days, intense positivity in the formation of osteoid tissue among the adipose cells (notched arrows) and positivity of fat cells to the protein analyzed, suggesting genetic reprogramming of mature cells. in this group, there was a crescent number of cells that showed positivity to wnt10b in relation to the period of time analyzed, even when the bone matrix was deposited (figure 3-b,d). the table 02 compare the means of wnt10b proteins concentrations in the groups. discussion bone repair is a complex biological process characterized by controlled bone recovery, through osteogenesis and osteoinduction, so that the functional and structural characteristics of injured tissue can be restored in the areas where there is a bone defect15. we chose to perform a critical size defect of 15 mm according to the study of maiti in 201616, because we believe it is a defect size sufficient to hinder bone formation by the normal cicatrization process from the clot. allowing the comparison of different levels of bone formation to correlate with the presence of the wnt10b protein . an ideal grafting material for bone repair is still a big challenge, and object of various researches. adipose tissue has proven to be rich in adult stem cells, and as a result, it has been the focus of extensive research on the regeneration of lost tissue17. therapies based on stem cells for the repair and regeneration of different kinds of tissue and organs have been considered, at least in theory, a therapeutic alternative with satisfactory solutions for a great number of diseases. stem cells have been widely used in experimental trials in order to develop new techniques for the repair of bone tissue18. in this regard, the autogenous adipose tissue can theoretically serve as an important and abundant source of multipotent adult stem cells, since it contains approximately 100 times more stem cells than the bone marrow do, with suitable properties for tissue engineering and tissue regeneration with orthopedic purposes19. moreover, new findings show that subcutaneous adipose tissue and bone marrow tissue share the same stem cell phenotype. studies have demonstrated the intimate relation between these two types of tissue (since bone and fat have the same cell clone). in addition, adipose tissue cannot only be genetically reprogrammed but can also transdifferentiate into bone tissue20. table 2. means and standard deviations of the number of wnt10/β + cells/mm2 in both groups in each postoperative period. postoperative period groups group c group atg 15 days 71.93 ± 22.28 bcd 189.08 ± 28.36 ab 40 days 26.23 ± 6.97d 305.33 ± 37.41 a according to the non-parametric statistical test of kruskal-wallis, the number followed by the same letter shows statistical similarities (p <0.05). 9 klug et al. our data suggest that the subcutaneous fat tissue graft could be a plausible osteoconductive biomaterial. the outcome has shown that the use of fat tissue increased the volume of tissue inside the bone bed providing a satisfactory bone formation. however, there is a need for new studies with histomorphometric analysis comparing fat graft with autogenous graft and with bone substitutes. when adipose tissue grafting was used (group atg), the histological results showed moderate bone deposition in the peripheral regions of the defect delimiting periosteal and endosteal areas, mimicking a ‘sandwich’ of intramembranous bone formation, and trabecular formation, with areas of de neovascularization as well as a reorganization of adipose tissue in bone graft areas. later, a higher bone density (in the period of 40 days) and substantial loss of grafted adipose tissue could be observed. thus, there were two independent, distinct processes of bone neoformation in group atg and group c. in group c, the bone neoformation was achieved more slowly and more concentrated around the remaining bone area (the edges of the defect), where there were medullar fragments and periosteum. the ossification process occurred in the regions where there was a higher concentration of cells marked by wnt10b. nowadays, it is well-known that regulatory factors that induce and control the development of skeletal structures arise from regulatory factors that include the proteins wnts and all cascades of events produced by their presence9. in this work, mineralization and bone tissue formation occurred primarily in the regions of fibrous matrix associated with fusiform-shaped cells, which also comprised the wnt10b protein. the expression of this protein was identified by bennett et al.7 in cell lineages inside the bone marrow of mice during a process of bone remodeling. the authors identified a direct correlation between the presence of the protein and the growth of the metafisial bone volume. furthermore, the authors also identified that the bone that had larger amounts of wn10b was characterized by a mechanically stronger bone tissue21. a study in vitro has shown that the wnt/β-catenin signaling pathway inhibited the adipogenic differentiation potential and altered the cell fate, from adipocytes to osteoblasts. the activation of the wnt/β-catenin signaled a cell fate change, from adipose-tissue derived mesenchymal stem cells to osteoblastogenesis, at cost to adipogenesis. our results presented in this study showed, in the initial periods, higher amounts of this protein concentrated in cells that were lodged in areas with connective tissue adjacent to grafted adipose tissue. later, this protein was also expressed in mature adipose tissue, which decreased in volume while the bone matrix increased. therefore, the expression of wnt10b appears to be an important path towards osteogenic differentiation when adipose tissue is grafted onto a craniofacial bone site. this result suggests a hypothesy of mineralization through cell transdifferentiation22. the term transdifferentiation refers to a phenotypic change from one kind of differentiated cell to another kind that is functionally different in terms of lineage. studies in vitro have demonstrated that such transdifferentiation occurs mainly between adipocytes and osteoblasts due to growth and hormone factors, similarly to the effect of osteoporosis, or “pseudo-osteoporosis”23. studies conducted by gimble and guilak22 have demonstrated that the transdifferentiation phenomenon has important therapeutic implications, though it should be 10 klug et al. regarded with caution since a prior commitment to differentiation, from one or another particular lineage, is a random event. however, this random aspect seems not to exist when subcutaneous adipose tissue is grafted onto rabbit calvaria, since the increase in bone tissue and in osteoid tissue was also associated with the increase in wnt10b. the concentration of the protein wnt10b in atg group was significant higher, comparing with c group, suggesting that this protein could play a role in the signaling of bone formation in bone defects treated with fragmented autogenous adipose tissue graft. new studies with measurement of newly bone formed area to compare with histological and immuhistochemical data should be perform to comprove this hypothese. references 1. orciani m, fini m, di primio r, mattioli-belmonte m. biofabrication and bone tissue regeneration: cell source, approaches, and challenges. front bioeng biotechnol. 2017 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adipose-derived stem cells (adscs): current and future applications. j plast reconstr aesthet surg. 2010 nov;63(11):1886-92. doi: 10.1016/j.bjps.2009.10.028. 18. trofin ea, monsarrat p, kémoun p. cell therapy of periodontium: from animal to human? front physiol. 2013 nov 15;4:325. doi: 10.3389/fphys.2013.00325. 19. gomes sp, deliberador tm, gonzaga cc, klug lg, oliveira lc, urban cd, et al. bone healing in criticalsize defects treated with immediate transplant of fragmented autogenous white adipose tissue. j craniofac surg. 2012 sep;23(5):1239-44. 20. gomillion ct, burg kjl. stem cells and adipose tissue engineering. biomaterials. 2006 dec;27(36):6052-63. 21. longo ka, wright ws, kang s, gerin i, chiang sh, lucas pc, et al. wnt10b inhibits development of white and brown adipose tissues. j biol chem. 2004 aug 20;279(34):35503-9. 22. gimble j, guilak f. adipose-derived adult stem cells: isolation, characterization, and differentiation potential. cytotherapy. 2003;5(5):362-9. 23. song l, tuan rs. transdifferentiation potential of human mesenchymal stem cells derived from bone marrow. faseb j. 2004 jun;18(9):980-2. untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8652637 volume 17 2018 e18027 original article 1 dds, department of restorative dentistry, faculty of dentistry, federal university of alfenas (unifal-mg), alfenas mg, brazil. 2 dds, msc, phd, department of dental materials and prosthodontics, aracatuba dental school, são paulo state university (unesp), araçatuba – sp, brazil. 3 dds, msc, phd, department of oral surgery, pathology and clinical dentistry, school of dentistry, federal university of minas gerais (ufmg), belo horizonte, mg, brazil 4 dds, msc, phd, department of restorative dentistry, faculty of dentistry, federal university of alfenas (unifal-mg), alfenas mg, brazil. corresponding author: marcela filié haddad department of restorative dentistry and prosthodontics, faculty of dentistry, federal university of alfenas (unifal-mg) rua gabriel monteiro da silva, 700, centro, alfenas – mg, brazil. cep: 37.130-000 phone / fax: (35) 3299-1464 e-mail: marcela.haddad@unifal-mg.edu.br received: october 02, 2017 accepted: march 28, 2018 effect of different methods of hygiene on the color stability of extrinsically pigmented facial silicone yasmin morais cabral1, aldiéris alves pesqueira2, amália moreno3, marcelo coelho goiato2, marcela filié haddad4 aim: evaluate the color stability of facial silicone pigmented extrinsically under the influence of the hygiene process. methods: 160 samples were prepared and divided into 8 groups (n = 20) according to the pigmentation technique used: group 1: colorless silicone; group 2: pigmented exclusively with oil ink; group 3: pigmented with oil ink + opacifier; group 4: colorless, applying prime; group 5: pigmented with oil ink covered with prime; group 6: pigmented with oil ink + opacifier and covered with prime; group 7: pigmented with oil ink diluted in prime; group 8: pigmented with oil ink + opacifier diluted in prime. then the samples were distributed into two subgropus (n=10): 1: neutral soap and 2: 1% hypochlorite solution. the color readings occurred in the initial period and 60 days after the hygiene procedures. for this, it was used a spectrophotometer reflection and cie-lab program. the data was tabulated and submitted to analysis of variance (anova) followed by tukey test (p <0.05). results: the groups of samples disinfected with soap showed significantly lower color change values than those presented by the samples disinfected with hypochlorite. the best results were presented by the group of samples pigmented with oil ink diluted in prime and sanitized with neutral soap (δe=1.21, without opacifier and δe=0.82, with opacifier). conclusions: the association of oil ink diluted in prime and hygiene technique with soap promotes the lower color change of facial silicone pigmented extrinsically. keywords: maxillofacial prosthesis, prosthesis coloring, disinfection. 2 cabral et al. introduction losses in the maxillofacial region are very embarrassing to the patients because they affect a region of the body so important for the interpersonal relationship, where there are contained organ of the senses, necessary for the interaction of the individual with the neighbor and with the environment. thus, the loss of such organs often leads mutilated individuals to social exclusion. maxillofacial prostheses aim to rehabilitate individuals who have suffered loss of structures in the maxillofacial region and that can not be surgically corrected, restoring lost esthetics and partially restoring function1. the material of choice for making these prostheses is facial silicone, which is available in the market in colorless form and must be pigmented in order to mimic the patient’s skin. the pigmentation of maxillofacial prostheses is carried out basically by the use of powdered or oil-based pigments associated or not with opacifiers2. there are two pigmentation techniques: the intrinsic technique, in which the pigments are added to the silicone prior to their polymerization in order to provide an uniform color base; and the extrinsic one, which consists of the characterization of the prostheses through the application of a thin layer of paint on the surface of the pigmented silicone that is intrinsically and already polymerized, in order to reproduce spots and small vessels present in the skin3. using these two techniques of pigmentation combined with the technical and artistic skill of the professional and considering the advancement of materials available for making maxillofacial prostheses, it is possible to obtain satisfactory aesthetic results, however, such results can not be maintained for the long term, since the prostheses undergo color changes in a short period of time, implying the need for retouching of extrinsic pigmentation or even making a new prosthesis2. it is a consensus in the literature that the main causal factor for the color change of maxillofacial prostheses is the exposure to climatic intemperatures (temperature variation, humidity and ultraviolet radiation exposure). however, clinically, it is possible to notice changes in the extrinsic pigmentation pattern due to the patient’s constant handling of the prosthesis and its exposure to the hygiene process with different substances and techniques. deficiency in hygiene of facial prostheses contributes to the underlying tissues being susceptible to infections. thus, it is of fundamental importance to perform the disinfection of the prostheses to maintain the health of the tissues. however, one should keep in mind the possible changes that this disinfection may cause in the physical properties of facial silicone4. maxillofacial prostheses hygiene is little explored in the literature in clinical studies. there are researches that evaluate the action of substances commonly used for complete dentures hygiene (such as neutral soap2,4-9, hypochlorite7, chlorhexidine5, effervescent tablets2,4,8,9, ethanol6, microwave energy7 and plant extracts5) and the methods of hygiene (immersion2,4-6,8,9 or friction 2,4-6,7-9) on physical properties of non-pigmented or pigmented intrinsically with different substances (makeup powders8,10, ceramic pigments8,10,11, oil paint1 and/or opacifiers9) used in the manufacture of facial prostheses. 3 cabral et al. the literature presents studies that evaluate the amount of pigment boundary that can be used for the intrinsic pigmentation of facial silicones12; which pigments are more stable chromatically, what physical properties of the silicone can be altered from the addition of pigments, what changes in optical, mechanical and physical properties occur in the pigmented silicone under the influence of changes in temperature, humidity and disinfection procedures. in addition, it is important to note that there is a strong correlation between the number of species and the number of species that can be identified. however, more information is needed contemplating extrinsic pigmentation, which is still performed empirically and artistically in clinical practice. thus, the aim of this study was to evaluate the color stability of facial silicone pigmented extrinsically under the influence of the hygiene process. the work hypothesis is that the different techniques of extrinsic pigmentation and hygiene procedures cause different discoloration patterns of facial silicone. material and methods silastic mdx4-4210 (dow corning corporation, midland, mi, eua) is a material used to make maxillofacial prostheses and was used to fabricate 160 specimens, that was divided in 8 groups (n=20) acoording the pigmentation, as shown in the table 1. a metallic cylindrical matrix (30-mm diameter, 3-mm thick) was used to obtain the specimens2. the silicone were weighed using a precision digital scale (bel analytical equipments, piracicaba, brazil). the opacifier weight was equivalent to 0.2% of the total weight of the silicone11. the silicone was manipulated according to manufacturer’s instructions, on a glass plate until a homogeneous mixture was obtained. the silicone was then inserted in the master mold, and the excess was removed with a spatula to maintain a regular thickness. silastic mdx4-4210 material was confined in the matrix with the external surface exposed to the environment for 3 days, according to the manufacturer’s instructions. the material is partially cured after 24 hours, allowing its handling, but final cure following the release of formaldehyde occurs within approximately 3 days, according to the manufacturer13. after this period, each specimen was carefully removed from the metallic matrix2,9,14 and the extrinsic pigmentation procedure started. the samples were pigmented according the division groups presented in table 1, using oil paint (color baked siena gato preto, sorocaba, sp, brazil); titanium dioxide opacifier (naturativa, araçatuba, sp, brazil) and dc 1205 prime (dow corning corporation, midland, mi, eua). table 1. test groups. group pigmentation 1 colorless 2 oil paint 3 oil paint + opacifier 4 prime 5 oil paint + prime 6 oil paint + opacifier + prime 7 oil paint diluted in prime 8 oil paint + opacifier diluted in prime 4 cabral et al. a protocol for extrinsic pigmentation was not found in the literature, so the techniques used in the present study were developed from pilot studies and varied according to the group, as explained below: • group 2, pigmented exclusively with oil paint, a thin layer of paint was applied on the surface of the samples with the aid of a flat brush, drying was expected and a new layer was applied. • group 3, which associated ink and opacifier, initially a portion of the ink was weighed on a precision digital scale and 0.2% (by weight) of opacifier was added to this portion. the two materials were mixed and the blend was applied on the surface of the samples in two layers as discussed above. • group 4, which received application exclusively of prime, a layer of prime was applied on the surface of the samples with the aid of a flat brush, drying was expected and a new layer was applied. • group 5, the same exposed paint technique was used for group 2, and after drying the two layers of paint, 2 layers of prime were applied, as shown for group 4. • group 6, the same painting technique presented for group 3 was used and, after drying of the second layer of the paint and opacifier mixture, two layers of prime were applied, as shown for group 5. • group 7, which consisted of mixing the paint to prime, initially a portion of the paint was weighed on a precision digital scale and 0.2% (by weight) of prime was added. these two substances were mixed and applied in two layers on the surface of the samples, as shown for all pigmented groups presented above. • group 8, initially, a portion of the ink was weighed on a precision digital scale and 0.1% (by weight) opacifier and 0.1% (by weight) prime were added. the substances were mixed and applied on the surface of the samples in two layers, as already explained for all the pigmented groups presented previously. all the test specimens obtained were submitted to initial color analysis by means of a visible ultraviolet reflection spectrophotometer, model uv-2450 (shimadzu, kyoto, japan). to perform this test, each sample was positioned in a holder which was positioned in the spectrophotometer and the color reading occurred in the central portion of the sample. the use of this holder guarantees that all samples are placed in the same position and that the same sample receives the initial and final color reading also in the same location (in its central portion). the color alterations were calculated using the cie l∗a∗b∗ system, established by the commission internationale de l’eclairage—cie2,9. this system allows calculation of the mean value of e (color variation) between two readings by the formula: δe = [(δl)2 + (δa)2 + (δb)2 ]1/2 after the initial color test, all specimens were stored in a black box without light2,9,14. the specimens were disinfected three times per week for 2 months2,9,14 after reading the initial color. all samples were submitted to the hygiene procedures. half of them was sanitized with neutral soap (johnsons baby, johnson & johnson, são josé dos campos, sp, brazil), and the other half disinfected with 1% hypochlorite (musgo, alfenas, mg, brazil). 5 cabral et al. the specimens were cleaned with neutral soap and rubbed with the fingertips for 30 seconds, then rinsed with water for 30 seconds2,9,14. the specimens were immersed in a solution of 1% hypochlorite for 10 minutes and rinsed in running water2,9,14. after simulated hygiene procedures, a new color analysis was performed2,9,14. the color change (∆e) values were analyzed by two-way anova, and means were compared by tukey’s test (p < 0.05). results the results obtained are shown in tables 2 and 3. it was observed that the pigmentation and disinfectant factors, associated or not, produced a statistically significant difference (p <0.0001) in the color change of facial silicone samples (table 2). table 3 shows the mean values and standard deviation of color change of the facial silicone samples for each group evaluated. it is verified that all the samples, pigmented or not, presented color change (δe>0), after hygiene procedure. the groups of samples disinfected with soap had lower color change values, statistically significant (p<0.05) compared to disinfection with hypochlorite, with the exception of groups 1 and 4 that did not present a statistically significant difference between neutral soap and hypochlorite. table 2. analysis of variance (anova) two factors. variation factors gl sq qm f value p value pigmentation 7 13286,001 1898,000 315,706 < 0,0001* disinfectant 1 8848,601 8848,601 1471,840 < 0,0001* pigmentation x disinfectant 7 6035,203 862,172 143,410 < 0,0001* error 144 865,718 6,012 total 159 29035,524 *p < 0,05 denotes significant statistical difference. table 3. mean values of color change δe (standard deviation) of facial silicone samples for each technique of pigmentation and disinfectant used. pigmentation color change – δe neutral soap hypochlorite colorless 1,92 (0,76) aa 5,42 (2,02) aa oil paint 29,89 (2,99) ba 35,43 (8,56) bdb oil paint + opacifier 11,64 (0,35) ca 35,98 (0,84) cb colorless + prime 3,42 (1,33) ada 4,70 (1,10) aa oil paint + prime 10,45 (0,48) cea 32,38 (1,00) bcb oil paint + opacifier + prime 6,66 (0,22) dea 24,88 (1,88) eb oil paint diluted in prime 1,21 (0,60) aa 28,96 (0,27) db oil paint + opacifier diluted in prime 0,82 (0,16) aa 28,33 (0,47) deb *means followed by the same capital letter in the column and the same lowercase letter in the row do not differ at the 5% level of significance (p <0.05) by the tukey test. 6 cabral et al. the lowest δe values were presented by the groups of oil paint pigmented samples diluted in prime and disinfected with soap (δe=1.21, without opacifier and δe=0.82, with opacifier), being statistically significant (p<0,0001) to the other groups, and without significant statistical difference (p>0.05) of the respective colorless control groups. in addition, the oil-pigmented samples in combination of primer and opacifier presented lower color change value (δe=6.66) statistically significant (p=0.001), compared to the oil-pigmented samples combined with the use opacifier only (δe=11.64). discussion the material of choice for the facial prostheses manufacture is silicone. it needs to be pigmented to mimic the patient’s skin characteristics. in addition, it is known that deficiency in hygiene of facial prostheses contributes to the underlying tissues being susceptible to infections. thus, it is fundamental to perform the disinfection of the prostheses to maintain the tissues health. however, materials and techniques used for pigmentation and hygiene can cause changes in the facial silicone4. the literature is scarce in relation to clinical studies that evaluate different methods of hygiene for maxillofacial prostheses, however, it offers researches that evaluate the interaction between the substances used for hygiene with the base material and the pigments added intrinsically, used to make these prostheses. in this context, several substances and methods of hygiene / disinfection have already been tested (neutral soap2,4-9, chlorhexidine5, hypochlorite7, effervescent tablets2,4,8,9, plant extracts5, ethanol6, microwave energy7) and none of them are considered ideal on all aspects (bactericidal potential and preservation of all properties of silicon and pigments). there are no studies evaluating the behavior of pigments added extrinsically to the facial silicone when submitted to the hygienization process. according to the national bureau of standards15, the color change is considered to be very small when the δe is less than 1. the situation is clinically acceptable if the color change is between 1 and 3; and is considered clinically perceptible if δe is greater than 3. in the present study, the observed color change varied from 0.82 to 35.98 (table 3), which allows to affirm that the color change would not be clinically perceptible only for the groups pigmented with oil paint + opacifier diluted in prime and oil paint diluted in prime, both disinfected with neutral soap. the null hypothesis was accepted. it can be observed in table 2 that pigmentation and disinfectant factors, associated or not, produced a statistically significant difference in the color change of facial silicone samples. it is noted that all samples, pigmented or not, showed color alteration after hygiene procedures (table 3). these results corroborate with those obtained by goiato et al.16, which concluded that even without the disinfection process, the facial silicone undergoes a color change independent of the pigment type and the pigmentation technique used, since the material itself undergoes aging and, consequently, changes in its physical and chemical properties. in addition, other factors may contribute to the silicone color change, such as exposure to solar radiation, temperature variation and humidity. these factors were not considered in the present study as the accelerated aging test was not performed and, as a means of prevention, the samples were stored in a black box while not undergoing tests to reduce their exposure to light17. 7 cabral et al. it is also known that different substances and disinfection techniques can promote color change in intrinsically pigmented test pieces. typically, this intrinsic discolouration occurs with aging of the material due to various physico-chemical conditions, such as thermal changes and humidity. extrinsic factors such as absorption and adsorption of substances may also cause discoloration4,18. in the present study, the groups of samples disinfected with soap presented statistically lower color change values than hypochlorite disinfected groups (table 3). these different types of substances should be considered as requiring different disinfection techniques. although the neutral soap hygiene process required friction of the samples, it was less aggressive than the immersion technique used for 1% hypochlorite. in the study of goiato et al.16, the neutral soap corresponds to the control product because it is considered chemically inert, however, they used intrinsic pigmentation of the silicone. with the results obtained here, it can be considered that the hygiene procedure with neutral soap is less harmful to facial silicone when compared to 1% hypochlorite even when using extrinsic pigmentation. it is known that hypochlorite has disinfectant action being able to eliminate bacteria, but also has a bleaching action causing the discoloration where it is made from the reaction of chlorine with calcium hydroxide. this fact justifies the color change of the pigmented samples extrinsically and immersed in 1% hypochlorite19. eleni et al.7 (2013) evaluated the effect of different disinfecting procedures (microwave exposure and immersion in three solutions, sodium hypochlorite, neutral soap and a commercial disinfecting soap) on the hardness and color stability of two maxillofacial elastomers (an experimental chlorinated polyethylene (cpe) and a commercial polydimethyl siloxane (pdms)). it was concluded that the  disinfection  procedures caused alterations in color and hardness of the examined materials. the most suitable  disinfection  procedure for the pdms material is microwave exposure, while  disinfection  with sodium hypochlorite solution is not recommended. the cpe material is suggested to be disinfected with sodium hypochlorite solution and the use of neutral soap is not recommended. comparing the two materials, the pdms material is most color stable, while the cpe material presented fewer changes in hardness. the lowest values of color change were presented by groups of oil-stained pigment samples diluted in prime and disinfected with soap, similar to the respective colorless control groups (table 3). in addition, oil-pigmented samples in combination of primer and opacifier showed lower values of color change compared to oil-pigmented samples combined with the use of opacifiers alone. it is known that pigment particles can readily separate from the silicone when poorly bonded to the polymer chain of the material and also when exposed to the repeated chemical disinfection process. it is believed that the association between different materials may interfere with the bond formed between pigment and silicone. in studies involving the intrinsic pigmentation of silicone, pigment and opacifier, when associated, can promote the formation of stronger bonding of these pigments to the silicon matrix, avoiding their removal with the disinfection process2,10,11. this assertion suggests that the same occurred in the present study when they were associated with oil-based pigment, opacifier and prime. 8 cabral et al. regarding the use of prime, when associated with the paint, it preserved the same of degradation, as it was coated with dilute solutions of silane coupling agents and other active ingredients. this fact allows us to believe that, when in use, silicone color protection would be even greater because, in the same way that this prime protects the paint, it also impermeabilizes the silicone, preventing it from absorbing external coloring agents that would degrade the prosthesis. based on the results obtained and considering the limitations of an in vitro study, it was concluded that: the association of oil ink diluted in prime and hygiene technique with soap promotes the lower color change of facial silicone pigmented extrinsically. acknowledgments financial support: programas institucionais de iniciação científica, fundação de amparo à pesquisa de minas gerais (pibict – fapemig – edital prppg 021/2015), alfenas, minas gerais, brasil. 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https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1507/1160 6/9 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1507/1160 7/9 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1507/1160 8/9 28/01/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1507/1160 9/9 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1533/1211 1/10 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1533/1211 2/10 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1533/1211 3/10 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1533/1211 4/10 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1533/1211 5/10 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1533/1211 6/10 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1533/1211 7/10 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1533/1211 8/10 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1533/1211 9/10 21/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1533/1211 10/10 1http://dx.doi.org/10.20396/bjos.v18i0.8657259 volume 18 2019 e191620 original article 1 school of dentistry, faculdade são leopoldo mandic. campinas, sp, brasil. 2 department of implantology, school of dentistry, universidade santo amaro. são paulo, sp, brasil. corresponding author: edelcio de souza rendohl department of implantology, school of dentistry, universidade santo amaro, 340 rua prof. enéas de siqueira neto, jardim das imbuias, são paulo sp, 04829-300, brasil. tel.: +55 0800171796, e-mail: edelcio.rendohl@gmail.com received: april 11, 2019 accepted: august 23, 2019 influence of angulation and height of tooth preparation on the distribution of tensions in prosthetic crowns for upper central incisor: in silico analysis marcos teixeira machado1, edelcio de souza rendohl2,*, karina andrea novaes olivieri1, milton edson miranda1, william cunha brandt2 aim: it was to use tridimensional finite element analysis (fea) to analyze the effect of height and angulation of prosthetic preparations on the distribution of stresses for lithium disilicate prosthetic crowns, the underlying resin cement, and the prosthetic preparation of a superior central incisor. methods: a cad modeling software, solidworks 2013, was used to generate three-dimensional virtual models comprising the dimensions of the preparation parameters. three angles (6, 12 and 16 degrees) were simulated on the prepared walls and two wall heights were utilized (4 and 5 mm), for a total of six model groups according to the height and angulation of the walls. a vertical line in the y-axis was used as a reference for determining the degrees of convergence (inclination of the preparations). the chamfer finish and preparation width were standardized for all groups. results: the 4 mm preparations behaved more appropriately when the axial wall convergence was approximately 6 degrees. the 5 mm preparations required 12 degrees of angulation. in relation to resin cement, there was better stress distribution when the angle of incisal convergence was between 6 and 12 degrees. an increase to 16 degrees led to a considerable increase in peak stress at the preparation margin. conclusion: it was concluded that the convergence of the axial walls of coronal preparations with 4 and 5 mm heights should be 6 and 12 degrees, respectively, to avoid high tension spikes in the underlying resin cement. keywords: dental prosthesis. tooth preparation. finite element analysis. 2 machado et al. introduction ceramics are the materials that most mimic the natural tooth. ceramics have excellent biocompatibility and high ability to replicate and restore coronal stiffness1. however, these materials are friable and susceptible to fractures2. thus, repetitive occlusal loads, coupled with the geometry of the restorations, intensity, direction, and properties of the materials used, can cause their longevity to be altered2. another important factor that influences the strength and durability of dental ceramics is the dental preparation, due to its relation with the distribution of stresses to the dental remnant and restoration3. according to rosentritt et al.4, clinical failures in ceramic restorations occur mainly due to the geometry and the characteristics of the tooth preparation, such as the angle of preparation of the axial walls, finishing line of the preparation, internal adaptation, thickness of the cement, internal draw of the crown and possible occlusal variations. the patterns of preparation used today were largely defined by studies carried out many years ago5 and with few recent modifications6. however, the properties of the materials used and the technology used to achieve those historical results differed from the materials currently used; therefore, further research and study on the ideal characteristics for a dental preparation are required. mainly due to the increase of using metal free ceramic restorations, which have the ability to bond to the dental substrate. likewise, the ability of various ceramic systems to adhere to the dentin substrate has led to a reduced need for tooth preparation and, often, the original preparation principles are no longer followed as initially proposed. however, it is worth noting that retention and the stability of dental preparations remain important when performing clinical procedures, as they influence stress distribution and can decisively contribute to the longevity of prosthetic procedures7-9. in general, dental preparations with lower angulation of axial walls are recommended. however, the height of dental preparation may influence the seating of the ceramic restorations, due to the impossibility of the resin cement flowing. in such cases, increased angulation of the dental preparation may be necessary9. several authors6,8-11 studied the angulation of axial walls of dental preparations. however, there has been no consensus in the literature on the interrelationship of height and angulation of axial walls and how these variables influence the distribution of stress in the dental preparation, resin cement, and prosthetic crown. therefore, it is important to establish clinical protocols involving height and axial inclination to create an adequate distribution of stress in the tooth preparation, resin cement and the prosthetic crown of lithium disilicate, with a view on the longevity of ceramic restorations. therefore, the objective of this study was to evaluate the influence of the angulation and the height of the dental preparation on the mechanical behavior of lithium disilicate prosthetic crowns, the underlying resin cement, and the coronal preparation of a superior central incisor submitted to an oblique load on its palatal face. 3 machado et al. materials and methods three-dimensional virtual models comprising the preparation dimensions were generated using the cad modeling software, solidworks (dassault systems solid-works corp., waltham, ma, usa). three angles (6, 12 and 16 degrees) and two heights (4 and 5 mm) were simulated for the prepared walls, totaling six models. a vertical line in the y-axis was used as the reference for determining the degrees of convergence (inclination of the preparations). a bevel edge, as well as the width of the preparation, was standardized for all groups in order to isolate only the factors under study (figure 1). with regards to simulating the resin cement, all faces of the preparation were converted to a zero thickness entity offset, which enabled a three-dimensional contour of the preparation. thereafter, a thickening of 60 μm was performed to simulate the cement layer. the behavior of the material was characterized during the subsequent phase of analysis. for modeling the lithium disilicate crown, we used the anatomical dimensions of a central, maxillary incisor. adjustments were made in the contours of the crown to fit in all the preparations, modifying only the internal region that was in contact with the preparation. the crown was positioned concentrically (both axes aligned) to the preparation and to the cement. the crown was adapted to the margin of the preparation, using the loft rebound tool, which enables copying the preparation margin and the margin of the crown to allow perfect adaptation between the two pieces. at this stage, the gaps and interference between the parts that could occur later during the 4 mm x 6 degrees 4 mm x 12 degrees 4 mm x 16 degrees 5 mm x 6 degrees 5 mm x 12 degrees 5 mm x 16 degrees figure 1. preparations according to the height of the remnant. 4 machado et al. analysis were verified. the crown was combined with the cementation line by means of boolean combination operations, which allowed the crown to be adapted to the cement and to the preparation. the set was exported to the ansys workbench software 14.0 (ansys inc., canonsburg, pa, usa), to perform the mathematical analysis. the characterization of the mechanical behavior of the materials was simulated based on data related to the modulus of elasticity and poisson’s coefficient, according to previously published works12-14. the data used are shown in table 1. the model was divided into small elements, which were interconnected through a mesh so that the software calculated the tensions generated in each piece. in the present study, a mesh of 0.50 mm tetrahedral elements was used. this type of element was selected because it is best suited to curved surfaces, as in the case of the tooth preparation. the number of nodes and elements obtained for each model are listed in table 2. in addition, the model was fixed on both lateral sides so that displacement did not occur during load application. the simulation of masticatory contact occurred by applying a load of 100 n on the palatal surface of the crown, at a slope of 45 degrees (figure 2). the data obtained were evaluated according to the von-mises criterion for the crown and the preparation, as it shows active compression tensile stresses. the shear stress was used to evaluate the cement since this type of test is often used for evaluation of cement in “in vitro” research. table 2. numbers of nodes and elements obtained for each model. 4 mm preparations 5 mm preparations 6 degrees 12 degrees 16 degrees 6 degrees 12 degrees 16 degrees nodes 24.139 24.085 23.077 25.637 25.280 24.279 elements 13.356 13.423 12.853 14.220 14.013 13.503 table 1. modulus of elasticity and poisson’s coefficient of the materials used. material modulus of elasticity poisson’s coefficient reference enamel 84 gpa 0.30 ichim et al. (2007) dentin 18.6 gpa 0.31 lin et al. (2006), ozçelik & ersoy (2007) periodontal ligament 69 mpa 0.45 ozçelik & ersoy (2007) resin cement 18.3 gpa 0.3 lin et al. (2006), ozçelik & ersoy (2007) lithium dissilicate 95 gpa 0.3 lin et al. (2006), ozçelik & ersoy (2007) 5 machado et al. results the maximum stress values, expressed in megapascal (mpa), are shown in table 3. with regards to the crown, an increase in stress concentration was observed when more convergence (12 and 16 degrees) was used in comparison to 6 degrees for the 4 mm preparations. the preparation of 4 mm x 6 degrees showed the lowest von-mises tension value of the crown (197.9 mpa), while the 12 and 16-degree preparations presented values of 250.26 and 247.26 mpa, respectively. when the 5 mm preparation was used, 6 degrees of slope showed the highest values of tension (243.83 mpa), followed by 16 degrees (227.78 mpa) and 12 degrees (220.31 mpa). the peak of concentration was located where the crown met the chamfer margin. at this site, compression stress predominated due to the nature of the applied load (oblique loading) (figure 3). table 3. the maximum stress value (expressed in megapascal – mpa) obtained. 4 mm preparations 5 mm preparations 6 degrees 12 degrees 16 degrees 6 degrees 12 degrees 16 degrees von-mises crown 197.9 250.26 247.26 243.83 220.31 227.78 resin cement 27.54 25.61 42.00 28.41 26.56 36.76 von-mises preparation 58.81 59.05 59.57 79.46 76.48 75.22 figure 2. application of loading site of 100n. 6 machado et al. when analyzing the resin cement, the peak shear stress concentration was located at the cementation line near the margin with the prosthetic crown. for the 4 mm preparations, the 6 and 12-degree angles resulted in similar tension values of 27.54 and 25.61 mpa, respectively. the preparation for the 16-degree angle presented the highest value of tension, being distant of the other groups (42 mpa). for the 5 mm preparations, a similar behavior occurred, with shear stress values of 28.41 mpa and 26.56 mpa for the 6 and 12-degree preparations, respectively. the 5 mm x 16-degree preparation produced the highest stress value of 36.76 mpa. for the 16 degrees angulation, the height increase decreased the tension values when comparing the 4 mm (42 mpa) and 5 mm (36.76 mpa) heights (figure 4). 4 mm x 6 degrees 4 mm x 12 degrees 4 mm x 16 degrees 5 mm x 6 degrees 5 mm x 12 degrees 5 mm x 16 degrees 250,26 max 232,39 214,52 196,65 178,78 160,91 143,04 125,16 107,29 89,421 71,549 53,678 35,806 17,935 0,06344 min figure 3. shear stress in the prosthetic crown. figure 4. shear stress in the cementation line. 4 mm x 6 degrees 4 mm x 12 degrees 4 mm x 16 degrees 5 mm x 6 degrees 5 mm x 12 degrees 5 mm x 16 degrees 42,009 max 39,059 36,11 33,161 30,211 27,262 24,313 21,363 18,414 15,465 12,515 9,5661 6,6168 3,6674 0,7181 min 7 machado et al. the von mises strain values in the preparation were similar regardless of the height or angulation of the preparation. the peak stress concentration was located in the chamfer region, external to the transition region between the chamfer and the outer surface. the 4 mm preparations presented tension values of 58.81 mpa, 59.05 mpa, and 59.57 mpa for the 6, 12 and 16 degrees, respectively. for the 5 mm preparations, a similar behavior occurred with higher values when compared to the 4 mm preparations. the tensile values were 79.46 mpa, 76.48 mpa and 75.22 mpa for the 6, 12 and 16-degree preparations, respectively (figure 5). discussion the methodology used in the present study (fea) provided relevant explanations of how tensions behave in the prosthetic crowns of lithium disilicate, the underlying resin cement, and the actual preparation, especially as to the interrelationship that exists between the height of the preparations and the angles of convergence. as shown in table 3, dental preparation tension has been higher due to the increase in the height of the preparation. this probably occurs because of the increased surface area in contact with the prosthesis. in addition, it has known that how lower convergence angle greater the retention and stability of the prosthesis, therefore, greater the initial tension9. these subjects also explain the fact of considering 4 and 5 mm preparations and the stress distributions in the lithium disilicate crowns, the results achieved adequate when the axial wall convergence was between 6 and 12 degrees. the results showed that the increase of the axial inclination increases the tension, therefore 6 degrees would be the most indicated. however, the height of the tooth preparation (from 4 to 5 mm) increased the surface area and allowed the axial inclination, it has also been showed to increase from 6 to 12 degrees. in fact, the use of axial inclination with 6 degrees showed the highest tension in the lithium disilicate crows when the height of 5 mm was performed. due to the higher height of the clinical crown, bigger convergence angle should allow adequate cementation. the greater axial inclination is important as it can decrease the hydrostatic pressure on the cement at the cementation and to facilitate the complete settling the prosthesis to the tooth. figure 5. von-mises stress for the preparation values. 4 mm x 6 degrees 4 mm x 12 degrees 4 mm x 16 degrees 5 mm x 6 degrees 5 mm x 12 degrees 5 mm x 16 degrees 79,469 max 73,793 68,116 62,44 56,764 51,087 45,411 39,734 34,058 28,382 22,705 17,029 11,353 5,6764 8,4983 min 8 machado et al. the findings of the present study also agree with the results of shillingburg et al.9, but those results were limited to an axial angulation of 6 degrees. those authors concluded that this angulation would allow a better distribution of stresses in the underlying cement. rosenstiel et al.6 likewise recommend an angle of 6 degrees to improve retention. bowley and kieser15 reached similar conclusions to those found in the present study, indicating that the axial inclination should not be greater than 10 degrees in crowns of 3 to 4 mm in height. beuer et al.16 agreed that 12 degrees of convergence would be a good limit for total crowns, confirming the data of the present research. the works of tiu et al.17 corroborate the present research, indicating a similar scope of axial angulation. however, according to gilboe and teteruck8, axial angulation must be limited between 2 and 5 degrees, which may be justified by the fact that those authors did not consider the height of the preparations and their interrelationship with the axial angulation in their conclusions. although the cited studies had similar results to this current research, they were performed using axial forces to evaluate retention and resistance, which does not simulate the functional components of the oral cavity. thus, the oblique force at 45 degrees is the most appropriate for studies of tension in the buccal cavity, since its physical components can be decomposed into horizontal and axial forces, more naturally simulating the forces that occur on the tooth, as used in the present study. in addition, those studies based their conclusions only on the retention factor, without taking into account the stress distribution in the prosthetic crowns, the underlying cement, and the prosthetic preparation. those studies also lack an evaluation of the interrelationships between the height and axial angulation of the preparations and the distribution of stresses during functional movements. the axial angulation of 6 and 12 degrees is not accepted throughout the literature. weed and baez18 have suggested an angulation of 16 degrees for 3.5mm preparations. however, the results of the present research showed that a slope of 16 degrees was undesirable for 4 and 5 mm preparations, as it induced an exaggerated increase of tensions in the underlying cement on the order of 61.5% and 33.7%, respectively. it should also be noted that when these authors published their results, most of the fixed prostheses were cemented with zinc phosphate or glass ionomer cement, which have compression strength and tensions ranging from 3.1 mpa to 5.3 mpa19. the level of stress concentration in the resin cement, at the preparation margins of 4 mm and 6 and 12-degree angulation, was 27.54 mpa and 25.61 mpa; while the concentration of the tensions in preparations of 5 mm and 6 and 12 degrees of axial angulation was 28.41 mpa and 26.56 mpa, respectively. these stress levels were already very high for the cementing agents used at that time. thus, an increase of tensions on the order of 33.7% to 61.5% would make the prognosis of these prostheses even more critical if the axial inclination is increased to 16 degrees. additionally, zidan and ferguson20 concluded that retentive values for preparations with 24-degree convergence using adhesive resin cement had higher retention than cemented crowns with conventional cement and 6-degree axial convergence angles. however, these authors did not take into account the resistance of the resin cement to the tension, but only the retention obtained by the inclination of the axial walls. according to lad et al.19, the resin cement showed a tensile strength of 34 to 41 mpa, 9 machado et al. according to the current results, a slope of 16 degrees and height of 4 mm presented 42 mpa, which exceeds the maximum resistance limit of the resin cement. additionally, 5 mm preparations with 16 degrees of inclination produced tensions of 36.76 mpa. thus, preparations with an angulation of 16 degrees or greater could lead to a failure of the cementing agent due to the excess tensions, especially in 4 mm preparations. therefore, it would be important to establish a safety margin in the resin cement of at least 50% in their tensile strength in order to make the prosthetic procedures more secure, as well as to use appropriate protocols regarding the height and angulation of axial walls so as to provide better stress distribution. another factor that must be taken into account is the surface area of the preparation21. according to shillingburg jr. et al.9, the larger the area of the cement fixed to the internal part of the restoration, the greater the retention. this factor partially explains why preparations with higher height allow greater angulation of the axial walls since they have a larger area of contact. larger vertical walls increase the area of the preparation as well as the arc of rotation of the prosthetic crown, reducing the concentration of stresses in the cement of the preparation margins. the results of the current research corroborate the findings of shillingburg jr. et al.9; however, new studies should be proposed to evaluate the relationship between the surface area and the height of the preparation. therefore, based on the findings of the present study, prosthetic preparations with 4 and 5 mm high walls should limit the angulation of the axial walls to 6 and 12 degrees respectively, in order to avoid overloading the underlying cement, which may lead to a decrease in the survival rates of prostheses. however, it is worth emphasizing that the reality found in clinical practice does not always respect the desired axial inclination22,23. according to the present research, the critical point to be observed in the distribution of tensions in fixed prosthodontics is found in the underlying resin cement. therefore, it is recommended to limit the inclination of the axial walls of the coronal preparations of 4 and 5 mm in height to 6 and 12 degrees, respectively, to avoid high tension spikes which could lead to a failure in the prosthetic treatment. references 1. magne p, belser u. bonded porcelain restorations in the anterior dentition: a biomimetic approach. carol stream: quintessence publishing; 2002. 2. zhang, y., sailer, i., lawn, b.r., 2013. fatigue of dental ceramics. j dent. 2013 dec;41(12):1135-47. doi: 10.1016/j.jdent.2013.10.007. 3. oyar p, ulusoy m, eskitascioglu g. finite element analysis of stress distribution of 2 different tooth preparation designs in porcelainfused-to-metal crowns. int j prosthodont. 2006 janfeb;19(1):85-91. 4. rosentritt m, steiger d, behr m, handel g, kolbeck c. influence of substructure design and spacer settings on the in vitro performance of molar zirconia crowns. j dent. 2009 dec;37(12):978-83. doi: 10.1016/j.jdent.2009.08.003. 10 machado et al. 5. prothero jh. prosthetic dentistry. chicago: medico-dental publishing; 1923. 6. rosenstiel sf, land mf, fujimoto j. contemporary fixed prosthodontics. saint louis: mosby elsevier; 2016. 7. rekow ed, harsono m, jadal m, thompson vp, zhang g. factorial analysis of variables influencing stress in all-ceramic crowns. dent mater. 2006 feb;22(2):125-32. 8. gilboe db, teteruck wr. fundamentals of extracoronal tooth preparation.part i. retention and resistance form. j prosthet dent. 1974 dec;32(6):651-6. 9. shillingburg jr ht, jacobi r, brackett se. fundamentals of tooth preparations for cast metal and porcelain restorations. chicago: quintessence books; 1987. 10. goodacre cj, campagni wv, aquino sa. tooth preparations for complete crowns. an art form based on scientific principles. j prosthet dent. 2001 apr;85(4):363-76. 11. goodacre cj. designing tooth preparations for optimal success. dent clin north am. 2004 apr;48(2):v, 359-85. 12. ichim i, schmidlin pr, kieser ja, swain mv. mechanical evaluation of cervical glass-ionomer restorations: 3d finite element study. j dent. 2007 jan;35(1):28-35. 13. lin cl, wang jc, kuo yc. numerical simulation on the biomechanical interactions of tooth/implant-supported system under various occlusal forces with rigid/non-rigid connections. j biomech. 2006;39(3):453-63. 14. ozçelik t, ersoy ae. an investigation of tooth/implant-supported fixed prosthesis designs with two different stress analysis methods: an in vitro study. j prosthodont. 2007 mar-apr;16(2):107-16. 15. bowley jf, kieser j. axial-wall inclination angle and vertical height interactions in molar full crown preparations. j dent. 2007 feb;35(2):117-23. 16. beuer f, edelhoff d, gernet w, naumann m. effect of preparation angles on the precision of zirconia crown copings fabricated by cad/cam system. dent mater j. 2008 nov;27(6):814-20. 17. tiu j, al-amleh, b, waddell jn, duncan wj. reporting numeric values of complete crowns. part 1. clinical preparations parameters. j prosthet dent. 2015 jul;114(1):67-74. doi: 10.1016/j.prosdent.2015.01.006. 18. weed rm, baez rj. a method for determining adequate resistance form of complete cast crown preparations. j prosthet dent. 1984 sep;52(3):330-4. 19. lad pp, kamath m, tarale k, kusugal pb. practical clinical considerations of luting cements: a review. j int oral health. 2014 feb;6(1):116-20. 20. zidan o, ferguson gc. the retention of complete crowns prepared with three different tapers luted with four different cements. j prosthet dent. 2003 jun;89(6):565-71. 21. o’kray h, marshall ts, braun tm. supplementing retention through crown/preparation modification. an in vitro study. j prosthet dent. 2012 mar;107(3):186-90. doi: 10.1016/s0022-3913(12)60054-7. 22. parker mh, malone kh 3rd, trier ac, striano ts. evaluation of resistance form for prepared teeth. j prosthet dent. 1991 dec;66(6):730-3. 23. ghafoor r, rahman m, siddiqui aa. comparison of convergence angle of prepared teeth for full veneer metal ceramic crowns. j coll physicians surg pak. 2011 jan;21(1):15-8. doi: 01.2011/jcpsp.1518. braz j oral sci. 15(3):181-184 influence of dentistry procedures on masticatory function of dentate patients lucas do nascimento tavares, dds, msc1, karla zancope, dds, msc, phd1, frederick khalil karam, dds, msc1, wilson mestriner júnior, dds, msc, phd2, flávio domingues das neves, dds, msc, phd1 1department of occlusion, fixed prosthodontics and dental materials, school of dentistry, federal university of uberlândia, uberlândia, mg, brazil 2department of pediatric clinics, preventive and social dentistry, faculty of dentistry of ribeirão preto, university of são paulo, ribeirão preto, sp, brazil correspondence to: prof. dr. flávio domingues das neves av. pará, 1720, bloco 4l, anexo a sala 4la-42 campus umuarama, cep: 38405-320 uberlândia, minas gerais, brazil e-mail: flaviodominguesneves@gmail.com phone: +55-34-3225-8105 fax: +55-34-3225-8105 abstract aim: evaluate the masticatory performance in patients with different clinical situations: patients with natural dentition without restorative and/or orthodontic intervention (group cg) and patients with restorations and/or orthodontic intervention (group tg). subjects received instruction before the experiment, related to masticatory movements. methods: three capsules containing the granules were delivered separately to chew for 20 seconds each, controlled by the examiner. one capsule was chewed for 20 seconds only on the left side; the second capsule was chewed for 20 seconds only on the right side; and the third capsule was chewed for 20 seconds to simulate each patient’s mastication. the mean data and standard deviation of masticatory function of each patient was calculated. results: the results obtained in this study showed that there were no statistically significant differences (p < 0.05) between both tested groups (0.0320 ± 0.00716, 0.0436 ± 0.00974). conclusions: patients who were submitted to orthodontic and/or restorative procedures, with balanced occlusion could be used as a control group, making easier the patient recruitment. clinicians and researchers on masticatory performance evaluation could apply the colorimetric method. the colorimetric method was a standardize method, effective and easy to execute. keywords: mastication. colorimetry. dentition, permanent. introduction the objective of oral rehabilitation is to reestablish phonetics, aesthetics and regular function. a healthy individual must have harmony between the stomatognathic system components; therefore, the knowledge of components of this complex system is extremely important to restore one of the most significant human function, the mastication1-3. it is crucial to evaluate correctly chewing function to determinate the quality of a treatment4-8. the orthodontic treatment prioritizes function and occlusion for treatment the patient that has malocclusion, and this should be explained before initiating the treatment9. previous study10 demonstrated that 80 per cent of the individuals that attend orthodontic practices disregard structural or functional consideration, could influence in your chewing, masticatory performance, including bite force, occlusal contact area, number of functional tooth units and malocclusion severity11. this treatment will assist the patient during your chewing, because the mastication is the first step in the process of digestion12,13. received for publication: february 20, 2017 accepted: may 3, 2017 original article braz j oral sci. july | september 2016 volume 15, number 3 http://dx.doi.org/10.20396/bjos.v15i3.8649603 182 obtaining the mean values of masticatory performance of a control group is important for defining a deadline. previous literature6 described the control group as total dentate patients without signs or symptoms of traumatic occlusion, which have not undergone restorative and/or orthodontic procedures. however, even with advances in preventive dentistry, it has been difficult to recruit patients with these specific characteristics, especially the patients that have not undergone restorative and/ or orthodontic procedures, because most of these patients today already passed by these procedures. the aim of this study was to evaluate the colorimetric method and the masticatory performance in patients with different clinical situations: patients with natural dentition without restorative and/or orthodontic intervention and patients with restorations and/or orthodontic intervention, using visible spectrophotometry to check for differences between groups. the tested hypothesis was that there is no difference in the masticatory performance when restorative and/or orthodontic interventions are performed. material and methods the study was submitted to the ethics committee in research of the federal university of uberlândia (307.001/13 cep/ufu) and approved. twenty patients were selected for this study, after authorization from each patient, expressed in term of informed consent. the sample calculation was conducted (sigmaplot 12.0 for windows program, systat software inc, usa), considering the expectation of the mean difference equal to 0.70, the expectation of a standard deviation of 0.80, the study power (1-β) of 0.80 and type i error (α) equal to 0.0514. the test estimated a minimum sample size of 20 units (n = 20) in each group. inclusion and exclusion criteria were developed in order to properly select the patients who would go through the research. the control group (group cg) was composed by adult patients with good general health, complete natural dentition (up to 2nd molar), without any restorative and/ or orthodontic procedures and without signs or symptoms of traumatic occlusion or malocclusion, angle class i and mesofacial. the test group (group tg) was composed by adult patients with good general health, complete natural dentition, but submitted to restorative (maximum extension until resin restorative for class ii mod) and/ or orthodontic intervention and without signs or symptoms of traumatic occlusion or malocclusion, angle class i and mesofacial. the simulator food15 used was standardized with 1mm diameter size (fig. 1). approximately 245-250 mg of pigmented granules are placed in a 10mm pvc rectangular capsule, welded by radiofrequency. subjects received instruction before the experiment, related to masticatory movements. three capsules containing the granules were delivered separately to chew for 20 seconds each, controlled by the examiner (fig. 2). one capsule was chewed for 20 seconds only on the left side; the second capsule was chewed for 20 seconds only on the right side; and the third capsule was chewed for 20 seconds to simulate each patient’s mastication. if the capsule ruptured during the experiment, it was discarded and a new capsule was performed. during mastication, the granules were broken and fuchsin dye was diffused into the capsule, according to the masticatory performance. the contents of the capsule were dissolved in 5 ml of water under continuous vibratory motion for 30 seconds. the solution was then filtered to remove the unbroken granules and the fuchsin solution was analyzed, based on the linear equation of the calibration curve. the fuchsin concentration was measured using a spectrophotometer (beckman du-7 uv, beckman inc., palo alto, ca, usa). the viewable area of the absorbance spectrum at 546 nm with fuchsin wavelength was defined as the standard calibration curve (0 12.5 mg/ ml). the mean data and standard deviation of masticatory function of each patient was calculated. the results were tabulated and analyzed by t-test (α=.05). statistical correlation analysis was performed using a statistical program (sigmaplot 12.0 for windows program, systat software inc, san jose; c.a.; usa). results the results obtained in this study demonstrated that both tested groups were not statistically different (p < 0.108), according to figure 3. for each group, the mean data and standard deviation was described in table 1. influence of dentistry procedures on masticatory function of dentate patients braz j oral sci. 15(3):181-184 fig.1. rectangular capsule containing an acid fuchsin pigment. fig.2. completely toothed patient chewing the capsule. 183 discussion the tested hypothesis was confirmed, since there was no difference in the masticatory performance when patients were submitted to restorative (maximum extension until resin restorative for class ii mod) and/ or orthodontic intervention and without signs or symptoms of traumatic occlusion or malocclusion, angle class i and mesofacial. this study confirms that restorative and/or orthodontic procedures could be performed, and do not affect the masticatory function. it is important to emphases that all tested patients did not present signs or symptoms of traumatic occlusion or malocclusion. a correctly balanced/adjusted occlusion is essential to determine the mean data of the control group. the colorimetric method was effective, easily to execute and the fuchsin capsule is standardize. all capsules have the same containing. it is possible to storage the capsule for a period of time, even after the masticatory test. it is an advantage over other objective masticatory function methods16-18. the application of this standardized methodology is suggested to perform comparison between future studies19. to accomplish some types of clinical studies it is necessary select patients for the control group, where they will be considered standard gold, was composed by adult patients with good general health, complete natural dentition (up to 2nd molar), without any influence of dentistry procedures on masticatory function of dentate patients braz j oral sci. 15(3):181-184 fig.3. mean data graphic and standard deviation of the groups and level of absorbance of fuchsin. restorative and/ or orthodontic procedures and without signs or symptoms of traumatic occlusion or malocclusion12, but this type of patient is rare. therefor, the results showed that adult patients who participated the study with good general health, complete natural dentition, but submitted to restorative (maximum extension until resin restorative for class ii mod) and/ or orthodontic intervention and without signs or symptoms of traumatic occlusion or malocclusion, does not affect your performance masticatory. within the limitations of this study, restorative (maximum extension until resin restorative for class ii mod) and/ or orthodontic intervention in patients without signs or symptoms of traumatic occlusion or malocclusion, angle class i and mesofacial patients, do not masticatory performance evaluation. the method used was effective and easy to execute. references 1. boretti g, bickel m, geering ah. a review of masticatory ability and efficiency. j prosthet dent. 1995 oct;74(4):400-3. 2. scudine kg, pedroni-pereira a, araujo ds, prado dg, rossi ac, castelo pm. assessment of the differences in masticatory behavior between male and female adolescents. physiol behav. 2016 sep 1;163:115-22. doi: 10.1016/j.physbeh.2016.04.053. 3. laird mf, vogel er, pontzer h. chewing efficiency and occlusal functional morphology in modern humans. j hum evol. 2016 apr;93:1-11. doi: 10.1016/j.jhevol.2015.11.005. 4. jacobsen hc, wahnschaff f, trenkle t, sieg p, hakim sg. oral rehabilitation with dental implants and quality of life following mandibular reconstruction with free fibular flap. clin oral investig. 2016 jan;20(1):187-92. doi: 10.1007/s00784-015-1487-3. 5. mendonça db, prado mm, mendes fa, borges tf, mendonça g, prado cj, et al. comparison of masticatory function between subjects with three types of dentition. int j prosthodont. 2009 jul-aug;22(4):399-404. 6. toman m, toksavul s, saracoglu a, cura c, hatipoglu a. masticatory performanceand mandibular movement patterns of patients with natural dentitions, complete dentures, and implant-supported overdentures. int j prosthodont. 2012 mar-apr;25(2):135-7. 7. giannakopoulos nn, wirth a, braun s, eberhard l, schindler hj, hellmann d. effect of the occlusal profile on the masticatory performance of healthy dentate subjects. int j prosthodont. 2014 jul-aug;27(4):383-9. doi: 10.11607/ijp.3793. 8. pereira lj, van der bilt a. the influence of oral processing, food perception and social aspects on food consumption: a review. j oral rehabil. 2016 aug;43(8):630-48. doi: 10.1111/joor.12395. 9. josefsson e, bjerklin k, lindsten r. factors determining perceived orthodontic treatment need in adolescents of swedish and immigrant background. eur j orthod. 2009 feb;31(1):95-102. doi: 10.1093/ejo/ cjn069. 10. baldwin dc. appearance and aesthetics in oral health. community dent oral epidemiol. 1980;8(5):244-56. 11. n'gom pi, woda a. influence of impaired mastication on nutrition. j prosthet dent. 2002 jun;87(6):667-73. 12. van der bilt a, engelen l, pereira lj, van der glas hw, abbink jh. oral physiology and mastication. physiol behav. 2006 aug;89(1):22-7. 13. olthoff lw, van der bilt a, bosman f, kleizen hh. distribution of particle sizes in food comminuted by human mastication. arch oral biol. 1984;29(11):899-903. 14. paolantonio m, perinetti g, dolci m, perfetti g, tetè s, sammartino g, et al. surgical treatment of periodontal intrabony defectswith calcium sulfate implant and barrier versus collagen barrier or open flapdebridement alone: a 12-month randomized controlled clinical trial. j periodontol. table 1 mean data and standard deviation of absorbance of fuchsin release of the tested groups. group cg group tg 0.0320 ± 0.00716 a 0.0436 ± 0.00974 a different upper case letters represent significant differences (α=0.05). 184 influence of dentistry procedures on masticatory function of dentate patients 2008 oct;79(10):1886-93. doi: 10.1902/jop.2008.080076. 15. escudeiro santos c, de freitas o, spadaro ac, mestriner-junior w. development of a colorimetric system for evaluation of the masticatory efficiency. braz dent j. 2006;17(2):95-9. 16. van der bilt a, fontijn-tekamp fa. comparison of single and multiple sieve methods for the determination of masticatory performance. arch oral biol. 2004 mar;49(3):193-8. 17. schimmel m, christou p, herrmann f, müller f. a two-colour chewing gum test for masticatory efficiency: development of different assessment methods. j oral rehabil. 2007 sep;34(9):671-8. 18. tarkowska a, katzer l, ahlers mo. assessment of masticatory performance by means of a color-changeable chewing gum. j prosthodont res. 2017 jan;61(1):9-19. doi: 10.1016/j.jpor.2016.04.004. 19. silva om, zancopé k, mestriner júnior w, prado cj, neves fd, simamoto-junior pc. masticatory function evaluation by two methods: colorimetric and sifters. rev odontol bras central. 2011;20(53):125-8. braz j oral sci. 15(3):181-184 28/01/2019 pdf.js viewer 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https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1514/1167 11/11 1 volume 16 2017 e17059 original article influence of enamel acid-etching on mechanical properties and nanoleakage of resin composite after aging erick kamiya coppini1*, lúcia trazzi prieto 2, josué junior araujo pierote3, cíntia tereza pimenta de araújo4, dayane carvalho ramos salles de oliveira5, luís alexandre maffei sartini paulillo6 corresponding author: piracicaba dental school state university of campinas – unicamp department of restorative dentistry – operative dentistry area avenida limeira, 901areião, piracicaba, são paulo brasil cep:13414-018 telephone: +55 19 98408-8800 e-mail: lucinhatrazzi@hotmail.com received: june 5, 2017 accepted: july 26, 2017 aim: the aim of this study was to evaluate how acid-etching of the cavosurface enamel in class i resin composite restorations influences the bond strength to the pulpal wall and the restoration, knoop microhardness and nanoleakage after thermomechanical aging. for this research 76 fresh human molars were selected and restored with silorane or clearfil sebond/z350xt composite divided in 4 groups (silorane system restored with or without enamel cavosurface acid-etching and clearfil sebond/z350xt with or without enamel cavosurface acid-etching). to induce artificial aging, samples were subjected to thermomechanical cycling through 200,000 and thermal cycling between 5 and 55 °c with 30 second filling and 15-second drainage steps. microhardness and microtensile bond strength were evaluated in 32 teeth (n=8) each and nanoleakage evaluation was performed in 12 teeth (n=3). samples restored by clearfil sebond/z350 xt without cavosurface acid-etching showed significantly lower microtensile bond strength results. the resin composite z350xt presented higher values of knoop microhardness. it was observed little or no infiltration for silorane groups and moderate infiltration for clearfil se bond groups. acid-etching of the cavosurface enamel during restoration procedure with clearfil se bond resulted in a stronger bond after thermomechanical cycling. silorane groups showed less infiltration than clearfil se bond groups. keywords: dental cavity preparation; tensile strength; silorane resins; adhesives. 1 ms, phd student in restorative dentistry, department of restorative dentistry, piracicaba dental school, state university of campinas, sp‐brazil. 2 dds, ms, phd in restorative dentistry, department of restorative dentistry, piracicaba dental school, state university of campinas, sp‐brazil. 3 dds, msc student in restorative dentistry, department of restorative dentistry, piracicaba dental school, state university of campinas, sp‐brazil. 4 dds, ms, phd, assistant professor, department of dentistry, faculty of sciences of health, federal university of jequitinhonha and mucuri valley ‐ ufvjm, diamantina, mg, brazil. 5 dds, ms, phd student in restorative dentistry, department of restorative dentistry, piracicaba dental school, state university of campinas, sp‐brazil. 6 dds, ms, phd, titular professor of department of restorative dentistry, piracicaba dental school, state university of campinas, sp-brazil. http://dx.doi.org/10.20396/bjos.v16i0.8650500 mailto:dayoli87@gmail.com 2 coppini et al. introduction shrinkage of dental composites is an ongoing challenge in dentistry. composite shrinkage can cause adhesive failure and loss of marginal sealing1, resulting in post operative sensitivity, marginal staining, and secondary caries2. silorane is a low-shrinkage, low-interfacial stress resin composite composed of a matrix of siloxane and oxirane. it is polymerized through a cationic reaction involving the cycloalyfatic oxirane ring opening. the resulting composite exhibits less than 1% shrinkage3, which leads to better marginal integrity4 and less microleakage compared to methacrylate composites5. in addition to the monomer composition, factors that influence the stresses created by polymerization include the photo initiator, number and type of filler, c-factor, restorative technique, and light source1,6. the composites are used with adhesive systems. compared to total-etch adhesives, self-etch adhesive systems are easier to manipulate, can be applied more rapidly6, and are less technique-sensitive, leading to fewer errors during application7. however, self-etch adhesives can present deficient penetration of the enamel8, leading to faster degradation of the interface9-11 and subsequent infiltration of the hybrid layer. acid-etching of the cavosurface enamel has been suggested as a means to improve the bond strength of the enamel, as well as the longevity and quality of the interface12,13. however, the vector of polymerization shrinkage is directed towards the walls with the highest bond strength. an overly large increase of the enamel bond strength could cause a decrease of the dentin bond strength, even when a layering technique is used, because the stress of polymerization shrinkage would be concentrated on this area14. in clinical evaluation, the microhardness of a composite material is an important indirect means for estimating the degree of conversion (dc) of the polymer15. a polymer that presents a relatively low dc will exert less stress after polymerization shrinkage. although this condition leads to better marginal stability, it could also masks the bond strength results. in this study, the knoop microhardness was tested to evaluate the dc and to ascertain (indirectly) the stress at the restoration interface. the aim was to evaluate how acid-etching of the cavosurface enamel during a class i restoration influences the knoop microhardness of the composite, the microtensile bond strength between the pulpal wall and the restoration composite, and the nanoleakage after thermomechanical aging. the hypotheses of this study were as follows: (1) acid-etching would influence the quality of the bond strength on the pulpal wall/restoration after aging, (2) silorane would present superior mechanical properties, and (3) nanoleakage would not differ between the two tested restorative systems. material and methods this research was approved by the ethics research committee of piracicaba dental school – university of campinas (number n-089/2012). the study consisted of three tests, measuring the microtensile bond strength, the microhardness, and the nanoleakage. the materials used are described in table 1. 3 coppini et al. briefly, 76 freshly extracted human third molars were stored for 24 hours in a buffered 0.1% thymol solution at 37 °c. after cleaning, the teeth were stored in distilled water until cavity preparation. the teeth were embedded in polystyrene resin. their occlusal surfaces were planed in a polishing machine (arotec ind., são paulo, brazil) with 400-grit sandpaper (3m 411q, sumaré brazil, sp, brazil). a standard cavity preparation machine was used to create a class i cavity in each polished tooth. each cavity had the following dimensions: 5 mm in the mesiodistal direction, 4 mm in the buccolingual direction, and 3 mm in depth. cavities were made with a #56 carbide bur (kg sorensen ind. e com ltda., barueri, sp, brazil), which was replaced after every 5 cavities. for the restorative procedure, half of the samples (38 teeth) were randomly selected and subjected to acid-etching of cavosurface enamel with 35% phosphoric acid for 30 seconds. the surface was washed thoroughly with water for 30 seconds and dried with air jets. half of these acid-etched teeth (19 teeth) were restored with silorane systems (3m espe, st. paul, minnesota, usa) (capn) and the other half (19 teeth) with clearfil se bond (kuraray medical inc. okayama, japan)/z350 xt (3m espe, st. paul, minnesota, usa) (cacz). similarly, for the samples that had not been acid-etched (38 teeth), 19 teeth were restored with silorane systems (sapn) and 19 teeth with clearfil se bond/z350 xt (sacz). the restorative procedure is described in table 2. after being kept in an environment at 37 °c with relative humidity for 24 hours, samples were placed in a thermomechanical cycling machine (msft, elquip, são table 1. composition and manufacturers of materials used on this study. material composition manufacturer scotchbond acid • phosforic acid; -water; • poli(éter vinil). 3m espe, st. paul, minnesota, usa silorane primer • 2-hidroxiethil metacrilate (hema); • bisphenol-a-diglicidil éter dimetacrilate (bis-gma); • water; -etanol; -silic treated with silane; • phosforic acid-metaciloxic-hexilester; • 1.6 hexanodiol dimetacrilate; • copolimer acrilic and itaconic acid; (dimetalimine) etil metacrilate; • dl-canforoquinone; -phosfine acid. 3m espe, st. paul, minnesota, usa silorane bond • dimetacrilate; -silic treated with silane; • trietilene glycol dimetacrilate (tegdma); • phosforic metacriloxi-hexilesters acid; • dl-canforouinone; • 1.6hexanodiol dimetacrilate 3m espe, st. paul, minnesota, usa silorane composite filtek p90 a3 • 3,4epoxiciclo hexiletilciclopolimetil siloxane; • bis-3,4 – epóxi ciclohexiletilfenilmetil silane; • silanizaded quartz; -itriumfluoride; • canforoquinone (3m espe, st. paul, minnesota, usa clearfil se bond primer • 10-metacriloiloxi -decil dihidrogenade fosfatase (mdp); • hema; -dimetacrilate hidrofílic; • canforoquinone; -terciary amine; -water kuraray medical inc. okayama, japan clearfil se bond bond • hema; -10-metacriloiloxi -decil dihidrogenado fosfatase (mdp); • bis-gma; -dimetacrilate hidrofílic; • terciary amine; • sílic coloidal silanizaded; -canforoquinone. kuraray medical inc. okayama, japan composite filtek z350 xt a3 • bis-gma; -bis-ema6; • udma; -tegdma; -silic; -canforoquinone (3m espe, st. paul, minnesota, usa 4 coppini et al. carlos, sp, brazil). each sample underwent 200,000 cycles of loading at 80n and 2 cycles/second. loading was applied perpendicular to and in the centre of the restoration. thermal cycling was performed by 30-second water filling and 15-second drainage steps, with temperatures ranging between 5 and 55 °c. microhardness and microtensile bond strength were evaluated in 32 teeth (n=8) each. nanoleakage evaluation was performed in 12 teeth (n=3) (figure 1). for the microtensile bond strength test, dental crowns were separated from the root portion, perpendicular to the long-axis of the tooth, with a double-sided diamond disc (kg sorensen). the crowns were set in a metallographic precision cutter (isomet 1000, buehler ltd., lake buff, il, usa). serial sections perpendicular to the long-axis of the crowns were cut with a high-concentration diamond disc (extec corp., enfield, ct, usa), used at low speed and under constant irrigation. this process resulted in stick-shaped samples (0.9 × 0.9 mm), where each stick included a portion of the bonding interface to the pulpal wall. the sticks were kept in an environment with relative humidity until the microtensile test. the fracture mode of each sample was evaluated on scanning electron microscopy (sem) and classified as adhesive, mixed, or cohesive in dentin or resin. for the knoop microhardness test, the crowns were separated from the root portions of the teeth as described above. the crowns were placed in a metallographic cutter and sliced into two parts through the centre of the restoration, in the mesiodistal direction parallel to the long-axis of the tooth. both parts of the tooth were embedded in the same cylinder of polystyrene resin, to facilitate microhardness testing. the restoration was finished with silicon carbide sandpaper (600-, 1200-, and 2000-grit) and polished by diamond paste (3-, 1-, and 0.25-mm granulations), applied with felt disks on a polisher underwater cooling. between each sandpaper treatment, the samples were ultrasonicated in distilled water for 5 minutes. to measure the microhardness, three indentations were made for 20 seconds each under a 25g load (hmv-2000, table 2. list of the groups of this study where ca = presence of acid etching and sa = absence of acid etching. pn = silorane and cz = clearfil se bond/z350 group enamel acid etching restorative systems restorative procedures capn present silorane systems after drying with air jets, actively primer was applied to enamel and dentin for 15 seconds with a microbrush, gentle air for 10 seconds of 10 centimeters apart and curing for 10 seconds. then the adhesive was applied with a microbrush across the cavity and gentle air jet for 10 seconds to 10 centimeters apart and curing for 10 seconds with halogen light unit qth lamp (bisco, schaumburg, illinois, usa). the teeth were restored with silorane composite resin in six increments cured for 40 seconds each with the same unit. sapn absence silorane systems cacz present clearfil se bond/z350xt it was actively applied the primer with a microbrush on enamel and dentin for 20 seconds and dried with gentle air for 10 seconds to 10 centimeters distance. then applied an even layer of adhesive to enamel and dentin with a microbrush for 20 seconds and cured for 10 seconds. the teeth were restored with composite resin z350xt in six increments and light cured for 40 seconds each with the same halogen light system used on silorane composite groups. sacz absence clearfil se bond/z350xt 5 coppini et al. shimadzu, japan) at three depths measured from the top of the tooth: 100 µm (top), 1500 µm (middle), and 2900 µm (base), at 25 µm from the mesial or distal wall. nanoleakage analysis was performed on 12 teeth (n=3). using a metallographic cutter machine, three mesiodistal cuts were made in each tooth, resulting in two 1-mm-thickslices. slices were immersed in a solution of ammoniacal silver nitrate for 24 hours, washed in distilled water, immersed in light developer for 8 hours, and then embedded in polystyrene resin16. embedded samples were polished with silicon carbide sandpaper and diamond paste (3 , 1-, and 0.25-µm granulations). samples were demineralized and deproteinated with 85% phosphoric acid and 2% hypochlorite. a b c d figure. asem photomicrography showing no silver deposits for the silorane systems with previous acid-etching; bsem photomicrography for the silorane systems without acid etching (arrows show a little infiltration of silver nitrate); cnanoleakage for the group clearfil/z350 xt with acid etching (arrows showing the silver nitrate moderate infiltration in the hybrid layer); dnanoleakage for the group clearfil/z350 xt without acid etching (arrows showing the silver nitrate moderate infiltration in the hybrid layer). 6 coppini et al. samples were dehydrated in serial ethanol solutions (25%, 50%, 75%, 90%, and 100%). dehydration was maintained by silica until the samples were ready to be coated with carbon (baltec sputtercoater scd – 050) for viewing by phenom microscope (dillenburgstraat 9e – netherland). results microtensile test results etching (p = 0.0015) and the interaction between the restorative system and etching had significant effects according to anova two-way. therefore, the post-hoc tukey test was applied for interaction (table 3), which revealed that samples restored by clearfil se bond/z350 without cavosurface enamel acid-etching had the lowest microtensile bond strength results among the groups (p < 0.05) (table 3). no other significant differences in microtensile bond strength were observed. all of the samples exhibited a mixed fracture pattern, with the adhesive showing the largest fracture areas and fractures also observed in the dentin. knoop hardness test for the microhardness test results, anova two-way only revealed statistically significant differences for the composite resin (p = 0.0001) and the interaction between the composite and cavosurface acid-etching (p = 0.0109). interactions between other factors or interactions had no statistical significance. therefore, the post-hoc tukey test was applied to the interaction between the composite and cavosurface acid-etching (table 4), which showed significant differences in microhardness between the z350 xt and silorane composite resins, independent of whether cavosurface acid-etching was performed and the depth at which the microhardness was measured. table 4. results of knoop hardness test (khn) for composite x cavosuperficial enamel etching. group mean (khn)/tukey sacz 96,398 (±3,54)a cacz 95,152 (±6,76)a capn 66,613 (±6,10)b sapn 64,194(±4,63)b table 3. results of tukey test to multiple comparations for microtensile test. group mean (mpa)/tukey cacz 25,66 (±5,62)a sapn 22,69 (±5,52)a capn 21,71 (±5,78)a sacz 12,16 (±4,33)b 7 coppini et al. nanoleakage results all of the samples were analysed for silver infiltration via phenom microscope. samples restored with silorane systems and clearfil se bond/z350xt with previous acid-etching showed low infiltration (figure 1a,b e c) of silver nitrate, whereas samples restored with clearfil se bond/z350xt without previous acid-etching showed moderate infiltration (figure 1d). discussion the first hypothesis of this study, that acid-etching would influence the quality of the bond strength on the pulpal wall, was found to be partially correct. statistically lower microtensile bond strength results were observed only for the groups restored with clearfil se bond/z350 xt without acid etching. the two-step self-etching adhesive system has good clinical performance when the adhesive is bound to dentin, but not to enamel6,17. selective etching has been shown to increase the bond strength of enamel18, improve the marginal sealing, and reduce the incidence of cracks19. acid-etching of the substrate increases the longevity of the restoration, by reducing degradation of the tooth/restoration interface, compensating for the characteristically poor penetration of the adhesive into the enamel12,13, and reducing the infiltration into and degradation of the internal walls. these previous findings are consistent with our observation that the bond strength of dentin after artificial aging was reduced in the absence of acid-etching of the cavosurface enamel. the study of the restoration under simulated aging showed that enamel etching was necessary for optimal use of the clearfil se bond, to prevent the hybrid layer from degrading. clearfil se bond can form a dense polymer network that imposes a certain resistance to water penetration20. compared to other adhesive systems, clearfil se bond reportedly provides satisfactory wettability in smear layer-covered dentin and the lowest contact angle in smear layer-free dentin21. nevertheless, these properties were not sufficient to promote good marginal sealing after artificial aging without acid-etching, probably due to lower interfacial stability caused by infiltration of the hybrid layer. in contrast, the silorane adhesive composite showed no difference in behaviour regardless of whether the enamel was conditioned. this two-step bonding system consists of a separately photoactivated primer and adhesive, which form a hybrid layer of 10 to 20 µm22. the primer and the bond exhibit conversions of over 90% and nearly 70%, respectively23. these high dcs lead to a more robust polymeric structure compared to partially polymerized adhesives, which are more permeable to fluid movement24. although clearfil se bond also has a high dc23, the highly hydrophobic nature of the silorane bond likely provides better sealing to dentin25 and creates an interface that is less prone to weakening26. the thick hybrid layer formed by the silorane adhesive system may act as an elastic buffer27, which would compensate for the polymerization shrinkage of the composite and the stress generated by artificial aging. due to its thinner hybrid layer and lower elasticity, clearfil se bond is less able to resist these stresses. the second hypothesis of this study, that silorane would present superior mechanical properties, was rejected. the silorane restorative system showed significantly lower microhardness values than the clearfil/z350 xt system, regardless of enamel etching 8 coppini et al. and the depth at which the microhardness was tested. the difference between the composites is mainly due to the type and amount of filler and the composition of the organic matrix28. the silorane composite resin has fewer filler particles22,28, which are irregular in shape but consistent in size, with a homogeneous distribution29, which explains the lower values of hardness. in contrast, the z350 xt composite has higher hardness due to its high content of inorganic nanoparticulate filler29, which undergoes strong intermolecular interactions within the monomer mixture30. our third hypothesis, that nanoleakage would not differ between the two tested restorative systems, was rejected. these images showed little infiltration of silver nitrate in the silorane restorative system and clearfil/z350 xt group with previous acid-etching, but moderate infiltration in the clearfil/z350 xt group without acid-etching. infiltration of silver ions in the latter group can be explained by the artificial aging, which increased the degradation of the hybrid layer. in contrast, the previous acid-etching for clearfil/z350 xt system promote a good marginal sealing, which prevented the infiltration of hybrid layer and increased the interfacial stability. for silorane system, the elastic buffering effect27 of the thick hybrid layer may have protected it from artificial aging, lowering the degradation. conclusion whereas acid-etching of the enamel substrate did not influence the behaviour of the silorane restorative system, it did increase the microtensile bond strength of the clearfil se plus/z350 xt restorative system, improving its performance under thermo mechanical aging. the z350 xt composite resin also had greater hardness than the silorane composite resin. silorane restorative system and clearfil se bond/z350 xt with previous acid-etching showed little infiltration whereas clearfil se bond/z350 xt without acid-etching showed moderate infiltration within the hybrid layer. 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different restorative composites. j biomed mater res b appl biomater. 2007 aug;82(2):346-51. 6. van meerbeek b, de munck j, yoshida y, inoue s, vargas m, vijay p, et al. adhesion to enamel and dentin: current status and future challenges. oper dent. 2003 may-jun;28(3):215-35. 9 coppini et al. 7. de munck j, van landuyt k, peumans m, poitevin a, lambrechts p, braem m, et al. a critical review of the durability of adhesion to tooth tissue: methods and results. j dent res. 2005 feb;84(2):118-32. 8. van landuyt kl, kanumilli p, de munch j, peumans m, lambrechts p, van meerbeek b. bond strength of a mild self-etch adhesive with and without prior acid-etching. j dent. 2006 jan;34(1):77-85. 9. knobloch la, gailey d, azer s, johnston wm, clelland n, kerby re. bond strengths of oneand two-step self-etch adhesive systems. j prosthet dent. 2007 apr;97(4):216-22. 10. uekusa s, tsubota k, tonegawa m, tsuchiya h, iwasa m, kawamoto r, et al. microtensile bond strengths of single-step self-etch adhesive systems to bovine teeth. j oral sci. 2007 sep;49(3):183-9. 11. bagis b, turkarslan s, tezvergil-mutluay a, uctasli s, vallittu pk, lassila lv. effect of ultrasonic agitation on bond strength of self-etching adhesives to dentin. j adhes dent. 2008 dec;10(6):441-5. 12. alexandre rs, sundfeld rh, giannini m, lovadino jr. the influence of temperature of three adhesive systems on bonding to ground enamel. oper dent. 2008 may-jun;33(3):272-8. 13. watanabe t, taubota k, takamizawa t, kurokawa h, rikuta a, ando s, et al. effect of prior acid etching on bonding durability of single-step adhesives. oper dent. 2008 jul-aug;33(4):426-33. doi: 10.2341/07-110. 14. soares gp, ambrosano gmb, lima danl, marchi gm, correr-sobrinho l, lovadino jr, et al. effect of light polymerization time, mode, and thermal and mechanical load cycling on microleakage in resin composite restorations. lasers med sci. 2014 mar;29(2):545-50. doi: 10.1007/s10103-012-1244-7. 15. porto iccm, aguiar fhb, brandt wc, liporoni pcs. mechanical and physical properties of silorane and methacrylate-based composites. j dent. 2013 aug;41(8):732-9. doi: 10.1016/j.jdent.2013.05.012. 16. tay fr, pashley dh, suh bi, carvalho rm, itthagarun a. single-step adhesives are permeable membranes. j dent. 2002 sep-nov;30(7-8):371-82. 17. perdigão j, lopes mm, gomes g. in vitro bonding performance of self-etch adhesives: ii—ultramorphological evaluation. oper dent. 2008 sep-oct;33(5):534-49. doi: 10.2341/07-133. 18. lima af, silva vb, soares gp, marchi gm, aguiar fhb, lovadino jr. influence of previous acid etching on interface morphology and bond strength of self-etching adhesive to cavosurface enamel. eur j dent. 2012 jan;6(1):56-62. 19. ermis rb, temel ub, celik eu, kam o. clinical performance of a two-step self-etch adhesive with additional enamel etching in class iii cavities. oper dent. 2010 mar-apr;35(2):147-55. doi: 10.2341/09-089-c. 20. malacarne j, carvalho rm, de goes mf, svizero n, pashley dh, tay fr, et al. water sorption/solubility of dental adhesive resins. dent mater. 2006 oct;22(10):973-80. 21. aguilar-mendoza já, rosales-leal ji, rodriguez-valverde ma, cabrerizo-vilchez ma. effect of acid etching on dentin wettability and roughness: self-etching primers versus phosphoric acid. j biomed mater res b appl biomater. 2008 jan;84(1):277-85. 22. mine a, de munck j, van ende a, cardoso mv, kuboki t, yoshida y, et al. tem characterization of a silorane composite bonded to enamel/dentin. dent mater. 2010 jun;26(6):524-32. doi: 10.1016/j.dental.2010.01.010. 23. navarra co, cadenaro m, armstrong sr, jessop j, antoniolli f, sergo v, et al. degree of conversion of filtek silorane adhesive system and clearfil se bond within the hybrid layer and adhesive layer: an in situ raman analysis. dent mater. 2009 sep;25(9):1178-85. doi: 10.1016/j.dental.2009.05.009. 24. tay fr, pashley dh, hiraishi n, imazato s, ruggeberg fa, salz u, et al. tubular occlusion prevents water-treeing and through-and-through fluid movement in a single-bottle, one-step self-etch adhesive model. j dent res. 2005 oct;84(10):891-6. 25. mine a, de munck j, van ende a, cardoso mv, kuboki t, yoshida y, et al. tem characterization of a silorane composite bonded to enamel/dentin. dent mater. 2010 jun;26(6):524-32. doi: 10.1016/j.dental.2010.01.010. 10 coppini et al. 26. sauro s, pashley dh, mannocci f, tay fr, pilecki ip, sherriff m, et al. micropermeability of current self-etching and etchand-rinse adhesives bonded to deep dentine: a comparison study using double-staining confocal microscopy technique. eur j oral sci. 2008 apr;116(2):184-93. doi: 10.1111/j.1600-0722.2007.00518.x. 27. van ende a, mine a, de munck j, poitevin a, van meerbeek b. bonding of low-shrinking composites in high c-factor cavities. j dent. 2012 apr;40(4):295-303. doi: 10.1016/j.jdent.2012.01.004. 28. tchorz jp, doll r, wolkewitz m, hellwig e, hannig c. microhardness of composite materials with different organic phases in deep class ii cavities: an in vitro study. oper dent. 2011 sep-oct;36(5):502-11. doi: 10.2341/10-325-l. 29. lien w, vandewalle ks. physical properties of a new silorane-based restorative system. dent mater. 2010 apr;26(4):337-44. doi: 10.1016/j.dental.2009.12.004. 30. sideridou i, tserki v, papanastasiou g. effect of chemicalstructure on degree of conversion in light-cureddimethacrylate-based dental resins. biomaterials. 2002 apr;23(8):1819-29. braz j oral sci. 15(1):57-61 original article braz j oral sci. january | march 2016 volume 15, number 1 salivary cortisol level and uncooperative behavior in pediatric dental practice ludmila tavares costa ercolin1, gláucia maria bovi ambrosano1, livia fernandes probst1, margarete c. ribeiro-dasilva2, scott l. tomar2, rosana de fátima possobon1 1universidade estadual de campinas – unicamp, piracicaba dental school, department of community dentistry, piracicaba, sp, brazil 2university of florida, college of dentistry, department of community dentistry & behavioral science, gainesville, fl, united states of america correspondence to: rosana de fátima possobon faculdade de odontologia de piracicaba unicamp av. limeira, 901 – cp.52 cep: 13414-903 – piracicaba – sp – brasil phone: +55 19 2106 5275 – fax: +55 19 2106 5218 e-mail: possobon@fop.unicamp.br abstract aim: to investigate the relation between uncooperative behavior and salivary cortisol level in children who underwent preventive dental care. methods: the sample was composed by 10 children of both sexes aged 40 to 52 months, presenting uncooperative behavior during dental preventive treatments. the saliva collection was performed using a cotton wheel and an eppendorf tube (sarstedt salivete®) in 3 different moments: a) at home, on a day without dental treatment and at the same time on the day of the sessions treatment; b) 30 min after the end of the session, when there was manifestation of uncooperative behavior; c) 30 min after the end of the session, when there was a cooperative behavior of the child. a sample of saliva was centrifuged for 5 minutes at 2400 rpm, 1 of ml of saliva was pipetted in an eppendorf tube and stored in a freezer at -20 ° c. for the determination of the levels of salivary cortisol was used an active® kit for cortisol enzyme immunoassay (eia) dsl-1067100, composed of specific rabbit antibody anti-cortisol. data were analyzed statistically for the uncooperative behavior issued in the beginning and at the end of sessions, using the paired t test (p<0.05) and for cortisol levels in saliva samples at home, after the beginning and at the end of sessions, using repeated-measures anova and tukey’s test (p<0.05). results: during expression of uncooperative behavior in preventive dental care sessions the salivary cortisol level was significantly higher (0.65 ± 0.25 µg/dl) compared with expression of collaborative behavior (0.24 ± 0.10 µg/dl). conclusions: it is possible to conclude that, even under preventive intervention, the stress must be controlled in order to reduce dental anxiety and fear. keywords: dental anxiety. salivary elimination. dental care. introduction anxiety and stress are deeply related to dental treatment. behaviors such as avoidance or escape during the treatment are commonly expressed by anxious patients, representing a barrier to receiving dental care, even when very necessary, compromising the individual oral health1,2. indeed, dental pediatric patients have no choice when they are taken by parents for dental treatment3. thus, children usually express their fears through behavior such as crying, refusal to open the mouth and physical aggression in an attempt to avoid the dental care4,5. in some cases, the child is referred to the dental office with a condition that does not require oral curative intervention but, even if only preventive procedures are attempted with low potential for generating pain and discomfort, manifesting a behavior of uncooperative with treatment. this experience can be very stressful for both, the dentist, children and their caregivers6. http://dx.doi.org/10.20396/bjos.v15i1.8647125 received for publication: july 14, 2016 accepted: august 02, 2016 58 some studies suggest that about 25% of children have bad behavior in the dental chair, which must be managed to ensure a minimum of technical quality of treatment7,8. in these circumstances, the professional must use strategies to teach children how to behave during the session, to minimize the aversive situation5. besides the observation of behavioral manifestations, an objective way to investigate the presence of stress is by measuring the levels of salivary cortisol, which usually change about 30 min after a stressor stimulus due to the activation of the hypothalamic-pituitary-suprarenal9,10. the involvement of glucocorticoids in the stress reaction was evidenced by selye11 (1936) that focused on the pituitaryadrenocortical axis as the effectors of this response. in humans, at least 95% of glucocorticoid activity of adrenal-cortical secretions is attributed to the cortisol hormone, also known as hydrocortisone, and its fundamental role in stress response been recognized ever since12,13. the plasma analysis of cortisol is considered a fair biomarker indicator of stress induced by the activity of the hypothalamic-pituitary-adrenal14. the assessment of cortisol in saliva is correlated positively with its blood concentration, reflecting about 5 to 10% of serum concentration15 and can perfectly substitute plasma for analysis. the salivary measure concentration of cortisol is technically simple, effective, noninvasive, free of stress, which is independent of salivary flow and fluctuations of transcortine with great potential for application16,17. kandemir et al.18 (1997) evaluated the level of salivary cortisol in children who had never had previous dental experience and concluded that the situation of curative treatment can be stressful for these patients. akyuz, pince and hekin19 (1996) showed that there is an increased level of children salivary cortisol passing through dental restorative procedures. the authors reported that cavity preparation was the procedure with greater stressor potential among children in their sample. however, there are no reports of such scientific research related to non-invasive dental procedures. therefore, this study investigated the level of salivary cortisol among children undergoing preventive dental treatment. material and methods the study design was reviewed and approved by the committee on human research from piracicaba dental school – state university of campinas, under the protocol: 124/2005. the sample were composed by 10 children of both sexes aged 40 to 52 months, presenting uncooperative behavior during dental preventive treatments (removal of dental biofilm, brushing training done by the mother and the dentist, and clinical examination). in order to reduce children’s stress and increase their cooperation, experimental sessions were planned with successive steps of approach, with presentation of clinical procedures and gradual invasiveness. thus, respecting the pace of each child, the dentist showed the brushing technique on a dummy, took the salivary cortisol level and uncooperative behavior in pediatric dental practice braz j oral sci. 15(1):57-61 child to the sink to brush his/her own teeth, applied a colorful plaque disclosing agent, brush the child’s teeth, led him/her to the dental chair to conduct the clinical examination using intraoral mirror and air jet under reflector light and prophylaxis with pumice and rotating robson brushes. during these sessions, the dentist employed behavior management strategies, such as distraction (songs and stories) and positive reinforcement (with compliments and gifts)5. all sessions were filmed and subsequently examined to determine uncooperative behaviors of the children (crying, movements of body and head in order to prevent or stop the procedure and verbal refusal). the researchers divided the total session time at intervals of 15 s and recorded all behaviors expressed during each interval. the saliva collection was performed using a cotton wheel and a tube of the type eppendorf (sarstedt salivete®) at 3 moments: a) the child’s at home, on a day without dental treatment and at the same time on the day of the treatment sessions, b) 30 min after the end of the session, when there was manifestation of uncooperative behavior, c) 30 min after end of the session, when there was the collaboration of the child. these sessions were always scheduled in the afternoon to avoid the influence of circadian cycle on the cortisol secreted level. a sample of saliva was centrifuged for 5 min at 2400 rpm, 1 ml was pipetted saliva in an eppendorf tube and stored in a freezer at -20 °c. for the determination of the levels of salivary cortisol was used an active® kit for cortisol enzyme immunoassay (eia) dsl-10-67100, composed of specific rabbit antibody anti-cortisol. the procedure followed the basic principle of enzyme immunoassay where there is a competition between an antigen enzyme not labeled with a certain number of antibody binding sites. the amount of antigen labeled with enzyme is inversely proportional to the concentration of analyte present unchecked. the unbound material is removed by decanting and washing the cavities. the absorbance of the solution was read in an elisa microplate reader with 450 nm and the ability to fix the double wavelength adjusted to 600 nm. data were analyzed statistically for the uncooperative behavior issued in the beginning and at the end of sessions, using the paired t test (p<0.05) and for cortisol levels in saliva samples at home, after the beginning and at the end of sessions, using repeated-measures anova and tukey’s test (p<0.05). results statistically significant differences were found between the average of the absolute frequency of uncooperative behavior by the children during the beginning and at the end of session (table 1). the level of salivary cortisol also showed significant variation among the collections made at home, after the beginning and at the end of sessions (table 2). the relationship between the salivary cortisol level and the frequency of uncooperative behavior can be verified in figure 1. this figure shows that in child presenting aversion to treatment, session frequency of behaviors per intervention beginning 44.6 ± 16.72a end 5.40 ± 3.92b table 1 absolute frequency average and standard deviation of uncooperative behavior by the children during the beginning and at the end of intervention session. averages followed by different letters differ vertically by paired t-test (p<0.05). 59salivary cortisol level and uncooperative behavior in pediatric dental practice braz j oral sci. 15(1):57-61 session salivary cortisol level home 0.22 ± 0.11a beginning 0.65 ± 0.25b end 0.24 ± 0.10a table 2 salivary cortisol levels (µg/dl) averages and standard deviations from collections made in the child’s home, after the beginning and at the end of dental intervention session. averages followed by different letters in the vertical differ by tukey test (p <0.05). the salivary cortisol level were higher when compared to session in which the child cooperated. discussion the determination of salivary cortisol has been evaluated in relation to deprivation of sleep in patients who are night workers20, in patients with chronic fatigue21 and for assessment of stress during dental treatment in adults22,24. however, few studies have evaluated the stress related to levels of cortisol among children in the dental situation, and those who have been, investigated the reaction of child during curative treatment18,19. these studies indicated that some invasive procedures, such as oral anesthesia, cavity preparation with high-speed handpiece and third molar extraction surgery are stressful for patients, with significant variation in salivary cortisol levels before and after the intervention. studies on the behavioral manifestations of the child in fearful dental situation also highlight certain clinical procedures as more aversive suggesting psychological approaches and/or pharmacological intervention to decrease patient stress5,6,25,26. however, as studies with determination of salivary cortisol18,19, this work investigated the conduct issued by children exposed only to the healing of dental treatment. this seems to be due to the fact that non-cooperation is a greater challenge than the curative action of preventive dentistry-whose performance depends directly on the clinical behavior of the patient. fig.1. immunohistochemical expression of timp-2 / oscc negative control. 60 this study showed that child who exhibit uncooperative behavior, or express fear of dental treatment, by crying or refusal to allow the dentist actions, also show high levels of cortisol in the context of preventive dental treatment. simple and less invasive, such as application of dye biofilm-brushing and clinical survey can trigger child fearful stress reactions. several authors have reported that stress in patients leads to the manifestation of non-cooperation pediatric dentistry behavior that hinder or prevent the completion of dental procedures27. some authors suggest that fearful children have often a painful history of treatments and/or unpleasant events in their first visits to the dentist26-28. the children in this study had not experienced dental curative treatments, but they had been exposed several times to preventive procedures. however, the children were not adapted to routine dental care because the goal in this study was to perform the preventive dental procedures and not to decrease the dental fear. as possobon et al.27 (2007) claim, when the professional is basically focused on technical procedures, child manifestation of fear and stress may not be noted or understood, depriving the necessary physiological support to the patient. the authors state that dental professionals might implement strategies to identify and then minimize the stress commonly generated by dental intervention, monitoring behaviors indicative of stress. thus, during the experimental sessions, in which psychological strategies were employed for managing behavior, there was a gradual decrease in the frequency of children’s behavior, and at the same time, a reduction in salivary cortisol level was noted, showing the relationship between behavioral and physiological manifestations of stress. even though curative interventions might eventually be necessary, preventive procedures must occur periodically over a child’s life. therefore, dentists must adopt measures to manage behaviors indicative of stress and anxiety in order to reduce escape and avoidance from routine dental visits29, allowing a good quality of intervention and also preventing unnecessary stressful situations to children. references 1. appukuttan dp. strategies to manage 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[portuguese]. braz j oral sci. 15(1):57-61 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1543/1196 1/9 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1543/1196 2/9 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1543/1196 3/9 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1543/1196 4/9 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1543/1196 5/9 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1543/1196 6/9 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1543/1196 7/9 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1543/1196 8/9 28/05/2019 pdf.js viewer https://www.fop.unicamp.br/bjos/index.php/bjos/article/view/1543/1196 9/9 1http://dx.doi.org/10.20396/bjos.v18i0.8657250 volume 18 2019 e191499 original article 1 department of surgery, stomatology, pathology and radiology. são paulo university, bauru dental school (fob/usp), bauru, sao paulo, brazil 2 federal university of ceará, fortaleza dental school, fortaleza, ceará, brazil corresponding author: eduardo sanches gonçales. fob/usp. departamento de cirurgia, estomatologia, patologia e radiologia. al. otávio pinheiro brizolla, 9-75. cep: 17017901. tel: +55 14 32358258. e-mail:eduardogoncales@usp.br conflict of interest disclosure: authors declare no conflict of interest. funding: fundação de amparo à pesquisa do estado de são paulo (fapesp) process: 201106280-0. received: january 10, 2019 accepted: june 13, 2019 condylar positioning in orthognathic surgery: a cone beam computed tomography-based in vitro analysis of a positioning method victor tieghi neto1, andréa guedes barreto gonçales1, alexandre simões nogueira2, osny ferreira júnior1, eduardo sanches gonçales1,*. aim: orthognathic surgery aims to correct facial skeletal deformities and the correct condylar positioning is very important for stable results. the aim of the present study was to verify the occurrence of changes in the postoperative condylar positioning in artificial skulls with a skeletal class ii maxillomandibular relationship submitted to bilateral sagittal split osteotomy when the method of cephalometric data transfer was used. methods: ten skeletal angle class ii polyurethane skulls were used with metallic markers in the articular surfaces of the temporomandibular joint and mandibular condyles. the skulls were submitted to preoperative and postoperative cone beam computed tomography before and after the bilateral sagittal split osteotomy. to verify the condylar positioning, measurements between the distances of the markers at the temporal bones and mandibular condyles were taken in the coronal and sagittal views by the distance tool of the icat vision software. all measurements were obtained by one examiner in the preoperative and postoperative cbcts, tabulated and submitted to statistical analysis by the wilcoxon test with a level of significance of 5% (p<0,05). after 15 days of the completion of the first data collection, all measurements were redone to determine the random and systematic error by the intraclass correlation coefficient. results: with the exception of the average of the lateral-medial distance (from the measurements between the medium left markers only), the averages of the anterior-posterior distances (only in the left posterior and lateral right markers) and the vertical 2 neto et al. introduction facial skeletal deformities can be characterized by the underdevelopment or hyper-development of the facial bones, especially in the maxilla and mandible; such changes might occur in the transverse, anterior-posterior and vertical directions1. the aim of orthognathic surgery is the correction of these deformities2,3 and it is performed with the use of le fort i osteotomy (in cases where the maxilla is involved), bilateral sagittal split osteotomy of the mandible (in cases involving the mandible) or the association of both osteotomies (in cases where the maxilla and the mandible are involved)4. there are effects on the condyles following the correction of facial skeletal deformities even in isolated maxillary deformities5 and during the execution of mandibular orthognathic surgery, the correct positioning of the condyle, and consequently the mandibular proximal segment, is essential for better and stable results. the inappropriate positioning of this segment is undesired6 and may result in recurrence, loss of mandibular angle, condylar displacement, pain, temporomandibular joint (tmj) dysfunction and disability7. several methods and devices to control the positioning of the proximal segment have been suggested over the years8 with good results9-18or not19,20, maintaining unanswered questions on this topic21. there is no scientific evidence supporting the routine use of condylar positioning devices (cpd) in orthognathic surgery19,22. in this context, there is no scientific evidence that desired postoperative condilar position is the same pre-operative condylar position, and there are many reasons for this17. however, it seems to be acceptable that if there aren’t pre-operative signs and symptoms of tmj problems, the pre-operative condyle position is good and it is desired in the postoperative moment. according to perez and liddell23, there are few reliable data regarding the possibility of the cpd maintaining the condyle in the desired position during orthognathic surgery or if this is relevant for success. more important than which cpd is the best, is to be certain with respect to the passive position of the proximal segment23. the aim of the present study was to verify the occurrence of changes in postoperative condylar positioning in artificial skulls with a skeletal class ii maxillomandibular relation submitted to bilateral sagittal split osteotomy when the method of cephalometric data and surgical plan transfer were used13,14. average (only in the central markers) showed no statistically significant differences between the preoperative and postoperative distances of the metallic markers. conclusion: even when using the method of cephalometric data transfer, variation of the condylar positioning occurred between the preoperative and postoperative periods. this variation occurred only in a few points of the mandibular condyles. keywords: orthognathic surgery. mandibular condyle. computed tomography. 3 neto et al. materials and methods ten artificial skulls developed in hard polyurethane with barium (nacional ossos, jau sp) were used. they consisted of skeletal class ii types and had the muscles of mastication and presented metallic markers (titanium screw with 5mm diameter and 5mm length) at the medial, anterior, posterior and lateral ends of the joint cavity and in the center of the joint cavity. there were also markers at the lateral, medial, anterior and posterior poles of the condyle joint surface and in the center of the mandibular condyle joint surface. such markers were installed bilaterally by the team of researchers, seeking to install them in a coincidental manner between the glenoid fossa and the condyle (figure 1). all screws were inserted with the use of a cylindrical drill 1.1mm in diameter and a manual screw driver. after the installation of the markers, the skulls were submitted to preoperative cbct (group 1: control). the scans were performed on an i-cat classic (imaging science international, hatfield, pennsylvania, usa) using the following protocol: 0.3mm voxel and extended height 20/20sec. next, they were submitted to the mandibular advancement procedure, which was performed by means of bilateral sagittal split osteotomy of the mandible with advancement of 10mm using the cephalometric data and surgical plan transfer method13,14, which can be summarized as follows: sagittal osteotomy was performed on the mandible on one side without the split. the 2.0 system four role plate was positioned in the oblique line, parallel to the mandible occlusal plane and screwed with 2 screws only in the proximal fragment. with a 1/2 size spherical bur, the insertion points of the screws on the plate in the distal fragment were drilled and the plate was removed. new holes were done with a surgical ruler 10mm distal from the holes done with the 1/2 size spherical bur. next, new holes were drilled again with the 1.5 drill to the 2.0 plate system. we proceeded with the sagittal split osteotomy of the mandible, put the plate again in the proximal segment with screws in the same holes that were first screwed in the distal fragment of the mandible in the holes that were screwed with figure 1. metallic markers at the medial (1), anterior (2), posterior (3) lateral (4) and central (5) positions of the glenoid glenoid fossa and condyle. 2 5 1 3 4 1 3 2 5 4 4 neto et al. the 1.5 drill. this automatically advanced the mandible 10mm (figure 2 and 3). this sequence was done bilaterally. after completion of the mandibular advancement (figure 3), the same 10 skulls were again submitted to postoperative cbcts (group 2: experimental) to verify the condylar positioning of both groups (control and experimental) via the measurement of the lateral-medial distances (in coronal reformatting), anterior-posterior and vertical distances (both in the sagittal reformatting) that were measured between the long axis of the metallic markers installed in the glenoid fossa and in the mandibular condyle, figure 2. view of the sagittal split osteotomy (without the split at this moment) in a mandible prototype with a plate and 02 screws in the proximal fragment and the holes done with a number 1/2 spherical bur inside the plate holes at the distal fragment (a). after removal of the plate, the digital caliper measured 5mm to the posterior position from the two holes done with the 1/2 spherical bur. these two news holes will be the screw holes for the plate in the distal fragment (b). then the split is done and the distal fragment moves forward and the plate and the four screws return to their original position (two in the holes of the proximal fragment and two in the new holes 5mm back in the distal fragment). in this example, the mandible will automatically be advanced 5mm (c) a b c figure 3. lateral view of artificial skull after 10mm mandibular advancement. 5 neto et al. always at the level of the head of the screw located in the condyle and perpendicular to the long axis of the same. all scans were performed on an i-cat classic (imaging science international, hatfield, pennsylvania, usa) using the following protocol: 0.3mm voxel and extended height 20/20sec. the cone beam computed tomography (cbct) was filed on a portable hard drive and the linear measurement tool (distance) of the i-cat vision program was used for obtaining the measurements as described below; the measurement of the changes between the distances of the metallic markers (screws) on the coronal reformatting were done via the demarcation of the lines that matches the long axis of the screws with the use of the distance tool of the software at the level of the screw head, perpendicular to the lines previously marked and then the distance between the lines were measured (figure 4). the measurement of the changes between the anterior-posterior distances of the metallic markers (screws) on the sagittal reformatting began with the demarcation of the lines in blue and red, which represented the long axis of the screws and were done using the distance tool of the software. at the top of the screw head in the condyle, the distance between the lines were measured perpendicularly (figure 5). the measurement of the changes between the vertical distances of the metallic markers (screws) on the sagittal reformatting were done via the demarcation of perpendicular lines from the top of the head of the screws using the distance tool of the software and then the distance between them were measured (figure 6). all measurements were obtained by one examiner in the preoperative and postoperative cbcts, tabulated and submitted to statistical analysis by the wilcoxon test with figure 4. measuring the changes between the distances of the metallic markers (screws) on the coronal reformatting. lines (blue, red, pink and light pink) representing the long axis of the screws were done using the distance tool of the software at the level of the screw head of the condyle, perpendicular to the lines previously marked, representing the long axes of the screws, and the distance between the lines were measured. in this picture, the distances between the lines of the long axes of the screws of the glenoid fossa and condyle were 1.34 mm and 0.90 mm (green and purple lines). flat d = 22.45 mm flat d = 17.33 mm flat d = 1.34 mm flat d = 0.00 mm flat d = 17.70 mm flat d = 12.24 mm flat d = 0.90 mm 6 neto et al. a level of significance of 5% (p<0,05). after 15 days of the completion of the first data collection24, all measurements were redone to determine the random and systematic error by the intraclass correlation coefficient (icc)25. results the results showed that, with the exception of the average of the medial-lateral and medial distances (only between the metallic markers on the left side), and the anterior-posterior middle distances (only in the positions of the left posterior and lateral right figure 5. measuring changes between the anterior-posterior distances of the metallic markers (screws) on the sagittal reformatting. lines (blue and red) representing the long axis of the screws and were done using the distance tool of the software. at the top of the screw head of the condyle, the distance between the lines (blue and red) were measured perpendicularly (green result that is showed at the picture upper left corner = 0.0mm). flat d = 12.26 mm flat d = 10.15 mm flat d = 0.00 mm p i figure 6. measuring changes between the vertical distances of the metallic markers (screws) on the sagittal reformatting. perpendicular lines (blue and red) from the top of the head of the screws were done using the distance tool of the software and the distance between them were measured (green vertical line). in this picture, the green line represents the vertical distance between the upper and lower lines and it was 2.70 mm flat d = 12.90 mm flat d = 11.40 mm flat d = 2.70 mm p t 7 neto et al. side markers) and the vertical average (only in the positions of the central markers), there were no statistically significant differences between the pre and post distances of the screws (metallic markers). tables 1 to 3 show the averages, standard deviation (sd) and the result of the statistical test of the distances between the screws at the glenoid fossa and mandibular condyle in the preoperative and postoperative periods of both sides in the coronal reformatting (medial-lateral measures) and sagittal reformatting (anterior-posterior and vertical measures). for error analysis, the icc was used; all measures were carried out by the same examiner, respecting the time of no less than 15 days. the icc was excellent since all their values were greater than 0.75. table 1. averages, standard deviation (sd) and statistical test result of the distances between the screws at the glenoid fossa and mandibular condyle in the preoperative and postoperative periods of both sides. *significant (p <0.05). side screw pre post lateral 1.47 (1.18) 1.5 (0.75) central 1.39 (0.52) 1.5 (0.97) right medial 2.52 (1.09) 2.58 (0.79) anterior 1.41 (0.93) 1.32 (1.23) posterior 1.26 (0.78) 1.86 (0.93) lateral 1.74 (0.83) 2.28 (1.13) central 0.63 (0.63) 0.84 (0.85) left medial * 0.87 (0.81) 1.83 (0.98) anterior 0.72 (0.8) 1.08 (0.79) posterior 0.69 (0.74) 0.69 (0.8) table 2. averages, standard deviation (sd) and statistical test result of the anterio-posterior distance (sagittal reformatting) between the screws of the glenoid fossa and mandibular condyle in the preoperative and postoperative periods of both sides. *significant (p<0.05). side screw pre post lateral* 1.11(0.69) 1.98(0.91) posterior 1.98(1.04) 2.25(1.07) right central 0.9(0.69) 1.17(0.57) anterior 0.48(0.47) 0.57(0.6) medial 1.08(1.19) 1.02(1.02) lateral 0.96(1.39) 1.83(1.42) posterior* 2.19(1.44) 2.85(1.9) left central 1.32(0.95) 1.65(1.16) anterior 0.9(1.24) 1.38(1.19) medial 1.95(0.82) 2(0.83) 8 neto et al. discussion according to ueki et al.26, mandibular bilateral sagittal split osteotomy is a procedure indicated for the correction of dentofacial deformities, however, changes in the mandibular condylar position arising from surgery may lead to malocclusion, higher risk of relapse and the development of temporomandibular joint disorders. epker and wylie27, believed that the maintenance of the preoperative anatomical position of the condyle after surgery was important and luhr28, defended the use of cpd for maintaining the centric relation and the preoperative condylar position in the postoperative period. therefore, the use of cpd in some studies was9-19 considered beneficia9-18 while in others it wasn`t19,20 ,allowing ellis21 to emphasize that questions related to the use of cpds remain unanswered. the review conducted by costa et al.22 found that there was no scientific evidence to support the routine use of cpds in orthognathic surgery. in 1997 and 2007, puricelli13,14 published a method based on the use of the fixation plate to transfer the cephalometric data and surgical plan during orthognathic surgery that might keep the original position (preoperative) of the mandibular condyle. the method in that in vitro study seems to be effective in maintaining the preoperative position of the mandibular condyle, because, even with few exceptions, no other portion (either left or right condyle) showed statistically significant variations between the preoperative and postoperative periods in a 10mm mandibular advancement. however, standing out in this context, the methodology employed in that study may have interfered in the results because it was an “in vitro” study done on an artificial skull without the muscles that obviously reproduce the characteristics of living human tissue. the tmj employed, for example, did not have a capsule and articular disk. in addition, the artificial muscle texture differed from the natural musculature. in this context, puricelli et al.29 analyses by finite element analysis (fem), the same osteotomy used in that study, stated that their results suggest, in vivo, larger and table 3. averages, standard deviation (sd) and statistical test result of the vertical distances (sagittal reformatting) between the screws of the glenoid fossa and mandibular condyle in the preoperative and postoperative periods of both sides. *significant (p<0.05). side screw pre post lateral 1.6(0.9) 1.17(0.69) posterior 0.81(1) 0.42(0.42) right central 0.24(0.30) 0.48(0.45) anterior 1.2(0.95) 1.14(0.54) medial 0.6(0.58) 0.6(0.5) lateral 2.52(0.82) 2.58(1.6) posterior 0.39(0.56) 0.93(1.1) left central* 0.3(0.4) 0.93(0.76) anterior 1.5(0.76) 1.71(0.77) medial 0.54(0.41) 1.26(1.20) 9 neto et al. more adjusted bone contact among bone fragments and decreased displacement due to muscle activity. nevertheless, the method employed in that study proved to be simple and applicable and may give us important information regarding the positioning of the mandibular condyle in orthognathic surgery, with the use or not of cpd. it should be noted that the experimental model used has limitations as exposed above, and it becomes a necessary caution to extrapolate these findings to real-life situations, and that further studies with experimental models closer to living humans appear to be necessary. a good question would be: why did only 4 points measured showed statistically significant changes between the preoperative and postoperative periods? it is believed that this occurred because of the rotational asymmetric condylar movements that interfered solely with the positioning of the markers in specific positions (medial markers on the left side in the coronal reformatting, lateral-medial measures, lateral right and posterior left markers in the anterior-posterior measures and central left in the vertical measures at sagittal reformatting). in addition, it could be due to mistakes in measurements between the markers in this region, however, even if this was considered true, it seems not to have interfered because 4 significant differences represent 8 points that could be mistakenly measured among the 60 points measured, which represent 13.33% of error. another question is: can the significant changes in the condylar positioning be translated into clinical problems? this is a question impossible to answer with the present study because there’s no clinical data here, however, it is believed that because they are small changes and in a minority of the points studied, they probably will not be clinical problems. another point to discuss regards the occurrence of any metal-related artifact from the screw in the tmj; and in the present study, probably because of the cbct image and the size of the screw, we didn’t have a metal artifact that could hinder the analysis of the condylar position, as can be seen in figures 4, 5 and 6. although some studies26,27 advocated the maintenance of the preoperative condylar positioning in the postoperative period, another current issue is which is the desirable postoperative position of the mandibular condyle in orthognathic surgery22? the present study, for reasons already exposed above, is limited to answering this question though it seems to agree with epker and wylie27 and luhr28, since the preoperative position of the mandibular condyles in most studied points are maintained (86.67%). it is lawful to believe that, in individuals with tmj disorders in the preoperative time, the same preoperative condylar position may not be desirable for the postoperative time. as a result, in individuals free of tmj disorders, the preoperative position seems to be desirable, and the use of the technique presented here might be useful. in conclusion, bilateral sagittal split osteotomy of the mandible associated with the method of cephalometric data and surgical plan transfer changed the condylar positioning in a few specific points at the postoperative time. acknowledgements fundação de amparo à pesquisa do estado de são paulo (fapesp) and nacional ossos for supporting this project. financial support: fapesp. process: 201106280-0. 10 neto et al. references 1. gonçales es, duarte mah, palmieri jr c, zakhary gm, ghali eg. retrospective analysis of the effects of orthognathic surgery on the pharyngeal airway space. j oral maxillofac surg. 2014 nov;72(11):2227-40. doi: 10.1016/j.joms.2014.04.006. 2. stricker m, van der meulen jc, raphael b. craniofacial 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11 neto et al. 19. bettega g, dessenne v. computer-assisted mandibular condyle positioning in orthogna thic surgery. j oral maxillofac surg. 1996 may;54(5):553-8. 20. renzi g, becelli r, paolo c, iannetti g. indications to the use of condylar repositioning devices in the surgical treatment of dental-skeletal class iii. j oral maxillofac surg. 2003 mar;61(3):304-9. 21. ellis e. condylar positioning for ortognathic surgery: are they necessary?. j oral maxillofac surg. 1994 jun;52(6):536-54. 22. costa f, robiony m, toro c, sembronio s, polini f, politi m. condylar positioning devices for orthognathic surgery: a literature review. oral surg oral med oral pathol oral radiol endod. 2008 aug;106(2):179-90. doi: 10.1016/j.tripleo.2007.11.027. 23. perez de, liddell a. controversies in orthognathic surgery. oral maxillofac surg clin north am. 2017 nov;29(4):425-440. doi: 10.1016/j.coms.2017.07.008. 24. houston wjb. the analysis of errors in orthodontic measurements. am j orthod. 1983 may;83(5):382-90. 25. fleiss jl. analysis of data from multiclinic trials. control clin trials. 1986 dec;7(4):267-75. 26. ueki k, moroi a, sotobori m, marukawa m, takatsuka s, yoshizawa k et al. a hypothesis on the desired postoperative position of the condyle in orthognathic surgery: a review. oral surg oral med oral pathol oral radiol. 2012 nov;114(5):567-76. doi: 10.1016/j.oooo.2011.12.026. 27. epker bn, wylie ga. control of the condylar-proximal mandibular segments after sagittal split osteotomies to advance the mandible. oral surg oral med oral pathol. 1986 dec;62(6):613-7. 28. luhr hg. the significance of condylar position using rigid fixation in orthognathic surgery. clin plast surg. 1989 jan;16(1):147-56. 29. puricelli e, fonseca jso, paris mf, sant’anna h. applied mechanics of the puricelli osteotomy: a linear elastic analysis with the finite element method. head face med. 2007 nov;3:38-45. revista fop n 13 1559 braz j oral sci. april/june 2008 vol. 7 number 25 cytogenetic damage in khaini users of tamilnadu,cytogenetic damage in khaini users of tamilnadu,cytogenetic damage in khaini users of tamilnadu,cytogenetic damage in khaini users of tamilnadu,cytogenetic damage in khaini users of tamilnadu, southern indiasouthern indiasouthern indiasouthern indiasouthern india vellingiri balachandar1; balasubramaniam lakshmankumar2; kuppanan suresh3; pappusamy manikantan3; raman sangeetha2; subramaniam mohanadevi3; keshavarao sasikala4 1m.sc., m.phil., phd., research scholar 2m.sc., ph.d, research scholar 3m.sc., m.phil, research scholar 4m.sc., ph.d., professor and head human genetics laboratory, school of life sciences, bharathiar university, coimbatore. tamilnadu, india received for publication: december 06, 2007 accepted: june 10, 2008 correspondence to: vellingiri balachandar human genetics laboratory, department of zoology, school of life sciences bharathiar university, coimbatore – 641046 tamilnadu, índia tel: +91-422-2422222 (extn: 482). fax: +91-422-2423837 e-mail: geneticbala@yahoo.co.in a b s t r a c t aim: the smokeless tobacco (st) has a strong association with the risk of oral leukoplakia (ol), oral submucous fibrosis (osf) and oral cancer (oc). st components exhibit genotoxicity and may alter the structure of dna, proteins and lipids, resulting in the production of antigenicity. in this study, an attempt was made to estimate the cytogenetic damage [chromosomal aberrations (ca) and micronucleus (mn)] in people habituated to consume khaini (st), which is one of the major forms of tobacco consumption in tamilnadu, india, and believed to be a major risk factor for oc. methods: after signing a consent form, volunteers provided blood samples (108 samples from including experimental and control subjects) to establish cell cultures at 52 h. for ca analysis, 100 complete metaphase cells per subject were evaluated. chromatidand chromosomaltype aberrations were identified in experimental and control subjects, where the latter showed a very minimal number of ca in age wise manner. results: statistically significant results were obtained in experimental subjects when compared to controls as confirmed by chi-square test. exfoliated cells from the buccal mucosa of khaini users were examined by using the micronucleus assay. the difference in mean micronucleated cell count for buccal mucosa between cases and controls were significant (p<0.01). hence, specific biomarkers on cytogenetic endpoints might help in establishing preventive measures to reduce cancer risks. conclusion: the genotoxic effect of smokeless tobacco should be considered in addition to other known hazards for assessing health risks. key words: oral cancer, smokeless tobacco, khaini, chromosomal aberration, micronucleus. i n t r o d u c t i o n cancer is currently one of the most dreaded diseases of which head and neck squamous cell carcinoma is the fifth most common. oral cancer affects as many as 274,000 people worldwide annually, and the frequency is often indicative of the patterns of tobacco use1,2, which is a major contributor to deaths from chronic diseases. smokeless tobacco (st) contains considerable nicotine much more than is contained in cigarette tobacco3. st perceived as a safer alternative to smoking, also contains 28 carcinogenic agents, including nitrites and alkylating agents4-5. numerous different forms of st have been used worldwide. khaini, an interesting kind of st, is commonly used instead of cigarettes in the tamilnadu region, southern india, especially in ooty and coimbatore. tobacco, slaked lime paste and areca nut are the major components in khaini, and a small amount of this mixture (approximately 1 g) is applied to the mucosa of the lower lip for 10-15 min and then it is spit out. this procedure is repeated many times during the day. pharmacological studies have shown that this kind of st is a form of buccal nicotine use6,7. the present study underscores the concepts of a molecular epidemiology to detect humans with a high cancer risk with the aid of valid biomarkers, such as biomonitoring. specific biomarkers on cytogenetic endpoints may help in establishing preventive measures to reduce cancer risks8. cytogenetic analysis has also been applied to the biological monitoring of human populations exposed to mutagenic and carcinogenic agents. the mutagenic and carcinogenic effects of st have been previously demonstrated in habitual users9,10. the frequency of chromosomal aberrations (ca) in peripheral blood lymphocytes is a relevant biomarker 1560 for cancer risk in humans, reflecting either early biological effects of genotoxic carcinogens or individual cancer susceptibility. micronucleus (mn) in buccal cells is a sensitive method for monitoring genetic damage in human populations11-13. in the present study, the assessment of cytogenetic damage might help understanding the mode of action of these obnoxious agents, which provide an inexpensive alternative to the smoking types, and the cancer risk involved. despite several earlier reports regarding the use of st from various regions around the world, tamilnadu being home to a more diverse number of such khaini products might aid in the assessment of chromosomal damage leading to tumorigenesis. therefore, this study investigated whether individuals with khaini habit present more ca with the increase in the exposure period and assessed whether prolonged habitual st leads to an increase in the number of buccal epithelial cells, by analysis using the mn assay. although st has been extensively investigated, to the best of our knowledge, this work is the first to perform a cytogenetic analysis in khaini users in this region. material and methods subject recruitment and sample collection the subjects for this study comprised 104 randomly selected male subjects with no immediate history of viral disease (i.e., during the past 3-4 months). the study groups comprised 52 individuals habituated to st consumption but without any other habits like smoking, pan chewing or alcohol use. an equal number of healthy subjects who did not use any form of tobacco or alcohol and had not been exposed to any kind of chemicals or radiation were selected as controls. cases and controls were omnivores, with no apparent nutritional deficiencies. a questionnaire arguing on lifestyle factors, smoking, alcohol consumption, personal information, age, health status, use of medication and exposure parameters, was used to interview the subjects. age matched controls (±2 years) were recruited to the respective subjects. all cases were exclusively khaini users at the time of the study, consumers for a period of 5 years or more. the cutoff of at least 4 cans/ pouches per week was established to ensure subject safety considering that the use of nicotine patch doses up to 63 g/ day in st users had only been previously reported in a case series14. the subjects were grouped as follows on the basis of age: group i d”30 years and group ii e”31 years. venous blood samples (5 ml) were drawn in heparinized syringes from each subject for the chromosomal analysis. the work was carried out in accordance with the ethical standards laid down in the 1964 declaration of helsinki. chromosomal aberration assay all chemical reagents were purchased from sigma chemicals, except for colcemid, which was obtained from gibco laboratory. blood samples were set up to establish leukocyte cultures in our laboratory following standard procedures15. 0.5 ml blood was added to 4.5 ml rpmi 1640 medium supplemented with 10% calf fetal serum, 2 mm l-glutamine, 1% streptomycin-penicillin, 0.2 ml reagent grade phytohemagglutinin, and was incubated at 37 ºc. after 50 h, cultures were treated with 0.1 g/ml colcemid to arrest the cells at metaphase in mitosis. lymphocytes were harvested after 52 h by centrifuging cell suspension to remove culture medium (800-1000 rpm), addition of hypotonic solution (kcl 0.075 m) at 37 ºc for 20 min to swell the cells, and treated twice with carnoy’s fixative (3:1 ratio of methanol: acetic acid). slides were carefully dried on a hot plate (56ºc, 2 min). three days later, the slides were stained using the giemsa technique. the observations were made by a researcher for ca on a routine basis. all authors in the research team made critical observations and recorded the results. for the ca analysis, 100 well spread complete metaphase cells in first cell cycle were evaluated per subject under a microscope at ×100 magnification to identify numerical and structural ca. chromatid-type cas: (chromatid gaps; chromatid breaks) chromosome-type cas: (break; gap; exchange) were observed. the collected data were registered on master tables and later transferred to a computer file. collection of cells and micronucleus assay subjects were asked to rinse their mouths with water and a premoistened wooden spatula was used to sample cells from the buccal mucosa. the spatula was applied to a pre-cleaned microscope slide and, after drying, it was fixed with methanol-acetic acid solution (3:1). to visualize the dna, specimens were subjected to giemsa (2%) staining for 10 min, when the slides had dried. finally, the slides were cleaned under running water and air dried. slides were evaluated microscopically (×400) by an observer not in possession of the results of the questionnaire. mn was reported per 1,000 counted cells. the results were controlled by a second experienced independent observer to ensure the quality of the examination. criteria used for identification of micronuclei were according to those described by countryman and heddle16. r e s u l t s individual data of the 104 subjects selected for the present study are presented in table 1. in groups i and ii, 28 (53.84%) and 24 (46.15%) subjects were enrolled in each category (experimental and control subjects). the mean percentage of mn cells in the experimental group i was 1.86 ± 0.63 in users whereas in group ii was 2.91 ± 0.80. in the controls, the mean percentages of mn cells were 0.46 ± 0.27 for group i and 0.97 ± 0.45 for group ii. statistically significant results were obtained in experimental subjects compared to controls (p < 0.01), confirmed by chi-square test. the mean ± sd of ca in experimental and control subjects in group i was 3.32 ± 1.86 and 0.42 ± 0.57, respectively; in group ii it was 9.54 ± 3.38 and 1.33 ± 1.04, respectively. chisquare tests with ca data showed significant values with 0.730 for group i and 0.267for group ii. d i s c u s s i o n in group i (d”30 years), 28 subjects were selected as this age group has been reported to present concentrate st consumers, comprising students, athletes and young workers. adolescent use of all tobacco products has increased (usdhhs)17. the national collegiate athletic association (ncha)18 has found that use of smokeless tobacco among braz j oral sci. 7(25):1559-1562 cytogenetic damage in khaini users of tamilnadu, southern india 1561 0.97 ± 0.45 1.33 ± 1.04 0.46 ± 0.660.96 ± 0.91na 40.41 ± 6.50 24group ii 0.46 ± 0.27 0.42 ± 0.57 0.07 ± 0.260.36 ± 0.56na 24.67 ± 4.14 28group i controls2 2.91 ± 0.80 9.54 ± 3.38 3.29 ± 1.086.25 ± 2.8811.62 ± 2.55 40.20 ± 6.15 24group ii 1.86 ± 0.63 3.32 ± 1.86 1.29 ± 1.122.04 ± 1.266.75 ± 1.62 24.57 ± 3.63 28group i experimen tal 1 chromosom al aberrations chromatid type aberration cells with micronuclei (mn) (mean%) total karyotyping with g banding chromosomal aberration (ca) period of exposure (yrs) age (yrs) total of subject s casess.no 0.97 ± 0.45 1.33 ± 1.04 0.46 ± 0.660.96 ± 0.91na 40.41 ± 6.50 24group ii 0.46 ± 0.27 0.42 ± 0.57 0.07 ± 0.260.36 ± 0.56na 24.67 ± 4.14 28group i controls2 2.91 ± 0.80 9.54 ± 3.38 3.29 ± 1.086.25 ± 2.8811.62 ± 2.55 40.20 ± 6.15 24group ii 1.86 ± 0.63 3.32 ± 1.86 1.29 ± 1.122.04 ± 1.266.75 ± 1.62 24.57 ± 3.63 28group i experimen tal 1 chromosom al aberrations chromatid type aberration cells with micronuclei (mn) (mean%) total karyotyping with g banding chromosomal aberration (ca) period of exposure (yrs) age (yrs) total of subject s casess.no table 1 chromosomal aberrations (ca) and micronucleus (mn) frequency in experimental and control subjects experimental subjects: (khaini users); control subjects: (normal, healthy subjects); group i d”30 years and group ii e”31 years ca=chromosomal aberrations mn= micronucleus na=not applicable. college athletes increased by 40% from 1985 to 1989. the majority of college users had begun “dipping” snuff and chewing tobacco before going to college (ncha)18. there is sufficient evidence that oral use of st is carcinogenic to humans. khaini addiction is frequent in some places of tamilnadu region, india. in order to elicit the above issues, the present study has been carried out to determine the chromosomal damage in khaini users in tamilnadu state. assessment of normal levels of cytogenetic damage in the general population is essential for proper interpretation of data obtained by monitoring of populations that are occupationally or accidentally exposed to known or potentially genotoxic agents. ca have for many years been applied as biomarkers of genotoxic exposure and early effects of genotoxic carcinogens 19,20.the micronucleus test has received increasing attention as a simple and sensitive short-term assay for the detection of environmental genotoxicants21. hence, the assessment of cytogenetic damage performed in the present study shall aid understanding the mode of action of these obnoxious agents, which are a cheap alternative to the available smoking types, as well as the cancer risk involved in using them. the data presented in table 1 show a larger number of ca in the experimental subjects compared to controls. chromosomal instability has been described in many human dysplastic lesions and is considered a primary event in neoplastic transformation as well as a marker of progression to cancer22,23.a significant increase in the mortality ratio for all types of cancer in subjects with increased levels of ca in their lymphocytes has been found8,24-25. the control subjects of groups i and ii showed minimum number of ca when compared to the experimental subjects (table 1). the ca in controls might have been due to factors like their age and lifestyle. several studies have reported the relationship between aging and structural ca. some studies have found a significant influence of age on ca frequency, whereas others have found no association at all. recently, age and lifestyle factors have been found to be strongly associated with the frequency of ca measured by the chromosome painting technique26,27. the present study also confirmed that age along with duration of exposure plays a major role in genetic damage as presented in group i and group ii experimental subjects, the latter presenting a larger number of ca and mn. as seen in the results section, the binucleated mn cells are significantly higher in khaini users than controls (table 1). similarly, the incidence of micronuclei increased in the buccal mucosa cells of smokeless tobacco users28. carcinogenic and mutagenic compounds, including tobacco-specific nitrosamines present in st forms29 are believed to be responsible for the induction of micronuclei. moreover, the carcinogenic and mutagenic effects of st forms have been attributed to tobacco-specific nitrosamines30,31. the ash that is mixed with tobacco leaf powder permits the absorption of alkaloids into the buccal mucosa by transforming them into a basic form6-7. these compounds are produced from nicotine by bacterial or enzymatic activity. the same formation occurs in the mouth under the influence of saliva32. controls showed a minimal number of mn when compared to experimental subjects because, in the present study, controls were healthy normal and omnivores, with no apparent nutritional deficiencies. finally, the present study showed that the mn assay is a cost-effective procedure, accurate and easy to carry out for population-based studies. furthermore, in vivo evaluations allow for considering the influence of the individual susceptibility in screened humans. previous reports have established that buccal cells are useful not only for characterizing the molecular mechanisms underlying tobacco-associated oral cancers, but also as exfoliative cells that express diverse changes that appear promising as candidate biomarkers for the early detection of oral cancer. braz j oral sci. 7(25):1559-1562 cytogenetic damage in khaini users of tamilnadu, southern india 1562 in conclusion, the experimental group ii presented a larger number of ca and mn when compared to the experimental group i. on the other hand, the control groups i and ii presented negligible numbers of ca and mn. the remarkably greater presence of ca and mn in the experimental subjects was exclusive from khaini users who are highly predisposed to oral carcinoma and head and neck squamous cell carcinoma. it is evident that cytogenetic assays can be used as a primary screening test for ones susceptibility to oral carcinoma. the consumption of khaini as a form of st poses serious risks as a genotoxic and carcinogenic substance. the accompanying analyses of trends and modalities of induction of cancers associated with the consumption of tobacco in all forms point firmly to the understanding that the only way to eliminate completely the cancer risk linked to tobacco use is not changing from one type of consumption to another, but rather stop using tobacco in any form or, even better, never to start. references 1. stewart, bw, kleihues p. world cancer report . lyon: iarc; 2003. 3251p. 2. parkin dm, bray f, ferlay j, pisani p. global cancer statistics. ca cancer j clin. 2005; 55 : 74-108. 3. benowitz nl, ferrence r, slade j, room r, pope m. nicotine toxicit. nicotine and public health. washington: american public health association; 2000. p.65-75. 4. chakradeo pp, nair j, bhide sv. endogenous formation of nnitrosoproline and other n-nitrosamino acids in tobacco users. cancer lett. 1994; 86: 187-94. 5. niphadkar mp, contractor qq, bhisey ra. mutagenic activity of gastric fluid from chewers of tobacco with lime. carcinogenesis. 1994; 15: 927-31. 6. erenmemisoglu a, tekol y, kartal m, kurucu s. the use of a smokeless tobacco in our country “maras powder”. in: proceedings of the 2nd international symposium on pharmaceutical science. ankara, turkey; june 1991. 7. erenmemisoglu a, tekoly, kartal m, kurucu s. yurdumuzda dumansýz bir tütün kullanýmý: maraþ otu. doða. turkish j med sci. 1992; 16: 867-76. 8. hagmar l, brogger a, hansteen il. cancer risk in humans predicted by increased levels of chromosomal aberrations in lymphocytes. nordic study group on the health risk of chromosome damage. cancer res. 54: 1994; 2919-22. 9. stich hf, stich w, parida jb. elevated frequency of micronucleated cells in the buccal mucosa of individuals at high risk for oral cancer betel quid chewers. cancer lett. 1982; 125-34. 10. shah as, sarodc av bhide sv. experimental studies on mutagenic and carcinogenic effect of tobacco chewing. j cancer res. 1985; 109: 203-7. 11. foiles pg, miglietta lm, quart am, quart e, kabat gc, hecht ss. evaluation of 32 p-postlabeling analysis of dna from exfoliated oral mucosa cells as a means of monitoring exposure of the oral cavity to genotoxic agents. carcinogenesis. 1989; 10: 1429-34. 12. sarto f, tomanin r, giacomelli l, canova a, raimondi f, guiotto c et al. evaluation of chromosomal aberrations in lymphocytes and micronuclei in lymphocytes oral mucosa and hair root cells of patients. mutat res. 1990; 228: 157-69. 13. kayal jj, trivedi ah, dave bj, nair j, nair uj, bhide sv et al. incidence of micronuclei in oral mucosa of users of tobacco products singly or various combinations. mutagenesis. 1993; 5: 31-3. 14. ebbert jo, dale lc, vickers ks, gauvin tr, bunge ne, hurt rd. residential treatment for smokeless tobacco use a case series. j. subst. abuse treat. 2004; 26: 261-7. 15. hoyos ls, carvajal s, solano l, rodriguez j, orozco l, lopez y et al. cytogenetic monitoring of farmers exposed to pesticides in colombia, environ. health perspect. 1996; 104: 535-8. 16. countryman pi and heddle aj. the production of micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. mutat res. 1976; 41: 321-32. 17. u.s. department of health and human services (us-dhhs). public health service centers for disease control and prevention (cdc). selected tobacco. youth risk behavior surveillance united states. atlanta, georgia: mmwr. 1995; 44: 1. 18. national collegiate health association (ncha). replication of the national study of the substance use and abuse habits of college student-athletes. college of human medicine, michigan state university; 1989. 19. albertini rj, anderson d, douglas gr, hagmar l, hemminki k, merlo f et al. ipcs guidelines for the monitoring of genotoxic effects of carcinogens in humans. mutat. res. 2000; 463: 11172. 20. norppa h. genetic susceptibility, biomarker responses, and cancer. mutat. res. 2003; 544: 339-48. 21. stitch hf, rosin mp. quantitating the synergistic effect of smoking and alcohol consumption with the micronucleus test on human buccal mucosa cells. int. j. cancer 1983; 31: 305-8. 22. rabinovitch ps, dziadon s, brentnall ta. pancolonic chromosomal instability precedes dysplasia and cancer in ulcerative colitis. cancer res. 1999; 59: 5148-53. 23. burt ec, james la, greaves mj, birch jm, boyle lm, varley jm. genomic alterations associated with loss of heterozygosity for tp53 in li-fraumeni syndrome fibroblasts. br j cancer. 2000; 83: 467-72. 24. bonassi s, abbondandolo a. are chromosome aberrations in circulating lymphocytes predictive of future cancer onset in humans?. cancer genet cytogenet. 1995; 79: 133-5. 25. balachandar v, lakshman kumar b, sasikala k, manikantan p, sangeetha r, mohana devi s. identification of a high frequency of chromosomal rearrangements in the centromeric regions of prostate cancer patients. j zhejiang univ sci b (springer) 2007; 8: 638-46. 26. ramsey mj, moore dh, briner jf, lee da, olsen la, senft jr et al. the effects of age and lifestyle factors on the accumulation of cytogenetic damage as measured by chromosome painting. mutat ret. 1995; 338: 95-106. 27. tucker jd, moore dh. the importance of age and smoking in evaluating adverse cytogenetic effects of exposure to environmental agents. environ health perspect. 1996: 104: 489-92. 28. das rk, dash bc. genotoxicity of gudakhu a tobacco preparation ii. in habitual users. food chem toxicol . 1992; 30: 1045-9. 29. hecht ss, hoffmann d. tobacco-specific nitrosamines. an important group of carcinogens in tobacco and tobacco smoke. carcinogenesis. 1981; 9: 875-84. 30. erenmemisoglu a, lystun h, kartal m. carcinoma of buccal mucosa in smokeless tobacco users a preliminary study of the use of cytology for early detection. cyiopathoh. 1995: 6: 403-40. 31. ozkul y, erenmemisoglu a, cqcerjs, menevse a, saatci c. sisterchromatid exchange inducing effect of smokeless tobacco using on t-lymphocyte chromosomes. mulat res. 1995; 334: 209-12. 32. winn dm, blot wj, shy cm, pickle xw, toledo a, fraumeni jf. snuff dipping and oral cancer among women in the southern united states. n engl j. med. 1981; 304: 745-8. braz j oral sci. 7(25):1559-1562 cytogenetic damage in khaini users of tamilnadu, southern india oral sciences n3 braz j oral sci. 14(4):256-261 original article braz j oral sci. october | december 2015 volume 14, number 4 dentists’ actions about oral health of individuals with down syndrome ana de lourdes sá de lira1, claudio inácio reis da silva1, sylvana thereza de castro pires rebelo1 1universidade estadual do piauí – uespi, school of dentistry, department of clinical dentistry, area of integrated clinic, parnaíba, pi, brazil correspondence to: ana de lourdes sá de lira universidade estadual do piauí, faculdade de odontologia rua senador joaquim pires 2076 ininga cep: 64049-590 teresina, pi, brasil phone: +55 86 999595004 e-mail: anadelourdessl@hotmail.com abstract aim: to investigate the knowledge and actions of dentists for treatment of individuals with down syndrome. methods: a questionnaire was applied to all the dentists (n=90) working at the fhs (family health strategy) modules in the urban limits of parnaíba, pi, brazil. four of the questions in the questionnaire were written according to the theory of planned behavior table and likert scale (questions 6,7,9 and 15), in order to analyze the professionals’ intentions. sixteen objective questions were elaborated with the purpose of collecting information about the degree of the dentists’ knowledge as regards the intention of attending courses in the patients with special needs area including ds, and interaction with other professionals and families. the option was to use a questionnaire applied to the dentists of the region, from august to november 2014. results: it was found that most professionals were women and they considered themselves able to identify these patients. among the professionals, 70% showed they had no difficulty in identifying the patient with ds, and 5.2% had no opinion about the subject. only 6.6% of the professionals showed to be certain about their aptitude to attend to these patients; 70% were partially apt, that is, they were not absolutely sure about their aptness. there was a statistical relationship between the variables understanding and difficulty in the treatment. there was no statistical relationship between the variable capacity to identify, understanding of the needs and fitness variable in attendance. conclusions: patients with down syndrome need more attention and care of dentists, they must also be involved in a multidisciplinary approach. most of the professionals do not follow the procedures laid down by the ministry of health, but showed interest in attending a course in this area and there is a low number of sd patients being cared in parnaíba, pi. keywords: down syndrome; oral health; quality of life. introduction down syndrome, a chromosomal anomaly in chromosome 21 was described in the 19th century and was the first chromosomal anomaly detected in the human species. those affected by the syndrome may present various alterations1. oral health is an important aspect for social inclusion of persons with disabilities. oral diseases and oral malformations rarely result in risk of death, but they cause conditions of pain, infections, respiratory complications and masticatory problems. from the esthetic point of few, characteristics such as bad breath, poorly positioned teeth, traumatisms, gingival bleeding, the habit of keeping the mouth open and drooling may mobilize feelings of compassion, repulsion and/or prejudice, accentuating attitudes of social rejection2-5. there is a set of alterations associated with down syndrome (ds), which demand special attention, requiring special exams for their identification, as well as general and oral implications, which must be known by the dentist. this is to http://dx.doi.org/10.1590/1677-3225v14n4a01 received for publication: october 21, 2015 accepted: december 09, 2015 257257257257257 enable the dentist both to identify them and handle the patient with regard to aspects of psychological, anatomical and medical interactions6-10. it is imperative that the dentists be aware about the diagnosis of signs and symptoms inherent to these patients and the possible complications that may arise at the time of intervention. the treatment demands the work of a multiprofessional team that must stay in communication in order to provide better treatment, associated with the participation of family members, in order to improve quality of life for these individuals11-16. dental attention provided as early as possible is of the utmost importance to this population, in order to prevent future wider problems and to help patients create habits that will perpetuate through their lifetime17-22. the present philosophy of dental treatment in basic care, directed towards early preventive and restorative actions, did not consider this part of special individuals, who find themselves faced with predominantly surgical and mutilating treatments. the development of health promotion activities, with a multidisciplinary health team, directed towards patients with ds and their caregivers has become indispensable in view of the precarious oral condition of such patients10-22. the aim of this research was to investigate the knowledge and actions of dentists in the treatment of individuals with down syndrome. material and methods the epidemiological study design was of the observational, transversal, non-probabilistic type, with a sample composed by dentists of the public base care network in the city of parnaíba, pi, brazil. the option was to use a questionnaire applied to the dentists of the region, from august to november 2014. the pilot project, performed in june, 2014, was carried out by a single researcher, with the aim of verifying the suitability of the questionnaires to be used in the research. the participants were 25 students of the 10th semester at the state university of piaui, brazil. the questionnaire was applied to all the dentists (n=90) working at the fhs (family health strategy) modules in the city of parnaíba, in the northeastern region of brazil (figure 1). the following exclusion criteria were considered in this study: refusal by the professional to answer the questionnaire, incomplete filling out of the applied questionnaires. the participants signed an informed consent form, after being informed that their participation was optional, confidential, the information would not be used to harm them, there was no possibility of any risks and they could step out from the research at any time. the present study was submitted to the ethics committee on research with human beings, of the state university of piauí – uespi, protocol caae (36950714.6.0000.5209), and report no. 1.125.322, fig. 1. questionnaire dentists’ actions about oral health of individuals with down syndrome braz j oral sci. 14(4):256-261 258258258258258 which complied with its requisites and requests, and was in accordance with the national health council resolution no.196/96, which rules the ethics in research with human beings in brazil. data were collected by application of the research form. the professional answered the questionnaire after selecting one or more options for each question, which was checked by the researcher. on conclusion of the interview, the interviewed professional signed the form of questions, evaluating the veracity of his/her responses. four of the questions in the questionnaire were elaborated according to the theory of planned behavior table, and likert scale (questions 6, 7, 9 and 15), in order to analyze the professional’s intents. to analyze the questions with reference to identification, aptitude, understanding of the limitations and difficulties in treatment, the likert scale was used in the area of social sciences, which is considered easy to understand 9. the questionnaire was divided according subjective norms, behavioral beliefs and internal control beliefs, with questions ordered by categories and relevant to the topic, which considers the aspects of belief, values and attitude. the fivepoint scale ranged from “i fully agree”, (meaning being certain as to the answer), “i partially agree”, (meaning not sure), “completely disagree”, (showing negative certainty), “i partially disagree” (partial negative certainty, doubt) and to “i do not agree or disagree” (the interviewed professional has no opinion on the subject). sixteen objective questions were elaborated for collecting information about the degree of the dentist’s knowledge as regards the intention of attending courses in patients with special needs area, including ds and interaction with other professionals and families. the research included aspects such as gender, time since graduation, frequency of attendance to patients with ds, the used form of treatment, taking into consideration the ease of identifying the patient, personal aptitude in attendance, understanding of the needs and whether they had any difficulty with the treatment. the responses were tabulated in the excel program according to the individual questions and answers, for statistical analysis in the “biostats 5.3” program. multiple linear regression tests were used to analyze the determinant and independent variables, and simple logistic test to evaluate the dichotomic variables. an inter-rater agreement statistic (kappa) was calculated to evaluate the agreement for nominal scales between two observers that performed the research and verify if the methodology can be applied in other population or in other city. the value of k was 0.89, a very strong agreement. results there was no significant difference as regards gender, remembering that among the interviewed professionals, 26 were women. the professionals had a mean 6 years since graduation and aged between 21-30 years; 39 had specialization and refresher courses; 35 only refresher courses; 15 only specialization, and only 1 academic master’s degree, but there was no post-graduation in patients with special needs. all professionals informed they performed fewer than 3 attendances per month to patients with ds. out of the professionals, 70% showed they had no difficulty in identifying the patient with ds, and 5.2% had no opinion about the subject. only 6.6% of the professionals have shown to be certain about their aptness to attend these patients; 70% were partially apt, that is, they were not absolutely certain about their aptitude. the other parts showed no significant values with regards to the question. out of the professionals 13.3% were found to have complete understanding of the needs and limitations of patients with ds; 52.3% of the participants understood partly these needs and limitations and 20% did not understand. 56.8% affirmed they had difficulties with the treatment of patients with ds; 26.6% had a certain amount of difficulty and 5.5% had no opinion (figure 2). among the participants, 66.7% answered they had difficulties with treatment; 20% attributed this difficulty to insufficient knowledge for providing attendance; 40% to difficulty in handling the patient; 40% because the patient was non-cooperative and 13.3% because the time of attendance did not allow the process to be performed step by step. for this question only the participant could choose more than one alternative (figure 3). about the procedure on the first consultation and time of attendance, 20% reported performing anamnesis as the initial procedure, 60% anamnesis and clinical exam; 13% psychological conditioning, and 7% the clinical procedure dentists’ actions about oral health of individuals with down syndrome braz j oral sci. 14(4):256-261 fig. 2. likert scale. source: direct research parnaíba 2014 fig. 3. difficulty with attending to patients with ds. source: direct research parnaíba 2014 259259259259259 only, with the majority (80%) spending a long time to provide the attendance. as regards handling/management of the patient, 60% answered that they did so in a multidisciplinary manner, but in conjunction with the family. the remaining 40%, referred the patient to a specialist. out of the professionals, 60% showed interest in qualifying themselves in the area and 40% did not consider this option. of these 60% who showed interest, the reason was the knowledge to be acquired during the course for 60%, and for the remaining 40%, it was the difficulty of dealing with patients with ds. as regards the difficulty of treating the patients with ds and understanding their needs and limitations, a significant correlation was verified by the multiple linear regression test (p=0.005). thus, 50% of the dentists were found to agree partially about having difficulty with treatment, and not having a good basis on the patient’s needs and limitations. statistical analysis was performed among the variables “difficulty with identifying the patients with ds”, “aptness for attendance”, “understanding of the needs of the patient with ds” and “degree of difficulty with treatment of the patient with ds” by the multiple logistic regression test, where no statistical relationship was found (p>0.05). most professionals had difficulty with handling the patient (40%) due to lack of cooperation from the patient (60%), a relationship proved by multiple logistic regression test (p=0.0346). simple logistic analysis between the difficulty with identifying the patient with ds and the difficulty with treatment showed there was no correlation (p=0.4269), revealing that in spite of the professionals having no difficulty with identifying the patient with ds, they appeared to have a certain impasse with regards to treatment. there was correlation between the interest in attending courses in the area of patients with special needs and knowledge to be acquired during the course (p=0.0023). discussion most of the professionals who participated in the research were women, aged between 21-30 years, none was older than 40 years, and had a mean of 6 years since graduation. this distribution, which is in agreement with the national mean value, was 26-35 years6. the fact that most of the professionals were postgraduates, does not mean to say they were able to care for the patient with ds, considering they were not specialists in the area of patients with special needs, which includes these patients. according the the federal council of dentistry (cfo) there were 480 registered specialists in patients with special needs throughout brazil as of 16/08/2015, out of a total number of 237.872 dentists. according to data of the federal council of dentistry, the piauí state has 2.488 dentists, with only one registered as professional in the abovementioned specialty. the low number of patients attended to per month is cause for concern, as the importance of dental attendance for the patient with ds starting at early childhood is known, so that preventive, interceptive and curative activities may be provided11-12. the lack of entry to dental assistance by patients with special needs, a group comprising ds, may be attributed to different factors: the professionals’ lack of knowledge and preparedness for attendance, changed information about the oral health conditions and dental needs, negligence of dental treatment provided by public and private services and the parents’ disbelief in the importance of oral health13. in spite of the high number of professionals able to identify the patient with ds (73.3%), there are few who fully understand the needs of the patient with ds (13.7%), in addition to showing difficulty with treatment (63.7%). these data show the dentists’ lack of preparation, or lack of attention to the oral health of these patients with ds, a common fact observed by other authors16-17,20,22 when they affirmed that lack of dental care is a health problem frequently found in children with ds. the fact that dentists had no difficulty in identifying these patients, but had difficulty in handling them, is probably due to the fact that the professional has not been prepared during his/her academic life for providing attendance to patients with special needs, as has also been suggested by some authors15-16. this also suggests that while attending the patient with ds and after obtaining the guardian’s consent and signature, meticulous questioning must be carried out, and whenever possible, observations about the patient’s systemic condition, such as: verifying the use of medications that may interfere in dental treatment; referring the patient to the physician when doubt arises about systemic changes observed in the interview; during the interview, observe possible evidence of maltreatment, lack of care or neglect. at the end of the research, a folder was delivered with guidance about oral health promotion, in accordance with the advice of authors9,11,14,20-21, in which preference should be given to preventive methods for oral health of patients ds, beginning right in the first year of life and annually, performing clinical and radiographic exams. and also to instruct the family about the importance of good oral hygiene and daily use of dental floss, of observing the daily amount of fluorides and dentifrices used, due to the risk of swallowing them and explaining to parents about delay in the eruption of teeth. however, this research found that only 20% of dentists perform only anamnesis; 60% perform anamnesis and clinical exam; 13% psychological conditioning and 7% perform only the clinical procedures, which is not recommended by some authors17-19 who are unanimous stating that such procedures may generate discomfort to patients and an unfavorable experience, thereby compromising healthy treatment. for some authors9,12,14,17-21 the dental attention to these patients is extremely important and is influenced by the attitude of the dentist in care of them, so that it is crucial a behavior that transmits confidence to patients. moreover, dentists’ actions about oral health of individuals with down syndrome braz j oral sci. 14(4):256-261 the time of consultation for each patient should be short and strictly calculated, so it does not exceed 30 minutes, differing from the results found in the research, in which 80% of the professionals reported spending a long time on the attendance of these patients. the oral health of patients with ds may be compromised due to professional´s lack of specific knowledge about treating special patients, in addition to the negligence of their parents2-5. with regard to the mode of attendance to these patients, analysis of the data showed that 60% of the professionals did not use a multidisciplinary approach, but one in contact with the family, explaining about the oral aspects of the syndrome, and 40% of the professionals reported referring the patient to a specialist. however, as previously described, piaui has only one professional in the specialty registered with the cfo, responsible for this population. therefore, these patients probably were not attended by a specialized professional6,10-12,14,20,22. none of the professionals worked in a multidisciplinary manner; they did not interact with other professionals and did not perform multidisciplinary management along with the family as observed by other authors7-8,11-13. however, this negative fact does not mean non-fulfillment of professional duties. a multiprofessional approach is described as the one in which there is reference and counter-reference, however, if the patient does not achieve one of the points the cycle is broken. this fact may frequently be explained by the distance, or difficulty with the patient’s residence from the center of reference. various studies have pointed out the importance of the manner of approaching these patients, thus the multidisciplinary manner prevails. since the term syndrome signifies “a set of signs and symptoms”, it should be approached at multidisciplinary level, which along with the family, offers better conditions for the patient’s quality of life 20-22. the importance of the professional’s interaction was pointed out, with the purpose of elucidating thise issue, emphasizing the involvement of family members in the treatment of patients with ds. differently from what was observed in the research, some authors11-13,19-21 have found the absence of family involvement in this process, probably due to the limitations of multidisciplinary integration in the area of health. the shared care of the patient with ds should be in conjunction with the diagnosis, constructing the therapeutic project, therapeutic goals, re-evaluating and following up progress. in addition, the discourse was about the importance of co-responsibility for the process of care among the professionals, the subject and the family, according to some authors6-7,11,22. on conclusion, the majority of dentists showed interest in qualifying themselves in this area, with a view to increase knowledge, with the possibility of reflecting in the improvement in attendance of patients with ds, who need dental care in a multidisciplinary context. most of them in parnaíba, pi, do not follow the clinical conduct recommended by the ministry of health for the attendance of these patients. a low number of attendances to the patients with ds were recorded, but with positive family involvement. nevertheless, professionals from other areas required for overall attendance of the patient were not inserted, which is unfavorable to the concept of a multidisciplinary approach. our hope is that the results of this study may encourage providing a larger number of training and qualification courses to dentists and members of the hmf, who intend providing with patients down syndrome a higher quality of care, and for the elaboration of preventive and educational programs. the interpretation of the present results should consider some limitations inherent to this study. it is very important that future studies assess if individuals with down syndrome keep being assisted by the fhs. the city where the study was conducted is also considered an undeveloped city in brazil, both economically and socially, which may explain the small number of patients that use fhs. references 1. hennequim m, faulks d, roux d. accuracy of estimation of dental treatment need in special care patients. j dent. 2000; 28: 131-6. 2. lee sr, kwon hk, song kb, choi yh. dental caries and salivary immunoglobulin a in down syndrome children. j paediatr child health. 2004; 40: 530-3. 3. fung k, allison p. a comparison of caries rates in non-institutionalized individuals with and without down syndrome. spec care dentist 2005; 25: 302-10. 4. frydman a, nowzari h. down syndrome-associated periodontitis: a critical review of the literature. compend contin educ dent. 2012; 33: 356-61. 5. kaye pl, fiske j, bower ej, newton jt, fenlon m. views and experiences of parents and siblings of adults with down syndrome regarding oral health care: a qualitative and quantitative study. br dent j. 2005; 198: 571-8. 6. atunes jlf, narvai pc, nugent zj. measuring inequalities in the distribution of dental caries. community dent oral epidemiol. 2004; 32: 41-8. 7. minnes p, steiner k. parent views on enhancing the quality of health care for their children with fragile x syndrome, autism or down syndrome. child care health dev. 2009; 35: 250-6. 8. wong df, lam ay, chan sk, chan sf: quality of life of caregivers with relatives suffering from mental illness in hong kong: roles of caregiver characteristics, caregiving burdens, and satisfaction with psychiatric services. health qual life outcomes. 2012; 10:15. doi: 10.1186/14777525-10-15. 9. shore s, lightfood t, ansell p. oral disease in children with ds: causes and prevention. community pract. 2010; 83(2): 18-21. 10. areias cm, sampaio-maia b, guimarães h, melo p, andrade d. caries in portuguese down syndrome children. clinics (sao paulo). 2011; 66: 1183-6. 11. khocht a, janal m, turner b. periodontal health in down syndrome: contributions of mental disability, personal, and professional dental care. spec care dent. 2010; 30: 118-23. 12. wilson md. special considerations for the dental professional for patients with down’s syndrome. j okla dent assoc. 1984; 84: 24-6. 13. anders p, davis el: oral health of patients with intellectual disabilities: a systematic review. spec care dent. 2010; 30: 101-17. 14. khocht a. subgingival microbiota in adult down syndrome periodontitis. j periodont res. 2012; 47: 500-7. 260260260260260dentists’ actions about oral health of individuals with down syndrome braz j oral sci. 14(4):256-261 261261261261261 15. sousa e, alberman e, morris jk. down syndrome and paternal age, a new analysis of case–control data collected in the 1960s. am j med genet a. 2009; 149: 1205-8. 16. mathias mf, simionato mr, guare ro. some factors associated with dental caries in the primary dentition of children with down syndrome. eur j paediatr dent. 2011; 12: 37-42. 17. vijayaprasad ke, ravichandra ks, vasa a, suzan s. relation of salivary calcium, phosphorus and alkaline phosphatase with the incidence of dental caries in children. j indian soc pedod prev dent. 2010; 28: 156-61. 18. oliveira ac, pordeus ia, torres cs, martins mt, paiva sm. feeding and nonnutritive sucking habits and prevalence of open bite and crossbite in children/adolescents with down syndrome. angle orthod. 2010; 80: 749-53. 19. vilella pgdp, costa pp. periodontite na síndrome de down. rev odontol planalto cent. 2013; 3: 71-6. 20. davidovich e, aframian dj, shapira j, peretz b. a comparison of the sialochemistry, oral ph, and oral health status of down syndrome children to healthy children. int j paediatr dent. 2010; 20: 235-41. 21. bonanato k, pordeus ia, compart t, oliveira ac, allison pj, paiva sm. cross-cultural adaptation and validation of a brazilian version of an instrument to assess impairments related to oral functioning of people with down syndrome. health qual life outcomes 2013; 11: 4. doi: 10.1186/ 1477-7525-11-4. 22. oliveira ac, pordeus ia, luz cl, paiva sm. mothers’ perceptions concerning oral health of children and adolescents with down syndrome: a qualitative approach. eur j paediatr dent. 2010; 11: 27-30. dentists’ actions about oral health of individuals with down syndrome braz j oral sci. 14(4):256-261 braz j oral sci. 15(1):8-15 original article braz j oral sci. january | march 2016 volume 15, number 1 factors influencing dental appearance satisfaction in adolescents: a cross-sectional study conducted in southern brazil gregori franco boeira1, mabel miluska suca salas1, diogo conceição araújo1, alexandre severo masotti1, marcos britto correa1, flávio fernando demarco1,2 1universidade federal de pelotas – ufpel, pelotas dental school, department of restorative dentistry, pelotas, rs, brazil 2universidade federal de pelotas – ufpel, dental school, area of epidemiology, pelotas, rs, brazil correspondence to: flávio fernando demarco universidade federal de pelotas. faculdade de odontologia rua gonçalves chaves, 457 – bairro: centro cep: 96015-568 – pelotas – rs, brazil phone: +55 53 3222 6690 – fax: +55 53 3222 6690 e-mail: flavio.demarco@pq.cnpq.br; ffdemarco@gmail.com abstract the perception of dental aesthetic appearance may affect social interaction and psychological status, influencing dental needs and the search for treatments. aim: to investigate the satisfaction with dental appearance and influencing factors among adolescents. methods: the study was carried out among adolescents aged 14 to 19 years attending a private high school in brazil. data on demographic information, the perception of dental appearance, previous aesthetic treatments and wish to perform dental treatments were collected in the school. data were analyzed using pearson’s chi-square test or linear trend. multivariate analysis was performed using the poisson regression. results: a total of 531 adolescents (response rate = 98.3%) answered the questionnaire. the prevalence of dissatisfaction with dental appearance was 17.4%. almost 65% had history of previous orthodontic treatment and 16% performed dental bleaching. approximately 45% of children wished to undergo orthodontics and 54.8% to bleach their teeth. dissatisfaction with dental appearance was associated with individuals unsatisfied with dental color (95% ic[1.73;4.32]), those perceiving poor dental alignment (pr3.16 95% ic[2.11;4.72]) and those wishing orthodontic treatment (pr2.9; 95% ic[1.79; 4.70]). conclusions: the prevalence of dissatisfaction was considerable and was associated with aesthetic concerns such as tooth color, dental alignment and with the wish for orthodontics. in this young population, a large part of adolescents had already performed orthodontic and bleaching treatments and wished to perform those treatments again. satisfaction with dental appearance could affect the adolescents’ behavior regarding search for dental treatment, thus causing possible overtreatment. keywords: patient satisfaction. esthetics. self concept. color. tooth. malocclusion. adolescent. introduction concerns in relation to esthetics are more present in modern society1 and have caused an increased demand for aesthetic treatments2. facial aesthetics has been associated with facial and smile harmony1. smile harmony is dependent on several factors such as tooth color, shape, size and position; lip position allowing tooth visibility and gingival disposition3. the lack of a proportional and beautiful smile could impact the self-esteem of a person, influencing the psychological and physical health4. this http://dx.doi.org/10.20396/bjos.v15i1.8647091 received for publication: march 04, 2016 accepted: may 05, 2016 9 situation can affect socio-emotional aspects of well-being and may influence social interaction5. studies have found that the satisfaction with dental appearance is greatly influenced by tooth color and malocclusion3-4,6-7. those parameters could be affected by psychosocial, cultural and sociodemographic factors8-10. white teeth have been associated with higher scores of social competence, intellectual ability, psychological balance and social status11 and may impact the quality of life of the subject12. in adolescence, facial attractiveness is an important social norm among adolescent groups, the dental appearance being the first factor related to attractiveness13. the social interactions with negative self-concept and peer-group acceptance have been associated with unacceptable dental appearance14. among adolescents, studies have shown that girls emphasize attractiveness15. age was found to influence the satisfaction with dental appearance and older individuals tend to be more satisfied with dental appearance than the younger ones8,16. the presence of untreated caries, stained anterior restorations and missing teeth can lead to dissatisfaction with dental appearance11. it seems that individuals with higher socioeconomic level are more concerned in relation to their attractiveness17 and with aesthetic appearance, and more inclined to undergo aesthetic treatments, like orthodontic appliances, compared with those in deprived situation18-19. moreover, studies reported that the performance of dental treatments, such as orthodontics and tooth bleaching, could improve the satisfaction with dental appearance, perception of attractiveness and the quality of life of patients7,20-21. the aim of this study was to investigate the self-reported satisfaction with dental appearance and the association with demographic factors, self-perceived dental problems and the wish to undergo aesthetic treatments among adolescents from a private school in southern brazil. material and methods the present study had approval of the ethics committee, federal university of pelotas (nº 196/96). formal permissions were obtained from the school’s director board. parents or legal guardians of the adolescents received and signed an informed consent form allowing the adolescents’ participation. this cross-sectional study was carried out in pelotas, an affluent city (350,000 inhabitants), located in southern brazil. according to the department of education of rio grande do sul state, pelotas has 19 states, 1 municipal and 9 private schools offering secondary education. the number of children enrolled in secondary schools of the city was 9,237 in public and 1,711 in private schools. the study population consisted of a sample of adolescents 14-19 years old, attending a private school. this population was selected due to the high number of children attending the school, considering that sample size can influence the presence of studied outcomes22. there were 540 students in this age range attending the school in 2012, the year when the fieldwork was carried out. the quality of the structured questionnaire based on previous investigations, was previously tested in adolescents with similar ages, not included in the study. adolescents were asked about their perceptions of tooth color, satisfaction with tooth appearance, tooth alignment, previous or actual use of orthodontic appliances and the wish to carry out treatments like orthodontics and tooth whitening. also questioned were the presence of tooth pain or impairments in tooth appearance, including tooth crowding, dental caries, misalignment, tooth fractures and gingival bleeding. satisfaction with facial, tooth and color appearance was initially recorded as very satisfied, satisfied, unsatisfied and very unsatisfied, later dichotomized in satisfied and dissatisfied. concern with aesthetics was categorized in never, sometimes, frequently and always. the questionnaire was conducted in a schoolroom by two trained examiners (post-graduate students) with previous experience in epidemiological studies. individuals were considered as losses if they could not be found after two times recall. data were recorded in the epi info 6.0 software and the analyses were carried out in stata 12.0. descriptive and bivariate analyses were performed to assess the association between studied factors and satisfaction with tooth appearance, using pearson’s chi-square test or linear trend, depending on the type of variable. all variables with p-value <0.25 in the bivariate analysis were considered potential confounders and included in the multivariate analysis, obtaining prevalence ratios (pr) and 95% confidence intervals for the outcome. multivariate analysis was performed by the poisson regression. explanatory variables were selected for the final models only if they had a p-value ≤0.05 after adjustment. results the rate of participation was 98.3% and 531 adolescents participated in the study. perception and characteristics of adolescents regarding dental aesthetics are described in table 1; with most of them being female (57.4%). the prevalence of actual or past use of orthodontic appliance was 64.7%. dissatisfaction with dental appearance was reported by 91 (17.4%) teenagers. the perception of dental protrusion, alignment and crowding were reported by 11.0%, 25.4% and 10.0% of the adolescents, respectively. tooth pain in the last 6 months, gingival bleeding and dental trauma were indicated respectively by 30.7%, 20.3% and 18.9%. most of the individuals (54.8%) wished to perform tooth whitening and 45.2% an orthodontic appliance. sixty-five adolescents (12.6%) were dissatisfied with their appearance and 226 (43.4%) felt unattractive or relatively attractive. adolescents reported to be concerned about others’ opinion regarding their dental appearance sometimes (54%), frequently (9.4%) or always (1.9%). bivariate analysis of the association between the satisfaction with dental appearance and the investigated factors is shown in table 2. the perception of an unpleasant dental appearance was associated with dissatisfaction with dental color and facial aesthetics, perception of dental crowding, dental alignment and tooth pain in the last 6 months, lack of use of orthodontic appliance, wish to carry out orthodontic treatment, tooth bleaching or to perform a corrective facial surgery. it was associated with lack or little perception of attractiveness, while the preoccupation with others’ opinion regarding the own adolescent’s dental appearance showed a tendency to increase in those unhappier with their appearance. factors influencing dental appearance satisfaction in adolescents: a cross-sectional study conducted in southern brazil braz j oral sci. 15(1):8-15 braz j oral sci. 15(1):8-15 *n may vary in difference questions and it is related to the number of each individuals that have answered it. variables/category n % sex 531 male 226 42.6 female 305 57.4 satisfaction with dental appearance 523 yes 432 82.6 no 91 17.4 satisfaction with tooth color 524 very satisfied/ satisfied 370 70.6 dissatisfied/ very dissatisfied 154 29.4 performed tooth bleaching 523 no 439 83.9 yes 84 16.1 used or is using orthodontic appliances 524 no 185 35.3 yes 339 64.7 perception of dental crowding 522 no 470 90.0 yes 52 10.0 perception of dental alignment 523 no 390 74.6 yes 133 25.4 perception of dental protrusion 519 no 462 89.0 yes 57 11.0 wish for orthodontic appliances 518 no /don`t know 284 54.8 yes 234 45.2 wish for tooth bleaching 520 no/ don`t know 236 45.2 yes 286 54.8 wish for composites 520 no /don`t know 400 76.9 yes 120 23.1 tooth pain (last 6 months) 521 no 361 69.3 yes 160 30.7 gingival bleeding 522 no 416 79.7 yes 106 20.3 dental trauma 523 no 424 81.1 yes 99 18.9 facial satisfaction 517 very satisfied/ satisfied 452 87.4 dissatisfied/ very dissatisfied 65 12.6 perception of attractiveness 521 no 105 20.2 relative 121 23.2 yes 295 56.6 concerned with others’ opinions regarding dental appearance 524 no 182 34.7 sometimes 283 54.0 frequently 49 9.4 always 10 1.9 table 1 dental aesthetic perceptions of schoolchildren from a private school in pelotas, brazil, 2012 (n=524).* 10factors influencing dental appearance satisfaction in adolescents: a cross-sectional study conducted in southern brazil variables/category dental appearance p valuesatisfied dissatisfied n (%) total + n (%) total + sex 432 91 0.955 male 179 (82.49) 38 (17.51) female 253 (82.68) 53 (17.32) satisfaction with dental color 432 91 <0.001* satisfied 337 (78.0) 95 (22.0) dissatisfied 32 (35.2) 59 (64.8) tooth bleaching 432 91 0.145* no 357 (81.5) 81 (18.5) yes 74 (88.1) 10 (11.9) perception of dental crowding 431 90 <0.001* no 400 (80.4) 69 (14.7) yes 31 (59.6) 21 (40.4) perception of dental alignment 431 91 <0.001* no 358 (91.8) 32 (8.2) yes 73 (55.3) 59 (44.7) perception of dental protrusion 427 91 0.066* no 385 (83.5) 76 (16.5) yes 42 (73.7) 15 (26.3) use or in use of orthodontic appliances 432 91 0.030* no 143 (77.7) 41 (22.2) yes 289 (85.6) 50 (14.8) desire of orthodontic appliances 284 233 <0.001* no /don`t know 264 (61.8) 163 (38.2) yes 20 (22.2) 70 (77.8) wish for tooth bleaching 235 289 0.002* no /don`t know 207 (48.1) 223 (51.9) yes 28 (30.8) 63 (69.2) wish for composites 429 91 0.002* no /don`t know 341 (85.5) 53 (14.5) yes 88 (73.3) 32 (26.7) tooth pain (last 6 months) 429 91 0.006* no 308 (85.56) 52 (14.44) yes 121 (75.63) 39 (24.38) gingival bleeding 430 91 0.670* no 344 (82.89) 71 (17.1) yes 86 (81.13 ) 20 (18.87) dental trauma 432 91 0.271* no 353 (83.45) 70 (16.55) yes 78 (78.79) 21 (21.21) facial satisfaction 452 65 <0.001** very satisfied /satisfied 386 (90.6) 40 (9.4) dissatisfied/ very dissatisfied 66 (72.5) 25 (27.5) perception of attractiveness 430 90 0.061** no 78 (75.0) 26 (25.0) relative 104 (86.0) 17 (14.0) yes 248 (84.1) 47 (16.0) concerned with others’ opinions regarding dental appearance 452 65 <0.001** no 163 (90.6) 17 (9.4) sometimes 252 (90.7) 26 (9.4) frequently/ always 37 (62.7) 22 (37.3) table 2 bivariate analysis between satisfaction with dental appearance and aestheticsrelated factors of schoolchildren from a private school in pelotas, brazil, 2012 (n=523). * chi-square (χ²) test or fisher ** χ² test for linear trend + values lower than 531 due incomplete data braz j oral sci. 15(1):8-15 11 factors influencing dental appearance satisfaction in adolescents: a cross-sectional study conducted in southern brazil multivariate analysis showed that adolescents dissatisfied with dental appearance were 2.73 times more unsatisfied with dental color (95%ic [1.73;4.32]) and perceived poor dental alignment (pr 3.16 95%ic [2.11;4.72]). adolescents wishing to perform orthodontic treatment had 2.9 times more prevalence of dissatisfaction with their dental appearance (95%ic [1.79; 4.70]) (table 3). discussion the present study showed that the prevalence of dissatisfaction with dental appearance among adolescents from a private school in southern brazil was 17.4%. dissatisfaction with dental appearance was associated with dissatisfaction with dental color and selfreported poor dental alignment. individuals wishing to undergo orthodontic treatment were more dissatisfied with dental appearance. dental appearance dissatisfaction can vary in different populations. studies have reported that the prevalence of dissatisfaction among adolescents 10-18 years old ranged from 11.4% to 42.8%23-24. evaluating the dissatisfaction with dental appearance in a birth cohort at 15 years of age in pelotas, peres et al. (2008)25 observed a prevalence of 29.8% for boys and 46.5% for girls (p<0.001) and the dissatisfaction was associated to the presence of malocclusion. a national oral health survey in brazil26 demonstrated among adolescents between 15-19 years a negative impact caused by malocclusion in oral health related quality of life and the impact was more prevalent in girls27. however, in our study, there was no significant difference in dissatisfaction between boys and girls. it was found that younger people tend to be less satisfied with their dental appearance4,8 and considered healthy and well aligned teeth as important factors in facial appearance28. adolescents usually attribute high importance to an attractive dental appearance3. our study was conducted among adolescents from middle and high-income families and probably in these individuals the demands for aesthetics are high, independent of sex. cultural factors and individual preferences may determine the perception of aesthetics and dental appearance could influence such perception6. this is the reason why perceptions in relation to dental appearance may differ from one population or individual to another29. society dictates the tendency of aesthetics and media is an important form of aesthetic norms dissemination, able to influence satisfaction11. younger people may be influenced more than middle or older aged groups by media and as consequence, their aesthetic awareness can be increased, influencing their satisfaction8. on the other hand, dental satisfaction may decrease with some dental conditions such as dental stains, fluorosis, malocclusion, caries, trauma, gingival diseases and missing teeth4,11. tooth color was reported to be an important factor for dental satisfaction and may be considered a proxy for aesthetic value8. in our study, dissatisfaction with dental appearance was associated with tooth color dissatisfaction. this result was also previously reported4,6,30-31. younger people preferred whiter teeth29 and adolescents tended to express dissatisfaction with color specially if someone called their attention to it31. the desire for beauty increases the pressure for aesthetics, due to the link between appearance and social status and acceptability8. adolescents dissatisfied with tooth color mentioned constraints for answering questions and interacting with people, had been associated with psychosocial effects produced by problems with tooth color31. it was also demonstrated that individuals who underwent tooth bleaching claimed to have an improvement in their ohrqol12. it is important to highlight that despite the young age of the evaluated individuals, 15% had already performed tooth bleaching and 54% wish to perform tooth bleaching, demonstrating the importance of tooth color for these young individuals. however, we should emphasize that individuals exhibiting normal tooth color have higher expectations for tooth color and are potential clients for sometimes unnecessary bleaching treatment9. malocclusion could also determinate dental appearance satisfaction. in our study, children who considered themselves with poor dental alignment were dissatisfied with their dental appearance. other studies reported similar findings4,32. the arrangement of teeth is correlated to a harmonious smile and attractiveness3. negative psychosocial effects were reported by the presence of anterior crowding in adolescents32. adolescents exhibited an aesthetic impact in daily living due to malocclusion32, which could impact directly on their quality of life28, interfering with the self-esteem33. studies found that diverse types of malocclusion could produce dissatisfaction with dental appearance32 severe malocclusions being the most likely to produce higher rates of dissatisfaction with dental appearance23. malocclusion may affect other people’s judgment and self-judgments. for instances, it was reported that the arrangement of the teeth may affect teachers’ judgments of students34. the aforementioned agrees with our findings of positive wish for orthodontic treatment observed in children with higher prevalence of dental appearance dissatisfaction, as similarly observed in other studies4,6,30. children with higher perception of malocclusion tend to wish more frequently orthodontic treatment35. regarding the malocclusion aspects investigated (dental crowding, poor alignment, and dental protrusion), the highest reported problem was poor alignment (25.4%). to note, despite the fact that around 65% of the evaluated individuals had already undergone orthodontic treatment, 45.2% of the sample had the wish to perform orthodontic treatment. studies showed that performing some aesthetic treatments may enhance self-esteem and improve the appearance satisfaction and in consequence better results of quality of live are32,36. adolescents completing orthodontic treatments could have social benefits37. a longitudinal study reported that the use of orthodontic appliances promoted psychosocial effects due to the improvement of aesthetics, increasing self-esteem and self-confidence, improving social skills, which may influence future behavior38. our sample included children from private schools. in brazil the type of school is a proxy for socioeconomic level39, and adolescents from private schools can be also considered in better socioeconomic situation. it was reported that individuals with higher socioeconomic status are more concerned with aesthetics than the ones in lower levels17. also, in private schools dental caries prevalence is lower than among adolescents attending public schools40 and use more orthodontic appliances compared with those with deprived situation18. thus, it was expected a higher proportion of children satisfied with their aesthetics. braz j oral sci. 15(1):8-15 12factors influencing dental appearance satisfaction in adolescents: a cross-sectional study conducted in southern brazil *heterogeneity test. variables that presented p>0.25 in bivariate analysis were not included in multivariate analysis model. variables/category dissatisfaction with dental appearance prc (95%ci) p* value pra(95%ci) p* value satisfaction with dental color <0.001 <0.001 satisfied 1.0 1.0 dissatisfied 2.75(1.71;4.41) 2.73 ( 1.73; 4.32) sex 0.143 0.114 male 1.0 1.0 female 0.76 (0.52;1.11) 0.75(0.53; 1.08) tooth bleaching 0.821 no 1.0 yes 0.95(0.53; 1.69) dental crowding 0.213 0.098 no 1.0 1.0 yes 1.36 (0.85; 2.18) 1.34( 0.96; 2.00) dental alignment <0.001 <0.001 no 1.0 1.0 yes 3.18 (2.06;4.92) 3.16( 2.11; 4.72) dental protrusion 0.330 no 1.0 yes 0.83(0.56; 1.23) use or in use of orthodontic appliances 0.879 no 1.0 yes 0.96(0.64; 1.45) wish for orthodontic appliances <0.001 <0.001 no don`t know 1.0 1.0 yes 2.76 (1.72; 4.42) 2.90(1.79; 4.70) wish for tooth bleaching 0.222 0.184 no -don`t know 1.0 1.0 yes 0.76 (0.49;1.19) 0.76 (0.50;1.15) facial satisfaction 0.234 0.101 satisfied 1.0 1.0 dissatisfied 1.27(0.86;1.87) 0.76 (0.95 1.87) perception of attractiveness 0.146 0.166 no 1.0 1.0 relative 0.86 (0.54; 1.39) 0.85(0.53; 1.35) yes 0.76 (0.50;1.12) 0.77(0.52;1.12) concerned with opinions regarding dental appearance 0.051 0.068 no 1.0 1.0 sometimes 1.54( 0.96;2.47) 1.43(0.90;2.27) frequently/always 1.71 (0.95;3.06) 1.64(0.94; 2.89) wish for composites 0.846 no/ don`t know 1.0 yes 1.04 (0.72; 1.48) pain(6 months before) 0.444 no/ don`t know 1.0 yes 1.17 (0.78; 1.75) table 3 crude (c) and adjusted (a) prevalence ratios (pr) for dissatisfaction with dental appearance in schoolchildren, according to independent variables. pelotas, rs, brazil. 2012. braz j oral sci. 15(1):8-15 13 factors influencing dental appearance satisfaction in adolescents: a cross-sectional study conducted in southern brazil this study has some limitations. it was a cross-sectional study, thus the data were collected at a particular moment and causality cannot be established. our population was a convenience sample and only children from a private school participated. in our study children from low socioeconomic status that generally attend public schools were not included. it has been demonstrated that children from private schools have a lower charge of dental disease compared to those from public school40, but they could have more concerns regarding aesthetic appearance, therefore our results could overestimate the problem. the external validity of our findings is limited. finally, even though the instruments for data collection were not previously validated, some questions were posed to be accurate to report dental appearance satisfaction and the perception of dental crowding23. the present study was based on perceptions of satisfactions and the presence of some tooth conditions such as malocclusion, dental discoloration and trauma. perception assessments are subjected to individual judgments and cannot be measured. for instance, subjective self-assessment of tooth color and objective evaluations presented different outcomes between patients and dentists, since patients could report abnormal tooth color when objective measurements indicated the contrary9. however, the assessment of self-perception can translate the feelings of a person and individual way of self-evaluation. adolescence is a transformational phase, and physical changes and alterations in attitude and self-perception take place, but adolescents’ capacity to cope and adapt with these changes often declines during this period. consciousness of body image increases during childhood and adolescence24. on the other hand, peer groups play a major role in adolescents’ emotional stability and adolescents may place high value on physical attractiveness under peer influence34,37. the understanding of factors collaborating to construct aesthetic perceptions could aid the planning and provision of care that addresses the individual needs and demands. thus unnecessary services could be discarded, providing more effective ones9. however, considering that patients’ psychological wellbeing is fundamental, a balance between patient expectations and cosmetic treatment performance are important challenges for dentistry11. dentists should dialogue with patients, taking into account their desires and expectations and considering the functional needs. the prevalence of self-reported appearance dissatisfaction in adolescents in the present study was 17.4% and it was associated with tooth color, perception of poor alignment and the wish for orthodontics. a large amount of the studied population had already undergone aesthetic treatments (orthodontics and bleaching). acknowledgements the authors are grateful to the state secretary of education, the municipal secretary of education and the direction of private schools, which allowed the study to be performed, to the brazilian national council for scientific and technological development (cnpq) for the funding (#308624/2013-0) provided to the principal investigator (ffd), to the twas/cnpq (process 83903402087/ 190268/2010-7) for the full-time postgraduate fellowship provided for a co-author (mmss) and to the brazilian government fellowships -capes for the phd scholarship given to the first author (gfb). references 1. poonam. dental aesthetics and patient satisfaction, a hospital based survey. arch oral sci res. 2011; 1: 1-3. 2. singh v, hamdan a, rock p. the perception of dental aesthetics and orthodontic treatment need by 10to 11-year-old children. eur j orthod. 2012; 34: 646-51. 3. van der geld p, oosterveld p, van heck g, kuijpers-jagtman am. smile attractiveness. self-perception and influence on personality. angle orthod. 2007; 77: 759-65. 4. samorodnitzky-naveh gr, geiger sb, levin l. patients’ satisfaction with dental esthetics. j am dent assoc. 2007; 138: 805-8. 5. agou s, locker d, muirhead v, tompson b, streiner dl. does psychological well-being influence oral-health-related quality of life reports in children receiving orthodontic treatment? am j orthod dentofacial orthop. 2011; 139: 369-77. 6. tin-oo m, saddki n, hassan n. factors influencing patient satisfaction with dental appearance and treatments they desire to improve aesthetics. bmc oral health. 2011; 11: 1-8. 7. silvola as, varimo m, tolvanen m, rusanen j, lahti s, pirttiniemi p. dental esthetics and quality of life in adults with severe malocclusion before and after treatment. angle orthod. 2014; 84: 594-9. 8. alkhatib mn, holt r, bedi r. age and perception of dental appearance and tooth colour. gerodontology. 2005; 22: 32-6. 9. xiao j, zhou xd, zhu wc, zhang b, li jy, xu x. the prevalence of tooth discolouration and the self-satisfaction with tooth colour in a chinese urban population. j oral rehabil. 2007; 34: 351-60. 10. mehl c, wolfart s, vollrath o, wenz hj, kern m. perception of dental esthetics in different cultures. int j prosthodont. 2014; 27: 523-9. 11. kershaw s, newton jt, williams dm. the influence of tooth colour on the perceptions of personal characteristics among female dental patients: comparisons of unmodified, decayed and ‘whitened’ teeth. br dent j. 2008; 204: e9; discussion 256-7. 12. meireles ss, goettems ml, dantas rv, bona ad, santos is, demarco ff. changes in oral health related quality of life after dental bleaching in a double-blind randomized clinical trial. j dent. 2014; 42: 114-21. 13. josefsson e, lindsten r, hallberg lr. a qualitative study of the influence of poor dental aesthetics on the lives of young adults. acta odontol scand. 2010; 68: 19-26. 14. onyeaso co, sanu oo. perception of personal dental appearance in nigerian adolescents. am j orthod dentofacial orthop. 2005; 127: 700-6. 15. boyatzis cj, baloff p, durieux c. effects of perceived attractiveness and academic success on early adolescent peer popularity. j genet psychol. 1998; 159: 337-44. 16. damyanov nd, witter dj, bronkhorst em, creugers nh. satisfaction with the dentition related to dental functional status and tooth replacement in an adult bulgarian population: a cross-sectional study. clin oral investig. 2013; 17: 2139-50. 17. macintyre s, west p. social, developmental and health correlates of ‘attractiveness’ in adolescence. sociol health illness. 1991; 13: 149-67. 18. chestnutt ig, burden dj, steele jg, pitts nb, nuttall nm, morris aj. the orthodontic condition of children in the united kingdom, 2003. br dent j. 2006; 200: 609-12;quiz 38. 19. van wezel na, bos a, prahl c. expectations of treatment and satisfaction with dentofacial appearance in patients applying for braz j oral sci. 15(1):8-15 14factors influencing dental appearance satisfaction in adolescents: a cross-sectional study conducted in southern brazil orthodontic treatment. am j orthod dentofacial orthop. 2015; 147: 698-703. 20. john mt, slade gd, szentpetery a, setz jm. oral health-related quality of life in patients treated with fixed, removable, and complete dentures 1 month and 6 to 12 months after treatment. int j prosthodont. 2004; 17: 503-11. 21. mehl c, harder s, lin j, vollrath o, kern m. perception of dental esthetics: influence of restoration type, symmetry, and color in four different countries. int j prosthodont. 2015; 28: 60-4. 22. salas mm, nascimento gg, huysmans mc, demarco ff. estimated prevalence of erosive tooth wear in permanent teeth of children and adolescents: an epidemiological systematic review and meta-regression analysis. j dent. 2015; 43: 42-50. 23. graber lw, lucker gw. dental esthetic self-evaluation and satisfaction. am j orthod. 1980; 77: 163-73. 24. espeland lv, stenvik a. perception of personal dental appearance in young adults: relationship between occlusion, awareness, and satisfaction. am j orthod dentofacial orthop. 1991; 100: 234-41. 25. peres kg, barros aj, anselmi l, peres ma, barros fc. does malocclusion influence the adolescent’s satisfaction with appearance? a cross-sectional study nested in a brazilian birth cohort. community dent oral epidemiol. 2008; 36: 137-43. 26. brazil. sb brazil 2010: national survey of oral health. main results. brasilia: ministry of health; 2011. p. 92. 27. p e r e s k g , c a s c a e s a m , l e a o at, c o r t e s m i , ve t t o r e m v. [sociodemographic and clinical aspects of quality of life related to oral health in adolescents]. rev saude publica. 2013; 47 suppl 3: 19-28. 28. de paula junior df, santos nc, da silva et, nunes mf, leles cr. psychosocial impact of dental esthetics on quality of life in adolescents. angle orthod. 2009; 79: 1188-93. 29. odioso ll, gibb rd, gerlach rw. impact of demographic, behavioral, and dental care utilization parameters on tooth color and personal satisfaction. compend contin educ dent suppl. 2000: s35-41; quiz s3. 30. al-zarea bk. satisfaction with appearance and the desired treatment to improve aesthetics. int j dent.2013; 2013: 912368. 31. ibiyemi o, taiwo jo. psychosocial aspect of anterior tooth discoloration among adolescents in igbo-ora, southwestern nigeria. annals ibadan postgraduate med. 2011; 9: 94-9. 32. marques ls, filogônio ca, filogônio cb, pereira lj, pordeus ia, paiva sm et al. aesthetic impact of malocclusion in the daily living of brazilian adolescents. j orthod. 2009; 36: 152-9. 33. agou s, locker d, streiner dl, tompson b. impact of self-esteem on the oral-health-related quality of life of children with malocclusion. am j orthod dentofacial orthop. 2008; 134: 484-9. 34. shaw wc. the influence of children’s dentofacial appearance on their social attractiveness as judged by peers and lay adults. am j orthod. 1981; 79: 399-415. 35. gosney mb. an investigation into some of the factors influencing the desire for orthodontic treatment. br j orthod. 1986; 13: 87-94. 36. klages u, claus n, wehrbein h, zentner a. development of a questionnaire for assessment of the psychosocial impact of dental aesthetics in young adults. european j orthod. 2006; 28: 103-11. 37. henson st, lindauer sj, gardner wg, shroff b, tufekci e, best am. influence of dental esthetics on social perceptions of adolescents judged by peers. am j orthod dentofacial orthop. 2011; 140: 389-95. 38. birkeland k, boe oe, wisth pj. relationship between occlusion and satisfaction with dental appearance in orthodontically treated and untreated groups. a longitudinal study. eur j orthod. 2000; 22: 509-18. 39. piovesan c, padua mc, ardenghi tm, mendes fm, bonini gc. can type of school be used as an alternative indicator of socioeconomic status in dental caries studies? a cross-sectional study. bmc med res methodol 2011; 11: 37. 40. goettems ml, correa mb, vargas-ferreira f, torriani dd, marques m, domingues mr, et al. methods and logistics of a multidisciplinary survey of schoolchildren from pelotas, in the southern region of brazil. cad saude publica. 2013; 29: 867-78. braz j oral sci. 15(1):8-15 15 factors influencing dental appearance satisfaction in adolescents: a cross-sectional study conducted in southern brazil 1http://dx.doi.org/10.20396/bjos.v18i0.8655468 volume 18 2019 e191581 original article 1 associate professor department of prosthodontics, college of dentistry, prince sattam bin abdulaziz university, al-kharj 11942, saudi-arabia. 2 specialist, department of prosthetic dental sciences, ministry of health asser region, saudi arabia. corresponding author: fawaz alqahtani, 3603 imam abdullah bin saud road,unit # 646, 8356-13225 riyadh. e-mail: implantologist@yahoo.com received: february 19, 2019 accepted: may 08, 2019 the effect of surface treatment and thermocycling on the shear bond strength of porcelain laminate veneering material cemented with different luting cements fawaz alqahtani1,*, mohammed alkhurays 2 aim: the study aimed to evaluate and compare the effect of different surface treatment and thermocycling on the shear bond strength (sbs) of different dual-/light-cure cements bonding porcelain laminate veneers (plv). methods: one hundred and twenty a2 shade lithium disilicate discs were divided into three groups based on the resin cement used and on the pretreatment received and then divided into two subgroups: thermocycling and control. the surface treatment were either micro-etched with aluminium trioxide and 10% hydrofluoric acid or etched with 10% hydrofluoric acid only before cementation. three dual-cure (variolink esthetic (i), relyx ultimate (ii), and relyx unicem (iii)) and three light-cure (variolink veneer (iv), variolink esthetic (v), relyx veneer (vi)) resin cements were used for cementation. the sbs of the samples was evaluated and analysed using three -way anova with statistical significant set at α=0.05. results: for all resin cements tested with different surface treatments, there was a statistically significant difference within resin cements per surface treatment (p<0.05). the shear bond strength in the micro-etch group was significant higher than the acid-etch group (p<0.05) there was statistically significant interaction observed between the surface treatment and thermocycling (p<0.05) as well as the cement and thermocycling(p<0.05). it was observed that the reduction in shear bond strength after thermocycling was more pronounced in the acid etch subgroup as compared to the microetch subgroup. however, the interaction between the three factors: surface treatments, thermocycling and 2 alqahtani and alkhurays introduction the most desirable characteristics of a dental restoration are good esthetics, strength and chemical stability, and ceramics inarguably possess these qualities1-3. the ceramic restorations is the first choice to be used for indirect restoration1,3. the sbs is getting more promising due to the newly developed resin cements with new composition of ceramic materials3. resin cements are the most used materials for the cementation of indirect restorations. the advantages of resin cements include improved marginal seal, reduced risk of postoperative sensitivity, low solubility, and superior mechanical properties, compared to zinc phosphate and glass-ionomer cements4-6. however, the success of ceramic restorations depends on factors, like the composition of the ceramic material and the cementation procedure7-9. therefore, for the long-term success of ceramics, selection of the appropriate resin cement as well as the bonding procedure is imperative. the efforts to improve resin bonding to ceramic include the application of different ceramic surface treatments. lithium disilicate glass ceramic (ips e.max press, ivoclar vivadent, schaan, liechtenstein) may be adhesively cemented, but the retention may be inadequate when the retentive area is small. etching with hydrofluoric acid roughens the surface on the bonding area of the ceramic material to enhance bonding by micromechanical interlocking between the ceramic and resin cement. it also creates irregularities within the lithium disilicate crystals by removing the glass matrix and the second crystalline phase3,9-11. airborne particle abrasion with 50-lm aluminum oxide (al2o3) particles is another surface treatment recommended for ceramic surfaces to aid in mechanical retention10-14. it leads to the coating of the ceramic surface must with a suitable silane, thereby resulting in the formation of chemical bonds between the inorganic phase of the ceramic and the organic phase of the resin cement11,15-17. clinically, when ceramic restorations are cemented and exposed to the oral environment, factors that could result in fatigue may influence their physical and mechanical properties. fatigue fracture is a form of failure that occurs in structures with microscopic cracks subjected to dynamic and fluctuating stresses9. thermal variations and the evaluation of fatigue resistance of dental ceramics could provide a more detailed understanding of clinical failures18. long-term water storage and thermocycling of bonded specimens are accepted methods to simulate aging and to stress the bondresin cements did not demonstrate statistically significant differences between and within groups (p=0.087). conclusions: within the limitations of the present study, it acan be concluded that dual cure resin cements showed a higher shear bond strength as compared to light cure resin cements. thermal cycling significantly decreased the shear bond strength for both ceramic surface treatments. after thermocycling, the specimens with 10% hf surface treatment showed lower shear bond strength values when compared to those treated by sandblasting with al2o3 particles. keywords: resin cements. dental cements. dental porcelain. shear strength. 3 alqahtani and alkhurays ing interface19. most studies that apply these methods reveal significant differences between early and late bond strength values20. microtensile, shear or tensile testing methods with and without simulated aging and/or thermocycling have been used and conflicting results are reported regarding the effect on bond strength after aging in water and thermocycling21-25. hence, there is a need to examine the effect of thermocycling and restoration surface treatment on the longevity of restorations estimated using shear bond strength. the aim of this study was to evaluate and compare the effect of different surface treatment and thermocycling on the sbs of different dual-/light-cure cements bonding plv. the null hypothesis was that there is no difference in the bond strength of differently pre-treated and thermocycled plv cemented using different light-/dual-cure resin cements. material and methods an in vitro experimental study was conducted to evaluate the effect of thermocycling and two different surface treatments on the shear bond strength of plv cemented with three light cure and three dual cure cements. lithium disilicate computer aided design/computer aided manufacturing (cad/cam) blocks (ivoclar vivadent, schaan, liechtenstein) were used to prepare one hundred and twenty a2 shade digitally calibrated discs (3mm × 10 mm) according to the manufacturer’s instructions. the specimens were designed using the 3d builder software and saved as stereolithography (stl) file. subsequently, milling was done with cam 5-s1(vhf, ammerbuch, germany)21,26. to ensure surface standardisation, the ceramic surfaces were finished and polished using the manufacturers’ recommended kit (lus80, meisinger, usa). the firing of the specimens was done at 850°c followed by embedding in the autopolymerising acrylic resin. the discs were sanded with 400-grit followed by 600-grit wet silicon carbide paper until the ceramic discs were perfectly flush with the acrylic resin. to clean off the abrasive particles, the specimens were rinsed, dried, and subsequently treated with 37% phosphoric acid for 1 minute. all specimens were again rinsed under running water and dried. the specimens were randomly divided into three light cure and three dual cure groups according to the cements used. three dual-cure variolink esthetic (i), relyx ultimate (ii), and relyx unicem (iii) and three light-cure variolink veneer (iv), variolink esthetic (v), relyx veneer (vi) resin cements were used. each group was further divided into two subgroups according to the surface treatment – micro-etch and acid-etch. the specimens were further divided into control and thermocycled subgroups. (fig. 1) the two surface treatments were micro-etching with al2o3 with particles size of 40 µm followed by etching with 10 % hydrofluoric acid (micro-etch) for two minutes and only etching with 10 % hydrofluoric acid (acid-etch) for two minutes. the debris was rinsed off and a special mould to provide a uniform area for cementation was placed at the center of each specimen. all resin cements were applied directly from a syringe on to the treated surface of the specimens. a 1-kg weight was placed on the top to form a uniform cemented layer. the specimens were then light cured for 40 seconds. 4 alqahtani and alkhurays sixty specimens (5 from each subgroup) were subjected to thermocycling, 3500 times between 5ºc and 55ºc, with a dwell time of 30 seconds at each temperature and a transfer time of 15 seconds. the other 60 specimens that were not subjected to thermocycling served as the control group. the specimens were tested for shear bond strength using a universal testing machine (instron corp, canton, mass., usa). the specimens were fixed by using a jig, and the interface between the specimens and resin was loaded at a crosshead speed of 1 mm/min. a knife-edge stainless steel chisel with a thickness 0.34 mm and diameter of 10 mm was used for loading. the shear load at failure was recorded by the software and the values were converted to stress in mpa. statistical analysis the data was analyzed using software ibm spss v. 20.0 (ibm statistics, spss, chicago, usa). the normality of the data was assessed using the shapiro wilk test while levene’s test for equality of error variances was used to analyze the homogeneity of error variances. thre-way anova with bonferroni’s correction for multiple group comparisons was used to test the interaction between factors: resin cement, surface treatment and thermocycling and its effect on the shear bond strength (mpa). p value less than 0.05 was considered statistically significant. results the mean and standard deviation for the shear bond strength at maximum load in mpa were compared using three-way anova (table 1). there was a statistically significant difference observed in the shear bond strength between the cements (p<0.05). there was a statistically significant difference seen in the shear bond strength of figure 1. flowchart showing the distribution of the study groups 5 alqahtani and alkhurays cements treated by different surface treatment (p < 0.05). within the acid etch group, the highest shear bond strength was observed by the dual cure cements iii whereas the lowest shear bond strength was for light cure cement v followed by i which were significantly different from the other resin cements (p < 0.05). within the micro-etch group, the highest shear bond strength was observed for vi, whereas the lowest shear bond strength was for the v followed by i which were significantly different from the other resin cements (p < 0.05). the shear bond strength in the micro-etch group was significantly higher across all the cements tested as compared to the acid-etch group (p < 0.05) also, there was a statistically significant difference observed between the thermocycled and non thermocycled subgroups (p<0.05). across all the resin cement groups, thermocycling significantly reduced the shear bond strength of the resin cements for both the surface treatments. however, the interaction between the three factors: surface treatments, thermocycling and resin cements did not demonstrate statistically significant differences between and within groups (p = 0.087). there was statistically significant interaction observed between the surface treatment and thermocycling (p<0.05). it was observed that the reduction in shear bond strength after thermocycling was more pronounced in the acid etch subgroup as compared to the microetch subgroup. for all resin cements tested with different surface treatments, there was a statistically significant difference within resin cements per surface treatment (p < 0.05). discussion the results of this study show that there are significant differences in bond strength between the thermocycled and non-treated specimens. aging in water significantly affected the bond strength of the pre-treated specimens. this effect was more protable 1. shear bond strength of the tested cements per surface treatment and thermocycling surface treatment/ cement shear bond strength (mpa) (mean ± sd) light-cure cements dual-cure cements v iv vi i iii ii thermocycling acid 11.36 ± 0.06a 6.95 ± 0.10b 12.00 ± 0.12a 9.42 ± 0.11c 13.21 ± 0.16d 12.31 ± 0.19ad acid + microetch 15.11 ± 0.35a 8.50 ± 0.35b 15.50 ± 0.96a 11.15 ± 0.32c 14.30 ± 0.74ae 13.53 ± 0.44de no thermocycling acid 12.13 ± 0.04i 8.26 ± 0.06ii 14.08 ± 0.79iii 11.65 ± 0.26i 13.37 ± 0.10iii 12.58 ± 0.53i, iii acid + microetch 13.85 ± 0.56αδ 9.12 ± 0.19β 15.96 ± 0.67χ 12.94 ± 0.23α 14.30 ± 0.89δ 13.67 ± 0.26αδ three-way anova; p < 0.05 is significant. factor 1: cement; p <0.001 factor 2: surface treatment; p <0.001 factor 3: thermocycling; p <0.001 factor 1*2; p <0.001 factor 1*3; p <0.001 factor 2*3; p <0.001 factor 1*2*3; p = 0.087 different superscripts indicate significant differences across significant groups. 6 alqahtani and alkhurays nounced for the specimens that received additional air abrasion with acid etching. therefore, the null hypothesis tested in the study was that there is no difference in the bond strength of differently pre-treated and aged plv cemented using different light-/dual-cure resin cements has been rejected. the adhesive porcelain veneer complex has been proven to be very strong in vitro and in vivo. an optimal bonded restoration can be obtained especially if the preparation is done properly, correct adhesive treatment procedures are performed, and a suitable resin cement is chosen. the mediumto long-term esthetic maintenance of porcelain veneers is excellent, resulting in high patient satisfaction. also, there are no adverse effects on the gingival health in patients with an optimum oral hygiene. however, the performance of the adhesive resin cements the natural oral habitat has been a topic of debate amongst clinicians27. the accepted methods to simulate aging and to stress the bonding interface are long-term water storage and thermocycling of the bonded specimens28-30. this is typically performed with temperatures between 5°c and 55°c28-32. in several studies, thermocycling was combined with a second treatment, such as dynamic loading, which was termed artificial aging31,33. most studies that used these methods revealed significant differences between early and late bond strength values34. white et al.35 showed that immersion of ceramics in water decreased their static strength and increased the crack velocity. subramanian et al.36 reported a decrease in flexural strength of aluminous and feldspathic porcelains when tested in water. they observed that the failure of the restorations and postoperative cracking can arise because of thermal variations. moreover, the resin cement used for luting the laminate veneer may impose surface changes on the veneer when it is subjected to thermocycling. stacey37 (1993) investigated the relative bond strength surface-treated porcelain and the effectiveness of silane treatment of the etched porcelain. he found that after thermocycling, etching the porcelain surface with acid did not create a sufficiently reliable bond with enamel for plvs. thermocycling did not significantly reduce the strength of etched enamel/composite resin/etched porcelain bonding when the porcelain was treated with silane. silane treatment of the etched porcelain surface can be considered a practical and a necessary procedure. in the present study, it was observed that though thermocycling reduced the sbs of the resin cenments, the reduction was significantly higher in the specimens which underwent only acid etching. hence, it can be inferred that microetching is an advantageous procedure which helps in improvement of the durability of plvs. understanding the mechanism behind the effects of water on the mechanical properties of polymers is of utmost imprtance. the sensitivity of resin-based materials to water depends on a multitude of factors suc as the degree of monomer conversion, degree of polymer crosslinking, volume fraction of intrinsic nanometer-sized pores, and the quantity and presence of fillers9,38,39. one study found that an increase in water sorption was observed by increasing the ratio of triethyleneglycol dimethacrylate (tegdma) and urethane dimethacrylate to bisphenol-a-glycidyl dimethacrylate40-41. this possible effect of tedgma in the resin cement on water sorption and thereby 7 alqahtani and alkhurays its effects on the mechanical properties of the resin cement after fatigue testing and thermocycling need to be investigated in future studies. this could provide a basis for future research for improving the stability of resin cements in the oral environment, therreby 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titley k, caldwell r, kulkarni g. factors that affect the shear bond strength of multiple component and single bottle adhesives to dentin. am j dent. 2003 apr;16(2):120-4. 26. della bona a, van noort r. shear vs. tensile bond strength of resin composite bonded to ceramic. j dent res. 1995 sep;74(9):1591-6. 27. peumans m, van meerbeek b, lambrechts p, vanherle g. porcelain veneers: a review of the literature. j dent. 2000 mar;28(3):163-77. 28. palacios rp, johnson gh, phillips km, raigrodski aj. retention of zirconium oxide ceramic crowns with three types of cement. j prosthet dent. 2006 aug;96(2):104-14. 29. johnson gh, hazelton lr, bales dj, lepe x. the effect of a resin-based sealer on crown retention for three types of cement. j prosthet dent. 2004 may;91(5):428-35. 30. ernst cp, cohnen u, stender e, willershausen b. in vitro retentive strength of zirconium oxide ceramic crowns using different luting agents. j prosthet dent. 2005 jun;93(6):551-8. 31. karimipour-saryazdi m, sadid-zadeh r, givan d, burgess jo, ramp lc, liu pr. influence of surface treatment of yttrium-stabilized tetragonal zirconium oxides and cement type on crown retention after artificial aging. j prosthet dent. 2014 may;111(5):395-403. doi: 10.1016/j.prosdent.2013.09.034. 32. seto kb, mclaren ea, caputo aa, white sn. fatigue behavior of the resinous cement to zirconia bond. j prosthet dent. 2014 may;111(5):395-403. doi: 10.1016/j.prosdent.2013.09.034. 9 alqahtani and alkhurays 33. shahin r, kern m. effect of air-abrasion on the retention of zirconia ceramic crowns luted with different cements before and after artificial aging. dent mater. 2010 sep;26(9):922-8. doi: 10.1016/j.dental.2010.06.006. 34. blatz mb, sadan a, kern m. resin-ceramic bonding: a review of the literature. j prosthet dent. 2003 mar;89(3):268-74. 35. white sn, zhao xy, zhaokun y, li zc. cyclic mechanical fatigue of a feldspathic dental porcelain. int j prosthodont. 1995 sep-oct;8(5):413-20. 36. subramanian d, sivagami 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applicability of bolton’s tooth size ratios in mediterranean, japanese and japanesebrazilian populations karine laskos sakoda1, arnaldo pinzan2, guilherme janson3, sérgio elias neves cury1 1dds, msc, graduate student. department of orthodontics, bauru dental school, university of são paulo, bauru, brazil 2dds, msc, phd. associate professor. department of orthodontics, bauru dental school, university of são paulo, bauru, brazil 3dds, msc, phd, mrcdc. professor and head. department of orthodontics, bauru dental school, university of são paulo, bauru, brazil correspondence to: dr. karine l. sakoda department of orthodontics bauru dental school university of são paulo alameda octávio pinheiro brisolla 9-75 bauru sp 17012-901 brazil business phone/fax: 55 14 32358217 e-mail: karine_vaz@hotmail.com abstract objective: the aim of this study was to determine if bolton’s tooth size ratios can be applied to mediterranean, japanese and japanese-brazilian populations. materials and methods: the sample comprised 90 pairs of dental casts of untreated individuals with normal occlusion, divided into 3 groups according to ethnical characteristics: white (30 mediterranean descendant subjects, with a mean age of 13.64 years), japanese (30 subjects with japanese ancestry, with a mean age of 15.63 years) and japanese-brazilian (30 japanese-brazilian subjects, with a mean age of 13.96 years). a digital caliper was used to measure the maxillary and mandibular mesiodistal widths from first molar to first molar on each dental cast. the anterior and overall tooth size ratios were calculated. t test was applied for comparisons between bolton standards and the ethnical groups for anterior and overall ratios. results: only the japanese-brazilian group showed significantly greater ratios than bolton standards. conclusion: it was concluded that bolton’s ratios are not applicable to the japanese-brazilian population. therefore, it is suggested that bolton’s ratios may not be suitable for different populations. keywords: dental occlusion. ethnic groups. orthodontics. received for publication: december 13, 2016 accepted: july 26, 2017 original article braz j oral sci. october | december 2016 volume 15, number 4 http://dx.doi.org/10.20396/bjos.v15i4.8650038 introduction tooth size proportion is an important factor in orthodontic diagnosis and treatment planning1. appropriate tooth width proportion between maxillary and mandibular teeth is required to achieve ideal occlusal interdigitation, overjet and overbite at the end of orthodontic treatment2. when a discrepancy is not detected, it may take longer to finish treatment and result in compromised outcome1. thus, it seems prudent for clinicians to include routinely a tooth size analysis during treatment planning3. although different methods of measuring tooth size proportions have been developed4,5, bolton’s tooth size ratio2 is the most commonly accepted and recognized as an important diagnostic tool for detecting interarch tooth size discrepancies. ideal number, size and shape, normal overbite and overjet relationships, absence of large restorations or caries that could affect the teeth’s mesiodistal diameter, dental casts in good conditions and ethnicity verified by photographs and subjects’ history obtained in surveys filled out by themselves or by their guardians. data collection the measurements were directly performed on the dental models, by one examiner (k. l. s.). a digital caliper (mitutoyo co, kanagawa, japan) accurate to 0.01 mm was used for measurements. the mesiodistal widths of each tooth from the maxillary and mandibular right first molar to the left first molar were measured at the largest distance between the contact points on the proximal surfaces. the caliper was positioned by the vestibular surface, parallel to the occlusal surface and perpendicular to the long axis of the crown. the anterior and overall bolton ratios were calculated, according to the formulas: (sum mandibular “6”/sum maxillary “6”)*100=anterior ratio (%) (sum mandibular “12”/sum maxillary “12”)*100=overall ratio (%) error study to evaluate intra examiner errors, the measurements were repeated 30 days after the first assessment in 30 pairs of randomly selected study casts. random errors were estimated with dahlberg’s formula and systematic errors were evaluated with paired t tests. statistical analysis since all variables presented normal distribution according to kolmogorov-smirnov tests, t test was applied to evaluate intersex differences and for comparisons between bolton standards and the ethnical groups for anterior and overall tooth size ratios. statistical analyses were performed with statistica software (statistica for windows 7.0 copyright statsoft, inc. tulsa, okla, usa). results were considered significant at p<0.05 results there were no significant systematic errors, and the random errors of the mesiodistal diameters of each tooth were within acceptable limits14,15, ranging from 0.10 (canines) to 0.31 mm (molars). there were no statistically significant intersex differences within each ethnicity for both anterior and overall ratios (table 1). only the japanese-brazilian group showed significantly greater anterior and overall ratios than bolton standards (table 2). for the anterior ratio, japanese-brazilians presented more subjects outside bolton standards ±2sd, followed by white and japanese subjects. for the overall ratio, japanese-brazilians presented more patients outside bolton standards ±2sd, followed by japanese subjects (table 3). the white subjects were within the overall standards ±2sd. xx anterior and overall ratios for proper harmony of maxillary and mandibular teeth were established with mean values of 77.2% (sd=1.65%) and 91.3% (sd=1.91%) respectively2. despite its importance in the clinical setting to guide the orthodontist in cases with extreme tooth size discrepancies, bolton’s ratios have limitations, since the population and sex proportions of his sample were not specified, which implies potential selection bias6. there is evidence in the literature pointing towards ethnic differences in tooth size ratios6-8. it is reported that people with african ethnic backgrounds have larger teeth than do japanese and caucasians9. because population differences in maxillary tooth size are not the same as the differences in mandibular tooth size, different interarch relationships might be expected6. currently, the information regarding tooth size discrepancy prevalence among ethnicities is controversial. although some studies report that bolton’s ratios can be applied for different populations10,11, others claim the opposite6-8,12,13. smith et al.6 (2000) found significant differences in interarch ratios between whites, blacks and hispanics. uysal and sari12 (2005) concluded that bolton’s original data do not represent turkish people. endo et al.7 (2007) detected a high prevalence rate of anterior tooth size discrepancy of more than 2sd above bolton’s mean and concluded that bolton anterior ratio is not applicable to the japanese population. paredes et al.8 (2011) noticed that anterior ratios for peruvian and spanish people are greater than bolton’s. subbarao et al.13 (2014) also found significant differences for the anterior and overall ratios compared to bolton's ratios in an indian population. these studies highlight the need for population-specific standards for clinical assessments of tooth size ratios. based on the controversy of the evidence available, studies are needed to assess the applicability of bolton’s ratios across different ethnicities. therefore, the purposes of this study are to determine anterior and overall ratios in mediterranean, japanese and japanese-brazilian populations and to compare them with bolton’s standard ratios. materials and methods sample selection the sample comprised 90 pairs of dental casts of untreated brazilian individuals, with normal occlusion and harmonious face, obtained from the files of the orthodontic department at bauru dental school, university of são paulo, bauru, brazil. the sample was divided into 3 groups, according to ethnical characteristics. the white group consisted of 30 mediterranean descendant subjects (15 male and 15 female), with a mean age of 13.64 years. the japanese group consisted of 30 subjects with japanese ancestry only (15 male and 15 female), with a mean age of 15.63 years. the japanese-brazilian group consisted of 30 japanese-brazilian subjects (japanese blended with white mediterranean subjects 15 male and 15 female), with a mean age of 13.96 years. the selection criteria were complete permanent dentition from first molar to first molar in both arches, angle class i molar relationship, with no crowding, no dental anomalies of applicability of bolton’s tooth size ratios in mediterranean, japanese and japanese-brazilian populations braz j oral sci. 15(4):269-272 xx discussion bolton’s2 (1962) sample consisted of 55 dental models with excellent occlusion, most of them orthodontically treated (44 cases). he did not specify the population and sex distribution of his sample. our sample consisted of dental models of untreated subjects with optimal occlusion, and the ethnic groups should be as pure as possible. the difficulties to obtain a sample with those characteristics explain the rather small sample size of the present study. it is generally agreed that there is no sex difference in tooth size ratios, as many studies report absence of sexual dimorphism in anterior and overall ratios1,7,10,11,16-19. since we didn’t find any statistically significant intersex difference in either anterior or overall ratios, and, also, bolton2 (1962) did not specify the sex composition of the sample used in his study, we found it reasonable to combine male and female ratios to compare our tooth size ratios with his results. we did not find significant differences for the anterior and overall ratios in mediterranean nor in japanese, indicating that bolton standards can be used in these populations. similarly, other studies did not report differences in both anterior and overall ratios comparing different populations with bolton standards10. on the other hand, contrasting results were reported by endo et al.7 (2007), who found anterior and overall ratios from a japanese population greater than those from bolton’s american population. although we did not find significant differences, the japanese group in our sample presented smaller values for anterior and total ratios. a possible explanation for this tendency may be the high prevalence of shovel-shaped maxillary incisors in the japanese population20,21, in which the pronounced marginal ridges increase applicability of bolton’s tooth size ratios in mediterranean, japanese and japanese-brazilian populations incisor thickness. bolton2 (1962) pointed out that the anterior ratio could be affected by tooth thickness. rudolph et al.22 (1998) stated that the prediction of the ideal intermaxillary ratio is more accurate when both tooth thickness and mesiodistal tooth width are considered. according to halazonetis23 (1996), each 0.2 mm change in tooth thickness represents 1% change in bolton anterior ratio, i.e., a 1 mm change in incisal edge thickness may affect bolton anterior ratio in up to 5%, which can represent almost 3 mm of tooth size discrepancy. therefore, the ideal ratios may need adjustments, depending on the tooth thickness or the overjet that will remain at the end of treatment23. in order to obtain proper interdigitation and an ideal overjet and overbite relationship, the intermaxillary ratio must be smaller for dental arches with large labiolingual incisor thickness22,23. the japanese-brazilian group showed significantly greater anterior and overall ratios than bolton standards. other studies also reported significantly greater anterior ratio8,7,13,24 and overall ratio6,13,25 than bolton standards for different populations. since the japanese-brazilian subjects represent a mixed race, it could be expected that they presented ratios between mediterranean and japanese. curiously, the japanese subjects showed smaller values, followed by mediterranean and japanesebrazilians. further studies should be undertaken to investigate this issue. according to bolton2 (1962), a value greater than 1 sd from his means indicates the need for diagnostic consideration, but its clinical relevance is questionable. more recently, a clinically significant tooth size ratio discrepancy has been defined as 2 sd outside bolton’s original mean ratios7,12. following the same rationale from other studies7,12, we also defined ratios outside of 2 sd from bolton’s means as values indicating a clinically significant table 1 intersex differences for anterior and overall tooth size ratios (t tests). table 3 number and percentage of subjects outside bolton standards ± 2 sd. white japanese japanese-brazilian anterior ratio overall ratio anterior ratio overall ratio anterior ratio overall ratio male female male female male female male female male female male female mean 77.943 77.224 92.048 91.292 76.933 76.133 90.669 90.685 78.663 78.528 93.027 92.067 sd 2.930 2.186 1.857 1.627 1.641 1.844 1.637 2.045 2.861 2.132 1.877 1.646 p 0.453 0.246 0.220 0.981 0.885 0.148 anterior ratio overall ratio total < 2 sd > 2 sd total < 2 sd > 2 sd n % n % n % n % n % n % white 6 20 2 6.67 4 13.33 0 0 0 japanese 3 10 3 10 0 2 6.67 2 6.67 0 japanese-brazilian 9 30 1 3.33 8 26.67 3 10 0 3 10 bolton (n=55) white (n=30) japanese (n=30) japanese-brazilian (n=30) mean sd mean sd p mean sd p mean sd p anterior ratio 77.2 1.65 77.584 2.566 0.410 76.533 1.763 0.084 78.596 2.480 0.003* overall ratio 91.3 1.91 91.670 1.759 0.383 90.677 1.820 0.150 92.547 1.802 0.004* table 2 comparisons between bolton standards and the ethnical groups for anterior and overall tooth size ratios (t test). * statistically significant at p<.05 braz j oral sci. 15(4):269-272 xx braz j oral sci. 15(4):269-272 discrepancy, as approximately 95% of bolton’s cases were within this range. we observed a higher prevalence of discrepancy in the anterior segment of the dental arches. significant tooth size discrepancies for anterior ratio were found in 10% of the japanese, 20% of the mediterranean and 30% of the japanese-brazilian. no discrepancy was found in overall ratio in mediterranean, while it varied from 6.67% in japanese to 10% in japanese-brazilian. these data corroborate with results reported in other studies, in which discrepancies of 21.3%12, 28.18%1 and 30.6%3 for anterior ratio, and 13.5%3 and 13.64%1 for overall ratio have been reported. most of the discrepancy found in this study was characterized by mandibular excess, confirming the results presented by freeman et al.3 (1996) and endo et al.7 (2007). according to the results obtained in this study in comparison to others, we can speculate that the values of tooth size ratios can vary not only between sex or populations. surprisingly, the same population can show contrasting results. therefore, generalized use of bolton’s ratios is questionable and may not be suitable for different populations. acknowledgements the authors would like to acknowledge the financial support from the capes foundation. references 1. tadesse p, zhang h, long x, chen l. a clinical analysis of tooth size discrepancy (bolton index) among orthodontic patients in wuhan of central china. j huazhong univ sci technolog med sci. 2008 aug;28(4):491-4. doi: 10.1007/s11596-008-0427-8. 2. bolton wa. the clinical application of a tooth-size analysis. am j orthod. 1962 jul;48(7):504-29. 3. freeman je, maskeroni aj, lorton l. frequency of bolton tooth-size discrepancies among orthodontic patients. am j orthod dentofacial orthop. 1996 jul;110(1):24-7. 4. bailey e, nelson g, miller aj, andrews l, johnson e. predicting tooth-size discrepancy: a new formula utilizing revised landmarks and 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med oral patol oral cir bucal. 2011 jul 1;16(4):e593-9. 9. fernandes tm, sathler r, natalicio gl, henriques jf, pinzan a. comparison of mesiodistal tooth widths in caucasian, african and japanese individuals with brazilian ancestry and normal occlusion. dental press j orthod. 2013 may-jun;18(3):130-5. 10. nourallah aw, splieth ch, schwahn c, khurdaji m. standardizing interarch tooth-size harmony in a syrian population. angle orthod. 2015 jul-sep;4(3):77-82. doi: 10.4103/2278-0203.160240. 11. abdalla hashim ah, eldin ah, hashim ha. bolton tooth size ratio among sudanese population sample: a preliminary study. j orthod sci. 2015 jul-sep;4(3):77-82. doi: 10.4103/2278-0203.160240. 12. uysal t, sari z. intermaxillary tooth size discrepancy and mesiodistal crown dimensions for a turkish population. am j orthod dentofacial orthop. 2005 aug;128(2):226-30. 13. subbarao vv, regalla rr, santi v, anita g, kattimani vs. interarch tooth size relationship of indian population: does bolton's analysis apply? j contemp dent pract. 2014 jan;15(1):103-7. 14. burhan as, nawaya fr. prediction of unerupted canines and premolars in a syrian sample. prog orthod. 2014 jan 6;15:4. doi: 10.1186/21961042-15-4. 15. wedrychowska-szulc b, janiszewska-olszowska j, stepien p. overall and anterior bolton ratio in class i, ii, and iii orthodontic patients. eur j orthod. 2010 jun;32(3):313-8. doi: 10.1093/ejo/cjp114. 16. kachoei m, ahangar-atashi mh, pourkhamneh s. bolton's intermaxillary tooth size ratios among iranian schoolchildren. med oral patol oral cir bucal. 2011 jul;16(4):e568-72. 17. kumar p, singh v, sharma p, sharma r. effects of premolar extractions on bolton overall ratios and tooth-size discrepancies in a north indian population. j orthod sci. 2013 jan;2(1):23-7. doi: 10.4103/22780203.110329. 18. alam mk, shahid f, purmal k, ahmad b, khamis mf. bolton tooth size ratio and its relation with arch widths, arch length and arch perimeter: a cone beam computed tomography (cbct) study. acta odontol scand. 2014 nov;72(8):1047-53. doi: 10.3109/00016357.2014.946967. 19. alam mk, iida j. overjet, overbite and dental midline shift as predictors of tooth size discrepancy in a bangladeshi population and a graphical overview of global tooth size ratios. 2013 nov;71(6):1520-31. doi: 10.3109/00016357.2013.775336. 20. tsai pl, hsu jw, lin lm, liu km. logistic analysis of the effects of shovel trait on carabelli's trait in a mongoloid population. am j phys anthropol. 1996 aug;100(4):523-30. 21. kimura r, yamaguchi t, takeda m, kondo o, toma t, haneji k, et al. a common variation in edar is a genetic determinant of shovel-shaped incisors. am j hum genet. 2009 oct;85(4):528-35. doi: 10.1016/j. ajhg.2009.09.006. 22. rudolph dj, dominguez pd, ahn k, thinh t. the use of tooth thickness in predicting intermaxillary tooth-size discrepancies. angle orthod. 1998 apr;68(2):133-8; discussion 139-40. 23. halazonetis dj. the bolton ratio studied with the use of spreadsheets. am j orthod dentofacial orthop. 1996 feb;109(2):215-9. 24. bernabe e, major pw, flores-mir c. tooth-width ratio discrepancies in a sample of peruvian adolescents. am j orthod dentofacial orthop. 2004 mar;125(3):361-5. 25. singh s, hlongwa p, khan mi. bolton ratios in a sample of black south africans. sadj. 2011 aug;66(7):336-9. untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8652924 volume 17 2018 e18325 original article 1 dds, department of dentistry, federal university of rio grande do norte (ufrn), natal, rio grande do norte, brazil. 2 phd, associate professor, department of dentistry, federal university of rio grande do norte (ufrn), natal, rio grande do norte, brazil. 3 phd, associate professor, institute of chemistry, federal university of rio grande do norte (ufrn), natal, rio grande do norte, brazil. corresponding author: ana margarida dos santos melo, 35 president juscelino kubitschek street, natal-rn 59069-170, brazil; tel.: +55-84-98600-3561; fax: +55-84-3215-4101; email: anamdsmelo@gmail.com received: february 27, 2018 accepted: june 24, 2018 degree of conversion, translucency and intrinsic color stability of composites during surface modeling with lubricants ana margarida dos santos melo1, thiago jonathan silva dos santos1, matheus dantas tertulino1, maria cristina dos santos medeiros2, ademir oliveira da silva3, boniek castillo dutra borges2 the tackiness presented by resins causes problems in performing the incremental technique, which can be improved by using lubricants in handling. aim: to evaluate the influence of two adhesive systems as brush lubricants on the degree of conversion, translucency and intrinsic color stability of composite resins. methods: samples (n=10) were fabricated according to the composites (filtek z350 xt, ips empress direct, and esthet x hd), shades (bleach shade and a2), and lubricants used (adper single bond 2, adper scotchbond multi-purpose bonding agent, and no lubricant). single composite increments were inserted into a teflon mold. the composite surface was then modeled using a brush dipped in an adhesive system. the control group was fabricated with no additional modeling. the surface degree of conversion (dc) was measured using fourier transform infrared spectroscopy; translucency and intrinsic color stability were accessed using a spectrophotometer. data were analyzed using three-way anova and the tukey test (p<0.05). results: scotchbond multi-purpose decreased the dc of all composites and shades in comparison with the control group (p<0.05), although it did not decrease color stability (p<0.05). the bleach shade of ips empress direct and esthet x hd showed higher translucency than the a2 shade when lubricated with the adhesive systems (p<0.05). conclusion: therefore, the two tested adhesive systems used as brush lubricants on composites promoted changes to the surface degree of conversion and aesthetic properties. keywords: composite resins. dentin-bonding agents. surface properties. 2 melo et al. introduction the current emphasis in aesthetic dentistry has placed great importance on the achievement of a perfect smile. due to the request for white teeth, individuals often require tooth bleaching with carbamide or hydrogen peroxides. the use of bleach shade composite resins is then of great relevance, since restorations that were completed prior to tooth bleaching should be replaced, as the peroxides do not change the composite color1. therefore, obtaining adequate physical and aesthetic properties of bleach shade composite resins can prolong the lifetime of tooth restorations using bleach shade composite resins. bleach shade composites can present changes in chemical composition when compared with composites of traditional shades, especially regarding the photoinitiator system. camphorquinone is unstable and tends to yellow over time due to its combination with tertiary amines2. therefore, it has been replaced with less yellow photoinitiators, such as phenylpropanedione (ppd), trimethylbenzoyl-diphenyl-phosphine oxide (tpo), and phenylbis (2,4,6-trimethylbenzoyl)-phosphine oxide (bapo)3,4 to decrease yellowing over time. on the other hand, the organic matrix of bleach shade composites has not changed in comparison with those traditional shades. stickiness between the composite and the dental instrument still remains due to the presence of viscous monomers5, causing difficulty in modeling the material6. instrument lubricants have been used to reduce stickiness between the composite and the instruments such as brushes to facilitate composite modeling and prevent drag7,8. however, the effects of these lubricants on the physical properties of the composites and the longevity of the restorations are not yet fully clarified, especially for bleach shade composites. laboratory studies have indicated that the use of lubricants on instruments during composite insertion does not negatively affect the cohesive and flexural properties6-8. in relation to the surface properties, the impregnation of adhesives and alcohol can alter the degree of conversion and the crosslink density of some nanocomposites with traditional shades9. it is known that a low degree of conversion is associated with increased growth of biofilm, the release of monomers, composite solubility, and greater susceptibility to staining10. when considering optical properties, translucency can be described as a state between complete transparency and complete opacity11, and is one of the factors responsible for influencing the final color of the composite12. these optical properties must be faithfully reproduced by the restorations for a natural comparison with natural teeth13. when taking into account the emerging relevance of restorative procedures after tooth bleaching using bleach shade composites, the present study aimed to evaluate the influence of different brush lubricants on the degree of conversion, translucency, and intrinsic color stability of composite resins of bleach and conventional a2 shades. the null hypothesis tested is that the lubricants do not affect the degree of conversion, the intrinsic color stability, or the translucency of the composite resins. 3 melo et al. materials and methods experimental design and preparation of samples this study implemented a 3 x 2 x 3 factorial design. three commercial brands of composites (filtek z350xt [xt], 3m espe, st paul, mn, usa; ips empress direct [ed], ivoclar vivadent, ag, schaan, liechtenstein; and esthet x hd [hd], dentsply caulk, milford, de, usa), in two shades (bleach and a2) and three lubricants (adper single bond 2 [sb], 3m espe, st paul, mn, usa; or adper scotchbond multi-purpose adhesive [mp], 3m espe, st paul, mn, usa), or no lubricant were the factors under study. the degree of conversion, intrinsic color stability, 24-h translucency, and 30-day translucency were the response variables. the compositions of the materials used in this study are listed in table 1. a total of 180 specimens were prepared according to different groups (n = 10 per group) (figure 1). composites were inserted into teflon molds (5 mm diameter × 2 mm height) with a spatula (thompson no. 1, thompson dental products, houston, tx, usa) in a single increment. the composite surface was then modeled using a flat composite brush (nº 1021, hot spot design, cugy, switzerland) coated with an table 1. lot and composition of the materials used in the study. product manufacturer lot composition (%wt) filtek z350 xt 3m espe, st. paul, mn, usa 1520800607 udma (1-10%), bis-ema (1-10%), bis-gma (1-10%), tegdma (<5%), polyethylene glycol dimethacrylate (<5%), silane treated zirconia (1-10%), 2,6-dibutyl-p-cresol (bht) (<0.5%), silane treated ceramics (60-80%), nanofiller (1-10%) ips empress direct ivoclar vivadent ag, schaanm, liechtenstein s10171 t28435 udma (10-25%), ytterbium trifluoride (3-10%), tricyclodocane dimethanol dimethacrylate (3-10%), bisgma (2.5-3%) esthet x hd dentsply caulk, milford, de, usa 025210g 005649g glass fibers loose special purpose (<50%), frits chemical (<30%), tegdma (<20%), urethane modified bisgma dimethacrylate (<10%), silica amorphous – fumed (<10%), silica amorphous (<10%) adper single bond 2 adhesive 3m espe, st. paul, mn, usa n508311 ethyl alcohol (25-35%), bisgma (10-20%), udma (1-5%), hema (5-15%), glycerol 1,3-dimethacrylate (5-10%), nanofiller (20%), copolymer of acrylic acid and itaconic (5-10%), water (<5%), diphenylodonium hexafluorophosphate (<1%), edmab (<1%) adper scotchbond multi-purpose adhesive 3m espe, st. paul, mn, usa n571827 bisgma (60-70%), hema (30-40%), triphenyl antimony (<1%) legend: udma: diurethane dimethacrylate bisema: bisphenol a polyethylene glycol diether dimethacrylate bisgma: bisphenol a diglycidyl ether dimethacrylate tegdma: triethylene glycol dimethacrylates hema: 2-hydroxyethyl methacrylate nanofiller: silane treated silica edmab: ethyl 4-dimethylaminobenzoate 4 melo et al. adhesive system sb and mp. no material was impregnated in the brush for the control group (ct). one drop of the material was applied to the brush head to coat the brush with adhesive systems, and excess was removed by stroking onto a glass plate for 3 s on each side of the brush. sculpting consisted of three sweeping motions of the adhesive-dampened instrument against each half of the circular composite surface (a total of six motions for each sample)9. the composite was photoactivated for 20 seconds using a light emitting diode (led) (coltolux led; coltène whaledent, altstätten, switzerland) with a light intensity of 1200 mw/cm2. a microscopy slide was pressed on the top of the samples to obtain a smooth surface, thus standardizing the distance between the light source and the composite resin during photoactivation. the tip of the light emitting diode was placed in contact and perpendicular to the slide’s surface, covering the entire surface of the sample, since the tip diameter was larger than the sample diameter. after photoactivation, the specimens were removed and the excess material was removed with a scalpel blade. the specimens were stored in dry, light-proof containers in an oven at 37ºc for 24 hours. degree of conversion (dc) the dc was evaluated 24 hours after curing using a fourier transform infrared spectrometer (ftir) (spectrum100 ftir/atr; perkin elmer, shelton, ct, usa) coupled to an attenlegend: a2: composites in color a2 bleached teeth: composites for bleached teeth xt: filtek z350 xt ed: ips empress direct hd: esthet x hd ct: control group sb: adper single bond 2 adhesive mp: adper scotchbond multi-purpose adhesive (nº3) figure 1. distribution of the samples according to the type and shade of composite resins and lubricant used. samples n=180 a2 shade n=90 xt n=30 ct n=10 sb n=10 mp n=10 ed n=30 ct n=10 sb n=10 mp n=10 hd n=30 ct n=10 sb n=10 mp n=10 bleach shade n=90 xt n=30 ct n=10 sb n=10 mp n=10 ed n=30 ct n=10 sb n=10 mp n=10 hd n=30 ct n=10 sb n=10 mp n=10 5 melo et al. uated total reflectance device. absorption spectra of the unpolymerized and the polymerized composite resins were obtained from the region between 4000 and 650 cm−1, with 32 scans at 4 cm−1. the 1590–1660 cm−1 interval was chosen to observe the absorbance at 1608 and 1638 cm−1, indicating the absorption peaks of the bisphenol aromatic vinyl bonds and the aliphatic bonds of the methacrylate functional group, respectively. the dc (%) was calculated using the following equation: dc (%) = 100 × (1 − [r polymerized /r unpolymerized ]), where r is the ratio between the absorbance peak at 1638 cm−1 and 1608 cm−1 14. samples were then stored in water at 37ºc for 24 hours. intrinsic color stability (δe) and translucency the intrinsic color stability and translucency evaluations were performed using a spectrophotometer (vita easyshade, vita zahnfabrik, bad sackingen, germany). following 24 h and 30 days of water storage, the initial (ti) and final (tf) color values, respectively, were taken using standard white and black ceramic backgrounds. each specimen and surface was assessed five times at the same assessment time and the average value was calculated. a silicon (express xt, 3m espe, st. paul, mn, usa) barrier was fabricated to surround the interface between the specimen and the spectrophotometer tip to avoid interference from external light. the silicon barrier allowed the tip to be placed on the same area of the specimen for both color measurements. the color was measured according to the ciel*a*b* color scale, as recommended by the international commission on illumination. spectrophotometers collect spectral data from the reflected light and automatically translate these data into the three-color coordinates (l* = lightness, +a* = red, -a* = green, +b* = yellow, -b* = blue). the intrinsic color stability (δe) was calculated between tf and ti for readings made on the white background using the following equation: δe = [(δl*)2 + (δa*)2 + (δb*)2]1/2, in which δl* represents l* final l* initial; δa* represents a* final a* initial; e δb* represents b* final b* initial15. the 24-h and 30-day translucencies were calculated using the translucency formula (t) for each period using the following equation: t = (l w * l b *)² + (a w * a b *)² + (b w * b b *)1/2, where the subscripts ‘w’ and ‘b’ refer to the ciel*a*b* values for each specimen on white background and black background, respectively16. statistical analysis the data of the dc, intrinsic color stability, 24-h and 30-day translucencies were analyzed statistically by three-way anova and tukey’s post-test with a significance level of 5% (p<0.05). all analyses were performed using assistat 7.7 beta software. results dc there was a statistically significant difference between composites (p<0.05), between lubricants (p<0.05) and the interaction between composites and lubricants (p<0.05). the comparisons between the groups are listed in table 2. when com6 melo et al. pared to the control group, mp reduced the dc of all composites. sb decreased the dc for both ed composite shades, and only increased the dc of the hd composite in a2 shade. there was no statistically significant difference in the control groups for either shade. there was a statistically significant difference between the shades for the xt composite when interacting with all the lubricants, and only for the hd when lubricated with mp. intrinsic color stability there was a statistically significant difference between composites (p<0.05), between lubricants (p<0.05) and in the interaction between composites and lubricants (p<0.05). the comparisons between the groups are listed in table 3. mp promoted higher color change values for the hd composite in the a2 shade when compared to the control group. there was a statistically significant difference between the shades for the ed and hd composites in the control group, with higher color change values for the a2 shade. table 2. means (standard deviations) of the degree of conversion (%) according to the shade, composite and lubricant used. shade composite lubricant none single bond 2 scotchbond a2 z350 xt 73.8 (4.7) aa* 71.1 (6.2) ab* 51.2 (7.4) ba* empress direct 71.3 (12.2) aa 58.2 (5.0) bc 41.4 (8.3) bb esthet x hd 78.3 (8.6) ba 88.6 (4.1) aa 57.5 (7.0) ca* bleached teeth z350 xt 84.1 (7.5) aa 79.4 (4.5) aa 63.1 (10.7) ba empress direct 77.6 (8.2) aa 64.3 (7.6) bb 41.5 (6.1) cb esthet x hd 78.4 (8.5) aa 83.8 (5.2) aa 66.5 (8.4) ba means followed by distinct upper case letters denote statistically significant differences between lubricants for the same shade and composite (p<0.05). means followed by lower case letters denote statistically significant differences between composites for the same shade and lubricant (p<0.05). means followed by an asterisk (*) denote statistically significant differences between shades for the same composite and lubricant (p<0.05). table 3. means (standard deviations) of the intrinsic color stability (δe) according to the colors, resins and lubricants used. color composite lubricant none single bond 2 scotchbond a2 x350 xt 3.0 (1.0) aa 3.0 (0.9) aa 2.3 (1.1) ab empress direct 4.0 (1.6) aa* 2.0 (0.8) ab 2.8 (0.9) ab esthet x hd 2.6 (1.1) ba* 2.4 (0.9) bab 4.6 (1.8) aa bleached teeth x350 xt 2.0 (0.6) aba 3.0 (1.5) aa 3.4 (1.8) bb empress direct 2.8 (1.4) aa 2.8 (1.7) aa 4.0 (2.0) aab esthet x hd 1.4 (0.8) aa 2.6 (1.2) aa 4.3 (1.6) aa means followed by distinct upper case letters denote statistically significant differences between lubricants for the same shade and composite (p<0.05). means followed by lower case letters denote statistically significant differences between composites for the same shade and lubricant (p<0.05). means followed by an asterisk (*) denote statistically significant differences between shades for the same composite and lubricant (p<0.05). 7 melo et al. 24-h translucency there was a statistically significant difference between composites (p<0.05), between lubricants (p<0.05) and in the interaction between composites and lubricants (p<0.05). the comparisons among the groups are listed in table 4. with the exception of composites for bleached teeth lubricated with mp, there was a statistically significant difference in translucency for all composites, including the control groups. only the hd composite in the a2 shade showed no statistically significant difference between the lubricants used. there was an increase in the translucency with the use of lubricants for the xt and a decrease for the ed in both shades, whereas there was only a decrease in the hd composite. there was also a difference between the shades for all sb-lubricated composites and only for the mp-lubricated hd composite, as well as the control group for xt and hd composites. 30-day translucency there was a statistically significant difference between composites (p<0.05), between lubricants (p<0.05) and in the interaction between composites and lubricants (p<0.05). comparisons between the groups are listed in table 5. only the xt composite in the a2 shade showed an increase in translucency when lubricated with sb and mp, table 4. means (standard deviations) of the translucency parameter after 24 hours of immersion in distilled water according to the colors, resins and lubricants used. color composite lubricant none single bond 2 scotchbond a2 filtek z350xt 5.5 (1.0) cb* 7.0 (0.8) aa* 6.3 (0.7) ba empress direct 7.8 (0.9) aa 6.1 (0.6) cb* 7.2 (0.8) ba esthet x hd 4.6 (1.0) ac* 4.6 (0.6) ac* 4.3 (0.5) ab* bleached teeth filtek z350xt 6.2 (0.3) cc 8.1 (0.9) aa 6.9 (0.8) ba empress direct 8.4 (0.9) aa 7.4 (0.5) bb 7.4 (0.4) ba esthet x hd 7.3 (0.9) ab 6.2 (0.3) bc 6.7 (0.9) ba means followed by distinct upper case letters denote statistically significant differences between lubricants for the same shade and composite (p<0.05). means followed by lower case letters denote statistically significant differences between composites for the same shade and lubricant (p<0.05). means followed by an asterisk (*) denote statistically significant differences between shades for the same composite and lubricant (p<0.05). table 5. means (standard deviations) of the translucency parameter after 30 days of immersion in distilled water according to the colors, resins and lubricants used. color composite lubricant none single bond 2 scotchbond a2 x350 xt 5.2 (1.0) bb* 7.7 (1.0) aa 7.2 (0.9) aa empress direct 6.8 (0.6) aa* 6.3 (0.5) ab* 6.5 (0.6) aa* esthet x hd 5.3 (1.0) ab* 4.5 (0.6) ac* 4.5 (1.0) ab* bleach x350 xt 6.9 (0.7) aa 7.7 (0.9) aa 7.1 (0.7) aa empress direct 7.9 (0.9) aa 7.9 (0.9) aa 7.4 (0.9) aa esthet x hd 7.2 (0.6) aa 5.8 (0.8) bb 7.6 (1.0) aa means followed by distinct upper case letters denote statistically significant differences between lubricants for the same shade and composite (p<0.05). means followed by lower case letters denote statistically significant differences between composites for the same shade and lubricant (p<0.05). means followed by an asterisk (*) denote statistically significant differences between shades for the same composite and lubricant (p<0.05). 8 melo et al. whereas only the hd composite in the bleach shade decreased its translucency when lubricated with sb. the comparison between the shades showed higher translucency values for all the composites in the control groups, while only the xt showed no difference for the other lubricants. discussion the null hypothesis tested in this study was rejected, since the use of lubricants promoted changes in the dc and optical properties of the tested composites in their different shades. dc is the percentage of monomers that were converted into polymer during polymerization17. as a consequence, its deficit implies weakening of the mechanical properties of the material, thereby affecting the quality of the formed resinous polymer18,19 and the restoration’s longevity4. in fact, dc depends on the material components such as the type and concentration of monomers, photoinitiators, pigments, and filler content14,20,21. light intensity of the photoactivation device, photoactivation time, and distance between the light source and composite also influence the final dc of the composite20. in this study, mp decreased the dc for all the composites, regardless of the tested shades. mp is a bonding agent that does not contain solvent and has 60 to 70% of bisphenol a glycidyl dimethacrylate (bis-gma), a high molecular weight monomer that promotes decreased dc in composite resins19 a higher incorporation of bisgma at the composite surfaces that were lubricated with mp likely decreased the dc in comparison with the control group. in contrast, it is likely that more hydrophilic low molecular weight monomers remained in the brush after the solvent evaporation of sb due to its chemical composition. as low molecular weight monomers increase the dc of polymer-based materials19, it can presume that a positive interaction between the low molecular weight monomers of sb, the low molecular weight monomers and the pigments of the a2 shade hd occurred, which increased the dc of hd only for the a2 shade. on the other hand, the absence of tegdma in ed may have impaired a chemically positive interaction between the residual hydrophilic monomers that remained from sb on the brush with those of the composite, leading to decreased dc for ed. however, further chemical analysis should be performed to confirm these assumptions. the fact that the bleach shade of xt presented a higher dc when compared to a2 for all the tested lubricants can probably be attributed to the difference in the nature and color of the pigments. lighter pigments may be present in the bleach shade of xt than in the bleach shade of ed and hd, favoring greater absorption of light by photoinitiators and higher dc for xt22. decreased dc is directly related to an intrinsic color change of composite resins due to the release of some coloring substances that can induce material discoloration23. however, in this study, composites lubricated with mp showed decreased dc, while only hd in the a2 shade presented a greater intrinsic color change. in fact, because dc was measured on the surface of the samples, it is likely that the color change of the entire sample was not only dependent on the color alteration caused on the surface of the samples. 9 melo et al. tooth restoration color is directly influenced by the translucency and opacity of the composites1, which should be reproduced to better mimic the optical properties of dental tissues13. composite translucency is most influenced by the organic matrix24 and the filler load content25. while the former is related to the absorption and reflection of light, the latter is responsible for the dispersion effect that may occur within the restoration structure26. therefore, the way that light is absorbed, reflected, and scattered influences the composite translucency27. the increased translucency after 24 hours observed for xt when lubricated by mp and sb is probably due to the absence of oxidation products on the composite due to a greater synergetic interaction between the monomers from the same manufacturer. on the other hand, it is likely the interaction between the monomers from mp and sb with those from ed and hd might have generated oxidation products, which can increase the opacity of the composites27. however, further chemical analysis should be performed to confirm this assumption. studies have shown that storage in water or solutions with pigments can alter the translucency of composites, mainly due to the degradation of the resinous monomers and leaching of the charged particles28. because translucency involves the amount of light transmitted through a material or body11, a less translucent material has fewer color pigments which can obstruct light penetration29. thus, it is reasonable to assume that both lubricants might have promoted an increase in monomer leaching to ed and hd after 30 days of water storage, thereby increasing the translucency. some studies have shown that the use of adhesives between composite layers during the incremental technique does not negatively interfere with physical properties such as flexural and cohesive resistance7,8 or even that they can increase the physical stability of some composite resins30. however, the results of the present study did find a positive influence of the adhesive system used as brush lubricants on the surface and aesthetic properties of composite resins, since the application of a bonding agent as a brush lubricant induced reduced polymerization on the surface and color alterations. in fact, adhesive systems can not randomly be used as a brush lubricant for just any composite resin, so clinicians should be aware of this point. further studies should be performed to analyze the influence of conventional brush lubricants on the surface properties of conventional and bleach shade composite resins. the unavailability of details about the chemical composition of the products by the manufacturers was a limitation in this study, because knowing what kind of photoinitiators and pigments are present in the composites would help to understand the behavior of the composite resins and its interaction with the adhesive systems used in the performed analyses. in conclusion, the two tested adhesive systems used as brush lubricants on composite surfaces in a bleach shade promoted changes to the degree of conversion, intrinsic color stability, 24-h translucency and 30-day translucency of all composites tested. disclosure the authors do not have any financial interest in the companies whose materials are included in this article. 10 melo et al. references 1. irawan ba, irawan sn, masudi sm, sukminingrum n, alam mk. 3d surface profile and color stability of tooth colored filling materials after bleaching. biomed res int. 2015;2015:327289. doi: 10.1155/2015/327289. 2. krämer n, reinelt c, richter g, petschelt a, frankenberger r. 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fractureinstrument fractureinstrument fracture rhonan ferreira da silva1; sávio domingos da rocha pereira2; eduardo daruge júnior3; luiz francesquini júnior3; cláudia daniela moreira portilho4; carlos estrela5 ldds, msc, postgraduate student in morphology anatomy division, piracicaba dental school, university of campinas, brazil 2dds, msc, professor of forensic dentistry, espírito-santense integrated schools, brazil 3dds, msc, phd, professor of forensic dentistry, piracicaba dental school, university of campinas, brazil 4dds, endodontist, dental school, federal university of goiás, brazil 5dds, msc, phd, professor of endodontics, dental school, federal university of goiás, brazil received for publication: march 23, 2008 accepted: june 30, 2008 a b s t r a c t aim: the aim of this study was to address several ethical aspects concerning the behavior of endodontists and general dentists regarding endodontic instrument fracture during root canal treatment. methods: the responses of a group of professionals (endodontists and general dentists) to a questionnaire were reviewed and analyzed statistically by fisher’s exact and chi-square tests at 5% significance level. results: forty-six percent of the interviewees responded that they would try to solve the problem without informing the patient about the accident. only 28.1% of the participants affirmed that they would let the patient know right at the moment of occurrence. conclusions: the outcomes of this survey demonstrate that most professionals are afraid of informing their patient about an accidental endodontic instrument breakage during treatment and might be subject to lawsuits. key words: ethics, accidents, dental instruments, endodontics. i n t r o d u c t i o n endodontics is a branch of dentistry that is in continuing development. recent findings in this field have allowed higher predictable success rates. nevertheless, dentistry as well as medicine is not an exact science, and treatment success is directly related to biological factors1. endodontic treatment success relies on the combination of several factors that include an accurate diagnosis, treatment method, technical difficulties, available approach and operator’s skills and knowledge. in some cases, procedural failures or accidents during root canal treatment are not the dentist’s fault, but this is usually not well accepted by patients, mainly because accidents frequently lead to an unfavorable prognosis. at this point, relationship problems rise between the patient and the dentist, and may evolve to lawsuits. correspondence to: rhonan ferreira da silva. departamento de odontologia legal, faculdade de odontologia de piracicaba, unicamp av. limeira, 901, bairro areião, 13414-903, piracicaba, sp, brasil fone: +55-19-2106-5282/83 fax: +55-19-2106-5218 e-mail: rhonanfs@terra.com.br as stated by cohen and burns2, even the most careful and skilled dentist can fracture an endodontic instrument during root canal preparation eventually. in case it happens, the patient must be warned at the moment of accident and dully informed on the real situation and case prognosis. explanations must be given in full, but in a proper manner not to alarm the patient and cause misunderstandings. dentists must work in compliance with the legal principals of dentistry and having an ethical behavior at all times in order to build a sound relationship with the people that seek their professional services. when an accident occurs during treatment, dental ethics must guide case management in all instances. frank3 called the attention to the fact that endodontists must be prepared for different patient reactions after being informed about endodontic accidents, like instrument fracture in the root canal. those reactions are related to fear, worry, anger and retaliation. although some reactions may seem irrational in a first moment, the dental staff must be prepared to assist the patient. the patient must be informed if periodical recalls are need for case follow up. the dentist must be honest with the patient when instrument breakage occurs and be aware that the fragment of a broken file in a root canal does not necessarily implicate in treatment failure4. prevention is the best way to reduce 1536 accident rate during endodontic treatment, but whenever the fracture of an endodontic instrument is perceived, is the dentist’s responsibility to inform the patient about the occurrence, possible consequences, treatment sequence and prognosis, and to provide a full documentation of the case (e.g.: dental records, radiographs)5-6. in a previous survey7 that assessed basic questions regarding intracanal breakage of instruments, among other issues, a questionnaire was sent to 300 endodontists listed in the membership board of the american association of endodontists. only 85 replies were received. from this total, 95.3% of the respondents (n=81) would inform the patient if intracanal breakage occurred, and 78.8% (n=67) would also inform in those cases where fragment removal was not possible. ree et al.8 discussed the factors influencing referral for specialist endodontic treatment among dutch general dentists. the authors handed out 593 questionnaires and had a response rate of 41%. of the respondents, 93% felt the need to refer cases to specialists. the majority of dentists preferred to refer to an endodontist rather than an oral surgeon. root canal obstruction (obliteration, calcification and fractured instruments) was the major factor for referring patients; 37% considered that important and 54% that very important situations caused referral to specialists. given that some problems in endodontics are not predictable, it is important to address ethical aspects of dentists’ behavior in cases of endodontic instrument fracture, since it is well known that endodontic treatment success relies on the combination of several factors, including those related to the patient, the tooth, the root canal system, the instruments and materials, as well as the operator9. this study addressed several ethical aspects concerning the behavior of endodontists and general dentists regarding endodontic instrument fracture during root canal treatment. material and methods the research protocol was reviewed and approved by the research ethics committee of the dental school of piracicaba, state university of campinas, brazil. a questionnaire with structured open questions was used. the questions argued about ethical aspects of dentists’ conduct in endodontics. some questions allowed more than one answer. an informed consent form was filled out warranting the confidentiality of the collected data and their use for research purpose only. three hundred questionnaires were submitted to dentists in the cities of goiânia and aparecida de goiânia, go, brazil. the collected data were reviewed and analyzed statistically by fisher’s exact and chi-square tests. the null hypothesis was that there was no association between the variables assessed in the questionnaire. significance level was set at 5% and statistical analysis system (sas) statistical software was used. r e s u l t s from the total of 300 questionnaires, a response rate of 66.7% (n=200) was obtained. based on data collected from the replied questionnaires, it was observed that 76% (152) of the respondents were general dentists (no specialization), 16% (32) were endodontists and 8% (16) were specialists in other areas. when asked if they had already fractured any type of endodontic instrument during root canal preparation 56% (112) of the participants answered affirmatively. when a correlation was made between professional qualification and fracture occurrence, 87.5% (28) of endodontists and 47.7% (72) of general dentists answered that they had already experienced instrument breakage during endodontic procedures (p<0.0001). the other 12 participants that mentioned experience with instrument breakage were of other specialties and did not represent statistical significance. when questioned about their first conduct in case of instrument fracture, 53% (106) stated that they would inform the patient about the accident, 46% (92) would try to solve the problem without telling the patient, and 1% (2) did not answer this question. the participants were also asked whether they would inform the patient if an endodontic instrument had broken during treatment with no possibility of fragment removal. concerning this question, 54% (108) answered that they would inform the patient about the accident and would schedule another appointment to try again, 29% (58) would tell the patient about the accident and would refer them to an endodontist, and 13% (26) would inform the patient and continue the endodontic treatment (figure 1). a statistically significant association (p=0.0028) was observed when both possibilities of professional conduct (either inform or not the patients about the accident) were evaluated considering the interviewees’ professional qualifications. the null hypothesis was thus rejected. the percentage of general dentists who would inform the patient n = 200 13% 29% 2% 54% 2% inform the patient and finish it in another appointment inform the patient and continue the treatment inform the patient and refer to another professional do not inform the patient and finish the treatment other conducts fig. 1 professional conduct in case of endodontic instrument fracture braz j oral sci. 7(25):1535-1538 ethical aspects concerning endodontic instrument fracture 1537 general dentist 92 (60.5) 59 (38.8) 1 (0.7) endodontist 9 (28.1) 22 (68.8) 1 (3.1) other specialization 6 (37.5) 10 (62.5) 0 (0) inform patient solve the problem without informing no response table 1 frequency of professional qualification [n(%)] related to the conduct after endodontic instrument fracture. p=0.0028 at the moment of accident (60.5%) was significantly higher than that of endodontists (28.1%) and other specialists (37.5%). accordingly, most endodontists and other specialists would attempt to solve the problem without informing the patient (table 1). data crossing regarding to the type of conduct towards the patient concerning the impossibility of removing the fragment with professional qualification demonstrated that there was a statistically significant association between these variables, but only when considering general dentists’ responses (p=0.012). in this group of professionals, only 4% (6) reported that they would finish the treatment without informing the patient about the instrument fractured. the great majority of general dentists (96%; n=146) would inform the patient, but they would take other conduct after that (figure 2). d i s c u s s i o n endodontic failure can lead to problems in the professional-patient relationship, and instrument fracture is considered one of the most unpleasant accidents during endodontic therapy. in the present study this type of accident was reported by 56% of participants. among the endodontists, 87.5% affirmed to have already fractured some type of endodontic instrument, which shows that even a specialist, who is presumably more skilled and technically prepared than a general dentist might experience this type of accident. a fractured instrument within root canal is a critical situation that requires a positive and calm attitude in order to establish the most appropriate plan to achieve the best result. in addition, the patient must be promptly informed as soon as the accident is confirmed and should be given full information about case sequence and prognosis1,4-5,9. patients are not always ready to receive this type of news, but the professional must be prepared to assist them3. informing the patient was the conduct chosen by 53.5% of the participants. nevertheless, a relevant part of the surveyed professionals is afraid of properly communication accidents to their patients, which may lead to an inappropriate conduction of the case. in addition, this result indicates that 46.5% of participants would not have an ethical behavior, keeping their patients unaware of the instrument breakage. an even higher percentage (95.3%) was reported in a previous study. analyzing the decision to inform the patient about the accident versus the qualification, it was observed that from the total number of specialist, only 28.1% stated that they would inform the patient about the fracture. this may be justified by the fact that the specialists would feel more capable of resolving the problem themselves because they believe to have more knowledge and are used to dealing with cases like those. even so, the recommended ethical conduct is to inform the patient about instrument breakage or any other type of accident occurred during endodontic therapy2-5,10. among general dentists it was observed a more ethical conduct compared to specialists, since 60.5% of them responded that they would inform the patient right after confirmation of instrument fracture. it was also asked which would be their conduct if the fragment could not be removed. results showed that almost all general dentists (96%) would inform the patient about the situation. in the study by itoh et al.7, the percentage of professionals that would have the same behavior was considerably smaller (78.8%). the association between the impossibility of removing the fragment in the same session and the professional qualification showed that, among all general dentists, 48.6% would inform the patient and would finish the treatment in a subsequent session. these findings demonstrate that after an unsuccessful attempt to remove the fragment in the same session, the conduct of trying to solve the problem in another moment can be a viable alternative that avoids session prolongation and minimizes physical and emotional distress both to the patient and to the dentist. it was verified that 61.2% of general dentists would try to resolve the problem according to their technical capacity n = 152 finish without informing the patient 4% inform and refer to an endodontist 34.8% inform and finish in the same session 12.5% inform and finish in another session 48.7% fig. 2 conduct of general dentists in case of impossibility of removing the instrument fragment from the root canal braz j oral sci. 7(25):1535-1538 ethical aspects concerning endodontic instrument fracture 1538 in the same appointment or in another one. however, almost one third of those professionals (34.8%) would refer the patient to a specialist. this shows the limitation of their technical capacity and also indicates their concern to provide the best care possible to the patient, since the case would be handled by a specialist. according to this study, instrument fracture was considered an important aspect to refer a case to a specialist by 34.8% of dentists, which is in accordance with the findings of a previous study8. endodontic instrument fracture may bring forth problems to patients and dentists, in different ways. immediate notification of such an occurrence to the patient is a desired and proper conduct to be followed by dentists. the best way to prevent lawsuits in dentistry is having an ethical and clear attitude towards the patient mainly in situations involving accidents related to dental treatment. it is also necessary to keep accurate and updated dental records (e.g.: radiographs, contracts, prescriptions, casts). based on the findings of the present investigation, it may be concluded that 53.5% of the participants reported that they would have a proper ethical conduct in case of intracanal instrument breakage. when fragment removal was not possible, 96% of general dentists would inform the patient about the situation. this article is part of mastership thesis in forensic dentistry fop/unicamp – piracicaba (sp) – brazil. r e f e r e n c e s 1. leite vg. odontologia legal. bahia: era nova; 1962. 2. coehn s, burns rc. caminhos da polpa. 2.ed. rio de janeiro: guanabara koogan; 1980. 3. frank al. the dilemma of the fractured instrument. j endod. 1983; 9: 515-6. 4. cohen s. endodontic treatment: avoid these malpractice traps. dent manag. 1988; 28: 36-8,40. 5. imura n, zuolo ml. procedimentos clínicos em endodontia. são paulo: pancast; 1988. 6. walton re, torabinejad m. princípios e prática em endodontia. 2.ed. são paulo: santos; 1997. 7. itoh a, higuchi n, minami g, yasue t, yoshida t, maseki t. a survey of filling methods, intracanal medications, and instruments breakage. j endod. 1999; 25: 823-4. 8. ree mh, timmerman mf, wesselink pr. factors influencing referral for specialist endodontic treatment amongst a group of dutch general practitioners. int endod j. 2003; 36: 129-34. 9. de deus qd. endodontia. 5.ed. rio de janeiro: medsi; 1992. 10. leonardo mr, leal jm. endodontia: tratamento dos canais radiculares. 3.ed. são paulo: panamericana; 1998. braz j oral sci. 7(25):1535-1538 ethical aspects concerning endodontic instrument fracture revista fop n 13 1682 an overview of caries risk assessment in 0-18 year-olds over the last ten years (1997-2007) elaine pereira da silva tagliaferro1;vanessa pardi1,2; gláucia maria bovi ambrosano3; marcelo de castro meneghim4; antonio carlos pereira5 1dds, ms, phd, postdoctoral student 2dds, ms, phd, professor, graduate program in community health, dental school, university of sagrado coração, brazil 3agr.eng., ms, phd professor, department of community dentistry, dental school of piracicaba, state university of campinas, brazil 4 dds, ms, phd, professor, department of community dentistry 5 dds, mph, drph, professor, department of community dentistry department of community dentistry, piracicaba dental school, university of campinas, brazil received for publication: september 05, 2008 accepted: october 15, 2008 correspondence to: prof. dr. antonio carlos pereira av. limeira 901 13414-903, piracicaba, sp, brasil phone: +55-19-2106-5209. fax: +55-19-2106-5218 e-mail: apereira@fop.unicamp.br a b s t r a c t this study aimed to review the dental literature about caries risk assessment over the last 10 years in order to show which variables have been considered risk predictors and risk factors of dental caries in infants, preschool-, schoolchildren and adolescents. a medline search of the published english language literature from 1997 to 2007 was made for papers of longitudinal studies that reported on caries risk assessment. a total of 39 papers were included in this review. most studies were conducted in schoolchildren (n=19), followed by preschool children (n=9), adolescents (n=7) and infants (n=4). variables such as caries experience, gingival status, microbiological counts, oral hygiene, plaque mineral concentration, fluoride history, socioeconomic and educational level, demographic, anthropometrical, oral, dietary and toothbrushing habits were studied. past caries experience has been the predominant predictor for future caries in 0-18year-old subjects. other variables, such as dietary habits, including sugar intake, and toothbrushing habits may also help identifying high-risk individuals. in conclusion, the variables related to caries experience continue to be the main predictor of caries increment. key words: dental caries; risk; assessment; prediction i n t r o d u c t i o n one of the main goals of dentistry has been to prevent dental caries, which has been, according to aoba and fejerskov1, the predominant cause of tooth loss in all populations worldwide. in general, two preventive strategies can be used to prevent and/or control caries disease: the “high-risk strategy”, which is directed towards individuals particularly susceptible to developing dental caries2, and the “population strategy”, which endeavors to protect all the people, including high and low caries risk individuals. burt3 has referred to the “geographically targeted strategy”, in which the preventive measures are targeted to a subgroup or a specific area of the city/country, such as schools in deprived areas of the city or an area of immigrant population. however, all strategies have the same goal: to prevent and/or to control the development of new carious lesions or to arrest the progression of preexisting lesions. in spite of increase in the adoption of preventive measures, it seems that for a minority of children these procedures have been insufficient for preventing and controlling the disease, as the majority of carious lesions are concentrating in this group. therefore, oral health care providers have been adopted the “high risk strategy” for these individuals. if individuals with a risk for developing dental caries are correctly identified, planning specific measures for caries control and prevention may become a biological and socioeconomic measure, increasing the efficiency of preventive programs, as emphasized by giannoni et al.4. moreover, identifying caries risk factors/predictors allows selecting the individuals or population groups that will really benefit from preventive measures. this makes it possible to use specific and appropriate preventive measures in target people and may work as an alert for conducting a more detailed dental examination. in addition to the aforementioned advantages, knowing caries risk factors is decisive in detecting those with initial carious lesions, who may benefit from novel and emergent preventive braz j oral sci. october/december 2008 vol. 7 number 27 1683 technologies5. fontana and zero6 discussing caries risk assessment in private practice have recommended that factors such as caries experience, dietary habits, fluoride exposure, presence of cariogenic bacteria, salivary status, general medical history and sociodemographic characteristics should be evaluated when assessing the patient’s caries risk. assessing caries risk consists of determining which individuals are more or less likely to prevent or to control dental caries in the future by means of knowing the variables associated with the disease5. caries risk assessment studies can be performed using cross-sectional data, in which the subjects’ data, usually about a disease prevalence or severity, are collected once or longitudinal data, in which the individuals are examined repeatedly over time. in addition to clinical variables, several others such as, socioeconomics, demographics, and behavioral characteristics can be used to assess their effects on caries levels (cross-sectional studies) or in caries incidence and/or increment (longitudinal studies). although longitudinal studies are expensive, difficult to conduct and depend on the participants´ willingness, their results are stronger than those obtained in cross-sectional studies7. moreover, when assessing caries risk, the use of multiple regression analysis is preferable8-9 since the etiology of dental caries is multifactorial. the aim of this work was to review the dental literature about caries risk assessment over the last 10 years (19972007), in order to show which variables have been considered caries risk factors/predictors in longitudinal studies conducted in infants, preschool-, schoolchildren and adolescents. concepts and terminology as one of the study’s aims is to review the dental literature about risk factors and risk predictors in longitudinal studies of dental caries, it is important to consider the following terms. risk factor has been defined by beck8 as “an environmental, behavioral or biologic factor confirmed by temporal sequence, usually in longitudinal studies, which if present directly increases the probability of a disease occurring, and if absent or removed reduces the probability. risk factors are part of the causal chain, or expose the host to the causal chain. once disease occurs, removal of a risk factor may not result in a cure”. risk predictor is also named by beck8 as a risk marker, and is defined as a characteristic associated with a high risk for the disease. the risk predictor predicts well but it is not thought to be part of the causal chain. as a good example, past caries experience has been strongly associated with a high risk for caries increment in the future. however, this variable is not part of the causal chain and is therefore considered a risk predictor. moreover, it has been reported that in case of preventive measures being introduced in the studied caries risk group to reduce the disease activity, past caries experience becomes a risk predictor with reduced worth. in this study, the expressions “risk factor” (rf) and “risk predictor” (rp) will be used for classifying those variables statistically associated with caries increment in multivariate regression analyses, if they were either part (rf) or not (rp) of the causal chain of dental caries. material and methods a search of the english language literature published from 1997 to 2007 was made in the medline database for articles that reported on caries risk assessment using the following descriptors: longitudinal caries risk. furthermore, mesh database pubmed service was used with some of the cited terms: “longitudinal studies”[mesh] and “dental caries”[mesh] and “risk”[mesh]. the limits for the search included: “publication date from 1997 to 2007”; language: english; ages: “all child: 0-18 years”. studies were selected if they met the following criteria: longitudinal study conducted with children aged 0-18 years, providing information on sample size, age at initial examination (baseline), variables collected at baseline, study lasting, statistical tests used and a measure of caries increment/ incidence as an outcome variable. in accordance with the search criteria, 189 papers were retrieved. a total of 59 were selected by reading the title and among them 30 were selected by reading the abstract and/or the full text. twenty-nine papers were excluded because they did not report the outcome variable as caries incidence/increment (n=9), were review articles (n=2), validation study (n=1) evaluation/comparative studies (n=11) or used crosssectional data (n=6). moreover, hand searching was performed from the citations of the identified reports (n=3) and other key papers (n= 6). therefore, a total of 39 papers were included in this review. a variable that is part or expose the host to the causal chain of dental caries and remained statistically significant in multivariate regression models was considered risk factor. risk predictor was considered the variable that is not part of the causal chain of dental caries and remained statistically significant in multivariate regression models. results and discussion researches on caries risk assessment have been conducted since the 1980’s, focused on developing an easy tool for identifying high-caries-risk individuals10. published studies in general have studied clinical, microbiological, salivary, socioeconomic and demographic data, medical history, dietary habits, fluoride history, use of dental services and dental health behaviors, separately and in combination to identify high-caries-risk individuals. according to the dental literature, the use of caries increment during a period of time is the primary outcome measure11 and statistical analysis based on logistic regression with multiple factors are preferable because of the complex and multifactorial etiology of the caries process8. braz j oral sci. 7(27):1682-1690 an overview of caries risk assessment in 0-18 year-olds over the last ten years (1997-2007) r f : la c k o f d a il y to o th b ru s h in g , c o n s u m p ti o n o f c a n d ie s t im in g o f c a ri e s o n s e t o n p ri m a ry a n d p e rm a n e n t m o la rs s a 7 v b : c a ri e s (i n it ia l: re s tr ic te d to th e e n a m e l + m a n if e s t c a ri e s : i n d e n ti n n e e d in g re s to ra ti v e d e n ta l c a re ), q u e s ti o n n a ir e (c o n s u m p ti o n o f c a n d ie s , u s e o f fl u o ri d e ta b le ts , to o th b ru s h in g , p a c if ie r s u c k in g , u s e o f a n u rs in g b o tt le a t n ig h t, p ro lo n g e d b re a s tf e e d in g ) e x : l o c a l h e a lt h c e n te r d e n ti s ts (n o rm a l ro u ti n e s ) 2 1 8 3 o ll il a a n d l a rm a s 1 3 , 2 0 0 7 (o u lu ), f in la n d r p : in c ip ie n t c a ri e s le s io n s ; r f : m s s tr ip , u s e o f c a n d ie s n o n e o f th e s tu d ie d v a ri a b le re a c h e d a n a c c u ra c y o f 8 0 % in c re m e n t o f c a v it a te d c a ri o u s le s io n s a n d o r fi ll in g s > 0 m l r , r o c 3 v b : c a ri e s – d m fs (i n c ip ie n t c a ri e s le s io n s in e n a m e l + d e n ti n a l le s io n s ), s e a la n ts (s u rf a c e le v e l) , p re s e n c e o f v is ib le p la q u e (v p ), g in g iv a l b le e d in g (g b ) o n b u c c a l to o th s u rf a c e s , m u ta n s s tr e p to c o c c i (m s ) fr o m p ro x im a l s u rf a c e o f a c e n tr a l m a x il la ry in c is o r a n d o f th e m o s t d is ta l m a n d ib u la r ri g h t m o la r, q u e s ti o n n a ir e (c o n s u m p ti o n o f fl u o ri d e s a n d c a n d ie s ) e x : d e n ta l u n it w it h g o o d li g h t a n d c o m p re s s e d a ir , m ir ro r w it h 1 .6 -f o ld m a g n if ic a ti o n , b lu n t p e ri o d o n ta l p ro b e a n d fi b e ro p ti c tr a n s il lu m in a ti o n 2 2 2 6 • p ie n ih ä k k in e n e t a l. 1 2 , 2 0 0 4 (s a a ri jä rv i) , f in la n d m e a n n e w le s io n s : 1 .7 (9 % d e v e lo p e d � 3 m a n if e s t c a ri e s ) c a ri e s in c id e n c e a s s o c ia te d w it h h ig h m s le v e ls a n d p o s it iv e ly c o rr e la te d w it h w ig s y n th e s is c a ri e s in c id e n c e (h ig h ri s k : � 3 m a n if e s t c a ri e s ) m w , c s , c a 1 v b : c a ri e s (i n it ia l + m a n if e s t le s io n s ), p la q u e o n th e la b ia l s u rf a c e s o f u p p e r in c is o rs , m u ta n s s tr e p to c o c c i v ir u le n c e fa c to rs (m s a c id o g e n e s is , w a te rin s o lu b le g lu c a n s y n th e s is w ig , a d h e re n c e a n a ly s is ) e x : d e n ta l s u rf a c e s b ru s h e d a n d d ri e d w it h g a u z e 1 2 .5 1 4 2 /1 0 1 m a tt o s -g ra n e r e t a l. 1 9 , 2 0 0 0 (p ir a c ic a b a ), b ra z il d e v e lo p in g c a ri e s b e fo re th e a g e o f 2 in d ic a te d th a t a c h il d is a t ri s k fo r d e n ta l c a ri e s d if fe re n c e s in d ft s c o re s c s , s t 1 .5 v b : c a ri e s (c a v it a ti o n ): d e c a y e d o r fi ll e d p ri m a ry te e th (d ft ) e x : m ir ro r, e x p lo re r, o p ti m a l li g h t a ft e r d ry in g (c o tt o n /a ir ) 1 .5 3 7 4 y o n e z u a n d m a c h id a 1 8 , 1 9 9 8 (t o k y o ), j a p a n m a in re s u lt s r p /r f § o u tc o m e v a ri a b le d a ta a n a ly s is ¶ t im e ‡ v a ri a b le s c o ll e c te d a t b a s e li n e (v b ) e x a m in a ti o n (e x ) a g e † s a m p l e * a u th o r, y e a r a n d lo c a l r f : la c k o f d a il y to o th b ru s h in g , c o n s u m p ti o n o f c a n d ie s t im in g o f c a ri e s o n s e t o n p ri m a ry a n d p e rm a n e n t m o la rs s a 7 v b : c a ri e s (i n it ia l: re s tr ic te d to th e e n a m e l + m a n if e s t c a ri e s : i n d e n ti n n e e d in g re s to ra ti v e d e n ta l c a re ), q u e s ti o n n a ir e (c o n s u m p ti o n o f c a n d ie s , u s e o f fl u o ri d e ta b le ts , to o th b ru s h in g , p a c if ie r s u c k in g , u s e o f a n u rs in g b o tt le a t n ig h t, p ro lo n g e d b re a s tf e e d in g ) e x : l o c a l h e a lt h c e n te r d e n ti s ts (n o rm a l ro u ti n e s ) 2 1 8 3 o ll il a a n d l a rm a s 1 3 , 2 0 0 7 (o u lu ), f in la n d r p : in c ip ie n t c a ri e s le s io n s ; r f : m s s tr ip , u s e o f c a n d ie s n o n e o f th e s tu d ie d v a ri a b le re a c h e d a n a c c u ra c y o f 8 0 % in c re m e n t o f c a v it a te d c a ri o u s le s io n s a n d o r fi ll in g s > 0 m l r , r o c 3 v b : c a ri e s – d m fs (i n c ip ie n t c a ri e s le s io n s in e n a m e l + d e n ti n a l le s io n s ), s e a la n ts (s u rf a c e le v e l) , p re s e n c e o f v is ib le p la q u e (v p ), g in g iv a l b le e d in g (g b ) o n b u c c a l to o th s u rf a c e s , m u ta n s s tr e p to c o c c i (m s ) fr o m p ro x im a l s u rf a c e o f a c e n tr a l m a x il la ry in c is o r a n d o f th e m o s t d is ta l m a n d ib u la r ri g h t m o la r, q u e s ti o n n a ir e (c o n s u m p ti o n o f fl u o ri d e s a n d c a n d ie s ) e x : d e n ta l u n it w it h g o o d li g h t a n d c o m p re s s e d a ir , m ir ro r w it h 1 .6 -f o ld m a g n if ic a ti o n , b lu n t p e ri o d o n ta l p ro b e a n d fi b e ro p ti c tr a n s il lu m in a ti o n 2 2 2 6 • p ie n ih ä k k in e n e t a l. 1 2 , 2 0 0 4 (s a a ri jä rv i) , f in la n d m e a n n e w le s io n s : 1 .7 (9 % d e v e lo p e d � 3 m a n if e s t c a ri e s ) c a ri e s in c id e n c e a s s o c ia te d w it h h ig h m s le v e ls a n d p o s it iv e ly c o rr e la te d w it h w ig s y n th e s is c a ri e s in c id e n c e (h ig h ri s k : � 3 m a n if e s t c a ri e s ) m w , c s , c a 1 v b : c a ri e s (i n it ia l + m a n if e s t le s io n s ), p la q u e o n th e la b ia l s u rf a c e s o f u p p e r in c is o rs , m u ta n s s tr e p to c o c c i v ir u le n c e fa c to rs (m s a c id o g e n e s is , w a te rin s o lu b le g lu c a n s y n th e s is w ig , a d h e re n c e a n a ly s is ) e x : d e n ta l s u rf a c e s b ru s h e d a n d d ri e d w it h g a u z e 1 2 .5 1 4 2 /1 0 1 m a tt o s -g ra n e r e t a l. 1 9 , 2 0 0 0 (p ir a c ic a b a ), b ra z il d e v e lo p in g c a ri e s b e fo re th e a g e o f 2 in d ic a te d th a t a c h il d is a t ri s k fo r d e n ta l c a ri e s d if fe re n c e s in d ft s c o re s c s , s t 1 .5 v b : c a ri e s (c a v it a ti o n ): d e c a y e d o r fi ll e d p ri m a ry te e th (d ft ) e x : m ir ro r, e x p lo re r, o p ti m a l li g h t a ft e r d ry in g (c o tt o n /a ir ) 1 .5 3 7 4 y o n e z u a n d m a c h id a 1 8 , 1 9 9 8 (t o k y o ), j a p a n m a in re s u lt s r p /r f § o u tc o m e v a ri a b le d a ta a n a ly s is ¶ t im e ‡ v a ri a b le s c o ll e c te d a t b a s e li n e (v b ) e x a m in a ti o n (e x ) a g e † s a m p l e * a u th o r, y e a r a n d lo c a l 1684 tables 1 to 4 present a detailed review of the papers published over the last 10 years (1997-2007) about caries risks assessment in infants (<2 years), preschool children (2-5 years), schoolchildren (6-12 years) and adolescents (13-18 years) is presented. the age groups were established based on those of medline. the studies on caries risk assessment during the last 10 years were conducted mainly in schoolchildren (n=19), followed by preschool children (n=9), adolescents (n=7), and infants (n=4). the majority of them were related to data collected in finland (n=7), followed by brazil (n=4), sweden (n=3), china (n=3), norway (n=3), usa (n=3), the netherlands (n=3), belgium (n=2), greece (n=2), japan (n=2), australia (n=1), denmark (n=1), germany (n=1), israel (n=1), italy (n=1), mexico (n=1) and new zealand (n=1). as one can see, european countries have contributed a great deal to the dental literature on caries risk assessment over the last decade. for classifying a variable as a risk factor (rf) or risk predictor (rp), the study had to use multivariate regression analyses including several variables in the regression models. there are few studies targeting infants at baseline examination, and these collected mainly dental variables (table 1). only one study12 used logistic regression models with multiple variables for identifying risk predictors/ factors (rp/rf) for caries development, which are the preferable model for this type of study, as dental caries presents a multifactorial and complex etiology8. in the pienihäkkinen’s et al.12 study the mutans streptococcus counts, the presence of incipient caries lesions, and the use of candies were predictors for caries increment after a 3-year-follow-up. another study13 used survival analysis and identified the consumption of candies and the lack of daily toothbrushing as the variables that impacted on caries onset. as one can see, sugar consumption is an important variable that may identify children at risk of caries in this age group. it is an important finding since according to zero14, the relationship between sugar consumption and dental caries is less strong in comparison to that from the prefluoride era. therefore dietary counseling is highly recommended to mothers and should be part of oral health preventive programs in public health services. considering the small number of studies in this age group during the last 10 years, further studies should be conducted and make use of more appropriate statistical analysis. as regards studies concerning preschool children (table 2), 9 papers published over the last 10 years were selected. variables such as dental, socioeconomic, behavioral, dietary, microbiological, medical and demographic data have been collected in study periods ranging from 0.5 to 10 years. most studies (n=7) used regression models as statistical analyses and showed that the main risk predictor was caries experience and the risk factors were sugar consumption and the presence of plaque/toothbrushing related habits. caries experience detected at baseline has t a b le 1 r ev ie w o f li te ra tu re o n c ar ie s ri sk a ss es sm en t in i n fa n ts ( 0 -2 y ea rs o ld ) o v er t h e la st 1 0 y ea rs ( 1 9 9 7 -2 0 0 7 ) braz j oral sci. 7(27):1682-1690 an overview of caries risk assessment in 0-18 year-olds over the last ten years (1997-2007) 1685 t a b le 2 r ev ie w o f li te ra tu re o n c ar ie s ri sk a ss es sm en t in p re sc h o o l ch il d re n ( 2 -5 y ea rs o ld ) o v er t h e la st 1 0 y ea rs ( 1 9 9 7 -2 0 0 7 ) * s am p le s iz e at i n it ia l/ fi n al e x am in at io n ; † a g e at b as el in e (y ea rs ); ‡ s tu d y l as ti n g ( y ea rs ); ¶ s ta ti st ic al t es ts : a n o v a = a n al y si s o f v ar ia n ce ; c a = c o rr el at io n a n al y si s; c s = c h isq u ar e; k w = k ru sk al l w al li s; m l r = m u lt ip le l o g is ti c re g re ss io n ; m n = m cn em ar ; m w = m an n -w h it n ey ; p v = p re d ic ti v e v al u es ; r o c = r ec ei v er o p er at o r ch ar ac te ri st ic c u rv es ; s ch = s ch ef fe ; s n = s en si ti v it y ; s p = s p ec if ic it y ; s t= s tu d en t’ s tte st ; w = w il co x o n ; § r p /r f : r is k p re d ic to rs /r is k f ac to rs o b ta in ed i n r eg re ss io n a n al y se s braz j oral sci. 7(27):1682-1690 an overview of caries risk assessment in 0-18 year-olds over the last ten years (1997-2007) s ig n if ic a n t c o rr e la ti o n b e tw e e n c a ri e s in th e p ri m a ry d e n ti ti o n a n d in p e rm a n e n t te e th r p : � 1 c a ri o u s le s io n o n p ri m a ry s e c o n d m o la rs h ig h e s t s u m o fs n a n d s p (1 4 8 % ): p re d ic to r “m o re th a n tw o le s io n s in p ri m a ry s e c o n d m o la rs ” t a k in g p a rt o f a ri s k g ro u p (b a s e d o n c a ri e s in c re m e n t in p e rm a n e n t te e th ) c s , s t, c a , m l r , s n , s p , p v , r o c 5 v b : c a ri e s (e n a m e l + d e n ti n c a ri e s ; v is u a l in s p e c ti o n ) e x : t e e th p o li s h e d w it h p ro p h y la c ti c p a s te , a ir -d ri e d /c o tt o n ro ll s , o p e ra ti n g li g h ts , b it e w in g ra d io g ra p h s 5 2 1 7 /1 8 6 s k e ie e t a l. 2 8 , 2 0 0 6 (b e rg e n ), n o rw a y m e a n in c re m e n t (e n a m e l + d e n ti n le s io n s ): 3 .0 5 r p : � 1 le s io n s o n p ro x im a l s u rf a c e s o f th e p ri m a ry m o la rs t a k in g p a rt o f a ri s k g ro u p (c a ri e s in c re m e n t in p ri m a ry te e th ) s t, m l r 5 v b : c a ri e s (e n a m e l + d e n ti n e le s io n s ) e x : t e e th p o li s h e d a n d d ri e d , p ro b e s , p la in , m ir ro rs , fa v o ra b le lig h t c o n d it io n s , b it e w in g ra d io g ra p h s 5 2 1 7 /1 8 6 s k e ie e t a l. 2 7 , 2 0 0 4 (b e rg e n ), n o rw a y m e a n d m fs : 4 .4 3 a t b a s e li n e a n d 6 .7 8 a t fo ll o w -u p . g ro u p s w it h h ig h m u ta n s s tr e p to c o c c i (m s ) a n d /o r h ig h p la q u e m s s c o re s : s ig n if ic a n tl y h ig h e r c a ri e s in c id e n c e r p : c a ri e s e x p e ri e n c e a t b a s e li n e ; r f : h ig h p la q u e m s s c o re � 1 n e w c a ri o u s s u rf a c e s n , s p , p v , c s , m l r 0 .5 v b : c a ri e s (w h o c ri te ri a ), d e n ta l p la q u e (v is u a ll y o n th e la b ia l su rf a c e o f th e m a x il la ry in c is o rs w it h o u t d is c lo s in g s o lu ti o n ), o ra l m ic ro b io lo g y (u n s ti m u la te d s a li v a a n d p la q u e in fo u r p ro x im a l s u rf a c e s : 5 4 -d , 5 5 -m , 7 4 -d , 7 5 -m ) e x : v is u a l in s p e c ti o n 1 .5 5 1 4 7 /1 2 9 s e k i e t a l.2 6 , 2 0 0 3 (t o k y o ), j a p a n c h il d re n w it h e c c h a d th e h ig h e s t n u m b e r o f n e w a ff e c te d s u rf a c e s p e r y e a r, fo ll o w e d b y th e c h il d re n w it h p o s te ri o r c a ri e s a n d th e c a ri e sfr e e c h il d re n . e c c g ro u p s d if fe re d s ta ti s ti c a ll y fr o m c a ri e sfr e e g ro u p a n d p o s te ri o r c a ri e s g ro u p n e w a ff e c te d s u rf a c e s p e r y e a r a n o v a , s c h 7 1 0 v b : c a ri e s : 3 g ro u p s w it h 5 0 c h il d re n e a c h b e in g c a ri e s fr e e c h il dr e n (c f ), c h il d re n w it h e a rl y c h il d h o o d c a ri e s (e c c ), a n d c h il d re n w it h p o s te ri o r c a ri e s o n ly (p c ) e x : f il e s o f tw o p ri v a te p e d ia tr ic d e n ta l c li n ic s , ra d io g ra p h ic e xa m in a ti o n s 3 -5 1 5 0 p e re tz e t a l. 2 5 , 2 0 0 3 , (j e ru s a le m a n d p e ta h t ik v a ), is ra e l r p : c a ri e s in p ri m a ry te e th (c a ri e s o n p ri m a ry m o la rs : h ig h e s t p re d ic ti v e v a lu e 8 5 .4 % ) d e v e lo p in g c a ri e s in p e rm a n e n t d e n ti ti o n m l r , c a , p v 8 v b : c a ri e s (d m f/ d m f w h o c ri te ri a ) o n e n ti re d e n ti ti o n a n d o n s u b s e ts o f te e th : m a x ill a ry in c is o rs , m a x il la ry a n te ri o r te e th , m a x il la ry fi rs t a n d s e c o n d m o la rs , m a n d ib u la r fi rs t a n d s e c o n d m o la rs , a ll p ri m a ry m o la rs e x : c la s s ro o m s e tt in g , n a tu ra l li g h t, m o u th m ir ro rs , e x p lo re rs 3 5 5 0 4 /3 6 2 l i a n d w a n g 2 4 , 2 0 0 2 (b e ij in g ), c h in a r p : p re v io u s c a ri e s e x p e ri e n c e (m o s t im p o rt a n t fa c to r) , fl u o ri d e u s a g e ; r f : n o t a tt e n d in g n u rs e ri e s w it h g u id e li n e s fo r s u g a r in ta k e , fr e q u e n c y a n d a m o u n t o f s u g a r in ta k e , to o th b ru s h in g re la te d h a b it s c a ri e s in c re m e n t m l r 1 v b : c a ri e s (w h o c ri te ri a ), e n a m e l h y p o p la s ia (d d e in d e x – d e v e lo p m e n ta l d e fe c ts o f e n a m e l) , n u tr it io n a l s ta tu s , s u g a r in ta k e d u ri n g 3 n o nc o n s e c u ti v e d a y s , d a il y fr e q u e n c y o f s u g a r in ta k e , 2 4 -h p e ri o d d ie ta ry re c a ll a t h o m e , q u e s ti o n n a ir e (s o c io e c o n o m ic , m e d ic a l, d e m o g ra p h ic , d ie ta ry h is to ry a n d d e n ta l-r e la te d in fo rm a ti o n ) e x : c la s s ro o m , h e a d la m p , m o u th m ir ro r, p ro b e s 3 6 5 0 /5 1 0 r o d ri g u e s a n d s h e ih a m 2 3 , 2 0 0 0 (r e c if e ), b ra z il h ig h c a ri e s ri s k c h il d re n a t b a s e li n e (> 5 0 c o lo n y fo rm in g u n it s ): s ig n if ic a n tl y g re a te r c a ri e s s c o re s a t fi n a l e x a m in a ti o n d if fe re n c e s in c a ri e s s c o re s k w , m w 6 v b : c a ri e s (r a d ik e m e th o d ), m u ta n s s tr e p to c o c c i c o u n ts in s a li v a e x : p o rt a b le d e n ta l c h a ir , m ir ro r, # 2 3 e x p lo re r fo c u s a b le fl a s h li gh ts 3 .8 (m e a n ) 8 5 /8 3 t h ib o d e a u a n d o ’s u ll iv a n 2 2 , 1 9 9 9 (h a rt fo rd ), u s a r p : p a c if ie rs u c k in g � 2 y e a rs ; r f : u s e o f n u rs in g b o tt le a t n ig h t o c c u rr e n c e o f c a ri e s (y e s /n o ) c s , m l r 2 v b : c a ri e s (i n it ia l + m a n if e s t c a ri e s ), s a li v a ry la c to b a c il li a n d c a n d id a , q u e s ti o n n a ir e (p a c if ie ra n d th u m b -s u c k in g , b re a s tf e e d in g , b o tt le -f e e d in g a t n ig h t, s o c ia l c la s s o f th e fa m il y ) e x : a t d e n ta l h e a lt h c e n te rs 2 .5 (m e a n ) 1 6 6 /1 5 2 o ll il a e t a l. 2 1 , 1 9 9 8 (o u lu ), f in la n d r p : m o th e rs ’e d u c a ti o n (u p to n in e y e a rs o f b a s ic e d u c a ti o n ), p re s e n c e o f c a ri e s ; r f : p re s e n c e o f p la q u e c h a n g e s in d m f in d e x c s , m w , k w , w , m l r 2 v b : c a ri e s (e n a m e l le s io n s + d m f in d e x ), d e n ta l c le a n li n e s s (n o d is c lo s in g s o lu ti o n ), q u e s ti o n n a ir e (b a s ic e d u c a ti o n a l le v e l, o c c u p a ti o n a l e d u c a ti o n ,o c c u p a ti o n , to o th b ru s h in g b e h a v io rs , u s e o f x y li to l c h e w in g g u m , s n a c k in g o n s w e e ts a n d fre q u e n c y o f s n a c k in g , d ri n k in g s o m e th in g o th e r th a n p u re w a te r) e x : p u b li c d e n ta l c e n te rs 3 1 2 9 2 /1 0 0 3 m a tt il a e t a l.2 0 , 1 9 9 8 (t u rk u ), f in la n d m a in re s u lt s r p /r f § o u tc o m e v a ri a b le d a ta a n a ly s is ¶ t im e ‡ v a ri a b le s c o ll e c te d a t b a s e li n e (v b ) e x a m in a ti o n (e x ) a g e † s a m p l e * a u th o r, y e a r a n d lo c a l s ig n if ic a n t c o rr e la ti o n b e tw e e n c a ri e s in th e p ri m a ry d e n ti ti o n a n d in p e rm a n e n t te e th r p : � 1 c a ri o u s le s io n o n p ri m a ry s e c o n d m o la rs h ig h e s t s u m o fs n a n d s p (1 4 8 % ): p re d ic to r “m o re th a n tw o le s io n s in p ri m a ry s e c o n d m o la rs ” t a k in g p a rt o f a ri s k g ro u p (b a s e d o n c a ri e s in c re m e n t in p e rm a n e n t te e th ) c s , s t, c a , m l r , s n , s p , p v , r o c 5 v b : c a ri e s (e n a m e l + d e n ti n c a ri e s ; v is u a l in s p e c ti o n ) e x : t e e th p o li s h e d w it h p ro p h y la c ti c p a s te , a ir -d ri e d /c o tt o n ro ll s , o p e ra ti n g li g h ts , b it e w in g ra d io g ra p h s 5 2 1 7 /1 8 6 s k e ie e t a l. 2 8 , 2 0 0 6 (b e rg e n ), n o rw a y m e a n in c re m e n t (e n a m e l + d e n ti n le s io n s ): 3 .0 5 r p : � 1 le s io n s o n p ro x im a l s u rf a c e s o f th e p ri m a ry m o la rs t a k in g p a rt o f a ri s k g ro u p (c a ri e s in c re m e n t in p ri m a ry te e th ) s t, m l r 5 v b : c a ri e s (e n a m e l + d e n ti n e le s io n s ) e x : t e e th p o li s h e d a n d d ri e d , p ro b e s , p la in , m ir ro rs , fa v o ra b le lig h t c o n d it io n s , b it e w in g ra d io g ra p h s 5 2 1 7 /1 8 6 s k e ie e t a l. 2 7 , 2 0 0 4 (b e rg e n ), n o rw a y m e a n d m fs : 4 .4 3 a t b a s e li n e a n d 6 .7 8 a t fo ll o w -u p . g ro u p s w it h h ig h m u ta n s s tr e p to c o c c i (m s ) a n d /o r h ig h p la q u e m s s c o re s : s ig n if ic a n tl y h ig h e r c a ri e s in c id e n c e r p : c a ri e s e x p e ri e n c e a t b a s e li n e ; r f : h ig h p la q u e m s s c o re � 1 n e w c a ri o u s s u rf a c e s n , s p , p v , c s , m l r 0 .5 v b : c a ri e s (w h o c ri te ri a ), d e n ta l p la q u e (v is u a ll y o n th e la b ia l su rf a c e o f th e m a x il la ry in c is o rs w it h o u t d is c lo s in g s o lu ti o n ), o ra l m ic ro b io lo g y (u n s ti m u la te d s a li v a a n d p la q u e in fo u r p ro x im a l s u rf a c e s : 5 4 -d , 5 5 -m , 7 4 -d , 7 5 -m ) e x : v is u a l in s p e c ti o n 1 .5 5 1 4 7 /1 2 9 s e k i e t a l.2 6 , 2 0 0 3 (t o k y o ), j a p a n c h il d re n w it h e c c h a d th e h ig h e s t n u m b e r o f n e w a ff e c te d s u rf a c e s p e r y e a r, fo ll o w e d b y th e c h il d re n w it h p o s te ri o r c a ri e s a n d th e c a ri e sfr e e c h il d re n . e c c g ro u p s d if fe re d s ta ti s ti c a ll y fr o m c a ri e sfr e e g ro u p a n d p o s te ri o r c a ri e s g ro u p n e w a ff e c te d s u rf a c e s p e r y e a r a n o v a , s c h 7 1 0 v b : c a ri e s : 3 g ro u p s w it h 5 0 c h il d re n e a c h b e in g c a ri e s fr e e c h il dr e n (c f ), c h il d re n w it h e a rl y c h il d h o o d c a ri e s (e c c ), a n d c h il d re n w it h p o s te ri o r c a ri e s o n ly (p c ) e x : f il e s o f tw o p ri v a te p e d ia tr ic d e n ta l c li n ic s , ra d io g ra p h ic e xa m in a ti o n s 3 -5 1 5 0 p e re tz e t a l. 2 5 , 2 0 0 3 , (j e ru s a le m a n d p e ta h t ik v a ), is ra e l r p : c a ri e s in p ri m a ry te e th (c a ri e s o n p ri m a ry m o la rs : h ig h e s t p re d ic ti v e v a lu e 8 5 .4 % ) d e v e lo p in g c a ri e s in p e rm a n e n t d e n ti ti o n m l r , c a , p v 8 v b : c a ri e s (d m f/ d m f w h o c ri te ri a ) o n e n ti re d e n ti ti o n a n d o n s u b s e ts o f te e th : m a x ill a ry in c is o rs , m a x il la ry a n te ri o r te e th , m a x il la ry fi rs t a n d s e c o n d m o la rs , m a n d ib u la r fi rs t a n d s e c o n d m o la rs , a ll p ri m a ry m o la rs e x : c la s s ro o m s e tt in g , n a tu ra l li g h t, m o u th m ir ro rs , e x p lo re rs 3 5 5 0 4 /3 6 2 l i a n d w a n g 2 4 , 2 0 0 2 (b e ij in g ), c h in a r p : p re v io u s c a ri e s e x p e ri e n c e (m o s t im p o rt a n t fa c to r) , fl u o ri d e u s a g e ; r f : n o t a tt e n d in g n u rs e ri e s w it h g u id e li n e s fo r s u g a r in ta k e , fr e q u e n c y a n d a m o u n t o f s u g a r in ta k e , to o th b ru s h in g re la te d h a b it s c a ri e s in c re m e n t m l r 1 v b : c a ri e s (w h o c ri te ri a ), e n a m e l h y p o p la s ia (d d e in d e x – d e v e lo p m e n ta l d e fe c ts o f e n a m e l) , n u tr it io n a l s ta tu s , s u g a r in ta k e d u ri n g 3 n o nc o n s e c u ti v e d a y s , d a il y fr e q u e n c y o f s u g a r in ta k e , 2 4 -h p e ri o d d ie ta ry re c a ll a t h o m e , q u e s ti o n n a ir e (s o c io e c o n o m ic , m e d ic a l, d e m o g ra p h ic , d ie ta ry h is to ry a n d d e n ta l-r e la te d in fo rm a ti o n ) e x : c la s s ro o m , h e a d la m p , m o u th m ir ro r, p ro b e s 3 6 5 0 /5 1 0 r o d ri g u e s a n d s h e ih a m 2 3 , 2 0 0 0 (r e c if e ), b ra z il h ig h c a ri e s ri s k c h il d re n a t b a s e li n e (> 5 0 c o lo n y fo rm in g u n it s ): s ig n if ic a n tl y g re a te r c a ri e s s c o re s a t fi n a l e x a m in a ti o n d if fe re n c e s in c a ri e s s c o re s k w , m w 6 v b : c a ri e s (r a d ik e m e th o d ), m u ta n s s tr e p to c o c c i c o u n ts in s a li v a e x : p o rt a b le d e n ta l c h a ir , m ir ro r, # 2 3 e x p lo re r fo c u s a b le fl a s h li gh ts 3 .8 (m e a n ) 8 5 /8 3 t h ib o d e a u a n d o ’s u ll iv a n 2 2 , 1 9 9 9 (h a rt fo rd ), u s a r p : p a c if ie rs u c k in g � 2 y e a rs ; r f : u s e o f n u rs in g b o tt le a t n ig h t o c c u rr e n c e o f c a ri e s (y e s /n o ) c s , m l r 2 v b : c a ri e s (i n it ia l + m a n if e s t c a ri e s ), s a li v a ry la c to b a c il li a n d c a n d id a , q u e s ti o n n a ir e (p a c if ie ra n d th u m b -s u c k in g , b re a s tf e e d in g , b o tt le -f e e d in g a t n ig h t, s o c ia l c la s s o f th e fa m il y ) e x : a t d e n ta l h e a lt h c e n te rs 2 .5 (m e a n ) 1 6 6 /1 5 2 o ll il a e t a l. 2 1 , 1 9 9 8 (o u lu ), f in la n d r p : m o th e rs ’e d u c a ti o n (u p to n in e y e a rs o f b a s ic e d u c a ti o n ), p re s e n c e o f c a ri e s ; r f : p re s e n c e o f p la q u e c h a n g e s in d m f in d e x c s , m w , k w , w , m l r 2 v b : c a ri e s (e n a m e l le s io n s + d m f in d e x ), d e n ta l c le a n li n e s s (n o d is c lo s in g s o lu ti o n ), q u e s ti o n n a ir e (b a s ic e d u c a ti o n a l le v e l, o c c u p a ti o n a l e d u c a ti o n ,o c c u p a ti o n , to o th b ru s h in g b e h a v io rs , u s e o f x y li to l c h e w in g g u m , s n a c k in g o n s w e e ts a n d fre q u e n c y o f s n a c k in g , d ri n k in g s o m e th in g o th e r th a n p u re w a te r) e x : p u b li c d e n ta l c e n te rs 3 1 2 9 2 /1 0 0 3 m a tt il a e t a l.2 0 , 1 9 9 8 (t u rk u ), f in la n d m a in re s u lt s r p /r f § o u tc o m e v a ri a b le d a ta a n a ly s is ¶ t im e ‡ v a ri a b le s c o ll e c te d a t b a s e li n e (v b ) e x a m in a ti o n (e x ) a g e † s a m p l e * a u th o r, y e a r a n d lo c a l m e a n d m f s i n c re m e n t: 1 .1 4 r p : p la q u e c a c o n c e n tr a ti o n , b a s e li n e d m f s s c o re ( u s e fu l p re d ic to r) ; r f : to o th b ru s h in g fr e q u e n c y d m f s i n c re m e n t � 3 m w , w , m l r 2 v b : c a ri e s ( c a v it a ti o n le v e l) , p la q u e s c o re ( q u ig le y a n d h e in i n d e x ), fl u o ro s is s c o re ( t f i n d e x ), i n o rg a n ic c o m p o u n d s i n d e n ta l p la q u e ( s u p ra g in g iv a l p la q u e c o ll e c ti o n f ro m b u c c a l a n d l in g u a l s u rf a c e s a ft e r 3 d a y s w it h n o o ra l h y g ie n e ), to o th b ru s h in g fr e q u e n c y , s n a c k s u s e , p a re n ts ‘ o c c u p a ti o n e x : c h il d s e a te d i n a n u p ri g h t c h a ir , a d e q u a te i ll u m in a ti o n , s h a rp p ro b e 1 2 1 7 5 /1 6 4 p e a rc e e t a l. 3 7 , 2 0 0 2 ( b e ij in g ), c h in a n o s ta ti s ti c a ll y s ig n if ic a n t p re d ic to r (p > 0 .0 5 ) c a ri e s i n c re m e n t c a , m l r 4 v b : c a ri e s ( in c ip ie n t + c a v it a te d le s io n s + f il li n g s ), m u ta n s s tr e p to c o c c i m s a n d l a c to b a c il li ( l b ) c o u n ts (s ti m u la te d s a li v a ) e x : v is u a l o b s e rv a ti o n , d ri e d s u rf a c e s , d e n ta l la m p , s m a ll m o u th -l ig h t, r a d io g ra p h s 7 .5 6 9 /6 2 v a n p a le n s te in h e ld e rm a n e t a l. 3 6 , 2 0 0 1 (c u le m b o rg ), t h e n e th e rl a n d s r p : n u m b e r o f c a v it a te d a n d n o n -c a v it a te d fi s s u re s o f th e n e w ly e ru p te d p e rm a n e n t fi rs t m o la r h ig h c a ri e s i n c re m e n t (� d 3 s u rf a c e s > 0 ; > 2 ; > 4 , > 7 ) c a , m l r , r o c 8 v b : c a ri e s ( n o n c a v it a te d a n d c a v it a te d c a ri e s , fi ll in g ) a n d s e a la n ts e x : v is u a l e x a m in a ti o n , d e n ta l p ro b e , d e n ta l la m p a n d s m a ll m o u th -l ig h t, b it e w in g r a d io g ra p h s 7 .5 3 1 8 /2 8 7 v a n p a le n s te in h e ld e rm a n e t a l. 3 5 , 2 0 0 1 ( t ie l, c u le m b o rg ) t h e n e th e rl a n d s r p : d m fs , e d u c a ti o n a l s y s te m ; r f : fr e q u e n c y o f b ru s h in g , d a il y u s e o f s u g a r -c o n ta in in g d ri n k s d m f s i n c re m e n t � 2 o n p e rm a n e n t 1 s t m o la rs m l r , s n , s p , r o c , c s 3 v b : c a ri e s ( b a s c d c ri te ri a – c a v it a ti o n le v e l) , o ra l h y g ie n e ( s il n e s s a n d l ö e ’s p la q u e i n d e x a n d p la q u e in d e x o n o c c lu s a l s u rf a c e s – c a rv a lh o ’s in d e x ), e ru p ti o n s ta g e , q u e s ti o n n a ir e ( o ra l h y g ie n e , d ie ta ry h a b it s , fl u o ri d e e x p o s u re , a c c e s s t o o ra l h e a lt h c a re s e rv ic e s , m e d ic a l h is to ry , s o c io e c o n o m ic l e v e l) e x : m o b il e d e n ta l c li n ic , w h o /c p it n p ro b e 7 3 3 0 3 /3 0 0 2 v a n o b b e rg e n e t a l. 3 4 , 2 0 0 1 (f la n d e rs ), b e lg iu m m e a n c a ri e s i n c re m e n t: 0 .4 5 d m ft /d m f t r p : m o th e r ’s p re v io u s c a ri e s ( d e c id u o u s t e e th ); r f : to o th b ru s h in g o n ly o c c a s io n a ll y , c h il d f re q u e n t u s e o f s w e e ts a n d c h il d ’s b e d ti m e a ft e r 9 p . m . (p e rm a n e n t te e th ), e a t s w e e ts f re q u e n tl y a t 3 y e a rs o f a g e ( b o th d e n ti ti o n s ) c a ri e s i n c re m e n t c s , m l r 3 v b : c a ri e s ( d m f t / d m ft ), q u e s ti o n n a ir e ( s o c io e c o n o m ic , d e m o g ra p h ic , fa m il y f a c to rs , d i e t, d e n ta l h y g ie n e , p a re n ts ’ o w n e a rl ie r d e n ta l h e a lt h h a b it s , p a re n ts ´ p re v io u s d e n ta l h e a lt h , c h il d re n ’s d is e a s e s a n d p h y s ic a l s y m p to m s ) e x : m ir ro r, p ro b e , fi b e r -o p ti c l ig h t 7 1 0 7 4 m a tt il a e t a l. 3 3 , 2 0 0 1 ( t u rk u ), f in la n d r p : p re s e n c e o f d e c a y e d t e e th a n d d e c a lc if ic a ti o n s ( m o s t s ig n if ic a n t) ; r f : d ie ta ry f a c to rs , p o o r o ra l h y g ie n e c u m u la ti v e f u tu re c a ri e s le s io n s ( h ig h r is k : � 5 n e w s u rf a c e l e s io n s ) c s , m l r , a n o v a 2 .5 v b : c h a rt s o f p a ti e n ts c o n ta in in g : m e d ic a l, d e n ta l a n d f lu o ri d e h i s to ry , d ie t, o ra l h y g ie n e , re te n ti v e p it s a n d f is s u re s , e x is ti n g r e s to ra ti o n s , n e w ly e ru p te d t e e th , c a ri e s /d e c a lc if ic a ti o n s , p u lp it s /a b s c e s s , g in g iv it is , c ro w d in g , b e h a v io r, a g e , g e n d e r e x : v a ri a b le s c o ll e c te d a t p a ti e n t ’s c h a rt s ( re tr o s p e c ti v e l o n g it u d in a l s tu d y ) 0 .9 -1 1 1 4 0 w a n d e ra e t a l. 3 2 , 2 0 0 0 ( m ic h ig a n ), u s a r p : e p in e p h ri n e l e v e ls c a ri e s i n c re m e n t s t, l ir a , m l r 1 v b : c a ri e s ( d m f / d m f) , d e n ta l p la q u e , c a te c h o la m in e c o n te n t in u ri n e s a m p le , b o d y w e ig h t, p a re n ta l a g e , e d u c a ti o n a n d p ro fe s s io n , m e d ic a l h is to ry a n d m e d ic a ti o n s e x : b it e w in g r a d io g ra p h s 6 -8 3 1 4 /2 7 0 v a n d e ra s e t a l. 3 1 , 2 0 0 0 , g re e c e m e a n c a ri e s i n c re m e n t: 0 .6 8 t e e th t h e m o re o ft e n t h e m s t e s t p o s it iv e , th e h ig h e r th e p ro p o rt io n o f c h il d re n w h o d e v e lo p e d c a ri e s l e s io n r p : fl u o ri d e ; r f : m s d m f t in c re m e n t � 1 c s , s n , s p , p v , m l r , c a 2 v b : c a ri e s ( w h o c ri te ri a ), p la q u e i n d e x ( s il n e s s a n d l ö e ), m ic ro b io lo g ic a l a n a ly s is o f m u ta n s s tr e p to c o c c i m s , (n o n -s ti m u la te d s a li v a s a m p le s ), q u e s ti o n n a ir e ( s u c ro s e i n ta k e , fl u o r id e e x p o s u re ) e x : c li n ic a l e x a m in a ti o n , v is u a l in s p e c ti o n , b it e w in g r a d io g ra p h s 6 -7 3 1 4 /3 0 4 (c a ri e s fr e e ) p e tt i a n d h a u s e n 1 5 , 2 0 0 0 (r o m e ), i ta ly m e a n c a ri e s i n c id e n c e : 0 .6 9 d m f s s ig n if ic a n t c o rr e la ti o n b e tw e e n i n it ia l m s s c o re s a n d c a ri e s d e v e lo p m e n t c a ri e s d e v e lo p m e n t c a , k w , m w , s n , s p 2 v b : c a ri e s ( d m f s / d m fs – w h o c ri te ri a , in it ia l le s io n s ), s e a la n ts f o r a ll m o la r fi s s u re s , o c c lu s a l p la q u e s a m p le s f ro m t e e th 1 6 a n d 3 6 ( m u ta n s s tr e p to c o c c i te s ts ), p la q u e i n d e x ( q u ig le y -h e in ) e x : e x p lo re r w it h o u t p re s s u re , li g h t s o u rc e 6 -7 2 3 0 /1 6 9 s p li e th a n d b e rn h a rd t 3 0 , 1 9 9 9 , g e rm a n y m e a n c a ri e s i n c re m e n t: 0 .9 5 d m f s c a ri e s i n c re m e n t: n e g a ti v e c o rr e la ti o n s w it h b a s e li n e la c to fe rr in , to ta l ig g a n d t o ta l a n a e ro b e s ; p o s it iv e c o rr e la ti o n w it h s p e c if ic a n ti -s . m u ta n s , ig g a n ti b o d y l e v e ls , m u ta n s s tr e p to c o c c i, l a c to b a c il li a n d s p e c if ic a n ti -s . m u ta n s ig g . c h il d re n w it h s ig n if ic a n tl y h ig h e r b a s e li n e c o n c e n tr a ti o n s o f h y p o th io c y a n it e , to ta l ig g a n ti b o d ie s a n d t o ta l a n a e ro b e s : n o n e w c a ri e s l e s io n s d m f i n c re m e n t > 0 s t, c a 2 v b : c a ri e s ( d m f / d m f w h o c ri te ri a , w h it e s p o ts l e s io n s ), p e ri o d o n ta l s ta tu s ( b le e d in g a n d c a lc u lu s ), u s a g e o f fl u o ri d a te d d e n ti fr ic e s , s a li v a s a m p le s ( b u ff e r c a p a c it y , h y p o th io c y a n it e a s s a y , to ta l s tr e p to c o c c i a n d m u ta n s s tr e p to c o c c i, la c to fe rr in a n d ly s o z y m e a n a ly s is , a g g lu ti n a ti o n a s s a y , to ta l s a li v a ry p e ro x id a s e a c ti v it y , to ta l a n d s p e c if ic ig a a n d ig g a n ti b o d ie s ) e x : v is u a l -t a c ti le m e th o d + f o t i 1 2 6 9 /6 3 k ir s ti lä e t a l. 2 9 , 1 9 9 8 ( t u rk u ), f in la n d m a in r e s u lt s r p /r f § o u tc o m e v a ri a b le d a ta a n a ly s is ¶ t im e ‡ v a ri a b le s c o ll e c te d a t b a s e li n e ( v b ) e x a m in a ti o n ( e x ) a g e † s a m p le * a u th o r , y e a r a n d lo c a l m e a n d m f s i n c re m e n t: 1 .1 4 r p : p la q u e c a c o n c e n tr a ti o n , b a s e li n e d m f s s c o re ( u s e fu l p re d ic to r) ; r f : to o th b ru s h in g fr e q u e n c y d m f s i n c re m e n t � 3 m w , w , m l r 2 v b : c a ri e s ( c a v it a ti o n le v e l) , p la q u e s c o re ( q u ig le y a n d h e in i n d e x ), fl u o ro s is s c o re ( t f i n d e x ), i n o rg a n ic c o m p o u n d s i n d e n ta l p la q u e ( s u p ra g in g iv a l p la q u e c o ll e c ti o n f ro m b u c c a l a n d l in g u a l s u rf a c e s a ft e r 3 d a y s w it h n o o ra l h y g ie n e ), to o th b ru s h in g fr e q u e n c y , s n a c k s u s e , p a re n ts ‘ o c c u p a ti o n e x : c h il d s e a te d i n a n u p ri g h t c h a ir , a d e q u a te i ll u m in a ti o n , s h a rp p ro b e 1 2 1 7 5 /1 6 4 p e a rc e e t a l. 3 7 , 2 0 0 2 ( b e ij in g ), c h in a n o s ta ti s ti c a ll y s ig n if ic a n t p re d ic to r (p > 0 .0 5 ) c a ri e s i n c re m e n t c a , m l r 4 v b : c a ri e s ( in c ip ie n t + c a v it a te d le s io n s + f il li n g s ), m u ta n s s tr e p to c o c c i m s a n d l a c to b a c il li ( l b ) c o u n ts (s ti m u la te d s a li v a ) e x : v is u a l o b s e rv a ti o n , d ri e d s u rf a c e s , d e n ta l la m p , s m a ll m o u th -l ig h t, r a d io g ra p h s 7 .5 6 9 /6 2 v a n p a le n s te in h e ld e rm a n e t a l. 3 6 , 2 0 0 1 (c u le m b o rg ), t h e n e th e rl a n d s r p : n u m b e r o f c a v it a te d a n d n o n -c a v it a te d fi s s u re s o f th e n e w ly e ru p te d p e rm a n e n t fi rs t m o la r h ig h c a ri e s i n c re m e n t (� d 3 s u rf a c e s > 0 ; > 2 ; > 4 , > 7 ) c a , m l r , r o c 8 v b : c a ri e s ( n o n c a v it a te d a n d c a v it a te d c a ri e s , fi ll in g ) a n d s e a la n ts e x : v is u a l e x a m in a ti o n , d e n ta l p ro b e , d e n ta l la m p a n d s m a ll m o u th -l ig h t, b it e w in g r a d io g ra p h s 7 .5 3 1 8 /2 8 7 v a n p a le n s te in h e ld e rm a n e t a l. 3 5 , 2 0 0 1 ( t ie l, c u le m b o rg ) t h e n e th e rl a n d s r p : d m fs , e d u c a ti o n a l s y s te m ; r f : fr e q u e n c y o f b ru s h in g , d a il y u s e o f s u g a r -c o n ta in in g d ri n k s d m f s i n c re m e n t � 2 o n p e rm a n e n t 1 s t m o la rs m l r , s n , s p , r o c , c s 3 v b : c a ri e s ( b a s c d c ri te ri a – c a v it a ti o n le v e l) , o ra l h y g ie n e ( s il n e s s a n d l ö e ’s p la q u e i n d e x a n d p la q u e in d e x o n o c c lu s a l s u rf a c e s – c a rv a lh o ’s in d e x ), e ru p ti o n s ta g e , q u e s ti o n n a ir e ( o ra l h y g ie n e , d ie ta ry h a b it s , fl u o ri d e e x p o s u re , a c c e s s t o o ra l h e a lt h c a re s e rv ic e s , m e d ic a l h is to ry , s o c io e c o n o m ic l e v e l) e x : m o b il e d e n ta l c li n ic , w h o /c p it n p ro b e 7 3 3 0 3 /3 0 0 2 v a n o b b e rg e n e t a l. 3 4 , 2 0 0 1 (f la n d e rs ), b e lg iu m m e a n c a ri e s i n c re m e n t: 0 .4 5 d m ft /d m f t r p : m o th e r ’s p re v io u s c a ri e s ( d e c id u o u s t e e th ); r f : to o th b ru s h in g o n ly o c c a s io n a ll y , c h il d f re q u e n t u s e o f s w e e ts a n d c h il d ’s b e d ti m e a ft e r 9 p . m . (p e rm a n e n t te e th ), e a t s w e e ts f re q u e n tl y a t 3 y e a rs o f a g e ( b o th d e n ti ti o n s ) c a ri e s i n c re m e n t c s , m l r 3 v b : c a ri e s ( d m f t / d m ft ), q u e s ti o n n a ir e ( s o c io e c o n o m ic , d e m o g ra p h ic , fa m il y f a c to rs , d i e t, d e n ta l h y g ie n e , p a re n ts ’ o w n e a rl ie r d e n ta l h e a lt h h a b it s , p a re n ts ´ p re v io u s d e n ta l h e a lt h , c h il d re n ’s d is e a s e s a n d p h y s ic a l s y m p to m s ) e x : m ir ro r, p ro b e , fi b e r -o p ti c l ig h t 7 1 0 7 4 m a tt il a e t a l. 3 3 , 2 0 0 1 ( t u rk u ), f in la n d r p : p re s e n c e o f d e c a y e d t e e th a n d d e c a lc if ic a ti o n s ( m o s t s ig n if ic a n t) ; r f : d ie ta ry f a c to rs , p o o r o ra l h y g ie n e c u m u la ti v e f u tu re c a ri e s le s io n s ( h ig h r is k : � 5 n e w s u rf a c e l e s io n s ) c s , m l r , a n o v a 2 .5 v b : c h a rt s o f p a ti e n ts c o n ta in in g : m e d ic a l, d e n ta l a n d f lu o ri d e h i s to ry , d ie t, o ra l h y g ie n e , re te n ti v e p it s a n d f is s u re s , e x is ti n g r e s to ra ti o n s , n e w ly e ru p te d t e e th , c a ri e s /d e c a lc if ic a ti o n s , p u lp it s /a b s c e s s , g in g iv it is , c ro w d in g , b e h a v io r, a g e , g e n d e r e x : v a ri a b le s c o ll e c te d a t p a ti e n t ’s c h a rt s ( re tr o s p e c ti v e l o n g it u d in a l s tu d y ) 0 .9 -1 1 1 4 0 w a n d e ra e t a l. 3 2 , 2 0 0 0 ( m ic h ig a n ), u s a r p : e p in e p h ri n e l e v e ls c a ri e s i n c re m e n t s t, l ir a , m l r 1 v b : c a ri e s ( d m f / d m f) , d e n ta l p la q u e , c a te c h o la m in e c o n te n t in u ri n e s a m p le , b o d y w e ig h t, p a re n ta l a g e , e d u c a ti o n a n d p ro fe s s io n , m e d ic a l h is to ry a n d m e d ic a ti o n s e x : b it e w in g r a d io g ra p h s 6 -8 3 1 4 /2 7 0 v a n d e ra s e t a l. 3 1 , 2 0 0 0 , g re e c e m e a n c a ri e s i n c re m e n t: 0 .6 8 t e e th t h e m o re o ft e n t h e m s t e s t p o s it iv e , th e h ig h e r th e p ro p o rt io n o f c h il d re n w h o d e v e lo p e d c a ri e s l e s io n r p : fl u o ri d e ; r f : m s d m f t in c re m e n t � 1 c s , s n , s p , p v , m l r , c a 2 v b : c a ri e s ( w h o c ri te ri a ), p la q u e i n d e x ( s il n e s s a n d l ö e ), m ic ro b io lo g ic a l a n a ly s is o f m u ta n s s tr e p to c o c c i m s , (n o n -s ti m u la te d s a li v a s a m p le s ), q u e s ti o n n a ir e ( s u c ro s e i n ta k e , fl u o r id e e x p o s u re ) e x : c li n ic a l e x a m in a ti o n , v is u a l in s p e c ti o n , b it e w in g r a d io g ra p h s 6 -7 3 1 4 /3 0 4 (c a ri e s fr e e ) p e tt i a n d h a u s e n 1 5 , 2 0 0 0 (r o m e ), i ta ly m e a n c a ri e s i n c id e n c e : 0 .6 9 d m f s s ig n if ic a n t c o rr e la ti o n b e tw e e n i n it ia l m s s c o re s a n d c a ri e s d e v e lo p m e n t c a ri e s d e v e lo p m e n t c a , k w , m w , s n , s p 2 v b : c a ri e s ( d m f s / d m fs – w h o c ri te ri a , in it ia l le s io n s ), s e a la n ts f o r a ll m o la r fi s s u re s , o c c lu s a l p la q u e s a m p le s f ro m t e e th 1 6 a n d 3 6 ( m u ta n s s tr e p to c o c c i te s ts ), p la q u e i n d e x ( q u ig le y -h e in ) e x : e x p lo re r w it h o u t p re s s u re , li g h t s o u rc e 6 -7 2 3 0 /1 6 9 s p li e th a n d b e rn h a rd t 3 0 , 1 9 9 9 , g e rm a n y m e a n c a ri e s i n c re m e n t: 0 .9 5 d m f s c a ri e s i n c re m e n t: n e g a ti v e c o rr e la ti o n s w it h b a s e li n e la c to fe rr in , to ta l ig g a n d t o ta l a n a e ro b e s ; p o s it iv e c o rr e la ti o n w it h s p e c if ic a n ti -s . m u ta n s , ig g a n ti b o d y l e v e ls , m u ta n s s tr e p to c o c c i, l a c to b a c il li a n d s p e c if ic a n ti -s . m u ta n s ig g . c h il d re n w it h s ig n if ic a n tl y h ig h e r b a s e li n e c o n c e n tr a ti o n s o f h y p o th io c y a n it e , to ta l ig g a n ti b o d ie s a n d t o ta l a n a e ro b e s : n o n e w c a ri e s l e s io n s d m f i n c re m e n t > 0 s t, c a 2 v b : c a ri e s ( d m f / d m f w h o c ri te ri a , w h it e s p o ts l e s io n s ), p e ri o d o n ta l s ta tu s ( b le e d in g a n d c a lc u lu s ), u s a g e o f fl u o ri d a te d d e n ti fr ic e s , s a li v a s a m p le s ( b u ff e r c a p a c it y , h y p o th io c y a n it e a s s a y , to ta l s tr e p to c o c c i a n d m u ta n s s tr e p to c o c c i, la c to fe rr in a n d ly s o z y m e a n a ly s is , a g g lu ti n a ti o n a s s a y , to ta l s a li v a ry p e ro x id a s e a c ti v it y , to ta l a n d s p e c if ic ig a a n d ig g a n ti b o d ie s ) e x : v is u a l -t a c ti le m e th o d + f o t i 1 2 6 9 /6 3 k ir s ti lä e t a l. 2 9 , 1 9 9 8 ( t u rk u ), f in la n d m a in r e s u lt s r p /r f § o u tc o m e v a ri a b le d a ta a n a ly s is ¶ t im e ‡ v a ri a b le s c o ll e c te d a t b a s e li n e ( v b ) e x a m in a ti o n ( e x ) a g e † s a m p le * a u th o r , y e a r a n d lo c a l 1686 t a b le 3 r ev ie w o f li te ra tu re o n c ar ie s ri sk a ss es sm en t in s ch o o lc h il d re n ( 6 -1 2 y ea rs o ld ) o v er t h e la st 1 0 y ea rs ( 1 9 9 7 -2 0 0 7 ) braz j oral sci. 7(27):1682-1690 an overview of caries risk assessment in 0-18 year-olds over the last ten years (1997-2007) s ig n if ic a n t, b u t w e a k , c o rr e la ti o n b e tw e e n m s c o u n ts a n d c a ri e s r p : p a s t c a ri e s e x p e ri e n c e c a ri e s i n c re m e n t (d e n ti n le v e l) c a , m r a 2 v b : c a ri e s ( e n a m e l + d e n ti n l e s io n s ), m u ta n s s tr e p to c o c c i (m s ) c o u n ts i n s a li v a e x : t e e th d ri e d , fi b e r -o p ti c l ig h t o n a m o u th m ir ro r, d e n ta l p ro b e ( re m o v a l o f p la q u e , d e te c ti o n o f fi s s u re s e a la n ts ) 6 -7 6 5 0 /4 3 3 z h a n g e t a l. 4 5 , 2 0 0 7 ( w u h a n ), c h in a t o ta l d m f t in c re m e n t: 0 .5 2 r p : c a ri e s i n p e rm a n e n t m o la rs , d m f t , c a ri e s s e v e ri ty d m f t � 1 i n c re m e n t c s , m w , k w , c a , w , m r a 2 v b : c a ri e s ( d m f t , d m ft – w h o c ri te ri a , c a ri e s i n a n y f ir s t p e rm a n e n t m o la r, c a ri e s i n a n y p e rm a n e n t u p p e r m o la rs , c a ri e s i n a n y p e rm a n e n t lo w e r m o la rs , c a ri e s s e v e r it y ), a g e , g e n d e r e x : d e n ta l m ir ro r, t e e th d ri e d w it h g a u z e , n a tu ra l d a y li g h t, a t s c h o o ls 6 -9 5 8 0 /4 5 2 v a ll e jo s -s á n c h e z e t a l. 4 4 , 2 0 0 6 (c a m p e c h e ), m é x ic o m e a n d m f s i n c re m e n t: 2 .6 3 r p : d m fs , d m f s , m o th e r ’s e d u c a ti o n d m fs > 0 : h ig h e s t s n (6 9 % ) d m f s > 0 : h ig h e s t s p ( 9 2 % ). d m f s � 1 i n c re m e n t c s , m n , m l r , s n , s p , p v , r o c 7 v b : c a ri e s ( d m f / d m f -w h o c ri te ri a , in it ia l c a ri e s l e s io n s ), p la q u e s c o re ( s im p li fi e d o ra l h y g ie n e i n d e x ), d e n ta l fl u o ro s is (d e a n i n d e x ), q u e s ti o n n a ir e ( to o th b ru s h in g fr e q u e n c y, t y p e o f p re v e n ti v e t o p ic a l m e th o d , fa th e r’ s a n d m o th e r ’s e d u c a ti o n a l le v e l, g e n d e r, r a c e , n u m b e r o f w o rk in g p e o p le l iv i n g i n t h e h o u s e h o ld , m o n th ly f a m il y i n c o m e , d e n ta l v is it s i n t h e y e a r p ri o r to b a s e li n e , re a s o n f o r d e n ta l v is it , d a il y s u g a r c o n s u m p ti o n , n u m b e r o f s u g a r s p o o n s i n b e v e ra g e s , n u m b e r o f b e tw e e n -m e a l s n a c k s o ra l h y g ie n e h a b it s ) e x : d e n ta l p ro b e a n d m ir ro r, u n d e r n a tu ra l li g h t in o u td o o r s e tt in g a t s c h o o ls 6 -8 4 8 0 /2 0 6 t a g li a fe rr o e t a l. 4 3 , 2 0 0 6 (p ir a c ic a b a ), b ra z il d m f s i n c re a s e : p o s it iv e ly a s s o c ia te d w it h d m fs in c re a s e a n d w it h i n it ia l c a ri e s r p : p a s t c a ri e s e x p e ri e n c e a re a u n d e r r o c c u rv e : 7 6 % c a ri e s i n c id e n c e c s , m l r , s n , s p , p v , r o c 1 v b : c a ri e s ( d m fs , d m f s , n u m b e r o f s u rf a c e s w it h i n it ia l c a ri e s , n º o f s u rf a c e s w it h p ri m a ry a n d s e c o n d a ry c a ri e s , n u m b e r o f fi ll e d s u rf a c e s , n u m b e r o f s u rf a c e s m is s in g d u e t o c a ri e s ), g e n d e r, y e a r o f b ir th , n u m b e r o f c h il d re n i n h o u s e h o ld , n u m b e r o f a d u lt s i n h o u s e h o ld , c it iz e n s h ip e x : d a n is h r o u ti n e d e n ta l a n d s o c io e c o n o m ic r e g is te rs 7 1 2 3 7 0 5 j e p p e s e n a n d f o ld s p a n g 4 2 , 2 0 0 6 ( a a rh u s ), d e n m a rk m e a n c a ri e s i n c re m e n t: 4 .2 ( e n a m e l + d e n ti n a l le s io n s ) h ig h e s t p re d ic ti v e p o w e r: p ro x im a l le s io n s i n p re m o la rs a n d s e c o n d m o la rs c a ri e s ( d e n ti n l e v e l) in c re m e n t o n p ro x im a l s u rf a c e s ( d f s > 0 , 1 , 2 , 3 a n d 4 ) s t, s n , s p 6 v b : c a ri e s ( e n a m e l + d e n ti n l e s io n s ), q u e s ti o n n a ir e ( g e n d e r, m o th e r’ s e d u c a ti o n ) e x : t e e th p o li s h e d a n d d ri e d , p la n e m o u th m ir ro r, p ro b e , b it e w in g ra d io g ra p h s i n e q u ip p e d d e n ta l c li n ic s 1 2 1 5 9 /1 1 2 d a v id e t a l. 4 1 , 2 0 0 6 ( b e rg e n ), n o rw a y r p : g e n d e r (g ir ls fo r lo w e r m o la rs ), c a v it y e x p e ri e n c e i n t h e d e c id u o u s d e n ti ti o n ; r f : o c c lu s a l p la q u e a c c u m u la ti o n , re p o rt e d b ru s h in g f re q u e n c y s u rv iv a l ti m e o f a p f m s a 6 v b : c a ri e s ( b a s c d c ri te ri a ), p re s e n c e o f p la q u e o n t h e o c c lu s a l s u rf a c e s o f p e rm a n e n t fi rs t m o la rs (p f m ), t im in g o f to o th e m e rg e n c e , g e n d e r , q u e s ti o n n a ir e ( o ra l h y g ie n e a n d d ie ta ry h a b it s ) e x : v is u a l in s p e c ti o n 6 4 4 6 8 l e ro y e t a l. 4 0 , 2 0 0 5 ( f la n d e rs ), b e lg iu m r p : p re s e n c e o f c a ri e s o n p s m d s s n = 4 5 % t o 9 7 % ; s p = 8 0 % t o 8 9 % c a ri e s i n c id e n c e o n m e s ia l s u rf a c e s o f p e rm a n e n t 1 º m o la rs (m s p f m ) m l r , s n , s p , p v 4 v b : c a ri e s ( p re s e n c e o f c a ri e s o n t h e p ri m a ry s e c o n d m o la rs ’ d is ta l s u rf a c e s p s m d s ), a g e , g e n d e r e x : b it e w in g r a d io g ra p h s 6 -8 3 1 4 /1 9 6 v a n d e ra s e t a l. 3 9 , 2 0 0 4 , g re e c e r f : h ig h m s l e v e ls l o w m s l e v e ls : s ig n if ic a n t e ff e c t o n t h e l o n g e r s u rv iv a l ti m e s t im e t o c a ri e s o n s e t s a , w 6 v b : c a ri e s ( v is u a l -t a c ti le c ri te ri a o f r a d ik e ), m ic ro b io lo g ic a l (m s c o u n ts i n w h o le s ti m u la te d s a li v a : if � 1 0 6 c f u /m l h ig h l e v e ls o f m s ) e x : f ib e r -o p ti c l ig h ts , p la n e m ir ro rs , # 2 3 e x p lo re rs ( c le a n in g o f s u rf a c e s a n d d e te c ti n g o f s e a la n ts ) 6 -7 4 6 4 */ 1 6 0 *c a ri e s fr e e k o p y c k a k e d z ie ra w s k i a n d b il li n g s 1 6 , 2 0 0 4 (n e w y o rk ), u s a m e a n d m f s i n c re m e n t: 1 .0 ( in c lu d in g e n a m e l le s io n = 1 .8 8 ) r p : p re v io u s e x p e ri e n c e o f c a ri e s , s o c io e c o n o m ic l e v e l c a ri e s i n c re m e n t p r 2 v b : c a ri e s ( d m f + e n a m e l c a ri e s ), s e a la n ts , q u e s ti o n n a ir e ( e th n ic i ty , re s id e n ti a l a re a , s o c io e c o n o m ic le v e l) e x : c li n ic a l s e tt in g : m ir ro r, g o o d o p e ra ti n g l ig h t, c o m p re s s e d a ir , c o tt o n r o ll s , tw o b it e w in g s r a d io g ra p h s 1 2 3 3 7 3 /3 1 0 7 k ä ll e s t å l a n d w a ll 3 8 , 2 0 0 2 , s w e d e n m a in r e s u lt s r p /r f § o u tc o m e v a ri a b le d a ta a n a ly s is ¶ t im e ‡ v a ri a b le s c o ll e c te d a t b a s e li n e ( v b ) e x a m in a ti o n ( e x ) a g e † s a m p le * a u th o r , y e a r a n d lo c a l t a b le 3 – c o n t. s ig n if ic a n t, b u t w e a k , c o rr e la ti o n b e tw e e n m s c o u n ts a n d c a ri e s r p : p a s t c a ri e s e x p e ri e n c e c a ri e s i n c re m e n t (d e n ti n le v e l) c a , m r a 2 v b : c a ri e s ( e n a m e l + d e n ti n l e s io n s ), m u ta n s s tr e p to c o c c i (m s ) c o u n ts i n s a li v a e x : t e e th d ri e d , fi b e r -o p ti c l ig h t o n a m o u th m ir ro r, d e n ta l p ro b e ( re m o v a l o f p la q u e , d e te c ti o n o f fi s s u re s e a la n ts ) 6 -7 6 5 0 /4 3 3 z h a n g e t a l. 4 5 , 2 0 0 7 ( w u h a n ), c h in a t o ta l d m f t in c re m e n t: 0 .5 2 r p : c a ri e s i n p e rm a n e n t m o la rs , d m f t , c a ri e s s e v e ri ty d m f t � 1 i n c re m e n t c s , m w , k w , c a , w , m r a 2 v b : c a ri e s ( d m f t , d m ft – w h o c ri te ri a , c a ri e s i n a n y f ir s t p e rm a n e n t m o la r, c a ri e s i n a n y p e rm a n e n t u p p e r m o la rs , c a ri e s i n a n y p e rm a n e n t lo w e r m o la rs , c a ri e s s e v e r it y ), a g e , g e n d e r e x : d e n ta l m ir ro r, t e e th d ri e d w it h g a u z e , n a tu ra l d a y li g h t, a t s c h o o ls 6 -9 5 8 0 /4 5 2 v a ll e jo s -s á n c h e z e t a l. 4 4 , 2 0 0 6 (c a m p e c h e ), m é x ic o m e a n d m f s i n c re m e n t: 2 .6 3 r p : d m fs , d m f s , m o th e r ’s e d u c a ti o n d m fs > 0 : h ig h e s t s n (6 9 % ) d m f s > 0 : h ig h e s t s p ( 9 2 % ). d m f s � 1 i n c re m e n t c s , m n , m l r , s n , s p , p v , r o c 7 v b : c a ri e s ( d m f / d m f -w h o c ri te ri a , in it ia l c a ri e s l e s io n s ), p la q u e s c o re ( s im p li fi e d o ra l h y g ie n e i n d e x ), d e n ta l fl u o ro s is (d e a n i n d e x ), q u e s ti o n n a ir e ( to o th b ru s h in g fr e q u e n c y, t y p e o f p re v e n ti v e t o p ic a l m e th o d , fa th e r’ s a n d m o th e r ’s e d u c a ti o n a l le v e l, g e n d e r, r a c e , n u m b e r o f w o rk in g p e o p le l iv i n g i n t h e h o u s e h o ld , m o n th ly f a m il y i n c o m e , d e n ta l v is it s i n t h e y e a r p ri o r to b a s e li n e , re a s o n f o r d e n ta l v is it , d a il y s u g a r c o n s u m p ti o n , n u m b e r o f s u g a r s p o o n s i n b e v e ra g e s , n u m b e r o f b e tw e e n -m e a l s n a c k s o ra l h y g ie n e h a b it s ) e x : d e n ta l p ro b e a n d m ir ro r, u n d e r n a tu ra l li g h t in o u td o o r s e tt in g a t s c h o o ls 6 -8 4 8 0 /2 0 6 t a g li a fe rr o e t a l. 4 3 , 2 0 0 6 (p ir a c ic a b a ), b ra z il d m f s i n c re a s e : p o s it iv e ly a s s o c ia te d w it h d m fs in c re a s e a n d w it h i n it ia l c a ri e s r p : p a s t c a ri e s e x p e ri e n c e a re a u n d e r r o c c u rv e : 7 6 % c a ri e s i n c id e n c e c s , m l r , s n , s p , p v , r o c 1 v b : c a ri e s ( d m fs , d m f s , n u m b e r o f s u rf a c e s w it h i n it ia l c a ri e s , n º o f s u rf a c e s w it h p ri m a ry a n d s e c o n d a ry c a ri e s , n u m b e r o f fi ll e d s u rf a c e s , n u m b e r o f s u rf a c e s m is s in g d u e t o c a ri e s ), g e n d e r, y e a r o f b ir th , n u m b e r o f c h il d re n i n h o u s e h o ld , n u m b e r o f a d u lt s i n h o u s e h o ld , c it iz e n s h ip e x : d a n is h r o u ti n e d e n ta l a n d s o c io e c o n o m ic r e g is te rs 7 1 2 3 7 0 5 j e p p e s e n a n d f o ld s p a n g 4 2 , 2 0 0 6 ( a a rh u s ), d e n m a rk m e a n c a ri e s i n c re m e n t: 4 .2 ( e n a m e l + d e n ti n a l le s io n s ) h ig h e s t p re d ic ti v e p o w e r: p ro x im a l le s io n s i n p re m o la rs a n d s e c o n d m o la rs c a ri e s ( d e n ti n l e v e l) in c re m e n t o n p ro x im a l s u rf a c e s ( d f s > 0 , 1 , 2 , 3 a n d 4 ) s t, s n , s p 6 v b : c a ri e s ( e n a m e l + d e n ti n l e s io n s ), q u e s ti o n n a ir e ( g e n d e r, m o th e r’ s e d u c a ti o n ) e x : t e e th p o li s h e d a n d d ri e d , p la n e m o u th m ir ro r, p ro b e , b it e w in g ra d io g ra p h s i n e q u ip p e d d e n ta l c li n ic s 1 2 1 5 9 /1 1 2 d a v id e t a l. 4 1 , 2 0 0 6 ( b e rg e n ), n o rw a y r p : g e n d e r (g ir ls fo r lo w e r m o la rs ), c a v it y e x p e ri e n c e i n t h e d e c id u o u s d e n ti ti o n ; r f : o c c lu s a l p la q u e a c c u m u la ti o n , re p o rt e d b ru s h in g f re q u e n c y s u rv iv a l ti m e o f a p f m s a 6 v b : c a ri e s ( b a s c d c ri te ri a ), p re s e n c e o f p la q u e o n t h e o c c lu s a l s u rf a c e s o f p e rm a n e n t fi rs t m o la rs (p f m ), t im in g o f to o th e m e rg e n c e , g e n d e r , q u e s ti o n n a ir e ( o ra l h y g ie n e a n d d ie ta ry h a b it s ) e x : v is u a l in s p e c ti o n 6 4 4 6 8 l e ro y e t a l. 4 0 , 2 0 0 5 ( f la n d e rs ), b e lg iu m r p : p re s e n c e o f c a ri e s o n p s m d s s n = 4 5 % t o 9 7 % ; s p = 8 0 % t o 8 9 % c a ri e s i n c id e n c e o n m e s ia l s u rf a c e s o f p e rm a n e n t 1 º m o la rs (m s p f m ) m l r , s n , s p , p v 4 v b : c a ri e s ( p re s e n c e o f c a ri e s o n t h e p ri m a ry s e c o n d m o la rs ’ d is ta l s u rf a c e s p s m d s ), a g e , g e n d e r e x : b it e w in g r a d io g ra p h s 6 -8 3 1 4 /1 9 6 v a n d e ra s e t a l. 3 9 , 2 0 0 4 , g re e c e r f : h ig h m s l e v e ls l o w m s l e v e ls : s ig n if ic a n t e ff e c t o n t h e l o n g e r s u rv iv a l ti m e s t im e t o c a ri e s o n s e t s a , w 6 v b : c a ri e s ( v is u a l -t a c ti le c ri te ri a o f r a d ik e ), m ic ro b io lo g ic a l (m s c o u n ts i n w h o le s ti m u la te d s a li v a : if � 1 0 6 c f u /m l h ig h l e v e ls o f m s ) e x : f ib e r -o p ti c l ig h ts , p la n e m ir ro rs , # 2 3 e x p lo re rs ( c le a n in g o f s u rf a c e s a n d d e te c ti n g o f s e a la n ts ) 6 -7 4 6 4 */ 1 6 0 *c a ri e s fr e e k o p y c k a k e d z ie ra w s k i a n d b il li n g s 1 6 , 2 0 0 4 (n e w y o rk ), u s a m e a n d m f s i n c re m e n t: 1 .0 ( in c lu d in g e n a m e l le s io n = 1 .8 8 ) r p : p re v io u s e x p e ri e n c e o f c a ri e s , s o c io e c o n o m ic l e v e l c a ri e s i n c re m e n t p r 2 v b : c a ri e s ( d m f + e n a m e l c a ri e s ), s e a la n ts , q u e s ti o n n a ir e ( e th n ic i ty , re s id e n ti a l a re a , s o c io e c o n o m ic le v e l) e x : c li n ic a l s e tt in g : m ir ro r, g o o d o p e ra ti n g l ig h t, c o m p re s s e d a ir , c o tt o n r o ll s , tw o b it e w in g s r a d io g ra p h s 1 2 3 3 7 3 /3 1 0 7 k ä ll e s t å l a n d w a ll 3 8 , 2 0 0 2 , s w e d e n m a in r e s u lt s r p /r f § o u tc o m e v a ri a b le d a ta a n a ly s is ¶ t im e ‡ v a ri a b le s c o ll e c te d a t b a s e li n e ( v b ) e x a m in a ti o n ( e x ) a g e † s a m p le * a u th o r , y e a r a n d lo c a l t a b le 3 – c o n t. 1687 * s am p le s iz e at i n it ia l/ fi n al e x am in at io n ; † a g e at b as el in e (y ea rs ); ‡ s tu d y l as ti n g ( y ea rs ); ¶ s ta ti st ic al t es ts : a n o v a = a n al y si s o f v ar ia n ce ; c a = c o rr el at io n a n al y si s; c s = c h isq u ar e; k w = k ru sk al l w al li s; l ir a = l in ea r re g re ss io n a n al y si s; m l r = m u lt ip le l o g is ti c re g re ss io n ; m n = m cn em ar ; m r a = m u lt ip le r eg re ss io n a n al y si s; m w = m an n -w h it n ey ; p r = p o is so n r eg re ss io n ; p v = p re d ic ti v e v al u es ; r o c = r ec ei v er o p er at o r ch ar ac te ri st ic c u rv es ; s a = s u rv iv al a n al y si s; s n = s en si ti v it y ; s p = s p ec if ic it y ; s t= s tu d en t’ s tte st ; w = w il co x o n ; § r p /r f : r is k p re d ic to rs /r is k f ac to rs o b ta in ed i n r eg re ss io n a n al y se s braz j oral sci. 7(27):1682-1690 an overview of caries risk assessment in 0-18 year-olds over the last ten years (1997-2007) 1688 * s am p le s iz e at i n it ia l/ fi n al e x am in at io n ; † a g e at b as el in e (y ea rs ); ‡ s tu d y l as ti n g ( y ea rs ); ¶ s ta ti st ic al t es ts : a n o v a = a n al y si s o f v ar ia n ce ; c a = c o rr el at io n a n al y si s; c s = c h isq u ar e; m l r = m u lt ip le l o g is ti c re g re ss io n ; m r a = m u lt ip le r eg re ss io n a n al y si s; m w = m an n -w h it n ey ; p r = p o is so n r eg re ss io n ; p v = p re d ic ti v e v al u es ; r r = r el at iv e ri sk ; s n = s en si ti v it y ; s p = s p ec if ic it y ; s t= s tu d en t’ s tte st ; w = w il co x o n ; § r p /r f : r is k p re d ic to rs /r is k f ac to rs o b ta in ed i n r eg re ss io n a n al y se s t a b le 4 r ev ie w o f li te ra tu re o n c ar ie s ri sk a ss es sm en t in a d o le sc en ts ( 1 3 -1 8 y ea rs o ld ) o v er t h e la st 1 0 y ea rs ( 1 9 9 7 -2 0 0 7 ) braz j oral sci. 7(27):1682-1690 an overview of caries risk assessment in 0-18 year-olds over the last ten years (1997-2007) r p : o ra l h e a lt h c o n c e rn ; r f : o ra l h e a lt h b e h a v io r (f o r o n ly th o s e a g e d 1 7 y e a rs a t b a s e li n e ) d if fe re n c e s b e tw e e n s c o re s fr o m b a s e li n e a n d fi n a l e x a m in a ti o n s t, m l r 3 v b : c a ri e s , q u e s ti o n n a ir e (d e n ta l k n o w le d g e , o ra l h e a lt h b e h a v io r, o ra l h e a lt h c a re a tt it u d e s ) e x : c li n ic a l a n d ra d io g ra p h ic e x a m in a ti o n 1 4 , 1 7 , 2 0 2 0 2 p o o rt e rm a n e t a l. 5 2 , 2 0 0 3 , t h e n e th e rl a n d s m e a n d f s a n d d m f s in c re m e n t: 0 .9 8 a n d 1 .1 0 , re s p e c ti v e ly r f : lo w s u g a rh ig h s ta rc h fo o d s fo r c a ri e s in c re m e n t o n a ll s u rf a c e s a n d p it a n d fi s s u re s s u rf a c e s t o ta l d m f s in c re m e n t; p it a n d fi s s u re d m f in c re m e n t; s m o o th s u rf a c e d m f in c re m e n t � 1 m l r 2 v b : c a ri e s (w h o c ri te ri a ), h e ig h t a n d w e ig h t ( “e s ti m a ti o n o f s u b je c ts ’ b a s a l m e ta b o li c ra te ”) , fo u rd a y d ie t re c o rd s , q u e s ti o n n a ir e (h o u s e h o ld in c o m e , e d u c a ti o n le v e l, o c c u p a ti o n a n d e th n ic it y ) e x : f ib e ro p ti c li g h t s o u rc e , p la n e m o u th m ir ro r, s ic k le p ro b e , a t s c h o o ls 1 2 1 3 6 4 5 /5 0 4 c a m p a in e t a l. 5 1 , 2 0 0 3 (m e lb o u rn e ), a u s tr a li a m e d ia n ti m e to th e fi rs t n e w p ro x im a l c a ri e s le s io n : 2 y e a rs in d iv id u a ls w it h n o p ro x im a l le s io n s a t b a s e li n e : 0 .0 3 1 s u rf a c e /y e a r in d iv id u a ls w it h 3 p ro x im a l le s io n s a t b a s e li n e : 0 .0 7 7 s u rf a c e /y e a r in c id e n c e o f th e fi rs t n e w p ro x im a l c a ri e s le s io n s a , p r 1 0 v b : p ro x im a l s u rf a c e s s ta tu s : c a ri e sfr e e o r in a c a ri e s s ta tu s e x : o n ly ra d io g ra p h ic e x a m in a ti o n 1 1 1 3 5 3 6 /5 3 4 s te n lu n d e t a l. 5 0 , 2 0 0 2 (s to c k h o lm ), s w e d e n m e a n d f s in c re m e n t: 2 .0 6 a s th m a ti c g ro u p s : n o s ig n if ic a n tl y h ig h e r c a ri e s in c re m e n t th a n th e n o n -a s th m a ti c g ro u p d f s in c re m e n t c s , m w 3 v b : c a ri e s (w h o c ri te ri a ), p a re n ta l s o c io e c o n o m ic le v e l, a s th m a s t a tu s . ( n o te : 2 0 6 in d iv id u a ls h a v in g n o h is to ry o f a s th m a w e re u s e d a s th e c o m p a ris o n g ro u p ) e x : f ib e ro p ti c li g h t, p la n e d e n ta l m ir ro r a n d s ic k le e x p lo re r 1 5 9 7 6 /7 8 1 m e ld ru m e t a l. 4 9 , 2 0 0 1 (o ta g o ), n e w z e a la n d s u b je c ts w it h n o c a v it ie s a t b a s e li n e : 6 0 % re m a in e d c a v it ie s -f re e s u b je c ts w it h g in g iv a l h e a lt h a t b a s e li n e : 4 7 % re m a in e d h e a lt h y a t th e fi n a l e x a m in a ti o n r p : d m f t > 2 , d t > 0 , g re a t g in g iv a l in fe c ti o n , g e n d e r (m a le s ) n u m b e r o f c a v it a te d te e th d u ri n g th e p e ri o d m l r 2 v b : c a ri e s (d m f in d e x , d c o m p o n e n t) , g in g iv it is (c p it n in d e x ), q u es ti o n n a ir e (i n fo rm a ti o n o n s m o k in g ) e x : a n n u a l d e n ta l e x a m in a ti o n s in h e a lt h c e n te rs a n d a t s c h o o ls , r a d io g ra p h ic e x a m in a ti o n s 1 3 2 4 2 2 /1 4 7 2 u tr ia in e n e t a l. 4 8 , 1 9 9 8 (k o k o la , p ie ta rs a a ri , v a a s a , s e in ä jo k i) f in la n d r p : f lu o ri d e le v e l in th e d ri n k in g w a te r, c a ri e s p re v a le n c e , n u m b e r o f c a v it a te d c a ri o u s le s io n s ; r f : to o th b ru s h in g fr e q u e n c y c a ri e s p ro g re s s io n s t, w , c s , m l r 1 v b : c a ri e s (d m f s in d e x – w h o c ri te ri a ), d e n ta l p la q u e (p a ti e n t h y g ie n e p e rf o rm a n c e p h p in d e x ), m a lo c c lu s io n (w h o c ri te ri a ), e n a m e l d e fe c ts (d e v e lo p m e n ta l d e fe c ts o f e n a m e ld d e in d e x ), a t b a s e li n e a n d fi n a l e x a m in a ti o n , in te rv ie w (a g e , g e n d e r, s o c io e c o n o m ic s ta tu s , ra c e , fl u o ri d e e x p o s u re , re s id e n c e h is to ri e s , p re v e n ti v e d e n ta l h e a lt h b e h a v io rs , u s e o f d e n ta l s e rv ic e s , to o th b ru s h in g fr e q u e n c y , to o th p a s te b ra n d u s e d , p ro fe s s io n a ll y a p p li e d fl u o ri d e g e l ri n s e s , v a rn is h e s o r s e a la n t s , h o m e u s e o f fl u o ri d e m o u th ri n s e s , u s e o f fl u o ri d e s u p p le m e n ts d u ri n g e a rl y c h il d h o o d , n u m b e r o f y e a rs o f re s id e n c e in th e re s e a rc h a re a , s o u rc e s o f d o m e s ti c d ri n k in g w a te r) e x : h e a d la m p , p la n e m ir ro r, c a ri e s e x p lo re r, c p it n p ro b e , d ry in g a t d e n ta l s u rg e ri e s o r c la s s ro o m s a t s c h o o ls , p o s te ri o r b it e w in g s 1 2 1 6 4 2 0 /2 9 0 l a w re n c e a n d s h e ih a m 4 7 , 1 9 9 7 (r io d e j a n e ir o , m a n g a ra ti b a a n d a n g ra d o s r e is ), b ra z il r p : in c ip ie n t c a ri e s e x p e ri e n c e ; r f : s a li v a ry m ic ro o rg a n is m s in c ip ie n t + m a n if e s t le s io n s : c o m b in e d v a lu e s o fs n a n d s p a ll o w e d to p re d ic t c a ri e s d e v e lo p m e n t in th e m a jo ri ty o f in d iv id u a ls d f s in c re m e n t � 5 d f s in c re m e n t � 3 c a , m r a , s n , s p , p v 3 v b : c a ri e s ( c a v it a ti o n a n d in c ip ie n t le s io n s ), m u ta n s s tr e p to c o c c i (m s ) a n d la c to b a c il lu s (l b ) c o u n ts in s ti m u la te d s a li v a e x : c li n ic a l + ra d io g ra p h ic e x a m in a ti o n 1 5 1 6 1 5 5 /8 7 b ja rn a s o n a n d k ö h le r4 6 , 1 9 9 7 , s w e d e n m a in re s u lt s r p /r f § o u tc o m e v a ri a b le d a ta a n a ly s is ¶ t im e ‡ v a ri a b le s c o ll e c te d a t b a s e li n e (v b ) e x a m in a ti o n (e x ) a g e † s a m p le * a u th o r, y e a r a n d lo c a l r p : o ra l h e a lt h c o n c e rn ; r f : o ra l h e a lt h b e h a v io r (f o r o n ly th o s e a g e d 1 7 y e a rs a t b a s e li n e ) d if fe re n c e s b e tw e e n s c o re s fr o m b a s e li n e a n d fi n a l e x a m in a ti o n s t, m l r 3 v b : c a ri e s , q u e s ti o n n a ir e (d e n ta l k n o w le d g e , o ra l h e a lt h b e h a v io r, o ra l h e a lt h c a re a tt it u d e s ) e x : c li n ic a l a n d ra d io g ra p h ic e x a m in a ti o n 1 4 , 1 7 , 2 0 2 0 2 p o o rt e rm a n e t a l. 5 2 , 2 0 0 3 , t h e n e th e rl a n d s m e a n d f s a n d d m f s in c re m e n t: 0 .9 8 a n d 1 .1 0 , re s p e c ti v e ly r f : lo w s u g a rh ig h s ta rc h fo o d s fo r c a ri e s in c re m e n t o n a ll s u rf a c e s a n d p it a n d fi s s u re s s u rf a c e s t o ta l d m f s in c re m e n t; p it a n d fi s s u re d m f in c re m e n t; s m o o th s u rf a c e d m f in c re m e n t � 1 m l r 2 v b : c a ri e s (w h o c ri te ri a ), h e ig h t a n d w e ig h t ( “e s ti m a ti o n o f s u b je c ts ’ b a s a l m e ta b o li c ra te ”) , fo u rd a y d ie t re c o rd s , q u e s ti o n n a ir e (h o u s e h o ld in c o m e , e d u c a ti o n le v e l, o c c u p a ti o n a n d e th n ic it y ) e x : f ib e ro p ti c li g h t s o u rc e , p la n e m o u th m ir ro r, s ic k le p ro b e , a t s c h o o ls 1 2 1 3 6 4 5 /5 0 4 c a m p a in e t a l. 5 1 , 2 0 0 3 (m e lb o u rn e ), a u s tr a li a m e d ia n ti m e to th e fi rs t n e w p ro x im a l c a ri e s le s io n : 2 y e a rs in d iv id u a ls w it h n o p ro x im a l le s io n s a t b a s e li n e : 0 .0 3 1 s u rf a c e /y e a r in d iv id u a ls w it h 3 p ro x im a l le s io n s a t b a s e li n e : 0 .0 7 7 s u rf a c e /y e a r in c id e n c e o f th e fi rs t n e w p ro x im a l c a ri e s le s io n s a , p r 1 0 v b : p ro x im a l s u rf a c e s s ta tu s : c a ri e sfr e e o r in a c a ri e s s ta tu s e x : o n ly ra d io g ra p h ic e x a m in a ti o n 1 1 1 3 5 3 6 /5 3 4 s te n lu n d e t a l. 5 0 , 2 0 0 2 (s to c k h o lm ), s w e d e n m e a n d f s in c re m e n t: 2 .0 6 a s th m a ti c g ro u p s : n o s ig n if ic a n tl y h ig h e r c a ri e s in c re m e n t th a n th e n o n -a s th m a ti c g ro u p d f s in c re m e n t c s , m w 3 v b : c a ri e s (w h o c ri te ri a ), p a re n ta l s o c io e c o n o m ic le v e l, a s th m a s t a tu s . ( n o te : 2 0 6 in d iv id u a ls h a v in g n o h is to ry o f a s th m a w e re u s e d a s th e c o m p a ris o n g ro u p ) e x : f ib e ro p ti c li g h t, p la n e d e n ta l m ir ro r a n d s ic k le e x p lo re r 1 5 9 7 6 /7 8 1 m e ld ru m e t a l. 4 9 , 2 0 0 1 (o ta g o ), n e w z e a la n d s u b je c ts w it h n o c a v it ie s a t b a s e li n e : 6 0 % re m a in e d c a v it ie s -f re e s u b je c ts w it h g in g iv a l h e a lt h a t b a s e li n e : 4 7 % re m a in e d h e a lt h y a t th e fi n a l e x a m in a ti o n r p : d m f t > 2 , d t > 0 , g re a t g in g iv a l in fe c ti o n , g e n d e r (m a le s ) n u m b e r o f c a v it a te d te e th d u ri n g th e p e ri o d m l r 2 v b : c a ri e s (d m f in d e x , d c o m p o n e n t) , g in g iv it is (c p it n in d e x ), q u es ti o n n a ir e (i n fo rm a ti o n o n s m o k in g ) e x : a n n u a l d e n ta l e x a m in a ti o n s in h e a lt h c e n te rs a n d a t s c h o o ls , r a d io g ra p h ic e x a m in a ti o n s 1 3 2 4 2 2 /1 4 7 2 u tr ia in e n e t a l. 4 8 , 1 9 9 8 (k o k o la , p ie ta rs a a ri , v a a s a , s e in ä jo k i) f in la n d r p : f lu o ri d e le v e l in th e d ri n k in g w a te r, c a ri e s p re v a le n c e , n u m b e r o f c a v it a te d c a ri o u s le s io n s ; r f : to o th b ru s h in g fr e q u e n c y c a ri e s p ro g re s s io n s t, w , c s , m l r 1 v b : c a ri e s (d m f s in d e x – w h o c ri te ri a ), d e n ta l p la q u e (p a ti e n t h y g ie n e p e rf o rm a n c e p h p in d e x ), m a lo c c lu s io n (w h o c ri te ri a ), e n a m e l d e fe c ts (d e v e lo p m e n ta l d e fe c ts o f e n a m e ld d e in d e x ), a t b a s e li n e a n d fi n a l e x a m in a ti o n , in te rv ie w (a g e , g e n d e r, s o c io e c o n o m ic s ta tu s , ra c e , fl u o ri d e e x p o s u re , re s id e n c e h is to ri e s , p re v e n ti v e d e n ta l h e a lt h b e h a v io rs , u s e o f d e n ta l s e rv ic e s , to o th b ru s h in g fr e q u e n c y , to o th p a s te b ra n d u s e d , p ro fe s s io n a ll y a p p li e d fl u o ri d e g e l ri n s e s , v a rn is h e s o r s e a la n t s , h o m e u s e o f fl u o ri d e m o u th ri n s e s , u s e o f fl u o ri d e s u p p le m e n ts d u ri n g e a rl y c h il d h o o d , n u m b e r o f y e a rs o f re s id e n c e in th e re s e a rc h a re a , s o u rc e s o f d o m e s ti c d ri n k in g w a te r) e x : h e a d la m p , p la n e m ir ro r, c a ri e s e x p lo re r, c p it n p ro b e , d ry in g a t d e n ta l s u rg e ri e s o r c la s s ro o m s a t s c h o o ls , p o s te ri o r b it e w in g s 1 2 1 6 4 2 0 /2 9 0 l a w re n c e a n d s h e ih a m 4 7 , 1 9 9 7 (r io d e j a n e ir o , m a n g a ra ti b a a n d a n g ra d o s r e is ), b ra z il r p : in c ip ie n t c a ri e s e x p e ri e n c e ; r f : s a li v a ry m ic ro o rg a n is m s in c ip ie n t + m a n if e s t le s io n s : c o m b in e d v a lu e s o fs n a n d s p a ll o w e d to p re d ic t c a ri e s d e v e lo p m e n t in th e m a jo ri ty o f in d iv id u a ls d f s in c re m e n t � 5 d f s in c re m e n t � 3 c a , m r a , s n , s p , p v 3 v b : c a ri e s ( c a v it a ti o n a n d in c ip ie n t le s io n s ), m u ta n s s tr e p to c o c c i (m s ) a n d la c to b a c il lu s (l b ) c o u n ts in s ti m u la te d s a li v a e x : c li n ic a l + ra d io g ra p h ic e x a m in a ti o n 1 5 1 6 1 5 5 /8 7 b ja rn a s o n a n d k ö h le r4 6 , 1 9 9 7 , s w e d e n m a in re s u lt s r p /r f § o u tc o m e v a ri a b le d a ta a n a ly s is ¶ t im e ‡ v a ri a b le s c o ll e c te d a t b a s e li n e (v b ) e x a m in a ti o n (e x ) a g e † s a m p le * a u th o r, y e a r a n d lo c a l 1689 been a strong variable in identifying children at risk because it shows that the oral environment was prone to develop caries. sugar consumption has played an important role in caries risk assessment in young children, as previously described for infants. others variables related to oral hygiene also showed their significance in identifying children at risk. the presence of plaque on teeth, due to the lack and/or deficiency in toothbrushing, offers substrates to cariogenic bacteria favoring caries development. schoolchildren have been the most studied group in caries risk assessment (table 3). from 1997 to 2007, 19 papers were selected and reviewed. study duration ranged from 1 to 8 years and, as usual, dental variables were collected at baseline in all the papers. other variables that also were collected in a considerable number of studies were: socioeconomic, microbiological and behavioral characteristics. among the studies that used regression techniques (n=16) in statistical analysis the predominant rp was past caries experience followed by others related to socioeconomic level. the main rfs were the variables related to oral hygiene. as previously reported, past caries experience detected at baseline is the variable that best indicates those at risk for developing new lesions and poor oral hygiene increases the probability of caries increment. seven studies involving caries risk assessment in adolescents (table 4) were found from 1997 to 2007. the researchers followed-up the participants from 1 to 10 years, and collected data on dental, behavioral, demographic, socioeconomic, anthropometric, medical and microbiological variables and dietary habits. caries experience at baseline was the main rp obtained in regression techniques. in general, review of the papers demonstrated that past dental caries was the risk predictor of the future disease for all age groups. others important rp include socioeconomic level and fluoride usage. the risk factors obtained in regression analyses were variables related to oral hygiene, sugar consumption and microbiological counts. collecting data on dental caries is very easy and may help in caries risk assessment. however, the disease has to be present. others risk factors such as sugar consumption and oral hygiene related characteristics are also not difficult to gather information on them from a community point of view and help dental professionals in selecting those cariesfree individuals at risk. in fact, it has been suggested that in caries risk assessment, variables such as caries experience and severity, plaque index, fluoride use, socioeconomic level status should be collected before the application of the test for e.g. mutans streptococcus15. as reported by kopycka-kedzierawski and billings16, “a caries risk assessment protocol must involve the use of measures that are easily obtained, widely accepted, simple to use, reproducible and cost-effective”. it is important to take into consideration that this study presents some limitations such as the absence of quality criteria for selecting the papers (no score for papers), and the selection of studies mainly from medline database. in spite of its limitations, by reviewing the published papers over the last 10 years, this study could clearly demonstrate that past caries experience has been the predominant predictor for future caries in subjects from 0 to 18 years of age. therefore, those with previous contact with the disease should receive good oral health education, preventive measures and should be made aware that they are subjects at risk for developing caries. continuous monitored is necessary to prevent the onset of new lesions. on the other hand, as discussed by tinanoff17, it would be unwise to wait for the presence of caries to know which subject will be more susceptible to develop lesions in the future. further studies involving a large number of cariesfree individuals should be conducted on caries risk assessment. nevertheless, the use of other variables such as dietary habits, including sugar consumption, and toothbrushing habits or presence of dental plaque may help identifying those caries-free subjects who might be more prone to have new carious lesions in the future. in 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caries progression in 12to 16year-old schoolchildren in fluoridated and fluoride-deficient areas in brazil. community dent oral epidemiol. 1997; 25: 402-11. 48. utriainen p, pahkala k, kentala j, laippala p, mattila k. changes in the oral health of adolescents treated by the finnish public dental services between the ages of 13 and 15 years. community dent oral epidemiol. 1998; 26: 149-54. 49. meldrum am, thomson wm, drummond bk, sears mr. is asthma a risk factor for dental caries? findings from a cohort study. caries res. 2001; 35: 235-9. 50. stenlund h, mejàre i, källestål c. caries rates related to approximal caries at ages 11-13: a 10-year follow-up study in sweden. j dent res. 2002; 81: 455-8. 51. campain ac, morgan mv, evans rw, ugoni a, adams gg, conn ja et al. sugar-starch combinations in food and the relationship to dental caries in low-risk adolescents. eur j oral sci. 2003; 111: 316-25. 52. poorterman jhg, aartman iha, kieft ja, kalsbeek h. approximal caries increment: a three-year longitudinal radiographic study. int dent j. 2003; 53: 269-74. braz j oral sci. 7(27):1682-1690 an overview of caries risk assessment in 0-18 year-olds over the last ten years (1997-2007) 1http://dx.doi.org/10.20396/bjos.v18i0.8657331 volume 18 2019 e191667 original article 1 department of prosthodontics, são leopoldo mandic and dental research center, campinas, brazil. 2 department of prosthodontics and periodontology, piracicaba dental school, university of campinas, piracicaba, brazil. corresponding author: dr marcelo ferraz mesquita department of prosthodontics and periodontology piracicaba dental school, university of campinas (unicamp) av limeira, 901, piracicaba, são paulo, 13414-903, brazil. e-mail: mesquita@unicamp.br received: may 21, 2019 accepted: october 23, 2019 biomechanical behavior of overdentures supported by different implant position and angulation using micro era® system: a finite element analysis study felipe franco ferreira1, guilherme almeida borges2, letícia del rio silva2, daniele valente velôso2, thaís barbin2, marcelo ferraz mesquita2,* aim: the aim of this study was to investigate the biomechanical behavior of implant-retained mandibular overdentures using micro era® system with different implant position and angulation by finite element analysis (fea). methods: four 3d finite element models of simplified mandibular overdentures were constructed, using one bränemark implant with a micro era® attachment. the implant was positioned on the canine or lateral incisor area with an angulation of either 0º (c-0º; li-0º) or 17º (c-17º, li-17º) to the vertical axis. a 100 n axial load was applied in one side simultaneously, from first premolar to second molar. in all models it was analyzed the overdenture displacement, compressive/tensile stress in the bone-implant interface, and also the von mises equivalent stress for the nylon component of the housing. the stresses were obtained (numerically and color-coded) for further comparison among all the groups. results: the displacement on the overdenture was higher at the posterior surface for all groups, especially in the c-17º group. when comparing the compressive/tensile stress in the boneimplant interface, the lateral-incisor groups (li-0º and li-17º) had the highest compressive and lowest tensile stress compared to the canine groups (c-0º and c-17º). the von mises stress on the nylon component generated higher stress value for the li-0º among all groups. conclusions: the inclination and positioning of the implant in mandibular overdenture interferes directly in the stress distribution. the results showed that angulated implants had the highest displacement. while the implants placed in the lateral incisor position presented lower compressive and higher tensile stress respectively. for the attachment the canine groups had the lowest stress. keywords: dental implants. denture, overlay. finite element analysis. 2 ferreira et al. introduction the high predictability and survival rate of dental implants made the rehabilitation of fully edentulous patients a possible treatment1. the use of an implant-supported mandibular overdenture (ismo) has been regarded as effective and the standard option of care for edentulous patients2-4. this method also provides higher positive impact in oral health related quality of life, satisfaction, comfort, and masticatory function in elderly patients when compared with conventional dentures5-7. those clinical findings supported the mcgill and york consensus statement on ismo that two-implant is the first and minimum treatment choice for the edentulous mandible8,9. besides that, the ismo retained by two implants is also in agreement with the requirements of schmitt and zarb for patients’ treatment that must be less invasive, complex, and expensive10. the majority of clinical and biomechanical studies choose the interforaminal region as the location of choice for the two implants placement5,11. however, if the patient presents insufficient alveolar bone at the canine region, switching implants to the lateral incisor remains a treatment option. at present moment there is insufficient scientific evidence available regarding the preferable locations for the implants. a previous study reported the lowest stress for implants inserted in the lateral incisor area compared to the other two groups located in the canine and premolar sites12. contrarily, other research found the lower stress levels with implants at the first premolar site compared with lateral incisor and canine sites9. the ideal implant placement in ismo should be as parallel as possible to one another and perpendicular to the occlusal plane13. nevertheless, the surgical procedure is limited by the anatomical structure, bone morphology and clinical practice, which tend to change the implant inclination toward the ideal position14. biomechanical studies have suggested that the lowest stress and the best stability of ismo were obtained when implants were placed parallel to the long axes of the teeth12,15. a previous study has demonstrated that individual implants angulations with a lingual inclination (≥6º) and a buccal inclination (<6.5º) were associated with more prosthesis repairs, in addition to a higher tendency for implants to demonstrate greater inclination when placed by less experienced surgeons16. according to the anchorage system, ismo is generally classified into splinted (bar) or unsplinted (stud) attachments. the retentive forces of paired stud attachments, as ball (range: 34.6–2.39 n), locator (range: 37.2–5.2 n), and era (range: 35.24–8.4 n) have been determined with different values for axial and non-axial directions in dislodging studies17,18. however, previous clinical trials have noted no considerable differences between bar and stud attachments for patients’ maximum bite forces, chewing efficiency, and satisfaction19,20. in a clinical application, the most relevant aspect is to understand the advantages and limitations of the attachment system to enhance the patient’s quality of life and success of the treatment. biomechanical behavior analysis of implant-supported prostheses can be made by strain gauges, photoelastic analysis, or finite element analysis (fea)21-23. the choice of fea allows the investigation not only the stress distribution for the ductile (implant 3 ferreira et al. and prosthetic components), but also nonductile (cancellous and cortical bone) materials. thus, according to its advantage in generating computational models, fea has been used outside of the clinical scenario to compare the biomechanical behavior in different ismo. therefore, the purpose of this in silico study was to evaluate the biomechanical behavior of implant-retained overdentures using micro era® system with different implant position (canine and lateral incisor) and inclination (0º and 17º to the vertical axis). the null hypothesis was that the different implant position and angulation would not affect the biomechanical behavior of implant retained mandibular overdentures using micro era® system. materials and methods four 3-dimensional (3d) finite element models of a simplified edentulous mandible were constructed to simulate an implant-retained overdenture with micro era® attachment. an external hexagon screw-shaped implant (3.75 × 11.5 mm, conexão sistemas de prótese, arujá, brasil) was placed with two different locations (canine or lateral incisor) and angulations (0º or 17º to the vertical axis) (fig 1). in addition, the direction of the inclination for the groups c-17º, li-17º was the posterior region. for the retention system, two micro era® attachments (ridgefield park, nj, usa; dental milestones guaranteed) were used, according to the implant angulation (fig 2a,b). c-0° c-17° li-0° li-17° mucosa cortical bone cancellous bone figure 1. virtual models. c-0º, implant positioned in the canine region 0º to the vertical axis. c-17º, implant positioned in the canine region 17º to the vertical axis. li-0º, implant positioned in the lateral incisor region 0º to the vertical axis. li-17º, implant positioned in the lateral incisor region 17º to the vertical axis. 4 ferreira et al. in the pre-processing phase for models construction, the implants and prosthetic components (attachment, housing, and overdenture) were created into the rhinoceros® 5.0 software (robert mcneel & associates, usa). the virtual mandible was made with bone quality type ii, according to the lekholm and zarb classification24, surrounded by 2 mm of cortical bone22, with 1 mm thick mucosa25. for this study it was modeled only half part of the jaw structure, because it was assumed that both mandibular sides would present the same biomechanical behavior25-29. the implant threads geometry were simplified for further computational analysis30. based on the original size of the micro era® (dmg dental milestones guaranteed), a reverse engineering technique was used by spark-erosion of thread (actspark® model xenon25, beijing agie charmiles industrial eletronics ltda) and a profile projector (mitutoyo® model pj 300h, mitutoyo sul americana ltda) to achieve the precise attachment dimension. after computer-aided design (cad) modeling, the structures were assembled to provide the 3d models. afterwards, hypermesh® software was used to promote the division of the structures into a geometric mesh with a finite number of elements. in addition, the geometric mesh was formed with parabolic tetrahedral interpolation solid elements, characterized by 10 nodes per element. as a result, the total (elements nodes) in each model was c-0º (486 794 – 793 872), c-17º (448 529 – 737 231), li-0º (1 865 301– 2 616 051), li-17º (487 159 – 753 906). the meshed virtual models were exported to the finite element analysis software optstruct® for mathematical solution. the bone tissues were considered isotropic, linear, homogeneous, and totally osseointegrated to the implants26. in addition, to correctly calculate the results for all the study variables, it was add boundary condition at the posterior region of the jaw for each of the four models (c-0º, c-17º, li-0º, li-17º) into three dimensions (x, y, z)22,26. the properties of each material (young modulus and poisson’s ratio) are presented in table 1. subsequently, the structures contact were a b figure 2. a, micro era® attachment – 0º to the vertical axis. b, micro era® attachment – 17º to the vertical axis. 5 ferreira et al. considered fixed, representing a perfectly united interaction, except between the housing (nylon)/attachment, and also the overdenture/mucosa in which a sliding contact is possible22. in order to simulate what happens in the clinical scenario, a 100 n occlusal load was divided in 4 application points simultaneously, from the second premolar to second molar31. a mirror condition was applied into the midline section of the model, assuming that both sides would show the same biomechanical behavior22. in all models it was analyzed the overdenture displacement, compressive/tensile stress in the bone-implant interface, and also the von mises equivalent stress for the nylon component of the housing32. the stresses were obtained (numerically and color-coded) for further comparison among all the groups. finally, the models were sent to hyperview® software to investigate the stress distribution. results under the axial load on the mandibular premolar and molars, the highest displacement was observed for both angulated groups (c-17º and li-17º) (fig 3a). the c-17º group exhibited the highest displacement among all the other groups. when comparing the compressive/tensile stress in the bone-implant interface the lateral-incisor groups (li-0º and li-17º) demonstrated the highest compressive stress (fig 3b), while the canine groups (c-0º and c-17º) presented the lowest tensile stress (fig 3c). the von mises stress on the nylon component for the group li-0º generated higher stress value among all groups (fig 3d). in fig 4, the same pattern of stress maps was observed for all models, but with different intensity. the stress maps indicated the highest displacement stress for c-17º at the posterior region of the overdenture (fig 4b). regarding the compressive/tensile stress in the bone/implant interface the groups with canine implants (c-0º and c-17º) presented the highest stress on the neck region, running through its first threads (fig 5a and 5b). the group li-0º (fig 5c) presented stress located only in the distal region. the group li-17º (fig 5d) showed similar distribution with c-0º and c-17º groups. the micro era® nylon component exhibited similar stress in the canine groups c-0º (fig 6a) and c-17º (fig 6b). the stresses were concentrated in the seating interface table 1. mechanical properties of materials used for fea analysis. material young modulus (mpa) poisson’s ratio (v) reference cortical bone 13 700 0,30 liu, 201330 cancellous bone 1370 0,30 liu, 201330 mucosa 1 0,37 liu, 201330 titanium (grade iv) 103 400 0,35 barão, 200826 nylon 2400 0,39 barão, 200826 stainless steel 190 000 0,31 barão, 200826 acrylic resin 8300 0,28 barão, 200826 6 ferreira et al. d is pl ac em en t s tr es s (m p a) c-0° c-17° li-0° li-17° 0 475 950 1425 1900 1372 1899 1487 1641 c om pr es si ve s tr es s (m p a) c-0° c-17° li-0° li-17° -4000 -3000 -2000 -1000 0 -1995 -1498 -3649 -2214 t en si le s tr es s (m p a) c-0° c-17° li-0° li-17° 0 1750 3500 5250 7000 4519 3572 6279 4870 m ax im um v on m is es s tr es s (m p a) c-0° c-17° li-0° li-17° 0 125 250 375 500 358 349 497 403 a b c d figure 3. a, displacement (mpa) combined in the overdenture and jaw. b, compressive strength in the bone/implant (mpa). c, tensile strength in the bone/implant (mpa). d, maximum von mises stress on micro era® nylon. c-0º, implant positioned in the canine region 0º to the vertical axis. c-17º, implant positioned in the canine region 17º to the vertical axis. li-0º, implant positioned in the lateral incisor region 0º to the vertical axis. li-17º, implant positioned in the lateral incisor region 17º to the vertical axis. 1.641e-01 1.200e-01 1.050e-01 9.000e-02 7.500e-02 6.000e-02 4.500e-02 3.000e-02 1.500e-02 0.000e+00 1.899e-01 1.200e-01 1.050e-01 9.000e-02 7.500e-02 6.000e-02 4.500e-02 3.000e-02 1.500e-02 0.000e+00 1.372e-01 1.200e-01 1.050e-01 9.000e-02 7.500e-02 6.000e-02 4.500e-02 3.000e-02 1.500e-02 0.000e+00 1.487e-01 1.200e-01 1.050e-01 9.000e-02 7.500e-02 6.000e-02 4.500e-02 3.000e-02 1.500e-02 0.000e+00 a b c d figure 4. stress maps (displacement) combined in the overdenture and jaw. a, group c-0º. b, group c-17º. c, group li-0º. d, group li-17º. color stress scale in mpa. c-0º, implant positioned in the canine region 0º to the vertical axis. c-17º, implant positioned in the canine region 17º to the vertical axis. li-0º, implant positioned in the lateral incisor region 0º to the vertical axis. li-17º, implant positioned in the lateral incisor region 17º to the vertical axis. 7 ferreira et al. 4.519e+00 3.700e+00 2.643e+00 1.586e+00 5.286e+00 -5.286e+00 -1.586e+00 -2.643e+00 -3.700e-00 -4.757e+00 a b c d tensile stress max compressive stress max 4.757e+00 3.700e+00 2.643e+00 1.586e+00 5.286e+00 -5.286e+00 -1.586e+00 -2.643e+00 -3.700e-00 -4.757e+00 tensile stress max compressive stress max 6.279e+00 3.700e+00 2.643e+00 1.586e+00 5.286e+00 -5.286e+00 -1.586e+00 -2.643e+00 -3.700e-00 -4.757e+00 tensile stress max compressive stress max 4.870e+00 3.700e+00 2.643e+00 1.586e+00 5.286e+00 -5.286e+00 -1.586e+00 -2.643e+00 -3.700e-00 -4.757e+00 tensile stress max compressive stress max figure 5. stress maps (compressive/tensile stress) on implant/bone interface. a, group c-0º. b, group c-17º. c, group li-0º. d, group li-17º. color stress scale in mpa. c-0º, implant positioned in the canine region 0º to the vertical axis. c-17º, implant positioned in the canine region 17º to the vertical axis. li-0º, implant positioned in the lateral incisor region 0º to the vertical axis. li-17º, implant positioned in the lateral incisor region 17º to the vertical axis. figure 6. stress maps (von mises stress) on micro era® nylon. a, group c-0º. b, group c-17º. c, group li-0º. d, group li-17º. color stress scale in mpa. c-0º, implant positioned in the canine region 0º to the vertical axis. c-17º, implant positioned in the canine region 17º to the vertical axis. li-0º, implant positioned in the lateral incisor region 0º to the vertical axis. li-17º, implant positioned in the lateral incisor region 17º to the vertical axis. 3.580e+01 3.400e+01 2.916e+01 2.431e+01 1.947e+00 1.463e+00 9.786e+00 4.943e+00 1.000e-01 -4.743e+00 a b c d 3.497e+01 3.400e+01 2.916e+01 2.431e+01 1.947e+00 1.463e+00 9.786e+00 4.943e+00 1.000e-01 -4.743e+00 4.570e+01 3.400e+01 2.916e+01 2.431e+01 1.947e+00 1.463e+00 9.786e+00 4.943e+00 1.000e-01 -2.171e+02 4.037e+01 3.400e+01 2.916e+01 2.431e+01 1.947e+00 1.463e+00 9.786e+00 4.943e+00 1.000e-01 -4.743e+00 8 ferreira et al. between the nylon/attachment. as observed for the li-0º (fig 6c) and li-17º group (fig 6d), the stress was located in the same position, but the group li-0º also concentrated stress on the superior portion of the housing and were more pronounced than the li-17º group. discussion this study found that inclined implants caused an increase in overdenture displacement for canine and lateral incisor regions, rejecting the null hypothesis, which different implant position and angulation would not affect the biomechanical behavior of implant retained mandibular overdentures using micro era® system. this may imply that changes in implant inclination from 0º to 17º can compromise the overdenture retention. similarly, previous studies have observed a reduction in the retentive force for the attachment as the implant inclination increases11,15,33. however, besides the inclination the number of implants can also take place in creating a more stable system with reduced displacement, and denture rotation around the fulcrum line34. under the clinical scenario, denture base rotation has a negative effect on masticatory ability and can influence patient satisfaction35. thus, an experienced surgeon should be aware of choosing the most parallel implant position and favorable biomechanical scenario as possible. the non-angulated implant in the lateral incisor position presented lower compressive stress values for peri-implant bone tissue. thus, the peri-implant stress caused by implant location can influence the bone response and should be evaluated when planning patient treatment12. similarly, previous studies stated that the optimum location for implant placement is the mandibular lateral incisor area on both sides with implants placed parallel to the axes of the missing teeth axis12,36. regarding the tensile stress, which will assume the most likely region to suffer resorption, the higher values was presented for the li-0º group, in which the stress was located in the cortical layers around the implant first treads (fig 4c)22,37. the results suggest that overdentures with lateral incisor implants is the worst design in terms of biomechanical environment for the bone, moreover, it may favor much greater stress registered into the attachment component38. for fea, when two bodies with different young modulus (cortical bone and titanium implant) come into contact, the highest stress is presented at the beginning of the contact surface9,39. this finding indicates that the loading applied to the implant is also transmitted to the cortical bone, which explains the clinical marginal bone loss found around the implants in other studies39,40. the models with implants in the lateral incisor position presented the highest von mises stress in the micro era® nylon. moreover, it was noted that the non-inclinated implants in the lateral incisor position presented a stress concentration in top of the housing, suggesting an implant intrusion. this finding shows that the location mentioned may be the one with greatest nylon wear, promoting retention loss and a higher maintenance costs. equally, a fea study38 compared the stress distribution in mandibular two-implant overdentures according to implant locations (lateral incisors and canines), and observed the worst biomechanical environment for the lateral incisor position. in addition to be the worst model for the attachment components38. for the 9 ferreira et al. different angulations, a previous study found that implant/attachments perpendicular to the occlusal plane were appropriately retentive in the first year and the retentive capacity of the nylon component was affected by implant inclinations41. however, in this study the angulated implant groups presented the lowest stress compared to the parallel implants and, according to this, would take more time to lose retention. this can be explained by the angled attachment to compensate inclined implants, favoring biomechanically the system. finite element analysis is a useful method in dentistry to estimate the stress distribution in the peri-implant bone, prostheses, and prosthetic components in different scenarios. however, in a clinical situation it would not be possible to control bone density, soft tissue resilience, implant inclination and osseointegration12,22. therefore, it has to be assumed simplifications related to material properties and geometry which sometimes limit the data to be extrapolated into the clinical scenario26,30. in addition, the absence of bilateral loading is a limitation of this study, since the loading orientation can change the tension patterns. despite the fact that mechanical load and stress distribution are directly related to the implant longevity, further studies and clinical trials should be performed to better understand the difference implant location, validate the results of this fea study and provide guidance for clinicians. from this in silico study it can be concluded, despite the fact 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oral maxillofac surg. 2012 sep;41(9):1090-6. doi: 10.1016/j.ijom.2011.12.026. 40. arat bilhan s, baykasoglu c, bilhan h, kutay o, mugan a. effect of attachment types and number of implants supporting mandibular overdentures on stress distribution: a computed tomography-based 3d finite element analysis. j biomech. 2015 jan 2;48(1):130-7. doi: 10.1016/j.jbiomech.2014.10.022. 41. rodrigues rc, faria ac, macedo ap, sartori ia, de mattos mda g, ribeiro rf. an in vitro study of non-axial forces upon the retention of an o-ring attachment. clin oral implants res. 2009 dec;20(12):1314-9. doi: 10.1111/j.1600-0501.2009.01742.x. 1http://dx.doi.org/10.20396/bjos.v16i0.8650493 volume 16, number 3 2017 e17033 original articlebjos 1 dds, mds student, department of orthodontics, graduate program in orthodontics, university of araras uniararas, araras, sp, brazil. 2 dds, mds, phd, associated researcher, department of restorative dentistry, dental materials division, piracicaba dental school, university of campinas – unicamp, piracicaba, sp, brazil. 3 dds, mds, phd, professor, department of restorative dentistry, dental materials division, piracicaba dental school, university of campinas – unicamp, piracicaba, sp, brazil. 4 dds, mds, phd, professor, institute of higher education, faipe, cuiaba, mt, brazil. 5 dds, mds, phd, professor, department of restorative dentistry, dental materials division, piracicaba dental school, university of campinas – unicamp, piracicaba, sp, brazil. 6 dds, mds, phd, professor, department of orthodontics, graduate program in orthodontics, university of araras uniararas, araras, sp, brazil. influence of adhesive and bonding material on the bond strength of bracket to bovine tooth bruna lorena dos santos oliveira1*, ana rosa costa2, américo bortolazzo correr3, marcus vinicius crepaldi4, lourenço correr-sobrinho5, julio cesar bento dos santos6 *corresponding author: department of orthodontics, graduate program in orthodontics, university of araras uniararas. av. dr. maximiliano baruto, 500 jd. universitário, araras, sp, brasil, cep: 13607-339, +55 19 3543-1423. e-mail: ortodontiavedovello@gmail.com received: april 13, 2017 accepted: august 14, 2017 aim: to evaluate, in vitro, the effect of adhesive and bonding materials on the shear bond strength (sbs) of metallic brackets bonded to bovine teeth. methods: bovine teeth were embedded with acrylic resin, etched with 35% phosphoric acid for 20 s, rinsed, dried for 20 s and divided into 8 groups (n=20), according to the bonding materials and adhesive: groups 1 and 5 – transbond xt; groups 2 and 6 – fill magic; groups 3 and 7 – biofix; and, groups 4 and 8 – orthocem. one layer of adhesive 3 m unitek (group 1), and one layer of single bond universal adhesive (groups 2, 3 and 4) were applied and light-cured with led for 10 s. brackets were bonded to the buccal surface with four bonding materials and light-cured with led for 40 s. sbs was carried out after 24 h and thermocycling (7,000 cycles 5°/55 °c). data were submitted to two way anova and tukey’s post hoc test (α=0.05). the adhesive remaining index (ari) was evaluated at 8×. results: the adhesive was effective in increasing the sbs for all bonding materials (p<0.05). significant difference (p<0.05) on the sbs was observed between bonding materials with or without adhesive. the ari showed a predominance of scores 0 for all groups. conclusions: the adhesive improved significantly the sbs of the brackets to teeth. different sbs values occurred among the bonding materials. ari index showed predominance of score 0 for all groups. keywords: shear strength. orthodontic brackets. adhesives. 2 oliveira et al. introduction the bracket bonding in teeth for mounting orthodontic appliances was one of the most significant advances in orthodontics. the bonding system has been based on the acid-etching technique1. the acid etching allowed improvement in the bond between the bracket and the tooth, enabling the perfectioning of direct fixation techniques of orthodontic appliances to teeth, with the advantage of technical simplification, reduced chair time, more comfort to the patient, better appearance, and less aggression to periodontal tissues2. after the acid etching of enamel, bonding systems have been used to promote increased durability and longevity for orthodontic brackets. the bonding procedure is based on clinical adhesive steps, as etch-and-rinse, self-etching, and self-adhesive3,4. conventional adhesive associates pretreatment of the tooth with an etched-and-rinse or self-etching adhesive. the adhesive penetrates the etched enamel, in order to increase the bond strength between the tooth and the composite resin5. the light-activated composite resins are used to attach orthodontic brackets to the teeth6. the composite resins are densely loaded with reinforcing filler particles for strength and wear resistance7. the advantage of light-activated composite resins is that the clinician has time to place the brackets in the position, may remove excess material before the light activation8, and support adequately the loads applied during orthodontic treatment resulting from chewing9. however, failures with composite resins can occur and has been attribute to humidity contamination10 or incomplete polymerization when considering the light exposure time or limited depth polymerization11, which changes with the light penetration into the material12. recently, some composites resins have been applied for orthodontic use by many clinicians immediately after acid etching of enamel without adhesive. according to the manufacturers’ instructions, the number of steps during bonding is reduced, decreased chair time for patients and less contamination during bonding procedures. however, a study showed that the adhesive promoted a significant effect on the sbs13. however, the literature is still not conclusive regarding the use of adhesive and bonding materials on the bond strength between orthodontic brackets and teeth. therefore, the aim of this study was to evaluate the effects of adhesive and bonding materials on the sbs of metallic brackets bonded to bovine teeth. the hypotheses tested were as follows: 1) the adhesive application would not influence the sbs; 2) no significant difference would occur between the bonding materials. material and methods preparation of the specimens and light-curing procedures bovine mandibular incisors teeth were embedded in rigid polyvinyl chloride tubes (tigre, joinvile, sc, brazil) with autopolymerizing acrylic resin (classico dental products, sao paulo, sp, brazil). the buccal surface of the teeth were parallel to the tube height, with the cementoenamel junction located approximately 3 mm above the 3 oliveira et al. acrylic resin. the buccal surface of the teeth was cleaned with pumice-water slurry (s.s. white, petropolis, rj, brazil) using a rubber cup (kg sorensen, cotia, sp, brazil) for 20 s, rinsed with air-water spray for 20 s and dried with air for 20 s. the rubber cup was replaced after each five teeth. the middle third of the buccal surface of the one hundred sixty bovine incisors were etched using 35% phosphoric acid gel (ultrandent, south jordan, ut, usa) for 20 s, rinsed with air-water spray for 20 s and dried with air for 20 s. after, the teeth were divided into 8 groups (n=20) according to bonding materials and adhesive: groups 1 and 5 – transbond xt (3m unitek, morovia, califórnia – usa); groups 2 and 6 – fill magic (vigodent, rio de janeiro, rj, br); groups 3 and 7 – biofix (biodinamics, ibiporapr, br); and, groups 4 and 8 – orthocem (fgm orthodontics products, joinville, sc,br). one layer of a light cured adhesive primer (3m unitek, monrovia, ca, usa – group 1) and one layer of single bond universal adhesive (3m espe, st. paul, mn, usa – groups 2, 3 and 4) were applied on the etched area of buccal surface of the teeth and exposed a blue led (radii-cal, sdi limited, bayswater, victoria, australian) for 20s with an irradiance of 1,200 mw/cm2 measured by curing radiometer model 100 (demetron research corporation, danbury, ct). stainless steel standard premolar brackets (abzil, 3m do brazil, sao jose do rio preto, sp, brazil) were positioned and firmly bonded of each tooth with transbond xt (3m unitek), fill magic (vigodent), biofix (biodinamica) or orthocem (fgm) light-cured bonding materials, following the manufacturers’ instructions. excess bonding resin was removed with microbrush. light-activation was carried out with four exposures on each side of the bracket with total exposure time of 40 s, using a blue led (radii-cal). storage and bonding testing after the bonding procedures, all samples were stored in deionized water at 37 °c for 24 h. twenty brackets were bonded in each group, totalizing 160 bonded brackets. after this period, all samples were subjected to a thermal cycles regimen in a thermal cycling (msct 3; marnucci me, sao carlos, sp, brazil) with deionized water between 5 °c and 55 °c (dwell time of 30 s) and transfer time of 10 s between baths. shear bond strength testing after thermal cycling, a mounting jig was used to align the tooth-bracket interface parallel to the testing device and sbs was performed in a universal mechanical testing machine (model 4411; instron, canton, ma, usa) with a knife-edged rod at a crosshead speed of 1.0 mm/min until failure. the sbs data were calculated in mpa and submitted to two-way anova and tukey’s post hoc test (α=0.05). failure analysis after debonding, the bracket and tooth surfaces were observed under optical microscopy (olympus corp, tokyo, japan) at 8× magnification. the adhesive remaining index (ari) was used to classify the failure modes14 as follows: score 0: indicated that no bonding resin remained on the tooth; score 1: indicated that less than half of the bonding resin remained on the tooth; score 2: indicated that more than half the 4 oliveira et al. bonding resin remained on the tooth; and score 3: indicated that all bonding resin remained on the tooth, with a clear impression of the bracket mesh. results table 1 shows the mean values of sbs. significant differences between the bonding materials (p<0.00001) and adhesive (p<0.00001) were detected. the interaction between bonding materials and adhesive factors (p<0.0003) were also significant. the use of adhesive had a significant effect on sbs for all bonding materials. the transbond xt showed sbs statistically higher than the other bonding materials (p<0.05), and the orthocem had the lowest sbs values (p<0.05) when adhesive was used. for the group without adhesive, the transbond xt showed sbs statistically higher than fill magic, biofix and orthocem (p<0.05). no statistically difference was found among fill magic, biofix and orthocem (p>0.05) figure 1 shows the results for ari. a predominance of score 0 was observed for all groups. table 1. mean shear bond strength values (sd) in mpa. bonding material adhesive with without transbond xt 10.3 (1.2) a,a 6.9 (1.3) a,b fill magic 9.0 (0.8) b,a 5.0 (1.0) b,b biofix 8.3 (1.4) bc,a 4.3 (0.7) b,b orthocem 7.8 (1.3) c,a 4.2 (0.8) b,b different capital letters in each row indicate significant difference for adhesive, and different small letters in each column indicate significant difference for bonding materials (p<0.05). figure 1. ari scores after debonding of brackets. fr eq ue nc y di st rib ut io n a r i ( % ) with adhesive without adhesive 100% 80% 60% 40% 20% 0% trans b. fill magic biofix orthocem trans b. fill magic biofix orthocem 3 2 1 0 5 oliveira et al. discussion the first hypothesis, which stated that the adhesive application do not influence the sbs was rejected. the results showed that significant differences were shown between the groups with adhesive and without adhesive. these results are in line with previous study, which also found significant differences between the groups with and without adhesives13. however, a recent study showed that brackets fixed without adhesive presented a debonding rate after 12 months similar to that of brackets fixed with adhesive15,16. other study showed that the penetration of the liquids into narrow capillaries, such as microporosities of etched enamel was influenced by properties of the liquid, such as viscosity and the surface free energy of the capillary wall17. besides, viscosity of composite resin is a parameter that can influence the penetration of restorative resins into enamel conditioned18. in the current study, the data showed significant differences between the bonding materials (p<0.0001). then, the second hypothesis was rejected. the transbond xt produced significantly higher sbs than another bonding material for both conditions with or without application of the adhesive. these findings are in agreement with previous studies, which also found significant differences between these bonding materials, when brackets were bonded to enamel surface13,19,20. however, other study showed that when the adhesive was not applied, sbs of bonding materials did not differ among themselves, except for transbond supreme lv that showed sbs values significantly higher than other bonding materials13. although transbond xt has a high filler concentration (77 wt%), it showed sbs values of 6.9 mpa adequate for clinical use. previous studies showed that the filler concentration may clinically influence the viscosity of bonding materials21, but the higher viscosity can not limit the free flow of the resin into the enamel pores and the formation of resin tags22. other studies showed that flowable resins can flow easily into an etched enamel structure adequately and onto a bracket base without need of an intermediate bonding resin, thus an enhance in the level of mechanical properties are expected13. depth of resin penetration into enamel decreases slightly with increasing viscosity23. others studies showed that, when applied to acid-enamel, there are no significant differences between low-viscosity and high-viscosity resins in terms of adaptation and depth of penetration24. bond strength values between 6 to 8 mpa are adequate for orthodontic applications in oral environment25. in this study, sbs values lower than 6 mpa were obtained for fill magic, biofix and orthocem bonding materials where adhesive was not applied. although the manufacturers of fill magic, biofix and orthocem bonding resins do not recommend the use of adhesives, the results of this study showed that the adhesive is necessary to obtain adequate sbs values. thus, care should be taken by clinicians when adhesive is not used especially when the fill magic, biofix or orthocem are used because these bonding resins have not been acceptable clinically sbs to resist forces during orthodontic treatment. the ari values indicated a predominance of failures with score 0 when no bonding resin on the ceramic surface was observed. this may be clinically advantageous because there is less bonding resin to remove from the bovine teeth after bracket debonding26. it may be concluded that the sbs values for bonding materials were influenced by the adhesives application. different sbs values occurred among the bonding materials. the ira index showed predominance of score 0 for all groups. 6 oliveira et al. references 1. buonocore mg. a simple method of increasing the adhesion of acrylic filling materials to enamel surfaces. j dent res. 1955 dec;34(6):849-53. 2. busato mca, busato pmr, dotto dv, pedrotti s, gasparello cr. evaluation of shear bond strength of backets bonded with different orthodontic resins. rev clin orthodon dental press. 2013 mar-apr;12(2):94-9. 3. silami fd, tonani r, alandia-roman cc, pires-de-souza fde c. influence of different types of resin luting agents on color stability of ceramic laminate veneers subjected to accelerated artificial aging. braz dent j. 2016 jan-feb;27(1):95-100. doi: 10.1590/0103-6440201600348. 4. lenzi tl, soares fz, raggio dp, pereira gk, rocha ro. dry-bonding etch-and-rinse strategy improves bond longevity of a universal adhesive to sound and artificially-induced caries-affected primary dentin. j adhes dent. 2016 nov-dec;18(6):475-82. doi: 10.3290/j.jad.a36670. 5. antoniazzi bf, nicoloso gf, lenzi tl, soares fz, rocha ro. selective acid etching improves the bond strength of universal adhesive to sound and demineralized enamel of primary teeth. j adhes dent. 2016 jul-aug;18(4):311-6. doi: 10.3290/j.jad.a36154. 6. bengassem aa, georgiou g, jones sp. initial and fatigue bond strengths of nanofilled and conventional composite bonding adhesives. j orthod. 2013 jun;40(2):137-44. doi: 10.1179/1465313312y.0000000034. 7. wang rl, bao s, liu fw, jiang xz, zhang qh, sun b, et al. wear behavior of light-cured resin composites with bimodal silica nanostructures as fillers. mater sci eng c mater biol appl. 2013 dec;33(8):4759-66. doi: 10.1016/j.msec.2013.07.039. 8. costa ar, correr ab, puppin-rontani rm, vedovello sa, valdrighi hc, correr-sobrinho l, et al. effects of thermocycling and light source on the bond strength of metallic brackets to bovine teeth. braz dent j. 2011 nov-dec;22(6):486-9. doi: http://dx.doi.org/10.1590/s0103-64402011000600008. 9. romano fl, correr ab, correr-sobrinho l, magnani mb, ruellas ac. clinical evaluation of the failure rates of metallic brackets. j appl oral sci. 2012 mar-apr;20(2):228-34. doi: http://dx.doi.org/10.1590/s1678-77572012000200018. 10. roelofs t, merkens n, roelofs j, bronkhorst e, breuning h. a retrospective survey of the causes of bracketand tube-bonding failures. angle orthod. 2017 jan;87(1):111-7. doi: 10.2319/021616-136.1.  11. correr ab, sinhoreti ma, sobrinho lc, tango rn, schneider lf, consani s. effect of the increase of energy density on knoop hardness of dental composites light-cured by conventional qth, led and xenon plasma arc. braz dent j. 2005 sep-dec;16(3):218-24. doi: http://dx.doi.org/10.1590/s0103-64402005000300009. 12. dalli’magro e, sinhoreti ma, correr ab, consani rl, sicoli ea, mendoca mj, et al. effect of different modes of light modulation on the bond strength and knoop hardness of a dental composite. braz dent j. 2008 oct-dec;19(4):334-40. doi: http://dx.doi.org/10.1590/s0103-64402008000400009. 13. albaladejo a, montero j, gomez de diego r, lopez-valverde a. effect of adhesive application prior to bracket bonding with flowable composites. angle orthod. 2011 jul;81(4):716-20. doi: 10.2319/062310-344.1. 14. artun j, bergland s. clinical trials with crystal growth conditioning as an alternative to acid-etch enamel pretreatment. am j orthod. 1984 apr;85(4):333-40. 15. nandhra ss, littlewood sj, houghton n, luther f, prabhu j, munyombwe t, et al. do we need primer for orthodontic bonding? a randomized controlled trial. eur j orthod. 2015 apr;37(2):147-55. doi: 10.1093/ejo/cju024. 16. altmann as, degrazia fw, celeste rk, leitune vc, samuel sm, collares fm. orthodontic bracket bonding without previous adhesive priming: a meta-regression analysis. angle orthod. 2016 may;86(3):391-8. doi: 10.2319/041615-255.1.  17. newman s. kinetics of wetting of surfaces by polymers capillary flow. j colloid and interface sci. 1968 feb;26(2):209-13. doi: 10.1016/0021-9797(68)90313-5. 7 oliveira et al. 18. asmussen e. penetration of restorative resins into acid etched enamel. i. viscosity, surface tension and contact angle of restorative resin monomers. acta odontol scand. 1977 feb;35(4):175-82. 19. correr sobrinho l, correr gm, consani s, sinhoreti ma, consani rl. [influence of post-fixation time on shear bond strength of brackets fixed with different materials]. pesqui odontol bras. 2002 jan-mar;16(1):43-9. doi: http://dx.doi.org/10.1590/s1517-74912002000100008. portuguese. 20. pepe mm, alucato as, dos santos jcb, tubel cam, valdrighi hc, correr ab. [shear bond strength to enamel after application of adhesive on the mesh of brackets]. rev ortod spo. 2015 sep-oct;48(5):441-5. portuguese. 21. collares fm, klein m, santos pd, portella ff, ogliari f, leitune vcb, et al. influence of radiopaque fillers on physicochemical properties of a model epoxy resin-based root canal sealer. j appl oral sci. 2013 nov-dec;21(6):533-9. doi: 10.1590/1679-775720130334. 22. mehrabkhani m, mazhari f, sadeghi s, ebrahimi m. effects of sealant, viscosity, and bonding agents on microleakage of fissure sealants: an in vitro study. eur j dent. 2015 oct-dec;9(4):558-63. doi: 10.4103/1305-7456.172631. 23. asmussen e. penetration of restorative resins into acid etched enamel. ii. dissolution of entrapped air in restorative resin monomers. acta odontol scand. 1977;35(4):183-91. 24. pahlavan a, dennison jb, charbeneau gt. penetration of restorative resins into acid-etched human enamel. j am dent assoc. 1976 dec;93(6):1170-6. 25. reynolds ir. composite filling materials as adhesives in orthodontics. br dent j 1975 feb;138(3):83. 26. costa ar, correr ab, puppin-rontani rm, vedovello sa, valdrighi hc, correr-sobrinho l, et al. effect of bonding material, etching time and silane on the bond strength of metallic orthodontic brackets to ceramic. braz dent j. 2012 jul-aug;23(3):223-7. doi: http://dx.doi.org/10.1590/s0103-64402012000300007. untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8652653 volume 17 2018 e18149 original article 1 ph.d. in periodontology. department of periodontology. university of passo fundo, brazil. br-285, são josé. 99052-900 – passo fundo – rs – brazil. 2 department of community dentistry. federal university of rio grande do sul, brazil. ramiro barcelos, 2492. 90035-003 – porto alegre – rs – brazil. 3 ph.d. in periodontology. department of periodontology. federal university of rio grande do sul, brazil. ramiro barcelos, 2492. 90035-003 – porto alegre – rs – brazil. corresponding author: francisco wilker mustafa gomes muniz rua ramiro barcelos, 2492 porto alegre/rs, brazil zip code: 90035-002 phone: +55 51 3308 5318 email: wilkermustafa@gmail.com received: december 22, 2017 accepted: february 14, 2018 tooth loss and associated factors in adolescents – impact of extractions for orthodontic reason paulo roberto grafitti colussi1, fernando neves hugo2, francisco wilker mustafa gomes muniz3, cassiano kuchenbecker rösing3 aim: to investigate tooth loss and its determinants in adolescents, considering the effect of extractions due to orthodontics reasons. methods: this cross-sectional study was performed in students from public and private schools aged 15-19 years old from passo fundo, brazil. the proportional randomly chosen sample included 736 adolescents. clinical examinations and interviews were performed. associations between prevalence of tooth loss and exposure variables studied were analysed by poisson regression with uniand multivariate robust variance in two models. one model comprised students who had experienced tooth loss without orthodontic reasons and the other with all of the subjects presenting tooth loss. results: prevalence of tooth loss was 21.1% (mean of 0.42). higher chances for tooth loss were found in the following features: non-whites (pr=1.72; ci95%:1.15-2.60), poorly schooling mothers (pr=2.2; ci95%:0.96-5.02), from public schools (pr=4.16; ci95%:0.9817.59), smokers (pr=1.91; ci95%:1.15-3.17). conclusion: demographic, socioeconomic and behavioural conditions were strongly associated with tooth loss. these associations were more evident when extractions for orthodontic reasons were not included in the analytical models. keywords: adolescence; dental health surveys; tooth number; epidemiology. 2 colussi et al. introduction tooth loss has been decreasing among brazilian adolescents. the national epidemiological surveys of 1986 and 2010 have indicated a dmft (decayed, missing, filled teeth) reduction, from 6.651 to 2.072, respectively, for the 12-year-olds. mean of tooth loss for the 15 to 19-year-old age group in the 1980’s ranged between 1.2 and 1.8 with prevalence of at least one lost tooth in almost 70% of adolescents1,3. however, in 2003, the national survey indicated a decrease in mean and prevalence of at least one tooth loss to 0.9 teeth and 38.9%, respectively4. in another study with a representative sample of the metropolitan region of porto alegre, state of rio grande do sul, mean and prevalence of at least one lost tooth were 0.5 teeth and 26%, respectively5. despite this improvement, studies showed that the dmft usually doubles from 12 year-olds to the 15 to 19-year-old age group2,6,7, as well as in studies showing high prevalence of periodontal problems in the final stages of adolescence2,8,9. in this life stage, dental caries is still the main cause of tooth loss4,5,10, considering that periodontal diseases still have low impact. adolescence is when behaviours and attitudes regarding health style usually become definitive, bringing consequences for life11. moreover, tooth loss caused by trauma and orthodontic indication is often considered the second greater cause of tooth loss in this life cycle, and it may usually be observed in studies reporting the premolar as the most lost tooth9. epidemiological studies associating extractions for orthodontic reasons, socioeconomic and cultural aspects and tooth loss are scarce in adolescents, while in other age groups such factors are strongly associated with oral health negative outcomes12. hence, it is important to discuss the adolescent social context, including school, mother’s schooling, and school delay. likewise, ethnicity/skin colour as a proxy of social inequalities has been used13. among the intra oral aspects, extractions for orthodontic reasons are also important. the high prevalence of malocclusion and orthodontic treatment need among adolescents is noteworthy14. the estimates of tooth loss comprise two situations as follows: those who have suffered from oral diseases and those related to orthodontic needs. thus, in order to avoid confusion bias, it should be noted the uniqueness of this type of approach in studies related to adolescents’ tooth loss. therefore, this study aimed to investigate tooth loss and its determinants in brazilian adolescents, considering the effect of extractions due to orthodontic treatment. material and methods study design and location this cross-sectional study examined adolescents aged from 15 to 19 years old; students enrolled in both public and private high schools in the city of passo fundo, state of rio grande do sul, brazil. in 2012, 7,558 students were enrolled in regular high school in 23 schools divided into 16 public schools (6,256 students – 82.78%) and 7 private schools (1,302 students – 17.22%). 3 colussi et al. ethical considerations the institutional review board of the university of passo fundo reviewed under protocol #016/2014 and approved the present study following the authorization by the 7th regional office of education to carry out the study in public schools as well as formal approval by the principals of the private schools. all selected students presented the informed consent signed by the parents or legally responsible person. sample the study coordinator invited all of the 23 high schools to participate and 30% of the high school students from each school that accepted to be part of the study were invited to participate. they were randomly chosen by draw from the lists of all high school students aged 15 to 19 years from each participating school, regardless of their school schedule. according to the distribution of male and female students, two blocks of randomization were performed. all selected students received the informed consent form to be signed by their responsible persons. in case of absence, a later contact was made. clinical examination and interview a structured questionnaire including demographic data, socioeconomic condition, general health behaviour, health record, and oral health self-perception was applied with the use of a group of questions from the pca tool-sb brazil adult version, validated in brazil15. moreover, other questions of interest were included regarding oral health at this age, such as orthodontic history and oral hygiene habits. all present teeth (except for third molars) were counted with the help of a wooden spatula. we used the world health organization (who) basis without looking at dental caries, just for tooth loss. analysis of dental caries, fillings etc. was not performed. the adolescents were examined and interviewed between april and july, 2012 by trained researchers. teeth that could be somehow restored were considered in the counting. teeth or roots indicated for extraction were considered absent. the training was performed in high school students who were not selected to participate in the study and 10% of the students were randomly chosen by draw and were re-examined and re-interviewed by the study coordinator to assess reproducibility. an agreement rate of 98% was observed for number of teeth between examinations. statistical analysis the dependent variable of the present study was the prevalence of tooth loss, assessed by the rate of individuals with one or more permanent lost teeth, and severity was assessed by the number of teeth lost per person. the explanatory variables for tooth loss outcome in adolescents were divided into two groups (figure 1)16. self-reported ethnicity/skin colour was classified as either white or non-white. the socioeconomic condition was assessed by a series of information on income and schooling. the level of parents’ schooling was classified in three groups – one group with complete or incomplete higher education, another one with complete or incomplete high school, and a third group gathering everyone who finished elementary 4 colussi et al. school at the most. having an unemployed person at home and having money for medical expenses were used as income proxy and dichotomized as either yes or no. housing at 12 years old was obtained with the question: “when you were 12 years old, did you live in the urban area, rural area, or state capital?” the adolescent’s occupation was classified in two groups one group in which the adolescent only studies, and another one where the adolescent studies and works at least one shift. adolescent’s school delay was defined whenever 16, 17, 18, or 19-year-old adolescents were still in the first year of high school; when 17, 18, and 19-year-old adolescents were still in the second year; and when 18 and 19-year-old adolescents were still in the third year of high school. public or private school was used as income proxy, as students from public schools are considered to come from lower income families. smoking was classified in two groups: adolescents who had never smoked and another one with adolescents that either are currently or former smokers. having a health problem was classified in two groups: those without a health problem or not being aware of it, and those who referred having a health problem that had lasted or will probably last more than one year. self-reported toothbrushing frequency was classified in three groups – more than three times a day, three times a day, and less than three times a day. the use of dental floss was dichotomized as either yes or no. data analysis was performed using the statistical package spss 18 (spss inc., chicago, united states). associations between the dependent variable and independent variables were assessed by the chi-square test or fisher’s exact test, and presented by frequency distribution. poisson regression models with uniand multivariate robust tooth loss sociodemographic factors age gender ethnicity/skin color socioeconomic condition mother’s level of education father’s level of education unemployed person at home money for medical expenses housing at 12 years old occupation of the adolescent school delay of the adolescent type of school behavioral, biological, and oral factors smoking presentiing health problems toothbrushing frequency use of dental floss extraction by orthodontic indication figure 1. explanatory variables for tooth loss. 5 colussi et al. variance were used to assess the association between dependent and independent variables. the significance level applied was 5%. the results from exposures regarding prevalence of tooth loss outcome were analysed in two different contexts. the first one only analysed subjects without extraction for orthodontic reasons, and the second context analysed all of the subjects with any tooth loss. results out of the 23 schools invited, 20 accepted to be part of the study, of which 16 were public and 4 were private schools. from the total of 6,122 students eligible for the study in the 20 schools, 1,836 students were chosen by draw and invited to participate, and 736 (40.08%) accepted the invitation. from these, 323 (43.9%) were males and 413 (56.1%) were females. the reasons for non-participation and the number of subjects in both private and public schools are expressed in figure 2. the prevalence of at least 1 lost tooth was 21.1% with mean of lost teeth of 0.42. total of students enrolled in the 23 selected high schools, regardless of age: 7,558 16 public schools 6,256 students (82.78%) 7 private schools 1,302 students (17.22%) schools participating in the study: 20 (86.95%) 16 public schools (100%) 4 private schools (57.14%) total of eligible students aged 15-19 years, in the 20 schools selected: 6,122 total of students selected by draw: 1,836 (30%) participants of the study: 736 (40.08%) (59.92%) non-participants of the study 1,100 public school: 620 (84.2%) private school: 116 (15.8%) school dropout: 190 (10.34%) non-respondents: 865 (47.12%) other reasons (transfer, health conditions, maternity leave, military service): 45 (2.46%) figure 2. study flowchart. 6 colussi et al. figure 3a and 3b shows the most absent teeth in the sample. the 1st upper premolar, the 1st lower molar, and the 1st lower premolar were the most absent teeth with rates of 21.75, 16.89, and 11.03%, respectively, representing almost 50% of the total lost teeth (figure 3c). age, gender, toothbrushing frequency, and dental floss usage were not associated with the prevalence of tooth loss in both analytical models used. ethnicity, mother’s level of schooling, school delay, having money for medical expenses, type of school, and smoking showed association with the prevalence of tooth losses (table 1). when the univariate model was analysed, non-white adolescents, those with mothers with low level of education, from families with no money for medical expenses, school delay, students from public schools, and students with smoking history were more likely to present tooth loss (table 2). the associations are similar; however the extent considerably increases when analysing only adolescents presenting tooth loss without orthodontic reason. when performing the multivariate analysis with all subjects, higher chances for tooth loss were found in adolescents from mothers with lower level of education, adolescents with school delay, and with history of smoking exposure (table 2). adolescents from mothmaxilla tooth number 40 25 15 10 5 35 30 20 0 17 1216 1115 2114 2213 23 24 25 26 27 mandible tooth number 40 25 15 10 5 35 30 20 0 47 4246 4145 3144 3243 33 34 35 36 37 maxilla/mandible lower lateral incisor upper lateral incisor 1st lower premolar 2nd lower premolar 1st lower molar upper central incisor lower central incisor lower canine 2nd lower molar 2nd upper molar 1st upper premolar 1st upper molar 2nd upper premolar upper canine 0% 5% 10% 15% 20% 25% (a) (b) (c) figure 3. total number of lost teeth according to the type and location in the sample (n=736) (a, b) and frequency distribution of lost teeth, according to the type of tooth (c). 7 colussi et al. table 1. frequency distribution of exposures regarding prevalence of tooth loss among 15-19 year-old adolescents. only subjects without extractions for orthodontic reasons all subjects yes – n (%) no – n (%) p-value* yes – n (%) no – n (%) p-value* age 15 23 (26.7%) 186 (34.1%) 0.443 40 (25.8%) 196 (33.7%) 0.239 16 27 (31.4%) 182 (33.4 %) 52 (33.5%) 199 (34.3%) 17 24 (27.9%) 124 (22.8%) 46 (29.7%) 129 (22.2%) 18 9 (10.5%) 43 (7.9%) 13 (8.4%) 45 (7.7%) 19 3 (3.5%) 10 (1.8%) 4 (2.6%) 12 (2.1%) gender male 34 (39.5%) 248 (45.5%) 0.351 60 (38.7%) 263 (45.3%) 0.146 female 52 (60.5%) 297 (54.5%) 95 (61.3%) 318 (54.7%) ethnicity white 42 (48.8%) 392 (71.9%) <0.001 93 (60.0%) 418 (71.9%) 0.006 non-white 44 (51.2%) 153 (28.1%) 62 (40.0%) 163 (28.1%) mother’s level of education complete or incomplete higher education 7 (8.1%) 135 (24.8%) <0.001 21 (13.5%) 143 (24.6%) 0.002 complete or incomplete high school 23 (26.7%) 199 (36.5%) 53 (34.2%) 212 (36.5%) finished up to elementary school 56 (65.1%) 211 (38.7%) 81 (52.3%) 226 (38.9%) father’s level of education complete or incomplete higher education 6 (7.0%) 98 (18.0%) 0.005 17 (11.0%) 107 (18.4%) 0.082 complete or incomplete high school 26 (30.2%) 196 (36.0%) 58 (37.4%) 208 (35.8%) finished up to elementary school 54 (62.8%) 251 (46.0%) 80 (51.6%) 266 (45.8%) unemployed person at home yes 19 (22.4%) 89 (16.5%) 0.215 34 (22.1%) 91 (15.8%) 0.071 no 66 (77.6%) 452 (83.5%) 120 (77.9%) 486 (84.2%) money for medical expenses yes 57 (66.3%) 439 (80.6%) 0.004 112 (72.3%) 469 (80.7%) 0.026 no 29 (33.7%) 106 (19.4%) 43 (27.7%) 112 (19.3%) housing at 12 years old urban 79 (91.9%) 514 (94.3%) 0.337 141 (91.0%) 548 (94.3%) 0.140 rural 7 (8.1%) 31 (5.7%) 14 (9.0%) 33 (5.7%) occupation of the adolescent studies only 48 (55.8%) 361 (66.2%) 0.068 97 (62.6%) 387 (66.6%) 0.391 studies and works 38 (44.2%) 184 (33.8%) 58 (37.4%) 194 (33.4%) school delay of the adolescent yes 40 (46.5%) 138 (25.3%) <0.001 95 (61.3%) 436 (75.0%) 0.001 no 46 (53.5%) 407 (70.7%) 60 (38.7%) 145 (25.0%) type of school public 84 (97.7%) 448 (82.2%) <0.001 140 (90.3%) 480 (82.6%) 0.018 private 2 (2.3%) 97 (17.8%) 15 (9.7%) 101 (17.4%) smoking never smoked 72 (83.7%) 519 (95.2%) <0.001 138 (89.0%) 555 (95.5%) 0.006 former smoker or currently smokes 14 (16.3%) 26 (4.8%) 17 (11.0%) 26 (4.5%) having health problems yes 15 (17.6%) 65 (12.1%) 0.164 21 (13.6%) 70 (12.2%) 0.681 no 70 (82.4%) 471 (87.9%) 133 (86.4%) 502 (87.8%) toothbrushing frequency >3x a day 27 (31.4%) 132 (24.2%) 0.161 44 (28.4%) 142 (24.4%) 0.467 3x a day 42 (48.8%) 326 (59.8%) 85 (54.8%) 350 (60.2%) <3x a day 17 (19.8%) 87 (16.0%) 26 (16.8%) 89 (15.4%) use of dental floss yes 38 (44.2%) 286 (52.5%) 0.165 79 (51.0%) 311 (53.5%) 0.588 no 48 (55.8%) 259 (47.5%) 76 (49.0%) 270 (6.5%) 8 colussi et al. table 2. univariate and multivariate analyses models associating exposures regarding prevalence of tooth loss outcome among adolescents from 15 to 19 years old. univariate analysis multivariate analysis only subjects without extraction for orthodontic reason all of the subjects only subjects without extraction for orthodontic reason all of the subjects pr ci 95% (p-value) pr ci 95% (p-value) pr ci 95% (p-value) pr ci 95% (p-value) age 19 1 (ref.) 1 (ref.) 15 0.47 0.16-1.40 (0.173) 0.67 0.27-1.65 (0.394) 16 0.56 0.20-1.60 (0.280) 0.82 0.34-2.00 (0.676) 17 0.70 0.24-2.02 (0.513) 1.05 0.43-2.54 (0.911) 18 0.75 0.24-2.40 (0.626) 0.90 0.34-2.38 (0.826) gender male 0.81 0.54-1.21 (0.303) 0.80 0.60-1.07 (0.147) ethnicity non-white 2.31 1.57-3.4 (<0.001) 1.51 1.14-2.00 (0.004) 1.72 1.15-2.60 (0.009) 1.27 0.94-1.71 (0.100) mother’s level of education higher education 1 (ref.) 1 (ref.) 1 (ref.) 1 (ref.) high school 2.1 0.93-4.77 (0.076) 1.56 0.98-2.50 (0.061) 1.32 0.56-3.11 (0.500) 1.39 0.86-2.24 (0.170) illiterate and complete elementary school 4.25 1.99-9.09 (<0.001) 2.06 1.33-3.20 (0.001) 2.20 0.96-5.02 (0.060) 1.67 1.05-2.67 (0.029) unemployed person at home yes 1.38 0.87-2.2 (0.175) 1.37 0.99-1.90 (0.060) money for medical expenses no 1.87 1.25-2.8 (0.002) 1.44 1.06-1.95 (0.019) housing at 12 years old urban 0.72 0.36-1.46 (0.364) 0.68 0.43-1.09 (0.112) occupation of the adolescent studies and works 0.69 0.46-1.02 (0.060) 0.87 0.65-1.16 (0.345) school delay lower level of education 2.2 1.50-3.26 (<0.001) 1.63 1.23-2.16 (0.001) 1.41 0.95-2.09 (0.086) 1.36 1.05-2.32 (0.028) type of school public 7.82 1.95-31.24 (0.004) 1.75 1.06-2.90 (0.027) 4.16 0.98-17.59 (0.050) smoking yes 2.87 1.79-4.62 (<0.001) 1.98 1.33-2.95 (0.001) 1.91 1.15-3.17 (0.011) 1.56 1.05-2.32 (0.034) having health problems yes 1.45 0.87-2.4 (0.151) 1.10 0.74-1.65 (0.638) 1.83 1.14-2.98 (0.013) toothbrushing frequency >3x a day 1 (ref.) 1 (ref.) 3x a day 0.67 0.43-1.05 (0.081) 0.96 0.18-0.30 (0.835) <3x a day 0.96 0.55-1.67 (0.893) 0.83 0.60-1.14 (0.243) use of dental floss no 1.33 0.9-1.98 (0.155) 1.08 0.82-1.43 (0.570) 9 colussi et al. ers with low level of education, and with school delay presented 67% (p=0.029) and 36% (p=0.028) more chances of having already lost teeth, respectively. demographic, socioeconomic, behavioural, and health aspects become more evident in the multivariate analysis including only adolescents without extraction for orthodontic reasons. the ones presenting more chances of having tooth loss are non-white, students from mothers with low level of education, students from public schools, with smoking history, and with any health problem (table 2). non-white students had 72% (p=0.009) higher chances of having experienced tooth loss. furthermore, studying in public schools increased the chances in 4.16 (p=0.05) times. having some history of smoking or reporting health problems increased in 91% (p=0.011) and 83% (p=0.013) the chance of teeth loss, respectively. discussion this study aimed to determine the occurrence and factors associated with tooth loss in adolescents in a medium-sized city in southern brazil. additionally, it sought to understand the occasional differences in the profile of tooth loss, considering the history of orthodontic treatment. when individuals experiencing extractions due to orthodontic indication, the magnitude of the associations found in this study were higher, reinforcing the role of social determinants in tooth loss in adolescents. in the final sample, the rate of socio-demographic variable, such as gender and ethnicity, and type of school were similar to the city population. the results of the present study show a relatively high prevalence of tooth loss in adolescents (21.1%), and mean of 0.42 lost teeth. the national 1986 and 20031,4 surveys show means of 1.2 and 0.9, respectively. the same situation may be observed for prevalence of tooth loss; rates decreasing from 38.9% in 20034 to 26% in 20065, and to 21.1% in the present study. despite the results of the present study have suggested a decrease in prevalence of tooth loss, they are higher than the ones observed in the last national survey in 2010, which shows mean and prevalence of tooth loss of 0.38 and 8.9%, respectively2. most studies in brazil indicate the first lower molar as the most frequently lost tooth3,4,5. however, this study showed that the first upper premolar was the most absent tooth. these results are in accordance with other studies9,12,17. this tendency may be explained by the change in the epidemiological profile of dental caries mainly observed by the reduction of dmft, and by the extraction of premolars for orthodontic reasons. however, epidemiological studies should confirm these findings. tooth loss was assessed by two distinct models. this separation in two models aimed to avoid extractions for orthodontic reasons to be considered an outcome, especially in the analysis of demographic and socioeconomic variables. age, gender, toothbrushing frequency, and dental floss usage were not associated with tooth loss in both models. age is considered a risk indicator for tooth loss, especially in adults and the elderly5,18. in this study it was included only 15 to 19-year-old adolescents, and this is a short period to detect differences in tooth loss. several other studies show that female adolescents present higher prevalence of tooth loss. however, these results are controversial, considering that some of these studies do not show significant differences3,5,12, such as the present study, while others reveal statistically significant differences4,19. 10 colussi et al. the socioeconomic conditions are strong determinants for tooth loss20. data from the present study clearly show that being a white adolescent is a protective factor against tooth loss. the question of ethnicity should be understood more as a proxy of socioeconomic status than a biological variable. white individuals in brazil have higher level of education and income than other ethnicities, leading to more access to dental services, more knowledge and behaviour regarding oral hygiene care. similarly, adolescents from mothers with low level of education had more chances of presenting tooth loss. the relation between mother’s level of education and oral health of children and adolescents is clear21,22, mainly regarding dental caries. likewise, adolescents with lower level of education presented higher chances of having experienced tooth loss. results similar to other studies show that lower level of education was related to worse oral health conditions4,23. students from public schools who participated in the present study had significantly higher chances of having tooth loss. this is compatible with results that show significant differences in oral health, among students from schools where the socioeconomic differences are highlighted8,24. adolescents who reported some history with smoking presented higher risk of having tooth loss. however, only 5.8% of adolescents have reported this, which is a low rate and it was also observed in other studies25,26. either way, smoking usually starts in adolescence and is associated to school problems26. regarding the limitations of the present study, the claimed representativeness is strict to adolescents who are studying. furthermore, a sample size calculation was not performed. analytical epidemiological studies need a minimum amount of 40-50 individuals for each variable in test. as a census would not be possible, we calculated that an invitation of 30% of the adolescents would suffice for the different analyses that would be performed, which is the case of this study. in the literature, other studies in this field have used smaller and similar sample sizes27,28. additionally, the response rate tends to be diminishing in adolescent studies, mainly because of the lack of signature in the informed consent. however, the analytical approach, with the relatively high number of individuals, strengthens the encountered associations, making possible to extrapolate the results regarding higher chances of presenting such an important and impacting event such as tooth loss. the results of the present study show that by removing extractions for orthodontic reason from the analysis, the demographic and socioeconomic differences become more evident while increasing the extent of associations. moreover, it is necessary to measure the impact that extractions for orthodontic reasons brings on the mean and prevalence of tooth loss in adolescents, considering that such distinct causes of tooth losses seem to be part of the same process. moreover, it is necessary to measure the impact that extractions for orthodontic reasons brings on the mean and prevalence of tooth loss in adolescents, considering that both variables seem to be part of the same process. in conclusion, demographic, socioeconomic, and behavioural conditions were strongly associated with tooth loss. extractions for orthodontic reasons have impact on tooth loss estimates. 11 colussi et al. acknowledgement this study was self-funded and the authors declare no conflict of interest related to this study. references 1. 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[dissertation]. porto alegre: faculdade de medicina, universidade federal do rio grande do sul; 2011. portuguese. 16. corraini p, baelum v, pannuti cm, pustiglioni an, romito ga, pustiglioni fe. tooth loss prevalence and risk indicators in an isolated population of brazil. acta odontol scand. 2009;67(5):297-303. doi: 10.1080/00016350903029107. 12 colussi et al. 17. akhlaghi f, yavari as, eshaghi sm. clinical prevalence of missing teeth (except third molar) in girl students at rasht high schools (1999-2000). j dent sch. 2006;24:155-62. 18. george b, john j, saravanan s, arumugham jm. prevalence of permanent tooth loss among children and adults in a suburban area of chennai. indian j dent res. 2011 mar-apr;22(2):364. doi: 10.4103/0970-9290.84284. 19. lópez r, baelum v. gender differences in tooth loss among chilean adolescents: socio-economic and behavioral correlates. acta odontol scand. 2006 jun;64(3):169-76. 20. gilbert gh, duncan rp, shelton bj. social determinants of tooth loss. health serv res. 2003 dec; 38 (6 pt2):1843-62. 21. mendes lga, biazevic mgh, michel-crosato e, mendes moa. dental caries and associated factors among brazilian adolescents: a longitudinal study. braz j oral sci. 2008;7(26):1614-9. doi: 10.20396/bjos.v7i26.8642814. 22. perera i, ekanayake l. social gradient in dental caries among adolescents in sri lanka. caries res. 2008;42(2):105-11. doi: 10.1159/000116874. 23. bastos jld, nomura lh, peres ma. dental caries and associated factors among young male adults between 1999 and 2003 in southern brazil. community dent health. 2007 jun;24(2):122-7. 24. campus g, cagetti mg, senna a, spano g, benedicenti s, sacco g. differences in oral health among italian adolescents related to the type of secondary school attended. oral health preventive dentistry. 2009;7(4):323-30. 25. malbergier a, cardoso lrd, amaral ra. adolescent substance use and family problems. cad saude publica. 2012 apr;28(4):678-88. 26. park yd, patton ll, kim hy. clustering of oral and general health risk behaviors in korean adolescents: a national representative sample. j adolesc health. 2010 sep;47(3):277-81. doi: 10.1016/j.jadohealth.2010.02.003. 27. murshid sa, al-labani ma, aldhorae ka, rodis om. prevalence of prematurely lost primary teeth in 5-10-year-old children in thamar city, yemen: a cross-sectional study. j int soc prev community dent. 2016 aug;6(suppl 2):s126-30. doi: 10.4103/2231-0762.189739. 28. susin c, haas an, opermann rv, albandar jm. tooth loss in a young population from south brazil. j public health dent. 2006 spring;66(2):110-5. untitled 1 volume 16 2017 e17034 original article 1 dentistry department, federal university of vales do jequitinhonha e mucuri, diamantina – mg, brazil. 2 dentistry school, federal university of sergipe, aracaju – se, brazil. 3 periodontology department, federal university of minas gerais, belo horizonte – mg, brazil. 4 oral pathology post graduate program, federal university of rio grande do norte, natal – rn, brazil. corresponding author: prof. joão luiz de miranda rua da glória, 187, centro, diamantina, minas gerais, brazil. zip code: 39100-000 phone:+55 38 35326082 joao.miranda.ufvjm@gmail.com received: april 17, 2017 accepted: september 14, 2017 immunohistochemical study of fibronectin, tenascin and type i collagen in tongue and lip carcinomas joão luiz de miranda, phd 1, felipe rodrigues de matos, phd 2, frederico santos lages, msc 3, dhelfeson willya douglas-de-oliveira, msc 3, roseana de almeida freitas, phd 4 aim: the aim was to compare the immunoexpression of extracellular matrix proteins in squamous cell carcinomas of tongue (sccto) and lower lip (sccli). methods: eleven sccto and 11 sccli were selected and examined according to bryne’s method (1998). for immunohistochemical study utilized antibodies to fibronectin, tenascin and type i collagen. histopathologic and immunohistochemical analysis were performed on the tumor invasive front. results: all sccto were classified in high score malignant grade and all sccli in lower score. fibronectin showed strong immunorreactivity in the peritumoral basement membrane (bm) in 91% of sccto and all cases of sccli, while in the tumor stroma (ts) all cases of sccto and sccli had strong intensity. tenascin had strong expression in bm of 91% cases of sccto and 63.4% of sccli and in ts had strong expression in 91% cases of sccto and 54.6% of sccli. type i collagen demonstrated weak immunoreactivity in the ts of 72.7% cases of sccto and 63.4% of sccli. conclusion: these results may suggest that the strong expression of fibronectin and tenascin proteins and the weak expression of type i collagen could play a role in the invasive process of oral scc. keywords: immunohistochemistry; oral cancer; tenascin; fibronectin; collagen. http://dx.doi.org/10.20396/bjos.v16i0.8651061 2 de miranda et al. introduction oral squamous cell carcinoma (scc) is the most common type of oral cancer, and it continues to have a poor 5 years survival rate1,2. identification of specific molecules associated with malignant transformation has led to identify an increasing number of molecular markers related to tumor stage and grading, and it may have a prognostic value for the disease. in addition, there is significant knowledge about key molecules that regulate the cell cycle, apoptosis, immunologic tumor defense, and extracellular matrix interactions and breakdown in oral cancer3,4. it has been postulated that tumorigenesis of oral scc is not dependent only on the type, duration and level of exposure to a specific carcinogen, but rather on the genetic sensitivity of the individual5. studies of clinical, morphological and immunohistochemical features have been conducted to demonstrate that the scc with poor prognosis show more proliferative activities6,7, less differentiation6, more vascularization8 and more invasive7 and spreading potential9,10 in the adjacent and distant tissues. the scc dissemination is possible elsewhere, and they lose the intercellular adhesion and initiate the migration mechanism into the basal lamina11,12. this migratory mechanism depends on the disrupting of the basal membrane11-13. in similar manner, the carcinoma cells migrated into tumor stroma degrading the interstitial collagen14. at this stage, these carcinoma cells expressed superficial receptors to extracellular matrix (ecm) components aiming to penetrate more freely the adjacent connective tissue11,12,14. fibronectin is a ubiquitous protein present in tissues and body fluids, including plasma that engages in these cellular functions and provides architectural scaffolding for cells and tissues, and it may be associated with invasion and metastasis. therefore, fibronectin variants could be used as possible prognostic factor15. tenascin is another protein of ecm that is a glycoprotein expressed in epithelial-mesenchymal interactions during embryogenesis and tumorigenesis of several tissues16. tenascin plays an important role as a molecular mediator in proliferation and progression in neoplastic processes17. additionally, increased synthesis and deposition of type i collagen by stromal cells has been described in mammary, skin, colon, and prostate carcinomas18-21. sccs of the tongue (sccto) and lower lip (sccli) reveal differences in their biological behavior. sccto has a great predisposition to produce metastasis in lymph nodes (incidence 15-75%) depending on the extension of the primary lesion22,23. the aim of this study was to verify the extracellular matrix protein expression (fibronectin, tenascin and type i collagen) in squamous cell carcinoma of tongue and lower lip, in an attempt to establish some comparison between these protein expressions and the biological behavior of the oral scc. materials and methods 2.1 sample size sample size was calculated based on the prevalence (1,4%) of squamous cell carcinomas24 with the margin of error set at 5%. the minimum sample size required was 21 sccs considering a 95% confidence level. 3 de miranda et al. 2.2 patients the study was approved by the ethics committee of the federal university of vales do jequitinhonha e mucuri under protocol number 122/2010. it was conducted according to the declaration of helsinki 1975, revised in 2013. eleven cases of tongue squamous cell carcinoma and eleven of lower lip squamous cell carcinoma were obtained from the files of laboratory of pathology of federal university of vales do jequitinhonha e mucuri. patients were surgically treated without radiotherapy or chemotherapy prior. samples were excluded from the study in case of incision biopsy, specimens with inadequate material or extensive areas of necrosis. each patient assigned a written informed consent at the moment of biopsy. sections (5 μm) were cut from paraffin-embedded tumor specimens and stained with hematoxylin and eosin for the grading of histological malignancy as proposed by bryne’s method25, utilizing the histological parameters as degree of keratinization (dk), nuclear polymorphism (np), pattern of invasion (pi) and lymphoplasmatic infiltration (li). a score of 0 to 4 was attributed to each parameter and cases with a total score of 8 or lower were classified as low-grade malignancy and those scoring higher than 8 were classified as high-grade malignancy26. 2.3 immunohistochemistry for the immunohistochemical study, the tissue sections were deparaffinized and immersed in 3% hydrogen peroxide to block endogenous peroxidase activity. the tissue sections were then washed in phosphate-buffered-saline (pbs). the antigen retrieval, antibody dilution and clone type for fibronectin, tenascin and type i collagen are shown in table 1. after treatment with normal serum, the tissue sections were incubated in a moist chamber with primary antibodies. the tissue sections were then washed twice in pbs and treated with streptoavidin-biotin-peroxidase complex (dako, carpinteria, ca, usa) at room temperature in order to bind the primary antibodies. peroxidase activity was visualized by immersing tissue sections in diaminobenzidine (d5637; sigma chemical, st. louis, mo), resulting in a brown reaction product. finally, tissue sections were counterstained with mayer’s hematoxylin and coverslipped. positive controls for fibronectin and tenascin were sections of normal oral mucosa and for type i collagen were sections of human placenta. as negative controls, samples table 1. specificity, dilution, antigen retrieval, and incubation of the antibody (ab) clones. ab clone specificity dilution antigen retrieval incubation a-245 ‡ fibronectin 1:500 pepsin 1% , ph1.8, oven 37°c, 60 min 120 min tn2 ‡ tenascin 1:100 pepsin 0.4% , ph 1.8, oven 37°c, 30 min overnight (18 h) ncl-coll-ip * type i collagen 1:40 citrate ph 6.0, steamer, 5min overnight (18 h) * novocastra laboratories. ‡ dako 4 de miranda et al. were treated as above, except that the primary antibody was replaced by a solution of bovine serum albumin (bsa) in pbs. 2.4 immunohistochemical analysis the immunohistochemical analysis for fibronectin, tenascin and collagen i was performed using light microscope at ×400 magnification for its location (basement membrane and stroma peritumoral) and intensity. the thickness of basement membrane was measured as 5 µm at the tumor front. the intensity of immunoreactivity was semiquantitatively assessed by two independent observers (kappa = 0.89) to remove any possible bias using a modification of criteria indicated as: weak (absence or weak staining) and strong (moderate or intense staining). the results obtained were submitted to statistical analysis. computations were made using the statistical package for the social sciences (version 22.0; spss inc., chicago, il). the association of the fibronectin, tenascin and collagen expression between sccto and sccli was performed by fisher’s exact test. for all tests, significance level was set p<0.05. results the histological gradation system utilized demonstrated that all sccli (n=11) had low malignant grade and all cases of sccto (n=11) have high malignant grade (table 2). there was significant association between the location and the grade of the scc (p < 0.001). all cases of sccto (fig. 1) and sccli (fig. 2) exhibited fibronectin immunoexpression throughout peritumoral basement membrane (bm) and tumor stroma (ts), which was mainly linear and thin in the bm and diffuse and fibrillar in the ts. in the bm, all cases of sccli and 10 (91%) cases of sccto showed strong intensity without significant difference between groups (table 3). in the ts, this protein demonstrated strong intensity in all sccto and sccli (table 3). tenascin immunoexpression was observed in bm of 10 cases of sccto (fig. 3a and 3b) and 8 cases of sccli (fig. 4), characterized by linear and thin pattern. ten (91%) cases of sccto and 7 (63.4%) of sccli had strong expression with no significant difference (p > .05) (table 3). at the ts, all cases of sccto and sccli was immunopositive and showed diffuse and fibrillar expression of this protein. ten table 2. malignancy grading of the squamous cell carcinomas of the lower lip and tongue. high malignant grade lower malignant grade p* n % n % lower lip 0 0.0 11 100 <0.001 tongue 11 100 0 0.0 5 de miranda et al. figure 1. immunoexpression of fibronectin in sccto throughout peritumoral basement membrane (arrow) and tumor stroma (arrowhead) (labelled streptavidin biotin (lsab) method, original magnification ×400). figure 2. immunoexpression of fibronectin in sccli throughout peritumoral basement membrane (arrow) and tumor stroma (arrowhead) (lsab method, original magnification ×400). table 3. distribution of percentages of the intensity of fibronectin, tenascin and type i collagen in sccto and sccli, according localization. localization intensity fibronectin p tenascin p type i collagen p sccto sccli sccto sccli sccto sccli basement membrane weak n (%) 1 (9%) 0 (0.0%) 0.999 1 (9%) 4 (36.3%) 0.311 11 (100%) 11 (100%) na strong n (%) 10 (91%) 11 (100%) 10 (91%) 7 (63.4%) 0 (0.0%) 0 (0.0%) tumor stroma weak n (%) 0 (0.0%) 0 (0.0%) na 1 (9%) 5 (45.4%) 0.149 8 (72.7%) 7 (63.4%) 0.999 strong n (%) 11 (100%) 11 (100%) 10 (91%) 6 (54.6%) 3 (27.3%) 4 (36.3%) na = not applied 6 de miranda et al. (91%) cases of sccto and 6 (54.6%) of sccli had strong expression with no significant difference (p > .05) (table 3). all cases of sccto and sccli were non-reactives in bm for type i collagen (table 3). at the ts, all cases of sccto (fig. 5) and sccli (fig. 6) had immunoexpression of this protein characterized by predominant fibrillar pattern. eight (72.7%) cases of sccto and 7 (63.4%) of sccli (fig. 5) had weak expression with no significant difference (p > .05) (table 3). discussion many studies about the biological behavior of the oral squamous cell carcinoma have been developed, seeking histomorphological parameters for the malignant grading systems for this carcinoma and aiming to find new prognostic indicators25-27. a b figure 3. a) immunoexpression of tenascin in sccto throughout peritumoral basement membrane (arrow) in linear and thin pattern and tumor stroma (arrowhead) (lsab method, original magnification ×400). b) intense cytoplasmic immunoexpression of tenascin in deepest nests (arrow) (lsab method, original magnification ×400). figure 4. immunoexpression of tenascin in sccli throughout peritumoral basement membrane (arrow) in linear and thin pattern and tumor stroma (arrowhead) (lsab method, original magnification ×400). 7 de miranda et al. the histomorphologic results of our study suggest that the sccto have more aggressive biological behavior than the sccli, since we observed that all sccto were high malignant grade compared to the sccli. these observations also have been demonstrated by several other studies27-29. the tongue is located in the floor of the oral cavity, which has a rich supply of lymphatic drainage and neurovascular bundles, thus increasing the probability that neck nodal metastasis could occur in tongue cancer patients30. but not only supplies of lymphatic drainage and neurovascular bundles are responsible for aggressiveness and invasion of the tumor cells. it has been clearly established in the literature that tumor stroma is directly related with biological behavior of neoplasm, despite the fact that for a long time, neoplasm cells were the main focus of cancer studies. the capability malignant cells have to destroy basal membrane and the other figure 5. immunoexpression of type i collagen in tumor stroma of sccto in fibrillar pattern (lsab method, original magnification ×400). figure 6. immunoexpression of type i collagen in tumor stroma of sccli in fibrillar pattern (lsab method, original magnification ×400). 8 de miranda et al. components of extracellular matrix (ecm) have been related to invasive potential of the scc12. ecm constituents contribute directly or indirectly to the tumor process, owing to the fact that the structure has potentially anti-adhesive components, modulators of adhesion, proliferation and cell migration. ecm may regulate cell behavior using different mechanisms: first through composition of proteins in a specific tissue and second in synergic interactions between growth factors and adhesion molecules or cell receptors that mediate adhesion of components31. recent studies have shown that the ecm does not only act passively as a support for cells, but also provides information to these cells modifying their behavior, an event that might be responsible for tumorigenesis32,33. some studies using markers for different constituents of emc, like fibronectin34,35, tenascin36,37 and collagen38, were conducted to clarify the role of this structure in the process of tumorigenesis and progression of head and neck tumors. fibronectin is a high-molecular-mass glycoprotein with additional domain in the iiics region39 and is not restricted to the basement membrane but is ubiquitously distributed throughout the cell matrix, and a soluble plasma fibronectin is found in body fluids40. fibronectin plays an important signaling function in cell adhesion and migration and interacts with a number of integrins, including αvβ6. its expression has been related to poor prognosis of head and neck tumors12,40, since cell migration on a fibronectin matrix is significantly faster in those cell lines expressing higher levels of αvβ6 integrin 40. hypoxia promotes cancer cell invasion by inducing the expression of mesenchymal cell markers such as fibronectin41. through the immunohistochemical evaluation we observed strong expression of fibronectin in the basement membrane of the neoplastic epithelial nests of all sccli. in the tumor stroma of the invasion front, the fibronectin exhibited strong immunorreactivity in all sccto and sccli. the expression of fibronectin was significantly increased in the stromal tissue of 50% of the scc but only in 10% of cases of verrucous carcinoma (vc) that is an uncommon rare low-grade variant of scc42. the results of our study suggest that the intense expression of the fibronectin may be involved in the migratory and invasive mechanisms of the neoplastic cells of the scc studied, since cancer cells degrade the ecm and invade the surrounding stroma by producing proteases and the degraded ecm may be replaced by fibronectin41. this may be indicative that tumor cells and not only stromal cells may synthesize fibronectin, building up on the basal membrane and peritumor matrix to probably facilitate adherence and posterior migration through tumor stroma. this data helps to better comprehend the molecular mechanisms involved in the cellular invasion process. whereas fibronectin induces the cell to adhere to the substrate strengthening and spreading, tenascin has the opposite effect of promoting rounding and detachment. tenascin has a modulatory effect on cell-interactions and in cell culture that antagonizes the adhesive effects of fibronectin. although tenascin and fibronectin are usually found together, regulatory mechanisms for each of them are quite different43. tenascin is another glycoprotein synthesized at specific time points and sites during embryogenesis, it is absent or greatly reduced in most adult tissue, but it increased in some pathological conditions, inclucing inflammation, wound healing and in a variety of neoplasias44. as observed in the present study, tenascin expression was associated with invasiveness and malignancy. 9 de miranda et al. tenascin is an extracellular matrix protein composed of six monomers linked at their n-termini with disulfide bounds to form a 1080-1500-kda hexamer and various solid tumors express high level of tenascin45. tenascin is secreted from both tumor cells and fibroblasts. the most prominent effects of tenascin are anti-adhesion and inhabitation of cell attachment, both of which favor cancer cell motility and invasion46. furthermore, tenascin promotes malignant transformation, uncontrolled proliferation, metastasis, angiogenesis, drug resistance and escape from tumor immunosurveillance33. for this reason, there was difference in the amount of tenascin, which was more intense in the stroma of tongue (91%) than is lip (55%) cases. the highest expression of tenascin is observed in unstable environments such as during cell migration, in active areas of epithelial–mesenchymal interactions, and in neoplastic stroma. this protein had many domains of binding, mediating cell-cell adhesion, cell migration, as well as cell adhesion close to matrix through fibronectin binding to proteoglicans. tenascin may have a complex structure with domains that are capable of interacting with a variety of cell-surface receptors, including integrins and extracellular matrix proteins40. when this protein is produced by malignant neoplastic cells, there seems to be an increase in proliferation and migration, probably owing to the fact that it has antiadhesive properties, because it blocks binding of fibronectin to cells12,47. the higher expression of tenascin in sccto in basement membrane and tumor stroma when compared to sccli might suggest a strong interaction between parenchyma and stroma in order to create a microenvironment permissive to the high proliferative activity and invasive capacity of tumor cells. probably, these findings were associated with the fact that the sccto demonstrated high malignant score and a diffuse cellular invasion pattern. during the initiation of oral cancer, tenascin could be directly produced by cancer cells and is responsible for the acquisition of an invasive phenotype48. tenascin and laminin in oral scc at the tumor margins by a laser scanning microscopy-based quantitative co-localization and verified the extent of tenascin and laminin arrangement into reorganized basement membrane structures correlated with malignancy grade47. thus, these results suggest that the tenascin contributes to a major migration and spreading of the malignant neoplastic cells. collagen represents a family of characteristic proteins with over 20 known types, present in all multicellular animals, being the most abundant components in all ecm12. over 20 genetically distinct collagens have been identified. type i collagen is found throughout the connective tissue and is one of the most abundant components of the interstitial ecm, being highly resistant to proteases due to its unique supercoiled triple helix structure. paradoxically, despite being the commonest molecule of the matrix, few tumor-associated changes are apparent and these seem to be of little prognostic significance40, although some studies have demonstrated discontinuity of different types of collagen during the process of tissue invasion by neoplastic cells38,49. type i collagen presented weak intensity predominantly in tumor stroma of sccto and sccli. recent studies have also documented a significant higher frequency of less packed collagen fibers at the sites of invasion, whereas the well packed ones predominated at the noninvaded sites. it was assumed that the less packed collagen fibers, together with other molecular events, could facilitate invasion into adjacent tis10 de miranda et al. sues and development of metastasis50. the type i collagen was deposited in a minor quantity in the peritumoral stroma of the oral scc38. these results are consistent with previous studies reporting that during the cellular invasion processes, the malignant neoplastic cells6,7,9,10, and stromal fibroblasts51,52 degrade the collagen by the production of proteolytic enzymes, stimulated by transforming growth factor-beta (tgf-β) production53. however, other studies verified that the type i collagen is a good substrate in the adherence and migration of the malignant neoplastic cells, suggesting that this collagen plays an important promoter role in tumoral metastasis52,53. the present results may suggest that the strong expression of fibronectin and tenascin proteins and the weak expression of type i collagen could play a role in the invasive process. over the years, oral cancer diagnosis and therapy have not changed significantly the survival rate54. thus, the results of our study help to comprehend the migration and invasion by the neoplastic cells of the oral scc by dynamic interactions between tumor cells and extracellular matrix. experimental studies with whole scc section using more careful analyses are needed to corroborate or not the present findings. the present histologic study concluded that fibronectin, tenascin and type i collagen were produced and deposited in the stromal space and in the basement membrane of scc. the more important conclusion from the present in vitro study was that scc cells produced tenascin molecules much more in the stroma of tongue than in the lip. acknowledgements this study was supported by the national council of scientific and technological development from the ministry of the science and technology (mct/cnpq), government of brazil. conflict of interest statement the authors declare no conflict of interest. references 1. aparna m, rao l, kunhikatta v, radhakrishnan r. the role of mmp-2 and mmp-9 as prognostic markers in the early stages of tongue squamous cell carcinoma. j 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expression in oral squamous cell carcinomas: correlation with tumor proliferation and patient survival. biomed res int. 2014;2014:905680. doi: 10.1155/2014/905680. braz j oral sci. 15(3):221-225 quantitative transportation assessment in simulated curved canals after large apical preparations vânia cristina gomes vieira1, felipe gonçalves belladonna2, erick miranda souza3, gustavo de-deus2, aline de almeida neves4, edson jorge lima moreira1, emmanuel joão nogueira leal silva1 1department of endodontics, school of dentistry, grande rio university (unigranrio), duque de caxias, rj, brazil 2department of endodontics, fluminense federal university (uff), niterói, rj, brazil 3department of restorative dentistry ii, federal university of maranhão (ufma), são luis, ma, brazil 4department of paediatric dentistry, rio de janeiro federal university (ufrj), rio de janeiro, rj, brazil correspondence to: prof. dr. emmanuel joão nogueira leal silva department of endodontics, school of dentistry, grande rio university (unigranrio) rua herotides de oliveira, 61/902, icaraí niterói rj brazil, zipcode: 24230-230 phone: (55) 21 83575757 e-mail: nogueiraemmanuel@hotmail.com abstract aim: to evaluate the ability of rotary (protaper universal [ptu] and protaper next [ptn]), reciprocating (reciproc [r] and waveone [wo]) and adaptive (twisted file adaptive [tfa]) systems in maintaining the original canal profile in straight and curved parts after apical preparations up to size 40. methods: resin blocks with simulated curved canals were randomly assigned to five groups: ptu, ptn, r, wo and tfa. images were captured from each block before and after canal preparation (n=10). assessment of canal transportation was obtained for the straight and curved parts of the canal. anova followed by tukey’s test was used (α = 5%). results: transportation values were increased at the curved part (p = .00). for both canal levels, tfa system induced the lowest mean of canal transportation followed by ptn, r, wo and ptu systems. at the straight portion, transportation for r and tfa systems were similar (p > .05), and these values were significantly lower than for wo, ptn and ptu (p = .00). at the curved portion, tfa resulted in less canal transportation, followed by ptn, r, wo and ptu systems (p = .00). conclusions: tfa system produced less canal transportation than other systems tested during large apical preparations. keywords: endodontics. root canal preparation. instrumentation. introduction root canal shaping is considered one of the most challenging tasks during endodontic treatment. although several techniques were developed to minimize errors arising from root canal instrumentation1, accidents such as zips, ledges, root perforation and canal/apex transportation can occur, especially in narrow and curved canals2,3. overall, nickel-titanium (niti) instruments have improved canal preparation procedures and reduced the odds of iatrogenic defects4,5. briefly, niti files have improved mechanical canal preparation by offering better centering ability, less extrusion of debris and a reduced learning curve for the clinician4,5. further developments of novel nitibased root canal preparation systems have been primarily centered on modifications in instruments design, alloy and shaping movements. received for publication: october 3, 2016 accepted: april 24, 2017 original article braz j oral sci. july | september 2016 volume 15, number 3 http://dx.doi.org/10.20396/bjos.v15i3.8649985 222 canal preparation with reciprocating movement has been shown to reduce file fracture6-8. the reciprocation-based systems available on the market reciproc (r) (vdw, munich, germany) and waveone (wo) (dentsply maillefer, ballaigues, switzerland)] allow single-file preparation for the entire root canal, thus requiring less time when compared to multi-file rotary systems. these files are made of a new niti alloy (m-wire), that in conjunction with the reciprocating kinematic, provided an increase in flexibility and an improved resistance to cyclic fatigue8. recently, the twisted file adaptive system (tfa) (sybronendo; orange, ca, usa) has been introduced onto the market. in theory, this system claims to maximize the advantages of using the reciprocation movement while downgrading possible disadvantages associated with this kinematic. the tfa system uses a patented unique motion technology, which automatically adapts the movement according to the instrumentation stress input to the file. according to the manufacturer, when stresses are imposed to the tfa instrument inside the canal, the motor performs a conventional clockwise movement, allowing better cutting efficiency and removal of debris. in contrast, during increased torsional stress, the movement automatically changes into a reciprocation mode. furthermore, tfa files have three unique design features: a special surface conditioning, an r-phase heat treatment and a twisting of the metal9,10. recent scientific evidence shows that larger apical canal preparations promote more effective irrigation into the apical area, improved infection control and better quality of the root fillings11-13. however, limited data regarding canal transportation after larger apical preparations using these new niti systems are available. thus, the aim of this study was to assess the ability of rotary [protaper universal (ptu) (dentsply maillefer) and protaper next (ptn) (dentsply maillefer)], reciprocating (r and wo) and adaptive (tfa) systems in maintaining the original profile in straight and curved parts of the canal after apical preparations up to size 40. the null hypothesis tested was that there would be no differences in canal transportation values among the tested systems. material and methods digital image acquisition a total of sixty endo training iso 15 simulated curved canals in clear resin blocks (dentsply maillefer) with 2% taper, 70° angle of curvature, 10-mm radius of curvature and 17-mm length were assigned to five experimental groups and one control group (n = 10) according to the system used for canal preparation: ptu, ptn, r, wo and tfa. a circular base with a rectangular slot matching the resin block dimensions was inserted into the microscope base used to record the digital images (1005t opticam stereomicroscope; opticam, são paulo, brazil). after that, each specimen was positioned again in the slot, and color images were obtained and saved in tiff format using a dedicated digital camera (cmos 10 megapixels; opticam). after the preparation procedures, new images were taken from each block following the same described protocol. in order to check the accuracy of the repositioning method, ten resin blocks were used as a control group where no canal preparation was performed. in this group, two stereoscopic images of each block were taken after consecutively inserting and removing each specimen from the silicon slot14. canal preparation in all experimental groups, a 15 k-file (dentsply maillefer) was used to scout the canal up to the working length (wl) in order to create an initial and standardized glide path. after that, each system was used in the following manner: ptu. ptu instruments were used at 300 rpm and 2 ncm torque (silver reciproc; vdw). the following sequence was used: sx file (19/0.04; 1/2 of the wl), s1 file (18/0.02; 2/3 of the wl), s2 file (20/0.04; 2/3 of the wl), f1 (20/0.07; full wl), f2 (25/0.08; full wl), f3 (30/0.09; full wl) and f4 (40/0.06; full wl) files. ptn. ptn instruments were used at 300 rpm and 2 ncm torque. the following sequence was used: x1 (17/0.04; full wl), x2 (25/0.06; full wl), x3 (30/0.07; full wl) and x4 (40/0.06; full wl) files. r. r40 (40/0.06) instruments were used at the pre-setting program (reciproc all) powered by a torque-controlled motor (silver reciproc). the instrument was gradually advanced in the root canal using pecking motion with a 3 mm amplitude limit. after 3 complete pecking movements, the instrument was removed from the simulated canal and were cleaned using a sponge. wo. wo large (40/0.08) files were used similarly to the r group, under waveone all pre-setting program. tfa. the instruments were used under tfa motion (elements adaptive motor; sybronendo). the following sequence was used: sm1 (20/0.04; full wl), sm2 (25/0.06; full wl), sm3 (30/0.06; full wl), sm4 (35/0.06; full wl) and sm5 (40/0.04; full wl) files. a single experienced operator performed all preparation procedures, and only new instruments were used. apical patency was established with a 10 k-file (dentsply maillefer) just beyond the wl between each preparation step. canals were irrigated with 1.0 ml sterile water using 30g max-i-probe needle (dentsply rinn; elgin, il, usa) placed to a depth immediately short of binding. after the completion of each preparation, the canal was irrigated with 1.0 ml sterile water and post preparation images were acquired, as described previously. image processing and analysis filtering, registration, segmentation and extraction of attributes from the acquired images were performed using an open source software (fiji) and its associated plugins15. image processing and analysis was based on a previously published methodology14. in short, the images were first converted to 8-bit grayscale. then, each pair of image (before and after canal preparation) was registered using the “rigid registration” plugin. an iterative polygon tracing tool was used as a threshold method to segment each canal (sound and instrumented) from the background. canal boundaries were visually determined by the quantitative transportation assessment in simulated curved canals after large apical preparations braz j oral sci. 15(3):221-225 223quantitative transportation assessment in simulated curved canals after large apical preparations braz j oral sci. 15(3):221-225 user aided by an automatic edge-segmentation algorithm. after tracing, a simple binarization scheme (0 for background, 255 for the defined polygon) was applied and, after that, a skeletonization algorithm was applied to the segmented images. this algorithm finds the centerlines (skeleton) in segmented images by applying binary thinning procedures (symmetrical erosion)16. the distance (in mm) between each xy coordinate in the sound and in the instrumented skeleton images were calculated using the following formula: where xb and yb are coordinates for the sound canal and xi and yi are coordinates for the instrumented canal. figure 1 shows images taken before and after preparation and the corresponding centerlines obtained after the analysis. the transportation measurements were converted from pixels to millimeters (mm) with the aid of a microscope magnification scale. after that, the transportation values were quantified for the complete canal or for two independent regions (straight and curved parts), as depicted in figure 2. statistical analysis quantification of deviation by pixels resulted in a great number of data points (straight canal part = 21,960 and curved canal part = 33,600). in this study, each pixel was considered as a unit for statistical analysis. considering the data size, a bell-shaped distribution has been assumed, and a univariate analysis of variance (two-way) procedure, with a significance level of α = 5%, has been selected considering root canal portion and instrumentation systems as independent variables and canal transportation (in mm) as the dependent. tukey honestly significant difference test was used for pair-wise comparisons. results the control group showed no canal transportation, confirming the consistency and the reliability of the current methodology. as seen in table 1, canal transportation was significantly influenced by the different instrumentation systems (p = .00). compared with the straight part of the root canal, transportation values were increased at the curved canal parts (p = .00) for all instrumentation systems. when all canal extension was considered, tfa system induced the lowest mean of canal transportation followed by ptn, r, wo and ptu systems. however, a significant interaction between canal part and instrumentation system was found. at the straight portion, r and tfa systems produced similar transportation (p > .05), which was significantly lower than wo, ptn and ptu systems (p = .00); at the curved part, tfa system resulted in the lowest canal transportation followed by ptn, r, wo and ptu systems (p = .00). figure 3 illustrates transportation values for each used system in each part of the simulated canal. √(xb‒xi)2 + (yb‒yi)2 table 1 canal transportation after large apical preparations using different instrumentation systems. system straight curved all ptu (n = 10) 0.087 ± 0.149a 0.199 ± 0.244a 0.160 ± 0.221a ptn (n = 10) 0.056 ± 0.050b 0.059 ± 0.047c 0.058 ± 0.048d r (n = 10) 0.025 ± 0.016d 0.111 ± 0.065b 0.080 ± 0.067c wo (n = 10) 0.040 ± 0.034c 0.110 ± 0.067b 0.085 ± 0.066b tfa (n = 10) 0.026 ± 0.014d 0.047 ± 0.042d 0.039 ± 0.036e mean (mm) ± standard deviation. different lowercase letters indicate a significant difference (p < 0.05) in columns confirmed by tukey honestly significant difference for both straight and curved canal portions and for all canal extension. fig. 1 image of a simulated canal block taken before (a) and after (b) preparation and corresponding centerlines obtained after the analysis. (c) superposition of baseline and final centerlines showing that transportation occurred after instrumentation. fig. 2 determination of straight and curved parts of one simulated canal before and after instrumentation. 224 quantitative transportation assessment in simulated curved canals after large apical preparations discussion biomechanical preparation during root canal treatment is aimed to prevent or eliminate apical periodontitis, contributing to higher endodontic predictability17. recent scientific evidence shows that larger apical preparations presents several advantages, such as increasing the irrigant solution volume at the apical third of the root canal system13,18, more debris elimination and a reduction of non-instrumented areas in the root canals19,20. moreover, the increase in apical diameter is related to a reduction in bacterial population and improved canal filling procedures11-13. however, it is certainly not an easy clinical task to achieve a larger apical diameter, especially in curved, narrow and long canals. one major point of concern is associated with the higher incidence of mishaps such as canal transportation, which can occur during preparation of curved canals2,3. thus, it is relevant to assess the efficacy of root canal preparation instruments. therefore, the current study compared the ability of ptu, ptn, r, wo and tfa systems in maintaining the original canal anatomy after large apical preparations. the standardization of the experimental design is important during the evaluation of the shaping ability of different niti systems. however, due to the great number of confounding variables present in the experimental design of the commercially available instruments such as the manufacturing process, the number of files and kinematics, it is not always possible to isolate the influence of each variable on the obtained results. overall, the findings of the present study showed that the tfa system presented lower canal transportation than the other systems tested. the null hypothesis was then rejected. previous studies have already shown that the tfa system induced lower canal transportation and produced a better centering ability when compared to rotary14 and reciprocating systems10,21. this result may be explained by the tfa system unique design features (special surface conditioning, r-phase heat treatment and twisting of the metal)9,10 associated with its lower taper (0.04). regarding the adaptive motion, silva et al.14 (2015) evaluated canal transportation using twisted file system both in adaptive and in rotary motions and concluded that the latter produced overall lower canal transportation10. therefore, this new kinematics cannot be braz j oral sci. 15(3):221-225 considered as the solely factor influencing the good results of tfa system herein. on the other hand, the ptu system exhibited the worst results, showing higher canal transportation, which are in agreement with previous studies5,14. some aspects might give some rationale to support these results. first, the ptu system used seven instruments to prepare the canals. in addition, it is conceived that the ptu system has a tendency to straighten curved canals causing transportation toward the furcation at the middle-coronal thirds and toward the outer aspect of the curvature at the apical third22. although r and wo systems have some similarities, such as the reciprocating motion and the m-wire niti alloy, r system showed less transportation in the straight portion and when all canal was considered. these results may be explained by differences in the cross-sectional design: the larger cross-sectional area of w system influences the bending resistance of the instrument, making it less flexible and increasing the straightening tend in curved canals23. the results obtained by the ptn system may be explained by the use of a progressive tapers on a single file and the unique offset mass of rotation. this design serves to minimize the contact between a file and dentine, decreasing dangerous taper lock, screw effect and root canal transportation. simulated curved canals in clear resin blocks were used in the current study. this method has been previously validated in order to evaluate the centering ability and canal transportation provided by endodontic instruments3,14. in addition, it is particularly attractive due to the possibility of standardizing the full canal anatomy. nevertheless, the use of resin blocks has a few disadvantages such as micro-hardness differences between the resin material and the root dentin, and the possible side effects created by heat generated during preparation procedures, which may soften the resin, leading to binding of the cutting blades and enhancing the chance of instrument fracture24. thus, care should be taken before extrapolating these results directly to a clinical situation. the present investigation used a recent described methodology to study transportation in simulated curved canals by confronting images before and after canal preparation14. this procedure has the potential to reduce the bias related to the subjective operatorbased image superimposition schemes and evaluation of canal transportation, once it is almost not dependent on user input and also provides information from the whole canal length, and not only from selected slices. although the bi-dimensional approach can be considered a limitation of this method, it is of outmost importance to state that current three-dimensional-based techniques used to assess canal transportation have not yet provided fully quantitative volumetric data10,25, which again results in the evaluation of limited slices and manual selection of gravity center points. conclusions under the conditions of this study, it can be concluded that tfa system produced less canal transportation than the other systems tested during large apical preparations. references 1. roane jb, sabala cl, duncanson jg. the balanced force concept for instrumentation of curved canals. j endod. 1985 may;11(5):203-11. fig. 3 transportation in the straight and curved portions for all instrumentation systems. 2. peters ao, peters ic. cleaning and shaping of the root canal system. in: cohen s, hargreaves mk, editors. pathways of the pulp. 9. ed. saint louis: mosby inc; 2006. p. 290-357. 3. weine fs, kelly rf, lio pj. the effect of preparation procedures on original canal shape and on apical foramen shape. j endod. 1975 aug;1(8):255-62. 4. bergmans l, van cleynenbreugel j, wevers m, lambrechts p. mechanical root canal preparation with niti rotary instruments: rationale performance and safety status report for the american journal of dentistry. am j dent. 2001 oct;14(5):324-33. 5. gergi r, rjeily ja, sader j, naaman a. comparison of canal transportation and centering ability of twisted files, pathfile-protaper system, and stainless steel hand k-files by using computed tomography. j endod. 2010 may;36(5):904-7. doi: 10.1016/j.joen.2009.12.038. 6. de-deus g, moreira ej, lopes hp, elias cn. extended cyclic fatigue life of f2 protaper instruments used in reciprocating movement. int endod j. 2010 dec;43(12):1063-8. 7. kiefner p, ban m, de-deus g. is the reciprocating movement per se able to improve the cyclic fatigue resistance of instruments? int endod j. 2014 may;47(5):430-6. doi: 10.1111/iej.12166. 8. gambarini g, rubini ag, al sudani d, gergi r, culla a, de angelis f, et al. influence of different angles of reciprocation on the cyclic fatigue of nickel-titanium endodontic instruments. j endod. 2012 oct;38(10):140811. doi: 10.1016/j.joen.2012.05.019. 9. gambarini g, gergi r, naaman a, osta n, al sudani d. cyclic fatigue of twisted file rotary niti instruments used in reciprocating motion. int endod j. 2012 sep;45(9):802-6. doi: 10.1111/j.1365-2591.2012.02036.x. 10. gergi r, osta n, bourbouze g, zgheib c, arbab-chirani r, naaman a. effects of three nickel titanium instrument systems on root canal geometry assessed by micro-computed tomography. int endod j. 2015 feb;48(2):162-70. doi: 10.1111/iej.12296. 11. van der sluis lw, shemesh h, wu mk, wesselink pr. an evaluation of the influence of passive ultrasonic irrigation on the seal of root canal fillings. int endod j. 2007 may;40(5):356-61. 12. elayouti a, kiefner p, hecker h, chu a, löst c, weiger r. homogeneity and adaptation of endodontic fillings in root canals with enlarged apical preparation. oral surg oral med oral pathol oral radiol endod. 2009 sep;108(3):e141-6. doi: 10.1016/j.tripleo.2009.04.022. 13. brunson m, heilborn c, johnson dj, cohenca n. effect of apical preparation size and preparation taper on irrigant volume delivered by using negative pressure irrigation system. j endod. 2010 apr;36(4):721-4. 225quantitative transportation assessment in simulated curved canals after large apical preparations braz j oral sci. 15(3):221-225 doi: 10.1016/j.joen.2009.11.028. 14. silva ej, tameirão md, belladonna fg, neves aa, souza em, dedeus g. quantitative transportation assessment in simulated curved canals prepared with an adaptive movement system. j endod. 2015 jul;41(7):1125-9. doi: 10.1016/j.joen.2015.02.028. 15. schneider ca, rasband ws, eliceiri kw. nih image to imagej: 25 years of image analysis. nature methods. 2012 jul;9(7):671-5. 16. lee tc, kashyap rl, chu cn. building skeleton models via 3-d medial surface/axis thinning algorithms. comput vis graph image process.1994;56:462-78. 17. schilder h. cleaning and shaping the root canal. dent clin north am. 1974 apr;18(2):269-96. 18. boutsioukis c, gogos c, verhaagen b, versluis m, kastrinakis e, van der sluis lw. the effect of root canal taper on the irrigant flow: evaluation using an unsteady computational fluid dynamics model. int endod j. 2010 oct;43(10):909-16. doi: 10.1111/j.1365-2591.2010.01767.x. 19. fornari vj, silva-sousa yt, vanni jr, pécora jd, versiani ma, sousaneto md. histological evaluation of the effectiveness of increased apical enlargement for cleaning the apical third of curved canals. int endod j. 2010 nov;43(11):988-94. doi: 10.1111/j.1365-2591.2010.01724.x. 20. de-deus g, marins j, silva ej, souza e, belladonna fg, reis c, et al. accumulated hard tissue debris produced during reciprocating and rotary nickel-titanium canal preparation. j endod. 2015 may;41(5):676-81. doi: 10.1016/j.joen.2014.11.028. 21. ordinola-zapata r, bramante cm, duarte ma, cavenago bc, jaramillo d, versiani ma. shaping ability of reciproc and tf adaptive systems in severely curved canals of rapid microct-based prototyping molar replicas. j appl oral sci. 2014 nov-dec;22(6):509-15. doi: 10.1590/1678775720130705. 22. bürklein s, schäfer e. critical evaluation of root canal transportation by instrumentation. endod topics. 2013;29:110-24. 23. de-deus g, vieira vtl, silva ejnl, lopes hp, elias cn, moreira ej. bending resistance and dynamic and static cyclic fatigue life of reciproc and waveone large instruments. j endod. 2014 apr;40(4):575-9. doi: 10.1016/j.joen.2013.10.013. 24. yoo ys, cho yb. a comparison of the shaping ability of reciprocating niti instruments in simulated curved canals. restor dent endod. 2012 nov;37(4):220-7. doi: 10.5395/rde.2012.37.4.220. 25. freire lg, gavini g, cunha rs, santos md. assessing apical transportation in curved canals: comparison between cross-sections and micro-computed tomography. braz oral res. 2012 may-jun;26(3):222-7. braz j oral sci. 15(4):315-319 fungal and viral oral infections in individuals with onco-hematologic neoplasms in a university hospital thaiane dantas dias dos santos,1 patrícia leite ribeiro,2 renata portela de rezende,1 davi silva carvalho curi,3 joão frank carvalho dantas de oliveira,4 viviane almeida sarmento5 1student of federal university of bahia postgraduate program in dentistry 2doctor, associate professor of dentistry college of federal university of bahia 3student of federal university of pernambuco postgraduate program in child and adolescent health 4post doctor, assistant teacher of dentistry college of federal university of bahia 5post doctor, associate professor of dentistry college of federal university of bahia correspondence to: thaiane dantas dias dos santos address: rua maria dos reis silva nº212, loteamento miragem, lauro de freitas, bahia, brasil. 42.700-000. telephone: +55 (71) 99276-8390 e-mail: thaianedantas@yahoo.com.br abstract introduction: individuals undergoing onco-hematologic treatment present higher risk for developing oral and/or systemic infections, due to the intense immunosuppression and compromise of the mucosal barriers during treatment. the aim of this study was to identify the fungal and viral oral infections that most frequently affected patients undergoing onco-hematologic treatment in a university hospital, and relate them to the biopsychosocial data, underlying disease and treatment. methods: this was a crosssectional study in which descriptive analysis of the data was performed, and the exact fisher test was applied to verify the association between the infections and the variables: sex, age group, educational level, underlying disease and treatment, considering a 5% probability of error. results and discussion: the clinical exam revealed that oral candidiasis was present in 8.92% of the individuals, and herpes simplex, in 4.5%. no significant associations were found between the variables and infections. the fungal and viral oral infections were little prevalente in individuals with hematologic neoplasms in the present study, suggesting that the action of dental surgeons in caring for the onco-hematologic patients at the institution evaluated and the protocols use were efficient. keywords: hematological diseases, oral candidiasis, herpes simplex. received for publication: may 03, 2017 accepted: june 15, 2017 original article braz j oral sci. october | december 2016 volume 15, number 4 http://dx.doi.org/10.20396/bjos.v15i4.8650048 introduction oral health care must be included in the treatment of individuals with oncohematologic neoplasms, since they may present deficient oral hygiene or infectious foci that predispose them to higher risk of developing oral and/or systemic infections when submitted to chemotherapy and/or bone marrow transplantation (bmt)1-3. furthermore, oral manifestations resulting from the direct action of onco-hematologic therapy on the oral tissues, or indirectly due to immunosuppression may increase the risk of mortality, time of hospitalization and treatment costs3-5. among the oral manifestations caused by immunosuppression, fungal and viral infections are outstanding, especially candidiasis and herpes simplex, respectively. chart, clinical chart from the dental service). all the patients received the necessary care suited to their oral requirements, including guidance on diet and oral hygiene, elimination of infectious foci and control of oral biofilm. moreover, they continued to receive dental follow-up or treat possible oral complications. descriptive analysis of the data was performed, and the mean and frequency were calculated. in addition the exact fisher test was used to verify the association between the infections and the variables: sex, age group, educational level, underlying disease and treatment, considering a 5% probability of error. results considering the eligibility criteria, 112 individuals with onco-hematologic neoplasms formed part of the sample. in relation to educational level, 40 individuals had no records, and analysis was performed in the 72 remaining individuals. table 1 shows the sociodemographic characteristics, underlying disease, oncological treatment instituted, and those of the oral infections of the sample. of these, 54 (48.2%) were of the male sex and 58 (51.8%) were women. their ages ranged from 15 to 85 years, with a mean age of 41.08 (+17.26)years, and there was discrete predominance of the age group 43-54 years. of the 72 individuals who had records of schooling, those who had up to 8 years of schooling (45.8%) predominated, followed by those we were from nine to 11 years of schooling (41.7%) (table 1). 316 candidiasis normally presents in the pseudomembranous6 form, with formation of whitened, scrapable plaques on the oral mucosa, capable of causing the patient discomfort. topical antifungal agents, such as nystatin, are the first line of treatment for candidiasis7,8, however, systemic antifungal medications are more effective in immunosuppressed individuals8. in relation to viral infections, immunosuppression may reactivate latent viral infections, generally with more serious atypical signs than those manifesting in immunocompetent patients9,10. in the oral mucosa, herpes simplex is the most common viral infection, characterized by the formation of vesicles that rupture and form painful ulcers in the oral cavity and oropharynx11. the use of systemic antiviral agents such as aciclovir and valaciclovir, is indicated for prophylaxis and treatment of these lesions12, in addition to complementary therapies, used for pain relief and reduction in the period of clinical manifestation, such as laser photobiomodulation13. the authors therefore suggest the inclusion of the dental surgeon (ds) in the multiprofessional team that cares for individuals with onco-hematologic neoplasms to prevent, diagnose and treat these manifestations, with a view to minimizing suffering and improve the quality of life during oncological treatment. nevertheless, there is a scarcity of studies that show the frequency of these infections in centers with the presence of the dental surgeon. therefore, the aim of this study was to evaluate the frequency of fungal and viral oral infections in individuals submitted to onco-hematologic treatment, in a university hospital, and associated them with the age, sex, educational level, underlying disease and oncological treatments performed. the hypothesis tested was that the frequency of these infections would be low, irrespective of the sociodemographic factors, disease and oncological treatment instituted, due to the regular dental care offered to this group of patients. methods this was an observational, cross-sectional, descriptive and quantitative study approved by cep/hupes (caee 1.158.496). included in the study were individuals undergoing oncohematologic treatment in a public university hospital, followedup by the dental service of the same hospital, in the period from november, 2013, to february, 2016, who accepted to participate in this research by signing the term of free and informed consent. the patients who did not wish to participate, and those who were in a physical condition to undergo the dental exam were excluded from the sample. identification data were collected and registered (age, gender and educational level), in addition to information about the underlying disease (diagnosis and treatment instituted) and subsequently the intraoral exam was performed with a dental mirror and oral retractors, under lighting suitable for identifying lesions of fungal or viral origin. the data of patients who had been discharged from hospital, or who were already being followed-up by the dental service prior to the research were collected from secondary sources (record fungal and viral oral infections in individuals with onco-hematologic neoplasms in a university hospital braz j oral sci. 15(4):315-319 table 1 sociodemographic characteristics of the base disease of the oncological treatment instituted, and the oral infections in onco-hematologic individuals at hupes/ufba, salvador, brazil, 2013-2016. categorical variables n % age 15-24 years 28 25.0 25-42 years 27 24.1 43-54 years 30 26.8 > 54 years 27 24.1 sex male 54 48.2 female 58 51.8 educational levela up to 08 years 33 45.8 9-11 years 30 41.7 > 11 years 09 12.5 underlying disease: acute leukemias chronic leukemias 10 8.9 lymphomas 27 24.1 multiple myeloma 32 28.6 mds 02 1.8 oncological treatment qtx alone 62 55.4 qtx + other treatments 40 35.7 bmt alone 10 8.9 oral infections without infections 97 86.6 candidiasis 10 8.9 herpes simplex 05 4.5 athe absolute and relative frequencies of this variable were calculated for 72 individuals. the 40 remaining subjects had no record of schooling. mds=myelodysplastic syndrome; qtx=chemotherapy; bmt=bone marrow transplant. 317 in relation to the underlying disease, acute leukemias (36.6%) and multiple myeloma (28.6%) were the most frequent onco-hematologic neoplasms, respectively. as regards the oncological treatment instituted, the majority of the individuals were submitted to chemotherapy alone (55.4%). in relation to the fungal and viral oral infections, candidiasis was present in 8.92% of the individuals, and herpes simplex in 5,35% (table 1). fungal and viral oral infections in individuals with onco-hematologic neoplasms in a university hospital braz j oral sci. 15(4):315-319 table 2 shows the association between oral candidiasis, age, sex, educational level, underlying disease and oncological treatment instituted. there were a higher number of cases in the age group from 15-24 years (50%), in the female sex (60%)and in individuals who had between nine and 11 years of schooling (57.1%), however, without statistically significant difference for the three variables (p>0.05). table 2 association between oral candidiasis and age, sex, educational level, underlying disease and oncological treatment instituted in onco-hematologic individuals of hupes/ufba, salvador, brazil, 2013-2016. oral candidiasis paindependent variables yes (n=10) no (n=102) n % n % age 15-24 years 5 50.0 23 22.5 0.35 25-42 years 2 20.0 25 24.5 43-54 years 2 20.0 28 27.5 > 54 years 1 10.0 26 25.5 sex male 4 40.0 50 49.0 0.41 female 6 60.0 52 51.0 educational levelb up to 08 years 3 42.9 30 46.2 0.649-11 years 4 57.1 26 40.0 > 11 years 0 0.0 09 13.8 underlying disease: acute leukemias 2 20.0 39 38.2 0.10 chronic leukemias 2 20.0 08 7.8 lymphomas 5 50.0 22 21.6 multiple myeloma 1 10.0 31 30.4 mds 0 0.0 02 2.0 oncological treatment qtx alone 7 70.0 55 53.9 0.68qtx + other treatments 3 30.0 37 36.3 bmt 0 0.0 10 9.8 mds=myelodysplastic syndrome; qtx=chemotherapy; bmt=bone marrow transplant. aexact fisher test level of significance p < 0.05. bthe absolute and relative frequencies of this variable were calculated for 72 individuals. the 40 remaining subjects had no record of schooling. in relation to the underlying disease, oral candidiasis was most frequent in those with diagnosis of lymphoma (5), although without statistically significant difference (p>0.05). as regards the oncological treatment instituted, the majority of individuals who developed candidiasis received only chemotherapy, however, without statistically significant difference (p>0.05) (table 2). table 3 shows the association between herpes simplex, age, sex, educational level, underlying disease and oncological treatment instituted. this infection was more frequent in the female sex although there was no statistically significant difference (p>0.05). there was no predilection for age group and educational level (p> 0.05), but the individuals from 15-24 years (40.0%) and 25-42 years (40.0%) and those who had up to eight years of schooling (50%) and between nine and 11 years of schooling (50%) were those most affected. in relation to the underlying disease, herpes simplex was most frequent in those with diagnosis of multiple myeloma (40%), and acute leukemias (40%) although without statistically significant difference (p>0.05). as regards the oncological treatment instituted, the majority of individuals who had herpes simplex received chemotherapy associated with other therapies (60%), followed by those who were submitted to chemotherapy alone (40%), however, not presenting any statistically significant difference (p> 0.05) (table 3). 318 in the study of gomez et al.17 (2001) the incidence of hsv in patients who were submitted to bmt was 26.9%. in the present study infection by the hsv affected only 4.5% of the individuals. this frequency was low, compared with those of other studies10,17, suggesting that the prophylactic regimes for viral infections15, adopted at the institution evaluated were effective. these prophylactic regimes may vary according to the underlying disease, chemotherapy regime and patient’s clinical condition. however, in general, the antiviral prophylaxis used by the service consists of the administration of aciclovir, beginning concomitantly with chemotherapy treatment. these findings corroborated controlled clinical trial studies with the use of aciclovir in patients with hematologic diseases, in whom a reduction of 80-90% in risk of infection by hsv was observed12,18. in relation to fungal infections it is known that in individuals with leukopenia due to myelosuppression, the main fungal infection were caused by candida albicans, resulting not only from neutropenia, but also from the use of broad spectrum antibiotics and inadequate oral hygiene19. this susceptibility increased when medications with high aggressive potential against the defense cells and oral mucosa cells are administered, such as chemotherapy drugs that induce epithelial changes and will favor the adhesion and proliferation of the microorganism20. in the study of xu et al.21 (2013) oral candidiasis affected 52% of the 850 patients studied, while in the present study candidiasis was present in only 8.9% of the patients. this difference may be explained by the fact that in the cited study, discussion individuals undergoing onco-hematologic therapy are more susceptible to viral, bacterial and fungal infections, due to the intense immunosuppression and compromise of mucosal barriers1-3, in addition to quantitative and qualitative changes in the oral microbioma, capable of occurring in over 70% of immunosuppressed patients14. in the present study, only 15 individuals (13.4%) presented fungal or viral infections. this low rate may be explained by the infectious prophylaxis protocols instituted by the oncohematologic service and by the dental care that patients receive during antineoplastic treatment. at this treatment center, in addition to receiving adequate oral hygiene guidance, patients are submitted to dental procedures suited to the oral medium, such as removal of root remainders, root scaling and planing, endodontic treatment, carious tissue removal and dental cavity sealing. these actions effectively tend to reduce the pathogenic oral microbioma and possibly influenced the results here observed. among the viral infections in the oral mucosa, infection by the herpes simplex virus (hsv) has been pointed out as the most common in immunocompromised patients. the reactivation of hsv affected up to 80% of the patients with hematological diseases before the use of antiviral prophylaxis15. the labial region most frequently being affected, followed by the keratinized mucosas, such as the palate, dorsum of the tongue and gingiva16. fungal and viral oral infections in individuals with onco-hematologic neoplasms in a university hospital table 3 association between herpes simplex and age, sex, educational level, underlying disease and oncological treatment instituted in onco-hematologic individuals of hupes/ufba, salvador, brazil, 2013-2016. oral candidiasis paindependent variables yes (n=10) no (n=102) n % n % age 15-24 years 2 40.0 26 24.3 0.5625-42 years 2 40.0 25 23.443-54 years 1 20.0 29 27.1 > 54 years 0 0.0 27 25.2 sex male 1 20.0 53 49.5 0.36female 4 80.0 54 50.5 educational levelb up to 08 years 2 50.0 31 45.6 1.009-11 years 2 50.0 28 41.2 > 11 years 0 0.0 09 13.2 underlying disease: acute leukemias 2 40.0 39 36.4 1.00 chronic leukemias 0 0.0 10 9.3 lymphomas 1 20.0 26 24.3 multiple myeloma 2 40.0 30 93.8 mds 0 0.0 02 1.9 oncological treatment qtx alone 2 40.0 60 56.1 0.61qtx + other treatments 3 60.0 37 34.6 bmt 0 0.0 10 9.3 mds=myelodysplastic syndrome; qtx=chemotherapy; bmt=bone marrow transplant. aexact fisher test level of significance p < 0.05. bthe absolute and relative frequencies of this variable were calculated for 72 individuals. the 40 remaining subjects had no record of schooling. braz j oral sci. 15(4):315-319 319fungal and viral oral infections in individuals with onco-hematologic neoplasms in a university hospital the author affirmed that the patients were in an advanced stage of the disease, with inadequate nutritional and oral hygiene status, whereas in the present study, the patients were on nutritional support therapy, antifungal prophylaxis with a systemic antifungal agent22, received oral hygiene instructions and underwent dental follow-up during antineoplastic therapy, therefore presenting adequate oral hygiene. in view of the findings, the authors perceived that the presence of the dental surgeon in the care of individuals with onco-hematologic neoplasms was related to a low frequency of fungal and viral infections, irrespective of the sociodemographic factors, disease and oncological treatment instituted. this guaranteed prevention, diagnosis and early treatment of infections and other complications secondary to the neoplasm and oncological treatment. references 1. rizzo jd, wingard jr, tichelli a, lee sj, van lint mt, burns lj, et al. recommended screening and preventive practices for long-term survivors after hematopoietic cell transplantation: joint recommendations of the european group for blood and marrow transplantation, the center for international blood and marrow transplant research, and the american society of blood and marrow transplantation. biol blood marrow transplant. 2006 feb;12(2):138-51. 2. mcguire db, correa mep, johnson j, wienandts p. the role of basic oral care and good clinical practice principles in the management of oral mucositis. support care cancer. 2006 jun;14(6):541-7. 3. brennan mt, elting ls, spijkervet fkl. systematic reviews of oral complications from cancer therapies, oral care study group, mascc/ isoo: methodology and quality of the literature. support care cancer. 2010 aug;18(8):979-84. doi: 10.1007/s00520-010-0856-3. 4. majorana a, schubert mm, porta f, ugazio ag, sapelli pl. oral complications of pediatric hematopoietic cell transplantation: diagnosis and management. support care cancer. 2000 sep;8(5):353-65. 5. da silva santos ps, guaré ro, fachin lv, correa msnp. [bone marrow transplant: general considerations and oral manifestations in pediatric patients]. rev assoc paul cir dent. 2008;62(6):450-4. portuguese. 6. lalla rv, latortue mc, hong ch, ariyawardana a, d'amato-palumbo s, fischer dj, et al. a systematic review of oral fungal infections in patients receiving cancer therapy. support care cancer. 2010 aug;18(8):985-92. doi: 10.1007/s00520-010-0892-z. 7. gligorov j, bastit l, gervais h, henni m, kahila w, lepille d, et al. prevalence and treatment management of oropharyngeal candidiasis in cancer patients: results of the french candidoscope study. int j radiat oncol biol phys. 2011 jun 1;80(2):532-9. doi: 10.1016/j. ijrobp.2010.02.006. 8. reboli ac, shorr af, rotstein c, pappas pg, kett dh, schlamm ht, et al. anidulafungin compared with fluconazole for treatment of candidemia and other forms of invasive candidiasis caused by candida albicans: a multivariate analysis of factors associated with improved outcome. bmc infect dis. 2011 sep 30;11:261. doi: 10.1186/1471-2334-11-261. 9. gürgan ca, özcan m, karaku ş ö, zincircio ğlu g, arat m, soydan e, et al. periodontal status and post ‐transplantation complications following intensive periodontal treatment in patients underwent allogenic hematopoietic stem cell transplantation conditioned with myeloablative regimen. int j dent hyg. 2013 may;11(2):84-90. doi: 10.1111/j.16015037.2012.00550.x. 10. palmason s, marty fm, treister ns. how do we manage oral infections in allogeneic stem cell transplantation and other severely immunocompromised patients? oral maxillofac surg clin north am. 2011 nov;23(4):579-99, vii. doi: 10.1016/j.coms.2011.07.012. 11. chen yk, hou ha, chow jm, chen yc, hsueh pr, tien hf. the impact of oral herpes simplex virus infection and candidiasis on chemotherapyinduced oral mucositis among patients with hematological malignancies. eur j clin microbiol infect dis. 2011 jun;30(6):753-9. doi: 10.1007/ s10096-010-1148-z. 12. glenny am, fernandez mauleffinch lm, pavitt s, walsh t. interventions for the prevention and treatment of herpes simplex virus in patients being treated for cancer. cochrane database syst rev. 2009 jan 21;(1):cd006706. doi: 10.1002/14651858.cd006706.pub2. 13. bello-silva ms, de freitas pm, aranha ac, lage-marques jl, simões a, de paula eduardo c. low-and high-intensity lasers in the treatment of herpes simplex virus 1 infection. photomed laser surg. 2010 feb;28(1):135-9. doi: 10.1089/pho.2008.2458. 14. naylor gd, marino gg, shumway rc. glossodynia after radiation therapy and chemotherapy. ear nose throat j. 1989 oct;68(10):751-7. 15. teh bw, slavin ma, harrison sj, worth lj. prevention of viral infections in patients with multiple myeloma: the role of antiviral prophylaxis and immunization. expert rev anti infect ther. 2015;13(11):1325-36. doi: 10.1586/14787210.2015.1083858. 16. neville bw, damm dd, chi ac, allen cm. oral and maxillofacial pathology. saint louis: elsevier health sciences; 2015. 17. gomez rs, carneiro ma, souza ln, victória jm, de azevedo wm, de marco l, et al. oral recurrent human herpes virus infection and bone marrow transplantation survival. oral surg oral med oral pathol oral radiol endod. 2001 may;91(5):552-6. 18. yahav d, gafter-gvili a, muchtar e, skalsky k, kariv g, yeshurun m, et al. antiviral prophylaxis in haematological patients: systematic review and meta-analysis. eur j cancer. 2009 dec;45(18):3131-48. doi: 10.1016/j. ejca.2009.08.010. 19. braunwald, e, harrison tr. harrison's principles of internal medicine. 2001. new york: mcgraw-hill professional publishing; 2001. v.1. 20. soysa ns, samaranayake lp, ellepola anb. cytotoxic drugs, radiotherapy and oral candidiasis. oral oncology. 2004 nov;40(10):9718. 21. xu l, zhang h, liu j, chen x. investigation of the oral infections and manifestations seen in patients with advanced cancer. pak j med sci. 2013 sep;29(5):1112-5. 22. tacke d, buchheidt d, karthaus m, krause sw, maschmeyer g, neumann s, et al. primary prophylaxis of invasive fungal infections in patients with haematologic malignancies. 2014 update of the recommendations of the infectious diseases working party of the german society for haematology and oncology. ann hematol. 2014 sep;93(9):1449-56. doi: 10.1007/s00277-014-2108-y. braz j oral sci. 15(4):315-319 1http://dx.doi.org/10.20396/bjos.v19i0.8658890 volume 19 2020 e200001 letter to the editor 1 oral diagnosis, dental school, state university of montes claros (unimontes), montes claros, minas gerais, brazil. 2 center for rehabilitation of craniofacial anomalies, dental school, university of alfenas, minas gerais, brazil. 3 department of oral diagnosis, school of dentistry, university of campinas, piracicaba, são paulo, brazil. 4 hospital for rehabilitation of craniofacial anomalies, university of são paulo, bauru, são paulo, brazil. corresponding author: renato assis machado department of oral diagnosis, school of dentistry, university of campinas (fop/unicamp) limeira avenue, 901, areião, zip code:13414-018, piracicaba, sp, brazil phone: +55 35 991792635 renatoassismachado@yahoo.com.br received: march 24, 2020 accepted: march 25, 2020 oral medicine, oral pathology and coronavirus (2019-ncov): current challenges hercílio martelli-júnior1,2, renato assis machado3,4 , daniella r. barbosa martelli1, ricardo della coletta3 letter to the editor in late december 2019, a cluster of unexplained pneumonia cases was diagnosed in wuhan, china, and few days later, the causative agent of this mysterious pneumonia was identified as a novel coronavirus. this causative virus has been temporarily named as severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and the relevant infected disease has been named as coronavirus disease 2019 (covid-19) by the world health organization respectively. the covid-19 epidemic is spreading in china and all over the world now. as of march 1st, 2020, outside china, another 58 countries had confirmed covid-19 cases. now, on march 21h, there are already 170 countries, in addition to china. in the world we have already 292,142 confirmed cases and 12,784 deaths (https://www.who.int/docs/defaultsource/coronaviruse/situation-reports/20200322-sitrep-62-covid-19.pdf?sfvrsn=f7764c46_2). in brazil, by march 22th, 2020, the ministry of health has already confirmed 1546 cases of covid-19 infection and 25 deaths (https://saude.gov.br/). coronaviruses are enveloped, single-stranded, positive-sense rna viruses that are phenotypically and genotypically diverse, and widespread in bats around https://orcid.org/0000-0002-1697-3662 https://orcid.org/0000-0001-5285-3046 https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200322-sitrep-62-covid-19.pdf?sfvrsn=f7764c46_2 https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200322-sitrep-62-covid-19.pdf?sfvrsn=f7764c46_2 https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200322-sitrep-62-covid-19.pdf?sfvrsn=f7764c46_2 https://saude.gov.br/ 2 martelli-júnior et al. the world. coronaviruses can also be found in many other species as well, including humans, other mammals, and birds. they may cause respiratory, enteric, hepatic, or neurologic diseases. zhu et al. (2020)1 have isolated a novel coronavirus from human airway epithelial cells, which was named 2019-ncov5. after, zhou et al. (2020)2 demonstrated that the angiotensin-converting enzyme ii (ace2) is likely its cell receptor as well as the receptor for sars-cov and hcov-nl63. to assess the potential routes of 2019-ncov infection on the oral cavity mucosa, xu et al. (2020)3 showed that ace2 highly expressed in epithelial cells, and among different oral sites, ace2 expression was higher in tongue than buccal mucosa and gingival tissues. these findings indicate that the mucosa of oral cavity may be a potentially high risk route of 2019-ncov infection. dental patients and professionals can be exposed to pathogenic microorganisms, including viruses and bacteria that infect the oral cavity and respiratory tract. dental care settings invariably carry the risk of 2019-ncov infection due to the specificity of its procedures, which involves face-to-face communication with patients, frequent exposure to saliva, and blood, and the handling of sharp instruments. the pathogenic microorganisms can be transmitted in dental settings through inhalation of airborne microorganisms that can also remain suspended in the air for long periods4. scully et al. (2016)5 in an extensive paper evaluating the growth and performance of oral medicine, highlight the importance of the specialist in handle viral infections including those involving enterovirus, herpesvirus human immunodeficiency virus and human papillomavirus. however, in recent years we have observed the important participation of dentistry, particularly oral medicine and oral pathology, in infections caused by zika virus, chikungunya, and now with the covid-19. in the current scenario of outbreaks, epidemics, endemics and pandemics, the idea of multiprofessional, interdisciplinary and transdiciplinar action is reinforced. the participation of oral medicine and pathology are essential for the better understanding, diagnosis and management of these patients. acknowledgment the minas gerais state research foundation (fapemig, minas gerais, brazil), the national council for scientific and technological development (cnpq, brazil), and the coordination of training of higher education graduate foundation (capes, brasilia, brazil). conflict of interest the authors declare that they have no conflict of interest. references 1. zhu n, zhang d, wang w, li x, yang b, song j, et al. a novel coronavirus from patients with pneumonia in china, 2019. n engl j med. 2020;382:727-33. doi: 10.1056/nejmoa2001017. 2. zhou p, yang xl, wang xg, hu b, zhang l, si hr, et al. a pneumonia outbreak associated with a new coronavirus of probable bat origin. nature. 2020;579:270-3. doi: 10.1038/s41586-020-2012-7. 3 martelli-júnior et al. 3. xu h, zhong l, deng j, peng j, dan h, zeng x, et al. high expression of ace2 receptor of 2019cov on the epithelial cells of oral mucosa. int j oral sci. 2020;24:1-8. doi: 10.1038/s41368-020-0074-x. 4. kampf g, todt d, pfaender s, steinmann e. persistence of coronaviruses on inanimate surfaces and its inactivation with biocidal agents. j hosp infect. 2020;104:246-51. doi: 10.1016/j.jhin.2020.01.022. 5. scully c, miller cs, aguirre urizar jm, alajbeg i, almeida op, bagan jv, et al. oral medicine (stomatology) across the globe: birth, growth, and future. oral surg oral med oral pathol oral radiol. 2016;121:149-57. doi: 10.1016/j.oooo.2015.10.009. https://www.ncbi.nlm.nih.gov/pubmed/?term=scully c%5bauthor%5d&cauthor=true&cauthor_uid=26792755 1http://dx.doi.org/10.20396/bjos.v18i0.8656598 volume 18 2019 e191208 original article 1 department of restorative dentistry, faculty of dentistry, university of são paulo, são paulo, brazil. 2 universitary center unichistus, fortaleza, ceará, brazil. corresponding author: giulio gavini dept. of restorative dentistry of faculty of dentistry, university of são paulo av. prof. lineu prestes, 2227 – cidade universitária, são paulo, sp, brasil e-mail: ggavini@gmail.com phone: 55-11-30917839 received: june 04, 2018 accepted: april 28, 2019 influence of cross-section and number of use in cyclic fatigue resistance of rotary instruments luís césar brisighello1, george táccio de miranda candeiro1,2, luiza riomar paz1, hermano camelo paiva1, giulio gavini1,* aim: the aim of present study was to evaluate if the crosssection and the number of use have influence in cyclic fatigue resistance of rotary files. methods: k3 endo (group k) and endosequence (group e) files, 25/.04, 25 mm, were subdivided into 3 subgroups (n=12) according to the number of uses, 1, 3 and 5 uses, totalizing 72 files. the files were submitted to dynamic assays device moved by an electric engine, using 300 rpm of speed that permitted the reproduction of pecking motion. the files run within a temperate steel ring’s groove, simulating an instrumentation of a curved root canal with 40-degrees and 5-mm of curvature radius. the fracture of file was detected by sensor of device and the time and the number of cycles was acquired. the data were analyzed statistically by two-way anova (p<0.05). results: there were no statistical significant differences in regard to the number of uses (p>0.05). k3 endo files showed greater resistance to cyclic fatigue than endosequence instruments (p<0.01). conclusion: it may be concluded that the cross-section of instruments presented significant influence in cyclic fatigue resistance and the number of uses up to 5 times had no influence in cyclic fatigue resistance in both niti rotation systems evaluated. keywords: fatigue. nickel. titanium. dental instruments. 2 brisighello et al. introduction the major aim of endodontic treatment is to maintain or to promote the periapical health, with the maximum elimination of microorganisms of the root canals system. therefore, the chemomechanical preparation plays an important role in the cleaning and shaping of root canals with the use of several instruments and chemical auxiliary substances. in curved canals, there are some physical and mechanical challenges that difficult the correct modeling and the effective bacterial elimination. to carry out a better instrumentation of these root canals, rotary niti files are employed due its superelasticity and shape memory effects. these instruments allow the dentist to efficiently prepare the root canal with significantly less zipping and ledging and with minimal transportation towards the outer aspect of the curve1-3. the vast majority of the studies uniformly describe good maintenance of curvature even in severely curved root canals3-5. despite greater flexibility and torsion resistance, fracture is the major concern in niti files, especially after prolonged use6. unfortunately, most of these fractures occur unexpectedly, with no sign of permanent deformation. cyclic fatigue of the alloy, with successive tension and compression loads on the curved areas of the root canal, can be the most destructive form of cyclic load7. therefore, most cases of mechanical failure of niti rotary files during clinical use have been associated with cyclic fatigue8. the frequency of use of rotary niti files and the file cross-section are parameters that might affect the cyclic fatigue resistance of these instruments9,10. few studies have investigated dynamic cyclic fatigue using an apparatus11-13 that simulates the pecking motion even as the use of this movement during instrumentation by niti rotary files appears to significantly extend the life of the instrument. therefore, the purpose of this study was to assess the cyclic fatigue behavior of two niti rotary files with different cross-section, k3 endo (sybronendo, orange, usa) and endosequence (brasseler usa, savannah, ga, usa), submitted to repeatedly used in simulated curved canals, using an experimental cyclic fatigue testing apparatus that simulates the pecking motion in curved canals. thus, the null hypothesis is that rotary niti systems with different cross-section and that the repeat uses of instruments have no influence in resistance to cyclic fatigue during instrumentation in curved canals performing pecking motion. materials and methods three hundred twenty simulated canals were constructed using size 15 silver points as templates. the annealed silver points were pre curved to create artificial canals with angle of curvature of 40º, 5 mm of radius curvature and 21 mm of length, and the beginning of the curve was positioned 14 mm from the canal orifice. these simulated canals were constructed using self-curing epoxy resin (araldyte ly 1316 ciba, são paulo, sp, brazil) in the proportion of 100g of resin to 13g of catalyst (hy 1208 ciba, são paulo, sp, brazil). to prevent the formation of bubbles, the mixing of the resin with the catalyst was carried out by the vacuum spatulate model a 300 3 brisighello et al. (polidental, são paulo, sp, brazil). clear spectrophotometer cuvettes (starna, uk) retained the epoxic resin that was poured around the silver points14. a total of thirty-six k3 endo (group k) and thirty-six endosequence (group e) files, size #25, taper 0.04 were divided into four subgroups of 12 specimens each, based on number of repetitions (1, 3 and 5 times) to which the files would be submitted. the files were used with an electric motor at 300 rpm with 2.0 n.cm of torque. a 16:1 reduction contra angle was attached to the electric motor. to reduce interoperator variables, each preparation was conducted by the same operator, with wide experience of the preparation technique for shaping root canals with the rotary niti files, prepared all the simulated curved root canals using the pecking motion, that consists in exerting enough pressure so that the instrument will move forward millimeter by millimeter, followed by light backward movements until the desired depth is reached. during the preparation of resin blocks canals, the operator was unaware of the number of times that the instrument was being used, nor the driven torque. prior to preparation and after the use of each instrument, copious irrigation with 3 ml of 1% naocl solution was performed using disposable syringes and endo-eze irrigation needle (ultradent products inc., south jordan, ut, usa). during the instrumentation procedure, approximately 12 ml of irrigant was used per block. after each cycle of use the files tested were examined under a magnification of 10x. if distortions or breakage in the flutes were observed, the instrument was discarded. based on this evaluation, no instrument has been discarded. then they were washed with tap water and soap, dried with paper towels, placed in an envelope and autoclaved for 20 minutes at 121ºc temperatures. cyclic fatigue testing was performed with a custom-made apparatus (fig. 1) specifically designed to allow dynamic testing by simulating the pecking motion, made essentially of aluminum, according to gavini et al.15 (2012). an electric motor handpiece (driller, são paulo, brazil) was used with a contra-angle of 16:1 (nsk, kanuma, japan). firstly, the micromotor/contra-angle handpiece was secured to the support arm in a parallel position to the apparatus base. then, the file was secured to the contra-angle handpiece, ensuring correct locking. the electric motor was calibrated to run at a speed of 300 rpm and torque identical to that used during the preparation of simulated canals. platforms were moved using the grading rings until reaching a position that allowed the file to remain curved and free to rotate between the cylinder and the steel jig, thus simulating rotary instrumentation of a canal with a 40-degree, 5-mm radius curvature. care was taken to ensure that the instrument was well positioned in the cylinder groove, so as to avoid file displacement. the instrument tip remained visible throughout the experiment, touching the sensor when the maximum displacement of the pneumatic system was achieved. with the file adequately positioned, the main switch turned on, the electric motor was powered, and simultaneously turned on the pneumatic switch. with that, the whole set micromotor/contra-angle/file were powered by the pneumatic system, reproduc4 brisighello et al. ing the pecking motion, with a 2 mm each movement forward and backward, where the file slide in the groove created on the ring made of tempered steel. this movement was repeated at a speed of one cycle per second. the fracture of the instrument was easily detected by the sensor, at moment which the counter and timer were stopped. testing time was registered with a digital stopwatch (casio, tokyo, japan), started at the moment the motor was turned on and stopped at fracture detection. this procedure was sequentially repeated for all groups. after completion of all tests, the mean time to failure observed in each group was recorded in seconds. because our study included independent set of samples with normal distribution and equal variances, the anova and the student t test were employed to assess the presence of statistically significant differences (p<0.01). statistical analysis was performed with spss 17.0 statistical software (spss, chicago, il, usa) results fatigue resistance data were assessed with regard to central tendency (means) and dispersion (standard deviation). the effect of number of uses of the files on cyclic fatigue was not statistically significant (p=0.3592) in both rotation systems. the instruments with 1, 3 and 5 numbers of uses, behave similarly in regard to cyclic figure 1. cyclic fatigue testing apparatus. letters a, b and c – rectangular platforms; d – grading rings; e – mechanical arm with locking ring to support micromotor/contra-angle/file; f – pneumatic cylinder to reproduce reproducing the pecking motion. a b c d b c d e f 5 brisighello et al. fatigue. on the other hand, k3 endo files showed significantly greater resistance to cyclic fatigue (p=0.0146) than endosequence files. table 1 shows fracture data obtained within each group. discussion in present study, the null hypothesis was accepted in regard to number of uses of instruments, however was rejected in regard to rotary systems. the present study assessed the cyclic cyclic fatigue resistance of k3 endo and endosequence files size #25, taper 0.04 submitted to different number of uses, using an experimental cyclic fatigue testing apparatus that simulates the pecking motion. file size (# 25) and taper (0.04) were chosen for being different from those established in the iso standard and compatible with the clinical instrumentation of apical thirds in curved canals. the option for k3 endo and endosequence files was based on idea that the cross-sectional area of the files may influence strongly the cyclic fatigue resistance16, fact that happens throughout the 16 mm of the working portion of these instruments. k3 endo files present a positive rake angle with three radial lands and a relatively large cross-sectional area17. endosequence has a triangular cross-section, without radial lands and with alternating contact points (acp) along the instrument’s shank. the use of acp allows the file to remain centered in the canal, while simultaneously reducing the torque requirements. the lack of radial lands provides a sharper instrument as a result of a decreased thickness of metal, thereby providing a more flexible file. combined with a precision tip, the alternating contact points provide an efficient instrument that will not transport the canal. the endosequence file undergoes electropolishing and the result is visible in its mirror-like finish that remains sharper longer and stays cleaner during use. some authors observed that electrochemical polishing did not inhibit the development of microcracks in endosequence niti files18 and k3 endo files19. the design of the cross-sectional will influence the file’s flexibility and how much lateral resistance is generated when the file is working within the canal9,20,21. file designs that incorporate radial lands, in an attempt to reinforce the cross-section of the file and thereby decrease file separations. this fact also will increase significantly the percentage of contact with the canal wall and subsequently increase lateral resistable 1. mean of cycles to occur the fracture (standard deviation), according to the number of uses and type of file used in each experimental group. uses k group e group 1 674.60a (78.25) 333.35b (70.90) 3 597.50a (64.60) 317.90b (83.05) 5 547.90a (105.20) 255.85b (68.95) different letters indicate the presence of statistically significant differences (p<0.01). 6 brisighello et al. tance. radial lands that increase the stiffness of a file decrease its flexibility in curved canals. the developers state that removing radial lands increases cutting efficiency, increases flexibility, and reduces “drag,” therefore lowering the torque requirements of the files20,21. files ground from triangular blanks will have greater flexibility than those with wide radial lands, but may transport the canal if they lack a centering device. furthermore, those files with a constant pitch have a tendency to create “suck-down”, particularly in larger sizes. suck-down, refers to the tendency of the file to be pulled apically as it engages the canal walls. this results in an increased potential for file separation. some authors reported a higher rate of fracture in files without radial lands than those instruments with radial lands9,18, agreeing with the present research. in a similar study, ray et al.9 (2007) also observed that endosequence files presented significantly lower resistance to fracture than k3 endo files, with used under 300 and 600 rpm. mechanical stress of niti files is strongly related to the curvature of the root canal and dentin hardness22, but it is also proportional to motor torque14,23, thus the instrument’s cyclic fatigue resistance should decrease with prolonged clinical use23,24. cyclic fatigue occurs at the instrument maximal flexure, when rotating freely inside curved canals, without prior indication of failure15. continuous traction and compression cycles in curved canals are the most destructive form of cyclic fatigue and fracture in endodontic files11,14,25-27. although many studies have assessed cyclic fatigue and the dynamics of niti rotary files14,23,28, the relationship between force exerted during preparation of the root canal and clinical risk of distortion and fracture of the files has not been properly studied yet. this study attempted to investigate the mechanisms of pecking motion associated cyclic fatigue test in the breakage of two niti rotary instruments. the methodology allowed the instruments to rotate freely at a standardized curvature. other studies23,24,27 have also indicated that these methodological characteristics are the most appropriate ones for the assessment of cyclic fatigue in rotary niti instruments, since static tests do not reproduce the real conditions faced in the clinical practice: automated instrumentation systems have been designed to enter the root canal in motion, with previously determined torque and speed values, whereas, the distribution of the load over a large area prolonged the useful life of the instrument. the occurrence of maximum flexion in the same location, in the same point, will decrease the lifetime of the instrument. the continuous strength of tension and compression in the curved area of the root canal can promote a destructive load of niti rotary instruments23,26. during the pecking motion, the instruments were always stressed in the curved canals, but the pecking distance gives the instruments a time interval before it once again passes through the highest stress area. according to li et al.12, the pecking motion may be a crucial factor in preventing the breakage of niti rotary instruments. thus, the pecking motion minimizes the stress on instruments into curves, decreasing the chance of occur a fracture. li et al.12 (2002) still recommended that to avoid breakage of a niti rotary instrument, appropriate rotational speed and continuous pecking motion in root canals are necessary. 7 brisighello et al. this study was conducted in simulated canals to reduce the variation in the instrumentation technique and limit the variability of parameters, such as length, width, anatomy, radius and angle of curvature of the canal. the handpiece was never forced apically during instrumentation. with regard to the angle and radius of curvature used, the option for 40º and 5 mm, respectively, better represents the clinical conditions of a root canal with gradual curvature. an analysis of the studies that investigated the impact of torque and number of uses on the cyclic fatigue behavior of niti rotary files allowed concluding that fracture can be avoided by regularly disposing of files after a few uses, and by using low torque motors, operating below the maximum torque limit of each different niti file. the results of this in vitro study must be interpreted critically, and comparisons with the clinical practice must be drawn carefully, because only two of the many variables of root canal preparation were assessed. during this procedure, there are different types of stress from different mechanisms, which are correlated and can affect the useful life of niti rotary files. although there is still no consensus regarding the number of uses or the maximum torque permitted for each file system. it may be affirmed according to results of this study that k3 endo and endosequence files, size #25, taper 0.04, can be used at up to 5 times and with a maximum torque of 2 n.cm, without affecting their cyclic fatigue behavior. it may be concluded that the cross-section of instruments presented significant influence in cyclic fatigue resistance and the number of use, up to 5 times, had no influence in cyclic fatigue resistance in both niti rotary systems analyzed. references 1. schäfer e, florek h. efficiency of rotary nickel-titanium k3 instruments compared with stainless steel hand k-flexofile. part 1. shaping ability in simulated curved canals. int endod j. 2003 mar;36(3):199-207. 2. schäfer e, schulz-bongert u, tulus g. comparison of hand stainless steel and nickel-titanium rotary instrumentation: a clinical study. j endod. 2004 jun;30(6):432-5. 3. schirrmeister jf, strohl c, altenburger mj, wrbas kt, hellwig e. shaping ability and safety of five different rotary nickel-titanium instruments compared with stainless steel hand instrumentation in simulated curved root canals. oral surg oral med oral pathol oral radiol endod. 2006 jun;101(6):807-13. 4. thompson sa, dummer pmh. shaping ability of quantec series 2000 rotary nickel-titanium instruments in simulated root canals: part 2. int endod j. 1998 jul;31(4): 268-74. 5. thompson sa, dummer pmh. shaping ability of quantec series 2000 rotary nickel-titanium instruments in simulated root canals: part 1. int endod j. 1998 jul;31(4):259-67. 6. yared g. in vitro study of the torsional properties of new and used profile nickel-titanium rotary files. j endod. 2004 jun;30(6):410-2. 7. serene tp, adams jd, saxena a. nickel-titanium instruments: applications in endodontics. st lois: ishiyaku euroamerica; 1995. 112 p. 8 brisighello et al. 8. spanaki-voreadi ap, kerezoudis np, zinelis s. failure mechanism of protaper ni-ti rotary instruments during clinical use: fractographic analysis. int endod j. 2006 mar;39(3):171-8. 9. ray jj, kirkpatrick tc, rutledge re. cyclic fatigue of endosequence and k3 rotary files in a dynamic model. j endod. 2007 dec;33(12):1469-72. 10. tygeson ya, steiman r, ciavarro c. comparison of distortion and separation utilizing profile and pow-r nickel-titanium rotary files. j endod. 2001 dec;27(12):762-4. 11. haïkel y, serfaty r, bateman g, senger b, allemann c. dynamic and cyclic fatigue of engine-driven rotary nickel-titanium endodontic instruments. j endod. 1999 jun;25(6):434-40. 12. li um, lee bs, shih ct, lan wh, lin cp. cyclic fatigue of endodontic nickel-titanium rotary instruments: static and dynamic tests. j endod. 2002 jun;28(6):448-51. 13. kawakami da, candeiro gt, akisue e, caldeira cl, gavini g. effect of different torques in cyclic fatigue resistance of k3 rotary instruments. braz j oral sci. 2015 jun;14(2):122-5. 14. zelada g, varela p, martín b, bahillo jg, magán f, ahn s. the effect of rotational speed and the curvature of root canals on the breakage of rotary endodontic instruments. j endod. 2002 jul;28(7):540-2. 15. gavini g, caldeira cl, akisue e, candeiro gt, kawakami da. resistance to flexural fatigue of reciproc r25 files under continuous rotation and reciprocating movement. j endod. 2012 may;38(5):684-7. 16. sattapan b, nervo gj, palamara jea, messer hh. defects in rotary nickel-titanium files after clinical use. j endod. 2000 mar;26(3):161-5. 17. mounce r. the k3 rotary nickel-titanium file system. dent clin north am. 2004 jan;48(1):137-57. 18. herold ks, johnson br, wenckus cs. a scanning electron microscopy evaluation of microfractures, deformation and separation in endosequence and profile nickel-titanium rotary files using an extracted molar tooth model. j endod. 2007 jun;33(6):712-4. 19. barbosa fog, gomes jacp, araújo mcp. influence of electrochemical polishing on the mechanical properties of k3 nickel-titanium rotary instruments. j endod. 2008 dec;34(12):1533-6. doi: 10.1016/j.joen.2008.08.023.. 20. koch ka, brave dg. real world endo sequence file. dent clin north am. 2004 jan;48(1):159-82. 21. koch k, brave d. the endosequence file: a guide to clinical use. compend contin educ dent. 2004 oct;25(10a):811-3. 22. mesgouez c, rilliard f, matossian l, nassiri k, mandel e. influence of operator experience on canal preparation time when using the rotary ni-ti profile system in simulated curved canals. int endod j. 2003 mar;36(3):161-5. 23. pruett jp, clement dj, carnes-jr dl. cyclic fatigue testing of nickeltitanium endodontic instruments. j endod. 1997 feb;23(2):77-85. 24. yared gm, bou dagher fe, machtou p. cyclic fatigue of profile rotary instruments after clinical use. int endod j. 2000 may;33(3):204-7. 25. melo mcc, bahia mga, buono vtl. fatigue resistance of enginedriven rotary nickel-titanium endodontic instruments. j endod. 2002 nov;28(11):765-9. 26. eggert c, peters o, barbakow f. wear of nickel-titanium lightspeed instruments evaluated by scanning electron microscopy. j endod. 1999 jul;25(7):494-7. 27. pessoa of, silva jm, gavini g. cyclic fatigue resistance of rotary niti instruments after simulated clinical use in curved root canals. braz dent j. 2013;24(2):117-20. doi: 10.1590/0103-6440201302164. 28. cho oi, versluis a, cheung gs, ha jh, hur b, kim hc. cyclic fatigue resistance tests of nickel-titanium rotary files using simulated canal and weight loading conditions. restor dent endod. 2013 feb;38(1):31-5. doi: 10.5395/rde.2013.38.1.31. braz j oral sci. 15(2):167-170 parental age is related to the occurrence of cleft lip and palate in brazilian populations paulo henrique pimenta de carvalho1, renato assis machado2, silvia regina de almeida reis3, daniella reis barbosa martelli1, verônica oliveira dias1, hercílio martelli-júnior1,4 1universidade estadual de montes claros unimontes, center of biological and health sciences, montes claros, mg, brazil 2universidade estadual de campinas – unicamp, piracicaba dental school, department of oral diagnosis, piracicaba, sp, brazil 3escola bahiana de medicina e saúde pública bahiana, department of basic science, salvador, ba, brazil 4universidade josé do rosário vellano – unifenas, center for the rehabilitation of craniofacial anomalies, alfenas, mg, brazil correspondence to: verônica oliveira dias universidade estadual de montes claros / unimontes – programa ciências da saúde rua olegário da silveira, 125/201 cep 39400-000 montes claros mg, brazil phone: +55 38 999862934 e-mail: veronicaunimontes@yahoo.com.br abstract aim: to evaluate the association of environmental risk factors, particularly paternal and maternal age, with gender and type of oral cleft in newborn with nonsyndromic cleft lip with or without cleft palate (nscl/p). methods: this study included 1,346 children with nscl/p of two brazilian services for treatment of craniofacial deformities. parental ages were classified into the following groups: maternal age <35, 36-39, and ≥40 years; paternal age <39 and ≥40 years. the data was analyzed with chi-square test and multinomial logistic regression analysis. the odds ratios were estimated with a 95% confidence interval. results: of the 1,346 children included in this study, clp was the type of nscl/p with highest prevalence, followed by, respectively, cl and cp. there was a greater occurrence of nscl/p in males compared to females (55.8% versus 44.2%). clp was more common in men, while the cl and cp were more prevalent in women (p=0.000). no association between maternal age and clefts was observed (p=0.747). however, there was evidence of association between father’s aged ≥40 years old and nscl/p (p=0.031). when patients with cp were analyzed separately, no association between the father’s age and the child’s gender (p=0.728) was observed, i.e. the female gender prevails among patients with cp, regardless of the father’s age. conclusions: this study showed that there were differences in the distribution of the non-syndromic cleft lip and/ or palate and the gender, and fathers aged ≥40 years old may have increased risk of oral cleft. further studies involving different populations are needed for a better understanding of the effect of maternal and paternal ages as a risk factor for the occurrence of oral clefts. keywords: cleft palate; cleft lip; paternal age; maternal age; risk factors. introduction orofacial malformations are the most common form of congenital anomalies in the world1. nonsyndromic cleft lip with or without cleft palate (nscl/p) is the most common facial birth defect with lifelong distressing consequences for the patient2. the prevalence of nscl/p varies among different populations. it has an estimated prevalence between 0.36 and 1.54 per 1,000 live births in brazil3,4. the risk factors associated with nscl/p are not completely understood, but there is a clear interaction between genetic and environmental factors in the etiology of this complex defect5. despite the advances in the identification of risk factors for nscl/p, there are gaps in the existing knowledge6. as far as embryology is concerned, nscl/p results from primary defects in the craniofacial fusion, which forms the primary and secondary palates in the first trimester of the intrauterine development7. these clinical fissures can be classified, having the received for publication: november 05, 2016 accepted: march 07, 2017 original article braz j oral sci. april | june 2016 volume 15, number 2 http://dx.doi.org/10.20396/bjos.v15i2.8648758 168 incisive foramen as an anatomical basis, into four groups: preincisive foramen or cleft lip (cl), post-incisive foramen fissures or cleft palate (cp), trans-incisive foramen fissures or cleft lip and palate (clp), and rare facial fissures8. clp is more common in males, while cp is more common in females9,10. males with clp tend to have a more severe cleft than females and familial clp is often less severe than sporadic cases9. the gender ratio of clp in the caucasian population is 1.7:1 (male:female)11. however, the predominance in males is lower for syndromic forms, which are defined as having other abnormalities present in addition to clp12. curiously, the frequency in females is higher when the father’s age is greater than 40 years13. to understand the developmental mechanisms underlying nscl/p, it is essential to review a large and varied field of research14. the environmental risk factors for nscl/p include maternal use of medications such as antiepileptic agents or corticosteroids, smoking and alcohol consumption during pregnancy15, intrapartum interval10, parity16, folic acid deficiencies17 and maternal and paternal age5. however, there is no consensus on the association of parental age and nscl/p18. thus, the purpose of this study was to evaluate the association of environmental risk factors, especially paternal and maternal age, with gender and type of cleft of the newborn. material and methods this study included 1,346 children with nscl/p, born between the years 2009 and 2013, who visited two brazilian services for treatment of craniofacial deformities. the first service was the center for rehabilitation of craniofacial anomalies, dental school, university of alfenas, minas gerais state, which is located in the southeastern of brazil. the second service was the santo antonio hospital, bahia state, which is located in the northeastern region of brazil. all patients were carefully examined and screened for the presence of associated anomalies or syndromes by a team of specialists from each center, and the clefts were classified with the incisive foramen as reference8. paternal ages were classified into the following groups: maternal age <35, 36-39, and ≥40 years; paternal age <39 and ≥40 years18. patients with congenital malformations (other than oral cleft), history of consanguinity or history of familial nscl/p were not included in this study. the populations of the two services have been previously analyzed in respect to ancestry19. in the two services mentioned, the treatment is exclusively done by the brazilian public health system. all information was collected after approval of the human research ethics committee of both services. informed consent was obtained from the parents or guardians of children. in order to assess the risk factors, an individual instrument (guided questionnaire) was used in both clinics, highlighting the variables of interest to the study. after deploying the questionnaires, the information collected was filed in a data bank and analyzed by the spss® version 19.0 statistical software (spss inc., chicago, usa). the data were analyzed with chisquare test and multinomial logistic regression analysis. the odds ratios (or) were estimated with a 95% confidence interval. results of the 1,346 children included in this study, clp had the highest prevalence (n=750; 55.7%), followed by cl (n=338; 25.1%) and cp (n=258; 19.2%). table 1 shows the distribution of nscl/p according to children’s gender. there was a greater occurrence of nscl/p in males compared with females (55.8% versus 44.2%). clp was more common in male, while the cl and cp were more prevalent in females (p=0.000). parental age is related to the occurrence of cleft lip and palate in brazilian populations braz j oral sci. 15(2):167-170 p=0.000 table 1 distribution of non-syndromic cleft lip and/or palate according to type and gender. cleft palate cleft lip cleft lip and palate n (%) n (%) n (%) gender male 93 12.4 167 22.2 491 65.4 female 165 27.7 171 28.8 259 43.5 total 258 338 750 p=0.747 table 2 distribution of non-syndromic cleft lip and/or palate according to type and maternal age. cleft palate cleft lip cleft lip and palate n (%) n (%) n (%) maternal age (year) ≤35 229 19.3 304 25.7 652 55.0 ≥36 ≤39 14 22.2 13 20.6 36 57.1 ≥40 5 14.7 11 32.4 18 52.9 total 248 328 706 with respect to distribution of nscl/p according to type and maternal age (table 2), a prevalence of mothers under the age of 35 years (92.4%) was observed, but no association was found between maternal age and clefts (p=0.747). in this table, information is found from 1,282 mothers, a different number from the total children in the study (n=1,346). this is due to the number of adopted children or subjects without the mother’s information. table 3 shows the distribution of nscl/p according to type and father’s age. it was observed that most of the men were less than 39 years old (88.5%). there was evidence of association between father’s aged ≥40 years old and nscl/p (p=0.031). table 4 showed the multinomial logistic regression analysis. it turns out that the chance of occurrence of cp in relation the clp was 3.89 times higher (p=0.00) in females compared with males. the chance of cl in relation clp was 2.15 times higher (p=0.00) in females compared to males. also, it was noted 169 in table 4 that there is less chance of occurrence of cp when compared with clp in father’s aged ≥40 years old (p=0.004). patients with cp were analyzed separately. no association between the father’s age and the gender was observed (p=0.728). for example, female gender prevailed among patients with cp, regardless of the father’s age (data not shown in the table). in brazil involving 126 patients with nscl/p, we demonstrated a caucasian predilection and a 1.3 ratio of males to females. males were 2.57-fold more affected by clp than females. clp, with a prevalence of 39.68%, and cl, with a prevalence of 38.09%, were the most common anomalies, followed by cp (22.23%)3. the findings of the present study reveal that, of the 1,346 patients with nscl/p, the prevalence of clp (55.7%) was significantly higher that of cl (25.1%) and cp (19.2%) (p=0.000). it turns out that there was a greater occurrence of nscl/p in males compared with females (55.8% versus 44.2%) and a 1.26 male-to-female ratio. when isolated cp is evaluated, the proportion of occurrence was 2.23 of female to male. there is also a female predominance with isolated cl (1.29 of female to male). however, when clp is assessed, the proportion of occurrence was 1.50 male to female. with respect to maternal age as a risk factor for the occurrence of nscl/p, we adopted the categories (maternal age <35, 36-39 and ≥40 years) recently cited in a meta-analysis study18. in the present study, we found no association between maternal age and clefts (p=0.747). a previous meta-analysis study16 showed that the probability of mothers aged between 35 and 39 years having a child with cleft palate was 20% higher in comparison with those between 20 and 29 years old. for mothers aged 40 years or older, this probability was 28% higher compared to those aged between 20 and 29 years old. mothers aged 40 years or older were 1.56 times more likely to have a newborn with cleft lip with or without palate compared to those aged between 20 and 29 years. in a previous study10 (2006-2008) with a limited population, it was found that the temporal intervals from 26 to 35 years and older than 35 years had reduced risk of having clp when compared with women with ages lower than 26 years. in another meta-analysis22, no general association between maternal age and clp was reported. there was a relation between cp and women between 20 and 24 years and older than 30 years old. it is well known that advanced paternal age (>40 years) is associated with an increased risk of different diseases, such as achondroplasia, apert syndrome, and neurofibromatosis, and a relation with clp is possible22. in the meta-analysis study18, fathers aged 40 years or more showed an increased risk of having a child with cp compared to their peers between 20 and 39 years old. an extensive study suggested predominance in females when the father is age 40 years or older13. in a previous study(2006-2008)10, paternal age did not show statistically significant association with clp (or: 0.7; 95% ci: 0.36-1.35). in the present study, the occurrence of cp in relation the clp was 3.89 times higher (p=0.00) in females compared to males, while the chance of cl in relation clp was 2.15 times higher (p=0.00) in females compared to males (table 4). it was also observed that there is lower a chance of cp compared with clp in father’s aged ≥40 years old (p=0.004). when patients with cp were analyzed separately, parental age is related to the occurrence of cleft lip and palate in brazilian populations p=0.031 table 3 distribution of non-syndromic cleft lip and/or palate according to type and paternal age. cleft palate cleft lip cleft lip and palate n (%) n (%) n (%) paternal age (year) ≤39 225 20.6 276 25.2 593 54.2 ≥40 16 11.3 41 28.9 85 59.9 total 241 317 678 table 4 multinomial logistic regression analysis. distribution of cleft lip and cleft palate according to gender and paternal age, with reference to the cleft lip and cleft palate. cleft lip cleft palate or (ic95%) p value or (ic95%) p value gender male 1.00 1.00 female 2.15 (1.64-2.82) 0.00 3.89 (2.855.32) 0.00 paternal age (year) ≤39 1.00 1.00 ≥40 0.95 (0.64-1.43) 0.821 0.43 (0.24-0.76) 0.004 among female patients with clefts, there was an association between the father’s age and the type of cleft (p=0.003). among the male patients, there was no association between the father’s age and the type of cleft (p=0.092) (data not shown in the table). discussion clp and cl are most frequent in males, and isolated cp is most typical in females across various ethnic groups. the gender ratio varies with the severity of the cleft and presence of additional malformations, number of affected siblings in the family, ethnic origin, and, possibly, paternal age11,13. in white populations, the gender ratio for clp and cl is about 2:1 (male:female)11. moreover, in some studies were observed differences in the distribution of nscl/p between males and females9,10. investigating the epidemiological features of nscl/p patients treated at a reference association in cascavel-parana, brazil, moreira et al.20 revealed that clo, clp and cpo prevailed in males. studies on brazilian populations showed a predominance of clp, followed by isolated cl, and cp9,21. in another study done braz j oral sci. 15(2):167-170 no association between the father’s age and the child’s gender (p=0.728) was observed. among female patients with clefts, there was an association between the father’s age and the type of cleft (p=0.003). among male patients, there was no association between the father’s age and the type of cleft (p=0.092). the variations of the results presented here need to be confronted with studies in different populations in order to better understand the influence of maternal and paternal age on the risk of occurrence of oral clefts. in summary, in this study evaluating 1,346 brazilian children born between the years 2009 and 2013, demonstrated a prevalence of clp, followed, respectively by cl and cp. in the general distribution, a greater occurrence of clefts was found in males compared with females. clp was more common in males, while cl and cp were more prevalent in females. the maternal age, when analyzed in three different ranges, was not considered as a risk factor for the clefts. when patients with cp were analyzed separately, no association between the father’s age and the gender (p=0.728) was observed. further studies involving different populations are needed for a better understanding of the effect of maternal and paternal ages as a risk factor on the occurrence of oral clefts. acknowledgments this work was supported by grants from the minas gerais state research foundation, fapemig, brazil, from the program casadinho/procad (cnpq/capes), and from the national council for scientific and technological development-cnpq, brazil (hmj). references 1. leslie ej, marazita ml. genetics of cleft lip and cleft palate. am j med genet c semin med genet. 2013 nov;163c(4):246-58. 2. rahimov f, jugessur a, murray jc. genetics of nonsyndromic orofacial clefts. cleft palate craniofac j. 2012;49(3):73-91. 3. martelli-junior h, porto lc, martelli drb, bonan pr, freitas ab, coletta rd. prevalence of nonsyndromic oral clefts in a reference hospital in minas gerais state, between 2000-2005. braz oral res. 2007;21(4):3147. 4. rodrigues k, sena mf, roncalli ag, ferreira ma. prevalence of orofacial clefts and social factors in brazil. braz oral res. 2009;23(4):38-42. 5. dixon mj, marazita ml, beaty ht, murray jc. cleft 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multi-malformed infants with cleft lip and palate: an epidemiologic study of nearly 6 million births in 23 eurocat registries. am j med genet a. 2007 mar 15;143a(6):528-37. 12. mossey p, castillia e. global registry and database on craniofacial anomalies. geneva: world health organization, 2003. 13. berg e, lie rt, sivertsen å, haaland øa.parental age and the risk of isolated cleft lip: a registry-based study. ann epidemiol. 2015 dec;25(12):942-7.e1. 14. setó-salvia n, stainer p. genetics of cleft lip and/or cleft palate: association with other common anomalies. eur j med genet. 2014;57(8):381-93. 15. zhang b, jiao x, mao l, xue j. maternal cigarette smoking and the associated risk of having a child with orofacial clefts in china: a case control study. j craniomaxillofac surg. 2011 jul;39(5):313-8. 16. luo yl, cheng yl, gao xh, tan sq, wang w, chen q. maternal age, parity and isolated birth defects: a population-based case-control study in shenzhen, china. plos. 2013;8(11):e81369. 17. bufalino a, paranaiba lmr, aquino sn, martelli júnior h, swerts mso, coletta rd. maternal polymorphisms in folic acid metabolic genes are associated with nonsyndromic cleft lip and/or palate in the brazilian population. birth defects res a clin mol teratol. 2010;88(7):980-6. 18. herkrath apcq, herkrath fj, rebelo mab, vettore mv. parental age as a risk factor for non-syndromic oral clefts: a meta-analysis. journal of dentistry. 2012;40(6):3-14. 19. aquino sn, messeti ac, hoshi r, borges a, viena cs, reis sa et al. analysis of susceptibility polymorphisms for nonsyndromic cleft lip with or without cleft palate in the brazilian population. birth defects res a clin mol teratol. 2014;100(2):36-42. 20. moreira hsb, machado ra, de aquino sn, rangel alc, martelli-júnior h, coletta rd. epidemiological features of patients with nonsyndromic cleft lip and/or palate in western parana. braz j oral sci. 2016;15(1):3944. 21. campos neves at, volpato le, espinosa mm, aranha am, borges ah. environmental factors related to the occurrence of oral clefts in a brazilian subpopulation. niger med j. 2016 may-jun;57(3):167-72. 22. vieira ar, orioli im, murray jc. maternal age and oral clefts: a reappraisal. oral surg oral med oral pathol oral radiol endod. 2002;94(5):530-5. braz j oral sci. 15(2):167-170 170 parental age is related to the occurrence of cleft lip and palate in brazilian populations oral sciences n3 original article braz j oral sci. october | december 2015 volume 14, number 4 impact of hypomineralized teeth and sociobehavioral aspects on caries development: a prospective cohort study cristiane maria da costa silva1, glaucia maria bovi ambrosano2, fábio luiz mialhe2 1prefeitura municipal de poços de caldas, health’s secretary, oral health coordinator, poços de caldas, mg, brazil 2universidade estadual de campinas – unicamp, piracicaba dental school, department of community dentistry, piracicaba, sp, brazil correspondence to: fábio luiz mialhe faculdade de odontologia de piracicaba unicamp departamento de odontologia social avenida limeira 901, bairro areão cep 13414-903 piracicaba, sp, brazil phone: +55 19 21065279/fax: +55 19 21065248 e-mail: mialhe@fop.unicamp.br abstract aim: this prospective cohort study was to evaluate the independent and mutual effects of socioeconomic, oral health behaviors and individual clinical factors, including enamel hypomineralization, as possible risk factors for increase in caries experience in second primary molar (spm) over a period of 2-years. methods: children (n=216) aged 4-6 years were examined for hypomineralized second primary molar (hspm) and dental caries in school settings and were recalled every 6 months. the caregivers filled out a semi-structured questionnaire about their socio-demographic and oral health-related behaviors. data analysis was performed using a hierarchical model with three levels. multiple analyses were performed at each level and variables with p<0.20 were tested by stepwise multiple generalized estimating equation. results: at final examination, 33.3% of the children had developed new caries lesions in spm. the model showed that the number of years of mother’s schooling and the caregiver´s perception about their children’s caries experience played a protective role in the incidence of dental caries. children who had white spot lesions were more likely to develop new carious lesions in spm. children with hspm showed no higher incidence of caries in their spm than those without hspm. conclusions: clinical, socioeconomic and behavioral factors impacted on caries development in primary second molars. however, further studies are required to better understand the role of hspm in caries development in other age groups. keywords: dental caries; tooth hypomineralization; socioeconomic factors. introduction despite the reduction in the incidence of dental caries, it continues to be the most prevalent oral disease in childhood1-2. concern about the high prevalence of caries in primary dentition has led the researchers around the world to conduct studies about the influence of various risk factors on the onset of disease, among which enamel hypomineralizations are outstanding3-4. in this context, second primary molars (spm) have attracted the attention, not only because of their high risk for caries development but also because of the prevalence of idiopathic demarcated enamel hypomineralization, called hypomineralized second primary molar (hspm)3-6. studies indicate that the microstructure of enamel hypomineralization favors the development of caries lesions due to its lower mechanical strength, which often shows structural losses and provides retention niches for dental biofilm7-8. another factor that could contribute to the higher caries experience among children http://dx.doi.org/10.1590/1677-3225v14n4a09 received for publication: november 09, 2015 accepted: december 13, 2015 braz j oral sci. 14(4):299-305 300300300300300 with hypomineralized teeth is the great and repeated need for restorative treatment of the affected teeth, as a result of the low mechanical resistance of the affected enamel7,9-10. however, the results of studies among children with enamel hypomineralizations may be controversial. while some studies point towards a supposed influence of enamel defects in the development of carious lesions, others have found no associations between hypomineralized enamel and dental caries4,6,10-12. this demonstrates the need to evaluate the influence of other variables, such as socioeconomic status and oral health behavior of children on posteruptive behavior of these enamel hypomineralizations and their influence on the development of dental caries in the affected teeth. in addition, studies have shown that mothers’ untreated caries almost doubled the odds of children’s untreated caries, showing the multiple determinants involved in the disease13. hence, the evidence of the risk factors involved in the development of dental caries may only be obtained from cohort studies. the aim of this study was to evaluate the plausibility of environmental, behavioral and clinical factors, especially the presence of hspm, as potential risk factors for the development of new caries lesions in spm tooth surfaces over a period of 2-years. material and methods approval for this assessment was obtained from the ethics committee of the piracicaba dental school, são paulo, brazil (protocol 037/2010). a population-based study was conducted, in which all children from 4 6 years (n=230) of age, resident in the rural and urban zones of botelhos, minas gerais, brazil, were invited to participate. the study was conducted from 2010 to 2012. the city has slightly over 15.000 inhabitants and has a human development index of 0.7, which is considered high and the municipal water system adjusts the level of fluoride to 0.7 ppmf. at baseline (2010), 216 children aged 4-to-6-years-old were examined (93.9% of all eligible children). the exam was carried out in the school environment by the first author. the training and calibration process included theoretical information, preliminary diagnostic training with photographs and clinical evaluation. to determine intraexaminer agreement, about 10% of the examined children were randomly selected and re-examined in the field work, on a separate occasion, after a 24 h interval. the kappa values for intra-examiner reliability with regard to the presence of dental caries and hspm were 0.92, and 0.94, respectively. before the examination, the participating children received a toothbrush and fluoridated toothpaste (1.100 ppmf) to brush their teeth. children were recalled and examined every 6 months from 2010 to 2012. the follow-up exams were carried out under the same conditions as those at baseline. the prevalence of dental caries was assessed by using the dmft index (number of decayed, missing and filled primary teeth) in accordance with the world health organization, with a mirror and dental probe14. dental caries on spm was assessed by the dmft index14. a carious lesion was recorded in a spm, when the surface had an unmistakable cavity, undermined enamel, or a detectably softened floor or wall on their coronal surface. no radiographs were taken. the presence of white spot lesions on spm surfaces was recorded. hspm was diagnosed when the child had one or more spm with demarcated opacities or post-eruptive break-down of the hypomineralized enamel or/and atypical restorations according to the eapd criteria, which have already been presented in other studies conducted by the authors9,11,15. demarcated enamel opacities were differentiated from initial caries lesions (white spot), according to their location in the teeth. white spot carious lesions present an enamel surface with a whitish/yellowish opaque coloration, occur under dental plaque, are located adjacent to the gingival margin and extend along the buccal or lingual surfaces and/or in pits or fissures9,14. in contrast, demarcated enamel opacities have no preferential location on the tooth 9. clinically detectable dental plaque on the buccal surfaces of the anterior primary teeth was recorded. the caregivers filled out a semi-structured questionnaire about their socio-demographic and oral health-related behaviors, considered independent variables that could be related to their children’s oral health status. the theoretical model for this study comprised three levels, based on a hierarchical approach16. level 1 of the model comprised socioeconomic and demographic variables concerning the child’s environment at baseline. the lowest level of parents’ education represents functional illiteracy in brazil16. the head of the household’s occupation was established by the question: what is the head of the household’s occupation? occupation was classified in accordance with the brazilian occupation classification (bco), regrouped into 2 categories: unskilled labor (for example: sales persons at stores and markets, maintenance workers, persons working in agricultural, forestry, hunting and fishing activities) and workers at technical or higher levels (science and art professionals, medium level technicians, workers at administrative level)17. level 2 of the model included factors involved in children’s oral health-related behaviors (frequency of tooth brushing; mother’s helping the child with tooth brushing; frequency of consuming sweets by children; dental care visits in children´s life; mothers’ perception of dental caries in their children through question “do you think your child has had cavities?”). level 3 involved the children’s clinical variables at baseline (caries experience measured by dmft index; presence of visible plaque; presence of hspm; presence of white spot lesions on spm). at follow-ups, a calibrated dentist (cmcs) evaluated the children for increment of dental caries in spm allowing direct comparisons to be made after a period of two years. the number of new decayed (d), filled (f) and missing (m) surfaces due to dental caries were calculated for the sample and was dichotomized into yes new lesion of caries and no no new caries lesions14. impact of hypomineralized teeth and sociobehavioral aspects on caries development: a prospective cohort study braz j oral sci. 14(4):299-305 301301301301301 analysis was performed using the generalized estimating equation (gee) to evaluate the possible risk factors for increase in caries experience. the hierarchical model was used, with the demographic and socioeconomic variables considered as the first level, behavioral variables the second level and clinical variables the third level. multiple analyses were performed at each level and the variables that presented values of p<0.25 were tested in the hierarchical model for stepwise multiple gee, selecting those with values of p≤0.05. all analyses were performed with the statistical software program sas (sas version 9.2). explanatory variables were selected for the final models only if they had a p-value of <0.05 after adjustment for variables from the same or previous levels of determinants. results in this study the data from 216 children were initially used (47.22% boys; mean age 5.22, sd±0.86). the response rates at each of the 6-month follow-ups were 204 (94.44%), 196 (90.74%), 186 (86.1%) and 174 (80.55%), respectively. the dropout rate was mainly due to participants who moved from the city. at baseline, the mean dmft index of the children was 2.45 and 81 children (37.5%) presented dental caries in one of their spm according to who criteria. at tooth level, among all 864 spm included in the analysis, 22.15% corresponded to caries experience in children at age 4 (median dfs = 0.20 per tooth), 26.36% at age 5 (median dfs = 0.26 per tooth), 24.0% at age 6 (median dfs = 0.24 per tooth). in relation to the hspm, the prevalence of children with hspm was 22.2% (48/216) at baseline and it was observed that children with hspm had more dental caries lesions in their spm than those without hspm (χ2 test, p<0.0001; or=2.57, ic [1.20-5.53]). at tooth level, the prevalence was of 7.4% (64/864), with a total of 76 surfaces with this enamel defect. no statistical differences were found in the presence of hspm between the mandible and maxilla, or between the left and right sides. demarcated enamel opacities (89%) were the most frequently scored characteristics of hspm, followed by atypical restorations and post-eruptive enamel loss. in relation to the hspm color, 4.7% were brown, 35.9 were yellow and 59.4 were white. at the final examination after 2 years, 31.8% (14/44) of children initially aged 4 years; 36.3% (20/55) at 5 years and 32.0% (24/75) at 6 years respectively, developed news caries lesions in their spm. the caries increment was mainly due to the incidence of decayed surfaces (92%), followed by fillings (7%) and extracted teeth (1%). among the 48 children presenting hspm at baseline, only 2 presented caries increment in their hspm after 2 years. multivariable analyses at each level the analyses were performed at subject-level. income levels were low for most children’s families, with 36% (89/ 174) of them living on an income of up to 1 minimum monthly wage. the majority of parents also had low educational levels, with only 22.1% (38/172) of the fathers and 28.1% (52/185) of mothers completed more than 8 years of formal education. for level 1 (socio-demographic factors), the mothers’ higher educational level was a protective factor for caries incidence in spm (table 1). for level 2, the mothers’ perception of the presence or absence of dental caries in their children was a protective factor for incidence of new caries lesions in spm (table 2). as regards the variables at level 3, only the variable presence of white spot lesions at baseline was associated with increased risk of caries development in spm after two years (table 3). stepwise multivariable analysis the multivariable stepwise gee analysis generated models 1 through 3. the models (model 1-3) are shown in table 4. the final model (model 3) showed that a high level of schooling and the caregiver’s positive perception of child’s dental caries, played a protective role in the incidence of dental caries. moreover, children with white spot lesions (wsl) at baseline were more likely to develop new dental caries lesions in their spm. discussion dental caries is a chronic childhood disease that can cause pain and impact the quality of life of younger children, affecting mainly the second primary molars1,3-6. thus, it is important that health professionals know the risk factors associated with the development of the disease in these teeth, in order to implement measures to maintain and promote the oral health of this population. the disease has a multifactorial etiology including social, economic, behavioral and oral variables that must be accounted for in the process of diagnosis and treatment. in the present study, instead of using socioeconomic status as a single index for evaluating the impact of social determinants on caries incidence, a set of socioenvironmental variables was included, comprising family environment (parents’ education and family income), number of children, access to fluoridated community water and rural or urban residence, which could have an influence on caries development2. we observed that children whose mothers had a higher level of schooling and had the perception of the presence of dental caries in their children were less likely to develop new caries lesions in their spm after two years. mothers are usually more directly involved in child feeding than fathers in the first five years of life and the maternal educational level has been identified as a factor associated with cariogenic feeding practices18-19. studies have indicated that the children whose mothers had a higher socioeconomic level, brushed their teeth more frequently, consumed fewer cariogenic foods, visited the dentist more frequently showed lower prevalence of caries in comparison with children whose mothers had a lower educational level2,20-21. these behaviors are related to caries experience: the more frequently they are performed, the lower the children’s dmfs index19,21. likewise, the mothers’ perception of the presence of impact of hypomineralized teeth and sociobehavioral aspects on caries development: a prospective cohort study braz j oral sci. 14(4):299-305 302302302302302 caries in their children was inversely associated with dental caries increment in spm over two years. a hypothesis is that the mother´s perception of oral disease in their children may have influenced their attitudes regarding the need for greater oral health care attention on their kids. to our knowledge, this is the first longitudinal study that verified that perception of parents about the oral condition of their children might be a factor of protection for caries increment in their spm. in relation to clinical variables, the presence of white spot lesions at baseline was a risk factor associated with caries development in spm in children 4-6 years old over the two years follow-up. white spot lesions are considered a high risk for caries since the precavitated lesions are indicative of caries activity22 and the findings found in the present study reinforce the evidence in the literature of the importance of past caries experience as an important risk predictor for disease. although enamel hypomineralization is a phenomenon described in other studies, there are still few studies investigating the prevalence of hspm in populations6,23-24. the results of the present investigation demonstrated a prevalence of 22.2%, which was higher than values found by other authors like elfrink et al.23, who found a prevalence rate of 4.9% at child level in a sample of 386 dutch 5-yearold children. among iraqi children, a prevalence of 6.6% was found in a sample of 809 children aged 7to 9 years old6. mittal and sharma found an overall prevalence of 5.6 % in a sample of 978, 6-8-year-old school children in in gautam budh nagar, uttar pradesh, india 24. therefore, differences in the age of the sample, contextual and environmental variables could impact on the prevalence of disease. associations between hspm and dental caries experience impact of hypomineralized teeth and sociobehavioral aspects on caries development: a prospective cohort study braz j oral sci. 14(4):299-305 table 1. table 1. table 1. table 1. table 1. association between socioeconomic and demographic factors and incidence of dental caries. * rr = relative risk; ci = confidence interval. 303303303303303 impact of hypomineralized teeth and sociobehavioral aspects on caries development: a prospective cohort study braz j oral sci. 14(4):299-305 table 2.table 2.table 2.table 2.table 2. association between children’s and parent’s oral health-related behaviors and incidence of dental caries. * rr = relative risk; ci = confidence interval table 3.table 3.table 3.table 3.table 3. association between the children’s clinical variables and incidence of dental caries. 1 hspm=hypomineralised second primary; 2 wsl=white spot lesion. have been observed in cross-sectional studies, in which children with hspm have greater caries experience in their spm4,6. information from the baseline of the present study showed the same tendency, i.e. children with hspm had 2.57 times greater chance of showing caries lesions in their spm, indicating that hspm could be considered an important indicator of risk for caries in spm. on the other hand, when we observed the outcomes of the prospective evaluation of these children, it was verified that enamel hypomineralization was not a risk factor for the development of new carious lesions in spm. although other studies have observed that enamel defects were predictors of caries development in primary dentition25-26, direct comparisons should be made with caution, due to various reasons. firstly, the age of sample and the follow up varies across the studies, for example, from 12 to 36 months of age25; ages 5 to 926 and from birth to 54 months27 which could interfere in the risk of caries development. we suppose that there is a time-dependency ratio between enamel hypomineralization and dental caries. hspm may have an early influence on dental caries, but the risk effect may not continue to drive subjects to an increased risk for caries many years after tooth eruption23. in the newly erupted teeth, enamel hypomineralization is more susceptible to the development of caries due to greater enamel immaturity9. moreover, in the early childhood, parents have more difficulties to brushing their children’s teeth, which certainly interferes in the amount of biofilm8. this fact suggests that further studies should be conducted in populations of a lower age than that of the present sample, preferably in subjects in whom the spm have erupted recently. secondly, the criteria utilized to diagnose enamel defects differ among studies as to the number of teeth examined for defects and caries development25-27. in our study, only the spm were examined for enamel defects, because our objective was to investigate what was the impact of an enamel defect on caries development in those teeth, not in others in oral cavity, as observed in other studies. this methodological approach could impact on the probability of associations between hspm and caries. thirdly, the structure and color of enamel defect could interfere in the risk of developing caries25,28. opacity plus hypoplasia was most significantly associated with caries than diffuse opacity alone25. moreover, elfrind et al.28 found that the mineral density in white opacities was not different from that in the enamel of sound molars, but deciduous molar hypomineralization opacities with yellow or brown color had a 20–22% lower mineral density compared with sound molars. in the present study, among the 48 children presenting hspm at baseline, only 2 children presented caries increment in their hspm after 2 years: one of them presented 4 brown opacities at baseline and the other presented one yellow opacity. costa-silva et al. observed in permanent molars that brown and yellow opacities were at higher risk for posteruptive enamel breakdown and caries15. furthermore, the color of opacity could also be a factor that contributes to the development or progression of caries in spm. we agree with elfrink et al. that there is a need for standardized studies on hspm29, however, to our knowledge, this is the first prospective study to evaluate the relationship between hspm and caries increment in spm, using the diagnostic criteria proposed by the eapd.. therefore, further studies are required to better understand the role of hspm in caries development, including studies of longer duration, with larger and diverse samples, and in populations with diversified level of caries experience, in order to enable the clarification of the time-dependent relationship between hspm, covariables and caries in primary dentition29. acknowledgements grant # 2010/13187-3, são paulo research foundation (fapesp). table 4. table 4. table 4. table 4. table 4. risk factors associated with incidence of dental caries analyzed by generalized estimating equation. * rr = relative risk; ci = confidence interval. 304304304304304impact of hypomineralized teeth and sociobehavioral aspects on caries development: a prospective cohort study braz j oral sci. 14(4):299-305 305305305305305 references 1. casamassimo ps, thikkurissy s, edelstein bl, maiorini e. beyond the dmft: the human and economic cost of early childhood caries. j am dent assoc. 2009; 140: 650-7. 2. li y, zhang ye, yang r, zhang q, zou j, kang d. associations of social and behavioral factors with early childhood caries in xiamen city in china. int j paediatr dent. 2011; 21: 103-11. 3. elfrink mec, veerkamp jsj, 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need. eur arch paediatr dent. 2015; 16: 247-55. impact of hypomineralized teeth and sociobehavioral aspects on caries development: a prospective cohort study braz j oral sci. 14(4):299-305 revista fop n 13 1563 braz j oral sci. april/june 2008 vol. 7 number 25 in vitroin vitroin vitroin vitroin vitro evaluation of torsional strength of evaluation of torsional strength of evaluation of torsional strength of evaluation of torsional strength of evaluation of torsional strength of orthodontic mini-implantsorthodontic mini-implantsorthodontic mini-implantsorthodontic mini-implantsorthodontic mini-implants matheus melo pithon1; rogério lacerda dos santos1; carla d’agostini derech2; carlos nelson elias3; antônio carlos de oliveira ruellas4; lincoln issamu nojima4 1dds, specialist in orthodontics, dental school, federal university of alfenas, brazil; student of the doctor’s degree program in orthodontics, dental school, federal university of rio de janeiro, brazil. 2dds, student of the master’s degree program in orthodontics, dental school, federal university of rio de janeiro, brazil. 3dds, msc, phd, professor, department of metallurgical engineering, fluminense federal university, brazil. 4dds, msc, phd, adjunct professor of orthodontics, dental school, federal university of rio de janeiro, brazil. received for publication: april 16, 2008 accepted: august 23, 2008 correspondence to: matheus melo pithon rua méxico, 78, 45020-390 vitória da conquista, ba, brazil. e-mail:matheuspithon@bol.com.br abstract aim: the aim of this study was to assess the maximum torsional strength of orthodontic mini-implants of different diameters. methods: eighteen mini-implants measuring 10 mm in length were divided into three groups (n = 6). g1, g2 and g3 had miniimplants of 1.2, 1.4, and 1.6 mm in diameter, respectively. mini-implants manufactured by sin (sistema de implantes, são paulo, sp, brazil) were used in all groups. results: the results showed statistically significant differences (p<0.0001) among the groups. the torsional strength was found to be higher in the mini-implants from the same manufacturer that had greater diameter (g3>g2>g1). conclusion: mini-implants with greater diameter should be used if increased torque is needed during orthodontic procedures. key words: mini-implants, anchorage, orthodontics. i n t r o d u c t i o n anchorage is an issue of great relevance in the orthodontic planning. crucial decisions are made depending on how orthodontic anchorage is delineated for a given treatment plan, that is: whether permanent teeth will be extracted, whether orthognathic surgery is needed, whether soft tissues are changed, whether the patient is cooperative, and whether the treatment will be simplified and shortened1. literature in recent years has described several advantages from dental implantology such as mini-plates2-3, onplants4, conventional osseointegrated implants5-6 and miniimplants7-9, all proved to be efficient as orthodontic anchorage. the use of mini-implants, however, has recently received great attention compared to other devices as a new concept of orthodontic anchorage because it is based on the absolute lack of movement of the anchorage unit due to orthodontic mechanics10. similarly to the conventional dental implant systems, those practitioners inserting mini-implants should take special care both during the surgery itself and during the phase of orthodontic force application, since deformation or even fracture of such mini-implants is more likely to occur when inserting or removing them11-12. mini-implants of reduced size provide greater variability in relation to the installation sites and decrease the risk of root damage13-14. on the other hand, mini-implants with reduced dimensions have less mechanical strength15, thus impending the application of maximum torsion force without causing deformation and fracture11,16. based on such a supposition, the present work aimed at quantifying the maximum fracturing torque of orthodontic mini-implants of different diameters. material and methods a total of 18 commercially available orthodontic screwable mini-implants made from ti6al4v alloy and measuring 10 mm in length were allocated into three groups, according to their diameter: g1, g2 and g3 had miniimplants of 1.2, 1.4, and 1.6 mm in diameter, respectively. mini-implants manufactured by sin (sistema de implantes, são paulo, sp, brazil) were used in all groups. the mini-implants were mounted onto devices specially designed for this study according to elias and lopes15. the torsional strength testing device consisted of a head containing a mandrel to hold the sample’s extremities, a hook to hold the opposite end of the mini-implant, and an axis from which a thread was attached to a battery cell (500 n) aimed to measure the force exerted on the miniimplants15. 1564 group n mean sd median significance* 1 (1.2mm) 6 20.44 2.06 20.16 a 2 (1.4mm) 6 43.76 4.67 44.89 b 3 (1.6mm) 6 58.06 2.52 58.15 c table 1 descriptive statistical analysis of the maximum fracturing torque regarding the mini-implants (n.cm2) *equal letters = no statistically significant difference (p> .05). once the specimens were attached to the torsional strength testing device, a universal testing machine (emic dl 10.000; emic, equipamentos e sistemas ltda.,são josé dos pinhais, pr, brazil) was used running at a speed of 1 mm/s. the thread passing through the pulley system was pulled to rotate the mandrel so that the mini-implants were consequently twisted until they fracture. at this moment the maximum torsional strength exerted on the mini-implant was recorded by a software (mtest program 1.01 version). the torsional strength test was carried out in the laboratory of biomaterials and mechanical assays of the military institute of engineering (rio de janeiro, brazil). the values of maximum fracture torques were analyzed by analysis of variance (anova) and tukey’s test at a level of significance of 0.5 %. r e s u l t s statistically significant differences (p<0.0001) in the torsional strength values were observed among the three groups (table 1). those mini-implants with greater diameter (group 3) had the highest mean torsional values, whereas those with smaller diameter (group 1) had the lowest ones (figure 1). fig.1 box plot showing values obtained from torsional strength tests performed within the groups evaluated. d i s c u s s i o n size reduction and immediate load are required for optimising and simplifying the methodology of a rigid intra-osseous anchorage using mini-implants. the reduced diameter of the mini-implants provides greater variability in relation to insertion locals and decreases the risks of root lesion as well. however, such a reduced size also decreases the mechanical strength of the mini-implant, thus reducing the maximum torsional strength and resulting in deformation and fracture11. mini-implants are more likely to break when osseous-integration occurs, since they have to be removed following orthodontic treatment16. as a result, new surgical procedures for removing or “entombing” the mini-implant are needed13. when the maximum torque strength of a given material is to be assessed, one can use the mechanical torsional essay in which a force is applied on the samples or finished products in order to induce rotational movement around their strength centre15. the objective of the present study was to assess the torsional strength of orthodontic mini-implants of different diameters. it was used the methodology proposed by elias and lopes15, who developed a specific device for mechanical torsional essays. orthodontic mini-implants of different diameters produced by the same manufacturer were compared, it was found that their torsional strength values increased as their diameters also increased. this means that insertion torques for installing small diameter mini-implants into high-density bones is near the fracture torque, thus requiring more attention on the part of the practitioners. the clinical importance in determining the optimal torque for a given mini-implant is based on selecting specific screws for certain areas of the oral cavity, since they have different osseous density. this clinical finding is corroborated by songa et al.14, who showed torque variations in both insertion and removal of several types of mini-implants for different bone densities. despite being useful in certain situations (e.g. cases involving high-density bones), insertion torques should not be excessive. motoyoshi et al.17 have demonstrated that increase in insertion torque of mini-implants is directly related to failure rates. the authors show a direct correlation between mini-implants successfully inserted and insertion torques ranging from 5 to 10 ncm2. elias et al.11 have stated that the greater the diameter of a mini-implant the greater the insertion torque, since torque is proportional to the contact area between mini-implant and bone. therefore, increased torque is necessary for larger areas. the results found in the present study meet satisfactorily the clinical needs as, according to elias et al.11 insertion torque for high-density bones ranges from 12.6 to 23.2 n cm2 and for low-density bones ranges around 9.6 n cm2. as can be seen, the values cited above are below the braz j oral sci. 7(25):1563-1565 in vitro evaluation of torsional strength of orthodontic mini-implants 1565 minimum ones found in the present study on fracture strength of mini-implants. the torsional strength essay for mini-implants has showed that fracture torque is relatively high compared to that used for mini-implants inserted in osseous substrates. furthermore, the use of greater-diameter mini-implants provides safer conditions regarding fracture. r e f e r e n c e s 1. freitas jc, castro js. the evaluation of frequence of using implantes as orthodontic anchorage. j bras ortodon ortop facial. 2004; 9: 474-9. 2. chung kr, kim ys, linton jl, lee yj. the miniplate with tube for skeletal anchorage. j clin orthod. 2002; 36: 407-12. 3. de clerck h, geerinckx v, siciliano s. the zygoma anchorage system. j clin orthod. 2002; 36: 455-9. 4. block ms, hoffman dr. a new device for absolute anchorage for orthodontics. am j orthod dentofacial orthop. 1995; 107: 251-8. 5. roberts we, smith rk, zilberman y, mozsary pg, smith rs. osseous adaptation to continuous loading of rigid endosseous implants. am j orthod. 1984; 86: 95-111. 6. favero l, brollo p, bressan e. orthodontic anchorage with specific fixtures: related study analysis. am j orthod dentofacial orthop. 2002; 122: 84-94. 7. lim s, cha j, hwang c. insertion torque of orthodontic miniscrews according to changes in shape, diameter and length. angle orthod. 2008; 78: 234-40. 8. chaddad k, ferreira a, geurs n, reddyd m. influence of surface characteristics on survival rates of mini-implants. angle orthod. 2008; 78: 107-13. 9. moon c, lee d, lee h, im j, baek s. factors associated with the success rate of orthodontic miniscrews placed in the upper and lower posterior buccal region. angle orthod. 2008; 78: 101-6. 10. southard te, buckley mj, spivey jd, krizan ke, casko js. intrusion anchorage potential of teeth versus rigid endosseous implants: a clinical and radiographic evaluation. am j orthod dentofacial orthop. 1995; 107: 115-20. 11. elias cn, serra gg, muller ca. insertion and remotion torque of mini screw orthodontic implant. rev bras implantod, 2005; 11: 5-8. 12. carano a, velo s, leone p, siciliani g. clinical applications of the miniscrew anchorage system. j clin orthod, 2005; 39: 924; quiz 29-30. 13. araújo tm, nascimento mha, bezerra f, sobral mc. skeletal anchorage in orthodontics with mini-implants. rev dent press ortod ortop facial. 2006; 11: 126-56. 14. song yy, chab jy, hwangc cj. mechanical characteristics of various orthodontic mini-screws in relation to artificial cortical bone thickness. angle orthod. 2007; 77: 979-85. 15. elias cn, lopes hp. materiais dentários: ensaios mecânicos. são paulo: santos editora; 2007. 16. serra gg, morais ls, elias cn, andrade l, muller ca. orthodontic mini-implants immediately loaded in vivo study. rev matéria 2007; 12: 111-9. 17. motoyoshi m, hirabayashi m, miwa uemura m, shimizu n. recommended placement torque when tightening an orthodontic mini-implant. clin oral impl res. 2005; 17: 109-14. braz j oral sci. 7(25):1563-1565 in vitro evaluation of torsional strength of orthodontic mini-implants 404 not found untitled 1 volume 16 2017 e17072 original article 1 phd in dentistry, departament of periodontology, school of dentistry, university center of pará, belém, pa, brazil 2 phd in biology, departament of patients with special needs, school of dentistry, federal university of pará, belém, pa, brazil 3 phd in genetics, departament of periodontology, school of dentistry, university center of pará, belém, pa, brazil 4 dds, departament of endodontics, school of dentistry, university center of pará, belém, pa, brazil 5 departament of periodontology, student of post graduate program in dentistry, school of dentistry, university center of pará, belém, pa, brazil corresponding author: silvio augusto fernandes de menezes university center of state of pará, school of dentistry. nine of january street; nº 927; neighborhood: são braz; zip code: 66037000; belém, pa – brazil; telephone: (55) 091984126230/ 09132662044; email: menezesperio@gmail.com. received: july 03, 2017 accepted: october 20, 201 analysis of il-10 in hiv-1 patients with chronic periodontitis in northern brazil silvio augusto fernandes de menezes1, tatiany oliveira de alencar menezes2, tânia maria de souza rodrigues3, brenna magdalena lima nogueira4, ricardo roberto de souza fonseca5 aim: the objective of this study was to investigate the levels of il-10 in the gingival crevicular fluid in hiv-1 positive patients with chronic periodontitis and to compare with hiv-1 negative patients with chronic periodontitis, also to correlate clinical periodontal parameters, viral load and count of cd4 + and cd8 + lymphocytes (ltcd4 + and ltcd8 +). methods: 33 patients were selected and splitted into two groups: 16 hiv-1 positive patients and 17 hiv-1 negative patients and all with chronic periodontitis. the clinical periodontal parameters recorded were: probing depth (pd) and clinical attachment level (cal); the sistemical parameters ltcd4 +, ltcd8 + and viral load were analized by the gingival crevicular fluid collected from all patients. enzymelinked immunosorbent assay (elisa) was used to determine the concentrations of interleukin (il)-10. for the statistical analysis the student t, mann-whitney and spearman tests were performed. il-10 levels were significantly lower in both patients groups. results: there was statistical difference betwen groups for probing depth (p=0.015) and clinical attachment level (p=0.011), no significant correlation was found among the analyzed variables. conclusion: the il-10 levels in hiv-1 positive patients had no influence in periodontal and medical parameters. keywords: interleukin-10. hiv and chronic periodontitis. valentim adelino ricardo barão� http://dx.doi.org/10.20396/bjos.v16i0.8651052 2 de menezes et al. introduction acquired immuno deficiency syndrome (aids) is characterized by an advanced state of immunodepression, is still considered a serious global public health problem, estimated at around 34 million people infected with hiv worldwide1. despite the reduction in the morbidity and mortality of patients who use antiretroviral therapy (art), there are still a large number of individuals carrying the human immunodeficiency virus 1 (hiv-1)2,3. the hiv-1 infects tcd4 + lymphocytes (ltcd4 +), also known as lymphocyte t helper (lth) and the decrease in the number of these cells may contribute to the appearance of several opportunistic infections and several pathologies, such as periodontal disease4. according to chin5 (2017), immunodeficiency caused by hiv-1 may have a direct influence on the pathology of periodontal disease. the periodontal manifestations are recognized as an important characteristic and are widely associated with the immunity caused by the virus, and can be considered one of the first clinical signs of hiv infection, which can be mitigated by the use of art6. according to elizondo et al.7 (2017) the presence of gingivitis and the severity of periodontitis, is indicated by the bone loss and increased pd, is directly related to hiv infection. periodontitis, when installed, has in its gingival crevicular fluid (gfc) several proteins linked to the inflammatory process, such as cytokines, which may be beneficial to diagnose the current status of the periodontium, as well as the effects of periodontal therapy8. among the cytokines present in the gfc, there is the presence of interleukin (il) 10. il-10 is an important cytokine suppressor of inflammatory activity, modulating the production and secretion of other cytokines. the lth 2 activated secretes il-10 that act inhibiting the cytotoxic tcd8 + lymphocyte (ltcd8 +), causing immunosuppression of the response. it also inhibits the production of interferon (ifn) by t lymphocytes, co-stimulates the proliferation and differentiation of b lymphocytes and suppresses the production and secretion of pro-inflammatory cytokines, constituting an important suppressor of cellular immunity8,9. the susceptibility and extention of tissue destruction seem to be determined by the complex cytokine balance produced by the presence of numerous associations between periodontal microorganisms. when the host’s response is exacerbated, it can lead to tissue damage, causing loss of periodontal support. the study of inflammatory mediators associated with periodontal disease, by immunological or biochemical methods, allows the evaluation of the host’s response to this disease10,11. so, the knowledge of the cytokines involved in the progression or not of periodontal disease, especially in hiv-infected individuals, is of fundamental importance for a better therapeutic behavior and, consequently, the quality of life of these patients11. in order to contribute to the characterization of the cytokine profile presented by patients with hiv-1 with chronic periodontitis, the objective of this study was to investigate the levels of il-10 in the gingival crevicular fluid in hiv-1 positive patients with chronic periodontitis and to compare with hiv-1 negative patients with chronic periodontitis, also to correlate clinical periodontal parameters, viral load and count of cd4 + and cd8 + lymphocytes. 3 de menezes et al. material and methods study population the sample size was calculated according to the pd (mean and standard deviation) of both groups by student’s sample t-test. the level of significance was 5%, with an effect of 0.80. considering a statistical power of 95%, the sample size was fixed in 16 patients per group. thirty-three individuals with chronic periodontitis, aged between 34 and 60 years were selected. of these, 16 patients were hiv-1 positive and used art regularly, all the patients selected used the same drug at the same frequency. the other 17 patients have no medical conditions. all subjects presented at least 20 teeth and were without periodontal therapy for about 1 year. the sites had a pd greater than or equal to 5 mm and radiographically must presented a great bone destruction. the teeth selected for the collection of gfc were all natural, intact, without prosthesis and did not present any dysfunction in relation to the occlusion12. the periodontitis was diagnosed based on the study of armitage13 (1996). the samples were collected by a single calibrated researcher (c.r), who had previously experience in clinical studies. the assessment was made using a williams periodontal probe (hu-friedy, chicago, il, usa), a mouth mirror, and clinical tweezers, all of which were sterile, consisted of disposable materials, and were used under natural lighting. the following parameters were assessed: cal and pd. the exams were carried out randomly only in upper posterior molars belonging to two different quadrants, the williams periodontal probe was introduced gently in each of the sites. the collection sites were choosen by c.r based on analysis of periodontal parameters and presence of periodontal pocket ≥ 5 mm. to confirm that the individuals in the control group were hiv-1 negative, blood samples were collected and they were sent to the clinical analysis laboratory for diagnostic purposes. an elisa type immunoenzyme assay were perfomed (diasorin, anti-hiv tetra elisa, biotest, germany), which includes a recombinant antigens, one of the envelope and two antigens of the viral capsid7. in the probability of a hiv-1 positive sample, the blood samples would be submitted to serological screening for anti-hiv antibodies with the microparticle immunoenzymatic method (axsym-system-abbott, germany), followed by indirect immunofluorescence confirmation. were excluded from this study: pregnant, lactating women, diabetic, smokers, patients on systemic or local antimicrobial therapy, hormone therapy or any other analgesic and anti-inflammatory drug with at least a 30-day interval until sample collection12. collect of samples clinical measurement clinical parameters were evaluated in all teeth, excluding third molars, and included the following: pd and cal. six sites were examined for each tooth: mesiobuccal, buccal, distobuccal, distolingual, lingual, and mesiolingual. one calibrated examiner monitored the patients and collected the clinical reports. data were collected and averaged between the sites collected divided by the number of teeth examined per patient. 4 de menezes et al. collection of gingival crevicular fluid (gcf) gcf samples were obtained from 2 sites in the periodontally affected sites at the mesiobuccal gingival sulci at teeth 16 and 26. after isolating the tooth with a cotton roll, supragingival plaque was removed with curettes (hu friedy, gracey, il, usa), without touching the marginal gingiva. the crevicular site was then dried gently with an air syringe. gcf was collected with paper strips (proflow, amityville, ny, usa). strips were placed into the sulci/ pocket until mild resistance was sensed and left in place for 30 seconds. strips contaminated by saliva or blood were excluded from the sampled group. each tip was placed in a sterile polystyrene tube (eppendorf, sigma, ca, usa) which was sealed and identified with patient data and the site where the sample was collected12. elisa test for quantification of il-10 the concentration of the immunoinflammatory mediator present in the gfc was evaluated by the enzyme-linked immunosorbent assay (elisa), following the manufacturer’s instructions (ebioscience, 10240 science center, san diego, ca, usa). all experiments were performed in duplicates for each biological triplicate. lymphocyte count and viral load the grade of impairment of the immune system was assessed through cd4 + and cd8 + levels, as well as the viral load present in the medical records of hiv-1 patients involved in the research. the exams were mandatory, at least 30 days before the samples were collected. the counts were recorded in patient medical records, individually, to relate with the il-10 found in gfc, as well as the level of clinical impairment of the periodontium. quantification of tcd4 + and tcd8 + lymphocytes the blood samples from hiv-1 subjects were quantified by t-lymphocyte count using the flow cytometry technique (facscalibur, becton & dickinson, usa), using the bd trucount tmtubes and bd multitest kit according the standard protocol recommended by the manufacturer (becton dickinson, usa) and used in the national network for quantification of tcd4 + and tcd8 + lymphocytes12. quantification of hiv-1 plasmatic viral load the plasma viral load in hiv-1 patients was determined by the branched dna (bdna) method using the versant® hiv-1 rna 3.0 assay bdna kit (bayer corporation, massachusetts, usa), using the system 340 bdna analyzer (siemens, deerfield, usa). statistical analysis the data on il-10 level, serological indicators and clinical parameters of chronic periodontitis were submitted to descriptive and inferential analyzes. to determine the proportion between patients of the different gender that composed the two groups, hiv-1 positive and negative, chi-square test was used. in order to verify any age difference between the patients of both groups, the t student test was used. to compare the clinical parameters of chronic periodontitis (cal and pd) and the level of il-10, 5 de menezes et al. between patients hiv-1 positive and negative, t student or mann-whitney tests were applied. spearman’s tests were used to verify correlations between clinical parameters of periodontitis and levels of interleukin. these tests were also used to assess the correlation between viral load and serologic indicators (ltcd4 + and ltcd8 +) with il-10 levels. the significance level adopted was 5%, and statistical calculations were conducted in the spss 20 program (spss inc., chicago, il, usa). results of the 33 patients, the number of hiv-1 carriers were 9 males (58.3%) and 7 females (41.6%), with the average of 47.1 (± 6.7) years. the hiv-1 negative patients, 9 belonged to the male gender (52.9%) and 8 to the female (47.1%), with the average of 47.0 (± 5.4) years. chi-square test showed no significant difference between the groups regarding the proportion of male and female participants (p = 1,000). the t student test indicated that there was no significant difference in age between the patients belonging to the groups of hiv-1 carriers and hiv-1 non-carriers (p = 0.971). t student test showed that the cal (p=0.011) and pd (p=0.015) were significantly different and higher in patients with hiv-1 positive. mann-whitney test revealed that the level of il-10 did not present significant difference between the two studied groups, as demonstrated in table 1. no significant correlation was found between il-10 and cal in hiv-1 patients group and control group. il-10 levels also did not correlate with pd in both groups. the amount of ltcd4 + showed no significant correlation with the level of il-10. as for ltcd8 +, its quantity had no correlation with the cytokine concentration studied. viral load did not correlate with il-10 level. these results are demonstrated in table 2. table 2. results of the spearman correlation test between il-10 and the clinical parameters of chronic periodontitis, serology and viral load in hiv-1 and non-hiv-1 carriers. parameters clinical level of insertion probing depth ltcd4+ ltcd8+ viral load hiv+ p = 0.794 p = 0.514 p = 0.569 p = 0.629 p = 0.894 hivp = 1.000 p = 1.000 * significant at the 5% level. table 1. mean values and standard deviation of cytokine and clinical parameters of chronic periodontitis among hiv-1 seropositive and seronegative patients. parameters p value hiv+ hivclinical level of insertion (mm) 7.1 (0.7) 6.4 (0.8) p = 0.011* probing depth (mm) 6.6 (0.7) 5.9 (0.7) p = 0.015* il-10 (pg/ml) 1.23 (1.97) 0.60 (0.07) p = 0.452** * values of p based on t-student test. ** values of p based on mann-whitney test *** significant at the 5% level. 6 de menezes et al. discussion usually seropositive patients presents a more severe periodontitis. this sistemical condition enhance the cal, the pd, gingival recession leading to a tooth loss. due to patient’s immunosupression, a increased viral load and a diffused invasion into the gingival tissue of opportunistic bacteria, fungi and viruses, present in the oral cavity cause a greater inflammatory response in tissues and a considerable bone destruction13. in seronegative patients when periodontitis is installed, in the gcf several cytokines linked to the inflammatory process, such as il-10, these cytokines act in immunological activities and in pro-inflammatory and anti-inflammatory processes in periodontal tissues to protect against microbial activity14. however, in seropositive individuals, these immunological reactions are weakened leading to a greater severity and progression of periodontitis, as the progression of the disease is determined by factors linked to the host’s immune response and the virulence of the bacteria. for this reason, biological characteristics such as cytokine profile have been studied in an attempt to better assess the behavior of the immune system in seropositive patients15. interleukin-10 is an anti-inflammatory cytokine that inhibits the production of proinflammatory cytokines15. the il-10 can attenuate the inflammatory response and exert an immunosuppressive and immunostimulatory effect, operating on a great variety of cellular types16. as a regulator of the cell-mediated immune response, il-10 can generally suppress the production of proinflammatory cytokines and chemokines. il-10 induce the proliferation of b lymphocytes and the proliferation and activation of natural killer cells16,17. based on its biological activities, it is evident that the reduction of il-10 production is associated, with higher susceptibility, to any type of infectious disease15. for this reason, some studies have analyzed the participation of this cytokine in infectious processes, such as periodontal diseases and peri-implants16,17. periodontitis is probably the most common chronic disease in adults. several studies have shown that the profile of locally produced cytokines may be relevant for periodontal destruction18. in the study, the cal as well as the pd were significantly higher in hiv-1 patients than hiv-1 seronegative patients, which may demonstrate the interference of hiv infection in the natural history of periodontal disease10,18. however, this relationship was not always found19. the present study used traditional periodontal clinical parameters to determine the level of il-10 in seronegative hiv-1 patients with chronic periodontitis. the clinical parameters chosen for the analysis consider the reality of public or private clinics, where simple and rapid methods are used for clinical follow-up. the il-10 evaluated in the present study, did not present significant differences within the studied groups in relation to the clinical parameters15-20. il-10 suppresses the immune response and, thus, modulates the production of other cytokines and, according to ujiie et al.21 (2016) and jaradat et al.22 (2012), there is an increase in the levels of il-10 in the presence of hiv infection. in contrast, teles et al.23 (2009) did not obtain relevant levels of il-10, which agrees with the present research that obtained low levels of il-10 in both hiv-1 and non-hiv-1 carriers. 7 de menezes et al. in this study, il-10 showed low levels in both hiv-1 and non-carrier patients. these lower levels of il-10 are characteristic of the decline in th2 response, were also observed by silveira et al.17 (2016), cullinan et al.24 (2008) and lappin et al.25 (2001), which may suggest a deficiency in the control of the immune response directed against pathogens, resulting in an exacerbated inflammation. according to silveira et al.17 (2016), the chemoattraction characteristics of il-10 producing cells could control the destructive potential of the disease. as il-10 is associated with suppression of bone resorption, the low expression in patients with chronic periodontitis would be related to the more severe form of the disease. the il-10 low levels were found in both groups. the studied cytokine is involved in the regulation of inflammatory responses, it is suggested that the low levels of this molecule, observed among patients with chronic periodontitis, contribute to the development of the disease. also as a result, il-10 showed no significant difference with viral load23,24. the analysis of the cytokine profile in chronic periodontitis does not aim to replace the routinely used clinical exams, but could complement the clinical evaluation, however would assist the diagnosis and possible therapeutic interventions. thus, clinical data related to cytokine levels in the gingival crevicular fluid may elucidate the pattern of local immune response, supporting a better understanding of the pathogenesis of periodontitis, especially in individuals with other chronic infections, such as that caused by hiv-124-26. the periodontal disease being proven to be more severe in seropositive patients with periodontitis, the il-10 levels were expected to be lower because it is a cytokine with innate and immune antiinflammatory activity. however, the results in the present mansucript conclude that this cytokine does not interfere in the severity of periodontal disease in these immunodepressed patients. the il-10 levels in the gingival crevicular fluid had no influence on the periodontal clinical parameters of seropositive patients, according to our results we could 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2001;123(2):294-300. 26. noro filho ga, salgado dm, casarin rc, casati mz, costa c, giovani em. anti-infective periodontal therapy promoting improvement in systemic markers of hiv infection. aids res hum retroviruses. 2013 jul;29(7):1040-4. https://www.ncbi.nlm.nih.gov/pubmed/?term=noro%20filho%20ga%5bauthor%5d&cauthor=true&cauthor_uid=23530962 https://www.ncbi.nlm.nih.gov/pubmed/?term=salgado%20dm%5bauthor%5d&cauthor=true&cauthor_uid=23530962 https://www.ncbi.nlm.nih.gov/pubmed/?term=casarin%20rc%5bauthor%5d&cauthor=true&cauthor_uid=23530962 https://www.ncbi.nlm.nih.gov/pubmed/?term=casati%20mz%5bauthor%5d&cauthor=true&cauthor_uid=23530962 https://www.ncbi.nlm.nih.gov/pubmed/?term=costa%20c%5bauthor%5d&cauthor=true&cauthor_uid=23530962 https://www.ncbi.nlm.nih.gov/pubmed/?term=giovani%20em%5bauthor%5d&cauthor=true&cauthor_uid=23530962 effect of referral for dental service on dentalservice utilization by primary school children aged 8 to 11 years in enugu, nigeria nneka kate onyejaka1, morenike oluwatoyin folayan2, nkiruka folaranmi1 1university of nigeria, department of child dental health, enugu, enugu state, nigeria 2obafemi awolowo university, faculty of dentistry, oral habit study group, ile-ife, osun state, nigeria correspondence to: onyejaka nneka kate department of child dental health, university of nigeria, enugu, nigeria. phone: +2348037449279 e-mail: nnekaonyejaka@yahoo.com abstract aim: to determine how one dental education session and referral of study participants aged 8-11 years would affect utilization of oral-health care services. methods: this descriptive prospective study recruited 1,406 pupils aged 8-11 years from randomly selected primary schools in enugu metropolis. all pupils received one oral-health education and referral letters for treatment. data were collected on the pupils’ socio-demographic profile, family structure, and history of oral-health care utilization in the 12 months preceding the study and within 12 months of receipt of referral letter. the effect of these factors as predictors of past and recent dental service utilization was determined using logistic regression. results: only 4.3% of the study participants had ever used oral-health services in the 12 months prior to the study. within 12 months of issuing the referral letters, 9.0% of pupils used the oral-health services. children from middle (aor: 0.46; ci: 0.29-0.73; p=0.001) and low socioeconomic strata (aor: 0.21; ci: 0.11-0.39; p<0.001) and those living with relatives/ guardians (aor: 0.08, ci: 0.01-0.56; p=0.01) were still less likely to have utilized oral-health services. conclusions: referral of children for oral-health care increased the number of children who utilized oral health care services. keywords: physician self-referral. oral health. utilization. introduction regular preventive dental attendance is a contributor to the oral-health status of people of all ages1. many factors affect utilization of oral-health care services: socioeconomic status2,3, attitude towards dental care4, family structure5, proximity to oral-healthcare centers6, being an immigrant7, and ability to take time out for dental visits8. these factors interplay, resulting in a synergistic effect that may worsen the effect of independent factors on the risk of poor utilization of oral-healthcare services. however, when primary care services are delivered for children using the one-stop-shop model, it increases dental service utilization for both preventive and curative care9. underutilization of oral health care services can lead to poor oral health and impact negatively on the oral health quality of life10. untreated dental diseases like dental caries may lead to dental pain and impact on the child’s daily activities like playing, sleeping, eating and school activities11. reports from sub-saharan africa show very low utilization of oral healthcare services and visits are mostly after symptoms have developed12. similarly, studies in the western region of nigeria show low utilization of oral-health services by children, with visits often prompted by oral symptoms like pain13,14. efforts at promoting utilization of dental service by children in nigeria received for publication: july 19, 2016 accepted: november 16, 2016 braz j oral sci. 15(2):151-157 original article braz j oral sci. april | june 2016 volume 15, number 2 http://dx.doi.org/10.20396/bjos.v15i2.8648767 152 have resulted in significant, but still low, dental service visits. moreover, these visits continued to be for curative rather than preventive purposes15. the need to promote children’s access to oral-health services in nigeria has been the major focus of many public oral health interventions15,16. there is dearth of information on any project that breaks barrier of utilization of oral health care services in southeastern nigeria. this study is an effort towards identifying mechanisms that can help improve dental service utilization by schoolchildren in enugu metropolis, nigeria. specifically, the study sought to identify if one dental educational session and referral of study participants 8-11 years old for both preventive and curative treatment. it assessed if it would increase the number of pupils who utilized oral health care services over a 12 months period when compared to the number of pupils that used oral health care services in prior years. material and methods study design: this was an observational prospective study. for this were enrolled pupils aged 8-11 years, schooling in the three lgas in the enugu metropolis of enugu state. enugu state is one of the 36 states in nigeria. its population is mostly the igbo ethnic group of nigeria. most of the enugu state inhabitants have monogamous families17 with an average of 4.8 children per family17. there is no public data on the oral health service utilization by the study population. to calculate the sample size using the formula of araoye18 based on a 15% prevalence of oral-health centre utilization by children in lagos12, a 5% margin of error and a confidence level of 95%, we estimated that, after referrals, approximately 200 pupils would eventually visit the dental clinics. to get a referred population of 200 pupils, the total sample size targ would be 1,333.3, rounded up to 1,400 pupils. a total of 30 primary schools to visit in order to recruit 1,400 pupils aged 8 to 11 years from a total school population of 41,853 pupils19 of the same age group was 30. a multistage stratified sampling technique was used to enroll study participants. the first stage involved selection of a proportional representation of the schools per lga. the second stage involved the random selection of classes with large populations of children aged 8-11 years. the class registration list, which showed the age of the students, was used to determine the classes with the highest number of pupils aged 8 to 11 years. the third stage involved the selection of 47 study participants from two classes in each school. study tool a three-part form was developed for the study. the first part contained questions that elicited information on age at last birthday, gender, residential address and the socioeconomic status. the socioeconomic status of each child’s family was derived by the multiple indices obtained from a scoring index, which combined with the mother’s level of education and the father’s occupation; social stratification based on this combination increases its validity20. the distance of the student’s residence to the closest dental service point was determined by use of a handheld global positioning system (gps) garmin map 76csx, which determined distances in degrees, minutes and seconds (dms). this device used point data and coordinate details of the residence and the closest dental clinic based on data collected in the field. the coordinated data were converted by use of tatuk geographic information system (gis) calculator from dms to decimal degrees to enable the details to be inserted into the arc gis software. the data were then exported from arc gis to quantum gis for determining the distance matrix (point data analysis). the second section contained questions that elicited information on the family structure. it included information on the type of family (monogamous, polygamous), number of siblings, birth position and family structure (living with parents, single parent, stepmother or other relatives). the third section elicited information on past utilization of oral-healthcare service, including the past dental visits (yes, no), date of last visit, reasons for last visit (pain, routine), type of treatment given (scaling and polishing, oral hygiene education, restorations, extractions), and address of the visited dental clinic. study procedure dentists working at all the registered dental clinics in enugu metropolis were contacted and the aims and objectives of the study were explained to them. dentists were encouraged to ask parents of children 8-11 years old if they were attending the dental clinic after the referrals ask for their referral letters and collect them. details of the dental treatment given for each child were required to be filled in specific sections of the referral letter. where referral letters could not be produced by the parent of the child, the details of the child and the dental treatment offered were to be entered into a form provided for the dentists. three dentists were enrolled as field workers and trained on the data collection procedure and details of the study collection tool. discussions and clarifications about content of the questionnaire were also made during the review of the outcomes of the field testing. pupils recruited for the study provided responses to the questions. all questionnaires were administered by the trained field workers. information about the type of family (monogamous, polygamous), number of siblings, birth position and family structure (living with parents, single parent, step mother or other relatives) of the child was also sought from the teachers of the study participants when they could not readily provide these information. missing data was obtained through telephone interviews of the parents using phone numbers provided by the study participants. oral-health education was provided to all pupils in the class, irrespective of whether they were enrolled for the study or not, after filling the study form. all study participants were given referral letters to visit any registered oral-health center of their choice within the enugu metropolis. the list of registered dental clinics and their addresses was attached to the referral letter addressed to their parents/guardians. children were encouraged to take the referral letters with the demand to leave them at the clinics they visited. effect of referral for dental service on dental-service utilization by primary school children aged 8 to 11 years in enugu, nigeria braz j oral sci. 15(2):151-157 153 the study’s principal investigator visited the registered dental clinics within enugu metropolis every month for 12 months to retrieve the referral letters and collect data on the types of dental treatment each child received. twelve months after the school visit, the principal investigator re-visited the schools and obtained information about the purpose of the dental visit(s) from pupils who visited dental clinics. clarifying information was obtained from the parents of the study participants when they could not provide the required information. the information from the child and the parent was needed to corroborate the information coming from the dental clinics and to identify other patients who may have made visits to dental clinics and whose details were not captured through the visits of the principal investigator to the dental clinics to retrieve referral letters. oral-health education was also provided to all pupils in their classrooms at this second visit, using the same oral-health education curriculum. data analysis statistical package of social science (spss) version 15 was used to analyze the collected data. descriptive analysis was conducted by use of a wide variety of measures of location (mean and mode) and dispersion (deviation). these data are represented as tables. bivariate analysis was conducted to test the association between the child’s socioeconomic status, type of family, family structure, birth position, family size, distance from child’s residence to the closest dental clinic, and history of oral-health service utilization before and after study intervention. a model was developed that included those factors that were significantly associated with health-service utilization and logistic regression conducted to identify the factors that played specific roles in utilization for the study population. the level of statistical significance was set at p<0.05. ethical consideration ethical approval for the study was obtained from the university of nigeria health research ethics committee (irb 00002323). permission was also obtained from school authorities in the enugu metropolis prior to the commencement of the study. written informed consent was obtained from the mothers of children who participated in the study and assent was also obtained from the children. results a total of 1,408 pupils were eligible to participate in the study. two children refused to continue with study participation following recruitment, leaving the total number of participants at 1,406. their mean age ± (sd) was 9.32±(1.08) years; 9.42± (1.09) years for the boys and 9.23±(1.07) years for the girls. table 1 shows that the study participants included 52.2% females, 37.1% of children from the higher socioeconomic stratum, 95.4% of children from monogamous families, and 84.4% of children living with both parents. also, 40.5% of study participants had 3-4 siblings. effect of referral for dental service on dental-service utilization by primary school children aged 8 to 11 years in enugu, nigeria table 1 general characteristics of the study participants (n=1,406). variables frequency n(%) age (years) 8 418(29.7) 9 372(26.5) 10 363(25.8) 11 253(18.0) gender male 672(47.8) female 734(52.2) socioeconomic status high 521(37.1) middle 439(31.2) low 446(31.7) type of family monogamy 1341(95.4) polygamy 65(4.6) family structure both parents 1186(84.4) one parent 46(3.2) relative/guardian 174(12.4) birth position only child 38(2.7) first child 326(23.2) last child 313(22.3) others 729(51.8) number of siblings 0 38(2.7) 1-2 245(17.4) 3-4 570(40.5) >4 553(39.3) utilization of dental services 1 year ago 60(4.3) 2 years ago 41(3.0) 3 years ago 18(1.3) ≥4 years ago 13(0.9) no visit 1274(90.5) table 2 illustrates the socio-demographic profile of the 132 (9.4%) pupils who had ever visited a dental clinic for oral-health services. more pupils of high socio-economic status than those with middle and low socio-economic status used the services (p<0.001). also, those living with both parents compared with those living with one parent or guardian/relatives (p=0.003), and those who lived within 1-1.9 km from an oral-healthcare center compared with those who lived 2.0 km or more from an oral-health care center (p<0.001) used oral-health care services. there was no significant difference between the type of family (p=0.41), birth rank (p=0.53) or number of siblings (p=0.10) of pupils who had previously used the services. braz j oral sci. 15(2):151-157 154 effect of referral for dental service on dental-service utilization by primary school children aged 8 to 11 years in enugu, nigeria table 3 illustrates the socio-demographic profile of the 126 (9.0%) pupils who visited an oral-health center after the school visit. just as observed before the issuance of referrals, significantly more pupils from the high socioeconomic strata than those from the middle and low socio-economic strata utilized the oral-health care centers (p<0.001). family living structure was also a significant factor affecting oral health service utilization: all the study participants who utilized oral-healthcare services were living with both parents, whereas none of those living with one parent or guardian visited a center (p<0.001). again there was no statistical difference in the number of study participants from monogamous and polygamous families who utilized the oral-health care centers (p=0.21) and birth rank did not significantly affect use of the services. in contrast to the findings before the school visit, the number of siblings was related to frequency of oral-health care visits: children who had three or more siblings visited the centers more often than those who had two siblings or fewer (p=0.002). also, the distance of participants’ residences from the oral-health care centers was not a factor in oral-health care utilization, whereas it was a factor before the use of referrals. table 4 illustrates the distribution of the study participants who had either previously utilized and/or newly utilized dental services. most of the pupils (77.8%) who utilized the dental services after issuance of referral letters were those who had never utilized dental services prior to the issuance of referrals (p<0.001). table 2 distribution of study participants who had ever used oral health care services in the past (n=132). variable frequencyn(%) p socioeconomic status <0.001 high 85(64.4) middle 31(23.5) low 16(12.1) type of family 0.41 monogamy 124(93.9) polygamy 8(6.1) familystructure 0.003 both parents 125(94.7) one parent 1(0.8) guardian 6(4.5) birth rank 0.53 only child 5(3.8) first child 26(19.7) last child 33(25.0) others 68(51.5) number of siblings 0.10 0 5(3.8) 1-2 28(21.2) 3-4 60(45.5) >4 39(29.5) distance of facility to participants’ residence(km) <0.001 <1.0 47(35.6) 1.0-1.9 48(36.4) 2.03.9 30(22.7) ≥4.0 7(5.3) table 3 distribution of study participants who utilized the oralhealth care services after referral (n=126). variable frequencyn(%) p value socioeconomic status <0.001 high 85(67.5) middle 33(26.2) low 8(6.3) type of family 0.21 monogamy 123(97.6) polygamy 3(2.4) familystructure <0.001 both parents 126(100.0) one parent 0(0.0) guardian 0(0.0) birth rank 0.80 only child 4(3.2) first child 31(24.6) last child 31(24.6) others 60(47.6) number of siblings 0.002 0 4(3.2) 1-2 36(28.6) 3-4 52(41.3) >4 34(27.0) distance of facility to participants’ residence(km) 0.66 <1.0 34(27.0) 1.0-1.9 34(27.0) 2.0-3.9 44(34.9) ≥4.0 14(11.1) table 4 distribution of study participants who had visited dental centres before and/or after referral. visited after referral visited before referral no yes total p n(%) n(%) n(%) no 1.176(91.9) 98(77.8) 1,274(90.6) <0.001 yes 104(8.1) 28(22.2) 132(9.4) total 1,280(100.0) 126(100.0) 1,406(100.0) figure 1 shows the profile of the pupils who received preventive or curative treatment before and after the issuance of braz j oral sci. 15(2):151-157 referrals. before the issuance of referrals, 54 (40.9%) of the 132 pupils received preventive treatment, while 78 (59.1%) received curative treatment (p=0.003). in contrast, after referral, 84 (66.7%) of the 126 pupils received preventive treatment, whereas 42 (33.3%) received curative treatment (p<0.001). 155effect of referral for dental service on dental-service utilization by primary school children aged 8 to 11 years in enugu, nigeria ohcc: oral-health-care centres fig.1. percentage of preventive and curative treatment received before and after referral. table 5 illustrates the results of the logistic regression analysis for the best predictor of participants’ utilization of oral-health care centers before the issuance of referrals. socioeconomic status, type of family and family structure were significant predictors of dental service use. children from middle (aor: 0.40; ci: 0.26-0.62; p<0.001) and low socioeconomic strata (aor: 0.19; ci: 0.11-0.34; p<0.001), those living with relatives/guardians (aor: 0.33, ci: 0.13-0.78; p=0.01), and those who lived 2.0-3.9km (aor: 0.41, ci: 0.25-0.68; p<0.001) and more than 4km (aor: 0.24, ci: 0.10-0.55; p=0.001) from a health care center were less likely to have utilized oral-health services. children from polygamous homes were 2.6 times more likely than those from monogamous families to have utilized oral-health services in the past (ci: 1.12-5.98; p=0.03). the birth position and the number of siblings participanting in the study did not significantly affect oral health service utilization. table 6 shows the logistic regression analysis for the best predictor of study participants’ utilization of oral-health care centers in the 12 months after the issuance of referrals. after the issuance of referrals, children from middle (aor: 0.46; ci: 0.29-0.73; p=0.001) and low socioeconomic strata (aor: 0.21; ci: 0.11-0.39; p<0.001) and those living with relatives/ guardians (aor: 0.08, ci: 0.01-0.56; p=0.01) still were less likely to have utilized oral-health services. families with 3-4 children were 2.21 times more likely to have utilized oral-health services (ci: 1.23-3.96; p=0.01) than the families with either fewer or more children. the significant effect of type of family and distance of residential location to closest dental clinic was lost after the issuance of referrals. variable multivariate adjusted or 95% c.i p value socioeconomic status high 1.00 middle 0.40 0.26-0.62 <0.001 low 0.19 0.11-0.34 <0.001 type of family monogamy 1.00 polygamy 2.6 1.12-5.98 0.03 family structure with both parents 1.00 with one of the parents 0.17 0.022-1.29 0.09 with relative/guardian 0.33 0.13-0.78 0.01 birth position first child 1.00 last child 1.65 0.93-2.93 0.08 only child 2.62 0.84-8.22 0.10 others 1.61 0.96-2.70 0.07 number of siblings <3 1.00 3-4 siblings 1.75 0.99-3.09 0.54 >4 siblings 0.47 0.94-2.30 0.09 distance of facility to participants residence(km) <1 1.00 1.0-1.9 1.03 0.66-1.63 0.87 2.0-3.9 0.41 0.25-0.68 <0.001 ≥4.0 0.24 0.10-0.55 0.001 table 5 logistic regression analysis of effect of factors on utilization of oral health-care services before education and referral intervention. or=odd ratio; ci=confidence interval; reference category=1 discussion the goal of our study was to explore how delivering referrals affected the utilization of oral health services by pupils attending schools in enugu metropolis, enugu state, nigeria. the study showed that socio-economic factors and family structure were significant factors that affected access of pupils to oral-health services. before the study intervention, pupils with low and middle socioeconomic status and those who lived with relatives/ guardians were less likely to have ever used a dental service than the pupils from high socioeconomic status or those living with parents. after referral for oral-health care, pupils from low and middle socioeconomic status and those living with relatives/ guardians were still less likely to use dental service. following referral, more pupils who never utilized a dental service visited an oral health facility. the use of referral letters to promote access to both prevention and curative treatment had four significant effects in this study. first, the number of pupils who visited the dental clinic within 12 months of issuing them a referral letter increased from 4.3% to 9.0%. second, the number of children who attended the dental clinic for preventive treatment as opposed to curative treatment, increased significantly. third, a lot more pupils who had never visited the dental clinic were motivated to do so following receipt of a referral letter albeit most of these pupils were from the high socioeconomic class. fourth, the distance between residential homes and dental service centers were no longer a barrier to dental service utilization. braz j oral sci. 15(2):151-157 156 effect of referral for dental service on dental-service utilization by primary school children aged 8 to 11 years in enugu, nigeria the increase in the number of pupils who visited the dental clinic for preventive oral-health treatment within 12 months of issuing them a referral letter may have resulted in part from the design of the study: all children were referred for oral-health care services whether they had dental symptoms or not. in the past, although school-based education programs had often conducted screening exercises, only children with treatment needs were referred to oral-health care services. this approach reinforced the emphasis on the children’s curative dental treatment, not the prevention treatment. referring children for both preventive and curative treatment encourages dentists to consider preventive oral-health care as important as curative care. sending children home with referrals for oral-health care may also have helped the parents to overcome their inertia to visiting dental clinics. a pointer to this is the significant high number of pupils who had never attended a dental clinic in the past who then visited a dental clinic following the issuance of a referral letter. the letter seemed also to serve as a motivator to overcome the limitations distance may otherwise place on a child’s access to dental care. this study therefore reinforces the importance of referrals for improved dental service utilization by children, as highlighted by a prior study by folayan et al.15. however, unlike the postulation of those authors, this study showed that referrals might increase access to preventive dental treatment and not just curative treatment. a child’s socioeconomic status is a risk factor for inequity in dental service access and utilization3,11. socioeconomic status is a reflection of family’s disposable income; those from the poor socio-economic status have little disposable income to pay for oral-health care, especially for preventive care11,20. the indirect costs associated with visiting the dentist (transport, off work time) may also be a barrier to use of oral-health services. this study reinforces the impact that a child’s socioeconomic status has to its access to oral-health services. strategic interventions are therefore required to help improve utilization of oral-health care services by children from the middle and low socio-economic strata. we believe that similar strategic thinking would also be required to promote access of children living with relatives/guardians to oralhealth services, even though newacheck21 reported that family structure had no impact on oral-health service utilization after controlling the socioeconomic status. despite the success recorded by the use of referral letters to increase the number of pupils who utilized dental care services, oral-health service utilization by the study population was still very low. this finding is similar to findings in other developing countries22,23, except kenya, where higher figures have been reported24. prior reports from ile-ife15,25, ibadan26 and enugu27 in nigeria all located in southern nigeria had reported higher proportions of children utilizing dental care services. children’s utilization of oral-health care services is, however, lower in north central nigeria28 and among children with special needs11. further studies are required to help identify factors that would increase oral-health service utilization by pupils in the study population to both preventive and curative dental care. a mixed-method study design may be most helpful in this respect. our study had limitations. first, the number of pupils who had previously visited oral-health clinics may have been underreported as some pupils may have utilized dental clinics outside the state where this study was conducted. also, data of students who did not return to the schools where the study was conducted in the following academic year might not be collected. second, the opportunity for some parents to visit the dental clinic may have been undermined if study participants did not give their parents the referral letter, which is a possibility with children. these limitations are, however, beyond the capacity of the study to address. the study therefore reflects the reality of what would happen in real life settings. thus, the limitations identified for this study did not detract from the validity of our findings. in conclusion, this study in a nigerian metropolis demonstrated that school-based education and referral programs overall increased pupils’ use of oral-healthcare services for both preventive and curative care. however, the intervention did not improve dental service utilization by children of middle and low socioeconomic status and children who lived with relatives/ guardians. future efforts should address factors that can reduce the inequity in access of these children to oral-health care services. or=odds ratio; ci=confidence interval; reference category=1.00 variable multivariate adjusted or 95% c.i p value socioeconomic status high 1.00 middle 0.46 0.29-0.73 0.001 low 0.21 0.11-0.39 <0.001 type of family monogamy 1.00 polygamy 0.76 0.17-3.54 0.73 family structure with both parents 1.00 with one of the parents 0.00 0.00 1.00 with relative/guardian/ step parent 0.08 0.01-0.56 0.01 birth position first child 1.00 last child 1.35 0.73-2.50 0.34 only child 1.70 0.46-6.33 0.43 others 1.32 0.77-2.25 0.32 number of siblings <3 1.00 3-4 2.21 1.23-3.96 0.01 >4 1.01 0.62-1.63 0.99 distance of facility to participants residence (km) <1.0 1.00 1.0-1.9 0.73 0.47-1.15 0.18 2.0-3.9 0.81 0.41-1.59 0.54 ≥4.0 1.98 0.60-6.51 0.24 table 6 logistic regression analysis of effect of factors on utilization of oral-health care services after school-based education and referral. braz j oral sci. 15(2):151-157 abbreviations lga: local government area; spss: statistical package of social science; gps: global positioning system; dms: degrees, minutes, seconds; gis: geographic information system references 1. luzzi l, spencer aj. factors 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graduate program in dentistry, positivo -up, curitiba, pr, brazil 5professor, graduate program in dentistry, positivoup, curitiba, pr, brazil corresponding author: dilcele silva moreira dziedzic rua prof. pedro viriato parigot de souza, 5300; 81280-330 curitiba, paraná, brazil phone: +55 41 3317-3180 fax: +55 41 3317-3082 email: dilceledz@gmail.com abstract aim: this study investigated the effect of an in-office bleaching technique on lightness, color and surface roughness of two commercially available materials: a resin-modified glass-ionomer cement and a nanohybrid resin composite. methods: twelve disk-shaped specimens were prepared with both materials. the samples were bleached with 35% hydrogen peroxide. bleaching was tested initially onto a smooth surface and later onto a polished one of the same specimens. the effect of the treatments on lightness and color was verified with a spectrophotometer. surface roughness was measured with a digital surface roughness tester. the data were statistically analyzed by repeated measures anova and post hoc tukey’s test (alpha = 0.05). results: significant variation in lightness and color was observed on the resin-modified glass-ionomer cement after the first bleaching procedure. roughness increased significantly only after polishing the resin-modified glass-ionomer cement surface. composite color variation was evident in the last observation period, but roughness and lightness variation due to bleaching and polishing was not significant. conclusion: the bleaching treatment caused significant color alterations on the materials tested. this study observed that the application of in-office bleaching onto the glass-ionomer cement promoted clinically observable color alteration, and polishing after bleaching is contraindicated for this material. key words: resin-modified glass-ionomer cement. composite. in vitro bleaching. color behaviour. surface roughness. introduction due to increased demand for aesthetic treatments, tooth bleaching has become a very popular procedure. dental bleaching is considered an effective treatment, and various concentrations of carbamide peroxide (cp) or hydrogen peroxide (hp) are used during at-home and in-office techniques. the immediate result of dental bleaching is directly related to the agent’s concentration and exposure time, despite the result after completion of the treatment being similar, clinically1 and in vitro2. patients should be advised that after the conclusion of the bleaching treatment restorations might require replacement, especially in anterior teeth, for not matching received for publication: january 22, 2017 accepted: june 12, 2017 original article braz j oral sci. july | september 2016 volume 15, number 3 http://dx.doi.org/10.20396/bjos.v15i3.8649978 186 the tooth color achieved after the treatment. the restorations may darken more or less than the teeth after exposure to components in the oral environment, and may not respond to the same degree to the bleaching treatment. it is also recommended to carefully examine the restorations before a bleaching treatment and replace the ones without adequate sealing, in order to reduce the risks of adverse effects3. restoration before the bleaching treatment is indicated to prevent sensitivity in non-carious cervical lesions and restoration replacement is recommended for those with marginal leakages. the effect of bleaching agents on restorative materials and adhesive/tooth interface has been investigated4, but has not been fully resolved because of the diverse material composition and experimental setting. composite resins and resin-modified glass-ionomer cements present organic resin matrix and inorganic filler particles. the bleaching agents can react differently with each phase or their interface. bleaching treatments of restorative materials may affect surface gloss5, microhardness6,8, roughness6,7,9-12, color13-16, and the amount of elutable components17,18. surface polishing after bleaching may not restore the physical properties of the restorative material because subsurface layers up to 2.0 mm can be affected8. the interaction between the bleaching products with restorative materials is of clinical significance, and therefore needs to be evaluated. according to wang et al. (2011), it is important to investigate the alteration of restorative materials to bleaching in order to minimize the need for replacement of restorations after this treatment9. the aim of this in vitro study was to investigate the effect of an in-office bleaching material onto two tooth-colored restorative materials, analyzing roughness, lightness and intrinsic color change. the null hypothesis of this study was that 35% hp would not result in significant roughness and color differences in the materials tested. material and methods disk-shaped specimens of a nanohybrid composite (filtek z250 xt, shade a3, table 1) and of a resin-modified glassionomer cement (vitremer, shade a3, table 1) were prepared according to manufacturer’s instructions (n = 12). each specimen (1.0 mm thick and 10.0 mm in diameter) was prepared after inserting the restorative material into a cylindrical nylon matrix and pressing between two polyester strips and two glass slides. the material was initially light-cured through the polyester strip; to ensure thickness uniformity. specimens were polymerized by a led unit (optilight max, dabiatlante, brazil) for 40 seconds (1000 mw/cm2). the protective gloss included in the vitremer kit was applied and polymerized onto both sides of vitremer disks. effect of hydrogen peroxide application on color and surface roughness of two restorative materials braz j oral sci. 15(3):185-189 table 1 chemical composition and manufacturer of materials used in the present study. material composition * manufacturer batch number filtek z250 xt bis-gma, udma, bis-ema, pegdma, tegdma zircônia/ silica particles ≤3 microns, silica particles 20 nanometer 3m espe, dental products, st. paul, mn, usa 1501300548 vitremer powder: fluoroaluminosilicate glass, potassium persulfate gloss: tegdma, bis-gma liquid: modified polycarboxylic acids (methacrylate groups), water, hema 3m espe, dental products, st. paul, mn, usa 1421100285 whiteness hp maxx 35% hydrogen peroxide, thickener, dye, propylene glycol, filler, water fgm produtos odontológicos, joinville, sc, brazil 131014 *information provided by manufacturers. abbreviations: bis-gma, bisphenol-a-glycidyl methacrylate; bis-ema, ethoxylated bisphenol-a-glycidyl methacrylate; hema, 2-hydroxyethyl methacrylate, pegdma, polyethylene glycol dimethacrylate; tegdma, triethylene glycol dimethacrylate; udma, urethane dimethacrylate. the nonirradiated surfaces were identified and the specimens were stored in distilled water at 37°c, with a weekly change of water. two weeks after preparation, the specimens received the first bleaching treatment with whiteness hp maxx (35% hp, table 1). the bleaching protocol used onto the specimen’s irradiated surfaces followed the manufacturer recommendation: three consecutive applications of the gel for 15 minutes each, after freshly mixing the bleaching component with the thickener, without light acceleration. after each bleaching application the specimens were wiped with gauze. the disks were rinsed with water for 1 minute after finishing the bleaching procedure, and stored in distilled water at 37°c. two weeks after the first bleaching procedure the specimens were polished manually with 600 grit silicon carbide paper and water irrigation, and then thoroughly rinsed with tap water to remove any debris. two weeks after polishing, the specimens received the second bleaching treatment. bleaching was performed initially onto a smooth surface and later onto a polished one, of each specimen. the roughness of the disks was verified with a digital handheld surface roughness tester (surftest sj210p, mitutoyo, japan). the roughness value was calculated by averaging three readings of ra (average roughness, measured in µm) of each specimen from three different orientations. cielab color parameters of the specimens were measured with a spectrophotometer (vita easyshade advance 4.0, vita zanfabrik, germany) calibrated with a white reflectance standard tile supplied by manufacturer and the specimens were placed against a white background. color variation (δe) was calculated using the following equation (1)19. δe= [(δl)2+ (δa)2+ (δb)2]1/2 (1) 187 where, the l* represents the degree of lightness (ranges from 0 black, to 100 white), the a* coordinate represents the degree of green/red color (-a* green, +a red), and the b* coordinate represents the degree of blue/yellow (-b* blue, +b* yellow). δe* values ≤ 3.3 were considered clinically acceptable for color variation, not relevant and not perceptible13,20. roughness, lightness and color parameters were collected in five observation periods: t0 = baseline (one week after disk preparation); t1 = one week after the first bleaching treatment; t2 = one week after polishing, t3 = one week after the second bleaching treatment; t4 = two weeks after the second bleaching treatment. the parameter’s variations were calculated between each observation period and baseline, and between every two consecutive observation periods. the data were statistically analyzed using statistica software (version 10, statsoft inc., usa). before the light variation analysis, the values were squared for handling the negative values. a repeated measures analysis of variance (anova) was used to analyze the significant differences between the variables with a significance level of 0.05. whenever there was a significant interaction between them, a post hoc tukey’s test was used to detect specific differences with a significance level of 0.05. results the mean values and standard deviations of color variation, lightness variation and roughness for the resin-modified glassionomer cement and the nanohybrid resin composite, after bleaching and polishing are summarized in tables 2, 3 and 4. the analysis of variance (anova) revealed a highly significant difference (p = 0.000) between the independent variables for color and lightness variation. these indicated that bleaching and polishing had a significant effect on the materials investigated. effect of hydrogen peroxide application on color and surface roughness of two restorative materials braz j oral sci. 15(3):185-189 table 2 mean values (standard deviation) of color variation δe* and lightness variation δl* of vitremer and z250 xt, between each time period and baseline. material δe*1 δe*2 δe*3 δe*4 vitremer 17.42 (±1.84)e 16.14 (±1.83)b 15.87 (±1.75)b 14.41 (±1.86)d z250xt 1.48 (±0.73)a 1.70 (±0.74)a 1.46 (±0.94)a 2.37 (±0.49)c δl*1 δl*2 δl*3 δl*4 vitremer 41.11 (±17.57)d 19.25 (±10.27)c 13.31 (±8.20)b, c 8.29 (±4.80)a,b z250xt 1.07 (±1.27)a 1.41 (±1.79)a 1.57 (±2.44)a 1.27 (±1.61)a values with the same superscript letters in each group were not statistically significant differences at p ≥ 0.05, n= 12. table 3 mean values (standard deviation) of and color variation δe* and lightness variation δl* of vitremer and z250 xt between each time period: δ1 (between t1 and t0), δ2 (between t2 and t1), δ3 (between t3 and t2), δ4 (between t4 and t3). material δe*1 δe*2 δe*3 δe*4 vitremer 17.42 (±1.84)c 2.31 (±0.78)a 1.38 (±0.91)b 2.13 (± 0.85)a z250xt 1.48 (±0.73)a, b 0.51 (±0.14)b 0.73 (±0.49)a, b 2.03 (±0.63)a δl*1 δl*2 δl*3 δl*4 vitremer 41.11 (±17.57)b 5.13 (±3.48 )a 1.03 (±1.49)a 2.26 (±2.21)a z250xt 1.07 (±1.27)a 0.11 (±0.09)a 0.39 (±0.97)a 1.41 (±2.15)a values with the same superscript letters in each group were not statistically significant differences at p ≥ 0.05, n= 12. table 4 mean values (standard deviation) of roughness (µm) of vitremer and z250 xt observed in the periods t0, t1, t2, t3, and t4 material ra0 ra1 ra2 ra3 ra4 vitremer 0.31 (±0.17)a, b 0.27 (±0.15)a, b 0.48 (±0.20)c, d 0.66 (±0.31)d 0.61 (±0.27)c, d z250xt 0.16 (±0.11)a 0.24 (±0.22)a, b 0.36 (±0.36)a, b,c 0.31 (±0.12)a, b 0.34 (±0.08)a, b,c values with the same superscript letters were not statistically significant differences at p ≥ 0.05, n= 12. the analyses of color variation (δe*) and lightness variation (δl*) for vitremer, between baseline and observation periods (table 2), and between observation periods (table 3) showed that alterations promoted by the bleaching procedure were significant after the first bleaching procedure (t1), on the material protected by the gloss. significant color variation for filtek z250 xt was detected in the last observation period (t4) compared to baseline, after polishing, bleaching and storage of the material (table 2). the analyses of lightness variation for filtek z250 xt showed that alterations promoted by bleaching and polishing, between baseline 188 effect of hydrogen peroxide application on color and surface roughness of two restorative materials and observation periods (table 2), and between observation periods (table 3), were not significant. the difference of roughness between the materials and observation periods was significant (p < 0.000). the analyses of the roughness values (ra) for vitremer demonstrated that the superficial modification promoted by the polishing procedure was significant and increased roughness, comparing both initial periods with the three final ones (table 4). however, the bleaching effect onto roughness was not significant (table 4). the analyses of roughness for filtek z250 xt revealed that the superficial alteration promoted by the bleaching and polishing procedures was not significant, as well as the variation of roughness promoted by bleaching onto smooth surface and roughened surface (table 4). discussion the null hypothesis was rejected for z250 xt, because the color alteration of the nanohybrid composite was significant, but in a clinically acceptable range of color change (δe*4 = 2.37). the δe* value is considered clinically undetectable if less than 1, acceptable if between 1 and 3.3 and unacceptable if greater than 3.313,20. the roughness variation due to bleaching onto smooth surface and polished composite surface was not significant. the color alteration observed on the resin-modified glassionomer, due to the first bleaching treatment with 35% hp, was significant and clinically detectable (δe*1 = 17.42). it was not due to superficial color alteration of the specimens, but intrinsic color change, because it persisted with time, even after the wear promoted by the polishing procedure. the substantial and positive δl* for vitremer, after the first bleaching treatment would de clinically unacceptable, if the natural dental structure did not lighten to the same degree. the color change in experimental subsequent periods was acceptable (δe* ≤ 2.31), with a tendency for recuperation towards the original color and lightness, but without matching them. vitremer roughness increased after polishing, but was not affected significantly after the bleaching agent was applied onto the more irregular surface. the choice of these parameters should be elucidated, because roughness can promote greater accumulation of food debris, biofilm formation, and increased colonization by cariogenic bacteria21; and alteration of color may impair the aesthetics. unacceptable color match of restorations, which may discolor more easily than teeth after prolonged exposure to the oral environment, is one reason for replacement of restorations. the present study analysed the color change of two restorative materials without any previous discoloration, both shade a3 as provided by the manufacturer. the in-office bleaching procedure used was recommended by the manufacturer, in order to represent the clinical conditions without continuous exposure or simulating the cumulative effect over a period of time. the gloss applied onto the resin-modified glass-ionomer, immediately after restoration, protects the material from syneresis and imbibition. surface protection is also recommended onto earlier restorations of glass-ionomer, when using rubber dam isolation and topical application of fluoride. the initial smooth surface with the gloss did not protect the material from intrinsic color change caused by the bleaching. the significant color alteration of the resin-modified glass-ionomer from the first bleaching treatment could be clinically detectable, and continued throughout of the experiments. the present study corroborates with a previous one, which compared the color stability of fluoridecontaining restorative materials and composites, and observed significant difference in color of ionomer cements22. the bleaching effect difference between the two shade a3 products might be due to water affinity, because the hydrophilic resin modified glassionomer cement showed lower intrinsic color stability than the hydrophobic nanohybrid composite22. some studies affirm that bleaching agents can remove the extrinsic staining from composite resins, but not promote intrinsic color change13,23. the intrinsic color alteration of the nanohybrid composite tested in this study, two weeks after the second bleaching, was significant, but considered clinically undetectable (mean δe* value < 3.3). one limitation of this study is that no control was used to verify the color or roughness change caused by the storage of the specimens, immersed in distilled water at 37°c during the study period of 9 weeks. the superficial alteration promoted by the bleaching and polishing procedures onto filtek z250 xt was not significant. studies with composites reported no observable or significant roughness alteration after bleaching9,24, and no significant effect on the microhardness14,24,25; agreeing that no replacement of restorations may be required after bleaching. it is recommended caution in bleaching treatments when restorations are present, in order to minimize the impact of bleaching therapies on restorative materials and restorations3,5. bleaching treatments may affect the color and surface roughness of existing restorations, and even though the alterations may not be clinically relevant to indicate the replacement of the restoration, they require close monitoring by the dentist and should not be used indiscriminately when restorations are present. color matching may be a problem when the intrinsic restoration color matches the dental element before bleaching. in the case of a composite restoration, it will remain unchanged, contrasting with the altered dental color after bleaching. however, resin-modified glass ionomer restoration will present color alteration with the bleaching. conclusions within the limits of the present study, it can be concluded that the in-office bleaching material caused significant and clinically detectable alterations on color of a resin-modified glass-ionomer cement. therefore, this study contraindicates the application of in-office bleaching products on resin-modified glass ionomer restorations, if the color shade is to be maintained, and also polishing after bleaching. the 35% hp bleaching material also caused significant alteration on the intrinsic color of a nanohybrid composite, but it is considered clinically undetectable. therefore, this study does not contraindicate the application of bleaching gel on the composite, or polishing the restoration after bleaching. even though there is no sufficient reason to indicate the braz j oral sci. 15(3):185-189 replacement of restorations after bleaching, this may be necessary to match the color variation of the dental elements. references 1. basting rt, amaral fl, franca fm, florio fm. clinical comparative study of the effectiveness of and tooth sensitivity to 10% and 20% carbamide peroxide home-use and 35% and 38% hydrogen peroxide in-office bleaching materials containing desensitizing agents. oper dent. 2012 sep-oct;37(5):464-73. doi: 10.2341/11-337-c. 2. meireles ss, fontes st, coimbra la, della bona a, demarco ff. effectiveness of different carbamide peroxide concentrations used for tooth bleaching: an in vitro study. j appl oral sci. 2012 marapr;20(2):186-91. 3. attin t, hannig c, wiegand a, attin r. effect of bleaching on restorative materials and restorations--a systematic review. 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il-6.total sialic acid and its fraction, and salivary total proteins statistical analysis was done by statistical package for social sciences. results: statistical analysis showed a significant (p≤ 0.01) highest value of total sialic acid in periodontitis. while the highest value of il-6 was in partial edentulous group. old ages increased salivary il-6 significantly. in periodontitis there was significantly association of il-6 with probe pocket depth, mobility scores of teeth, protein bonund sialic acid and significant negative association with lipid bound sialic acid in the precipitate. while in partial edentulous il-6 associated significantly with gingival index and free sialic acid in precipitate and negatively with probe pocket depth. conclusions: salivary sialic acid and il-6 are periodontitis biomarkers in dentate. pleiotropic role of il-6 can be diagnosed by sialic acid levels. it depends on age, (which affects number of teeth and salivary flow rate), and treatment conditions. keywords: sialic acid. periodontal inflammation and il-6. mailto:jwanjaw@yahoo.com mailto:jwanjawzali@gmail.com 2 jwan ibrahim introduction periodontitis is the major chronic inflammatory oral disease in dentate people. it is a multi -factorial influenced by genetics as well as by the environment1. it is initiated by gram negative bacteria that secrete various cytokines from lipo-polysaccharides2. improper or exuberant immune response lead directly toward over production of inflammatory cytokines that lead to deleterious inflammatory processes and destruction of the periodontal tissue and alveolar bone, consequently periodontal attachment loss3. interleukin-6 (il-6) is an important cytokine involved in the regulation of host response to tissue injury and infection4. il-6, produced locally in bone following stimulation by il-1 and tumor necrosis factor (tnf)5, by a variety of cells, such as monocytes, fibroblasts, osteoblasts, and vascular endothelial cells in response to inflammatory challenges. il-6 activity in inflammation is considered double-edged (a pleiotropic cytokine), that may enhance or suppress inflammatory bone destruction. traditionally il-6 has been considered to be a pro-inflammatory mediator, because it is induced by il-1 and tnf-α early in the inflammatory cascade and stimulates expression of acute-phase proteins that have anti-inflammatory properties6. previous studies reported anti-inflammatory properties of il-6; it can increase the production of tissue inhibitors of matrix metalloproteinases (timp), suppresses il-1 expression, can induce the synthesis of il-1 receptor antagonist (il-1ra) and the release of soluble tnf receptors4. recent studies demonstrate that il-6 deficient in many typical pro-inflammatory properties and exert a number of anti-inflammatory activities such as; indirect stimulation and production of; collagenase, and matrix metalloproteinase, or stromelysin, inhibition superoxide production, and suppression spontaneous il-1-mediated degradation of cartilage matrix5. therefore individual variability in the ability to synthesize and release of il-6 may modulate the susceptibility, development, and progression of a number of autoimmune and inflammatory diseases (periodontal diseases) which are recently reported to be associated with il-6 deregulation6. sialic acids are a family of nine carbon acidic monosaccharide. an important function of host sialic acid is to regulate innate immunity7. bacteria with capsular polysaccharide (such as gram negative bacteria) containing sialic acid interacts with component of the hosts nonspecific immune response, may modulate the ability of the host to mediate an immune response by affecting the release of cytokine molecules thereby disrupting the coordination of the hosts cell-mediated immune response8. pro-inflammatory cytokines such as interleukin-1, interleukin-6, and tumor necrosis factor-α stimulated to produce acute-phase glycol-proteins with sialic acid as a component of the oligosaccharide side chain9. sialic acid is a stable and representative marker of the overall acute-phase response10. the current criteria for assessment of periodontal tissues surrounding teeth were based on clinical and radiographic changes. saliva is a non-invasive suitable environment for biochemical and immunological analysis and can be an early indicator for detection of active periodontal disease11. 3 jwan ibrahim imbalance between proand anti-inflammatory cytokines could be involved in the initiation and progression of chronic periodontitis12.there are conflicting reports on differences in salivary il-6 levels between chronic periodontitis and healthy subjects. studies are required for clarifications the roles of il-6 in periodontitis progression and identifying factors that may affect the role of il-6. therefore this study conducted to evaluate interleukin-6 levels in periodontitis and its relation to clinical features and inflammable salivary biomarkers; total sialic acid and its fraction and total proteins to identify its role in disease progression. methods and patients: the study was observational case-control study. it was arried out in the dental teaching clinic college of dentistry / hawler medical university. during the period of 1st august 2009 up to 30th september 2009. participants (study populations): a total  of 60 convenient samples recruited in this study, with age ranged between 18 and 70 years of both genders. they were screened for periodontal health status and divided into three groups; first were 20 participants free from periodontal disease and regarded as control group, second 32 dentate out patients group, diagnosed with chronic periodontitis disease,(had >3-≥7mm pockets depth), according to the classification of the periodontal diseases issued by the american academy of periodontology13, in 1999, third group were 8 partial edentulous periodontitis patients with history of treatment. inclusion criteria: healthy participants systemically and with no history of periodontal and antibiotic treatment prior to the study for three month. exclusion criteria: pregnant and lactating women and patients with systemic diseases. sampling technique: a structured questionnaire (interview form) was used to collect data by asking the studied population about social and behavior factors included; age, sex, occupation, educational levels, smoking status, oral hygiene habits (frequency of tooth brushing, use of dental floss), and use of medications. all participants were informed on the procedure and their consents had been taken. clinical measurement: the clinical periodontal examinations were carried out by the trained dentist and calibrated by the supervisor of the teaching clinic. the measurements included; bleeding on probing by saxer and mühlemann14, probe pocket depth (pd) was measured with (williams probe), for all teeth from gingival margin to the base of pocket at four sides (labial/buccal, lingual/palatal, mesial and distal surfaces), per teeth, mobility of teeth classified by carranza and takei15, and miss of teeth. classifications of the (aap)13 were used for identifying the severity of disease; probing depths >3 & <5 mm (mild pockets), ≥ 5 & <7 mm (moderate pockets) and ≥ 7 mm (sever pockets). the gingival index (gi) and plaque index (pi) scores were recorded, using criteria of (löe, 1987)16. saliva collection saliva samples (prior to the clinical measurements) were collected from all subjects between 9 and 11 hours am. spitting method17 was used for collection un-stimulated whole saliva. patients were prevented from eating, drinking, and oral hygiene for 2 h before collection. the samples were stored at -200 c for one hour, then centrifuged immediately at (10000) g and at 40c for 20 minutes to obtain supernatant and precipitate. both fractions were stored at -20c0 for analysis. 4 jwan ibrahim biochemical tests: this included salivary il-6 enzyme-linked immunosorbent assay (elisa) kits (abeckman coulter manufacture). total sialic acid (tsa) and free sialic acids (fsa) were measured with modified thiobarbituric acid method of skoza and mohos18. extraction and determination of lipid -bound sialic acid (lsa) by masami19. determination of sialic acid bound to total proteins (psa) by shetty and pattabiraman20, salivary total proteins by lowry method davidson college21. statistical analysis: the statistical package for social sciences (spss, version 18) was used for data analysis by parametric and non parametric tests for normal and abnormal distribution of variables checked by shapiro-wilk test. analysis of variance (anova-test) to compare between three or more means and t-test to compare between two means. correlation coefficient for measuring correlation between quantitative data and eta test for measuring association between nominal and interval. binary logistic regression for odds ratios measurement. a p-value of ≤ 0.05, and ≤ 0.01 were considered statistically significant and high significant respectively. results the studied population composed of 32 (53.3%) periodontitis, 20 (33.3%) participant as control and 8 (13.3%) partial edentulous patients with teeth miss more than (10). salivary mean levels of il-6, total sialic acid and total protein statistical analysis showed a significant (p≤ 0.01) difference in the means of total sialic acid and total protein between periodontitis, control, and partial edentulous group. post hoc least significant difference (lsd) test showed significant (p≤ 0.01) difference of periodontitis with control and partial edentulous groups in sialic acid, while partial edentulous showed similarity with control group. total salivary proteins, in periodontitis differ significantly (p≤ 0.05) with control but not with partial edentulous. there was also a significant (p≤ 0.01) difference in the means of il-6 between the groups. post hoc (lsd) test showed significant highest value (p≤ 0.01) value of il-6 in partial edentulous compare to periodontitis and control groups. table 1. mean levels of salivary; il-6, protein, and total sialic acid groups no. il-6 (pg/ml) mean ± s.e f-value p-value periodontitis 32 26.13± 7.4 18.7 0.00** control 20 1.7 ± 8.3 partial edentulous 8 66.35± 8.3 groups no salivary protein mean ± s.e. f-value p-value periodontitis 32 6.6± 0.34 11.5 0.00** control 20 4.12 ±0..27 partial edentulous 8 5.7± 0.47 groups no. total sialic acid (mg/l) mean ± s.e. f value p-value periodontitis 32 156.84± 3.9 16.96 0.00** control 20 96.80± 0.32 partial edentulous 8 100.36± 19.5 **(p≤ 0.01), *(p≤ 0.05), no = number 5 jwan ibrahim relation of ages with salivary il-6 and cases: statistical analysis showed a significant correlation of il-6 with ages and significant differences of il-6 among age groups. there was also significant relation and differences of ages among cases. binary logistic regression showed a significant little increase risk of il-6 levels in old ages and in partial edentulous, table (2). relation between il-6 and periodontal health status in periodontitis: table (3) shows significant positive correlations of il-6 and protein bound to sialic acid (psa) with probe pocket depth (pd). there was also high relations of (il-6) and total sialic acid with mobility of teeth. statistical analysis showed significant differences in the means of il-6 and psa among groups of probe depth (pd), and tsa among teeth mobility scores. table 2. relation and differences of ages with salivary (il-6) and cases age groups (years) il-6 (pg/ml) mean± s.e. no. value of r p-value f-value p-value b 95% ci 18-44 14.9 ± 3.7 36 0.37 0.003** 2.3 0.028* 1.024* 1.0 1.1 45-75 35.9 ± 8.3 24 groups of the study (cases) mean of ages ± se no eta test degree f-value p-value b 95% ci periodontitis 39.5 ± 2.23 0.79 strong 3.5 0.037* 1.03* 1.0 1.06 control 34.9 ± 3.5 partial ed. 49.5 ± 2.8 total 39.3 ± 1.8 (p≤ 0.01)**,(p≤ 0.05)*, r=pearson’s correlation coefficient, b = odds ratio, ci = confident interval, ed. = edentulous table 3. association of il-6 and sialic acid with probe pocket depth and teeth mobility in periodontitis prop depth of teeth (pd) in mm il-6 (pg/ml) means± s.e. no. value of r p-value f-test value p-value of difference mild > 3< 5 20.7 ± 4.9 17 0.37 0.039* 6.5 0.005* moderate ≥5-6 26.1 ± 4.9 13 sever ≥ 7 53.0 ± 37.75 2 total 26.14 ± 3.9 32 prop depth (pd) in mm psa means± s.e.(mg/l) no. value of r p-value f-test value p-value of differences mild > 3< 5 26.5 ± 2.6 17 0.52 0.002** 15.1 0.001** moderate ≥5-6 30.2. ± 2.9 13 sever ≥ 7 79.5 ± 29.4 2 total 31.3 ± 17.9 32 mobility scores il-6 (pg/ml) means± s.e. no. value of r p-value f-value p-value of difference. 0 16.44±5.3 9 0.37 0.038* 1.76 0.176 1 23.9±21.6 14 2 40.3± 10.9 7 3 36.0± 15.0 2 total 26.13±3.9 32 mobility scores total sialic acid (mg/l) means± s.e. no. value of r p-value f-value p-value of difference. 0 100.6 ± 28.9 9 0.35 0.047* 6.0 0.003** 1 87.7 ± 4.4 14 2 130.3 ± 15.5 7 3 145.1 ± 18.7 2 total 156.8 ± 41.6 32 * = (p≤ 0.05), ** = (p≤ 0.01), rpearson’s correlation 6 jwan ibrahim assosiation of il-6 in partially edentulous with periodontal health status table (4) shows a negative correlation of il-6 with probe pocket depth, and significant positive correlation of il-6 with gingival index gi and significant difference of il-6 among gi groups. there was also negative correlation of probe pocket depth with tsa (-0.85, p value = 0.1) and its fractions, while gi showed positive significant correlation (0.78, p value = 0.05*) with fsa in sediment of saliva. correlation of il-6 with total sialic acid and its fractions among groups of studied population: statistical pearson’s correlation showed a significant correlation of (il-6) with protein bound sialic in supernatant (r=0.468**), (fig 1) and total salivary protein (r=0.451**) in periodontitis,. while lipid bound sialic acid in sediment showed significant (p≤ 0.05) negative correlation (r= -0.382*) with (il-6) in periodontitis (fig 2) as well as free sialic acid in sediment of saliva showed a significant positive correlation (spearman correlation value= 0.820*) with salivary il-6 among partial edentulous group. table 4. association of il-6 with probe pocket depth, and gingival scores prop depth of teeth (pd) in mm il-6 (pg/ml) means± s.e. no. value of spearman p-value of r kruskal-test value p-value sig. of difference mild > 3< 5 79.5± 35.7 4 0.22 no sig. 0.33 no sigmoderate ≥5-6 53.3±19.9 4 total 66.4± 19.5 8 groups of gingival index means± s.e spearman p-value of spearman mann-whitney moderate (1.1-2) 42.8 ± 14.2 6 0.93 0.01** 0.047*sever (2.1-3) 136.9 ± 31.9 2 total 66.4 ± 19.5 8 * = (p≤ 0.05); ** = (p≤ 0.01) figure 1. correlation between salivary il-6 and protein bound sialic acid in supernatant of saliva of periodontitis 120.00 le ve ls o f p ro te in b ou nd s ia lic a ci d in su pe rn at an t o f s al iv e (m g/ l) r2 linear = 0.219 100.00 80.00 60.00 40.00 20.00 0.00 0.00 20.00 40.00 60.00 levels of salivary il-6 in periodotitis (pg/ml) 80.00 100.00 7 jwan ibrahim discussion salivary levels of il-6, total sialic acid and total protein in periodontitise and controls total salivary; sialic acid, protein and il-6 showed significant high concentration in periodontitis patients compare to controls. this view pathogenesis role of increased salivary tsa levels in periodontal disease suggested by shinohara et al.22 and can differentiate between periodontal disease and normal condition. high level of il-6 is in line with previous studies (elbersol and cappelli23, miller et al.24) who found that periodontitis patients have higher (il-6) levels when compared to the periodontally healthy population. this result is in contrast with studies of nibali et al.6 and shaker and hashem12 who found no significant difference in the levels of il-6 between chronic periodontitis and periodontally healthy subjects. significant difference in the level of total; protein, sialic acid and (il-6) between periodontitis and control may be returned to virulence type of bacteria and its products in periodontitis, and change in biosynthesis and post translational glycosylation processes of the acute-phase glycoprotein in the liver as indicated a by significant positive and negative correlation of (il-6) with, protein bound to sialic acid (psa), and lipid bound sialic acid (lsa) in sediment respectively. bacteria with virulence factor included either serotype lipopolysaccharide or specific antigen stimulates secretion of cytokines from monocytes that are modulated with sialic acid. this was in agreement with soell et al.25 who stated that structurally related cell surface proteins from streptococcus mutans (major bacteria of oral cavity) binds to monocyte surface receptors via sialic acid residues and exerts immune-modulator effects on human monocytes like induction of (tnf-α), il-1β, and il-6). mean level of salivary (il-6) in control in this study was in agreement with other investigators26,27 who found low levels (1.4±1.0 pg/ml) and (1.8± 4.25 pg/ml) for salivary (il-6) respectively. figure 2. inverse correlation between salivary il-6 and lipid bound sialic acid in sediment of saliva of periodontitis patients 60.00 li pi d bo un d si al ic a ci d in s ed im en t of s al iv a (m g/ l) r2 linear = 0.146 50.00 40.00 30.00 20.00 10.00 0.00 0.00 20.00 40.00 60.00 levels of salivary ii-6 in periodotitis (pg/ml) 80.00 100.00 8 jwan ibrahim salivary levels of il-6, total sialic acid and total protein in partial edentulous majority (75%) of partial edentulous group were female former smokers, in age group (45-70) years. low number of teeth caused lower intensity of inflammation compared to dentate periodontitis group as indicated by lower levels of total salivary sialic acid. this result conforms the hypothesis that intensity of periodontal inflammation has been associated with the number of teeth affected28 and ensured by no significant difference in the level of sialic acid between partial edentulous group and controls. similarity of partial edentulous group in total protein with periodontitis. may relate to history of accumulation effects of periodontitis and treatments process and increase in salivary antimicrobial agents. this result consistence with jawzaly1 and shetty and pattabiraman20 who found high concentration of total protein in periodontitis and gingivitis. high value of il-6 may due to; sex and old ages, that affect salivary secretion and flow rate29. this result consistence with alwan et al.30 who found significant difference in the volume of gingival crevicular fluid between chronic periodontitis and healthy control and with slade et al.31 who suggested that c-reactive protein levels among edentulous not similar to periodontal healthy individuals and could be raised by other risk factors; ages, and smoking, additionally history of periodontitis and accumulative effects of treatments may affect the volume of saliva and change the equilibrium between the activities of pro-inflammatory and anti-inflammatory cytokines and determine the stage of severity and dissolution of inflammation as stated by shaker and hashem12 and reported that the total amount of cytokine might be more representative of the disease condition as compared to its concentration. this result also accompanied with nibali et al.6 who reported that il-6 increase associated to the short-term inflammatory response to therapy and long-term reductions when a clinical improvement in the periodontal status is obtained. this idea was more abundant among former smokers who had history of compromised outcome of periodontal therapy and conform goutoudi et al.4 who found higher concentration of il-6 in gingival cervicular fluid in diseased sites following treatment, and better clinical result in nonsmokers following treatment of periodontitis. relation between ages and interleukin 6 (il-6) poor periodontal status in old ages reveals a cumulative effects of periodontal microbial challenges and periodontal treatment, which cause severe diseases in old ages as indicated by high pd and mobility scores in periodontitis and more teeth miss in partial edentulous. this result agrees with goutoudi et al.32 who reported deterioted periodontal status (according to periodontal indices) with age. also jawzaly et al.1 reported that old age individuals had received more therapy with selective extractions of teeth affected by periodontitis relation between (il-6) and periodontal health status among periodontitis and partial edentulous association both il-6 and sialic acid with periodontitis indices reveals predictor roles of both for severity of periodontitis. results of il-6 consistence with shaker and hashem12 who found a significant positive correlation between periodontal param9 jwan ibrahim eters and serum il-6, also alwan et al.30 who found a positive significant correlation between quantities of il-6 in crevicular fluid and tissues inflammation (gi) and destruction (pd), and with ng et al.33 who identified significant correlation between alveolar bone loss score and (il-6). accompanying association of (il-6) and sialic acid with its fraction with indices of periodontitis can be explained by interaction of sialic acid of salivary glycoprotein in pellicle and products of bacteria and immune system. this view the finding of murray et al.34 that tissues destruction and pocket formation and mobility are the result of long accumulation of plaque, and bacteria toxic products and immune system mediators; such as collagenase, metalloproteinase, that are stimulated and produced by il-6. it is online also with gani et al.35 who proposed releases of inflammatory cytokines, including interleukins il 1α and il-6 and tumor necrosis factorα, as a result of the recruitment and activation of the monocyte/t-lymphocyte axis by bacterial proliferation and / or bacterial products in periodontal pockets. this in turn leads to periodontal tissue destruction. negative correlation of il-6 and tsa with pd and significant positive correlation of il-6 with gi in partial edentulous conforms moderate to severe gingivitis and history of irreversible tissues destruction and pocket formation among partial edentulous group. this result supports geivelis et al.36 who found significant positive correlations between gcf il-6 levels in sites with gingivitis than in healthy ones and goutoudi et al.4 who found negative correlation between total il-6 in gcf and pd among patients with chronic periodontitis, and murata et al.37 who found no association between severity of periodontitis and the number of teeth and circulating il-6 in the elderly. however it is in contrast with previous results suggested a positive correlation of total il-6 with disease activity and bleeding as well as pd (lin et al.38). different results in different studies support the idea that the production of inflammatory mediators differs by type of sample and from subject to subject and other several factors; genetic and bacterial composition4. correlation of il-6 with total sialic acid and its fractions in periodontitis and partial edentulous high correlation of (il-6) with psa and total salivary protein may due to the role of (il-6) in inducing synthesis of other mediator and enzymes and agrees with gani et al.35 who reported that (il-6) increased hepatic protein synthesis of acute phase proteins (such as richily sialylated α-acid glycoprotein, carbon reactive protein, and others) and decreased synthesis of negative reactant proteins. lsa and fsa in sediment of saliva represent fraction of cells, and high molecular weight mucin (mg1)39. negative correlations lsa and positive fsa with (il-6) in periodontitis and partial edentulous respectively may explore significant role of terminal sialic acid mucin in both adherence and aggregation by cleaving sialc acid and using it by bacteria depending on the severity of inflammation. salivary sediment lsa in periodontitis may represent incorporation terminal sialic acid of mg1 with lipo-polysaccharide of gram negative bacteria that can hinder the function of the host defenses as reported by1. additionally degradation of mg1 causes precipitation of its glycoprotein which has sequestering effect for soluble (il-6)33, and may cause decrease in the level of soluble (il-6). these results combine also with gibbons et al.40 that terminal sialic acid mucine glycoprotein cleavage create a variety of carbohydrate linkages. 10 jwan ibrahim correlation of salivary il-6 with fsa in sediment of partial edentulous may reveal role of il-6 in regulation of the innate immune response to inflammation by change in glycosylation of glycoprotein of free mucin that induce il-6 secretion. this result explores the suggestion of41 that terminal sialic acid of free glycoprotein mucin is important component of interaction with bacteria protein and prevents colonization by aggregation and swallowing and mcbride and gisslow42 who showed a correlation between the amounts of sialic acid released from normal saliva and its aggregating activity. association may due to il-6 in modulation protein secretion and glycosylation as suggested by previous studies groux-degroote et al.43, and chaudhury et al.44 that changes in glycosylation of mucin may induce interleukin 6 secretion. these observations may reveal anti-inflammatory role of il-6 and consistence with tilg et al.45 that (il-6) regulated acute phase proteins that have anti-inflammatory and immuno-suppressive properties, and may regulate the acute phase response negatively. also46 concluded that il-6 suppresses il-iβ and tnf production induced by lps and may provide negative feedback effect. limitation of the study: small sample size and lack of longitudinal monitoring the changes in salivary biomarkers from onset of periodontitis, progression and treatment the findings concluded that salivary sialic acid and il-6 are oral inflammatory biomarkers in dentate periodontitis. direction balance of il-6 toward pro and anti-inflammation can be diagnosed by sialic acid fraction, and affected by age (which determine the number of teeth, and salivary; 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file:///s:/__revistas/bjos/v017/editora%c3%a7%c3%a3o/produ%c3%a7%c3%a3o/0015/consolidado/javascript:void(0); file:///s:/__revistas/bjos/v017/editora%c3%a7%c3%a3o/produ%c3%a7%c3%a3o/0015/consolidado/javascript:void(0); file:///s:/__revistas/bjos/v017/editora%c3%a7%c3%a3o/produ%c3%a7%c3%a3o/0015/consolidado/javascript:void(0); untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8652645 volume 17 2018 e18030 original article 1 master in orthodontics. united colleges of north of minas funorte, maceió – al, brazil. 2 phd in biothecnology. federal university of alagoas ufal, arapiraca – al, brazil. 3 master in orthodontics. araraquara university uniara, school of dentistry, departament of orthodontics, araraquara – sp, brazil. 4 phd in orthodontics. araraquara university uniara, school of dentistry, departament of orthodontics, araraquara – sp, brazil. corresponding author: wanderson roberto dos santos azevedo wandersongrfc@hotmail.com received: october 08, 2017 accepted: february 02, 2018 changes in head posture, hyoid bone position and airway dimensions after orthognatic surgery wanderson roberto dos santos azevedo1; christiane cavalcante feitoza2; carlos sanches vargas jr.3; karina eiras dela coleta pizzol4 abstract: mandibular/bimaxillary advancement surgery is described as a potential means of increasing the oropharyngeal airspace, provided a significant improvement in breathing capacity. aim: to evaluate postural changes suffered in the positioning of the head and the hyoid bone, dentofacial deformity patients undergoing orthognathic surgery with consequent dimensional changes of oropharyngeal airspace. methods: we evaluated the archived records of patients with postoperative 6 months minimum, being used as criteria for selecting individuals with dental class ii malocclusion and facial pattern ii, jaw or maxilomandibular deficiency, patients undergoing orthodontic-surgical treatment through mandibular advancement or of both bony bases, associated or not to genioplasty. twenty-eight patients were part of the sample and were evaluated by means of lateral radiographs in lateral standard digitized, in three distinct periods: pre-operative, immediate, postoperative period and late postoperative period (minimum 6 months). 12 linear measures and two angular cephalometric analysis were used in this research. results: with the surgery, there was an average of mandibular advancement 6.76 mm, while remaining stable in the long-term follow-up period; the hyoid bone moved onward and upward, extending your movement in the post-operative. the cervical region presented minimal movement of the head extension in the immediate post operative with almost total returns the position of the head in the post-operative. the surgical movement of oropharyngeal air space was in the same direction of the mandibular movement, but to a lesser extent (1.88 -2.76 mm). in the post-operative period was a late partial reduction of the diameter of the air space between 34-56% of gain, representing an apparent accommodation of this anatomical region soft tissue. conclusion: orthognathic surgery of mandibular advancement or bimaxillary promotes significant changes in aesthetics, in the position of the hyoid bone and upper airway dimensions, getting better quality of life to these patients. keywords: posture; hyoid bone; oral surgical procedures. 2 azevedo et al. introduction among the morphofunctional deviations which affect the population, class ii with skeletal involvement, also known as facial pattern ii, deserves special attention because of the significant changes it exerts in facial aesthetics and in masticatory and respiratory functions1,2. studies2-6 involving radiographic cephalometry in pattern ii individuals with mandibular deficiency and clockwise rotation have demonstrated that these patients frequently have higher anteroinferior facial height, class ii occlusion, height reduction of the mandibular branch, retraction of the base of the skull, frequent tmj problems, narrow posterior air space, as well as an inferiorly positioned hyoid bone and maxillary retroposition. the inadequate respiratory pattern in these individuals induces functional adaptations, promoting muscular imbalance of the face, postural changes such as parted lips and posterior head extension. the vital need to maintain the airspace pattern at the base of the tongue can explain the rotation in these pattern ii patients, who develop over the years a long face, due to mandibular retrusion, requiring orthognathic surgery for its correction7. mandibular advancement surgery is described as a potential means of increasing oropharyngeal airspace8, while, the airspace amplitude, obtained from it, is directly related to the amount of surgical advancement performed9,10. the first 10 mm of mandibular advancement tend to present a higher percentage of increase in the oropharyngeal air dimensions. for numbers above, the space continues to increase in size, but at a lower rate considering the amount of mandibular advancement. the position of the head and neck are also directly related to the dimensions of the pharyngeal air space acquired after maxillomandibular advancement surgery, as it has been demonstrated in clinical and experimental studies11,12. as the studies referring to the morphofunctional alterations of facial-pattern-ii patients represent an open field in science, the present study aimed at examining the cephalometric relationship between the hyoid bone, mandible, skull and cervical spine, trying to establish a relationship between such variables and the size of the pharyngeal airspace before and after orthognathic surgery. materials and methods ethics committee this study was submitted and approved by the human research ethics committee of the university of araraquara (cep) under number caae 32629314.3.0000.5383. sample size this retrospective study was carried out by evaluating the cephalograms of 28 patients from the centro de pesquisa e tratamento das deformidades bucofaciais (center for research and treatment of buccal deformities – cedeface, araraquara_s.p., brazil), 20 of which were female and 8 male, with an average 30 years of age. 3 azevedo et al. eligibility criteria a minimum postoperative period of 6 months, class ii and standard ii malocclusion due to mandibular deficiency, submitted to orthodontic-surgical treatment of mandibular or maxillomandibular advancement, associated or not to advanced genioplasty and / or anti-clockwise rotation of the occlusal plane. initially, 686 medical records of individuals treated between 1997 and 2014 were evaluated, of which, according to inclusion and exclusion criteria, the medical records of 34 individuals were selected. during the sample selection, 6 cases were excluded because of any of the following factors: transverse maxillary discrepancy requiring surgical transverse intervention, presence of intercurrences during orthodontic-surgical treatment or need for a new surgical intervention, absence of complete documentation or of unsatisfactory quality, thus leaving a final sample of 28 individuals. calibration the examiner was previously calibrated by repetition until the procedures were considered consistent by a second examiner. the intraclass correlation coefficient was always higher than 0.9. a pilot study was performed in order to minimize possible errors of the method. study design the radiographs were evaluated in three distinct periods: preoperative (t1), up to 1 month before surgery; immediate postoperative period (t2), within 15 days after surgery; and late postoperative (t3), at least 6 months after surgery. for the evaluations at the proposed times, digital lateral cephalometric teleradiographs obtained from the same digital x-ray imaging system (vatech® pax-400c, korea) were used, on which the cephalometric analysis uniara, created by radio memory (radio memory®, belo horizonte_m.g., brasil), based on the works of coleta et al.4 and dela coleta et al.13, to meet the proposed objectives of the research. for its construction and greater position specificity, special tools were developed to help locate the 19 cephalometric landmarks used in the scientific work (figure 1). like the examiner, the method was previously tested in a pilot study. a horizontal reference line (lhr), drawn from sella and at a seven-degree angle from s-n line and a perpendicular vertical reference line (lvr) were used as reference for the quantification of the surgical movement, evaluation of the postural changes of the head and movement of the hyoid bone. linear distances (millimeters) were measured from each point to the vertical and horizontal reference lines, thus obtaining two measurements for each cephalometric landmark (figure 1). after the marking of the stitches, the pre and post-operative radiographs and tracings (immediate and late) were superimposed to check if the reference lines were coincident in all three surgical times. based on the cephalometric tracings, it was possible to quantify the changes promoted by the surgery (t2-t1), as well as the stability of this movement (t3-t2). the cephalometric measurements used in the research are described in figure 1. statistical analysis the data obtained in the study were divided into groups according to the type of surgery performed (maxillomandibular or mandibular) and also, according to the amount 4 azevedo et al. of mandibular advancement (≤10mm or> 10mm), for better interpretation of the data and comparison with findings of literature. the statistical analyzes used were the parametric student’s t-test for the data with normal distribution and the pearson correlation coefficient to measure the degree of linear correlation between two variables. results all 28 patients in the sample underwent mandibular advancement (measured at point b), with a minimum movement of 2mm and a maximum of 15mm. approximately half of the patients (13) were also submitted to anti-clockwise rotation of the mandible and 9 received advancement / impaction mentoplasty as a complement to the mandibular advancement. the bimaxillary surgeries were performed in 9 patients, all characterized by the advancement, impaction and / or inclination of the palatal plane. the values of the changes promoted by orthognathic surgery and long-term stability are described in table 1 (total sample); while in tables 2 and 3, it is possible to visualize, in a comparative way, the surgical changes that occurred according to the type of surgery (maxillomandibular or mandibular) or the amount of movement executed (≤10mm or> 10mm). pearson’s correlation was applied to the airspace in the 3 areas measured and to the position of point b, the hyoid bone, the head and the neck produced low values for most variables. table 4 shows pearson’s correlation according to the type of surgery performed and the amount of surgical movement. lhr n a b me hgn h’ lvr h cvtopt c4i c3 c2i bl u ea3 ea2 pm go ea1c2s s figure 1. uniara cephalometric tracing, with cephalometric landmarks, reference lines and linear and angular cephalometric magnitudes. 5 azevedo et al. table 1. mean and standard deviation (sd) of the initial radiographs (t1), the surgical movement performed (t2-t1) and its stability (t3-t2). factors t1 t2-t1 t3-t2 mean sd mean sd mean sd linear (mm) a-lhr 47.84 3.25 -0.54 2.52 -0.12 1.08 a-lvr 71.54 6.39 0.7 2.79 -0.42 1.36 b-lhr 90.44 7.57 0.68 3.05 -0.91 1.52 b-lvr 61.59 8.47 6.76 4.81 -0.62 2.28 h-lhr 109.4 10.86 -1.05 4.82 -3.05 4.14 h-lvr 15.88 7.75 2.48 6.7 0.98 4.24 h-h’’ 9.11 6.56 -2.14 4.75 -1.26 4.04 c3-h 34.98 4.13 1.02 2.1 -0.83 2.21 h-rgn 35.43 6.88 4.11 6.86 -1.76 4.87 eas 10.28 3.11 1.88 2.66 -1.06 2.15 eam 9.97 3.59 2.06 2.05 -0.81 2.52 eai 9.94 3.78 2.76 2.32 -0.95 2.64 angular (o) sn.go-me 37.1 7.79 -1.56 3.43 -0.18 2.54 opt.cvt 10.76 5.34 0.44 2.64 -0.42 2.92 table 2. mean, standard deviation (sd) and significance level (p) of surgical movement (t2-t1) and longterm stability (t3-t2), according to the type of surgery performed.   maxilomandibular surgery   mandibular surgery factors t2-t1 t3-t2 t2-t1 t3-t2 mean sd p mean sd p   mean sd p mean sd p a-lhr -2.24 3.13 0.40 1.34   0.26 1.74 -0.37 0.87 a-lvr 0.98 2.63 -0.38 1.92   0.57 2.93 -0.44 1.07 b-lhr -0.85 3.96 -0.48 0.92   1.4 2.30 -1.11 1.72 b-lvr 10.89 4.84 -0.84 3.08   4.81 3.43 * -0.52 1.88 h-lhr -0.87 5.96 -5.71 5.72   -1.13 4.37 -1.78 2.44 h-lvr 3.68 6.13 1.23 4.64   1.91 6.13 0.86 4.64 h-h’’ -1.00 3.97 -2.87 2.76   -2.68 3.97 -0.5 2.76 c3-h 1.08 1.86 -0.61 3.05   0.99 1.86 -0.94 1.78 h-rgn 7.79 5.06 * -3.40 4.75   2.37 5.06 -0.97 4.75 eas 1.86 2.27 0.01 2.11   1.89 2.27 -1.57 2.11 eam 2.40 2.09 0.43 2.71   1.91 2.09 -1.4 2.71 eai 3.52 2.25 -0.33 2.66   2.41 2.25 -1.24 2.66 sn.go-me -4.43 2.72 0.71 2.91   -0.2 2.72 -0.6 2.91 opt.cvt 1.15 2.88 0.20 3.12   0.11 2.88 -0.71 3.12 * p<0.05 6 azevedo et al. table 3. mean, standard deviation (sd) and significance level (p) of surgical movement (t2-t1) and longterm stability (t3-t2), according to the amount of mandibular advancement.   advancement ≤10mm   advancement >10mm factors t2-t1 t3-t2 t2-t1 t3-t2 mean sd p mean sd p   mean sd p mean sd p a-lhr -0.17 2.46 -0.19 1.12   -1.65 2.55 0.09 2.02 a-lvr 0.46 2.75 -0.26 1.09   1.45 3.00 -0.91 0.73 b-lhr 1.25 2.89 -0.97 1.72   -1.05 3.08 -0.71 2.93 b-lvr 4.51 2.67 * -0.09 1.80   13.51 3.09 -2.22 4.54 h-lhr -1.68 4.55 -2.21 3.75   0.84 5.48 -5.56 3.88 h-lvr 1.65 5.69 1.04 4.44   4.97 9.18 0.79 4.51 h-h’’ -2.85 3.94 -0.87 3.92   -0.02 6.54 -2.42 1.23 c3-h 0.81 2.08 -0.67 2.46   1.64 2.20 -1.32 5.17 h-rgn 2.59 5.24 -1.07 4.69   8.69 9.37 -3.81 1.69 eas 1.86 2.35 -1.22 2.30   1.95 3.66 -0.58 1.32 eam 1.87 2.21 -1.12 2.76   2.64 1.44 0.14 2.39 eai 2.51 2.13 * -1.01 2.77   3.51 2.85 -0.78 0.99 sn.go-me -0.67 3.22 -0.37 2.81   -4.8 1.72 0.63 1.97 opt.cvt 0.7 2.40 -0.35 3.21   -0.34 3.37 -0.63 0.00 * p<0.05 table 4. pearson’s correlation to airspace and position of the head, hyoid bone, mandibular advancement. factors maxilomandibular surgery mandibular surgery eas eam eai   eas eam   eai b-lhr 0.48 * 0.33 0.27 -0.23 0.08 0.27 b-lvr -0.23 0.22 0.16 -0.06 0.10 0.16 h-lhr -0.19 0.17 0.17 -0.14 0.15 0.17 h-lvr -0.27 -0.44 * -0.62 * -0.48 * -0.38 -0.62 * opt.cvt 0.05   -0.17 0.21 0.07 0.03 0.21 factors advancement ≤ 10mm advancement >10mm eas eam eai eas eam   eai b-lhr 0.19 0.20 -0.18 0.48 * 0.17 0.34 * b-lvr 0.05 0.03 0.02 -0.26 0.33 * -0.02 h-lhr -0.04 0.22 -0.14 -0.42 * -0.26 0.10 h-lvr -0.34 * -0.52 * -0.47 -0.04 -0.27 -0.69 ** opt.cvt 0.17   0.17   0.14 0.12 0.63 * 0.60 * * weak to moderate correlation ** strong correlation 7 azevedo et al. discussion a variety of ways of assessing oropharyngeal airway changes as a result of orthognathic surgery, such as lateral cephalometry, computed tomography, polysomnography, and nasopharyngoscopy14,15 are described in relevant literature. in our study, we chose to measure the airspace through lateral normalized cephalometric radiographs, taken while patients were sitting and in the natural position of the head. this technique has the advantage of, conveniently, being part of the orthodontic documentation usually required for planning and post-surgical follow-up4. some limitations were observed in the present study as the sample size, and post-surgical follow-up (minimum of 6 months). although half of the sample had a follow-up of 12 months or more, in the case of airspace assessment. when evaluating the effects of orthognathic surgery on the airway in patients with obstructive sleep apnea, recurrence of the soft tissues of the airways during the first year has been observed, especially in advances fewer than 10 mm. thus, it would be interesting that the follow-up of all patients was greater than 12 months. another important factor is that linear analysis is based on craniometric points that undergo changes in anatomic location when observed in the preoperative and immediate postoperative periods. likewise, the accommodation of soft tissues of the palate and tongue in the late postoperative period causes changes in the location of measurements of craniometric points. for this reason, in the cephalometric analysis of this study, the morphometric quantification of the airspace area16 was performed. according to the literature3,8,17, maxillomandibular advancement movements promote a substantial increase of the airways in all dimensions, be it antero-posterior or lateromedial. in the present study, 9 patients underwent bimaxillary advancement surgery, with an average sagittal movement of 9.80 mm in the maxilla and 10.89 mm in the mandible. in these cases, there was a linearly quantified increase from cephalometry, 1.86 mm of airspace in the upper region, 2.40 mm in the middle region and 3.52 mm in the lower region, stable in the long run. the maxillo-mandibular advancement values achieved by the patients studied are compatible with other studies in the literature16,18. in the cases in which only the mandibular advancement was performed, which was the greater part of the sample studied19, the mean value found for point b was lower compared to that of the maxillomandibular surgery group (4.81 mm, sd 3, 43mm), although both values are within those reported in the literature. this fact proves that the movements observed in the studied sample are comparable to the ones from studies already published, acting as a parameter for the evaluation of the modifications occurred in the oropharyngeal airspace. another movement capable of providing an increase in oropharyngeal airspace is the anticlockwise rotation of the occlusal plane, due to the lower and anterior positioning it provides to the soft tissues of the palate, promoting an increase in space for the tongue that is now more anterior9,16,18,19. keeping the supra-hyoid muscles attached to the anterior region of the mandible promotes an anterior tension of the tongue and hyoid bone, pulling them forward, and consequently, significantly increasing the pharyngeal airspace. in the present study, 13 patients were submitted to anti-clockwise rotation of the mandible, and 9 of them also received advancement / impaction 8 azevedo et al. mentoplasty both as a complement to the mandibular advancement and as an additional movement. in these patients, the results of oropharyngeal airway gain were slightly higher than in the others, which reinforces the importance of this movement to increase the span of the upper airways. soft tissue edema is a factor that should be considered when assessing air space and head posture, especially in the immediate postoperative period of bimaxillary advancement surgery. this fact may justify the small average gain of oropharyngeal airspace in our sample, which ranged from 27.81% to 40.82% of the mandibular advancement, since 8 patients presented some reduction of the initial air space in one of the measured areas (upper, middle or lower) in the immediate postoperative period. carvalho16 also observed this event in his sample (n = 20), with a reduction in both area and volume of airspace of four patients in the immediate postoperative period but who had a substantial gain in the late postoperative period. these findings corroborate the study by coleta et al.4, who obtained an increase of 28% in the upper air space related to mandibular advancement. apparently, airway edema may have camouflaged the actual gain of airspace in the immediate postoperative period in such cases. in addition to postoperative soft tissue edema, the size of the airspace is also subject to the influence of the type and the amplitude of the surgical movement performed. authors such as li et al.20 considered that for a 10mm skeletal advancement a 5mm gain was obtained in the oropharyngeal space in its anteroposterior diameter, predicting a relation between the movement of the bone bases and the linear airway gain of about 2: 1. although the present study recorded an average linear airway gain of less than 50% of the surgical movement performed, when the sample was divided into two groups, according to the amount of mandibular advancement performed, different proportions of airspace gain became evident. in the group where mandibular advancement was >10mm (mean value of 13.51mm), the percentage of airspace increase in the three areas analyzed ranged from 14.43% to 25.98% for the measure obtained for b-lvr. in the group with mandibular advancement ≤10mm (mean value of 4.51mm), the linear airspace gain varied from 41.24% to 55.65% of the mandibular surgical movement. in both groups, there was a substantial reduction in airspace in the 3 points measured in the late postoperative period. this difference between the proportions of the linear gain of the oropharyngeal airway observed between the two groups of the present study can be justified by the work of reiche-fischel and wolford21, in which the oropharyngeal space changes of 72 patients with mandibular advancement were evaluated. in this study, the authors demonstrated that there was a higher percentage of airspace size gain for the first 10 mm of mandibular advancement (66%). for advancements greater than 10 mm, the air space continues to increase in size, but at a lower rate relative to the amount of mandibular advancement (between 10 and 15 mm of mandibular advancement, the air space increased 56% and for advances >15 mm the space gain was 41%). considering that the normal size of the upper airways is 11 ± 2 mm22 when measured on lateral radiography, our results showed mean values for airspace always higher than 12 mm in the immediate postoperative period, stabilizing above 11 mm in the late postoperative period, within the limits of normality. another area that plays an important role in the maintenance of oropharyngeal airspace and that is influenced by the surgical movement of the mandible is the hyoid bone7,12,14,23,24. 9 azevedo et al. the antero-posterior position of the hyoid bone is determined by the joint action of the muscles that are attached to the structures above and below it and by the resistance offered by the elastic membranes of the larynx and trachea, which forces it towards a return to, almost, its original position after a certain postoperative period12,14,23,25. the vast majority of studies describe changes in the position of the hyoid bone and in the size of the pharyngeal space 1-2 years after surgery7,12,13,23-25. contrary to these findings, we observed a forward and upward movement of the hyoid bone following the surgical movement of the mandible. although the distances between the h-c3 and h-rgn points were reduced in the late postoperative period, the linear distance of the hyoid bone in relation to the vertical and horizontal reference lines clearly showed an enhancement of the movement obtained in the hyoid bone soon after the surgery without relapse. the relation between the position of the hyoid bone and the posture of the head has also been studied in some scientific articles, in which it was verified that the position of the head promotes antero-posterior adaptation of the hyoid bone and interferes in the pre and post-surgical size of airspace11,26,27. patients with long face and facial pattern ii tend to exhibit an extended head posture as a way to improve respiratory capacity. however, after surgery, there is a strong tendency for the head to flex, after most surgical procedures, acquiring a more centralized position. the cervical skull position acquired after surgery is influenced by supra-hyoid post-surgical muscle tension, by the direction and distance of the surgical movement, and by changes in the upper airways. the angle formed by the cvt and opt lines, used to observe the changes in the cervical region resulting from the surgery, showed a minimal movement of cervical flexion in the immediate postoperative period (0.44º), directing the head upward, with an almost complete return, to the previous position, of the head in the late postoperative period (-0.42° of cervical extension). coleta et al.4 observed that patients flexed their heads after surgery (5.6º) and extended it 1.8º during long-term follow-up. other authors have reported a 0.65º to 3.4º of flexion of the head after mandibular advancement surgery24,28. the lack of standardization in the positioning of the head and neck during x-ray imaging may justify the wide variety of values obtained for the cervical skull region in different scientific studies4. although most of the changes observed in the present study were similar to those reported in the literature, few variables showed statistically significant changes, just as the correlations observed between airspace and other areas were not strong, with the exception of h-lvr and iea (>10mm). this can probably be explained by the fact that, although the sample had a reasonable number of individuals, when divided into groups, they became relatively low in number for statistical analysis, interfering with the results. thus, it would be prudent to continue the study increasing the size of the sample for greater fidelity of the statistical data. in conclusion, orthognathic surgery of mandibular / bimaxillary advancement promoted favorable changes in position of the hyoid bone and in the dimensions of the upper airways. the upper airways showed an increase in their dimensions after surgery, and this gain was proportionally greater in cases of bimaxillary surgery and mandibular advancement of less than 10 mm. statistical analysis showed, in most cases, little correlation between changes in the mandible, hyoid bone and / or head with the increase in oropharynx airspace. 10 azevedo et al. references 1. pizzol kedc, barbeiro rh, coleta rd, marcantonio e. 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[cephalometric and tridimensional avaliation of posterior airway space after maxillomandibular advancement] [tese]. araçatuba: faculdade de odontologia, universidade estadual paulista; 2011. portuguese. 17. zhao x, liu y, gao y. three-dimensional upper-airway changes associated with various amounts of mandibular advancement in awake apnea patients. am j orthod dentofacial orthop. 2008 may;133(5):661-8. doi: 10.1016/j.ajodo.2006.06.024. 18. gonçalves jr, buschang ph, gonçalves dg, wolford lm. postsurgical stability of oropharyngeal airway changes following counter-clockwise maxillo-mandibular advancement surgery. j oral maxillofac surg. 2006 may;64(5):755-62. 19. wei s, zhangy, guo x, yu w, wang m, yao k et al. counterclockwise maxillomandibular advancement: a choice for chinese patients with severe obstrutive sleep apnea. sleep breath. 2017 dec;21(4):853-860. doi: 10.1007/s11325-017-1484-7. 20. li kk, powel nb, riley rw, troell rj, guilleminault c. long term results of maxillomandibular advancement surgery. sleep breath. 2000;4(3):137-140. 21. reiche-fischel o, wolford lm. posterior airway space changes after double jaw surgery with counter-clockwise rotation. aaoms 78th annual meeting and scientific sessions. j oral maxillofac surg 1996;54:96. 22. riley rw, powell nb, guilleminault c. obstructive sleep apnea syndrome: a surgical protocol for dynamic airway reconstruction. j oral maxillofac surg. 1993 jul;51(7):742-7; discussion 748-9. 23. chung dh, hatch jp, dolce c, van sickels je, bays ra, rugh jd. positional change of the hyoid bone after bilateral sagittal split osteotomy with rigid and wire fixation. am j orthod dentofacial orthop. 2001 apr;119(4):382-9. 24. gale a, kilpeläinen pv, laine-alava mt. hyoid bone position after surgical mandibular advancement. eur j orthod. 2001 dec;23(6):695-701. 25. wessberg ga, schendel sa, epker bn. the role of suprahyoid myotomy in surgical advancement of the mandible via sagittal split ramus osteotomies. j oral maxillofac surg. 1982 may;40(5):273-7. 26. winnberg a, pancherz h,westesson pl. head posture and hyomandibular function in man. a synchronized electromyographic and videofluorographic study of the open-close-clench cycle. am j orthod dentofacial orthop. 1988 nov;94(5):393-404. 27. hedayati z, paknahad m, zorriasatine f. comparison of natural head position in different anteroposterior malocclusions. j dent (tehran). 2013 may;10(3):210-20. 28. phillips c, snow md, turvey ta, proffit wr. the effect of orthognathic surgery on head posture. eur j orthod. 1991 oct;13(5):397-403. 1http://dx.doi.org/10.20396/bjos.v18i0. 8657260 volume 18 2019 e191626 original article 1 department of health education & health promotion, faculty of medical sciences,tarbiat modares university corresponding author: fatemeh zarei department of health education and health promotion, faculty of medical sciences, tarbiat modares university, tehran, iran. email: f.zarei@modares.ac.ir received: april 16, 2019 accepted: september 10, 2019 comparison of peer led and teacher led oral health educational program among students afsaneh karami1, alireza heidarnia1, fatemeh zarei1,* aim: the purpose of this study was to determine the impact of peer-led education (ple)comparing with the teacher-led education(tle) approach about oral health behavior of female schoolstudent aged at 6-12 in district 6 of tehran, iran. methods: this is a quasi-experimental study. two primary public schools in tehran district 6, were selected by simple random sampling and from each school one class considered for ple and tle. 120 fourth grade female students of the 6th district of tehran were enrolled for study. all participants were assessed about knowledge, attitude, and practice in oral health by pre-test one month before the intervention program. a valid questionnaire in persin version used for data gathering. two representatives including teacher and students from both groups trained under the supervision of a health educator. in ple each of the six students trained 10 other students in the middle of the day per three consecutive sessions. in tle one teacher trained one class with about 30students. both education program set up at the same time and duration. the educational materials included educational short films, animation, and booklet powered driven by oral health burea, iran health ministry. apart from, the intervention included role play and storytelling too. the evaluation had done by post-test with the same tool one month later after the intervention. results: the average mean score of the knowledge, attitude, and practice in both group after educational intervention significantly increased (p<0.05).the mean score of knowledge, attitude, and practice in ple have been effective than tle. conclusion: health education with peer education approach is effective in improving oral health behavior in children at school age. keywords: oral health. health education. peer education. 2 karami et al. introduction oral hygiene is one of the important branches of public health that has a noteworthy impact on the health of the people1. the world health organization (who) considers oral health as a necessity and part of overall health throughout life, saying that poor oral health and oral untreated diseases can have a profound impact on quality of life2. one of the most common chronic diseases of oral health is tooth decay, which is a major health problem in most countries of the world, especially in developing countries3. despite the importance of oral and dental hygiene among children and adolescents, we continue to see the prevalence of dental caries among them4. oral diseases can lead to irreversible injuries and consequences such as pain, depression, decreased self-esteem, low quality of life, acute and chronic infections, and so on. in addition, oral and dental illness can restrict individual activities at school, at work, or at home, causing millions of hours to die every year around the world5. one of the goals of the world health organization for oral health is that the dmft (the number of stuffed, rotten or drawn teeth) should reach less than one6. evaluation of dmft in students of yazd in 2012, this amount is estimated at 7.17. also, according to the world health organization dmft index for 12-year-old children in america 8/2, 6/2 in europe and in africa is 3.1.in iran, the average of this index is 1.73. nutritional behaviors in children such as high consumption of sugar, lack of dairy consumption, unfulfilled habits, such as the tendency to brush and use floss, increase the prevalence of caries in these children8. in this regard, the findings indicate that the health of oral and dental tissues of the population is related to the level of knowledge and information about oral hygiene behaviors9. effective techniques for the prevention of dental caries and periodontal diseases include self-care oral health (using dental floss and brushing) and fluoride therapy10. in order to change habits, interventions in the field of health education are essential. in this regard, sattel believes health education is an essential element in the advancement of health, and one of its important roles is to prepare the people of the community through knowledge and information to them, and to demonstrate their skills and health practices11. the age group of 126 years old is one of the priority groups of oral health programs due to the high prevalence of tooth decay and the valuable position of age in terms of the development of permanent teeth on the one hand and the formation of beliefs, habits,and lifestyle on the other hand. any change or improvement in the health behavior of this group can have a lasting and significant impact on the health of the future generation of the community12. meanwhile, the school is the easiest and most cost-effective way to reach this age group13. one of the approaches used in teaching is the childto-child education approach. the original idea of this approach was presented in 1978 by hove hawz and morley. the program has been officially launched since 1979 (world children’s year), and since then many countries, including iran, have implemented the program. the main feature of the child-child approach is the involvement of children directly in the education process and promoting health14. innovative methods to education for health are essential to gain the benefits. child to child approach education method is a new way of providing health education to 3 karami et al. school children and the use of this method maximizes the spread of health messages1. it is an active method that encourages learning by easy manner. the child has the power to spread the health messages, therefore, this method links what is educated in school room with what to do out of the class and at home. a child passing health messages to younger brothers and sisters, friends and so jointly collaborating to become a positive power for health15. considering the necessity of prevention of oral and dental illness in students, and the weakness of the results of the current training peer-led education (ple), we aimed to use the alternative peer-led education to make it effective approach. therefore, the present study designed to determine the effectiveness of ple approach in comparison with the teacher-led education (tle) approach in the field of oral and dental care in primary school girls in district 6 of tehran. materials and method study design and eligibility criteria the study was a quasi-experimental study that will be conducted on 120 female primary schoolchildren from two elementary schools in tehran’s district 6 in 2018. the study conducted before and after interventions with two interventional groups, including ple and tle. the inclusion criteria were studying at grade 4, living in tehran, willingness to participate in the study and the parents, satisfaction. the following flow diagram shows the methodology design in brief (figure 1). sampling the study samples were elementary school students who were selected from two public schools in the 6th district of tehran and randomly divided into two groups of intervention, including peer education and classroom education (coaching assistance). regarding the mean and standard deviation in the two groups based on the previous studies16 and considering confidence level of 95% and the test power of 80% the sample size of 46 students were achieved in each group and with considering an excess number of study sample due to withdrawal (missing) about 60 students were chosen to participate in each study group. the two schools were randomly selected from the list of all public schools in district 6 in total, 120 students at fourth-grade are selected to participate in the study and will be measured before and one month after the intervention. from each school, a class of 30 is selected as ple and another class as tle. in ple group 30 students in the 10-member group received educational intervention from 3 trained student who was at fifth-grade. instrument data were collected through a standard questionnaire in persian17 including four sections: demographic information, knowledge, attitude, and behavior before and after the intervention. demographic questions included age, residency, parenting, parenting. students’ attitudes are measured according to the 5-likert scale,i.e. completely 4 karami et al. agree, agree, uncertain, disagree, and completely disagree. the questionnaire validity and validity were confirmed. the questionnaire validity was checked by 10 experts in health promotion fields and the content validity was evaluated by two indexes of cvr and cvi that were 0.90 % and 0.85 %, respectively. to determine the questionnaire reliability, test-re-test and the cronbach’s alpha coefficient were calculated and cronbach’s alpha was calculated to be 0.76. pre-test before intervention at the entrance to each of the primary and before completing the questionnaires, the study goals and objectives will be explained. the questionnaires will be distributed and explained on how to complete them. the completed anonymous questionnaires were remarked by code by the students themselves to ensure them that data was collected confidentially. development and implementation of training program according to the results obtained in the pre-test and the needs assessment that was done based on the objectives of the study, training programs are prepared for the figure 1. flow diagram of methodology design assessed for eligibility (n=130) randomomized (n=120) allocation allocated to peer led education group (ple) (n=60) pre-intervention (n=120) intervention 1 (education by peers) intervention 2 (education by teacher) post-intervention one month later (n=120) comparison (n=120) allocated to teacher led education group (tle) (n=60) excluded (n=10) not meeting inclusion criteria (n=10) 5 karami et al. two groups. training will be provided through educational pamphlets, posters and 10-minute animations, and practical training for brushing and flossing. it should be noted that all the materials (educational content) used in both ple and tle groups are accurately trained by the health educator. apart from, all educational contents were powered driven by oral health burea, iran ministry of health. evaluation of training program to evaluate the educational programs, the study groups will be assessed in two different time periods, before and one month after the educational intervention through a questionnaire. data analysis the data analysis was conducted using spss software to assess the impact of peer education and classroom education. the descriptive statistics (such as means, standard deviation, and frequency tables) and chi-squire (for demographic variables) were used. to analyze the data, before and after the interventions, ankova were performed using spss version 18 (spss inc., chicago, illinois, usa). the significance level was set at p<0.05. ethics of research this study approved by an ethical code (ir.tmu.rec.1396.665). in this study participation is voluntary and the students participate in the study with consent. the study objectives are explained to students and their parents, teachers, educational authorities, and schools’ administrators. during the educational programs, the authors tried to avoid any interruption on the student’s classes. results according to the finding 120, fourth-grade students from two schools with an average age of 10 years were enrolled in the study. demographic variables include student’s age, father’s job, father education level, mother’s job, mother education level. the results showed that none of the demographic variables, except the father’s job (p=0.039), were significantly different between the two groups of teacher led and peer-led education. (table 1) according to table 2, the mean score of knowledge after intervention in the education group by the teacher (tle) and the education group by peers (ple) and also in the groups with different father’s job were not significant (p>0.05). furthermore, the score of knowledge in different groups has not the same effect. (p<0.05). (table 2) according to the partial eta squared, group covers 1.8% of knowledge chenges in post test, 0.7% of the father’s job, and 6.7% of knowledge chenges in pre-test. (table 3) according to table 4, the mean score of attitude after intervention in the education group by the teacher (tle) and the education group by peers(ple) was significant (p<0.05). there was no signifact meaning in the groups with different father’s job (p>0.05). furthermore, the score of attitude in different groups has not the same effect (p<0.05). (table 4) 6 karami et al. according to the partial eta squared, group covers 22.0 % of attitude chenges in post test, 0.4% of the father’s job, and 7.0% of attitude chenges in pre-test. (table 5) according to table 6, the mean score of practice after intervention in the education group by the teacher (tle) and the education group by peers (ple) was significant (p<0.05). table 1. demographic variables between the two groups of ple and tle teacherled n (%) peer –led n (%) p-value age 9 6 (10) 2 (3.3) 0.136 10 54 (90) 58 (96.7) mother education level under the diploma 18 (30) 19 (31.7) 0.650diploma 6 (10) 9 (15) academic 36 (60) 32 (53.3) father education level under the diploma 9 (15.5) 6 (10.2) 0.244diploma 12 (2.07) 20 (33.9) academic 37 (63.8) 33 (55.9) mother’s job housekeeper 22 (36.7) 28 (46.7) 0.528 employee 25 (41.7) 18 (30) unemployment 7 (11.7) 6 (10) others 6 (10) 8 (13.3) father’s job worker 3 (5) 5 (8.3) 0.039 employee 28 (46.7) 16 (26.7) unemployment 19 (31.7) 17 (28.3) others 10 (16.7) 22 (36.7) table 2. analysis of covariance for the effect of education on knowledge source df ss ms f partial eta squared group 1 7.540 7.540 2.116 0.018 father’s job 3 2.852 0.951 0.267 0.007 knowledg 1 29.317 29.317 **8.227 0.067 error 114 406.241 3.564 total 119 440.992 **p<0.01 * p<0.05 table 3. the mean and standard deviation of the knowledge scores after intervention in terms of education type and father’s job father’s job tle ple m sd m sd worker 7.33 2.31 7.80 .45 employee 7.46 1.48 7.00 1.46 unemployment 7.21 2.18 6.71 2.87 others 6.90 1.20 7.68 2.12 total 7.28 1.70 7.23 2.14 7 karami et al. there was no signifact meaning in the groups with different father’s job (p>0.05). furthermore, the score of practice in different groups has not the same effect (p<0.05). (table 6) according to the partial eta squared, group covers 11.3% of practice chenges in post test, 0.8% of the father’s job, and 3.4% of practice chenges in pre-test. (table 7) table 7. the mean and standard deviation of the practice scores after intervention in terms of education type and father’s job. father’s job tle ple m sd m sd worker 2.67 .58 2.60 .55 employee 2.18 1.63 2.94 1.48 unemployment 2.21 1.90 3.18 1.13 others 1.80 1.48 3.50 1.37 total 2.15 1.64 3.18 1.30 table 6. analysis of covariance for the effect of education on practice source df ss ms f partial eta squared group 1 31.697 31.697 **14.518 0.113 father’s job 3 2.001 0.667 0.306 0.008 practice 1 8.839 8.839 4.049* 0.034 error 114 248.905 2.183 total 119 290.667 **p<0. 01 * p<0.05 table 5. the mean and standard deviation of attitude scores after intervention in terms of education type and father’s job father’s job tle ple m sd m sd worker 30.67 8.08 34.80 2.59 employee 30.96 4.60 34.94 3.11 unemployment 30.63 5.48 34.47 4.32 others 30.60 4.50 35.09 3.15 total 30.78 4.92 34.85 3.40 table 4. analysis of covariance for the effect of education on attitude source df ss ms f partial eta squared group 1 550.310 550.310 **32.074 0.220 father’s job 3 8.086 2.695 0.157 0.004 attitude 1 148.103 148.103 **8.632 0.070 error 114 1955.939 17.157 total 119 2603.967 **p<0.01 * p<0.05 8 karami et al. discussion the effectiveness of peer education approach is based on the theory that reveal sensitive information will be transmitted more easily between people of the same situation16. in this regard, the present study aimed to compare two methods included peer-led education (ple) and teacher-led education (tle) on improving oral health behaviors of iranian female students. the findings indicated that the knowledge was increased in both tle and ple groups but the comparison of the difference between the mean score of knowledge before and after the intervention between tle and ple indicated that the level of knowledg increased differently. in other words, ple was more effective than tle to increase the knowledge about oral health among participants. this finding was confirmed by other similar studies17. the study of najjar lashgari et al.16 (2013) revealed that a child-to-child education had been effective on improving the health awareness in students. in more clarification, the education by peer groups not only adds to student’s awareness, but also health behaviour would be eased for any cognitive changes18. to our knowledg, some studies do not confirm effects of child-to-child education or peer education posetivley. in line with this, moeini et al.19 (2013) proved that there was no significant difference in the knowledge, attitude, and practice levels after educational intervention which adressed the child coach and teacher coach group19. along with this study, kargar et al.20 (2013) concluded that there was no significant difference between the peers and adults-led education program before, immediately and one month later20. in our study low growth in knowledge after intervention among students who coached with peer might be due to the lack of information of fellows. apart from the knowledge level of a peer, characteristically it seems that, choosing a proper educator as a fellow is another considerable point in this type of educational program. the peer students should have autonomy and be acceptable to other students and should be explaining the lesson that have learned to other students appropriately. however, according to the results of the current study the level of student’s knowledge who trained as peers were increased after post-intervention. it may be due to peer accountability and emphasis on the most frequently asked questions by students. in this matter, peer educators seemed to be more effective and successful than the tle because of their familiarity with the needs of the target group. in the present study, our findings indicated that the attitude was increased in both tle and ple groups. the results showed that the difference in mean score of attitude between both groups before and after the intervention was also significant (p<0.005). therefore education by peers had a greater impact on students’ attitudes than education by teachers. several studies indicated that peer-led educational programs have had a greater impact on children’s attitude and understanding of health issues compared to children who did not recived these programs21. in the study of akbarzadeh et al.22 (2009), the attitudes of trained students by peer groups after intervention improved compared to health staff which represents the effect of education by peers22. moreover, in the study of noorisistani et al.23 (2010), there was a significant 9 karami et al. difference between the mean scores of attitude in two groups of peers and the lecture-based education on attitude improvement in health issues23. regarding the significance of the difference in attitude before and after intervention in peer group, it seems that there were some reasons to belive why a peer-led educational program was more effective and succsessful method for health education among students. the way of communicating with students, as an acceptable and believable role model, the simplicity in explanning for the educational contents, are some of these effective reasons to greatly influence on changing the attitude. in this study, the students’ practice in brushing, dental floss, using mouthwash and regular referral to the dentist significantly has been increased before and after the education in the peerled and the teacher-led. furthuremore, the mean performance score for oral health behaviors before training between the two groups was statistically significant (p<0.05). however, this difference after training was significant between the two groups (p<0.05) perhaps the conclusion can be interpreted that students who have a high level of knowledge and attitude in the field of oral health behaviour have a better performance than the others. this result was confirmed in several studies, including kaveh et al. in 201624 and leana and d’souza in 201725. a study by walvekar et al showed that the child-to-child education program had a significant impact on increasing knowledge, changing in attitude and behaviors in relation to diarrhea in students in child-to-child group compared to the control group26; in contrast, kaveh et al, showed that there was no significant difference in the mean scores of nutritional behaviors in the control group24. additionally, tolli27 (2012) concluded that there was no evidence of the effectiveness of the peer education method in relation to the desired behaviors in peer groups compared to the control group27. in general, the results of this study showed that the peer group’s approach to improving the knowledge, attitude and oral health behaviors was more successful than teacher-led education,by adjusting the father’s job variable.to put it simple, the oral health behavior including the knowledge, attitude and practice after intervention is affected by both the father’s job and also the type of education approach. therefore, due to the serious shortage of health educators in the country’s schools, proper planning and holding of better and longer-term training courses for students can fill up the vacancies of health education in schools. for an effective oral health education addressing school student age group, as well as peer-led educational program, is suggested to be used in the oral health promotion program. strengths and limitations despite all the strong points of the present research, it had some limitations; firstly, the outcomes were evaluated only for one month after the educational intervention. thus, future studies with longer followup periods are recommended to be conducted for better evaluation. in addition, the final evaluation in this study was based on the students self-reports, which could result in bias. hence, future studies can use a combination of self-report, direct observation of the behavior, and report by parents. as a final limitation. for the present study, 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health educ res. 2012 oct;27(5):904-13. doi: 10.1093/her/cys055. braz j oral sci. 15(1):62-65 original article braz j oral sci. january | march 2016 volume 15, number 1 influence of adhesive system on quartz fiber post dislocation resistance in endodontically treated teeth luis francisco maglione garcía1, vicente castelo branco leitune2, stéfani becker rodrigues2, susana maria werner samuel2, fabrício mezzomo collares2 1universidad de la república udelar, facultad de odontología, area of operative dentistry, montevideo, uruguay 2universidade federal do rio grande do sul ufrgs, department of conservative dentistry, porto alegre, rs, brazil correspondence to: fabrício mezzomo collares rua ramiro barcelos 2492 laboratório de materiais dentários universidade federal do rio grande do sul ufrgs porto alegre, brazil. 90035-003 phone/fax: +55 51 3308 5198. e-mail: fabricio.collares@ufrgs.br abstract aim: to evaluate the dislocation resistance of the quartz fiber post/cement/dentin interface after different adhesion strategies. methods: forty bovine lower central incisors were selected and prepared with k-files using the step-back technique, and irrigated with 3 ml of distilled water preceding the use of each instrument. prepared teeth were stored at 37ºc and 100% humidity for 7 days. the roots were prepared and randomized into 4 groups. the quartz fiber post was cemented with an adhesion strategy according to the following groups: gbiscembiscem; gonestep±c&bone step ± c&b; gallbond±c&ballbond3 ± c&b; gallbondse±c&ballbondse ±c&b with a quartz fiber post. cross-sectional root slices of 0.7 mm were produced and stored for 24 h at 37° c before being submitted to push-out bond strength. results: the mean and standard deviation values of dislocation resistance were gbiscem: 1.12 (± 0.23) mpa, gonestep±c&b: 0.81 (± 0.31) mpa, gallbond±c&b: 0.98 (± 0.14) mpa, and gallbondse±c&b: 1.57 (± 0.04) mpa. gallbondse±c&b showed significantly higher values of dislocation resistance than the other groups. conclusions: based on this study design, it may be concluded that adhesion strategies showed different results of quartz post dislocation resistance. simplified adhesive system with sodium benzene sulphinate incorporation provided superior dislocation resistance. keywords: tooth, endodontically-treated. post and core technique. resin cements. dentinbonding agents. introduction the objectives of the restorative treatment of endodontically treated teeth with large crown destruction are giving the teeth more resistance to masticatory forces, reestablishing function, and esthetics1. thus, sometimes the use of intraradicular post and cores is necessary2,3. ideally, the intraradicular post and cores should not move into the root canal and forces are distributed in the teeth homogeneously, decreasing the risk of tooth fractures4,5. cast metal posts have been used for a long time in clinical practice and have presented high survival rates6. metal posts have high elastic modulus, which increases fracture and catastrophic root failure. therefore, glass fiber posts have been introduced as an alternative. due to their low elastic modulus compared with cast metal posts, glass fiber posts have decreased the number of catastrophic failures7. in addition, they are more aesthetic and result in less clinical time3,8,9. http://dx.doi.org/10.20396/bjos.v15i1.8647126 received for publication: april 25, 2016 accepted: august 03, 2016 63 carbon-fiber posts were introduced in the 1990s, and after that other types were produced such as glass fiber posts and zirconia and quartz posts. fiber posts are cemented with a polymer luting cement to dentin due to chemistry affinity for its own epoxy resin matrix with the resin cement and quartz posts presenting a 98% survival rate after 9 years9. however, difficulty in adhesive cementation is observed and debonding is the main type of failure10-15. it is important to know which adhesive strategy should be used to decide the best choice for fiber post cementation16. thus, the objective of this study was to evaluate the dislocation resistance of the quartz fiber post/ cement/dentin interface after different adhesion strategies. the null hypothesis was that the different adhesion strategies would not affect the dislocation resistance. material and methods teeth preparation forty bovine incisors were selected for this study. to be included, the following criteria had to be met: straight roots and a root length of at least 15 mm. external debris were removed with a periodontal curette and scalpel blade. the teeth were sectioned transversely 15 mm from the apex using a slow-speed diamond disc under water coolant, the pulp tissue was removed, and chemomechanical preparation was performed according described previously17,18. the root canals were prepared with k-files using the step-back technique and irrigated with 3 ml of distilled water preceding the use of each instrument. after that, they were stored at 37ºc and 100% humidity for 7 days. subsequently, the roots were prepared (figure 1) with a specific bisco burr (bisco int., schaumburg, illinois, usa), and randomized into 4 groups. quartz fiber post was cemented according to the adhesive system and cements (table 1). fiber posts had 12 mm of length, 1.8 mm of cervical diameter and 1.0 mm of apical diameter. influence of adhesive system on quartz fiber post dislocation resistance in endodontically treated teeth braz j oral sci. 15(1):62-65 push-out test after cementation of the quartz post, the teeth were sectioned transversely into 7 slices that were approximately 0.7 mm thick using a low-speed disc (isomet, buehler ltd, lake bluff, il, usa) with constant water cooling. the internal diameter of the canal of each slice was measured with a digital caliper (digimess, 100.174bl, digimess instrumentos de precisão ltda, são paulo, sp, brazil) and the contact area between the filling and dentin of each slice was calculated. each slice was placed with the apical side up on a mechanical testing machine (dl-2000, emic equipamentos e sistemas de ensaio ltda, são josé dos pinhais, pr, brazil). a force was applied to the shutter toward the apical neck using a 500 n load cell and a crosshead speed of 1 mm/min with a 0.8-mm diameter cylindrical device. the dislocation resistance (mpa) was obtained by dividing the force (n) required to displace the filling material by the adhesive area (mm2). the adhesive area was calculated using (1 and 2): g = (h2 ± (r2 r1)2)½ (1) a = π.g.(r1 ± r2) (2) where, g is the root taper, r1 represents the apical radius, r2 represents the coronal radius, h is the thickness of the slice, and a is the adhesive area. apical and coronal radii were obtained from photographs and measured with image tool software 10 times from different sides to obtain the radii means (figure 2). statistical analysis data normality was checked by the kolmogorov-smirnov test. data were analyzed by anova and the tukey post-hoc test. a significance level of 5% was used for analysis. fig.1. roots prepared with specific bur. group material method gbiscem biscem cement (bisco) mixed for 15 s, applied to the post and root canal; photoactivated for 30 s. gonestep±c&b phosphoric acid 35% (coltene/whaledent, cuyahoga falls, ohio, usa) one step (bisco) c&b cement (bisco) applied for 15 s; rinsed with distilled water; dried with absorbent paper cones. applied for 20 s; dried with absorbent paper cones; photoactivated for 20 s. mixed for 15 s; applied in the post and root canal. gallbond±c&b phosphoric acid 35% (coltene/whaledent) all bond 3 a & b (bisco) c&b cement (bisco) applied for 15 s; rinsed with distilled water; dried with absorbent paper cones. mixed primer a and b for 5s; applied for 20 s; dried with absorbent paper cones; applied adhesive resin and photoactivated for 20 s. mixed for 15 s; applied in the post and root canal. gallbondse±c&b phosphoric acid 35% (coltene/ whaledent) all bond se i and ii (bisco) c&b cement (bisco) applied for 15 s; rinsed with distilled water; dried with absorbent paper cones. mixed part i and part ii for 5 s; applied for 20 s dried with absorbent paper cones; photoactivated for 20 s; mixed for 15 s; applied in the post and root canal. table 1 groups, materials and methods used in the study. 64influence of adhesive system on quartz fiber post dislocation resistance in endodontically treated teeth braz j oral sci. 15(1):62-65 fig.2. photograph of canal slice and measurement of internal diameter with software in 10 different regions. groups dr (mpa) gbiscem 1.12 (± 0.23) b* gonestep±c&b 0.81 (± 0.31) b gallbond±c&b 0.98 (± 0.14) b gallbondse±c&b 1.57 (± 0.04) a table 2 results of mean and standard deviation, in mpa, of dislocation resistance (dr) with different adhesive cements. *different capital letters indicates statistically significant difference within the column (p<0.05). fig.3. push-out (mpa) values of groups tested (gbiscem, gonestep±c&b, gallbond±c&b and gallbondse±c&b). results the results of the dislocation resistance are shown in table 2 and figure 3. the gallbondse±c&b group presented the highest dislocation resistance value (1.57 ± 0.04 mpa), with a significant difference from the other groups, p < 0.05. gbiscem (1.12 ± 0.23 mpa), gonestep±c&b (0.81 ± 0.31 mpa), and gallbondse±c&b (0.98 ± 0.14 mpa) did not show statistically significant differences between each other, p > 0.05. discussion in this study, the dislocation resistance of root dentin/cement/ post was evaluated after different adhesive strategies. the materials in this study showed easy clinical use, furthermore, the decreased catastrophic failure could have occurred as result of correct dissipation of forces in the middle third of teeth. the use of fiber post with resin cement leads to a block formation with dental tissue since the elastic modulus is similar to dentin (18 gpa), once the values for fiber post is between 16-40 gpa and resin cement between 6.8-10.8 gpa19. however, the adhesive system strategies used in this study showed different results for dislocation resistance, and the highest values were found in the gallbondse±c&b group. difficulty in hybrid layer formation results in ineffective adhesion and a decrease in dislocation resistance due to: high factor cavity configuration, high polymerization shrinkage, and difficulty in the homogeneous acid condition of the root walls8. in the gbiscem group, self-adhesive cement was used. this cement compounds the acid monomers, which demineralize the dentin substrate while infiltrating the monomers to form a hybrid layer. it is likely that the lower dislocation resistance occurred due to the fact that there was no formation of a homogeneous hybrid layer14. the simplified adhesive systems (primer with adhesive) showed statistically significant lower dislocation resistances between groups (gonestep±c&b and gallbondse±c&b), p < 0.05%. one step adhesive is a universal self-adhesive of one plot that presents hydrophilic monomers (hydroxyethyl methacrylate), acid monomers, and acetone as solvent. hydroxyethyl methacrylate presents low hydrolytic stability when in the same environment with acid monomers and degrades in little time. furthermore, acetone solvents have high volatility, which makes the operatory technique difficult and justifies the low value of dislocation resistance20. however, in this study, a significant difference between the gonestep±c&b (universal adhesive), 0.81 (± 0.31), and gallbond±c&b (etch-and-rinse adhesive), 0.98 (± 0.14), groups was not observed, p > 0.05%. conventional adhesive systems, with 3 steps etch-and-rinse, present difficulties related to incomplete acid filling of the root and incorrect evaporation of the primer solvent, compromising the adhesion process, mainly in the medium and apical thirds13,14,21,22. the highest values of dislocation resistance were found in the gallbondse±c&b group. it is likely that the compound present in part i of the adhesive, sodium benzene sulphinate, acts as a co65 braz j oral sci. 15(1):62-65 initiator in the acid-base reaction between benzoyl peroxide and tertiary amine of the chemically cured resin cements, increasing the results of dislocation resistance23-25. the sodium benzene sulphinate allows the acidity of the adhesive system to demineralize the dentin substrate without interfering in the resin cement degree of conversion. thus, it is possible that the incorporation of the coinitiator avoids the chemical incompatibility between simplified adhesives and chemically cured resin cements. based on this study design, it is licit to conclude that the adhesion strategies showed different results of quartz post dislocation resistance. the simplified adhesive system with sodium benzene sulphinate incorporation presented superior dislocation resistance. acknowledgements this study received no specific grant from any funding agency in the public, commercial or not-for-profit sector. no conflits of interest are present. the authors gratefully acknowledge capes for the scholarship (sbr). references 1. sequeira-byron p, fedorowicz z, carter b, nasser m, alrowaili ef. single crowns versus conventional fillings for the restoration of root-filed teeth. cochrane database syst rev. 2015 sep 25;(9):cd009109. doi: 10.1002/14651858.cd009109.pub3. 2. perdigao j, gomes g, augusto v. the effect of dowel space on the bond strengths of fiber posts. j prosthodont. 2007 may-jun;16(3):154-6. 3. lamichhane a, xu c, zhang fq. dental fiber-post resin base material: a review. j adv prosthodont. 2014 feb;6(1):60-5. doi: 10.4047/jap.2014.6.1.60. 4. bosso k, gonini júnior a, guiraldo rd, berger sb, lopes mb. stress generated by customized glass fiber posts and other types by photoelastic analysis. braz dent j. 2015 may-jun;26(3):222-7. doi: 10.1590/01036440201300256. 5. sterzenbach g, franke a, naumann m. rigid versus flexible dentine-like endodontic posts--clinical testing of a biomechanical concept: 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department of odontological clinic, school of dentistry, university center of the state of pará, belém, pará, brazil. 2 department of materials engineering, school of mechanical engineering, federal university of pará, belém, pará, brazil. 3 department of dental materials science, school of dentistry, federal university of pará, belém, pará, brazil. corresponding author: maria helena rossy borges tv. benjamin constant 845, 66053-040, belém-pa, brazil; e-mail: mariahelena.rb@hotmail.com; tel 55+ 91 981053100. received: january 27, 2020 accepted: august 16, 2020 evaluation of physical-mechanical properties of self-adhesive versus conventional resin cements maria helena rossy borges1,* , carmen gilda barroso tavares dias2 , cristiane de melo alencar3 , cecy martins silva3 , renata antunes esteves1 aim: the purpose of this study was to compare the microhardness, diametral tensile strength, compressive strength and the rheological properties of self-adhesive versus conventional resin cements. methods: specimens of a conventional (relyx arc) and 3 self-adhesive (relyx u200, maxcem elite, bifix se) types of resin cements were prepared. the knoop test was used to assess the microhardness, using a microhardness tester fm 700. for the diametral tensile strength test, a tensile strength was applied at a speed of 0.6 mm/ minute. a universal testing machine was used for the analysis of compressive strength and a thermo-controlled oscillating rheometer was used for the rheology test. one-way anova and tukey’s test (α=0.05) were used for data analysis. results: according to microhardness analysis, all the cements were statistically similar (p>0.05), except for maxcem that presented lower hardness compared with the other cements in relation to the top surface (p<0.05). in the diametral tensile strength test, relyx u200 and relyx arc cements were statistically similar (p>0.05), presented higher value when compared to the maxcem and bifix cements (p<0.05). the compressive strength of relyx arc and maxcem elite cements was statistically higher than relyx u200 and bifix cements (p<0.05). regarding the rheology test, maxcem elite and relyx arc cements showed a high modulus of elasticity. conclusions: the self-adhesive cements presented poorer mechanical properties than conventional resin cement. chemical structure and types of monomers employed interfere directly in the mechanical properties of resin cements. keywords: cementation. dental materials. flexural strength. longevity. resin cements. https://orcid.org/0000-0002-2132-4827 https://orcid.org/0000-0002-8834-8364 https://orcid.org/0000-0002-0694-6863 https://orcid.org/0000-0002-7870-8848 https://orcid.org/0000-0002-3421-2365 2 borges et al. introduction the performance of resin cement on the luting procedure and their mechanical properties are essential requirements for the clinical success of indirect restorations1. moreover, due to its low solubility, high bonding strength, better physical and mechanical properties such as high values of fracture toughness, tensile strength and compression, resin cements are frequently used in the cementation of ceramics2-4. nevertheless, the broad variety of brands and types of resin cements makes the selection of material difficult for the dentist. resin cements were developed to be used in the cementation of indirect restorations and intra-radicular pins; thus, they contain different types of monomers that connect to each other during the polymerization reaction5. therefore, the cement composition is generally a mixture of dimethacrylate monomers, such as: bisgma (bisphenol a diglycidil dimethacrylate), tegdma (triethylene glycol dimethacrylate), udma (urethane dimethacrylate) and inorganic fillers which vary according to the trademarks and initiator. silica or high-molecular-weight oligomers can also be added to modify rheological properties and achieve optimum handling characteristics6. however, the manufacturers of several materials often do not entirely disclose details of cement composition. the dual-cure resin cements are available on the dental market and are considered practical to use, because they combine benefits such as working time and the mechanical properties of both light cure and chemical cure resin cements7. however, the chemical polymerization associated with photopolymerization provides better monomer conversion8. furthermore, these cements can be classified in two categories according to adhesive cementing technique: conventional and self-adhesive resin cements. the self-adhesive resin cements are considered to be easier to use, once do not require any pretreatment of dental substrate9. the bonding mechanism of conventional resin cement depends on the type of adhesive used in combination with this system, whereas the bonding mechanism of self-adhesive resin cements to dental tissues depends on chemical reactions among acid monomer or phosphoric acid ester with calcium of enamel and dentin10. in addition, the clinical indications of self-adhesive and conventional resin cements are resembling. however, differences in chemical compositions may lead to dissimilar mechanical properties11. these cements must be selected according to the clinical conditions of each case, the physical properties of the indirect restorative material and the physical and biological characteristics of the cementitious materials, such as adhesiveness, solubility, resistance and biocompatibility12. hence, for clinical success with long term follow-up, it is important that the cementitious material exhibits mechanical stability, since the filling is subjected daily to mechanical forces such as mastication, and also to parafunctional habits such as bruxism and tightening13. therefore, in order to assess the ability of the material to withstand these types of stress, mechanical tests such as compressive strength and diametral traction are increasingly applied in research. in addition, to evaluate the surface of the material, 3 borges et al. degree of conversion, rate of wear and other properties, a microhardness mechanical test is performed. tests such as rheology are extremely important to understand the behavior of the material. thus, the purpose of this study was to evaluate the microhardness, diametral tensile strength, compressive strength and the rheological properties of self-adhesive versus conventional resin cements. the null hypothesis tested was: there are no significant differences in physical-mechanical properties between the conventional resin cement and the self-adhesive resin cements. materials and methods in order to carry out the experiment, samples were prepared from specific matrices, which were made with resin cements (table 1). for the microhardness analysis, the samples were made from a circular teflon matrix with 2 mm of height and 8 mm of diameter. for the preparation of each sample (total of 4 samples), the cement was introduced into the single-increment circular matrix which was pressed between two polyester strips and glass coverslips, so the surfaces would be smooth. afterwards, photoactivation was carried out with a led dental curing light device (elipar freelight 2, 3m espe, usa) with irradiance of 800 w² for 20 seconds. immediately after being made, the samples were removed from the matrix and incubated at 37°c for 24 hours. after this period, the knoop microhardness test was performed on the top and base surfaces, using an fm 700 microhardness tester (future teck kanagawa, japan), applying a load of 25 grams for 30 seconds. by activating the penetrator of the equipment, a compression was applied to the sample, which generated an indentation (diamond-shaped geometric print) on its surface. three indentations were made on each top and base surface of each sample. then, the knoop hardness average was obtained for each sample by applying the values found with the indentations with the equation: knh = c x c / d2. table 1. the materials used in the study and their composition according to their manufacturers resin cements (lote no) manufactures type resin matrix relyx arc (n502901) 3m espe (3m/espe,st paul, mn, usa) dual-cured conventional cement bis-gma, tegdma relyx u200 (1518200193) 3m espe (3m/espe,st paul, mn, usa) dual-cured self-adhesive bis-phenol-a-bis-(2-hydroxy-3 methacryloxypropyl), ether bis-gma, tegdma maxcem elite (5925082) kerr (orange,ca, usa) dual-cured self-adhesive gpdm, hema bifix se (1621136) voco gmbh, (cuxhaven. germany) dual-cured self-adhesive bis-gma,udma,gly-dma, phosphatemonomers bis-gma bisphenol a dimethacrylate; tegdma triethylen glycol dimethacrylate; gpdm glycerol phosphate dimethacrylate; hema hydroxyethyl methacrylate; udma urethane dimethacrylate; gly-dma glycerol dimethacrylate. 4 borges et al. where: knh is the knoop hardness value; c (constant) = 14.230; c = 25 grams; d is the length of the longest diagonal of the indentation. a cylindrical matrix (1 ml insulin syringe, sr insulin u-100 luer slip) with cylindrical specimen (8 mm high and 2 mm diameter) was used to test the mechanical resistance to diametral tensile. the material was handled properly and inserted into the matrix with a plastic spatula. the test was performed 24 hours after the preparation. after the resin cement was inserted into the tube, the photopolymerization was carried out, following the manufacturer’s instructions. each pick was then removed from the syringe with an exploratory probe that pushed it out of the tube. all samples were previously assessed in a 30x magnification optical microscope (opmi pico®, carl zeiss, oberkochen, germany) to verify their structural integrity. ten samples of each resin cement and its respective control and experimental groups were made for each group. the samples were individually attached to the ends of a special traction device and mounted on the instron universal testing machine, model 4411 (instron inc. canton, ma, usa). the strength was tested by applying tensile forces at a speed of 0.6 mm/min. the axial compression test used a cylindrical matrix (1 ml insulin syringe, sr insulin u-100 luer slip) whose sample was cylindrical in shape (8 mm height and 2 mm diameter). for each group, 10 samples of each resin cement and its respective control and experimental groups were made. the material was handled properly and inserted in the matrix with a plastic spatula. the test was performed 24 hours after the preparation. samples were taken to the instron universal testing machine, model 4411 (instron inc. canton, ma, usa). after obtaining the necessary loads for the rupture of the samples, the compressive strength was calculated: compressive strength = load/ π.r2 (mpa), where: π= 3.14 (constant) and r = cylinder base radius. the rheology test used a thermo-controlled oscillating rheometer (thermo scientific haake rheostress 6000 design) driver, version 13. the parallel plate model with a diameter of 35mm was used to measure the rheological properties of the materials. the space between the plates was 1 mm. the material was handled according to the manufacturer’s instructions and inserted into the plate. the average initial temperature was 25°c and the final temperature reached 250°c. the test time ranged from 0 to 600 seconds. therefore, the test was performed at an angular frequency of 100 to 0.01 rad.s-1, determining, due to sinusoidal voltage, the viscosity and the modulus of viscosity. in a dynamic oscillatory shear test with an oscillating frequency (ω) and the phase difference (δ) between stress (σ) and strain (y), the strain and the stress in a complex formula are as follows: strain y(t) = y0 e i(ωt), stress σ(t) = σ0 e i(ωt+δ). the complex shear modulus, g*, is defined as stress over strain g* = eiδ == (cos δ + i sin δ)= g’+ ig’’ σ(t) γ(t) σ0 γ0 σ0 γ0 . where g’ is the real (storage) shear modulus and g” is the imaginary (loss) shear modulus. the magnitude of the complex modulus is given by: |g*| = σ0/γ0 = (g’) 2 + (g”)2 and the complex viscosity n* = g*/ = ω. g’ is a measure of stored energy without phase difference between the stress and strain, and represents the elastic component of the material. in contrast, g” represents the viscosity of the materials, and it is a measure of the energy lost as heat. the ratio g”/g’ is the loss tangent, tan δ, which represents the ratio of the viscous part to the 5 borges et al. elastic part (energy loss/energy stored) of the materials. the g’, g”, n*, and tan δ of the composite specimens were measured, and the relationships between these measured values and the resin matrix formulations of the experimental composites were investigated14. after reaching the results, the values were tabulated and submitted to a statistical analysis. first, it was evaluated whether the data presented a normal distribution and homoscedasticity, so that the one-way analysis of variance (anova) and tukey’s range test could be applied. the level of significance was 5% (p <0.05). results table 2 shows the microhardness values of all the cements analyzed. in this test, all the cements were statistically similar (p>0.05), except for maxcem that presented lower hardness compared with the other cements in relation to the top surface (p<0.05). for all cements, the microhardness values of the top surface were higher than the basal area. table 3 brings the diametral tensile values of all the cements analyzed. the relyx u200 and relyx arc presented the highest values (p>0.05). a significant difference was observed between bifix and maxcem in comparison to the other cements analyzed (p<0.05). table 2. averages (standard deviation) of microhardness of resin cements cement microhardness top microhardness base relyx u200 41.2 (1.3)a 35.7 (1.4)b relyx arc 43.9 (2.1)a 37.8 (0.8)b bifix se 42.0 (0.7)a 35.4 (2.1)b maxcem elite 36.6 (0.3)b 34.3 (1.3)b means followed by the same capital letter in the column do not present significant statistical difference (p>0.05). table 3. averages (standard deviation) of diametral tensile strength of resin cements. cement diametral tensile strength relyx u200 155.6 (19.7)a relyx arc 144.3 (12.4)a bifix se 97.2 (10.6)c maxcem elite 116.8 (11.8)b means followed by the same capital letter in the column do not present significant statistical difference. (p>0.05). 6 borges et al. table 4 presents the compression values of all the cements analyzed. the relyx arc showed the highest compressive strength without statistical difference to maxcem (p>0.05). the lowest value was obtained for bifix with significant difference for the other cements analyzed (p<0.05). according to the rheology test, was verified that the resin cements relyx arc and maxcem elite presented high elasticity modulus (em). both presented high em at the beginning of the test. in addition, the four cements had similar behavioral characteristics over the period of 100 seconds, remaining practically until the end of the rheological test (figure 1,2). table 4. averages (standard deviation) of compressive strength of resin cements cement compressive strength relyx u200 190.8 (17.3)b relyx arc 261.3 (16.9)a bifix se 176.9 (18.1)c maxcem elite 233.5 (19.1)a means followed by the same capital letter in the column do not present significant statistical difference. (p>0.05). figure 1. rheological behavior of the four cements analyzed. em(mpa) and time in seconds(s) relyx arc maxcem elite bifix relyx u200 t (s) 0 100 200 300 400 500 600 -2 -1 0 1 2 3 4 5 6 7 8 9 g ’ ( m p a) 7 borges et al. discussion the physical and mechanical properties investigated were different among the tested resin cements. the results could be related to the different chemical composition of the materials. thus, in the present study, the null hypothesis that there are no significant differences in physical-mechanical properties between the conventional resin cement and the self-adhesive resin cements was rejected. high values of the knoop microhardness test may be related to high conversion degree values and to other factors such as high crosslink density in the polymer matrix, chemical composition and material translucency15.regarding the microhardness test, in the present study, all the cements had a top surface value higher than the base surface value, 24 hours after being made. this superiority from the top to the base was also demonstrated in the study by arraias et al.16 (2010). this superiority of the top over the base is related to the absorption of light during the photopolymerization process. the top receives higher incidence of light and consequently acquires higher hardness values. in addition, some studies point to a figure 2. (a) rheological behavior of relyx u200 cement. em(mpa) and time in seconds(s); (b) rheological behavior of relyx arc cement. em(mpa) and time in seconds(s); (c) rheological behavior of bifix se cement. em(mpa) and time in seconds(s); (d) rheological behavior of maxcem elite cement. em(mpa) and time in seconds(s) a 6 5 4 3 2 1 0 0 100 200 300 400 500 600 t (s) g ’ ( m p a) g’ in mpa polynomial fit of relyx u200 model adj. r-squar relyx u200 relyx u200 relyx u200 polynomial 0,37102 intercept b1 b2 value 3,26047 -0,01626 1,91481estandard erro 0,77816 0,00601 9,64084e-6 b 14 12 10 6 4 2 0 0 100 200 300 400 500 600 t (s) g ’ ( m p a) g’ in mpa polynomial fit of relyx arc model adj. r-squar relyx arc relyx arc relyx arc polynomial 0,30907 intercept b1 b2 value 5,90068 -0,03228 4,19014estandard erro 1,5112 0,01165 1,86826e-6 8 c 4,0 3,5 3,0 2,0 1,5 1,0 0,5 0 100 200 300 400 500 600 t (s) g ’ ( m p a) g’ in mpa polynomial fit of bifix model adj. r-squar bifix bifix bifix polynomial 0,1206 intercept b1 b2 value 1,37577 -0,00758 1,07108estandard erro 0,48145 0,00371 5,93742e-6 d 30 25 15 10 5 0 0 100 200 300 400 500 600 t (s) g ’ ( m p a) g’ in mpa polynomial fit of maxcem elite model adj. r-squar maxcem elit maxcem elit maxcem elit polynomial 0,13276 intercept b1 b2 value 8,51688 -0,0499 6,45674estandard erro 3,4254 0,0264 4,22431e-5 20 0,0 -0,5 2,5 8 borges et al. reduction of hardness due to the partial absorption of light caused by the thickness and opacity of aesthetic restorative materials, such as porcelain and metal crowns, for example17,18. diametral tensile strength is a simple method of evaluating tensile forces in cements19,20. in relation to the present study, the diametral tensile test found that the relyx u200 resin cement presented higher value in relation to the maxcem elite resin cement, coinciding with the study by kim et al.20 (2016). in addition, this difference in behavior can be explained by the varied formulation of the materials, related to the quality of the inorganic polymer phases. therefore, the resin matrix, inorganic fillers and other components, influence the high mechanical and physical properties of resin cements. filler particles incorporated in their composition improve their properties, such as elastic modulus, compressive and tensile strength 21,22. the high em of the cement is related to the transfer of strain from the restoration to the tooth and demonstrates the capacity of strength against elastic deformation23. in the study in question, the cement that presented the highest em under traction was relyx arc. this demonstrates its high mechanical resistance when compared to the other cements analyzed. one of the hypotheses to explain the high mechanical property of relyx arc may be due to this cement contains spherical shape filler of different sizes, while other cements have mainly irregular-shaped particles in their composition24. moreover, the high em of the conventional relyx arc cement may be related to the bonding strength. higher values of bond strength of conventional cements may be due pretreatment of the dentin. this process provides the creation of a real hybrid layer, raising a bonding performances to dentin25. compressive strength of cement is an important factor to predict a restorations resistance against masticatory forces26. according to piwowarczy and lauer27 (2003), the evaluation of the degree of compression has been used as an instrument for analyzing the behavior of cement. in their study, the degree of compression of the self-adhesive cements varied between 198.3 mpa and 240.6 mpa, a lower value when compared with the conventional resin cements that were evaluated, which ranged from 244.2 mpa to 325.8 mpa. the study in question found a similar behavior in relation to the superiority of the conventional cement relyx arc. this superiority of conventional cement relative to self-adhesive can be explained by the amount of monomer diluent, which is different in the two types of material21. the composition of the resinous material directly interferes with its viscosity28. it is known that rigid monomers such as bisgma and udma, for example, are fundamental in the formation of more homogeneous and mechanically resistant polymers. furthermore, due to the high hardness, the density of crosslinks of the polymers increases29. however, in addition to the monomers, the content, shape, size distribution and treatment of silane, inorganic filler, filler particles and other factors interfere in the rheological and mechanical properties of the composite28. the maximum value of the shear storage modulus achieved for each cement was different. moreover, this method of the rheology test provides information about how a material changes with time. thus, was observed the highest g’ of the relyx arc and maxcem in relation to the other cements analyzed from the beginning of the test. 9 borges et al. the increase of g’ means the progression of the cross-linking and entanglements of polymer chains29. this is an important factor, because the lower elastic modulus and resilience of cements may compromise the longevity of brittle restorations, such as all-ceramic restorations30. the viscosity of resin cements has an influence on the handling properties of the material. this material has time-dependent properties that affect the working time, setting time and the quality of the cementation31. change of viscosity was monitored according to the increasing shear rate and temperature. however, the viscosity of cements varies considerably among the brands though they were nominally of the same class28. the results of this study indicate that relyx arc cement showed peaks during the rheometric test. in general, this demonstrates the need for a more cautious handling of this cement, possibly related to its monomeric constitution and viscosity. the monomeric composition of self-adhesive cements differs from conventional ones. they are composed of acid-functional adhesive resinous monomers, which are a type of monomeric methacrylate that has a phosphoric acid or carboxylic acid grouping in their molecular structure29. the presence of those functional monomers may be interfere with the amine initiator and compromises the mechanical properties5. therefore, the inferiority of some self-adhesive cements analyzed in relation to conventional cement may be related to several factors, such as a low capacity of acid monomer corrosion, inorganic polymer phases, reducing surface demineralization; incomplete removal of the smear layer, which promotes weak bonding with the resin intermediate layer and mineral buffering effect on the dentin that neutralizes the cement ph32-34 . with the limitations of this study and based on the results obtained, it can be concluded that the physical and mechanical properties investigated were different among the tested resin cements. the self-adhesive cements presented poorer mechanical properties than 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27(5):537-42. doi: 10.1590/0103-6440201600445. 1http://dx.doi.org/10.20396/bjos.v19i0.8658556 volume 19 2020 e208556 original article 1 restorative dentistry department, tehran university of medical sciences school of dentistry, tehran, iran 2 dentist, tehran, iran 3 department of resin and additives, institute for color science and technology, tehran, iran corresponding author: fariba motevasselian orcid number:0000-0002-3856-5096 address: restorative dentistry department, school of dentistry, tehran university of medical sciences, north karegar street, tehran, iran. postal code: 1439955991 tel: + 98-21-88015801 e-mail:f-motevasselian@sina.tums. ac.ir fariba.motevaselian@yahoo.com received: february 29, 2020 accepted: november 24, 2020 degree of conversion and water sorption of self-adhesive and conventional flowable composites: an in vitro study ladan ranjbar omrani1 , mahdi abbasi1 , fariba motevasselian1,* , mohmad amin yektaei2, farhood najafi3 aim: self-adhesive flowable composite resins have been recently introduced to the market. degree of conversion (dc) and water sorption (ws) are two important parameters affecting the properties of restorative materials. this study aimed to assess the dc and ws of a self-adhesive flowable composite resin in comparison with two conventional flowable composite resins. methods: vertise flow (vf) self-adhesive and tetric-n flow (tf) and grandio flow (gf) conventional flowable composites were evaluated in this in vitro, experimental study. the dc (n=3) was determined by attenuated total reflectance-fourier transform infrared spectroscopy (atr-ftir). the ws (n=7) was measured after 7 days of immersion in artificial saliva according  to iso 4049  specifications. data were analyzed by one-way anova and a post-hoc test (p<0.05). results: vf showed the highest dc percentage (84.3%) followed by gf (72.79%) and tf (68.7%). the latter two had no significant difference (p=0.8). ws was the highest in vf (55.2 µg/mm3), and the two conventional flowable composites had a significant difference in ws (19.5 µg/mm3 in tf and 11 µg/mm3 in gf; p<0.001). conclusions: flowable composite resins had significant differences in dc and ws, and vf demonstrated the highest dc and ws. keywords: absorption. composite resins. polymerization. vertise flow. https://orcid.org/0000-0001-8304-038x https://orcid.org/0000-0003-1155-1409 https://orcid.org/0000-0002-3856-5096 https://orcid.org/0000-0003-3094-1322 2 omrani et al. introduction composite resins are widely used due to the increased esthetic demands of patients and improvements in their mechanical properties. composite resins can be classified into putty-like and flowable composite resins according to their consistency1. dental composites consist of two main components namely the organic matrix and the inorganic fillers. the organic phase of composite resins is dominantly composed of dimethacrylate resins. the monomers that are most widely used in commercially available composite resins include bisphenol-a glycidyl methacrylate (bis-gma) and urethane dimethacrylate (udma), which are high molecular weight monomers1. low-viscosity monomers, such as triethylene glycol dimethacrylate (tegdma), are added as diluents to the highly viscous resin matrix in various concentrations to improve the degree of conversion (dc) and increase the filler content1,2. flowable composites were designed for enhanced adaptation to the cavity walls and easy handling and application. they either have less filler loading or high proportion of diluent monomers added to viscous monomers1,2. researchers have focused on simplifying the adhesive and composite application steps by developing self-adhesive restorative materials with favorable handling properties3. self-adhesive flowable composite resins have been recently introduced to the market, which are similar to the solvent-free self-etch adhesive systems4. they combine the merits of adhesion to tooth structure and tooth restoration in one product5. they bond to tooth structure by mechanical interlocking and chemical interactions3,6. their chemical formulation contains acidic functional monomers such as 4-methacryloxyethyltrimetellitic acid as in fusio liquid dentin (pentron clinical, orange, ca, usa) or glycerophosphate dimethacrylate as in vertise flow (vf; kerr, orange, ca, usa) self-adhesive flowable composite. the acidity of monomers varies from 1.9 to 33. the composition of resin matrix also includes hydroxyethyl methacrylate (hema)3. hema is responsible for improvement of miscibility of hydrophilic and hydrophobic components, and prevention of phase separation7. it also improves the wetting of dentin substrate, and enhances resin penetration into dentin6,7. the self-adhesive flowable restorative materials are indicated for small class i and class v cavities, and for sealing of the pits and fissures3,8. in the hostile oral environment, the restorative materials need to meet some certain specifications to ensure optimal product quality9. dc is one of the most important parameters affecting the properties of restorative materials. the dc of carbon-carbon double bonds to single bonds determines the dc percentage (dc%) of composite resins10. after the polymerization reaction of monomers, a cross-linked structure is formed10,11. the dc and cross-linking density are important factors affecting the physical and mechanical properties of composite resins10,11. however, the monomer conversion is never complete, and a number of double bonds often remain10,11. when the polymerized material is exposed to oral fluids, the unreacted monomers can leach out of the resin mass and cause adverse effects such as allergic reactions10,11. in addition, a correlation has been reported between the residual double bonds and internal color change of light-cure composite resins10. the 3 omrani et al. possible causes include oxidation of unreacted double bonds, and chemical degradation and formation of colored degradation products10,12. several studies have demonstrated that the dc and density of polymer network are important factors affecting the polymer/solvent diffusion behavior13. the solvent permeability of polymer networks decreases as the dc and cross-linking between the polymer chains increase14,15. on the other hand, water sorption (ws) is a diffusion-controlled process14,15. it has been demonstrated that water sorption highly depends on the hydrophilicity/hydrophobicity of monomers in the resin matrix14-16. ws can cause several detrimental effects. its major effect is polymer plasticization, mediated by different mechanisms. the absorbed water may cause hydrolytic breakdown of the resin matrix-filler interface and cause filler debonding from the resin matrix, hydrolytic cleavage of polymer chains, elution of unreacted monomers and leachable components, and deterioration of the mechanical properties of polymer such as its wear resistance10,11,17. in addition, ws is of particular importance in external discoloration of composite resins18. it could be related to different mechanisms including penetration of staining agents into the surface and subsurface layers of composite resin, and superficial degradation18. resin matrix hydrophilicity is also associated with external color change14,18. therefore, dc and ws are important parameters affecting the physical and mechanical properties of composite resins, and longevity and clinical service of adhesive restorations10,11. the purpose of this study was to compare vf (kerr, orange, ca, usa) a self-adhesive flowable composite with two conventional flowable composites namely tetric-n flow (tf; ivoclar vivadent ag, schaan, liechtenstein) and grandio flow (gf; vocco, cuxhaven, germany) in terms of dc and ws. the null hypotheses were that the dc and ws of the three composite resins would not be significantly different. materials and methods sample size calculation: one-way anova of pass 11 software was used for sample size calculation, assuming type one error of 0.05, statistical power of 80%, and a standard deviation of 2.87. accordingly, the minimum sample size to ensure detecting a difference of at least 10% between the groups was found to be 3 samples for the dc test8. for the ws test, assuming a standard deviation of 4.86, the minimum sample size to ensure detecting a difference of at least 8 μg/mm3 between the groups was found to be 7 samples18. specimen preparation for the dc test table 1 lists the materials used in this study. the a2 shade of composite resins was used. for preparation of composite specimens (4 mm in diameter, 1 mm in thickness, n = 3 of each material), the flowable composite resins were dispensed into plexiglass molds, pressed between two glass slides (0.5 mm thick), and covered with a polyester strip form a flat surface and to prevent the formation of oxygen inhibited layer 4 omrani et al. upon polymerization. each specimen was polymerized for 20 s using a led curing unit (bluephase; ivoclar vivadent, schaan, liechtenstein). the tip of the light curing unit was in contact with the top glass slide. the light intensity was 1100 mw/cm2. the specimens were demounted, and the dc was measured at the center of the top surface of the specimen immediately after polymerization by attenuated total reflectance-fourier transform infrared spectroscopy (atr-ftir; nicolet is10; thermo fisher scientific, waltham, ma, usa). the ir spectra of the specimens were determined in the wave number range of 650-4000 cm−1 at a resolution of 4 cm−1 and ir depth of ~2 μm. uncured resin specimens were also subjected to atr-ftir, to serve as non-cured references. unpolymerized composite was directly placed on the device’s atr crystal. the dc was determined according to the changes in the absorbance values of the aliphatic c-c bonds (1635 cm−1), which are consumed during polymerization, and the aromatic c…c bonds (1608 cm−1), which are not affected during the curing process; this peak is almost constant and therefore serves as an internal standard. the ratios of the absorbance peak intensities ascribed to (c=c) before and after polymerization, representing the unreacted carbon-carbon double bonds, were compared and accordingly, the dc was calculated by the following equation: dc% = 1 ×100 (1635 cm-1 / 1608 cm – 1) peak after curing) (1635 cm-1 / 1608 cm – 1) peak before curing)[ ] specimen preparation for the ws test the same flowable composite resins were used for the ws test according to iso 4049:2009. seven disc-shaped specimens of each composite were fabricated using plexiglass molds (15 ± 1 mm in diameter and 1.0 ± 1 mm in thickness). composite resins were applied into the molds, their surface was covered with a polyester strip and a glass slide, and compressed to prevent voids and porosities. table 1. resin composites used and their classification, manufacturer, and composition product composite type manufacturer resin composition filler system filler load (wt%) vertise flow nanohybrid kerr, orange, ca, usa gpdma, hema, bisgma, mehq prepolymerized filler, barium glass, nanosized colloidal silica, nanosized ytterbium fluoride 70 grandio flow nanohybrid voco, cuxhaven, germany bis-gma, tegdma, hedma inorganic fillers: silicate oxide, silicate glass pigment 80 tetric-n flow nanohybrid ivoclar vivadent, schaan liechtenstein bis-gma, udma, tegdma barium glass, ytterbium fluoride, silica 63.8 gpdma: glycerol prosphate dimethacrylate; hema: hydroxyethyl methacrylate; bis-gma: bisphenol a glycidyl dimethacrylate; mehq: monomethyl ether hydroquinone; tegdma: triethylene glycol dimethacrylate; hddma: 1,6-hexanediol dimethacrylate; udma: urethane dimethacrylate 5 omrani et al. the composites were then light-cured from both sides for 20 s through the glass slide/polyester strip with a led curing unit (bluephase; ivoclar vivadent, schaan, liechtenstein) with a light intensity of 1100 mw/cm2  and a tip diameter of 10 mm using the overlapping exposure technique to ensure adequate exposure of all areas. the mean output intensity of the light curing unit as assessed with a calibrated radiometer (bluephase meter; ivoclar- vivadent), was 1100 mw/cm2. the periphery of all specimens was ground with 1200-grit silicon carbide abrasive paper. water sorption test the specimens were placed vertically on a metal rack to avoid sample-to-sample contact. the specimens were placed in a desiccator containing dehydrated silica gel. the desiccators were placed in an incubator (kavosh mega; kavosh azma co, tehran, iran) at 37°±1° c for 22 h followed by an additional 2 h at 23°±1°c. each specimen was then weighed on a scale (gf-3000; a&d, tokyo, japan) with 0.1 mg accuracy. this drying cycle was repeated until the weight loss of each specimen was lower than 0.1 mg within a 24-h period. at this point, the weight was considered constant (w1) and reported in micrograms (µg). next, the specimens were immersed in distilled water for 7 days at 37º±1ºc. the water was changed every day. after completion of the storage period, the specimens were removed from the water with tweezers, and blotted dry with a clean tissue paper and weighed again (w2). next, the specimens were subjected to the same conditioning cycle until achieving a constant weight (w3). after final drying, the thickness of each specimen was measured using a digital caliper (mitutoyo corp., kawasaki, japan) at four equally spaced points on the circumference. the thickness at the center of the specimens was also measured. the diameter was measured in two perpendicular lines as well. the average volume (v) of specimens was calculated in cubic millimeters (mm3). the ws value of each specimen (in µg/mm3) was calculated using the following formula: ws (µg/mm3) = (w2 –w3)/v statistical analysis: normal distribution of data was evaluated using the shapiro-wilk and kolmogorov-smirnov tests. the data were analyzed using one-way anova. the tukey’s test and the games-howell tests were chosen according to the homogeneity of variances, which was checked by the levene’s test. multiple comparisons were performed by the tukey’s test for dc (since the homogeneity of variances was met for this variable) while the games-howell test was used for ws (since the homogeneity of variances was not met for this variable). p-values < 0.05 were considered significant. all statistical analyses were carried out using spss version 20 (spss inc., chicago, il, usa). results table 2 presents the mean values of dc and ws. the tested materials had statistically significant differences in both ws (p=0.001) and dc (p=0.002). vf presented significantly higher ws than other composites (p<0.001); while, gf had the lowest ws 6 omrani et al. (p<0.001).vf showed significantly higher dc than other composite resins; other composites had no significant difference with each other in this respect (p=0.8). discussion this study evaluated the ws and dc of a self-adhesive flowable composite compared with two conventional flowable composite resins. the results demonstrated statistically significant differences among the materials in ws and dc (table 2). therefore, the null hypotheses were rejected. in the present study, the dc was quite high in the three flowable composites, ranging from 71.5% to 84.3%; vf showed the highest dc. the minimum dc for a clinically acceptable composite restoration has not been exactly determined. however, a dc ranging from 55% to 85% has been reported in the literature10,19. the level of polymerization is an important indicator for the quality and characteristics of polymers10. in the current study, dc was measured 5 min after light polymerization. several studies revealed that the dc values measured 24 h after light irradiation were significantly higher than the dc values measured immediately or 5 min after curing20,21. however, the dc measured after 5 min is of great importance because the restorative material is exposed to the water spray of low-speed and high-speed handpiece for contouring or occlusal adjustment and is also exposed to the saliva and oral fluids shortly after light irradiation22. this description could legitimize our methodology regarding measuring the dc 5 min after light irradiation. furthermore, ftir in the current study showed that most of the polymerization occurred within 5 min after light exposure, and it can be assumed that no further statistical increase in the dc values can be attained in up to 24 h. several factors affect the dc including the material-related factors. it has been demonstrated that the type, volume and viscosity of monomers in the resin matrix composition could affect the dc. monomers are selected based on their viscosity and their contribution to the crosslink density of the polymer network11,23. the highly viscous monomers have lower mobility, which affects the propagation of free radicals in the resin matrix11,23. increased viscosity causes the termination of diffusion-controlled mechanism at earlier stages of the polymerization process, leading to a low dc23. to improve the conversion of the network, low viscosity monomers are required to dilute the highly viscous monomers and provide an optimal monomer ratio. it has an important effect on the mobility of molecules in the resin matrix and polymerization process, and providing the best performance in terms of required parameters such as high table 2. mean (standard deviation) of the degree of conversion (dc%) and water sorption (µg / mm3) data vertise-flow (vs) grand flow (gf) tetric-n flow (tn) vs vs. gf mean difference (95%ci) vs vs. tn mean difference (95%ci) gf vs. tn mean difference (95%ci) dc 84.39±1.21a 72.79±3.21b 71.50±2.91b 11.59 (5.08 to 18.11) p-value=0.004 12.88 (6.37 to 19.40) value=0.002 1.29 (5.23 to 7.81) value=0.82 ws 55.21±4.92a 11.06±2.37b 19.53±3.15c 44.15 (38.34 to 49.96) p-value<0.001 35.68 (29.65 to 41.72) p-value<0.001 -8.46 (-12.49 to -4.44) p-value<0.001 means with the same superscript letters indicate non-significant difference (p≥0.05) 7 omrani et al. mechanical properties and ws10,22. also, various types of photo-initiators can affect the dc of monomers. filler type, filler size, and loading level can affect the monomer conversion by altering light transmission and viscosity of composite resins as well1. visible light-cured dental composite resins are usually polymerized by the camphorquinone (cq)/amine photo-initiator system1. free radical polymerization is started upon blue light exposure and excitation of cq and reduction of tertiary amine electron donor1. however, it has been documented that proper polymerization is inhibited by acidic monomers through neutralization of tertiary basic amine as a reducing agent24,25. the inhibition of photopolymerization depends on the type, concentration, and ph of the acidic functional monomers25. however, neutralization depends on the type of photo-initiator system24,25. therefore, a suitable initiator system is critical for polymerization of acidic photopolymerizable composite resins24,25. the possible explanations for the high dc of vf self-adhesive composite resin are as follows: (a) higher amounts of initiator systems: vf may contain alternative co-initiators as amine substitutes to compensate for inactivation of the amine co‐initiator, which improve the dc, (b) since bis-gma is highly viscous, some of this monomer is probably replaced with low molecular weight co-monomers to obtain lower viscosity. it allows better monomeric mobility, which can enhance polymerization8,24-26. however, these effects cannot be assessed since detailed information about the chemical composition of vf has not been disclosed by the manufacturer22. the tf and gf composites had no significant difference in dc, although they have a different filler content. the filler content is 80 wt.% for gf and 63.8 wt.% for tf. an inverse correlation exists between the dc and filler loading27. composites with higher filler content usually have a high viscosity and this factor may interfere with monomer conversion and limit the propagation of polymerization process1,3,10. however, we did not find such a causal relationship in the current study. the similar values observed in this study could be related to the differences in the resin matrix composition, and type and proportion of monomers9. gf does not contain high molecular weight udma (571 g/mol), and is composed of low viscosity monomers such as hexanediol dimethacrylate resin, a hydrophobic low viscosity monomer (η=0.007 pa.s) with low molecular weight (254 g/mol), and tegdma (mw=286 g.mol and η=0.008 pa.s) as diluents11,28. the tf is composed of high molecular weight monomers, bis-gma, and udma. tegdma is the only low viscosity monomer in its formulation (table 1). thus, the composition of gf may affect the dc in spite of higher filler content. vf exhibited the highest amount of water sorption during 1 week of immersion in distilled water (55.2 µg/mm3), while gf had the lowest water sorption (11 µg/mm3) during this period. these results agree with other studies15,18. several factors can affect water sorption such as the chemical formulation of matrix monomers and the matrix filler content18. the three flowable composites tested in the present study were nano-hybrid composites. one difference between the conventional flowable composites and vf is that they do not contain hema. in addition, bis-gma and udma, which are highly hydrophobic, are incorporated in the resin matrix of conventional flowable composites. however, self-adhesive flowable composites contain an acidic monomer, gpdm, 8 omrani et al. which is a quite hydrophilic monomer due to the presence of one phosphate acidic functional group, as well as hema in their resin matrix, which affects the hydrophilicity of the resulting polymer and makes it more susceptible to ws3,18. another factor that may contribute to higher water sorption of vf is the type of filler particles18. vf is composed of four types of fillers including pre-polymerized fillers (ppfs). ppfs are ground, cured composite resins initially filled with sub-micron particles29. the rate of water sorption depends on the quality of resin-filler bond. if the filler-matrix interface is uncoupled, it can provide paths for water diffusion15,29. integration of ppfs in the resin matrix is weak because the functional groups have already reacted during their preparation, and silanization is difficult; thus, breakdown of the interface may occur29. gf had the lowest water sorption in this study, which can be attributed to its filler content. an inverse correlation exists between the filler loading and water sorption16. this can be explained by lower free space in the resin matrix as the filler content increases30. in the current study, ws behavior of the materials was not inversely correlated with their dc as demonstrated in another study16. the self-adhesive composite showed the greatest ws with high dc. it implies that the cross-linking nature of the polymer network does not necessarily decrease ws of the polymer. it can indicate the important role of chemical composition and hydrophilicity of resin monomers in ws30. the ws level of vf in the current study did not comply with the requirements of iso 4049:2009 for ws31, as the value was above 40 µg/mm3. it may cause fast intraoral degradation, and lead to decreased physical and mechanical properties, and reduced longevity of composite restorations, questioning their routine clinical application. future studies are recommended to assess the effects of resin composition, filler content, filler type, and polymer network of these composites on their ws, solubility, color 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adhesive. acta biomater. 2012 may;8(5):1928-34. doi: 10.1016/j.actbio.2012.01.013. 26. tadin a, marovic d, galic n, kovacic i, zeljezic d. composite-induced toxicity in human gingival and pulp fibroblast cells. acta odontol scand. 2014 may;72(4):304-11. doi: 10.3109/00016357.2013.824607. 27. barron d, rueggeberg f, schuster g. a comparison of monomer conversion and inorganic filler content in visible light-cured denture resins. dent mater. 1992 jul;8(4):274-7. doi: 10.1016/0109-5641(92)90099-x. 28. trujillo‐lemon m, ge j, lu h, tanaka j, stansbury jw. dimethacrylate derivatives of dimer acid. j polym sci part a: polym chem. 2006 may;44(12):3921-9. doi.org/10.1002/pola.21493. 29. randolph ld, palin wm, leloup g, leprince jg. filler characteristics of modern dental resin composites and their influence on physico-mechanical properties. den mater dec. 2016;32(12):1586-99. doi: 10.1016/j.dental.2016.09.034. 30. bociong k, szczesio a, sokolowski k, domarecka m, sokolowski j, krasowski m, et al. the influence of water sorption of dental light-cured composites on shrinkage stress. materials (basel). 2017 sep;10(10):1142. doi: 10.3390/ma10101142. 31. online browsing platform (obp). iso 4049:2009(en). dentistry — polymer-based restorative materials. 2009 [cited 2020 jan 20]. available from: https://www.iso.org/obp/ui/#iso:std:iso:4049:ed-4:v1:en. https://doi.org/10.1002/pola.21493 1 volume 16 2017 e17060 original article 1 department of orthodontics and radiology, unicid (university of são paulo city), são paulo, sp/ brazil 2 neuroimaging laboratory, department of neurology, unicamp (university of campinas) campinas – sp/brazil. corresponding author: ana carla raphaelli nahás-scocate, department of orthodontics and radiology, unicid, rua cesário galeno 448, bloco a. tatuapé, são paulo, spcep 03071-000 email: carlanahas@yahoo.com.br received: june 6, 2017 accepted: july 26, 2017 evaluation of the skeletal maturation of cervical vertebrae with magnetic resonance imaging: a piloty study camila chaparin baldin1, dds, m.sc; marjorie kitt1, ugs; andré luiz ferreira costa1,2, dds, phd; clarissa lin yasuda2, md, phd; fernando cendes2, md, phd; ana carla raphaelli nahás-scocate1, dds, phd aims: the objective of the present study was to assess the skeletal maturation by means of three-dimensional models of the cervical vertebrae generated through segmentation of the magnetic resonance (mr) images by using medical software. methods: twenty mr images of the skull of male and female individuals aged between 8 and 22 years old were selected. assessment of the images was performed by using the itk-snap software, consisting of three steps: 1) vertebral segmentation; 2) three-dimensional reconstruction; and 3) classification of skeletal maturation. two specialists in orthodontics and two specialists in dentomaxillofacial radiology assessed the images. results: analysis of reproducibility and repeatability were performed by using the rr method, with paired t-test also being applied to the repeatability factor together with lin’s concordance coefficient. the significance level was set at 5%. it was found that there was no difference in the inter-rater reliability (p-value = 0.625), but without statistical repeatability. conclusions: new tools, as 3d reconstruction software, enabled us to build an effective and friendly 3d-reconstruction system for classification of the skeletal maturation stages of cervical vertebrae. keywords: maturation; cervical vertebrae; magnetic resonance image; 3d reconstruction http://dx.doi.org/10.20396/bjos.v16i0.8650501 2 skeletal maturation and mri baldin et al. introduction in medicine and dentistry it is fundamental to determine the growth process of an individual in order to know the related disorders in endocrinology and orthopaedics, as well as to assist in the diagnosis and planning of the orthodontic treatment1,2. this growth process has a great variability from individual to individual, being influenced by several factors3. the term “skeletal maturation age” was introduced in order to analyse and differentiate with greater accuracy the biological age, skeletal age and skeletal maturation found in the same individual4. there are different forms of estimating the maturational process. however, despite the reliability of the different forms of assessment, the one regarding skeletal maturation is the most reliable and efficient, since the variations in the form and density of the bone allow to gauge the growth trajectory during development5. the study of skeletal maturation is performed by means of radiographs, which serve to evaluate ossification centres based on the emergence or absence of certain structures and development of others6. with this aim in mind, radiographs of various parts of the human body are used, with hand and wrist bones being the most useful and recognised7-9. despite being a simple and cheap method, there is a great inconvenience in using this method because of the extra ionising radiation to the patient10. nowadays, there is an increasing tendency to use other structures to estimate the skeletal age, such as cervical vertebrae visualised on lateral teleradiographs. studies by franchi et al.11 and baccetti et al.12 corroborate that this is an adequate method for assessing the skeletal maturation as they found a positive relationship between cervical vertebrae and mandibular growth during puberty. in this methodology, the stages representing different morphological characteristics of the cervical vertebrae are defined, with each of these stages reflecting the progressive levels of skeletal maturation4. however, the method is considered subjective, since it is based on visual exams of the morphological changes in the cervical vertebrae. additionally, the evaluator is required to have experience in order to establish a correct diagnosis13,14. according to mendelson and rubin15, the advances in the computing field allows for the development of better software packages. therefore, these software packages are of great value for radiological diagnosis16. there are currently software packages enabling both visualisation and segmentation of images from magnetic resonance (mr) image and computed tomography (ct), including rendering and classification of 3d models17-21. this process provides important information both didactically and diagnostically, allowing better localisation of structures, surgical planning and exam analysis22. there is no study in the literature showing assessment of cervical vertebrae by means of mr imaging. therefore, we have proposed to develop and implement a semi-automatic methodology for assessing the skeletal maturation of cervical vertebrae from mr images of the skull by using free software. 3 skeletal maturation and mri baldin et al. material & methods this study was approved by the local research ethics committee according to protocol number 999.190. the images were originally taken as part of previous mr images of the central nerve system and were obtained from the laboratory of neuroimaging of school of medicine of university of campinas (unicamp). there was no need to recruit further subjects for conduction of mr exam. only images of subjects who had no disease which might result in oromaxillofacial changes were included for study. twenty images of male and female individuals comprised the sample, which was divided into four groups of five subjects each: g1 (8-10 years old); g2 (12-14 years old); g3 (16-18 years old); and g4 (20-22 years old). the inclusion criteria were the following: mr images of the skull of individuals aged between 8 and 22 years old. on the other hand, the exclusion criteria were: history of facial injury due to accidents; previous orthodontic treatment; and any pathology making interpretation of the results difficult. the mr images were obtained by using a 3-tesla scanner (phillips achieva-intera). the image acquisition parameters were: spin echo t1 weighted sagittal images (6-mm thickness, tip angle of 180o, tr = 430, te = 12, 200x350 matrix, and fov = 25x25 cm). these images were used to guide the acquisition plane for axial and coronal images. the mr images were acquired in analyze format and then converted into dicom format by using the mipav software (mipav.cit.nih.gov). the itk-snap software (www.itksnap.org)23 was used to perform the image segmentation and to obtain 3d models. this software also allows for semi-automatic segmentation in which several colours can be chosen for the structures delineated. in the segmentation, the similarity in the shades of gray among equal-density tissues allows to locate image edges and consequently the total mapping of the structure being studied24. for obtaining the 3d models from each mr image, 10 slices (84 to 94) were selected before thresholding of each image of the vertebrae (i.e. c2, c3, c4). after delineation, the vertebrae were filled with the selected colours. in the end of this process, the models were generated for analysis. the method used in this work was the interactive thresholding by manipulating the image histogram. this technique is based on determining two values termed minimum and maximum thresholds, which delineate in the histogram the region of interest (roi) to be segmented. the segmentation was performed by grouping neighbour pixels with similar values at a tolerance level. next, the segmented image is extracted from other surrounding structures, reconstructed and visualised three-dimensionally17,24. our regions of interest were the cervical vertebrae c2, c3 and c4. the software threshold was used for segmentation of the images based on defining density intervals expressing, for example, only voxels which correspond to the desired cervical vertebra (fig. 1a). for doing so, a detection algorithm is applied to each transversal slice to define each contour. after some tests with several detection algorithms, we found the most satisfactory one for the purposes of this study. each object was assigned a label for identification. after mapping the vertebra, the 3d reconstruction of the object was performed (fig. 1b). image assessment was conducted by two orthodontists and two oral radiologists, previously calibrated, who used supporting material containing skeletal maturation stages http://www.itksnap.org 4 skeletal maturation and mri baldin et al. and description of each stage as suggested by hassel & farman (1995). the evaluators analysed and evaluated the twenty 3d images and then answered a 3-item questionnaire on the method used. they re-evaluated the images after 1-week interval. exploratory analysis of the data was carried out based on resumed measurements (i.e. mean, standard deviation, minimum, median, maximum values) and graph construction. analysis of repeatability and reproducibility of the method was performed by using the rr method. for assessment of repeatability, the paired t-test for each evaluator and li’s concordance coefficient were used at significance level of 5% by using the minitab statistical software, version 16 r302. results table 1 shows the measurements of position and dispersion per evaluator and repetition; the elaboration and conduction of the study on repeatability and reproducibility of the method. in the graph of variation components, the contribution percentage of the patients is higher than that of study, demonstrating that the largest part of the variation found in the study is due to differences between patients, but more than 30% (63.02) of the variation is due to measurement system (fig. 2). table 1. measurements of position and dispersion of skeletal maturation per evaluator and repetition. evaluater repetition n mean sd minimum median maximum 1 1 20 3.2 1.2 1.0 3.5 5.0 1 2 20 3.5 1.3 2.0 3.5 6.0 2 1 20 2.8 1.7 1.0 2.0 6.0 2 2 20 3.4 1.9 1.0 3.0 6.0 3 1 20 3.0 1.3 1.0 3.0 5.0 3 2 20 3.7 1.2 2.0 3.0 6.0 4 1 20 3.3 1.4 1.0 3.5 5.0 4 2 20 3.2 1.4 1.0 3.0 6.0 figure 1. a) segmentation of vertebra c3 by using the software itk-snap; b) 3d model of vertebrae c2, c3 and c4 generated by segmentation. a ba r l p 1 cm 5 skeletal maturation and mri baldin et al. in the graph per patient, there are differences between patients as expected and shown by the non-linear straight line linking their mean values. in the graph per evaluator, there is a small difference between the evaluators as also expected and shown by the almost non-linear straight line linking their mean values (fig. 2). in the x-bar graph per evaluator, the majority of the points are within the limits, indicating a variation in the measurement system. the graph evaluator*patient shows an interaction between these two variables (p-value = 0.017), indicating that there was a difference in the process of assessment made by each evaluator regarding each patient (fig. 2). it was found that there was no difference between the evaluators (p-value = 0.625), despite the poor repeatability. paired t-test was performed for each evaluator in order to compare their answers between the repetitions. no statistically significant differences were found between the repetitions performed by evaluators 1 (p-value = 0.110) and 4 (p-value = 0.847). there was found no statistically significant difference between the two repetitions performed by evaluator 2 (p-value = 0.077). on the other hand, a significant difference was observed between the repetitions performed by evaluator 3 (p-value = 0.004). the lin’s concordance coefficient showed around a coefficient of 0.7. p er ce nt components of variation 80 40 0 gage r&r repeat % contribution % study var reprod part-to-part sa m pl e r an ge r chart by evaluatot 4 2 0 ucl = 2,491 r = 0,763 lcl = 0 nome 1 2 3 4 sa m pl e m ea n r chart by evaluatot 6 4 2 ucl = 4,768 lcl = 1,810 1 2 3 4 x = 3,244 maturation by evaluator 6 4 2 1 2 evaluator 3 4 maturation by patient 6 2 4 all iss on 17 an a i za be l 1 2 be atr iz 17 br un ar 21 cr ist ian ob 13 da nie le 17 fe rn an da 21 fe rn an do r 21 fr an cie lly 17 gu sta vo a1 4 gu sta vo h 9 he nr i b 12 he nr iqu e 2 1 la ur a 9 lu cia na 21 m ag ab rie laz 10 pr isc ila ap 17 m ga br iel a 1 0 vic tor e m ate us 14 patient * evaluator interaction a ve ra ge evaluator 6 2 4 all iss on 17 an a i za be l 1 2 be atr iz 17 br un ar 21 cr ist ian ob 13 da nie le 17 fe rn an da 21 fe rn an do r 21 fr an cie lly 17 gu sta vo a1 4 gu sta vo h 9 he nr i b 12 he nr iqu e 2 1 la ur a 9 lu cia na 21 m ag ab rie laz 10 pr isc ila ap 17 m ga br iel a 1 0 vic tor e m ate us 14 1 2 3 4 figure 2. study of repeatability and reproducibility of the method. 6 skeletal maturation and mri baldin et al. discussion hand-wrist radiograph has been used in the orthodontic practice for assessment of the ossification centres, as it is possible to find distinct modifications in each one during their development. these changes are used to inform the orthodontist about the period of skeletal maturation in which the patient is7-9. fishman7 observed a synchronism in the growth of several body structures, showing that the information on hand and wrist structures can be used for assessing the general growth of the body and face as well1,10,25. although the hand-wrist radiograph is consecrated in the orthodontic practice, there is currently a trend towards the use of alternative methods for determination of the growth spurt, such as the observation of cervical vertebrae6,8,26. according to hassel and farman4, the cervical vertebrae are viewed on lateral teleradiographs comprising the routine documentation of patients who are submitted to orthodontic and/or functional orthopaedic treatment, which can reduce the amount of ionising radiation to the patient9,13. the efficacy in using cervical vertebrae by means of lateral teleradiography was also evaluated by wong et al.1, kucukkles et al.6, flores et al.8, danaei et al.9, caldas et al.13, gandini et al.25 and mahajan27, who concluded that this alternative method is a reliable parameter to assess the skeletal maturation as it helps to determine the best time to initiate the orthopaedic intervention in patients in the stage of craniofacial growth. the range of information provided by the assessment of cervical vertebrae can replace the traditional method based on hand-wrist radiographs, thus simplifying the orthodontic routines the patients face and preventing additional dose of ionising radiation to them. assessing the cervical vertebral maturation using mr images is suggested by the present study, since there is no work in the literature relating visualisation methods to interpretation of the skeletal maturation. we consider that our work is a pilot study because it addresses an innovative method in the dentistry field, favouring the patients as they are less exposed to ionising radiation. however, it is natural that many biases emerge during the development of a study method. according to the results found, it is suggested that the number of evaluators should be higher in further studies and that they should be rigorously trained, since it is known that assessment of 3d models requires a qualified evaluator. on the other hand, this method is easy and quick to understand and this has encouraged us to improve it in further studies. the fact that the evaluator can also manipulate the image of the vertebrae by using software makes this method dynamic and non-static. the method of assessing the cervical vertebrae by means of magnetic resonance imaging, as suggested in our study, has innovated the dental and medical scopes scientifically as the patient is less exposed to radiation. demystifying the use of 3d image in the final diagnosis is part of our ongoing achievement for advance of imagenology. conclusion according to the methodology applied and the results obtained, one can conclude that there was no difference between the evaluators despite the poor repeatability. the method’s reproducibility depends on systematisation of the measurements, since 7 skeletal maturation and mri baldin et al. the assessment of 3d images requires well-trained evaluators. the skeletal maturation stages of the cervical vertebrae followed the same pattern, suggesting that this proposed methodology deserves to be improved as no ionising radiation is involved. acknowledgements this work was supported by são paulo research foundation – fapesp (grant number 12/16953-4). declaration of conflicting interests the authors declare that there is no conflict of interest. references 1. wong rw, alkhal ha, rabie ab. use of cervical vertebral maturation to determine skeletal age. am j orthod dentofacial orthop. 2009 oct;136(4):484.e1-6; discussion 484-5. doi: 10.1016/j.ajodo.2007.08.033. 2. crawford b, kim dg, moon es, johnson e, fields hw, palomo jm, et al. cervical vertebral bone mineral density changes in adolescents during orthodontic treatment. am j orthod dentofacial orthop. 2014 aug;146(2):183-9. doi: 10.1016/j.ajodo.2014.04.019. 3. 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skeletal age? a comparison of radiation dose of two strategies for skeletal age estimation. eur j orthod. 2013 oct;35(5):604-9. doi: 10.1093/ejo/cjs043. 27. mahajan s. evaluation of skeletal maturation by comparing the hand wrist radiograph and cervical vertebrae as seen in lateral cephalogram. indian j dent res. 2011 mar-apr;22(2):309-16. doi: 10.4103/0970-9290.84310. 1http://dx.doi.org/10.20396/bjos.v18i0.8657251 volume 18 2019 e191502 original article 1 universidade estadual do piauí – uespi, school of dentistry, department of clinical dentistry, area of integrated clinic, parnaíba, pi, brazil corresponding author: ana de lourdes sá de lira universidade estadual do piauí, faculdade de odontologia rua senador joaquim pires 2076 ininga. fone (86) 999595004 cep: 64049-590 teresina-pi-brasil email: anadelourdessl@hotmail.com received: january 17, 2019 accepted: may 25, 2019 anterior crossbite malocclusion: prevalence and treatment with afixed inclined plane orthodontic appliance ana de lourdes sá de lira1,*, guilherme henrique alves da fonseca1 aim: to evaluate the prevalence of anterior crossbite and to verify the effectiveness of the orthodontic appliance inclined plane in the correction of this malocclusion. methods: the clinical examination was performed 702 children in the deciduous or mixed dentition of 7 schools and in those found the anterior crossbite was performed treatment with fixed inclined plane. results: the prevalence of the anterior crossbite was 2.14%, characterizing 15 of the 702 children evaluated, of which 60% were female and 40% male, all of which were dental crossbites. only 12 accepted the treatment with an average duration of 4.4 weeks. conclusion: the prevalence of anterior crossbite was 2.14%. the inclined plane proved to be a viable and effective therapy in the correction of anterior crossbite. it is one of the options of the orthodontic treatment in patients in the deciduous or mixed dentition, propitiating greater possibility of dentoskeletal development, since the malocclusion is corrected. however this method needs to be correctly indicated and its execution technique rigorously followed. keywords: orthodontics. dentistry. pediatric dentistry. 2 lira et al. introduction anterior crossbites are malocclusions observed relatively frequently during the period of deciduous dentition and onset of mixed dentition, with a prevalence of around 7.6% of children1,2. it stands out due to its functional and aesthetic alterations, being defined as an abnormal lingual vestibular relation of one or more teeth of the maxilla in relation to the mandible, the two arches being in occlusion, with the anterior superior teeth occluding by lingual in relation to the inferior3. in functional anterior crossbite, or pseudoclasse iii, a protrusion of the mandible occurs during occlusion that can be attributed to inclinations of the upper incisors for palatine and vestibularization of the mandibular incisors, forcing an anterior positioning of the mandible. it can be caused by hypertrophy of the tonsils and or adenoids, digital sucking habits, pacifiers or even the upper lip. it can also be caused by an interference in the trajectory of the mandibular closure, where premature contacts during centric occlusion lead the child to adopt a deviant mandibular posture by accommodation4,5. in the case of anterior crossbite is observed the involvement of a single tooth or set of teeth, with the upper incisors inclined to the palate and / or the lower incisors to the vestibular, maintaining a good positioning of the apical bases in relation to the base of the cranium6. the skeletal anterior crossbite is characterized by mandibular protrusion, maxillary retrusion or the combination of both. it can be derived from the genetic inheritance of the individual, or due to an endocrine disorder, such as, for example, acromegaly7,8. early treatment of dental, functional or skeletal crossbite ensures that craniofacial development and occlusion occur normally, as obstacles are removed during the active phase of growth9,10. class iii malocclusion is of a relevant aesthetic and functional impairment. it is believed that early diagnosis, during deciduous or mixed dentition, and orthodontic intervention with one of the innumerable interceptive orthodontic appliances, specifically the inclined plane, will enable the harmonic growth of the jaws, even in skeletal class iii of functional or environmental origin, once the dental interference has been corrected. it is justified to perform this clinical research to prove that it is feasible to intercept the evolution of class iii malocclusion, be it functional, dental or skeletal, if the orthodontic intervention with specific devices, especially the inclined plane, is performed in deciduous dentitions or mixed, prior to the maxillary growth spurt. in view of the functional and aesthetic problems that the anterior crossbite can generate to the patient, the objective of this study was to evaluate the prevalence of anterior crossbite and to verify the efficacy of the inclined plan orthodontic appliance in the correction of this malocclusion. material and methods this study was approved by the research ethics committee of the state university of piauí cep / uespi, under number 2.199.979, being of the transverse type, 3 lira et al. non-random clinical intervention. it was a cross-sectional, quantitative, intervention study. the sample calculation was based on the target population: children between 3 and 5 years or between 6 and 8 years old, specifically in the school phase, of the city of parnaíba in 2017, totalizing 5.087 students. the estimated sample size was 702 children, who were selected, according to the inclusion criteria, as the population representative of the municipality of parnaíba-pi. the seventeen schools of the municipal network of the city of parnaíba-pi were chosen by lot to obtain the sample. this minimum number of participants is considered sufficient considering the proposed analyzes, the sampling error of 5%, and a 95% confidence level, indicating that the probability of the mistake made by the survey does not exceed 5%11. inclusion criteria were children in the complete deciduous dentition (between 3 and 5 years of age) and those in the initial phase of the mixed dentition, with one to eight permanent incisors (between 6 and 8 years of age) of both genders, with anterior crossbite, without posterior crossbite. children above this age range (even with only previous crossbite) and children presenting with posterior crossbite or other type of malocclusion associated with anterior crossbite were excluded from this study because orthodontic intervention in these cases is more complex and should be orthopedic and orthodontic, without indication of fixed inclined plane (fip), device to be used in the research. the researchers were trained in the clinical school of dentistry (ceo) of the state university of piauí by means of calibration exercises with 10 children not participating in the sample plan, who received dental care at the ceo during their routine operation. the training consisted of identifying the crossbite anterior by means of midline evaluation and clinical examination of the occlusion, observing if the upper incisors were occluding behind the lower incisors and this analysis according to the methodology described by peres et al.11. first, a superficial clinical examination of the children’s occlusion was performed, observing whether the upper incisors were occluding behind the lower incisors, characterizing the anterior crossbite. in children in whom this situation was present, a thorough examination was performed to classify the crossbite into functional, dental or skeletal. the midline evaluation was performed because possible functional deviations could occur during complete dental occlusion. for the diagnosis of functional cross-bite in maximal habitual intercuspation (mih), the patient presents a class iii dental relationship with anterior crossbite, while in the centric relation (cr), a class i interarcos relationship (normoclusion) , with top relationship between the upper and lower incisors. the inclination of the upper incisors to the lingual and / or lower incisors to the vestibular are the main factors responsible for the occlusal interference that lead the patient to occlude with the mandible designed for anterior12. in cases of dental anterior crossbite, a localized inclination of one or more teeth is observed at the level of the alveolar process, without affecting the size or shape of the bone bases. the teeth are not centralized in the alveolar process and the most important diagnostic factor is an asymmetry of the dentoalveolar arch. in cases of skeletal 4 lira et al. crossbite, the concave profile is observed, deficiency of the maxilla associated or not to the excessive jaw in relation to the skull and anterior crossbite. for the three types of anterior crossbite mentioned, the fip was implanted because it would correct the dental relationships in the anteroposterior direction, favoring the normal development of the bone bases down and forward. after a conversation with the parents and the explanation about the treatment alternatives, the reasons for choosing the fip, which were due to the patient’s age and the patient’s cooperation difficulty, fast result and low cost. the pilot study involving 10 children participating in the sample had the objective of testing the proposed methodology. as a result, its viability was observed without adjustments. to measure intraand inter-examiner diagnostic reproducibility, 10% of the total sample was double checked by each of the examiners, with the kappa coefficient for intraand inter-examiner agreement of 0.98 and 0.99, respectively. the children who presented the malocclusion were referred to the orthodontics department of the clinical school of dentistry (ceo) of the state university of piauí (uespi) for their correct treatment. from each patient, two periapical radiographs were taken from the upper and lower incisors, respectively, to visualize the germs of the permanent successors and to verify if there was any extra tooth included in the region of the incisors (mesiodentes) and if present, it would be extracted prior to placement the appliance. for the confection of the appliance, a pair of working models was obtained for each patient. the lower model was isolated with thin layer of wax utility and then applied a layer of self-curing acrylic resin on the middle and incisal thirds, buccal and lingual side of canine to lower canine at a 45 ° angle, without making contact with the gingival tissue to prevent inflammation (figure 1). subsequently, the finishing and polishing of the appliance was performed12. for each tooth to be uncrossed, two teeth were used as support in the lower arch. for the installation the “say-show-do” conditioning technique was used, obtaining, in this way, patient acceptance and collaboration. then, the adjustment of the apparatus was carried out and soon after cementation with glass ionomer, due to its advantages figure 1. appliance used in the research. 5 lira et al. such as fluoride release, good adhesion, biological compatibility, low volumetric and thermal coefficient of change, low solubility in the mouth. during the cementation, relative insulation and suction system were performed, essential for an effective cementation of the fip12,13. the children returned to the clinic for control examination on a weekly basis. if it were observed that the bite had not yet been uncrossed, wear on the vestibular of the apparatus could be accomplished by maintaining the 45 degree angulation so that only the tooth (s) to be uncrossed touched the acrylic. removal of the tooth would occur after the uncrossing and the realignment of the teeth, using as a criterion the obtainment of 2mm of overjet13. the spss statistical package, version 23, was used to calculate proportion and percentage measures to characterize the prevalence. the degree of association between the prevalence between genders was determined using the chi-square test and the comparison between groups with mann-whitney test and kruskal-wallis test, considering a significance level of 5%. results the prevalence of anterior dental crossbite was 2.14%, characterizing 15 of the 702 children evaluated, with mean and standard deviation of age of 5.3 years ± 2.2, which 60.11% were female (n= 422) and 39.89% male (n=280) with mean and standard deviation of age of 5.5 years ± 2.3 for female and 5.1 years ± 2 for male respectively. table 1 shows data on treated children. with the result of the statistical calculation χ² (chi-square), χ² (1) = 0.601, p = 0.44, it was verified that there was no difference of statistically significant association between the genders of the participants. table 1. data of children who placed the plane inclined device case gender age crossed incisors treatment time (weeks) 1 male 5 anos 51, 52, 61, 62 4 2 male 8 anos 11, 21 4 3 female 5 anos 51, 52, 61, 62 3 4 male 7 anos 11, 21 5 5 female 8 anos 11, 21 5 6 female 5 anos 51, 52, 61, 62 4 7 female 8 anos 11 6 8 male 4 anos 51, 52, 61, 62 4 9 female 4 anos 51, 52, 61, 62 4 10 female 8 anos 11, 21 5 11 female 8 anos 11 6 12 male 4 anos 51, 52, 61, 62 4 6 lira et al. among the 12 children who were treated, there were no differences between genders, chi-square, χ² (1) = 0.331, p = 0.56, considering a significance level of 5% with mean age of 5.6 years for male gender and of 5.8 year for female gender . as for the treatment time, the chi-square test was also performed, and it was verified that no statistically significant differences were also found (χ² (chi-square), χ² (1) = 0.601, p = 0.44), even estimating that 50% of the cases were treated in 4 weeks, 33.3% in 5 weeks, 8.3% in 6 weeks and 8.3% also in 3 weeks. a non-parametric mann-whitney test was performed, which found that there was no statistically significant difference between the genders and the treatment time (u=15, p = 0.65). the kruskal-wallis non-parametric test showed statistically significant differences between the three groups of crossed teeth (p = 0.02), suggesting that the group with 1 crossed tooth was the one that took the most time to correct (9.5 weeks), followed by 2 teeth (8.8 weeks) and 4 teeth (3.9 weeks) respectively (table 2). discussion in this cross-sectional study, a prevalence of lower anterior crossbite of 2.14% was observed. this data corroborates those found in the studies of morais et al.1 (2014) and fernandes et al.13 (2007), whose prevalence was 2.2% and 3.10% respectively. the prevalence of anterior crossbite has been shown to be lower than that of posterior crossbite13-15. this fact is understandable by the complex etiological factor of the previous crossed bites that according to lee15 (1978) is the result of traumatic injuries, bone sclerosis or fibrous tissue barrier, inadequate bow length and upper lip interposition habits. when analyzing the gender in this study there was a higher prevalence for the female gender (60%). this finding distances itself from that found by woitchunas et al.14 (2001), which presented a tendency for males (56%). regarding the treatment of this malocclusion, dias et al.16 (2018) affirmed that the performance of the pediatric dentist in the early diagnosis constitutes a clinical instrument of relevance because it allows the interceptive treatment, besides minimizing future damages to the patients. table 2. variables studied in the 12 treated children variables test valor p valor significance difference between the genders chi-square test χ² (1) = 0.33 0.56 n.s treatment time chi-square test χ² (1) = 0.60 0.44 n.s relationship between treatment time and gender mann-whitney test u= 0.15 0.65 n.s relationship between number of corrected teeth and time of treatment kruskal-wallis test 1 tooth (9.5 weeks) 2 teeth (8.8 weeks) 4 teeth (3.9 weeks) 0.02 * n.s (not significant); *(p ≤ 05) 7 lira et al. for figueiredo et al.10 (2014) a good treatment option is given with the flat inclined flat appliance because it is a quick and low cost technique, corroborating this work, which was adopted in 12 schoolchildren in the deciduous dentures and mixed. when the treatment time was analyzed, half of the cases (6) were corrected in 4 weeks, 33.3% (4 cases) in 5 weeks, 8.3% (1 case) in 3 weeks and 8.3 (1 case) at 6 weeks, differing from manjarrés and silva17 (2017) who corrected ten cases of anterior crossbite at 7 weeks. analyzing the groups of teeth that were crossed in this study it was verified that the treatment became faster in descending order in the cases, where four crossed teeth were found, followed by the two teeth crossed and with a crossed tooth. this fact suggest that the more crossed teeth are supported on the plateau of the inclined plane the shorter the treatment time. prakash and durgesh18 (2011) treated two cases, the first with a crossed incisor tooth and the second with four crossed incisors in three weeks. dias et al.16 (2018), when treating a crossbite that affected a group of six teeth (53 to 63), obtained a four-week uncrossing and araujo et al.19 (2017) when treating a single-crossover case tooth got its complete uncrossing in 2 weeks. these findings show that the treatment time is relative to each patient, but it is configured as a rapid treatment. the treatment of the anterior crossbite should be started as soon as possible, so that it allows adequate growth of the jaws and correct dental positioning. the treated cases have been followed up and no relapse was observed. guzzo et al.20 (2014) elucidated that 51.6% of the interviewees in the city of florianopolis-sc considered the correction of crossbite at the basic health units (bhu) necessary. it was concluded that the prevalence of anterior crossbite was 2.14%. the inclined plane proved to be a viable and effective therapy in the correction of anterior crossbite. it is one of the options of the orthodontic treatment in patients in the deciduous or mixed dentition, propitiating greater possibility of dentoskeletal development, since the malocclusion is corrected. because it is low cost, easy to make, effective and with reduced treatment time, it can be used in undergraduate courses and in ubs. however this method needs to be correctly indicated and its execution technique rigorously followed. references 1. moraes spt, mota ela, amorim ldaf. [factors associated with the incidence of malocclusion in the deciduous dentition of children in a public hospital cohort from northeast brazil]. rev bras saude mater infant. 2014;14(4):371-82. doi: 10.1590/s1519-38292014000400007. portuguese. 2. pino román im, concepción olv, garcia vega pa. [malocclusions according to the dental aesthetic index in seventhgrade students from santa clara]. rev cient villa clara. 2014;18(4):177-9. spanish. 3. manoharan m, disha p, nagaveni nb, roshan nm, poornima p. correction of anterior crossbite with different approaches: a series of three cases. int j oral health med res. 2016 sep-oct;3(3):41-3. 4. paul s, rao a, chalakkal p, mony s. fixed appliance with expansion scren for the treatment of primary anterior crossbite. j clin diagn res. 2016 may;10(5):zd03-4. doi: 10.7860/jcdr/2016/17986.7752. 8 lira et al. 5. wiedel ap, bondemark l. fixed versus removable orthodontic appliances to correct anterior crossbite in the mixed dentition a randomized controlled trial. eur j orthod. 2015 apr; 37(2):123-7. doi:10.1093/ejo/cju005. 6. leite kcfl, pires skc, leite fcf, benetti pvn, barbosa, olc, barbosa ccn. [intercept of anterior crossbite in the deciduos dentition using fixed inclined plane: case report]. bjscr. 2017 jun-aug;19(1):96-100. portuguese. 7. de clerck hj, proffit wr. growth modification of the face: a current perspective with emphasis on class iii treatment. am j orthod dentofacial orthop. 2015 jul; 148(1):37-46. doi: 10.1016/j.ajodo.2015.04.017. 8. oltramari pvp, garib dg, conti accf, henriques jfc, freitas mr. [orthopedical treatment of class iii in different facial patterns]. rev dent press ortod ortop facial. 2005 sep-oct;10(5):72-82. portuguese. doi:10.1590/s1415-54192005000500008. 9. oliveira ple, emmerich a. [the early diagnosis importance in the treatment of angle class iii malocclusion]. rev bras pesq saude. 2010;2(2):75-81. portuguese. 10. figueiredo pba, ferraz rp, silva vc, pinheiro junior jm, silva arq, silva ai. [treatment with inclined plane for anterior crossbite: clinical case report]. rfo. 2014;19(2):229-33. portuguese. 11. peres ma, traebert j, marcenes w. [calibration of examiners for dental caries epidemiology studies]. cad saude publica 2001; 17(1):153-59. portuguese. 12. 12.tanaka om, maciel jvb, kreia tb, ávila alr, pithon mm. the anterior dental crossbite: the paradigm of interception in orthodontics. rev clin pesq odontol. 2016;6(1):71-8. 13. 13.fernandes kp, amaral mat, monico ma. [malocclusion and the need for its orthodontic treatment in deciduous dentition]. rgo. 2007 jul/set;53(3):223-7. portuguese. 14. 14.woitchunas dr, busato c, tocheto lr, brockstedt rh. [anterior cross-bite: diagnosis and treatment of pseudoclass iii clinical case report]. rev fac odontol upe. 2001;6(2):23-8. doi: 10.5335/rfo.v6i2.1572. portuguese. 15. 15.lee bd. correction of cross-bite. dent clin north am. 1978;22(4):647-8. 16. 16.dias gf, alberton lp, santos mb, fernandes knt, alves fbt. [the relevance of the role of pediatric dentistry in the diagnosis and early treatment of the anterior cross bite in children case report]. rev odontol araçatuba. 2018 may-aug;39(2):47-53. portuguese. 17. 17.manjarrés cr, silva jah. [treatment of anterior cross bite with anterior inclined plane. effector on the dental arches]. rev odontop latinoameric. 2017;7(1):44-53. spanish. 18. 18.prakash p, durgesh bh. anterior crossbite correction in early mixed dentition period using catlan’s appliance: a case report. isrn dent. 2011;29(8):1-5. doi: 10.5402/2011/298931. 19. 19.araujo ec, silva te, santos rss, brum sc, barbosa ccn, barbosa olc. [previous cross bite treatment using pif: a vibrant aternative case report]. braz j surg clin research. 2017 sep-nov;20(2):116-119. portuguese. 20. 20.guzzo sc, finkler m, junior cr, reibnitz mt. [preventive and interceptive orthodontics in the primary health care network of the brazilian unified health system: the viewpoint of the dental surgeons of florianopolis city hall]. cienc saud colet. 2014 jan;19(2):449-60. doi:10.1590/1413-81232014192.22852012. portuguese. https://doi.org/10.1093/ejo/cju005 1http://dx.doi.org/10.20396/bjos.v19i0.8656977 volume 19 2020 e206977 original article 1 department of preventive and social dentistry, federal university of rio grande do sul, porto alegre, rs, brazil. 2 federal university of pelotas, pelotas, rs, brazil. 3 department of stomatology, federal university of de santa maria (ufsm), santa maria, rs, brazil. 4 dental materials laboratory, school of dentistry, federal university of rio grande do sul, porto alegre, rs, brazil. 5 department of periodontology, federal university of rio grande do sul, porto alegre, rs, brazil. corresponding author: roger keller celeste department of preventive and social dentistry, federal university of rio grande do sul, porto alegre, rs, brazil. email: roger.keller@ufrgs.br received: october 06, 2019 accepted: december 02, 2019 brazilian dentistry research productivity: state level socioeconomic, educational and structural factors roger keller celeste1,* , francisco wilker mustafa gomes muniz2, thiago machado ardenghi3, fabrício mezzomo collares4, cassiano kuchenbecker rösing5 aim: to explore socioeconomic, educational and research factors associated with dental research productivity at the state level in brazil. methods: the authors used the scopus database to identify dental articles published from 2006 to 2016 associated with brazilian universities at the state level. several social, economic, educational and research structure variables were obtained from the census and national research council to predict the rate of articles per 100 thousand inhabitants among the 27 brazilian states. rates were fitted in linear weighted least-squared regression with stepwise technique. twenty-two variables were grouped in six blocks (social, economic, general education, dental education, research workforce and structure). results: a total of 21189 articles were published, and the state of são paulo accounted for 46%, followed by rio grande do sul with 9.4%; four states did not publish any articles. there were an average (± standard deviation) of 2.6 (±1.98) published articles per 100 researchers and 13.4 (±9.6) articles per 100 thousand inhabitants. research structure and workforce explained 92.4% and 87.2% of state variability, respectively, while the final model explained 94.5%. one extra phd and one extra undergraduate researcher per 100 thousand inhabitants were associated with 11.3 more and 3.5 fewer articles, respectively, while every 10 points (range 0-100) on the human development index (education component) was associated with 3.3 more articles. conclusion: state scientific output has several associated factors, but research workforce and general education variables seem to be good predictors. large disparities among state research outputs have been described and must be addressed by research and development policies. keywords: bibliometrics. dentistry. research. science. https://orcid.org/0000-0002-2468-6655 2 celeste et al. introduction sustainable development goals in relation to educational processes highlight the necessity of expansion in higher education and scientific programmes, especially in less developed countries1. one of the reasons is that investment in science and technology has long been accepted as a way to generate knowledge and a cornerstone of social and economic development2. regarding investment in research and development (r&d), brazil, an upper middle-income country, has fallen behind the average for upper income countries, with only 1.2% of gross domestic product (gdp) expenditures and 698 full-time researchers per million inhabitants3. on the other hand, estimates related to dental research in brazil have been recognized as outstanding, and current brazilian research productivity is higher than many high-income countries4-7 as it has a strong increment since late 1990’s8. research productivity as an output of investments has been assessed based on the number of scientific articles. the main determinants have been studied at individual (researcher) and institutional/organizational levels9. for example, male researchers of higher rank and those awarded large research grants were reported to have higher productivity in the usa10, although younger researchers supervising graduate students tend to publish more in saudi arabia11. also, higher education institutions (hei) have pivotal roles, as they account for 23.7% of r&d expenditure and 64.3% of all scientific publications2. an important fraction of research from hei comes from graduate programs; hence, it is not surprising that the ratio of graduate students to staff has been described as an important factor for departmental productivity12. few studies have assessed the impact of undergraduate students in these figures; however results show some effect on faculty productivity13. on a macro-level, gdp has also been associated with performance14 and productivity, and the human development index (hdi) an important predictor of country rate of article publication7. although it could be hypothesised that socioeconomic factors are associated with research productivity in brazil, it is unclear if this association will remain after controlling for direct factors such as the rate of researchers per inhabitants. in addition, no study has described this phenomenon in the brazilian context, which will be important in explaining state differences. in the mid-1990s, brazil implemented an evaluation system for graduate study programs with a strong emphasis on scientific productivity in high-impact journals. this policy was proposed from a national perspective with few incentives for state-level research agencies, with some exceptions. variability among brazilian states has not been described or explained to the best of our knowledge. however, analysing such variability will foster equitable development and should be pursued, as the capacity to produce contextualized knowledge at local levels is key for sustainable development. in addition, taking the continental size of brazil into consideration, understanding local/state conditions may shed some light on possible contextual predictors affecting scientific productivity elsewhere. therefore, the objectives of this study are to explore socioeconomic, educational and specific research factors associated with state-level output for brazilian dental research. 3 celeste et al. materials and methods this is an ecological study in which the units of observation were all 27 brazilian states. the number of published articles (dependent variable) was obtained from the scopus database, and the other 20 potential predictors were obtained from several sources. scopus was chosen because it is used by the brazilian higher education assessment council (capes) to assess institutional proposals. it also allows the identification of authors’ institutional addresses, making it possible to count the number of articles per state. outcome variable the dependent variable was the rate of articles per 100 thousand inhabitants at the state level. this was established by dividing the total number of articles from each state in an 11-year-period (2006-2016) by their population according to the 2010 census. the number of publications was retrieved by combining a search strategy for dental articles with an additional search for universities with undergraduate programs in dentistry (using authors’ address identification filters at scopus). the search strategy used to identify dental articles was obtained from a previous publication5 and is available only in the original publication. the search to identify universities was based on the names of 219 undergraduate programs available on the website of the federal dental council (cfo). universities with more than one program in the same state counted as one, because such differences are not distinguished by scopus. independent variables seven social and economic variables were obtained from the instituto brasileiro de geografia e estatísica and atlas de desenvolvimento humano do brasil15. they are based on the 2010 census data: gini coefficient of income inequality, proportion of individuals with inadequate sanitation, proportion of individuals living in urban areas, hdi, mean per capita income, proportion of individuals living in poverty and gdp per capita. those variables are considered distal contextual factors that may indirectly affect research productivity. we selected five variables that represent the general and dental educational context at the state level. the mean number of years of education at the age of 18 and the education component of the hdi were obtained from atlas de desenvolvimento humano do brasil. the rate of dental schools with undergraduate programs per million inhabitants was calculated using the number of programs available from the cfo website. two other variables were obtained from official government data (instituto nacional de estudos e pesquisas educacionais anísio teixeira [inep]) from http:// inep.gov.br/enade: mean score on the national board for final-year dental students (years: 2007, 2010, 2013) and percentage of members of dental school teaching staff holding a phd. finally, we collated data to calculate eight variables concerning research structure and workforce. based on information from the research-groups census (2010) for different types of researchers at the national research council (cnpq) website (http://dgp. cnpq.br/planotabular/index.jsp), we created eight rates per state level inhabitants: rate http://inep.gov.br/enade http://inep.gov.br/enade http://dgp.cnpq.br/planotabular/index.jsp http://dgp.cnpq.br/planotabular/index.jsp 4 celeste et al. of phd researchers, rate of phd students, rate of undergraduate students in research, rate of research groups and research lines. data from the research-groups census (2010) are provided by group leaders and certified by their institutions. data about the number of graduate programs in dentistry (master and phd levels) for each state was obtained from the capes website (https://sucupira.capes.gov.br/sucupira/) and overall, there were 101 active graduate programs. statistical analysis descriptive data on the overall profiles of articles were presented by institution, journal and country of co-authors. bivariate analyses were presented with categorized covariates, and differences were tested using the kruskal-wallis non-parametric rank test. categorization was necessary for descriptive purposes, and states were grouped in tertiles in the case of a gradient or median/specific cut-off point if the bivariate relation was non-linear. the rate of publication was modelled using linear regression with ordinary weighted least squares by population size16. all 20 variables were grouped in 6 blocks (see table 2) and modelled using a within-block stepwise forward technique, taking p < 0.20 as a variable to enter into the model. those significant variables within each block were transferred to the full model, also modelled with a stepwise forward technique with p < 0.10 to enter into the model. this two-step approach was needed due to the high degree of collinearity among variables; the final model was evaluated based on r-squared fit index, variance inflator factor (vif), homoscedasticity (cook-weisberg test for heteroscedasticity) and normality of residuals (shapiro-wilk test). in multiple linear regression, variables were entered into the model as continuous and non-categorized. all analyses were carried out using stata 13.1. results in an 11-year period, brazilian dental researchers published 21189 articles indexed in the scopus database. the state of são paulo accounted for 46% of all articles (n = 11767), followed by rio grande do sul with 9.4% (n = 2395). four states (acre, roraima, amapá and rondônia) did not publish any articles. overall, during the study period (2006-2016), there were an average (± standard deviation) of 2.6 (±1.98) published articles per 100 researchers and 13.4 (±9.6) articles per 100 thousand inhabitants. the state with the highest rate was são paulo, with 28.5 articles per 100 thousand inhabitants, followed by rio grande do sul (22.4), rio grande do norte (15.0), paraíba (14.9) and paraná (14.8). productivity per 100 researchers was highest in the federal district with 17.1 articles per 100 researchers, followed by mato grosso (6.1), ceará (3.6), sergipe (3.3) and five states (maranhão, santa catarina, goias, rio de janeiro and são paulo) with 3.1. articles are concentrated in a limited number of journals, institutions and co-autors’ countries. the top 10 journals accounted for 24.5% of all articles published by brazilian researchers (table 1), and brazilian journals summed 6 of the top 10. while the whole list reaches almost 1000 journals, about 150 journals published 80% of all papers. the top 10 institutions were associated with 80.4% of all published articles: universidade de são paulo published 28.2% (the three campuses cannot be distinhttps://sucupira.capes.gov.br/sucupira/ 5 celeste et al. guished in scopus), followed by unesp (the same as universidade de são paulo, n = 3) with 14.7% and unicamp with 12.8%. the top 10 co-author countries summed table 1. percentages of articles published (n=21189) with at least one brazilian author among journals indexed in scopus between 2006 and 2016. publication institution n % universidade de sao paulo usp 5966 28.2% universidade estadual paulista – unesp 3114 14.7% universidade estadual de campinas 2720 12.8% universidade federal do rio de janeiro 1029 4.9% universidade federal de minas gerais 969 4.6% universidade federal do rio grande do sul 867 4.1% universidade federal de pelotas 656 3.1% universidade federal de santa catarina 614 2.9% universidade federal fluminense 560 2.6% universidade federal de sao paulo 546 2.6% subtotal 17041 80.4% total 21189 100.00% country united states 2229 10.5% italy 492 2.3% united kingdom 455 2.1% canada 437 2.1% germany 266 1.3% switzerland 255 1.2% spain 249 1.2% netherlands 203 1.0% sweden 169 0.8% france 155 0.7% subtotal 4910 23.2% total 21189 100.00% journals brazilian dental journal 870 4.1% journal of applied oral science 709 3.3% dental press journal of orthodontics 625 2.9% journal of endodontics 617 2.9% brazilian oral research 569 2.7% pesquisa brasileira em odontopediatria e clinica integrada 401 1.9% brazilian journal of oral sciences 373 1.8% american journal of orthodontics and dentofacial orthopedics 346 1.6% journal of periodontology 344 1.6% operative dentistry 344 1.6% subtotal 5198 24.5% total 21189 100.00% 6 celeste et al. table 2. mean rate of articles per 100 thousand inhabitant in the period of 2006-2016 among brazilian states (n=27). mean std. dev. n p-value* social indicators gini coefficient lower level 14.47 6.93 5 0.15 middle level 16.92 10.35 12 higher level 5.12 2.94 10 proportion of people with inadequate sanitation up to 10% 15.99 9.58 15 0.03 11% or more 5.54 3.83 12 urbanization lower half 5.49 4.23 13 0.07 upper half 16.69 9.35 14 idh lower half 5.79 3.84 13 0.48 upper half 17.47 9.31 14 economic indicators mean individual income lower half 5.81 3.82 13 0.51 upper half 17.44 9.34 14 proportion of poverty up to 10% 17.81 9.16 11 0.02 11% or more 5.60 3.87 16 gdp per capita lower tertile 5.29 3.19 9 0.10 middle tertitle 8.64 4.05 9 highest tertile 20.03 9.02 9 general education mean number of year of education lower tertile 7.14 4.52 9 0.18 middle tertitle 8.34 4.52 9 highest tertile 19.88 9.95 9 hdi education component lower tertile 5.06 3.75 9 0.01 middle tertitle 5.99 4.02 9 highest tertile 18.49 8.84 9 dental education % of undergrad lectures with phd lower level 5.15 4.27 9 <0.01 middle level 8.21 4.21 9 higher level 20.18 9.28 9 rate of dental schools per 1 million inhabit <=1 school 5.67 3.09 9 0.76 >1 school 16.93 9.48 18 mean enad score 2007-2013 <=2 points 2.40 2.20 5 <0.01 >2 points 13.85 9.52 22 research structure graduate programs per million inhabit lower tertile 6.24 5.06 9 <0.01 middle tertitle 4.92 1.45 9 highest tertile 19.34 8.45 9 research groups per 100 thousand inhabit lower tertile 4.39 3.70 9 <0.01 middle tertitle 6.88 3.79 9 highest tertile 19.35 9.05 9 research lines per 100 thousand inhabit lower tertile 4.65 3.78 9 <0.01 middle tertitle 7.65 3.92 9 highest tertile 21.45 8.53 9 research workforce phd researchers per 100 thousand inhabit lower tertile 4.35 3.51 9 <0.01 middle tertitle 6.28 3.15 9 highest tertile 20.03 8.23 9 graduate student researchers per 100 thousand inhabit lower tertile 4.10 3.84 9 <0.01 middle tertitle 4.75 1.96 9 highest tertile 18.61 8.44 9 undergraduate researchers per 100 thousand inhabit upper half 4.48 2.79 13 <0.01 lower half 16.15 9.34 14 % of researcher >50 year-old upper half 7.53 4.53 11 0.76 lower half 16.16 10.07 12 % of male researchers upper half 7.71 4.45 11 0.80 lower half 16.14 10.15 12 total 13.40 9.57 27 * kruskal-wallis ranking test 7 celeste et al. 23.2% out of 133 countries; the usa accounted for 10.5%, followed by italy with 2.3% and the uk/canada with 2.1% each. in bivariate analysis, several variables were significantly related to state productivity and showed high degrees of correlation (table 2 and table 3). nonetheless, in the final regression model, only three variables explained 94.5% of state variability (table 4): the educational component of the hdi, rate of undergraduate students in research and rate of phd researchers. every 10 points in the educational component of hdi was associated with 3.3 more articles per 100 thousand inhabitants (95% confidence interval – 1.0: 5.5), while every additional phd researcher per 100 thousand inhabitants was associated with 11.3 more articles (95% confidence interval – 8.8: 13.8), and one additional undergraduate researcher was associated with 3.5 fewer articles (95% confidence interval – 6.2: -0.7). in the final model (table 4), no variables were heteroscedastic; the highest vif was associated with the rate of phds (vif = 4.9). graphic analysis of residuals showed that they were normally distributed (shapiro-wilk test, p = 0.06). discussion state scientific output has several associated social and economic factors, but three seem to be good predictors: rate of phd researchers, rate of undergraduate students (involved in research) and general education level (hdi-education). in addition, our findings showed striking state disparities in total research output. taking the size of brazil into consideration, as this mirror other large disparities in social, economic and cultural aspects. the use of such associations is of interest to understand which factors can predict better or worse research productivity rates. only three variables remained in the final model, and the most influential was the rate of phd researchers, confirming a previous study12, followed by the educational component of hdi and rate of undergraduate students involved in research. the rate of phd researchers was highly correlated to other variables and may have affected some of them. for example, graduate programs educate phds and may be indirectly responsible for their scientific output. furthermore, the presence of a graduate program is an interesting indicator of phd students, research grants and other resources, such as laboratory infrastructure. states with lower levels of competitiveness may fall behind and try to offset with more undergraduate researchers than expected. in contrast to our results, another study showed that undergraduate students may increase overall productivity13. nonetheless, such papers may be published in journals not indexed by scopus and thus did not appear in our work. to our knowledge, this is the first study to include social and educational indicators, with hdi-education showing a statistically significant effect. we speculate that it may have a direct effect on research productivity by boosting critical thinking in lower education, but it is also likely to be a general marker of social development and investments in education at basic and higher levels. state disparities were found in total research output, with são paulo having 46% of all papers, as the university of são paulo (usp) accounts for 28% of the whole country. usp’s superiority over other brazilian institutions has also been confirmed in previ8 celeste et al. ta bl e 3. s pe ar m an c or re la tio n co effi ci en t m at rix in cl ud in g al l v ar ia bl es a m on g b ra zi lia n st at es (n =2 7) . v 1 v 2 v 3 v 4 v 5 v 6 v 7 v 8 v 9 v 10 v 11 v 12 v 13 v 14 v 15 v 16 v 17 v 18 v 19 v 20 v 21 v 22 v 1 a rt ic le r at e pe r 1 00 th ou sa nd /h ab 1 v 2 g in i -0 .3 7 1 v 3 in ad eq ua te s an ita tio n -0 .6 2 0. 66 1 v 4 u rb an iz at io n 0. 40 -0 .5 1 -0 .7 0 1 v 5 h d i 0. 42 -0 .6 3 -0 .8 3 0. 89 1 v 6 % o f p ov er ty -0 .4 8 0. 72 0. 86 -0 .8 3 -0 .9 6 1 v 7 m ea n in di vi du al in co m e 0. 40 -0 .6 1 -0 .8 1 0. 87 0. 98 -0 .9 7 1 v 8 g n p p er c ap ita 0. 38 -0 .5 4 -0 .7 7 0. 83 0. 95 -0 .9 2 0. 97 1 v 9 h d i-e du ca tio n 0. 42 -0 .6 3 -0 .8 1 0. 87 0. 98 -0 .9 2 0. 93 0. 89 1 v 10 m ea n ye ar s of ed uc at io n 0. 46 -0 .7 1 -0 .7 2 0. 62 0. 69 -0 .7 0 0. 61 0. 52 0. 73 1 v 11 m ea n en a d s co re 0. 39 -0 .2 1 -0 .2 1 -0 .0 8 -0 .0 4 -0 .0 4 -0 .0 8 -0 .0 6 -0 .0 3 0. 24 1 v 12 r at e of d en ta l s ch oo ls -0 .2 2 -0 .0 9 -0 .1 4 0. 33 0. 39 -0 .3 4 0. 41 0. 40 0. 32 0. 19 -0 .5 9 1 v 13 % o f p hd in u nd er gr ad pr og ra m s 0. 67 -0 .0 4 -0 .3 1 0. 30 0. 20 -0 .2 4 0. 18 0. 15 0. 19 0. 30 0. 54 -0 .3 8 1 v 14 r at e of g ra du at e pr og ra m s 0. 72 -0 .3 5 -0 .3 8 0. 22 0. 12 -0 .2 0 0. 15 0. 16 0. 11 0. 25 0. 38 -0 .3 0 0. 56 1 v 15 r at e of r es ea rc h li ne s 0. 82 -0 .2 6 -0 .4 2 0. 17 0. 15 -0 .2 1 0. 16 0. 19 0. 11 0. 23 0. 40 -0 .2 3 0. 52 0. 84 1 v 16 r at e of r es ea rc h g ro up s 0. 79 -0 .2 9 -0 .4 8 0. 27 0. 21 -0 .2 6 0. 21 0. 22 0. 15 0. 29 0. 40 -0 .1 8 0. 50 0. 77 0. 93 1 v 17 r at e of r es ea rc he rs (t ot al ) 0. 82 -0 .2 6 -0 .4 2 0. 17 0. 15 -0 .2 1 0. 16 0. 19 0. 11 0. 23 0. 40 -0 .2 3 0. 52 0. 84 1. 00 0. 93 1 v 18 r at e of p hd r es ea rc he rs 0. 86 -0 .3 9 -0 .5 1 0. 27 0. 24 -0 .3 4 0. 26 0. 26 0. 22 0. 30 0. 54 -0 .3 5 0. 66 0. 89 0. 94 0. 87 0. 94 1 v 19 r at e of u nd er gr ad ua te r es ea rc he rs 0. 76 -0 .1 5 -0 .3 2 0. 14 0. 01 -0 .1 1 0. 04 0. 02 -0 .0 4 0. 15 0. 46 -0 .3 1 0. 57 0. 77 0. 85 0. 90 0. 85 0. 84 1 v 20 r at e of p hd s tu de nt s 0. 85 -0 .4 7 -0 .5 6 0. 36 0. 34 -0 .4 0 0. 32 0. 29 0. 32 0. 41 0. 53 -0 .3 3 0. 61 0. 77 0. 87 0. 87 0. 87 0. 92 0. 84 1 v 21 % o f m al e r es ea rc he rs -0 .1 0 -0 .3 6 -0 .1 0 0. 09 -0 .0 1 -0 .0 1 -0 .0 2 0. 01 -0 .0 6 0. 07 -0 .0 4 -0 .1 0 -0 .1 6 0. 31 0. 03 0. 19 0. 03 0. 06 0. 15 0. 04 1 v 22 % o f s en io r r es ea rc he rs 0. 00 -0 .1 5 -0 .2 0 0. 24 0. 08 -0 .1 3 0. 12 0. 15 0. 06 -0 .0 3 0. 26 -0 .3 1 0. 20 0. 33 0. 24 0. 31 0. 24 0. 41 0. 30 0. 34 0. 39 1 n o te : i n sh ad e co effi ci en ts o f v ar ia bl es re la te d to s am e th eo re tic al b lo ck 9 celeste et al. ta bl e 4. c oe ffi ci en ts fr om li ne ar re gr es si on m od el s of a rt ic le s pe r 1 00 0 00 /i nh ab ita nt s am on g b ra zi lia n st at es (n =2 7) , 2 00 620 16 . b lo ck v ar ia bl e w ith in b lo ck s te pw is e* re gr es si on fi na l m od el s te pw is e* re gr es si on co effi ci en t (i c 95 % ) ad ju st ed r 2 co effi ci en t (i c 95 % ) ad ju st ed r 2 so ci al in di ca to rs h d i* * (e ve ry 1 0 po in ts in cr ea se ) 14 .2 (9 .5 : 19 .0 ) 58 .6 % 94 .5 % ec on om ic in di ca to rs m ea n in di vi du al in co m e (e ve ry r $1 00 0 in cr ea se ) 25 .8 (1 6. 4 : 3 5. 1) 54 .4 % g en er al e du ca tio n in di ca to rs h d i* * ed uc at io n co m po ne nt (e ve ry 1 0 po in ts in cr ea se ) 11 .9 (8 .2 : 15 .5 ) 62 .4 % 3. 3 (1 .0 : 5. 5) d en ta l e du ca tio n in di ca to rs % o f u nd er gr ad le ct ur es w ith p hd (e ve ry 1 0 pe rc en t p oi nt s in cr ea se ) 7. 1 (5 .1 9. 1) 76 .8 % r at e of d en ta l s ch oo ls p er 1 m ill io n in ha bi t ( >1 s ch oo l) 9. 2 (5 .2 : 13 .2 ) r es ea rc h st ru ct ur e in di ca to rs r es ea rc h gr ou ps p er 1 00 th ou sa nd in ha bi t ( ev er y on e m or e) 35 .0 (2 0. 4 4 9. 6) 87 .2 % g ra du at e pr og ra m s pe r 1 m ill io n in ha bi t ( ev er y on e m or e) 8. 4 (1 .1 : 15 .8 ) r es ea rc h w or kf or ce p hd r es ea rc he rs p er 1 00 th ou sa nd in ha bi t ( ev er y on e m or e) 14 .2 (1 2. 1 : 1 6. 2) 92 .4 % 11 .3 (8 .8 : 13 .8 ) u nd er gr ad ua te re se ar ch er s pe r 1 00 th ou sa nd in ha bi t ( ev er y on e m or e) -5 .7 (8. 5 : 2. 9) -3 .5 (6. 2 : 0. 7) * fo rw ar d st ep w is e re gr es si on w ith e nt ry v al ue p <0 .1 0 (o rd in ar y w ei gh te d le as tsq ua re s by p op ul at io n si ze ) ** h d i= h um an d ev el op m en t i nd ex v ar ie s fr om 0 to 1 00 . 10 celeste et al. ous studies5,17, and it has been estimated to contribute more than 20% in all research areas18. indeed, são paulo is the only state in brazil where the dentistry field is the most productive in all research fields in brazil18; therefore, state differences are likely to be larger in dentistry than other areas. a similar concentration of publications in a few places has been reported in the african continent, where nigeria and south africa account for over two-thirds of all oral health-related research19. on one hand, são paulo has the highest percentage of investments in r&d regarding gdp20, the são paulo research foundation (fapesp) plays an important traditional role21. on the other hand, there seems to be a trend to decentralize researchers, relocating them to other areas of brazil from são paulo22. although the role of national r&d agencies in compensating regional disparities is not clear from our study, the concentration of graduate programmes in the southeast declined from 73% to 51% between 1980 and 2010 as part of the capes policy23. that policy increased the number of graduate programs in other regions, decreasing the share of programs in already developed areas. one important aspect is that the regulatory system of evaluating higher education in brazil likely triggers of the development of scientific communication and dissemination; the field of dentistry is an example. our analysis shows a steep increase in publications in high-impact journals (data not shown). over the period observed, the increase was not uniform countrywide; social, economic and cultural variables probably accounted for the differences. the increase in funding in r&d must also explain part of the increase in productivity in the last decades. the present study confirms the virtuous cycle of investment and development output. in addition, the association between the increase in phds among teaching staff in dental education and better research output should be highlighted. the results encountered herein should encompass the increase in dental programs in states where there were few or none. a limitation of this study is the use of university names as surrogates for states. there may be other institutions contributing scientific output that were not included, although we have no reason to think our conclusions would be different in that case, as very few papers would be lost. a second point concerns the quality of the data, a common issue in ecological studies, as validity and reliability are usually lower when information is not designed for scientific purposes. data from cnpq and other sources are administrative in nature with some degree of measurement error. nonetheless, we believe that such measurement errors are likely to be random and do not invalidate our findings. another limitation is that our study cannot identify inter-state institutional collaboration; this would require a different approach to find all authors’ addresses. the strengths of this work include the large geographical coverage and a search strategy with good sensitivity and specificity5. the generalization of our results is limited to brazilian scenarios but may hold true for other countries with similar contexts. in conclusion, this study demonstrates the role of important factors associated with dental research productivity at the state level in brazil. the rate of researchers, the most influential variable, is likely to be a consequence of other structural determinants of research productivity. state disparities were found not only in total output but also in per capita productivity. this research may assist agencies and researchers to better understand macro-determinants of scientific research and foster future policies. 11 celeste et al. acknowledgements rkc, tma, fmc and ckr hold cnpq pq felowship. 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[spacial mobility of másters and doctors in brazil: 1975-2010] [thesi]. institute of philosophy and human sciences, the university of campinas; 2015. oral sciences n3 original article braz j oral sci. october | december 2015 volume 14, number 4 effect of bleaching agents containing fluoride or calcium on enamel microhardness, roughness and permeability charles alex rauen1, júlio cezar chidoski filho1, bruna fortes bittencourt1, giovana mongruel gomes1, joão carlos gomes1, osnara maria mongruel gomes1 1universidade estadual de ponta grossa uepg, school of dentistry, area of dental materials, ponta grossa, pr – brazil correspondence to: bruna fortes bittencourt rua paula xavier, 1399 – ap 142, centro cep: 84010-270, ponta grossa, pr, brasil phone: +55 42 32245164 fax: +55 42 32241166 e-mail: brubita@hotmail.com abstract aim: to evaluate the effect of different in-office bleaching agents on the permeability, roughness and surface microhardness of human enamel. methods: for evaluation of roughness and microhardness, 40 hemi-faces of 20 premolars were subjected to initial roughness (ra parameter) and microhardness (vhn) measurements. thirty-two premolar’s crowns were used for permeability test. then, all specimens were randomly divided into four groups: c without bleaching (control), hp35 bleaching with 35% hydrogen peroxide (hp), hpf38 38% hp+fluoride, hpc35 35% hp+calcium. final roughness (fr) and microhardness (fm) measurements were evaluated. for permeability, the 32 crowns were immersed in 1% sodium hypochlorite (20 min) and silver nitrate solutions (2 h) and subjected to developing solution under fluorescent light (16 h). three sections from the crowns were analyzed in light microscope (100x) to evaluate the scores of permeability: score 0 no tracer agent penetration; score 1 less than half the thickness of enamel penetration; score 2 tracer agent reaching half the enamel thickness; score 3 entire enamel depth penetration, without reaching dentin and score 4 tracer agent reaching dentin. for roughness and microhardness evaluation were used one-way anova and dunnet post-test for independent samples, and t test for paired samples. for permeability, the data were analyzed by kruskal wallis and dunn tests. results: a significantly higher permeability and surface roughness were observed in groups hp35, hpf38 and hpc35 compared to the c group, as well as decreased microhardness (p<0.05). conclusions: all bleaching agents increased permeability and surface roughness, and decreased microhardness of human enamel; thus, the addition of fluoride or calcium was not beneficial. keywords: tooth bleaching; dental enamel permeability; hydrogen peroxide; surface properties. introduction bleaching technique, either with hydrogen peroxide or carbamide peroxide, has become a popular procedure, mainly due to guaranteed benefits such as the ability to promote color change and lighter shades of teeth 1. the reaction mechanism of hydrogen peroxide is based on the degradation of these substances and release of reactive oxygen species, which diffuse through the enamel surface, acting primarily on dentin, breaking down high-molecular carbon rings of the chromophore molecules into smaller molecules; which, in turn, diffuse to the surface, providing the desired color change2. there is concern that bleaching agents (hydrogen peroxide and carbamide received for publication: september 09, 2015 accepted: december 10, 2015 braz j oral sci. 14(4):262-266 http://dx.doi.org/10.1590/1677-3225v14n4a02 263263263263263 peroxide) bring some damage to enamel surface, like increased roughness3-4 and permeability5-6. this effect was demonstrated in a previous study 7 where silver nitrate penetration in tooth structure after bleaching was performed and it was found that these agents might diffuse into dentin by opening channels in tooth structure. two distinct paths were observed when the silver nitrate particles penetrated through enamel and reached dentin: through the prisms and through the microcracks typical of the enamel structure8. if nitrate penetrate these channels, oxygen may penetrate more readily due to its atomic weight (seven times less than silver nitrate) 8. probably, mineral loss may be detected after bleaching procedures9-10 and this has been pointed as the cause of microhardness decrease11-12. it likely that one of the causes of these adverse effects are the free oxygen radicals released during the reaction, which do not act in a specific way and may act on the organic matrix of dental structures13, breaking up the lipids and proteins of dental tissues14. physiologically, any bleaching therapy has the ability to increase free radical levels in the pulp–dentin complex, which may induce oxidative stress in odontoblasts and tertiary dentin formation. this fact may explain the numerous reports of sensitivity observed during sessions15. also characteristic of these changes are decreases in calcium and phosphate matrices9,16. these changes are detrimental to dental health because along with this process, there is an increased susceptibility to caries formation due to the ease of plaque adherence on the previously demineralized structure10,17. fluoride has proven to be effective in promoting remineralization and inhibiting enamel demineralization8. fluoride is not the only way to ensure this remineralization, as calcium compounds also exhibit efficient recovery of lost mineral content18. with this objective, fluoride and calcium have been added to bleaching gels as alternatives to reduce these adverse effects caused by enamel bleaching19. however, controversial results are found in literature, as authors report that adding fluoride to bleaching gels shows no beneficial results in terms of reduction of enamel demineralization after bleaching20-21. therefore, the aim of this study was to evaluate in vitro changes in surface roughness, microhardness and permeability of human enamel when exposed to in-office bleaching agents containing fluoride or calcium in their composition. the null hypothesis tested was that bleaching agents do not influence enamel surface roughness, microhardness and permeability of the specimens. material and methods the research project was approved by the institutional review board state university of ponta grossa: report number 25/2011 and protocol number 18741/10. specimen preparation fifty-two human premolars were used in this study. the crowns of the selected teeth were separated from their roots at the cementoenamel junction (cej) using a low-speed diamond saw (isomet 1000, buehler, lake bluff, il, usa) under water cooling at 300 rpm. twenty crowns were used for roughness and microhardness evaluation. for permeability, 32 crowns were sealed at the cej with adhesive system (adper single bond, 3m espe, st. paul, mn, usa) and resin composite (z100, 3m espe, st. paul, mn, usa) to prevent further penetration of the tracer agent (nitrate solution; vetec química fina, xerém, rj, brazil) in this area. for microhardness and roughness tests, the crowns were divided mesiodistally into two portions with a low-speed diamond saw (isomet 1000, buehler) at 300 rpm under constant water cooling. forty enamel specimens (2 mm x 2 mm x 2 mm) were obtained. study design is displayed in figure 1. fig. 1 – study design flow chart. surface roughness analysis after 48 h, the initial roughness (ir) of all specimens was measured with a digital roughness meter (mitutoyo surftest-301; mitutoyo-kawasaki, kanagawa, japan) in all 40 specimens. the specimens were divided into four quadrants, and one measurement was made in each quadrant. from these values, the arithmetic mean was considered as the ir. the used parameters were ra, 0.25 mm cutoff and 1.25 mm reading length. microhardness analysis after the roughness analysis, the specimens were flattened with wet #600, 1000 and 1200 grit aluminum oxide abrasive papers and polished with 6, 3, ½ and ¼ µm-grit diamond pastes in a polishing machine (arotec, cotia, sp, brazil) and subjected to vickers microhardness test (shimadzu, kyoto, effect of bleaching agents containing fluoride or calcium on enamel microhardness, roughness and permeability braz j oral sci. 14(4):262-266 material whiteness hpmaxx opalescence boost pf hpblue artificial saliva manufacturer fgm, joinville, santa catarina brazil ultradent products inc., salt lake city, utah, usa fgm, joinville, santa catarina brazil eficácia pharmacy, ponta grossa, paraná, brazil application mode 3-15 min applications 3-15 min applications one-45 min application ———composition 35% hydrogen peroxide, thickening agent, colorant, glycol, inorganic filler and deionized water 38% hydrogen peroxide with 1.1% sodium fluoride and 2.6% potassium nitrate 35% hydrogen peroxide, thickening agent, colorant, neutralizing agents, glycol calcium gluconate and deionized water benzoate 1g; cmc 10g; magnesium chloride 0.05g; potassium chloride 0.62g, sodium chloride 0.025g; sorbithol 42.74g, distilledwater 944.53 ml; dibasic potassium phosphate 0.8g; monobasic potassium phosphate 0.3g. table 1 –table 1 –table 1 –table 1 –table 1 – manufacturer, application mode and composition of each material used in the study. japan). the specimen’s surface was divided into 4 quadrants, and 5 indentations were made with a 50 g load for 15 s each. the obtained arithmetic mean was considered as the initial microhardness (im) of each specimen. experimental groups bleaching treatments all specimens were assigned as follows: for permeability, 32 crowns were randomly divided into 4 groups (n=8), and for roughness and microhardness, 40 specimens were divided into 4 groups (n=10): group c no bleaching (control); group hp35 bleaching with 35% hydrogen peroxide (whiteness hpmaxx, fgm, joinville, sc, brazil); group hpf38 bleaching with 38% hydrogen peroxide containing fluoride (opalescence boost pf, ultradent, south jordan, ut, usa) and group hpc35 bleaching with 35% hydrogen peroxide containing calcium (hp blue, fgm, joinville, sc, brazil). bleaching procedures were performed according to manufacturer‘s instructions (table 1). the hemi-teeth were randomized to different experimental groups in a way that specimens from the same hemi-tooth were never assigned to the same experimental group. the control group (c) was maintained in artificial saliva and was not subjected to any bleaching treatment. all specimens were maintained in artificial saliva during all experiments. final roughness and microhardness evaluation after 48 h of the last bleaching procedure, the specimens (n=40) were subjected to final roughness (fr) and final microhardness (fm) evaluations, in the same way as for the initial evaluations. permeability analysis the methodology of permeability analysis was based on previous studies5,7,22. after bleaching, all specimens, including the control group (n=32), were stored in a dark environment in artificial saliva for 48 h at 37oc. the lingual and proximal surfaces and the cej were sealed with cyanoacrylate resin (super bonder loctite, henkel ltda, são paulo, sp, brazil) and a nail varnish to prevent dye penetration of silver nitrate (tracer agent) through this area. the nail varnish was also used to delimit an area of 4 x 4 mm in the middle third of the labial surface. specimens were then immersed in 50% aqueous silver nitrate solution (vetec química fina, xerém, rj, brazil) for 2 h in a dark and closed environment. subsequently, the specimens were photodeveloped for 16 h (developing solution, kodak, eastman kodak company, rochester, ny, usa). after this, the specimens were washed in tap water and the nail varnish layer was removed with manual cutting instruments. the specimens were then embedded in a polyvinyl chloride (pvc) tube with acrylic resin (duralay, reliance, dental mfg. co., worth, il, usa), and three longitudinal slices of each tooth up to approximately 0.4 mm thick were obtained, in a buccolingual direction, with a low-speed diamond saw (isomet 1000, buehler) under water cooling. the slices were then photographed under an optical microscope (leica®, olympus bx41-u-ca, tokyo, japan) with 100x magnification. the images were made with a digital camera at a 5.1 megapixels resolution. the dye penetration degree was analyzed by three previously calibrated evaluators, using a 0 to 4 score system: score 0 no dye penetration; score 1 less than half the enamel thickness; score 2 half the enamel thickness; score 3 full extent of enamel without reaching the dentin and score 4 tracer agent reaching dentin. this evaluation was made visualizing the middle third of the dental crowns. if there was any disagreement between the evaluators, the sample under discussion was jointly analyzed until a consensus was reached. the median scores of the images from the same tooth were considered for statistical analysis using the kruskal wallis and dunn tests (α=0.05). statistical analysis roughness and microhardness data were analyzed by one-way anova and dunnet post-test (α=0.05) for independent samples, and t test for paired samples (initial 264264264264264effect of bleaching agents containing fluoride or calcium on enamel microhardness, roughness and permeability braz j oral sci. 14(4):262-266 265265265265265 and final values from each group). for permeability, qualitative data were analyzed by non-parametric kruskall wallis test and dunn post-test (α=0.05 and confidence interval of 95%). results mean values and standard deviations of initial and final roughness (µm) for each experimental group are in table 2. anova showed no significant differences between ir of all groups (p=0.9312). for fr values, significant differences (dunnet, p=0.0383) were demonstrated between group c and the other groups. the t test verified that all initial values were statistically different from final values, except for group c. means and standard deviations of initial and final microhardness values are in table 3. one-way anova showed no statistical differences among the initial values of all groups (p=0.2849). however, the fm of group c and group hp35 were statistically different from hpf38 and hpc35 (p<0.05), and t test demonstrated significant differences between im and fm (p<0.05) for all groups except group c. medians (1st/3rd interquartiles) for permeability obtained from each experimental group are in table 4. group c was statistically different from the other groups (p<0.05), although different medians were found in each group. the predominant score in each group is shown in figure 2. discussion the main focus of this study was to evaluate the effect of calcium and fluoride addition to bleaching agents as an alternative to reduce the adverse effects on human enamel. groups initial final c 376.1 ± 78.6 aa 370.2 ± 78.6 aa hp35 413.1 ± 74.6 aa 369.7 ± 47.1 ab hpf38 406.1 ± 80.3 aa 284.4 ± 22.2 bb hpc35 342.1 ± 34.1 aa 258.1 ± 34.6 bb table 3 -table 3 -table 3 -table 3 -table 3 mean values (vhn) and standard deviations of initial (im) and final microhardness (fm) for each experimental group* * lowercase letters establish relationship between columns and capital letters establish relationship between rows. different letters show statistically significant differences (p≤0.05). groups median significance (1st/3rd interquartiles) c 1 (1/1) a hp35 3 (2/3) b hpf38 2 (1.75/2.25) b hpc35 1.5 (1/3) b table 4 –table 4 –table 4 –table 4 –table 4 – median (1st/3rd interquartiles) for permeability degree and significance for each experimental group* * different letters show statistically significant differences (p≤0.05). fig. 2 – illustrative image of the predominant score found in each group. the arrows show the dye penetration in enamel and/or dentin. changes in roughness, microhardness and permeability were similar, with variations occurring between bleached samples and control groups (without bleaching), regardless of calcium or fluoride content in their composition. similar results were found by horning et al.5 (2013), who compared human enamel permeability after exposure to athome bleaching agent (6% hydrogen peroxide containing calcium) and in-office bleaching (35% hydrogen peroxide). no significant differences between bleached groups were found, but all these groups showed a significantly increased permeability, resulting in no benefit of calcium addition to the bleaching agents. otherwise, better results with calciumand fluoride-containing carbamide peroxide gels were found in another study19 that demonstrated that these gels promoted mineral loss minimization. one hypothesis that may explain this result is related to the lower concentrations of carbamide peroxide in relation to hydrogen peroxide used in this study. several factors such as concentration10, application time23 and ph24 of the bleaching agent may predict the influence of these substances on dental structures. according to bistey et al.23 (2007), higher concentration and longer treatment result in more severe changes on human enamel. another study10 groups initial final c 0.31 ± 0.08 aa 0.31 ± 0.08 aa hp35 0.29 ± 0.12 aa 0.40 ± 0.09 bb hpf38 0.29 ± 0.08 aa 0.41 ± 0.05 bb hpc35 0.31 ± 0.09 aa 0.40 ± 0.08 bb table 2 –table 2 –table 2 –table 2 –table 2 – mean values (µm) and standard deviations of initial (ir) and final roughness (fr) for each experimental group* * lowercase letters establish relationship between columns and capital letters establish relationship between rows. different letters show statistically significant differences (p≤0.05). effect of bleaching agents containing fluoride or calcium on enamel microhardness, roughness and permeability braz j oral sci. 14(4):262-266 266266266266266 showed a proportional relationship between mineral loss and bleaching agent concentration, pointing out that bleaching agents based on 35-38% hydrogen peroxide showed significantly higher values of calcium loss compared to athome gels (10% carbamide peroxide). the mineral loss also provides decreased microhardness values, which was found in this study and corroborated by other authors24. the reasons for these discrepancies between the results of this study with those already published on this issue may be the above-mentioned variants. not only the concentration and application time, but also the ph of the bleaching agents, may have an important role in mineral loss. a similar study25 investigated the effect of the bleaching agents’ ph on enamel surface. there was no morphological or chemical change of the enamel surface in neutral or alkaline bleaching solutions. in the present study, care was taken to select products with similar concentrations (35 and 38%), and the total time that the gels remained in contact with enamel was the same (45 min), varying only the instructions proposed by each manufacturer, as shown in table 1. the ph of each bleaching gel had a slight variation (hpmaxx ~ 6; opalescence boost pf ~ 7; hpblue ~ 8), without significance with respect to our results. artificial saliva was used for specimen storage. a recent study26 demonstrated that artificial saliva is suitable to simulate the oral conditions, being closest to natural saliva. however, as this is a laboratory study; further in vivo studies are required to confirm this hypothesis, since in the oral cavity there is dentinal fluid movement coming from the pulp chamber of the tooth, which may turn enamel less permeable in the in vitro conditions27. this study showed that all bleaching agents decreased microhardness, and increased permeability and surface roughness of human enamel; thus, the addition of fluoride or calcium was not beneficial. it is worth mentioning that clinicians should be aware of the indiscriminate use these agents, as the remineralizing compounds were not able to reverse the adverse effects in enamel tissue. references 1. haywood vb, hook v, heymann h. nightguard vital bleaching effects of various solutions on enamel surface texture and color. quintessence int. 1991; 22: 775-82. 2. dietschi d, rossier s, krejci i. in vitro colorometric evaluation of the efficacy of various bleaching methods and products. quintessence int. 2006; 37: 515-26. 3. dominguez ja, bittencourt b, michel m, sabino n, gomes jc, gomes om. ultrastructural evaluation of enamel after dental bleaching associated with fluoride. microsc res tech. 2012; 75: 1093-8. 4. markovic l, jordan ra, lakota n, gaengler p. micromorphology of enamel surface after vital tooth bleaching. j endod. 2007; 33: 607-10. 5. horning d, gomes gm, bittencourt bf, ruiz lm, reis a, gomes omm. evaluation of human enamel permeability exposed to bleaching agents. braz j oral sci. 2013; 12: 114-8. 6. soares dgs, ribeiro apd, sacono nt, coldebella cr, hebling j, souza costa ca. transenamel and transdentinal cytotoxicity of carbamide peroxide bleaching gels on odontoblast-like mdpc-23 cells. int endod j. 2011; 44: 116-25. 7. mendonça lc, naves lz, garcia lfr, correr-sobrinho l, soares cj, quagliatto ps. permeability, roughness and topography of enamel after bleaching: tracking channels of penetration with silver nitrate. braz j oral sci. 2011; 10: 1-6. 8. ten cate jm, buijs mj, miller cc, exterkate ram. elevated fluoride products enhance remineralization of enamel. j dent res. 2008; 87: 943–7. 9. soares dg, ribeiro ap, sacono nt, loguércio ad, hebling j, costa ca. mineral loss and morphological changes in dental enamel induced by a 16% carbamide peroxide bleaching gel. braz dent j. 2013; 24: 517-21. 10. al-salehi sk, wood dj, hatton pv. the effect of 24 h non-stop hydrogen peroxide concentration on bovine enamel and dentine mineral content and microhardness. j dent. 2007; 35: 845-50. 11. klaric e, rakic m, sever i, milat o, par m, tarle z. enamel and dentin microhardness and chemical composition after experimental light-activated bleaching. oper dent. 2015; 40: e132-41. 12. magalhães jg, marimoto ar, torres cr, pagani c, teixeira sc, barcellos dc. microhardness change of enamel due to bleaching with in-office bleaching gels of different acidity. acta odontol scand. 2012; 70: 122-6. 13. hegedüs c, bistey t, flora-nagy e, keszthelyi g, jenei a. an atomic force microscopy study on the effect of bleaching agents on enamel surface. j dent. 1999; 27: 509-15. 14. minoux m, serfaty r. vital tooth bleaching: biologic adverse effects a review. quintessence int. 2008; 39: 645-59. 15. leonard rh jr, haywood vb, phillips c. risk factors for developing tooth sensitivity and gingival irritation associated with nightguard vital bleaching. quintessence int. 1997; 28: 527-34. 16. soares dg, ribeiro ap, sacono nt, loguércio ad, hebling j, costa ca. mineral loss and morphological changes in dental enamel induced by a 16% carbamide peroxide bleaching gel. braz dent j. 2013; 24: 517-21. 17. hosoya n, honda k, iino f, arai t. changes in enamel surface roughness and adhesion of streptococcus mutans to enamel after vital bleaching. j dent. 2003; 31: 543-8. 18. langhorst se, o’donnell jn, skrtic d. in vitro remineralization of enamel by polymeric amorphous calcium phosphate composite: quantitative microradiographic study. dent mater. 2009; 25: 884-91. 19. cavalli v, rodrigues lka, paes-leme af, soares les, martin aa, berger sb, et al. effects of the addition of fluoride and calcium to lowconcentrated carbamide peroxide agents on the enamel surface and subsurface. photomed laser surg. 2011; 29: 319-25. 20. attin t, kocabiyik m, buchalla w, hannig c, becker k. susceptibility of enamel surfaces to demineralization after application of fluoridated carbamide peroxide gels. caries res. 2003; 37: 93-9. 21. tschoppe p, neumann k, mueller j, kielbassa am. effect of fluoridated bleaching gels on the remineralization of predemineralized bovine enamel in vitro. j dent. 2009; 37: 156-62. 22. muñoz ma, luque i, hass v, reis a, loguercio ad, bombarda nhc. immediate bonding properties of universal adhesives to dentine. j dent. 2013; 41: 404-11. 23. bistey t, nagy ip, simó a, hegedus c. in vitro ft-ir study of the effects of hydrogen peroxide on superficial tooth enamel. j dent. 2007; 35: 32530. 24. lia mondelli rf, garrido gabriel tr, piola rizzante fa, magalhães ac, soares bombonatti jf, ishikiriama sk. do different bleaching protocols affect the enamel microhardness? eur j dent. 2015; 9: 25-30. 25. xu b, li q, wang y. effects of ph values of hydrogen peroxide bleaching agents on enamel surface properties. oper dent. 2011; 36: 554-62. 26. zeczkowski m, tenuta lm, ambrosano gm, aguiar fh, lima da. effect of different storage conditions on the physical properties of bleached enamel: an in vitro vs. in situ study. j dent. 2015; 43: 1154-61. 27. vongsavan n, matthews b. the permeability of cat dentin in vivo and in vitro. arch oral biol. 1991; 36: 641-6. effect of bleaching agents containing fluoride or calcium on enamel microhardness, roughness and permeability braz j oral sci. 14(4):262-266 1http://dx.doi.org/10.20396/bjos.v19i0.8659296 volume 19 2020 e209296 original article 1 dentist and clinical psychologist. invited assistant & member of the prosthodontics research group of the removable prosthodontics department of the faculty of dental medicine of the university of porto, porto, portugal 2 dentist. invited assistant of the institute of health sciences – viseu, portuguese catholic university, viseu, portugal. ph.d. student at faculty of dental medicine of the university of porto, porto, portugal. 3 dentist. associate professor of the institute of health sciences – viseu, portuguese catholic university, viseu, portugal. 4 dentist. professor with tenure of removable prosthodontics of the faculty of dental medicine of the university of porto, porto, portugal. corresponding author: fernando filipe dos santos dantas departamento de prótese removível faculdade de medicina dentária da universidade do porto rua dr. manuel pereira da silva, 4200-393, porto portugal received: april 25, 2020 accepted: september 27, 2020 impact of personality traits on prosthodontic patients fernando filipe dos santos dantas1 , carlos emanuel natividade ferreira de almeida2 , andré ricardo maia correia3 , josé carlos reis campos4,* determining oral health-related quality of life in elderly patients with removable prostheses who have increased difficulty in reporting oral issues is imperative for a successful rehabilitation. aim: our objective is to assess the extent to which a trace, or personality dimension, can influence oral health-related quality of life (ohrqol) in rehabilitated patients. methods: 135 participants rehabilitated with removable prosthesis were evaluated in the clinical unit of removable prosthodontics of the faculty of dental medicine of porto university, portugal. the oral health impact profile (ohip-14) questionnaire was applied to evaluate oral health-related quality of life, along with the eysenck personality inventory (e.p.i.) to determine a personality trait. statistical analysis was carried out using mann-whitney, student’s t-test, spearman’s rho and linear regression. results: no statistical correlation was found concerning age, gender and oral health-related quality of life. regarding the latter, the highest agreement was found on the question involving “pain in teeth”. the prevalence of acrylic partial dentures, as opposed to partial dentures with metallic framework, was substantially higher. no significant association between both extraversion/neuroticism (e.p.i.) and oral health-related quality of life (ohip-14) upon rehabilitation with removable prostheses was found (p>0,05). in our study, personality traits neuroticism and extraversion did not influence oral health-related quality of life with removable prostheses. however, we should not overlook the importance of other psychological factors (such as motivation or perception) and their role in determining oral health-related quality of life. conclusion: personality traits “extraversion” and “neuroticism” did not influence oral health-related quality of life. keywords: oral health. quality of life. aged. dental prosthesis. personality assessment. https://orcid.org/0000-0003-4954-5979 https://orcid.org/0000-0002-6464-8282 https://orcid.org/0000-0002-0119-9790 https://orcid.org/0000-0002-4006-3589 2 dantas et al. introduction health-care responsibility involves more than the mastery of technical skills1. in this sense, the field of psychology should be seen as a vast source of knowledge for current dentistry practice. presently, specific guidelines defined by the association for dental education in europe stress out the importance of developing a particular set of psychological skills to further improve doctor-patient relationship. accordingly, some researchers2-4 have been trying to investigate the association between certain psychological traits and oral health-related quality of life (ohrqol). roughly 80 years ago, m. house5,6 devised a revolutionary psychological assessment tool for dentists, thus linking psychology and dentistry for the first time. it consisted of four levels or “mental attitudes”: philosophical, exacting, hysterical, and indifferent minds. according to him, patients should be classified before prosthetic rehabilitation to guide dentists in their diagnosis and treatment plan. his ideas were drafted by predicting the adaptive response of aging patients to the daily use of complete removable prostheses7. in psychology, there has been a long tradition of studying the concept of personality8-9. one of the best approaches to the comprehensive study of human personality is the “big five personality traits” theory. researchers supporting this theory measure trends, which may be defined as “normal patterns of behavior, thought, and emotion”10. for allport11, the basic unit of personality is a “trait.” the sum of individual traits provides a detailed notion of a person’s personality. eysenck12 and catell13 added that personality traits are reasonably stable over time, differ from one individual to another, and influence their behavior. moreover, they tend to evolve with experience and adaptive responses to the environment. within this field of expertise, researchers14 from different schools of thought have studied several personality dimensions, resulting in a remarkable convergence of ideas. although some scientific terminology may differ, five personality traits have been consistently pointed out. some authors propose that these five traits are an integral part of the “ocean of human personality” and are often considered the essential traits upon which all other aspects of personality fall. this theory is also called “personality traits theory” or “five-factor model,” and includes five “factors”: openness to experience; conscientiousness; extraversion-introversion; agreeableness and neuroticism-emotional stability. among these, the traits extraversion-introversion and neuroticism-emotional stability are perceived as central dimensions in human personality. both are recognized as a continuum belonging to opposite poles, thus taking on paramount importance in the study of human behavior. in line with the aforementioned, upon oral rehabilitation, when trying to assess the vast complexity of individual responses, it becomes imperative to assess personality in a clinical setting. over the years, different authors have been building a bridge between psychology and ohrqol. some15 have found a significant relationship between patients’ personality and emotional responses to new dentures, while others16 have concluded that patients with higher neuroticism scores have a greater tendency to complain due to prosthetic errors. nevertheless, although psychological 3 dantas et al. variables play a significant role in rehabilitation, prosthetic faults may also be responsible for acceptance17. more recently, researchers18 have highlighted that personality accounted for around 38% of functional limitation. since research involving oral health in the elderly is quite scarce in portugal, the authors saw fit to conduct further research on the topic. so being, this study has two major aims: 1. understand the impact of removable prostheses (including dentures) in oral health-related quality of life; 2. analyze the relationship between personality and oral health-related quality of life upon removable prosthesis rehabilitation. materials and methods this research was accepted and followed by the fmdup ethics committee, assuring confidentiality in data processing and complete privacy of all recorded information. all participants were examined in the faculty of dental medicine of the university of porto clinic (fmdup). before the study, all participants were asked to sign informed consent forms. participants with sensory impairments (blindness, deafness, hearing impairment) and illiterates were excluded from this study. also, only complete surveys were accepted. no dropouts or non-responders were reported. the institutional sample accurately represents its target population. two types of removable prosthesis were included in this study: acrylic dentures (partial and complete) and partial dentures with metallic framework. all prostheses were produced at fmdup. variables related to prostheses quality or anatomical factors (such as alveolar ridge or jaw morphology) were not assessed. to determine ohrqol, we applied the oral health impact profile questionnaire (ohip-14), which measures the acceptance of dentures by assessing quality of life. the scoring is obtained by employing a likert scale and features five different response categories ranging from 1 (strongly disagree) to 5 (strongly agree). the ohip-14 is an accurate and reliable instrument, validated for the american19, spanish20, and brazilian21 populations. it has also been employed in several other studies. personality traits were assessed by using the eysenck personality inventory (epi) to measure neuroticism-emotional stability and extraversion-introversion. to simplify, the dimension neuroticism-emotional stability will be simply addressed as “neuroticism” whereas extraversion-introversion will be addressed as “extraversion.” since personality is virtually impossible to be measured, it must first be broken down into its prime elements. hence, an individual can be either considered an introvert with low neuroticism, or an introvert with high neuroticism (same for extraversion). in short, epi is a trustworthy instrument with two variants – a and b – validated for the portuguese language and population22. form b was chosen due to the nature of the questions, which are far more suitable for the elderly. prior to the testing, we employed a short screening to inquire about gender, age, type and use of dental prostheses. 4 dantas et al. statistical analysis was carried out with the spss® v.23.0 (statistical package for the social sciences) software. appropriate descriptive statistics were also employed, namely, the mann-whitney test, student’s t-test, as well as the linear regression method. spearman’s rho was employed to correlate ohip-14 scores and age. categorical variables were described through absolute and relative frequencies (in percentage), age (in years) and depending on their distribution. continuous variables were described using average, median, standard deviation, p05 (5th percentile) and p95 (95th percentile). values were recorded as absolute and relative frequencies (%). the independent variable was age. in all hypotheses, a significance level of 0.05 was considered. the null hypothesis was the following: “there is no correlation between personality and ohrqol”. results our sample consisted of one hundred thirty-five participants with removable prostheses, of which 68 (50.4%) were males and 67 (49.6%) females. 62.5% of the study subjects had a single denture, while 37.5% had both dentures. the sample was split into two separate groups according to their age reference: 61 (45%) nonelderly participants (<65) and 74 (55%) elderly participants (≥65) [age range between 40 and 86yo; (x)=64; (s)=11]. a total of 228 removable prostheses were assessed: 64 skeletal prostheses, 118 acrylic prostheses, and 46 complete dentures. in our sample, acrylic prostheses were substantially more prevalent than metallic framework (skeletal) prostheses (fig. 1). no statistical correlation was conducted between type of prosthesis and ohip-14 test results. 140 120 100 80 60 40 20 0 64 118 46 type of prosthesis n um be r o f p ro st he si s skeletal prosthesis acrylic prosthesis complete denture figure 1. types of prosthesis. for the purpose of this study, “continuous use,” or “daily wear,” is defined as the act of wearing a removable prosthesis typically either during the whole day or during day 5 dantas et al. and night-time. on the other hand, “intermittent use” is defined as the act of wearing a removable prosthesis during short intervals of time (minutes to hours) and only during specific tasks (such as eating or public speaking). a considerable amount of patients acknowledged using prostheses continuously rather than intermittently (fig. 2). 115 20 140 120 100 80 60 40 20 0 n um be r o f p ar tic ip an ts continuous use intermittent use figure 2. prosthetics use. the ohip-14 test results (fig. 3) showed no association with age (p>0.05). 60 50 40 30 20 10 0 40 50 60 70 80 90 age o h ip 1 4 a very slight relationship was observed between the ohip-14 test results and gender (p=0.297) as females generally obtained higher results than males (average of 26.1 versus 24.0 “negative points,” in a total of 70). please note that higher scores in this test indicate less ohrqol. figure 3. relationship between age and ohip-14 test results after applying the linear regression method (univariable linear regression model for each result). regarding the ohrqol index (ohip-14), each answer was registered on a likert scale (minimum score of 1 and maximum of 5) (fig. 4). 6 dantas et al. 26.1 24 70 60 50 40 30 20 10 0 o h ip -1 4 t es t s co re s female male figure 4. relationship between gender and ohip-14 test scores. the ohip-14 test consists of 14 questions (maximum total score of 70). since all ohip-14 questions deal with negative experiences, a virtual score of 70 would imply the lowest ohrqol recorded, as well as an extremely unsatisfied patient. on average, the question with the highest agreement was number 3: “have you had painful aching in your mouth?”. conversely, the least agreed question was number 14: “have you been unable to complete any daily chores?” (fig. 5). 0 1 2 3 1.9 1.8 1.7 1.2 1.2 1.2 1.6 1.4 1.4 2.5 2.5 2.2 2.1 2.3 ohip-14 1. have you had trouble pronouncing any words? 2. have you felt that your sense of taste has worsened? 3. have you had painful aching in your mouth? 4. felt uncomfortable eatinh something? 5. have you been self-conscious? 6. have you felt tense? 7. has your diet been unsatisfactory? 8. have you had to interrupt meals? 9. have you found it difficult to relax? 10. have you been a bit embarrassed? 11. have you been a bit irritable with other people? 12. have you had difficulty doing your usual jobs? 13. have you felt that life in general was less satisfying? 14. have you been unable to complete any daily chores? figure 5. mean agreement of each ohip-14 question (total of 14 questions). the maximum score per question is 5. higher scores indicate less ohrqol (1 strongly disagree / 5 strongly agree). 7 dantas et al. the ohip-14 test scores were obtained by summing the total points scored by each participant on all 14 questions (likert scale). for the personality inventory (epi), each participant was scored by summing their extraversion and neuroticism levels into two distinct table charts. the ohip-14 test scores were then overlapped with the epi ones to study their association (fig. 6 and 7). 60 50 40 30 20 10 0 2 4 6 8 10 12 epi neuroticism o h ip -1 4 r=0.156; p=0.071 figure 6. ratio between neuroticism and ohip-14 test results (r=0.156; p=0.071). 60 50 40 30 20 10 0 2.5 5 7.5 10 12.5 epi extraversion r=0.143; p=0.099 o h ip -1 4 figure 7. ratio between extraversion and ohip-14 test results (r=0.143; p=0.099). 8 dantas et al. no linear correlation was found between epi neuroticism and ohip-14 (r = 0.156; p = 0.071), or between epi extraversion and ohip-14 (r = 0.143; p = 0.099) (table 1). table 1. spearman’s rho between all scores and age (n=135). ohip-14 epi neuroticism epi extraversion epi n+e age ohip-14 rho 1.000 0.156 0.143 0.282 -0.148 p 0.071 0.099 0.001 0.087 epi neuroticism rho 0.156 1.000 -0.350** 0.648 0.012 p 0.071 <0.001 <0.001 0.891 epi extraversion rho 0.143 -0.350** 1.000 0.456 0.033 p 0.099 <0.001 <0.001 0.707 epi n+e rho 0.282** 0.648** 0.456** 1.000 0.020 p 0.001 <0.001 <0.001 0.818 age rho -0.148 0.012 0.033 0.020 1.000 p 0.087 0.891 0.707 0.818 discussion relationship between aging and oral health-related quality of life (ohrqol) is currently a fast-growing notion. in the field of dentistry, the concept of ohrqol is particularly significant within three distinct areas: clinical practice, dental research, and dental education23. when applied to the field of prosthodontics, ohrqol can roughly be defined as the acceptance or rejection of a given prosthesis. our study had two major goals. the first involved understanding the impact of oral rehabilitation on ohrqol in both gender and age. the second, analyzing the impact of personality in ohrqol. upon statistical analysis the authors, alongside smith17, berg et al.24 and smith and hughes25, found that quality of life was not influenced by gender or age. also, no association between personality traits and ohrqol (while using prosthesis) was found, meaning that neither neuroticism nor extraversion is associated with ohrqol. these findings suggest that personality does not affect the overall success of removable prosthesis rehabilitation. according to recent scientific discoveries in the field of psychology, m. house’s classification has been considered obsolete. a few authors have pointed it out by stating that personality is far more lasting and stable than a given “mental attitude,” as was repeated by gamer et al.26. even more recently, some efforts have been made to investigate the association between personality and the acceptance of removable prostheses. however, most 9 dantas et al. investigations are conflicting since several authors27 have found an inverse relationship between neuroticism and ohrqol (higher neuroticism levels with less ohrqol), while others28 refute it. interestingly, studies highlighting such association had larger samples than those with no association. the institutional sample in this study is similar, in size, to that of sobolik and larson15, but yielded different results. on the other hand, our results are consistent with those of smith17, berg et al.24 and smith and hughes25. bolender et al.28 also found no association between personality and post-insertion control sessions. a few studies have reported that three months after a removable prosthesis rehabilitation, personality becomes the dominant factor, as was later confirmed by klages et al.18. according to these authors, a three months adaptation period is a mandatory step for personality to set in. hereupon, even though some studies focus on the acceptance of removable prostheses, there is still little research linking it with psychological variables. in addition, although support for the “big five personality traits” theory is quite robust, it is still unclear whether or not these five traits are the best possible measure of personality for all cultures. accordingly, some researchers have stressed out that essential aspects of certain societies are not embraced by this theory. we believe that cultural assessment of multiple personality types is hard to carry out since each country, or region, has its underlying characteristics and intrinsic values. thus, trying to predict a behavior pattern based solely on personality attributes may lead to misleading conclusions because, despite some similarities in personality traits between cultures, differences always arise. thus, it may be productive to think of this particular model as a framework to begin exploring differences between cultures. present day findings suggest that patients’ satisfaction with their oral condition following a removable prosthetic rehabilitation improves if certain conditions are met, such as motivation, conscientiousness, openness, among others. it is also important to provide a detailed explanation of issues that might arise by providing small tips about the daily use and maintenance of their prosthesis. the psychological impact of oral diseases on our daily life has taken on critical significance over the last few years. in the field of dentistry, whether by providing valuable information, whether by predicting the outcome of current treatments, psychological factors play an important role and must never be overlooked. therefore, ohrqol should be further investigated. the authors encourage further studies in this field, particularly to assess the influence of the factor “time” and subsequent adaptive behavior of patients to a removable prosthesis. in our study, there was no significant association between personality traits and oral health-related quality of life. also personality traits “extraversion” and “neuroticism” did not influence oral health-related quality of life upon rehabilitation with removable prostheses. in the ohip-14 assessment profile, “pain in teeth” was reported as the most prevalent, implicating less oral health-related quality of life (ohrqol) whenever dental pain occurs. the results suggest no statistical meaning between the stated variables, therefore implying the acceptance of the null hypothesis. 10 dantas et al. acknowledgments we want to thank dr. orquídea ribeiro, from the department of information science and decision in health (cides) of the faculty of medicine of the university of porto, for her support in the statistical analysis. funding: there was no external or internal source of funding for this study. ethical approval: all procedures involving human participants were in accordance with the ethical standards of the institutional committee, as well as with the 1964 helsinki declaration and its later amendments or comparable ethical standards. informed consent: informed consent was obtained from all participants included in this study, in full accordance with all current ethical principles. conflict of interest: the authors declare they have no conflict of interest. no personal gain was sought by either researchers or participants. references 1. mostofsky di, forgione ag, giddon db. behavioral dentistry. ames, io: wiley-blackwell; 2006. 2. house je. complete denture department--review and future. alumni bull sch dent indiana univ. 1967 fall:8-9, 38. 3. house je. promoting a denture practice. alumni bull sch dent indiana univ. 1970 spring:42-6. 4. winkler s. house mental classification system of denture patients: the contribution of milus m house. j oral implantol. 2005;31(6):301-3. doi: 10.1563/0-783.1. 5. house mm. studies in prosthesis. j am dent assoc. 1931;18:827-52. 6. house mm. art -fundamental in denture prosthesis. j am dent assoc. 1938;24:406-22. 7. winkler s. psychological aspects of treating complete denture patients: their relation to prosthodontic success. j geriatr psychiatry neurol. 1989 jan-mar;2(1):48-51. doi: 10.1177/089198878900200110. 8. allport gw. the open system in personality theory. j abnorm soc psychol. 1960 nov;61:301-10. doi: 10.1037/h0043619. 9. ewen rb. an introduction to theories of personality. 7. ed): psychology press; 2009. 10. eysenck sb, eysenck hj, claridge g. dimensions of personality, psychiatric syndromes, and mathematical models. j ment sci. 1960 apr;106:581-9. doi: 10.1192/bjp.106.443.581. 11. allport gw. personalistic psychology as science; a reply. psychol rev. 1946 mar;53:132-5. doi: 10.1037/h0058477. 12. eysenck hj. biological basis of personality. nature. 1963 sep 14;199:1031-4. doi: 10.1038/1991031a0. 13. cattell rb. advances in the measurement of neuroticism and anxiety in a conceptual framework of unitary-trait theory. ann n y acad sci. 1962 oct;93:815-39. doi: 10.1111/j.1749-6632.1962.tb30509.x. 14. hall cs, lindzey g, loehlin jc, manosevitz m, locke vo. introduction to theories of personality. john wiley & sons; 1985. 15. sobolik c, larson h. predicting denture acceptance through psychotechnics. j dent educ. 1968;32:67-72. 11 dantas et al. 16. nairn ri, brunello dl. the relationship of denture complaints and level of neuroticism. dent pract dent rec. 1971 jan;21(5):156-8. 17. smith m. measurement of personality traits and their relation to patient satisfaction with complete dentures. j prosthet dent. 1976 may;35(5):492-503. doi: 10.1016/0022-3913(76)90043-3. 18. klages u, esch m, wehrbein h. oral health impact in patients wearing removable prostheses: relations to somatization, pain sensitivity, and body consciousness. int j prosthodont. 2005 mar-apr;18(2):106-11. 19. scott bj, forgie ah, davis dm. a study to compare the oral health impact profile and satisfaction before and after having replacement complete dentures constructed by either the copy or the conventional technique. gerodontology. 2006 jun;23(2):79-86. doi: 10.1111/j.1741-2358.2006.00112.x. 20. zani sr, rivaldo eg, frasca lc, caye lf. oral health impact profile and prosthetic condition in edentulous patients rehabilitated with implant-supported overdentures and fixed prostheses. j oral sci. 2009 dec;51(4):535-43. doi: 10.2334/josnusd.51.535. 21. serra as, ponciano e, freitas jf. [results of applying the eysenck personality inventory to a sample of the portuguese population]. psiquiat clin. 1980;1(2):127-32. portuguese. 22. serra as, gomes fe. [results of applying the maudsley personality inventory to a portuguese sample of normal individuals]. coimbra med. 1973;20(8):859-73. portuguese. 23. al shamrany m. oral health-related quality of life: a broader perspective. east mediterr health j. 2006 nov;12(6):894-901. 24. berg e, johnsen tb, ingebretsen r. psychological variables and patient acceptance of complete dentures. acta odontol scand. 1986 feb;44(1):17-22. doi: 10.3109/00016358609041293. 25. smith jp, hughes d. a survey of referred patients experiencing problems with complete dentures. j prosthet dent. 1988 nov;60(5):583-6. doi: 10.1016/0022-3913(88)90218-1. 26. gamer s, tuch r, garcia ltmm. house mental classification revisited: intersection of particular patient types and particular dentist’s needs. j prosthet dent. 2003 mar;89(3):297-302. doi: 10.1067/mpr.2003.49. 27. al quran f, clifford t, cooper c, lamey pj. influence of psychological factors on the acceptance of complete dentures. gerodontology. 2001 jul;18(1):35-40. doi: 10.1111/j.1741-2358.2001.00035.x. 28. bolender cl, swoope cc, smith de. the cornell medical index as a prognostic aid for complete denture patients. j prosthet dent. 1969 jul;22(1):20-9. doi: 10.1016/0022-3913(69)90268-6. revista fop n 13 1591 appraisal of oral lesions status of hiv/aids patients in south western uganda ezera agwu1; john cletus ihongbe2; jackson francis tirwomwe3; victoria pazos4; michael tirwomwe5; luis casadesus6 1msc, phd, student, lecturer and head, department of medical microbiology 2bsc, phd, associate professor, department of medical laboratory sciences, faculty of basic medical sciences, college of medicine, ambrose alli university, nigeria 3bds, dpd, senior lecturer and head, department of dentistry 4bsc, phd, professor, department of medical microbiology 5bsc, teaching assistant, department of biochemistry 6bsc, msc, professor, department of medical microbiology school of health sciences, kampala international university, western campus, bushenyi, uganda received for publication: may 12, 2008 accepted: september 09, 2008 correspondence to: agwu ezera, department of medical microbiology school of health sciences kampala international university, western campus, ishaka, box 71, bushenyi, uganda. phone: +256782101486 e-mail: agwuezera@yahoo.com a b s t r a c t aim: to establish a regional surveillance data that may contribute to improvement of oral health prevention/control programs in south western uganda. methods: using who guidelines, hiv-associated oral lesions were identified among 605 hiv/aids patients (469 females and 136 males), selected at random from 4 districts of south western uganda. pearson’s chi square test of independence (α=0.01), was used to compare results. results: prevalence of oral lesions was 73.2%. pseudomembranous candidiasis (pc) showed the highest overall prevalence of 71.1% (34.9% single and 36.2% mixed); followed by 39.9% prevalence of erythematous candidiasis (ec) (13.2%) single; 26.7% mixed) and 18.8% prevalence of angular chelitis (ac) (3.1% single and 15.7% mixed). there was: 23.6% co-infection of pc and ec; 12.6% pc and ac, 3.1% were ec and ac, and 17.0% intra-oral pigmentation. prevalence of oral lesions was significantly (p<0.01) dependent on host pre-exposure to antimicrobials. conclusions: oral infections are still highly prevalent among the studied hiv/aids patients in south western uganda. this observation may be due to inadequate antimicrobial agents and oral/dental care. there’s urgent need to train primary health care workers in management of oral lesions associated with hiv/aids. more studies are needed to evaluate hiv/aids oral lesions in the populace. key words: oral lesions, hiv/aids, uganda i n t r o d u c t i o n once infected with human immunodeficiency virus (hiv), progression to acquired immunodeficiency syndrome (aids) and death is the reality for most people in resource poor countries1. the impact of hiv on individuals and families in sub-saharan africa is still very great2 and therefore increasing research attention is being given to prevention, control and management hiv/aids victims3. in uganda, and most lowincome settings, the relationships of these parameters are far less clear, partly because of the scarcity of cohorts with complete laboratory testing and diagnostic capabilities4. the oral cavity is an essential part of the body and contributes to total health and well-being. it is evident that poor oral health affects general health and that some systemic diseases can affect oral health. since hiv infection was first described in 1981, a variety of oral conditions associated with hiv disease have been documented. studies have shown that 70 to 90 percent of people infected with hiv will develop at least one oral manifestation during the course of the disease5-6. oral lesions correspond to the decline in numbers of cd4+ cells and an increase in viral load and are also independent indicators of disease progression 7-8. bacteria are the predominant component of resident oral flora and its high species diversity reflects many endogenous derived nutrients and the varied types of habitat for colonization. the composition of the oral bacterial flora in disease and in health may not be different and any adverse changes resulting from immunosuppression predispose to infections9. conditions at the sites and compositions of oral microorganisms vary with respect to oxygen level, braz j oral sci. july/september 2008 vol. 7 number 26 1592 oral lesions number (%) positive a. pseudomembranous candidiasis (pc) 211 (34.9) b. erythematous candidiasis (ec) 80 (13.2) c. angular chelitis alone (ac) 19 (3.1) d. pc and ec 143 (23.6) e. pc and ac 76 (12.6) f. ec and ac 19 (3.1) g. kaposi sarcoma (ks) 28 (4.6) h. recurrent aphthous ulceration (rau) 15 (2.5) i. acute necrotizing ulcerative gingivitis (anug) 7 (1.2) j. linear gingival erythematous banding (lgeb) 6 (1.0) k. necrotizing ulcerative gingivitis (nug) 1 (0.2) l. intra-oral pigmentation 103 (17.0) m. xerostomia 206 (34.0) table 1 distribution of oral lesions identified among the hiv infected and aids taso clients surveyed using pearson’s chi square test (á=0.05), there was a statistical significant difference (p<0.05) when the relationship between prevalence of oral lesions of the studied population was compared availability of nutrients and exposure to salivary secretions, immunosuppression, and oral hygiene10. despite many african reports on oral lesions associated with hiv/aids, similar reports from uganda are highly limited1011. in this study, we appraised the current status of oral lesions among hiv patients presenting to “the aids support organization-(taso)” centers in south-west uganda. the ultimate goal of this study is to obtain a regional surveillance data on oral lesions among hiv patients so as to determine the role of oral lesion surveillance in hiv prevention programs. this is expected to improve our existing diagnostic and management protocols of oral lesions among people living with hiv in this region. materials and methods sample area, size and inclusion criteria three main centers of the aids support organization “taso”, located in three districts (masaka, rukunguri and mbarara), and five outreach taso centers (katungu, motoma, kigarama, ibwanda and nyihanga), located in two districts (bushenyi and mbarara) all in south western uganda were used for this investigation. the inclusion criterion for taso centers was based on center participation in a previews study12 in a similar cohort which revealed different kinds of oral manifestations of hiv/aids infection. the conditions for which patients were included in this study were that patients: must have tested positive to hiv using an enzyme linked immunosobent assay (elisa) and any other immuno-serological method; must have had his/her hiv/aids clinical staging done not earlier than one month prior to time of sample collection; must be a current taso client above 18 years of age although 826 patients have consented to participate in this study, 605 were qualified. this was guided by the upper limit required to give 95% level of confidence at an expected prevalence of about 55%12 using the precise prevalence formula: sample size (n) = z2p (100-p)/d2 (epi-info version 3.2 data-base; 1995), where z is a constant given as (1.96), p is expected prevalence (55%), and d is acceptable error (5%). informed consent was sought and obtained from the following: uganda national council of science and technology, kampala international university research and ethics committee, the aids support organizations (taso) at both local and national level and taso clients (patients) through their informed consent. mouth examination and sample collection the diagnostic criterion for the oral lesion was according to world health organization’s established clinical criteria for hiv-associated oral lesions13. the mouth examination of the taso clients was done by a previously trained and calibrated11 oral clinicians. the three oral clinicians included a dental surgeon, a clinical microbiologist and a public dental health officer. prior to field survey, the three oral clinicians were standardized for consistencies in examination of oral lesions to minimize inter examiners variability using guidelines reported by muwazi et al11. briefly, a gold standard (benchmark) examiner showed the examiners some photographic slides with clinical examples of classical oral lesions expected to be seen during the survey. this was done to determine the examiners’ ability to uniformly identify oral lesions with minimum inconsistency. the clients were examined in a room with a natural light while seated on an office chair and facing the window. the mouth was examined using natural light and a mouth mirror. a disposable wooden tongue depressor was used to retract the cheeks. some clinical photographs of classical examples of the lesions were taken using digital camera. r e s u l t s the overall prevalence of individuals with oral lesions was 73.2%, occurring both as single and double manifestations, as showed in table 1. pseudomembranous candidiasis (pc) showed the highest overall prevalence of 71.1% (34.9% single and 36.2% mixed oral manifestations), followed by 39.9% prevalence of erythematous candidiasis (ec), (13.2% single and 26.7% mixed oral manifestations); 18.8% prevalence of angular chelitis (ac) (3.1% was single and 15.7% was mixed oral manifestations). out of 605 oral manifestations diagnosed from single and mixed infections, 143 (23.6%) were pc and ec; 76 (12.6%) were pc and ac and 19 (3.1%) were ec and ac. other oral lesions also observed in this study included: 4.6% kaposi sarcoma (ks); 2.5% recurrent aphthous ulceration (rau); 1.2% acute necrotizing ulcerative gingivitis (anug); 1.0% linear gingival braz j oral sci. 7(26):1591-1595 appraisal of oral lesions status of hiv/aids patients in south western uganda 1593 erythematous banding (lgeb); and 0.2% necrotizing ulcerative gingivitis (nug). intra-oral pigmentation was 103 (17.0%) prevalent among the studied population. using pearson’s chi square test of independence (x2; á=0.05, 0.01), there was a statistical significant difference (p<0.05; 0.01) when the relationship between prevalences of oral lesions of the studied population were compared. out of the 605 patients diagnosed with oral lesions, the result of pre-exposure of patients to antimicrobial agents was as follows: clotrimazole 44 (7.3%); fluconazole 27 (4.5%); ketoconazole 62 (10.2%); nystatine 68 (11.2%); anti-retroviral agent (unspecified) 48 (7.9%) and patients not exposed to anti-fungal/viral agents 356 (58.8%). there was a significant statistical difference (p<0.01) when chi square (x2; á=0.01) was used to test for independence of the different antimicrobial agents used. d i s c u s s i o n in resource poor countries, sporadic prevalence reports with no longitudinal cohort studies have made oral lesions an increasing concern, mainly in immunocompromised patients14. the observed 73.2% prevalence of oral lesions among the consenting population of hiv infected and aids taso patients, is in line with the 30-80% oral lesions among hiv/aids patients in developing countries, reported by arendiof et al.5 and patton et al.15, as well as with a survey conducted on a subset of taso clients in the same region in the year 200512. moreover, tirwomwe et al.12 found similar prevalence of oral lesions, meaning that hiv/ aids patients benefiting from taso services need essential oral health care. in this region more effort is therefore required to reduce the current prevalence of oral lesions among taso patients and other hiv patients to the barest minimum. on the other hand, the found prevalence in the present study was different from a former survey in a rural uganda community, which showed 42% manifestation of oral lesions16, probably due to the inclusion of non hiv/ aids individuals. latiff et al.17 reported a prevalence of 75% in india and a higher prevalence, 90%, was found by fidel18 in the wester18. lower result should have been expected from india and the western because of expected increased support from government and donor agencies and better strategies for management and control of this infection. umedevi et al.19 in johannesburg, south africa, found significant differences in the occurrence of oral lesions between hiv-positive and -negative women, thus highlights the usefulness of oral lesions in the detection of early and unknown hiv infections. the wide range of oral manifestation (table 1), from 0.1% (nug) to 71.1% (pc), is slightly lower than reports in south africa7 and the united states20 (1.5 % to 94%). slightly lower range of oral manifestation observed in uganda taso hiv population points to a positive impact of taso services to the people living with hiv in uganda. hodgson et al.8 had considered that the wide range found in african studies could be due to the improper calibration in methodologies. lack of adequate resources to warrant in-depth qualitative laboratory study so as to confidently and more accurately formation of lesion groups, as recommended by e-c clearinghouse 17, may be a contributing factor for the wide range in prevalence of different oral lesions found in african studies. meanwhile, it is interesting to note that the 17.0% prevalence of unique iop found in this study is lower than the 26.3% reported in south indian hiv population21. in documenting the population with oral pigmentation seen after hiv infection, 48 (7.9%) patients with oral lesions and who were taking unspecified antiretroviral therapy, including those who possessed oral pigmentation from birth, were excluded. having ruled out possible racial melanination by hereditary and pigmentation due to side effect of antiretroviral therapy, the next most probable cause of oral pigmentation among the patients studied could be increased release of α-melanocyte stimulating hormone (α-msh) due to deregulated release of cytokines in hiv disease and less likely due to use of antifungals2122. this observation warrants further investigation so as to hypothesize that hiv disease is more likely to cause oral pigmentation in indians than in africans. the observation of 40.9% mixed infection of pc with other oral lesions (pc with ec: 29.3% and pc with ac: 11.6%; table 1) gives this study another unique importance because such finding is rare in many african studies. there was a statistical significant difference (p<0.05 and p<0.01) when the relationship between prevalence of different oral lesions identified among the studied population were compared. this implies that the interaction of different oral lesions noted in this study was due to other factors (such as change in cd4 level and viral load) and not by chance alone. therefore, in addition to the reported possible correlation of single or grouped oral lesions to cd4 cell levels23, we hereby suggest that cd4 cell levels could also be correlated with the levels of mixed manifestations of oral lesions in severely immunosuppressed patients. thus the level of mixed oral infection may herald emergence and progression of hiv disease. despite the fact that the critical immunological defects, which are responsible for the onset and maintenance of mucosal candidiasis in patients with hiv infection, has not been elucidated, the devastating impact of hiv infection on mucosal langerhans’ cell and cd4+ cell populations may most probably be central to the pathogenesis of mucosal candidiasis in hiv-infected patients24. however, these defects may be partly compensated by preserved host defense mechanisms (calprotectin, keratinocytes, cd8+ t cells, and phagocytes), which individually or together, may limit candida albicans proliferation to the superficial mucosa25. a close comparison with a tirwomwe et al.12 study reveals minor improvement in some lesions and worsening condition in other ones. oral hairy leukoplakia which was braz j oral sci. 7(26):1591-1595 appraisal of oral lesions status of hiv/aids patients in south western uganda 1594 0.3%12 was not seen in the present study. lgeb was reduced by 6.8% (7.8%: 1.0%), while kaposi sarcoma was reduced by 0.2% (4.8%: 4.6%) in the present study. the slight reduction in the relative prevalence of oral lesions, two years after the lesions were identified and recommendations made on the need to follow improved management protocols, shows that the goal of improvement of oral health condition among people living with hiv in uganda may be achievable. therefore, more efforts should be made to increase the reduction rate of these oral lesions observed among the people with hiv in uganda. on the other hand, the incidence of pc among hiv patients diagnosed with oral lesions increased by 30.7% (40.8%: 71.1%), angular chelitis increased by 1.2% (17.6%: 18.8%), ec increased by 1.0% (38.9%: 39.9) and nug was increased by 0.9% (0.3%: 1.2%) respectively. information obtained from this study (table 1) shows that from 71.1% individuals with pc, 58.8% were not taking any antifungal and antiviral therapy, whereas 11.2% were taking nystatine at the time of this investigation. furthermore, from the 39.9% individuals with ec, about 35% were not taking any antifungal and antiviral therapy while 4.9% were on azoles. finally, from 18.8% patients with ac, 7.9% were on unspecified anti-retroviral therapy and 10.9% were on azoles antifungals. therefore, observed increase in the relative prevalence of candidiasis in this study may be due to antifungal resistance and inadequate treatment. proper oral care of patients especially in remote villages where taso render their services once in a month or two were clearly lacking. this is because taso services do not include routine oral inspection and care of their hiv/aids patients. inclusion of trained dental officers among taso healthcare team is hereby recommended. this recommendation is paramount to the overall effort in reduction of oral infection among people living with hiv in uganda and other developing resource poor countries. in conclusion, oral infections are still highly prevalent among hiv/aids patients in south western uganda. inadequate provision of antimicrobial agents and absence of oral and dental care were suggested to have contributed to upsurge of oral infections among taso patients in this region. increasing prevalence of oral candidiasis among hiv/ aids patients stresses the fact that hiv/aids is still a terminal disease especially among people living in resource poor countries. provision of adequate resources and qualified personnel to warrant in-depth clinical and qualitative laboratory study is supreme to achieving the goal of overall improvement of oral health condition of these patients. there is an urgent need to train primary health care workers in management of oral lesions associated with hiv/aids. more studies are needed to evaluate oral lesions and to assist health care providers in the early detection of hiv/aids in the studied population. a c k n o w l e d g e m e n t this study was supported in part by kampala international university, western campus. we are grateful to management and staff of the aids support organization both at the headquarters in kampala and in the three centers located in south western uganda for their support. we also thank all taso clients whose consent and participation made this investigation possible. r e f e r e n c e s 1. morgan d, whitworth j. the natural history of hiv-1 infection in africa. nat med. 2001; 7: 143-5. 2. dixon s, mcdonald s, roberts j. the impact of hiv and aids on africa’s economic development. bmj. 2002; 324: 232-4. 3. farmer p, leandre f, mukherjee js. community-based approaches to hiv treatment in resource-poor settings. lancet. 2001; 358: 404-9. 4. grant ad, djomand g, de cock km. natural history and spectrum of disease in adults with hiv/aids in africa. aids 1997; 11: s43–54. 5. arendorf tm, bredekamp b, cloete cac, sauer g. oral manifestations of hiv infection in 600 south african patients. j oral pathol med. 1998; 27: 176-9. 6. patton ll, phelan ja, ramos-gomez fj, nittayananta w, shiboski ch, mbuguye tl. prevalence and classification of hivassociated oral lesions. oral dis. 2002; 82:98-109. 7. greenspan js, greenspan d. the epidemiology of the oral lesions of hiv infection in the developed world. oral dis. 2002, 8 suppl 2: 34-9. 8. hodgson ta, naidoo s, chidzonga m, ramos gomez f, shiboski c. identification of oral health care needs in children and adults, management of oral diseases. proceedings of the 5th world workshop on oral health and disease in aids, july 6–9, 2004, phuket, thailand. advances in dental research; 2005. 9. scully c, laskaris g, porter sr. oral manifestations of hiv infection and their management. i: more common lesions. oral surg oral med oral pathol 1991, 71: 158-66. 10. marsh, pd, martin mv. oral microbiology. 3rd ed.. london: charpman and hall; 1992. 11. 11, muwazi lm, rwenyonyi cm, tirwomwe jf, ssali c, kasangaki a, nkamba me et al. prevalence of oral diseases/ conditions in uganda. afr health sci. 2005; 5: 227-33. 12. tirwomwe jf, rwenyonyi cm, muwazi lm, besigye b, amboli f. oral manifestation of hiv/aids in clients attending taso clinics in uganda. clin oral investig. 2007; 11: 289-92. 13. ec-clearinghouse on oral problems related to hiv infection and who collaborating centre on oral manifestations of the human immunodeficiency virus. classification and diagnostic criteria for oral lesions in hiv infection. j oral pathol med. 1993, 22: 289-291. 14. hodgson ta, rachanis cc. oral fungal and bacterial infection in hiv infected individuals, an overview in africa. oral dis. 2002, 8(suppl 2): 80-7. 15. patton ll, mckaig r, straauss r, rogers d, enron jj jr. changing prevalence of oral manifestations of human immunodeficiency virus in the era of protease inhibitor therapy. oral surg oral med oral pathol oral radiol endod. 2000, 90:299-304. 16. mayanja b, morgan d, ross a, whitworth j. the burden of mucocutaneous conditions and the association with hiv-1 infection in a rural community in uganda. trop med int health 1999, 4: 349-34. 17. lattif aa, banerjee u, prasad r, biswas a, wig n, sharma n, haque et al. susceptibility pattern and molecular type of braz j oral sci. 7(26):1591-1595 appraisal of oral lesions status of hiv/aids patients in south western uganda 1595 species-specific candida in oropharyngeal lesions of indian human immunodeficiency virus-positive patients. j clin microbiol 2004, 42: 1260-2. 18. fidel pl jr. immunity to candida. oral dis. 2002, 8:69-75 19. umadevi m., adeyemi pm, reichart pa, robinson pg. periodontal diseases and other bacterial infections. adv dent res. 2006, 19: 139-45. 20. jonsson n, zimmerman m, chidzonga mm. oral manifestations in 100 zimbabwean hiv/aids patients referred to a specialist centre. cent afr j med 1998, 44: 31-4. 21. ec-clearinghouse on oral problems related to hiv infection and who collaborating centre on oral manifestations of the immunodeficiency virus. an update of the classification and diagnostic criteria of oral lesions in hiv infection. j oral pathol med 1991; 20:97-100. 22. ranganathan k, umadevi m, saraswathi tr, n kumarasamy n, solomon s, johnson n. oral lesions and conditions associated with human immunodeficiency virus infection in 1000 south indian patients. ann acad med singapore; 2004, 33: 37s-42s. 23. eisen d. disorders of pigmentation in the oral cavity. clin dermatol. 2000, 18:579-87. 24. begg md, lamster ib, panageas ks, mitchell-lewis d, phelan ja, grbic jt. a prospective study of oral lesions and their predictive value for progression of hiv disease. oral dis. 1997, 3:176-83. 25. repentigny l, daniel lewandowski d, jolicoeur p. immunopathogenesis of oropharyngeal candidiasis in human immunodeficiency virus infection. clin microbiol rev. 2004, 17: 729–59. braz j oral sci. 7(26):1591-1595 appraisal of oral lesions status of hiv/aids patients in south western uganda 1http://dx.doi.org/10.20396/bjos.v16i0.8650494 volume 16 2017 e17045 original articlebjos current trends in technological armamentarium and treatment among brazilian endodontists ana cristina garcia ferreira1 marcos frozoni2, maíra prado3, brenda gomes4, fernanda signoretti5, adriana de-jesus-soares6. correspondence: dr maira do prado praça da bandeira, 149 praça da bandeira, rio de janeiro rj, postal code 20270-150– brazi phone/ fax: (0055) (21) 2502-3238 e-mail: maira.prado@uva.br received: may 6, 2017 accepted: august 09, 2017 1 post graduate student, são leopoldo mandic dental research center, campinas, são paulo, brazil. 2 phd, professor, são leopoldo mandic dental research center, campinas, são paulo, brazil. 3 phd, professor, department of restorative dentistry, veiga de almeida university, rio de janeiro, rj, brazil. 4 phd, professor, department of restorative dentistry, piracicaba dental school, state university of campinas unicamp, piracicaba, sp, brazil. 5 phd, professor, são leopoldo mandic dental research center, campinas, são paulo, brazil. 6 phd, professor, department of restorative dentistry, piracicaba dental school, state university of campinas unicamp, piracicaba, sp, brazil. aim: to determine the current trends in technological armamentarium and endodontic treatment among brazilian endodontists. methods: a total of 279 endodontists answered a web-based survey questionnaire about their region of activity in brazil and years as a specialist, average number of endodontic cases treated per month, number of visits to complete the treatment, use of rubber dam for isolation, type of irrigant, obturation technique and device used for this purpose, temporary filling materials, and greater difficulty encountered during treatment and technological armamentarium. a descriptive analysis, expressed in terms of frequency and percentage, was performed and the data were correlated using the chi-square test (p<0.05). results: most of the respondents had up to 10 years as specialists. more than 50% of endodontists preferred to complete the endodontic treatment in a single visit. ninety-nine percent of endodontists used rubber dam for isolation. naocl was the most widely used irrigant. most of the respondents associated different techniques for root canal filling. lateral condensation and continuous wave of condensation were the isolated technique most reported. filling devices (thermocompactors) were used by 53% of endodontists. glass ionomer was the preferred temporary filling material. the answers for the use of technological armamentarium revealing that 94% of endodontists used an apex locator; 67.38% utilized magnification (loupe: 23.66%; microscope: 35.48%; microscope and loupe: 8.24%); 58% reported to digital radiography; and 47.31% used computed tomography as a complementary tool. about mechanized instrumentation, 44.44% endodontists employed rotary and reciprocating files. the difficulties encountered during endodontic treatment were classified as preparation > access > obturation > anesthesia > isolation. conclusions: most endodontists have implemented new technologies, such as mechanical instrumentation, apex locators, magnification, digital radiography, computed tomography, ultrasound, and obturation tools, in their clinical practice. keywords: endodontics. root canal irrigants. root canal obturation. root canal therapy. trends. mailto:ajsoares.endo@uol.com.br 2 ferreira et al. introduction in the last decades, new trends and technologies in dentistry, specifically in endodontics, have been introduced in the clinical practice of specialists and general practitioners, providing benefits to the quality, efficiency, and safety of endodontic treatment1,2. technological innovations such as apex locator, electric motor with controlled speed and torque, nickel titanium files, irrigation systems, ultrasound, clinical microscope, thermoplastic filling devices, digital radiography, and computed tomography have brought about a revolution in endodontic treatment1-4. these new technologies and materials have allowed obtaining a more accurate working length and root canal patency, with consequently better preparation and filling, contributing to better predictability of endodontic treatment5,6. additionally, the length of treatment was reduced. according to bjørndal and reit7, in the past, conventional endodontic treatments were performed in five, six, or up to seven sessions. with technological advancements, the number of sessions declined and endodontic treatment is usually performed in a single session. questionnaires to assess trends in endodontic treatment and new technologies are commonly applied in other countries8-18. in brazil, in 1999, gurgel-filho et al.19 conducted a study on the philosophy of single-session endodontic treatment in brazilian and north american universities. afterwards, in 2001, santos et al.20 evaluated the conception of endodontists regarding new technologies and teaching in the area. the most recent study found in the literature dates back to 2011, when pedrosa et al.21 evaluated the panorama of tooth isolation in dental clinics in belo horizonte. in view of the new technologies incorporated into the endodontic arsenal in recent years and the limited knowledge about endodontic treatment trends in brazil, the present study aimed to determine the current trends in technological armamentarium and endodontic treatment among brazilian endodontists. material and methods this study was approved by the local research ethics committee (protocol no. 47780415.2.0000.5374). an invitation to participate in a web-based survey was sent to members of the brazilian forum on endodontics (http://www.forumdeendodontia.com.br), an online group of clinical specialists and researchers in endodontics. a total of 2,000 endodontists from the southeast, south, and northeast regions of brazil were invited to participate. these regions were selected because, according to the brazilian federal dental council, they concentrate the largest number of endodontists. the survey consists of a questionnaire with 17 multiple-choice questions with multiple selections about region of activity in brazil and years as a specialist, average number of endodontic cases treated per month, number of visits to complete the treatment, use of rubber dam for isolation, type of irrigant, obturation technique and device used for this purpose, temporary filling materials, and greater difficulty encountered during treatment and technological armamentarium (use of apex locator, mechanized instrumentation, 3 ferreira et al. magnification, digital radiography, cone beam computed tomography (cbct), and ultrasonic system). the questionnaire (translated into english) is shown in table 1. table 1. questionnaire 1. region of activity 2. years of activity as a specialist 3. on how many teeth do you perform endodontic treatment on a monthly basis? ( ) northeast ( ) 1-5 years ( ) 1-10 ( ) southeast ( ) 6-10 years ( ) 11-20 ( ) south ( ) 11-15 years ( ) > 20   ( ) 16-20 years     ( ) 21-25 years     ( ) 26-30 years     ( ) > 30 years   4. in how many sessions do you perform endodontic treatment? 5. do you use rubber dam for tooth isolation? 6. why do you not use rubber dam for tooth isolation? ( ) one visit ( ) yes ( ) difficulty ( ) multiple visits ( ) no ( ) cost ( ) mostly in one visit   ( ) delay ( ) mostly in multiple visits     7. which irrigant(s) do you use? 8. which obturation technique(s) do you use? 9. do you use any filling device (thermocompactor)? ( ) sodium hypochlorite ( ) lateral condensation ( ) yes ( ) saline ( ) schilder technique ( ) no ( ) distilled water ( ) continuous wave of condensation (buchanan)   ( ) anesthetic ( ) mcspadden technique   ( ) hydrogen peroxide ( ) tagger’s hybrid technique   ( ) edta ( ) other   ( ) 2% chlorhexidine gel     ( ) combination of two irrigants     ( ) other     10. what material(s) do you use for temporary sealing after endodontic treatment? 11. do you use an apex locator? 12. do you use ultrasound? ( ) irm ( ) yes ( ) yes ( ) zinc oxide/eugenol ( ) no ( ) no ( ) glass ionomer     ( ) composite resin     ( ) temporary restorative material (coltosol)     13. do you use magnification? 14. do you use digital radiography? 15. do you use routine exams such as cone beam computed tomography to aid in the diagnosis? ( ) microscope ( ) yes ( ) yes ( ) loupe ( ) no ( ) no ( ) no     16. do you use mechanized instrumentation? 17. which step(s) is(are) most difficult during endodontic treatment?   ( ) rotary techniques ( ) anesthesia ( ) reciprocating techniques ( ) access ( ) oscillating techniques ( ) isolation ( ) no ( ) preparation   ( ) obturation 4 ferreira et al. the survey was closed 3 months after the questionnaire was sent. data were collected, exported to excel (microsoft, seattle, usa), and analyzed by the ibm spss 2.1 software. a descriptive analysis, expressed in terms of frequency and percentage, was performed and the data were compared using the chi-square test (p<0.05). results a total of 279 complete questionnaires were received (response rate of 13.95%). figure 1 showed the region of activity and years of activity as specialist of the respondents. the most of respondents work in the southeast region. with respect to years of activity as a specialist, it was ranked as: 1-5 years > 6-10 years > 11-15 years > 16-20 years > 21-25 years > 26-30 years = >30 years. most respondents (61.75%) had up to 10 years’ experience as endodontists. regarding the number of monthly endodontic treatments, most specialists (82%) treated more than 11 teeth. as to the number of visits to conclude the treatment, it was ranked as: mostly in one visit > mostly in multiple visits > only in single visit > only in multiple sessions (figure 2). regarding rubber dam for tooth isolation, 99% of respondents said they used it. the remaining 1% who did not use it reported it was either due to the cost or time required for isolation (treatment delay). figure 1. percentage of respondents according to their region of activity and years as a specialist. region of activity (%) years of activity as specialist (%) northeast southeast south 1-5 years 6-10 years 11-15 years 16-20 years 21-25 years 26-30 years 63.44 20.79 13.98 8.24 6.09 5.02 5.02 40.86 20.7915.77 figure 2. number of monthly endodontic treatments and number of visits to conclude the treatment (%). number of endodontic tratment performed per month (%) years of activity as specialist (%) 1-10 11-20 > 20 one-visit multiple-visit the most in one-visit the most in multiple-visit 33 53.05 23.66 15.77 7.53 18 49 5 ferreira et al. concerning irrigants (table 2), they were ranked as: naocl/edta > others > only naocl > naocl/ edta/ 2% chx gel/ saline solution > naocl/ edta/ 2% chx gel = naocl/ edta/saline solution > saline solution/ edta/ 2% chx gel > saline solution/2% chx gel = only 2% chx gel > saline solution. the techniques for root canal filling, used either in isolation or combined, are displayed in table 3. the most of participants associated different techniques. lateral condensation and continuous wave of condensation (buchanan) were the techniques most cited in isolation. filling devices (thermocompactors) were used by 53% of endodontists. regarding temporary sealing materials after endodontic treatment, 20.07% of the respondents used only glass ionomer, 17.56% used only provisional restorative material (coltosol®), 17.20% participants answered others, 16.13% used composite resin only; 10.75% used glass ionomer and coltosol®; 8.24% used glass ionomer and composite resin; 6.09% used composite resin and coltosol®; 2.87% used irm and 1.08% zinc oxide/eugenol. the answers for the use of technological armamentarium revealing that 94% of endodontists used an apex locator; 67.38% utilized magnification (loupe: 23.66%; microscope: 35.48%; microscope and loupe: 8.24%); 58% resorted to digital radiography; and 52.69% did not use computed tomography as a complementary tool. table 2. irrigants used in isolation or combined. irrigants used isolated and in association. number of respondents (%) 2% chx gel 12 (4.30%) naocl 55 (19.71%) naocl/ edta 69 (24.73%) naocl/ edta/ 2% chx gel 17 (6.09%) naocl/ edta/ saline solution 15 (5.38%) naocl/ edta/ 2% chx gel/ saline solution 26 (9.32%) saline solution 1 (0.36%) saline solution/2% chx gel 12 (4.30%) saline solution/ edta/ 2% chx gel 15 (5.38%) others 57(20.43%) total 279 (100%) table 3. root canal filling techniques used either in isolation or combined. root canal filling techinique number of respondents (%) lateral condensation 34 (12.19%) tagger’s hybrid technique 23 (8.24%) mc spadden 19 (6.81%) continuous wave of condensation (buchanan) 34 (12.19%) schilder 24 (8.60%) other 57 (20.43%) associations 88 (31.54%) total 279 (100%) 6 ferreira et al. about mechanized instrumentation, 44.44% employed rotary and reciprocating files; 3.94% associated rotary and oscillating tools; 14.7% associated rotary, reciprocating, and oscillating tools; 28.32% used only rotary instruments; 5.73% used only reciprocating instruments; 1.43% utilized only oscillating files; and 1.43% did not use mechanized instrumentation at all. the difficulties encountered during endodontic treatment were classified as preparation (44.8%) > access (22.94%) > obturation (13.98%) > anesthesia (9.32%) > isolation (2.15%). more than one item was reported as a difficulty by 6.81%. there was statistical difference in mechanical instrumentation across different regions (p = 0.009). all respondents from the south of brazil reported using some type of mechanical instrumentation compared with the southeast and northeast where a small percentage (1.7%) did not use any mechanical instrumentation. in the south, there was a higher percentage (29.5%) of professionals who combined rotating, oscillating, and reciprocating instruments compared to 15.5% in the northeast and only 10.7% in the southeast. on the other hand, in the association of rotary and reciprocating tools, there was a lower percentage in the south (29.5%) than in the southeast (48%) and northeast (44.8%) regions. a statistical difference was observed between the use of digital radiography and time of activity as a specialist (p = 0.000). among endodontists with 1 to 5 years’ experience, 74.5% did not use digital radiography, and among those with 11 to 15 years of practice, 61.5% used it. however, among endodontists with over 21 years of experience, the use of digital radiography was greater than 50%. years as a specialist significantly influenced (p = 0.007) the use of cbct. with up to 10 years of practice, most endodontists did not use cbct as a complementary tool, but there was a predominance of cbct use between 16 and 30 years of practice. among endodontists who used digital radiography, 60.5% utilized cbct as a complementary tool – which was statistically significant (0.000). the number of years as a specialist influenced the use of magnification (p = 0.021) and ultrasound (p = 0.021). as time as a specialist increases, endodontists use more magnification and ultrasound. when comparing mechanical instrumentation and magnification, four endodontists did not use mechanical instrumentation and also did not adopt any type of magnification. among those who used rotary tools, 24.1% used a microscope, 10.1% microscope and loupe, 13.9% loupe, and 51.9% did not use any magnification. discussion a questionnaire was employed in the present study to identify the materials and techniques most widely used in endodontics as well as the new technologies and trends incorporated into the daily practice of brazilian endodontists. most respondents had 1 to 5 years’ experience whereas quite a small number had 26 to 30 years or more of practice. this finding is consistent with that of other studies conducted elsewhere1,10,22. however, lee et al.4, in the united states, and slaus and bottenberg18, in belgium, observed the highest number of professionals with 11 years or more of clinical practice, while whitten et al.23 showed similar percentages for all groups. 7 ferreira et al. regarding the number of monthly endodontic treatments, almost half of the endodontists treated 20 teeth or more. locke et al.15, after evaluating general practitioners in wales, observed that 83% of them performed endodontic treatment on 1 to 5 teeth every week, totaling 5 to 20 teeth per month. savani et al.17 showed that most general practitioners (58%) treated 1 to 5 teeth and that only 2% treated more than 20 teeth per month. the difference in the results may be due to the professionals evaluated. in the present study, only endodontists answered the questionnaire while in other studies, general practitioners were also evaluated. the present study indicates that the monthly number of teeth treated by endodontists exceeds the number of teeth treated by general practitioners. concerning the number of sessions required to complete endodontic treatment, the most of respondents said they performed most of the treatments in a single visit in accordance with savani et al.17 who observed that most interviewees preferred to perform the treatment in a single session (63%) and only 21% in multiple sessions. preference is related to the shorter length of treatment after the advent of nickel titanium files. however, other authors7,12,18,22 found that most of the surveyed dentists preferred multiple endodontic treatment sessions. whitten et al.23 argue that specialists opted for a single session while general practitioners preferred multiple sessions. with respect to tooth isolation, in the present study, 99% of endodontists used a rubber dam, a superior incidence than found by zou et al.1 and whitten et al. 23. also, these studies1,23 observed that the percentage of specialists who used a rubber dam during endodontic practice was higher than that obtained for general practitioners. additionally, other previous studies7,10,11-13,18,22 that evaluated general practitioners observed that the most of them did not use rubber dam routinely during root canal treatment. the reasons why some participants did not to use a rubber dam included treatment delay and costs, as described in the present study. as to the types of irrigants, most professionals used naocl and a combination of sodium hypochlorite and edta. compared with other studies, sodium hypochlorite is, in fact, the most widely used irrigant in most countries9,11,23. gurgel filho et al.19 observed that edta was employed in 67% of brazilian dental schools universities against only 8% in the united states. however, sodium hypochlorite at higher concentrations (2.5% to 5.25%) was more widely used in the united states (94%) than in brazil (43%). in countries such as india10 and saudi arabia12, saline solution is far more common than sodium hypochlorite. in nigeria22, the use of saline solution (28.8%) is similar to that of sodium hypochlorite (32.5%). regarding root canal filling, a great variation was observed in the combination of techniques, revealing the use of heterogeneous protocols by brazilian specialists. lateral condensation and continuous wave of condensation were the most frequently adopted techniques isolated, in accordance with lee et al. 4 findings in the united states. other studies, in denmark7 and in north of saudi arabia12 revealed that lateral condensation was the most commonly used technique. in the present study, endodontists preferred glass ionomer, followed by temporary restorative material (coltosol®) and composite resin as temporary sealing materials. other professionals employed more than one material. slaus and bottenberg18 and lee et al.4 showed that provisional restorative material cavit® was the material of choice, followed 8 ferreira et al. by irm® and zinc oxide eugenol cements. gupta and rai10 also described extensive use of cavit® and eugenol zinc oxide cement. kaptan et al.13 also found that cavit® was the preferred, followed by zinc phosphate cement, and zinc oxide eugenol and irm® cements. composite resin was not cited as a temporary restorative material. among technological innovations, an apex locator was widely employed by the respondents. this percentage was significantly higher than that reported in previous studies1,4,11,22. this can be explained because the present study evaluated endodontists participating in a forum that discusses innovations and exchanges of experiences in endodontic practice. also, the difference can be associated with the culture, the academic formation (in the graduation), the socioeconomic condition found among the dentists of different countries. additionally, according to scientific evidence, previous studies report increasing use of this tool, as it provides greater accuracy in the determination of working length and a decrease of operative time5,6. regarding magnification, 23.66% of the endodontists used a loupe, 35.48% adopted the clinical microscope, 8.24% associated the microscope with a loupe, and 32.62% did not use any magnification instrument. kersten et al.14 showed that the use of magnification accounted for 90% while in 1999 it was only 52%. when compared with the present study, their rate was higher than that obtained here. by evaluating different groups, the use of the clinical microscope as a function of years of activity was 22.8% in the group with 1-5 years’ experience and 43.6% among those with 11-15 years of practice. endodontists with 16-20 years of activity were those who used magnification most frequently (65.2%). the findings also show that the adoption of new technologies by the professionals requires a certain time of activity, continuing education and training, with a long learning curve10,12. digital radiography is a faster, non-polluting method that provides less radiation exposure5. in this study, it was employed by 42% of the endodontists. gupta and rai10, in india, observed that digital radiography was used by 17% of the respondents and a significantly higher proportion of respondents had postgraduate qualifications as compared to respondents who did not have postgraduate qualifications. savani et al.17, in usa, found that 72% of general dentists used this technological innovation. the difference in the results can be associated with the culture, the academic formation (in the graduation) and the socioeconomic condition found among the dentists of different countries in the present study, most specialists used mechanical instrumentation, choosing more than one system. in most recent surveys, the results vary across countries. in nigeria22 and in saudi arabia12, the use of mechanical instrumentation is low (2.5% and 17.5%, respectively). in india10 and in turkey13, the utilization of nickel titanium files accounts for 61% and 43.9%, respectively. in the united states17 and in wales15, the rate is higher (74% and 71%, respectively), even among general practitioners. the use of rotary instrumentation associated with some other type (oscillating, reciprocating, or both) demonstrated a significant increase in the number of teeth treated per month. in the present study, endodontists who used manual instrumentation answered to treat a maximum of 10 teeth per month while those who employed mechanical instrumentation treated, on average, 11 teeth or more. those who combined different types of instrumentation treated 20 teeth or more. our study consolidates the literature findings about the efficiency and speed of mechanical systems5,24,25. 9 ferreira et al. a positive correlation was observed between mechanical instrumentation and magnification. these findings indicate that professionals working with varied instrumentation systems are more prone to implement technological innovations in their clinical practice. regarding the greatest difficulties encountered during endodontic treatment, preparation was mentioned by 44.8%, access by 22.94%, obturation by 13.98%, and anesthesia by 9.32%. these findings are consistent with those of other studies, such as the one conducted by udoye et al.22, who detected difficulties in access and preparation among nigerian dentists, leading to a high rate of perforations. gupta and rai10 and hommez et al.11 suggested continuing education and training as ways to solve these problems. in conclusion, most endodontists affiliated with the brazilian forum on endodontics from the southeast, south, and northeast regions of brazil have implemented new technologies, such as mechanical instrumentation, apex locators, magnification, digital radiography, computed tomography, ultrasound, and obturation tools, in their clinical practice. more than 50% of endodontists preferred to complete the endodontic treatment in a single visit. ninety-nine percent of endodontists used rubber dam for isolation. naocl was the most widely used irrigant. most of the respondents associated different techniques for root canal filling. lateral condensation and continuous wave of condensation were the isolated technique most reported. glass ionomer was the preferred temporary filling material. the most of endodontists used an apex locator; 67.38% utilized magnification (loupe: 23.66%; microscope: 35.48%; microscope and loupe: 8.24%); 58% reported to digital radiography; and 47.31% used 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dent assoc. 2000 jun;131 suppl:39s-46s. 6. bahcall j. today’s endodontic therapy driven by advances in technology, changes in thinking. compend contin educ dent. 2015 may;36(5):378-9. 7. bjørndal l, reit c. the adoption of new endodontic technology amongst danish general dental practitioners. int dent j. 2005 jan;38(1):52-8. 8. clarkson rm, podlich hm, savage nw, moule aj. a survey of sodium hypochlorite use by general dental practitioners and endodontists in australia. aust dent j. 2003 mar;48(1):20-6. 10 ferreira et al. 9. dutner j, mines p, anderson a. irrigation trends among american association of endodontists members: a web-based survey. j endod. 2012 jan;38(1):37-40. doi: 10.1016/j.joen.2011.08.013. 10. gupta r, rai r. the adoption of new endodontic technology by indian dental practitioners: a questionnaire survey. j clin diagn res. 2013 nov; 7(11):2610-4. doi: 10.7860/jcdr/2013/5817.3628. 11. hommez gmg, braem m, de moor rjg. root canal treatment performed by flemish dentists. part 1. cleaning and shaping. int dent j. 2003 mar;36(3):166-73. 12. iqbal a, akbar i, qureshi b, sghaireen mg, al-omiri mk. a survey of standard protocols for endodontic treatment in north of ksa. isrn dent. 2014 may 4;2014:865780. doi: 10.1155/2014/865780. 13. kaptan rf, haznedaroglu f, kayahan mb, basturk fb. an investigation of current endodontic practice in turkey. scientificworldjournal. 2012;2012:565413. doi: 10.1100/2012/565413. 14. kersten dd, mines p, sweet m. use of the microscope in endodontics: results of a questionnaire. j endod. 2008 jul;34(7) 804-7. doi: 10.1016/j.joen.2008.04.002. 15. locke m, thomas mb, dummer pmh. a survey of adoption of endodontic nickel-titanium rotary instrumentation part 1: general dental practitioners in wales. br dent j. 2013 feb;214(3):e6. doi: 10.1038/sj.bdj.2013.108. 16. lynch cd, mcconnell rj. attitudes and use of rubber dam by irish general dental practitioners. int endod j. 2007 jun;40(6):427-32. 17. savani gm, sabbah w, sedgley cm, whitten b. current trends in endodontic treatment by general dental practitioners: report of a united states national survey. j endod. 2014 may;40(5):618-24. doi: 10.1016/j.joen.2014.01.029. 18. slaus g, bottenberg p. a survey of endodontic practice amongst flemish dentists. int endod j. 2002 sep;35(9):759-67. 19. gurgel filho ed, coutinho filho t, de deus g, krebs rl. [critical study of the philosophy of endodontic treatment in a single session in brazilian and north american universities]. rev bras odontol. 1999;56(4):148-52. portuguese. 20. santos ks, oliveira mrns, moraes vr. [conception of endodontists regarding new technologies and teaching in the area]. rev bras odontol. 2001;58(6):368-72. portuguese. 21. pedrosa fas, silveira rr, yamauti m, castro cdl, freitas abda. [isolation of the operative field: panorama of use in private clinics and clinics of belo horizonte. mg, brazil]. pesq bras odontoped clin integr. 2011;11(3):443-9. portuguese. 22. udoye ci, sede ma, jafarzadeh h, abbott pv. a survey of endodontic practices among dentists in nigeria. j contemp dent pract. 2013 mar;14(2):293-8 . 23. whitten bh, gardiner dl, jeansonne bg, lemon rr. current trends in endodontic treatment: report of a national survey. j am dent assoc. 1996 sep;127(9):1333-41. 24. gutmann jl. revisting the scope of contemporary endodontics. dent today. 2015 may;34(5):14-5. 25. kishen a, peters oa, zehnder m, diogenes ar, nair mk. advances in endodontics: potential applications in clinical practice. j conserv dent. 2016 may-jun;19(3):199-206. doi: 10.4103/0972-0707.181925. untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8652654 volume 17 2018 e18153 original article 1 specialist in dental prosthesis – bahiana school of medicine and public health, salvador, bahia, brazil 2 adjunct professor – bahiana school of medicine and public health and faculty of dentistry of federal university of bahia, salvador, bahia, brazil corresponding author: emilena maria castor xisto lima address: rua waldemar falcão, n. 1906, torre paysage, apt. 1402 horto florestal salvador – bahia cep – 40295-010 brazil tel: +55 (71) 99194-6656 e-mail: emilenalima@gmail.com received: december 28, 2017 accepted: march 28, 2018 effect of colorant solutions on the color stability of provisional prosthetic materials ítalo alisson da fonsêca costa1, emilena maria castor xisto lima2 aim: to evaluate the color stability of acrylic and bis-acrylic resins after immersion in 3 staining solutions. methods: forty-eight samples (10 x 2 mm) of each provisional restorative material (duralay, dencrilay, structur 2 and protemp 4) were fabricated and distributed into four groups (n = 12): g1 – distilled water (control group); g2 – a cola flavored soft drink; g3 – wine and g4 – coffee. the specimens were immersed for seven days at 37°c in the solutions, which were changed every 24 hours. the color of all specimens was measured with a spectrophotometer (vita easyshade advance) before (t0) and after immersion (t1), and the color changes (δe) were calculated. nonparametric kruskal-wallis tests were used, followed by dunn’s test with a significance level of 5%. results: for the acrylic resins (duralay and dencrilay), the largest color change values were obtained in group g4 – coffee, whereas in the bis-acrylic resins (protemp 4 and structur 2), the largest color difference was observed in groups g3 – wine and g4 – coffee. the acrylic resins showed statistically significantly less color change than the bis-acrylic resins. conclusions: the coffee and the wine promoted larger color changes in the provisional prosthetic materials tested in this study. the acrylic resins showed more color stability than the bis-acrylic resins. keywords: color. solutions. dental restoration, temporary. 2 costa and lima introduction the provisional phase of oral rehabilitation is the longest and decisive stage of prosthetic treatment. therefore, temporary crowns and fixed prostheses should be adequate to provide comfort, hygiene, health and aesthetic satisfaction to the patient during this period. for this, both the material and its fabrication method must be carefully chosen to maintain surface smoothness, mechanical resistance and color stability properties that are favorable to the success of the treatment. the dental material most used in the manufacturing of temporary fixed prostheses are acrylic resins due to their resistance, color stability, polishing properties and, especially, low cost1. additionally, bis-acrylic resins have been developed to minimize the adverse effects of acrylic resins; thus, they have improved mechanical stability, are biocompatible, are easy to handle (available in base paste / catalyst), have a less exothermic reaction and release fewer monomer residues2. therefore, these resins cause less pulpal and periodontal irritation, which favors a better contour and the marginal adaptation of the provisional prostheses3,4. regardless of the chosen material, the aesthetic quality of provisional prostheses is fundamentally important for patient satisfaction and should approach the full form and color of the natural teeth5. despite the quality of dental materials that are available in the dental market, none of them can exactly maintain their original color as they are subjected to various chemical substances, foods and dying solutions during treatment. the color change may still be affected by the material type that is used, incomplete polymerization and oral hygiene of the patient. such a change may result in poor cosmetic appearance and subsequently cause patient dissatisfaction and even additional costs for its replacement6,7. according to reis et al. 8 (2003) the intrinsic color of aesthetic materials can be altered when these materials are aged under various physical-chemical conditions, such as thermal and humidity changes. extrinsic factors are related to the adsorption or absorption of dyes from food or substance intake. in practice, the use of provisional restorations could extend from several weeks to a half a year9. the ability of the tooth-colored restorative materials used for provisional restorations to maintain their color stability is important when temporary prostheses are worn for a long period10. as a rule, the longer the material is exposed to various surrounding factors, the greater the chance for color alteration and material wear3. although studies1,5,7 have evaluated the color stability of resin-based materials used for provisional prosthodontic restorations, these materials were subjected to coffee, tea or wine as the staining solutions. however, the effect of soft drink, wine and coffee on color difference of provisional restorative materials has not been completely clarified. these staining solutions that served as means for storage of the specimens were chosen based on the frequency of ingestion of the population and the potential for staining. thus, the aim of this study was to evaluate the color stability of acrylic (duralay and dencrilay) and bis-acrylic resins (protemp 4 and structur 2) after immersion in different solutions: water, cola-flavored soft drink, wine and coffee. 3 costa and lima material and methods provisional prosthetic materials used are listed in table 1. a total of 192 samples were fabricated: 48 samples of each material using circular stainless steel molds that were 10 mm in diameter and 2 mm in high. each material was mixed and polymerized according to the manufacturer’s instructions. the acrylic resin was inserted in a single increment in the mold. the insertion of bis-acrylic resins in the mold was achieved using the respective dispenser and selfmixer tips of each manufacturer. a polyester strip and a glass plate were placed over the stainless steel mold to promote removal of the excess material and to ensure a flat and parallel surface to facilitate the reading of the samples. after the curing period, the samples were removed, and according to the manufacturer’s instructions, the bis-acrylic resin samples were subjected to rubbing with a gauze soaked in alcohol (for removal of the inhibiting layer) for 20 s. specimens were stored in distilled water for 24 hours at 37°c. the rehydration was simulated during the first day of service for provisional restorations in an oral environment3,6,7. baseline cie (commission internationale de l’eclairage) l*a*b* color coordinates were established using a spectrophotometer vita easyshade® (vita zahnfabrik, bäd sackingen, germany) for every specimen before its exposure to a staining agent. the l*a*b* coordinates are relative to a standard illuminant a against a white background. l* refers to the lightness coordinate with a value ranging from zero (black) to 100 (white). the values a* and b* are chromaticity coordinates in the red-green and yellow-blue axes, respectively. positive a* values indicate a shift to red, and negative values indicate a shift to green. similarly, positive b* values indicate the yellow color range, and negative values indicate the blue color range11. the measurements were repeated 3 times for each specimen, and the mean values of the l*, a*, and b* data were calculated. to obtain the baseline color data, a custom-made silicone (elite hd + putty soft normal – zhermach) holder7 was used to hold the specimen, and the tip of the spectrophotometer was surrounded by a pvc pipe containing table 1. provisional prosthetic materials used in the study. material composition color manufacturer duralay copolymer of plasticizable methacrylate, methylmethacrylate monomer, paraffin, mineral oil 62 reliance, cotia-sp, brazil dencrilay methyl methacrylate, butyl acrylate, benzoyl peroxide, ethylene glycol, pigments 62 dencril, são paulo-sp, brazil protemp 4 dimethacrylate polymer. bis-gma, zirconium particles, silica and silane, pigments a2 3 m espe ag, seefeld, germany structur 2 bis-gma, bht, amines, benzoyl peroxide, dimethacrylates, glass particles a2 voco, cuxhaven, germany 4 costa and lima addition silicone during the color measurement to minimize the influence of external light through the edge of the specimen. furthermore, the tip of the spectrophotometer rested on the same point of the specimens during every color measurement. after the baseline color measurements were made, the specimens were distributed into 4 groups (n = 12): g1 (control) – distilled water (amazonas comércio e indústria de produtos químicos ltda., feira de santana, bahia, brazil); g2 – a cola-flavored soft drink (coca cola, curitiba, pr, brazil); g3 – wine (dry red table, campo largo, campo largo, campo largo, curitiba, pr, brazil); and g4 – coffee (nescafé tradicional instantâneo, nestlé brasil ltda., araras, sp, brazil) (table 2). the coffee (3.6 g) was dissolved in 300 ml of boiling distilled water according to the manufacturer’s suggested concentration12. after 10 min of stirring, the solution was filtered through a filter paper. the specimens were suspended by orthodontic wires in 24-well plates containing 2.5 ml of solution in each well so that all their surfaces remained in contact with the staining solution. grouped specimens were immersed into specified liquids for 7 days and stored in bath at 37°c6,7. every 24 hours the solutions were changed. after 7 days, the specimens were rinsed with water and blotted dry with tissue paper3,10 before the color measurement. each specimen’s color was evaluated in the same manner prior to exposure. the calculation of the color variation δe* between the 2 color measurements (after 7 days storage and baseline) was performed through the standard formula of the cie lab model: δe l * a * b * = [(δl *) 2+ (δa *) 2+ (δb *) 2]7. the data were checked to ensure normality and homogeneity in the variance by the shapiro-wilk and levene tests. afterward, the non-parametric kruskal-wallis, followed by the dunn test, were used at a 5% level of significance. the statistical calculations were carried out with spss – statistical package for social sciences (spss inc., chicago, il). results the color changes (mean δe values) of provisional materials after exposure to different staining solutions are presented in table 2. table 2. mean and standard deviation of color changes (δe) of acrylic and bis-acrylic resins submitted to different staining solutions. g1 – control (water) g 2 – cola-flavored soft drink g 3 – wine g4 – coffee p-value duralay mean δe dp 0.52a a 0.74 1.08a a 1.12 0.94 aa 0.84 3.92b a 2.43 <0.001 dencrilay mean δe dp 0.82a a 0.77 1.15ab a 0.93 2.61ab a 2.10 5.17b a 4.38 0.002 protemp 4 mean δe dp 1.95a b 1.36 4.71a b 3.36 11.73b b 3.47 10.60b b 1.66 <0.001 structur 2 mean δe dp 2.69a b 1.76 3.58a b 0.75 10.70b b 3.59 11.07b b 1.82 <0.001 p-value 0.002 <0,001 <0.001 <0.001 different letters (lowercase in columns and uppercase in rows) indicate statistically significant differences (p<0.05. kruskal-wallis, dunn). 5 costa and lima when comparing the different solutions, for the acrylic resin duralay, the largest color-change values were observed in group 4 – coffee, which was found to be significantly different compared with values obtained in the other groups. for the acrylic resin dencrilay, there was no statistically significant difference between groups 2, 3 and 4. for the bis-acrylic resins (protemp 4 and structur 2), the largest color differences were observed in groups 3 and 4, which were not significantly different from each other. when comparing the four different provisional prosthetic materials, the acrylic resins demonstrated statistically significant less color change compared with the bis-acrylic resins. the acrylic resins (duralay and dencrilay) were not significantly different from each other as well as bis-acrylic resins (protemp 4 and structur 2). discussion the staining of dental materials is the result of both extrinsic (surface roughness, poor oral hygiene, nutrition, and material wear) and intrinsic (filler and monomer composition, residual unpolymerized monomers resulting from incomplete polymerization) factors8,13,14. extrinsic factors for color alteration include staining by colorant adsorption or absorption because of contamination from exogenous sources15. additionally, extrinsic factors for color alteration are known to cause staining of oral tissues and restorations, especially in combination with dietary factors. among these, coffee, tea, nicotine, and beverages have been reported15,16. the solutions used in the study (a soft drink, coffee, wine) were chosen because of the high frequency of ingestion by the population and high potential for staining. according to koishi et al.17 (2001), the thickness and smoothness of the specimen surface also affect color. in the present study, the thickness of provisional restorative material specimens was 2 mm2. however, because the calculation of the color variation (δe*) between 2 color positions (7-day storage and baseline) in the 3-d l*a*b* color space was investigated, the thickness of the specimens was not important. in practice, the use of a provisional prosthesis may extend from a few days to six months or more, indicating that a low quality of provisional restorations can bring complications, dissatisfaction and even additional costs for its replacement6,7,9,10. as a rule, the longer the material is exposed to various factors (diet, oral hygiene, water sorption, and chemical reactivity), the greater the chances for color alteration and increased roughness7. the period of immersion in the solutions was 7 days, and according to guler et al.3 (2005), the coffee manufacturer purports that the average time for consumption of 1 cup of a drink is 15 minutes, and among coffee drinkers, the average consumption of coffee is 3.2 cups per day. therefore, 24 hours of immersion of samples in coffee simulate consumption of the drink over 1 month. the storage for 7 days was selected as a standard time and correspond to seven months of provisional phase treatment. in this study, a color change was verified in all the resins regardless of the solution used, but g3 – wine and g4 – coffee promoted greater variable color changes in the 6 costa and lima samples. these solutions elevated the δe of the samples to values that are higher than clinically acceptable values. according to johnston and kao18 (1989), the size of the color difference, which is larger than δe 3.7 e units, is the highest value that has been determined as clinically acceptable. the influence of coffee and wine on the color change was observed in other studies. rutkunas et al.7 (2010) verified that bis-acryl composite resins exhibited more significant color changes in coffee with sugar and in red wine compared with methyl/ethyl methacrylates. these results agree with the bis-acryl resin results in this study. the color stability in acrylic and bis-acrylic resins was also evaluated by haselton et al.1 (2005). this group analysed in vitro 12 prosthetic materials that were immersed in pure artificial saliva and coffee at 37°c after 1, 2 and 4 weeks. the results verified that the luxatemp, protemp and temphase bis-acrylic resins obtained more impactful color change results in coffee than in pure saliva. in the present study, group 4 – coffee promoted a significant color change compared with wine in the acrylic resin duralay, but there was no difference between coffee and wine in the other materials. in the present study, the bis-acrylic resins demonstrated statistically significantly more color change compared with the acrylic resins. these results agree with the study of rutkunas et al. 7 (2010). they investigated the effects of different polishing techniques on the color stability of provisional prosthetic materials upon exposure to different staining agents (distilled water (control), food colorant, coffee and red wine) and verified that bis-acryl composite resins compared with methyl/ethyl methacrylates exhibited more significant color changes in coffee with sugar and in red wine. additionally, givens et al.5 (2008) evaluated the color stability of two autocured bis-acryl materials. one dual-cure bis-acryl material and one pema control material were immersed cyclically in tea for 1 week, and the results verified that of the four materials, only protemp garant, an autocured bis-acryl composite, exhibited a clinically noticeable change in shade. several studies have reported that water absorption is influenced by factors such as filler content19,20, the presence of residual unpolymerized monomers, the inclusion of air bubbles21,22 and the cross-linking degree of resin molecules23,24. in particular, incomplete polymerization might cause a deterioration in physical properties of the resin material and an increase in microleakage, thereby inducing color changes. in the present study, the results showed a greater vulnerability of the bis-acryl resins in relation to color changes. several authors1,5,10 associate this characteristic to the composition of the bis-acryl resins. according to haselton et al.1 (2005), most bis-acryl polymers are more polar than pmma polymers and therefore have a greater affinity towards water and other polar liquids. thus, there is a greater absorption of liquids in bisacrylic resins that favors the incorporation of pigments that are contained in these liquids. this phenomenon is probably the reason for the larger color changes in bis-acryl resins that were found in this study. the results suggest that during provisional phase of treatment, the patient should avoid staining drinks. when provisional fixed prosthodontic materials are used for long periods, the acrylic resin tested may be preferred over the bisacrylic resins for 7 costa and lima areas that aesthetic is important and the long time of provisional phase is necessary. in other hand, bis-acrylic resin is the option for short time provisional phase or in case of long time in area that aesthetic is no relevant. in view of the diversity of information available in the scientific literature on the aesthetic characteristics of provisional prosthetic materials, further studies are necessary, since the color change of these materials is a reality that must be carefully observed and monitored by the professional to obtain dental rehabilitation success. within the limitations of this study, it could be concluded that: both coffee and wine promoted larger color changes in the provisional prosthetic materials tested in this study. the acrylic resins showed more color stability than the bis-acrylic resins. references 1. haselton dr, diazarnald am, dawson dv. color stability of provisional crown and fixed partial denture resins. j prosthet dent. 2005 jan;93(1):70-5. 2. strassler he, lowe ra. chairside resin-based provisional restorative materials for fixed prosthodontics. compend contin educ dent. 2011 nov-dec;32(9):10, 12, 14 passim; quiz 20, 38. 3. guler au, yilmaz f, kulunk t, guler e, kurt s. effects of different drinks on stainability of resin composite provisional restorative materials. j prosthet dent. 2005 aug;94(2):118-24. 4. perry rd, magnuson b. provisional materials: key components of interim fixed restorations. compend contin educ dent. 2012 jan;33(1):59-60, 62. 5. givens ej jr, neiva g, yaman p, dennison jb. marginal adaption and color stability of four provisional materials. j prosthodont. 2008 feb;17(2):97-101. doi: 10.1111/j.1532-849x.2007.00256.x. 6. rutkünas v, sabaliauskas v. effects of different repolishing techniques of colour change of provisional prosthetic materials. stomatologija. 2009;11(4):105-12. 7. rutkunas v, sabaliauskas v, mizutani h. effects of different food colorants and polishing techniques on color stability of provisional prosthetic materials. dent mater. 2010 mar;29(2):167-76. 8. reis af, giannini m, lovadino jr, ambrosano gm. effects of various finishing systems on the surface roughness and staining susceptibility of packable composite resins. dent mater. 2003 jan;19(1):12-8. 9. scotti r, mascellani sc, forniti f. the in vitro color stability of acrylic resins for provisional restorations. int j prosthodont. 1997 mar-apr;10(2):164-8. 10. sham ask, chu fcs, chai j, chow tw. color stability of provisional prosthodontic materials. j prosthet dent. 2004 may;91(5):447-52. 11. commission internationale de l’eclairage. technical report. colorimetry. 3rd ed. vienna: cie central bureau; 2004. cie 15. 12. guler au, kurt s, kulunk t. effects of various finishing procedures on the staining of provisional restorative materials. j prosthet dent. 2005 may;93(5):453-8. 13. yap au, lee hk, sabapathy r. release of methacrylic acid from dental composites. dent mater. 2000 may;16(3):172-9. 14. oréfice rl, discacciati jac, neves ad, mansur hs, jansen wc. in situ evaluation of the polymerization kinetics and corresponding evolution of the mechanical properties of dental composites. polym test. 2003 feb;22(1):77-81. doi: 10.1016/s0142-9418(02)00052-1. 8 costa and lima 15. um cm, ruyteri e. staining of resin-based veneering materials with coffee and tea. quintessence int. 1991 may;22(5):377-86. 16. prayitno s, addy m. an in vitro study of factors affecting the development of staining associated with the use of chlorhexidine. j periodont res. 1979 sep;14(5):397-402. 17. koishi y, tanoue n, matsumura h, atsuta m. colour reproducibility of a photo-activated prosthetic composite with different thicknesses. j oral rehabil. 2001 sep;28(9):799-804. 18. johnston wm, kao ec. assessment of appearance match by visual observation and clinical colorimetry. j dent res. 1989 may;68(5):819-22. 19. braden m, clarke rl. water absorption characteristics of dental microfine composite filling materials. i. proprietary materials. biomaterials. 1984 nov;5(6):369-72. 20. oysaed h, ruyter ie. water sorption and ller characteristics of composites for use in posterior teeth. j dent res. 1986 nov;65(11):1315-8. 21. akashi a, matsuya y, unemori m, akamine a. the relationship between water absorption characteristics and the mechanical strength of resin-modified glass-ionomer cements in long-term water storage. biomaterials. 1999 sep;20(17):1573-8. 22. pascual b, gurruchaga m, ginebra mp, gil fj, planell ja, goñi i. infuence of the modification of p/l ratio on a new formulation of acrylic bone cement. biomaterials. 1999 mar;20(5):465-74. 23. arima t, murata h, hamada t. properties of highly cross-linked autopolymerizing reline acrylic resins. j prosthet dent. 1995 jan;73(1):55-9. 24. arima t, murata h, hamada t. the effects of cross-linking agents on the water sorption and solubility characteristics of denture base resin. j oral rehabil. 1996 jul;23(7):476-80. untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8652926 volume 17 2018 e18336 original article 1 phd student, msc, dds, department of restorative dentistry, piracicaba dental school, university of campinas, piracicaba, sp, brazil. 2 professor, phd, msc, dds, department of restorative dentistry, piracicaba dental school, university of campinas, piracicaba, sp, brazil corresponding author: giselle maria marchi. department of restorative dentistry, piracicaba dental school, unicamp. av. limeira, 901 areião p.o. box 52 zip code 13414-903 piracicaba, sp, brazil. phone: +55 19 2106-5341 fax: +55 19 2106-5337 e-mail address: gimarchi@ unicamp.br received: march 04, 2018 accepted: may 30, 2018 treatment of non-carious lesions: diagnosis, restorative materials and techniques caroline mathias1, laura nobre ferraz1, débora alves nunes leite lima2, giselle maria marchi2* management of non-carious cervical lesions (nccls) still is a challenge in clinical practice. the diagnosis is the key to treat these lesions successfully. aim/case report: therefore, the aim of this case report is to describe the diagnosis and treatment of generalized abfraction lesions associated to abrasion lesions of a 43 years old patient and to discuss considerations about the technique and materials to be used appropriately. after the diagnosis, a treatment plan with preventive and restorative approach was elaborated. the occlusal adjustment was performed to distribute the contact points and the patient was instructed in relation to tooth brushing habits. it was also recommended that the patient use a dentifrice with potassium nitrate for the control of sensitivity. for lesions over 1mm deep, the restorative treatment was performed using the self-etching adhesive system and nanohybrid composite by the stratification technique. unsatisfactory restorations have been replaced. occlusal splint was done and the patient was instructed to use the device every day overnight. one year after treatment, follow-up was performed. the appearance of new nccls was not observed. there was no progression of lesions smaller than 1 mm. there was no loss of restorations. all restorations were fully satisfactory and the patient reported absence of dentin hypersensitivity. conclusion: the treatment performed besides being aesthetically satisfactory after 1 year resulted in the control of the disease, preventing the appearance of new lesions and guaranteeing a better quality of life to the patient. keywords: tooth wear. diagnosis. dental restoration. 2 mathias et al. introduction the term non-carious cervical lesions (nccls) refers to the loss of dental tissue caused by processes that do not involve bacteria1. its etiology is considered multifactorial and complex involving processes such as biocorrosion, abrasion and abfraction2. dental biocorrosion is characterized by chemical dissolution of mineralized dental tissues by acids of non-bacterial origin3. abrasion, occurs by objects or substances that are in frequent contact with dental surfaces, such as toothbrushing and abrasive dentifrices, resulting in mechanical wear4,5. the abfraction theory is based mainly on the concentration of tensions in the cervical areas, which cause flexion of the tooth resulting in microfractures and loss of dental6,7. considering that occlusal forces may cause stress concentration, occlusal interferences, premature contact, bruxism and clenching could contribute to the etiology of nccls8. however, some studies have proposed a combination of occlusal tensions with abrasion and biocorrosion in the development of lesions, leading to a conclusion that the progression of abfraction may be multifactorial9,10. clinical appearance of nccls can vary from soft depressions to large wedges or discshaped lesions, characterized by uniform loss of hard tissue, predominantly on the buccal surfaces, independently of the affected teeth, near the cement-enamel junction (cej)11. these lesions may lead to biofilm growth and are often associated with irritation and gingival recession12, causing structural fragilities reflected in a poor rootcrown relationship and aesthetic complaints13. also, nccls are a predisposing factor for dentin hypersensitivity (dh)14. dh is a painful condition that occurs when the dentin is exposed to the oral environment. dh is certainly a reflection of the cumulative exposure to causal agents, which is strongly related to age, with a higher incidence in the age group of 31-50 years, and, in older age groups, dh tends to decrease due to the repair processes of the pulp14. successful prevention and management of abfraction or any nccls requires an understanding of the risk factors and how these factors change over time in individual patients15. preventive interven tions may include counseling for changes in patient’s behav ior, such as diet, brushing technique, use of protective night guards to reduce clenching or bruxism, use of chewing gums to increase salivary flow, and/or to seek therapy or medical attention if there is a potential, intrinsic medical or mental condition15. other treatment options include the following: monitoring of lesion progression, occlusal adjustments, occlusal splints, techniques to alleviate hypersensitity, placement of restorations, and root coverage surgical procedures in combination with restorations15. since nccls often involve the simultaneous loss of tooth structure and gingival recession the diagnostic assessment and treatment should be based on a multidisciplinary approach to periodontal and restorative aspects13. as treatment strategies may vary according to the type of gingival recession, marginal level and extent of nccl, the clinical characteristics of each defect should be considered prior to treatment13. the nccls restorative treatment improves oral hygiene maintenance by the patient. it also helps to decrease thermal sensitivity, improving aesthetics and strengthening teeth16. the most important criterion for restoration is retention and restoring these lesions represents a challenge in dentistry16,17. in most cases, lesions involve margins 3 mathias et al. on enamel and dentin and also due to the increased amount of scleral dentin, nccls present low permeability and hypermineralization, resulting in a surface that is not favorable for adhesion15,16. along with restoration, a variety of treatment strategies have also been proposed as occlusal adjustments, occlusal plaques and elimination of parafunctional habits18. therefore, the aim of the present case report is to describe and discuss the current understanding of treatment for nccls  with a  preventive and  restorative approach, and to describe the techniques and materials indicated for such treatments. case report a 43 year old female presented to the clinic of piracicaba dental school, state university of campinas são paulo, with aesthetic complaint of teeth and dental hypersensitivity to thermal (cold) and air stimulus. for the diagnosis and treatment planning, full-mouth radiographs, a periodontal examination, and the fabrication of study casts were performed, and a full-mouth photograph was taken. the patient present nccls on teeth 11 and 21 on the buccal surface, at a lower depth, polished and with no defined contour (figure 1). also presented nccls with dentin exposure, wedge-shaped or v-shaped with clearly defined internal and external angles located in the cervical region of the buccal surface of the teeth 13, 16, 24, 26, 34, 35, 36, 44, 45 , 46, 47 (figure 2a and 2b). furthermore, the teeth 12, 14, 15, 23, 25 and 27 had unsatisfactory class v restorations due to bad adaptation at the margins of the underlying lesion (figure 2a and 2b). in addition, all teeth had a highly polished buccal surface. multiple gingival recessions were present in all teeth that presented cervical lesion. the patient reported the use of a rigid dental brush and the use of excessive force during brushing. also, occlusal interferences, premature contact and the presence of unsatisfactory occlusal restorations on teeth 36, 45, 46 and 47 were observed (figure 2c and 2d). the patient reported clenching in situations of stress and especially during the night. figure 1. preoperative frontal view. note the presence of a non-carious cervical lesion on the maxillary central incisor, the right lateral incisor, and the left premolar. old resin restorations on right canines, left and right premolars showed ill-fitting margins. 4 mathias et al. the operative dentistry team diagnosed chronic sensitivity due to loss of enamel tissue and dentin exposure. the etiology of cervical lesions of the posterior teeth was considered multifactorial due to the association of abfraction and abrasion factors and characteristics. the cervical lesions of the anterior teeth were probably caused only by the abrasion process. after the diagnosis, a treatment plan with preventive and restorative approach was elaborated. preventive intervention and control of dental sensitivity preventive intervention was performed to aim controlling the etiological agent avoiding the appearance of new lesions and the progression of existing lesions. thus, the patient was instructed to perform brushing with vertical movements, using little force and avoiding brushing for very long periods. for the treatment of sensitivity was indicated brushing 3 times daily using potassium nitrate dentifrice (sensodyne extra fresh, smithkline beecham consumer heathcare, berkshire, united kingdom). to improve occlusal harmony and distribution of contact points, occlusal adjustment was performed by reduce heavy contacts and removing of premature contacts. restorative treatment for the nccls of the teeth 11 and 21 only the monitoring of the lesion at regular intervals is sufficient because they present less than 1 mm in depth. for the teeth 13, 16, 24, 26, 34, 35, 36, 44, 45, 46, 47 the restorative treatment for rehabilitation of aesthetics and figure 2. initial clinical appearance of the posterior teeth. (a) nccls in the lower molars and premolars, superior canine and unsatisfactory restorations in the upper molar teeth. (b) nccls in the molars and premolars and unsatisfactory restorations in the upper second molar. (c) view of the occlusal contact points of maxillary teeth with excessive points of contact on the left side. (d) view of the occlusal contact points of the mandibular teeth with excessive points of contact on the posterior teeth on the left side. a b c d 5 mathias et al. health due to structural loss was indicated. for this case report, the nanohybrid composite empress direct (ivoclar-vivadent, liechtenstein) was chosen. firstly, dental prophylaxis was performed using a mixture of stone powders and water, for the removal of the biofilm. after that, the choice of color was made by photopolymerization of small increments of composite resin on the vestibular surface of the tooth, close to the region to be restored and the color a3.5 was chosen for the dentin and a3 for enamel. relative isolation of operative field was performed. a retracting wire was inserted into the gingival sulcus to expose the margin of the lesion and also to control the inflow of the crevicular fluid (figure 3a). also, were used high-power suckers, cotton rollers and front lip retractor. a self-etching adhesive system (clearfil se bond, kurary, tokyo, japan) was used. for this, just the enamel was conditioned with 35% phosphoric acid (ultra-etch, ultradent products inc.) for 30 seconds (figure 3b). the tooth was then washed with a water spray for 30 seconds and dried with cotton ball. the primer of the adhesive system was actively applied for the time of 20 seconds (figure 3c) followed by slight air drying and the application of two bond layers (figure 3d). the photopolymerization was performed for 40 seconds using a led light device (radii plus, sdi, melbourne, victoria, australia). in order to reduce the tension generated by the polymerization, the stratification technique was performed using small increments of composite resin with dentin characteristics, first the cervical margin and then the occlusal margin of the cavity (figure 3e). after the dentin reconstruction, a layer of composite with enamel characteristics was applied to the cavity with the aid of a brush to reduce the excesses and obtain a smooth and uniform surface (figure 3f). after insertion, each increment was photopolymerized for 20 seconds, according to the manufacturer’s recommendation. after the final polymerization, the retraction wire was removed and the finishing procedures started. adhesive and resin excesses on the restoration margins were detected with an exploratory probe and removed with no. 12 scalpel blades and 2200f and 2200ff diamond figure 3. restorative procedures on the righ maxillary canine. (a) retracting wire insertion. (b) selective phosphoric acid etching of the enamel. (c) self-etching adhesive system application (primer). (d) selfetching adhesive system application (bond). (e) resin increase for dentin. (f) resin increase for enamel. a b c d e f 6 mathias et al. tips. the diamond tip 3082 was also used to remove superficial excesses. after 48 hours, the finishing and polishing procedures were performed, using flexible disks with decreasing granulation (figure 4a, 4b, 4c and 4d) and abrasive rubbers (figure 5a, 5b and 5c) until obtaining a highly smooth surface. for the polishing, silicon carbide brushes (figure 5d) and polishing paste were used on felt disks (figure 5e). in figure 5f the final result of such procedures are shown, where was obtained a perfectly adapted, smooth, shiny and polished restoration, which mimics the natural tooth. figure 4. finishing procedure of restoration. (a, b, c and d) use of flexible disks with decreasing granulation. a b c d figure 5. finishing and polishing procedures. (a, b and c) finishing with abrasive rubbers. (d) polishing with silicon carbide brush. (e) polishing with paste on felt disk. (f) final aspect of restoration. a b c d e f 7 mathias et al. after restoration of nccls, class v restorations were replaced for teeth 12, 14, 15, 23, 25 and 27. class i restorations of the teeth 36 and 47 and the class ii restoration of the tooth 45 were also changed. the tooth 46 presented a great loss of dental tissue, thus indicating the placement of a total crown. endodontic retreatment, fiberglass pin cementation and a monolithic zirconia crown were performed. class iii restoration of the tooth 11 and class iv of the tooth 21 were replaced due to staining. at the end of all restorative procedures, occlusal adjustment was again performed (figure 6a and 6b). occlusal splints after the end of all the restorations occlusal splint was done and the patient was instructed to use the device every day overnight (figure 6d). follow-up one year after the end of restorations, the patient was contacted for follow-up. when questioned about oral health the patient reported that the dental treatment performed resulted in a better quality of life mainly due to the absence of hypersensitivity to thermal (cold) and air stimulus. the patient changed her brushing habits, performed the oral hygiene according to instruction and used the occlusal splint every day during the night. during the clinical examination, the presence of new cervical lesions was not detected. it was observed that there was no progression of the lesions smaller than 1 mm of the teeth 11 and 21. all restorative procedures performed were aesthetically satisfactory. figure 6. final aspect of the restorations. (a) occlusal view of maxillary teeth after termination of treatment showing well-distributed occlusal contact points (b) occlusal view of mandibular teeth after treatment showing well-distributed occlusal contact points (c) frontal view after treatment (d) installation of the occlusal splints. a b c d 8 mathias et al. there were no losses of the restorations. all class v restorations were fully satisfactory with perfectly matched edges and good surface polishing. class i restorations of teeth 36 and 47 and tooth ii class ii also remained satisfactory without any maladjustment, fracture or excessive wear of the restorative material. the total crown of the tooth 46 also remained intact (figure 7). discussion the nccls etiology is still controversial, abfraction lesions are believed to be caused by tooth flexure arising from cyclic and eccentric occlusal forces, besides parafunction8,19. other modifying factors must be considered in the abfraction etiology as the composition, buffering capacity, flow rate, viscosity and ph of saliva8. these lesions are considered multifactorial, once according to traditional theory of abfraction etiology, the enamel micro cracks, formed by tensile stresses under non-axial loading, can predisposed the tooth tissue to biocorrosion and abrasion8. however, the lack of clinical studies and the fact that nccls are often found in buccal faces of teeth, it reinforces the belief of some authors that the cause-effect relationship between the abfraction lesions and occlusal loading can not be confirmed15,16,20. therefore, the clinicians often have difficulties in correctly diagnosing the lesions, which require a careful anamnesis to treat according to their etiology. the first step on treatment of nccls lesions is to stop or prevent further progression of the lesions by controlling all potential etiologic factors that can be associated to figure 7. aspects of the restorations after 1-year follow up. (a) frontal view of restoration in function after 1-year placement. (b) left view of restorations. the crown and class v restorations well adapted. (c) right view of restorations. class v restorations without margin cracks and presenting good adaptation. a b c 9 mathias et al. these lesions21. for this, the patient should be instructed to avoid the consumption of acid fruits and to perform brushing with vertical movements, using little force during short periods. vertical brushing promotes three times less wear and tear than horizontal brushing22. also, a proper occlusal adjustment should be performed to eliminate premature contacts and promote a balanced occlusion, often requiring the replacement of deficient occlusal restorations that do not allow adequate contact with the opposing tooth23. when the treatment is not based on the etiological agent, even though the lesion is restored, probably it can occurs the progression of the underlying lesion and consequently misalignment of the composite resin at the margins of the lesion, leading it to failure15. dh is associated to ageing, presence of nccls, premature contacts and frequent consumption of erosive fruit juices24. the treatment of dh still lacks a standard protocol to solve the patient discomfort. because of this, recurrences are common25. freitas et al.25 reported that is more efficient to restore a tooth with moderate or severe dh as soon as possible instead of trying to desensitize it before the filling procedure. besides, the choice of the desensitizing agent is important if the tooth will posteriorly be restored, because agents that contain sodium and calcium fluoride may reduce the bond strength of adhesives26. therefore, no desensitizing agent was recommended for the patient in this study. there are no consensus or guidelines in the literature about when abfraction lesions should be restored15. however, when these lesions are less than 1 mm in depth, according to shetty et al.27 no restorative procedure is necessary, only monitoring at regular intervals. because of this, the nccls of the patient’s teeth 11 and 21 were not restored. the advantages in restoring the nccls are: better maintenance of oral hygiene by the patiente, decreasing of thermal sensitivity, improvement of esthetics and strengthening of the teeth27. one of the challenges in restoring these lesions is the difficulty with moisture control15. relative isolation of operative field was preferred in this case report, because it was necessary to completely visualize the dentogengival regions, which would not be possible with the absolute isolation. in addition, the teeth to be restored presented lesions at the gingival level and did not present hard tissue for the retention of the staples. clinical studies have shown that nccls restorations have higher rate of failure in the cervical area, due to constant deformation of the tooth caused by parafunctional habits of the patient28. also, nccls lesions have large amount of sclerotic and hypermineralized dentin that affect it permeability for adhesives agents15,16. therefore, another important criterion for these kind of restoration is the retention16. while some studies have shown no difference between retention rates of etch-and-rinse and self-etch adhesives after short and long-term clinical29,30, others have shown that self-etch adhesives should be preferred as long as the enamel is conditioned with phosphoric acid15,28. restorative materials with low modulus of elasticity should be the first choice to restore abfraction lesions15,16,31. when the aesthetic is not a concern, glass ionomer should be used, because these restorations revealed the best results in the context of clinical effectiveness15,27. however, methacrylate-based composites are usually the first choice to restore these lesions, being the gold-standard, due to their mechanical 10 mathias et al. properties and better esthetic compared to glass ionomers32. microfilled composites demonstrate a greater elasticity than hybrid composites, even presenting excellent polishing properties16. because of this, a nanohybrid composite was selected to restore the patient’s lesions, due to its excellent mechanical properties and good polishing. the polishing procedure is important to decrease the biofilm adhesion on these restored surfaces, avoiding inflammation and gingival recession13. because of the association between occlusal stress and abfraction lesions, occlusal splints have been proposed as an alternative and conservative treatment for the management of abfraction lesions, to reduce the amount of nocturnal bruxism or clenching and to preserve the restorations33. besides, the use of occlusal splints can avoid other damages for dental, periodontal and musculoskeletal tissues, caused by nocturnal parafunctional activity33. to achieve the success of nccl lesions treatment it is necessary to identify correctly the etiology of the lesion, to act on them and to carefully select the restorative materials to be used. also, it is important to consider the patient’s oral hygiene and follow-up should be performed for long-term maintenance of restorations. the follow-up session was performed 1 year after treatment. it is advisable to monitor the progression of these lesions at regular intervals without any treatment intervention15. the assessment of lesion activity can be performed every 6 months to 12 months and during regular hygiene visits15. there was no progression of lesions smaller than 1 mm and no new lesions appeared. the orientation of brushing habits, occlusal adjustment and occlusal splints were determinant for this. there were no losses of any of the class v restorations performed. cervical lesions do not provide micromechanical retention and an ineffective adhesion may result in the loss of the restoration. this type of substrate is a single substrate probably not found in any other region of the mouth34. lesions are often characterized by the presence of physiologically and pathologically altered scleral dentin, resulting in partial or complete obliteration of the dentinal tubules. these lesions present a complex structure with high variability of tubule occlusion34. because of such characteristics, bond strengths to nccl have consistently been reported to be 20% -50% lower than bonds made to sound dentin34. nevertheless, in this clinical case the restorative protocol used resulted in a satisfactory performance after 1 year with perfectly adapted restorations and with good surface polishing, due to the focus on nccls etiology and adequate restoration technique performed. in addition, the restorative procedure resulted in the absence of dh, since restorative treatments of nccls help to decrease the thermal sensitivity16. nccls still represent a challenge for clinical practice and the accurate diagnosis and the choice of proper treatment is the key of success for theses lesions. the restorative treatment must be considered for dentin hypersensibility and for the re-establishing of dental esthetics. however, a good finishing and polishing is essential for gingival health. 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in non-carious cervical lesions. dent mater. 2010 sep;26(9):940-6. doi: 10.1016/j.dental.2010.05.009. 31. srirekha a, bashetty k. a comparative analysis of restorative materials used in abfraction lesions in tooth with and without occlusal restoration: three-dimensional finite element analysis. j conserv dent. 2013 mar;16(2):157-61. doi: 10.4103/0972-0707.108200. 32. cieplik f, scholz kj, tabenski i, may s, hiller ka, schmalz g, et al. flowable composites for restoration of non-carious cervical lesions: results after five years. dent mater. 2017 dec;33(12):e428-e437. doi: 10.1016/j.dental.2017.09.012. 33. tsiggos n, tortopidis d, hatzikyriakos a, menexes g. association between self-reported bruxism activity and occurrence of dental attrition, abfraction, and occlusal pits on natural teeth. j prosthet dent. 2008 jul;100(1):41-6. doi: 10.1016/s0022-3913(08)60135-3. 34. carvalho rm, manso ap, geraldeli s, tay fr, pashley dh. durability of bonds and clinical success of adhesive restorations. dent mater. 2012 jan;28(1):72-86. doi: 10.1016/j.dental.2011.09.011. braz j oral sci. 15(1):16-20 original article braz j oral sci. january | march 2016 volume 15, number 1 influence of ultrasonic activation in association with different final irrigants on intracanal smear layer removal matheus albino souza1, felipe trentin motter1, tobias pitton fontana1, marlos barbosa ribeiro2, daniela cristina miyagaki1, doglas cecchin1 1universidade de passo fundo – upf, school of dentistry, department of restorative dentistry, passo fundo, rs, brazil 2universidade de campinas – unicamp, piracicaba dental school, department of restorative dentistry, piracicaba, sp, brazil correspondence to: matheus albino souza. programa de pós-graduação – universidade de passo fundo br 285, são josé, prédio a7, sala 2 cep: 99052-900. passo fundo rs-brazil phone: +55 54 3316-8402 e-mail: matheus292@yahoo.com.br abstract aim: to evaluate the influence of ultrasonic activation (us) with different irrigant regimens in smear layer removal. methods: one hundred bovine incisors were instrumented and divided into ten groups (n=10) according to final irrigation protocols: distilled water (dw); dw+us; 17% edta; qmix; 10% citric acid; 37% phosphoric acid; 17% edta+us; qmix+us; 10% citric acid+us; 37% phosphoric acid+us. the samples were then submitted to scanning electron microscopy where a score system was used to evaluate the images and effectiveness of proposed treatments. the data were statistically analyzed by kruskal-wallis and mann-whitney u tests for intergroup comparisons as well as the wilcoxon and friedman tests for intragroup comparisons at 5% significance level. results: in the cervical third, groups 17% edta, qmix, 10% citric acid, 17% edta+us, qmix+us and 10% citric acid+us were more effective in smear layer removal (p<0.05); in the middle third, groups 17% edta+us and qmix+us were more effective in smear layer removal (p<0.05); in the apical third, groups 17% edta,17% edta+us and qmix+us were more effective in smear layer removal (p<0.05). conclusions: us can aid 17% edta and qmix in smear layer removal at the middle third and qmix at the apical third, contributing to the cleaning of root canal system. keywords: root canal irrigants. smear layer. ultrasonics. introduction microorganisms and their products are the main etiological factor of pulp and periapical pathology. they play a significant role in the induction and progression of the disease1. therefore, chemomechanical procedures are necessary to control microbial agents, where the chemical action of auxiliary chemical substances and mechanical action of endodontic instruments contribute to adequate cleanliness during the root canal therapy. however, an agglomeration of dentin chips, irrigant solutions, microorganisms and organic matter, called smear layer, forms during root canal preparation and remains adhered to the root canal walls2. the presence of smear layer represents a barrier to intracanal dentin that limits penetration of auxiliary chemical substances and intracanal dressings into infected dentinal tubules3. furthermore, it also increases microleakage through usual root canal sealers and decreases the bond strength of resin-based materials4. several substances have been used in root canal therapy in order to promote smear layer removal, like edta, citric acid and phosphoric acid5. more recently, a novel endodontic irrigant called qmix (dentsply tulsa dental specialties, tulsa, ok, usa), received for publication: march 29, 2016 accepted: may 16, 2016 http://dx.doi.org/10.20396/bjos.v15i1.8647092 17 which contains edta, chlorhexidine and surfactant agent in its composition, have also been used as a final irrigation protocol in endodontics6. however, due to the anatomical complexity of the root canal system, inorganic and organic components cannot always be reached by irrigants7, requiring the use of auxiliary techniques to promote an effective smear layer removal. the ultrasonic activation (us) is the activation of an endodontic instrument by an ultrasonic device placed inside the root canal. this promotes mechanical agitation of a chemical substance without instrument contact with the root canal wall8. some studies have recommended the use of us to improve smear layer removal upon delivery of edta with needles without agitation9,10. despite the relevant studies concerning the benefits of this technique and edta on smear layer removal, there is no consensus in the literature regarding the use of us with other agents, such as qmix, citric acid and phosphoric acid. it limits the ability to make valid comparisons between the products, especially when considering the use of us. thus, the purpose of present study was to evaluate, in vitro, the influence of us with different final irrigants in intracanal smear layer removal, using scanning electron microscopy (sem). the tested null hypothesis was that the use of us with four tested irrigants would not promote better removal of the smear layer. material and methods this study was approved by the ethics committee of the school of dentistry of university of passo fundo (passo fundo, rs, brazil), protocol 016/2014. smear layer production and irrigation protocols one hundred bovine incisors from animals killed for commercial reasons were used. the teeth were extracted from the jaws immediately after the death of the animals and stored in vials containing 4% formalin (royal plás, curitiba, pr, brazil), in order to preserve their properties, for a period up to 72 h. dental crowns were sectioned with a rotating diamond saw (#911h brasseler, savannah, ga, usa) set at 20,000 rpm under water-coolant, so that all of the roots were 15 mm long. all samples were prepared using the same protocol in order to remove pulp tissue, standardize the canal diameter and produce smear layer. the cervical third was prepared using #4 and #5 largo drills (dentsply-maillefer, ballaigues, switzerland). the working length was established by introducing a #10 k-file (dentsply-maillefer) in the canal until its tip was visualised at the apical foramen. one millimeter was subtracted from this measurement, establishing the working length. after that, only straight roots with apical diameters equal to the k-file size 25 (dentsply-maillefer) were included in this study. the roots were enlarged up to instrument #60 (dentsply-maillefer), by serial instrumentation, using 2.5% sodium hypochlorite (naocl) (decloquimis, são paulo, sp, brazil) as irrigant, in order to remove organic components from dentin. the samples were completely filled with 2.5% naocl and the root canals were irrigated with 2 ml of the same solution using disposable 5 ml syringes (ultradent products, south jordan, ut, usa) and a 30-gauge needle (navi tip, ultradent products) after each change of instrument. after complete root canal instrumentation, the teeth were irrigated with 5 ml of naocl (decloquimis). following preparation, each root was fixed with putty-c silicone for impression (silon2aps – dentsply, petrópolis, rj, brazil) in a plastic micro-tube (axygen inc., union city, ca, usa), to prevent the flow of chelating agents through the apical foramen. the tested substances were 17% edta (biodinâmica, ibiporã, pr, brazil), qmix (dentsply tulsa dental specialties, tulsa, ok, usa), 10% citric acid (biodinâmica) and 37% phosphoric acid solution (biodinâmica). the samples were randomly divided into 10 groups (n=10) according to the protocol of final irrigation, as follows: distilled water (dw),control group; dw + us; 17% edta; qmix; 10% citric acid; 37% phosphoric acid; 17% edta + us; qmix + us; 10% citric acid + us; 37% phosphoric acid + us. the groups with no us were treated using the same protocol. first, the root canals were completely filled with 2 ml of the tested solution. then, the tested solution remained in contact with root canal walls for 3 min. after that, irrigation with 3 ml distilled water was performed, concluding the smear layer removal procedure. using the same protocol, groups with us were prepared. first, the root canals were filled with 2 ml of tested solution. then, the tested solution remained in contact with root canal walls for 2 min. next, us was performed using an ultrasonic device (nac plus ultrasonics adiel, ribeirão preto, sp, brazil). the stainlesssteel endodontic tip to a size et40 (satelec-acteon, mount laurel, nj, usa) was inserted 1 mm short of the working length and activated for 1 min, resulting in the same 3 min of contact with root canals, as performed in groups with no us. scale power 3 for endodontics (75% power) was used to promote the ultrasonic activation. after that, irrigation with 3 ml of distilled water was performed, concluding the smear layer removal procedure. scanning electron microscopy (sem) all root canals were dried with #60 size paper points (tanari, manaus, am, brazil) after procedures of smear layer removal. two longitudinal grooves were prepared on the external root surface by diamond disc without reaching the canal space. subsequently, the roots were split into two halves with a hammer and chisel. for each root, the half containing the most visible part of the apex was used for sem preparation and analysis. the samples were dehydrated in increasing ethanol concentrations up to 100% and mounted on aluminum stubs. next, they were coated with gold palladium and examined in a scanning electron microscope (jsm 6460 lv; jeol, tokyo, japan) operated at 15 kv. all samples were numbered, and the images were obtained without disclosing the tested group. first, a scan of all samples was made at 30x of magnification for each group, in order to identify each third. then, an area of each third of each tooth was randomly selected and magnified at 100x. each 100x image was scanned, and three areas were magnified at 1000x in order to perform the capture of images. therefore, three images of each third were obtained for each tooth, providing 9 images per tooth and 90 images per group (n=10). in the end, each group had 30 images for each third of root canal. influence of ultrasonic activation in association with different final irrigants on intracanal smear layer removal braz j oral sci. 15(1):16-20 18 sem evaluation the effectiveness of tested protocols on smear layer removal was evaluated by the scoring system described by prado et al.11, where each micrograph was scored using a semi-quantitative analysis with a four step scale as follows: score 1 no smear layer, with all tubules cleaned and opened; score 2 = few areas covered by smear layer, with most tubules cleaned and open; score 3 = smear layer covering almost all the surface, with few open tubules; and score 4 = smear layer covering all the surfaces. two blinded observers performed this evaluation. figure 1 provides illustration of representative photomicrographs of the scoring system used to analyze the sem results. statistical analysis the weighted kappa coefficient test was performed in order to verify the reliability for the sem evaluation between the readings of observers. the data were analyzed using the kruskalwallis and mann-whitney u tests for intergroup comparisons (p<0.05) as well as the wilcoxon and friedman tests for intragroup comparisons (p<0.05). results the kappa test showed good agreement between observers, with values of 0.9 or above. the mean and standard deviation of smear layer scores for each group are in table 1. the intergroup analysis revealed that the us did not promote better results to tested substances in the cervical third. in the middle and apical third, groups 17% edta+us and qmix+us were more effective on smear layer removal compared with all other groups, with no statistically significant difference between them (p<0.05), and similar to group 17% edta in the apical third. furthermore, the intragroup analysis revealed that groups 17% edta and qmix+us were more effective in the cervical third than in the apical third, being similar to medium third; whereas qmix, 10% citric acid and 10% citric acid+us were more effective in the cervical third when compared to medium and apical third (p<0.05). influence of ultrasonic activation in association with different final irrigants on intracanal smear layer removal fig.1. photomicrographs by sem of the scoring system used to analyze the effectiveness of each group in smear layer removal. (a) score 1; (b) score 2; (c) score 3; (d) score 4. * different capital letters indicate significant differences between groups. different small letters indicate significant differences between root thirds (p<0.05) ** dw=distilled water; us=ultrasonic activation table 1 mean and standard deviation of smear layer scores for each group. group cervical middle apical 1. dw 3.50 ± 0.52 a,a 3.50 ± 0.52a,a 3.50 ± 0.52a,a 2. dw+us 3.45 ± 0.48 a,a 3.48 ± 0.51a,a 3.49 ± 0.50a,a 3.edta 1.70 ± 0.67 b,a 2.10 ± 0.73b,ab 2.10 ± 0.50b,b 4. qmix 1.40 ± 0.51 b,a 2.40 ± 0.96b,b 2.80 ± 0.78a,b 5. citric acid 1.70 ± 0.48 b,a 2.50 ± 0.52b,b 2.70 ± 0.48a,b 6. phosphoric acid 3.20 ± 0.78 a,a 3.30 ± 0.67a,a 3.30 ± 0.82a,a 7. edta+us 1.60 ± 0.51 b,a 1.50 ± 0.52c,a 2.00 ± 0.53b,a 8. qmix+us 1.20 ± 0.42 b,a 1.50 ± 0.52c,ab 2.00 ± 0.61b,b 9. citric acid+us 1.90 ± 0.31 b,a 2.40 ± 0.80b,b 3.30 ± 0.82a,b 10. phosphoric acid+us 3.30 ± 0.67 a,a 3.30 ± 0.48a,a 3.30 ± 0.67a,a discussion the action time of auxiliary chemical substances used inside the root canal as final irrigants is variable in the literature. according to a previous study by prado et al.11, 17% edta was used for 30 s. however, this chelating agent used for a short period resulted in low performance in smear layer removal, compared with results obtained by distilled water (control group). this results underscores that 17% edta is ineffective for smear layer removal after 30 s of contact with root canal walls. çalt and serper12 found similar results, where the use of chelating agents for 1 min did not result in effective removal of smear layer produced over radicular dentin. the action time of final irrigation protocols tested in the present study was set at 3-min contact with the root canal walls. this time was based on a previous study of scelza et al.13, where the results showed that final irrigation protocols were effective after 3 min and did not show better results for smear layer removal after increasing the contact time of chemical agents with the root canal walls. the null hypothesis of present study, that us over four chelating agents would not promote better removal of the smear layer was not confirmed, since the use of us has not improved only the ability of 10% citric acid and 37% phosphoric acid to promote smear layer removal in every third of root canal. the use of ultrasonic activation (us) as an auxiliary technique in endodontic therapy has been suggested as a method to increase cleaning and disinfection of the root canal system8,14. however, its use has been limited to endodontic irrigants such as naocl and calcium hypochlorite15,16. our goals were to investigate the influence of ultrasonic activation on improvement of four chelating agents in smear layer removal. according to the results of the present study, the use of us with the four tested chelating agents in the cervical third did not improve the smear layer removal capacity compared with groups where us was not used. however, in the middle third, groups 17% edta+us and qmix+us have shown a higher ability to promote smear layer removal compared with all other groups. however, in the apical third, the group braz j oral sci. 15(1):16-20 19 qmix+us has shown a greater ability to promote smear layer removal when compared with group qmix, where us was not performed. these findings are in accordance with those of with previous studies, which reported that smear layer removal was enhanced with the adjunctive use of ultrasonic irrigation9,10,17. the action of ultrasonic devices induces hydrodynamic turbulence in the solution inside the root canal, producing cavitation and bubbles that collide against the walls. these elements increase the temperature and hydrostatic pressure, producing waves to remove the smear layer by continuous irrigation with ultrasonic device18. the higher effectiveness of qmix+us on smear layer removal in the cervical third compared with the apical third may be explained by the higher volume of contact of the tested chelating agents in the cervical third and by the difficulty of effectively reaching the remaining thirds of the root canals. due that oscillation amplitude is greatest on the tip of the instrument, any interference may significantly affect the apical portion19. the current results confirm these concepts, because us has shown less influence on qmix and 10% citric acid in smear layer removal in the apical portion of the root canals. the 37% phosphoric acid did not demonstrate effectiveness in smear layer removal, according to the results of the present study, even with us. the results of groups 37% phosphoric acid and 37% phosphoric acid+us were similar to control group (dw), revealing high amounts of smear layer after the tested protocols. the present results disagree with those of a previous study11 in which phosphoric acid solution showed excellent results on smear layer removal after a 3-min exposure, even in the apical third. however, the use of high-concentration phosphoric acid is related to dentinal erosion11 and may carry a higher risk of cytotoxicity, especially when used in the apical third of the root canal. the lowest means of smear layer removal were observed in the groups 17% edta+us and qmix+us, even with no statistically significant difference between these groups and group 17% edta, in the cervical and apical thirds. however, 17% edta may promote erosion of peritubular and intertubular dentin, which may compromise the fracture strength of tooth20-22. despite the presence of edta in its composition, qmix has not shown the ability to promote dentinal erosion20. qmix also comprises chlorhexidine and a detergent (surfactant agent). chlorhexidine has a broad spectrum of antimicrobial activity23, substantivity24 and promotes a reduction of dentinal microhardness25, while the surfactant agent decreases the surface tension and increases the wettability26, increasing the effectiveness in penetrating deeper into the dentinal tubules, thus contributing to decontamination of root canal system. for these reasons and according to current results, the present study suggests that the qmix with us may be a good protocol for final irrigation, reinforcing the findings of niu et al.27, where activation of qmix appears to maximize the smear layer removal, bringing significant benefits to endodontic therapy. considering such results, we believe that ultrasonic activation can aid 17% edta and qmix in smear layer removal, contributing in a significant way to the cleaning of root canal system, since better results were found for 17% edta and qmix in the middle third, and for qmix in apical third when ultrasonic activation of these substances was performed. further studies are required to evaluate the depth of demineralization caused by us with chelating agents and its influence on dentinal adhesion of filling materials in order to optimize these protocols in endodontics. references 1. kakehashi s, stanley hr, fitzgerald rj. the effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats. oral surg oral med oral pathol . 1965 sep;20:340-9. 2. teixeira cs, felippe mc, felippe wt. the effect of application time of edta and naocl on intracanal smear layer removal: an sem analysis. int endod j. 2005 may;38(5):285-90. 3. wang z, shen y, haapasalo m. effect of smear layer against disinfection protocols on enterococcus faecalis-infected dentin. j endod. 2013 nov;39(11):1395-400. doi: 10.1016/j.joen.2013.05.007. 4. shahravan a, haghdoost aa, adl a, rahimi h, shadifar f. effect of smear layer on sealing ability of canal obturation: a systematic review and meta-analysis. j endod. 2007 feb;33(2):96-105. 5. pérez-heredia m, ferrer-luque cm, gonzález-rodríguez mp, martínpeinado fj, gonzález-lópez s. decalcifying effect of 15% edta, 15% citric acid, 5% phosphoric acid and 2.5% sodium hypochlorite on root canal dentine. int endod j. 2008 may;41(5):418-23. doi: 10.1111/j.1365-2591.2007.01371.x. 6. stojicic s, shen y, qian w, johnson b, haapasalo m. antibacterial and smear layer removal ability of a novel irrigant, qmix. int endod j. 2012 apr;45(4):363-71. doi: 10.1111/j.1365-2591.2011.01985.x. 7. george s, kishen a, song kp. the role of environmental changes on monospecies biofilm formation on root canal wall by enterococcus faecalis. j endod. 2005 dec;31(12):867-72. 8. van der sluis lw, versluis m, wu mk, wesselink pr. passive ultrasonic irrigation of the root canal: a review of the literature. int endod j. 2007 jun;40(6):415-26. epub 2007 apr 17. 9. caron g, nham k, bronnec f, machtou p. effectiveness of different final irrigant activation protocols on smear layer removal in curved canals. j endod. 2010 aug;36(8):1361-6. doi: 10.1016/j.joen.2010.03.037. 10. blank-gonçalves lm, nabeshima ck, martins gh, machado me. qualitative analysis of the removal of the smear layer in the apical third of curved roots: conventional irrigation versus activation systems. j endod. 2011 sep;37(9):1268-71. doi: 10.1016/j.joen.2011.06.009. 11. prado m, gusman h, gomes bp, simão ra. scanning electron microscopic investigation of the effectiveness of phosphoric acid in smear layer removal when compared with edta and citric acid. j endod. 2011 feb;37(2):255-8. doi: 10.1016/j.joen.2010.11.011. 12. calt s, serper a. time-dependent effects of edta on dentin structures. j endod. 2002 jan;28(1):17-9. 13. scelza mf, pierro v, scelza p, pereira m. effect of three different time periods of irrigation with edta-t, edta, and citric acid on smear layer removal. oral surg oral med oral pathol oral radiol endod. 2004 oct;98(4):499-503. 14. plotino g, pameijer ch, grande nm, somma f. ultrasonics in endodontics: a review of the literature. j endod. 2007 feb;33(2):81-95. 15. gründling gl, zechin jg, jardim wm, de oliveira sd, de figueiredo ja. effect of ultrasonics on enterococcus faecalis biofilm in a bovine tooth model. j endod. 2011 aug;37(8):1128-33. doi: 10.1016/j. joen.2011.05.006. 16. de almeida ap, souza ma, miyagaki dc, dal bello y, cecchin d, farina ap. comparative evaluation of calcium hypochlorite and sodium hypochlorite associated with passive ultrasonic irrigation on antimicrobial activity of a root canal system infected with enterococcus braz j oral sci. 15(1):16-20 influence of ultrasonic activation in association with different final irrigants on intracanal smear layer removal faecalis: an in vitro study. j endod. 2014 dec;40(12):1953-7. doi: 10.1016/j.joen.2014.08.025. 17. rödig t, döllmann s, konietschke f, drebenstedt s, hülsmann m. effectiveness of different irrigant agitation techniques on debris and smear layer removal in curved root canals: a scanning electron microscopy study. j endod. 2010 dec;36(12):1983-7. doi: 10.1016/j. joen.2010.08.056. 18. ribeiro em, silva-sousa yt, souza-gabriel ae, sousa-neto md, lorencetti kt, silva sr. debris and smear removal in flattened root canals after use of different irrigant agitation protocols. microsc res tech. 2012 jun;75(6):781-90. doi: 10.1002/jemt.21125. 19. jensen sa, walker tl, hutter jw, nicoll bk. comparison of the cleaning efficacy of passive sonic activation and passive ultrasonic activation after hand instrumentation in molar root canals. j endod. 1999 nov;25(11):735-8. 20. saghiri ma, delvarani a, mehrvarzfar p, malganji g, lotfi m, dadresanfar b, et al. a study of the relation between erosion and microhardness of root canal dentin. oral surg oral med oral pathol oral radiol endod. 2009 dec;108(6):e29-34. doi: 10.1016/j. tripleo.2009.07.056. 21. qian w, shen y, haapasalo m. quantitative analysis of the effect of irrigant solution sequences on dentin erosion. j endod. 2011 oct;37(10):1437-41. doi: 10.1016/j.joen.2011.06.005. 22. aranda-garcia aj, kuga mc, chavéz-andrade gm, kalatzis-sousa ng, hungaro duarte ma, faria g, et al. effect of final irrigation protocols on microhardness and erosion of root canal dentin. microsc res tech. 2013 oct;76(10):1079-83. doi: 10.1002/jemt.22268. 23. ferraz cc, gomes bp, zaia aa, teixeira fb, souza-filho fj. in vitro assessment of the antimicrobial action and the mechanical ability of chlorhexidine gel as an endodontic irrigant. j endod. 2001 jul;27(7):452-5. 24. böttcher de, sehnem nt, montagner f, fatturi parolo cc, grecca fs. evaluation of the effect of enterococcus faecalis biofilm on the 2% chlorhexidine substantivity: an in vitro study. j endod. 2015 aug;41(8):1364-70. doi: 10.1016/j.joen.2015.04.016. 25. oliveira ld, carvalho ca, nunes w, valera mc, camargo ch, jorge ao. effects of chlorhexidine and sodium hypochlorite on the microhardness of root canal dentin. oral surg oral med oral pathol oral radiol endod. 2007 oct;104(4):e125-8. 26. giardino l, ambu e, becce c, rimondini l, morra m. surface tension comparison of four common root canal irrigants and two new irrigants containing antibiotic. j endod. 2006 nov;32(11):1091-3. 27. niu ln, luo xj, li gh, bortoluzzi ea, mao j, chen jh, et al. effects of different sonic activation protocols on debridement efficacy in teeth with single-rooted canals. j dent. 2014 aug;42(8):1001-9. doi: 10.1016/j. jdent.2014.05.007. 20 braz j oral sci. 15(1):16-20 influence of ultrasonic activation in association with different final irrigants on intracanal smear layer removal untitled 1http://dx.doi.org/10.20396/bjos.v16i0.8651062 volume 16 2017 e17040 original article 1 msc student, department of orthodontics, school of dentistry, universidade federal fluminense, niterói, rio de janeiro, brazil. 2 adjunct professor, department of orthodontics, school of dentistry, federal fluminense university, niterói, rio de janeiro, brazil. 3 professor and chair, department of orthodontics, school of dentistry, universidade federal fluminense, niterói, rio de janeiro, brazil. corresponding author: taísa figueiredo chagas. disciplina de ortodontia. faculdade de odontologia. rua mário santos braga, 30, 2º andar, sala 214cep 24.020-140. niterói/rjbrazil. tel: (21) 2622-1621 / (21) 2629-9812. chagastaisa@hotmail.com received: april 21, 2017 accepted: september 14, 2017 the facial profile in brazilian adults taísa figueiredo chagas1, mariana martins e martins2, josé nelson mucha3 aim: to investigate if there is agreement between measurement values obtained for brazilian adults and the values recommended by holdaway, merrifield, burstone, steiner and ricketts, for facial profile soft tissue analysis. methods: a sample of 30 cephalometric radiographs was used, consisting of 15 women and 15 men, aged 18 to 31 years, all exhibiting excellent occlusion and balanced facial profile. comparisons were made with the measurement values proposed by the aforementioned authors using student’s t-test and to determine the correlation between the anb and â-h measurement values described by holdaway, using pearson’s correlation coefficient. results: among the measures evaluated, 4 showed statistically significant differences: â.z (merrifield), s-ls and s-li (steiner), and e-li (ricketts) relative to the standards recommended by the authors. the measurement values advocated by merrifield, steiner and ricketts showed statistical differences, and as for the z-angle, brazilians feature a slightly more convex profile, which appeared slightly concave according to steiner, and according to the e-plane (ricketts), it meant an increased protrusion of the lower lip. conclusion: it could be asserted that adult brazilians have a slightly more convex facial profile than us standards, but these differences should be viewed with caution, as they are clinically unimportant. keywords: cephalometry; diagnostic imaging, radiography https://www.ncbi.nlm.nih.gov/mesh/68011859 2 chagas et al. introduction variables such as gender and ethnic origin have different characteristics of skeletal, dental and soft tissue normality, which require knowledge of what is considered normal or standard in each ethnic group1-4. in addition, patients expect results to be in accordance with social and cultural standards of beauty prevalent in their reference group, the zeitgeist, as well as in society in general5. the standards for hard and soft tissues should be considered in establishing a balanced facial aesthetics and an ideal functional occlusion. however, the most widely accepted normative values are based on studies of untreated subjects of european6,7, or north american stock8. thus, white brazilian adults with excellent occlusion might display the position of the lips in the contour of the facial profile in a different relationship from those advocated by various authors for a population of north american or european origin, which would necessarily entail different clinical decisions when planning the orthodontic treatment. although the issue has been addressed in the literature, no single work has hitherto evaluated the major facial profile measurements for white brazilian adults with a balanced facial profile and established an ideal occlusion. the aim of this study was to investigate if there is agreement between the measurement values obtained for white brazilian adults and the values recommended by steiner6, ricketts8, holdaway9, burstone10, e merrifield11 for an analysis of facial profile soft tissues. the tested null hypothesis was that there would be no statistically significant difference between the measurement values obtained and the values recommended by these authors. material and methods cephalometric measurements of facial profile soft tissues were obtained from 30 cephalometric radiographs from the archives of the department of orthodontics, federal fluminense university, composed of white brazilian adults aged 18 to 31 years old with all teeth, in established normal or excellent occlusion and balanced facial profile. the sample comprised 15 women (mean age = 22.67 ± 3.48) and 15 men (mean age = 23.93 ± 3.47). inclusion criteria were: subjects with all teeth in normal or excellent occlusion and balanced facial profile. controversial occlusion cases were examined by three orthodontists and removed from the sample, as were patients with a history of trauma and previous orthodontic treatment. cephalometric radiographs were taking in natural head position and the cephalograms were traced manually by a single calibrated operator. the anatomical details, points, lines and planes that make up the analysis of soft tissue described by steiner6, ricketts8, holdaway9, burstone10, e merrifield11 and were marked (figure 1). 3 chagas et al. in order to assess intraexaminer method error measurements from 10 radiographs were evaluated in two stages, with a minimum interval of one week. after the initial evaluation, new tracings were carried out, determining the new points, lines and planes that make up the analysis of the soft tissues proposed for the study. moreover, intraclass correlation coefficient (icc) was applied. the means and variations around the means were obtained and comparisons with the measurement values advanced by the aforementioned authors were made6,8-11. a pilot study was done for the accomplish of sample calculation in which we selected 10 participants randomly, indicating the need to include 30 individuals. the differences between genders and between ages were compared, and the shapiro-wilk test was used to analyze data normality. student’s t-test was applied to those variables whose data distribution was normal in order to compare the means for gender characteristics. the purpose was to determine whether or not sexual dimorphism was present. furthermore, the mann-whitney test was applied to those variables whose data distribution was abnormal. the mean measurement values found in this study were compared to the values proposed by the authors6,8-11 using t-test. the correlation between anb and â-h measurement values as described by holdaway9 was tested using pearson’s correlation coefficient. figure 1. measures used (1) h-angle9; (2) anb angle9; (3) z-angle11; (4) linear measurement from the most anterior point of the upper lip to the line joining the subnasal and pogonion points10; (5) linear measurement from the most anterior point of the lower lip to the line joining the subnasal and pogonion points10; (6) linear measurement from the most anterior point of the upper lip to the line joining the midpoints of the base of the nose to the pogonion6; (7) linear measurement from the most anterior point of the lower lip to the line joining the midpoints of the base of the nose to the pogonion6; (8) linear measurement from the most anterior point of the upper lip to the line connecting the tip of the nose and pogonion points8; (9) linear measurement from the most anterior point of the lower lip to the line joining the tip of the nose and pogonion points8 8 97 64 5 1 2 3 4 chagas et al. biostat 5.3 (belém, pará, brazil) software was employed for statistical analysis and significance level of 5% was adopted for all tests (p<0.05). results in evaluating icc, excellent reproducibility was found in most measures (â.h, anb, â-z lb-ls and lb-li), as well as an average to good reproducibility in 4 other measures (s-ls, s-li, e-ls and e-li). age in the total sample ranged from 18 to 31 years, with a mean value of 23.30 (sd ± 3.48). in males, the mean value was 23.93 (sd ± 3.47), and in females, 22.67 (sd ± 3.48), with no statistically significant difference between the groups as a result of the mann-whitney test (p-value = 0.2998). all individuals included have a balanced facial profile. twenty eight were skeletal class i (anb between 0 and 4.5) and only one take anb=-1 and another take anb=5. each of the nine measures was evaluated to elicit differences between genders. distribution was tested by the shapiro-wilk test. the five measurements showed abnormal distribution (a-h, lb, ls, li s-e-e-ls, li). the mann-whitney test was applied to these measurements and independent t test was used for other measurements with normal distribution. given that no significant differences were found between the genders, the subgroups were gathered in a single sample group (n = 30) and statistical analysis was performed with independent t test (table 1). no statistically significant differences were found between the means of the anb and â.h measurement values using the parameters provided by holdaway9. the same proved true for the lb-ls and lb-li mean values using the parameters provided by burstone10. table 1. statistical results for measurement values obtained for white brazilian adults and the values recommended by authors for an analysis of facial profile soft tissues measure mean (sd) standard error range author’s mean diferences between mean and author’s mean p holdaway â.h (0) 8.11 (2.42) 0.44 1.5/13.9 8 0.11 0.7939 anb (0) 1.68 (1.17) 0.21 -1/5 2 0.32 0.1491 merrifield âz (0) 77.93 (4.85) 0.89 66/89 80 2.07 0.0267* burstone lb-ls mm 3.41(1.75) 0.32 -2.5/6 3.5 0.09 0.7963 lb-li mm 2.28 (1.56) 0.29 -1/5 2.2 0,08 0.7761 steiner s-ls mm -1.48(1.6) 0.29 -5/2 0 -1.48 <0.0001* s-li mm -1.01(1.6) 0.29 -4/1.5 0 -1.01 0.0016* ricketts e-ls mm -4.85 (2.58) 0.48 -9/5 _ _ e-li mm -2.78 (2.71) 0.50 -6/5 -4 -1.22 0.0203* *significant difference (p < 0.05) based on independent t test 5 chagas et al. holdaway9 described a correlation between anb and â-h. thus, this correlation was tested in this sample using the pearson correlation coefficient, and a positive (r=0.4419) and significant (p=0.0144) correlation was found. due to the dependence among these measures a simple linear regression was performed and found the equation: y=6.5766x + 0.9149x, where y=a.h and x=anb. a statistically significant difference was found between mean â.z values in the sample and those provided by merrifield11. differences were also found between the means of the s-ls and s-li measurement values compared to the standards provided by steiner6; and between the mean values for e-li relative to the norms provided by ricketts8. only a descriptive analysis of the e-ls was performed since ricketts8 failed to provide a reference value for this measure. discussion regarding holdaway’s9 analysis, given that statistically significant differences were not observed among the measures of this sample and the above measures, one can consider that the standards and correlations of this analysis can be applied to white brazilian adults. for the z-angle a mean of 77.93 degrees was found, 2.07 degrees less than the value recommended by merrifield11, suggesting a slightly larger convexity in the profile of white brazilian adults. although it is a small difference it proved statistically significant. but clinically, this difference cannot be considered relevant. leichsenring et al.12 found a mean of 71.75 degrees and yu et al.13 found a mean of 68.33 for the z-angle in a study conducted with chinese patients with normal occlusion and balanced profiles, values lower than the 77.93 degrees found in this study. this is probably due to age differences between the samples and can be attributed to the early maturation of girls14. it is to be expected that due to mandibular growth there should be an increase in this angle. in measuring the subnasal-pogonion line in the soft tissues relative to the lips10 the values for lb-ls=3,41mm and lb-li=2,28mm, appeared very close to the standards. however, this applied only to adolescents with normal occlusion (lb-ls=3.50 and lb-li=2.20). one can therefore consider that the measurement values for american teenagers resemble those of brazilian adults. the s-ls (-1,48mm) and s-li (-1.01 mm) measures in this study showed slightly more retruded upper and lower lips compared to the standard established by steiner6. these differences were significant. moreover, the lower lip protruded more than the upper lip. the differences found in this study can be attributed to the growth of the nose and chin regions, causing the lips to take on a more retruded position relative to this line15. these differences may be explained by ethnic differences in each group. erbay, caniklioğlu, erbay et al.16 evaluated 96 turkish adults aged between 21.63 and 22.45 years, with normal occlusion, and also found lips that were more retruded than the values recommended by steiner6 (women: s-ls=2.7mm and s-li=-2.0mm, and men s-ls=3.3mm and s-li=-2.7mm). isiekwe et al.17 in evaluating 100 adult nige6 chagas et al. rian individuals (ages 18 to 25 years) with normal occlusion found the values of s-ls=5.89 mm and s-li=8.19 mm, indicating lips that were much more protruded than the values established by steiner.6 sharma18 also found upper and lower lips that were more protruded than steiner’s standards6 in assessing 121 nepalis with normal occlusion and well balanced faces (s-ls=2.1mm and s-li=2.2mm). regarding the measurement values found for ricketts’ aesthetic plane8, the e-ls averaged -4.85 mm and could not be compared quantitatively owing to the lack of a reference value. the difference was attributed to the early maturation of girls and their increased nose growth compared to boys. freitas et al.19 also found different results in white brazilians with normal occlusion (e-ls = -4.23mm), and although a sample of adolescents was used, the results were similar to those found in this study. in measuring e-li, the mean was -2.78mm, with the lower lip positioned 1.22mm more anteriorly in relation to the standards established by ricketts8, suggesting that the lower lip in white brazilian adults is more protruded than advocated by the author, and with a significant difference. nobuyasu et al.20 in evaluating brazilian individuals aged between 12 and 15 years with normal occlusion also found more protruded lower lips (-0.95mm ± 2,37mm). freitas et al.19 found -1.96mm for e-li in white brazilian adolescents with normal occlusion. however, the nose and chin positions were not evaluated separately, and those with a more developed nose and chin can provide a good aesthetic appearance due to their greater labial protrusion21. lahlou et al.22 also found more retruded upper and lower lips (e-ls=-1.23mm and e-li=-0.05mm) after evaluating 102 moroccan adults with normal occlusion and mean age of 21 years and 6 months. cephalometric measurements should not be used strictly for face evaluation and/or clinical examination. furthermore, extraoral photographic analysis is also extremely important. however, it is still through measurements that we can quantify patient changes and guide our treatment plans. neglecting soft tissue analysis and evaluating dental and skeletal relationships separately can produce misleading results since the soft tissues of the face vary in thickness, length and postural tonus in different individuals6. it is important to consider the specific variations in different populations and establish standards for each group, which should be treated according to their own characteristics23. in the present study, a sample group with ages ranging from 18 to 31 years was selected because we wanted to establish an excellent occlusion involving no changes, or at most minimal growth–driven changes in the facial profile so as to allow comparisons to be made. but caution should be exercised in growing patients, and the changes in nose, chin and lip growth should be considered. nose growth is greater in boys than in girls, and the convexity of the profile soft tissue increases with age, influenced by the position of the nose24. holdaway’s9 was the best analysis for relating the position of the lips with the other structures of the facial profile. besides, it yielded similar results to this sample. burstone’s10 analysis uses an area of stable growth, i.e., the subnasal point, and also showed results that were similar to this sample. 7 chagas et al. although this study was concerned with establishing standards for white brazilian adults, one should take into account the fact that brazil is a huge country with an interbred population, making it difficult to establish a single diagnosis and planning standard. one must also take into account each individual’s ascendency19,25, as there are differences in dentofacial relationship depending on the ethnic variability of each racial group19. this study was concerned with setting standards for white brazilian adults, and by including only whites, one should take into account the fact that brazil is a huge country with an interbred population, making it difficult to establish a single diagnosis and planning standard. the authors consider a limitation of this study the fact that the ancenstry of each individual included in the sample was not determined and evaluated as there are differences in the dentofacial relationship depending on the ethnic variability of each racial group19,25. contemporary orthodontics recognizes the ethnic diversity of human facial contours. however, we still use cephalometric measurements that were designed for specific populations. thus, the present work contributes to clinical practice as it establishes which cephalometric measures of soft tissue evaluation can be used for white brazilian adults and which should be evaluated with caution. even though some of the measurement values presented statistically significant differences compared to the standards or means recommended by different authors one can consider, from a clinical point of view, that the differences were of approximately 1 to 1.5mm, and that these values are therefore very close to the standards, enabling us to use these measurements as a parameter for white brazilian adults. it should also be emphasized that there is a wide variety of facial and ethnic types among individuals seeking treatment to improve the aesthetic appearance of their facial profile, thereby rendering these small differences of little clinical significance. in conclusion, no statistically significant differences were found between the mean values obtained in this sample and the mean values recommended by holdaway and burstone. the measurement values advocated by merrifield, steiner and ricketts showed statistical differences. nevertheless, regarding the z-angle, brazilians feature a more convex profile, which was slightly more concave compared to steiner’s, and showed an increased protrusion of the lower lip compared to the e-plane (ricketts). it could be asserted that white brazilian adults have a slightly more convex facial profile than us standards, but these differences should be viewed with caution, as they are clinically unimportant. references 1. tikku t, khanna r, maurya rp, verma s l, srivastava k.,kadu, m. cephalometric norms for orthognathic surgery in north indian population using nemoceph software. j oral biol craniofac res. 2014;4(2):94-103. 2. aldrees am. lateral cephalometric norms for saudi adults: a meta-analysis. saudi dent j. 2011;23(1):3-7. 3. haskell bs, segal es. ethnic and ethical challenges in treatment planning: dealing with diversity in the 21st century. angle orthod. 2014;84(2):380-2. 8 chagas et al. 4. scavone h, zahn-silva w, do valle-corotti km, nahás ac. soft tissue profile in white brazilian adults with normal occlusions and well-balanced faces. angle orthod. 2008;78(1):58-63. 5. kiekens rm, maltha jc, hof mat, kuijpers-jagtman am. objective measures as indicators for facial esthetics in white adolescents.  angle orthod. 2006;76(4):551-6. 6. steiner cc. cephalometrics for you and me. am j orthod 1953;39(10):729-55. 7. tweed ch. the frankfort mandibular incisal angle (fmia) in orthodontic diagnosis, treatment planning and prognosis. angle orthod 1954;24(3):121-69. 8. ricketts rm. esthetics, enviroments, and the law of lip relation. am j orthod. 1968;54(4):272-89. 9. holdaway ra. soft-tissue cephalometric analysis and its use in orthodontic treatment planning. am j orthod. 1983;84(1):1-28. 10. burstone cj. lip posture and its significance in treatment planning. am j orthod 1967;53(4):262-84 11. merrifield ll. the profile line as an aid in critically evaluating facial esthetics. am j orthod 1966;52(11):804-22 12. leichsenring a, invernici s, maruo it, maruo h, ignácio sa, tanaka o. evaluation of the merrifield´s “z” angle in the mixed dentition. rev. de clín. pesq. odontol 2004;1(2):9-14. 13. yu xn, bai d, feng x, liu yh, chen wj, li s et al. correlation between cephalometric measures and end-of-treatment facial attractiveness. journal of craniofacial surgery. 2016;27(2), 405-409. 14. bishara se, treder je, jakobsen jr. facial and dental changes in adulthood. am j orthod dentofacial orthop 1994;106(2):175-86. 15. chaconas sj; bratroff jd. prediction of normal soft-tissue facial changes. angle orthod. 1975;45(1):12-25. 16. erbay ef, caniklioğlu cm, erbay şk. soft tissue profile in anatolian turkish adults: part i. evaluation of horizontal lip position using different soft tissue analyses. am j orthod dentofacial orthop 2002;121(1):57-64. 17. isiekwe gi, olatokunbo co, chukwudi im. a cephalometric investigation of horizontal lip position in adult nigerians. j orthod 2012;39(3):160-9. 18. sharma jn. steiner’s cephalometric norms for the nepalese population. j orthod. 2011;38(1):21-31. 19. freitas lmad, freitas kmsd, pinzan a, janson g, freitas mrd. a comparison of skeletal, dentoalveolar and soft tissue characteristics in white and black brazilian subjects. j appl oral sci 2010;18(2):135-42. 20. nobuyasu m, myahara m, takahashi t, attizzani a, maruo h, rino w, carvalho smrd. padrões cefalométricos de ricketts aplicados a indivíduos brasileiros com oclusão excelente. rev dental press ortod ortop facial 2007;12(1):125-56. 21. czarnecki st, nanda rs, currier gf. perceptions of a balanced facial profile. am j orthod dentofacial orthop 1993;104(2):180-7. 22. lahlou k, bahoum a, makhoukhi mb, aalloula eh. comparison of dentoalveolar protrusion values in moroccans and other populations. eur j orthod 2010;32(4):430-4. 23. hwang hs, kim ws, mcnamara jr ja. ethnic differences in the soft tissue profile of korean and european-american adults with normal occlusions and well-balanced faces. angle orthod 2002;72(1):72-80. 24. chaconas sj. a statistical evaluation of nasal growth. amj orthod 1969;56(4):403-14. 25. fernandes tmf, pinzan a, sathler r, freitas mrd, janson g, vieira fp. comparative study of the soft tissue of young japanese-brazilian, caucasian and mongoloid patients. dental press j orthod. 2013;18(2):116-24. 1http://dx.doi.org/10.20396/bjos.v19i0.8656155 volume 19 2020 e206155 original article ¹ department of restorative dentistry, school of dentistry, state university of ponta grossa, pr, brazil. corresponding author: giovana mongruel gomes avenida carlos cavalcanti 4748 – uvaranas, ponta grossa, paraná, brazil. 84030-900 telephone: 005542988463753 email: giomongruel@gmail.com received: august 06, 2019 accepted: march 28, 2020 effect of surface treatments on repair strength, roughness and morphology in aged metal-free crowns yançanã luizy gruber¹, thaís emanuelle bakaus¹, bruna fortes bittencourt¹, joão carlos gomes1, alessandra reis1, giovana mongruel gomes1,* aim: the roughness and micromorphology of various surface treatments in aged metal-free crowns and the bond strength of these crowns repaired with composite resin (cr) was evaluated in vitro. methods: a cr core build-up was confectioned in 60 premolars and prepared for metal-free crowns. prepared teeth were molded with the addition of silicone, and the laboratory ceromer/ fiber-reinforced crowns (sr adoro/fibrex lab) were fabricated. subsequently, the crowns were cemented and artificially aged in a mechanical fatigue device (1.2 x 106 cycles), then divided into 4 groups (n = 15) according to the surface treatment: 1) phosphoric acid etching (pa); 2) pa + silane application; 3) roughening with a diamond bur + pa; and 4) sandblasting with al2o3 + pa. after the treatments, the crowns (n = 2) were qualitatively analyzed by scanning electron microscope (sem) and surface roughness (n = 5) was analyzed before and after the surface treatment (ra parameter). the remaining crowns (n = 8) received standard repair with an adhesive system (tetric n-bond) and a nanohybrid cr (tetric n-ceram), and the microshear bond strength (sbs) test was performed (0.5 mm/min). roughness and sbs data were analyzed by oneand two-way anova, respectively, as well as tukey’s post-test (α = 0.05). results: sandblasting with al2o3 + pa resulted in the highest final roughness and sbs values. the lowest results were observed in the pa group, whereas the silane and diamond bur groups showed intermediate values. conclusion: it may be concluded that indirect ceromer crowns sandblasted with aluminum oxide prior to pa etching promote increased roughness surface and bond strength values. keywords: ceramics. composite resins. electron microscope tomography. shear strength. surface properties. https://orcid.org/0000-0001-6603-5239 2 gruber et al. introduction indirect restorations, also known as “ceromer,” “polymeric glass porcelain,” or “second-generation laboratory cr,” are widely used in clinical practice because they minimize the adverse effects of direct restorations, such as polymerization shrinkage1, poor marginal adaptation, and postoperative sensitivity2 in addition, they can provide better standards of translucency and can be low-cost alternatives to all-ceramic restorations3. although indirect resins possess high mechanical strength, these restorations are subject to fractures as any other material. this type of failure should be carefully evaluated to define the best treatment. clinically, the affected crowns can be classified according to the extent of the fracture. a fracture can be minimal (e.g., cracks) or extensive (e.g., displacement of more than half of the crown)4,5. corroborating in vitro studies6-8, clinical studies show that most cases of crown fractures are repairable9,10. this is advantageous because complete replacement of indirect restorations may present more disadvantages than advantages, such as the treatment complexity and expense11. with the evolution of adhesive techniques, adhesive repair has been widely used and can be considered beneficial, allowing good longevity in this type of dental restoration12,13. for proper repair, the surface of the indirect restoration should be subjected to a pre-treatment to create micromechanical retention with the repair material14. in the available literature, several surface treatments techniques are described for the repair of composites. roughening with diamond burs, sandblasting with aluminum oxide, conditioning with hydrofluoric acid etching or pa etching, and silanization are the most frequently reported11,15,16. the current literature presents several studies comparing different surface treatments; however, the best pre-treatment technique still generates controversial results17-19. thus, the present study aimed to evaluate the surface roughness, morphology, and repair strength of aged indirect resin restorations with sem, microshear bond strength test, and digital roughness meter. the tested hypothesis was that differences would exist in morphology, surface roughness, and bond strength after various surface treatments. material and methods sixty extracted human mandibular premolars, with the protocol number 1871/10 from the research ethics committee of the state university of ponta grossa (brazil), were stored in distilled water at 4oc and used within 6 months after extraction. to be included in the study sample, teeth should be sound, without cracks, and not submitted to previous endodontic treatment. teeth were transversally sectioned 2 mm above the cement-enamel junction using a low-speed diamond saw (isomet 1000, buehler, lake bluff, il, usa) and received a standardized endodontic treatment. after 1 week, the root canals were prepared to receive glass fiber posts (whitepost dc # 0.5, fgm, joinville, sc, brazil), which were cemented with the excite dsc (ivoclar-vivadent, schaan, liechtenstein) adhesive system and variolink ii (ivoclar-vivadent) resin cement in accordance with the manufacturer’s instructions. 3 gruber et al. after the post-luting procedures, cores were built-up with a nanohybrid cr (tetric n-ceram, ivoclar-vivadent). an incremental technique was used to place the cr, and each 2 mm increment was light cured for 20 s. indirect composite crowns cementation the composite cores were prepared to receive a full indirect composite restoration using a high-speed hand piece under water cooling. in all roots a ferrule was made in the coronal ending with 2.0 mm height, 1.2 mm depth, and 1.5 mm occlusal reduction. full indirect composite restorations were fabricated with the sr adoro (ivoclar-vivadent) restorative system reinforced by fibers (fibrex-lab coronal, angelus, londrina, pr, brazil). after fitting and adjustment, the restorations were adhesively cemented with excite dsc and variolink ii according to the manufacturers’ recommendations. teeth were then embedded in acrylic resin (duralay, reliance, worth, il, usa) and periodontal ligament was simulated using a polyether impression material (impregum™ soft, 3m espe, st paul, mn, usa), according to the method described by soares et al. 20052. mechanical aging to increase the study’s reliability20, the samples were subjected to mechanical fatigue in a controlled chewing simulator (elquip, são carlos, sp, brazil). the samples were placed at the base of a material-fatigue-testing machine at a 90o angle in relation to the horizontal plane and were subjected to repetitive impacts directed on the occlusal surface of the crown. a lower force of 40 n (to avoid possible fractures) at a frequency of 2 hz was applied for 1.2 x 106 cycles, which represents 5 years of clinical service21,22. during the cycles, the samples were kept at 37oc in relative humidity. surface treatments of the indirect restorations and experimental groups the specimens were then randomly divided into 4 groups, according to the surface treatments. each treatment was performed on a square delimited area (3 mm x 3 mm) on the buccal surface of each crown. in the pa group, the buccal surfaces of the indirect cr were treated with 35% pa for 2 min according the manufacture’s recommendation, washed for 2 min with distilled water, and gently air dried for 5 s at 2 cm. for the silane group—after pa treatment as reported above—a silane coupling agent (prosil, fgm, joinvile, sc, brazil) was applied for 1 min with a disposable applicator, and the surface was dried with compressed air for 5 s at 2 cm. the buccal surfaces of the diamond bur group were roughened with a diamond bur (# 3195, kg sorensen, são paulo, sp, brazil) using a high-speed hand piece under water cooling for 5 s, with weak movements and minimal wear. then, the surface was conditioned with pa as reported in the first group. for the sandblasting group, the surfaces were sandblasted with 50 μm al2o3 (microblaster standard model, bio-art, são carlos, sp, brazil) for 10 s and then conditioned with pa as reported in the first group. 4 gruber et al. surface roughness test after mechanical aging, the initial roughness (ir) of five random buccal surface restorations per group was obtained with a digital roughness meter (mitutoyo surftest-301, mitutoyo-kawasaki, kanagawa, japan). three measures were performed on each specimen, and the arithmetic mean was obtained from these values. the mean represents the ir. surface roughness reading was performed using the ra parameter (µm) and the iso 2001 measuring profile23, a 0.25 mm cut-off, 1.25 mm in length and 0.1 mm/s speed. afterward, the specimens were submitted to the abovementioned surface treatments and stored at 37oc in artificial saliva, simulating oral condition. after 48 h of the surface treatments procedures, we measured the final roughness (fr) in the same way as the initial evaluation. sem analysis two restorations per group were prepared for the sem (ssx – 550; shimadzu, tokyo, japan). the surfaces were sputter coated with gold in a vacuum evaporator (belzers scd 050 sputtercoater, bal-tec, germany) and photomicrographs of representative areas were taken at 1.000x magnification. bond strength test after surface treatment, eight crowns per group were submitted to microshear bond strength test. for this purpose, one coat of the adhesive system (tetric n-bond, ivoclarvivadent) was applied on the delimited area (3 mm x 3 mm) of the treated buccal surfaces and then gently air dried for 5 s and light-cured for 10 s (table 1). three tygon tubes, approximately 0.75 mm in diameter and 1 mm high, were used for each crown. the tubes were positioned on the flattest areas of the treated buccal surface (3 mm x 3 mm) of the indirect restorations, filled with cr (tetric n-ceram, ivoclarvivadent), and individually photoactivated for 40 s. each light-cured specimen was protected with aluminum strip to afford protection from additional polymerization, as well as the unpolymerized specimens. all specimens were checked with an optical microscope (olympus-bx 51, olympus, tokyo, japan) at 10x magnification to discard any specimens with air bubbles or evident gaps at the interface. table 1. manufacturer, composition and instructions for each material used in the study. material (manufacturer) composition instructions for use tetric n-bond (ivoclar vivadent) phosphoric acid acrylate, hema, bisgma, urethane dimethacrylate, ethanol, film-forming agent, catalysts and stabilizers. apply a thick layer of tetric n-bond for at least 10 seconds. remove excess material and the solvent by a gentle stream of air and light-cure for 10 seconds. tetric n-ceram (ivoclar vivadent) dimethacrylates (19-20 wt.%); barium glass, ytterbium trifluoride, mixed oxide, copolymers (80-81 wt.%); additives, catalysts, stabilizers and pigments are additional contents (< 1 wt.%). the total content of inorganic fillers is 55–57 vol.%. the particle size of inorganic fillers is between 40 nm and 3000 nm apply tetric n-ceram in layers of max. 2 mm or 1. polymerize each layer individually for 40 seconds. 5 gruber et al. all light-curing procedures of this study were performed with a led light-curing device (radii plus, sdi limited, victoria, australia) using a 1200 mw/cm2 power density. the specimens were mounted in acrylic resin and placed in a universal testing machine (kratos, são paulo, sp, brazil), and a microshear force was applied using a shearing blade as close as possible to the adhesive interface. the load was applied to the interface at a crosshead speed of 1 mm/min until failure, and the bond strength values were recorded in mpa. statistical analysis before running parametric statistical analysis, we tested whether the assumptions of normality of the data and equality of variances were valid, using the shapiro-wilk and barlett tests at an alpha of 5%. the data from surface roughness and bond strength were statistically analyzed by oneand two-way anova, respectively, and tukey’s test was used for pairwise comparisons at a 5% significance level. all calculations were performed using spss® statistical software (statistical package for the social sciences, version 21.0 mac, spss inc., chicago, il, usa). results the means and standard deviations of surface roughness (ra parameter) and microshear bond strength values (mpa) for the experimental groups are demonstrated in table 2. in relation to the surface roughness, two-way anova showed that the cross-product interaction between the factors time and experimental groups were statistically significant (p < 0.001). at baseline, all groups were statistically similar (p > 0.05). roughness increased significantly after the treatments in all groups (p < 0.001). the final roughness was higher in the sandblasting group and lower in the pa group, whereas the silane and diamond bur groups showed intermediate values. for the microshear bond strength, one-way anova showed significant statistical differences between the experimental groups (p < 0.0001). the lowest repair strength was observed for the pa group and the highest was observed in the sandblasting group. the silane and diamond bur groups were statistically similar and had an intermediate performance. in the sem images (figure 1), the diamond bur and sandblasting groups showed very irregular surfaces. however, they differed in the direction of the grooves and deprestable 2. mean values ± standard deviation of roughness (ra parameter) and microshear bond strength (mpa) for each experimental group (*). experimental groups roughness shear bond strength baseline post treatment phosphoric acid 0.24 ± 0.08 e 0.42 ± 0.14 d 9.4 ± 3.1 c silane 0.21 ± 0.07 e 0.64 ± 0.15 c 20.3 ± 6.1 b diamond bur 0.25 ± 0.07 e 0.86 ± 0.11 b 18.3 ± 4.9 b sandblasting 0.21 ± 0.09 e 1.28 ± 0.13 a 37.1 ± 8.7 a * comparisons are valid just for the same property. distinct letters show significant differences (p < 0.05). 6 gruber et al. sions. grooves are unidirectional in the diamond bur group, probably resulting from the direction of the bur roughening, whereas in the sandblasting group they do not follow a pattern due to the abrasion of aluminum oxide particles on the surface. discussion fatigue studies can mimic the effect of mechanical and thermal cycles, as well as a wet oral environment24. mechanical aging better reproduces the clinical reality, because failures and fractures in indirect cr restorations occur only after years of clinical service13. although previous studies have already investigated different surface treatment techniques for the repair of indirect restorations25-27, most did not simfigure 1. images of the surface roughness obtained by the sem (x 1,000). a: phosphoric acid group; b: silane group; c: diamond bur group: d: sandblasting group. sem mag: 1.00 kx c-labmu uepg view field: 138 μm 20 μm sem hv: 15.0 kv date(m/d/y): 11/23/15 det: se wd: 15.22 mm vega3 tescan sem mag: 1.00 kx c-labmu uepg view field: 138 μm 20 μm sem hv: 15.0 kv date(m/d/y): 11/23/15 det: se wd: 15.53 mm vega3 tescan sem mag: 1.00 kx c-labmu uepg view field: 138 μm 20 μm sem hv: 15.0 kv date(m/d/y): 11/23/15 det: se wd: 14.74 mm vega3 tescan sem mag: 1.00 kx c-labmu uepg view field: 138 μm 20 μm sem hv: 15.0 kv date(m/d/y): 11/23/15 det: se wd: 15.06 mm vega3 tescan a c b d 7 gruber et al. ulate three important clinical features found in this study’s protocol: cementation of a fiber post to stabilize the final restoration, simulation of the periodontal ligament, and simulation of mechanical aging. in a clinical scenario, the core and post are placed to retain the final restoration in endodontically treated teeth, improving their integrity28. the presence of the periodontal ligament and tooth-supporting structures partially absorb the masticatory loads; therefore, studies that did not simulate these structures may have obtained unreliable values21. finally, post-retained indirect crowns are subject to repetitive ordinary chewing forces over time, as well as other environmental challenging factors29. thus, mechanical aging is essential to simulate closely the clinical conditions to which these indirect restorations are subjected. in this study, all specimens were submitted to 1.2 x 106 cycles of mechanical fatigue, which is commonly assumed to correspond to 5 years of clinical service22. in this study, the results showed that air abrasion with aluminum oxide promoted the highest bond strength values30,31 and higher roughness. although some authors have reported that pre-treatment with diamond burs can yield higher repair strengths than air abrasion with aluminum oxide32,33, other studies30,34,35 have shown the opposite, with results similar to our observations. the higher surface roughness produced by aluminum air abrasion increases the surface area and wetting for the adhesive penetration36,37, which may have yielded the highest bond strength values. this positive correlation between increased surface roughness and improved repair strength has already been demonstrated in other studies38 39. indeed, the bond repair strength observed in the pa etching group might be lowest because this procedure produced the lowest surface roughening on the aged resin surface. previous studies have demonstrated that acid etching alone is not enough to guarantee adequate repair strength40. although surface treatment with silane does not generate the roughest surfaces, this group presented intermediate bond strength values, similar to that achieved by the asperization with diamond bur. the chemical bond produced by this bifunctional molecule between the inorganic particles of glassy substrates (silica filler particles) and the adhesive cr matrix19 probably compensated for the reduced surface area. this bonding agent has a general chemical structure, r′si(or)3, where r′ is the organ functional group (typically a methacrylate) that reacts to the adhesive system or the composite cement, creating a covalent bond after polymerization. the alkyl group (r) is hydrolyzed to a silanol (sioh), creating a covalent bond with the inorganic silicon particles41. this study has some limitations, due to which not all clinical conditions could be reproduced. in addition, only a resin cement was used, the chewing forces of an occlusion were not applied in the mechanical aging, and the treatments were performed on a flat surface rather than a cusp area. in summary, the results of the present study demonstrated that aged indirect crs should be pre-treated with aluminum oxide + pa prior to repair to increase the surface roughness and consequently the bond strength repair. thus, the tested hypothesis was accepted. 8 gruber et al. sandblasting aged indirect resin restorations with aluminum oxide prior to pa etching increases the surface roughness and repair bond strength values. conclusions sandblasting aged indirect resin restorations with aluminum oxide prior to pa etching increases the surface roughness and repair bond strength values. references 1. chun kj, lee jy. comparative study of mechanical properties of dental restorative materials and dental hard tissues in compressive loads. j dent biomech. 2014 oct 11;5:1758736014555246. doi: 10.1177/1758736014555246. 2. soares cj, pizi ec, fonseca rb, martins lr. influence of root embedment material and periodontal ligament simulation on fracture resistance tests. braz oral res. 2005 jan-mar;19(1):11-6. 3. likai w, yanan l, yan z, pingping l. [color stability of ceromer of different thicknesses and resin adhesive materials of different types after accelerated aging]. hua xi kou qiang yi xue za zhi. 2015 apr;33(2):201-5. chinese. 4. mak m, qualtrough aj, burke fj. the effect of different ceramic materials on the fracture resistance of dentin-bonded crowns. quintessence int. 1997 mar;28(3):197-203. 5. heintze sd, rousson v. fracture rates of ips empress all-ceramic crowns--a systematic review. int j prosthodont. 2010 mar-apr;23(2):129-33. 6. aggarwal v, singla m, miglani s, kohli s. comparative evaluation of fracture resistance of structurally compromised canals restored with different dowel methods. j prosthodont. 2012 jun;21(4):312-6. doi: 10.1111/j.1532-849x.2011.00827.x. 7. hurst d. indirect or direct restorations for heavily restored posterior adult teeth? evid based dent. 2010;11(4):116-7. doi: 10.1038/sj.ebd.6400760. 8. koczarski mj. utilization of ceromer inlays/onlays for replacement of amalgam restorations. pract periodontics aesthet dent. 1998 may;10(4):405-12; quiz 414. 9. sequeira-byron p, fedorowicz z, carter b, nasser m, alrowaili ef. single crowns versus conventional fillings for the restoration of root-filled teeth. cochrane database syst rev. 2015 sep 25;(9):cd009109. doi: 10.1002/14651858.cd009109.pub3. 10. fennis wm, kuijs rh, roeters fj, creugers nh, kreulen cm. randomized control trial of composite cuspal restorations: five-year results. j dent res. 2014 jan;93(1):36-41. doi: 10.1177/0022034513510946. 11. loomans ba, mesko me, moraes rr, ruben j, bronkhorst em, pereira-cenci t, et al. effect of different surface treatment techniques on the repair strength of indirect composites. j dent. 2017 apr;59:18-25. doi: 10.1016/j.jdent.2017.01.010. 12. opdam nj, bronkhorst em, loomans ba, huysmans mc. longevity of repaired restorations: a practice based study. j dent. 2012 oct;40(10):829-35. doi: 10.1016/j.jdent.2012.06.007. 13. demarco ff, correa mb, cenci ms, moraes rr, opdam nj. longevity of posterior composite restorations: not only a matter of materials. dent mater. 2012 jan;28(1):87-101. doi: 10.1016/j.dental.2011.09.003. 14. arami s, hasani tabatabaei m, namdar f, safavi n, chiniforush n. shear bond strength of the repair composite resin to zirconia ceramic by different surface treatment. j lasers med sci. 2014 fall;5(4):171-5. 9 gruber et al. 15. komine f, kobayashi k, saito a, fushiki r, koizumi h, matsumura h. shear bond strength between an indirect composite veneering material and zirconia ceramics after thermocycling. j oral sci. 2009 dec;51(4):629-34. 16. loomans b, ozcan m. intraoral repair of direct and indirect restorations: procedures and guidelines. oper dent. 2016 sep;41(s7):s68-s78. 17. campos f, almeida cs, rippe mp, de melo rm, valandro lf, bottino ma. resin bonding to a hybrid ceramic: effects of surface treatments and aging. oper dent. 2016 mar-apr;41(2):171-8. doi: 10.2341/15-057-l. 18. eslamian l, borzabadi-farahani a, mousavi n, ghasemi a. the effects of various surface treatments on the shear bond strengths of stainless steel brackets to artificially-aged composite restorations. aust orthod j. 2011 may;27(1):28-32. 19. foxton rm, nakajima m, hiraishi n, kitasako y, tagami j, nomura s, et al. relationship between ceramic primer and ceramic surface ph on the bonding of dual-cure resin cement to ceramic. dent mater. 2003 dec;19(8):779-89. 20. kelly jr. clinically relevant approach to failure testing of all-ceramic restorations. j prosthet dent. 1999 jun;81(6):652-61. 21. naumann m, sterzenbach g, rosentritt m, beuer f, frankenberger r. in vitro performance of self-adhesive resin cements for post-and-core build-ups: influence of chewing simulation or 1-year storage in 0.5% chloramine solution. acta biomater. 2010 nov;6(11):4389-95. doi: 10.1016/j.actbio.2010.05.023. 22. gomes gm, gomes om, gomes jc, loguercio ad, calixto al, reis a. evaluation of different restorative techniques for filling flared root canals: fracture resistance and bond strength after mechanical fatigue. j adhes dent. 2014 jun;16(3):267-76. doi: 10.3290/j.jad.a31940. 23. bittencourt bf, gomes gm, trentini fa, azevedo mrd, gomes jc, gomes omm. effect of finishing and polishing on surface roughness of composite resins after bleaching. j braz j oral sci. 2014;13(2):158-62. doi: 10.1590/1677-3225v13n2a15. 24. amaral fl, colucci v, palma-dibb rg, corona sa. assessment of in vitro methods used to promote adhesive interface degradation: a critical review. j esthet restor dent. 2007;19(6):340-53; discussion 354. 25. barragan g, chasqueira f, arantes-oliveira s, portugal j. ceramic repair: influence of chemical and mechanical surface conditioning on adhesion to zirconia. oral health dent manag. 2014 jun;13(2):155-8. 26. lee sj, cheong cw, wright rf, chang bm. bond strength of the porcelain repair system to allceramic copings and porcelain. j prosthodont. 2014 feb;23(2):112-6. doi: 10.1111/jopr.12064. 27. cotes c, cardoso m, melo rm, valandro lf, bottino ma. effect of composite surface treatment and aging on the bond strength between a core build-up composite and a luting agent. j appl oral sci. 2015 jan-feb;23(1):71-8. doi: 10.1590/1678-775720140113. 28. skupien ja, cenci ms, opdam nj, kreulen cm, huysmans mc, pereira-cenci t. crown vs. composite for post-retained restorations: a randomized clinical trial. j dent. 2016 may;48:34-9. doi: 10.1016/j.jdent.2016.03.007. 29. ferro mc, colucci v, marques ag, ribeiro rf, silva-sousa yt, gomes ea. fracture strength of weakened anterior teeth associated to different reconstructive techniques. braz dent j. 2016 sepoct;27(5):556-61. doi: 10.1590/0103-6440201602452. 30. costa tr, ferreira sq, klein-junior ca, loguercio ad, reis a. durability of surface treatments and intermediate agents used for repair of a polished composite. oper dent. 2010 mar-apr;35(2):231-7. doi: 10.2341/09-216-l. 31. su n, yue l, liao y, liu w, zhang h, li x, et al. the effect of various sandblasting conditions on surface changes of dental zirconia and shear bond strength between zirconia core and indirect composite resin. j adv prosthodont. 2015 jun;7(3):214-23. doi: 10.4047/jap.2015.7.3.214. 10 gruber et al. 32. petridis h, garefis p, hirayama h, kafantaris nm, koidis pt. bonding indirect resin composites to metal: part 1. comparison of shear bond strengths between different metal-resin bonding systems and a metal-ceramic system. int j prosthodont. 2003 nov-dec;16(6):635-9. 33. ozcan m, van der sleen jm, kurunmaki h, vallittu pk. comparison of repair methods for ceramicfused-to-metal crowns. j prosthodont. 2006 sep-oct;15(5):283-8. 34. al-shehri ez, al-zain ao, sabrah ah, al-angari ss, al dehailan l, eckert gj, et al. effects of air-abrasion pressure on the resin bond strength to zirconia: a combined cyclic loading and thermocycling aging study. restor dent endod. 2017 aug;42(3):206-15. doi: 10.5395/rde.2017.42.3.206. 35. grasel r, santos m, rêgo hc, rippe m, valandro ljod. effect of resin luting systems and alumina particle air abrasion on bond strength to zirconia. oper dent. 2018 may/jun;43(3):282-90. doi: 10.2341/15-352-l. 36. hallmann l, ulmer p, reusser e, hammerle ch. surface characterization of dental y-tzp ceramic after air abrasion treatment. j dent. 2012 sep;40(9):723-35. doi: 10.1016/j.jdent.2012.05.003. 37. medvedev ae, ng hp, lapovok r, estrin y, lowe tc, anumalasetty vn. effect of bulk microstructure of commercially pure titanium on surface characteristics and fatigue properties after surface modification by sand blasting and acid-etching. j mech behav biomed mater. 2016 apr;57:55-68. doi: 10.1016/j.jmbbm.2015.11.035. 38. jain s, parkash h, gupta s, bhargava a. to evaluate the effect of various surface treatments on the shear bond strength of three different intraoral ceramic repair systems: an in vitro study. j indian prosthodont soc. 2013 sep;13(3):315-20. doi: 10.1007/s13191-013-0270-x. 39. da costa tr, serrano am, atman ap, loguercio ad, reis a. durability of composite repair using different surface treatments. j dent. 2012 jun;40(6):513-21. doi: 10.1016/j.jdent.2012.03.001. 40. lucena-martin c, gonzalez-lopez s, navajas-rodriguez de mondelo jm. the effect of various surface treatments and bonding agents on the repaired strength of heat-treated composites. j prosthet dent. 2001 nov;86(5):481-8. 41. anusavice kj, shen c, rawls hr. phillips’ science of dental materials. 12.ed. elsevier health sciences; 2012. braz j oral sci. 15(4):320-325 dental caries in children on deciduous dentition: art and socioeconomic aspect ana de lourdes sá de lira,1 sabrynna gonçalves candeira portela1 1universidade estadual do piauí – uespi, school of dentistry, department of clinical dentistry, area of integrated clinic, parnaíba, pi, brazil correspondence to: ana de lourdes sá de lira universidade estadual do piauí faculdade de odontologia rua senador joaquim pires 2076 ininga fone (86) 999595004 cep: 64049-590 teresina-pi-brasil email: anadelourdessl@hotmail.com abstract aim: determine the prevalence and incidence of dental caries in children´s deciduous teeth, who were treated with art technique. methods: in t1, 62 children were examined for being between 3 and 6 years of age. a questionnaire was applied to the parents or guardians of the children involved in the research, to analyze their knowledge about eating habits and oral hygiene. an intra-oral examination of children from 3 to 6 years old was carried out and dental treatment was performed by the art technique. six months after,(t2), the clinical examination was redone to investigate the restorations´stability. results: in t1, 94 carie cavities were found in 19 children and 86 were recommended for restoration with art. in t2, 58 restorations were damaged or lost and 15 children had 20 new carie cavities. conclusion: the prevalence of dental caries in the deciduous dentition was 38.8% and the incidence was 31.9%. in t2, the majority restorations class i and ii were damaged or lost. art technique is an option, despite the high failure in art, especially for child population with deciduous teeth that has limited access to dental services. however, restorations should be supervised and healthy eating habits implemented. keywords: pediatric dentistry. dental caries. oral hygiene. received for publication: june 18, 2017 accepted: july 11, 2017 original article braz j oral sci. october | december 2016 volume 15, number 4 http://dx.doi.org/10.20396/bjos.v15i4.8650049 introduction the prevalence of caries has declined, but it is more significant in the most favored populations, which have a lower cpo-d and a higher number of children without caries1-3. maintaining good oral hygiene is essential so that the individual does not acquire caries disease. this is justified by the dental biofilm, which is the determining factor for the disease to occur4,5. but also, to reduce this situation, it is important to take greater care with the children through health promotion measures and prevention6-7. the world health organization (who) has stated that health is also related to schooling, so it is clear that increasing school performance leads to improvements in the health of individuals8. the definition of early caries can be determined by the presence of decayed teeth (even those without cavitations), dental elements lost due to caries or restorations, in individuals with a maximum age of 71 months. in addition, any carious surface is defined as severe precarious caries in children under three years of age9. seeking to change reality, promotion measures should be instituted together with other collective health actions, such as preventive, curative, health education and control10. the individuals involved were oriented on the objectives of the research and their methods through the informed consent term (tcle) and the informed consent term (tale), signed by parents / guardians and children, respectively. prior to the execution of the research, the questionnaire was applied to 10 parents, after validation, having been previously applied to 10 parents, and the clinical examination was carried out in 10 children, aged between 3 and 6 years, in the deciduous dentition, attended at the odontopediatrics clinic of the dental uespi, in parnaíba, chosen randomly for calibration of the researcher student. at the first moment of the research (t1) a lecture was given to the 49 parents or guardians for awareness and instruction according to the research objectives. the questionnaire was applied. the researcher performed the intra-oral examination of the sample, children enrolled in the preschools and the municipal school of early childhood education tio zeca, in search of caries lesions with cavitations, the majority (n = 26) between 5 and 6 years of age. the individual examination was carried out in the schoolyard, under natural light, using wooden spatula and mouth mirror. when present, the teeth and faces were recorded in the odontogram. teeth with evident pulp involvement or painful symptomatology and / or presence of edema or fistula were excluded from the study and immediately referred to the clinical school of dentistry of uespi for endodontic and restorative treatment. the prevalence (t1) and later the incidence of caries (t2) in the sample studied were determined. subsequently dental treatment was performed by the art technique, in the school environment, under natural light. vitro molar (nova dfl, rio de janeiro, brazil) was used as the glass ionomer cement (civ), because it is one of the materials of choice for this technique, because it has high viscosity, better strength and good mechanical properties13. the technique of art11 consisted in detail in: a) cleaning of the dental surface with friction with a cotton dressing; b) access to caries lesion, if there is enamel without support, with axes, hoe or gingival margin trimmer; c) removal of carious dentin with sharp curettes, starting from the edges of the cavity, maintaining the dentin that leaves in flakes or scales in the portion closest to the pulp (affected dentin); d) cleaning the cavity with the liquid of the restorative material, with cotton dressing for 15 seconds on the surface to remove the dentin sludge; e) washing with cotton swabs moistened with water (minimum 3); (f) drying with dry cotton; g) dosing and manipulation of the civ for 30 seconds according to the manufacturer's recommendation; h) insertion of the restorative material into the cavity, when it was in the consistency of shiny wire, with insertion spatula, in increments. leaving with slight excess; i) with finger and vaseline, the surface of the restoration was pressed for 30 seconds; j) use of hollemback spatula or sharp curette to remove excess; k) protection of the surface of the restoration with solid 321 one option for caries disease control is atraumatic restorative treatment (art), which was devised in the 1980s by dr. jo e. frencken. this method is of minimal intervention that aims to paralyze the carious process and provide favorable conditions to restore oral health11. in view of the current goals of dentistry (prevention, promotion and early and conservative intervention in the carious process), the art technique should not be considered only an alternative restorative technique, but rather to be evaluated within its complexity as a promotion strategy oral health that aims to take the dental treatment to the non-assisted populations, adapting, however, to the reality of these. therefore, this treatment associates the educational actions and preventive health care for the population, as well as curative actions through the removal of carious tissue with instruments, and restoration of the cavity12. it is believed that early dental caries, in the deciduous denture is a public health problem with high prevalence and incidence rates, and that few children have easy access to public dental care. this type of treatment also seeks to respect one of the main current concepts of dental practice, which is to create a favorable environment that halts the caries disease process by means of minimum intervention and maximum preservation of dental structures. in addition to the curative aspect of cavity preparations and restorations, there are the added advantages of concern focused on health promotion, education and patient motivation. so it was decided to carry out this research to determine the prevalence and incidence of dental caries in children in the deciduous denture, who were treated during this research with art technique. material and methods this study was approved by the research ethics committee of the state university of piauí cep / uespi, under caae number: 51011015.4.0000.5209. the principle of ethics was based on the norms contained in resolution no. 466/12 (cns/ms). the researcher was provided with the consent term for the directors of the school units, authorizing the development of the research after ethical appreciation of the research ethics committee of the state university of piauí – cep/ uespi. inclusion criteria were children with caries in the deciduous dentition, in the age range of 3 to 6 years, whose parents or guardians accepted the research. this age group of research was chosen due to the greater probability of the children being with the deciduous complete dentition and attending the preschool. children less than 3 years of age or older than 6 years of age or who were already in the mixed dentition were excluded from the study because the focus was on the deciduous dentition and those that the parents or guardians did not authorize to participate in the study. according to the sample calculation, only 45 children should be examined, but it was decided that all 62 children would be examined for being between 3 and 6 years of age. however, 13 were excluded because they were in the mixed dentition. preventive activities of supervised brushing and topical application of fluoride were performed for children who were excluded from the study, since no oral pathology was present. dental caries in children on deciduous dentition: art and socioeconomic aspect braz j oral sci. 15(4):320-325 322 petroleum jelly; l) use of carbon paper for checking the occlusion; m) removal of premature contacts with hollemback or sharp curette. caries lesions included in the art intervention, according to the nomenclature of black13, comprised those that were in teeth: 1) later class i and ii; 2) previous class iii; 3) with loss of cusps; 4) posterior and anterior class v after 6 months (t2), 47 children were again examined to see if there was improvement in oral health, two of them no longer studied in one of the schools. the evaluation criteria for the control of oral health status were based on ferreira et al.11. as explicit in table 1, in which 0 was considered as indicative of successful treatment and 1 to 3 to indicate intervention failure. dental caries in children on deciduous dentition: art and socioeconomic aspect in the first intervention period (t1) by art, 19 children out of 49 in the sample had caries lesions, 9 of which were female and 10 were male. the prevalence of dental caries in the deciduous dentition was 38.8%. in these 19 children, 94 decayed teeth were diagnosed, of which 86 were restored (8 teeth presented great destruction by caries, with indication of exodontia, and were later performed in the uespi school of dentistry clinic), corresponding to 42 class i restorations, 22 of class ii, 17 of class v and 5 of class iii. according to figures 1 and 2 it was observed in t1 that the second deciduous molars were the ones that presented the highest number of carious lesions. table 1 criteria for evaluation of restorations with art11 satisfactory art 0 present, minor defects in the margin and / or wear of 0.5mm depth 1 partial loss of restorative material 2 total loss of restorative material 3 absent or loss of tooth by exfoliation x the data were tabulated and analyzed by the spss statistical program, version 21. the chi-square association test was performed to verify if there were significant differences in the results found. descriptive statistics (measures of central tendency and dispersion) and construction of graphs were used in order to illustrate the findings of this research. results the parents were on average 30 years and 10 months old, the majority were single (n = 25). in terms of schooling, there were similar numbers who declared that they only had incomplete secondary education (n = 16), and those with up to the 4th grade (n = 16). the family income of 1 to 2 minimum wages was informed by 39 parents. at the first moment, when asked if they had already participated in any lecture on oral health, 34 answered yes. upon receiving oral hygiene instructions from the children, 37 reported having received some guidance. of these, 23 were passed on by dentists and others by other professionals, relatives and / or friends. in relation to the habit of doing the oral hygiene of the children, the majority answered yes (n = 42), being executed 3 times a day, according to 26 of these. in addition, he was asked if his children used to sleep without doing oral hygiene. of this, 33 answered to do it and 18 reported not doing the hygiene of the child before bed. information about the children's diet, milk consumption and sucking habit were collected. in this way, 41 children do not use sugary pacifiers, most have healthy meals, but they included sugary foods in the snack, in addition to the intake of artificial milk. after the lectures, parents acknowledged that oral hygiene and healthy eating may decrease the chances of developing caries disease. fig. 1 upper deciduous teeth with carious lesions. parnaíba-pi. 2016. fig. 2 lower deciduous teeth with caries lesions. parnaíba-pi. 2016. braz j oral sci. 15(4):320-325 323 affected by early caries, probably because they used sugar in the snack, both in t1 and t2. according to almeida et al.16, it was not possible to notice a direct and expressive relationship between the daily frequency in the consumption of sweet foods with the caries index. in the same way, it cannot be said that the experience of caries and the number of toothbrushes are inversely proportional. in the study by ribeiro et al.17 224 children were part of a control for four years, and it was found that brushing was not an expressive factor for individuals not to have caries, since 40.4% of children who brush their teeth and more than half (53.4%) of those who did not brush had caries. early caries brings physical and psychological consequences, since it interferes in the quality of life. this can be explained by the progression of the disease, as abscesses may appear, damage of permanent teeth, painful symptomatology, feeding, swallowing, breathing damages18,19. initially, it manifests itself through white and opaque patches, but with progression the clinical aspects become cavitations. as it is acute, if there is no intervention, there will be total loss of the dental crown. the most affected elements are the upper and lower anterosuperior and molars. anteroposterior teeth are included in more advanced cases of caries disease20. the second deciduous molars were the teeth most affected by caries (figures 1 and 2). these findings are in agreement with rigo et al.21 when affirming that dental elements 55, 65, 75 and 85 presented the highest caries in 56%, 55%, 46% and 45% of the cases, respectively. as an alternative treatment in situations of low socioeconomic level and high occurrence of caries, art has good results because it includes, among others, less invasive care in established caries lesions and oral health guidelines and it is well accepted by children22. according to data collected 6 months after the intervention with art, there was a high number of total loss of the restorations (figure 3). a similar result was found by pazuch et al.23 in which more than half of the restorations (62.5%) performed needed repair after 6 months. these results occurred with civ, which is the material of choice in art practice, due to its properties, such as: fluoride release and physicochemical adhesion. it is believed that the high failure rate in treatment and the increase in the number of carious lesions may have been due to the absence of cavitary preparation, such as removal of carious tissue by means of drills, associated with insufficient relative isolation to avoid contact of the civ with fluids and inadequate conditioning of the children. sales et al.24 also justified their failure to condition and control the child during care. the incidence of caries in t2 occurred in children whose parents reported that they did not brush their teeth before bed, although most did so after meals. probably the consumption of sweets in the intervals of meals, even at low frequency, associated with the absence of brushing at bedtime have favored the appearance of new caries. failure to remove bacterial biofilm and low salivation during sleep predispose to the appearance of carious lesion25,26. a greater number of brushing provides a better chance of avoiding the development of the carious process27. there twenty new cavitated lesions were observed in 15 of these 17 children, being 11 of class i and 9 of class ii, corresponding to incidence of dental caries of 31.9%. among these 15 preschoolers, it was found that in 12 of them only a new carious lesion was found, but 2 presented with two lesions and one with 4. according to figure 3, it was verified that 58 of the 86 restorations performed on t1 had failed after 6 months (t2). of the 58 compromised restorations, there were partial loss of 6 class i and 2 class ii restorations, and total loss of 15 class ii and 23 class i restorations. all 17 class iii and class iii restorations performed remained satisfactory. five of class ii and 7 of class i presented defects of 0.5mm. the failures in art restorations were highly significant when the chi-square test = 27.3002; gl = 3; with p <0.001. therefore, if other restorations are made, under the same conditions, teeth are expected to be divided equally into each of the scores adopted in the survey. the number of teeth in each score did not happen at random. dental caries in children on deciduous dentition: art and socioeconomic aspect fig. 3 evaluation criteria of art control. parnaíba-pi. 2016. discussion in this study it was observed that families had low monthly schooling and income. these data corroborate with those found in another study, 15 in which a family income of up to two minimum wages predominated (98.3%), and 76.6% of the parents / guardians declared having only complete elementary education. it is essential to make parents or caregivers aware of the importance of oral health of children with guidelines on how to maintain it, including oral hygiene before bed, dental care before 12 months of age to limit or prevent cases of early decay, as well as the integrated assistance of health professionals: surgeon-dentist and pediatrician13,14. in the study carried out by robles et al.15, the mothers reported having knowledge about the importance of brushing after meals, the relationship between the high carbohydrate diet and the development of caries and its consequences. the guidelines received from pediatricians, nurses and other mothers diverged from this research, in which it was found that the dentist was the one most responsible for disseminating these instructions. the children had a diet based on healthy foods, the parents did the hygiene of the children, mostly three times a day. but despite this, there were a large number of children braz j oral sci. 15(4):320-325 324 are factors that favor the installation of early caries, such as: sucrose-rich diet, cariogenic microbiota, oral hygiene efficiency and the time the food stagnates on the teeth28. the restorations that failed after 6 months were those of class i and class ii, corroborating with the findings of other authors25-28, who verified that occlusal-proximal restorations (class ii) have higher rates of failure, since they are more favorable to fractures and complete loss. it can be explained by the failure to adapt the civ to the cavity due to its viscous texture and low flow, allowing the appearance of spaces inside the restoration, consequently reducing its longevity. other physical characteristics of this cement also influence the success of art as low mechanical properties, high solubility and porosity28. it was noted that the results of the questionnaires diverged in some questions from what had been found in the intra-oral examination of preschool children. although the parents / guardians had notions of hygiene and eating habits, there was a high prevalence and incidence of caries and failure of the restorations performed with civ by the art technique. it was concluded that there was a high prevalence of caries in the deciduous dentition (38.8%), which may be related to socioeconomic factors and the need for greater efforts to expand dental care with oral health promotion and prevention actions. the parents were advised on the measures to be taken to prevent tooth decay. in t2, six months after restorative art treatment, most class i and ii restorations were damaged or lost, probably due to the large cavities and the limitation of the technique. class iii and v restorations remained satisfactory. there was a high incidence of class i and ii caries (31.9%) in 15 of 17 children in whom oral hygiene was not performed before bedtime. art technique is an option, despite the high failure in art, especially for child population with deciduous teeth that has limited access to dental services. however, restorations should be supervised and healthy eating habits implemented. the interpretation of the results should consider some limitations inherent in this study. it is important that future studies be carried out on the treatment and control of dental caries in the deciduous dentition. references 1. rezende ln, santos fcs, neto ms, santos f. [feeding rampant caries in children aged 2 to 5 years: literature review]. j manag prim health care. 2014;5(2):219-29. portuguese. 2. oliveira lb, moreira rs, reis scgb, freire mcm. dental caries in 12-year-old schoolchildren: multilevel analysis of individual and school environment factors in goiânia. rev bras epidemiol. 2015 julsep;18(3):642-54. doi:10.1590/1980-5497201500030010. 3. scalioni far, figueiredo sr, curcio wb, alves rt, leite icg, ribeiro ra. [early childhood dietary and caries habits in children attended by brazilian dentistry school]. pesq bras odontoped clin integr. 2012;12(3):399-404. doi: 10.4034/pboci.2012.123.16. portuguese. 4. santos app, soviero vm. [assessment of the quality of oral hygiene in infants and preschool children: importance and methods: literature review]. rev clín pesq odontol. 2008;4(2):87-92. portuguese. 5. moura lfa, moura, m. s, toledo oa. [knowledge and practices in oral health of mothers who attended a dental program of maternal and child care]. cienc saude colet. 2007;12(4):1079-86. doi: 0.1590/s141381232007000400029. portuguese. 6. aquilante ag, almeida bs, castro rfm, xavier crg, peres shcs, bastos jrm. [the importance of dental health education for preschool children]. rev odontol unesp. 2003;32(1):39-45. portuguese. 7. souza lm, macedo a, gusmão rcmp, athayde acr, costa led, queiroz fs, et al. [oral health in school and family: from autonomy to social transformation]. rev bras educ med. 2015;39(3):426-32. portuguese. 8. navarro mfl, leal sc, molina gf, villena rs. [atraumatic restorative treatment: news and perspectives]. rev assoc paul cir dent. 2015;69(3):289-301. portuguese. 9. garbin cas, sundfeld rh, santos kt, cardoso jd. [current aspects of atraumatic restorative treatment]. rfo. 2008;13(1):25-9. portuguese. 10. santos mmpr, mathias if, diniz mb, bresciani e. [evaluation of the surface hardness of glass ionomer cements reinforced by carbon nanotubes]. rev odontol unesp. 2015;44(2):108-12. doi: 10.1590/18072577.1060. portuguese. 11. ferreira ll, ferreira-nóbilo np, gibilini c, sousa mlr. longevity of atraumatic restorations performed by undergraduate dentistry students. rev odontol unesp.2014;43(4):241-4. doi: 10.1590/rou.2014.039 12. aguiar ypc, dantas dcre, ribeiro aiam, lima rf, sousa yc, guênes gmt. [clinical evaluation of glass ionomer restorations in children]. rfo. 2014;19(1):70-6. doi: 10.5335/rfo.v19i1.3628. portuguese. 13. castilho arf, mialhe fl, barbosa ts, puppin-rontani rm. [influence of the family environment on the oral health of children: a systematic review]. j pediatr (rio j). 2013;(2):116-3. doi: 10.1016/j.jped.2013.03.014. portuguese. 14. lemos lvfm, zuanon acc, myaki si, walter lrf. [experience of dental caries in children attended in a program of dentistry for babies]. einstein. 2011;9(4):503-7. doi: 10.1590/s1679-45082011ao2184. portuguese. 15. robles acc, grosseman s, bosco vl. [practices and meanings of oral health: a qualitative study with mothers of children attended at the federal university of santa catarina]. cienc saude coletiva. 2010;15(supl. 2):3271-81. doi: 10.1590/s1413-81232010000800033. portuguese. 16. almeida al, barbosa amf, menezes va, granville-garcia af. [caries experience among mothers and children: influence of socioeconomic and behavioral factors]. odontol clín cient. 2011;10(4):373-9. portuguese. 17. ribeiro ag, oliveira a f, rosenblatt a. [early childhood caries: prevalence and risk factors in preschool children at 48 months in the city of joão pessoa, paraíba, brazil]. cad saude publica. 2005;21(6):1695-700. doi: 10.1590/s0102-311x2005000600016. portuguese. 18. lemos lvfm, correia mf, spolidório dmp, myaki dmp, zuanon acc. [cariogenicity of breast milk: myth or scientific evidence]. pesq bras odontoped clin integr. 2012;12(2):273-8. doi: 10.4034/ pboci.2012.122.18. portuguese. 19. feitosa s, colares v. [the repercussions of early childhood caries in the quality of life of pre-school children]. rev ibero-am odontopediatr odontol bebe. 2003;6(34):542-8. portuguese. 20. losso em, tavares mcr, silva jyb, urban ca. [early and severe childhood caries: an integral approach]. j pediatr. 2009;85(4):295-300. doi: 10.1590/s0021-75572009000400005. portuguese. 21. rigo l, souza ea, junior, afc. [experience of dental caries in the first dentition in a municipality with water fluoridation]. rev bras saude matern infant. 2009;9(4):435-42. doi: 10.1590/s1519-38292009000400008. portuguese. 22. raggio dp, imperato jcp, politlano gt, echeverria sr, uemura st, ferreira ems. [atraumatic restorative treatment]. rgo. 2004;52(5):3558. portuguese. 23. pazuch j, zottis m, perussolo b, patussi eg, pavinato lcb, bervian j. [evaluation of the clinical performance of art restorations (atraumatic restorative treatment)]. rfo. 2014;19(1):88-93. doi: 10.5335/rfo. dental caries in children on deciduous dentition: art and socioeconomic aspect braz j oral sci. 15(4):320-325 325 dental caries in children on deciduous dentition: art and socioeconomic aspect v19i1.3694. portuguese. 24. sales peres shc, hussne r, perer as. [atraumatic restorative treatment (art) in children aged 4 to 7 years: clinical evaluation after six months]. rev inst cienc saude. 2005;23(4):275-80. portuguese. 25. eleutério asl, cota als, kobayashi ty, silva smb. [clinical evaluation of oral health of children of the municipalities of alfenas and areado, minas gerais, brazil]. pesq bras odontoped clin integr. 2012;12(2):195201. doi: 10.4034/pboci.2012.122.07. portuguese. 26. deliberali fd, brusco ehc, brusco l, perussolo b, patussi eg. [behavioral factors involved in the development of early caries in children attended at the pediatric dentistry clinic of the school of dentistry of passo fundo rs, brazil]. rfo. 2009;14(3):197-202. portuguese. 27. goes mf, martins al, sartori cg, sinhoreti mac. [solubility of glass ionomer cements indicated for atraumatic restorative treatment]. rev assoc paul cir dent. 2015;69(3):272-8. portuguese. 28. yip hk, smales rj. tay fr. chu fcs. selection of restorative material for the atraumatic restorative treatment (art) approach: a review. spec care dentist. 2001;21:216-21. doi: 10.1111/j.1754-4505.2001. braz j oral sci. 15(4):320-325 braz j oral sci. 15(4):273-279 multi detector computerized tomography scans aid in the staging of head and neck cancers sigbeku opeyemi1, kolude bamidele1, adeniji-sofoluwe adenike2, adeosun aderemi3 1dept of oral pathology, university of ibadan/university college hospital 2dept of radiology, university of ibadan/university college hospital 3dept of otorhinolaryngology, university of ibadan/university college hospital correspondence to: dr. bamidele kolude, dept of oral pathology, faculty of dentistry, uch, ibadan. delekolude2003@yahoo.co.uk +2348055309574 abstract introduction/objectives: to assess the efficacy and correlation of mdct scans in the clinical staging of patients with hncs prior to therapeutic intervention. methodology: thirty-four hncs were studied according to the 2005 who. clinical ajcc 6th edition & radiological staging. results: 14 squamous cell carcinoma (scc 41.2%) mean age 49.4 + 14.7 years, 13 nasopharyngeal carcinoma (npc 38.2%) mean age 37.1 + 20.5 years, 3 odontogenic carcinoma (odc 8.8% made up of 2 cases ameloblastic carcinoma 5.9% and 1 case of ameloblastic carcinosarcoma 2.9%). others cases were 3 adenocarcinoma (8.8%) and 1 sinonasal carcinoma nc (2.9%). mean age insignificant according to gender (p = 0.342). sensitivity, specificity, positive & negative predictive values and accuracy of clinical and radiological nodal involvements were: (47.4%; 80%; 61.8%; 75%; 54.5%) & (78.9%; 93.3%; 85.3%; 93.8%; 77.8%) respectively. difference between clinical and radiological stages was statistically significant (x2= 260.8; p=0.01). there was a low but positive correlation between the clinical and radiologic stages (pearson’s correlation r = 0.6). conclusion: mdct was significantly more accurate than clinical examination in the tnm of hncs using ajcc/uicc tnm guidelines. authors recommend mdct as first line imaging technique in resource limited settings. keywords: multi-detector ct, staging, head and neck cancers. received for publication: december 27, 2016 accepted: july 01, 2017 original article braz j oral sci. october | december 2016 volume 15, number 4 http://dx.doi.org/10.20396/bjos.v15i4.8650039 introduction recent cancer estimates show that out of 14.1 million new cancer cases diagnosed annually more than 0.7 million patients suffer from head and neck cancers (hncs) and 60% of all hncs occur in developing countries1. hncs are particularly distressing because the head and neck region constitute the most complex functional anatomy in the human body2. the stage of disease at presentation is the most important prognostic factor as early stage of presentation increase the survival rate and improves the quality of life in head and neck cancer because less aggressive and mutilating treatment options are offered3. in addition, early stage in hnc has up to a 60% chance of cure with local treatment alone whereas advanced stage disease have greater than 50% risk of recurrence and development of distant metastasis4. unfortunately, most patients present with late stage of disease that requires radical treatment and often result in considerable morbidity and mortality with attending poor prognosis5. since the evolution of linear tomography, faster and better revelations and delineation of the extent of neoplasm and lymph nodes involvement became obvious when compared the assignment of a higher clinical stage. authors envisage that the advantage of this forward step in ajcc guideline will result in better patient stratification for appropriate therapy and better report of treatment outcome. a 64-slice toshiba aquillon mdct machine was used to acquire the images of all enrolled patients and mpr images of the tumor were viewed interactively in all imaging plane with radiological staging performed according to madison et al.16 (1998). patients with incomplete radiographic images, incomplete data for clinical staging of head and neck cancer, lack or inconclusive histological findings, non-consenting patients and patients with primary tumors of the brain, eye, thyroid and salivary glands were excluded from the study. data was analyzed using version 21 software of ibm statistical package for social sciences (spss-21). proportional distributions of various clinical and radiological stages were expressed as percentages and compared using chi-square statistics. correlation of individual histological and radiological stages was conducted using pearson’s correlation statistics. significant level was set at p < 0.05. ethical clearance was obtained. (ui/ ec/12/0238). results a total of 34 cases of head and neck cancers were enrolled in the present study. among these, 21 were male (61.8%) while 13 were female (38.2%) giving a male to female ratio of 1.6:1 and the overall mean age was 42.9+ 17. 1years.the mean ages according to gender were 45.1+15.7 for males and 39.2 +19.5 for females; there was no statistically significant difference in the mean ages according to gender (p = 0.342). the histology and site distribution (icdo-9)17 of enrolled hnc cases are as depicted in table 1. more squamous cell carcinoma cases occurred among females (69.2%) while majority of the nasopharyngeal carcinoma cases occurred among males (76.9%). the four nasal malignancies occurred in males, two of the odontogenic carcinomas cases were males but the third was a female. there was no significant difference in the site distribution of hnc according to gender (x2 = 8.1, p = 0.524). among the scc group, the most common histological type was the keratinizing (k) type which occurred most frequently at three sites; the maxillary sinuses see comment above (35.7%), paranasal sinuses (21.4%) and the pharynx (21.4%). the most common histological type in the npc group was the nonkeratinizing (n-k) variety (table 1). the clinical staging revealed that a minority of the tumors (47%) were less than 6cm in diameter at the time of clinical presentation, among this group, 8.8% were t2 tumors while the remaining cases were t3 tumors (38.2%). however, majority (53%) of the hncs cases were t4 clinical tumors which comprised of 47.1% t4a tumors and 5.9% of t4b tumors. clinically, more than half (55.9%) of the hncs cases were n0 while 23.5% were n1, 8.8% of the cases were n2a but there was no clinical tumor case of n2b or n2c. approximately 274 with the era of conventional radiography only6. the development of the helical multi detector computerized tomography (mdct) has the advantage of faster ct scans with rapid acquisition of numerous thin (0.5 to 1mm thickness) images in axial, coronal and sagittal planes. furthermore, excellent soft tissue delineation of tumor extent and reconstructed shaded surface display ssd of bony structures is possible and more accurate images than linear ct and conventional spiral ct are obtainable7. tumor volume, lymph node infiltration distant metastases are the most important factors that influence the therapeutic approach and the prognosis of the patient with hnc5,8. exact tumor staging is necessary for treatment planning, leading to reduced postoperative morbidity and tumor recurrence-associated mortality9. sub mucosal extension cannot be sufficiently assessed by endoscopy and physical examination but can be evaluated with magnetic resonance imaging mri and ct to a lesser extent10,11. clinical examination alone frequently underestimates the extent of disease. mdct technology is capable of acquiring high-resolution (sub millimeter) studies in less than 20 seconds, although axial images with a slice thickness of 3–5 mm were previously advocated in various imaging protocols. thick slice thickness reduces resolution and makes multi-planar reformation mpr sub-optimal. when overlapping images are reconstructed from raw data with a nominal slice thickness of 0.5–1.25 mm, (mpr) images of the tumor can be viewed interactively in any arbitrarily chosen imaging plane12. due to better delineation of bony extent and lymph node evaluation by contrast enhanced, ct scan (cect) has become an essential part of the workup of hncs patients. the present study is an assessment of the efficacy and correlation of mdct scan in the evaluation and as an adjunct to clinical staging of patients with hncs prior to therapeutic intervention. rationale for the study: the study aims to assess the efficacy of mdct as an efficient low-cost 3d imaging technique for the evaluation of hncs in a low economic resource setting where mri and pet-ct are not affordable and readily available. materials and methodology a two-year study that, included 34 patients with histological diagnosis of hnc according to the 2005 who criteria for head and neck tumors13. tumors at presentation were staged clinically according to 6th edition of american joint committee on cancer ajcc classification (ajcc, 2002)14 for head and neck sites which featured some improvements on the 5th edition15. these improvements were in (a) uniform description of advanced tumors whereby t4 lesions were divided into t4a (resectable) and t4b (unresectable) and (b) advanced stage diseases in patients were further assigned into three categories. the new categories were stage iva (advanced resectable disease); stage ivb (advanced unresectable disease); and stage ivc (advanced distant metastatic disease). the 6th edition of ajcc guidelines for clinical tnm staging include “collaborative staging” aimed at increased accuracy of diagnostic test by the use of newer clinical and radiologic diagnostic techniques. techniques such as mdct scan, mri, positron emission tomography pet or pet-ct may result in multi detector computerized tomography scans aid in the staging of head and neck cancers braz j oral sci. 15(4):273-279 275 3.0%2.9% of the tumor cases were assessed clinically as n3. another 8.8% of the cases were clinical nx (inaccessible lymph nodes). furthermore, clinical assessment of metastasis multi detector computerized tomography scans aid in the staging of head and neck cancers revealed that 70.6% were m0 cases while only 8.8% were m1, the remaining 20.6% presented with tumor metastasis that were not discernible (mx). table 1 site distribution of hncs according to histological types. tumour sites ic do c o de histological types of hncs scc npc odc snc adc total k nk ba sa lo id pa pi lla ry k nk ba sa lo id am c am cs base of the tongue c01.9 1 1 maxillary alveolar/ gum c03.0 1 1 mandibular alveolar/ gum c03.1 1 1 oropharynx, c10.9 1 3 4 nasopharynx c11.0 2 1 1 4 laryngopharynx/ hypopharynx, c13.9 2 1 3 pharynx, c14.0 1 1 nasal cavity c30.0 1 2 1 1 1 6 maxillary sinus c31.0 2 1 1 1 5 paranasal sinus c31.9 2 2 1 2 1 8 total 7 5 1 1 5 7 1 2 1 1 3 34 scc = squamous cell carcinoma; npc = nasopharyngeal carcinoma; odc = odontogenic carcinoma ; snc = sinonasal carcinoma; adc = adenocarcinoma ; amc ameloblastic carcinoma; amcs = ameloblastic carcinosarcoma ; k = keratinizing; nk = non-keratinizing. braz j oral sci. 15(4):273-279 overall, only a minority of cases were clinical stage ii and stage iii tumors (5.9% and 38.2%) respectively while the majority of the hncs cases were stage iv tumors (55.9%). among the stage iv tumors, majority (84.2%) were clinical stage iva while 15.8% were stage ivc with metastasis to distant sites such as brain, lungs and visceral organs. there was no significant difference in the clinical stage of hnc according to sex and site (p= 0.153 kruskalwallis non-parametric test & x2 = 26.6; p=0.49 respectively) mdct imaging also revealed that a minority of the head and neck cancers were t3 and t4a (11.8% each) while the majority belong to t4b (76.5%) with radiological evidence of tumor invasion into vital structures and surrounding tissues. the proportional changes from clinical tumour size to radiological tumour size of hncs cases are as shown in table 2. the clinical tumor size and the radiological tumor size showed a statistical significant difference (x2=5.5, p=0.019) and a low correlation (pearson’s correlation r=0.498). mdct showed only a minority (38.2%) of the cases presented as radiological n0 tumors, unlike the clinical nodal staging with majority at n0 (55.9%). also, 14.7% and 11.8% presented as radiological n1 and n2a nodal involvement respectively. the only case of clinical n3 was also assessed as radiologic n3 nodal involvement. the proportional changes from clinical nodal size to radiological nodal size of hncs cases are as shown in table 2. there was a statistical significant difference between the clinical size and the radiological size of lymph nodes involvement (x2=53.01, p=0.000). however, there was a higher positive correlation between clinical and radiological assessment of lymph node involvement (pearson’s correlation r=0.690) when compared with the correlation of clinical and radiologic tumor size (table 2& figure 1). the sensitivity, specificity, positive & negative predictive values and accuracy of clinical and radiological nodal involvements are as shown in table 3. sixty two percent 61.8% of cases had radiologic assessment as m0 while 38.2% had metastasis to distant sites (m1). however, unlike the clinical tumor assessment that presented with some cases of indiscernible metastasis to distant site (mx=20.6%), there was no tumor case of indiscernible distant metastasis with mdct imaging because the clinical cases of mx were either regarded as m0 (5.9%) or upgraded to m1 (14.7%) (table 2). there was a statistical significant difference between the clinical assessment and the radiological assessment of metastasis (x2=11.19, p=0.004). an inverse correlation between clinical metastasis and radiologic metastasis to distant site was observed (pearson’s correlation r = -0.054). most of the clinical stages ii, iii & iv cases were upgraded to higher radiological stages as shown in table 4. there was a weak but positive correlation (pearson’s correlation r = 0.6, figure 2k) with statistically significant difference between the clinical stages and radiological stages of head and neck cancers cases (x2= 260.8 p=0.01). there was a significant difference in the final clinical and radiological staging of hncs in this study (x2 = 270.79; p = 0.00) but there was no significant difference in the radiological stages of hnc according to sex and site. (p= 0.445 kruskal-wallis non-parametric test; x2=33.8; p=0.17 respectively). 276 multi detector computerized tomography scans aid in the staging of head and neck cancers table 2 the relationship between clinical tnm and radiological tnm. table 4 relationship between the final clinical and radiological staging of hncs. tumor size (t) clinical tumor size radiologic tumor size (t) total t3 t4a t4b t2 1 (2.9%) 0 2 (5.9%) 3 (8.8%) (x2=5.5, p=0.019; r=0.498)t3 3 (8.8%) 3 (8.8%) 7 (20.6%) 13 (38.2%) t4a 0 (0.0%) 1 (2.9%) 15(44.1%) 16 (47.1%) t4b 0 (0.0%) 0 (0.0%) 2 (5.9%) 2 (5.9%) total 4(11.8%) 4(11.8%) 26 (76.5%) 34(100%) nodal size (n) radiological size of lymph node (x2=53.01, p=0.000; r =0.690) nx: no: n1: n2a: n2c: n3: total clinical size of lymph nodes nx: 1(2.9%) 0 (0.0%) 2 (5.9%) 0 (0.0%) 0(0.0%) 0(0.0%) 3 (8.8%) no: 3(8.8%) 13(38.2%) 2 (5.9%) 1 (2.9%) 0(0.0%) 0(0.0%) 19 (55.9%) n1: 1(2.9%) 0 (0.0%) 1 (2.9%) 2 (5.9%) 4(11.8%) 0(0.0%) 8 (23.5%) n2a: 0(0.0%) 0 (0.0%) 0 (0.0%) 1 (2.9%) 1 (2.9%) 1(2.9%) 3 (8.8%) n3: 0(0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 1(2.9%) 1 (2.9%) total 5(14.7%) 13(38.2%) 5 (14.7%) 4 (11.8%) 5(14.7%) 2(5.9%) 34(100.0%) metastasis (m) radiological metastasis mx m0 m1 total mx 0(0.0%) 2 (5.9%) 5(14.7%) 7 (20.6%) (x2=11.19, p=0.004; r = -0.054)m0 0(0.0%) 19 (55.9) 5(14.7%) 24(70.6%) m1 0(0.0%) 0 (0.0) 3 (8.8%) 3 (8.8%) total 0(0.0%) 21(61.8%) 13(38.2%) 34(100.0%) cl in ic al s ta g in g radiological staging stage 3 stage 4a stage 4b stage 4c total n % n % n % n % n % stage 2 1 2.9% 0 0.0% 1 2.9% 0 0.0% 2 5.9% stage 3 3 8.8% 2 5.9% 5 14.7% 3 8.8% 13 38.2% stage 4a 0 0.0% 2 5.9% 9 26.5% 5 14.7% 16 47.1% stage 4c 0 0.0% 0 0.0% 0 0.0% 3 8.8% 3 8.8% total 4 11.8% 4 11.8% 15 44.1% 11 32.4% 34 100.0% table 3 comparison of the accuracy of clinical and mdct detection of nodal involvement. comparison of clinical and histological results histology clinical positive negative total positive 9 3 12 negative 10 12 22 total 19 15 34 comparison of mdct and histological results histology mdct positive negative total positive 15 1 16 negative 4 14 18 total 19 15 34 analysis of the accuracy of clinical versus mdct examination modality sensitivity (%) specificity (%) accuracy (%) ppv (%) npv (%) clinical 9/19(47.4%) 12/15(80%) 21/34(61.8%) 9/12(75%) 12/22(54.5%) mdct 15/19(78.9%) 14/15(93.3%) 29/34(85.3%) 15/16(93.8%) 14/18(77.8%) ppv = positive predictive value, npv = negative predictive value. sensitivit = a/(a+c) = true positives true positives + false negatives specificity = d/(b+d) = true negatives false positives + true negatives positive predictive value = a/(a+b) = true positives true positives + false positives negative predictive value = d/(d+c) = true negatives true negatives + false negatives accuracy = a+d = true positives + true negatives (a+b+c+d) all positives + all negatives braz j oral sci. 15(4):273-279 discussion late stage hncs clinical presentation (stages iii and iv) is a management challenge with up to 50% cited in western 277multi detector computerized tomography scans aid in the staging of head and neck cancers fig. 1 ct of the head and neck showing multiple enhancing round-oval shaped iso-dense masses with central hypo-densities consistent with enlarged cervical lymph nodes (red arrow heads) with necrotic centers in the anterolateral and the left side of the neck demonstrated in a) axial view in non-enhanced contrast nect b) axial view in contrast enhanced cect c) sagittal view cect d) coronal view cect there is associated medial extension with right sided displacement of the trachea and compression of the laryngeal airway fig. 2 correlation between clinical stages and radiological stages of hnc. literature6,18,19. in developing countries late stage presentation of up to 90% in kenya, 79.1% in brazil and 65% in thailand20-22 have been reported. the findings in this study (94.2%) is in keeping with other resource limited economy. kolude et al.23 (2013) attributed the delayed presentation to poverty, lowawareness, poor health seeking attitudes of patients and health care professional delay. the present study went further to sub-classify the clinical tumor cases by utilizing mdct images for radiological staging.previous study by issacs et al.24 have shown upgrade of t2 to t4 laryngeal carcinoma with deep-spread pattern while gatenby et al.25 reported a series in which ct scan findings altered treatment planning in up to 35% of patients who presented with t2 and t3 stages. the low percentage of upstage in the later study may be partly due to the use of conventional 3-mm slices in axial ct imaging as against the more recent 0.5mm-1mm cuts inmultiplanar reconstructed ct imaging employed in this study. dillon and hamsberger26 in a study of hncs observed that ct imaging upgraded previous clinical t2 stages to higher grades. berker27 also pointed out the tendency of ct to up-stage malignant tumors in a large number of cases which was validated by lell et al.10 (2008). in another study by prehn et al.28 (1998) that utilized conventional ct, the majority of the hypopharyngeal tumors were upstaged to t4 but only a minority of oropharyngeal tumors were upstaged to t4. in our study, all hypopharyngeal t2, t3 and t4a clinical tumors were upstaged to t4b radiological tumors while the majority of oropharyngeal t3 cases were upstaged to t4a. the higher proportion of upgrade in the present study may be partly due to the predominant advanced stage of cases due to delay in patients’ presentation for cancer care; an additional factor may be the use of high resolution mdct. it is generally agreed that detection of hnc nodal metastasis is more accurately performed with imaging rather than with clinical palpation, therefore imaging is used in detection of nodal metastasis at presentation and in early detection of nodal tumor recurrence. ct was widely considered to be the gold standard imaging technique for identifying nodal metastasis and extra nodal spread29,30. however, mri provides several advantages like excellent soft-tissue contrast by being able to differentiate normal from pathologic tissues, permits the exact delineation of tumor margins, over ct in the evaluation of head and neck region tumors. mri is non-ionizing with multiplanar acquisition and might not require intravenous contrast administration. on the other hand, ct has the advantage of detection of mild bony changes, relatively unaffected by patient motion and other artifacts unlike mri which is also expensive, unavailable and or inaccessible for hnc patients in most institutions in our setting31. in the index study, it was observed that about 35.2% of the regional lymph node metastasis was revealed on clinical assessment in contrast with 47.1% revealed by the ct imaging. the sensitivity (78.9%), specificity (93.3%), ppv (93.8%), npv (77.8%) and accuracy (85.3%) indicate that mdct increased the potential of detecting metastatic cervical lymph nodes, this was evident in 6 cases in which mdct correctly changed the clinical staging of nodal involvement; clinical nodal stage was upgraded in 5 cases and downgraded in 1 case. previous works on the efficacy of ct in detecting nodal metastasis in hncs include those of king et al.29, branstetter et al.32 and anand et al.33 the braz j oral sci. 15(4):273-279 sensitivity, specificity, ppv, npv and accuracy of ct findings inf their studies were: (sensitivity 65%, 74% & 77.5%); (specificity 93%, 75% &92.4%); ppv (96%, 63% & 94.5%); (npv 50%, 83% & 71%); (accuracy 73%, 74% & 83%) respectively. the sensitivity in the present study is within the upper limit of the estimated range of 70 – 80% in previous literature, this is possibly due to the use of contrast enhancement and multiplanar reformatted images (as compared to conventional ct imaging techniques). krestan et al.34 stated that the efficacy of ct scans depend on the type of ct machine and other technical abilities such as contrast enhancement. anand et al.33 ascribed differences in the efiicacy and appearances of ct images to several factors such as the use of larger sections of scan, use of different criteria for positive node and use of different bolus or continuous contrast infusion by pump injectors. a contrary opinion by lell et al.10 observed no significant difference between the performance of the recent mdct (high resolution ct using 0.5-1mm cuts) and the older ct protocols utilized for conventional ct scans with 3mm slice thickness for the accurate estimation of nodal mettastsses. up to 32.8% of patients in the study had radiologic stage of m1 in contrast to clinical examination which revealed metastasis of 8.8%. most reports on hncs gave 10 – 20% prevalence of metastasis in hncs but de bree et al.35 stated that patients with ≥3metastatic lymph nodeshave up to a 50% risk of distance metastasis. majority of the cases in this study were late stage presentations that may have allowed enough time for multiple nodal involvement and distant metastasis. each tumor stage has inherent prognostic importance but of particular importance is the pre-operative prediction of margins of t4 tumor in determining resectability and the optimal extent and duration of such surgery. staging also provides information on the need for concomitant chemo-radiotherapy conley36, 2006 and petralia et al.37 the implication of alteration of changes from clinical to radiological staging plays a significant role in tumor resectability. because n this study, only 23.6% of stages iii and iva were resectable against the suggestion of 91.2% (stages ii,iii and iva) by clinical staging. furthermore, a sizeable proportion of clinically resectable tumors turned out to be radiologically advanced unresectable tumors. many of the cases in this study later required adjuvant surgery and chemo-radiotherapy (44.1%). in addition, a considerable proportion were inoperable and only benefited from palliative care (32.4%). early diagnosis is the most important determining factor for improving hncs with up to 80-90% survival rate stated for stages ihncs3. in developed countries, recent imaging investigation and evaluation of hncs involve the use of 5’fluro-deoxyglucose fdg-pet/ct with supportive mri or ultrasound as adjuncts. despite these facilities, ~50%of patients diagnosed with cancer die of advanced disease. indeveloping countries; this figure reaches up to 80%. by the year 2020, who estimates that 70% of new cancer cases will be in developing countries, with most patients presenting with late stages of cancer, even when patient seek early care, diagnosis and treatment may be delayed, unaffordable, or unavailable38. branstteter et al.32 compared pet scan, mdct and pet-ct scan, and observed that the proportion of extra lesions identified were 21%, 37% and 40% respectively which suggests better detection and evaluation of mdct than pet scan alone and a very close sensitivity of mdct compared withd pet-ct scan. pet and mri as imaging modalities have been available for over 30 years. inspired by the pet-ct combination, over 50 pet-mri machines have been developed and utilised in the usa and european countries. , though a hybrid form of imaging with promising clinical applications ,it has not yet been established as an imaging modality for clinical practice39. the findings in this study show the superiority of mdct imaging compared with clinical physical examination in staging of patients with hncs and it is in agreement with shah et al.40 (2008) who stated that mdct should be the first diagnostic imaging study for patients with head and neck cancers particularly with suspected oropharyngeal and/or laryngeal involvement. therefore, we recommend mdct as the first line image investigation for hncs in low economic resource setting where pet-ct may not be available. in conclusion mdct was significantly more accurate than clinical examination in the determination of tumor size, nodal involvement and tumor metastasis of hncs according to the ajcc/uicc tnm guidelines. authors recommend mdct as first line imaging technique in the evaluation of hncs in resource limited settings. references 1. shishodia np, divakar dd, al kheraif aa, ramakrishnaiah r, pathan aa, parine nr, et al. end stage palliative care of head and neck cancer: a case study. asian pac j cancer prev. 2015;16(3):1255-8. 2. chin d, boyle gm, porceddu s, theile dr, parsons pg, coman wb. head and neck cancer: past, present and future. expert rev anticancer ther. 2006 jul;6(7):1111-8. 3. lam dk, schmidt bl. orofacial pain onset predicts transition to head and neck cancer. pain. 2011 may;152(5):1206-9. doi: 10.1016/j. pain.2011.02.009. 4. brockstein b, haraf dj, rademaker aw, kies ms, stenson km, rosen f. et al. patterns of failure, prognostic factors and survival in loco regionally advanced head and neck cancer treated with concomitant chemo radiotherapy: a 9-year, 337-patient, multi-institutional experience. ann oncol. 2004 aug;15(8):1179-86. 5. worsham mj. identifying the risk factors for late stage head and neck cancer. expert rev anticancer ther. 2011 sep;11(9):1321-5. doi: 10.1586/era.11.135. 6. lopez-jornet p, camacho-alomso f: new barriers in oral cancer. patient accessibility to dental examination-a pilot study. oral oncol. 2006 nov;42(10):1022-5. 7. mccabe kj, rubinstein d: advances in head and neck imaging. otolaryngol clin north am. 2005 apr;38(2):307-19, vii. 8. forghani r, yu e, levental m, som pm, and curtin hd: imaging evaluation of lymphadenopathy and patterns of lymph node spread in head and neck cancer. expert rev anticancer ther. 2015 feb;15(2):20724. doi: 10.1586/14737140.2015.978862. 9. agarwal v, branstetter iv bf, johnson jt, indications for pet/ct in the head and neck. otolaryngol clin north am. 2008 feb;41(1):23-49, v. doi: 10.1016/j.otc.2007.10.005. 10. lell mm, gmelin c, panknin c, eckel kt, schmid m, bautz wa, et al. thin-slice mdct of the neck: impact on cancer staging. ajr am j roentgenol. 2008 mar;190(3):785-9. doi: 10.2214/ajr.07.3081. 278 multi detector computerized tomography scans aid in the staging of head and neck cancers braz j oral sci. 15(4):273-279 11. hsu wc, loevner la, karpati r, et al. accuracy of magnetic resonance imaging in predicting absence of fixation of head and neck cancer to the prevertebral space. head neck. 2005 feb;27(2):95-100. 12. lell mm, greess h, hothorn t, janka r, bautz wa, baum u. multiplanar functional imaging of the larynx and hypopharynx with multi-slice spiral ct. eur radiol. 2004 dec;14(12):2198-205. 13. barnes l, eveson j, reichart p, sidransky d, editors. world health organization classification of tumors. pathology and genetics of head and neck tumors. lyon: iarc press; 2005. 14. greene fl, page dl, fleming id, fritz ag, balch c, haller dg, et al., editors. the american joint committee on cancer staging manual. 6th. new york: springer; 2002. 15. fleming id, cooper js, henson de, hutter rvp, kennedy bj, murphy gp, et al., editors. ajcc cancer staging manual. 5th ed. philadelphia: lippincott-raven; 1997. 294p. 16. madison mt, md, remley kb, richard md. radiologic diagnosis and staging of head and neck squamous cell carcinoma. otolarygol clin north am. 1998 aug;31(4):727-54. 17. fritz a, percy c, jack a, shanmugaratmam k, sobin l, arkin dm, et al. editors. international classification of diseases for oncology (icd-o). 3rd ed. geneva: who; 2000. 18. zong. j, lin s, lin. j, tang l, chen b, zhang m, et al. impact of intensitymodulated radiotherapy on nasopharyngeal carcinoma: validation of the 7th edition ajcc staging system. oral oncol. 2015 mar;51(3):254-9. doi: 10.1016/j.oraloncology.2014.10.012. 19. warnakulasuriya s. global epidemiology of oral and oro-pharyngeal cancer. oral oncol. 2009 apr-may;45(4-5):309-16. doi: 10.1016/j. oraloncology.2008.06.002. 20. onyango jf, awange do, njiru a, macharia im. pattern of occurrence of head and neck cancer presenting at kenyatta national hospital, nairobi. east afr med j. 2006 may;83(5):288-91. 21. carvalho al, pintos j, schlecht nf, oliveira bv, fava sa, curado mp, et al. predictive factors for diagnosis of advanced-stage squamous cell carcinoma of the head and neck. arch otolaryngol head neck surg. 2002 mar;128(3):313-8. 22. kerdpon d, sriplung h. factors related to delay in diagnosis of oral squamous cell carcinoma in southern thailand. oral oncol. 2001 feb;37(2):127-31. 23. kolude b, adisa a, adeyemi b, lawal a. stages of delay in oral cancer care evaluated at a tertiary health centre. afr. j.med.med sci. 2013 dec;42(4):347-53. 24. issacs jj, mancuso aa, mendenhall wm, parsons jt. deep spread patterns in ct staging of t2-4 squamous cell laryngeal carcinoma. otolaryngol head neck surg. 1988 nov;99(5):455-64. 25. gatenby ra, mulhern cb, strawitz j, moldofsky pj. comparison of clinical and ct staging of head and neck tumors. ajnr am j neuroradiol. 1985 may-jun;6(3):399-401. 26. dillon wp, harnsberger hr. the impact of radiologic imaging on staging of cancer of the head and neck. semin oncol.1991 apr;18(2):64-79. 27. berker m. neoplastic invasion of laryngeal cartilage: radiologic diagnosis and therapeutic implications. eur. j radiol. 2000 mar;33(3):216-29. 28. prehn rb, pasic tr, harari pm, brown wd, ford cn. influence of computed tomography on pre-therapeutic tumor staging in head and neck cancer patients. otolaryngol head neck surg. 1998 dec;119(6): 628-33. 29. king ad, tse gm, yuen eh, to ew, vlantis ac, zee b, et al. comparison of ct and mr imaging for the detection of extra-nodal neoplastic spread in metastatic neck nodes. eur j radiol. 2004 dec;52(3):264-70. 30. sakai o, curtin hd, romo lv, som pm. lymph node pathology. benign proliferative. radiol clin n am. 2000 sep;38(5):979–98. 31. dillon wp. magnetic resonance imaging of head and neck tumors. cardiovasc intervent radiol. 1986;8(5-6):275-82. 32. branstetter bf, blodgett tm, zimmer la, snyderman ch, johnson jt, raman s, et al. head and neck malignancy: is pet/ct more accurate than pet or ct alone? radiology. 2005 may;235(2):580-6. 33. anand n, chaudhary n, mittal m.k, prasad r. comparison of the efficacy of clinical examination, ultrasound neck and computed tomography in detection and staging of cervical lymph node metastasis in head and neck cancers. indian j otolaryngol head neck surg. 2007 mar;59(1):19-23. doi: 10.1007/s12070-007-0005-6. 34. krestan c, herneth am, formanek m, czerny c. modern imaging lymph node staging of the head and neck region. eur j radiol. 2006 jun;58(3):360-6. 35. de bree r, deurloo ee, snow gb, leemans cr. screening for distant metastasis in patients with head and neck cancer. laryngoscope. 2000 mar;110(3 pt1):397-401. 36. conley ba. treatment of advanced head and neck cancer: what lessons have we learned? j clin oncol. 2006 mar;24(7):1023-5. 37. petralia g, preda l, giugliano g, jereczek-fossa ba, rocca a, d'andrea g, et al. perfusion computed tomography for monitoring induction chemotherapy with squamous cell carcinoma of the upper aero digestive tract: correlation between changes in tumor perfusion and tumor volume. j comput assist tomogr. 2009 jul-aug;33(4):552-9. doi: 10.1097/rct.0b013e31818d446e. 38. soyannwo so. how to make a difference in the developing world: organizing resources. in: paice ja, bell rf, kalso ea, soyannwo oa., editors. cancer pain from molecules to suffering. iasp press; 2010. p.333-45. 39. nensa f,beiderwellen k, heusch p, wette a. clinical applications of pet/mri: current status and future perspectives. diagn interv radiol. 2014 sep-oct;20(5):438-47. doi: 10.5152/dir.2014.14008. 40. shah gv, wesolowski jr, ansari sa, mukherji sk. new directions in head and neck imaging. j surg oncol. 2008 jun 15;97(8):644-8. doi: 10.1002/jso.21022. 279multi detector computerized tomography scans aid in the staging of head and neck cancers braz j oral sci. 15(4):273-279 1 volume 16 2017 e17075 original article 1graduate school of pediatric dentistry, faculty of dentistry universitas padjadjaran, indonesia. 2dept of pediatric dentistry faculty of dentistry universitas padjadjaran, indonesia corresponding author: lani manurung 232, pinus raya street,  bandung, indonesia elemem25@gmail.com received for publication: july 12, 2017 accepted: october 20, 2017 mic, mbic, mbec analyses of garlic extract (allium sativum) from indonesian variety against streptococcus mutans lani manurung1, eriska riyanti2, eka chemiawan2, mieke hemiawati satari2, meirina gartika2 aim: this study was designed to evaluate the inhibition and eradication ability for garlic against streptococcus mutans, the bacteria that cause dental caries. methods: garlic extract was obtained from ciwidey garlic maseration process in etanol 96%. mic was a test to measure the inhibition ability of garlic extract against streptococcus mutans in planktonic form, and mbic in biofilms form. mbec was a test to measure the eradication ability of garlic extract against streptococcus mutans in biofilms form. statistic analysis using anova followed by post hoc with ρ value <0.05. results: the garlic extract showed mic value at 9.39% and mbic value at 37.5% but was not observed to have eradication activity against streptococcus mutans in biofilms form up to concentration 37.5%. conclusion: garlic extract inhibit streptococcus mutans both in planktonic and biofilm form. garlic extract has no eradication activity against streptococcus mutans in biofilms form up to concentration 37.5%. keywords: garlic extract. streptococcus mutans. planktonic. biofilm. http://dx.doi.org/10.20396/bjos.v16i0.8651055 2 manurung et al. introduction nature provides many natural ingredients that are nutritious for health, such as garlic, turmeric, ginger and many others2. natural ingredients can be better option because of several advantages such as often having fewer side-effects, better patient tolerance, being relatively less expensive and acceptable due to a long history of use. garlic (allium sativum) has many uses such as an antioxidant, antiseptic, anti-viral, anti-fungal, anti-cancer, anti-aging, reduce the risk of heart disease, and antibacterial1. garlic contains allicin that well known has antibacterial properties that can inhibit the growth of bacteria which cause disease3. however, there is lack of studies regarding the exact value of garlic’s inhibition ability against bacteria. one of the disease that caused by bacteria is dental caries. dental caries is a multifactorial, chronic bacterial disease, that causes demineralization and destruction of the hard tissues, usually by production of acid by bacterial fermentation of the food debris accumulated on the tooth surface4. bacteria has two forms, which is planktonic form and biofilm form that have different ability, properties and resistance. indonesian still have high levels of tooth decay or caries according to riset kesehatan dasar5. garlic has two properties that are useful for reducing caries: antibacterial so it can reduce bacteria’s acid production and can stimulate saliva expenditure caused garlic flavor6,7. the aim of this study is to find out the inhibition and eradication ability of garlic extract (allium sativum) against streptococcus mutans atcc 25175 in planktonic and biofilm form. material and methods garlic extract the fresh ciwidey garlic were collected on march 2015. ciwidey is one of agricultural area in bandung, indonesia. the determination of the species was from taxonomy test in bidang sumber daya sekolah ilmu dan teknologi hayati institut teknologi bandung, indonesia. 500 gram fresh garlic peeled and cleaned, then soaked in 650 ml ethanol 96%. the next phase is filtered, followed by evaporation of the solvent with a rotary evaporator. maceration process is performed three times at the laboratory of chemistry, university of padjadjaran, bandung, indonesia. microbial strain microbial strain used was streptococcus mutans atcc 25175 obtained from unpad laboratory of chemistry, bandung, indonesia. bacteria were grown in brain heart infusion broth enriched with sucrose 2% for 48 h at 37°c both for planktonic and biofilm assay. determination of the minimum inhibitory concentration (mic) mics were determined by modified micro titer broth method in sterile flat bottom 96-well polystyrene plates8. prepared microwell plate formats: media + samples, media, media + sample + bacteria, media + bacteria (made duplo). 3 manurung et al. brain heart infusion (bhi) broth 150μl pipette into a microwell plate. then, pipette 150μl sample (garlic extract) in a microwell plate and performed a serial dilution. liquid culture of streptococcus mutans 10μl pipetted and put it in a microwell plate then incubated at 37°c for 48 hours. optical density read by bio-rad microplate reader at absorbance 595 nm. determination of the minimum biofilm inhibition concentration (mbic) mbics were determined by the adherence assay in sterile flat bottom 96-well plates. a similar serial dilution was as performed in antibacterial assay. after 48 h incubation, the supernatants were aspired from the wells, rinsed 3 times with phosphate buffered saline (pbs) and incubated at 70 0c for 15 min. then, 200 μl of 0.1% crystal violet was added to the wells and left for 30 min to stain the formed biofilm. the excess stain was rinsed off with pbs 3 times, followed by addition of 200μl of ethanol to the wells. optical density read by bio-rad microplate reader at absorbance 595 nm. determination of the minimum biofilm eradication concentration (mbec) mbecs were determined by the adherence assay in flat bottom 96-well plates as performed on biofilm formation inhibition assay9. after 48 h incubation of bacteria suspension in media used (without samples), the supernatants were aspired from the wells. a serial dilution of samples in media were then added to the wells and incubated for another 24 h in 37° c. further treatments were as performed on the biofilm formation inhibition assay. results determination of the minimum inhibitory concentration (mic) and minimum biofilm inhibitory concentration (mbic) results summary of the minimum inhibitory concentration (mic) is as described in table 1. results summary of the minimum biofilm inhibitory concentration (mbic) is as described in table 2. garlic extract concentration (% v/v) 37.5% 18.76% 9.38% 4.69% 2.34% 1.17% 0.59% 0.29% 0.15% 0.07% 0.04% m+s 0.266 0.305 0.273 0.245 0.240 0.2405 0.250 0.349 0.265 0.240 0.226 m+p 0.285 m+s+b 0.214 0.283 0.274 0.383 0.732 1.416 0.862 0.401 0.358 0.359 0.376 m+p+b 0.301 table 1. results of the minimum inhibitory concentration (mic). m = media s = samples (garlic extract) p = solvent b = bacteria (streptococcus mutans) 4 manurung et al. the result showed mic value at 9.39%. and mbic at 37.5%, so that garlic extract has inhibitory ability against streptococcus mutans atcc 25175 in planktonic and biofilm form. determination of the minimum biofilm eradication concentration (mbec) results summary of the minimum biofilm eradication concentration (mbec) is as described in table 3. the result showed that garlic extract not observed to have eradication activity against streptococcus mutans atcc 25175 in biofilms form up to concentration 37.5% which is the highest concentration on this study. discussion there are five stages in the process of biofilm formation (figure 1): at the first stage, planktonic cells create reversible attachment to a biomaterial surface and/or host cell surface. the cells are still susceptible to antimicrobial agents. at the second stage, the adhesion of subsequent microorganism to surface becomes irreversible and the cells begin to secrete eps. the eps reducing the amount of antimicrobial available to interact with biofilm, act as an adsorbent or reactant, as well as their structure physically reduces the penetration of antimicrobial agents by walling off access to regions of the biofilm10,11. table 2. results of the minimum biofilm inhibitory concentration (mbic). garlic extract concentration (% v/v) 37.5% 18.76% 9.38% 4.69% 2.34% 1.17% 0,59% 0,29% 0,15% 0,07% 0,04% 0,02% m+s 0.255 0.167 0.125 0.165 0.113 0.124 0.113 0.283 0.105 0.105 0.100 0.098 m+p 0.087 m+s+b 0.2545 0.419 0.266 0.311 0.292 0.271 0.243 0.2675 0.266 0.1795 0.093 0.091 m+p+b 0.2162 table 3. results of the minimum biofilm eradication concentration (mbec). garlic extract concentration (% v/v) 37.5% 18.76% 9.38% 4.69% 2.34% 1.17% 0.59% 0.29% 0.15% 0.07% m+s 0.1915 0.146 0.105 0.1055 0.1035 0.124 0.0975 0.106 0.107 0.172 m+p 0.087 m+s+b 0.2075 0.1505 0.1275 0.2335 0.159 1.6655 1.9935 0.113 0.115 0.103 m+p+b 0.1466 5 manurung et al. third stage is the maturation stage where the amount of ecm increases around the microcolonies, due a continued secretion of eps. it is possible to observe a mature biofilm containing cavities that serve as transport channels of water and planktonic cells throughout the biofilm community, at the fouth stage, and also provides a unique environment for optimum nutrient absorption and waste disposal. biofilms detachment can occurs in two ways (stage 5): continual dispersal of single cell or small portion of the biofilms (erosion); and, where large pieces of the biofilms are significantly lost (sloughing)12. this disruption can expand the infection once the microbial cells are liberated and can colonize another location/surface. all this five stages explain why bacteria in biofilm formation more difficult to inhibit or eradicate than bacteria in planktonic formation. in conclusion, as according to karygianni journal2, yu-ying journal6, and el-samarrai and rashad journal7, the result of this study revealed that garlic extract have inhibition ability against streptococcus mutans atcc 25175 in planktonic and biofilm form, but was not observed to have eradication activity against streptococcus mutans atcc 25 175 in biofilms form up to concentration 37.5%. acknowledgment the authors would like to thank ministry of research technology and higher education republic of indonesia for funding this study by means of superior research university grant, universitas padjadjaran (grant no 393/un6.r/pl/2015) references 1. harini k, babu s, ajila v, hegde s. garlic: it’s role in oral and systemic health. nujhs. 2013 dec;3(4):17-22. 2. karygianni l, al-ahmad a, argyropoulou a, hellwig e, anderson ac, skaltsounis al. natural antimicrobials and oral microorganisms: a systematic review on herbal interventions for the eradication of multispecies oral biofilms. front microbiol. 2016 jan 14;6:1529. doi: 10.3389/fmicb.2015.01529. 3. rajsekhar s, kuldeep b, chandaker a, upmanyu n. spices as antimikrobial agents: a review. irjp. 2012;3(2):4-9. 1 2 3 4 5 figure 1. stages in biofilm formation10. 6 manurung et al. 4. karpinski tm, szkaradkiewicz ak. microbiology of dental caries. j biol earth sci. 2013;3(1):m21-4. 5. ministry of health of indonesia. [basic health research 2013]. riset kesehatan dasar 2013 [cited 2015 feb 5]. available from: http://www.litbang.depkes.go.id/sites/download/rkd2013/laporan_ riskesdas2013.pdf. indonesian. 6. yu-ying c, chiu h, wang y. effect of garlic extract on acid production and growth of streptococcus mutans. j food drug anal. 2009;17(1):59-63. 7. el-samarrai sk, rashad jm. garlic extracts and acidogenicity of mutans streptococci. e-j dent. 2013 jan;3(1). 8. sasmita is, gartika m, satari mh, chairulfattah a, hilmanto d. antibacterial activity of papain againt streptococcus mutans atcc 25175 int j dev res. 2014 oct;4(10):2075-7. 9. hertiani t, pratiwi sut, irianto idk, febriana a. kaemferia galangal l. rhizome as a potential dental plaque preventive agent. indones j cancer chemoprevention. 2010;1(1):19-25. 10. melo wcma, perussi jr. strategies to overcome biofilm resistance. in: méndez-vilas, editor. microbial pathogens and strategies for combating them: science, technology and education. badajoz, spain: formatex research center; 2013. p. 179-87. 11. ccahuana-vasquez ra, cury ja. s.mutans biofilm model to evaluate antimicrobial substances and enamel demineralization. braz oral res. 2010. apr-jun;24(2):135-41. 12. gupta a. biofilm quantification and comparative analysis of mic (minimum inhibitory concentration) & mbic (minimum biofilm inhibitory concentration) value for different antibiotics against e. coli. int j current microbiol applied sci. 2015;4(2):198-224. http://www.litbang.depkes.go.id/sites/download/rkd2013/laporan_riskesdas2013.pdf http://www.litbang.depkes.go.id/sites/download/rkd2013/laporan_riskesdas2013.pdf 1http://dx.doi.org/10.20396/bjos.v18i0.8656599 volume 18 2019 e191443 original article 1 department of dental materials, dentistry college, federal university of pará (ufpa), belém, pará, brazil. 2 department of prosthodontics, dentistry college, federal university of pará (ufpa), belém, pará, brazil. corresponding author: laise pena braga monteiro universidade federal do pará instituto de ciências da saúde faculdade de odontologia avenida augusto corrêa, 01. belém pa, 66075-110 brazil phone number: 55-91-32017563 fax number: 55-91-32017563 e-mail: laisemonteiro@hotmail.com https://orcid.org/0000-0003-1406-2969 received: november 09, 2018 accepted: may 15, 2019 evaluation of ceramic flexural strength of a cobalt-chromium alloy subjected to airborne particle abrasion and tungsten inert gas welding laise pena braga monteiro1,*, issae sousa sano2, suelen reis cunha2, eliza burlamaqui klautau2, bruno pereira alves2 aim: the aim of this study was to evaluate the influence of tungsten inert gas (tig) welding and airborne particle abrasion using aluminum oxide particles on the flexural strength of a joint between ceramic and cobalt-chromium alloys. methods: the specimens were cast and welded using tig, then divided into 6 groups (n = 10) and subjected to blasting with 250 μm, 100 μm, and 50 μm aluminum oxide particles. ceramic systems were applied to the central part of all specimens. a three-point bending test using a velocity of 0.5 mm/m was performed on the specimens to measure flexural strength. data were analyzed using two-way analysis of variance and tukey’s test. results: tig welding demonstrated the lowest resistance compared with the non-welded groups. airborne particle abrasion using 250 μm aluminum oxide particles demonstrated greater resistance in the welded groups (p < 0.05). mixed faults were found in all specimens. conclusion: tig welding decreased the bond strength, and the particle size of aluminum oxide did not affect the metal-ceramic bond in groups without tig welding. keywords: dental materials. dental soldering. dental porcelain. https://orcid.org/0000-0003-1406-2969 2 monteiro et al. introduction although metal-free restorations are a growing trend in dentistry, metal-ceramic restorations have been widely used in dental practice due to the excellent clinical performance and feasibility for fabricating fixed partial dentures and single crowns. for this, metallic alloys, such as cobalt-chromium (co-cr), are used because of their biocompatibility and low cost compared with noble metallic alloys1-3. the formation of an adhesive layer between metal and ceramic is necessary for the longevity of restorations. there are three possibilities of retention, including van der waals forces, micromechanical retention and chemical bonding, which is the main factor, and is characterized by the exchange of oxides between the metal and the oxidizable elements of ceramics4-6. several surface treatments have been studied, such as airborne particle abrasion (apa) with aluminum oxide (al2o3) particles, which is a commonly used method for providing mechanical retention, resulting in the formation of an oxide layer that facilitates adhesion of the porcelain and increases the metal surface energy7-10. however, the size of the particles used in this process varies. therefore, it is important to define possible interactions between these different size particles and metal-ceramic adhesion in co-cr alloys. marginal adaptation and passive fit are one of the most important requirements of dental prosthesis. prosthetic infrastructure fused in a single piece may increase the probability of lack of fit. a solution to this problem is to section the metallic infrastructure and weld the abutments. this procedure is used to achieve the best adaptation and uniform distribution of forces, thus reducing trauma to the supporting teeth11-13. in the case of implants, this distribution becomes even more important, because they do not have the physiological mobility necessary to compensate for distortions resulting from adaptation errors14. figure 1. aspect of the metal surface without welding: apa a) 250 μm, b) 100 μm and c) 50 μm. aspect of the metal surface with tig welding: apa c) 250 μm, d) 100 μm and e) 50 μm. a b c c d e 1 mm 1 mm 1 mm 1 mm 1 mm 3 monteiro et al. although the conventional brazing technique is more used in dentistry, studies have demonstrated the superiority of tungsten inert gas (tig) welding15,16. this process uses an electric arc formed between a non-consumable tungsten electrode and the part to be fused and applies an inert gas (argon or helium) to provide local protection to prevent oxidation17-20. in this type of welding, heating is concentrated and disorders and deformation are minimized; furthermore, there is an increase in corrosion resistance due to the lack of galvanic effects in the joint17. although tig welding is widely used in engineering, the equipment has been adapted to enable its use in dentistry. therefore, it is an alternative in rehabilitation treatment with metal-ceramic restorations. however, there is still a lack of studies evaluating the adhesion of ceramics in areas that have been subjected to this type of welding, mainly with co-cr alloys. the purpose of this study was to evaluate the bonding strength between areas in co-cr alloys that undergo tig welding and apa using different sizes of al2o3 particles. material and methods a total of 90 patterns of thermopolymerizable acrylic resin (jet, artículos odontológicos clássico ltda, são paulo, sp, brazil) were obtained with the following dimensions: 25 mm in length, 3 mm in width and 1 mm in thickness (n=60); and 50 mm in length, 3 mm in width and 1 mm in thickness (n=30). the ends of the strips were joined using wax (wax in sprue kota, são paulo, brazil), and the feed conduits (wax in sprue kota, são paulo-sp, brazil) were positioned perpendicular to the standards (silicone ring ogp, são paulo-sp, brazil) for inclusion. a nº 5 silicone ring was filled with coating material (microfine 1700 coat talmax, curitiba-pr, brazil) at a ratio described by the manufacturer (90 g powder: 23 ml of liquid; the liquid solution comprised 18 ml of liquid and 5 ml of distilled water) mixed for 10 s, and vacuum spatulated (vrc-vrc equipment guarulhos – sp, brazil) for 30 s. the coating was placed in an electric oven (edgcon 5p, equipamentos e controlles ltda, são carlos, sp, brazil) at a temperature of 400°c for 30 min. subsequently, the temperature was raised to 950°c, and maintained for 20 min. after this, the temperature was reduced, and the ring was removed (900°c for co-cr alloys). fifteen grams of co-cr alloy were distributed inside the ceramic crucible for the induction centrifugation system (power cast red edg são carlos sp brazil). the specifications of the alloy are listed in table 1. after metal cooling, the strips were finished with polystyrene (arotec, cotia, sp, brazil) al2o3 sandpaper (120 grit; aixa abrasive, norton abrasive ltd, são paulo-sp, brazil). the group allocations are listed in table 2. a single operator performed the entire welding process according to standardized methods. tig welding was performed using tig nty 60c welding equipment (kernit indústria mecatrônica ltda, indaiatuba, sp, brazil). the apparatus was positioned perpendicular to the part to be welded. a grounding claw was attached to the part. the system was activated and argon flow was initiated, forming an oxygen-free region. an acrylic device containing a channel was used for accommodating the specimens, without any spaces between them19. 4 monteiro et al. all groups were subjected to apa using al2o3 (bio-art aluminum oxide, são carlos-sp, brazil) for 10–15 s, at a pressure of 60 lbs/in2 (table 2). for the control groups, a particle size of 250 μm was used, as recommended by the manufacturer of the ceramics. in addition, all strips were placed on a vibrator with distilled water for waste removal. an acrylic matrix was prepared to delimit the ceramic application area to 8 ± 0.1 mm in length, 3 ± 0.1 mm width and 1 ± 0.1 mm thickness. the ceramic, vita vm13 (vita ceramics, bad säckingenbaden-württemberg, germany) was applied to the center of the weld area on one side of the metal specimens. first, an opaque layer was applied, which was subjected to a firing cycle according to the manufacturer’s protocol. using the bounding matrix, dentin layers were applied to the established dimensions21. an evaluation of bond strength between the ceramic and metallic substrates was performed using a 3-point bending test, which used a 50 n load at a speed of 0.5 mm/min. all samples were evaluated in a test machine (kratos equipment, são paulo, sp, brazil) at the dental materials laboratory in the dental school of the federal university of pará, brazil. the flexural strength was calculated according to the following formula: ∑ = 3pi 2bd2 table 2. description of groups. groups alloy welding / apa ceramics g1(n = 10) co-cr without welding / apa (250μm) control vita vm13 g2(n = 10) co-cr tig / apa (250μm)control vita vm13 g3(n = 10) co-cr without welding / apa (100μm) vita vm13 g4(n = 10) co-cr tig / apa (100μm) vita vm13 g5(n = 10) co-cr without welding / apa (50μm) vita vm13 g6(n = 10) co-cr tig / apa (50μm) vita vm13 * apa: airborne-particle abrasion table 1. mechanical, physical and thermal properties of the co-cr alloy (fit cast talmax) available from the manufacturer. properties values expansion coefficient 14.0 (25 ºc 600 ºc) specific weight 8.2 (g/cc) stretching 2% elasticity 663mpa resistance 549 mpa fusion interval 1360 ºc 1410 ºc casting temperature 1460 ºc 5 monteiro et al. in which p is the maximum force (n), i is the distance between the supports (mm), b was the sample width (mm), d is the sample thickness (mm), and σ is the flexural strength (mpa)17. the specimens were analyzed using a stereomicroscope (leica m205a, meyer instruments, houston, tx, usa; collection of paleontology, the emilio goeldi museum, belém, pará, brazil), at 30× magnification. the fractured areas of porcelain were classified as follows4: adhesive failure, when most of the ceramic had been released from the metal; cohesive failure, when the ceramic detached from the structure without exposing the metal; and mixed failure, when part of the ceramic was released from the metal and a part remained attached to it. flexural strength data (in mpa) were analyzed using two-way analysis of variance (factor 1: use of welding; factor 2: size of al2o3 particle). the tukey test was performed to compare mean values among the groups (α = 0.05). statistical analysis was performed using spss version 20.0 (ibm corporation, armonk, ny, usa). the level of statistical significance level was set at 5%. results mean values (mpa) and standard deviation of flexural strength are summarized in table 3. the analysis revealed a significant difference in the bond strength of ceramic between the groups that used tig welding and those that did not, for all sizes of al2o3 (p < 0.01). the best flexural strength results were observed in all groups without welding. there was no statistical difference between the sizes of oxide particles among the groups without welding. however, there were significant differences between the tig 250 μm and tig 50 μm groups (p < 0.05), and the control group demonstrated better results. there were only mixed faults in all groups. the predominance of irregularities in the metal surface in the groups that underwent tig welding (g2, g4 and g6), such as internal defects and depressions, were clearly evident. discussion the primary requirement for success in metal-ceramic restorations is the effective attachment of the ceramic to the substructure of the metal alloy22. this bond depends table 3. mean (mpa) and standard deviation of the flexural strength. without tig tig welding apa 250μm 54.4 ± 4.07 aa 41.7 ± 5.09 bb apa 100μm 49.5 ± 2.32 aa 34.6 ± 2.38 b* apa 50μm 44.7 ± 3.01 aa 31.5 ± 2.44 ba * different lowercase letters in columns and different uppercase letters in rows represent significant statistical differences. the presence of * indicates no difference compared to the other groups in the same column. 6 monteiro et al. directly on the formation of an oxide layer on the surface of the metal before firing the ceramic4-6. the control of this layer can be facilitated by apa using al2o3, which also increases the area and the degree of wettability of the metal framework for the application of the ceramic8,9. several sizes of al2o3 particles have been used in the apa process in non-noble alloys. reports have claimed, however, that this process may compromise the marginal integrity of the metal, and that damage is directly proportional to the size of the al2o3 particle 23-25. in a study comparing metal-ceramic adhesion in co-cr and ni-cr alloys, there was a better bond strength in specimens that used al2o3 particles 110 μm in size8. in the present study, there were no significant statistical differences between the nonwelded groups. a possible explanation could be that the time used (10-15 s) was not sufficient, given that in a recent study, 30 s increased surface roughness, which increased mechanical retention, which in turn is a significant factor for achieving better metal-ceramic bond strength26. apa with variation in time and distance could be a useful analysis in a future study. welding is used to solve problems of adaptation. this technique reduces faults during the fabrication of the metallic structure and provides uniform distribution of forces27. tig welding has advantages over the conventional technique, such as lower heat production and the possibility of welding on the working model itself, thus reducing process time and failure rate28. tig welding resulted in a significant difference between the groups blasted with 250μmand 50-μm-al2o3 particles, with larger particles demonstrating greater resistance. these results are in agreement with those described by galo et al.29, who reported better results using 110-μm-al2o3 particles without welding, while the lowest value was found in the laser group and blasted with 50-μm-particles. it is possible that blasting with smaller particles influences the thickness of the oxide layer of metal surface required for the bond strength between metal and ceramic with tig welding, thus reducing flexural strength. another possibility is that blasting with 50-μm-particles does not result in an optimum variation in surface irregularities. thus, particles this size are too small to promote complete filling with liquid ceramics in a metal framework and supplying effective mechanical joints7. the present study demonstrated better performance in the groups without welding compared to those with tig welding. differences in chemical and physical composition between the metallic alloys, the weld area, and the weld contributed to normalize the structure and may have caused this decrease in flexural strength. previous studies have shown that this rapid solidification of the bound area could cause microstructural changes12. however, tig welding has the advantage of using a minimum amount of heat, reducing the amount of free oxygen and, when compared with brazing, yields greater flexural strength 27. the results of the present study are consistent with those reported by mehdi and ibrahim30, who found significantly lower resistance for laser-welded groups (11.7 mpa) 7 monteiro et al. and brazing groups (10.4 mpa) compared with the control group (25.4 mpa); thus, welding negatively affected the metal-ceramic bond strength. however, it is necessary to investigate differences in the coefficients of thermal expansion between the materials, because a minimum difference between these values is required30. the nature of the fractures was mixed in all the samples. this was considered an adequate bond strength because higher resistance is evident when the fracture occurs inside the ceramic and not at the interface (i.e., adhesive failures)31. although statistical results revealed the least resistance in the welded groups, no adhesive failures were observed, unlike a study using brazing and laser welding, in which adhesive failures predominated in the welded specimens12. nevertheless, further studies using scanning electron microscopy are necessary, and it is important to note that this was a limitation of the present study. within the limitations of the present study, we conclude that apa using 250-μm-al2o3 particles significantly increased the bond strength between metal and ceramic in tig-welded specimens. however, the size of the al2o3 particles exhibited no differences in the bond strength of ceramic in samples without the welding process. the welding process negatively interfered with the bond strength of ceramic to metal. acknowledgements fundação amazônia de amparo à estudos e pesquisas (fapespa) and emilio goeldi museum of the state of pará. declaration of conflicting interests the authors declares that there is no conflict of interest. references 1. patel ka, mathur s, upadhyay s. a comparative evaluation of bond strength of feldspathic porcelain to nickel-chromium alloy, when subjected to various surface treatments: an in vitro study. j indian prosthodont soc. 2015 jan-mar;15(1):53-7. doi: 10.4103/0972-4052.155036. 2. al bakkar h, spintzyk s, schille c, schweizer e, geis-gerstorfer j, rupp f. influence of a bonding agent on the bond strength between a dental co-cr alloy and nine different veneering porcelains. biomed tech. 2016 oct;61(5):509-17. doi: 10.1515/bmt-2015-0101. 3. joias rm, tango rn, de araujo jej, de araujo maj, de siqueira ferreira anzaloni saavedra g, de arruda paes-junior tj, et al. shear bond strength of a ceramic to co-cr alloys. j prosthet dent. 2008 jan;99(1):54-9. doi: 10.1016/s0022-3913(08)60009-8. 4. lombardo gh, nishioka rs, souza ro, michida sm, kojima an, mesquita am, buso l. influence of surface treatment on the shear bond strength of ceramics fused to cobalt-chromium. j prosthodont. 2010 feb;19(2):103-11. doi: 10.1111/j.1532-849x.2009.00546.x. 5. li j, ye x, li b, liao j, zhuang p, ye j. effect of oxidation heat treatment on the bond strength between a ceramic and cast and milled cobalt-chromium alloys. eur j oral 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of metal conditioner on bonding of porcelain to cobalt-chromium alloy. 2 j adv prosthodont. 2016 feb;8(1):1-8. doi: 10.4047/jap.2016.8.1.1. 11. martins je, bastos neto fvr, duarte da, araki ât. assessing different patterns of laser welding with nickel-chromium (ni-cr) alloy for structures applicable to fixed prosthesis. braz dent sci. 2017 apr-jun;20(2):62-9. 12. aladaǧ a, çömlekoǧlu me, dündar m, güngör ma, artunç c. effects of soldering and laser welding on bond strength of ceramic to metal. j prosthet dent. 2011 jan;105(1):28-34. doi: 10.1016/s0022-3913(10)60187-4. 13. ghadhanfari ha, khajah hm, monaco ea, kim h. effects of soldering methods on tensile strength of a gold-palladium metal ceramic alloy. j prosthet dent. 2014 oct;112(4):994-1000. doi: 10.1016/j.prosdent.2014.02.015. 14. karl m, winter w, taylor td, heckmann sm. in vitro study on passive fit in implant-supported 5-unit fixed partial dentures. int j oral maxillofac implants. 2004 jan-feb;19(1):30-7. 15. wang rr, welsch ge. joining titanium materials with tungsten inert gas welding, laser welding, and infrared brazing. j prosthet dent. 1995 nov;74(5):521-30. 16. bock jj, fraenzel w, bailly j, gernhardt cr, fuhrmann ra. influence of different brazing and welding methods on tensile strength and microhardness of orthodontic stainless steel wire. eur j orthod. 2008 aug;30(4):396-400. doi: 10.1093/ejo/cjn022. 17. matos ic, bastos in, diniz mg, de miranda ms. corrosion in artificial saliva of a ni-cr-based dental alloy joined by tig welding and conventional brazing. j prosthet dent. 2015 aug;114(2):278-85. doi: 10.1016/j.prosdent.2015.01.017. 18. graczyk h, lewinski n, zhao j, concha-lozano n, riediker m. characterization of tungsten inert gas (tig) welding fume generated by apprentice welders. ann occup hyg. 2016 mar;60(2):205-19. doi: 10.1093/annhyg/mev074. 19. atoui ja, felipucci dnb, pagnano vo, orsi ia, de arruda nóbilo ma, bezzon ol. tensile and flexural strength of commercially pure titanium submitted to laser and tungsten inert gas welds. braz dent j. 2013 nov-dec;24(6):630-4. doi: 10.1590/0103-6440201302241. 20. ting s, li kc, waddell jn, prior dj, van vuuren lj, swain m v. influence of a tungsten metal conditioner on the adhesion and residual stress of porcelain bonded to cobalt-chromium alloy. j prosthet dent. 2014 sep;112(3):584-90. doi: 10.1016/j.prosdent.2013.12.009. 21. pretti m, hilgert e, bottino ma, avelar rp. evaluation of the shear bond strength of the union between two cocr-alloys and a dental ceramic. j appl oral sci. 2004 dec;12(4):280-4. 22. simamoto júnior pc, resende novais v, rodrigues machado a, soares cj, araújo raposo lh. effect of joint design and welding type on the flexural strength and weld penetration of ti-6al-4v alloy bars. j prosthet dent. 2015 may;113(5):467-74. doi: 10.1016/j.prosdent.2014.10.010. 23. de melo rm, travassos ac, neisser mp. shear bond strengths of a ceramic system to alternative metal alloys. j prosthet dent. 2005 jan;93(1):64-9. 9 monteiro et al. 24. fischer j. ceramic bonding to a dental gold-titanium alloy. biomaterials. 2002 mar;23(5):1303-11. 25. anusavice kj, ringle rd, fairhurst cw. adherence controlling elements in ceramic-metal systems. ii. nonprecious alloys. j dent res. 1977 sep;56(9):1053-61. 26. coskun me, akar t, tugut f. airborne-particle abrasion; searching the right parameter. j dent sci. 2018 dec;13(4):293-300. doi: 10.1016/j.jds.2018.02.002. 27. matos, adaias oliveira et al. comparative analysis of ceramic flexural strength in co-cr and ni-cr alloys joined by tig welding and conventional brazing. braz j oral sci. 2018 mar;16:e17049. doi: 10.20396/bjos.v16i0.8650496. 28. rocha r, pinheiro alb, villaverde ab. flexural strength of pure ti, ni-cr and co-cr alloys submitted to nd:yag laser or tig welding. braz dent j. 2006 jan;17(1):20-3. 29. galo r, ribeiro rf, rodrigues rcs, pagnano vdeo, mattos mdagcde. effect of laser welding on the titanium composite tensile bond strength. braz dent j. 2009;20(5):403-9. 30. mehdi ag, ibrahim af. effect of soldering on shear bond strength of porcelain fused to metal (in vitro study). j baghdad coll dent. 2007;19(1):6-10. 31. miller ll. framework design in ceramo-metal restorations. dent clin north am. 1977 oct;21(4):699-716. 1http://dx.doi.org/10.20396/bjos.v18i0.8657393 volume 18 2019 e191600 original article 1 department of endodontics, faculty of dentistry, university of bolu abant izzet baysal, bolu, turkey 2 department of biostatistics, faculty of medicine, tokat gaziosmanpasa university, tokat, turkey corresponding author: hakan gokturk, department of endodontics, faculty of dentistry, abant izzet baysal university, bolu, turkey, 14100 phone: +90-374-2705353 ext-8453 fax: +90-374 2700066 e-mail: gokturk82@hotmail.com received: march 20, 2019 accepted: september 19, 2019 evaluation of the dislodgement resistance of bioceramic reparative cements placed in a retrograde cavity using a different technique hakan gokturk1,*, ismail ozkocak1, seda tan-ipek1, osman demir2 aim: calcium silicate-based fillings have been widely used in surgical endodontic treatment because of hard-tissue conductive and inductive properties. the aim of present study is to investigate the bond strength of different calcium silicate-based fillings in retrograde cavities. methods: forty-four maxillary single rooted teeth were endodontically treated. the apical portions of the teeth were removed and root-end cavities were prepared using an ultrasonic tip. the roots were randomly divided into four experimental groups (n = 11) according to the material used; (1) mta-fillapex, (2) mta repair hp, (3) mta-fillapex+ mta repair hp, and (4) mta plus. two horizontal cross sections (1±0.1 mm thick) from each specimen were resected from the apices. these sections were placed in a universal testing machine to evaluate the push-out bond strength force required for dislodgement of the root end filling was recorded. the failure type was also evaluated by using a stereomicroscope. the differences in bond strength were analyzed using the two-way analysis of variance (anova). results: mta-fillapex and mta plus displayed the lowest and highest dislocation resistance, respectively (p < 0.05). in the apical level, bond strength was significantly higher than the coronal level in all groups except for mta-fillapex. mixed failure was prevalent in all groups, except for mta-fillapex, which showed purely cohesive failures. conclusions: investigated calcium silicate-based filling materials showed different bond strength to the root-end cavity. the bond strength was significantly decreased when the prior application of mta-fillapex before delivery of mta repair hp. keywords: calcium compounds. silicates. root canal filling materials. retrograde obturation. endodontics. mailto:gokturk82@hotmail.com 2 gokturk et al. introduction in cases of orthograde root canal treatment failures or presence of persistent periradicular lesion, endodontic surgery is recommended. the aim of endodontic surgery is to remove periradicular lesion and irritants, 3-dimensional sealing of the retrograde cavity. hermetic sealing of retrograde cavities in 3-dimensions with biocompatible materials is crucial for the success of endodontic surgery1,2. mineral trioxide aggregate (mta) is one of the most used root-end filling material because of its superior sealing ability, antibacterial properties, biocompatibility, hard-tissue conductive and inductive properties and set in the presence of blood and moisture3,4. also, mta promotes the accumulation of calcium and phosphate crystals into the gaps between the filling material and dentin5. however, the slow setting time of mta is problematic especially when it used in the endodontic surgery because of the possibility of washout from the root-end cavity during irrigation2,6,7. another common drawbacks of mta are difficulties in handling and manipulation7. mta plus (prevest denpro limited, jammu, india) is another calcium silicate-based material which is composed of tricalcium and dicalcium silicate, calcium sulfate, silica, and bismuth oxide. bismuth oxide is the most used radiopacifying agent in traditional mta. it plays a crucial role in the hydration processes of calcium silicate8. however, it responsible for tooth discoloration especially when it contacts with sodium hypochlorite9. according to manufacture the particle size of mta plus is smaller than mta that improves its manipulation and handling characteristics10. mta repair hp (angelus, londrina, pr, brazil) is another newly formulated mta based material which is composed of calcium oxide, dicalcium silicate, tricalcium aluminate, tricalcium silicate, and calcium tungstate as radiopacifier. the plasticizer in the liquid facilitates manipulation and handling of material. also, this material has the same biological effects as the traditional mta11. mta-fillapex (angelus, brazil) is a bioceramic root canal sealer based on mta, composed of salicylate resin, natural resin, diluting resin, mta, nanoparticulated silica, and calcium tungstate as radiopacifier. when the set mta comes into contact with simulated body fluids, hydroxyl and calcium ions released from the material encourage the formation of calcium phosphates. in previous studies, mtafillapex displayed lower push-out bond strength to root dentin than ah plus and iroots sp12,13. the reason for this situation was attributed to the low adhesion capacity of tag-like structures which is produced within collagen fibrils13. adequate adhesion of the root-end filling material to the dentin is important to prevent the leakage which is critical for the long-term success of the endodontic surgery14. push-out testing is the most suitable test for evaluating the bond strength and the dislocation resistance of dental materials to root dentin. maximum bond strength of retrograde filling materials minimizes the consist of displacement that may cause voids and cracks, resulting in failure of endodontic surgery15. the aim of this study is to investigate the bond strength of various calcium silicate-based filling materials in retrograde cavities. mta plus was used as the reference material for comparison. the null hypothesis is that there will be no difference in the dislocation resistance between the investigated filling materials. 3 gokturk et al. material and methods sample size calculation a priori power analysis was performed to determine adequate number of samples to be included in the study. an effect size of 0.40 was added to a power b = 87% and a = 5% input into an f test family for analysis of variance, we needed 88 samples for four groups. sample selection and preparation after ethics committee approval (2018/36), 44 human, single-rooted teeth with single root canal were immersed in 1% thymol solution until they were used. a periodontal scaler was used to remove soft tissue and calculus from the root surfaces. then all teeth were examined under stereoscopic at ×25 magnification and teeth with free of root caries, fractures, resorption, calcifications and previous endodontic treatment were included. the included teeth were radiographed mesiodistally and buccolingually to confirm a single canal and other inclusion criteria. the coronal portion was removed under copious water irrigation with a diamond disc to obtain roots of 15 mm length. the working length was determined by using a k-file (vdw, munich, germany) which inserted into the root canal until it was visible at the apex. the working length was determined to be 1 mm short of this point. the root canals were shaped using reciproc rotary files (vdw) till size r40 according to the manufacturer’s instructions. after three pecking motions, 2 ml of 5.25% naocl solution was used for irrigation. five ml of 17% edta (imicryl ltd., konya, turkey) was used to remove the smear layer for 1 min. the prepared canals were dried with paper points and filled with cold lateral compaction of gutta-percha and an epoxy-resin based sealer (2seal, vdw, munchen, germany). the access of canals was filled with cavit (espe, seefeld, germany) and the roots were kept at 37°c in 100% humidity for 14 days. a 3 mm section of the apical part of the root was removed at 90 to the longitudinal axis of the teeth with a diamond bur using a high-speed handpiece under copious irrigation with saline (figure 1a). a retrograde cavity of 3 mm depth was prepared with ultrasonic retro tips (as3d, satelec acteon group, merignac cedex, france) coupled to the file holding adapter of a satelec p5 newtron xs ultrasonic system handpiece (satelec acteon group, france) at power setting 5 under copious irrigation with saline. the handpiece and sample were fixed on a device used to favor parallelism of the dentin walls. to obtain standard root-end cavity, residual gutta-percha was removed with a probe and the dimensions of the cavity were checked with a periodontal probe. figure 1. (a) resected root sample. (b) root-end cavity preparation and control of dimensions. (c) root-end cavity filled sample. (d) push-out bond strength test of 1 mm cross sections sample. a b c d 4 gokturk et al. the dimensions of the retrograde cavity were approximately 1.5 mm diameter and 3 mm deep (figure 1b). the preparation time was approximately 35 s. the cavities were dried with paper points. the samples were randomly divided into 4 groups based on root-end filling material (n = 11 per group) as follows; mta-fillapex, mta repair hp, mta-fillapex+mta repair hp, and mta plus. the materials were prepared according to the manufacturers. group 1 (mta-fillapex); an equal portion of base and catalyst paste (1:1 ratio) was mixed for 30 s to obtain homogeneous consistency. the mixed sealer was transported into a cleaned acid etching syringe with a luer lock-type delivery tip. the first 3 mm of the delivery tip was bent 90° and the suitability to retro-cavity was checked without excessive binding before use. the sealer was injected from the deep of the retro-cavity to the root end to avoid any voids. group 2 (mta repair hp); one package of mta repair hp powder (85 mg) and 2 drops of the liquid was mixed on the glass slap for 40 s to obtain a homogeneous mixture. a microcarrier was used to place the cement into the retro-cavity and a matching hand plugger was used to compact it. group 3 (mta-fillapex +mta repair hp); retro-cavities filled with mta-fillapex as in group 1 were then filled with mta repair hp as in group 2. group 4 (mta plus); according to the manufacturer; a 1:3 liquid: powder ratio by weight was mixed on a glass for 30 s to obtain a homogeneous consistency. a microcarrier was used to place the cement into the retro-cavity and a fitted hand plugger was used to compact it (figure 1c). excess materials on the resected root surface were removed with a microbrush and a plastic instrument. a radiograph was taken to confirm the quality of filling from all samples. all procedures were carried out by one endodontist who has seven years of clinical experience. after root-end filling procedures, the specimens were immersed in 8 ml of mgand cafree phosphate-buffered saline solution (pbs, ph = 7.2) at 37 °c for 14 days. pbs was renewed at 3 days intervals. push-out bond strength test following the restoration of root-end cavities two horizontal cross sections (1±0.1 mm thick) from each specimen (n = 22 slices/group) was created, from the apico-coronal direction, using a low-speed saw (isomet; buehler ltd, lake bluff, usa) with a water cooled diamond disc. the thickness of each section was controlled using a digital caliper (macrona, simetri teknik, i̇zmir, turkey) at an accuracy of 0.001 mm. the coronal and apical diameters of each root-end filling material were measured under a stereomicroscope (×20-30 magnification). the push-out test was performed using a mechanical testing machine (ags-x, shimadzu, kyoto, japan) with a load of 5 kn operating at 1 mm/min crosshead speed until dislodgement of the filling material occurred. the push-out force was applied from the coronal to apical the direction with a stainless steel plugger which its diameter covered the 90% of the diameter of the root-end filling material on the coronal 5 gokturk et al. surface without coming into contact with the surrounding dentin (figure 1d). the dislocation resistance value at failure in megapascals (mpa) was calculated from the maximum push-out force (n) divided by bonding surface area (mm). the bonding surface area (sa) for each slice was calculated according to the following formula: sa=, = 3.14; means the thickness of each root disc (1mm), is coronal radius or the smaller radius of the canal width, and is apical radius or the larger radius of the canal width. the failure modes of each slide were assessed under a stereomicroscope at ×40 magnification and classified into one of the three following categories: (1) cohesive failure inside the root-end filling, (2) adhesive failure between root-end filling and dentin, (3) mixed failure which is a combination of both. all statistical analyses were performed by using spss software (ibm spss statistics 19, spss inc., ibm co., somers, new york, usa). two-way analysis of variance (anova) and the post hoc bonferroni test was used to analyze the differences of the data with a 0.05 level of significance. results table 1 shows the mean and standard deviations of the bond strength for each group. no premature failure was observed (occurred) and all the specimens showed measurable debonding values. statistically significant differences were observed in the dislocation resistance of the retrograde filling materials regardless of the root regions (p < 0.05). the highest and lowest dislocation resistance was obtained for the mta plus (14.97±2.13) and the mta-fillapex (0.41±0.09), respectively (p < 0.05). a statistically significant ranking for dislocation resistance values was obtained as follows: mta plus >mta repair hp >mta-fillapex +mta repair hp >mta-fillapex (p < 0.05). a significant difference between each group in both regions were identified (p < 0.05), except for group 1 and 3 at the coronal region (p > 0.05). a statistically significant ranking for dislocation resistance values at apical and coronal level was obtained as follows: mta plus >mta repair hp > mta-fillapex + mta repair hp > mta-fillapex and mta plus >mta repair hp > mta-fillapex + mta repair hp ≥ mta-fillapex, respectively (p < 0.05). comparisons in terms of root regions revealed that the highest values were observed in the apical level of the all groups, but this difference was statistically significant for all investigated materials except for mta-fillapex (p < 0.05). table 1. the push out bond strength (mpa, mean ± standard deviation) of root filling materials in different root regions. material region p value total apical coronal mta-fillapex 0.55±0.10 a, a 0.41±0.09 a, a 0.746 0.48±0.12 a mta repair hp 7.21±0.93 b, a 6.35±0.98 b, b 0.048 6.78±1.03 b mta-fillapex +mta repair hp 2.38±0.29 c, a 1.06±0.14 a, b 0.003 1.72±0.72 c mta plus 14.97±2.13 d, a 13.28±1.22 c, b <0.001 14.12±1.90 d p value <0.001 <0.001 <0.001 two-way anova was used. lowercase letters indicate comparison among materials in column; uppercase letters indicate comparison among region in row. different letters indicate significance of the difference at a p < 0.05. 6 gokturk et al. failure modes are shown in figure 2. in all investigated materials expect for mta-fillapex, most common mode of failure was mixed failure, which means the presence of adhesive and cohesive failures at the same slice. mta-fillapex displayed purely cohesive failure in all samples. adhesive failure was seen only in the mta plus among all investigated materials. discussion in previous studies, it was reported that if a root canal filling material chemically bonds to root dentin, it resists the dislocation forces and improves the push-out bond strength of materials and contributes to the longevity and prognosis of endodontically treated teeth15-17. this study investigated the dislodgement resistance of different root-end filling materials to root canal dentin. in the light of the results of the present study, all investigated retrograde filling materials showed different dislocation resistance. therefore, the null hypothesis which there is no difference in the dislocation resistance between the investigated filling materials was rejected. also, the use of mta-fillapex to coat the root-end cavity walls before obturation with mta repair hp displayed a decrease in the push-out bond strength of mta repair hp. the push-out test is a reliable, easy, and widely used technique to investigate the bond strength of root canal filling materials and posts to the root dentin. the push-out strength also called dislodgement resistance defines the resistance to dislocation of materials applied to dental hard tissues. improved dislodgement resistance indicates that the material has good adhesion ability and represents the longevity of the restoration. the push-out test allows many sub-samples (or sections) to be taken from a sample12,13,18-20. soares et al.18 reported that the push-out test produces less variability and a more homogenous stress distribution than the microtensile bond test during dislocation resistance testing applied to intraradicular dentin. however, a recently published systematic review with meta-analysis demonstrated that some methodfigure 2. number and percentage of failure modes in each investigated groups. 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% mta-fillapex mta repair hp mta-fillapex+mta repair hp mta plus mixed cohesive adhesive 000 4 9 8 22 17 1314 1 7 gokturk et al. ological variables such as tooth portion, storage time, slice thickness, obturation technique, punch diameter, and load velocity influence the resistance to dislodgment of material21. similarly, storage environment (distilled water or pbs) influenced the dislocation resistance of the materials from retrograde cavities22. these variables may cause restrictions on the comparison of different study results. according to previous studies’ reports, in order to standardize all variables, 1±0.1 mm thickness slices21, and a plugger which cover at least 90% of the canal width19 should be used. all samples were stored in pbs to simulate clinical conditions23 in this study. ultrasonic tips, drills, and lasers have been used to prepare root-end cavities20,22,24,25. the use of ultrasonic tips allows obtaining parallel walls with improving retention for filling material and provides obtain deeper, cleaner, and more centered cavities with less opening of dentin tubules24,25. ultrasonic tips with different designs and angles facilitate the preparation of the root-end cavity at the posterior teeth. all of them have contributed to the increase in the success rate of periapical surgery1. thus, in this in vitro study, diamond ultrasonic tips (as3d, satelec acteon group, france) were chosen to prepare the root-end cavities. mta-fillapex composition is based on bioceramic materials which containing mta and resins. in a study, it was observed that when the set sealer contacts with phosphate-containing fluids it release calcium and hydroxyl ions and it creates apatite crystals. it was showed that created apatite produced by pbs and mta precipitate within collagen fibrils, promoting controlled mineral nucleation on dentin and this situation results in the formation of an interfacial layer with tag-like structures26. the results of the present study showed it has significantly lower bond strength values (0.48±0.12 mpa) than the other investigated materials. the obtained results may be explained by the low adhesion capacity of mta-fillapex due to the aforementioned interfacial layer. these results are in agreement with the results reported by sagsen et al.13 who reported that the bond strength of mta-fillapex to root dentine was lower than i root sp and ah plus. another explanation of low adhesion of mta-fillapex is related with the existing of smear layer on the dentin surface27. mta repair hp is a recently introduced mta-based material to improve physical properties of traditional mta. according to the manufacturer it is recommended for use as pulpotomy, pulp capping, apexification, apexogenesis, root-end filling, and to repair resorption and perforations. it contains calcium tungstate as a radiopacifier. calcium tungstate also prevents dental discoloration caused by bismuth oxide. silva et al.23 report that calcium tungstate contributes to higher calcium release, promoting higher biomineralization, and result in mta repair hp displayed higher dislocation resistance compared to white mta. however, in the present study mta repair hp, which containing calcium tungstate, showed significantly lower bond strength than mta plus, which containing bismuth oxide. this difference may be related to the small particle size of the mta plus. mta plus has a finer particle size (50 % of the particles/power thinner than 1 μm.) compared to traditional mta, which improves its handling characteristics and may increase the speed of hydration process. mta plus powder is mixed with water or gel. in a previous study, mta plus mixed with water, chemically curing resin, antiwashout gel, and light‐curing resin. investigators have reported that mta plus mixed with water had higher bond 8 gokturk et al. strength than the one mixed with gel20. in another study, mta plus mixed with polymer gel showed significantly lower bond strength than mta and biodentine in furcal perforation areas after a setting time of seven days28. consistent with the aforementioned study results, the present study showed that the mta plus represented a higher dislocation resistance than other investigated retrograde filling materials when mixed with water. following endodontic surgery, root-end filling materials contact directly with blood. however, the use of blood in in vitro studies during the setting time of cement is not possible. usually, the samples were stored at 100% humidity environment, in a pbs22,23 deionized water22 or hanks’ balanced salt solution5,20. results of the previous studies, demonstrated that the contact of calcium silicate containing materials with phosphate-based solutions results in the formation of apatite-like structures at the dentin-material interface and promote the formation of tag-like structures26, which subsequently result in improved dislocation resistance22 sealing ability by decreasing the interface voids5. in the present study, the resected root samples were stored in pbs to promote a condition closer to a clinical condition. regarding the type of failure, mixed failure was predominated, in accordance with the results of the previous studies20,29 which means failure is related with not only in the bond with the radicular dentin (adhesive failures) but also the material itself (cohesive failures). in line with the presented study results, formosa et al.20 comparing the bond strength of mta plus which was mixed with different liquids (light-curing resin, chemical curing resin, antiwashout gel, distilled water) have found failure was significantly higher in the distilled water mixing group. mta-fillapex which was investigated in the present study showed cohesive failure in all samples. because of mta-fillapex is a sealer, its consistency is more fluid than the other investigated filling materials. the lack of consistency may have caused more ion release during the setting period. this situation as stated in previous studies29,30 results in an uneven and porous microstructure, which disrupts the material structure and prevents material cohesion, and may be the cause of excess cohesive failure observed in the present study. to the best of our knowledge, studies evaluating the effect of the prior application of endodontic sealer on the bond strength of retrograde material to root-end cavity wall are limited. in a recent study, the marginal adaptation of bc rrm-fast set putty, and bc sealer + bc rrm-fast set putty was investigated after orthograde placement in teeth with open apices. the authors reported that the gap size in the root-end filling was significantly smaller when prior use of bc sealer before application of bc rrmfast set putty31. however, in the present study, the gap size in the root-end filling was not investigated, only bond strength was compared. so, these results could not be compared with those of the aforementioned study results. the investigated filling materials in this study showed different dislodgement resistance to the root-end cavity. prior application of mta-fillapex before delivery of mta repair hp, decreased the bond strength of mta repair hp. acknowledgements the authors declare that they have no conflict of interests. this study was not supported by any foundation. 9 gokturk et al. references 1. kim s, kratchman s. modern endodontic surgery concepts and practice: a review. j endod. 2006 jul;32(7):601-23. 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to root dentin. braz oral res. 2018 mar;32:e18. doi: 10.1590/1807-3107bor-2018.vol32.0018. 30. borges r, sousa‐neto m, versiani m, rached‐júnior f, de‐deus g, miranda c, et al. changes in the surface of four calcium silicate‐containing endodontic materials and an epoxy resin‐based sealer after a solubility test. int endod j. 2012 may;45(5):419-28. doi: 10.1111/j.1365-2591.2011.01992.x. 31. tran d, he j, glickman gn, woodmansey kf. comparative analysis of calcium silicate-based root filling materials using an open apex model. j endod. 2016 apr;42(4):654-8. doi: 10.1016/j.joen.2016.01.015. 1http://dx.doi.org/10.20396/bjos.v19i0.8658910 volume 19 2020 e208910 original article 1 department of prosthodontics and dental materials, school of dentistry, federal university of uberlandia, uberlandia, minas gerais, brazil. 2 school of dentistry, university of anápolis, anápolis, brazil. 3 department of prosthodontics and periodontology, piracicaba dental school, university of campinas (unicamp), piracicaba, são paulo, brazil. corresponding author: priscilla cardoso lazari school of dentistry, university of anápolis, anápolis, brazil department of prosthodontics centro universitário de anápolis, faculdade de odontologia. avenida universitária, km 3,5 cidade universitária 75083515 anápolis, go brasil telefone: (62) 999723755 e-mail: lazari.pcl@gmail.com received: march 27, 2020 accepted: july 24, 2020 3d finite element model based on ct images of tooth: a simplified method of modeling germana de villa camargos1, priscilla cardoso lazari-carvalho2,* , marco aurélio de carvalho2, mariane boaventura de castro2 , naysa wink neris2, altair antoninha del bel cury3 aim: this study aimed the description of a protocol to acquire a 3d finite element (fe) model of a human maxillary central incisor tooth restored with ceramic crowns with enhanced geometric detail through an easy-to-use and low-cost concept and validate it through finite element analysis (fea). methods: a human maxillary central incisor was digitalized using a cone beam computer tomography (cbct) scanner. the resulted tooth cbct dicom files were imported into a free medical imaging software (invesalius) for 3d surface/geometric reconstruction in stereolithographic file format (stl). the stl file was exported to a computer-aided-design (cad) software (solidworks), converted into a 3d solid model and edited to simulate different materials for full crown restorations. the obtained model was exported into a fea software to evaluate the influence of different core materials (zirconia zr, lithium disilicate ds or palladium/silver ps) on the mechanical behavior of the restorations under a 100 n applied to the palatal surface at 135 degrees to the long axis of the tooth, followed by a load of 25.5 n perpendicular to the incisal edge of the crown. the quantitative and qualitative analysis of maximum principal stress (ceramic veneer) and maximum principal strain (core) were obtained. results: the zr model presented lower stress and strain concentration in the ceramic veneer and core than ds and ps models. for all models, the stresses were concentrated in the external surface of the veneering ceramic and strains in the internal surface of core, both near to the loading area. conclusion: the described procedure is a quick, inexpensive and feasible protocol to obtain a highly detailed 3d fe model, and thus could be considered for future 3d fe analysis. the results of numerical simulation confirm that stiffer core materials result in a reduced stress concentration in ceramic veneer. keywords: ceramic. dental stress analysis. finite element analysis. three-dimensional imaging. https://orcid.org/0000-0002-5123-5358 https://orcid.org/0000-0003-0142-3980 https://orcid.org/0000-0002-4329-0437 2 camargos et al. introduction in dentistry, the mechanical behavior of restorative materials is a determinant factor for their clinical success. therefore, in vitro tests are not only important tools to determine materials properties and resistance but also to predict stresses that could lead to clinical failure 1,2. in these in vitro studies, when specimens that precisely simulate the restoration geometry are used, the mechanical behavior might be closer to the clinical situation3. however, the stress and strain distribution within complex geometries is difficult to be accessed using conventional in vitro approaches4. in this context, finite element analysis (fea) has been used successfully to investigate stress distribution in complex structures, such as restored human teeth. in the bioengineering field, the use of computer simulations is an important instrument to measure and test the best clinical option5. the use of fea in dental rehabilitation improves the understanding of biomechanical behavior of different dental restoration materials and designs, and therefore the optimal approaches that are expected to provide better clinical performance6,7. this experimental-numerical methodology was initially developed in the early 1960s to solve structural problems in the aerospace industry, but it has since been extended to solve problems in medical sciences, including dentistry8. currently, fea is a popular method and represents a comprehensive in silico method in dentistry. this method allows a better understanding of the mechanical behavior of dental restorations by testing them virtually under all conceivable loading conditions, designs and materials3. to conduct a fea, the start point is the construction of an accurate model, which is the key to the analysis outcome9,10. several methods have been used to generate fe models of teeth, such as the use of standard anatomical data in the literature11, manual tracing of tooth sections from histological12 or computer tomographic (ct) images of teeth13-16. these conventional methods of modeling are time-consuming and often demand labor-intensive efforts and highly skilled operators. also, they result in geometry over simplification that might induce false predictions in the fea3. to overcome this problem, previous studies have used sophisticated techniques with micro-ct images associated with costly specific medical imaging software (mimics, materialise, leuven, belgium)3,7,17,18. the generation of highly anatomically accurate 3d models of teeth was possible with this approach, minimizing errors in the following phases3,17,18. nevertheless, despite the optimal models obtained, the combination of micro-ct with costly imaging software reduce the accessibility of many researchers. in this context, public domain medical imaging software have emerged and are available for free download, allowing the free generation of 3d surface/geometric models from a sequence of 2d dicom files acquired through a cheaper exam, such as cone beam computer tomography (cbct). the software explored in this study is invesalius (renato archer information and technology center, campinas, brazil). the use of invesalius in combination with a computer aided-design (cad) software allows the generation of precise 3d solid models of dental geometry based on an unaltered tooth19-21. thus, this study aimed the description of a protocol to acquire a 3d fe model of a human maxillary central incisor tooth restored with ceramic crowns with enhanced geo3 camargos et al. metric detail through an easy to use and low-cost concept using invesalius and a cad software. to demonstrate the potential of its employability, the obtained 3d model was used for a fea of the influence of different core materials (zirconia, lithium disilicate or palladium/silver) on the mechanical behavior of veneering ceramic and core of crowns. materials and methods the 3d models of a crown, with different core material (zirconia zr, lithium disilicate – ds, or palladium/silver ps), were obtained through the 3d reconstruction from the cbct of a sound extracted human maxillary central incisor. the process required to obtain the 3d models and fea consists on the following stages: cbct acquisition, 3d surface/geometric reconstruction, 3d solid modeling and finally the fea. tooth fe modeling from cbct data this study was approved by the ethics committee in research of the piracicaba dental school – university of campinas (register number 106/2014). first, the cbct acquisition of an extracted central incisor in digital imaging communications in medicine (dicom format) was performed using a kodak 9000 3d extraoral imaging system (carestream dental llc, atlanta, ga, usa). the scanning parameters used were: tube voltage of 60 kv, tube current of 2 ma, and a slice thickness of 75 μm. a total of 180 slices were provided and used for the modeling. subsequently, the resulted tooth images in dicom format were imported into invesalius, for 3d surface/geometric reconstruction in solid-display stereolithographic file format (stl). in addition, this software presents segmentation functions based on image density thresholding that allows the creation of segmented models of mineralized (enamel and dentin) or non-mineralized tissues (pulp) in stl format (figure 1). figure 1. (a, b) cbct data as seen in the public domain medical imaging software invesalius. tooth is presented in different cross-sectional views. masks have been applied to mineralized tissues (enamel and dentin) according to the voxel density thresholding. (c, d) 3d surface model of incisor tooth in invesalius. a b c d 4 camargos et al. nevertheless, stl models are improper for use in fea because of the density and quality (aspect ratio and connectivity) of the mesh3 (figure 2a). moreover, using stl file prevent changes in the design or dimensions of the models, jeopardizing their use for different designs evaluation. for this reason, the 3d reconstructed surface of the tooth in stl format were exported to a cad software (solidworks 2011, concord, ma, usa) through its scanto-cad plugin function (scanto3d). in solidworks software, the file was opened as mesh files (stl) and converted into a 3d solid model of a maxillary central incisor with the aid of mesh prep wizard and surface wizard built-in tools. these tools allowed the simplification of the mesh (reduction of the number of triangles) and surface smoothing, improving the final mesh quality while the original model geometry is maintained (figure 2b). once the solid model is obtained and its surface smoothened, the tooth was edited by conventional cad modeling in solidworks, in order to simulate complete crowns supported by tooth. for this, the tooth crown was circumferentially reduced by 2.0 mm mesiodistally and buccolingually and 3.0 mm incisally. the veneering ceramic and core (2.0 mm and 0.4 mm of thickness, respectively) were created using boolean operations (addition, intersection or subtraction of volumes). the cementation of the crown was simulated using a resin cement (panavia f2.0, kuraray, tokyo, japan) with a 0.09 mm thick layer (figure 3). additionally, the root with its periodontal ligament (0.25 mm thick) was embedded in an acrylic resin cylinder, simulating the conditions of in vitro studies22. figure 2. (a) stereolithography triangulated (stl) file of incisor tooth obtained in invesalius. (b) incisor tooth stl file optimized for fea using mesh prep wizard within the cad software solidworks. a b 5 camargos et al. finally, the optimized 3d solid models of the segmented tooth (dentin and pulp), restorative crown (veneering ceramic and core), resin cement layer, periodontal ligament, and cylinder support were assembled and imported into a fea software (ansys workbench 13.0, swanson analysis inc., houston, usa) for the generation of a volumetric mesh, attribution of material properties, and mathematical solution (fea) (figure 4a). all structures were considered linearly elastic, isotropic and homogeneous. the mechanical properties of enamel23, dentin24, pulp12, periodontal ligament25, ceramic veneer12, lithium disilicate26, zirconia27, palladium-silver28, resin cement29 and polystyrene resin22 were taken from literature and are listed in table 1. the mesh was generated with tetrahedral elements of 0.8 mm after a convergence analysis (5%). as a result, the models presented a number of elements and nodes of 15,378 and 29,303, respectively. figure 3. tooth model with restorative crown (veneering ceramic and core), cement layer, dentin, dental pulp and periodontal ligament. figure 4. (a) model with tetrahedral element of 0.8 mm. (b) loading was performed in 2 steps: oblique load (135 degrees) of 100 n was applied to incisal third of lingual crown, and 25.5 n was applied perpendicularly to incisor crown. a b 6 camargos et al. boundary and loading conditions fixed zero-displacement in the three spatial dimensions was assigned to the nodes at the bottom surface of the cylinder support. the tooth and restorative materials were considered perfectly bonded. the crowns were loaded with 100 n applied to the palatal surface at 135 degrees to the long axis of the tooth22, followed by a load of 25.5 n perpendicular to the incisal edge of the crown19 (figure 4b). the maximum principal stress for the veneering ceramic and the maximum principal strain for the core were obtained as dependent variables for both quantitative and qualitative (color-coded) comparisons. results the zr model presented the lowest stress concentration in the ceramic veneer. the maximum principal stress (σmax) peak was higher in ds and ps models (24.728 mpa and 24.711 mpa, respectively), than in zr (23.395 mpa) even though the differences between the lowest and the highest was an increase of 5.69%. for all models, the stresses were predominantly concentrated on the external surface of the veneering ceramic, surrounding the loading area. the qualitative analysis suggests that the compression load, generated by the force application, resulted in tensile stresses also on the buccal surface and cervical area (interface with the core) (figure 5a). when comparing different core materials, those with lower elastic modulus presented higher values of maximum principal strain, which concentrated in the area directly below the loading site (figure 5b). these values were 0.4 μm for zr and 0.7 μm for ds and ps models. there was an increase of 75% in strain between the lowest to the highest models. table 1. mechanical properties of the materials included in finite element analysis. material yield strength (gpa) poisson’s ratio enamel 80 0.33 dentin 20 0.31 pulp 0.002 0.45 periodontal ligament 0.0689 0.45 ceramic veneer 70 0.30 litium-disilicate 95 0.30 zirconia 205 0.22 palladium-silver 95 0.33 resin cement 18.3 0.33 polystyrene resin 13.5 0.31 7 camargos et al. discussion fea represents a powerful tool to understand the mechanical behavior of all-ceramic crowns17. however, the analysis might be limited by difficulties related to model generation. teeth and dental restorations are difficult to model because of their complex anatomical shape and layered structure30. therefore, many studies used simplified 2d models for specific problems in dentistry, underestimating the influence of the complex geometry in the stress and strain distribution. although 2d models are simpler and easier to be constructed compared to 3d models, the biaxial state of 2d models may compromise the results reliability as it does not take into consideration some important biomechanical aspects clinically observed31. therefore, a 3d simulation should net be simplified when investigating the biomechanics of dental restorations32. to overcome this problem, the present study described an easy to use and lowcost modeling technique for the generation of an accurate 3d model of a maxillary central incisor using cbct images associated with a public domain medical imaging software, invesalius. this method is simpler when compared to other methods used in previous studies11-13, as it consists of going through few semi-automatic steps. furthermore, the use of public and free medical imaging software to generate 3d models from ct/cbct images might extend access to more researches in the biomechanical field, contributing to a better understanding of restorations’ mechanical behavior and consequently improving their clinical performance. previous studfigure 5. (a) maximum principal stress in ceramic veneer and (b) maximum principal strain in core of zr, ds and ps groups, respectively. zr ds ps maximum principal stress (mpa) 24,728 max 8,4225 -7,8827 -24,188 -40,493 -56,798 -73,103 -89,408 -105,71 -122,71 min zr ds ps maximum principal stress (mpa) 0,00077424 max 0,00068751 0,00060077 0,00051404 0,00042731 0,00034058 0,00025385 0,00016711 8,0381e-5 -6,3517e-6 min 8 camargos et al. ies used similar approaches with micro-ct images and another interactive medical image control software (mimics 9.0, materialise, leuven, belgium), also obtaining highly detailed and accurate 3d models of dental structures or restorations3,17,18, with the disadvantage of higher costs. particularly in this study, cbct images were used instead of micro-ct images once the latter is not suitable for human teeth in live patients, which prevents the generation of instant patient-specific models for more realistic simulations. although cbct images of an extracted sound incisor tooth were used for generating the fe model, a similar approach could be also performed using cbct images from living individuals in other studies. additionally, the kodak 9000 3d ct scanner was chosen because it provides a sub-millimeter isotropic voxel resolution that is the closest to that given by some microscale ct scanners33. despite the advantages of cbct, when this technique is applied to small structures like teeth (with thin anatomical details such as the enamel shell) it does not allow the fine control of internal boundaries (e.g. dentin-enamel junction), raising difficulties in the generation of precise 3d segmented models of enamel and dentin. it is expected that the rapid development of more precise dental cbct scanners, computer processing power, and interface friendliness will make this approach even faster, more accurate and fully automated in the near future3. fea was performed to demonstrate the applicability of the 3d fe model generated in this study. in this analysis, it was evaluated the influence of different core materials (zirconia, lithium disilicate or palladium/silver) on the mechanical behavior of the weak link of modern esthetic indirect restorations, the veneering ceramic. the introduction of zirconia and lithium disilicate as core materials makes possible to produce long-lasting all-ceramic restorations with improved esthetics with less invasiveness when compared to conventional metal-ceramic restorations. however, the stresses in the veneering ceramic could jeopardize the longevity of these bilayer ceramic restorations34, once ceramic chipping has been reported as the most frequent failure35. according to the results of this in silico simulation, the zr model presented lower stress concentration in veneering ceramic than ds and ps models. this result can be attributed to lower deformation of zirconia core, once that hard and stiff ceramic cores effectively prevents the flexure of veneering ceramic36, decreasing the stress in this structure and consequently its risk of fracture37. despite the zr model presents the best biomechanical behavior, the other materials evaluated (ds and ps) also showed stress values in the veneering ceramic below the critical values described for its fracture(31-38 mpa)38. however, it is worthwhile to mention that clinically, zirconia restorations can fracture under low-stress values and more frequently than metal-ceramic restorations due to poor bonding on the veneer-zirconia interface39. hence, future studies considering also the influence of ceramic-ceramic bonding conditions are necessary. besides, despite the effort on improving of reality of the present simulation, it is important to emphasize that the fea models still present limitations when compared to the clinical conditions, as the materials were considered homogeneous, isotropic and with a linear elastic behavior. disadvantages of fea are known as incorrect information about mechanical properties, statistics applied to the results, 9 camargos et al. and interpretation will yield misguiding results. also, researches need to have minimum computer knowledge and information about the mechanical behavior of human models.(5) in conclusion, the described protocol consists on a low-cost, fast and efficient method to obtain a highly detailed 3d fe model of a maxillary central incisor restored with full crowns. the numerical simulation outcomes confirm that stiffer core materials result in a reduced stress concentration in the veneering ceramic. acknowledgements this study was supported by são paulo research foundation (grants no. 2011/14001-3 and no. 2011/22231-9). the authors would like to thank dr. carlos murgel (campinas, brazil) for the tomographic images acquisition. references 1. rosentritt m, behr m, van der zel jm, feilzer aj. approach for valuating the influence of laboratory simulation. dent mater. 2009 mar;25(3):348-52. doi: 10.1016/j.dental.2008.08.009. 2. berger g, pereira lf de o, souza em 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liu y, liu g, wang y. failure modes and fracture origins of porcelain veneers on bilayer dental crowns. int j prosthodont. 2014 mar-apr;27(2):147-50. doi: 10.11607/ijp.3608. 38. taskonak b, yan j, mecholsky jj, sertgöz a, koçak a. fractographic analyses of zirconia-based fixed partial dentures. dent mater. 2008 aug;24(8):1077-82. doi: 10.1016/j.dental.2007.12.006. 39. silva nrfa, bonfante e, rafferty bt, zavanelli ra, martins ll, rekow ed, et al. conventional and modified veneered zirconia vs. metalloceramic: fatigue and finite element analysis. j prosthodont. 2012 aug;21(6):433-9. doi: 10.1111/j.1532-849x.2012.00861.x. oral sciences n3 braz j oral sci. 14(4):306-310 original article braz j oral sci. october | december 2015 volume 14, number 4 evaluation of biomaterials with and without platelet-rich plasma: a histometric study using beagle dogs brunamelia de oliveira1, wirley gonçalves assunção1, jacqueline nelisis zanoni2, edevaldo tadeu camarini2, roberta okamoto3 1universidade estadual paulista – unesp, araçatuba dental school, department of dental materials and prosthodontics, araçatuba, sp, brazil 2universidade estadual de maringá – uem, center of biological sciences, department of dentistry, area of morphological sciences, maringá, pr, brazil. 3universidade estadual paulista – unesp, araçatuba dental school, department of basic sciences, araçatuba, sp, brazil correspondence to: brunamélia de oliveira rua taioaba 125, mooca, são paulo cep: 03170-070 são paulo, sp, brasil phone: +55 44 9944-4984 / +55 11 99941-0010 e-mail: brunamoliveira@hotmail.com abstract aim: to compare the alveolar bone repair process using biomaterial in dogs with and without the incorporation of platelet-rich plasma. methods: six beagles were used. bilateral extractions of the three mandibular premolars were performed. bio-gen® was applied in the first alveolus, the clot was maintained in the second alveolus and genox® was applied to the third alveolus. prp was added to all alveoli on the left side only. the dogs were submitted to euthanasia after 30, 60 and 90 days and submitted to histological analysis for the determination of mean area of new bone formation. tukey’s post test was used in the statistical analysis. results: significant increase in bone formation occurred in bio-gen® + prp when compared with the other groups at 30 and 90 days. in the evaluation at 60 days, no statistically significant differences among the groups were found. conclusions: the bio-gen® biomaterial led to the best bone repair and the combination of platelet-rich plasma accelerated the repair process. keywords: biocompatible materials; platelet-rich plasma; bone regeneration. introduction the reconstruction of bone defects in the jaws has been widely studied1-3. the main objective is to achieve future oral rehabilitation with the use of dental implants. the correction of bone defects can be achieved with biomaterials, which are substances with combinations of a natural or synthetic origin indicated for the replacement of tissue. biomaterials are currently used more often than homogenous, xenogenous and autogenous grafts4-5. a xenogenous graft is obtained from a donor of a different species. bio-gen® (bioteckarcugnano vicenza, italy) is a natural osteoconductive material of equine origin without collagen with a high osteogenic capacity due to the absence of calcination in the mixing phase, which preserves a large portion of the biological properties of this material. complete resorption time ranges from four to 12 months6. genox® (baumer, são paulo, sp, brazil) is a product of bovine origin with an inorganic, freeze-dried matrix sterilized with gamma radiation. this product acts as an osteoconductor, conferring strength to the bone bed as it is resorbed7. autogenous grafts are considered the gold standard due to the absence of http://dx.doi.org/10.1590/1677-3225v14n4a10 received for publication: november 09, 2015 accepted: december 13, 2015 307307307307307 immune reactions and disease transmission, as well as their innate osteogenic, osteoinductive and osteoconductive properties8. however, the disadvantages are the need for two simultaneous surgical sites and the risk of infection9. platelet-rich plasma (prp) is an autogenous substance used in the form of a platelet gel extracted through the centrifugation of a blend of bovine thrombin, 10% calcium chloride and venous blood 10-11. prp has a significant concentration of growth factors that enhance bone formation and mineralization, induce stem cells to differentiate into osteoblasts, diminish bone resorption, promote angiogenesis and produce collagen through the activation of fibroblasts1213. a number of studies have demonstrated that the combination of prp and biomaterials achieves the best results in the bone repair process in comparison to groups in which prp was not employed14-15. the purpose of the present study was to perform bilateral tooth extractions in dogs and compare the alveolar bone repair process using bio-gen® (bioteck) and genox® (baumer) with and without the incorporation of platelet-rich plasma. materials and methods six beagles with a mean age of two years and weighing approximately 10 kg were maintained in appropriate confinement for the species (two dogs per kennel) in a clean, aerated environment with free access to food and water. the procedures employed in this study were approved by the ethics committee on animal experimentation (ceua/ emu) state university of maringá, process number 017/ 2010. preparation of prp forty milliliters of blood were taken from the jugular vein of each animal. sodium citrate 10% was used as anticoagulant. the blood was homogenized and centrifuged at 1200 rpm for 10 min (sin centrifuge: sin implant system, são paulo, sp, brazil). the first centrifugation resulted in the complete separation of the blood into two layers: a lower layer of red blood cells and an upper layer of plasma. the plasma layer had two parts: the upper portion (approximately 75% of the total plasma) had a greater concentration of platelet-poor plasma (ppp) and the lower portion (25%) had a greater concentration of prp. for the second centrifugation, the total plasma (ppp and prp) was pipetted and transferred to a new recipient, which was centrifuged at 1200 rpm for 10 min for better separation of both parts. the lower portion (containing prp rich in growth factors) was then pipetted1. the gelification process involved the use of a 10-ml syringe for each mixture, with 6 ml of prp, thrombin, calcium gluconate and air to assist in the mixture. the syringe was manually shaken for 6 to 10 s until the gel was formed. surgical procedure the animals were pre-medicated with acepromazine 0.2% at a dose of 0.03 mg/kg and fentanyl citrate at a dose of 3 µg/kg intravenously with the administration of ringer’s lactate solution at a volume of 10 ml/kg/h to maintain the venous access. anesthesia was performed with propofol at a dose of 3 mg/kg and 2 mg/kg of ketamine intravenously and a local block with 0.5% bupivacaine. systolic blood pressure was monitored using vascular doppler. intubation was performed using a number 7.5 tube and oxygen flow was maintained at 1 ml/kg/h. bilateral extraction of the mandibular three premolars was performed on all dogs without compromising the alveolar crest. on the right side, bio-gen®(bioteck) was applied in the first alveolus, the clot was maintained in the second alveolus and genox®(baumer) was applied to the third alveolus. on the left side, bio-gen®(bioteck) + prp was applied in the first alveolus, the clot was maintained and prp was applied to the second alveolus and genox®(baumer) + prp was applied to the third alveolus (figure 1a). the alveoli were completely filled with the biomaterials. when prp was added to the grafts, each was mixed individually. the suture was performed with vicryl 4.0 (ethicon, johnson & johnson, somerville, nj, usa) (figure 1b). fig. 1. left side of beagle mandible: a) 1first alveolus – bio-gen® + prp; 2second alveolus – clot + prp; 3third alveolus – genox® + prp; b) suture with single stitch recovering alveolar ridge. post-extraction care after the surgical procedures, the animals received an intramuscular injection of 1 ml of enrofloxacin 5% (5 mg/ kg, baytril®) and an intravenous dose of 5 ml of dipyrone 50%. over the next three days, the animals received 1 ml/ 10 kg oral doses of dipyrone 50%. at 30, 60 and 90 days postoperatively, two animals were euthanized with an intravenous dose of thiopental (25 mg/kg), fentanyl (3 µg/kg and potassium chloride (20 ml). the mandibles were dissected and each alveolus studied was submitted to histological analysis with hematoxylin-eosin staining. evaluation of biomaterials with and without platelet-rich plasma: a histometric study using beagle dogs braz j oral sci. 14(4):306-310 histomorphometric study four cuts/alveolus/animal/time were selected for the analysis of bone formation. five images were obtained for each cut through the center of each alveolus following the long axis and using the adjacent alveolar bone as reference (figure 2). the images (total area: 5.56 mm2) were captured using a camera (qcolor3; olympus, tokyo, japan) coupled to an optical microscope (bx41, olympus) with a 4x objective. the histomorphometric measures were made with the image pro plus® software, version 4.5 (media cybernetics, rockville, md, usa). a polygonal measuring tool was used to delimit the area of mature bone on each image. the mean area of newly formed bone was measured on each cut. bio-gen ® clot genox® bio-gen® + clot + genox® + prp prp prp 30 days 0.9 b 0.58 b 1.24 1.51 a 0.91 b 1.22 60 days 3.17 2.62 2.91 2.92 2.08 2.92 90 days 3.64 2.16 b 3.17 b 4.59 a 1.82 b 2.36 b table 1.table 1.table 1.table 1.table 1. mean area (mm2) of newly formed bone in different groups at different evaluation times means with different letters denote statistically significant differences among groups in each period. tukey post hoc test at 5% is considered significant level (p<0.05). fig. 2. images of center of each alveolus following long axis with adjacent alveolar bone as reference. statistical analysis the data were analyzed using the graphpad prism r 3.1 (graphpad software, inc. la jolla, ca, usa). nonparametric one-way analysis of variance (anova) was employed. tukey’s post hoc test was used for the comparison of means, with the level of significance set to 5% (p<0.05). results table 1 displays the mean area of newly formed bone in the alveoli of the different groups [clot, bio-gen® (bioteck), genox®(baumer), clot + prp, bio-gen® (bioteck) + prp and genox® (baumer) + prp] at the different evaluation times (30, 60 and 90 days). at 30 days, greater bone formation was found in all groups in comparison to the control (clot). the best result was achieved with bio-gen®(bioteck) + prp, with statistically significant differences in comparison to the clot, clot + prp and bio-gen®(baumer) groups (p<0.05) at 60 days, an increase in bone formation was found for all groups compared with the 30-day evaluation. however, no significant differences were found among the different groups (table 1). at 90 days, the lowest rates of bone formation were found in the clot groups and the best result was achieved with bio-gen® (bioteck) + prp, which differed significantly from all other groups (p<0.05), except the bio-gen® (bioteck) group (table 1). figure 3 shows the bone formation rate in the groups with prp at the different evaluation times. better results were achieved with bio-gen® (bioteck) + prp in comparison to clot + prp and genox® (baumer) + prp. however, the difference was only statistically significant at the 90-day evaluation (p<0.05). figure 4 displays the results of the groups without prp at the different evaluation times. figure 5 shows the histological cuts (4× magnification). greater bone formation was evident in the bio-gen® (bioteck) + prp in comparison to all other groups at the 90-day evaluation. fig. 3. mean area of bone formation (mm2) at 30, 60 and 90 days in the alveoli of clot + prp, genox® + prp and bio-gen® + prp groups. fig. 4. mean area of bone formation (mm2) at 30, 60 and 90 days in the alveoli of clot, genox® and bio-gen® groups. 308308308308308evaluation of biomaterials with and without platelet-rich plasma: a histometric study using beagle dogs braz j oral sci. 14(4):306-310 discussion a number of studies in the literature have demonstrated the biocompatibility of deproteinized animal matrices used 309309309309309 fig. 5. histological cuts (4´ magnification) of alveoli of mandibular premolars in beagles 90 days after extraction (hematoxylin-eosin stain): a) bio-gen®; b) clot; c) genox®; e) bio-gen® + prp; f) clot + prp; g) genox® + prp. evaluation of biomaterials with and without platelet-rich plasma: a histometric study using beagle dogs braz j oral sci. 14(4):306-310 as bone graft material16-17. in the present nvestigation, genox® (baumer) exhibited a tendency toward greater bone formation in comparison to bio-gen® (bioteck) at the 30-day evaluation. this finding may be explained by the fact that the surface of this material exerts an influence on cell colonization due to its preserved micro-architecture, which offers spaces for cells to fill, thereby enabling angiogenesis, cell migration, adhesion and the formation of new bone tissue. martins et al.18 (2004) report similar results regarding a bone graft of bovine origin. in the same evaluation period, the addition of prp to genox ® (baumer) did not enhance the bone formation process. this finding agrees with data reported by rocha et al.19 (2010), who report that the combination of prp and genox® (baumer) did not lead to a significant increase in bone formation. a possible explanation for this would be the fact that prp has a longer action time (up to seven days after preparation) and biomaterial of bovine origin is made by deproteinization at high temperatures, causing permanent changes, which may decrease the action of the growth factors in prp20. the best results at the 30-day evaluation were achieved when prp was added to bio-gen® (bioteck), with a significant increase in bone formation in comparison to the use of biogen® (bioteck) alone. this likely occurred due to the mixing process, which is performed at 37 °c, with decontamination occurring through enzymatic means and sterilization by gamma-radiation21. this process is less aggressive to the structure of the proteins in the biomaterial. thus, the more active growth factors in prp probably led to this finding. the worst results were found in the clot groups with and without prp at all evaluation times. this may be related to the progressive atrophy of the alveolar process that occurs following a tooth extraction22-23. similar findings are described by nevins et al.24 (2006), who evaluated the width of the alveolus 30 and 90 days after extraction using computerized tomography (transverse cut) and demonstrated progressive atrophy of the bone. the authors concluded that filling the post-extraction alveolus with a biomaterial is a way to avoid this atrophy. no statistically significant differences among the different groups were found at the 60-day evaluation. this may be explained by the high degree of repair activity in the initial days following an extraction, with a subsequent reduction in the repair rate over time25-26. at the 90-day evaluation, the best results were achieved with bio-gen® (bioteck) + prp, with a significantly greater rate of new bone formation in comparison to all other groups, except bio-gen® (bioteck) without prp. this finding may be explained by the hydrophilic nature of bio-gen® (bioteck) and its structure, which allows cell migration and angiogenesis, thereby facilitating bone regeneration. moreover, the production of this material at low temperature allows eliminating organic matter without causing changes in the structure of trabecular bone27. in contrast, genox® (baumer) is derived from de-mineralized, hydrophobic bovine bone composed only by inorganic matrix28-29. smieszek-wilczewska et al.30 (2010) compared bio-gen® (bioteck), bio-oss® and a control group (clot alone). the authors found that bio-gen® (bioteck) led to a significantly greater increase in radiological density compared with the other groups, confirming the efficacy of biomaterial of equine origin. considering the importance of preserving the postextraction alveolus for future interventions such as implants, the present findings demonstrate that bio-gen® (bioteck) is an excellent biomaterial for the repair and conservation of alveolar bone structures. moreover, the results were even more satisfactory when this product was used together with prp, as demonstrated by the accelerated bone formation process. further studies should compare the structure of biomaterials of equine and bovine origin to confirm the advantage of one material over the other. 310310310310310evaluation of biomaterials with and without platelet-rich plasma: a histometric study using beagle dogs braz j oral sci. 14(4):306-310 acknowledgements the authors would like to thank capes (coordenação de aperfeiçoamento de pessoal de nível superior)/brazil and area of morphological sciences at state university of maringá for their contributions to this research. the authors state that they have no conflicts of interest. references 1. camarini et, janoni jn, leite pcc, boos fbd. use of biomaterial with or without platelet-rich plasma in postextraction sites: a microscopic study in dogs. int j oral maxillofac implants. 2009; 24: 432-38. 2. jahanbin a, rashed r, alamdari dh, koohestanian n, ezzati a, kazemian m, et al. success of maxillary alveolar defect repair in rats using osteoblastdifferentiated human deciduous dental pulp stem cells. j oral maxillofac surg. 2015;.13: 1-9. 3. dolanmaz d, esen a, yýldýrým g, ýnan ö. the use of autogeneous mandibular bone block grafts for reconstruction of alveolar defects. ann maxillofac surg. 2015; 5: 71-6. 4. hench ll, thompson i. twenty-first century challenges for biomaterials. j r soc interface. 2010; 7: 379-91. 5. hench l, polak j. third-generation biomedical materials. science. 2002; 295: 1014-7. 6. bianchi sd, lojacono a, nevins m, ramieri g, corrente g, martuscelli g, et al. the use of replicate resin models in the treatment of maxillary sinus augmentation patients. int j periodontics restorative dent. 1997; 17: 348-57. 7. do desterro fp, sader ms, soares gda, vidigal gm jr. can inorganic bovine bone grafts present distinct properties? braz dent j. 2014; 25: 282-8. 8. khojasteh a, kheiri l, motamedian sr, nadjmi n. regenerative medicine in the treatment of alveolar cleft defect: a systematic review of the literature. j craniomaxillofac surg. 2015; 43: 1608-13. 9. borba am, borges ah, da silva cs, brozoski ma, naclério-homem m da g, miloro m. predictors of complication for alveolar cleft bone graft. br j oral maxillofac surg. 2014; 52: 174-8. 10. schmitz jp, hollinger jo. the biology of platelet-rich plasma. j oral maxillofac surg. 2001; 59: 1119-21. 11. al-hezaimi k, nevins m, kim sw, fateh a, kim dm. efficacy of growth factor in promoting early osseointegration. j oral implantol. 2014; 40: 543-8. 12. marx re, carlson er, eichstaedt rm, schimmele sr, strauss je, georgeff kr. platelet rich plasma: growth factor enhancement for bone grafts. oral surg oral med oral pathol oral radiol endod. 1998; 85: 638-46. 13. menezes dj, shibli ja, gehrke sa, beder am, sendyk wr. effect of platelet-rich plasma in alveolar distraction osteogenesis: a controlled clinical trial. br j oral maxillofac surg. 2016;54: 83-7 14. al-hezaimi k, nevins m, kim sw, fateh a, kim dm. efficacy of growth factor in promoting early osseointegration. j oral implantol. 2014; 40: 543-8. 15. zechner w, tangl s, tepper g, furst g, bernhart t, haas r, et al. influence of platelet-rich plasma on osseous healing of dental implants: a histologic and histomorphometric study in minipigs. int j oral maxillofac implants. 2003; 18: 15-22. 16. liu t, wu g, wismeijer d, gu z, liu y. deproteinized bovine bone functionalized with the slow delivery of bmp-2 for the repair of criticalsized bone defects in sheep. bone. 2013; 56: 110-8. 17. schmidlin pr, nicholls f, kruse a, zwahlen ra, weber fe evaluation of moldable, in situ hardening calcium phosphate bone graft substitutes. clin oral implants res. 2013; 24: 149-57 18. martins lv, cestari tm, sottovia ad, ganjeiro jm, taga r. radiographic and histological study of perennial bone defect repair in rat calvaria after treatment with blocks of porous bovine organic graft material. j appl oral sci. 2004; 12(1): 62-9. 19. rocha fs, ramos lm, batista jd, zanetta-barbosa d, ferro ea, dechichi p. bovine anorganic bone graft associated with platelet-rich plasma: histologic analysis in rabbit calvaria. j oral implantol. 2011; 37: 511-8. 20. stievano d, di stefano a, ludovichetti m, pagnutti s, gazzola f, boato c, et al. maxillary sinus lift through heterologous bone grafts and simultaneous acid-etched implants placement. five years follow-up. minerva chir. 2008; 63: 79-91. 21. islam mm, khan ma, rahman mm. preparation of gelatin based porous biocomposite for bone tissue engineering and evaluation of gamma irradiation effect on its properties. mater sci eng c mater biol appl. 2015; 49: 648-55. 22. xie q, ainamo a, tilvis r. association of residual ridge resorption with systemic factors in home-living elderly subjects. acta odontol scand. 1997; 55: 299-305. 23. jahangiri l, devlin h, ting k, nishimura i. current perspectives in residual ridge remodeling and its clinical implications: a review. j prosthet dent. 1998; 80: 224-37. 24. nevins m, camelo m, de paoli s, friedland b, schenk rk, parmabenefati s, et al. a study of the fate of the buccal wall of extraction sockets of teeth with prominent roots. int j periodontics restorative dent. 2006; 26: 19-29. 25. araújo m, linder e, lindhe j. effect of a xenograft on early bone formation in extraction sockets: an experimental study in dog. clin oral implants res. 2009; 20: 1-6. 26. misawa m, lindhe j, araújo mg. the alveolar process following singletooth extraction: a study of maxillary incisor and premolar sites in man. clin oral implants res. 2015 nov 14. doi: 10.1111/clr.12710. 27. carotenuto g, spagnuolo g, ambrosio l, nicolais l. macroporous hydroxyapatite as alloplastic material for dental applications. j master sci master med. 1999; 10: 671-6. 28. wener j, linner-krcmar b, friess w, greil p. mechanical properties and in vitro cell compatibility of hydroxyapatite ceramics with graded pore structure. biomaterials. 2002; 23: 4285-94. 29. kim es, park ej, choung ph. platelet concentration and its effect on bone formation in calvarial defects: an experimental study in rabbits. j prosthet dent. 2001; 86: 428-34. 30. smieszek-wilczewska j, koszowski r, pajak j. comparison of postoperation bone defects healing of alveolar processes of maxilla and mandible with the use of bio-gen and bio-oss. j clin exp dent. 2010; 2: 60-2. braz j oral sci. 15(2):185-190 perception of dental care among children cláudia lobelli rangel gomes1, manoelito ferreira silva-júnior2, ana lílian correia lopes3, symone fernandes de melo4, manuel antonio gordón-núnez5, isabelita duarte azevedo6 1universidade de campinas – unicamp, piracicaba dental school, department of pediatric dentistry, piracicaba, sp, brazil 2universidade de campinas – unicamp, piracicaba dental school, department of community dentistry, piracicaba, sp, brazil 3exército brasileiro and hospital geral de belém, belém, pa, brazil 4universidade federal do rio grande do norte – ufrn, department of psychology, area of psychology, natal, rn, brazil 5universidade estadual da paraiba – uepb (campus viii), departament of dentistry, area of pathological processes, araruna, pb, brazil 6universidade federal do rio grande do norte – ufrn, dental school, departament of dentistry, area of pediatric dentistry, natal, rn, brazil correspondence to: isabelita duarte azevedo departamento de odontologia (área de odontopediatria) avenida senador salgado filho, 1787, lagoa nova cep: 59.056-000 natal-rn, brazil phone: +55 84 3215-4100 e-mail : isabelitaduarte@hotmail.com abstract children that come to dental offices with fear and anxiety usually tend to resist conditioning mechanisms. aim: to evaluate children’s perception of dental treatment and to identify the factors influencing this perception. methods: for this study, we selected a random sample of 100 children of both genders aged 3 to 12, treated at the department of dentistry of a university (group i) and at a children’s hospital (group ii). a structured questionnaire about the child’s perception about dental care was applied and the children were asked to draw a picture of this topic. results: most children expressed a positive perception in the questionnaire and in the drawings (93.8%). this positive perception was more accentuated in group i (94%) and in children aged 3 to 5 years (100%), particularly in girls (78%). the main cause of fear was the use of needles (42.4%). many children (24.2%) reported to prefer the noninvasive procedures. conclusions: a positive perception of dental treatment was observed in most children. therefore, dental pediatricians must be aware of the children’s perception for establishing a better conditioning. keywords: psychology, child, fear, pediatric dentistry. introduction knowledge of the child’s perception about dental care helps understanding the causes of the fear and anxiety, as well as about what is happening in this universe during dental treatment. the false image of torture in the dentist chair seems to occupy the imagination of many children that arrive at dental offices with this pre-established concept and resist conditioning mechanisms. it is therefore important to make sure that children feel comfortable during their dental visit and consequently have a positive experience1-3. negative experiences are difficult to overcome, especially when they were the child’s first impression of dental care1,4,5. safe methods to identify the feelings of children are important to establish the adequate dental treatment. for this purpose, questionnaires are useful tools to evaluate a child’s perception of dental treatment by the professional, which use exclusively the verbal information provided by the patients3,5-7. drawings are better since they do not require direct responses and help express feelings which the child is not aware of or unable to express verbally8-10. identification of the causes of fear and anxiety is of utmost importance for the adequate management of pediatric patients. in this context, the aim this study was to evaluate children’s perception of dental treatment and to identify factors that influence this perception. received for publication: october 15, 2016 accepted: march 08, 2017 original article braz j oral sci. april | june 2016 volume 15, number 2 http://dx.doi.org/10.20396/bjos.v15i2.8648759 186 material and methods the study was approved by the ethics committee of the federal university of rio grande do norte (ufrn) (protocol 375/10). the parents or legal guardians received detailed information about the objectives and methods of the study and agreed to their children’s participation by signing the free informed consent form. the sample size calculation was performed using the literature data11 on the proportion of dental anxiety in children as being 84%, absolute precision of 10% and a significance level of 5%. the sample size was 52 for each group, totaling 104 individuals. there was a loss of 4 individuals from the sample size (3.84%). a random sample of 100 children of both genders was selected, with ages ranging from 3 to 12 years. fifty children were seen at the department of dentistry, ufrn (group i), and 50 were recruited from the varela santiago children’s hospital, natal, rn, brazil (group ii). a structured questionnaire with objective and subjective questions about the perception of dental care was applied to each child. the researcher filled out the questionnaire according to the verbal answers of the child. after application of the questionnaire, the child received a white paper, a box of wooden color pencils, a box of color crayons, a pencil and an eraser. the box of pencils was opened and all available colors were shown to the child. the child was then asked to make a drawing about the topic “how do you perceive dental care?” according to marshman and hall12, drawings may be used in several ways to investigate children and their relationship with dentistry. there was no time limit for this task. the child received no verbal encouragement during drawing and the researcher waited for the signal from the child when he/ she finished the task. the researcher then collected the drawing and recorded the caption dictated by the child. on the back of the sheet, the examiner recorded the child’s name and age, date, code corresponding to the sample number and description of the drawing as reported by the child, asking the child to confirm the contents. the figure caption contained the identification and description of each element drawn by the child, as well as the feelings present in the image. the perception of dental care expressed in the drawing was classified according to the child’s verbal interpretation of the drawing (figure 1) to psychological interpretation, categorized in four groups: positive, negative, ambivalent or neutral. the drawings were analyzed by the method proposed by taylor et al.13, based on the occurrence (frequency) of some items. it was classified as positive when the image and caption revealed positive feelings about dental care (presence of elements of a cheerful and calm environment, such as a smile on the face of the child or the dentist); negative, when these elements revealed negative feelings about dental care (presence of sad or fearful elements, such as when the child's face showed fear, crying, angry, sad or the dentist's figure as a torturer or a bad guy); ambivalent, when positive and negative elements were perceived during interpretation of the drawing (when the design presented elements both positive and negative, as in the previous categories and we were not able to determine the child's feeling); neutral (when it was not possible to establish the feeling, but it was categorized when we were unable to verify any well-defined characteristics; neither positive or negative) present in the drawing and especially in the explanation present in the legend given after the finalization of the drawing (figures 1a to 1c). perception of dental care among children braz j oral sci. 15(2):185-190 figure 1 classification of the drawings. a) drawing with ambivalent perception. the figure on the left side of the image was called by the child a “good dentist”. the one on the right side was called a “bad dentist”. note the light colors, harmony of lines and cheerful aspect of the figure on the left, whereas strong colors, irregular lines and a face with empty eyes and somber expression are seen on the right. b) drawing with positive perception. this drawing was collected from group ii. observe the harmony of smiling individuals. notice the influence of the hospital environment on the representation of the dental chair which resembles a gurney. c) drawing with negative perception. notice the sad expression on the face of the figure on the right, as well as the absence of color throughout the drawing. natal/ rn, brazil, 2011. 187 for the purpose of statistical and descriptive analysis of the child’s perception found in the drawing in relation to the results of the questionnaire, the question “do you like to go to the dentist?” was considered as indicative of the child’s perception, obtained with the questionnaire. the dichotomous answers “yes” or “no” corresponded to a positive or negative perception, respectively. the collected data were entered in a database and analyzed using the statistical package for the social sciences 18.0 (spss inc., chicago, il, usa). questionnaire data were compared with the psychological interpretation of the child’s perception of dental care obtained from the drawings. the results were reported as means, absolute and relative frequencies, compared by the chisquared or fisher’s exact test at a 5% statistical significance level. results most children expressed a positive perception of dental care both in the drawing (91%) and in the questionnaire (81%). this positive perception was also observed when the questionnaire and drawings were analyzed together (72%). table 1 shows the distribution of dental care perception expressed in the questionnaire according to gender, age group, institution and getting little gifts. no significant difference in the perception of dental care was observed between genders (p = 0.682). there was a significant association between the perception of dental care and the institution where the participants were enrolled (p = 0.005), with a higher frequency of positive perception among children from the department of dentistry of ufrn (group i). percentage of positive perception among boys compared to girls. as regards the age group, the results were similar to those obtained with the questionnaire, showing a higher frequency of positive perception in children aged 3-5 years, followed by those aged 6-8 years and 9-12 years. analysis of the institution from which the children were recruited showed a higher percentage of positive perception in group i compared to group ii. there was no association between the perception expressed in the drawing and getting or not little gifts, similar to the results obtained from the questionnaire. perception of dental care among children *chi-square test. **fisher’s exact test. table 1 distribution of dental care perception expressed in the questionnaire according to gender, age group, institution and getting little gifts. natal/rn, brazil, 2011. variable variable perception on dental carepositive – n (%) negative– n (%) p-value gender female 34 (82.9) 7 (17.1) 0.682*male 47 (79.7) 12 (20.3) total 81 (81.0) 19 (19.0) age 35 years 10 (100.0) 0 (0.0) 0.255**6 – 8 years 33 (80.5) 8 (19.5)9 -12 years 38 (77.6) 11 (22.4) total 81 (81.0) 19 (19.0) group group i 46 (56.8) 4 (21.1) 0.005**group ii 35 (43.2) 15 (78.9) total 81 (81.0) 19 (19.0) gifts yes 22 (27.2) 4 (21.1) 0.585**no 59 (72.8) 15 (78.9) total 81 (81.0) 19 (19.0) table 2 distribution of dental care perception expressed in the drawing according to gender, age group, institution and getting little gifts. natal/rn, brazil, 2011. variable variable perception on dental care positive negative neutral ambivalent n (%) n (%) n (%) n (%) gender female 54 (91.5) 1 (1.7) 1 (1.7) 3 (5.1) male 37 (90.2) 2 (4.9) 0 (0.0) 2 (4.9) total 91 (91.0) 3 (3.0) 1 (1.0) 5 (5.0) age 3 5 years 10 (100.0) 0 (0.0) 0 (0.0) 0 (0.0) 6 8 years 38 (92.7) 1 (2.4) 0 (0.0) 2 (4.9) 9 12 years 43 (87.8) 2 (4.1) 1 (2.0) 3 (6.1) total 91 (91.0) 3 (3.0) 1 (1.0) 5 (5.0) group group i 48 (96.0) 1 (2.0) 0 (0.0) 1 (2.0) group ii 43 (86.0) 2 (4.0) 1 (2.0) 4 (8.0) total 91 (91.0) 3 (3.0) 1 (1.0) 5 (5.0) gifts yes 23 (88.5) 0 (0.0) 1 (3.8) 2 (7.7) no 68 (91.9) 3 (4.1) 0 (0.0) 3 (4.1) total 91 (91.0) 3 (3.0) 1 (1.0) 5 (5.0) * attitudes and characteristics of the dentist, reported by the children, which provide more confidence during the visit, for example, patience of the dentist, an affectionate approach and the fact that the dentist explains the procedure before performing it. table 3 distribution of the sample according to preference of tooth removal in the dental office or at any other place. natal/ rn, brazil, 2011. reasons of preference for tooth extraction in the dental office or another place office other place 10 (37.03%) positive feelings to the dentist* 11 (44%) less pain 8 (29.62%) they feel less pain 7 (28%) less fear 3 (11.1%) they feel less afraid 4 (16%) fear of anesthesia 3 (11.1%) receive anesthesia 1 (4%) preferred dental floss 1 (3.7%) difficulty in getting elsewhere 1 (4%) fear of plier extraction 1 (3.7%) increased bleeding outside the office 1 (4%) not like the office 1 (3.7%) can cry without recrimination 28 (50.0%) total 24 (25.0%) total table 2 shows the distribution of dental care perception expressed in the drawings, according to gender, age group, institution and getting little gifts. descriptive analysis of the psychological interpretation of the drawings revealed a higher the frequency of dental treatment fear was higher among boys (63.6%) than girls (36.4%). fear was reported by 16 (39.0%) children aged 6-8 years and by 17 (34.7%) aged 9-12 years. with respect to the institution from where the children were recruited, the percentage of fear of dental treatment was higher in group ii (46.0%) than in group i (20.0%). the reasons for preference for tooth extraction in the dental office or in any other place are presented in table 3. attitudes of the dentist that children do not like are shown and attitudes that the dentist could use improve the visit are shown in table 4. braz j oral sci. 15(2):185-190 188 discussion the technical advances and increasing progress in prevention of oral diseases over the past decades led to the improvement of pain management in dental practice. however, many patients are still afraid of the dentist14. fear of dental treatment has been recognized as one of the leading problems in pediatric dentistry. children with fear tend to visit the dentist less often, compromising their oral health. however, the etiology of this fear is not completely understood15. the anxiety and fear of dental treatment in pediatric patients have been recognized as potential problems for the management of these patients. early recognition and management of dental fear are critical to win the child’s confidence and for efficient treatment16. in the present study, the prevalence of fear during dental treatment was 33%. similar results have been reported by nicolas et al.14, who observed a prevalence of fear of 24.3%, and by oliveira and colares17 who found a prevalence of dental anxiety of 34.7%. most of the children who reported to be afraid to go to the dentist (33.3%) were boys (63.6%) aged 6 to 8 years (39.0%). some studies have found higher prevalence of anxiety among girls3,14. according nicolas et al.14, this finding is likely to be associated with cultural influences such as the fact that boys are uncomfortable to admit their fear and anxiety since this is not the behavior of a “manly man”. on the other hand, studies observed no significant difference of influence in dental fear18 and dental anxiety1,9 between genders. a possible explanation could be the different factors able to trigger dental fear among boys and girls. a recent study using the children’s fear survey schedule dental subscale showed some differences between girls and boys. for girls, four factors were found (‘fear of usual dental procedures and injections’; ‘fear of strangers’; ‘fear of general medical aspects of treatment’; ‘fear of health care personnel’). however, for boys, five factors were identified (‘fear of usual dental procedures’; ‘fear of general medical aspects of treatment’; ‘fear of invasive dental procedures’; ‘fear of health care personnel and injections’; ‘fear of strangers’)6. thus, further studies are required to identify the gender peculiarities in relation to dental fear, and not just to find which gender has more prevalence of fear or which has a higher association with dental fear. studying the influence of age, the fear and anxiety appear to decrease with increasing age, due to the progressively greater number of experiences with oral problems and dental care5,7. dutt et al.16 found a predominance of dental fear among older children. in contrast, lee et al.15 reported that children younger than 4 years were significantly more anxious than children over 7 years. in another study, younger children cried more frequently during dental treatment than older children, a finding that can be explained by the fact that crying is usually a physiological expression of aggressive behavior in younger children4. these results disagree with the present study in which only children aged 3-5 years were not afraid to go to the dentist and children aged 9-12 years were less afraid than children of the 6-8 year age group. in contrast, the above studies did not observe significant association between age and dental anxiety3,19. the influence of age on anxiety during dental treatment is still unclear, since few studies have investigated this topic14. the results of the present study did not reveal a significant association between the child’s perception of dental care expressed in the drawing or questionnaire and the fact of getting or not little gifts. thus, this factor does not seem to influence the child’s perception of dental care. in the present study, the child’s perception of dental care was positive in most cases, as expressed by the drawings and questionnaires. this finding agrees with the results reported by alsarheed4 who used a questionnaire to evaluate children’s attitudes towards dentists and observed that most children reported liking their visit to the dentist, whereas a low percentage of the sample stated they did not like the visit to the dentist and feel afraid. few studies used children’s drawings as an instrument to evaluate subjective feelings. however, drawings have been shown to be a useful and valid tool to capture the emotional state of a child in dental settings, since drawing is easier, familiar and more enjoyable for the child patient9,10. children present difficulties perception of dental care among children table 4 the attitudes or procedures of the dentist that the child reported not to like and the opinion of the child about the attitudes the dentist could assume to improve the visit. natal/rn, brazil, 2011. the attitudes of the dentist reported not to like invasive procedures 29.0% use of needles 28.0% they couldn’t answer 11.0% negative behavior of the dentist 11.0% nothing unpleasant 6.0% high and low speed dental 5.0% non-invasive procedures 3.0% staying long with the mouth open 3.0% light reflector on the eyes 2.0% i do not like when the dentist messes in my teeth 2.0% attitudes the dentist could assume to improve the visit positive behavior of dentist 31.0% they couldn’t answer 22.0% receiving gifts 18.0% no anesthesia and no needle 8.0% getting braces put on 5.0% using anesthesia 4.0% x-rays 3.0% visit no anesthesia and extraction 2.0% use of triple syringe 2.0% extractions 2.0% oral prescription medication 1.0% toys in the office 1.0% don’t need nothing, the dentist is already nice 1.0% braz j oral sci. 15(2):185-190 with the abstract task of describing subjective experiences using verbal language and rather match internal states better with pictorial representations of their emotions20-23. studies suggested that projective techniques are a more adequate measure to collect data about a child’s perception21,23. children’s drawings and their description can provide a unique window into their inner experiences, particularly when they experience stress or anxiety24. in clinical practice, drawings have been used for decades by child psychiatrists and psychologists to analyze the subjectivity of children’s feelings, their fears, anxiety, concerns and anger23. the use of non-verbal assessment tools like drawings is more appealing since this method overcomes language limitations and cultural barriers that might be encountered with traditional verbal tests25. drawing helps children organize their narrative which, in turn, gives them the opportunity to find their voice9,26. the drawings of hospitalized children have been shown to be a valuable assessment tool to measure their emotional state20. in the present study, the frequency of dental fear was higher in the group of hospitalized children (group ii, 46%) than among children seen at the dentistry department of a university (group i, 20%). according to themessl-huber et al.27, dental fear in hospitalized children is usually associated with negative medical and dental experiences of these children. the frequency of dental fear in hospitalized children ranged from 5% to 20% in another study28. this percentage was 33% in the present investigation. the use of needles and tooth extraction were the main reasons of dental fear reported by most children of the sample. similar results have been reported, like local anesthesia, mainly the use of a needle and the tooth extraction associated with pain, were the most common reasons cited by children who did not like dental treatment4,7. when the children were asked about their preference of undergoing tooth extraction in the dental office or at any other place, half the children (50%) preferred to be treated in the dental office. the most frequently reported reason for this preference was the positive attitude of the dentist (37%). most children (31%) provided the same answer, i.e., a positive attitude of the dentist, when they were asked about what the dentist could do to improve the visit. according to alsarheed4, dental surgeons have to be aware of the perception, preferences and fears of the patient, since children who have a positive interaction with their dentist are less likely to develop fear during the visits. several factors, such as experience of oral problems, number of tooth extractions, number of invasive treatments, number of visits before the first invasive treatment, management of behavior problems, parents’ fear of dental treatment and classification of dental fear, are significantly associated with the level of the child’s fear of dental treatment1,5. studies have shown that visiting the dentist more regularly and without previous traumatic dental experiences made the patient less anxious1,3. berge et al.1 observed that children with more noninvasive visits are less likely to develop dental fear than those with a previous invasive experience. these findings agree with the present study where most children cited noninvasive procedures as the ones they liked most during dental treatment. tickle et al.2 also showed that a history of extractions and asymptomatic dental visits were significantly associated with anxiety, suggesting that the etiology of dental anxiety in children is multifactorial. a study about the understanding of children enrolled in an oral health education programs on tooth care showed satisfactory knowledge, mainly on positive and negative aspects related to the oral health-disease process10, and not associating with negative professional aspect and dental care. programs like this must be encouraged to disassociate oral care form negative aspects. besides that, the results of the present study indicate the need for strategies designed to promote constant asymptomatic visits to the dentist at an early age. dental professionals need to understand the etiology of dental anxiety in children and its development over time in order to provide a more effective care to their patients and therefore, to minimize the risk of developing anxiety2. employing scales that measure fear, as children’s fear survey schedule dental subscale, the dentist may recognize which item(s) of the scale reported by the child were related to a particular fear. accordingly, the dentist can approach the child and guide his/her behavior during the dental visit6. in most cases, anxiety towards dental treatment has its origin in childhood and, if not overcome, may extend into adult life, compromising oral health29. our study verified that most children of the sample expressed a positive perception of dental care in both the drawing and questionnaire. the diverse instruments used in our study for the evaluation of the child’s perception of dental care, are important tools for the investigation of perceptions, attitudes and behaviors of child patients designed to improve the quality of dental care in this population. besides that, dental professionals have to be aware of the perception, preferences and fears of the patient for better conditioning and for a good doctor-patient relationship. references 1. berge mt, veerkamp jsj, hoogstraten j. the etiology of childhood dental fear: the role of dental and conditioning experiences. j anxiety dis. 2002; 16(3): 321-9. 2. tickle m, jones c, buchannan k, milsom km, blinkhorn as, humphris gm. a prospective study of dental anxiety in a cohort of children followed from 5 to 9 years of age. int j paediatr dent. 2009 2009 jul;19(4):22532. doi: 10.1111/j.1365-263x.2009.00976.x. 3. saatchi m, abtahi m, mohammadi g, mirdamadi m, binandeh es. the prevalence of dental anxiety and fear in patients referred to isfahan dental school, iran. dent res j (isfahan). 2015 may-jun;12(3):248-53. 4. alsarheed m. children’s perception of their dentists. eur j dent. 2011 189perception of dental care among children braz j oral sci. 15(2):185-190 apr;5(2):186-90. 5. olak j, saag m, honkala s, nõmmela r, runnel r, honkala e, et al. children's dental fear in relation to dental health and parental dental fear. stomatologija. 2013;15(1):26-31. 6. el-housseiny aa, alamoudi nm, farsi nm, el derwi da. characteristics of dental fear among arabic-speaking children: a descriptive study. bmc oral health. 2014 sep;14:118. doi: 10.1186/1472-6831-14-118. 7. oliveira mg, ávila ra, silva rrc, gomes fv, moraes jfd, weber jbb. dental fear and anxiety among children and their caregivers. rev cienc med biol. 2014 may-aug;13(2):137-41. doi: http://dx.doi. org/10.9771/cmbio.v13i2.10113. 8. hamama l, ronen t. children’s drawings as a self-report measurement. child family social work. 2009;14(1):90-102. 9. torriani dd, goettems ml, cademartori mg, fernandez rr, bussoletti dm. representation of dental care and oral health in children’s drawings. br dent j. 2014 jun;216(12):e26. doi: 10.1038/ sj.bdj.2014.545. 10. pacheco kts, silva-junior mf, arcieri rm, garbin aji, garbin cas. the knowledge of children on tooth care: a qualitative and quantitative approuch. rev odonto cienc. 2016;31(2):49-53. 11. bottan er, oglio jd, araújo sm. dental anxiety in elementary schoolchildren. pesq bras odontoped clin integr. 2007;7(3):241-6. 12. marshman z, hall mj. oral health research with children. int j paediatr dent. 2008 jul;18(4):235-42. doi: 10.1111/j.1365-263x.2008.00922.x. 13. taylor d, roth g, mayberry w. children's drawings about dentistry. community dent oral epidemiol. 1976 jan;4(1):1-6. 14. nicolas e, bessadet m, collado v, carrasco p, rogerleroi v, hennequin m. factors affecting dental fear in french children aged 5–12 years. int j paediatr dent. 2010 sep 1;20(5):366-73. doi: 10.1111/j.1365263x.2010.01054.x. 15. lee c, chang y, huang s. the clinically related predictors of dental fear in taiwanese children. int j paediatr dent. 2008 nov;18(6):415-22. doi: 10.1111/j.1365-263x.2008.00924.x. 16. dutt k, nagar a, pandey r, singh p. reliability and factor analysis of children’s fear survey schedule-dental subscale in indian subjects. j indian soc pedod prev dent. 2010 jul-sep;28(3):151-5. doi: 10.4103/0970-4388.73788. 17. oliveira mmt, colares v. the relationship between dental anxiety and dental pain in children aged 18 to 59 months: a study in recife, pernambuco state, brazil. cad saude publica. 2009 apr;25(4):743-50. 18. klaassen ma, veerkamp jsj, hoogstraten j. dental fear, communication, and behavioural management problems in children referred for dental problems. int j paediatr dent. 2007 nov;17(6):469-77. 19. jones lm, buchanan h. assessing children’s dental anxiety in new zealand. n z dent j. 2010 dec;106(4):132-6. 20. clatworthy s, simon k, tiedeman m. child drawing: hospital – an instrument designed to measure the emotional status of hospitalized schoolaged children. j pediatr nurs. 1999 feb;14(1):2-9. 21. clatworthy s, simon k, tiedeman m. child drawing: hospital manual. j pediatr nurs. 1999 feb;14(1):10-8. 22. chambers ct, giesbrecht k, craig kd, bennet sm, huntsman e. a comparison of faces for the measurement of pediatric pain: children’s and parents’ ratings. pain 1999 oct;83(1):25-35. 23. skybo th, ryan-wenger n, su y. human figure drawings as a measure of children’s emotional status: critical review for practice. j pediatr nurs. 2007 feb; 22(1):15-28. 24. looman ws. a developmental approach to understanding drawings and narratives from children displaced by hurricane katrina. j pediatr health care. 2006 may-jun;20(3):158-66. 25. matto hc. drawing in clinical assessment of children and adolescent. in: smith sr, handler l. the clinical assessment of children and adolescents: a practitioner’s handbook. mahwah, nj: lawrence erlbaum associates; 2006. p.207-9. 26. driessnack m. children’s drawings as facilitators of communication: a meta-analysis. j pediatr nurs. 2005 dec;20(6):415-23. 27. themessl-huber m, freeman r, humphris g, macgillivray s, terzi n. empirical evidence of the relationship between parental and child dental fear: a structured review and meta-analysis. int j paediatr dent. 2010 mar;20(2):83-101. doi: 10.1111/j.1365-263x.2009.00998.x. 28. klingberg g, broberg ag. dental fear⁄anxiety and dental behaviour management problems in children and adolescents: a review of prevalence and concomitant psychological factors. int j paediatr dent. 2007 nov;17(6):391-406. 29. oliveira rs, torres lms, gomes is, nicoló rd. [evaluation of anxiety levels in children during odontological treatment]. ijd, int j dent. 2010 oct-dec;9(4):193-7. 190 perception of dental care among children braz j oral sci. 15(2):185-190 revista fop n 13 braz j oral sci. april/june 2008 vol. 7 number 25 prevalence of malocclusion in a brazilian schoolchildrenprevalence of malocclusion in a brazilian schoolchildrenprevalence of malocclusion in a brazilian schoolchildrenprevalence of malocclusion in a brazilian schoolchildrenprevalence of malocclusion in a brazilian schoolchildren population and its relationship with early tooth losspopulation and its relationship with early tooth losspopulation and its relationship with early tooth losspopulation and its relationship with early tooth losspopulation and its relationship with early tooth loss ricardo alves de souza1; maria beatriz borges de araújo magnani2; darcy flávio nouer2; fábio lourenço romano3; manuela ribeiro passos4 1dds, ms, assistant professor, department of heath, state university of bahia southwest, brazil 2dds, ms, phd, professor, department of pediatric dentistry, piracicaba dental school, university of campinas, brazil 3dds, ms, phd student of piracicaba dental school, university of campinas, brazil 4dds, auxiliary professor, department of biological science, state university of bahia southwest, brazil received for publication: september 23, 2007 accepted: june 12, 2008 correspondence to: ricardo alves de souza departamento de saúde, universidade estadual do sudoeste da bahia av. josé moreira sobrinho s/n, jequiezinho, 45200-000 jequié, bahia, brasil. phone:+55-73-3528-9623/9680. e-mail ricardoalves@fop.unicamp.br riacardoalves@ortodontista.com.br a b s t r a c t the purpose of this study was to evaluate the prevalence of malocclusion in a brazilian schoolchildren population in the mixed dentition, assessing its relationship with early tooth loss. the study population consisted of 1,014 7-11-year-old children of both genders, with low socioeconomic backgrounds and multiracial characteristics, living in the city of jequié, in the northeast region of brazil. the clinical examination was carried out by an adequately calibrated orthodontist in the children’s classrooms. data were analyzed statistically by either chi-square or fisher’s exact test (a=0.05). angle’s classification revealed that 22.3% of the population had normal occlusion. class i malocclusion was observed in 47.6%, class ii division 1 in 21%, class ii division 2 in 0.9% and class iii in 8.2% of the children. excluding the subjects (n=199) with early tooth loss, which is a condition that can modify malocclusion status, the distribution was as follows: class i (36.2%), class ii division 1 (17.5%), class ii division 2 (0.7%) and class iii (3.7%). early tooth loss was more commonly observed in boys (class i, class iii left side, and both sides simultaneously). anterior crossbite was observed in 5.7% of the subjects, posterior crossbite in 8.4%, anterior and posterior crossbite simultaneously in 2.6%, open bite in 13% and crowding in 49.6%. in conclusion, class i malocclusion was the most prevalent alteration. the occlusal pattern of class iii was more common than class ii division 2, among the examined individuals. key words: malocclusion, orthodontics, open bite, crossbite, tooth loss. i n t r o d u c t i o n orthodontic care in brazilian public dental services is still rare, probably due to the high treatment cost and the lack of specific public assistance policies. a systematic and well-organized dental care program for any target population in a community requires some basic information, such as the prevalence of the condition to be assessed1. jequié is a city in the state of bahia, in the northeast region of brazil, with a population from a low socioeconomic background. the population is mainly composed of biracial subjects with characteristics between white and black, which is a multiracial condition commonly observed in the state of bahia. however, few epidemiological studies have been conducted with regard to the prevalence of malocclusion in this region. as socioeconomic factors interfere significantly with oral health, developing nations still have problems with children suffering from early tooth loss mostly due to caries2-4. this situation is directly related to malocclusion being an important factor for its establishment as well as for changing the malocclusion classification interpretation due to tooth migration. the angle’s classification5 method has been widely used as a qualitative epidemiological tool for malocclusion assessment. moreover, the prevalence of malocclusion has been extensively investigated worldwide6-12 (table 1). the high prevalence of malocclusions implies that public health efforts are required, as such conditions affect negatively the individual’s quality of life, particularly in the case of children and adolescents, who are sensitive about their appearance13-18. a previous study with 395 canadian children also found a high caries prevalence in primary teeth, reporting percentages of 30.4% and 20.6% for children aged 6 and 9 years, respectively 19. an 1566 first author year normal occlusion class i class ii class iii american children and adolescents breham6 1961 16.6% 60.1% 22.8% 0.5% swedish children björk7 1974 26.4% 51.8% 18.9% 2.8% english children haynes8 1970 26.8% 50.3% 19.5% 2.5% brazilian children silva filho9 1989 11.4% 42.5% 42% 3% turkish children sayin & türkkahraman10 2004 64% 24% 12% brazilian children biázio et al...11 2005 23.4% 74.5% 20.2% 5.1% brazilian children schwertner et al...12 2007 72.9% 23.5% 3.6% table 1 literature regarding the prevalence of malocclusion worldwide epidemiological study with 493 nigerian children of different socioeconomic groups emphasized the need for treating crowded teeth (18.9%), carious lesions (14.8%), oral habits (7.3%), crossbite (10.3%), late primary tooth loss (6.9%), and early primary tooth loss (4.3%)20. the early loss of primary teeth can often result in malocclusion in a later moment21 involving the sagittal, vertical and transverse planes, thus becoming priority cases needing dental treatment22. therefore, the purpose of this study was to evaluate the prevalence of malocclusion in a brazilian schoolchildren population in the mixed dentition, assessing its relationship with early tooth loss. material and methods the study population consisted of 1,014 7-11-year-old children (mean age = 9.52 ± 1.16 years) of both genders, being 503 boys (49.6%) and 511 girls (50.4%), who lived in the city of jequié, in the northeast region of brazil. the population presented low socioeconomic backgrounds and multiracial characteristics. five public primary schools, representing different districts, were randomly selected from over 20 schools. after obtaining informed consent from their parents, and also approval from the school authority to conduct the study, one orthodontist (c.o.o.) examined the students clinically under natural illumination, using a sterile wooden spatula, mask and disposable gloves. assessment of the anteroposterior relationship of the dental arches was based on angle’s classification5. other occlusal problems, such as anterior and posterior crossbite, open bite and tooth crowding were observed in this study. data referring to early primary or permanent tooth loss, and patient identification, gender, age, address, and telephone number were also recorded by another dentist. in assessing the occlusal classification, the teeth were in maximal intercuspation, which was achieved by asking the subject to swallow and then to clench the teeth. occlusions with minor deviations from the hypothetical concept of the ideal in permanent dentition, which did not cause esthetic or functional problems, were classified as normal. according to the 2003 education census23, a total of 35,740 schoolchildren were attending local or state schools in the city of jequié (excluding private and federal institutions), the great majority (n = 32,933) being public elementary schoolchildren. as a result, the sample selected for the present survey (n = 1,014) had a high reliability for epidemiological studies. sampling error of 2.46% was calculated for the studied population with 95% confidence interval. intraexaminer reliability, tested by re-examining 40 subjects after an interval of 2 to 4 weeks, was very high (r = 0.97; p<.001). the research protocol was independently reviewed and approved (process #066/2005) by the human ethics research committee of the dental school of piracicaba (fop-unicamp). statistical analysis the data were submitted to statistical analysis by using either chi-square test or fisher’s exact test for bivariate analysis at 5% significance level (p<0.05). data analysis was performed using spss statistical software for windows v.10.0 (spss inc., chicago, il, usa), which also includes frequency distribution and test of association. r e s u l t s normal occlusion was found in 22.3% of subjects (table 2). the prevalence of malocclusion changed when the individuals with tooth loss were excluded. in class i and class iii individuals, malocclusion prevalence dropped from 47.6 to 36.2% and from 8.2 to 3.7%, respectively. as regards gender (table 3), no statistically significant difference (p>0.05) was observed between boys (75.6%) and girls (79.8%) regarding malocclusion prevalence. statistically significant difference (p<0.05) was observed in children with class ii division 1 right side subdivision (boys = 1.6% and girls = 6.7%) and class iii left subdivision (boys = 2.6% and girls = 0.2%) malocclusions. the distribution of early tooth loss (table 4) was statistically different (p<0.05) comparing class i (boys = 13.5% and girls = 9.4%), class iii left subdivision (boys = 1.8% and girls = 0.4%) and class iii both sides (boys = 2.3% and girls = 0.8%). table 5 shows the distribution of other occlusal problems observed in the studied population. anterior crossbite, posterior crossbite and open bite were found in 5.7, 8.4 and 13% of the subjects. statically significant by gender was observed only for tooth crowding (p<0.05). 1567 braz j oral sci. 7(25):1566-1570 prevalence of malocclusion in a brazilian schoolchildren population and its relationship with early tooth loss occlusal classifications n (%) malocclusion with early tooth loss excluded normal occlusion 226 (22.3%) class i 483 (47.6%) 367 (36.2%) class ii division 1 213 (21%) 177 (17.5%) class ii division 2 9 (0.9%) 7 (0.7%) class iii 83 (8.2%) 38 (3.7%) total 1014 (100%) 589 (58%) table 2 occlusal classifications and malocclusions with early tooth loss excluded early tooth loss boys girls p value * class i 68 (13.5%) 48(9.4%) 0.0391 right side 7 (1.4%) 8 (1.6%) 0.8186 left side 3 (0.6%) 7 (1.4%) 0.3415division 1 both sides 3 (0.6%) 8 (1.6%) 0.2242 right side 0 0 left side 1 (0.2%) 1 (0.2%) 1.000 class ii division 2 both sides 0 0 right side 5 (1.0%) 10 (2.0% ) 0.2041 left side 9 (1.8%) 2 (0.4%) 0.0362class iii both sides 15 (2.3% ) 4 (0.8%) 0.0105 subtotal 111 (10.9%) 88 (8.6% ) 0.029 total 199 (19.6%) table 4 distribution of children with early tooth loss *statistically significant if p<0.05 (chi-square test or fisher’s exact test) occlusal classifications boys girls p value * normal occlusion 123 (24.4%) 103 (20.2%) 0.1002 class i 242 (48.1%) 241 (47.2%) 0.7623 class ii division 1 67 (13.3%) 80(15.7%) 0.2909 right side 8 (1.6%) 31 (6.7%) < 0.0001class ii division 1 subdivision left side 14 (2.8%) 13 (2.5%) 0.8129 class ii division 2 3 (0.6%) 3 (0.6%) 1.000 right side 0 2 (0.4%) 0.4995class ii division 2 subdivision left side 0 1 (0.2%) 1.000 class iii 27 (5.4%) 26 (5.1%) 0.8414 class iii right subdivision 6 (1.2%) 10 (2.0%) 0.329 class iii left subdivision 13 (2.6%) 1 (0.2%) < 0.0001 table 3 distribution of normal occlusions and malocclusions by gender *statistically significant if p<0.05 (chi-square test or fisher’s exact test). 1568 braz j oral sci. 7(25):1566-1570 prevalence of malocclusion in a brazilian schoolchildren population and its relationship with early tooth loss total boys girls n % n % n % p value * anterior crossbite 58 5.7 25 2.4 33 3.2 0.4349 posterior crossbite 85 8.4 31 3.0 54 5.3 0.1643 anterior and posterior crossbit e 26 2.6 14 1.3 12 1.1 0.9661 open bite 132 13. 0 55 5.4 77 7.5 0.2265 tooth crowding 503 49. 6 231 22.7 272 26.8 0.0036 table 5 distribution of other types of malocclusions *statistically significant if p<0.05 (chi-square test or fisher’s exact test). d i s c u s s i o n the rate of normal occlusion observed in the present study (22.3%) differed from another study reported in the southern region of brazil (11.47%)9. however, the normal occlusion score in jequié was similar to that in swedish (26.4%)7 and english children (26.8%) 8. in africanamerican children, it has been reported that 17% of the children had normal occlusion24, while another study found a prevalence of 16.6% in white american children6. there are several etiologic factors (e.g.: early tooth loss, oral habits) for malocclusions that modify all occlusal development. these factors are more important than the racial characteristics. in the present study, class i malocclusion was found in 47.6% of the sample versus 55% found in a city of the southern brazil9. however, when the children with early tooth loss were excluded from the analysis, the percentage dropped to 36.2% in our study. exclusion of individuals with early tooth loss from malocclusion prevalence studies has been considered as relevant for reducing significantly the number of children with class i malocclusion. the findings of the present survey showed that early tooth loss interfered with malocclusion classification. tooth migrations also changed the occlusal characteristics of the subjects in class ii division 1, with a decrease from 21% to 17.5%, and in class iii malocclusion, from 8.2% to 3.7%. this finding does not agree with the results of a previous study involving 1,201 white brazilian children aged 6 to 12 years, which reported a percentage of 43.4% and 1.2% for class ii and class iii malocclusions, respectively25. in class ii division 1, differences also occurred from other studies: onyeaso1 (12.3%), haynes8 (12.5%) and foster and day26 (27.2%). the methodologies and/or the characteristic of the samples could explain the differences. in this study, the prevalence of class iii with early loss tooth agrees with the rates reported by foster and day26 (3.5%), but presented differences from those reported by haynes8 (2.5%) and goose et al....27 (2.91%). in this study, there was a higher percentage of class iii malocclusions (3.7%) than class ii division 2 (0.7%). onyeaso1 demonstrated a trend towards more class iii malocclusion in black children (11.8%), which seems to be related to the miscegenation of the schoolchildren in the jequié sample. in order to evaluate the differences between ethnic groups and malocclusion prevalence, a previous study28 involving low-socioeconomic level black and white 8-9-year-old children was carried out in pretoria, south africa and found a significantly higher percentage of white children presented with class ii malocclusions, while black children showed a higher tendency for the class iii malocclusion. according with table 4, early tooth loss in class iii left side was more significant for boys than girls (p=0.0362), which accounted for the significant differences in class iii subdivision left between genders in table 3 (boys = 2.6% and girls = 0.2%). the tooth early loss in class iii on both sides elevated the severity of class iii (mandibular early loss). if the migrations occurred in the maxillary arch, the malocclusions were probably changed into class i or class ii. the high differences in class i (table 2) promoted a significant value increase in the subtotal of early tooth loss (p=0.029); this condition would appear to be because of better oral hygiene care in girls than in boys. early tooth loss should be taken into account for research as it is an important etiological agent with severe consequences for occlusion22. the differences between boys and girls in the class ii division 1 subdivision right and class iii subdivision left seemed to be due the early loss (table 3). several studies1,7-9 showed that no statistically significant sex differences were founded in the prevalence of malocclusions. other occlusal problems (table 5), such as anterior crossbite (5.7%) and open bite (13%) were in agreement with the previous data of ramos et al....29, who observed that 6.7% of 218 children had anterior crossbite, and 15.4% of the sample presented open bite. however, differences were observed in the prevalence of posterior crossbite (8.4%), tooth crowding (49.6%) and early tooth loss (19.6%) compared to the results of another study29, which were 14.4%, 34% and 30.7% for posterior crossbite, tooth crowding and early tooth loss, respectively. a greater prevalence of crowding was observed in our study. 1569 braz j oral sci. 7(25):1566-1570 prevalence of malocclusion in a brazilian schoolchildren population and its relationship with early tooth loss the results of the present investigation demonstrated the importance of early tooth loss as an etiologic factor of malocclusions. moreover, further studies involving multiracial populations are necessary in the brazilian population, which requires increased attention from the public health system regarding dental assistance and preventive orthodontic care. a c k n o w l e d g e m e n t s the authors would like to thank the jequié authorities, particularly dr. tânia diniz correia leite de britto, secretary of health, and dr. domingos sávio perpétuo coelho, dentistry coordinator, for the endeavor to create an inedited database on the prevalence of malocclusion in this city. r e f e r e n c e s 1. onyeaso co. prevalence of malocclusion among adolescents in ibadan, nigeria. am j orthod dentofacial orthop. 2004; 126: 604-7. 2. kock g, martinsson t. socio-odontologic investigation of school children with high and low caries frequency. i socioeconomic background. odontol revy. 1970; 21: 207-16. 3. carmichael cl, rugg-gunn aj, french ad, cranage jd. the effect of fluoridation upon the relationship between caries experience and social class in 5-year-old children in newcastle and northumberland. br dent j. 1980; 149: 163-7. 4. fejerskov o, baelum v, luan wm, manji f. caries prevalence in africa and the people’s republic of china. int dent j. 1994; 44: 425-33. 5. angle eh. classification of malocclusion. dent cosmos. 1899; 41: 248-64. 6. breham h, jackson d. an investigation of the extent of need for orthodontic service. am j orthod. 1961; 47: 148-9. 7. björk a. the face in profile. sven tandl-tidskr. 1947; 40 (suppl 5b). 8. haynes s. the prevalence of malocclusion in english schoolchildren aged 11-12 years. eur orthod soc trans. 1970; 48: 89-98. 9. silva filho og, freitas sf, cavassan ao. prevalência de oclusão normal e má oclusão na dentadura mista em escolares da cidade de bauru (são paulo). rev assoc paul cir dent. 1989; 43: 287-90. 10. sayin mo, türkkahraman h. malocclusion and crowding in an orthodontically referred turkish population. angle orthod. 2004; 74: 635-9. 11. de biázio rc, costa gc, das virgens filho js. prevalência de má-oclusão na dentadura decídua e mista no distrito de entre rios, guarapuava-pr. publ. uepg ci. biol. saúde. 2005; 11: 29-38. 12. schwertner a, nouer pra, garbui iu, kuramae m. prevalência de maloclusão em crianças entre 7 e 11 anos em foz do iguaçu, pr. rgo. 2007; 55: 155-61. 13. soha j, sandhamb a, chanc yh. occlusal status in asian male adults: prevalence and ethnic variation. angle orthod. 2005; 75: 814-5. 14. thilander b, pena l, infante c, parada ss, de mayorga c. prevalence of malocclusion and orthodontic treatment need in children and adolescents in bogotá, colombia. an epidemiological study related to different stages of dental development. eur j orthod. 2001; 23: 153-67. 15. shaw wc, meek sc, jones ds. nicknames, teasing, harassment and the salience of dental features among school children. br j orthod. 1980; 7: 75-80. 16. hill pa. the prevalence and severity of malocclusion and the need for orthodontic treatment in 9-, 12-, and 15-yearold glasgow schoolchildren. br j orthod. 1992; 19: 87-96. 17. oliveira cm, sheiham a. orthodontic treatment and its impact on oral health-related quality of life in brazilian adolescents. j orthod. 2004; 31: 20-7. 18. shaw wc, lewis hg, robertson nre. perception of malocclusion. br dent j. 1975; 138: 211-7. 19. karaiskos n, wiltshire wa, odlum o, brothwell d, hassard th. preventive and interceptive orthodontic treatment needs of an inner-city group of 6and 9-year-old canadian children. j can dent assoc. 2005; 71: 649. 20. onyeaso co. need for preventive/interceptive orthodontic treatment among 7-10-year-old children in ibadan, nigeria: an epidemiological survey. odontostomatol trop. 2004; 27: 15-9. 21. lindsten r, ogaard b, larsson e. anterior space relations and lower incisor alignment in 9-year-old children born in the 1960s and 1980s. angle orthod. 2001; 71: 36-43. 22. pedersen j, stensgaard k, melsen b. prevalence of malocclusion in relation to premature loss of primary teeth. community dent oral epidemiol. 1978; 6: 204-9. 23. ministério da educação do brasil. instituto nacional de estudos e pesquisas educacionais (inep). censo educacional [homepage on the internet] [cited 2008 aug 11]. available from: http: // www.inep.gov.br/basica/censo/escolar/sinopse/sinopse.asp. 24. altemus l. the frequency of the incidence of malocclusion in american negro children aged 12-16. angle orthod. 1959; 29: 189-200. 25. gandini mreas, pinto as, gandini junior lg, martins jcr, mendes ajd. estudo da oclusão dentária de escolares da cidade de araraquara na fase de dentadura mista. ortodontia 1994; 27: 37-49. 26. foster td, day ajw. a survey of malocclusion and the need for orthodontic treatment in a shrophire school population. br j orthod. 1974; 1: 73-8. 27. goose dh, thompson dg, winter fc. malocclusion in school children of the west midlands. br dent j. 1957; 102: 174-8. 28. de muelenaere kr, wiltshire wa. the status of the developing occlusion of 8-9 year-old children from a lower socio-economic group in a developing country. j dent assoc s afr. 1995; 50: 113-8. 29. ramos al, gasparetto a, terada hh, furquim lz, basso p, meireles rp. assistência ortodôntica preventiva-interceptora em escolares do município de porto rico – parte 1: prevalência das más-oclusões. rev dental press ortodon ortop facial. 2000; 5: 9-13. 1570 braz j oral sci. 7(25):1566-1570 prevalence of malocclusion in a brazilian schoolchildren population and its relationship with early tooth loss oral sciences n3 001 effect of bapo on the degree conversion of experimental adhesives photoactivated by leds sinhoreti mac*, souza-junior ej, brandt wc, oliveira dcrs, alonso rcb, puppinrontani rm, correr-sobrinho l. piracicaba dental school unicamp sinhoreti@fop.unicamp.br the aim of this study was to evaluate the degree of conversion (dc) of experimental dental adhesives composed by bisalquylphosphine oxide (bapo) and/or camphorquinone (cq), photocured by led single peak (radii-cal sdi) or polywave (bluephase g2 ivoclar vivadent). experimental adhesives composed by bisgma and hema were handled with different photoinitiators system: cq/amine, bapo/ amine, bapo, cq/bapo/amine. the hydrophobic resin adhesive adper scotchbond multi-purpose was used as control. specimens of each adhesive type in bar-type (7mm x 2mm x 1mm) were prepared (n=5) and photocured by led single peak or polywave with 48j of energy density. after 24h, the gc was measured by ftiratr. the data were submitted to anova two-way and tukey test (p<.05). when the led polywave was used the adhesive bapo/amina showed higher gc (69.9 ± 3.2%) and was statistically different from the control group (61.5 ± 2.0%) and cq groups (58.2 ± 4.2%) and cq/bapo (59.6 ± 4.8%). however, did not differ from group with bapo (66.8 ± 3.76%) only. for the led single peak, all groups did not differ, except the control group (60.34 ± 5.72%) than group cq/bapo/ amine (49.89 ± 5.85%). regarding the apparatus, for the adhesives witth bapo the led ploywave showed statistically better average than led single peak. when associated with amine, the bapo showed satisfactory degree of conversion. the led polywave promoted better monomeric conversion than led single peak for the adhesives with bapo. 003 reliability of different y-tzps and identification of phase transformation by micro-raman spectroscopy ramos cm*, cesar pf, bonfante ea, tabata as, mondelli rfl, rodrigues rf, francisconi pas, borges afs bauru dental school usp carla_muller@yahoo.com.br the aim of this study was to evaluate the reliability of different y-tzps flexural strength and investigate the presence of monoclinic tetragonal phase transformation (t ’! m) after tension fracture.( the groups were: y-tzp experimental (ze), zirconcad (zca), ips e.max zircad (zmax) and in ceram yz (zyz). 120 specimens were constructed (n = 30) and subjected to testing in three point bending speed of 0.5 mm/min, according to iso 6872. for the analysis of variability of flexural strength, weibull statistical analyses by means of two parameters were used: the weibull modulus (m) and the characteristic resistance (ó0). the phases of y-tzps crystalline structure have been identified by the detection of characteristic bands of each phase.( for 95% intervals; there was no statistical difference for the parameter m between groups but zmax showed the highest numerical values and thus a higher reliability of the results. zca was statistically significant different in the values of ó0. micro-raman spectroscopy showed that there was phase transformation (t � m) for all y-tzps studied, and that the band characteristic of monoclinic phase was identified near the origin of the fracture. zmax showed the best reliability results of flexural strength and the presence of the monoclinic crystalline phase in the area of origin of the fracture indicates that the region subjected to high stress induced phase transformation. 005 biomechanical evaluation of the use of mouthguard and mode of impact simulation: finite element analysis veríssimo c*, costa pvm, santos-filho pc, fernandes-neto aj, soares cj. federal university of uberlandia crisnicaw@hotmail.com the use of mouthguard can minimize the effects of dental trauma by impact energy absorption. this study evaluated the effect of the use of mouthguards on stress distribution in the tooth-bone complex by finite element method (fem). four twodimensional models were generated from images of a tooth-bone complex by the association with the software’s of image manipulation image j and finite element analysis, marc/mentat-msc software, in accordance with the factors under study: (1) presence or absence of mouthguard, (2) load application without antagonist and with the antagonist in occlusion. in the software marc/mentat, the manual meshing process of each structure was made and the boundary conditions were defined. nonlinear analysis was performed with simulated friction contact between the mouthguard and tooth. was applied a loading of 1000 n on the labial surface of the incisor. the analysis parameters were the von mises criterion and total displacement in the x-axis. the use of mouthguard significantly reduced the stress generated in the tooth-bone complex. the presence of the antagonist at the time of load application accentuated the stress generated in the tooth and alveolar bone. the analysis of the total displacement (x-axis) demonstrated that the use of the mouthguard decreases the displacement of the tooth in front of load application. it was concluded that the use of mouthguards decreased the levels of stress in the tooth-bone complex. the presence of the antagonist at the time of load application increased the stress levels on the tooth structure. 002 restoring the optical properties and color change in the enamel infiltrated with icon araujo gsa*, naufel fs, lima danl, alonso rcb, puppin-rontani rm piracicaba dental school unicamp giovanaaraujo@hotmail.com the aim of this study was to evaluate the effect of optical properties of infiltrant applied in enamel carious lesions before and after staining. 30 blocks (4x4mm) were produced from bovine teeth crowns. these were divided into three groups (n = 10): control, decayed and artificially infiltrated with icon. color measurements by reflectance spectrophotometry (konica minolta cm 700d, cielab system) were performed before and after coffee cycling pigmentation (14 days, 3 times a day for 15 minutes). the color parameters used were l, a, b and äe. the results were subjected to anova one-way for comparison between groups and test t for comparison before and after pigmentation (p<0,05). after pigmentation, results present a significant reduction in l values for all groups, and the control and infiltrant group showed no significant difference in l values (control: 88.7; infiltrant: 87.59). the a and b values decreased after staining for all groups. the color variation (äe) was higher in infiltrant groups (41.10) and decayed (33.19) did not differ among themselves. the control group showed the lowest äe values (13,12).( conclusion: infiltrant application restored luminosity of decayed teeth similarly to control before staining. however, after staining, the infiltrant group presented color change similar to decayed and higher than control group. 004 effect of photo-activation protocols for resin cements on shrinkage stresses valdivia adcm*, pereira rd, bicalho aa, tantbirojn d, versluis a, soares cj federal university of uberlandia dolocorreia@hotmail.com finite element analysis (fea) was used to evaluate how time elapsed between mixing and polymerization affects the elastic modulus (e) and residual shrinkage stresses (shr) for different resin cements as luting agent in incisors restored with fiber-glass posts (fgp). post-gel shrinkage of ru-relyx unicem (3m-espe), bcbiscem (bisco), ra-relyx arc (3m-espe) and pf-panavia f (kuraray), was measured by strain gauge test (n=10). the e of the resin cements at 9 root depths was measured using dynamic indentation test. the photo-activation protocols were: ilight-curing immediate; 3m-three minutes and 5m-five minutes (n=3). 2d fea models were created of an incisor restored with fgp to assess the shr along the interface of the resin cement/root dentin. stress results were evaluated by modified von mises criterion. post-gel shrinkage decreased up to 70% with increasing photo-polymerization delay. the 5 minutes delayed photo-activation protocol decreased the shr and improved the e of all resin cements. 006 influence of the use of mouthguards and impact angulation on the strain of anterior teeth costa pvm*, veríssimo c, santos-filho pc, fernandes-neto aj, soares cj federal university of uberlandia pvmcosta@hotmail.com the occurrence of dental trauma more frequently affects the jaw anterior teeth. customfitted mouthguards are devices that decrease the frequency of dental trauma by the impact absorption capability. it was developed a custom pendulum device based on charpy test with a 210g steel ball to perform the tests in dentistry. five bovine jaws were selected with standard ages and dimensions. custom-fitted eva mouthguards were made with a thickness of 6mm.the jaws were selected and fixed on the pedulum device and the impacts were perfomed with 90, 60 and 45º angulation, with and without the mouthguard. straingauges of 350ù were attached at palatal surface of the tooth that would receive the impact. the strain and impact absorption capability of the custom-fitted mouthguards was calculated and data was analyzed with anova and tukey test (á=0.05). without mouthguards, the increase in impact angulation increases significantly the calculated strain. the average values (sd) obtained without mouthguards to the angulation was: 90°: 2562,6±828,7; 60°: 1546,3±272,6 and 45°: 101,8±33,8. with mouthguards, the average values (sd) to the strain (µs) obtained to the angulation was: 90°: 45,2±13,4; 60°: 40,9±11,1 and 45°: 37,6±10,9. the impact absorption capability of the custom-fitted mouthguards was 98,2; 97,4 and 63,1% to the angles of 90, 60 and 45°, respectively. it was concluded that the use of mouthguards had direct influence on strain and impact absorption capability of mouthguards in situation of impact application by the different angles. gbmd 2013 – 49th meeting of the brazilian group of dental materials238238238238238 braz j oral sci. 12(3):237-274 007 bond strength of self-adhesive cements in bovine enamel rodrigues rf*, ramos cm, soares af, honório hm, borges afs, francisconi pas bauru dental school usp raphinhafarias@gmail.com the objective of this study was to evaluate the bond strength of self-adhesive cements relyx u100 and relyx u200 to enamel (e) in relation different surface treatments and comparing them with resin cement relyx arc. one hundred and twenty bovine incisors were selected; their crowns were separated from the roots and embedded with epoxy resin in pvc tubes. it was used silicon carbide sandpaper for planning enamel, then were delimited areas aimed at cementing with adhesive tape; the sample was divided into 6 groups (n = 10) according to the cement and realization of acid etching with phosphoric acid 37% (p) or not on the enamel surface: e-u100, e-p-u100, e-u200, e-p-u200, e-arc and e-p-arc. a split teflon mold allowed manufacturing of cement cylinders, which coincide with delimited areas. the shear strength test was performed in a universal testing machine emic (0.5 mm / min) after 7 days in artificial saliva. the analysis of the fracture was performed with digital microscope. with illustrative purposes, two specimens of each group were observed in scanning electron microscopy. data were analyzed with anova-2 criteria and tukey (5%). no difference was observed between self-adhesive cements. when it was done phosphoric acid etching, selfadhesive cements were better than the resin cement. 53.3% of the fractures were adhesive. it was concluded that the self-adhesive cement associated with prior acid etching of the surface can be used as an alternative to resin cement in enamel. 009 photophysics of rhodamine-labeled dentin bonding agents bim junior o*, cebim ma, silva acs, francisconi lf, atta mt, borges afs, wang l bauru dental school usp odair.bim@gmail.com adhesive systems have been modified with rhodamine b (rb) in the interest of assessing bonded interfaces via confocal laser scanning microscopy (clsm). this study aimed (i) to investigate some characteristics of excitation of fluorophores and fluorescence emission from non-simplified adhesives labeled with rb at different concentrations, (ii) to estimate the lowest ranges of concentrations of rb for clsm. the adhesive systems adper scotchbond multi-purpose and clearfil se bond were modified with rb by means of concentrated ethanol-rb aliquots. thus, five films of each adhesive were prepared directly on microscope slides by adding different concentrations of rb (mg/ml): c1=0.50, c2=0.10, c3=0.02; c4=0.004 or c5=0.0008. the fluorescence from films was evaluated both by fluorescence spectroscopy and by clsm. the effectiveness of some concentrations was confirmed by applying them on the assessment of dentin bonded interfaces. fluorescence spectra showed differences in the excitation and emission bands, according to the concentrations of rb in each film. the maximum excitation wavelengths were set in the green region (553-563 nm), and the emission ones between 565-587 nm. preliminary microscopic analysis of the films allowed the pre-selection of the concentrations c2, c3 and c4 for the assessment of dentin bonded interfaces, which were best visualized with c3. investigating the behavior of rb-labeled adhesives allows the predetermination of relevant parameters for the optimization of the clsm technique. 011 influence of the cements and ceramic translucency on the luminosity of restoration of darkened teeth araújo dfg*, sanfelice a, chaves lp, naufel fs, schmitt vl, ueda jk, nahsan fps, alencar ms, mondelli rfl, wang l bauru dental school usp diana_gadelha@hotmail.com this study aimed to evaluate the masking ability of darkened substrate with ceramic with different levels of translucency (ips e-max press) and different cements (a1). the null hypotheses tested were that there were no difference in the restoration’s luminosity according to the level of translucency (high-ht and low translucency-lt), level of teeth darkening (normal-n or darkened-d) and cement (relyx arc-arc, variolink iiv and biodinâmica cement-b). sixty circular fragments of bovine enamel were prepared, in which half was submitted to the darkening through the immersion in bovine blood. circular specimens of the ceramic material (60) were obtained, half with high translucency (ht) and half with low translucency (lt). about the combination with the cements, 12 groups (n=5) were determined. the luminosity of the luted specimens were measured with spectrophotometer to determine the variation of luminosity (äe). the data was analyzed by three-way anova and tukey tests (p<0.05). the factors ceramic translucency and level of darkening were significant, as well as the interaction between the three factors. for the cement arc, the high translucency and the darkened substrate (ht-d-arc) were different from the other combinations, with higher äe. for the cement v, the low translucency of the normal substrate (lt-n-v) determined the smallest äe. the cement b did not reveal any differences among the combinations. the luminosity of restoration depends on the combination between the level of ceramic translucency, level of substrate darkness and cement. 008 surface treatment of dental porcelain with co2 laser – contrast ratio and masking ability sgura r*, taddeo f, maciel ses, reis mc, serinhano ba, medeiros is faculdade de odontologia da usp risgura@hotmail.com aim: co2 laser continuous irradiation applied over porcelain specimens produces a surface similar to that achieved after oven glaze regarding roughness and color aspects. (sgura r. et al., dent mat 2011, e72-e73). nevertheless, the influence of co2 laser in porcelain opacity was never evaluated. this study evaluated the opacity of dental porcelain after co2 laser irradiation by means of contrast ratio (cr) and making ability (ma) tests. materials and methods: 80 porcelain discs (diam 3,5 x 2,0mm) were oven-sintered and had one of their faces mirror polished (ecomet 3 – buehler). specimens were divided into groups: control (c), oven autoglaze (g) and laser in 45 and 50 w/cm2 irradiances (l45, l50) for 3, 4 or 5 minutes. cr and ma were measured in spectrophotometer (cm3370d konica minolta) by measuring reflectance and color difference (?e) with specimens placed over black and white backgrounds. data were subjected to anova and a tukey post-hoc test (p<0.05). a correlation test between cr and ma were conducted. results: opacity in g was higher (cr=0.82) than in c (cr=0.73). cr of laser groups ranged from 0.78 to 0.88. l45/5 minutes presented the higher masking ability (?e=4.5) when compared to other groups. cr and ma showed a linear correlation r = -0.76 (p=0.02). conclusions: cr of irradiated porcelains varied according to irradiance and time. the ma of the studied porcelain was not altered after co2 laser exposition. 010 influence of different protocols of activation in degree of conversion and knoop microhardness of a dual resin cement mainardi mcaj*, giorgi mcc, ambrosano gmb, lima danl, marchi gm, aguiar fhb piracicaba dental school unicamp camoajm@hotmail.com the aim of this study was to evaluate the degree of conversion and microhardness of dual-cured resin cement used for cementation of fiber posts. forty-five roots of bovine incisors were used for this purpose. these roots were submitted to endodontic treatment and random assigned to 8 experimental groups and a control, all of them with n=5. after seven days, the fiber post white post dc #3 was cemented with the dual-cured resin cement, relyx arc (3m-espe) in each root. following this step, the light curing was performed with the 3rd generation led device (valo-ultradent), with four different energy densities (7, 14, 20 or 28 j/cm²) and two different delay times (0 or 2 minutes). the control group was not light-cured. after the cementation, the roots were stored in distilled water at 37º c for 15 days, and the degree of conversion an knoop microhardness were obtained in three different root segments (cervical, medium and apical). data were submitted to three-way split-spot anova and tukey´s test. dunnett’s test was used for comparisons with control. results of degree of conversion showed that the cervical root segment obtained higher values than the others, at all experimental conditions. the degrees of conversion values for 2-minutes delay time were higher than 0-minute delay-time. the energy density of 28 j/cm² obtained the highest values, with significant differences from the others energy densities. for microhardness values there were not significant differences between the delay times. for the cervical and apical root segments, there were not statistical differences for the energy densities tested. the energy density of 14 j/cm² showed the higher values of microhardness at the medium root segment, while the 28 j/cm², showed the lowest. the apical root segment obtained similar microhardness values from the same third at the control. in conclusion, the degree of conversion was influenced by the energy densities and delay times, as well as the root segment. the microhadness was not affected by the delay time at all conditions, and the energy densities showed similar behavior at cervical and apical root segments. 012 push-out bond strength between glass fiber posts to intraradicular dentin with different luting agents suzuki tyu*, gomes-filho je, gallego j, briso alf, assunção wg, dos santos ph faculdade de odontologia de araçatuba – unesp tha.suzuki@gmail.com the aim of this study was to evaluate the bond strength between glass fiber posts to different regions of intraradicular dentin. forty extracted single-rooted human teeth were used in this study. after endodontic filling and mechanical preparation of root canals, teeth were divided into five groups (n=8) according to the technique adopted for the adhesive cementation: g1: single bond 2 + relyx arc; g2: excite dsc + relyx arc; g3: adper se plus + relyx arc; g4: relyx unicem; g5: set. the bond strength was measured using the push-out test in a universal testing machine in different areas of intraradicular dentin (cervical, middle, and apical). the data were subjected to 2-way analysis of variance and fisher’s plsd test. images of representative fractured sample were obtained by scanning electron microscopy. the highest values for the push-out bond strength were found for g3 in all experimental conditions, without a significant difference for the excite dsc in the middle and apical regions. the lowest values of bond strength were found for the other groups, with no statistical difference between them. there was a decrease in push-out bond strength in the cervico-apical direction for all groups except group 2, which did not show a difference among the different regions. the different interactions of the resin materials and the intraradicular depth influenced the bond strength of adhesives materials to dentin substrate. gbmd 2013 – 49th meeting of the brazilian group of dental materials 239239239239239 braz j oral sci. 12(3):237-274 013 effect of surface treatments on the flexural strength of ceramics in lithium disilicate pressed sato ts*, cotes c, yamamoto lt, kimpara et universidade estadual paulista – instituto de ciência e tecnologia de são josé dos campos tatapsique@gmail.com aimed to evaluate in this study the resistance of a ceramic lithium disilicate pressed, when subjected to different surface treatments and cementation. were made 60 bars (16x2x4mm), divided into subgroups consisting of 10 bars each, and subjected to the following surface treatments: c, no treatment (control); hf, application of 10% hydrofluoric acid (hf) for 20s + wash (60s), drying (30 s) application of silane agent and hold (60s), hfc, hf (20s) + wash (60s), drying (30s), application of silane agent and hold (60s) and cementation; hfn, hf (20s) + wash (60s) + neutralization with supersaturated solution of sodium bicarbonate (sb) + rinsing for 40s (5s) + drying (30s), application of silane agent and wait (60 seconds) and cementation; hfu, hf (20s ) + wash (60s) + ultrasonic cleaning in deionized water (u.s.) for 4 min + drying (30s), application of silane agent and hold (60s) and cementation; hfnu, hf (20s) + wash (60s) + neutralizing with bs (40s) + cleaning in u.s. (4 min), application of silane agent and hold (60s) and cementation. the specimens were tested for flexural strength of three points. the results showed that the hfn and hfnu groups showed lower flexural strength than the other groups (p = 0.00).thus it was concluded that the neutralization with supersaturated solution of sodium bicarbonate, followed or not by ultrasonic bath results in lower mechanical strength of the ceramic lithium disilicate pressed. 015 effect of surface treatments on the flexural strength in a lithium disilicate ceramic rossi nr*, cotes c, lima lc, macedo vc, kimpara et universidade estadual paulista – instituto de ciência e tecnologia de são josé dos campos ntlrossi@gmail.com the etching creates subproducts and precipitates on the ceramic surface, blocking the microporosity of ceramic, which difficults the penetration of the resin luting agent. thus, it is necessary to dispose of these wastes, to promote improved luting, and thus obtain a greater longevity of the restoration. therefore, the aim of this study was to evaluate the effect of different surface treatments on the flexural strength of lithium disilicate ceramics processed by cad / cam. 60 ceramic bars were prepared and divided into 6 groups (n = 10): a, no treatment (control), hf, application of 5% hydrofluoric acid (hf) for 20s, washing (40 seconds), drying (30s); hfc, hf (20s), washing (40s), drying (30s), silanization and cementation; hfu, hf (20s), washing (40s), ultrasonic cleaning in distilled water for 4 min (us), drying (30s) silanization and cementation; hfn, hf (20s), washing (40 seconds), neutralization with supersaturated solution of sodium bicarbonate (40s), washing (5s), drying (30s), silanization, cementing; and hfnu, hf (20s), washing (40s), neutralization with supersaturated solution of sodium bicarbonate (40s), us, silanization and cementation. after 24 hours, the bars were subjected to three-point bending test. there was no significant difference between the means of the groups according to the one-factor anova (p= 0.315). it was concluded that surface treatments did not influence the mechanical strength of lithium disilicate ceramic. 017 construction and validation of device for humidity and temperature control in measurement of post-gel shrinkage bicalho aa*, ruggiero jg, sousa sjb, barreto bcf, versluis a, soares cj federal university of uberlandia alinearedesbicalho@gmail.com the post gel shrinkage (shr) of composites causes strain and stress in the complex restorative and may be influenced by heat and temperature environmental. the aim of this study was to develop a device for measuring of shr that allows control of relative humidity (rh) and temperature environment (°c) and to validate testing six composites in three environments conditions of polymerization. the device is characterized by glass device with internal electrical resistance connected to a control panel. the internal temperature parameter is achieved with a sensor positioned near the test sample. the rh is controlled by humidification system with internal variation limit ± 1 ° c and ± 5% rh. for validation of the device were tested the composites filtek ls (ls), venus diamond (vd), aelite ls (ae), evolu-x (ex), filtek supreme (su) and filtek z100 (z) in three conditions (22 º c and 50% rh (22/50), 37 º c and 50% rh (37/50), 37 º c and 90% humidity (37/90). each sample was polymerized on the bi-directional gage with halogen light for 20s. shapirowilk test, two-way anova and tukey test were applied (p <0.05). a shr was directly proportional with the increase in rh and temperature for all materials tested. during the three environmental conditions, the materials presented shr ls. 014 influence of photoactivation time on water sorption and solubility of dental adhesives ruy lgm*, pomacóndor-hernández c, feitosa vp, sinhoreti mac, consani s piracicaba dental school unicamp lucasruy@terra.com.br the aim of this study was to evaluate the influence of different photoactivation times on water sorption (ws) and solubility (so) of dental adhesives. two singlebottle etch-and-rinse adhesives were used: single bond 2 (sb 3m espe) and ambar (am fgm). each adhesive was dispensed in silicon molds for confection of disc-shaped specimens (6.00 mm x 1.00 mm). specimens were divided in 4 groups according to different photoactivation times: g1: 5s (sub-photoactivation), g2: 10s (recommended), g3: 30s, e g4: 60s (n = 6). light-curing unit used was a led bluephase 2 (ivoclar vivadent) with 1050 mw/cm2 irradiance. ws and so were obtained after 7, 30, 60 and 90 days of storage in 1 ml of distilled water at 37°c. ws and so values were analyzed using two-way repeated measures anova and student-newman-keuls test (á = 0.05). after 90 days of storage, the prolonged photoactivation times resulted in higher ws for sb (g2: 159.7 µg/mm3 < g4: 204.6 µg/mm3) and am (g2: 95.9 µg/mm3 < g4: 121.3 µg/mm3). the influence of photoactivation time on so was material-dependent (sb g2: 82.1 µg/mm3 = g4: 85.9 µg/mm3; and am g2: 112.4 µg/mm3 > g4: 76.9 µg/mm3). both adhesives presented reduction in ws and increase in so when storage time was prolonged. it can be concluded that prolonged photoactivation times in dental adhesives produce increased ws and may reduce the so. prolonged storage times for etch-and-rinse adhesives reduce ws and increase so. 016 evaluation of tooth bleaching in enamel and opposite dentin after prior application of potassium nitrate públio jc*, d´arce mbf, ambrosano gmb, aguiar fhb, lovadino jr, paulillo lams, lima, danl piracicaba dental school unicamp jupublio@hotmail.com this study evaluated the efficacy of bleaching on enamel and opposite dentin surfaces using 35% hydrogen peroxide (hp) with prior application of a desensitizing agent (5% potassium nitrate with 2% sodium fluoride). bovine dental fragments, with thicknesses of 1.0 mm enamel and 1.75 mm dentin, were stained in a solution of black tea. the fragments were randomly divided into three groups (n=10) according to the following protocols: nf: 2% neutral fluoride (4min) + bleaching (45min), d: desensitizing agent (10min) + bleaching (45min), and wd: without desensitizer + bleaching(45min). the bleaching efficacy was evaluated at four times: after staining with tea (baseline) and after each of the 3 weeks of bleaching, by means of the cie lab method using a reflectance spectrophotometer. the data coordinate l* was evaluated by analysis of repeated-measures with proc mixed and tukey-kramer’s test. the äe values were subjected to analysis of variance anova and tukey’s test (á = 0.05). two samples from each group were randomly selected for analysis of enamel surfaces in scanning electron microscopy at 2000x. we analyzed the sample surface only with the application of 2% neutral fluoride and desensitizing. the 35% hp treatment showed greater efficacy on deep dentin after removal of enamel stains, with increasing means during all times in all treatments. the use of a desensitizing agent prior to the bleaching session did not affect the mechanism of action of 35% hydrogen peroxide with regard to tooth depth. more apparent topographic changes on the enamel surface were observed when using the ph35%. 018 effect of protocol restorative composite resin on the deformation of cusp molars ferreira ms*, bicalho aa, pereira ras, ruggiero jg, versluis a, soares cj federal university of uberlandia marianasantos2604@gmail.com the post gel shrinkage and filling technique of composite resins in posterior teeth may cause strain in teeth that can to manifest as broken enamel and post operative sensitive. the use of glass ionomer has been suggested to minimize this effect. the aim of this study was to evaluate the cusp strain (cs) of molars with large structural loss restored with composite resin (filtek supreme-3m espe) according to 2 factors under study: factor 1: presence (ion) or absence (nion) of glass ionomer (vitremer-3m espe); factor 2: filling technique starting from the occlusal box (co) filling technique starting from the proximal box (cp). twenty-eight human molars were divided into 4 groups (n = 7) with standardized mod cavities that were restored. cs was obtained with strain gages fixed on the basis of the buccal (b) and lingual (l) cusps. the tests were performed with temperature (37 ° c) and humidity (50%) controlled. the cs data were analyzed using factorial anova (2x2) with subplots factor cusp (b, l and buccal, lingual) and tukey test (p <0,05). the values of dc (ms) were ionco-v: 50.8 ± 14.5; g: 72.1 ± 21.8; ioncp-v: 60.9 ± 16.6; g: 65.6 ± 20 , 6; nionco-v: 65.0 ± 16.6, l: 101.9 ± 13.2; nioncp-v: 74.6 ± 17.0, l: 105.4 ± 25.6. the cs was significantly higher for the lingual cusp in all groups. the presence of ion results in lower co values, regardless of the restorative technique. the restorative technique did not influence cs regardless of the presence of ion. gbmd 2013 – 49th meeting of the brazilian group of dental materials240240240240240 braz j oral sci. 12(3):237-274 019 initiator system changes may affect the properties of resin cements brandt wc*, silveira lf, souza-junior ej, boaro lc, sendyk wr, kim yj, sinhoreti mac universidade de santo amaro – unisa williamcbrandt@yahoo.com.br the study examined the flexural strength (fs), flexural modulus (fm) and knoop hardness (kh) of experimental resin cements containing different photoinitiator systems. resin cements containing bisgma, tegdma and 65wt% of silanized filler particles were prepared with the use of photoinitiators: cq-camphorquinone, ppd-phenyl propanedione, bapobisacylphosphine oxide, dmaemadimethylaminoethyl and dfi-diphenyliodonium hexafluorphosphate. the combinations used were: g1-cq/dmaema, g2-cq/dmaema/dpi, g3-ppd/ dmaema, g4-ppd/dmaema/dpi, g5-bapo/dmaema and g6-bapo/dmaema/ dpi. the photoactivation was made during 20 s with a ceramic (ips emax a2 color, thickness 1 mm) between the led light curing (bluephase) and resin cement. fs and fm were tested in a universal testing machine (instron) and kh in microhardness (shimadzu). according to the results, the values of fs (mpa) were: g5 (102.0)a; g2 (101.4)a; g6 (98.8)a; g1 (81.5)a; g4 (75.5)a and g3 (0,0)b. in the fm test (gpa) values obtained were: g2 (2.9)a; g6 (2.7)a; g5 (2.6)a; g1 (1.4)b; g4 (1.3)b and g3 (0,0)c. for dk (khn) the values were: g6 (66.1)a; g5 (65.7)a; g2 (21.0)b; g4 (11.5)c; g1 (11.3)c; g3 (4.8)d. the resin cement containing the photoinitiator bapo showed the highest values in the properties tested and the combination of the dfi with all photoinitiators increased the properties of resin cements. 021 bond strength to dentin of adhesive systems irradiated with diode laser maenosono rm*, bim júnior o, cardia gs, wang l, palma-dibb rg, ishikiriama sk school of dentistry of bauru usp rafamaenosono@usp.br the aim of this study was to evaluate the effect of diode laser irradiation (ë = 970nm) on adhesive systems already applied to dentin, irradiated before light curing, on bond strength obtained by microtensile test. this study presented experimental design with two factors (adhesive system / laser) each divided into two subgroups, respectively: adper™ singlebond 2 (sb); adper™ easyone (eo) / no laser (nl); with laser (l). forty healthy human molars were randomly distributed in 4 groups (n=10) and prepared in accordance with the respective treatment for microtensile bond strength test. the test was carried out in a universal testing machine (instron 3342), with a 500n load cell, obtaining values of bond strength in mpa, analyzed through two-way anova, followed by tukey test for individual comparisons (p<0.05). mean values of bond strength (mpa) ± standard deviation were respectively: sb-nl 33.49 ± 6.77; sb-l 43.69 ± 8.15; eo-nl 19.67 ± 5.86; eo-l 29.87 ± 6.98. both factors analyzed were able to influence on bond strength values, with significant increase in bond strength of both adhesive systems irradiated with diode laser. the association of diode laser to simplified adhesive systems seems to be promising technic to achieve a more effective adhesion between dentin and adhesive systems. 023 bond strength between conventional resin cement and self-adhesive to dental ceramic godas agl*, couto ems, suzuki tyu, briso alf, assunção wg, dos santos ph faculdade de odontologia de araçatuba – unesp andrelgodas@gmail.com the aim of this study was to evaluate the microshear bond strength between conventional resin cement and self-adhesive to dental ceramic. twenty starlight ceramic discs were made with 15 mm in diameter and 2 mm in thickness. the bonding process of resin cements to ceramic was made by insertion of material into tubes of 0.7 mm internal diameter. four resin cements were used: relyx arc, panavia f, relyx unicem e set. the samples were stored for 24 hours or 6 months in distilled water at 37ºc. the samples were submitted to microshear bond strength in a universal testing machine at a crosshead speed of 1.0 mm/min. data was subjected to 2-way anova and a fisher’s plsd test. relyx arc conventional resin cement showed the highest bond strength with no statistical difference for relyx unicem and set at 24 hours. the lowest values were obtained for the panavia f, with statistically significant difference for the other cements in the two periods studied. at six months there was a significant increase in the values of bond strength for all cements, except for relyx arc. the bond strength is dependent on the type of resin cement, and self-adhesive resin cements behaved similarly to conventional resin cement. 020 influence of different concentration of photoinitiator system on the properties of experimental resin composites grohmann cvs*, souza-junior e, sinhoreti mac, puppin-rontani rm, ferracane jl, machado-santos l, brand wc piracicaba dental school unicamp caiov@hotmail.com the aim of this study was to determine the influence of the concentration of camphorquinone (cq) and tertiary amine (dabe) on properties of experimental composites (ec). we tested the degree of conversion (dc), yellowing (yl), elastic modulus (em), flexural strength (fs), sorption (sp) and solubility (sl). ten ec were handled, varying concentrations of cq/dabe (% by weight): 0.4/0.4, 0.4/0.8, 0.6/0.6, 0.6/1.2, 0.8/0.8, 0.8/1.6; 1/1, 1/2, 1.5/1.5 to 1.5/3. for dc, 8 specimens of each material were prepared. after 24 hours, were analyzed by fourier transform infrared spectroscopy. subsequently, for fs and em, the same samples were subjected to bending test in an universal testing machine. for testing yl, sp and sl, five samples were prepared for each material for each test. the yl was measured after 24 hours of storage in water, using a spectrophotometer. for testing sp and sl, the samples were dissected for 1 week and weighed daily until the maximum variation of weight was 0.0001 g of an overnight (m1). for m2, the samples were stored in water for 1 week and weighed one time only. and m3 were obtained in the same way as m1. the different concentrations of cq and dabe did not significantly alter the properties of fs and sp. the results of dc, yl and em were directly proportional to the concentration of cq/dabe. already sl was inversely proportional.( high concentrations of cq/dabe lead to the best properties, but the larger yl. the increases in the proportion of dabe not influence the properties of all ec. 022 influence of different surface treatments on bond strength in compositecomposite repairs almeida sa*, zawadzki v, pereira kf, poskus ltuff (universidade federal fluminense) sarahaquino@gmail.com this study evaluated the influence of different surface treatments on the bond strength (tbs) in composite-composite repairs (3m espe). 8 cylinders of each composite (z350/z3; z250/z2; p90/ p) were obtained using a metal matrix (10mm x 5mm). after thermocycling (1000 cycles at 5°c/55°c) and roughening the surface (320 grit sandpaper), the cylinders were distributed according to the following surface treatments: cwithout treatment (control); ssandblasting with al2o3 (50ìm), se-sandblasting with al2o3 (50ìm) + ethanol for 5 min, sc-silica coating. after silanization, the adhesive single bond 2 and silorane were applied to methacrylic and p90 composites, respectively. the cylinders were placed in a matrix with 12mm in height, which was filled with a new composite, similar to the old one. after thermo-mechanical cycling (1000 cycles at 5°c/37°c/55°c and 300 000 cycles at 2.5 hz and 98n), the cylinders were cut into beams (1mmx1mm) (n = 28) and subjected to the microtensile test in a universal testing machine with speed of 0.5 mm/min. data were submitted to anova and tukey’s test (5%), verifying that sandblasting and silica coating led to better values of tbs for the z2, while for z3, only silica coating was effective. for p, all treatments led to similar values of tbs. z2je showed lower values of tbs compared to others. it can be concluded that sandblasting and silica coating can be indicated as a pretreatment in composite repairs. ethanol after sandblasting should not be used because it is possible to damage the adhesive interface. 024 synthezis and characterization of composites with polymeric matrices modified by polyhedral oligomeric silsesquioxane bailly h*, netto lrc, andrade jss, ton rs, silva em universidade federal fluminense labiom-r helobailly@yahoo.com.br the aim of this work was to produce and characterize experimental composites with polymeric matrices (udma / tegdma 70/30 w/w%) modified by polyhedral oligomeric silsesquioxane (poss). six experimental composites were produced by partial replacement of udma by poss (w/w%): c poss 0%; p2 poss 2%; p5 poss 5%; p10 poss 10%; p25 poss 25% and p50 poss 50%. the photoinitiation system was composed by camphorquinone and edmab. the composites had 70% of barium borosilicate glass particles of 0.7 ìm. the follow properties were evaluated: conversion degree, flexural strength, flexural modulus, hardness, and crosslink density. the micromorphology was evaluated by sem. the obtained data were analyzed by analysis of variance and tukey hsd test. conversion degree: the lower result was presented by p50 and c, p2, p5 and p25 showed the highest values without statistical difference among them. flexural strength: the lowest value was presented by p50 and the highest by p2. it was not found statistical differences in flexural modulus among all composites (p>0.05). hardness: p50 showed the highest result and c, p2 and p5 presented the lowest. crosslink density: p25 and p5 showed the best statistical results while group c had the worst. it was concluded that the introduction of poss until the limit of 25% showed the overall best results. gbmd 2013 – 49th meeting of the brazilian group of dental materials 241241241241241 braz j oral sci. 12(3):237-274 025 chemical bonding to calcium/dentin and physicochemical properties of mdp comonomers with different chemical structures feitosa vp, sauro s, ogliari fa, watson tf, ogliari ao, correr-sobrinho l, sinhoreti mac, correr ab piracicaba dental school unicamp victorpfeitosa@hotmail.com the aim of this study was to evaluate the chemical bonding of functional monomers and their effects on the properties of all-in-one adhesives. we synthesized four comonomers of mdp (10-metacryloyloxi-decyl-dihidrogen-phosphate) with different hydrophilicities and length of the spacer carbon chain. the chemical interaction of the monomers with calcium/dentin was assessed by atomic absorption spectroscopy (aas) and ftir. the monomers were mixed in similar molar ratio in an adhesive blend to prepare the one-step self-etch adhesives. the ultimate tensile strength (uts) and degree of conversion of such adhesives were evaluated. the data was analyzed with anova and tukey’s test (p<0.05). aas showed better chemical bonding for monomers with long and hydrophobic spacer chains. the monomer with short spacer chain (mep, metacryloyloxi-ethyl-dihydrogenphosphate) achieved the worst chemical interaction. ftir showed that only the joint mep-dentin was not stable after water rinsing. the monomers with hydrophilic chains remained attached to the dentin. the uts was lower with mep (8,42±0,66 mpa) and metacryloyloxi-caprolactone-dihydrogen-phosphate (8,09±0,8 mpa) with intermediary hydrophilic spacer chain. all monomers attained similar degree of conversion. the results showed that the length of spacer carbon chain has more influence than the hydrophobicity on the chemical bonding of phosphate acidic monomers. monomers with shorter spacer chain induce lower uts. 027 in situ evaluation of surface roughness and microhardness of restorative materials submited to erosive challenges sahyon hbs*, guedes apa, suzuki tyu, sundfeld rh, briso alf, dos santos ph universidade estadual paulista “júlio de mesquita filho” unesp araçatubaico_strazzi@hotmail.com the aim of this study in situ was to evaluate the effect of different acidic solutions present in soft drinks and gastric juice in the microhardness and surface roughness of restorative materials.168 specimens were randomly divided into 3 groups with 8 volunteers. the volunteers wore palatal device with 7 specimens (ketac nano, ketac nano + biscover lv, esthet-x, esthet-x + biscover lv, supreme xt, supreme xt + biscover lv and bovine enamel). the group i was immersed in hcl; the group ii was immersed in soft drink and the group iii was subjected to saliva only. the microhardness and surface roughness data were analyzed by two-way anova and fisher’s plsd test (á = 0.05). the results were, supreme xt showed the highest values of microhardness before and after aging with a significant difference for the other materials. the lowest values were found for the materials sealed with biscover lv in all periods analyzed. the ketac nano glass-ionomer cement showed the highest values of surface roughness after exposure in acidic solutions. the application of the sealant did not reduce the roughness values for the composites studied except for esthet-x after the challenge in hydrochloric acid. the conclusion is the acidic solutions promoted changes in microhardness and surface roughness of restorative materials, except for the sealed materials. 029 bond strength of self-etching and self-adhesive resin cements to enamel and dentin lacerda rfs*, shinohara ms, de goes mf piracicaba dental school unicamp renatafernandeslacerda@hotmail.com it was evaluated the bond strength (bs) of resin cements to dentin and enamel surface. it was used 36 third molars. half of these had the oclusal face removed to exposed dentine and the other half was obtained hemi-sections of enamel. blocks of indirect composite resin were bonded to the surface of the substrates. the sets were divided into 4 groups: u2-relyx unicem 2 and max-maxcem elite (self-adhesive), u2s-relyx unicem 2 associated with the adhesive system scothbond universal and mu-multilink automix (self-etching). they were then stored in distilled water at 37°c for 24h, sectioned in shaped sticks with 0.8 ± 0.2mm2 and tensiled under speed 1mm/min (ez test shimadzu). the fractures were classified as scanning electron microscopy (sem) and data were submitted to anova and tukey test (p <0,05). u2 (13 ± 4.2 and 14.3 ± 6.8) had significantly higher value compared to max (12.2 ± 5.9 and 5.4 ± 3.1) when evaluated on dentin. and u2s (29.7 ± 12.1 and 50.6 ± 18.2) showed a statistically greater value than the presented by mu (20.5 ± 10.4 and 20.7 ± 10.5) to enamel and dentin. the fracture patterns in sem for both substrates showed predominance of cohesive cement fractures for most groups, except for group max in dentin, which showed a predominance of mixed fracture (cohesive cement and smear layer) and mu in enamel with predominance of fractures mixed (adhesive and cohesive cement). the self-etching cements had higher bs regarding self-adhesive and among these, u2 showed higher values. 026 internal and marginal misfit of crowns produced with a new resin composite processed by cad-cam silva lh*, arashiro ll, villaça mchca, lima e, maeda f, cesar pf faculdade de odontologia da university of sao paulo cdhian@gmail.com the objective of this study was to evaluate and compare the internal and marginal misfit of crowns machined by cad-cam of a new nanofilled resin composite reinforced with nano ceramic particles (lava ™ ultimate lu) with a lithium disilicate glass-ceramic (e.max cad ec). dentin analogous (g10) models were manufactured with crowns preparation and replicated in dental stone. these were digitized by the cad-cam for virtual 3d modeling of the crowns for the two materials (n = 5). after machining, the crowns were placed in their respectively g10 models interposed by a light body addition silicone layer, copying the cementation space. the polymerized light body silicone film was captured with a silicon putty to obtain 4 slices corresponding to the crown faces: buccal, mesial, lingual and distal. the misfit was measured at five points: pulp, axial-pulp angle, axial, axial-gingival angle and marginal. the data were submitted to 3-way anova (2 factors within groups) and tukey’s test (α = 0.05). lu (198.1 ± 102.6 µm) showed a lower misfit (p = 0.042) than the ec (287.2 ± 102.6 µm). the lowest misfit was observed in the axial point for both materials (p = 0.000). no difference was observed (p = 0.201) among the crowns faces. lu showed lower misfit (p = 0.001) in pulp point (lu: 318.8 ± 82.6 µm / ec: 450.2 ± 104.3 µm) and marginal point (lu: 221.5 ± 67.1µm / ec: 321.8 ± 136.4 µm) than the glass-ceramic. crowns milled from the new resin composite had a better fit than the glass-ceramic ones, indicating that there is an important difference between the machining ability of these materials. 028 evaluation of dimensional changes in alginates mixed by standard and dense technique alencar ms*, bombonatti jfs, bombonatti r, mondelli rfl, araújo dfg, soares af, bombonatti pe bauru dental school usp marinastudart@hotmail.com in orthodontics, it is a frequent practice using alginate with a heavier consistency in order to provide better molding of hard and soft tissues. this study aimed to evaluate the dimensional changes of three alginates (jeltrate-dentsply; avageldentsply-herpo; orthoprint-zhermack) mixed in two different ways: according to the manufacturer´s instructions (standard technique) and with 33% reduction of water (dense technique). a stainless steel testing device with two micro-spherical indentations on surface was molded with the different alginates and techniques (n=5). the dimensional changes were measured with a karl zeiss microscope with an accuracy of 5 µm, immediately and after 15, 30, 45 and 60 minutes from mixing. data were subjected to two-way anova and then tukey test was applied (p <0,05). there was no significant difference between the types of alginate, but the different manipulation techniques had significant differences in all periods. the dense technique presented higher dimensional changes compared to standard technique. 030 clinical evaluation of in-office bleaching, with and without hybrid light source activation: 36 month follow-up trentino ac*, cardia gs, soares af, mondelli rfl bauru dental school usp anatrentino@usp.br this in vivo study compared two techniques for whitening vital teeth, the degree of color change and the stability of treatment over a period of thirty six months. twenty patients were selected and subjected to in-office bleaching, and ten patients received 2 whitening protocols. g1a left side: 3x 15' applications (total 45') of 35% hydrogen peroxide gel (hp) (lase peroxide sensy, dmc equipment ltd.); g1b right side: 3x35% hp gel with hybrid light activation (led/laser diode, whitening lase ii, dmc equipments ltd.) for 3x2', with an interval of 30" (7’30"), was totaling 22’30". ten other patients received another two bleaching procedures: g2a left side: 3x15' applications (45') of 35% hp (whiteness hp maxx, fgm); g2b right side: 3x25% hp gel applications with hybrid light activation for 3x2', with an interval of 30' (7’30"), totaling 22’30". the color evaluation was performed with the vita easyshade (vita) spectrophotometer after 24 hours, 1, 12 and 36 months. two-way anova testing revealed no statistically significant differences between the groups (p<0.05). the tukey’s test (p<0.05) for individual comparisons showed significant differences between the groups g1a (f=18.28) and g2a (f=7.22). the g1b group showed lower color variation (“e) at the times studied when compared to the other groups. the group that received a 35% lase peroxide sensy activated with hybrid light source presented the same whitening effect but in half the time and more color stability over 36 months. gbmd 2013 – 49th meeting of the brazilian group of dental materials242242242242242 braz j oral sci. 12(3):237-274 031 comparative analysis in relation to color stability, sorption and solubility of different restorative materials rodrigues r*, felizardo kr, sinhoreti macpiracicaba dental school unicamp renatogd@gmail.com color stability (cs), sorption (so) and solubility (sol) of the materials natural look (nl), herculite classic (hc), filtek z250 (z250), natural flow (nf), tetric flow (tf), filtek z350 flow (z350f), vitro fill (vf), vitremer (v), gc gold 1 (gc1), gc gold 2 (gc2), gc gold 9 (gc9), vitro molar (vm), vitro cem (vc), ketac molar (km), ketac cem (kc) e ionofil plus (ip) were analyzed. forty five (45) samples (n=5) for cs and ninety (90) for so and sol were made. for the so and sol tests, iso 4049 specifications were followed. the samples for the accelerated aging test (aa) were exposed to uv radiation under condensation temperature (50ºc), triggered at different cycles (4h of light emission and 4h of condensation) in a total of 36 cycles (=348h). the cs was verified under spectrophotometer using cie l*a*b* software. the results were analyzed by anova and tukey (p<0,05). according to so results, the vc (258,0) and vf (138,8) presented higher values under distilled water (353,0) (175,6). when comparing the environments, only km achieved statistic difference. there were no statistic differences for sol under acidic environment. under distilled water, vc (226,30) was statistically different from gc1 (-51,40) and gc2 (-51,40). material z350f (10,47) presented the higher cs value, demonstrating statistic difference compared to the other materials. conclusion is that for so, materials vc and vf presented higher values, different to km which had the highest value under distilled water. for sol, only vc presented difference under distilled water. z350f showed the highest color alteration. 033 effect of laboratory test conditions on the behavior and properties of the composites pereira ras*, bicalho aa, sousa sjb, valdivia adcm, barreto bcf, versluis a, soares cj federal university of uberlandiarenatjval afonso@hotmail.com environmental conditions can alter the behavior and properties of composites.this study investigated the effect of heat and humidity in the pós-gel contraction (shr), deformation of cusp (dc) and composite resins shrinkage stresses. shr and dc were obtained with measurement equipment of contraction and extensometry. heat and humidity were simulated through control adapted to bellflower. six groups (n10) were divided according to ambient conditions 3 (22ºc and humidity 502250, 37° c and humidity 50-3750, 37° c and humidity 90-3790) and 2 types of resins (charisma diamond-cd and direct-ips empress). to obtain the shr, 4mm³ specimens were polymerized with halogen light on bi-directional extensometer for 20s. ten molars in each group with mod cavities were restored and dc obtained by extensometry. five were held in knoop indents depths of -4.5 0.5 mm from the cavity. the hardness data were used to calculate the modulus of elasticity (e). shr and values and were used in finite element models (mef). two-way anova and tukey test (p) were used.( this paper concludes that heat and humidity interfere in shr values obtained by mechanical testing, the dc and the stress distributions. 035 effect of indirect restorative material on the transmittance of light emitted by curing units pacheco rr*, di francescantonio m, oliveira mt, romanini jc, giannini m, rueggeberg fa piracicaba dental school unicamp pachecu@hotmail.com this study evaluated the influence of different types of restorative indirect materials (zirconium oxide and lithium disilicate) in transmittance of light emitted by different types of curing units. discs with 1 mm thickness and 10 mm diameter were prepared from the indirect restorative materials: ips e.max zircad (ivoclar vivadent) and ips e.max (ivoclar vivadent). a nanofluorapatite ceramic (ips e.max, ivoclar vivadent) was applied over the indirect materials with 1 mm thickness. five different light curing units were tested: arc light ii (air technologies), coltolux led (coltène whaledent), elipar free light 2 (3m espe), astralis 10 (ivoclar vivadent) and ultralume 5 (ultradent), which was used in regular mode, only light blue and only violet. the irradiance through different restorative materials was measured using a spectroradiometer (das 2100, labsphere inc.) associated with a specific software (spectra suite v5.1, ocean optics inc.). data were analyzed by two-way analysis of variance and tukey test (p<0,05). the plasma arc curing unit (arc light ii) showed the highest irradiance values through different restorative materials, while the light source ultralume 5, violet mode, showed the lowest irradiance values through diferente restorative materials. the zirconia allowed higher light intensity passed through it when compared to lithium disilicate. the indirect restorative materials tested showed distinct properties regarding the transmittance of light and the type of curing unit influenced the results significantly. 032 a simple method for improved bonding to zirconia oliveira-ogliari a*, collares fm, ogliari fa, moraes rr ufpel alineoliveiraogliari@gmail.com the aim of this study was to develop a simplified method for bonding to yttriastabilized zirconia ceramic. the method was based on the deposition of a silica reactive layer on the ceramic surface using si and zr alkoxide precursors followed by heat treatment for condensation of a siox layer. the study had six steps: (i) preparation of solutions using four concentrations of tetraethyl orthosilicate (teos) and zirconium tert-butoxide (ztb) diluted in hexane; (ii) preparing the zirconia substrate; (iii) application of the silica based treatments before (infiltration, inf) or after (coating, coa) full zirconia sintering; (iv) analysis by scanning electron microscopy and energy dispersive spectroscopy (sem-eds); (v) µ-raman confocal spectroscopy analysis; and (vi) shear bond strength of resin cement to zirconia after storage for 24h, 3 months, and 6 months. an untreated (control) and a commercial reference groups were tested (rocatec plus, 3m espe). quantitative data were analyzed using two-way anova and tukey’s post-hoc test (5%). sem micrographs showed that the zirconia surface was covered by nanoparticle clusters, while eds and µ-raman analyses confirmed that this layer was composed by silica. the bond strength results showed that most groups that received coa and inf silica-coating treatments presented a higher bonding potential than the control group. almost all experimental groups had results similar to the commercial reference. mixed failures were predominant. the storage time had no influence on the zirconia bonds. 034 evaluating of capacity of masking and opacity of ceramic systems according to different resin cements rodrigues rb*, roscoe mg, lima e, simamoto júnior pc, silveira júnior cd, césar pf, soares cj, novais vr federal university of uberlandia renataborges4@hotmail.com this study evaluated the capacity of masking (cm) and opacity (op) of two ceramics (feldspathic and lithium disilicate-reinforced), cemented with different resin cements, by calculating the color difference (äe) in the measurement of the coordinates l * a * b * reflectance on white and black background. were made 15 sample of each ceramic discs with 10 mm diameter and 1.5 mm thick that were divided into 3 subgroups according to the type of cement (n=5): ra, relyx arc, rv, variolink ii, ru, relyx u200. the color analysis was done on a spectrophotometer. two-way anova showed that for values of cm factor cement was statistically significant (p<0.002). there was no significance for the factor ceramic (p=0.737) and the interaction between the two factors (p=0.758). for op, it showed significant for the type of cement (p<0.004). however, ceramic type (p=0.555) and the interaction between the two factors (p=0.893) were not significant. tukey test (á=0.05) showed that values of cm and op showed significant differences between cements ra and ru, and ru had higher cm and lower op. the correlation between cm and op was determined by pearson correlation showed a coefficient r2=0.973, p<0.001, indicating that there is correlation between cm and op. it was concluded that none of the materials tested were able to mask the black background, as äe values were above the limit of clinically acceptable (äe<3). cm and op were affected only by the cement, and, ru showed a lower capacity masking and lower opacity than ra. 036 immediate and delayed photoactivation of self-adhesive resin cements and retention of glass-fiber posts silva fp*, menezes ms, peixoto ac, moraes rr, borges mg, faria-e-silva al federal university of uberlandia fer_nanda_ps@hotmail.com the aim of this study was to evaluate the effect of immediate and delayed photoactivation of self-adhesive resin cements (sarcs) on the retention of glassfiber posts luted into root canals. bovine incisors were endodontically treated and 9-mm-deep postholes were prepared. the fiber posts were luted using two sarcs (biscem – bisco, or relyx unicem clicker – 3m espe) or a regular (etch-and-rinse) resin cement (allcem – fgm). photoactivation was carried out 5 or 10 min after cementation. the root/post specimens were transversally sectioned 7 days after luting into 1-mm thick slices, which were submitted to push-out testing in a mechanical testing machine. bond strength data were analyzed by two-way anova and student-newman-keuls’ method (á = 0.05). immediate photoactivation resulted in the highest bond strength for unicem, while biscem had higher values when photoactivated after 10 min. for allcem, immediate photoactivation yielded the lowest bond strengths, while there was no difference between 5 and 10 min of delay. in conclusion, the moment of photoactivation of resin cement might interfere with the intra-radicular retention of fiber posts depending on the resin cement used for luting. gbmd 2013 – 49th meeting of the brazilian group of dental materials 243243243243243 braz j oral sci. 12(3):237-274 037 light transmission capacity of glass fiber post on self-adhesive resin cement polymerization rodrigues mp *, tavares rp, rf zanatta, valdivia adcm, dantas no, soares cj federal university of uberlandia monise_paula@hotmail.com this study evaluated the influence of translucency of glass fiber post (gfp) on activation of resin cement (rc) by the limitation of the light transmission. three gfp were tested (ex, exact post n.3; wp, whitepost n.3; and dt,dt translucent n.3) as regards the conversion degree (cd) of the rc (relyx unicem), simulating different depths of the root canal (2, 4, 6, 8, and 10 mm) depending on the light side lateral and apical of the root canal, and also the transmittance (tr) of the incident light in the gfp. a teflon device (td) composed of 5 disks allowed to measure depths of cd by ftir method. the td with side perforations in the depths allowed measuring the cd on the side of the gfp. the tr was measured with light emission sensorspectra physispositioned on apical and lateral in the gfp, using high potency laser with wave length (wl) of 514.5; 457.9 and 405.0nm. the apical cd on the depths 2 to 10 mm were: ex: 46/44/36/37/30; wp: 36/27/25/16/ 17; dt: 32/28/31/26/29. the cd lateral was: ex, 24/19/19/17/10; wp:29/23/18/ 15/16; dt: 23/23/18/17/12. the values tr wl were 514.5 apical;457.9 and 405, 0nm: ex, 0.6/0.3/0.2; wp: 0.4/0.5/0.1; dt: 0.6/0.3/0.1. lateral tr were: ex, 0.15/ 0.17/0.16; wp: 0.18/0.14/0.14; dt: 0.08 0.09 0.09. the data were analyzed by anova factorial (3 x 5) and tukey test. the apical cd was influenced significantly by the depth for ex and wp; and the lateral cd for all gfp. ex resulted in statistically apical cd greater than wp at all depths. it is concluded that the optical characteristic of translucency of the gfp significantly influences the properties of rc. 039 influence of chemical structure of mdp co-monomers on the physicochemical properties of experimental adhesives correr ab*, feitosa vp, ogliari fa, sauro s, ogliari ao, gotti vb, corrersobrinho l, sinhoreti mac piracicaba dental school unicamp amerbc@yahoo.com.br the aim was to evaluate the effects of acidic phosphate monomers with different length and hydrophilicity of the spacer chain on the properties of all-in-one adhesives. to a base blend (control) based on hema, udma, bisgma, tegdma, water, ethanol, edmab, camphoroquinone and iodonium salt, it was added equimolar percentages of mdp co-monomers with spacer chains with different length and hydrophilicities: mep (spacer chain with two carbons), mddp (twelve carbons), mtep (highly hydrophilic spacer chain) and cap-p (intermediate hydrophilicity). the ultimate tensile strength (uts) was assessed in dumbbell-shaped specimens. the degree of conversion was determined by ftir. disc-specimens were used to survey the water sorption (wsp). data was analyzed by anova and tukey’s test (á=0.05). the uts (mpa) of mtep (10.9), mddp (10.4), and mdp (10.2) were significantly higher than mep (8.4), cap-p (8.1) and control (6.1); control presented the lowest uts (p<0,05). all monomers attained similar degrees of conversion (%) (mtep-89.0; mddp-91.5; mdp-89.0; mep-82.7; cap-p-87.2; controle-83.5). wsp (µg/mm3) of mtep (101.0) and cap-p (85.5) were significantly higher than mep (67.1), mdp (61.0), mddp (59.4) and control (62.7). it can be concluded that monomers with short spacer chain provide lower uts; the water sorption was more influenced by the hydrophilicity than by the length of the spacer chain; the length and hydrophilicity did not influence the degree of conversion. 041 adhesion to zirconia: study of films deposition based on silica in the ytz-p surface druck cc*, pozzobon jl, dornelles ls, valandro lf federal university of santa maria carolinacdruck@gmail.com this study proposed to evaluate the influence of silica-based film coated on the y-tzp (in-ceram yz, vita) surface on the durability bond strength (bs) between ceramic and resin cement. eighty y-tzp blocks (4x4x3 mm) were obtained, included in acrylic resin and were divided into 4 groups according to the surface treatments (n=20): (tbs) tribochemical silica coating (cojet, 3m/espe), (f-5) 5 nm sio2 film and silanization, (f-500) 500 nm sio2 film and silanization, and (f-500hf) 500 nm sio2 film + hf + silanization. specimens of composite resin ( θ= 3,25 mm) were cemented with resin cement (relyx arc) to y-tzp blocks. half of the specimens of each treatment was tested 24 hours after adhesion (ddry), and another half were subjected to the aging (astorage for 90 days and 10,000 thermal cycles). the specimens were subjected to shear test (1mm/min). after debonding, the surfaces were analyzed by optical microscopy and sem. data were statistically analyzed for kruskalwallis/mann whitney (α=0,05) tests. the bond strength was influenced by the type of surface treatment, in conditions d (p=0.0001) and a (p=0.0000). means (sd) for bs data (mpa) were: tbs/d: 10.2 (5.1)ab; f-5/d: 12.0 (3.9)a; f-500/d: 14.9 (4.7)a; f-500hf/d: 4.1 (5.6)b; tbs/a: 9.1 (4.4)a; f-5/a: 7.8 (5.3)a; f-500/a: 0.01 (0.0)b; f500hf/a: 1.4 (2.3)b. it was concluded that adhesion to zirconia can be enhanced if the surface to receive the 5 nm sio2 film deposition or be subjected to sandblasting with silica particles followed by silanization. 038 evaluation of enamel wear due to etching and different bleaching gels soares af*, alencar ms, rodrigues rf, trentino ac, bombonatti jfs, ishikiriama sk, mondelli rfl bauru dental school usp anaflaviasoares@usp.br this in vitro study aimed to evaluate the wear of enamel due to etching and different concentrations of bleaching gels after simulated tooth brushing. fragments of standard bovine teeth were divided in half, one half determining the control area and another half the test area, subdivided and one side received etching prior to bleaching. one session (5x8') was held and activated with hybrid light source (led blue or violet/laser), determining 6 groups (n=10): control; 35%total blanc office; 35% lase peroxide sense; 25% lase peroxide sense, 15% lase peroxide lite and 10% lase peroxide lite (violet light). the specimens were stored in artificial saliva for seven days and then subjected to 100,000 cycles of simulated brushing. the surface wear after brushing and bleaching was determined by averaging (ìm) the three readings with a rugosimeter hommel tester t 1000 in a function profiler. two criteria anova and tukey testing (p<0.05) were employed. the results demonstrated a significantly lower wear in the control group, and significantly higher wear for the 25% group. the presence of acid caused greater wear in all groups except in the total blanc office group. the etching prior to the bleaching treatment increased the wear values. all gels caused increased wear and the 25%.gel was the most aggressive. 040 effect of the moment of fiber post cutting on post retention to root canal borges mg*, faria-e-silva al, santos-filho pcf, martins lrm, menezes ms federal university of uberlandia marcelaborgesufu@yahoo.com.br this study investigated the effect of the moment of fiber post cutting on push-out bond strength. glass-fiber posts were luted into 60 incisor roots using conventional resin cement relyx arc (arc) or self-adhesive cement relyx unicem (uni). the posts were cut prior to cementation, immediately after the luting, or after building up the core (n = 10). after storage for 24 h, each root was sectioned into 6 slabs, which were subject to a compressive load (0.5 mm/min) until the post was dislodged. data (n = 10) were submitted to two-way anova and tukey’s test (á = 0.05). the moment of fiber post sectioning did not affect the bond strength for uni, whereas immediate cutting reduced the bond strength for arc. in conclusion, the moment of fiber post cutting can influence post retention to root canal. 042 development of experimental 3-step dental adhesives formulated with bapo and ppd as photoinitiator systems souza-junior ej, brandt wc, grohmann cvs, hass v, oliveira dcrs, loguercio ad, rontani rm, sinhoreti mac piracicaba dental school unicamp edujcsj@gmail.com the aim of this study was to evaluate the dentin microtensile bond strength (mtbs), dentin permeability (perd), degree of conversion (gc), flexural strength (rf) and elastic modulus (me) of experimental adhesives formulated with alternative photoinitiators (camphorquinone – cq, ppd and bapo). thus, the adhesives contained bisgma/hema as organic matrix, with different photoinitiator content: g1cq/ amine, g2ppd/amine, g3cq/ppd/amine, g4bapo/amine, g5bapo, g6cq/ bapo/amine, g7ppd and g8the hydrophobic resin of adper scotchbond multipurpose as control. for the mtbs, 70 molars were restored and beams (1 x 1mm) were made and the mtbs was performed after 24h using the machine ez-test (0.5mm/min). the perd was measured by the dentin sealing method, using a dentin permeability device. for the gc, rf and me, specimens (7 x 2 x 1mm) were made and evaluated by ftir (gc) and then submitted to the three-point bending test for the measurement of rf and me. for all tests, the adhesives were photoactivated by led for 40s. data were submitted to anova and tukey’s test (alpha = 0.05). g4, g5 and g8 showed mtbs values similar to g1. in relation of the perd, there was a higher dentin sealing for the groups g4 and g8 (95.8% and 91% respectively). evaluating gc, rf and me, the groups containing bapo presented the higher means. camphorquinone can be replaced by bapo and ppd in hydrophobic adhesive resins, since the alternative photoinitiator systems promote higher bond performance, dentin sealing and physical properties. gbmd 2013 – 49th meeting of the brazilian group of dental materials244244244244244 braz j oral sci. 12(3):237-274 043 bleached enamel susceptibility to coffee and sports drink staining at different intervals elapsed from bleaching ayres apa*, de sá rbc, barros jong, ambrosano gmb, munin e, giannini mpiracicaba dental school unicamp anapaulaayres4@gmail.com the aim of this study was to analyze the enamel susceptibility to coffee and sports drink (gatorade) staining at different time intervals post-bleaching, using photoreflectance methodology. it was used the in-office bleaching technique with 38% hydrogen peroxide (38%hp). the buccal surfaces of seventy-two bovine incisors were prepared for initial measurements using a spectrophotometer, and after selection of the samples, they were divided into 9 groups (n = 8). all samples (except those of control group 3) were stained with sports drink or coffee in the time intervals: after 1 hour, 12 hours and 24 hours from the last session of bleaching and then stored in artificial saliva. the measurements were taken: before treatment; immediately after bleaching and after immersion in coffee or gatorade, according to the experimental groups. after exploratory analysis, data from experimental groups were analyzed using mixed models for repeated measures and tukey-kramer test. the level of significance was set at 5%. it was observed that the 38%hp was effective to increase reflectance values of the bleached samples, which were more susceptible to staining than non-bleached samples. coffee and gatorade lead to dental staining, but the results were dependent on the time post bleaching that the samples were exposed to staining. 045 influence of radiation and dentin pretreatment with doxycycline on dentin bond strength using different adhesives soares ef*, naves lz, correr ab, costa ar, sinhoreti mac, soares cj, garciagodoy f, correr-sobrinho l piracicaba dental school unicamp evelinesoares@live.com the purpose of this in vitro study was to evaluate the effect of radiotherapy on dentin bond strength mediated by adhesive systems, with or without doxycycline. the occlusal surface of 60 human third molars were worn and divided into 3 groups (n=20): control group, not submitted to radiation; group (rtre) underwent radiotherapy before being restored, and group (rert), underwent radiotherapy after being restored. in half of the samples were used adper scotchbond mp, 3m, and half clearfil se, kuraray, with or without the application of doxycycline. in the submitted to radiation samples, it underwent 60 gy in fractionated doses of 2gy/ day, for 6 weeks. a resin block of z250, 3m, 5 mm thickness was attached to the tooth, and light cured for 40 seconds. after 24 hours at 37 º c, the samples were sectioned perpendicular on union area to obtain beams with 1mm2 area and submitted to microtensile bond strength test in a speed of 0.5 mm/min. the data were submitted to anova and tukey’s test (5%). the values of bond strength, mpa, adper scotchbond mp (25.5 ± 11.1) and clearfil se (27.6 ± 9.1) were not statistically different from each other. the use of doxycycline (21.7 ± 7.6) significantly reduced bond strength compared to groups without doxycycline (33.6 ± 8.6). control group (30.5 ± 10.9) and rert group (29.2 ± 10.4) were significantly higher than the rtre group (23.1 ± 7.2). the radiation of the samples prior to the composite restoration procedure significantly decreased microtensile bond strength. the use of doxycycline significantly reduced bond strength. 047 influence of nanotube growth on the surface of the cpti dental implant: characterization and degree of wettability alves rezende mcr*, rosa jl, robin a, nakazato rz, schneider sg, de oliveira jag, capalbo bc, bensi mep, alves claro apr aracatuba dental school unesp rezende@foa.unesp.br the osseointegration of dental implants is dependent of the material used, the manufacturing process, drawing, machining conditions, the type of bone, surgical technique, and features of the prosthesis to be applied on the implant and loading conditions during chewing. above is strongly dependent of the interaction between cells and the surface of the implant. titanium oxide nanotubes have been the subject of recent studies regarding their ability to promote differentiation of various cell lineages. we evaluated the wetting of layer nanotubes formed on titanium oxide (tio2) prepared by anodic oxidation on the surface of commercially pure titanium implants (cp ti) astm f67/grade 4. nanotubes were grown by anodization using glycerol di-h2o (50-50 v / v) + nh4f (0.5-1,5% / 10-20v) for 3 hours at 37 ° c. after the formation of nanostructures the surface topography was evaluated measuring the amount, height and diameter of the nanotubes by scanning electron microscope of field emission (fesem).the wettability was evaluated by measuring the contact angle obtained in anodized surface using goniômetro300f1 (ramé inst.co-hard) mode sessile drop / 5 drops / ml. the control group not used anodized surface. the results showed values of 39.1 ° for nanotube surface and 75.9 ° for control surface. it was concluded that the growth of nanotubes was effective in the experimental conditions used and allowed a higher wettability and lower the surface tension of titanium. 044 evaluation of disinfection with electrolyzed acid water of elastomers paulus m*, arthur ra, parolo ccf, leitune vcb, samuel smw, collares fm federal university of rio grande do sul mariliapaulus@hotmail.com the aim of this study was to evaluate the efficacy of electrolyzed acid water (eaw) in the disinfection of elastomeric impressions. eaw was produced by electrolyzing an aqueous solution of sodium chloride, with ph reaching between 2 and 3 and a redox potential higher than 1100mv. two impression materials were used: vinyl polysiloxane (xtreme hydro) and polyether (impregum). 10x10x2mm specimens were sterilized in hydrogen peroxide plasma and transferred into a 24-well plate. biofilms of staphylococcus aureus were grown on these specimens for 24 hours at 37°c. after, the test specimens (n=3) were immersed for 10 minutes in the following solutions: sterile saline solution (negative control), 2% glutaraldehyde (positive control), and acid electrolyzed water. the biofilm present in each sample was aseptically collected, diluted and aliquots of the dilutions were inoculated onto blood agar. after 24 hours of incubation at 37°c, the number of bacterial colony forming units in each of the test specimens was counted, and the results expressed in ufc/ml. data were analyzed using one-way anova e tukey multiple comparisons at a significance level of 5%. the results showed that there was growth in the specimens immersed in sterile saline. there was no bacterial growth in specimens immersed in glutaraldehyde and electrolyzed acid water (p<0,001). it was concluded that electrolyzed acid water was efficient in the disinfection of the elastomers evaluated. 046 effect of grinding with resin-bonded diamond discs and diamond burs on the mechanical behavior of y-tzp ceramics pereira gkr, rocha gc, simoneti r, amaral m, valandro lf federal university of santa maria gabrielkrpereira@hotmail.com this study compared the effects of grinding on y-tzp ceramic performed by disc and diamond burs on the micromorphology of the surface, phase transformation (t m), biaxial flexural strength and structural reliability (weibull analysis). 170 discs (15 x 1.2mm) yz (lava) were made and divided into 5 groups, considering the surface treatment employed: without treatment (ctrl, as-sintered) extra-fine diamond bur (25um, xfine), diamond disc 600 grit(25um, d600), coarse diamond bur (181um, coarse) and diamond disc 120 grit (160um, d120). grinding with diamond burs was executed with a contra-angle multiplier (t2 revo,sirona) and a straight hand piece at low speed (kavo), while for the resin-bonded diamond discs (allied) a polishing machine (ecomet, buehler) was employed, both under water cooling. abrasion with diamond burs developed different micromorphological aspects (deeper scratches) if compared to diamond discs. higher grit sizes of the grinding tool resulted in an increase at monoclinic content. there was no statistically difference for the characteristic strength values between treatments with lower grit sizes (d600-1050,08 and xfine-1171,33) which were statistically higher than ctrl (917,58). as for higher grit sizes it was observed a statistically difference (coarse1136,32>d120-727,47) being d120 the lowest statistically values. weibull modules were statistically similar. thus for the treatments studied, under the conditions evaluated, only for lower grit sizes, grinding with diamond discs and burs promoted similar effects. 048 synthesis, characterization and photocuring of siloxane-oxirane monomers leal fb*, pereira cm, ogliari fa federal university of pelotas fernandableal@gmail.com the aim of this study was synthesize, characterize and photopolymerize an alternative monomers for use in dentistry. three siloxane-oxirane monomers were synthesized and the products conversion was followed by fourier-transform infrared spectroscopy. the products obtained were characterized by 1h and 13c nmr and evaluated for viscosity and refractive index. the polymerization was evaluated by formulating of two experimental photoinitiation systems wich varied for the presence of 1,2 ethanediol. a ternary system with camphorquinone (cq), ethyl 4dimethylaminobenzoate (edab) and diphenyliodonium hexafluorphosphate (dpi) was used as control. the degree of conversion was accessed by ftir and dsc-pca. the nmr confirmed the synthesis success with 75, 87 and 55% yield for the monomers synthesized. moreover, the presence of 1,2 ethanediol increase the degree conversion of the siloxane-oxirane monomers. this study showed simple and effective way to synthesize siloxane-oxirane monomers with a high potential for application in dental materials. gbmd 2013 – 49th meeting of the brazilian group of dental materials 245245245245245 braz j oral sci. 12(3):237-274 049 effect of a bleaching gel on pulp cells differentiation soares dg*, hebling j, de souza costa ca araraquara school of dentistry unesp diana_odonto@yahoo.com.br the effects of a bleaching gel with 17.5% hydrogen peroxide (hp) on pulp cell differentiation were assessed. enamel/dentin discs individually adapted to transwells devices were applied on odontoblast-like mdpc-23 and human dental pulp cells (hdpc) previously seeded in wells. the bleaching gel was applied or not on enamel for different periods, giving rise to the following groups: g1 – no treatment (negative control); g2 – 3x15 min; g3 – 1x15 min; g4 – 1x5 min. cell viability was assessed immediately after bleaching. mineralized nodule (mn) formation and alkaline phosphatase activity (alp) were evaluated at 7, 14 and 21 d post-bleaching periods (kruskal-wallis and mann-whitney; á=5%). for hdpcs, significant reduction on cell viability was observed in g2 (86.1%), g3 (76.6%) and g4 (65.1%). increase on alp activity with time was observed for all groups with no difference compared to g1. mn deposition in g2 was significantly lower than in g1 in all periods evaluated. however, no significant difference occurred when g3 and g4 were compared to g1 at 21 d. significant reduction on mdpc-23 viability was observed in g2 (33%) and g3 (25.4%). the alp activity observed in bleached groups was significantly lower than in g1 at 7 d, with significant increase at 14 and 21 d. no difference on mn formation occurred when g3 and g4 were compared to g1 at 14 d, and even for g2 at 21 d. it was concluded that pulp cells damaged with 17.5% hp gel maintained their differentiation ability up to 21 d postbleaching period. 051 influence of antioxidants incorporation on adhesive systems performance and durability of the bonded interface gotti vb*, feitosa vp, sauro s, correr-sobrinho l, sinhoreti mac, ogliari fa, leal fb, stansbury jw, correr ab piracicaba dental school unicamp val_bisinoto@hotmail.com the aim was to evaluate the effect of antioxidants (ao) incorporation into adhesive systems (as) on bond strength (ìtbs), nanoleakage (na), degree of conversion (dc) and polymerization rate (rp). 144 human premolars were used and divided into groups according to the as (adper single bond 2-sb, clearfil se bond-cs ou adper easy bond-eb) and ao incorporation (no antioxidant-co, ascorbic acidvc, α-tocopherol-ve and quercetin-q). the samples were restored with filtek z350xt and stored for 24 hours in distilled water or for 6 months under simulated pulpal pressure previously to obtain sticks for µtbs testing and qualitative na analysis. the µtbs values (mpa) were analyzed using anova and tukey’s test, at α 5%. for dc and rp, the data were plotted and a curve was obtained by non-linear regression. in 24h, sbco (63) and sbve (56) showed higher ìtbs than sbvc (40) e sbq (31). csco (69) showed higher µtbs than csvc (56), csve (41) and csq (40). ebco (48), ebve (41) e ebqe (46) showed higher µtbs than ebvc (14). in 6 months, only sbco (50) showed ìtbs decreased (sbvc 39; sbve 55; sbq 38; csco 69; csvc 61; csve 57; csq 69; ebco 51; ebvc 30; ebve 58; ebq 44). in general, the na decreased over time using ao while the inverse was observed in the co groups. less dc was obtained in ao groups compared to their co for sb and eb and polymerization kinetics revealed a reduced rp for as with ao. in conclusion, the ao performance varied according to the as and the ao incorporation did not increase the ìtbs compared to co groups, however, was able to maintain or increase the initial ìtbs. 053 influence of light exposure protocol on characterization of methacrylate resin-composite by dynamic mechanical analysis giorgi mcc*, pistor v, lima danl, marchi gm, mauler rs, aguiar fhb piracicaba dental school unicamp cecilia.giorgi@yahoo.com the aim of this study was to evaluate the degree of conversion (dc) and to identify the viscoelastic properties storage modulus (e’), loss modulus (e’’), tan ä and glass transition temperature (tg) of a microhybrid resin-composite light-activated with four different protocols. a filtek z250 (3m espe) shade a3 was inserted in a teflon mold (1.0 x 5 x 21mm) and light-activated according to the following light exposure protocols: qth (xl) 500 mw/cm2 x 38s, led (s) 1000 mw/cm2 x 19s, led (hp) 1400 mw/cm2 x 14s, led (pe) 3200 mw/cm2 x 6s, all set up to deliver 19 j/cm2. dynamic mechanical analysis (n=3) was performed in single cantilever clamped mode. dc (n=5) was measured by ftir on top (t) and bottom (b) surfaces and the data was submitted to a split-plot 1-way anova. there was a significant effect for surface; t showed higher dc than b. light exposure protocols did not affect dc. viscoelastic properties (e’, e’’, tan ä, tg) were not affected by light exposure protocols. it could be conclude that polymer structure was not influenced by the light exposure protocols. light exposure protocols delivering 19 j/cm2, applied in continuous mode with at least 500 mw/cm2 results in polymers with similar crosslink density. 050 action of different monomers on the degree of conversion, optical and physical properties of experimental composites albuquerque ppac*, moreira adl, cesar pf, pfeifer cs; cavalcante lma; schneider lf** university of sao paulo pedroalbuquerque2@gmail.com the aim of the present study was to evaluate the degree of conversion (dc), resistance to degradation by hydrolysis and the optical properties of experimental composites formulated with different monomers. groups were formulated containing monomers bisgma, bis-ema, bisema 30, udma and udma-modified (fit 852) in a proportion of 70%. to this mixture was added 30% diluent monomer (tegdma) and 60% of inorganic particles by final weight, and a photoinitiator system (cq + edmab). the optical properties were evaluated using a spectrophotometer, applying the cielab parameter. the polymerization kinetics and dc, were evaluated from the atr-ftir technique. absorption and solubility were analyzed from adaptation of standard iso4049. the results were submitted to anova and tukey 95%. result: the group formulated with bis-ema 30 had the highest dc, but with a slower onset of polymerization compared to the others groups. groups with udma and bis-ema showed less degradation. as for the optical properties, the groups formulated with udma and bis-ema had lower color changes. bis-gma generated materials with minor changes in brightness. based formulations of bis-ema and udma showed less degradation by hydrolysis and color change (äe). the bis-gma showed lower brightness variations. generally, bis-ema/tegdma provided the best mixture. 052 optic behavior of ceramic composites through the kubelka-munk method lima e*, pinto mm, takahashi dt, yoshimura hn, cesar pf university of sao paulo ericklima.usp@gmail.com the objective of this work was to determine the absorption (k) and the scattering (s) coefficients of three ceramic composites as a function of thickness, through the kubelka-munk method. three ceramic composites were used (ia-alumina/glass; isspinell/glass and iz-alumina/zircônia/glass). ten discs were prepared for each material and then serially worn out for optical analysis, in the following thickness: 2.0; 1.5; 1.0 e 0.5 mm. using a spectrophotometer, reflectance values were obtained from the materials on two different backgrounds (black and white), which were used for the calculation of the materials coefficients (k and s) by the kubelkamunk model. spectral graphs were constructed correlating the values of k and s as a function of wavelength (ë). all three composites have showed 0 as minimum value of k (mm-1) and maximum values of 0.7, 0.2 and 3.0 for ia, is and iz respectively. this coefficient increased with decreasing ë for all materials. the coefficient s (mm-1) has decreased with decreasing ë for all the materials and showed values ranging from 1.0 to 7.5 for ia, from 2.5 to 4.5 for is and from 5.0 to 80 for iz. within the range of visible light wavelengths, all the materials showed scattering values (s) significantly higher than the absorption values (k), indicating that the optical behavior of these ceramics is regulated by the scattering phenomenon. 054 fracture load, mechanical fatigue and finite element analysis of glass fiber posts wandscher vf*, oliveira a, bérgoli cd, borges als, limberger if, valandro lf federal university of santa maria viniwan@hotmail.com this study evaluated fracture resistance (fr), fatigue and stress distribution by finite element analysis (fea) of glass fiber posts with different coronal diameters. double-tapered posts (white post dc, fgm) with different diameters were tested (n=20): 0.5 (1.4mm), 0.5e (1.8mm), 1 (1.6mm), 1e (2mm), 2 (1.8mm), 2e (2.2mm), 3 (2mm) and 4 (2.2mm). each specimen was inserted in a pvc cylinder that was filled with epoxy resin. ten posts of each group (n= 10) were tested for fr (45°) and the other ten (n= 10) were submitted to mechanical fatigue (3,000,000 cycles, 45°, 50n, 4hz and 37 °c). the eight types of posts were modeled in 3d finite elements for stress distribution simulation and the load application was the average fr of each group. only 0.5 group did not survive mechanical fatigue. one-way anova showed that fr of posts with the same diameter was not different. according to failure analysis, 95% of specimens failed due to shear stress. fea showed similar values of shear stress in relation to the real values calculated. in relation to fr, larger posts had better performance compared to thinner posts. due to the failures observed, more studies are necessary for evaluation of new configurations of fiber posts to minimize the effects of shear stress. gbmd 2013 – 49th meeting of the brazilian group of dental materials246246246246246 braz j oral sci. 12(3):237-274 055 effect of specimen positioning and loading on microshear bonding outcomes: a non-linear finite element analysis raposo lha*, pereira ag, dantas lcm, barreto bcf, sinhoreti mac, corrersobrinho l, soares cj piracicaba dental school unicamp luisrfox@gmail.com this study compared the effect of different testing parameters on the stress distribution of microshear specimens. three-dimensional models consisting of ceramic plates with two resin cement cylinders were generated. the distances between the two cylinders were varied (1.0, 1.5, 2.0, 2.5, 3.0 mm). a 10 n load was applied on one cylinder using a 0.2 mm diameter orthodontic-looped wire in three different directions: y (perpendicular to adjacent cylinder), x+ (towards adjacent cylinder) and x(away from adjacent cylinder). additional three-dimensional microshear models with one resin cement cylinder were loaded (10 n) by: large stainless-steel tape (lt), small stainless-steel tape (st), chisel (ch), orthodonticlooped wire (ow), or customized chisel (cc). stress concentration arising from the loaded cylinder reached the adhesive region of the adjacent non-loaded cylinder for 1.0, 1.5 and 2.0 mm models for all loading directions. when 3.0 mm apart, no stress elevation was found at the non-loaded cylinder. for the ch and ow loading, tensile stresses were more dominant at the interface. the model loaded with the cc scheme presented lower tensile and shear stresses. a predominance of shear stresses was verified for the st and lt loading systems. a 3.0 mm separating space between cylinders is a safe distance to avoid unwanted stress to reach the non-loaded cylinders. loading the cylinders perpendicularly with straight-aligned wire appears the most suitable condition. loading specimens with small (st) and large (lt) stainless-steel tapes seem the better alternative. 057 surface roughness of composite resins composition and morphology of filler particles ruivo ma*, pacheco rr, giannini m, ambrosano gmb piracicaba dental school unicamp melissaruivo@gmail.com the aim of this study was to analyze the morphology and composition of filler particles of five composites and the surface of these materials in scanning electron microscopy (sem) and rugosimeter after finishing / polishing procedures (fp) and after 6 months of storage and brushing (sb). twenty discs were prepared (2mm thickness and 5mm diameter) from the materials: z100 (z1, 3m) z350xt (z3, 3m), estelite sigma (es, tokuyama), beautiful ii (be, shofu) and dyad flow (df, kerr). the sof-lex (3m) polishing system was used for the fp. after the fp half of each resin sample was stored in distilled water (37° c) for 1 week before rugosimeter analysis (se 1700, kosaka) and sem (5600lv, jeol). other half was subjected to sb (30,000 cycles) and stored in distilled water for 6 months (n=5). surface roughness data (ra, µm) were analyzed by anova two way and tukey’s test (5%). for particle composition and morphology analysis energy dispersive x-ray (link isis) and sem were used. at the evaluation of the initial roughness, the materials showed no statistical difference. after sb, resins es, be and df showed a higher ra. the resins showed particles with different size and shapes. all particles contain silica. z1 and z3 presented zirconia in the composition, while df presented sodium. sb increases the roughness depending on the material. resins vary in the size and particles’ morphology. silica was detected in all materials. 059 influence of cement layer thickness and variation of temperature in the mechanics of ceramics fragments martini ap*, anchieta rb, almeida eo, freitas-junior ac, kina s, rocha ep aracatuba dental school unesp martini.anapaula@gmail.com considering that the thickness of the resin cement and the variation of oral temperature are factors influencing the success of conventional ceramic restorations, the aim of this study was to analyze, through the finite element method, the behavior of the layer of cement and ceramics, varying the thickness of this layer and the temperature in a restoration with ceramic fragment. after obtaining the set of microtomography images of a maxillary central incisor, the tooth model was developed in the program mimics. from this model (m), five models were prepared in the solidworks program simulating the restoration of the angle distoincisal with the use of a ceramic fragment, varying the thickness of the cement layer: m1 0 mm in thickness, m2 50 mm to the fullest extent; m3 50 micron and 100 micron in margins distance; m4 50 um with the banks and 200 mm distance, and m5 100 micron with the banks and 200 mm distance. for all models were simulated by 4 temperature changes similar to the changes that occur in the oral cavity (5, 20, 37, and 50 °c). the finite element mesh was generated in the program ansys workbench. the maximum principal stress in the fragment models m1 to m5 in temperature was 5 °c (mpa): 7.81, 7.82, 27.3, 27.4 and 1.83, respectively. to 50 ºc, the values were 35.6, 35.6, 87.6, 79.2 and 25.9, respectively. the fragment had higher tensile stress as increased temperature and higher values of compressive stress with decreasing temperature, whereas the highest stress values were located on its banks. 056 effect of the bur grit on the flexural strength of leucite-reinforced glass-ceramic aurélio il*, may lg, kist pp, amaral m federal university of santa maria lamadridiana@hotmail.com the aims of this study were: 1) to determine the biaxial flexural strength (bfs) of a cad/cam leucite reinforced glass ceramic cutting by different diamond bur grit sizes; 2) to analyze the correlation of bfs and the roughness of the cut surface. a hundred four (104) tabs (12 x 10 x 1.5 mm) were obtained from cad/cam ceramic blocks (ips empress cad®, i12). the upper side of the tabs were polished and divided into 4 groups (n = 26). the lower side of the tabs were cut with tapered diamond burs (#4138, kg sorensen) according to the different grit size groups: extra-fine (ff), fine (f), medium (m) and coarse (c). roughness parameters (ra, rymáx) were measured and the tabs were kept dry for 7 days. the flexural test was carried out according the iso 6872 and the bfs was calculated. the bfs, ra and rymáx data were subjected to the kruskal-wallis analysis of variance and post-hoc lsd test. weibull analysis was used to compare characteristic strength and weibull modulus (reliability). regression analysis for ra, rymáx vs. bfs was performed. the grit size was found to have a negative effect on the ceramic bfs (116,71 mpa for ff and 82,65 mpa for g). the correlation (r) between surface roughness and bfs was 0,78 for rymáx and 0,73 for ra. the diamond grit size has a significative negative effect on bfs of leucite-reinforced glass-ceramics. this suggests that the cut of sintered glass-ceramic should be done or maybe completed using burs with the finest grit possible, in order to minimize the roughness of the internal surface and maximize the structural strength. 058 influence of different resin cements on the optical properties of ceramics: evaluation by means of spectroscopy ramos junior mjp*, rodrigues rb, roscoe mg, lima e, silva gr, simamoto junior pc, césar pf, soares cj, novais vr federal university of uberlandia mardengnr@hotmail.com the aim of this in vitro study was to evaluate the color of two ceramics (feldspathic and lithium disilicate-reinforced), cemented with different resin cements by calculating the color difference (∆e) by measuring the coordinates l * a * b * of transmittance. it was made 16 discs of each ceramic with 10 mm diameter and 1.5 mm thick. each group was randomly divided into 03 subgroups according to the type of cement used (n = 5): rv, variolink ii, ra, relyx arc; ru, relyx u200. a disc of each ceramic was evaluated without being cemented, which is the control sample. after preparation, the samples were stored in a dark bottle and dry at room temperature for 24 hours. subsequently, the samples were subjected to the test in a spectrophotometer with a wave length (ë) of 360-740. the data of ∆e obtained for each group were analyzed using analysis of variance factorial (2 x 3) (α = 0.05). there was no statistical significance for any of the factors analyzed: ceramic (p = 0.283), cement (p = 0.139), and interaction among ceramic and cement (p = 0.912). knowing that the transmittance is the fraction of light that passes through the sample and the more a material is translucent, the greater the fraction of transmitted light, one can conclude this work that light passed through the materials tested similarly, and both ceramics evaluated and resin cements showed similar transmittance optical property. 060 cationic polymer [qamp]: structural elucidation and antimicrobial potential after incorporation into adhesive system pupo ym*, farago pv, maluf df, nadal jm, esmerino la, gomes omm, gomes jc ponta grossa state university yasminemendes@hotmail.com the aim of this study was to elucidate the chemical structure of a new quaternary ammonium methacrylate polymer [qamp] by nuclear magnetic resonance spectroscopy [nmr] for incorporation into adhesive systems in order to provide antimicrobial effectiveness of these materials. the antimicrobial potential of qamp was assessed as minimal inhibitory concentration (mic) and minimum bactericidal concentration (mbc) against streptococcus mutans after incorporation into clearfil™ se bond. clearfil™ protect bond with the standard antimicrobial [mdpb] and clearfil™ se bond without qamp were used as controls. the release of quaternary ammonium compounds from the adhesive systems after 1, 7, 14, 21, and 30 days were also carried out. considering 1h nmr spectrum of qamp, 1h chemical shifts of methyl and methylene groups attached to the quaternary ammonium were assigned at ä 3.18 and 3.39 ppm, respectively. regarding the 13c-dept nmr spectrum, qamp showed a deshielding effect on its methyl groups and shifted the peak (ä 45.8 ppm) assigned to the methyl groups adjacent to nitrogen of eudragit™ e100 to downfield (ä 50.9). mic/mbc were 20, 10 and 80 ìl.ml-1 to clearfil™ se bond qamp, clearfil™ protect bond and negative control, respectively. clearfil™ se bond containing 5% qamp showed a cumulative release of quaternary ammonium compounds of only 5.1% while clearfil™ protect bond released 47.2% of mdpb. these data confirm the development of a new quaternary ammonium polymer with antimicrobial effect for adhesive systems. gbmd 2013 – 49th meeting of the brazilian group of dental materials 247247247247247 braz j oral sci. 12(3):237-274 061 influence of 3 or 4 points bending test on the elastic modulus of orthodontic wires okada cy, ambrósio rs*, faltin junior k, ortolani clf paulista university unip sunao_rsa@yahoo.com.br the aim of this study was to evaluate the influence of 3 or 4 point bending test and wire diameter on stainless steel (ss) orthodontic wires elastic modulus (e). 10 ss wires (morelli®), 45mm length, of each diameter (0.016" or 0.020") for each test configuration (3 or 4 point bending) were used. e was calculated from the linear portion of stress/strain graphics obtained from tests. e results were analyzed by factorial anova 2 x 2 and tukey’s test (alpha=0.05). elastic modulus of 0.016" and 0.020" wires obtained from 3 point bending test showed no statistical differences, 174,37(9,83)gpa and 178,45(9,07)gpa, respectively, and they were statistically lower than those obtained from 4 point bending test, 284,07(24,64)gpa to 0.016" wire and 241,39(14,17)gpa to 0.020" wire, statistically different with each other. it could be concluded that the wire diameter did not influence the e on 3 point bending test while it did on 4 point bending test, also, the test configuration influenced the e, so that 4 point bending presented higher e values. 063 influence of weakened cusp, composite resin and loading presence on behavior of premolars: laboratory and fea barreto bcf*, xavier ta, silva gr, soares pv, noritomi py, martins lrm, soares cj federal university of uberlandia bcfbarreto@gmail.com the study evaluated the influence of weakened cusp, composite resin type and axial loading presence, on the cusp deformation (cd), marginal integrity (mi), fracture strength (fs) and stress distribution of restored teeth. it was selected 40 maxillary premolars (mp), which received class ii mod cavities, and 20 of these teeth had both weakened cusps (v/p). were established 4 groups (n = 10) according to the factors under study: resin (z250/venus diamond-vd), weakened cusp (with/ without), and presence/absence of axial loading (100n). the specimens were submit to strain gauge test, thermal and mechanical cycling, epoxy resin replicas confection for analysis of mi using sem, and fs testing. a selected tooth was scanned and 8 finite element (fe) models were generated, representing the same factors of the laboratory testing. to characterize the shrinkage stress, post-gel shrinkage of the resins were measure by means of strain gauge method and the elastic modulus was calculated by micro hardness knoop testing. the laboratory results showed that the presence of weakened cusps not affected the cd, however decreased mi and fs. the z250 composite resin generated higher value for cd and smaller to mi and fs. the compressive load decreased cd, but according fe, the stress had a synergistic effect, greatly increasing its concentration in the marginal interface. it was concluded that the type of resin, the presence of weakened cusp and compressive loading, it can influence the biomechanical behavior of teeth with large composite restorations. 065 synthesis, characterization and sintering y-tzp/tio2 depending on the amount of tio2 and the sintering temperature miranda rbp*, ussui v, cesar pf, miranda wg, lazar drr, marchi j university of sao paulo ranulfoo7@gmail.com the objective of this work was to synthesize, characterize and sinter the composite ytzp/tio2 varying the amount of tio2 and the sintering temperature (st). the synthesis of the composite y-tzp/tio2 occurred with the precursors (zirconium oxychloride, titanium chloride and yttrium chloride) by coprecipitation technique with ammonium hydroxide, the following groups were produced: z (100% y: tzp), zt10 (90% y-tzp and 10% tio2) e zt30 (70% y: tzp and 30% tio2). the powders produced were characterized by x-ray diffraction (xrd), particle size (ps) by laser diffraction, gas adsorption for evaluation of specific surface area (sbet) and scanning electron microscopy (sem). discs (15 mm in diameter and 2.5 mm in thickness) were pressed (65 mpa) and sintered at 1400 ° c or 1500 ° c for 2 hours with subsequent measurement of the density (d) by the method of archimedes and microstructural analysis by sem. the sem and ps showed that the powders are formed of agglomerates and their average size (µm) ranged as follows: z (0.5 e 5.4), zt10 (0.7 e 17.6) e zt30 (0.7 e 11.4). the sbet values of the powders (m2/g) were: z (47.4), zt10 (42.3) e zt30 (58.0). the xrd analysis showed peaks of tetragonal and monoclinic zirconia in all groups. all the discs have a density exceeding 94% and sem showed that the higher st (1500 °c) and the greater amount of tio2 favored grain growth. the amount of tio2 affected sbet and ps of the powders. st did not affect d. the grain growth was higher in the samples sintered at 1500 ° c and tio2. 062 effect of bur set and machining order on the surface roughness of a sintered leucite glass ceramic may lg*, fraga s, bottino ma federal university of santa maria liligmay@gmail.com considering that machining can produce defects at the ceramic surface, the aim of this study was to evaluate the effect of the bur set and machining order on the surface roughness of leucite glass ceramic discs obtained by a cad/cam system (cerec inlab ® mc xl). six bur sets (a-f) were used for the machining of 155 ips empress cad® ceramic blocks (1 disc for each block). twenty-eight discs (n=28) were machined by the first 5 bur sets (a-e). the last bur set (f) was used for the machining of the remaining 14 blocks (n=14). a total of 144 discs had their surface roughness measured in a contact profilometer (ra and rz). spearman coefficient indicated a significant, moderate to strong, correlation between machining order and ra (rsra = -0.66) and rz (rsrz = -0.73). therefore, the f bur set was excluded from the variance analysis. kruskal-wallis analysis showed a significant difference in ra and rz values obtained from the first five bur sets (p<0,05). the rz values varied from 7.71±1.77 µm for the c bur set to 9.33±1.16 µm for the b bur set (statistically higher than those obtained for the c and a bur sets). in conclusion, different values of roughness can be expected even for identical burs, in geometry, composition and manufacturing. in addition, the surface roughness seems to reduce according the number of specimens produced by the same bur set increases. 064 effect of immersion in different alcoholic beverages on the roughness and microhardness of composite resins da silva mab*, vitti rp, spazzin ao, guarda gb, tonholo j, da silva júnior jg, santos lm, reis jil, consani rlx federal university of alagoas marcosbomfim21@hotmail.com the purpose of this study was to evaluate in vitro the microhardness and surface roughness of composite resin submitted to cycling 30 days in alcoholic solutions. three composites were selected for the study (durafill / kulzer, z250 xt and z350 xt). 120 samples were prepared for each material and after polymerization the samples were stored in 100% relative humidity for 24 h at 370c. the surface roughness was measured with atomic force microscope then the initial microhardness analysis. the samples were divided into four groups (n = 30) according to solutions: g1-artificial saliva;-g2 beer, g3 vodka, g4 whisky. the immersion were performed 3x for 15 minutes daily for 30 days. analysis of surface microhardness and roughness were repeated after 30 days of immersion. the values of roughness and hardness were tested using anova one way, complemented by tukey’s test at a significance level of 5%. the results of surface roughness revealed that all composites analyzed showed increased roughness after 30 days soaking in beer and whiskey. the results showed significant reduction in the surface hardness of the resins analyzed after immersion in alcoholic solutions for 30 days and more significant for composite durafill. based on the results it was observed that the surface degradation of composites depends on the composition, immersion time, alcohol content and ph of the solutions. 066 effects of antioxidants application time on bond strength of enamel after bleaching berger sb*, ozelin aa, contreras er, guiraldo rd, lopes mb, moura sk, carvalho rv university of north parana unopar berger.sandrine@gmail.com this study evaluated the effect of antioxidants application time on bond strength of enamel after enamel bleaching. were obtained enamel samples from 70 third molars and randomly divided into 7 experimental groups. g1 – bleached with 10% cabamide peroxide (cp); g2 – cp + 10% sodium ascorbate gel (sa) for 15min; g3 – cp + sa for 30min; g4 – cp + sa for 60min; g5: cp + 10% green tea gel (gt) for 15min; g6 – cp + gt for 30min; g7 – cp + gt for 60min. the cp was applied onto the enamel surface for 6 hours, every day for 14 days. the sa was applied in the groups 2, 3, and gt in the groups 5, 6 and 7 according the applications time as described above. immediately after treatment, the specimens were bonded with adper single bond 2 and filtek z350 xt. the specimens were sectioned perpendicular to the adhesive–tooth interface and microtensile bond strengths were measured with a universal testing machine. fracture mode analysis of the bonded enamel surface was performed using scanning electron microscopy. the data were statistically analyzed by two-way anova and dunnett’s test (á = 5%). the means (standard deviation) were: g1 – 23.29(3.20); g2 – 25.18 (3.95); g3 – 26.41(5.40); g4 – 30.17(4.46); g5 – 26.63(3.43); g6 – 22.02(5.41); g7 – 31.40(3.35). all groups had a higher percentage of adhesive failures. in conclusion, only when the antioxidants were applied for 60 minutes, the bond strength values were higher than bleached group. gbmd 2013 – 49th meeting of the brazilian group of dental materials248248248248248 braz j oral sci. 12(3):237-274 067 bond strength and contact angle of feldspathic ceramic subjected to different conditioning methods rambo dr*, prochnow c, venturini ab, valandro lf federal university of santa maria dagma.rambo@hotmail.com to evaluate the effect of different concentrations of hydrofluoric acid (hf) in the contact angle and bond strength between feldspathic ceramic and resin cement. in order to analyze the contact angle, 30 ceramics specimens (12x10x2,4mm) (vita blocks mark ii) were divided into 6 groups (n=5): cs1hf10%+wash/dry; cs2hf1%+wash/dry; cs3hf3%+wash/dry; cs4hf5%+wash/dry; cs5no conditioning; cs6silane only. the contact angle measurements were performed on a goniometer. for bond strength test, 40 ceramic blocks were made (12x10x4mm), which were subjected to the following surface treatments (n = 10): cs1hf10% 1min+wash/dry+silane; cs2-hf1% 1min+wash/dry+silane; cs3hf3% 1min+wash/dry+silane; cs4hf5% 1min+wash/dry+silane. the specimens were stored in distilled water (37ºc) for 24 hours and sectioned in the x and y directions. the microtensile test was performed in a universal testing machine (1mm/min). data obtained were submitted to one-way anova and tukey’s test (á=0,05). cs5 had the largest contact angle (61.4° ± 5.6°), whereas cs1 showed the smallest value (17.5° ± 4°). different concentrations hf promoted bond strenght statistically similar (14.2 to 15.1 mpa) (p<0.05). in terms of adhesion, the tested ceramic can be etched with hf in the evaluated concentrations. the modification of the micromorphologic pattern of the treated surfaces did not affect the bond strength, which may mean a strong importance of silanization in the adhesion. 069 influence of the incorporation of antimicrobial monomer in antibacterial activity of composite experimental de paula ab*, stipp rn, taparelli jr, alonso rcb, mei l, puppin-rontani rm piracicaba dental school unicamp andbol_63@hotmail.com the aim of this study was to evaluate bacterial inhibition promoted by triclosan methacrylate monomer added to an experimental resin composite. triclosan metacrylate monomer (tm) was synthesized by esterification chemical process. the materials were evaluated: a) experimental composite resin (c), control group, b) c+ 30% of mt (ctm). four specimens (5mm diameter x 1mm thick) of each material were prepared. cultures of streptococcus mutans ua159 were grown for 24 h, adjusted to an optical density (od550nm) of 1.0, and diluted 20-fold in brain heart infusion broth supplemented with 0.1% sucrose. biofilms were statically formed on the surface of the materials for 24 h. specimens were washed for 5 min and biofilms disrupted by vortexing. cell suspensions were serially diluted and plated onto mitis salivarius agar. after incubation for 48 h, cell counting was performed. four independent experiments were conducted in triplicate. data were analyzed with anova (p<0.01). bacterial counting (log) in the control group (c) was 8.9±0.29 while ctm reach 7.2±0.15. that difference was statistically significant (p<0.01), showing an antibacterial effect against streptococcus mutans of 1.7 log order reduction. the triclosan metacrylate monomer demonstrated inhibition effect against streptococcus mutans when added to experimental resin composite. 071 influence of adding phenathrenequinone optical properties of experimental composites bertolo mvl*, teles ya, albuquerque ppac, cavalcante lm, schneider lfj universidade federal fluminense marcusbertolo@hotmail.com the system camphorquinone/amine (cq/am) presents several drawbacks, like high yellowing effect caused by the excess of no reactive cq after polymerization and the potencial of oxidation by the addition of an amine in the system. phenathrenequinone (fq) could be an interesting possibility, because present low yellow colors than cq and theoretically, present a larger number of actives sites for the initiation of the polymerization process. the aim of the present study was to evaluate the optical properties, depth of cure and degree of conversion of experimental composites formulated with fq and cq with different co-initiators. experimental composites were formulated with bisgma and tegdma as organic matrix (50/50 wt%) and the following photoinitiator/co-initiator were added: cq + edmab, cq + edmab + dpi , fq, fq + edmab, fq + dpi and fq + edmab + dpi. inorganic particles were added in 60% by final weight. optical properties were evaluated using a spectrophotometer, apllaying the cielab parameter. depth of cure through the iso 4049 and degree of conversion was analized using ftir / atr. the results were submitted to anova and tukey 95%. cq groups showed lower äe than fq. the addition of dpi in groups with fq caused a drop in color grade. cq generated higher depth of cure than fq. fq alone, was able to promote satisfactory degree of conversion (51 ± 1 g3), but the addition of co-initiators promote better values (g4 = 54 ± 2 63 ± 2 = g5 and g6 = 62 ± 3). the cq was able to produce materials with improved optical properties and greater depth of cure than fq. however, fq was able to produce an affective polymerization even without the presence of co-initiators. 068 tooth structural reinforcement and sealing ability of temporary fillings fronza bm*, dal bello y, rosa l, barbizam jvb, rosa v passo fundo university bruna.fronza@hotmail.com the purpose was to evaluate the capability to reinforce remaining tooth structure and sealing ability of temporary filling materials. forty higid human premolars received root canal treatment and class ii mod cavities. restorations (n = 10) were placed with three temporary filling materials: pre-mixed non-eugenol cement (cim – cimpat n, septodont), glass ionomer cement (gic – vidrion r, sswhite) and a light curable composite (bio – bioplic, biodinâmica ltda). higid premolars (h) and endodontic treated premolars with mod cavities (c) were used as control. the samples were tested for compressive strength. for microleakage test teeth were prepared with mod cavities and after restoration placement (n = 6) the samples were soaked in methilen blue for 24 hours dye penetration and evaluated under optical microscope. beam-shaped specimens of each materials (n = 10) were facricated and tested for flexural strenght. means±standard deviations for maximum compressive load (n) were 10006.0±273.7; 790.3± 259.8; 622.1±157.2; 330.4±105.6 and 324.9±155.2 for h, gic, cim, bio and c, respectively. flexural strength (mpa) was 29.2±4.3 for gic; 5.9±2.0 for cim and 28.7±7.3 for bio. gic presented higher microleakage (2.1) compared to bio (0.5) and cim (0.3). bio and cim presented the lower microleakage scores and can be considered good choices to seal cavities; however gic presented the higher fracture resistance. 070 volumetric shrinkage and conversion of low-stress flowable dental composites nishida ac, yamasaki lc, pfeifer c, francci c university of sao paulo ale-nishida@ig.com.br recently, low-stress restorative technologies have been developed, both as regular consistency pastes and easier to place flowable materials, which have been proposed as bulk-fill composites. this study analyzes such materials compared to their conventional analogs in terms of their shrinkage and conversion, important determinants to final mechanical properties and stress development. six composites were selected: venus diamond (vd) and esthetxhd (ehd) as the conventional controls, venus diamond flow (vdf) and esthetx flow (esf) as conventional flowable composites, and venus bulk fill (vbf) and surefil sdr flow (sdr) as low-stress flowable composites. volumetric shrinkage (vs, n=3) was determined in a linometer (acta) for 30min. specimens (6mm x 1.5mm) were photoactivated (740mw/cm2) for 27s. conversion (dc) was determined on the same specimens right after the linometer run in near-ir (6165cm-1). results were analyzed with one-way anova/tukey’s test (á=5%). due to their lower filler content, flowable composites presented higher vs, in general accompanied by higher dc. vd presented the lowest vs due to its higher molecular weight compared to conventional counterparts. within the “low-stress” materials, sdr presented the lowest vs but also the lowest dc. in spite of their higher vs and conversion, a better modulus match with dentin may help reduce overall stress. provided that the occlusal layer is constructed with high inorganic content composites, bulk-filled flowable restorations may be a viable, less time consuming restorative alternative. 072 influence of non-carious cervical lesion geometry and occlusal loading on biomechanical behavior of maxillary premolars pereira ag*, machado ac, reis br, zeola lf, soares pv federal university of uberlandia analicegpereira@hotmail.com occlusal contact outside the long axis of tooth can be critical when associated with non-carious cervical lesions (nccl). this study aimed to analyze the influence of the nccl geometry, restored or not, and the loading condition on the biomechanical behavior of maxillary premolars. three-dimensional finite element method was used, and 7 linear, elastic, isotropic models were generated. besides the sound tooth (h), 3 different morphologies of nccl: wedge (cn), rounded (re), and mixed (mi), and their respective models restored with composite resin (r) were generated. the mechanical properties were set after volume definition tridimensional mesh was generated. models underwent to 3 loading conditions (100n): vertical (cv), buccal (cb) and palatal (cp); with displacement at the base and lateral of the models. data were analyzed by maximum principal stress. cp group showed the highest tensile stress values at the lesion center, while for cv and cb stresses were concentrated at the upper wall of the lcnc. the cn model showed the highest stress concentration. it was concluded that the lesions associated with acute angles, resulted in higher stress concentration when subjected to oblique loading. the cp was the most damaging to the tooth structure. nccls restored with composite resin favor similar stress pattern, to sound tooth models. gbmd 2013 – 49th meeting of the brazilian group of dental materials 249249249249249 braz j oral sci. 12(3):237-274 073 analysis of the degree of conversion of different resin cements polymerized under different ceramic systems lopes cca*, rodrigues rb, da silveira júnior cd, simamoto-júnior pc, faria-e-silva al, soares cj, novais vr federal university of uberlandia milalopes_81@hotmail.com this study aimed to evaluate the degree of conversion (dc) of polymerized resin cements under ceramic systems. were evaluated four resin cements (n = 5): one self-curing resin cement (multilink); and three dual-curing resin cements (variolink ii; allcem, e relyx u200), photocured after 5 minutes, under three types of ceramics (feldspathic, lithium disilicate glass-ceramic and reinforced by zirconia), for 120 seconds. the control group consisted of polymerization of the cement without the interposition of the ceramic disk. after 24 hours, the gc was measured by fourier transform infrared spectroscopy (ftir). data were analyzed by factorial anova with two factors studied (resin cement and ceramic), followed by tukey’s test (α = .05). it was significant for the cement factor (p <0.001). however, the factor ceramic (p = 0.540), and the interaction between the factors (p = 0.893) were not significant. means and standard deviations for the gc: allcem (67.2 ± 5.2) a; variolink ii (64.5 ± 1.7) ab; relyx u200 (63.7 ± 4.1) b; multilink (57.6 ± 4.2) c. was concluded that the ceramic system did not affect the gc of resin cements, while the chemically activated cement showed the lowest values for the gc. 075 influence of clip material and cross-section of the bar framework on the stress in implant-retained overdentures3d fea bacchi a, spazzin ao, dos santos mbf, anacleto fn, vitti rp, correr-sobrinho l, sinhoreti mac, consani rlx piracicaba dental school unicamp atais_bacchi@yahoo.com.br the aim of this study was to evaluate the stress concentration caused by different cross-sections of bar frameworks and clip materials used to retain overdentures. three-dimensional models of a severely resorbed jaw and an overdenture retained by two implants and bar-clip attachment system were modeled using specific 3d modeling software (solidworks 2010). a vertical misfit of 100 µm between the implant and the bar framework was made in the right side. a total of six models were made according to the cross-section of the bar framework (round, oval or hader®) and the clip material (gold or plastic). finite element models were obtained by importing the solid model into mechanical simulation software (ansys workbench 11). the base of the mandible was set to be the fixed support and a pressure (100 mpa) was applied to the right inferior canine. the analysis was made by means of von mises stress for the prosthetic components and microstrain to the bone tissue. round bars led to lower values of stress in the clip, prosthetic screw of the ill-fitted component and lower microstrain values in the peri-implant bone tissue. the lowest values of stress in the bar were observed in the hader® groups plastic clips reduced the stress concentration in all structures when compared to gold clips. the clip material and the cross-section of the bar framework presented relevant influence on the stress distribution in overdentures retained by a bar-clip system presenting vertical misfit. 077 influence of different ways and times of volatilization of solvent on the mechanical properties of adhesive systems barbosa if*, araújo ctp, prieto lt, araujo lsn, pereira gds, marchi gm, paulillo lams piracicaba dental school unicamp barbosa.isabelferreira@gmail.com union to dentin can be affected by the presence of residual water or excess solvent interfering in the mechanical properties of adhesive systems. this study evaluated the influence of time and form of solvent volatilization of ultimate tensile strength (uts) (n = 10), flexural strength (rf) (n = 5) and modulus of elasticity (me) (n = 5) of adhesive system scotchbond multi-smp and clearfil se-cse. for this shaped samples bar (2x1x7mm)were confectioned with 10ìl of primer, evaporating with jets of air at ± 23 ° c, ± 40 ° c and a negative control at the times 5, 20, 30 and 60 seconds according to the experimental group, and put up 20 ìl of bond on the primer, and homogenized for 60 seconds and light-cured for 60s. the result of the anova and tukey showed that volatilization to ± 40 º c resulted in better me, for both adhesives and in any time interval. there were no significant differences between the times and modes of volatilization for uts. the cse had higher rf and the time of 30s for solvent volatilization for cse increased the me. the results of this study indicate that the volatilization temperature of ± 40 ° c was able to promote the mechanical properties based on the relative stiffness of the material of both adhesives tested independent of the time of evaporation 074 biodegradation of restorative materials, in situ padovani gc*, fúcio sbp, araújo gsa, ambrosano gmb, sinhoreti mac, puppin-rontanni rm piracicaba dental school unicamp gcpadov@yahoo.com.br the aim of this study was to evaluate the influence of biofilm on the surface characteristics (roughness-ra, surface hardness-vhn, energy dispersive spectroscopyeds and scanning electron microscopy-sem) from different restorative materials, in situ. fifteen discs of each material [ips e.max (in), filtek supreme (fs); vitremer (vi); ketac molar easymix (km); amalgam gs-80 (am)] were prepared in a metallic mold (4.0 mm x 1.5 mm). ra, vhn, sem and eds were initially evaluated. fifteen healthy volunteers wore palatal devices containing 5 wells (one restorative material per well) for 7 days. after interacting with the biofilm, ra, vhn, sem and eds were again evaluated. data were statistically analyzed using the kolmogorov-smirnov and tukey-kramer tests (p<0.05). all esthetic restorative materials demonstrated a significant increase in ra values after biodegradation. observed increase in vhn for am, km and vi compared to vhn values before biodegradation. after biodegradation, the vhn values were significantly different: em>am>fs=km >vi. the sem showed porosities, cracks and surface irregularities in all materials tested. while the eds for fs showed accumulation on the surface of cl-, k+ ca2+ and a decrease of the fpeak for vi and km after biodegradation. under the conditions of this study, one can conclude that the effects of biofilm on the surface properties are material-dependent. 076 efficiency of electric currents applied to dentin resin adhesives abuna g*, vitti rp, feitosa vp, bacchi a, santos mbf, souza-junior ej, sinhoreti mac piracicaba dental school unicamp abuna@outlook.com the aim of this study was to assess the effect of the application of different electric currents on the tensile bond strength of dentin adhesives. there were used third molars, which were divided into groups according to the bonding agent and the electric current employed (n=5). there were assessed two self-etch adhesives (clearfil se bond, kuraray and adper easy one, 3m espe) and one etch-and-rinse adhesive (adper single bond 2, 3m espe). each adhesive was applied under different electric current (0µa, 5µa, 10µa, 15µa, 20µa, 25µa, 30µa, and 35µa). build-ups were constructed using the resin composite filtek z350 xt (3m espe). the microtensile bond strength (µtbs) was analyzed after 24h. the results of µtbs were assessed using a two factors anova test, and a tukey test (p<0.05). the results show significant differences between the electric currents and the adhesives systems evaluated, easy one and the 5µa – 20µa obtained the lowest µtbs, concluding that the µtbs could be improved with the application of adhesives under electric currents 078 interaction of surface between universal adhesives of one bottle and chemical and dual cure resin cements guimaraes ir*, shinohara m, de goes mf piracicaba dental school unicamp isaguimaraes_1@yahoo.com.br the aim of this study was to evaluate the morphology and bond strength (sb) of contemporary adhesive systems on dentin surface. the occlusal surfaces of 42 third molars were removed and dentin prepared with medium sandpaper 600 sic. the indirect restorations lava were blasted with aluminum oxide and silanized. the tooth were divided into 6 groups according to the cementation technique: group 1 all bond / c & b bond, group 2 single bond universal (sbu) photoactivated and relyx ultimate (rx) chemical group 3 sbu photoactivated / relyx ultimate photoactivated, group 4 chemical sbu / relyx ultimate photoactivated, group 5 chemical sbu / relyx ultimate chemical, and group 6 sbu + dca / relyx ultimate chemical. the tooth (n = 7) were stored in distilled water at 37 ° c for 24 hours and sectioned shaped sticks with area 0.8 ± 1.0 mm² for the tensile test using a testing machine ez-test. the fracture mode was observed by sem (scanning electron microscopy). the results were submitted to anova and tukey’s test (p <0,05). the results for sb were: 1to 59.9 (9.3); 2to 57.6 (15.9); 3to 63.9 (12.7); 4to 53.7 (13.9 ), 5to 16.2 (6.4) and 6-15.3 (4.3). all the materials that were light cure exhibited sb statistically higher when compared with the chemical polymerization method. the findings of this study support the concept that some materials depend significantly on light to reach polymerization effective and achieve the maximum mechanical properties. gbmd 2013 – 49th meeting of the brazilian group of dental materials250250250250250 braz j oral sci. 12(3):237-274 079 evaluation of different surface treatments with h2o2 in the bond strength of fiber posts to resin cement santos djs*, rodrigues rb, roscoe mg, santos filho pcf, menezes ms, simamoto-júnior pc, soares cj, novais vr federal university of uberlandia danda_777@hotmail.com this study evaluated the effect of surface treatment of fiber glass pin with different concentrations of hydrogen peroxide (h2o2) on the bond strength to the resin cement (relyx unicem). fifty bovine roots received endodontic treatment and then were randomly divided in five groups (n=10), according to the surface treatment performed: g1, negative control; g2 industrialized 24% h2o2, g3, 24% h2o2 manipulated; g4 35% h2o2, g5, h2o2 40%. the roots were sectioned perpendicularly to the long axis, resulting in slices of 1 mm thickness in different thirds (cervical, middle, and apical). the samples were subjected to mechanical testing of push-out. the surface treated pins was evaluated by scanning electron microscopy (sem). two-way anova showed no statistical difference for the surface treatment factors (p = 0.304), and interaction between it and the root region (p = 0.083). there was significant difference between the root thirds (p <0.001). one-way anova showed no statistical difference between the treated groups and the control group (p = 0.102). sem analysis showed that in g2 and g5 treatment with h2o2 was able to increase the surface roughness of the pin due to disruption of epoxy resin matrix. it was concluded that the surface treatment with different concentrations of h2o2 did not affect the bond strength between fiber post and resin cement in the root canal, and, regardless of treatment, cervical showed higher bond strength. 081 survival rate, load to fracture and fea of anterior teeth restored with laminate veneer varyng the design prepare bergoli cd*, meira jbc, valandro lf, bottino ma sao jose dos campos state university – unesp serginhobergoli@hotmail.com this study evaluated the survival rate, load to fracture and stress distribution in maxillary anterior teeth restored with ceramic veneers varying the design prepare. thirty maxillary central incisors (ci) and thirty canines (c) were selected. after standardizing the specimens, they were allocated in four groups (n = 15): gr1-ic with conservative preparation; gr2-ic with conventional preparation with palatal chamfer; gr3c with conservative preparation; gr4 c with conventional preparation with palatal chamfer. the specimens were restored with ceramic veneers based lithium di-silicate. the specimens were subjected to 4 million mechanical cycles (45 °, 37 ° c, 100 n, 4 hz) and evaluated at every 500,000 cycles. the specimens that survived were submitted to load to fracture test. 2d models, corresponding to each groups, were obtained (rhinoceros 4.0) and evaluated (msc.patrans e.msc.marc 2005r2 2005r2) by the values of maximum principal stress. the survival rates were calculated by kaplan-meier test and log-rank test (á = 0.05), and the fracture load values using t-student test (á = 0,05). the different designs of preparation had no influence on survival rates or on the loads to fracture values. the finite element analysis showed higher tensile stress concentration in veneers submitted to conventional preparation with palatal chamfer. we conclude that different designs of preparation does not influence the mechanical behavior of teeth restored with ceramic veneers. 083 evaluation of coconut water neutralized by different agents on the viability of fibroblasts rocha am, moura ccg, reis mvp, soares pbf, soares cj federal university of uberlandia aletheiarocha@yahoo.com.br coconut water (cw) is proposed as a storage medium for avulsed teeth by having nutrients which maintains cell viability. however, its acidic ph should be adjusted. the aim of this study was to compare the effect different ph adjustments of the cw on the fibroblasts viability (fb). natural and industrial cw (ducoco) were adjusted to ph 7.0 using: (1) naoh, (2) nahco3, (3) triethanolamine, (4) amp(amino methil propanol). fibroblasts were plated at 2x104/ well in 96 well plates in dmem 10%. after confluence, dmem was removed and replaced with a solution of 2, 4 and 6 hours. the positive control was represented by fb maintained in dmem and the negative one kept in tap water. then, we analyzed the viability by mtt formazan method. data were analyzed by grafpad. at 2 h, cw set with natural substances 2, 3 and 4 showed higher viability than its corresponding industrialized (p <0,05). at 4 h, natural cw neutralized by substances 1 and 3 were significantly higher than their corresponding industrialized (p <0,05). at 6 h, natural cw showed statistically higher absorbance than industrialized cw adjusted with the same agents. the results of this study indicate that ph adjusted natural cw showed better performance, independently of adjusted solution used. 080 influence of reline material type on stress distribution over ridge: finite element analyses garcia-silva tc*, dos santos mbf, correr-sobrinho l, consani rlx piracicaba dental school unicamp tales_candido@hotmail.com the process of mandibular ridge bone resorption promotes misfit between prosthesis base exacerbating bone resorption; in this case, relines have being used. this threedimensional finite elements method study aimed to evaluate the effect stress of a overdenture in models that simulated bone resorption (in installing prosthesis, 3, 5 and 10 years of use) and reline materials (soft or hard) on stress distribution in posterior ridge. through the computer aided design software (solidworks), threedimensional prosthetic components were built and jaw models resulting in four models. these geometric models were obtained through export to the specific mechanical simulation software (ansys workbench), and subjected to a load of 100 n in the first right molar region. the lowest observed values were in the period of installation prosthesis for all situations. the stress distributions in models with 3, 5 and 10 years of resorption were similar considering the location of tensions; however, the stress concentration increased values in bone, always on the side of load application. the longer resorption, the higher stress values were generated, with the influence of reline materials. it can be concluded that: the power increase of posterior ridge or peri-implant bone resorption promotes increased tensions in the prosthetic components in the bone tissue; the use of soft or hard relines, minimized the generated tensions; regardless the stuff resilience, the hard reline was more efficient in reducing tensions when compared to soft reline. 082 the role of resin cement on bond strength of glass-fiber posts (gfps): a systematic review and meta-analysis sarkis-onofre r*, skupien ja, cenci ms, moraes rr, pereira-cenci t federal university of pelotas rafaelonofre@terra.com.br a systematic review was conducted to verify if there is difference in bond strength to dentin between regular and self-adhesive resin cements and verify the influence of several variables on the retention of gfps. this report followed the prisma statement. in vitro studies that investigated the bond strength of gfps luted with self-adhesive and regular rcs were selected. searches were carried out in pubmed and scopus databases. no publication year or language limit was used, and the last search was made in october 2012. global comparison between self-adhesive and regular resin cements was performed. two subgroup analyses were performed: selfadhesive × regular resin cement + etch-and-rinse adhesive, and self-adhesive × regular resin cement + self-etch adhesive. the analyses were carried out using fixed-effect and random-effects model. the results showed heterogeneity in all comparisons and higher bond strength to dentin was identified for self-adhesive cements; self-adhesive cements also presented higher dentin bond strength than regular resin cements + etch-and-rinse or self-etch adhesives. the in vitro literature seems to suggest that the use of self-adhesive resin cement could improve the retention of gfps into root canals. 084 influence of polishing systems and chemical degradation on the color of the resin materials inagaki lt*, naufel fs, de paula ab, cardoso m, lima danl, puppin rontani rm piracicaba dental school unicamp luciana.inagaki@gmail.com this study evaluated the influence of different polishing systems (ps) and staining solution on color of composites. 72 cylindrical samples (7 mm diameter x 2 mm thick) were prepared with filtek z350xt (3m/espe) and vitalescense (ultradent) composites. after 24h at 37°c, the samples were polished with soflex (3m/espe) or jiffy (ultradent) systems. they were randomly divided into 8 groups (n= 9) in accordance with composite, ps and solution (coffee (cf) and artificial saliva (sa)control). initial color measurements were made using ciel*a*b* system. then, each sample was immersed in 4 ml of cf for 15 min, 3 times a day for 14 days. after that, the color was measured again and color variations were calculated (∆e). normal data distribution was verified (kolmogorov-smirnov test, p>0.05) and submitted to three way anova and holmom-sidak test (p<0.05). both composites showed some staining after degradation in cf and sa. differences were showed in triple interaction (p= 0.001). the composites polished by ps showed higher staining in cf than sa. the z350, in cf and sa, showed similar staining for soflex and jiffy. however, vitalescence in sa showed higher staining when polished with jiffy; and in cf, showed higher staining when polished with soflex. the staining was influenced by the three factors studied. the staining on z350 was not affected by ps. however, vitalescence showed less staining when immersed in sa and polished with soflex; and when immersed cf and polished with jiffy. it could be concluded that color stability is material, solution and ps dependent. gbmd 2013 – 49th meeting of the brazilian group of dental materials 251251251251251 braz j oral sci. 12(3):237-274 085 morphological and structural changes of y-tzp ceramics after different surface treatments tostes bo*, silva em, guimarães rb, sampaio filho hr, mondelli rfluff, labiom-r and fob-usp bhenya@hotmail.com the purpose of this study was to evaluate morphological and structural changes of three y-tzp dental ceramics (lava frame lf, lava plus lp and ips zircad iz) submitted to three surface treatments. plates obtained from sinterized blocks of each ceramic were divided into four groups according to the surface treatments: control – c – (as-sintered); 30 (abrasion with 30 ìm si-coated al2o3 particles); 50 (abrasion with 50 ìm al2o3 particles) and 150 (abrasion with 150 ìm al2o3 particles). after the surface treatments, the plates were submitted to the following analysis: surface roughness (sa ìm) by using a 3-d perfilometry; phasetransformation by using x-ray difratometry; surface morphology by using scanning electron microscopy (sem) and elemental composition by using energy dispersive x-ray spectroscopy (eds). the abrasion increased the roughness of all ceramics, with 150 group presenting the highest values. it was observed tetragonal to monoclinic and cubic phase transformation in all ceramic surfaces after abrasion. sem analysis showed changes in ceramic surface morphologies, with presence of grooves, after abrasion. the elemental composition analysis showed a decrease in zr content and a increasing in o and al elements after abrasion. the presence of si was also detected in 30 group. it was conclude that although the surface treatments increased the roughness, they also produced a tetragonal to monoclinic and cubic phase transformation which could compromise the mechanical behavior of materials. 087 linear polymerization shrinkage and shrinkage stress of silorane varying the photoactivation mode almeida gs*, carvalho ms, silva em, poskus lt labiom-r almeida.giselle@gmail.com the aim of this study was to analyze the linear of polymerization shrinkage (lps) and the shrinkage stress (ss) of different composites: durafill vs/heraeus kulzer (d), filtek z250/3m-espe (z2), filtek z350/3m-espe (z3), filtek p90 /3mespe(p). cylinders (2mm x 2mm) of each composite (n=5) were photocured with different methods and similar power density (24j/cm2): low (600mw/cm2 x 40s), high (1000mw/cm2 x 24s) and softstart (200mw/cm2 x 15s + 600mw/cm2 x 35s). for lps, each composite (n=5) was inserted between two glass plates treated with hydrofluoric acid and silano, and an extensometer (emic) recorded the polymerization shrinkage for 10 min. for ss, polymethylmethacrylate rods treated with methyl methacrylate liquid and coated with the single bond 2 (3m espe) for methacrylic composites and the silorano adhesive for p. the ss considered the values of compliance mountings. lps (%) and ss (mpa) were recorded for 10 min. data were statistically analyzed by two-way anova and tukey test (5%). for lps only individual factors were statically significant: p=z3=z2; d z3s = z3l; ps < ph = pl. conclusion: all the composites showed lower values for lps using the s mode and the kind of composite influenced data of lps. for ss, the s mode led to the lowest values for p and z3, with no influence to z2. 089 evaluation in vivo of the roughness and surface morphology of enamel after removal of brackets with different polishing guiraldo rd*, faria-júnior em, berger sb, lopes mb, carvalho rv, goninijúnior a, moura sk, contreras efr, correr ab, correr-sobrinho l university of north parana – unopar rdguiraldo@gmail.com the aim of this study was to evaluate the roughness and morphology of the enamel by surface roughness tester and scanning electron microscopy (sem) after removal of metal brackets. 10 patients were selected, they had no caries, restoration, trauma, bruxism or cracks on the upper incisors. after the conclusion of treatment, the brackets were removed. the teeth of the patients were randomly polished, to one side previously drawn was performed finishing and polishing with sof-lex or carbide bur multi-laminated (n = 10). replicas dental with polished teeth were obtained using epoxy resin. surface roughness measurements were performed. the data were statistically evaluated by student t test. after the roughness test, three specimens from each group were used for the sem analysis. student t test showed that the carbide bur group (0.31 ± 0.07 µm) had significantly greater irregularities when compared with the sof-lex group (0.25 ± 0.02 µm) after composite resin removal. the sof-lex polishing system showed the best polishing enamel. 086 effect of cooling protocol on biaxial flexural strength and failure behavior of a bilayer ceramic system amaral m*, freitas gs, benetti p, valandro lf, bottino ma sao jose dos campos dental school unesp marinamaral_85@yahoo.com.br the purpose of this study was to evaluate the biaxial flexural strength (óf) of a bilayer ceramic system submitted to cooling protocols of porcelain layer (vm9) applied on zirconia ceramic (yz). z discs were manufactured, received a layer of vm9 and were divided into groups according to cooling protocol from sintering to room temperature with furnace opening at: 200°c, 400°c, 600°c or 800°c. after surface finishing, samples were submitted to flexural strength (σf) test, which was interrupted at the first signal of fracture. failure mode was classified as crack of vm9 until system interface or catastrophic failure. flexural strength was influenced by the opening-temperature from sintering furnace (p=0.0283). the lowest strength values was found in g800 (62.91 mpa)b, and g400 presented the highest flexural strength values (112,99 mpa)a. remaining groups presented middle strength values: g200 (97.46 mpa)ab, g600 (95.88 mpa)ab. the main failure mode found was crack of vm9 until system interface for all tested groups, and in previous failures (vm9 presenting cracks after cooling). furnace opening is advised to be carried out on temperatures lower than 600°c, since an increase in bilayer ceramic system strength was observed under this condition. 088 analysis of the wettability of experimental monomers and icon ® benetello v*, inagaki lt, ozera eg, alonso rcb, pascon fm, puppin-rontani rm piracicaba dental school unicamp vanb09@hotmail.com the wettability of a liquid is determined by the contact angle once the greater is the wettability; the smaller is the contact angle. the aim of this study was to evaluate the wettability of four experimental resin materials (mer) over two different surfaces: smooth(s) and rough(r). the infiltrant icon® was used as commercial control group. the materials were distributed in the following groups (n=10): g1-tegdma (pure monomer); g2-(tegdmai) tegdma infiltrant with 0.5% of canphoroquinone, 1% of dmaema and 0.1% of bht; g3-tegdmai+0,1% of chlorhexidine (chx); g4-tegdmai+0,2%chx; g5-icon®. the wettability was evaluated by the left and right contact angles which were obtained by drop in the glass surface (s and r), using goniometer digidrop (labometric, lda). the data were submitted to two way anova and tukey test (p<0.05) to compare g1, g2, g3 and g4 with each other; and dunnet’s test (p<0.05) to compare g5 with the other groups. there were interaction between the surfaces and materials (p<0.01). in s surface, g1 (51.98 ± 5.50) showed higher contact angles and was statistically different when compared with groups g3(41,94 ± 4,69) and g4(41,92 ± 4,72). in r surface, g1 (47,28 ± 8,26) and g2(45,57 ± 8,59) showed higher contact angles, followed by g5(35,29 ± 9,32), g3(24,62 ± 6,69) and g4(20,94 ± 3,31). comparing the materials over different surfaces, g3, g4 and g5 showed higher contact angle than g1 and g2. according to the results, it was concluded that the materials with chx-added on rough surface showed the lowest contact angles; and influenced positively in the mer wettability property. 090 influence of the matrix for making cylinders to testing bond strength by microshear vieira hh*, catelan a, aguiar fhb, paulillo lams, lovadino jr, lima danl piracicaba dental school unicamp henriquefidi@hotmail.com the aim of this study was to compare bond strength of composite resin cylinders to dentin prepared with three types of matrices. composite cylinders were prepared on bovine dentin blocks, using three types of matrices (n = 20): 1 matrix made with addition silicone, 2 matrix made with drilled noodles; 3 matrix made with tygon tubes. the cylinders were made with simplified conventional adhesive (single bond / 3m espe) and flowable composite (filtek z-350 flow / 3m espe). 1 and 3 types of matrices were removed by cutting. noodles matrices were removed after cylinder immersion in water for 1h. dentin fragments who failed pre-test were excluded. the specimens were submitted to microshear (emic ddl 500, 1mm/ min). the fracture pattern was evaluated with a stereomicroscope (leica microsystems, 50x). data were analyzed by anova with a criterion (α = .05). there was no statistical difference between the types of matrices (p = 0.7427). the matrix of noodles showed the highest rate of cohesive failures in the substrate, and showed no failures pretest. the silicone matrix showed the highest rate of adhesive fractures. the matrix of tygon had the highest rate of fractures mixed, with showed most pretest flaws, followed by silicone matrix. all matrices were effective for making cylinders. the matrix of noodles simplified the process and this way pre-test failure does not occur. gbmd 2013 – 49th meeting of the brazilian group of dental materials252252252252252 braz j oral sci. 12(3):237-274 091 bactericidal activity evaluations of adhesive systems andré cb*, duque tm, chan d, ambrosano gmb, gomes bpfa, giannini m piracicaba dental school unicamp carolina.bosso@gmail.com this study evaluated the direct contact bactericidal activity of three adhesive systems containing antibacterial compounds against four facultative bacterial species (staphylococcus aureus, enterococcus faecalis, streptococcus mutans and lactobacillus casei). test adhesives: gluma 2bond (g2b heraeus), clearfil se protect (csp kuraray noritake), clearfil se protect primer (pcsp) and peak universal bond (pub ultradent). control adhesives: gluma comfort bond (gcb), clearfil se bond (csb) and peak lc bond (plb). positive controls: chlorhexidine 0.2% and 2% and glutaraldehyde 5%. negative controls: physiological saline solution or only the inoculum. after bacterial growing, isolated colonies were suspended until reaching 0.5 of the mcfarland scale (inoculum). thirty microliters of adhesive systems were dispensed in a sterile cylindrical device and photoactivated. each cylinder adhesive or control substance was added to a well (96 well plate) containing 90ìl of inoculum (test performed in triplicate). after contact, 5ìl of the inoculum from each well were plated at the following times: 5 min, 10 min, 30 min, 1 hour and 24 hours. after 24 hours it was verified the bacterial growth (not bactericidal). the g2b presented results similar to its negative control (gcb). the pcsp promoted antimicrobial activity against all oral pathogens the pub was not bactericidal against any bacteria tested. the g2b, csp and pcsp showed antimicrobial activity, while pub promoted no bactericidal potential. 093 effect of alteration of the dimensional elastic orthodontic stored in sodium fluoride and artificial saliva reges rv*, paranhos djc, bites c, castro fal, lenza ma, silva rf, botelho tl, campos bb paulista university unip vieirareges@yahoo.com.br the aim of this study was evaluation of the dimensional changes of orthodontic elastics, being subjected to storage in artificial saliva 37c (control) and sodium fluoride 0.05% during the immediate 24 hours and 30 days. the elastic split in five different colors with five samples each (grey, green, black, pink and transparent) and trademarks (morelli, 3m unitek, ormco). we used analysis machine dimension (profilometer) mitutoyo to evaluate the changes in size of each elastic as the factors involved. then the data were analyzed statistically, getting the results and therefore made tables and charts. the results showed that the orthodontic elastics in the middle of artificial saliva showed significant change after 30 days. amid sodium fluoride 0.05% had no significant dimensional change between periods. faced trademarks, dimensional stability both presented in accordance with the storage times for both synthetic saliva and sodium fluoride 0.05%. generally when compared to media solution, the sodium fluoride was higher dimensional change when compared with the artificial saliva. 095 restorative system and thermocycling effect on tooth/class v interface: oct evaluation sampaio cs*, rodrigues rv, pascon f.m, ambrosano gbm, correr-sobrinho l, souza-junior ej, freitas az, puppin-rontani rm piracicaba dental school unicamp camisobral@hotmail.com it was evaluated tooth/class v restoration interface accomplished by different adhesive systems -as (total and self-etching) and restorative composites –rc (low shrinkage and conventional), submitted to thermal cycling (tc), using optical coherence tomography (oct). 60 class v cavities were accomplished on extracted sound 3rd molars. and distributed into 6 groups according to as and rc (n=10): g1adper single bond 2 (sb2)+ aelite ls posterior (ap); g2sb2 + venus diamond (vd); g3sb2 filtek z250 + xt (z250); g4clearfil se bond (cse) + ap; g5cse + vd; g6cse + z250. specimens were analyzed by oct before/after tc (1000 cycles: 5°c and 55°c, dwell time of 30s in each bath). the interface dentin/restoration and enamel/restoration gap (%) was evaluated separately from one oct shot from each group using image j. statistical analysis used was of mixed model methodology for repeated and tukey kramer test (p<0.05). for dentin interfaces, it was observed a significant interaction between as and tc; the restorations with cse as, after tc, showed smaller percentages of gaps at the dentin/restoration interface, for all composites, comparing to the sb2 adhesive system. concerning tc, the results showed lower values of interfacial adaptation, when compared the same samples prior to their implementation. in enamel, it was not observed interface gaps (pre and post-tc), for all rc and as. depending on the as, tc can provide higher percentage of gaps at the dentin/restoration interface, for all rc. cse provided improved interfacial adaptation than sb2. oct was capable of evaluating the marginal integrity of class v resin composite restorations. 092 influence of the initiation system in performance and stability of an experimental self-adhesive composite resin meereis ctw*, leal fb, de almeida cm, ogliari fa federal university of pelotas carinemeereis@gmail.com this study simulated the shelf life to evaluate the stability of initiation systems in an experimental self-adhesive composite resin (sacr). as sacr model was used a mixture by bis-gma, tegdma, gdma-p and filler particles. five sacr were formulated changing the initiation system: cq, cq+edab, cq+edab+dpihfp, bapo e tpo. analysis of the polymerization kinetics in real time was carried out in triplicate by infrared spectroscopy (is). to simulate shelf life the sacr were maintained at 23 ° c and according to the storage time were evaluated for degree of conversion (dc) and microtensile bond strength (µtbs). analysis of dc by is was performed in triplicate after 0, 7, 14, 30, 60, 90 and 180 storage days of sacr. to evaluate the µtbs in dentin (n=10), restorations were build up after 0, 30, 60, 90 and 180 storage days of sacr, , which were sectioned and the beams was tested for ìtbs after 24h and 6m. data were analyzed by one-way anova and holmsidak (p <0.05). the initiation system cq+edab+dpihfp showed higher polymerization rate. the dc of cq+edab, cq+edab+dpihfp and bapo were similar (40%) and higher than tpo e cq (20 e 10%), maintaining stable up to 180 days. the cq, cq+edab e tpo did not presented adhesion. the aging of sacr (180 days) did not affect the adhesion. the immediate µtbs (mpa) cq+edab+dpihfp (8.4) was similar to bapo (10.5), however after 6m only cq+edab+dpihfp maintained bond strength. the choice of a suitable initiation system is crucial to the performance and stability of sacr. 094 effect of non-carious cervical lesion, restorative material and loading on the deformation of premolars-moiré method zeola lf*, milito ga, pereira fa, pereira ag, machado ac, riveira jlv, dantas no, soares pv federal university of uberlandia livia.zeola@terra.com.br the tooth structure reduction is modulating factor of the premolars biomechanical behavior. the aim of this study was to analyze the influence of non-carious cervical lesions (nccl), mesio-occlusal-distal (mod) cavity restorative material and occlusal loading variations on the deformation of premolars (pms), using the moiré projection technique. eighteen premolars (pms) were selected and randomly distributed into 6 groups (n = 3). group a: mod cavity restored with amalgam, al: mod amalgam + not restored nccl; alr: mod amalgam + restored nccl; r: composite resin mod; rl: composite resin mod + nccl not restored; rlr: composite resin mod + restored nccl. the samples were subjected to two types of occlusal loading: axial and oblique. the deformations were analyzed by moiré projection technique in mesiodistal (md), cervical occlusal (co) and bucco-lingual (bl) directions. after anova and tukey test (p <0.05), the results showed that the groups with not restored nccl presented higher strain values in both co and md directions, regardless of the occlusal loading type. the al group subjected to oblique loading showed the highest strain values for co and md directions. on bl direction the type of loading and mod cavity restorative material influenced the samples deformation. from the results, it was concluded that through the moiré projection technique it is possible to quantify the deformations in the tooth structure and that the presence of nccl, variations of occlusal loading and restorative material type influence the deformation pattern of premolars. 096 marginal adaptation between glass fiber post and primary root dentin iwamoto as*, alonso rcb, benetello v, hosoya y, puppin-rontani rm, pascon fm piracicaba dental school unicamp alek-xexe@hotmail.com the aim of this study was to evaluate the influence of filling materials and cleaning agents in marginal adaptation between glass fiber post and root canal dentin. roots of primary bovine teeth were endodontically treated and assigned into groups: control (no material c); calen® + zinc oxide (czo); vitapex® (v); calcipex ii® (cp). after 7 days, the filling materials were removed and the roots were subdivided according the groups (n=10): no cleaning (nc); 70% ethanol (e); tergenform® (t). glass fiber posts were luted using resin cement relyxtm arc after acid etching, rinsing and application of adhesive system adper single bond 2tm. specimens were sectioned (±1 mm), polished and replicas were made with epoxy resin. images obtained by scanning electron microscopy were measured using the software image j 1.45. the percentage of gap formation (%g) was analyzed by an examiner calibrated. the data were submitted to two-way anova and tukey’s test (α=5%). interaction was observed between the studied factors (p<0.05). c/e showed the lowest %g (29.45±15.65), significantly different from t (56.92±19.74). the highest %g was observed for v/e (59.13±21.34) and significantly different from t (35.25±19.89). czo and c showed no significant difference between cleaning agents. in conclusion, the filling materials and cleaning agents influenced the marginal adaptation of glass fiber post to root dentin. when vitapex® is used for root canal filling, its indicate cleaning with tergenform®, while calen®+oz and calcipex ii® not require cleaning agents. gbmd 2013 – 49th meeting of the brazilian group of dental materials 253253253253253 braz j oral sci. 12(3):237-274 097 effect of different tooth bleaching therapies on human dental pulp cells viability pontes ecv *, soares dg, hebling j, de souza costa ca araraquara dental school foar/unesp elainecvpontes@foar.unesp.br the aim of this study was to evaluate the influence of applications of a bleaching gel with different concentrations of hydrogen peroxide (h2o2) on human dental pulp cells (hdpcs) viability. enamel/dentin discs adapted to trans-wells devices were placed on cultured hdpcs, establishing the following groups: g1 no treatment (control); g235% h2o2 / 1x15 min.; g3– 35% h2o2 / 3x15 min.; g417,5% h2o2 / 1x15 min.; g5– 17,5% h2o2 / 3x15 min.; g610% h2o2 / 1x15 min.; g7– 10% h2o2 / 3x15 min.; g88,75% h2o2 / 1x15 min.; e g9 – 8,75% h2o2 / 3x15 min. cell viability (mtt assay) was evaluated immediately (t1) or 72 h post-bleaching (t2) (kruskal-wallis e mann-whitney; á=5%). the variables frequency of application and concentration of h2o2 in the bleaching gel had significant effect on hdpcs viability. in groups g2, g3, g4, g5, g6, g7, g8, and g9 the cell viability reduction (in t1) was: 92.8%; 96.7%; 75.1%; 85.1%; 37.2%; 72.7%; 36.2%; and 59.7%, respectively. g6 and g8 presented no significant difference compared to control (g1). in t2, all groups presented cell viability increase, except g3. cell viability greater than 100% at t2 period was observed in g6 (102.2%) and g8 (114.4%). it was concluded that bleaching gels with low concentrations of h2o2, such as 10% and 8.75%, applied for 15 min on enamel, cause discrete reduction in hpdcs viability, which was recovered at 72 h postbleaching period. 099 effect of dentin desensitizers on resin cement bond strengths garcia rn*, takagaki t, sato t, matsui n, nikaido t, tagami j univille, tmdu and univali rubensngarcia@gmail.com to evaluate the effect of dentin desensitizers on bond strengths of one resin cement. twenty bovine teeth were prepared until obtaining flat surfaces of median dentin. a standardized smear layer was created just before the adhesive procedures. twenty composite blocks, 3 mm thick (estenia c&b, kuraray noritake/kn) were used. the composite surfaces were abraded with aluminum oxide, and then silanized. the samples were randomly divided into the following four groups (n=5): no treatment (con, controle), gluma desensitizer (gd, heraeus kulzer), super seal (ss, phoenix dental) and teethmate desensitizer (tmd, kn). the dentin surfaces were then treated with ed primer ii (kn). the composite blocks were bonded to the dentin surfaces with a resin cement (panavia f2.0, kn) according to the manufacturer’s instructions. after 24-hour storage (37ºc, 100% rh), the bonded samples were cut into beam–shaped microtensile specimens and loaded in tension until failure. data were analyzed with anova and the dunnett’s test (á=0.05). sem was used to examine the fracture modes. the microtensile bond strengths (mpa±sd) were: con 24.4±3.2 b, gd 14.0±5.6 c, ss 8.6±4.7 d and tmd 34.7±4.6 a. the efficacy of the desensitizers agents is material-dependent. gd and ss decreased the µtbs, however the tmd improved. more studies about the mechanism of tmd are necessary in the future. 101 influence of cross-sections and vertical misfit in overdentures bar system in stress distribution nogueira mcf*, caetano cr, caldas ra, campana jt, zen bm, bacchi a, santos mbf, correr-sobrinho l, consani rlx piracicaba dental school unicamp moises.2303@gmail.com passive fit is very important on the long-term success of rehabilitation. however, obtaining this situation is not always a reality due to the inherent problems on manufacturing of infrastructure/prosthesis. when an ill-fitted framework is installed, the prosthetic screws are torqued and might propagate tensions to the whole system. 098 influence of post-thickness and material on the fracture strength of teeth with reduced coronal structure caldas ra*, caetano cr, campana jt, pimentel mj, vitti rp, bacchi a, santos mbf, consani rlx piracicaba dental school unicamp ricardoacaldas@hotmail.com to evaluate the fracture strength of endodontically treated teeth with reduced coronal structure reinforced with glass-fiber posts and cast posts and core (nickel– chromium alloy) with different thickness. forty maxillary central incisors were sectioned at 1 mm of the cement enamel junction and endodontically treated. the teeth were divided into four groups (n=10) and restored with cast post and core and glass-fiber posts with diameters of 1.5 mm and 1.1 mm. the fracture strength was evaluated using a universal testing machine (instron 1144) at 45° of angulation. the results were submitted to analysis of variance two-way and tukey’s test (p<0.05). the failure mode was also evaluated. cast post and core were statistically superior to the glass-fiber posts with the self-post diameter (p<0.001). when the self-post material was considered, no significant difference was observed between the two post-diameters (p=0.749). the glass-fiber post-groups presented more fractures in the cervical third than the cast post and core groups. teeth restored with cast post and cores present higher fracture strength than those reinforced with glass-fiber posts. an increased post-thickness does not increase the fracture strength. glass-fiber posts lead to less severe fractures. 100 influence of implant´s inclination on stress distribution in overdenture-retaining bar system with vertical misfit caetano cr*, nogueira mcf, caldas ra, zen bm, campana jt, bacchi a, santos mbf, correr-sobrinho l, consani rlx piracicaba dental school unicamp conradorc7@gmail.com a concern about implants inclination during clinical planning of an overdenture is always present, especially by the effects on the biomechanics of various restraint systems, such as bar-clip system. the present study evaluated the overdenture barclip system with: different latero-lateral inclinations in an implant (-10, -5, 0, +5, +10); vertical misfits on the second implant (50, 100, 200 ìm) and bar materials (au type iv, ag-pd, ti cp, co-cr) through finite element analysis. three-dimensional models of an overdenture retained by 2 implants and bar-clip system were modeled using 3-d modeling software. finite element models were obtained by importing the geometric model to mechanical simulation software. the inclination +10° showed the worst biomechanical behavior, presenting higher von mises stress in the bar and the highest values of maximum principal stress in peri-implant bone tissue. the group -5º with au bar showed the lowest values of tension on the prosthetic components (151 mpa; 9,37 mpa; bar and prosthetic screw, respectively), and -10° with au alloy, showed the best distribution tension in the peri-implant bone tissue (5,08 mpa). the increase in the vertical variation and hardness of the material of the bar caused an increase in the stress values measured in all structures. the implants’ inclinations have significant influence on the stress distribution at overdenture-retaining bar system. increase of the vertical variation and different materials bar leads to an increase in the stress values. 102 effect of 2% chx on dentin/resin bond strength at different dentin substrate conditions and storage time costa ar*, correr ab, naves lz, sinhoreti mac, correr-sobrinho l, garcia-godoy f, puppin-rontani rm piracicaba dental school unicamp anarosacosta_1@hotmail.com the aim of this study was to evaluate the effect of 2% chlorhexidine digluconate (chx) on microtensile bond strength (µtbs) and degradation between an adhesive system and dentin under 3 dentin conditions and storage times. the bonding interface was also analysed using sem. forty-eight dentin surfaces from sound third molar were divided into 3 groups, according to dentin substrates: sound dentin(sd), cariesinfected dentin(ci) and caries-affected dentin(ca). ca and ci were submitted to development of artificial caries using visual inspection with caries detector solution. it was considered as ci: soft and deeply pigmented dentin and ca: hard and slightly pigmented dentin. chx was applied on half of groups, just after etching with 35% phosphoric acid gel. afterwards, the dentin surfaces were bonded with adper single bond 2(3m espe) adhesive system according to manufacturer’s instructions. teeth were longitudinally sectioned across the bonded interface (1.0mm2). the specimens were stored in deionized water at 37°c for 24h, 6 months and 1 year later. two additional teeth were used to analyze the bonding interfaces by sem. data was submitted to anova and tukey’s test (α= 0.05). ci decreased the bond strength values, when compared to ca and sd. stored samples for 6 months and 1 year decreased the µtbs for all analyzed conditions. chx did not influence bond strength to dentin over time, regardless substrate conditions. time is the most important factor in the bond strength degradation. the bonding to caries-infected dentin decreased the bond strength values, when compared to caries-affected and sound dentin. gbmd 2013 – 49th meeting of the brazilian group of dental materials254254254254254 braz j oral sci. 12(3):237-274 103 evaluation of the abutment retention force in cone morse implants after removal cycles lopes mb*, santos amt, bertoncelo ca, guiraldo rd, berger sb, gonini-júnior a university of north parana unopar baenalopes@gmail.com the prosthetics interferences of morse cone are based on the mechanical principle of “cone inside cone”, which promotes great imbrication by contact between the surfaces. the objective was to evaluate the prosthetic retention of the cone morse implant system after innumerous activations and deactivations. 10 implant/abutment set were used, one with 2mm prosthetic interface (g1) and other with 3mm (g2). each set were sequentially activated for 3 times with 60n and 0 degrees. 10 abutmenst activation/removal were made for each set. the data in mpa was submitted to anova and tukey’s test (á=0.05). g1 showed no difference (p>0.05) from cycle 1 (81.05±14.73) to cycle 8 (87.51±11.55), however a higher traction force (p<0.05) were found in cycle 9 (109.08±16.32) and 10 (109.59±14.22). g2 showed no diference among the 10 activation/removal cycles (cycle 1 – 82.68±10.55, cycle 10 – 97.35±12.17). cone morse system showed no retention reduction after successively activations and removals, characterizing a stable system. 105 effect of silane application form on lithium disilicate ceramic and resin cement bond strength zanini mm*, favarão j, sicoli ea, mendonça mj, guarda gb, grando af, consani s state university of west of parana unioeste mauriciomattezanini@gmail.com the use of silanes as adhesion promoters agents have been routinely employed in dental clinics. in order to evaluate the influence of silanes application form in microshear resistance interface: ips e.max press ceramic relyx arc resin cement, 20 samples were manufactured in ceramics, etched with 10% hydrofluoric acid (fgm) for 20 seconds and separated into 5 groups of ceramic silanized with: asilane angelus, brelyx ceramic primer (3m espe), cbis-silane (bisco), dsilane dentsply, eprosil (fgm). the applications of silanes were performed according to manufacturers’ recommendations. on the ceramic, silicone rubber matrices with three perfurations, were positioned and fulfilled with relyx arc in all groups. the light activation was done using led bluephase (ivoclair vivadent) for 30 seconds. the samples were stored in environment at 37°c for 24 hours and positioned at instron testing machine to microshear test. the results were submitted to analysis of variance and tukey’s test (p <0,05). statistical analysis showed microshear bond strength value significantly lower for group d. the standard fracture analysis showed predominance of ceramic cohesive failure in groups c and e and adhesive problems in groups a and b. group d showed the same percentage of fractures adhesive, cohesive ceramic, and mixed. it was concluded that the manner of application of silane showed different effect on the bond strength between ceramic lithium disilicate and resin cement. 107 critical analyses of glass and quartz fiber post treatment surface protocols – literature review teixeira dnr*, machado ac, vilela alr, pereira ag, zeola lf, faria-e-silva al, menezes ms, soares pv federal university of uberlandia dnrteixeira@gmail.com the step of treating the glass fiber post surface, prior to adhesive cementation, is critical to the rehabilitation successful. the present study aimed, through literature review, elaborate the most suitable protocol for glass fiber post surface treating. as a method, it sought through the platform medline / pubmed, articles of associations with the following keywords: fiber post, fiber post and adhesion, fiber post and bond, e fiber post and surface treatment. it was found 705 articles of which 515 papers were excluded because they are not in english; was about others themes and materials or physic-mechanical properties tests; was literature review; was not completed available; and did not treated the post surface; remaining 190 articles. it was found that the silane was the most common material used, mentioned in 115 (60,52%) papers, followed by application of primer and/or adhesive and use of alcohol, consisting of 70 (36,84%). and 72 (38.29%) of the works, respectively. even mentioned by only 11 (5.78%) of the authors, hydrogen peroxide showed favorable results, unlike treatment with abrasive particles, cited in 34 (17.89%) of the studies, which can promote fractures and damage to the fibers. it was concluded that the surface treatment has no standard protocol established, and for most successful rehabilitation treatment, it is recommended the use of hydrogen peroxide as promoter of roughness and removal of epoxy resin, followed by silane for optimization of chemical bonding. 104 in vitro marginal adaptation evaluation of different restorative materials after thermal cycling girotto ac*, rodrigues rv, puppin-rontani rm, pascon fm piracicaba dental school unicamp liny_cq@hotmail.com despite advances in restorative materials marginal defects can appear as a result of secondary caries. the aim of this study was to evaluate in vitro the marginal adaptation of restorative materials submitted to thermal cycling. 60 cavities were prepared in enamel and dentin of bovine teeth and were randomly divided into 3 groups (n = 10): g1 ketactm molar easymix, g2 vitremertm, and g3 filtektm z350. after 24 hours, replicas were made and the specimens were submitted to thermal cycling (elquip, são carlos, sp, brazil 2880 cycles of 30s at temperatures of 5 ºc and 55 ºc, for 24 hours). after thermal cycling, new replicas were made and observed by scanning electron microscopy to measure the gaps formed (in percentage) using the software image j 1.45. it was calculated the difference between the final and initial gaps formed (äf). the data were analyzed using wilcoxon, kruskal-wallis and dunn (á = 0.05). no significant difference were observed between the materials studied (p>0.05), in enamel, regarding the gap formation. in dentin, significant difference between g1 and g2 (p = 0.0046) and between g1 and g3 (p=0.0145) were observed. g2 and g3 showed no difference between groups (p>0.05). it could be concluded that the enamel marginal adaptation was not affected by thermal cycling, however, the dentin restored with conventional glass ionomer cement showed a greater tendency to gap formation when compared to resin modified glass ionomer cement and composite resin. 106 evaluation of surface roughness of denture liners in different time periods poletto-neto v, luz ms, valentini f, sarkis-onofre r, boscato n, pereira-cenci t federal university of pelotas ufp elvixxtorio@hotmail.com the aim of this study was to clinically evaluate a possible change on the surface roughness of different denture liners. thirty complete denture wearers were selected to participate of this in situ study with 2 phases of 21 days. samples of 2 denture liners (silicone and acrylic based materials) and acrylic resin (control) were inserted inside the maxillary denture and the surface roughness was evaluated before and after their insertion. the samples were removed of the dentures after 7, 14 and 21 days. the denture liners showed higher surface roughness than acrylic resin (control) (p<0.001), with no difference between denture liners (p=0.109). surface roughness was lower on day 7 than on days 14 and 21 (p<0.001). we can conclude that the denture liners tested should be used with caution since they showed an increase of surface roughness over time. 108 influence of the model and brand of diamond bur in flexural strength and failure pattern galvão am*, pereira ag, machado ac, zeola lf, tolentino ab, spini, phr, gonzaga rcq, soares pv federal university of uberlandia alexiamgalvao@gmail.com the diamond burs are rotary abrasives instruments used to perform dental preparations and finishing restorations, and consist of shank, intermediate shaft and active tip. the objective of this study was to measure the flexural strength of the shank and intermediate shaft of six different diamond burs commercial brands, using threepoint flexural test and analysis of the failure pattern. it was used 60 diamond burs, 30 burs model 2200 (n = 5) and 30 burs model 2135 (n = 5), of brands kg sorensen, option, microdont, fava, zeep, vortex. the samples were subjected to the load applied to the center of the shank (2200) and intermediate (2135) by triangular device to the point of fracture, held at the universal testing machine (emic). two failure patterns were defined by analysis of possible fractures of each sample in a magnifying 40x, as follows: abends and b-total fractures. as result, it was observed that kg sorensen rotary instruments, stood out in all tests, showing high values of flexural strength for shank and intermediate shaft, without total fracture failure. option brand had the highest rate of total fracture failure. it is conclude that is not standardization between the flexural trademarks, and the use of instruments with low resistance may cause higher rates of failure, damaging the equipment or cause injuries to the tissues of the patient. gbmd 2013 – 49th meeting of the brazilian group of dental materials 255255255255255 braz j oral sci. 12(3):237-274 109 influence on compressive strength of a composite resin containing nanoplates, nanorods and nanospheres of zno and ag dias hb*, bernardi mib, souza cwo, faria aa, de souza anr, hernandes ac university of sao paulo são carlos hercules@ursa.ifsc.usp.br antimicrobial restorative materials may be one way to prevent the biofilm formation in dental composites and, therefore, secondary caries. nanoparticles (nps) of zno and ag have shown antimicrobial potential when incorporated into dental resins. the aim of this study was to evaluate the compressive strength of dental composite filtektmz350 xt (r) plus zno nps and ag. zno.ag nps were synthesized at room temperature (rt), by hydrothermal (hs) and pechini methods (ps), characterized by x-ray diffraction, thermogravimetry and scanning electron microscopy. composite specimens (ss) were prepared in stainless steel cylindrical molds (6x4mm) and stored in artificial saliva at 37 ° c for 24h prior the test. three groups (r control, r + 1% nps and 2% nps) with 8 ss (n = 24) underwent compressive strength test, determined with emic universal testing machine with 10kn load cell at 0, 5mm/min. the results were submitted to two-way analysis of variance (anova) followed by tukey test for multiple comparisons. the synthesis generated polycrystalline powders with nanoplates of ~ 10 nm agglomerated in microspheres (rt), hexagonal nanorods of ~ 345 nm (hs) and nanospheres with ~ 88 nm (ps). anova showed a significant difference (p> 0.05) between nps hs 2%, 1% ps, ps 2% and the control group. the compressive strength decreases with the increase of nps in resin, however, inclusion of 1%, 2% of nanoplates and 1% of nanorods do not change significantly the compressive strength of the resin. 111 evaluation of the behavior of colored alumina/feldspar based ceramics facing a new production protocol macêdo eod*, takimi as, bergmann cp, samuel smw ufrgs erikaodias@gmail.com the aim of this study was to characterize by evaluating the elastic modulus, hardness and roughness, the behavior of alumina / feldspar colored bodies after using a new production process, considering that the process initially used generated excessive porosity. the ceramic raw material was formed of pink and blue alumina powder, produced by a new process of ceramic pigment, combined with 20 wt% of feldspar resulting in groups r20 (pink alumina+ 20% feldspar) and a20 (blue alumina+ 20% feldspar). after mixing the pressing additives with the powders, the mixture was compacted and pressed into disc-shaped with 12.0(± 0.2)mm of diameter and 1.2(± 0.2)mm of thickness. the pieces were kept in a stove at 100°c for 24h before sintering. the first stage of sintering protocol used was similar for both groups, they were a heating using a rate of 100°c/h up to 400°c, remaining on this temperature for 4 hours. next, was used a heating rate of 150°c/h up to 1450°c for 8h to r20, and 1300ºc for 10h to a20. after sintering, the elastic modulus of the sintered pieces was evaluated by ultrasound, as well as the microhardness and roughness. the elastic modulus of the groups r20 and a20 were 198 mpa and 182 mpa respectively. the mean values of vickers hardness for the groups r20 and a20 were 419 (± 37) and 367 (± 29) and roughness mean values were 0.43 (± 0.1) and 0.49 (± 0.04). from the results obtained it can be concluded that the production protocol suggested produced pieces with the most promising properties. 113 influence of successive light-activation on degree of conversion and knoop hardness of the first composite increment theobaldo j*, giorgi mcc, lima danl, marchi gm, ambrosano gm, aguiar fhb piracicaba dental school unicamp jeh_theobaldo@hotmail.com the aim of this study was to evaluate the influence of light-activation of four successive increments on knoop hardness (khn) and degree of conversion (dc) of top (t) and bottom (b) surfaces of the first increment. samples were made in fouroverlapped teflon mold (2mm high each). a microhybrid composite was lightactivated according to the experimental groups (n=5). g1 to g4 (multiple irradiation) and g5 to g8 (single irradiation) were light-activated with qth xl (500 mw/cm2 x 38 s); and led s (1000 mw/cm2 x 19 s), hp (1400 mw/cm2 x 14 s) and pe (3200 mw/cm2 x 6 s). in groups 1 to 4, after each light-activation, t and b surfaces of the first increment were measured in dc and khn. in groups 5 to 8, only the first increment was made, and the four measurements of dc and khn were taken at 15 minutes interval. results for dc and khn were analyzed separately; the data were analyzed by using proc mixed for repeated measures and tukey test (á=0.05). for khn, b showed lower values than t. for both single and multiple irradiations, t and b of first measurement showed the lowest khn and the fourth measurement showed the highest, with significant difference between them. for dc, except qth, t presented higher dc than b. the light-activation of successive increments influenced the knh of the first increment but did not influence the dc. 110 edge chipping test on microhybrid and nanocomposites tanaka cb*, ballester ry, de souza g, zhang y, meira jbc university of sao paulo carina.tanaka@usp.br the aim of this study was to investigate the influence of indenter type on the “edge toughness (te)” of two different restorative composites. a microhybrid composite, (z250 filtek supreme xt, 3m-espe, mn) and a nanocomposite (z350 filtek supreme xt, 3m-espe, mn) were used to prepare 3 bars (5x32x2.5 mm) samples for each material. the specimens were light-cured using a led with an irradiance of 1200mw/cm2 (radii cal sdi, bayswater, aus). three base increments were photo-activated for 20s each and a final surface increment for 120s. specimens were storage for 7 days in distillated water at 37°c. the edge chipping tests were carried out in a universal testing machine, with a constant load (0.1 mm/min). rockwell (conical 120°) and vickers indenter were used. the load was applied in different distances between the upper longitudinal edge and the indenter (da). each specimen was chipped around twenty times, with five different da values, from 0.1 mm to 0.5 mm. an average force and standard deviation were calculated for each distance. the relationship between mean critical force (fc) and da was plotted. te was defined as the slope of the linear trend on these graphs. the results showed that highest values of te was found using vickers indenter. differences between composite (z250 te = 335.9 and z350 te = 277.5) were found with vickers indenter, but not with rockwell indenter (z250 te = 234.9 and z350 te = 235.0). the type of indenter influences on te value. vickers indenter was able to distinguish the different composite, while rockwell indenter showed similar toughness for them. 112 influence of the framework design on the stress distribution: finite element analysis roscoe mg*, reis br, jikihara an, chaves ks, karkuszewski sd, ballester ry, meira jb university of sao paulo marininharoscoe@yahoo.com.br the aim was to study the influence of the framework design on the thermal residual stresses (trt) at the porcelain, by the finite element method. axisymmetric finite element models of crowns were generated according to 4 framework designs: (1) cv: conventional coping, (2) cvc: cv with cervical brace, (3) cmod: coping modified; (4) cmodc: cmod with cervical brace. a cooling protocol of the model from 600oc to 25oc was simulated. the data of temperature in this step were incorporated into the structural analysis. the peak value and the distribution pattern of the maximum principal stress (ó1) were evaluated at the porcelain. it was observed that the presence of convex regions at the zirconia/porcelain interface reduces the tensile stresses in the porcelain. thus, two new models were generated: m1, with concave surface in the porcelain interface with zirconia, and m2 with convex surface. the analysis of trt was performed following the protocol described previously. the values of ó1 for each framework design were: cv = 22 mpa, cvc = 20 mpa, cmod = 11 mpa, cmodc = 10 mpa. for the models m1 and m2, the values of ó1 were 26 mpa and 7 mpa, respectively. it can be concluded that the presence of cervical brace did not cause a considerable decrease in the tensile stress values. the influence of the framework design in the generation of trt may be related to the change of curvature of the porcelain/zirconia interface. the trt is more favorable in framework designs which porcelain surfaces attached to zirconia is convex. 114 effect of non-carious cervical lesions morphology and loading on the biomechanical behavior of maxillary incisors machado ac*, naves mfl, pereira ag, zeola lf, veríssimo c, faria vlg, soares pv federal university of uberlandia alexandrecoelhomachado@gmail.com non-carious cervical lesions (nccls) are formed by loss of tooth structure in the cervical third of the crown and root surface, with multi-factorial origin. the aim of this study was to measure the effect of five morphological types of nccls, simulating two different loads on maxillary incisors, by quantifying the stress distributions. eleven virtual models of maxillary incisors were generated using the cad software rhinoceros 4.0. these models presented six nccls morphologies: sound (h), concave (co), irregular (ir), notched (no), shallow (sh) and wedged-shape (ws); unrestored and restored with composite resin. the models were exported to an analyses software (ansys workbench 12.0), considered homogeneous, linear and isotropic. then, the models were meshed and submitted to two types of load (100 mpa): palatine (p) and incisal (i). the displacement restriction was made on the base and on the sides of the bone. data summarizing the stress distributions were obtained in mpa using von mises criteria. as results, the models co, ws and ir showed higher stress concentration on the bottom of the lesion. models with the center of the nccl at acute angle (ws and ir) showed higher stress on the junction of the celling and floor walls. all the restored morphologies, independent of the load type, showed biomechanical behavior similar to the h. it was conclude that deeper nccls and with acute angles, shows higher stress on the deep of the lesion; and restore with composite resin is important, independent of the morphology. gbmd 2013 – 49th meeting of the brazilian group of dental materials256256256256256 braz j oral sci. 12(3):237-274 115 morphological characterization of bonding between dentin and resin cements antunes ang*, vaz rr, de goes mf, di hipólito vale do rio verde university – unincor antunes1978@gmail.com the aim of this study was to investigate the morphology of the interface between human dentin and resin cements rely x unicem (3m espe), set (sdi) and c&b cement (bisco). the occlusal surface and roots of six third molars were removed, producing 12 dentin discs with 2.0 mm thick. the dentine surface was prepared with abrasive sic granulation 600, cemented in pairs according to the manufacturers’ recommendations, mesio-distal and enclosed in pvc tubes with epoxy resin. sic sandpaper in grits 600, 1200, 2000 were used to plan each pair of disk cemented. then felt and diamond pastes, granules in 3, 1, 1 / 4, alternating with ultrasonic cleaning for 12 minutes were used to finish the polishing. each sample was etched with phosphoric acid 50% for 3 seconds and immersed in 2.5% naocl for 10 minutes. after this period, the samples were washed three times with distilled water, placed in solutions of increasing concentration of ethanol (25, 50, 75 and 100%) for 10 minutes, immersed in hmds for 10 minutes and stored dry for 12 hours. finally, the samples were metallized and observed under a scanning electron microscope (sem). the results of morphological analysis of the union showed no hybrid layer formation for self-adhesive cements rely x unicem and set. only chemically activated c&b cement prduced hybrid layer. 117 shear bond strength to cad/cam materials using four adhesive luting strategies bernades ko*, schauffert acb, pereira pnr, ribeiro apd, hilgert la brasília university – unb karinabernades@gmail.com the adhesive luting of indirect materials relies on a correct treatment of their surfaces in order to achieve a good interaction with the resin cements. this study evaluated the shear bond strength (sbs) to cad/cam materials [empress cad glass-ceramic (ec); lava ultimate indirect resin (lu)] using four adhesive protocols, including the use of an adhesive system that contains silane and the mdp monomer in its formula (scotchbond universal, sbu). plates of ec and lu materials received standard surface treatment (ec: sandblasting, hf 10% 60 sec, ultrasonic bath; lu: sandblasting, ultrasonic bath). for both materials four adhesive protocols were used: g1: silane (s) + singlebond2 + relyxarc; g2: s + u200; g3: sbu + relyx ultimate; g4: sbu + u200. for each combination of material and adhesive protocol 14 cement cylinders were built (area=4.15mm2). sbs was measured after 24 hours. results of anova and tukey (á=5%) showed that for ec protocols g4 and g2 were superior than g1 and equal to g3. for lu, g3 and g4 were higher than g1 and g2. sbs to lu was higher than to ec when using g3 and g4. use of sbu as a surface treatment acting both as silane and adhesive was effective, especially for lu. 119 influence of diameter for platform switching on implant biomechanics bordin d, lazari pc, camargos gdv, sotto-maior b, del bel cury aa, silva wj piracicaba dental school unicamp dimorvan_bordin@hotmail.com the aim of this study was to evaluate the influence of the reduction of abutment’s diameter for platform switching on stress distribution of single implant with external or internal connection using three dimensional finite element analyses. a total of 8 virtual 3d models were constructed containing one single implant (5.0 x 11mm) in a mandibular segment supporting a single first molar screwed crown. the implants present external or internal hexagon connection. it was used ucla abutment platforms with different diameters: 3.8; 4.2; 4.6 or 5.0 mm. all structures were considered perfectly bonded and each model received a 200 n oblique load on the occlusal surface distributed on 8 different points. the maximum principal stress and the maximum elastic strain were calculated for the cortical and trabecular bone and equivalent von-misses for dental implant and platform abutment using ansys workbench software. the reduction of abutment’s diameter produced a reduction of stress and strain values on bone tissue. however, the smallest diameter for external hexagon connection produced the highest stress values. on the other hand, the reduction of abutment’s diameter increased the stress and strain in abutment and implant, regardless of implant connection. the reduction of abutment’s platform diameter improves stress distribution in bone tissue, independently of the implant connection type. however, it increases the stresses within the implant and abutment, which could compromise their mechanical resistance. 116 effect of non-carious cervical lesion, root morphology and loading on the biomechanical behavior of premolar soares pv*, souza lv, veríssimo c, pereira ag, machado ac, zeola lf, pereira fa, reis br federal university of uberlandia paulovsoares@yahoo.com.br the premolars have a higher prevalence of non-carious cervical lesions (nccls). the present study aimed to investigate the biomechanical behavior of premolars, analyzing the root morphology, the nccls depth, and type of load; by the finite element analysis and strain gage test. six models virtual 3d models were generated: single-rooted (higid; lesion of 1.5 and 2.5 mm) and bi-rooted (healthy, 1.5 and 2.5 mm). each model was subjected to axial and oblique loading and data analysis was performed by the von mises criterion and maximum principal stress. for strain gage test, 14 premolars were selected and divided into 2 groups (single-rooted and bi-rooted), wich received strain gage in the buccal and mesial faces. the healthy samples received a compressive loading (0-100n). this was followed by the simulation of the 1.5 mm nccl, with 3118 diamond bur, and the samples subjected to new loading. then, the 2,5mm lesion was simulated and the load was applied again. as a result, the bi-rooted teeth associated with oblique loading showed higher strain values. the axial loads showed lower rates of deformation. nccls promoted greater depths with a higher concentration of stresses and strains. it is concluded that the deeper nccls associated with bi-rooted teeth and oblique loading, increase the concentration of stresses and deformation in the tooth structure. 118 stress distributions in different overdenture-retaining system spazzin ao*, bacchi a, consani rlx, correr-sobrinho l, santos mbf federal university of pelotas aospazzin@yahoo.com.br this study evaluated the influence of cross-section geometry of the bar framework on the distribution of static stresses in an overdenture-retaining bar system simulating horizontal misfit and bone loss. three-dimensional fe models were created including two titanium implants and three cross-section geometries (circular, ovoid or hader) of bar framework placed in the anterior part of a severely resorbed jaw. one model with 1.4-mm vertical loss of the peri-implant tissue was also created. the models set were exported to mechanical simulation software, where horizontal displacement (10, 50 or 100 ìm) was applied simulating the settling of the framework, which suffered shrinkage during the laboratory procedures. the bar material used for the bar framework was a cobalt–chromium alloy. for evaluation of bone loss effect, only the 50-ìm horizontal misfit was simulated. data were qualitatively and quantitatively evaluated using von mises stress for the mechanical part and maximum principal stress and ì-strain for peri-implant bone tissue given by the software. stresses were concentrated along the bar and in the join between the bar and cylinder. in the peri-implant bone tissue, the ì-strain was higher in the cervical third. higher stress levels and ì-strain were found for the models using the hader bar. the bone loss simulated presented considerable increase on maximum principal stresses and ì-strain in the peri-implant bone tissue. in addition, for the amplification of the horizontal misfit, the higher complexity of the bar cross-section geometry and bone loss increases the levels of static stresses in the peri-implant bone tissue. 120 effect of polishing systems and chemical degradation in surface roughness of resin-based materials cardoso m*, inagaki lt, de paula ab, naufel fs, correr-sobrinho l, puppin-rontani rm piracicaba dental school unicamp micaelazcardoso@hotmail.com this study evaluated the influence of polishing systems and coffee immersion on the surface roughness (ra) of composites. 72 cylindrical samples (7 x 2 mm) were prepared and divided into 8 groups (n= 9) in accordance with the factors (soflex and jiffy / filtek z350xt and vitalescense / coffee (cf) and artificial saliva (sa)control). the initial ra was measured. then, each sample was immersed in 4 ml of solution for 14 days (cf: for 15 min, three times a day). after that, the ra was measured again and the delta values (∆ra) were calculated. the results were submitted to tree way anova and tukey test (p< 0.05). after degradation in both solutions for both composites, there was a reduction in surface roughness. there were no statistically significant interaction between composites and polishing system (p < 0.001). independent of the solutions, vitalescence composite showed no significant change in surface roughness when polished with the soflex (∆ra = -0.05) and jiffy (∆ra = -0.09). however, the resin z350 showed differences for the polishing system, with greater ∆ra for the jiffy (∆ra = -0.25) compared with soflex (∆ra = -0.01). after the degradation, the soflex polishing system does not promote variations in ra for both composites. however, the jiffy promoted smaller ∆ra for the vitlescence (∆ra = % -0.09) than in z350 (∆ra = -0.25). the solutions did not affect the surface roughness of composites. however, the composition of the composite resins and the different polishing systems influenced the surface roughness of composite restorative after the degradation by the solutions. gbmd 2013 – 49th meeting of the brazilian group of dental materials 257257257257257 braz j oral sci. 12(3):237-274 6262626262 121 physical-mechanical properties and µct evaluation of low shrinkage resin composites oliveira dcrs, souza-júnior ej, rovaris k, haiter-neto f, brandt wc, sinhoreti mac piracicaba dental school unicamp dayoli87@gmail.com the aim of this study was to evaluate the physical-mechanical properties (pmp) of low shrinkage resin composites (lsrc). thus, commercial lsrc were evaluated (kalore gc corp and venus diamond heraeus kulzer) and compared to a conventional microhybrid composite (cmc) (filtek z250 3m). all specimens were photocured by led 16j. 5 specimens were scanned using a µct, before and after the photoactivation. the composite volume was measured before and after the curing process, and the % of shrinkage was measured and the volumetric shrinkage (vs) evaluated.10 bar specimens of 7x2x1 mm of each resin composite were performed to evaluate the pmp. after 24 hours, the degree of conversion (dc) was evaluated by ft-ir. the flexural strength (fs) and elastic modulus (em) were measured by a three-point bending test. data were submitted to one-way anova and tukey’s test (α = 0.05). the µct evaluation showed that the lsrc showed lower vs values (kalore = 1.8 % and venus diamond = 1.7%) compared to the z250 (2.0 %) (p < 0.05). for the fs and em, only kalore showed lower values compared to cmc. also, kalore presented higher dc (68,8 %) compared to the other lsrc (49,5 %) and also to the cmc (49,6 %). in conclusion, the low shrinkage composites showed lower vs without affecting the physical-mechanical properties. 123 influence of experimental adhesives on cell metabolism and mmp expression lima af*, marques mr, dias ribeiro ap, carrilho mr, pashley dh, hebling j, de souza costa ca nove de julho university – uninove lima.adf@uol.com.br to evaluate the effect of experimental adhesives with different hydrophilic characteristics on metabolism and expression of metalloproteinase-2 (mmp-2) by cultured odontoblast-like cells. sterile filter paper discs were impregnated with 10 ìl of different experimental adhesives: r1, r2, r3, r4 and r5 (in increasing order of hydrophilicity), and two commercial available adhesives (scotchbond-sc and single bond 2-sb2), and then light cured for 20 s. the specimens were individually immersed for 6 or 12 h in 1 ml of culture medium (dmem) to obtain the extracts (dmem+ components released from adhesives), which were applied on cultured mdpc-23 cells (3x104cells/cm2) for 8 h. discs not impregnated with adhesives (r0) were used as control. cell metabolism (mtt assay) and mmp-2 expression (zymography) were evaluated. the data were submitted to two-way anova complemented by tukey’s test (á=0.05). after 6-h elution, the cells exposed to the extracts obtained from r1, r5 and sb2 presented lower metabolism than the other groups. at 12-h elution, the extracts from all adhesives significantly decreased the cell metabolism compared to control group (p<0.05). the mmp-2 expression was statistically lower for r4 and sb2, after 6-h elution. at 12-h elution, the cells exposed to the extracts obtained from r5, sc and sb2 presented lower mmp expression than the other groups (p<0.05). it can be concluded that the adhesives can influence the metabolism and expression of mmp-2 by odontoblast-like cells. however, the effects of these resin-based materials on the cells activity are not modulated by their hydrophilic characteristics. 125 analysis of shear strenght of repairs after different treatments on ceramic surface bettinelli jd*, pagnoncelli rm, hirakata lm pontifical catholic university of rio grande do sul pucrs jbettinelli@terra.com.br the aim of this study was to determine the shear strength of repaired ceramic after different treatments. it was used forty ceramic discs noritake ex-3 (group e) and another forty discs with the system noritake cerabien (group c). group e1 and c1: laser application, group e2 and c2: etching with hydrofluoric acid 10%, group e3 and c3: sandblasting with aluminum oxide, group e4 and c4: roughened with silicon carbide grain # 400 (control). all discs were given applying a layer of coupling agent for ceramics and a layer of adhesive. in the center of the ceramic disk, a block was made in composite. each group was maintained at 100% humidity at 37 ° c for one to four weeks in the greenhouse cultivation. sandblasting with aluminum oxide showed the highest values of shear strength at the interface between the composite and ceramic ex3, and the same occurred in cerabien pottery, but pottery ex3 with the storage time of 1 week and the ceramic cerabien with the storage time of 4 weeks. sandblasting with aluminum oxide surface treatment was the one that showed better results in both types of ceramics. the storage time of 4 weeks did not have a significant influence in this study. 122 bonding effectiveness of universal self-etching adhesive systems to chlorhexidine-treated dentin salvio la*, andrade co, resende fo, devito kl, de goes mf federal university of juiz de fora luciana.salvio@ufjf.edu.br chlorhexidine (chx) is an antimicrobial agent with ability to detain the endogenous activity of the matrix metalloproteinases in phosphoric acid etched dentin substrate. however, it is speculated that the chlorine residues on dentin surface after chx treatment could interfere chemical and physically on immediate bond strength of universal self-etching adhesive systems. the aim was to evaluate the prior application of 2% chx influences on tensile bond strength (tbs) of universal self-etching adhesive systems. for tbs test, third molars were cut to expose a middle flat dentin surface and randomly assigned into 4 groups: (cse) clearfil se bond – hibridization according to manufacturer´s instructions; (cse+chx) chx applied during 20s following hybridization with cse; (sbu) scotchbond universal – hibridization according to manufacturer´s instructions at self-etching mode; (sbu+chx) chx applied during 20s following hibridization according sbu system. filtek z350 composite resin blocks were incrementally built up. after 24h, specimens were sectioned in 1mm2 beams and subjected to tensile test (n=18). the data were analyzed with two-way anova two-way and tukey´s test (p<0,05). groups (cse) 39,77±11,56mpa and (sbu) 38,43±12,49mpa presented the highest tbs values and were not statistically different among each other. (cse+chx) 22,61±5,18mpa and (sbu+chx) 22,25±5,58mpa presented the lowest tbs values and were not statistically different. in conclusion, it is suggests that pretreatment of dentin with 2% chx adversely affects the immediate bonding efficacy when associated with universal self-etching adhesive systems. 124 effect of polishing systems and staining solution on surface gloss of restorative materials medeiros b*, naufel fs, de paula ab, inagaki lt, cardoso m, correr-sobrinho l, rontani rmp state university of west of parana unioeste medeiros.bianca@hotmail.com this study aimed to evaluate the influence of two polishing systems in the gloss of composites after degradation in coffee. 72 cylindrical samples were made (7 mm diameter x 2 mm thick) of the composites filtek z350xt and vitalescense. after 24 h at 37 °c, the samples were polished with soflex systems (3m/espe) and jiffy (ultradent), and then randomly separated into 8 groups (n = 9) according to the composite, the gloss and the staining solution [coffee (cf) and artificial saliva (sa) – control]. initial measurements of gloss of surface were made. then, each sample was immersed in 4 ml of cf for 15 min, 3 times a day for 14 days. after that, the gloss of surface was measured again and variations of gloss (∆gloss) were calculated. the normal distribution of data was verified (kolmogorov-smirnov test, p>0.05) and then subjected to anova and holmom-sidak test (5%). there was interaction between restorative material and polishing system (p = 0.007). regardless of the solutions, vitalescence not exhibited significant ∆gloss differences in gloss values when polished by soflex systems (3.80) and jiffy (4.68). however, z350 showed significant differences with the greatest ∆gloss after polishing with soflex (10.32) when compared with jiffy (5.85). after degradation, regardless of polishing system employee, vitalescence did not show significant ∆gloss of surface in cf and sa. however, z350 showed less ägloss in sa (6.14) than in cf (10.03). it is concluded that after degradation in cf, the composite resin z350 showed higher variation in gloss when polished with the system soflex and immersed in cf. vitlescence not exhibited significant variations in gloss of surface in all the situations studied. 126 influence of photo-initiation system on the radical polymerization of elastomeric monomers münchow ea*, valente ll, barros gd, alves ls, cava ss, piva e, ogliari fa federal university of pelotas eliseumunchow@hotmail.com the aim of this study was to evaluate the degree of conversion (dc), the kinetic of polymerization (kp), and the rate of polymerization (rp) of elastomeric monomers (exothanes) containing different photo-initiation systems. five exothanes (8, 9, 10, 24, and 32) were mixed with the following photo-initiation systems: unitary [camphorquinone (cq); binary [cq + tertiary amine (edab); and ternary [cq + edab + diphenyliodonium (dpi)] at concentrations of 0.5%, 1% and 1% in weight for cq, edab, and dpi, respectively. udma was used as control. each resin blend was evaluated in infrared spectroscopy (rt-ftir, shimadzu prestige-23) using a diamond crystal (n=3). the dc data was analyzed by two-way anova (factors monomer type x photo-initiation system) and tukey’s test (p<0.05). independently of the photoinitiation system used, the exothanes 8, 9, and 32 demonstrated higher dc than the others. the exothane 24 resulted in the lowest dc values, although the ternary system presented similar dc when compared to the control and higher dc than the unitary and binary systems. the unitary system was effective as initiation system for the polymerization of the exothanes and the udma. regarding the kp, the exothane 32 was completely transformed in polymer from 12 to 17s of photo-activation. within the rp results, the binary and ternary systems increased the exothane’s reactivity, but not the control’s one. therefore, urethane derived monomers (udma and exothanes) performed as co-initiators of the radical polymerization of cq. the exothanes 8, 9, and 32 were the most reactive ones, reaching dcs higher than 80%. gbmd 2013 – 49th meeting of the brazilian group of dental materials258258258258258 braz j oral sci. 12(3):237-274 127 analysis of inflammatory markers and systemic cytotoxicity of endodontic pastes proença js*, kreling pf, ambrosio s, flaiban kkmc, ramos sp, contreras efr state university of londrina uel julah22@hotmail.com the aim of this study was to evaluate of the natural extract endodontic paste systemic toxicity. wistar rats were randomly divided into five groups (n=12). control group was not submitted to endodontic procedure. the remaining groups were submitted to pulpectomy in upper incisors and the root channel was fulfilled with vaseline (control positive); iodoform-based paste with rifocort® (guedespinto’s paste); iodoform-based paste with guaco extract and iodoform-based paste with copaiba oil. after 14 and 28 days, blood samples were collected for analysis of c reactive protein, alamine aminotransferase (alt), aspartate aminotransferase (ast) and creatinine. the statistical analysis was performed with kruskal-wallis and dunn’s tests (p< 0.05). mean body weight were not significantly different among groups after 14 days. liver weight and relative liver weight were decreased in vaseline group. mean spleen weight were increased in guaco group but this difference were relative to mean body weight. no significant increase in creatine kinase mb, alt, ast and creatinine serum levels were found on day 14. after 28 days, mean body weight were decreased in vaseline group and increased relative liver and kidney weights were found in rifocort® and guaco groups, respectively. a high rate of renal clearance was found in rifocort® group, as reduced creatine levels, compared to vaseline e copaibe groups. concluded that the iodoform-based paste with natural extracts were more promissory that positive control group and iodoform-based paste with rifocort®. 129 degree of conversion and hydrolitic degradation of experimental composites: influence of surface treatment filler system netto, lrc*, noronha filho, jd, garcia, mlb, silva, em federal fluminense university labiom-r lucianodentistica@yahoo.com.br the aim of this project was to analyze the influence of the surface treatment of inorganic fillers system on absorption, solubility and degree of conversion of experimental composites. the comercial avaiable composite z250 (3m espe) was used as a standard. in the experimental group e1, the particles were not treated. in e2 group, the particles were silanized with γ-mps. in groups e3 and e4, particles of barium-borosilicate were conditioned for 2 min with 10% hydrofluoric acid in order to create porosities and increase interaction with the organic matrix. finaly, the particles in group e4 were also silanized. regarding the degree of conversion, group e2 showed the lowest value (70,37% ± 0,88) and the group e3 showed the highest value (77,32% ± 0,73). the group e5, used as standard showed the value of 74,49% ± 6,00. regarding the absorption, group e1 had the lowest value (48,86 µg ± 5,78) and the experimental group e4 showed the highest value (83,64 µg ± 10,19). the group e5, used as standard showed the result of 37,51 µg ± 5,70. for the solubility the experimental group e1presented the lowest result (6,43 µg ± 1,40) and the experimental group e4 showed the highest result (24,69 µg ± 3,40). the group e5, showed the value of 5,37 µg ± 1,52. it is possible to conclude that the increasing in filler particles porosity increased the degree of conversion and the silanic layer and porosity could increase the composite degradation. 131 biological characterizations of implant surfaces in vitro study soares pbf*, moura ccg, coró cgc, reis mvp, zanetta-barbosa d, soares cj federal university of uberlandia pbfsoares@yahoo.com.br the aim of this study was to evaluate the biological performance of titanium alloys grade iv under different surface treatments: g1, sandblasting and etching (neoporos, neodent); g2, surface with wettability increase (acqua neodent) on response of preliminary differentiation and cell maturation. immortalized osteoblast cells were plated on g1 and g2 titanium discs. the polystyrene plate surface without disc was used as control group (c). cell viability was assessed by measuring mitochondrial activity (mtt) at 4 and 24 h (n = 5), cell attachment was performed using trypan blue exclusion within 4 hours (n = 5), serum total protein and alkaline phosphatase normalization was performed at 4, 7 and 14 days (n = 5). data were analyzed using one-way anova, kruskal-wallis and dunn’s tests. the values of cell viability were: 4h: c0.32±0.01a; g1 0.34±0.08a; g20.29±0.03a. 24h: c0.43±0.02a; g10.39±0.01a; g20.37±0.03a. the cell adhesion counting was: c85±10a; g135±5b; g220±2b. the amounts of serum total protein were 4d: c40±2b; g1120±10a; g2130±20a. 7d: c38±2b; g175±4a; g270±6a. 14 d: c100±3a; g1130±5a; g2137±9a. the values of alkaline phosphatase normalization were: 4d: c2.0±0.1c; g15.1±0.8b; g29.8±2.0a. 7d: c1.0±0.01c; g15.3±0.5a; g23.0±0.3b. 14 d: c4.1±0.3a; g14.4±0.8a; g22.2±0.2b. different letters related to statistical differences. the surfaces tested exhibit different behavior at dosage of alkaline phosphatase normalization showing that the g2 is more associated with induction of cell differentiation process and that g1 is more related to the mineralization process. 128 effect of 5% sodium thiosulfate in reestablishing the bond strengths to 5,25% sodium hypochlorite-treated dentin. corrêa acp*, zaia aa, gomes bpfa, souza-filho fj, almeida jf, ferraz ccr piracicaba dental school unicamp acpcorrea@hotmail.com evaluate the antioxidant efficiency of 5% sodium thiosulfate, at different times, in reestablishing the bond strengths to 5,25% sodium hypochlorite-treated dentin. thirty crowns of bovine incisors were cut to expose the pulp chamber. the dentin surfaces were treated as follows: group 1: 0.9% sodium chloride for 30 min; group 2: treated with 5.25% sodium hypochlorite (naocl) for 30 min, 17% edta for 3 min and additional 5.25% naocl for 1 min; groups 3: immersion in 0.9% sodium chloride for 10 min after the same treatment performed in group 2; group 4, 5, 6: followed the same treatment performed in group 2, adding the final applications of 5% sodium thiosulfate solution for 1 min (group 4), 5 min (group 5) and 10 min (group 6). after that, scotchbond multipurpose adhesive system was applied to pulp chamber dentin, followed by filtek z250 composite. after storage in water for 24h at 37oc, twenty-five rectangular sticks were obtained from the specimens of each group and subjected to the microtensile bond test. the data converted to mpa were analyzed by one-way anova and fisher’s test (p<0.05). the groups 2 and 3 had significantly lower bond strength than the control (group 1). there was no statistically significant difference between groups 1 and 6 (p = 0.944), but both are statistically different from the groups 2 and 3. the reversal effect on compromised bonding to naocl-treated dentin is obtained using 5% sodium thiosulfate for 10 min. on the other hand, the washing with 0.9% nacl does not reestablishes the bond strength values. 130 influence of thermal simulation on the stress distribution in ceramic veneers y-tzp crowns lazari pc*, carvalho ma, camargos gv, sotto-maior bs, del bel cury aa piracicaba dental school unicamp lazari.pcl@gmail.com the aim of this study was to evaluate the stress distribution in ceramic veneers ytzp crowns and evaluate the compatibility between the coefficients of thermal expansion (cte) the materials after the heating and cooling cycle using by threedimensional finite element analysis. virtual crowns were constructed using microtomography images of superior central incisor. the models were composed of ceramic veneer with a thickness of 2mm (feldspathic ceramic) and 0.4mm framework (zirconia, alumina or metal). the finite element software ansys workbench was used for thermal analysis and the loading was performed in 2 steps, simulating the heating and cooling manufacture cycle: 1st step – from 403o to 750o c, 2nd step from 750o to 25o c. the maximum principal (ómax) and minimum principal (ómin) stress was used to evaluate the ceramic veneer and ceramic frameworks. the equivalent von mises (óvm) used for the evaluation only the ceramic and metal frameworks. the results showed higher stress values on the ceramic veneer models with metal framework (376.76 mpa), followed by alumina (310.24 mpa) and zirconia models (213.32 mpa). compressive stresses followed the same behavior showing higher values in metal-ceramic prosthesis. the stresses were concentrated at the cervical margin for all models regardless of material used. the difference in stress distribution between systems shows that the compatibility between the cets is essential to keep mechanical performance of ceramic veneer. 132 dislodgement resistance of root canal filling materials: metaregression of push-out studies portella ff*, rodrigues sb, celeste rk, leitune vcb, collares fm, samuel smw federal university of rio grande do sul portellaff@yahoo.com.br the aim was verify the influence of technical variables, materials and parameters of push-out test on dislodgment resistance of root canal filling materials to root canal dentin. a systematic review was performed using the “pushout” and “push-out” terms to search at pubmed database. laboratorial studies published until 2013 were included. from the 639 identified studies, 39 met the inclusion criteria. four reviewers extracted data regarded origin country, year of publication, tooth type, smear layer removal, root canal sealer, core material, obturation technique, sample storage, tooth portion, velocity of test machine and slice thickness. a metaregression of 237 groups resultants was performed to analyze the influence of each variable on outcomes, in mpa. the regression model could explain 85% of the between-groups variance. tooth type (p=0.53), irrigant solution (p=0.16), sample storage time (p=0.47), radicular portion (p=0.22) and test machine velocity (p=0.92) did not influence the dislodgement resistance of materials. the other analyzed variables influenced the push-out values (p<0.05). the dislodgment resistance, measured by push-out assay, it is influenced by variations on technique used to prepare and filling the root canals and by parameters adopted on test. 259259259259259gbmd 2013 – 49th meeting of the brazilian group of dental materials braz j oral sci. 12(3):237-274 133 evaluation of the removal torque on different alloy/coating external hexagon prosthetic screws dantas lcm*, raposo lha, silva jpl, dantas lcm, davi l, neves fd, soares cj, correr-sobrinho l, consani s piracicaba dental school unicamp lucascmdantas@gmail.com the purpose of this study was to investigate the maintenance of the tightening torque in different types of screws for implant-supported single-tooth restorations by assessing the torque removal of each system. in addition, a new method for gold coating of abutment screws was also evaluated. hexagonal (hx) and square (sq) titanium screws types were selected and designated to eight experimental groups (n=5): hxau1 and sqau1screws subjected to gold-sputtering for 120 s; hxau2 and sqau2screws subjected to gold-sputtering for 240 s; hxti and sqtiscrews kept unchanged; hxdlchexagonal titanium screws with diamond-like carbon coating; hxgthexagonal gold screws. one assembly of implant and abutment was used to test each group. the screws were tightened with 32 n cm and loosed three times each, and the torque necessary to loose the screw was recorded. data were submitted to two-way anova repeated measures and tukey’s test. all groups showed lower torque removal values in comparison to the initial torque applied. no statistical differences were found between the tightening times. the screws with dry lubrication (hxgt and hxdlc) showed the lower torque removal values, being statistically similar to each other. the gold-sputtered screws hxau1, sqau1 and sqau2 were statistically similar to successive tightening did not influence the maintenance of the torque applied. the lubricant coating on the screws influences the maintenance of the applied torque. 135 preliminary comparative study of two conventional bleaching systems gentil fhu, pinheiro hb, capel cardoso pe university of sao paulo umeda1984@hotmail.com the objective of this work is to analyze and compare 2 in-office bleaching systems through the clinical application of these to groups of patients and further evaluation of color change during time: preoperative, immediate postoperative, 7, 14 and 30 days after the treatment. 22 patients were randomly divided into two groups (n = 11) and underwent one bleaching session according to the manufacturers’ instructions: boost, 38% hydrogen peroxide (hp) opalescence xtra boost (ultradent); pola, hp 35% pola office + (sdi). three different systems were used in color evaluation: vita classical shade guide (vc) bleachedguide vita 3dmaster (vb) and vita-easyshade spectrophotometer (sp), with the measurements being made in the central incisors and canines. statistical analyzes was performed by means of 3 way anova. comparisons of factor level means were performed through contrasts. for evaluation systems vc and vb, the two bleaching agents behaved similarly, with the bleaching effect being observed, for incisors and canines, from pre-operative to post immediate and maintaining the color obtained until the 30-day period. as for the sp evaluation the same difference was observed, however only in canines a second bleaching peak occurred between the post immediate and 7 days. therefore, the two systems promoted significant bleaching effect in all evaluations and none of the treatments relapsed to the initial shades. 137 feldspar as alternative filler for dental composites alonso rcb, piveta fb, rodriguez jmc, alves wj, di hipólito v, d’alpino php, anido-anido a, anauate netto c bandeirante anhanguera university robalonso@yahoo.com the aim of the study was to determine the effect of the filler type and size on the mechanical properties of experimental composites. knoop hardness and depth of cure of 10 experimental composites based on bisgma / tegdma with different types and sizes of filler particles (c1: silica; c2: barium glass/silica; c3: barium glass 1ìm; c4: barium glass 0.02ìm; c5: barium glass mixture; c6: feldspar 2ìm; c7: feldspar 1 ìm; c8: feldspar 0.8 ìm; c9: feldspar mixture; c10: feldspar / silica) were determined. cylindrical specimens were prepared (n=10) and light-cured for 40s. the uncured portion was removed and the remaining thickness of the specimen was measured with a digital caliper. after 24h, the knoop hardness was measured using a microhardness tester hmv 2000 shimadzu (3 indentations, load 50g, 30s). data were submitted to anova and tukey’s test (á=0.05). the hardness of the experimental composites ranged from 59.5 knh (c1) and 32.1 khn (c8). depth of cure ranged from 2.71mm (c1) and 5.05mm (c4). it was observed that the type and size of filler particles influenced the hardness and depth of cure of the composites. the feldspar appears to be a reliable alternative for mechanical reinforcement of composites, however, the use of 0.8ìm particles (c8) caused a reduction on hardness and depth of cure when compared with other particle sizes of feldspar. silica caused an increased on hardness, but reduced the depth of cure on the composite containing only silica (c1) and those in which it was combined with other types of fillers (c2 and c10). 134 effect of filling technique and composite on cuspal strain, bond strength, shrinkage and physical properties soares cj*, bicalho aa, pereira rd, valdivia adcm, barreto bcf, tantbirojn d, versluis a federal university of uberlandia carlosjsoares@umuarama.ufu.br to evaluate the effect of composite resins and filling techniques on cuspal strains (cs), bond strength (ìtbs), composite ultimate strength (uts), shrinkage stress and mechanical properties of the composites in molars. 135 human molars received standardized class ii mod and restorations with 3 composites (ls, filtek ls; ae, aelite ls; su, filtek supreme) using 3 filling techniques (bulk, 8 and 16 increments). cs was measured by strain gauges; the same restored teeth were used to assess ìtbs and uts. the elastic modulus (e) and vickers hardness (vh) at different depths were determined from microhardness. polymerization shrinkage was modeled by finite elements using post-gel shrinkage, measured using the strain gauge (n=10). the cs, ìtbs, uts, e and vh data were statistically analyzed using splitplot anova and tukey test (p = 0.05). the cs was higher for 16 increments. filtek ls caused lower cs. the ìtbs and uts were similar for 8 and 16 increments and higher when compared to the bulk filling in all composites. e and vh were constant through the depth when applied in 8 or 16 increments. post-gel shrinkage values were: ls < ae < su. the 16 inc filling caused substantially higher stresses and strains in the cervical enamel region. the 8 increments resulted in less cs with the same ìtbs and uts, without affecting e and vh through the depth of the composites. increasing the number of increments, and high post-gel shrinkage caused higher stresses. cuspal deformation measured by strain gauge validated the finite element analyses. 136 evaluation of flexural strength in ti-6al-4v alloy welded with different settings for tig welding segatto td*, martins po, de castro mg, da silveira júnior cd, novais vr, simamoto-júnior pc federal university of uberlandia thais.segatto@hotmail.com this study evaluated strength mechanics of ti-6al-4v alloy with 3.18 mm in diameter, through testing flexural strength, welded with different parameters for tig welding machine. it was made 40 samples, which were divided into 4 groups (n = 10): control, intact bars, g2, power 5 (a) and time 2 (ms); g3, power 5 (a) and time 3 (ms), g4, power 5 (a) and time 4 (ms). the samples were welded in a tig welding machine and evaluated radiographically for verification of bubbles and porosities. then test for liquid penetrant was applied to observe surface discontinuities. the samples were subjected to mechanical testing flexural strength and subsequently analyzed by stereomicroscopy by the program motic images plus 2.0ml, and the welded areas calculated for assessment of weld penetration. maximum values of flexion were analyzed by bending the formula for obtaining the flexural stress (mpa) for bodies of circular cross section and then subjected to statistical tests dunnett and tukey (p <0.05). most samples showed the presence of bubbles and internal porosity and no sample showed surface discontinuities. there was significant difference between the control group with the experimental groups for the parameter maximum stress. regarding the penetration area only g2 showed significant difference with others. it is concluded that changes in the regulation of the time for joining prosthetic infrastructure did not affect the flexural strength of the welded areas. 138 one year clinical evaluation of two in-office bleaching systems capel cardoso pe, pinheiro h, lopes ba, ballester ry, umeda gentil fh fousp paulocapel@gmail.com the aim of this study was to evaluate the efficacy and fadeback of two in-office bleaching systems. 40 patients were randomly divided into 2 groups (n = 20) and submitted to a bleaching treatment according to manufacturers instructions: 1. boost, 38% de hydrogen peroxide (hp) opalescence xtra boost (ultradent), 2. zoom, 25% hp-zoom 2 + zoom ap light (phillips oral healthcare). teeth shade was mesasured on superior central incisors and canines using 3 different measuring systems: vita bleachedguide 3d-master (vb), vita shade guide classic (vc) and vita-easyshade spectrophotometer (sp). measurements were made immediately before and after the tretamente anda t 7, 14, 30, 180 e 365 days. statistical analyzes of the results were made using 3-way anova. vc: showed that both systems promoted bleaching and color stability at 365 days. vb: for both experimental groups canine teeth presented a stable bleaching result at 365 days. bleaching results were also observed for incisive but at the 180 days measurement zoom group showed fadeback, and remained stable at 365 days. fadeback was also observed for incisive for boost group at 365 days measurement. sp: zoom group showed bleaching results immediate post op that improved at 7 days, maintaining it stable at 365 days. the same behavior was observed only for the canine teeth for boost group. we concluded that zoom group presented a superior bleaching result in all visual analyzes for both canines and incisive as well as superior delta e result for canine teeth. for all measurement methods used, neither system showed fadeback to the original shades after 365 days. gbmd 2013 – 49th meeting of the brazilian group of dental materials260260260260260 braz j oral sci. 12(3):237-274 139 development and characterization of a resin based endodontic sealer containing á-tricalcium phosphate and chlorhexidine santos pd*, portella ff, leitune vcb, parolo ccf, dos santos la, samuel smw, collares fm federal university of rio grande do sul dapper.santos@ufrgs.br the aim was to develop and characterize a methacrylate-based endodontic sealer containing á-tricalcium phosphate (á-tcp) and chlorhexidine (chx). a base resin was formulated using: 70% of udma, 15% of bisema and 15% of gdma. ytterbium trifluoride was added as a radiopacifier agent 60%wt. nine experimental groups were formulated adding different concentration, in weight, of á-tcp and chx: 0/0; 0/2.5; 0/5; 25/0; 25/2.5; 25/5; 50/0; 50/2.5; 50/5%. sealers were submitted to radiopacity, flow, and film thickness according iso 6876:2001; sorption and solubility according iso 4049:2009; ph; degree of conversion with ftir; microbial growth and diffusion disc tests. all groups presented radiopacity higher than 3mmal. the flow varied from 15.09 ± 0.11 to 17.33 ± 0.48mm. all sealers presented film thickness inferior than 50µm. the addition of chx increased the solubility, but did not interfere in sorption. all groups presented ph results were close to neutrality and degree of conversion higher than 60%. the addition of chx inhibited bacterial growth. the addition of chx and á-tcp appear a promising filler in develop of experimental endodontic sealers. 141 effect of non-carious cervical lesions and coronary structure loss on biomechanical behavior of premolars montes tc*, pereira ag, machado ac, zeola lf, faria vlg, souza lv, pereira fa,milito ga, soares pv federal university of uberlandia tatianacarvalhom@gmail.com dental lesions and cavity preparations can impact the biomechanical behavior of teeth due to tooth structure loss. the aim of this study was to analyze the influence of non carious cervical lesions (nccls) and coronary preparations on the biomechanical behavior of premolars, using finite element analysis (fea). it was generated 2d linear and elastics models, (auto-cad) simulating sound tooth (s); mod preparation (p); mod preparation restored with composite resin (pr), cervical lesion (l); cervical lesion restored with composite resin (lr), and combinations, pr + lr, p + l, l pr +, p + lr. the areas of each structure were meshed (ansys fea) with eight-node elements (plane183). all structures were asssumed isotropic except enamel and dentin, considered orthotropic. oblique load (45n) was applied on buccal and palatine cusps, simulating a sphere contact. data were analyzed by von mises (vm) and principal maximum strain (s1) criterion. vm showed similar pattern of stress distribution for s, pr, lr and pr + lr. models l, p + l, p + lr e pr + l had the highest strain at the center of the lesions with s1 levels of 0.22, 0.2, 0.25 and 0.31 mpa, respectively. for groups p e p + lr, vm stress concentration was observed at the internal angles of the preparations and at the cusps base. it can be concluded that nccls associated with loss of coronal tooth structure promotes greater strain values at the center of the lesion. models restored with composite resin presents similar biomechanical behavior to the s model and load application influenced in stress distribution. 143 biofilm formation in different materials used to restore non-carious cervical lesion carvalho vf*, soares pbf, flausino js, magalhães d, mello jdb, costa hl, junior wms, ribeiro sf, soares cj federal university of uberlandia valessa@outlook.com restoration of non-carious cervical lesions (lcnc) requires use of aesthetic materials such as composite resin and glass ionomer or possibly ionomer resin modified. this study evaluated the effect of the parameters of topography and hydrophobicity of these materials in biofilm formation. we analyzed four materials: conventional glass ionomer cement (kf), resin-modified glass ionomer cement (vt), nanofilled resin-modified glass ionomer (kn) nanofilled resin composite (fz). in the analysis using 3d laser profilometry (n = 10), we calculated the amplitude parameters (sa and sq), space (sds) and hybrid (ssc). hydrophobicity measured as the contact angle of water on the surface (n = 5). the biofilm was evaluated by confocal laser scanning microscopy, examining parameters and thickness of the biofilm biovolume (n = 5) after 24 hours of culture and staining with sodium fluorescein at 1%. all data were analyzed by anova for single factor followed by tukey’s test, and was used pearson correlation test between topography data with the parameters of the biofilm (á = .05). the parameters of topography showed significant direct correlation with biofilm formation. there were significant differences between the parameters of amplitude (fz=kn>vt>kf). kn showed the highest hydrophobicity. fz and kn showed less biofilm thickness and biovolume compared to vt and kf. the use of nanoparticulate materials results in better performance in the topography and biofilm formation. 140 the effect of abutment material on stress distribution in single anterior implant-supported restoration carvalho ma*; lazari pc; sotto-maior bs; cury aa, henriques gep piracicaba dental school unicamp marco_carv@hotmail.com the aim of this study was to evaluate the effect of the abutment material on stress distribution in single anterior implant-supported restorations, through the finite element method. three experimental groups were design from the combination three abutment material (titanium, zirconia and hybrid) on morse tapered (mt) implants: mtti, mtzr, mth. finite element models were obtained with the aid of modeling software and consisted of: titamax ex 4x13mm mt implants; mt anatomic abutment in titanium, zirconia and hybrid; lithium disilicate central incisor crown cemented over the abutment. the occlusal loading, consisted of a magnitude of 49n in 45 degrees to the implant long axis, was applied in six steps in order to simulate the incisal guidance. the equivalent von mises criterion (σvm) was used for both qualitative and quantitative evaluation of abutment. the maximum (σmax) and (ómin) minimum principal stresses were obtained for numerical comparison of zirconia abutment and zirconia abutment body. the highest abutment σvm (mpa) occurred in mtzr, followed by mth and mtti (315.61; 293.61; 289.36 respectively). the σmax and ómin values were lower in mth group than mtzr group. the stress distribution concentrated in the abutment/implant interface in all groups, regardless the abutment material. it was concluded that the hybrid abutment had similar mechanical performance to titanium one, and these were better than zirconia abutments. 142 photoelastic analysis of the stress distribution of different coating materials in prosthetic occlusal protocol mello cc*, mazaro jvq, silva cr, gennari-filho h, pellizzer ep araçatuba dental school unesp caroline.cantieri@gmail.com the aim of this study was to analyze, through the photoelasticity method, the tension generated in different situations of the branemark protocol prostheses retained by 4 implants, considering: 1. different lengths of cantilever; 2. different occlusal materials coating (acrylic resin – ra, metal – m, and porcelain p); 3. tilt posterior implants to 30o. two photoelastic models were made: model 1 4 implants external hexagon (conexão sistemas de próteses, brazil) (4.1x10mm) distributed perpendicular to the alveolar ridge; model 2 – medial implants (4.1x10mm) perpendicular to the alveolar ridge and posterior implants (4.1x13mm) tilt to the 30o. it was made three branemark protocol prostheses with ra, m and p occlusal coatings. axial loads of 100n were applied in the premolar and molar and conducted analysis photoelastic. in the model with straight implants, the stress distribution generated by the prosthesis with porcelain occlusal coating was close to the metal coating, and that acrylic resin coating showed a lower stress. loading the molar observed forming one more fringe order in the pattern of stress distribution compared to the loading premolar. the model 2, the premolar and molar loading showed no significant difference in the stress distribution among the different coating occlusal materials. the inclination of the implants decreased the extent of the cantilever and is biomechanically more favorable providing less stress on implants. in dentures retained by 4 implants with posterior tilt no difference in stress distribution among the differents occlusal coating materials. 144 fiber glass posts bond strength fixed with experimental resin cements containing an onium salt leite tv*, lima af, palialol arm, gonçalves l, lancelloti a, martins lrm piracicaba dental school unicamp thati_mds@hotmail.com cementation of fiber glass is still a challenge, mainly due to polymerization in the apical region. this study aimed to evaluate the bond strength (ru) of fiber glass fixed with experimental resin photo-activated resin cement (cre), containing different concentrations of the hexafluorophosphate of difeniliodonium salt (dfi) ranging tertiary amines edab and dmaema and compare them to a commercial resin cement (dual and photo-activatable). eighty bovine incisors roots 15 mm long and 2 mm diameter were divided into 8 groups (n = 10): g1 cre without addition of dfi 2 mol% of edab g2 cre, 0.5 mol% dfi, 2 mol% of edab; g3cre, 1 mol% of dfi, 2 mol% of edab; g4 cre without adding dfi, 2 mol% of dmaema g5 cre, 0.5 mol% of dfi, 2 mol% of dmaema and g6 cre, 1 mol% of dfi, 2 mol% of dmaema and g7 variolink ii, photo-activatable; g8 variolink ii dual. the adhesive protocol used was, etching, application of primer and bond scotchbond multipurpose. ru evaluated by the push-out speed of 1 mm / min and load cell of 200 n. analysis of variance was applied to a criterion with split plots and tukey test (á = 0,05). the amine type did not influence the ru (p> 0.05), the higher values of ru were found in g2, g3, g5, g6, g8, and lower values for g1, g4 and g7. invariably the apical region showed the lowest values of ru. the cre containing dfi proved able to cementation of fiber glass. the tertiary amine type and concentration of dfi had no effect on ru. gbmd 2013 – 49th meeting of the brazilian group of dental materials 261261261261261 braz j oral sci. 12(3):237-274 145 pre-heating experimental composites containing different filler loadings: evaluation of material´s sorption and solubility castro fla*, gomes krm, pereira ls, reges rv unip e unievangelica fabriciodcastro@yahoo.com.br the aim of this study was to investigate the effect of composite pre-heating on material’s sorption and solubility. an experimental composite was evaluated, which contains different filler loadings. forty seven specimens were obtained using this experimental composite (fgm, joinville, santa catarina, brazil). the specimens were built up using a stainless steel matrix with 8x2 mm. two temperatures (25°c and 60°c) and four composite filler loadings in %/weight (74.5, 75.5, 78.2; and 78.53) were investigated. the specimens were weighed at the following conditions: m1after drying at 37 ° c for 24h; m2after more 7 days of storage in ethanol / water (75% / 25%); m3after another day of drying. the specimen’s dimensions as well m1; m2 and m3 weights were used to calculate material´s sorption and solubility. data were analyzed using a two-way anova design (temperature and composite loading), considering α = 5%. the results of this study showed that the investigated factors as well their interaction did not influence the data obtained for both sorption and solubility (p> 0.05). it was concluded that within the limitations of this study, heating the composite at 60°c did not affect the sorption and the solubility of the material in comparison with the resin activated at 25°c. the results were not dependent of composite´s filler loading, at least for the experimental material evaluated. 147 differents mechanical tests to evaluate bond-strength on y-tzp prochnow c*, otani ac, may lg, cesar pf, valandro lf federal university of santa maria catinaprochnow@hotmail.com the objective of the present study was to evaluate the bond strength using different geometries of test, between a y-tzp ceramic and composite resin, according two types of surface treatments. for test execution were used 330 y-tzp blocks (size / shape according to each test) [yz cubes / in ceram vita], received one of the two surface treatments: utcontrol (untreated) (n = 180) and sssilica coating (cojet/espe) + silanization (relyx – ceramic primer 3m/espe) (n = 180). composite resin (opallis/fgm) of different geometries were made and cemented with resin cement (relyx u100-3m/espe) in the y-tzp. the 180 specimens was divided into 6 subgroups (n = 30) according to mechanical test: tbs: tensile; µtsb: microtensile; sbs: shear; µsbs: microshear; psh: push-out; µpsh: micropushout. one-way anova and kruskal-wallis were used for data analyzing. both the type of surface treatment as the size of the specimen showed a statistically significant difference between groups (p = 0.00). independent of the type of surface treatment, microtensile test (µtbs/ss: 37,245,63 mpa) and microshear (µsbs/ut: 9,255,45 mpa; µsbs/ss: 9,255,45 mpa) had higher values than their equivalent macro: (tbs/ut: 4,582,06 mpa; tbs/ss: 10,475,13 mpa; sbs/ut: 4,220,99 mpa; sbs/ss: 11,894,04 mpa), on the other hand, push-out found higher values in the macro tests (psh/ut: 41,479,92 mpa; psh/ss: 50,966,99 mpa), instead of micropush-out (µpsh/ut: 28,9310,69 mpa; µpsh/ss: 38,056,67 mpa). according to the results obtained, microshear, microtensile and shear tests have proved to be more satisfactory for evaluating bond strength. 149 influence of geometries and thickness of porcelain on the thermal residual stresses: finite element analysis chaves ks*, jikihara an, reis br, roscoe mg, ballester ry, meira jb university of sao paulo kianneschaves@gmail.com studies that evaluate the thermal residual stresses (trs) are performed using different specimen geometries. this study aimed to analyze, by the finite element method, the trs at porcelain generated in specimens with different geometries and porcelain thicknesses. axisymmetric finite element models of discs, cylinders and spheres were constructed by varying the porcelain thickness (1 mm, 2 mm and 3 mm). the zirconia thickness was kept at 0.7 mm for all models. the cooling protocol of the specimens (from the glass transition temperature porcelain of 600°c to room temperature) was simulated. the materials were considered homogeneous and linear elastic. the interface porcelain/zirconia was considered perfectly united. the peak and the distribution of the maximum principal stress (σ1) at the porcelain were analyzed. for the discs, the peaks of σ1 were 14 mpa, 23 mpa and 26 mpa for 1 mm, 2 mm and 3 mm respectively. the values of σ1 increased from the porcelain/zirconia interface to the outer surface, with no tensile stress in the central region (negative ó1), regardless of the thickness. for the cylinders, the peaks of σ1 were 13 mpa, 23 mpa and 28 mpa for 1 mm, 2 mm and 3 mm respectively. the values of σ1 decreased from the porcelain/zirconia interface to the outer surface, regardless of the porcelain thickness. for the spheres, the peaks of σ1 were 34 mpa for 1 mm and 35 mpa for 2 mm and 3 mm. the stress distribution pattern of spheres was similar to the cylinders. the extrapolation of the trt results obtained for simplified geometries to crowns is critical, since the geometry of the specimen influenced the distribution of stress in the veneering porcelain. 146 influence of the cementum presence and the periodontal ligament width on stress distribution: finite element analysis karkuszewski sd*, roscoe mg, tanaka cb, reis br, ballester ry, meira jc university of sao paulo ste_nkg@hotmail.com the aim of the present work was to investigate, by finite element analysis (fea), the influence of the cementum presence and the periodontal ligament (lp) width on the stress distribution by an intrusion loading. four axisymmetric fea models were created from a maxillary second premolar geometry: lpu-sc: uniform lp (0,25 mm of width) without cementum, lpu-c: lp uniform lp with cementum, lpnu-sc: nonuniform lp (0,3 mm of width at the cervical and apical third, and 0,2 mm at the middle third) without cementum, lpnu-c: lp nonuniform lp with cementum. lp, cementum, enamel, dentine, pulp, cortical and cancellous bone were considered elastic and isotropic. a total load of 10n was applied at the occlusal surface, simulating an intrusion movement. the displacement of the nodes at the cancellous bone base was restricted. the minimum principal stress (σ3) distributions were analyzed in the radicular dentin, lp and cortical bone. for the radicular dentin, the peak of σ3 was obtained at the cervical region (-0,57 mpa to lpu-sc and lpnu-sc; -0,58 mpa to lpu-c; -0,59 mpa to lpnu-c). for the periodontal ligament, the peak of σ3 was obtained at the apical region (-0,34 mpa to lpu-sc, lpnu-sc and lpnu-c; -0,43 mpa to lpu-c. for cortical bone, the peak of σ3 was obtained at the middle third radicular (-0,34 mpa to lpu-sc, lpnu-sc and lpnuc; -0,27 mpa to lpu-c. the fea analysis demonstrated that the cementum presence and the lp width did not affect the stress distribution in the radicular dentin, pdl and cortical bone. 148 leveraging the effect of hydrogen peroxide concentration 6% maximum dutra santos m*, antunes lopes b, rodrigues a, capel cardoso pe university of sao paulo mariana.dutra12@gmail.com the aim was to quantify the efficacy of hydrogen peroxide 6%, associated with fenton or photo-fenton technologies, and compare it to 38% conventional peroxide. an aqueous solution of the carmine cochineal dye (0.005%) was prepared,and submitted to different bleaching processes according to their experimental group: group 1 carmine cochineal dye (c), group 2 c + hydrogen peroxide 6% (h2o2 6%),;group 3 c + h2o2 6% + fe2+ + ,group 4 c + h2o2 6% + fe2+ + led irradiation for 15 min, group 5 c + h2o2 6% + fe2+ + uva light irradiation for 15 min, group 6 c + hydrogen peroxide 38% (h2o2 38%). for each experimental group 3 samples were performed and these were analyzed by a spectrophotometer, where the initial and final concentration level of dye was recorded. ,. results were analyzed statistically by anova and tukey test, where all groups were different. group 5 showed the greatest reduction compared to the initial concentration of dye (58.6%), followed by group 4 (49.8%), group 3 (20.9%), group 6 (14.9% ) and group 2 (10.8%). group 1 did not change. thus, we conclude that the reactions of fenton and photo-fenton influenced positively for decolorization. the lower concentration hydrogen peroxide (6%) associated with the fenton or the photo-fenton technology presented a superior result when compared to the hydrogen peroxide at higher concentration (38%). 150 effect of the type of die and infrastructure material on the final color of prosthetic crowns borba m*, vendruscolo tk, sonza qn passo fundo university marcia_borb@hotmail.com the objective of this study was to evaluate the effect of the type of die and infrastructure material on the final color and lightness of prosthetic crowns. a steel model simulating an abutment tooth was used to design prosthetic crowns. three types of infrastructure (is) material were evaluated (n=8): mc nicr alloy is; yz – y-tzp is (vita in-ceram yz); iz –in-ceram zirconia is (vita). all is were veneered with the same shade (2m2) and thickness of porcelain. the color difference (de) and lightness difference (dl) between the initial porcelain shade and the final crown were obtained using a clinical spectrophotometer (vita easyshade). two measurements were taken from each crown, one with the crown placed over a metal die and another over a resin composite die, using a black background. data were analyzed using two-way anova and tukey’s test with a significance level of 5%. for both parameters, only the factor material showed statistical significance (p=0,001). de was higher and similar for yz and iz groups. mc showed a significantly lower de value. iz group had the highest dl value, resulting in a whiter crown than mc and yz. it was concluded that the type of die had no influence on the color and lightness of the evaluated crowns. in addition, allceramic crowns showed a higher color difference then metal-ceramic crowns. yet, the color differences observed are within the clinical threshold (de lower than 3.3). gbmd 2013 – 49th meeting of the brazilian group of dental materials262262262262262 braz j oral sci. 12(3):237-274 151 influence of different surface treatments on the bond strength between composites and a resin cement vasconcelos l*, borges a, foscaldo t, da silva em, poskus lt fluminense federal university labiom-r vasconcelos.luisa@yahoo.com.br this study aims to evaluate the influence of different surface treatments on bond strength (bs) between different composite and resin cement. 75 composite discs was fabricated (10 x 2 mm) filtek p90, filtek z250 and filtek z350 xt (3m espe), were divided into 5 groups according to the surface treatment: c = control no treatment; sandblasting j = aluminum oxide (50ìm); sandblasting je = + 99.3% ethanol for 5 min; silica coating s = (3m-espe cojet 30 microns); ss = silica coating + silane. pvc tubes (0.5 x 0.80 mm) were attached on the composite disc, and then, inserted resin cement (3m espe-relyx arc). after 24 hours artificial saliva storage at 37oc, the specimens were tested for microshear crosshead speed of 1.0 mm / min. data were evaluated in two-way anova and tukey’s test (5%) for contrast. the sandblasting with aluminum oxide was efficient in increasing the bs for composites z350 and p90. the composite z250 was not influenced by the treatments studied, with the exception of ethanol treatment that reduced the values. however, silica coating followed by silane application showed similar results as control for all composites. in conclusion, bs values were dependent on the type of composite used and the surface treatment. the sandblasting with aluminum oxide was the best treatment leding to higher bs values, and should be suitable for surface treatment, while the use of ethanol is doubtful. 153 micro-raman characterization of hanano adhesive/dentin interface balbinot gs*, provenzi c, portella ff, leitune vcb, collares fm, samuel smw rio grande do sul federal university gabi_balbinot@hotmail.com the aim of this study is to characterize the adhesive interface of an experimental resin using micro-raman spectroscopy. human molars were cleaned and stored. the superficial enamel was removed and each tooth was divided in blocks. exposed dentin was etched and commercial primer was applied. adhesive resin was applied in the dentin in different concentrations of hanano: 0%; 0,5%; 1%; 2%; 5%; 10% and 20% . furthermore, two commercial adhesives were tested. above the adhesive, one resin increment was placed. interfaces were prepared by sectioning perpendicular to the flat adhesive–dentine surface. analysis was performed using raman microscope and one-dimensional mapping was performed over 150µm line across the adhesive– dentine interface at 1µm intervals using the xyz axes. one mapping was performed and processing allowed differentiating spectral components of the adhesive and dentine. one correspondent peak of each substance was used for integration: hydroxyapatite at 960cm-1 and resin at 1610cm-1. the presence of hydroxyapatite could be seen throughout the hybrid layer. however, in the groups of commercial adhesive system, any phosphates were observed. experimental adhesive resins with hanano seem to improve adhesive interfaces. 155 degradation of edc-biomodified collagen and mmp inactivation in situ scheffel dls*, scheffel rh, agee ka, de souza costa ca, pashley dh, hebling j araraquara dental school – unesp de_salles@yahoo.com.br this study evaluated the effect of edc application in short periods of time on dentin collagen degradation and matrix-bound mmp inactivation. two hundred beams (1x1x3mm) were obtained from mid-coronal dentin of sound third molars and completely demineralized in 10% phosphoric acid for 18 h at 25°c. the specimens were randomly divided into 5 groups and treated for 30 or 60s with the following solutions: deionized water (negative control); 0.5m edc, 1m edc, 2m edc and 10% glutaraldehyde (gd) (positive control) (n=10). ten beams of each group were submitted to a microcolorimetric assay (sensolyte) to evaluate the total mmp activity before and after the treaments. the remaining 10 beams were tested for hyp realease and dry mass loss after 1 week of artificial saliva storage at 37°c. data were analyzed by kruskal-wallis, mann-whitney, anova and tukey tests (α=0.05). all treatments were able to inactivate mmps and the best results were observed for the group treated with 2m edc for 60s (82.7%). edc was capable of reducing mass loss and hyp release irrespective of concentration and time of application. dentin treatment with edc is effective in reducing collagen degradation and may enhance the stability of resin-dentin bonds over time. this work was supported in parts by roi de 05306 from nidcr, capes 6937/11-0, cnpq 305204/2010-6 and fapesp 08866-4. 152 temperature and type of silanes effetc in bond strength of glass fiber post and composite resin core/resin cement rosatto cmp*, roscoe mg, silva gr, novais vr, soares cj federal university of uberlandia camilamaria_pr@hotmail.com the aim of this study was evaluate the effect of temperature (23º e 60ºc) and type of silane (3 prehydrolyzed: silano, angelus; prosil, fgm; and relyx ceramic primer, 3m espe; and 1 two-component, coupling agent, dentsply) on push-out bond strength (rups) of glass fiber posts (pfv) and resin cement (cr) or and composite resin core (np). pfv were treated with hydrogen peroxide 24% for 1 minute. the negative controls did not receive any silane. in other groups, it was applied 1 of the 4 tested silanes for 1 minute. then, on half the pfvs was used air jet (23ºc); on the other half hairdryer air jet (60ºc controlled by multimeter) was used, both for 5 s. the pfvs were divided in two groups to test rups on np and cr. np/pfv were embedded with composite (filtek z250) in circular plastic matrix. cr/pfv were cemented (relyx u100) into endodontically treated bovine roots. the samples were sectioned and rups evaluated. the failure pattern was analysed by confocal microscopy. two-way anova (2x4) and tukey’s test evaluated the effect of the study factors, with subdivided for root region in cr. the one-way anova and dunnett’s test compared the np and cr with control groups. values of rups (mpa) cr: between 27.5±10.5 and 12.2±2.7; in np: 13.2±2.7 and 16.9±2.6. it is possible to clonclude that higher temperature (60ºc) improves rups only cr (resin cement). temperature had no signficative influence on silanes used on np (composite resin). the two-component silane had greater variability (p=0.003) in 23º and 60ºc. the pre-hydrolyzed silanes had good performance in 23º and 60ºc regardless of the root region. 154 effect of cutting with rotatory instruments in the integrity, morphology and flexural strength of fiber posts scheffel dls*, scheffel rh, agee ka, de souza costa ca, pashley dh, hebling j federal university of santa maria andressa.venturini@hotmail.com to evaluate the effect of cutting with different rotary instruments on the 3-point (n=10) and 2-point (n=10) flexural strength, and the surface micromorphology (n=2). one hundred and thirty-two posts (white post, fgm, brazil) were allocated into 12 groups: ctrl – without cutting, db – coarse diamond bur (kg sorensen), dbff – extra fine coarse diamond bur (kg sorensen), cb – carbide bur (kg sorensen), cd – carborundum disc (komet), dd – diamond disc (kg sorensen). after specimen preparation, all cutting procedures were performed with abundant irrigation. the data obtained from the flexural strength tests were inserted into specific formulas for calculating the recommended resistance. micromorphological analysis of cutting surfaces was made by sem. one-way anova indicated no statistical significant difference among the groups (p=0.0968) for 3-point bending test, though statistical difference was found (p=0.0233) for the 2-point bending. tukey’s test indicated statistical difference between dc and ctrl, with higher values for cd group. micromorphological analysis showed superficial changes generated by the cutting instruments assessed, but insufficient to affect the flexural strength of the polymer. the cutting instruments used with simultaneous cooling not affected intrinsic strength of the fiber post tested. therefore, these methods seem to be suitable for cutting the assessed composite. 156 flexural strength of monolithic and trylayer ceramics structures basso gr*, moraes rr, griggs j, borba m, della bona a federal university of pelotas gabybasso@yahoo.com.br the aim of this study was to evaluate the flexural strength (ó) and weibull modulus of monolithic (m) and trilayer (t) ceramics structures used for the cad-on (ivoclar) technology. bar-shaped m (ips e.max zircad ivoclar vivadent) and t specimens (ips e.max zircad – ips e.max cad crystall./connect ips e.max cad ivoclar vivadent) with 1.8 mm x 4 mm x 16 mm were fabricated (n=30). all specimens were flexural strength tested in 37°c distilled water using a universal testing machine at a crosshead speed of 0.5 mm/min. the failure load was recorded and the flexural strength values were calculated. fractographic analysis was performed using optical and scanning electron microscopes (sem) to examine the fractured surfaces identifying fracture markings and the critical crack. results were statistically analyzed using student t test (α=0.05). mean flexural strength (σ) and standard deviation (sd) values for m= 915.55±143.77a, and t=763.07±208.24 b, weibull modulus m= 7.6 (5.7 – 10.1)a, and t= 4.1 (3.1 – 5.3)b, characteristic strength (σ0) m= 975 (928 – 1025)a, and t= 841 (766 – 923)b, 5% failure probability (σ5%) m=660 (577 – 756)a and t=405 (317 – 518)b. the 95% confidence intervals are in parentheses. the monolithic structures (m) showed significantly higher mean values than the t for all parameters evaluated. gbmd 2013 – 49th meeting of the brazilian group of dental materials 263263263263263 braz j oral sci. 12(3):237-274 157 influence of ceramic thickness on sorption and solubility of dual resin cements silva jpl, furtado ls, dantas lcm, raposo lha, sinhoreti mac, costa ar, correr ab, correr-sobrinho l piracicaba dental school unicamp joaodf22@hotmail.com the aim of this study was to evaluate the sorption (so) and solubility (sol) of 3 resin cements light-cured beneath a dental ceramic. five specimens with 6 mm diameter and 0.5 mm of thickness (relyx arc, relyx u100 and variolink ii-var) were carried out using a metallic mold and light-cured beneath a ceramic disk with 12 mm diameter by 0.7 mm of thickness. the specimens were immersed in distilled water, lactic acid, and propionic acid to 37 oc and were weighed at intervals of 1, 24, 48, and 72 h to obtain masses (m1, m2, m3). the diameter and thickness of the specimens after final drying (m1) were measured to obtain the volume (v) of each specimen and calculate the rates of so and sol. data were submitted to anova and dunn’s test (á = 0.05). according to so data, var and arc had the highest values in lactic acid with statistical difference in relation to the propionic acid and water. no statistical difference was observed for u100 cement. for sol, propionic acid values were statistically superior to other solution storage, for the three cements, except for the arc into lactic acid. it was concluded that variolink ii had higher values of sorption and solubility in all solution media. 159 influence of candida albicans biofilm and mma surface treatment on adhesion of soft liners to acrylic resin cavalcanti yw*, bertolini mm, bordin d, silva wj, del bel cury aa piracicaba dental school unicamp yuri.wanderley@yahoo.com.br this study analyzed the influence of candida albicans biofilm on bond strength between soft denture liners and poly (methylmethacrylate) pmma resin, which previously received, or not, methylmethacrylate (mma) pretreatment. specimens were prepared and randomly divided into eight groups, according to study factors: pmma pretreatment (mma and no treatment), denture liner type (silicone-based and pmma-based denture liner), and c. albicans biofilm accumulation (with and without biofilm). pmma bars were prepared and had its surface treated. denture liners were applied between two treated pmma bars and specimens (n=10) were submitted to biofilm formation, or pbs storage, for 12 days, at 35º c, under agitation. afterwards, tensile bond strength test was performed and failure type was evaluated in stereomicroscope. highest tensile bond strength was observed in groups with silicone-based denture liner, with or without pmma pretreatment, stored in pbs (p<0.01). silicone-based specimens presented mostly adhesive failures, while pmma-based groups presented predominantly cohesive failures. in vitro exposure to c. albicans biofilm reduced the tensile bond strength of denture liners to pmma resin, and mma pretreatment of denture base may be recommended for relining procedures. 161 the effect of poly (methyl methacrylate) surface treatments on the adhesion of silicone-based resilient denture liners bertolini mm*, cavalcanti yw, del bel cury aa, silva wj piracicaba dental school unicamp martinnabertolini@gmail.com different surface treatment protocols of poly (methyl methacrylate) (pmma) have been proposed to improve the adhesion of silicone-based resilient denture liners to pmma surfaces. the aim was to o evaluate the effect of different pmma surface treatments on the adhesion of silicone-based resilient denture liners. pmma specimens were prepared and divided into 4 treatment groups: no treatment (control c), methyl methacrylate (mma) for 180 seconds, acetone (ac) for 30 seconds, or ethyl acetate (ea) for 60 seconds. pmma disks (30.0 mm in diameter × 5.0 mm thick; total specimen number=40, n=10) were evaluated regarding surface roughness (sr) and surface free energy (sfe). to evaluate tensile bond strength, the resilient material was applied between 2 treated pmma bars (60.0 × 5.0 × 5.0 mm; n=20 for each group) to form a 2-mm-thick layer. data were analyzed by 1-way anova and the tukey hsd tests (α<.05). failure type was assessed and the pmma surface treatment modifications were visualized with scanning electron microscopy (sem). the sr was increased (p<.05) by mma treatment. for groups ac and ea, the sfe decreased (p<.05). the tensile bond strength was higher for the mma and ea groups (p<.05). specimens treated with ac and mma presented a cleaner surface, while the ea treatment produced a porous topography. the mma and ea surface treatment protocols improved the adhesion of a silicone-based resilient denture liner to pmma. 158 discolored tooth substrate influence of thickness on the masking ability of laminate veneers kodama ab *, hauschild fb, basso gr, moraes rr, della bona a, kaizer mr, boscato n federal university of pelotas abkodama@hotmail.com the aim of this study was to evaluate the masking ability and optical effect of different thicknesses of the enamel and dentin layers on ceramic veneers in vitro, simulating dental substrate with higher (shade a2) and lower (color c4) value by using ceramic discs. the combination of ceramic discs of enamel (e) and dentine (d) of different thicknesses (0.5 mm, 0.8 mm and 1.00 mm) resulted in the following groups (n = 10): d1e1, d1e0.8; d1e0.5; d0.8e0.8; d0.8e0.5, d0.5e0.5. it was evaluated the translucency of mono and bilayer specimens, and the influence of the substrate on the final color of bilayer specimens both with ciel*a*b* (∆e), vita classical and vita 3d master. all measurements were performed with a spectrophotometer vita easyshade (vita zahnfabrik/bad saeckingen, germany). for the monolayer groups, both enamel and dentine ceramics, as lower the thickness the higher the translucency. for bilayer groups, both the translucency and the influence of the tooth-colored background were more sensitive to the dentin layer thickness. the color variation (∆e) with tooth-colored backgrounds was greater with the lower value background. in comparison to the higher value background, it was observed that for the lower value background the set thickness was more critical. the final color ranged around c3 and c2 for thicker specimens and around c3 and c4 for thinner specimens. it was concluded that the reduction in thickness of the dentin layer and a tooth-colored background with lower value had greater influence in the final color assessment for laminated veneer ceramics. 160 correlation between bond strength and nanomechanical properties of adhesive interface freitas ph*, correr ab, consani s piracicaba dental school unicamp freitasph@globo.com to evaluate the correlation between nanohardness and elastic modulus of the adhesive interface and microtensile bond strength of adhesive systems to dentin. 40 sound human third molars were randomly divided into four groups according to the adhesive system. correlation between bond strength and mechanical properties of adhesive interface was evaluated with spearman’s test. anova and tukey’s test was used to evaluate the influence of adhesive system on all variables (α=0.05). the spearman analysis did not showed significant correlation between microtensile bond strength and mechanical properties of adhesive interface (p>0.05). microtensile bond strength of clearfil se bond (62,57 ± 11,49) was significantly higher than single bond 2 (43,97 ± 9,73) and clearfill s3 (41,84 ± 6,52); adper scotchbond multipurpose (56,94 ± 9,90) showed bond strength statistically higher than clearfil s3 bond. adhesive systems did not influence significantly the nanohardness and elastic modulus of the hybrid layer. nanohardness and elastic modulus of adhesive layer using single bond 2 (ha=0,40 ± 0,03; ya=6,69 ± 0,57) was significantly higher than the clearfill se (ha=0,33 ± 0,03; ya=5,08 ± 0,35), clearfill s3 (ha=0,31 ± 0,04; ya=5,41 ± 0,55) and adper scotchbond multipurpose (ha=0,32 ± 0,02 ; ya=5,49 ± 0,19). the 4 adhesive systems tested haven’t shown correlation between nanohardness and elastic modulus of the adhesive interface and microtensile bond strength to dentin after 24h storage. 162 effect of surface treatment of composite resins on bond strength to adhesive resin cements vilela alr*; santos vh; figueiredo fed; menezes ms; faria-e-silva al federal university of uberlandia analaurarvilela@gmail.com the self-adhesive cements have been popularized by the ease use, without treatment of the substrate, but little is known about his union to composite resin commonly used as core. thus, this study aimed to evaluate the bond strength of two selfadhesive resin cements to composite resin, simulating the core of indirect restorations. the substrate used in this study was confectioned with the composite resin tetric ceram (ivoclar). the resin surface was treated with one of following treatments (n=10): 35% phosphoric acid for 30s (pa); silane; pa + silane; pa + adhesive; or pa + silane + adhesive. the absence of treatment was used as control. after the treatments, silicone mold containing a rounded orifice with 1 mm2 of diameter was placed over the composite resin and one of self-adhesive resin cements (relyx u100 – 3m espe; or biscem – bisco) was inserted into orifices and light-cured. the self-adhesive cement cylinders were submitted to shear load and data analyzed by two-way anova and tukey’s test (p<0.05). independently of cement, pa + silane + adhesive showed higher bond strength values than pa and pa + silane. there was no difference between the other treatments, while u-100 presented higher bond values than biscem for all experimental conditions. in conclusion, the pre-treatments of composite resin surface can to affect the bond strength of selfadhesive resin cements to this substrate, but no one of treatments evaluated differed from the control without treatment. 264264264264264 gbmd 2013 – 49th meeting of the brazilian group of dental materials braz j oral sci. 12(3):237-274 163 analysis of stress distribution at the interface of adhesive class v restorations pereira fa*, roscoe mg, soares pv, meira jbc fousp fabricia_pereira@hotmail.com the aim of this study was to analyze the finite element method, the influence of the elastic modulus (e) of the composite stress distribution in adhesive interfaces of class v restorations. two 2d models of premolar were created, representing enamel, dentin, pulp, adhesive and resin composite. for the model called “flow”, the composite was simulated with e 5.3 gpa for the “traditional” model, e was 16.6 gpa materials were homogeneous, and linear elastic. an oblique load of 100 n was distributed in 3 nodes of the external dimension of the buccal cusp. the displacement of the nodes of the external root surface located 5 mm below the cementoenamel junction has been restricted. it was observed values of principal maximum stress (ó1) along the interface adhesive / dentin and composite resin / adhesive to both models. the highest values were observed in ó1 cavosurface angle of interfaces. although the flow model has shown the highest peak tensile stress curve ó1 versus distance at the interface showed a sharp drop away when the cavosurface angle, so that a larger extent of the interfaces was with smaller values of tensile stress when compared the interfaces of the traditional model. the choice of composite type flow seems interesting to restore cervical lesions, by request unless the interface when the occlusal loading tends to lengthen the restoration towards cervical-occlusal. 165 bioactivity of a novel adhesive resin with incorporation of niobium pentoxide klein m*, leitune vcb, takimi a, samuel smw, collares fm federal university of rio grande do sul marianinha_klein@hotmail.com the purpose of this study was to evaluate, in vitro, the bioactivity of experimental adhesive resins with incorporation of niobium pentoxide (nb2o5). an experimental adhesive resin was formulated with different concentrations of nb2o5 (0, 5, 10 and 20 wt%). three specimens for each concentration were produced. after photoactivation, the specimens were immersed at simulated body fluid (sbf) for 7, 14 and 28 days. after each immersion period, the specimens were evaluated by micro raman, scanning electron microscopy (sem) and energy dispersive spectroscopy (eds). an area of 20736 µm2 for each specimen was analyzed by micro raman, performing one analysis each 9.6 µm. it was used a 785 nm laser for 10 s with 2 co-additions. sem images were obtained with a low vacuum electron microscope. eds analysis were performed using 2000x magnification images. despite the concentration of nb2o5 and immersion time at sbf was possible to observe at raman, sem and eds analysis the deposition of material compatible with po4 at the surface of the specimens with nb2o5. at control group, it was not possible to detect the presence of po4 deposition. therefore, nb2o5 incorporation presented in vitro bioactivity for experimental adhesive resins. 167 evaluation of surface roughness of ionomer cement after manipulation in conventional glass plate and paper block panarello p*, reges rv, castro fal, botelho tl, silva rf paulista university paola_panarello@hotmail.com the aim of this study was to evaluate the surface roughness of conventional glass ionomer cement (gic) (c vitro cem a3 dfl), with a profilometer (mitutoyo), after manipulation in a glass plate and a paper pad. 5 samples were used in the mic test handled in conventional glass plate and pad, with a plastic spatula, according to the manufacturer’s recommendations. manipulated ionomers were divided into two groups (n = 5) according to the protocols: s1 glass plate; s2 pad. after the setting time of approximately 5 minutes, the samples were subjected to quantitative analysis of surface roughness (ra) measured by profilomete. there were five readings on each specimen, with a speed of 0.5 mm/s and obtained the statistical difference of roughness between the glass plate and pad. by statistical analysis it was concluded that the surface roughness of the ivc when handled in paper pad is higher than when manipulated on the glass plate, the authors concluded therefore that it is most suitable to use the glass plate associated with a plastic spatula. 164 effect of saliva on enamel after microabrasion: in situ study pini np*, lima danl, ambrosando gmb, aguiar fhb, lovadino jr piracicaba dental school unicamp nubiapini01@gmail.com aim: to evaluate, in situ, the effect of saliva on enamel after microabrasion. bovine enamel blocks were divided into 9 groups (n=19), being: 1 control group, without treatment and 7 days of salivary exposition; 4 groups treated with 35% phosphoric acid + pumice and 4 groups with 6.6% hydrochloric acid + silica. the groups with microabrasion were subdivided in accordance with the in situ protocol: without, with 1 hour, 24 hours or 7 days salivary exposition. nineteen volunteers used an intraoral appliance. the surface microhardness (smh) and roughness (ra) were evaluated before and after the microabrasion, and after salivary exposition. representative specimens were evaluated by scanning electron microscopy (sem). the results were analyzed with proc mixed, tukey-kramer and dunnet tests (p<0.05). for smh and ra, all groups presented reduction of mean values after microabrasion with statistical differences in relation to the control. for smh, the treatment with hcl + silica presented the lower reduction. after the in situ regimen, the results showed that the microabrasion with hcl + silica was more prone to action of saliva, once with 1 hour the smh present increase in its value with difference in relation to the analysis after microabrasion. only for smh, hcl + silica was able to reestablish this property as similar found in the control. sem analysis showed, for both treatments, the saliva effect on different time observed. the microabrasion treatment with hcl + silica resulted in an enamel surface more prone to remineralization. 166 different design tests for the evaluation of the bond strength between resin cement and zirconia rodrigues va*, corazza ph, bergoli cd, borges als sao jose dos campos state university unesp vinicius.rodrigues@fosjc.unesp.br this study evaluated the effect of different methods of shear (wire or knife) and different specimens (cement cylinders or composite resin cylinders) on the bond strength between resin cement and zirconia by bond strength test and finite element analysis (fea). for the study, twenty zirconia blocks (ytzp vita zahnfabrik, germany), with dimensions of 10 x 10 x 5 mm were obtained and sandblasted with cojet (3m espe, usa). on the zirconia block was build up a resin cement cylinder (panavia f, kuraray, japan) and cemented a composite resin cylinder (z 250, 3m espe, usa). both cylinders were obtained with a plastic matrix (θ = 3 mm). half of the specimens were subjected to shear test with a wire (θ = 0.4 mm) and the other half to shear test with a knife (1 mm / min) (emic dl 2000, pr, brazil). the values of the bond strength were subjected to anova-two way and tukey test (á = 0.05). the specimens were submitted to failure analysis. statistical analysis showed no influence of the shear method (p = 0.933) or the material used to fabricate the cylinder (p = 0.806). however, the failure analysis showed predominance of adhesive failure between the cement and zirconia submitted to wire and for the resin cement cylinders. the fea distribution showed more favorable tensile stress for the occurrence of adhesive failure between the cement / zirconia for the wire shear test. thus, the current study conclude that the use of wire and resin cement cylinders enable a better evaluation between the resin cement / zirconia interface. 168 assessment of enamel microabrasion technique and their effects over time: case study franco lm*, machado ls, sundfeld neto d, sundfeld rh araçatuba state university – unesp lauramf3@hotmail.com the presence of stains and irregularities on dental enamel may compromise dental esthetics, but when localized in most superficial layers of the enamel, they can be removed by enamel microabrasion technique. the aim of this study was to demonstrate through different clinical reports, the steps of the technique, the indications and limitations, and longitudinal clinical following of more than 20 years of observation. with the presentation of cases, it was observed, after performing microabrasion technique, a significant improvement of dental esthetics with the removal of stains, which over time, still show surface smoothness, brightness, anatomy and proper esthetics. it may be concluded that the enamel microabrasion technique is a safe clinical procedure, simple to perform, with excellent esthetic resolution. 265265265265265gbmd 2013 – 49th meeting of the brazilian group of dental materials braz j oral sci. 12(3):237-274 169 influence of delayed photoactivation time at self-adhesive resin cements augusto cr*, collares fm federal university of rio grande do sul carolrocha13@yahoo.com.br the aim of this study was to evaluate the influence of immediate and delayed photoactivation time at the degree of conversion, depth of cure and solvent degradation of dual-cured self-adhesive resin cements (relyx u100 and biscem). according to the time for photoactivation, five groups were evaluated: g30s, light activation after 30 sec; g60s, after 60 sec; g150s, after 150 sec; g5m, after 5 min; g10m, after 10 min. the degree of conversion was evaluated by fourier transform infrared (ftir) spectroscopy (bruker), using an attenuated total reflectance (atr) device. the depth of cure was evaluated using spectroscopic micro raman and solvent degradation by knoop microhardness before and after immersion in ethanol for 4 hours. the degree of conversion increased in all groups after photoactivation, regardless of the delayed time (p<0.05). the depth of cure, after 7 days, showed no statistically significant difference in both cements (p>0.05). the cement biscem with 5 and 10 minutes delayed photoactivation showed degradation in ethanol (p> 0.05). the delayed photoactivation of 5 minutes of biscem increased degradation the material degradation in solvent. therefore, the delayed photoactivation time did not improved the degree of conversion and depth of cure of the self-adhesive resin cements tested. 171 comparasion of vertical missfit of fixed implant frameworks: conventional casted x cnc milled barros vm, fontoura dc*, discacciati jac, vasconcellos wa, júnior cso, vaz rr. fo-ufmg cunhafontoura@yahoo.com.br passive fit of an implant framework is one of the mechanical parameters can affect the longevity of the prosthesis. several complications in treatment with dental implants may be due to misfit of metallic frameworks and can be aggravated by the absence of periodontal ligament. the objective of this study is to compare the misfit of a one-piece casted and a metallic milled by cad cam system implant frameworks. on a master aluminum model were installed four analogues of conical abutments. on these, were fabricated two frameworks: a casted in cocr and a milled by cad cam system. the vertical misfits in the framework analogues interface were measured using an optical microscope. the mean misfit in each of the pillars (45, 43, 33 and 35) and the mean misfit of each framework were measured. in milled framework there was a reduction in the values of vertical misfit in all pillars. the casted framework showed a mean vertical misfit (108.6 µm) approximately 10 times larger than the milled framework (9.6 µm). 173 s. mutans adherence on the surface of toothpaste -treated resinbased materials almeida lfd*, paula jf, santos tfc, cavalcant yw, almeida-marques rvd, hebling j araraquara dental school unesp leopoldinalmeida@hotmail.com the aim of this study was to evaluate the adherence of streptococcus mutans on resin-based materials treated with fluoride and non-fluoride containing dentifrice. cylindrical specimens (n=9) (5.0 mm diameter x 2.5 mm thickness) of composite resin (cr) and resin sealant (rs) were fabricated and immersed in brain heart infusion (bhi) broth supplemented with 10% sucrose and inoculated with 0.1 ml of s. mutans suspension (1 x 107 cfu/ml). specimens were incubated for 24 h (t1) or 7 days (t2) after being treated with the supernatant from fluoride toothpaste (ft), a non-fluoride, phytotherapic toothpaste (nfp) or saline (c – control). adhered cells were serially diluted and analyzed according to the number of viable microorganisms (ufc/ml x 105). data were analyzed by three-way anova and holm-sidak multiple comparison (α=0.05). analysis of variance showed a statistically significant influence of “treatment” while no effect was detected for period and material as well as for any possible interplay (p>0.05). for cr, only the ft treatment was effective in reducing the number of adhered microrganisms (p<0.01), with no difference between nfp and c (p>0.05). for rs, both toothpaste treatments significantly reduced the number of s. mutans on the surface of the material. both resin-based materials contributed similarly to s. mutans adherence after 24 h and 7 days and only the ft treatment was effective for both materials. 170 analysis of wear of diamond burs by scanning electron microscopy spini phr*, zeola lf, machado ac, gonzaga rcq, tolentino ab, pereira ag, soares pv federal university of uberlandia pedrospini@hotmail.com diamond burs are abrasives rotary instruments used in restorative dentistry. these tips may differ in granulation diamond, resulting in damage to the quality and standardization of preparations. the aim of this study was to analyze and compare by scanning electron microscopy the quality of the diamond beads impregnated and the amount of wear experienced by diamond burs. it was used 70 diamond burs, model 1014, of seven trademarks: kg sorensen, option, microdont, fava, vortex, zeep and kerr. the instruments were tested in a nanohibrid composite resin block (n = 5) and lithium disilicate (n = 5). the samples were subjected to three sequential wear of 3 minutes. at the beginning and end of each stage, sem was performed. as a result, it was observed that microdont and fava presented few diamond impregnated or irregular distribution of the granules initially. after periods of wear, the brands presented partial loss or large wear of the diamond. after testing, it was concluded that there was a change of shape, besides the loss of diamonds. thus, it emphasizes the importance of replacing rotary instruments in clinical activity. 172 degradation of primary caries-affected dentin bonding to mdpbcontaining adhesive system after s.mutans storage carvalho fg*, carlo hl, santos rl, guenes gmt, costa chm, puppin-rontani rm federal university of campina grande fabigalbi@yahoo.com.br the aim of this study was to evaluate the effect of s. mutans culture and water storage on the durability of bonding of fluoride/mdpb monomer-containing adhesive system to primary artificial caries-affected dentin (cad). twelve primary molars were selected. flat surfaces of dentin were submitted to artificial caries development in s. mutans and bhi broth. caries-infected dentin was removed with burs according to clinical criteria and cad cavities were restored with adper scotchbond multi-purpose (sbm) or clearfil protect bond (cpb) (n=6). nontrimmed resin-dentin bonded interfaces (1mm2) were stored in s.mutans + bhi for 3 days, in deionized water for 3 months and afterwards subjected to microtensile bond strength (µtbs) test. the control group was not submitted to storage and immediate µtbs testing was performed. fractographic analysis was performed after µtbs testing. two-way anova with split-plot design and tukey’s tests were performed. there was a significant difference between µtbs values of sbm (25.2 ± 8.5 mpa) and cpb (15.6 ± 6.1 mpa) only for control group. a significant decrease in µtbs values after s. mutans culture and water storage was observed for sbm (18.7 ± 5.7 mpa and 17.4 ± 4.1 mpa, respectively) and cpb (13.9 ± 5.2 mpa and 13.7 ± 4.8 mpa, respectively), but no difference was found between them. the fluoride/mdpbcontaining adhesive system did not prevent the degradation of primary cad bond strength in both degradation methods. 174 influence of hydrogen peroxide-based bleaching agents on the bond strength of resin-enamel/dentin interfaces carlo hl*, didier vf, batista aud, fonseca rb, montenegro rv, carvalho fg, bonan prf, santo, rl federal university of paraíba hugo@ccs.ufpb.br this study evaluated the effect of different bleaching techniques on the bond strength of pre-existing adhesive restorations in enamel and dentin. hydrogen peroxidebased bleaching gels with different concentrations (7.5% and 35%) were used on composite restorations of adper single bond 2 (3m/espe, st. paul, usa) and filtek z250 (3m/espe, st. paul, usa). twenty human third molars were randomly divided into 8 groups: ge – enamel control; ge7.5 – bleaching using 7.5% hydrogen peroxide; ge35 – bleaching using 35% hydrogen peroxide; ge 7.5+35 – bleaching using 7.5% and 35% hydrogen peroxide; gd – dentin control; gd7.5 – 7.5% hydrogen peroxide; gd35 – 35% hydrogen peroxide; and gd 7.5+35 –7.5% and 35% hydrogen peroxide. bleaching was performed using long clinical applicationtime to low concentration gel, and short clinical application-time to high concentration gel. unbleached specimens were stored in artificial saliva for 14 days. specimens subject to micro-shear testing and data were analyzed by analysis of variance and tukey´s test (p=0.05). enamel micro-shear bond strength was reduced after 7.5% hydrogen peroxide and after association of 7.5% and 35% hydrogen peroxide. bleaching treatment altered dentin bond strength only when using 7.5% hydrogen peroxide. the results suggest that the bond strength of the restorations was influenced by the clinical extent of bleaching-gel application time and was not dependent on bleaching-gel concentration. gbmd 2013 – 49th meeting of the brazilian group of dental materials266266266266266 braz j oral sci. 12(3):237-274 175 tribocorrosion behaviour of dental alloys in contact with human teeth pupim d*, galo r, mattos mgc, rocha la ribeirão preto dental school – usp denisepupim@gmail.com the complexity of contact in the mouth leads to an interplay of sliding wear, abrasion, corrosion and fatigue, irrespective of the surfaces in contact, which involve either tooth-to-tooth or tooth-to-restoration. the aim of this study was focused on in vitro tribocorrosion tests of dental alloys under reciprocating sliding. tests were done involving four different dental alloys (ni-cr, ni-cr-ti, co-cr and commercially pure ti) tested against human teeth in artificial saliva. the main idea was to characterize the dental alloys emphasizing the influence on human teeth. a normal load of 3 and 10 n, reciprocating amplitude of 4 mm, and frequency of 1 hz were used. tests lasting up to 900 cycles were conducted. an increase in normal force induces an increase in current and a decrease in potential accelerating the depassivation rate of the tested dental alloys. sliding wear affects the repassivation behaviour of the tested materials by increasing the anodic current in the wear track area. 177 evaluation of chlorhexidine release from microcapsules incorporated into a resin sealant nojosa js*, alencar caa, pires apm, de sousa ffo, rodrigues lka, yamauti m federal university of ceará jacquesantiago@yahoo.com.br the aim was to evaluate the feasibility of the incorporation of microparticles loaded with chlorhexidine into a dental sealant. a commercial resin sealant (bioseal, biodynamic, brazil) was used. in its pure form, the sealant was used as control. test formulations were prepared by incorporating 10% (w/w) of microparticles, which were loaded either with chlorhexidine diacetate (da) or digluconate (dg). specimens (2 cm diameter x thickness 1 mm, n = 3) were prepared for each formulation. each specimen was placed in a polystyrene tube containing 1.0 ml of distilled water at 37° c. for quantification of chlorhexidine release, aliquots (1.0 ml) were collected, and this volume was immediately replaced. quantification was through uv-visible spectrophotometry (ë = 255 nm) and concentrations were obtained based on the calibration curves for each salt of chlorhexidine. readings were taken after 6 h, 24 h and at weekly intervals for 90 days. the release of chlorhexidine in all groups occurred after a latency period. the release of the group containing da started after 24 h, though this was more evident from 40 days. after 90 days, this formulation released about 11.31% (168.17 µg) of chlorhexidine. the group containing dg showed a further latent release, which initiated and was maintained under control only after 24 days. however, after 90 days, this group presented a release rate of approximately 12.89% (230.85 µg). the results suggest a delayed and controlled release of chlorhexidine when they are microencapsulated and incorporated into this commercial sealant. 179 influence of addiction of tantalum oxide in an experimental adhesive resin garcia im, genari b, balbinot gs, leitune vc, samuel sm, collares fm federal university of rio grande do sul isadora.mgarcia@hotmail.com the aim of the study was to evaluate the influence of tantalum oxide (ta2o5) in an experimental adhesive resin. a resin was formulated with 50%wt bisgma, 25%wt tegdma, 25%wt hema and 1% mol of camphorquinone and edab as initiator system. ta2o5 was incorporated to resin in different concentrations, by weight: 0, 1, 2, 5 and 10%. the resins with ta2o5 addition were evaluated by degree of conversion, radiopacity and color parameters. for degree of conversion, three specimens from each group were evaluated by ftir-atr before and after light curing for 20 seconds. the radiopacity was evaluated according to iso 4049 standards by a digital system. the color parameters were evaluated in the cielab system by a spectrophotometer. statistical analysis was performed using one-way anova and tukey’s test at the 0,05 level of significance. the degree of conversion ranged from 70 to 62%, decreasing significantly compared to control (0%) from the incorporation of 5% of the inorganic filler (p <0,05). the addition of 5% and 10% showed significant difference in radiopacity (p <0,05), increasing with the addition of the filler. the reflectance increased significantly (p <0,05) for all wavelengths (400, 500, 600 and 700 nm) with increasing concentration of the filler. the ∆e of the samples with addition of ta2o5 showed color parameters alteration at all concentrations according to the cielab system. it is concluded that ta2o5 seems to be promising filler for adhesive resins. 176 effect of different light-activation times on physicochemical properties of an experimental dental adhesive pomacóndor-hernández c*, ruy lgm, feitosa vp, ogliari fa, sinhoreti ma, consani s piracicaba dental school unicamp cesarpomacondor@hotmail.com this study evaluated the effect of different light-activation times on water sorption (ws), solubility (so), modulus of elasticity (me) and flexural strength (fs) of an experimental self-etch adhesive. the blend was prepared with bis-ema (40%), tegdma (10%), hema (10%), udma (10%), gdma-p (15%), water (10%), camphorquinone (1%), edab (1.5%), diphenyl iodonium (1.5%), and phenylpropanodione (1%), weight percentages. the blend was dispensed in molds to prepare disc-shaped specimens (6mm x 1mm) for ws and so surveys (after 7 and 90 days), and bar-shaped specimens (1mm x 1mm x 7mm) for three-point bending test. specimens were divided in 4 groups according to different light-activation times: g1: 5s, g2: 10s, g3: 30s, e g4: 60s (n=5). light-activation was performed with led bluephase 2 (ivoclar vivadent) with 1050 mw/cm2 irradiance. values were analyzed using one-way anova and student-newman-keuls test (á=0.05). ws increased when prolonged light-activation time was performed (7d g1: 88.7µg/mm3 < g4: 138.5µg/mm3, and 90d g1: 88.1µg/mm3 < g4: 149.9µg/ mm3). the so reduced by increasing light-activation time only after 90d-storage (7d g1: 6.9µg/mm3 = g4: 4.1µg/mm3, and 90d g1: 17.6µg/mm3 > g4: 5.4µg/mm3). the me and fs augmented by prolonging light-activation time; however, there was no statistical difference between g3 and g4 (me g1: 0.91gpa < g4: 1.26gpa, and fs g1: 61.6mpa < g4: 81.5mpa). it can be concluded that prolonged light-activation time of an experimental self-etch adhesive results in increase of ws, me, and fs, and reduction of so. 178 color change, diffusion of hydrogen peroxide and cytotoxicity caused by in-office bleaching protocols gonçalves rs*, de almeida lcag, soares dg, gallinari mo, pontes ecv, costa cads, dos santos ph, briso alf araçatuba dental school – unesp rafael895@hotmail.com the present study evaluated color change, cytotoxicity and diffusion of hydrogen peroxide (h2o2) in bovine teeth with different protocols of in-office whitening. group 1the specimens were not whitened; group 2(3×15 minutes) 3 consecutive applications, the bleaching agent was refreshed every 15 minutes. group 3(1×45 minutes) one 45 minutes-application of hydrogen peroxide 35%. this procedure was done 3 times at weekly intervals. the analysis of the color change was carried out by spectrophotometry reflection. the penetration of h2o2 into enamel during bleaching was measured by placing the specimens into artificial pulp chambers (apcs), which contained acetate buffer solution that stabilizes the h2o2 that has permeated the tooth structure. immediately after bleaching, this solution was collected, processed and submitted to optical density analysis by spectrophotometry. for cytotoxicity analysis, the specimens were placed into apcs, and the diffusion of the bleaching products together with the culture medium were applied on mdpc-23 odontoblast cells culture for 1 hour. the cell morphology and its viability (cytotoxicity) were evaluated by mtt analysis and sem, respectively. both whitened groups had similar results considering color change and diffusion of h2o2. in addition, both groups caused not only decrease in cell metabolism but also alterations in cells morphology. it can be concluded that despite the effectiveness of whitening, bleaching with high concentrations of peroxide is potentially aggressive to odontoblast cells. 180 evaluation and comparison of rnickel-titanium rotatory endodontic instruments before and after clinical use fracasso lm*, nogarett lm, weis a, mota eg pontifical catholic university of rio grande do sul pucrs lisi_mf@hotmail.com the aim of this study was evaluate the resistance of niti instruments to cyclic fatigue before and after clinical use. twenty four groups of rotary endodontic instruments protaper system were divided into two groups randomly. the instruments of group 1 (n=12) were stored while the instruments of group 2 (n=12) were performed by the same endodontist which used to prepare ten root canals. the instruments were tested for their resistance to cyclic fatigue with a simulator of curved canal. the time for occurs the fracture was analyzed and the size of the fragments. data were analyzed using kolmogorov-smirnov test at the level of significance of 1% and two-way anova (rpm and fragment size), followed by tukey’s test at a significance level of 5%. the results showed statistically significant difference for the rpm factor (p = 0.008) but not significant for the factor size of fragment (p = 0.12). all instruments were analyzed in a side view and fracture surface in scanning electron microscopy for qualitative analysis of fractured instruments. in front of the limitations of this study, it is concluded that all instruments analyzed (s1, s2 and f1) showed a lower resistance to cyclic fatigue after being used clinically in preparing ten root canals when compared with the new instruments. and regarding the size of these fragments niti rotary instruments, there was no relationship between the use or do not use of them. gbmd 2013 – 49th meeting of the brazilian group of dental materials 267267267267267 braz j oral sci. 12(3):237-274 181 evaluation of physicochemical properties of experimental root canal sealers based on mta and salicylate moreno mbp*, vitti rp, silva ejn, feitosa vp, bacchi a, santos mbf, corrersobrinho l, sinhoreti mac piracicaba dental school unicamp marinabpmoreno@hotmail.com the aim of this study was to develop and evaluate physicochemical properties of three experimental root canal sealers based on mta and a salicylate resin with different calcium phosphates. the sealers were composed by base and catalyst pastes mixed in 1:1. the base paste was made with bismuth oxide and butylene glycol disalicylate. three different catalyst pastes were formulated: (1) mta, resimpol 8 and titanium dioxide, (2) mta, resimpol 8, hydroxyapatite and titanium dioxide, (3) mta, resimpol 8, dibasic calcium phosphate dehydrate and titanium dioxide. mta fillapex (angelus) was used as control. working time and flow were tested according to iso 6876 and setting time according to astm c266 (n = 3). the materials were placed in pvc molds (8 mm diameter x 1.6 mm thick) and stored in 20 ml of deionized water at 37°c for solubility and water sorption tests (n = 10 for each material and test). after 1, 7, 14 e 28 days the samples were removed from the solutions and blotted dry for solubility and water absorption tests. data were analyzed using anova and tukey’s test (p<.05). mta fillapex showed the highest values of flow (29.04 mm) working (30 min) and setting time (218 min). mta fillapex had the lowest solubility values (18.10%) and water sorption (12.65%). mta fillapex and experimental sealers showed values in accordance with iso 6876:2001. the experimental sealers exhibited satisfactory physicochemical properties. 183 a dye degradation by exposure to hydrogen peroxide associated with catalysts according to the time lopes ba*, ballester ry, teixeira acsc, cardoso pec university of sao paulo brunolopes13@yahoo.com.br the objective of this study was to quantify in vitro degradation of a food dye according to the time of exposure to bleaching agents with or without the addition of catalysts and irradiation with led or uva/visible. a solution was prepared of 0.01% carmine dye (c) and various solutions of h2o2 (25% h2o2) and ferrous gluconate 0.03% (fe2+). the experimental groups were: a 0.5 ml of “c” + 0.5 ml distilled water + 0.005 ml of “ h2o2”; b 0.5 ml “c” + 0.5 ml of “ fe2+” + 0.005 ml “ h2o2”; c 0.5 ml “c” + 0.5 ml of “ fe2+” + 0.005 ml of “ h2o2” + led irradiation; d 0.5 ml “c” + 0.5 ml of “ fe2+” + 0.005 ml of “h2o2”+ uva / visible irradiation. was obtained the dye concentration by spectrophotometric analysis, before the preparation of the groups (c) immediately after the preparation (time 0) and subsequently every 3 minutes up to a total of 15 minutes of reaction. the concentration was determined from a straight of concentration / absorbance previously defined. for each test, three replicates were performed. the data were submitted to the test factorial anova where the variables, treatment, time, and their interaction were significant (p <0.000). group 1 was not statistically different in 0-15 minutes, group 2 was statistically different after 0-15 minutes and groups 3 and 4 were statistically different starting from 0-3 minutes. the major degradation over time was found for the experimental group “d” followed by the experimental groups respectively “c”, “b” and “a”. 185 profile of stress distribution at the interface of restorative materials and dental cements miranda glp*, pereira cnb, silveira rr, silva nrfa federal university of minas gerais – ufmg glpm80@hotmail.com the knowledge about the mode of stress distribution in dental cements is poorly described when referring to the elastic modulus of the material. the objective was to evaluate the elastic modulus (e) of different cementing agents, using mathematical models, the distribution of the stress generated by the restorative material. generated a solid three layers (10x10mm) with a height that varies according to the material, the first from the bottom up to the substrate (4mm), the second cement (100ìm) and the third for restoring material (1.5 mm ) it generated a mesh with a 200n load applied at the center of the third layer. the cements used were zinc phosphate (sswhite), relyx u200 clicker and automix (3m espe), ketac-cem (3m espe); materials and lava ultimate restorative lava (3m espe), and e.maxpress e.maxcad (ivoclar), and gold. for properties, (e) and poisson’s ratio, we used values reported in the literature. 40 groups were created by combining cement / restorative material. it captured the image of the cement / restorative material and deployed. we obtained values for the voltage spikes along the interface. these values were modeled via a normal distribution and generated a distribution curve. each cement was fixed to observe the behavior of ceramics and vice versa. lava ultimate absorbed more tension between all restorative materials. gold, e.maxpress and e.maxcad showed similar behavior. the lava showed lower absorption voltage. observing cements, zinc phosphate lowest absorbed less tension while the other cements had a similar profile. the analysis of the finite element method suggested by observations only the modulus appears to influence the stress distribution profile. 182 effect of cure mode on biaxial properties of resin cements sartori cg*, de goes mf, rueggeberg fa piracicaba dental school unicamp cristianagsa@gmail.com to determine the effect of curing mode on 1-hour biaxial modulus and strength values of commercial and experimental dual-cured resin cements. paste components of four commercial, dual-cured resin cements [relyx unicem2, relyx arc and relyx ultimate (3m/espe); panavia f2.0 (kuraray)] were dispensed and mixed according to manufacturers instructions and placed into teflon molds (6.5 x 0.5 mm). the material was immediately light-cured (s10, 3m espe) or allowed to selfcure. specimens were isothermally conditioned at 35°c for 1 hour immediately after placement into mold. cured specimens were retrieved, trimmed of flash, and tested. ten disc-shaped specimens from each test group were placed into a biaxial-flexure jig and a vertical load was applied (1.27 mm/min) on a universal testing machine (model 5844, instron corp., norwood, ma) until specimen fracture. software calculated biaxial flexural strength from the recorded data. statistical analysis consisted of a 2-way anova and tukey’s post-hoc test at 0.05 pre-set alpha among products and cure modes within each test parameter.anova for modulus indicated significant effects of product and cure mode (p<0.001), but not their interaction (p=0.212). anova for strength revealed significant effect of product and cure more with significant interaction ((p<0.001). providing direct light curing of resin cements significantly improved flexural modulus and strength for most products, while strength of one product remained unchanged. 184 evaluation of physical properties and antibacterial activity of a modified composite resin with tio2 nanoparticles faria aa*, diashb, bernardi mib, garcia ppns, hernandes ac, basso kcfj, rastelli ans araraquara dental school foarunesp dri.a.f@hotmail.com the aim of this study was to evaluate the influence of tio2 nanoparticles incorporation in a composite resin (filtektm z350 xt) at 1.0 and 2.0% concentrations in color stability (äe*), compressive strength (cs) and antibacterial activity (aa). 50 specimens (10x2mm) were divided into 6 groups for ∆e* test: g1 (control – resin (r) without nano, articial saliva (as)), g2 (rwithout nano, coffee), g3 (r + 1% nano, as), g4 (r + 1% nano, coffee), g5 (r + 2% nano, as), g6 (r + 2% nano, coffee). the ∆e* was obtained by cie-l*a*b* with a spectrophotometer-45/0 immediately, 30 and 60 days after storage at 37 °c (± 1 ° c). 24 specimens (6x4mm) were used for the cs test. 18 specimens were prepared (4x2mm) for aa of s. mutans (sm). the sm biofilm was induced on the specimens and the survival % was calculated. contact inhibition was observed by sem. äe* values greater or equal to 3.3 showed clinically unacceptable color change and detection by eye. anova and tukey’s post-test were performed for cs and aa. the lowest and highest values of ∆e* were g1: 1.47 (± 0.84), 30 days in as and g6: 32.22 (± 1.22) 60 days in coffee (p> 0:05). the addition of tio2 nanoparticles did not change the color in as after 30 days at 1% and 2% concentrations, 60 days with addition of 1%. for the cs test, the lower and higher mean values were g3 (2%) 194.58 (± 35.26), g2 (1%) 227.67 (± 49.41) and g1 (p> 0.05), however, the tio2 at 1 and 2% did not provided significant changes in the cs. the survival test, g1: 100%, g2 (1%): 54% and g3 (2%): 91% indicated a significant antimicrobial effect mainly at 2% (p <0,05). the tio2 incorporation showed significant aa, without change in color and cs and may be an option as antibacterial agent in composite resins. 186 influence of filler’s surface area over optical and surface properties of resin composites salgado ve*, cavalcante lma, schneider lfj federal fluminense university salgadouff@gmail.com the aim of this study investigates the influence of nanoscale filler’s surface area over optical and surface properties of model resin composites before and after aging. three model composites were formulated with different silica filler sizes, inserted in different masses in order to equalize the surface area (bet method): 7nm-15% (g1), 12nm-26% (g2) and 16nm-35,5% (g3), in a matrix of bisgma/ tegdma 1:1. the ciel*a*b* parameters, the color difference (∆e*), the translucency parameter (tp), the surface gloss (sg) and surface roughness (sr) were determined before and after aging procedures of immersion in water and toothbrush abrasion. results were submitted to two-way anova followed by tukey’s post-hoc test performed at a pre-set alpha of 0.05. results: the immersion in water leads to increase cie a* (p<0,05) in g2 and decrease cie b* (p<0,05) in g3, did not lead significante difference in g1, cie l* and tp of any group. the toothbrush abrasion leads to decrease sg (p<0,05) and to increase sr (p<0,05) in all groups, despite it wasn’t observed any difference between groups. conclusion: the optical properties were influenced by filler’s size, smaller the filler greater the color stability. when equalizing the filler’s surface area, it was observed no significant difference in surface properties between groups. 268268268268268 gbmd 2013 – 49th meeting of the brazilian group of dental materials braz j oral sci. 12(3):237-274 187 water degradation of resin-dentin interfaces subjected to direct and indirect exposure silva ia, abuná g, felizardo kr, bacchi a; vitti rp; correr-sobrinho l; sinhoreti mac; correr ab; feitosa vp piracicaba dental school unicamp igoralvesmd@gmail.com the aim of this study was to assess the effects direct or indirect water exposure on the 3 months hydrolytic degradation of three dentin bonding agents. the samples were divided in three groups: clearfil se bond, clearfil s3, adper singlebond 2; and the samples were restored with filtek z350. subsequent to the restorative procedures, the specimens of each group were divided into three subgroups (immersed in water deionised): control (24h-37ºc), direct water exposure dwe for 3 months (37º), indirect water exposure (iwe-3m) with enamel margins for 3 months (37ºc). after the storage the samples were sectioned into sticks and µtbs testing (ez test) and scanning electronic microscopy assessed the failure mode. the µtbs data were statistically analysed using two-way anova and tukey’s test at á=0.05%. the samples were processed for nanoleakage evaluation immersed in 50 wt% ammoniacal silver nitrate (24 h), rinsed and immersed in a photo-developing solution for 8 h. after 3 months cse was the least affected by water degradation regardless the aging strategy. iwe afforded very little variation on µtbs after 3 m. intense nanoleakage was observed with dwe groups with increases incidence of mixed failures instead bonded dentin margins are more prone to hydrolytic degradation than resin-enamel interfaces. the increased nanoleakage and the drop of bond strength showed this. 189 influence of fatigue and thermocycling on bond strength to ceramic correr-sobrinho l, costa ar, correr ab, sinhoreti mac, puppin-rontani rm, versluis a, garcia-godoy f piracicaba dental school unicamp sobrinho@fop.unicamp.br the purpose of this study was to evaluate the influence of surface treatments with 5% and 10% hydrofluoric acid on the microtensile bond strength of ceramic/ cement/composite, after thermocycling and fatigue. twenty-four brocks were made with ceramic ips empress esthetic and divided into 6 groups (n=4): groups 1, 2 and 3 acid etching with 5% hydrofluoric acid for 60 s; groups 4, 5 and 6 – acid etching with 10% hydrofluoric acid for 60 s. silane was applied on treated ceramic surfaces and the blocks of the ceramic were bonded to a block of composite with relyx arc and photoactivated for 160 s. all specimens were stored in distilled water at 37oc for 24 hours and specimens of groups 2 and 5 were submitted to 3,000 thermal cycles and groups 3 and 6 were submitted to a fatigue test of 250,000 cycles. after, the specimens were sectioned perpendicular to obtain beams with area of 1mm2 and submitted to a microtensile at a crosshead speed of 0.5mm/ min. data were submitted to anova and tukey’s test (p<0.05). the bond strength values (mpa; mean±standard deviation) for the specimens stored 24 hours: groups 1 (50.84±4.34) and 4 (51.11±4.70) were significantly higher than thermocycled groups 2 (33.80±2.84) and 5 (32.52±2.23) and fatigue groups 3 (31.13±3.19) and 6 (30.09±2.65). the thermocycled groups were significantly stronger than the fatigue groups. no significant difference was found between the two surface treatments. in conclusion the thermocycling and fatigue significantly decreased the microtensile bond strength for both ceramic surface treatments in relation to control groups. 191 18-months clinical evaluation of previous etching with edta using one-step adhesive hass v*, luque-martinez iv, muñoz ma, mena-serrano ap, reis a, loguercio ad ponta grossa state university vivikl_hass@hotmail.com the benefits of previous etching with edta have only been demonstrated in an in vitro setting. this 18-months randomized clinical trial study evaluated the performance of adper easy one (3m espe) in non-cervical caries lesions applied according to the manufacturer’s directions (eo) or with previous etching with edta. forty-eight patients with at least two nccl with similar size participated in this study. a total of 96 composite resin restorations were placed by two operators. in half of the restorations the adhesive was applied as per manufacturer’s directions (eo) while in the other half the adhesive was applied after etching with edta (edta+eo). the restorations were placed incrementally using the composite resin z 350 (3m espe). they were evaluated at baseline and after 6, 12 and 18 months following the fdi criteria. statistical differences between the groups at each period were tested using with mcnemar’s test and the clinical performance over time for each group with the fisher’s exact test (á= 0.05). the retention rates for eo+edta were 100%, 95.2% and 88.1% and for eo were 95.7%, 93.1% and 76.4%, respectively for 6, 12 and 18months of clinical evaluation (p<0.05). despite 63.5% of patients reported tooth sensitivity, this occurrence did not occur in the immediate postoperative and only 17.5% after 18-months clinical evaluation. no statistically differences regarding the other fdi items were detected. the previous etching of dentin with edta can improve the retention rates of composite resin restorations in nccls when bonded with the one-step adhesive adper easy one. 188 evaluation of mechanical properties of composite resins araujo tgf*, quiles hk, feitosa vp, sinhoreti mac, correr ab piracicaba dental school unicamp tatygfa@yahoo.com.br the aim is to evaluate the mechanical properties of composite resins, three flowable and seven conventional. we prepared three shapes of specimens, bar-shaped for bending test, cylinder-shaped for compression test and hourglass-shaped for ultimate tensile strength (uts), knoop hardness, and crosslink density (cld) surveys. the composites tested were: filtek z100, tph, natural look, opallis fill magic, llis, charisma, fill magic flow, opallisflow and natural flow. data were submitted to anova and tukey test (p <0.05). z100 obtained higher knoop hardness both in the top and the base while lowest hardness were detected with flowable resins that were significantly similar to fill magic. in the compression test, z100 achieved the highest outcomes which were similar to fill magic, llis and tph. natural flow attained the lowest compressive outcomes. tph and z100 obtained the greatest flexural strength whereas all other resins had statistically similar results. the elastic modulus was higher with natural flow and lower and fill magic flow. uts of tph and z100 were higher than that of charisma and all other resins were similar overall. cld was higher for flowable resins, except for fill magic flow. in conclusion, filtek z100 promotes better mechanical strength against different strains. with lower elastic modulus, natural flow and fill magic flow are the most adequate as “liners”. 190 development and evaluation of experimental resin primers for repair resin composite restorations valente ll*, münchow ea, da silva mf, manso is, cenci ms, moraes rr federal university pelotas lisialorea@hotmail.com the aim of this study was to evaluate the microshear bond strength (µsbs) and the degree of conversion (dc) of experimental resin primers bonded to repaired resin composite. five primers were prepared with silane, solvent, udma, hema, and gdma-p, varying only the concentration of gdma-p in: p1=10%; p2=20%; p3=30%; p4=40%; and p5=0%. resin composite blocks were thermo-cycled in 1000 cycles, embedded in acrylic resin and polished with #600silicon carbide papers. each primer was actively applied in two layers for 20s, followed by the solvent drying for 20s. an elastomeric matrix was placed over the block surface, which was photoactivated for 20s and filled with the correspondent resin composite (n=16). after 24h, each specimen was tested in the µsbs test; the failure pattern was determined under magnification of 40x. the dc (%) (n=3) was measured after the solvent drying (20s) using infrared spectroscopy. data were statistically analyzed with one-way anova and tukey’s test (p<0.05). the mean±sd values were: p1=41.7±1.4; p2=27.2±10.9; p3=26.8±3.4; p4=23.0±8.2; and p5=43.0±0.8 for dc analysis; and p1=19.0±9.3; p2=25.3±8.7; p3=19.78.2; p4=26.8±7.2; and p5=26.5±11.7 for µsbs analysis; no statistical differences were verified between the groups for both dc and µsbs analysis. regarding the failure pattern results, the percentage of cohesive failure in the thermo-cycled resin composite was: p1=60%; p2=73%; p3=33%; p4=80%; and p5=50% of cohesive failure in thermo-cycled resin. it can be concluded that the concentration of acidic monomer seems to not influence the composite’s repairing procedure. 192 effect of the radiant exposure on the physical properties of methacrylateand silorane-based composites santos-caldeira mmp*, catelan a, kawano y, ambrosano gmb, martins, lrm, aguiar fhb piracicaba dental school unicamp milenapierre@yahoo.com.br the objective of this study was to evaluate the effect of different radiant exposures on the degree of conversion (dc), knoop hardness number (khn), plasticization (p), water sorption (ws), and solubility (s) of different monomer resin-based. circular specimens (5 x 2 mm) were carried out of the methacrylate (filtek z250, 3m espe) and silorane (filtek p90, 3m espe) composite resins, and light-cured at 19.8, 27.8, 39.6, and 55.6 j/cm2, using second-generation led at 1390 mw/cm2. after 24 h, dc (n = 5) was obtained using a ft-raman spectrometer, khn (n = 10) was measured with 50-g load for 15 s, and p (n = 10) was evaluated by percentage reduction of the hardness after 24 h immersed in absolute alcohol at top and bottom surfaces. ws and s (n = 5) were determined according to iso 4049. data were subjected to 2-way anova and tukey´s test (alpha = 0.05). methacrylate material presented higher dc, khn, p, and ws than silorane (p < 0.05). there was no difference in the s values (p > 0.05). in general, top surface showed higher dc and khn than bottom, for both materials (p < 0.05). the increase of the radiant exposure did not improve most physical properties of the composites and were monomer-based dependent. 269269269269269gbmd 2013 – 49th meeting of the brazilian group of dental materials braz j oral sci. 12(3):237-274 193 bond strength of glass fiber post to root dentin after different treatment with filling materials and cleaning agentes rodrigues rv*, iwamoto as, banzi ecf, benetello v, hosoya y, puppin-rontani rm, pascon fm piracicaba dental school unicamp rquelrodrigues@hotmail.com the aim of the study was to evaluate the bond strength and failure patterns in adhesive interface of glass fiber posts to root dentin after treatment with filling materials and cleaning agents. one-hundred and twenty roots from bovine primary teeth were endodontically treated and assigned into groups: control (no material c); calen® + zinc oxide (czo); vitapex® (v); calcipex ii® (cp). after 7 days, filling materials were removed and subdivided in: no cleaning (nc); 70% ethanol (e); tergenform® (t). posts were luted, specimens were sectioned (±1 mm), prepared for push-out test (n=10) and the failure pattern was analyzed and classified by sem (40x and 250x). data from push-out test were submitted to two-way anova and tukey test’s (α=.05) and failure patterns were calculated in percentage and analyzed descriptively. interaction was observed between the studied factors (p=.004). czo/e showed the highest bond strength values (12.21±2.73), significantly different from nc (6.85 ± 0.98). v and cp showed no significant difference between cleaning agents. the lowest bond strength values were found for c/t (4.37±3.07). the failure adhesive-type between dentin/resin cement was the most frequently observed in the groups c/nc (60%), c/e (70%), czo/e (40%), v/nc (80%), v/t (80%) e cp/nc (70%). cohesive failure was not observed in czo/nc and v. mixed failure between adhesive/cohesive was found in all groups. in conclusion, the filling materials and cleaning agents influenced the bond strength and failure pattern. calen®+oz showed the best associated with 70% ethanol. 195 influence of hydrofluoric acid concentrations on the microshear bond strenght between glass ceramics and resin cement sundfeld-neto d; naves lz, costa ar, correr ab, sinhoreti mac, sundfeld rh, correr-sobrinho l piracicaba dental school unicamp sundfeldneto@gmail.com this study evaluated the influence of hydrofluoric acid (hf) concentration on the surface and microshear bond strenght on ceramics: ips empress esthetic (est), ips e.max press (emx), bonded to a resin cement (variolink ii). each type of ceramic blocks with 8 mm × 8mm × 2 mm was separeted into 12 groups (n=6), according to the acid concentration: 1%, 2.5%, 5%, 7.5%, 10% and 15%. etching time was fixed in 60 seconds for est and 20 seconds for emx. all the groups were silanated after etching and the acronym ‘ur’ on the designated groups received a layer of an unfilled resin after silane application. characterization of the etching patterns was conducted by sem. for the microshear bond test, resin cilinders were built on the ceramic surface, photoactivated during 40 seconds and stored in distilled water during 24 hours, 37°c. the data were submitted to three-way anova and tukey’s test (p<0.05). sem images showed that poor etching was detected when using hf 1% on est and emx groups. hf 2.5% resulted in large remnants of vitreous phase and shallow grooves on ceramic surface. similar etching patterns were detected for hf 7.5 and 10%. deep channels were extensively observed on surfaces etched with hf 15%. the results showed no statistical difference among est and emx. groups with unfilled resin showed statistical higher microshear bond values and hf10% and hf15% showed higher values compared to hf1% and hf2.5%. conclusion: hydrofluoric acid concentration influenced the ceramic topography and microsher bond strength values and the unfilled resin resulted in higher bond strength. 197 effect of intrinsic nanoparticle pigmentation on the color stability of denture base acrylic resins sônego mv*, goiato mc, zuccolotti bc, dos santos dm, sinhoreti mac, moreno a aracatuba dental school unesp mah_vs@hotmail.com to investigate the effect of intrinsic nanoparticle pigmentation on the color stability of acrylic resins, onda cryl, qc 20, classico, and lucitone resins were evaluated. a total of 21 specimens (30 mm-diameter, 3 mm-thick) were fabricated from each resin. seven were colored with 3% poli-cor intrinsic pigment used to color denture base, 7 were colored with 7% pigment, and 7 were not pigmented. in addition, 7 specimens were fabricated containing only pigment. the specimens were thermally cycled 2000 times between 5°c and 55°c with a 30-second dwell time at each temperature. the specimen colors were measured with a spectrophotometer and evaluated with the cie l*a*b* system before (b) and after thermal cycling (t). the pigment morphology was analyzed by using a scanning electron microscope (sem) and energy-dispersive x-ray spectroscopy (eds) techniques. the results were analyzed with the anova and tukey hsd tests (α=.05). classico acrylic resin with and without pigment underwent the least color change, followed in order by lucitone, onda cryl, and qc-20. the presence of pigments reduced the color change of the acrylic resins, with statistical significance (p<.05) for the specimens containing 7% pigment (0.32 ± 0.18 ∆e). titanium was the sole metallic component present in the pigment, probably in the oxide form (tio2). nanoparticle pigments enhanced the color stability of denture base acrylic resins. 194 biomechanical analyses of impla partial prostheses made from different materials and levels of vertical misfit campana jt*, caetano cr, nogueira mcf, caldas ra, zen bm, bacchi a, dos santos mbf, consani rlx, correr-sobrinho l piracicaba dental school unicamp juliatcampana@hotmail.com currently, the literature has suggested the use of various materials for making prosthetic infrastructures. regardless of the material, the presence of mismatches in the prosthesis is considered an important factor in the long-term success. the aim of this study was to evaluate the influence of the material infrastructure and vertical misfit on stress distribution over implant fixed partial prosthesis. a model representing the posterior region of a mandible with two implants in the second premolar and second molar was made using specific software. finite element models were obtained by importing the solid model mechanical simulation software. the groups were divided according to the material infrastructure of the prosthesis (type iv au, ag-pd, cp ti, co-cr, and zi) and level of vertical misfit (10, 50 and 100ìm). an offset was made in the region of misfit simulating the screw tightening. the zirconia caused higher stress concentration in infrastructure (3458.5 mpa) and the screw (95.9 mpa). however, au type iv showed the highest values in the veneering porcelain (1376.0 mpa). bone tissue showed no significant changes with different material infrastructure. a considerable increase in the stress concentration was observed in all the frames with the mismatch amplification evaluated. the material infrastructure influenced the stress concentration in the prosthetic components, but no difference in bone tissue. all structures have been significantly influenced by increased levels of vertical misfit. 196 study of the influence of radiation in ionising diametral tensile ionomer cements glass nogarett lm*, fracasso lm, trinca w, mota eg pucrs nogarett@yahoo.com.br the aim was evaluate the influence of ionizing radiation under three glass ionomer (conventional, resin modified and silver added) on the diametral tensile strength. samples were made with into a cylindrical bipartite mould of ptfe high according to manufacturer’s instructions. all samples were stored in plastic tubes protected from light for 24 h at 37° c in a culture stove. after that, each group was divided in a control (n=20) or submitted to ionizing radiation (n=20), in 35 daily applications of 2 gy by a linear accelerator. the diametral tensile strength test was performed in a universal testing machine, with 1 mm/min of cross-head speed. data were submitted to analysis of variance and multiple comparison test of tukey (α=0.05). significant differences were recorded (p<0.001) between the glass ionomer tested for diametral tensile strength. comparing groups with the same classification, submitted or not to radiation, there was no significant difference. riva light cure control showed an average (mpa) of 31.83 in comparison to 24.82 of irradiated group. riva silver control showed an average of 12.06 in comparison to 17.29 for the irradiated group. riva self cure control showed an average of 14.05 in comparison to 26.04 irradiated group. comparing all groups of different classification submitted to ionizing radiation, there were significant difference between them (p<0.001). riva silver control showed the lowest average (12.03) in comparison to riva light control (31.83). the radiotherapy applied as protocol to head and neck cancer therapy do not act negatively in the diametral tensile strength of the three glass ionomer tested. 198 effect of photoactivation time in degree of conversion of resin cements phtoactivaded through fiber post reis gr*, menezes ms, oliveira aca, tavares cm, borges mg, silva fp, faria-e-silva al federal university of uberlandia rodrigues.giselle@yahoo.com.br the aim of this study was to evaluate the effect of the moment of photoactivation in the degree of conversion (dc) of selfadhesive resin cements light-cured of through glass-fiber post. a cast with a 2x2mm ²orifice was used to make specimens. cylinders of acrylic resin containing three posts were prepared for the polymerization could be made through of posts; these cylinders had 7, 10 and 13 mm in height, representing the cervical, middle and apical thirds of root canal, respectively. samples were prepared with relyx u-100 and biscem, these being photoactivated in these times: immediately; after 5, and 10 min of its insertion into the cast. the dc was measured after 24 h using a spectrometer fourier transform infrared spectrometer ft-ir with attenuated total reflectance (atr) coupled. data were analyzed by three-way anova and tukey’s test (α=0.05). the degree of conversion of biscem showed greater than u-100 in all experimental conditions, with a tendency for reduction of degree of conversion with the increasing of the distance. the moment of light curing did not affect the degree of conversion of the u-100, while the biscem showed higher degree of conversion with immediate photoactivation. the curing time have more influence with increasing the photoactivation distance. it was concluded that both the photoactivation time as well as the distance from the light may influence the degree of conversion of adhe resin cement through fiberglass posts. 270270270270270 gbmd 2013 – 49th meeting of the brazilian group of dental materials braz j oral sci. 12(3):237-274 199 chemical analysis of enamel after bleaching and acid etching cura m, fuentes mv, yamauti m, ceballos l rey juan carlos university maria.cupe@hotmail.com the objective was to determine the effect of in-office and commercial free for sale bleaching agents on the chemical composition of enamel. bovine incisors were used after properly cleaning and were stored in thymol solution. the buccal surfaces of the teeth were polished to obtain flat areas in enamel, which were divided in halves. one half served as a control and the others were divided into 3 groups according to the bleaching agent used: opalescence 10% (o10, ultradent); whitekin (wk, kin); clysiden expess kit (cke, ern sa). the products were applied according to manufacturers’ instructions for 4 weeks. teeth were stored in artificial saliva at 37° c. the enamel composition was analyzed by x-ray spectroscopy. after initial analysis, control and bleached surfaces were conditioned with phosphoric acid and again observed. data were analyzed with student t-test (p <0,05). in mineralized enamel, there was no difference in the levels of calcium and phosphorus for the control and bleached substrates. after acid etching, there was a decrease in the levels of calcium and phosphorus for cke compared to control. comparing the groups treated with bleaching agents after conditioning, there was an increase in calcium levels, but no difference in phosphorus levels. the application of bleaching agents did not alter the chemical structure of mineralized enamel. in bleached enamel after acid etching, there was an increase of free calcium on the surface of teeth bleached with all products. 201 influence of storage medium on the vickers microhardness of acrylic denture base wingert a*, mota eg, lehuger g, nogarett lm, fracasso lm pontifical catholic university of rio grande do sul-pucrs dr.awingert@gmail.com the aim of this study was to evaluate the influence of three storage media in knoop microhardness for two acrylic denture bases. sixty samples were fabricated in acrylic with 10 mm in diameter and 4 mm high and divided into two groups according to the method of activation (thermal activated and microwave). after determining the initial knoop hardness, were divided into three subgroups within each activation method according to the storage medium (distilled water, wine and cola). two impressions were made by specimens after 7 days storage at 37° c. data were submitted to anova and tukey (α = 0.05). the values recorded (khn) were: heat-polymerized initial microhardness 4.12; heat-polymerized wine 4.06; heat-polymerized distilled water 5.53; heat-polymerized coca-cola 4.68; microwave initial microhardness 4.56; microwaves wine 4.97; microwave distilled water 5.02, and microwave coca-cola 5.05.the tests showed a statistically significant difference in hardness between both resins activated by heat or microwave, and among the storage media used. 203 effect of dentifrices associated to 10% carbamide peroxide on superficial/cross-sectional enamel microhardness santos ale*, fogaça jf, pessan jp, delbem ac, shinohara ms araçatuba dental school – unesp analaurasantos@hotmail.com dental enamel surface alterations after the bleaching treatment (bt) could be clinically significant or not. the aim of this study was to evaluate the action of 10% carbamide peroxide (cp) associated or not to a dentifrice with 1100ppm/f (denf) or placebo (den), after each daily session of bt. sixty blocks (4x4mm) of bovine enamel were cut, polished, selected from superficial microhardness (sh) and randomly divided into 6 groups (n=10): g1-cp+denf; g2-cp+den; g3-cp; g4-non bleached, control (co)+denf; g5-co+den and, g6-co. the groups submitted to bt, the cp was applied for 4h/daily during 21 days. the slurry was applied for 1min after the bt. during the experiment, all specimens were stored in artificial saliva at 37oc. in the end, the superficial and cross-sectional microhardness were determined to calculate the % of sh loss and the hardness integrated area (hia), respectively. the data of %sh and hia were submitted to two-way analysis of variance followed by fisher’s plsd and student-newman-keuls, respectively. the groups submitted to bt presented superior mineral loss (%sh and hia) when compared to groups non bleached. the denf reduced the mineral loss of the bleached enamel and demonstrated similar %sh loss to non bleached groups. therefore, it can be conclude that the immediate application of denf after each daily session of bleaching treatment with 10% cp may decrease mineral loss of enamel structure. 200 effects of different polishing systems on the surface roughness and microhardness of a silorane-based composite ruschel vc*, basso gr, stolf sc, andrada mac, maia hp federal university of santa catarina ufsc vane_ruschel@hotmail.com the aim of this study was to investigate the effects of different polishing systems on the surface roughness and microhardness of a silorane-based resin composite. forty disks were fabricated (ø 12 mm x 2.5 mm) of a silorane-based resin composite (filtek p90 3m espe, usa). the specimens were divided into four groups (n = 10), according to the polishing system: g1 mylar strip (control); g2 felt-disc + diamond paste, g3 sandpaper discs; g4 rubber tips. the specimens were stored in distilled water at 37ºc for 24 h. the external surface roughness was determined through measuring the ra of the specimens. the vickers microhardness was measured using a microhardness tester. the values of surface roughness and microhardness of each specimen were statistically analyzed using one-way anova, games-howell and ryan-einot-gabriel-welsch (regw-q), and setting the statistical significance at p < 0.05. the results observed were that g2 (0.42 µm) and g4 (0.43 µm) showed statistically significant differences when compared to groups g1 (0.25 µm) and g3 (0.19 µm) (p <0.05). there was no statistical difference between groups regarding microhardness (p> 0.05). we concluded that polishing systems altered the surface roughness of a silorane-based resin composite, but did not influence the microhardness values. 202 comparison of two application techniques of luting agent for retention of fiberglass posts pereira cnb*, daleprane b, silva eh, moreira an, magalhães cs federal university of minas gerais ufmg carolnemesio@oi.com.br the aim of this study was to evaluate the bond strength of intracanal fiberglass posts (pfv) and dentin, using two techniques of insertion of self-adhesive resin cement. we selected 28 bovine teeth whose crowns were sectioned, leaving the roots to 18mm in length. the conduits were instrumented and prepared for the attachment of pfv reforpost # 3 (angelus) leaving 4 mm apical shutter. the roots were randomly divided into 2 groups (n = 14) according to the technique for insertion of resin cement: (b) lentulo drill and (s) centrix ® syringe with needle tip. the relyx u200 (3m) was photoactivated (bluephase, ivoclar vivadent, 1340mw/mm2, 40sec) under the load of 10n. after 7 days, roots were sectioned perpendicular to the long axis of the tooth obtaining two specimens of 1 mm thickness in each third root: coronal (c), medium (m) and apical (a). the bond strength was measured by push-out test (mpa). the cement line obtained was qualitatively assessed with a stereomicroscope (40x). the strength values, in mpa, of the c, m and a thirds in group b were 10.6 ± 3.1, 9.4 ± 3.5 and 9.4 ± 3.4 respectively. in group s, the mean (± sd) in thirds c, m and a were 10.3 ± 3.2, 9.8 ± 3.8, 11.6 ± 2.9, respectively. statistical analysis (anova and tukey test) showed no significant difference between groups b and s (p = 0.278) or between the root thirds (p = 0.521) in each group. it was concluded that the technique of insertion of relyx u200 did not influence the values of bond strength between pfv and bovine root dentin. 204 kinetics and degree of conversion of light-cured temporary restorative materials oliveira hl*, peralta sl, meereis ctw, dutra al, leles sb, piva e, lund rg federal university of pelotas hellen.loli@gmail.com this study aimed to evaluate the kinetics and degree conversion of of light-cured temporary restorative materials. four materials were tested: bioplic biodinâmica(b), fermit inlay ivoclar vivadent™ (f), fill magic tempo vigodent™ (fm) and revotek lc gc américa (r). the test was performed by fourier transform infrared spectroscopy (ftir, spectrometer shimadzu prestige21) with an attenuated total reflectance (atr), composed of a diamond crystal (smiths detection, danbury, ct). a support was connected with the purpose of fixing the led photo-activating (radii™ curing light, sdi, bayswater, victória, austrália) to the spectrophotometer enabling standardized distance between the tip end of the optical fiber and the sample. the degree of conversion after 60s was (mean ± standard deviation): b (69,4±1,5), fm tempo (59,7±7,3), r (59,3±5,5) and f (33,7±14,0). the percent conversion after 20 sec with respect to the end of the polymerization was: b (84.5%), fm (93.8%), r (81.9%) and f (62.1%). the maximum rate of polymerization (rpmax) of b, fm and r occurred at a maximum time (tmax) of about 5 sec, while the f showed a low rpmax in a tmax around 12s. it is concluded that after 20 sec the polymerization fill magic tempo, bioplic and revotek converted more than 80% and the fill magic tempo showed higher polymerization rate. 271271271271271gbmd 2013 – 49th meeting of the brazilian group of dental materials braz j oral sci. 12(3):237-274 205 dentin bond stability and antibacterial effect of an experimental adhesive with butiá oil ribeiro js, peralta sl,de leles sb, dutra al, piva e, lund rg federal university of pelotas jujusilvaribeiro@gmail.com the objective of this study was to evaluate the stability for microtensile bond strength (ìtbs) and the antibacterial effect by direct contact test (dct) of an experimental adhesive containing butiá oil (butia capitata). the materials used were experimental adhesive with oil (ao), experimental adhesive control (ac), clearfil protect bond (cpb) and clearfil se bond (cseb). for ìtbs, bovine teeth were restored and stored in distilled water at 37 ° c for 24 h. then dental sticks were obtained and tested in the testing machine (speed of 0.5 mm / min and load cell of 100n). specimens were also evaluated after 24h, 6 m, 1 and 2 years of storage. for tcd adhesive systems were applied to the sidewalls of a 96-well plate then was placed 10µl of the suspension (bhi grow + s. mutans) and stored for 1, 3 and 6h, then were taken for reading of the absorbance in espectrofometer. data were subjected to two-way anova followed by fisher lsd test (p <0.001). for ìtbs had statistical difference both time (p=0,001) and material (p=0,001), presented interaction between two variables. the cpb showed better stability up to 1 year, in two years all had similar performance. tcd showed statistic difference for material but not for time, presented interaction between time and material. conclusion the experimental adhesive with oil showed antimicrobial effect and the stability similar than others after two years. 207 degree of polymerization and optical properties of self-adhesive flowable composites formulated with initiation systems. teles ya*, bertolo mv, salgado ve, gwinner f, pfeifer cs, schneider lfj federal fluminense university yanaffonso@hotmail.com to evaluate the influence of the photoinitiator system on the curing efficiency and optical properties of experimental self-adhesive composites formulated with different initiators. ten experimental groups were formulated, which varied in relation to the organic matrix content– (1) bisgma-hema (50/50%, control group), and (2) the self-adhesive 2mp-hema (70/30%) and the photoinitiator system: cq, tpo, bapo, cq and cq + tpo + bapo. degree of conversion (dc) and maximum rate of polymerization (rpmax) was determined by spectroscopy (ftir). the yellowing effect (b *) was measured before and after polymerization with a spectrophotometer. 24 hours after polymerization and after 10 days of immersion in water, knoop hardness (khn) was determined on the irradiated surface. the results were submitted to anova and tukey’s tests(95% confidence). with the exception of the cq group, all other composites showed higher dc in the self-adhesive formulation than conventional. the rpmax did not differ for the groups with tpo regardless of the monomer serie. cq had lower rpmax in the self-adhesive mode than the traditional, whereas for bapo it was observe an opposite situation. self-adhesive materials tend to present lower b* values than conventional ones. cq had the lowest khn values of all composites evaluated. there was no significant difference between those composites formulated with tpo. dc, rpmax, khn and yellowing were dependent on the photoinitiator system and the type of monomeric system usedtraditional or self-etching. 209 influence of solvent type on the physicochemical properties of experimental dental adhesives sisti icg, adabo hd, pomacóndor-hernández c, feitosa vp, ogliari fa, sinhoreti ma, correr ab piracicaba dental school unicamp victorpfeitosa@hotmail.com the aim of this study was to evaluate the influence of different solvents (ethanol, acetone, tetrahydrofuran [thf] and dimethyl sulfoxide [dmso]) on physicochemical properties of experimental dental adhesives. two model resin blends (etch-andrinse [e&r], and self-etch [se]) were prepared and afterwards 10wt% of each solvent was added. in order to determine the modulus of elasticity (me) and flexural strength (fs), bar-shaped specimens (n=10) with 1mm x 1mm x 7mm were made and subjected to three-point bending test. the water sorption (ws) and solubility (so) were determined by preparing disc-shaped specimens for each experimental adhesive (n=10). the results were analyzed with one-way anova and tukey’s test (α=0.05). thf (me: 0.88 gpa; fs: 81.3 mpa) and dmso (me: 0.89 gpa; fs: 54.7 mpa) presented the highest mechanical properties in e&r and se respectively. dmso obtained the highest ws (e&r: 66.9 µg/mm3; se: 194.7 µg/mm3) and so (e&r: 93.8 µg/mm3; se: 107.4 µg/mm3) in both experimental resins. it can be concluded that incorporation of alternative solvents as dmso and thf into dental adhesives may improve their mechanical properties. 206 physical and mechanical properties of light-cured temporary restorative materials peralta sl*, leles sb, dutra al, ribeiro js, piva e, lund rg federal university of pelotas solupe@gmail.com there are many temporary light-cured temporary restorative materials. however, little is known about their physical and mechanical properties. the objective of this study was to evaluate the cohesive strength (cs), sorption (ws) and solubility (sl) of five commercial temporary restorative materials. the materials tested were: bioplic biodinâmica(b), fermit inlay ivoclar vivadent™(f), fill magic tempo vigodent™ (fm) e revotek lc gc américa (r) e luxatemp inlay dmg™ (l). for the cs, hourglass shaped specimens were made (n = 10), polymerized for 20s and taken to the emic universal testing machine with load cell of 100 n and speed 1.0 mm/min. the sw and sl test was performed according to iso 4049. data were evaluated by kruskall-wallis test followed by fisher’s lsd (p <0.05). cs (mean ± standard deviation): r (32,8±3,1)a, f (9,5±1,9)b, b (8,9±2,4)b, fm (5,7±0,9)c and l (4,4±1,4)c. para sw: l (3,0±0,1)ab, f (2,2±0,0)bc, fm (2,3±0,0)abc, b (4,2±1,0)a e r (1,6±0,9)c. sl: l (0,9±0,0)a, f (0,5±0,0)ab, fm (0,1±0,0)bc, b (0,1±0,7)bc and r (0,0±0,0)c. materials with greater cs (p <0.001) were r, followed by f and b. b, l, and fm showed higher sorption, whereas l and f presented higher solubility. r was the material with significantly lower sorption and solubility. it was concluded that the revotek lc showed better performance on trials of cohesive strength and sorption and solubility. 208 evaluation ph and calcium release of experimental root canal sealers based on mta and salicylate modified by calcium vitti rp*, prati c, bacchi a, santos mbf, souza e silva mg, sinhoreti mac, zanchi ch, ogliari fa, piva e, gandolfi mg piracicaba dental school unicamp rafapvitti@gmail.com the objective of this study were to develop and evaluate the ph and calcium release of three experimental root canal sealers based mta and resin salicylate with different calcium phosphate (cap). the sealers were composed by bases and catalyst pastes mixed in 1:1. the base paste was made with bismuth oxide and butylene glycol disalicylate. three different catalyst pastes were formulated: (1) mta, resimpol 8 and titanium dioxide, (2) mta, resimpol 8, hydroxyapatite and titanium dioxide, (3) mta, resimpol 8, dibasic calcium phosphate dehydrate and titanium dioxide. mta fillapex (angelus) was used as control. the materials were placed in pvc molds (8 mm diameter x 1.6 mm thick) and immersed in 10 ml of deionized water in cylindrical polystyrene-sealed container stored at 37°c for the ph and calcium release tests (n=10 for each material and test). after 3 and 24 hours and 4, 7, 14 28 days the soaking water was collected for ca and ph analysis. data were analyzed using anova and tukey’s test (p<.05).the higher ph values were found in the initial times (up to 24h), except for mta fillapex. the experimental sealer 1 showed higher ph values in 3h-4 days period, and mta fillapex in 7-28 days period. high calcium release occurred at 28 days for all sealers except for the experimental sealer 3 (14 days). generally, the experimental sealer 1 released more calcium than others sealers. all experimental sealers and mta fillapex showed basic ph and calcium ion release in the times analyzed. 210 effect of different bleaching agents on roughness, surface gloss, speed and longevity of tooth whitening maas m*, salgado ve, albuquerque pp, schneider lfj, cavalcante lm federal fluminense universitys algadouff@gmail.com the aim was to evaluate the surface propertiesm, speed and longevity of whitening treatments performed with two different agents. 40 bovine incisors were darkened with coffee solution for 30 days. then, they were subjected to hydrogen peroxide 7.5% (ph) or carbamide peroxide 22% (pc) up to 10 days. color parameters cie l * a * b *, gloss (bs) and surface roughness (sr) were determined before and after the bleaching agents. after bleaching treatment, the specimens were again immersed in coffee and their optical properties evaluated for up to 30 days to establish the longevity of the treatment according to each experimental group. the results were submitted to anova and tukey’s test (95%). the bleaching results led to a significant increase in cie l *, a significant reduction in a *, b * and bs in both groups, with no significant differences between them. darkening, after bleaching led to a significant decrease in cie l *, increase in a *, b *, bs and sr in both groups. at the end, rs was greater in ph than pc. there were no significant differences in speed and longevity of the whitening treatment when hydrogen peroxide and carbamide peroxide were tested. however, hydrogen peroxide promoted greater surface roughness. 272272272272272 gbmd 2013 – 49th meeting of the brazilian group of dental materials braz j oral sci. 12(3):237-274 211 contact angle of zirconia pretreated with atmospheric plasma bellotti l, bellotti b, ayres ap, giannini m piracicaba dental school unicamp bbellotti51@hotmail.com this study investigated the effect of atmospheric pressure plasma application (ap) on contact angle of 2 zirconia ceramics. 5 sintered zirconia plates (10 x 10 x 1 mm) of katana (kuraray noritake) and lava (3m espe) were obtained using zirconium dioxide stabilized by yttrium oxide. the plasma torch (surface plasma tool model: sap lab applications) ran at room temperature (22°c) and 20 mm long, using argon gas (praxair 4.8) with 1.0 liter per minute output. the distance between the nozzle and the samples was 10 mm and the time of plasma exposure was 1 minute for each treatment. immediately after the exposition, a water drop of approximately 15 to 20 ìl was placed at zirconia surfaces. contact angle data were analyzed by two-way anova and tukey test (5%). measurements to evaluate the hydrophobic recovery of katana and lava zirconia were executed acquiring hourly the contact angle by image j software (national institutes of health). profile images were acquired with a digital 300x microscope. the contact angles of both materials decreased around 50% if compared to the initial one and became constant in approximately 12 hours after treatment. lava’s got a faster hydrophobic recovery. ap pretreatment shows a possible technique to improve the bond strength between zirconia and polar resins, since the surface wettability increased after plasma exposure. 213 characterization of topography of a an yttrium stabilized zirconia after silica-coating at the pre-sintered state salazar marocho sm*, manarão ds, cesar pf university of sao paulo salazar.marocho@gmail.com characterize the microstructure and determine the surface roughness (ra) and phase transformation of y-tzp structures after the following surface treatments (st): (a) control (without st, as sintered surfaces); (b) silica-coating using 30µm aluminum oxide particles modified by silica (cojet-sand) after final sintering; and (c) silicacoating before final sintering. y-tzp bar-shaped specimens (1.2 mm x 4 mm x 20 mm) were fabricated and divided into 3 groups according to the st described above. silica-coating was performed perpendicular to the y-tzp surface, at 10 mm for 15 s at a pressure of 2.8 bars. an optical profilometer was used to examine the surface roughness (ra), a scanning electron microscopy (sem) to examine the topography of y-tzp after st, and x-ray diffraction (xrd) for phase transformations determination. ra values were analyzed by one-way anova and tukey post-hoc test (5%). group c) presented significantly lower surface roughness (0.26) in comparison to group a) (0.56) and c) (0.82). when the silica-coating was performed at the pre-sintered state, xrd peaks for the monoclinic phase were observed (17%), however after final sintering the monoclinic phase decreased to 0%. in group b), the monoclinic phase content was 7%. group c) showed a rough surface topography consisting of rounded depressions and projections, while group b) showed sharp peaks. group c) also revealed large areas with loss of structure at the treated surface that resulted in a reduction in the specimen’s original thickness ranging from 0.50 to 4 µm. the ts is preponderant on the final topography, and eliminates the problem of phase transformation which is advantageous from the clinical point of view because it would be expected a longer lifetime. 215 surface treatments: effect on bond strength and analysis by confocal scanning laser guarda g.b., guarda m.b., fugolin a.p.p., consani s., sinhoreti m.a.c., corrersobrinho l piracicaba dental school unicamp gui_guarda@hotmail.com this study aimed to evaluate the influence of methods of surface treatment of indirect composite resin on the microtensile bond strength and analyze the infiltration of resin cement through confocal scanning laser. were made 50 blocks (5.0 x 5.0 x 2.0 mm) divided into 5 groups (n = 10), according to the surface treatments: group a spherical diamond bur, b bur spherical diamond, and application of silane bond, c etching with hydrofluoric acid 10%, d etching with hydrofluoric acid 10%, and application of silane bond and e (control) – none. the resin blocks were cemented in bovine teeth etched with 37% phosphoric acid and adhesive system. the samples were subjected to the test of the microtensile bond strength in a universal testing machine (ez test). other samples (n = 3) prepared in the same manner were analyzed in confocal laser scanning for analysis of cement penetration in composite resin blocks. the resin cement was mixed dye (rhodamine). the data were submitted to anova and tukey test (p <0,05). results: the values of bond strength microtraction (mpa) were: group a 27.41 ± (7.40) b, b 28.48 ± (7.64) b, c 26.62 ± (3.03) b, d 39.46 ± (7.40) a, e 33.71 ± (2.78) ab. the best surface treatment for cementation of indirect composite resin prostheses seems that combines the application of hydrofluoric acid, and silane bond. and, it is possible to establish a correlation between penetration of cement and the microtensile bond strength. 212 long term effect of chlorhexidine on the dentin microtensile bond strength of resin cements: a two-year in vitro study yanikian crf, stape ths, quagliatto ps, martins lrm piracicaba dental school unicamp cristiane@fyodontologia.com.br this study investigated the effect of dentin pre-treatment with chlorhexidine on the long-term bond strength of resin cements. composite blocks were luted to caries-free human coronal dentin using a conventional (relyx arc, 3m espe: arc) or a self-adhesive (relyx u100, 3m espe: u100) cement with/without dentin pretreatment by 2% chlorhexidine. chlorhexidine was applied for 60s on etched-dentine for arc and on the smear layer covered dentin for u100. bonded teeth (n=10) were stored in water for 24h and sectioned in 0.9mm x 0.9mm sticks for microtensile bond strength (?mtbs) test. composite-dentin sticks from each bonded tooth were randomly divided to be tested immediately or after two-years of storage in artificial saliva. fracture failures were determine by scanning electron microscopy. repeated measures factorial anova and tukey kramer test (α=0.05) revealed that resin cement, time, the interaction between resin cement, time and dentin pre-treatment (p<0.001) had significant effects on the dentin µmtbs. u100 provided reduced bond strengths at 24h and two-year storage periods (p<0.05); two-year storage did not cause u100 bond-strength reduction (p>0.05). pretreatment with 2% chlorhexidine reduced dentin bond strength loss of arc after storage (p<0.05); there was no adverse effect on the 24h and two-year storage of u100 µmtbs (p>0.05). chlorhexidine is effective to reduce long-term bond strength loss of conventional resin cements contributing to increase indirect restorations longevity. chlorhexidine use with self-adhesive cements does not impair immediate or aged dentin bond strength. its use to increase durability of restorations luted with self-adhesive cements seems irrelevant. 214 effect of light sources with different wavelengths in micro and ultramicrohardness of a nanohybrid composite resin araujo j.l., silva c.m., turbino m.l* university of sao paulo miturbin@usp.br this study evaluated in vitro the hardness (micro and ultramicro) and of a nanohybrid composite resin (tetric n-ceram/ivoclar-vivadent) with different colors, light sources and thickness. the specimens were divided into groups (n=5): khnknoop microhardness, duh-nanohardness; colors: a2 and bleach-m (bm); light sources: ledblue elipar free light 2/3m-espe (750mw/cm²/ 25s/430-480nm(fl)), ledblue/violet bluephase/ivoclar-vivadent (1.200mw/cm²/15s/380-515nm) (b15) and 30s (b30); thickness: 1mm, 2mm and 3mm, compared to the irradiated surface (0mm). specimens were stored dry for 24hours at 37ºc after photo curing. assays were performed at khn in hmv-2000/ shimadzu with load of 25gf at 40s and duh/me in duh 211s/shimadzu with force of 10mn and no hold time (0s). 5 indentations of each test were made in the surfaces opposites to the photo curing. a statistical analysis was performed by anova, tukey and pearson correlation (p<0.01%). pearson’s test showed a direct correlation between khn and duh. comparison between khn and duh in the studied resins indicated that the light source fl promoted higher values of khn and duh to both resins tested. regarding thickness 0mm and 1mm showed higher khn and duh than 2 and 3mm thickness. a2 color showed higher khn and duh than bm. conclusion: the ledblue/ violet was not better for curing the light color of the nanohybrid resin, than ledblue. 216 deproteinized dentin bond strength evaluation of etch-and-rinse adhesive systems bacelar-sá r*, bermejo gn, ambrosano gmb, giannini m piracicaba dental school unicamp renatabcs@hotmail.com the aim of this in vitro study was to evaluate the microtensile bond strength (mbs) of two adhesive systems (as) after differents strategies of adhesion: acid conditioning (ac) or prior application of 10% sodium hypochlorite (sh), and water storage or sh storage. two as was performed: gluma 2bond (heraeus) and one step (bisco). twenty-eight human third molars was used and as were applied following the instructions of each manufacturer with prior application of sh and storade for 1 year. after as applied, a composite were incrementally built on dentin surfaces and the teeth were stored for 24hours (h). the teeth were prepared to microtensile bond strength test (ez test, shimadzu), whereas 1/3 were immediately tested (t1), 1/3 were stored in sh for 3h and washed for 10min before the test (t2) and the remaining of samples were water stored for 1 year (t3). data were analyzed by two-way anova and tukey test (5%). the bond strength means (sd) were (mpa): gluma 2bond t1: 48,4(13,1) and sh36,7(6,7); t2: 36,4(8,5) and sh26,3(2,9); t3: 43,0(5,7) and sh31,1(4,7); one step t1: 54,2(6,2) and sh49,9(9,8); t2: 41,4(5,8) and sh39,1(7,9); t3: 49,1(7,7) and sh45,2(19,5). the application of sh before ac decrease mbs only for gluma 2bond. after 1 year of water storage and sh storage showed lower mbs for all as but sh showed less mbs. the mbs of as did not differ among them when they were used according manufacturers. 273273273273273gbmd 2013 – 49th meeting of the brazilian group of dental materials braz j oral sci. 12(3):237-274 217 ytzp crowns retention varying the substrate, surface treatment and cement rippe mp*, amaral r, saraiva f, cesar pf, lf valandro, bottino ma são josé dos campos dental school – unesp mariliarip@hotmail.com to compare the retention of ytzp zirconia crowns cemented to different substrates considering different cementation protocols. 216 extracted molars were prepared for full crown (n=12); 108 were reconstructed with glass fiber post and composite resin core. ytzp zirconia copings were milled and 144 specimens were divided into 12 groups according to the factors: dentin (d) and composite resin (c); cements: multilink (m) and relyx arc (r) and zirconia surface treatments: alcohol (a) silica coating (s) and vitrification (v). the remaining 72 specimens were divided into 6 groups cemented with relyx luting (civ), relyx u100 (aut) and zinc phosphate (fo) in dentine (d) and composite resin (c) on untreated zirconia. the specimens were thermally cycled (5-55°c, 6000 cycles) and data from the tensile test were analyzed by kruskal wallis and dunn test. the retention values were: dma: 44.8 (+9.3); dms: 55.7 (+16.5); dmv: 53.8 (+11.9); cma: 20.5 (+3.5); cms: 29.9 (+13.1); cmv: 38.8 (+19.6); dra: 16.6 (+10.3); drs: 37.6 (+26.3); drv: 42.4 (+9.7); cra: 22.8 (+5.3); crs: 25.2 (+7.8); crv: 25.9 (+5.3). dciv: 11.4 (+6.3); daut: 42.9 (+18.7); dfo: 0 (0); cciv: 5.4 (+5.1); caut: 4.9 (+4.2); cfo: 8.6 (+7.5). for the zirconia surface treatment groups the cement type was important for the dentine substrate. for groups without zirconia surface treatment the cement type was only important for the zinc phosphate group in dentine. 218 collagen biomodification by edc enhances the stability of resindentin bonds delgado cc*, scheffel dls, de souza costa ca, pashely dh, hebling j araraquara dental school unesp claudia.ng@hotmail.com the aim of this study was to evaluate the effect of dentin treatment with edc on immediate and long-term bond strength of single bond 2 (sb). forty-eight sound third molars were divided into 3 groups (n=16) according to dentin treatment: deionized water (control), 0.5m edc applied for 30s and 0.5m edc applied for 60s. flat dentin surfaces were etched with 37% phosphoric acid for 15 s, rinsed and blot-dried with absorbent paper. the treatment solutions were passively applied on etched dentin by the pre-determined period of time, also followed by rinsing and drying. sb was used according to manufacturer’s instructions and crowns were reconstructed with composite resin. specimens with adhesive area of 0.81mm2 were produced and submitted to microtensile test 24h, 6 or 12 months after artificial saliva storage at 37oc. bond strength (tbs) data were submitted to anova and tukey tests (á =0 .05). the dentin treatment with 0.5m edc for 30s (25.4±4.6 mpa) and 60s (27.4±6.2 mpa) did not interfere on immediate tbs compared to the control group (26.1±4.6 mpa). after 12 months, the highest tbs were observed for the group treated with edc for 60s (29.2±6.5 mpa) followed by the group treated with edc for 30s (22,2±5,1 mpa). the lowest tbs values were seen for the control group (19,9±6,3 mpa). edc was capable of preventing resin-dentin bond degradation after 12 months of artificial saliva storage. 274274274274274 gbmd 2013 – 49th meeting of the brazilian group of dental materials braz j oral sci. 12(3):237-274 braz j oral sci. 15(3):226-233 periodontopathogens, candida spp. and immunological aspects in type 2 diabetes mellitus patients with chronic periodontitis gabriela alessandra da cruz galhardo camargo1, natalia linhares coutinho silva2, ana luísa palhares de miranda2, jorge luiz mendonça tributino3, natália helena colombo4, cristiane duque4 1department of periodontology, fluminense federal university, nova friburgo, rio de janeiro, brazil. doutor sylvio henrique braune, 22, centro, 28625-650, nova friburgo, rio de janeiro, brazil. phone number 5522-981496066 2lassbio, faculty of pharmacy, federal university of rio de janeiro, rio de janeiro, brazil. carlos chagas filho, 373, ccs, bloco bss, sala 22, 21941-902, rio de janeiro, brazil. phone number 55-21-25626503 3institute of biomedical sciences, federal university of rio de janeiro, rio de janeiro, brazil. carlos chagas filho 373, ccs, bloco j sala j01-029, ilha do fundão, 21941-902, rio de janeiro, brazil. phone number 55-21-22805694 4department of pediatric dentistry and public health, araçatuba dental school, univ estadual paulista (unesp), araçatuba, brazil. josé bonifácio, 1193, vila mendonça, 16015050, araçatuba, são paulo, brazil. phone number 5518-36363315 correspondence to: profa. dra. cristiane duque department of pediatric dentistry and public health, araçatuba dental school, univ estadual paulista (unesp), araçatuba, brazil. josé bonifácio, 1193, vila mendonça, 16015050, araçatuba, são paulo, brazil. phone number 5518-36363315 e-mail: cduque@foa.unesp.br, cristianeduque@yahoo.com.br abstract this study aimed to evaluate clinical, microbiological and immunological parameters in type 2 diabetes mellitus (dm) in comparison with normoglycemic patients (ndm). glycemic and lipid profiles and periodontal clinical status were determined for thirty-three patients (17 dm and 16 ndm). the presence of periodontopathogens and species of candida in subgingival sites were determined by polymerase chain reaction and immunological parameters by elisa assays. all glycemic and clinical parameters evaluated were higher in the dm group, with statistical difference for fasting glucose, glycated-hemoglobin, and periodontal parameters. lipid profile (except triglycerides), levels of tnf-α and myeloperoxidase and the prevalence of the tested microorganisms were similar between the groups, except for candida albicans and candida glabrata, which was higher in the dm group. in conclusion, although microbiological and immunological parameters were similar in the dm and ndm groups, periodontitis and the levels of some species of candida were more severe in dm patients. keywords: diabetes mellitus, periodontal disease, candida spp., tnf-α, myeloperoxidase. introduction type 2 diabetes mellitus (dm) is a metabolic disease characterized by insulin resistance and relative or absolute insulin deficiency1. this type of diabetes represents 85-90% of the diabetic group and is associated with lifestyle factors, mainly obesity and lack of physical activity, and genetic susceptibility. blood glucose levels can be controlled with dietary changes and body fat reduction2. the prevalence of diabetes mellitus has increased significantly and it is estimated that by the year 2030 the world population of people with diabetes will be around 552 million, and in brazil will reach 19 million3. patients with chronic hyperglycemia have increased susceptibility to opportunistic infections, as well as oral infections4. periodontitis is the most common chronic oral received for publication: september 19, 2016 accepted: april 24, 2017 original article braz j oral sci. july | september 2016 volume 15, number 3 http://dx.doi.org/10.20396/bjos.v15i3.8649993 227 infection, after dental caries, that causes tooth loss in adults and has been considered the sixth most common complication of diabetes mellitus3. periodontal disease is more prevalent and more severe in diabetes mellitus than in normoglycemic patients and periodontal infection may impair glycemic control, since it is able to activate a systemic inflammatory response4. thus, investigators have considered the mechanism of both diseases to be bidirectional, suggesting that one influences the control of the other5. there are conflicting data regarding whether dm individuals with chronic periodontitis present an altered subgingival microbiota compared with nondiabetic patients. ebersole et al.6 reported that periodontitis sites in dm individuals showed a higher frequency of aggregatibacter actinomycetemcomitans, porphyromonas gingivalis and campylobacter spp. when compared with nondiabetic patients. field et al.7 quantified the subgingival levels of aggregatibacter actinomycetemcomitans, fusobacterium nucleatum and porphyromonas gingivalis in patients with periodontitis and found no significant differences between the subgingival microbiota of dm patients compared with non-diabetic subjects. a report demonstrated that the periodontal pockets of dm patients harbored a higher prevalence of candida spp., mainly c. albicans and c. dubliniensis, compared with periodontal sites from non-diabetic patients8. the same study reported a lower prevalence of tannerella forsythia in periodontitis sites of dm patients compared with non-diabetic patients8. the presence of bacteria is an essential primary factor for the onset of periodontal disease, however, considering the presence of this alone is not sufficient to predict the onset or severity of periodontal disease, it is also necessary to consider the susceptibility of the host which contributes significantly to the appearance of pathological changes in the periodontium; an imbalance between host and microorganisms that may be caused by local or systemic factors, such as diabetes mellitus, is required8. studies have pointed out that tissue destruction in periodontal disease results from the interaction of bacteria and their products (such as lipopolysaccharide lps) with macrophage activation, triggering the local secretion of inflammatory mediators, especially tumor necrosis factor α (tnf-α), interleukin 1β (il-1β), prostaglandin e2 (pge2) and interleukin 6 (il-6)9,10. these mediators are responsible for periodontal breakdown, leading to the clinical signs and symptoms of periodontal disease10. in diabetes mellitus, these mechanisms seem to be accomplished through a low resistance to infection, exaggerated inflammatory response and a deficiency in healing, justifying the severity of periodontal disease in these patients4. in periodontal disease, neutrophils degranulation can release myeloperoxidase (mpo), among other inflammatory mediators, and mpo has been appointed as a promising marker of periodontal disease activity11. in systemically healthy patients, mpo levels were higher in periodontitis sites compared to gingivitis and healthy sites12. gonçalves et al.13 analyzed mpo activity in the gingival crevicular fluid of dm with inadequate metabolic control and non-diabetic patients, both with chronic periodontitis, and observed that mpo activity was lower in dm compared to the control group before and after periodontal treatment. furthermore, after the periodontal therapy the mpo activity was decreased in both groups13. the aim of this study was to evaluate clinical parameters, the presence of putative periodontopathogenic bacteria and candida spp. and the levels of tnf-α and mpo in subgingival sites of non-diabetic and type 2 diabetes mellitus patients with chronic periodontitis. material and methods thirty-three patients (17 with type 2 diabetes mellitus (dm) aged 53.41 + 9.48, 16 non-diabetes mellitus patients (ndm) aged 47.87±10.37 were selected to participate in this study. all subjects were recruited from the department of periodontology, school of dentistry, fluminense federal university, nova friburgo, rio de janeiro state, brazil, over a period of 6 months between 2011 and 2012. the study protocol was approved (protocol number: caae – 0434.0.258.000-11) by the ethics committee of the school of medicine, fluminense federal university. prior to participation, the purpose and procedures of the study were fully explained to all patients, who consequently gave written informed consent in accordance with the helsinki declaration. medical and dental histories were taken and patients received clinical evaluation at prescreening visits. the inclusion criteria were: presence of advanced periodontitis defined by bleeding on probing in sites where probing depth was ≥ 5 mm in a minimum of two teeth in different arches; radiographic bone loss ranging from 30% to 50%14,15. the exclusion criteria were: patients submitted to periodontal treatment in the last 6 months; patients with systemic diseases; osteoporosis; pregnant lactating females; use of immune suppressive medication, phenytoin, cyclosporine, calcium channel blockers or any use of antibiotics or nonsteroidal anti-inflammatory drugs in the previous 3 months; and any medical conditions requiring immunotherapy or a diagnosis of hiv+ or aids, which could interfere with the periodontium status. glycemic and lipid profile parameters a specialized professional collected blood samples from the peripherical vein (cubital fossa) of the individuals who had fasted overnight. samples were collected in vacuum collection tubes and sent to the raul sertã hospital laboratory at nova friburgo/rj for clinical analysis of the following glycemic and lipid parameters: fasting glucose levels (fgl), glycosylated hemoglobin (hba1c), triglycerides (trg), high–density lipoprotein (hdl) and low-density lipoprotein (ldl), using specific kits (gold analisa, belo horizonte/mg). clinical examination an experienced periodontist evaluated the clinical parameters and selected two unirradicular teeth for the protocol procedure. each selected tooth was measured according to the following periodontal parameters: plaque index (pi), bleeding on probe (bop), pocket probing depth (ppd), gingival recession type 2 diabetes mellitus and periodontitis braz j oral sci. 15(3):226-233 228 purified using chloroform: isoamil-alcohol, followed by dna precipitation with isopropanol and 70% ethanol. the dna was ressuspended in te buffer (10 mm tris–hcl, 0.1 mm edta, ph 7.5, with 10 μg/ml rnase). microbial molecular identification was carried out through pcr with specific primers for aggregatibacter actinomycetemcomitans, porphyromonas gingivalis, tannerella forsythia, prevotella intermedia, campylobacter rectus, candida albicans, candida glabrata, candida tropicalis and candida dubliniensis (table 1). pcr amplification was performed with a tgradient 96 pcr system (tx96, amplitherm, usa) under thermal conditions specific for each pair of primers. the pcr products were separated by electrophoresis in 2% agarose gels and tris-borate-edta running buffer (ph 8.0). the molecular mass ladder (100 bp dna ladder, gibco, grand island, ny, usa) was included for running in the agarose gel. the dna was stained with 0.1μl of sybr safe/ml (invitrogen, ca, usa) and visualized under uv illumination (pharmacia lkb-macrovue, san gabriel, ca, usa). photographs of the images were taken (image mater – liscap, vds, pharmacia biotech piscataway, nj, usa) and analyzed. type 2 diabetes mellitus and periodontitis braz j oral sci. 15(3):226-233 (gr), clinical attachment level (cal) using a periodontal probe pcp15 (pcp-unc15, hu-friedy, chicago, il); six sites (mesio-buccal, mediobuccal, disto-buccal, mesio-lingual, medio-lingual, disto-lingual)16 were recorded. two sites with probing depth (ppd) > 5mm were selected for microbiological and immunological analysis. after clinical measurements, the supragingival biofilm was removed with sterile gauze. gingival crevicular samples were taken from the 4 sites with the deepest ppd (≥ 5mm) in each patient, using a sterile paper point from the deepest pocket for 30s. pooled biofilms from each site were separated in two microtubes containing tris -edta buffer (10 mm tris–hcl, 0.1 mm edta, ph 7.5) and were stored at -20o c. the samples were analyzed microbiologically using pcr. microbiological assessment pcr assays dna was extracted using a protocol originally described by sardi et al.8, and quantified in a spectrophotometer at 260 nm (genesys 10uv, rochester, ny, usa), in order to obtain a standard concentration of 100 ng/ml and stored at −20°c for subsequent pcr reactions. briefly, samples were submitted to a lysing solution (extraction buffer and proteinase k) and then table 1 primers sequences for pcr assays. species (reference) sequences (5´-3´) amplicon length(bp) number of accession genbank a. actinomycetemcomitans (ashimoto et al.17) for: aaa ccc atc tct gac ttc ttc ttc rev: atg cca act tga cgt ta at 557 nc_014629.1 p. gingivalis (benkirane et al.42) for: aat cgt aac ggg cga cac ac rev: ggg ttg ctc ctt cat cat ac 593 nc_002950.2 t. forsythia (slots et al.43) for: gcg tat gta acc tgc ccg ca rev: tgc ttc agt gtc agt tat acc t 641 nc_016610.1 jaez01000014.1 p. intermedia (ashimoto et al.17) for: ttt gtt ggg gag taa agc ggg rev: tac aca tct ctg tat cct gcg t 575 nc_017861.1 c. rectus (ashimoto et al.17) for: tttcggagcgtaaactccttttc rev: tttctgcaagcagacactctt 595 acfu01000050.1 c. albicans (sardi et al.8) for:actgctcaaaccatctctgg rev: cacaaggcaaatgaaggaat 452 ajiq01000008.1 c. glabrata (sardi et al.8) for: ggagatagactgggcgttat rev: gttgttcaatggctttcttc 314 xp_448539.1 c. tropicalis (sardi et al.8) for: cacccaaacaattaccaagt rev: tgcaaactctttacctggat 253 nw_003020040.1 c. dubliniensis (donnelly et al.44) for: gtatttgtcgttcccctttc rev: gtgttgtgtgcactaacgtc 288 nc_012860.1 tumor necrose factor-α (tnf-α) and myeloperoxidase (mpo) level measurement gingival crevicular fluid (gcf) was sampled 1 week after clinical examination, by a researcher blinded to the clinical parameters. gcf samples were taken from two different sites labeled as 1 or 2, in both sites the deepest ppd was ≥ 5 mm and bop was chosen for sampling the same patient. the sites were isolated with sterile cotton rolls and dried with an air syringe to eliminate the possibility of contamination with saliva. gcf samples were obtained by placing a calibrated, volumetric microcapillary pipette with an internal diameter of 1.1 mm and a capacity of 5 μl. sites that did not express the appropriate volume of fluid and micropipettes, which were contaminated with blood and saliva, were not included in the study14. the gcf was immediately placed into separate tubes containing 250 μl phosphate-buffered saline. the samples were stored at -20°c and analyzed by a single, blinded examiner using commercial kits of tnf-α and mpo enzyme-linked immunosorbent assay (r&d systems, minneapolis, mn, usa). statistical analysis required sample size was determined by openepi (openepi, version 3.03a, 2015) and was calculated to detect a 0.05 difference between bop (ndm) and bop (dm) with a power level of 84%. 229 type 2 diabetes mellitus and periodontitis the samples size was based on previous studies in the literature18,19 using data related to differences in mean and standard deviation values, determining a minimum of 13 patients with type 2 diabetes mellitus required to detect significant differences in clinical and laboratorial variables between groups. the primary efficacy variables were whole-mouth mean bop (ndm) and bop (dm). statistical tests were performed using the software spss, version 17.0 (chicago, il) to compare differences between the dm and ndm groups. age and gender were compared between the groups using the student’s t-test. the mann whitney u test was performed to compare clinical and immunological parameters (pi, bop, ppd, gr, cal, fasting glucose and hba1c, ldl, hdl, trg, tnf-α and mpo). microbiological analysis of periodontopathogenic bacteria was performed using chi-square tests. spearman’s rho tests were applied to explore correlations between clinical, microbiological and immunological parameters, according to the presence of diabetes mellitus. statistical significance for all variables was defined at the 5% level. results study subjects no significant differences were observed between the dm and ndm groups, considering age (dm: 53.41±9.48 and ndm: 47.87±10.37, p=0.751) or gender (dm: male 52.9%, female 47.55 and ndm: male 37.5%, female 62.5%, p=0.292). the mean duration of diabetes was 7.76±7.61 years ranging from 1 to 30 years. glycemic and lipid profiles descriptive statistics with means/medians/quartiles of the glycemic and lipid variables are presented in table 2. fasting glucose levels and hba1c were statistically higher in dm compared to ndm showing glycemic differences between the groups. dm patients had a poorer glycemic control. positive correlations were found between hba1c and bop in diabetic patients (spearman’s rho, 0.502, p=0.04). ldl and hdl levels were similar between the groups and remained within the normal values. trg values were statistically higher (mann whitney test, p=0.03) in diabetic compared to non-diabetic patients. trg was also positively correlated with gr (spearman’s rho, 0.568, p=0.017) and cal (spearman’s rho, 0.516, p=0.034). periodontal parameters pi and bop indices were evaluated on all tooth surfaces and determined for the whole mouth (%). ppd, gr and cal were calculated in millimeters. means/medians/quartiles of the clinical parameters recorded for both groups are summarized in table 2. diabetic patients presented significantly higher values for pd, gr and cal compared to the control group. the other clinical parameters did not differ significantly between the groups. for both groups, positive correlations (0.491-0.910) were observed between ppd, gr and cal values, showing that these parameters are intrinsically associated with the periodontal status (spearman’s rho, p<0.05). braz j oral sci. 15(3):226-233 table 2 summary of clinical and laboratorial parameters and proinflammatory mediators levels for dm and ndm groups. data were expressed in medians (inter-quartiles). dm ndm p value cl in ica l p ar am et er s pi (%)** 69.84 (51.78-91.7) 61.94 (43.45-77.94) 0.363 bop (%) 46.66 (35.2-65.74) 51.83 (24.25-68.34) 0.958 ppd (mm) 5.5 (5.24-5.65) 5.19 (5-5.29) 0.045* gr (mm) 1.81 (1.33-3.11) 0 (0-2.28) 0.045* cal (mm) 6.1 (5.5-7.39) 5.2 (5-5,8) 0.014* la bo ra to ria l pa ra m et er s g lyc em ic pr ofi le fgl (mg/dl) 131 (94-172) 89 (78.75-97.25) 0.006* hba1c (%) 7.6 (6.85-8.3) 4.7 (4.5-5) 0.000* li pi d pr ofi le ldl (mg/dl) 125 (107-139) 93 (87.5-136.0) 0.165 hdl(mg/dl) 44 (38-48) 47.5 (40.75-55.5) 0.102 tgr (mg/dl) 146 (89-261) 74 (54.5-154) 0.033* pr oi nfl am m at or y m ed ia to rs le ve ls tnf-α (pg/ ml) 1.03 (0.76-1.22) 1.18 (0.9-1.49) 0.068 mpo (pg/ ml) 0.028 (0.01-0.07) 0.06 (0.026-0.079) 0.382 * statistically significant difference between dm and ndm groups (mann whitney u-test, p <0.05). **plaque index (pi), bleeding on probe (bop), pocket probing depth (ppd), gingival recession (gr), clinical attachment level (cal), fasting glucose levels (fgl), glycosylated hemoglobin (hba1c), triglycerides (trg), high –density lipoprotein (hdl) and low-density lipoprotein (ldl). microbiological analysis figure 1 shows the prevalence of a. actinomycetemcomitans (aa), p. gingivalis (pg), t. forsythia (tf), p. intermedia (pi), c. rectus (cr), c. albicans (ca), c. glabrata (cg), c. tropicalis (ct) and c. dubliniensis (cd) in the dm and ndm groups. a significant difference between the groups was observed only for c. albicans and c. glabrata. the prevalence of putative periodontopathogens (aa, pg, tf, pi and cr) was similar between dm and ndm patients. twenty-four of the 33 patients (17 dm and 7 ndm) were colonized by c. albicans and twenty (13 dm and 7 ndm) by c. glabrata. all patients harbored c. dubliniensis and none presented c. tropicalis. the percentage of sites harboring c. albicans, t. forsythensis and p. gingivalis simultaneously was statistically higher in dm compared to ndm patients (figure 2). the association of four or five periodontopathogens and c. albicans or c. glabrata was observed only in dm patients (figures 2 and 3). immunological analysis levels of tnf-α and mpo measured in gingival crevicular fluid are presented in table 2. no statistically significant differences were found for either proinflammatory marker between the dm and ndm groups. no correlations were verified between these biological markers and clinical, glycemic, lipid or microbiological profiles (spearman’s rho correlation, p>0.05). of the present study confirmed that patients with type 2 diabetes mellitus (dm) presented significantly higher values of ppd, gr and cal compared to the control group and consequently greater severity of pd. the 2009-meta-analysis, investigated whether or not diabetes is a risk factor for periodontitis and included papers published between 1980 and 2007. twenty-seven crosssectional studies were included and detected more periodontal disease in diabetic compared to non-diabetic subjects. although methodological flaws were found, the results showed a significant association between cal (difference 1.00, 95% ci 0.15-1.84), ppd (difference 0.46, 95% ci 0.01-0.91) and dm, confirming that dm is an important risk factor for periodontitis5. glycemic profile is usually measured in studies with diabetics to evaluate the influence of these parameters on the prevalence, extent and severity of periodontitis. generally, well-controlled diabetes with glycemic parameters within the normal values seems to have little effect on the risk of periodontitis, however, the contrary is observed in poorly controlled patients. in the current study, fasting glucose levels and hba1c were statistically higher in dm compared to ndm patients. our glycemic results are consistent with important systematic reviews that investigated associations between diabetes, glycemic control and complications, showing that pd was more prevalent among individuals with poorer glycemic control5,20. both studies supported the evidence that diabetes has an adverse effect on periodontal status and pd has an adverse effect on glycemic control. due to the crosssectional design of the present study, it is not possible to state that periodontal disease had an effect on glycemic control. this evidence could be provided by treatment and longitudinal studies. in poorly controlled diabetes, there is a nonspecific glycation of lipids and proteins that forms reactive oxygen species (ros). in the case of dm, ros are considered as a major risk for developing micro and macrovascular complications. they participate in the formation of advanced glycated end products (age) that induce crosslinkation processes in the structure of proteins, such as collagen, modifying blood vessel structure. after binding to their specific receptors (rage), they lead to cytokine production and proinflammatory effects21. all these factors can exacerbate the severity of periodontal disease, besides contributing to the development of systemic complications such as retinopathy, nephropathy, neuropathy and other diseases4. another occurrence concomitant to diabetes mellitus is dyslipidemia, defined as a high blood concentration of lipids, especially ldl and triglycerides (trg). studies have indicated an association between elevation in blood lipoproteins and alterations in periodontal disease2,22. however, other clinical trials failed to identify this relationship22,23. in the current study, both groups with periodontal disease, independent of the presence of diabetes, presented normal values of ldl. however, dm patients had high levels of trg, above normal values (>150 mg/dl), and were positively correlated with two important periodontal parameters, gr and cal. our results are in agreement with tu et al.24 who evaluated associations between lipid parameters and periodontitis and found a positive correlation for trg and c-reactive protein (crp). controversially, sora et al.25 evaluated the relationship of metabolic syndrome (five cardiovascular risk factors: abdominal obesity, hypertension, reduced hdl, elevated trg and elevated 230type 2 diabetes mellitus and periodontitis fig. 1 frequency of periodontal sites harboring aggregatibacter actinomycetemcomitans (aa), porphyromonas gingivalis (pg), tannerella forsythia (tf), prevotella intermedia (pi), campylobacter rectus (cr), candida albicans (ca), candida glabrata (cg), candida tropicalis (ct) and candida dubliniensis (cd) in the dm and ndm groups. *statistically significant differences were found between the groups for ca and cg, according to χ2 tests (test, p <0.05). fig. 2 frequency of periodontal sites (%) harboring combinations of putative periodontopathogens with candida albicans (ca) in the dm and ndm groups. *statistically significant differences between the dm and ndm groups, according to χ2 tests (p <0.05). aggregatibacter actinomycetemcomitans (aa), porphyromonas gingivalis (pg), tannerella forsythia (tf), prevotella intermedia, campylobacter rectus (cr). fig. 3 frequency of periodontal sites (%) harboring combinations of putative periodontopathogens with candida glabrata (cg) in the dm and ndm groups. *statistically significant differences between the dm and ndm groups, according to χ2 tests (p <0.05). aggregatibacter actinomycetemcomitans (aa), porphyromonas gingivalis (pg), tannerella forsythia (tf), prevotella intermedia, campylobacter rectus (cr). discussion although the majority of the published studies are crosssectional, providing a limited possibility of a causal-effect relationship10, substantial evidence has indicated diabetes mellitus (dm) as a risk factor for periodontal disease (pd)3. the results braz j oral sci. 15(3):226-233 fasting glucose or diabetes), and these factors individually, with the extent of severe periodontitis among patients with dm from gullah. they found a relationship between metabolic syndrome and periodontitis in this population; however, no results from the multivariable binomial regression demonstrated a relationship between the isolated components of metabolic syndrome, including hdl and trg, and periodontitis in these individuals with diabetes. the authors explained that more than one risk factor linked to diabetes is necessary to increase the extent of periodontitis and commented that their results may not apply to other populations with a different genetic and socio-cultural background. however, almeida abdo et al.26 found no associations between dyslipidemia and periodontal disease. they observed that diabetes, age and smoking had positive correlations with periodontitis (cal ≥ 3 and cal ≥ 5). periodontal health is dependent on a balance between the bacteria harboring the subgingival biofilm and the host response to them. environmental changes may modify the bacterial challenge or host immune response, as occurs in diabetes patients7. the prevalence of putative periodontal bacteria in dm compared to ndm has been previously investigated by some authors7,8,27,28. the bacterial species belonging to the “red and orange complexes” are the most frequently studied and there is no consensus whether specific periodontal pathogens can harbor subgingival sites in subjects with dm when compared with ndm. in the present study, the frequency of periodontal sites harboring a. actinomycetemcomitans, p. gingivalis, t. forsythia, p. intermedia and c. rectus (cr) was statistically similar between the groups. our results corroborate with those found by field et al.7, sardi et al.8 and yuan et al.28 and differ from other studies29-32. we focused on subgingival sites with periodontal disease, specifically deep pockets, for both groups, also reported by the majority of these studies. contrarily, the study conducted by aemaimanan et al.30 found high levels of t. forsythia in healthy sites and p. gingivalis and t. forsythia in gingivitis sites and a higher quantity of p. gingivalis only in periodontal sites of poorly controlled dm patients compared to the ndm group. recent studies have indicated a high prevalence of t. forsythia in dm patients with periodontitis and their possible role in the severity of periodontitis29,30. li et al.31 also demonstrated not only higher levels of t. forsythia but also t. denticola, however lower levels of p. intermedia in the subgingival plaque of chinese patients with type 2 diabetes mellitus. zhou et al.32 concluded that subjects with healthy periodontium harbored different genera (abundance of prevotella, pseudomonas and tannerella) compared to patients with periodontitis (abundance in actinobacteria, proteobacteria and bacteriodetes), and in both groups, the authors detected that diabetic and nondiabetic subjects harbored bacteria at several taxonomic levels with significant different prevalence or abundance. these discrepancies in the results could be related to the differences in the lifestyle of the populations, which interferes directly in the composition of the subgingival microbiota33. diabetic patients are known for their predisposition to oral infections caused by candida spp., particularly those with poor glycemic control. this condition may occur concomitantly with a higher incidence and greater severity of periodontitis34. in the current study, species of candida were detected from deep pockets in both groups with chronic periodontitis, regardless of the presence of diabetes. however, two of these species, c. albicans and c. glabrata, were more frequently found in dm patients. candida spp. have also been identified in periodontal sites by other investigators8,35,36, but only the study developed by melton et al.36 demonstrated higher levels of c. albicans (53%) followed by c. glabrata (20%), similar to those obtained in the present study. c. albicans has been described as the most prevalent and pathogenic species of the genus34 and plays a role in immune evasion and adherence to epithelium, causing inflammatory reactions37 and may be detrimental to the periodontal environment. c. glabrata has emerged as an important opportunist pathogen in oropharyngeal candidiasis, after c. albicans. the frequency of c. glabrata infections increased up to 50% in some populations, due to immunosuppressive therapies and broad spectrum antibiotics37. a significant correlation between poor glycemic control and periodontal candida colonization has been reported8,34,35 and is in agreement with our results. glucose level concentration in gingival crevicular fluid is known to be associated with blood glucose level9 and could partly explain the proliferation of candida in periodontal sites35. another suggested effect of poorly controlled glycemic status in dm is the aggregation of local cytokine response9. in the current study, tnf-alpha was chosen for its intrinsic relationship with the severity of periodontal disease, but this study aimed to evaluate if this cytokine could be more elevated in diabetic patients due to their glycemic status. however, there was no difference between the groups. the relevance of the cytokine profile in subjects with dm and chronic periodontitis has been explored and the levels of these inflammatory markers seem not to differ from normoglycemic patients with a similar periodontal condition9,38. javed et al.38 evaluated databases from 1988 to 2011 and concluded that gingival crevicular fluid cytokine profile, including tnf-alpha, in patients with or without dm seems to be related to the severity of periodontal inflammation and the diabetes is secondary. besides, the action of many of the biomarkers is short-lived and is likely to vary based upon stimulatory molecules, leading to a limited local response at affected sites. after activation of downstream biomarkers and/ or local cell activation, many biomarkers, such as il-6 and tnf-alpha, are degraded quickly39. the myeloperoxidase (mpo) activity was also evaluated in the present study. mpo is one of the peroxidase systems responsible for immunological defense in saliva and gingival crevicular fluid. studies have demonstrated an increase in the mpo level in systemically healthy patients with periodontitis40. for diabetic subjects, two studies showed different results, with greater mpo activity13,41. in our study, there was no statistical difference in the mpo levels obtained from gingival crevicular fluid between diabetic and non-diabetic individuals, both with periodontal disease. the presence of diabetes did not influence the level of this proinflammatory marker, in agreement with tenovuo et al.41. controversially, gonçalves et al.13 found lower mpo activity in diabetic patients with chronic periodontitis, before and after periodontal treatment. there are methodological differences between the present study and the above-mentioned investigations. the first one41 analyzed 231 type 2 diabetes mellitus and periodontitis braz j oral sci. 15(3):226-233 saliva samples from patients with different types of diabetes, not exclusively dm and the last one13 evaluated enzyme activity, not its concentration, as in the present study. these methodological differences make it difficult to compare the present study with the other investigations. within the limitations of this study, mainly the small sample size, it may be concluded that although chronic periodontitis was clinically more severe, the level of tnf–α, mpo and the prevalence of putative periodontal pathogens were not different in dm patients compared to non-diabetic individuals. an interesting fact was the high frequency of c. albicans and c. glabrata, which seem to have a preferential capacity for colonizing the periodontal pockets of diabetic patients. more longitudinal studies are necessary to confirm the influence of the species of candida on the progression of periodontal disease. acknowledgements this study 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nondiabetic patients with generalized chronic periodontitis. anaerobe. 2013 aug;22:64-8. doi: 10.1016/j.anaerobe.2013.06.010. 31. li c, liu j, tan l, yu n, lin l, geng f, et al. the sociodemographic characteristics, periodontal health status, and subgingival microbiota of patients with chronic periodontitis and type 2 diabetes mellitus: a casecontrol study in a chinese population. j periodontol. 2013 aug;84(8):105866. doi: 10.1902/jop.2012.120282. 32. zhou m1, rong r, munro d, zhu c, gao x, zhang q, et al. investigation of the effect of type 2 diabetes mellitus on subgingival plaque microbiota by high-throughput 16s rdna pyrosequencing. plos one. 2013 apr 22;8(4):e61516. doi: 10.1371/journal.pone.0061516. 33. haffajee ad, bogren a, hasturk h, feres m, lopez nj, socransky ss. subgingival microbiota of chronic periodontitis subjects from different geographic locations. j clin periodontol. 2004 nov;31(11):996-1002. 34. javed f, klingspor l, sundin u, altamash m, klinge b, engström pe. 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jan;12(1):80-96. 38. javed f, al-askar m, al-hezaimi k. cytokine profile in the gingival crevicular fluid of periodontitis patients with and without type 2 diabetes: a literature review. j periodontol. 2012 feb;83(2):156-61. doi: 10.1902/ jop.2011.110207. 39. geisinger ml, michalowicz bs, hou w, schoenfeld e, gelato m, engebretson sp, et al. systemic inflammatory biomarkers and their association with periodontal and diabetes-related factors in the diabetes and periodontal therapy trial, a randomized controlled trial. j periodontol. 2016 aug;87(8):900-13. doi: 10.1902/jop.2016.150727. 40. yamalik n, cağlayan f, kilinç k, kilinç a, tümer c. the importance of data presentation regarding gingival crevicular fluid myeloperoxidase and elastase-like activity in periodontal disease and health status. j periodontol. 2000 mar;71(3):460-7. 41. tenovuo j, lehtonen op, viikari j, larjava h, vilja p, tuohimaa p. immunoglobulins and innate antimicrobial factors in whole saliva of patients with insulin-dependent diabetes mellitus. j dent res. 1986 jan;65(1):62-6. 42. benkirane rm, guillot e, mouton c. immunomagnetic pcr and dna probe for detection and identification of porphyromonas gingivalis. j clin microbiol 1995;33(11):2908-12. 43. slots j, ashimoto a, flynn mj, li g, chen c. detection of putative periodontal pathogens in subgingival specimens by 16s ribosomal dna amplification with the polymerase chain reaction. clin infect dis 1995;20(suppl):s304-7. 44. donnelly sm, sullivan dj, shanley db, coleman dc. phylogenetic analysis and rapid identification of candida dubliniensis based on analysis of act1 intron and exon sequences. microbiology 1999;145(pt 8):1871182. braz j oral sci. 15(3):226-233 1http://dx.doi.org/10.20396/bjos.v18i0.8657261 volume 18 2019 e191635 original article 1 piracicaba dental school, university of campinas, piracicaba, são paulo, brazil 2 state university of ponta grossa, ponta grossa, paraná, brazil 3 department of dentistry at state university of ponta grossa, paraná, brazil corresponding author: marcos pomini department of prosthodontics and periodontics, piracicaba dental school, university of campinas avenida limeira, 901, areião, piracicaba-sp, brazil email: marcospomini@outlook.com tel: +55(45)988123993 received: april 23, 2019 accepted: july 17, 2019 in vitro fracture strength and modes of cast post and cores luted with self-adhesive resin-based cement marcos c pomini1,*, marina m machado2, giovanny de paula quadros2, lidia o b pinheiro,3 adriana p b samra3 cast post and core (cpc) remains one of the most used post type; however, the biomechanical behavior of cpcs luted with self-adhesive (sa) resin-based cement is unclear. aim: to evaluate the fracture resistance (fr) and fracture modes of teeth restored with cpcs luted with zinc-phosphate (zp) or resin-based sa cements, as well as the infuence of the coronal remnant. methods: twenty-four recently extracted human premolars were divided into four groups according to the cement used (zp or sa) and residual tooth structure (with or without 2 mm). we then tested fr using a universal machine and analyzed data using two-way anova and tukey hsd (α=.05). fracture modes were classifed according to the degree of dental destruction results: no difference was found in fr (p=.352); however, teeth without ferrules presented more irreparable fractures, especially in the zp group. conclusion: luting cpcs with sa resin-based cement does not enhance fr, but it reduces the number of irreparable fractures compared to zp cement. keywords: post and core technique. resin cements. adhesiveness. fractures, compression. 2 pomini et al. introduction post restoration choice and ferrule affect the prognosis of endodontically treated teeth1,2. although the use of fiber reinforced composite posts is increasing in contemporary dentistry2, cast post and cores (cpcs) remain one of the most used post type3,4. metal-based cpcs exhibit great long-term clinical results and are still used successfully5,6 because they present a better fit for root canal irregularities, avoid excessive canal preparation, and are preferred in situations involving insufficient ferrules or for supporting fixed partial dentures7. luting agents are expected to increase the retention of the post and core restoration and maintain its integrity5. on the other hand, zinc-phosphate cements (zp) have poor mechanical and biological properties, lower compressive strength, and high solubility8. self-adhesive (sa) resin-based cements were introduced with an organic multifunctional methacrylate matrix, which not only conditions the tooth surface, but also contributes to adhesion5. the adhesive cementation of posts and cores is of great significance to the stability and strength of the overall rehabilitation5, however, the mechanical behavior of sa adhesion to metal-based cpcs remains unclear in the literature. thus, in this in vitro study, we aimed to evaluate the fracture resistance (fr) and fracture modes of teeth with and without ferrules restored with cpcs luted with zp or sa resin-based cement. we tested the following hypothesis: self-adhesion enhances fr and reduces irreparable fractures in endodontic treated teeth restored with cpcs. materials and methods the ethics committee of the state university of ponta grossa approved this study’s experimental protocol. the sample consisted of twenty-four recently extracted human single-rooted premolars. half of the teeth (n=12) were decoronated at the cementenamel junction, and the other half decoronated 2 mm coronally. the roots received standard endodontic treatments and post space preparations. resin patterns were cast in copper-aluminum alloy9. prior to cementation, the roots were irrigated with 2.5% sodium hypochlorite, rinsed with distilled water, and dried gently with blown air, followed by absorbed points. twelve specimens were cemented with zp cement (ls, coltene, rio de janeiro, brazil), and the other 12 were cemented with a sa resin-based cement (relyx u200, 3m espe, st. paul, mn, usa). the roots were divided into four groups (n=6) according to coronal remnant (with or without 2 mm) and cementing agent (zp or sa). twenty-four hours and five minutes after cementation, the sa and zp roots were prepared for metal-free crowns, respectively, and received self-curing acrylic resin (vipiflash; vipi, pirassununga, brazil) crowns made using preformed acetate matrices (tdv dental, pomerode, brazil). the crowns were luted with relyx u200. to provide a more accurate assessment of human root fracture strength, the alveolar bone and periodontal ligament were simulated by dipping the root surfaces into melted wax (asfer, são caetano do sul, brazil) up to 2 mm below the cej, resulting in a wax layer approximately 0.3 mm thick. the roots were then placed in polyvinyl chloride tubes filled with self-curing acrylic resin (vipi). after 24 hours, the wax was removed 3 pomini et al. and replaced with polyether impression material (3m espe, st. paul, mn, usa). one week after cementation, the samples were submitted to a compressive load (ag-i; shimadzu, columbia, usa) at a crosshead speed of 0.5 mm/min and angulation of 150° until fracture10. the values were submitted to two-way anova, followed by tukey hsd (α=.05), and the fracture modes were classified based on the degree of dental destruction: reparable (crown displacement or fracture, or horizontal fracture in the cervical third) or irreparable (vertical or oblique root fracture, or horizontal fracture in the apical or middle third), which would require tooth extraction. results we found no significant difference in fr among the groups (p=.352) regardless of ferrule presence (table 1). however, table 2 shows that teeth with ferrules presented one irreparable fracture (8.3%), whereas teeth without ferrules presented three (25%). in the absence of ferrules, the zp group presented 3 irreparable fractures (50%), whereas the sa group presented 1 (16.7%). discussion our results show that the chemical adhesion of cpcs does not enhance fr, but it reduces the number of irreparable fractures, especially in the presence of 2mm coronal remnant. similarly, in a clinical setting, behr et al.8 found no difference when luting metal-based materials with zp and sa. because cpc retention with zp depends on friction along the root canal, it transmits the stress directly to the dentinal walls, increasing root fractures3. in addition, the adhesion of posts to the root canal has been described as reducing stress transmission to the root because it promotes table 1. mean and standard deviation (n) of the failure loads (n=6). coronal remnant cementing agent zinc-phosphate resin-based self-adhesive ferrule present 1141.2 ± 149.6a 1058.4 ± 188.3a ferrule absent 1102.3 ± 154.4a 967 ± 117.1a *similar letters indicate statistically similar values table 2. fracture modes and overall percentage of repairable and irreparable for the experimental groups (n=6) groups repairable irreparable displacement or fracture of the crown vertical or oblique root fracture horizontal root fracture in the apical or middle third zp/ferrule (%) 5 (83.3) 1 (16.7) zp/without ferrule (%) 3 (50) 2 (33.3) 1 (16.7) sa/ferrule (%) 6 (100) sa/without ferrule (%) 5 (83.3) 1 (16.7) total (%) 79.2% 8.3% 12.5% overall (%) 79.2% 20.8% *zp: zinc-phosphate cement; sa: resin-based self-adhesive resin cement. 4 pomini et al. increased retention and attenuates tooth weakening11, reducing the number of irreparable fractures. this corroborates our results. in addition, the most common cause of failure in cpcs luted with zp cement is retention loss12. the strong physical interaction between the methacrylates in sa resin-based cement5 may favor adhesion, creating a monoblock restoration. this seems to increase over time13 and may favor stress distribution. in a similar setting, pomini et al.13 demonstrated that the bond strength of cpc luted with sa increased after 6 months of storage, indicating that the biomechanical behavior of cpc luted with sa resin-based cement may improve over time, unlike zp, which is known for lower compressive strength and high solubility in the oral environment.8 in addition, the amount of coronal remnant may have influenced the number of root fractures because the remnant modifies the root stress distribution pattern. teeth with ferrules present higher resistance to functional forces, wedging effects, lateral forces, and greater strength1, which may reduce the number of fractures. ferrule presence is considered the most important factor affecting fr and post and core rehabilitation survival rates2,4,13. nonetheless, metal-based posts and cores have demonstrated the lowest complication rates, being root fractures the most commonly reported complication4. juloski et al.2 demonstrated that teeth restored with cpcs are more resistant to fractures than prefabricated posts not only in the absence of ferrules, but also in their presence. on the other hand, in the presence of ferrules, cpcs present similar fr compared to ceramic custom-fabricated posts2. therefore, the better outcomes found for cpcs in the absence of residual tooth structure maybe be the main factor that influences dentists to continue using cpcs instead of newer post types. however, our results should be evaluated carefully because not all clinical aspects can be reproduced in a laboratory setting. this study evaluated only an oblique incident force, but in the oral cavity, force direction varies. in addition, we used acrylic resin crowns for this study. however, these crowns are routinely used in temporary rehabilitation, often for considerable lengths of time. because we aimed at evaluating the posts’ fr, the use of crowns that do not influence resistance and only superficially dampen compressive forces during the test was considered. therefore, researchers should conduct additional multi-variable studies to evaluate the long-term performance of sa resin-based luted cpcs. conclusion the use of sa resin-based cement in cpcs does not promote enhanced fr, but it reduces the number of irreparable fractures considerably, especially in the presence of 2mm coronal remnant. thus, luting cpcs with sa resin-based cement seems to be the ideal choice for endodontically treated teeth. acknowledgements the authors are grateful to 3m espe and tdv, for the donation of the materials employed in this study and to the phd professor giovana mongruel gomes for reviewing the final article. 5 pomini et al. references 1. shamseddine l, chabaan f. impact of a core ferrule design on fracture resistance of teeth restored with cast post and core. adv med. 2016;2016: 5073459. doi:10.1155/2016/5073459. 2. juloski j, radovic i, goracci c, vulicevic zr, ferrari m. ferrule effect: a literature review. j endod. 2012;38(1):11-9. doi: 10.1016/j.joen.2011.09.024. 3. soares cj, valdivia adcm, silva gr, santana fr, menezes ms. longitudinal clinical evaluation of post systems: a literature review. braz dent j. 2012;23(2):135-740. doi:10.1590/s0103-64402012000200008. 4. said yh, sahib d. “the use of post and core in public and private swedish dental care : a questionnaire study.” (2018). available at: http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-143912. 5. capa n, ozkurt z, canpolat c, kazazoglu e. shear bond strength of luting agents to fixed prosthodontic restorative core materials. aust dent j. 2009 dec;54(4):334-40. 6. sarkis-onofre r, jacinto rc, boscato n, cenci ms, pereira-cenci t. cast metal vs. glass fibre posts: a randomized controlled trial with up to 3 years of follow up. j dent. 2014;42:582-7. 7. hendi ar, moharrami m, siadat h, hajmiragha h, alikhasi m. the effect of conventional, half-digital, and full-digital fabrication techniques on the retention and apical gap of post and core restorations. j prosthet dent. 2019;121(2):364.e1-364.e6. doi:10.1016/j.prosdent.2018.09.014. 8. behr m, rosentritt m, wimmer j, lang r, kolbeck c, bürgers r, handel g. self-adhesive resin cement versus zinc phosphate luting material: a prospective clinical trial begun 2003. dent mater. 2009;25(5):601-4. doi:10.1016/j.dental.2008.11.003. 9. pinto cl, bhering clb, de oliveira gr, maroli a, reginato vf, caldas ra, et al. the influence of post system design and material on the biomechanical behavior of teeth with little remaining coronal structure. j prosthodont. 2019;28(1):e350-e356. doi: 10.1111/jopr.12804. 10. gomes gm, gomes om, gomes jc, loguercio ad, calixto al, reis a. evaluation of different restorative techniques for filling flared root canals: fracture resistance and bond strength after mechanical fatigue. j adhes dent. 2014;16(3):267-76. doi:10.3290/j.jad.a31940. 11. balkaya mc, birdal is. effect of resin-based materials on fracture resistance of endodontically treated thin-walled teeth. j prosthet dent. 2013;109(5):296-303. 12. balkenhol m, wöstmann b, rein c, ferger p. survival time of cast post and cores: a 10-year retrospective study. j dent. 2007;35(1):50-8. 13. carvalho ma, lazari pc, gresnigt m, del bel cury aa, magne p. current options concerning the endodontically-treated teeth restoration with the adhesive approach. braz oral res. 2018;32(suppl 1):e74. doi:10.1590/1807-3107bor-2018.vol32.0074. 14. pomini mc, machado mm, de paula quadros g, gomes gm, pinheiro lob, samra apb. in vitro fracture resistance and bond strength of self-adhesively luted cast metal and fiber-reinforced composite posts and cores: influence of ferrule and storage time. int j prosthodont. 2019;32(2):205-7. doi:10.11607/ijp.5956. untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8651897 volume 17 2018 e18017 original article a department of dental materials and prosthodontics, dental school of ribeirão preto, university of são paulo, são paulo, brazil. av. do café, s/n, 14040-904 – ribeirão preto – sp – brazil. corresponding author: dra. adriana cláudia lapria faria, department of dental materials and prosthodontics, dental school of ribeirão preto, university of são paulo, av. do café, s/n, 14040-904 – ribeirão preto – sp – brazil. tel: + 55 16 33154130 fax: + 55 16 33150999 e-mail: adriclalf@forp.usp.br received: august 29, 2017 accepted: december 01, 2017 fracture toughness of three heat pressed ceramic systems lívia fiorin, ddsa; guilherme teixeira theodoro, ddsa; izabela cristina maurício moris, dds, ms, phda; renata cristina silveira rodrigues, dds, ms, phda; ricardo faria ribeiro, dds, ms, phda; adriana cláudia lapria faria, dds, ms, phda aim: the aim of this study was to evaluate fracture toughness by indentation method of three dental ceramics processed by heat pressing. the ceramics evaluated were fluorapatite glass ceramic (zir), glass ceramic containing leucite (pom) and leucite-reinforced glass ceramic (emp). materials and methods: ninety disks (13mm of diameter x 4mm of thickness) and nine rectangular specimens (25x4x2mm) were made to evaluate, respectively, microhardness/fracture toughness (n=30) and elastic modulus (n=3). samples were obtained by pressing ceramic into refractory molds. after polishing, vickers microhardness was evaluated under 4,904n load for 20s. elastic modulus was measured by impulse excitation technique. data from microhardness and elastic modulus were used to calculate fracture toughness, after measuring crack length under 19,6n load applied for 20s. results were evaluated by anova and tukey´s test. results: microhardness (vhn) of pom (637.9±53.6) was statistically greater (p<0.05) than zir (593.0±14.3), followed by emp (519.1±21.5); no significant difference (p=0.206) was noted for elastic modulus (gpa) (zir: 71.5±9.0; pom: 67.3±4.4; emp: 61.7±2.3). fracture toughness (mpa/m) of pom (0.873±0.066) was statistically lower (p<0.05) than zir (0.977±0.021) and emp (0.965±0.035). conclusion: the results suggest that fluorapatite glass ceramic (zir) and leucite-reinforced glass ceramic (emp) processed by heat pressing presented greater fracture toughness, improving clinical prognosis of metal free restorations. keywords: dental porcelain. hardness tests. elastic modulus. 2 florin et al. introduction the use of dental ceramics has increased because of aesthetics, biocompatibility, chemical stability, compression strength and color stability1-2. nevertheless, ceramics are brittle materials that are unable to absorb elastic energy and resists to crack propagation, fracturing. then, several all-ceramic materials and processing techniques have been introduced in the past decade3-4. heat pressing, slip-casting and computer aided design-computer aided machining (cad-cam) are the processing techniques developed to offer greater mechanical performance associated with higher crystalline content of new ceramic systems, extending the indications to posterior restorations and fixed partial dentures5. common defects, inherent in the processing, such as internal microcracks, pores, inclusions and second-phase clusters, can possibly propagate under tensile stresses until achieving a critical size, fracturing6. the methods used in the processing of materials may lead to different mechanical properties7. heat pressing involves the use of heat and pressure to inject molten ceramic in an investment mold8. several studies have evaluated the effect of heat pressing in the properties of different ceramic systems8-12. there is not a consensus about the effect of heat pressing at the mechanical properties and microstructure of ceramic. a previous study reported that repeated heat pressing increased the size of crystals and porosities and decreased density, hardness, flexural strength and fracture toughness of lithium disilicate12. other authors argued that mechanical properties of lithium disilicate were not significantly affected by repressing, although qualitative differences in the microstructure11. another study that evaluated lithium disilicate and leucite based ceramics reported a decrease in porosities and a better crystal distribution after heat pressing, increasing flexural strength although the similar fracture toughness9. veneering ceramics obtained by heat pressing and layering for metal and zirconia copings were also compared and authors reported that veneering ceramics to zirconia are affected by processing technique while the veneering ceramic for metal alloys, not8. fracture toughness is defined as the resistance of brittle materials to rapid crack propagation13. then, this property represents serviceability in the oral cavity14. there are several methods to evaluate fracture toughness: indentation fracture, single edge “v” notch beam, single edge precracked beam, surface crack in flexure and chevron notch beam, but the indentation method is popular because it is simple, non-destructive and permits evaluation at small samples15. fracture toughness is related to elastic modulus, crack length and microhardness16. thermal mismatch, chemical stress and mechanical process such as cyclic loading can lead to crack initiation at surface, and these microscopic cracks propagate trough bulk of the restorations, leading to catastrophic failures17-18. then, fracture toughness permits to predict the clinical performance (fracture and wear) of ceramic materials describing the ability of the material to withstand crack propagation7,19. although fracture toughness of some ceramic systems is sometimes provided by manufacturers, studies have reported that heat pressing has affected differently mechanical properties of ceramic systems. thus, the aim of the present study was to evaluate fracture toughness of three different ceramic systems processed by heat pressing. 3 florin et al. material and methods for fracture toughness evaluation by indentation technique, microhardness and elastic modulus were also evaluated. specimen preparation in the present study, three ceramic systems were evaluated (table 1). for this, thirty disks (13mm diameter x 4mm thickness) and three rectangular (25mm length x 4mm width x 2mm thickness) specimens of each ceramic were obtained dripping molten wax in teflon matrixes. then, wax patterns were invested (bellavest sh, bego bremer goldschlägerei, bremen, germany), submitted to the heating cycle (table 2) of the investment. after, ceramic ingots and plunger were positioned in the investment block and ceramic was heat pressed (table 3) in the ceramic furnace (alumini sinter press, edg equipamentos, são carlos, sp, brazil). after pressing, samples were divested using airborne-particle abrasion with 100µm glass beads (renfert, hilzingen, germany), and polished with silicon carbide papers (320-2000, norton, saint-gobain abrasivos ltda., igarassu, pernambuco, brazil). microhardness vickers microhardness was evaluated (n=30) at disks with a load of 4,904 n applied for 20 seconds (hmv-2, shimadzu corp., kyoto, japan)20. five randomly chosen sites per sample were evaluated and a mean value (vhn) was obtained. table 1. ceramic systems, indications and commercial names. materials indication commercial name (manufacturer) fluorapatite glass ceramic (zir) pressing onto zirconia copings and frameworks ips e.max zirpress (ivoclar vivadent) leucite-reinforced glass ceramic (emp) all-ceramic single tooth restorations ips empress esthetic (ivoclar vivadent) glass ceramic containing leucite (pom) pressing onto metallic copings and frameworks ips inline pom (ivoclar vivadent) table 2. heating cycle of investment blocks. initial temperature temperature increase rate 1 stage 1 temperature increase rate 2 stage 2 pressing room temperature 20°c/min 300°c (30 min) 25°c/min 800°c/850°c* (60min) 800°c/850°c* * investment blocks were heated until 850°c only for e pressing. table 3. firing parameters of each ingot pressing for all ceramic systems tested. material stand-by temperature temperature increase rate firing temperature holding time ips e.max zirpress (zir) 700°c 60°c/min 900°c 15 min ips empress esthetic (emp) 700°c 60°c/min 1075°c 20 min ips inline pom (pom) 700°c 60°c/min 940°c 20 min 4 florin et al. elastic modulus elastic modulus was evaluated (n=3) at rectangular specimens by impulse excitation technique (sonelastic, atcp physical engineering, ribeirão preto, sp, brazil)21. specimens were positioned and submitted to an impact by a pulsator. the acoustic response was captured by a transducer and translated into electric sign to read the resonance frequencies. elastic modulus was determined by bending excitation. fracture toughness fracture toughness was evaluated (n=30) by the indentation method proposed by anstis22, using a microhardness tester (hmv-2, shimadzu corp., kyoto, japan)4,11,18,20. a load of 19,6n was applied for 20 seconds. three randomly sites per sample were evaluated. crack length was measured from the center of indent and a mean value for cracks was used to calculate fracture toughness using the formula20, where kic is fracture toughness (mpa/m), e is elastic modulus (gpa), p is the load applied (n), h is vickers microhardness (gpa) and c is crack length(m): kic = 0,016 ( e h ) 0,5 ( p c1,5 ) the data of microhardness, elastic modulus and fracture toughness were analyzed by one-way anova and post-hoc tukey´s test using statistical software (spss 17.0, ibm spss software, ibm corporation). a significance level of 5% was used. results the results of vickers microhardness are shown in figure 1. microhardness of ceramic pom (621.9±78.1) was statistically greater than zir (593.0±14.3) and emp (519.1±21.5), and zir was statistically greater than emp (p<0.05). the results of elastic modulus are presented in figure 2. the elastic modulus of the ceramics zir (71.54±9.02), emp (61.67±2.26) and pom (67.27±4.42) was statistically similar (p=0.206). fracture toughness of zir (0.977±0.021) and emp (0.965±0.035) was statistically similar, and both zir and emp presented greater fracture toughness than pom (0.873±0.066) (p<0.05). the results are presented in figure 3. m ic ro ha rd ne ss (v h n ) 700 600 500 400 300 200 100 00 zir zir * & + emp pom emp pom figure 1. results of vickers microhardness (vhn) of the ceramics evaluated.different symbols represent statistically significant difference (α=5%). 5 florin et al. discussion the ability of a material to react to a load and/or impact is described by its mechanical properties. dental ceramics are brittle materials that fracture into two or more pieces when they are submitted to tensile forces23. thus, determination of fracture toughness of these materials is important to predict the clinical performance once measures the ability of a material to withstand crack propagation7,19. in the present study, fracture toughness was evaluated by indentation method. this method was chosen because it is fast and permits measuring cracks in a small sample. although there is difference in the results of fracture toughness evaluated by different methods15,24, this study aimed to compare fracture toughness of three ceramics using the indentation method. fracture toughness measured by indentation method is related to hardness and elastic modulus. when hardness increases, toughness and plasticity decreases, increasing fragility25. the ceramic emp is a leucite-reinforced glass ceramic. according to some authors, high leucite content, particle size and homogeneous particle distribution can improve ceramic strength6. then, high fracture toughness of emp can be attributed to its leucite content (35%), once there is a positive correlation between leucite content and fracture toughness because leucite increases the resistance to crack propagation19,26. additionally, fracture toughness increases with greater particle size6, corroborating the results of the present study where greater crystals (5 to 10 micrometer) are present in emp. a previous study reported that a homogeneous crystal distribution is achieved by fr ac tu re to ug hn es s (m pa /m ) 1.2 1.0 0.8 0.6 0.4 0.2 0.0 zir zir emp pom emp pom + + * figure 3. results of fracture toughness (mpa/m) of the ceramics evaluated. different symbols represent statistically significant difference (α=5%). el as tic m od ul us (g pa ) 80 70 60 50 40 30 20 10 00 zir zir emp pom emp pom * * * figure 2. results of elastic modulus (gpa) of the ceramics evaluated.similar symbols show that elastic modulus of the evaluated ceramics is statistically similar (α=5%). 6 florin et al. heat pressing5, possibly increasing fracture toughness because crack propagation is avoided. however, another study reported that fracture toughness of this ceramic is not affected by pressing and repressing even crystal distribution is more uniform, and pores and defects introduced during machining decrease in number and size after pressing9. pom is a glass ceramic containing leucite, but the concentration and size of leucite crystals present in this ceramic is not presented by the manufacturer. considering that pom presented lower fracture toughness of this study, the leucite content and crystal size are probably lower than in emp that is a leucite reinforced glass ceramic. in addition, the low fracture toughness of pom is directly affected by its high microhardness while the lowest microhardness of emp leads to greater fracture toughness than pom. some authors also argued that some surface flaws (microcracks, scratches and broken edges) of dental ceramics are caused by grinding or usage6. all samples were polished for microhardness and fracture toughness evaluation, and polishing may originate surface flaws, especially when materials present greater microhardness (pom) that make difficult polishing. then, low fracture toughness of pom can also be attributed to surface flaws originated from polishing. some authors argue that fracture toughness of leucite containing ceramic is greater than non-leucite glass ceramic26, but fracture toughness of emp (leucite reinforced glass ceramic) and zir (fluorapatite glass ceramic) was statistically similar and greater than pom (glass ceramic containing leucite) in the present study. zir is a ceramic whose microstructure is primarily amorphous with little evidence of crystalline phase by fluorapatite crystals26. according to some authors, the higher content of needle-like fluorapatite crystals increases vickers microhardness and fracture toughness27. then, high fracture toughness of this ceramic is attributed to its fluorapatite crystals, that measure 100nm by 300nm and 300nm by 2 to 5 micrometer, as manufacturer provides. the results of vickers microhardness (600vhn) and fracture toughness (0.9±0.3mpa/m) provided in scientific documentation of pom manufacturer´s is similar to the results (pom=0.873mpa/m) found in the present study. however, the fracture toughness results are lower than results of other study that evaluated the same ceramic8. this difference is probably caused by the difference of the method used to evaluate fracture toughness because the previous study used single edge notched beam method8 and there is difference in the results of fracture toughness evaluated by different methods15,24. the same difference was noted for fluorapatite glass ceramic (zir=0.977mpa/m) that presented lower fracture toughness in the present study than in the study mentioned before8. on the other hand, lower values of fracture toughness results were found in a study that evaluated this fluorapatite glass ceramic before and after pressing, using or not zirconia in association26. differences of results in the fracture toughness values of this study in relation to the literature can be caused by residual stress after heat pressing and polishing. some authors recommend slow cool at the last heating cycle, such as glazing, to reduce residual stress within ceramic system26. additionally, defects introduced during machining also affect ceramic strength9. considering that samples of this study were polished and not submitted to auto-glaze heating cycle after polishing, different fracture toughness results was probably affected by polishing that can introduce surface flaws and interfere with fracture toughness. 7 florin et al. it is important to consider that this study evaluated intrinsic fracture toughness of ceramics zir and pom once this ceramics were evaluated without zirconia or metallic copings, differently from their clinical application. some authors argued that fracture toughness of ceramic is greater than ceramic/zirconia samples because the last present residual stress in its interface because of thermal expansion coefficient mismatch10. differently, other study that evaluated residual stress at ceramic-metal and ceramic-zirconia systems, correlating residual stress with fracture toughness, reported that fracture toughness is higher where compressive residual stress is greater, especially close to surface and interface while tensile stresses found in the bulk of ceramic of ceramic-metal system decreases fracture toughness28. then, the compressive residual stresses created at the surface and interface of zir and pom associated to zirconia and metal, respectively, would increase fracture toughness of these ceramics. in addition, it is also necessary to consider the effect of the geometry of sample that can affect the stress and possibly fracture toughness28. although pom presented lower fracture toughness, it is important to consider that this ceramic is indicated for metal-ceramic restorations and metallic coping would improve mechanical behavior of the restorations19. the ceramics zir and emp presented higher fracture toughness but the association with zirconia copings can increase fracture toughness of zir, improving the clinical prognosis. in the present study, microstructure of ceramics were not evaluated, but further studies are necessary to evaluate microstructure of these ceramics before and after heat pressing, correlating these information with their mechanical properties. based on the results of the present study, it is possible to conclude that fluorapatite glass and leucite-reinforced glass ceramics processed by heat pressing presented greater fracture toughness, improving clinical prognosis of metal free restorations. acknowledgements the authors thank fapesp for the financial support (#2013/11926-1; #2014/08212-0). references 1. gomes ea, assunção wg, rocha ep, santos ph. [ceramic in dentistry: current state] cerâmica. 2008;54:319-25. portuguese. 2. kelly jr, benetti p. ceramic materials in dentistry: historical evolution and current practice. aust dent j. 2011 jun;56 suppl 1:84-96. doi: 10.1111/j.1834-7819.2010.01299.x. 3. bona ad, mecholsky jjj, anusavice kj. fracture behavior of lithia-disilicate and leucite-based ceramics. dent mater. 2004 dec;20(10):956-62. 4. denry il, holloway ja. elastic constants, vickers hardness, and fracture toughness of fluorrichteritebased glass-ceramics. dent mater. 2004 mar;20(3):213-9. 5. gurram r, krishna chv, reddy km, reddy gvkm, shastry ym. evaluating the fracture toughness and flexural strength of pressable dental ceramics: an in vitro study. j indian prosthodont soc. 2014 dec;14(4):358-62. doi: 10.1007/s13191-013-0331-1. 8 florin et al. 6. cesar pf, yoshimura hn, miranda jr wg, miyazaki cl, muta lm, rodrigues filho le. relationship between fracture toughness and flexural strength in dental porcelains. j biomed mater res b appl biomater. 2006 aug;78(2):265-73. 7. alkadi l, ruse d. fracture toughness of two lithium disilicate dental glass ceramics. j prosthet dent. 2016 oct;116(4):591-596. doi: 10.1016/j.prosdent.2016.02.009. 8. ansong r, flinn b, chung kh, mancl l, ishibe m, raigrodski aj. fracture toughness of heat-pressed and layered ceramics. j prosthet dent. 2013 apr;109(4):234-40. doi: 10.1016/s0022-3913(13)60051-7. 9. albakry m, guazzato m, swain mv. influence of hot pressing on the microstructure and fracture toughness of two pressable dental glass–ceramics. j biomed mater res b appl biomater. 2004 oct 15;71(1):99-107. 10. longhini d, rocha com, medeiros is, fonseca rg, adabo gl. effect of glaze cooling rate on mechanical properties of conventional and pressed porcelain on zirconia. braz dent j. 2016 sepoct;27(5):524-531. doi: 10.1590/0103-6440201600709. 11. gorman cm, horgan k, dollard rp, stanton kt. effects of repeated processing on the strength and microstructure of a heat-pressed dental ceramic. j prosthet dent. 2014 dec;112(6):1370-6. doi: 10.1016/j.prosdent.2014.06.015. 12. tang x, tang c, su h, luo h, nakamura t, yatani h. the effects of repeated heat-pressing on the mechanical properties and microstructure of ips e.max press. j mech behav biomed mater. 2014 dec;40:390-396. doi: 10.1016/j.jmbbm.2014.09.016. 13. amer r, kurklu d, johnston w. effect of simulated mastication on the surface roughness of three ceramic systems. j prosthet dent. 2015 aug;114(2):260-5. doi: 10.1016/j.prosdent.2015.02.018. 14. taira m, nomura y, wakasa k, yamaki m, matsui a. studies on fracture toughness of dental ceramics. j oral rehabil. 1990 nov;17(6):551-63. 15. cesar pf, bona ab, scherrer ss, tholey m, noort rv, vichi a, et al. adm guidance ceramics: fracture toughness testing and method selection. dent mater. 2017 jun;33(6):575-584. doi: 10.1016/j.dental.2017.03.006. 16. pagani c, miranda cb, bottino mc. [relative fracture toughness of differents dental ceramics]. j appl oral sci. 2003 mar;11(1):69-75. portuguese. 17. rosemblum ma, schulman a. a review of all-ceramic restorations. j am dent assoc. 1997 mar;128(3):297-307. 18. scherrer ss, kelly jr, quinn gd, xu k. fracture toughness of a dental porcelain determined by fractographic analysis. dent mater. 1999 sep;15(5):342-8. 19. cesar pf, yoshimura hn, júnior wgm, okada cy. correlation between fracture toughness and leucite content in dental porcelains. j dent. 2005 oct;33(9):721-9 20. fahmy nz, el guindy j, zamzam m. effect of artificial saliva storage on microhardness and fracture toughness of a hydrothermal glass-ceramic. j prosthodont. 2009 jun;18(4):324-31. doi: 10.1111/j.1532-849x.2009.00448.x. 21. suansuwan n, swain mv. determination of elastic properties of metal alloys and dental porcelains. j oral rehabil. 2001 feb;28(2):133-9. 22. anstis gr, chantikul p, lawn br, marshall db. a critical evaluation of indentation techniques for measuring fracture toughness: i, direct crack measurements. j am ceram soc. 1981;64(9):533-8. doi: 10.1111/j.1151-2916.1981.tb10320.x. 23. campbell sd. a comparative strength study of metal ceramic and all-ceramic esthetic materials: modulus of rupture. j prosthet dent. 1989 oct;62(4):476-9. 24. domingues nb, galvão br, ribeiro s, almeida aaj, longhini d, adabo gl. comparison of the indentation strength and single-edge-v-notched-beam methods for dental ceramic fracture toughness testing. dent mater. 1998 jul;14(4):246-55. 9 florin et al. 25. elias cn, lopes hp. [dental materials: mechanical testing]. são paulo: santos; 2007. portuguese. 26. choi jr, waddell jn, swain mv. pressed ceramics onto zirconia. part 2: indentation fracture and influence of cooling rate on residual stresses. dent mater. 2011 nov;27(11):1111-8. doi: 10.1016/j.dental.2011.08.003. 27. xiang q, liu y, sheng x, dan x. preparation of mica-based glass-ceramics with needle-like fluorapatite. dent mater. 2007 feb;23(2):251-8. 28. sebastiani m, massimi f, merlati g, bemporad e. residual micro-stress distributions in heat-pressed ceramic on zirconia and porcelain-fused to metal systems: analysis by fib-dic ring core method and correlation with fracture toughness. dent mater. 2015 nov;31(11):1396-405. doi: 10.1016/j.dental.2015.08.158. 1http://dx.doi.org/10.20396/bjos.v20i00.8660053 volume 20 2021 e210053 original article 1 department of dentistry, federal university of sergipe, são cristovão, sergipe, brazil 2 faculty of dentistry, university of vale do taquari, lajeado, rs, brazil 3 university of pelotas, pelotas, rs, brazil corresponding author: luiz alexandre chisini alexandrechisini@gmail.com editor: dr altair a. del bel cury received: june 14, 2020 accepted: march 22, 2021 laser photobiomodulation effect on fibroblasts viability exposed to endodontic medications gustavo danilo nascimento lima1 , luiz alexandre chisini2,3* , marcus cristian muniz conde2 , andré luís faria e silva1 , flávio fernando demarco3 , maria amália gonzaga ribeiro1 aim: the literature has not yet reported investigations about the effect of laser photobiomodulation (lpbm) over the cytotoxicity of drugs for endodontic treatments. thus, the aim of this study was to evaluate, in vitro, the effect of the association between lpbm and intracanal medications on fibroblasts viability in different exposure times. methods: calcium hydroxide (ca(oh)2) and iodoform (io) were used pure or associated to lpbm. eluates of medications were prepared and placed in contact with the cells in three different periods: 24h, 48h and 72h. laser irradiation (emitting radiation λ 660nm, power density of 10mw, energy density of 3 j/cm²) has been performed in two sessions within a six hour interval, for 12s per well. after each experimental time, the colorimetric assay (mtt) has been performed. statistical analysis was applied for mann-whitney test with 5% α error admitted test. results: at 24h, the use of lpbm did not increase cell viability while after 72h cell proliferation was stimulated in the group without medications. lpbm application did not increase cell viability in ca(oh)2 group and io at any tested time. ca(oh)2 cytotoxicity at 24h was higher than iodoform, while at 72h not difference was observed. therefore, after 72 hours was no statistical difference between the io and ca(oh)2 groups. conclusion: lpbm was able to increase cell viability in 72h in the group without medication, although no improvement was observed in the other groups. thus, lpbm was not able to reduce the cytotoxic effects of the materials on fibroblasts in vitro. keywords: endodontics. low-level light therapy. fibroblasts. https://orcid.org/0000-0002-4626-5173 https://orcid.org/0000-0002-3695-0361 https://orcid.org/0000-0003-2662-3305 https://orcid.org/0000-0003-3846-4786 https://orcid.org/0000-0003-2276-491x http://orcid.org/0000-0002-3682-4189 2 lima et al. introduction decontamination of the root canal system is the critical step to achieve success in endodontics, being performed through effective chemical-mechanical preparation1-3. with the introduction of rotatory systems, the working time has decreased and, consequently, the contact of disinfectant agents with the microorganisms presents in the root canal4. thus, several irrigation solutions in different concentrations has been applied to achieve quick and effective disinfection2. however, some microorganisms can remain in the root canal system1,2. in this way, decontaminant pastes, as calcium hydroxide – ca(oh)2 – and iodoform – io – are essential to combat the microorganisms resisting of root canal system5. these medications should be biocompatible, not carcinogenic, and/or genotoxic to periradicular tissue since they remain in contact with the periodontium6. products of materials degradation in contact with existing cells in periradicular tissue can result in chemical irritation and inflammation, with a huge variety of chemical inflammatory mediators, which in high levels can cause tissue destruction and delay on healing process7-9. therefore, these medications should present the ability to induce repair in the injured area without interfering with osteogenesis and cementogenesis9-11. searching strategies to minimize tissue damage, the association between intracanal medications and laser photobiomodulation (lpbm) has been proposed12,13. lpbm is the term applied to describe the wide range of laser applications with low-energy densities and based on photochemical mechanisms where the energy is transferred to the intracellular mitochondrial chromophores and respiratory chain components12,14. thus, lpbm is an electromagnetic radiation source with characteristics (monochromaticity, coherence, and one-pointedness) that differentiate it from other light fonts showing several clinical applications with anti-inflammatory, analgesic, and trophic-regenerative effects12,13. lpbm can induce cell’s metabolic changes through a series of cascading reactions through photochemical and photoelectric with primary and secondary effects on exposed tissue to laser irradiation12. its ability to stimulate the proliferation of various cell types such as fibroblasts, epithelial cells, lymphocytes, and odontoblasts, participating directly in the cell and tissue repair is directly linked to parameters used, including wavelength15-18. to the best of our knowledge, there is no report evaluating the effect of lpbm on fibroblasts viability exposed to different endodontic medications. thus, this study aimed to evaluate in vitro the effect of the association between different exposure times to lpbm and intracanal medications on the viability of fibroblasts. materials and methods cell culture: fibroblast cells 3t3/nih (previously cryopreserved19) were cultivated in dmem (dulbecco’s modified eagle mediumcultilab, campinas, sp, brasil) supplemented with 10% fetal bovine serum (fbs) (sigma chemical co. st. louis, mo, usa) and 1% of penicillin (105ui/ml) (gibcobrl, gibco-invitrogen, grand island, ny). the cells were maintained in an incubator (37°c, 5% co2, and 95% humidity) 20. the culture medium was changed every two days. after reaching subconfluence (80%), 3 lima et al. the medium has been removed and the cells were washed with phosphate-buffered saline (pbs). tripsine/edta (gibco brl) was added for five minutes to create a cell suspension and inactivated with dmem/fbs. cells were counted in the neubauer chamber and 2x104 cells dmem/fbs solution was applied in each well, in a 96 well plate. cells were stored in an incubator for 24h to allow cell adhesion. manufacture specimens: for the made the specimens of calcium hydroxide group (hc) was used 1g pa calcium hydroxide (biodynamic chemicals & pharmaceuticals ltd, pr, brazil) + 1.1ml of distilled water. for iodoform group (io) was used 1.5g of iodoform (k-dent, quimidrol sc, brazil) + 600 µl of distilled water. the materials from both groups were spatulate and placed in an array with the following formats iso guidelines for cytotoxicity assays (iso 10993-5: 2009). they were sterilized with ultraviolet (uv) radiation for 1 hour. eluate was produced by dipping the samples in a solution of 1 ml of dmem + 10% fbs and storing them in an oven at 37°c for 24 hours. after 24 hours of cell adhesion, the adhered cells on the plate were washed with pbs and 200 ul of the eluate was added to each well. each eluate filled 4 plate wells (n=8 for the group). after one and six hours were conducted at the experimental plates irradiation with the laser medium inserted directly into the cell monolayer and the different groups. laser application was performed with wave-length in the red spectrum 660nm – ingaalptwin-laser (mmoptics®, equipamentos ltda., são carlos, são paulo, brasil) and 3j/cm2 for 12 seconds18 (table 1). controls did not receive laser irradiation (n=8). cell viability assay: after irradiation, the plates returned to the humid atmosphere for cytotoxicity testing 24h, 48h, and 72h. cell viability was determined by colorimetric method mtt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] (mtt table 1. irradiation parameters for the groups ca(oh)2, io and control. irradiation parameters emission mode (cw) continuous length (nm) 660 active medium ingaalp optical power of the laser (output)(mw) 10 optical power of the laser (input) (mw) 40 field of beam spot (mm) 4 area (cm2 – a=π.r2)(cm2) 0,04 power density (pd)(w/cm2) 0,25 energy density (ed)(j/cm2) 3 time per point (s) 12 total energy (j) 0,24 energy per session (j) 0,12 j/well divergence of the beam perpendicular to the junction 17º point angle 50º 4 lima et al. 5 mg / ml dmem). the mtt was maintained in contact with cells for 4 hours to allow the cells to metabolize and consequently reduce the mtt salt by dehydrogenases present in mitochondria of fibroblasts, thus forming formazan crystals21. after this period, the medium was sucked, and formazan crystals resuspended in 200μl of dmso (dimethyl sulfoxide). the dmso was placed in contact with the cells for 15 minutes then the plate was placed for 5 minutes on a shaker (150 rpm). spectrophotometry universal reader elisa, at a wavelength of 540 nm, assessed the results which were considered the values of absorbance as an indicator of cell viability. assays were realized in triplicate. statistical analysis: data were analyzed for the mann-whitney rank sum test with 5% α error admitted. sigmastat 3.5® software was used in the analysis. results after 72hs lpbm group demonstrated the best results for cell viability maintenance, however, it has been observed exclusively in the group without medication (p < 0,05). in groups containing medications, lpbm was not able to increase cell viability (table 2). in contrast, lpbm combined with io demonstrated a decrease of absorbance in 24h. ca(oh)2 presented high initial toxicity which remained at all periods (24, 48 and 72h) while io showed more cell viability in 24h compared to ca(oh)2. in a longer period (72h), io showed toxicity similar to ca(oh)2. discussion lpbm has been attracting more interest in dentistry, with increasing applicability in this field. several approaches have incorporated lpbm as an adjuvant to reduce the inflammatory process, aiming a modulation effect, as well as a restorative stimulus for bone, muscular and neural lesions12,13,15,17,22,23. the lpbm was used in this study, with the perspective of minimizing possible cytotoxic effects of endodontic drugs in contact with cells in vitro. the irradiation parameters used in this study were chotable 2. median absorbance (nm) (25% / 75%) for the groups submitted to lbpm according to medications and time (n = 8) (mann-whitney test, p ≤ 0.05). time laser median absorbance (25% 75%) medications ca(oh)2 io control 24h no #aa 0.32 (0.26 / 0.35) #ba 1.49 (1.36 / 1.66) #ba1.17 (1.10 / 1.67) yes #aa 0.29 (0.22 / 0.35) *aac 0.90 (0.18 / 1.1) #ba 1.67 (1.23 / 1.96) 48h no #aa 0.29 (0.27 / 0.33) #bb 0.70 (0.55 / 0.85) #ca 1.08 (1.01 / 1.35) yes #aa 0.29 (0.27 0.32) #ba 0.66 (0.41 / 0.89) #ba 1.11 (0.54 / 1.70) 72h no #aa 0.28 (0.26 / 0.28) #ac 0.27 (0.25 / 0.29) #bb 0.39 (0.33 / 0.85) yes #aa 0.27 (0.26 / 0.32) #ac 0.29 (0.26 / 0.31) *ba 1.55 (1.31 / 1.90) symbols (#, *) represents statistical differences of laser application at the same time uppercase letters represent differences in a row (different medications) lowercase letters represent differences over time within the same laser application or not 5 lima et al. sen because the radiation in the visible wavelength is absorbed by the mitochondrial photoreceptor, resulting in photochemical effects, triggering a cascade of metabolic events resulting in a response to biomodulation16,18,24. previous studies18,25 also reported that the same energy density was able to show positive results in stimulation of fibroblasts’ metabolism increasing the number of viable cells and their proliferation. in this way, we chose a wavelength of 660nm because recent studies have reported that shorter wavelengths, ranging from 600 to 700 nm, could be considered best to treat superficial tissue18,26. on the other hand, wider wavelengths, ranging from 780 to 950nm, should be chosen to deeper tissues27,28. moreover, several studies18,29 propose that variations from 3 to 10 j/cm2 could produce the desired stimulation of metabolic activity. fibroblasts were used in this study due to the fact presented as the major constituent of connective tissue, being the most predominant cell type in the periodontal ligament and the largest producer of collagen, elastin, glycosaminoglycans and glycoproteins30,31. intracanal medications when in contact with the periapical tissues cause an inflammatory response and the lpbm could provide an enhanced cellular response in the affected area. although the lpbm proved better maintenance of the fibroblasts viability after 72h, it has not been observed in the presence of ca(oh)2 or io. the basic biological mechanisms behind the effects of lpbm occurs through absorption of red light by chromophores mitochondrial cytochrome c oxidase, which is contained in the respiratory chain of mitochondria. then, a cascade of events occurs, which leads to biostimulation of various processes, leading to increased enzymatic activity, mitochondrial respiration, transport of electrons, and production of adenosine triphosphate. the lpbm, in turn, alters the redox cell that induces the activation of many intracellular signaling pathways and alters the affinity of transcription factors related to cell proliferation, survival, tissue repair, and regeneration32. therefore, when light is applied to cells, mitochondria are the initial sites of absorption. the photon absorption leads to nitric oxide dissociation, e reactive oxygen species production, and increased atp synthesis28. thus, photons at the red and infrared wavelengths can interact with specific photoreceptors located within the cell presenting beneficial results in vivo models33. ca(oh)2 and io containing groups presented lower cell viability than the control, indicating the drugs’ cytotoxicity effect. ca(oh)2 showed to be more cytotoxic than io at 24 and 48 hours when compared to the group without medications. besides, when it has been associated with the lpbm, there was none positive changes in the results. this fact may be related to ph increases promoted by ca(oh)2 24,34,35 which promotes enzymatic denaturation and destruction of the cell membrane, causing the cell’s death30 neutralizing the possible positive effect of lpbm. after 72h, there was no difference between ca(oh)2 and io. in this way, a recent systematic review evaluating clinical studies showed that lpbm has a positive effect by avoiding dental pain after endodontic treatment since it reduces tissue inflammation22. delay in the onset of pain or reduction in the pain severity and duration were also related to lpbm in endodontic treatment with different laser parameters, despite the studies have not investigated the combination with intracanal medications as the outcome22,36. 6 lima et al. similar results were found by sarigol et al.37 (2010) in which an io-base substance showed increasing cytotoxicity in fibroblasts over time. in contrast, it was noted in 24h greater aggressiveness of iodoform when compared to calcium hydroxide38. the laser therapy’s modulator mechanisms are not yet fully understood. it is assumed that some effects from io association with lpbm may be related to increased production of hydrogen peroxide by modulating the redox activity of mitochondria and/ or the redox state of the cell can stimulate cell cycle and protein synthesis at lower concentrations, but which are very cytotoxic at higher concentrations39. in conclusion, lpbm was able to increase cell viability in 72h in the group without medication, whereas no improvement was observed in the other groups. thus, lpbm was not able to reduce the cytotoxic effects of the materials on fibroblasts in vitro. compliance with ethical standards conflict of interest: no conflict of interest. funding: none ethical approval: this article does not contain any studies with human participants or animals performed by any of the authors. references 1. conde mcm, chisini la, sarkis-onofre r, schuch hs, nör je, demarco ff. a scoping review of root canal revascularization: relevant aspects for clinical success and tissue formation. int endod j. 2017 sep;50(9):860-74. doi: 10.1111/iej.12711. 2. dioguardi m, gioia gd, illuzzi g, laneve e, cocco a, 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[comparative analisys of in vitro citotoxicity of iodoform and calcium hydroxide associated with two differents vehicles] [dissertation]. são paulo: faculdade de odontologia da universidade de são paulo; 1998. portuguese. 39. vladimirov ya, osipov an, klebanov gi. photobiological principles of therapeutic applications of laser radiation. biochemistry (mosc). 2004 jan;69(1):81-90. doi: 10.1023/b:biry.0000016356.93968.7e. 1http://dx.doi.org/10.20396/bjos.v18i0.8657252 volume 18 2019 e191505 original article 1 department of prosthodontics, school of dentistry, university of taubaté, taubaté, brazil. 2 department of materials and technology, school of engineering of guaratinguetá, são paulo state university, guaratinguetá, brazil. corresponding author: rafael pino vitti https://orcid.org/0000-0001-6366-5868 university of taubaté 09 rua dos operários, taubaté, sp cep: 12020-270 phone/fax: 55-12-3629-2130 e-mail: rafapvitti@gmail.com received: january 20, 2019 accepted: june 28, 2019 stress distribution in the peri-implant area of pure titanium and titanium-zirconium small implants tatiana de andrade sabino1, laís regiane da silva-concílio1, ana christina elias claro neves1, ana paula rosifini alves claro2, marina amaral1, rafael pino vitti1,*, cristiane aparecida de assis claro1 aim: in dental implant treatment, there is a demand for mechanically stronger implants. despite the existence of several studies showing the clinical success of narrow diameter implants, most of them are based on pure titanium (cpti) alloys. there is a few clinical evidences of the success rate of titanium-zirconium (tizr) narrow diameter implants. the aim of this study was to evaluate the stress distribution in the peri-implant area of narrow diameter cpti and tizr implants under axial and oblique loads. methods: photoelastic models were produced using epoxy resin (pl2, vishay precision group) from a master model. the implants (cpti and tizr; straumann ag) had 3.3 mm in diameter and 12 mm in height. loads of 100 n and 200 n were applied to the abutment at angles of 0° (axial), 10°, 20°, and 30° (oblique). a circular polariscope (eikonal) was used under dark field white-light configuration. the isochromatic fringes were analyzed in the peri-implant region in 5 areas, using astm table with isochromatic fringes; cervical-mesial, cervical-distal, mid-mesial, mid-distal and apical. results: in general, under axial and oblique loads, the stress in the tizr implant was lower than in the cpti implant. the load of 200 n produced the highest stress values in cpti and tizr implants. in both implants and loads, the fringes were located more in apical area at all angles evaluated. conclusion: it can be concluded that for small implants, the load inclination and intensity change the pattern of stress distribution and the cpti implant exhibited the highest peri-implant stress. keywords: dental implants. dental stress analysis. stress, mechanical. https://orcid.org/0000-0001-6366-5868 2 sabino et al. introduction narrow diameter implants are indicated as a clinical alternative for patients with a limited alveolar ridge or limited space. the osseointegration of commercially pure titanium (cpti) with surrounding bone emphasize its clinical success, however, its mechanical strength can be insufficient when narrow diameter implants (<3.5 mm) are used, since the diameter directly influences the fatigue strength1. one approach to overcome this problem is strengthening the mechanical properties of the titanium by alloying it with other materials, such as zirconium (zr). this strategy increased the elastic modulus, hardness as well as tensile and fatigue strength, maintaining the biocompatibility similar to cpti both in laboratory2,3 and clinical studies4,5. the cpti implants with a narrow diameter have a lower mechanical strength compared with titanium-zirconium (tizr) implants. these characteristics can influence the magnitudes of stress and consequently the outcomes of peri-implant therapy4. the addition of more than 50% zr to tizr alloy increases its resistance by two and a half fold5. zirconium also reduces the melting point of the alloy and associated costs6. another advantage of the addition of zirconium in titanium alloys is an improvement in the corrosion resistance with the formation of a stable oxide layer on the surface of the alloy7,8. in a study investigating the effect of the percentage by weight of zr on the mechanical properties of tizr alloy samples, it was found that the tizr samples had higher micro-hardness values than ti samples at all concentrations of zr9. the success rate of tizr narrow diameter implants has been shown to be similar to that of regular diameter cpti implants10. the tizr alloy has been classified as non-cytotoxic material11. furthermore, tizr alloy has a monophasic α-structure like titanium and it allows performing surface modification using the conventional sandblasting and acid etching procedures12. despite the reported benefits of narrow diameter tizr implants for use in narrow areas, particularly the upper resistance of tizr compared with that of cpti2-5, literature is scarce regarding the evaluation stress distribution patterns of tizr alloy implants in surrounding bone. thus, it was of great interest to understand if the microtextured tizr implant surface would present similar peri-implant stress compared with the cpti. the aim of this study was to assess the peri-implant stress of narrow diameter (3.3 mm) cpti and tizr implants under load (100 n and 200 n) in axial (0°) and oblique direction centralized (at 10°, 20°, and 30°) to the long axis of the implants. the hypothesis was that there will be difference in the number of high-intensity fringes between the type of dental implants and the loads applied. materials and methods two photoelastic models were produced using epoxy resin (pl2, vishay precision group, wendell, nc, usa) from a master model; one with a cpti implant and the other with a tizr implant. the cpti implant (institut straumann ag, peter merian, basel, switzerland) comprised the following: nitrogen 0:05%; carbon 0:08%; hydrogen 3 sabino et al. 0.015%; iron 0.50%; oxygen 0:40%; maximum waste 0.1% each; total maximum waste 0.4%, and ti (comprising the remaining balance). the chemical composition of the tizr implant (institut straumann ag, peter merian, basel, switzerland) was 85% ti and 15% zr. both implants were of the bone level type, 3.3 mm in diameter and 12 mm in height, slactive, and had a narrow connection. healing abutments were positioned on both implants. the circular polariscope (eikonal, são paulo, brazil) was used in the dark field configuration; therefore, the polarizer and analyzer were crossed. the optical axes of the quarter wave plates were also crossed and made an angle of 45° with those of the polarizer and the analyzer. the photoelastic models were observed before each test. the circular polariscope was utilized to verify the absence of residual stress and also for recording stresses (isochromatic fringes) during rehearsals. the polariscope was adapted to a universal test machine (ag-x 50 kn, shimadzu, tokyo, japan) with a 5 kn load cell for implementing centralized and oblique axial compressive load at a crosshead speed of 0.5 mm/min. eight trials were performed with simulated forces (100 n and 200 n) and at a central axial oblique of 0°,10°, 20°, and 30° to the long axis of the implants. stress was identified with the aid of a american society for testing and materials (astm) table which lists the sequence of colors and values for the orders of isochromatic fringes in the photoelastic material observed in a circular polariscope; in a dark field configuration with white light, under progressive increasing load (black=0; gray=0.28; light yellow=0.6; orange=0.79; intense red=0.9; red-blue transition=1.0; intense blue=1.06; blue-green=1.2; green-yellow=1.38; orange=1.62; pink-red=1.81; red-green transition=2.0; green=2.33; green-yellow=2.5; red=2.67; red-green transition=3.0; green=3.1; pink=3.6; pink-green transition=4.0; green=4.13)13. the following peri-implant areas were evaluated: cervical-mesial (cm), cervical-distal (cd), mid-mesial (mm), mid-distal (md) and apical (a). results the results (magnitude of stress) around the cpti and tizr implants (incline = 0°, 10°, 20°, and 30°), which illustrate the stresses resulting from the 100 n and 200 n loads, were based on the number of high-intensity fringes in the stress patterns in the photoelastic models (figures 1 and 2). table 1 shows that for the majority of the areas evaluated, the values of the isochromatic fringe order around the tizr implant were equal to or lower than those observed in case of the cpti implant. the differences in stress between the two implants were small, with the exception of a few regions such as the cm and cd under an axial force of 200 n. in these areas, the tizr implant demonstrated considerably lower stresses (0.45 and 0.45, respectively) compared with the cpti implant (1.2 in both areas). 4 sabino et al. discussion the proposed hypothesis was accepted since both implants increased stress. tizr implant showed the highest values of stresses (fringe orders). moreover, the apical peri-implant area received the greatest stress, except when the inclination of the implant increased to 30º. in this case the highest stresses were observed in the contralateral region of the application of force (cm). ticp 0° tizr 0° ticp 10° tizr 10° ticp 20° tizr 20° ticp 30° tizr 30° figure 2. photoelastic models with load of 200 n. ticp 0° tizr 0° ticp 10° tizr 10° ticp 20° tizr 20° ticp 30° tizr 30° figure 1. photoelastic models with load of 100 n. 5 sabino et al. several factors have been associated with changes in peri-implant stress14. these include bone density15, the bone ridge16, type of material and intermediate prosthetic, occlusal relationship, implant connections16,17 and the length and diameter of the implant18. the implants investigated in this study have characteristics that in favor of the reduction of stress. implants are intraosseous because they have lower stresses than those placed at the gingival level19, and their internal hexagon produces lower stresses due to its geometry and connection stability20. the tizr implant demonstrated superior tensile strength (953 mpa)21 and fatigue strength (230 n)22 compared with the cpti implant (680 mpa21 and 205 n22, respectively). this is because the modulus of elasticity of the tizr implant (100 gpa) is smaller than that of the cpti implant (110 gpa)19. the difference in the modulus of elasticity between the tizr and cpti implants has been attributed to minor stresses in tizr peri-implants observed using finite elements19 and strain gage23. this also supports the results of the present study, which identified lower stresses in the tizr implant through photoelasticity. the alloys used in implants have to combine high mechanical strength with low modulus, and should be located close to the bone at 30 to 40 gpa24 in order to avoid stress shielding and subsequent bone resorption. therefore, the fact that the tizr implant has a lower modulus of elasticity than the cpti implant might also explain the highest quality of bone observed around this implant when compared with the cpti implant25 and the minimal bone resorption within the first two years after implantation26. although the tizr implant has a lower elastic modulus than the cpti implant, it is still very different from that of bone. therefore, the future development of superior alloys requires those with similar characteristics to the alloys in this present study, but with a closer proximity to the bone’s modulus of elasticity. table 1. cpti and tizr peri-implant stresses (fringe orders) with loads of 100 n and 200 n. 100 n  area cpti 0° tizr 0° cpti 10° tizr 10° cpti 20° tizr 20° cpti 30° tizr 30° cm 0.28 0.28 0.60 0.45 0.79 0.45 1.38 1.20 mm 0.79 0.60 0.79 0.60 0.90 0.90 1.00 0.90 a 1.20 1.20 1.38 1.20 1.20 1.20 1.20 1.06 md 0.79 0.60 0.60 0.45 0.45 0.45 0.28 0.28 cd 0.45 0.28 0.45 0.28 0.28 0.28 0.28 0.28 200 n area cpti 0° tizr 0° cpti 10° tizr 10° cpti 20° tizr 20° cpti 30° tizr 30° cm 1.20 0.45 1.38 1.38 1.81 1.81 2.50 2.33 mm 1.38 1.20 1.38 1.38 1.81 1.81 2.33 1.81 a 2.33 2.00 2.33 2.00 2.00 2.00 2.33 1.81 md 1.38 1.20 1.06 0.60 0.60 0.60 0.45 0.45 cd 1.20 0.45 0.79 0.28 0.28 0.28 0.45 0.28 the tizr peri-implant stresses (fringe orders) that are lower than cpti peri-implant stresses are highlighted in bold. 6 sabino et al. qualitative analysis of the stresses indicated differences in stress between peri-implants of the same geometry in the majority of the analyzed areas. in 80% of the cases, at 0° and 10°, the stress was lower in the tizr implant than in the cpti implant, independent of the applied force. these cases exemplify situations similar to the angulation of the teeth in individuals with an average of 9° (mesiodistal) to the upper lateral incisors and 3° slope (buccolingual), according to andrews’ classification. the four lower incisors have an angulation (mesiodistal) average of 2° and a tilt (buccolingual) average of –1°27. the lower stress observed with the use of the tizr implant in this study is favorable because the lower stress in the peri-implant bone reduces the potential for bone resorption and increases the commitment of the short or long-term implant. the implant inclination in these regions should not exceed the specified value, however, in many cases implants are inserted at greater slopes to compensate for differences in bone sagittal maxillomandibular relationships. in the present study, the highest stress levels were recorded in the apical region, except at an angle of 30°. in this case the stress was higher in the cm region. when both implants were oriented at 10°, 20°, and 30°, stresses tended to increase gradually with increasing load, and were concentrated in the contralateral side (cm and mm) of the applied load. however, on the opposite side (cd and md), the application of load resulted in a gradual decrease in the stress under the same conditions. the findings of previous photoelastic analysis studies of peri-implant stresses applying oblique forces are consistent with the present findings and demonstrate that the higher stresses generated are due to oblique loading27. in the previous studies, the slopes varied accordingly: 0° and 10°1, 0° and 20°28, 0° and 30°29, and 0° and 45°27. the masticatory load of each edentulous region under consideration for rehabilitation should be considered when selecting the implant diameter. the bite force in the region of the incisors is 14 to 25 kgf and varies according to gender and age1. the present study utilized forces of 100 n and 200 n applied to the loads and these forces were consistent with those used in previous photoelastic analysis studies1,27-29. there is a few peri-implant stress analysis studies in literature that investigated narrow diameter cpti and tizr implants. while this present study results supports recently published outcomes from in vitro studies, clinical studies are still required to confirm these findings. in conclusion, compared with the cpti implant, the tizr (15% zr) implant is associated with lower stress in the majority of peri-implant regions when subjected to a variety of loads and angles. references 1. allum sr, tomlinson ra, joshi r. the impact of loads on standard diameter, small diameter and mini implants: a comparative laboratory study. clin oral implants res. 2008 jun;19(6):553-9. doi: 10.1111/j.1600-0501.2007.01395.x. 2. taddei eb, henriques var, silva crm, cairo caa. production of new titanium alloy for orthopedic implants. mater sci eng c. 2004 nov;24(5):683-7. 7 sabino et al. 3. li sj, yang r, niinomi m, hao yl, cui yy. formation and growth of calcium phosphate on the surface of oxidized ti-29nb-13ta-4.6zr alloy. biomaterials. 2004 jul; 25(13):2525-32. 4. grandin hm, berner s, dard m. a review of titanium zirconium (tizr) alloys for use in endosseous dental implants. materials. 2012 aug;5(8):1348-60. 5. kobayashi e, matsumoto s, doi h, yoneyama t, hamanaka h. mechanical properties of the binary titanium-zirconium alloys and their potential for biomedical materials. j biomed mater res. 1995 aug;29(8):943-50. 6. ho wf, chen wk, wu sc, hsu hc. structure, mechanical properties and grindability of dental ti-zr alloys. j mater sci mater med. 2008 oct;19(10):3179-86. doi: 10.1007/s10856-008-3454-x. 7. niinomi m. recent research and development in titanium alloys for biomedical applications and healthcare goods. sci technol adv mater. 2003 sep;4(5):445-54. 8. geetha m, singh ak, asokamani r, gogia ak. ti based biomaterials, the ultimate choice for orthopedic implants – a review. prog mater sci. 2009 may;54(3):397-425. 9. correa dr, vicente fb, donato ta, arana-chavez ve, buzalaf ma, grandini cr. the effect of the solute on the structure, selected mechanical properties, and biocompatibility of ti-zr system alloys for dental applications. mater sci eng c mater biol appl. 2014 jan;34:354-9. 10. sohrabi k, mushantat a, esfandiari s, feine j. how successful are small-diameter implants? a literature review. clin oral implants res. 2012 may;23(5):515-25. doi: 10.1111/j.1600-0501.2011.02410.x. 11. park yj, song yh, an jh, song hj, anusavice kj. cytocompatibility of pure metals and experimental binary titanium alloys for implant materials. j dent. 2013 dec;41(12):1251-8. doi: 10.1016/j.jdent.2013.09.003. 12. bernhard n, berner s, de wild m, wieland m. the binary tizr alloy a newly developed ti alloy for use in dental implants. forum implantol. 2009 jan;5:30-9. 13. claro caa, chagas rv, neves ac, silva-concílio lr. comparative photoelastic study of dental and skeletal anchorages in the canine retraction. dental press j orthod. 2014 jan-feb;19(1):100-5. 14. meijer hj, kuiper jh, starmans fj, bosman f. stress distribution around dental implants: influence of superstructure, length of implants, and height of mandible. j prosthet dent. 1992 jul;68(1):96-102. 15. tada s, stegaroiu r, kitamura e, miyakawa o, kusakari h. influence of implant design and bone quality on stress/strain distribution in bone around implants: a 3-dimensional finite element analysis. int j oral maxillofac implants. 2003 may-jun;18(3):357-68. 16. torres em, barbosa ga, bernardes sr, de mattos mda g, ribeiro rf. correlation between vertical misfits and stresses transmitted to implants from metal frameworks. j biomech. 2011 jun 3;44(9):1735-9. doi: 10.1016/j.jbiomech.2011.03.032. 17. goiato mc, sarauza arsufi g, de medeiros ra, pesqueira aa, da silva evf, sonego mv, et al. stress distribution of different implant connections associated with multiple implant-supported prostheses. j med eng technol. 2018 jul;42(5):359-67. doi: 10.1080/03091902.2018.1513575. 18. baggi l, cappelloni i, di girolamo m, maceri f, vairo g. the influence of implant diameter and length on stress distribution of osseointegrated implants related to crestal bone geometry: a three-dimensional finite element analysis. j prosthet dent. 2008 dec;100(6):422-31. doi: 10.1016/s0022-3913(08)60259-0. 19. eser a, tonuk e, akca k, dard mm, cehreli mc. predicting bone remodeling around tissueand bone-level dental implants used in reduced bone width. j biomech. 2013 sep 3;46(13):2250-7. doi: 10.1016/j.jbiomech.2013.06.025. 20. balfour a, o’brien gr. comparative study of antirotational single tooth abutments. j prosthet dent. 1995 jan;73(1):36-43. 8 sabino et al. 21. grandin hm, berner s, dard m. a review of titanium zirconium (tizr) alloys for use in endosseous dental implants. materials (basel). 2012 aug;5(8):1348-60. doi: 10.3390/ma5081348. 22. osman rb, swain mv. a critical review of dental implant materials with an emphasis on titanium versus zirconia. materials (basel). 2015 mar;8(3):932-58. doi: 10.3390/ma8030932. 23. wu ay, hsu jt, huang hl. an in vitro biomechanical evaluation of a new commercial titanium-zirconium alloy dental implant: a pilot study. implant dent. 2014 oct;23(5):534-8. doi: 10.1097/id.0000000000000108. 24. calderon-moreno jm, vasilescu c, drob si, ivanescu s, osiceanu p, drob p, et al. microstructural and mechanical properties, surface and electrochemical characterisation of a new ti–zr–nb alloy for implant applications. j alloys compd. 2014 nov;612:398-410. doi: 10.1016/j.jallcom.2014.05.159. 25. wen b, zhu f, li z, zhang p, lin x, dard m. the osseointegration behavior of titanium– zirconium implants in ovariectomized rabbits. clin oral implants res. 2014 jul;25(7):819-25. doi: 10.1111/clr.12141. 26. barter s, stone p, brägger u. a pilot study to evaluate the success and survival rate of titanium– zirconium implants in partially edentulous patients: results after 24 months of follow-up. clin oral implants res. 2012 jul;23(7):873-81. doi: 10.1111/j.1600-0501.2011.02231.x. 27. pellizzer ep, tonella bp, ferraço r, falcón-antenucci rm, de carvalho ps, alves-rezende mc. photoelastic stress analysis in screwed and cemented implant-supported dentures with external hexagon implants. j craniofac surg. 2010 jul;21(4):1110-3. doi: 10.1097/scs.0b013e3181e1b46e. 28. akça k, çehreli mc. a photoelastic and strain-gauge analysis of interface force transmission of internal-cone implants. int j periodontics restorative dent. 2008 aug;28(4):391-9. 29. sotto-maior bs, senna pm, da silva-neto jp, de arruda nóbilo ma, del bel cury aa. influence of crown-to-implant ratio on stress around single short-wide implants: a photoelastic stress analysis. j prosthodont. 2015 jan;24(1):52-6. doi: 10.1111/jopr.12171. braz j oral sci. 15(1):66-69 original article braz j oral sci. january | march 2016 volume 15, number 1 antimicrobial and bond strength properties of a dental adhesive containing zinc oxide nanoparticles mahshid saffarpour1, marjan rahmani2, mohammadreza tahriri3, amir peymani4 1alborz university of medical sciences, dental school, department of restorative dentistry, karaj, iran 2qazvin university of medical science, department of cosmetic & restorative dentistry, qazvin, iran 3tehran university of medical sciences, school of dentistry, department of dental biomaterials, tehran, iran 4qazvin university of medical science, department of microbiology, qazvin, iran correspondence to: marjan rahmani cosmetic & restorative dentistry department qazvin university of medical science, qazvin, iran phone: 00982633631614 e-mail: mrqazvin1355@gmail.com abstract aim: to assess the effect of adding zinc oxide nanoparticles to dental adhesives on their antimicrobial and bond strength properties. methods: 45 human premolars were cut at the cement enamel junction (cej) and the crowns were sliced into buccal and lingual halves. the specimens were classified into three groups, etched with 37% phosphoric acid for 15 s and rinsed for 30 s. single bond, single bond+5% zinc oxide and single bond+10% zinc oxide were used in the first, second and third groups. a cylinder of z250 composite was bonded and cured for 40 s. for antibacterial testing, 10 samples of each group were assessed by direct contact test; 10 μl of bacterial suspension was transferred into tubes containing adhesives and incubated for one hour; 300 μl of brain heart infusion (bhi) broth was added to each tube and after 12 h, 50 μl of bacteria and broth were spread on blood agar plates and incubated for 24 h. results: the colony count decreased significantly in the second and third groups compared to the first. conclusions: incorporation of zinc oxide nanoparticles into dental adhesives increases their anti-microbial properties without affecting their bond strength. keywords: zinc oxide. nanoparticles. dental cements. anti-infective agents. dental caries. introduction dental caries is a common infectious disease, in which enamel and dentin are demineralized due to the acids produced from the fermentation of carbohydrates by acidogenic bacteria1. at present, carious teeth are commonly restored with tooth-colored restorative materials such as composite resins due to their optimal esthetic properties2. occurrence of secondary caries is the most important reason for replacement of composite restorations3,4. nanotechnology is the manipulation of matter on a molecular scale, which has revolutionized the modern medicine5. zinc oxide has innate antibacterial properties. in the form of nanoparticles, it shows significantly higher antibacterial activity due to increased surface/volume ratio6. studies have shown that zinc oxide can inhibit acid production by streptococcus mutans and lactobacillus in dental plaque7. moreover, it has confirmed antibacterial effects on gram-negative and gram-positive bacteria and is commonly used as an antibacterial agent in dental hygiene products such as toothpastes and mouthwashes8. the purpose of this study was to assess the effect of adding zinc oxide http://dx.doi.org/10.20396/bjos.v15i1.8647127 received for publication: june 22, 2016 accepted: august 18, 2016 67 nanoparticles to single bond dental adhesive on its antibacterial and bond strength properties. material and methods this experimental study was conducted on 45 samples prepared from caries-free extracted human premolars. immediately after tooth extraction, the teeth were rinsed under running water and immersed in 12% formaldehyde for one week. then, they were stored in 0.9% nacl solution. using a high-speed hand piece under air and water spray, the crowns were cut at the cej and each crown was sliced into a buccal and a lingual half. the specimen surfaces were ground flat in order to achieve dentin surface covered with smear layer. the specimens were divided into three groups, etched with 37% phosphoric acid solution for 30 s, rinsed with water jet spray for 30 s and the excess water was removed by a cotton pellet without air spray so that the dentin surfaces remained slightly moist. bonding was performed using single bond (adper single bond plus; 3m espe, mn, usa) in the first group, single bond with 5% zinc oxide nanoparticles in the second group and single bond with 10% zinc oxide nanoparticles in the third group. zinc oxide nanoparticles (pishgaman nano-material co., mashhad, iran) had an average particle size of 20 nm with a crystalline structure. they were almost spherical in shape and had 99% purity. adhesive resins were applied to the teeth surfaces in two consecutive layers and were mildly agitated for 20 s followed by gentle air-drying for 20 s (in order to evaporate the solvent). prior to light curing, a cylindrical tube (0.75 mm internal diameter and 1.0 mm high) was placed on the dentin surface. after 20 s of irradiation using a light curing unit (optilux 501; kerr, orange, ca, usa) with a 300mw/cm2 light intensity, verified by a demetron radiometer (sybron, ca, usa), each tube was filled with a2 shade of z250 composite (3m espe) and cured at all directions for 40 s. the samples were stored in water at 37 °c for 24 h. then, shear bond strength was measured in a universal testing machine (zwick; roell, ulm, germany). the fracture surfaces were evaluated under a stereomicroscope (olympus, japan) in order to determine the mode of failure as adhesive, mixed or cohesive. for antibacterial testing in each group, 10 samples were evaluated by direct contact method; 10 μl of the bacterial suspension was added to the tubes containing adhesives and then the tubes were placed vertically in an incubator and incubated for one hour. then, 300 μl of bhi were added to each tube and after 12 h, 5 μl of the bacterial suspension mixture and bhi broth was spread on a blood agar plate. the plate was placed in an incubator for 24 h. then the number of bacterial colonies was counted and reported as colony forming units per milliliter (cfu/ml). data were analyzed using spss (release 18; spss inc, chicago, il, usa) and anova. a post-hoc test was applied for assessment of inter-group differences. the significance level was set at p<0.05. results table 1 shows the descriptive statistics of bond strength to dentin in the study groups. antimicrobial and bond strength properties of a dental adhesive containing zinc oxide nanoparticles braz j oral sci. 15(1):66-69 according to table 1, the mean bond strength was the highest in the control group (single bond) followed by single bond + 5% zinc oxide. the minimum bond strength was in single bond + 10% zinc oxide. however, the difference among the study groups was not significant (p>0.05). stereomicroscopic assessment for determination of the mode of failure showed that out of 45 studied samples, 84% had adhesive failure and 16% had mixed failure. no cohesive failure was observed among the samples (figure 1). table 2 shows descriptive statistics for microbial count in the study groups. fig.1. adhesive failure (a), mixed failure (b). mean±sd range f, p-value group 1 26.00±3.59 19.70-31.60 f= 0.575, group 2 25.47±2.98 19.50-30.00 p = 0.056 group 3 24.92±2.30 19.20-29.20 table 1 comparison of the mean bond strength to dentin in the study groups (in mpa). according to table 2, the mean colony count was significantly higher in the control group and the minimum colony count occurred in single bond + 5% zinc oxide and single bond + 10% zinc oxide groups. inter-group comparison by the post hoc test showed that the control group had a significant difference to single bond + 5% zinc oxide and single bond + 10% zinc oxide groups (p<0.05). the difference between the two latter groups was not significant (p>0.05). 68antimicrobial and bond strength properties of a dental adhesive containing zinc oxide nanoparticles braz j oral sci. 15(1):66-69 mean±sd minimum maximum f, p-value group 1 131.65 ± 1020×102 800×102 1200×102 f=568.083, group 2 6.68 ± 33.5×102 25×102 45×102 p<0.001 group 3 4.95 ±18.1×102 12×102 25×102 table 2 comparison of the microbial count among the study groups (cfu/ml). discussion considering the extensive use of dental composites due to their excellent esthetic properties and also to the high prevalence of secondary caries as the main reason for failure of composite restorations, attempts are made to confer antibacterial properties to these composites9. because of the suitable antibacterial property of zinc oxide, we assessed the effect of adding zinc oxide nanoparticles in two different concentrations to single bond adhesive on streptococcus mutans count (the main etiologic factor for dental caries) and bond strength to dentin. for antibacterial testing, the agar diffusion test is the most frequently used method for testing the antibacterial activity of dental materials10. however, the mechanism of action of this method is based on water-soluble components released from mass substances. thus, it is not feasible for evaluating the antibacterial efficacy of less water-soluble substances such as zinc oxide11. direct contact test is commonly used for assessment of antibacterial efficacy of solids in soluble substances. in this method, the bacteria are directly exposed to the substance in a controlled fashion and then their growth and proliferation are evaluated after certain time intervals12. in our study, this test showed that zinc oxide nanoparticles conferred antibacterial properties to single bond, and that increasing concentration of zinc oxide nanoparticles increased the antibacterial activity of the mixture (table 2). tavassoli et al.11 assessed physical, mechanical and antibacterial properties of flowable composites containing zinc oxide nanoparticles and showed that zinc oxide conferred antibacterial properties to resin and increased concentration of zinc oxide nanoparticles significantly decreased bacterial growth and proliferation, which concurs with the results of the present study. zinc oxide nanoparticles have been proven to provide significant antibacterial properties to resin13,14, as observed in the present study. niu et al.13 found that turbidity test was used to assess the antibacterial properties. but it has a limitation, as it examines both living and dead bacteria in liquid conditions. instead, in the our study, a colony counting method was used in which only the living bacteria are counted13. spencer et al.15 showed that adding zinc oxide to a light-cured resin-modified glass ionomer orthodontic bonding agent conferred antimicrobial properties to it without significantly altering the shear bond strength. they also demonstrated that increasing the concentration of zinc oxide, its antimicrobial properties increased and these results were in line with our findings15. moreover, kasraei et al.6 showed that the effects of zinc oxide on streptococcus mutans were significantly greater than those of silver. in terms of bond strength in our study, the mean bond strength was highest in the single bond group followed by the single bond + 5% zinc oxide and single bond + 10% zinc oxide, respectively (table 1). osorio et al.8 indicated that degeneration decreased collagen in demineralized dentin and the effect of zinc oxide remained for three weeks as opposed to that of chlorhexidine, which was short-term. larsen et al.16 showed that zinc, forming zinc mono hydroxide, links catalytic ions to the lateral chain in the active side of carboxy peptidase a and inhibits it. they showed that zinc not only has a catalytic role for enzymes, but it also plays a role in protein stability. zinc acts as a matrix metalloproteinase (mmp) competitive inhibitor, a protective effect by bonding to some regions of collagen that are sensitive to cleavage by metal proteinase matrix16. osorio et al.17, demonstrated that zinc decreased collagen degeneration in single bond hybrid layer and had no adverse effect on the bond strength; their results were in accordance with ours. in their study, single bond containing zinc decreased c-terminal telopeptide of type-i collagen concentration (collagen degeneration product) at one and four weeks and the best result was obtained when zinc oxide nanoparticles were added to the bonding agent17. etch and rinse adhesive systems are still the gold standard for bonding to enamel and if bonding by etch and rinse adhesive containing zinc decreases dentin collagen degeneration, a strong bond to dentin can be achieved18. in another study8, bond strength in single bond and single bond + 10% zinc oxide was higher than the value obtained in the current study after 24 h (37.5 and 36.26 mpa versus 26 and 24.92 mpa, respectively). this difference in results may be ascribed to the fact that they measured micro-tensile bond strength while we measured micro-shear bond strength. micro-tensile bond strength level is often higher than the micro-shear bond strength19. in our study, similar to a previous research11, the immediate bond strength of single bond plus zinc oxide was lower than that of single bond alone. this reduction may be attributed to the agglomeration phenomenon or to the increase in the opacity of composite and incomplete curing11. as mentioned earlier, the difference in bond strength among the three groups was not significant and all values were within or higher than the acceptable range of bond strength to dentin (20 mpa). dental restorative materials must be able to provide a strong and durable bond to tooth structure20,21. based on studies on previous investigations17,20, zinc provides a durable and strong bond at the resin/dentin interface by decreasing collagen degeneration. the results of the current study showed that adding zinc oxide nanoparticles to single bond inhibited the formation of streptococcus mutans colonies without significantly compromising the bond strength. since 10% zinc oxide had a greater anti-bacterial effect than 5% zinc oxide and considering the insignificant difference in immediate bond strength of these two groups, use of single bond + 10% zinc oxide is recommended. 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containing nanoparticles of silver and amorphous calcium phosphate. dent mater. 2013 feb;29(2):199-210. doi: 10.1016/j. dental.2012.10.005. 10. hendriksen rs, editor. global salm-surv. a global salmonella surveillance and laboratory support project of the world health organization. laboratory protocols. level 2 training course. mic susceptibility testing of salmonella and campylobacter. 4th ed. 2003. 11. hojati st, alaghemand h, hamze f, babaki fa, rajab-nia r, rezvani mb, et al. antibacterial, physical and mechanical properties of flowable resin composites containing zinc oxide nanoparticles. dent mater. 2013 may;29(5):495-505. doi: 10.1016/j.dental.2013.03.011. 12. osorio r, cabello i, toledano m. bioactivity of zinc-doped dental adhesives. j dent. 2014 apr;42(4):403-12. doi: 10.1016/j.jdent.2013.12.006. 13. niu l, fang m, jiao k, tang l, xiao y, shen l, et al. tetrapod-like zinc oxide whisker enhancement of resin composite. j dent res. 2010 jul;89(7):746-50. doi: 10.1177/0022034510366682. 14. aydin sevinç b, hanley l. antibacterial activity of dental composites containing zinc oxide nanoparticles. j biomed mater res b appl biomater. 2010 jul;94(1):22-31. doi: 10.1002/jbm.b.31620. 15. spencer cg, campbell pm, buschang ph, cai j, honeyman al. antimicrobial effects of zinc oxide in an orthodontic bonding agent. angle orthod. 2009 mar;79(2):317-22. doi: 10.2319/011408-19.1. 16. larsen ks, auld ds. characterization of an inhibitory metal binding site in carboxypeptidase a. biochemistry. 1991 mar 12;30(10):2613-8. 17. osorio r, yamauti m, osorio e, román js, toledano m. zinc‐doped dentin adhesive for collagen protection at the hybrid layer. eur j oral sci. 2011 oct;119(5):401-10. doi: 10.1111/j.1600-0722.2011.00853.x. 18. spero jm, devito b, theodore l. regulatory chemicals handbook. boca raton: crc press; 2000. 19. powers jm, sakaguchi rl. craig’s restorative dental materials. saint louis: mosby elsevier; 2012. 20. toledano m, yamauti m, ruiz-requena me, osorio r. a zno-doped adhesive reduced collagen degradation favouring dentine remineralization. j dent. 2012 sep;40(9):756-65. doi: 10.1016/j.jdent.2012.05.007. 21. p h a e c h a m u d t, m a h a d l e k j , a r o o n r e r k n , c h o o p u n c , charoenteeraboon j. antimicrobial activity of zno-doxycycline hyclate thermosensitive gel. scienceasia. 2012;38(1):64-74. doi: 10.2306/ scienceasia1513-1874.2012.38.064. antimicrobial and bond strength properties of a dental adhesive containing zinc oxide nanoparticles influence of different surface treatments of fiberglass posts on the bond strength to dentin bruna cristina do nascimento rechia1, ruth peggy bravo1, naylin danyele de oliveira1, flares baratto filho1, carla castiglia gonzaga1, carmen l. mueller storrer1 1universidade positivo – up, school of dentistry, department of operative dentistry, curitiba, pr, brazil correspondence to: carmen l. mueller storrer universidade positivo – programa de pósgraduação em odontologia rua. prof. pedro viriato parigot de souza, 5300. campo comprido – curitiba – pr – br. cep: 81280-330 phone: +55 41 3317-3454 fax: +55 41 3317-3000 e-mail: carmen.storrer@gmail.com abstract aim: to assess the influence of different fiberglass post surface treatments on the bond strength (bs) to root dentin. methods: thirty bovine root canals were endodontically treated and filled with gutta-percha and ah plus sealer. at 24 h after the endodontic filling, the post spaces were prepared with gates-glidden drills and #3 drills of the dc white post system, maintaining a 4 mm apical seal. the roots were randomly divided into three groups: s (fiberglass posts treated with silane), sa (fiberglass posts treated with silane and a hydrophobic adhesive system) and sha (fiberglass posts treated with silane, followed by drying with hot air). the posts were cemented with a selfadhesive cement (relyx u200). the specimens were stored in distilled water at 37 °c for 24 h and subjected to the push-out test (0.5 mm/min). data were statistically analyzed using one-way anova and tukey's test (α=0.05) results: sa and sha groups showed the highest bs mean values (11.29 and 10.85 mpa, respectively), while the s group presented the lowest bs mean value (7.21 mpa). s group was significantly different from sa and sah groups. conclusions: the surface treatment of fiberglass posts influenced bs values. keywords: dentin-bonding agents. dentin. denture retention. introduction the use of intraradicular posts for improving retention of coronal restoration and enabling coronary reconstruction is guided by the amount of remaining tooth structure after removal of carious tissue and endodontic treatment1-3. posts also help supporting fixed partial dentures4, where they dissipate and absorb forces during mastication, in a way that avoids damage to the root and the cementing film. fiberglass posts have mechanical, clinical and esthetic advantages, including improved corrosion resistance, biocompatibility, easy removal for endodontic retreatment, rapid insertability and an elastic modulus close to that of dentin; thus, these posts provide a better distribution of occlusal stresses5. they can be cemented to the root dentin, which makes the stress on the root more uniform, resulting in fewer root fractures or at least more favorable fractures6. failure in fiber-reinforced posts results mainly from problems in the luting procedures2,7. cementation of the posts has two functions: retention and stress distribution. stress distribution is important for preventing fractures due to the potential functional stress transmission of load to the dentin via cement, which becomes a cushion against forces8. received for publication: july 20, 2016 accepted: november 16, 2016 braz j oral sci. 15(2):158-162 original article braz j oral sci. april | june 2016 volume 15, number 2 http://dx.doi.org/10.20396/bjos.v15i2.8648768 159 a disadvantage of fiber-reinforced posts is that they cannot achieve intimate adaptation in non-circular, wide or extremely tapered root canals, which may compromise their retention in comparison to customized posts9. in an attempt to provide better retention of fiberglass posts, various surface treatment techniques have been suggested, such as cleaning the post surface with alcohol, conditioning with phosphoric or hydrofluoric acid, sandblasting with aluminum oxide, silicatization, or applying hydrogen peroxide, silane or a hydrophobic adhesive (unfilled resin) on the surface5,10-12. among the various surface treatments, silanization is one of the most widely used and has the advantage of being a simple procedure that can be performed by the dentist immediately prior to cementing the post, but its effect on the bond strength of the fiberglass post is inconclusive. some studies have shown that the bond strength between the post and the resin cement may be improved by silanizing the post before cementation13,14, while other studies reported that the use of silane alone did not increase bond strength15,16. silanization may be considered a very sensitive technique. the bond strength of resin cement to the fiberglass posts may be affected by different silane compositions and by the air-drying temperature17. solvent evaporation also plays an important role. different forms of solvent evaporation or unreacted excess silane may influence the bond strength of the fiberglass post. for feldspathic ceramics and glass ceramics, the use of a hot air jet was reported to help in solvent evaporation and reactions on silanized surfaces, improving bond strength18-21. however, there is yet no established protocol for the heat treatment of silane in the literature and results using this approach are controversial22. the aim of this study was to assess the influence of different surface treatments of fiberglass post on the push-out bond strength to root dentin. material and methods thirty freshly extracted bovine incisors from healthy cattle were obtained and kept under refrigeration (4oc) in distilled water changed weekly until the moment of use of the teeth. the crowns were sectioned perpendicular to the long axis of the tooth, below the level of the cemento‒enamel junction, using a diamond disc attached to a precision sectioning saw (isomet 1000, buehler, lake bluff, il, usa), under constant refrigeration, in order to create standardized access to the root canal and obtain 14-mm-long root portions. a single operator performed all root canal fillings. the working length was determined visually at 1 mm short of the root apex. rotary instrumentation was performed with protaper universal files (dentsply maillefer, ballaigues, switzerland) in the sx-s1-s2-f1-f2-f3 sequence, with 2.5 ml of 2.5% sodium hypochlorite irrigation during the preparation. the final irrigation was done with 17% edta for 3 min, followed irrigation with 2.5 ml 2.5% sodium hypochlorite and drying with tissue paper points (dentsply maillefer). the roots were filled with ah plus sealer (denstply detreykonstanz, germany) and gutta-percha according to the tagger's hybrid technique. twenty-four hours after root canal filling, post spaces were prepared using gates glidden drills #1, 2 and 3, and the #3 drill of the dc white post (fgm, joinville, sc, brazil) system, maintaining a 4 mm apical seal. the prepared post spaces were then irrigated with 2.5 ml 0.9% nacl solution and dried with paper points (dentsply maillefer). after preparation, the roots were randomly divided into the following three groups (n=10), according to the surface treatment of the fiberglass posts (#3, dc white post, fgm): group s (fiberglass posts treated with silane [ceramic primer, 3m espe, st paul, mn, usa]), group sa (fiberglass posts treated with silane and a hydrophobic adhesive system [adper scotchbond multi-purpose; 3m espe]) and group sha (fiberglass posts treated with silane, followed by drying with hot air [50oc for 60 s]). all posts were cleaned with 37% phosphoric acid for 15 s (scotchbond etchant, 3m espe), washed and dried with air jet and then the surface treatments were performed. in s group, the silane was applied with a microbrush for 60 s; in sa group, after silane treatment, a hydrophobic adhesive layer was also applied with a microbrush, followed by photoactivation for 10 s; in sha group, silane was applied followed by hot air drying for 60 s at 50 oc. the source of hot air was a hair dryer. the posts were cemented with a self-adhesive resin cement (relyx u200; 3m espe), with manual pressure, and excess cement was immediately removed. the cement was light-cured for 40 s, with a led curing unit at 1.100 mw/cm2 (poly wireless, kavo, joinville, sc, brazil). the specimens were stored in distilled water for 24 h at 37 °c, and were then subjected to the push-out bond strength test. two 1-mm thick slices of each root third of the specimens (coronal, medial and apical) were obtained using a diamond disc mounted on a precision sectioning saw (isomet 1000; buehler), under constant water cooling. the coronal side of each slice was identified, and each slice was subjected to a push-out bond strength test using a universal testing machine (emic dl 2000, são josé dos pinhais, pr, brazil) at a crosshead speed of 0.5 mm/ min. the load was applied in the apical-coronal direction until the dislodgment of the post. for all tests, the push-out pin on the center of the post surface was carefully placed and different sizes of punch pins were used to match the diameter of the post at the tested different root thirds. bond strength (mpa) was obtained by dividing the maximum force in n by the area of the bonded interface. the area of the truncated cone of each specimen was calculated by the equation: influence of different surface treatments of fiberglass posts on the bond strength to dentin al = π(r+r)√ h 2 + (r-r)2 where al is the lateral area of a truncated cone, π=3.14, r is the coronal post radius, r is the apical post radius, and h is the thickness of the root slice. the dimensions of the specimens were obtained with a digital caliper with an accuracy of 0.01 mm. f r a c t u r e s u r f a c e s w e r e a n a l y z e d u s i n g a 5 7 × stereomicroscope (szx9, olympus, tokyo, japan) to determine the failure mode. failures were classified as adhesive (between braz j oral sci. 15(2):158-162 160 post and cement or between cement and dentin), cohesive (on post or cement) or mixed. the mean bond strength of each specimen (root) was determined by the bond strength mean value obtained by the six slices, two of each root third. the results of the bond strength values were statistically analyzed using one-way anova and tukey’s test with a significance level of 5%. results table 1 shows the mean bond strength values and standard deviations of the evaluated groups as determined by the push-out test (in mpa). statistically significant differences were found in the bond strength values, according to the surface treatments performed on the fiberglass posts (p=0.038). sa and sha groups showed the highest bond strength values (11.29 and 10.85 mpa, respectively); these values were statistically similar to each other (p=0.961). s group presented the lowest bond strength values (7.21 mpa), and was statistically different from the sa (p=0.050) and sha (p=0.088) groups. discussion to mimic the restoration of endodontically treated teeth in this study were used fiberglass posts, which possess physical and mechanical properties similar to those of dentin. these posts are more aesthetic, can be rapidly inserted, have lower risk of radicular fractures, do not corrode and may be easily removed, allowing the operator more treatment options with greater efficiency and effectiveness. according to lamichhane et al.23, these posts outperform other materials and systems, and are the best choice for the treatment of teeth with severe damage. proper bonding of the post to the tooth structure is also essential to a successful outcome of treatment2,24-30. the results of the bond strength values found in this study agreed with the results described in the literature, and indicated that the use of hot air after application of silane may improve the strength of bonding of fiberglass posts to dentin31. about the use of silane and adhesive as a post-surface treatment, some studies have shown that there are no statistically significant differences in bond strength values using only silane or silane followed by adhesive, but the bond strength values are lower in the absence of silane32. goracci et al.33 evaluated the adhesion between different types of fiber posts and composites after silanization. the bond strength values increased after the application of silane. silane does not form a suitable bond with an epoxy resin matrix, resulting in poor bonding strength between fiber retainers and resin cement. thus, before silane application, the surface of the posts should be treated to expose glass fibers. in this study, the use of an additional adhesive layer after the silane treatment improved the strength of fiberglass post bonding to the root dentin, providing similar bond strength values to those of the group where the silane was hot air dried . thus, both methods used in the sa and sha groups may be used to achieve more effective bonding of cemented fiberglass posts. another possible explanation for the use of an additional adhesive layer after the silane application is the fact that this material presents mechanical interlocking and chemical reaction with fiberglass posts, improving bond strength. when silane is used alone, it can form a non-homogeneous layer on the post surface. when used in combination with an adhesive layer, both materials can infiltrate the post surface and reinforce the bond with resin cement34. the use of heat-treated silane on the post surfaces is less common than its use to treat feldpsathic or glass ceramics before cementation.35,36 the rationale for using hot air jets after silane application relies on the fact that it eliminates alcohol, water and other by-products of the reaction of silane with the substrate37 of alcohol evaporation may increase the local density of available connections38. in addition, after heat-treating the silane, higher bond strength values may be achieved due to the removal of the upper layers of silane, leaving a more stable silane layer at the bonding interface37. failure mode analysis showed that, in the three groups, mixed failure was predominant, indicating that there was a good interaction of the resin cement with the post and root dentin after the different surface treatments; thus, the adhesion process was effective31,39-42. previous studies have reported that clinically influence of different surface treatments of fiberglass posts on the bond strength to dentin table 1 means and standard deviations of bond strength values as determined using the push-out extrusion test. groups post treatment bond strength (mpa) s silane 7.21±2.69 b sa silane and adhesive 11.29±4.27 a sha silane and drying (hot air) 10.85±3.35 a values followed by the same superscript are statistically similar (p>0.05). fig.1. failure modes observed after push-out bond strength tests for the different groups. for all groups, mixed failures were the most frequent failure type (adhesive failures between the dentin and cement and cohesive failures in the cement (figure 1). braz j oral sci. 15(2):158-162 161 influence of different surface treatments of fiberglass posts on the bond strength to dentin the most frequent failures are adhesive ones, which leads to the displacement of the post2,13,17,43-46. as regards post retention in the root dentin, combined failures are considered favorable and even desirable when compared with adhesive failures, since it indicates that the adhesion of the retainer to the dentin was adequate. it may be concluded that the surface treatment of fiberglass posts influenced bond strength 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the adhesion of fiber post. j adv prosthodont 2014 aug;6(4):295-301. doi: 10.4047/jap.2014.6.4.295. 46. yilmaz sk, cengiz e, ozan o, ramoglu s, yilmaz hg. the effect of er,cr:ysgg laser application on the micropush-out bond strength of fiber posts to resin core material. photomed laser surg 2014 oct;32(10):574-81. doi: 10.1089/pho.2014.3780. 162influence of different surface treatments of fiberglass posts on the bond strength to dentin braz j oral sci. 15(2):158-162 1 volume 16 2017 e17064 original article 1 universidade estadual do piauí – uespi, school of dentistry, department of clinical dentistry, area of integrated clinic, parnaíba, pi, brazil corresponding author: ana de lourdes sá de lira universidade estadual do piauí, faculdade de odontologia rua senador joaquim pires 2076 ininga. fone (86) 999595004 cep: 64049-590 teresina-pi-brasil email: anadelourdessl@hotmail.com received: june 18, 2017 accepted: july 12, 2017 dental caries in children on deciduous dentition: art and socioeconomic aspect ana de lourdes sá de lira1*, sabrynna gonçalves candeira portela1 aim: determine the prevalence and incidence of dental caries in children´s deciduous teeth, who were treated with art technique. methods: in t1, 62 children were examined for being between 3 and 6 years of age. a questionnaire was applied to the parents or guardians of the children involved in the research, to analyze their knowledge about eating habits and oral hygiene. an intra-oral examination of children from 3 to 6 years old was carried out and dental treatment was performed by the art technique. six months after,(t2), the clinical examination was redone to investigate the stability of restorations. results: in t1, 94 caries cavities were found in 19 children and 86 were recommended for restoration with art. in t2, 58 restorations were damaged or lost and 15 children had 20 new caries cavities. conclusion: the prevalence of dental caries in the deciduous dentition was 38.8% and the incidence was 31.9%. in t2, the majority restorations class i and ii were damaged or lost. art technique is an option, despite the high failure in art, especially for child population with deciduous teeth that has limited access to dental services. however, restorations should be supervised and healthy eating habits implemented. keywords: pediatric dentistry. dental caries. oral hygiene. http://dx.doi.org/10.20396/bjos.v16i0.8650502 2 lira et al. introduction the prevalence of caries has declined, but it is more significant in the most favored populations, which have a lower cpo-d and a higher number of children without caries1-3. maintaining good oral hygiene is essential so that the individual does not acquire caries disease. this is justified by the dental biofilm, which is the determining factor for the disease to occur4,5. but also, to reduce this situation, it is important to take greater care with the children through health promotion measures and prevention6-7. the world health organization (who) has stated that health is also related to schooling, so it is clear that increasing school performance leads to improvements in the health of individuals8. the definition of early caries can be determined by the presence of decayed teeth (even those without cavity), dental elements lost due to caries or restorations, in individuals with a maximum age of 71 months. in addition, any carious surface is defined as severe precarious caries in children under three years of age9. seeking to change reality, promotion measures should be instituted together with other collective health actions, such as preventive, curative, health education and control10. one option for caries disease control is atraumatic restorative treatment (art), which was devised in the 1980s by dr. jo e. frencken. this method is of minimal intervention that aims to paralyze the carious process and provide favorable conditions to restore oral health11. in accordance with the current objectives of dentistry (prevention, promotion, early and conservative intervention in the carious lesions), the art technique should not be considered only an alternative restorative technique, but as a promotion strategy of oral health that aims to take the dental treatment to the non-assisted populations, adapting itself to the reality of them. therefore, this treatment associates educational actions and preventive health care for the population, as well as curative actions through the removal of carious tissue with instruments, and restoration of the cavity12. it is believed that early dental caries, in the deciduous denture is a public health problem with high prevalence and incidence rates, and that few children have easy access to public dental care. this type of treatment also seeks to respect one of the main current concepts of dental practice, which is to create a favorable environment that halts the caries disease process by means of minimum intervention and maximum preservation of dental structures. in addition to the curative aspect of cavity preparations and restorations, there are the added advantages of concern focused on health promotion, education and patient motivation. so it was decided to carry out this research to determine the prevalence and incidence of dental caries in children in the deciduous denture, who were treated during this research with art technique. material and methods this study was approved by the research ethics committee of the state university of piauí cep / uespi, under caae number: 51011015.4.0000.5209. the principle of ethics was based on the norms contained in resolution no. 466/12 (cns/ms). the researcher was provided with the consent term for the directors of the school 3 lira et al. units, authorizing the development of the research after ethical appreciation of the research ethics committee of the state university of piauí – cep/ uespi. inclusion criteria were children with caries in the deciduous dentition, in the age range of 3 to 6 years, whose parents or guardians accepted the research. this age group of research was chosen due to the greater probability of the children being with the deciduous complete dentition and attending the preschool. children less than 3 years of age or older than 6 years of age or who were already in the mixed dentition were excluded from the study because the focus was on the deciduous dentition and those that the parents or guardians did not authorize to participate in the study. according to the sample calculation, only 45 children should be examined, but it was decided that all 62 children would be examined for being between 3 and 6 years of age. however, 13 were excluded because they were in the mixed dentition. preventive activities of supervised brushing and topical application of fluoride were performed for children who were excluded from the study, since no oral pathology was present. the individuals involved were oriented on the objectives of the research and their methods through the informed consent term (tcle) and the informed consent term (tale), signed by parents / guardians and children, respectively. prior to the execution of the research, the questionnaire was applied to 10 parents, after validation, having been previously applied to 10 parents, and the clinical examination was carried out in 10 children, aged between 3 and 6 years, in the deciduous dentition, attended at the pediatric dentistry clinic of the dental uespi, in parnaíba, chosen randomly for calibration of the researcher student. at the first moment of the research (t1) a lecture was given to the 49 parents or guardians for awareness and instruction according to the research objectives. the questionnaire was applied. the researcher performed the intra-oral examination of the sample, children enrolled in the preschools and the municipal school of early childhood education tio zeca, in search of caries lesions with cavity, the majority (n = 26) between 5 and 6 years of age. the individual examination was carried out in the schoolyard, under natural light, using wooden spatula and mouth mirror. when present, the teeth and faces were recorded on the patient´s record cards. teeth with evident pulp involvement or painful symptomatology and / or presence of edema or fistula were excluded from the study and immediately referred to the clinical school of dentistry of uespi for endodontic and restorative treatment. the prevalence (t1) and later the incidence of caries (t2) in the sample studied were determined. subsequently dental treatment was performed by the art technique, in the school environment, under natural light. vitro molar (nova dfl, rio de janeiro, brazil) was used as the glass ionomer cement (civ), because it is one of the materials of choice for this technique, because it has high viscosity, better strength and good mechanical properties13. the technique of art11 consisted in detail in: a. cleaning of the dental surface with friction with a cotton dressing; b. access to caries lesion, if there is enamel without support, with axes, hoe or gingival margin trimmer; 4 lira et al. c. removal of carious dentin with sharp curettes, starting from the edges of the cavity, maintaining the dentin that leaves in flakes or scales in the portion closest to the pulp (affected dentin); d. cleaning the cavity with the liquid of the restorative material, with cotton dressing for 15 seconds on the surface to remove the dentin sludge; e. washing with cotton swabs moistened with water (minimum 3); f. drying with dry cotton; g. dosing and manipulation of the civ for 30 seconds according to the manufacturer’s recommendation; h. insertion of the restorative material into the cavity, when it was in the consistency of shiny wire, with insertion spatula, in increments. leaving with slight excess; i. solid vaseline was pressed for 30 seconds in the surface of the restoration; j. use of hollemback spatula or sharp curette to remove excess; k. protection of the surface of the restoration with solid petroleum jelly; l. use of carbon paper for checking the occlusion; m. removal of premature contacts with hollemback or sharp curette. caries lesions included in the art intervention, according to the nomenclature of black13, comprised those that were in teeth: 1. later class i and ii; 2. previous class iii; 3. with loss of cusps; 4. posterior and anterior class v after 6 months (t2), 47 children were again examined to see if there was improvement in oral health, two of them no longer studied in one of the schools. the evaluation criteria for the control of oral health status were based on ferreira et al.11. as explicit in table 1, in which 0 was considered as indicative of successful treatment and 1 to 3 to indicate intervention failure. the data were tabulated and analyzed by the spss statistical program, version 21. the chi-square association test was performed to verify if there were significant differences in the results found. descriptive statistics (measures of central tendency and dispersion) and construction of graphs were used in order to illustrate the findings of this research. table 1. criteria for evaluation of restorations with art11 satisfactory art 0 present, minor defects in the margin and / or wear of 0.5mm depth 1 partial loss of restorative material 2 total loss of restorative material 3 absent or loss of tooth by exfoliation x 5 lira et al. results the parents were on average 30 years and 10 months old, the majority were single (n = 25). in terms of schooling, there were similar numbers who declared that they only had incomplete secondary education (n = 16), and those with up to the 4th grade (n = 16). the family income of 1 to 2 minimum wages was informed by 39 parents. at the first moment, when asked if they had already participated in any lecture on oral health, 34 answered yes. upon receiving oral hygiene instructions from the children, 37 reported having received some guidance. of these, 23 were passed on by dentists and others by other professionals, relatives and / or friends. in relation to the habit of doing the oral hygiene of the children, the majority answered yes (n = 42), being executed 3 times a day, according to 26 of these. in addition, he was asked if his children used to sleep without doing oral hygiene. of this, 33 answered to do it and 18 reported not doing the hygiene of the child before bed. information about the children’s diet, milk consumption and sucking habit were collected. in this way, 41 children do not use sugary pacifiers, most have healthy meals, but they included sugary foods in the snack, in addition to the intake of artificial milk. after the lectures, parents acknowledged that oral hygiene and healthy eating may decrease the chances of developing caries disease. in the first intervention period (t1) by art, 19 children out of 49 in the sample had caries lesions, 9 of which were female and 10 were male. the prevalence of dental caries in the deciduous dentition was 38.8%. in these 19 children, 94 decayed teeth were diagnosed, of which 86 were restored (8 teeth presented great destruction by caries, with indication of exodontia, and were later performed in the uespi school of dentistry clinic), corresponding to 42 class i restorations, 22 of class ii, 17 of class v and 5 of class iii. according to figures 1 and 2 it was observed in t1 that the second deciduous molars were the ones that presented the highest number of carious lesions. t1 11 8 2 11 13 13 11 2 13 17 t2 central incisor lateral incisor canine first molar second molar 18 16 14 12 10 8 6 4 2 0 figure 1. upper deciduous teeth with carious lesions. parnaíba-pi. 2016. 6 lira et al. twenty new cavity lesions were observed in 15 of these 17 children, being 11 of class i and 9 of class ii, corresponding to incidence of dental caries of 31.9%. among these 15 preschoolers, it was found that in 12 of them only a new carious lesion was found, but 2 presented with two lesions and one with 4. according to figure 3, it was verified that 58 of the 86 restorations performed on t1 had failed after 6 months (t2). of the 58 compromised restorations, there were partial loss of 6 class i and 2 class ii restorations, and total loss of 15 class ii and 23 class i restorations. all 17 class iii and class iii restorations performed remained satisfactory. five of class ii and 7 of class i presented defects of 0.5mm. the failures in art restorations were highly significant when the chi-square test = 27.3002; gl = 3; with p <0.001. therefore, if other dental restorations have been made under the same conditions, teeth are expected to be divided equally into each of the scores adopted in the survey. the number of teeth in each score did not happen at random. t1 222 20 23 222 25 27 t2 central incisor lateral incisor canine first molar second molar 30 25 20 15 10 5 0 figure 2. lower deciduous teeth with caries lesions. parnaíba-pi. 2016. figure 3. evaluation criteria of art control. parnaíba-pi. 2016. 8 12 20 38 8 satisfactory art partial loss present, with defects of 0.5 mm total loss absent tooth 20 40 0 7 lira et al. discussion in this study it was observed that families had low monthly schooling and income. these data corroborate with those found in another study, 15 in which a family income of up to two minimum wages predominated (98.3%), and 76.6% of the parents / guardians declared having only complete elementary education. it is essential to make parents or caregivers aware of the importance of oral health of children with guidelines on how to maintain it, including oral hygiene before bed, dental care before 12 months of age to limit or prevent cases of early decay, as well as the integrated assistance of health professionals: surgeon-dentist and pediatrician13,14. in the study carried out by robles et al.15, the mothers reported having knowledge about the importance of brushing after meals, the relationship between the high carbohydrate diet and the development of caries and its consequences. the guidelines received from pediatricians, nurses and other mothers diverged from this research, in which it was found that the dentist was the one most responsible for disseminating these instructions. the children had a diet based on healthy foods, the parents did the hygiene of the children, mostly three times a day. but despite this, there were a large number of children affected by early caries, probably because they used sugar in the snack, both in t1 and t2. according to almeida et al.16, it was not possible to notice a direct and expressive relationship between the daily frequency in the consumption of sweet foods with the caries index. in the same way, it could not be said that the experience of caries and the number of toothbrushes are inversely proportional. in the study by ribeiro et al.17 224 children were part of a control for four years, and it was found that brushing was not an expressive factor for individuals not to have caries, since 40.4% of children who brush their teeth and more than half (53.4%) of those who did not brush had caries. early caries brings physical and psychological consequences, since it interferes in the quality of life18. this can be explained by the progression of the disease, as abscesses may appear, damage of permanent teeth, painful symptomatology, feeding, swallowing, breathing damages19. initially, it manifests itself through white and opaque patches, but with progress, the clinical aspects become cavities. as it is acute, if there is no intervention, there will be total loss of the dental crown. the most affected elements are the upper and lower anterosuperior and molars. anteroposterior teeth are included in more advanced cases of caries disease20. the second deciduous molars were the teeth most affected by caries (figures 1 and 2). these findings are in agreement with rigo et al.21 when affirming that dental elements 55, 65, 75 and 85 presented the highest caries in 56%, 55%, 46% and 45% of the cases, respectively. as an alternative treatment in situations of low socioeconomic level and high occurrence of caries, art has good results because it includes, among others, less invasive care in established caries lesions and oral health guidelines and it is well accepted by children22. according to data collected 6 months after the intervention with art, there was a high number of total loss of the restorations (figure 3). a similar result was found by pazuch et al.23 in which more than half of the restorations (62.5%) performed 8 lira et al. needed repair after 6 months. these results occurred with civ, which is the material of choice in art practice, due to its properties, such as: fluoride release and physicochemical adhesion. it is believed that the high failure rate in treatment and the increase in the number of carious lesions may have been due to the absence of cavity preparation, such as removal of carious tissue by means of drills, associated with insufficient relative isolation to avoid contact of the civ with fluids and inadequate conditioning of the children. sales et al.24 also justified their failure to condition and control the child during care. the incidence of caries in t2 occurred in children whose parents reported that they did not brush their teeth before bed, although most did so after meals. probably the consumption of sweets in the intervals of meals, even at low frequency, associated with the absence of brushing at bedtime have favored the appearance of new caries. failure to remove bacterial biofilm and low salivation during sleep predispose to the appearance of carious lesion25,26. a greater number of brushing provides a better chance of avoiding the development of the carious process27. there are factors that favor the installation of early caries, such as: sucrose-rich diet, cariogenic microbiota, oral hygiene efficiency and the time the food stagnates on the teeth28. the restorations that failed after 6 months were those of class i and class ii, corroborating with the findings of other authors25-28, who verified that occlusal-proximal restorations (class ii) have higher rates of failure, since they are more favorable to fractures and complete loss. it can be explained by the failure to adapt the civ to the cavity due to its viscous texture and low flow, allowing the appearance of spaces inside the restoration, consequently reducing its longevity. other physical characteristics of this cement also influence the success of art as low mechanical properties, high solubility and porosity28. it was noted that the results of the questionnaires diverged in some questions from what had been found in the intra-oral examination of preschool children. although the parents / guardians had notions of hygiene and eating habits, there was a high prevalence and incidence of caries and failure of the restorations performed with civ by the art technique. it was concluded that there was a high prevalence of caries in the deciduous dentition (38.8%), which may be related to socioeconomic factors and the need for greater efforts to expand dental care with oral health promotion and prevention actions. the parents were advised on the measures to be taken to prevent tooth decay. in t2, six months after restorative art treatment, most class i and ii restorations were damaged or lost, probably due to the large cavities and the limitation of the technique. class iii and v restorations remained satisfactory. there was a high incidence of class i and ii caries (31.9%) in 15 of 17 children in whom oral hygiene was not performed before bedtime. art technique is an option, despite the high failure in art, especially for child population with deciduous teeth that has limited access to dental services. however, restorations should be supervised and healthy eating habits implemented. 9 lira et al. the interpretation of the results should consider some limitations inherent in this 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[evaluation of the clinical performance of art restorations (atraumatic restorative treatment)]. rfo. 2014;19(1):88-93. doi: 10.5335/rfo.v19i1.3694. portuguese. 24. sales peres shc, hussne r, perer as. [atraumatic restorative treatment (art) in children aged 4 to 7 years: clinical evaluation after six months]. rev. inst ciênc saúde. 2005;23(4):275-80. portuguese. 25. eleutério asl, cota als, kobayashi ty, silva smb. [clinical evaluation of oral health of children of the municipalities of alfenas and areado, minas gerais, brazil]. pesq bras odontoped clin integr. 2012;12(2):195-201. doi: 10.4034/pboci.2012.122.07. portuguese. 26. deliberali fd, brusco ehc, brusco l, perussolo b, patussi eg. [behavioral factors involved in the development of early caries in children attended at the pediatric dentistry clinic of the school of dentistry of passo fundo rs, brazil]. rfo. 2009;14(3):197-202. portuguese. 27. goes mf, martins al, sartori cg, sinhoreti mac. [solubility of glass ionomer cements indicated for atraumatic restorative treatment]. rev assoc paul cir dent. 2015;69(3):272-8. portuguese. 28. yip hk, smales rj. tay fr. chu fcs. selection of restorative material for the atraumatic restorative treatment (art) approach: a review. spec care dentist. 2001;21(6):216-21. doi: 10.1111/j.1754-4505.2001. braz j oral sci. 15(3):190-195 musculoskeletal disorders among dentists in northwest area of the state of são paulo, brazil ana carolina da graça fagundes freire1, gabriella barreto soares2, tânia adas saliba rovida3, cléa adas saliba garbin4, artênio josé ísper garbin5 1phd student of preventive and social dentistry araçatuba school of dentistrypaulista state university / unesp, jose bonifácio street, 1193, araçatuba, são paulo, brazil 2phd student of preventive and social dentistry araçatuba school of dentistrypaulista state university / unesp, jose bonifácio street, 1193, araçatuba, são paulo, brazil. e-mail: gabriella.barreto@yahoo.com.br , phone: +55(027)981000511 3professor of the program of preventive and social dentistry araçatuba school of dentistrypaulista state university / unesp. jose bonifácio street, 1193, araçatuba, são paulo, brazil 4professor of the program of preventive and social dentistry araçatuba school of dentistrypaulista state university / unesp. jose bonifácio street, 1193, araçatuba, são paulo, brazil 5professor of the program of preventive and social dentistry araçatuba school of dentistrypaulista state university / unesp. jose bonifácio street, 1193, araçatuba, são paulo, brazil corresponding author: gabriella barreto soares 1193, jose bonifacio st araçatuba – spbrazil phone : +55-027-981000511 gabriella.barreto@yahoo.com.br abstract objective: to determine the prevalence of musculoskeletal disorders in dentists who attend postgraduate courses in various specialties and establish possible relationships with sociodemographic and occupational factors. methodology: this is a cross-sectional study where 91 professionals responded to validated instruments: “work-related activities that may contribute to job-related pain and / or injury” and nmq “nordic musculoskeletal questionnaire”, and variables were also collected regarding sociodemographic and occupational characteristics. results: wmsds were often reported in the neck, shoulder, lower/upper back, and hand/wrists. logistic regression analysis revealed that there was a correlation between reports of wmsd in lower back and work related (or=13.40). moreover, associations were found between wmsds and the occupational factors that can contribute to musculoskeletal disorders. conclusion: there was a high prevalence of musculoskeletal disorders among brazilian dentists, and that the work-related activities contributed to musculoskeletal disorders in dentists. keywords: musculoskeletal pain; occupational health; risk factors; dentistry. introduction musculoskeletal disorders are generally defined as a group of injuries affecting the bones, muscles and tendons of the body, and can occur after a single event or due to an accumulation of traumas related to labor activity1,2. the dentists highlighted by the vulnerability and problems of various natures in the occupational context. among these, those related to the specific postures during their clinical activity are evident3-5. since dentistry is a profession that demands attention and precision in movements where the professional must constantly interact with tools and received for publication: december 29, 2016 accepted: may 31, 2017 original article braz j oral sci. july | september 2016 volume 15, number 3 http://dx.doi.org/10.20396/bjos.v15i3.8649979 191 equipment to assist in the care, it is natural to experience increased muscle tension exacerbated by the number of hours worked1,3,4. in this context, the professional end up sharply bending the body or performing rotations of the regions of the neck and spine, and abduction of the shoulder to improve the field of view and obtain easier access to the oral cavity3-9. the static and sometimes clumsy posture combined with repetitive movements and compression of musculoskeletal structures increase the risk for developing injuries among dental professionals, jeopardizing their health and may lead to the removal from the career1,5,9. to help prevent the installation of musculoskeletal injuries and minimize the negative effects, the professionals should orientate themselves about the risks to which they are exposed. studies shows a high prevalence of lesions, but few have explored and discussed how early musculoskeletal disorders develop in dentists, thus undermining the implementation of preventive methods5,10. most musculoskeletal disorders may be alleviated with appropriate preventive attitudes, especially before chronic damage is installed1,5-9. furthermore, the adopted work postures and their duration are closely related to musculoskeletal disorders and have their variations among the specialties11. despite the high prevalence of musculoskeletal complaints described, very little is known about these disorders among dental professionals and their associated factors. in this sense, the objective was to study the prevalence of musculoskeletal disorders and their possible relationship to sociodemographic and occupational variables in dentists who attend postgraduate courses and verify their perception in relation to risk factors for development of musculoskeletal disorders. materials and methods this is an epidemiological study, following the ethical standards proposed for studies in humans. the sample consisted of dentists enrolled in postgraduate courses of various specialties (dentistry, endodontic, periodontic, surgery, prosthodontics, implant dentistry, pediatric dentistry and orthodontic) of a medium-sized municipality in the state of são paulo, brazil, during 2016. the dental class board advocates that each specialization class is comprised of 12 students (n = 96). dentists with physical limitations and pregnant women were excluded from the study. data collection was carried out during 4 months in 2016. the data variables related to sociodemographic characteristics, work and health of the dentists were acquired through a structured questionnaire designed specifically for this study. socio-demographic variables included age, gender and marital status. health-related variables included: body mass index (bmi), practice of physical exercises, diagnosis of any disease in the last 12 months and the use of pain medications. to evaluate the musculoskeletal symptoms, the nordic questionnaire developed by kuorinka was used with the goal of standardizing the measuring of the musculoskeletal symptoms. brazilian version of the nordic questionnaire was validated and adapted by barros and alexandre in 200312. this tool consists of a posterior view of a human figure, which is subdivided in 9 anatomical regions: neck, shoulders, upper and lower back, elbows, wrists/hands, hips/thighs, knees and ankles/feet, and questions about the presence of musculoskeletal pain in any of the 9 anatomical areas, inability to perform normal activities and necessity to consult a health professional. the respondent has to signal with an x an affirmative or negative response (yes or no). a 12-month period prior to the time of the study was considered for the occurrence of musculoskeletal symptoms. the questionnaire on risk factors at work that may contribute to musculoskeletal disorders (work-related) was also applied to identify how much work activities contribute to the development of musculoskeletal disorders. this tool was originally developed in the united states by rosecrance et al. in 2009 it was adapted and validated for brazilian culture by colucci and alexandre13. this instrument comprises 15 items that evaluate individuals’ awareness of job environment factors and their potential contribution to musculoskeletal disorders. the individuals were asked to indicate on a scale from 0 to 10 (with 0 = nothing, and 10 = a lot) how much each variable contributed to the development of pain and lesions related to their current work tasks. responses were classified in a scale of 3 categories, which indicated severity of symptoms in crescent order: the first, from 0–1, indicated absence of symptoms; the second, from 2–7, indicated minimal to moderate symptoms; and the last, from 8–10, indicated severe symptoms. data were entered into the database and analyzes were performed using the spss version 21.0. to check if the pain interferes with the perception of risk factors for musculoskeletal disorders, professionals were divided into two groups, the group of symptomatic subjects (with pain) and the group of asymptomatic subjects (no pain) to be later compared. this diagnosis was made with the aid of the “nordic musculoskeletal questionnaire” (nmq). the comparison of the means between groups (symptomatic and asymptomatic) was performed using the mann whitney test. the logistic regression was used to analyze the relationship between the sociodemographic variables and the presence of pain. also performed was an association by reason of the maximum like hood performed by associating the general questions of the “nordic musculoskeletal questionnaire” (nmq) (question a: over the last 12 months have you had any problems such as pain, tingling or numbness; b question: in the last 12 months, were you unable to perform normal daily activities; c question: in the last 12 months have you consulted a health professional because of this condition; d question: over the past seven days have you had any problems) with the sociodemographic and occupational variables. a statistical significance level of 5% was considered for all tests. the study was approved by the committee on ethics for research on human beings of the college of odontology of araçatuba of the paulista state university, and performed with the understanding and a written consent of every participant. results more than half of the dentists were women (63.8%) with musculoskeletal disorders among dentists in northwest area of the state of são paulo, brazil braz j oral sci. 15(3):190-195 192 average age of 30 years old (sd 6.16) and single (60.6%). the majority had practiced for an average of 21 years (sd 9.48). a large part of the sample worked in public and private services (55.3%), with a workload of more than 8 hours per day (36.2%), and 41.5% reported not taking breaks between appointments. an half of the study subjects had normal bmi (50.0%), however, there is a considerable part that was overweight (31.9%). alarge number of the dentists (62.8%) had practiced physical activity for more than 1 year. the table 2 indicates that 90.4% of the sample reported pain in the last 12 months. more than a half of the dentists reported neck pain (58.5%), lower back (57.4%), upper back (55.3%), shoulders (46.8%), hands/wrist (44.7%). table 1 – sociodemographic and work characteristics of dentists. são paulo, brazil, in 2016. variables n % gender female 60 63.8 male 34 36.2 age 20 to 24 years 12 12.8 25 to 29 years 34 36.1 30 to 34 years 22 23.4 35 to 39 years 14 15.0 40 to 53 years 9 9.7 no information 3 3.2 marital status married 37 39.4 single 57 60.6 bmi underweight 5 5.3 normal weight 47 50.0 overweight 30 31.9 obesity 12 12.8 workplace public 22 23.4 private 52 55.3 both 18 19.1 no information 2 2.1 work hours / day 6 hours 12 12,8 8 hours 28 29.8 > 8 hours 34 36.2 no information 20 21.2 breaks yes 52 55.3 no 39 41.5 table 2 distribution of musculoskeletal symptoms (12 months and 7 days), disability and demand for professional healthcare among dentists. são paulo, brazil, in 2016. body area symptoms in the last 12 months impediment to perform normal activities bacause of this problem over the past year consultation with a professional in the area of health because of this condition in the past 12 months symptoms in the last 7 days n % n % n % n % neck 41.5 58.5 93.6 6.4 81.9 18.1 74.5 25.5 shoulder 53.2 46.8 94.7 5.3 86.2 13.8 85.1 14.9 upper back 44.7 55.3 95.7 4.3 81.9 18.1 80.9 19.1 elbow 87.2 12.8 98.9 1.1 96.8 3.2 96.8 3.2 wrist/hand 55.3 44.7 96.8 3.2 87.2 12.8 85.1 14.9 lower back 42.6 57.4 89.4 10.6 78.7 21.3 80.9 19.1 hip/haunch 87.2 12.8 97.9 2.1 96.8 3.2 94.7 5.3 knee 71.3 28.7 94.7 5.3 90.4 9.6 87.2 12.8 ankle/feet 81.9 18.1 93.6 6.4 94.7 5.3 94.7 5.3 the association test was performed by the maximum likelihood of the general questions of the “nordic musculoskeletal questionnaire” (nmq) and the sociodemographic and work related variables. a statistically significant association was only observed for the daily working hours, when it was presented as very prolonged, hence contributing to the onset of musculoskeletal pain, interfering in performing normal daily activities such as housework, work and leisure (p=0.015) (table 3). table 3 association between general questions of the nmq and daily work day among dentists. são paulo, brazil, in 2016. nmq daily work day p-value 6 hours 8 hours 8 hours more than n % n % n % ga yes 10 83.3 26 92.9 32 94.1 0.546** no 2 16.7 2 7.1 2 5.9 gb yes 1 8.3 5 17.9 15 44.1 0.015** no 11 91.7 23 82.1 19 55.9 gc yes 4 33.3 11 39.3 22 64.7 0.062 no 8 66.7 17 60.7 12 35.3 gd yes 7 58.3 18 64.3 20 58.8 0.891** no 5 41.7 10 35.7 14 41.2 total 12 100 28 100 34 100 **maximumlikelihoodratio musculoskeletal disorders among dentists in northwest area of the state of são paulo, brazil braz j oral sci. 15(3):190-195 193 musculoskeletal disorders among dentists in northwest area of the state of são paulo, brazil the comparison of the symptomatic and asymptomatic groups was performed by the non-parametric mann-whitney test and a statistical significance was observed regarding the presence of pain when the dentist finds himself in an uncomfortable working situation, such as working in a very tight location inappropriately furnished (p= 0.005) (table 4). the situations listed in the “work-related activities that may contribute to job-related pain and / or injury” questionnaire that showed greater significance in relation to the pain experience were: performing the same task repeatedly (p= 0.001); work fast for short periods (p= 0.026); have to handle or hold small objects (p= 0.006); intervals or insufficient breaks during the workday (p= 0.003), working in uncomfortable positions (p= 0.001); working in the same position for long periods (p=value 0.001); bending or twisting one`s back uncomfortably (p= 0.001); long working hours (p= 0.004) (table 4). symptomatic professional has a greater awareness of the risk factors for the development of musculoskeletal symptoms. braz j oral sci. 15(3):190-195 table 4 comparison between the presence of pain and the 15 job factors that can contribute to musculoskeletal symptoms featured in the “work-related activities that may contribute to job-related pain and / or injury”. são paulo, brazil, in 2016. instrument regarding job factors that can contribute to musculoskeletal symptoms symptoms of pain p-valueyes no (sd) (sd) perform the same task repeatedly. 5.0(±3.33) 1.11(±1.83) 0.001* work fast for short periods. 3.74(±3.27) 1.22(±1.92) 0.026 have to handle or hold small objects. 3.51(±3.54) 0.78(±2.33) 0.006* intervals or insufficient breaks during the workday. 4.69(±3.38) 1.22(±2.73) 0.003* working in uncomfortable positions / very tight space. 6.47(±2.89) 2.44(±3.09) 0.001* working in the same position for long periods. 6.78(±2.71) 2.67(±3.32) 0.001* bending or twisting of the back uncomfortably. 6.66(±2.88) 2.67(±2.60) 0.001* working near or at their physical limit. 6.00(±2.93) 3.44(±4.28) 0.059 reaching or working above head level or away from the body. 4.53(±3.56) 2.56(±3.68) 0.119 working in the heat, cold, damporwet. 4.08(±3.26) 3.33(±4.44) 0.433 continue working with any pain or injury. 6.81(±2.85) 5.67(±3.20) 0.275 carry, liftor move materials or heavy equipment. 4.26(±3.50) 3.67(±3.97) 0.597 workday 4.91(±3.30) 1.56(±2.19) 0.004* use tools (format, vibration). 3.04(±3,16) 2.44(±2.92) 0.627 work without receiving training. 3.32(±3.59) 2.78(±3.23) 0.893 general 4.94(±2.23) 2.44(±2.24) 0.005* *mann whitney test in the proposed model, individuals with greater awareness of the risk factors were classified with serious problems, and presented a risk of pain in the lower back 13.40 times higher than the asymptomatic subjects (table 5). table 5 multivariate analysis of the relationship of the regions lower back and “work-related activities that may contribute to job-related pain and / or injury” são paulo, 2016. instrument regarding job factors that can contribute to musculoskeletal symptoms symptoms of pain p-valueyes no (sd) (sd) perform the same task repeatedly. 5.0(±3.33) 1.11(±1.83) 0.001* work fast for short periods. 3.74(±3.27) 1.22(±1.92) 0.026 have to handle or hold small objects. 3.51(±3.54) 0.78(±2.33) 0.006* discussion in this study, the majority of respondents were female (63.8%), but this condition was not a risk factor for the presence of pain. dentistry has shown a gradual increase in female workers in recent years11,14-18. the correlation between gender and pain becomes more evident with each passing year, this factor must be the mechanical overload caused by double shifts to which women are subjected, thus establishing an association between the female gender and musculoskeletal disorders11,15,19. thus the average age in these surveys showed professionals over 35 years of age5,9,15,16 and in our study, the average age was 30.68 years, this variable also did not appear as a risk factor or statistical association with reports of pain. musculoskeletal disorders can be prevented by adopting a healthier lifestyle such as: nutritional care, practice of sports, performing daily stretches and adoption of ergonomic principles20. physical exercise on a regular basis causes circulatory and metabolic adaptations beneficial to musculoskeletal structures, helping to maintain the static and dynamic posture, thus reducing the risk of musculoskeletal injuries11,15,20. although most have reported engaging in physical activity (62.8%) with frequency and the body mass index (bmi) found was considered normal (bmi = 25.02), these factors did not help in the prevention of musculoskeletal disorders, a plausible reflection would be a late start to a healthier life style coupled with excessive workload and lack of breaks. musculoskeletal disorders have increasingly become a matter of concern, research and discussion worldwide. it is fundamental importance to the quality of the professional and personal life of the dentist, knowing its causes, manifestations and ways to prevent and treat these lesions9. in this study, the presence of musculoskeletal disorders was observed in 90.4% of the professionals, and this is a very high prevalence, and similar results were found in other studies14-16,19,21,22. the regions most committed to musculoskeletal disorders were the neck, upper back, shoulders, wrists / hands and lower back, and similar findings were found in studies of dental professionals14-17,19-21. the dentist performs flexion and abduction of the shoulder to serve as a support base for the fine and precise movements performed with the hands, so the more overloaded regions by static muscular effort of these professionals are the neck, shoulder and lower back10,15,21. the pain is manifested in higher and lower grades in accordance with the daily demands of static overload that each professional undergoes15. the lengthening of the working day ends up requesting more maintenance on the static posture of the body, causing muscle fatigue, thereby the professional ends up adopting compensatory postures, leading to the onset of muscle pain and decreased strength of the upper limbs9,15,16. this fact can be seen in the results of this study in which the painful manifestation presented a statistical significance with the work hours and insufficient interval / breaks during the labor activity. the daily working hours of most dentists was over 8 hours, and the breaks was reported by just over half of the subjects. the workload can be considered a pre-determining factor for the development of musculoskeletal disorders, but does not act in isolation. therefore, professionals who work ergonomically, perform pauses between calls, maintain a workload that does not exceed 8 hours / day are less likely to develop musculoskeletal disorders15,20,21. one must also be aware of the ergonomics afforded by the furnishings in the workplace, such as tables and chairs, in the case of dentists, whose design often does not meet the biotype of the users, since they are manufactured in a standardized manner, and working on a device to which their body cannot adapt comfortably and ergonomically can affect the performance of the professional at work23. among the surveyed correlations, situations of discomfort at work get highlighted in this and other studies14-16,21, presenting an intimate relation to the painful events. in the analysis of the situations considered uncomfortable, the ones most significant in relation to the pain experience were: performing the same task repeatedly; work fast for short periods; have to handle or hold small objects; working in uncomfortable positions; working in the same position for long periods; bending or twisting of the back uncomfortably. this can be explained by the overloading of the muscles of the upper body (including shoulder, neck, hands) that the dentist does when performing their work activities, coupled with the stress and excessive workload5,16. in the proposed model, individuals classified with severe problems presented greater perception of risk factors for the development of musculoskeletal disorders. thus, dentists with musculoskeletal disorders have 13:40 times greater risk of developing pain in the lower back than asymptomatic individuals. the lack of stability and alignment of the spine associated to work for an extended time in the same position can generate decreased muscle flexibility and joint mobility, leading to muscle fatigue15,24. professionals who complain of pain first use some form of drug therapy to relieve this sensation, seeking professional help from a specialist only after the persistence of symptoms15,19,24,25. the pain can cause limitations in performing daily professional activities in individuals with low back pain or other musculoskeletal disorders15. when performing the association of general questions of the “nordic musculoskeletal questionnaire” (nmq) with the sociodemographic and occupational variables, a statistical significance was observed only in the hours worked / day, demonstrating that the daily working hours, when more than 8 hours, contributing to the onset of musculoskeletal disorders, interfering in performing normal daily activities such as housework, work and leisure. so the professionals who work long continuous hours in uncomfortable positions, with a high volume of calls, without holding breaks may present some kind of pain or discomfort15,16,21. the short breaks are as important as the regimented labor stretches or long breaks, since they allow the professional to conduct some postures to reduce muscle tension, without interrupting the pace of work during short breaks, like drinking water, going to the bathroom, or between servicing of patients. these short breaks bring a release of accumulated lactic acid by prolonged postures, improving tissue oxygenation26. performing rest breaks is not a vital need of the body, but the introduction of this practice in daily life can reduce mental and physical fatigue, especially for workers such as dentists who perform activities that require much of the nervous system, in mental work, where finger dexterity and the requirement of the sense organs is of utmost importance27. the results of this study were a high prevalence of musculoskeletal disorders among dentists and the region of the upper limbs and lower back region were the most affected. the extension of daily working hours proved to be a factor that interferes with pain, disabling the professional to perform their daily activities. the perception of professionals in relation to risk for the onset of painful symptom factors was considered severe in symptomatic individuals. more research on the subject becomes interesting for the purpose of further analysis on the installation, demonstration and prevention of musculoskeletal disorders in dentists, including the identification of risk factors and their impact on labor activity. 194musculoskeletal disorders among dentists in northwest area of the state of são paulo, brazil braz j oral sci. 15(3):190-195 195 acknowledgement we appreciate the funding from the fundação de amparo a pesquisa de são paulo (foundation for support and research of 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[work related musculoskeletal disorders in dentists]. rev bras epidemiol. 2006;9(3):346-59. portuguese. musculoskeletal disorders among dentists in northwest area of the state of são paulo, brazil braz j oral sci. 15(3):190-195 untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8652646 volume 17 2018 e18038 original article a department of oral diagnosis, division of oral radiology. piracicaba dental school, university of campinas. piracicaba, são paulo, brazil. b department of physiological science. piracicaba dental school, university of campinas. piracicaba, são paulo, brazil. *corresponding author: liana matos ferreira. university of campinas, piracicaba dental school, department of oral diagnosis. av. limeira, 901 zip code 13414-903 piracicaba, sao paulo, brazil. phone: +55 – 19 -2106-5327. e-mail: liana.rj@gmail.com received: october 17, 2017 accepted: march 17, 2018 the fundaments of cbct and its use for evaluation of paranasal sinuses: review of literature liana matos ferreira, dds, msa, yuri nejaim, dds, ms, phda, deborah queiroz freitas, dds, ms, phda, cínthia pereira machado tabchoury, dds, ms, phdb image methods such as magnetic resonance imaging (mri), computed tomography (ct) and cone beam computed tomography (cbct) are powerful tools to help clinicians on diagnosis and preoperative planning. they provide an accurate view of regional anatomy, anatomical variations and the presence of diseases. compared to ct, cbct produces images with adequate spatial resolution with smaller fields of view at lower radiation doses. it has emerged as a potential alternative for obtaining 3d evaluation of the paranasal sinus at relatively modest costs. the aim of this review was to verify whether cbct images offer an additional value to the evaluation of paranasal sinus. keywords: paranasal sinuses. diagnostic imaging. cone beam computed tomography. 2 ferreira et al. introduction the purpose of radiological evaluation of the paranasal sinuses and related structures is to provide an accurate description of the regional anatomy, any osseous changes or variations, sinus mucosa, fluid levels and to establish the presence and extent of diseases1,2. available imaging techniques that might be used in this situation include two-dimensional x-rays, like waters’ and panoramic, computed tomography (ct), magnetic resonance imaging (mri), and more recently cone beam computed tomography (cbct)1,3 – these are summarized in table 1. plain films are widely available; however, provide insufficient detail to allow surgical planning. at best, they give only an overview of the anatomy and underlying pathology, as they are limited to displaying three-dimensional structures in a two-dimensional plane. the technological advances in radiological imaging from 2d projection radiography towards 3d and interactive imaging applications have made an enormous impact in head imaging and have increased surgeon’s ability to depict accurately the status of structures within the paranasal sinus region and to delineate the location and extent of pathology4,5. multidetector ct (mdct) and mri have the advantage of being able to show fine anatomic detail in serial tomographic sections1,6. mri allows excellent visualization of soft tissues, but does not adequately represent the bone walls and paranasal sinuses ostia; on the other hand, mdct provides a lot of information, both about the bony part as soft tissue, remaining as technique of choice for assessing the presence and extent of disease in the paranasal sinuses. additionally, the coronal sections perpendicular to the hard palate allow optimal viewing ostiomeatal complex1. introduced in 1998, cbct is increasingly used for 3-dimensional imaging in maxillofacial radiology, generates high-resolution isotropic volume data and could, therefore, show benefits for evaluating the bony aspects of the maxillary sinus by using a lower dose of radiation2. although ct is considered as the “gold standard” in imaging for visualization of the paranasal sinus, cbct is gaining increasing popularity in this respect4. even though, a large dose of ionizing radiation is generally delivered by medical computed tomography; in this way, cbct technology has achieved considerable reduction of absorbed radiation doses, with equal image qualities and less artifacts for visualizing the maxillofacial bone structures compared to mdct imaging7. paranasal sinus 3d images are relevant for the planning of procedures, since it allows the direct visualization of anatomical variations and pathological conditions, which when combined with the clinical examination, can provide to the patient treatment options or referral to specialists, in cases that are not directly linked to dentistry. therefore, this review of literature aims to present the fundaments of cbct as well its application on evaluation of paranasal sinuses. paranasal sinus image techniques – comparative aspects for many years, conventional x-rays, like panoramic and water`s radiography, have been used to investigate the paranasal sinuses3. however, 2d radiographic images are difficult to interpret because of the overlapping of ostiomeatal complex and osseous structure8,9. generally, they are efficient to display the regional morphology, character3 ferreira et al. table 1. summary of literature review of diagnostic imaging modalities in paranasal sinuses. author n objectives imaging modality findings konen et al. 2000 134 diagnosis of paranasal sinusitis mdct and water´s the diagnosis by water´s view was very poor. mdct with a low dose resolution is recommended. rafferty et al. 2005 12 assist surgical approach to the frontal recess endoscopy and cbct cbct increased surgical confidence in accessing the frontal recess, resolved ambiguities with anatomical variations and provided valuable teaching information to surgeons in training in preoperative planning daly et al. 2006 performance as a function of dose and other acquisition/ reconstruction parameters cbct cbct was sufficient for guidance of head and neck procedures. the dose was comparable to or less than the effective dose of a typical diagnostic mdct. bremke et al. 2009 23 to analyze the anterior skull base cbct the surgical key landmarks were possible in all patients. ritter et al. 2011 129 to assess the prevalence of pathologic findings in the maxillary sinus cbct pathologies in the maxillary sinus are frequently found in cbct imaging. cbct is applicable for diagnosis and treatment planning. minni et al. 2012 500 study of frontal recess and especially its anatomical variants in a youth population cbct cbct may be used in the analysis of frontal recess pathologies. göçmez et al. 2013 50 to evaluate the anatomy of the sphenoid ostium mdct with mdct, surgeons can make a pre-operative 3d evaluation of the sphenoid ostium. bui et al. 2014 10 to create a 3d model of the nasal cavity and paranasal sinuses cbct automated cbct segmentation of the airway and paranasal sinuses was highly accurate. demeslay et al. 2015 15 to assess the morphological concordance between cbct and ct in the sinonasal anatomy cbct and mdct cbct represents a valid, reproducible and safe technique zojaji et al. 2015 64 to evaluate the agreement of image modalities in patients with chronic rhinosinusitis endoscopy and cbct cbct has nearly the same diagnostic accuracy as sinus endoscopy. al abduwani et al. 2016 121 to compare the absorbed dose and image quality cbct and mdct the dose of cbcts was approximately 40% lower when compared to standard mdct examinations and 30% lower when compared to low dose sinus mdct scans. the visualization of high-contrast bone morphology on cbct was comparable to standard sinus mdct. soft tissue visibility was limited. rani et al. 2017 60 to estimate age and sex using the dimensions and volume of the maxillary sinus mri mri measurements of maxillary sinuses may be useful to support gender and age estimation in forensic radiology. mdct, multidetector computed tomography; cbct, cone beam computed tomography; mri, magnetic resonance imaging 4 ferreira et al. ize the extent and localization of disease and describe anatomical variants of paranasal sinuses10; however, radiographic images allows limited value in the diagnosis of maxillary sinusitis and is less sensitive for detecting abnormalities in other sinuses3. mri is ideal for assessing soft-tissue masses, mucosa and extension of infectious/malignant disease processes beyond the paranasal sinuses. imaging of the paranasal sinuses must include high-resolution (3 mm) t1weighted and t2-weighted images, not only of the sinonasal cavity but also of the orbit, skull base, and the adjacent intracranial compartment1, which is provided by mri. the use of non-ionising radiation is an advantage of this technique1,10. while offering excellent soft tissue definition, mri provides poor bony definition, which is so critical in the frontal sinus and anterior skull base11. mdct is a valuable tool10. for confirmation the clinical diagnosis of the paranasal sinuses, provides detailed images of the sinuses and gives the examiner a clear view of the areas that are key in the pathogenesis of rhinosinusitis. mdct also reveals the anatomical details of the nose and paranasal sinuses in relation to vital adjacent structures3 and allows 3d observation and clear visualization of the inflammatory changes and pathologic status in the nasal and paranasal sinus mucosa12. the treatment of choice of chronically infected sinuses is the surgical clearance that maintains the ventilation and drainage. to achieve this goal, there should be some diagnostic modalities, which guide towards exact diagnosis and safe intervention. over the past few decades, both mdct and nasal endoscopy have been used successfully as diagnostic modalities in sinus disease5,13. moreover, mdct imaging of sinonasal region has become the gold standard in the evaluation of patients with chronic sinusitis. its ability to accurately map out the bony and soft tissue anatomy of the paranasal sinuses has proven invaluable to the endoscopic surgeon ability to depict accurately the status of structures within the paranasal sinus region and to delineate the location and extent of pathology5. despite the fact that mdct scan of the paranasal sinuses can be recommended in case of normality and abnormality of the paranasal sinuses or in patients with chronic sinusitis, the high radiation dose and costs do not allow its usage routinely8,12,14. after all, the mdct cannot stand alone as a gold standard for the diagnosis of rhinosinusitis because it may be positive in the absence of clinical disease. history and physical examination should be taken into consideration when evaluating the mdct scan. if mdct findings are not interpreted in light of signs and symptoms, a person with incidental abnormal findings may be labeled as having a sinus condition. in such cases, the diagnosis is incorrect, and inappropriate treatment is often initiated3. nevertheless, in the last two decades, cbct has been emerging, and now, it is widely used in dentistry, due to its high image resolution, low radiation dose and low costs, compared to mdct. moreover, the boundaries between empty spaces and soft tissues or bones are well defined12,13. because of these advantages, cbct currently has become a valuable method for the evaluation paranasal sinus. further prospective studies are required to confirm that. cbct cbct is a 3-dimensional (3d) x-ray-based volume acquisition imaging modality, first introduced in 199814. offering the advantage of lower radiation dose4,10,14-16, cbct 5 ferreira et al. has been widely used in dental practice for various purposes such as maxillary sinus evaluation, oral surgery, evaluation of temporomandibular joint, orthodontic evaluation, implant planning, and craniofacial trauma evaluation and treatment2,8,14. after these primary applications, cbct has gained popularity and is now increasingly being used for the diagnostic imaging of the head and neck region and the ear, nose, and throat area, mucosal thickness, nasal septum deviation, conchal hypertrophy, bullous concha, and retention cysts in these areas2,14,16. in cbct systems, the x-ray beam forms a conical geometry between the source and the detectors; in addition, digital flat-panel detectors replace the row(s) of detectors in mdct. as result, a major difference is the isotropic nature of acquisition and reconstruction that is used in cbct systems (i.e., cubic voxels). the fact that each voxel is isotropic explains the high fidelity of the reconstructions in any plane used in cbct imaging4,16. the main advantages of cbct over mdct scanning are lower radiation dose (around 10 times lower), lower costs, shorter scanning time, providing very thin slices in any plane, automatic generation of surface and volume reconstructions, easy access, and higher spatial resolution4,8,10,12,14,16-18. technical aspects of cbct the cone beam technique involves a single scan of 360° for the majority of machines, in which the x-ray source and a reciprocating area detector synchronously move around the patient’s head, which is stabilized with a head holder19. during the rotation, multiple (from 150 to more than 1000) sequential planar projection images of the field of view (fov) are acquired. the dimensions of the fov or scan volume able to be covered depend primarily on the detector size and shape, the beam projection geometry, and the ability to collimate the beam. the shape of the scan volume can be either cylindric or spherical (eg, newtom 3g). collimation of the primary x-ray beam limits x-radiation exposure to the region of interest selected by the professional. field size limitation, therefore, ensures that an optimal fov can be selected for each patient, based on disease presentation and the region designated to be imaged. cbct systems can be categorized according to the available fov or selected scan volume height as follows: localized region: approximately 5 cm or less (eg, dentoalveolar, temporomandibular joint); single arch: 5 to 7 cm (eg, maxilla or mandible); interarch: 7 to 10 cm (eg, mandible and superiorly to include the inferior concha); maxillofacial: 10 to 15 cm (eg, mandible and extending to nasion); craniofacial: greater than 15 cm (eg, from the lower border of the mandible to the vertex of the head)19. in general, small fov and high-resolution scans are optimal for detailed diagnostic tasks (e.g. endodontics), while large volume scans will be able to deliver better 3d models and a comprehensive radiologic view of the maxillofacial skeleton and partly of the soft tissue therein2,8. effective dose of cbct the effective dose takes into account the radiation dose produced by the imaging system and the radiation sensitivity of the tissues that the x-ray beam is passing through during the exposure sequence. effective dose is measured in sieverts (sv) and is often expressed in microsieverts (sv)20. the radiation dose produced by a cbct system is dependent on a number of factors: the nature of the x-ray beam i.e. whether 6 ferreira et al. it is continuous or pulsatile, the degree of rotation of the x-ray source and detector and the size of the fov. moreover, the amount and type of beam filtration and the kv, ma and voxel size settings may also influence21. although mdct is the gold standard for radiologic examination of the paranasal sinuses10, cbct in dental and sinus applications is generally considered as a low-dose alternative to mdct scanners2,4. this dose reduction is significant because radiosensitive organs are present in the field explored during sinus imaging, particularly of pediatric patients16. advantages of cbct as exposed previously, cbct technology has emerged as a potential alternative for obtaining 3d evaluation of the paranasal sinus at relatively modest costs, with easy access and a short scanning time compared with mdct and mri4,8,10,14,22,23. cbct exposes the patient to substantially lower radiation compared with standard mdct24 and, although mri is still superior in soft tissue rendering, its use is limited by its cost and restricted accessibility15. cbct has become a diagnostic method to analyze airways characteristics, craniofacial growth, dentomaxillofacial pathology and obstructive sleep apnea15, considering its capacity to define the boundaries between soft tissue and empty spaces (air) accurately. the advantage of reduced cbct exposure over mdct can be explained due to the conical geometry of the x-ray beam and to the pulsed rather than continuous emission in majority of the machines22, which means that actual exposure time is markedly less than scanning time. this technique considerably reduces patient radiation dose19. with correct patient positioning, a selected volume of 10 x 10 cm is sufficient to display the nasal cavity, lateral nasal wall, paranasal sinuses and adjacent vital structures25. cbct generally acquires all basis projection images in a single rotation, so scan time can be minimized. an entire head sometimes can be scanned in 10 s or less4, with realistic representation. added to this, cbct imaging of the sinuses provides excellent contrast between air and mucosa16. these advantages make the system attractive for scanning paranasal sinus. limitations of cbct the main drawback of cbct is its dynamic range, which is insufficient for displaying contrast within soft tissue and the presence of metal artefact10,12,26. the contrast resolution is limited by scattered radiation and the divergence of the x-ray beam over the area detector that produces a large variation in, or no uniformity of, the incident x-ray beam on the patient. these factors contribute to increased image noise. with regard to metal, an artifact is any distortion or error in the image, unrelated to the subject being studied, that can impair the diagnostic19. it happens when the cbct x-ray beam encounters an object of very high density (eg, metallic restorations, dental implants), with absorption of lower energy photons in the beam by the structure rather than higher energy photons; then, the mean energy of the x-ray beam increases. this is called ‘beam hardening’ and the phenomenon produces two types of artifact: distortion of metallic structures and the emergence of streaks and dark bands between two dense structures21,27. 7 ferreira et al. applications of cbct in paranasal sinus intraoperative guidance cbct generates images in the coronal, axial, sagittal, parasagittal (figure 1) and any other planes that the professional needs. these three dimensional information can be used to assist the surgeon in the preoperative planning endoscopic sinus surgery and allow the surgeon to correlate positional information regarding the patient’s anatomy as it is observed intraoperatively with a radiological image obtained preoperatively, reducing the risk of serious complication11,25,28. endoscopy of the paranasal sinuses allows the observation of anatomical areas and the evaluation of sinonasal lesions and their relationship with endonasal structures. however, endoscopy is an invasive and costly method, needs local or general anesthesia, cannot be applied to all patients and may be associated with severe complications. regarding these limitations, finding an alternative diagnostic modality is beneficial. cbct may be an alternative modality for diagnostic sinus endoscopy14. intraoperative imaging offers the potential to improve surgical performance in existing procedures, extend the applicability of surgery to cases that would be otherwise inoperable, and has great potential utility in training surgeons, facilitating advancing the novice surgeon from a 2d to a more complete 3d11,17,28,29. besides that, it is especially desirable in areas that are close to vital anatomical structures, distorted anatomy, extensive sino-nasal polyposis and increased risk of intraoperative bleeding17. a b c d figure 1. (a) coronal, (b) axial, (c) sagital and (d) parasagittal planes 8 ferreira et al. inflammatory pathology periapical inflammation was shown to be capable of affecting the maxillary sinus mucosa with and without perforation of the cortical bone of the sinus floor30 (figure 2). untreated dental condition can cause odontogenic sinusitis that can be presented in various ways, and they are particularly characterized by inflammation and localized mucosal thickening23,30. the accurate identification of changes in the maxillary sinus with cbct could provide the size and location of the periapical lesion, and also would help deciding if the teeth need to be treated, retreated or surgical procedure yet30. regarding the frontal sinus, some cells can block it at the level of frontal recess, causing frontal sinusitis, mainly because of inadequate removal of agger nasi and frontal recess cells during endoscopic sinus surgery31. cbct multiplanar reconstruction could be used to identify potential causes of frontal recess stenosis and evaluates all of the cell anatomical variable with a lower use of radiating energy11. data gained from the cbct scans, in addition to clinical impression and endoscopy, suggest that such images provide useful radiologic documentation for the diagnosis of chronic rhinosinusitis4, effusion, mucosal thickening and ostial obstruction are perfectly visible, with precision equal to or greater than that of mdct. any inflammatory or infectious sinus pathology is accessible to cbct examination, with complete topographic exploration23. implant placement for dental implant site assessment in the maxilla, the configuration and status of the maxillary sinus is important to assess the available amount of bone (figure 3), principally if a sinus lift is indicated2,32. incidental findings such as mucosal thickening can be associated with a sinus outflow obstruction which can impact on the clinician’s treatment decisions26. maxillary sinus septa are barriers of cortical bone that divide the maxillary sinus floor into figure 2. periapical inflammation 9 ferreira et al. multiple compartments, known as recesses33. it seems that an antral septa, detected in almost half of the cbct exams, might increase the risk of sinus membrane perforation during the maxillary sinus floor elevation surgery33. mdct and cbct are definitely the preferred imaging techniques for the assessment of this anatomic variation32. anatomical variations the imaging investigation of anatomical variations (figure 4) of the paranasal sinuses is important in assessing the predisposing factors for inflammatory changes of the paranasal sinuses. these changes in the sinuses are a common problem encountered in clinical practice. the most encountered variations are the concha bullosa, figure 4. (a) concha bullosa, (b) hipertrophy of the uncinate process, (c) haller cell, (d) nasal septum deviation a b c d figure 3. relationship between dental implant and sinus floor. 10 ferreira et al. haller cell presence and the modifications of the uncinate process morphology and positions. the haller cell is an asymptomatic maxilla ethmoidal cell and, in some situations, may narrow the osteo-meatal complex, inhibiting the ciliary function and leading to obstruction of the ostium. the uncinate process allows air flow and mucus drainage. morphological variations of this hook-like process might be a factor of narrowing the unit and, thus, blocking the drainage and consequently producing inflammation13. concha bullosa may be implicated as a possible etiological factor in the causation of recurrent chronic sinusitis, due to its negative influence on paranasal sinus ventilation and mucociliary clearance in the middle meatus region13. the nasal septum deviation is also among the most observed anatomical variations13. this condition may cause compression of the nasal concha laterally, with consequent obstruction of the infundibulum, presenting clinical importance in the approach of recurrent sinusopathy5. volumetric evaluation cbct has become a widely used imaging modality for evaluating maxillary sinus volume. it is used to investigate changes before and after rapid maxillary expansion34, gender assessment15 and the effects of long-term oral breathing7. additionally, a simulated system for medical training in upper air way related surgery can be built from the surface model. a cbct air way segmentation scheme will provide extra information in the case of patients who have already undergone cbct scans for other treatments such as orthodontics without the need of a high radiation dose of mdct18. furthermore, the frontal sinus cavity can be segmented and reconstructed for determining sex and person identification35. final considerations this review paper highlights the potential uses of cbct in the assessment of paranasal sinuses and confirms that it is an accurate and reliable tool. plain films offer limited information about the paranasal sinuses, with the inherent errors of a 2d representation of a 3d structure and the lack of information about cross-sectional area and volume. cbct will eventually become the gold standard in routine sinus exploration, because it combines good image quality, even at low radiation exposure, short examination time, easy use and low cost in relation to mdct and mri. besides, nowadays the volumetric evaluation of the paranasal sinus has been easily achieved by several open-access software. the technique’s limitations, however, need to be borne in mind. it is remarkably good for bone evaluation, with excellent bone/mucosa/air contrast, but its poor density resolution is a drawback for soft-tissue contrast studies. in case of tumoral, septic or hematic soft-tissue infiltration, mdct or mri is mandatory. in addition, 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dimorphism in 3d cone beam ct images and can be used for sex determination. j forensic sci. 2018 may;63(3):692-698. doi: 10.1111/1556-4029.13601. 1http://dx.doi.org/10.20396/bjos.v19i0.8658569 volume 19 2020 e208569 original article 1 centro universitário newton paiva, silva lobo avenue, 1730 – grajaú. zip code 30411-335. belo horizonte, mg – brazil. corresponding author: lidiane cristina machado costa department of periodontics, school of dentistry, centro universitário newton paiva silva lobo avenue, 1730 – grajaú zip code 30411-335 belo horizonte, mg – brazil phone: 55 31 991836684 e-mail: lidiane.cmachadocosta@gmail.com received: march 02, 2020 accepted: june 13, 2020 comparative analysis of the surface roughness of conventional resins and filling after immersion in mouthwashes juliana de souza silva zica1 , isabela araújo fernandes1 , fernanda barcellos ameno faria1 , fernanda cruz ferreira1 , nívea aparecida reis albuquerque1 , josé flávio batista gabrich giovannini1 , lidiane cristina machado costa1,* aim: bulk fill composite resins were released on the market in order to reduce the time in clinical sessions by using increments of up to 5.0 mm thickness. the aim of this study was to evaluate the effect of the rinsing solutions on the surface roughness of the conventional composite and bulk fill composite resins. methods: 40 specimens were prepared from a 4.0mm x 10.0mm teflon matrix and photoactivated for 20 seconds, with 20 specimens made of filtek bulk-fill composite resin (3m espe) and 20 specimens made with filtek™ z350 xt composite resin (3m espe). each group was subdivided into 2 subgroups: g1 (filtek™ z350 xt-3m espe-immersed in colgate plaxwhitening®); g2 (filtek™ z350 xt-3m espe-immersed in plaxfreshmint®); g3 (filtek™ bulk-fill-3m espe-immersed in colgate plaxwhitening®) and g4 (filtek™ bulk-fill-3m espe-immersed in colgate plaxfreshmint®). the surface roughness test was performed initially and after immersion in rinses by the time group inc.tr200® rugosimeter apparatus and the data were submitted to statistical analysis (two-way repeated measures anova). results: surface roughness values of the filtek™ bulk-fill composite resin (3m espe) were significantly higher than the filtek™ z350 xt composite resin (3m espe) (p <0.0001). however, no differences were identified before and after immersion in rinses with or without alcohol. conclusion: the use of mouthwashes does not interfere with the surface roughness of the tested resins, but the composite resin filtek z350 xt (3m espe) presents a surface with less roughness. keywords: composite resins. materials testing. mouthwashes. surface properties. https://orcid.org/0000-0001-9846-9613 https://orcid.org/0000-0001-8382-1151 https://orcid.org/0000-0001-5337-061x https://orcid.org/0000-0002-5855-2241 https://orcid.org/0000-0003-0126-3307 https://orcid.org/0000-0001-9156-2225 https://orcid.org/0000-0002-3843-1186 2 zica et al. introduction composite resins are restorative materials extensively used in dentistry due to their excellent aesthetic1 physical and mechanical properties, which allows the reproduction of characteristics similar to dental structures2 as well as minimal removal of healthy dental structure. the popularity of the composite resins arises from their outstanding adhesiveness and the ability to mimic oral tissues. however, the insertion of composite resins into the cavity requires more clinical time, since the incremental technique is the most appropriate3. typically, this technique consists of adding composite resin increments up to 2.0 mm thick, followed by exposure to the photoactivator until the cavity is entirely filled4. driven by consumer demand for faster, more straightforward procedures and reduced clinical time, the market has recently launched a new category of fillers for posterior teeth know as bulk-fill composite resin. said resin allows the insertion of up to 4.0 mm thick increments without too much polymerization shrinkage5. said decreased polymerization shrinkage derives from properties capable of reducing the contraction stress and increasing the pre-gel phase, which is characterized by more flexible polymer chains, allowing the material to flow freely through the cavity surface6. moreover, these materials provide higher transmission of light, thus allowing the reach of greater polymerization depth6. notwithstanding the recommended optimization of the clinical time, some deficiencies of composite resins concerning aesthetic properties, such as color change and translucency, must be considered. these aesthetic properties can be influenced by the surface roughness of the restorations as well as by the oral conditions in which they are inserted7. as a result of this interaction, mainly by contact with substances containing dyes, extrinsic staining occurs through the absorption of pigments from exogenous factors associated with individuals’ habits, food, and use of mouthwashes8. thus, the properties of the composite resins can be changed by environmental conditions, considering that the exposure to acid solutions contained in the buccal cavity can influence the surface gloss and hardness, which causes degradation of the materials and reduces their clinical longevity. this process allows plaque retention, wear and staining of the restorations9. mouthwashes help to control the biofilm and serve as a complement for the patient’s toothbrushing. the mouthwashes have been widely used, even without professional prescription10,11. such products have varied compositions and ingredients that can also cause degradation, softening, and wear of composite resins7, making the surface irregular and exposed to bacterial plaque retention. the composition of these products consists of water, antimicrobial agents, salts, and, in some cases, alcohol. the antiseptics ph may be affected as a result of the different concentrations of these substances. despite the frequent use of these products, the effects of such components on the composite resin polymer matrix have not been widely discussed9. besides, changes along the inorganic phase may decrease the physical properties of the material, such as microhardness and roughness11. in contrast, the effect of rinses on wear and hardness also depends on the material that is analyzed. differences in chemical composition, type, and filler content are accountable for this variation. more3 zica et al. over, the chemical alteration of the restoration surface cannot be attributed to a single chemical component, but it is otherwise the result of complex reactions between the different chemical composites12. the literature on the surface roughness of bulk-fill composite resins is still scarce. the literature does not indicate whether changes in the composition of materials tend to affect the surface roughness and whether chemical compounds, such as mouthwashes, can adversely affect the integrity of their surface. further researches should be carried out about changes in the surface of these composite resins. these composite resins must have a smooth surface to increase durability, improve aesthetic appearance, and avoid color changes in the restoration13-18. therefore, this study aims to evaluate the effect of the rinsing solutions on the surface roughness of the conventional composite and bulk-fill composite resins. materials and methods this experiment consists of the surface roughness comparison test between filtek™ bulk-fill composite resins (3m espe) and filtek™ z350 xt composite resins (3m espe), both with the same nanoparticle technology, after immersion in mouthwashes, either with or without alcohol. table 1 provides the characteristics of the composite resins used. the analyses were carried out in the material engineering laboratory of the centro universitário newton paiva, in the state of belo horizonte, brazil. the specimens were prepared using a polished teflon™ matrix of 4.0 mm depth and 10.0 mm internal diameter15,19-22 in which the composite resin was added. thus, 20 specimens were prepared using filtek™ bulk-fill composite resin (3m espe), and the other 20 specimens used filtek™ z350 xt composite resin (3m espe). these specimens were divided into four groups: (i) g1filtek™ bulk-fill composite resins table 1. characteristics of the composite resins used. material type/color organic composition inorganic load average size manufacturer filtek z350 xt a2 enamel bis-gma*, udma**, tegdma***, bis-ema**** 78.5% by weight or 63.3% by volume 10 to 20 nanometers 3m espe filtek bulk-fill a2 audma ¶, afm ¶¶, udma ddma¶¶¶ 76.5% by weight or 58.4% by volume 10 to 20 nanometers 3m espe * bisphenolglycidyl methacrylate ** urethane dimethacrylate *** triethylene glycol dimethacrylate **** bisphenol a polyethylene glycol di-di-methacrylate ¶ aromatic dimethacrylate urethane ¶¶ additional fragmentation monomer ¶¶¶ dodecanedimetacrylate 4 zica et al. (3m espe) immersed in the colgate plaxwhitening™ mouthwash; (ii) g2filtek™ bulk-fill composite resins (3m espe) immersed in colgate plaxfreshmint™ mouthwash; (iii) g3filtek™ z350 xt composite resin (3m espe) immersed in the colgateplaxwhitening™ mouthwash; and (iv) g4filtek™ z350 xt composite resins (3m espe) immersed in the colgate plaxfreshmint™ mouthwash. table 2 provides the characteristics of the mouthwashes used in tests. the composite resin was added into the teflon matrix with a particular instrument and condensed against the sidewalls and the bottom of the matrix. subsequently, the composite resin was settled with a glass microscope slide approximately 1.0 mm thick. then, the increment was immediately photoactivated for 20 seconds23 using the led photoactivator device (led 3m espe elipar™ deep cure-l) with an irradiance of 1470mw/cm2 and wavelength from 430 to 480nm at distance zero from the specimen. the light intensity of the photoactivation device was gauged by the ecel radiometer model rd, and read the light with predetermined intensity. the specimens were wrapped in aluminum foil for 24h to allow late polymerization and, afterwards, they were immersed in different antiseptic solutions with the aid of a universal holder (metalic). the sample was immersed in a glass beaker containing 100.0ml of mouthwash on a magnetic stirrer (fisatom). the specimens were maintained using an orthodontic strand attached to the support that embraced its entire diameter. the specimens were immersed in the respective mouthwashes for 12 hours, which is equivalent to one year of daily use of the solution for two minutes24,25. before and after their immersion in the mouthwash, the specimens were submitted to the surface roughness test. the time group inc.-tr200™ portable roughness test was used in all specimens of each group, totalizing ten analyses of each group in this step. a utility wax was used to fix the specimen in each roughness test performed. the surface roughness values were obtained before and after immersion in mouthwashes with alcohol (colgate plaxwhitening™) and without alcohol (colgate plaxfreshmint™) for the analysis. initially, a descriptive analysis of the sample was carried out, and the findings are provided in table 3. table 2. characteristics of the used mouthwashes. brand type composition manufacturer colgate plax whitening solution antiseptic hydrogen peroxide 1,5%, water, sorbitol, ethyl alcohol 8%, poloxamer 338, polysorbate 20, methyl salicylate, menthol, sodium saccharin colgate-palmolive industrial ltda colgate plax freshmint solution antiseptic sodium fluoride 0,05%, cetylpyridinium chloride 0,075, water, glycerin, propylene glycol, sorbitol, poloxamer 407, potassium sorbate, sodium saccharin, citric acid, contains 225 ppm of fluorine colgate-palmolive industrial ltda 5 zica et al. in order to evaluate possible differences between groups, a two-way repeated measures anova (linear mixed model) was fitted (data provided in table 4). both the main effects and the interactions of the predictor variables were evaluated. pairwise testing was performed using tukey’s procedure. normality and homoscedasticity assumptions were checked graphically. all tests considered a 5%-significance level. a priori, the size of the sample to achieve a minimum power of 80% was established based on a 95%-confidence level to detect an effect size of 0.1 mm considering a conservative standard deviation of 0.1mm. the formula resulted in at least 16 samples per group. this number was rounded to 20 samples per group to increase precision. after the experiment, the post hoc statistical power of the observed effect size for each predictor variable was estimated through simulation with 2000 replications26. all statistical analyzes were performed using software r version 3.6.127. results a significant difference was found only between composite resins (p <0.001). the effect of the type of mouthwash and time (before and after) were only marginally table 4. p-values of the main effects and interactions of the predictor variables variáveis valor p resin <0,001 alcohol 0,065 time 0,075 resin : alcohol 0,715 resin : time 0,148 alcohol : time 0,843 resin : alcohol : time 0,743 : interações entre as variáveis table 3. descriptive statistics of the measurements for each type of resin. z350 xt* bf** numberofvalues 38 40 minimum 0,106 0,104 25% percentile 0,174 0,393 median 0,234 0,443 75% percentile 0,322 0,551 maximum 0,614 0,813 mean 0,246 0,474 std. deviation 0,101 0,142 std. error 0,016 0,022 lower 95% ci ofmean 0,214 0,430 upper 95% ci ofmean 0,278 0,518 * filtek™ z350 xt (3m espe) ** filtek ™ bulk fill (3m espe) 6 zica et al. significant. none of the interactions between the predictor variables were significant, as shown in table 4. it was noted that all filtek™ bulk-fill composite resins (3m espe) groups have significantly higher averages than the filtek™ z350 xt composite resin (3m espe) groups. however, for the same composite resin, the groups do not differ from each other (figure 1). the average difference in roughness between the two resins was 0.208μm, with a 95% confidence interval between 0.103μm and 0.314μm. discussion the bulk-fill composite resins have been launched to shorten the time in clinical sessions, inserting increments of up to 5.0 mm in thickness. thus, we intend to justify the clinical use of these new materials based on studies that assess their microhardness, polymerization shrinkage, and the contraction stress generated in the cavity walls28. however, the clinical longevity of restorations is directly related to several factors, including their surface roughness, and few works analyze the surface layer of bulk-fill composites. according to the results of this study, the first null hypothesis that the use of mouthwashes does not interfere with the surface roughness of filtek™ bulk-fill compounds (3m espe) and filtek™ z350 xt (3m espe) has been confirmed. the second null hypothesis, which provides that differences in the chemical composition of filtek™ bulk-fill composite resin (3m espe) did not increase its surface roughness compared to conventional composite resins, was rejected. the surface smoothness of the test specimens of this work was achieved with a glass slide, thus avoiding interference of any polishing techniques on results. the roughness tester measures high-frequency irregularities on the surface of a sample. the average roughness (ra) is the parameter used to analyze the surface13. under the particular circumstances of this study, an outstanding difference was evidenced bf – bulk-fill figure 1. roughness averages for each of the combinations of the three predictor variables. the error bars represent the 95% confidence interval for the population average. the letters above the groups specify multiple comparisons. groups that share at least one letter are not significantly different from each other at 5% significance. r ou gh ne ss (μ m ) 0.6 0.4 0.2 0.0 z350 with alcohol before after a ab a a c c bc c z350 no alcohol bf with alcohol bf no alcohol before after before after before after 7 zica et al. in the initial surface roughness of filtek™ bulk-fill composite resin (3m espe) compared to the filtek™ composite resin z350 xt (3m espe) before immersion in mouthwashes. bulk-fill composite resins have been developed for exclusive use on posterior teeth and have differentiated monomers so that they can be used in single increments without causing damage to the bonding layer with the tooth structure. studies show that non-fluid filled composite resins exhibit similar performance to conventional composites, making them a promising alternative in terms of mechanical performance29. bulk-fill flow composites have the advantage of filling hard-to-reach angles in deep and narrow cavities, while larger cavities can be restored easily and more quickly using high-viscosity bulk-fill composites28. according to the manufacturer, the two composite resins we evaluated in this study are classified as nanoparticles. however, filtek™ composite resin z350 xt (3m espe) has medium size particles of 10 to 20nm, while filtek™ bulk-fill composite resin (3m espe) has, among its particles, ytterbium trifluoride with an average size of 10 to 100nm, which would justify the increased surface roughness by the increase of the particle size30. the findings in this study suggest that the composition of the organic matrix could influence surface roughness. in the case of filtek™ bulk-fill composite resin (3m espe), this influence can be explained by the incorporation of two novel methacrylate monomers: audma (high molecular weight urethane dimethacrylate) and afm (additional fragmentation monomer), which promote relief of the polymerization shrinkage stress30. both methacrylate monomers seem to attribute high viscosity to the bulk-fill composite so that it is easily inserted into the cavity, which may have contributed to its increased surface roughness. filtek™ composite resin z350 xt (3m espe) has the highest percentage weight and volume filler compared to filtek™ bulk-fill (3m espe). according to silva et al. (2013)23, the physical and mechanical properties of composite resins are determined, among other factors, by the size, volume, and distribution of the filler particles in the matrix. in its organic matrix, filtek™ composite resin z350 xt (3m espe) presents pegdma, which, together with tegdma, is used to adjust viscosity. the lower the viscosity of the organic matrix, the higher the amount of charge that can be incorporated, resulting in improved mechanical strength. this justifies the low roughness and excellent surface smoothness of filtek™ composite resin z350 xt (3m espe) before immersion in rinses23. the use of mouthwashes is an excellent tool for biofilm control. often, the chemical resource used may be extended10. to simulate the clinical use of mouthwash for 2 minutes daily for one year, we immersed the specimens in both types of mouthwash for 12h24,25. the changes that mouthwashes can cause in the surface roughness of a restorative material depend on their composition. the causes of the changes to chemical structure and molecules of the polymer chains are critical to determine the degree of alteration by the aqueous environment on the restorative material12. 8 zica et al. although oral antiseptics are widely recommended for plaque control, their excessive use can damage restorative materials due to the low ph and alcohol present in the solutions9. the excessive use causes sorption and hygroscopic expansion phenomena, derived from acid production methacrylate as a consequence of the degradation process of enzymatic hydrolysis9. such factors can interfere in the polymeric matrix of the composite resins by the catalysis of the ester groups of the dimethacrylate monomers present in its composition11. however, the mouthwashes tested did not significantly interfere in the increase of the surface roughness of the tested composite resins, probably due to their high degree of conversion and consequent reduction in solubility31. in particular, the composite resin filtek™ bulk-fill (3m espe) has a large part of its composition with low solubility monomers, such as audma, afm, and ddma30, which may also have contributed to the smallest change in its surface after immersion in the mouthwashes tested. the 0.1µm difference used to estimate the sample size is considered a relatively small effect size when compared to other studies that found effect sizes larger than 1µm9. (in this study, the effect size for the difference between composite resins was 0.208mm and, therefore, we had a statistical power of almost 100% to detect this difference. however, the difference between the periods and the types of mouthwash was less than 0.04mm, which results in the statistical power of less than 50%. for this insignificant difference, it is not possible to distinguish whether the lack of a significant result is due to the sample size or if the difference does not exist. regardless of the cause, this difference may not have clinical significance. the results of this in-vitro work are consistent with other studies, such as that of lucena et al. (2010)12, in which the filtek™ composite resin z350 xt (3m espe) did not present a significant difference in surface roughness between mouthwashes either with or without alcohol. similarly, in another study, the surface roughness of restorative materials was evaluated after immersion in mouthwashes, leading to the conclusion that the mouthwashes do not promote a significant change in the surface roughness of filtek™ composite resin z350 xt (3m espe)9. the clinical effects of mouthwashes on composite resins may depend on some other reasons, such as plaque, beverages, eating habits, and mouthwash, which cannot be reproduced in vitro. this is a limitation of this study. these factors, whether acting together or separately, can influence the mechanical and physical characteristics and interfere in the longevity of the restorative treatment11. in this study, the samples were adequately polymerized in contact with the tip of the photoactivator, whereas in clinical practice, especially in posterior teeth, this is not possible. furthermore, as a limitation of the present study, the flat surface of the specimens cannot reproduce the clinical situation, such as the occlusal region, which has concave and convex areas. besides, there was difficulty in carrying out the stabilization of the specimens and the agitation of the mouthwashes so that the clinical situation of daily rinsing was simulated correctly. the results of this in-vitro study indicated intermaterial relationships, but they cannot be fully extrapolated to clinical practice. additional in-vitro studies using scanning electron microscopy, 9 zica et al. mechanical cycling, and in-vivo longitudinal studies should be performed to predict the clinical longevity of bulk-fill composites. since there are no findings in literature similar to this study, the behavior of the high viscosity filtek™ bulk-fill composite resin (3m espe) could not be clinically predicted. further studies comparing bulk-fill composite resins are required to assess whether the surface roughness found is acceptable for clinical use. in view of the limitations of the study, the use of mouthwashes does not interfere with the surface roughness of the tested composite resins, but the composite resin filtek z350 xt (3m espe) presents a surface with less roughness, being, therefore, more suitable for clinical use. acknowledgements we thank the technicians of the material engineering laboratory of the centro universitário newton paiva belo horizonte. references 1. ali z, eliyas s, vere jw. choosing the right dental material and making sense of the options: evidence and clinical recommendations. eur j prosthodont restor dent. 2015 sep;23(3):p150-62. 2. khalaf me, alomari qd, omar r. factors relating to usage patterns of amalgamand resin composite for posterior restorations a prospective analysis. j dent. 2014 jul;42(7):785-92. doi: 10.1016/j.jdent.2014.04.010. 3. kapoor n, bahuguna n, anand s. influence of composite insertion technique on gap formation. j conserv dent. 2016;19(1):77-81. doi: 10.4103/0972-0707.173205. 4. chandrasekhar v, rudrapati l, badami l, tummala m. incremental techniques in direct composite restoration. j conserv dent. 2017;20(6):386-91. doi: 10.4103/jcd.jcd_157_16. 5. rizzante fap, mondelli frfl, furuse ay, borges afs, mendonça g, ishikiriama sk. shrinkage stress and elastic modulus assessment of bulk-fill composites. j appl oral sci. 2019 jan 7;27:e20180132. doi: 10.1590/1678-7757-2018-0132. 6. van ende a, de munck j, lise dp, van meerbeek b. bulk fill composites: a review of the current literature. j adhes dent. 2017;19(2):95-109. doi: 10.3290/j.jad.a38141. 7. festuccia mscc, garcia lf, cruvinel dr, pires-de-souza fcp. color stability, surface roughness and microhardness of composites submitted to mouthrinsing action. j appl oral sci. 2012;20(2):200-5. doi: 10.1590/s1678-77572012000200013.  8. sadaghiani l, wilson ma, wilson nh. effect of selected mouthwashes on the surface roughness of resin modified glass-ionomer restorative materials. dent mater. 2007 mar;23(3):325-34. doi: 10.1016/j.dental.2006.01.024.  9. bohener lol, godoi apt, ahmed as, tortamano neto p, catirse abceb. surface roughness of restorative materials after immersion in mouthwashes. eu j gen dent. 2016;5(3):111-4. doi: 10.4103/2278-9626.189255. 10. borges ah, pedro flm, semenoff tdv, porto n, semenoff-segundo a, buzelle sl. antimicrobial effectiveness of different trademarks mouthwashes with and without alcohol against different organisms: in vitro study. rev odonto cienc. 2010; 25(2):178-81. doi: 10.1590/s1980-65232010000200014. 10 zica et al. 11. miranda da, bertoldo ces, aguiar fhb, lima danl, lovadino jr. effects of mouthwashes on knoop hardness and surface roughness of dental composites after different immersion times. braz oral res. 2011;25(2):168-73. doi: 10.1590/s1806-83242011000200012. 12. lucena mcm, gomes rvs, santos mcms. 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[influence of mouthwashes on surface roughness of a composite resin]. rev bahiana odontol. 2016;7(4):243-52. doi: 10.17267/2596-3368dentistry.v7i4.936. portuguese. 26. green p, macleod cj. simr: an r package for power analysis of generalized linear mixed models by simulation. methods ecol evol. 2016;7(4):493-8. doi: 10.1111/2041-210x.12504. 27. r core team. the r project for statistical computing. r: a language and environment for statistical computing. 2019. available from: https://www.r-project.org. https://pubmed.ncbi.nlm.nih.gov/31172875/?from_term=mouthwashes+%2c+surface+roughness+of+bulk-fill+composite+resins&from_pos=2 https://pubmed.ncbi.nlm.nih.gov/31172875/?from_term=mouthwashes+%2c+surface+roughness+of+bulk-fill+composite+resins&from_pos=2 https://pubmed.ncbi.nlm.nih.gov/29178729/?from_term=+surface+roughness+of+bulk-fill+composite+resins&from_pos=4 https://pubmed.ncbi.nlm.nih.gov/29178729/?from_term=+surface+roughness+of+bulk-fill+composite+resins&from_pos=4 https://pubmed.ncbi.nlm.nih.gov/31774724/?from_term=+surface+roughness+of+bulk-fill+composite+resins https://pubmed.ncbi.nlm.nih.gov/31774724/?from_term=+surface+roughness+of+bulk-fill+composite+resins https://pubmed.ncbi.nlm.nih.gov/31774724/?from_term=+surface+roughness+of+bulk-fill+composite+resins file:///d:/%5bjobs%5d/diagrama%c3%87%c3%95es/8569/consolidado/javascript:void(0) file:///d:/%5bjobs%5d/diagrama%c3%87%c3%95es/8569/consolidado/javascript:void(0) 11 zica et al. 28. ilie n, schöner c, bücher k, hickel r. an in-vitro assessment of the shear bond strength of bulk-fill resin composites to permanent and deciduous teeth. j dent. 2014 jul;42(7):850-5. doi: 10.1016/j.jdent.2014.03.013. 29. papadogiannis d, tolidisk, gerasimou p, lakes r, papadogiannis y. viscoelastic properties, creep behavior and degree of conversion of bulk fill composite resins. dent mater. 2015 dec;31(12):1533-41. doi: 10.1016/j.dental.2015.09.022. 30. 3m science. applied to lifetm. available from: http://solutions.3m.com.br/wps/portal/3m/pt_br/skinwoundcare/home/healthcareacademy/odontologia/materiaistecnicos?wt.mc_id=www.3 m.com. br/healthcareacademy. 31. cebe ma, cebe f, cengiz mf, cetin ar, arpag of, ozturk b. elution of monomer from different bulk fill dental composite resins. dent mater. 2015 jul;31(7):e141-9. doi: 10.1016/j.dental.2015.04.008. oral sciences n3 original article braz j oral sci. october | december 2015 volume 14, number 4 color stability and surface roughness of artificial teeth brushed with an experimental ricinus communis toothpaste lourenço de moraes rego roselino¹, carla cecília alandia-román¹, vanessa maría fagundes leite¹, cláudia helena silva-lovato¹, fernanda de carvalho panzeri pires-de-souza¹ ¹universidade de são paulo – usp, ribeirão preto school of dentistry, department of dental materials and prosthodontics, ribeirao preto, sp, brazil correspondence to: fernanda de carvalho panzeri pires-de-souza faculdade de odontologia de ribeirão preto usp departamento de materiais dentários e prótese av. do café, s/n bairro monte alegre cep: 14040-904 ribeirão preto, sp, brasil phone: +55 16 33153973 fax: +55 16 36330999 e-mail: ferpanzeri@usp.br abstract aim: to evaluate, in vitro, the effect of brushing with a ricinus communis-based experimental toothpaste on color stability and surface roughness of artificial teeth. methods: ninety artificial teeth (maxillary central incisors) in different shades, light and dark (natusdent triple pressing, dentbras) were used. initial color (spectrophotometer easyshade, vita) and surface roughness (rugosimeter surfcorder se 1700, kosakalab) readouts were performed. after baseline measurements, samples were assigned to 10 groups (n=9) according to the artificial tooth shade and type of toothpaste used during the mechanical brushing test (pepsodent, mavtec): sorriso dentes brancos – sdb, colgate luminous white clw (colgate-palmolive), close up white now cwn (unilever), trihydral thl (perland pharmacos) and ricinus communis rce (experimental). after 29,200 cycles of brushing, corresponding to 2 years of brushing by a healthy individual, new color and roughness readouts of the specimens were performed. data (before and after the tests) were statistically analyzed (2-way repeated measures anova, tukey, p<0.05). results: rce toothpaste produced the greatest color stability for dark tooth shade and the second best color stability for light tooth shade. for surface roughness alteration, there was no difference (p>0.05) for any tested toothpaste regardless of tooth shade. conclusions: the experimental ricinus communis toothpaste did not cause color and surface roughness alteration in the artificial teeth, and it may be considered a suitable option for denture cleaning. keywords: oral hygiene; toothpastes; castor oil; color. introduction with the increasing life expectancy and consequent ageing of the worldwide population, wear and stain of artificial teeth have become more and more common, jeopardizing the aesthetic appearance of removable prosthesis, since artificial teeth are a significant factor for the aesthetic outcome. one of the most important cautions that the edentulous patients must have is the maintenance of adequate complete denture hygiene1, which is important to increase denture longevity2. methacrylate-based resins (acrylic resin) are the most commonly used material for artificial denture teeth in prosthetic dentistry, due to many advantages, such as the chemical bond to denture base, lower susceptibility to fracture, easy handling and limited biofilm accumulation3-4. however, one disadvantage is the increased wear and discoloration after certain time of use1. to overcome this shortcoming, highly resistant resin teeth with an interpenetrating polymer network was developed received for publication: november 18, 2015 accepted: december 11, 2015 braz j oral sci. 14(4):267-271 http://dx.doi.org/10.1590/1677-3225v14n4a03 268268268268268 increasing the amount of cross-linked agents or blending special polymers and co-polymers. improved mechanical and physical properties have been achieved5-6, but susceptibility to staining and wear7 still remains. different from patients with remaining natural teeth, edentulous patients have no periodontal ligament receptors that regulate occlusal forces8. this is an aggravating factor for artificial teeth wear, because despite the reduced maximum occlusal force when compared with natural teeth, edentulous patients have a higher masticatory frequency9. cleaning methods for dentures include daily brushing and immersion in denture cleaners10. there is a large number of chemical dental cleansers for immersion, containig substances like alkaline peroxides, alkaline hypochlorites, acids, enzymes and disinfectants, which are widely used for denture cleaning due to their bactericidal and fungicidal properties11. however, some of these solutions may cause alterations in the physical and mechanical properties of denture base resin 12 and artificial teeth 13 due to the penetration of these agents into the material. brushing is the most commonly used method for denture hygiene, since it is a simple, inexpensive and effective hygiene method 14. furthermore, brushing has the same efficiency as a chemical method for biofilm removal14. the brushing method is combined with the use of toothpastes, which have a complex composition including water, detergents, flavoring, abrasive and therapeutic agents. due to its characteristics, many studies have shown that brushing is the cleaning method of choice for complete dentures wearers15. however, if improperly used, the acrylic resin may become rough16, leading to microorganism and organic matter retention17, reducing cleaning and increasing stain. the addition of antimicrobial agents facilitates the cleaning and sanitizing procedures for denture users18. many plants are known to possess antimicrobial properties, and several innovative attempts have been made to incorporate them into daily oral hygiene products. ricinus communis, also known as castor plant, is found in tropical climates, as in brazil19. its oil has been used to produce detergent agents and could be an alternative for denture cleaning, as it causes cell destruction by breaking the sugar leakage of the cellular wall of pathogenic microorganisms20. recently, a study has demonstrated the antimicrobial potential of a ricinus communis-based experimental dentifrice for denture hygiene which seems to be related to the presence of a strong surfactant18. due to the constant pursuit of aesthetics, and the importance of durability, staining resistance and color-stability of denture teeth21, the objective of this study was to evaluate in vitro the effect of brushing with an experimental ricinus communis-based toothpaste on color stability and surface roughness of artificial teeth. the tested null hypothesis was that the experimental dentifrice will not produce color change and surface roughness in the artificial teeth. material and methods the present study used 90 artificial teeth (maxillary central incisors) in different shades, light (60) and dark (69) (natusdent tripla prensagem dentbras ind, pirassununga, sp, brazil). teeth were embedded in pvc cylinders (1 cm high x 2 cm diameter) with self-cured acrylic resin (vipiflash, ltda. pirassununga, sp, brazil) using a paralellometer to stabilize their buccal surface facing upwards. initial color (spectrophotometer easyshade, vita zahnfabrik, badsäckingen, germany) and surface roughness (rugosimeter surfcorder se 1700, kosakalab, tokyo, japan) readouts of the specimens were taken. for color readouts, the observation pattern followed the cie l*a*b* system (comission internationale de i’e’ clairage) which simulates a measurement of 45/0 geometry, d65 standard illuminant, and observer pattern of 2o. the specular component was excluded in order to prevent interference by surface brightness. the l*a*b* system consists of two axes, a* and b*, at right angle and represent the dimension of tonality or color. the third axis is luminosity l*, perpendicular to the a* b* plane. for color readings, the digital tip of the device (6 mm diameter) was positioned perpendicular to the buccal surface of artificial teeth and three measurements were taken; the mean of them was considered the baseline color value. for surface roughness measurements, a needle with a very fine diamond at its tip was driven at constant speed over a distance of 4.8 mm with a 0.8 mm cut-off and a 0.25 mm/s speed . three readouts were taken at different sites on the sample surface to obtain the mean roughness (ra). after color and roughness baseline readings, samples were assigned to 10 groups (n=9) according to the shade of the artificial tooth and type of toothpaste used during the mechanical brushing test (table 1). mechanical brushing (pepsodent, mavtec – com parts, acc., and serv. ltda. me, ribeirão preto, sp, brazil) was performed with soft toothbrushes (tek, johnson & johnson ind. com ltda., são josé dos campos, sp, brazil) – one per specimen. the toothbrush heads were separed from stem and then fitted into the clamp of the equipment. each brush/ clamp set resulted in a final weight of 200 g. each specimen was fitted into a central circular hole (of equal dimensions) of a plexiglass slide (acrilpress artefacts ltda., ribeirão preto, sp, brazil) to allow adaptation and fixation during mechanical brushing (course=3.8 cm; speed=356 rpm) with toothpaste diluted in distilled water (1:1). specimens were subjected to 29,200 cycles of mechanical brushing, corresponding to 2 years of exposure to brushing by a healthy individual22. after brushing, new color and roughness readouts were performed. color alteration (∆e) was calculated according to the formula: ∆e = [(“l*)2 + (“a*)2 (“b*)2]1/2 where ∆e=color alteration; ∆l=lightness difference (l*), so that the greater l* values, the higher the brightness of the sample; ∆a=axis a* difference; so that positive values for ∆a means redder samples and negative values, greener; ∆b=axis b* difference; so that positive values for ∆b mean yellower samples and negative values bluer, being: color stability and surface roughness of artificial teeth brushed with an experimental ricinus communis toothpaste braz j oral sci. 14(4):267-271 groups 1 2 3 4 5 6 7 8 9 10 shades light dark light dark light dark light dark light dark toothpaste sorriso dentes brancos (palmolive ltda, são bernardo do campo, sp, brazil) colgate luminous white (palmolive ltda, são bernardo do campo, sp, brazil close up white now (unilever ltda, ipojuca, pe, brazil) trihydral (perland pharmacos ltda, cornélio procopio, pr, brazil) ricinus communis (experimental) composition calcium carbonate, water, glycerin, sodium lauryl sulfate, sodium monofluorophosphate (1450 ppm fluoride), cellulose gum, tetrasodium pyrophosphate, sodium bicarbonate, benzyl alcohol, sodium saccharin, sodium hydroxide and limonene. water, hydrated silica, glycerin, pentasodium triphosphate, peg-12, tetrapotassium pyrophosphate, sodium lauryl sulfate, cellulose gum, polyethylene, cocamidopropyl betaine, xanthan gum, sodium saccharin, sodium hydroxide, titanium dioxide, d & c blue 1 aluminum lake (ci 42090) and sodium fluoride (1100 ppm fluoride). sorbitol, water, silica, peg-32, sodium lauryl sulfate, cellulose gum, sodium fluoride (1450 ppm), saccharin sodium, pvm / ma copolymer, trisodium phosphate, mica, cl 74160 and limonene. sodium fluoride (1500 ppm), sodium monofluorophosphate, 0.2% chloramine-t, calcium carbonate, propylene glycol, sorbitol, hydroxyethylcellulose, tetrasodium pyrophosphate, sodium laurylsulfate, decyl polyglucose, metilpabenzeno and water. hydroxyethylcellulose, glycerine, edta, sodium saccharin, ricinoleic acid ester, silica (sident 9) silica (sident 22s), titanium dioxide, methylparaben, sodium lauryl sulfate and water. table 1. table 1. table 1. table 1. table 1. groups formed according to tooth shade and dentifrice used in the study. toothpastes light shade (60) dark shade (69) sorriso dentes brancos (sdb) 2.57(0.93)aa 1.50(0.43)bab colgate luminous white (clw) 2.17(0.60)aa 2.27(0.62)aa close up white now (cwn) 0.68(0.35)ab 1.17(0.65)ab trihydral (thl) 0.84(0.43)ab 1.13(0.88)ab experimental (rce) 0.72(0.29)ab 0.83(0.51)ab table 2. table 2. table 2. table 2. table 2. means (standard deviation) of color stability (∆e) of artificial teeth (light and dark) subjected to mechanical brushing with different toothpastes (2-way anova, bonferroni test, p<0.05). different letters, lowercase in rows and uppercase in columns, indicate statistically significant difference (p<0.05). toothpastes light shade dark shade sorriso dentes brancos (sdb) 0.349(0.32) 0.156(0.46) colgate luminous white (clw) 0.402(0.22) 0.217(0.28) close up white now (cwn) -0.181(0.48) -0.131(0.35) trihydral (thl) -0.134(0.49) -0.036(0.79) experimental (rce) -0.056(0.38) -0.286(0.68) table 3. table 3. table 3. table 3. table 3. means (standard deviation) of surface roughness alteration ∆ra of artificial teeth (light and dark) subjected to mechanical brushing with different dentifrices (2-way anova, bonferroni test, p<0.05). there was no statistically significant difference among the groups (p>0.05). ∆l=lf – li ∆a=af – ai ∆b=bf – bi where l*i, a*i e b*i are referred to as the initial color measurement and l*f, a*f e b*f as the final color measurement. color alteration values greater than 3.3 were considered clinically unacceptable10. surface roughness alteration (∆ra) was calculated according to the formula: ∆ra=raf rai where rai is the initial roughness measurement and raf the final roughness measurement. after verifying the normality of the results, the values were analyzed by 2-way repeated measures anova and by the multiple-comparisons bonferroni test with 95% significance level. results the means of color alteration (∆e) and surface roughness (∆ra) were compared (2-way repeated measures anova, bonferroni test, p<0.05), considering treatments and tooth shade as variation factors. (table 2 and 3 respectively). for light teeth shade, sdb and clw toothpastes presented greater (p<0.05) color change compared with the other toothpastes, which showed no difference (p>0.05) between them. regarding dark teeth shade, the greatest color change (p<0.05) occurred after brushing with clw, a result similar (p>0.05) to sdb, which showed no difference (p>0.05) compared with other toothpastes. rce toothpaste produced the greatest color stability for dark teeth shade and the second best color stability for light teeth shade. when the effect of toothpastes was compared for light and dark teeth shade, only sdb produced significant color change (p<0.05), greater for light teeth shade. there was no statistical significance ( p>0.05) for the other comparisons. the comparison of surface roughness alterations showed 269269269269269 color stability and surface roughness of artificial teeth brushed with an experimental ricinus communis toothpaste braz j oral sci. 14(4):267-271 270270270270270 no significant difference (p>0.05) for any tested toothpaste regardless of tooth shade. discussion this study evaluated the effect of mechanical brushing (29,200 cycles = 2 years) on the color stability (∆e) and surface roughness (∆ra) of two shades (60 and 69) of artificial teeth brushed with different toothpastes. the null hypothesis stated that there would be no difference in the studied properties regardless the tested toothpastes. considering the results, it was possible to accept partially the tested hypothesis, since there was a greater color alteration for teeth brushed with two types of toothpaste (sdb and clw), this alteration was greater when sdb was used on light tooth shade. however, there was no surface roughness alteration regardless of the tested toothpaste or tooth shades. when ∆e was analyzed, light shade samples brushed with rce (experimental), cwn and thl toothpastes, showed less color alteration than those brushed with sdb and clw. the same occurred for dark shade samples, except for those brushed with sdb, which showed a ∆e similar to the samples brushed with the other toothpastes used in the study. the color alteration of a material is expressed in units of ∆e, which represent the color change after different treatments. in dental literature, several authors23 have used ∆e values to evaluate the “““““perceptibility ” ” ” ” ” of color differences. color difference thresholds constitute the lowest perceptual limit to estimate the maximum number of discernible colors by the human visual system. there are two m ajor thresholds for assessing color differences: perceptible and acceptable24, passing from “just noticeable differences” to “color tolerances”25. however, it is noteworthy to underline that the criteria of perceptibility adopted by each author are different. in this study, the threshold used as perceptible was ∆e=2.7 25, above this value, color changes would be considered aesthetically unacceptable. thus, analyzing the results, it was found that all samples showed acceptable levels of color change, regardless the used type of dentifrice. the denture teeth used in this study are composed by triple-pressed acrylic layers of pmma (polymethyl metacrylate) and edma (dimethacrylate of polymerized ethylene glycol), which is a cross-linking agent. however, even the more resistant teeth are subject to water sorption, demonstrated by microhardness decrease26. the water or aqueous cleansers can act as resin plasticizers, since the small molecules of water diffuse into the polymer mass, causing the relaxation of polymer chains27. thus, when penetrating the artificial tooth, solvents may also be responsible for the color change of teeth. furthermore, artificial teeth have small amounts of dibenzoyl peroxide, a heat-sensitive initiator added to the polymer, which remains after polymerization and may also alter the material’s optical properties28. even though the color stability may have been compromised by the above-mentioned factors, in this study the teeth showed color changes within the acceptable limits and the experimental ricinus communis-based dentifrice showed the smallest color change values. this may be justified by the effect of polishing and wear produced by the dentifrice abrasiveness, as evidenced by the negative values found for roughness changes. increased values of surface roughness caused by the formation of a ricinus communis pellicle on the specimen surface have been reported in a previous study12 that used ricinus communis in an experimental immersion cleaning solution. this pellicle was not observed in the present study, since brushing and subsequent polishing of the tooth surface kept teeth free of this pellicle, with non-significant surface roughness alteration, maintaining the color stability. besides the polishing effect, the composition of toothpastes may have also contributed to these findings. sdb has calcium carbonate in its composition, unlike the other tested dentifrices, which have silica as abrasive agent. calcium carbonate has a lower abrasive potential than silica, but this component is not the sole responsible for dentifrice abrasiveness. clw is more abrasive than sdb as it presents highly abrasive components in its composition, such as tetra potassium pyrophosphate. one could expect higher levels of color alteration because of the dentifrice abrasiveness; however, color changes occurred within the acceptable limits, which might be justified by the presence of a bleaching agent, sodium hydroxide, in the dentifrice composition. it is important to highlight the research and efforts undertaken worldwide to develop new plant-derived materials. the experimental resinus communis -based dentifrice produced similar color alteration when compared with most commercial toothpastes, including the specific dentifrice for denture cleaning (trihydral), and did not increase the surface roughness of teeth. these are important findings for the validation of the new experimental material. it may be concluded that artificial acrylic resin teeth brushed with the experimental ricinus communis-based dentifrice did not undergo color or surface roughness alteration, thus the experimental dentifrice may be considered a suitable option for denture cleaning. acknowledgements the authors would like to thank the são paulo research foundation (brazil), for their financial support, protocol no. 2012/13342-4. the authors have no conflict of interests to disclose. references 1. andrade im, andrade km, pisani mx, silva-lovato ch, de souza rf, paranhos h de f. trial of an experimental castor oil solution for cleaning dentures. braz dent j. 2014; 25: 43-7. 2. pisani mx, da silva ch, paranhos hf, souza rf, macedo ap. evaluation of experimental cleanser solution of ricinus communis: effect on soft denture liner properties. gerodontology. 2012; 29: 179-85. 3. heintze sd, zellweger g, sbicego s, rousson v, munoz-viveros c, stober t. wear of two 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properties of acrylic resins subjected to chemical polishing. j oral rehabil. 2003; 30: 91-8. 28. assuncao wg, barao va, pita ms, goiato mc. effect of polymerization methods and thermal cycling on color stability of acrylic resin denture teeth. j prosthet dent. 2009; 102: 385-92. color stability and surface roughness of artificial teeth brushed with an experimental ricinus communis toothpaste braz j oral sci. 14(4):267-271 1http://dx.doi.org/10.20396/bjos.v20i00.8659320 volume 20 2021 e219320 original article 1 school of dentistry, community university of chapecó region (unochapecó), chapecó, sc, brazil. 2 private practice, chapecó, sc, brazil. 3 health sciences post-graduate program, community university of chapecó region (unochapecó), chapecó, sc, brazil. corresponding author: prof. dr. sinval adalberto rodrigues-junior, community university of chapecó region – unochapecó, área de ciências da saúde – caixa postal 1141, servidão anjo da guarda 295-d – efapi, cep 89809-000 – chapecó – sc – brazil; phone number: +55 (49) 3321-8069; e-mail: rodriguesjunior.sa@ unochapeco.edu.br received: april 28, 2020 accepted: december 9, 2020 effect of whitening mouth rinses on the chemical and physical properties of a nanofilled composite laura carolina kepler1 , ana paula morona rodrigues2 , mauro antonio dall agnol1 , sinval adalberto rodrigues-junior3,* aim: this study analyzed the effect of whitening mouth rinses on water sorption (ws), solubility (sl), color change, and surface roughness of a nanofilled composite. whitening perceptibility and acceptability (wid) were also studied. methods: forty specimens of filtek z350xt, shade ea2 were produced and randomly distributed (n=8) to as – artificial saliva (control); lwe – listerine whitening extreme; clw – colgate luminous white; lcm – listerine cool mint; and cp – colgate plax. they were immersed in the mouth rinses 2x/day, for one minute, during 28 days. the color was assessed using an easyshade spectrophotometer (cie-l*a*b* system). surface roughness (ra-µm) was measured with three parallel measures, using an rp-200 roughness meter. the ws and sl (μg/mm-3) were analyzed based on the iso 4049 recommendations. the data were analyzed using oneand two-way anova/tukey tests (α=0.05). results: surface roughness significantly increased after immersion in as and lcm, with no significant differences between the groups either before or after immersion. the ∆e* was not significantly different between the groups. all substances produced a ∆wid higher than the 50%:50% perceptibility and acceptability thresholds. the ws and sl were not significantly affected by the mouth rinses. conclusion: whitening mouth rinses did not affect ws, sl, surface roughness, and color stability of a nanofilled composite, regardless of the presence of ethanol in the composition. keywords: tooth bleaching agents. nonprescription drugs. mouthwashes. color. composite resins. mailto:rodriguesjunior.sa@unochapeco.edu.br mailto:rodriguesjunior.sa@unochapeco.edu.br http://orcid.org/0000-0002-8304-5671 http://orcid.org/0000-0003-0761-7284 http://orcid.org/0000-0002-5581-105x http://orcid.org/0000-0002-4475-1725 2 kepler et al. introduction mouth rinse solutions are adjunct products for oral self-care. as such, most mouth rinses have an anti-microbial effect1 or a fluoride-releasing effect accounting for the control of the demineralization-remineralization process2. mouth rinses also have become, since the 2000s, a means of attending to the growing cosmetic demand for tooth whitening. these over-the-counter products are acquired and administered by the patient, not requiring professional prescription and supervision during application3. whitening mouth rinses may present up to 2% of hydrogen peroxide4. other substances, such as sodium hexametaphosphate, may help to prevent tooth surface staining5. whitening mouth rinses also share similar substances with conventional mouth rinses, such as detergents, dyes, organic acids, emulsifiers, and alcohol6. alcohol is not present in every mouth rinse but those containing it present antiseptic and preserving functions, besides dissolving active ingredients7. additionally, alcohol softens dimethacrylate-based resin composites, accelerating their degradation8. anterior composite restorations have failed mostly due to esthetic reasons such as color change and surface staining9, and the esthetic appearance of dental restorations and its decrease are related to the hydrolytic degradation of the material10. the effect of an alcohol-containing mouth rinse on the solubility of a dimethacrylate-based composite has been shown but in conditions that do not emulate the clinical use of the mouth rinse11. therefore, the extent of degradation triggered by the alcohol present in mouth rinses in daily mouthwashes of one to two minutes remains unclear. the hydrogen peroxide present in some whitening mouth rinses has produced a whitening effect on previously stained composites12. this occurs when the whitener breaks dark complex molecules of pigment into simpler structures with lighter optical properties12. it is noteworthy that a significant whitening effect only occurs in the presence of acquired extrinsic stains and it is not an issue when the composite is not extrinsically pigmented12,13. according to goldberg et al.14 (2010), hydrogen peroxide increases the leaching of components from amalgam and fluoride-containing restorative materials, affecting the bond interface between composite and bleached dentin. nevertheless, the effect of hydrogen peroxide on composite degradation is not fully established, requiring further research. additionally, the level of perceptibility and acceptability of the composite color change from the contact with mouth rinses, from a viewer standpoint, must be explained13. considering the likelihood of substances contained in whitening mouth rinses to potentially help the degradation of composites, expectations are that surface and bulk properties such as color change and surface roughness are affected by contact with these oral hygiene products. therefore, this study aimed to test the hypothesis that whitening mouth rinses affect the chemical and physical properties of a nanofilled universal composite resin. materials and methods forty disc-shaped specimens with 10 mm of diameter (d) and 2 mm of height (h) were produced with the filtek z350xt nanofilled composite (shade ea2, 3m/espe, st. paul, mn, usa) using a circular stainless steel mold (odeme, luzerna, sc, brazil). the specimens were light-cured with 20-second overlapped light exposures on the top and 3 kepler et al. bottom surfaces, according to the iso 404915 recommendations. an led light-curing unit (ultraled, dabi atlante, ribeirão preto, sp, brazil) was used and had its light irradiance monitored at the beginning and end of each specimen build-up session using an led radiometer (ecel, ribeirão preto, sp, brazil) – mean irradiance of 1193mw/cm2. the specimens were finished with a #400 sandpaper and had their dimensions measured with a digital caliper (mitutoyo corporation, kanagawa, japan) and averaged based on the iso 4049 recommendations15. the diameter was cross-measured and averaged from the two measures, while the height was measured in five points (four extremities and one in the center). these measures were used to calculate the volume of each specimen: v = π. r2. h, where r is the specimen radius (d/2). intervention protocol (mouth rinses) the specimens were randomly allocated into five groups (n=8)16 using an online random number generator (www.random.org). grouping involved two whitening mouth rinses and two conventional (non-whitening) mouth rinses, along with a negative control group: as– artificial saliva (control); lwe– listerine whitening extreme; clw– colgate luminous white; lcm– listerine cool mint; and cp– colgate plax. the artificial saliva was prepared in the institutional pharmacology laboratory, by a pharmacology technician, based on the formulation developed by the school of pharmaceutical sciences of the são paulo state university (table 1). the ph of the substances was measured in the same laboratory, in duplicate, using a ph meter (q-400a, quimis aparelhos científicos ltda., diadema, sp, brazil) previously calibrated with standard solutions. table 1 presents the compositions of the substances used in the study. the specimens were immersed daily, twice a day, for one minute in the solutions, for 28 days. during the treatment, the specimens were stored in artificial saliva at 37oc, which was changed once a day. table 1. materials used in the study product manufacturer components ph filtek z350xt 3m/espe, st. paul, mn, usa bis-gma*, bis-ema (6)*, udma*, tegdma*, 78.5% of filler particles in weight (clusters of 0.6-1.4µm – individual particle size of 5-20nm) – silica and zirconia artificial saliva (santos17 2008) institutional pharmacology laboratory potassium chloride, sodium chloride, magnesium chloride, potassium phosphate, calcium chloride, nipagin, nipasol, carboxymethylcellulose, sorbitol, distilled water 5.3 colgate plax colgate-palmolive, são bernardo do campo, sp, brazil water, glycerin, propylene glycol, sorbitol, poloxamer 338, poloxamer 407, aroma, peg-40 hydrogenated castor oil, cetylpyridinium chloride, potassium sorbate, sodium fluoride, sodium saccharin, citric acid, sucralose, ci 42053 4.8 listerine cool mint johnson & johnson industrial ltda., yumbo, valle, colombia water, sorbitol, alcohol, poloxamer 407, benzoic acid, sodium saccharin, eucalyptol, aroma (d-limonene), thymol, methyl salicylate, sodium benzoate, menthol, ci 42053 4.2 colgate luminous white colgate-palmolive, são bernardo do campo, sp, brazil water, glycerin, propylene glycol, sorbitol, tetrapotassium pyrophosphate, polysorbate 20, tetrasodium pyrophosphate, zinc citrate, pvm/ma copolymer, aroma, benzyl alcohol, sodium fluoride, sodium saccharin, ci 42051 7.2 listerine whitening extreme johnson & johnson industrial ltda., yumbo, valle, colombia water, alcohol, 2.5% hydrogen peroxide, aroma, poloxamer 407, sodium saccharin, menthol, phosphoric acid, disodium phosphate, sodium fluoride, sucralose 3.5 * bis-gma=bisphenol a-glycidyl methacrylate, bis-ema=bisphenol a diglycidyl methacrylate ethoxylated, tegdma=triethylene glycol dimethacrylate, udma=urethane dimethacrylate http://www.random.org 4 kepler et al. water sorption and solubility test to determine water sorption and the solubility of the composite after the mouth rinse protocol, the specimens were first dried, stored in a desiccator containing silica gel at 37oc, and weighted daily to check for mass stabilization (dry mass, m1). then, the specimens were submitted to the mouth rinse application protocols and stored in artificial saliva. at the end of the 28-day application period, the specimens were reweighted to obtain the wet mass (m2). finally, the specimens were stored in the desiccator at 37oc again and weighted until they reached a new stable dry mass (m3). an analytical balance with 0.1-mg accuracy (auw220d; shimadzu corporation, kyoto, japan) was used to weigh the specimens. water sorption (ws) and solubility (sl) were expressed as μg/mm-3 and calculated as follows: ws = m2 − m3/v and sl = m1 − m3/v 15. surface roughness test the mean surface roughness (ra) was measured by three parallel surface readings along a 4-mm length (cut-off=0.8mm) in the top surface of the specimen. a roughness meter rp200 (instrutherm, são paulo, sp, brazil) was used to read the surface roughness before and after the mouth rinse application protocol18. color change analysis the color was read before and after the 28-day application protocol, using an easyshade portable digital spectrophotometer (vita zahnfabrik, bad säckingen, germany). the specimens were positioned over a white background under the same lighting conditions, and the color was read by positioning the spectrophotometer probe perpendicular to the specimen’s surface. the color results were expressed based on the three-dimensional ciel*a*b* system. this system considers three color parameters, represented by three axes. the l* axis represents luminosity and varies from 0 (black) to 100 (white), the a* axis represents the color variation between red (a* positive) and green (a* negative), and the b* axis varies between yellow (b* positive) and blue (b* negative). color change in these axes is represented by ∆l*, ∆a*, and ∆b*, which are used to calculate the ultimate general color change (∆e*) through the equation: ∆e*ab = (l * 2 l * 1) 2 + (a*2 a * 1) 2 + (b*2 b * 1) 2 the 50%:50% perceptibility and acceptability thresholds for color change of 1.2 and 2.7 were used, respectively19. also based on the ciel*a*b* three-dimensional color space, the whitening index for dentistry (wid) 20 was calculated to generate the ∆wid, which represents the difference of whiteness before and after treatment with the mouth rinses. wid = 0.511l * 2.324a* 1.100b* for the ∆wid, 50%:50% perceptibility and acceptability thresholds of 0.61 and 2.90 were used, respectively19. statistical analysis first, the data were verified for normal distribution and equal variances. outliers were recognized using grubb’s test and removed from the dataset. the data of surface 5 kepler et al. roughness, water sorption, solubility, ∆a*, ∆b*, ∆l*, ∆e*, and ∆wid were analyzed with one-way analysis of variance (anova). the data of a*, b*, l*, and wid were analyzed with two-way anova, considering the immersion substances and time as factors. for these analyses, tukey’s complementary test was applied and the level of significance of all analyses was α=0.05. results the mouth rinse application protocol did not show a significant effect either for composite water sorption (p=0.656) or solubility (p=0.207) (figure 1). surface roughness significantly increased after immersion in saliva (p=0.023) and listerine cool mint (p=0.011) (figure 2). figure 1. water sorption and solubility of the nanocomposite filtek z350 after application protocol in whitening and conventional mouth rinses ∆ e* 3.0 2.5 2.0 1.5 1.0 0.5 0.0 control (as) lwe clw lcm cp 2.13 2.04 2.19 2.33 2.24 figure 3. color change (∆e*) of the nanocomposite filtek z350 after application protocol in whitening and conventional mouth rinses su rf ac e ro ug hn es s (r a) µm 0.6 0.5 0.4 0.3 0.2 0.1 0.0 lwe clw lcm cp 0.264 0.481 * * 0.294 0.323 0.255 0.273 0.211 0.442 0.244 0.396 *p<0.05 before after before after before after before after before after control (as) 6 kepler et al. table 2 presents the color results considering the l*, a*, and b* parameters, and color change was expressed as ∆l*, ∆a*, and ∆b*. luminosity (l*) was significantly affected by the immersion substances (p=0.018) but not by time (p=0.654) or the interaction between both (p=0.915). the ∆l* was not significantly affected by the immersion substances (p=0.667). the a* was significantly affected by the immersion substances (p<0.001), time (p<0.001), and the interaction between both (p=0.009). the a* reduced after the 28-day immersion period and ∆a* was significantly affected by the immersion substances (p=0.004), with colgate plax producing the highest ∆a*. as for b*, it was significantly affected by the immersion substances (p=0.024) and time (p<0.001) but not the interaction between both (p=0.692). immersion time reduced b* and artificial saliva produced the lowest b*. no significant difference was observed in ∆b* between the groups (p=0.945). overall color change (∆e*) was not significantly different between the groups (p=0.805) (figure 3). the color change results of all groups were higher than the perceptibility threshold but lower than the acceptability threshold. figure 3. color change (∆e*) of the nanocomposite filtek z350 after application protocol in whitening and conventional mouth rinses µg m m -3 40 35 30 25 20 15 10 5 0.0 lwe clw lcm cp 30.4 30.2 31.1 30.5 30.5 6.0 6.9 5.7 6.1 8.0 control (as) lwe clw water sorption solubility lcm cpcontrol (as) table 2. color parameters l*, a* and b* of the nanofilled composite filtek z350xt before and after application protocol in whitening and conventional mouth rinses color parameter baseline mean (sd) 28-day mean (sd) ∆ mean (sd) l* as 81.2 (0.8) a,a 81.1 (0.7) a,a -0.025 (0.315) a lwe 81.7 (1.0) a,ab 81.6 (0.7) a,ab -0.050 (1.016) a clw 81.7 (0.7) a,ab 81.8 (0.6) a,ab 0.100 (0.674) a lcm 81.9 (0.4) a,b 81.9 (0.5) a,b -0.037 (0.498) a cp 81.1 (0.8) a,ab 81.5 (0.8) a,ab 0.386 (0.445) a continue 7 kepler et al. table 3 presents the results of wid and ∆wid. the statistical analysis of the wid data revealed that the immersion substance (p<0.001), time (p<0.001), and the interaction between both (p=0.001) significantly affected wid. the wid significantly increased in all groups after the 28-day immersion protocol. the highest ∆wid was observed in the colgate plax conventional mouth rinse. all substances, including saliva, produced a ∆wid higher than the 50%:50% perceptibility and acceptability thresholds. discussion mouth rinses are adjunct over-the-counter substances for mouth cleansing. whitening mouth rinses supposedly present a tooth whitening effect, mainly produced by substances such as hydrogen peroxide5,21. considering the acquisition of mouth rinses does not require a professional prescription, anybody can use them, including individuals with esthetic dental restorations. therefore, understanding the effect of whitening mouth rinses in tooth-colored restorations is important to correctly advise a* as 1.2 (0.1) a,a 0.8 (0.1) b,a -0.438 (0.092) a lwe 1.2 (0.3) a,a 0.8 (0.1) b,a -0.314 (0.146) a clw 1.2 (0.3) a,a 0.7 (0.1) b,a -0.500 (0.330) ab lcm 1.3 (0.3) a,a 0.7 (0.1) b,a -0.587 (0.394) ab cp 0.9 (0.1) a,b -0.01 (0.1) b,b -0.871 (0.160) b b* as 20.9 (0.2) a,a 18.8 (0.3) a,a -2.062 (0.374) a lwe 21.2 (0.3) a,b 19.5 (0.3) a,b -1.829 (0.150) a clw 21.0 (0.6) a,ab 19.1 (0.5) a,ab -1.900 (1.009) a lcm 21.0 (0.6) a,ab 18.9 (0.5) a,ab -2.063 (0.918) a cp 21.0 (0.3) a,ab 19.0 (0.3) a,ab -1.986 (0.146) a lower case letters represent the comparison of groups in lines; capital letters represent the comparison of groups in columns; different letters reveal statistically significant differences in comparisons continuation table 3. wid and ∆wid of the nanofilled composite filtek z350xt before and after application protocol in whitening and conventional mouth rinses whiteness parameter wid ∆wid mean (sd) baseline mean (sd) 28-day mean (sd) as 15.7 (0.4) a,ab 19.0 (0.6) b,ab -3.3 (0.6) a lwe 15.6 (0.6) a,a 18.5 (0.5) b,a -2.9 (0.4) a clw 15.9 (0.4) a,ab 19.2 (0.5) b,ab -3.3 (0.9) a lcm 15.8 (0.4) a,b 19.4 (0.7) b,b -3.6 (0.6) ab cp 16.3 (0.5) a,c 20.8 (0.5) b,c -4.4 (0.6) b lower case letters represent the comparison of groups in lines; capital letters represent the comparison of groups in columns; different letters reveal statistically significant differences in comparisons 8 kepler et al. those who have them about possible impacting adverse effects such as increased roughness or restoration color change. our results revealed no significant effect of conventional and whitening mouth rinses on water sorption and solubility of the composite. moreover, the greatest changes in surface roughness occurred in the control group and with one of the conventional mouth rinses, also revealing no significant effect of whitening mouth rinses on this property. finally, the overall color change was not significantly affected by either mouth rinse. therefore, the study hypothesis that whitening mouth rinses would affect the chemical and physical properties of a nanofilled composite resin was rejected. no difference was observed in the water sorption and solubility of the composite resin in the different groups (figure 1). both are related to hydrolytic degradation, which mediates the deterioration of other properties of composite resins over time, affecting surface and bulk properties of the restoration10,22. water uptake within the polymer chains through voids leads to the cleavage of links that form polymers, producing smaller-sized molecules such as oligomers and monomers, which may be leached to the environment18. additionally, it causes a hygroscopic expansion of the material, ultimately resulting in the solubilization of other molecules such as fillers, catalysts, and unreacted monomers10. in this study, neither the presence of whiteners from some mouth rinses nor alcohol from others affected the extension of polymer degradation, as expressed by water sorption and solubility. the latter could have affected polymer chains differently, as it has been shown that aqueous solutions that associate water and ethanol potentiate polymer degradation due to the similarity of the solubility parameter with the polymer10. an ethanol-containing mouth rinse was previously identified for increasing the solubility of a dimethacrylate-based composite. however, these results were obtained with the authors storing the specimens for two days in contact with the mouth rinse11, while our study limited the contact with the mouth rinse to two minutes daily. under these conditions, which simulate one month of mouth rinse application twice a day, water sorption results were considered acceptable for all groups, considering the parameters set by the iso 4049. regarding solubility, the conventional mouth rinse group was the only one to produce solubility results above those considered acceptable by the iso 4049 standards (7.5 µg mm-3)15, suggesting a higher solubilizing effect on the composite resin. surface smoothness is an important aspect of esthetic dental restorations. it affects color by reflecting light differently and mechanically retaining surface stains6. polishing with a #400 sandpaper ensured a baseline surface roughness lower than 0.3 µm, which in turn increased to almost 0.5 µm in the negative control group consisting of artificial saliva and one of the conventional mouth rinses containing alcohol (figure 2). the presence of hydrogen peroxide or ethanol in the mouth rinses did not seem to have affected surface roughness results. therefore, using whitening or alcohol-containing mouth rinses does not seem an issue for the surface roughness of dental restorations, considering the total daily application time of two minutes. moreover, the presence of acidic substances such as phosphoric acid has been considered to affect the organic matrix of the composite23. except for colgate luminous white, all other substances presented a low ph. curiously, the substance with the lowest ph and the presence of phosphoric acid (listerine whitening extreme) did not affect significantly 9 kepler et al. the surface roughness of the composite studied. munchow et al.24 (2014), testing the effect of substances with varying acidic potentials on the surface degradation of composite resins, observed that all the substances affected similarly the surface roughness of the composite. according to the authors, factors such as the solubility parameter of the solvent and material, the cross-linked nature of the resin matrix, and solvent uptake may explain better the surface deterioration of the composite resin. the last two factors were similar in this study, leaving the first factor to explain the differences found for surface roughness in some of the groups tested. the results of this study revealed no significant overall color change in the nanofilled composite after mouth rinse immersions, including the whitening ones (figure 3). the color of tooth-colored restorations is usually a concern after tooth whitening, requiring a restoration replacement after tooth whitening is finished13. recent studies showed that perceptible color change (∆e*) might be induced by tooth whitening substances when extrinsically acquired stains are impregnated in the restoration12,25. however, lower thresholds were set recently for 50%:50% perceptibility and acceptability of color change. these thresholds aid dental patients and set values of 1.2 for perceptibility and 2.7 for acceptability based on the perception of a layperson19. in this recent scenario, even the color change results by farinon et al. from water storage would be considered perceptible by a layperson standpoint, as well as our color change results after immersions in mouth rinses and artificial saliva. these values would also be considered acceptable taking the recent 50%:50% threshold for acceptability19, making it unlikely to require a restoration replacement after applying the mouth rinse twice a day for one month, regardless of the type of mouth rinse. it is noteworthy that the presence of 2.5% hydrogen-peroxide in listerine whitening extreme did not play a role in color change, producing the lowest nominal ∆e* value of all groups. this lack of significant influence of a whitening substance in the mouth rinse may again be explained by the absence of extrinsic staining agents in the composite resin12,25, causing minimum color changes and unlikely presenting clinical significance25. the overall color change was mainly driven by the reduction in a* and b* axes values, which leads to color changes towards green in the a* axis and towards blue in the b* axis. both sets of mouth rinses (colgate and listerine) present pigments with a blueish/greenish color that could have caused color changes in these axes, despite the low contact time with the composite23. the wid significantly increased after the 28-day immersion protocol in all groups (table 3). according to pérez et al.20 (2016), the wid is the best approximation of the visual perception of whiteness based on a set of psychophysical experiments. it depends on the l*, a*, and b* coordinates of the three-dimensional color space. expectations were that a reduced variation in l* would produce an insignificant perception of whiteness before and after the immersion protocol, as previously observed22. however, our results showed that variations of a* and b* produced ∆wid results varying from 2.9 to 4.4 towards a whiter material. most importantly, the ∆wid results correspond to values higher than the 50%:50% perceptibility and acceptability thresholds for whiteness perception22, meaning that dental restorations may be perceived whiter after contacting the substances studied. 10 kepler et al. we methodologically decided to include a control group constituted by artificial saliva to reveal possible changes in properties that could have been caused by common substances in conventional and whitening mouth rinses. we also attempted to simulate a relevant clinical regimen of mouth rinse application involving daily applications, twice a day, for one minute26. the methodological limitations involved the light-curing protocol, which followed the recommendations of the iso 4049 and did not reflect the real light-curing conditions when a dental restoration is built-up. müller et al.27 (2017) highlight the role of product standards in providing in vitro reproducible test designs to characterize materials. they also call attention to the limited evidence on the clinical performance of these materials, suggesting further clinical approaches to confirm the in vitro results obtained. the results of this study suggest that whitening mouth rinses would interact with a composite resin similar to regular mouth rinses, not producing any additional deleterious effect on restorations. the study hypothesis was rejected because water sorption, solubility, surface roughness, and color of a densely polymerized nanofilled composite are not affected by a one-month mouth rinse application protocol with either whitening or conventional mouth rinses. acknowledgements this study was funded by fapesc, grant no. 06/2017. references 1. devore lr. antimicrobial mouthrinses: impact on dental hygiene. j am dent assoc. 1994 aug;125 suppl 2:23s-28s. doi: 10.1016/s0002-8177(94)14004-5. 2. marinho vcc, chong ly, worthington hv, walsh t. fluoride mouthrinses for preventing dental caries in children and adolescents. cochrane database syst rev. 2016 jul 29;7(7):cd002284. doi: 10.1002/14651858.cd002284.pub2. 3. ada council on scientific affairs. tooth whitening/bleaching: treatment considerations for dentists and their patients. chicago: ada; 2009 sep. p.1-12. 4. demarco ff, meireles ss, masotti as. over-the-counter whitening agents: critical assessment of the otc products for home-use bleaching. braz oral res. 2009 jun;23(suppl 1):64-70. doi: 10.1590/s1806-83242009000500010. 5. lima fg, rotta ta, penso s, meireles ss, demarco ff. in vitro evaluation of the whitening effect of mouth rinses containing hydrogen peroxide. braz oral res. 2012 may-jun;26(3):269-74. doi: 10.1590/s1806-83242012000300014. 6. cengiz s, yuzbasioglu e, cengiz mi, velioglu n, sevimli g. color stability and surface roughness of a laboratory-processed 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composite resins. gen dent. 2017 jan-feb;65(1):68-74. 17. santos pa. [infuence of the light source on color stability of a compoiste resin. effect of storage medium and storage times] [thesis]. araraquara: são paulo state university “júlio de mesquita filho”; 2008. portuguese. 18. biazuz j, zardo p, rodrigues-junior sa. water sorption, solubility and surface roughness of resin surface sealants. braz j oral sci. 2015;14(1):27-30. doi: 10.1590/1677-3225v14n1a06. 19. pérez mm, pecho oe, ghinea r, pulgar r, della bona a. recent advances in color and whiteness evaluations in dentistry. cur dent. 2019;1(1):23-9. doi: 10.2174/2542579x01666180719125137. 20. pérez mm, ghinea r, rivas mj, yebra a, ionescu am, paravina rd, et al. development of a customized whiteness index for dentistry based on cielab color space. dent mater. 2016 mar;32(3):461-7. doi: 10.1016/j.dental.2015.12.008. 21. jorge os, de arruda cnf, torrieri rt, vivanco rg, pires-de-souza fcp. over-the-counter bleaching agentes can help with tooth whitening maintenance. j esthet restor dent. 2020 jul 6. doi: 10.1111/jerd.12617. 22. drummond jl. degradation, fatigue and failure of resin dental composite materials. j dent res. 2008 aug;87(8):710-9. doi: 10.1177/154405910808700802. 23. oliveira albm, botta ac, campos jadb, garcia ppns. influence of light curing units and fluoride mouthrinse on morphological surface and color stability of a nanofilled composite resin. microsc res tech. 2014 nov;77(11):941-6. doi: 10.1002/jemt.22421. 24. münchow ea, ferreira aca, machado rmm, ramos ts, rodrigues-junior sa, et al. effect of acidic solutions on the surface degradation of a micro-hybrid composite resin. braz dent j. 2014;25(4):321-6. doi: 10.1590/0103-6440201300058. 25. lago m, mozzaquatro lr, rodrigues c, kaizer mr, mallmann a, jacques lb. influence of bleaching agentes on color and translucency of aged resin composites. j esthet restor dent. 2017 sep;29(5):368-77. doi: 10.1111/jerd.12261.. 26. rodrigues apm, klein mc, dall agnol ma, rodrigues-junior sa. in vitro tooth whitening effectiveness of whitening mouth rinses. braz j oral sci. 2020;19:e206779. doi.org/10.20396/bjos.v19i0.8656779. 27. müller ja, rohr n, fischer j. evaluation of iso 4049: water sorption and water solubility of resin cements. eur j oral sci. 2017 apr;125(2):141-50. doi: 10.1111/eos.12339. 1http://dx.doi.org/10.20396/bjos.v19i0.8658220 volume 19 2020 e201662 original article 1 department of dental materials and prosthodontics, universidade estadual de são paulo (unesp), school of dentistry, araraquara, sp, brazil. 2 department of restorative dentistry, universidade federal da paraíba, joão pessoa, pb, brazil. 3 department of restorative dentistry, piracicaba dental school, universidade de campinas (unicamp), piracicaba, são paulo, brazil corresponding author: ana karina maciel de andrade https://orcid.org/0000-0003-4520-5176 department of restorative dentistry, federal university of paraíba, joão pessoa, pb, brazil. cidade universitária, s/n, castelo branco, joão pessoa, pb, brazil. postal code: 58051 900. phone: +55 83 999411191. e-mail: kamandrade@hotmail.com received: may 05, 2019 accepted: october 10, 2019 bond strength of universal adhesive applied to dry and wet dentin: one-year in vitro evaluation maria luísa de alencar e silva leite1, caroline de farias charamba2, renally bezerra wanderley e lima3, sônia saeger meireles2, rosângela marques duarte2, ana karina maciel de andrade2,* aim: this study evaluated the influence of dentin wettability on the immediate and extended microtensile bond strength (mtbs) of a universal adhesive system used in the etch-and-rinse strategy. methods: twenty human third molars were selected and divided into four groups according to the adhesive system and dentin wettability. the mtbs values of each group were registered 24 h and one year after adhesive system application and resin composite block build-up (n=30). data were analyzed by the t-test (p<0.05). results: when both adhesive systems were compared, there was no statistically significant difference when they were applied following wet bonding (p>0.05). however, the dry bonding reduced µtbs values of the adper single bond 2 adhesive (p<0.05). regarding storage time, both groups presented similar µtbs values at 24 h and one year (p>0.05). conclusions: therefore, the scotchbond universal adhesive can be applied to dry or wet dentin without compromising the etch-and-rinse bonding quality and the durability of the restorations. keywords: dental cements. dentin. tensile strength. https://orcid.org/0000-0003-4520-5176 2 leite et al. introduction due to the high patient demand for esthetic restorations and minimally invasive treatments, adhesive systems have become a fundamental material for achieving the stable and long-term bonding effectiveness of aesthetic restorations to mineralized tooth tissues1. adhesion of restorative materials to hard tissues (enamel and dentin) is challenging due to the differences in these tissues2. dental enamel presents a structure more homogenous than that of dentin, resulting in reliable and long-term durable adhesive bonds between the enamel and the restorative material. in contrast, dentin substrate is characterized by a wide variety of inorganic and organic components; consequently, adhesive bonding to dentin is more sensitive2. in the past, the adhesive systems available on the market have been classified into two categories: etch-and-rinse (er) and self-etch strategies (se)3,4. in the er strategy, a phosphoric acid gel is first applied to the dentin substrate, followed by application of the primer and the bond resin separately or in a single solution3,4. a major disadvantage of this strategy is susceptibility to variations in the degree of dentin moisture, which is subjective and depends on operator skills5. excessive residual moisture may hinder the impregnation of monomers into demineralized substrate by dilution of these components6. conversely, the overdrying of the dentin surface may promote collagen fibril collapse and, consequently, the incomplete impregnation of resin monomers into the collagen fibers, decreasing the bond strength7. in addition, the dentin etching with phosphoric acid can lead to collagen matrix degradation in the dentin as a result of the activation of endogenous dentin collagenolytic enzymes by acidity8,9. this process can result in impaired bond integrity of the adhesive interface8,9. to overcome the limitations of the er strategy, se adhesives were developed, characterized by acidic functional monomers that simultaneously etch and prime the tooth substrate for bonding10. previous studies have demonstrated that the laboratory and clinical performance of traditional se adhesives has not been satisfactory, mainly in the enamel11,12. selective enamel etching has been suggested to improve the adhesive bond to enamel13,14. however, the application of phosphoric acid to enamel may inadvertently etch dentin, decreasing the bond strength of se adhesives to this tissue15. the latest trends in adhesive systems are multi-mode or universal one-bottle adhesives that may be used as either er or se adhesives or as se adhesives on dentin and er adhesives on enamel (selective enamel-etching)16-18. most of the multi-mode or universal adhesives are characterized by 10-methacryloyloxydecyl dihydrogen phosphate (10-mdp), which bonds ionically to dentin, forming hydrolytically stable calcium salts on hydroxyapatite in the form of “nano-layering”16. the manufacturers claim that multi-mode adhesives may also be applied to dentin under different bonding strategies (dry or wet bonding)15,19. however, there are few studies of the bonding performance and reliability of those adhesives over the short or long term when applied to dry or wet dentin15,19,20. thus, more information is necessary to predict the long-term bonding durability of universal adhesives. therefore, the aim of this in vitro study was to evaluate whether the bond strength of a universal adhesive to dentin is affected by application mode (dryand wet-bonding), 3 leite et al. when the etch-and-rinse strategy is used, after 24 h and one year of water storage. three null hypotheses were set: 1) bond strength to dentin would not be affected by application mode (dryand wet-bonding) when the etch-and-rinse protocol is used; 2) bond strength to dentin would not be affected by storage period; and 3) there would be no difference in bond strength to dentin between etch-and-rinse and universal adhesives. materials and methods tooth selection and preparation twenty healthy human third molars were used in this study, after approval from the research ethics committee of the university of paraiba (protocol n. 17665613.2.0000.5188). the teeth were cleaned, stored in 0.2% thymol solution, and used within one month after extraction. all tooth roots were embedded in self-curing acrylic resin. then, the occlusal enamel was removed by means of a diamond disc (extec, enfield, ct, usa) under water-cooling. the exposed occlusal dentin surfaces were wet-abraded with silicon carbide paper (600 grit) under water-cooling for 60 s by means of a polishing machine (politriz erios – 27000, são paulo, sp, brasil) to standardize the smear layer21. experimental design the teeth were randomly assigned among four groups according to the different bonding strategies of the selected adhesive systems (n = 5). the two-step etch-and-rinse adhesive, adper single bond 2 (as) (3m espe, st. paul, mn, usa), and the universal adhesive, scotchbond universal adhesive (su) (3m espe, st. paul, mn, usa), were applied to dentin surfaces following a dryor a wet-bonding etch-and-rinse adhesive protocol. composition, batch number of each material, and adhesive strategies are shown in table 1. table 1. brand, batch number, composition, and adhesive strategies of materials used. adhesive system manufacturer/ batch number type composition etch-and-rinse strategy scotchbond universal 3m/espe, st. paul, mn, usa (526247) universal adhesive system 10-mdp, phosphate monomer, dimethacrylate resins, hema, methacrylate-modified polyalkenoic acid copolymer, filler, ethanol, water, initiators, silane i. apply etchant for 15 s. ii. rinse for 10 s. iii. air-dry to remove excess water. iv. keep dentin moist (wet-bonding approach) or keep dentin dry. do not overdry (dry-bonding approach). v. apply 2 consecutive coats of adhesive. vi. gently air-dry for 5 s. vii. light-polymerize for 10 s. adper single bond 2 3m/espe, st. paul, mn, usa (n49344) etch-and-rinse adhesive system 1. etchant: 35% phosphoric acid (scotchbond etchant). 2. adhesive: bis-gma, hema, dimethacrylates, ethanol, water, photoinitiator, methacrylate functional copolymer of polyacrylic and poly(itaconic) acids, 10% by weight of 5nm-diameter spherical silica particles i. apply etchant for 15 s. ii. rinse for 10 s. iii. air-dry to remove excess water. iv. keep dentin moist (wet-bonding approach) or keep dentin dry. do not overdry (dry-bonding approach). v. apply 2 consecutive coats of adhesive. vi. gently air-dry for 5 s. vii. light-polymerize for 10 s. 4 leite et al. restorative procedure and specimen preparation after the bonding process, three resin composite increments (z100-3m espe, st. paul, mn, usa) of 1.5 mm were placed on the dentin surface, and each increment was light-cured for 40 s by means of a led light-curing unit set at 400 mw/cm2 (gnatus, são paulo, sp, brazil). the restored teeth were then stored in distilled water at 37°c (± 1°c) for 24 h22. after this storage period, the specimens were sectioned longitudinally in the mesio-distal and buccal-lingual directions across the bonded interface, by means of a slow-speed diamond disc (labcut 1010, extec, enfield, ct, usa). then, the specimens were sectioned transversely in the cervical region to obtain bars measuring 1 mm2 x 10 mm21. half of the bars obtained from each tooth were used immediately (24 h) for the micro-tensile bond strength (μtbs) test, while the other half were stored in distilled water at 37°c (± 1°c) for one year22 and then subjected to μtbs testing. for μtbs testing, the bars were fixed to a testing jig with cyanoacrylate glue (super bonder gel – loctite brasil ltda) and subjected to tensile load at a crosshead speed of 0.5 mm/min until failure (microtensor om-100 machine, odeme, luzerna, sc, brasil)23. the μtbs values (mpa) were calculated by dividing the load at failure by the cross-sectional bonding area. the fractured surfaces of all specimens were observed by means of an optical microscope (xjm-400, kozo, nanjing, china) at a magnification of 100x, and fracture patterns were classified as (1) cohesive failure in adhesive, (2) cohesive failure in dentin, (3) cohesive failure in the hybrid layer, or (4) mixed failure (cohesive failure in adhesive and cohesive failure in the hybrid layer). the data from fracture patterns were analyzed by descriptive statistics. the experimental unit in the current study was the bar. an average of 10 to 15 bars was obtained from each tooth, with the experimental group (n = 30) having the smallest number of test specimens, the experimental groups are in the figure 1. thus, the µtbs value of each bar was used for statistical analysis. the data from µtbs were analyzed by the t-test for independent samples (α = 0.05). in addition, the reliability of the bond figure 1. sample diagram. n = 20 third molars n = 5 n = 5 n = 5 n = 5 adper single bond 2 – dry dentin scotchbond universal adhesive – dry dentin adper single bond 2 – wet dentin scotchbond universal adhesive – wet dentin n = 60 por grupo cui in bars experimental unit immediate tested (n =30) storage 1 year in distilled water (n =30) 5 leite et al. strength for each group was analyzed by weibull analysis. the weibull moduli (shape parameter) (slope of the line relating applied stress and the probability of specimen failure, m) were calculated, applying maximum likelihood estimation. the 95% upper and lower confidence intervals were calculated using the likelihood ratio (minitab 17.0, state college, pennsylvania, usa). differences between the paired values for m were considered significant when the 95% confidence intervals did not overlap. results the bond strength means (mpa) and standard deviations for all experimental groups are presented in table 2. the su adhesive showed no difference in µtbs values between dryand wet-bonding etch-and-rinse strategies in 24 hours and after one year (p=0.48). conversely, the as adhesive applied to a dry dentin surface presented significantly lower µtbs values than that measured when the adhesive was applied to the dentin following a wet-bonding technique (p< 0.001). when both adhesive systems were compared, there was no statistically significant difference between them when they were applied following a wet-bonding strategy in 24 hours (p=0.74) and after one year (p=0.26). however, when the adhesive systems were applied to dry dentin, the su showed significantly higher µtbs values (p< 0.001) for both storage times. regarding storage time, both adhesive systems presented similar µtbs values at 24 hours and one year (p> 0.05), regardless of the etch-and rinse strategy (dry or wet dentin). the weibull analysis indicated no change in the weibull parameter for su adhesive for all study conditions. on the other hand, as adhesive showed the lowest m values applied to the dentin following a wet-bonding (24 hours) and dry-bonding (one year) technique. comparing the adhesive systems, su demonstrated higher m values than table 2. microtensile bond strength (mpa) values of adhesive systems to dentin among the test groups, comparing materials and different etch-and-rinse strategies. storage time etch-and-rinse strategy adhesive system scotchbond universal (su) adper single bond 2 (as) 24 hours wet-bonding 49.08 (15.23)aa 47.38 (23.23)aa dry-bonding 52.28 (19.43)aa 26.91 (10.25)bb one year wet-bonding 50.22 (16.36)aa 45.13 (18.61)aa dry-bonding 53.87 (23.51)aa 21.63 (13.91)bb means followed by different uppercase letters in the same row indicate statistically significant differences between adhesive systems within the same etch-and-rinse strategy and evaluation time. means followed by different lowercase letters on the same column indicate statistically significant differences between etch-andrinse strategy within the same adhesive system and evaluation time (p < 0.05). 6 leite et al. as for wet-bonding strategy in 24 hours and dry-bonding strategy in one year. this finding suggests a more predictable, consistent performance of su product than seen when using the as adhesive (table 3, figure 2, and 3). the percentages of specimens according to fracture mode for all experimental groups at 24 h and one year are summarized in tables 4 and 5, respectively. the predominant failure patterns observed were mixed failure, cohesive failure in adhesive, and cohesive failure in the hybrid layer (iv). table 3. weibull moduli (m) values, among the experimental groups comparing different adhesive system, storage time, and etch-and-rinse strategy. storage time etch-and-rinse strategy adhesive system scotchbond universal (su) adper single bond 2 (as) 24 hours wet-bonding 3.6 (2.73-4.74)aa 2.23 (1.67-2.98)bab dry-bonding 3.12 (2.32-4.2)aa 2.80 (2.16-3.63)aa one year wet-bonding 3.37 (2.57-4.42)aa 2.68 (2.02-3.56)aa dry-bonding 2.57 (1.9-3.46)aa 1.68 (1.28-2.19)bb means followed by different uppercase letters in the same row indicate statistically significant differences between adhesive systems within the same etch-and-rinse strategy and evaluation time. means followed by different lowercase letters on the same column indicate statistically significant differences between etch-andrinse strategy within the same adhesive system and evaluation time (p < 0.05). figure 2. weibull distribution plots of microtensile bond strength data for different etch-and-rinse strategy and evaluation time within the same adhesive system. aswadper single bond 2 wet-bonding; asdadper single bond 2 dry-bonding. 99 90 80 70 60 50 40 30 20 10 5 3 2 1 1 5 10 15 20 40 60 80 100 asw-24h asd-24h asw-1 year asd-1 year p ro ba bi lit y of fa ilu re (% ), f( t) =1 – r (t ) microtensile bond strength (mpa) 7 leite et al. table 4. percentages (%) of specimens according to the fracture mode of all test groups in 24 hours’ storage time. adhesive systems etch-and-rinse strategy fracture patterns (%) scotchbond universal wet-bonding i ii iii iv 23.3 0 3.3 73.4 dry-bonding i ii iii iv 13.3 0 13.3 73.4 adper single bond 2 wet-bonding i ii iii iv 16.5 0 16.25 66.7 dry-bonding i ii iii iv 13.3 0 20 66.7 table 5. percentages (%) of specimens according to the fracture mode of all test groups in one-year storage time. adhesive systems etch-and-rinse strategy fracture patterns (%) scotchbond universal wet-bonding i ii iii iv 0 0 0 100 dry-bonding i ii iii iv 20 0 0 80 adper single bond 2 wet-bonding i ii iii iv 13.3 0 0 86.7 dry-bonding i ii iii iv 50 0 0 50 figure 3. weibull distribution plots of microtensile bond strength data for different etch-and-rinse strategy and evaluation time for scotchbond universal adhesive system. suwscotchbond universal wet-bonding; sudscotchbond universal dry-bonding. 99 90 80 70 60 50 40 30 20 10 5 3 2 1 1 5 10 15 20 40 60 80 100 suw-24h sud-24h suw-1 year sud-1 year p ro ba bi lit y of fa ilu re (% ), f( t) =1 – r (t ) microtensile bond strength (mpa) 8 leite et al. discussion based on the results of this in vitro study, the universal adhesive etch-and-rinse strategy can be applied to dry and wet dentin surfaces, since dryand wet-bonding strategies demonstrated suitable bond strength to dentin in short (24 h) and long periods of aging (one year). in contrast, the dry-bonding mode had a negative impact on dentin bond quality of the two-step adhesive, and, consequently, this material presented the worst bond strength values when applied to dry dentin. thus, the null hypotheses of the present study were rejected. the universal adhesives were introduced to simplify and optimize the adhesive procedures. these materials can be used in different types of adherent substrate and etching modes (etch-and-rinse or self-etch). a recent systematic review and meta-analysis showed reveled that the mild universal adhesives seem stable materials, in both etch-and-rinse or self-etch strategies. in this study, the universal adhesive was used in etch-and-rinse mode. as is known, the su adhesive tested in this study contains 10-mdp monomer, which provides chemical bonding to hydroxyapatite, forming hydrolytically stable calcium salts. additionally, this adhesive contains a polyalkenoic acid copolymer that also interacts ionically with the hydroxyapatite through the carboxylic groups24,25. to trigger the ionization of the phosphate monomer and polyalkenoic acid copolymer, a certain amount of water (10–15% by wt) (3m espe) was added to the universal adhesive26. thus, the unique composition of the universal adhesive (su) can explain the results observed in this study, which revealed no statistically significant difference for µtbs values between the dry or wet-bonding etch-and-rinse strategy after 24 hours and one year. the chemical components and the water contained in su adhesives may be able to rehydrate collagen fibrils, allowing for the re-expansion of the interfibrillar spaces for the infiltration of resin monomers and the formation of resin tags branching out profusely into dentin tubules27,28, permitting a satisfactory dentin seal and performance of those adhesives, even with dry-demineralized dentin15,19,29. the results of this study are in agreement with those of other reports15,19,25,28-31, which concluded that the bonding durability of the universal adhesive was acceptable and did not seem to vary depending on the etching and application mode (dryor wet-bonding). this outcome was confirmed by weibull analysis that revealed how reliable a given treatment is32. the high m values observed for su adhesive (table 3, figure 1), mainly with dry-demineralized dentin, suggest that the universal adhesive investigated can be considered reliable over time. conversely, µtbs values for the conventional two-step etch-and-rinse adhesive (as) decreased when this adhesive was applied to a dry-dentin surface in the etch-andrinse protocol after 24 h and one year. this can be attributed to an insufficient amount of water within the dentin structure, which can lead to a collapsed collagen network7. consequently, reduced resin monomer penetration into the entire depth of the decalcified dentin can occur when the as adhesive is used as the etch-and-rinse adhesive to dry dentin, corroborating with other studies7. the low m values observed for the as adhesive with dry dentin (table 3, figure 2) ratify that the application of this material to wet dentin is recommended to obtain mechanical stability of the bond to dentin. 9 leite et al. regarding failure patterns, mixed failure (cohesive failure in adhesive and cohesive failure in the hybrid layer) was the predominant mode for all experimental groups. this finding is in agreement with other studies5,33-36 and suggests that the bonding agents interacted with dental substrates during monomer infiltration by forming a hybrid layer. however, this layer fractured due to concentrated tension at the adhesive interface. one of the main degradation mechanisms of dentin bonding is the hydrolysis of the collagen fibrils and the polymerized resin matrix in the adhesive layer37. however, the patterns of degradation of resin-dentin bonding depend on the type of adhesive system37. in this study, µtbs values for both adhesives tested did not decrease significantly after one year of storage in distilled water. therefore, the storage medium used in this research did not influence the long-term stability of the bond-adhesive interface. therefore, according to the present study, the su adhesive may be an effective alternative approach for dentin restorations. nevertheless, other factors related to the oral environment may have a more complex influence on the bonding performance of adhesives. thus, future investigations should be conducted for the clinical evaluation of the su adhesive to confirm the results presented in this research. in conclusion, the universal adhesive tested in this study can be applied to dry or wet demineralized dentin without compromising the etch-and-rinse bonding quality and the durability of the restorations. on the other hand, the dry-bonding etch-and-rinse adhesive protocol influenced negatively the dentin bond quality of the conventional two-step adhesive. acknowledgments the authors would like to acknowledge the financial support from the conselho nacional de desenvolvimento científico e tecnológico (cnpq, brazil). references 1. takamizawa t, barkmeier ww, tsujimoto a, berry tp, watanabe h, erickson rl, et al. influence of different etching 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doi: 10.3290/j.jad.a19741. 11. peumans m, kanumilli p, de munck j,  van landuyt k, lambrechts p, van meerbeek b. clinical effectiveness of contemporary adhesives: a systematic review of current clinical trials. dent mater. 2005 sep;21(9):864-81. 12. perdigão j, carmo ar, anauate-netto c, amore r, lewgoy hr, cordeiro hj, et al. clinical performance of a self-etching adhesive at 18 months. am j dent. 2005 apr;18(2):135-40. 13. frankenberger r, lohbauer u, roggendorf mj, naumann m, taschner m. selective enamel etching reconsidered: better than etch-and-rinse and self-etch? j adhes dent. 2008 oct;10(5):339-44. 14. jacker-guhr s, sander j, luehrs ak. how “universal” is adhesion? shear bond strength of multi-mode adhesives to enamel and dentin. j adhes dent. 2019;21(1):87-95. doi: 10.3290/j.jad.a41974. 15. mena-serrano a, kose c, de paula ea, tay ly, reis a, loguercio ad, et al. a new universal simplified adhesive: 6-month clinical evaluation. j esthet restor dent. 2013 feb;25(1):55-69. doi: 10.1111/jerd.12005. 16. yoshida y, yoshihara k, nagaoka n,  hayakawa s, torii y, ogawa t, et al. selfassembled nano-layering at the adhesive interface. j dent res. 2012 apr;91(4):376-81. doi: 10.1177/0022034512437375. 17. lenzi tl, soares fz, raggio dp, pereira gk, rocha ro. dry-bonding etch-and-rinse strategy improves bond longevity of a universal adhesive to sound and artificially-induced caries-affected primary dentin. j adhes dent. 2016;18(6):475-82. doi: 10.3290/j.jad.a36670. 18. cuevas-suárez ce, da rosa wlo, lund rg, da silva af, piva e. bonding performance of universal adhesives: an updated systematic review and meta-analysis. j adhes dent. 2019;21(1):7-26. doi: 10.3290/j.jad.a41975. 19. hanabusa m, mine a, kuboki t, momoi y, van ende a, van meerbeek b, et al. bonding effectiveness of a new ‘multi-mode’ adhesive to enamel and dentine. j dent. 2012 jun;40(6):475-84. doi: 10.1016/j.jdent.2012.02.012. 20. perdigão j, kose c, mena-serrano ap, de paula ea, tay ly, reis a, et al. a new universal simplified adhesive: 18month clinical evolution. oper dent. 2014 mar-apr;39(2):113-27. doi: 10.2341/13-045-c. 21. orellana n, ramírez r, roig m, giner l, mercade m, durán f, et al. comparative study of the microtensile bond strength of three different total etch adhesives with different solvents to wet and dry dentin. (in vitro test). acta odontol latinoam. 2009;22(1):47-56. 22. shinohara ms, de goes mf, schneider lf,  ferracane jl, pereira pn, di hipólito v, et al. fluoride-containing adhesive: durability on dentin bonding. dent mater. 2009 nov;25(11):1383-91. doi: 10.1016/j.dental.2009.06.011. 23. di hipólito v, reis af, mitra sb, de goes mf. interaction morphology and bond strength of nanofilled simplified-step adhesives to acid etched dentin. eur j dent. 2012 oct;6(4):349-60. https://www.ncbi.nlm.nih.gov/pubmed/?term=pagani c%5bauthor%5d&cauthor=true&cauthor_uid=23579885 https://www.ncbi.nlm.nih.gov/pubmed/?term=rocha gomes torres c%5bauthor%5d&cauthor=true&cauthor_uid=23579885 https://www.ncbi.nlm.nih.gov/pubmed/?term=tj%c3%a4derhane l%5bauthor%5d&cauthor=true&cauthor_uid=23128110 https://www.ncbi.nlm.nih.gov/pubmed/?term=polimeni a%5bauthor%5d&cauthor=true&cauthor_uid=23128110 https://www.ncbi.nlm.nih.gov/pubmed/?term=mazzoni a%5bauthor%5d&cauthor=true&cauthor_uid=23953737 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https://www.ncbi.nlm.nih.gov/pubmed/?term=mercade m%5bauthor%5d&cauthor=true&cauthor_uid=19601496 https://www.ncbi.nlm.nih.gov/pubmed/?term=dur%c3%a1n f%5bauthor%5d&cauthor=true&cauthor_uid=19601496 https://www.ncbi.nlm.nih.gov/pubmed/?term=ferracane jl%5bauthor%5d&cauthor=true&cauthor_uid=19646747 https://www.ncbi.nlm.nih.gov/pubmed/?term=pereira pn%5bauthor%5d&cauthor=true&cauthor_uid=19646747 https://www.ncbi.nlm.nih.gov/pubmed/?term=di hip%c3%b3lito v%5bauthor%5d&cauthor=true&cauthor_uid=19646747 11 leite et al. 24. yoshida y, van meerbeek b, nakayama y,  snauwaert j, hellemans l, lambrechts p, et al. evidence of chemical bonding at biomaterial-hard tissue interface. j dent res. 2000 feb;79(2):709-14. 25. vermelho pm, reis af, ambrosano gmb, giannini m. adhesion of multimode adhesives to enamel and dentin after one year of water storage. clin oral investig. 2017 jun;21(5):1707-5. doi: 10.1007/s00784-016-1966-1. 26. luque-martinez iv, perdigão j, muñoz ma,  sezinando a, reis a, loguercio ad. effects of solvent evaporation time on immediate adhesive properties of universal adhesives to dentin. dent mater. 2014 oct;30(10):1126-35. doi: 10.1016/j.dental.2014.07.002. 27. dal-bianco k, pellizzaro a, patzlaft r, de oliveira bauer jr, loguercio ad, reis a. effects of moisture degree and rubbing action on the immediate resin-dentin bond strength. dent mater. 2006 dec;22(12):1150-6. 28. lenzi tl, soares fzm, de oliveira rr. does bonding approach influence the bond strength of universal adhesive to dentin of primary teeth? j clin pediatr dent. 2017;41(3):214-8. doi: 10.17796/1053-4628-41.3.214. 29. loguercio ad, de paula ea, hass v, luque-martinez i, reis a, perdigão j. a new universal simplified adhesive: 36-month randomized double-blind clinical trial. j dent. 2015 sep;43(9):1083-92. doi: 10.1016/j.jdent.2015.07.005. 30. tsujimoto a, shimatani y, nojiri k, barkmeier ww, markham md, takamizawa t, et al. influence of surface wetness on bonding effectiveness of universal adhesives in etch-and-rinse mode. eur j oral sci. 2019 apr;127(2):162-9. doi: 10.1111/eos.12596. 31. sugimura r, tsujimoto a, hosoya y, fischer ng, barkmeier ww, takamizawa t, et al. surface moisture influence on etch-and rinse universal adhesive bonding. am j dent. 2019 feb;32(1):33-8. 32. weibull w. a statistical distribution function of wide applicability. j appl mech. 1951 set;18:293-7. 33. hass v, dobrovolski m, zander-grande c, martins gc, gordillo la, rodrigues accorinte mde l, et al. correlation between degree of conversion, resin–dentin bond strength and nanoleakage of simplified etch-and-rinse adhesives. dent mater. 2013 sep;29(9):921-8. doi: 10.1016/j.dental.2013.05.001. 34. marchesi g, frassetto a, mazzoni a,  apolonio f, diolosà m, cadenaro m, et al. adhesive performance of a multi-mode adhesive system: 1-year in vitro study. j dent. 2014 may;42(5):603-12. doi: 10.1016/j.jdent.2013.12.008. 35. munoz ma, luque i, hass v, reis a, loguercio ad, bombarda nh. immediate bonding properties of universal adhesives to dentine. j dent. 2013 may;41(5):404-11. doi: 10.1016/j.jdent.2013.03.001. 36. takai t, hosaka k, kambara k, thitthaweerat s, matsui n, takahashi m, et al. effect of air-drying dentin surfaces on dentin bond strength of a solvent-free one-step adhesive. dent mater j. 2012;31(4):558-63. 37. breschi l, mazzoni a, ruggeri a, cadenaro m, di lenarda r, de stefano dorigo e. dental adhesion review: aging and stability of the bonded interface. dent mater. 2008 jan;24(1):90-101. https://www.ncbi.nlm.nih.gov/pubmed/?term=snauwaert j%5bauthor%5d&cauthor=true&cauthor_uid=10728971 https://www.ncbi.nlm.nih.gov/pubmed/?term=hellemans l%5bauthor%5d&cauthor=true&cauthor_uid=10728971 https://www.ncbi.nlm.nih.gov/pubmed/?term=lambrechts p%5bauthor%5d&cauthor=true&cauthor_uid=10728971 https://www.ncbi.nlm.nih.gov/pubmed/?term=giannini m%5bauthor%5d&cauthor=true&cauthor_uid=27714528 https://www.ncbi.nlm.nih.gov/pubmed/?term=sezinando a%5bauthor%5d&cauthor=true&cauthor_uid=25139815 https://www.ncbi.nlm.nih.gov/pubmed/?term=reis a%5bauthor%5d&cauthor=true&cauthor_uid=25139815 https://www.ncbi.nlm.nih.gov/pubmed/?term=loguercio ad%5bauthor%5d&cauthor=true&cauthor_uid=25139815 https://www.ncbi.nlm.nih.gov/pubmed/?term=de oliveira bauer jr%5bauthor%5d&cauthor=true&cauthor_uid=16405988 https://www.ncbi.nlm.nih.gov/pubmed/?term=loguercio ad%5bauthor%5d&cauthor=true&cauthor_uid=16405988 https://www.ncbi.nlm.nih.gov/pubmed/?term=reis a%5bauthor%5d&cauthor=true&cauthor_uid=16405988 https://www.ncbi.nlm.nih.gov/pubmed/?term=luque-martinez i%5bauthor%5d&cauthor=true&cauthor_uid=26159382 https://www.ncbi.nlm.nih.gov/pubmed/?term=reis a%5bauthor%5d&cauthor=true&cauthor_uid=26159382 https://www.ncbi.nlm.nih.gov/pubmed/?term=perdig%c3%a3o j%5bauthor%5d&cauthor=true&cauthor_uid=26159382 https://www.ncbi.nlm.nih.gov/pubmed/?term=tsujimoto a%5bauthor%5d&cauthor=true&cauthor_uid=30536451 https://www.ncbi.nlm.nih.gov/pubmed/?term=shimatani y%5bauthor%5d&cauthor=true&cauthor_uid=30536451 https://www.ncbi.nlm.nih.gov/pubmed/?term=nojiri k%5bauthor%5d&cauthor=true&cauthor_uid=30536451 https://www.ncbi.nlm.nih.gov/pubmed/?term=barkmeier ww%5bauthor%5d&cauthor=true&cauthor_uid=30536451 https://www.ncbi.nlm.nih.gov/pubmed/?term=markham md%5bauthor%5d&cauthor=true&cauthor_uid=30536451 https://www.ncbi.nlm.nih.gov/pubmed/?term=takamizawa t%5bauthor%5d&cauthor=true&cauthor_uid=30536451 https://www.ncbi.nlm.nih.gov/pubmed/?term=sugimura r%5bauthor%5d&cauthor=true&cauthor_uid=30834729 https://www.ncbi.nlm.nih.gov/pubmed/?term=tsujimoto a%5bauthor%5d&cauthor=true&cauthor_uid=30834729 https://www.ncbi.nlm.nih.gov/pubmed/?term=hosoya y%5bauthor%5d&cauthor=true&cauthor_uid=30834729 https://www.ncbi.nlm.nih.gov/pubmed/?term=fischer ng%5bauthor%5d&cauthor=true&cauthor_uid=30834729 https://www.ncbi.nlm.nih.gov/pubmed/?term=barkmeier ww%5bauthor%5d&cauthor=true&cauthor_uid=30834729 https://www.ncbi.nlm.nih.gov/pubmed/?term=takamizawa t%5bauthor%5d&cauthor=true&cauthor_uid=30834729 https://www.ncbi.nlm.nih.gov/pubmed/?term=sugimura%2c+2019+univeral+adhesive https://www.ncbi.nlm.nih.gov/pubmed/?term=martins gc%5bauthor%5d&cauthor=true&cauthor_uid=23830512 https://www.ncbi.nlm.nih.gov/pubmed/?term=gordillo la%5bauthor%5d&cauthor=true&cauthor_uid=23830512 https://www.ncbi.nlm.nih.gov/pubmed/?term=rodrigues accorinte mde l%5bauthor%5d&cauthor=true&cauthor_uid=23830512 https://www.ncbi.nlm.nih.gov/pubmed/?term=apolonio f%5bauthor%5d&cauthor=true&cauthor_uid=24373855 https://www.ncbi.nlm.nih.gov/pubmed/?term=diolos%c3%a0 m%5bauthor%5d&cauthor=true&cauthor_uid=24373855 https://www.ncbi.nlm.nih.gov/pubmed/?term=cadenaro m%5bauthor%5d&cauthor=true&cauthor_uid=24373855 https://www.ncbi.nlm.nih.gov/pubmed/?term=reis a%5bauthor%5d&cauthor=true&cauthor_uid=23499568 https://www.ncbi.nlm.nih.gov/pubmed/?term=loguercio ad%5bauthor%5d&cauthor=true&cauthor_uid=23499568 https://www.ncbi.nlm.nih.gov/pubmed/?term=bombarda nh%5bauthor%5d&cauthor=true&cauthor_uid=23499568 https://www.ncbi.nlm.nih.gov/pubmed/?term=thitthaweerat s%5bauthor%5d&cauthor=true&cauthor_uid=22864208 https://www.ncbi.nlm.nih.gov/pubmed/?term=matsui n%5bauthor%5d&cauthor=true&cauthor_uid=22864208 https://www.ncbi.nlm.nih.gov/pubmed/?term=takahashi m%5bauthor%5d&cauthor=true&cauthor_uid=22864208 1http://dx.doi.org/10.20396/bjos.v19i0.8657039 volume 19 2020 e207039 original article 1 universidade católica de brasília ucb, centro de análises proteômicas e bioquímicas. post-graduation program in biotechnology and genomic sciences, brasília, brazil. 2 universidade católica de brasília ucb, pharmacy department, brasília, distrito federal, brazil 3 universidade católica de brasília ucb, dental course, brasília, distrito federal, brazil 4 universidade federal de mato grosso do sul ufms, programa de pós graduação em saúde e desenvolvimento na região centro oeste. faculdade de medicina, campo grande, mato grosso do sul, brazil. 5 universidade católica dom bosco ucdb, s-inova biotech, post-graduation program in biotechnology, campo grande, mato grosso do sul, brazil 6 universidade de brasília unb, post-graduation program in molecular phatology, brasília, distrito federal, brazil 7 universidade de brasília unb, post-graduation program in health sciences, brasília, distrito federal, brazil corresponding author: taia maria berto rezende universidade católica de brasília ucb post-graduation program in biotechnology and genomic sciences sgan 916n – av. w5 – campus ii – modulo c, room c-221 brasília-df, brazil fone: + 55-61-981349001 fax: + 55-61-3347-4797 e-mail: taiambr@gmail.com / taia@ucb.br received: october 17, 2019 accepted: february 24, 2020 enterococcus faecalis and staphylococcus aureus stimulate nitric oxide production in macrophages and fibroblasts in vitro maurício gonçalves da costa sousa1, patrícia diniz xavier2, stella maris de freitas lima3, jeeser alves de almeida4, octávio luiz franco1,5,6, taia maria berto rezende1,3,7,* aim: nitric oxide (no) is an important mediator related to damage of the pulp tissue and at the same time to regenerative pulp processes. however, it is not clear how common endodontic microorganisms can regulate this mediator. this study aimed to investigate no production by macrophages and fibroblasts against enterococcus faecalisand staphylococcus aureus-antigens. methods: raw 264.7 macrophages and l929 fibroblast cell lines were stimulated with different heatkilled (hk) antigen concentrations (105-108 colony forming units cfu) from e. faecalis and s. aureus with or without interferon-gamma (ifn-γ). cell viability by mtt colorimetric assay and no production from the culture supernatants were evaluated after 72 h. results: data here reported demonstrated that none of the antigen concentrations decreased cell viability in macrophages and fibroblasts. the presence of hk-s. aureus and hk-e. faecalis antigenstimulated no production with or without ifn-γ on raw 264.7. the hk-s. aureus antigen stimulated no production in l929 fibroblasts with or without ifn-γ, and the highest concentration of hk-e. faecalis with ifn-γ also stimulated no production by these cells. conclusion: the amount of no produced by macrophages and fibroblasts may be involved in the concentration and type of prevalent endodontic microorganisms, generating new answers for the understanding of pulpal revascularization/regeneration processes. keywords: enterococcus faecalis. staphylococcus aureus. fibroblasts. macrophages. nitric oxide. 2 sousa et al. introduction regenerative endodontic therapies have become widespread, especially in immature teeth with open apex1. difficulties during root canal instrumentation and disadvantages of the conventional apexification technique motivate pulp revascularization therapy. briefly, this process consists of accessing the root canal system (rcs), irrigating it and subsequently using an intracanal medication aiming to remove the largest number of microorganisms in the rcs. this aseptic environment for blood clot formation is essential for the new tissue formation1,2. the success of regenerative therapies will depend on three important factors, namely the presence of stem cells, growth factors and scaffold3. several mesenchymal stem cells, originating from the apical papilla (scap), dental pulp stem cells (dpscs) or from exfoliated deciduous teeth (shed) can contribute to pulp tissue regeneration4,5. however, the biomarkers needed for tissue reconstruction are still unknown. in addition, many clinical studies have reported the formation of fibroblast-rich scar tissue, without free nerve endings6. until now, the role of other cells such as macrophages and fibroblasts in this process is unclear7. moreover, the presence of some gram-positive bacterial species such as enterococcus faecalis has been found in revascularized tissues, triggering the production of mediators and pro-inflammatory cytokines, which may hamper tissue repair8. among all the mediators produced by macrophages and fibroblasts, nitric oxide (no) can act both in the elimination of invading agents and in the formation or destruction of tissues9. in this way, in a previous study, no was upregulated in the presence of e. faecalis in vitro10. in relation to the formation of new tissues, low concentrations of no may stimulate new vessel formation during pulpal regeneration / revascularization processes11. however, on the other hand, high concentrations can cause pulp tissue damage and particularly hinder new tissue formation9. this happens because no may be associated with odontoblast differentiation and the production of enzymes and proteins related with bone and dentin formation, including alkaline phosphatase and calcitonin12,13. therefore, considering the difficulty of promoting an aseptic environment, the purpose of this study was to evaluate in vitro cell viability and the production of no in two cell lines. these cells were stimulated with different concentrations of heat-killed antigens from prevalent endodontic bacterial e. faecalis and s. aureus, mimicking the environment related to pulp revascularization. materials and methods raw 256.7 and l929 fibroblast cultures raw 264.7 osteoclast precursor monocyte cells (cr108, rio de janeiro cell bank, rio de janeiro, brazil) were cultured at 1x105 cells per well in 96-well culture plates (tpp, usa). l929 fibroblasts (atcc 929) were cultivated at 1x105 per well in 96-well culture plates (tpp, usa). both cells were cultured in dulbecco modified eagle medium (gibco, usa) supplemented with 10 % fetal bovine serum (gibco, usa), 1 % 3 sousa et al. penicillin / streptomycin (1000 u.ml-1) (invitrogen, grand island, ny), 1 % nonessential amino acids (invitrogen), 1% l-glutamine and 0.1% gentamicin (invitrogen)14,15. these cell cultures were stimulated in vitro with different concentrations of heatkilled antigens (hk) from e. faecalis (atcc 19433) and s. aureus (atcc 25923) with or without recombinant (r) ifn-γ (10 u per well, peprotech, usa), mimetizing the endodontic environment in the necrosis of incomplete rhizogenesis processes. as a control, both cells were also stimulated with lipopolysaccharide (lps) (3 µg.ml-1, sigma-aldrich, usa)16. cell viability assay and no production were assessed after 72 h of incubation. hk antigen preparations experimental groups determined for cytotoxicity and no production analyses were stimulated with hk-e. faecalis and -s. aureus. heat-killed antigens were prepared, as previously described10. briefly, colonies were grown in luria bertani agar (lb; kasvi, ph 7.3; usa) and subsequently resuspended in sterile phosphate-buffered saline solution, followed by their quantification by optical density. then, they were heated at 121 °c, for 50 min10. different concentrations of antigens from both microorganisms (105-108 colony-forming units/well) were tested. death of microorganisms was confirmed by the absence of colonies, after 24 hours of incubation in luria bertani agar (lb; kasvi, ph 7.3; usa). optical microscopy images (inverted microscope axiovert 40 cfl, usa; objective 20x) were obtained after 72h of incubation, from the experimental groups stimulated with rifn-γ, lps, and rifn-γ, hk-s. aureus (106 cfus) with or without rifn-γ and hk-e. faecalis (106 cfus) with or without rifn-γ. viability assay cell viability assays were performed on both cells with antigen stimulus after a period of 72 h incubation at 37 °c, 5 % co2 and 95 % humidity, with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenylte-trazolium (mtt) bromide (0.25 mg.ml-1). the absorbance was measured at 595 nm in a microplate spectrophotometer (bio-tek, winooski, vt). the results were compared to a positive control (unstimulated cells) and negative controls (cell culture in lysis buffer solution 10 mmol.l-1 tris, ph 7.4, 1 mmol.l-1 edta and 0.1% triton x-100) and plotted as mean±standard error of absorbance16. nitric oxide production no levels in culture supernatant from both stimulated cell lines were determined by griess reaction. after 72h of incubation, the supernatant was mixed with an equal volume of griess reagent (1 % sulfanilamide and 0.1 % naphtylethylene in 2.5 % ortho-phosphoric acid; sigma-aldrich, gb, brazil). the absorbance was measured by a microplate spectrophotometer (bio-tek, winooski, vt) at 490 nm. the nitrite concentration was determined according to a standard curve (0–200 mmol.l-1 sodium nitrite)17. statistical analysis all experiments were carried out in technical and biological triplicates. statistical analyses were performed by kolmogorov-smirnov test followed by one-way analysis of 4 sousa et al. variance (anova) and bonferroni post hoc by using graphpad prism 6 (graphpad software, san diego, ca); p<0.05 was considered statistically significant. results cellular viability related to microbial antigen and controls first, the cell viability of both chosen cell lines was evaluated, in the presence of hk-e. faecalis or hk-s. aureus. in this context, antigen concentration may be determinant for cell viability. thus, raw 264.7 stimulated with different concentrations of s. aureus did not diminish cell viability and the lps-stimulated group induced cell proliferation (fig. 1a). otherwise, the addition of rifn-γ and 108 cfu.ml-1 of s. aureus upregulated cell proliferation (fig. 1c). regarding e. faecalis, this antigen did not reduce cell viability and, at 108 cfu.ml-1, it was also able to increase cell viability (fig. 1b). furthermore, the lps-control group induced cell proliferation, while the presence of rifn-γ did not represent an additional stimulus to alter cell viability (fig. 1c and 1d). in order to relate our viability results with the morphological characteristics presented by these cells, the optical microscopy images (fig. 1a-l) were observed, and these showed that after three days’ incubation, the groups containing hk-s. aureus (fig. 1i and 1j) and hk-e. faecalis (fig. 1k and 1l) antigens with or without rifn-γ significantly altered the cellular morphology of raw 264.7, when compared to the control group (fig. 1e). regarding l929 fibroblasts cell viability, none of the concentrations studied was cytotoxic. therefore, neither l929 fibroblasts stimulated with different concentrations of s. aureus, nor lps, diminished cell viability (fig. 2a). besides, the highest concentration of this hk upregulated cell proliferation (fig. 2c). a similar relationship was observed when the rifn-γ was added to hk-s. aureus. these stimuli did not reduce cell viability and, at 106 cfu.ml-1, they also stimulated cell proliferation (fig. 2c). hk-e. faecalis did not reduce cell viability, and at 106, 107 and 108 cfu.ml-1, it again increased cell viability (fig. 2b). the rifn-γ stimulus was not able to alter cell viability in any of the tested groups (fig. 2d). concerning the morphological alterations by optical microscopy, the images demonstrated the structural differences under stress that both cells may present in the presence of both tested antigens. thus, the optical microscopy images (fig. 1a-l) showed that after three days’ incubation, groups containing lps (fig. 1g), hk-s. aureus (fig. 1i and 1j) and hk-e. faecalis (fig. 1k and 1l) antigens with or without rifn-γ significantly altered cellular morphology of l929, when compared to the control group (fig. 1e). no production related to microbial antigen and controls the evaluation of no production was performed after 72h in both cells studied. assembling a system involving both the different antigens and the presence of the rifn-γ recombinant, it was possible to mimic an in vitro infection. in this way, in raw 264.7 cultures, the presence of lps and different concentrations of hk-s. aureus was able to upregulate the production of sodium nitrite, after 72 hours of incubation, except for the group containing 108 cfu.ml-1 (fig. 3a). therefore, the presence of ifn-γ increased nitrite production by these cells in different concentra5 sousa et al. tions of s. aureus or lps, except for the group stimulated by 108 cfu.ml-1 (fig. 3c). only the 108 cfu.ml-1 of e. faecalis stimulus was able to induce the production of sodium nitrite (fig. 3b). the addition of rifn-γ led to an increase in sodium nitrite production, except for the group containing 108 cfu.ml-1 of e. faecalis (fig. 3d). regarding the l929 cultures, these cells may represent the most abundant cells on the pulp tissue: fibroblasts. then, the presence of lps or different hk-s. aureus figure 1. raw 264.7 cell viability. graphs represent cell cultures with different concentrations (105-108 cfu.ml-1) of hk-s. aureus antigen (a) with rifn-γ (c) or hk-e. faecalis antigen (b) with rifn-γ (d), after 72h of cell incubation. bars represent mean and standard error of cellular absorbance (595 nm) carried out in technical and biological triplicates. statistical differences by one-way anova test and bonferroni post hoc were represented by * (p < 0.05), ** (p < 0.01) and *** (p< 0.001). optical microscopy (20x) shows the initial (day 1) and final (day 3) cell morphology aspects (e-k) of raw 264.7 stimulated with rifn-γ (f), lps (g), lps and rifn-γ (h), hk-s. aureus 106 cfus without (i) or with rifn-γ (j), hk-e. faecalis 106 cfus without (k) or with rifn-γ (l) compared to the cell control group (e). +ifn-γ +lps +ifn-γ +lps +hk s. aureus +hk s. aureus +ifn-γ +hk e. faecalis +hk e. faecalis +ifn-γ d ay 1 d ay 3 (e) (f) (g) (k)(j)(i)(h) (l)control (a) (b) (c) (d) a bs or ba nc e (5 95 n m ) 1.0 0.8 0.6 0.4 0.2 0.0 ** ** ** *** ** ** hk – s. aureus hk – e. faecalis hk – s. aureus + rifn-γ hk – e. faecalis + rifn-γ raw lps + ifn-γ 105 106 107 108 a bs or ba nc e (5 95 n m ) 1.0 0.8 0.6 0.4 0.2 0.0 raw lps + ifn-γ 105 106 107 108 a bs or ba nc e (5 95 n m ) 1.0 0.8 0.6 0.4 0.2 0.0 raw lps + ifn-γ 105 106 107 108 a bs or ba nc e (5 95 n m ) 1.0 0.8 0.6 0.4 0.2 0.0 raw lps + ifn-γ 105 106 107 108 user highlight conferimos as provas anteriores e não encontramos a indicação da correção para (μm) nas figuras 1 e 2. devemos corrigir? 6 sousa et al. concentrations were capable of stimulating the production of sodium nitrite, with or without rifn-γ (fig. 4a and 4c). only the highest concentration of hk-e. faecalis was able to stimulate the production of sodium nitrite without rifn-γ (fig. 4b). nevertheless, when the rifn-γ was added to all groups, sodium nitrite production was upregulated (fig. 4d). figure 2. l929 cell viability. graphs represent cell cultures with different concentrations (105-108 cfu. ml-1) of hk-s. aureus antigen (a) with rifn-γ (c) or hk-e. faecalis antigen (b) with rifn-γ (d), after 72h of cell incubation. bars represent mean and standard error of cellular absorbance (595 nm) carried out in technical and biological triplicates. statistical differences by one-way anova test and bonferroni post hoc were represented by * (p < 0.05). optical microscopy (20x) shows the initial (day 1) and final (day 3) cell morphology aspects (e-k) of l929 stimulated with rifn-γ (f), lps (g), lps and rifn-γ (h), hk-s. aureus 106 cfus without (i) or with rifn-γ (j), hk-e. faecalis 106 cfus without (k) or with rifn-γ (l) compared to the cell control group (e). a bs or ba nc e (5 95 n m ) 1.0 0.8 0.6 0.4 0.2 0.0 * * * * * hk – s. aureus hk – e. faecalis hk – s. aureus + rifn-γ hk – e. faecalis + rifn-γ l929 lps + ifn-γ 105 106 107 108 a bs or ba nc e (5 95 n m ) 1.0 0.8 0.6 0.4 0.2 0.0 l929 lps + ifn-γ 105 106 107 108 a bs or ba nc e (5 95 n m ) 1.0 0.8 0.6 0.4 0.2 0.0 l929 lps + ifn-γ 105 106 107 108 a bs or ba nc e (5 95 n m ) 1.0 0.8 0.6 0.4 0.2 0.0 l929 lps + ifn-γ 105 106 107 108 +ifn-γ +lps +ifn-γ +lps +hk s. aureus +hk s. aureus +ifn-γ +hk e. faecalis +hk e. faecalis +ifn-γ d ay 1 d ay 3 (e) (f) (g) (k)(j)(i)(h) (l)control (a) (b) (c) (d) 7 sousa et al. figure 3. no production by raw 264.7 cells. graphs represent values of sodium nitrite with different concentrations (105-108 cfu.ml-1) of hk-s. aureus antigen (a) with rifn-γ (c) or hk-e. faecalis antigen (b) with rifn-γ (d), after 72h of cell incubation. bars represent mean and standard error of sodium nitrite production in µm carried out in technical and biologic triplicates. statistical differences by one-way anova test and bonferroni post hoc were represented by *** (p< 0.001) and **** (p < 0.0001). + *** *** **** **** -+ **** **** + + + ++ + **** **** **** **** --++ + + + + **** **** *** **** 105 106 107 108 n itr ite (µ m ) 0 1 2 3 lps 3 µg.ml-1 hk – e. faecalis n itr ite (µ m ) 0 1 2 3 lps 3 µg.ml-1 hk – e. faecalis 105 106 107 108 n itr ite (µ m ) 0 1 2 3 lps 3 µg.ml-1 rifn-g (10u per well) hk – e. faecalis 105 106 107 108 0 1 2 3 lps 3 µg.ml-1 rifn-g (10u per well) hk – e. faecalis 105 106 107 108 (a) (b) (c) (d) n itr ite (µ m ) figure 4. no production by l929 fibroblasts. graphs represent values of sodium nitrite with different concentrations (105-108 cfu.ml-1) of hk-s. aureus antigen (a) with rifn-γ (c) or hk-e. faecalis antigen (b) with rifn-γ (d), after 72h of cell incubation. bars represent mean and standard error of sodium nitrite production in µm carried out in technical and biologic triplicates. statistical differences by one-way anova test and bonferroni post hoc (p<0.05) were represented by * (p < 0.05), ** (p < 0.01) *** (p< 0.001) and **** (p < 0.0001). n itr ite (µ m ) 0 1 2 3 lps 3 µg.ml-1 hk – e. faecalis ----105 106 107 108 + **** **** * ** **** n itr ite (µ m ) 0 1 2 3 lps 3 µg.ml-1 hk – e. faecalis ------+ 105 106 107 108 *** * n itr ite (µ m ) 0 1 2 3 lps 3 µg.ml-1 rifn-g (10u per well) hk – e. faecalis ---+ ++++ 105 106 107 108+ **** * ** ** * n itr ite (µ m ) 0 1 2 3 lps 3 µg.ml-1 rifn-g (10u per well) hk – e. faecalis + ---------105 106 107 108 ++ ++ + **** * (a) (b) (c) (d) 8 sousa et al. discussion regenerative therapies may contribute to endodontic treatment in immature teeth with open-apex3,18. however, both the absence of microorganisms and the presence of mediators and growth factors are essential for the construction of new pulp tissue18. thus, the polymicrobial infected root canal system is composed of gram-positive and gram-negative bacteria. among the microorganisms, e. faecalis is prevalent in infected immature permanent teeth19. moreover, this microorganism is associated with different forms of periradicular disease, including primary endodontic infections as well as persistent periapical lesions20. in the category of primary endodontic infections, e. faecalis is present in 40% of them20. s. aureus might be another bacterium found in pulpitis and may have quorum-sensing as its main mechanism of virulence21. this factor contributes to the control of the pathogenesis of this microorganism, which is involved with the density that occurs through cellular communications22. the presence of microorganisms may inhibit the development of new tissue, modifying the normal function of these cells23. in an in vitro study, lps from pseudomonas aeruginosa did not reduce cell viability, but reduced the ability of periodontal ligament stem cells to differentiate into osteoblasts; in addition, it upregulated the production of proinflammatory cytokines such as il-1β, il-6, and il-8 by these cells23. in the same way, the activation of immune system cells, metalloproteinase, reactive oxygen species (ros) and bacterial endotoxins, for instance lps and lipoteichoic acid (lta), may compromise the development of loose connective tissue24. it has been described that the presence of s. aureus antigens downregulated the bone marrow stem cell and human fibroblast adhesion factors, by blocking tlr225. in addition, lta from s. aureus walls was related with the production of no in raw 264.7 macrophages, via tlr226. the antigen-fighting process of resistant microorganisms is mostly associated with the first line of immune response, represented by cytokines and lysosomal enzyme-macrophage producers, related to tissue destruction27. however, fibroblasts are the main cells present in connective tissue, deploying a structural and repair role, including the release of tissue repair mediators28. thus, for this study, raw 264.7 macrophages and l929 fibroblasts were chosen. in this context, ifn-γ may be responsible for upregulating the class i and ii major histocompatibility complexes and activating reduced nicotinamide adenine dinucleotide phosphate–dependent phagocyte oxidase and no production in macrophages, besides exacerbating the response to the production of no in fibroblasts29,30. in this study, the s. aureus and e. faecalis stimuli in raw 264.7 macrophages were not able to decrease cell viability, even at higher tested concentrations. however, the hk antigens altered cell morphology at all tested concentrations. these results were also observed in a previous study, in which raw 264.7 cells remained viable even at higher s. aureus antigen concentrations, after 48 h of incubation31. the presence of different concentrations of s. aureus and e. faecalis antigens also did not diminish the l929 fibroblast viability. this is the first study, according to our knowledge, that has evaluated the effects of gram-positive bacteria on the l929 cell line. 9 sousa et al. the presence of heat-killed porphyromonas gingivalis in fibroblasts from periodontal ligament was not cytotoxic, after 48 h of incubation, even at the highest cells: bacteria proportion (1:100)32. the main events reported with the presence of antigens in the root canal systems are related to changes in the pattern of response and production of mediators by these cells10. among these mediators, no is a gaseous free radical produced by no-synthase, by converting l-arginine to l-citrulline33. the action of inducible nitric oxide (no2) on pulp tissue can contribute to the destruction of microorganisms, but at the same time, high concentrations (500 µm) were able to cause apoptosis of macrophages and osteoblasts in an in vitro periapical lesion model34. the beneficial or malignant action of no may be related to the levels of no produced. low concentrations of no in pulp space can contribute to tissue formation and regeneration processes, since the formation of new vessels may be essential for the construction of new tissue11. and because it is lipophilic, no can easily be permeable to biological membranes, causing vasodilation35. in addition, no (100 µm) may upregulate the vascular endothelial growth factor (vegf), which is essential for angiogenesis36. as the pulp tissue have a higher concentration of blood vessels, the synthesis of this free radical becomes essential in the support and establishment of their physiology11. an in vitro study demonstrated an increase in no synthase expression in pulp cells derived from immature permanent teeth when compared to third molar pulp cells37. this study showed that both stimuli (s. aureus and e. faecalis) were able to induce the production of no in raw macrophage 264.7, based on a standard curve of sodium nitrite. no production in macrophages in the presence of s. aureus stimuli seems to be dose dependent. however, at the highest concentration the abundance of this specific mediator was not improved. in this context, macrophages are the first defense line and, when in contact with antigens, are specialized in producing inducible no38. macrophage polarization (m1) may perpetuate an inflammatory response, whereas a macrophage response (m2) may contribute to the formation of new tissues. an in vitro study associated the autocrine action of no on lps-stimulated macrophages with the polarization in profile m139. virulence factors such as lta from gram-positive bacteria, peptide glycol and adhesion factors are generally associated with the induction of no synthase in macrophages32,40. here, the higher antigen concentrations, in the presence of ifnγ, did not stimulate no production in raw 264.7 cells. in situations of high antigen concentrations, the immune cells can lose their response pattern and may act unresponsively due to the mechanisms of immune regulation mediated by regulatory t lymphocytes39,41. the presence of s. aureus antigens stimulated no production at all tested concentrations, with or without ifn-γ in l929 fibroblasts. in the presence of e. faecalis, the highest concentration of antigens was significantly important in inducing no production. fibroblasts are known to have an important role in tissue repair; however, they also respond to the antigen by producing il-6, mcs-f, tgf-β, and no42,43. until then, the classical stimuli studied for the evaluation of no production in l929 are ifn-γ, lps or both30. in this way, no production in human pulp fibroblasts in response to heat-killed 10 sousa et al. antigens from e. faecalis may increase alkaline phosphatase production in fibroblasts and, consequently, pulp calcification44. in addition, the production of no and other proinflammatory cytokines in fibroblasts may favor the expression of opg in these cells and consequently the formation of calcified pulp tissue45. besides, fibroblasts may be susceptible to no. an in vitro study demonstrated that 3 mmol.l-1 of no were responsible for the apoptosis of gingival fibroblasts. this action was associated with the c-jun n-terminal kinase signaling pathway46. in conclusion, no production by raw 264.7 monocytes and l929 fibroblasts against the pathogens presented in this study may contribute to the understanding of how microorganisms prevalent in the root canal system lead to a pro-inflammatory response, increasing no. this is an initial study and in view of the real role of this mediator, new studies with human cells must be carried out to establish its action both in the elimination of microorganisms and in the formation of new tissues during pulp revascularization/regeneration processes. acknowledgements this study was supported by the universidade católica de brasília (ucb), conselho nacional de desenvolvimento tecnológico (cnpq), coordenação de aperfeiçoamento de pessoal de nível superior (capes), fundação de amparo do distrito federal (fapdf) and fundação de apoio ao desenvolvimento do ensino, ciência e tecnologia do estado de mato grosso do sul (fundect). the authors deny any conflicts of interest. references 1. yang j, yuan g, chen z. pulp regeneration: current approaches and future challenges. front physiol. 2016 mar 7;7:58. doi: 10.3389/fphys.2016.00058. ecollection 2016. 2. galler, km clinical procedures for revitalization: current knowledge and considerations. int endod j. 2016 oct;49(10):926-36. doi: 10.1111/iej.12606. 3. dhillon h., kaushik m, sharma r. regenerative endodontics creating new horizons. j biomed mater res b appl biomater. 2016 may;104(4):676-85. doi: 10.1002/jbm.b.33587. 4. gronthos s, brahim j, li w, fisher lw, cherman n, boyde a, et al. stem cell properties of human dental pulp stem cells. j dent res. 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bg, et al. calcium hydroxide inactivates lipoteichoic acid from enterococcus faecalis through deacylation of the lipid moiety. j endod. 2011 feb;37(2):191-6. doi: 10.1016/j.joen.2010.11.007. 41. majka g, więcek g, śróttek m, śpiewak k, brindell m, koziel j, et al. the impact of lactoferrin with different levels of metal saturation on the intestinal epithelial barrier function and mucosal inflammation. biometals. 2016 dec;29(6):1019-1033. doi: 10.1007/s10534-016-9973-x. 42. gao yl, chai yf, qi al, yao y, liu yc, dong n, et al. neuropilin-1highcd4(+)cd25(+) regulatory t cells exhibit primary negative immunoregulation in sepsis. mediators inflamm. 2016;2016:7132158. doi: 10.1155/2016/7132158. 13 sousa et al. 43. chaves, ca., vergani ce, thomas d, young a, costa ca, salih vm, et al. biological effects of soft denture reline materials on l929 cells in vitro. j tissue eng. 2014 jun 23;5:2041731414540911. doi: 10.1177/2041731414540911. 44. miljkovic d, cvetkovic i, sajic m, vuckovic o, harhaji l, markovic m, et al. 5-aza-2’-deoxycytidine and paclitaxel inhibit inducible nitric oxide synthase activation in fibrosarcoma cells. eur j pharmacol. 2004 feb 6;485(1-3):81-8. doi: 10.1016/j.ejphar.2003.11.057. 45. sipert cr, moraes ig, bernardinelli n, garcia rb, bramante cm, gasparoto th, et al. heatkilled enterococcus faecalis alters nitric oxide and cxcl12 production but not cxcl8 and ccl3 production by cultured human dental pulp fibroblasts. j endod. 2010 jan;36(1):91-4. doi: 10.1016/j.joen.2009.10.014. 46. zhang x, aubin je, kim th, payne u, chiu b, inman rd. synovial fibroblasts infected with salmonella enterica serovar typhimurium mediate osteoclast differentiation and activation. infect immun. 2004 dec;72(12):7183-9. doi: 10.1128/iai.72.12.7183-7189.2004. braz j oral sci. 15(1):21-26 original article braz j oral sci. january | march 2016 volume 15, number 1 removable orthodontic appliances: frequency and cleaning agents used by students and recommended by dentists rita regina souza lamas1, mabel miluska suca salas1, tatiana pereira cenci2,marcos britto corrêa1, rafael guerra lund1 1universidade federal de pelotas – ufpel, pelotas dental school, department of restorative dentistry, pelotas, rs, brazil 2universidade federal de pelotas – ufpel, pelotas dental school, area of prosthetic dentistry, pelotas, rs, brazil correspondence to: rafael guerra lund universidade federal de pelotas, faculdade de odontologia, programa de pós-graduação em odontologia, área de concentração em dentística rua gonçalves chaves, 457 / sala 503 – centro cep: 96015568, pelotas, rs, brasil phone: + 55 53 3225-6741 /3222-4305 / 3222-4162 e-mail: rafael.lund@gmail.com abstract aim: to evaluate the prevalence and hygiene habits of 13-19 years-old adolescent users of removable orthodontic appliances (roa) and to determine hygiene methods for the appliances prescribed by dentists, in the city of pelotas. methods: the study had two stages. the first stage was a telephone interview with dentists. dentists were interview by telephone calls in order to obtain information regarding the hygiene methods for cleaning acrylic appliances. second stage was a cross-sectional study performed with schoolchildren. children from public and private schools with secondary level were included in the sample. a questionnaire was applied to the students using any type of roa. questionnaires included demographic information and behavioral characteristics. data collected were subjected to chi-square test and logistic regression. results: the prevalence of children using roa was 5.4%. students (89.7%) and dentists (47.2%) reported to prefer mechanical methods to clean their roa. cleaning with soup, hydrogen peroxide or effervescent tabs were less used. high frequency of use was associated with higher frequency of hygiene on the roa. conclusions: the prevalence of schoolchildren using removable appliances was low. the common cleaning method used by children and prescribed by dentists was mechanical. hygiene frequency was significantly associated with the routine of use of the appliance and with the type of hygiene method. keywords: adolescent. disinfection. hygiene. orthodontic appliance design. introduction concern for aesthetics has increased in the population1 and during the last three decades there has been a great demand for orthodontic treatments2. children in treatment with removable orthodontic appliances (roa) have higher risk for proximal caries, gingivitis and halitosis than children without roa3. a proper hygiene of the orthodontic appliances could reduce bacteria in the oral environment4. studies have investigated the efficacy of diverse hygiene methods to control the presence of microorganism in removable acrylic appliances5-9. most of them showed that the combination of a mechanical and chemical methods reduced microorganisms on the appliance surface, compared with other methods7-9. the role of the dentist in oral health promotion is important since dentists have evidence-based knowledge of oral health and could influence patient’s behavior10. few studies investigated the attitudes of dentists toward oral preventive measures. habits and characteristics of orthodontic patients including hygiene and attitudes toward roa are scarce. moreover, studies assessing the hygiene methods used by children to clean received for publication: march 09, 2016 accepted: may 17, 2016 http://dx.doi.org/10.20396/bjos.v15i1.8647093 22 their roa are inexistent. knowledge of children’s characteristics treated with orthodontic appliances is important. dentist’s recommendation should include effective alternatives to reduce microbiota and be able to prevent oral diseases. the present study had two objectives: (1) to determine the prevalence of the roa use in schoolchildren and its possible association with some socio-demographic factors and hygiene habits; and (2) to verify the hygiene methods prescribed by dentists. material and methods the study was approved by the ethics committee of the federal university of pelotas (protocol number 216/2011 ufpel/rs). a cross-sectional study was performed among schoolchildren and dentists in pelotas, a mid-sized city of the southern brazil, 260 km from porto alegre, the capital of rio grande do sul state. the study was performed in two stages. 1) dentists interview. a full list of the dentists registered in the state was obtained from the regional council of dentistry of rio grande do sul state (cro – rs) and the brazilian dental association – region of pelotas (abo-pelotas). eligibility criteria for the enrollment of dentists for this study: dentists living in the city of pelotas, actively working in the clinical practice with treatments involving roa and that agreed to participate were included in the study. dental specialties of dentists were not asked. interviews were conducted by phone calls. the sequence to obtain the final dentists sample is shown in figure 1. the questionnaire included two open question regarding the recommended methods and frequency (once, twice and three times a day) for roa cleaning. dentists not contacted on the first call, were re-called. after the third attempt without contact, dentists were considered as losses. removable orthodontic appliances: frequency and cleaning agents used by students and recommended by dentists fig.1. flowchart of dentist’s selection for telephone survey, pelotas, rs, brazil. 2) students interview according to the department of education of rio grande do sul state, pelotas has 19 states, 1 municipal and 9 private schools with secondary education. the number of children enrolled in secondary education of the city was 9,237 in public schools and 1,711 in private schools. stratification by type of school (private and public) was carried out from the number of high schools. four private schools and 16 public schools were randomly selected by the method of proportionality, where the probability of selection is proportional to the number of students in the school. the number of 20 schools was adequate to ensure good variability of the sample11. students from secondary education were initially eligible for the study. schoolchildren enrolled regularly in the selected high schools of the city, users of roa and able to answer the questionnaires were included in the study. parents or legal guardians and school tutors signed a written consent form to allow the participation of adolescents. previously developed questionnaires were administered to children by an interviewer in the classrooms. the interviewer was a previously trained post-graduate student. questionnaires included socio-demographic and behavioral data of children, such as age (complete years and later categorized in 13-14, 15-17, 18-19), sex, type of school, mother’s education in years (categorized in ≤ 8, 9-12, ≥13), frequency of roa use (categorized in sometimes and frequently), roa time of use, reason for the orthodontic treatment, usage discomfort and cleaning method of roa. the frequency of hygiene of the roa was categorized in low one or less times during a week and high every day. statistical analysis data were recorded in duplicate in the epidata 3.1 database (version 3.1, release 2006, epidata association, denmark). statistical analyses were carried out in stata 10.0 (statacorp. 2007. stata statistical software: release 10; statacorp lp college station, tx, usa). descriptive analysis was performed to describe the dentists’ recommendations and the children’s studied characteristics. bivariate analyses were performed to assess the association between frequency of hygiene (outcome) and the investigated variables (age, sex, type of school, frequency of roa use, reason for treatment, maternal education and cleaning habits) of children using roa. pearson’s chi-square test or linear trend were used depending on the type of variable. variables with p-value <0.30 were included in the multivariate analysis. for multivariate analysis, the poisson regression was used, considering a p value <0.05 as statistically significant with 95% confidence interval. results in the city of pelotas 1,056 dentists were on the abo-rs list. after the application of the eligibility criteria, 354 dentists remained as potential participants. two hundred and thirty five dentists participated in the study. the response rate was 66.4%. the selection process of the dentists is in figure 1. table 1 describes dentists’ recommendations. dentists recommended mainly to brush the acrylic appliances with toothpaste braz j oral sci. 15(1):21-26 23 (74.4%), 9.6% indicated to brush the roa with soap, 8.0% advised immersions in hypochlorite, chlorhexidine or peroxide solutions, 6.0% suggested other cleaning materials, 1.6% suggested corega tabs®, and 0.4% to brush with water. most dentists recommended cleaning the removable appliances 3 times per day (64.0%). removable orthodontic appliances: frequency and cleaning agents used by students and recommended by dentists * values lower than 235 are due to incomplete data. table 1 methods and frequency of hygiene recommended for removable orthodontic appliances (roa) by dentists. pelotas, brazil (n=235) * variables/categories n* % methods 235 brushing with common toothpaste 186 (74.4) brushing with soap 24 (9.6) mouthwash 20 (8.0) corega tabs® 4 (1.6) water 1 (0.4) hygiene frequency 208 once a day 16 (9.0) twice a day 48 (27.0) three times a day 144 (64.0) distribution of the children according to the outcome and the investigated factors is presented in table 2. our study included a total of 6,158 schoolchildren from high schools. the prevalence of the roa users in the schools was 5.4% (n=334). in private schools, the frequency of roa users was 14.3% and in public schools it was 3.0%. data showed that most users of the removable appliances were female (73.6%). a larger number of 15-17-yearold children (84.8%) used roa as the main orthodontic treatment. most children (35.1%) were using their roa for 6 months or less. children used their roa daily (71.3%). the roa were used mostly after the fixed appliances as adjuvant to orthodontic treatments (88.9%). most students (85.2%) cleaned their roa daily. the method most commonly used was brushing with toothpaste (89.8%). bivariate analysis is described in table 3. adolescents who used their roa only occasionally also cleaned them less frequently (p<0.001). the type of cleaning method was associated with the hygiene frequency (p<0.001) and children with lower frequency of hygiene used less frequently brushing tooth with soap. multivariate analysis (table 4) showed that children that used their roa frequently had less chances to clean less their appliances (or = 0.33; 95% ci: 0.19 to 0.57). discussion in our study, the prevalence of schoolchildren from high schools using roa was 5.4%. this result was lower than other studies12,13. krey and hirsch13 (2012) found that 16% of 11-14-year-old children used roa in germany. in england, chestnutt et al.12 (2006) observed that 28% of 12-year-old children and 18% of 15-year-old children used roa. this difference may be explained by the fact that in other countries such as germany13 and england12, orthodontic treatments were included in the health insurance, allowing orthodontic treatment for children. in brazil, the public health services do not include the orthodontic treatment as primary care policy, situation that could produce some social inequalities regarding the need and the actual used of an orthodontic appliance. in the present study, most children using an orthodontic appliance came from private schools. however, these results were not statistically different. demographic characteristics of the population can also influence the access to orthodontic treatment. the present study found a higher frequency of girls (73.6%) using removable appliances. similar results were found by o’brien et al.14 (1996). this situation was attributed to a higher demand of esthetic treatments by women15. table 2 socio-demographic and behavioral characteristics of students users of removable orthodontic appliances (roa). pelotas, brazil (n=334) * variables/categories n* % sex 334 male 88 (26.4) female 246 (73.6) type of school 334 private 173 (14.4) public 161 (3.0) age (years) 329 13-14 34 (10.3) 15-17 280 (84.8) 18-19 15 (4.8) mother’s education (years) 330 >13 143 (43.3) 9-12 117 (35.5) <8 70 (21.2) habits regarding the roa time of treatment 332 <6months 117 (35.2) 6-1year 104 (31.3) 1-2years 70 (21.1) >2years 41 (12.4) frequency of use 334 sometimes 96 (28.7) frequently 238 (71.3) reason for use 334 adjuvant of braces treatment 297 (88.9) dental retainer 28 (8.4) muscular retainer 9 (2.7) frequency of hygiene 332 low (<1 time/week) 49 (14.8) high (daily) 283 (85.2) cleaning method 332 brushing with common toothpaste 298 (89.8) brushing with soap 6 (1.8) hydrogen peroxide 7 (2.1) corega tabs® 8 (2.4) other 13 (3.9) * values lower than 334 are due to incomplete data. regarding the hygiene methods, schoolchildren mostly used toothbrush and toothpaste to clean their roa (89.9%). patients often clean their acrylic removable appliances with toothbrush and toothpaste12. this method was reported to be effective to maintain the health of the mucosa in contact with the acrylic appliance16. the use of a single hygiene method, without any chemical disinfectant, has been criticized, due to its poor biofilm removal from acrylic surface and improper control of microbial load8. in fact, several cleaning methods were investigated, including mechanical, chemical or the combination of both methods. the use of chlorhexidine6, mechanical brushing with water8 or effervescing tabs5 has shown a reduction of microbial load, but the comparison was with surfaces without any cleaning. studies have observed that the combination between a mechanical and a chemical method reduced significantly the presence of microorganisms7-9 from removable appliances compared with other methods. braz j oral sci. 15(1):21-26 24 braz j oral sci. 15(1):21-26 removable orthodontic appliances: frequency and cleaning agents used by students and recommended by dentists variables/categories hygiene frequency p valuelow high n % total n % total type of school 49 283 0.174* private 28 (17.5) 132 (82.5) public 21 (12.2) 151 (87.8) sex 49 283 0.160* male 17 (19.3) 71 (80.7) female 32 (13.1) 212 (86.9) age (years) 48 280 0.467** 13-14 3 (8.8) 31 (91.2) 15-17 43 (15.4) 236 (84.6) 18-21 2 (13.3) 13 (86.7) maternal education (years) 48 280 0.790** >13 22 (15.5) 120 (84.5) 9-12 16 (13.7) 101 (86.3) <8 10 (14.5) 59 (85.5) time of treatment 48 282 0.087** <6 months 15 (12.8) 102 (87.2) 6months-1 year 11 (10.7) 92 (89.3) 1-2 years 13 (18.6) 57 (81.4) >2 years 9 (22.5) 31 (77.5) frequency of use 49 283 0.001* rarely 25 (26.6) 69 (73.4) frequently 24 (10.1) 214 (89.9) reason for use 49 283 0.215* adjuvant of orthodontic fixed appliances 40 (13.6) 255 (86.4) dental retainer 7 (25.0) 21 (75.0) muscular retainer 2 (22.2) 7 (77.8) hygiene method 47 283 0.010* brushing with common toothpaste 36 (12.2) 260 (87.8) brushing with soap 2 (33.3) 4 (66.7) hydrogen peroxide 2 (28.6) 5 (71.4) corega tabs® 4 (50.0) 4 (50.0) other 3 (23.1) 10 (76.9) * chi-square (χ²) test ** χ² test for linear trend table 3 bivariate analysis of hygiene frequency of removable appliances wore by the students according to socio-demographic and behavioral characteristics of students. pelotas, brazil (n=334). our results showed that dentists that usually work with acrylic appliances recommended mainly brushing the acrylic appliances with toothpaste (74.4%). eichenauer et al.17 (2012) reported similar results regarding dentists’ attitudes regarding hygiene method for roa. these results contradict those of several studies8-9 with the most effective method to remove oral microbiota from surfaces. the dental practice should be related to these findings. studies have shown that some dental recommendations and examination protocols used by dentists are partially18 or totally in disagreement with scientific evidence19. the clinician’s decisions seem to be influenced by their knowledge and attitudes, as well as the dentist’s feelings and conscience10. the cost of the hygiene materials seems to be an important factor that determines dentists’ recommendations, as well as the facility for cleaning the roa by patients17. 25 braz j oral sci. 15(1):21-26 removable orthodontic appliances: frequency and cleaning agents used by students and recommended by dentists * variables with p< 0.30 in the bivariate analysis were included in the final analysis. table 4 odds ratio (or) for hygiene frequency of removable orthodontic appliances wore by the students according to sociodemographic and behavioral factors. pelotas, rs, brazil. variables/categories low hygiene frequency or (95%ci) p value* sex 0.393 male 1.0 female 0.73 (0.40-1.32) school /category 0.823 private 1.0 public 0.97 (0.56-1.68) time of treatment 0.069 <6 months 1.0 6 months-1 year 0.93 (0.44-1.93) 1-2 years 1.49 (0.74-3.02) >2 years 1.46 (0.64-3.30) frequency of use 0.001 sometimes 1.0 frequently 0.33 (0.19-0.57) reason for use 0.678 adjuvant of braces treatment 1.0 dental retainer 1.64 (0.72-3.67) muscular retainer 0.73 (0.11-4.73) hygiene method 0.002 brushing with common toothpaste 1.0 brushing with soap 1.50 (0.33-6.79) hydrogen peroxide 1.73 (0.53-5.66) corega tabs® 5.30 (2.27-12.36) other 2.80 (0.78-9.97) oral hygiene instructions for patient self-care and for roa cleaning are important activities to promote oral health and prevent diseases. healthy behaviors can prevent future oral problems. dentists could influence the patient behaviors10. for instance, the schoolchildren in our study seem to follow dentists’ recommendations regarding roa care. the importance of a correct hygiene relies on adequate control of the biofilm on surfaces20, especially for children undergoing orthodontic treatment. adolescence also could modulate and promote the increment of microorganism in the oral environment21 increasing the risk for caries and gingivitis3. it is necessary to learn the habits of children’s at risk to implement adequate oral health education programs. some limitations of this study can be pointed out. it had a cross-sectional design which is limited to the moment of data collection. the response rate of dentist was intermediate and was obtained by telephone survey. surveys have used telephone contact to obtain information in a fast way, with low cost22, especially if used as complement of other methods23. however, the telephone method has some limitations and could induce biased answers, increasing non-responses and lack of representativeness. to minimize bias, we used telephone survey and the internet to contact and interview dentists. even though in our study it was observed descriptively that adolescents are using the same cleaning method recommended by dentists, both observations were conducted in two different populations. hence, it is not possible to make predictions using any correlation. further research should be conducted to define the profile of children in orthodontic treatment and the optimal methods for cleaning roas, and most important to communicate this protocol to orthodontists/dentists and the population. in conclusion, the prevalence of schoolchildren using roa is low. children frequently clean the roa and frequently use them. the cleaning method prescribed by dentist and used by adolescents are different from the most effective method showed in the literature. acknowledgements the authors are grateful to the state secretary of education, the municipal secretary of education and the direction of private and public schools, which allowed the study to be performed. references 1. de couto nascimento v, de castro ferreira conti ac, de almeida cardoso m, valarelli dp, de almeida-pedrin rr. impact of orthodontic treatment on self-esteem and quality of life of adult patients requiring oral rehabilitation. angle orthodont. 2016 feb. 2. dimberg l, lennartsson b, arnrup k, bondemark l. prevalence and change of malocclusions from primary to early permanent dentition: a longitudinal study. angle orthod. 2015 sep;85(5):728-34. doi: 10.2319/080414-542.1. 3. pathak ak, sharma ds. biofilm associated microorganisms on removable oral orthodontic appliances in children in the mixed dentition. j clin pediatr dent. 2013 spring;37(3):335-9. 4. chauhan p, dua vs, kainth n, tosh a, tomar a. the effect of various oral hygiene products on the microbial flora in patients undergoing orthodontic treatment. apos trends orthod. 2015;5(2):63-9. 5. nisayif dh. the effects of removable orthodontic appliance hygiene on oral flora. j bagh college dentistry. 2009;21(2):105-8. 6. friedman m, harari d, raz h, golomb g, brayer l. plaque inhibition by sustained release of chlorhexidine from removable appliances. j dent res. 1985 nov;64(11):1319-21. 7. salas mms, lamas rrs, cenci tp, lund rg. how are children and adolescents cleaning their orthodontic appliances? a cross-sectional study in private schools. braz j oral sci. 2014 jan/mar;13(1):34-6. 8. paranhos hf, silva-lovato ch, de souza rf, cruz pc, de freitas-pontes km, watanabe e, et al. effect of three methods for cleaning dentures on biofilms formed in vitro on acrylic resin. j prosthodont. 2009 jul;18(5):42731. doi: 10.1111/j.1532-849x.2009.00450.x. 9. peixoto it, enoki c, ito iy, matsumoto ma, nelson-filho p. evaluation of home disinfection protocols for acrylic baseplates of removable orthodontic appliances: a randomized clinical investigation. am j orthod dentofacial orthop. 2011 jul;140(1):51-7. doi: 10.1016/j. ajodo.2009.12.036. 10. madan c, arora k, chadha vs, manjunath bc, chandrashekar br, rama moorthy vr. a knowledge, attitude, and practices study regarding dental floss among dentists in india. j indian soc periodontol. 2014 may;18(3):361-8. doi: 10.4103/0972-124x.134578. 11. bennett s, woods t, liyanage wm, smith dl. a simplified general method for cluster-sample surveys of health in developing countries. world health stat q. 1991;44(3):98-106. 12. chestnutt ig, burden dj, steele jg, pitts nb, nuttall nm, morris aj. the orthodontic condition of children in the united kingdom, 2003. br dent j. 2006 jun 10;200(11):609-12;quiz 638. 13. krey kf, hirsch c. frequency of orthodontic treatment in german children and adolescents: influence of age, gender, and socio-economic status. eur j orthod. 2012 apr;34(2):152-7. doi: 10.1093/ejo/cjq155. 14. o’brien k, mccomb jl, fox n, wright j. factors influencing the uptake of orthodontic treatment. br j orthod. 1996 nov;23(4):331-4. 15. zhang yf, xiao l, li j, peng yr, zhao z. young people’s esthetic perception of dental midline deviation. angle orthod. 2010 may;80(3):51520. doi: 10.2319/052209-286.1. 16. rossato mb, unfer b, may lg, braun ko. analysis of the effectiveness of different hygiene procedures used in dental prostheses. oral health prev dent. 2011;9(3):221-7. 17. eichenauer j, serbesis c, ruf s. cleaning removable orthodontic appliances: a survey. j orofac orthop. 2011 oct;72(5):389-95. doi: 10.1007/s00056-011-0043-2. 18. norton we, funkhouser e, makhija sk, gordan vv, bader jd, rindal db, et al. concordance between clinical practice and published evidence: findings from the national dental practice-based research network. j am dent assoc. 2014 jan;145(1):22-31. doi: 10.14219/jada.2013.21. 19. frame ps, sawai r, bowen wh, meyerowitz c. preventive dentistry: practitioners’ recommendations for low-risk patients compared with scientific evidence and practice guidelines. am j prev med. 2000 feb;18(2):159-62. 20. duyck j, vandamme k, krausch-hofmann s, boon l, de keersmaecker k, jalon e, et al. impact of denture cleaning method and overnight storage condition on denture biofilm mass and composition: a crossover randomized clinical trial. plos one. 2016 jan 5;11(1):e0145837. doi: 10.1371/journal.pone.0145837. 21. sampaio-maia b, monteiro-silva f. acquisition and maturation of oral microbiome throughout childhood: an update. dent res j (isfahan). 2014 may;11(3):291-301. 22. mokdad ah, remington pl. measuring health behaviors in populations. prev chronic dis. 2010 jul;7(4):a75. epub 2010 jun 15. 23. moura ec, claro rm, bernal r, ribeiro j, malta dc, morais neto o. a feasibility study of cell phone and landline phone interviews for monitoring of risk and protection factors for chronic diseases in brazil. cad saude publica. 2011 feb;27(2):277-86. 26 braz j oral sci. 15(1):21-26 removable orthodontic appliances: frequency and cleaning agents used by students and recommended by dentists untitled 1 volume 16 2017 e17058 original article 1 department of occlusion, fixed prosthodontics and dental materials, school of dentistry, federal university of uberlândia, uberlândia, mg, brazil. corresponding author: prof. dr. flávio domingues das neves av. pará, 1720, bloco 4l, anexo a sala 4la-42, campus umuarama, cep: 38405-320, uberlândia, minas gerais, brazil e-mail: neves@triang. com.br phone: +55-34-3218-2222 fax: +55-34-3218-2626 received: june 02, 2017 accepted: september 20, 2017 comparative analysis of optical microscopy, scanning electron microscopy, and micro-computed tomography on measurements frederick khalil karam, dds, msc1, karla zancope, dds, msc, phd1, thiago de almeida prado naves carneiro, dds, msc, phd1, murilo navarro de oliviera, dds candidate, caio césar dias resende, dds, msc1, flávio domingues das neves, dds, msc, phd1 abstract: microscopic measurements are widely used in scientific research and the correct equipment to perform these evaluations could be critical to determine study results. regarding microscopic measurements, three of the most used methods are: optical microscopy (om), scanning electron microscopy (sem), and micro-computed tomography (mct). it is important to select the best method for assessing diverse parameters, considering operational characteristics of the method, the equipment efficiency, and the machinery cost. aim: therefore, the main objective of this study was to define which is the most useful measurement method for assessing magnitudes below 0.4 mm. methods: ten dental implants, with known dimensions as defined by the manufacturer were randomly distributed. two blinded observers assessed the distance between the second and the third screw vortex of the implants using three suggested methods. the true distance was defined to be 0.5 mm. results: the assessed distances were: 0.597±0.007mm for om, 0.578±0.017mm for sem, and 0.613±0.006mm for mct. the assessed distances were significantly different when the methods were compared (p>0.01). all measurements were into the cad tolerances. conclusion: it was possible to conclude that linear measurements between 595 and 605 μm could be performed by any of the described technologies. keywords: measurements, dental implants, optical microscope, micro-ct, electron microscope. http://dx.doi.org/10.20396/bjos.v16i0.8651056 2 karam et al. introduction the application of the correct measurement method for microscopic analysis on scientific research is crucial to obtain reliable values, which will not cause distortions on investigation results1,2. the choose of the correct method will diverge according to the study objectives and the material to be evaluated3. some other variables should be considered to final decision, like operator dexterity with the equipment, apparatus disposition and machinery operation cost; however these aspects could not interfere on results trustworthiness4,5. before adopting any technology to measure samples, the operator needs to consider the main objective of the study and what part of the sample must be analysed6,7. the election of an inadequate measurement technique may lead to an unnecessary use of method, with consequently depreciation of the equipment and waste of time. lack of necessary data could make an inconclusive study8. among diversified researches, dentistry, and specifically implant dentistry requires micrometric examinations to define security measurements. implant manufacturing, requires meticulous and preciseregulations9,10. measurements and surface analysis at lower scales of the dental implants and their fitting components during their fabrication should be severally reliable to ensure a satisfactory piece seating and consequently, the rehabilitation biomechanical success11,12. therefore, the correct methodology application during the implant manufacturing by the producer are directly connected to the therapy prosperity13. in dispersion through the most used approaches to realize micrometric analysis are: optical microscope (om), a two-dimensional evaluation method that uses a series of glass lenses to create a limited magnification, is widely used for surface quantitative mensuration12,14. scanning electron microscope (sem), an electronic microscope that could provide surface images with high resolution and magnification15,16 and micro-computed tomography (micro-ct), a non-destructive method that allows high-resolution tridimensional analysis, without damaging the sample17. therefore, the aim of this study was to evaluate the three method’s precision, comparing to o.m., s.e.m. and micro-ct on dental implants mensuration, checking for possible images distortions, according to manufacturer tolerance limits. the null hypothesis was there was no statistic difference between the three evaluated methodologies, presenting all values into a confidence interval. material and methods initially, two evaluators were randomly recruited to perform the measurements of all tested methodologies. these evaluators did not know what they would evaluate and were trainee to perform ruler measurements. ten dental implants (titamax 3.75mm x 13mm; neodent, curitiba, brazil) were selected for this study, and distance between vertex to vertex from the second to the third screw was chosen to be measured in all methods described below. an implant, to be manufactured, is design first in cad, and then sent to the milling machine. the cad used to produce these implants was requested, in order to establish what is the real distance from vertex 3 karam et al. to vertex of these implants. therefore, the cad of the implant itself was used as a control. the samples were positioned on a device to standardize the position of the implant to each method. optical microscope (om) the samples were fixed in an optical microscope (mitutoyo tm-500, tokyo, japan) to perform the implant mensuration. this is a monocular microscope with two digital micrometers and0.001 resolution. the equipment has objective lenses with 2x magnification and ocular lenses with 20x magnification, resulting in a 40x enlargement. the pixel resolution obtained is 5 mp (mega pixel). the microscope has a holder over all the samples that allow the positioned sample to move over the x and y-axes, during the mensuration (fig. 1). scanning electron microscope (sem) the sem used in this study (hitachi, chiyoda-ku, tokyo, japan) operates in different pressure conditions, controlled by a computer using the windows operational system (loquif software, leo user interface, carl zeiss, oberkochen, germany). magnification of 150x was applied. the pixel resolution obtained is 5 mp. the measurements were realized directly on the equipment software (fig. 2). micro-computed tomography (micro-ct) the implants were scanned using a computed microtomography (skyscan 1272, konith, belgium). the used parameters were: 100kv voltage source, 100a chain source, 2452 x 1640 resolution, 20 pixels, 18.0 cu filter, 0.2 degrees rotation step, 20 aleatory movements and images average of 2, rotation step (deg) 1000, averagfigure 1. optical microscope obtained image, and vortex marking to the distance calculation. 4 karam et al. ing frames of 2, random movement (pix) of 20, pixel size 18.0 mp and the standard scan in central camera position. after these parameters definition, the samples were removed of the tomography and a flat-field was requested to generate smaller artifacts in the final image. the scanning time was set to 26 minutes, for this kind of evaluation (fig. 3). figure 2. sem surface image and software vortex to vortex measurement. 560um figure 3. micro-ct constructed image and measure tool utilization. distance = 0.609 mm 5 karam et al. two blinded evaluators performed all measurements, and the mean values of each sample were considered for statistical evaluation (sigmaplot 12.0, systat software inc, usa). the data were initially submitted to the variance homogeneity and normality tests (α=0.05). to compare all groups, the anova one way statistical method was applied before the tukey test. to compare each group with the cad (control group), the t-test was applied. a qualitative evaluation was also applied, to define if the values were into a confidence interval, defined by implant’ manufacturer. the control group was considered by the cad image of the tested implant, provided by manufacturer. results the mean values of all methodologies, between both evaluators, are shown in table 1. there were statistical differences between all tested methodologies. however, according to a qualitative evaluation, all methodologies presented acceptable values, when comparing to the cad (control group), according to table 2. discussion the null hypothesis that, there was no statistic difference between all evaluated methodologies presenting all values into a confidence interval, was reject. the results demonstrated in table 1 prove that there is statics difference between the three tested groups. however, all values were into a fabrication tolerance, defined by manufacturer. this qualitative evaluation define the values into the cad’s tolerance, produced by the milling machine, but do not affect the implant’s quality. the values of the cad were between 595μm and 605μm. any value into this interval was considered acceptable. table 1. dental implants vertex to vertex average and standard deviation evaluation. letters between parentheses demonstrate statistical differences. group samples average standard deviation p value m.c.t. 10 0.613 (a) 0.00624 <0.001 o.m. 10 0.597 (b) 0.00664 <0.001 s.e.m. 10 0.578 (c) 0.00168 <0.001 table 2. evaluations average. the provided company cad (control) was inserted on the first line. the second, third and fourth line were the groups values. group minimal value maximal value control 0.550 0.650 o.p.t.(a) 0.588 0.607 m.e.v.(a) 0.560 0.611 m.c.t.(a) 0.604 0.617 6 karam et al. regardless of the methodology, the operator factor could directly influence any study that uses measurements. two blind evaluators were used to determine the distance between two determined points. they were instructed only to measure the distance between the vortices of the second and third threads of dental implants. the evaluators were not influenced in any way about the location of the selected point. the operator factor is directly related to the results of any measurement and therefore was discussed. that’s why the authors used ten implants on each methodology: to decrease operators’ bias (mean±sd). it is uncommon, in literature, studies that indicate the specific criteria used for evaluation in a measurement methodology; it is only reported which methodology was used for evaluation. several studies18-22 evaluated marginal fit using different methodologies for measurements. but to decrease operators’ bias, the studies used more then one operator to perform measurements. according to a previous study23, the om is the best methodology to superficially measure the spaces. the om demonstrates lower spatial resolutions, making it difficult the using for analysis of ultrafine deformations and strain measurements24. it is the cheaper technology compared to the other methods, and is easy handling equipment, which does not, requires a special training to use it. for these reason this methodology could be indicated technology to external analysis with values between 595μm and 605μm. however, it is important to emphases that all methodologies allow the visualization in two dimensions, and could be an important limitation. some studies18,20 that performed internal measurements, these methodologies could not be used. the sem is an alternative methodology that produces high-resolution images down to the nanometer scale and provides reliable measurement data using image analysis methods, such as digital image correlation16. in meantime, alerts that sem images are usually contaminated with distortions and drift aberrations that could disturb the accuracy of imaging and measurement24. to analyze organic materials, such as the needing of a specialized apparatus and the risk of damage to the samples caused by the vacuum pressure in tem sem3, could interfere on studies with human tissues measurements, for example. in the present study, the evaluators related a complexity to use this equipment, once the samples preparing and readout were more complicated compared with the other technologies. the price of the equipment would not justify the using for linear analysis above 605 μm, due all methodologies are statistical equal. however, for measurements below this value, some studies presents advantages to this equipment7,8,12,16,25. according to the literature, micro-ct is a faster method that limits the manual error that occurs using the om, which is an optically based measurement, being susceptible to human failures26. beside that, the micro-ct technology can extract 3d information, different of the 2d information obtained in optical measurements. the quality of micro-ct images, and the advantages of being a non-damage technique proposing this technology utilization to quantify mistakes on techniques with less image quality27. but is a methodology used only on in vitro studies, despites the high radiation and the time spent on scanning, reconstruction and measurement; making this technology impracticable during the clinical routine28. instead the related works affirms26, our study shows that micro-ct method takes more scanning time than the other methodologies, moreover, is an expensive equipment, which limits the utilization for 7 karam et al. large scale measurements. the convenience observed using microtomography is the possibility to realize internal analysis without damaging the samples29. in this work there was no advantage with this equipment utilization, once this technology requires specifically preparation and is an expensive machinery compared with the om. the statistical differences found at the present study makes it impossible to compare the three methodologies, although all measurements obtained are in the manufacturing tolerance, it could not be considered as accurate. considering the results of this study, any methodology can be used to measure values larger than 0.5mm. so, authors should choose the methodology passed in two different parameters: first, the most familiar methodology to researcher and second, less cost and time to be done. in addition, the sem image quality and the non-destruction of samples in micro-ct are determinant in the results. future studies could evaluate the influence of methodologies in smaller measurements with values below 0.5mm. in conclusion, all measurements were into the cad tolerances. it was possible to conclude that linear measurements between 595 and 605 μm could be performed by any of the described technologies. acknowledgements the authors would like to thank the researchers rubens spin neto, marcel santana prudente, neodent, fapemig, capes and research center for biomechanics, biomaterials and cell biology (cpbio ufu) references 1. chatburn rl. evaluation of instrument error and method agreement. aana j. 1996 jun;64(3):261-8. 2. ludbrook j. statistical techniques for 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dentistry, area of forensic dentistry, piracicaba, sp, brazil. 3 dds, ms, phd universidade de campinas unicamp, piracicaba dental school, department of fisiology sciences, area of pharmacology, piracicaba, são paulo, brazil. 4 dds, ms, phd, universidade de campinas unicamp, piracicaba dental school, department of social dentistry, area of forensic dentistry, piracicaba, sp, brazil. 5 dds, msph, phd, universidade de campinas unicamp, piracicaba dental school, department of social dentistry, piracicaba, sp, brazil. corresponding author: luiz francesquini júnior faculdade de odontologia de piracicaba – unicamp departamento de odontologia social avenida limeira, 901 cep: 13.414-903 piracicaba, sp, brasil e-mail: francesq@unicamp.br phone: +55 19 98304-6173 received: may 11, 2017 accepted: august 10, 2017 sex estimation in brazilian sample: qualitative or quantitative methodology? viviane ulbricht1, cristiane martins schmidt2, francisco carlos groppo3, eduardo daruge júnior4, dagmar de paula queluz5, luiz francesquini júnior4* aim: this study carried out cranial measurements (in mm) [zygion-zygion (zy-zy); zygion-glabella-right side (zy-ga-right); zygion-glabella-left side (zy-ga-left); zygion-glabella-mean (zy-ga-mean); rhinion-anterior nasal spine (rhi-ans); nasal width (na wid); nasion-anterior nasal spine (na-ans); glabella-anterior nasal spine (ga-ans); glabella-prosthion (ga-pr)], to verify whether they are dimorphic. methods: we used skulls from the eduardo daruge laboratory of forensic physical anthropometry, which did not present growth abnormalities and belonged to the age range of 18 to 100 years. linear measurements were performed by digital caliper, properly calibrated. inter and intra-calibrator calibration was performed by obtaining as result the value of 0.98 (considered excellent). results: we found that all measures carried out are dimorphic, and we were able to create a logistic regression model (logit: sex = −33.6 + (0.15 × zy-zy) + (0.21 × rhi-ans) + (0.16 × na-ans)) to estimation the sex. conclusions: we concluded that the developed quantitative method results in 85.2% sensitivity, 76.2% specificity, and 81.1% accuracy, being, therefore, more effective in the prediction of sex than the mere random hit. keywords: forensic dentistry. forensic anthropology. sex characteristics. skull. 2 ulbricht et al. introduction the identification process has forensic physical anthropometry as its tool, which, although not permitting to establish the identity of a corpse (bones), allows to determine sex1 and ancestry and estimate age and height2. although the international literature recommends that ancestry must be established before sex, in brazil, such rule has been modified, due to the large number of miscegenation in the brazilian population growth2. based in the publications review of the last fifteen years3 on the methods of anthropometric identification used by various authors, researchers have found a distribution in six categories: visual examination of bones; anthropometric measurements of bones; anthropometric measurements with subsequent statistical analysis in the form of discriminant function analysis; time and sequence of teeth eruption; radiographic examination of the internal structure of bones; and microscopic examination of the inner structure of the bone. reviewing the situations that created the need for performing bone anthropometry, from 1971 to 1981, authors4 have verified that the bones most found in disaster situations and/or mass accidents were the skull, femur, and jaw. in situations with large number of bones, such as: explosions, wars, other mass disasters, and aviation accidents, the sex determination (separation into male and female) allows a considerable time gain in the process of identity establishment1, which is important in the daily activities of an institute of medicine, legal dentistry, and forensic physical anthropometry. sex estimation is a primary component for identifying skeletonized individuals in forensic physical anthropology5. it can be qualitative and/or quantitative (using logistic regression mathematical formulas), and both must be done judiciously, considering the ethnic or regional group of the sample, the time period in which it was held, among other situations. for the qualitative method, a study6 evaluated the conduct of several authors and stated that the bizygomatic width, mastoid length, zygomatic process width, and mastoid height, in that order, are the most important features for sex determination. rogers7 (2005) studying 17 qualitative characteristics to determine sex, authors have concluded that the anterior nasal aperture, zygomatic extension, size and roughness of the maxilla and supraorbital ridge are the most dimorphic ones, followed by the mentum and nasal crest shapes and mastoid size. verifying the sex in known population groups by qualitative methodology is possible, provided that the expert has prior knowledge of the population group to which this belongs8. currently, several authors9,10 have reported in their studies that the metric method (qualitative) cannot be replaced only by the visual method (qualitative), but, in practice, some mathematical models that were created by national sample do not faithfully reproduce the results found by its idealizers, which puts the expert in state of alert, because one can make mistakes in the application of such regression models2. by the quantitative method, one can determine the sex in the skull by numerous structures and several linear measures, namely: 3 ulbricht et al. • bicondylar width, mandibular angle, minimum ramus width11; • bizygomatic width, ramus height, face depth12; however, the mandibular angle is not very useful for determining the sex of africans from pretoria13. because of the miscegenation that occurred per region in our population, we think that the mathematical models created should be properly validated in other samples of population groups by brazilian geographic region, to verify the real hit and viability of using this technique in the brazilian territory. in the same research line, one should also look at cases in which the deoxyribonucleic acid (adn) testing does not allow to establish the identity of a corpse. a study14 analyzed mandibles of pre-hispanics from the canary islands using three identification methods, namely: • visual inspection; • osteometric measures; • analysis of dna amelogenin. they obtained, respectively, 66.04% hit, followed by 72.2% and 73.78%. from this result, one can infer the need to associate methodologies, depending on the financial availability, to achieve greater security in the identification process. material and methods this study is in accordance with the brazilian resolution no. 466/12 of the national health council, ministry of health, which regulates research involving human beings, and had prior approval of the research ethics committee at the piracicaba dental school, university of campinas (caae: 38522714.6.0000.5418). this is a cross-sectional analytical observational study based on file of human bones of both sexes, with 136 from females and 184 from males, aged between 18 and 100 years, and races. to start the measurements, we promoted inter and intra-examiner calibration. linear measurements were performed (zygion-zygion; zygion-glabella-right side; zygion-glabella-left side; zygion-glabella (mean); rhinion-anterior nasal spine; nasal width; nasion-anterior nasal spine; glabella-anterior nasal spine; glabella-prosthion) in three different time periods in 25 bones, with an interval of a month between them. the choice for linear measures used bone structures that are indicated in the literature10,15 as dimorphic. ended this step and with tools duly calibrated, we measured the rest to reach 185 skulls of the eduardo daruge laboratory of forensic physical anthropology, with known sex, age, and race. to carry out the measurements, we used a digital caliper (stainless-hardened® 150 mm, mauá, são paulo, brazil) data were submitted to the shapiro–wilk and levene’s tests to assess, respectively, the distribution and equality of variances (homoscedasticity) of the variables under study. student’s t test and chi-squared test also were conducted, as well as logistic regression (backward stepwise – wald), hosmer–lemeshow and nagelkerke tests. 4 ulbricht et al. results the calibration result was considered excellent (icc ≥ 0.75) for all calibrated measurements. table 1 shows the means (± standard error) of the variables studied according to the sexes and races. there was normal distribution (p>0.05), and variances were similar (p>0.05) for all variables. data analysis showed balance in the number of male and female samples (chi-squared, p=0.24), and race did not differ between sexes. however, all measures were significantly (p<0.01) higher in males. to observe if there was dependency between sexes with the other measures, a logistic regression (backward stepwise – wald) was performed, considering males as “1” and females as “0” for calculation purposes, as table 2 shows. at random, the chance to correctly predict the sex in the study sample would be 54.6%. the proposed model was significantly (chi-squared=94.5; p=6.4×10−20, for cutoff value of 0.5) better than chance to predict sex. hosmer–lemeshow test showed that the model was properly adjusted (chi-square=6.12, p=0.63) to the data. in addition, the nagelkerke r2 showed that the variables account for 52.6% of the variation found in sex. the logit was, therefore: sex = −33.6 + (0.15 × zy-zy) + (0.21 × rhi-ans) + (0.16 × na-ans). values higher and lower than 0.5 (cutoff), respectively, would be regarded as “male” and “female.” table 3 shows the prediction considering this relationship. table 1. means (± standard error) of the variables studied according to the sex. female (n=84) male (n=101) p-value1 race white 47 (56%) 62 (73.8%) 0.51black 16 (19%) 13 (15.5%) brown 21 (25%) 26 (31%) measures (in mm) zy-zy 121.4 (±0.63) 128.7 (±0.61) 2.3×10−14 zy-ga-right 90.2 (±0.55) 95.5 (±0.45) 2.1×10−12 zy-ga-left 91.1 (±0.55) 96.6 (±0.49) 1.9×10−12 zy-ga (mean) 90.6 (±0.53) 96.1 (±0.45) 2.5×10−13 rhi-ans 29.5 (±0.37) 33.2 (±0.31) 1.2×10−12 na wid 24.2 (±0.23) 25.2 (±0.23) 0.0025* na-ans 47.4 (±0.34) 51.3 (±0.33) 3.4×10−14 ga-ans 57.9 (±0.41) 61.6 (±0.4) 1.1×10−09 ga-pr 71.6 (±0.67) 76.7 (±0.57) 2.6×10−08 *statistically significant difference (p-value<0.05). 1race – chi-squared; measures – unpaired student’s t test. 5 ulbricht et al. table 3 shows that the method results in 85.2% sensitivity, 76.2% specificity, and 81.1% accuracy, being, therefore, more effective in the prediction of sex than the mere random hit. the regression also showed that the variables race (p=0.64), zy-ga-right (p=0.35), zy-ga-left (p=0.46), zy-ga-mean (p=0.34), na wid (p=0.40), ga-ans (p=0.49), and ga-pr (p=0.55) were not relevant for the model. discussion it was verified that all linear measurements were dimorphic and it was possible to create a logistic regression model [logit: sex = 33.6 + (0.15 x zi-zi) + (0.21 x ri-ena) + (0.16 x na-ena )]; to determine the sex obtaining 85.2% of sensitivity, 76.2% of specificity and 81.1% of accuracy. brazilian anthropometry only gained prominence in the nineteenth century, where rodrigues began studies in brazilians in order to prove the superiority of races (greater intellect, better strength and general health, besides anthropometrically determining the criminality of each individual)16. it was intended to predict the possibility that some item of the phenotype (skin color, hair type, etc.) and anatomical aspects (broad and low head, etc.) could indicate whether or not an individual would be a criminal16. these studies16 have proved imprecise and even prejudiced, and are not, in fact, studies that prove the brazilian reality. according to penna16 (2002), this fact was studied by roquete pinto, who analyzed brazilian mixed-race individuals (white individuals x black individuals x aborigens individuals), proved that they were intelligent, strong and healthy. table 3. prediction of sex according to the logistic regression. prediction by formula female male correct percentage real sex female 64 20 76.2 male 15 86 85.2 correct overall percentage 81.1 table 2. logistic regression mode/for sex determination. measures coefficient standard error wald p-value standardized coefficient zy-zy 0.15 0.035 19.3 1.1×10−05 1.17 rhi-ans 0.21 0.074 8.4 0.0037 1.24 na-ans 0.16 0.075 4.5 0.0332 1.17 constant −33.6 4.91 46.7 8.2×10−12 2.7×10−15 6 ulbricht et al. this fact breaks the theory of the superiority of the “races”, which considered the brazilian “ mixed-race individuals” to be “unfeasible” as part of a promising nation, recommending the whitening of the population, seeking the incorporation of attributes of the white individuals. in brazil, only after 1990, began anthropometric studies of real interest to identify a particular bone9. however, these studies occurred in a sparse way, with different methodology and the mathematical models obtained were in small, old samples, which could present a large margin of error when applied9. however, the incessant quest for quality has generated the need for methodological standardization. such a feat became world-wide by interpol17 (2014), who classified forensic anthropometry as a secondary method to be used in identification. this situation, according to jurda and urbanová18 (2016), have demanded from the experts involved in the process an analysis of the need to improve existing mathematical models through the validation of developed models, as well as more complete qualitative evaluations and with greater reliability, and certainty about the achievement of the results (sex, age, race and height). anthropometric, quantitative (with linear and angular measurements) in teeth19, skulls20 and other bones of the human body21 have been stimulated, mainly because they generate discriminant functions and logistic regression, which are the result of scientific research that was duly evaluated by the editorial staff. most of the authors9 who studied the sexual dimorphism by logistic regression indicate that the skull alone allows the establishment of sex with approximately 77% or more of certainty and if the pelvis is also used it reaches 95%. however, it should be noted that in our country, similar to the european countries, there is a percentage of 15 to 20% of undifferentiated individuals (individuals with few differential characteristics)10. another problem frequently reported by anthropologists is that the of estimation sex by the qualitative method has generated the impression that these are more reliable than the quantitative method. it happens that such anthropologists forget that unintentionally, when analyzing anatomical aspects, they are actually measuring them, as an example is the pubic angle, which if open is female and if closed is male. in this way, the performance of a qualitative evaluation can be in fact quantitative and in a certain plausible way of being measured, allowing the estimation of sex in a safe way. this study has already been carried out by photogrammetry using 3d laser scanner22. in addition to choosing a reproducible methodology, it is also necessary to clearly specify how the sample to be evaluated will be composed, taking care to specify the age of the sample, its general race and sex. the sample studied is inserted into the bone biobank, composed of 320 complete bones, of which 184 are male and 136 are female. of these, 58.75% are white individuals, 27.81% are mixed-race individuals, 13.12% black individuals and 0.32% (n = 1) aborigens individuals. the cause of death is still further information. 7 ulbricht et al. another situation to be highlighted is the fact that it is necessary for the individual who comes to perform the measurements for anthropometric purposes to be initially calibrated (intra and inter-examiner calibration) and the assistance of an experienced anthropologist in order to avoid problems of errors in the location of the anthropometric points to be measured. the national literature9,23 and international10 are unanimous and affirm that the qualitative method when used alone is flawed, with only the exception for cases where even not measuring the structure analyzed, the evaluator can do it visually. the result found in the present quantitative study is in agreement with the findings of other researchers24-27 in other countries and allows the estimation of sex in bones of missing individuals. another interesting situation is the use of computed tomography scans that allow internal structures to be measured with or without craniometric points28,29. computed tomography scanners must have minimum contrast, brightness and sharpness requirements and the software should allow visualization in axial, coronal and sagittal sections. in general, these software have a high cost and depend on a minimum knowledge of sectional anatomy. it should be noted that there are 3d (three-dimensional) software that can measure even situations that are apparently immeasurable, such as rougher, more prominent, more prominent, so-called photogrammetry areas using 3d laser scanners, both of which are being developed by our group and soon the results will be released. it was concluded that all measures performed are dimorphic and it was possible to create a logistic regression model to estimated the sex. it was concluded that the developed logistic regression model obtained 81.1% accuracy, which is therefore more effective in predicting sex than a simple visual qualitative test. acknowledgements the authors are thankful to the cnpq for financial support. references 1. kanthem rk, guttikonda vr, yeluri s, kumari g. sex determination using maxillary sinus. j forensic dent sci. 2015 may-aug;7(2):163-7. doi: 10.4103/0975-1475.154595. 2. francesquini júnior l, francesquini ma, de la cruz bm, pereira sd, ambrosano gm, barbosa cm, et al. identification of sex using cranial base measurements. j forensic odontostomatol. 2007 jun;25(1):7-11. 3. bass wm. recent developments in the identification of human skeletal material. am j phys anthrop. 1969 may;30:459-62. 4. bass wm, driscoll pa. summary of skeletal identification in tennessee: 1971-1981, j forensic sci. 1983 jun;28(1):159-68. 5. isaza j, díaz ca, bedoya jf, monsalve t, botella mc. assessment of sex from endocranial cavity using volume-rendered ct scans in a sample from medellin, colombia. forensic sci int. 2014 jan;234:186 e1-10. doi: 10.1016/j.forsciint.2013.10.023 8 ulbricht et al. 6. krogman wm, iscan my. the human skeleton in forensic medicine. ilinois: cc thomas publisher; 1986. 7. rogers tl. determining the sex of human, remains trough cranial morphology. j forensic sci. 2005 may;50(3):493-500. 8. keen ja. a study of the differences between male and female skulls. am j phys anthrop. 1950 mar;8(1):65-79. 9. daruge e, daruge júnior e, francesquini júnior l. [treaty of legal dentistry and deontology]. rio de janeiro: guanabara koogan; 2017. portuguese. 10. coma jmr. [forensic antropology]. madrid: ministerio da justicia; 1999. spanish. 11. kharoshah maa, almadani o, ghaleb ss, zaki mk, fattah yaa. sexual dimorphism of the mandible in a modern egyptian population. j forensic leg med. 2010 may;17(4):213-5. doi: 10.1016/j.jflm.2010.02.005. 12. naikmasur vg, shrivastava r, mutalik s. determination of sex in south indians and immigrant tibetans from cephalometric analysis and discriminant functions. forensic sci int. 2010;197:122.e1-122.e6. 13. oettle ac, becker pj, villiers e, steyn m. the influence of age, sex, population group, and dentition on the mandibular angle as measured on a south african sample. am j phys anthropol. 2009 aug;139(4):505-11. doi: 10.1002/ajpa.21009. 14. rosa ma, gonzáles e, fregel r, velasco j, delgado t, gonzáles am, et al. canary islands aborigin sex determination based on mandible parameters contrasted by amelogenin analysis. j archaeol sci. 2007 sept;34(9):1515-22. doi: http://dx.doi.org/10.1016/j.jas.2006.11.008 15. vanrell j. [legal dentistry and forensic anthropology]. rio de janeiro: guanabara koogan; 2009. portuguese. 16. penna, sdj, organizator. [homo brasilis: genetic, linguistic, historical and socioanthropological aspects of the brazilian people’s formation]. ribeirão preto: funpec; 2002. 191 p. portuguese. 17. interpol – disaster victim identification guide – 2014 [accessed 2017 feb 7]. available from: https:// www.interpol.int/interpol-expertise/forensics/dvi. 18. jurda m, urbanová p. sex and ancestry assessment of brazilian crania using semi-automatic mesh processing tools. leg med (tokyo). 2016 nov;23:34-43. doi: 10.1016/j.legalmed.2016.09.004. 19. iqbal r, zhang s, mi c. reability of mandibular canine and mandibular canine index in sex determination: a study using uwghur population. j forensic leg med. 2015 jul;33:9-13. doi: 10.1016/j.jflm.2015.03.007. 20. garvin hm, sholts sb, mosca la. sexual dimosphism in human cranial trait scores: effects of population, age, and body size. am j phys anthropol. 2014 jun;154(2):259-69. doi: 10.1002/ajpa.22502. 21. dabbs gr, moore-jansen ph. a method for estimating sex using metric analysis of the scapula. j forensic sci. 2010 jan;55(1):149-52. doi: 10.1111/j.1556-4029.2009.01232.x. 22. urbanova p, ross ah, jurda m, nogueira m. testing the reliability of software tools in sex and ancestry estimation in a multi-ancestral brazilian sample. leg med (tokyo). 2014 sep;16(5):264-73. doi: 10.1016/j.legalmed.2014.06.002. 23. silva m. [compendium of legal dentistry]. são paulo: medsi; 1997. portuguese. 24. asghar a, dixit a, rani m. morphometric study of nasal bone and piriform aperture in human dry skull of indian origin. j clin diagn res. 2016 jan;10(1):ac05-7. doi: 10.7860/jcdr/2016/15677.7148. 9 ulbricht et al. 25. mahakkanukrauh p, sinthubua a, prasitwattanaseree s, ruengdit s, singsuwan p, praneatpolgrang s, et al. craniometric study for sex determination in a thai population. anat cell biol. 2015 dec;48(4):275-83. doi: 10.5115/acb.2015.48.4.275. 26. zaki me, soliman ma, el-bassyouni ht. a cephalometric study of skulls from the bahriyah oasis. j forensic dent sci. 2012 jul;4(2):88-92. doi:10.4103/0975-1475.109895. 27. moreddu e, puymerail l, michel j, achache m, dessi p, adalian p. morphometric measurements and sexual dimorphism of the piriform aperture in adults. surg radiol anat. 2013 dec;35(10):917-24. doi: 10.1007/s00276-013-1116-2. 28. mana md, adalian p, lynnerup n. lateral angle and cranial base sexual dimorphism: a morphometric evaluation using computerised tomography scans of a modern documented autopsy population from denmark. anthropol anz. 2016;73(2). doi: 10.1127/anthranz/2016/0424. 29. tambawala ss, karjodkar fr, sansare k, prakash n, dora ac. sexual dimorphism of foramen magnum using cone beam computed tomography. j forensic leg med. 2016 nov;44:29-34. doi: 10.1016/j.jflm.2016.08.005. untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8652928 volume 17 2018 e18370 original article 1 universidade federal do rio grande do norte (ufrn), departamento de dentística, av sen salgado filho 1787 – lagoa nova, natal, rn, 59056-000, brazil. 2 faipe school, rua dos girassóis 86 – jardim cuiabá, cuiabá, mt, 78043-132, brazil. corresponding author: rodolfo xavier de sousa-lima av sen salgado filho 1787, natal, rn, 59056-000, brazil. tel./fax: +55 84 3215 4101. e-mail address: rodolfo_xsl@hotmail.com received: march 06, 2018 accepted: july 09, 2018 polymerization capability of simplified dental adhesives with camphorquinone, phenylpropanedione and bis-alkyl phosphine photoinitiators boniek castillo dutra borges1, rodolfo xavier de sousa-lima1, géssica dandara medeiros de souza1, ana carla bezerra de carvalho justo-fernandes1, letícia virgínia de freitas chaves1, eduardo josé carvalho souza-junior2, isauremi vieira de assunção1 aim: this study aimed to evaluate the degree of conversion (dc) exhibited by novel formulations of dental adhesive systems including camphorquinone (cq), phenyl-propanedione (ppd), and bis-alkyl phosphine oxide (bapo) when cured by monoor polywave light emitting diodes (leds). methods: an adhesive model was formulated by mixing hydroxyethyl methacrylate (hema, 40 wt%) and bisphenol a glycidyl dimethacrylate (bisgma, 60 wt%) in ethanol (30 wt%). five materials were then formulated by adding the following photoinitiators: cq (1 mol%), cq/ppd (0.5/0.5 mol%), cq/bapo (0.5/0.5 mol%), ppd (1 mol%), and bapo (1 mol%). the dc for each material was measured with fourier transform infrared spectroscopy. analysis of variance and tukey’s post-hoc test were used to analyze the data (p < 0.05). results: except for cq, the photoinitiators provided a significantly higher dc in the adhesive systems following photoactivation with a polywave led. conclusion: the use of alternative photoinitiators and a polywave led improved the dc of the adhesive systems examined. keywords: spectroscopy, fourier transform infrared. dental materials. dental cements. 2 borges et al. introduction dental adhesives are materials composed of monomers with both hydrophilic and hydrophobic groups, photoinitiators, inhibitors or stabilizers, solvent and, in some cases, inorganic fillers1. the classical model of dental adhesives is available in three application steps (acid etching, priming and bonding). over time, the need to reduce the number of clinical steps during application of dental adhesives required the emergence of simplified materials to reduce the chair time. in this way, adhesives systems have gone through several changes in recent years, with the creation of new monomers and photoinitiating molecules, in an attempt to simplify bonding procedures without compromising adhesion to tooth substrates2. in general, the adhesive performance depends on the degree of conversion (dc), so that a high dc is fundamental to improving resistance of material degradation under in vivo clinical conditions. low dc of dental adhesives is associated with high water sorption/solubility, as well as low bond strength values, low mechanical properties, increased permeability, and even the occurrence of phase separation3. this conversion could be affected by many factors, including the photoinitiator systems and light wavelength of the curing unit used4. thus, the development of simplified adhesive systems capable to show increased dc is detrimental. the most contemporary adhesive systems are activated by light within the blue band of the spectrum (400–500 nm) and they use camphorquinone (cq) as a photoinitiator5. cq is a solid yellow compound with an unbleachable chromophore group that can absorb light in the spectral range of approximately 400–500 nm, with a peak near 470 nm6,7. however, the yellow hue characteristic of cq compromises its aesthetic performance and photoinitiators eventually degrade over time5,8. therefore, alternative photoinitiators such as phenyl propanodione (ppd), and bis-alkyl phosphine oxide (bapo) have been investigated in an attempt to replace cq or decrease the amount of cq into dental materials without compromising the dc6,9,10. most of the alternative photoinitiators that have been studied have an absorption peak in the ultraviolet region which extends slightly into the visible light spectrum (380–420 nm)5,11. both the spectrum emitted by a light source and the absorption capacity of a photoinitiator have an effect on the polymerization process of composites, thereby influencing their properties5,11,12. as a result, cure efficiency can be compromised when narrowband light-emitting diodes (leds), such as conventional monowave leds are used, since these leds do not have light emission in the violet wavelength range5,11,13. thus, the ability of conventional leds to activate photoinitiators that respond to ultraviolet light is limited. however, polywave leds emit dual peaks, with one additional peak being near 405 nm14-16, and this allows these leds to activate photoinitiators such as ppd and bapo. in this way, the aim of this study was to evaluate the degree of conversion (dc) of novel formulations of dental adhesive systems including cq, ppd, and bapo when cured by monoor polywave leds in order to test the hypothesis that the use of alternative photoinitiators and photoactivation with a polywave led can lead to an increased dc. 3 borges et al. materials and methods experimental design the response variable evaluated in this in vitro study was dc. five photoinitiators (cq, cq/ppd, cq/bapo, ppd, and bapo) (table 1) and two types of leds (monowave and polywave) (table 2) were tested. formulation of the experimental adhesive systems bisphenol a glycidyl dimethacrylate (bisgma) and hydroxyethyl methacrylate (hema) (60:40 wt%) (sigma-aldrich, st. louis, mo, usa) were mixed with ethanol (30 wt%)17. then, five different materials were generated with the addition of these various photoinitiators: cq (1 mol%), cq/ppd (0.5/0.5 mol%), cq/bapo (0.5/0.5 mol%), ppd (1 mol%), and bapo (1 mol%). ethyl 4-(dimethylamino)benzoate (edmab) (sigma-aldrich) (1 mol%) was added to all of the prepared formulations to serve as a co-initiator. dc evaluation dc was evaluated with a fourier transform infrared/attenuated total reflectance instrument (ftir/atr) (spectrum 100, perkinelmer, shelton, ct, usa) at 24 ºc under 64% relative humidity. one drop of each adhesive system (n = 10 per photoinitiator and led) was applied to the atr surface and the solvent was evaporated for 10 s. then, a thin glass plate (0.5 mm thick) was placed on the material and it was photoactivated for 10 s using a led. the irradiance of the leds were measured by using a computer-controlled spectrometer (usb2000, ocean optics, dunedin, usa) and was integrated using origin 6.0 software (originlab, northampton, usa). the absorption spectra of both the nonpolymerized and polymerized adhesive systems prepared were obtained between 4000 and 650 cm-1 with 32 scans at 4 cm-1. intensities of the aliphatic carbon-to-carbon double-bond absorbance peak (located table 1. characteristics of photoinitiators used in this study. photoinitiator absorption spectrum range (nm) absorption intensity peak (nm) [7] molar extinction coeficiente (l/mol cm) [7] cq* 400 – 500 [7] 470 28±2 ppd** 350 – 480 [17] 398 150±10 bapo*** 365 – 416 [7] 370 300±10 *camphorquinone; **phenyl-propanedione; ***bis-alkyl phosphine oxide table 2. technical details of light emitting diodes used in this study according to the manufacturers. comercial name/ manufacturer classification spectrum range intensity peaks irradiance bluephase g2, ivoclar vivadent, schaan, liechtenstein polywave 385 515 nm (380 420; 420 490) 405 nm 460 nm 1200mw/cm2 radii cal, sdi, victoria, australia. monowave 440 480 nm 460 nm 1313 mw/cm2 4 borges et al. at 1638 cm-1) and the aromatic component (located at 1608 cm-1; reference peak) were recorded. dc (%) was calculated using the following equation18: r polymerized r nonpolymerized], dc (%) = 100 x [1 ( )], where r represents the ratio between the absorbance peaks at 1638 cm-1 and 1608 cm-1. statistical analysis two-way analysis of variance (anova) and tukey’s post-hoc test were used (p < 0.05). results and discussion there were statistically significant differences in the interaction between photoinitiators x leds (p < 0.01). table 3 shows the intergroup comparisons. only the cq adhesive system achieved a similar mean dc as the samples were photoactivated by radii cal and bluephase g2, so that bluephase g2 provided a higher mean dc than radii cal to the other adhesive systems. bluephase g2 provided similar mean dc between adhesive systems. radii cal provided the highest dc to the cq adhesive system, while the lowest dc was observed for the cq/bapo and bapo adhesive systems. thus, the hypothesis that the use of alternative photoinitiators that and photoactivation with a polywave led can lead to an increased dc was accepted. the dc of an adhesive system is influenced by the activity of photoinitiators and the wavelength and intensity of the curing light that is applied19. in this study, only the adhesive systems that included cq exhibited a similar dc between the samples that were photoactivated by radii cal and bluephase g2. conventional monowave leds, such as radii cal, have an emission band in the visible region which results in the emission of a single peak in a narrow spectral band20. in contrast, bluephase g2 is a dual peak led that provides additional light with a spectrum that nearly includes 405 nm14,15. cq is activated within the visible light spectrum and has a peak absorbance near 470 nm6,7. based on the data collected, it appears that both monowave and polywave leds are able to excite cq. this corroborates with that found by segreto et al.21 (2016) who tested different photoinitiator units and photoinitiator systems and concluded that both types of light (mono and polywave) are capable of activating cq and ppd. table 3. degree of conversion (%) means (standard-deviation) of dental adhesive systems according to the photoinitiator system and the curing light. photoinitiator system light emitting diode radii cal bluephase g2 cq 77.8 (6.8) aa 77.3 (14.1) aa cq/ppd 48.8 (7.4) bb 71.6 (7.7) aa cq/bapo 31.5 (11.5) cb 74.1 (6.9) aa ppd 47.2 (4.0) bb 74.2 (6.4) aa bapo 27.6 (3.9) cb 81.6 (6.5) aa means followed by different capital letters indicate statistically significant differences between curing lights for the same photoinitiator (p<0.05). means followed by different lower case letters indicate statistically significant differences among photoinitiator systems for the same curing light (p<0.05). 5 borges et al. unlike cq, the adhesive systems formulated with cq/ppd, cq/bapo, ppd, and bapo exhibited a higher dc when they were photoactivated by bluephase g2 than with radii cal. alternative photoinitiators such as ppd and bapo have an absorption peak in the ultraviolet region (100–400 nm)22, specifically at 398 nm and 370 nm, respectively5. thus, photoactivation with a polywave led could promote an increased excitation of these photoinitiators, thereby increasing the generation of free radicals that initiate the polymerization reaction. however, the monowave led, radii cal, provided a higher dc for the ppd adhesive system than the bapo system. thus, it is likely that ppd can also absorb light in the visible range of the light spectrum20, thereby accounting for the greater excitation of ppd by radii cal compared with bapo. the results indicated that ppd was a viable alternative in the formulation of experimental adhesives, observing that it presents greater reactivity independent of the type of photoinitiator unit21. despite the fact that bapo is a norirish type i photoinitiator which generates free radicals via a photocleavage process that does not require a co-initiator23, a tertiary amine edmab was added to the bapo-containing materials in the present study. edmab is capable of reacting with the oxygen that is dissolved in the monomer, thereby reducing an oxygen-mediated inhibition of polymerization23. since cq employs a mechanism that predominantly involves abstraction of a proton from the amine hydrogen, and ppd can undergo photocleavage and proton abstraction of the amine24, edmab was included with all of the photoinitiators tested so the same conditions would be compared. the findings obtained in this study are of great relevance, since dc is the main physical property related to other biological, physical and mechanical properties such as sorption and solubility, long-term stability of the hybrid layer25, liberation of residual monomers and preservation of the complex dentin pulp4, bond strength to dentin9, elastic modulus and flexural strength of dental adhesives26. confirming this statement, schneider et al.27 (2009) evaluated the effect of the photoinitiator type on the maximum rate of polymerization (r(p)(max)), stress development (final stress and maximum rate, r(stress)(max)), dc and cross-link density (cld) of materials containing cq, ppd or cq/ppd and conclude that cq/ppd reduced the r(p)(max) and r(stress)(max) without a reduction in dc and cld. in this way, the use of alternative photoinitiator systems could be a promising way to reduce the stress developed during the composite’s polymerization without affecting the final properties. thus, to be able to show that the insertion of alternative photoinitiators in conjunction with third generation leds are able to increase the degree of conversion is a positive and relevant result for adhesive dentistry. the literature states, therefore, that the combination of alternative photoinitiators with the traditional camphorquinone/amine system improved the color stability of the model resin composites and maintaining their mechanical properties28,29. despite the important finding obtained in this study regarding dc, further physical, mechanical and biological properties should be investigated to strength the effect of alternative photoinitiators on the performance of dental adhesives. indeed, since acidic monomers such as methacryloyloxydecyl hydrogen phosphate (mdp), and glycerol dimethacrylate phosphate (gdma-p) have been included in dental adhesive systems with bis-gma and/ or hema30,31 further studies should be conducted to evaluate the dc exhibited by other formulations including acidic monomers and alternative photoinitiators. 6 borges et al. in conclusion, the use of alternative photoinitiators and polywave led was found to improve the dc and decrease the yellowing effect of the experimental dental adhesive systems tested. acknowledgements this study was not supported by any funding agency. all expenses were made through own initiative. references 1. sofan e, sofan a, palaia g, tenore g, romeo u, migliau g. classification review of dental adhesive systems: from the iv generation to the universal type. ann stomatol (roma). 2017 jul 3;8(1):1-17. doi: 10.11138/ads/2017.8.1.001. 2. pena ce, rodrigues ja, ely c, giannini m, reis af. two-year randomized clinical trial of selfetching adhesives and selective enamel etching. oper dent. 2016 may-jun;41(3):249-57. doi: 10.2341/15-130-c. 3. vale mrl, afonso fac, borges bcd, freitas jr ac, farias-neto a, almeida eo, et al. preheating impact on the degree of conversion and water sorption/solubility of selected single-bottle adhesive systems. oper dent. 2014 nov-dec;39(6):637-43. doi: 10.2341/13-201-l. 4. barbosa mo, carvalho rv, demarco ff, ogliari fa, zanchi ch, piva e, et al. experimental self-etching hema-free adhesive systems: cytotoxicity and degree of conversion. j mater sci mater med. 2015 jan;26(1):5370. doi: 10.1007/s10856-014-5370-6. 5. neumann mg, miranda jr wg, schmitt cc, rueggeberg fa, correa ic. molar extinction coefficients and the photon absorption efficiency of dental photoinitiators and light curing units. j dent. 2005 jul;33(6):525-32. 6. stansburry jw. curing dental resins and composites by photopolymerization. j esthet dent. 2000;12(6):300-8. 7. rueggeberg fa. comtemporary issues in photocuring. compend contin educ dent suppl. 1999;(25):s4-15; quiz s73. 8. ilie n, hickel r. can cq be completely replaced by alternative initiators in dental adhesives?. dent mater j. 2008 mar;27(2):221-8. 9. borges bcd, sousa-lima rx, moreno gbp, moreira dgl, oliveira dcrs, sousa-junior ej, sinhoreti mac. polymerization and adhesion behavior of experimental dental bonding materials with different initiator systems. j adhes sci 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five composites cured with single-peak and polywave led curing lights. quintessence int. 2010 nov-dec;41(10):e181-91. 16. schroeder w, arenas g, vallo c. monomer conversion in a light-cured dental resin containing 1-phenyl-1,2propanedione photosensitizer. polym int. 2007 sep;56(9):1099-105. doi: 10.1002/pi.2239. 17. daood u, swee heng c, neo chiew lian j, fawzy as. in vitro analysis of riboflavin-modified, experimental, two-step etch-and-rinse dentin adhesive: fourier transform infrared spectroscopy and micro-raman studies. int j oral sci. 2015 jun 26;7(2):110-24. doi: 10.1038/ijos.2014.49. 18. ye q, park j, topp e, spencer p. effect of photoinitiators on the in vitro performance of adentin adhesive exposed to simulated oral environment. dent mater. 2009 apr;25(4):452-8. doi: 10.1016/j.dental.2008.09.011. 19. ikemura k, endo t. a review of the development of radical photopolymerization initiators used for designing light-curing dental adhesives and resin composites. dent mater j. 2010 oct;29(5):481-501. 20. sim js, seo hj, park jk, garcia-godoy f, kim hi, kwon yh. interaction of led lights with coinitiatorcontaining composite resins: effect of dual peaks. j dent. 2012 oct;40(10):836-42. doi: 10.1016/j. jdent.2012.06.008. epub 2012 jul 4. 21. segreto dr, naufel fs, brandt wc, guiraldo rd, correr-sobrinho l, sinhoreti ma. influence of photoinitiator and light-curing source on bond strength of experimental resin cements to dentin. braz dent j. 2016 jan-feb;27(1):83-9. doi: 10.1590/0103-6440201600387. 22. price rb, felix ca. effect of delivering light in specific narrow bandwidths from 394 to 515 nm on the microhardness of resin composites. dent mater. 2009 jul;25(7):899-908. doi: 10.1016/j.dental.2009.01.098. 23. rueggeberg fa, margeson dh. the effect of oxygen inhibition on an unfilled/filled composite system. j dent res. 1990 oct;69(10):1652-8. 24. park yj, chae kh, rawls hr. development of a new photoinitiation system for dental light-cure composite resins. dent mater. 1999 mar;15(2):120-7. 25. borges bcd, sousa-junior ej, brandt wc, loguercio ad, montes majr, puppin-rontani rm, et al. degree of conversion of simplified contemporary adhesive systems as influenced by extended air-activated or passive solvent volatilization modes. oper dent. 2012 may-jun;37(3):246-52. doi: 10.2341/11-248-l. 26. leal fb, madruga fc, prochnow ep, lima gs, ogliari fa, piva e, et al. effect of acidic monomer concentration on the dentin bond stability of self-etch adhesives. int j adhes adhes. 2011 sep;31(6):571-4. doi: 10.1016/j.ijadhadh.2011.05.007. 27. schneider lf, consani s, sakaguchi rl, ferracane jl. alternative photoinitiator system reduces the rate of stress development without compromising the final properties of the dental composite. dent mater. 2009 may;25(5):566-72. doi: 10.1016/j.dental.2008.10.007. 28. salgado ve, borba mm, cavalcante lm, moraes rr, schneider lf. effect of photoinitiator combinations on hardness, depth of cure, and color of model resin composites. j esthet restor dent. 2015 mar-apr;27 suppl 1:s41-8. doi: 10.1111/jerd.12146. 29. albuquerque pp, moreira ad, moraes rr, cavalcante lm, schneider lf. color stability, conversion, water sorption and solubility of dental composites formulated with different photoinitiator systems. j dent. 2013 aug;41 suppl 3:e67-72. doi: 10.1016/j.jdent.2012.11.020. 30. ganglione la, lima af, gonçalves ls, cavalcanti an, aguiar fhb, marchi gm. mechanical properties and degree of conversion of etch-and-rinse and self-etch adhesive systems cured by a quartz tungsten halogen lamp and a light-emitting diode. j mech behav biomed mater. 2012 aug;12:139-43. doi: 10.1016/j.jmbbm.2012.01.018. 31. anchieta rb, machado ls, martini ap, santos ph, giannini m, janal m, et al. effect of long-term storage on nanomechanical and morphological properties of dentin-adhesive interfaces. dent mater. 2015 feb;31(2):141-53. doi: 10.1016/j.dental.2014.11.010. untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8652655 volume 17 2018 e18486 original article 1 professor of biochemistry. department of physiological sciences, piracicaba dental school, university of campinas, piracicaba, sp, brazil. jcury@unicamp.br tel. 19 2106 5302 2 msc. in cariology. department of physiological sciences, piracicaba dental school, university of campinas, piracicaba, sp, brazil. kwalsh31@hotmail.com tel. 505 83204966. 3 technical assistant. department of physiological sciences, piracicaba dental school, university of campinas, piracicaba, sp, brazil. wvfilho@unicamp.br tel. 19 2106 5303 4 assistant professor at social dentistry department at universidad peruana cayetano heredia, lima-perú. jenniferricaldi@gmail.com tel. +1 206 960 3760 corresponding author: prof. jaime aparecido cury piracicaba dental school, po box 52 13414-903, piracicaba, sp, brazil tel. +55 19 2106 5303, fax +55 19 2106 5302 e-mail: jcury@unicamp.br received: march 28, 2018 accepted: april 23, 2018 fluoride concentration in peruvian salts and the standardization of analysis with specific electrode by the direct method jaime aparecido cury 1 , karla irina walsh 2 , waldomiro vieira 3 , jennifer ricaldi 4 according to the peruvian legislation, salt for human consumption should contain 200-250 mg f/kg, but there is limited data showing whether this requirement is being accomplished. aim: in this pilot study, we evaluated the fluoride concentration in samples of salt marketed in lima, peru, using a standardized protocol with ion-specific electrode by direct method (ise). methods: seven 1 kg salt packages of four brands were purchased in two supermarkets of lima. six aliquots of each package were weighed and dissolved in the proportion of 0.025 g/ml of water. duplicates of 1.0 ml of these solutions were mixed with 1.0 ml of tisab ii and fluoride concentration was determined with ise calibrated with standards fluoride solutions ranging from 0.25 to 16.0 µg f/ ml. the ionic strength of the standards was adjusted with p.a nacl (25 mg/ml). in addition, triplicates of 15 g of each salt package were fractionated in a set of sieves (0.590 to 0.177 mm) to determine the homogeneity of fluoride concentration in salt. results: in four packages the mean fluoride (mean±sd,n=6) concentration (mg f/kg) was in agreement to the peruvian regulation (214.5±10.4; 221.8±14.3; 226.9±19.1 and 237.2±52.0 mg f/kg), but in 3 packages it was lower (145.2±7.9; 145.7±23.3 and 158.4±20.6 mg f/kg). variability in fluoride concentration was observed within the same brand and among brands. also, the fluoride concentration was not homogeneous in none of the salt samples, ranging from 72.0 to 1449.7 mg f/kg. conclusion: the findings suggest that the manufacturing and sanitary surveillance of fluoridated salt in peru should be improved. keywords: fluoridation. dental caries. ion-selective electrodes mailto:kwalsh31@hotmail.com mailto:wvfilho@unicamp.br 2 cury et al. introduction oral health approaches based on the safe use of fluorides have contributed worldwide to the reduction of dental caries1. among the ways of use of fluoride, the community based is considered the one with greater coverage, allowing the benefit of all social classes. salt fluoridation is a community based strategy to prevent tooth decay and it has been recommended as an effective alternative to water fluoridation2-5. in addition, salt fluoridation has been implemented in several latin american countries, reaching nearly 200 million people6,7. on the other hand, an adequate fluoride surveillance system is required to guarantee that any program of fluoride use reaches the maximum anticaries benefits with the minimal risk to develop undesirable side effects, such as dental fluorosis8. one of the advantages of salt fluoridation compared to water would be the facility to monitor the fluoride concentration, because in any country the number of salt producers to be monitored is very smaller than the number of water treatment plants. despite this advantage, fluoride concentrations below or above those established by local legislations have been found in salt brands sold in el salvador, méxico, colombia and peru9-15. in peru, the fluoridated salt program was set in 198416 and the fluoride concentration in salt for human consumption should be from 200 to 250 mg f/kg17. official data about the fluoride concentration in peruvian salts have not been reported and searching the literature it was found only one evaluation carried out 12 years ago15. ten brands of salt were purchased in 34 markets from the province of trujillo, north coast of peru. however, only one brand was fluoridated and the concentration found was below the legislation (152 ppm f). considering the scarce and limited data found, the aim of this study was to report updated findings about the fluoride concentration in salts brands sold in peru. material and methods this exploratory in vitro study, blind regarding the analyst, determined the fluoride concentration in four fluoridated salt brands of human consumption found in lima, peru. seven 1 kg packages were purchased in 2013, in two supermarkets of lima city. it was recorded the producer, batch, validity, type of granulation, ingredients and fluoride concentration declared on the package label (table 1). standardization of the sample salt weight taken for analysis the fluoride concentration in salt could be influenced by the amount of salt sample taken for analysis if the mixture of nacl and the fluoride salt used is not homogeneous. therefore, to obtain more accurate results, it was investigated which would be the weight of salt aliquots representative of the mean fluoride concentration in the salt package. for analysis, salt aliquots from 0.025 to 2.5 g (±0.01) were dissolved in purified water at a ratio of 1:40 (w/v). fluoride concentration was determined using ion-specific electrode (ise) by the direct method and the concentration of nacl in fluoride standard solutions was adjusted according to the sample18. the variation coef3 cury et al. ficient of the fluoride concentration in the salt samples weighing less than 2.5 g was high (up to 30 %), while in samples weighing around 2.5 g it was lower (10 %). thus, salt aliquots with weight of around 2.5 g were taken for this study. standardization of fluoride determination with ion selective electrode (ise) by the direct method the analysis of the fluoride concentration with ise by direct method is subjected to interferers19,20. calcium-ferrocyanide, calcium carbonate, calcium triphosphate, magnesium oxide, magnesium hydroxide and magnesium carbonate are usually added to salt as anticaking agents21. ca++ and mg++ ions form complexes with fluoride that could interfere with the determination of fluoride in salt using ise by the direct method22. another problem is the ionic strength of the salt solutions prepared for analysis because it decreases the activity of the fluoride ion in solution. the interference of ca++ and mg++ could be controlled by the presence of chelating agents added in the buffer solution used for the analysis, such as cdta presents in tisab ii (total ionic strength adjustment buffer). the effect of the ionic strength could be controlled by preparing a very diluted salt solution for analysis. however, as described in the previous item, it is necessary to weigh a representative amount table 1. producer, code for analysis, salt brands, batch and validity, granulation type, fluoride concentration and ingredients declared on the label producer code salt brands batch and validity type of granulation fluoride concentration declared (mg f/kg) ingredients quimpac s.a. 1 pura sal yodada cocina lt.ln 10910131 fv 091015_17:19_3 fine 200-250 kf, kio 3 , sio 2 (max1%) idem 2 pura sal yodada cocina lt.ln 11 210 131 fv 121015_01:24_2 fine idem idem idem 3 em sal yodada cocina lt. ln11109131 fv 1109154_06:15_1 fine idem idem idem 4 em sal yodada cocina lt. ln 12710131 fv. 271015_02:38_1 fine idem idem idem 5 sal extra refinada marina lt.ln11310131 fv 131015_04:06_1 refined idem idem idem 6 sal extra refinada marina lt.ln127101311 fv 271015_04:18_3 refined idem idem idem 7 em sal yodada mesa p 16.09.13 v 16.09.15 13:47 b2 fine idem idem 4 cury et al. of salt to obtain a more accurate fluoride concentration in salt. thus, the concentration of salt in the solution prepared may surpass the concentration of nacl (1.0 m) contained in the tisab ii buffer. this problem has been solved adding pure nacl in the tisab ii buffer to prepare the fluoride standard solutions used in the calibration curve18, as is described further. to test the effect of the ionic strength on the accuracy of the fluoride concentration determination, salt sample solutions from 0.5% to 20% (w/v) were prepared. fluoride concentration was determined using calibration curves made with standard fluoride solutions adjusted or not (raw data) with pure nacl (sigma aldrich, lot # 70m027330v) according to the percentage of salt in the solution prepared for analysis. figure 1 shows fluoride concentration (mg f/kg) found in salt according to the amount of salt (g) dissolved in 100 ml of purified water, determined from calibration curves made with standard fluoride solutions not adjusted (raw data) or adjusted (data normalized) according to the percentage of nacl in the samples. if the salt concentration in the standard solutions is not adjusted (below curve), the fluoride concentration is underestimated from 4% to 15%. also, the data shows that a more accurate fluoride concentration is obtained when salt is prepared at proportion of around 2 g of salt in 100 ml of water and the standard solutions are adjusted with pure nacl. below and above this weight, the concentration of fluoride is underestimated. thus, a final proportion around 2.5 g of salt dissolved in 100 ml of purified water was used for the analysis of salt samples. m g f/ kg % (g salt/100 ml) 250.0 225.0 200.0 175.0 150.0 normalyzed raw 125.0 100.0 75.0 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 fluoride concentration (mg/kg) in the salt mean ± sd (n=6) from ~ 2.3% salt soln raw data = 213.8 ± 5.4 normalyzed = 225.8 ± 5.6 figure 1. concentration of fluoride found in salt (mg f/kg) according to the amount (g) of salt dissolved in 100 ml of purified water, determined from calibration curves made with standards fluoride solutions adjusted or not with p.a nacl according to the % of salt in the samples. 5 cury et al. salt sample harvest and preparation for analysis the salt packages were manually agitated for 2 min for homogenization. six aliquots weighting 2.5 g (± 0.01) were harvested from the top of each package and dissolved in the proportion of 0.025 g/ml of purified water. to each duplicate of 1 ml of the prepared solutions, 1 ml of tisab ii was added (1:1; v/v). fluoride analysis was carried out as is described further. assessment of the fluoride concentration homogeneity in the salt package to determine the homogeneity of fluoride in the salt packages, triplicates of 15 g of each package were fractionated using a set of sieves (telatest) with tyler of 28 (0.59 mm), 35 (0.42 mm), 60 (0.250 mm) and 80 (0.177 mm). after 1 min of manual agitation, the fractions of salt that passed through the 28-60 tyler as well as the retained in the tyler 80 (particles from < 0.177 to > 0.590) were collected and dissolved in the proportion of 0.025 g/ml of purified water. solutions were prepared as described before for fluoride determination. fluoride analysis the fluoride concentration in the salt solutions was determined using ion-specific electrode (orion 96-09, thermo scientific, cambridge, ma, eua) coupled to an ion analyzer (orion star a214; thermo scientific, eua). this equipment was previously calibrated with standard fluoride solutions ranging from 0.25 to 16.0 µg f/ml, mixed in ratio (1:1; v/v) with tisab ii added of 0.025 g nacl (p.a) /ml18. microsoft excel software was used to estimate by linear regression the relationship between the logarithm of the fluoride concentration in the standards and the mv. the linear correlation coefficient was of 0.999. the regression was used to determine the fluoride concentration in the samples and the average coefficient of variation for the duplicate analysis was of 1.3 %. for the data analysis, it was calculated the mean (±sd; n=6) fluoride concentration in every package and the mean (±sd; n=3) and range of fluoride concentration in the fractionated samples of salt. the results were expressed in mg f/kg and compared with the range of fluoride concentration demanded by the peruvian legislation. results only 4 brands of fluoridated salt were found in two supermarkets of lima, all from the same manufacturer, quimpac, that is responsible for 60% of salt marketed in peru. from 3 brands were purchased 2 packages, one in each supermarket, but one brand was found in only one supermarket. seven salt packages from different batches were purchased and analyzed (table 1). five out of the seven (71%) salt samples analyzed were fine granulated and the remaining two (29%) refined granulated. all the samples were iodized and had silicon dioxide (max 1%) as the anticaking agent. the fluoride agent declared by the manufacturer in all the samples was kf and the final fluoride concentration declared was in the range of 200 to 250 mg f/kg. 6 cury et al. results showed that the mean (± sd; n=6) of fluoride concentration (mg f/kg) of the salt samples ranged from 145.2 to 237.2 mg f/kg (table 2). in four packages, the fluoride concentration was according to the peruvian legislation (salts code 2,4,6 and 7), but, in three of them (salts code 1,3 and 5) it was lower. the fluoride concentration (±sd; n=3) found (mg f/kg) in the fractionated samples of salt is shown in table 3. the concentration in the fractions of the salt code 2 was the most heterogeneous (from 116.2 to 1449.7), while in salts 3 and 5 it was the most homogeneous (from 164.6 to 221.3 and 164.6 to 221.3, respectively). discussion the monitoring of the fluoride concentration in salt is necessary to guarantee the maximum anticaries benefits with the minimal risk to develop unacceptable dental table 3. mean (± sd; n=3) of fluoride concentration (mg f/kg) found in the fractions of the salt samples and the range found code salt brands particle size (mm) range < 0.177 0.1770-250 0.250-0.420 0.420-0.590 > 0.590 1 purasal yodada cocina 332.4 ± 228.6 181.6 ± 32.2 137.0 ± 11.8 98.6 ± 27.9 126.6 ± 23.2 98.6-332.4 2 purasal yodada cocina 125.6 ± 11.0 116.2 ± 3.1 202.5 ± 65.3 1449.7 ± 1496.1 271.0 ± 12.4 116.2-1449.7 3 emsal yodada cocina 115.1 ± 19.0 155.8 ± 39.1 127.1 ± 46.3 159.4 ± 49.5 167.5 ± 14.3 115.1-167.5 4 emsal yodada cocina 141.6 ± 7.1 72.0 ± 16.5 152.3 ± 39.7 420.9 ± 25.9 234.8 ± 9.8 72.0-420.9 5 sal extra refinada-marina 218.7 ± 35.8 221.3 ± 65.4 178.7 ± 24.3 164.6 ± 14.4 167.5 ± 53.3 164.6-221.3 6 sal extra refinada-marina 382.0 ± 62.7 297.3 ± 91.7 159.8 ± 11.0 120.9 ± 12.6 275.3 ± 112.7 120.9-382.0 7 emsal yodada mesa 375.2 ± 28.6 290.7 ± 40.4 211.4 ± 10.7 121.9 ± 12.1 93.7 ± 13.6 93.7-375.2 table 2. code, salt brands and mean (± sd; n= 6) of fluoride concentration (mg f/kg) found in the samples of salt code salt brands *mg f/kg 1 purasal yodada cocina 145.7 ± 23.3 2 purasal yodada cocina 214.5 ± 10.4 3 emsal yodada cocina 145.2 ± 7.9 4 emsal yodada cocina 221.8 ±14.3 5 sal extra refinada-marina 158.4 ± 20.6 6 sal extra refinada-marina 226.9 ± 19.1 7 emsal yodada mesa 237.2 ± 52.0 * expected: 200-250 mg f/kg, according to peruvian regulation 7 cury et al. fluorosis in the population. to determine an accurate and precise fluoride concentration in salt is essential to have a valid methodology of analysis. additionally, the analysis should be feasible to be conducted in any country where salt fluoridation programs were implemented. our findings showed that accurate fluoride concentration in salt was found when salt sample solutions were prepared at concentration of around 2.5 % (w/v) and the ionic strength of the fluoride standards were adjusted with pure nacl (fig 1). these results were obtained using ion specific electrode (ise) by direct analysis but a publication has suggested that the determination of fluoride in salt using ise should be made after microdifussion and not by the direct analysis19. however, according to this publication suggesting that microdifussion should be used19, fluoride concentration determined in the salt by the direct analysis was made from a 10% salt solution and the ionic strength of the samples was not adjusted. as fig 1 shows, if a salt sample solution is prepared at 10% and the electrode is calibrated without adjustment of the ionic strength, the fluoride concentration would be underestimated in 27%, compared with the concentration found from analysis of 2.5% salt solution. it should be emphasized that even with the adjustment of the ionic strength, the fluoride concentration in the salt from analysis of a 10% solution would still be underestimated in 18% (fig.1). in conclusion, to obtain accurate results is necessary to weigh an amount of salt that is representative of the fluoride concentration in the salt (at around 2.5 g) which may be dissolved in the proportion of 0.025 g/ml of water. also, the ionic strength of the fluoride standards must be properly adjusted with nacl chemically pure. regarding the results found and the surveillance system of the salt fluoridation program in peru, the local legislation establishes that the adequate range of fluoride in salt for human consumption should be from 200 to 250 mg f/kg17. thus, fluoride concentration in salt below or above this range would not have a maximum anticaries benefit or would provoke unacceptable fluorosis, respectively. according to the findings (tab 2), 57% of the samples analyzed showed fluoride concentration according to the legislation and the remaining salt samples (43%) showed concentration lower than the minimum recommended. additionally, it was observed variability of the mean fluoride concentration within the same brand (tab 2). as described in the results item, all the seven packages of salt analyzed were produced by the same manufacturer but were products of 4 brands (table 1). moreover, three of the brands analyzed (purasal yodada cocina, emsal yodada cocina and sal extra refinada-marina) were purchased in duplicate but from different batches (tab 1). table 2 shows that one sample from each brand presented adequate fluoride concentration, but its duplicate did not. considering that all salts were produced by the same manufacturer and probably by the same process of fluoride addition, the findings suggest that the program of quality control of fluoride concentration during salt production is deficient. moreover, when the data of mean fluoride concentration in the salts (table 2) are confronted with the data of fluoride concentration in the fractions of salts sieved (table 3), other problems are noticed. thus, although the mean fluoride concentration found in salts code 2, 4 and 6 (table 2) are according the peruvian legislation, they were not the most homogeneous (table 3) compared with salts batches 1, 3 and 5. fluoride concentration in the fractions of salts 2, 4 and 6 ranged respectively from 116.2 to 8 cury et al. 1449.7 (12-fold), 72.0 to 420.9 (6-fold) and 120.9 to 380.2 (3-fold), while in salts 1, 3 and 5 ranged from 98.6 to 332.4 (3-fold), 115.1 to 167.5 (1.4-fold) and 164.6 to 221.3 (1.3-fold). therefore, for example, when people use a small amount of salts code 2, 4 or 6 in a salad than a larger amount to cook foods, they were subjected to greater variability of fluoride ingestion. although the sampling of salts analyzed in our study was limited to only 7 packages of salt, we compared our findings (table 2) with reports from peru and other countries, where the program of salt fluoridation was implemented10-15. sunohara15 analyzed in 2005 ten brands of peruvian salts. fluoride was found in only one brand (emsal yodada de mesa) but fluoride concentration was below the legislation (152 ppm f). in this same brand (table 2, code 7), we found 237.2 ppm f. this difference found in the same brand shows deficiency of a program of quality control of fluoride concentration during the production of salt. the results that we found in peruvian salts (table 2) are similar to findings for 75 mexican salts analyzed in 2002 and 2003 because 50% of the samples were in agreement with the local legislation (200-250 mg f/kg)10. however, other analyses conducted in mexican salts have found worst results. analyses made in199511 and in 200812 showed respectively that only 1 and 7% of the salt samples presented fluoride concentration in accordance with the local legislation. regarding labeling, all the peruvian salt brands selected are in agreement with the peruvian norm of salt for human consumption17 in terms of the declaration of the fluoride concentration in mg/kg of salt, method used for fluoride addition, sanitary registration number, batch, date of production and expiration. the discrepancy between the mean fluoride concentration found (table 2) and that declared by the manufacturer (table 1) on the label of samples code 1, 3 and 5 is due to the deficiency of quality control of fluoride concentration during salt production, as described before. although the present pilot study was limited to samples of salts found in lima, the impact of fluoride concentration found (table 2) for the peruvian population, considering the anticaries benefits and the risks of dental fluorosis of the salt fluoridation program implemented in peru may be discussed. people who use salt brands 2, 4, 6 and 7 would have the maximum anticaries effect but who use salts 1, 3 and 5 would be less protected. however, dental caries is a chronic disease and the effect of fluoride on caries is local, not systemic23. therefore, the time of maintenance of an effective fluoride concentration in the mouth to interfere with the caries process should be considered than “optimal” concentration for short time. also, it is not known whether salts with concentration of around 150 mg f/kg (tab 2) would be less effective in terms of caries prevention regarding salts with concentration of 200-250 mg f/kg. concerning dental fluorosis, the risk is not increased because none of the salt samples presented mean fluoride concentration above the established range (table 2). however, when the salt was fractioned it was found in the sample code 2 a fraction with 1449.7 mg f/kg (tab 3). although this concentration is around 6-fold the upper limit, there is a few concern because it was found in very small salt particle (0.420-0.59 mm). a small particle could be used by chance for example to add salt to a tomato slice but not to cook, when larger amount of salt is used. therefore, assuming that a 20 kg child added 9 cury et al. 0.1 g of salt with concentration of 1449.7 mg f/kg to a tomato slice, he would intake 0.15 mg of fluoride. thus, the child would be subjected to the dose of 0.0075 mg f/kg of body weight, which is 10 times lower the upper limit dose for unacceptable risks of fluorosis24. indeed, peruvian children are not exposed to dose of risks of fluorosis from foods cooked with fluoridated salt25. in addition, dental fluorosis is a chronic disease and the time of duration of an “optimun” dose is more relevant than sporadic higher dose. however, this discussion about fluorosis risks of the peruvian fluoridated salt program is not considering the effect of fluoridated salt for people living in areas containing natural fluoride. in fact, the peruvian regulation set that “the population who is exposed to water containing natural fluoride at concentration of 0.7 ppm or greater does not need consume fluoridated salt”. however, it does not establish any mechanism to avoid that fluoridated salt is market in these areas. the overall findings of this pilot study about fluoride concentration in salts marketed in lima, peru, and the publication cited15, alert about the need to improve the monitoring of the fluoride concentration in salt that is responsibility of manufacturers and governmental entities. it is reasonable to think that the balance between the anticaries potential and low risk of fluorosis requires an adequate concentration of fluoride in salt. based on this pilot finding, we concluded that the manufacturing and surveillance system of fluoridated salt in peru should be reinforced. acknowledgments a preliminary report of this study was presented at the 92nd iadr general session and exhibition, 2014. references 1. petersen pe. the world oral health report 2003: continuous improvement of oral health in the 21st century the approach of the who global oral health programme. community dent oral epidemiol. 2003 dec;31 suppl 1:3-23. 2. petersen pe, lennon m. effective use of fluorides for the prevention of dental caries in the 21st century: the who approach. community dent oral epidemiol. 2004 oct;32(5):319-21. 3. jones s, burt ba, petersen pe, lennon ma. the effective use of fluorides in public health. bull world health organ. 2005 sep;83(9):670-6. 4. marthaler tm, petersen pe. salt fluoridation an alternative in automatic prevention of dental caries. int dent j. 2005 dec;55(6):351-8. 5. o’mullane dm, baez rj, jones s, lennon ma, petersen pe, rugg-gunn aj. fluoride and oral health. community dent health. 2016 jun;33(2):69-99. 6. gillespie gm, baez r. development of salt fluoridation in the americas. schweiz monatsschr zahnmed. 2005;115(8):663-9. 7. pollick hf. salt fluoridation: a review. j calif dent assoc. 2013 jun;41(6):395-7, 400-4. 8. sosa rosales m de la c, garcía melian m, gómez a, gonzález i, mojáiber de la peña a. [surveillance system of the program of salt fluoridation of human consumption in cuba]. rev cubana salud pública. 2004 sep-dic [cited 2017  sep  29]; 30(4). available from: http://scielo.sld.cu/scielo. php?script=sci_arttext&pid=s0864-34662004000400011. spanish. 10 cury et al. 9. girón álvarez be, márquez hernández rv, sermeño camacho kj. [presence and concentration of fluoride in salt brands marketed in el salvador] crea cienc. 2005 [cited 2017  sep  29];5(2)5-9. available from: http://dsuees.uees.edu.sv/xmlui/handle/20.500.11885/104. spanish. 10. martínez-mier ea, soto rojas ae, buckley cm, stookey gk, zero dt, margineda j. [evaluation of fluoride content in fluoridated table salt]. salud publica mex. 2004 may-jun;46(3):197-8. spanish. 11. maupomé carvantes g, lanchero jaramillo d, andrade delgado lc, juárez reyes pl, pérez lópez r, navarro sánchez w, et al. [fluoride contained in salt for human consumption distributed in mexico city] bol of sanit panam. 1995 sep;119(3):195-201. spanish 12. hernández-guerrero jc, de la fuente-hernández j, jiménez md, ledesma-montes c, castañedacastaneira e, molina-frechero n. fluoride content in table salt distributed in mexico city, mexico. j public health dent. 2008;68(4):242–5. spanish 13. franco am, saldarriaga a, gonzalez mc, martignon s, arbelaez mi, ocampo a. [fluoride concetration in kitchen salt in four colombian cities] revista ces odontología vol. 16. 2003. p. 21–6. spanish 14. tovar valencia s, misnaza castrillon s. perspectiva del uso del flúor vs caries y fluorosis dental [technical document on the internet] colombia: minsalud; 2016 [cited 2017 sep 29] available from: http://www.minsalud.gov.co/sites/rid/lists/bibliotecadigital/ride//vs/pp/ent/ perspectiva-uso-fluor.pdf 15. sunohara asa. [mapping of fluoridated salt in markets of the province of trujillo using the geographic information system]. rev estomatol hered. 2006;16(1):5–8. spanish 16. ministerio de salud perú [homepage]. lima: minsa resolución ministerial no 0131-85-sa-dvm; 1985 [cited 2017 set 29] available from: ftp://ftp2.minsa.gob.pe/descargas/prevencion_salud/salud_ bucal/rm0131-85-sa-dvm.pdf 17. ministerio de salud perú [homepage] lima: minsa norma técnica peruana sal para consumo humano. 2006 [cited 2017 set 29] available from: ftp://minsa.gob.pe/normaslegales/2006 18. cury ja, guimarães lo, moreira bh [fluoridation of cooking salt: a method for familiar use]. rev ass paul cirurg dent. 1983;37(5):452-455. portuguese 19. martínez-mier ea, soto-rojas ae, buckley cm, margineda j, zero dt. evaluation of the direct and diffusion methods for the determination of fluoride content in table salt. community dent health. 2009 dec;26(4):204-10 20. martínez-mier ea, cury ja, heilman jr, katz bp, levy sm, li y. development of gold standard ionselective electrode-based methods for fluoride analysis. caries res. 2011;45(1):3-12 21. european salt producers association. sodium chloride analytical standard determination of fluorides potentiometric method [technical document on the internet]. european salt producers association 2005 [cited 2017 sep 29]. available from:http://eusalt.com/sites/www.eusalt.com/files/ pagedocuments/eusalt%20as0172005%20fluorides%20%20potentiometric%20method.pdf 22. campbell ad. determination of fluoride in various matrices. pure appl chem. 1987;59(5):695–702 23. cury ja, tenuta lma. how to maintain a cariostatic fluoride concentration in the oral environment. adv dent res. 2008 jul 1;20(1):13–6 24. burt ba. the changing patterns of systemic fluoride intake. j dent res. 1992 may 1;71(5):1228–37. review. 25. rodrigues mh, leite al, arana a, villena rs, forte fd, sampaio fc, buzalaf ma. dietary fluoride intake by children receiving different sources of systemic fluoride. j dent res. 2009 feb;88(2):142-5. oral sciences n3 original article braz j oral sci. october | december 2015 volume 14, number 4 received for publication: november 15, 2015 accepted: december 13, 2015 distinguishing predisposing factors for enamel hypoplasia and molar-incisor hypomineralization in children in ile-ife, nigeria oluwaseyi dada temilola1, morenike oluwatoyin folayan2 1obafemi awolowo university, teaching hospitals complex, department of child dental health, ile-ife, osun state, nigeria 2obafemi awolowo university, faculty of dentistry, oral habit study group, ile-ife, osun state, nigeria correspondence to: morenike oluwatoyin folayan faculty of dentistry, obafemi awolowo university ile-ife, osun state, nigeria phone: +23-470-6292-0394 e-mail: toyinukpong@yahoo.co.uk abstract aim: to determine if the prevalence of enamel hypoplasia, molar-incisor hypomineralisation (mih) and deciduous molar hypomineralisation (dmh) is associated with the socioeconomic status of the child and to determine the prevalence of enamel hypoplasia and mih/dmh comorbidity in the study population. methods: information was collected on the sex and socioeconomic status of the 1,169 study participants’ resident in ile-ife, nigeria, recruited through a household survey. the children were clinically examined to assess for the presence of enamel hypoplasia, mih and dmh. associations between sex, socioeconomic status and the prevalence of enamel hypoplasia, mih and dmh were determined. the proportion of children with enamel hypoplasia and mih/dmh co-morbidity was also determined. results: among the 1,169 study participants, 47(4.0%) had mih, 15 (1.3%) had dmh and 161 (13.8%) had enamel hypoplasia. one (0.09%) study participant had mih/dmh co-morbidity, 12 (1.0%) had dmh/enamel hypoplasia co-morbidity, and 9 (0.8%) had mih/hypoplasia co-morbidity. there was no significant association between the socioeconomic status and presence of enamel hypoplasia (p=0.22), mih (p=0.78) or dmh (p=1.00). conclusions: the socioeconomic status cannot be used as a distinguishing factor for enamel hypoplasia, mih and dmh. the possibility of co-existence of enamel hypoplasia and mih/dmh makes it imperative to find ways to distinguish between the lesions. keywords: dental enamel hypoplasia; child; nigeria; social class. introduction developmental defects of enamel (dde) are well recognized in the dental literature and defined as any alteration resulting from diverse disturbances during the process of odontogenesis caused by hereditary, local or systemic factors1. the primary, permanent or both dentitions may be affected. lesions may appear opaque due to hypo-mineralization, which causes alteration in the translucency of enamel. these opacities may be white, cream, yellow or brown in color. lesions may also appear as grooves, pits, partial or total loss of surface enamel, due to quantitative defect in enamel called hypoplasia2-3. it may be difficult to distinguish between hypoplasia and post-eruptive enamel loss4. this challenge may be real in regions where the prevalence of enamel hypoplasia and enamel hypomineralization is high. nigeria is one such country: the prevalence of dde is approximately 4% in the primary dentition5 and 6.0 11.7% in the permanent dentition 6 -7. the prevalence of molar-incisor http://dx.doi.org/10.1590/1677-3225v14n4a12 braz j oral sci. 14(4):318-322 hypomineralization (mih) ranges between 9.7% and 17.7%89 and that of deciduous-molar hypomineralization (dmh) is 4.6%8. the possibility of co-existence of these lesions is therefore high. little is known or discussed in the literature about the co-existence of enamel hypoplasia and enamel hypomineralization and the implications of these comorbidities for patients’ health and welfare. the etiological factors for dde are diverse, ranging from birth prematurity to low birth weight, infections, malnutrition or metabolic disorders3,10. although the precise cause and effect mechanism has not been clearly elucidated, these enamel defects can have a significant impact on esthetics, tooth sensitivity and occlusal function2,11-13. many of the aetiological factors have a higher incidence in families with low socioeconomic status3,10. the low prevalence of dde in developed countries with good nutrition 10 and the high prevalence in developing countries highlight the possible role of socioeconomic factors as aetiological factor for dde. a previous study on dde in children from nigeria showed a strong association between socioeconomic status and prevalence of different forms of dde6. similar findings have been reported in tanzania14. another study has also suggested that children from higher socioeconomic groups tend to have more diffuse mottling than children from low socioeconomic group7. however, the only study retrievable on the association between mih and socioeconomic status showed no significant association9. these data suggest that the socioeconomic status may be a distinguishing predisposing factor between enamel hypoplasia and mih. there is however, no study conducted to determine if the socioeconomic status of a particular population could be used as a distinguishing feature for enamel hypoplasia, mih and dmh. this study therefore aimed to determine if the prevalence of enamel hypoplasia, mih and dmh is associated with the socioeconomic status of the child. it also would determine the prevalence of enamel hypoplasia/ mih/dmh co-morbidities in the study population. material and methods this is a secondary analysis of a data collected to determine the prevalence of developmental dental hard tissue anomalies in the mixed dentition of children resident in ile-ife, central local government area (lga), osun state, nigeria. the data was collected during the july and august, 2013. the study population, sample size, sampling techniques for determining the prevalence of enamel hypoplasia, mih and dmh have been described in great details in prior study reports8,15. data were collected by a household survey administered to 1,169 children aged 1 to 19 years old. children excluded from the study were those who had a medical condition or syndrome that could increase the risk for tooth anomalies such as those who had cleft palate, and those with a history of diseases that could increase the risk for developing dental anomalies, such as maternal syphilis. sample size: the sample size required to determine if there was an association between different socioeconomic status and enamel hypoplasia using a prevalence of 18.9%15, to determine if there was an association between different socioeconomic status and mih using a prevalence of 19.2%9 and to determine if there was an association between different socioeconomic status and dmh using a prevalence of 4.6%8 is 1,169. data collection tool: data were collected by personal interview method, using a structured questionnaire. a dentist experienced with normal and pathological dental features and who had been engaged in a similar household dental survey in the same lga, was engaged as field worker for the study. data collected included information on the child’s socio-demographic characteristics (sex and socioeconomic status). socioeconomic status for the purpose of this study was obtained by a multiple item scoring index16 used in prior studies in nigeria17,18. the status designation combines the mother’s level of education with the occupation of the father; each child was allocated to a social class i to v, with class v being the lowest. each social class was classified as class i (upper class), class ii (upper middle class), class iii (middle class), class iv (lower middle class) and class v (lower class). clinical examination: all children eligible to participate in the study had an oral examination. the examinations were conducted under natural light, with the children sitting on a chair. the teeth were examined wet, after debris had been removed by use of a piece of gauze, and sterile dental mirrors and probes. the dental mirror was used to further provide illumination of the tooth surfaces through reflection of light and sun. each fully emerged tooth in the mouth was screened for enamel defects (hypoplasia and hypomineralization) using the modified dde index19. distinction was made between defects that appear as changes in the translucency of enamel (enamel opacity/hypomineralization), or as deficiencies in the quantity of enamel (enamel hypoplasia). the diagnosis of enamel hypoplasia was made when there was evidence of deficiency in enamel formation seen clinically as pits, grooves or generalized20. the diagnosis of mih was established as mih or dmh using the criteria described by kemoli21. the coronal part of the second primary molars, permanent first molars and permanent incisors were thoroughly examined for evidence of enamel hypomineralization. a tooth was considered to have mih or dmh when there was a demarcated opacity of about 2 mm associated with or without postoperative defects of deficiency in the enamel, large and extensive restorations on any of these teeth and suspected to be a result of hypomineralization. dmh and mih were diagnosed according to the european academy of pediatric dentistry (eapd) criteria22. a first permanent molar or a second primary molar was diagnosed as having mih or dmh when at least one of these criteria or a combination was found23. enamel defects were differentiated from carious lesions by their clinical appearance and locations (usually not related to gingival margins or occlusal fissures)2. distinguishing predisposing factors for enamel hypoplasia and molar-incisor hypomineralization in children in ile-ife, nigeria319319319319319 braz j oral sci. 14(4):318-322 standardization of examiner: an inter-examiner reliability test was done to calibrate the principal investigator on consistency of diagnosis for structural dental defects. the calibration was done by a consultant paedodontist who had worked extensively on mih and dmh over the last five years. the test was done by examining photographs of hypoplastic teeth and hypomineralized molars and incisors. the inter-examiner reliability score for means mih was 0.90 and enamel hypoplasia was 0.90. the inter-examiner reliability score for enamel hypoplasia was 0.75. data analysis: the socioeconomic status of children was re-categorized into three classes: social classes i and ii, high socioeconomic status; social class iii, middle socioeconomic status; and social classes iv and v, low socioeconomic status. descriptive and bivariate analyses were conducted to test the association between dependent variables (enamel hypoplasia, mih/dmh) and the child’s socioeconomic status and sex. where appropriate, the pearson’s chi-squared test or fisher’s exact test was used to test associations. univariate logistic regression was also conducted to determine the odds of having enamel hypoplasia, mih and dmh adjusting for sex and socioeconomic status of the child. statistical analysis was done with intercooled stata (release 12) for windows. simple proportions were computed. statistical significance was inferred at p<0.05. ethical consideration: ethical approval was obtained from the obafemi awolowo university teaching hospital complex ile-ife (erc/2013/07/14) on the 27th of june 2013. approval for community entry was obtained from the lga office. written informed consent was obtained from a parent or legal guardian of each study participant prior to enrollment. information on the socio-demographic profile of the children was obtained from either the consenting parent or legal guardian and written assent for children 8 years and above. variables enamel hypoplasia absent present m i h absent present d m h absent present socioeconomic status n=1,169 h i g h n=250 n (%) 208 (83.2%) 42 (16.8%) 238 (95.2%) 12 (4.8%) 247 (98.9%) 3 (1.2%) middle n=402 n (%) 354 (80.1%) 48 (11.9%) 387 (96.3%) 15 (3.7%) 397 (98.8%) 5 (1.2%) l o w n=516 n (%) 445 (86.2%) 71 (13.8%) 496 (96.1%) 20 (3.9%) 509 (98.6%) 7 (1.4%) p value 0.22 0.78 1.00 tatatatatable 1:ble 1:ble 1:ble 1:ble 1: association between socioeconomic status, hypoplasia, mih and dmh of 1169 study participants resident in nigeria. results there were 575 (49.2%) male and 594 (50.8%) female participants. the mean age of the study participants was 7.2+4.3 years. the median age was 6 years. two hundred and fifty (21.4%), 402 (34.4%) and 516 (44.2%) of the respondents were in the high, middle and low socio-economic strata, respectively. of the 1,169 study participants, 47 (4.0%) had mih, 15 (1.3%) had dmh and 161 (13.8%) had enamel hypoplasia. one (0.09%) of the study participant had mih/dmh comorbidity, 12 (1.0%) had dmh/hypoplasia co-morbidity, and 9 (0.8%) had mih/hypoplasia co-morbidity. table 1 shows the association between socioeconomic status, mih, dmh and enamel hypoplasia. there was no significant association between socioeconomic status and presence of enamel hypoplasia (p=0.22), mih (p=0.78) or dmh (p=1.00). table 2 shows the multivariate logistic regression analysis determining how sex and socioeconomic status predicted presence of enamel hypoplasia, mih and dmh. the odds of having enamel hypoplasia (aor: 0.83; ci: 0.591.16; p=0.27) and dmh (aor: 0.69; ci: 0.24 – 1.94; p=0.48) was lower for females when compared with males though these findings were not significant. the odds of having mih was higher for females (aor: 1.07; ci: 0.60 – 1.93; p=0.81) when compared with males though this finding was also not significant. the odds of having enamel hypoplasia (aor: 1.36; ci: 0.93-2.00; p=0.11) and mih (aor: 1.27; ci: 0.65 – 2.18; p=0.48) was higher for those with lower socioeconomic status when compared with those with high socioeconomic status though these findings were not significant. the odds of having dmh was lower for those with lower socioeconomic status when compared with those with high socioeconomic status (aor: 0.93; ci: 0.26 – 3.32; p=0.91) though this finding was also not significant. distinguishing predisposing factors for enamel hypoplasia and molar-incisor hypomineralization in children in ile-ife, nigeria mih: molar-incisor hypomineralisation dmh: deciduous molar hypomineralisation 320320320320320 braz j oral sci. 14(4):318-322 independent variables sex male female socioeconomic status high l o w sex male female socioeconomic status high l o w sex male female socioeconomic status high l o w yes 73 (45.3%) 88 (54.7%) 42 (26.1%) 119 (73.9%) 24 (50.0%) 24 (50.0%) 35 (72.9%)1 3 (27.1%) 6 (40.0%) 9 (60.0%) 3 (20.0%)12 (80.0%) n o 501 (49.7%) 507 (50.3%) 208 (20.6%) 780 (79.4%) 550 (49.1%) 571 (50.9%) 238 (21.2%) 883 (78.8%) 569 (49.3%) 585 (50.7%) 247 (21.4%) 907 (78.6%) adjusted odds ratio 0.83 1.36 1.07 1.27 0.69 0.93 96% ci 0.59 1.16 0.93 2.00 0.60 1.93 0.65 2.18 0.24 1.94 0.26 3.32 p value 0.27 0.11 0.81 0.49 0.48 0.91 multiple regression analysisdependent variables n(%) enamel hypoplasia table 2. table 2. table 2. table 2. table 2. logistic regression analysis for the association between sex, socioeconomic status, enamel hypoplasia, mih and dmh of 1,169 study participants resident in nigeria discussion the study highlights the non-significant role of sex and socioeconomic status as distinguishing predisposing factors for enamel hypoplasia, mih and dmh: sex and socioeconomic status were not associated with presence of enamel hypoplasia, mih or dmh. neither was sex and socioeconomic status a significant predictive factor for enamel hypoplasia, mih or dmh. however, the possibility of having both enamel hypoplasia and mih/dmh comorbidities reinforces the need to identify ways of differentiating the lesions. the prevalence of these comorbidities is low in this study population. in a study conducted in the same environment, oyedele et al 9 had earlier highlighted that the non-statistical association between socioeconomic status and mih may be a distinctive feature of mih and enamel hypoplasia, since prior studies conducted in the country 5,6 highlighted an association between socioeconomic status and enamel hypoplasia. however, the present study was not able to make such distinction through the tests of associations nor could it establi sh that the socioeconomic status of study participants could serve as a significant predictor for enamel hypoplasia, mih or dmh. one of the strengths of the study is the ability to generalize the findings to the study environment as the participants were recruited by a household survey. this increased the chances of including all segments of the study population in the sampling framework. however, this study was a secondary data analysis not specifically powered to test the hypothesis. as the sample size used for this secondary data analysis exceeded the minimum sample size required to conduct such primary analysis, the study findings are valid. the finding is specific for the study environment and the outcome cannot be generalized beyond the study population. it can be therefore concluded that for this study population, sex and socioeconomic status are not associated with nor are they predictors of enamel hypoplasia, mih or dmh. the possibility of co-existence of enamel hypoplasia and mih, and enamel hypoplasia and dmh makes it imperative to find ways to distinguish between the lesions, especially since the predisposing factors for mih/dmh are poorly understood. acknowledgements we are grateful to dr. t owoeye for his contributions towards the collection of the data for this study. distinguishing predisposing factors for enamel hypoplasia and molar-incisor hypomineralization in children in ile-ife, nigeria mih dmh mih: molar-incisor hypomineralisation dmh: deciduous molar hypomineralisation 321321321321321 braz j oral sci. 14(4):318-322 references 1. robles mj, ruiz m, bravo-perez m, gonzález e, peñalver ma. prevalence of enamel defects in primary and permanent teeth in a group of 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ten cate jm, jaddoe vw, hofman a, moll ha, veerkamp js. deciduous molar hypomineralization and molar incisor hypomineralization. j dent res. 2012; 91: 551. distinguishing predisposing factors for enamel hypoplasia and molar-incisor hypomineralization in children in ile-ife, nigeria 322322322322322 braz j oral sci. 14(4):318-322 revista fop n 13 1602 stromal changes in apparently normal mucosa of smokers and pan chewers – a multi-parametric approach ramalingam karthikeyan1; basireddy siva reddy2; herald.j.sherlin1; anuja n3; pratibha ramani3; thiruvengadam chandrasekar4; s ganesan5; pushpa viswanathan6 1 senior lecturer, 2 post graduate student 3assistant professor, 4 professor and head, department of oral and maxillofacial pathology, college of dental surgery, saveetha university, chennai 5 professor, department of medical physics, anna university, chennai 6 assistant professor, department of electron microscopy, cancer institute (wia), chennai received for publication: may 26, 2008 accepted: september 09, 2008 correspondence to: r. karthikeyan department of oral and maxillofacial pathology, college of dental surgery, saveetha university, 162, p.h. road, velappanchavadi, chennai 600077. phone:+ 9144-9840851467, +9144-26801580 fax: +9144-26800892 email: drrkn@rediffmail.com a b s t r a c t aim: to compare and contrast the various changes in the connective tissue among patients with the habits of smoking, pan chewing and controls by using fluorescence spectroscopy, histopathology and transmission electron microscopy. method: thirty subjects were categorized into three groups: pan chewers, smokers and controls without any oral lesions. fluorescence spectroscopy was carried out using fluoromax-2. excitation spectroscopy was performed at 280 and 320nm respectively and emission spectroscopy was performed at 340 and 390nm excitation. subsequently, histopathological evaluation and transmission electron microscopy was done for biopsies taken from test groups and controls. results: the mean, standard deviation and test of significance of mean values between different groups for intensity-380nm, intensity–420nm and intensity – 460nm at 320 nm excitations, showed that the mean values in group i and group ii were significantly higher than the mean value in group iii with a p-value of less than 0.001. considering the histopathological parameters, the pattern of the sub-epithelial connective tissue, the presence of chronic inflammatory cells and lysis of connective tissue was significant. the ultrastructural analysis revealed the presence of epithelioid, spindle or elongated and stellate shaped fibroblasts in the connective tissue. conclusion: in this pilot study, we could find variations in emission characteristics of various amino acids which correlated with histopathology and electron microscopy. our study suggests that there are connective tissue changes in oral mucosa among smokers and pan chewers, though it is apparently normal in clinical presentation. these initial connective tissue changes could determine the progression of altered mucosa to a pre-cancer or cancer, which is further related to other complex interactions. elaborate studies are required to evaluate the significance of our hypothesis. k e y w o r d s : autofluorescence, normal mucosa, smokers, pan chewers i n t r o d u c t i o n oral cancer is dominated by squamous cell carcinoma, which represents 90%-95% of all oral cancers. the role of tobacco in the development of oral squamous cell carcinoma is well recognized. the long-term prognosis is quite poor, and treatment can lead to further functional and cosmetic problems1-2. tobacco use, including smoking of cigarettes, cigars and pipes, reverse smoking (smoking with the lit end inside the mouth), chewing of betel quid (a mixture of areca nut, slaked lime, and tobacco wrapped in betel leaf), and use of smokeless tobacco increases the risk of cancers of the upper aerodigestive tract3-4. on population-based case-control studies, cigarette smokers have risk from two to five times than that of non-smokers for developing oral cancer. the risk increases with rise in number of cigarettes smoked and duration of smoking5. tobacco is chewed predominantly as an ingredient of betel quid or pan, which is a combination of betel leaf, areca nut, and lime. based on analysis that excluded smokers, smokeless tobacco users experienced about four to six times the risk of oral and pharyngeal cancer than non-users1-2. in the indian scenario, the proportion attributed to tobacco use in the form of smoking and chewing comprises about 61-70% of cancer incidence. among indians, alcohol does not emerge as a strong risk factor6. there is increasing evidence that sub-epithelial connective braz j oral sci. july/september 2008 vol. 7 number 26 1603 tissue can modify the phenotypic expression of the overlying epithelium. various tissue culture studies suggest that the histodifferentiation of the epithelium, including its phenotype and keratin expression could be extrinsically modified by mesenchymal fibroblasts. it is possible to elucidate that the underlying connective tissue plays a pivotal role on maintenance of the integrity of overlying epithelium, as per several studies on precancer and cancer7-9. thus this study was designed for evaluating different parameters of the stromal changes in apparently normal oral mucosa of smokers, pan chewers and controls using fluorescence spectroscopy. moreover, histopathological examination using hematoxylin and eosin stain and van gieson stain was performed, as well as the evaluation of ultrastructural changes in fibroblasts using transmission electron microscopy (tem). materials and methods thirty subjects aged from 16 to 60 years (mean age 45.6 years) without any oral lesions from the department of oral medicine and radiology, college of dental surgery, saveetha university, chennai were considered for the present study. the subjects were categorized into the following groups: group i, with 10 subjects without the habits of smoking or alcoholism or pan chewing, as the controls, group ii: 10 subjects with the habit of pan chewing (10-15 quid’s per day) for about 5-10 years; group iii: 10 subjects with the habits of smoking more than 10 cigarettes per day for about 5-10 years,. fluorescence spectroscopy was carried out using fluoromax-2 (usa, 1996) in the department of medical physics, anna university, chennai, with a fiber-optic probe attached to spectrofluorometer. the probe was placed in contact with the mucosa to record the data. for group ii, readings were taken from the area where the quid was placed and an area adjacent to the premolars at the occlusal level for groups i and iii. excitation spectroscopy was performed at 280 and 320nm respectively and emission spectroscopy was performed at 340 and 390nm. corrections were done for the non-uniform spectral response and for the variations in the intensity of illumination source of the spectrometer. the spot size of the illumination was approximately 1 cm in diameter and photo-bleaching was not observed in the test site after the procedure. subsequent to the autofluorescence spectroscopy, after taking informed consent, incisional biopsy was performed in the subjects of the three groups, under local anesthesia at the same sites used for fluorescent spectroscopy. the biopsy samples were rinsed in saline and then fixed with 10% buffered formalin. the tissue was processed routinely and embedded in paraffin. 4µ sections were prepared with a leica semi-automated microtome (rm 2245). routine hematoxylin and eosin staining was done for microscopic examination of the sections. sections were also stained with van gieson stain to demonstrate collagen. the connective tissue in the sub-epithelial region and deeper region were assessed for the following parameters, density – minimal/moderate/ increased, pattern – wavy/ bundles/haphazard/parallel/stream, number of fibroblasts minimal/ moderate/ increased, number of inflammatory cells absent/minimal/moderate/ intense, hyalinization– present/absent and amount of vascularity minimal/ moderate/intense, connective tissue status homogenous/ lysis and intensity of van gieson stain minimal/ moderate/ intense. the epithelial parameters that were assessed include keratinization – ortho/para/non-keratinized, thickness– atrophy/hyperplastic/normal, epithelium-connective tissue interface: normal/ thickened/discontinuous. samples were also fixed in 2.5% buffered glutaraldehyde, processed and subjected to transmission electron microscopy. all the parameters were tabulated and the statistical significance was assessed with spss version 10. r e s u l t s native fluorescence spectroscopy of groups i, ii and iii at different wavelengths, corresponds to the native fluorophores present in cells and extracellular matrix, such as tryptophan, collagen, elastin and nadh. the average emission spectrum was performed at 280 nm excitation corresponding to the amino acid residue, tryptophan. normal oral mucosa and pan chewer’s mucosa showed a prominent peak at 338 nm and a small peak at 440 nm. it is worth to mention that the emission at 280 nm mostly is due to tryptophan, as the emission efficiency (quantum yield) of other two amino acids, phenyl alanine and tyrosine is very minimal as that of tryptophan. smoker’s mucosa did not show any peaks at 338 nm and 440 nm. in order to overcome the artifacts of instruments and tissue heterogeneity, normalization of spectra was performed in relation to its peak wavelength or any wavelength of interest. the averaged emission spectra of normal mucosa, mucosa of pan chewers and smokers at 280nm excitation showed maximum emission intensity at 340 nm (fig1). the normal tissues showed a lesser intensity than pan chewers and smokers, at higher wavelengths of 400-500 nm. it clearly indicates that nadh/nad(p)h and collagen fig. 1 averaged fluorescence emission spectra of normal, pan chewers and tobacco smokers at 280nm excitation braz j oral sci. 7(26):1602-1608 stromal changes in apparently normal mucosa of smokers and pan chewers – a multi-parametric approach 1604 distribution may be altered under various tissue transformation conditions. the averaged emission spectrums at 320 nm excitation corresponding to the collagen/elastin are shown in fig. 2. the emission spectra corresponding to normal mucosa and pan chewer’s mucosa showed two prominent peaks at 390 nm and 460 nm, respectively. the first peak which is at 390 nm corresponds to the collagen intensity (around 390) and the second peak corresponds to the nadh (around 460 nm), which are almost equal. however, surprisingly the smoker’s mucosa did not show any increase in intensity at 390nm and 460nm. the average excitation spectra at 340 nm emission corresponding to the tryptophan are shown in fig. 3. the emission spectra corresponding to normal mucosa showed prominent intensity at 295 nm whereas the pan chewer’s mucosa showed a prominent peak at 296 nm. tobacco smoker’s mucosa showed a small peak, with maximum intensity at 292 nm wavelength. the normalized excitation spectra were performed at 34 0nm emission. pan chewer’s mucosa did not show significant peak shift compared to the normal individuals, both of them showed maximum intensity at 295nm. surprisingly, smoker’s mucosa showed maximum intensity at 292 nm showing a shift to the left, i.e., towards shorter wavelength. this shift to the left could probably attributed to any conformational changes in the tissue protein sequences and/or partial folding of protein structures. the average excitation spectra at 390 nm excitation corresponding to the collagen are shown in fig 4. the excitation spectra corresponding to normal mucosa and pan chewer’s mucosa showed relatively a prominent peak at 295 nm, whereas smoker’s mucosa showed a small peak at the same wavelength. the excitation intensity of pan chewer’s mucosa was much less compared to normal at this emission spectrum. the normalized emission spectra were performed at 390 nm emission. the emission spectral findings at 390 nm are similar to that of 340 nm emission. however, a slight shift to the left (towards shorter wavelength) was seen in pan chewer’s mucosa when compared to the normal individuals. smoker’s mucosa showed a gross shift to the left, as seen in 340 nm emission. the maximum intensity values at this emission spectrum for normal, pan chewer’s mucosa and smoker’s mucosa are 298, 297 and 294 respectively. this clearly indicates that the absorption bands of collagen vary with tissue conditions. in order to improve the diagnostic interpretation, different parameters were introduced at the emission peak of 320 nm excitation. we selected 320 nm excitation because the presence of fluorophores – collagen, hemoglobin and nadh that were altered in smokers. the wavelengths were selected at points where maximum intensity of corresponding fluorophores was observed. based on these intensity values, we performed the statistical analysis. fig. 2 averaged fluorescence emission spectra of normal, pan chewers and tobacco smokers at 320nm excitation fig. 3 averaged fluorescence excitation spectra of normal, pan chewers and tobacco smokers at 340nm emission fig. 4averaged fluorescence excitation spectra of normal, pan chewers and tobacco smokers at 390nm emission there was a considerable difference in the fluorescence intensity at i-380 between normal mucosa and smoker’s mucosa (fig. 5, table 1). it was also observed that this intensity value discriminated normal from smoker’s mucosa with a sensitivity of 100% and specificity of 100%. there was a considerable overlap in the fluorescence intensity at i-380 between normal and pan chewer’s mucosa. this intensity value discriminated normal from pan chewer’s braz j oral sci. 7(26):1602-1608 stromal changes in apparently normal mucosa of smokers and pan chewers – a multi-parametric approach 1605 mucosa with a sensitivity of 78% and specificity of 12%. there was considerable difference in the fluorescence intensity at i-380 between pan chewer’s mucosa and smoker’s mucosa (fig. 6). this intensity value discriminated smoker’s mucosa from pan chewer’s mucosa with a sensitivity of 100% and specificity of 100%. the data were compared between groups for intensity-380 nm, intensity–420 nm and intensity – 460 nm at 320 nm excitations, showing that the mean values in group i and group ii were similar (p > 0.05) and both significantly higher than group iii (p < 0.001). in the histopathological assessment (table 2) all the subjects of group i and ii did not show lysis in the connective tissue. from group iii, 6 cases showed lysis groups sensitivity specificity group i vs.group iii 100 100 group ii vs. group iii 100 100 group i vs. group ii 78 12 table 1 – fluorescence intensity at i-380 fig. 5 scatter plot of intensity value at 380nm vs sample number for normal and tobacco smokers from fluorescence emission at 320nm excitation fig. 6 scatter plot of intensity value at 380nm vs sample number for pan chewers and tobacco smokers from fluorescence emission at 320nm excitation criteria group i group ii group iii lysis in the connective tissue 0/10 0/10 6/10 intense inflammatory cell infiltrate 0/10 0/10 2/10 moderate inflammatory cell infiltrate 0/10 8/10 6/10 table 2 – histopathological assessment fig. 7 lysis of connective tissue h&e – 10x (fig. 7) and 4 cases did not show. presence of lysis in the connective tissue and presence of inflammatory cells in the deeper region of the connective tissue were significant (p=0.024 and p=0.036, respectively). it was observed in group i 6 cases with minimal inflammatory cells and 4 cases with mild inflammatory infiltrate, whereas in group iii, 2 subjects presented mild inflammatory infiltrate and 8 subjects moderate inflammatory infiltrate (fig. 8). in group ii, mild inflammatory infiltrate was observed in 2 subjects, moderate inflammatory infiltrate in 6, and intense inflammatory infiltrate in 2. the other histopathological parameters did not achieve statistical significance. the ultrastructural analysis of the oral mucosa of controls by tem showed presence of continuous, homogeneous, fig. 8 inflammatory cells in the connective tissue h&e – 10x braz j oral sci. 7(26):1602-1608 stromal changes in apparently normal mucosa of smokers and pan chewers – a multi-parametric approach 1606 thick collagen bundles with occasional blood vessels and numerous fibroblasts. the fibroblasts were spindle / elongated in shape with elongated nucleus, dense chromatin, numerous endoplasmic reticulum and extended cisternae. the smoker’s mucosa showed the presence of numerous discontinuous bundles and few dense granules. there were few fibroblasts actively secreting collagen with increased bundles of collagen showing prominent banding. the fibroblasts were elongated to spindle shaped, with elongated nucleus pushed to one end, numerous endoplasmic reticulums, extended cisternae, granules filled with proteinaceous material. the mucosa of pan chewers showed the presence of immature, thin filaments and the banding was not prominent. the fibroblasts were epithelioid in shape with a round nucleus, prominent nucleolus and endoplasmic reticulum along with increased cellularity. highly vacuolated cells containing some debris, degenerating cells or apoptotic cells with fragmented organelles and apoptotic bodies were seen. inflammatory cells, predominantly granulocytes and mast cells were also evident in both the study groups. d i s c u s s i o n autofluorescence spectroscopy is a non-invasive and easily applicable tool for the detection of alterations in the structural and chemical compositions of cells, which may indicate the presence of diseased tissue10. when tissue is illuminated with specific wavelengths of ultraviolet (uv) or visible (vis) light (excitation), fluorescent biological molecules (fluorophores) will absorb the energy and emit it as fluorescent light at longer wavelengths (emission). furthermore, there are non-fluorescent light absorbers (such as hemoglobin) and scatterers (cells and sub-cellular organelles) in tissues that modulate the tissue fluorescence intensity at the excitation and emission wavelengths. endogenous fluorophores in tissue include amino acids, structural proteins, enzymes and coenzymes, vitamins, lipids, and porphyrins. each of these molecules has unique excitation and emission spectra in the uv/vis spectral region. the excitation wavelengths used, ranging from 300 to 460 nm, allow for characterization of a number of biologic fluorophores, including tryptophan, nicotinamide adenine dinucleotide, flavoproteins, and collagen, all of which are present in tissue systems. tryptophan shows maximum intensity at 280nm, collagen at 320nm, nadh at 340nm and endogenous porphyrin at 405nm excitation11. accordingly, autofluorescence spectra were performed corresponding to these wavelengths for normal mucosa and mucosa of pan chewers and smokers. though there are studies on autofluorescence spectra in sub mucous fibrosis, leukoplakia and squamous cell carcinoma10-13, our pilot study is the first of its kind in english literature to assess the autofluorescence in apparently normal mucosa of smokers and pan chewers. in the present study, the averaged fluorescence emission spectrum of normal mucosa, mucosa of pan chewers and smokers, at 280 nm and 320 nm excitation wavelengths were performed. although the normalized spectral signature of smoker’s mucosa is similar to that of pan chewers and normal mucosa, the absolute intensity is 8 folds lesser than that of normal mucosa. the major peak around 338 nm may be attributed to tryptophan emission. most proteins are endowed with an intrinsic uv fluorescence because they contain aromatic amino acids, particularly phenylalanine, tyrosine and tryptophan. of these three aromatic amino acids tryptophan has the highest fluorescence quantum yield overshadowing markedly the emissions of the other two. tryptophan emission maxima in proteins can vary from 332 nm to 342 nm depending on the protein. free tryptophan has a characteristic fluorescence emission at 350–360 nm14. this decrease in tryptophan levels may be attributed to the sparse cellularity or it may also be due to the distortion of fluorescent intensity. analysis using fluorescent excitation spectra is a complimentary technique which is sensitive to any conformational changes that take place during the process of tissue transformation. it will provide the changes in absorption band of molecules. in order to get the emission spectrum, the molecule of interest should be excited at its exact absorption wavelength. generally it is very difficult to measure absorption spectra of turbid media like cells and tissues. under such conditions, fluorescence excitation spectra can indirectly measure the absorption band. the peak emission of the fluorophores is monitored at different excitation wavelengths. the maximum intensity corresponding to a particular wavelength of excitation is the absorption wavelength of the molecule. hence, we also monitored the fluorescence excitation spectra for all the tissues, for their emission at 340 nm and 390 nm to confirm whether the emission at 340 nm is due to tryptophan and to check whether there is any change in the absorption band, when normal tissues undergo various transformations. a shift to the right, i.e., to higher wavelength is called red shift. similarly a shift to the left is called blue shift. it is found that normal and pan chewer’s mucosa have similar absorption bands at 300nm. howeve,r smoker’s mucosa had very minimal absorption, which was 30 times lesser than normal. this may be due to the scattering or reflection of light due to decreased blood supply. though there are many controversies behind the decrease in fluorescence, our results are in line with tsai et al.12, who attributed the decrease in fluorescence to the distortion of fluorescence caused by collagen excess in the sub-mucosa. the average excitation spectra of normal, mucosa of pan chewers and smokers at 340 nm emission corresponding to tryptophan, smoker’s mucosa showed the left shift compared to normal, indicating that there is a considerable rearrangement in the protein sequences of amino acids. the autofluorescence emission spectra at 320 nm excitation, corresponding to collagen, revealed a first peak around 390 nm, which corresponds to emission from collagen and elastin. the second peak at 460 nm corresponds to nadh present in epithelial cells of the surface mucosa. the valley between the two peaks corresponds to the hemoglobin absorption of braz j oral sci. 7(26):1602-1608 stromal changes in apparently normal mucosa of smokers and pan chewers – a multi-parametric approach 1607 the tissues. the emission spectra of normal mucosa and pan chewers showed two prominent peaks centered at 390 nm and 460 nm with a valley around 420 nm. however, the relative intensity of emission of pan chewers is slightly lower than that of normal individuals. the smoker’s mucosa had only one broad peak in the region from 370 nm to 530 nm without any dip at 420 nm, indicating less vascularity. it is surprising to observe that smokers displayed decreased fluorescent intensity compared to that of normal mucosa and pan chewer’s mucosa at this particular wavelength. this observation is contradictory to that of chen et al.13 who found increased intensity for collagen in oral sub mucous fibrosis. however, our results are similar to findings of tsai et al.12, since decreased intensity for collagen in sub mucous fibrosis was observed. the changes in epithelium may result in increased nadh intensity. however, the nadh intensity in pan chewers was slightly less compared to that of normal. this might be due to minimal scattering caused by collagen that is beginning to accumulate in the sub-mucosa. in smoker’s mucosa, no peak was observed at 440 nm, which probably could be attributed to the distortion of fluorescence caused by collagen. the valley that is observed around 420 nm corresponds to hemoglobin absorption. the valley indicates the amount of vascularity in the tissues. normal mucosa and pan chewer’s mucosa showed a prominent dip around 420 nm. but smokers did not show any dip at this wavelength. it is also interesting to note that spectral signature of smokers was entirely different from normal and pan chewers. the spectral band of normal and pan chewers showed a valley around 420nm and 580nm and this indicates a considerable vascularity in these tissues. however, the valley at 420nm and 580nm are completely absent in smokers indicating minimal vascularity. liang et al.15 explained that the left shift by photo-physical characteristics of tryptophan depends on the microenvironmental conditions. in particular, the emission of tryptophan depends upon its solvent polarity. the shift to shorter wavelength occurs as the solvents surrounding the tryptophan residues decrease13. this explanation could be applied to smoker’s mucosa, as there is stabilization of collagen, making them less soluble to collagenases when compared to normal collagen. another explanation for the left shift or the decrease in tryptophan intensity could be due to the sparse cellularity in the dense, collagenized sub mucosa. another reason for the decrease in collagen fluorescence, in spite of having more amounts of collagen and elastin, is that there may be a reflection of light at the epithelium-connective tissue interface without any absorption. this may be due to the change in the refractive index of the tissues. fluorescence intensity can be influenced by inter-subject variability in the amount of blood, with absorption leading to a wavelength-dependent decrease in fluorescence intensity. besides the biological variation, varying experimental circumstances can influence the total fluorescence intensity10. the present non-invasive in-vivo autofluorescence spectroscopy helps to differentiate smoker’s mucosa from normal with a significant difference. however, further studies with more samples are necessary to identify the exact cause of decreased fluorescence in smokers, though we attributed it to the distortion of fluorescence as mentioned by tsai et al.12. the attempt to differentiate pan chewers from normal was also successful, since early changes with decreased fluorescent intensity compared to normal were noted. subsequently, biopsy samples were taken from the representative site as detected by autofluorescence. keratinization of mucosal epithelium is believed to be interrelated with the inflammatory cell infiltrate. in our study, the samples predominantly showed hyper-parakeratinization. the epithelial thickness may depend on the tobacco habit and the location. the epithelial hyperplasia could be an adaptive response to local irritants, to provide a greater degree of protection to the underlying connective tissue. the collagen fibers were wavy among the controls and majority of the pan chewer’s mucosa. considering the amount and nature of collagen in the sub-epithelial region and deeper region, the haphazard and thickened arrangement of connective tissue seen in smokers along with lysis, correlates with the connective tissue changes seen in the early stages of sub mucous fibrosis. the presence of inflammatory cells also has a role to play in the fibrosis of connective tissue stroma. the production of inflammatory mediators is controlled by various enzymes such as cyclo-oxygenases. increased expression of fibrogenic cytokines like tgf-beta, pdgf, bfgf and pro-inflammatory cytokines like il-1 & 6 plays a role in fibrosis16. subsequently, to confirm to our histopathological findings, tem was performed. in smoker’s mucosa, numerous discontinuous bundles were present along with few dense granules. there was an increase in collagen bundles and prominent banding was also evident. the fibroblasts were elongated to spindle shaped, elongated nucleus pushed to one end along with numerous endoplasmic reticulums, extended cisternae and granules filled with proteinaceous material. mast cells were also present. there were few fibroblasts actively secreting collagen. our study also revealed the presence of epithelioid, spindle or elongated and stellate shaped fibroblasts in accordance with literature, mollenhauer and bayreuther17 described three distinct fibroblast cell forms in rat connective tissue that can be identified on the basis of their morphology. they can also be distinguished from one another by the amount and type of collagen synthesized. the f1 fibroblast is spindle shaped, highly proliferative and secretes low levels of type i and iii collagen. the f2 fibroblast is more epitheloid, less proliferative and synthesizes relatively more collagen. the f3 fibroblast is a large stellate cell and the least proliferative, produces four to eight times more types i and iii collagen than f1. according to these workers, f2 cells sequentially arise from f1 cells and f3 cells sequentially arise from f2 cells. the lysis and fragmentation of collagen fibres was also considered as one of the change especially authenticated as an ultrastructural change in sub mucous fibrosis. braz j oral sci. 7(26):1602-1608 stromal changes in apparently normal mucosa of smokers and pan chewers – a multi-parametric approach 1608 binnie and cawson18 found an excess of fine, immature fibrils and inter-fibrillar matrix in sub mucous fibrosis. the collagen fibrils were observed to be fragmented, some of them showed frayed and bent ends with apparent partial degeneration into amorphous material. rajendran et al.19 also reported similar ultra-structural findings in sub mucous fibrosis. thus the ultrastructural changes of increased fibrosis in smoker’s mucosa, is in accordance with the findings of autofluorescence and histopathology and these changes are comparable to oral sub mucous fibrosis. our study showed that smokers had connective tissue changes similar to sub mucous fibrosis more than that seen in pan chewers. literature states that sub mucous fibrosis is more prevalent in pan chewers and smokers have increased tendency to develop leukoplakia, though both are caused by tobacco products. hence, we have hypothesized that the response of connective tissue in relevance to smoking differs from pan chewing. in smoking, apart from the tobacco component, heat also acts as an additional source of insult and may aid in faster diffusion. the initial response of collagen fibres in smoking could be due to the synergistic effect of heat and tobacco. the collagen response would prevent the further diffusion, resulting in changes in epithelium and may thereby progress to leukoplakia and other changes. tilakaratne et al.16 have hypothesized that dense fibrosis and less vascularity of the corium, in the presence of altered cytokine activity creates a unique environment for the carcinogens of tobacco and areca nut to accumulate over a long period of time either on or below the epithelium. but in pan chewing, diffusion may be slow in comparison to smoking, thereby producing an initial, protective inflammatory response in the connective tissue. the fibroblasts would initially produce collagen as a defensive reaction and subsequent insults would result in the morphologic change of fibroblasts and thus progress to sub mucous fibrosis. hence, our study suggests that there are connective tissue changes in oral mucosa among smokers and pan chewers, though it is apparently normal in clinical presentation. these initial connective tissue changes could determine the progression of altered mucosa to a pre-cancer or cancer, which is further related to other complex interactions. elaborate studies are required to evaluate the significance of our hypothesis. a c k n o w l e d g e m e n t s we would like to thank dr. m.r. muthusekar, professor and head and dr. vinod narayanan, professor, and postgraduates of the department of oral and maxillofacial surgery, college of dental surgery, saveetha university for their biopsies. we would like also to thank the postgraduates of our department, dr. richa goel, dr. gheena.s and dr.k.jeyanthi and my lab technician, mrs. uma sankari for their help in histological evaluation. i would like to thank the research fellows in department of medical physics, anna university for their help in assessment of autofluorescence. r e f e r e n c e s 1. gupta pc, bhonsle rb, murty pr, mehta fs, pindborg jj. epidemiologic characteristics of treated oral cancer patients detected in a house tohouse survey in kerala, india. indian j cancer. 1986; 23: 206-11. 2. zavras ai, douglass cw, joshipura k, wu t, laskaris g, petridou e et al. smoking and alcohol in the etiology of oral cancer: gender-specific risk profiles in the south of greece. oral oncol. 2001; 37: 28-35. 3. franceschi s, talamani r, barra s, baron ae, negri e, bidoli e et al. smoking and drinking in relation to cancers of the oral cavity, pharynx, larynx and oesophagus in northern italy. cancer re. 1990; 50: 6502-7. 4. lyon. tobacco smoking. iarc monogr eval carcinog risk chem hum. 1986; 38: 35-394. 5. bartsch h, nair u, risch a, rojas m, wikman h, alexandrov k. genetic polymorphism of cyp genes, alone or in combination, as a risk modifier of tobacco related cancers. cancer epidemiol biomarkers prev. 2000; 9: 3-28. 6. gupta pc, mehta fs, daftary dk, pindborg jj, bhonsle rb, jalnawalla pn et al. incidence rates of oral cancer and natural history of oral precancerous lesions in a 10-year follow-up study of indian villagers. community dent oral epidemiol. 1980; 8: 283-333. 7. mackenzie ic, hill mw. connective tissue influences on patterns of epithelial architecture and keratinization in skin and oral mucosa of the adult mouse. cell tissue res. 1984; 235: 551-9. 8. hill mw, mackenzie ic. the influence of subepithelial connective tissues on epithelial proliferation in the adult mouse. cell tissue res. 1989; 255: 179-82. 9. squier ca, kammeyer ga. the role of connective tissue in the maintenance of epithelial differentiation in the adult. cell tissue res. 1983; 230: 615-30. 10. de veld dc, skurichina m, witjes mj, duin rp, sterenborg dj, star wm et al. autofluorescence characteristics of healthy oral mucosa at different anatomical sites, lasers surg med. 2003; 32: 367-76. 11. breslin tm, xu f, palmer gm, zhu c, gilchrist kw, ramanujam n. autofluorescence and diffuse reflectance properties of malignant and benign breast tissues, ann surg oncol. 2003; 11: 65-70. 12. tsai t, chen hm, wang cy, tsai jc, chen ct, chiang cp. in vivo autofluorescence spectroscopy of oral premalignant and malignant lesions: distortion of fluorescence intensity by submucous fibrosis. lasers surg med. 2003; 33: 40-7. 13. chen hm, wang cy, chen ct, yang h, kuo ys, lan wh. auto-fluorescence spectra of oral submucous fibrosis. j oral pathol med. 2003; 32: 337-43. 14. siik s, airaksinen pj, tuulonen a, nieminen h. autofluorescence in cataractous human lens and its relationship to light scatter. acta ophthalmol (copenh). 1993; 71: 388-92. 15. liang jn. heat induced conformational change of lens recombination, a & b crystallization. mol vis. 2000; 6: 10-4. 16. tilakaratne wm, klinikowski mf, saku t, peters tj, warnakulasuriya s. oral submucous fibrosis: review on aetiology and pathogenesis. oral oncol. 2006; 42: 561-68. 17. mollenhauer j, bayreuther k. donor age related changes in the morphology, growth potential and collagen biosynthesis in rat fibroblast population in vitro. differentiation. 1986; 32: 165-72. 18. binnie wh, cawson ra. a new ultrastructural finding in oral submucous fibrosis. br j dermatol, 1972; 86: 286-90. 19. rajendran r, sugathan ck., reman ip, ankathil r, vijayakumar t. cell mediated and humoral immune responses in osf. cancer1986; 58: 2628-31. braz j oral sci. 7(26):1602-1608 stromal changes in apparently normal mucosa of smokers and pan chewers – a multi-parametric approach 1http://dx.doi.org/10.20396/bjos.v18i0.8657417 volume 18 2019 e191647 original article 1 departament of orthodontics, araras dental school, uniararas, araras, sp, brazil. 2 department of restorative dentistry dental materials division, piracicaba dental school, unicamp, piracicaba, sp, brazil. corresponding author: heloisa c valdrighi av. dr. maximiliano baruto, 500 jardim universitário. araras, sp – brazil. 13607-339 19 3543-1423 e-mail: heloisavaldrighi@gmail.com https://orcid.org/0000-0001-7567-1990 received: may 03, 2019 accepted: october 19, 2019 can the chlorhexidine gluconate solution potentiate the staining of polycrystalline ceramic brackets? catharina e silva monteiro barros1, jose guilherme neves2, ana paula terossi de godoi1, ana rosa costa2, mario vedovello filho1, heloisa cristina valdrighi1,* aim: the present study aimed to assess, in vitro, the effect of chlorhexidine on the potentiation of polycrystalline ceramic bracket staining. methods: seventy-two polycrystalline ceramic brackets of upper right central incisors were divided into six groups (n=12) according to immersion solution. the groups were g1 distilled water (control); g2 chlorhexidine; g3 coffee; g4 red wine; g5 chlorhexidine associated with coffee; and g6 chlorhexidine associated with red wine. the samples were analyzed by means of a spectrophotometer according to the ciel*a*b* system, and color change (δe*) was calculated. the readings were performed at the following times: t0 after package removal and t1 after staining. the data were analyzed by kruskal wallis and t tests (p<0.05) at 5% significance level. results: the total color variation (δe*) was greater in the group that received chlorhexidine associated with red wine (p<0.05) and lower in the groups that received distilled water. all other groups showed greater value variations when compared to g1 and g2. group g6 showed a greater color change due to the potentiation of chlorhexidine with the dye substance. conclusion: it is concluded that chlorhexidine potentiates the staining caused by red wine in polycrystalline ceramic brackets. keywords: orthodontic brackets. mouthwashs. ceramics. https://orcid.org/0000-0001-7567-1990 2 barros et al. introduction over the last years, patients seeking orthodontic treatment have not only been demanding an effective occlusal result, but also looking for the satisfaction of a harmonious appearance, aiming to resemble the color of tooth enamel1,2. the use of esthetic devices is mainly indicated for adult patients who wish to maintain a natural aspect to their smile and correct its positioning at the same time3. the first esthetic orthodontic devices were polycarbonate brackets, which present negative characteristics such as high deformation rate, structural fragility, low adhesion, and low stain resistance, thus compromising clinical performance and not reaching the esthetic objective4. attempting to solve such problems, a few structural changes were performed, such as the reinforcement with ceramic loads and glass fiber, which originated the esthetic ceramic brackets5. according to the fabrication process, there are two forms of ceramic brackets: polycrystalline and monocrystalline6. the polycrystalline ceramic or polycrystalline alumina brackets are made of aluminum oxide crystals fused at high temperatures that allow molding several brackets simultaneously. among esthetic brackets, these are the most common and popular, due to material quality and the ease of production in comparison to monocrystalline alumina brackets7. the ceramic brackets, although desired by patients, still deteriorate in the oral cavity and present some limitations when exposed to certain substances such as coffee, black tea, and red wine, causing color change and consequently the loss of the esthetic standard desired8-10. this impregnation of food and beverage pigments is considered one of the main extrinsic staining factors of such devices11. in the clinical procedure during orthodontic treatment, the use of mouthwashes is rather common12. however, few studies show the effects of mouthwashes on the color stability of polycrystalline ceramic orthodontic brackets13 and composite resins14. chlorhexidine gluconate, from the biguanide family, is considered the gold standard in therapies for controlling plaque15, thus preventing potential inflammation of the periodontal tissue adjacent to the orthodontic appliances. on the other hand, the continuous use of chlorhexidine can generate the appearance of dark spots on dental surfaces, presenting variation according to the frequency of use and concentration of the product used15. due to the presence of pigmentation in esthetic orthodontic brackets caused by the consumption of coloring foods and mouthwashes, the present study aimed to assess, in vitro, the effect of chlorhexidine on the potentiation of ceramic bracket staining. the null hypothesis in this study was that chlorhexidine does not have the capacity to potentiate staining in ceramic brackets material and methods the sample size considered the previous literature9,13, significance level of 0.05, and test power of 0.80. therefore, the final sample included 72 polycrystalline ceramic brackets (iceram, orthometric, marília, são paulo, brazil; batch 037268001) of upper 3 barros et al. right central incisors, which were divided into six groups (n=12) according to dye solution: g1 distilled water (control group, asfer, indústria química, são caetano do sul, sp, brazil; batch 274); g2 0.12% chlorhexidine (periogard, colgate; batch 7125br121a); g3 instant coffee (nescafé, nestlé, são paulo, brazil; batch 80301210); g4 red wine (reservado, carbenet sauvignon 12.5% alcohol volume, concha y toro/ santiago do chile; batch: 1110716); g5 chlorhexidine and coffee; g6 chlorhexidine and wine, as figure 1 shows. pigmentation process the instant coffee solution was prepared according to the concentration suggested by the manufacturer, with 100 ml of boiling water to two teaspoons of powder. the other solutions did not require previous preparations. the specimens were immersed in staining solutions and maintained under agitation (ultrasonic tank) according to each group described. the samples were immersed for three minutes, once a day for five days of the week during 28 days16-18. then, the solufigure 1. experimental design of the study. polycrystalline ceramic bracket (n=72) color analysis: cielab spectrophotometer tt0 – reading of initial color of brackets g1 distilled water (n=12) g2 chlorhexidine (n=12) g3 coffee (n=12) g4 wine (n=12) g5 chlorhexidine and coffee (n=12) g6 chlorhexidine and wine (n=12) t1 – after staining with dye solutions color reading and measuring: spectrophotometer statistical analysis 4 barros et al. tions were removed and the specimens were washed with distilled water and dried with absorbent paper. next, the specimens were stored in distilled water at 37°c until the next staining protocol18,19. on the other hand, the control group remained in distilled water for the entire experimental period. all groups were immersed in 3 ml of each dye solution, stored in units in a lidded polypropylene recipient to prevent the evaporation of solutions and identify the group name in order to to codify it for individualization. color reading the samples were subjected to color reading using a sp62s x-rite spectrophotometer (grand rapids, michigan, usa) with the qa master software. this reading was performed at the times described, that is, initial time (t0), immediately after package removal, immediately after water storage, and at the end of the entire pigmentation process (t1). this spectrophotometer presents focal aperture of 4 mm and diffuse geometry of d/8°. it emits light with wavelength from 400 to 700 nm on the object and measures the reflection of such spectrum. color was measured with the ciel*a*b* color system. the δe*, that is, the total difference between two color stimuli, was calculated with the following formula: δe* = √(δl*)² + (δa*)² + (δb*)². for the color readings, the brackets were supported on a white surface properly standardized for the reading so it was not affected by the background color. for the readings, the brackets were manipulated with clinical tweezers and procedure gloves. the positioning for the reading was always the center of the spectrophotometer, with the incisal side toward the center of the device and the buccal surface upward, and it was determined by tagging the bracket to standardize the position for color reading in the different axes (l*a*b*). the l* parameter corresponds to the value or degree of lightness or brightness and a*b* values of chroma, where +a* is red and -a* is green, +b* is yellow and -b* is blue. this process was performed once per bracket and time, always repositioning the bracket in the center of the device. figure 2. color reading analysis. 5 barros et al. statistical analysis the exploratory analysis showed the data do not meet the assumptions of an analysis of variance. then, generalized linear models were adjusted considering the outline of measures repeated in time for l*, a*, and b* values. the variation data (deltas) were analyzed with kruskal wallis and dunn’s non-parametric tests. the analyses were performed in the r and sas software (institute inc., cary, nc, usa, release 9.3, 2011) at 5% significance level. the numerical data were analyzed with the kruskal wallis test at p<0.05. the difference of final values observed for l*, a* and b* of each sample from their initial values was considered a variable. a non-parametric test was selected for the statistical analysis of the data observed in the experiment. results there was a statistically significant decrease in the l* value after the immersion in all solutions and distilled water (p= 0.0002). the highest l* value was observed in the groups with distilled water and chlorhexidine (p<0.05) and the lowest values in the group with chlorhexidine associated with wine (p<0.05). as for the a* values, there was a significant increase after the immersion in all solutions and distilled water (p<0.0001). at the final time, the highest values were observed for the groups that received wine and chlorhexidine associated with wine (p<0.05), and the lowest values were observed for distilled water and chlorhexidine (p<0.05). the b* value also showed a significant increase at the final time relative to the initial time in all groups (p<0.0001). at the final time, the highest values were observed for instant coffee and the lowest values for distilled water, chlorhexidine, and wine (p<0.05). table 1 presents the analysis of variation for l*, a*, and b* values (δl*, δa*, and δb*) and total color variation (δe*). it is observed that when immersed in chlorhexidine associated with wine, the brackets presented greater variations in the l* value than when immersed in chlorhexidine and distilled water (p<0.0001). except for the brackets immersed in chlorhexidine, all the other groups presented greater variation of table 1. median (minimum and maximum values) of the variation of l*, a*, and b* (delta l, delta a, and delta b) and total color (delta e) values according to solution, at the final time relative to the initial time (tfinal-t0). solution delta l* delta a* delta b* delta e* distilled water -2.51 (-8.79; -0.99) a 0.18 (0.13; 0.29) bc 1.28 (0.03; 2.74) c 2.89 (1.00; 8.85) c 0.12% chlorhexidine -3.47 (-4.93; -2.83) ab 0.16 (0.01; 0.29) c 1.67 (1.15; 2.49) bc 3.90 (3.25; 5.06) c instant coffee -6.23 (-11.50; -2.60) bc 1.06 (0.68; 2.42) a 7.87 (4.30; 11.33) a 9.91 (5.07; 16.32) b red wine -7.16 (-11.76; -5.81) c 2.09 (0.77; 2.76) a 2.02 (0.86; 5.29) bc 7.89 (6.40; 13.09) b chlorhexidine and coffee -6.24 (-9.42; -3.93) bc 1.02 (0.47; 2.14) ab 6.55 (3.54; 8.97) a 9.31 (5.31; 12.95) b chlorhexidine and wine -14.32 (-19.86; -4.12) c 2.13 (0.83; 3.06) a 6.00 (0.81; 7.58) ab 15.68 (4.28; 21.32) a medians followed by different letters vertically differed from each other (≤0.05). 6 barros et al. the a* value when compared with the control group, which received distilled water (p<0.05), according to table 1. the groups that received instant coffee, chlorhexidine associated with coffee, and chlorhexidine associated with wine presented greater variation in the b* value than the control group (p=0.0007). table 1 also shows that δe* (color variation) was greater in the group that received chlorhexidine associated with wine and lower in the groups that received distilled water and chlorhexidine (p<0.05). discussion in this study, the null hypothesis was rejected because that chlorhexidine potentiates staining when associated with dye substances, and chlorhexidine associated with red wine promotes greater color change than the other substances and associations tested. in dentistry, the antibacterial agents can perform a bactericidal action that is rather important for controlling bacterial colonies, thus safely promoting anticariogenic benefits. in this context, the current gold standard of antibacterial agent is chlorhexidine, because it is a cationic detergent from the class of bisbiguanides and available in the forms of acetate, hydrochloride, and gluconate. the latter is the salt most commonly used in formulas and products20,21. however, chlorhexidine has some undesirable effects such as the appearance of extrinsic coloration in tooth enamel. in the present study, the polycrystalline ceramic brackets were subjected to treatments with chlorhexidine associated or not with dye substances, which were previously used in studies on staining9,10. the spectrophotometer measures a wavelength by time from the reflective property or object transfer22. this study assessed the samples immersed in staining solution for three minutes under agitation, once a day for five days of the week, during 28 days, according to the protocol used by godoi et al.16 (2011). these 28 days represent clinically 4 indications of use of this mouthwash for 5 days. it is noteworthy that these 4 indications are present within the average clinical time of orthodontic treatment which is approximately 1.5 years. the l*, a*, and b* values were used for each bracket, in which l* corresponds to luminosity and a* and b* correspond to chromaticity. if l* is positive, the color will change toward white and if negative, the color will change toward black. positive a* changes toward red and negative a* toward green, while positive b* changes toward yellow and negative b* toward blue. color change is obtained with the following calculus: δe*=[(δl*)2 + (δa*)2 + (δb*)2]½22. the δe* value considers the total color change relative to the l*, a*, and b* axes. according to a previous study, if δe*<1.0, color change is nor visibly perceptible; if 1<δe*<3.3, color change is considered clinically acceptable; and if δe*>3.3, color change has clinical significance23. several hypotheses were investigated for the mechanisms associated with staining. this finding may be explained by the fact that the cationic molecule of chlorhexidine is rapidly attracted by the negative load of the cell membrane and it is adsorbed to the cell membrane by electrostatic interactions, potentially by hydrophobic ligation or hydrogen bridges. such adsorption is concentration-dependent. thus, at high doses, it causes the precipitation and coagulation of cytoplasmic proteins and bacterial death. at low doses, the integrity of the cell membrane changes, resulting in an over7 barros et al. flow of the bacterial components of low molecular weight24. this study showed that among the δe* (color variation) results, the group of chlorhexidine associated with wine presented statistical difference when compared with other experimental groups. the explanation of the potentiation of chlorhexidine staining when associated with chromogenic foods may lie in the fact that cationic mouthwashes such as chlorhexidine may precipitate or ligate to anionic dyes contained in foods and beverages25. when considering the δl*, the groups of distilled water and chlorhexidine did not show statistically significant difference (p<0.05). despite the little influence on color change, water seems to change the brightness of brackets, even though such change is not significant, because the color change is considered clinically acceptable12. the groups of chlorhexidine and chlorhexidine associated with coffee did not present statistical difference in value when compared with the control group that received distilled water (p<0.05). groups of instant coffee, red wine, and chlorhexidine associated with red wine were likely to increase the red color with greater variation of the a* value, which may occur due to the excessive consumption of red wine, causing extrinsic pigmentation due to the high content of tannin compounds that are highly denaturing26. similarly, a previous study showed higher staining potential in red wine, because its composition contains a high concentration of pigments and it presents low ph and a reasonable amount of alcohol27. the groups that received instant coffee, chlorhexidine associated with coffee, and chlorhexidine associated with wine presented greater variation for the b* value than the control group (p<0.05) and they were likely to increase the yellow color, which agrees with a previous study that observed that the yellow pigment in coffee is less polar and therefore less hydrophilic. in studies with resin composites, the discoloration by coffee is processed by absorption and adsorption of polar dyes, potentially due to the compatibility of the organic phase of the polymer with this specific dye28, and the staining increased when the surface of the specimen tested was subjected to the use of chlorhexidine. the group chlorhexidine associated with red wine has potentiated the effect of the staining in relation to the other substances, because the red wine has in its composition a tannin, a polyphenol, with high molecular weight, generally between 500 and 3000 daltons, soluble in water and in addition, may still be favored by the use of cationic antiseptics such as chlorhexidine29, precipitating or bind to anionic coloring agents contained in foods and beverages30. in studies with ceramic brackets, in which the major component is inorganic, the literature has not yet explained the interaction among dyes from solutions and the components of such materials. hence, the development of further studies in this field would be important. whitin this, it is concluded that chlorhexidine potentiates the staining caused by red wine in polycrystalline ceramic brackets. references 1. lee y-k, bin y. translucency and color match with a shade guide of esthetic brackets with the aid of a spectroradiometer. dent press j orthod. 2016 mar-apr;21(2):81-7. doi: 10.1590/2177-6709.21.2.081-087.oar. 8 barros et al. 2. dos santos pr, meneghim mc, ambrosano gm, vedovello filho m, vedovello sa influence of quality of life, self-perception, and self-esteem on orthodontic treatment need. am j orthod dentofacial orthop. 2017 jan;151(1):143-7. doi: 10.1016/j.ajodo.2016.06.028. 3. gkantidis n, zinelis s, karamolegkou m, eliades t, topouzelis n. comparative assessment of clinical performance of esthetic bracket materials. angle orthod. 2012 jul;82(4):691-7. doi: 10.2319/092511-605. 4. kukiattrakoon b, samruajbenjakul b. shear bond strength of ceramic brackets with various base designs bonded to aluminous and fluorapatite ceramics.eur j orthod. 2010 feb;32(1):87-93. 5. alexopoulou e, polychronis g, konstantonis d, sifakakis i, zinelis s, eliades t. a study of the mechanical properties of as-received and intraorally exposed single-crystal and polycrystalline orthodontic ceramic brackets. eur j orthod. 2019 apr 23. pii: cjz024. doi: 10.1093/ejo/cjz024. 6. maltagliati la, feres r, figueiredo ma, siqueira df. [aesthetic brackets: clinical considerations] rev clín ortod dental press. 2006 jun-jul;5(3):89-95. portuguese. 7. karamouzos a, athanasiou ae, papadopoulos ma. clinical characteristics and properties of ceramic brackets: a comprehensive review. am j orthod dentofacial orthop. 1997 jul;112(1):34-40. 8. oliveira cb, maia lg, santos-pinto a, gandini júnior lg. in vitro study of color stability of polycrystalline and monocrystalline ceramic brackets. dental press j orthod. 2014 jul-aug;19(4):114-21. 9. guignone bc, silva lk, soares rv, akaki e, goiato mc, pithon mm, et al. color stability of ceramic brackets immersed in potentially staining solutions. dental press j orthod. 2015 jul-aug;20(4):32-8. doi: 10.1590/2176-9451.20.4.032-038.oar. 10. lima filho hl, maia lh, araújo mv, eliast cn, ruellas ac. colour stability of aesthetic brackets: ceramic and plastic. aust orthod j. 2013 may;29(1):13-20. 11. vivek aithal pr, akshai shetty kr, dinesh mr, amarnath bc, prashanth cs, roopak md. in vitro evaluation of microbial contamination and the disinfecting efficacy of chlorhexidine on orthodontic brackets. prog orthod. 2019 may 1;20(1):17. doi: 10.1186/s40510-019-0270-4. 12. mendonça mr, fabre af, goiatto mc, cuoghi oa, martins lp, verri acg. spectrophotometric evaluation of color changes of esthetic brackets stored in potentially staining solutions. rpg rev pos grad. 2011;18(1):20-7.  13. gürdal p, akdeniz bg, hakan sen b. the effects of mouthrinses on microhardness and colour stability of aesthetic restorative materials. j oral rehabil 2002 sep;29(9):895-901. 14. mathur s, tanu m, rahul s. chlorhexidine: the gold standard in chemical plaque control. natl j physiol pharm pharmacol. 2011;1(2):45-50. 15. colucci v, dos santos cd, amaral fl, corona sa, catirse ab. influence of nahco3 powder on translucency of microfilled composite resin immersed in different mouthrinses. j esthet restor dent. 2009;21(4):242-8. doi: 10.1111/j.1708-8240.2009.00269.x. 16. godoi apt, freitas db, trauth kgs, colucci v, catirse ab. combined effect of the association between chlorhexidine and a diet protein on color stability of resin composites. int j clin dent. 2011 jan;4(3):113-121. 17. nagem filho h, d’azevedo mtfs, nagem hd, marsola fp. surface roughness of composite resins after finishing and polishing. braz dent j 2003;14(1):37-41. 18. brandão rb, martin c, catirse abce, de castro e silva m, evison mp, guimarães ma. heat induced changes to dental resin composites: a reference in forensic investigations? j forensic sci. 2007 jul;52(4):913-9. 9 barros et al. 19. cui t, luo w, xu l, yang b, zhao w, cang h. progress of antimicrobial discovery against the major cariogenic pathogen streptococcus mutans. curr issues mol biol. 2019 jun 5;32:601-44. doi: 10.21775/cimb.032.601. 20. zanatta fb, rosing ck. chlorhexidine: action’s mechanisms and recent evidences of its efficacy over supragingival biofilm context. scientific-a 2007;1(2):35-43. 21. joiner a. tooth colour: a review of the literature. j dent. 2004;32(suppl 1):3-12. 22. guignone bc, silva lk, soares rv, akaki e, goiato mc, pithon mm, et al. color stability of ceramic brackets immersed in potentially staining solutions. dental press j orthod. 2015 jul-aug;20(4):32-8. doi: 10.1590/2176-9451.20.4.032-038.oar. 23. hjeljord lg, rolla g, bonesvoll p. chlorhexidine-protein interactions. j periodontal res suppl. 1973;12:11-6. 24. leard a, addy m. the propensity of different brands of tea and coffee to cause staining associated with chlorhexidine. j clin periodontol. 1997 feb;24(2):115-8.  25. norbdo h. discoloration of dental pellicle by tannic acid. acta odontol scand. 1977;35(6):305-10. 26. bagheri r, burrow mf, tyas m. influence of food-simulating solutions and surface finish on susceptibility to staining of aesthetic restorative materials. j dent. 2005 may;33(5):389-98. 27. um cm, ruyter ie. staining of resin-based veneering materials with coffee and tea. quintessence int. 1991 may;22(5):377-86. 28. pontefract h, courtney m, newcombe rg, addy m. development of methods to enhace extrinsic tooth discoloration for comparison of toothpaste. 1. studies in vitro. j clin periodontol. 2004 jan;31(1):1-6. 29. lee yk, powers jm. combined effect of staining substances on the discoloration of esthetic class v dental restorative materials. j mater sci mater med. 2007 jan;18(1):165-70. 30. shree roja rj, sriman n, prabhakar v, minu k, subha a, ambalavanan p. comparative evaluation of color stability of three composite resins in mouthrinse: an in vitro study. j conserv dent. 2019 mar-apr;22(2):175-80. doi: 10.4103/jcd.jcd_241_18. untitled 1http://dx.doi.org/10.20396/bjos.v16i0.8651063 volume 16 2017 e17035 original article a phd student, faculdade de odontologia de araraquara, são paulo state university (unesp), araraquara, sp, brazil. b associate professor, faculdade de odontologia de araraquara, são paulo state university (unesp), araraquara, sp, brazil, department of orthodontics and pediatric dentistry. c assistant professor, faculdade de odontologia de araraquara, são paulo state university (unesp), araraquara, sp, brazil, department of orthodontics and pediatric dentistry. corresponding author: ana carolina de oliveira becci email: acbecci@gmail.com faculdade de odontologia de araraquara unesp rua humaitá nº 1680 araraquara, sp, brasil. cep: 14801-903 tel: + 55 16 33016336 fax: + 55 16 33016329 received: april 19, 2017 accepted: september 14, 2017 long-term influence of associating an antibacterial agent with gic on bond strength to caries-affected dentin ana carolina de oliveira beccia, luana mafra martia, angela cristina cilense zuanonb, fernanda lourenção brighentic, elisa maria aparecida girob abstract aim: to evaluate the bond strength of a gic associated with chlorhexidine (chx) to sound and caries-affected dentin, immediately and after six months of storage. methods: sixty molars were assigned to two groups of 30 teeth. one had flat dentin surfaces produced and submitted to caries induction to obtain a caries-affected dentin. in the other group dentin was maintained sound. teeth of each group were randomly reassigned to three subgroups (n=10) according to the concentration of chx added to the gic (0%, 1% and 2% by weight). two specimens (1mm diameter x 1 mm high) of the same material were constructed on each dentin surface. one was submitted to the microshear bond strength (µsbs) test after 24 hours and the other after 6 months of storage in water at 37oc. failure modes were analyzed under a stereomicroscope. bond strength data were analyzed by three-way anova followed by games-howell tests for multiple comparisons, and failure modes by the chi-square test (α = 0.05). results: the µsbs values obtained to sound dentin were higher compared with those to caries-affected dentin (p≤0.001). in sound dentin, the group with 2% chx showed lower µsbs values compared with 0% and 1% chx after 24 hours (p=0.005 and p=0.032 respectively). in caries-affected dentin, after 24 hours, µsbs in group with 1% chx was statistically higher than the values in groups with 2% chx after 24 hours (p=0.001) and 1% chx after 6 months (p=0.024). irrespective of the condition of substrate, comparisons showed no statistically significant differences between the other groups (p≥0.053). cohesive in material and mixed failures prevailed for all groups. conclusions: the addition of chx at concentrations of up to 2% to the gic did not affect the bond strength of the material to sound and caries-affected dentin in a long-term evaluation. keywords: glass ionomer cements. chlorhexidine. dentin 2 becci et al. introduction contemporary restorative dentistry concepts characterize a less invasive approach to the treatment of carious lesions1. studies have recommended that infected dentin should be removed and caries-affected dentin should be preserved. these substrates show outstanding differences in their characteristics. infected dentin is defined as the necrotic surface area of a highly demineralized substrate2-4 in which there are degenerated collagen fibrils and presence of a bacterial biomass2,4. the affected dentin, capable of remineralization, is considered a variation of reactive dentin produced in response to soft stimuli such as caries, presenting small changes in the cross-linking of its collagen fibrils2-4. in this scenario, the use of glass ionomer cements (gics) is interesting because of their capacity for releasing ions especially fluoride5-7 that may improve the remineralization of caries-affected dentin5. this feature along with low initial ph of gics gives them their antibacterial property. the release of ions from gics, however, decreases rapidly over time making it interesting to associate this material with antimicrobial compounds to reduce recurrent caries at restoration margins; inhibit biofilm formation on the restored surface; reduce the number of microorganisms in oral cavity fluids, and inhibit microbial growth under the restoration8,9. previous in vitro studies have shown that the incorporation of chlorhexidine (chx) into gics reduces streptococcus mutans, lactobacillus spp, candida albicans and actinomyces naeslundii8-11. however, its antibacterial effect is concentration-dependent and in high concentrations chx may interfere with the mechanical properties of gic10. as the longevity of tooth restorations is partly related to mechanical properties, among them a good bond to tooth structures, the antibacterial agent should be added at a concentration that will not impair these properties of gics11,12. to date, there are few studies that have investigated how gic bonds are affected by time13,14 or by the presence of caries-affected dentin1,11. moreover, there are no studies that demonstrate the influence of storage time and the type of substrate on gics with incorporation of the chx concentrations that were used in this study. thus, the purpose of this study was to evaluate the bond strength of a gic associated with chx to sound and caries-affected dentin, immediately and after six months of storage. the null hypotheses were that the bond strength of the gic to dentin would not be altered by the condition of the substrate, concentration of chx associated with gic, or storage time of the specimens. material and methods the experimental procedure flowchart is shown in figure 1. sample size was estimated using the pspower and sample size program software, version 3.0.43. the microshear bond strength values obtained in a pilot study were normally distributed with standard deviation 1.4 and the true difference between the experimental and control group mean values was 2.1. to be able to reject the null hypothesis with a power of 80% and a type i error probability of 0.05 it was necessary to have at least 8 specimens in each group. considering possible losses during the experiment, 10 specimens were used in each group. 3 becci et al. after approval by the research ethics committee (protocol 68.388), sixty sound extracted human third molars were obtained from the tooth bank of the araraquara school of dentistry unesp. teeth without anatomical and structural defects were selected and a flat surface was produced in dentin by sectioning the teeth at the level of the occlusal third of the crown; by means of a diamond disc (no.11-4254, buehler ltd., lake bluff, il, usa) mounted in a metallographic cutter (isomet 1000, buehler ltd., lake bluff, il, usa). the teeth were divided into two groups of 30 teeth using simple random probabilistic sampling by chance. in one group they were submitted to artificial caries induction, and in the other dentin was maintained sound. the teeth allocated to the group with caries-affected dentin were sealed with two layers of acid resistant enamel, leaving only the dentin surface exposed. the specimens were sterilized with ethylene oxide, and then suspended in a cariogenic solution (bhi broth supplemented with 2% sucrose, 1% glucose and 0.5% yeast extract; 25 ml/tooth) inoculated with 105 cfu/ml of streptoccocus mutans atcc 25175 (tropical culture collection – andré toselo foundation) (figure 2a). the set was incubated under microaerophilic conditions at 37 ºc for 14 days. the cariogenic solution was changed every 48 hours, without inoculating new microorganisms. after this, the biofilm formed (figure 2b) was removed, teeth were washed in deionized water. the resulting dentin surface was found to be darkened and softened when touched with a sharp explorer without pressure. subsequently, the softened carious dentin (infected dentin) was manually removed using 320-grit silicon carbide abrasive paper under running water, until a touch resistant dentin (caries-affected dentin) was obtained15 (figure 2c). the teeth with dentin surfaces that were maintained sound were also worn with the same type of abrasive paper, in an attempt to obtain a dentin depth similar to that of the caries-affected teeth. the teeth were cleaned in an ultrasonic bath. afterwards, they were embedded in self-polymerizing acrylic resin, by using a cylindrical pvc tube (20 mm x 18 mm) as matrix, so that the dentin surface would be centralized and parallel to the base of the tube (figure 2d). finally, the teeth of each group (sound dentin and caries-affected dentin) were reassigned, using simple random probabilistic sampling by chance, to three subgroups (n=10) according to the chlorhexidine diacetate (sigma aldrish, steinheim, germany) concentration (0%, 1% and 2% by weight) added to the glass ionomer 0% chx (10 teeth; 20 specimens) 1% chx (10 teeth; 20 specimens) 2% chx (10 teeth; 20 specimens) 0% chx (10 teeth; 20 specimens) 1% chx (10 teeth; 20 specimens) 2% chx (10 teeth; 20 specimens) sound dentin (30 teeth) caries affected dentin (30 teeth) 60 sounds third molars 10 specimens from each group were analyzed after 24 hours, and the remaining 10 specimens after 6 months of storage in distilled water at 37°c figure. flowchart of the distribution of groups 4 becci et al. cement (ketac molar easymix, 3m-espe dental products, st. paul, mn, usa). two specimens (1mm diameter x 1 mm high) of the same material were prepared on the dentin surface of each tooth. briefly, the bond area was delimited with double-faced acid resistant adhesive tape (3m brazil, sumaré, sp, brazil) with a perforation measuring 1.0 mm in diameter (figure 2e). ketac molar liquid (3m – espe dental products, st. paul, mn, usa) was applied to dentin for 10 seconds (figure 2f), washed with a jet of water-air for 10 seconds and the dentin surface was dried with cotton wool balls. a cylindrical silicone matrix (embramed, são paulo, sp, brazil) with an orifice 1 mm in diameter and 1 mm high was placed so that its internal diameter would coincide with the delimited adhesive area (figure 2g). the amount of chx diacetate required to obtain final concentrations of 1% and 2% by weight was determined based on the average weight of a spoonful of the gic powder. chx diacetate was incorporated into the gic powder immediately before manipulating the cement. the powder-liquid weight ratio recommended by the glass ionomer cement manufacturer was maintained. the material was manipulated at a room temperature of 24 ± 1 ºc, in accordance with the manufacturer’s recommendations (3m – espe dental products, st. paul, mn, usa), and then inserted into the matrix with the aid of a centrix syringe (dfl, indústria e comércio s.a, jacarepaguá, rj, brazil) (figure 2h). the tops of the test specimens were protected with vaseline, and they were stored at 37 ºc in 100% humidity for 24 hours, avoiding their direct contact with the humidity. subsequently, the matrixes were removed and the specimens were observed under a stereomicroscope (olympus szx7, olympus corporation, tokyo, japan) at 40x magnification to certify the absence of defects at the bond interface. all the procedures were carried out by a single experience and previously trained operator. figure 2. a: teeth immersed in cariogenic solution. b: tooth after 14 days of immersion in cariogenic solution. c: tooth after removal of infected dentin. d: tooth included in pvc tube with acrylic resin. e: delimitation of the adhesive area with double-sided tape. f: dentin conditioning with gic liquid. g: positioning of the microtubes over the perforations in the adhesive tape. h: insertion of the materials into the microtubes. i: die positioned for the performance of the microshear test 5 becci et al. microshear bond strength test and failure mode analysis the microshear bond strength (µsbs) test was performed in one of the two specimens prepared on each tooth 24 hours after their fabrication, and the other specimen prepared on the same tooth was protected with vaseline and remained stored in distilled water (ph = 6.8-7.4) at 37oc for six months to evaluate the bond strength. during this period, distilled water ph was monitored once a week with the aid of a ph meter (model q400as, quimis aparelhos científicos, diadema, sp, brazil). in case of change in ph, the distilled water was replaced with a fresh amount. a mechanical testing machine (dl-digital line, emic, são josé dos pinhais, pr, brazil), adjusted for tensile forces was used for the tests. a wire 0.2 mm in diameter was looped around the specimen as closely as possible to the interface (figure 2i). force was applied with a load cell of 100 n at a crosshead speed of 0.5 mm/min until failure occurred, and the maximum stress values in megapascal (mpa) withstood by the dentin/material bond were recorded. fractured surfaces were observed under a stereomicroscope (olympus szx7, olympus corporation, tokyo, japan) at 40x magnification. failures were classified as adhesive (at substrate/restorative material interface), cohesive in dentin or in the material (fracture within the dentin or material, respectively), and mixed (partially adhesive and partially cohesive failures). pre-testing failures were recorded, but were not included in the statistical analysis. a single trained examiner, who did not know to which group each test specimen belonged, performed the µsbs test and the failure mode assessments. statistical analysis the bond strength data (in mpa) passed the tests of normality (shapiro wilk; p≥0.056), but the assumption of homoscedasticity was not obeyed (levene, p=0.009). thus, three-way anova was used and multiple comparisons, when necessary, were made by the games-howell post-test for heteroscedastic data. the chi-square test was used to compare failure modes between conditions of substrate, storage periods and materials. all statistical tests were performed by the pasw statistics software (v.22, spss inc, chicago, il) and the level of significance adopted for decision-making was 5%. results the mean (sd) microshear bond strength (µsbs) values are shown in table 1. according to three-way anova, the µsbs values were significantly higher in sound dentin (3.62 ± 1.69 mpa) than in caries-affected dentin (2.52 ± 1.14 mpa) (p≤0.001. storage periods and different concentrations of chx diacetate added to the gic, also had a significant influence on the µsbs values (p=0.002 and p=0.001, respectively), and there was a significant interaction between these two factors (p≤0.036). thus, the multiple comparisons showed that in sound dentin, µsbs of group gic + chx 2% was statistically lower than the values found for groups gic and gic + chx 1% at 24 hours (p=0.005 and p=0.032 respectively). in caries-affected dentin, µsbs in group gic + 1% chx at 24 hours was statistically higher than that observed for groups gic + 2% chx at 24 hours (p=0.001) and gic + 1% chx at 6 months (p=0.024). irrespective of the substrate condition, comparisons showed no statistically significant differences between the other groups (p≥0.053). 6 becci et al. table 2 shows the percentage of failure modes and pre-testing failures. only condition of substrate and storage period had significant influence on the failure modes (p=0.028 and p=0.017 respectively). irrespective of substrate condition and storage period, different concentrations of chx diacetate showed no statistically significant influence on failure modes (p=0.633). the following percentage of failure modes were found: gic and gic + chx 1% (40.0% cohesive in material, 17.5% adhesive and 32.5% mixed), and gic + chx 2% (25.0 % cohesive in material, 22.5% adhesive and 40.0 % mixed) no cohesive failures in dentin were observed. pre-testing failures were more frequent in caries-affected dentin and in the storage period of 6 months, and represented 10% in group gic, 2.5% in group gic + chx 1% and 12.5% in group gic + chx 1%. discussion the findings of this study showed that the bond strength of the gic to dentin was influenced by the condition of the substrate; concentration of chx associated with gic, and storage time of the specimens, so the null hypotheses were rejected. bonding to tooth structure is one of the most important mechanical properties of gics9. several factors may influence bond strength tests: test device; biological substrate; table 1. microshear bond strength (µsbs) of gic to dentin, depending on the chlorhexidine diacetate concentration added and storage period substrate material µsbs (mpa) ϯ storage period 24 hours 6 months sound dentin gic 4.5 ± 1.4a 3.7 ± 1.7ab gic+ chx 1% 4.7 ±1.8a 3.4 ± 1.7ab gic + chx 2% 2.4 ± 0.9b 2.5 ± 1.4ab caries-affected dentin gic 3.3 ± 1.2ab 2.1 ± 1.0ab gic + chx 1% 3.6 ± 0.7a 1.9 ± 1.1b gic + chx 2% 2.0 ± 0.6b 2.1 ± 1.2ab ϯvalues correspond to mean and standard deviation different superscript capital letters(a,b) denote statistically significant differences between groups for sound dentin (games howell test; p≤0.032) and different superscript lowercase letters(a,b) denote statistically significant differences between groups for caries-affected dentin (games howell test; p≤0.024). table 2. number and percentage of specimens (%) in according with failure mode, considering substrate conditions, storage period and concentration of chlorhexidine diacetate added to the gic failure modes cohesive in material adhesive mixed pre-testing p value* substrate sound dentin 17(28.33) 17(28.33) 26(43.33) 0(0.00) 0.028 caries-affected dentin 27(45.00) 7(11.67) 19(31.67) 7(11.67) storage period 24 hours 16(26.67) 15(25.00) 29(48.33) 0(0.00) 0.017 6 months 28(46.67) 9(15.00) 16(26.67) 7(11.67) material gic 16(40.00) 7(17.50) 13(32.50) 4(10.00) 0.633gic + chx 1% 16(40.00) 7(17.50) 16(40.00) 1(2.50) gic + chx 2% 10(25.00) 9(25.71) 16(40.00) 5(12.50) * chi-square test results of failure mode proportions (significant difference: p<0.05). 7 becci et al. position of the specimens on the tooth, and storage time. in this study, the microshear bond strength test was performed, in which the area of the specimens is reduced and there is no great need to manipulate them during their preparation as there is in the microtensile bond strength test16. the bond of gics to caries-affected dentin is more complicated than to sound dentin because of its porosity and the presence of lactic acid involved in the caries lesion17,18 . several studies have supported the use of artificially induced carious dentin to test new materials and techniques14,15,19, because it is difficult to standardize natural dentinal caries, since there are structural differences within the different carious zones. the size and shape of the naturally formed carious lesion are also characteristics that make it difficult to form a standardized flat substrate surface for bond strength tests. in addition, criteria commonly used to guide excavation of the lesion, such as color and tactile hardness, are subjective19. in this study, the microbiological method of caries induction was applied to produce caries-affected dentin because lesions in dentin seem to have a molecular and structural arrangement that is more similar to that of natural lesions21. regarding storage time, in the present study only the group containing chx 1% in period of 24 hours had µsbs values that were statistically significant and higher than those of group gic + chx 1% after the 6-month period when the bond was produced in caries affected dentin. colluci et al.13 (2014) analyzing the long-term water storage of gic specimens observed a reduction in shear bond strength only when dentin surfaces were prepared with rotatory instruments. on the other hand, azevedo et al.21 (2011) observed that after specimens were stored in water for 90 days, the bond strength improved, which was attributed to the acid-base reaction that occurs in a slow and continuous manner. as observed in the present study, group gic + 2% chx, showed a decrease in the bonding capacity to sound dentin in the 24-hour period compared with the control group and gic + 1% chx. takahashi et al.22 (2006) also observed that in sound dentin the addition of chx in concentration of 2% or higher caused a significant reduction in dentin bond strength after 24 hours of storage. other antibacterial agents such as cetrimide, ciprofloxacin, metronidazole and minocycline associated with gics at high concentrations have also shown decrease in bonding capacity of these materials23,24. these results can be explained by the fact that a higher concentration of the antibacterial agent may interfere with the reaction between glass particles and liquid of the cement, thereby increasing the number of unreacted particles in the structure and reducing the mechanical properties24. for bond strength tests, it has also been hypothesized that antibacterial agents may affect the polar and ionic attraction force between carboxyl groups and inorganic ions in the dentin. in a previous study becci et al.25 (2014) compared the bond strength of a gic with different concentrations of chx to sound and caries-affected dentin only in the period of 24 hours. although in both the cited study and the present study, the infected dentin was manually removed using 320-grit silicon carbide abrasive, the authors showed that the condition of the substrate had no influence on the immediate bond strength values. this was attributed to the excessive removal of carious dentin resulting in a 8 becci et al. caries-affected dentin with characteristics very close to those of sound dentin, and the loss of calcium ions was insufficient to determine bonding differences in the cited study. in the present study, the carious dentin was removed more carefully, taking care not to wear too much caries-affected dentin. the results showed that the bond strength values of the gic to this dentine were statistically lower than those to sound dentin. no other studies using caries-affected dentin and antibacterial agents associated with gics were found so that no comparison with this study could be made. the literature has described that specimen storage in water at 37 oc decreased the bond strength because water degrades the restorative material/dentin interface. an interesting finding of the present study was that despite the remarkably low bond strength values found for group gic + 2% chx, the bond strength to caries-affected dentin did not decrease after 6 months, as it did for groups gic and gic + 1% chx. this is an important result, since 6 months is a considerable period of the gic restoration permanence on caries-affected dentin, depending on the clinical conditions found in the mouth. the addition of chlorhexidine to the material is particularly important when performing bonding to caries-affected dentin since chlorhexidine is an inhibitor of metalloproteinases and cysteine cathepsins26. thus, addition of 2% chx could be considered a promising way to preserve the bond interface and increase the bond durability in clinical practice, as previously suggested14,27. with respect to the failure modes, cohesive in material and mixed failures were predominant for all groups. the high frequency of cohesive failures within material were related to the low resistance of the tested material itself rather than its true bond strength to dentin16,29, and in the majority of instances this did not represent the real bond strength of the material to dentin29. a higher tendency towards cohesive fractures may also occur because of numerous porosities in the structure of the material, which may act as stress points30. in the present study, cohesive defects in material were observed in 25% to 40 % of the specimens, and mixed fractures were present in 32.5% to 40 % of the specimens for all materials irrespective of the substrate and storage time. in summary, the current study demonstrated that time and substrate condition were important factors that should be considered when using gic associated with antibacterial agents. furthermore, although the addition of 2% chx affected the early bond strength, it seems to have protected the caries-affected dentin/ gic bond during the storage period in water for 6 months. although the association of chx with gics seemed to be a promising alternative to increase their anticariogenic properties, further studies evaluating the physicochemical and antibacterial properties should be conducted with the purpose of determining the optimal concentration that would provide better antibacterial power and less degradation of the material over time. acknowledgements the authors acknowledge the financial support provided by the brazilian federal agency for the support and evaluation of graduate education 9 becci et al. references 1. alves fb, hesse d, lenzi tl, guglielmi cde a, reis a, loguercio ad, et al. the bonding of glass ionomer cements to caries-affected primary tooth dentin. pediatr dent. 2013 jul-aug;35(4):3204. 2. banerjee a, kellow s, mannocci f, cook rj, watson tf. an in vitro evaluation of microtensile bond strengths of two adhesive bonding agents to residual dentine after caries removal using three excavation techniques. j dent. 2010 jun;38(6):480-9. 3. almeida neves a, coutinho e, cardoso mv, lambrechts p, van meerbeek b. current concepts and techniques for caries excavation and adhesion to residual dentin. j adhes dent. 2011 feb;13(1):7-22. 4. costa ar, garcia-godoy f, correr-sobrinho l, naves lz, raposo lh, carvalho fg, et al. influence of different dentin substrate (caries-affected, caries-infected, sound) on long-term μtbs. braz dent j. 2017 jan-feb;28(1):16-23. 5. ngo h. glass-ionomer cements as restorative and preventive materials. dent clin north am. 2010 jul;54(3):551-63. 6. türkün ls, türkün m, ertuğrul f, ateş m, brugger s. long-term antibacterial effects and physical properties of a chlorhexidine-containing glass ionomer cement. j esthet restor dent. 2008;20(1):29-44. 7. tüzüner t, kuşgöz a, er k, taşdemir t, buruk k, kemer b. antibacterial activity and physical properties of conventional glass-ionomer cements containing chlorhexidine diacetate/cetrimide mixtures. j esthet restor dent. 2011 feb;23(1):46-55. 8. hoszek a, ericson d. in vitro fluoride release and the antibacterial effect of glass ionomers containing chlorhexidine gluconate. oper dent. 2008 nov-dec;33(6):696-701. 9. tyas mj, burrow mf. adhesive restorative materials: a review. aust dent j. 2004 sep;49(3):112-21. 10. deepalakshmi m, poorni s, miglani r, rajamani i, ramachandran s. indian. evaluation of the antibacterial and physical properties of glass ionomer cements containing chlorhexidine and cetrimide: an in-vitro study. j dent res. 2010 oct-dec;21(4):552-6 11. tenuta lm, ribeiro cc, goncalves nc, del bel cury aa, aires cp, tengan c, et al. the shortterm in situ model to evaluate the anticariogenic potential of ionomeric materials. j dent. 2005 jul;33(6):491-7. 12. reis kr, spyrides gm, oliveira ja, jnoub aa, dias kr, bonfantes g. effect of cement type and water storage time on the push-out bond strength of a glass fiber post. braz dent j. 2011;22(5):359-64. 13. colucci v, de araújo loiola ab, da motta ds, do amaral fl, pécora jd, corona sa. influence of longterm water storage and thermocycling on shear bond strength of glass-ionomer cement to er:yag laser-prepared dentin. j adhes dent. 2014 feb;16(1):35-9. 14. sanabe me, costa ca, hebling j. exposed collagen in aged resin-dentin bonds produced on sound and caries-affected dentin in the presence of chlorhexidine. j adhes dent. 2011 apr;13(2):117-24. 15. ricci ha, scheffel dl, de souza costa ca, dos santos fj, jafelicci m jr, hebling j. wettability of chlorhexidine treated non-carious and caries-affected dentin. aust dent j. 2014 mar;59(1):37-42. 16. bonifácio cc, shimaoka am, de andrade ap, raggio dp, van amerongen we, de carvalho rc. micromechanical bond strength tests for the assessment of the adhesion of gic to dentine. acta odontol scand. 2012 dec;70(6):555-63. 17. czarnecka b, deregowska-nosowicz p, limanowska-shaw h, nicholson jw. shear bond strengths of glass-ionomer cements to sound and to prepared carious dentine. j mater sci: mater med. 2007 may;18(5):845-9. 18. nicholson jw, aggarwal a, czarnecka b, limanowska-shaw h. the rate of change of ph of lactic acid exposed to glass-ionomer dental cements. biomaterials. 2000 oct;21(19):1989-93. 10 becci et al. 19. lenzi tl, tedesco tk, calvo af, ricci ha, hebling j, raggio dp. does the method of caries induction influence the bond strength to dentin of primary teeth? j adhes dent. 2014 aug;16(4):333-8. 20. pacheco lf, banzi ev, rodrigues e, soares le, pascon fm, correr-sobrinho l, et al. molecular and structural evaluation of dentin caries-like lesions produced by different artificial models. braz dent j. 2013 nov-dec;24(6):610-8. 21. azevedo er, coldebella cr, zuanon ac. effect of ultrasonic excitation on the microtensile bond strength of glass ionomer cements to dentin after different water storage times. ultrasound med biol. 2011 dec;37(12):2133-8. 22. takahashi y, imazato s, kaneshiro av, ebisu s, frencken je, tay fr. antibacterial effects and physical properties of glass-ionomer cements containing chlorhexidine for the art approach. dent mat. 2006 jul;22(7):647-52. 23. prabhakar ar, prahlad d, kumar sr. antibacterial activity, fluoride release, and physical properties of an antibiotic-modified glass ionomer cement. pediatr dent. 2013 sep-oct;35(5):411-5. 24. yesilyurt c, er k, tasdemir t, buruk k, celik d. antibacterial activity and physical properties of glassionomer cements containing antibiotics. oper dent. 2009 janfeb;34(1):18-23. 25. becci aco, marti lmm, zuanon acc, brighenti fl, giro ema. influence of the addition of chlorhexidine diacetate on bond strength of a high-viscosity glass ionomer cement to sound and artificial caries-affected dentin. rev odontol unesp. 2014;43(1):1-7. 26. hebling j, pashley dh, tjäderhane l, tay fr. chlorhexidine arrests subclinical degradation of dentin hybrid layers in vivo. j dent res. 2005 aug;84(8):741-6. 27. tjäderhane l, nascimento fd, breschi l, mazzoni a, tersariol il, geraldeli s, et al. strategies to prevent hydrolytic degradation of the hybrid layer-a review. dent mater. 2013;29(10):999-1011. 28. peez r, frank s. the physical-mechanical performance of the new ketac molar easymix compared to commercially available glass ionomer restoratives. j dent. 2006 sep;34(8):582-7. 29. choi k, oshida y, platt ja, cochran ma, matis ba, yi k. microtensile bond strength of glass ionomer cements to artificially created carious dentin. dent. 2006 sep-oct;31(5):590-7. 30. hoshika s, de munck j, sano h, sidhu sk, van meerbeek b. effect of conditioning and aging on the bond strength and interfacial morphology of glass-ionomer cement bonded to dentin. j adhes dent. 2015;17(2):141-6. 1http://dx.doi.org/10.20396/bjos.v20i00.8659022 volume 20 2021 e219022 original article 1 department of restorative dentistry, operative dentistry division, piracicaba dental school, university of campinas. corresponding author: janaina emanuela damasceno department of restorative dentistry, piracicaba dental school, university of campinas av. limeira, 901 13414-903 piracicaba sp brazil e-mail: janaina.damasceno. santos@gmail.com received: july 6, 2020 accepted: march 03, 2021 editor: dr. altair a. del bel cury conservative treatment of interproximal incipient caries lesions by resin infiltration priscila regis pedreira1 , janaina emanuela damasceno1,* , ana ferreira souza1 , gabriela alves de cerqueira1, flávio henrique baggio aguiar1 , giselle maria marchi1 minimally invasive dentistry is based on conservative techniques for the treatment of initial caries lesions, the so-called white spot lesions. one of the conservative maneuvers includes the use of enamel resin infiltrant: a low viscosity material that penetrates the enamel pores to stop lesion progression. aim: therefore, this case report aimed to describe the clinical case of a 28-year-old female patient under routine consultation, in which a radiographic examination showed the presence of two incipient caries lesions in the left upper premolars. methods: the application of a resin infiltrating agent (icon®, dmg – hamburg, germany) was chosen as treatment to stop caries lesions progression. conclusion: the use of this conservative technique can be considered a promising approach for the prevention of dental tissue wear, and the resin infiltrant is considered an effective material. keywords: dental caries. conservative treatment. composite resins. mailto:janaina.damasceno.santos@gmail.com mailto:janaina.damasceno.santos@gmail.com https://orcid.org/0000-0003-4398-9085 https://orcid.org/0000-0003-2656-2434 https://orcid.org/0000-0002-7961-4886 https://orcid.org/0000-0003-3389-5536 https://orcid.org/0000-0002-0945-1305 2 pedreira et al. introduction behavioral habits, such as maintenance of oral hygiene and dietary control, are effective in the process of controlling caries disease progression, in addition to local fluoridation maneuvers1,2. however, despite their effectiveness, these maneuvers are more strongly indicated for lesions restricted to enamel in patients with less risk of caries, due to the great dependence on collaboration on the part of these1,2. minimally invasive dentistry recommends early intervention approaches in the initial manifestations of caries disease to inhibit its progress, as well as acting preventively, aiming at the treatment of signs of the disease with maximum preservation of dental tissue3. dental caries is a multifactorial disease that has as dependent factors a diet rich in carbohydrates, in addition to the susceptibility of the host and the presence of a cariogenic microbial flora4. the process of constant reductions in oral ph disrupts the demineralization-remineralization cycle (de-re), which constantly occurs in a balanced way in an oral health situation5. acid by-products generated by bacterial activity cause localized destruction of dental tissues as they penetrate and diffuse through tissues, and, if this process is not controlled, it progresses to the first clinically visible manifestation of caries disease: white spot lesions6. these lesions appear morphologically with an apparently intact external surface, followed by a demineralized subsurface6. it begins with a small superficial demineralization and, with the advance of the etiological process, the acids reach the subsurface layer, while the external surface remains “intact”6. within the philosophy of conservative treatment, materials such as fissure sealants are used successfully, being indicated for application on newly erupted molars of young patients, due to the retentive anatomy of these teeth, which favors the bacterial accumulation on the occlusal surface7,8. a limitation of these materials, however, are interproximal caries lesions, which do not present any form of retention for sealants. before this issue, the use of resin infiltrants has been proposed in dentistry9. infiltrants are resinous materials of low viscosity, indicated for application in initial caries lesions located on flat dental surfaces10. due to the characteristics of these lesions, which appear as a porous subsurface tissue under a mineralized pseudo-intact enamel layer6, these materials represent a promising alternative to prevent the wear of sound tooth tissue11. the mechanism of action of resin infiltrants occurs by the obliteration of the pores of the demineralized subsurface enamel, controlling the progression of the lesions by preventing the diffusion of bacterial products into these12, being considered materials of satisfactory effectiveness12,13. its advantage, compared to conventional restorative treatment, is the preservation of dental tissue, to the detriment of the wear commonly necessary to gain access to interproximal caries lesions14, since its penetration into the interior of the lesion is due to capillarity forces10, in addition to avoiding the beginning of the restorative cycle, since restorative procedures may require, in some cases, repair or replacement, especially in patients with a history of high caries activity15. in view of the above, this article aimed to describe a clinical case using icon® (dmg – hamburg, germany) resin infiltrant in two interproximal caries lesions, focusing on the application protocol of the material used, discussing its inherent aspects. 3 pedreira et al. case report a 28-year-old female patient sought the graduate clinic of the faculty of dentistry of piracicaba for a routine consultation. the case reported was submitted to the ethics and research committee of the same faculty, being approved under number: caae – 30541620.8.0000.5418. the clinical examination began and, after prophylaxis and visual inspection, no changes in the dental elements were observed (fig.1). interproximal radiography was then performed, and radiolucent lesions restricted to tooth enamel could be seen in two elements – on the distal 24 and mesial 25 surfaces (fig.2). due to the characteristics of the lesions, the treatment with enamel resin infiltrant was chosen. figure 1. initial clinical appearance figure 2. diagnosis by interproximal radiography 4 pedreira et al. the procedure was started with absolute isolation. then, the interproximal wedge was inserted (fig.3a) to create space between the dental elements 24 and 25 and allow the stabilization of the applicator tips. it was decided to start the procedures on the distal face of element 24. thus, following all the manufacturer’s recommendations, icon® etch (composed of 15% hydrochloric acid – hcl) was applied (fig.3b) to increase the permeability of the enamel surface. it remained on the surface for 2 minutes and was then removed with a jet of water for 30 seconds, followed by drying with air for 30 seconds (fig,3c). then, icon® dry was applied for 30 seconds (fig.3d), twice, followed by drying with air for 30 seconds. finally, the application of the infiltrant itself, icon® infiltrant, was carried out, which remained in contact with the dental surface for 3 minutes (fig.3e). the matrix was removed, and the excess material was removed with dental floss, followed by photoactivation for 40 seconds (radii plus, sdi brasil industria e comercio ltda, são paulo, sp – brazil) on each dental face (fig.3f). as indicated by the manufacturer, a second application of icon® infiltrant was performed, this time for only 1 minute, without the need to repeat the previous steps, to ensure the diffusion of the material throughout the extension of the lesion and, consequently, to seal marginal surface pores. figure 3. protocol of the application for the resinous infiltrant icon® a d b e c f 5 pedreira et al. the same protocol applied for element 24 was performed for the treatment of element 25. follow-up was performed by an interproximal radiographic examination, after 18 months of application (fig.4). discussion the trend towards minimally invasive dentistry has expanded the treatment possibilities for carious dental lesions and the knowledge currently available. the advancement of products and techniques allows for the use of more conservative strategies for incipient and non-cavitated lesions16, including the application of icon® infiltrant. in view of the above, the treatment for initial carious lesions with icon® infiltrant was proposed to the patient of this clinical case, which does not require cavity preparation. regarding the development of caries disease, a balanced de-re cycle is responsible for maintaining the integrity of hard dental tissues because, in this situation, there is no loss or gain of minerals5. however, the bacteria contained in the biofilm are metabolically active all the time and, because of the disposition of fermentable carbohydrates, they generate by-products that alter the oral ph to intensify demineralization and hinder remineralization, leading to the development of caries lesions5. lesions of dental tissues are considered signs of caries disease and may be present at an ultrastructural level, i.e., not be visible on clinical examination until total destruction of dental tissues6,17. in this clinical case, the patient did not present symptoms or detectable clinical signs, and it was possible to visualize incipient caries lesions only by radiographic examinations during the routine consultation. the philosophy of minimal intervention advocates the use of conservative treatments to minimize the loss of dental tissues. thus, prevention and early intervention in caries injury are the first approaches18. the treatment of incipient lesions begins with etiological factors, i.e., oral hygiene and dietary control, to reestablish the de-re balance figure 4. follow-up after 18 months of the radiographic aspect. 6 pedreira et al. and enable the remineralization of the lesions19. the patient in this case reported a balanced diet concerning carbohydrates and presented good oral hygiene, without plaque accumulation; however, there was a deficiency in the use of dental floss. therefore, corroborating the literature19, the dentist reinforced to the patient all care about oral feeding and hygiene, motivating her regarding the need for regular flossing. however, only the control of etiological factors may not be able to promote the remineralization of initial caries lesions in some situations, especially in the case of interproximal regions, which represent areas that require greater discipline from patients concerning hygiene20. another treatment option is the topical applications of fluoride, which is also highly dependent on the patient’s adherence to the return visits to the dental office21. in this context, it was decided to treat the incipient caries lesions with resin infiltrant, since the material is able to interrupt the progression of the lesions, as well as seal the marginal surface, besides having its application performed in a single clinical session and not requiring return visits over a short period of time, as is the case with topical fluoride application22. the literature reports several advantages of the resin infiltration technique: mechanical stabilization of demineralized enamel, sealing of surface pores and enamel areas with deeper demineralization, preservation of dental enamel and paralysis of progression of the lesion, increased resistance to demineralization, minimal risk of development of secondary caries, and high patient acceptance23,24. a limitation of commercially available resin infiltrant, however, is the fact that it does not present radiopacity, which is an essential property for detecting the contour of the restorations and the depth of penetration of the infiltrant in the injury, for example25. thus, the control of the stabilization of the lesion is possible by the performance of radiographic control takes. in this case, the follow-up of the treatment of white spot lesions infiltrated with icon® was performed by interproximal radiographic examination 18 months after the application of the product. the paralysis of caries lesions was observed by comparing the final and initial radiographic images (diagnosis) of the treatment, which showed no progression of the lesions. several reports in the literature indicate the efficacy of infiltrants in controlling the progression of caries disease. a clinical study11, which compared the evolution of interproximal enamel lesions treated or not with resin infiltrant, in which all patients received guidance regarding oral hygiene care, obtained as a result, after 18 months follow-up, a reduced progression of caries in treated groups compared to the untreated control group, concluding that infiltration is an effective treatment to reduce lesion progression. another investigation26 obtained satisfactory penetration depth results and a statistically significant increase in microhardness values of enamel surface lesions after infiltration, indicating infiltrants as a valid treatment option for non-cavitated lesions. it is known that white spot injury is porous and, when exposed to constant acid challenges, can become cavitated. however, this type of injury can also be paralyzed or remineralized16,27, and thus modern dentistry has focused on early intervention to reduce patient damage and loss of dental tissue, succeeding in these new approaches. in conclusion, the treatment of incipient caries lesions by the application of the enamel resin infiltrant icon® has achieved positive results, corroborating minimally invasive dentistry, besides presenting good acceptance by the patient. 7 pedreira et al. acknowledgments the authors thank espaço da escrita – pró-reitoria de pesquisa – unicamp for the language services provided. conflicts of interest statement the authors report no conflicts of interest in this case report. references 1. vanderas ap, gizani s, papagiannoulis l. progression of proximal caries in children with different caries indices: a 4-year radiographic study. eur arch paediatr dent. 2006 sep;7(3):148-52. doi: 10.1007/bf03262556. 2. vanderas ap, manetas c, koulatzidou m pl, papagiannoulis l. progression of proximal caries in the mixed dentition: a 4-year prospective study. pediatr dent. 2003 may-jun;25(3):229-34. 3. skucha-nowak m, gibas m, tanasiewicz m, twardawa h, szklarski t. natural and controlled demineralization for study purposes in minimally invasive dentistry. adv clin exp med. 2015;24(5):891-8. doi: 10.17219/acem/28903. 4. cury, ja; tenuta lm. enamel remineralization: controlling the caries disease or treating early caries lesions? dental caries: the disease and its signals enamel remineralization. braz oral res. 2009;23 suppl 1:23-30. doi: 10.1590/s1806-83242009000500005. 5. moreno ec, zahradnik rt. demineralization and remineralization of dental enamel. j dent res. 1979 mar;58(spec issue b):896-903. doi: 10.1177/00220345790580024301. 6. kidd eam, fejerskov o. what constitutes dental caries? histopathology of carious enamel and dentin related to the action of cariogenic biofilms. j dent res. 2004;83 spec no c:c35-8. doi: 10.1177/154405910408301s07. 7. ahovuo-saloranta a, forss h, walsh t, nordblad a, mäkelä m, worthington h v. pit and fissure sealants for preventing dental decay in permanent teeth. cochrane database syst rev. 2017 jul;7(7):cd001830. doi: 10.1002/14651858.cd001830.pub5. 8. berger s, goddon i, chen cm, senkel h, hickel r, stösser l, et al. are pit and fissure sealants needed in children with a higher caries risk? clin oral investig. 2010 oct;14(5):613-20. doi: 10.1007/s00784-009-0343-8. 9. paris s, soviero vm, seddig s, meyer-lueckel h. penetration depths of an infiltrant into proximal caries lesions in primary molars after different application times in vitro. int j paediatr dent. 2012 sep;22(5):349-55. doi: 10.1111/j.1365-263x.2011.01204.x. 10. meyer-lueckel h, paris s. progression of artificial enamel caries lesions after infiltration with experimental light curing resins. caries res. 2008;42(2):117-24. doi: 10.1159/000118631. 11. paris s, hopfenmuller w, meyer-lueckel h. resin infiltration of caries lesions: an efficacy randomized trial. j dent res. 2010 aug;89(8):823-6. doi: 10.1177/0022034510369289. 12. paris s, meyer-lueckel h. infiltrants inhibit progression of natural caries lesions in vitro. j dent res. 2010 nov;89(11):1276-80. doi: 10.1177/0022034510376040. 13. meyer-lueckel h, bitter k, paris s. randomized controlled clinical trial on proximal caries infiltration: three-year. caries res. 2012;46(6):544-8. doi: 10.1159/000341807. 14. vila verde a, ramos mmd, stoneham am. benefits in cost and reduced discomfort of new techniques of minimally invasive cavity treatment. j dent res. 2009 apr;88(4):297-9. doi: 10.1177/0022034509334157. 8 pedreira et al. 15. qvist v. longevity of restorations: the ‘death spiral’. in: fejerskov oke, editor. dental caries: the disease and its clinical management. 2nd ed. oxford: blackwell munksgaard; 2008. p.443-56. 16. kidd e. the implications of the new paradigm of dental caries. j dent. 2011 dec;39 suppl 2:s3-8. doi: 10.1016/j.jdent.2011.11.004. 17. clark ca, fintz jb, taylor r. effects of the control of plaque on the progression of dental caries: results after 19 months. j dent res. 1974;53(6):1468-74. doi: 10.1177/00220345740530062901. 18. peters mc. strategies for noninvasive demineralized tissue repair. dent clin north am. 2010 jul;54(3):507-25. doi: 10.1016/j.cden.2010.03.005. 19. paula abp, fernandes ar, coelho as, marto cm, ferreira mm, caramelo f, et al. therapies for white spot lesions—a systematic review. j evid based dent pract. 2017 mar;17(1):23-38. doi: 10.1016/j. jebdp.2016.10.003. 20. ashwath b, vijayalakshmi r, arun d, kumar v. site-based plaque removal efficacy of four branded toothbrushes and the effect of dental floss in interproximal plaque removal: a randomized examiner-blind controlled study. quintessence int. 2014;45(7):577-84. doi: 10.3290/j.qi.a31960. 21. lindhe j, axelsson p. the effect of controlled oral hygiene and topical euoride application on caries and gingivitis in swedish schoolchildren. community dent oral epidemiol. 1973;1(1):9-16. doi: 10.1111/j.1600-0528.1973.tb01056.x. 22. lasfargues jj, bonte e, guerrieri a, fezzani l. minimal intervention dentistry: part 6. caries inhibition by resin infiltration. br dent j. 2013 jan;214(2):53-9. doi: 10.1038/sj.bdj.2013.54. 23. schwendicke f, meyer-lueckel h, stolpe m, dorfer ce, paris s. costs and effectiveness of treatment alternatives for proximal caries lesions. plos one. 2014 jan 27;9(1):e86992. doi: 10.1371/journal. pone.0086992. 24. anauate-netto c, borelli l neto, amore r, di hipólito v, d’alpino php. caries progression in non-cavitated fissures after infiltrant application: a 3-year follow-up of a randomized controlled clinical trial. j appl oral sci. 2017;25(4):442-54. doi: 10.1590/1678-7757-2016-0633. 25. saridag s, helvacioglu-yigit d, alniacik g, özcan m. radiopacity measurements of direct and indirect resin composites at different thicknesses using digital image analysis. dent mater j. 2015;34(1):13-8. doi: 10.4012/dmj.2014-181. 26. prajapati d, nayak r, pai d, upadhya n, bhaskar vk, kamath p. effect of resin infiltration on artificial caries: an in vitro evaluation of resin penetration and microhardness. int j clin pediatr dent. 2017 jul-sep;10(3):250-6. doi: 10.5005/jp-journals-10005-1445. 27. ferreira zandoná a, santiago e, eckert gj, katz bp, pereira de oliveira s, capin or, et al. the natural history of dental caries lesions: a 4-year observational study. j dent res. 2012 sep;91(9):841-6. doi: 10.1177/0022034512455030. 1http://dx.doi.org/10.20396/bjos.v19i0.8660266 volume 19 2020 e200266 original article 1 departmant of oral diagnosis, piracicaba dental school, university of campinas (unicamp), piracicaba, são paulo, brazil. 2 private office and uniodonto, piracicaba, são paulo, brazil. 3 ilumina hospital, piracicaba, são paulo, brazil. corresponding author: márcio ajudarte lopes av limeira 901, piracicaba, são paulo, brazil, 13414-903 email: marcioajudartelopes@gmail. com received: june 29, 2020 accepted: october 16, 2020 causes of death in brazil: analysis by geographic regions and in the highest populated cities of são paulo matheus ferreira linares1 , silvia maria paparotto lopes2 , adriana eliza brasil moreira3, pablo agustin vargas1 , alan roger santos-silva1 , márcio ajudarte lopes1,* aim: in this study we described the causes of mortality in brazil, its 5 geographic regions, and in the most populated cities of sao paulo state in order to contribute for development of prevention and intervention strategies. methods: data on causes of death and age distribution of the populations were collected from online public databases and then submitted to the 2001 world health organization age standardization of rates for better assessment. results: data showed that the main causes of death in brazil and in all 5 geographic regions were diseases of the circulatory system. neoplasms were the second most frequent cause of death in brazil and in 3 regions (south, southeast and midwest). however, in the other 2 regions (north and northeast) the second most common was associated to external causes, being neoplasms the third most often. additionally, in the south and southeast the third cause of deaths were from diseases of the respiratory system and from the external causes occupied the fourth position. analyzing the most populated cities of sao paulo state it was observed that all of them have the same profile of the country. on the other hand, as speculated previously, in piracicaba city, the most common cause of mortality was neoplasm. conclusions: these findings showed that brazil has a large spectrum of causes of death and methods to decrease the mortality rates should be implemented in a local scenario rather than a nation-wide approach, where each location has to focus on its most urging problem. keywords: mortality. cause of death. neoplasms. https://orcid.org/0000-0001-5250-1294 https://orcid.org/0000-0003-2711-3147 https://orcid.org/0000-0003-1840-4911 https://orcid.org/0000-0003-2040-6617 https://orcid.org/0000-0001-6677-0065 2 linares et al. introduction causes of mortality data is an important tool for developing guidelines and policies for prevention and treatment of diseases. these data in brazil have been published by the brazilian health ministry since the year of 1975 by using the mortality information system (sim), which provides the possibility to assess all causes of death from each city, state, region and the whole country1. it has already been reported by prestes et al.2 (2018), that sim is a viable and precise tool when analyzing causes of death in a local scenario. according to bray et al.3 (2018), noncommunicable diseases is currently the major cause of death worldwide, but it is expected that cancer will become the most common death cause. therefore, it is possible to observe that the trends of cancer in several countries are slowly changing, not just in mortality, but also in prevalence levels4-6. in some countries it is already the main cause of death4-6. this fact is occurring in part because of the aging and the population growth in association with habits and life style3,7. however, these levels in mortality could be reduced, if education, prevention methods, and strategies for precocious diagnosis were applied, since, successful outcome is achieved particularly when the tumor is treated at the early stages4,8. thus, the aim of this study was to assess the mortality rates in brazil, in its 5 geographic regions, and in the most populated cities of sao paulo state in order to identify the main causes of death, which could help for better understanding the scenario and consequently for developing strategies of prevention and intervention, particularly at the public primary care health centers. materials and methods to obtain the mortality data it was used the sim, which is available by the computing department of the unified health system (datasus), and the international classification of diseases 10th revision (icd – 10), through www.datasus.saude.gov.br1 and oncocentro foundation database system available at http://www.fosp.saude. sp.gov.br9. the data on the age distribution from the determined populations was acquired through the brazilian institute of geography and statistics (ibge) available at www.ibge.gov.br10 and seade foundation, available at www.seade.gov.br11. in addition, the crude mortality rate was calculated according to whole nation, region, state and city level and then the data was submitted to numbers of death to the 2001 world health organization (who) age standardization of rates to age-adjust standardized mortality rates (asmr), for better assessment, using piracicaba city population as reference. to calculate the asmr, initially is necessary to calculate the crude mortality rate for age group, for each disease that is going to be evaluated. this rate is obtained by dividing the number of deaths for that specific disease by the city’s population in that age group, and then multiplying the results by 100,000. then we multiply, individually, the crude mortality rate of each age group by the proportion of habitants of the assessed city of that same age group. after obtaining the result of each group, we add them to have the asmr. http://www.datasus.saude.gov.br http://www.fosp.saude.sp.gov.br/ http://www.fosp.saude.sp.gov.br/ http://www.ibge.gov.br 3 linares et al. results assessing brazil as a whole, the main cause of death, according to the chapters of the icd – 10, were the diseases of the circulatory system (dcs), with a total of 358.882 deaths (27.3%), followed by neoplasms, external causes of morbidity and mortality (ecmm), and diseases of the respiratory system (drs) accounting for 221.821 (17%), 158.657 (12%) and 155.620 (11.9%) deaths, respectively. the external causes of morbidity and mortality is used to classify deaths caused by environmental events and circumstances that are not related to diseases, such as traffic accident, suicide and murder. when the causes of death were observed by region, it was possible to find some differences among them. for instance, in the north and northeast regions the second most common cause of death was of ecmm with, respectively, 15.789 (19%) and 52.983 (15%) deaths, instead of neoplasms. however, when asmr was applied, the five geographic regions had similar ranking to brazil, excluding north and northeast regions, where ecmm were still high (figure 1). regarding sao paulo state, the number of ecmm was higher than most of the regions. however, when its asmr was analyzed, it was the lowest of all. it was also observed the asmr for deaths caused by dcs was higher than in any other region of the country, with a rate of 205.5 (table 1). figure 1. asmr’s in brazil and its geographic regions according to icd 10 chapters. brazil north northeast southeast south midwest asmr's in brazil and regions diseases from the circulatory system neoplasms external causes of morbidity and mortality diseases from the respiratory system others 19 0. 40 18 4. 80 21 4. 50 20 3. 60 18 4. 30 18 9. 20 12 2. 10 10 4. 60 10 8. 60 12 4. 60 14 4. 00 11 6. 90 79 .6 0 96 .8 0 97 .6 0 64 .3 0 76 .6 0 71 .9 0 86 .4 0 76 .2 0 80 .9 0 92 .4 0 81 .5 0 84 .7 0 22 2. 70 23 3. 90 25 0. 00 22 2. 80 19 2. 20 19 9. 40 table 1. causes of death in sao paulo state according to icd 10 chapters. icd – 10 chapters causes of death numbers crude rate asmr % diseases of the circulatory system 87.725 194,3 205,5 29.8% neoplasms 55.256 122,4 129 18.7% continua 4 linares et al. assessing the most populated cities of sao paulo state, it was observed that all of them had the same profile of the whole state (table 2). however, as speculated previously, in the city of piracicaba, the main cause of death was by neoplasms (table 3). it was interesting that the top three causes of death had almost the same percentage. when the neoplasms were classified into the icd – 10 groups, it was observed that the most frequent were malignant neoplasms of the digestive organs followed by malignant neoplasms of respiratory and intrathoracic organs, with 172 (34.1%) and 91 deaths (18%), respectively. cause of deaths by malignant neoplasms of lip, oral cavity and pharynx, corresponded to 19 (3.8%), being the eighth most common (table 4); the majority of them were diagnosed at stages iii or iv (57.9%). table 2. asmr’s in the 17 most populous cities from sao paulo state according to icd – 10 chapters. cities with more than 380,000 habitants. number of habitants diseases from the circulatory system neoplasms diseases from the respiratory system external causes of morbidity and mortality others sao paulo 11.696.088 246,9 137,6 105,7 43,6 184 campinas 1.150.753 193,3 127,1 101,1 54,9 167,4 guarulhos 1.313.169 261,4 130,4 115,7 58,8 171,7 sao bernardo do campo 799.645 200,7 131,6 99,2 41 154,7 santo andre 688.899 247,1 151,2 100,5 56,1 201,3 sao jose dos campos 687.544 163,6 128,3 74,6 35,7 211,9 osasco 676.149 275,2 145,3 103,8 62,7 180 ribeirao preto 661.997 198,3 137 104,6 62,5 199 sorocaba 637.437 179 133,1 116,3 56,5 240,6 mogi das cruzes 491.486 265 141,5 87,8 55,2 197,1 maua 447.911 237,2 107,6 89,3 39,5 149,4 sao jose do rio preto 437.273 195,5 135,3 117 70 192,3 santos 425.621 248 160,6 92,7 36,2 220,2 diadema 399.510 267 139,1 96,4 56,7 176,5 jundiai 397.353 209 168,3 94,6 41,8 163,1 carapicuiba 387.735 228,4 108,5 73,5 46,8 146,3 piracicaba 382.817 124,1 126,8 82,8 47,8 163,1 icd – 10: international classification of diseases 10th revision, asmr – age-adjust standardized mortality rates continuação diseases of the respiratory system 40.676 90,1 95,3 13.8% external causes of morbidity and mortality 22.194 49,1 50,5 7.5% others 88.902 196,9 205,6 30.2% total 294.753 652,8 100% icd – 10: international classification of diseases 10th revision 5 linares et al. discussion in overall analysis, it could be observed that the main cause of death in brazil and in its all 5 geographic regions was dcs. this profile is similar to other developing countries where most of the people die because of dcs4,6,12. on the other hand, in some developed countries the main cause of death is related to neoplasms5,12. however, it seems that in developing countries the scenario is also changing with trends in mortality associated with dcs declining, while mortality caused by neoplasms are remaining table 3. causes of death in the city of piracicaba according to icd 10 chapters. icd – 10 chapters causes of death numbers crude rate asmr % neoplasms 504 126,8 126,8 18.9% symptoms, sings and abnormal clinical and laboratory findings, not elsewhere classified 496 124,8 124,8 18.7% diseases from the circulatory system 493 124,1 124,1 18.5% diseases from the respiratory system 329 82,8 82,8 12.4% external causes of morbidity and mortality 190 47,8 47,8 7.1% others 648 163,1 163,1 24.4% total 2.660 669,4 100% icd – 10: international classification of diseases 10th revision table 4. causes of death in the city of piracicaba according to icd – 10 groups. icd – 10 groups causes of death numbers asmr % 126,8 100% malignant neoplasms of the digestive organs 172 43,3 34.1% malignant neoplasms of respiratory and intrathoracic organs 91 22,9 18% malignant neoplasm of breast 47 11,8 9.3% malignant neoplasms, stated or presumed to be primary of lymphoid, hematopoietic and related tissues 37 9,3 7.3% malignant neoplasms of male genital organs 31 7,8 6.2% malignant neoplasms of female genital organs 27 6,8 5.4% malignant neoplasms of urinary tract 25 6,3 5% malignant neoplasms of lip, oral cavity and pharynx 19 4,8 3.8% malignant neoplasms of eye, brain and other parts of central nervous system 17 4,3 3.4% malignant neoplasms of ill-defined, secondary and unspecified sites 14 3,5 2.8% melanoma and other malignant neoplasms of skin 13 3,3 2.5% malignant neoplasms of mesothelial and soft tissue 05 1,3 1% benign neoplasms 3 0,7 0.6% neoplasms of uncertain or unknown behavior 3 0,7 0.6% total 504 126,8 100 icd – 10: international classification of diseases 10th revision 6 linares et al. stable. in a near future it is possible that eventually neoplasms may become the leading cause of death also in developing countries13-16. although in the entire brazilian territory most of deaths were caused by dcs, there are important differences in mortality profile according to geographic regions. in the current study asmr was used to standardize the mortality rates according to age for better assessment. it was observed that in the north and northeast regions ecmm was the third most common cause of death, however it presented the highest asmr in ecmm when compared to any other regions. in contrast, the south and southeast regions had less number of deaths caused by ecmm and with a lower asmr in ecmm. these data may be in part because the north and northeast regions are less developed and are the more violent areas of brazil. in addition, the mortality profile of the south and southeast regions of brazil was similar to developed countries, where the population is older with a very low asmr in ecmm and higher asmr in neoplasms and in drs. consequently, they are more likely to die from neoplasms and drs than ecmm17-19. analyzing sao paulo state, the most populated and developed state of brazil, the mortality profile followed the same pattern of the southeast, the region where it is inserted, with dcs being the first and neoplasms the second main causes of death. in addition, it was also analyzed the mortality profile of the most populated cities of sao paulo state and all of them had the same profile of mortality causes comparing to the state. however, in the city of piracicaba, which is located in the interior of sao paulo state, with approximately 380,000 habitants and where the dental school of university of campinas is located, the main cause of death was by neoplasms. interestingly, that this mortality profile was similar to developed countries, such as the united kingdom, spain, and netherlands where the neoplasms are the main cause of death4-6,12. this can be related to degree of instructions of the population, access to health care system and the age distribution in piracicaba. therefore, in this city is more likely that people die of diseases, which are related to aging process, such as neoplasms and dcs. when assessing the neoplasms by icd-10 groups, it was noticed that malignant neoplasms of the digestive organs with an asmr of 43.3, followed by malignant neoplasms of respiratory and intrathoracic organs with asmr of 22.9 were the main sites. despite being a city in brazil where cancer is the main cause of death, it is interesting to know that these rates in mortality are quite low compared to studies performed by bray et al.3 (2018) and ferlay et al.20 (2015). the asmr for the worldwide mortality in these types of cancers is almost the double compared to that observed in piracicaba. malignant neoplasms of lip, oral cavity and pharynx were the eighth in the city and with an asmr of 4.8. when compared to the worldwide mortality for this type of cancer, in the studies by bray et al.3 (2018) and ferlay et al.20 (2015), it was possible to observe that the amsr in piracicaba was higher and this can be related to these group of neoplasms being diagnosed at late stage, as reported in this study. it is well known that prevention, early diagnosis and adequate treatment are essential to decrease deaths. screening for high risk patients is one of the methods that can 7 linares et al. be effective in reducing mortality rates. although some difficulty has been already reported, this strategy may allow to diagnose cancer at early stages21-25. although to our knowledge the current study has valuable information, the data collection from online databases of public services could be considered a limitation since the authors did not have access to the original reports. in conclusion, these data reinforced that brazil is a huge country with a wide variation of mortality profiles. therefore, having information of the main cause of deaths according to the specific geographic region, state or city is essential to establish appropriate public policy. in the city of piracicaba, a project aiming to inform the population of how to prevent the main types of cancer and screening has been conducted in order to establish diagnosis at early stage and consequently decrease the mortality rate associate to malignant tumors. references 1. 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[cited 2020 feb 2]. available from: www.seade.gov.br. portuguese. 8 linares et al. 12. araújo f, gouvinhas c, fontes f, la vecchia c, azevedo a, lunet n. trends in cardiovascular diseases and cancer mortality in 45 countries from five continents (1980-2010). eur j prev cardiol. 2013 jul;21(8):1004-17. doi: 10.1177/2047487313497864. 13. ribeiro al, duncan bb, brant lc, lotufo pa, mill jg, barreto sm. cardiovascular health in brazil: trends and perspectives. circulation. 2016 jan 26;133(4):422-33. doi: 10.1161/circulationaha.114.008727. 14. brant lcc, nascimento br, passos vma, duncan bb, benseñor ijm, malta dc, et al. variations and particularities in cardiovascular disease mortality in brazil and brazilian states in 1990 and 2015: estimates from the global burden of disease. rev bras epidemiol. 2017 may;20(suppl 1):116-8. doi: 10.1590/1980-5497201700050010. 15. barbosa ir, de souza dl, bernal mm, do c c costa í. cancer mortality in brazil: temporal trends and predictions for the year 2030. medicine (baltimore). 2015 apr;94(16):e746. doi: 10.1097/md.0000000000000746. 16. guerra mr, bustamante-teixeira mt, corrêa csl, de abreu dmx, curado mp, mooney m, et al. magnitude and variation of the burden of cancer mortality in brazil and federation units, 1990 and 2015. rev bras epidemiol. 2017 may;20(suppl 1):102-15. doi: 10.1590/1980-5497201700050009. 17. kochanek kd, murphy sl, xu j, arias e. mortality in the united states, 2013. nchs data brief. 2014 dec;(178):1-8. 18. ringard å, sagan a, sperre saunes i, lindahl ak. norway: health system review. health syst transit. 2013;15(8):1-162. 19. malta dc, oliveira tp, luz m, stopa sr, da silva junior jb, dos reis aa. smoking trend indicators in brazilian capitals, 2006-2013. cien saude colet. 2015 mar;20(3):631-40. doi: 10.1590/1413-81232015203.15232014. 20. ferlay j, soerjomataram i, dikshit r, eser s, mathers c, rebelo m, et al. cancer incidence and mortality worldwide: sources, methods and major patterns in globocan 2012. int j cancer. 2015 mar;136(5):e359-86. doi: 10.1002/ijc.29210. 21. sankaranarayanan r, mathew b, jacob bj, thomas j, somanathan t, pisani p, et al. early findings from a community-based, cluster-randomized, controlled oral cancer screening trial in kerala, india. the trivandrum oral cancer screening study group. cancer. 2000 nov 20;88(3):664-73. 22. ramadas k, sankaranarayanan r, jacob bj, thomas g, somanathan t, mahé c, et al. interim results from a cluster randomized controlled oral cancer screening trial in kerala, india. oral oncol. 2003 sep;39(6):580-8. doi: 10.1016/s1368-8375(03)00041-1. 23. sankaranarayanan r, ramadas k, thomas g, muwonge r, thara s, mathew b, et al. effect of screening on oral cancer mortality in kerala, india: a cluster-randomised controlled trial. lancet. 2005 jun 4;365(9475):1927-33. doi: 10.1016/s0140-6736(05)66658-5. 24. sankaranarayanan r, ramadas k, thara s, muwonge r, thomas g, anju g, et al. long term effect of visual screening on oral cancer incidence and mortality in a randomized trial in kerala, india. oral oncol. 2013 apr;49(4):314-21. doi: 10.1016/j.oraloncology.2012.11.004. 25. de andrade mac. [active search of malignant lesions and potentially malignant disorders in four family health untis (usf) in the city of piracicaba] [dissertation]. piracicaba: piracicaba dental school, university of campinas; 2010. portuguese. 1http://dx.doi.org/10.20396/bjos.v18i0.8656600 volume 18 2019 e191445 original article 1 department of dentistry. pontifical catholic university of minas gerais, belo horizonte, minas gerais, brazil. hospital público regional de betim. betim, minas gerais, brazil. 2 department of oral surgery and pathology. school of dentistry. universidade federal de minas gerais, belo horizonte, minas gerais, brazil. 3 private clinic. faculdade arnaldo de odontologia belo horizonte, minas gerais brazil. 4 d’or institute for research and education. rio de janeiro, rio de janeiro brazil. 5 department of paediatric dentistry, school of dentistry, universidade federal de minas gerais, belo horizonte, mg, brazil. conflicts of interest: none corresponding author: giovanna ribeiro souto departamento de odontologia – icbs/puc minas laboratório de biologia oral rua dom josé gaspar, 500 – prédio 45 – sala 111 coração eucarístico belo horizonte mg, brasil. 30.535-610 voice: +55-31-33194341 e-mail: grsouto@hotmail.com https://orcid.org/0000-0003-3617-8794 received: november 12, 2018 accepted: may 23, 2019 mast cell degranulation in periodontal disease from hiv-infected individuals giovanna ribeiro souto1, alessandro oliveira de jesus2, takeshi kato segundo3, paôlla freitas perdigão4, lucas guimarães abreu5, fernando oliveira costa2, ricardo alves mesquita2 aim: the objective was to compare the density and degranulation of mast cells on specimens obtained from individuals diagnosed with gingivitis or chronic periodontitis who were either non-hiv-infected or hiv-infected patients treated with highly active antiretroviral therapy (haart). methods: gingival samples were taken from 16 nonhiv-infected individuals and 17 hiv-infected individuals diagnosed with gingivitis and chronic periodontitis. the samples were processed and stained with 0.3 percent o-toluidine blue. densities (cells/mm²) and percentages of intact and degranulated mast cells were obtained. results: no statistically significant differences were observed in the mast cell density and the percentage of degranulated mast cells between non-hiv-infected and hiv-infected individuals diagnosed with gingivitis and chronic periodontitis. mononuclear inflammatory infiltrate was weakly correlated with the percentage of mast cells degranulated for both groups. conclusions: there are no differences of the density and degranulation of mast cells in gingival tissue between non-hiv-infected and hiv-infected patients undergoing haart, both groups with diagnosis of gingivitis or chronic periodontitis. this may be a result of the recovery of the immunologic system by haart treatment. keywords: mast cells. infection. inflammation. humans. mouth mucosa. https://orcid.org/0000-0003-3617-8794 2 souto et al. introduction mast cells were first described in 1876 by paul ehrlich1. the key role of mast cells in the pathophysiology of different diseases such as atherosclerosis, autoimmune disorders, intestinal diseases, cancer, oral lichen planus and periodontal disease was demonstrated in experimental and clinical studies1-7. the function of mast cells was associated with the degranulation of cytotoxic mediators responsible for producing deleterious effects in different tissues8. in periodontal disease, mast cells seem to contribute with modulation of humoral and cellular response9,10. according to steinsvoll et al.11 (2004) evidence attributes to mast cells in natural immunity to bacteria a role in the local differentiation of monocytes into macrophages and dendritic cells. when mast cells are properly activated, the release of mast cells mediators in the tissue may be contributing to the maintenance of the periodontal disease12. degranulation of the granule-associated mediators is a result of mast cell activation. the granules of these inflammatory cells contain histamine, heparin, serotonin, chemotactic factors and various proteases such as peroxidase, tryptase, chymase, carboxidase and beta glucuronidase as primary mediators1,13. marshall et al.14 (2003) demonstrated that mast cells can be infected with several viruses, including the human immunodeficiency virus (hiv), dengue virus, cytomegalovirus and adenovirus. bacterial plaque is the etiologic factor most important in inflammatory periodontal disease, and mast cells were found in high numbers in gingival samples taken from patients having chronic periodontitis in comparisons with controls2. huang et al.7 (2013) demonstrated a significant correlation between mast cell density, the degree of their degranulation, and human periodontitis severity. in the same context, an association between mast cells, hiv and periodontal disease also has been observed in patients undergoing treatment for hiv infection, but none had received highly active anti-retroviral therapy (haart)15,16. patients in all stages of hiv infection showed increased numbers of mast cells on gingival tissue with periodontal disease15. furthermore, a significantly higher proportion of matrix metalloproteinases (mmp) expressed by mast cells was displayed in samples of hiv-infected individuals diagnosed with chronic periodontitis in comparisons with controls16. however, mast cell degranulation was not evaluated. in a prior analysis of our research group17, the results did not demonstrate a statistically significant difference in the mast cell densities in hiv-infected individuals with periodontal disease undergoing haart in comparison with non-hiv-infected individuals with periodontal disease. it was suggested that this fact may well be the result of the application of haart in individuals with hiv17. considering the importance of the mast cell in the immune response, it was evaluated the hypothesis of an alteration on the mast cell degranulation in gingival tissue of hiv-infected individuals. therefore, this study compared the mast cell density and degranulation percentage between hiv-infected undergoing haart and non-hiv-infected individuals diagnosed with gingivitis or chronic periodontitis. also, the effect of mononuclear inflammatory infiltrate on mast cell degranulation was evaluated. 3 souto et al. materials and methods the present study was approved by the research ethics committee from the federal university of minas gerais (ufmg) under protocol number 514/07. all experiments were conducted in accordance with the declaration of helsinki. clinical evaluation individuals with plaque-induced periodontal disease, classified as gingivitis or chronic periodontitis, were selected for the study18. periodontal disease status was determined with the probing depth (pd), clinical attachment level (cal), and gingival bleeding (gb). a full-mouth periodontal examination was performed by a single trained examiner (tks) in four sites per tooth in all teeth, in a circumferential mode. the presence of four or more teeth with one or more sites containing pd ≥4 mm and cal ≥3 mm within the same site constituted a diagnosis of chronic periodontitis19. individuals who presented >25% of sites with gb, and an absence of pd ≥4 mm and cal ≥3 mm were diagnosed as having gingivitis19,20. the patients ranged in age from 30 to 60 years old and included both men and women. the hiv-infected individuals were recruited from the orestes diniz center (belo horizonte, brazil) during a period of one year. hiv infection was determined by applying the western-blot test, and all positive patients had been undergoing haart for a period of five to 13 years. the non-hiv-infected individuals were recruited from the periodontology clinic of the school of dentistry and were sent to the anonymous test center (belo horizonte, brazil) to confirm their hiv-negative status. cd4 and cd8 t-lymphocyte levels and viral loads were obtained from the medical records of the hiv-infected individuals up to two months prior to performing the biopsy of the gingival tissues. procedures for tissue collection for both groups 1. individuals diagnosed with gingivitis: for group diagnosed with gingivitis, gingival tissue samples were obtained from teeth with absence of pd ≥4 mm and cal ≥3 mm removed for prosthetic or orthodontic reasons2. 2. individuals with chronic periodontitis: for group diagnosed with chronic periodontitis, samples were obtained during the modified widman surgery. these individuals were provided with oral hygiene instruction as well as sub-gingival scaling and root planning prior to surgery. after 45 to 60 days, for patients presenting with both pd > 5 mm and bleeding on probing, modified widman surgery was recommended. all gingival tissue samples were removed at sites with higher pd21. sample selection flowchart can be observed in the fig. 1. the group diagnosed with gingivitis included seven samples from hiv-infected individuals and six samples from non-hiv-infected individuals. the group diagnosed with chronic periodontitis included 10 samples from hiv-infected individuals and 10 samples from non-hivinfected individuals. 4 souto et al. exclusion and inclusion criteria individuals hiv-infected and non-hiv-infected diagnosed with gingivitis and chronic periodontitis were included in the study. individuals from the non-hiv-infected group that reported systemic diseases or immunologic abnormalities were excluded. hiv-infected group that reported a non-controlled immunological system and the presence of others systemic diseases were also excluded from the study. the patients have not used any medication, except to hiv infection condition. staining and grading the specimens were fixed in 10 percent neutral-buffered formaldehyde solution. sections stained with hematoxylin-eosin (h&e) were digitized using a microscope (axioskop 2 plus, carl zeiss, göttingen, germany) at 400x magnification, interfaced to a computer. mononuclear inflammatory cell count was determined by a trained investigator (aoj) using a software program (image tool, v.3.0, university of texas health science center, san antonio, tx, usa), and the count was performed in eight to 10 consecutive fields. serial sections of 4 µm from paraffin-embedded blocks were deparaffinized and dehydrated. mast cells were detectable with o-toluidine blue 0.3 percent stain. the toluidine blue gave a light blue background to the section and allowed easy mapping of metachromatically-stained mast cells4. the degranulated mast cells were determined at a magnification of 100x under a light microscope (axioskop 2 plus, carl zeiss, göttingen, germany). mast cells were clearly identified by o-toluidine blue stain, and they presented in the lamina propria (lp) subjacent to the oral epithelium (oe) and sulcular epithelium (se) in samples of the gingival tissue (fig. 2a). mast cell densities (per mm2) were calculated. mast cells were counted only when the nucleus was clearly visible (fig. 2b and c). according to ghalayani et al.6 (2012), the mast cells were categorized into two groups: figure 1. sample selection flowchart selection of the sample individuals hiv-infected non-individuals hiv-infected gingivitis n=10 chronic periodontitis n=15 gingivitis n=10 chronic periodontitis n=15 samples lost during processing of the specimens gingivitis n=7 chronic periodontitis n=10 gingivitis n=6 chronic periodontitis n=10 5 souto et al. 1. degranulated: less intense metachromasia or stainability and an obvious clear outline of the nucleus and/or free granules in close proximity to the cell membrane (fig. 2b). 2. intact: intense and dense metachromasia or stainability in which the nucleus was not apparent and/or no granule extrusion around the cell was present (fig. 2c). mast cell degranulation was determined in the inflammatory infiltrate subjacent to the sucular and oral gingival epithelium. all gingival specimens were evaluated, so eight to 10 fields were viewed via a microscope (axioskop 2 plus, carl zeiss, göttingen, germany) at 400x magnification using a meshwork eyepiece (0.1024 mm²). mast cell densities and the percentage of mast cell degranulation were obtained, and the results were expressed as means and were compared. the densities of degranulated mast cells [the cell number per millimeters squared (per mm2)] were correlated with the densities of mononuclear inflammatory infiltrate cells in hiv-infected and in non-hiv-infected individuals diagnosed with gingivitis or chronic periodontitis, because in this phase of the inflammation process, the mast cell has already released its granules. statistical analysis the statistical analysis was performed using the software program (bioestat, version 5.0, pa, belém, pará, brazil). the sample distribution was tested using the shapiro-wilks procedure, and the samples presented a non-normal distribution; therefore, the mann-whitney test and spearman correlation were applied. the α level was figure 2. gingival sample stained with o-toluidine blue. a) the oral (oe) and sulcular (se) epithelium and lamina propria (lp) in gingival tissue sample were showed in toluidine blue stain (o-toluidine blue, original magnification 50x). b) degranulated mast cells it is observed less intense metachromasia and obvious clear outline of the nucleus (asterisk) (o-toluidine blue, 400x original magnification). c) intacted mast cellsit is observed intense and dense metachromasia and nucleus not apparent (arrow) (o-toluidine blue, 400x original magnification). a b c * * * se lp oe 6 souto et al. set to 0.05. the correlation was graded according to the cohen classification as weak (< 0.30), moderate (0.30 to 0.50), or strong (> 0.50)22. the reliability of the measurements was assessed by the intraclass correlation coefficient (icc). an icc > 0.91 was considered to be a very good correlation; 0.71 < icc < 0.91 was a good correlation; 0.51 < icc < 0.71 was a moderate correlation; 0.31 < icc < 0.51 was considered to be a bad correlation; and an icc < 0.31 was a very bad correlation23. sample size calculation was carried out with the power and sample size program (ps, version 3.0, nashville, usa) and considered both type i and ii errors. for this, we assumed a 95% confidence interval, 80% power of test and parameters of values of densities of mast cells obtained in study of periodontal disease of huang et al.7 results data clinical and periodontal clinical parameters of the hiv-infected and non-hivinfected individuals are presented in table 1. there were no statistically significant differences in clinical periodontal parameters between the groups. individuals who were hiv-infected presented a blood level of cd4 t cells/mm3 of 521 (117-1,054) and 450 (28-815), and a blood level of cd8 t cells/mm3 of 850 (267-1140) and 1,048 (406-1,565) for gingivitis or chronic periodontitis, respectively. cell counts were performed throughout the sections by two masked examiners at two different times, two weeks apart. the reliability of the measurements was assessed, and the icc was 0.85. statistically significant differences were not observed in the mast cell density between hiv-infected and non-hiv-infected individuals diagnosed with gingivitis (p=0.43) or chronic periodontitis (p=0.76). in addition, there were no statistically significant differences in degranulation of the mast cells between hiv-infected and non-hiv-infected individuals diagnosed with gingivitis (p=0.18) or table 1. data clinical and periodontal clinical parameters of the hiv-infected and non-hiv-infected individuals. clinical data gingivitis chronic periodontitis hiv-infected (n = 7) non-hiv-infected (n = 6) hiv-infected (n = 10) non-hiv-infected (n = 10) age (years)* 38 (34 to 50) 38 (30 to 45) 46 (40 to 60) 42 (32 to 52) sex (n) males 3 3 4 6 females 4 3 6 4 gb (mean** ±sd) 0.94 ±0.91 1.69 ±0.83 1.05 ±0.29 1.35 ±0.65 pd > 4 mm (% of sites) nd nd 8.2 6.0 cal > 3 mm (% of sites) nd nd 14.9 18.6 * median. **mean hiv= human immunodeficiency virus (hiv); sd= standard deviation; gb= gingival bleeding; pd = probing depth; cal= clinical attachment level; nd = not determined; mann-whitney test (p>0.05) 7 souto et al. chronic periodontitis (p=0.20) (table 2). it was observed that the densities of the mononuclear inflammatory infiltrate were weakly correlated with the density of mast cell degranulation for both groups–hiv-infected and non-hiv-infected with gingivitis (p=0.21) or chronic periodontitis (p=0.87). discussion in human periodontal disease, there is an increase in the number of mast cells that may be participating either in the destructive events or in the defense mechanism of periodontal disease via secretion of cytokines2. it was suggested that hiv infection may increase or be associated with an augmented mast cell count in the cervixes of women, even without treatment information8. in addition, some authors think that mast cells represent a potential reservoir for infectious hiv-124,25. however, in the present study, statistically significant differences were not observed in the mast cell density and degranulation percentage between hiv-infected individuals undergoing haart and non-hiv infected individuals diagnosed with gingivitis or chronic periodontitis. the mast cells degranulation represents their functional status26. this occur in response to various stimuli including chemicals, drugs, allergen-bound immunoglobulin-e (ige) or to non-immunological stimuli, as well as in response to bacterial products or cytokines26,27. huang et al.7 (2013) showed strong mast cell degranulation in both the moderate and advanced periodontitis groups, suggesting that periodontal mast cell accumulation and degranulation may play a central role in the pathogenesis of human periodontal disease. to date, the role of mast cells degranulation in periodontal disease from hiv-infected individuals it was not evaluated. in the present study, mast cells were clearly identified by o-toluidine blue stain. the evaluation of mast cell activation by degranulation using o-toluidine blue stain was considered to be a reliable method in comparison with immunohistochemical reaction7. histological analysis performed by huang et al.7 (2013) demonstrated that inflammatory infiltrate present in chronic periodontitis samples is significantly increased in comparison to clinically healthy gingival tissues. this infiltrate is composed by predominantly mononuclear cells and focally distributed with large presence of lymphocytes and plasma cells, as well as a discrete presence of macrophage-like cells and foci of polymorphonuclear cells7. in the current study, inflammatory infiltrate density table 2. mast cell densities and degranulation in gingival samples of individuals presenting with gingivitis or chronic periodontitis. gingivitis chronic periodontitis hiv-infected (n = 7) non-hiv-infected (n = 6) hiv-infected (n = 10) non-hiv-infected (n = 10) mean ± sd of mast cell densities (cell/mm2) 46.19±47.64 53.80±52.35 48.21±51.68 51.78±48.32 mast cell degranulation (%) 52.74 62.54 52.88 47.11 hiv= human immunodeficiency virus (hiv); sd= standard deviation; mann-whitney test (p>0.05) 8 souto et al. was not significantly correlated to mast cell density and degranulation. these results contrast with the significant correlation demonstrated between inflammatory infiltrate and dendritic cells densities28. therefore, it is possible that the mast cells recruitment and activation in periodontal disease is more related to secretion of inflammatory cytokines and chemokines by inflammatory infiltrate cells than number of this cells. the clinical examiner was a specialist in periodontics with more than 10 years of experience and therefore we considered that the clinical parameters used in the study were reliable. however, the absence of intra-examiner calibration may be a limitation of the study. in addition, the absence of mean and standard deviation of pd and cal of teeth whose gingival tissues were obtained was also a limitation of the present study. the severity of the disease could impact the analysis performed in this study. however, we considered no significant variation was found in this parameters, since the gingival tissues were obtained from teeth with of pd <4 mm and cal <3 mm for gingivitis group, and sites with higher pd for chronic periodontitis group. studies performed before the introduction of haart presented an accelerated progression of periodontitis existing previously29. with haart, studies have demonstrated a decrease in the oral manifestations of kaposi’s sarcoma, oral candidiasis, and hairy leukoplakia in hiv-infected patients30. however, in these studies, the findings are not clear about periodontal disease, mainly due to the lack of consistent criteria to define disease. it has been difficult to determine the true prevalence of the periodontal disease in patients with hiv-infection30-32. a study of the frequency of oral lesions in hiv-positive patients undergoing haart, compared with a non-hiv-infected control group, showed that periodontal diseases were the second most frequent oral lesions (25 percent) found in these patients33. in contrast with peppes et al.33 (2013), gonçalves et al.30 (2013) showed no statistically significant differences in the prevalence of oral manifestations, including periodontal disease, between patients who were hiv-positive and undergoing haart, in comparison with those not on haart. recently, to assess the prevalence and severity of chronic periodontitis in patients with hiv-infection, groenewegen et al.34 (2019) compared 258 patients with hiv-infection and undergoing haart with 539 controls and observed severe chronic periodontitis more prevalent in infected patients (66%) than in controls (36%). in the present study, the samples had no significant differences in the severity of periodontal disease. this could be a possible explanation for similar recruitment and activation of mast cells observed between groups. however, cytokines and other inflammatory mediators expressed by mast cells from gingival samples could explain the role of these cells in the periodontal disease of patients with hiv-infection. a potential function in hiv-1 dissemination was demonstrated for gut mucosal mast cells. they expressed a variety of hiv-1 attachment factors (hafs) and mast cell surface-bound viruses were efficiently transferred to target t cells. strategies futures to prevent viral capture and transfer mediated by mast cells could be beneficial in combating primary hiv-1 infection 35. the present study concluded that hiv-infected individuals undergoing haart did not present differences in mast cell density and degranulation in comparison with 9 souto et al. non-hiv-infected. this mast cell accumulation and degranulation similar may be a result of the recovery of the immunologic system by haart treatment. from the clinical point of view, this could explain because, in the last years, the progression of periodontal disease has been shown similar between the hiv-infected and non-hiv-infected groups. acknowledgments: the authors would like to thank the national council for scientific and technological development (cnpq), brasília, df, brazil (grant numbers 309322/2015-4). reference 1. anand p, singh b, jaggi as, singh n. mast cells: an expanding pathophysiological role from allergy to other disorders. naunyn schmiedebergs arch pharmacol. 2012 jul;385(7):657-70. doi: 10.1007/s00210-012-0757-8. 2. batista ac, rodini co, lara vs. quantification of mast cells in different stages of human periodontal disease. oral dis. 2005 jul;11(4):249-54. 3. juneja m, mahajan s, rao nn, george t, boaz k. histochemical analysis of pathological alterations in oral lichen planus and oral lichenoid lesions. j oral sci. 2006 dec;48(4):185-93. 4. rao kn, brown ma. mast cells: multifaceted immune cells with diverse roles in health and 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periodontal therapy may reduce the risk of preterm low birth weight in women with periodontal disease: a randomized controlled trial. j periodontol. 2002 aug;73(8):911-24. 20. lópez nj, da silva i, ipinza j, gutiérrez j. periodontal therapy reduces the rate of preterm low birth weight in women with pregnancy-associated gingivitis. j periodontol. 2005 nov;76 suppl 11s:2144-2153. doi: 10.1902/jop.2005.76.11-s.2144. 21. salvi ge, lindhe j, lang np. plano de tratamento de pacientes com doenças periodontais. 5th ed. rio de janeiro: guanabara koogan; 2008.p. 629. 22. cohen j. statistical power analysis for the behavioral sciences. 2nd ed. hillsdale, nj: lawrence erlbaum associates; 1988. p. 40-151. 23. bressenot a, salleron j, bastien c, danese s, boulagnon-rombi c, peyrin-biroulet l. comparing histological activity indexes in uc. gut. 2015 sep;64(9):1412-8. doi: 10.1136/gutjnl-2014-307477. 24. crowe s, zhu t, muller wa. the contribution of monocyte infection and trafficking to viral persistence, and maintenance of the viral reservoir in hiv infection. j leukoc biol. 2003 nov;74(5):635-41. 25. sundstrom jb, little dm, villinger f, ellis je, ansari aa. signaling through toll-like receptors triggers hiv-1 replication in latently infected mast cells. j immunol. 2004 apr 1;172(7):4391-401. 26. maurer m, theoharides t, granstein rd, bischoff sc, bienenstock j, henz b, et al. what is the physiological function of mast cells? exp dermatol. 2003 dec;12(6):886-910. 27. caughey gh. mast cell tryptases and chymases in inflammation and host defense. immunol rev. 2007 jun;217:141-54. 28. souto gr, queiroz-junior cm, de abreu mh, costa fo, mesquita ra. pro-inflammatory, th1, th2, th17 cytokines and dendritic cells: a cross-sectional study in chronic periodontitis. plos one. 2014 mar 26;9(3):e91636. doi: 10.1371/journal.pone.0091636. ecollection 2014. 29. lamster ib, grbic jt, bucklan rs, mitchell-lewis d, reynolds hs, zambon jj. epidemiology and diagnosis of hiv-associated periodontal diseases. oral dis. 1997 may;3 suppl 1:s141-8. 30. gonçalves ls, gonçalves bm, fontes tv. periodontal disease in hiv-infected adults in the haart era: clinical, immunological, and microbiological aspects. arch oral biol. 2013 oct;58(10):1385-96. doi: 10.1016/j.archoralbio.2013.05.002. 31. mataftsi m, skoura l, sakellari d. hiv infection and periodontal diseases: an overview of the post-haart era. oral dis. 2011 jan;17(1):13-25. doi: 10.1111/j.1601-0825.2010.01727.x. 32. ryder mi, nittayananta w, coogan m, greenspan d, greenspan js. periodontal disease in hiv/aids. periodontol 2000. 2012 oct;60(1):78-97. doi: 10.1111/j.1600-0757.2012.00445.x. 33. peppes cp, lemos asp, araujo rlf, portugal meg, buffon mcm, raboni sm. oral lesions frequency in hiv-positive patients at a tertiary hospital, southern brazil. bra j of oral sci. 2013;12(3):216-22. doi: 10.1590/s1677-32252013000300012. 11 souto et al. 34. groenewegen h, bierman wfw, delli k, dijkstra pu, nesse w, vissink a, et al. severe periodontitis is more common in hivinfected patients. j infect. 2019 mar;78(3):171-177. doi: 10.1016/j.jinf.2018.11.008. 35. jiang ap, jiang jf, wei jf, guo mg, qin y, guo qq, et al. human mucosal mast cells capture hiv-1 and mediate viral trans-infection of cd4+ t cells. j virol. 2015 dec 30;90(6):2928-37. doi: 10.1128/jvi.03008-15. oral sciences n3 braz j oral sci. 11(3):352-356 diagnosis of oral manifestations in hiv/aids patients who used haart and developed diabetes mellitus adam de mello silva1, camila correia dos santos1, elcio magdalena giovani2 1dds, msc, paulista university (unip) ceape (center of study and care of patients with special needs) oral diagnosis, sp,brazil 2dds,msc phd, professor at the integrated and special needs patients clinic and professor at the master’s graduate program, paulista university (unip), sp, brazil correspondence to: adam de melo silva avenida itamarati 1855, curuça, santo andré sp brasil phone: +55 11 86179286 e-mail: businesska@hotmail.com abstract aim: to assess the prevalence of oral manifestations in hiv/aids patients and a possible correlation of the development of diabetes mellitus due to highly active antiretroviral therapy (haart). methods: 56 patients were examined, divided into two groups, group 1, the hiv group, with 28 patients known to be hiv+, who developed diabetes mellitus due to haart, and group 2, the control group, with 28 patients with hivdiagnosed with diabetes mellitus. results: in group 1, normal salivary flow rate and buffering capacity were observed in 18 (64.3%) patients, but the bleeding index high (46.53%) and higher incidence of periodontal disease was found in this group. in group 2, 11 (39.2%) and 15 (53.5%) patients had low and normal salivary flow rate, respectively. high t-cd4 indices with mean value of 22.46 for each patient, showing xerostomia in 9 (32.1%) patients and dental caries in 11 (39.3%). conclusions: regarding the oral and general manifestations, the hiv group showed higher rates of pathologies when compared with the control group. the hiv group and the control group were diagnosed with diabetes, but this condition in the hiv group presented earlier than in the control group, suggesting a possible association with haart. keywords: aids, hiv, diabetes mellitus. introduction acquired immunodeficiency syndrome (aids) was first described in 1981 in the united states, and its causative agent is the human immunodeficiency virus (hiv). since 1996, with the advent and introduction of highly active antiretroviral therapy (haart), a profound impact on the natural history of hiv infection was observed, with a remarkable increase in the survival of hiv-infected individuals, transforming the panorama of the disease. today, the morbidity and mortality ratios become significantly smaller in those adhering to the treatment. however, although the substantial benefits of haart far outweigh its potential risks, it is known that prolonged treatment may result in a variety of disorders related to compliance and toxicity. long-term haart is accompanied by undesirable adverse side effects such as cardiovascular and metabolic alterations that are risk factors for cardiovascular disease and diabetes mellitus (dm). metabolic alterations include dyslipidemia, diabetes mellitus type 2 (dm2), insulin resistance, hyperlactatemia, hyperlipidemia. a morphological, anatomical, metabolic change in body fat and bone metabolism is the set of changes known as lipodystrophy syndrome1-8. original article braz j oral sci. july | september 2012 volume 11, number 3 received for publication: october 18, 2011 accepted: february 18, 2012 braz j oral sci. 11(3):352-356 the anatomical changes include lipoatrophy in the region of the face (facial fat loss), loss of limb fat (upper and lower), especially of the superficial veins, loss of fat from the buttocks associated or not with the accumulation of fat in the abdomen mainly due to fat deposition, visceral fat accumulation in the posterior cervical region (giba), accumulation of fat in the breasts that can occur in both women and men. metabolic changes include the increase of serum lipids and peripheral insulin resistance resulting in dm9-12. insulin resistance (ir) is common in hiv-seropositive patients, particularly among those receiving protease inhibitor (pi) and is more prevalent among those with lipoatrophy or fat accumulation in the visceral region. the prevalence of hyperglycemia and diabetes is significantly higher in those receiving haart, compared to the general population. the occurrence of diabetes is four times greater in hivseropositive patients than in hiv-seronegative patients matched by age and body mass10,13-16. according to the american diabetes association17, dm is a disease in which the body does not produce insulin or does not produce it correctly. diabetes is latent when a blood glucose level of 100-126 mg/dl is detected on fasting. diabetes is relatively similar in hiv-positive and negative individuals. however, the association with haart has shown an increased risk for developing dm, cardiovascular disease and metabolic syndrome. there is a relationship between diabetes and re-exposure to haart, especially among individuals under therapy with stavudine, zidovudine, didanosine, and indinavir, while the risk is reduced in case of exposure to ritonavir and nevirapine. there is a clear cumulative effect of exposure to different classes of drugs on the incidence of diabetes, exposure to nrti (nucleoside reverse transcriptase inhibitors), a combination of nrti with pi, or combination with nrti, pi, nnrti increasing the risk of developing dm 6,18-21. the most common oral manifestations associated with hiv are candidiasis, gun (necrotizing ulcerative gingivitis), pun (necrotizing ulcerative periodontitis), hairy leukoplakia, herpes simplex, hpv (human papillomavirus) and kaposi’s sarcoma22-25, but haart is a associated with a significant reduction in the occurrence of oral manifestations. currently, the oral manifestations of candidiasis, severe and rapid progression periodontal disease, hairy leukoplakia are important indicators, highlighting the increase of immunosuppression and/or the failure of haart. however, these manifestations are more evident and exuberant with the development of dm as a result of the adverse effects of haart26-29. the aim of this study was to determine the prevalence of oral manifestations in hiv/aids patients and a possible correlation of the development of dm due to haart. material and methods the research protocol was approved by the institutional ethics committee (process# 015/09 cep / ics / unip) and all patients that agreed to participate in the study signed an informed consent form. individuals of both genders aged 27 to 75 years were tested for hiv/aids and distributed into two groups: group i – hiv-positive patients referred to the ceape (center of study and care of patients with special needs) who were under haart and had developed dm as an adverse effect of the antiretroviral therapy and/or their own immunosuppression; group ii (control) hiv-negative patients with dm referred to the ceape. the tests to confirm dm control of glycosylated hemoglobin or glucose were performed by the physician who referred the patient and not by ceape, so some patients could not inform how long they had the disease. for both groups, relevant information on age, race, education, general and oral manifestations, and habits was collected. for group i, information was collected on the probable means of contamination by hiv, the t lymphocytecd4 count, viral load, and haart in use. for both groups, the following tests and examinations were performed: salivary flow rate, bacterial plaque index, gingival bleeding, periodontal pockets and dmft (decayed missing and filled tooth) index. stimulated saliva of all patients was collected for 5 min and a saliva test method (dentobuff kit®; inodon, porto alegre, brazil) was used to assess salivary flow, daily secretion of saliva, buffering capacity, salivary ph. normal salivary flow: 1.6 to 2.3 ml/ min, intermediate: between 1.0 and 1.5 ml/min and low: less than 1.0 ml/min. normal buffering capacity, ph greater than 5.5, intermediate: between 4.5 and 5.5 and low: less than 4.5. a plaque disclosing agent (erythrosine tablet) was used to obtain bacterial plaque index. the result was evaluated using the ainamo and bay test (1975)30 to determine the presence or absence of plaque in a standard binomial (dichotomous score). the visible plaque received a visible marking “1” while the non-visible plaque received marking “0” for the analysis of the dmft index. after pumice prophylaxis, clinical examination was performed using a dental mirror under relative isolation and artificial lighting to evaluate the presence of caries, missing and filled teeth, and the bleeding index, recorded by visible bleeding points within 15 s after probing, and periodontal pocket using williams 1-10 mm periodontal probe. the probe was placed on the buccal and proximal gingival margin, around each tooth to measure sulcus/pocket depth. for quantitative variables, the analysis was made through observation of minimum and maximum values, and calculation of means, standard deviations and medians. for qualitative variables, absolute and relative frequencies were calculated and the student’s t-test was used for comparison of the groups. when normality assumption was rejected, the nonparametric mann-whitney test was used. to test the homogeneity between the ratios, we used the chi-square test or fisher’s exact test (when we had frequencies below 5). significance level used for the tests was 5%. the groups showed no significant differences regarding gender (p = 0.109) and race (p = 1.000), but they differed significantly 353353353353353diagnosis of oral manifestations in hiv/aids patients who used haart and developed diabetes mellitus 354354354354354 braz j oral sci. 11(3):352-356 in age (p<0.05). the hiv group presented age (p <0.001) significantly lower than that of the control group. regarding habit, the percentage of patients who did not state habits was practically the same in both groups. results fifty-six patients between 27 and 75 years of age (mean age of 53.96 years with standard deviation of 11.22 years and median of 54 years) were evaluated. of the 28 patients examined in the hiv group, 17 (60.7%) were male and 11 (39.3%) female, 21 (75.0%) were caucasian, 6 (21.4%) african descendants and 1 (3.6%) oriental descendants and the mean age was 47 years. regarding the count of cd4 t lymphocytes, 1 (3.5%) presented t-cd4 inferior to 199 cells/mm ³, while 14 (50.0%) had t-cd4 between 200 to 499 cells/mm ³ and 13 (46.5%) t-cd4 e” 500 cells/mm ³. about the viral load (vl), 8 (28.5%) had less than 4999 virus, 2 (7.2%) between 5000 to 9999 virus, 3 (10.8%) e” 10 000 virus and 8 (28.5%) undetectable. all patients used haart; of these, 20 (71.4%) used biovir, 15 (53.6%) efavirenz, 7 (25.0%) lamivudine, 7 (25.0 %) stavudine, 5 (17.9%) zidovudine, 2 (7.1%) tenofovir, 2 (7.1%) kaletra, 2 (7.1%) abacavir, 1 (3.6%) atazanavir, a (3.6%) zalcitabine, 1 (3.6%) and a didanosine (3.6%) ritonavir. for the variable means of contamination of the hiv group, 17 (60.7%) were infected through heterosexual contact, 9 (32.3%) msm (homosexual), 1 (3.5%) bisexual and 1 (3.5%) by transfusion. from the 28 patients of the control group, 11 (39.3%) were male and 17 (60.7%) female, 21 (75.0%) were caucasian, 5 (17.8%) african descendants and 2 (7 2%) oriental descendants, and the mean age was 60.11 years. initially all variables were analyzed descriptively. the most prevalent habits in both groups were smoking and alcohol, being present in 64.3% of patients in the control group and 67.9% of patients in the hiv group. table 1 shows the frequency distributions between distribution of frequencies of the variable salivary flow and buffering capacity. table 2 presents the means, standard deviations, medians and p values for the variable dmft index, plaque index and bleeding index. table 3 presents the joint frequency distribution for the variable oral manifestations. these tables also present the descriptive p value of fisher’s exact test. it was observed that for these variables there was no significant difference. the groups did not differ significantly regarding plaque index. the most prevalent oral manifestations in the hiv group were oral candidiasis, herpes simplex and periodontal disease, and the control group caries, bleeding on probing and xerostomia. the percentage of patients who had no oral manifestation in the hiv group is 32.1% higher than in the control group that is 21.4%. the groups differ in relation to caries (p = 0.004), xerostomia (p = 0.005), oral candidiasis (p = 0.001) and herpes simplex (p = 0.010). the hiv group had a higher percentage of cases with oral candidiasis (35.7%) and herpes simplex (25.0%) and lower percentage of cases with caries (7.1%) and xerostomia (3.6%) compared to control group. the groups did not show significant difference compared with other oral manifestations. discussion regarding the oral manifestations present in the hiv group, the most common manifestation was oral candidiasis, affecting 35.7% patients, which is in accordance with the literature 22-25,28-29. besides oral candidiasis, other oral manifestations were found, such as herpes simplex (25%), periodontal disease (21.4%), oral ulcer (14.3%), hairy leukoplakia (10.7%), caries and kaposi’s sarcoma (7.1%), xerostomia, nicotinic stomatitis and lichen planus (3.6%). this is consistent with the findings of coogan et al. (2005)23 and giovani et al. (2007) 24, who reported that the most frequent dmft index plaque index bleeding index group mean standard median mean standard median mean standard median deviation (min-max) deviation (min-max) deviation (min-max) control 22.46 5.12 23.5(11-32) 49.19 29.12 52.0(0-100) 32.14 26.76 24.0(0-98) hiv 17.54 8.11 16.0(2-32) 54.80 24.98 52.0(17-100) 46.53 25.21 45.0(0-100) p*=0.019 p*=0.533 p*=0.017 table 2 mean, standard deviation, median and descriptive p level for dmft index, plaque index and bleeding index (*)descriptive level of probability of mann-whitney’s non-parametric test. (*) descriptive level of probability of fisher’s exact test. salivary flow buffering capacity group l o w normal total l o w n o r m a l total intermediate intermediate control 11(39.2) 2(7.14) 15(53.5) 28(100.0) 0(0.0) 11(39.3) 17(60.7) 28(100.0) hiv 6(21.4) 4(14.3) 18(64.3) 28(100.0) 2(7.2) 8(28.5) 18(64.3) 28(100.0) total 17(30.3) 6(10.8) 33(58.9) 56(100.0) 2(3.6) 20(35.7) 34(60.7) 56(100.0) p*=0.332 p*=0.413 table 1distribution of frequencies of salivary flow rate and buffering capacity diagnosis of oral manifestations in hiv/aids patients who used haart and developed diabetes mellitus braz j oral sci. 11(3):352-356 355355355355355 oral manifestations in hiv were candidiasis, gun (necrotizing ulcerative gingivitis), pun (necrotizing ulcerative periodontitis), hairy leukoplakia, herpes simplex, hpv (human papillomavirus) and kaposi’s sarcoma. in the control group, the most frequent oral manifestation were caries (39.3%), followed by periodontal disease (35.7%), xerostomia (32.1%) and radicular cyst (3.6%); 21.4% did not show any kind of utterance. thus, the group hiv had more oral manifestations than the control group, demonstrating the susceptibility of patients to opportunistic infections because of the immunosuppression of hiv-positive individuals. according to de wit et al. (2008)6, with the use of stavudine, the risk of developing diabetes was higher, as well as the use of zidovudine and didanosine, disagreeing with this research, which revealed that the highest risk of developing diabetes comes with prolonged use of combivir (71.4%) and efavirenz (53.6%). it was also observed in this study that the use of zalcitabine (3.6%) and didanosine (3.6%) was associated with low risk for developing diabetes. among the most common manifestations observed in patients with diabetes are xerostomia, caries and periodontal disease29, in agreement with our research, which revealed that these diseases were most prevalent in the control group (39.3% for caries, 35.7% for periodontal disease and 32.1% for xerostomia). for the analysis of dmft index in our sample, it was found a statistically significant difference (p = 0.019) between groups. for the control group, the mean was 22 for each patient and for the hiv group, the mean was 17 for each patient. in addition to the high dmft presented index, the groups also had high plaque index. the control group had a mean of 49% and 54% hiv group, which indicates that the control group is more prone to a higher prevalence of caries due to poor hygiene, the possible intake of foods rich in sucrose, presence of cariogenic bacteria and even the longest living with the disease diabetes than the patients who developed diabetes due to adverse effects of haart. so, patients should be counseled about oral hygiene and should maintain regularity in the treatments and have a lowsucrose diet. thus, oral pathologies can be minimized by maintaining the oral health of patients. the buffering capacity was also evaluated and represented as follows: low (ph<4.5), intermediate (ph between 4.5 and 5.5) and normal (ph>5.5). in the control group, 60.7% patients had normal buffering capacity, 39.3% had intermediate buffering capacity and no patient had low buffering capacity. in the hiv group, 64.3% patients had normal buffering capacity, 28.5% intermediate buffering capacity and 7.2% low buffering capacity. these results show that the control group had better buffering capacity of saliva than the hiv group, which is the capacity of saliva to maintain the a constant ph in the oral cavity, thus maintaining the health of the oral mucosa and teeth, unlike the two patients of the hiv group who had low buffer capacity. if the buffering capacity is low, the ph is not constant and the action of saliva against acid attacks to the oral cavity becomes ineffective, making these patients more susceptible to oral pathologies. in the present study, the bleeding index score was determined by a dichotomous count, marking the presence or absence of bleeding. significant differences (p = 0.017) were found between groups. in the control group, the mean of bleeding tooth surfaces was 32% and 46% in the control and hiv group, respectively. the hiv group showed higher bleeding index than the control group, and also higher prevalence of periodontal diseases, with the formation of periodontal pockets with faster evolution, which may be caused by the body’s own immunosuppression by hiv or even haart. the hiv group and the control group were diagnosed with diabetes, but this condition in the hiv group presented earlier than in the control group, suggesting a possible association with haart. there is an increased prevalence of oral manifestations in immunocompromised patients and general. the study showed a correlation between the administration of haart with the development of diabetes mellitus, as well as a increased prevalence of oral manifestations, suggesting a more pronounced worsening immunosuppression in these patients. table 3 joint frequency distribution for oral manifestations (1) descriptive level of probability of the chi-square test. (2) descriptive level of probability of fisher’s exact test. oral manifestations control % hiv % p oral candidiasis 0 0.0 10 21.2 0.001(1) n o 6 21.4 9 19.1 0.365(1) herpes simplex 0 0.0 7 14.9 0.010(2) periodontal disease 10 35.7 6 12.8 0.237(1) oral ulcer 0 0.0 4 8.6 0.111(2) hairy leukoplakia 0 0.0 3 6.4 0.236(1) kaposi‘s sarcoma 0 0.0 2 4.3 0.491(2) caries 11 39.3 2 4.3 0.004(1) xerostomia 9 32.1 1 2.1 0.005(1) nicotinic stomatitis 0 0.0 1 2.1 1.000(2) sinus oral communication 0 0.0 1 2.1 1.000(2) lichen planus 0 0.0 1 2.1 1.000(2) radicular cyst 1 3.6 0 0.0 1.000(2) diagnosis of oral manifestations in hiv/aids patients who used haart and developed diabetes mellitus braz j oral sci. 11(3):352-356 356356356356356 references 1. rinkman k, smeitink ja, romijn ja, reiss p. mitochondrial toxicity induced by nucleoside-analogue reverse-transcriptase inhibitors is a key factor in the pathogenesis of antiretroviral-therapy-related lipodystrophy. lancet. 1999; 354: 1112-5. 2. boubaker k, 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developed diabetes mellitus braz j oral sci. 15(2):109-112 comparison of the indentation strength and single-edge-v-notched beam methods for dental ceramic fracture toughness testing natália bertolo domingues1, beatriz regalado galvão2, sebastião ribeiro3, antonio alves de almeida junior4, diogo longhini5, gelson luís adabo5 1universidade estadual paulista unesp, araraquara dental school, department of pediatric dentistry and orthodontics, araraquara, sp, brazil 2universidade potiguar laureate international universities – unp, dental school, department of dental materials and prostodonthics, natal, rn, brazil 3universidade de são paulo – usp, lorena engineering school, department of engineering materials, lorena, sp, brazil 4universidade tiradentes – unit, dental school, department of dental materials and prostodonthics, aracaju, se, brazil 5universidade estadual paulista unesp, araraquara dental school, department of dental materials and prostodonthics, araraquara, sp, brazil correspondence to: gelson luís adabo faculdade de odontologia de araraquara unesp cep: 14801-903 rua humaitá, 1680 araraquara, sp, brasil phone: +55 16 3301-6415 e-mail: adabo@foar.unesp.br abstract aim: to study influence of the cooling rate after sintering a veneering porcelain (vita vm9) on fracture toughness by indentation strength (is) and single-edge-v-notched beam (sevnb) methods. methods: vita vm9 bars were sintered according to the manufacturer’s recommendation and cooled under three conditions: slow (inside the furnace from sintering temperature to room temperature); normal (inside the furnace from sintering temperature to 500 ºc and outside the furnace from 500 ºc to room temperature); and fast (outside the furnace from sintering temperature to room temperature). fracture toughness was measured by is (n=10) and sevnb (n=10) methods. data were analyzed by two-way anova (α=0.05). results: the fracture toughness obtained from sevnb (slow 1.02±0.10; normal 1.09±0.13; and fast 1,02±0.18 mpa.m1/2 cooling techniques) was significantly lower than is (slow 1.19±0.13; normal 1.17±0.07; and fast 1.16±0.06 mpa. m1/2 cooling techniques). there was no significant influence of the cooling technique (p=0.012). conclusions: the measurement technique influenced the fracture toughness values . is method overestimated the fracture toughness values. irrespective of the measuring method, cooling rate did not influence the vita vm9 veneering porcelain fracture toughness. keywords: ceramics. mechanical processes. dental materials. introduction yttrium-stabilized tetragonal zirconia polycrystals (y-tzp) for dental prosthesis became more popular because of its high mechanical performance, but it usually demands veneering with feldsphatic porcelain to improve esthetics. however, chipping the veneering porcelain in y-tzp prosthesis is more prevalent than for porcelain fused to metal1-5. a possible cause of porcelain chipping in y-tzp restorations is the difference between thermal properties of y-tzp and porcelain. y-tzp thermal conductivity and diffusivity is lower than those of porcelain veneer, and temperature gradient may interfere in residual stress development3, which depends directly on the cooling rate. it is likely that slow cooling can partially release the residual stresses, but it may reduce the beneficial compressive stress on porcelain surface4. on the other hand, a fast cooling rate does not allow for the residual stress relaxation; conversely, it reinforces porcelain on surface by compressive tempering stress. the effects of received for publication: july 06, 2016 accepted: august 25, 2016 original article braz j oral sci. april | june 2016 volume 15, number 2 http://dx.doi.org/10.20396/bjos.v15i2.8648760 110 different cooling rates have been studied by computer simulations and mechanical tests, such as flexural strength and fracture toughness. in addition to residual stresses developed because of the different ceramic association, there is a possible effect of cooling rate on the mechanical properties of porcelain, in spite of its attachment to the substructure6,7. fracture toughness, defined as the resistance to fast crack propagation at a critical stress level (kic), is widely used for mechanical characterization of dental ceramics8-10. several techniques for measuring fracture toughness of ceramics have been described, such as single-edge-v-notched beam (sevnb), indentation strength (is) and indentation fracture (if). these techniques differ by two different modalities, notch and indentation9,11-13. the sevnb, is considered the most reliable, accurate and reproducible method12,14. it is the test advised by iso 6872:200815, which specifies the requirements and the test methods for dental ceramic materials. however, this technique demands caution to be performed, because it requires the preparation of large bar shaped specimens (4 mm x 3 mm x 22 mm), straight-through notch, precise notch length measurement, and determination of the critical crack tip radius9,12. moreover, the ceramic microstructure may affect the notch preparation, and the result may be underestimated due to the pre-cracks16,17. alternatively, the is method is simple, economical and requires fewer specimens14,18. to calculate the fracture toughness, it is necessary to know the material’s elastic modulus and to measure vickers hardness in a small sample18,19. nevertheless, for running this method, special care is required. specimens should have a flat and polished surface with no crack chipping or branching. it is important to highlight that problems with high rejection rate of vickers indentations may affect the reliability of fracture toughness data, due to route deviations in cracks and chipping around the indent, errors in length measurements of crack and progressive slow crack growth9. although it does not provide exact fracture toughness values, there is some agreement with other mechanical tests for dental materials9. because of these characteristics, some authors named this method as apparent fracture toughness. the sevnb method is recommended by the iso 6872:200815, but presents some disadvantages regarding specimen size and difficulty to prepare, while the is method is easier and faster to perform, the purpose of this study was to compare fracture toughness of a veneering porcelain subjected to different cooling rates measured by is and sevnb methods. material and methods monolayer bar shaped specimens of vita vm9 (vita zahnfabrik, bad säckingen, baden-württemberg, germany) feldspathic porcelain were prepared by mixing the powder and liquid (vita modeling liquid, vita zahnfabrik,). the slurry was inserted into a polyether mold (impregum f, 3m espe, seefeld, baviera, germany) of 4.5 mm x 5.5 mm x 25 mm for sevnb or 2.4 mm x 6 mm x 14.5 mm for is, 20% larger than the expected final dimensions because of the sintering shrinkage. the excess liquid was blotted with absorbent paper before the specimen’s removal from the mold. the specimens were sintered in an oven (edg, são carlos, sp, brazil) according to the manufacturer’s instructions and cooled at three different rates: fast, slow and regular (table 1). comparison of the indentation strength and single-edge-v-notched beam methods for dental ceramic fracture toughness testing table 1 cooling methods applied on the specimens, according to the group. groups method of cooling approximate time to the cooling slow samples were left inside the closed, turned-off furnace until they reached room temperature. 8 h normal the elevator of the furnace was lowered, and when the temperature inside the furnace reached 500 ºc, the samples were removed and cooled at room temperature. 20 min fast samples were removed from the furnace immediately after the holding time and blasted by compressed air. 5 s fracture toughness by sevnb after cooling, the specimens (n=10) were bonded on a metal plate and rectified by a grinding machine (rectifier flat tangential model ta42; ferdimat, são paulo, sp, brazil) until reaching the final dimensions of 4±0.25 mm width, 1.2 0.2 mm thickness and 22 mm length. a cut was made across the surface of the specimens, with a diamond cutting disc (0.3 mm thick), perpendicular to the length of the bars, as a starter notch 0.5 mm deep. the produced notches were filled with diamond polishing pastes (3 µm) and it were finished using a razor blade attached to a custom machine with an uniform backand-forth movements to achieve a controlled and smooth notch. the notches were examined in an optical microscope (m80; leica microsystems ltd., heerbrugg, st. gallen, switzerland) to check the depth, which ranged from 0.8 mm to 1.2 mm. the specimens were cleaned with acetone in an ultrasonic bath15. the specimens were placed with the v-shaped notch down and loaded at the speed of 0.5 mm/min, at room temperature, in a universal testing machine (mts 810 material test system, eden prairie, mn, usa). the thickness (b), and width (w) of each specimen were measured with a caliper. the depths of the v-shaped notches were measured in three positions (a1, a2, a3) using a stereomicroscope (m80, leica) at 50x magnification. to accept each test, an optical microscope (m80, leica microsystems) verified if the fracture started at the bottom of the v-shaped notch and continued for the entire extent. the fracture toughness, kic, was calculated by the following equations: braz j oral sci. 15(2):109-112 111 where: kic = sevnb fracture toughness (mpa.m 1/2). σ = fracture strength (mpa) p = fracture load (mn) b = specimen thickness (mm) w = specimen width (mm) s1 – s2 = difference between the span’s upper and lower support (mm) y = form factor of stress intensity fracture toughness by indentation strength (is) for indentation strength (is) test, the bars (12 mm x 5 mm x 2 mm) were embedded along their axis in acrylic resin (jet artigos odontológicos clássico ltda., são paulo, sp, brazil), and ground by 120to 1500-grit wet sic paper discs for 3 min each at 400 rpm speed. the polish was made by monocrystalline diamond suspension in an aqueous base, particle size of 3 µm (metadi; buehler, lake bluff, il, usa) with polishing cloth (microcloth; buehler) for 10 min. the specimens were cleaned by ultrasound in distilled water for 12 min (n=10). vickers hardness was measured in the buehler model 16006300 (buehler) hardness tester with a 9.8 n load for 20 s on a 5 mm x 2 mm cross-sectional area. cracks resulting from the vickers indentation were measured immediately to avoid slow crack growth after printing, started by the stress field that acts upon loading. the images of the cracks were made with a digital video camera model tk-c1380u (jvc, tokyo, honshu, japan) coupled to the hardness tester. the crack extent was measured with an image analysis software (leica application suite ez; leica microsystems ltd). the fracture toughness (kic) was calculated by the following equation: statistics statistical analyzes were performed using two-way anova and tukey’s post hoc test (α<0.05). results the is method showed significantly higher values of fracture toughness compared to the sevnb method (p=0.012). however, two-way anova was not significant for cooling rate (p=0.144) and interactions between cooling rate and fracture toughness method (p=0.091) (table 2). comparison of the indentation strength and single-edge-v-notched beam methods for dental ceramic fracture toughness testing kic = p s1 s2 3√ × × b√ w w 2 (1 α)1,5 yα y = 1.988 7-1.326α (3.49-0.68α+1.35α 2)α(1-α) (1+ α)2 kic = 0.016 e p× 1 2 h c( (( ( 3 2 where: kic: is fracture toughness (mpa.m 1/2) e = modulus of elasticity of the tested material (gpa) p = force at applied for the hardness test (n) h = vickers hardness (gpa) c = largest lateral extension of the crack (m) table 2 comparison of is and sevnb methods according to the cooling protocol (mean ± sd). cooling protocol method slow normal fast is 1.19 ± 0.13aa 1.17 ± 0.07aa 1.16 ± 0.06aa sevnb 1.02 ± 0.13ab 1.09 ± 0.09ab 1.01 ± 0.32ab different uppercase letters indicate significant differences in columns (p<0.05) different lowercase letters indicate significant differences in rows (p<0.05). discussion fracture toughness is a useful property to assess the reliability of a material subjected to stresses of mechanical nature20. the value of fracture toughness can be affected by factors such as the type of tested dental materials and chosen technique9,21. the is and sevnb methods were chosen to compare the fracture toughness of dental ceramics because they are the most widely used in the literature. in this study, it was found that there was difference between sevnb and is, but cooling rate was not significant irrespective of the measurement method. sevnb method is considered the “golden standard” to determine fracture toughness values in ceramics12,15. our sevnb data are in agreement with other studies9,13,21, indicating that this method may reflect the actual material properties due to precise and controlled fracture induced by the v-notch in the specimens22. the sevnb method has been considered as more reliable and accurate, because it provides reproducible toughness values14. on the other hand, the sevnb method is quite sensitive, especially because it requires meticulous notch preparation9. if the critical notch length is underestimated; the toughness will be lower than the actual fracture toughness9,14 and if the notch root radius is larger, it may produce overestimated results12. with regard to is technique, overestimated fracture toughness values were found. chai and lawn23 suggested that is method could be a simple and fast way to evaluate the fracture toughness of glassy materials, as few specimens are required for the test9. however, according to fisher and marx14, this technique is not an appropriate tool to determine exactly the fracture toughness of an unknown ceramic material, because the term e =0.018 is a constant value in the equation, but it should not be, since it depends on specific characteristics of each tested ceramic material. in braz j oral sci. 15(2):109-112 112 comparison of the indentation strength and single-edge-v-notched beam methods for dental ceramic fracture toughness testing addition, scherrer et al.9 pointed out that there is greater dispersion of data by is method, for being a superficial measure that may be influenced by residual stresses on the surface. moreover, if the indenter produces chipping, the measurement of hardness mark and resulting cracks may produce inaccurate parameters to introduce in the equation. actually, it is difficult to measure precisely the crack length24 and the crack measurement may vary because of slow crack growth9. conversely, our results exhibited low standard deviation. this may be due to the close finishing protocol that allowed clear visualization of the mark and cracks, as well as the time standardization for measuring the cracks. regardless of this, is is an affordable alternative method14 and it should be used only when the standard sevnb method cannot be performed, such as for specimens obtained from small dental ceramic blocks. additionally, the manufacturers recommended slow cooling to reduce clinical failure rates and there is no standardization in the different cooling methods25. the ideal cooling protocol is not yet established. the effect of thermal properties of zirconia and feldspathic porcelain on veneer chipping has been addressed extensively in recent years, but the possible effect solely on the porcelain was not studied, in spite of being applied on zirconia. in theory, cooling rate may potentially induce phase transformation in leucite, and it may interfere on the mechanical properties2,6,7,26,27. however, in the present study, different methods of cooling did not differ from each other and there was no interaction with the fracture toughness techniques. in summary, cooling rates did not influence fracture toughness of the studied porcelain, irrespective of the measurement method, but the is method overestimates the fracture toughness values. acknowledgements the authors acknowledge the support of the sao paulo research foundation (fapesp) grants 2010/05469-9 and 2010/05395-5. references 1. sailer i, feher a, filser f, gauckler lj, luthy h, hammerle ch. five year clinical results of zirconia frameworks for posterior fixed partial dentures. int j prosthodont. 2007 jul-aug;20(4):383-8. 2. choi je, waddell jn, swain mv. pressed ceramics onto zirconia. part 2: indentation fracture and influence of cooling rate on residual stresses. dent mater. 2011 nov;27(11):1111-8. doi: 10.1016/j.dental.2011.08.003. 3. swain mv. unstable cracking (chipping) of veneering porcelain on allceramic dental crowns and fixed partial dentures. acta biomater. 2009 jun;5(5):1668-77. doi: 10.1016/j.actbio.2008.12.016. 4. göstemeyer g, jendras m, dittmer mp, bach f, stiesch m, kohorst p. influence of cooling rate on zirconia/veneer interfacial adhesion. acta biomater. 2010 dec;6(12):4532-8. doi: 10.1016/j.actbio.2010.06.026. 5. pjetursson be, sailer i, makarov na, zwahlen m, thoma ds. all-ceramic or metal-ceramic tooth-supported fixed dental prostheses (fdps)? a systematic review of the survival and complication rates. part ii: multiple-unit fdps. dent mater. 2015 jun;31(6):624-39. doi: 10.1016/j. dental.2015.02.013. 6. mackert jr, jr., evans al. effect of cooling rate on leucite volume fraction in dental porcelains. j dent res. 1991 feb;70(2):137-9. 7. mackert jr, jr., twiggs sw, russell cm, williams al. evidence of a critical leucite particle size for microcracking in dental porcelains. j dent res. 2001 jun;80(6):1574-9. 8. mecholsky jj jr. fracture mechanics principles. dent mater. 1995 mar;11(2):111-2. 9. scherrer ss, denry il, wiskott hw. comparison of three fracture toughness testing techniques using a dental glass and a dental ceramic. dent mater. 1998 jul;14(4):246-55. 10. wang rr, lu cl, wang g, zhang ds. influence of cyclic loading on the fracture toughness and load bearing capacities of all-ceramic crowns. int j oral sci. 2014 jun;6(2):99-104. doi: 10.1038/ijos.2013.94. 11. wang h, pallav p, isgro g, feilzer aj. fracture toughness comparison of three test methods with four dental porcelains. dent mater. 2007 jul;23(7):905-10. 12. fischer h, waindich a, telle r. influence of preparation of ceramic sevnb specimens on fracture toughness testing results. dent mater. 2008 may;24(5):618-22. 13. triwatana p, srinuan p, suputtamongkol k. comparison of two fracture toughness testing methods using a glass-infiltrated and a zirconia dental ceramic. j adv prosthodont. 2013 feb;5(1):36-43. doi: 10.4047/ jap.2013.5.1.36. 14. fischer h, marx r. fracture toughness of dental ceramics: comparison of bending and indentation method. dent mater. 2002 jan;18(1):12-9. 15. international organization for standardization. iso 6872: 2008: dental ceramics, switzerland; 2008. 16. hermann r, moffatt j, plumbridge wj. a comparison of crack initiation techniques for ceramics. j mater sci lett. 1995;14:282-4. 17. quinn jb, sundar v, lloyd ik. influence of microstructure and chemistry on the fracture toughness of dental ceramics. dent mater. 2003 nov;19(7):603-11. 18. anstis gr, chantikul p, lawn br, marshall bd. a critical evaluation of indentation techniques for measuring fracture toughness: i, direct crack measurement. j am ceram soc. 1981;64(9):533-8. 19. evans ag, charles ea. fracture toughness determinations by indentation. j am ceram soc. 1976;59:371-2. 20. pecnik cm, courty d, muff d, spolenak r. fracture toughness of esthetic dental coating systems by nanoindentation and fib sectional analysis. j mech behav biomed mater. 2015 jul;47:1-11. doi: 10.1016/j. jmbbm.2015.03.006. 21. coldea a, swain mv, thiel n. in-vitro strength degradation of dental ceramics and novel picn material by sharp indentation. j mech behav biomed mater. 2013 oct;26:34-42. doi: 10.1016/j.jmbbm.2013.05.004. 22. strecker k, ribeiro s, hoffmann mj. fracture toughness measurements of lps-sic: a comparison of the indentation technique and the sevnb method. mater res. 2005 feb;8(2):121-4. 23. chai h, lawn br. a universal relation for edge chipping from sharp contacts in brittle materials: a simple means of toughness evaluation. acta mater. 2007 apr;55:2555-61. 24. guazzato m, albakry m, swain mv, ironside j. mechanical properties of in-ceram alumina and in-ceram zirconia. int j prosthodont. 2002 jul-aug;15(4):339-46. 25. almeida aa jr, longhini d, domingues nb, santos c, adabo gl. effects of extreme cooling methods on mechanical properties and shear bond strength of bilayered porcelain/3y-tzp specimens. j dent. 2013 apr;41(4):356-62. doi: 10.1016/j.jdent.2013.01.005. 26. cesar pf, yoshimura hn, miranda junior wg, okada cy. correlation between fracture toughness and leucite content in dental porcelains. j dent 2005 oct;33(9):721-9. 27. ong jl, farley dw, norling bk. quantification of leucite concentration using x-ray diffraction. dent mater. 2000 jan;16(1):20-5. braz j oral sci. 15(2):109-112 braz j oral sci. 15(3):234-237 evaluation of antioxidant efficacy of purslane extract in patients with recurrent aphthous stomatitis: a randomized, placebo-controlled, triple-blinded, clinical trial mahsa mohammadzadeh1, nima rezaei2, elnaz kamran1, faraneh abdolhoseinpour3, mohsen mohammadzadeh4 isaac firooze moqadam5,shamsolmoulouk najafi6,7, mohsen hoseini ahmadabadi8 1postgraduate student of orthodontics, dental branch, islamic azad university, tehran, iran 2research center for immunodeficiencies, children’s medical center, tehran university of medical sciences, tehran, iran 3postgraduate student of pediatric dentistry, dental branch, islamic azad university, tehran, iran 4d.d.s, msc,ophtalmologist, north-khorasan university of medical sciences,bojnourd,iran. 5d.d.s, msc, periodontist, private practice. 6dental research center, school of dentistry, tehran university of medical sciences, tehran,iran. 7department of oral medicine, school of dentistry, tehran university of medical sciences, tehran, iran. 8chimist,pharmacy tehran university of medical sciences,tehran,iran. corresponding author: shamsolmolouk najafi, dds, ms address: department of oral medicine, school of dentistry, tehran university of medical sciences. e-mail: najafi_drsh@yahoo.com running title: purslane efficacy in treatment of aphthous stomatitis abstract background: this herbal medicine is considered a rich source of antioxidants with anti-inflammatory effects. the purpose of this study was to evaluate the effectiveness of purslane in treatment of recurrent aphthous stomatitis (ras) and also it ̓ s effect on antioxidant level. materials and methods: 50 patients were selected for this randomized triple-blind placebo-controlled trial. all subjects were randomly divided in to two groups, one group received purslane (n=25) and another group, placebo (n=25) for 3 month. superoxide dismutase (sod), glutathione peroxidase (gshpx) and total antioxidant status (tas) was measured in plasma at baseline and after 3 month of treatment. also pain intensity based on the visual analogue scale (vas), the mean interval between lesion, number of lesions and the mean duration of complete healing at baseline and in month 1, 2 and 3 were recorded. statistical analysis was performed by using mann-whitney and t-test. results: a significant decrease in pain intensity in vas scores was seen after treatment in intervention group (p<0.001). the mean duration of complete healing showed significant differences (p<0.001) between the two groups. the mean interval between lesions also showed significant differences (p<0.001) among the intervention group (33.12 days) compared with the placebo group (17.88 days). no significant differences were found regarding the number of lesions, level of erythrocyte gshpx, tas and sod. no serious side-effects occurred in either of groups. conclusions: according to this study, purslane is clinically effective in treatment of ras (number of lesions, pain intensity and duration of healing) although it is unable to change the level of antioxidants. keywords: recurrent aphthous stomatitis, antioxidants, purslane, treatment received for publication: april 7, 2017 accepted: may 27, 2017 original article braz j oral sci. july | september 2016 volume 15, number 3 http://dx.doi.org/10.20396/bjos.v15i3.8649994 introduction one of the most common oral inflammatory ulcerative diseases, involving more than 20% of population worldwide, is recurrent aphthous stomatitis (ras)1. the disease is 235 characterized by painful, round or ovoid ulcers with circumscribed margins, white-gray pseudomembrane and erythematous haloes in non-keratinized mucosa specially the lips, the wall of the cheeks, soft palate and ventral surface of the tongue and floor of the mouth2. three clinical forms of the disease has been recognized: minor (miras), major (maras) and herpetic form ulcers. miras accounts for 80% of ras patients3. the etiology of ras is unknown, but several immunologic, hematologic, allergic and psychologic disorders have been proposed as causative agents. such malnutritions as iron, folic acid and b12 vitamin deficiencies, local trauma, emotional stress, hormonal changes, and infectious agents have also been suggested as etiologic factors2,4. recently it has been proposed that free radicals may lead to ras through oxidative stress pathway5. decreasing antioxidant defenses in the body or increasing free radicals level, known as oxidative stress, has several destructive effects and often leads to tissue breakdown, which is proposed to play a significant role in development and progression of ras5. purslane or portulaca oleracea is from the portulacaceae family, which possesses antioxidant and anti-inflammatory properties6, and it has been clinically effective in the treatment of oral mucosal disorders such as oral lichen planus7. the aims of this study were to evaluate the efficacy of purslane in the treatment of ras, and its antioxidant efficacy in the serum of patients with ras. materials and methods subjects and study design the present study was a randomized, triple blinded, placebocontrolled clinical trial. fifty patients with miras comprised of 22 male and 28 female (range from 19 to 55 years) from the department of oral medicine, school of dentistry, tehran university of medical sciences were enrolled in this study based on the inclusion and exclusion criteria (table 1). table 1 selection criteria inclusion criteria 1. history of presenting at least three recurrences per year 2. clinically being in active phase 3. educated enough to understand the method and sign the informed consent form (older than 18) exclusion criteria 1. history of any local or systemic non-aphthous diseases such as diabetes, hepatitis, hiv, blood pressure, cardiovascular or neurological or respiratory disorders 2. treatment with immunosuppressive drugs 1 month prior to the study 3. treatment with iron or vitamins 3 months prior to the study 4. patients with behcet disease and any other active inflammatory bullous diseases all the patients were randomly divided into two groups (balance block randomized): 25 patients in intervention group and 25 patients in placebo group. gender, age and medical history of patients were recorded. research ethics committee of tehran university of medical sciences approved the study. written informed consent was obtained from all subjects. plant collection and extraction we collected plants from the southern part of tehran, capital city of iran. it was authenticated in faculty of pharmacy, tehran university of medical sciences. aerial parts of the plant and some of the seeds were stabilized in boiling alcohol and were extracted by continuous extractor in environmental temperature with 96˚c ethanol. this method effectively prevents any enzymatic destruction. the resulting extract was filtered by filter and centrifuge. then a vaccume-distiller was used to produce a very viscose residue. a dosage form was prepared and the extraction was granulated with an appropriate amount of lactose. the granules were filled in hard capsules, each containing 235mg extract, based on previous studies7. the capsules had the same color, with the same shape and size and were coded. intervention patients in intervention group received the capsules containing purslane extract, and patients in control group received the same capsules in shape, size and color but containing placebo. the subjects were told to take 2 capsules each day (in morning and at night) for 3 months. the investigator and subjects and analyzer were blind to the code record. outcome measures the number of lesions, the mean interval between lesions (in days), and the mean duration of complete healing (in days), at baseline and in month 1,2 and 3 were measured. moreover, the following outcome measures were recorded: pain severity: to evaluate the severity of pain, a visual analogue scale (vas) ranged from 0, showing no pain; to 10, showing extreme pain8 was used at baseline and in month1, 2 and 3. antioxidant status: in brief, blood samples were collected from subjects based on venous puncture method at baseline and in month 1, 2 and 3 in order to detect the level of total antioxidant status (tas) and suoer oxide dismutase (sod) and glutathione peroxidase (gshpx). the blood was centrifuged at 3000 rpm for 5 minutes at . ransel kit was used to measure the activity rate of sod. the activity rate of sod was measured based on inhibition of the reaction between superoxide radical and lodophenyl nitrophenol phenyl tetrazoliu-chloride (int) by sod. one unit of enzyme was defined as the amount of enzyme that inhibits 50% of int reduction rate. ransel kit was used to measure the activities of gshpx, based on reduction in absorption degree at 340 nm (after the oxidation of nadph). the measurement of tas was based on spectrophotometry. statistical analysis t-test and mann-whitney test served for comparisons of pain severity, the mean interval between lesion occurrence, and serum level of antioxidant between the two groups. statistical significance was set at 0.05. results a significant difference existed in pain severity between the two groups after 3 months, with more pain relief occurring in evaluation of antioxidant efficacy of purslane extract in patients with recurrent aphthous stomatitis: a randomized, placebo-controlled, triple-blinded, clinical trial braz j oral sci. 15(3):234-237 236 intervention group (p< 0.001) (table 2). table 2 comparison of pain relief between the two groups pain relief placebo (%) intervention (%) 1 ( 1 degree worsening) 4 0 0 ( no change) 48 4 -4 ( 4 degrees improvement) 28 40 -3 ( 3 degrees improvement) 8 20 -2( 2 degrees improvement) 8 20 -1 ( 1 degree improvement) 4 16 patients in the intervention group experienced significantly longer interval between lesion recurrence compared to the patients in control group at 3 months follow up (33.12 vs. 17.88 days respectively, p< 0.001) (table 3). at the same follow up, the decrease in healing duration was also significantly more among patients in intervention group compared to that in patients in control group (6.56±4.50 vs. 1.52±4.07 day respectively, p<0.001) (table 3). the differences in number of lesions (table 3), tas levels, gshpx levels, and sod levels (table 4) between the two groups at 3 months follow up remained insignificant (p>0.05). table 3 comparison of number of lesions, healing duration and interval between lesions recurrences intervention placebo p value healing duration 6.56±4.50 1.52±4.07 p< 0.001* interval between lesions 33.12 17.88 p< 0.001* number of lesions 1.28±1.07 0.8±1.65 p= 0.23 table 4 comparison of mean and standard deviation of tas, sod and gshpx between the two groups case n=25 control n=25 p value tas 0.01±0.23 0.04±0.2 0.62 gshpx 18.87±29.78 19.20±53.31 0.98 sod 14.6±48.91 42.44±70.53 0.11 discussion studies have shown that ras prevalence is higher among women than men9 especially between the ages of 20 to 30 years4. in our study the average age of participants was 35 years, and the majority of them were women (28 women vs. 22 men). although azizi et al.10 and gudaz et al.11 found no significant differences in antioxidant levels between ras patients and control group10,11, disorders in antioxidant system in patients with ras has been reported by several studies1,12-14. to the best of our knowledge, the present study is the first triple-blind placebo-controlled clinical trial on efficacy of antioxidant-rich purslane and its anti-inflammatory effects15,16 in treatment of ras. according to the results, no significant changes in the levels of sod, gshpx, and tas occurred in the purslanetreated patients. however, our study demonstrated a beneficial effect of purslane in healing or controlling symptoms of ras. this effect might be related to other components of this plant, such as: phosphate, zinc, iron, etc. efficacy of various drugs in treatment of ras such as rebamipide, colchicine and petonxyfilline has been studied previously9,17. none of them, however, has been as effective as purslane. however, many adverse side effects were reported for colchicine and talidomide17-19, none of which occurred in purslane-treated patients up to 3 months of treatment. in the present study we demonstrated that an oral tablet with purslane can reduce pain severity, healing duration, and can increase the interval between lesions recurrences with no adverse side effects at 3 months follow up. further studies on larger groups of patients with longer follow-up might be needed to confirm these results. aknowledgement the authors are very thankful to all patients and their families for their kind collaboration in this study. this research has been supported by tehran university of medical sciences, faculty of dentistry. conflict of interest the authors declare that they have no conflict of interests. references 1. momen-beitollahi j, mansourian a, momen-heravi f, amanlou m, obradov s, sahebjamee m. assessment of salivary and serum antioxidant status in patients with recurrent aphthous stomatitis. med oral patol oral cir bucal. 2010 jul 1;15(4):e557-61. 2. koybasi s, parlak ah, serin e, yilmaz f, serin d. recurrent aphthous stomatitis: investigation of possible etiologic factors. am j otolaryngol. 2006 jul-aug;27(4):229-32. 3. altenburg a, zouboulis cc. current concepts in the treatment of recurrent aphthous stomatitis. skin therapy lett. 2008 sep;13(7):1-4. 4. jurge s, kuffer r, scully c, porter sr. number vi recurrent aphthous stomatitis. oral dis. 2006 jan 1;12(1):1-21. 5. gurel a, altinyazar hc, unalacak m, armutcu f, koca r. purine catabolic enzymes and nitric oxide in patients with recurrent aphthous ulceration. oral dis. 2007 nov 1;13(6):570-4. 6. movahedian a, ghannadi a, vashirnia m. hypocholesterolemic effects of purslane extract on serum lipids in rabbits fed with high cholesterol levels. int j pharmacol. 2007;3(3):285-9. 7. agha hosseini f, borhan mojabi k, monsef esfahani hr, mirzaii dizgah i, etemad moghadam s, karagah a. efficacy of purslane in the treatment of oral lichen planus. phytotherapy research. 2010 feb 1;24(2):240-4. 8. scott j, huskisson ec. graphic representation of pain. pain. 1976 jun 1;2(2):175-84. 9. mimura ma, hirota sk, sugaya nn, sanches jr ja, migliari da. systemic treatment in severe cases of recurrent aphthous stomatitis: an open trial. clinics. 2009 mar;64(3):193-8. evaluation of antioxidant efficacy of purslane extract in patients with recurrent aphthous stomatitis: a randomized, placebo-controlled, triple-blinded, clinical trial braz j oral sci. 15(3):234-237 237 evaluation of antioxidant efficacy of purslane extract in patients with recurrent aphthous stomatitis: a randomized, placebo-controlled, triple-blinded, clinical trial 10. azizi a, shah siah s, madhani a. comparison of amount of salivary total antioxidant in patients with recurrent aphtous stomatitis. j dent med. 2012 apr 15;25(1):14-8. 11. gunduz k, ozturk g, sozmen ey. erythrocyte superoxide dismutase, catalase activities and plasma nitrite and nitrate levels in patients with behcet disease and recurrent aphthous stomatitis. clinical and experimental dermatology. 2004 mar 1;29(2):176-9. 12. saral y, coskun bk, ozturk p, karatas f, ayar a. assessment of salivary and serum antioxidant vitamins and lipid peroxidation in patients with recurrent aphthous ulceration. the tohoku journal of experimental medicine. 2005;206(4):305-12. 13. karincaoglu y, batcioglu k, erdem t, esrefoglu m, genc m. the levels of plasma and salivary antioxidants in the patient with recurrent aphthous stomatitis. j oral pathol med. 2005 jan 1;34(1):7-12. 14. arikan s, durusoy c, akalin n, haberal a, seckin d. oxidant/antioxidant status in recurrent aphthous stomatitis. oral dis. 2009 oct 1;15(7):512-5. 15. ezekwe mo, omara-alwala tr, membrahtu t. nutritive characterization of purslane accessions as influenced by planting date. plant foods hum nutr. 1999;54(3):183-91. 16. simopoulos ap, norman ha, gillaspy je, duke ja. common purslane: a source of omega-3 fatty acids and antioxidants. j am coll nutr. 1992 aug;11(4):374-82. 17. liu c, zhou z, liu g, wang q, chen j, wang l, et al. efficacy and safety of dexamethasone ointment on recurrent aphthous ulceration. am j med. 2012 mar 31;125(3):292-301. 18. stephen j. challacombe, surab alsahaf, anwar tappuni. recurrent aphthous stomatitis: towards evidence-based treatment?. curr oral health rep. 2015. 2: 158. 19. leung yy, hui ll, kraus vb. colchicine—update on mechanisms of action and therapeutic uses. inseminars in arthritis and rheumatism 2015 dec 31: 45(3): 341-350. braz j oral sci. 15(3):234-237 braz j oral sci. 15(4):280-286 profiling of pro-inflammatory cytokines in radiation induced oral mucositis (riom) among indian patients akanksha marwah1 #, zafar iqbal2#, md. zafaryab3, mahesh verma1, m.m.a rizvi2, sunita gupta1 1department of oral medicine & radiology, maulana azad institute of dental sciences, bsz marg, new delhi – 110002, india 2department of biosciences, jamia millia islamia, new delhi-110025, india #both authors contributed equaly correspondence to: dr. m.m.a rizvi, professor department of biosciences, jamia millia islamia, new delhi-110025, india. email id: rizvijmi@gmail.com contact number: +91-9911661657 abstract radiation-induced oral mucositis (riom) is aimed at evaluating the expression of nf-κβ, il-1α, il-6, il-8 and tnf-α in patients with riom so as to validate their role in the pathobiology of the disease. blood samples were collected and serum of 45 patients isolated with clinical signs and symptoms of mucositis and 10 healthy controls were also included in the study. the expression level of nf-κβ, il-1α, il-6, il-8, tnf-α was investigated using elisa. mann whitney u test was applied to find the significance of the expression of these markers in riom patients as compared to normal healthy controls and significant expression (p< 0.05) for nf-κβ, il-6, tnf-α and non-significant expression (p > 0.05) il-1α and il-8 was found. no significant change in the expression level of the cytokines was observed for patients undergoing chemotherapy and radiation therapy as well as those receiving only the radiation therapy as a part of their treatment. we have also found less expression in grade 1 of mucositis as compared to grade 4. proinflammatory cytokines indeed play a vital role in the pathogenesis as well as progression of riom. keywords: mucositis, nf-κβ, proinflammatory cytokines, radiation therapy received for publication: december 29, 2016 accepted: july 26, 2017 original article braz j oral sci. october | december 2016 volume 15, number 4 http://dx.doi.org/10.20396/bjos.v15i4.8650040 introduction incidence of various cancer types have been reported to increase multifold in recent times1,2 with oral mucositis being one of the most recurrent, symptomatic, and troubling complication of the conventional cancer treatments, viz., radiation and/or chemotherapy3,4. it can be defined as inflammatory and/or ulcerative lesions of the oral and/or gastrointestinal tract usually caused by cancer therapies5. it is mostly associated with pain and increased risk of infection thus leading to impaired nutritional status and inadequate hydration3,6. it may also be associated with increased risk for poorer outcome of cancer treatment due to the need for treatment interruption in some patient cohorts6,7. the incidence of oral mucositis can be close to 80% in patients receiving radiation therapy, with or without chemotherapy having head and neck cancer8,9 with 25-45% of the patients reporting with grade 3 or 4 mucositis10. the process of mucositis occurs in five stages or phases: initiation, message generation, signal amplification, ulceration, and healing11,12. there are numerous transcription factors involved in the establishment of mucositis, one of the most important is nuclear factor-kappa β (nf-κβ). nf-κβ activation can upregulate the expression of pro-inflammatory cytokines including tumor necrosis factor-α (tnf-α), assay employs the quantitative sandwich enzyme immunoassay technique performed using human recombinant standards according to the manufacturer’s instructions. a monoclonal antibody specific for the respective cytokines was precoated onto the microplate. standards and samples (100 μl each) were pipetted into the wells in duplicate and incubated for 2.5 hrs. so that any of the proteins, i.e., nf-κβ, il-1α, il-6, il-8 and tnf-α, if present in the sample can bind to the immobilized antibody. after washing away any unbound substances biotinylated antibody (100 μl) was added to the wells and incubated again for an hour. after washing 100 μl of hrp-streptavidin solution was added to each well followed by 45 min. incubation. following a wash to remove any unbound antibody-enzyme reagent a substrate solution (100 μl) was added to the wells and incubated for 30 min. the color developed in proportion to the amount of protein bound to the immobilized antibody in the initial step. the yellow color development was stopped and the intensity of the blue color was measured by determining its absorbance at 450 nm using an elisa plate reader (bio-rad). the concentration of proteins in the samples was calculated from the standard curve and the results were presented in picogram per milliliter (pg/ml) for il-1α, il-6, il-8 and tnf-α and in nanogram per milliliter (ng/ml) for nf-κβ. the standard curves for nf-κβ, il-1α, il-6, il-8 and tnf-α ranged from (0–500 ng/ml), (0–300 pg/ml), (0–1000 pg/ml), (0–1000 pg/ml) and (0–600 pg/ml) respectively. statistical analysis data were analyzed with descriptive statistical methods (frequency percentage and mean ± standard deviation) and mann–whitney u test (a non-parametric method) using spss (statistical package for social sciences) software version 19.0 for windows. statistical significance was defined at p < 0.05. results serum levels of nf-κβ and pro-inflammatory cytokines detectable levels of nf-κβ, il-1α, il-6, il-8 and tnf-α proteins were present in serum samples obtained from the control group (group ii). serum levels of nf-κβ, il-1α, il-6, il-8 and tnf-α were elevated in 100% (45/45), 60% (27/45), 100% (45/45), 53.34 % (24/45), 100% (45/45) of the patients respectively as compared to their concentration in the serum of healthy subjects. the mean values of nf-κβ (262.65±169.91 ng/ ml), il-6 (112.56±309.07 pg/ml) and tnf-α (372.28±472.39 pg/ml) in serum of the patients were significantly (p<0.05) higher in comparison with the values obtained from control subjects that were 25.66±9.502 ng/ml, 4.880±2.95 pg/ml and 12.00±7.0590 pg/ml for nf-κβ, il-6 and tnf-α, respectively, as revealed by mann–whitney u test2 (figure 1) mean values for il-1α (38.919± 69.99 pg/ml) and il-8 (53.38±137.88 pg/ml) were also increased as compared to their values in the control group (il-1α=26.790±10.055 pg/ml and il-8=8.690±2.534pg/ml) but statistically a non-significant difference was obtained between the two groups (figure 1). 281 interleukin-1α (il-1α), il-6 and il-813,14. the increased levels of these cytokines induce inflammatory reactions in oral mucosa and promote the damage of the underlying connective tissues reduce epithelial oxygenation and ultimately result in epithelial basal cell death and injury4,15. since tnf-α, il-1α and il-6 are efficient activators of nf-κβ and the repeated nf-κβ activation by them may amplify the mucosal damage in a vicious circle4,13,14 which hence would ultimately lead to the development of mucositis as an after effect of radiation therapy. the aim of this study is to analyze the cytokine expression of nf-κβ and its associated cytokines (il-1α, il-6, il-8, tnf-α) in the serum of patients with radiation induced oral mucositis after undergoing radiation therapy for head and neck cancers so as to validate their role in the pathobiology of the disease. materials and methods sample selection 45 patients (group i) reported to department of radiation therapy, lnjp hospital, delhi with head & neck cancer undergoing radiation therapy with/without chemotherapy and showing clinical symptoms of oral mucositis were selected for the study. around 2 ml blood was collected and centrifuged at 5000rpm. their serum was separated and stored at -80oc until further use. serum samples from 10 healthy volunteers with no history of cancer or previous radiation therapy served as the control group (group ii). approval from institutional ethical committee & prior informed consent was taken from all the patients. patient characteristics in group i 33 male and 12 female with a mean age of 49.82 years were enrolled. group ii included 10 healthy controls (5 male and 5 female) with a mean age of 44.9 years with no prior history of undergoing any radiation treatment. 39 out of 45 patients developed symptoms of mucositis during /after the treatment therapy while about 6 patients showed no visible signs of any such complications (grade 0). 27 out of 39 patients had developed early signs of mucositis (grades 1 &2) with symptomatic redness and erythema, loss of taste, mouth dryness. 12 out of 39 patients had developed severe signs of mucositis (grades 3 & 4) during the course of their treatment, with painful contiguous pseudo membraneous lesions developed along with associated dysphagia and decreased oral intake. 26 out of 45 patients had undergone chemotherapy along with the radiation treatment, while 19 out of 45 patients undertook just the radiation therapy as a part of their cancer treatment. detection of serum levels of nf-κβ, il-1α, il-6, il-8 and tnf-α the concentration of the studied biomarkers were assessed in the serum collected from patients and healthy subjects by elisa by using a commercially available enzyme-linked immunosorbent assay (elisa) kit (ray biotech, inc.). this cytokine expression in riom patients braz j oral sci. 15(4):280-286 282cytokine expression in riom patients table 1 clinical detail of patients. s.no. sex age tumour type radiation therapy chemo-therapy mucositis grade 1 f 40 tongue yes no no 0 2 m 30 tongue & fossa yes yes yes 4 3 f 64 larynx yes yes yes 1 4 m 60 tonsil-r yes yes yes 1 5 f 55 tonsil-l yes yes yes 1 6 m 26 buccal mucosa yes no yes 2 7 m 40 gingivae yes no yes 2 8 m 65 base of tongue yes yes yes 4 9 m 50 soft palate yes no yes 3 10 m 48 larynx yes yes no 0 11 f 60 base of tongue yes yes yes 3 12 m 35 base of tongue yes no yes 3 13 m 76 base of tongue yes no no 0 14 f 45 tongue yes yes yes 3 15 m 65 body of tongue yes yes yes 1 16 m 40 pharynx yes yes yes 1 17 m 68 tonsil yes yes yes 1 18 m 32 buccal mucosa & border of tongue yes yes yes 1 19 m 63 soft palate yes no no 0 20 f 45 larynx yes yes yes 1 21 m 28 tongue yes yes yes 3 22 m 45 nasopharynx yes yes yes 1 23 m 36 buccoalveolar region-r yes no yes 4 24 m 22 buccal mucosa & border of tongue yes yes yes 1 25 m 40 larynx yes yes yes 1 26 m 62 larynx yes yes yes 1 27 f 50 right tonsil yes yes yes 1 28 m 68 left tonsil yes no no 0 29 m 70 buccal mucosa-left yes no yes 1 30 m 63 buccal muco-sa-right yes no yes 3 31 m 55 base of tongue yes yes yes 1 32 m 54 buccal mucosa-left yes no yes 3 33 f 55 recurrent adenoid cystic carcinoma with secondary metastasis yes yes yes 1 34 m 62 base of tongue yes no no 0 35 f 48 tonsil yes no yes 1 36 m 27 buccal mucosa yes yes yes 3 37 f 55 tongue yes yes yes 1 38 m 71 tongue yes yes yes 1 39 f 43 larynx yes no yes 1 40 f 45 tonsilleft yes no yes 2 41 m 42 floor of mouth yes yes yes 2 42 m 65 soft palate yes no yes 2 43 m 40 base of tongue yes yes yes 2 44 m 53 larynx yes no yes 2 45 m 36 base of tongue yes no yes 4 mean values of cytokine concentration in serum of mucositis patients and healthy controls. cytokines mean±s.d. median percentage of patients showing upregulation in mucositis group as compared to control group p-value riom patients (group i) control group (group ii) riom patients (group i) control group (group ii) nf-κβ (ng/ml) 262.65±169.91 25.66±9.502 240 28.00 100% (45/45) .001 (s) il-1α(pg/ml) 38.919±69.99 26.7907±10.5 29 25.7 60% (27/45) .115(ns) il-6 (pg/ml) 112.56±309.7 4.880±2.95 45.7 5.450 100%(45/45) .001(s) il-8 (pg/ml) 53.38±137.88 8.690±2.354 9.8 9.050 53.34%(24/45) .952(ns) tnf-α (pg/ml) 372.28±472.39 12.00±7.0590 256 9.100 100% (45/45) .001(s) braz j oral sci. 15(4):280-286 283 on comparing the expression level of different proinflammatory cytokines of the patients receiving chemotherapy along with the radiation therapy and those undergoing just the radiation therapy no significant difference (p>0.05) was observed in the expression of these cytokines as depicted in figure 2 thus implying that radiation and chemotherapy treatments whether given alone or in combination as a part of treatment regimen for head and neck cancer have almost the same effect on the expression level of the studied pro-inflammatory cytokines. cytokine expression in riom patients furthermore, analyzing the expression of different cytokines (tnf-α, nf-κβ, il-8, il-6, il-α) with respect to the different grades of mucositis we have found that the expression level of studied cytokines increases with the progression of the mucositis (grade 1 to grade 4) as depicted in figure 3. fig. 1 mean serum concentrations of pro-inflammatory cytokines in control subjects and mucositis patients. fig. 2 graph depicting the mean of expression of different pro-inflammatory cytokines for patients undergoing radiation therapy along with chemotherapy (ct+rt) and those undergoing only the radiation therapy (rt). fig. 3 comparison of expression of different cytokines-nf-κβ, il-1α, il-6, il-8 and tnf-α in different grades of mucositis. discussion oral mucositis is an undesirable painful outcome endured by patients receiving radiation and/or chemotherapy for head and neck cancer treatment. radiation and chemotherapy are effective activators of several pathways in endothelia, fibroblasts and epithelia thus leading to the production and upregulation of certain pro inflammatory cytokines16,17 which have been reported to play an important role during inflammation and tissue damage18. the present study examined the expression level of nf-κβ and its associated pro-inflammatory cytokines (il-6, tnf-α, il-1α and il-8) in head and neck cancer patients undergoing radiation and/or chemotherapy as a part of their treatment. our results showed a significant increase in the expression of nf-κβ, il-6 and tnf-α (p value<0.05) in the serum of all the patients enrolled for the study as compared to the control group. however, nearly 60 % and 52.5 % of the patients showed an increase in the expression of il-1α and il-8 (p value>0.05) as compared to the control group (healthy volunteers). a marginal increase has been seen in the expression of nf-κβ due to the exposure of the tissue to chemotherapy or radiation therapy causing the production of reactive oxygen species (ros) that ultimately results in tissue injury and cell death4,11,14,16. previous studies have reported the transcription factor nf-κβ as being a regulator of inflammation signal and immunity as well as also being involved in the progression of mucositis4,11. nf-κβ regulates the expression of approximately 200 genes many of which may play the role in the pathogenesis of mucositis4,13,19 including those encoding cox-215,19-21 and proinflammatory cytokines such as tnf-α, il-6, and il-1β4,11,14,22. cox-2 intensifies and prolongs mucositis by interacting with a variety of apoptotic pathways and by providing positive feedback to nf-κβ. the increase in the expression of cox-2 can be an braz j oral sci. 15(4):280-286 initial sign of the inflammatory cascade which leads to the production of prostaglandins and further tissue damage4,11,15,19. further, upregulation in the expression of proinflammatory cytokines by the action of nf-κβ triggers the initiation of various pathways that damage epithelial cells and surrounding fibroblasts. the feedback loop formed between various proinflammatory cytokines such as tnf-α and nf-κβ promotes the cycle of inflammation, pain, and functional impairment11,13,14,17,23,24. the presence of tnf-α has been reported by previous studies related to the stimulation of early damage to connective tissue and endothelium thus initiating mesenchymal-epithelial signaling and reducing epithelial oxygenation ultimately resulting in epithelial basal cell death and injury11,15. several studies have also reported the increase in the expression of il-6 and il-8 as a downstream product of nf-κβ activation. the upregulation of these proinflammatory cytokines has been known to mediate neutrophil extravasation and tissue infiltration during inflammation thereby playing an important role in the migration of leukocytes into the site of inflammation15,25-27. the amplification of these biological events via positive feedback loops as well as stimulation by bacterial cell wall products results in the widespread tissue damage as seen in the clinical setting as ulceration. this ulcerative phase is primarily responsible for the main clinical symptoms of mucositis (pain, inflammation, and loss of function)11,17,28. a study by ong et al., in 2010 partially related to our study was done to characterize the expression of pro-inflammatory cytokines in the gastrointestinal tract using a rat model of fractionated radiotherapy-induced toxicity. ong found that a significant upregulation of il-1β, il-6 and tnf mrna levels in the jejunum and colon thus concluding the role of pro-inflammatory cytokines in radiotherapy-induced gastrointestinal mucositis22. another study29 implicated the role of nf-κβ, proinflammatory cytokines, cox and mmps in the pathogenesis of mucositis as these factors were being expressed at elevated levels in both serum and tissue following radiotherapy and/or chemotherapy29. our findings have also been supported by studies done on an animal model by logan et al that reported the changes in the expression of nf-κβ, tnf, il-1β and il-6 in the mucosa and serum following chemotherapy orated a statistically increased oral mucosal stain17,30. another study done by logan et al., on a human biopsy demonstrated the increased level of nf-κβ and cox-2 in patients with chemotherapy as well as radiotherapy17. however, ikebe et al., observed the expression of nf-κβ to be reduced after chemo radiotherapy and concluded that the vulnerability of oral mucosa undergoing chemo radiation may be associated with reduced nf-κβ expression and impaired growth activity31. the role of systemic inflammation in the development of mucositis was further supported by studies showing that the therapy targeted to alter cytokine expression is able to modify the course of mucositis32. animal33 and human studies34 have demonstrated a decrease in the occurrence or severity of mucositis following the administration of tnf inhibitors. the cytokines are reported to be unregulated when individually subjected to radiation treatment11,15,19,20,22,35 and chemotherapy24,30,36-41. however, no such studies have been made on patients undergoing chemotherapy and radiation therapy simultaneously. we evaluated the level of cytokines (tnf-α, nfκβ, il-8, il-6, il-α) in patients receiving radiation treatment and those receiving chemotherapy along with the radiation treatment. this study for the first time reveals that there is no significant variation in the levels of cytokines in the patients undergoing radiation as well as chemotherapy and those undergoing just the radiation treatment. this finding demonstrated that the severity of mucositis does not depend on the type of treatment (radiation and/or chemotherapy) being administered to the patient and that both the treatments have more or less the same effect on the level of these cytokines. we further correlate our study to the clinic-pathological parameters (grading of mucositis). interestingly we found that advanced stage (grade 4) mucositis demonstrated the increase in the expression of cytokines (tnf-α, nf-κβ, il-8, il-6, il-α) as compared to early stage (grade o & 1) of mucositis as well as healthy control. this finding was supported by ong et al., who also reported the importance of these proinflammatory cytokines in the development and severity of radiotherapy induced gastrointestinal mucositis in rats22. it is therefore very clear to understand that inflammatory cytokines (tnf-α, nf-κβ, il-8, il-6, il-α) play a vital role in the progression of radiation induced oral mucositis. conclusion on the basis of above findings, we are able to conclude that nf-κβ and its associated cytokines (tnf-α, il-8, il-6, il-α) are involved in the pathogenesis of radiation induced oral mucositis. we further provide evidence for the association of these pro-inflammatory cytokines with the progression of this disease into more advanced stages. continued research will undoubtedly provide a better understanding of the dynamics leading to mucosal injury and to more effective measures for prevention and treatment. refinement of the five-stage model of mucositis pathogenesis may aid in the rational use of therapies to maximize their efficacy. further studies are necessary to elucidate the exact role of these cytokines as biomarkers for this disease. acknowledgement this work has been supported by grants from university grants commission (ugc), bahadur shah zafar marg, delhi-110 002, india. conflict of interest none declared. references 1. rizvi mm, alam ms, ali a, mehdi sj, batra s, mandal ak. aberrant promoter methylation and inactivation of pten gene in cervical carcinoma from indian population. j cancer res clin oncol. 2011 aug;137(8):1255-62. doi: 10.1007/s00432-011-0994-0. 2. rizvi mm, ali a, mehdi sj, saluja ss, mishra pk. association of 284cytokine expression in riom patients braz j oral sci. 15(4):280-286 285 cytokine expression in riom patients epigenetic alteration in pten gene with colorectal cancer progression among indian population. int j colorectal dis. 2013 feb;28(2):283-4. doi: 10.1007/s00384-012-1482-y. 3. dirix p, nuyts s, van den bogaert w. radiation-induced xerostomia in patients with head and neck cancer: a literature review. cancer. 2006 dec;107(1):2525-34. 4. sonis st, elting ls, keefe d, peterson de, schubert m, hauer-jensen m, et al., perspectives on cancer therapy-induced mucosal injury: pathogen¬esis, measurement, epidemiology, and consequences for patients. cancer. 2004 may;100(9 suppl):1995-2025. 5. peterson de, bensadoun rj, roila f; esmo guidelines working group. management of oral and gastrointestinal mucositis: esmo clinical practice guidelines. ann oncol. 2011 sep;22 suppl 6:vi78-84. doi: 10.1093/annonc/mdr391. erratum in: ann oncol. 2012 mar;23(3):810. 6. peterson de. new strategies for management of oral mucositis in cancer patients. j support oncol. 2006 feb;4(2 suppl 1):9-13. 7. pico jl, avila-garavito a, naccache p. mucositis: its occurrence, consequences, and treatment in the oncology setting. oncologist. 1998;3(6):446-51. 8. trotti a, bellm la, epstein jb, frame d, fuchs hj, gwede ck, et al., mucositis incidence, severity and associated outcomes in patients with head and neck cancer receiving radiotherapy with or without chemotherapy: a systematic literature review. radiother oncol. 2003 mar;66(3):253-62. 9. silverman s jr. diagnosis and management of oral mucositis. j support oncol. 2007 feb;5(2 suppl 1):13-21. 10. rubenstein eb, peterson de, schubert m, keefe d, mcguire d, epstein j, elting ls, fox pc, cooksley c, sonis st; mucositis study section of the multinational association for supportive care in cancer; international society for oral oncology. clinical practice guidelines for the prevention and treatment of cancer therapy-induced oral and gastrointestinal mucositis. cancer. 2004 may 1;100(9 suppl):2026-46. 11. sonis st, o’donnell ke, popat r, bragdon c, phelan s, cocks d, et al. the relationship between mucosal cyclooxygenase-2 (cox-2) expression and experimental radiation-induced mucositis. oral oncol. 2004 feb;40(2):170-6. 12. dorr w, hamilton cs, boyd t, reed b, denham jw. radiation-induced changes in cellularity and proliferation in human oral mucosa. int j radiat oncol biol phys. 2002 mar;52(4):911-7. 13. sonis st. the biologic role for nuclear factor-kappab in disease and its potential involvement in mucosal injury associated with anti-neoplastic therapy. crit rev oral biol med. 2002;13(5):380-9. 14. sonis st. pathobiology of oral mucositis: novel insights and opportunities. j support oncol. 2007 oct;5(9 suppl 4):3-11. 15. yeoh asj, bowen jm, gibson rj, keefe dm. nuclear factor κb (nfκb) and cyclooxygenase-2 (cox-2) expression in the irradiated colorectum is associated with subsequent histopathological changes. int. j. radiation oncology biol. phys. 2005 dec;63(5):1295-303. 16. satheesh kumar ps. radiation mucositis. in: racz gb, noe ce, editors. pain management current issues and opinions. intech. chapter 22. isbn: 2012.978-953-307-813-7. available from: https://www.intechopen. com/books/pain-management-current-issues-and-opinions/radiationmucositis. 17. logan rm, gibson rj, sonis st, keefe dm. nuclear factor-κappab (nfkappab) and cyclooxygenase-2 (cox-2) expression in the oral mucosa following cancer chemotherapy. oral oncol. 2007 apr;43(4):395-401. 18. sultani m, stringer am, bowen jm, gibson rj. anti-inflammatory cytokines: important immunoregulatory factors contributing to chemotherapy-induced gastrointestinal mucositis. chemother res pract. 2012;2012:490804. doi: 10.1155/2012/490804. 19. yeoh a, gibson r, yeoh e, bowen j, stringer a, giam k, et al. radiation therapy-induced mucositis: relationships between fractionated radiation, nf-κb, cox-1, and cox-2. cancer treat rev. 2006 dec;32(8):645-51. 20. brach ma, hass r, sherman ml, gunji h, weichselbaum r, kufe d. ionizing radiation induces expression and binding activity of the nuclear factor, kappa b. j clin invest. 1991 aug;88(2):691-5. 21. lee sj, dimtchev a, lavin mf, dritschilo a, jung m. a novel ionizing radiation-induced signaling pathway that activates the transcription factor nf-kappab. oncogene. 1998 oct;17(14):1821-6. 22. ong zy, gibson j, bowen jm, stringer am, darbyjm, logan rm, et al. pro-inflammatory cytokines play a key role in the development of radiotherapy-induced gastrointestinal mucositis. radiat oncol. 2010 mar;5:22. doi: 10.1186/1748-717x-5-22. 23. sonis st. mucositis as a biological process: a new hypothesis for the development of chemotherapy-induced stomatotoxicity. oral oncol. 1998 jan;34(1):39-43. 24. logan rm, stringer am, bowen jm, gibson rj, sonis st, keefe dm. serum levels of nfkappab and pro-inflammatory cytokines following administration of mucotoxic drugs. cancer biol ther. 2008 jul;7(7):113945. 25. kelly cp, keates s, siegenberg d, linevsky jk, pothoulakis c, brady hr. il-8 secretion and neutrophil activation by ht-29 colonic epithelial cells. am j physiol. 1994 dec;267(6 pt 1):991-7. 26. campbell bj, yu lg, rhodes jm. altered glycosylation in inflammatory bowel disease: a possible role in cancer development. glycoconj. 2001 nov-dec;18(11-12):851-8. 27. ikeda y, ito m, matsuu m, shichijo k, fukuda e, nakayama t et al., expression of icam-1 and acute inflammatory cell infiltration in the early phase of radiation colitis in rats. j radiat res. 2000 sep;41(3):279-91. 28. duncan m, grant g. oral and intestinal mu¬cositis—causes and possible treatments. aliment pharmacol ther. 2003 nov;18(9):853-74. 29. scully c, sonis s, diz pd. oral mucositis. oral dis. 2006 may;12(3):22941. 30. logan rm, gibson rj, bowen jm, stringer am, sonis st, keefe dm. characterisation of mucosal changes in the alimentary tract following administration of irinotecan: implications for the pathobiology of mucositis. cancer chemother pharmacol. 2008 jun;62(1):33-41. 31. ikebe t, yamasakia k, takamunea y, nakayamaa h, shinohara m. reduced expression of nuclear factor kb in oral mucosa undergoing preoperative chemoradiotherapy. oral science international. 2012 nov;992):33-7. doi:10.1016/s1348-8643(12)00008-0. 32. khaw a, logan r, keefe d, bartold m. radiation-induced oral mucositis and periodontitis – proposal for an inter-relationship. 2014 apr;20(3):e7-18. doi: 10.1111/odi.12199. 33. lima v, brito ga, cunha fq, rebouças cg, falcão ba, augusto rf, et al., effects of the tumour necrosis factor-alpha inhibitors pentoxifylline and thalidomide in short-term experimental oral mucositis in hamsters. eur j oral sci. 2005 jun;113(3):210-7. 34. ferra c, de sanjose s, lastra cf, martí f, mariño el, sureda a, et al. pentoxifylline, ciprofloxacin and prednisone failed to prevent transplant related toxicities in bone marrow transplant recipients and were associated with an increased incidence of infectious complications. bone marrow transplant. 1997 dec;20(12):1075-80. 35. yeoh asj, gibson rj, yeoh e, bowen jm, stringer am, giam ka, et al. a novel animal model to investigate fractionated radiotherapy-induced alimentary mucositis: the role of apoptosis, p53, nuclear factor-kb, cox-1, and cox-2. mol cancer ther. 2007 aug;6(8):2319-27. 36. logan rm, stringer am, bowen jm, yeoh as, gibson rj, sonis st, et al. the role of pro-inflammatory cytokines in cancer treatment-induced alimentary tract mucositis: pathobiology, animal models and cytotoxic drugs. cancer treat rev. 2007 aug;33(5):448-60. 37. gibson rj, keefe dm, thompson fm, clarke jm, goland gj, cummins ag. effect of interleukin-11 on ameliorating intestinal damage after methotrexate treatment of breastcancer in rats. dig dis sci. 2002 dec;47(12):2751-7. 38. gibson rj, bowen jm, inglis mr, cummins ag, keefe dm. irinotecan braz j oral sci. 15(4):280-286 286cytokine expression in riom patients causes severe small intestinal damage, as well as colonic damage, in the rat with implanted breast cancer. j gastroenterol hepatol. 2003 sep;18(9):1095-100. 39. gibson rj, bowen jm, cummins ag, keefe dm. relationship between dose of methotrexate, apoptosis, p53/p21 expression and intestinal crypt proliferation in the rat. clin exp med. 2005 mar;4(4):188-95. 40. soares pmg, mota j, souza ep, justino pf, franco ax, cunha fq, et al. inflammatory intestinal damage induced by 5-fluorouracil requires il-4. cytokine. 2013 jan;61(1):46-9. doi: 10.1016/j.cyto.2012.10.003. 41. logan rm, stringer am, bowen jm, gibson rj, sonis st, keefe dm. is the pathobiology of chemotherapy-induced alimentary tract mucositis influenced by the type of mucotoxic drug administered? cancer chemother pharmacol. 2009 jan;63(2):239-51. doi: 10.1007/ s00280-008-0732-8. braz j oral sci. 15(4):280-286 1 volume 16 2017 e17065 original article 1 msc department of pediatric dentistry, university of são paulo school of dentistry of ribeirão preto, ribeirão preto, sp, brazil. 2 dds department of pediatric dentistry, university of são paulo school of dentistry of ribeirão preto, ribeirão preto, sp, brazil. 3 associate professor department of pediatric dentistry, university of são paulo school of dentistry of ribeirão preto, ribeirão preto, sp, brazil. 4 full professor department of pediatric dentistry, university of são paulo school of dentistry of ribeirão preto, ribeirão preto, sp, brazil. 5 phd department of pediatric dentistry, university of são paulo school of dentistry of ribeirão preto, ribeirão preto, sp, brazil. corresponding author: juliana arid university of são paulo school of dentistry of ribeirão preto avenida do café s/n, 14040-904 ribeirão preto, sp, brazil tel: +55-16-3315-3995 fax: +55-16-3633-0999 e-mail: juliana_arid@hotmail.com received: june 19, 2017 accepted: august 25, 2017 nutritional status is associated with permanent tooth eruption chronology juliana arid1*, mariana cecília vitiello2, raquel assed bezerra da silva3, léa assed bezerra da silva4, alexandra mussolino de queiroz3, erika calvano küchler5, paulo nelson-filho4 either obesity and underweight are public health concerns that affect the development of children. aim: the aim of this study was to evaluate whether the nutritional status affects permanent tooth eruption chronology in brazilian children. methods: a hundred sixty children were examined by a pediatric dentistry and at the clinical examination, the number of erupted permanent teeth was evaluated. the anthropometric measurements of the children were determined, and they were divided into groups: underweight, eutrophic, overweight and obese. the association between delayed tooth eruption and nutritional status groups was evaluated using chi-square test. the shapiro–wilk test was used to verify the normality of the data. to compare the mean number of delayed teeth according to nutritional status’ groups kruskal-wallis test with multiple comparison by dunn’s test was performed. results: fifty-six children had delayed tooth eruption in at least one permanent teeth and delayed tooth eruption was more common in underweight children than in eutrophic children (p=0.0091). conclusion: in conclusion, our study suggests that underweight brazilian children have a higher incidence of delayed eruption. keywords: tooth eruption. nutritional status. children. dentistry http://dx.doi.org/10.20396/bjos.v16i0.8650503 2 arid et al. introduction childhood obesity is a public health concern and has been increasing over the years1. according to the brazilian institute of geography and statistics (ibge), the obesity in brazilian children ranges from 8.2% and 11.9%, depending on the gender and the geographic region2. it is well known that obesity is associated to adversely health issues, including timing of puberty3 and overweight children are taller than non-overweight children at the same age4. nutritional deprivation in children affects the development of most body systems5. some studies have hypothesized that some systemic conditions, such as celiac disease, diabetes mellitus, congenital abnormalities and cancer affect tooth eruption1,6-8. tooth eruption consists in the movement of the tooth from the osseous crypt into the oral cavity. this phenomenon could be affected by many factors, including molecular signing9, and hormonal and growth mediators10. overweight and obese children have hormonal metabolism alterations11, so it is possible that they have alterations in tooth eruption. it has been demonstrated that obese and overweight children have more erupted teeth when compared to their age-matched peers, suggesting that obese and overweight children´s teeth erupt earlier1,11. although a recent study demonstrated that malnutrition has no effects on the time of tooth formation5, previous studies showed an association with delayed eruption of the primary dentition12 and of the permanent dentition in underweight children6. nevertheless, this relationship between effects on eruption time and childhood nutritional status is still unclear. therefore, the aim of this study was to evaluate an association between childhood nutritional status and permanent tooth eruption chronology. materials and methods ethical and sample this study was submitted and approved by ethics committee of school of ribeirão preto of university of são paulo (#35323314.7.0000.5419). all parents or caregivers were informed about the study and signed an informed consent. the sample was collected in the school of dentistry of ribeirão preto. the studied population is a convenience sample, with no sample size calculation, and thus, reflect the characteristics of the pediatric population that sought for dental treatment from 2014 to 2016. the parents/caregivers answered a sociodemographic questionnaire, and provided information about the children’s characteristics and habits. clinical examination a hundred sixty children of both sexes, aged 6-13 years old were clinically examined by an experienced pediatric dentist. clinical examinations were performed in sitting chairs in the dental office under artificial light. at the clinical examination, the number of erupted permanent teeth was evaluated. tooth eruption was defined as having occurred if any tooth surface had emerged the alveolar mucosa6. children that presented factors that could alter the tooth eruption, leading to dental impaction such as odontoma, cysts, dental agenesis and supernumerary teeth were 3 arid et al. excluded after dental radiographs were taken. children with syndromes or oral cleft were also excluded. canines were not included on the analysis regarding their high rate of impaction13,14. the delayed permanent tooth eruption was considered according to a scale designed for brazilian children according to the gender15. tooth delayed was considered when the children had at least one delayed permanent tooth eruption according to the suggested chronology. anthropometric measurement the children’s heights were determined in meters. also, a digital weighing machine was used to determine the children’s weight in kilograms while they were wearing light clothes and no shoes. the body mass index (bmi) z-score was calculated by the pediatric z-score calculator of the children´s hospital of philadelphia (http://zscore. research.chop.edu/index.php) using individual height, weight, age and gender as variables. the nutritional status was classified according to the world health organization (2006) as follows: • bmi z-score <-2; percentile o <3: underweight; • bmi z-score ≥ -2 and ≤ +1; percentile ≥ 3 and ≤85: eutrophic; • bmi z-score > +1 and ≤ +2; percentile > 85 and ≤ 97: overweight; • bmi z-score > +2; percentile > 97: obese. statistical analysis data were analyzed using epi info 7 (epi info 7 software, atlanta, ga, usa). comparisons were performed between ‘eutrophic’, ‘underweight’, ‘overweight’ and ‘obesity’ groups. chi-square or fisher´s exact tests for the dichotomous variables. odds ratio was used to calculate the relative risk among these groups the shapiro–wilk test was used to verify the normality of the data. to compare the mean number of delayed teeth according to nutritional status’ groups kruskal-wallis test with multiple comparison by dunn’s test was used. the established alpha was 5%. results a hundred sixty children were evaluated, 82 boys (51.25%) and 78 girls (48.75%). the mean age was 8.88 (sd 2.02). the characteristics of the studied population is presented on the table 1. table 1. characteristics of the studied children. characteristics groups p-value eutrophic underweight overweight obese age mean (sd) 8.97 (2.08) 8.00 (1.63) 8.92 (1.92) 8.25 (2.00) 0.54 gender n (%) male 57 (69.5%) 2 (2.5%) 15 (18.2%) 8 (9.7%) 0.19 female 47 (60.2%) 2 (2.4%) 25 (32.0%) 4 (5.1%) http://zscore.research.chop.edu/index.php http://zscore.research.chop.edu/index.php 4 arid et al. the z-score of the evaluated children ranged from -2.93 to 2.62, and the percentile from <1 to >99. four (2.5%) of the children were underweight; 104 (65%) were eutrophic; 40 (25%) were overweight, and 12 (7.5%) were obese. fifty-six children (35%) had delayed tooth eruption in at least one permanent teeth, out of those 26 (46.4%) were girls and 30 (53.6%) boys, and there was no statistical difference (p=0.33). the association between tooth eruption and the nutritional status is presented on table 2. delayed tooth eruption was more common in underweight children than in eutrophic children (p=0.0091). the number of delayed erupted teeth ranged from 1 to 10, with the mean of 2.63 (sd 1.83). figure 1 shows the mean number of delayed erupted teeth according to each group. the mean number of delayed teeth in eutrophic children was 0.92 (sd 1.50), for underweight children 1.75 (sd 0.50), for overweight children 1.07 (sd 2.18) and for obese children 0.16 (sd 0.38). underweight children had more delayed teeth and a statistical difference was observed betwwen underweight and children (p<0.05). table 2. association between delayed tooth emergence and the nutritional status nutritional status without delayed tooth eruption n (%) with delayed tooth eruption n (%) p-value or (95% ic) eutrophic 67 (64.4%) 37 (35.6%) reference reference underweight 0 (0%) 4 (100%) 0.0091* undefined overweight 27 (67.5%) 13 (32.5%) 0.72 0.87 (0.40–1.89) obesity 10 (83.33%) 2 (16.67%) 0.33 0.36 (0.07–1.14) note: *indicates statistics significance; all comparisons were related to eutrophic figure 1. mean number of delayed teeth according to the nutritional status. * indicates statistical significance. eu tro ph ic un de rw eig ht ov er we igh t ob es ity 2.5 1.5 2.0 1.0 0.5 0.0 * 5 arid et al. discussion the health burden from obesity and malnutrition is the driving factor behind several research studies regarding the impact of nutritional status alterations on oral health. many studies attempted to evaluate associations between oral issues and overweight/obesity, but studies evaluating those issues associated to undernutrition are uncommon. it was previously demonstrated a positive correlation between bmi and decayed, missing and filled teeth (dmft) in adults16 and in children17,18, although it has also been demonstrated that overweight children have lower caries index11,19, which might be explained by the fact that those children consume more fatty acids and sugar than eutrophic or underweight children20. some studies have also proved that obesity is a risk factor for periodontitis21,22 and that obese children are more likely to have dental erosion when compared to healthy children23. the relationship between the nutritional status and tooth eruption chronology has been evaluated by few studies1,6,11,24,25. our study found an association between permanent tooth eruption and the nutritional status, in which delayed permanent tooth eruption was more common in underweight children. this association between delayed permanent tooth eruption is supported by other studies6,24,25, and underweight is also associated with delayed eruption of primary teeth12. in contrast the literature supports that obese children have teeth erupted earlier than non-obese1, which can be due to their earlier puberty3. in our study, although earlier teeth eruption was more frequent in obese children, we were not able to observe a statistical difference. this might be explained by the low frequency of obesity in our population. it is possible that our result is a false-negative. it is well known that results from small samples sizes may lead more commonly to falsely negative leading to a type ii error26, in which accepted the null hypothesis regarding the association between tooth eruption and obesity. our results also demonstrated that the number of teeth with delayed eruption differs according to the nutritional status, in which the mean number of delayed tooth eruption was higher, suggesting that the nutritional status is an important etiological factor involved in the chronology of permanent tooth eruption. however, it is important to emphasize that our study has some obvious limitations. some other factors could be involved in tooth eruption9,10,11. in addition, some aspects involved in the nutritional status, such as dietary intake were not evaluated in this study and should not be taken into consideration in future studies. in conclusion, underweight brazilian children have a higher incidence of delayed eruption and a higher number of affected teeth. acknowledgements the authors thank mariana bezamat c. lucas for the english revision of this manuscript. references 1. must a, phillips sm, tybor dj, lividini k, hayes c. the association between childhood obesity and tooth eruption. obesity (silver spring). 2012 oct;20(10):2070-4. doi: 10.1038/oby.2012.23. 6 arid et al. 2. (ibge) biogas. pof 2008-2009: desnutrição cai e peso das crianças brasileiras ultrapassa padrão internacional. nov 2010 [cited 2017 mar 10]. available from: http://censo2010.ibge.gov.br/noticiascenso.html?view=noticia&id=1&idnoticia=1699&busca=1&t=pof-20082009-desnutricao-cai-pesocriancas-brasileiras-ultrapassa-padrao-internacional. portuguese. [pof 2008-2009: malnutrition falls and the weight of brazilian children exceeds international standard] 3. aksglaede l, juul a, olsen lw, sorensen ti. age at puberty and the emerging obesity epidemic. plos one. 2009 dec 24;4(12):e8450. doi: 10.1371/journal.pone.0008450. 4. he q, karlberg j. bmi in childhood and its association with height gain, timing of puberty, and final height. pediatr res. 2001 feb;49(2):244-51. 5. elamin f, liversidge hm. malnutrition has no effect on the timing of human tooth formation. plos one. 2013 aug 30;8(8):e72274. doi: 10.1371/journal.pone.0072274. 6. heinrich-weltzien r, zorn c, monse b, kromeyer-hauschild k. relationship between malnutrition and the number of permanent teeth in filipino 10to 13-year-olds. biomed res int. 2013;2013:205950. doi: 10.1155/2013/205950. 7. hernandez m, phulpin b, mansuy l, droz d. use of new targeted cancer therapies in children: effects on dental development and risk of jaw osteonecrosis: a review. j oral pathol med. 2017 may;46(5):321-326. doi: 10.1111/jop.12516. 8. rivera e, assiri a, guandalini s. celiac disease. oral dis. 2013 oct;19(7):635-41. doi: 10.1111/odi.12091. 9. wise ge. cellular and molecular basis of tooth eruption. orthod craniofac res. 2009 may;12(2):67-73. doi: 10.1111/j.1601-6343.2009.01439.x. 10. barberia leache e, maranes pallardo jp, mourelle martinez mr, moreno gonzalez jp. tooth eruption in children with growth deficit. j int assoc dent child. 1988 dec;19(2):29-35. 11. sanchez-perez l, irigoyen me, zepeda m. dental caries, tooth eruption timing and obesity: a longitudinal study in a group of mexican schoolchildren. acta odontol scand. 2010 jan;68(1):57-64. doi: 10.3109/00016350903449367. 12. psoter wj, reid bc, katz rv. malnutrition and dental caries: a review of the literature. caries res. 2005 nov-dec;39(6):441-7. 13. yan b, sun z, fields h, wang l, luo l. etiologic factors for buccal and palatal maxillary canine impaction: a perspective based on cone-beam computed tomography analyses. am j orthod dentofacial orthop. 2013 apr;143(4):527-34. doi: 10.1016/j.ajodo.2012.11.021. 14. uribe p, ransjo m, westerlund a. clinical predictors of maxillary canine impaction: a novel approach using multivariate analysis. eur j orthod. 2017 apr 1;39(2):153-160. doi: 10.1093/ejo/cjw042. 15. marques gd, guedes-pinto ac, abramowicz m. [the eruption sequence of permanent teeth in children from the city of sao paolo]. rev fac odontol sao paulo. 1978;16(2):187-94. portuguese. 16. alswat k, mohamed ws, wahab ma, aboelil aa. the association between body mass index and dental caries: cross-sectional study. j clin med res. 2016 feb;8(2):147-52. doi: 10.14740/jocmr2433w. 17. willerhausen b, blettner m, kasaj a, hohenfellner k. association between body mass index and dental health in 1,290 children of elementary schools in a german city. clin oral investig. 2007 sep;11(3):195-200. 18. gerdin ew, angbratt m, aronsson k, eriksson e, johansson i. dental caries and body mass index by socio-economic status in swedish children. community dent oral epidemiol. 2008 oct;36(5):459-65. doi: 10.1111/j.1600-0528.2007.00421.x. 19. liang jj, zhang zq, chen yj, mai jc, ma j, yang wh, et al. dental caries is negatively correlated with body mass index among 7-9 years old children in guangzhou, china. bmc public health. 2016 jul 26;16:638. doi: 10.1186/s12889-016-3295-3. 7 arid et al. 20. werner sl, phillips c, koroluk ld. association between childhood obesity and dental caries. pediatr dent. 2012 jan-feb;34(1):23-7. 21. suvan j, d’aiuto f, moles dr, petrie a, donos n. association between overweight/obesity and periodontitis in adults. a systematic review. obes rev. 2011 may;12(5):e381-404. doi: 10.1111/j.1467-789x.2010.00808.x 22. gaio ej, haas an, rosing ck, oppermann rv, albandar jm, susin c. effect of obesity on periodontal attachment loss progression: a 5-year population-based prospective study. j clin periodontol. 2016 jul;43(7):557-65. doi: 10.1111/jcpe.12544. j clin periodontol. 2016;43(7):557-65. 23. tong hj, rudolf mc, muyombwe t, duggal ms, balmer r. an investigation into the dental health of children with obesity: an analysis of dental erosion and caries status. eur arch paediatr dent. 2014 jun;15(3):203-10. doi: 10.1007/s40368-013-0100-1. 24. psoter w, gebrian b, prophete s, reid b, katz r. effect of early childhood malnutrition on tooth eruption in haitian adolescents. community dent oral epidemiol. 2008 apr;36(2):179-89. doi: 10.1111/j.1600-0528.2007.00386.x. 25. gaur r, boparai g, saini k. effect of under-nutrition on permanent tooth emergence among rajputs of himachal pradesh, india. ann hum biol. 2011 jan;38(1):84-92. doi: 10.3109/03014460.2010.497499. 26. nayak bk. understanding the relevance of sample size calculation. indian j ophthalmol. 2010 nov-dec;58(6):469-70. doi: 10.4103/0301-4738.71673. socioeconomic level and the parents’ perception of the impact of oral diseases on their children’s quality of life naiana de melo belila1, ronald jefferson martins1, cléa adas saliba garbin1, wanilda maria meire costa borghi2 1universidade estadual paulista – unesp, school of dentistry of araçatuba, department of children and social dentistry, araçatuba, sp, brazil correspondence to: ronald jefferson martins nepesco – center for research in public health faculdade de odontologia de araçatuba unesp departmento de odontologia infantil e social rua josé bonifácio, nº 1193 vila mendonça cep. 16015-050 araçatuba-sp brasil phone: +55 18 3636-2824 e-mail: rojema@foa.unesp.br abstract aim: to analyze how parents of different socioeconomic levels perceive the impact of oral diseases on their children’s quality of life. methods: all parents or guardians of students aged 11 to 14 years old, regularly enrolled in fundamental schooling at public schools of two towns in the northwestern region of the state of são paulo, brazil, were enrolled in the study. the questionnaire of “socioeconomic assessment tool” was used to classify the families with regards to socioeconomic class and the “parental-caregiver perceptions questionnaire (p-cpq)” was used to verify the parents’ perception of the impact of oral diseases on their children’s quality of life. results: 172 (41.8%) individuals answered the survey. among them, most belonged to the upper low class (61%). 21.5% of the individuals answered that they considered their children’s oral health “regular or bad” and 71.5% answered that their child’s general well-being was not or was little affected by the condition of his/ her teeth, lips, jaws or mouth. there was an association between the quality of life sub-scales, especially “oral symptoms”, with all socioeconomic classes. conclusions: there is a relationship between parents’ socioeconomic class and the perception of the impact of oral disease on their children’s quality of life. keywords: social class. oral health. quality of life. introduction good oral health condition is critical to maintaining the general welfare of the individual, allowing him or her to perform their daily functions normally with a healthy quality of life1. the effects of tooth decay and other oral diseases reflect many negative aspects in the lives of people who are still in the infancy stage and adolescence, such as difficulties to socialize, chew, swallow, speak, sleep, lack of appetite, low self-esteem and behavior changes; that can harm even school performance2. parents’ knowledge and perception about oral health originates from their culture, beliefs, habits and environment. this in turn, influences directly their children’s behavior and oral conditions3. hygiene habits and healthy eating habits tend to be a family characteristic. thus the influence of parents on their children’s oral health, from childhood to adolescence, is unequivocal4. the income and low levels of education are closely related to poor hygiene and nutrition of the families, as well as unpleasant experiences of early childhood caries, both by the parents and the children, which consequently influences the quality of life of individuals3,5. received for publication: november 08, 2016 accepted: march 08, 2017 braz j oral sci. 15(2):171-175 original article braz j oral sci. april | june 2016 volume 15, number 2 http://dx.doi.org/10.20396/bjos.v15i2.8648777 172 the quality of life is defined, according to the world health organization as "the individual's perception of their position in life in the context of culture and value system in which they live, and in relation to their goals, expectations, standards and concerns"6. this concept is multidimensional and includes both positive and negative factors in relation to the overall well-being due to social, economic and cultural aspects of the individual2. for a long time, oral health had been measured only through clinical examinations, which does not allow for the evaluation of the impact that oral diseases cause on individuals, requiring new tools for a more accurate analysis2. in this context, this study aimed to analyze how parents of different socioeconomic levels perceived the impact of oral diseases on the quality of life of their children, who were public school students of two small towns in the northwestern region of the state of são paulo, in brazil. material and methods the study consisted of a descriptive transversal survey, with a quali-quantitative approach. it was carried out from july to september 2015, in fundamental schools in two towns in the northwestern region of the state of são paulo, brazil: américo de campos and pontes gestal. these cities were chosen because they have small, predominantly urban population and a similar human development index (hdi), income (hdi income) and education (hdi education). all parents or guardians of students aged 11 to 14 years old regularly enrolled in fundamental schooling of public schools of the towns participated. those who did not answer the questionnaire completely and those who did not sign the informed consent form were excluded. the sample size should be 134 individuals, when calculated with sampling error of 7% and confidence level of 93%, so the sample obtained was 22.1% greater than the sample size. initially, the municipal secretaries of education and the principals of the schools were informed about the aim of the survey and the future use of the data collected, in order to obtain their support for the study. the same information was passed on to the students’ parents at the parent-teacher conference. later, two questionnaires were handed to the parents: a) socioeconomic assessment tool, to classify the families with regards to socioeconomic class. in the scale in this tool, the family’s economic status scores from 1 to 21 points (from gross income up to ½ minimum wage to over 100 minimum wages), the number of family members living in the household from 1 to 6 points (from over 8 to 1 to 2 members), the education level of the members from 0 to 7 points (from illiterate to graduate diploma), the housing condition / situation from 0 to 10 points, and the occupation of the head of household from 1 to 13 points. the sum of these indicators classifies the families in six classes, lower lower class (ll), upper lower class (ul), lower middle class (lm), middle class (mi), upper middle class (um) and upper class (up), classification in which the “upper class” (from 55 to 57 points) is the is the most affluent and the “lower lower class” (from 0 to 20 points) the least affluent7. b) parental-caregiver perceptions questionnaire (p-cpq). a questionnaire consisting of 35 questions to assess parents or guardians’ perception of the impact of oral diseases (cavities, malocclusion, etc, for instance) on the quality of life of their children. questions 1 and 2 refer to the global perception of caregivers about oral health and general well-being of the child. the possible answers to question 1 range from “excellent” to “bad” and to question 2 range from “not at all” to “very much”. the other questions are subdivided into four broad categories: oral symptoms (questions 3 to 8), functional limitations (questions 9 to 16), emotional well-being (questions 17 to 24), and social well-being (questions 25 to 35). the answer options are presented in a likert-type scale, which ranges from zero to four points (0 = never; 1 = once or twice; 2 = sometimes; 3 = frequently; 4 = every day or nearly every day). the answer option “i don’t know” was marked as “0” (zero), based on the studies of jokovic et al.8, as the data indicate that the children “never” reported that item to their parents. the total score is obtained by the sum of the scores of all questions. the greater the score, the greater the impact of oral diseases on the quality of life9. p-cpq was originally developed in english, in toronto, canada, by jokovic et al.8,10 and transculturally adapted in brazilian portuguese and validated by barbosa et al.2. it has shown to be valid and reliable to assess parents’ perception9. regarding p-cpq, a descriptive analysis of the results of the first question, about socioeconomic classes and how parents or guardians considered their children’s oral health, and the second, about socioeconomic classes and how much the parents thought that their child’s general well-being was affected by his/her oral health, was made because they cannot be included in the sum of the subscale scores. the scales of this questionnaire were analyzed by biostat 5.0 software11. as the score was not evenly distributed, kruskal wallis non parametric test with significance level of 5% was used to assess the difference of mean scores among the groups of the different socioeconomic classes. as a significant difference was detected among the socioeconomic classes, dunn’s test of multiple comparisons was performed. the study was approved by the research in humans ethics committee, within the standards required by resolution 466/12, caae process no. 39094214.2.0000.5420. all participants signed an informed consent form. results the universe of the research comprised 412 parents or guardians, from which 172 (41.8%) answered the survey. based on the socioeconomic assessment tool, 20.4% of the heads of household belonged to ll class, 61% to ul class, 14.5% to lm class and 4.1% to mi class. as only one participant belonged to um class, that one was included in the mi class. concerning p-cpq, the reliability of internal consistency of subscales among the participants’ responses was estimated by the cronbach alpha coefficient (n= 0,887). the first question was how the caregiver would classify his/her child’s health with regards to teeth, lips, jaws and mouth, and 21.5% of the individuals socioeconomic level and the parents’ perception of the impact of oral diseases on their children’s quality of life braz j oral sci. 15(2):171-175 173 answered that considered it “regular or bad”. the second question was how much his/her child’s general well being was affected by the condition of his/her teeth, lips, jaws or mouth, and 71.5% answered “not at all” or “just a little” (tables 1 and 2). socioeconomic level and the parents’ perception of the impact of oral diseases on their children’s quality of life table 1 number and percentage of parents or guardians with regards to the perception of their children’s oral health and socioeconomic classes, américo de campos/pontes gestal, brazil, 2015. how would you classify the health of child’s teeth, lips, jaws and mouth? excellent very good good regular bad total n % n % n % n % n % n % lower lower (ll) 6 3.5 5 2.9 16 9.3 9 5.2 1 0.6 37 21.5 upper lower (ul) 16 9.3 23 13.4 48 27.9 14 8.1 2 1.2 103 59.9 lower middle (lm) 4 2.3 4 2.3 10 5.8 6 3.5 1 0.6 25 14.5 middle (mi) 1 0.6 1 0.6 1 0.6 3 1.7 1 0.6 7 4.1 total 27 15.7 33 19.2 75 43.6 32 18.6 5 2.9 172 100 table 2 number and percentage of parents or guardians according to the perception of the general well-being due to their children’s oral health and socioeconomic classes, américo de campos/pontes gestal, brazil, 2015. how much is your child’s general well-being affected by the condition of his/her teeth, lips, jaws or mouth? not at all just a little more or less a lot very much total n % n % n % n % n % n % lower lower (ll) 20 11.6 7 4.1 8 4.6 2 1.2 0 0 37 21.5 upper lower (ul) 59 34.3 23 13.4 15 8.7 5 2.9 1 0.6 103 59.9 lower middle (lm) 8 4.6 3 1.7 6 3.5 8 4.7 0 0 25 14.5 middle (mi) 2 1.2 1 0.6 2 1.2 2 1.1 0 0 7 4.1 total 89 51.7 34 19.8 31 18 17 9.9 1 0.6 172 100 fig.1. relationship between the subscales of p-cpq index and socioeconomic classes, américo de campos/pontes gestal, brazil, 2015. specifically in relation to the children feeling any pain, 62.8% of parents or guardians said that the children have had this experience. kruskal-wallis test result was highly significant in all subscales of pcp-q, with p<0.0001, reason for which the analysis was furthered with dunn’s multiple comparison test. concerning subscales “oral symptoms”, “functional limitations”, “social well-being” and “emotional well-being”, dunn test showed mean scores among social classes ll x lm, ll x mi, ul x lm and ul x mi with p values less than the alpha level of 0.05, being thus considered very significant. the subscale “oral symptoms” showed the greatest impact on the quality of life of all socioeconomic classes (figure 1). discussion quality of life encompasses meanings that reflect the individual’s knowledge, experiences, expectations and values and is related to the factors that lead to health. its main focus is the capacity to live disease-free or to overcome morbidity, pain and discomfort12. in this context, a close relationship is observed among social level, oral health and quality of life13,14. parents are greatly responsible for their children’s health. therefore, it is of utmost importance to assess their perceptions of the oral health related to children and teenagers’ well being and quality of life2. oral diseases present negative impact on the quality of life of children and teenagers because they lead to mastication difficulties, appetite decrease, weight loss, sleeping problems, behavioral changes, low self-esteem and decrease in academic performance1. even knowing all the consequences and damages that unsatisfactory oral health can bring, most parents in this study believed that the general well being of their children was little affected by their oral health, corroborating the finding of a study conducted with parents of children with brain disturbances, in which most participants reported that the general well-being of the children was “not at all” affected by their oral health. however, the responses might have been influenced by the specific conditions of those individuals for whom the oral health was not considered a priority15. on the other hand, in a qualitative study the parents reported their great concern about their children’s oral health, due to the possible negative interference in their future16. in another study, braz j oral sci. 15(2):171-175 174 in which a questionnaire about quality of life and oral health was answered before and after the individuals had been submitted to dental treatment, the responses changed after the end of the treatment. this result shows that most participants considered that the general well-being is very much affected by the oral health, which influences both general health and social life17. oral disturbances have little impact on the quality of life of children and teenagers whose families belong to the upper socioeconomic classes. nevertheless, they present a strong impact on the low income individuals, showing a significant relationship between lower social classes and the impact of oral diseases on the quality of life18. this study presented statistically significant differences between socioeconomic classes and the parents’ perception of the impact of oral diseases on the quality of life of their children. in all socioeconomic classes, the oral symptoms subscale showed the greatest impact. toothache caused by dental cavities and periodontal diseases are the main responsible for the impact of oral health on the individual’s quality of life. this is highly present in the brazilian population because the lower classes do not see oral health as a priority, as they have other urgencies, such food issues19-22. a study with individuals in an area encompassed by the family health strategy program showed that the participants who had seen a dentist three or more years ago, the ones with total prostheses and those with unsatisfactory oral hygiene belonged to the lower social classes and reported that they only sought dental care when they had a toothache, thus, not taking regular care of their dental health23. pain is the main reason parents seek dental care for their children13,22,24,25. in this study, pain was reported by a great portion of the participants, corroborating the finding of another study, in which the parents also reported the frequent occurrence of this symptom in their children22. public policies should be implemented to facilitate the access of the population to health care through preventive, educational and curative actions and activities. this will broaden the parents view about the importance of maintaining good oral health so that their children have quality of life. new studies should be carried out with populations of higher social classes in order to confirm the relationship between socioeconomic level and parental perception of the impact of oral diseases on children's quality of life. it may be concluded that there is an association between socioeconomic class and the individual's perception of parents or guardians about the impact of oral diseases on the quality of life of children. references 1. bica i, duarte j, camilo a, jesus a, ferreira, c; oliveira f. [oral healthrelated with quality of life: parental perceptions of childhood]. millenium 2014 jun-dec;47:141-52. portuguese. 2. barbosa t, mialhe fl, castilho arf, gavião mbd. [quality of life and oral health in children and adolescents: conceptual and methodological aspects]. physis 2010;20(1):283-300. portuguese. 3. castilho ar, mialhe fl, barbosa ts, puppin-rontani rm. influence of family environment on children’s oral health: a systematic review. j pediatr (rio j). 2013 mar-apr;89(2):116-23. doi: 10.1016/j.jped.2013.03.014. 4. camargo mbj, barros ajd, frazão p, matijasevich a, santos is, peres ma, et al. [predictors of dental visits for routine check-ups and for the resolution of problems among preschool children]. rev saude publica. 2012 feb;46(1):87-97. portuguese. 5. paredes so, galvão rn, fonseca fra. [influence of oral health on the life quality of preschool children]. rev baiana saúde pública 2014;38(1):12539. portuguese. 6. world health organization, division of mental health. measurement of quality of life in children. geneva: world health organization; 1993. 7. graciano mig, lehfeld nas. [socioeconomic study: indicators and methodology in a contemporary approach]. rev serv soc saúde. 2010;9(9):157-85. portuguese. 8. jokovic a, locker d, stephens m, kenny d, tompson b, guyatt g. measuring parental perceptions of child oral health-related quality of life. j public health dent 2003 apring;63(2):67-72. 9. barbosa td, gavião mb. validation of the parental-caregiver perceptions questionnaire: agreement between parental and child reports. j public health dent. 2015 fall;75(4):255-64. doi: 10.1111/j.17527325.2012.00371.x. 10. jokovic a, locker d, stephens m, kenny d, tompson b, guyatt g. validity and reliability of a questionnaire for measuring child oral-health-related quality of life. j dent res 2002 jul;81(7):459-63. 11. ayres m, ayres jr m, ayres dl, santos as. [bioestat 5.0: statistics applications in the areas of biomedical sciences [computer program]]. belém: ong mamieraua; 2007. portuguese. 12. minayo mcs, hartz zma, buss pm. [quality of life and health: a necessary debate]. cienc saude colet. 2000;5(1):7-18. portuguese. 13. pandolfi m, barcellos la, miotto mhmb, goés psa. [oral health and dental services users' quality of life]. pesq bras odontoped clin integr. 2011 jul-sep;11(3):311-6. portuguese. 14. vagetti gc, moreira nb, barbosa filho vc, oliveira v, cancian cf, mazzardo o, et al. [aspects of quality of life associated with self-rated health: a study of elderly women from a physical activity program in lowincome neighborhoods in curitiba in the state of paraná, brazil]. cienc saude colet. 2013;18(12):3483-93. portuguese. 15. abanto j, carvalho ts, bonecker m, ortega aol, ciamponi al, raggio dp. parental reports of the oral health-related quality of life of children with cerebral palsy. bmc oral health 2012 jun;12(15)1-8. doi: 10.1186/14726831-12-15. 16. abreu mhng, pordeus ia, modena cm. [a social representation study of oral health among mothers in rural areas, itaúna (mg), 2002]. cienc saude colet. 2005;10(1):245-59. portuguese. 17. thomson wm, foster page la, gaynor wn, malden pe. short-form versions of the parental-caregivers perceptions questionnaire and the family impact scale. community dent oral epidemiol 2013 oct;41(5):44150. 18. locker d. disparities in oral health-related quality of life in a population of canadian children. community dent oral epidemiol 2007 oct;35(5):34856. 19. góes psa. the prevalence and impact of dental pain in brazilian schoolchildren and their families [thesis]. london: university college london; 2001. 20. brazil. ministry of health of brazil. [department of primary care. project sb brazil 2010: national survey of oral health, the main results]. brasília: ministry of health; 2011 [cited 2015 out 12]. available in: http://dab. saude.gov.br/cnsb/sbbrasil/arquivos/projeto_sb2010_relatorio_final. pdf. portuguese. 21. costa sm, vasconcelos m, abreu mhng. [impact of dental caries on quality of life among adults resident in greater belo horizonte, state of minas gerais, brazil]. cienc saude colet. 2013;18(7):1971-80. portuguese. 22. silveira mf, marôco jp, freire rs, martins amebl, marcopito lf. [impact socioeconomic level and the parents’ perception of the impact of oral diseases on their children’s quality of life braz j oral sci. 15(2):171-175 175socioeconomic level and the parents’ perception of the impact of oral diseases on their children’s quality of life of oral health on physical and psychosocial dimensions: an analysis using structural equation modeling]. cad saude publica. 2014 jun;30(6):116982. portuguese. 23. baldani mh, brito wh, lawder jac, mendes ybe, silva ffm, antunes jlf. [individual determinants of dental care utilization among low-income adult and elderly individuals]. rev bras epidemiol 2010;13(1):150-62. portuguese. 24. edelstein bl. disparities in oral health and access to care: findings of national surveys. ambul pediatr 2002;2(2):141-7. 25. cohen-carneiro f, souza-santos r, rebelo mab. quality of life related to oral health: contribution from social factors. cienc saude colet. 2011;16(suppl 1):1007-15. braz j oral sci. 15(2):171-175 original article braz j oral sci. january | march 2016 volume 15, number 1 analysis of root canal organic tissue dissolution capacity according to the type of irrigation solution and agitation technique kiany scarssi nunes1, letícia feron1, francisco montagner2, tiago andré fontoura de melo1 1faculdade da serra gaúcha – fsg, dental school, department of clinical, caxias do sul, rs, brazil 2universidade federal do rio grande do sul – ufrgs, dental school, department of conservative dentistry, porto alegre, rs, brazil correspondence to: tiago andré fontoura de melo curso de odontologia – faculdade da serra gaúcha – fsg rua os dezoito do forte, 2366 – são pelegrino cep 95020-472 – caxias do sul/rs – brazil phone: +55 54 2101 6000 e-mail: tafmelo@gmail.com abstract aim: to analyze the root canal organic tissue dissolution capacity promoted by irrigating solutions, with or without the use of different agitation techniques. methods: bovine pulp tissue fragments were initially weighed. the following irrigating solutions were tested: 2.5% sodium hypochlorite, 2% chlorhexidine digluconate solution, and distilled water. the irrigating protocols were: immersion, mechanical agitation with endodontic files, and ultrasonic or sonic systems (endoactivactor® and easy clean®). at the end of the protocols, the pulps were weighed to determine their final weight. for comparison, the average percentage of tissue dissolution in relation to the groups was analyzed using the kruskal-wallis nonparametric test complemented by multiple comparisons test. the significance level was set at 5%. results: among the irrigation solutions, 2.5% sodium hypochlorite showed a higher dissolving power than 2% chlorhexidine digluconate and distilled water. furthermore, ultrasonic and sonic systems were more effective irrigating protocols than immersion and mechanical agitation with endodontic files. conclusions: the combination of sodium hypochlorite with an agitation system promotes a greater degree of tissue degradation. keywords: endodontics. dissolution. sodium hypochlorite. chlorhexidine. vibration. introduction because of the anatomical complexity of the root canal system, approximately 50% of the root canal walls remain untouched by instruments during preparation, which results in insufficient cleaning1. necrotic tissue debris may provide a nutrient source for the surviving bacteria2. sodium hypochlorite is widely used to irrigate the root canal during endodontic procedures; its properties are low surface tension, antimicrobial capacity3, and ability to dissolve organic tissue4. however, because of its harmful action on periapical tissues and its cytotoxicity5, other substances have been used as alternatives, such as chlorhexidine digluconate. chlorhexidine has antimicrobial properties, power of substantivity, and is less harmful to vital tissues compared with sodium hypochlorite6. many studies have reported the significant influence of a variety of factors such as concentration, time, temperature, contact area of tissue irrigation, and mechanical action of the irrigation process on tissue dissolution7,8. currently, there have several resources to assist in the agitation process of an irrigating solution in the root canal. sonic and ultrasonic systems with different tips and inserts and with different protocols have been tested. unlike ultrasonic systems, which function at a high frequency and with a range of inserts for different areas of dentistry and with very well-defined http://dx.doi.org/10.20396/bjos.v15i1.8647128 received for publication: august 05, 2016 accepted: august 25, 2016 braz j oral sci. 15(1):70-74 71 purposes, the sonic systems such as the endoactivator® (dentsply/ maillefer, ballaigues, switzerland) and easy clean® (easy equipamento odontológicos, belo horizonte, mg, brazil) provide a specific mechanism of action and applicability for its use in the endodontic irrigation process. the tips of these two devices are flexible and have a small caliber in all extensions, which helps improve the action of the irrigation solution in areas of difficult access inside the root canal. the aim of this study was to analyze the dissolution capacity of pulp tissue according to the type of the irrigating solution and the agitation technique employed in the chemical protocol of endodontic instrumentation. material and methods overall, 150 fragments of bovine pulp tissue, obtained from extracted mandibular central incisors, with an initial mean weight of 3.5 ± 0.5 mg, were selected and divided into 15 experimental groups (table 1). analysis of root canal organic tissue dissolution capacity according to the type of irrigation solution and agitation technique the irrigating solutions used were 2.5% sodium hypochlorite (bellafarma farmácia de manipulação, caxias do sul, rs, brazil), 2% digluconate chlorhexidine (bellafarma farmácia de manipulação, caxias do sul, rs, brazil), and distilled water (iodontosul industrial odontológica do sul ltda., porto alegre, rs, brazil) as control. the type of agitation protocols included mechanical with endodontic files, ultrasonic system, and sonic systems (endoactivator® and easy clean®). the immersion of pulp tissue only in distilled water solution was used as the control group. for the experiment, 10 bovine incisor roots were prepared to simulate the conditions of the pulp tissue within the root canal during the execution of the protocols, as reported in a recent experimental group n irrigation solution agitation protocol g1 10 distilled water immersion g2 10 distilled water mechanical agitation g3 10 distilled water sonic system (easy endo®) g4 10 distilled water sonic system (endoactivactor®) g5 10 distilled water ultrasonic system g6 10 2.5% sodium hypochlorite immersion g7 10 2.5% sodium hypochlorite mechanical agitation g8 10 2.5% sodium hypochlorite sonic system (easy endo®) g9 10 2.5% sodium hypochlorite sonic system (endoactivactor®) g10 10 2.5% sodium hypochlorite ultrasonic system g11 10 2% chlorhexidine digluconate immersion g12 10 2% chlorhexidine digluconate mechanical agitation g13 10 2% chlorhexidine digluconate sonic system (easy endo®) g14 10 2% chlorhexidine digluconate sonic system (endoactivactor®) g15 10 2% chlorhexidine digluconate ultrasonic system table 1 experimental groups. study9. roots were standardized at 16 mm in length and the root canal was dilated to an equivalent of a number 4137 bur tip (kg sorensen indústria e comércio ltda., barueri, sp, brazil). the dental models were fixed on a metal tray with cyanoacrylate (loctite, hartford, ct, usa) to enable the solution agitation protocols. in order to firm the pulp fragment in the apical third of the root canal, a surgical steel needle 0.20 mm in diameter was transfixed in the mesiodistal direction (moxom acupuncture, budapest, hungary) approximately 4 mm short of the apex (figure 1). initially, the pulp fragment was weighed on an electronic scale (exacta série basic/ bl-1200as, curitiba, pr, brazil). afterwards, the bovine tissue was fixed within the canal and the canal was filled with 3 ml of irrigation solution. fig.1. image of pulp tissue fragment fixed inside the root canal: a (coronal view of the tooth), b (axial view of the tooth), and c (surgical steel needle). before the solution agitation protocol inside the root canal, the endodontic instrument for the manual-mechanical agitation and the sonic and ultrasonic tips were calibrated on 8 mm from the root edge. for the immersion (control) group, there was no agitation protocol. the pulp tissue fragment remained immersed in the solution (distilled water) for 2 min. for the mechanical agitation with endodontic files, sonic (endoactivator® and easy clean®), and ultrasonic groups, the proposed protocol was to intersperse three immersing cycles of the pulp fragment inside the root canal containing irrigating solution (20 s) followed by agitation (20 s), totaling 2 min, according to the protocol suggested by the systems manufacturers. for mechanical agitation with endodontic files, an endodontic instrument type k file number 25 was used (dentsply/ maillefer). for the ultrasonic system, an insert gt40 was used (gnatus equipamentos médico-odontólogicos, ribeirão preto, sp, brazil) and activated with an ultrasonic vibration of 50% potency of the jet sonic device 30,000 hz (dabi atlante®, são paulo, sp, brazil), without irrigation. for the sonic systems endoactivator® and easy clean®, their respective flexible tips with diameters of 0.25 and 0.04 braz j oral sci. 15(1):70-74 72analysis of root canal organic tissue dissolution capacity according to the type of irrigation solution and agitation technique irrigation solution agitation protocol distilled water 2% chlorhexidine digluconate 2.5% sodium hypochlorite initial weight final weight p initial weight final weight p initial weight final weight p immersion 0.033±0.015 0.033±0.015 1.000 0.029±0.011 0.029±0.011 1.000 0.039±0.017 0.036±0.016 0.081 mechanical agitation 0.040±0.009 0.037±0.011 0.081 0.029±0.011 0.026±0.009 0.081 0.036±0.014 0.031±0.011 0.015 ultrasonic system 0.040±0.014 0.035±0.014 0.015 0.032±0.010 0.027±0.011 0.015 0.032±0.009 0.018±0.009 0.001 endoactivactor® system 0.040±0.010 0.030±0.009 0.008 0.039±0.011 0.029±0.011 0.008 0.039±0.015 0.012±0.004 0.001 easy clean® system 0.032±0.009 0.027±0.009 0.015 0.031±0.007 0.026±0.007 0.015 0.037±0.017 0.023±0.010 0.001 table 2 comparison of irrigating solutions and agitation protocols as to the tissue dissolution capacity. taper were used. the easy clean® system was activated with the aid of a handpiece (kavo in brazil industria e comercio ltda, joinville, sp, brazil) linked to a micromotor (kavo in brazil industria e comercio ltda). after completion of the agitation protocols associated with irrigating solutions, the pulp fragments were removed, dried with absorbent paper, and reweighed on a precision scale. the t test was used to obtain the mean and standard deviation of the experimental groups. to compare the tissue dissolution capacity regarding the association of irrigating solutions and agitation protocols, the nonparametric kruskalwallis test was used, complemented by a multiple comparisons test. the significance level used in the study was set at 5%. results table 2 shows the comparison between the initial and final weight for pulp tissue dissolution according to the type of the irrigant and agitation protocol. regardless of the irrigating solution, the ultrasonic system and the sonic systems endoactivator® and easy clean® differed significantly from immersion and mechanical agitation with endodontic files. in addition, there was a significant difference for mechanical agitation with endodontic files combined with 2.5% sodium hypochlorite. table 3 shows the comparison of the agitation protocols with each of the irrigating solutions. there was no significant difference in the tissue dissolution capacity between the irrigants in the immersion (p = 0.418) and mechanical agitation with endodontic files (p = 0.665). however, the use of ultrasonic system (p = 0.041) and endoactivator® (p = 0.013) and easy clean® systems (p = 0.040) resulted in a statistically significant difference. the combination of these devices with 2.5% sodium hypochlorite promoted greater dissolution capacity compared with the other two tested irrigators. when comparing the solutions in relation to the agitation protocols (table 4), it was observed that regardless of the irrigant, the use of sonic and ultrasonic systems promoted a greater capacity of tissue dissolution. discussion several studies10-12 have tested the outcomes of tissue dissolution during the process of endodontic irrigation. however, it is known that the capacity and efficiency of organic tissue degradation processes are related to the type, concentration, and quantity of irrigating, and how the solution is agitated7,8. thus, this study aimed to analyze the capacity for tissue dissolution according to the combination of different types of irrigating solutions and agitation methods. agitation protocol irrigation solution p destilled water 2% chlorhexidine digluconate 2.5% sodium hypochlorite immersion average (p25;p75) 0.000 (0.000; 0.000) 14.05a 0.000 (0.000; 0.000) 14.10a 0.000 (0.000; 0.010) 18.50a 0.418 rank medium mechanical agitation average (p25;p75) 0.000 (0.000; 0.010) 14.45a 0.000 (0.000; 0.010) 14.50a 0.005 (0.000; 0.010) 17.50a 0.665 rank medium ultrasonic system average (p25;p75) 0.005 (0.000; 0.010) 14.75b/ 0.005 (0.000; 0.010) 14.50b 0.015 (0.007; 0.020) 21.00a 0.041 rank medium endoactivactor® system average (p25;p75) 0.010 (0.000; 0.012) 12.30b 0.010 (0.000; 0.012) 12.40b 0.020 (0.010; 0.042) 21.90a 0.013 rank medium easy clean® system average (p25;p75) 0.005 (0.000; 0.011) 13.50b 0.005 (0.000; 0.011) 13.20b 0.015 (0.007; 0.020) 21.10a 0.040 rank medium table 3 average and medium ranks of the differences between the values of initial and final weight of the pulp fragments after the irrigation protocols. average followed by different capital letters in rows differ significantly by the nonparametric kruskal-wallis test, at a significance level of 5%. braz j oral sci. 15(1):70-74 73 braz j oral sci. 15(1):70-74 bovine pulp tissue fragments were used according to methods reported in recent studies13,14. the ease of obtaining and standardizing the length and weight of the pulp bovine tissue is an advantage because variation in these measures may influence the dissolution ability15. bovine pulp tissue has been shown to be similar to the human pulp tissue2. the delineation of an approximate initial weight of 0.03 g bovine tissue fragment is in agreement with the mass of the pulp tissue in a human molar tooth (0.04 ml × 1 g/ml = 0.04 g)16. in order to approximate this experiment to a more credible clinical condition, the dissolution capacity of the pulp tissue was measured within a tooth structure previously prepared for use, as previously reported in studies by slutzky-goldberg et al.17 (2013) and arslan et al.18 (2015). other studies13,14 used the same methodology, but immersed on a culture plate. there are a variety of experimental methods for measuring the dissolution capacity, such as measuring the time of dissolution19 and visual examination12. in this study, the initial and final weights of the sample were compared, as reported in other studies4,14. the definition and standardization of an experimental time of 2 min for each sample in all groups followed the manufacturers’ recommendations for use of the endoactivactor® and easy clean® systems. among the tested solutions, 2.5% sodium hypochlorite showed a higher tissue dissolution capacity than 2% chlorhexidine digluconate and distilled water, as reported elsewhere20 (1981). one of the known properties of sodium hypochlorite is its capacity of degradation organic tissue21. on the other hand, chlorhexidine digluconate and distilled water showed no significant difference in their ability to dissolve tissue, and none of them was effective in the degradation of pulp tissue, which has been observed previously4,22. according to marley et al.23 (2001), chlorhexidine digluconate has no solvent action on organic tissues, and neither does distilled water. regarding the agitation protocols, the combined use of sonic (endoactivactor® and easy clean®) and ultrasonic systems with irrigating solution has been shown to increase the power to dissolve tissue, as seen in other studies24–26. in the present study, the power of tissue dissolution with the combination of these systems with chlorhexidine digluconate and distilled water was more efficient compared with the use of the immersion and mechanical agitation with endodontic files methods. it is believed that this result was possibly due to the direct action afforded by the vibration equipment: ultrasound (28.000 hz), endoactivactor® (190 hz), and easy clean® (approximately 4000 hz). although at different frequencies, the similarity in results between the sonic and ultrasonic devices may have been provided by the small diameter of the tips used for operating the sonic devices. both the endoactivactor® and easy clean® systems have tips with a diameter of 0.25 and 0.04 taper. however, the ultrasonic insert used in this study has a pointed shape with initial diameter of 0.40, which is therefore greater. the fact that none of the protocols tested was fully effective in pulp tissue degradation can be associated with and justified by the agitation time used in the study. in a way, this condition may be of concern if we take into account faster preparations. however, it is believed that the process of agitation associated with the appropriate irrigating solution and not just the action of irrigating leverage the power of tissue dissolution. the repetition of several cycles of agitating the irrigating solution during chemomechanical preparation can promote the complete dissolution of the pulp tissue. references 1. peters oa, laib a, göhring tn, barbakow f. changes in root canal geometry after preparation assessed by high resolution computed tomography. j endod. 2001 jan;27(1):1-6. 2. vera j, siqueira jf jr, ricucci d, loghin s, fernández n, flores b, et al. oneversus two-visit endodontic treatment of teeth with apical periodontitis: a histobacteriologic study. j endod. 2012 aug;38(8):1040-52. doi: 10.1016/j. joen.2012.04.010. 3. giardino l, estrela c, mohammadi z, palazzi f. antibacterial power of sodium hypochlorite combined with surfactants and acetic acid. braz dent j. 2014;25(4):289-94. 4. tartari t, guimarães bm, amoras ls, duarte ma, silva e souza pa, bramante cm. etidronate causes minimal changes in the ability of sodium hypochlorite to dissolve organic matter. int endod j. 2015 apr;48(4):399404. doi: 10.1111/iej.12329. 5. mirhadi h, abbaszadegan a, ranjbar ma, azar mr, geramizadeh b, torabi s, et al. antibacterial and toxic effect of hydrogen peroxide combined with different concentrations of chlorhexidine in comparison with sodium hypochlorite. j dent (shiraz). 2015 dec;16(4):349-55. 6. gomes bp, vianna me, zaia aa, almeida jf, souza-filho fj, ferraz cc. chlorhexidine in endodontics. braz dent j. 2013;24(2):89-102. doi: analysis of root canal organic tissue dissolution capacity according to the type of irrigation solution and agitation technique stirring agitation irrigation solution destilled water 2% chlorhexidine digluconate 2.5% sodium hypochlorite immersion 15.4b 15.5b 13.5c mechanical agitation 22.7bc 22.8bc 16.8bc ultrasonic system 27.3ab 27.5ab 29.4ab endoactivactor® system 34.3ac 34.6ac 38.6a easy clean® system 27.5ab 27.8ab 30.2ab p 0.013 0.015 < 0.001 table 4 comparative analysis of the power to dissolve tissue of each irrigating solution for the different agitation protocols. average followed by different capital letters in columns differ significantly by the nonparametric kruskal-wallis test, at a significance level of 5%. 74 braz j oral sci. 15(1):70-74 10.1590/0103-6440201302188. 7. clarkson rm, moule aj, podlich h, kellaway r, macfarlane r, lewis d, et al. dissolution of porcine incisor pulps in sodium hypochlorite solutions of varying compositions andconcentrations. aust dent j. 2006 sep;51(3):24551. 8. del carpio-perochena ae, bramante cm, duarte ma, cavenago bc, villas-boas mh, graeff ms, et al. biofilm dissolution and cleaning ability of different irrigant solutions on intraorally infected dentin. j endod. 2011 aug;37(8):1134-8. doi: 10.1016/j.joen.2011.04.013. 9. andersen m, lund a, andreasen jo, andreasen fm. in vitro solubility of human pulp tissue in calcium hydroxide and sodium hypochlorite. endod dent traumatol. 1992 jun;8(3):104-8. 10. cobankara fk, ozkan hb, terlemez a. comparison of organic tissue dissolution capacities of sodium hypochlorite and chlorine dioxide. j endod. 2010 feb;36(2):272-4. doi: 10.1016/j.joen.2009.10.027. 11. stojicic s, zivkovic s, qian w, zhang h, haapasalo m. tissue dissolution by sodium hypochlorite: effect of concentration, temperature, agitation, and surfactant. j endod. 2010 sep;36(9):1558-62. doi: 10.1016/j. joen.2010.06.021. 12. taneja s, mishra n, malik s. comparative evaluation of human pulp tissue dissolution by different concentrations of chlorine dioxide, calcium hypochlorite and sodium hypochlorite: an in vitro study. j conserv dent. 2014 nov;17(6):541-5. doi: 10.4103/0972-0707.144590. 13. irala led, soares rg, salles aa, munari az, pereira js. dissolution of bovine pulp tissue in solutions consisting of varying naocl concentrations and combined with edta. braz oral res. 2010 jul-sep;24(3):271-6. 14. só mvr, cemim a, pereira ep, irala led. tissue dissolution ability of sodium hypochlorite from different manufacturers. braz endod j. 2010 sep;36(9):1558-62. doi: 10.1016/j.joen.2010.06.021. 15. niewierowski rs, scalzilli lr, morgental rd, figueiredo ja, vier-pelisser fv, borba mg, et al. bovine pulp tissue dissolution ability of irrigants associated or not to ultrasonic agitation. braz dent j. 2015 oct;26(5):53740. doi: 10.1590/0103-6440201300243. 16. morgan rw, carnes dl jr, montgomery s. the solvent effects of calcium hydroxide irrigating solution on bovine pulp tissue. j endod. 1991 apr;17(4):165-8. 17. slutzky-goldberg i, hanut a, matalon s, baev v, slutzky h. the effect of dentin on the pulp tissue dissolution capacity of sodium hypochlorite and calcium hydroxide. j endod. 2013 aug;39(8):980-3. doi: 10.1016/j. joen.2013.04.040. 18. arslan d, guneser mb, kustarci a, er k, siso sh. pulp tissue dissolution capacity of qmix 2in1 irrigation solution. eur j dent. 2015 julsep;9(3):423-7. doi: 10.4103/1305-7456.163229. 19. johnson br, remeikis na. effective shelf-life of prepared sodium hypochlorite solution. j endod. 1993 jan;19(1):40-3. 20. gordon tm, damato d, christner p. solvent effect of various dilutions of sodium hypochlorite on vital and necrotic tissue. j endod. 1981 oct;7(10):466-9. 21. estrela c, estrela cr, barbin el, spanó jc, marchesan ma, pécora jd. mechanism of action of sodium hypochlorite. braz dent j. 2002;13(2):113-7. 22. okino la, siqueira el, santos m, bombana ac, figueiredo jap. dissolution of pulp tissue by aqueous solution of chlorhexidine digluconate and chlorhexidine digluconate gel. int endod j. 2004 jan;37(1):38-41. 23. marley jt, ferguson db, hartwell gr. effects of chlorhexidine gluconate as an endodontic irrigant on the apical seal: short-term results. j endod. 2001 dec;27(12):775-8. 24. sabins ra, johnson jd, hellstein jw. a comparison of the cleaning efficacy of short term sonic and ultrasonic passive irrigation after hand instrumentation in molar root canals. j endod. 2003 oct;29(10):674-8. 25. paragliola r, franco v, fabiani c, mazzoni a, nato f, tay fr, et al. final rinse optimization: influence of different agitation protocols. j endod. 2010 feb;36(2):282-5. doi: 10.1016/j.joen.2009.10.004. 26. haapasalo m, wang z, shen y, curtis a, patel p, khakpour m. tissue dissolution by a novel multisonic ultracleaning system and sodium hypochlorite. j endod. 2014 aug;40(8):1178-81. doi: 10.1016/j. joen.2013.12.029. analysis of root canal organic tissue dissolution capacity according to the type of irrigation solution and agitation technique 1http://dx.doi.org/10.20396/bjos.v18i0.8657272 volume 18 2019 e191679 original article 1 department of orthodontics, centro universitário hermínio ometto-uniararas, araras, são paulo, brazil. corresponding author: william custodio av. dr. maximiliano baruto, 500 jd. universitário araras, sp, br. zip code: 13607-339 phone: 55 19 38610472 e-mail: wcust@hotmail.com https://orcid.org/0000-0003-1416-1414 received: june 07, 2019 accepted: august 29, 2019 comorbidity of tmd and malocclusion: impacts on quality of life, masticatory capacity and emotional features beatriz moraes d’avilla1, michelle cristina berbet pimenta1, vivian fernades furletti1, mario vedovello filho1, giovana cherubini venezian1, william custodio1,* aim: to evaluate the synergic impact of muscular tmd and malocclusion on quality of life, masticatory capacity and emotional features of young adults. methods: this cross-sectional study comprised 4 groups (n= 15): g1, individuals without tmd or malocclusion; g2, with tmd and malocclusion; g3, with tmd and without malocclusion, and g4, without tmd and with malocclusion. muscular tmd was diagnosed by rcd/tmd. data included quality of life (ohip-14), masticatory capacity test (x50), emotional stress (pss-14), depression (mdi), pain intensity and salivary cortisol. comparative statistical analysis included one-way anova and tukey post hoc test (x50, stress and cortisol) and genmod followed by wald test (ohip-14 and pain data). fisher’s and pearson’s association analysis were carried out. results: comorbidity of muscular tmd and malocclusion leads to significant lower masticatory capacity (p<0.05).  tmd groups independently of the occlusal condition had considerably lower ohip-14 scores and higher stress levels (self-perceived and hormonal) (p<0.05). there was no statistically significant difference of emotional depression among groups. a significant positive correlation was observed among quality of life, stress and pain perception. conclusion: muscular tmd in the overlap of malocclusion potentializes their negative effect on masticatory capacity. in addition, the hindering effect of the comorbidity is variable, however, tmd has a greater negative impact on quality of life and stress, whilst malocclusion on mastication. keywords: temporomandibular joint disorders. mastication. malocclusion. quality of life. stress, psychological. https://orcid.org/0000-0003-1416-1414 2 d’avilla et al. introduction association of temporomandibular disorders (tmd) and occlusal characteristics is controversial and evidences, at present, does not support a cause-effect relationship between these highly prevalent conditions of the population1. in addition, comorbid occlusal disturbances and self-perceived psychological conditions make difficult to draw the real impact of tmd on patients’ quality of life and on the functional response of their masticatory system2. structural variations in components of the masticatory system, such as muscular disturbances, have now been characterized as main etiological factors for tmd3. however, occlusal disturbances have been considered as triggers, perpetuating and even contributory factors for tmd4,5. this lack of concordance may be related with the individual capacity of the body for adaptation to occlusal discrepancies overlapped with the predisposition to chronic pain conditions1,6. the pain related to tmd may generate changes in the mandibular kinematics, increasing the number of occlusal interferences and consequently, changing the masticatory pattern6. furthermore, this may lead to compensation of muscles and other associated structures for the purpose of bearing excessive functional loads however, in a cyclic manner, this adaptation may accentuate the pain, and if not treated, lead to important tissue damage. masticatory function in individuals with tmd has been suggested to be limited because the functional response of the masticatory system is subject to a complex interaction between physical and emotional aspects. the number of occlusal contacts, dimension of the grinding area, sagittal and transverse changes in the teeth and bony bases modulate masticatory capacity7,8. in addition to the occlusal factor, malocclusion may correlate with a reduction in masticatory capacity by hindering the ability of the muscle to work9,10. since malocclusion and tmd may lead to functional harm, self-perception of oral health may determine a decline in the quality of life9. however, the pain component of tmd and compromised esthetic appearance resulting from malocclusion are subjective factors that may be influenced by the emotional state10. therefore, the levels of stress and depression are capable of changing the self-perception of the impact of these conditions on quality of life11. thus, in patients with tmd or occlusal discrepancies, we may recognize an poorer masticatory function with concomitant impairment of quality of life. however, the direction of a mutual influence remains obscure. thus, the aim of this study was to evaluate the role of tmd, malocclusion and their interaction in quality of life, masticatory capacity, and emotional aspects. materials and methods experimental design and participants this double-blind cross-sectional study had a parallel-group design, in which four independent groups were evaluated. malocclusion and muscular tmd were considered 3 d’avilla et al. independent variables while the dependent variables were masticatory efficiency, quality of life, self-perception of pain, emotional stress, depression and salivary cortisol level. the subjects entry order for the different experimental tests was randomized (simple randomization by lottery method). the participants were not informed about their diagnoses regarding muscular tmd and malocclusion till the end of the study. the investigator who performed the examination for muscular tmd diagnoses and malocclusion assessment was blinded regarding the study objectives the other investigator that carried out the experimental tests was blinded to the patient allocation group. this study was approved by the research ethics committee of the university center of the hermínio ometto foundation fho under protocol number 1.329.422. all participants gave their informed consent prior to their inclusion in the study. the participants, 210 young adults with the same educational level, were recruited at a university dental clinic (araras, brazil). the exclusion criteria were: missing teeth, use of orthodontic appliance, periodontal disease, subjects taking antidepressant, anti-inflammatory, analgesic medications or those with the potential to change salivary flow, subjects undergoing treatment for tmd, and those with a history of trauma or surgery in the region of the head and neck. a total of 60 participants (mean age = 25.3, s.d. = 5.1) met the criteria and were selected and allocated to four gender paired groups (n=15). group i (control) comprised individuals who were asymptomatic for tmd and with clinically normal occlusion; group ii, subjects with tmd and malocclusion; group iii, made up of individuals with tmd and with clinically normal occlusion; and group iv, asymptomatic for tmd and with malocclusion. to diagnose the presence of tmd, the rdc/tmd (research diagnostic criteria for ttemporomandibular disorders) was used12. individuals with muscular tmd (rdc/tmd groups ia and ib) were included in the sample. occlusion that was considered clinically normal was based on the angle class i dental relationship, with a positive horizontal overjet and vertical overlap of less than 3 mm, a normal transverse relationship and class i molar and canine relationship, among others characteristics as described previously by manfredini et al.13 (2016). subjects that didn’t presented two or more of the stablished occlusal features were considered with malocclusion. the sample size of 15 participants per group, provided a test power of over 80% to detect differences of 5 in imc, 2 in ohip, 8 in pss-14, 3 in cortisol, 0.7 in masticatory efficiency (assessed by the particle median (x50)), and 10 in the self-perception of pain (as assessed using a visual analog scale (vas)), between means of the groups, with a level of significance of 5%. masticatory capacity the masticatory capacity was determined using the masticatory performance test (x50)14. briefly, the participants were instructed to chew, in the habitual manner, a portion of 17 standardized cubes of a chewable test material (optosil plus, heraeuz kulzer, hanau, germany) (weight = 3.4 g; edges = 5.6 mm) in a total of 20 masticatory cycles. the produced particles were fractionated by a system of 10 sieves with meshes with openings that ranged between 5.6 mm and 0.5 mm, coupled to a vibratory table for 20 minutes. the amount accumulated in each sieve was weighed on an analytical balance with a precision of 0.0001 g (mark, 2060, bel engineering, milan, italy). masticatory performance was evaluated by calculating the median particle size (x50) by means of the 4 d’avilla et al. rosin-rammler equation15. in the equation, x50 é is the opening in a hypothetical sieve through which 50% of the weight is capable of passing. thus, the lowest mean values of the particles (x50) denote the highest masticatory performance. self-evaluation scales the impact of oral health conditions on quality of life was evaluated by the validated brazilian version of the oral health impact profile (ohip-14) questionnaire. this self-applicable questionnaire consists of 14 questions about oral-health related problems experienced over the past year, with answers based on a likert-type scale of 0 (never), 1 (hardly ever), 2 (occasionally), 3 (almost always) and 4 (always). the sum scores of responses obtained may vary from 0 to 56 points, and the higher the score, the greater the negative impact on the quality of life16. the self-applicable questionnaire pss-14 (perceived stress scale) was used to determine the impact of the level of emotional stress perceived17. this is made up of 14 questions that must be answered by considering only the last month. the responses are as follows: 0 (never), 1 (hardly ever), 2 (occasionally), 3 (almost always) and 4 (always). to calculate the total score, answers to questions with a positive connotation (4, 5, 6, 7, 9, 10 e and 13) were inverted. the result of the sum of the instrument score varies from zero to 56. the higher the final score, the higher will be the level of emotional stress perceived. the level of depression was determined using the validated brazilian version of the major depression inventory (mdi) questionnaire18. this comprises 10 questions with reference to the past weeks. as a measurer of severity, the score of the mdi varies from 0 to 50, because each of the 10 items may receive a score ranging from 0 (never) to 5 points (all the time). thus a score between 20 and 24 classifies the depression as light, from 25 to 29 as moderate, and over 30 points, as severe depression19. self-perception of pain intensity, immediately before and after the masticatory performance test, was evaluated using a visual analogue scale (vas)20. vas score was determined by using a 10-cm line presenting in one extremity the “no pain” anchor and in the other “worst pain imaginable”. the participant was asked to place a line perpendicular to the vas line at the point that represents their pain intensity. the distance (mm) from the vertical line traced from the value zero (absence of pain) was considered the pain intensity present. all tests were conducted by the same examiner, who was trained for to apply the experimental instruments and collecting the data in a standardized manner. salivary cortisol as a biomarker of self‐reported mental stress, the salivary cortisol level was determined. stimulated whole saliva was collected with the use of salivette® (sarstedt ag & co, nümbrecht, germany). salivary collections were carried out under the same sampling strategy respecting the avoidance of recent food intake, exposure to mental stressors and the circadian rhythms of glucocorticoid21. the samples were stored in accordance with the manufacturer’s specifications, and evaluated in a laboratory with iso 9001 certification. the exam was performed after the salivette tubes were centrifuged at 1000g to separate the saliva, and subsequently submitted to chemiluminescence analysis. the results were expressed in nmol/l. 5 d’avilla et al. statistical analysis the mean pss14 score, salivary cortisol and masticatory performance (x50) values were submitted to analysis of variance (one-way anova), followed by the tukey test. the cortisol data were transformed into log base 10. the ohip data were analysed using generalized linear models (genmod) and the pain intensity scores using generalized linear models for repeated measures, followed by the wald test. the association of tmd and malocclusion with depression (mdi) was analysed using the fisher exact test. the pearson correlation analysis was used to correlate the other variables. all analyses were performed using the sas (sas institute inc., cary, nc, usa, release 9.2, 2010) and r (r core team (r foundation for statistical computing, vienna, austria) programs, with a level of significance of 5%. results the comparisons between groups regarding body mass index (bmi), masticatory performance (x50), impact of oral health conditions on quality of life (ohip-14), perceived level of emotional stress (pss14) and salivary cortisol, are presented in table 1. the sample did not differ with regard to bmi (p=0.1337). the worst masticatory performance (highest x50 score) was observed in the comorbidity group (g2) (p<0.05). the higher masticatory performance were showed by individuals without malocclusion (g1 and g3), independently of muscular tmd presence (p<0.05). compared with the negative control group (g1), the impact of oral health conditions on quality of life (ohip-14), self-perceived emotional stress (pss14) and salivary cortisol level was significantly higher in individuals with tmd (g2 and g3), independently of the presence/absence of malocclusion (p<0.05). individuals whose overlapped muscular tmd and malocclusion (g2) and those with muscular tmd without malocclusion (g3) showed higher mean values of self-perceived pain even before mastication (p<0.05). after the masticatory function muscular tmd implies higher pain levels independently of occlusal condition (g2 and g3) (p<0.05) (figure 1). table 1. groups comparisons for bmi, x50, ohip-14, pss14 and salivary cortisol. (mean ± sd) group condition bmi x50 ohip -14 pss14 cortisol † tmd malocclusion g1 absence absence 21.8 ± 2.6 a 2.71 ± 0.65 c 0.53 ± 1.3 c 16.27 ± 762 b 3,83 ± 2,72 c g2 present present 20.3 ± 4.4 a 6.09 ± 0.89 a 6.53 ± 5.9 a 26.13 ± 6.33 a 7,45 ± 4,93 ab g3 present absence 23.0 ± 3.5 a 3.22 ± 0.62 c 5.20 ± 4.0 ab 23.53 ± 5.07a 7,87 ± 3,52 a g4 absence present 23.4 ± 4.8 a 4.90 ± 0.30 b 2.07 ± 2.5 bc 21.00 ± 5.59 ab 4,35 ± 2,59 bc p-value 0.1337 ≤0.05 ≤0.05 ≤0.05 ≤0,05 different lowercase letters in the columns indicate the statistical difference. †salivary cortisol level in micrograms per deciliter (μg / dl). 6 d’avilla et al. figure 2 presents the distribution of frequency of the degree of depression (mdi) in the groups. there was no significant differences among groups (p=0.5686). the majority of the volunteers presented no depression. table 2 shows the correlations between the variables. a significant moderately positive correlation was observed (p<0.05) between the impact of oral health conditions and the quality of life, s stress (r=0.40), and self-perception of pain before (r=0.39) and after mastication (r=0.44). self-perception of pain before mastication presented a significant positive correlation (p<0.05) with x50 (r=0.64); that is, the higher the pain level before mastication, the worse was the masticatory performance. in contrast, the median particle size arising from mastication was not correlated with pain after the test (p=0.8879). figure 2. relative frequency (%) of the degree of self-perception of depression (mdi) according to the different groups. malocclusion absence malocclusion presence tmd absence tmd presence absence mild moderate severe 0 20 40 60 80 100 malocclusion absence malocclusion presence figure 1. pain self-perception by visual analogue scale (vas) in as a function of groups and time. malocclusion absence malocclusion presence tmd absence tmd presence before mastication after mastication 70 60 50 40 30 20 10 0 malocclusion absence malocclusion presence malocclusion absence malocclusion presence tmd absence tmd presence malocclusion absence malocclusion presence 7 d’avilla et al. discussion in this study the different carried out tests provided a substantial amount of information to suggest the impairment in masticatory function and quality of life due to muscular tmd, malocclusion, pain and emotional stress. the masticatory performance can be considered as an important method for evaluating the functionality of the stomatognathic system and it is capable of measuring food comminution potential, by quantifying the particle size. patients with malocclusion evaluated during food chewing, showed significant lower masticatory performance when compared to controls (g1 e g3). similar results were obtained in previous studies, showing that malocclusion had a negative impact on masticatory capacity22,23. the literature shows that patients with malocclusion usually have less occlusal contacts and reductions in the occlusal functional areas of teeth during mastication24. greater masticatory function impairment was observed in muscular tmd co-occurrence patients (g2) (table1). possibly, the limitation of masticatory performance aims to avoid the pain exacerbation, which could explain the lower food comminution rates in patients with muscular tmd. patients with tmd (g2 and g3) presented higher levels of pain before and after mastication, irrespective of the presence or absence of malocclusion. however, the pain generated by mastication was not necessarily correlated with the particle size obtained. the pain of patients with tmd probably limited their masticatory performance, leading to compensatory mechanisms such as a larger number of cycles, slower rate of mastication and accessory muscle recruitment22-24,25. therefore, despite the presence of pain during mastication in the groups with tmd, the presence of malocclusion had a greater impact on masticatory capacity. it must also be remarked that static or dynamic occlusal characteristics were not associated with tmd pain conditions. pain or the structural changes associated with tmd limit or even deviate mandibular kinetics, and consequently affect the masticatory pattern4,6. however, it is important to emphasize that masticatory capacity may not be affected, even in individuals with tmd. the authors suggest that compensatory mechanisms that lead to greater table 2. pearson correlation coefficient (p-value) among assessed variables quality of life emotional stress pain before mastication pain after mastication salivary cortisol x50 bmi -0.04 (0.7340) -0.10 (0.4307) -0.10 (0.4470) -0.04 (0.7647) -0.05 (0.6844) -0.14 (0.2661) quality of life 0,40 (0.0014) 0.39 (0.0022) 0.44 (0.0004) 0.13 (0.3343) 0.22 (0.0964) emotional stress 0.31 (0.0145) 0.13 (0.3137) 0.13 (0.3309) 0.29 (0.0245) pain before mastication 0.24 (0.0701) 0.20 (0.1251) 0.64 (<0.001) pain after mastication 0.12 (0.3646) 0.02 (0.8879) salivary cortisol 0.15 (0.2517) 8 d’avilla et al. recruitment of other masticatory muscles, such as the temporal muscles, may compensate for debilities resulting from tmd11,26. thus, the results demonstrated that the impact on the functional response only became relevant when associated with the presence of malocclusion. although tmd had no impact on masticatory capacity, we observed a negative impact on to the oral health-related quality of life (ohip-14). pain, a common symptom of tmd is believed to be the main factor related to compromised social behaviour, and the emotional and psychological state of the individual12,27. the impact of the oral health condition on quality of life was higher when tmd was associated with the presence of malocclusion. nevertheless, malocclusion alone did not determine greater compromise of quality of life, corroborating the findings of other studies25,28. the behavioural variations of each individual and non-experience of a clinically adequate occlusion made it difficult to evaluate the real impact on to the quality of life due to malocclusion28,29. studies have suggested that when malocclusion has an impact on quality of life, this is due to the aesthetic-emotional component, and not due to the functional impact25,28. moreover, both quality of life and tmd may be modulated by emotional aspects such as stress. in the present study, individuals with tmd not only presented self-perception about stress, but also had higher salivary cortisol levels, a biomarker of physiological stress, corroborating with previous studies30. emotional stress may be associated with muscle hyperactivity and overload of the stomatognathic system, causing inflammation in the retrodiscal tissues, muscle fatigue and muscular tmd. the results demonstrated no direct correlation between stress and pain. it can be due to the fact that individuals with chronic stress, even with pain, may present lower levels of salivary cortisol as a result of the reduction in hormone secretion by the adrenal gland31,32. in the present study, no statistical difference in self-perception of depression was observed. besides depression has an influence on tmd11,33, the onset time of the tmd of the included volunteers may be responsible for the non-association. the limitations of the present study included lack in information regarding the time-onset and frequency of tmd related pain, the non-selection of participants at random from the population and the absence of other emotional variables such as anxiety that may be related to tmd. however, sample homogeneity regarding to gender, age and bmi avoided biases. clinically, the results indicate that although malocclusion has more effect on masticatory capacity, this functional impairment has less influence on the quality of life. thus, the impact on quality of life is probably not related to the reduction in masticatory function, but rather to the pain component of muscular tmd. according to the evaluated parameters, it is possible to conclude that co-occurring conditions, muscular tmd and malocclusion had a greater negative influence on masticatory capacity. quality of life, pain and emotional stress are associated and seems to be impaired by the tmd condition, regardless of malocclusion presence. acknowledgements the masticatory capacity test was carried out at the department of prosthodontics and periodontology, piracicaba dental school, state university of campinas (brazil), and we acknowledge prof. altair antoninha del bel cury for her generous support. 9 d’avilla et al. references 1. manfredini d, lombardo l, siciliani g. temporomandibular disorders and dental occlusion. a systematic review of association studies: end of an era? j oral rehabil. 2017 nov;44(11):908-923. doi: 10.1111/joor.12531. 2. okeson jp, de leeuw r. differential diagnosis of temporomandibular disorders and other orofacial pain disorders. dent clin north am. 2011 jan;55(1):105-20. doi: 10.1016/j.cden.2010.08.007. 3. slade gd, ohrbach r, greenspan jd, 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joint disorder pain. j electromyogr kinesiol. 2011 aug;21(4):659-64. doi: 10.1016/j.jelekin.2011.03.003. 27. lobbezoo f, drangsholt m, peck c, sato h, kopp s, svensson p. topical review: new insights into the pathology and diagnosis of disorders of the temporomandibular joint. j orofac pain. 2004 summer;18(3):181-91. 28. feu d, de oliveira bh, de oliveira almeida ma, kiyak ha, miguel ja. oral health-related quality of life and orthodontic treatment seeking. am j orthod dentofacial orthop. 2010 aug;138(2):152-9. doi: 10.1016/j.ajodo.2008.09.033. 29. liu z, mcgrath c, hägg u. the impact of malocclusion/orthodontic treatment need on the quality of life. a systematic review. angle orthod. 2009 may;79(3):585-91. doi: 10.2319/042108-224.1. 30. dahlström l, carlsson ge. temporomandibular disorders and oral health-related quality of life. a systematic review. acta odontol scand. 2010 mar;68(2):80-5. doi: 10.3109/00016350903431118. 31. rosmond r, björntorp p. low cortisol production in chronic stress. the connection stress-somatic disease is a challenge for future research. lakartidningen. 2000 sep;97(38):4120-4. 32. kanehira h, agariguchi a, kato h, yoshimine s, inoue h. association between stress and temporomandibular disorder. nihon hotetsu shika gakkai zasshi. 2008 jul;52(3):375-80. 33. reiter s, emodi-perlman a, goldsmith c, friedman-rubin p, winocur e. comorbidity between depression and anxiety in patients with temporomandibular disorders according to the research diagnostic criteria for temporomandibular disorders. j oral facial pain headache. 2015 29(2):135-43. doi: 10.11607/ofph.1297. 1http://dx.doi.org/10.20396/bjos.v19i0.8656624 volume 19 2020 e206624 original article 1 department of community dentistry, são paulo state university (unesp), school of dentistry, araraquara, sp, brazil. 2 university of florida college of dentistry, director, pain clinical research unit, uf ctsi, deputy director, south atlantic region, dental practice-based research network. clinical and translational research building (ctrb), gainesville, fl, usa. 3 department of clinical & community sciences, school of dentistry, university of alabama at birmingham, birmingham, al, usa. 4 university of florida, college of dentistry, gainesville, fl, usa. corresponding author: elaine pereira da silva tagliaferro school of dentistry, araraquara, são paulo state university (unesp) rua humaitá, 1680 centro 14801-903 araraquara, sp, brasil tel: +55 16 3301-6343 fax: +55 16 3301-6343 elaine.tagliaferro@unesp.br received: august 14, 2019 accepted: january 24, 2020 methods for caries prevention in adults among dentists from a brazilian community elaine pereira da silva tagliaferro1,*, silvio rocha correa da silva1, fernanda lopez rosell1, aylton valsecki junior1, joseph l. riley iii2, gregg h. gilbert3, valeria veiga gordan4 little is known about dental practice patterns of caries prevention in adults among brazilian dentists. aim: to quantify procedures used for caries prevention for adult patients among dentists from a brazilian community. methods: dentists (n=197) who reported that at least 10% of their patients are more than 18 years old participated in the first brazilian study that used a translated version of the “assessment of caries diagnosis and caries treatment” from the u.s. national dental practice-based research network. a questionnaire about characteristics of their practice and patient population were also completed by the dentists. generalized linear regression models and a hierarchal clustering procedure were used (p<0.05). results: in-office fluoride application was the preventive method most often reported. the main predictors for recommending some preventive agent were: female dentist (dental sealant; in-office fluoride; non-prescription fluoride) and percentage of patients interested in caries prevention (dental sealant; in-office fluoride; non-prescription fluoride). other predictors included private practice (dental sealant), percentage of patients 65 years or older (in-office fluoride), graduation from a private dental school (non-prescription fluoride), years since dental school graduation (chlorhexidine rinse) and using a preventive method (recommending sealant/fluoride/chlorhexidine rinse/sugarless, xylitol gum). cluster analysis showed that dentists in the largest subgroup seldom used any of the preventive agents. conclusion: dentists most often reported in-office fluoride as a method for caries prevention in adults. some practitioner, practice and patients’ characteristics were positively associated with morefrequent use of a preventive agent. keywords: dental caries. practice patterns, physicians. preventive dentistry. 2 tagliaferro et al. introduction caries prevalence in adults is high worldwide. more than 90% of adults experience caries at some point in their lifetimes1-4. mean dmft scores for 35to 44-year-old adults ranged from 6.6 to 17.6 among twenty-three european countries3 and is 16.75 among brazilian adults, according to the last national epidemiological survey1. a recent report estimated that about 25 percent of adults in the u.s. had untreated caries5. treatment needs were reported by 75% and 47% of brazilian adults and elderly, respectively1. these findings may be related to the fact that dental caries prevention efforts historically have focused on children rather than adults6. a major increase in the focus of public health efforts in adults should be on those who are transitioning into higher caries risk status7. therefore, dentists and dental health managers should direct efforts to improve adults’ oral health and research should assess the oral health status as well as which preventive strategies the adult population is receiving from their dentists. members of the dental practice-based research network (dental pbrn) from the united states, denmark, norway, and sweden reported applying in-office fluoride on 37% of their adult patients8. a minority (21%) of dentists in the japan dental pbrn recommended in-office fluoride application to most of their patients over 18 years old9. the most-frequent users of caries prevention were recently-graduated dentists, those who perform caries risk assessment or who practice individualized caries prevention8. japanese dentists whose patients are interested in caries prevention or those who believe in the effectiveness of caries risk assessment were more likely to recommend in-office fluoride to 50% or more of their patients9. dentists from the brazilian community of araraquara, são paulo state, participated in the first brazilian study to use the same questionnaire (after translation and cultural adaptation) used in the u.s. and japanese studies described above to assess dental preventive practices. the current study aims to quantify procedures used for caries prevention for adult patients among these brazilian dentists. material and methods study design this research is part of a major cross-sectional study that was performed to assess dental practices related to diagnosis and treatment of dental caries by means of two paper questionnaires: (1) one about characteristics of their practice and patient population; and (2) a translated version of the “assessment of caries diagnosis and caries treatment” from the u.s national dental practice-based research network. in the present paper, we present the results from the caries prevention section of the questionnaire. ethical aspects the major cross-sectional study was reviewed and approved by the institutional review board (research ethics committee; protocol number #78/11). all participants provided informed consent prior to participation in the study. 3 tagliaferro et al. participants and data collection questionnaires were sent by mail to 801 dentists for whom we had address/contact information. during study planning, we received a list of 722 dentists registered at the regional council of dentistry of são paulo state – araraquara region, in 2011. because data were collected in 2014-2015, we updated the list by consulting internet sources, which increased the list to 801 dentists. after using several strategies to increase the response rate (pre-paid return envelope, questionnaires sent to work address; a second copy of the questionnaire to non-respondents; precontact by telephone; collection of completed questionnaires at work address)10, a total of 217 dentists who met all inclusion criteria (currently practices in araraquara, treats dental caries; not retired; and provided signed informed consent) participated in the major study, providing an overall response rate of 27% (217/801). in the present paper, participants were 197 dentists among the 217 dentists who reported that at least 10% of their patients are more than 18 years of age. measures participant dentists received two paper questionnaires: (1) one about demographic data and information about their clinical training and individual practices and (2) a translated version of the “assessment of caries diagnosis and caries treatment” from the u.s national dental practice-based research network. the brazilian version of the questionnaire was produced by conducting the following steps: initial translation, back-translation, committee review11 and pre-testing, during which comprehension of questions was tested with 21 dentists and test-retest reliability was estimated with 17 dentists, with a mean time between test and retest of seven days. results from this process showed the intraclass correlation coefficients (icc) as follows: 22 (42%) questions with satisfactory correlation (0.40≤icc<0.75) and 31 (58%) with excellent correlation (icc≥0.75), according to szklo and nieto12 (2000). considering that the translated questionnaire does not measure psychometric data and had been previously validated13, no additional validation was needed. detailed information on the process is published elsewhere10. table 1 presents the series of questions asked about the use of caries preventive agents in adult patients as well as questions about caries diagnosis, caries risk assessment, and individualized caries preventive treatment regimen. statistical analyses descriptive statistics were calculated for all study variables. when reporting ordinal variables representing the percentage of patients receiving caries-related procedures and prevention, ordinal responses are presented as the 25th, 50th (median) and 75th percentile. in addition, these ordinal data were transformed to the average of each endpoint for each category as follows: 0%=0%, 1-24%=12.5%, 25-49%=37%, 50-74%=62%, 75-99%=87%, 100%=100%. the distance between ordinal categories, although not equal, can be estimated in this way with moderate precision. consequently, we believe the data transformed to percentages in this manner for presentation in the tables can aid readers in interpretation. 4 tagliaferro et al. next, practitioner and practice characteristics were tested as predictors of use for each caries prevention agent for adult patients using generalized linear models and an ordinal response model. these variables included dentist gender (male=0 and female=1), years since dental school graduation, whether the dental school from which the dentist graduated was private or public (public=0 and private=1), completed specialization training (general dentistry=0 and specialization=1), an advanced degree (no advanced degree=0, master’s or doctorate=1), percentage of patients who are 65 years of age or older, the dentist’s practice is exclusively a private practice model (public health or hybrid private/public health models=0, private=1). practitioner and practice characteristics that were significant at p<0.10 were included in the first step of subsequent model testing for each individual caries prevention agent. next, these items were included in a second step: caries-related practice patterns; percent of patients for whom a dental explorer is used to diagnose an occlusal caries lesion; whether caries risk was assessed (not performing caries risk assessment=0 and performing caries risk assessment=1); and percentage of patients who desire individualized caries prevention and who receive an individualized caries prevention regimen. in the final step, frequencies of the other preventive agents were entered to test for associations between use of agents. a backward elimination approach was used for step two and three that removed the least-significant variable from the model in subsequent steps until all remaining variables were significant using p<0.10 for retention14. the change in the chi-square (x2) statistic as well as the differences in degrees of freedom (x2diff = x 2 s x 2 1 and dfdiff = dfs df1 where s denotes the “smaller” model with less parameters) were used to test for significance and reflects the improvement in prediction following each step15. to identify subgroups of dentists with a similar preventive orientation, a hierarchal clustering procedure was used. the sugarless or xylitol gum variable was not included as it was table 1. questions asked about caries prevention, assessment, caries risk assessment, and individualized preventive treatment. instructions: of patients more than 18 years old, for what percentage do you: caries prevention apply dental sealants on the occlusal surface of at least one of their permanent teeth? administer an in-office fluoride application, such as fluoride gel, fluoride varnish, or fluoride rinse? recommend a non-prescription (over-the-counter) fluoride rinse? provide a prescription for some form of fluoride? recommend an at-home regimen of chlorhexidine rinse? recommend sugarless chewing gum or xylitol chewing gum? caries assessment, risk assessment, individualized preventive treatment when you examine patients to determine if they have a primary occlusal caries lesion, on what percent of these patients do you use a dental explorer to diagnose the lesion? do you assess caries risk for individual patients in any way? do you use a special form for caries risk assessment? what percent of patients in your practice are interested enough in caries prevention to justify you recommending to them an individualized caries preventive regimen? for what percent of patients do you give individualized preventive treatment specifically for their needs? participants had the following answering choices: 1 – never or 0% 2 – 1 to 24% 3 – 25 to 49% 4 – 50 to 74% 5 – 75 to 99% 6 – every time or 100% 5 tagliaferro et al. considered an adjunctive rather than a primary prevention agent. ward’s clustering method with squared euclidean distances as the similarity measure was chosen in order to be sensitive to differences in elevation as well as profile shape16. dentist and practice characteristics were tested for differences across the preventive clusters using anova or chi-square as appropriate. pair-wise comparisons were performed using a bonferroni correction. results table 2 shows the practitioner and practice characteristics for eligible dentists. most of them were females (59%), working in a private hybrid (private + public) model (78%), graduated from a public institution (77%), and had received specialty training (63%). table 3 summarizes the frequency of use of each caries prevention agent for adult patients. in-office fluoride application was the preventive method most reported by dentists for caries prevention in adults. table 4 shows results of the generalized linear regression modeling, specifically the statistical significance at each step in the analysis and the parameter estimates for the predictors of the frequency of use of each caries prevention agent. dental sealants. female dentists and those in private practice apply dental sealants to a higher percentage of adult patients compared to dentists in other practice models (p=0.001) and male dentists (p=0.044). in step 2, dentists who have a greater percentage of patients interested in a caries prevention regimen apply dental sealants to a significantly higher percentage of adult patients (p=0.048). in step 3, dentists who apply dental sealants to a higher percentage of adult patients are also more likely to administer an in-office fluoride (p=0.042) and recommend sugarless/xylitol gum (p=0.012) more often to their adult patients. the overall model was a good fit for the data [x2 (5) = 21.645, p=0.001]. in-office fluoride. female dentists administer an in-office fluoride application to a higher percentage of adult patients compared to male dentists (p=0.014). in addition, dentists with a higher percentage of patients who are 65 years of age or older were more likely to use in-office fluoride (p=0.042). in step 2, dentists who have a greater percentage of patients interested in a caries prevention regimen are more likely to administer an in-office fluoride to their adult patients (p=0.009). in step 3, dentists who more frequently administer an in-office fluoride to their adult patients are also more likely to apply dental sealants (p=0.006) and recommend a non-prescription fluoride (p=0.001). the overall model was a good fit for the data [x2 (6) = 26.972, p<0.001]. non-prescription fluoride. female dentists recommend an over-the-counter (otc) fluoride rinse to a higher percentage of adult patients compared to male dentists (p=0.009). dentists who graduated from a private dental school recommend an otc fluoride rinse to a larger percentage of their adult patients than dentists who graduated from a public dental school (p=0.017). in step 2, dentists who have a greater percentage of patients interested in a caries prevention regimen (p=0.012) are more likely to administer an otc fluoride to their adult patients compared to dentists who have a smaller percentage of patients interested in a caries prevention regimen. in step 3, dentists who are more likely to recommend an otc fluoride rinse are significantly more likely to apply in in-office fluoride (p<0.001) and recommend an at-home regimen of chlorhexidine rinse (p<0.001). the overall model was a good fit for the data [x2 (6) = 35.518, p<0.001]. 6 tagliaferro et al. prescription fluoride. in step 3, dentists who more frequently provide a prescription for some form of fluoride are significantly more likely to apply in in-office fluoride (p=0.042) and recommend an at-home regimen of chlorhexidine rinse (p=0.012). the overall model was a good fit for the data [x2 (2) = 9.484, p=0.009]. table 2. dentist’s and practice’s characteristics characteristic percentage (n) mean (sd) mean % ∆, percentiles (25th, 50th, 75th) ф age of dentist 42.2 (sd=11.4) gender of dentist (female) 59% (n=116) type of practice private practice 50% (n=98) private/public hybrid 28% (n=55) public health 17% (n=34) other 5% (n=10) years since dental school graduation 19.7 (sd=11.1) type of dental school from which the dentist graduated public institution 77% (n=151) private institution 23% (n=46) specialization not completed specialization training 37% (n=72) specialization training 63% (n=125) advanced degree no advanced degree 70% (n=138) master’s degree 6% (n=11) phd degree 25% (n=48) percent of patients by age cohort pediatric patients (1-18 years) 19% (sd=19) adults (19-44 years) 36% (sd=17) adults (45-64 years) 32% (sd=16) adults (65 years or older) 13% (sd=10) percent of patients a dental explorer is used to diagnose an occlusal caries lesion? 65% 2, 5, 6 assess caries risk for individual patients 34% (n=63) ¥ use a special form for caries risk assessment (of the 63 who perform caries risk assessment) 38% (n=24) percent of patients who are interested in a caries prevention regimen 44% 2, 4, 4 percent of patients who receive a caries risk prevention regimen 54% 2, 4, 5 ∆ percentage when the ordinal values were transformed as follows to category median: 0%=0%, 1-24%=12.5%, 25-49%=37%, 50-74%=62%, 75-99%=87%, 100%=100% ф 25th, 50th (median) and 75th percentile for ordinal categories scaled as 1 – never or 0%, 2 – 1 to 24%, 3 – 25 to 49%, 4 – 50 to 74%, 5 – 75 to 99%, 6 – every time or 100% ¥ nine practitioners did not indicate whether they assess for caries risk 7 tagliaferro et al. chlorhexidine rinse. dentists who had more years since graduation from dental school were less likely to recommend an at-home regimen of chlorhexidine rinse (p=0.016). in step 3, dentists who are more likely to recommend an at-home regimen of chlorhexidine rinse are also more likely to also recommend an otc fluoride (p<0.001), provide a prescription for some form of fluoride (p=0.015), and recommend sugarless/xylitol gum (p<0.001) to their adult patients. the overall model was a good fit for the data [x2 (5) = 46.467, p<0.001]. sugarless or xylitol gum. in step 3, dentists who more frequently recommend sugarless/xylitol gum are significantly more likely to apply dental sealants (p=0.001) and recommend an at-home regimen of chlorhexidine rinse (p=0.001) to their adult patients. the overall model was a good fit for the data [x2 (2) = 31.862, p<0.001]. dentists grouped by preventive profile inspection of the agglomeration coefficients from the cluster analysis showed that the percentage increase between the four-cluster and the three-cluster solutions was nearly twice the increase for the preceding steps. this suggests that the final four clusters are sufficiently dissimilar and that the four-cluster solution is the most appropriate16. means and sd for the six caries prevention agents for each of the three-cluster subgroups are presented in table 5. dentists in the largest subgroup (n=99) seldom used any of the preventive agents and we labeled this group as “infrequent users of prevention”. consistent with this, they also had the lowest percentage of patients who receive individual caries prevention (46% of patients) and lowest percentage of patients who desire individual caries prevention (36%). they were also among the subgroups least likely to assess caries risk. this subgroup also contained the lowest percentage of female dentists (51%). these dentists had the lowest percentage of patients 18-44 years of age (34%) and the highest percentage of patients in the 45-64 age group (35%). table 3. mean percent of adult patients within a practice who receive each caries preventive agent. preventive agent mean % ∆ (95% ci) € percentiles (25, 50, 75) ф in-office fluoride 51% (46,56) 2, 3, 6 chlorhexidine rinse 27% (23,30) 2, 2, 3 non-prescription fluoride 22% (20,24) 1, 1, 3 xylitol gum 18% (16,21) 1, 1, 3 prescription fluoride 15% (14,17) 1, 2, 2 dental sealant 14% (12,16) 1, 2, 2 ∆ percentage when the ordinal values were transformed as follows to category median: 0%=0%, 1-24%=12.5%, 25-49%=37%, 50-74%=62%, 75-99%=87%, 100%=100%. € 95% confidence interval for mean %. ф 25th, 50th (median) and 75th percentile for ordinal categories scaled as 1 – never or 0%, 2 – 1 to 24%, 3 – 25 to 49%, 4 – 50 to 74%, 5 – 75 to 99%, 6 – every time or 100% 8 tagliaferro et al. the second largest group (n=38 dentists) consistently applied in-office fluoride (91%) and made frequent recommendations for an otc fluoride rinse (76%). they were also the group most likely to provide a prescription for chlorhexidine rinse (42%). overall, they consistently used the full range of preventive agents and we have labeled this subgroup “comprehensive use of prevention”. however, they were among the subgroups least likely to assess caries risk (27%). this subgroup contained the highest percentage of female dentists (74%). table 4. modeling to explain the use of preventive agents among adult patients preventive agent final model fit b (se) p. value dental sealant private practice 0.965 (0.292) 0.001 dentist gender (female) x2 (2) = 9.354, p=0.008* 0.522 (0.250) 0.044 % patients interested in caries prevention ∆x2 (1) = 3.865, p=0.049** 0.143 (0.706) 0.048 in-office fluoride 0.150 (0.066) 0.042 sugarless/xylitol gum ∆x2 (2) = 8.426, p=0.013*** 0.219 (0.084) 0.012 in-office fluoride private practice -0.251 (0.134) 0.056 dentist gender (female) 0.606 (0.246) 0.014 patients 65 years of age or older x2 (3) = 8.187, p=0.039* 0.027 (0.012) 0.042 % patients interested in caries prevention ∆x2 (1) = 6.578, p=0.011** 0.255 (0.116) 0.009 dental sealants 0.347 (0.127) 0.006 non-prescription fluoride ∆x2 (2) = 12.207, p=0.003*** 0.268 (0.103) 0.001 non-prescription fluoride dentist gender (female) 0.571 (0.207) 0.009 patients 65 years of age or older 0.019 (0.011) 0.070 type of dental school (private) x2 (3) = 13.354, p=0.004* 0.631 (0.264) 0.017 % patients interested in caries prevention ∆x2 (1) = 5.354, p=0.014** 0.212 (0.101) 0.012 in-office fluoride 4.592 (1.179) < 0.001 chlorhexidine rinse ∆x2 (2) = 18.810, p<0.011*** 6.572 (1.964) < 0.001 prescription fluoride in-office fluoride 0.99 (0.043) 0.042 chlorhexidine rinse x2 (2) = 9.484, p=0.009** 0.144 (0.068) 0.012 chlorhexidine rinse years since dental school graduation -0.016 (0.007) 0.016 type of dental school (private) ∆x2 (2) = 10.431, p=0.005* -0.338 (0.197) 0.066 non-prescription fluoride 0.187 (0.046) <0.001 prescription fluoride 0.158 (0.065) 0.015 sugarless/xylitol gum ∆x2 (3) = 36.036, p<0.001*** 0.157 (0.044) <0.001 sugarless/xylitol gum dental sealants 0.262 (0.089) 0.001 chlorhexidine rinse x2 (2) = 31.862, p<0.001**** 0.291 (0.097) 0.001 * chi-square (x2) for model with practice/practitioner variables ** ∆x2 for model with step 2 variables (caries-related practice patterns) added *** ∆x2 for model with step 3 variables (other preventive agents) added **** ∆x2 for model with only step 3 variables (other preventive agents) as no variables were significant in the baseline model or following step 2. 9 tagliaferro et al. the next group also consisted of 38 dentists; these dentists tend to focus on the use of in-office fluoride (97%) and seldom recommend at-home use of prescription or otc fluoride. this group was labeled “in-office fluoride preference”. along with the “in-office sealant and fluoride preference” subgroup discussed below, they were the most likely to assess caries (47%). in addition, they had the highest percentage of patients who desired (53%) and received (68%) individual caries prevention. table 5. use of preventive agents for adult patients by preventive subgroups and dentist’s, patient’s, and practice’s characteristics use of preventive agent infrequent users of prevention comprehensive use of prevention in-office fluoride preference in-office sealant and fluoride caries prevention (n=197) n=99 n=38 n=38 n=22 dental sealant 4% ∆ (4, 6) € a 1, 1, 2 ф 24% (19,30) b 1, 1, 4 7% (5,9) a 1, 1, 2 51% (44,59) c 2, 4, 5 in-office fluoride 20% (18,22) a 2, 2, 3 91% (88,94) b 5, 5, 6 97% (96,98) b 6, 6, 6; 48% (41,56) c 2, 3, 5 non-prescription fluoride 12% (10,14) a 1, 1, 2 76% (72, 81) b 4, 5, 6 3% (0,6) a 1, 1, 1 7% (4,11) a 1, 1, 2 prescription fluoride 9% (8,11) a 1, 1, 2 24% (19,29) b 1, 2, 3 10% (7,12) a 1, 1, 2 33% (26,41) c 1, 2, 4 chlorhexidine rinse 23% (21,25) a 2, 2, 3 42% (37,48) b 2, 3, 4 27% (23,31) a 2, 2, 3 22% (18,26) a 1, 2, 3 sugarless/xylitol gum 14% (11,18) 1, 1, 2 21% (14,27) 1, 1, 3 21% (14,27) 1, 1, 2 17% (9,25) 1, 1, 2 practice characteristics dentist gender (females) 51% a 74% b 60% 59% type of practice (private) 52% 47% 38% a 75% b years since dental school graduation 19.9 (sd=12) 18.5 (sd=9) 19.7 (sd=11) 20.3 (sd=11) type of dental school from which the dentist graduated (public institution) 79% 68% 87% 76% specialization (specialization training) 60% 68% 68% 60% advanced degree (master’s or doctorate) 31% 24% 30% 40% percent of patients by age cohort pediatric patients 18% 16% 24% 20% adults (19-44 years) 34% a 35% 38% 45% b adults (45-64 years) 35% a 34% 25% b 25% b adults (65 years and older) 13% 15% 13% 9% assess caries risk for individual patients 29% a 27% a 47% b 42% b patients who desire individual caries prevention (%) 36% b 2, 3, 4 51% a 3, 4, 4 53% a 3, 3, 4 49% a 3, 4, 4 patients who receive individual caries prevention (%) 46% a 2, 3, 5 55% 2, 4, 5; 68% b 3, 5, 6 60% 3, 4, 5 groups with different superscripts are different using a bonferroni correction (p=0.01) for that variable and groups without superscripts or that share superscripts are not significantly different. subgroups did not differ on other practice characteristics. ∆ percentage when the ordinal values were transformed as follows to category median: 0%=0%, 1-24%=12.5%, 25-49%=37%, 50-74%=62%, 75-99%=87%, 100%=100%. € 95% confidence interval for mean %. ф 25th, 50th (median) and 75th percentile for ordinal categories scaled as 1 – never or 0%, 2 – 1 to 24%, 3 – 25 to 49%, 4 – 50 to 74%, 5 – 75 to 99%, 6 – every time or 100% 10 tagliaferro et al. the final group was the smallest (n=22) and had the most frequent use of dental sealants (51%) and in-office fluoride (47%). they were also the ones most likely to recommend fluoride prescription. this group was labeled “in-office sealant and fluoride preference” and had the higher percentage of practitioners using a private practice model. along with the “in-office fluoride preference” subgroup discussed above, they were the most likely to assess caries risk in their patients (42%). discussion to our knowledge this current work is the first report in the literature about caries prevention in adults by brazilian dentists as assessed using the translated version of the “assessment of caries diagnosis and caries treatment” questionnaire from the practice-based research network in the united states of america. dentists participating in this study were primarily middle-aged (42.2 years) females (59%), working in a private hybrid (private + public) model (78%), graduated from a public institution (77%), and had received specialty training (63%). demographic data from the regional council of dentistry of são paulo state showed that 57% of dentists from araraquara were female and 66% were younger than 50 years of age17. national data show that most brazilian dentists are female (51.2%), are up to 40 years-old (57.4%), graduated from private schools (65.0%) and had no specialty training (75%)18. therefore, in spite of using a convenience sample, demographic data from participant dentists were quite similar to dentists from araraquara and a lesser extent to brazilian dentists. dentists in the current study reported that in-office fluoride application was the preventive method most commonly used (51% of patients) for caries prevention in adults. participating dentists also reported recommending non-prescription fluoride to 22% of their adult patients (table 3). dentists from japanese9 and us8 dental pbrns recommended in-office fluoride application to 21% and 37% of their adult patients, respectively. brazilian dentists reported recommending in-office fluoride at more than twice the rate of japanese dentists. yokoyama et al.9 (2016) mentioned that the focus of the current japanese health insurance system is disease treatment and that it does not cover most preventive dental care services. as a result, the percentage of japanese dentists providing preventive treatment may be reduced9. brazilian oral health insurance system and public service offer several preventive measures, including in-office fluoride application. taking into account that 98% of dentists in the current study reported working in a private and/or public health service, and that participant dentists believe that 44% of their adult patients are interested in a caries prevention regimen, their first choice for caries prevention dentists was in-office fluoride application, which was significantly higher than those reported by dentists from the japanese and us dental pbrns. chlorhexidine rinse was also reported frequently by brazilian dentists as a caries preventive agent for their adult patients (table 3). scientific evidence for chlorhexidine as a caries preventive agent is not consensual. some authors found statistically significant differences in streptococcus mutans levels during and after the use of a chlorhexidine mouthwash on patients with moderate to high caries risk. however, they suggested the need for additional studies in order to assess whether the results con11 tagliaferro et al. firm the reduction in dental caries and, consequently, whether or not these products should be incorporated into existing prevention protocols19. others studies have not found good evidence of the effectiveness of chlorhexidine for caries prevention20,21. results from our regression analyses suggest that the main predictor for recommending in-office fluoride application or other preventive methods was dentist gender, with females recommending more often than male dentists (table 4). these findings are in agreement with those of riley et al.22 (2011), who found that female dentists had a greater overall preventive orientation than male dentists for both adult and pediatric patients. however, the scientific literature is not consensual about gender differences on attitudes on prevention and treatment of dental caries. some researchers have found a more-conservative approach towards prevention and treatment of dental caries among female dentists23-26. in contrast, other studies have found no statistically significant relationship between dentist gender and choices for caries prevention or treatment27-29. a previous brazilian study has also found no association between dentists’ gender and decision making for restoring dental caries as seen in radiographs30. further studies are needed to clarify this issue. other predictors for recommending some type of preventive method showed that patients 65 years of age or older are more likely to receive in-office fluoride (table 4). this finding could be related to dentists being concerned with the prevention of root caries. root caries is most commonly found in the elderly population, with four out of ten adults being affected31. brazilian elderly presented a mean dmft of 27.5, with a mean of 0.2 decayed roots and 0.1 filled roots1. the overall prevalence of root caries was estimated at 41.5% in a systematic review and meta-analysis published by pentapati et al.31 (2019). the authors related that the number of adults with root caries might expand in the future because of the increase in aging population and dentition longevity, and suggested that preventive measures should be the focus of policy-makers and health care professionals to reduce the burden of disease among the elderly. it is relevant to emphasize that the percentage of patients interested in caries prevention may be predictive of dentists being more likely to provide dental sealants, in-office fluoride application or non-prescription fluoride to their adult patients. although there is lack of literature supporting the cost-effectiveness of use of fluorides and sealants for caries prevention in adult patients32, one can speculate that patient interest in caries prevention may stimulate dentists to adopt a more-preventive approach, perhaps influenced more strongly by the patients’ interest than by the patients´ caries risk. in the current study only 34% of dentists assessed caries risk for individual patients (table 1). another possible explanation is that dentists are working in a person-centered care environment, employing the principles of shared decision-making33 in which the patient can act as a partner who co-designs his/her care delivery34. further studies are needed to assess these assumptions. modern caries management emphasizes a conservative and preventive evidence-based philosophy, with personalized disease management, monitoring of caries lesions, and efforts to remineralize and/or arrest lesions32. as we consider the above-mentioned evidence in caries prevention, it is worth discussing the results of cluster analysis that showed a clear agglomeration of dentists in the largest subgroup (n=99) characterized by infrequent use of prevention and associated with the 12 tagliaferro et al. following profile: 1) lowest percentage of patients who receive or desire individual caries prevention, 2) least likely to assess caries risk, 3) lowest percentage of female dentists, and 4) the highest percentage of patients in the 45-64 age group (table 5). the results of the current study showed a gap between evidence-based dentistry and dental practice for half of participating dentists. although there are limitations associated with the study, it clearly indicates that additional means to translate current evidence-based findings for caries prevention into clinical practice is needed and it may be targeted to the above-mentioned practice characteristics. this study did have these limitations: a) we cannot infer causality from a cross-sectional design; b) it used a convenience sample and singular characteristics of the region (access to a dental school, preventive practices taught in the region, etc.) may have strongly influenced the results; c) the assumption that the reported overall preventive measures are actually what the responding dentists perform routinely and not related to individual patient recommendations for single or multiple treatments22; and that they may be influenced by social desirability and recall bias8. the study strengths include the similarity of the demographic characteristics between the participating dentists and those from araraquara, and the feasibility of the questionnaire to assess and to compare dental practice patterns among dentist populations35. in conclusion, in-office fluoride application was the most commonly reported preventive method for caries prevention in adults. some practitioner, practice and patients’ characteristics were positively associated with more-frequent use of a preventive agent. acknowledgements financial support was provided by the foundation for the development of the são paulo state university (fundunesp; grant 0170/004/13-prope/cdc) and by the são paulo research foundation (fapesp; grant 2012/10397-2). certain components of this work were supported by national institutes of health grants u01de-16746, u01-de-16747, u19-de-22516, and u19-de-28717. opinions and assertions contained herein are those of the authors and are not to be construed as necessarily representing the views of the respective organizations or of the national institutes of health. nidcr had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. the authors thank claudia huck, fabiano jeremias, juliana alvares duarte bonini campos, mariana de matos, luana moreira loures ridolfi, wilson chediek, elina mara da silva marcomini, rita de cassia prando, márcia santana, luis alberto da silva, ivanete correa macieira, célia regina de freitas rocha, olavo bergamaschi barros and karina antunes for their assistance and dentists who participated in this study. references 1. sb brazil 2010: [national research 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and meta-analysis. saudi dent j. 2019 jan;31(1):3-15. doi: 10.1016/j.sdentj.2018.11.008. 32. fontana m, gonzalez-cabezas c. evidence-based dentistry caries risk assessment and disease management. dent clin north am. 2019 jan;63(1):119-128. doi: 10.1016/j.cden.2018.08.007. 33. slayton rl, fontana m, young d, tinanoff n, nový b, lipman rd, et al. dental caries management in children and adults. discussion paper. washington: national academy of medicine; 2016 sep 14. doi: 10.31478/201609d. 34. lee h, chalmers ni, brow a, boynes s, monopoli m, doherty m, et al. person-centered care model in dentistry. bmc oral health. 2018 nov 29;18(1):198. doi: 10.1186/s12903-018-0661-9. 35. tagliaferro eps, valsecki junior a, rosell fl, silva src, riley jl, gilbert gh, et al. caries diagnosis in dental practices: results from dentists in a brazilian community. oper dent. 2019 jan/feb;44(1):e23-e31. doi: 10.2341/18-034-c. 1http://dx.doi.org/10.20396/bjos.v18i0.8657253 volume 18 2019 e191561 original article 1 school of dentistry, franciscan university, santa maria, rio grande do sul, brazil. 2 department of biomedical and neuromotor sciences, unit of odontostomatological sciences, division of prosthodontics, alma mater studiorum, university of bologna, bologna, italy. 3 division of conservative dentistry, school of dentistry, franciscan university, santa maria, rio grande do sul, brazil. 4 division of prosthodontics, school of dentistry, franciscan unviersity, santa maria, rio grande do sul, brazil. corresponding author: vinícius felipe wandscher, d.d.s., m.sci.d., ph.d., adjunct professor, franciscan university faculty of odontology prosthodontics-biomaterials unit r. silva jardim, 1175, 97010-491, rio grande do sul state, santa maria, brazil. phone/fax: +55 55 30251202/ 30259002 e-mail: viniwan@hotmail.com (dr vinicius) received: january 28, 2019 accepted: august 20, 2019 fracture load and shear stress of prefabricated glass fiber posts helder callegaro velho1, paolo baldissara2, jovito adiel skupien3, vinícius felipe wandscher4,* aim: this study evaluated the fracture load and pattern failure of different prefabricated glass fiber posts (gfps) of the same diameter. methods: seventy-eight (n=13 for six groups) gfps of 1.6 mm coronal diameter of different brands were evaluated— exacto (angelus), power post (bm4), white post dc (fgm), hirem (overfibers), maquira fiber post (maquira), and superpost (supordont). the posts were subjected to fracture load testing (45° of inclination and 1 mm/min until fracture). each factor (load (n) and shear stress (mpa)) was analyzed separately using one-way anova followed by the tukey test (α=0.05). results: the type of failure was evaluated on a stereomicroscope (×10). the power post samples presented higher values of fracture load (p<0.001) followed by maquira fiber post, white post, hirem, superpost, and the exacto posts. the failure pattern observed was intralaminar mode ii in-plane shear, such as a failure occur parallel to fibers. conclusion: despite the same diameter of gfps, the fracture load and shear resistance were brand-dependent. keywords: glass. materials testing. stress, mechanical. flexural strength. 2 velho et al. introduction after endodontic treatment on teeth (ett) with extensive mineral tissue loss, an intraradicular post can improve the retention of the coronal restoration1, considering the quantity and quality of the dental remaining2. when the remaining is higher than 2 mm, prefabricated glass fiber posts (gfps) can be used; otherwise, a cast post and core are preferred3. glass fiber posts are usually used in dentistry because they present adequate adhesion to monoand dimethacrylates4, good aesthetic properties, and clinically higher survival rates5-7. the gfps are composed of unidirectional fibers, lying parallel to each other (carbon, quartz, or glass), and a polymer matrix. the polymer matrix is a highly cross-linked structure composed of epoxy polymers of a high conversion degree8. the performance of fibers is influenced by the type, orientation, adhesion to the polymer matrix, and impregnation of the polymer matrix on the fibers9. the fibers are responsible for tensile resistance, whereas the matrix supports the compression stresses10. the union between the fibers and the polymer matrix is an important factor that can influence gfp resistance8. during the manufacturing process, a silane layer is applied on the fibers (inorganic portion) to improve the adhesion with the resin matrix (organic portion)11. another step in the manufacture of gfps that improves the mechanical properties is the pretension of fibers. this process occurs before matrix incorporation, enabling fiber resistance at tensile stresses when the post is exposed to bending forces10. wandscher et al.12 (2015) showed that the shear stress was similar among fiber posts of different diameters of the same brand when tested with a 45° loading (except for 1.4 mm). besides, wandscher et al.13 (2016) showed that an endodontically treated tooth restored with fiber post and matalloceramic crown failure with similar characteristics (scallops, consequences by shear stress) at in vitro studies12,14,15. so, a sequence of events can lead to the ultimate failure in anterior ett restored with posts due to shear stresses that is present in the central region of the restorer assembly during the load application, inducing adhesive failures in the post/cement/dentine interfaces16. thus, the question is, which fiber post is less flexible to minimize the shear stresses? gfps from different manufacturers exhibit a wide variation because of different fabrication processes. thus, the aim of the present study was to evaluate the fracture load and the pattern failure of different prefabricated gfps of the same diameter commercially available in the brazil. the null hypothesis tested was that there is no difference between brands. materials and methods seventy-eight gfps of 1.6 mm coronal diameter were allocated to six groups (n=13) based on their manufacturer (table 1). the coronal distance was set to 6 mm with a digital caliper. the posts were fixed in a parallelometer and embedded in pvc cylinders with a self-cure acrylic resin (vipi flash, vipi, pirassununga, sp, brazil) to the reference of 6 mm. 3 velho et al. fracture loading test the specimens were positioned at 45° (in relation to the horizontal plane) in a universal testing machine (dl-1000, emic, são josé dos pinhais, brazil), and a cylindrical piston of planar tip (ø: 6 mm) applied a load at the rate of 1 mm/min on the more coronal portion of the post until fracture. the force (n) on the fracture moment was recorded, and the shear stress (mpa) was calculated according to wandscher et al.12 (2015): t = 16fmax cos 45 / 3πd 2 (1) where t = shear stress, fmax = maximum force to fracture (n), p = 3.14, d p= diameter of the specimen. failure analysis after the fracture loading tests, the samples were submitted to failure analysis on a stereomicroscope (discovery v20; carl zeiss, germany) on x10. representative images were observed in scanning electron microscope (sem) (vega3, tescan). statistical analysis the mean values of the force/load (n) and the shear stresses (mpa) were analyzed using one-way anova and the tukey test (α=0.05), after to prove the normal distribution and equality of the data with shapiro-wilk and levene tests, respectively. results the statistical analysis showed differences between groups, in terms of force values and shear stresses, wherein the pw group exhibited the highest values followed by fp, wp, hr, sp and ex groups (p<0.0001). the fracture load and shear stress values are listed in table 2. the pattern of failure is presented in figure 1 and 2 (stereomicroscope and sem respectively). it was possible to observe that all specimens fractured with a central longitudinal crack perpendicular to the direction of the applied force. table 1. experimental groups groups/posts brands composition* ex angelus, londrina, pr, brazil glass fiber (80%) epoxy resin (20%) pw bm4, maringá, pr, brazil n/a wp fgm, joinville, sc, brazil glass fiber (80%) epoxy resin (20%) hr overfibers, mordano, bo, italy fiber glass (65–72%) epoxy resin (35–28%) fp maquira, maringá, pr, brazil n/a sp superdont, rio de janeiro, rj, brazil n/a * according to the manufacturers’ brochures. n/a—not available. 4 velho et al. table 2. fracture load (n) and shear stress (mpa) of the glass fiber posts. groups load/resistance ± standard deviation* fracture (n) shear (mpa) pw 137.1 ± 9.5a 63.7 ± 4.4a fp 119.5 ± 19.6b 55.5 ± 9.1b wp 112.2 ± 15.7b,c 52.1 ± 7.3b,c hr 110.15 ± 20.4b,c 51.2 ± 9.5b,c spt 103.5 ± 11.6b,c 48.1 ± 5.4b,c ex 100.1 ± 6.5c,d 46.5 ± 3c,d p-value <0.0001 <0.0001 *different letters indicate statistically significant differences. one-way anova was performed separately for fracture and shear stress. figure 1. representative images of the failure pattern. we can observe that the cracks occurs in the center of the specimen (red arrows), which is a characteristic of failure by shear stresses. the white arrow indicates the load application sense. exacto fiber post hi rem power post super post white post 5 velho et al. discussion the results of the current study showed statistical difference between the tested groups; thus, the hypothesis that there is no difference between brands was rejected. the pw group exhibited higher values, and the ex group exhibited smaller values. as the samples were of same diameter and, basically, of the same composition (of glass fiber and epoxy matrix), the difference between groups should be explained by factors such as fiber/matrix proportion, the diameter and pretensioning of the fibers, and fiber/matrix union. in addition, possible errors in the manufacturing process such as bubble incorporation and empty spaces can jeopardize the mechanical properties of gfps. it may be concluded that the shear stress and diameter are not correlated because the shear values did not change for the different diameters12. however, one factor makes the discussion of the results difficult: the exact composition of the gfp. the difficulty arises especially because some manufacturers do not present this information explicitly. the brochures present only the basic composition (glass fiber and epoxy resin). data such as fiber diameters, type of glass fibers, pretension of the fibers, and application of a coupling agent (silane) on the fiber are not disclosed. a higher fiber/matrix ratio is reported to generate a higher loading fracture8,17. however, if the link between fibers and the matrix is not adequate, no improvement occurs8. this fact can explain the small value of the loading found for the ex group, although posts from this group contain 80% of fibers. bubbles and empty spaces left during the fabrication process may cause inadequate fiber/matrix union, consequently leading to the formation of structural defects propagating failure18. on the other hand, during the fabrication process, fiber treatment can improve the mechanical properties of gfp. pretension of fibers is one way to achieve it. in this process, the fibers are pretensioned, incorporated into the matrix, and liberated only figure 2. sem images of the shear failures in the gfps. a. representative sem of the wp group x500. b. sem (x1000) of the wp group presenting the f: glass fiber, m: epoxy matrix and s: scallops (undulations on the epoxy matrix consequences of the shear stresses). m m f f m f s s s a b 6 velho et al. after the complete cure of the epoxy resin. thus, the fibers are compressed and are able to absorb tensile stresses, when an oblique force is applied10. another possibility is the application of a couple agent on fibers before the matrix incorporation, promoting a better chemical union between the fibers (inorganic portion) and the matrix (organic portion)19. despite the diameter of the posts being the same, the diameter of the fiber might have influenced our results. larger diameters may improve the mechanical properties of gfps20, since the distribution of these fibers in the matrix are homogeneous21. with respect to failure pattern, most of the specimens presented a longitudinal crack in the center of the coronal portion (figure 1) because of shear stresses. these failures, classified as intralaminar mode ii in-plane shear, are consequences of two important factors: fiber arrangement (parallel, 0°) and shear stress. an important feature are the scallops in the epoxy matrix (figure 2) consequences of the shear stresses in the gfps. these scallops were founded too in other studies12,13,15. the fracture loading test at 45° evaluated the resistance of fiber-reinforced polymers by simulating the load application in an anterior tooth17. an oblique force (45°) applied on the post generated tensile stresses on the outer surface and compression stresses on the interior surface causing a dislodgement of the fiber/matrix interface in the center of the specimen and consequently failure by shear stresses12,15. besides, using finite element analysis, wandscher et al.12 (2015) showed that shear stress on fiber-reinforced polymers is the highest in the center of the specimens and null on the extremities, justifying the failure pattern obtained in the current study. furthermore, marchionatti et al.14 (2014) evaluated the fracture resistance of teeth restored with fiber posts and composite cores and showed that this type of failure also occurs when teeth are restored with fiber posts. another factor that could influence this type of failure (fiber/matrix interface) is the different elastic moduli between glass fibers and the matrix, because when a load is applied, the stresses converge to this interface8. an important limitation of the current study is that the test evaluated only the mechanical properties of the posts, thus, the clinical significance is to better know the mechanical properties of gfps. in addition, the posts were submitted only to static loads and this does not occur clinically. fatigue tests simulate the clinical situation better, because cyclic loads are applied simulating the chewing22,23. until now, studies are not available that show how much load support an endodontically treated tooth restored clinically with fiber post. in vitro studies as well as clinical studies evaluating the fracture and fatigue of endodontically treated teeth restored with posts are recommended. in conclusion, despite the diameter of the glass fiber posts being the same, fracture resistance is brand-dependent. acknowledgements the authors state that there is no conflict of interests. we thank to dental store dental cremer for materials donation. 7 velho et al. references 1. assif d, gorfil c. biomechanical considerations in restoring endodontically treated teeth. j prosthet dent. 1994;71:565-7. doi:10.1016/0022-3913(94)90438-3. 2. baldissara p. mechanical properties and in vitro studies. in: ferrari m, scotti r, editors. fiber posts: characteristic and clinical aplication. milan: masson; 2003. p. 39-51. 3. newman mp, yaman p, dennison j, rafter m, billy e. fracture resistance of endodontically treated teeth restored with composite posts. j prosthet dent. 2003 apr;89(4):360-7. doi:10.1067/mpr.2003.75. 4. vallittu pk. curing of a silane coupling agent and its effect on the transverse strength of autopolymerizing polymethylmethacrylate-glass fibre composite. j oral rehabil. 1997 feb;24(2):124-30. 5. bergoli cd, brondani lp, wandscher vf, pereira g, cenci ms, pereira-cenci t, et al. a multicenter randomized double-blind controlled clinical trial of fiber post cementation strategies. oper dent. 2018 mar/apr;43(2):128-35. doi:10.2341/16-278-c. 6. gómez-polo m, llidó b, rivero a, del río j, celemín a. a 10-year retrospective study of the survival rate of teeth restored with metal prefabricated posts versus cast metal posts and cores. j dent. 2010;38:916-20. doi:10.1016/j.jdent.2010.08.006. 7. sarkis-onofre r, fergusson d, cenci ms, moher d, pereira-cenci t. performance of post-retained single crowns: a systematic review of related risk factors. j endod. 2017;43:175-83. doi:10.1016/j.joen.2016.10.025. 8. lassila lvj, tanner j, le bell a-m, narva k, vallittu pk. flexural properties of fiber reinforced root canal posts. dent mater. 2004 jan;20(1):29-36. 9. vallittu pk. the effect of glass fiber reinforcement on the fracture resistance of a provisional fixed partial denture. j prosthet dent. 1998 feb;79(2):125-30. 10. seefeld f, wenz h-j, ludwig k, kern m. resistance to fracture and structural characteristics of different fiber reinforced post systems. dent mater. 2007 mar;23(3):265-71. doi:10.1016/j.dental.2006.01.018. 11. söderholm kj, shang sw. molecular orientation of silane at the surface of colloidal silica. j dent res. 1993;72(6):1050–4. doi:10.1177/00220345930720061001. 12. wandscher vf, bergoli cd, de oliveira af, kaizer ob, souto borges al, limberguer if, et al. fatigue surviving, fracture resistance, shear stress and finite element analysis of glass fiber posts with different diameters. j mech behav biomed mater. 2015 mar;43:69-77. doi:10.1016/j.jmbbm.2014.11.016. 13. wandscher v, bergoli c, limberger i, cenci t, baldissara p, valandro l. fractographical analysis and biomechanical considerations of a tooth restored with intracanal fiber post: report of the fracture and importance of the fiber arrangements. oper dent. 2016;41:e149-58. doi:10.2341/15-262-s. 14. marchionatti ame, wandscher vf, broch j, bergoli cd, maier j, valandro lf, et al. influence of periodontal ligament simulation on bond strength and fracture resistance of roots restored with fiber posts. j appl oral sci. 2014 sep-oct;22(5):450-8. 15. pereira gkr, lançanova m, wandscher vf, kaizer ob, limberger i, özcan m, et al. fiber-matrix integrity, micromorphology and flexural strength of glass fiber posts: evaluation of the impact of rotary instruments. j mech behav biomed mater. 2015;48:192-9. doi:10.1016/j.jmbbm.2015.04.008. 8 velho et al. 16. wandscher v, bergoli c, limberger i, ardenghi t, valandro l. preliminary results of the survival and fracture load of roots restored with intracanal posts: weakened vs nonweakened roots. oper dent. 2014;39:541-55. doi:10.2341/12-465. 17. asmussen e, peutzfeldt a, heitmann t. stiffness, elastic limit, and strength of newer types of endodontic posts. j dent. 1999;27(4):275-8. 18. grandini s, goracci c, monticelli f, tay fr, ferrari m. fatigue resistance and structural characteristics of fiber posts: three-point bending test and sem evaluation. dent mater. 2005;21(2):75-82. doi:10.1016/j.dental.2004.02.012. 19. monticelli f, toledano m, osorio r, ferrari m. effect of temperature on the silane coupling agents when bonding core resin to quartz fiber posts. dent mater. 2006;22:1024-8. doi:10.1016/j.dental.2005.11.024. 20. obukuro m, takahashi y, shimizu h. effect of diameter of glass fibers on flexural properties of fiber-reinforced composites. dent mater j. 2008;27(4):541-8. 21. cheleux n, sharrock pj. mechanical properties of glass fiber-reinforced endodontic posts. acta biomater. 2009;5(8):3224-30. doi:10.1016/j.actbio.2009.04.008. 22. cohen bi, deutsch as, musikant bl. cyclic fatigue testing of six endodontic post systems. j prosthodont. 1993;2(1):28-32. 23. wiskott hw, nicholls ji, belser uc. stress fatigue: basic principles and prosthodontic implications. int j prosthodont. 1995;8(2):105-16. bjos-v017-0018-p2.indd 1http://dx.doi.org/10.20396/bjos.v17i0.8651898 volume 17 2018 e18018 original article 1 dds, ms, phd student in restorative dentistry, department of restorative dentistry, piracicaba dental school, state university of campinas, piracicaba, sp, brazil. 2 dds, ms, phd in restorative dentistry, department of restorative dentistry, piracicaba dental school, state university of campinas, piracicaba, sp, brazil. 3 dds, ms, phd student in restorative dentistry, department of restorative dentistry, piracicaba dental school, state university of campinas, piracicaba, sp, brazil. 4 dds, ms, phd student in restorative dentistry, department of restorative dentistry, piracicaba dental school, state university of campinas, piracicaba, sp, brazil. 5 dds, ms, phd, titular professor of department of exact sciences, luiz de queiroz college of agriculture, university of são paulo, piracicaba, sp, brazil 6 dds, ms, phd, titular professor of department of restorative dentistry, piracicaba dental school, state university of campinas, piracicaba, sp-brazil. corresponding author: lúcia trazzi prieto piracicaba dental school state university of campinas – unicamp department of restorative dentistry – dental materials area 901 avenida limeira, areião, piracicaba, sao paulo, brazil. 13414-018. telephone: +55 19 98408-8800 e-mail: lucinhatrazzi@hotmail.com received: september 05, 2017 accepted: december 01, 2017 influence of whitening dentifrices and mechanical brushing on color change and surface roughness of bulk fill resins mayara zaghi dal picolo1, lúcia trazzi prieto2, josué junior araujo pierote3, suelem chasse barreto4, carlos tadeu dos santos dias5, luís alexandre maffei sartini paulillo6 the use of whitening dentifrices during oral hygiene may cause surface modifications such as color change (δe) and increase surface roughness (ra) of composite resin. aim: this study aimed to evaluate the effect of whitening dentifrices and mechanical brushing on color change and surface roughness of bulk fill (filtek bulk fill f) and (aura bulk fill a) composite resins. materials and methods: sixty cylindrical specimens were fabricated of each composite resin. after initial color evaluations (konica minolta cm-700d), with d65 standard illuminant, and the surface roughness (surfcorder se 1700, kosalab), the specimens were assigned (n=10) according to the whitening dentifrice used: true white (t), colgate total 12 professional whitening (d), luminous white advanced (l). specimens were submitted to mechanical brushing (10,000 cycles); new color and surface roughness evaluations were taken. data were analyzed by anova, duncan test and dunnet test were used to identify differences between groups (α=5%). results: the results showed that the tooth brushing time promoted a significant increase in δe of the ft group. the fd group exhibited intermediate values of δe and was similar to all groups tested. there was a significant increase in the surface roughness of the ad and at groups after the mechanical tooth brushing. conclusion: the results showed no significant changes were observed in surface roughness for f groups after toothbrushing. it be concluded that the color change and surface roughness of the composite resins after toothbrushing are dependent on the interaction between the composition of the composite resin and the characteristics of the dentifrice. keywords: composite resins. toothbrushing. dentifrices. color. 2 picolo et al. introduction composite resin has been widely used in procedures that require the restoration of the shape, function and aesthetics of the dental element, due to its physical and chemical properties. in addition, composites mimic the color, translucence and dental texture, and provide mechanical resistance similar to the healthy dental tissues1. the most used technique in direct restorations with composite resins is the incremental technique, since it minimizes the effects of the polymerization contraction. nevertheless, this technique considerably increases the clinical time to perform the restorative procedure2,3. with the aim of simplifying the restorative procedure, with no damages to the composite performance, bulk fill composite resins were introduced in the market4,5. this composite category has alternative photoinitiators, such as monoacylphosphine oxide or dibenzoyl germanium derivatives that, when exposed to light, promote an unimolecular reaction more efficient than camphorquinone and have the potential to increase the cure depth, allowing the use of increments 4 to 5 mm6,7. on the other hand, composite resins present limitations, especially regarding the maintenance of their characteristics over time. the decrease of the composites mechanical properties, due to buccal environment adverse conditions, such as temperature and ph changing, wear promoted by masticatory movements and brushing affect mainly the surface smoothness and the original color of the composites8. the use of dentifrice is essential for maintenance of oral health. besides having therapeutic function, acting as a vehicle for the incorporation of fluoride in buccal environment, which has proven action in the reduction of caries, dental brushing, when associated with dentifrice, favors the mechanical removal of biofilm, as well as the removal of pigments from the teeth and restorations9. when dental brushing is associated with a whitening dentifrice, dental tissues and restorations polishing may be damaged, due to higher concentration of abrasive particles responsible for the superficial removal of stains and enzymatic disintegration of organic molecules present in the biofilm. additionally, some whitening dentifrices have concentrations of hydrogen peroxide (1 to 2%). changes on the composite surface may be related to this agent10,11. dental toothbrushing with dentifrices affects the color and surface roughness of conventional composite resins8,12. its effect on the composites roughness is significant in determining the performance of the material, once the dentifrice relative abrasiveness may increase the surface porosity and remove the composite fillers, inducing water sorption. this results in color changing and loss of brightness and influences the surface smoothness. however, there is little evidence in the literature regarding the action of whitening dentifrices on the surface roughness, color changing of bulk fill composites, and the aging of the composites through simulated mechanical brushing proves to be a valid method to evaluate the behavior of this class of restorative material8,13. therefore, this in vitro study aims to evaluate the behavior of two bulk fill composites when subjected to simulated mechanical brushing using whitening dentifrices. 3 picolo et al. materials and methods in the present study two bulk fill composite resins, shade a3, and three whitening dentifrices were used for the brushing of the samples (table 1). twenty specimens of each bulk-fill resin (f or a) were obtained using a bipartite teflon matrix (7.0mm diameter and 5.0mm tick). the cavity mold was filled in a single increment. after inserting the composite in the mold, the increment was covered with a glass slide with minor digital pressure was placed on the composite resin increment and a weight of 500 grams for 1 minute to obtain a flat and regular surface. the specimens were photoactivated for 40 seconds using a led light-emitting source (valo, ultradent products inc., s. jordan, ut, usa-1400mw / cm2) according to manufacturers recommendations. after, the specimen was detached from the mold and the excess resin runoff with a 15-scalpel blade and stored in distilled water at 37oc for table 1. composition and manufacturers of bulk fill composites and dentifrices used in color change and surface roughness tests. material manufacturer composition particle size % filler (by vol) filtek bulk fill 3m espe st paul, mn, usa ceramics treated with silane, udma, aromatic dimethacrylate uretane, silica treated with silane, itibérbio fluoride, ddma, zirconia treated with silane, water, monomer afm-1, edmab, benzotriazole, doxido of titanium. 20nm silica filler, 4 to 11nm zirconia filler, and 100nm 76.5%/ 58.4% aura bulk fill sdi bayswater, australia barium aluminosilicate pre-polymerized filler; improved optical properties; amorphous silicon dioxide; udma/ bisema/bisgma 0.02 micron 0.4 micron fillers 81% wt filled, 65% vol true white sensodyne glaxosmithkline sodium fluoride; potassium nitrate 5%; triofosfato pentasódio 5%; sorbitol; glycerine; hydrated silica; water; peg-6; cocamidopropyl bataine; (dlimonene e cinnamal) arome; dióxido de titânio; xantana gun; sodium saccharin; sodium hydroxid. ______ _____ colgate total 12 professional whitening colgate palmolive company,osasco, sp, brasil water; sorbitole; hydrated silica; glicerine; sodium lauryl sulfate; fluoruro sodium; pvm/ma copolymer; sodium hydroxid; propilenglicol; cellulose gun; triclosano; sodium saccharin; carrageenin; titanium dioxide; sodium fluoride. ______ _____ luminous white advanced colgate palmolive company, osasco, sp, brasil hydrogen peroxide 1%; sodium monofluorephosphate 0,76%; propylene glicol; calico pirephosphatte; glicerine, peg; ppg116; 66 copolymer; peg -12; pvphydrogen peroxide; pvp; silica; tetrasodium pirophosphate ; sodium lauril sulfate; disodium pirophosphate ; sodium sacarine; sucralose; bht; eugenolpirofosfato dissódico; sacarina sódica; sucralose; bht; eugenol ______ ______ 4 picolo et al. 24 hours. afterwards, the surface of each specimen was polished (polishing machine, apl-4; arotec, sp, brazil) with silicon carbide (sic) papers of decreasing abrasiveness (#2000 and #4000 grit) (carbimet paper discs; buehler, il, usa) for 1 minute. subsequently, initial color readouts (baseline) were (konica minolta cm-700d, konica minolta investment ltd. sensing business division, shanghai, china) was then calibrated according to the manufacturer’s instructions. the specimens were placed in a teflon device (sample port), with d65 standard illuminant, to standardize the environment during the readings. next, surface roughness (baseline) was obtained in three measurements (surfcorder se 1700, kosalab), cut-off of 0.8mm and speed of 0.25mm/s. the specimens were positioned parallel to the surface of the equipment, each sample being fixed to an acrylic base and the measuring tip positioned on the sample surface. after each reading, the sample was rotate 120o, thus the three readings would be passed over the same point in the center of the sample. the initial (baseline) roughness of bulk fill filtek and aura were used as controls for the statistical analysis after the initial readings of color and roughness, the 30 specimens of each bulk fill resin were randomly divided according to the interaction bulk fill x whitening dentifrice used: true white (t), colgate total 12 professional whitening (d) and luminous white advanced (l), totaling 6 experimental groups (n = 10): filtek bulk fill / true white (ft), filtek bulk fill / total 12 professional whitening (fd), aura bulk fill / total 12 professional whitening (ad), filtek bulk fill / luminous white advanced (fl), aura bulk fill / luminous white advanced (al) and aura bulk fill / true white (at). mechanical brushing was performed with 60 soft toothbrushes (oral b indicator plus procter & gamble) one per specimen. the toothbrush head were cut off and attached to the brush holder device of the brushing machine mset (marcelo nucci me, são carlos, brazil), by means of thermal glue (brascola, são bernardo do campo, sp, brazil) so that the toothbrush head was parallel and in contact with the surface of the specimen. in this equipment it was possible to perform the simultaneous brushing of ten specimens at the same time. for brushing of each test specimen, a quantity of 8g of dentifrice was mixed with 24ml of distilled water, measured on analytical balance and precision pipette, forming slurry with a ratio of 1: 3 by mass for the dilution of the dentifrice. each specimen was submitted to a total of 10,000 cycles of linear brushing movements, at a frequency of 4 hz, under a load of 200g, to simulate the force used during oral hygiene procedures13. after this, the machine was switched off, the specimens were removed from the machine, rinsed in distilled water and dried with absorbent paper to remove surface debris (kleenex kimberly-clark, são paulo, sp, brazil) and submitted to a new color and surface roughness measurements. the color measurements were taken and the color change for each different group was calculated by cie lab system in three coordinates to allow the calculation of the color variation (δe), using the following formula: δe = [l1 l0)2 + (a1 a0)2 + (b1 b0)2]1/2. 5 picolo et al. surface roughness alteration (ra) was calculated using the formula: ra= raf rai where rai is the initial and raf the final roughness measurement. results statistical analysis: after checking the normality of the results, for the color variation was applied one-way anova and duncan’s test. for the surface roughness the dunnet’s test (sas) was applied and the 5% probability limit was adopted for decision making for both statistical analyzes. color change the data obtained in the color variation test (δe) were submitted to the normality test (shapiro wilk), which indicated discrepant values, so the data were transformed, increasing to the 0.2 powers that corrected the problems. thus anova one-way was applied and showed that there was significant statistical difference between the studied groups and duncan’s test was applied to evidence this result that is presented in table 2. the results for the duncan test showed that the filtek bulk fill resin brushed with the sensodyne true white (ft) showed higher color variation compared to the fl, at, ad and al groups. the fd group presented intermediate values, with no statistical difference in relation to all the experimental groups. the results of δl, δa and δb did not differ statistically between the different groups. surface roughness the filtek bulk fill and aura bulk fill were not compared with each other. they were compared only with to control (the same composite resin without treatment). the results of the dunnet test for the roughness test showed that only a when brushed with d and t presented significant statistical difference for its control, that is, for the roughness before brushing. while f presented no significant statistical difference for its control when brushed with the whitening toothpaste (table 3). table 2. mean (m), standard deviation (dv) and duncan (d) test result (5%) for the color variation (δe), δl, δa and δb of bulk fill composite resins. groups n δe δl δa δb ft 10 2.57 (1.42)* 0.61(0.03)a 0.11(0.04)b 1.26(0.29)c fd 10 1.53 (0.78)*/** 0.77(0.05)a 0.28(0.10)b 0.35(0.11)c fl 10 1.46 (0.75)*** 0.54(0.05)a 0.15(0.02)b 0.09(0.14)c at 10 1.17 (0.54)*** 0.07(0.40)a 0.35(0.07)b 0.25(0.04)c ad 10 1.16 (0.67)*** 0.51(0.07)a 0.10(0.01)b 0.55(0.07)c al 10 1.12 (0.74)*** 0.14(0.02)a 0.12(0.05)b 0,53(0.06)c a,b,c equal letters show that there is no significant difference between the means and standard deviation of δl, δa and δb compared by lines. */**/***statistically different from baseline groups (control), according to dunnet test (p<0.05) f= filtek bulk fill; a= aura bulk fill; t= true white; d= colgate total 12 professional whitening; l= luminous white advanced 6 picolo et al. discussion in buccal environment the composite resin is subjected to chemical and mechanical challenges that can alter its properties and expose its filler particles. this allows the incorporation of exogenous pigments, modifying their coloration and surface roughness8. it is imposed that dental brushing may cause chemical-mechanical challenge to the composites, so this study evaluated the color change and surface roughness of two brands of bulk fill composites resin after mechanical brushing with three different whitening dentifrices, totaling 10,000 cycles, which are clinically equivalent to a period of approximately one year of dental brushing13. the abrasiveness of the paste created by the dentifrice during brushing is influenced by physical characteristics of the abrasive particles, such as shape, size, and hardness. consequently, thicker and irregular particles produce rougher surfaces and, since there are more abrasives in the whitening dentifrices responsible for the bleaching effect and, considering the brushing period, the surface color and surface roughness of the composites tested will be affected on a larger scale8. the results of color variation showed that filtek brushed with true white showed the highest color variation without statistical difference when the same resin was brushed with colgate total 12 professional whitening and with difference for the other groups. the aura bulk fill resin did not show color variation when brushed with different whitening dentifrices. this result may be related to the composition of filtek and dentifrices true white and colgate total 12 professional whitening. filtek resin contains benzotriazole, an antibacterial monomer with a light yellow coloration. in a previous study, was observed that benzotriazole has antibacterial action on an experimental resin, however, the increase in the concentration of this monomer, decreased the degree conversion of the resin. as a consequence, there is greater water absorption, which degrades the ester bond of the methacrylate polymers, promoting color change of the material. the same may have occurred with filtek resin causing damage on the performance of this material14. true white and colgate total 12 professional whitening dentifrices have titanium dioxide (tio2) as their composition. it was observed that the incorporation table 3. results of the dunnet test for the surface roughness test of bulk fill composites after brushing with bleaching dentifrices. groups n mean (dv) dunnett baseline a 30 0.079 (0.007) ad 10 0.143 (0.035) *** al 10 0.098 (0.026) at 10 0.124 (0.039) *** baseline f 30 0.071 (0.006) fd 10 0.088 (0.021) fl 10 0.075 (0.009) ft 10 0.084 (0.044) ***statistically different from baseline groups (control), according to dunnet test (p<0.05) f= filtek bulk fill; a= aura bulk fill; t= true white; d= colgate total 12 professional whitening; l= luminous white advanced 7 picolo et al. of tio2 nanoparticles significantly increased the opalescence of composite resins15. thus; it is believed that during the brushing there was deposition of tio2 on the surface of filtek changing the opacity of the material and the initial color. however, it is emphasized that the color variation values obtained are less than 3.3. according to the literature this value of δe although perceived by skilled operators are clinically acceptable16. the results of the surface roughness test showed that the aura bulk fill resin showed a significant statistical difference between the initial roughness (control) and final roughness after brushing with colgate total 12 professional whitening and sensodyne true white. dental brushing can degrade the composite surface through a three-body wear process by removing the polymer matrix layer that is a smoother layer, exposing the filler particles, which are stiffer and more uneven. the toothbrush can also increase this effect of abrasion, since the brush bristles do not wear out the surface of the material as evenly as flat discs or rubber cups would do, in finishing and polishing procedures17. the organic matrix of filtek bulk fill has udma in its composition, which is a less viscous and more flexible functional monomer than bisgma, present in the aura bulk fill resin composition. because it is stiffer and less flexible than udma, bis-gma produces fewer crosslinks; with the result that the copolymers containing bisgma tend to have lower hardness, which is directly related to the degree of conversion and greater sorption of water. it can be concluded that the udma present in the organic matrix of filtek bulk fill resin plays an important role for wear resistance and lower surface roughness18-21.  the filler / matrix interface present in the composite resin is designed to chemically bond the matrix to the filler particles by means of a silane bonding agent. this location is subject to the formation of microcracks in which water can penetrate, causing degradation of the composite. the surface of the composites becomes susceptible to the formation of cracks resulting from chewing. therefore, it is assumed that the exposure of the filler particles, caused by the brushing with whitening dentifrices, accelerates the degradation of the composites, compromising the polishing, and increasing the roughness22,23. the difference in behavior between the filtek and aura resins in the surface roughness test can be explained by the composition. filtek is a nanoparticulate resin formed by nanomers and nanoclusters, which are slightly nano-sized charge-free agglomerates, which reduce the interstitial spacing between the particles. therefore, there is greater amount of charge in this composite, and consequently, better physical properties and better surface smoothness. during mechanical brushing, only the nano-sized particles are displaced while the nanoclusters remain in the resin matrix. in this way, it can be assumed that the composition of filtek favored its better performance in relation to surface roughness24,25. in conclusion, it was concluded that the color changing and surface roughness of bulk fill resins after brushing with whitening dentifrices are dependent on the interaction between the composition of the composite resin and the characteristics of the dentifrice. 8 picolo et al. references 1. ferracane jl. resin composite--state of the art. dent mater. 2011 jan;27(1):29-38. doi: 10.1016/j.dental.2010.10.020. 2. park j, chang j, ferracane jl, ib lee. how should composite be layered to reduce shrinkage stress: incremental or bulk filling? dent mater. 2008 nov;24(11):1501-5. doi: 10.1016/j.dental.2008.03.013. 3. kapoor n, bahuguna n, anand s. influence of composite insertion technique on gap formation. j conserv dent. 2016 jan-feb;19(1):77-81. doi: 10.4103/0972-0707.173205. 4. bacuta s, ilie n. light transmittance and micromechanical properties of bulk fill vs. conventionl resin based composites. clin oral investig. 2014 nov;18(8):1991-2000. doi: 10.1007/s00784-013-1177-y. 5. ilie n, bacuta s, draenert m. bulk-fill resin-based composites: an in vitro assessment of their mechanical performance. oper dent. 2013 nov-dec;38(6):618-25. doi: 10.2341/12-395-l. 6. kalliecharan d, germscheid w, price rb, stansbury j, labrie d. shrinkage stress kinetics of bulk fill resin-based composites at tooth temperature and long time. dent mater. 2016 nov;32(11):1322-1331. doi: 10.1016/j.dental.2016.07.015. 7. harlow je, rueggeberg fa, labrie d, sullivan b, price rb. transmission of violet and blue light through conventional (layered) and bulk cured resin-based composites. j dent. 2016 oct;53:44-50. doi: 10.1016/j.jdent.2016.06.007. 8. roselino lde m, chinelatti ma, alandia román cc, pires-de-souza fde c. effect of brushing time and dentifrice abrasiveness on color change and surface roughness of resin composites. braz dent j. 2015 oct;26(5):507-13. doi: 10.1590/0103-6440201300399. 9. barbieri gm, mota eg, rodrigues-junior sa, burnett lh jr. effect of whitening dentifrices on the surface roughness of commercial composites. j esthet restor dent. 2011 oct;23(5):338-45. doi: 10.1111/j.1708-8240.2011.00426.x. 10. amaral cm, rodrigues ja, erhardt mc, araujo mw, marchi gm, heymann ho, et al. effect of whitening dentifrices on the superficial roughness of esthetic restorative materials. j esthet restor dent. 2006;18(2):102-8; discussion 109. 11. heintze sd, forjanic m, ohmiti k, rousson v. surface deterioration of dental materials after simulated toothbrushing in relation to brushing time and load. dent mater. 2010 apr;26(4):306-19. doi: 10.1016/j.dental.2009.11.152. 12. roselino lde m, cruvinel dr, chinelatti ma, pires-de-souza fde c. effect of brushing and accelerated ageing on color stability and surface roughness of composites. j dent. 2013 nov;41 suppl 5:e54-61. doi: 10.1016/j.jdent.2013.07.005. 13. wang l, garcia fc, amarante araujo p, franco eb, mondelli rf. wear resistance of packable resin composites after simulated toothbrushing test. j esthet restor dent. 2004;16(5):303-14; discussion 314-5. 14. centenaro cc, rostirolla fv, leitune vc, parolo cf, oligari fa, saumel sm, et al. influence of addition of 2-[3-(2h-benzotriazol-2-yl)- 4-hydroxyphenyl] ethyl methacrylate to an experimental adhesive system. acta odontol latinoam. 2015 apr;28(1):72-8. doi: 10.1590/s1852-48342015000100010. 15. yu b, ahn js, lim ji, lee yk. influence of tio2 nanoparticles on the optical properties of resin composites. dent mater. 2009 sep;25(9):1142-7. doi: 10.1016/j.dental.2009.03.012. 16. shamszadeh s, sheikh-al-eslamian sm, hasani e, abrandabadi an, panahandeh n. color stability of the bulk-fill composite resins with different thickness in response to coffee/water immersion. int j dent. 2016;2016:7186140. doi: 10.1155/2016/7186140. 17. bagheri r, tyas mj, burrow mf. subsurface degradation of resin-based composites. dent mater. 2007 aug;23(8):944-51. 9 picolo et al. 18. asmussen e, peutzfeldt a. influence of uedma, bisgma e tegdma on selected mechanical properties of experimental resin composites. dent mater. 1998 jan;14(1):51-6. 19. barszczewska-rybarek i, jurczyk s. structure-property relationships in dimethacrylate networks based on bis-gma, udma and tegdma. dent mater. 2009 sep;25(9):1082-9. doi: 10.1016/j.dental.2009.01.106. 20. sideridou i, tserki v, papanastasiou g. study of water sorption, solubility and modulus of elasticity of light-cured dimethacrylate-based dental resins. biomaterials. 2003 feb;24(4):655-65. 21. rodrigues-junior sa, scherrer ss, ferracane jl, della bona a. microstructural characterization and fracture behavior of a microhybrid and a nanofill composite. dent mater. 2008 sep;24(9):1281-8. doi: 10.1016/j.dental.2008.02.006. 22. ferracane jl. hygroscopic and hydrolytic effects in dental polymer networks. dent mater. 2006 mar;22(3):211-22. 23. whitehead sa, shearer ac, watts dc, wilson nhf. surface texture changes of a composite brushed with tooth whitening dentifrices. dent mater. 1996 sep;12(5):315-8. 24. attar n. the effect of finishing and polishing procedures on the surface roughness of composite resin materials. j contemp dent pract. 2007 jan 1;8(1):27-35. 25. yadav rd, raisingani d, jindal d, mathur r. a comparative analysis of different finishing and polishing devices on nanofilled, microfilled, and hybrid composite: a scanning electron microscopy and profilometric study. int j clin pediatr dent. 2016 jul-sep;9(3):201-8. 1http://dx.doi.org/10.20396/bjos.v19i0.8660119 volume 19 2020 e200119 original article 1 undergraduate dental clinic, university of campinas – unicamp, piracicaba dental school – fop, piracicaba, sp, brazil. 2 department of prosthodontics and periodontology, university of campinas unicamp, piracicaba dental school – fop, piracicaba, sp, brazil. 3 dental research division, school of dentistry, ibirapuera university, sp, brazil corresponding author: victor ricardo m. muñoz-lora, department of prosthodontics and periodontology, piracicaba dental school, university of campinas. avenida limeira 901, areão, piracicaba, são paulo, brazil, cep: 13414-903. phone: +55(19)999370977, e-mail: victor_9874@hotmail.com; victormunoz2512@gmail.com received: june 19, 2020 accepted: september 18, 2020 evaluation of pain intensity of the masticatory muscles after occlusal appliance and combined therapy: a 6-months follow-up pilot study tainá queiroz dos santos1, giancarlo de la torre canales2 , celia marisa rizzatti-barbosa2 , victor ricardo manuel muñoz-lora2,3 masticatory muscle pain (mmp) is a common type of orofacial pain. occlusal appliance (oa) is contemplated as a first-line conservative approach for chronic mmp, however, integrated biopsychosocial approaches such as counseling and self-care therapies (csg) are also considered essential. aim: this pilot study aimed to compare the use of a combined therapy (gsg + oa) and solely oa treatment on pain intensity related to chronic mmp over a 6-month follow-up. methods: for this, 20 patients diagnosed with chronic mmp using the diagnostic criteria for temporomandibular disorders (dc/tmd) were divided into 2 groups (n=10) and treated with oa or combined therapy (cot; oa + csg). electromyographic muscle activity (emg), visual analogue scale (vas) and pressure pain threshold (ppt) were recorded at baseline, 1, 3 and 6 months after treatment. data was collected and statistical analysis were applied at a significance level of 5%. results: results showed no significant differences at baseline among groups for any assessment. vas showed that both treatments decreased subjective pain in volunteers over time, but no significant differences among both groups were observed at any evaluation time. for electromyography, cot and oa presented no significant differences throughout the experiment neither on relaxed muscle position or maximum volunteer contraction. finally, a significantly higher ppt for cot was found for all muscles at the last assessment point (p<0.05). conclusion: these findings suggest that both treatments are effective for the reduction of pain perception (vas) in patients with chronic mmp. however, the addition of csg to an oa therapy may be more beneficial for the improvement of tenderness on the same patients, at least in a long-term basis (> 3 months). notwithstanding, a larger study should be performed to substantiate these findings. keywords: facial pain. myofascial pain syndromes. occlusal splints. https://orcid.org/0000-0002-0921-342x https://orcid.org/0000-0002-8747-0034 https://orcid.org/0000-0002-1134-8542 2 santos et al. introduction temporomandibular disorders (tmds) embrace a range of painful and non-painful conditions involving the temporomandibular joint (tmj), masticatory muscles, and associated structures1-3. among tmds, masticatory muscle pain (mmp) is one of the most common types of orofacial pain conditions4 and , in chronic states, is frequently associated with symptoms like tmj sounds and uncoordinated mandibular movements, which affect the social, vocational, and emotional life of patients3,5. even though numerous treatments such as pharmacotherapy, physiotherapy, oral appliances (oa), botulinum toxin, surgical approaches, and counseling and self-care therapies (csg) are widely used to treat chronic mmp, they usually have a high, but not total, success rate1,6,7. within these treatments, systematic reviews confirmed an effective performance of oa to control pain related to different tmds8,9 and to decrease the frequently associated psychosocial impairments10,11. for these reasons, oa is contemplated as a first-line conservative approach for chronic mmp, although it involves clinical and laboratorial steps, which represent a manufacturing cost that may differ depending on the employed material12-15. however, even though oa is stablished as a beneficial treatment for chronic mmp at a short-term basis (< 3 months), its benefits seemed to be equalized or even lesser than other therapeutic modalities at a long-term (> 3 months)16. moreover, few studies have reported the shortor long-term effects of solely oa as a treatment for chronic mmp17, since it is usually associated to other therapies14,18,19. on the other side, it is well known that emotions play an important role in the perception of pain and are contemplated as perpetuating factors20,21. focusing only on a mechanical approach may be insufficient to promote long-term control of the pain associated to tmds, since negative emotions are frequently involved causing anxiety, depression, stress and fatigue22. negative emotional states of patients are often present in severe clinical conditions, significantly affecting the progression of chronic pain21,23. for these reasons, integrated biopsychosocial approaches with conservative and reversible characteristics such as csg, which involves self-care strategies and behavioral therapies, are essential in the treatment of chronic tmds24. csg are used to stimulate patients to change their behavior and stress11, and are contemplated as very powerful tools in the control of chronic mmp. although both therapies (oa and csg) have been broadly studied, the possible benefits on improving pain intensity that csg may offer to an exclusively oa treatment for chronic mmp, whether at short(< 3 months) or/and at a long-term basis (> 3 months), are still not completely elucidated. therefore, this pilot study aimed to compare the benefits of a combined therapy (cot; gsg + oa) versus oa on pain intensity of chronic mmp over a 6-months period. we assumed that a cot will be more effective to control the pain associated to chronic mmp than oa itself at short and long-term assessments. methodology patients the present study was approved by the ethics committee of the piracicaba dental school, university of campinas, são paulo, brazil (caae# 70654317.2.0000.5418). all 3 santos et al. patients received a consent form before involvement. a total of 112 female patients who attended to the clinics of the piracicaba dental school unicamp, são paulo, brazil were evaluated using the diagnostic criteria for temporomandibular disorders (dc/tmd)25 by an experienced examiner. patients diagnosed with chronic mmp (myofascial pain on masticatory muscles lasting > 3 months) greater than 50mm in the visual analogue scale (vas), and under contraceptive intake (in an attempt to control hormonal imbalance and better standardize our sample) were included for this experiment. exclusion criteria (figure 1) comprised patients with a history of face or tmj trauma, probable diagnosis of bruxism (clinical findings + self-report), partial removable or total prosthesis users, daily use of anti-inflammatory, analgesic or myorelaxant treatment, patients with unrealistic expectation for treatment results, presence of arthritis, osteoarthritis, diabetes, fibromyalgia or neurological disorders, presence of primary headache diagnosed by the international classification of headache disorders questionnaire26, and those who did not agreed to sign the consent form. after applying inclusion and exclusion criteria, 20 patients were randomly allocated into two different groups (n=10) using the random allocation software27: cot, with patients treated with csg and oa; and oa, with patients treated with oa only. 112 screened patients inclusion criteria female patients; diagnosed with mop according to the dc/tmd; initial pain of more than 5 mm (vas); use of contraceptive. exclusion criteria face or tmj trauma; bruxism; use of removable partial or total prosthesis; daily use of anti-inflammatory, analgesic or myorelaxant treatment; unrealistic expectations for treatment results; arthritis, osteoarthritis, diabetes, fibromialgia, or neurological disorders; presence of primary headache; no agreed to sign the consent form. 92 excluded patients 20 included patients oa (n=10) ct (n=10) day -7 stone casts acquisition baseline oc installation vas, ppt, and emg baseline oc installation + counseling vas, ppt, and emg 1, 3, 6 months vas, ppt, and emg 1, 3, 6 months vas, ppt, and emg figure 1. flowchart of the study showing the screened patients, inclusion and exclusion criteria, enrolled patients, and sequence of treatments application and assessment. 4 santos et al. treatments all treatments were performed by an experienced clinician who was not involved in the outcomes assessment. counseling and self-care techniques (csg) for csg, patients received extensive verbal and written instructions from the same clinician (table 1). the aim of the therapy was to learn about the anatomy and physiology of the stomatognathic system as well as the etiology and prognosis of tmds. also, patients received self-care strategies to control parafunctions and relieve pain (e.g. practicing physical exercises and how to relax jaw muscles), along with information to improve sleep (e.g. reducing alcohol or coffee consumption before sleeping, maintain a regular sleep schedule, among others), correct body posture, and the importance of dietary habits17,28. counseling was reinforced every time the patient returned for evaluation and with weekly mobile text message reminders. table 1. counseling and self-care strategies given to the patients. instructions provided to the patients • explanation about anatomy and physiology stomatognathic system • explanation about the genesis and evolution of mmp • at rest position of jaw, there should be no contact of teeth • exercise regularly • use relaxation techniques during stressing situations (e.g. deep breathing and progressive muscle relaxation)29 • try to avoid hard food and eat small pieces at each time • avoid parafunctional habits (biting things, nails, leaning on the jaw, etc) • reduce alcohol or coffee consumption before sleeping • maintain regular sleep schedule occlusal appliance (oa) to make the oa, maxillary and mandibular stone casts were obtained from the patients one week before baseline session. both casts were mounted on a semi-adjustable articulator in a position of maximal intercuspidation and considering a 3mm posterior disocclusion. the oa consisted in a rigid 3-mm flat splint covering all maxillary teeth, made of transparent thermo-polymerized acrylic resin (vipicril plus, vipi®, brazil). oa was adjusted to the centric position of each patient´s jaw and using a bilateral balanced occlusion design (i.e. simultaneous contacts on all teeth during excursive movements)30. patients received the oa and were instructed to use it every night while sleeping. baseline was considered as the day patients received the splint and follow-up period started after the splint installation. adjustments were made every time they were required until a comfortable fit of the oa and the correct centric position were achieved. 5 santos et al. outcomes assessments were performed by an experimenter who was blinded to treatment assignments. outcomes (pain intensity and muscle electric activity) were measured in all participants at four different times, as described in figure 1: baseline (before application of the corresponding treatment), and 1, 3and 6-months following oa installation and/or after providing counselling instructions to the patients. visual analog scale (vas) vas consists of a 100 mm horizontal line in which the left end is labeled with the words “no pain” and the right end is written “worst pain imaginable”. volunteers had to mark a point on the line according to the level of their current pain. the media of vas from all patients in a group was considered for data analysis7. pressure pain threshold (ppt) a digital algometer with 1 cm2 circular at rod (kratos ddk-20. são paulo, brazil) was used to assess ppt by a single calibrated operator (kappa = 0.89). pressure was applied perpendicular to the surface of the skin with relaxed muscles at a rate of 0,3kg/ cm2 according to the following muscle sequence: right anterior temporal (rt), right masseter (rm), left masseter (lm), and left anterior temporal (lt) muscle; after 5 min, a second series of stimuli were applied at inverse order. pressure stopped when patients started to feel pain-like perceptions31. electromyography (emg) the ads 1200 (lynx electronic technology ltd, sao paulo, brazil) equipment with eight channels was used to record the electromyographic signal of the evaluated muscles. the electrodes were fixed in the most prominent part of the muscle at maximal contraction; also, a personalized acetate plate was elaborated for each patient in order to locate the electrodes in the same muscle part every time. the relaxed muscle position (rmp) and maximum volunteer contraction (mvc) were evaluated. to record maximum electrical muscle activity a parafilm m (american national can, chicago, il, usa) was bitten bilaterally in the molar region for five seconds. this process was recorded for three different times and the arithmetic mean was calculated32. the softwares lynx aqdados 7.02 and lynx aqd analysis 7.0 (lynx electronic technology ltd, sao paulo, brazil) were used for the acquisition of simultaneous signals and to process the root mean square (rms) values, expressed in mv. statistical analysis the results were analyzed using the analysis of variance (anova) for repeated measures (rm) to test the effect of time (4 periods) and treatment (oa and cot) on the assessed outcomes (vas, ppt, and emg), followed by tukey´s multiple comparisons test to compare the paired inter-groups effect. intra-group analysis was assessed using one-way anova rm, followed by tukey´s multiple comparisons test. all data 6 santos et al. was processed using the jamovi® statistic software, version 1.2 (the jamovi project, 2020). the significance level of 5% was set for all data. results the sample was composed of women with a mean age of 27±4 years, diagnosed with mmp according to the dc/tmd. vas-baseline values showed no differences among groups. post-treatment assessments of vas (figure 2) showed that both, oa and cot, decreased pain intensity in volunteers over time (1, 3 and 6 months). in addition, we observed no significant differences among both groups (cot and oa) at any evaluation period (p=0.56; effect size £0.10). 0 1 2 3 4 5 6 7 8 baseline p a in (m m ) visual analogue scale oa cot 6m3m1m + + 6.99 (±0.79) 2.24 (±0.75) 1.81 (±0.60) 1.53 (±0.81) 7.65 (±1.81) 1.42 (±1.22) 1.36 (±2.14) 0.73 (±0.99) figure 2. visual analogue scale (vas). occlusal appliance (oa) and combined therapy (cot) decreased pain intensity assessed by vas after 1 month and maintained up to 6-months. mean ± standard deviation; +=p<0.001 within group (one-way anova followed by tukey´s multiple comparisons test). however, no significant differences were found among groups throughout the experiment (p>0.05; two-way repeated measures anova followed by tukey´s multiple comparisons test). for electromyography, the relaxed muscle position (rmp) and maximum volunteer contraction (mvc) of muscles were evaluated. the within group analysis showed no differences on oa or cot over-time (p>0.05; one-way repeated measures anova followed by tukey´s multiple comparisons test). additionally, cot and oa groups presented no significant differences among them throughout the experiment (i.e. from baseline to 6-month assessment) neither on rmp (p>0.05; effect size=0.13) nor mvc (p>0.05; effect size= 0.16), as observed in figure 3. 7 santos et al. 0 1 2 3 4 5 6 7 8 r el ax ed p os iti on (u v ) electromyography 0 50 100 150 200 250 300 b as el in e 1m 3m 6m b as el in e 1m 3m 6m b as el in e 1m 3m 6m b as el in e 1m 3m 6m m ax im un v ol un te er c on tr ac tio n (u v ) electromyography oa cot lmltrmrt b as el in e 1m 3m 6m b as el in e 1m 3m 6m b as el in e 1m 3m 6m b as el in e 1m 3m 6m lmltrmrt figure 3. the electromyographic assessment of masseter and temporalis muscles showed no significant differences within groups (p>0.05; one-way repeated measures anova followed by tukey´s multiple comparisons test). among groups comparisons also showed no differences (p>0.05; three-way repeated measures anova followed by tukey´s multiple comparisons test) neither on (a) relaxed muscular position nor (b) maximum volunteer contraction. oa, occlusal appliance; cot, combined therapy; rt, right temporalis; rm, right masseter; lt, left temporalis; lm, left masseter. regarding ppt (figure 4), cot, but not oa, showed a significant relieve of tenderness after 3 months on the left side muscles (p<0.01; one-way repeated measures anova followed by tukey´s multiple comparisons test) and an improvement in all muscles after 6 months (p<0.05; one-way repeated measures anova followed by tukey´s 8 santos et al. multiple comparisons test). moreover, overall differences among groups were found (p<0.001; effect size = 0.28). the statistical analysis showed no differences between treatments at baseline or at 1-month assessment. however, after 3 months, differences became notorious with higher ppt values for cot rather than oa, and a clear significant difference on the left temporalis and masseter among groups (p<0.05; lt: 1.37± 0.11 for cot vs 0.73± 0.12 for oa; and lm: 1.53±0.24 for cot vs 0.73±0.13 for oa). finally, a significantly higher ppt for cot over oa was found for all muscles at the last assessment point (p<0.05; rt: 1.02± 0.29 for cot vs 0.79± 0.23 for oa; and rm: 0.95±0.27 for cot vs 0.77±0.95 for oa; lt: 1.92± 0.07 for cot vs 0.76± 0.18 for oa; and lm: 1.79±0.17 for cot vs 0.73±0.16 for oa). 0.68 (±0.16) 0.78 (±0.22) 0.76 (±0.14) 0.79 (±0.23) 0.63 (±0.27) 0.59 (±0.24) 0.92 (±0.34) 1.02 (±0.29) 0.69 (±0.16) 0.75 (±0.22) 0.73 (±0.16) 0.77 (±0.19) 0.68 (±0.11) 0.62 (±0.23) 0.79 (±0.24) 0.99 (±0.27) 0.72 (±0.14) 0.80 (±0.19) 0.73 (±0.12) 0.76 (±0.18) 0.62 (±0.19) 0.95 (±0.04) 1.37 (±0.11) 1.92 (±0.07) 0.64 (±0.14) 0.78 (±0.17) 0.73 (±0.13) 0.73 (±0.16) 0.59 (±0.21) 0.96 (±0.05) 1.53 (±0.24) 1.79 (±0.17) * * * * * * 0.0 0.5 1.0 1.5 2.0 2.5 rt a 0.5 1.0 1.5 2.0 2.5 rm b 0.0 0.5 1.0 1.5 2.0 2.5 baseline lt c oa cot 0.0 0.5 1.0 1.5 2.0 2.5 lm d 6m3m1m baseline 6m3m1m baseline 6m3m1m baseline 6m3m1m 0.0 figure 4. pressure pain threshold (ppt) of (a) right temporalis (rt), (b) right masseter (rm), (c) left temporalis (rm), and (d) left masseter (lm). mean ± standard deviation; *=p<0.5 among inter-group paired variables (three-way repeated measures anova followed by tukey´s multiple comparisons test). oa, occlusal appliance; cot, combined therapy. discussion in this pilot study, we aimed to compare the effectiveness of an oa treatment against cot (oa plus csg) on the reduction of pain intensity related to chronic mmp, over a 6-month evaluation period. our results suggested that both therapeutic approaches (oa and cot) had a similar positive effect on pain perception (vas) throughout the 6-month follow-up. however, significantly higher ppt values can be seen after 6 months just for cot, which may suggest a positive influence of csg over a solely oa therapy for the improvement of tenderness on patients with chronic mmp, at least at a long-term evaluation. 9 santos et al. both, oa and csg, had previously demonstrated a positive effect for the treatment of pain related to tmds, including mmp16,28. however, different from previous studies14,17-19, we assessed the benefits of including csg to a solely oa approach on patients with chronic mmp. it is important to consider that csg is a non-invasive treatment that can be used by any health-care professional, because of its simple technique that requires no ample experience and no profound knowledge of psychological domains28. additionally, csg is a low-cost strategy with proven beneficial effects when used alone17,33 or in combination with other treatments like physical therapy34. similar to our results, other studies have shown an improvement on reported pain (vas) after the use of oa16, csg14,17 or cot (i.e. different types of oa plus csg)18,19. on the other side, the results of ppt from our study suggested no improvement on tenderness for solely oa, and an improvement just on the left-side muscles for cot after 3 months. these findings are comparable to the ones presented by conti et.al 2012, where none of the assessed groups (full coverage splint plus csg, anterior trigeminal inhibitory nociceptive device plus csg, and csg alone) showed an increase on ppt levels at 3-month evaluation19. the different effects among these two pain-rating methods (vas and ppt) may be due to their weak correlation for assessing pain intensity in myogenic-tmd populations35,36, proving that vas may not be an adequate ppt predictor. additionally, following the aforementioned results, it may be suitable to suggest that both, oa and csg, have a higher impact on patient´s perception of pain rather than on the physiological domain, at least in a short-term basis (< 3 months). as it is known, pain is a complex and multidimensional experience that receives influence from physiological, emotional, and cognitive dimensions37. for this reason, it is difficult to describe pain intensity based just on a self-reported scale (vas) or a mechanically-stimulated pain test (ppt) without assessing other pain domains (e.g. cognitive or emotional), which is a limitation of our pilot study. interestingly, we found a significant long-term (>3 months) improvement on ppt for cot compared to solely oa. it is important to remark that few studies have reported long-term effects of csg or oa as singular treatments, as they are usually studied together or in combination with other therapies14,16,28. truelove et al.14, 2006, observed no differences among two cot groups (conventional flat plane acrylic splint plus csg, and soft vinyl splint plus counseling) and csg alone after a 12-month evaluation period. it was concluded that oa provides no additional benefits to csg and makes the treatment more expensive14. additionally, a systematic review and metanalysis concluded that oa may have a higher short-time benefit for reducing pain related to tmds when compared to other therapeutic modalities. though, this effect seemed matched or even lesser than other therapies at a long-term16. the better performance of cot may be attributed to the knowledge and, consequently, better control and awareness of the possible causes related to mmp, which are shown during csg. for these reasons, the concomitant use of csg and oa (i.e. cot) may be more favorable than a merely oa intervention for chronic pain of the masticatory muscles, at least at a long-term basis. regarding emg, our results showed no differences within and among groups over time on electric muscle activity for rmp and mvc muscle positions. previous 10 santos et al. experiments evaluated oa electromyographic effects of masticatory muscles and found a decreased electromyographic activity8,38,39. however, with the exception of one study39, this reduction is frequently transient and may be attributed to the presence of an unusual object inside the mouth (e.g. the oa) which produces an avoidance conduct13. finally, some shortcomings need to be reported. first, the use of a small sample size is clearly a limitation of this pilot, so larger studies are required to substantiate the results from this experiment. second, our sample was restricted to a female population, due to their higher prevalence of tmds3. also, the use of contraceptives was an attempt to avoid unstable pain periods associated with hormonal fluctuation throughout menstrual cycle40; however, since hormones play an important role on pain outcomes from myofascial pain patients41, different results in other populations cannot be disregarded. third, pain is a multidimensional experience42 with highly prevalent psychosocial impairments5, so the assessment of cognitive and emotional features should be considered in future experiments. conclusion the use of oa and cot seems to have a similar positive effect on patient´s perception of pain over a 6-month evaluation period. however, even though both treatments reported a slightly, but no significant, improvement on tenderness at a short-term basis (< 3 months), the use of a cot presented an increased beneficial effect after 6 months on patients diagnosed with chronic mmp. due to the aforementioned reasons and considering that csg is a no-cost simple treatment modality, its concomitant use with oa therapy may be a better option for patients suffering from chronic mmp rather than oa alone. yet, larger studies including different populations and assessing other pain dimensions should be encouraged. acknowledgements: the work was supported by piracicaba dental school, university of campinas, são paulo, brazil. tainá queiroz dos santos was an undergraduate student with a scholarship from pibic/cnpq. victor ricardo manuel muñoz lora received a scholarship from the são paulo research foundation (fapesp; 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change assessment of co-cr alloy subjected to immersion in denture cleansers pedro carlos cortiana borsa1, mariana marquezan2, liliana gressler may3, katia olmedo braun4 1msc in prosthodontics, e-mail: pedroborsa@gmail.com phone (55) 9938-5312, private practiner, santa maria, rs, brazil 2phd in orthodontics, e-mail: marianamarquezan@gmail.com phone: (55) 9674-4678, professor of the department of stomatology at universidade federal de santa maria (ufsm), santa maria, rs, brazil 3phd in prosthodontics, e-mail: liligmay@gmail.com phone: (55) 9141-8101, professor of the department of restorative dentistry at universidade federal de santa maria (ufsm), santa maria, rs, brazil 4phd in prosthodontics, e-mail: aitakbraun@gmail.com phone: (55) 9971-3099, professor of the department of restorative dentistry at universidade federal de santa maria (ufsm), santa maria, rs, brazil abstract choosing the right chemical cleanser for removable partial dentures is a challenge, because they present an acrylic and a metallic portion, which should be cleaned and not damaged. aim: the aim of this study was to assess surface changes of cobalt chromium alloys immersed in different cleaners solutions: 0.05% sodium hypochlorite, 4.2% acetic acid, 0.05% sodium salicylate, sodium perborate (corega tabs®) and 0.2% peracetic acid. material and methods: one hundred and twenty circular specimens (10 mm in diameter) of two commercial available co-cr alloys were tested: gm 800 ® (dentaurum) and co-cr® (degudent). the samples were randomly divided into ten experimental groups (n=10), according to the trend mark of alloy and cleaners solutions in which they were immersed, and two control groups, in which the samples of the two alloys were immersed in distilled water. evaluations were performed through roughness measurement (rugosimeter surftest 211, mitutoyo), visual evaluation with stereomicroscope (stereo discovery 20, carl zeiss) and scanning electron microscope surface (jsm, 6360 sem, jeol), at experimental times t0 before immersions, t1 after one immersion, and t2 after 90 immersions. intergroup comparison for the effect of immersion in the different cleanser agents was evaluated through anova/tukey tests (p≤0.05). the effect of the time in the immersion of each alloy was evaluated by t-pared test (p≤0.05). the two alloys were compared using the t-student test. results: the analysis of roughness and microscopy showed that surface changes were significantly greater in groups submitted to 0.05% sodium hypochlorite after 90 immersions (t2). when comparing the two alloys, a similar behavior of roughness was observed for the cleaning agents. however, alloy gm 800® showed significant statistical difference for roughness variations in experimental times (δ1 and δ2), when immersed in sodium 0.05% hypochlorite. the number of exposures of the alloys to the cleaning agents showed a negative influence when using sodium hypochlorite solution. conclusions: it is possible to conclude that 0.05% sodium hypochlorite has caused the greatest apparent damage to alloy surface. keywords: dental alloys, chromium alloys; denture cleansers; sodium hypochlorite 1. introduction despite advances in materials and techniques in dental rehabilitation, removable partial dentures (rpd) remain as an important tool for public health, because they are a less costly option1. upon its installation in the oral cavity of patients, it is a dentist’s received for publication: december 21, 2016 accepted: may 27, 2017 original article braz j oral sci. july | september 2016 volume 15, number 3 http://dx.doi.org/10.20396/bjos.v15i3.8649980 197 duty to instruct them about hygiene2 to avoid the accumulation of biofilm, which is an etiological factor of oral diseases, such as caries and stomatitis. patients can make use of mechanical and chemical cleaning methods. their association has been reported in the literature as the best choice2-7, especially for special and geriatric patients, who find it difficult to properly brush their dentures1. techniques and materials should be effective in cleaning, and should not affect the components of the prosthesis. of the chemical cleansers used for full dentures, sodium hypochlorite solutions deserve special attention, since they degrade mucin and allow for greater removal of bacterial biofilm in depth2,8. solutions of sodium salicylate, sodium perborate, and acid peracetic are also used due to their antimicrobial potential8-10. a home-made option is vinegar (4.2% acetic acid) capable of reducing the number of bacteria on the surface11. removable partial denture, however, present metallic components on their composition, normally cobalt chromium alloy12. choosing the right cleanser is a challenge, because solutions containing hypochlorites can cause some corrosion, staining, and even loss of physical properties13, 14. thus, the aim of this study was to assess surface changes in cobalt chromium alloys subjected to immersion in different cleanser solutions: 0.05% sodium hypochlorite, 4.2% acetic acid, 0.05% sodium salicylate, sodium perborate (corega tabs®) and 0.2% peracetic acid. 2. material and methods 2.1 sample one hundred and twenty circular specimens (10mm in diameter) of two different alloys of cr-co – remanium gm 800 ® (dentaurum, pforzheim, germany) and co-cr® degudent (dentsply ind. and co. ltd, são paulo, sp, brazil) – were obtained through casting, polished with a sequence of wet sandpaper (600 to 2500) and diamond polishing paste with felt disk (master diamond ferramentas, são paulo, sp, brazil) in polisher (arotec apl-4, arotec, cotia, são paulo, brazil), numbered and randomly divided into 12 groups (n=10). ten experimental groups were designed, according to cleanser solutions – 0.05% sodium hypochlorite, 4.2% acetic acid, 0.05% sodium salicylate, sodium perborate (corega tabs®) and 0.2% peracetic acid; the type of alloy used (gm 800 ® or co-cr®); and two control groups, where the bodies and specimens were immersed in distilled water (control). the alloys used have more than 85% of chromium and cobalt in their compositions: gm 800® (63.3% co; 30% cr; 5% mo; 1% si; 0.5% mn; 0.4% c); cocr® (64.8% co; 28.5% cr; 5.3% mo; 0.5% si; <1% mn; <1% c; <1% n). sample size calculation was performed for mean difference of roughness and it was found that at least seven samples should be used to achieve 80% power. thus, 8 specimens were used for roughness assessment, and 2 were intended for microscopic analysis. 2.2 immersion in cleansers the cleanser solutions were prepared and 15 ml were poured into test tubes (pyrex no. 9820, corning inc., usa), in which the specimens of each group were fully immersed. for 0.05% sodium hypochlorite solution preparation, 5 ml of 2.5% sodium hypochlorite solution (q-boa®, anhembi s/a osasco, são paulo, brazil) was diluted in 200 ml of distilled water6. immersion time for this solution was 10 min per cycle15. the 4.2% acetic acid solution consisted of pure white vinegar (wms supermercados do brasil s/a, porto alegre, rs, brazil). immersion time for this solution was 10 min16. the 0.05% sodium salicylate solution was prepared by diluting 1/4 teaspoon (0.25g) of sodium salicylate pa (c7h5nao3) (vetec química fina ltda, rio de janeiro, rj, brazil) in 250 ml of distilled water. immersion time for this solution was 15 minutes2. effervescent sodium perborate solution was prepared by diluting one tablet of corega tabs® (stafford-miller ind., rio de janeiro, rj, brazil) in 150 ml of water at 45°c, as recommended by the manufacturer. its immersion time was 15 minutes17. the 0.2% peracetic acid solution was prepared by diluting 13.4 ml of 15% peracetic acid (sigmasul, cachoeirinha, rs, brazil) in one liter of distilled water. immersion time for this solution was 15 minutes. surface assessments were held before immersions (t0), after one immersion (t1), and after 90 immersion cycles (t2), simulating the daily use of these solutions for three months. the samples were cleaned with spray of distilled water and dried on absorbent sheet, between each immersion, and the interval between consecutive immersions were merely the time necessary to wash and dry the specimens. 2.3 surface roughness surface roughness was measured using a rugosimeter (surftest sj 211, mitutoyo corp., kanagawa, japan), with 6 readings with cut-off of 0.25 mm in each specimen, 3 on the x-axis (x) and 3 on the ordinate axis (y). the ra parameter, which provides the means of peaks and valleys, was assessed in all experimental time intervals (t0, t1, and t2), using the center of the sample. 2.4 microscopic assessment two microscopic analyses were performed. initially, a stereomicroscope was used with magnification of 8.5x to assess samples in 3 experimental times. a damage index was created for the surface, where 0 indicates the absence of any signs of changes; 1, the loss of brightness and light surface deposition; 2, the occurrence of spots in more than two thirds of the surface of the specimens; and 3, the total darkening of the specimens. after that, scanning electron microscopy (sem) was performed with magnification of 500x, in order to view the topographic surface appearance of alloys. with the use of x-ray energy dispersive spectroscopy (eds), it was possible to determine which chemical elements were present on the surface. 2.5 statistical analysis data were tabulated and statistically analyzed using spss (statistical package for social sciences, version 13.0). normality was verified by the shapiro-wilk test. surface roughness after the application of cleaning protocols at times t1 and t2, as well as the difference found by subtracting roughness after immersion (in both surface change assessment of co-cr alloy subjected to immersion in denture cleansers braz j oral sci. 15(3):196-200 198 times) by the initial roughness (baseline), were compared between different experimental groups by analysis of variance and multiple comparison tukey test (p≤0.05). roughness data after immersion in cleanser solutions were compared to initial (baseline) by paired t test (p≤0.05). the two alloys were compared with respect to roughness in the various protocols through t-student test (p≤0.05). the captured images of sample surface changes were visually assessed twice in an optical stereomicroscope by one observer, to yield a kappa coefficient of 0.87. the scores to visual changes underwent transformation “rank” to then be compared between the different experimental groups by analysis of variance and multiple comparison test of tukey (p≤0.05). the two alloys were compared in scores of visual changes on different protocols by mann-whitney test (p≤0.05). 3. results 3.1 surface roughness results showed no statistically significant difference between the methods of cleaning after the first immersion as to roughness (t1). after 90 immersions (t2), the means of ra (µm) in the groups submitted to 0.05% sodium hypochlorite were significantly higher (table 1). other cleansers did not cause surface roughness changes in the alloy over time (table 1). when comparing the two alloys, we have found similar behavior in roughness for cleansers. however, alloy gm 800® showed significant statistical difference between δ1 and δ2 when immersed in 0.05% sodium hypochlorite (table 2). surface change assessment of co-cr alloy subjected to immersion in denture cleansers table 1 mean and standard deviation (sd) of surface roughness ra (µm) of alloys immersed in various cleansers in experimental times. alloy solution ra (µm) t0 ra (µm) t1 ra (µm) t2 co-cr® 0.05% sodium hypochlorite 0.050 (0.009)aa 0.063 (0.011)ab 0.494 (0.083)bc 4.2% acetic acid 0.061 (0.017)aa 0.070 (0.027)aa 0.059 (0.021)aa 0.05% sodium salicylate 0.061 (0.016)aa 0.068 (0.035)aa 0.083 (0.037)aa sodium perborate (corega tabs®) 0.047 (0.006)aa 0.060 (0.019)ab 0.067 (0.010)ab 0.2% peracetic acid 0.052 (0.013)aa 0.067 (0.029)aab 0.080 (0.032)ab distilled water control 0.062 (0.021)aa 0.069 (0.018)aa 0.060 (0.011)aa gm 800® 0.05% sodium hypochlorite 0.053 (0.015)aa 0.077 (0.021)ab 1.254 (0.191)bc 4.2% acetic acid 0.073 (0.019)aa 0.088 (0.023)aa 0.103 (0.048)aa 0.05% sodium salicylate 0.062 (0.026)aa 0.068 (0.026)aa 0.072 (0.027)aa sodium perborate (corega tabs®) 0.062 (0.017)aa 0.074 (0.021)ab 0.074 (0.018)aab 0.2% peracetic acid 0.085 (0.034)aa 0.099 (0.042)aa 0.084 (0.032)aa distilled water control 0.072 (0.023)aa 0.087 (0.038)aa 0.075 (0.033)aa different lowercase letters indicate statistically significant difference between the immersion solutions, with the same alloy and time (anova/tukey, p≤0.05). different capital letters indicate statistically significant difference among immersion times (t-pared test, p≤0.05). table 2 mean and standard deviation (sd) of the variation of roughness after 1 (δ1) and 90 immersions (δ2). solutions co-cr® gm 800® 0.05% sodium hypochlorite ∆1 0.013 (0.012)a 0.024 (0.017)a ∆2 0.446 (0.085)a 1.202 (0.194)b 4.2% acetic acid ∆1 0.008 (0.030)a 0.014 (0.024)a ∆2 0.000 (0.028)a 0.030 (0.042)a 0.05% sodium salicylate ∆1 0.007 (0.041)a 0.007 (0.034)a ∆2 0.021 (0.037)a 0.010 (0.021)a sodium perborate (corega tabs®) ∆1 0.014 (0.017)a 0.010 (0.013)a ∆2 0.022 (0.014)a 0.010 (0.029)a 0.2% peracetic acid ∆1 0.013 (0.025)a 0.015 (0.045)a ∆2 0.028 (0.034)a 0.000 (0.037)a distilled water control ∆1 0.002 (0.021)a 0.003 (0.020)a ∆2 0.071 (0.030)a 0.083 (0.036)a different letters indicate statistically significant differences among alloys for roughness variation δ1 and δ2 with the same cleansers (p≤0.05). 3.2 microscopic assessment after one immersion (t1) no clear visual change was noted on the surface of any of the groups. however, after 90 immersions (t2), the two alloys submitted to 0.05% sodium hypochlorite presented the highest scores, pointing to the major changes (table 3). table 3 mode of visual scores after 90 immersions (t2). solutions co-cr® gm 800® 0.05% sodium hypochlorite 2a a 3a b 4.2% acetic acid 0b b 0c b 0.05% sodium salicylate 1b b 1bb sodium perborate (corega tabs®) 0b b 0c b 0.2% peracetic acid 0b b 1b c distilled water control 1b b 0c b different lowercase letters indicate statistically significant difference between the columns (difference between the alloys mann-whitney test, α=0.05). different capital letters indicate statistically significant difference between the lines (difference between immersion solution anova/tukey, α=0.05). braz j oral sci. 15(3):196-200 199 surface change assessment of co-cr alloy subjected to immersion in denture cleansers in further analysis by sem, it was possible to observe sharpening occurrence, suggesting a slight texturing of the surface of the co-cr® alloy after the first immersion. however, after the ninetieth immersion, widespread surface change was noticed, with the presence of protruding clusters and occasional depressions (figure 1). for the gm 800® alloy, after the first immersion, minimal superficial change was noticed. however, after the ninetieth immersion, the image shows more abrupt changes in the structure of the sample (figure 2). eds surface analysis of both alloys, when immersed in 0.05% sodium hypochlorite, showed the presence of oxygen and chlorine, which indicates corrosion. iron and tungsten were also found in the composition of alloy gm 800®, not reported by the manufacturer. fig. 1 co-cr® alloy specimen immersed in 0.05% sodium hypochlorite solution in the three experimental times (a: t0; b: t1; c: t2). above there is the stereomicroscope image (magnification of 8.5x). below, sem images (magnification of 500x). t1 shows little change in surface brightness (score 1), while t2 shows a widespread staining on the surface (score 2). fig. 2 gm 800® alloy specimen immersed in 0.05% sodium hypochlorite solution in all three experimental days (a: t0; b: t1; c: t2). above there is the stereomicroscope image (magnification 8.5x). below, sem images (magnification 500x). t1 shows little change in the surface brightness (score 1), while t2 is observed to such darkening the surface and abrupt changes in relief. 4. discussion when choosing a metal for facing different challenges in hostile environments, its corrosion behavior its corrosion behavior is the most important factor to be considered18. thus, this work has compared the co-cr® e gm 800® metal cobalt chromium alloys, after being immersed in 5 cleaners. regarding cleanser comparison, 0.05% sodium hypochlorite solution caused more obvious changes to alloy, generating higher roughness values and higher scores on the analysis with stereomicroscope. analysis of roughness after immersion of the alloy in 4.2% acetic acid solution, 0.05% sodium salicylate, sodium perborate (corega tabs®) and 0.2% peracetic acid, showed no statistically significant difference between experimental periods with no increased roughness over time. it was also observed that water-immersed alloys (control) had scores of 0 and 1, with slight loss of brightness (score 1) only seen with a microscope and not to the naked eye. therefore, we decided to consider these two as having no damage. thus, 4.2% acetic acid solution, 0.05% sodium salicylate, sodium perborate (corega tabs®), and 0.2% peracetic acid did not cause visible damage to alloys at different experimental times. when the alloys were compared, it was observed that, with regard to roughness, the nominal values of ra (µm) were higher for gm 800®, but with no statistically significant difference. however, statistically significant difference has been found for δ1 and δ2 for this alloy. with the stereomicroscope, clearer changes were observed for alloy gm 800® after 90 immersions (t2). the evaluation by sem confirmed most surface changes for this alloy at t2. as for the cocr® alloy, it showed superficial changes similar to those that occur when superficial electrochemical attack is conducted with acid solution to metallographic analysis19, with the view of protruding beads on the surface of the alloy. gm 800® alloy showed greater degree of change with suggestive image of detachment of surface oxidation plates. it is believed that the observed difference for the two alloys at t2 may be related to the fact that gm 800® has shown iron and tungsten in its composition, which was identified by eds, since the presence of other metals in the alloy can modify its corrosion resistance and increase the speed of etching20. it is believed that surface roughness of the alloy reaches clinical significance when reaching 0.2 µm, which favors the adhesion of biofilm. thus, values higher than this cannot be clinically accepted21. in this study, the two alloys exceeded this cut-off point after 90 immersion in 0.05% sodium hypochlorite (co-cr®=0.446 µm; gm 800®=1.202 µm), which suggests that 0.05% sodium hypochlorite may cause damage the co-cr alloys used in rpd, which agrees with the literature2,13,14,22. although sodium hypochlorite has fungicidal8,16,23 and bactericide effect, and is able to penetrate up to 3 mm in the resin, not only eliminating the surface bacteria, but also those in depth, if allowed to act for ten minutes, at a concentration of 0.525%15, its use in rpd should be cautious due to the deleterious effects on metal infrastructure. recent studies have demonstrated the damaging effect of sodium hypochlorite on the alloy co-cr by weight and ion loss22, and by reducing the modulus of elasticity and ultimate strength. in the latter study, however, the property of bending was found satisfactory according to ada specification no.1424. with respect to the quantity of infused over time, only groups exposed to sodium hypochlorite 0.05% showed obvious changes after the first immersion. comparing evaluations in sem’s first exposure to hypochlorite (t1) with the evaluation after 90 exposures (t2), it is clear that there was a real deterioration of the surface of the two alloys, which is higher in the alloy (gm braz j oral sci. 15(3):196-200 800 ®). the visual scores evaluation showed scores 2 and 3 after dipping 90 cycles, while after the first immersion the score was 0, agreeing with results of previous studies25, 26. with the exception of the groups submitted to 0.05% sodium hypochlorite solution, there was no occurrence of surface damage to the alloy. therefore, it is possible to perform removable partial denture cleaning with most solutions used in the study. however, further studies are needed for evaluating the mechanical properties of alloys, as well as evaluate more immersions. 5. conclusion the solution of 0.05% sodium hypochlorite showed significant surface changes, suggestive corrosion, while other solutions did not present such deleterious effects. both alloys showed similar surface changes after 90 immersion cycles for different cleansers. increased contact with cleansers caused greater surface changes on the alloy only when 0.05% sodium hypochlorite solution was used. references 1. sesma n, takada ks, laganá dc, jaeger rg, azambuja 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of dental alloys submitted to disinfection. rev odontol unesp. 2004;33(3):143-8. 200surface change assessment of co-cr alloy subjected to immersion in denture cleansers braz j oral sci. 15(3):196-200 1http://dx.doi.org/10.20396/bjos.v16i0.8650445 volume 16 2017 e17006 original articlebjos 1 master degree student, universidade são francisco. rua santa cruz, 683, santa libania, bragança paulista, são paulo, brasil. tel. +55 (11) 9-5228-0358. karina_f_r@hotmail.com 2 laboratory of microbiology and molecular biology, universidade são francisco, av. são francisco de assis 218, bragança paulista, são paulo, brasil. 3 odontology, hospital das clínicas samuel libanio, r. comendador josé garcía, 777 centro, pouso alegre, minas gerais, brasil. 4 department of pediatric dentistry, universidade são francisco, av. são francisco de assis 218, bragança paulista, são paulo, brasil. 5 department of dentistry, universidade são francisco, av. são francisco de assis 218, bragança paulista, são paulo, brasil. corresponding author: cirurgiã-dentista: karina ferreira rizzardi. rua santa cruz, 683, santa libania, bragança paulistasp. tel +55 (11) 9-5228-0358. karina_f_r@hotmail.com received: january 20, 2017 accepted: july 26, 2017 do aesthetics dental needs interfere in the oral health-related quality of life and in the self-steem of patients seeking for treatment at university são francisco dental school? karina ferreira rizzardi1, 2, leonardo caldas vieira3, thais manzano parisotto2, 4, cristiane franco pinto5 aim: the objective was to evaluate oral health-related quality of life (ohrqol) in patients aging 18 60 years, considering oral health, dental aesthetic impact and self-esteem. methods: the sample comprised 81 patients, regardless gender/ ethnicity, seeking for dental aesthetic treatment at university são francisco, bragança paulista-sp. the instruments used to assess the ohrqol were the questionnaires: 1. rosenberg self-esteem scale (rss); 2. oral health impact profile-14 brazil (ohip) and 3. psychosocial impact and aesthetic dental questionnaire-brazil (piadq). data were analyzed by spearman correlation (α=5%) and descriptive statistics. results: the older the patient the worse the oral and general health conditions found (p<0.05). moreover, the age showed significant correlation with oihp and piadq questionnaires scores (p=0.000). the three questionnaires showed moderate positive correlations (p<0.05 r=0.461–0.685) among them. in addition, ohip and qiped questionnaires correlated with general health and oral health (p<0.05 r=0.230–0.558). conclusion: it could be concluded that aesthetic dental needs interfere, in fact, in the oral health-related quality of life and in the self-steem of patients seeking for treatment university são francisco dental school. keywords: quality of life. esthetics dental. oral health. mailto:karina_f_r@hotmail.com 2 rizzardi et al. introduction quality of life refers to the individual’s perception of their everyday position, their goals, expectations, concerns, experiences and culture to which the individual belongs1,2. quality of life research in the health area contributes to the evaluation of the cost/benefit ratio of a service, which contributes to the improvement of the treatment offered2,3. according to the study of castro et al.4, health is not restricted to the absence of diseases/injuries, but also to the repercussion of the health problems in the daily activities. in the same way as general health, oral health is essential for quality of life, and pain and discomfort are closely related to the difficulty of eating, socializing and working, for example5. thus, the objective of medical/dental care for most patients is to achieve a better quality of life with good functional capacity and well-being6. the importance of the patient’s opinion about the results of the interventions, and not only the professional opinion, is of great importance. this way, it is crucial that researchers could be able to measure the individual’s health perception in order to evaluate the benefit of their interventions in the patient’s life7. successful measurement within oral health is essential toward more patient-centered oral health care8. oral health is influenced by many factors generating positive and negative emotions9 that change over time. the oral health operation, include daily life activities such as eating, talking and smiling10. oral health-related quality of life (ohrqol) can be associated with psychological variables, social interactions and general well-being11. it was already shown that low ohrqol conditions in adults living in england could be associated with depressive symptoms12. furthermore, people may be judged by their teeth: adults with ideal smiles were considered more intelligent, whereas those with poor dental esthetics were associated with lower intelligence. additionally, subjects with good oral health have greater chances of finding a job, than of those with unpleasant smiles13. in this context, the application of instruments to quantify physical, emotional and social well-being, which are closely related to oral health-related quality of life, has been recognized as an important outcome measure6. thus, the objective of the present study was to evaluate the oral health-related quality of life of individuals aged 18 to 60 years, who sought for aesthetic treatment in the dentist sector of the university of são francisco usf, considering self-esteem, oral health and the impact of aesthetic dentistry. material and methods a convenience sample comprised 100 individuals, aged 18-60, of all genders and ethnicities, with aesthetic needs, who attended the integrated clinic of dentistry of the university of são francisco-usf. patients with dental aesthetic needs, especially in the upper incisors, were included in this study. on the other hand, unlettered patients were excluded, because they could not be able to read and answer the questionnaires. the period of data collection was august 2016 – december 2016 in bragança paulista. 3 rizzardi et al. the city of bragança paulista is located at 88 kilometers from the capital (são paulo) and has a population of approximately 159,000 inhabitants (ibge 2014), most living in the urban area. the city shows a relatively high human development index (hdi = 0.776), and offers the following oral health programs: primary dental care and dental specialty center (ceo/university of são francisco-usf). the aesthetic needs included: direct facets, restorations and temporary crowns involving anterior teeth, as well as dental whitening. the identification of the aesthetic needs was performed by the students enrolled in the integrated dental clinic of usf. theoretical instructions about aesthetic needs were given by the professors of the dental clinic to the students and clinical photographs were also used in the calibration process. the patients were invited to fill three questionnaires in the waiting room of the dental clinic. the questionnaires were related to self-esteem14, oral health-related quality of life15 and the influence of dental aesthetics on the quality of life16 of the individuals in the waiting room of the dental clinic”. the instruments used in the present research were: 1. rosenberg self-esteem scale (rss) unifesp/epm14, a specific questionnaire for the evaluation of self-esteem, feelings, personal care and appreciation, comprising 10 questions, scored from 0 to 3. thus, the final score of this questionnaire ranged from 0 to 30, and the closer to 0, the better the self-esteem. 2. oral health impact profile (ohip) 14 brazil15. a generic questionnaire about the oral health-related quality of life with 14 questions assessing problems with teeth, mouth or dental prosthesis. responses were scored from 0 to 4 and the final score of all questions resulted in scores between 0 and 56, where the higher the score, the greater the individual perception and the worse the quality of life. 3. psychosocial impact and aesthetics dental questionnaire (piadq) brazil/unifesp16. a specific questionnaire to evaluate the influence of dental aesthetics on the quality of life of the individuals, with 23 items containing four dimensions: dental self-confidence, social impact, psychological impact and aesthetic impact. the items were scored from 0 4, ranging from 0 to 92, and lower values indicated a better quality of life. all instruments were self-applied and involved closed questions. together with the three questionnaires, the oral health conditions were also assessed by closed questions answered by the patients themselves. still, patients were asked to complete a form about the following data: age, gender, family income, general and oral health. in this form about 10 questions were asked, such as: “do you have systemic disease?”, “are you in medical treatment?”, “do you take medicine for chronic disease?” “have you ever had a tooth/teeth extracted by dentist(s)?”, “do you wear dental prosthesis?” “have you ever used orthodontic appliance?”, “have you ever had a cosmetic treatment in your teeth?”. the answers were: yes or no.” the project was approved by the ethics committee in reasearch of the university são francisco (caae: 58766516.6.0000.5514) and only the individuals who agreed and signed the informed consent form took part in the research. the data were analyzed by spearman correlation, using statistical package for social science – spss 16.0 (usa), considering a 5% level of significance. secondary data were analyzed by descriptive statistics. 4 rizzardi et al. results from a total of 100 patients selected for the study, 19 individuals did not fill a certain question of the evaluation instruments and, therefore, were excluded from the study. this way, the final sample consisted of 81 individuals, the minimum age was 18 years and the maximum age was 60 years. table 1 shows the mean age of the participants in the study, which was 35,25 years. similar values were found when men’s age was compared to women’s age. the mean and standard error of the scores of the questionnaires were also displayed in this table. the worse the oral health conditions, the higher the impact of the dental clinical condition, resulting in higher questionnaires scores. figure 1 shows that the majority of the sample was comprised by female. information about general and oral health of the individuals enrolled in the present research indicated that the general conditions were good, whereas oral conditions were regular or poor. these data were obtained through a questionnaire including only closed questions. table 1. sample characterization regarding age, income and questionnaires’ scores. characteristics mean standard error women`s age (years) 34.12 ±1.40 men` age (years) 27.57 ±1.35 women`s and men`s age (years) 35.25 ±1.54 rosenberg self-esteem scale (rss) scores 7.10 ±0.54 oral health impact profile-14 brazil (ohip) scores 11.00 ±1.05 psychosocial impact and aesthetic dental questionnaire-brazil (piadq) scores 29.30 ±2.12 the higher the questionnaires `scores, the higher the impact of the dental clinical condition figure 1. sample characterization considering income, gender, general health and dental health conditions obtained by questionnaires. 120.00% 80.00% 60.00% 40.00% 20.00% 0.00% 100.00% inc om e ge nd er 0= m /1 =f do yo u h av e s ys tem ic dis ea se ? do yo u t ak e m ed ici ne fo r c hr on ic dis ea se ? ar e y ou in m ed ica l tr ea tm en t? ha ve yo u e ve r h ad te eth ex tra cte d? ha ve yo u e ve r h ad ae sth eti c p ro ce du re ? ha ve yo u e ve r h ad te eth ae sth eti c p ro ble m ? ha ve yo u e ve r u se d o rth od on tic ap pli an ce ? do yo u w ea r d en ta l p ro sth es is? w ou ld yo u c ha ng e y ou r t ee th ? ha ve yo u e ve r h ad to ot h w hit en ing ? 0 1 2 3 5 rizzardi et al. table 2 shows the correlations between the three oral health-related quality of life questionnaires (rss, ohip and piadq), age, gender, income, and general/oral health conditions in the patients studied. the higher the patient’s age, the higher were the ohip and piadq scores, indicating that the oral health-related quality of life and the dental aesthetics worsened with age (p=0.000). positive correlations (p<0.05, r=0.299 0.468) were also observed between the patient’s age and the variables: general health (systemic disease and medical treatment) and oral health (extracted tooth, aesthetic problem in the tooth, dental prosthesis, appearance of the teeth). negative correlations (p=0.000, r=-0.399 -0.468) were observed between age and income, as well as the use of orthodontic appliances. the three quality of life questionnaires: rss, ohip and piadq presented: 1. moderate positive correlations (p=0.000, r=0.331 0.685) with each other and with the individual wishing to change the appearance of the teeth; 2. negative correlations (p<0.05, r=-0.237 -0.447) with income and the following oral health conditions: orthodontic appliance usage and performance of aesthetic procedures in teeth, indicating that the worse the oral health-related quality of life the less the access to aesthetic treatments. the ohip and piadq questionnaires showed correlations with the following variables: systemic disease, aesthetic problems in the teeth and prosthesis usage (p<0.05, r=0.292 0.345). considering the oral health conditions, the presence of extracted teeth correlated with the ear and piadq questionnaires, with already having an aesthetic problem on the teeth, dental prosthesis usage, income and orthodontic appliance usage (p<0.05, r=-0.239 0.255). the use of dental prosthesis showed positive correlations with general health, desire to change the appearance of the teeth and aesthetic dental problem (p<0.05, r=0.257 0.458); and negative correlations with income, use of orthodontic appliance and teeth bleaching (p<0.05, r=-0.247 -0.381). the desire to change the teeth appearance showed significant positive correlations with medical treatment and aesthetic problems in the teeth (p<0.05, r=0.230 0.291); and significant negative correlations with the income and use of orthodontic appliance (p<0.05, r=-0.344 -0.352). the question “have you ever had an aesthetic procedure in the teeth?” had positive correlations with the use of appliance and bleaching (p=0.000, r=0.408 0.441). the use of orthodontic appliance had positive correlations with income and bleaching (p<0.05, r=0.252 0.381), and negative correlations with systemic diseases and medical treatment (p<0.05, r= -0.277 -0.341). discussion in the present study, it was pointed out that the older the patient who require aesthetic dental procedures, the worse the oral health-related quality of life. the higher the patient’s age, the worse the scores obtained in the specific questionnaires related to oral health (ohip) and dental aesthetics (piadq), showing that patients with higher age probably had less access to the dentist and to preventive measures at 6 rizzardi et al. ta bl e 2. c or re la tio n be tw ee n th e th re e qu es tio nn ai re s of s el fes te em , o ra l h ea lth -r el at ed q ua lit y of li fe a nd t he in flu en ce o f de nt al a es th et ic s on t he q ua lit y of li fe , i nc om e, ge ne ra l a nd o ra l h ea lth c on di tio ns in th e st ud y po pu la tio n. q ue st io nn ai re s g en er al h ea lth o ra l h ea lth in co m e r s s o h ip p ia d q s ys te m ic d is ea se m ed ic al tr ea tm en t ex tr ac te d te et h a es th et ic de nt al p ro bl em p ro st he si s us ag e o rt ho do nt ic ap pl ia nc e us ag e d es ire to c ha ng e te et h ap pe ar an ce d en ta l w hi te ni ng g en de r r =0, 01 5 p =0 ,8 93 r =+ 0, 00 9 p =0 ,9 39 r =+ 0, 02 2 p =0 ,8 44 r =+ 0, 03 5 p =0 ,7 54 r =0, 04 2 p =0 ,7 06 r =+ 0, 09 6 p =0 ,3 89 r =0, 00 9 p =0 ,9 36 r =0, 01 8 p =0 ,8 69 r =+ 0, 06 5 p =0 ,5 65 r =+ 0, 14 7 p =0 ,1 90 r =+ 0, 02 8 p =0 ,8 01 r =0, 06 0 p =0 ,5 92 a ge r =0, 39 9 p =0 ,0 00 * r =+ 0, 20 5 p =0 ,0 65 r =+ 0, 50 1 p =0 ,0 00 * r =+ 0, 53 0 p =0 ,0 00 * r =+ 0, 44 7 p =0 ,0 00 * r =+ 0, 45 9 p =0 ,0 00 * r =+ 0, 41 5 p =0 ,0 00 * r =+ 0, 29 9 p =0 ,0 06 * r =+ 0, 44 8 p =0 ,0 00 * r =0, 46 8 p =0 ,0 00 * r =+ 0, 37 7 p =0 ,0 01 * r =0, 13 2 p =0 ,2 39 r ss r =0, 23 7 p =0 ,0 32 * r =+ 0, 46 1 p =0 ,0 00 * r =+ 0, 46 7 p =0 ,0 00 * r =+ 0, 16 6 p =0 ,1 35 r =+ 0, 08 5 p =0 ,4 48 r += 0, 25 5 p =0 ,0 21 * r =+ 0, 09 0 p =0 ,4 19 r =+ 0, 14 3 p =0 ,1 99 r =0, 26 5 p =0 ,0 17 * r =+ 0, 33 1 p =0 ,0 03 * r =0, 31 5 p =0 ,0 04 * o h ip r =0, 32 1 p =0 ,0 03 * r =+ 0, 46 1 p =0 ,0 00 * r =+ 0, 68 5 p =0 ,0 00 * r =+ 0, 30 1 p =0 ,0 06 * r =+ 0, 17 3 p =0 ,1 20 r =+ 0, 20 1 p =0 ,0 71 r =+ 0, 29 7 p =0 ,0 07 * r =+ 0, 29 9 p =0 ,0 06 * r =0, 40 2 p =0 ,0 00 * r =+ 0, 42 7 p =0 ,0 00 * r =0, 35 8 p =0 ,0 01 * p ia d q r =0, 33 0 p =0 ,0 02 * r =+ 0, 46 7 p =0 ,0 00 * r =+ 0, 68 5 p =0 ,0 00 * r =+ 0, 29 7 p =0 ,0 07 * r =+ 0, 21 1 p =0 ,0 57 r += 0, 23 0 p =0 ,0 37 * r =+ 0, 29 2 p =0 ,0 08 * r =+ 0, 34 5 p =0 ,0 01 * r =0, 44 7 p =0 ,0 00 * r =+ 0, 55 8 p =0 ,0 00 * r =0, 39 9 p =0 ,0 00 * sy st em ic d is ea se r =0, 38 5 p =0 ,0 00 r =+ 0, 16 6 p =0 ,1 35 r =+ 0, 30 1 p =0 ,0 06 * r =+ 0, 29 7 p =0 ,0 07 * r =+ 0, 67 4 p =0 ,0 00 * r =+ 0, 20 5 p =0 ,0 64 r =+ 0, 24 5 p =0 ,0 27 * r =+ 0, 45 8 p =0 ,0 00 * r =0, 34 1 p =0 ,0 02 * r =+ 0, 15 9 p =0 ,1 56 r =0, 14 6 p =0 ,1 93 c hr on ic u se o f m ed ic at io n r =0, 11 4 p =0 ,3 06 r =0, 06 9 p =0 ,5 39 r =0, 05 0 p =0 ,6 57 r =0, 11 9 p =0 ,2 85 r =+ 0, 39 7 p =0 ,0 00 * r =+ 0, 59 1 p =0 ,0 00 * r =+ 0, 12 6 p =0 ,2 60 r =+ 0, 04 1 p =0 ,7 14 r =+ 0, 14 6 p =0 ,1 91 r =+ 0, 13 1 p =0 ,2 42 r =0, 05 1 p =0 ,6 50 r =+ 0, 15 6 p =0 ,1 64 m ed ic al tr ea tm en t r =0, 41 1 p =0 ,0 00 * r =+ 0, 08 5 p =0 ,4 48 r =+ 0, 17 3 p =0 ,1 20 r =+ 0, 21 1 p =0 ,0 57 r =+ 0, 67 4 p =0 ,0 00 * r =+ 0, 20 5 p =0 ,0 64 r =+ 0, 31 3 p =0 ,0 04  * r =+ 0, 45 8 p =0 ,0 00 * r =0, 27 7 p =0 ,0 12 * r =+ 0, 23 0 p =0 ,0 39 * r =0, 01 2 p =0 ,9 13 a es th et ic pr oc ed ur e on te et h r =0, 00 4 p =0 ,9 72 r =0, 34 4 p =0 ,0 02 * r =0, 25 5 p =0 ,0 21 * r =0, 27 5 p =0 ,0 12 * r =+ 0, 10 1 p =0 ,3 68 r =+ 0, 31 3 p =0 ,0 04 * r =0, 01 2 p =0 ,9 12 r =+ 0, 16 1 p =0 ,1 47 r =+ 0, 08 9 p =0 42 8 r =+ 0, 40 8 p =0 ,0 00 * r =0, 06 7 p =0 ,5 50 r =+ 0, 44 1 p =0 ,0 00 * ex tr ac te d te et h r =0, 23 8 p =0 ,0 31 * r += 0, 25 5 p =0 ,0 21 * r =+ 0, 20 1 p =0 ,0 71 r += 0, 23 0 p =0 ,0 37 * r =+ 0, 20 5 p =0 ,0 64 r =+ 0, 20 5 p =0 ,0 64 r =+ 0, 21 8 p =0 ,0 49 * r =+ 0, 23 1 p =0 ,0 37 * r =0, 23 9 p =0 ,0 32 * r =+ 0, 12 7 p =0 ,2 58 r =+ 0, 01 5 p =0 ,8 92 a es th et ic d en ta l pr ob le m r =0, 17 9 p =0 ,1 08 r =0 ,0 90 p =0 ,4 19 r =+ 0, 29 7 p =0 ,0 07 * r =+ 0, 29 2 p =0 ,0 08 * r =+ 0, 24 5 p =0 ,0 27 * r =+ 0, 31 3 p =0 ,0 04  * r =+ 0, 21 8 p =0 ,0 49 * r =+ 0, 27 3 p =0 ,0 13 * r =0, 02 7 p =0 ,8 08 r =+ 0, 29 1 p =0 ,0 09 * r =0, 02 0 p =0 ,8 59 p ro th es is u sa ge r =0, 38 1 p =0 ,0 00 * r =+ 0, 14 3 p =0 ,1 99 r =+ 0, 29 9 p =0 ,0 06 * r =+ 0, 34 5 p =0 ,0 01 * r =+ 0, 45 8 p =0 ,0 00 * r =+ 0, 45 8 p =0 ,0 00 * r =+ 0, 23 1 p =0 ,0 37 * r =+ 0, 27 3 p =0 ,0 13 * r =0, 25 4 p =0 ,0 22 * r =+ 0, 25 7 p =0 ,0 20 * r =0, 24 7 p =0 ,0 26 * o rt ho do nt ic ap pl ia nc e us ag e r =+ 0, 25 2 p =0 ,0 23 * r =0, 26 5 p =0 ,0 17 * r =0, 40 2 p =0 ,0 00 * r =0, 44 7 p =0 ,0 00 * r =0, 34 1 p =0 ,0 02 * r =0, 27 7 p =0 ,0 12 * r =0, 23 9 p =0 ,0 32 * r =0, 02 7 p =0 ,8 08 r =0, 25 4 p =0 ,0 22 * r =0, 35 2 p =0 ,0 01 * r =+ 0, 38 1 p =0 ,0 00 * d es ire to c ha ng e te et h ap pe ar an ce r =0, 34 4 p =0 ,0 02 * r =+ 0, 33 1 p =0 ,0 03 * r =+ 0, 42 7 p =0 ,0 00 * r =+ 0, 55 8 p =0 ,0 00 * r =+ 0, 15 9 p =0 ,1 56 r =+ 0, 23 0 p =0 ,0 39 * r =+ 0, 12 7 p= 0, 25 8 r =+ 0, 29 1 p =0 ,0 09 * r =+ 0, 25 7 p =0 ,0 20 * r =0, 35 2 p =0 ,0 01 * r =0, 06 0 p =0 ,5 92 r ss = r os en be rg s el fes te em s ca le o h ip = o ra l h ea lth im pa ct p ro fil e p ia d q = p sy ch os oc ia l i m pa ct a nd a es th et ic d en ta l q ue st io nn ai re -b ra zi l th e as te ris k “* ” in di ca te s st at is tic al s ig ni fic an ce (α = 5 % ) a nd h at ch in g ar ea s sh ow th is s am e st at is tic al s ig ni fic an ce w ith ou t c on si de rin g re pe at ed d at a. 7 rizzardi et al. population level. in line with these findings, the study of colussi and freitas17 confirms that the oral health conditions of the old people are extremely precarious in brazil. regarding the aging process, there is a great probability of failure in the biological systems of the human organism and older people in the present study showed more systemic diseases, more medical treatments and more dental problems (extracted teeth/dental prosthesis usage), corroborating the study of sáez-prado et al.18. moreover, with aging, teeth might become yellowish with the decrease in enamel thickness, root tissue could be exposed, and greater susceptibility to the repetitive restorative cycle become evident. it should be emphasized that the poor oral health can lead to serious complications, since dental and gingival infections until coronary problems19,20, and the oral health is an inseparable part of the general health of our organism18,21. it was also found that the older the person the lower the income, which may be related to retirement. this way, older people can have less financial conditions for having their teeth treated by a dentist, especially due to the high value of the prosthetic procedures, which are their main need. the three questionnaires (rss, ohip and piadq) showed positive correlations among themselves. moreover, all of them were effective in showing the impact of oral health conditions on the quality of life, reinforcing that a bad oral health and a low self-esteem are in fact associated with quality of life. a negative correlation was obtained among the scores of the three questionnaires and income, dental aesthetic procedure, orthodontic appliance and whitening, which may suggest that bad financial conditions could lead to difficulties in the access to aesthetic procedures. thus, most of the time, procedures of high cost and complexity could only be obtained in the dental schools, highlighting the importance of a straight relationship between university and community. this is in line with the study of maciel and kornis22. specific oral health (ohip) and dental aesthetic questionnaires (piadq) revealed higher and consequently worse scores, which correlated positively with systemic disease, dental aesthetic problems, wear of dental prosthesis and desire to change the appearance of the tooth. there is already scientific evidence associating oral diseases with systemic diseases, for example, the individuals with diabetes mellitus are prone to develop periodontitis with aggressive progression23, closely related to teeth loss. the loss of the teeth influences not only aesthetics, but also the quality of life and the food trituration24, which is the first step in the digestive process, prejudicing the absorption of nutrients, which are essential for a good health. the more the teeth were lost, the higher the scores of the self-esteem (rss) and dental aesthetic questionnaire (piadq), indicating a worse quality of life. in addition, patients with more extracted teeth by dentist also had lower income and a frequency of orthodontic appliance usage. thus, it is observed that this kind of patient, ends up looking for the public service, because they cannot afford a particular treatment. according to the study of vargas and paixão25 in the public service only basic care attention is offered and therefore, extraction is the only procedure that can be performed when the dental situation is critical. this is a very invasive procedure performed in cases of: extensive caries, periodontal disease, iatrogeny involving dental prosthesis of poor quality26. the loss of the dental element causes great personal and social damages, for example: difficulties in employment, communication and shaming, which significantly reduces the self-esteem. 8 rizzardi et al. although the dental prosthesis minimizes this loss, just a few cities in brazil are able to offer this treatment in the public service, limiting the reestablishment of aesthetics and function25 to individuals with greater economic conditions. the ibge 2015 survey shows that 11% of brazilians do not have teeth at all in the mouth, which corresponds to 16 million people, and about 33.3% of the population uses dental prosthesis. it was observed in the present research that even using dental prosthesis, the self-esteem has still been poor in certain patients, because although the prosthesis minimizes the loss, it never replaces identically the dental element. our results also indicate that individuals with dental prosthetic needs have more often systemic diseases, medical treatments, aesthetic problems in teeth, would like to change their teeth appearance, have low purchasing power, hadn’t used orthodontic device and have never whitened the teeth. thus, it is clear that the factors are closely linked, that is, one condition leads to another, culminating in worse conditions of oral health-related quality of life in the population. it is worth remembering that older patients lived in a time when dentistry was focused on curative treatments. nowadays, dentistry is focused on prevention and health promotion, as well as minimally invasive treatments27, aiming at maintaining the dental element as long as possible in the oral cavity, favoring self-esteem and consequently a better the oral health-related quality of life. the use of orthodontic appliance showed positive correlations with a higher income, bleaching performance, and less needs for medical treatments and systemic diseases. the fact that they have a better financial condition is usually linked to a better social condition, so they could afford preventive treatments, as observed in baldani’s et al. research28, that identified the regular visit to the dentist as a strong factor of protection against oral diseases. in brazil the social difference in the population is high, there is a big difference between the percentage of the rich and the poor people, and the access to public treatment is time-consuming because of the demand. the big point is that the curative treatment is much more expensive than the preventive ones, leading to more costs to the government. nowadays, the higher cost is a significant factor in our country, therefore, effective preventive programs should be stimulated, beginning with childhood29. in the present study, the patients who seeked for a treatment at usf dental clinic had a large difference in the family income, indicating that patients who make more money as well as those who make less money looked for dental care in the university. this finding suggests that treatment within the universities have teacher’s supervision and therefore, have usually good quality, being even better than those performed in private clinics, especially when considered the popular ones. moreover, all the patients in this study present aesthetic problems, wishing to reestablish the harmony of a smile and also good masticatory and phonetic conditions, and a multidisciplinary treatment could be often found at universities30. in addition, these procedures have high costs in private practices, and in the university most of the aesthetic treatments are for free. the limitation of this research includes the size of the sample, as many patients were excluded because of unanswered questions in the questionnaires, narrowing the extrapolation of our results to the entire population. 9 rizzardi et al. the data analysis of the present research suggests that aesthetic dental needs actually interfere with patients’ quality of life, and that the needs become worse with aging and with low family income. acknowledgements this work was supported 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[aesthetic rehabilitation with porcelain laminate] [monography]. federal university of rio grande do sul. school of dentistry; 2011 [cited 2017 jul 10]. available from: http://hdl.handle.net/10183/37714. https://www.ncbi.nlm.nih.gov/pubmed/?term=friedman%20pk%5bauthor%5d&cauthor=true&cauthor_uid=27501497 https://www.ncbi.nlm.nih.gov/pubmed/?term=lamster%20ib%5bauthor%5d&cauthor=true&cauthor_uid=27501497 https://www.ncbi.nlm.nih.gov/pubmed/27501497 https://www.ncbi.nlm.nih.gov/pubmed/?term=luo%20xp%5bauthor%5d&cauthor=true&cauthor_uid=27719699 https://www.ncbi.nlm.nih.gov/pubmed/27719699 https://www.ncbi.nlm.nih.gov/pubmed/?term=natapov%20l%5bauthor%5d&cauthor=true&cauthor_uid=27708769 https://www.ncbi.nlm.nih.gov/pubmed/?term=sasson%20a%5bauthor%5d&cauthor=true&cauthor_uid=27708769 https://www.ncbi.nlm.nih.gov/pubmed/?term=zusman%20sp%5bauthor%5d&cauthor=true&cauthor_uid=27708769 http://hdl.handle.net/10183/37714 1http://dx.doi.org/10.20396/bjos.v19i0.8660656 volume 19 2020 e200656 original article 1 department of restorative dentistry, faculty of dentistry, federal university of minas gerais (ufmg), belo horizonte, minas gerais, brazil 2 department of chemistry, paulista state university (unesp), araraquara, são paulo, brazil corresponding author: hugo henriques alvim, dds, ms, phd professor, department of restorative dentistry, faculty of dentistry, federal university of minas gerais (ufmg), av. presidente antônio carlos 6627, belo horizonte, cep 31270-901, mg, brazil e-mail: hugoalvim@gmail.com, tel: +55 31-3409-2440, fax: +55 31-3409-2440 received: july 29, 2020 accepted: september 30, 2020 cytotoxicity and degree of conversion of methacrylate and silorane virgínia angélica silva1 , sávio morato de lacerda gontijo1 , alexandre gatti2 , luiz thadeu de abreu poletto1 , hugo henriques alvim1,* abstract: composites have been proven to have a cytotoxic effect on a variety of tissues and cells. aim: the aim of this study was to analyse the degree of conversion of resins and its correlation with the cell viability in primary gingival fibroblasts. methods: resin-based silorane (filtek p90) and conventional methacrylate resins (filtek z100, filtek z250 and filtek z350xt) were used to evaluate cell viability and the degree of conversion. the resins were light-cured by a led for 20 and 40 seconds. the degree of conversion was analysed by fourier transform infrared spectroscopy. cellular metabolism was evaluated after 24 hours by the mtt assay (n = 6) using the storage solution of composite resin for either 24 hours or 12 days. variance analysis (anova) with a bonferroni correction (p < 0.05) was performed to compare the groups. results: the composite filtek p90 showed a higher degree of conversion when polymerised for 40 or 20 seconds, while the composites filtek z100, filtek z250 and filtek z350xt showed similar degree of conversion. only the filtek z100 resin was cytotoxic. conclusion: we found no statistically significant correlation between cell viability and the degree of conversion. keywords: composite resins. cytotoxins. fibroblasts. https://orcid.org/0000-0001-5700-1512 https://orcid.org/0000-0002-7803-4345 https://orcid.org/0000-0002-9943-2983 https://orcid.org/0000-0002-2767-3833 https://orcid.org/0000-0003-1861-226x 2 silva et al. introduction light-cured composite materials have been used as fillers in restorative dentistry for a number of years and are an established alternative to amalgam1. however, it has been demonstrated that composite restorative materials continue to release resin monomers and other components even after polymerisation. degradation can lead to resin-based dental material components leaching into the oral environment and initiating adverse effects2. after polymerisation, unbound monomers and additives are extracted by solvents, e.g. saliva and/or digestive solvents, especially during the first 24 hours. previous studies revealed a variety of potential cytotoxic and metabolic effects due to the leaching of these methacrylates from the restorations, such as teeth sensitivity, local immunological effects, chronic inflammatory reactions of human pulps, genotoxicity and apoptosis2-4. by varying the composite compounds, the manufacturers can influence the physical and chemical properties to suit special requirements regarding handling, clinical acceptance and the toxicity of these materials. due to the severe cytotoxicity of some traditional composites like methacrylate, industries are searching and developing new materials and new strategies. materials based on modern chemistry (e.g. silorane, ormocer) have been shown to be the most biocompatible5. resins containing the monomer silorane (filtek p90) are based on the replacement of the methacrylate monomers system for an epoxy ring-opening-silorane. the monomer silorane is obtained by the reaction between siloxane and oxirane and has shown to have a shrinkage percentage of only 0.99 vol%3,6 while methacrylate has been shown to have an average contraction of 2.33% vol over time7. the siloxane component brings a higher hydrophobicity to the material, while the oxirane component has a higher reactivity and a lower polymerisation shrinkage8. in addition, the degree of conversion of silorane-based resin has been shown to be greater than that of methacrylate-based resin compounds9. composites have been proven to have a cytotoxic effect on a variety of tissues and cells. cell culture studies are frequently used to assess the cytotoxicity of resin-based materials and their components10. among these cells include gingival fibroblasts that are closely related to restorative materials, especially in class ii restorations4. the cytotoxicity of a composite is related both to its chemical composition and to the conversion percentage of its monomers. as it is known, a high conversion percentage is vital for good mechanical properties and biocompatibility11,12. low values of double bond conversion involve a large number of residual monomers trapped in the polymeric matrix, reducing its biocompatibility. however, the cytotoxicity presented by the filtek z350xt resin in gingival fibroblasts did not correlate with the degree of conversion13. there are few studies in the literature that have evaluated the relationship between cytotoxicity and the degree of conversion12,13. thus, the present study aimed to evaluate the cytotoxic effect on primary gingival fibroblasts from different materials used 3 silva et al. in restorative dentistry and to analyse the degree of conversion of these materials and its possible correlation. the null hypothesis tested was that a lower degree of conversion of the resin would result in lower cytotoxicity in gingival fibroblasts. materials and methods materials and sample preparation the resin composites used in this study were obtained from 3m espe (table 1). table 1. resin composite used in this study with respective matrix and inorganic filler products resin matrix % inorganic filler filtek p90 silorano 76 filtek z350xt bis-gma, bis-ema, udma, tegdma 78,5 filtek z100 bis-gma, tegdma 71 filtek z250 bis-gma, bis-ema, udma, tegdma 60 the specimens were fabricated in a glass matrix, (4 mm diameter x 2 mm height) under aseptic conditions. the moulds were placed on a glass plate, and the restorative materials were condensed into them from above. a mylar strip was applied to the surface. the specimens were divided into two groups which were light-cured from above for 20 or 40 seconds, with the curing tip placed 2 mm away from the material surface, simulating the distance between the composite and the pulp wall. a diode light source emitting blue light (flash lite, discus dental, culver city, usa) was used to cure the resin composites. irradiation intensities (570-600 mw/cm2) were monitored during the test by an external radiometer (model 100 curing radiometer, demetron/ kerr, danbury, ct, usa). excess flash was trimmed away with a sterile scalpel, and the discs were removed by extrusion of the glass matrix and immediately stored in a dry and protected from the light environment. cell culture primary human gingival fibroblasts (hgfs) were purchased from american type culture collection (atcc; manassas, va, usa). the hgfs were cultivated in dulbecco’s modified eagle medium (dmem) with 10% foetal bovine serum (fbs) and 0.1% antibiotic-antimycotic solution at 37ºc and 5% co2. mtt assay the sterilised specimens by ethylene oxide, were placed in 1ml of dmem without fbs and incubated at 37ºc in a 5% co2 air atmosphere for two periods: 24 hours or 12 days (n = 6). fresh dmem was used as the control group. exponentially growing cells were seeded in 96-well plates at a density of 5.0 x 103 cells/well and were cultured for 24 hours. after this, the culture medium was replaced with 100 µl of culture medium containing the material extracts of the resin-based restoratives and incubated for 24 hours. 4 silva et al. cell viability was evaluated according to the reduction of tetrazolium salt to formazan crystals. briefly, mtt solution (10 μl; 5 mg.ml-1) plus 100 μl of growth medium were added to each well, and then the plates were incubated at 37°c in a 5% co2 atmosphere for 4 hours. sodium dodecyl sulphate (sds; 100 μl) was added to dissolve the formazan, and the absorbance was measured at 570 nm by a spectrophotometer (thermo scientific multiscan spectrum, vantaa, finland) after 4 hours of incubation. cell survival was calculated as the percentage of the dye accumulated in the untreated controls14. ftir assay the degree of conversion (%dc) was assessed by micro-attenuated total reflectance fourier transform infrared spectrometry (micro-atr ftir). the ftir spectrometer (spectrum 2000 perkin elmer, boston, ma, usa) was operated under 4000–400 cm−1 range, 2 cm−1 resolution and 32 scans per sample9. three specimens of each material were prepared as described above and placed immediately in the sample holder of the device, and the spectra were recorded. for the methacrylate resin-based composites (filtek z100, filtek z250 and filtek z350xt), the peak intensity ratio of aliphatic c=c to aromatic c=c (1638 and 1609 cm-1, respectively) was evaluated before and after irradiation to determine the percentage of unsaturated aliphatic c=c bonds remaining9. the absorption of the aromatic c=c stretching band remains constant during polymerisation and serves as an internal standard (figure 1). then, the dc of each methacrylate specimen was calculated according to eq. (1). (%c=c) = (aliphatic c=c/aromatic c=c) polymer (aliphatic c=c/aromatic c=c) monomer x 100 figure 1. ftir spectra exhibiting the reduction in the peak height at 1638 cm-1 associated with the saturation of aliphatic c=c within the resin-based methacrylates (z100, z250 and z350xt). absorption of the aromatic c=c stretching band (1609 cm-1) remains constant during polymerisation. 1600 1610 1620 1630 1640 1650 1660 z100 z250 z350 non polymerized polymerized 20 s polymerized 40 s a bs or ba nc e / a. u. wavenumber / cm-1 analitycal band 1638 cm-1 internal standard 1609 cm-1 5 silva et al. the monomer of the silorane resin-based composites (filtek p90) does not contain aliphatic c=c groups (1610 cm-1), remaining constant during polymerisation. the mean %dc of silorane specimens were identified through the ftir spectra in 883 cm-1, which corresponded with the oxirane ring-opening regions (figure 2). then, the %dc of silorane specimen was calculated according to eq. (2). (%c-o-c) = (oxirane (c-o-c)/aromatic c=c) polymer (oxirane (c-o-c)/aromatic c=c) monomer x 100 statistical analysis statistical analyses were performed using an analysis of variance (anova) followed by the bonferroni test. the level of significance was set at p < 0.05. results the cell viability the mitochondrial reducing activity assessed with the mtt assay was inhibited only by filtek z100 (20 and 40 seconds) in both periods of pre-incubation (24 hours or 12 days). for all the resins evaluated, the variation of the polymerisation time did not affect the percentage of cell viability (figure 3). figure 2. ftir spectra exhibiting the (a) reduction in peak height at 883 cm-1 associated with the saturation of the oxirane rings within the resin-based oxirane (filtek p90); (b) absorption of the aromatic c=c stretching band (1610 cm-1) remains constant during polymerisation. a bs or ba nc e / a. u. a bs or ba nc e / a. u. wavenumber / cm-1 non polymerizeda b polymerized 20 s polymerized 40 s non polymerized polymerized 20 s polymerized 40 s 1660 1640 1620 1600 1580 1560890 885 880 875 870 wavenumber / cm-1 analytical band 883 cm-1 internal standard 1610 cm-1 6 silva et al. degree of conversion among the assessed resins, filtek p90 showed the highest %dc. statistical comparisons showed a significant increase in the %dc of filtek z100 and filtek p90 when cured for 40s compared to cured for 20s. filtek z250 and filtek z350xt did not reveal significant differences between %dc when cured for 20 or 40 seconds (table 2). no correlation was found between the degree of conversion and cell viability. table 2. degree of conversion (dc) of resin composites after irradiation by 20 or 40 s (n=3). standard deviation in parentheses. composite polymerization time %dc filtek z100 20 s 62.9 (0.7)a filtek z100 40 s 68.5 (0.7)c filtek z250 20 s 60.1 (0.8)a,f filtek z250 40 s 57.2 (0.8)b,f,g filtek z350xt 20 s 60.7 (0.9)a,g filtek z350xt 40 s 59.2 (0.9)a,g filtek p90 20 s 81.7 (1.1)d filtek p90 40 s 87.0 (1.1)e values followed by the same superscript are not statistically different, p > 0.05. discussion the present study investigated the cytotoxicity and the degree of conversion of currently used resin composites. one major interest factor when making a clinical decision about resin composites is based on their potential for adverse biological effects. the cytotoxicity of the resin composites has been mainly attributed to the release of monomers such as udma, hema and tegma, which are frequently added to the chemical composition of resins15. figure 3. percentage gingival fibroblast viability after incubation with composite extracts after (a) 24 hours or (b) 12 days. (fib = fibroblasts; the same letters indicated similar groups). mtt/24h % c el l v ia bi lit y % c el l v ia bi lit y z1 00 20 ’’ z1 00 40 ’’ z2 50 20 ’’ z2 50 40 ’’ z3 50 xt 20 ’’ z3 50 xt 40 ’’ p9 0 2 0’’ p9 0 4 0’’ fib z1 00 20 ’’ z1 00 40 ’’ z2 50 20 ’’ z2 50 40 ’’ z3 50 xt 20 ’’ z3 50 xt 40 ’’ p9 0 2 0’’ p9 0 4 0’’ fib 100 a a aa ab ab ab ab ab ab b b b b b b b b 80 60 40 20 0 100 80 60 40 20 0 mtt/12 daysa b 7 silva et al. both the resin content and percentage of monomer conversion of dental materials were considered as potential causes of cytotoxicity11. these unconverted monomers, such as tegma and udma, and photo-initiators, such as camphoroquinone, are known to be cytotoxic for cells16. the mtt results showed that only the resin filtek z100 was cytotoxic. considering the degree of conversion of the resin filtek z100, it wouldn’t be expected to present high cytotoxicity. by presenting a lower account of the residual monomers, a smaller release of dimethacrylates into the storage medium should be expected. by comparing it with filtek z250, which presents more components and organic solvents (table 1), the latter would present higher cytotoxicity. the literature has shown that the monomer bis-gma, is known as the most cytotoxic one among all other monomers6,16. it should, however, discuss the characteristics of the polymer chains formed. while some composites have in their composition different amounts of components, possibly generated by chain reactions, they are shown to be more stable and, therefore, release a smaller number of monomers into the environment. despite the cytotoxicity presented by bis-gma, they are important in maximizing the conversion of the monomer into methacrylate groups17. the filtek z100 resin, composed of bis-gma and tegdma chains, was the methacrylate resin that showed the highest degree of conversion when polymerized for 40 seconds. a previous study by ferracane and condon18 reported that the majority of toxic effects from resin composites occur during the first 24 hours. unlike these authors18, the present study showed that the release of the unreacted toxic components from the composite materials probably continues. the formulation of dental materials affects the substances that are released and thus their cytotoxicity. because of the severe cytotoxicity of traditional methacrylates, manufacturers have developed new materials and filling strategies. in the present study, the silorane base composite filtek p90 showed no cytotoxic effect6. regarding the degree of conversion, the composite with the highest value was filtek p90. according to palin et al.19 the polymerisation of silorane-based composites generates reactive species with higher mobility than the free radicals generated in the polymerisation of the composite methacrylate, and this is responsible for the highest degree of conversion. by containing higher amounts of the organic matrix, it was expected that filtek z250 had a higher degree of conversion compared to the other composites (table 1 and 2). however, it did not show that. it should, however, be considered that the concentration of the materials and its chemistry has a strong influence on the characteristics of the polymerisation18. the monomer udma, presented in the z250 and z350xt resins, has a long linear chain, without aromatic rings, which gives greater flexibility and hence also a higher degree of monomer conversion20,21 that contradict our findings. the particle size was also related to the degree of conversion of methacrylate resins. resins with larger particles would hinder the passage of light, increasing dispersion and reducing the conversion of the monomer22. therefore, it was expected that 8 silva et al. filtek z100 and z250 resins had a lower degree of polymerization compared to filtek z350xt. however, the results were similar. a possible explanation would be the presence of bis-gma in the methacrylate resins evaluated, which have a high refractive index, or due to the significant reduction of tegdma in filtek z350xt, which is related to a lower degree of conversion23. the composites filtek z250 and filtek z350xt showed no differences when they were polymerised for 20 or 40 seconds. these findings confirm the information provided by the manufacturer that 20 seconds was enough for sufficient polymerisation of the structure of these resins when using a curing light (led flash lite disculs). according to calheiros et al.24 by increasing the time of light exposure, one does not increase the degree of conversion due to the fact that a saturation of the polymer chains of the composite is obtained. we found no statistically significant correlation between cell viability and degree of conversion. thus, the null hypothesis is accepted. however, more studies are needed to evaluate the cytotoxicity effect and the degree of conversion in other materials. conclusions in conclusion, we observed that the levels of cytotoxicity of restorative resin materials should vary with the number and type of constituent components. as the density and stability of the connection polymer formed; this can influence the release of unreacted components. conflict of interest no potential conflict of interest relevant to this article was reported. references 1. vieira ar, silva mb, souza kka, filho ava, rosenblatt a, modesto a. a pragmatic study shows failure of dental composite fillings is genetically determined: a contribution to the discussion on dental amalgams. front med (lausanne). 2017 nov 6;4:186. doi: 10.3389/fmed.2017.00186. 2. santerre jp, shajii l, leung bw. relation of dental composite formulations to their degradation and the release of hydrolyzed polymeric-resin-derived products. crit rev oral biol med. 2001;12(2):136-51. doi: 10.1177/10454411010120020401. 3. al-boni r, raja om. microleakage evaluation of silorane based composite versus methacrylate based composite. j conserv dent. 2010 jul;13(3):152-5. doi: 10.4103/0972-0707.71649.. 4. jerg a, schulz sd, tomakidi p, hellwig e, polydorou o. modulation of gingival cell response towards dental composites. dent mater. 2018 mar;34(3):412-26. doi: 10.1016/j.dental.2017.11.025. 5. franz a, könig f, lucas t, watts dc, schedle a. cytotoxic effects of dental bonding substances as a function of degree of conversion. dent mater. 2009 feb;25(2):232-9. doi: 10.1016/j.dental.2008.07.003. 6. eick jd, barragan-adjemian c, rosser j, melander jr, dusevich v, weiler ra, met al. silorane resin supports proliferation, differentiation, and mineralization of mlo-a5 bone cells in vitro and bone formation in vivo. j biomed mater res b appl biomater. 2012 apr;100(3):850-61. doi: 10.1002/jbm.b.32649. 7. duarte s jr, botta ac, phark jh, sadan a. selected mechanical and physical properties and clinical application of a new low-shrinkage composite restoration. quintessence int. 2009 sep;40(8):631-8. 9 silva et al. 8. yesilyurt c, yoldas o, altintas sh, kusgoz a. effects of food-simulating liquids on the mechanical properties of a silorane-based dental composite. dent mater j. 2009 may;28(3):362-7. doi: 10.4012/dmj.28.362. 9. mousavinasab sm, atai m, salehi n, salehi a. effect of shade and light curing mode on the degree of conversion of silorane-based and methacrylate-based resin composites. j dent biomater. 2016 dec;3(4):299-305. 10. longo dl, paula-silva fw, faccioli lh, gatón-hernández pm, queiroz am, silva la. cytotoxicity and cytokine expression induced by silorane and methacrylate-based composite resins. j appl oral sci. 2016 jul-aug;24(4):338-43. doi: 10.1590/1678-775720150449. 11. małkiewicz k, wychowański p, olkowska-truchanowicz j, tykarska m, czerwiński m, wilczko m, et al. uncompleted polymerization and cytotoxicity of dental restorative materials as potential health risk factors. ann agric environ med. 2017 dec 23;24(4):618-23. doi: 10.5604/12321966.1235159. 12. johnsen gf, thieu mk, hussain b, pamuła e, reseland je, lyngstadaas sp, et al. own brand label restorative materials-a false bargain? j dent. 2017 jan;56:84-98. doi: 10.1016/j.jdent.2016.11.004. 13. gonçalves f, campos lmp, rodrigues-júnior ec, costa fv, marques pa, francci ce, et al. a comparative study of bulk-fill composites: degree of conversion, post-gel shrinkage and cytotoxicity. braz oral res. 2018 mar 8;32:e17. doi: 10.1590/1807-3107bor-2018.vol32.0017. 14. gontijo sml, gomes adm, gala-garcía a, sinisterra rd, esperanza cortés m. evaluation of antimicrobial activity and cell viability of aloe vera sponges. electron j biotechnol. 2013;16(1):1-10. doi: 10.2225/vol16-issue1-fulltext-2. 15. volk j, engelmann j, leyhausen g, geurtsen w. effects of three resin monomers on the cellular glutathione concentration of cultured human gingival fibroblasts. dent mater. 2006 jun;22(6):499-505. doi: 10.1016/j.dental.2005.06.002. 16. kong n, jiang t, zhou z, fu j. cytotoxicity of polymerized resin cements on human dental pulp cells in vitro. dent mater. 2009 nov;25(11):1371-5. doi: 10.1016/j.dental.2009.06.008. 17. magne p, malta da, enciso r, monteiro-junior s. heat treatment influences monomer conversion and bond strength of indirect composite resin restorations. j adhes dent. 2015 dec;17(6):559-66. doi: 10.3290/j.jad.a35258. 18. ferracane jl, condon jr. rate of elution of leachable components from composite. dent mater. 1990 oct;6(4):282-7. doi: 10.1016/s0109-5641(05)80012-0. 19. palin wm, fleming gj, nathwani h, burke fj, randall rc. in vitro cuspal deflection and microleakage of maxillary premolars restored with novel low-shrink dental composites. dent mater. 2005 apr;21(4):324-35. doi: 10.1016/j.dental.2004.05.005. 20. r ruyter ie, oysaed h. composites for use in posterior teeth: composition and conversion. j biomed mater res. 1987 jan;21(1):11-23. doi: 10.1002/jbm.820210107. 21. monte alto rv, guimarães jg, poskus lt, da silva em. depth of cure of dental composites submitted to different light-curing modes. j appl oral sci. 2006 apr;14(2):71-6. doi: 10.1590/s1678-77572006000200002. 22. balbinot edca, pereira mfcc, skupien ja, balbinot cea, da rocha g, vieira s. analysis of transmittance and degree of conversion of composite resins. microsc res tech. 2019 nov;82(11):1953-61. doi: 10.1002/jemt.23364. 23. lin gss, abdul ghani nrn, ismail nh, singbal kp, yusuff nmm. polymerization shrinkage and degree of conversion of new zirconia-reinforced rice husk nanohybrid composite. eur j dent. 2020 jul;14(3):448-55. doi: 10.1055/s-0040-1713951. 24. calheiros fc, braga rr, kawano y, ballester ry. relationship between contraction stress and degree of conversion in restorative composites. dent mater. 2004 dec;20(10):939-46. doi: 10.1016/j.dental.2004.03.003. 1http://dx.doi.org/10.20396/bjos.v19i0.8661109 volume 19 2020 e201109 letter to the editor 1 department of restorative dentistry, piracicaba dental school, university of campinas (unicamp), piracicaba, são paulo, brazil corresponding author: brenda p. f. a. gomes university of campinas (unicamp) av limeira, 901, piracicaba – sp, brazil, 13414-903 email: bpgomes@fop.unicamp.br received: september 01, 2020 accepted: october 04, 2020 teaching experiences during the sars-cov-2 pandemic in a brazilian school of dentistry brenda p. f. a. gomes1,* , rodrigo arruda-vasconcelos1 , lidiane m. louzada1 , marina a. marciano1 , adriana de-jesus-soares1 abstract: the coronavirus outbreak (2019) represents a public health emergency of global concern. several measures have been taken to minimise the risk of infection among the population, including social distancing, working from home, closure of non-essential activities since the detection of the first case in brazil. this study describes the teaching experiences during the sars-cov-2 pandemic in a brazilian school of dentistry. the state university of campinas (unicamp) was the first public university in brazil to stop all the classroom activities on 13th of march 2020 due to covid-19, followed by other universities. unicamp developed several initiatives and created a special support page for digital teaching, where it is possible to obtain guidance, support materials for teachers and a space for exchanging messages (e-mail and chat) for specific guidelines. unicamp has started lending computer equipment to undergraduate and graduate students according to socioeconomic criteria. along with the equipment, 500 chips have been delivered with 10 gb of internet so that these students are able to access the network and carry out the remote activities related to their courses. in conclusion, quality education is the key-element in forming high-quality professionals that will in a near future provide health care for the community, be part in international research groups and become lecturers. keywords: coronavirus. dentistry. infections. sars virus. https://orcid.org/0000-0002-8449-0646 https://orcid.org/0000-0002-0968-0212 https://orcid.org/0000-0002-3480-8433 https://orcid.org/0000-0001-6244-2531 https://orcid.org/0000-0002-8078-1606 2 gomes et al. the coronavirus outbreak (2019) represents a public health emergency of global concern1 due to its high transmission among the population and that 5% of the infected people will need intensive hospital care, which puts additional pressure on the lack of available infrastructure to treat the serious cases2. the outbreak officially started in december 2019 in wuhan, capital of the province of hubei, china, and it was characterised as a pandemic on 11th march 2020 by the world health organization (who) when 6,315 were affected (https://www.who.int/emergencies/diseases/novel-coronavirus-2019/events-as-they-happen). international centres for disease control and prevention are monitoring the covid-19 outbreak, which presents the main following symptoms: fever, dry cough, absence of nasal congestion, persistent headache, anorexia, dizziness, asthenia (lack of energy and strength), chills, myalgia, conjunctivitis, diarrhoea and nausea/vomiting in the early stages, and more distinctive symptoms including anosmia (loss of smell), ageusia (loss of taste), dyspnoea, (shortness of breath), chest pain, haemoptysis, vascular lesions in the later stages3-10. the mortality rate due to covid-19 is higher among people who suffer from chronic health problems (comorbidities), such as hypertension, diabetes or chronic kidney and obstructive pulmonary diseases5,11,12. the novel coronavirus continues to circulate with over 33 million affected worldwide (28th september 2020) (https://covid19.who.int/). however, brazil has become the new epicentre of the pandemic as it has accumulated over 4.7 million infected people (28th september 2020) with 14,318 new cases in the last 24 hours and a total of 141,741 deaths (335 in the last 24 hours) (https://covid.saude.gov.br/). since the detection of the first case of coronavirus in brazil, several measures have been taken to minimise the risk of infection among the population, including social distancing, working from home, closure of non-essential activities. however, delivery options have remained available. additionally, schools and universities have ceased their activities due to the high risk of infection with agglomeration. dentists are directly impacted by the new coronavirus as they work with the oral cavity that is irrigated by the saliva and receive the respiratory aerosols and droplets from the patient. the fact that sars-cov-2 remains and replicates in the upper respiratory tract (mouth and nose) for approximately a week before reaching the lungs, trachea and bronchi, and before the classical symptoms of the disease are apparent, makes the disease even worse for dentists, who may have close contact with patients in this phase during treatment. with regard to dental practice and biosafety in dental teaching, the association of dental schools in brazil (abeno) (http://www.abeno.org.br/arquivos/downloads/ retomada_de_praticas_seguras_no_ensino_odontologico.pdf) and the brazilian dental federal council (cfo) (http://website.cfo.org.br/cfo-reforca-necessidade-do-ministerio-da-educacao-suspender-autorizacoes-para-abertura-de-novos-cursos-de-odontologia/), based on the american dental association (ada) guidelines (https://www.ada. org/en/publications/ada-news/2020-archive/march/ada-developsguidance-on-dental-emergency-nonemergency-care), has advised dental practitioners to only attend dental emergencies and urgent care, with elective treatment not being recommended at the moment. http://www.abeno.org.br/arquivos/downloads/retomada_de_praticas_seguras_no_ensino_odontologico.pdf http://www.abeno.org.br/arquivos/downloads/retomada_de_praticas_seguras_no_ensino_odontologico.pdf 3 gomes et al. the state university of campinas (unicamp) was the first public university in brazil to stop all the classroom activities on 13th of march 2020 due to covid-19, followed by other universities. however, in são paulo state the quarantine period officially started only on the 24th march 2020. at the moment towns are opening/closing their commercial centres according to municipal decrees, but most of the schools/universities are closed until the beginning of september. several international web seminars have been held among the universities in brazil, south and central america and portugal to address several topics including the need for an emergency change to remote education addressing such topics as: i. how to improve the remote activities being offered; ii. how to deal with disciplines with practical activities, internships and laboratory experience; iii. the semi-annual evaluations; iv. and lessons to be given. the most important thing is the need to recognize the exceptionality of the moment, anticipating situations, and realising that we are all more vulnerable and need support. webinars should be attended by teaching staff, employees, lecturers, under and post graduate students from unicamp and other universities unicamp also developed several initiatives and created a special support page for digital teaching, where it is possible to obtain guidance, support materials for teachers and a space for exchanging messages (e-mail and chat) for specific guidelines. the material recorded and made accessible in the form of a webinar are also publicly available. importantly, unicamp has a policy of admitting students from low income families under a quota system, varying from 9.6 to 13.5% in the most competitive courses (i.e. medicine, biological sciences and computer sciences). therefore, it is natural to expect that these students present a lower potential to invest in educational tools such as a personal computer and have broad-band internet access. in this context, institutional support is crucial to support such heterogenicity. it was also necessary that the students have access to the necessary equipment to use the internet, as well as being trained to use virtual learning environments and to study in the virtual context. in addition to special mobile phone internet packages, available to all students, unicamp has also started lending computer equipment to undergraduate and graduate students according to socioeconomic criteria. along with the equipment, 500 chips have been delivered with 10 gb of internet so that these students are able to access the network and carry out the remote activities related to their courses. a covid-19 committee has been implemented in the school of dentistry – unicamp to discuss important measures for the return of undergraduate and postgraduate students to research and pre-clinical laboratories, and to the dental clinics, setting rules for biosafety and well-being. furthermore, protocols for surfaces and dental instruments disinfection, and the use of personal protective equipment (including how to 4 gomes et al. put it on and how to remove it) is being discussed. overall, these rules aim to provide a healthy environment for both professionals and patients from the community. unicamp has published a gradual return plan for the technical-administrative and academic activities, which should be followed under strict measures for control and prevention of covid-19. remote activities will be kept during the second semester of the current year. as for postgraduate courses, in most of the cases ongoing research is based on surveys and data/samples processing. for the dental school, clinical activities are still suspended. it is important to emphasise that the reflections presented in this study are valid because the covid-19 pandemic is still in progress; however, as the scenario changes novel measures/strategies may be considered according to the evolution of the disease. moreover, this work should be interpreted as a portrait of the current pandemic state in order to guide future generations in case of new outbreaks. this article reports the experience of a single group in a brazilian school of dentistry. therefore, evaluation of the local situation as well as the careful reading of any published guidelines should be considered before other institutions adopt guidelines regarding their activities/plans for a gradual return to full activity. above all, education during the pandemic with social isolation and all its complications, should focus on the students, not only on their professional training, which is in the essence of the university, but also to train them as responsible citizens and how to interact with the community. we are living a moment with a need for innovation in all areas. currently, in the field of dentistry, and especially in endodontics, several conferences are being held remotely which give students/professionals opportunities to participate and give presentations in events including the brazilian society of endodontics (sbendo) and brazilian division of the international association for dental research (sbpqo), as well as others. in this new format conferences, professionals across brazil will meet online removing the need to travel long distances. in conclusion, quality education is the key-element in forming high-quality professionals that will in a near future provide health care for the community, be part in international research groups and become lecturers. acknowledgements the authors would like to thank david henry moon for english proofreading and technical support. this work was supported by the brazilian agencies são paulo research foundation (fapesp, grant no. 2015/23479-5, 2017/25242-8, 2019/19300-0), national scientific and technological development council (cnpq 308162/2014-5, 303852/2019-4); coordination for improvement of higher education personnel (capes finance code 001) and university of campinas research, teaching and extension support fund (faepex 2036/17). they also thank the pro-rectory of undergraduate studies and the space to support teaching and learning at unicamp (ea2) for organizing several webinars to discuss issues related to the pandemic. 5 gomes et al. conflict of interest the authors declare that they have no conflict of interest. references 1. the lancet. emerging understandings of 2019-ncov. lancet. 2020 feb 1;395(10221):311. doi: 10.1016/s0140-6736(20)30186-0. 2. sood s, aggarwal v, aggarwal d, upadhyay sk, sak k, tuli hs, et al. covid-19 pandemic: from molecular biology, pathogenesis, detection, and treatment to global societal impact. curr pharmacol rep. 2020 jul 27:1-16. doi: 10.1007/s40495-020-00229-2. 3. ather a, patel b, ruparel nb, diogenes a, hargreaves km. coronavirus disease 19 (covid-19): implications for clinical dental care. j endod. 2020 may;46(5):584-95. doi: 10.1016/j. joen.2020.03.008. 4. carlos wg, dela cruz cs, cao b, pasnick s, jamil s. novel wuhan (2019-ncov) coronavirus. am j respir crit care med. 2020 feb 15;201(4):p7-p8. doi: 10.1164/rccm.2014p7. 5. huang c, wang y, li x, ren l, zhao j, hu y, et al. clinical features of patients infected with 2019 novel coronavirus in wuhan, china. lancet. 2020 feb 15;395(10223):497-506. doi: 10.1016/s0140-6736(20)30183-5. erratum in: lancet. 2020 jan 30. 6. lee y, min p, lee s, kim sw. prevalence and duration of acute loss of smell or taste in covid-19 patients. j korean med sci. 2020 may 11;35(18):e174. doi: 10.3346/jkms.2020.35.e174. 7. ren ll, wang ym, wu zq, xiang zc, guo l, xu t, et al. identification of a novel coronavirus causing severe pneumonia in human: a descriptive study. chin med j (engl). 2020 may 5;133(9):1015-24. doi: 10.1097/cm9.0000000000000722. 8. rothan ha, byrareddy sn. the epidemiology and pathogenesis of coronavirus disease (covid-19) outbreak. j autoimmun. 2020 may;109:102433. doi: 10.1016/j.jaut.2020.102433. 9. wang w, tang j, wei f. updated understanding of the outbreak of 2019 novel coronavirus (2019-ncov) in wuhan, china. j med virol. 2020 apr;92(4):441-7. doi: 10.1002/jmv.25689. 10. zhu n, zhang d, wang w, li x, yang b, song j, et al. a novel coronavirus from patients with pneumonia in china, 2019. n engl j med. 2020 feb 20;382(8):727-33. doi: 10.1056/nejmoa2001017. 11. chen n, zhou m, dong x, qu j, gong f, han y, et al. epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study. lancet. 2020 feb 15;395(10223):507-13. doi: 10.1016/s0140-6736(20)30211-7. 12. wang d, hu b, hu c, zhu f, liu x, zhang j, et al. clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china. jama. 2020 mar 17;323(11):1061-9. doi: 10.1001/jama.2020.1585. 404 not found 1http://dx.doi.org/10.20396/bjos.v19i0.8658591 volume 19 2020 e208591 original article 1 graduate program in dentistry, federal university of pelotas (ufpel), pelotas, rs, brazil. 2 graduate program in dentistry, catolic university of pelotas (ucpel), pelotas, rs, brazil. corresponding author: catarina borges da fonseca cumerlato, dds, msc; graduate program in dentistry, federal university of pelotas rua gonçalves chaves 457, 96015-560, pelotas-rs, brazil tel./fax: +55-53-3222.6690 e-mail: catarinacumerlato@hotmail.com received: march 04, 2020 accepted: july 11, 2020 #dentalpain: what do the brazilian instagram® users want to mean? catarina borges da fonseca cumerlato1,* , rodrigo nunes rotta2 , luísa jardim corrêa de oliveira2 , marcos britto corrêa1 aim: the aim of this study was to evaluate what brazilian instagram® users manifest when they use #dordedente (#dentalpain in portuguese). methods: the content of 1,000 publications related to dental pain was evaluated. through print screens posts were collected in two periods (2015 and 2020) and organized into files. variables related to characteristics of users, publication’s type, cause of pain, impact on daily life and actions take to relieve pain were collected. data analysis was realized through the qualitative methodology of conventional content analysis and a descriptive analysis (95% ci) was performed. results: in the first assessment period 76% of the posts referred to self-declaration of pain. regarding to the users’ characteristics, 54% were female, 20.4% male, and the remaining 25.6% were clinic/ company profiles, or it was not possible to identify the user’s gender. apparently, 34.4% of users were adults, 18.6% were adolescents and only 1.2% were children. on the other hand, in the second evaluation the vast majority (99.6%) were clinic/company profiles. in addition, 90.2% were informative posts and only 3 posts (0.6%) were related to self-declaration of pain. conclusions: the social network instagram® serves as an efficient means of communication for informative and advertising purposes being an interesting alternative for the dissemination of health information, and as an instrument of health surveillance. keywords: dentistry. pain. quality of life. social media. toothache. https://orcid.org/0000-0001-5943-6958 https://orcid.org/0000-0003-0401-1543 https://orcid.org/0000-0001-7738-4338 https://orcid.org/0000-0002-1797-3541 2 cumerlato et al. introduction social networks are social structures that can be formed by people, companies, or organizations, connected to each other, in order to exchange common values, experiences and information through horizontal and non-hierarchical relationships among the participants. in brazil, according to the we are social study, there were 139.1 million internet users in 2017 (66% of the brazilian population), of which 122 million have profiles in social networks1. a recent research realized in 2019 has shown that the number of internet users in brazil has grown to 149.1 million and the number of active social media users has also grown to 140 million, demonstrating a exponential increase of social media users in the last few years2. according to literature the most common acute manifestation of orofacial pain is odontalgia or toothache, a stressful and intensely painful experience, often leading to absences from school or work, affecting quality of life, being considered as an important public health problem3,4. a research performed in twitter® demonstrated that social networks are used by users to communicate experiences of dental pain, including actions taken to relieve pain and impact on daily life5, suggesting that social networks can be an interesting instrument for behaviors analysis and dissemination of health information. instagram® is a social media that allows users to share photos and videos online to their followers, and at the same time enables the use of hashtags. this social media was created in october 2010 and since april 2012 is part of facebook®6. a recent research has shown that facebook® monthly active users is declining in some demographics, in contrast, instagram® users who visit the platform daily are growing rapidly since 20167. this mobile social network had reached 1 billion monthly active users in 2020, up from 800 million in january of 20188,9. as a relatively new social network, no study analyzed aspects related to toothache manifested by it users. in this sense, the aim of this study was to evaluate what brazilian instagram® users manifest when they use #dordedente (#dentalpain in portuguese). material and methods we evaluated the content of a sample of 1,000 publications related to dental pain utilizing publically available data from instagram®. in order to carry out the search and reduce the “algorithmic bias” which is nothing more than a term used to describe how we may be influenced by recommendations based on our personal data on the internet10, we created a profile on the network totally empty and without any connection with another user. research was carried out in two assessment periods. the first was realized on july 3, 2015 at 6:00 pm, where were collected the 500 most recent posts containing “#dordedente”, which means dental pain in portuguese, through print screens for further review of their content. the second assessment also collected the 500 most recent publications containing “#dordedente” and it was performed on june 23, 2020 at 6:00 pm. the images were recorded and organized into ten files, each file containing 100 posts, in order to facilitate their analysis. 3 cumerlato et al. the first part of our data analysis was performed through the qualitative methodology of conventional content analysis, which is one of the methods used to analyze text data and that is used generally with a study design who aims to describe and interpret a phenomenon as emotional reactions. in this method, the study starts with an observation of the content that we have collected and coding categories are defined during the data analysis11,12. as the social network instagram® does not provide the characteristics of users, information such as sex and age were recorded according to the appearance of people in photographs (when possible). the variable age was categorized as adult, adolescent, child, and others (company or clinic profiles, or when it was not possible to identify). and the variable sex was categorized as male, female and others (company or clinic profiles, or when it was not possible to identify). cause of pain, impact on daily life, and action taken to relieve pain were considered when they were explicitly declared by the user through the photo or subtitle. the variable pain´s cause was categorized as third molar, orthodontic appliance, caries, trauma, infection, and dental eruption. impact on daily life was categorized as impacting feeding, sleep, leisure, study, and family. and if the user has taken an action to relieve pain, this action was categorized as going to the dentist, taking medications, and resting. feelings about pain were interpreted through images, statements and emojis, which are specific figures of characters used in social networks. these feelings was categorized as sadness, resignation, faith, irritation, irony, and happiness. the data of interest were collected and organized into a database created in software excel, so that the posts could be classified into categories according to their content through the conventional content analysis method. in addition, a descriptive analysis was performed to verify the relative and absolute frequencies of the information and 95% confidence intervals using the software stata 12.0. results until the date of the first search, there was 6,101 publications related to the term #dordedente on instagram®. in the second assessment, which was realized in 2020, the number of posts sharply increased to 40,500 (figure 1). figure 1. flowchart of the publications included. 2015 assessment publications on the instagram related to #dentalpain (n = 6,101) posts evaluated (n = 500) 2020 assessment publications on the instagram related to #dentalpain (n = 40,500) posts evaluated (n = 500) 4 cumerlato et al. from the 500 analyzed posts in 2015, 76% referred to self-declaration of pain, where the user usually published a photo of him/her in which one could notice through the physiognomy of the person feelings. in addition, the text that followed just below the picture describing the photo often made explicit the annoyance that the dental pain causes and how much it affects the daily routine of people. figure 2 illustrates changing of type of posts on five-year period. in 2015, more than 80% of posts were classified as personal, while in 2020 90.2% of posts were informative. table 1 presents the descriptive analysis of the variables sex, age, self-declaration of pain, and declaration of pain’s cause, collected from the publications. in the first assessment, 54% were female, 20.4% male, and the remaining 25.6% were clinic/ company profiles, or it was not possible to identify the user’s gender. apparently, 34.4% of users were adults, 18.6% were adolescents and only 1.2% were children. on the other hand, in the second evaluation the vast majority (99.6%) were clinic/company profiles. in addition, from the personal profiles we could identify that two of them were posted by adult women (0.4%). in 2015, only 70 users have reported the cause of their pain, being the most frequent reason related to third molars (67%), followed by the use of orthodontic appliances (20%). the eruption of third molars was perceived through the pictures and subtitles that contained hashtags like “#wisdomtooth #thirdmolar #wisdom #wisdomteeth”. about orthodontic appliances some users made explicit the pain through phrases like “the thing was so tense that the mouth guard tore with the orthodontic appliance!”, and “enough of hiding the smile and orthodontic appliance, toothache!”. figure 2. type of posts containing #dordedente on instagram® by year of analysis. 17.2 82.8 2015 90.2 9.8 2020 informative personal 100 90 80 70 60 50 40 30 20 10 0 5 cumerlato et al. table 1. descriptive analysis of the variables sex, age, self-declaration of pain, and declaration of pain’s cause, collected from the 1,000 posts evaluated that contained the term #dordedente in the social network instagram® by year of analysis. variable/category 2015 2020 n % 95% ci n % 95% ci sex (1,000) male 102 20.4 17.0-24.2 0 0 0.0-0.1 female 270 54.0 49.5-58.4 2 0.4 0.2-0.6 others 128 25.6 21.8-29.7 498 99.6 98.6-99.9 age (1,000) child 6 1.2 0.4-2.6 0 0 0.0-0.1 adolescent 93 18.6 15.3-22.3 0 0 0.0-0.1 adult 172 34.4 30.2-38.7 2 0.4 0.2-0.6 others 229 45.8 41.4-50.3 498 99.6 98.6-99.9 self-declaration of pain (1,000) yes 380 76.0 72.0-79.7 3 0.6 0.4-0.8 no 120 24.0 20.3-28.0 497 99.4 98.3-99.9 declaration of pain’s cause (1,000) yes 70 14.0 11.1-17.4 0 0 0.0-0.1 no 430 86.0 82.6-88.9 500 100.0 99.9-100 pain’s cause (70) third molar 47 67.0 54.9-77.9 0 0 0.0-0.1 orthodontic appliance 14 20.0 11.4-31.3 0 0 0.0-0.1 caries 1 1.5 0.03-7.7 0 0 0.0-0.1 trauma 1 1.5 0.03-7.7 0 0 0.0-0.1 infection 3 4.3 0.9-12.0 0 0 0.0-0.1 dental eruption 4 5.7 1.6-14.0 0 0 0.0-0.1 table 2 presents the descriptive analysis of the variables related to impact on daily life, action taken to relieve pain, and feelings about pain. from the total analyzed posts, 8.7% of the users manifested some impact of dental pain on their daily routine, where feeding (46%), sleep (26.4%) and leisure (19.5%) were the predominant factors. the impact on feeding was manifested mainly through images of special foods like soups and creams, and was perceived also through sentences like “i cannot chew anything… so soup of potato and carrot made by mom!”. there were also sentences that showed the impact on sleep and leisure time, for example:” from the series i hate my wisdom tooth, i want to sleep and i cannot!”, and “starting the holiday at home with style!”. from the total of 1,000 publications, 10.3% of users reported having taken some measure to get around the pain such as going to the dentist (48%), resting (42%) and taking medications (38%). we identified these actions mainly by images of medicines, dental clinics and people resting, and through sentences like “let’s go to the dentist!”, “at home lying down… toothache.”, and “about my friday, medicine and more medicine!”. 6 cumerlato et al. in almost one third of the publications (26.1%) it was possible to observe the user’s feelings regarding toothache. the feeling of sadness was manifested in 46.7% of the posts, through photos of the users with a sad face, crying and no smiles and due to the use of crying emoticons in the subtitle. about the feeling of resignation, where the user was resigned to the pain appeared in 23.4% of the posts, by means of phrases like “hey pain, i do not listen to you anymore.”, “a toast to the pains!”, and “and today was like this ... weekend at home ... all that remains is to enjoy some music !!”. and 15.3% of people expressed a feeling of faith and hope in front of toothache, which was perceived by subtitle as “even if i have no strength left, i will still praise you.”, “thank you jesus for everything.”, and “luck was cast, the cards are on the table, focus, strength, faith and a single certainty!”. table 2. descriptive analysis of the variables related to impact on daily life, action taken to relieve pain, and feelings about pain, collected from the 1,000 publications containing the term #dordedente in the social network instagram®. variable/category (n) n % 95% ci impact on daily life (1,000) yes 87 8.7 7.0-10.6 no 913 91.3 89.4-93.0 impact type (87) feeding 40 46.0 35.2-57.0 sleep 23 26.4 17.6-36.7 leisure 17 19.5 11.8-29.4 study 3 3.4 0.7-9.7 family 4 4.6 1.3-11.4 action taken to relieve pain (1,000) yes 103 10.3 8.5-12.4 no 897 89.7 87.6-81.5 type of action taken (128) going to the dentist 48 46.7 36.7-56.7 taking medications 38 36.9 27.6-47.0 resting 42 40.8 31.2-50.9 feelings about pain (1,000) yes 261 26.1 23.4-28.9 no 739 73.9 71.1-76.6 type of manifested fellings (261) sadness 122 46.7 40.6-53.0 resignation 61 23.4 18.4-29.0 faith 40 15.3 11.2-20.3 irritation 22 8.4 5.4-12.5 irony 10 3.8 1.9-6.9 happiness 6 2.3 0.8-4.9 7 cumerlato et al. discussion the present study was the first to analyze contents related to #dentalpain (#dordente) in the social network instagram®. our study found that brazilian users of instagram® utilize this social media to express feelings/experiences of toothache, including the cause of pain, the impact of this pain on daily life and measures taken to get around the dental pain. some findings about our research are similar to the results of a study realized in 2009, that found through telephone interviews that the most frequently reported impacts from dental pain on daily life of the participants are difficulty to eating, worry and disruptions in sleep3. another similarity with this study is related with the measures taken to relieve pain by users. both studies found that the users reported have used a medicine and/or have gone to the dentist to deal with pain. the association of toothache and its impacts in many aspects of normal functioning and daily living – eating, drinking, sleeping, talking and socializing – is well documented on literature, as well as the use of nonprescription medicine and home remedies for toothache pain relief13,14. these similarities demonstrate that the findings of a survey on instagram® are corresponding with results of studies realized by traditional methods of search, suggesting that this social media could be used for surveillance proposes, with special interest for public health. besides that, instagram® serves as an efficient means of communication between the user and his followers, with informative and / or advertising purposes. our results demonstrated that users also utilize the instagram® social media in order to inform their followers about dentistry content with the purpose of advertising and publicity. one interesting result that should be highlighted and discussed is the drastic change in pattern of publications from 2015 to 2020. in 2015, 82.8% of posts were personal and mostly about declaration of pain. while in 2020 more than 90% of publications were informative for advertising purposes publicated by clinic/company profiles. in the recent digital transformation, social media has offered a social communication space with people in timely and cost-efficient way, resulting in an efficient manner to establish relationships with patients and do marketing15. in this sense, instagram® plays an important role in dentistry and it has been extremely used by dentists with advertising purposes, which explains this great change in pattern of publications over time, where we have a lot more publications related to information and marketing than personal manifestations. another aspect that could direct influenced the change over time is the big number of dentists in brazil, with almost 350,000 professionals in 202016. this high number leads to a very competitive market, where professionals put their best efforts to attract patients for the private practice. another research that has results corroborating with ours, was published in 2011 and evaluated the content of tweets relating to dental pain from twitter5. they found that the majority of tweets analyzed were statements suggesting that the users were experiencing a toothache and the vast majority of these statements connoted some negative or even catastrophic association with the experience. an example of a tweet published by a user that express a negative and suffer experience is: “ugh!! my toothache is killing me!! pain go away!!”5. as well as in our study, where in 8 cumerlato et al. more than half of the posts it was possible to observe the user’s feelings regarding toothache. the feeling of sadness was the most manifested in analyzed posts and resignation’s feeling was the second, reinforcing the relation of the experience of dental pain and negative impacts in the user’s quality of life expressed in different social networks. other important and similar result to our study was the most frequent actions taken to relieve pain reported by the users of twitter, that were going to the dentist, taking medications including analgesics and antibiotics, and seeking advice from the twitter community5. these results suggest that social networks are not only efficient means of communication but also a good method to evaluate the activities that are being impacted and the measures that are being taken by the people in front of some situation, reinforcing the idea that they could be used as an instrument of health surveillance. in this same network, another study conducted in 2014, through the qualitative analysis of posts related to orthodontics, demonstrated that online social networks can become a powerful complement to traditional sources of health information, and in addition, the study has shown that the users of the research use the twitter® social network for the purpose of expression of feelings17. given these findings, we can see that social networks can be beneficial and useful for both professionals and patients. the social network instagram® has several advantages. first, is the abundance of real-time data. the use of real-time updates averts retrospective reports and creates greater accuracy and sensitivity in the measurement of behavioral responses to pain. another advantage of instagram® is that the users can access the network on smartphones or tablets, increasing the frequency of use and posting. moreover, in the case of professionals of health, instagram® could be useful to disseminate health information, do marketing, and guide their followers how to face some kind of difficulty in the face of dental pain or any other health problem. health surveillance actions which have as principle to observe and analyze the health situation of the population articulating themselves in a set of measures aimed to control determinants, risks and damages to health18, could also be put into practice in social networks, where one could analyze in part the health situation and behaviors of the population and thus, to promote virtual actions aiming, besides controlling determinants, to disseminate health information. dental caries is still a major health public problem in most countries, in which 60–90% of children and the vast majority of adults are affected19, being also considered one of the main causes of dental pain20,21. however, in our study the main responsible for pain were the third molar and the use of orthodontic appliances. dental caries was one of the causative factors that appeared less frequently in the posts. this divergence of results can be explained by the individual socioeconomic characteristics of the users. the social determinants experienced throughout life are strongly associated with the occurrence of caries disease22,23. however, people who have access to smartphones and computers are people with greater purchasing power, having more access to information and knowledge, and consequently having a lower risk factors for the development of caries disease. on the other hand, the 9 cumerlato et al. use of orthodontic appliances and third molar extraction involves the person having a higher purchasing power to access these treatments, and it may be for these reasons that these two factors appeared more frequently as causes of dental pain in our study. another possible explanation for these findings could be related to the impressions that users would like to manifest to their followers. social networking has become an increasingly efficient mean for people to exhibit themselves, mainly through the selfies, where the users tend to expose their “media identities” exalting perfect images of themselves and avoiding sharing those more modest24. in this way, pain related to third molar and orthodontic appliances could be interpreted as more socially accepted events, because are not associated with disease or lack of self-care and can be considered as unavoidable. on the other hand, causes of caries are well known by population, referring to absence of hygiene and diet rich in sugars, which denotes bad impressions of individuals. however, our study has some important limitations that have to be discussed. first, our research was carried out limiting the language to search only portuguese posts affecting the representativeness of the study population and limiting the extrapolation of the results. on the other hand, brazil is one of the countries in the world with the largest number of social media users and this must be taken into account2. besides that, posts without the hashtag were not evaluated, so the manifestations about dental pain on instagram® may have been underestimated. in relation to the use of social networks as an instrument of health surveillance is interesting, but we have to consider that not all users who feel pain will manifest it in the networks. this is also important in terms of interpretation and extrapolation of data. the use of social media and websurveys is dramatically growing in the recent time, being an accessible alternative to do research during pandemic of covid-1925. due to these limitations, is necessary that future studies be performed to determine the validity of instagram® of a data source. in this study, we have shown that instagram® users share online their experiences of toothache, involving the cause´s pain, feelings about the pain, and measures taken to relieve pain. in this way, the social network instagram® serves as an efficient means of communication between the user and his followers. in addition, this social network is also used by dentists for informative and advertising purposes being an interesting alternative for the dissemination of health information, and as an instrument of health surveillance. acknowledgements this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior brasil (capes) finance code 001. references 1. we are social ltd. new york: we are social; 2017 [cited 2018 may 9]. available from: https://wearesocial.com/special-reports/digital-in-2017-global-overview. 10 cumerlato et al. 2. we are social ltd. digital 2019. globo digital yearbook: essential digital data for every country in the world. new york: we are social; 2019 [cited 2020 june 26]. available from: https://datareportal.com/ reports/digital-2019-global-digital-yearbook?rq=global%20digital%20yearbook. 3. cohen la, bonito aj, akin dr, manski rj, macek md, edwards rr, et al. toothache pain: behavioral impact and self-care strategies. spec care dentist. 2009 mar-apr;29(2):85-95. doi: 10.1111/j.1754-4505.2008.00068.x. 4. pau a, croucher re, marcenes w. demographic and socio-economic correlates of dental pain among adults in the united kingdom, 1998. br dent j. 2007 may 12;202(9):e21; discussion 548-9. doi: 10.1038/bdj.2007.171. 5. heaivilin n, gerbert b, page je, gibbs jl. public health surveillance of dental pain via twitter. j dent res. 2011 sep;90(9):1047-51. doi: 10.1177/0022034511415273. 6. instagram press page. new york: instagram press page; 2015 [cited 2018 may 9]. available from: <https://instagram-press.com/our-story/>. 7. smith a, anderson m. social media use in 2018. washington: pew research center; 2018 [cited 2020 jul 5]. available from: https://www.pewresearch.org/internet/wp-content/uploads/sites/9/2018/02/ pi_2018.03.01_social-media_final.pdf 8. the statistics portal. most popular social networks worldwide as of july 2020, ranked by number of active users (in millions). hamburg: statitista; 2018 [cited 2018 apr 12]. available from: https://www.statista.com/statistics/272014/global-social-networks-ranked-bynumber-of-users. 9. instagram press page. a quick walk through our history as a company. new york: instagram; 2020 [cited 2020 june 26]. available from: https://instagram-press.com/our-story/2 10. bozdag e. bias in algorithmic filtering and personalization. ethics inf technol. 2013 jun;15:209-27. doi: 10.1007/s10676-013-9321-6. 11. hsieh hf, shannon se. three approaches to qualitative content analysis. qual health res. 2005 nov;15(9):1277-88. doi: 10.1177/1049732305276687. 12. kondracki nl, wellman ns, amundson dr. content analysis: review of methods and their applications in nutrition education. j nutr educ behav. 2002;34(4):224-30. doi: 10.1016/s1499-4046(06)60097-3. 13. riley jl, gilbert gh, heft mw. oralfacial pain: racial and sex differences among older adults. j public health dent. 2002;62(3):132-9. doi: 10.1111/j.1752-7325.2002.tb03434.x. 14. stoller ep, gilbert gh, pyle ma, duncan rp. coping with tooth pain: a qualitative study of lay management strategies and professional consultation. spec care dentist. 2001;21(6):208-15. doi: 10.1111/j.1754-4505.2001.tb00256.x. 15. keller b, lambrique a, jain km, pekosz a, levine o. mind the gap: social media engagement by public health researchers. j med internet res. 2014 jan;16(1):e8. doi: 10.2196/jmir.2982. 16. federal council of dentistry. brazil. [registered professionals and entities]. 2020; [cited 2020 june 3]. available from: http://website.cfo.org.br/profissionais-cadastrados. portuguese. 17. henzell mr, knight am, morgaine kc, antoun js, farella m. a qualitative-analysis of orthodonticrelated posts on twitter. angle orthod. 2014 mar;84(2):203-7. doi: 10.2319/051013-355.1. 18. ministry of health; brazil. [health surveillance]. brasília: ministry of health; 2007. portuguese. 19. petersen pe, bourgeois d, ogawa h, estupinan-day s, ndiaye c. the global burden of oral diseases and risks to oral health. bull world health organ. 2005 sep;83(9):661-9. 20. pitts nb, zero dt, marsh pd, ekstrand k, weintraub ja, ramos-gomez f, et al. dental caries. nat rev dis primers. 2017 may;3:17030. doi: 10.1038/nrdp.2017.30. https://instagram-press.com/our-story/ https://www.statista.com/statistics/272014/global-social-networks-ranked-by-number-of-users/ https://www.statista.com/statistics/272014/global-social-networks-ranked-by-number-of-users/ https://instagram-press.com/our-story/2/ 11 cumerlato et al. 21. boeira gf, correa mb, peres kg, peres ma, santos is, matijasevich a, et al. caries is the main cause for dental pain in childhood: findings from a birth cohort. caries res. 2012;46(5):488-95. doi: 10.1159/000339491. 22. thomson wm, poulton r, milne bj, caspi a, broughton jr, ayers km. socioeconomic inequalities in oral health in childhood and adulthood in a birth cohort. community dent oral epidemiol. 2004 oct;32(5):345-53. doi: 10.1111/j.1600-0528.2004.00173.x. 23. peres ma, peres kg, barros ajd, victora cg. the relation between family socioeconomic trajectories from childhood to adolescence and dental caries and associated oral behaviours. j epidemiol community health. 2007 feb;61(2):141-5. doi: 10.1136/jech.2005.044818. 24. jerônimo sobrinho p. [my selfie: body representation in the social network facebook.] artefactum rev est ling tecnol. 2014;8(1):1-13. portuguese. 25. de boni rb. web surveys in days of covid-19. cad saúde pública. 2020;36(7):e00155820. doi: 10.1590/0102-311x00155820. untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8652647 volume 17 2018 e18063 original article 1 dds, university of campinas, piracicaba dental school, department of community dentistry, piracicaba, sp, brazil. 2 dds, msc student, university of campinas, piracicaba dental school, department of community dentistry, piracicaba, sp, brazil. 3 dds, msc, phd, professor, university of campinas, piracicaba dental school, department of community dentistry, piracicaba, sp, brazil. 4 dds, msc, phd, full professor, university of campinas, piracicaba dental school, department of community dentistry, piracicaba, sp, brazil. corresponding author: maria da luz rosário de sousa piracicaba dental school, university of campinas. limeira avenue, 901, areião. zip code 13414-018. piracicaba-sp, brazil. e-mail: luzsousa@fop.unicamp.br received: october 26, 2017 accepted: april 23, 2018 effectiveness of a preventive oral health program in preschool children caroline zeeberg1, sthefanie del carmen perez puello2, marília jesus batista3, maria da luz rosário de sousa4 aim: to evaluate the effectiveness of an educational preventive program in oral health on preschoolers. methods: the final sample was 71 children in the test group and 48 in the control group. intraoral exams were conducted for caries experience (dmf-s), white spot lesions (wsl) diagnosis, dental biofilm and treatment needs (before and after interventionthe interval was 18 months). caregivers answered a questionnaire about socioeconomic data and health behavior. the educational preventive program consisted of supervised brushing, education in oral health, fluoride application and lectures to caregivers. mann whitney and wilcoxon tests (p <0.05) were used to compare data between groups. results: mean caries experience was 0.94 (± 3.42) and 0.94 (± 2.87) in test and control groups, respectively. baseline mean for dental biofilm was 4.95, and final mean was 0.21 in test group (p = 0.047). conversely, the same variables were 4.11 and 0.84 in the control group (p = 0.047). the program was evaluated as very good (54.9% of caregivers), improvement of brushing was related by 62%, and more children went to the dentist (p <0.01). conclusion: the educational preventive program seems to be effective for dental biofilm reduction, improved brushing and dental visits, being an important strategy for oral health maintenance in children. keywords: health promotion, oral health, preschool, child health, epidemiology. 2 zeeberg et al. introduction behavior factors are acquired in early childhood and there is a strong mother or caregivers influence according to their examples related to eating and oral hygiene habits that may have a positive or negative influence on child primary dentition1. considering the fact that habits are acquired at this age and that caries experience in primary dentition is a predictor of this disease in permanent teeth, it is important to emphasize in maintenance of preschoolers oral health to constitute healthy habits and improve the quality of life2. dental caries is a multifactorial disease, including aspects that go beyond to those that determine hard dental tissue demineralization. for example, modifying factors such as income, education, behavior factors, knowledge, schooling, attitudes indirectly influence or not the individual to have a higher risk to develop the disease3.in addition, health education is considered an important strategy for health promotion, not only by the impact and voluntary positive changes in the individual’s lifestyle and health habits; also, it improves familiar and community habits, generating political actions that allow the development of new strategies to promote health and improve the quality of life of the population4. in this way, the development of promotion and prevention strategies promotes the acquisition of knowledge in general and oral health, however this will not always generate good habits and behaviors considering the factors involved in the change of health habits. therefore, the implementation of education and prevention strategies in oral health are useful tools for the empowerment of population groups, knowledge acquisition and identification of the active role of the individual in the protection and conservation of their oral health4. for the development of educational and preventive strategies in preschools, educational institutions are important spaces that allow their implementation and greater interaction with the community. for example, children from an early age come in contact with information and models beyond the family due to they are included in the primary socialization process. therefore, preschools will be an ideal space to interact with children and their parents, trying to improve and transfer all health information as well as to include the practice of healthy habits by interventions5. from the evidence-based dentistry approach, the implementation and effectiveness of educational preventive programs (epp) have become relevant due to the interception of risk factors for oral diseases, knowledge acquisition and consequently behavioral changes. in addition, epp have been of great help to improve oral health in different population groups, provide important information for the design of preventive public policies and community interventions and monitor the incidence and prevalence of oral diseases in the population4. the verification of caries experience has become more important in epidemiological surveys, since its early diagnosis and the evaluation of disease predictor factors help in the caries risk assessment. this turns the treatment, simpler, less invasive and lower cost, involving preventive strategies such as fluoride use and behavioral changes regarding to diet and oral hygiene6,7. 3 zeeberg et al. caries study in children population is extremely relevant, since it is the most common chronic disease in this age group8,9. in addition, some studies have shown that when healthy behaviours and oral hygiene habits are taught to children from an early age, they can keep them until being adults10,11. in this way, the aim of the present study was to evaluate the effectiveness of a educational preventive program in preschool children. material and methods the methodology implemented in this study was intervention in children and their parents in the city of piracicaba, são paulo, brazil, from 2013 to 2015. it was evaluated and approved by the research ethics committee of the school of dentistry from university of campinas, according to resolution 196/96 of 10/10/1996 of the national health council, ministry of health from brazil. the data collection was carried out in the following preschools “antônio boldrin municipal school” (test group) and “profa bernadete de fátima oliveira municipal school” (control group), where studied children who had the same sociodemographic. the test group consisted of 233 children aged between 3 months and 6 years from the preschool “antônio boldrin municipal school”. on the other hand, sample from the control group was constituted by 201 children who had the same age group of “profa bernadete de fátima oliveira municipal school”. children´s parents or caregivers were approached and clarified about the research during parents meeting of the preschool, where they received information by researchers about research development. the preschool children were examined before and after the implementation of the educational preventive program, evaluating the following clinical conditions: caries experience measured by decayed, missing and filled teeth (dmf-t) and dmf-t indexes according to world health organization criteria8,12. the international caries detection and assessment system (icdas ii)13 was used to evaluate the presence of white spot lesions (wsl), also dental biofilm and treatment need were evaluated. oral examinations were performed in a light place, using a flat mirror, prolonged air drying and special cpi periodontal probe (dental plaque removal) by a postgraduate dentist who had been previously calibrated. in children from the control group were conducted only initial and final clinical examinations (after 18 months). children who required dental treatment were referred for care in the family health unit (fhu). a flow chart of enrollment and attrition of participants during the 18 months of the study is present in the figure 1. after participation study approval, a questionnaire was applied to parents or caregivers of the children to obtain data related to 1) sociodemographic and information about the family environment 2) socioeconomic 3) access to the general health service and oral health 4) feeding and deleterious habits of the child 4) oral hygiene habits and caregivers performance. from the clinical exams, caries activity risk was determined and the epp in oral health was implemented, initially in children from test group, by monthly visits for 12 months (12 visits) where supervised brushing was done. in 7 from these visits, playful materials such as macromodels, books, puppets, poster, wheel play and storyteller were presented before supervised brushing. later, play activities and lectures were addressed 4 zeeberg et al. to parents or caregivers. fluoride varnish (5% naf, duraphat®, colgate) application was done every 6 and 3 months for children who presented medium or high caries risk, respectively14. in addition, a lecture regarding oral health condition and prevention of caries disease and gingivitis was presented during the bimonthly parents meeting. it was discussed about oral health conditions, dental caries, disease stages, gingivitis, and consequences of harmful habits, prevention methods and techniques in different age groups. the activity was developed to improve the knowledge of parents or caregivers regarding oral health to generate changes related to oral hygiene habits in their children. at the end of the preventive educational program in oral health, parents or caregivers from test group were invited to answer a second questionnaire about oral health habits and opinions about the preventive program categorized as follows: 1) oral hygiene habits child independence during tooth brushing, routine supervision, parent or caregivers position, tooth brushing frequency, amount of toothpaste, its ingestion and orientations about oral hygiene on primary dentition. 2) opinions about the preventive program evaluation of child oral health, improvements of home brushing routine, last visit to the dentist, prevention program evaluation, importance of the program for child oral health. the data were tabulated and analyzed in the statistical package for the social sciences (spss), version 20.0 and in microsoft office excel 2016® (microsoft corporation, redmond, washintong, usa) by means of descriptive analysis, obtaining data distribution in percentage, mean, median and standard deviation. the statistical tests used were: chi-square to verify if the groups were equal to each other; student’s t-test to detect differences in mean caries experience and plaque index between both groups. paired t-test was used to determine intergroup differences related to dmf-t, dmf-t and icdas scores between the beginning and the end of the program. in addition, mann-whitney test was used to compare the data between the groups and mcneen ro llm en t a llo ca tio n in te rv en tio n a na ly si s total eligibible n=202 • move to school, n=15 • leaving school for unknown reasons, n=1 • move to school, n=38 • leaving school for unknown reasons, n=29 control n=64 intervention n=138 18 months without interventions n=48 18 months with interventions n=71 after 18 months final exam, n=48 after 18 months final exam, n=71 fig 1. flow chart of enrollment and attrition of participants during the 18 months of the study 5 zeeberg et al. mar’s test to compare data related to health habits before and after the epp. finally, a regression analysis was performed to verify the association of risk factors with caries experience. for all statistical tests, the level of significance was set at 5%. results after 18 months of epp, the final sample was 71 children in the test group and 48 in the control group. in the test group, 52.1% were female, 28.2% were 4 years old and the income with the highest prevalence was over r $ 1000.00 (320 usd) (74.6%). conversely, in the control group 58.3% of participants were female with 4 years and 62.5% of the parents had an income greater than r $ 1000.00 (320 usd). regarding mother’s scholar level, 59.2% had incomplete high school education or higher in the test group but, 52.1% had completed higher education in the control group. socioeconomic and demographic characteristics are shown in table 1. regarding caries experience in children, the initial mean was 0.44 (1.66) and the final mean was 0.83 (3.73) for the test group. however, for the control group, mean number of decayed surfaces was 0.81 (1.66) and the final mean was 1.33 (2.65). there was no statistical difference for the caries variable in the test and control groups. in relation wsl, table 1. socioeconomic and demographic characteristics of preschool children, piracicaba 2015. variables study groups p valueteste (n=71) control (n=48) n (%) n (%) age up to 1 year 5(7) 6 (12.5) 0.051 2 years 16 (22.5) 3 (6.3) 3 years 15 (21.1) 8 (16.7) 4 years 20 (28.2) 15 (31.3) 5 years 13 (18.3) 5 (10.4) missing 2 (2.8) 11 (22.9) sex boys 34 (47.9) 20 (41.7) 0.316 girls 37 (52.1) 28 (58.3) monthly income (usd) <$160 1 (1.4) 2 (4.2) 0.496 from $160$320 15 (21.1) 14 (29.2) >$320 53 (74.6) 30 (62.5) missing 2 (2.8) 2 (4.20) maternal education < 4 years 0 (0) 0 (0) 0.158 4 years 3 (4.2) 1 (2.1) 8 years 14 (19.7) 6 (12.5) up to 11 years 42 (59.2) 13 (27.1) university 10 (14.1) 25 (52.1) missing 2 (2.8) 3 (6.3) 6 zeeberg et al. the test group had an initial mean of 0.14 (0.66) and a final mean of 0.15 (0.69). in the preschool (control), an initial mean of 0.13 (0.61) and final mean of 0.15 (0.41) was observed. regarding restored surfaces, the initial mean of the test group was 0.07 (0.39) and the final mean was 0.13 (0.41). for the control group, the initial and final mean of restored surfaces was 0.58 (2.91) and 0.71 (1.61) respectively. there was no difference for the variables of caries experience between the beginning and the end of the epp. however, there was difference in relation to average of restored surfaces between both groups. the number of dental biofilm surfaces was also verified in the study through the vpi (visible plaque index). the initial mean of vpi was 4.95 (19.91) and the final mean was 0.21 (0.42) in the test group. the initial and final mean of vpi values in the control group were 4.11 (13.99) and 0.84 (0.59), respectively. the student’s t-test and the paired t test were used to show statistically significant differences between the test and control groups at the end of the epp in relation to vpi means. also, there were vpi differences in the test group between when compared the beginning and final of the preventive program, which represented a reduction of surfaces with biofilm. (table 2). in addition, the preschoolers from the test group were classified according to caries risk, 69.02% had low risk, 5.63% moderate risk and 25.35% high risk. after classification, one fluoride varnish application for middle-risk children and two applications for children who presented high risk for caries were done. a second questionnaire was administered to the parents or caregivers after the end of the preventive educational program. from the 71 parents or caregivers from the test group, table 2. mean and standard deviation of the caries experience, wsl (white spot lesions) and biofilm for the test group and control groups at the beginning and end of the preventive educational program, piracicaba, 2015 study groups baseline final p-value mean (se) mean (se) teste dmf-t 0.54 (1.85) 1.03 (3.91) 0. 152 dmft 0 (0) 0 (0) n=71 decayed 0.44 (1.66) 0.83 (3.73) 0. 216 missing* 0.07a (0.39) 0.13a (0.41) 0. 375 filled 0 (0) 0.070 (0.59) 0. 321 wsl 0.14 (0.66) 0.15 (0.69) 0. 885 dental plaque** 4.95 a (19.91) 0.21 b (0.42) 0. 047 control dmf-t 1.48 (3.40) 2.10 (3.44) 0. 136 dmft 0 (0) 0.06 (0.32) 0. 182 decayed 0.81 (1.66) 1.33 (2.65) 0. 071 n=48 missing* 0.58b(2.91) 0.71b (1.61) 0. 782 filled 0 (0) 0.10 (0.72) 0. 322 wsl 0.13 (0.61) 0.15 (0.41) 0. 821 dental plaque** 4.11 a (13.99) 0.84 a (0.59) 0. 117 note: different capital letters in the column mean difference between the test and control groups according clinical variables respectively, as described: * difference among control group and test regards missing teeth in the baseline and final evaluation. ** difference among control group and test regards dental plaque just at final clinical evaluation. student’s t test (p <0.05). se: standard error. 7 zeeberg et al. only 44 parents returned the completed questionnaire. regarding the information about health practices of examined preschool children, using the mcnemar’s statistical test, it was stated that most parents reported having received oral hygiene instructions at the end of the program when compared to the beginning of the program (p < 0.04) and more children went to the dentist at the end of the intervention (p <0.01). there was a statistically significant decrease in the children who performed their own oral hygiene. at the end of the program, from 88.4%, 66.0% still performed oral hygiene alone. (table 3) about the opinion of parents and caregivers about the preventive educational program, 45.1% evaluated the oral health of their child as good and only 2.8% as bad. regarding the time of the last visit to the dentist, 38.0% answered that the last visit was done in the last month. the epp was evaluated by 54.9% as very good. all parents or caregivers who submitted the questionnaire answered that the home brushing routine had improved and this preventive educational program has been important to their child health. (table 4) table 3. practices in health of the test group, before and after the preventive educational program, piracicaba, 2015. variable baseline n (%) final n (%) p-value child performs oral hygiene yes 122 (88.4) 44 (62.0) p<0.01 no 7 (5.1) 0 (0) child went to the dentist. yes 50 (36.2) 35 (49.3) p<0.01 no 80 (58.0) 9 (12.7) parents with instruction on oral hygiene yes 78 (56.5) 41 (57.7) p<0.04 no 47 (34.1) 3 (4.2) note: mcnemar’s test, significance p <0.05 * some questions have not been answered, so they do not add 100% to the table values. table 4. frequency and percentage of the opinion of the parents / caregivers from the test group regarding the preventive educational program, piracicaba, 2015. variable n (%) evaluation of the oral health of the child very good 10 (14.1) good 32 (45.1) bad 2 (2.8) improved brushing routine yes 44 (62.0) no 0 (0.0) last visit to the dentist last month 27 (38.0) last 6 months 7 (9.9) last 1 year 5 (7.0) never 4 (7.0) evaluation of the prevention program very good 39 (54.9) good 5 (7.0) bad 0 (0.0) very bad 0 (0.0) the program was important to the child’s health. yes 44 (62.0) no 0 (0.0) 8 zeeberg et al. discussion it is important to maintain oral health in early childhood because caries experience in primary dentition is considered as the strongest predictor of this disease in permanent teeth, besides the impact in the quality of life. therefore, the development of epp in oral health applied to this population is relevant15. dental biofilm contains acidogenic microorganisms that together with other factors such as time, poor oral hygiene habits and carbohydrate-rich diet can cause the development of caries disease16,17. therefore, caries is a disease mediated by demineralization and remineralization processes of hard dental tissues that will be carbohydrate-biofilm dependent18. the consumption of carbohydrates leads to a decrease in the ph of saliva which causes changes in the biofilm, becoming more cariogenic19. according to damle et al.20, dental biofilm reduction in preventive educational programs are very important to predict caries disease. the decrease is mainly related to frequent oral examinations and motivational activities with different methodologies that improve brushing techniques in children. based on the result of the present study, it was verified that the preventive program in oral health has positive results in the reduction of dental biofilm which would be related to the improvement in oral hygiene habits, even with sample loss21. this limitation was presented because in brazil there is a transition from the place of study of preschool to elementary school in this age. probably, this limitation did not allow to have results in relation to caries experience. rong et al.22, in a similar research, developed a dental caries preventive program for children with the help of teachers, parents or caregivers. it was demonstrated that there is a reduction in the indices of caries experience in children due to changes in oral hygiene habits in conjunction with the increase of oral health knowledge of their parents or caregivers. the positive result of the study was related to biofilm reduction (vpi index) in the test group, when compared to the end of the program in relation to control group. these positive results could be attributed to the educational and preventive intervention conducted on children, the improvement in their habits and parents or caregivers help during brushing technique. on this sense, the result evidenced from the intervention program was very important for child oral health due to the fact that the presence of biofilm is considered an etiological factor for dental caries and periodontal disease23. in addition, another study conducted by sánchez-huamán and sence-campos24 (2012) showed that a preventive educational program in school-age children, teachers and parents was effective. the annual program approached supervised brushing, educational sessions and workshops on oral health, treatment and application of fluoride gel, improving the oral hygiene condition and reducing plaque index, as in the present study24. from this fact, the participation in the preventive program of the child, the help offered by their parents or caregivers and/or support of the school personnel allowed the development of activities that produced changes in their habits and behaviors which will be very effective to improve their oral health. regarding habits and behaviors in children and their parents or caregivers before and after the epp, an improvement in the percentage of children who performed 9 zeeberg et al. self-oral hygiene was observed, which represented an improvement in routine brushing and parental help during its performance. despite the fact that there was no data comparison with control group, our results revealed that parents and caregivers perceived the importance of epp to improve their children health. the change in the habits and behaviors of children and in the perception of oral health of parents and caregivers was evidenced by the increase of dental visits. thus, parents and caregivers play a key role in changing the habits and behaviors of their children, acting as facilitators and positive reinforces in the process. the results could be consistent to those observed in the study conducted by yekaninejad et al.25, who reported it is necessary to integrate parents, schools, and community to improve the effectiveness of epp in oral health. in addition, to promote the acquisition of knowledge by parents who will be responsible for reinforcing good practices of the child at home. according to castilho et al.26 (2013), pre-school health habits begin at home, mainly because the mother has influence on the child’s oral health and health habits. in addition, parents should be aware of the strong influence that they have on their child’s habits and how they might affect their quality of life. from this fact, we could stay that health education programs involving the family could improve their quality of life26. the link with parents and guardians was made during a bimonthly parents meeting, offered by school staff, with a lecture presentation regarding caries disease and oral hygiene habits of the children. in the present study, parents and responsible participation was important for the awareness about the health condition of the child’s mouth. this was positive for the development of the project, because at the end of the epp, responsible reported that they considered their child’s oral health as good and that home brushing routine had improved. autonomy to prevent oral diseases is relevant and this process begins in childhood. parents can become aware to choose good health habits for themselves and their children by transferring the information they have learned. one of the reflexes of this awareness by parents/caregivers was to increase the percentage of brushing supervision in children by approximately 26% in the test group. in conclusion, the epp was effective in these children presenting reduction of dental biofilm in the test group after the intervention. in addition, improvements in health habits and behavioral changes such as lower percentage of children who perform their oral hygiene alone, parents’ help during tooth brushing, increased visits to the dentist, and a higher prevalence of parents who reported having received oral hygiene information were observed. therefore, the educational program may be an important strategy for the maintenance of oral health and dental caries prevention. a more extensive educational program and a longer-term monitoring are needed to show greater differences. acknowledgements the authors thanks fapesp (fundação de amparo à pesquisa do estado de são paulo) process (2012/25205-1) for financial support in this study. in addition, we thank the school directors, children and their caregivers. 10 zeeberg et al. references 1. lourenço cb, saintrain mv, vieira ap. child, neglect and oral health. bmc pediatr. 2013 nov 18;13:188. doi: 10.1186/1471-2431-13-188. 2. srinivasan d, louis cj. evaluation of caries in deciduous second molar and adjacent permanent molar in mixed dentition. j pharm bioallied sci. 2015 aug;7(suppl 2):s572-5. doi: 10.4103/0975-7406.163544. 3. chen x, zhan jy, lu hx, ye w, zhang w, yang wj, et al. factors associated with black tooth stain in chinese preschool children. clin oral investig. 2014 dec;18(9):2059-66. doi: 10.1007/s00784-013-1184-z. 4. nakre pd, harikiran ag. effectiveness of oral health education programs: a systematic review. j int soc prev community dent. 2013 jul;3(2):103-15. doi:10.4103/2231-0762.127810. 5. garbin c, garbin a, dos santos k, lima d. oral health education in schools: promoting health agents. int j dent hyg. 2009 aug;7(3):212-6. doi: 10.1111/j.1601-5037.2009.00394.x. 6. batchelor p. what do we mean by population health? community dent oral epidemiol. 2012 oct;40 suppl 2:12-5. doi: 10.1111/j.1600-0528.2012.00713.x 7. prakash p, subramaniam p, durgesh bh, konde s. prevalence of early childhood caries and associated risk factors in preschool children of urban bangalore, india: a cross-sectional study. eur j dent. 2012 apr;6(2):141-52. 8. colak h, dülgergil ct, dalli m, hamidi mm. early childhood caries update: a review of causes, diagnoses, and treatments. j nat sci biol med. 2013 jan;4(1):29-38. doi: 10.4103/0976-9668.107257. 9. krol dm, nedley mp. dental caries: state of the science for the most common chronic disease of childhood. adv pediatr. 2007;54:215-39. 10. inglehart mr, tedesco la. the role of the family in preventing oral diseases. in: cohen lk, gift hc, editor. disease prevention and oral health promotion: sociodental sceinces in action. copenhagen: munksgaard; 1995. p. 271-305. 11. bartley m, blane d, montgomery s. health and the life course: why safety nets matter. bmj. 1997 apr 19;314(7088):1194-6. 12. wigen ti, wang nj. maternal health and lifestyle, and caries experience in preschool children. a longitudinal study from pregnancy to age 5 yr. eur j oral sci. 2011 dec;119(6):463-8. doi: 10.1111/j.1600-0722.2011.00862.x. 13. de amorim rg, figueiredo mj, leal sc, mulder j, frencken je. caries experience in a child population in a deprived area of brazil, using icdas ii. clin oral investig. 2012 apr;16(2):513-20. doi: 10.1007/s00784-011-0528-9. 14. american dental association council on scientific affairs; 2006. evidenced in cortelazzi kl, tonello as, mialhe fl, pereira ac. [professional methods, autouso and fluoride combinations: an evidencebased approach]. in: pereira ac, et al. [treaty of public health dentistry]. nova odessa: napoleão; 2009. p.508-28. portuguese. 15. aquilante ag, almeida bs, martins de castro rf, xavier cr, sales sh, bastos jr. [the importance of dental health education for preschool children]. rev odontol unesp. 2003; 32:39-45. portuguese. 16. borgström mk, edwardsson s, sullivan a, svensäter g. dental plaque mass and acid production activity of the microbiota on teeth. eur j oral sci. 2000 oct;108(5):412-7. 17. oliveira lb, sheiham a, bönecker m. exploring the association of dental caries with social factors and nutritional status in brazilian preschool children. eur j oral sci. 2008 feb;116(1):37-43. doi: 10.1111/j.1600-0722.2007.00507.x. 18. ccahuana-vásquez ra, tabchoury cp, tenuta lm, del bel cury aa, vale gc, cury ja. effect of frequency of sucrose exposure on dental biofilm composition and enamel demineralization in the presence of fluoride. caries res. 2007;41(1):9-15. 11 zeeberg et al. 19. cury ja, tenuta lm. how to maintain a cariostatic fluoride concentration in the oral environment. adv dent res. 2008 jul 1;20(1):13-6. 20. damle sg, patil a, jain s, damle d, chopal n. effectiveness of supervised toothbrushing and oral health education in improving oral hygiene status and practices of urban and rural school children: a comparative study. j int soc prev community dent. 2014 sep;4(3):175-81. doi: 10.4103/2231-0762.142021. 21. manchanda k, sampath n, sarkar ad. evaluating the effectiveness of oral health education program among mothers with 6-18 months children in prevention of early childhood caries. contemp clin dent. 2014 oct;5(4):478-83. doi:10.4103/0976-237x.142815. 22. rong ws, bian jy, wang wj, wang jd. effectiveness of an oral health education and caries prevention program in kindergartens in china. community dent oral epidemiol. 2003 dec;31(6):412-6. 23. boka v, trikaliotis a, kotsanos n, karagiannis v. dental caries and oral health-related factors in a sample of greek preschool children. eur arch paediatr dent. 2013 dec;14(6):363-8. doi: 10.1007/s40368-013-0097-5. 24. sánchez-huamán y, sence-campos r. [effectiveness of an educational and preventive program to improve hygiene habits and oral hygiene condition in schoolchildren]. kiru. 2012;9 (1): 21-33. spanish. 25. yekaninejad ms, eshraghian mr, nourijelyani k, mohammad k, foroushani ar, zayeri f, et al. effect of a school-based oral health-education program on iranian children: results from a group randomized trial. eur j oral sci. 2012 oct;120(5):429-37. doi: 10.1111/j.1600-0722.2012.00993.x. 26. castilho ar, mialhe fl, barbosa tde s, puppin-rontani rm. influence of family environment on children’s oral health: a systematic review. j pediatr (rio j). 2013 mar-apr;89(2):116-23. doi: 10.1016/j.jped.2013.03.014. untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8652883 volume 17 2018 e18138 original article 1 dds, ms, department of orthodontics, são paulo state university (unesp), school of dentistry, araraquara, brazil. 2 professor, graduate program in orthodontics, school of health and biosciences, pontificial catholic university of paraná, brazil. 3 adjunct professor of orthodontics, são paulo state university (unesp), school of dentistry, araraquara, brazil. corresponding author: dr. luís filipe siu lon rua ébano pereira, 60, sl 1105 curitiba – paraná – cep: 80410-902 brazil phone: 55-41-99838-1718/ fax number: 55-41 3322-5776 e-mail: filipelon@hotmail.com received: december 05, 2017 accepted: june 02, 2018 shear bond strength of three diferent bonding systems for orthodontic brackets luís filipe siu lon1, luegya amorim henriques knop1, ricardo lima shintcovsk1, odilon guariza filho2, dirceu barnabé raveli3 aim: the objective of this study was to compare the efficiencies of different adhesive systems used to bond orthodontic brackets as well as the fracture pattern during debonding on bovine teeth. methods: the sample included 45 specimens assigned to 3 groups according to the adhesive system applied: group i: transbond xt (3m unitek®, monrovia/ca usa), group ii: orthocem (fgm® joinville/ sc-br), and group iii: orthobond (morelli®, sorocaba/sp br). for this purpose, metal brackets were bonded to bovine teeth following the instructions from each manufacturer. the specimens were subjected to a shear test to assess bond strength (bs). finally, after debonding, the adhesive remnant index (ari) was estimated. results: the average shear strength for a tooth bonded using the adhesive system transbond xt was 16.39 mpa, while it was 18.08 mpa for orthocem and 7.28 mpa for orthobond; the tukey test revealed no statistically significant differences between groups i and ii (p < 0.01) and group iii differed statistically from groups i and ii. conclusion: in conclusion, both adhesive systems transbond xt and orthocem attained higher bond strength values than orthobond; the fracture pattern was similar for all adhesive systems applied. keywords: dental bonding. shear strength. dental enamel. 2 lon et al. introduction brackets are essential pieces of orthodontic appliances. direct bonding is an effective alternative to the procedure of welding bands, which represents a true revolution in orthodontic treatments. direct bonding features several advantages, such as better appearance, easy manipulation, enhanced comfort of the patient, absence of pain, decreased irritation of the soft tissue, easier oral hygiene, decreased incidence of gingivitis, better caries detection, possibility of exact bracket positioning, and no space closure required after the treatment. the primary disadvantage is decreased retention area, which directly interferes with masticatory force resistance resulting in accidental debonding and subsequent professional frustration, longer procedures and higher costs1-4. previous studies have commonly explored factors regarding the optimization of bond strength. retention mechanisms present in the brackets, the adhesive system, and the acid conditioning of the enamel can influence bond strength. insufficient adhesive strength is considered a cause of bracket debonding1,2,5-8. for the past few years, significant scientific and technological advances have brought several benefits to orthodontics especially in the area of direct bonding of the accessories. new techniques and materials have been incorporated into clinical practice leading to simpler procedures and improved comfort of a patient. despite the great advances in the development of orthodontic adhesives, studies have shown a failure rate of 5 to 7% in resin-based bonding5. orthodontic clinics have been applying fluidic systems immediately after the acid etching of enamel using no intermediate adhesives. as the number of steps during bonding is reduced, orthodontists can decrease chair time and potential errors associated with the contamination during bonding procedures9. one example of fluidic systems is orthocem (fgm®, joinville/sc). the most frequently used adhesive systems for bonding orthodontic brackets in the brazilian dental market are transbond xt (3m unitek®, monrovia/usa), orthocem (fgm®, joinville/sc-br), and orthobond (morelli®, sorocaba/sp-br). although published scientific studies comparing either orthocem or orthobond with other adhesive systems are available in the literature1,10-15, to our knowledge, no experiments comparing these two adhesive systems to each other have been conducted to date. the objective of this study was to compare the shear bond strength (sbs) and the adhesive remnant index (ari) between 3 different adhesive systems: transbond xt (3m unitek®, monrovia/usa), orthocem (fgm®, joinville/sc-br), and orthobond (morelli®, sorocaba/sp-br). the hypothesis tested in this study was that there are no significant differences in bond strength among the bonding materials. material and methods this study was approved by the ethics committee in the use of animals (ceua) of the pontificia universidade catolica do paraná (protocol number: 204/07). 3 lon et al. the sample was composed of 45 freshly extracted bovine permanent incisors randomly divided into 3 groups (n=15): group i (control) 15 brackets by miniature twin (3m unitek®, monrovia/usa) bonded with resin transbond xt (3m unitek®, monrovia/usa); group ii 15 brackets by miniature twin (3m unitek®, monrovia/ca usa) bonded with resin orthocem (fgm®, joinville/sc br); group iii 15 brackets by miniature twin (3m unitek®, monrovia/ ca-usa) bonded with resin orthobond (morelli®, sorocaba/sp-br). the coronary portion of the teeth selected for this study was characterized by integrity of the crowns, the absence of decay, cracks or fractures to ensure even quality of the sample. to prepare the specimens, each tooth presenting flat surface was selected and attached to a mechanical workbench by clamping (metalsul®, joinville/sc-br). each randomly selected tooth was submitted to prophylaxis with water and pumice stone using a rubber cup (microdont, são paulo/sp–br), and the enamel surface was rinsed with distilled water and dried with oil-free compressed air for 20 seconds. a new rubber cup was used every 5 prophylaxis cycles. a 37% phosphoric acid gel (condac, fgm®, joinville/sc-br) was applied to the buccal surface of each tooth for 30 seconds for enamel etching, followed by air/water spray rinse for 10 seconds and gentle drying with airflow for 10 seconds. the adhesive system was applied following the manufacturer instructions. groups i and iii had a thin layer of primer applied on the tooth surface using disposable applicator microbrush (vigodente®, rio de janeiro/ rj-br) and group ii used a simplified system that did not require primer application. the bonding resin was inserted on the bracket base and the bracket was positioned on the tooth surface under pressure of 400 grams measured with the tensiometer (morelli®, sorocaba/sp-br) to standardize the adhesive layer thickness. the excess of the resin was removed using a small scaler (ss white®, rio de janeiro/rj-br). in all of the groups, the specimens were light cured for 40 seconds using optilight ld max (gnatus®, são paulo/sp-br). a stainless steel device was made with a rectangular .021” x.025” wire (morelli®, sorocaba/sp-br) to ensure that the bracket was perpendicular in a horizontal plane. then, the teeth were fixed on the device by an elastomeric ring holding it to the wire. after fixation, the root was embedded in chemically cured acrylic resin and placed in metal rings. that way, the bonding surface was perpendicular to the horizontal plane and parallel to the direction of the force to be applied in the sbs test. all bonded specimens were stored in distilled water at 37º c16. twenty-four hours after the bonding procedures, the sbs test was performed using a universal testing machine emic dl 2000 (emic equipamentos e sistemas de ensaio ltda., são josé dos pinhais/pr-br) at 0.5 mm/min crosshead speed, until the bracket failure. as the bracket was moved, the required strength was calculated using tesc software version 3.01. the shear strength was measured as newton (n) divided by the bracket base area in mm2 and converted to megapascal (mpa). subsequently, the ari was assessed with stereoscopic magnifying glass (722 schwenningen, waldmann-leuchterz, germany) under 20× magnification. a modification of the ari originally proposed by artun and bergland17 was used. according to the modified ari the following scores are assigned: score 0, no adhesive bonded 4 lon et al. on the bracket; score 1, 50% of the adhesive bonded on the bracket; score 2, 75% of the adhesive bonded on the bracket and score 3, the whole adhesive bonded on the bracket. the values were entered into excel for the statistical analysis. analysis of variance (anova) was applied to estimate shear strength and verify statistically significant differences between groups i, ii and iii. the tukey honestly significant difference (hsd) post hoc test was used to compare the group means. the kruskal-wallis test was applied to compare the ari scores. results the descriptive statistics for sbs in megapascals (mpa) is shown in table 1. statistically significant differences were observed among groups according to anova. the tukey test revealed that group iii differed from groups i and ii. no statistically significant differences between groups i and ii were observed (p < 0.01). table 1. descriptive statistics in megapascals (mpa) of shear bond strengths of experimental groups studied groups n mean standard deviation group i 15 16.39 a 3.78 group ii 15 18.08 a 8.50 group iii 15 7.28 b 2.51 different letters indicate significant difference by tukey test (p < 0.001). the ari scores are presented in table 2. the kruskal-wallis test showed no significant differences among groups regarding the ari score (p < 0.05). most specimens from groups presented ari scores ranging from 2 to 3. table 2. frequency distribution of adhesive remnant index (ari) of groups studied groups ari scores 0 1 2 3 gi frequency 0(0%) 0(0%) 2(13.3%) 13(86.7%) gii frequency 0(0%) 1(6.7%) 5(33.3%) 9(60%) giii frequency 0(0%) 1(6.7%) 1(6.7%) 13(86.7%) ari 0, no adhesive bonded on the bracket; 1: 50% of the adhesive bonded on the bracket; 2: 75% of the adhesive bonded on the bracket and 3: the whole adhesive bonded on the bracket. there were no significant differences among groups (p-value < 0.05). discussion the flat surface of human maxillary central incisor is ideal for studies on shear strength18. however, improved health conditions for the population in general, advanced restoration techniques and greater access to health services have led to a 5 lon et al. decrease in the number of tooth extractions and a subsequent decrease in the offer of human teeth for studies in vitro. in our study, we used the bovine incisors because they are easily obtained, have low cost, and are similar to human teeth, which makes them an option to replace human teeth in research18. bonding orthodontic brackets onto tooth surface remains a debatable subject. a fast and effective bonding of orthodontic appliances is a clinical challenge regarding the use of adhesive systems since the procedures are generally carried out in humid environments with relative isolation of operative field. therefore, it is important to apply simplified systems to reduce steps of the procedure and clinical time to ensure a secure bonding19. the ideal properties of luting agents for orthodontic brackets are as follows: sufficient adhesive strength to tolerate orthodontic forces during treatment; fluidity required to penetrate the material during bonding; viscosity to maintain the bracket positioned before cement polymerization; proper time to reach the correct bracket positioning and remove the excess material; possibility of working in a humid environment to decrease posterior teeth deviation index, fluoride release and risks of white spots; removal with no damages to the enamel surface19. the configuration of light cure adhesives requires primer and adhesive separately or unified. the configuration of orthocem presents adhesive and primer unified. the mono-component configuration of bracket bonding reduces the process to only 2 steps. the orthocem adhesive system presents the following characteristics: simplified technique, proper viscosity, the presence of fluoride, high shear strength, easy removal, and enamel polishing. in this study, the transbond xt adhesive system was used as a control group because it was shown to be efficient and exhaustively tested with proven characteristics to resist masticatory forces1,6,11-15. ryou et al.6 (2008) compared fluidic systems that do not require intermediate steps with transbond xt observing that, although transbond xt was superior to the other systems in terms of bond strength, their adhesive strength was sufficient, and therefore suitable for clinical application. although, there are studies that compared orthocem or orthobond to other adhesive systems 1,10,11-15, in our knowledge these work would be the first one to compare these 2 adhesive systems. the null hypothesis stating that there are no significant differences in sbs among the bonding materials was rejected. no statistically significant differences were observed between systems transbond xt and orthocem; however, both of them showed statistically significant increases in adhesive strength (p<0.001) when compared to orthobond. in the present study, the mean adhesive strength of orthocem (18.08 ± 8.5 mpa) was higher than the values reported in previous studies11,12 which were 8.31 ± 3.5 mpa and 4.2 ± 0.8 mpa, respectively under the same conditions. the difference between these values is probably due to different bracket base mesh used. 6 lon et al. our results for orthobond were quite similar to those obtained by other researchersl13-15, who evaluated the sbs of the orthobond system. according to reynolds3, the adhesive strength values between 5.9 and 7.8 mpa, which would be adequate to support masticatory forces, are acceptable for clinical use and show satisfactory performance. in our study, all of the experimental groups presented values compatible with clinical requirements. in the present investigation, evaluation of the ari scores showed no significant differences between groups i, ii, and iii. the ari scores showed a predominance of scores 2 and 3, which means that most of the adhesive remained attached to the bracket base and minimum amount remained on the enamel surface. clinically, this implies that a shorter time is required to remove the adhesive after debonding. our results for ari scores are in agreement with some studies4,20 that state that the resin should remain on the bracket after the debonding, claiming it would require fewer steps to remove the adhesive from enamel4,20. besides, it indicates that the cohesive strength of enamel is superior to the bond strength of bracket base. on the other hand, the residual adhesive remaining on enamel surface reduces enamel fractures; in addition, the residual adhesive is removed using special drill without any damage to tooth enamel21. in conclusion, both of the adhesive systems orthocem and transbond xt had higher sbs than orthobond. all 3 systems examined presented similar enamel fracture pattern. references 1. busato mca, busato pmr, dotto dv, pedrotti s, gasparello, cr. evaluation of shear bond strength of brackets bonded with different orthodontic resins. rev clin ortodon dental press. 2013 apr-may;12(2):94-9. portuguese. 2. newman gv. epoxy adhesives for orthodontic attachments: progress report. am j orthod dentofacial orthop. 1965 dec;51(12):901-12. 3. reynolds ir. a review of direct orthodontic bonding. br j orthod. 1975;2:171-8. 4. sorel o, el-alam r, chagnedy f, gathelineau g. comparison of bond strength between simple foil and laser structured base retention brackets. am j orthod dentofac orthop. 2002 sep;122(3):260-6. 5. chung ch, friedman sd, mante fk. shear bond strength of rebounded mechanically retentive ceramic brackets. am j orthod dentofacial orthop. 2002 sep;122(3):282-7. 6. ryou db, park hs, kim kh, kwon ty. use of flowable composites for orthodontic bracket bonding. angle orthod. 2008 nov;78(6):1105-9. doi: 10.2319/013008-51.1. 7. sant’anna ef, monnerat me, chevitarese o, stuani mb. bonding brackets to porcelain in vitro study. braz dent j. 2002;13(3):191-6. 8. tavares sw, consani s, nouer df, magnani mb, nouer pr, martins lm. shear bond strength of new and recycled brackets to enamel. braz dent j. 2006;17(1):44-8. 9. meehan pm, foley tf, mamandras ah. a comparison of the shear bond strengths of two glass ionomer cements. am j orthd dentofacial orthop. 1999 feb;115(2):125-32. 10. teixeira cm, roya rr, oliveira mt. influence of variation on polymerization time in the shear bond strength for different cements to orthodontic brackets. rev bras odontol. 2012 jul/dez;69(2):220-3. portuguese. 7 lon et al. 11. scribante a, sfondrini mf, fraticelli d, daina p, tamagnone a, gandini, p. the influence of no-primer adhesives and anchor pylons bracket bases on shear bond strength of orthodontic brackets. biomed res int. 2013;2013:315023. doi: 10.1155/2013/315023. 12. oliveira bls, costa, ar, correr ab, crepaldi mv, correr-sobrinho l, santos jcb. influence of adhesive and bonding material on the bond strength of bracket to bovine tooth. braz j oral sci. 2017 jul/sep;16(3):1-7. e17033. 13. pithon mm, santos rl, oliveira mv, sant’anna ef, ruellas aco. evaluation of the shear bond strength of the orthobond composite under different conditions. rgo 2008 oct/dez;56(4):405-10. portuguese. 14. vilar rv, souza, nf, cal-neto jp, galvão m, sampaio-filho h, mendes ade m. shear bond strength of brackets bonded with two light-curing orthodontic adhesives. j adhes dent. 2009 aug;11(4):259-62. 15. pithon mm, santos rl, oliveira mv, sant’anna ef, ruellas aco. evaluation of the shear bond strength of two composites bonded to conditioned surface with self-etching primer. dental press j orthod 2011 ar/apr;16(2):94-9. doi: 10.1590/s2176-94512011000200012. portuguese. 16. cacciafesta v, sfondrini mf, de angelis m, scribante a, klersy c. effect of water and saliva contamination on shear bond strength of brackets bonded with conventional, hydrophilic, and selfetching primers. am j orthod dentofacial orthop. 2003 jun;123(6):633-40. 17. artun j, bergland s. clinical trials with crystal growth conditioning as an alternative to acid-etch enamel pretreatment. am j orthod. 1984 apr;85(4):333-40. 18. oesterle lj, shellhart wc, elanger gk. the use of bovine enamel in bonding studies. am j orthod dentofacial orthop. 1998 nov;114(5):514-9. 19. buonocore mg. a simple method of increasing the adhesion of acrylic filling materials to enamel surface. j dent res. 1955 dec;34(6):849-53. 20. costa ar, correr ab, puppin-rontani rm, vedovello sa, valdrighi hc, correr-sobrinho l, et al. effect of bonding material, etching time and silane on the bond strength of metallic orthodontic brackets to ceramic. braz dent j. 2012 jul-aug;23(3):223-7. doi: http://dx.doi.org/10.1590/s0103-64402012000300007. 21. maccoll ga, rossouw pe, titley kc, yamin c. the relationship between bond strength and orthodontic bracket base surface area with conventional and microetched foil-mesh bases. am j orthod dentofacial orthop. 1998 mar;113(3):276-81. 1http://dx.doi.org/10.20396/bjos.v16i0.8650496 volume 16 2017 e17049 original articlebjos 1 doctoral student, department of prosthodontics and periodontology, piracicaba dental school, university of campinas (unicamp), av limeira, 901, piracicaba, são paulo 13414-903, brazil. 2 master degree student, department of prosthodontics, dentistry college, federal university of pará (ufpa), rua augusto corrêa, 01, belém, pará 66075-110, brazil. 3 permanent professor, department of prosthodontics, dentistry college, federal university of pará (ufpa), rua augusto corrêa, 01, belém, pará 66075-110, brazil. corresponding author: av. limeira, 901, piracicaba, sp, brazil 13414-903, tel.: + 55-19-984333415; e-mail: adaiasmatos@hotmail.com received: may 16 2017 accepted: august 03, 2017 comparative analysis of ceramic flexural strength in co-cr and ni-cr alloys joined by tig welding and conventional brazing adaias oliveira matos1*, cristiane de castro c. castelo branco2, eliza burlamaqui klautau3, bruno pereira alves3. aims: the purpose of the present study was to evaluate the flexural strength of specimens made of nickel-chromium (ni-cr) and cobalt-chromium (co-cr) alloys and joined by tungsten inert gas (tig) welding and conventional brazing. ni–cr and co–cr base metal specimens (n = 40, each) were cast and welded by tig or brazing. the specimens were divided into six groups (2 base metals, four welded specimens). ceramic systems were applied to the central part of all the specimens. a three-point bending test with a velocity of 0.5 mm/m was performed on the specimens up to the point of the first ceramic bond failure by measuring the flexural strength. data were analyzed using two-way anova and bonferroni’s tests. conventional welding showed the lowest flexural strength results for both alloys, while the tig weld and the control group presented with varying bond strengths for the alloys studied. we concluded that tig welding was superior to the conventional welding method for both ni–cr and co–cr alloys with regard to the flexural strength of the ceramic. keywords: ceramics, welding, dental alloys 2 matos et al. introduction despite several ongoing studies on the development and improvement of pure ceramic systems, metal-ceramic fixed prostheses continue to be of great clinical importance owing to their versatility and feasibility in various therapeutic modalities and affordable cost1-3. the metal-ceramic system produces a restoration in which, the physical properties of porcelain and metal are used for mutual reinforcement, but if the ceramic gets detached, metal exposure is inevitable leading to the loss of esthetics4,5. thus, the union between metal and ceramic is essential for successful restoration that involves longevity in the oral cavity6. basic metal alloys have been used as alternatives for metal-ceramic prosthetics owing to their low cost compared to other alloys7. this has enabled the employment of high quality treatment for a large number of patients, especially those with low purchasing power2,7. however, difficulty in handling, susceptibility to oxidation, high melting temperature, and inferior finishing are the main disadvantages of these alloys3,8. the advantages of basic metal alloys include high fracture strength, high modulus of elasticity, rigidity, and resistance to permanent deformation9,10.an additional advantage of the metal structure of a fixed prosthesis is its resistance to plastic deformation11. in addition to mechanical properties, metals must be biocompatible, resistant to corrosion, and easy to manipulate during the preparation of prostheses. nickel–chromium (ni–cr) alloys are the most commonly used basic metal alloys for metal-ceramic restorations12,13. however, because of the potential health problems associated with beryllium and nickel, the cobalt–chromium (co–cr) alloy has been used as an alternative despite its inferiority to ni-cr alloys3. when the metal framework of the prosthesis does not fit perfectly on the abutments, the metallic infrastructure is sectioned, and the welding process is carried out after adjusting to the abutments; this procedure aims to improve the adaptation of prostheses to abutments or implants, thereby restoring the force previously lost following the sectioning of the metallic infrastructure14. the joining of metals in prosthetic structures can be achieved by brazing or via laser or tungsten inert gas (tig) welding9,15. in the brazing process, the bonding between metals is produced by heating an additional metal that has a melting point lower than that of the base metal to a suitable temperature. tig welding process involves joining metal structures by heating and melting them through an electric arc established between a tungsten electrode and the parts to be fused16. the most commonly used gases during tig welding are helium and argon10,17. although the use of tig welding in dentistry is uncommon, some studies show the superiority of this technique compared to conventional brazing18,19. several studies have evaluated the bond strength of porcelain in different alloys7,20-22. however, few studies have evaluated the bond strength of ceramics in regard with tig welding or brazing. therefore, the aim of the present study was to compare the ceramic bonding strength between areas in the co–cr and ni–cr alloys that underwent tig welding and conventional brazing. the null hypothesis was that there would be no significant differences between the co–cr and ni–cr alloys. 3 matos et al. material and methods experimental design co–cr (fit cast cobalt-talladium do brasil) and ni–cr (fit cast sb plus-talladium do brasil) alloys were joined by tig welding or conventional brazing, and ceramic coated (vita vm13-germany) in the center of the specimens23. two control groups with co–cr and ni–cr alloys (no welding), and four test groups (two co–cr alloys and two ni–cr alloys) joined by tig welding and conventional brazing (10 samples in each group) were used in this study. obtaining patterns for casting initial patterns of thermopolymerizable acrylic resin (jet, artículos odontológicos clássico ltda, são paulo, sp, brazil) were made using the following dimensions: 25 mm in length, 3 mm in width and 1 mm in thickness; and 50 mm in length, 3 mm in width and 1 mm in thickness. the inclusion was performed by arranging the resin patterns parallel to each other and joining them using wax (kota, indústria e comércio, são paulo, sp, brazil). the assembly was mounted on a silicone ring for inclusion before coating. elimination of acrylic a nº5 silicone ring was filled with coating (microfine1700, talladium, curitiba, pr, brazil) at a ratio described by the manufacturer (powder:liquid, 90 g of powder to 23 ml of liquid; the liquid solution comprised 18 ml of liquid and 5 ml of distilled water), mixed for 10 s, and vacuum-spatulated (vrc-vrc equipamentos-guarulhos-sp-brazil) for 30 s. after 20 min in the ring, the hot coating was placed in an electric oven (edgcon 5p, equipamentos e controlles ltda, são carlos, sp, brazil) at an initial temperature of 400°c for 30 min. subsequently, the temperature was raised to 950°c, and maintained for 20 min. after the heating cycle, the temperature was reduced and the ring was removed (at 850°c for ni–cr alloys and 900°c for co–cr alloys). induction casting of co-cr and ni-cr alloys forty patterns from the 25-mm co–cr alloy casting and 10 patterns of the 50-mm casting were used. likewise, 40 and 10 patterns from the 25-mm and 50-mm ni–cr co-cr ni-cr control tig welding conventional brazing ceramics flexural strength figure 1. flowchart of experimental procedure. 4 matos et al. alloy casting, respectively, were used in this study. the co–cr and ni–cr alloys were weighed (v.h equipamentos, araraquara, sp, brazil). initially, the alloys were distributed inside the ceramic crucible following which, the induction centrifugation system (power cast red edg são carlos sp brazil) was activated. after centrifugation and metal cooling, the strips were finished with carbide tungsten minicut drills. conventional brazing in order to carry out the brazing process, an acrylic device was fabricated to position the metal specimens without any spaces between them. the specimens were attached with chemically activated resin (duralay, reliance dental company, usa) through the hole in the device, and the resin-wrapped area was covered with utility wax (wilson, polidental indústria, cotia, sp, brazil). the high-temperature coating (easy-stack-stage) was prepared according to the manufacturer’s instructions and spatulated with water. it was hand-manipulated for 40 s, and the hardening reaction was expected to occur within 20 min. the coating block was placed in the oven (edg equipment) along with the specimens at 540°c for 20 min. after heating, the block was removed from the oven and cooled slowly to room temperature. the flame was regulated until it achieved a 15-mm long blue cone using a gas torch (liquefied petroleum gas; edg equipment) with a single-hole nozzle. the central parts of the strips were heated until bright red. this coloration was obtained by placing the strips on to the side of the torch (tilite-talladium) following which, the flame was rapidly passed throughout the region of the weld24. tig welding tig welding was performed using tig nty 60c welding equipment (kernit indústria mecatrônica ltda, indaiatuba, sp, brazil). argon flow was released when the system was activated, forming an oxygen-free region and triggering the electric current. the specimens were positioned in an acrylic device containing a channel in which they could be accommodated without any spaces between them17. application of ceramics after the welding processes, the specimens were finished using drills, ground in polystyrene (arotec, cotia, sp, brazil), blasted with aluminum oxide at a particle size of 110 μm and compression of 5.1 kgf/cm, and they were placed on a vibrator with isopropyl alcohol for waste removal. an acrylic matrix was prepared to delimit the area of application of the ceramic to 8 ± 0.1 mm in length, 3 ± 0.1 mm in width, and 1 ± 0.1 mm in thickness. ceramic was applied to the center of the weld area, on one side of the metal specimens. initially, the specimens were pre-oxidized in an oven (ceramsinter-edg). following the application of an opaque layer, the specimens were subjected to firing; this process was performed twice. dentin layers were applied to the established dimensions and glazing of the ceramic surface was finally achieved3. 5 matos et al. flexural test the specimens were submitted to a 3-point bending test in a test machine (kratos equipamentos, são paulo, sp, brazil) equipped with a 50 n load and a speed of 0.5 mm/min. the coated ceramic face was positioned down toward the rupture between the ceramic and metal substructure. the maximum force (n) was recorded until the first fault, for each test body. the flexural strength was calculated according to the following formula: ∑ = 3pi 2bd2 , where p is the maximum force (n), i is the distance between the supports (mm), b the sample width (mm), d the sample thickness (mm), and σ is the the flexural strength (mpa)3. statistical analysis flexural strength (mpa) data were analyzed by two-way anova (factor 1: framework type; factor 2: welding type). bonferroni tests were performed to compare the mean values among groups (α = 0.05; spss version 20.0 statistical package for the social sciences, inc.). a p value of 0.05 was considered significant. results two-way anova revealed a significant difference in the bond strength of ceramic between the welding types and framework types when subjected to flexural strength test. figure 2 displays the results of flexural strength between the ceramic and areas tig welded areas and brazing in co–cr and ni–cr alloys. the best flexural strength results were numerically observed in the welding type for tig welding and framework type for ni–cr alloy. tig welding in the co–cr alloy (p < 0.05) and ni–cr alloy (p < 0.05) increased the bond strength between the ceramic and tig welded area when compared with the group with welding to brazing. flexural strengths in the control (p = 0.000) and tig groups (p= 0.011) were significantly affected by the figure 2. flexural strength assay flexural strength metalic sample ceramic f 6 matos et al. type of framework used. on the one hand, welding types were significantly different for the co–cr alloy in all groups (p < 0.05), whereas, on the other hand, the ni–cr alloy demonstrated significant decrease in flexural strength for conventional brazing when compared to welding (p < 0.05). discussion dental ceramics possess biocompatibility, color stability, and wear resistance; however, they also have low flexural strength, and one method to address this disadvantage is to attach the ceramic to a substructure of metal alloys24. the welding process is widely used during the manufacture of fixed dental prostheses. currently, several techniques are employed, and each has its own advantages and disadvantages14,25. the conventional technique involves several critical steps including temperature control and exposure time of the metal to the flame for proper solder flow; the technique is even more challenging when ni–cr or co–cr alloys are involved because of difficulty in the removal of oxides21. both temperature and flame control of the torch can influence the conventional brazing method by altering the oxide layer formed on the surface of these alloys, which interfere with bonding of the ceramic to the metal during the welding process; the incorporation of oxygen results in porosity in the welded area, making it less resistant to flexion18. thus, the results presented in the brazing group indicate that this welding method reduces of the flexural strength of co–cr and ni–cr alloys by interfering with the bonfigure 3. mean flexural strengths (mpa) in the control, tig, and brazing groups based on the different welding and framework types. the different lower case letters represent significant differences among the same type of alloy associated with the different types of welding methods used. the capital letters represent significant differences among the different structures related to the same of type welding method used. fl ex ur al s tr an gt h (m p a) 40 20 15 10 5 0 35 30 25 control tig brazing a a a b b a a b c a b a co-cr ni-cr 7 matos et al. ding of the ceramic to the metal. brazed areas showed less flexural strength when compared to the welded areas of tig. in the case of ni–cr and co–cr alloys, bending mainly occurs between the metal and ceramic coating in the passive layers. the co–cr alloy is less resistant to bending and has higher elasticity compared to the ni–cr alloy, which possesses comparatively higher bending strength and lower elasticity. flexural strength was higher in the ni–cr alloy than in the co–cr alloy in the control and tig groups. this may be explained by the fact that this process has the advantage of using the minimum amount of heat, thereby reducing the area affected by heat and the production of free oxygen in the region; this results in a decrease in oxide residues formed during welding, and facilitates the maintenance of alloy properties25. the advantages of tig welding over conventional welding include the realization of the weld in the working model itself, reduction in working time and consequently, occurrence of inherent failures during the welding process, and production of less heat, which allows for welding after the application of ceramics25. the tig weld used for bonding ceramic to metal presented with more resistance to flexion only in the co–cr alloy group compared to the control groups. both ni–cr and co–cr alloys can be satisfactorily used in the clinics. nevertheless, further in vitro and in vivo studies simulating the application of masticatory loads and using biocompatibility tests on these welds are warranted. conclusion within the limitations of the present in vitro study, we draw the following conclusions: the bond strength of ceramic to metal using the tig welding process was superior to that using the conventional brazing process. different alloys with the same type of weld showed no differences in the bond strength of ceramic only for conventional brazing process. co–cr alloys may be used as an alternative to ni–cr alloys. acknowledgements fapespa references 1. joias rm, tango rn, junho de araujo je, junho de araujo ma, ferreira anzaloni saavedra gde s, paes-junior tj et al. shear bond strength of a ceramic to co-cr alloys. j prosthet dent. 2008 jan;99(1):54-9. doi: 10.1016/s0022-3913(08)60009-8. 2. fischer j, zbaren c, stawarczyk b, hämmerle ch. the effect of thermal cycling on metal-ceramic bond strength. j dent. 2009 jul;37(7):549-53. doi: 10.1016/j.jdent.2009.03.014. 3. pretti m, hilgert e, bottino ma, avelar rp. evaluation of the shear bond strength of the union between two cocr-alloys and a dental ceramic. j appl oral sci. 2004 dec;12(4):280-4. 8 matos et al. 4. malo p, de araujo nobre m, borges j, almeida r. retrievable metal ceramic implant-supported fixed prostheses with milled titanium frameworks and all-ceramic crowns: retrospective clinical study with up to 10 years of follow-up. j prosthodont. 2012 jun;21(4):256-64. doi: 10.1111/j.1532-849x.2011.00824.x. 5. dhima m, paulusova v, carr ab, rieck kl, lohse c, salinas tj. practice-based clinical evaluation of ceramic single crowns after at least five years. j prosthet dent. 2014 feb;111(2):124-30. doi: 10.1016/j.prosdent.2013.06.015. 6. scolaro jm, pereira jr, do valle al, bonfante g, pegoraro lf. comparative study of ceramic-to-metal bonding. br dent j. 2007; 18(3):240-3. 7. hong jt, shin sy. a comparative study on the bond strength of porcelain to the millingable pd-ag alloy. j adv prosthodont. 2014 oct;6(5):372-8. doi: 10.4047/jap.2014.6.5.372. 8. sipahi c, ozcan m. interfacial shear bond strength between different base metal alloys and five low fusing feldspathic ceramic systems. dent materj. 2012;31(3):333-7. 9. vasquez v, ozcan m, nishioka r, souza r, mesquita a, pavanelli c. mechanical and thermal cycling effects on the flexural strength of glass ceramics fused to titanium. dent materj. 2008 jan;27(1):715. 10. galo r, ribeiro rf, rodrigues rc, pagnano vde o, mattos mda g. effect of laser welding on the titanium ceramic tensile bond strength. j appl oral sci. 2011 aug;19(4):301-5. 11. saito h, hisanaga n, okada y, hirai s, arito h. thorium-232 exposure during tungsten inert gas arc welding and electrode sharpening. ind health. 2003 jul;41(3):273-8. 12. bauer j, costa jf, carvalho cn, grande rh, loguercio ad, reis a. characterization of two ni-cr dental alloys and the influence of casting mode on mechanical properties. j prosthodont res. 2012 oct;56(4):264-71. doi: 10.1016/j.jpor.2012.02.004. 13. bumgardner jd, lucas lc: surface analysis of nickel-chromium dental alloys. dent mater. 1993 jul;9(4):252-9. 14. costa em, neisser mp, bottino ma. multiple-unit implant frames: one-piece casting vs. laser welding and brazing. j appl oral sci. 2004 sep;12(3):227-31. 15. orsi ia, raimundo lb, bezzon ol, nóbilo ma, kuri se, rovere ca, et al. evaluation of anodic behavior of commercially pure titanium in tungsten inert gas and laser welds. j prosthodont. 2011 dec;20(8):628-31. doi: 10.1111/j.1532-849x.2011.00771.x. 16. fattahi m, nabhani n, rashidkhani e, fattahi y, akhavan s, arabian n. a new technique for the strengthening of aluminum tungsten inert gas weld metals: using carbon nanotube/aluminum composite as a filler metal. micron. 2013 nov-dec;54-55:28-35. doi: 10.1016/j.micron.2013.07.004. 17. atoui ja, felipucci dn, pagnano vo, orsi ia, nóbilo ma, bezzon ol. tensile and flexural strength of commercially pure titanium submitted to laser and tungsten inert gas welds. braz dent j. 2013 nov-dec;24(6):630-4. doi: 10.1590/0103-6440201302241. 18. wang rr, welsch ge. joining titanium materials with tungsten inert gas welding, laser welding, and infrared brazing. j prosthet dent. 1995 nov;74(5):521-30. 19. bock jj, fraenzel w, bailly j, gernhardt cr, fuhrmann ra. influence of different brazing and welding methods on tensile strength and microhardness of orthodontic stainless steel wire. eur j orthod. 2008 aug;30(4):396-400. doi: 10.1093/ejo/cjn022. 20. atluri kr, vallabhaneni tt, tadi dp, vadapalli sb, tripuraneni sc, averneni p. comparative evaluation of metal-ceramic bond strengths of nickel chromium and cobalt chromium alloys on repeated castings: an in vitro study. j int oral health. 2014 sep;6(5):99-103. 21. wu sc, ho wf, lin cw, kikuchi h, lin ft, hsu hc. surface characterization and bond strengths between ti-20cr-1x alloys and low-fusing porcelain. dent mater j. 2011;30(3):368-73. 9 matos et al. 22. fernandes neto aj, panzeri h, neves fd, prado ra, mendonça g. bond strength of three dental porcelains to ni-cr and co-cr-ti alloys. braz dent j. 2006;17(1):24-8. 23. iso 9693-1:2012. dentistry—compatibility testing—part 1: metal-ceramic systems. geneva, switzerland: iso; 2012. 24. lee wv, nicholls ji, butson tj, daly ch. fatigue life of a nd:yag laser-welded metal ceramic alloy. int j prosthodont. 1997 sep-oct;10(5):434-9. 25. rocha r, pinheiro al, villaverde ab. flexural strength of pure ti, ni-cr and co-cr alloys submitted to nd:yag laser or tig welding. braz dent j. 2006;17(1):20-3. untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8652929 volume 17 2018 e18373 original article 1 dds, graduate student, department of dental materials and prosthodontics, ribeirao preto school of dentistry, university of são paulo, ribeirao preto, sp, brazil. 2 bacs, technician, department of dental materials and prosthodontics, ribeirao preto school of dentistry, university of são paulo, ribeirao preto, sp, brazil. 3 dds, master student, department of restorative dentistry, ribeirao preto scholl of dentistry, university of sao paulo, ribeirao preto, sp, brazil. 4 phd, researcher, department of dental materials and prosthodontics, ribeirao preto school of dentistry, university of são paulo, ribeirao preto, sp, brazil. 5 phd student, department of dental materials and prosthodontics, ribeirao preto school of dentistry, university of são paulo, ribeirao preto, sp, brazil. 6 cdt, technician, department of dental materials and prosthodontics, ribeirao preto school of dentistry, university of são paulo, ribeirao preto, sp, brazil. 7 phd, associate professor, department of dental materials and prosthodontics, ribeirao preto school of dentistry, university of são paulo, ribeirao preto, sp, brazil. corresponding author: fernanda de carvalho panzeri pires-de-souza university of são paulo, ribeirão preto school of dentistry, department of dental materials and prosthodontics. av. do café, s/nº. bairro monte alegre. cep 14040-904 ribeirão preto – sp. brazil phone: +55 16 3315 3973 e-mail: ferpanzeri@usp.br received: march 07, 2018 accepted: may 11, 2018 influence of aging on bond strength of artificial teeth to denture base acrylic resins etiene faria aguiar 1 , rafaella tonani 2 , fabiana de goes paiola 3 , michelle alexandra chinelatti 4 , carolina noronha ferraz de arruda 5 , julio césar souza da matta 6 , fernanda de carvalho panzeri pires-de-souza 7 aim: the aim of this study was to evaluate the bond strength of artificial teeth to different types of denture base resins when submitted to thermomechanical cycling (tmc). methods: sixty artificial mandibular first molars (trilux, vipi) were randomly divided into 3 groups according to denture base acrylic resins (vipi wave, vipi cril, and vipi cril plus, vipi). the teeth were fixed onto self-polymerizing acrylic resin bars (0.5 cm2 cross-section x 2 cm height), and the set was included in a metal flask using dental stone/silicone. after the dental stone was set, the bar was removed, and the denture base resin was packed and processed according to the group studied (vipi wave: 180 w/20 minutes + 540w/5 minutes; vipi cril and vipi cril plus: water bath at 74ºc for 9h). after polymerization, the samples were divided into 2 groups (n=10), according to the tmc treatment received (simulation of 5 years of mastication or not). the samples were submitted to tensile bond strength test (1 mm/min), and the data (mpa) were statistically analyzed (2-way anova, bonferroni, α=0.05). the fracture interfaces were evaluated using a stereomicroscope (50x). results: the bond strength results showed no statistically significant difference (p>0.05) between the resins studied. tmc was significant (p<0.05), demonstrating lower values for the bond strength of artificial teeth to vipi cril plus. the predominant fracture type was cohesive in resin. conclusions: it was concluded that there is no difference in bond strength between artificial teeth and the resins used for denture base. however, tmc decreases the bond strength values of artificial teeth and crosslink thermo-polymerizable acrylic resin. keywords: cyclic loading. acrylic resins. denture, complete. bond strength. 2 aguiar et al. introduction complete dentures have a positive effect on the quality of life of patients, not only for provides esthetics, but also function, once edentulism harms masticatory efficiency, the concentration of nutrients required by the body and the maintenance of the individual’s intermaxillary relationship1, regardless in conventional or implant supported prostheses. the dental material most used for fabrication of denture bases and tooth over the last 60 years has been acrylic resin, due to its low cost and easy processing2,3. unfortunately, dental prostheses are subject to failures and the debonding of artificial teeth accounts for approximately 22% to 30% of the repairs carried out4,5. previous studies reported that the main causes of prosthetic failures are: tooth position in the alveolar ridge, incorrect occlusion, incorrect processing, different methods of preparation, contamination of the surfaces between the artificial tooth and denture base, and excessive forces during mastication1,4,5. considering that artificial teeth are essential parts of dentures, the bond between the teeth and denture base resin must be adequate, because this factor increases the strength and durability of the dental prosthesis6, but the debonding of denture teeth may become a greater clinical problem and increases laboratory costs7. the use of acrylic artificial teeth is preferable than other materials as ceramic teeth, due to the possibility of a chemical bond to the denture base resin by polymethyl methacrylate (pmma) that is copolymerized with the cross-linking substances8. methods that may be used to increase the artificial tooth bond to the denture base resin include performing mechanical retentions, wear on the denture base surface9, and chemical treatment with monomer10, solvent, pmma or silanization6,7. studies2,7,11 conducted about the bond strength of artificial teeth and denture base regarding the effect of static compression or tensile loads12,13 and thermalcycling2. however, the dynamic forces of mastication, the influence of fatigue loads and thermomechanical on the bond strength of artificial teeth, have not been considered or limited14,15. accelerated aging conditions can indicate a performance degradation of the materials and bonding interface involved16. so, thermomechanical cycling can be used to simulate the oral condition and to evaluate the durability of the bond between the materials17. therefore, the aim of this study was to evaluate the influence of thermomechanical cycling on the bond strength of artificial teeth to different types of denture base resins. the study hypothesis was that the type of denture base resin and the thermomechanical cycling would not influence the bond strength of the artificial teeth to resins. material and methods sixty artificial mandibular first molars and 3 thermoactived acrylic resins for denture base were selected (table 1). 3 aguiar et al. sample preparation rectangular bars (cross-section of 0.5 cm2 x 2.0 cm height) were prepared with self-cured acrylic resin (vipi flash, vipi dental products, pirassununga, sp, brazil), according to methodology adapted from consani et al.18 (2012). acrylic artificial teeth were fixed on the bars with soft wax (wilson, polidental ind&com. ltda, cotia, sp, brazil). the tooth-wax bar set was included in dental stone (gesso pedra creme, gesso-rio, rio claro, sp, brazil) proportioned and mixed according to the manufacturer`s instructions and included in a metal flask (mac artigos odontologicos e protese ltda., sao paulo, sp, brazil). before hardening, five rectangular wax bars (1.5 x 3.5 x 0.5 cm) were included on the dental stone. after the dental stone hardening, the wax bars were removed and the stone molds were filled with laboratory silicone putty (zetalabor, zhermack, rovigo, italy). the artificial teeth were partially included into the silicone layer (figure 1a), and then covered with another layer of silicone (figure 1b). after dental stone insulation with petroleum jelly, the flask was filled with dental stone. a b figure 1. initial preparation of samples. a) the artificial tooth was put into the silicone layer. b) another layer of silicone covered the tooth. after stone set, the acrylic resin was included on the hole in silicon table 1. acrylic resins for denture base used in the study and their respective characteristics. material manufacturer composition processing method polymerization method (recommended by manufacturer) vipi wave microwave polymerized resin vipi ind. com. exp. imp. de produtos odontológicos ltda, pirassununga, sp, brazil powder: polymethylmethacrylate, benzoyl peroxide, biocompatible pigments; liquid: methylmethacrylate, edma, inhibitor pressing microwave oven 180 w for 20 min/540 w for 5 min vipi cril plus heatpolymerized crosslinked resin powder: polymethylmethacrylate, benzoyl peroxide, biocompatible pigments; liquid: methylmethacrylate, inhibitor fluorescent, edma water bath 74ºc for 9h vipi cril heatpolymerized resin powder: polymethyl methacrylate, benzoyl peroxide, biocompatible pigments; liquid: methylmethacrylate, inhibitor, edma water bath 74ºc for 9h edma – ethylenedimethacrylate 4 aguiar et al. once the dental stone had set, the flask was opened. the wax bars were removed and the wax residues on the ridge lap surface was removed with tap water. to improve the bond strength of the teeth to base resin, mechanical retentions were made in the surface of the ridge laps using a tungsten carbide bur (# 6, kg carbide; kg sorensen, cotia, sp, brazil) at low speed of rotation (n270, dabi atlante, ribeirao preto, sp, brazil). the operator used one bur for each ten teeth. after cleaning with compressed air, the mechanical retentions were etched with methyl methacrylate monomer19. inclusion of denture base acrylic resins the teeth were randomly separated into three groups according to the denture resin used as the base. specimens (figure 2) were made with the tooth ridge lap surface attached to the acrylic resin, which was proportioned and manipulated according to the manufacturer’s instructions. after inclusion, the flasks were submitted to 750 kgf load hydraulic pressure to remove resin excesses and, after, to 1000 kgf load for approximately 20 minutes. the flasks were polymerized as described in table 1. twenty-four hours after polymerization, the specimens were deflasked after flask cooling at room temperature. before testing, the resin bars were finished with abrasive stones, and the specimens stored in distilled water at 37°c for 7 days, to release the internal stress. thermomechanical cycling (tmc) the specimens of each group were divided into two subgroups (n=10) according to the treatment performed; with or without thermomechanical cycling (tmc). specimens that were not submitted to tmc (control group) were immediately tested after stored in water for 7 days. prior to the tmc, the specimens were fixed into rigid pvc rings (16 mm height x 21 mm in diameter) using putty silicone, and a parallelometer to verified if the specimens were perpendicular to the horizontal plane. the tmc (er 37000; erios, sp, figure 2. sample prepared. 5 aguiar et al. brazil) was performed at 1,200,000 cycles, with a load of 98 n (10 kg), and frequency of 2 hz/s. a rounded tip with 6mm in diameter was used as antagonist. the frequency used corresponds to 2 chewing movements per second20, which simulated 5 years of chewing21. bond strength test after cycling, the specimens were fitted to a device with two adjustable clips: one adapted to the portion adjacent to the tooth, simulating a hook; and the other fixed to the end of the bar. the specimens were submitted to the bond strength test (mechanical test machine, emic 1l-2000, sao jose dos pinhais, pr, brazil) with a tensile load speed of 1 mm/min. the bond strength values were recorded in mpa. the bonding area was 25 mm2 and the strength (mpa) was calculated by dividing the maximum force applied before fracture (n) by the area (mm2). statistical analysis the values of bond strength (mpa) were analyzed using 2-way anova, and bonferroni’s test at a significance level of 5% (software graphpad prism 4.0®, graphpad software, inc., la jolla, ca, usa). fracture pattern analysis the fracture patterns were analyzed with a stereomicroscope (keyence brazil, sao paulo, sp, brazil) at 50x magnification, and classified as: a) adhesive when there was debonding between resin and tooth; b) cohesive in the tooth or in the resin; c) mixed – adhesive and cohesive fractures. results bond strength the mean values of bond strength (mpa) of the artificial teeth to the denture base resins are described in table 2. none of the specimens failed during tmc test. when the resins were compared, there was no statistical difference (p>0.05) in the bond strength. there was significant reduction (p<0.05) in the bond strength values for the vipi cril plus resin, and no effect (p>0.05) on the other studied acrylic resins when submitted to tmc. there was no interaction between the factors (p=0.5062). table 2. comparison of bond strength mean values (mpa) (± standard deviations) of artificial teeth to denture base resins as a function of the thermomechanical cycling (tmc) vipi wave vipi cril plus vipi cril control 16.27 (±7.4) aa 21.52 (±8.8) aa 17.60 (6.2) aa with tmc 12.13 (±5.6) aa 12.75 (±5.5) ab 10.70 (3.4) aa different letters, lowercase in line and uppercase in column, indicate statistically significant difference (p<0.05). 6 aguiar et al. fracture type analysis figure 3 presents the fracture mode for each group. the cohesive fracture in the resin was predominant in the specimens with and without tmc. the control group for microwaved resin presented the same occurrence for mixed fractures (50%). the vipi cril plus resin presented twice the mixed fracture on the group submitted to tmc (20%) when compared with the group with no cycling (10%). discussion the aim of this study was to evaluate the tooth/resin bonding after tmc. the study hypothesis was that the type of denture base resin and the thermomechanical cycling would not influence the bond strength of the artificial teeth to resins. the results showed that there was no difference in the bond strength when the resins were compared. however, tmc reduced the bond strength of the artificial teeth to the vipi cril plus resin, which allowed the hypotheses to be rejected. the reduction in bond strength values for the vipi cril plus resin after tmc may be explained by the degradation of the interface of the ridge lap/resin bond, as the mechanical stress associated with thermal changes can induce crack propagation through bonded interface22, explaining the lower bond strength found in this group. however, the bond strength depends on the level of penetration of the monomer that plasticizes the surface and diffuses into the tooth acrylic resin13. the tmc cycles could change the ratio of monomer inter-penetration the denture tooth resulting in less strength of polymer networks formed23,24. on the other hand, no influence of tmc was observed on the bond strength values between ridge laps and the other studied resins. this can be attributed to the lower levels of residual monomer verified in these resins25. beside this, the water sorption microwave polymerized resin 90% 80% 60% 40% 20% 0% cohesive in resin cohesive in tooth mixed adhesive 70% 50% 30% 10% with tmc heat-polymerized cross-linked resin heath-polymerized resin control cohesive in resin cohesive in tooth mixed adhesive figure 3. distribution, in percentage, of the fracture types for each group. (tmc = thermomechanical cycling) 7 aguiar et al. and thermal expansion coefficient are different and inherent to each material26. based on this, the diffusion of water molecules at the interface between the artificial tooth and acrylic resin27 and different thermal expansion coefficients of the resins may result in differences in the bonding ability. the tmc was used in this study because it can simulate intraoral conditions more closely. the heating–cooling process associated to simulated chewing can result in repeated expansion and contraction of the tooth and acrylic resin, stressing the bonding area, producing fatigue of the denture tooth/ridge laps interface, and decreasing the bond strength. all groups and resins presented a higher prevalence of the cohesive fractures in the resin bars irrespective of submission to tmc test. this can be explained by the ridge laps mechanical retentions, which were able to improve the retentiveness. the thermomechanical cycling is a suitable method for simulation of degradation of bond strength because the mechanical stress can induce crack propagation through bonded interfaces and the thermal changes can speed up this process28. however, there is no evidence that bonding failures in clinical practice occur as a result of thermomechanical stress and whether failures occur because of leakage in one or another layer in the bonded structure, which must be dependent on the glass transition temperatures of the bonded material28. this study has several limitations. one is that it is not possible to know the exact difference in composition of the heat-polymerized and cross-linked heat-polymerized resin due to the lack of available manufacturer’s information. further study is needed of the chemical changes in the bonding surface between the resin and ridge laps surface. to overcome the limitations of in vitro tests, the bonding between denture teeth and acrylic resins should be evaluated using different methods. it was concluded that there is no difference in bond strength between artificial teeth and the resins used for denture base. however, tmc decreases the bond strength values of artificial teeth and crosslink thermo-polymerizable acrylic resin. list of abbreviations: tmc – thermomechanical cycling pmma – polymethyl methacrylate pvc – polyvinyl chloride conflict of interest disclosure all authors deny any financial and personal relationships with other people or organizations that could inappropriately influence the study. acknowledgements this study was supported with scholarship by sao paulo state research foundation fapesp (grant number: 13/13529-0). 8 aguiar et al. references 1. tôrres acsp, maciel aq, de farias db, de medeiros akb, vieira fptv, carreiro adfp. technical quality of complete dentures: influence on masticatory efficiency and quality of life. j prosthodont. 2017 nov 9. doi: 10.1111/jopr.12703. 2. barbosa db, barão var, monteiro dr, compagnoni ma, marra j. bond strength of denture teeth to acrylic resin: effect of thermocycling and polymerisation methods. gerodontology. 2008 dec;25(4):237-44. doi: 10.1111/j.1741-2358.2008.00218.x. 3. cunningham jl. shear bond strength of resin teeth to heat-cured and light-cured denture base resin. j oral rehabil. 2000 apr;27(4):312-6. 4. kurt m, sarac ys, ural c, sarac d. effect of pre-processing methods on bond strength between acrylic resin teeth and acrylic denture base resin. gerodontology. 2012 jun;29(2):e357-62. doi: 10.1111/j.1741-2358.2011.00480.x. 5. patil sb, naveen bh, patil np. bonding acrylic teeth to acrylic resin denture bases: a review. gerodontology. 2006 sep;23(3):131-9. 6. chung k-h, chung cy, chung cy, chan dcn. effect of pre-processing surface treatments of acrylic teeth on bonding to the denture base. j oral rehabil. 2008 apr;35(4):268-75. doi: 10.1111/j.13652842.2007.01779.x. 7. meng gk, chung kh, fletcher-stark ml, zhang h. effect of surface treatments and cyclic loading on the bond strength of acrylic resin denture teeth with autopolymerized repair acrylic resin. j prosthet dent. 2010 apr;103(4):245-52. doi: 10.1016/s0022-3913(10)60038-8. 8. yanikoglu dn, duymus dz, bayindir df. comparative bond strengths of autopolymerising denture resin and light cured composite resin to denture teeth. int dent j. 2002 feb;52(1):20-4. 9. zuckerman gr. a reliable method for securing anterior denture teeth in denture bases. j prosthet dent. 2003 jun;89(6):603-7. 10. saavedra g, valandro lf, leite fpp, amaral r, ozcan m, bottino ma, et al. bond strength of acrylic teeth to denture base resin after various surface conditioning methods before and after thermocycling. int j prosthodont. 2007 mar-apr;20(2):199-201. 11. de chaves cal, regis rr, machado al, de souza rf. effect of ridge lap surface treatment and thermocycling on microtensile bond strength of acrylic teeth to denture base resins. braz dent j. 2009;20(2):127-31. 12. sanders jl, levin b, reitz p v. comparison of the adaptation of acrylic resin cured by microwave energy and conventional water bath. quintessence int. 1991 mar;22(3):181-6. 13. takahashi y, chai j, takahashi t, habu t. bond strength of denture teeth to denture base resins. int j prosthodont. 2000 jan-feb;13(1):59-65. 14. drummond jl, bapna ms. static and cyclic loading of fiber-reinforced dental resin. dent mater. 2003 may;19(3):226-31. 15. garoushi s, lassila lvj, tezvergil a, vallittu pk. static and fatigue compression test for particulate filler composite resin with fiber-reinforced composite substructure. dent mater. 2007 jan;23(1):17-23. 16. kappert pf, kelly jr. cyclic fatigue testing of denture teeth for bulk fracture. dent mater. 2013 oct;29(10):1012-9. doi: 10.1016/j.dental.2013.07.001. 17. schneider rl, curtis er, clancy jms. tensile bond strength of acrylic resin denture teeth to a microwaveor heat-processed denture base. j prosthet dent. 2002 aug;88(2):145-50. 18. consani rlx, soave t, mesquita mf, sinhoreti mac, mendes wb, guiraldo rd. effect of repeated microwave disinfections on bonding of different commercial teeth to resin denture base. gerodontology. 2012 jun;29(2):e553-9. doi: 10.1111/j.1741-2358.2011.00516.x. https://www.ncbi.nlm.nih.gov/pubmed/29120095 9 aguiar et al. 19. lagouvardos pe, polyzois gl. shear bond strength between composite resin and denture teeth: effect of tooth type and surface treatments. int j prosthodont. 2003 sep-oct;16(5):499-504. 20. fontijn-tekamp a, slagter ap, van der bilt a, van’t hof ma, witter dl, kalk w, et al. biting and chewing in overdentures, full dentures, and natural dentitions. j dent res. 2000 jul;79(7):1519-24. 21. delong r, sakaguchi rl, douglas wh, pintado mr. the wear of dental amalgam in an artificial mouth: a clinical correlation. dent mater. 1985 dec;1(6):238-42. 22. chai j, takahashi y, takahashi t, habu t. bonding durability of conventional resinous denture teeth and highly crosslinked denture teeth to a pour-type denture base resin. int j prosthodont. 2000 marapr;13(2):112-6. 23. vallittu pk. fibre-reinforced composites in root canal anchoring. int dent south africa. 2006;8:20-7. 24. barbosa db, monteiro dr, barão var, pero ac, compagnoni ma. effect of monomer treatment and polymerisation methods on the bond strength of resin teeth to denture base material. gerodontology. 2009 sep;26(3):225-31. doi: 10.1111/j.1741-2358.2008.00262.x. 25. yunus n, harrison a, huggett r. effect of microwave irradiation on the flexural strength and residual monomer levels of an acrylic resin repair material. j oral rehabil. 1994 nov;21(6):641-8. 26. amin wm. durability of acrylic tooth bond to polymeric denture base resins. eur j prosthodont restor dent. 2002 jun;10(2):57-61. 27. marra j, de souza rf, barbosa db, pero ac, compagnoni ma. evaluation of the bond strength of denture base resins to acrylic resin teeth: effect of thermocycling. j prosthodont. 2009 jul;18(5):438-43. doi: 10.1111/j.1532-849x.2009.00478.x. 28. gale ms, darvell bw. thermal cycling procedures for laboratory testing of dental restorations. j dent. 1999 feb;27(2):89-99. untitled 1 volume 16 2017 e17070 original article 1 universidade federal de uberlândia – ufu, school of dentistry, department of endodontics, school of dentistry, uberlândia, mg, brazil. 2 universidade federal de uberlândia, school of dentistry, department of occlusion, fixed prosthodontics and dental materials, uberlândia, mg, brazil. 3 universidade federal de uberlândia, school of math, department of statistic, uberlândia, mg, brazil. corresponding author: maria antonieta v. c. de oliveira, department of endodontics, school of dentistry, federal university of uberlândia avenida pará 1720, campus umuarama, bloco 4l, sala 4la 28 uberlândia, mg, brazil. zip-code: 38400-902. phone: 55 34 32258103 e-mail: mariaoliveira@ufu.br received: june 26, 2017 accepted: november 23, 2017 analysis of the presence of filling material in oval root canals using computed microtomography after endodontic retreatment performed by different techniques maria antonieta veloso carvalho oliveira1, jady karine borelli1, kellen cristina mendes azevedo1, luís henrique araújo raposo2, lúcio borges de araújo3, nayara rodrigues nascimento oliveira tavares1 aim: the aim of this study was to evaluate the presence of filling material in oval root canals after endodontic retreatment performed by different techniques, considering the area (mm2), location and root third using computed microtomography (µ-ct). methods: thirty human lower central incisor underwent biomechanical preparation, root filling and filling removal using two techniques (n=15): mnmanual retreatment technique (gates glidden burs and stainless steel manual files); and rtrotary retreatment technique (protaper universal and protaper retreatment systems). cross-sectional images of the teeth were made using µ-ct to identify the presence of remaining filling in all root thirds of the canal walls. the remaining material detected in 150 µ-ct sections was identified and its area quantified (mm2) for each root third individually. results: data analysis showed no difference in the remaining area of filling material (p=0.8611) for the both techniques. higher frequency of remaining material was verified in the lingual wall of the root canals. regardless of the retreatment technique, the apical third showed lager areas of remaining filling material. more areas of remaining material were detected in the cervical third of the rt group, whereas for the mn group, most areas were observed in the middle and apical thirds. conclusion: according to our results, no significant differences were verified between the efficiency of the rotary and manual techniques for removing filling material due to the interferences caused by the root canal anatomy. keywords: retreatment. micro-computed tomography. root canal preparation http://dx.doi.org/10.20396/bjos.v16i0.8651185 mailto:mariaoliveira@ufu.br 2 oliveira et al. introduction the removal of obturation materials from the root canal system is a primary objective in  root  canal  retreatment  procedures, to eliminate or at least reduce the amount of microorganisms in the root canals. in addition to the complete removal of the material filling, it is essencial the cleaning and shaping of root canal system and other obturation for a favorable prognosis1. there are several techniques that can be used to remove filling materials of root canal during endodontic retreatments, such as, hand files, reciprocating systems, ultrasonic instruments, rotary files, solvents, lasers and combinations of these methods1-11. several studies have demonstrated that the complete removal of the root filling from the canal walls is not always possible1-5,7-12. the remaining filling material after retreatment has been described by its presence8,9, area3,5,7,10,12 and volume11,13, through different analytical techniques using microscopy9, radiographies3,8,10, photographies5,12, and computed tomography4,6,11,13. however, the studies do not report in which canal walls filling material remained in larger amounts5. this specific information would allow for better understanding of the limitations involving different retreatment techniques and instruments. thus, the aim of this study was to evaluate the presence of filling material in oval root canal after endodontic retreatment performed by different techniques, considering the area (mm2) and location (root aspect and third) of the remaining material using micro-computed tomography (µ-ct). the null-hypothesis tested was that no differences would be detected between the efficiency of the manual and rotary instrumentation techniques in the removal of filling material from oval root canals. material and methods thirty human lower central incisors with single root canal were selected (following informed consent approved by the committee for ethics in research of the federal university of uberlândia #887.517). the selected teeth had no previous endodontic treatment, intraradicular post, fractures or extensive damage in crowns. teeth showing apical curvature, incomplete root formation, calcification or more than one root canal were excluded, using periapical radiograph. coronal opening was performed using round diamond burs #1016 (kgsoresen, barueri, brazil) and tapered carbide burs with non-cutting tip, endo-z (dentsply-maillefer, ballaigues, switzerland). the root canal was located and explored with #10 k-file (dentsply-maillefer) and the working length determined subtracting 1 mm from the length measured when the tip of the file was first observed emerging from the apical foramen. teeth were instrumented with a step-down technique, using k-files (dentsply-maillefer) up to #30 memory file. irrigation was performed with 1.0 ml of 1% sodium hypochlorite after each instrument and 5.0 ml of physiological saline for final rinse. canals were dried with paper points and filled with gutta-percha and mineral trioxide aggregate-based sealer, mta fillapex (ângelus, londrina, brazil), by the lateral condensation technique. coronal access was provisionally restored with zinc-oxide temporary cement (biodinâmica, ibiporã, brazil). the specimens were then stored (100% humidity at 37ºc) for 3 weeks to allow for complete setting of the sealer. 3 oliveira et al. the teeth were randomly assigned into two experimental groups (n=15): mnmanual retreatment technique; and rtrotary retreatment technique. for mn group, #2 and 3 gates-glidden burs (dentsply-maillefer) were used to initially remove the filling material from the cervical and middle root thirds. then, eucalyptol-based solvent (biodinâmica) was inserted in the filling with #15 and #20 k-files (dentsply-maillefer) using oscillatory movements and the material was laterally removed with hedströem files (dentsply-maillefer). canal repreparation was performed with k-files up to #30 file, in order to obtain 0.30 mm final apical diameter. for rt group, d1-d3 protaper universal retreatment (dentsply-maillefer), were used to prepare the cervical, middle and apical thirds, respectively. the finishing of the canal repreparation was done with f1-f3 protaper universal (dentsply-maillefer) to obtain a final apical diameter compatible to that produced by the #30 k-file, since the last file from the retreatment system (d3) correspond only to the diameter of a #20 k-file. the instrument was used in electric motor x-smart (dentsply – maillefer) five times each. the removal procedure was interrupted when filling material was no longer detected in the instruments5,7,8,10. canal irrigation and drying was performed as described before. one experienced operator conducted all endodontic procedures. afterwards, teeth were mounted on a custom attachment base and scanned with the µ-ct scanner (skyscan 1174v2; skyscan, kontich, belgium) at an isotropic pixel size of 19.6 µm, 90 kv, 112 µa, resulting in the acquisition of 800-1.000 transverse cross sections per tooth. the scanning procedure was carried out by 360º rotation around the vertical axis; camera exposure time of 2.600 ms, rotation step of 0.6°, frame averaging of 2 and medium filtering of the data were applied. x-rays were filtered with aluminum (500 μm) and copper (38 μm) filters and a flat field correction were performed prior to scanning to correct for variations in the pixel sensitivity of the camera. a thousand µ-ct cross-sections were individually analyzed for each specimen, seeking for the presence of remaining filling material and identification of its location in the canal walls. the sections were then divided according to the root thirds (cervical, middle, and apical) for each specimen (fig. 1). after, 150 sections were selected for each specimen, with 50 sections per each third. the criterion for selection was defined by picking the sections following the first 100 sections of each root third. the selected sections were then employed for quantifying the remaining filling material area in the root thirds using the imagej software (national institutes of health, eua) (fig. 2). the normality test was not applied during data processing because of the large sampling (n=2250), thus the estimators approached a normal distribution by the central limit theorem14. the t test for two independent samples was applied for comparisons between the techniques, and one-way analysis of variance (anova) was used for comparisons among thirds. a multiple comparison test (tukey test) was applied to check the differences among thirds. all tests were conducted using 5% significance level in the spss statistics software for windows, version 20.0 (ibm copr. releases in 2011, armonk, ny, usa). results remaining filling material was found after retreatment in 1.918 sections for the rt group (85.24%) and in 1.462 sections for the mn group (64.98%). higher frequency of remaining filling material was observed in the buccal wall of the root canal for the both groups (table 1). 4 oliveira et al. no significant differences (p=0.861) were detected on the total area of remaining filling material between the both experimental groups (table 2). however, significant differences were verified between the groups when comparing the remaining filling in the root thirds (table 3). more areas of remaining filling material were observed in the apical third of specimens from rt group, whereas for mn group, remaining filling was detected most in the middle and apical thirds. the apical third showed the largest amount of remaining filling material for both groups. discussion the hypothesis tested was accepted since no significant differences were observed between the efficiency of the manual and rotary instrumentation techniques in the removal of filling material from oval root canals. although no significant differences were observed between the both experimental groups, the rotary technique would be expected to remove more filling material since these instruments are designed to remove filling towards the apex-crown direction1,5 and because the frictional heat generated by the files can soften the gutta-percha, allowing to reach the working length more easily5. adding the two analyzed groups the remaining filling material was observed in 75% of all the µ-ct sections analyzed in this study. according to our results, none of the retreatment techniques tested was able to completely remove filling material from the root table 1. frequency distribution (%) of remaining filling material presence after retreatment in the root canal walls according to the experimental groups group/wall distal (%) mesial (%) buccal (%) lingual (%) p value rt 14.81d 19.56c 45.25a 20.39b < 0.001 mn 18.10b 9.99d 13.17c 58.74a < 0.001 p value 0.005 < 0.001 < 0.001 < 0.001 lowercase letters indicate differences between columns (between groups). table 2. mean (±sd), minimum and maximum area (mm²) of remaining filling material in the sections according to the experimental groups group mean (mm²) minimum (mm²) maximum (mm²) rt 0.08+0.06 0.00 0.31 mn 0.08+0.08 0.00 0.50 table 3. mean (±sd) area (mm2) of remaining filling material in the root thirds according to the experimental groups root third rt (mm²) mn (mm²) p value cervical 0.06+0.06aa 0.02+0.04ba 0.0001 middle 0.07+0.06bb 0.08+0,07ab 0.0040 apical 0.10+0.06bc 0.12+0.07ac 0.0001 p value 0.0001 0.0001 uppercase letters indicate statistical difference between lines (within root thirds) and lowercase letters indicate differences between columns (between groups). tukey test (p< 0.05). 5 oliveira et al. canals. some endodontic treatment failures are related to the permanence of microorganisms in the root canals, so it is very important to assure complete removal of root filling material during endodontic retreatment in order to allow proper canal cleaning11. the possible cause for the permanence of remaining filling material in the root canals after the use of both retreatment techniques may be credited to the failure of instruments to completely reach all canals walls due to anatomic interferences5,7. the interference of anatomical complexities in the capacity of instrumenting root canals was evidenced by a previous study that analyzed human mandibular incisors before and after biomechanical preparation using digital radiographs taken in the buccolingual and mesiodistal direction and micro-computed tomography sections15. this study has reported that after instrumentation, the canal remained without action of files in a region described as critical instrumentation area15. in some cases, these unprepared areas represented 64.2% of the root canal area, being located in the lingual and buccal walls of the root canal. the critical instrumentation area previously described corresponds to the same regions that presented the a b c figure 1. sections of a rt specimen before removal of the filling material: cervical (a), middle (b) and apical (c) thirds. m d l b figure 2. section of the middle third mn specimen after removal of the filling material, showing remaining material (yellow arrows) in the root canal walls (m-mesial, d-distal, bbuccal and l-lingual aspects). 6 oliveira et al. greatest amount of remaining filling material in the present study. it was clearly possible to observe that the filling remained in the areas where the canal has not been instrumented during the retreatment procedure (figures 3a and 3b). this fact probably occurred when trying to remove the filling material during retreatment, which was probably, pressed to these areas which are inaccessible to files or where the instruments had limited action15. the critical instrumentation area of human mandibular incisors occurs on the buccal and lingual walls because the root canal is not conical as observed in conventional buccolingual radiographs, but it presents irregular shape when analyzed in the mesiodistal view by x-ray or tomography exams15. the classical periapical x-ray gives poor information about root canal morphology. in this study the micro-computed tomography was chosen because provides detailed three-dimensional reconstructions of root canal, and accurate images of the endodontic space. micro-ct can also be used to evaluate the ability of endodontic instruments to clean the root canal system16. endodontic hand and rotary files are commonly designed to work in canals with conical shape6, what affects the action of files in regions presenting irregular configurations. a previous study, observed that the files could be deviated from the original canal path to the buccal aspect when instrumenting lower incisors with oval canals17. consequently, non-instrumented areas may remain on the lingual wall of the root canal. the non-instrumented regions reported in the previous study18, correspond to the critical instrumentation area formerly described15, and to the zones presenting remaining filling material found in our study. these anatomical interferences are not present only in mandibular incisors, but in all teeth with roots presenting oval shaped canals, as the distal root of mandibular molars11. additionally, it was found that irrespective of the retreatment technique, the apical third showed the largest areas of remaining filling material. other studies have also presented similar results8,9,19, as the apical third is a critical area and requires enlargement for proper cleaning and shaping of canals17. the canal enlargement in this region may lead to apical deviations, which can play an important role in the retention of filling material during canal retreatment18,19. thus, in order to reduce the limitations imposed by anatomical interferences of teeth with oval root canals, it is suggested to a b figure 3.post–instrumentation sections of the middle third showing: area file action (blue line), and remaining filling material (yellow arrows) in the rt (a) and mn (b) groups. 7 oliveira et al. perform an enlargement of the canal entrance during retreatments, allowing to direct the files to the buccal and lingual walls. additionally, active ultrasonic irrigation with a straight edge tip should be used, since ultrasound devices are effective in areas difficult to be accessed20, due to its great irrigation capacity through oscillation in canal limits, thereby eliminating pulp tissue, filling material and dentin debris21. according to the results of the present study, no significant differences were observed between the efficiency of filling material removal for the manual and rotary retreatment techniques evaluated, probably due to the interferences caused by the root canal anatomy. in addition, clinicians should take into account that large amounts of filling material may remain on the buccal and lingual walls when performing retreatment in teeth presenting oval canals. efforts should be focused on removing the filling material of these areas through the association of techniques and instruments. acknowledgments the authors are indebted the fundação de amparo à pesquisa do estado de minas gerais (fapemig) for the financial support of the project. references 1. iriboz e, sazak öveçoğlu h. comparison of protaper and mtwo retreatment systems in the removal of resin-based root canal obturation materials during retreatment. aust endod j. 2014 apr;40(1):6-11. doi: 10.1111/aej.12011. 2. joseph m, ahlawat j, malhotra a, murali-rao h, sharma a, talwar s. in vitro evaluation of efficacy of different rotary instrument systems for gutta percha removal during root anal retreatment. j clin exp dent. 2016 oct 1;8(4):e355-e360. 3. crozeta bm, silva-sousa yt, leoni gb, mazzi-chaves jf, fantinato t, baratto-filho f, et al. micro-computed tomography study of filling material removal from oval-shaped canals by using rotary, reciprocating, and adaptive motion systems. j endod. 2016;42(5):793-797. 4. ersev h, yilmaz b, dinçol me, dağlaroğlu r. the efficacy of protaper universal rotary retreatment instrumentation to remove single gutta-percha cones cemented with several 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reciproc file systems in the removal of root filling material: an ex vivo micro-computed tomography study. j endod. 2016 feb;42(2):315-9. doi: 10.1016/j.joen.2015.11.005. 14. bussab wo, morettin pa. [basic statistics]. 5th ed. são paulo: saraiva; 2006. p 526. portuguese. 15. oliveira mav, venâncio jf, pereira ag, raposo lha, biffi jcg. critical instrumentation area: influence of root canal anatomy on the endodontic preparation. braz dent j. 2014;25(3):232-6. 16. crăciunescu el, boariu m, ioniţă c, pop dm, sinescu c, romînu m, et al. micro-ct and optical microscopy imagistic investigations of root canal morphology. rom j morphol embryol. 2016;57(3):1069-1073. 17. duarte mah, só mvr, cimadon vb, zucatto c, vier-pelisser fv, kuga fv. effectiveness of rotary or manual techniques for removing a 6-year-old filling material. braz dent j. 2010;21(2):148-52. 18. oliveira mav, alves ld, pereira lha, biffi jcg. influence of flexion angle of files on the decentralization of oval canals during instrumentation. braz oral res. 2015;29. pii: s180683242015000100273. doi: 10.1590/1807-3107bor-2015.vol29.0078. 19. topçuoğlu hs, düzgün s, kesim b, tuncay o. incidence of apical crack initiation and propagation during the removal of root canal filling material with protaper and mtwo rotary nickel-titanium retreatment instruments and hand files. j endod. 2014 jul;40(7):1009-12. doi: 10.1016/j.joen.2013.12.020. 20. grischke j, müller-heine a, hülsmann m. the effect of four different irrigation systems in the removal of a root canal sealer. clin oral investig. 2014 sep;18(7):1845-51. doi: 10.1007/s00784-013-1161-6. 21. souza ma, motter ft, fontana tp, ribeiro mb, miyagaki dc, cecchin d. influence of ultrasonic activation in association with different final irrigants on intracanal smear layer removal. braz j oral sci. 2016 jan-mar;15(1):16-20. revista fop n 13 1609 the effects of age and gender on cd3+ and cd19+ lymphocyte in gingival tissue and peripheral blood: an animal study turgut demir1; varol canakci1; cankat kara1; cenk fatih canakci1; fuat erdem2; mustafa atasever3 1department of periodontology, atatürk university, faculty of dentistry, erzurum, turkey 2department of internal medicine, atatürk university, faculty of medicine, erzurum, turkey 3department of “food science and technology”, atatürk university, faculty of veterinary science, erzurum, turkey received for publication: may 26, 2008 accepted: september 10, 2008 correspondence to: turgut demir atatürk üniversitesi dis hekimligi fakültesi periodontoloji anabilim dali 25240, erzurum,turkey e-mail: turgdemir@hotmail.com a b s t r a c t aim: the aim of the present study was to determine ageand gender-related values for healthy mice of cd3+ t and cd19+ b lymphocytes and cd3+/ cd19+ (t/b) ratios in peripheral blood and gingival tissue by the flow cytometry technique. methods: the study was carried out on periodontally healthy 60 balb/c mice. they were divided into five groups according to the their age (newborn, weaning, puberty, adult and elder). males and females were equally represented in each group. cd3+ and cd19+ t lymphocytes and cd3+/cd19+ ratio values in gingival tissue and peripheral blood were determined using flow cytometry in the biopsy samples. results: there were no significant differences in the cd3+ and cd19+ lymphocytes, and cd3+/ cd19+ ratios in gingival tissue for all age groups (p>0.05). mean relative number of peripheral blood cd3+ t lymphocyte indicated a decrease in puberty group compared to the other groups (p<0.05), while relative number of peripheral blood cd19+ b lymphocyte increased in adult and aged group. the peripheral blood cd3+/ cd19+ t/b lymphocytes ratios decreased in adult and aged group. conclusions: the results of the present study showed that differences were present in periods of life and gender in peripheral blood of mice. moreover, significantly differences were found between genders in gingival tissue. key words: lymphocytes, cd3+, cd19+, age, gender, gingiva, peripheral blood, mice. i n t r o d u c t i o n although periodontal bacteria are the causative agents in periodontal diseases, subsequent progression and disease severity are thought to be determined by the host immune response1. social and behaviour modulations, and genetic or epigenetic traits of the host, each of which influenced and/or modulated by the host’s immune and inflammatory responses. the clinical entity of the periodontal disease results from the interaction of periodontopathic plaque bacteria and host immune response mechanisms2-3. the infiltrate in periodontal disease contains mononuclear cells, which are mainly transmigrated mononuclear phagocytes and lymphocytes. whereas t lymphocytes predominate in the established chronic lesion, the proportion of b cells and plasma cells increases with the progression of the disease4-7. the immune response is promoted by secretion of bacterial products and involves t and b lympho cytes and macrophages. in fact, clear differences in the degree of inflammation and secreted cyto-kines were observed when periodontitis patients were compared with normal healthy individuals8-10. some studies have suggested that b cells occur in larger numbers than t cells in chronic periodontitis 11-12 whereas in a study on progressive periodontitis, t cells and b cells were found to occupy similar proportions13. recently study show that the cd3+/ cd19+ (t/b) ratio in gingival tissue was about 4.2 times higher in the adult periodontitis than in the localized prepubertal periodontitis units. the corresponding ratio for peripheral blood was 1.614. it is suggested that certain differences may existed in the local defense mechanisms in different periodontal diseases. in order to understand the pathogenesis of periodontal diseases, the systemic factors and conditions that will affect the prevalence, progression and severity of periodontal diseases should be well observed and considered15. age, sex, stress levels, nutrition, systemic diseases, exercise, smoking and/or alcohol consumption habits play an important role that can affect the pathogenesis of different periodontal diseases15-21. however, the systemic immune braz j oral sci. july/september 2008 vol. 7 number 26 1610 response is affected by age, sex and other factors19-22. if confounding factors such as stress levels, nutrition, systemic diseases, exercise, smoking and/or alcohol consumption habits are eliminated by means of periodontally healthy animal model, the study would standardize and we are able to clarify effects of age and gender on relative numbers of t and b lymphocyte, thanks to the short lifetime of the these animals. to our knowledge, there is no published study about effects of age and gender on relative number of cd3+ t cd19+ b lymphocytes in both gingival tissue and peripheral blood of periodontally healthy animals. the aim of the present study was to determine ageand gender-related values for healthy mice of cd3+ t and cd19+ b lymphocytes and cd3+/ cd19+ (t/b) ratios in peripheral blood and gingival tissue by the flow cytometry technique. material and methods animals healthy 60 balb/c mice were obtained from medical experimental practice and research centre, atatürk university. the animals were maintained under a 12/12 h dark/light cycle at constant temperature 21±2 ºc. each mouse was placed to a separate cage. the mice’s diet was stored at 4.5 ± 0.50c in plastic containers and handled with plastic gloves and appropriated with utensils to avoid contamination. the diet was placed in shallow glass food cups with stainless steel follow-through disks to reduce food spills. the average of life time of the mice were 2-3 years (19).they were separated into five groups according to the life expectancy23: group i (newborn, 1-10 days old), group ii (age at weaning, 21-28 days old), group iii (age of sexual maturity, puberty ,7-8 weeks old), group iv (adult , 8 months old), and group v (the-aged, 14 and over). males and females were equally represented in each group. ethics: the study protocol was reviewed and approved by atatürk university medical experimental practice and research centre ethical committee. clinical evaluation the clinical evaluation consisted of plaque index 24, gingival index25 and probing pocket depths to determine gingival or periodontal health. the measurements were made in the medical experimental practice and research centre, atatürk university, by the same periodontist. the numerical scores of the plaque index and gingival index were obtained according to the formula per mice = sum of individual scores /number of anterior teeth present for each mouse, and subsequently group score was calculated by adding together the individual scores and dividing the total into the number of mice included. the pocket depths were recorded by measuring the distance from the free gingival margin to the base of the pocket with a thin wire exerting a constant force of 1 gr. examiner variability in using the dental examination criteria were tested by performing duplicate examinations on 12 randomly selected mice on consecutive days. corresponding percentages of agreement were 97 % for plaque index and gingival index and 99% for probing depth. thus, these clinical measurements were used to determine periodontal health of mice. collection of blood and tissue samples all of the mice were killed by cardiac puncture after anaesthesia with thiopental. blood was removed directly from the heart by injection. blood (minimal 0.5 ml) collected into edta-containing hemogram tubes. they were sent to haematology laboratory at once. gingiva was peeled from around the mandibular and maxillary incisors in upper and lower jaw and separated from tongue with scalpel and obtained gingiva cut into very small pieces with a scalpel and was resuspended in phosphate buffered solution (pbs). lymphocyte subset identification t cells were defined as those cells express-ing the cd3+ antigen, and b cells were defined as those cells expressing cd19+. lymphocytes levels blood (100 µl) was incubated with antibody (10µl) for 15 min at room temperature. after incubation the samples lysed and fixed with 500 µl optilyse® c. finally, the cells were washed and resuspended in 500 ml of phosphate buffered solution (pbs) for flow cytometry analysis. gingiva was peeled from upper and lower jaw with scalpel and obtained gingiva cut into very small pieces with a scalpel. tissues were transformed into suspension through filtration. for lymphocyte subset analysis a flow cytometer (coulter epics®xl-mcl) was used. the computer-assisted evaluation was made with a commercially available software program (coulter system ii software version 2.0). with the use of volume and side scatter, lymphocytes were gated and specific fluorescence dot blot. the absolute and the relative (proportional) count of each lymphocytes subpopulation were calculated. monoclonal antibodies the following monoclonal antibodies used: rat antimouse cd19-fitc (pn im3233), rat anti-mouse cd3fitc (pn im2768). igg2a (rat) isotypic control (im1271) was used as control. (data sheet immunotech, beckman coulter). statistical analysis the significance of differences in number of t and b lymphocytes in groups was determined by two factor analysis. duncan’s multiple range test was used to determine differences among groups. student’s t-test was braz j oral sci. 7(26):1609-1613 the effects of age and gender on cd3+ and cd19+ lymphocyte in gingival tissue and peripheral blood: an animal study 1611 used for determine difference between male and female in each group. a value of p<0.05 was considered to be significant. all values are expressed as mean ± standard deviation. for these procedures, spss for windows (version 11.0) was used. r e s u l t s the mean relative number of cd3+ t lymphocytes and cd19+ b lymphocytes and cd3+/ cd19+ (t/b) ratios in gingival tissue and peripheral blood of periodontally healthy balb/c mice according to age group and gender are shown in table 1. there were no significant differences in the total number of cd3+ and cd19+ in gingival tissue for all age groups (p>0.05). in addition, the cd3+/ cd19+ t/b lymphocytes ratios in gingival tissue did not differ in all age groups (p>0.05).the mean relative number of cd3 + t lymphocytes in gingival tissue for group i (newborn) (p<0.05), group iii (puberty) (p<0.05), and group iv (adult) (p<0.05) in male was higher than in female. the relative number of cd19+ b lymphocytes in gingival tissue for group ii (weaning) (p<0.05), group iii (puberty) (p<0.05), and group iv (adult) (p<0.05) in male was higher than in female. but, our data show that cd3+/ cd19+ ratios in gingival tissue were similar for all age table 1 ageand gender-related change the relative number of tand b-lymphocytes in gingival tissue and peripheral blood ( n= 12 each, female n=6 and male n=6). group when considering the gender differences (p>0.05). mean relative number of peripheral blood cd3 + t lymphocyte indicated a decrease in group ii (weaning) and group iii (puberty) compared to the other groups (p<0.05), while relative number of peripheral blood cd19+ b lymphocyte increased in group iv (adult) and group v (aged). the peripheral blood cd3+/ cd19+ t/b lymphocytes ratios decreased in group iv (adult) and group v (aged) mice. significant distinction occurring with regard to statistically and decrease at this rate is stemmed from the fact that the increase in cd19+ b lymphocytes is larger than that of cd3+ t lymphocytes. the mean relative number of cd3+ t lymphocytes in peripheral blood for group i (newborn) (p<0.05), and group ii (weaning) (p<0.05) in male was lower than in females, while for group iii (puberty) (p<0.05), group iv (adult) (p>0.05), and group v (aged) (p>0.05) in male higher than in female. the mean relative number of cd19 + b lymphocytes and cd3+/ cd19+ (t/b) ratios in peripheral blood were similar for all age group when considering the gender differences (p>0.05). d i s c u s s i o n it is known that during the transition from gingivitis to periodontitis, a characteristic change in the predominant a,b,c,d; p < 0.05, significant differences among group i, ii, iii, iv and v. x, y; p< 0.05. significant differences between gender in the same group braz j oral sci. 7(26):1609-1613 the effects of age and gender on cd3+ and cd19+ lymphocyte in gingival tissue and peripheral blood: an animal study group i ( newborn) group ii (weaning) group iii (puberty) group iv (adult) group v (aged) p value gi ngival ti ssue cd3 + total male (n=6) female (n=6) cd19 + total male (n=6) female (n=6) cd3 + /cd19 + total male (n=6) female (n=6) 30.6 ± 4.1 33.7 ± 3.0 x 27.5 ± 1.9 y 9.9 ± 1.5 9.2 ± 1.7 9.0 ± 1.4 3.4 ± 0.8 3.8 ± 0.9 3.1 ± 0.5 28.9 ± 2.7 28.8 ± 2.8 29.0 ± 2.8 9.3 ± 1.8 10.3 ± 1.4 x 8.3 ± 1.6 y 3.2 ± 0.8 2.9 ± 0.7 3.6 ± 0.8 31.2 ± 2.7 32.7 ± 2.9 x 29.7 ± 1.8 y 10.5 ± 1.7 11.3 ± 1.0 x 9.7 ± 1.8 y 3.0 ± 0.5 2.9 ± 0.4 3.1 ± 0.5 32.7 ± 4.5 35.8 ± 3.1 x 29.5 ± 3.4 y 9.0 ± 1.9 10.2 ± 1.7 x 7.8 ± 1.3 y 3.8 ± 0.9 3.6 ± 0.8 3.9 ± 1.1 30.5 ± 3.6 31.0 ± 4.7 30.2 ± 2.4 8.6 ± 1.4 8.5 ± 1.9 8.7 ± 0.8 3.6 ± 0.5 3.7 ± 0.7 3.5 ± 0.2 p>0.05 p>0.05 p>0.05 p>0.05 p>0.05 p>0.05 p>0.05 p>0.05 p>0.05 peripheral blood cd3 + total male (n=6) female (n=6) cd19 + total male (n=6) female (n=6) cd3 + /cd19 + total male (n=6) female (n=6) 51.7 ± 7.8 a 45.8 ± 6.4 a x 57.7 ± 3.2 a y 10.4 ± 2.9 a 9.7 ± 1.9 a 11.2 ± 3.8 a 5.2 ± 1.3 a 4.9 ± 1.1 a 5.6 ± 1.5 a 46.7 ± 3.9 b 43.8 ± 3.3 a x 49.5 ± 1.9 b y 13.7 ± 2.15 a 12.3 ± 1.21 b 15.0 ± 2.1 b 3.5 ± 0.5 b 3.6 ± 0.4 b 3.4 ± 0.6 b 42.6 ± 4.9 b 46.0 ± 4.1 a x 39.2 ± 3.1 c y 11.5 ± 1.9 a 12.2 ± 1.3 b 10.8 ± 2.4 a 3.8 ± 0.7 b 3.8 ± 0.4 b 3.8 ± 1.0 b 56.2 ± 8.2 c 58.5 ± 7.6 b 53.8 ± 8.7 a 37.1 ± 8.1 b 39.3 ± 2.6 c 34.8 ± 11.2 c 1.6 ± 0.4 c 1.5 ± 0.3 c 1.7 ± 0.6 c 56.8 ± 4.2 c 57.3 ± 2.3 b 56.3 ± 5.8 a 27.5 ± 9.2 c 28.5 ± 11.3 d 26.5 ± 7.6 d 2.5 ± 1.5 d 2.6 ± 2.0 d 2..4 ± 1.0 d p<0.05 p<0.05 p<0.05 p<0.05 p<0.05 p<0.05 p<0.05 p<0.05 p<0.05 1612 lymphocyte subpopulation occurs. in cases of mild gingivitis there is a slight preponderance of t lymphocytes, where b lymphocytes and plasma cells predominate in the sulcus tissue in cases of periodontitis. a study carried out by gemmel et al demostrates that the mean percent cd3 lymophocytes did not vary significantly between the tissue from healthy/gingivitis and periodontitis subjects, while the percent b lymphocytes was increased in group with periodontitis tissue comparison with healthy/gingivitis tissue26. while most studies have focused t lymphocytes subgroups from the periodontally diseased tissue and peripheral blood, potential ageand gender-related changes of t and b lymphocytes from periodontally healthy tissues are not still characterized. in this study, we analysed the effects of age and sex on relative number of cd3+ t and cd19+ b lymphocyte and cd3+/ cd19+ ratios in gingival tissue and peripheral blood of periodontally healthy balb/c mice with flowcytometric analysis. lymphocyte can be analyzed at the level of one cell through flow cytometry, and physical and biological features of the cells can be accurately and quantitatively measured27. because of all these qualities, the flow cytometry method in present study is preferred. our data revealed no difference in the relative number of cd3+ t and cd19+ b lymphocytes in gingival tissue for all age groups. however, the relative number of cd3+ t lymphocyte in gingival tissue was higher in males than in females in newborn group, puberty group and adult group. the relative number of cd19+ b lymphocyte in gingival tissue was significantly differences between male and female in weaning group, puberty group and adult group. the cd3+/ cd19+ ratios in gingival tissue were same in all age group and gender. a study carried out by lazuardi et al.28 revealed no differences in number and size or percentage of b lymphocytes between young and old individuals. in another study, fransson et al.29 used the experimental model to evaluate the host response to plaque in periodontally healthy young (20-25 years) and old subjects (65-80 years), and gingival biopsies were obtained at days 0, 7 and 21 of plaque formation. authors concluded that although the ratio between t-cell and bcell markers (cd3+/cd19+) was found to be higher in the young (14.8) than in the old subjects (5.9) on day 0 (healthly gingiva), the ratio decreased during the course of the experiment in both groups. they also reported that during the 3-week period of plaque accumulation, older subjects developed a larger sized inflammatory cell infiltrate (ict) with a greater proportion of b cells than younger subjects. their study suggested that differences exist between young and old periodontally-healthy individuals in the inflammatory response during the course of experimental gingivitis in another study, zitzmann et al.30 reported sites treated with non-resective methods (open flap debridement; ‘‘old gingiva’’) resulted in larger ict and higher b-cell proportions than at sites exposed to resective procedures (gingivectomy; ‘‘young gingiva’’). the findings of our study are not consistent with findings reported by fransson et al.29 and zitzmann et al.30 but in agreement with lazuardi et al.28. on the other hand, the data of the present study revealed difference in the relative number of cd3+ t and cd19+ b lymphocytes in peripheral blood for all age groups. while the relative number of cd3+ t lymphocyte in peripheral blood was higher in newborn group than in weaning and in puberty group, it was lower in newborn, in weaning and in puberty group than in adult and in the aged group significiantly. although the relative number of cd3+ t lymphocyte in peripheral blood in females was higher than males in newborn and weaning group, it was significantly lower in females than males in puberty group. the relative number of cd19+ b lymphocyte in peripheral blood significantly elevated in adult group, it was decrease in aged group. previously, weksler31 revived a decrease in b lymphocytes with aging. our finding comfirms the data of weksler me. the cd3+/ cd19+ ratios in peripheral blood decreased gradually from newborn group to the aged group. the results of the present study suggest that significant differences observed with periods of life and gender in periferal blood of peridontally healthy mice, but significant differences exist with only gender in gingival tissue of periodontally healthy mice. these ageand sex-related differences may have a bearing on the natural pro-gression of periodontal disease with aging in humans. however, further studies are required to establish whether these mechanisms are important during the normal course of periodontal disease. this kind of information could represent a useful tool both in clinical practice and in the understanding the physiological process of immunescence. a c k n o w l e d g e m e n t s this study was supported by a grant (pn-2003 / 274) from ataturk university,turkey. r e f e r e n c e s 1. seymour gj, gemmell e, reinhardt ra, eastcott j, taubman ma. immunopathogenesis of chronic inflammatory periodontal disease: cellular and molecular mechanisms. j periodontal res. 1993; 28: 478-86. 2. haffajee ad, socransky ss. microbial etiological agents of destructive periodontal diseases. periodontol 2000. 1994; 5: 78-111. 3. salvi ge, lang np. host response modulation in the management of periodontal diseases. j clin peridontol. 2005; 32: 108-29. 4. zappa u, reinking-zappa m, graf h, espeland m. cell populations and episodic periodontal attachment loss in humans. j clin periodontol. 1991; 18: 508-15. 5. saglie fr, pertuiset j, rezende mt, nester m, marfany a, cheng j. 1988.in situ correlative immuno-identification of mononuclear infiltrates and invasive bacteria in diseased gingiva. j periodontol. 1988; 59: 688-96. 6. seymour gj. possible mechanism involved in the immunoregulation of chronic inflammatory periodontal disease. j dent res. 1987; 66: 2-9. braz j oral sci. 7(26):1609-1613 the effects of age and gender on cd3+ and cd19+ lymphocyte in gingival tissue and peripheral blood: an animal study 1613 7. seymour gj. importance of the host response in the periodontium. j clin periodontol. 1991; 18: 421-6. 8. page rc, schroeder h. periodontitis in man and other animals. a comparative review. basel and new york: s. karger; 1982. 9. page rc. gingivitis. j clin periodontol. 1986; 13: 345-59. 10. payne wa, page rc, ogilvie al, hall wb: 1975. histopathology features of the initial and early stages of experimental gin¬givitis in man. j periodontal res 1975; 10:51-64. 11. mackler bf, waldrop tl, schur p, ro¬bertson pb, levy bm: immunoglobulin bearing lymphocytes and plasma cells in human periodontal disease. j periodontal res 1977; 12:37-45. 12. seymour gj, greespan js: the phenotypic characterization of lympho¬cyte subpopulations in established human periodontal disease. j periodontal res 1979; 14: 39-46. 13. liljenberg b, lindhe j, berglundh t, dahlen g, jonsson r: some microbiological, histopathological and im-munohistochemical characteristics of pro¬ gressive periodontal disease. j clin periodontol 1994; 21: 720-727. 14. berglundh t, wellfelt b, liljenberg b, lindhe j: some local and systemic immunological features of prepubertal periodontitis. j clin periodontol 2001; 28: 113-120. 15. lang np, kiel ra, anderhalden k. clinical and microbiological effects of subgingival restorations with overhanging or clinically perfect margins. j clin periodontol 1983; 10: 563-578. 16. haffajee a, socransky ss, lindhe j,kent rl,okamoto h, yoneyama t. clinical risk indicators for periodontal attachment loss. j clin periodontol. 1991; 18: 117-25. 17. norderyd o, hugoson a, grusovin g. risk of severe periodontal disease in swedish adult population. a longitudinal study. j clin periodontol. 1999; 26: 608-15. 18. faddy mj, cullinan mp, palmer je, westerman b, seymour gj. ante-dependence modelling in a longitudinal study of periodontal disease: the effect of age, gender, and smoking status. j periodontol. 2000; 71: 454-9. 19. salonen lw, frithiof l, wouters fr, hellden lb. marginal alveolar bone height in an adult swedish population. a radiographic cross-sectional epidemiologic study. j clin periodontol. 1991; 18: 223-32. 20. grossi sg, genco rj, machtei ee, ho aw, koch g, dunford r, et al. assessment of risk for periodontal disease. ii. risk indicators for alveolar bone loss. j periodontol. 1995; 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1987. p.67-108. 28. lazuardi l, jenewien b, wolf am, pfisherg, tzankov a, grubeck-loebenstein b. age-related loss of naïve t cells and dysregulation of t-cell/b-cell interactions in human lymph nodes. immunology. 2005; 114: 37-43. 29. fransson c, mooney j, kinane df, berglundh t. differences in the inflammatory response in young and old human subjects during the course of experimental gingivitis. j clin periodontol. 1999; 26: 453-60. 30. zitzmann nu, lindhe j, berglundh t. host response to microbial challenge following resective/non-resective periodontal therapy. j clin peridontol. 2005; 32: 1175-80. 31. weksler me. changes in the b-cell repertoire with age. vaccine. 2000; 8: 1624-8. braz j oral sci. 7(26):1609-1613 the effects of age and gender on cd3+ and cd19+ lymphocyte in gingival tissue and peripheral blood: an animal study 1http://dx.doi.org/10.20396/bjos.v18i0.8657273 volume 18 2019 e191692 original article 1 department of dental materials and prosthodontics, araraquara dental school, unesp – sao paulo state university, araraquara, são paulo, brazil. 2 division of oral health and society, faculty of dentistry, mcgill university, montreal, quebec, canada. corresponding author: josé maurício dos santos nunes reis humaitá street, 1680 araraquara /spbrazil zip code: 14801-903 pabx: +55 16 33016300 e-mail: jm.reis@unesp.br received: june 28, 2019 accepted: september 11, 2019 comparison of impression techniques and double pouring by dental cast’s accuracy. preliminary clinical-lab trial aion mangino messias1,*, stephania caroline rodolfo silva1, filipe de oliveira abi-rached1, raphael de freitas souza2, josé maurício dos santos nunes reis1 aim: this study compared impression techniques and double pouring by means of cast’s accuracy. methods: for each patient (n=10), impressions from right maxillary canine to first molar were made with acrylic resin trays and vinyl-polysiloxane using one single-step, and four two-steps techniques: relief with poly(vinyl chloride) film; tungstencarbide bur/scalpel blade; small movements of the tray; non-relief. total visible buccal surface area of crowns was measured three times using photographs from patients (baseline) and casts. mean area values (mm2) between baseline and casts differences were analyzed by two-way repeated-measures anova (α=.05; 1-β=85%). results: no significant differences were observed for impression techniques (p=.525), double pouring (p=.281), and their interaction (p=.809). conclusion: all impression techniques and double pouring produced casts with similar accuracy. keywords: dental impression materials. dental impression technique. photography, dental. dimensional measurement accuracy. 2 messias et al. introduction dimensionally accurate impression is an integral step for fabricating well-fitting restorations. among elastomeric materials, vinyl-polysiloxane (vps) stands out due to its excellent chemical and physical properties1. although impressions can be made with custom or stock trays, the optimum accuracy is obtained with the custom ones2. however, regarding the techniques, there is no consensus with respect to the best one. to fabricate fixed prostheses, stone dies must be made for improving marginal fit of crowns. although current techniques for making removable dies have become more accurate, cutting a stone die out may result in dimensional change between abutments3. therefore, producing more than one cast from the same impression is an option for preserving marginal fit. this study aimed to compare impression techniques and double pouring by means of cast’s accuracy. materials and methods for each recruited patient (n=10; table 1), impressions from right maxillary canine to first molar were made (chart 1) with partial trays (figure 1) and vps material table 1. criteria used for patients’ recruitment accepted by the araraquara dental school research and ethics committee (#75/11-foar/unesp). criteria of inclusion criteria of exclusion age between 18-80 years pregnancy absence of caries and/or periodontal disease in the maxillary right quadrant. allergic reaction known and informed of any material used. teeth of the right maxillary quadrant healthy or with satisfactory direct restorations. periodontal disease or impaired by caries / trauma / unsatisfactory restorations of the teeth of interest. use of orthodontic braces. concurrent or recent participation in another clinical study. chart 1. impression techniques. impression techniques codes descriptions single-step ss 1) putty and light body materials were used simultaneously. poly(vinyl chloride) (pvc) film pvc 1) a sheet of pvc film covered the putty body material and it was removed after the impression has been taken off from the oral cavity. 2) putty body material was relined with light body material. tungsten carbide bur / scalpel blade bur 1) after impression with putty body material, the axial region of the teeth was worn (5 s) with a slow-speed tungsten carbide bur (maxicut #1520; edenta ag,). a scalpel blade (15c; swann morton ltd.) was used to cut the inter-proximal embrasures. 2) putty body material was relined with light body material. small movements of the tray mov 1) putty body material was inserted in the oral cavity and compressed in the interested area. buccal-lingual small movements of the tray were made (5 s) until material’s polymerization. 2) after impression was removed from the oral cavity, putty body material was relined with the light body material. non-relief nr 1) putty body material was compressed in the interested area. 2) the impression was removed from the oral cavity and relined with the light body material. 3 messias et al. (express xt, 3m espe). a single operator randomly made the impressions following the consort statement. after the waiting time (120 min) recommended by the vps manufacturer, casts were poured using vacuum mixed (turbo mix, edg equipment and controls ltd) type-iv gypsum (gc fuji rock ep, gc europe), following the recommended water/powder ratio by its manufacturer. after removal of the first cast, the second pouring employed these same parameters, waiting 120 min as an elastic recovery time. three intra-oral photographs (raw extension, 300 dpi) of each patient were taken in lateral view with digital camera (d7000, nikon corporation) coupled to a ring flash (sigma em-140dg, sigma corporation) (figure 2). the images (figure 3) were imported into the imagej software, and the total visible buccal surface area of crowns were measured three times by a single and blind examiner to obtain the means and standard deviations. a tool of the software was used to contour the perimeter of the teeth. a maximum variance of 4% was established for the reliability use of the intra-oral images measurements (baseline)4. these same procedures were performed to obtain experimental casts’ images (figure 4). the average area of each cast was compared with the baseline values and the difference between them was expressed in mm2. figure 1. custom acrylic resin partial tray. to standardize the thickness of the impression material (2.0-mm relief), the seating position, and to limit the pressure over the tray, extensions were made on the right maxillary lateral incisor and second molar (arrows). the trays were obtained and maintained in distilled water at 37ºc one week before impressions. figure 2. view of the standardizing device/radiographic positioner with occlusal registration coupled to the camera lens to standardize the angle, focal length and framing. the occlusal registration (pattern resin ls, gc america) was made over the positioner, not compromising the area to be digitized. 4 messias et al. data were submitted to shapiro-wilk’s and levene’s tests, followed by two-way repeated-measures anova (α=.05). results no statistically significant differences were observed for the impression techniques (p=.525), double pouring (p=.281), and their interaction (p=.809) (table 2). for the nature of this investigation, since the power was 85%, the sample size was considered adequate. figure 3. intra-oral photograph. note the ends of the digital caliper (arrow) fixed with opening of 1.0 mm for the calibration of the imagej software (version 1.47a). calibration was performed informing how 1.0 mm corresponded to pixels in each image, calculating the total visible surface area (mm2) of the buccal surface through the perimeter contour of the teeth (clinical crowns). table 2. mean area (mm2) from the differences between baseline and casts’ values for each impression technique and pouring. impression techniques 1st pouring 2nd pouring ss 1.18 -0.91 pvc -2.68 -3.00 bur -2.33 -3.22 mov -3.77 -2.45 nr -2.02 -3.24 figure 4. cast photograph following the same standardization used intra-orally. the respective radiographic positioner was positioned over each cast. the area measurements from intra-oral photographs (baseline) was compared with those obtained on the casts photographs of each respective patient. 5 messias et al. discussion vps material allowed double pouring without impairing cast’s accuracy. probably, the tray’s rigidity and its positioning with controlled pressure reduced the bending and residual stresses in the tray’s walls2. the proximity between baseline and ss technique may be attributed to the thinner layer of the light-body material in comparison to that of two-step techniques. as higher as the viscosity of the material, smaller dimensional change would be expected5. however, a higher volume of filler content means that there is less elasticity and fluidity, resulting in lower detail reproduction1. thus, it is highly recommended to use small thickness of light-body material in combination with putty-body one. despite correcting laboratory bias and minimizing often-clinical steps, multiple pouring can provide errors. however, in this study, there were no statistically differences between the casts, regardless of impression techniques and double pouring. kumar et al.6 also observed no dimensional changes between multiple casts when the elastic recovery time is respected. one of the limitations of this study was to analyze only the buccal surface of the teeth by 2d-measurements. conversely, this sort of evaluation is supported by authors7, who observed no differences between 2d and 3d-analyzes. although the major problem of most 2d-techniques is the limitation to single measurement points, both techniques (2d and 3d) can show comparable results and are in the range of the values of former studies8,9, which used well-established methods like direct view technique used in this study. impression techniques and double pouring did not influence the cast’s accuracy. ss presented the closest absolute values to baseline ones. acknowledgments this study was granted by the “fundação de amparo à pesquisa do estado de são paulo – fapesp” (grants 2011/19165-4 and 2011/19314-0). references 1. balkenhol m, ferger p, wostmann b. dimensional accuracy of 2-stage putty-wash impressions: influence of impression trays and viscosity. int j prosthodont. 2007 nov-dec;20(6):573-5. 2. davis rd, schwartz rs. dual-arch and custom tray accuracy. am j dent. 1991 apr;4(2):89-92. 3. al-abidi k, ellakwa a. the effect of adding a stone base on the accuracy of working casts using different types of dental stone. j contemp dent pract. 2006 sep 1;7(4):17-28. 4. silva sc, messias am, abi-rached fo, de souza rf, reis jm. accuracy of gypsum casts after different impression techniques and double pouring. plos one. 2016 oct 13;11(10):e0164825. doi: 10.1371/journal.pone.0164825. 5. fano v, gennari pu, ortalli i. dimensional stability of silicone-base impression materials. dent mater. 1992 mar;8(2):105-9. 6. kumar d, madihalli au, reddy kr, rastogi n, pradeep nt. elastomeric impression materials: a comparison of accuracy of multiple pours. j contemp dent pract. 2011 jul;12(4):272-8. 6 messias et al. 7. anadioti e, aquilino sa, gratton dg, holloway ja, denry i, thomas gw, qian f. 3d and 2d marginal fit of pressed and cad/cam lithium disilicate crowns made from digital and conventional impressions. j prosthodont. 2014 dec;23(8):610-7. doi: 10.1111/jopr.12180. 8. contreras ef, henriques ge, giolo sr, nobilo ma. fit of cast commercially pure titanium and ti–6al–4v alloy crowns before and after marginal refinement by electrical discharge machining. j prosthet dent. 2002 nov; 88(5):467-72. 9. romeo e, iorio m, storelli s, camandona m, abati s. marginal adapatation of full-coverage cad/cam restorations: in vitro study using a non-destructive method. minerva stomatol. 2009 mar;58(3):61-72. oral sciences n3 prevalence and type of gingival recession in adults in the city of divinópolis, mg, brazil original article braz j oral sci. july | september 2012 volume 11, number 3 geraldo muzzi guimarães1, evandro guimarães aguiar2 1master and specialist in periodontology, coordinator of the periodontal plastic surgery course of the brazilin dental association, divinópolis, mg, brazil 2phd in oral and maxillofacial surgery and specialist in periodontology, professor of oral and maxillofacial surgery, federal university of minas gerais, belo horizonte, mg, brazil correspondence to: geraldo muzzi guimarães rua paraíba, 225, 1301 a 1303 cep: 35500-160 divinópolis, mg, brasil fax: +55 37 32210657 e-mail: gmuzzi@ig.com.br abstract aim: to evaluate the prevalence of gingival recession (gr), its classification according to miller’s classification, and its relationship with gender, age, income and level of education, in the population of the city of divinópolis, mg, brazil. methods: two questionnaires were distributed to the local dentists. one of them was directed to the patients and had questions referring to gender, age and socioeconomic conditions; the other was directed to dentists and had questions about the type of gr found in each patient. results: 245 patients were included in the study. gr prevalence was higher in women. gr prevalence increased with age and seemed to stabilize after the age of 30. there was no relationship between gr and patient’s socioeconomic status. a higher gr prevalence was found in premolars with no statistically significant difference among them. there is a higher prevalence of miller’s class i gr. conclusions: as the prevalence of gr increases with age, a frequency of 81.40% of gr was obtained. no correlation was found between socioeconomic level and gr. in addition, there was no significant difference between genders on gr prevalence. a higher miller class i (p=0.000) prevalence was observed. there was no significant difference between mandibular and maxillary molars. however, the mandibular premolars were the most affected and there was a higher gr prevalence in maxillary teeth than in mandibular teeth. keywords: gingival recession, epidemiology, gingival recession classification. introduction due to increasing patients’ demand for a harmonious smile, a common concern in periodontology is solving gingival aesthetic problems, mainly gingival recession (gr), which has gained the status of discomfort in cosmetic dentistry. as much as 25.6% of people feel unpleased with their gingival or dental esthetics in the anterior region. gr has the highest index of complaints1. this could be verified in a previous study in which questionnaires were sent to periodontists to investigate patients’ main complains on the periodontal esthetics condition, and the correction of denuded roots was the most requested treatment2. data on gr prevalence, most affected teeth, patients’ age and socioeconomic conditions are scarce in the brazilian literature, especially referring to the population living in the minas gerais state, which justifies the present study. gr can be related to mechanical factors (hygiene trauma) and to periodontal disease processes and it is therefore not possible to identify a single factor, but rather a combination of factors3. gr is multifactorial and can occur in patients with periodontal disease as well as in those with high levels of oral hygiene4. gr received for publication: february 22, 2012 accepted: may 14, 2012 braz j oral sci. 11(3):357-361 affects all ages but is more frequent with ageing, and can reach 100% of individuals5. the highest gr frequency was found on mandibular incisors and maxillary first molars at the age of 206. on patients over 43 years of age, 68% had at least one tooth with gr being the mandibular teeth more affected than the maxillary7. it is very uncommon to find an individual over 45 years with thoroughly healthy periodontal tissues8. between 20 and 34 years of age, gr has a 32% prevalence. between 45 and 64, the prevalence raises to 64%, being the maxillary teeth more affected than the mandibular teeth, with a greater prevalence on the maxillary first molars9. gr in left-handed patients has a higher prevalence in the maxillary premolars and canines, with no statistically significant difference between the right and the left side10. the premolars, followed by incisors and canines both maxillary and mandibular, show a higher gr prevalence, and women show less prevalence than men11. in a brazilian adult population aged between 35 and 59 years, a gr prevalence of 98.9% was found, with no correlation with gender; there also is an increase in lesion severity with age12. a previous questionnaire-based study7 with a sample of 1,460 individuals aged 25 to 50 years reveled that gr had no statistically significant association to patient socioeconomic status. when the impact of miller’s classification was observed13, class l gr was more prevalent and a gradual decrease for class ll, lll and lv was noticed10 . the objective of this study was to obtain data on gr prevalence using questionnaires aimed at 20-49-year-old patients living in the city of divinópolis, mg and to the dentists enrolled in the regional council of dentistry of minas gerais that worked in that city. material and methods the population of this study was 20-49-year-old individuals living in the city of divinópolis, mg, brazil. data on gr prevalence, most affected age, and teeth, it’s possible relation to the patient’s socioeconomic status, along with the gr classification according with miller’s classes13 i, ii, iii, iv were obtained from the questionnaire. nine hundred and ninety nine questionnaires were sent to be filled by the dentists working in divinópolis. these questionnaires were personally handed to the dentists at their offices, with purpose of explaining how to fill them. for this work, in order to avoid misinterpretation on the gr prevalence, some exclusion criteria were established: gr less than 1 mm, gr not in the buccal region, smokers, patients under periodontal treatment, individuals using anticonvulsant drugs, cyclosporine or calcium blockers, patients out of the age group, patients with missing teeth (except for the third molars), and specialists in pediatric dentists and periodontics. the questionnaire directed to patients had questions referring to gender, age, education level and income, following the ibge methodology14. the patients of the sample were divided into group a, with patients from 20 to 29 years; group b from 30 to 39 years, and group c from 40 to 49 years. the educational level was subdivided into: illiterate, elementary education, incomplete middle level, complete middle level, incomplete high school, complete high school, college and post graduate14. the income was subdivided into values corresponding to the minimum wage: no income, 1 minimum wage, 2 to 3, 3 to 5, 5 to 9, 9 to 15, 15 to 20, and above 20 minimum wages14. the studied teeth (n=6,020) were divided in groups of anatomical denominations (except molars that were divided into first and second molars) without defining which quadrant. they were divided in mandibular incisors, maxillary incisors, mandibular canines, maxillary canines, mandibular premolars, maxillary premolars, mandibular first molars, maxillary first molars, mandibular second molars and maxillary second molars. after the participating dentists received the forms, they handed them over to their patients with questions about age, gender, education level and income range. after receiving the answers from the patients, the dentists examined the patients for gr. in positive cases, they answered a specific questionnaire with data on the affected teeth and classification of gr according to miller’s classification13. this evaluation was supported by figures illustrating this classification. nonrandom, nonprobability sampling was used15, since not all the population was examined, but all available cases were included. descriptive analyses were performed using tables of distribution of frequencies and proportions calculation. the pearson chi-square test was used, which is appropriate for proportion comparisons16. from the sample obtained, a significant power of 99.6% was achieved, allowing us to detect every existing associations16. results a 23.02% response rate was obtained, as 230 out of 999 questionnaires sent to eligible participants were returned. from these, 15 were excluded because they were not correctly filled. results according to gender are presented in figure 1. as a non-random, probabilistic sample was used, the choice of gender for research followed the same principle, i.e., the frequency was determined by the gender that more commonly attended the clinics. therefore, females were more present to the office than males. standard deviation (s) = 13.5. no statistically significant differences were found between genders (p=0.066). out of 215 patients (100% of sample) included in this work, 21.40% belong to group a (20 to 29 years), 39.53% belong to group b (30 to 39 years) and 39.07% belong to group c (40 to 49 years). gr frequency related to each age group is presented in figure 2. (s)= 18.15. no statistically significant differences were found between groups b and c (p=0.871), but statistically significant differences were detected between group a and b, and between group a and 358358358358358 prevalence and type of gingival recession in adults in the city of divinópolis, mg, brazil braz j oral sci. 11(3):357-361 359359359359359 fig. 1: gender evaluated fig. 2: age group fig. 3: gingival recession in age group fig. 4: tooth group fig. 5: education level fig. 6: incomec (p=0.000). the relative percentage for each age group is presented in figure 3. (s)= 24.36. no significant differences could be detected between groups b and c (p=0.458), but statistically significant differences between groups a and b, and between groups a and c (p=0.000). gr prevalence, according to the tooth groups is on figure 4. (s)= 31.48. there were statistically significant differences between mandibular incisors and maxillary incisors (p=0.047), between mandibular canines and maxillary canines (p=0.012), but no statistically significant differences were found between mandibular premolars and maxillary premolars. the same result was found by other authors17 (p=0.892) and between mandibular second molars and maxillary second molars (p=0.206). there were statistically significant differences between mandibular first molars and maxillary prevalence and type of gingival recession in adults in the city of divinópolis, mg, brazil braz j oral sci. 11(3):357-361 360360360360360 first molars (p=0.000). there were no statistically significant differences between maxillary and mandibular teeth (p=0.177). gr classification according to miller was as follows: 76.86% class i, 19.39% class ii, 2.84% class iii and 0.91% class iv. there were statistically significant differences between class ii and iii (p=0.000), and between class iii and iv (p=0.002). there were statistically significant differences between class i and class ii, between class i and class iii, and class i and iv (p=0.000). the results for educational level are given in figure 5. (s)= 25.27. no statistically significant differences could be found between illi teracy and elementary education (p=0.059), between elementary education and incomplete middle level (p=0.089), between incomplete middle level and complete middle level (p=0.369), between complete middle level and incomplete high school (p=0.746), or between complete high school and higher education (p=0.146). statistically significant differences were detected between incomplete high school and complete high school and between higher school and postgraduate education (p=0.000). the results for income are given in figure 6. (s)= 15.04. no statistically significant differences could be found between 2 to 3 minimum wages and 3 to 5 minimum wages (p=0.622), between 3 to 5 minimum wages and 5 to 9 minimum wages (p=0.166), between 5 to 9 minimum wages and 9 to 15 minimum wages (p=0.752), 15 to 20 minimum wages, and above 20 minimum wages (p=0.072). there were statistically significant differences between no income and minimum wage (p=0.012), between minimum wage and 1 to 2 minimum wages (p=0.000), 1 to 2 minimum wages and 2 to 3 minimum wages (p=0.017), and between 9 to15 minimum wages and 15 to 20 minimum wages (p=0.034). discussion in city of divinópolis, 48.11% of the population is between 20 and 49 years old. according to most studies on gr frequency, there is a higher frequency of this condition in young adults and elderly5-6,12,18-19, and that is the reason why this city and this age group were chosen in the present study. the exclusion criteria were used to prevent bias from our sample, which could compromise the reliability of the results. the sample was separated into groups with the purpose of analyzing gr evolution and the amount of researched individuals. based on the literature, which considers gr a highprevalence event, the sample of this study allowed an improved statistical treatment ensuring detection of all existing associations. therefore, the sample of this survey was considered representative, with a 5% significance, error type i, power 99.6%, 1-error type ii, allowing a lower percentage error in the collected data. regarding the prevalence of the patient’s gender, there was no statistically significant difference between both genders (p=0.066), which is in agreement with a previous study12 that did not correlate gender with gr. gr was found in all assessed age groups, with a gradual increase with age , reaching 81.40% of frequency between 20 and 49 years, which may relate age to gr. significant differences were found between groups a and b, and a and c, but not between groups b and c. thus, it was found that there was a significant increase in frequency between groups a and b, and a and c, consistent with the findings of other authors1,11-12,18. however, this increase stabilized for groups b and c, with no statistically significant difference between these groups (p=0.871). it may be verified that after 30 years of age the frequency of gr stabilizes. the increase of gr prevalence in patients over 43 years is due to a long exposure to etiological factors that cause gr7. however, when patients are well educated with reference to their oral hygiene, the prevalence can be minimized. a correlation between income and prevalence of gr or between frequency of gr and educational level could not be observed. this was due to the large number of income categories/levels of education surveyed and the small number of study subjects in each category, thus failing to get enough power to obtain associations and verify statistical differences between these categories. a better option to this could be a regrouping of the wage categories, which would lead to find significance between groups16. however, it was considered too risky, thereby remaining an inconclusive result in relation to income categories and gr frequency, as found by other authors20. significant differences were found between first molars (p=0.000), with a higher gr frequency in the maxillary first molars than in the mandibular first molars, which is also consistent with other findings18. if we sum the gr-affected teeth on this work, 52.94% are in the maxilla and 47.06% are in the mandible. these data are in agreement with those of other authors9 who reported a higher gr prevalence in maxillary teeth, with no statistically significant difference between arches (p=0.177). it is interesting to notice that the sum of gr prevalence in the maxillary incisors, canines and premolars, which compound the smile line affecting the aesthetics of the smile, represents 36.62%, which explains the great importance given by patients to this type of gingival alteration. miller’s class i13 was the most frequent type, followed by class ii, iii and class iv, which are statistically significant results (p=0.000). similar results have been reported elsewhere3. in order to understand this finding, a class i frequency of 76.86% was obtained, which may be attributed to the fact that the plaque acts easily in the thin and delicate gingival tissue and in the thin alveolar bone19. thus, in the cervical region of the teeth, the bone and gingiva are thinner and therefore more likely to be resorbed, leading to class i or even class ii gr formation. as the bone volume increases on the alveolar ridge region and in the most apical region, it becomes less vulnerable to bacterial attack and trauma, and therefore less prone to changes21. in view of the results of this study, further research may prevalence and type of gingival recession in adults in the city of divinópolis, mg, brazil braz j oral sci. 11(3):357-361 361361361361361 be directed to elucidate more relations of gr, such as: i) the reason behind the higher gr prevalence on premolars, ii) studies with different methodologies as used in the present study19; with fewer income and education level groups to be studied. we recommend this due to the many income and education level groups included in the present methodology. the patient sample size was insufficient to verify whether there is a correlation between gr and brazilian socioeconomic factors, iii) a significant difference between groups a and b, when it comes to age are minimum, this can lead to the suspicion that gr ceases after 30 years of age, and therefore a study to that suspicion would be of great value to the scientific community. we also recommend that before periodontal therapy or orthodontic movement, a detailed study of periodontal conditions should be performed, thereby, preventing gr. prevention should be the focus, as adequate hygiene leads to minimal gr frequency. based on the results, it may concluded that there has been a gradual increase on the gingival recession frequency with age, there was no correlation between gr increase and income, due to the large number of study categories included in this work, most of the gr cases were miller’s class i, the most affected teeth were premolars with no significant difference between maxillary and mandibular teeth, no significant difference was found on the gingival recession frequency between maxillary and mandibular teeth and no gender preference was found on the patients’ gr. references 1. brunsvold ma, nair p, oates tw. queixas principais de pacientes que procuram tratamento para periodontite. j am dent assoc. 1999; 2: 46-51. 2. zaher ca, hachen j, puban ma, mombelli a. interest in periodontology and preferences for treatment of localized gingival recessions. j clin periodontol. 2005; 32: 375-82. 3. löe h, anerud a, boysen h. the natural history of periodontal disease in man: prevalence, severity and extent of gingival recession. j periodontol. 1992; 63: 489-95. 4. litonjua la, andreana s, bush pj, cohen re. toothbrushing and gingival recession. int dent j. 2003; 53: 67-72. 5. carranza fa. periodontia clínica. rio de janeiro: guanabara koogan; 1992. 6. akapata es, jackson d. the prevalence and distribution of gingivitis and gingival recession in children and young adults in lagos , nigéria. j periodontol. 1979; 50: 79-83. 7. vehkalahti m. occurrence of gingival recession in adults. j periodontol. 1989; 60: 599-603. 8. pilot t, miyazakih h, leclerq mh, barnes de. profiles of periodontal conditions in older age cohorts, measured by cpitn. int dent j. 1992; 42: 23-30. 9. palenstein heldermann wh, lembarti bs, weijden ga, hof ma. gingival recession and its association with calculus in subjects deprived of prophylactic dental care. j clin periodontol. 1998; 25: 106-11. 10. tezel a, çanakçi v, siçek y, demir t. evalution of gingival recession in left-and right-handed adults. int j neurosci. 2001; 110: 135-46. 11. careño res, salazar v, rosa c, gudiño p. factores precipitantes en el desarrollo de recessión gingival. acta odontol venez. 2002; 40: 122-36. 12. watanabe mgc. root caries prevalence in a group of brazilian adult dental patients. braz dent j. 2003, 14: 153-6. 13. miller jr. pd. a classification of marginal tissue recession. int j period rest dent. 1985; 5: 9-13. 14. instituto brasileiro de geografia e estatística (ibge). divinópolis population; 2001. [cited 2007 mar 3]. available from: http://www.ibge.org.br. 15. martins jp, santos gp. metodologia da pesquisa científica. rio de janeiro: grupo palestra; 2003. 16. medeiros ml. g4 consulting. query and aid the statistic [email]; 2009. g4-consultoria@hotmail.com 17. marini mg, greghi sla, passanezi e, passanezi acs. gingival recession: prevalence, extension and severity in adults. j appl oral sci. 2004; 12: 250-5. 18. gormann wj. prevalence and aethiology of gingival recession. j periodontol. 1967; 38: 316-22. 19. lindhe j,okamoto h, yoneyama t, haffajee a, socransky ss. periodontal loser sites in untreated adult subjects. j clin periodontol. 1989; 16: 671-8. 20. susin c, hass c, oppermann rv, hangejorden o, albandar jm. gingival recession: epidemiology and risk indicators in a representative urban brazilian population. j periodontol. 2004; 75: 1377-86. 21. yared kfg, zenóbio eg, pacheco w. a etiologia multifatorial da recessão gengival. rev dent press ortod facial. 2006; 11: 45-51. prevalence and type of gingival recession in adults in the city of divinópolis, mg, brazil braz j oral sci. 11(3):357-361 1http://dx.doi.org/10.20396/bjos.v19i0.8659191 volume 19 2020 e209191 original article 1 department of biosciences, piracicaba dental school, university of campinas, são paulo, brazil. 2 department of prosthodontics and periodontology, piracicaba dental school, university of campinas, são paulo, brazil. 3 department of structural and functional biology, institute of biology, university of campinas, são paulo, brazil corresponding author: paulo henrique ferreira caria department of structural and functional biology, institute of biology, university of campinas, monteiro lobato st, 255 – zip code: 13083-862 campinas, são paulo, brazil. phone/fax;+55193521-6184 e-mail address: phcaria@unicamp.br received: april 16, 2020 accepted: august 31, 2020 how many implants are needed for mandibular full-arch rehabilitation? karina giovanetti1 , ricardo armini caldas2 , paulo henrique ferreira caria3,* aim: to analyze the stress distribution at the peri-implant bone tissue of mandible in full-arch implant-supported rehabilitation using a different number of implants as support. methods: three-dimensional finite element models of full-arch prosthesis with 3, 4 and 5 implants and those respective mandibular bone, screws and structure were built. ansys workbench software was used to analyze the maximum and minimum principal stresses (quantitative analysis) and modified von mises stress (qualitative analysis) in peri-implant bone tissue after vertical and oblique forces (100n) applied to the structure at the cantilever site (region of the first molars). results: the peak of tensile stress values were at the bone tissue around to the distal implant in all models. the model with 3 implants presented the maximum principal stress, in the surrounding bone tissue, higher (~14%) than the other models. the difference of maximum principal stress for model with 4 and 5 implants was not relevant (~1%). the first medial implant of the model with 5 implants presented the lower (17%) stress values in bone than model with 3 implants. it was also not different from model with 4 implants. conclusion: three regular implants might present a slight higher chance of failure than rehabilitations with four or five implants. the use of four implants showed to be an adequate alternative to the use of classical five implants. keywords: dental implants. finite element analysis. mouth rehabilitation. prosthodontics. http://orcid.org/0000-0001-6279-7121 https://orcid.org/0000-0002-5362-4744 https://orcid.org/0000-0001-8829-6704 2 giovanetti et al. introduction the implant-supported prostheses are a successful form of treatment, presenting a high survival rate and favorable biomechanical conditions1. among the possibilities of treatment for edentulous patients, the full-arch fixed prosthesis presents better stability and masticatory efficiency when compared to complete denture or overdentures2. the brånemark novum concept (oral implant protocol) indicates the use of three wide implants to support a full-arch fixed prosthesis in the edentulous mandible3. from this, technical variations were developed by changing the size, position, and numbers of implants4. the success of new protocols depends on several factors, such as implant inclination, bone quality, bone quantity, and distribution of masticatory loads to the prosthetic system (prosthesis, framework, and prosthetic components)5. as an alternative to improve the biomechanical behavior, it has been suggested to increase the implant diameter and tilting the distal implants to reduce the cantilever length6. also, the stress distribution in the periimplant bone is directly related to occlusion, masticatory force, number, and position of the implants7. short and medium-term clinical reports have demonstrated the successful use of four implants, even inclined or parallel8. thus, simplified protocols become cheaper and provide less morbidity to patients. full-arch rehabilitation in the edentulous mandible supported by 3, 4 or 5 regular implants has been described by several studies showing high success rates9,10. however, long-term studies are necessary to evaluate the biological complications, survival rates, implant failures, and technical complications of these rehabilitations9. to understand the bone behavior in rehabilitations with dental implants, several studies with different methods have been performed. the use of finite element method (fem) allows investigating the biomechanical behavior on specific three-dimensional models, making it possible to predict and quantify the stresses induced throughout the biological system11. therefore, this study aimed to evaluate the stresses transmitted to the peri-implant bone tissue in 3d finite elements models with three, four, or five dental implants built for the oral rehabilitation of the mandible full-arch fixed prosthesis. materials and methods three-dimensional models of full-arch prosthesis were constructed, varying from 3 to 5 implants (figure 1). c b a d c b a e d c b a model 1 model 2 model 3 figure 1. three-dimensional computer models of three full-arch prosthesis evaluated, varying from 3 to 5 implants. 3 giovanetti et al. the groups: model 1: total of 3 implants, two positioned 5 mm mesial to each mental foramen (15 degrees angulated to distal) and the third implant vertically at midline; model 2: total of 4 implants, two positioned 5 mm mesial to each mental foramen (15 degrees angulated to distal) and two implants vertically, 9 mm from midline to each side; model 3: total of 5 implants, two positioned 5 mm mesial to each mental foramen (15 degrees angulated to distal), two implants vertically 9 mm from midline and one implant vertically at midline. in all models, the framework was 4 mm distant from the alveolar process, with a cross section of 4.3 mm width x 3.6 mm height, and 8 mm in each side cantilever. the bone geometry was constructed based on an edentulous mandible of an approximately 60-year-old man from a cone bean computerized tomography (images with 0.25 mm range) mimics 17.0 (materialise, leuven, belgium). the desired bone density was selected to create the image mask and then regularized the remaining alveolar ridge of the mandible. furthermore, the model was simplified by removing mandibular ramus, with no influence on the results. also, models of external hexagonal cylindrical implants with dimensions of ø4.1 mm x 11.5 mm in length nobel biocare (yorba linda, ca, usa), screws, prosthesis framework and complete assemblies were made in the software solidworks® (dassaul systeme, waltham, ma, usa). the 3d models are available at the supplementary data (file format: .obj). the 3d models were imported to software ansys workbench® 14, (canonsburg, pa, usa). all the materials were set to homogeneous, isotropic and linear elastic. the material properties are shown in table 1, as previous studies12. in addition, the contact between implants and framework was set to frictional (µ = 0.3)13 and all other contacts between different materials were set to be bonded. then, the meshes were set as 10-node tetrahedrons and refined to a point where it does not considerably affect the obtained results. the mesh was checked for element quality and refined in the regions of interest, resulting in about 400,000 elements and 600,000 nodes per model. the posterior surface of mandible was set to fixed (zero degrees of freedom). the mechanical loading was performed with vertical or oblique (45° to vestibular) forces of 100 n applied at the framework’s cantilever in different analysis, simulating bite forces14. the data of the maximum and minimum principal stresses (quantitative analysis) and modified von mises stress (qualitative analysis) were realized by the ansys worbench® 14 software in the 3d finite element models15. results at surrounding bone tissue of implant a, the model 1 presented the maximum principal stress ~14% higher than model 2 and 3 (table 1). the maximum principal stress difference for model 3 to 2 was not relevant (~1%). at implant b, the model 3 present the lower stress values in bone, being 17% lower than model 1, and also not relevant different from model 2 (table 1). 4 giovanetti et al. table 1. comparison of vertical loading (maximum and minimum principal stress) to implant a and b, on the three full-arch prosthesis evaluated. 20 10 0 mpa -10 -20 -30 -40 model 1 model 2 model 3 max principal min principal implant a max principal min principal implant b the compressive stress values (minimum principal stresses) at vertical loading showed inverse relationship to number of implants. however, these differences of stress values were not relevant (≤1%) (table 1). for oblique loading, model 1 presented minimum principal stress 10% higher than model 2 and 3 at implant a (table 2). table 2. comparison of oblique loading (maximum and minimum principal stress) to implant a and b, on the three full-arch prosthesis evaluated. 20 10 0 mpa -10 -20 -30 -40 model 1 model 2 model 3 max principal min principal implant a max principal min principal implant b no relevant differences were observed for maximum principal stress at implant a. the stress field presented by modified von mises indicates a similar behavior around implant a for all models (figure 2). 5 giovanetti et al. vertical oblique y z x 15.6 13.9 12.1 10.4 8.7 6.9 5.2 3.5 1.7 0.0 mpamodel 1 model 2 model 3 figure 2. the stress field by vertical and oblique loading, represented by von mises analysis, around implant a, for the three models of full-arch prosthesis evaluated. the highest values were at distal side of implant a for vertical loading and directed to buccal side for oblique loading. as the higher stress values concentrated at implant a and b, the implants c, d and e were not included in the comparison. for a better understanding of stress location at bone tissue (tensile and compressive), the figure 3 indicates the movements of the cantilever after the mechanical loading. the vectors indicate the movement direction and magnitude. vertical oblique y y z z xx deformation max min figure 3. the deformation of the cantilever during the vertical and oblique loading. the vectors indicate the movement direction and the respective magnitude. 6 giovanetti et al. discussion this study evaluated the peri-implant bone stresses with a different number of implants supporting a full-arch oral rehabilitation. the results showed different stresses values for the studied cases. the decreasing in the number of implants used for fullarch rehabilitation has been subject to laboratory and clinical studies9,16,17, aiming to minimize treatment costs and patient morbidity8. literature data have shown high success rates for total rehabilitations with 3, 4 and 5 dental implants8-10,17. in an observational study with 33 patients with an 18-month follow-up of a complete fixed mandibular prosthesis supported by three implants, an adequate option was developed with peri-implant bone loss that has been described for prostheses of the same type supported by larger numbers of implants9. in a systematic review, it was observed survival rate for more than 24 months of 99.8% in full arch 4 mandibular implants10. however, there is a lack of sufficient long-term data with follow-ups of at least 5 years to evaluate the biological complications, survival rates, implant failures, and technical complications of these rehabilitations with three implants9,18. also, some studies concerns about the influence of reducing implants’ number in the survival rate of prosthetic components9,19. the present study did not evaluated the influence of reduced number of implants at prosthetic components and its long-term consequence. nevertheless, it is reported that the use of a lower number of implants could overload the prosthetic components, resulting in higher rates of screw loosening and leading to a larger number of follow-up appointments17. according to the literature17, the tensile and compressive stresses generated during function were not sufficient to immediately damage the bone, presenting stress values lower than ultimate compressive (167 mpa)20 and tensile (100 mpa)21 strengths. however, these stresses can be harmful at the long-term analysis9,19. in a full-arch rehabilitation, the greatest loads are received by the distal implants, regardless of the total numbers of implants22. in the present study, the stresses generated in the peri-implant bone (implant a) are in agreement with silva-neto et al.17; they reported that reducing the number from 5 to 3 implants relevantly increased the stress in the peri-implant bone. in our study, during axial forces the rehabilitation with 3 implants showed higher tensile stresses (~14% higher) at peri-implant bone tissue than other analyzed models. the compressive stresses followed the same pattern, but with no relevant difference at stress values among models (≤1%). also, for oblique loading, the compressive stress was 10% higher at the model with three implants17. the stresses at implant b presented influence of the implant number, with the reduction of number of implants increasing the bone stress. the rehabilitation with 5 implants showed a tensile stress 17% lower than the 3-implant rehabilitation. our data did not present relevant differences between the use of four or five implants. even with differences among models, all results were lower than critical for bone fracture, and lower than the stress in implant a. as the risk of implant failure is directly related to peri-implant stresses, the main concern relies at implant a. as one of the alternatives to number of implants, literature suggests that increasing in stresses provided by three regular implants could be avoided placing wider implants3,17. 7 giovanetti et al. the results of this study suggest that it is not necessary to use of the traditional 5 implants to support a mandibular full-arch rehabilitation9,17. other studies have demonstrated satisfactory success rates with the use of 4 implants for full-arch fixed mandibular prosthesis10,23. a longitudinal study of the survival of all-on-4 implants in the mandible with up to 10 years of follow-up showed prostheses’ survival rate was 99.2 percent and implant-related success rates 93.8 percent23. although studies have demonstrated satisfactory survival rates of full-arch prosthesis fixed by 3 implants9, the use of 4 implants is in general safer, since in case of loss of some implant other than the distal one, there will still be a favorable biomechanical condition, not requiring a new surgical procedure. moreover, if the implant is lost after function, it is possible to use an existing prosthesis8,10. the use of osseointegrated implants in dentistry provides stability and comfort to denture wearers, especially in the mandibular arch8. implants are used as pillars and when exposed to excessive functional loading can transmit harmful stresses directly to the periimplant bone, which may cause failure in osseointegration8. around two thousand implant options are available for any clinical situation. studies about implants use have to be done to orientate the clinic procedures form dentists. in vitro tests can be divided into surface analysis and mechanical assessment. different methodologies can present results that dentists may find difficult to understand their clinical application. the in vitro testing has limitations, however, current evidence presents new analysis as scanning electron microscopy useful to inform about the implant surface topography. atomic force microscopy, single-cell tests, 3d imaging, and gene expression tests also could be used to the assessment of cellular and physio-biochemical properties of the implants24. as well as 3d finite element analysis has been used in the evaluation of mechanical properties of dental implants, as on the distribution of stresses in bone regions and prosthetic components25. the fem allows to evaluate similarly to what happens in vivo by specific three-dimensional models, enabling prediction and quantification of stresses induced by the entire system11. the fem is a computational analysis with restrictions because it cannot reproduce some variables from the oral environment. the results obtained with that analysis can be useful to understand the behavior of prosthesis supported by implants, which could not be done in vivo. in a study similar to ours26, the authors concluded that the greater amount of implants supporting a complete arch prosthesis, promotes less stress concentration during simulated loading, and that decreasing the number of implants in a rehabilitation is harmful. further experimental studies and clinical trials should be performed to verify the effects of such arrangements on the longevity of this type of rehabilitation. the present results suggest that full-arch rehabilitations in the edentulous mandible with 3 regular implants present a slightly higher chance to failure than rehabilitations with 4 or 5 implants because of the higher stress concentration presented in some analysis. the use of 4 implants presented promising results suggesting to be adequate substitute to the classical technique that uses 5 implants. in addition, 8 giovanetti et al. further clinical long-term analyses are recommended as the literature suggests that the use of wider implants could bypass this problem of higher stresses with 3 implants. conflicts of interest the authors declare that there is no conflict of interest regarding the publication of this paper. acknowledgements the authors thank prof. altair antoninha del bel cury for providing the fea facility. references 1. wennerberg a, albrektsson t. current challenges in successful rehabilitation with oral implants. j oral rehabil. 2011 apr;38(4):286-94. doi: 10.1111/j.1365-2842.2010.02170.x. 2. mendonca db, prado mm, mendes fa, borges tf, mendonça g, prado cj, et al. comparison of masticatory function between subjects with three types of dentition. int j prosthodont. 2009 jul-aug;22(4):399-404. 3. brånemark pi, engstrand p, ohrnell lo, gröndahl k, nilsson p, hagberg k, et al. brånemark novum: a new treatment concept for rehabilitation of the edentulous mandible. preliminary results from a prospective clinical follow-up study. clin implant dent relat res. 1999;1(1):2-16. doi: 10.1111/j.1708-8208.1999.tb00086.x. 4. capelli m, zuffetti f, del fabbro m, testori t. immediate rehabilitation of the completely edentulous jaw with fixed prostheses supported by either upright or tilted implants: a multicenter clinical study. int j oral maxillofac implants. 2007 jul-aug;22(4):639-44. 5. de almeida eo, rocha ep, freitas ac, freitas mm. finite element stress analysis of edentulous mandibles with different bone types supporting multiple-implant superstructures. int j oral maxillofac implants. 2010 nov-dec;25(6):1108-14. 6. bevilacqua m, tealdo t, pera f, menini m, mossolov a, drago c, et al. three-dimensional finite element analysis of load transmission using different implant inclinations and cantilever lengths. int j prosthodont. 2008 nov-dec;21(6):539-42. 7. himmlová l, dostálová t, kácovský a, konvicková s. influence of implant length and diameter on stress distribution: a finite element analysis. j prosthet dent. 2004 jan;91(1):20-5. doi: 10.1016/j.prosdent.2003.08.008. 8. crespi r, vinci r, capparé p, romanos ge, gherlone e. a clinical study of edentulous patients rehabilitated according to the “all on four” immediate function protocol. int j oral maxillofac implants. 2012 mar-apr;27(2):428-34. 9. rivaldo eg, montagner a, nary h, da fontoura frasca lc, brånemark pi. assessment of rehabilitation in edentulous patients treated with an immediately loaded complete fixed mandibular prosthesis supported by three implants. int j oral maxillofac implants. 2012 may-jun;27(3):695-702. 10. soto-penaloza d, zaragozí-alonso r, penarrocha-diago m, penarrocha-diago m. the all-on-four treatment concept: systematic review. j clin exp dent. 2017 mar 1;9(3):e474-88. doi: 10.4317/jced.53613. 11. taddei f, cristofolini l, martelli s, gill hs, viceconti m. subject-specific finite element models of long bones: an in vitro evaluation of the overall accuracy. j biomech. 2006;39(13):2457-67. doi: 10.1016/j.jbiomech.2005.07.018. https://www.ncbi.nlm.nih.gov/pubmed/?term=gherlone e%5bauthor%5d&cauthor=true&cauthor_uid=22442784 9 giovanetti et al. 12. spazzin ao, costa ar, correr ab, consani rl, correr-sobrinho l, dos santos mb. effect of bar crosssection geometry on stress distribution in overdenture-retaining system simulating horizontal misfit and bone loss. j biomech. 2013 aug 9;46(12):2039-44. doi: 10.1016/j.jbiomech.2013.05.025. 13. deslis a, hasan i, bourauel c, bayer s, stark h, keilig l. numerical investigations of the loading behaviour of a prefabricated non-rigid bar system. ann anat. 2012 nov;194(6):538-44. doi: 10.1016/j.aanat.2012.04.004. 14. ferreira mb, barão va, delben ja, faverani lp, hipólito ac, assunção wg. non-linear 3d finite element analysis of full-arch implant-supported fixed dentures. mater sci eng c mater biol appl. 2014 may;38:306-14. doi: 10.1016/j.msec.2014.02.021. 15. bicalho aa, tantbirojn d, versluis a, soares cj. effect of occlusal loading and mechanical properties of resin composite on stress generated in posterior restorations. am j dent. 2014 jun;27(3):129-33. 16. naconecy mm, geremia t, cervieri a, teixeira er, shinkai rs. effect of the number of abutments on biomechanics of branemark prosthesis with straight and tilted distal implants. j appl oral sci. 2010 mar-apr;18(2):178-85. doi: 10.1590/s1678-77572010000200013. 17. silva-neto jp, pimentel mj, neves fd, consani rl, santos mb. stress analysis of different configurations of 3 implants to support a fixed prosthesis in an edentulous jaw. braz oral res. 2014;28:67-73. doi: 10.1590/s1806-83242013005000028. 18. patzelt sb, bahat o, reynolds ma, strub jr. the all-on-four treatment concept: a systematic review. clin implant dent relat res. 2014 dec;16(6):836-55. doi: 10.1111/cid.12068. 19. hatano n, yamaguchi m, yaita t, ishibashi t, sennerby l. new approach for immediate prosthetic rehabilitation of the edentulous mandible with three implants: a retrospective study. clin oral implants res. 2011 nov;22(11):1265-9. doi: 10.1111/j.1600-0501.2010.02101.x. 20. teixeira mf, ramalho sa, de mattias sartori ia, lehmann rb. finite element analysis of 2 immediate loading systems in edentulous mandible: rigid and semirigid splinting of implants. implant dent. 2010 feb;19(1):39-49. doi: 10.1097/id.0b013e3181cc7ffc. 21. martin rb, burr db, sharkey n. skeletal tissue mechanics. new york: springer-verlag; 1998. 22. duyck j, van oosterwyck h, vander sloten j, de cooman m, puers r, naert i. magnitude and distribution of occlusal forces on oral implants supporting fixed prostheses: an in vivo study. clin oral implants res. 2000 oct;11(5):465-75. doi: 10.1034/j.1600-0501.2000.011005465.x. 23. malo p, de araújo nobre m, lopes a, moss sm, molina gj. a longitudinal study of the survival of all-on-4 implants in the mandible with up to 10 years of follow-up. j am dent assoc. 2011 mar;142(3):310-20. doi: 10.14219/jada.archive.2011.0170. 24. bhatavadekar nb, gharpure as, balasubramanium n, scheyer et. in vitro surface testing methods for dental implants-interpretation and clinical relevance: a review. compend contin educ dent. 2020 mar;41(3):e1-e9. 25. dos santos mb, caldas ra, zen bm, bacchi a, correr-sobrinho l. adaptation of overdenture-bars casted in different metals and their influence on the stress distribution: a laboratory and 3d fea. j biomech. 2015 jan;48(1):8-13. doi: 10.1016/j.jbiomech.2014.11.015. 26. tribst jpm, dal piva amo, borges als, bottino ma. effect of implant number and height on the biomechanics of full arch prosthesis. braz j oral sci. 2018;17:e18222. doi: 10.20396/bjos.v17i0.8653837. braz j oral sci. 15(1):27-34 original article braz j oral sci. january | march 2016 volume 15, number 1 influence of factors in the oral mucosa maturation pattern: a cross-sectional study applying multivariate analyses cristina da silva baumgart1, natália batista daroit1, bruna jalfim maraschin1, alex haas2, fernanda visioli1, pantelis varvaki rados1 1universidade federal do rio grande do sul ufrs, school of dentistry, area of oral pathology, porto alegre, rs, brazil 2universidade federal do rio grande do sul ufrs, school of dentistry, area of periodontology, porto alegre, rs, brazil correspondence to: pantelis varvaki rados universidade federal do rio grande do sul faculdade de odontologia, area de patologia oral. rua ramiro barcelos, 2492/503 – cep 90035-003 porto alegre, rio grande do sul, brazil e-mail: pantelis@ufrgs.br abstract aim: to evaluate the association between oral health status, socio-demographic and behavioral factors with the pattern of maturity of normal epithelial oral mucosa. methods: exfoliative cytology specimens were collected from 117 men from the border of the tongue and floor of the mouth on opposite sides. cells were stained with the papanicolaou method and classified into: anucleated, superficial cells with nuclei, intermediate and parabasal cells. quantification was made by selecting the first 100 cells in each glass slide. sociodemographic and behavioral variables were collected from a structured questionnaire. oral health was analyzed by clinical examination, recording decayed, missing and filled teeth index (dmft) and use of prostheses. multivariable linear regression models were applied. results: no significant differences for all studied variables influenced the pattern of maturation of the oral mucosa except for alcohol consumption. there was an increase of cell surface layers of the epithelium with the chronic use of alcohol. conclusions: it is appropriate to use papanicolaou cytopathological technique to analyze the maturation pattern of exposed subjects, with a strong recommendation for those who use alcohol a risk factor for oral cancer, in which a change in the proportion of cell types is easily detected. keywords: papanicolaou test. mouth mucosa. oral health. multivariate analysis. introduction cytopathology is a diagnostic method that involves removing superficial mucosal cells by exfoliation for subsequent microscopic analysis1. with the introduction of quantitative techniques, oral cytology has become an important preventive exam for monitoring the oral mucosa exposed to carcinogens2-6. from these studies, it became apparent the individual nature of this type of evaluation, suggesting that some individuals are more susceptible than others to oral cancer development. the cell type quantification of exfoliated oral mucosa by papanicoloau staining is one of the mentioned quantitative methods associated with cytopathology; this procedure allows the assessment of epithelial maturation process. over time, many studies have sought to evaluate the influence of extrinsic factors (tobacco and alcohol consumption) and intrinsic factors such as sex and age in the process of oral epithelium maturation2,7. however, it remains uncertain whether the consumption of alcohol and tobacco, in addition to individual parameters modify the normal process of epithelial maturation of the oral cavity. other factors that may change the pattern of oral epithelial maturation are oral hygiene status and socioeconomic factors, since they have been suggested as risk factors for the development of oral cancer8-9. http://dx.doi.org/10.20396/bjos.v15i1.8647094 received for publication: may 05, 2016 accepted: may 17, 2016 28 thus, the aim of this study was to evaluate the influence of the oral health status, sociodemographic and behavioral factors on the maturation pattern of normal oral mucosa applying multivariate models. material and methods study design and sample this was a cross-sectional observational study that evaluated male patients aged 25 years or older undergoing treatment at the school of dentistry of the federal university of rio grande do sul, brazil, who were considered eligible for the study. visible lesions in the oral mucosa (except for periodontal disease), previous or current histories of malignant or benign tumors, radiotherapy and/ or chemotherapy, and use of fixed orthodontic appliances were the exclusion criteria. the sample size was estimated from data obtained in a previous study2. the final sample comprised 117 men. means and standard deviations of the number of superficial cells with nuclei in smokers/drinkers and non-smokers/non-drinkers were considered for sample calculation (23.17±13.78 and 18.00±11.91, respectively). by estimation, 98 patients should be included to conduct the study with 80% power and alpha of 5%. ethical considerations the present study was conducted in accordance to the ethical guidelines set forth in the declaration of helsinki. the local ethics committee approved the study protocol and all patients signed an informed consent form prior to their inclusion in the study. interview and clinical examination participants were interviewed using a structured questionnaire to gather demographic data, information about tobacco and alcohol habits, oral hygiene conditions, educational status, socio-economic variables and history of oral and systemic diseases. a single researcher performed the oral examination, with the participant sitting in the proper position, artificial light, buccal mirror and explorer. the first physical examination was performed to detect visible oral lesions for exclusion. in their absence, the dmft was recorded index according to the who criteria. next, the prosthetic conditions were assessed. dentures and removable appliances were removed for inspection. the number or lost teeth in each individual was derived from the dmft index. sample collection and cytopathological analysis exfoliative cytology smears were collected from the border of tongue and floor of the mouth on opposite sides with four uniform brush turns by cytobrush plus®. smears were applied on a glass slide and then fixed in 99.6% alcohol. samples were stained by the modified papanicolaou technique2. one blinded observer examined the maturation pattern in all fields of each slide. one hundred wellformed and isolated cells were counted on each slide horizontally, from left to right, at 400x magnification. cells were classified as anucleated, superficial with nuclei, intermediate and parabasal (figure 1). intra-examiner reproducibility for classification of cell types was influence of factors in the oral mucosa maturation pattern: a cross-sectional study applying multivariate analyses braz j oral sci. 15(1):27-34 measured by the kappa statistic, considered acceptable when kappa is above 0.7. prior to the study, intra-examiner reproducibility was tested for quantification of 20 slides. during the study 10% of the sample was reassessed for reproducibility analysis. fig.1. cellular types of normal oral mucosa epithelium: 1) parabasal; 2) intermediate; 3) superficial with nuclei and 4) anucleated. (papanicolaou method, 400x). statistical methods independent variables: socio-demographic, behavioral and oral variables were evaluated in the present study. age was categorized as <50, 50-59 and ≥60 years old. skin color was dichotomized into white and non-white individuals. marital status was categorized as married, single/divorced and widowed. the total number of packs of cigarettes consumed in a lifetime was calculated by multiplying the number of cigarettes smoked per day by the number of years of habit, divided by 20 (1 pack). subjects were classified into three groups: non-smokers (0 packyears), level 1 exposure (≤20 pack-years) and level 2 exposure (>20 pack-years). daily alcohol consumption was calculated by multiplying the number of drinks consumed weekly by the average alcohol content of a glass of beer, wine or cachaça (a typical brazilian drink distilled from sugarcane), divided by 7 days. the alcohol contents were estimated as 8 g in a glass of beer, 9.6 g in a glass of wine, and 8 g in a drink of cachaça. drinkers were categorized into three groups: level 1 (≤3 g/day), level 2 (>3 g/day) and non-drinker. oral health conditions of the subjects were analyzed by dmft index (decay, missing, filled teeth) and were classified in three categories according to the number of lost teeth: level 1 (1-19 teeth lost), level 2 (≥20 teeth lost) and no tooth loss. for caries experience, individuals were grouped in two categories: level 1 (dmft=0) and level 2 (dmft≥1). socio-economic status was assessed using a standard brazilian economy classification (cceb), which quantifies the home consumer goods. the subjects were grouped as low status or medium/high status and the cut off was 12 points. educational level was categorized according to the number of education years, as follows: low level (≤4 years), intermediate level (5-10 years of education), and high level (≥11 years of education). statistical analyses: cell types were expressed in mean percentage and standard error separately for the border of the tongue and floor of the mouth. comparisons between means according to the independent variables (socio-demographic, behavioral and oral) were performed using wald tests, adjusting for multiple comparisons if required. predictive models of multivariable linear regression were 29 applied by entering all independent variables of interest in the model. interactions and multicollinearity were evaluated and were not found. data analysis was conducted using stata software (statacorp., version 10 for macintosh). the individual was the analytical unit. the level of significance was set at 5%. results the characteristics of the participants of the study are presented in table 1. the study sample comprised mostly caucasian men under 50 years of age, married, from low socioeconomic status and medium educational level. regarding oral status, the majority of individuals reported brushing teeth at least once a day and did not use mouthwashes. almost half the patients said they had never smoked. among smokers or former smokers, the percentage of heavy smokers was high. regarding alcohol consumption, almost 40% of the patients never ingested alcohol beverages and equal amount use more than 3 g of alcohol per day (table 1). tables 2 and 3 present the means and standard errors of exfoliated squamous cells percentage from the border of the tongue and the floor of the mouth, respectively, according to the independent variables. on the border of tongue, 75% of the cells were intermediate, 20% were superficial cells with nuclei, 5% were anucleated cells. parabasal cells were seldom observed. a significantly higher percentage of superficial and anucleated cells was observed in drinker subjects compared to non-drinker individuals. for intermediate cells, the percentage was significantly lower in drinkers compared to those who did not drink alcoholic beverages (table 2). table 3 presents the pattern of maturation of the floor of the mouth, where approximately 82.5% of the cells were intermediate, 15% were superficial cells with nuclei, 2.5% were anucleated, whereas parabasal cells were rarely observed. evaluating the studied variables, again only alcohol consumption was statistically significant, with differences between drinkers and non-drinkers. similar to the tongue, intermediate cells were found in smaller proportions and anucleated cells in larger number in individuals who drank alcohol often. tables 4 and 5 show the multivariate linear regression models for the association between the percentage of cell types on the border of tongue and floor of mouth, respectively, as well as the independent variables. significant associations were observed for consumption of alcoholic beverages, which showed increase of anucleated cells and superficial cells with nuclei, as well as decrease of intermediate cells with increased alcohol consumption. discussion some risk factors contributing to oral cancer, such as smoking and alcohol, are already well established in the literature10-11. analysis of individuals exposed to these factors using papanicolaou test showed alterations in maturation pattern of cells2,12. this study aimed to evaluate other possible variables that could influence the pattern of maturation of oral epithelial cells, such as oral health and sociodemographic factors. to our influence of factors in the oral mucosa maturation pattern: a cross-sectional study applying multivariate analyses braz j oral sci. 15(1):27-34 table 1 absolute distribution and percentage of sociodemographic and behavioral status of the participants. variables n % age <50 years 61 52.1 50-59 years 30 25.7 ≥60 years 26 22.2 skin color white 81 69.2 non-white 36 30.8 marital status married 67 57.3 single/divorced 48 41.0 widowed 2 1.7 socio-economic status medium/high 53 45.3 low 64 54.7 educational level high (≥11 education years) 44 37.6 intermediate (5-10 education years) 50 42.7 low (≤4 education years) 23 19.7 brush teeth < 1 time/day 3 2.6 ≥1 time/day 114 97.4 use mouthwashes yes 25 21.4 no 92 78.6 visited the dentist <1year 57 48.7 ≥1 year 60 51.3 smoke current smoker 25 21.4 former smoker 30 25.6 never smoked 62 53.0 exposed smoke never smoked 62 53.0 smoked ≤ 20 pack-years 34 29.1 smoked > 20 pack-years 21 17.9 alcohol consumption never ingested alcohol 46 39.3 drink ≤ 3g 24 20.5 drink > 3g 47 40.2 total 117 100.0 knowledge, this study is the first to assess by cytopathology, the influence of oral health, behavioral and sociodemographic factors on maturation pattern of oral mucosa using multilevel modeling. in order to assist the early detection of this altered tissue even before clinical lesion, when a biopsy contraindicated, oral exfoliative cytopathology can be used, performing tests such as papanicolaou stain (pap). oral cytopathology is a fast, easy to perform, non-invasive and inexpensive technique; which allows to evaluate changes in cytological pattern maturation of normal oral mucosa, inferring risk groups and assisting in the prevention of these factors1. 30 in these studies, the only statistically significant variable associated with alterations in maturation pattern was the use of alcohol, which resulted in an increase of surface cell layers of the epithelium (anucleated and superficial with nuclei) in individuals exposed to alcohol. our results contradict the previous literature. one study evaluated the cell maturation pattern of individuals exposed to tobacco and alcohol observed a smaller number of superficial cells with nuclei compared to the non-exposed group2. we consider that these results differ from our study for some reasons: first, because alcohol consumption was not evaluated alone, but synergistically with tobacco; further, in our study was performed a multivariate analysis, thus, it can control other variables that could be biases. another reason is that in our study a greater number of participants was assessed compared with the above-mentioned study. influence of factors in the oral mucosa maturation pattern: a cross-sectional study applying multivariate analyses variables anucleated p* supnuclei** p* intermediate p* parabasal p* age <50 years 4.97±0.33 ref. 20.37±0.38 ref. 74.84±0.53 ref. 0.00±0.00 ref. 50-59 years 5.37±0.51 0.51 20.10±0.52 0.67 74.53±0.81 0.76 0.00±0.00 60+ years 5.57±0.53 0.33 19.77±0.66 0.43 74.58±0.84 0.80 0.08±0.05 0.15 skin color white 5.35±0.31 ref. 20.37±0.34 ref. 74.40±0.50 ref. 0.01±0.01 ref. non-white 4.89±0.38 0.35 19.72±0.48 0.27 75.36±0.59 0.22 0.03±0.03 0.61 socio-economic status high 5.15±0.38 ref. 20.34±0.40 ref. 74.51±0.58 ref. 0.02±0.02 ref. low 5.25±0.32 0.84 20.03±0.39 0.58 74.86±0.53 0.66 0.02±0.02 0.89 educational level high 5.39±0.38 ref. 20.36±0.41 ref. 74.48±0.58 ref. 0.00±0.00 ref. intermediate 5.04±0.38 0.52 20.28±0.46 0.89 74.66±0.62 0.83 0.04±0.03 0.16 low 5.22±0.56 0.80 19.56±0.64 0.30 75.22±0.94 0.51 0.00±0.00 alcohol consumption never ingested alcohol 4.43±0.36 ref. 19.35±0.45 ref. 76.22±0.58 ref. 0.02±0.02 ref. drink ≤3g 6.04±0.56 0.02 20.42±0.68 0.19 73.96±0.85 0.03 0.00±0.00 0.32 drink > 3g 5.53±0.38 0.04 20.85±0.39 0.01 73.60±0.60 0.01 0.02±0.02 0.99 exposed smoke never smoked 5.00±0.33 ref. 20.24±0.40 ref. 74.74±0.52 ref. 0.02±0.02 ref. smoked ≤20 pack-years 5.47±0.42 0.38 20.21±0.49 0.95 74.35±0.72 0.66 0.00±0.00 0.32 smoked >20 pack-years 5.38±0.67 0.61 19.90±0.65 0.66 75.14±1.04 0.73 0.05±0.05 0.53 smoke + alcohol never smoked/never drink 4.82±0.49 ref. 19.71±0.58 ref. 75.43±0.74 ref. 0.04±0.04 ref. smoke or drink intermediate 5.29±0.48 0.50 19.75±0.65 0.97 75.00±0.81 0.70 0.00±0.00 0.32 smoke or drink hard 5.34±0.35 0.39 20.57±0.35 0.21 74.23±0.55 0.20 0.02±0.02 0.62 use mouthwashes no 5.17±0.27 ref. 20.17±0.31 ref. 74.74±0.43 ref. 0.02±0.02 ref. yes 5.32±0.56 0.82 20.16±0.67 0.98 74.56±0.94 0.86 0.00±0.00 0.16 teeth lost 0 tooth 4.08±0.45 ref. 20.00±0.47 ref. 75.19±0.70 ref. 0.00±0.00 ref. 1-19 teeth 5.38±0.30 0.30 20.31±0.37 0.61 74.46±0.50 0.39 0.01±0.01 0.32 20+ teeth 5.21±0.87 0.68 19.86±0.88 0.89 74.86±1.25 0.81 0.07±0.07 0.32 caries experience dmft≤8 4.88±0.38 ref. 20.32±0.39 ref. 74.98±0.64 ref. 0.02±0.02 ref. dmft≥9 5.46±0.31 0.24 20.04±0.38 0.61 74.47±0.47 0.53 0.02±0.02 0.87 use of oral prosthesis not use 5.17±0.28 ref. 20.43±0.32 ref. 74.40±0.46 ref. 0.01±0.01 ref. partial removable prosthesis 5.47±0.56 0.63 19.63±0.63 0.26 75.89±0.79 0.59 0.00±0.00 0.31 total prosthesis 5.07±0.86 0.92 19.36±0.95 0.29 76.21±1.38 0.22 0.07±0.07 0.42 total 5.21±0.24 20.17±0.28 74.70±0.39 0.02±0.01 * comparisons of the reference group (ref.). ** superficial cells with nuclei. table 2 mean distribution (standard error) the percentage of the type of cells on the border of tongue according to independent variables. braz j oral sci. 15(1):27-34 another study evaluated the proportion of cell types of the oral mucosa of individuals who used daily listerine® mouthwash with 26.9% of alcohol for 6 months compared to the group that used the mouthwash without alcohol. only outer cells of the epithelium (superficial and intermediate) were found in both groups, with no statistical differences between the pattern 31influence of factors in the oral mucosa maturation pattern: a cross-sectional study applying multivariate analyses * comparisons of the reference group (ref.). ** superficial cells with nuclei. variables anucleated p* supnuclei** p* intermediate p* parabasal p* age <50 years 2.31±0.30 ref. 15.46±0.53 ref. 82.06±0.65 ref. 0.00±0.00 ref. 50-59 years 2.17±0.35 0.75 14.33±0.81 0.25 83.53±0.97 0.21 0.03±0.03 0.31 60+ years 2.40±0.39 0.82 15.88±0.87 0.68 81.65±0.97 0.72 0.04±0.04 0.32 skin color white 2.21±0.24 ref. 15.17±0.48 ref. 82.48±0.56 ref. 0.03±0.02 ref. non-white 2.50±0.37 0.51 15.47±0.71 0.72 82.06±0.89 0.69 0.00±0.00 0.16 socio-economic status high 2.06±0.25 ref. 15.64±0.55 ref. 82.30±0.59 ref. 0.04±0.03 ref. low 2.50±0.30 0.26 14.95±0.56 0.38 82.39±0.71 0.93 0.00±0.00 0.16 educational level high 2.43±0.33 ref. 15.41±0.68 ref. 82.16±0.73 ref. 0.02±0.02 ref. intermediate 2.34±0.33 0.84 15.14±0.55 0.76 82.34±0.74 0.86 0.02±0.02 0.93 low 1.96±0.35 0.32 15.26±1.01 0.90 82.73±1.14 0.67 0.00±0.00 0.32 alcohol consumption never ingested alcohol 1.63±0.25 ref. 14.74±0.68 ref. 83.61±0.75 ref. 0.02±0.02 ref. drink ≤3g 2.75±0.49 0.04 15.08±0.72 0.73 81.79±1.04 0.16 0.04±0.04 0.67 drink > 3g 2.72±0.33 0.01 15.87±0.63 0.22 81.40±0.72 0.04 0.00±0.00 0.32 exposed smoke never smoked 2.18±0.28 ref. 15.18±0.54 ref. 82.65±0.65 ref. 0.02±0.02 ref smoked ≤20 pack-years 2.47±0.38 0.53 16.47±0.70 0.15 80.76±0.80 0.07 0.00±0.00 0.32 smoked >20 pack-years 2.38±0.47 0.71 13.57±0.90 0.13 84.05±1.13 0.29 0.05±0.05 0.53 smoke + alcohol never smoked/never drink 1.82±0.35 ref. 15.46±0.86 ref. 82.64±0.98 ref. 0.04±0.04 ref. smoke or drink intermediate 2.29±0.42 0.40 15.14±0.70 0.77 82.28±0.93 0.79 0.00±0.00 0.32 smoke or drink hard 2.52±0.28 0.12 15.23±0.57 0.82 82.25±0.67 0.74 0.02±0.02 0.62 use mouthwashes no 2.39±0.23 ref. 15.04±0.42 ref. 82.43±0.53 ref. 0.02±0.02 ref. yes 1.96±0.34 0.31 16.08±1.04 0.36 82.04±1.05 0.74 0.00±0.00 0.16 teeth lost 0 tooth 1.81±0.32 ref. 15.45±0.65 ref. 82.74±0.73 ref. 0.00±0.00 ref. 1-19 teeth 2.44±0.27 0.13 15.29±0.55 0.85 82.11±0.66 0.52 0.03±0.02 0.16 20+ teeth 2.64±0.58 0.21 14.71±1.00 0.54 82.71±1.31 0.99 0.00±0.00 caries experience dmft≤8 2.32±0.27 ref. 15.15±0.60 ref. 82.51±0.68 ref. 0±0 ref. dmft≥9 2.27±0.28 0.89 15.35±0.60 0.80 82.21±0.65 0.75 -0.30±-0.21 0.15 use of oral prosthesis not use 2.35±0.25 ref. 15.51±0.48 ref. 82.12±0.56 ref. 0.02±0.02 ref. partial removable prosthesis 1.68±0.36 0.13 15.26±1.04 0.83 82.53±1.26 0.76 0.00±0.00 0.16 total prosthesis 2.79±0.53 0.47 13.79±0.90 0.09 83.5±1.26 0.32 0.00±0.00 0.16 total 2.30±0.20 15.26±0.40 82.35±0.47 0.02±0.01 table 3 mean distribution (standard error) the percentage of the type of cells on the floor of the mouth according to independent variables. of epithelium maturation using a mouthwash with or without alcohol13. however, that study13 does not detail the criteria used for cell type analysis, nor how many cells were analyzed. another factor that explains the difference observed in our study was that individuals evaluated in our study were chronic alcohol users for years and several times a day. braz j oral sci. 15(1):27-34 32 influence of factors in the oral mucosa maturation pattern: a cross-sectional study applying multivariate analyses variables anucleated supnuclei* intermediate parabasal β±se** p β±se** p β±se** p β±se** p age 0.02±0.01 0.21 0.06±0.03 0.07 -0.08±0.04 0.05 0.01±0.01 0.25 skin color white ref. ref. ref. ref. non-white 0.41±0.45 0.36 0.48±0.91 0.59 -0.63±1.08 0.55 -0.01±0.02 0.53 exposed smoke never smoked ref. ref. ref. ref. smoked ≤20 pack-years 0.21±0.47 0.64 1.07±0.95 0.26 -1.49±1.12 0.18 -0.01±0.02 0.51 smoked >20 pack-years 0.02±0.61 0.97 -1.68±1.23 0.17 1.77±1.45 0.22 0.03±0.03 0.35 alcohol consumption never ingested alcohol ref. ref. ref. ref. drink ≤3g 1.10±0.54 0.04 0.26±1.10 0.80 -1.70±1.30 0.19 0.02±0.03 0.51 drink > 3g 1.16±0.47 0.01 1.24±0.94 0.19 -2.29±1.12 0.04 -0.01±0.02 0.92 use mouthwashes no ref. ref. ref. ref. yes -0.32±0.49 0.51 0.69±0.99 0.48 -0.05±1.17 0.96 -0.02±0.03 0.47 dmft index -0.01±0.03 0.68 -0.02±0.06 0.72 0.04±0.07 0.60 0.01±0.01 0.12 use of oral prosthesis not use ref. ref. ref ref partial removable prosthesis -0.89±0.63 0.16 -1.06±1.28 0.41 1.12±1.51 0.42 -0.06±0.03 0.47 total prosthesis 0.20±0.72 0.77 -1.63±1.46 0.26 1.30±1.73 0.45 -0.08±0.04 0.06 r2 0.02 0.01 0.03 0.01 table 5 multivariate linear regression model of the association between the percentage of cells on the floor of the mouth and the independent variables. * superficial cells with nuclei. ** beta ± standard error. table 4 multivariate linear regression model of the association between the percentage of cells at the border of tongue and the independent variables. * superficial cells with nuclei. ** beta ± standard error. variables anucleated supnuclei* intermediate parabasal β±se** p β±se** p β±se** p β±se** p age 0.01±0.02 0.85 0.01±0.02 0.67 -0.27±0.03 0.44 0.01±0.01 0.16 skin color white ref. ref. ref. ref. non-white -0.25±0.56 0.66 -0.57±0.65 0.37 0.61±0.88 0.49 0.01±0.02 0.61 exposed smoke never smoked ref. ref. ref. ref. smoked ≤20 pack-years 0.05±0.58 0.92 -0.33±0.68 0.62 0.27±0.92 0.76 -0.01±0.02 0.68 smoked >20 pack-years 0.14±0.75 0.84 -0.16±0.88 0.85 0.29±1.19 0.80 alcohol consumption never ingested alcohol ref. ref. ref. ref. drink ≤3g 1.69±0.67 0.01 1.03±0.78 0.19 -2.31±1.07 0.03 -0.01±0.03 0.66 drink > 3g 1.40±0.58 0.01 1.37±0.67 0.04 -2.92±0.92 0.00 0.01±0.02 0.69 use mouthwashes no ref. ref. ref. ref. yes 0.24±0.61 0.69 -0.04±0.70 0.95 -0.19±0.96 0.84 -0.01±0.03 0.59 dmft index 0.06±0.04 0.09 -0.02±0.04 0.53 -0.05±0.06 0.38 -0.01±0.01 0.84 use of oral prosthesis not use ref. ref. ref. ref. partial removable prosthesis -0.03±0.78 0.96 -0.46±0.91 0.61 0.82±1.24 0.50 -0.03±0.03 0.33 total prosthesis -0.33±0.89 0.71 -0.85±1.04 0.41 2.05±1.42 0.15 0.02±0.04 0.63 r2 0.02 -0.01 0.03 -0.03 braz j oral sci. 15(1):27-34 some researches using cytopathology observed the influence of the chronic use of ethanol on the cells of oral mucosa. reis et al.14 (2006) showed a significant increase in micronucleus, abnormal nucleus/cytoplasm ratio, pyknosis, karyorrhexis and karyolysis in exposed mucosa. webber et al.6 (2016) evaluated by feulgen staining nuclear changes in cells from the border of the tongue and the floor of the mouth of alcoholics. the last cited site showed a higher frequency of karyorrhexis and this suggests higher degrees of keratinization. this statement agrees with our results, taking into account the alcohol variable, we found more cells of the superficial layers, which suggests an organism reaction as protection against aggression, maybe with increased keratinization. in contradiction, smoking, the greatest risk factor for oral cancer alone did not significantly affect the maturation pattern of the oral mucosa. the results are conflicting in the literature on this subject. burzlaff and gedoz2-3 found an increased number of surface nucleated cells in patients exposed to tobacco smoke. the sites chosen for the collection of smears were tongue and floor of the mouth because in brazil these locations are the most frequently affected by oral squamous cell carcinoma15-16. in our results it is possible to see a clear distinction of cell types (anucleated, superficial cells with nuclei, intermediate and parabasal) at each site, approximately with 5%, 20%, 75%, 0% on the tongue, and 2.5 %, 15%, 82.5%, 0% on the floor of the mouth, respectively (tables 2 and 3). it is important to know the particular pattern of each site – under normal conditions the floor of mouth is less keratinized than the border of tongue, so it presents a lower amount of surface and more intermediate cells6,17 to detect possible changes in this cellular types proportion when exposed to a carcinogenic factor. some studies correlated low income with higher incidence of oral cancer18-19,for this reason sociodemographic factors were included as independent variables. because cytopathology has been used as a monitoring tool of cytogenetic changes, we decided to include these factors in our study. madani19 reported that individuals with low educational level have 3.3 more chances of developing the disease than those with higher schooling. regarding the results of our study, the pattern of the oral epithelium maturation had no significant differences comparing the different ranges of economic and educational level. the association between the status of oral health and head and neck cancer has been widely studied; these studies suggest that poor oral hygiene can contribute to an increased risk of this pathology20-21. in the present study there was no change in cell maturation pattern in relation to oral health status, which disagrees with the idea that oral microorganisms can increase inflammatory cytokines level and change the complex metabolic pathways, triggering the process of carcinogenesis22. the use of papanicolaou cytopathological method is appropriate to analyze the proportion of exfoliated cells as the maturation pattern of healthy male subjects. the maturation pattern of the buccal mucosa cells was not affected by the studied variables, except by the intake of alcoholic beverages. references 1. kazanowska k, hałoń a, radwan-oczko m. the role and application of exfoliative cytology in the diagnosis of oral mucosa pathology – contemporary knowledge with review of the literature. adv clin exp med. 2014; 23: 299-305. 2. burzlaff jb, bohrer pl, paiva rl, visioli f, sant’ana filho m, da silva vd, et al. exposure to alcohol or tobacco affects the pattern of maturation in oral mucosal cells: a cytohistological study. cytopathology. 2007; 18: 367-75. 3. gedoz l, lauxen i, sant’ana filho m, rados pv. proliferative activity in clinically healthy oral mucosa exposed to tobacco smoking and alcohol: a longitudinal study using the agnor staining technique. anal quant cytol histol. 2007; 29: 231-8. 4. ahmed hg, omer as, abd algaffar sa. cytological study of exfoliative buccal mucosal cells of qat chewers in yemen. diagn cytopathol. 2011; 39: 796-800. 5. oliveira lu, lima cf, salgado ma, balducci i, almeida jd. comparative study of oral mucosa micronuclei in smokers and alcoholic smokers. anal quant cytol histol. 2012; 34: 9-14. 6. webber lp, pellicioli a, magnusson as, danilevicz ck, bueno cc, sant’ana filho m, et al. nuclear changes in oral mucosa of alcoholics and crack cocaine users. hum exp toxicol. 2016; 35: 184-93. 7. cowpe jg, longmore rb, green mw. quantitative exfoliative cytology of normal oral squames: an age, site and sex-related survey. j r soc med. 1985; 78: 995-1004. 8. cowpe jg, longmore rb, green mw. quantitative exfoliative cytology of normal oral squames: an age, site and sex-related survey. j r soc med. 1985; 78: 995-1004. 9. ahrens w, pohlabeln h, foraita r, nelis m, lagiou p, lagiou a, et al. oral health, dental care and mouthwash associated with upper aerodigestive tract cancer risk in europe: the arcage study. oral oncol. 2014; 50: 616-25. 10. andrade jo, santos ca, oliveira mc. associated factors with oral cancer: a study of case control in a population of the brazil’s northeast. rev bras epidemiol. 2015; 18: 894-905. 11. sanner t, grimsrud tk. nicotine: carcinogenicity and effects on response to cancer treatment a review. front oncol. 2015; 5: 196. 12. abdelaziz ms, osman te. detection of cytomorphological changes in oral mucosa among alcoholics and cigarette smokers. oman med j. 2011; 26: 349-52. 13. bagan jv, vera-sempere f, marzal c, carcelén ap, martí-bonmati e, bagan l. cytological changes in the oral mucosa after use of a mouth rinse with alcohol: a prospective double blind control study. med oral patol oral cir bucal. 2012; 17: e956-e961. 14. reis sr, do espírito santo ar, andrade mg, sadigursky m. cytologic alterations in the oral mucosa after chronic exposure to ethanol. braz oral res. 2006; 20: 97-102. 15. gervásio ol, dutra ra, tartaglia sm, vasconcellos wa, barbosa aa, aguiar mc. oral squamous cell carcinoma: a retrospective study of 740 cases in a brazilian population. braz dent j. 2001; 12: 57-61. 16. durazzo md, de araujo ce, brandão neto js, potenza as, costa p, takeda f, et al. clinical and epidemiological features of oral cancer in a medical school teaching hospital from 1994 to 2002: increasing incidence in women, predominance of advanced local disease, and low incidence of neck metastases. clinics. 2005; 60: 293-8. 17. braga fl, meneguzzi rd, paiva rl, rados pv. [cytopathology evaluation of the oral mucosa of smokers and nonsmokers]. odonto cien. 2004; 19: 157-63. [portuguese]. 18. boing af, antunes jl. [socioeconomic conditions and head and neck cancer: a systematic literature review]. cien saude colet. 2011; 16: 33influence of factors in the oral mucosa maturation pattern: a cross-sectional study applying multivariate analyses braz j oral sci. 15(1):27-34 615-22. [portuguese]. 19. madani ah, dikshit m, bhaduri d, jahromi as, aghamolaei t. relationship between selected socio-demographic factors and cancer of oral cavity a case control study. cancer inform. 2010; 9: 163-8. 20. meyer ms, joshipura k, giovannucci e, michaud ds. a review of the relationship between tooth loss, periodontal disease, and cancer. cancer causes control. 2008; 19: 895-907. 21. coussens lm, werb z. inflammation and cancer. nature 2002; 420: 860-7. 22. rajeev r, choudhary k, panda s, gandhi n. role of bacteria in oral carcinogenesis. south asian j cancer. 2012; 1: 78-83. 34 braz j oral sci. 15(1):27-34 influence of factors in the oral mucosa maturation pattern: a cross-sectional study applying multivariate analyses 1http://dx.doi.org/10.20396/bjos.v20i00.8659342 volume 20 2021 e219342 original article 1 department of operative dentistry, endodontics and dental materials, bauru school of dentistry, university of são paulo, bauru, são paulo, brazil. corresponding author: genine guimarães email: genine_mg@hotmail.com received: april 29, 2020 accepted: december 14, 2020 dentin bond strength evaluation between a conventional and universal adhesive using etch-and-rinse strategy genine moreira de freitas guimarães1,* , karin cristina da silva modena1 , carolina yoshi campos sugio1 , tamires de luccas bueno1 , maria angélica silvério agulhari1 , maria teresa atta1 aim: the aim of this study was to compare the microtensile bond strength (μtbs) and the characteristics of the adhesive interface of scotchbond universal su – etch-and-rise mode (3m espe) and adper scotchbond multi-purpose mp (3m espe) to dentin over time. methods: class i cavity preparations were performed in 60 human molars that were randomly divided according to the dentin bonding system (dbs) used (n=30): (1) acid conditioning + su and (2) acid conditioning + mp. for bonding strength (bs) analysis, 30 teeth (n = 15) were sectioned into sticks and submitted to the microtensile test in a universal testing machine after 24 hours and 12 months. the adhesive interface of the others 30 teeth was analyzed in a confocal microscope after 24 hours and 12 months. the data of μtbs were analyzed by two-way repeated measures anova and tukey’s hsd (α = 0.05). results: su presented the lowest dbs compared to mp (p=0.000). time did not influenced dbs for both adhesive systems (p=0.177). confocal microscopy analysis showed no cracks between both adhesive systems tested. conclusion: the results indicate that mp μtbs showed a better performance compared to su in total-etch mode. keywords: dentin-bonding agents. dentin. methacrylates. microscopy, confocal. https://orcid.org/0000-0002-4415-850x https://orcid.org/0000-0002-0926-1253 https://orcid.org/0000-0001-8934-4472 https://orcid.org/0000-0002-4912-5846 https://orcid.org/0000-0003-3945-4205 https://orcid.org/0000-0002-2449-5728 2 guimarães et al. introduction since the introduction of the adhesive systems, over 50 years ago, the union interface to dentin remains the weakest link in restorative treatment1-4. although many studies have shown excellent short-term and immediate adhesion effectiveness5,6, the durability and stability of the adhesive interface on dentin remain questionable7,8 due to its inherent characteristics. the failure of adhesion may lead marginal infiltration, which may cause discoloration, secondary caries, and subsequent loss of retention3,9,10. in order to minimize adhesive failures, universal adhesives have become a trend in dentistry due to its effectiveness and longevity, and a simplified operative technique. conventional three-step adhesive systems are considered the “gold standard”. however, as a disadvantage, if all the collagen exposed after acid etch is not completely covered by the adhesive systems, matrix metalloproteinases (mmps) are activated, when they have free access to water, causing restoration failures and post-operative sensitivity8,11,12. the first simplified adhesive system ‘’one bottle’’ introduced on the market was the scotchbond universal (su) adhesive (3m espe, saint paul mn, usa), which can be used as an etch-and-rinse or a self-etch mode13,14, according to the clinical conditions. the self-etch mode eliminate previous application of phosphoric acid, that is a sensitive part of the technique. thus, acid monomers have the ability to demineralize and penetrate the dentin substrate simultaneously, decreasing the chances of demineralized zones without hybridization, as can happen in etch-and-rinse mode15. however, the different applications of su (etch-and-rise and self-etch mode) show different behaviors and reflect on the bond strength and quality of the hybrid layer16-19. the difference from su to the others adhesive systems is the substitution of methacrylate monomers (udma and gdma) or phosphorylated methacrylate monomer (mhp) by 10-methacryloyloxydecyl dihydrogenpho-sphate (10-mdp)15,18,20-23. functional monomers, such as 10-mdp, contain carboxylic groups and phosphates that are capable of chemically interact with the calcium of the hydroxyapatite by means of primary ionic bonds, forming stable salts of calcium-phosphate and calcium-carboxalate together with a limited effect of descaling24. the chemical adhesion promoted by 10-mdp seems to be not only effective but also more stable in water than that promoted by other functional monomers such as 4-met and phenyl p23. despite the favorable chemical reaction from mdp, the factors that interfere with the longevity of the bonding interface are still complex. there are several mechanisms that favor the degradation of the hybrid layer. one of the most relevant factors is related to simplified adhesive systems that have hydrophilic characteristics25-27. in addition, there is a big difference in μtbs for su when used in the etch-and-rise or self-etch strategy9-16. therefore, the objective of this study is to evaluate the difference between mechanisms of adhesion and to compare the dentin bonding system (dbs), as well as to evaluate the characteristic of the bonding interface between a universal and a conventional three-step adhesive system with using etch-and-rise mode. 3 guimarães et al. the null hypothesis of this study is that both dbs evaluated did not show differences between the adhesives and through the time. material and methods specimens preparation sixty sound human molars with no fracture, cracks or caries lesions extracted due to periodontal or orthodontic reasons were used according to the protocol of ethics and research committee of the bauru school of dentistry, university of são paulo (caae nº 336.286). teeth were cleaned removing any residue of periodontal and gingival tissues adhered to the dental surface with manual curettes and stored in a 0.1% thymol solution at room temperature for less than 6 months. using a low-speed diamond saw (isomet low speed saw; buehler ltda., lake bluff/il eua) under water lubrication, the crowns were separated from the roots. class i cavities28-34 were prepared 4.0 mm deep in dentin, with 3.0 mm buccal extension and 5.0 mm mesiodistal extension using carbide drills (#245, kg sorensen). microtensile bond strength test (μtbs) bonding procedures the experimental unit considered was the tooth, so the sticks of each tooth (n=30) were randomly divided according to dbs (n=15): 1 su or 2 mp using excel’s “randomization” tool. each class i cavity were etch-and-rinse with 35% phosphoric acid etchant (condac, fgm, brazil) for 30s (enamel) and 15s (dentin). the two adhesives (su and mp) were carefully applied according to the manufacturer’s instructions (table 1). all teeth were restored by incremental technique with filtek™ z250 resin composite (3m espe, saint paul mn, usa) and photoactivated for 40s with 1200mv/cm2 of irradiation (radii-cal®, sdi, sp, brazil). after the restorative procedures, the specimens were immersed in deionized water at 37 ° c for 24h or 12 months, according to the tested group. table 1. adhesive systems: composition and protocol. material composition protocol adper single bond universal (su) (n=15) 3m espe saint paul mn, usa 10-mdp phosphate monomer, vitrebond copolymer hema bisgma, dimethacrylate resins filler, silane, initiators ethanol, water 1. acid etch (scotchbond etchant 35%condac, fgm, brazil) of the enamel for 30s and dentin for 15s followed by washing with “spray” air/water for 30s. excess water removed with absorbent paper. 2. application of the adhesive for 20s with slight movements with the application applicator. light dry with air for 5s to evaporate the solvent. polymerization for 10s. adper scotchbond multi-purpose (mp) (n=15) 3m espe saint paul mn, usa primer: hema, polyalkanoic acid copolymer, water adhesive: bis-gma, hema, camphorquinone 1. acid etch (scotchbond etchant 35%) of the enamel for 30s and dentin for 15s followed by washing with “spray” air/water for 30s and removing excess water with absorbent paper. 2. application of a primer layer and light drying for 5s 3. application of the adhesive and polymerization for 10s. 4 guimarães et al. bonding test the restored teeth were sectioned buccal-lingually into slices with a double-sided diamond disc (extec corp., enfield / ct usa), cooled with deionized water, at a 150 rpm speed in a sectioning machine (isomet low speed saw; buehler ltda., lake bluff / il usa). subsequently, each slice was cutted into sticks with a cross-sectional area of approximately 0.64 mm2 that were separated into 2 groups according to the test period: 24h (baseline) and 12 months and stored in water at 37ºc with frequent water exchange. after the storage, each stick was individually fixed with cyanoacrylate-based adhesive (loctite super bonder flex gel, henkel ltda., são paulo/sp brazil) in a bencor multi-t device (danville engeneering, danville/ca usa) and submitted to the microtensile test in a universal testing machine (instron model 3342, instroncorp., canton, ma) at a constant speed of 0.5 mm/min, with maximum load of 500n. the mann whitney test was realized and the results were analyzed by two-way repeated measures anova and tukey’s hsd (α = 0.05). the average µtbs value for each tooth and time based on all the sticks was calculated and the premature failures were considered as zero for calculating the mean values. analysis of the adhesive interface confocal laser scanning microscopy the remained 30 teeth were prepared as describe above, but the adhesive systems were labeled with rhodamine b (0.02 μg/ml for su and 0.1 μg/ml for mp)35, in order to allow the analysis of the micromorphology of the adhesive interface with greater accuracy. a confocal laser scanning microscopy (leica tcs spe, leica microsystems cms, mannheim, germany) at 40x magnification microscopy software (leica application suite advanced fluorescence, leica microsystems cms) (1.0 mm, 1024 pixels and 0.976μm in resolution) was used to evaluate the quality of the hybrid layer, through the analysis of the presence or absence of cracks after storage in water at 37ºc for 24h and 12 months. as it is a qualitative evaluation, no statistical analysis was performed. results microtensile bond strength (μtbs) test the means and respective standard deviations of bond strength (mpa) in the periods of 24h (baseline) and 12 months are shown in table 2. only dbs was a statistically significant factor (p= 0.000). non-significant differences were detected in the microtensile bond strength among the adhesives tested and the periods evaluated (p = 0.1772) as well as the interaction dbs/time (p=0.570). mp dbs presented the highest values of bs. both adhesives were able to maintain dbs after 12 months. fracture analysis revealed that the most predominant failure pattern was ‘adhesive’. the fracture pattern of each specimen (stick) was evaluated and the results obtained are listed in table 3. 5 guimarães et al. adhesive interface analysis (hybrid layer quality confocal microscopy) the results showed that at 24h and 12 months, no difference was observed between the two adhesive systems su and mp. no gaps were observed at the bonding interface (figure 1). table 2. means and standard deviations of tested groups (mpa). adhesives baseline (24h) 12 months su 24.09±8.46ab (n=15) 22.58±7.35ab (n=13) mp 29.96±9.76aa (n=15) 28.09±11.54aa (n=15) different uppercase letters indicate differences between time (columns) (p≤0.05). different lowercase letters indicate differences between μtbs (rows) (p≤0.05). table 3. type of fracture in each group. type of fracture baseline (24h) 12 months n (%) n (%) adhesive 33 44.59 27 51.92 mixed 28 37.83 19 36.53 cohesive in resin 11 14.86 4 7.69 cohesive in dentin 2 2.70 2 3.84 total 74 100 52 100 there was no statistical significant difference between (significance level of 5%). figure 1. confocal microscopy images show the hybrid layer with no gaps at all periods observed. a) 24h-su; b) 24h-mp; c) 12 months-su; d) 12 months-mp. a c b d 6 guimarães et al. discussion the microtensile bond strength test is frequently performed to evaluate in vitro adhesive systems. this study compared the μtbs of dentin, using a universal (su) and a conventional adhesive (mp) in the etch-and-rinse mode. this study also evaluated the quality of the bonding interface of both adhesive systems in a confocal microscopy. the proposal was to observe the behavior of su and mp adhesives under similar conditions. the specimens were obtained from class i cavities, representing high c-factor which influence the values of μtbs29,31-33,36. specimens obtained from cavities showed a statistically significant reduction in bond strength values due to the high c-factor32. so, the objective was to evaluate the behavior of those adhesives in an extremely situation28-34. also was performed class i cavities because, unlike posterior restorations, non-carious cervical lesions usually have sclerotic dentin and could reflect different results37. studies show that cavities with low c-factor, as in non-carious cervical lesions, have underestimated values compared to values presented clinically, as it appears in class i cavities32, and the reliability of dentin adhesives is dependent upon the quality of the dentin36. another factor to consider is the presence of enamel on the cavity margins, which theoretically provides a good seal against the ingress of bacteria and oral fluids and thus protects the most vulnerable adhesive bonding of the underlying dentin16,38. without enamel protection on the periphery of the restoration, water promotes adhesive interface degradation resulting in decreased bond strength over time16,25,39. this was considered mainly because of the 12 months specimens. with the limitations of this study, the μtbs results showed that the mp values was significantly higher compared to su, with no significance between the two times tested. besides the universal single bond promotes chemical bonding to the hydroxyapatite crystals present on the enamel and dentine6,40,41, some authors claim that there are no differences in the performance of adhesive systems containing 10-mdp13. one hypothesis suggests that prior acid etch may remove hydroxyapatite and hinder chemical bonding, which is the main benefit of mdp. in contrast, hidari et al.42 (2020) show that the presence of the functional monomer mdp, even with previous phosphoric acid conditioning, produces greater bond strength results in dentin than the absence of this functional monomer19,42,43. however, it is concluded that although the functional monomer mdp has an important role in the quality of the bonding interface, the removal of the smear layer and hydroxyapatite through prior acid conditioning can be disadvantageous related to the long-term bond strength durability. a systematic review with meta-analysis concluded that universal adhesives with etchand-rinse strategy is more effective and produces higher values of μtbs in enamel44, and, on dentin, self-etch mode can produce better values18,45,46. however, in this study, the su was used only with the etch-and-rinse strategy and can explain the results of the present study which showed lower statistical values (22.58±7.35) compared to mp (28.09±11.54). the quality of the hybrid layer is necessary to prevent microleakage and gap formation47. therefore, the confocal interface analysis supported the μtbs data. regarding the interface durability of both adhesives tested, there were no statistical differences 7 guimarães et al. on μtbs in the two periods tested (24h and 12 months). this shows that both adhesives were able to maintain the hybrid layer quality with no cracks (figure 1). adhesive systems without the application of hydrophobic compound as last step tends to present higher hydrolytic degradation and bond instability because they are semipermeable membranes48. although the su acquires hydrophobic characteristics due to the presence of mdp after its polymerization, the adhesive still absorbs more water compared to the two-step self-adhesives (separate bottles) because they have better hydrophobic characteristics in contrast to the one-step adhesives44. in order to assess the state of deterioration or to predict the longevity of dental adhesives, clinical studies are clearly the best methods25,49. however, due to the difficulty of standardizing clinical studies, in-vitro tests are performed to simulate the clinical conditions. therefore, by observing several different methods and comparing the results, it may be useful to understand the degradation process that occurs in intraoral conditions. thus, methods such as water storage17,50 and thermal cycle42,51,52, are the most used forms of artificial aging. hidari et al.42 (2020) compared water storage and thermal cycle methods and assessed statistical differences between the adhesives. the water storage has an accelerated aging potential due to the hydrolysis capacity of hydrophilic components of the adhesive and the host-derived proteases with collagenolytic activity53,54. therefore, water storage for 12 months may show results that reflect what happens clinically. the null hypothesis of this study that there was no difference on dbs between the adhesives and through the time was partially rejected and these results are related to the variables adopted. therefore, it is necessary to carry out further tests and evaluate different adhesives and their different application steps clinically. the need for longterm evaluations is also needed. under the limitations of this in vitro study, it was possible to conclude that the mp adhesive showed higher values of μtbs compared to su in both times of storage tested. clinical significances the integrity of the hybrid layer is important to the longevity of resin-based restorations. testing different adhesive systems clarifies the mechanisms involved on the effectiveness of the bonding interface and allows better choice for the clinician. acknowledgements this work was 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mine a, van landuyt kl, poitevin a, opdenakker g, et al. inhibition of enzymatic degradation of adhesive-dentin interfaces. j dent res. 2009 dec;88(12):1101-6. doi: 10.1177/0022034509346952. 54. pashley dh, tay fr, yiu c, hashimoto m, breschi l, carvalho rm, et al. collagen degradation by host-derived enzymes during aging. j dent res. 2004 mar;83(3):216-21. doi: 10.1177/154405910408300306. braz j oral sci. 15(3):238-241 effect of different impression techniques and elastomeric impression materials on the dimensional accuracy of partially edentulous mandibular arch rafael pino vitti¹, adriano relvas barreira de oliveira², mário alexandre coelho sinhoreti³ ¹dds, msc, phd, associate professor, universidade de taubaté (unitau), school of dentistry, department of prosthodontics, taubaté, sp, brazil ²dds, msc student, universidade de taubaté (unitau), school of dentistry, department of prosthodontics, taubaté, sp, brazil ³dds, msc, phd, full professor, universidade de campinas (unicamp), piracicaba dental school, department of restorative dentistry, piracicaba, sp, brazil corresponding author: adriano relvas barreira de oliveira university of taubaté rua dos operários, 09 taubaté, sp, cep: 12020-270 phone/fax: 55-12-3629-2130 e-mail: <relvasrj@yahoo.com.br abstract aim: evaluate the dimensional accuracy of stone casts of a partially edentulous mandibular arch made by two impression techniques (1-step putty/light-body and 2-step putty/light-body) using addition and condensation silicones. methods: a partially edentulous steel stainless cast with four markings on teeth 33, 37, 43, and 47 was used to obtain the impressions. the transverse (33-43 and 37-47) and anteroposterior (33-37 and 43-47) distances were measured by measuring microscope (30x magnification; 0.5µm accuracy). for the 1-step putty/light-body technique, both viscosities of the impression materials were handled together. for the 2-step putty/light-body technique, the impression materials of different viscosities were handled separately and a polypropylene spacer (2 mm thick) was used to create a relief. the same distances were measured on stone casts (n = 5). the values were submitted to normality test (kolmogorov-smirnov) and analyzed statistically by three-way anova and tukey’s (5%). results: all distances showed shrinkage (negative linear changes). the addition silicones showed better accuracy than condensation silicones (p<0.05) and no significant difference was found between the impression techniques (p>0.05). the edentulous zone (43-47) presented worst dimensional accuracy results. conclusions: the accuracy of the casts is more related to the impression material than impression technique. keywords: dimensional accuracy. silicone elastomers. dental impression technique. introduction impression technique is a frequently performed procedure in the dental office that requires selection of an appropriate impression material1. dental impression presents a negative imprint of buccal structures2. it is usually a first step during fabrication of indirect restorations3,4 that have to be seated in or on prepared teeth. dimensional accuracy during making impressions is crucial to the quality of fixed prosthodontic treatment and impression technique is a critical factor affecting this accuracy, since that an accurate impression has a significant role in the success of treatment2,5,6. knowing the physical and biological properties as well as the advantages and disadvantages of received for publication: november 17, 2016 accepted: june 7, 2017 original article braz j oral sci. july | september 2016 volume 15, number 3 http://dx.doi.org/10.20396/bjos.v15i3.8649995 239 different impression materials, is a prerequisite for adequate practical application of dental materials and contributes to the success of prosthetic therapy7. in dentistry there are four groups of impression materials: polysulfides, polyethers, condensation, and addition silicones. these materials present four viscosities: putty(type 0), heavy (type 1), medium/regular(type 2), and light-body (type 3). for the clinical use the most important difference between these materials is their dimensional stability8. factors such as viscosity, hydrophilicity, thickness, soaking, polymerization shrinkage, incomplete elastic recovery of the impression materials, type of the adhesive used in tray, pouring time, thermal shrinkage between buccal cavity and room temperature9 may affect the dimensional accuracy of the molds. the different viscosities of the impression materials allows use several impressions techniques, such as 1-step putty/light-body technique and 2-step putty/light-body technique. the 1-step putty/ light-body technique is performed using two impression materials with different viscosities at the same time. both viscosities of the impression materials are handled and placed in the tray together10,11. on other hand, the 2-step putty/light-body technique was created to minimize the shrinkage of the condensation silicones12. in this technique, two impressions procedures are realized. the prior impression is performed using a tray with putty-material. after its polymerization a relief is performed in the initial mold and the light-body material is handled and placed on the putty material10,11,13. clinically these techniques have some differences as simplicity, number of operators to handle the impression materials, chair time, and the control of the impression materials thickness2,13,14. there is no consensus in literature on the best method or ideal impression technique. furthermore, if the same type of the impression material could have different behavior with the different impression techniques. the aim of this study was to evaluate the dimensional accuracy of stone casts made by two impression techniques (1step putty/light-body and 2-step putty/light-body) using addition and condensation silicones. material and methods table 1 shows the materials used in this study. two viscosities of silicone impression material were used during the impressions: putty(type 0) and light-body (type 3). table 1 impression materials used. brand names manufacturers clonage dfl, rio de janeiro, rj, brazil zetaplus/oranwash l zhermack, rovigo, italy optosil/xantopren vl plus heraeus kulzer gmbh, hanau, germany silon 2 aps dentsply ind. e com. ltda., petrópolis, rj, brazil futura ad dfl, rio de janeiro, rj, brazil express regular set 3m unitek, monrovia, ca, usa elite hd+ normal setting zhermack, rovigo, italy aquasil ultra regular set dentsply gmbh, konstanz, germany a steel stainless model simulating a partially edentulous mandible with absence of the teeth 44, 45, and 46 and with four markings on buccal cusps of the teeth 33, 37, 43, and 47 was used to perform the impressions techniques. using a measuring microscope (olympus® measuring microscope stm, olympus optical co., japan) at 30x magnification the anteroposterior (3337; 43-47) and transversal distances (33-43; 37-47) were measured. a stock tray i-3 (tecnodent, casalecchio di reno, italy) was used for 1-step putty/light-body and 2-step putty/light-body techniques. the putty-body silicones were handled using plastic gloves to avoid the inhibition of polymerization reaction by contaminants as zinc diethyl dithiocarbamate present in latex gloves15. for the 1-step putty/light-body technique the puttyand lightbody materials were handled together. thus, a second calibrated operator handled the light-body material. both impression materials were placed together on the stock tray. for the 2-step putty/light-body technique a polypropylene spacer (2 mm thick) was used on the stainless steel cast to form a relief. the puttybody material was manipulated and inserted on the stock tray to perform a first impression. then, the spacer was removed and the light-body material was manipulated and inserted on the first mold. a second impression was made to obtain the final mold. all impression materials used in this study were handled according to the manufacturer’s instructions. the set tray/impression material was positioned and seated manually on the stainless steel model, from posterior to anterior direction. after the setting time of the impression material the tray was removed from stainless steel model by a pneumatic equipment with 3 bar pressure. this movement standardized was used to avoid distortions caused in the mold by vertical movement. all impressions procedures were performed in a room with temperature and relative humidity controlled (23°c ± 2°c and 50% ± 10%). dental stone type iv (durone, dentsply, petrópolis, rj, brazil) was used in a water/powder ratio of 28.5 ml/150 g for stone cast pouring (n = 5). the molds were poured after 30 min of the tray detachment in order to allow the elastic recovery of the impression material. for each distance between the teeth three readings were made by a single calibrated operator as in the stainless steel cast. the means were calculated and compared with those obtained from the stainless steel cast (%): negative values indicate a decrease in the distances (shrinkage) and positive values indicate an increase in the distances (expansion). the values were submitted to normality test (kolmogorovsmirnov). the data were analyzed statistically by three-way anova and the means compared by tukey’s test (α=0.05) (bioestat 5.0, instituto mamiraua, am, brazil). results table 2 shows that all distances showed shrinkage. the anteroposterior distances showed greater dimensional changes than transverse distances. the edentulous region (43-47) presented the greatest dimensional change (p<0.05), except for express (1-step putty/light-body technique) and elite hd+ (2-step putty/lightbody technique) where no significant difference was found between effect of different impression techniques and elastomeric impression materials on the dimensional accuracy of partially edentulous mandibular arch braz j oral sci. 15(3):238-241 240 both anteroposterior distances (33-37 and 43-47) (p>0.05). no significant difference was found between transversal distances (p>0.05). in general, the addition silicones showed better accuracy than condensation silicones (p<0.05). no significant difference was found (p>0.05) between impression techniques. effect of different impression techniques and elastomeric impression materials on the dimensional accuracy of partially edentulous mandibular arch braz j oral sci. 15(3):238-241 table 2 dimensional accuracy of the impression materials, techniques and distances evaluated. distances materials 33-43 37-47 33-37 43-47 1-step putty/light-body clonage -.244 (.025) b,c -.200 (.019) b,c -.462 (.022) a,b -.566 (.033) b,a zetaplus/oranwash -.238 (.028) b,c -.195 (.022) b,c -.353 (.037) b,b -.512 (.020) bc,a optosil/xantopren -.231 (.027) b,c -.212 (.020) b,c -.340 (.028) b,b -.489 (.042) c,a silon 2 aps -.321 (.024) a,c -.297 (.031) a,c -.444 (.030) a,b -.645 (.028) a,a futura ad -.056 (.010) c,c -.062 (.018) c,c -.157 (.025) c,b -.277 (.026) d,a express -.032 (.008) c,c -.024 (.006) d,c -.132 (.026) c,a -.192 (.022) e,a elite hd+ -.039 (.006) c,c -.043 (.012) cd,c -.160 (.018) c,b -.251 (.032) d,a aquasil -.043 (.006) c,c -.039 (.011) d,c -.158 (.024) c,b -.246 (.017) d,a 2-step putty/light-body clonage -.210 (.032) b,c -.241 (.033) b,c -.403 (.026) ab,b -.602 (.026) a,a zetaplus/oranwash -.215 (.023) b,c -.180 (.024) c,c -.355 (.032) b,b -.555 (.032) b,a optosil/xantopren -.201 (.022) b,c -.193 (.027) c,c -.364 (.024) b,b -.471 (.040) c,a silon 2 aps -.298 (.019) a,c -.306 (.030) a,c -465 (.033) a,b -.633 (.018) a,a futura ad -.062 (.018) c,c -.042 (.015) d,c -.182 (.020) c,b -.254 (.058) d,a express -.030 (.014) c,c -.016 (.011) e,c -.119 (.019) e,b -.201 (.020) e,a elite hd+ -.046 (.009) c,c -.028 (.016) e,c -.142 (.016) d,a -.196 (.028) e,a aquasil -.051 (.012) c,c -.020 (.015) e,c -.148 (.025) d,b -.228 (.008) de,a different letters indicate statistically significant difference: lowercase letters for comparison between impression materials (in columns) and capital letters for comparison between distances within each impression techniques (in rows) (p<0.05). the greek letters indicate comparison between impression techniques. discussion the results of this study showed that addition silicone provided greater accuracy in the stone casts and greater reliability in impression structures than condensation silicone in both impression techniques evaluated. these results are consequence of the excellent physical and mechanical properties of the addition silicone, such as no formation of byproducts during and after the set reaction by the terminal group ethylene or vinyl with hydride groups11,16. these properties provide the obtainment of more than one accurate stone casts from the same mold due to excellent elastic recovery (approximately 99%)17 and tear strength of the addition silicone18. the dimensional changes that occur with the silicone-based impression materials are related to the temperature differences between the buccal environment and the room where the mold will be stored (thermal shrinkage)19 and their polymerization shrinkage, besides the incomplete elastic recovery (approximately 1%)17. hung et al. (1992)12 reported that the small differences found in the dimensional accuracy among the addition silicone materials can be due to the variability in the composition of each brand name, especially in the matrix-filler ratio, which can provide different levels of shrinkage polymerization and elastic recovery. the condensation silicones showed the largest dimensional change values corroborating with others studies in both impression techniques20. silon 2 aps and clonage were similar and showed the worst results of all impression materials tested. the strong and continuous setting reaction of this category of impression materials form volatile byproducts, such as ethyl alcohol. these byproducts cause greater shrinkage affecting the dimensional stability and, consequently, the accuracy of the condensation silicone16,20. in this study, some silon 2 aps molds were discarded after mold-cast separation, since its light-body viscosity has low cohesive strength. the elastomers undergo shrinkage during the polymerization in direction to center of the mold21-23, while the gypsum undergoes expansion during its setting process. so, the negative linear changes in the stone casts showed in table 2 show that the expansion of dental stone type iv is not enough to minimize the shrinkage occurred in the elastomers. comparing all percentages of the dimensional changes occurred in the studied distances, it was found that all transversal distances suffered less change than anteroposterior distances. this fact can be explained by the bilateral adherence of the casting to tray when considered the transversal direction and just an unilateral adherence due the presence of free end of the tray (anteroposterior distance). this free end could offer less restrictive resistance to shrinkage of the impression material, allowing dimensional changes in these distances. the partially edentulous region (43-47) showed greater dimensional changes values except for express (1-step putty/ light-body technique) and elite hd+ (2-step putty/light-body technique). the greater volume of impression material due to the absence of three teeth on this side may be the reason for the greater shrinkage by volume of mold’s mass and, consequently, greater dimensional change. the results of this study showed no statistical differences 241 between impression techniques, which corroborates with other studies(16-18). however, different results were showed in studies where matrices11,24,25 or fully dented casts26,27 were used instead partially edentulous casts, polypropylene with different spacer thick27, different temperature24,25,27 and storage times25,27 and when the casting were made under water24. these different protocols may explain the different results. thus, the accuracy of the casts is more related to the impression material used than to the impression technique chosen12. from the results obtained, it is recommended to use addition silicone. based on the results of this study, some clinical factors, such as the correct replacement of mold in the patient’s mouth and the longer time required in 2-step putty/ light-body technique and the simultaneous shrinkage of different materials with different viscosities, details reproduction by the putty material caused by excessive pressure applied during the impression and consequent flow of the light-body material and the need for a second person to handle the putty-body material in 1-step putty/light-body technique, should be the most important factors to influence the professional’s choice. therefore, as there was no statistical difference between the impression techniques, it is recommended that dentists use impression technique that they are most familiar considering the limitations of each technique and checking all variables, since some procedures as impression material handling, and its removal from the buccal cavity and pouring are under control in laboratory studies. the choice of the impression material is the primarily responsable for dimensional precision of the stone casts obtained. the addition silicones produce more accurate stone casts than the condensation silicones, while there are no significative differences when the same impression material are used with different impression techniques. references 1. thota kk, jasthi s, ravuri r, tella s. comparative evaluation of dimensional stability of three different elastomeric impression materials – an invitro study. j clin diagn res. 2014 oct;8(10):zc48-50. doi: 10.7860/ jcdr/2014/9768.5036. 2. varvara g, murmura g, sinjari b. evaluation of defects in surface detail for impression techniques: an in vitro study. j prosthet dent. 2015 feb;113(2):108-13. doi: 10.1016/j.prosdent.2014.08.007. 3. vadapalli sb, atluri k, putcha ms, kondreddi s, kumar ns. evaluation of surface detail reproduction, dimensional stability and gypsum compatibility of monophase polyvinyl-siloxane and polyether elastomeric impression materials under dry and moist conditions. j int soc prev community dent. 2016 jul-aug;6(4):302-8. doi: 10.4103/2231-0762.186795. 4. shembesh m, ali a, finkelman m, weber hp, zandparsa r. an in vitro comparison of the marginal adaptation accuracy of cad/cam restorations using different impression systems. j prosthodont. 2016 feb 8. doi: 10.1111/jopr.12446. 5. kotha sb, ramakrishnaiah r, devang divakar d, celur sl, qasim s, matinlinna jp. effect of disinfection and sterilization on the tensile strength, surface roughness, and wettability of elastomers. j investig clin dent. 2016 oct 26. doi: 10.1111/jicd.12244. 6. caputi s, varvara g. dimensional accuracy of resultant casts made by a monophase, one-step and two-step, and a novel two-step putty/ light-body impression technique: an in vitro study. j prosthet dent. 2008 apr;99(4):274-81. doi: 10.1016/s0022-3913(08)60061-x. 7. surapaneni h, samatha p, shankar r, attili s. polyvinylsiloxanes in dentistry: an overview. trends biomater artif organs. 2013 jul;27(3):115-23. 8. daou e. the elastomers for complete denture impression: a review of the literature. saudi dent j. 2010 oct;22(4):153-60. doi: 10.1016/j. sdentj.2010.07.005. 9. reisbick mh. effect of viscosity on the accuracy and stability of elastic impression materials. j dent res. 1973 may-jun;52(3):407-17. 10. myers ge, stockman dg. the thiokol rubber impression materials. j prosthet dent. 1958;8(2):330-9. 11. dugal r, railkar b, musani s. comparative evaluation of dimensional accuracy of different polyvinyl siloxane putty-wash impression techniques-in vitro study. j int oral health. 2013 oct;5(5):85-94. 12. hung sh, purk jh, tira de. accuracy of one-step versus two-step putty wash addition silicone impression technique. j prosthet dent. 1992 may;67(5):583-9. 13. mann k, davids a, range u, richter g, boening k, reitemeier b. experimental study on the use of spacer foils in two-step putty and wash impression procedures using silicone impression materials. j prosthet dent. 2015 apr;113(4):316-22. doi: 10.1016/j. prosdent.2014.09.014. 14. chee wwl, donovan te. polyvinyl siloxane impression materials: a review of properties and techiniques. j prosthet dent. 1992 nov;68(5):728-32. 15. craig rg, urquiola nj, liu cc. comparison of commercial elastomeric impression materials. oper dent. 1990 mayjun;15(3):94-104. 16. giordano r. impression materials: basic properties. gen dent. 2000 sep-oct;48(5):510-6. 17. custer f, updegrove l, ward m. accuracy and dimensional stability of a silicone rubber base impression material. j prosthet dent. 1964;14(6):1115-21. 18. christensen gj. what category of impression material is best for your practice? j am dent assoc. 1997 jul;128(7):1026-8. 19. mccabe jf, wilson hj. addition cure silicone rubber impression materials. br dent j. 1978;145(4):17-20. 20. stackhouse jr. ja. the accuracy of stones die made from rubber impression materials. j prosthet dent. 1970 oct;24(4):377-86. 21. valderhaug j, fløystrand f. dimensional stability of elastomeric impression materials in custom made and stock trays. j prosthet dent. 1984 oct;52(4):514-7. 22. johnson gh, craig rg. accuracy of addition silicones as a function of technique. j prosthet dent. 1986 feb;55(2):197-203. 23. pande na, parkhedkar. an evaluation of dimensional accuracy of one-step and two-step impression technique using addition silicone impression material: an in vitro study. j indian prosthodont soc. 2013 sep;13(3):254-9. doi: 10.1007/s13191-012-0182-1. 24. nirmala k, nandeeshwar b. the dimensional accuracy of polivinyl siloxane impression materials using two different impression techniques: an in vitro study. j indian prosthodont soc. 2015 julsep;15(3):211-7. doi: 10.4103/0972-4052.158074. 25. nissan j, rosner o, bukhari ma, ghelfan o, pilo r. effect of various putty-wash impression techniques on marginal fito of cast crowns. int j periodontics restorative dent. 2013 jan-feb;33(1):e37-42. 26. abbade da, nakandakari s, mendes aj. [moulds in fixed partial prosthesis: comparative amalysis of two mouldding techmiques using sylicon based material to verify oclusal areas with dimens anal alterations in tee prepared to receive the prosthesis]. rgo. 1983 janmar;31(1):11-4. portuguese. 27. singh k, sahoo s, prasad kd. effect of different impressions techniques on the dimensional accuracy of impresions using various elastomeric impression materials: an in vitro study. j contemp dent pract. 2012 jan;13(1):98-106. braz j oral sci. 15(3):238-241 effect of different impression techniques and elastomeric impression materials on the dimensional accuracy of partially edentulous mandibular arch 1http://dx.doi.org/10.20396/bjos.v19i0.8657468 volume 19 2020 e207468 original article 1 state university of piauí – uespi, school of dentistry, department of clinical dentistry, area of integrated clinic, parnaíba, pi, brazil corresponding author: *corresponding author: ana de lourdes sá de lira state university of piauí, school of dentistry rua senador joaquim pires 2076 ininga. fone (86) 999595004 cep: 64049-590 teresina-pi-brasil email: anadelourdessl@hotmail.com received: november 12, 2019 accepted: may 02, 2020 influence of non-nutritive sucking habits on anterior open bite ana de lourdes sá de lira1,* , alice rodrigues santos1 aim: to evaluate the clinical behavior of sucking habits in children between 2 to 6 years old in a private (a1) and a public school (a2) in the state of piauí. methods: it was cross-sectional and quantitative study in 340 participants, 169 in a1 and 171 in a2. the researchers asked the children evaluated to keep their teeth occluded while analyzing whether there was no contact between the anterior teeth and no lip sealing, characterizing the anterior openbite for g1 or if there was contact between the incisors, with lip sealing, characterizing the control group (g2). results: there was no statistically significant difference between groups regarding bottle feeding at main meals (χ2 = 3.03; p = 0.08). however, regarding the use of a pacifier, there was a statistically significant association (χ2 = 17.99; p <0.01) between pacifier use and the presence of anterior openbite. such association was also observed between digital sucking habit and malocclusion (χ2 = 8.99; p = 0.01). only the parents of the children with anterior openbite noticed the disharmony in the occlusion. it can be deduced that there was an awareness of parents /guardians about the disharmony generated by nonnutritive sucking habits. conclusion: nonnutritive sucking habits influenced the appearance of the anterior open bite in children with deciduous dentition. nonnutritive sucking habits, such as digital sucking and pacifiers, are significantly associated with the presence of anterior open bite. breastfeeding is important in preventing this malocclusion. keywords: open bite. malocclusion. sucking behavior. https://orcid.org/0000-0002-9299-1416 2 lira et al. introduction open bite can be defined as a deficiency in normal vertical contact between antagonist teeth and may manifest in a limited region or, more rarely, throughout the dental arch. if the lack of contact of the teeth is located in the incisors and / or canines region, and when the occlusion is in centric relation, it is renamed anterior open bite1-3. this malocclusion has a multifactorial origin and is almost always associated with an orofacial myofunctional disharmony, either due to hereditary or environmental factors. among the environmental factors we can mention the presence of harmful sucking habits1,4. basically there are two types of suction, the first for nutritional purposes, which refers to breastfeeding itself, providing essential nutrients for the child’s growth, and the second for non-nutritive purposes, such as the sucking of fingers and pacifiers, which provides a sensation pleasant safety and welfare5. suction is a natural reflection of the human being and has been present since intrauterine life. this maneuver involves various structures such as the tongue, cheeks and lips, and stimulates the normal growth of the jaws and stomatognathic structures. in early life, this habit is related to the child’s diet and nutrition, as well as psychological and emotional development aspects5,6. however, this habit can become deleterious if sustained for a prolonged period, which may alter the normal growth pattern and damage the occlusion. damage will be determined by the frequency, duration, intensity and the object used, as well as the age of the individual at the time of its installation6,7. regarding the endless nutritional habit, the most found in children are those of digital suction and sprinkles, being prevalent in the first years of the child’s life tending to decrease with age. both forms of suction promote similar changes in peribucal musculature and occlusion, however it is suggested that due to the difficulty in interrupting digital suction, this habit may result in greater deleterious effects. however, if the deleterious oral habit persists until three years of age, there is a chance of self-correction of possible occlusal disharmonies present6. children with non-nutritive sucking habits have a high prevalence of anterior openbite, showing a significant association between the presence of deleterious sucking habits and the occurrence of malocclusion8,9. in 2004 the prevalence of this malocclusion in preschoolers in brazil was 24%10. on the other hand, in 2012 the prevalence in a city in the northeast was 20.3%11. it is expected to find out in this study low prevalence of anterior openbite due to the guidance received by their prenatal mothers and medical and dental consultations on the importance of breastfeeding and preventive measures on nonnutritive sucking habits. based on this context, it belived that the non-nutritive sucking habit, such as finger and pacifier sucking, may predispose the installation of the anterior open bite, taking into account that the longer the duration and frequency of the habit, as well as the intensity of the force applied, the greater the severity of the malocclusion. hence, it is 3 lira et al. important to increase our understanding of the association of non-nutritive sucking habit with alterations in occlusion and oral myofunction in preschool children who are being introduced to oral health care. this would provide pediatric dentists with clinical evidence to guide the children’s parents and guardians. we wanted to conduct this research to investigate whether there is an association between non-nutritive sucking habits and anterior open bite. this research aimed to evaluate the clinical behavior of sucking habits in children between 2 to 6 years old in a private (a1) and a public school (a2) in the state of piauí. materials and methods this study was approved by the research ethics committee of the state university of piauí cep / uespi, under number 2.537.169. it was cross-sectional observational study, from august to december 2018, whose sample size calculation was based on the target population: children between 2 and 6 years old, specifically in the preschool phase of parnaíba in 2018, totaling 4.087 students. the required sample size was 350 participants, according to the inclusion criteria, as the population representative of the municipality of parnaíba-pi. this minimum number of participants was considered sufficient considering the proposed analyzes, the 5% sampling error, and the 95% confidence level, indicating that the probability of the research error does not exceed 5%12. however due to the parents’ lack of interest in answering the questionnaire and signing the consent form, only 340 children were allowed to participate, 169 in private school (a1) and 171 in municipal public school (a2). inclusion criteria were children in primary dentition, with or without non-nutritive sucking habit (pacifier or digital). those who presented anterior open bite were named g1 and those without this malocclusion were classified as g2, between 2 and 6 years old, whose parents or guardians and the children accepted the research. as exclusion criteria, children under 2 years, because the deciduous dentition would not be fully formed, and those over 6 years old, because they were already in mixed dentition and children whose parents or guardians did not authorize the research. the anterior open bite is characterized by the lack of normal vertical contact between the antagonistic teeth, located in the region of incisors and / or canines4. to the parents or guardians of the children were sent a questionnaire prepared by the researchers about non-nutritive oral habits, such as pacifier use and / or digital sucking, as well as breastfeeding history. the questionnaire was based on studies made for the elaboration of this research, containing questions related to the type of breastfeeding (natural and / or artificial), non-nutritive oral habits (pacifier and / or digital sucking), frequency and duration of habits and perception of breastfeeding and parents or guardians perception of the presence of anterior openbite in the child (figure 1). the questionnaire used in this research was previously tested and applied by ling et al.7 to carry out an integrative review of the literature on the clinical behavior of sucking habits in children between 2 to 6 years. two researchers were calibrated with the pilot study by examining ten children who sought care at the clinic school of dentistry (ceo) of the state university of piauí 4 lira et al. (uespi) campus of parnaíba, to identify the presence of anterior open bite and this analysis according to the methodology described by peres et al.13. the pilot study involving 10 children not participating in the sample, randomly chosen 5 for each gender attended at the ceo, aimed to test the proposed methodology. two examinations separated by an interval of 10 days were performed for each child. intra-examiner reliability for all variables was evaluated using the kappa statistic. the kappa coefficient for intraand inter-examiner agreement being 0.87 and 0.85, respectively. for clinical evaluation, each evaluated child was asked to keep the teeth occluded while the evaluators separated the lips with the index finger and thumb, observing if there was no contact between the anterior teeth and absence of lip sealing, characterizing the anterior opebite for g1. if there was contact between the incisors, with lip sealing, the child would participate in the control group g2. the biosafety norms recommended, such as the use of gloves and mask, were taken into consideration, and the examination was performed under natural light, with the child comfortably positioned in front of the examiners. statistical methodology the results were stored in the excel windows 2010 microsoft® database and arranged in graphs and tables for better interpretation and discussion. averages, dispersion and chi-square test were applied to compare the results between a1 and a2 and between g1 and g2 and to identify occlusion that was significantly associated with non-nutritive sucking habits, with 95% statistical significance. results from the tabulation of the participants’ data, it was observed that the average age was 3.87 years (sd = 1.46). it was possible to verify the association between figure 1. questionnaire applied to the survey respond according to the characteristics you observe in the child. 1. regarding breastfeeding: a ( ) exclusive breastfeeding up to six months b ( ) breastfeeding combined with bottle feeding c ( ) exclusive use of bottle 2. do you currently use a bottle to supplement your diet? a ( ) yes b ( ) no 3. had/are you in the habit of using a pacifier? a ( ) yes b ( ) no 4. had/have a digital sucking habit (finger sucking)? a ( ) yes b ( ) no 5. if yes to questions 3 and/or 4, what is the frequency and duration of these habits? a ( ) all day long b ( ) at long intervals during the day c ( ) at short intervals during the day d ( ) all night long e ( ) at long intervals during the night f ( ) at short intervals at night 6. do you notice any disharmony in your child's smile? a ( ) yes b ( ) no 5 lira et al. presence/absence of maa and sample feeding habits. for this, a chi-square association test was performed, with a statistical significance level of 5%, with its results presented in cross-tables, described below. from the results it was observed that most children in g1 used the combination of bottle feeding and breastfeeding for up to six months (n=30), and those in g2 had exclusive breastfeeding (n=217). this result was statistically significant (χ2 = 22.95; p <0.01). as seen in table 1, there was no statistically significant difference between the groups regarding bottle feeding at main meals (χ2 = 3.03; p = 0.08). however, regarding pacifier use, there was a statistically significant association (χ2 = 17.99; p <0.01) between pacifier use and the presence of anterior openbite. such association was also observed between digital sucking habit and malocclusion (χ2 = 8.99; p = 0.01). regarding the perception of some disharmony in their child´s smile, the most parents who did not have the malocclusion (g2) did not notice it, while the parents of those who presented it (g1) reported noticing the disharmonic smile (χ2 = 17.65 ; p <0.01). the comparison between g1 and g2 regarding these variables was shown in figure 2. table 1. comparison between g1 and g2 regarding the studied variables variables answers g1 g2 χ2 p valor bottle at meals yes 34 122 3.03 0.08 no 17 167 pacifier use yes 37 57 17.99 0.01 no 14 231 digital suction yes 17 20 8.99 0.01 no 34 269 smile disharmony perception yes 31 38 17.65 0.01 figure 2. graphical representation of the comparison between g1 and g2 in relation to the studied variables. pacifier use digital suction smile disharmony perception feeding bottle g1 g2 300 250 200 150 100 50 0 noyesnoyesnoyesnoyes 34 122 167 17 37 14 231 269 252 57 17 20 31 38 20 34 6 lira et al. as represented in figure 3, for those participants who answered positively to questions about pacifier and digital sucking habits, they were asked to state the frequency. the results presented indicated that the majority (33.3%) presented this habit during the day, with short intervals. according to table 2 in both a1 and a2, the number of children who had exclusive breastfeeding up to six months without openbite was significant. based on table 3 in both schools (a1 and a2) there was an association between non-nutritive sucking habits and anterior openbite (g1). only the parents were able to perceive the disharmonious smile of g1 (χ2 = 7.12; p <0.00). however, there was no significant difference between a1 and a2 when only g1 and g2 were evaluated separately. there was no significant difference between genders in both schools when g1 and g2 were evaluated. however, when genders were evaluated separately for g1 and g2, it was found that for both genders the number of children without anterior openbite was significantly higher for those who were exclusively breastfed until 6 months of age (table 4). table 5 shows that bottle feeding at the main meals did not significantly influence the anterior openbite in both genders and that the parents did not statistically perceive the disharmony of the smile in female children. figure 3. graphical representation of frequency and duration of sucking habits 1 the whole day 2 long breaks in the day 3 short breaks in the day 4 all night long 6 short breaks at night options 2 and 4 options 2 and 5 options 2 and 6 options 3 and 6 35 30 25 20 15 10 5 0 11.90% 21.40% 33.30% 16.70% 7.10% 2.40% 2.40% 2.40% 2.40% table 2. type of food up to six months old between g1 and g2 of a1 and a2. food up to six months old groups a1 χ2 p valor a2 χ2 p valor g1 g2 g1 g2 breastfeed only 0 107 12.35 0.00 7 110 11.18 0.00combined feeding 17 38 13 20 bottle only 4 4 10 10 7 lira et al. discussion there was immediate benefit to the children who participated in the research, when their parents / guardians became aware of the existence of anterior openbite and the harm caused by non-nutritive sucking habits. it has been reported that children under 3 years of age may have their anterior open bite corrected if deleterious sucking habits are removed14. it was found by moimaz et al.14 (2013), in similar studies of sucking habits 44.4% of the evaluated children had a pacifier sucking habit and did not receive breast milk exclusively until six months of age. these findings reinforce the data found in this table 3. analysis of sucking habits and perception of disharmony between g1 and g2 of a1 and a2. variables answers a1 χ2 p valor a2 χ2 p valor g1 g2 g1 g2 bottle at meals yes 10 45 0.93 0.33 23 77 1.62 0.20 no 10 103 7 64 pacifier use yes 14 24 7.67 0.00 23 33 9.71 0.00 no 7 124 7 107 digital suction yes 10 10 9.07 0.00 7 10 4.90 0.05 no 10 138 23 131 smile disharmony perception yes 10 14 7.12 0.00 21 23 9.75 0.00 no 10 134 10 117 table 4. comparison of feeding up to six months between g1 and g2 in male and female. food up to six months old groups male χ2 p valor female χ2 p valor g1 g2 g1 g2 breastfeed only 0 119 3.01 0.00 7 99 7.32 0.02combined feeding 17 37 13 20 bottle only 7 0 7 14 table 5. evaluation of smile sucking and disharmony habits between g1 and g2 regarding gender. variables answers male χ2 p valor female χ2 p valor g1 g2 g1 g2 bottle at meals yes 17 51 3.89 0.06 17 71 0.20 0.65 no 7 105 10 61 pacifier use yes 20 27 14.59 0.00 17 30 4.93 0.02 no 3 129 10 102 digital suction yes 3 7 4.12 0.02 13 14 7.42 0.00 no 20 150 13 119 smile disharmony perception yes 24 10 34.68 0.00 7 27 4.08 0.05 no 0 146 20 105 8 lira et al. research, in which there was a predominance of breastfeeding combined with bottle-feeding or bottle-feeding up to six months of age in children with anterior openbite. of the 224 children who received exclusive breastfeeding by the age of six months, only 7 developed the malocclusion because they used a pacifier. similar results were obtained in the study by pereira et al.15 (2018). it was found that 96.6% of preschool children who had some habit of deleterious sucking, exclusively fed on the bottle. they also found a decrease in the number of children who developed finger and / or pacifier sucking when breastfed for more than 6 months, confirming that this practice is a protective factor for the development of conditions that may negatively affect child occlusion. the present study showed the direct relationship between non-nutritive sucking habits and the development of the malocclusion. this was confirmed because all children with maa used pacifiers or digital suction. bottle feeding at the main meals did not influence the onset of malocclusion, since there was no significant difference between g1 and g2. this was due to the fact that of the 51 children in g1, 17 did not use the bottle, but had the deleterious habit of digital sucking (table 1 and figure 2). these findings corroborate those found in studies by eslamipour et al.3 (2018), conducted in children aged 3 to 5 years, whose prevalence of open bite was 20%. the risk of anterior openbite was 3 times higher in children who did digital sucking and almost 5 times higher than do used pacifiers, when compared to those who did not have such habits. in a study by macho et al.16 (2012), it was found that 34.1% of the sample with deleterious oral habits had malocclusions. thus, 51.8% of children with pacifier sucking habits and 19.6% of children with digital sucking habit had this malocclusion. according to studies by bona et al.17 (2016), the non-nutritive sucking habit does not always cause occlusion anomalies. in their research, despite the high incidence of malocclusions in the sucking habit group, it was observed that a small percentage of children who presented occlusion changes did not have sucking habits, contrary to the findings of our study. however, the same author stated that the severity of malocclusion depends on the frequency, intensity and duration of the habit, corroborating our research, in which children who presented with openbite and a habit of digital sucking and / or pacifiers maintained these habits in high frequency and intensity. (figure 3). in studies by garbin et al.18 (2014) which was performed on preschool children, it has been showed 42.7%of the children used pacifiers for more than 6 hours daily. these evidences were similar to those found in our study, in which 33% of participants using pacifiers and / or digital sucking had these habits during the day, with short intervals, followed by 21.4% with long intervals during the day (figure 3). only the parents of the children with anterior openbite noticed the disharmony in the occlusion. it can be deduced that there was an awareness of parents / guardians about the disharmony generated by non-nutritive sucking habits (table 1). these data corroborate another literature study by garbin et al.18 (2014), in which 97.1% of parents / guardians believed that non-nutritive sucking habits could cause harm to children’s teeth. 42.25% of them reported that they knew the possible damage, but only 33.3% pointed out that they had already tried to remove the habit. 9 lira et al. when g1 and g2 from schools a1 and a2 were compared, it was found that in both schools was significant the number of children from g2 who were exclusively breastfed until 6 months of age, emphasizing the importance of breastfeeding in preventing malocclusions. this was reinforced by the fact that in a2 only 7 of the 51 children with anterior openbite were fed to the breast, but used pacifiers (table 2). continuing this comparison, there was a significant difference between g1 and g2 in both schools regarding deleterious oral habits, suggesting their importance in the installation of the malocclusion (table 3), as observed by other authors5,7,17,19-21 by stating that the frequency, duration and intensity of non-nutritive sucking habits are determining factors for the installation of it. when genders were assessed separately, the number of g2 children in both genders who were exclusively breastfed until 6 months of age and did not develop the malocclusion was significant statistically (table 4). similar results were observed by other authors16,22,23, emphasizing that the benefits of breastfeeding include the promotion of stomatognathic system development. cavalcanti et al.24 (2007), in their research found that the frequency of sucking habits was higher among children with artificial feeding than in children with natural feeding, reinforcing the importance of breastfeeding. according to table 5, there was a significant difference between g1 and g2 regarding the use of pacifiers and digital sucking, demonstrating that they favored the development of maa in both genders indistinctly, corroborating the findings of other authors20,25-30 by emphasizing that such habits may compromise the stomatognathic system similarly in both genders. there is a higher prevalence and risk of developing malocclusions, such as class ii canine relationship, a posterior crossbite and anterior open bite, among children with primary dentition who have a history of suboptimal breast-feeding31. the results of the present study found that the non-nutritive sucking habit, such as finger and pacifier sucking, may predispose the installation of the anterior open bite. it should be emphasized the importance of recognizing early occlusal and myofunctional changes caused by non-nutritive sucking habit in young children. as a limitation of the study, the need to carry out a larger sample in several schools is emphasized in order to test the reproducibility of the findings. future studies on the relationship between nonnutritive sucking habits and anterior open bite are important. conclusion nonnutritive sucking habits influenced the appearance of the anterior open bite in children with deciduous dentition. nonnutritive sucking habits, such as digital sucking and pacifiers, are significantly associated with the presence of anterior open bite. breastfeeding is important in preventing this malocclusion. 10 lira et al. references 1. antunes ls, teixeira ec, gomes if, almeida mh, mendes pp, antunes laa. [causal relationship between the oral habits, feeding and malocclusion in children deciduous teeth]. cient cienc biol saúde. 2015;17(2):75-80. portuguese. 2. wajid ma, chandra p, kulshrestha r, singh k, rastogi r, umale v. open bite malocclusion: an overview. j oral health craniofac sci. 2018;3(3):11-20. doi: 10.29328/journal.johcs.1001022. 3. eslamipour f, afshari z, najimi a. prevalence of malocclusion inpermanent dentition of iranian population: a review article. iran j public health. 2018 feb;47(2):178-87. 4. artese a, drummond s, nascimento jm, artese f. criteria for diagnosing and treating anterior open bite with stability. dental press j orthod. 2011 may-june;16(3):136-61. portuguese. doi: 10.1590/s2176-94512011000300016.  5. grochentz jbg, laginski mcs, dalledone m, bruzamolin cd. marques, fr. 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[calculation of sample size in administration research]. curitiba: autor’s edition; 2011. portuguese. 13. peres ma, traebert j, marcenes w. [calibration of examiners for dental caries epidemiology studies]. cad saúde pública. 2001 jan-feb;17(1):153-9. doi: 10.1590/s0102-311x2001000100016. portuguese. 14. moimaz sas, rocha nb da, garbin aji, saliba o. [the effect of breastfeeding in the acquisition of non-nutritive sucking habits and malocclusion prevention]. rev odontol unesp 2013;42(1):31-6. doi: 10.1590/s1807-25772013000100006. portuguese. 15. pereira mbb, ferreira ap, bastos ncs, avelino mag, pacheco jf, duarte mdl, et al. [association between breastfeeding duration, non-nutritive sucking habits and deglutition in preschool children]. robrac. 2018;27(83):223-8. portuguese. 16. macho v, andrade d, areia c, norton a, coelho a, macedo p. [prevalence of deleterious oral habits and occlusal anomalies in a population aged 3-13 years]. rev port estomatol med dent cir maxilofac. 2012;53(3):143-7. doi: 10.1016/j.rpemd.2012.04.002. portuguese. 17. bona ap, moreira kms, tedesco tk, imparato jcp, ferreira bd, reis jb. [multidisciplinary performance to anterior open bite associated with finger sucking: case report]. rev assoc paul cir dent. 2016;70(1):58-63. portuguese. https://dx.doi.org/10.29328/journal.johcs.1001022 11 lira et al. 18. garbin cas, garbin ají, martins rj, de souza np, moimaz sas. [prevalence of non-nutritive sucking habits in preschoolers and parents’ perception of its relationship with malocclusions]. cien saude colet. 2014;19(2):553-8. doi: 10.1590/1413-81232014192.23212012. portuguese. 19. tomita ne, bijella vt, franco lj. [the relationship between oral habits and malocclusion in preschool children]. rev saude publica. 2000;34(3):299-303. doi: 10.1590/s0034-89102000000300014. portuguese. 20. pizzol kedc, montanha ss, fazan et, boeck em, rastelli ans. [prevalence of nonnutritive sucking habits and their relationship to age, gender and type of feeding in preschool children from araraquara-brazil]. rev cefac. 2012 may-jun;14(3):506-15. doi: 10.1590/s1516-18462012005000001. portuguese. 21. romagosa der, gamboa mrp, muñiz ya, oliva lmp, oliva de, naranjo st. risk factors associated with deforming oral habits in children aged 5 to 11: a casecontrol study. medwave. 2014 mar;14(2):e5927. doi: 10.5867/medwave.2014.02.5927. 22. oakley ll, henderson j, redshaw m, quigley ma. the role of support and other factors in early breastfeeding cessation: an analysis of data from a maternity survey in england. bmc pregnancy and childbirth. 2014 feb 26;14:88. doi: 10.1186/1471-2393-14-88. 23. victora cg, bahl r, barros ajd, frança gva, horton s, krasevec j et al. breastfeeding in the 21st century: epidemiology, mechanisms, and lifelong effect. lancet. 2016 jan;387(30):475-90. doi: 10.1016/s0140-6736(15)01024-7. 24. cavalcanti al, medeiros pkb, moura c. [breast-feeding, bottle-feeding, sucking habits and malocclusion in brazilian preschool children]. rev salud publica (bogota). 2007 apr-jun;9(2):194-204. doi: 10.1590/s0124-00642007000200004. portuguese. 25. pithon mm, magno mb, coqueiro rs, paiva sm, marques ls, paranhus lr, et al. oral health–related quality of life of children before, during, and after anterior open bite correction: a singleblinded randomized controlled trial. am j orthod dentofacial orthop. 2019 sep;156(3):303-11. doi: 10.1016/j.ajodo.2019.04.022. 26. gomes mc, clementino ma, pinto-sarmento tca, costa emmb, martins cc, granville af, et al. parental perceptions of oral health status in preschool children and associated factors. braz dent j. 2015;26(4):428-34. doi: 10.1590/0103-6440201300245. 27. nihi vsc, maciel sm, jarrus me, nihi fm, salles clf, pascotto rc, et al. pacifier-sucking habit duration and frequency on occlusal and myofunctional alterations in preschool children. braz oral res. 2015;29(1):1-7. doi: 10.1590/1807-3107bor. 28. lopes freire gm, espasa suarez de deza je, rodrigues da silva ic, butini oliveira l, ustrell torrent jm, boj quesada jr. non-nutritive sucking habits and their effects on the occlusion in the deciduous dentition in children. eur j paediatr dent. 2016;17(4):301-6. 29. schmid km, kugler r, nalabothu p, bosch c, verna c. the effect of pacifier sucking on orofacial structures: a systematic literature review. prog orthod. 2018 mar;19(8):1-11. doi: 10.1186/s40510-018-0206-4. 30. lima aadsj, alves cmc, ribeiro ccc, pereira alp, da silva aam, silva lfge, et al. effects of conventional and orthodontic pacifiers on the dental occlusion of children aged 24-36 months old. int j paediatr dent. 2017 mar;27(2):108-19. doi: 10.1111/ipd.12227. 31. doğramacı ej, rossi-fedele g, dreyer cw. malocclusions in young children: does breast-feeding really reduce the risk? a systematic review and meta-analysis. j am dent assoc. 2017 aug;148(8):566-74.e6. doi: 1010.1016/j.adaj.2017.05.018. https://sci-hub.tw/10.1016/j.adaj.2017.05.018 1http://dx.doi.org/10.20396/bjos.v18i0.8657170 volume 18 2019 e191007 original article 1 department of prosthodontics and periodontics, division of periodontics, piracicaba dental school, university of campinas unicamp, piracicaba, são paulo, brazil. 2 department of community dentistry, division of biostatistics, piracicaba dental school, university of campinas – unicamp, piracicaba, são paulo, brazil. 3 department of community dentistry, piracicaba dental school, university of campinas – unicamp, piracicaba, são paulo, brazil. corresponding author: karina gonzales silvério phone/fax number (it can be published): +55 19 2106 5301 email address (it can be published): kgsilverio@fop.unicamp.br postal address: department of prosthodontics and periodontics, division of periodontics, piracicaba dental school, university of campinas – unicamp. av. limeira, 901, cep 13414-901, piracicaba, são paulo, brazil. received: may 11, 2018 accepted: august 20, 2019 periodontal disease and associated factors in the adult and elderly population from jundiaí city, brazil marcela di moura barbosa1, marília jesus batista2, evely sartorti da silva morgan3, enilson antonio sallum1, marcio zaffalon casati1, karina gonzales silvério1,* aim: this study assessed the prevalence of periodontal disease in the adult and elderly populations from jundiaí city, and its association with individual social inequalities in a conceptual framework approach. methods: the survey was conducted with a sample of 342 adults and 145 elderly, and periodontal disease was assessed based on the community periodontal index (cpi) and clinical attachment loss (cal). a questionnaire addressing socio-demographic and behavioral variables, smoking and diabetes was included. bivariate and multivariate analyses, using binary regression analyses, were carried out in a hierarchical approach with conceptual framework to reveal association among periodontal disease and social-demographic, smoking and diabetes variables. results: one adult and fifty-six elderly who had lost all teeth were excluded from the sample. mild periodontal disease (cal ≤3 mm) was the condition more prevalent in the adult (74%) and elderly populations (60.6%). adjusted analysis revealed that low educational level (or 2.65, 95% ci 1.19-5.88), irregular use of tooth floss (or 1.9, 95% ci 1.06-3.40), and smoking (or 2.14, 95% ci 1.04-4.42) were independently associated with moderate/ severe periodontal disease (cal and probing depth ≥4 mm) in the adult group. for the elderly group, low educational levels (or 0.16, 95% ci 0.04-0.58), use of public dental service (or 5.32, 95% ci 1.23-23.03), and diabetes condition (or 3.78, 95% ci 1.2011.91) were significantly associated with periodontal disease. conclusion: in conclusion, the data showed that education level, smoking habits, diabetes, use of dental floss and type of dental service are factors associated to moderate/severe periodontal disease among brazilians from jundiaí city. keywords: periodontal disease. epidemiology. oral hygiene. smoking. diabetes mellitus. 2 barbosa et al. introduction periodontal diseases are one of the most important oral health conditions contributing to the global burden of chronic diseases1. along with severe dental caries, periodontal diseases are a major cause of tooth loss, particularly among the elderly, which directly affects the quality of life of people in terms of reduced functional capacity, self-esteem and social relationships, representing a public health problem1. there are different clinical manifestations of periodontal diseases, in which gingivitis is the most prevalent form, found in large proportions in all populations2. regarding chronic periodontitis, in which breakdown of supporting tissues of teeth occurs, epidemiological surveys have shown that this condition varies significantly between ages and countries2. according to world oral health report (who), severe periodontitis affects between 5-15% of most adult populations (35-44 years) worldwide3. data from the 2009-2010 national health and nutrition examination survey (nhanes)4 showed that the prevalence of periodontitis in the adult population was 36.6% while in the elderly it was 70.1% 4. in england, the prevalence of periodontitis was 42% between individuals of 35-44 years, and 70% in individuals of 55-64 years5. in brazil, a representative study conducted in porto alegre, involving only adults (>30 years), showed a clinical attachment loss ≥ 5 mm in 79% of adults and ≥ 7mm in 52% of the population6. further, data from the last brazilian oral health survey showed that 15.3% of the adult population had “moderate to severe” periodontal disease and that 5.8% had a “severe” form of disease7. compared with the 2003 epidemiological survey, it was observed a 6.3% increase in the prevalence of “moderate to severe” periodontal disease8. this increasing of the prevalence of periodontal disease may be a consequence of improvements in life expectancy and the growth of brazilian population, which may result in an increased number of people with a higher number of natural teeth9. the etiology of the inflammatory periodontal disease is associated with the accumulation of supraand sub-gingival microflora of dental biofilms, mainly due to poor oral hygiene10. in addition, tobacco smoking and diabetes mellitus have been recognized as true risk factors for the development of this inflammatory process11. further, some studies have also suggested that socioeconomic inequalities play a significant role in the occurrence of periodontal diseases10,12,13. in this context, the aim of the present study was to assess the prevalence of periodontal disease in the adult and elderly population from jundiaí city and its association with individual social inequalities in a conceptual framework approach. material and methods study design and location this cross-sectional study was conducted in the city of jundiaí, são paulo state, brazil, with a household probability sample. in 2014, the population of jundiaí consisted 3 barbosa et al. of 397.965 residents. a group of adult and elderly subjects, 35 to 44 and 65 to 74 years old were respectively, 56.569 and 20.431 inhabitants14. the present study is part of a major study for “oral health conditions of the population from jundiaí”. ethical considerations the study was approved by the research ethics committee of the campinas state university – unicamp (#077/2013). individuals who agreed to participate signed the informed consent form. at the end of the examination, the participants were provided with a report about their oral status and diagnosed diseases. patients with diagnosed periodontal diseases were advised to seek oral health consultation and treatment. the study was conducted in between the month of april to september 2014. sample for the purpose of this study, adults aged 35 to 44 years old, and elderly aged 65 to 74 years old residing in jundiaí were eligible to participate. the sample size was calculated in order to obtain a representative sample of the adult population of this municipality. the prevalence of periodontal disease adjusted for the jundiaí population size for adults and elderly individuals, of 70.2% and 90.9% respectively, was the basis of the calculation7. a confidence interval of 95%, an accuracy of 10% and a design effect of 2 were adopted. a 30% increase was added to this total in order to compensate the possible loss, thereby resulting in an estimate of 204 adults 35–44 years old and 27 elders 65–74 years old, to be representative for periodontal disease. however, sample size considered caries disease in order to obtain oral health conditions data for the major study. the sample size adopted for the study was 300 adults and 71 elderly. to select the houses, considering the possibility of refusals, we added 30% of this sample size, which comprised 428 houses for adults and 101 for elderly. the total was divided by the 30 census tracts selected for the study (figure 1). 517 census tract (132.028 houses) 32 census tract randomly selected 2 substitutes census tract 30 census tract 428 houses for adults 101 houses for elders 342 adults 145 elders 1 adults completely edentulous were excluded 341 adults 89 elders 56 elders completely edentulous were excluded figure 1. flowchart of study sample. 4 barbosa et al. sample selection was carried out in two stages. in the first stage, the unit of selection was the census tract and from 517 census tracts, 30 were randomly selected (plus 2 in case substitutions were needed). the second stage consisted of the selection of households, and a 30% increase in the probabilistic sample size to select the houses was used to compensate for non-responses. this resulted in a total of 342 houses, divided by the 30 census tracts selected for the study, resulting in a fraction of 11.4 houses per census tract. based on the average population size of each census tract, 11 houses per tract for adults and 3 for elderly were randomly selected and then one adult or elderly, per house was also randomly selected. interview and clinical examination a team of five dentists, two dental assistants, and twenty local community health agent conducted the fieldwork. dentists using a written questionnaire, which included 66 questions about demographics, socioeconomics, behavioral, dental services and diabetes mellitus data, interviewed participants. all clinical examinations were performed with individuals seated on a regular chair, in a well-illuminated part of the house, using an intraoral mirror and a ball point probe. periodontal diseases were assessed based on the community periodontal index (cpi) as proposed at oral health surveys by world health organization in 201315. in addition, clinical attachment loss (cal) was performed in all sextants using the following categories: (0) up to 3mm, (1) 4-5mm, (2) 6-8mm, (3) 9-11mm, (4) 12mm or more and (x) excluded sextant. a sextant should be examined only if there are two or more teeth present which are not indicated to extraction. all examiners and interviews were trained and calibrated by a researcher with experience in this type of epidemiological study. the calibration process consisted of two processes: firstly, a theoretical phase where diagnostic criteria were discussed and secondly, a practical phase, in which 20 individuals were examined twice, in order to calculate intra and inter-examiner reliability indexes. further, the calibration process was performed during the fieldwork to ensure the inter-examiner reliability. the intra and inter-examiner reproducibility was calculated using kappa test. for periodontal conditions, the values vary from 0.63 to 0.91 (mean value = 0.87). the intra-examiner values vary from 0.63 to 0.87. data analysis data were analyzed using the statistical package for the social sciences (spss), version 19.0 software program. descriptive weighted analyses were performed to obtain the frequency, mean, median, and standard deviation (sd) of variables which were the clinical conditions examined. the independent variables studied were selected according to a validated conceptual framework adapted from batista mj et al.16 (2014) (figure 2). after a descriptive analysis, the variables selected were categorized and/or dichotomized for statistical analysis. 5 barbosa et al. the outcome of this study was mild and moderate-severe periodontal disease. it was considered mild periodontal disease for individuals who presented at least one sextant with bleeding, calculus and clinical attachment loss up to 3mm. for periodontal disease moderate to severe, it was considered individuals who presented clinical attachment loss and periodontal pocket ≥ 4 mm. individuals who presented the six sextants without any sign of periodontal disease (cpi=0) was excluded from the sample of affected individuals. bivariate and multivariate analyses were performed for adults and elderly separately, using binary regression analysis in a hierarchical approach according to the conceptual model in figure 2 16. first, a preliminary analysis was performed using univariate model, and all variables showing associations with p<0.25 were included in a multivariable model. at first level, age was the exogenous variable, analysed as a discrete variable. for this study, at second level, the primary determinants of health were: oral health service (public, private and insurance), sex (male and female), marital status (dichotomized in those who lived with a partner and not), family income ($405 or less, $405 to $810 or more than $810), educational level (less than 8 years, 8 to 12 and more than 12 years), and economic status in childhood (rich/ middle class or poor/very poor). at third level, oral health behaviors were tooth brushing (one/two or three or more times/ day), tooth flossing (yes or no), smoking (yes or no). the use of dental services was characterized by the frequency of use (once a year, less than once a year and urgency), the type of service (public, dental insurance or private) and the time since last visit (3 or more years, 1 to 2 years and less than one year ago. at fourth level, systemic disease was assessed as having or not diabetes mellitus. at fifth level, clinical conditions such as, gingival bleeding, biofilm, and calculus were considered. the outcome variable was the presence of periodontal disease. exogenous variables primary determinants of oral health oral health behaviors systemic disease results in oral health diabetes age oral health services type of service personal characteristics marital status sex family income educational level economic status in childhood use of dental services time frequency personal health practices tooth brushing tooth flossing smoking clinical conditions gingival bleeding biofilm calculus p eriodontal disease figure 2. conceptual framework for oral health-related qualify of life adapted from batista mj et al., 2014. 6 barbosa et al. results a total of 342 individuals aged 35 to 44 years and 145 aged 65 to 74 years were examined. one adult and 56 elderly completely edentulous were excluded from the analyses. then, the study sample included 341 adults and 89 elderly. mild periodontal disease was the condition more prevalent between adult (74%) and elderly (60.6%) population. when the prevalence of moderate to severe disease was assessed, this condition was higher among elderly (39.3%) compared to adult (25.8%) individuals. the distribution of periodontal conditions by independent variables are displayed in tables 1 and 2. the prevalence of moderate to severe disease was higher in adult self-declared as white (65.9%) and in non-whites elderly individuals (80.0%). mild periodontal disease was more frequently in adults and elderly who lived in houses with four or fewer individuals. in addition, elderly individual who lived a poor or very poor childhood had the highest prevalence of moderate to severe periodontal disease (table 1). both mild and moderate/severe periodontal diseases were more prevalent in individuals who had used dental services at less than one year and who reported table 1. distribution of periodontal conditions according to demographic and socioeconomic factors in adult and elderly populations from jundiaí city. variables adults (35-44 years) elderly (65-74 years) early periodontal disease moderate/ severe periodontal disease early periodontal disease moderate/ severe periodontal disease n (%) n (%) n (%) n (%) sex male 75 (29.6) 36 (40.9) 23 (42.6) 22 (62.9) female 178 (70.4) 52 (59.1) 31 (57.4) 13 (37.1) race non white 70 (27.8) 30 (34.1) 37 (68.5) 28 (80.0) white 182 (72.2) 58 (65.9) 17 (31.5) 7 (20.0) marital status married/ living common law 172 (69.4) 61 (69.3) 39 (72.2) 24 (68.6) not living common law 76 (30.6) 27 (30.7) 14 (25.9) 11 (31.4) household income < r$1620.00 ($405.00) 45 (18.3) 20 (23.0) 8 (15.4) 6 (17.1) r$1620 to r$3240 ($405 to $810) 67 (27.2) 30 (34.5) 23 (44.2) 9 (25.7) > r$3240 ($810) 134 (54.5) 37 (42.5) 21 (40.4) 20 (57.1) individuals per household 4 or less individuals 194 (77.6) 60 (69.0) 47 (88.7) 31 (88.6) more than 4 individuals 56 (22.4) 27 (31.0) 6 (11.3) 4 (11.4) education less than 8 years 68 (26.4) 25 (34.7) 32 (59.3) 16 (44.4) 8 to 12 years 94 (36.0) 35 (48.6) 10 (18.5) 7 (19.4) more than 12 years 88 (34.1) 10 (13.9) 9 (16.7) 13 (36.1) economic status in childhood poor or very poor 114 (45.6) 51 (58.6) 26 (48.1) 21 (60.0) rich or middle class 136 (54.4) 36 (41.4) 28 (51.9) 14 (40.0) current situation compared to childhood better 176 (70.1) 65 (73.9) 41 (75.9) 32 (91.4) same or worse 75 (29.9) 23 (26.1) 13 (24.1) 3 (8.6) 7 barbosa et al. brushing their teeth three or more times a day. the prevalence of moderate to severe periodontal disease was higher among non-smokers adult and elderly individuals who reported to have already received periodontal treatment and who did not use dental floss. diabetes mellitus was a systemic disease present in 33.0% of adult and 91.4% of the elderly population with moderate to severe periodontal disease (table 2). the results of multivariate analysis of periodontal diseases are displayed in tables 3 and 4. in the adult group, education level, smoking, and use of dental floss were significantly associated with periodontal disease (table 3). further analysis showed table 2. distribution of periodontal conditions according to dental service, behavioral and diabetes factors in adult and elderly populations from jundiaí city. variables adults (35-44 years) elderly (65-74 years) early periodontal disease moderate/ severe periodontal disease early periodontal disease moderate/ severe periodontal disease n (%) n (%) n (%) n (%) time since last visit 3 or more years 43 (17.3) 17 (19.3) 19 (35.2) 7 (20.0) 1 to 2 years 68 (27.4) 27 (30.7) 15 (27.8) 5 (14.3) less than one year 137 (55.2) 44 (50.0) 20 (37.0) 23 (65.7) type of service public 33 (13.3) 13 (14.8) 5 (9.3) 8 (22.9) dental insurance/ others 50 (20.1) 15 (17.0) 9 (16.7) 3 (8.6) private 166 (66.7) 60 (68.2) 40 (74.1) 24 (68.6) service rating great/ good 222 (89.9) 72 (82.8) 50 (92.6) 31 (88.6) regular/ bad 25 (10.1) 15 (17.2) 4 (7.4) 4 (11.4) knows what periodontal disease is? no 153 (60.5) 58 (65.9) 37 (68.5) 18 (51.4) yes 98 (38.7) 29 (33.0) 17 (31.5) 17 (48.6) has received periodontal treatment? no 108 (42.7) 34 (38.6) 26 (48.1) 10 (28.6) yes 142 (56.1) 54 (61.4) 27 (50.0) 25 (71.4) how many times do you brush your teeth? one or two times/ day 66 (25.6) 24 (33.3) 22 (41.5) 14 (38.9) 3 or more times/ day 192 (74.4) 48 (66.7) 31 (58.5) 22 (61.5) use of dental floss no 83 (32.8) 47 (53.4) 30 (55.6) 19 (54.3) yes 168 (66.4) 41 (46.6) 23 (42.6) 16 (45.7) have you ever received information about preventing dental problems? no 44 (17.4) 18 (20.5) 15 (27.8) 8 (22.9) yes 206 (81.4) 70 (79.5) 38 (70.4) 27 (77.1) smoker yes 27 (10.7) 19 (21.6) 1 (1.9) 3 (8.6) no 224 (88.5) 69 (78.4) 53 (98.1) 32 (91.4) former-smoker no 205 (81.0) 75 (85.2) 41 (75.9) 17 (48.6) yes 46 (18.2) 12 (13.6) 13 (24.1) 18 (51.4) diabetes no 162 (64.0) 58 (65.9) 9 (16.7) 3 (8.6) yes 89 (35.2) 29 (33.0) 44 (81.5) 32 (91.4) 8 barbosa et al. table 3. multivariate analyses of factors associated with moderate to severe periodontal disease in adults from jundiaí city . variables adults (35-44 years) or 95% ci p or adjusted 95% ci p early periodontal disease moderate to severe periodontal disease n (%) n (%) sex male 75 (29.6) 36 (40.9) 0.67 0.39-1,14 0,41 female 178 (70.4) 52 (59.1) race non white 70 (27.8) 30 (34.1) 1.07 0.60-1.89 0.821 white 182 (72.2) 58 (65.9) marital status living common law 172 (69.4) 61 (69.3) 0.77 0.44-1.35 0.363 not living common law 76 (30.6) 27 (30.7) household income < r$1620.00 ($405.00) 45 (18.3) 20 (23.0) 1.49 0.81-2.74 0.198 r$1620 to r$3240 ($405 to $810) 67 (27.2) 30 (34.5) 1.68 0.85-3.32 0.138 > r$3240 ($810) 134 (54.5) 37 (42.5) individuals per household 4 or less individuals 194 (77.6) 60 (69.0) 1.83 1.03-3.25 0.039 more than 4 individuals 56 (22.4) 27 (31.0) education less than 8 years 18 (7.2) 7 (8.0) 3.31 1.55-7.09 0.002 2.65 1.19-5.88 0.017 8 to 12 years 141 (56.6) 66 (75.0) 3.24 1.46-7.19 0.04 2.36 0.98-5.69 0.056 more than 12 years 90 (36.1) 15 (17.0) economic status in childhood rich or middle class 136 (54.4) 36 (41.1) 1.33 0.78-2.25 0.292 poor or very poor 144 (45.6) 51 (58.6) current situation compared to childhood better 176 (70.1) 65 (73.9) 0.98 0.55-1.74 0.936 same or worse 75 (29.9) 23 (26.1) frequency of visit to dentist urgency 81 (32.3) 44 (50.0) 2.21 1.22-4.00 0.009 1.4 0.72-2.71 0.325 less than once a year 46 (18.3) 12 (13.6) 1.09 0.49-2.45 0.834 1.12 0.46-2.59 0.78 once or more a year 124 (49.4) 32 (36.4) time since last visit 3 or more years 43 (17.3) 17 (19.3) 1.69 0.95-3.16 0.073 1 to 2 years 68 (27.4) 27 (30.7) 1.69 0.85-3.37 0.137 less than one year 137 (55.2) 44 (50.0) continue 9 barbosa et al. that smoking exposure, the absence of use of dental floss and low education level (£8 years) increased approximately two times the risk of experiencing moderate/ severe periodontal disease (table 3). for the elderly population, education level, type of dental service and diabetes mellitus condition were significantly associated with periodontal disease (table 4). adjusted analysis in the elderly population revealed that presence of diabetes mellitus increased almost four times the risk for moderate to severe periodontal disease. also, elderly population who reported to use public dental service had five times higher risk of having moderate/severe disease than individuals that used private or dental insurance services (table 4). discussion and conclusion the present population-based study assessed the prevalence of periodontal disease in the southeast brazilian adult and elderly population and its association with demographic, socioeconomic, behavioral and systemic factors. compared to the last survey conducted in the city of jundiaí in 1998 (unpublished data), it was observed an increase in the prevalence of periodontal disease (90.4% versus 98% in 1998 and 2014, respectively) in the adult population. these data are higher compared to the national epidemiological survey performed in brazil in 2010, in which 82.2% of adults were cpi> 0 7. according to vettore et al. 8, this rise may be associated in part, to a decline in tooth loss over the last few years. regarding the elderly population, it was not possible to assess whether there was any change in the periodontal status of the population from jundiaí, since this track age was not considered in the survey of 1998. however, compared with the data of sbbrasil 20107, the prevalence of periodontal disease found in the elderly population was lower, 59% versus 63.6%, respectively. considering the total population from jundiaí, the prevalence of periodontal disease was lower in elderly individuals, probably continuation type of service public 33 (13.3) 13 (14.8) 1.34 0.64-2.81 0.432 dental insurance/ others 50 (20.1) 15 (17.0) 0.89 0.44-1.82 0.761 private 166 (66.7) 60 (68.2) how many times do you brush your teeth? one or two times/ day 66 (25.6) 24 (33.3) 1.46 0.83-2.56 0.193 3 or more times/ day 192 (74.4) 48 (66.7) use of dental floss no 83 (32.8) 47 (53.4) 2.39 1.40-4.06 0.001 1.9 1.06-3.40 0.03 yes 168 (66.4) 41 (46.6) smoker yes 27 (10.7) 19 (21.6) 2.44 1.25-4.76 0.009 2.14 1.04-4.42 0.039 no 224 (88.5) 69 (78.4) 1 diabetes no 162 (64.0) 58 (65.9) 0.36 0.08-1.58 0.176 yes 89 (35.2) 29 (33.0) 10 barbosa et al. table 4. multivariate analyses of factors associated with moderate to severe periodontal disease in elderly from jundiaí city. variables elderly (65-74 years) or ic 95% p or adjusted ic 95% p early periodontal disease moderate to severe periodontal disease n (%) n (%) sex male 23 (42.6) 22 (62.9) 0.47 0.20-1.12 0.087 female 31 (57.4) 13 (37.1) race white 37 (68.5) 28 (80.0) 1.26 0.49-3.28 0.631 non white 17 (31.5) 7 (20.0) marital status living common law 39 (72.2) 24 (68.6) 0.77 0.31-1.92 0.574 not living common law 14 (25.9) 11 (31.4) household income < r$1620.00 ($405.00) 8 (15.4) 6 (17.1) 1.33 0.40-3.51 0.644 r$1620 to r$3240 ($405 to $810) 23 (44.2) 9 (25.7) 0.52 0.1411.932 0.33 > r$3240 ($810) 21 (40.4) 20 (57.1) individuals per household 4 or less individuals 47 (88.7) 31 (88.6) 1.02 0.27-3.91 0.975 more than 4 individuals 6 (11.3) 4 (11.4) education less than 8 years 32 (62.7) 16 (44.4) 0.35 0.12-0.98 0.046 0.5 0.12-2.04 0.332 8 to 12 years 10 (19.6) 7(19.4) 0.49 0.14-1.75 0.27 0.16 0.04-0.58 0.005 more than 12 years 9 (17.6) 13 (36.2) economic status in childhood poor or very poor 26 (48.1) 21 (60.0) 1.25 0.054-2.92 0.605 rich or middle class 28 (51.9) 14 (40.0) current situation compared to childhood same or worse 13 (24.1) 3 (8.6) 0.44 0.13-1.48 0.185 better 41 (75.9) 32 (91.4) frequency of visit to dentist urgency 25 (46.3) 13 (37.1) 0.64 0.24-1.71 0.376 less than once a year 11 (20.4) 7 (20.0) 0.72 0.22-2.29 0.573 once or more a year 18 (33.3) 15 (42.9) time since last visit 3 or more years 19 (35.2) 7 (20.0) 0.31 0.11-0.88 0.028 1 to 2 years 15 (27.8) 5 (14.3) 0.28 0.086-0.09 0.032 less than one year 20 (37.0) 23 (65.7) type of service public 5 (9.3) 8 (22.9) 2.56 0.75-8.71 0.132 5.32 1.2323.03 0.025 dental insurance/ others 9 (16.7) 3 (8.6) 0.46 0.09-2.38 0.352 0.35 0.06-2.21 0.265 private 40 (74.1) 24 (68.6) continue 11 barbosa et al. due to high rates of tooth loss. in fact, the percentage of tooth loss among adults was 19.1 whereas elderly individuals presented a rate of 72,8% of teeth loss. as reported by peres et .17, the rate of tooth loss among adolescents and adults brazilian seems to decline from 2003 to 2010, which was not observed among elderly population. this findings point to the existence of inequalities of the brazilian national policy of oral health, also known as smiling brazil. this national policy gives priority to particular community actions such as, water fluoridation, which has a significant impact to reduce the rate of decayed, missing, or filled teeth (dmft) among adolescents and adults18. therefore, there was not a community actions to prevent periodontal disease, which could have a significant impact on the reduction of tooth loss among elderly population, and consequently, reduce the prosthodontics needs. it is important to highlight that, 41% of the elderly from jundiaí had all their sextants excluded according to the criteria established for the cpi index (more than two teeth should be present for the sextant to be considered). this exclusion criteria may have led to an underestimation of the prevalence of periodontal disease among elderly population. community periodontal index is recommended by who in oral health surveys, although has some limitations such as, the underestimation of the prevalence of periodontal diseases due to use of index teeth19. the standard protocol for assessing periodontal disease status in periodontal research and periodontal practice involves a full-mouth clinical examinations conducted on a six sites per tooth20,21. however, the application of this protocol in population surveys may not be feasible, mainly when the data collection is performed at home under natural light. in this case, full-mouth examination could trigger patient and examiner fatigue, which may potentially increase measurement errors20,21. consequently, the present study was outlined using a partial record protocol to define the prevalence and severity of periodontal disease. then, to estimate the severity of periodontal disease it was considered the association of probing depth and clinical attachment loss by the use of cpi and cal indexes. this association of clinical parameters made possible the definition of periodontal disease both for the cumulative of periodontal attachment loss and for the current disease. during this epidemiological survey, adult and elderly populations were characterized in terms of socioeconomic determinants, type of dental services, oral hygiene continuation how many times do you brush your teeth? one or two times/ day 22 (41.5) 14(38.9) 0.90 0.38-2.13 0.805 3 or more times/ day 31 (58.5) 22 (61.5) use of dental floss no 30 (55.6) 19 (54.3) 1.41 0.60-3.30 0.437 yes 23 (42.6) 16 (45.7) smoker yes 1 (1.9) 3 (8.6) 0.82 0.48-48.20 0.181 3.2 0.26-38.71 0.36 no 53 (98.1) 32 (91.4) diabetes yes 11 (20.4) 13 (36.1) 2.21 0.86-5.71 0.102 3.78 1.20-11.91 0.023 no 43 (79.6) 23 (63.9) 12 barbosa et al. habits, smoking and diabetes mellitus condition. the findings showed an association between the prevalence of moderate/severe periodontal disease and low educational level for both population. this data is comparable with the most recent national survey performed in brazil and in uruguay, which identified a higher prevalence of moderate/severe periodontal disease among adults with lower educational level7,13. further, in a study conducted in china, the severe periodontal disease was more prevalent among illiterate adults or that they had not completed six years of schooling22. educational level is a important determinant of employment and income13. so, it is expected that the socioeconomic status of families could be influenced by the educational level of their members13. according to some studies, the impact of socioeconomic status in periodontal disease may be explained by psychosocial stress caused by poverty, unemployment and poor living conditions23. stress can negatively alter the immune-inflammatory response to periodontal disease, and also affect behaviors associated with periodontal diseases, such as oral hygiene and smoking habits13. oral hygiene practices such as, tooth brushing and flossing, play an important role in the prevention of periodontal diseases. in this study, the absence of the flossing habit was associated with a higher prevalence of periodontal diseases in the adult population. this finding is in agreement with a recent brazilian cross-sectional study24, in which individual who never perform interproximal cleaning had 2.19 times higher chance of having gingivitis than those performed interproximal cleaning. diabetes mellitus is considered one of the major risk factors for destructive periodontal disease11,25. in this survey, elderly diabetic individuals had four times higher risk to have moderate/severe periodontal disease than non-diabetics in the multivariate analysis after adjusting for other risk factors. in the adult population, this statistical association was not observed, probably because of the low number of adults self-reported diabetics. another true risk factor for periodontal disease is smoking habits as there are a higher prevalence and severity of periodontal disease in smokers regardless of oral hygiene26,27. the association between smoking and increased risk for periodontal disease (moderate/severe) was only found in the adult population from the city of jundiaí, probably because of low numbers of elderly smokers. the literature has been shown a dose-dependent association between smoking and periodontitis using the number of cigarettes smoked per day27,28. however, in the present study, it was not possible to associate the severity of periodontal disease with the number of cigarettes/day, because of the subjects only self-reported as smoker or non-smoker. an important aspect to be considered in this epidemiological survey is the type of dental service used by the population. the elderly population that reported to use public dental service had five times higher risk of having moderate/severe periodontal disease than individuals who reported to use dental insurance or private dental clinics. this fact could be associated to the absence of oral health care coverage for the elderly population in jundiaí, which is focused on emergency demands only. to conclude, this study showed that education level, smoking habits, diabetes, use of dental floss and type of dental service are factors associated to moderate/severe periodontal disease in a population from jundiaí city. moreover, these findings sug13 barbosa et al. gest that local government from jundiaí requires action on reducing inequalities and improving the accessibility of dental care to socially disadvantaged communities, particularly to elderly population. references 1. petersen p, ogawa h. the global burden of periodontal disease: towards integration with chronic disease prevention and control. periodontol 2000. 2012 oct;60(1):15-39. doi: 10.1111/j.1600-0757.2011.00425.x. 2. albandar jm. periodontal diseases in north america. periodontol 2000. 2002;29:31-69. 3. petersen pe. the world oral health report 2003 who global oral health programme. community dent oral epidemiol. 2003 dec;31 suppl 1:3-23.. 4. eke pi, dye ba, wei l, thornton-evans go, genco rj, cdc periodontal 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49: 491–516. 27. haas an, wagner mc, oppermann rv, rösing ck, albandar jm, susin c. risk factors for the progression of periodontal attachm j clin periodontol. 2014 mar;41(3):215-23. doi: 10.1111/jcpe.12213. 28. genco rj, borgnakke ws. risk factors for periodontal disease. periodontol 2000. 2013 jun;62(1):59-94. doi: 10.1111/j.1600-0757.2012.00457.x. https://www.ncbi.nlm.nih.gov/pubmed/?term=prado r%5bauthor%5d&cauthor=true&cauthor_uid=31141035 https://www.ncbi.nlm.nih.gov/pubmed/?term=rios fs%5bauthor%5d&cauthor=true&cauthor_uid=31141035 https://www.ncbi.nlm.nih.gov/pubmed/?term=costa rdsa%5bauthor%5d&cauthor=true&cauthor_uid=31141035 https://www.ncbi.nlm.nih.gov/pubmed/?term=angst pdm%5bauthor%5d&cauthor=true&cauthor_uid=31141035 https://www.ncbi.nlm.nih.gov/pubmed/?term=moura mds%5bauthor%5d&cauthor=true&cauthor_uid=31141035 https://www.ncbi.nlm.nih.gov/pubmed/?term=maltz m%5bauthor%5d&cauthor=true&cauthor_uid=31141035 https://www.ncbi.nlm.nih.gov/pubmed/?term=jardim jj%5bauthor%5d&cauthor=true&cauthor_uid=31141035 braz j oral sci. 15(2):176-179 influence of adhesive and thermal cycling on the bond strength of ceramic brackets to dental ceramic patricia de fátima fraga1, ana paula terossi de godoi1, ana rosa costa2, lourenço correr-sobrinho2, mario vedovello filho1, heloisa cristina valdrighi1, 1fundação hérminio ometto fho/uniararas, school of dentistry, department of orthodontics, araras, sp, brazil 2universidade estadual de campinas – unicamp, piracicaba dental school, department of restorative dentistry, area of dental materials, piracicaba, sp, brazil correspondence to: mario vedovello filho fundação hérminio ometto fho/uniararas departmento de ortodontia av. dr. maximiliano baruto, 500 jd. universitário, cep: 13607-339 araras, sp, brasil phone: +55 19 35431423 e-mail: mario@vedovelloeassociados.com.br abstract aim: this in vitro study investigated the effect of the application of an adhesive, silane and thermal cycling (tc) on the shear bond strength (sbs) of ceramic brackets to feldspathic ceramic. methods: 16 cylinders of feldspathic ceramic were etched with hydrofluoric acid and divided into four groups (n=4): g1 silane, without tc; g2 – silane, with tc; g3 adhesive, without tc; g4 adhesive, with tc. one layer of silane was applied on the surface of cylinders in g1 and g2 e one layer of photo-activated adhesive single bond universal was used in g3 and g4. ceramic brackets were bonded using transbond xt. the sbs data were subjected to two-way anova and tukey’s post hoc test (α=0.05). the adhesive remnant index (ari) was evaluated at 40× magnification. results: silane was more effective than adhesive on the sbs of the brackets to ceramic (p<0.05). tc decreased significantly the sbs values compared with the groups without tc (p<0.05). the ari results showed predominance of score 0. conclusions: groups with silane showed higher sbs than groups with adhesive. tc influence significantly on the bond strength. regarding ari, score 0 predominated in all groups. keywords: shear strength. adhesives. ceramics. introduction ceramics have been used routinely for dental restorations because they provide optimal characteristics such as biocompatibility, mechanical resistance, esthetic similar to natural teeth, color stability, radiopacity and low thermal conductivity1-2. in addition to teeth, ceramic materials may serve as substrates for bonding of orthodontic brackets under clinical conditions. etching with hydrofluoric acid (hf) promotes dissolution the glass ceramic creating irregularities on the ceramic surface and greater contact surface in the ceramic bonding area, promoting a stronger bond between dental ceramics and composite resin3-6. bonding materials need sufficient wettability to completely infiltrate the irregularities of ceramic surface. normally, silane has been used on the internal ceramic surface prior to applying bonding material because they are capable of forming chemical bonding to the resin material, which improves the durability and bonding strength7-10. however, it is questionable if the silane and resin cement are efficient in wetting ceramic surface3. on the other hand, some clinicians have applied a layer of adhesive on the ceramic surface after the silane, but the literature has little information about received for publication: november 15, 2016 accepted: march 08, 2017 original article braz j oral sci. april | june 2016 volume 15, number 2 http://dx.doi.org/10.20396/bjos.v15i2.8648779 177 luting purposes11. naves et al. (2010)3 and sundfeld et al. (2015)11 showed that the use of an adhesive improve bond strength and adaptation of substrates along the ceramic/resin cement interface. recently, a new adhesive was released on the market to be used without prior application of silane because this material also acts as silane according to the manufacturer. however, the literature has no information about its action for bonding brackets. clinically, orthodontic brackets are subjected to physical and mechanical challenges when bonded to ceramic surface in the oral environment. they are exposed to thermal changes, chemical and physical and due to the contact with drinks and food12. thus, failure can occur at the interface among ceramic, bonding material and orthodontic brackets due to heavy forces produced by an archwire during the orthodontic movement and thermal changes13. thermal cycling use temperature variations and regimens between 500 and 7,000 cycles to promoted stresses at the interface between bonded materials causing their deteriorations under simulated oral conditions12-17. however, the literature is still not conclusive about silane, adhesive and thermal cycling. the aim of this present in vitro study was to investigate the effect of the application of the universal adhesive, silane and thermal cycling on the bond strength of ceramic brackets to feldspathic ceramic. the hypotheses tested were: 1) the adhesive is not higher than the silane on the bond strength; and, 2) thermal cycling does not affect the bond strength. material and methods the surface of 16 feldspathic ceramic glazed cylinders (certec advanced ceramics, barueri, sp, brazil; 20 mm high x 13 mm diameter) were cleaned with a rubber cup (kg sorensen, cotia, sp, brazil) and pumice-water slurry (s.s. white, petropolis, rj, brazil) for 30 s, rinsed with air-water spray for 30 s and dried with air for 30 s before testing. the rubber cup was replaced after each cylinder. the cylinders were randomly assigned into four groups (n=4) according to the treatment of surface: g1 silane, without thermal cycling; g2 – silane, with thermal cycling; g3 adhesive, without thermal cycling; and, g4 adhesive, with thermal cycling. the surface of all the cylinders were etched with 10% hydrofluoric acid gel (condac; fgm, joinvile, sc, brasil)) for 60 s, rinsed with oil-free compressed air/water spray for 60 s and dried with air for 60 s. one layer of a silane coupling agent relyx ceramic primer (3m espe, st. paul, mn, usa) was applied onto the cylinders surface of the g1 and g2, left in contact for 60 s and dried for 60 s. g3 and g4 received a layer of photo-activated adhesive single bond universal (3m espe, st. paul, mn, usa) and light-cured for 20 s using a led device radii plus (sdi limited, bayswater, victoria, australia) having an irradiance of 1,200 mw/cm2 as measured using a curing radiometer (model 100, demetron research corporation, danbury, ct). after that, gemini clear ceramic brackets, standard maxillary premolar (3m unitek, monrovia, ca, usa) were positioned and bonded to the curved area of the ceramic cylinders surface using light-cured bonding resin transbond xt (3m unitek, monrovia, ca, usa), according to the manufacturer’s instructions. the brackets were seated and positioned firmly on the ceramic surface. excess of light-cured bonding resin was removed using a microbrush and light-activation was carried out with 4 exposures, one on each side of the bracket, totalizing 40 s using led device radii plus (sdi limited). five ceramic brackets were bonded to each ceramic cylinder (n=5) for each treatment of surface and thermal cycling, totalized 80 bonded brackets. all samples were stored in deionized water for 24 h at 37 oc. after this period, specimens of g2 and g4 were submitted to a 7,000 thermal cycles in a thermal cycler (msct 3, marnucci me, são carlos, sp, brazil) with deionized water between 5 °c and 55 °c (dwell time of 30 s) and transfer time of 10 s between baths. the sbs test was performed in a universal mechanical testing machine (model 4411; instron, canton, ma, usa) using a knife-edged rod at a crosshead speed of 1.0 mm/min until failure. a mounting jig was used for the parallel alignment of the ceramicbracket interface to the testing device. results of sbs were submitted to two-way anova and tukey’s post hoc test (α=0.05). the fractured specimens were observed under optical microscopy (olympus corp, tokyo, japan) at 40× magnification and the adhesive remnant index (ari) was used to classify the mode of failure as follows18: score 0: no resin was left on the ceramic; score 1: less than half of the resin was left on the ceramic; score 2: more than half of the resin was left on the ceramic ; and score 3: all resin was left on the ceramic, with a clear impression of the bracket mesh. results the mean sbs values are shown in table 1. the interaction between thermal cycling and treatment was significant (p<0.0001). the thermal cycling (p<0.0001) and treatment (p<0.0001) directly influenced the sbs values. influence of adhesive and thermal cycling on the bond strength of ceramic brackets to dental ceramic braz j oral sci. 15(2):176-179 means followed by different uppercase letters in the same row and lowercase letters in the same column indicate statistically significant difference (p<0.05). table 1 mean shear bond strength values (s.d.) in mpa for treatment of surface, with and without thermal cycling. treatment of surface thermal cycling without with silane 14.7 ± 1.2 a, a 7.4 ± 1.1 a, b adhesive 9.9 ± 1.1 b, a 5.3 ± 0.8 b, b specimens treated with silane provided significantly higher sbs values than those treated with adhesive alone, with or without thermal cycling (p<0.05). groups submitted to thermal cycling demonstrated lower sbs values than those without thermal cycling, regardless of the treatment of surface (p<0.05). 178 figure 1 shows the distribution frequency of ari. a predominance of score 0 was detected in all groups. without prior application of silane, because the material has silane function. however, in this study it was observed that the adhesive without silane was not able to penetrate completely into the irregularities of the ceramic, probably because in these groups the silane was not used. when, the silane is used the groups monovalent hydrolysable bond chemically to silicon contained in the glass matrix and lithium disilicate3,8. thus, the results showed that the adhesive was not able to promote the expected union. the clinical success, quality and durability of the bond is determined by the mechanisms of the bond strength among ceramic, bonding materials and orthodontic bracket and may be influenced by some factors such as mechanical properties of composite resin, silane, adhesive, mechanical fatigue and thermal cycling7,9,17. the thermal cycling test has been used to verifiy if changes in temperature can interfere on the reduction on the bond strength among bracket, bonding material and substrate. probably, the reduction of the mechanical properties of bonding materials is a function of a continuous action of water on the interface among orthodontic bracket, bonding material and substrate. the decrease of bond strength could be caused by hydrolytic degradation of the interface components22 or by the abrupt fall of temperature of the bonding materials with different coefficient of thermal expansion, which can promote thermal stresses at the interface brackets, bonding material and ceramic14. in relation to the thermal cycling, significant difference was found between thermal cycling and water storage (24 h), regardless of the treatment of ceramic surface. the results indicate that the second hypothesis was rejected. this finding is in agreement with those of previous study showing significant differences in bond strength for specimens subjected to thermal cycling3. however, some studies have found no significant difference for bond strength after thermal cycling5,12,17,23,24. the results of this study suggest that the absence of difference might be explained by the fact that in these studies, the specimens were subjected to a small number of cycles, while in the current study was used a larger number of cycles. according to gale and darvell12 (1999) a larger number of cycles are necessary to permit accelerated simulation. a previous study showed that bond strength values in the range of 6 to 8 mpa are necessary for orthodontic procedures in oral environment25. in this study, brackets bonding to ceramic with strength values lower than 6 mpa were obtained for groups where adhesive was applied without silane after thermal cycling. therefore, care should be taken when adhesive is used without silane because it has not been acceptable potential to resist clinically bond strengths. the analysis of failure modes (ari scores) showed a predominance of failures with score 0 in all groups, with no influence of adhesive and thermal cycling on the bond strength of ceramic brackets to dental ceramic fig.1. distribution frequency of adhesive remnant index (ari) scores. discussion silane is a monomeric species in which silicon is linked to hydrolysable ester groups and reactive organic radicals. the monovalent hydrolysable groups bond chemically to the silicon contained in the glass matrix and lithium disilicate8,1 9. according to naves et al.11, the effectiveness of bonding when using only silane depends on the ability of the resin cement to fill the irregularities and to promoted contact between the resin cement and the ceramic surface. in the present study, when a silane was used, the sbs values were significantly higher than with adhesive alone, regardless of the thermal cycling. therefore, the first hypothesis, which stated that the adhesive is not superior to silane in increasing the bond strength was accept. the results of the present study are in agreement with those of a previous study, which also found significant differences when silane was used7,8,20,21. as silane are usually monomeric species, in which silicon is linked to reactive organic radicals and hydrolysable ester groups, the reactive organic groups become chemically bonded to the resin molecules. on the other hand, the hydrolysable groups bond chemically to silicon contained in the glass matrix or lithium disilicate8 and a chemical bond is formed between the silica layer and silane agent on the ceramic surface or the bonding materials. on the other hand, when the adhesive was applied on the ceramic surface without silane, the sbs decreased significantly compared when silane was applied. according to the manufacturer’s instructions, the adhesive could be used braz j oral sci. 15(2):176-179 179 bonding resin on the ceramic surface. clinically it may be advantageous because there is less bonding material to remove from the ceramic surface after bracket debonding7,13. in this context, the present study showed that thermal cycling decreased significantly the bond strength and the use of the silane is decisive factor to obtain improved bond strength of orthodontic brackets to ceramic surfaces. clinicians should take care during bonding procedures, irrespective of the use of adhesive without silane after thermal cycling. therefore, the silane should be used after etching ceramic surface with hydrofluoric acid. thus, additional studies must be performed to investigate other possible factors affecting the clinical performance of bracket bonding to ceramic such as the types of silane and bonding materials. it may be concluded that the application of silane increased significantly the sbs of brackets to ceramic surface in relation to adhesive alone, with or without thermal cycling. thermal cycling decreased significantly the sbs in all groups. the ari results showed predominance of score 0 in all groups. references 1. silva nr, thompson vp, valverde gb, coelho pg, powers jm, farah jw et al. comparative reliability analyses of zirconium oxide and 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strength of ceramic brackets to dental ceramic braz j oral sci. 15(2):176-179 untitled 1 volume 16 2017 e17077 original article a department of dental materials and prosthodontics, araçatuba dental school, unesp – univ estadual paulista, araçatuba school of dentistry, araçatuba, são paulo, brazil. b department of restorative dentistry, araçatuba dental school, unesp – univ estadual paulista, araçatuba, são paulo, brazil c department of dental materials and prosthodontics, university of são paulo – usp, ribeirão preto, são paulo, brazil. d department of operative dentistry, bauru dental school – university of são paulo – usp, bauru, são paulo, brazil. surface roughness, gloss and color change of different composites after exposure to ultimate challenges cleidiel aparecido araujo lemosa, silvio josé maurob, andré luiz fraga brisob, fernanda de carvalho panzeri pires de souzac, maria fidela de lima navarrod, ticiane cestari fagundesb correspondence: profa. ticiane cestari fagundes; departamento de materiais odontológicos e prótese; faculdade de odontologia de araçatuba, unesp josé bonifácio, 1193, araçatuba, sp, brasil / cep 16015-050 telephone: +55 18 3636-3253 / fax: +55 18 3636-3253 e-mail: ticiane@foa.unesp.br received: july 23, 2017 accepted: september 20, 2017 aim: the study aimed to investigate the effect of the association of chemical and mechanical degradation on the surface vsyklriww��kpsww�erh�gspsv�sj�rers�erh�qmgvsƽppih�gsqtswmxiw�� method: disc-shaped specimens (n=10) were prepared for three nanocomposites (filtek z350xt, ips empress direct, charisma (meqsrh � erh� xlvii� qmgvsƽppih� gsqtswmxiw� �)wxipmxi� ·� 5ymgo�� (yveƽpp�:7��6ireqip ��%jxiv�tspmwlmrk��fewipmri�wyvjegi�vsyklriww�� gloss and color measurements were obtained. specimens were submitted subsequently to the following challenges: chemical for 1 week (hydrochloric acid, coca-cola and red wine) and mechanical (toothbrushing). surface roughness and gloss data were analyzed by kruskal-wallis followed by dunn’s test. color difference (') � [ew� erep]^ih� f]� sri�[e]� %23:%� erh� 8yoi]� test. results: the initial data were compared with those after challenges using the wilcoxon test (p<0.05). all composites wls[ih� e� wmkrmƽgerx� mrgviewi� �t ���� � sj� wyvjegi� vsyklriww�� ejxiv�szivwxviww��*mpxio�>����<8�erh�6ireqip�leh�xli�ps[iwx�ƽrep� surface roughness values and roughness increase (p<0.05). the gsqtevexmzi�erep]wmw�viziepih�xlex�)wxipmxi�·�5ymgo�erh�6ireqip� hmh�rsx�wmkrmƽgerxp]�glerki�xli�kpsww�ejxiv�gleppirkiw��t!����� �� filtek z350 xt and ips empress direct had higher color variations, after the challenges than the other resins (p<0.05). conclusion:�'liqmgep�erh�qiglermgep�gleppirkiw�mrƽyirgi�sr� glevegxivmwxmgw�sj�epp�gsqtswmxiw��i\gitx�jsv�kpsww�sr�)wxipmxi�·� 5ymgo�erh�6ireqip�gsqtswmxiw�� keywords: dental resin; physical properties; immersion; toothbrushing. http://dx.doi.org/10.20396/bjos.v16i0.8651057 2 lemos et al. introduction changes in surface roughness, gloss, and color can compromise the longevity and the clinical success of restorations1. the composites undergo the action of intrinsic factors due to the physicochemical reactions in the inner portions of the restorative material, and also suffer the action from extrinsic factors, such as acidic substances that degrade organic matrix, exposing the material to coloring agents present in foods and beverages2,3, and abrasive substances, for example, from the brushing process4��mrƽyirgmrk�xli�gsqtswmxi�glevegxivmwxmgw��wygl�ew�wyvface roughness5 and gloss. surface roughness has been recognized as high clinical relevance parameter that contributes to dental plaque accumulation6. it is known that composite roughness mw�mrƽyirgih�f]�xli�wm^i��hmwxvmfyxmsr�erh�zspyqi�sj�xli�ƽppiv�gsrxirx�tviwirx�mr�xli� restorative material. furthermore, the increase of roughness is correlated with the characteristics of gloss and color of the composites restoration because of the possifmpmx]�sj�e�ps[iv�pmklx�viƽigxergi�erh�lmkliv�vixirxmsr�sj�tmkqirxw1. it has been observed that the better the polish obtained at the end of the composite restoration, the greater the scattering of light inside the material and consequently this can result in a gloss increase7. in a condition that is necessary to produce highly aesthetic restorations, the gloss has an important role, since it needs to reproduce the optical properties of the enamel surface, making the restorations imperceptible to the human eye8. the visible differences in color between teeth and esthetic materials are the major cause of patient discontentment with restorative treatments9. in this context, to obtain a composites restoration that reproduce colors of the tooth structure is considered a gleppirki��qemrp]�mr�erxivmsv�xiixl�[livi�wpmklx�gspsv�glerkiw�sziv�xmqi�ger�mrƽyirgi� patient compliance9. 8li�qmgvsƽppih�gsqtswmxiw�lezi�lmkl�wyvjegi�wqssxlriww�fyx�tviwirx�ps[�qiglermcal strength in comparison with universal composites and therefore can be indicated as surface resins for anterior teeth4��8li�ehzirx�sj�xli�rersxiglrspsk]�ƽiph�lew�pih� to the development of nanocomposites with smoothness characteristics similar to xlswi�sj�qmgvsƽppih�viwmrw��erh�epws�[mxl�lmkl�qiglermgep�wxvirkxl4. in vitro studmiw�lezi�erep]^ih�xli�tivjsvqergi�sj�xliwi�x]tiw�sj�gsqtswmxiw�[mxl�xli�mrƽyirgi� of toothbrushing and chemical degradation separately1-3,8,10. thus, it is important to note that in the oral cavity, there is the interaction of these mechanical and chemical challenges4,5. however, there is a lack of studies evaluating the effects of chemicals immersions in association with toothbrushing. the objective of this study was to evaluate the surface roughness, gloss and color glerkiw�sj�rersgsqtswmxiw�erh�qmgvsƽppih�viwmrw�ejxiv�xli�ewwsgmexmsr�sj�gliqmgep� and mechanical challenges. the null hypotheses tested were: 1) there is no difference before and after challenges for composites when surface roughness and gloss were izepyexih�� �xli�wm^i�erh�ƽppivw�sj�gsqtswmxiw�hs�rsx�mrƽyirgi�xli�wyvjegi�vsyklriww�� gloss and color change after chemical and mechanical challenges. 3 lemos et al. material and methods specimen preparation the composites used in this experiment are shown in table 1. sixty disc-shaped specimens (5.0 mm diameter and 1.5 mm height) for each composites (n=10) were built yt�ywmrk�e�8iƽsr�vmrk�qexvm\��%jxiv�xli�qexvm\�[ew�ƽppih��xli�qexivmep�[ew�gszivih� with a polyester strip and a glass slab. the specimens were then light-activated in the upper and lower surfaces for 40 seconds each, using a led light-curing unit (ultraled, dabi atlante, ribeirão preto, sp, brazil, irradiance of 500 mw/cm2, monitored by a radiometer (model 100; kerr, danbury, ct, usa). after 24 hours in water storage at 37ºc, all specimens were sequentially polished (aropol e, arotec ind. com. sa, cotia, sp, brazil) with the silicon carbide papers: 320-, 600-, 800and 1200-grit. uniform pressure and application time of 120 seconds were standardized, under constant water irrigation. the direction of polishing was from left to right and the rotation rate was set as 10.000 rpm. between each silicon carbide paper and at the end of polishing procedures the specimens were cleaned in an ultrasonic bath (cristófoli, campo mourão, 46��&vewmp �[mxl�hmwxmppih�[exiv�jsv���qmryxiw��8li�ƽrep�tspmwlmrk�[ew�qehi�ywmrk�e� diamond flex felt disks (fgm, joinville, sc, brazil) associated with polishing paste (enamelize, cosmedent inc., chicago, il, usa) manually, to better simulate clinical procedures. after polishing, the specimens were stored in relative humidity at 37°c for 24 hours before the initial readings of surface roughness, gloss and color. table 1. characteristics of composites materials analyzed in this study. composite resins manufacturer classification mean filler size and composition weight (%) organic matrix batch # estelite σ quick tokuyama dental co. tokyo, japan submicron filled spherical silica-zirconia filler 0.1-0.3 pm 71 bis-gma, teg-dma e536 durafill vs heraus kulzer, hanau, germany microfilled prepolymerized silica: 10-20 pm sílica: 0.02-2 pm 40 udma 010218 renamel cosmedent inc., chicago, il, usa microfilled pyrogenic silicic acid filler 0.02-0.04 pm 59 bis-gma bis-ema 104819j filtek z350xt 3m/espe, st. paul, mn, usa nanofilled zircônia and silica clusters:0.6-1.4 pm sílica: 5-20 nm 55 bis-gma bis-ema teg-dma udma 1210900268 ips empress direct ivoclar vivadent, schaan, liechtenstein nanohybrid barium alumina fluorosilicate glass, barium glass filler, mixed oxide,copolymer 0.04–3 pm 52-59 bis-gma teg-dma udma n21727 charisma diamond heraus kulzer, hanau, germany nanohybrid barium, aluminium, fluoride glass 5-20000 nm 64 tcd-di-hea udma 010041 4 lemos et al. surface roughness measurements 8lvii�iuymhmwxerx�viehmrkw�sj�wyvjegi�vsyklriww��6e �[ew�vikmwxivih��4vsƽpsqixiv�� sj-401, mitutoyo, kanagawa, japan) that represents the arithmetic mean of peaks and valleys of surface roughness on the specimens. the measurement was performed in each specimen individually, and the needle was positioned on the specimen surface and moved at a constant speed of 0.05 mm/s, using a cut-off of 0.25 mm. three readings were performed on each surface specimen in equidistant positions in approximately 120°11. the average of these three measurements was calculated as roughness value of the specimen. surface gloss measurements 8li�wyvjegi�kpsww�[ew�qiewyvih�ex�e���{�erkpi�sj�mrgmhirgi�erh�viƽigxmsr�ywmrk�e�gepibrated glossmeter (microgloss, byk gardner, geretsried, germany). the instrument qiewyviw�xli�mrxirwmx]�sj�e�viƽigxih�pmklx�fieq�ejxiv�wxvmomrk�xli�wyvjegi�erh�gsqpares it to a reference value. the device has a measuring window of 2 mm x 2 mm, sziv�[lmgl�xli�wtigmqir�mw�tpegih�erh�xlir�gszivih�[mxl�e�ƽpq�gsrxemriv�xs�ezsmh� external light exposure during the measurement. the average of three measurements was recorded for each specimen. color analysis baseline color was measured according to the cielab (commission internationale hi� -ƅ)gpemveki � gspsv� w]wxiq� ywmrk� e� viƽigxmsr� wtigxvstlsxsqixiv� �9:������� 7lmmadzu, kyoto japan) over a black background and standard illuminant d65. the cielab color space graph is a 3-d color measurement: l refers to the lightness coordinate, and its value ranges from 0 for perfect black to 100 for perfect white; “a” and “b” are chromaticity coordinates on the green–red (a = green; +a = red) and blue–yellow (b = blue; +b = yellow) axes11,12. color change was calculated between the color before challenges (baseline) and after challenges, measured by the formula: ²)�!��?²0�a���?²e�a���?²f�a2)½ 11. chemical and mechanical challenge after initial readings, the specimens were submitted to chemical challenge by immersion of the samples individually into 10 ml of hydrochloric acid (ph 1.6, hcl 0.01mol/l, apothicário, araçatuba, brazil) simulating the action of acid from the gastric juice, 10 ml of coca cola (ph 2.5, coca cola company, spaipa s.a., marília, sp, brazil) in order to simulate the action of the acid from soft drinks, and 10 ml of red wine (ph 3.4, concha y toro cabernet sauvignon, santiago, chile). the samples were individually stored in sealed tubes for one week in each acid solution at 37°c13,14. between each acidic challenge, the samples were washed and immersed in 10 ml of distilled water for 24 hours at 37°c. 7tigmqirw�[ivi�mrhmzmhyepp]�wyfnigxih�xs��������qiglermgep�fvywlmrk�g]gpiw��1):���3hiqi� biotechnology, joaçaba, sc, brazil) performed with toothbrushes (one for each specimen, colgate classic clean, colgate, palmolive co. osasco, sp, brazil) and toothpaste (colgate total 12, colgate palmolive, kolynos division of brazil ltd, osasco, sp, brazil) diluted in distilled water (ratio 1:2 by weight). after brushing, the specimens were rinsed with distilled 5 lemos et al. water for 2 minutes and submitted to ultrasonic bath for 10 minutes with distilled water to remove the abrasive particles from the toothpaste. the specimens were maintained in vipexmzi�lyqmhmx]�ex���q'�jsv����lsyvw�fijsvi�ƽrep�viehmrkw�sj�wyvjegi�vsyklriww��kpsww�erh� color, under the same conditions previously stated. color changes ('e) were calculated by the formula: ')�!�?�'0� 2 + ('e� 2 + ('f� 2a1/2. all analyses were blinded to the resins. scanning electronic microscopy (sem) analysis sem analysis was not performed initially because same specimens were used in all experimental procedures. two specimens of each material were mounted on aluminum stubs, sputter-coated with gold (balzers scd-050 sputter coater, oc oerlikon 'svtsvexmsr�%+��4jʞƾosr��7[mx^ivperh �erh�wyfqmxxih�xs�7)1�erep]wmw��)zs�07���� 'evp�>imww��3fivosglir���+ivqer] �ex�����\�qekrmƽgexmsr� statistical analysis the assumptions of equality of variances and normal distribution of data were checked using the shapiro–wilk test, once the homogeneity was not achieved, data of surface roughness and gloss were analyzed with nonparametric tests, kruskal-wallis followed by dunn’s test. however, the normal range was observed jsv�xli�gspsv�glerki��mr�[lmgl�xli�qierw�[ivi�izepyexih�f]�er�%23:%�erh�8yoi]ƅw� multiple comparisons. wilcoxon test was applied for surface roughness and gloss analysis considering two-time evaluation (before and after challenges). spearman test was used to check the correlation between the surface roughness and gloss teveqixivw��7xexmwxmgep�xiwxmrk�[ew�tivjsvqih�ywmrk�7477�:ivwmsr�����-&1�7477� statistics for windows, armonk, ny, usa). a p-value less than 0.05 was considered wxexmwxmgepp]�wmkrmƽgerx� results data of surface roughness are shown in figure 1. charisma diamond had the highiwx�mrmxmep�wyvjegi�vsyklriww�zepyi��[mxl�e�wmkrmƽgerx�hmjjivirgi�jsv�epp�viwmrw��i\gitx� jsv�)wxipmxi�·�5ymgo��t!����� ��%jxiv�gleppirkiw��'levmwqe�(meqsrh�erh�(yveƽpp�:7� showed the highest values of surface roughness (fig. 2b and f). all composites wls[ih�e�wmkrmƽgerx�mrgviewi�mr�eqsyrx�sj�wyvjegi�vsyklriww�ejxiv�xli�ewwsgmexmsr� of challenges. filtek z350 xt and renamel had the lower ra values when compared to other composites, as shown in fig. 2c and d. the initial analysis of the gloss revealed that the ips empress direct and charisma diamond had the highest and lowest gloss values, respectively, with statistical difference for the other composites (p<0.05). after association of the chalpirkiw�� mx� [ew� jsyrh� xlex� xli� qmgvsƽppih� viwmrw� leh� xli� ps[iwx� kpsww� vihygxmsr� zepyiw��*mk���%��&�erh�' ��8li�gsqtevexmzi�erep]wmw�viziepih�xlex�)wxipmxi�·�5ymgo� erh�6ireqip�hmh�rsx�wls[�wxexmwxmgepp]�wmkrmƽgerx�glerki�sj�kpsww�ejxiv�gleppirkiw� (p=0.185). these values can be seen in figure 3. the spearman correlation test showed positive correlation between surface roughness and gloss for the initial analysis (p=0.0198; coef. 0.8827), however, there was no correlation after the challenges (p=0.1107; coef. 0.7143). 6 lemos et al. su rf ac e ro ug hn es s (r a) roughness of composites before and after challenges estelite σ quick before bc/a ab/a ab/a a/a a/a c/a cd/b de/b ab/b a/b bc/b e/b after 0.18 0.15 0.12 0.09 0.06 0.03 0 durafill vs renamel filtek z350xt ips empress direct charisma diamond figure 1. box plots of surface roughness values (ra: pm) of resin composites before and after the challenges. different uppercase letters represent statistical significance among composites (p<0.05). different lowercase letters indicate statistical significance before and after challenges (p<0.05). a b c d e f eht = 20.00kv wd = 13.0 mm signal a = se1 photo no. = 2262 date = 15 sep 2014 time: 14:07:37 feis unesp mag = 5.00 k x eht = 20.00kv wd = 18.0 mm signal a = se1 photo no. = 2233 date = 15 sep 2014 time: 11:48:03 feis unesp mag = 5.00 k x eht = 20.00kv wd = 17.5 mm signal a = se1 photo no. = 2221 date = 15 sep 2014 time: 11:38:51 feis unesp mag = 5.00 k x eht = 20.00kv wd = 18.0 mm signal a = se1 photo no. = 2237 date = 15 sep 2014 time: 11:50:52 feis unesp mag = 5.00 k x eht = 20.00kv wd = 14.5 mm signal a = se1 photo no. = 2212 date = 15 sep 2014 time: 11:17:12 feis unesp mag = 5.00 k x eht = 20.00kv wd = 13.0 mm signal a = se1 photo no. = 2274 date = 15 sep 2014 time: 14:17:44 feis unesp mag = 5.00 k x 2µ 2µ 2µ 2µ 2µ 2µ figure 2. representative scanning electron micrographs of composites after submission challenges. (a) estelite σ quick, (b) durafill vs, (c) renamel, (d) filtek z350 xt, (e) ips empress direct, and (f) charisma diamond. note different patterns of corroded resin matrix provided by the association of chemical and mechanical challenges. original magnification 5000x. g lo ss u ni t ( g u ) gloss of composite before and after challenges estelite σ quick before b/a b/a b/a b/a a/a c/a ab/b b/b a/a b/b b/b c/b after 5 10 15 20 25 30 35 40 45 0 durafill vs renamel filtek z350xt ips empress direct charisma diamond figure 3. box plots of gloss (gu unit) of resin composites before and after challenges. different uppercase letters represent statistical significance among composites (p<0.05). different lowercase letters indicate statistical significance before and after challenges (p<0.05). 7 lemos et al. regarding color changes, after challenges it was found that filtek z350 xt and ips empress direct had higher color variations than the other resins (p<0.05) (table 2). discussion in vitro�wxyhmiw�lezi�erep]^ih�witevexip]�xli�mrƽyirgi�sj�gliqmgep�gleppirkiw2,14,15 and toothbrushing7,8,10 on surface changes of resin composites. however, it is well known that the degradation of materials in the oral environment is a complex process, which involves mechanical and chemical mechanisms5,15,16. thus, challenges were not performed separately in this present study to predict the performance of composites after ultimate challenge. the characteristics of surface roughness, gloss and color are considered important properties for the clinical success of restorative materials1. the chemical challenges tested in this study used acidic solutions and dyes, simypexmrk�xli� mrƽyirgi�sj� mrxvmrwmg�erh�i\xvmrwmg�jegxsvw� mr�viwxsvexmzi�qexivmepw17,18. the mechanical challenge was performed by brushing process that, over time, has caused wear on the resin surface, especially in the organic matrix promoting the inorganic particles display, and increasing the surface roughness of the composites4,7,10. twenty thousand brush cycles were used since it is considered superior to 3 years in vivo19. 8li�ƽvwx�rypp�l]tsxliwmw�izepyexih�[ew�vinigxih��%pp�sj�xli�xiwxih�gsqtswmxiw�wls[ih� statistically ra surfaces after challenges. these results corroborate with previous studies that found the increase in surface roughness after chemical challenge2 and mechanical challenges2,4,7,10. 6ikevhmrk�xli�kpsww��srp]�x[s�qmgvsƽppih�gsqtswmxiw�wxyhmih�hmh�rsx�wls[�wxexmwtical difference after the proposed challenges. lee et al.20 observed that renamel showed lowest change in gloss when compared with other composites studied, when a progressive number of toothbrushing cycles were applied, as it was observed in the present study. renamel also presented the best values of gloss when different ƽrmwlmrk�erh�tspmwlmrk�tvsgihyviw�[ivi�wxyhmih21. it is noteworthy that this resin showed the lowest value of gloss loss among all analyzed resins, changing only 1% of the initial gloss after exposure to ultimate challenge proposed in this present study (fig. 1c). table 2. comparisons of color change among different materials, 'l*, 'a*, 'b* e 'e. composite resins δl* δa* δb* δe estelite σ quick 0.69 0.72 -1.17 1.80 (0.42) b durafill vs -1.08 0.26 -0.85 1.50 (0.66) b renamel -0.24 -0.16 1.59 1.70 (0.46) b filtek z350 xt -2.08 0.94 -0.80 2.80 (1.08) a ips empress direct 1.63 2.40 1.70 3.54 (0.80) a charisma diamond 1.31 0.55 0.17 1.65 (0.74) b means followed by the same letters in columns are not statistically different for the anova and tukey test at p>0.05. 8 lemos et al. 8li� pego� sj� hmjjivirgi� mr� kpsww� ejxiv� xli� gleppirkiw� jsv� fsxl� )wxipmxi� ·� 5ymgo� erh� 6ireqip�ger�fi�nywxmƽih�f]�xli�wleti�erh�wm^i�sj�ƽppivw20��7mrgi�)wxipmxi�·�5ymgo�ywiw� wtlivmgep�ƽppivw��xliwi�tevxmgpiw�evi�efpi�xs�viƽigx�qsvi�pmklx�xler�mvvikypev�sriw20. although renamel have irregular shapes, the predominance of particles were of one micron or less22. however, there is no consensus regarding the change of gloss, since large loss of gloss was observed after performing challenges when various types of composites were evaluated7. therefore, it seemed that change in gloss was primarily mrƽyirgih�f]�xli�glevegxivmwxmgw�sj�xli�gsqtswmxiw��rsx�f]�eqsyrx�sj�[iev20. when comparing the composites in relation to the characteristics evaluated, the second null hypothesis was also rejected. the highest ra values for the charisma diamond resin were found before and after challenges. this composite resin, despite its nanometric particles, present large particle (fig. 1f) size in the range 0.005-20pm, [lmgl�qe]�mrƽyirgi�jsv�pevkiv�wyvjegi�vsyklriww�zepyiw�ejxiv�xli�hikvehexmsr�sj�xli� organic matrix, as also observed in a previous study23. the lowest value was found for ƽrep�wyvjegi�vsyklriww�erh�mrgviewih�wyvjegi�vsyklriww�sj�xli�rersƽppih�*mpxio�>���� xt. this can be explained by the greater resistance to chemical and mechanical wear found by this resin, due to less exposure of the organic matrix, favored by the presence of nanoclusters in its composition, which can be observed in fig. d2. renamel exhibited similar surface roughness to the filtek z350 xt, probably due to its small average particle size (fig. 1c)22. higher values of roughness are unacceptable, since it favors the accumulation of bacteria and consequently greater risk of carious lesion erh�kmrkmzep�mrƽeqqexmsr7. in this study, it was observed that after the challenge proposed, all composites presented surface roughness lower than the 0.2pm (ra), which is considered clinically acceptable6. gloss values, they vary according to the incidence of light on surface resin; the lack sj�viƽigxmsr�wls[w�zepyiw���+9��[lmpi�e�kpeww�wyvjegi�[mxl�e�lmkl�vijvegxmzi�mrhi\�lew� about 100 gu21. to avoid changes in the refractive index of the samples, they were erep]^ih�hv]��tvizirxmrk�xli�jsvqexmsr�sj�[exiv�ƽpq��[lmgl�[syph�epxiv�xli�fvmklxriww� sj�xli�viƽigxmsr�zepyi24. in the present study, the nanohybrid ips empress direct showed the highest initial values of gloss, as seen in a previous study, after performing the polishing25. takahashi, et al.10�vitsvxih�xlex�xli�viwmr�-47�)qtviww�(mvigx�gpewwmƽih�jsv�ireqip��gsrxemr�ƽri�fevmyq�kpeww�ƽppivw��[lmgl�qe]�jezsvefpi�lmkl�wyvjegi�pywxiv��'levmwqe�(meqsrh�viwmr�wxetistically had the lowest brightness values, probably due to its particular feature regardmrk�mrwivxmsr�sj�pevkiv�tevxmgpiw�erh�e�kviex�[imklx�sj�ƽppiv���� ��*yvxlivqsvi��'levmwqe� diamond present a distinct monomer called tcd-di-hea instead of bis-gma and this could also have contributed for this result. it is known that bis-gma presents a high resistance to degradation from effects of immersion media by the fact that bis-acryl resin composite materials contain bifunctional acrylatto provide increase d mechanical strength and resistance to weakening in the presence of solvents26,27. in this context, the cross-link nature of the resin matrix, and the solvent sorption ytxeoi�qe]�mrƽyirgi�qsvi�hmvigxp]�xli�tsp]qivƅw�hikvehexmsr�vexi3. toothbrushing after the immersion on food-simulating media resulted in the removal of part of the svkermg�qexvm\�evsyrh�xli�psswir�ƽppivw5,26. however, the effect of toothbrushing in the composites resins depends on different factors, such as the type of toothpaste, type 9 lemos et al. and shape of the brush bristles, the proportion of deionized-water solution as well as speed and weight applied during simulation process26. after association of challenges, the highest gloss values were observed for the microƽppih�viwmr�6ireqip�jspps[ih�f]�)wxipmxi�·�5ymgo��%pxlsykl��xli�viwmr�)wxipmxi�·�5ymgo� tviwirx�lmkl�vsyklriww�zepyiw��*mk���% ��8lmw�ƽrhmrk�qe]�mrhmgexi�xlex�wtlivmgep�ƽppivw� facilitate the abrasive media sliding from the surface of the specimen, instead of the ƽppivw�fimrk�viqszih�f]�xli�efvewmzi�qihme��i\tpemrmrk�[l]�xlmw�qexivmep�vixemrw�mxw� gloss after mechanical abrasion20��8lir��kpsww�mw�rsx�mrƽyirgih�srp]�f]�xli�wyvjegi� roughness, but also by other factors such as difference in the refractive indices of the viwmr�qexvm\�erh�xli�ƽppivw20. these factors may explain the existing initial correlation between the parameters surface roughness and gloss, since there is greater homogeneity of values in polished surfaces7,20��-qqihmexip]�ejxiv�ƽrmwlmrk�erh�tspmwlmrk�tvsgihyviw�mw�tswwmfpi�xs�pizip� the organic and inorganic phase by the regularly wear of rotary cutting instruments. however, when the samples are subjected to experimental challenges, such action mw�wipigxmzi�erh�qe]�lezi�mrƽyirgi�mr�xli�svkermg�tlewi�erh�mr�xli�hmjjivirx�x]tiw�sj� ƽppivw15. this explains the lack of correlation between surface roughness and gloss ejxiv�xli�gleppirkiw��[lmgl�qe]�geywi�e�hikvii�sj�verhsq�viƽigxmsr�xs�xli�i\xirx�xlex� there is loss of surface polishing26. regarding color results, despite all the composites are marketed as a2, differences were sfwivzih�fix[iir�xli�gsqtswmxiw�xiwxih�ejxiv�xli�gleppirkiw��8li�rersƽppih�*mpxio�>���� xt and ips empress direct showed greater variation in color when compared to the others. filtek z350 xt resin, despite having nanoparticles in its composition, showed higher wsvtxmsr�getegmx]�xler�xli�qmgvsl]fvmh�erh�qmgvsƽppih�viwmrw17��8lmw�jegxsv�qe]�lezi�mrƽyenced to reduce the color stability, favoring the absorption of the dyes that are in coke and wine. the nanohybrid ips empress direct presents small particles of barium, compromising the strength of the material4,10 and increasing the possibility of incorporation of color pigments27��3xliv�jegxsvw�xlex�qe]�mrƽyirgi�jsv�xli�gspsv�glerki�evi�xli�eqsyrx� sj�tlsxsmrmxmexsvw�ekirxw��ƽppivw�erh�tmkqirxw�ekirxw�mrgsvtsvexih�f]�qeryjegxyvivw28. however, it is important to remember that these differences in color changes only become clinically noticeable when 'e value is higher than 3.312. according to the present study, ips empress direct was the only resin in that category, since other composites showed good color stability after chemical degradation and brushing. ren et al.18 also reported that a nanocomposite (filtek supreme ultra) showed more color change than tph and renamel after the stain challenges and after brushings. an in vitro study cannot represent all the conditions and interactions acting on the restorative material in the oral cavity, since cycles of acid challenge and toothbrushing occurs in patients. in this sense, randomized clinical trials are required in order to ewwiww�xliwi�teveqixivw��erh�iwtigmepp]�xli�mrƽyirgi�sj�xliwi�sr�xli�uyepmx]�sj�pmji� of the patient, as it interferes directly with the patient’s self-esteem. therefore, information obtained with this present study should prove valuable for clinicians to make decisions in selecting the best materials for aesthetic restorations for their patients. however, none of studied composites had the best performance for all analysis performed in this study. 10 lemos et al. in conclusion, within the limitations of this study can be drawn that chemical and qiglermgep�gleppirkiw�mrƽyirgi�xli�wyvjegi�vsyklriww�sj�epp�gsqtswmxiw��erh�xli]� lezi�nywx�rs�mrƽyirgi�mr�kpsww�sj�)wxipmxi�·�5ymgo�erh�6ireqip�gsqtswmxiw��8li�gsqqivgmep�gpewwmƽgexmsr�sj�gsqtswmxi�viwmr��rersgsqtswmxi�sv�qmgvsƽppih �[ew�rsx�e� determining factor in relation to the analyzed characteristics. references 1. ,sws]e�=��7lmvemwlm�8��4yttmr�6srxerm�61��4s[ivw�.1��)jjigxw�sj�egmhypexih�tlswtlexi�ƽysvmhi� gel application on surface roughness, gloss and colour of different type resin composites. j dent. 2011;39(10):700-6. 2. hi�4eype�%&��hi�*ygms�7&��%psrws�6'��%qfvswers�+1��4yttmr�6srxerm�61��-rƽyirgi�sj�gliqmgep� degradation on the surface properties of nano restorative materials. oper dent. 2014;39(3):e109-17. 3. munchow ea, ferreira ac, machado rm, ramos ts, rodrigues-junior sa, zanchi ch. effect of acidic solutions on the surface degradation of a micro-hybrid composite resin. braz dent j. 2014;25(4):321-6. 4. da silva em, de sa rodrigues cu, dias da, da silva s, amaral cm, guimaraes jg. effect of xssxlfvywlmrk�qsyxlvmrwi�g]gpmrk�sr�wyvjegi�vsyklriww�erh�xstskvetl]�sj�rersƽppih��qmgvsƽppih�� and microhybrid resin composites. oper dent. 2014;39(5):521-9. 5. :spxevippm�*6��7erxsw�(evs^�'&��%pziw�1'��'ezepgerxm�%2��1evglm�+1��)jjigx�sj�gliqmgep�hikvehexmsr� followed by toothbrushing on the surface roughness of restorative composites. j appl oral sci. 2010;18(6):585-90. 6. &sppir�'1��0eqfviglxw�4��5ymv]rir�1��'sqtevmwsr�sj�wyvjegi�vsyklriww�sj�svep�levh�qexivmepw�xs� the threshold surface roughness for bacterial plaque retention: a review of the literature. dent mater. 1997;13(4):258-69. 7. ,imrx^i�7(��*svnermg�1��3lqmxm�/��6sywwsr�:��7yvjegi�hixivmsvexmsr�sj�hirxep�qexivmepw�ejxiv�wmqypexih� toothbrushing in relation to brushing time and load. dent mater. 2010;26(4):306-19. 8. lefever d, perakis n, roig m, krejci i, ardu s. the effect of toothbrushing on surface gloss of resin composites. am j dent. 2012;25(1):54-8. 9. sabatini c, campillo m, aref j. color stability of ten resin-based restorative materials. j esthet restor dent. 2012;24(3):185-99. 10. takahashi r, jin j, nikaido t, tagami j, hickel r, kunzelmann kh. surface characterization of current composites after toothbrush abrasion. dent mater j. 2013;32(1):75-82. 11. 'exiper�%��7y^yom�8=��&igoiv�*��.v���&vmws�%0��(sw�7erxsw�4,��-rƽyirgi�sj�wyvjegi�wiepmrk�sr�gspsv� stability and roughness of composite submitted to ultraviolet-accelerated aging. j investig clin dent. 2017 may;8(2). doi: 10.1111/jicd.12203. 12. schulze ka, marshall sj, gansky sa, marshall gw. color stability and hardness in dental composites after accelerated aging. dent mater. 2003;19(7):612-9. 13. briso al, caruzo lp, guedes ap, catelan a, dos santos ph. in vitro evaluation of surface roughness and microhardness of restorative materials submitted to erosive challenges. oper dent. 2011;36(4):397-402. 14. erdemir u, yildiz e, eren mm, ozel s. surface hardness evaluation of different composite resin qexivmepw��mrƽyirgi�sj�wtsvxw�erh�irivk]�hvmrow�mqqivwmsr�ejxiv�e�wlsvx�xivq�tivmsh��.�%ttp�3vep�7gm�� 2013;21(2):124-31. 15. carvalho fg, sampaio cs, fucio sb, carlo hl, correr-sobrinho l, puppin-rontani rm. effect of chemical and mechanical degradation on surface roughness of three glass ionomers and a rersƽppih�viwmr�gsqtswmxi��3tiv�(irx����������� �������� 11 lemos et al. 16. 0iqsw�'%��1eyvs�7.��(sw�7erxsw�4,��&vmws�%0��*ekyrhiw�8'��-rƽyirgi�sj�1iglermgep�erh�'liqmgep� degradation in the surface roughness, gloss, and color of microhybrid composites. j contemp dent pract. 2017;18(4):283-8. 17. rahim tn, mohamad d, md akil h, ab rahman i. water sorption characteristics of restorative dental composites immersed in acidic drinks. dent mater. 2012;28(6):e63-70. 18. ren yf, feng l, serban d, malmstrom hs. effects of common beverage colorants on color stability of dental composite resins: the utility of a thermocycling stain challenge model in vitro. j dent. 2012;40 suppl 1:e48-56. 19. heintze sd, forjanic m. surface roughness of different dental materials before and after simulated toothbrushing in vitro. oper dent. 2005;30(5):617-26. 20. lee yk, lu h, oguri m, powers jm. changes in gloss after simulated generalized wear of composite resins. j prosthet dent. 2005;94(4):370-6. 21. 4evezmre�6(��6sihiv�0��0y�,��:skip�/��4s[ivw�.1��)jjigx�sj�ƽrmwlmrk�erh�tspmwlmrk�tvsgihyviw�sr� surface roughness, gloss and color of resin-based composites. am j dent. 2004;17(4):262-6. 22. &ivkiv�7&��4epmepsp�%6��'ezeppm�:��+merrmrm�1��'levegxivm^exmsr�sj�[exiv�wsvtxmsr��wspyfmpmx]�erh�ƽppiv� particles of light-cured composite resins. braz dent j. 2009;20(4):314-8. 23. 7y^yom�8��/]sm^yqm�,��*mrkiv�;.��ix�ep��6iwmwxergi�sj�rersƽpp�erh�rersl]fvmh�viwmr�gsqtswmxiw�xs� toothbrush abrasion with calcium carbonate slurry. dent mater j. 2009;28(6):708-16. 24. silikas n, kavvadia k, eliades g, watts d. surface characterization of modern resin composites: a multitechnique approach. am j dent. 2005;18(2):95-100. 25. ereifej ns, oweis yg, eliades g. the effect of polishing technique on 3-d surface roughness and gloss of dental restorative resin composites. oper dent. 2013;38(1):e1-12. 26. 8svviw�'6+��(e�7mpze�81��7epiw�%007��4yggm�'6��&svkiw�%&��-rƽyirgi�sj�'liqmgep�(ikvehexmsr�erh� toothbrushing on surface of composites. world j dent 2015;6(2):65-70. 27. akova t, ozkomur a, uysal h. effect of food-simulating liquids on the mechanical properties of provisional restorative materials. dent mater 2006 dec;22(12):1130-4. 28. ;exerefi�8��1m]e^eom�1��1ssvi�&/��-rƽyirgi�sj�tspmwlmrk�mrwxvyqirxw�sr�xli�wyvjegi�xi\xyvi�sj�viwmr� gsqtswmxiw��5ymrxiwwirgi�-rx����������� ������ 1http://dx.doi.org/10.20396/bjos.v20i00.8660328 volume 20 2021 e210328 original article 1 são leopoldo mandic faculty, são leopoldo mandic research institute. corresponding author: prof. dr. flavia lucisano botelho do amaral flbamaral@gmail.com faculdade são leopoldo mandic, instituto de pesquisas são leopoldo mandic. josé rocha junqueira, 13, ponte preta, campinas, sp, brazil. zip code: 13045-755. phone: (55 19) 3211-3610 / fax: (55 19) 3211-3610. editor: dr altair a. del bel cury received: july 3, 2020 accepted: november 3, 2020 effect of different concentrations of green tea extract solutions on bonding durability of etchand-rinse adhesive system to caries affected dentin ricky rolim de moura1 , fabiana mantovani gomes frança1 , cecilia pedroso turssi1 , roberta tarkany basting1 , flavia lucisano botelho do amaral1,* aim: the in vitro study evaluated the effect of different concentrations of green tea extract solution (gt) on the bonding durability of etch-and-rinse adhesive system to caries dentin affected (cad). methods: dentinal surfaces of human third molars were polished and submitted to a microbiological caries induction protocol for 14 days. after removal of the infected dentin layer, the samples were randomly divided into 4 groups (n= 10), according to the concentration of gt solution applied in cad, after acid etching: 0.05%; 0.2%; 2% and nt (no treatment – control). after application of an etch-and-rinse adhesive system (adper single bond 2, 3m espe), composite resin restorations were performed on the dentin. after 24 hours, the resin-dentin blocks were sectioned 1mm2 specimens, which were subjected to the microtensile test immediately or after 6 months of storage in water. data were submitted to two-way anova for randomized blocks and tukey test (α= 5%). results: there was no effect of double interaction (p= 0.934). the application of 0.2% gt promoted a statistically significant increase in dentin bond strength values in comparison to the condition where gt was not used (p=0.012). there was a significant decrease of bond strength after 6 months of storage, regardless of dentin pretreatment (p = 0.007). the g test identified that there was no statistical difference regarding failure mode (p= 0.326). conclusion: the concentration of 0.2% improved the bond strength of an etch-and-rinse adhesive system to caries affected dentin, however, none of dentin pretreatments could prevent the decrease in bond strength over time. keywords: dentin. camellia sinensis. tensile strength. dental caries. mailto:flbamaral@gmail.com https://orcid.org/0000-0002-8902-7597 https://orcid.org/0000-0002-2877-6797 https://orcid.org/0000-0002-0078-9895 https://orcid.org/0000-0002-5345-5776 https://orcid.org/0000-0002-8934-6678 2 moura et al. introduction the longevity of adhesive restorations is directly related to the durability of the bond between adhesive systems and dental substrate. unlike dental enamel, which is highly inorganic, dentin has an organic content and is essentially moist1. during hybridization, it is important that the collagen exposed during the demineralization stage becomes fully encapsulated and protected by the polymerized composite monomer2, a more difficult condition with etch-and-rinse adhesive systems, for which the acid conditioning of the dental structure and subsequent application of the primer/adhesive are done in separate steps3. exposure and degradation of collagen can also occur over time, if hydrolytic degradation of the resinous components of the hybrid layer occurs, leaving the collagen exposed and unorganized4. this situation is more aggravated in adhesive interfaces produced in caries affected dentine5, a common and challenging dentin substrate that is available for adhesion in cavity preparations. collagen eventually exposed can be degraded by enzymes with collagenolytic activity present in the dentin matrix, such as matrix metalloproteinases (mmps)6 and cysteine cathepsins (cts)7,8. such degradation, along with the hydrolytic degradation of resin components, negatively affect the hybrid layer structure and lead to a loss of dentin bond strength over time2, and this can directly affect the durability of adhesive restorations. mmps activation occurs in moments when ph falls (denaturation) and subsequent dentin buffering (activation), as is the case due to dentin acid conditioning and subsequent application of the adhesive system9,10 or, yet, in the context of pathogenesis of dental caries, due to the drop in ph due to lactic acid fermentation by bacteria and subsequent neutralization by buffer systems11-12. in addition, cts, normally present in the dentin matrix, are also activated at low ph, and once activated, participate in the activation of mmps7,8. specially in caries-affected dentin, there is an abundance of mmps and cts13, what highlights the importance of studying this type of dentin substrate in the context of strategies that can prevent bond strength decrease over time and increase the stability of adhesive interface. green tea has been described as a natural inhibitor of mmps, due to the presence of a catechin, epigallocatechin gallate (egcg)14. it is a flavonoid with antioxidant properties14, without any reports of side effects15. another property is related to the ability of egcg of interacting with collagen fibers through hydrogen bonds and hydrophobic interactions and, thus, also considered a cross-linking agent, which improves the mechanical properties of collagen16. regarding the stability of the bond with regards to dentin pretreatments with green tea extract, he results appear to have been contradictory. on one hand, the application of diluted green tea from commercially available tea (approximately 2% concentration) to healthy dentin17 or even the application of green tea extract in low concentrations (0.05%-0.2%) promoted the maintenance or even increased bond strength of adhesive systems to healthy dentin18 or caries affected dentin19. however, opposite results have also been found with the application of 0.2% green tea solution20. although the green tea application may be a promising strategy of dentin pre-treatment, it is evident that there is still no defined protocol regarding the concentration of green tea extract solution in relation to the bond strength, mainly in caries affected dentin substrate. 3 moura et al. thus, considering the relevance of studying adhesion to dentin affected by caries and in an attempt to establish an efficient protocol for a therapeutic agent that can inhibit the degradation of the adhesive interface in this type of substrate, this in vitro study evaluated the effect of different concentrations of green tea extract solution on the bond strength immediately and after 6 months of storage in water, of an etch-and-rinse adhesive system to dentine affected by caries. materials and methods ethical aspects the present study was approved by the local research ethics committee (caae: 49027515.1.0000.5374). experimental design and sample size calculation the study involved a microbiological model of caries lesion induction, carried out according to the protocol of sanabe et al.21. the factors under study were: 1. type of solution/concentration used on dentine affected by caries after acid etching and before applying the etch-and-rinse adhesive system (adper single bond 2 3m espe, st. paul, mn, usa), at three levels: cv 0.05. 0.05% green tea extract solution; cv 0.2. 0.2% green tea extract solution; cv 2. 2% green tea extract solution. as a control level, an untreated group was added (nt). 2. moment of the bond strength test, in two levels: 24 hours (immediate) and 6 months (long-term). the experimental units were composed of 32 human third molars, restored with composite resin, according to the treatment assigned to each group (n=8). from a pilot study carried out with three specimens, means and standard deviations were used to calculate the effect size (f = 0.349). the software g*power 3.1.9.4 (heinrich-heine universitat, dusseldorf, germany) retrieved 7 specimens per group to detect difference among groups at a 0.05 alpha level and 80% power. the final sample size per group was fixed at 8 specimens to account for potential losses during the study. from each tooth-restoration set, eight longitudinal sections were obtained, which were divided according to the storage time, therefore replicates were formed. the variable of continuous quantitative response was obtained through microtensile bond strength tests (mpa). the materials cited in the experimental design, as well as their composition and mode of use, are described in table 1. table 1. materials, composition, application method and ph. material batch number# composition application method ph* condac 37 (fgm, joinville, sc, brasil) #230915 37% phosphoric acid applied to the dentin surface for 15 s, rinsed for 30 s and dried gently with absorbent paper. -green tea extract (farmácia natal, águas frias, sc, brasil) #185841 camellia sinensis twenty microliters of the solution were passively applied to the dentin for 60 s. the dentin was dried gently with absorbent paper. 0,05% 4.80 0,2% 4.50 2% 4.28 continue 4 moura et al. etch-and-rinse adhesive system adper single bond 2 (3m espe, st. paul, mn, eua) #612979 bis-gma, hema, copolymer of acrylic and itaconic acids, water, ethyl alcohol, glycerol 1,3-dimethacrylate, udma, silane treated silica. the adhesive system was applied in two consecutive layers; the remaining solvent was evaporated with a brief, gentle, dry air jet for 10 s and light cured for 20 s. 4.7 composite resin filtek™ z250 xt (3m espe, st. paul, mn, eua) #452516 aluminum oxide, silica, zirconium oxide, bis-gma, bis-ema, udma each 1-mm increment was light cured for 20 s. -hema (hydroxyethyl methacrylate); bis-gma (bisphenol a-glycidyl methacrylate), udma (urethane dimethacrylate); bis-ema (2,2-bis-4-2-(hydroxi-3methylacriloxietoxi)-phenylpropane). *ph measured in triplicate tooth selection and fragments preparation thirty-two human third molars were cleaned with a periodontal curette and kept in 0.1% aqueous thymol solution, at 4 °c, until the study was conducted. occlusal portions were removed with a high concentration diamond cutting disc (series 15 hc buehler ltd, lake bluff, illinois, usa) mounted on a precision electric cutter (isomet 1000 precision diamond saw (buehler ltd, lake bluff, illinois, usa) under constant cooling and speed. artificial caries induction for the induction of carious lesions, the protocol obtained in the study by sanabe et al.21 was used. the roots were sealed with composite resin and then waterproofed with a layer of epoxy adhesive (araldite, ciba especialidades quimicas ltda., são paulo, sp, brasil) and another one of nail polish (colorama, ltda ceil com. exp. ind. ltda, são paulo, brasil), leaving only the dentin surface exposed. all teeth were autoclaved for 20 minutes at 121 °c. the cariogenic solution consisted of 3.7 g of bhi broth (brain heart infusion broth, becton dickinson and company,sparks, md, eua), 2 g of sacarose (synth; labsynth, são paulo, sp, brasil), 1 g glucose and 0.5 g yeast extract (becton dickinson and company, sparks, md, eua) for every 100 ml of distilled water. this solution was autoclaved for 20 minutes at 121 ° c prior to inoculation of 2% strains of streptococcus mutans atcc25175 (andre tosello foundation tropical cultures collection) (108 ufc/ml). the teeth were suspended in the cariogenic environment and the set was taken to microaerophilia (co2 incubator, te 399, tecnal, piracicaba, sp, brazil) for 14 days. during this period, the cariogenic solution was replaced every 48 hours, but without the inoculation of new microorganisms. after the incubation period, the teeth were again autoclaved. the biofilm was removed with gauze and the insulating materials (epoxy adhesive and nail polish) were removed manually with scalpel blades and the teeth were then washed with deionized water. dentin surface was slightly darkened and softened, a condition that was measured with a non-cutting manual instrument. adhesive procedures the infected dentin was removed with an excavator until a more hardened and touch-resistant dentin to exploratory probe was obtained, without pressure. this continuation 5 moura et al. procedure was performed by a single, previously trained, operator. after that, the fragments were conditioned for 15 seconds with 37% phosphoric acid, washed and rinsed for 10 seconds and dried with absorbent paper. the dentin fragments were randomly divided into 4 groups, according to the treatment performed on the dentin surface: 0.05%, 0.2% and 2% green tea extract solution (groups cv 0.05; cv 0.2 and cv 2 respectively); and no treatment (nt). the green tea extract solution was diluted to concentrations of 0.05%, 0.2% and 2%. the initial concentration was based on the study by zheng et al.18 and the final concentration on the study by carvalho et al.19. the volume of the solution (cv) was 20μl per fragment17, that were passively applied for 60 seconds on the conditioned dentin and the excess removed with absorbent paper, keeping the tissue moist always. then, the adhesive system (adper single bond 2, 3m espe, st. paul, usa) was applied according to the manufacturer’s recommendations. the dentin surface was restored with a composite resin (filtek™ z250 xt, 3m espe, irvine, ca, usa color a2). each 1-mm resin composite increment was light cured for 20 seconds with a with led light device (sdi radii cal light curing unit, sdi limited bayswater, victoria 3153 australia), positioned as close as possible to the equipment. the final resin block was 4-mm high. microtensile test preparation and water storage the restorations were stored in distilled water, at 37°c, for 24 hours and then sectioned perpendicularly to the adhesive interface with a flexible diamond disk mounted on a precision electric cutter, obtaining stick-shaped specimens, with dimensions of 0.8 mm2. each tooth-restoration block provided, on average, 8 stick-shaped specimens that were randomly divided into two groups, according to the storage time in distilled and deionized water, 24 hours and 6 months. the specimens were kept in a bacteriological incubator (odontobrás, ribeirão preto, sp, brazil) at 37°c and the water changed every 2 days. microtensile bond strength testing each stick-shaped sample was fixed in a microtensile test device with the aid of instant adhesive (super bonder gel, henkel loctite ltda, são paulo, sp, brazil) and subsequently subjected to the microtensile test in a universal testing machine (mem-2000 model, emic, são josé dos pinhais, pr, brazil) with a speed of 0.5 mm/min. the load at the time of the fracture of each specimen was recorded in kgf and divided by the cross-sectional area of the stick (mm2), in order to obtain the microtensile bond strength values expressed in mpa. the comparison was made by the average of the sticks in each tooth, each tooth being considered an experimental unit. the surfaces of the fractured specimens were examined using a stereoscopic magnifying glass (eikonal equip. optical and analytical, model ek3st, são paulo, sp, brazil), with a 30-fold magnification, to classify the type of fracture that occurred. the fractures were classified as: adhesive (failure of adhesion), cohesive in dentin (failure of the dental substrate), cohesive in composite resin (failure of composite resin) or mixed (adhesive failure and cohesive in composite resin). 6 moura et al. statistical analysis the compliance with the presuppositions of normality and homoscedasticity was verified by the shapiro-wilk and levene tests, respectively, to compare the effect of applying green tea extract solution in different concentrations and the storage time, as well as the interaction of these two study factors, the bond strength data were subjected to analysis of variance at two criteria for randomized blocks. tukey’s test was used for multiple comparisons. the failure modes observed were subjected to descriptive approaches and g test. statistical calculations were performed using the spss 23 program (spss inc., chicago, il, usa), adopting a significance level of 5%. results the two-way analysis of variance for randomized blocks demonstrated that there was no statistically significant interaction between the concentration of the green tea extract solution and the storage time (p = 0.934). the bond strength was significantly influenced by the concentration of green tea extract from the solution that was applied to the dentin (p = 0.012). regardless of dentin pretreatment, it was observed a significant decrease in bond strength values after 6 months of water storage (p = 0.007) (table 2). table 2. average values and standard deviation of the bond strength (in mpa), according to the concentration of the green tea extract solution applied to the dentin and the storage time. green tea extract storage time grand mean 24 hours 6 months 0.05% 14.42 (6.20) 9.53 (4.83) 11.97 (5.91) ab 0.2% 17.80 (6.49) 13.25 (5.82) 15.52 (6.37) a 2% 11.04 (2.94) 7.09 (4.14) 9.07 (4.01) b absent (control) 11.29 (4.78) 8.82 (6.23) 10.05 (5.48) b grand mean 13.64 (5.72) a 9.67 (5.51) b – grand means followed by distinct uppercase letters indicate a significant difference between dentin pretreatments, regardless of storage period (p<0.05). grand means followed by distinct lowercase letters indicate a significant difference between dentin storage periods, regardless of dentin pretreatment (p<0.05). the g test identified that there was no statistically significant difference between the groups regarding the failure mode (p = 0.326). in all groups, there was a predominance of adhesive failures (figure 1). 7 moura et al. discussion considering knowledge of the degradative processes of the hybrid layer, it is important to search for and evaluate compounds that can delay the decrease in bond strength over time. in this study, we sought to evaluate the effect of pretreatment on dentine affected by caries with a solution containing green tea extract, in different concentrations, since this polyphenol contains catechins, such as egcg, with proven inhibitory activity against mmps22 and it is a crosslinking agent16, which improve the mechanical properties of collagen. it was found that, regardless of the storage time, the application of green tea extract at 0.2% concentration provided bond strength values higher than the other groups, except with respect to the 0.05% concentration, this one with intermediate bond strength values. the concentration of 2% obtained the lowest values of bond strength, compared to other treatments both in 24 hours and in 6 months of storage. therefore, the null hypothesis failed to be rejected. lower bond strength findings for green tea extract at a concentration of 2% may be related to the saturation of the extract in solution, which may have interfered negatively in the bond strength, similar to the tests in the immediate time of carvalho et al.19, who also observed bond strength to dentin affected by caries to be inferior when using a solution containing 2% green tea extract. gerhardt et al.23, using the same concentration of egcg (2%) for dental pre-treatment before applying a two-step self-etching adhesive system (clearfil se bond), reached results similar to the present study. although the solution containing green tea extract at a concentration of 0.05% did not differ from the 0.2% group, it also did not differ significantly from the group without dentin pre-treatment (nt). it is speculated that the 0.05% concentration may not have been sufficient, that is, the concentration of green tea extract was too low to produce any significant effect on bond strength in caries affected dentin. figure 1. bar diagram of the relative frequency (%) of failure modes, according to the concentration of the solution containing green tea extract applied to the dentin and the storage time. adhesive mixed cohesive in dentin cohesive in resin 100% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 0.05 % gt 0.2 % gt 2 % gt 0.05 % gt 0.2 % gt 2 % gtno treatment (control) no treatment (control) 75% 75% 69% 87% 90% 75% 71% 86% 16% 6% 3% 16% 9% 9% 22% 7% 3% 3% 3% 7% 13% 13% 26% 3% 10% 5% 24 hours 6 months 8 moura et al. the concentration of solution containing 0.2% tea extract seems to be, among all the studied, the best concentration to be applied to caries affected dentin, as it obtained the highest average bond strength, higher than the control group (nt), regardless of storage time. this result can be attributed to the concentration of catechins in the green tea extract, such as egcg, which is a cross-linking agent16 and may have improved the mechanical properties of collagen, besides of not impairing hybrid layer formation when the adhesive interface is evaluated under scanning electronic microscopy analysis20. however, it is worth noting that none of the dentin pretreatments prevented the decrease in bond strength over 6 months, similar to bond strength studies on sound dentin20. in fact, the dentin pretreatment with 0.2% egcg solution did not prevent the increase of nanoinfiltration in the hybrid layer formed in caries affected dentin, over a year of storage in water24. one explanation for this result may be related to the time that solutions were applied to dentin, which in the present study was 60 seconds as done in previous studies17,20,24. it has been observed that the increase in dentin elasticity modulus, interpreted as an increase in dentin mechanical strength, was achieved only after 60 minutes of application of green tea extract25. in fact, studies that applied other natural extracts on dentin, such as 6.5% grape seed extract have found a positive effect on the bond strength stability of the pre-treated dentin with the application time of 10 minutes26. it is hypothesized that the effect of pre-treatments with polyphenols, such as green tea, is time and concentration dependent. thus, future studies should assess whether longer application times of the green tea extract solution can increase the modulus of dentin elasticity (proving its cross-linking effect) and at the same time stabilize the bond strength over time. also, the decline in bond strength after 6 months of water storage can be explained by the hydrolytic degradation of the resin-dentin adhesive interface after immersion in water, that promotes loss of resinous material throughout the hybrid layer and micromorphological changes in the collagen fibrils4. these micromorphological changes seem to be responsible for the degradation of the hybrid layer and reduction of the bond strength. specially in caries affected dentin, the hybrid layer presents an increase in the exposed collagen zone and a decrease in the quality of the adhesive infiltration, and consequently, the adhesive interface is more prone to hydrolytic degradation8. in fact, the percentage of adhesive failures increased over time, which can be confirm the degradation that occurred at the adhesive interface. an important point is in regard to the use of water as a storage medium. based on the fact that mmps require zinc and calcium to be active27. it has been reported that the use of water underestimates the activity of dentin mmps, thereby promoting loss of calcium and zinc from dental matrices instead of restoring these ions25. in this sense, the use of solutions that simulate body fluids as a means of storing samples is of interest in future evaluations involving mmp inhibitors as dental pre-treatments. in terms of clinical application, the results found here allow for reflection and the need for a greater understanding of the action of the activity of collagen-degrading enzymes, and it is important to conduct further studies in order to assess the ideal concentration as well as time and form of application of green tea extract to dentin. also, clinically, the caries affected dentin is considered a challenging substrate, which can outweigh 9 moura et al. the benefits of pretreatments applied to it. so, this study revealed that, although the application of 0.2% green tea extract solution promoted greater bond strength than the other studied concentrations, none of the dentin pre-treatment solutions were sufficient to prevent the decrease in bond strength to caries affected dentin over time. in conclusion, dentin pre-treatment with aqueous solution containing 0.2% green tea extract promoted higher bond strength of etch-and-rinse adhesive system to caries-affected dentin than the concentrations of 0.05% and 2%. however, bond strength decreased 6 months of water storage, regardless of whether or not the dentin received pre-treatment solutions. disclosure statement no potential conflict of interest was reported by the authors. references 1. nakabayashi n, kojima k, masuhara e. the promotion of adhesion by the infiltration of monomers into tooth substrates. j biomed mater res. 1982 may;16(3):265-73. doi: 10.1002/jbm.820160307. 2. tjäderhane l, nascimento fd, breschi l, mazzoni a, tersariol il, geraldeli s, et al. strategies to prevent hydrolytic degradation of the hybrid layera review. dent mater. 2013;29(10):999-1011. doi: 10.1016/j.dental.2013.07.016. 3. van meerbeek b, de munck j, yoshida y, inoue s, vargas m, vijay p, et al. buonocore memorial lecture. adhesion to enamel and dentin: current status and future challenges. oper dent. 2003;28(3):215-35. 4. hashimoto m, tay fr, ohno h, sano h, kaga m, yiu c, et al. sem and tem analysis of water degradation of human dentinal collagen. j biomed mater res b appl biomater. 2003 jul;66(1):287-98. doi: 10.1002/jbm.b.10560. 5. erhardt mc, toledano m, osorio r, pimenta la. histomorphologic characterization and bond strength evaluation of caries-affected dentin/resin interfaces: effects of long-term water exposure. dent mater. 2008;24(6):786-98. doi:10.1016/j.dental.2007.09.007. 6. pashley dh, tay fr, yiu c, hashimoto m, breschi l, carvalho rm, et al. collagen degradation by host-derived enzymes during aging. j dent res. 2004;83(3):216-21. doi: 10.1177/154405910408300306. 7. nascimento fd, minciotti cl, geraldeli s, carrilho mr, pashley dh, tay fr, et al. cysteine cathepsins in human carious dentin. j dent res. 2011 apr;90(4):506-11. doi: 10.1177/0022034510391906. 8. tjäderhane l, nascimento fd, breschi l, mazzoni a, tersariol il, geraldeli s, et al. optimizing dentin bond durability: control of collagen degradation by matrix metalloproteinases and cysteine cathepsins. dent mater. 2013;29(1):116-35. doi: 10.1016/j.dental.2012.08.004. 9. mazzoni a, pashley dh, nishitani y, breschi l, mannello f, tjäderhane l, et al. reactivation of inactivated endogenous proteolytic activities in phosphoric acid-etched dentine by etch-and-rinse adhesives. biomaterials. 2006;27(25):4470-6. doi: 10.1016/j.biomaterials.2006.01.040. 10. mazzoni a, carrilho m, papa v, tjäderhane l, gobbi p, nucci c, et al. mmp-2 assay within the hybrid layer created by a two-step etch-and-rinse adhesive: biochemical and immunohistochemical analysis. j dent. 2011;39(7):470-7. doi: 10.1016/j.jdent.2011.04.004. 11. tjäderhane l, larjava h, sorsa t, uitto vj, larmas m, salo t. the activation and function of host matrix metalloproteinases in dentin matrix breakdown in caries lesions. j dent res. 1998;77(8):1622-9. doi: 10.1177/00220345980770081001. 10 moura et al. 12. chaussain-miller c, fioretti f, goldberg m, menashi s. the role of matrix metalloproteinases (mmps) in human caries. j dent res. 2006;85(1):22-32. doi: 10.1177/154405910608500104. 13. vidal cm, tjäderhane l, scaffa pm, tersariol il, pashley d, nader hb, et al. abundance of mmps and cysteine cathepsins in caries-affected dentin. j dent res. 2014;93(3):269-74. doi: 10.1177/0022034513516979. 14. kim hs, quon mj, kim ja. new insights into the mechanisms of polyphenols beyond antioxidant properties; lessons from the green tea polyphenol, epigallocatechin 3-gallate. redox biol. 2014;2:187-95. doi: 10.1016/j.redox.2013.12.022. 15. chow hh, cai y, hakim ia, crowell ja, shahi f, brooks ca, et al. pharmacokinetics and safety of green tea polyphenols after multiple-dose administration of epigallocatechin gallate and polyphenon e in healthy individuals. clin cancer res. 2003;9(9):312-9. 16. kwon y, kim h, hwang y, rosa v, yu m, min k. effects of epigallocatechin gallate, an antibacterial cross-linking agent, on proliferation and differentiation of human dental pulp cells cultured in collagen scaffolds. j endod. 2017;43(2):289-96. doi: 10.1016/j.joen.2016.10.017. 17. monteiro tma, basting rt, turssi cp, frança fmg, amaral flb. influence of natural and synthetic metalloproteinase inhibitors on bonding durability of an etch-and-rinse adhesive to dentin. int j adhes. 2013;47:83-8. doi: 10.1590/1678-775720150518. 18. zheng p, zaruba m, attin t, wiegand a. effect of different matrix metalloproteinase inhibitors on microtensile bond strength of an etch-and-rinse and a self-etching adhesive to dentin. oper dent. 2015;40(1):80-6. doi: 10.2341/13-162-l. 19. carvalho c, fernandes fp, freitas v da p, frança fm, basting rt, turssi cp, et al. effect of green tea extract on bonding durability of an etch-and-rinse adhesive system to caries-affected dentin. j appl oral sci. 2016;24(3):211-7. doi: 10.11607/ijp.6468. 20. barcellos dc, fonseca bm, bresciani e, pucci cr, araujo mam. effect of chlorhexidine, green tea and egcg as therapeutic primers to increase the durability of resin-dentin bond. braz dent sci. 2016;19(4):72-82. doi: 10.14295/bds.2016.v19i4.1316. 21. sanabe me, costa ca, hebling j. exposed collagen in aged resin-dentin bonds produced on sound and caries-affected dentin in the presence of chlorhexidine. j adhes dent. 2011;13(2):117-24. doi: 10.3290/j.jad.a19239. 22. demeule m, brossard m, pagé m, gingras d, béliveau r. matrix metalloproteinase inhibition by green tea catechins. biochim biophys acta. 2000;1478(1):51-60. doi: 10.1016/s0167-4838(00)00009-1. 23. gerhardt kmf, oliveira car, frança fmg, basting cp, turssi cp, amaral flb. effect of epigallocatechin gallate, green tea extract and chlorhexidine application on long-term bond strength of self-etch adhesive to dentin. int j adhes adhes. 2016 dec;71:23-7. doi: 10.1016/j.ijadhadh.2016.08.005. 24. fialho mpn, hass v, nogueira rp, frança fmg, turssi cp, basting rt, et al. effect of epigallocatechin-3gallate solutions on bond durability at the adhesive interface in caries-affected dentin. j mech behav biomed mater. 2019;91:398-405. doi: 10.1016/j.jmbbm.2018.11.022. 25. aguiar tr, vidal cm, phansalkar rs, todorova i, napolitano jg, mcalpine jb, et al. dentin biomodification potential depends on polyphenol source. j dent res. 2014 apr;93(4):417-22. doi: 10.1177/0022034514523783. 26. castellan sc, bedran-russo ak, antunes a, pereira nrp. effect of dentin biomodification using naturally derived collagen cross-linkers: one-year bond strength study. int j dent. 2013:918010. doi: 10.1155/2013/918010. 27. tezvergil-mutluay a, agee ka, hoshika t, carrilho m, breschi l, tjäderhane l, et al. the requirement of zinc and calcium ions for functional mmp activity in demineralized dentin matrices. dent mater. 2010;26(11):1059-67. doi: 10.1016/j.dental.2010.07.006. effectiveness of a single-tuft toothbrush for control of newly formed dental biofilm iohana hasegawa1, camila veríssimo1, victor angelo montalli1, marcelo sperandio1, daiane cristina peruzzo1 1são leopoldo mandic – slmandic, são leopoldo mandic dental school and research institute, department of oral pathology, campinas, sp, brazil correspondence to: daiane c. peruzzo são leopoldo mandic institute and research center rua josé rocha junqueira 13, ponte preta, cep: 13045-755 campinas sp brasil phone: +55 19 3211 3659 fax: +55 19 3211 3635 e-mail: daiaperuzzo@yahoo.com.br abstract aim: to compare the effectiveness of a single-tuft toothbrush (stb) with conventional toothbrushes (ct) to control dental biofilm neoformation in the dentogingival area. methods: for this cross-sectional prospective blind study, 20 periodontally healthy subjects were selected and randomly divided into 4 groups: stb; ct; chx chlorhexidine mouthwash (positive control) and ps placebo mouthwash (negative control). the subjects were instructed to use only the assigned care method for 72 h with a 7-day washout period between experiments. the evaluated parameters were visible and disclosed plaque indices (pi and dpi), gingival bleeding index (gbi) at baseline (t-0) and at the end of each experimental period (t-72). results: data analysis demonstrated that at t-0 no difference was observed for any of the parameters (p>0.05); after 72 h, ct, stb and chx showed equivalent effectiveness at controlling biofilm. when the pi data were analyzed, between t-0 and t-72, stb was similar to ct and chx (p<0.05), whereas for dpi, stb was significantly superior to the other methods. except for ps, all methods yielded similar results for gbi (p<0.05). conclusions: the tested stb was effective at controlling short-term dental biofilm neoformation on the dentogingival area. keywords: dental plaque. biofilms. oral hygiene. introduction gingivitis is primarily caused by dental biofilm, which must be controlled in order to achieve and maintain periodontal health1. the bacteria in biofilm are mostly in balance with the host, which denotes a state of persistent periodontal health. when such homeostasis is broken due to inadequate oral hygiene, gingivitis settles in, which may progress into periodontitis2. from the pathophysiological viewpoint, no individual is immune to gingivitis, provided biofilm is allowed to accumulate over time, breaking the gingival homeostasis. when intrasulcular homeostasis is broken, visible clinical changes begin to emerge, such as spontaneous gingival bleeding, bleeding on brushing, erythema, swelling and changes in gingival texture2,3. it is therefore paramount to concentrate efforts at tackling the root of the problem using methods of oral hygiene and consequently halting dental biofilm formation4. the gold standard for prevention of gingivitis is mechanical removal of biofilm by regular toothbrushing4-6. for hygiene to be performed according to the instructions by dental professionals, biofilm control strategies must be tailored to the needs of each individual. toothbrush and toothpaste are undoubtedly the most widespread tools for mechanical removal of plaque and debris from the tooth surface. thus, to meet specific individual needs several devices were developed, for instance, interdental brushes and dental floss for interdental areas, and single-tuft brushes, for intra-sulcular and even buccal/lingual/palatal areas7-10. in normal circumstances, teeth cleaning solely with a conventional toothbrush will not remove biofilm equally from all surfaces11. complementation is therefore received for publication: july 19, 2016 accepted: november 16, 2016 braz j oral sci. 15(2):113-118 original article braz j oral sci. april | june 2016 volume 15, number 2 http://dx.doi.org/10.20396/bjos.v15i2.8648761 114 required using auxiliary devices, such as dental floss or tape, interproximal brushes and/or single-tuft brushes, according to the shape, size and access to the cleaned site. single-tuft brushes are delicate and may be advised for specific areas. usually, they are recommended for difficult to access sites, such as furcations, distal surfaces of molars, areas of amputated roots, buccal or lingual surfaces with irregular gingival margin, crowded areas and proximal surfaces of isolated teeth12. additionally, because it is relatively uncomplicated to direct stb towards the gingival sulcus, they may be the most effective method to remove biofilm from deep pockets. the flowchart of evidence on the role of supragingival bacterial biofilm is complete when biofilm control leads to gingival health1. evidence derived from large cohort studies have demonstrated that high standards of oral hygiene will ensure stability of the periodontium13. both short-term and long-term cross-sectional as well as longitudinal studies have shown that the incidence of gingivitis and biofilm accumulation still seems to be high even among the adult population that brush their teeth frequently14. despite stb being highly recommended by periodontal specialists, there are few studies15-17 demonstrating the effectiveness of such tools at controlling dental biofilm at crevicular sites. the aim of this study was to compare the effectiveness of single-tuft brushes against the gold standard, namely conventional toothbrushes, at controlling newly formed biofilm at the dentogingival area of healthy individuals. material and methods sample selection this study was approved by the ethics committee of the são leopoldo mandic dental school, campinas / sp, protocol #356827/2013. sample size was based on previously published studies of similar design18,19 and consisted of 20 dental students from the são leopoldo mandic dental school. the inclusion criteria were: systemically and periodontally healthy subjects (probing depth ≤ 3mm and no gingival bleeding)20-21, aged between 18 and 30 years with a minimum of 20 remaining teeth, who agreed to participate in the study. exclusion criteria were: presence of cervical restorations, antimicrobial therapy for any medical or dental condition within 6 months prior to the trial, use of drugs known to affect the periodontal environment (anti-inflammatories, pain-killers, contraceptives, anticonvulsants, immunosuppressants, cyclosporin, anticoagulants and calcium channel blockers) also within 6 months prior to the baseline periodontal examination, orthodontic treatment or devices, pregnant women and breastfeeding mothers. study design two types of toothbrushes were compared: a single-tuft brush (bitufo® hypermarcas, senador canedo go, brazil) and a conventional toothbrush (bitufo® hypermarcas, senador canedo go, brazil). 0.12% chlorhexidine mouthwash (bitufo®, senador canedo go, brazil) was used every 12 h as a positive control, whereas a placebo solution (bitufo®hypermarcas, senador canedo go, brazil) with similar features as the chlorhexidine mouthwash, but without the active ingredient, was used as a negative control. the groups were defined as follows: stb – single-tuft brushes (test), n=20; ct conventional toothbrush (gold standard), n=20; chx chlorhexidine mouthwash (0.12% positive control), n=20; and ps placebo solution (negative control), n=20. during each experimental phase, the subjects were instructed to use solely the method designated to their group, excluding any other additional cleaning strategy. each experimental phase lasted 72 h with a 7-day washout period in between, in order to avoid a possible residual (carryover) effect of the previous treatment method. during this washout period, all volunteers used a standard toothbrush and toothpaste provided by the researchers. the mouthwash solutions were packed and coded in order to prevent identification of the used product. the codes were revealed only when the study was complete. clinical experimental phase following patient selection, a clinical oral examination was performed by a single examiner (ih), trained and calibrated to obtain the following initial clinical parameters: visible plaque index (pi), disclosed plaque index (dpi) and gingival bleeding index (gbi), according to ainamo and bay22, as shown in figure 1 (a, b and c, respectively). in addition, periodontal evaluation was performed, which included probing depth (pd), gingival recession and clinical attachment level, in order to assure absence of gingivitis clinically. subsequently, professional biofilm removal was performed on each volunteer using a rubber cup and prophylaxis paste. personalized instructions for toothbrushing were given individually and verbally by another researcher (cv), according to brush (conventional and stb) and solution (placebo and chlorhexidine). only during the washout period were the subjects encouraged to apply other conventional oral hygiene methods, such as dental floss or tape. following the trial phase, a second professional prophylaxis session was performed. the volunteers were then randomly assigned (using a computer list) to their respective sequence of oral hygiene methods, observing the 7-day washout period20. pi, dpi and gbi were recorded both at the beginning and at the end of each trial period. statistical analysis only the subjects who completed the study (n=18) were considered for statistical purposes. prior to the analysis, the kolmogorov-smirnov test was applied to assess normality. for intra-group analysis (between periods) of the data (pi, dpi and gbi), student’s t test was used. for inter-group analysis (between treatments), anova/tukey tests were applied. bioestat 5.0 (sustainable mamirauá institute, belém, pa, brazil) software was used for statistical calculations. for all analyzes, the significance level was set at 5%. effectiveness of a single-tuft toothbrush for control of newly formed dental biofilm braz j oral sci. 15(2):113-118 115 results twenty volunteers were selected from march to june 2013. the participants were aged between 18 and 30 years (mean age 21.1 years), 13 females and 7 males. among the 20 initially selected individuals , 18 completed the study and two were lost to follow-up. intergroup analysis at the early experimental stage (t-0) revealed no statistically significant difference between treatments (stb, ct, chx, ps) for any of the evaluated parameters (pi, dpi, gbi), demonstrating homogeneity between the groups (figure 2a). after 72 h (t-72) (figure 2b), a significant difference was observed (p<0.05) only for ps in terms of pi and dpi, but not gbi. in the intra-group comparison between t-0 and t-72, the percentage of accumulated visible plaque (pi) (figure 3) increased significantly only in the ps group (p<0.05). as shown in figure 4, assessing the percentage of disclosed plaque (dpi), a significant difference was observed in ct, chx and ps, while the stb group showed similar results between t-0 and t-72 (p<0.005). effectiveness of a single-tuft toothbrush for control of newly formed dental biofilm fig.1. intra-oral examination for the following clinical parameters: (a) visible plaque index (pi); (b) disclosed plaque index (dpi), and (c) gingival bleeding index (gbi). fig.2. mean (±sd) for the clinical parameters at baseline (t-0) (a), and after 72 h (b) for single-tuft brush (stb), conventional toothbrush (ct), chlorhexidine (chx) and placebo solution (sp). * indicate significant intragroup differences for the clinical parameters evaluated, by anova and tukey test (p <0.05). fig.3. mean (±sd) for visible plaque index (pi), for the treatments, at baseline (t-0) and after 72 h (t-72) to single-tuft brush (stb), conventional toothbrush (ct), chlorhexidine (chx) and placebo solution (sp). different lowercase letters indicate significant intragroup differences over time, by student t test (p<0.05). regarding gbi (figure 5), no significant differences were observed between t-0 and t-72 for all treatments. braz j oral sci. 15(2):113-118 116 effectiveness of a single-tuft toothbrush for control of newly formed dental biofilm discussion faced with the limitations of conventional hygiene methods, new types of brushes have been developed, including electric toothbrushes, single-tuft and interdental brushes7,8,23. tooth brushing per se is often insufficient to remove dental biofilm, particularly from interproximal and dentogingival areas. in turn, such scenario will demand complementary strategies to tackle biofilm disruption, including the use of dental floss/tape, interdental brushes, mouthwashes, etc. only a handful of methodologically sound studies have focused on the effectiveness of such methods, especially in the intra-sulcular area. in this context, the present cross-sectional and prospective study aimed to evaluate the effectiveness of single-tuft brushes to control the new formation of bacterial biofilm at this particular site. the methodological design of the present study allowed for testing all volunteers for all evaluated methods, thus reducing possible biases. additionally, the inclusion of washout periods minimized the residual effects of the methods used before each new treatment24-26. such study design may counterbalance carryover interferences and provide an estimate of treatment effect with minimal increase in variance, even if carryover is included in the model. at baseline (t-0), no difference was observed between treatments for any of the parameters evaluated, demonstrating sample homogeneity between groups. at t-72, however, differences were observed in the placebo group for pi, dpi, but not for the gbi. these results reflect the relatively low hygiene withdrawal period in this sample of periodontally healthy individuals. at 72 h, no clinically evident signs of gingival inflammation could be detected3,27, corroborated in the present study by the gbi values. additionally, scanning electron microscopy studies evaluating the initial stages of supragingival plaque formation confirmed the presence of a microbial deposit-free zone located between the biofilm layer and the gingival margin28. bergström29 and quirynen and coworkers30, when analyzing the initial stages of biofilm formation by sequential photographic records registered clinically this event. in their study, the biofilm-free zone was not stained by plaque-disclosing solutions. subsequently, studies have shown that a supragingival plaque-free zone persists for up to 96 h31,32. in this study, both disclosed and visible plaque indices were used to assess plaque scores and biofilm new formation. visible plaque index (pi) was used to demonstrate gross plaque accumulation, whereas dpi was used to detect low amounts of plaque, as it is a much more sensitive method than pi. although the use of disclosing solutions in the management of biofilm control can be somehow discouraging for some patients, when combined with index scales, they enable comparisons between new and existing oral hygiene products33. the choice for disclosing tablets over disclosing solutions was based on the fact that the former is widely used and is likely to be less disturbing of the biofilm, since the latter involves mechanical application of the solution with a cotton swab, which in turn may disrupt biofilm and risk false negatives34. when confronting the results between t-0 and t-72, only the placebo solution showed a significant difference to pi. it is important to stress again that this index requires a greater accumulation of biofilm on the tooth surface for clinical detection. as for dpi, greater biofilm accumulation for the conventional brush, placebo solution and chlorhexidine solution groups was present, which was not observed in the group that used the singletuft brush. this may reflect the macrostructural characteristics of the single-tuft brush, which has a small head with bristles directed towards the area to be cleaned and must necessarily be used on a single surface of the tooth at a time, thus resulting in thorough, slower and more rational brushing. these findings agree with those by ferraz et al.15, who compared mechanical biofilm control with conventional and single-tuft brushes and concluded that the singletuft brush group had a lower pi than the conventional brush groups after a 4-week period. the results obtained in the study by lee and moon26, which evaluated the effectiveness of single-tuft brushes on the buccal and lingual surfaces of molars also corroborate the findings of the present study. they concluded that difficult access areas could be best reached using this type of brush. in the present study, chlorhexidine was used as a positive control, as it is independent from an individual’s manual fig.4. mean (±sd) disclosed plaque index (dpi) for the treatments at baseline (t-0) and after 72 h (t-72) for single-tuft brush (stb), conventional toothbrush (ct), chlorhexidine (chx) and placebo solution (sp). different lowercase letters indicate significant intragroup differences over time, by student t test (p<0.05). fig.5. mean (±sd) gingival bleeding scores (dpi) for the treatments at baseline (t-0) and after 72 h (t-72) to single-tuft brush (stb), conventional toothbrush (ct), chlorexidine (chx) and placebo solution (sp). different lowercase letters indicate significant intragroup differences over time, by student t test (p<0.05). braz j oral sci. 15(2):113-118 dexterity and is regarded as a gold standard for chemical plaque control due to its bactericidal and bacteriostatic properties35-37. a concentration of 0.12% was selected based on a previous study38, which demonstrated that a lower concentration of chx was just as effective at reducing gingivitis as the traditional 0.20%. rinsing is easier than either brushing or flossing and takes less time, therefore requiring a shorter attention span. patients also tend to be more concerned with a ‘’fresh breath’’ than with plaque and gingivitis levels; consequently, patient adherence to rinsing may be higher in this case than to adequate brushing and flossing (or other cleaning dispositive)39. rapp and coworkers16 compared the bass technique (conventional brushes) using single-tuft brushes alone or in combination with dental flossing in interproximal areas. their results showed that, histomorphometrically, the bass technique and the combination of single-tuft brushes with floss yielded very similar results and slightly better than the bass-floss combination, while the use of single-tuft brushes without dental flossing showed poorer results. the findings from rapp et al.16 do not corroborate those from the present study. it is important to highlight that their analysis involved a histomorphometric evaluation of biopsies from interproximal areas after 28 days. such different methodological approaches are not directly comparable and any loose parallels can only be established based on extrapolation. franceschi and oppeman17 evaluated the interproximal cleaning capacity of dental flossing and toothpicking with that of a single-tuft brush and found that both were able to maintain adequate levels of hygiene and gingival health. under special circumstances, whenever the use of dental floss is not applicable, other methods can be applied due to their popularity, which can make plaque control more acceptable. as there was a combination of single-tuft brushes with toothpicks, the effectiveness of the former on its own cannot be verified, though it may be suggested that their results corroborate those from the present study, since the use of toothpicks alone is generally regarded as inefficient. in a randomized, single-blinded, controlled clinical trial10 performed with orthodontic patients, subjects wearing lingual fixed appliances were asked to brush with a triple head or an orthodontic toothbrush alone for one month. subsequently, they were instructed to brush in conjunction with a single-tufted toothbrush for an additional month. their teeth were professionally cleaned at baseline and one month later. similarly to the present study, the authors observed a positive effect of the single-tuft brush: when used alone, the triple-headed toothbrush seemed to have removed dental biofilm more effectively than the orthodontic toothbrush. the addition of a single-tuft brush, however, eliminated differences between groups. as far as the authors are aware, there have only been a few studies comparing single-tuft brushing with conventional brushing at the dentogingival areas. caution must be taken with the interpretation of the results from this study, to prevent a hasty notion that single-tuft brushes should be indicated as a sole method for oral hygiene. the present study did not aim to directly influence clinicians into recommending stb as a substitute to conventional mechanical biofilm control methods. additionally, lee & moon26 reported that participants in their study complained that using single-tuft brushes was rather tiresome. such drawbacks suggest that single-tuft brushes should be used as an additional tool and not as single method of oral hygiene. some limitations of this study include the short-term nature of the collected data, making it difficult to forecast long-term results. in addition, the age of the volunteers varied from 18 to 30 years, with occasional differences in motivational levels and possible inherent differences in the anatomy of their dentition, which may, to some extent, interfere with the results. longer follow-up studies should be performed to evaluate the longitudinal effects of the tested methods. in general, this study was able to demonstrate the short-term effectiveness of single-tuft brushes, though it must be stressed that they should only be used as an adjuvant strategy to conventional brushing to tackle crevicular areas of buccal and lingual surfaces in the same way as dental flossing is combined to conventional brushing to tackle interdental areas. in conclusion, the single-tuft brush tested in this study was effective at controlling short-term dental biofilm new formation at the dentogingival area. acknowledgements the authors wish to acknowledge bitufo hypermarcas for providing the materials tested in this study. references 1. loe h, theilade e, jensen sb. experimental gingivitis in man. j periodontol. 1965 may-jun;36:177-87. 2. lang np, schätzle ma, löe h. gingivitis as a risk factor in periodontal disease. j clin periodontol. 2009 jul;36 suppl 10:3-8. doi: 10.1111/j.1600051x.2009.01415.x. 3. peruzzo dc, gimenes jh, taiete t, casarin rc, feres m, sallum ea, et al. impact of smoking on experimental gingivitis. a clinical, microbiological and immunological prospective study. j periodontal res. 2016 mar 3. doi: 10.1111/jre.12363. 4. arweiler nb, henning g, reich e, netuschil l. effect of an aminefluoride-triclosan mouthrinse on plaque regrowth and biofilm vitality. j clin periodontol. 2002 apr;29(4):358-63. 5. deacon sa, glenny am, deery c, robinson pg, heanue m, walmsley ad, et al. different powered toothbrushes for plaque control and gingival health. cochrane database syst rev. 2010 dec 8;(12):cd004971. doi: 10.1002/14651858.cd004971.pub2. 6. van der weijden fa, slot de. efficacy of homecare regimens for mechanical plaque removal in managing gingivitis a meta review. j clin periodontol. 2015 apr;42 suppl 16:s77-91. doi: 10.1111/jcpe.12359. 7. gomes lk, sarmento cf, seabra fr, santos pb, pinheiro fh. randomized clinical controlled trial on the effectiveness of conventional and orthodontic manual toothbrushes. braz oral res. 2012 julaug;26(4):360-5. 8. sharma s, yeluri r, jain aa, munshi ak. effect of toothbrush grip on plaque removal during manual toothbrushing in children. j oral sci. 2012;54(2):183-90. 9. klukowska m, grender jm, conde e, ccahuana-vasquez ra, ram goyal c. a randomized clinical trial evaluating gingivitis and plaque reduction of an oscillating-rotating power brush with a new brush head with angled bristles versus a marketed sonic brush with self-adjusting technology. am j dent. 2014 aug;27(4):179-84. 10. ashkenazi m, salem nf, garon s, levin l. evaluation of orthodontic 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[clinical periodontology and implant dentstry]. 5. ed. rio de janeiro (rj): guanabara koogan; 2010. p.689. portuguese. 13. hujoel pp, cunha-cruz j, banting dw, loesche wj. dental flossing and interproximal caries: a systematic review. j dent res. 2006 apr;85(4):298-305. 14. bogren a, teles rp, torresyap g, haffajee ad, socransky ss, wennström jl. clinical and microbiologic changes associated with the combined use of a powered toothbrush and a triclosan/copolymer dentifrice: a 3-year prospective study. j periodontol. 2007 sep;78(9):1708-17. 15. ferraz c, gomes cas, gomes r, rached rsga, toleso bec. [mechanical control of bacterial plaque with conventional and single tuffed brushes]. rev assoc paul de cir dent.1987;41(4):206-9. portuguese. 16. rapp ge, toledo bec, neto cb, abi rached rsg. [effect of the intracrevicular techniques by bass and unitufo with or without dental flossing on the interproximal gingiva: histomorphological analysis in humans]. rev odontol unesp. 1997;26(2):325-36. portuguese. 17. franceschini c, oppermann rv. [effect of the intracrevicular techniques by bass and unitufo with or without dental flossing on the interproximal gingiva: histomorphological analysis in humans]. rev bras odontol 1987;44:2-7. portuguese. 18. erovic ademovski s, lingström p, winkel e, tangerman a, persson gr, renvert s. comparison of different treatment modalities for oral halitosis. acta odontol scand. 2012 may;70(3):224-33. doi: 10.3109/00016357.2011.635601. 19. ademovski se, persson gr, winkel e, tangerman a, lingström p, renvert s. the short-term treatment effects on the microbiota at the dorsum of the tongue in intra-oral halitosis patients--a randomized clinical trial. clin oral investig. 2013 mar;17(2):463-73. doi: 10.1007/ s00784-012-0728-y. 20. peruzzo dc, jandiroba pf, nogueira filho gda r. use of 0.1% chlorine dioxide to inhibit the formation of morning volatile sulphur compounds (vsc). braz oral res. 2007 jan-mar;21(1):70-4. 21. sälzer s, slot de, dörfer ce, van der weijden ga. comparison of triclosan and stannous fluoride dentifrices on parameters of gingival inflammation and plaque scores: a systematic review and meta-analysis. int j dent hyg. 2015 feb;13(1):1-17. doi: 10.1111/idh.12072. 22. ainamo j, bay i. problems and proposals for recording gingivitis and plaque. int dent j. 1975 dec;25(4):229-35. 23. re d, augusti g, battaglia d, giannì ab, augusti d. is a new sonic toothbrush more effective in plaque removal than a manual toothbrush? eur j paediatr dent. 2015 mar;16(1):13-8. 24. sharma nc, qaqish j, walters pa, grender j, biesbrock ar. a clinical evaluation of the plaque removal efficacy of five manual toothbrushes. j clin dent. 2010;21(1):8-12. 25. wilder rs, bray ks. improving periodontal outcomes: merging clinical and behavioral science. periodontol 2000. 2016 jun;71(1):65-81. doi: 10.1111/prd.12125. 26. lee dw, moon is. the plaque-removing efficacy of a single-tufted brush on the lingual and buccal surfaces of the molars. j periodontal implant sci. 2011 jun;41(3):131-4. doi: 10.5051/jpis.2011.41.3.131. 27. eberhard j, grote k, luchtefeld m, heuer w, schuett h, divchev d, et al. experimental gingivitis induces systemic inflammatory markers in young healthy individuals: a single-subject interventional study. plos one. 2013;8(2):e55265. doi: 10.1371/journal.pone.0055265. 28. friedman mt, barber pm, mordan nj, newman hn. the "plaque-free zone" in health and disease: a scanning electron microscope study. j periodontol. 1992 nov;63(11):890-6. 29. bergström j. photogrammetric registration of dental plaque accumulation in vivo. acta odontol scand. 1981;39(5):275-84. 30. quirynen m, van steenberghe d. is early plaque growth rate constant with time? j clin periodontol. 1989 may;16(5):278-83. 31. maliska an, weidlich p, gomes sc, oppermann rv. measuring early plaque formation clinically. oral health prev dent. 2006;4(4):273-8. 32. branco p, weidlich p, oppermann rv, rösing ck. early supraand subgingival plaque formation in experimental gingivitis in smokers and never-smokers. oral health prev dent. 2015;13(1):13-20. doi: 10.3290/j. ohpd.a32669. 33. pretty ia, edgar wm, smith pw, higham sm. quantification of dental plaque in the research environment. j dent. 2005 mar;33(3):193-207. 34. muniz fw, sena ks, de oliveira cc, veríssimo dm, carvalho rs, martins rs. efficacy of dental floss impregnated with chlorhexidine on reduction of supragingival biofilm: a randomized controlled trial. int j dent hyg. 2015 may;13(2):117-24. doi: 10.1111/idh.12112. 35. van strydonck da, slot de, van der velden u, van der weijden f. effect of a chlorhexidine mouthrinse on plaque, gingival inflammation and staining in gingivitis patients: a systematic review. j clin periodontol. 2012 nov;39(11):1042-55. doi: 10.1111/j.1600-051x.2012.01883.x. 36. herrera d, roldán s, santacruz i, santos s, masdevall m, sanz m. differences in antimicrobial activity of four commercial 0.12% chlorhexidine mouthrinse formulations: an in vitro contact test and salivary bacterial counts study. j clin periodontol. 2003 apr;30(4):307-14. 37. papaioannou w, vassilopoulos s, vrotsos i, margaritis v, panis v. a comparison of a new alcohol-free 0.2% chlorhexidine oral rinse to an established 0.2% chlorhexidine rinse with alcohol for the control of dental plaque accumulation. int j dent hyg. 2015 oct 9. doi: 10.1111/idh.12182. 38. segreto va, collins em, beigwanger bb, de la rosa m, issacs rl, lang np, et al. a comparison of mouthrinses containing two concentrations of chlorhexidine. j periodontol res. 1986;21(suppl):23-32. 39. arora v, tangade p, t l r, tirth a, pal s, tandon v. efficacy of dental floss and chlorhexidine mouth rinse as an adjunct to toothbrushing in removing plaque and gingival inflammation a three way cross over trial. j clin diagn res. 2014 oct;8(10):zc01-4. doi: 10.7860/jcdr/2014/8807.4943. braz j oral sci. 15(2):113-118 1http://dx.doi.org/10.20396/bjos.v18i0.8656601 volume 18 2019 e191462 original article 1 dentistry course, school of medicine and public health of bahia (bahiana). salvador-ba, brazil. 2 institute of health sciences, federal university of bahia (ufba). salvador-ba, brazil. 3 kings college london dental institute, king’s college london, london, uk. 4 school of dentistry, federal university of bahia (foufba). salvador-ba, brazil. corresponding author: luana mendonça dias school of medicine and public health of bahia (bahiana) av silveira martins, n.3386, cabula. 41150-100, salvador-ba, brazil. email: luanadias.1@hotmail.com received: november 28, 2018 accepted: may 23, 2019 can surface protection prevent damage in margins of composite resin restorations after simulated endogenous erosion? luana mendonça dias1,*, janaina emanuela damasceno1, patricia akemi nishitani shibasaki1, max josé pimenta lima2, roberto paulo correia de araújo2, richard mark foxton3, andrea nóbrega cavalcanti1,4 aim: the study investigated the effect of using surface protection agents in the adaptation of external and internal margins of restorations subjected to simulated erosion. methods: cavities with margins in dentin were prepared in bovine incisors (n=120). adhesive restorations were placed using a three-step etch&rinse adhesive system and nanofilled composite resin. the specimens were divided into four groups, according to the surface protection: negative control, topical application of fluoride (taf), resin sealant and resin-modified glass ionomer varnish (rmgi varnish). afterwards, they were divided into three sub-groups, according to the exposure to a simulated solution of gastric acid (des) (5% hcl, ph=2,2) and subsequent remineralization (re): negative control, 9 and 18 cycles of des-re. the evaluation of the tooth-restoration interface was performed on the internal and frontal images with the aid of a stereoscopic microscope (15x), and the percentage of continuous margins without adhesive failures was quantified. results: in the external margins, only those groups with surface protection using sealants (resin and glass-ionomer) did not exhibit a significant decrease in the percentage of continuous margins after the erosive challenges. after 18 cycles of des-re, the use of resin-modified glass ionomer varnish resulted in the highest percentage of continuous margins. conclusion: it was concluded that physically covering the surfaces with a sealing agent preserved the marginal adaptation of composite resin restorations exposed to endogenous erosive challenges. keywords: dentin; erosion; acid gastric. 2 dias et al. introduction erosion is defined as a localized loss of hard dental tissue caused by exposure of dental surfaces to extrinsic or intrinsic acids, unrelated to bacterial metabolism1. this demineralization by acid can have an endogenous etiology, characterized by intrinsic factors, such as gastric acid; an exogenous etiology, characterized by extrinsic factors, such as; sugar, dietary soft drinks, fruit juices, carbonated beverages; and an idiopathic etiology, when the diagnosis is not possible through anamnesis or clinical examination. the erosive challenge occurs in a progressive way and results in the loss of surface structure. it initially affects enamel followed by dentin, making the tooth brittle and more susceptible to wear by brushing or masticatory forces2, also compromising the dental anatomy and the vertical dimension of occlusion3. resin composite restorative materials represent one of the many biomaterial research successes4 and are frequently used for the rehabilitation of sequels resulting from endogenous erosion5. composite resin is considered to be less susceptible to erosive challenges when compared to dentin and other restorative materials, but the acid attack can still induces matrix degradation and loss of filler particles6. there is evidence that chemical degradation of the composite resins can occur because of the diffusion of un-reacted monomers molecules and ions7. an increase in surface roughness and softening can occur, resulting in alterations by ruptures of small fragments, as well as accumulation of bacterial plaque6. endogenous erosion is a condition, which is difficult to control, and preventive measures are necessary for the preservation of both the remaining dental structure and the surface of restorative materials used for rehabilitation. one option is to use fluoride ions in various sources, or physical barriers against the contact of acids, such as glass ionomer and resin sealants, however the most appropriate treatment has yet to be determined8-13. therefore the aim of the present study to evaluate the effectiveness of different methods of surface protection in maintaining the quality of the margins of restorations in the composite resin, submitted to acid challenges of endogenous origin, in order to extend the longevity of restorative materials in the oral environment. the hypothesis tested was that a resin-modified glass ionomer varnish, a resin sealant and a topical application of 2% neutral fluoride would have potential to prevent damages in the dentine-composite resin interface of adhesive restorations, in situations of endogenous dental erosion. materials and methods obtaining the test specimens 120 bovine incisors were obtained, cleaned with scalpel blades to remove organic debris and polished with pumice paste, using robson’s brushes in counter angle of low rotation. the teeth were visually examined for confirmation of the absence of physical damage that would compromise the study, such as discoloration, cavities, and cracks. 3 dias et al. the selected teeth were stored in 0.1% thymol solution (cromato produtos químicos ltda, diadema – sp – brazil) until the moment of use, when they were placed in saline solution. the teeth were cut with a double-sided diamond disc (n.7020, kg sorensen, cotia – sp – brazil), casting aside the root and sectioning the vestibular face in the mesial-distal and incisor-occlusal directions to obtain similar sized fragments. each fragment was individually mounted in polystyrene resin and the enamel of the vestibular face was ground until dentin was exposed using abrasive discs of descending grit (#400 e #600, vonder – odv, feira de santana – ba – brazil), in a polishing machine (arotec, s/a industria e comercio, cotia – sp – brazil) under constant cooling. the area exposed to erosion challenge was delimited using a 6mm diameter adhesive tape. the entire surface of the dentin, besides this delimitation was covered with 2 layers of nail polish in red color. after drying the varnish, the adhesive tape was removed and, at the center of the dentine delineation, 2x2 mm cylindrical cavities were made with diamond burs (n.2294, kg sorensen, cotia – sp – brazil), which were replaced every 10 cavity preparations. at the end, a limit of 2 mm around each cavity was established for the erosion challenge. the adhesive procedure was performed using a three-step etch & rinse system (scotchbond multipurpose plus, 3m-espe, sumaré – sp – brazil), according to the manufacturer’s recommendations. conditioning of the cavities was performed using 37% phosphoric acid for 15 seconds (biodinamica quim, e farm. ltda, ibiporã – pr – brazil), washing and drying with absorbent paper. the application of the primer was done actively, followed by drying with a light jet of air for 5 seconds at 10 cm. after application, the adhesive was photo-activated for 10 seconds with a light-curing unit of 1500 mw/cm² light intensity (radii plus, sdi brasil industria e comercio ltda, são paulo – sp – brazil). the composite resin restorations (filtekz350, color a3b, 3mespe, sumaré – sp – brazil) were performed in a single layer. after insertion of the composite, a polyester matrix tape was placed on the surface and photoactivation was performed. polishing of the restorations was performed 24 hours later using sandpaper discs in a descending sequence of abrasiveness (sof-lex pop-on, 3m-espe, sumaré – sp – brazil). the quality of the margin finishing was verified with a stereoscopic microscope (opton, parque industrial san josé, cotia – sp – brazil), with 15x magnification. after specimens were fabricated, they were randomly allocated into twelve experimental groups (n=10) in accordance with the method of preventing the effects of erosive challenge and the intensity of erosive simulation by gastric acid (figure 1). methods of preventing the effects of erosive challenge the methods were performed as described below: • negative control: the units belonging to this group did not suffer any changes in their restored surfaces, only maintained at relative humidity at 37oc. • topical application of fluoride (taf): on each surface, 1 ml of naf gel (neutral fluoride gel, 2%, dfl industria e comercio sa, jacarepaguá – rj – brazil) was ap4 dias et al. plied for 1 minute, then washed with distilled water in an ultrasonic bath (unique indústria and comercio ltda, são paulo – sp – brazil) for 2 minutes. afterwards, they were stored in relative humidity of 37oc. • resin-modified glass ionomer varnish (rmgi varnish): the application of the material (clinpro xt varnish, 3m-espe, st paul, mn, usa) followed the manufacturer’s recommendations. the components of the rmgi varnish were applied to a paper surface and quickly mixed for 15 seconds and applied in a thin layer on the surface with the aid of a disposable applicator. then, photoactivation was performed for 20 seconds, after which time, they were stored at relative humidity at 37oc. • resin sealant: the restored surface was conditioned with 37% phosphoric acid for 15 seconds, washed abundantly and dried with air jets, at a distance of 10 cm. a thin layer of the resin sealant (fortify, biosco inc., shaumburg usa) was actively applied to the surface of the composite resin, and then the surface was photo-activated for 10 seconds. simulation of erosion by gastric acid after applying the respective preventive method, the treated specimens were randomly distributed according to the conditions of the simulated erosion. therefore, the 4 experimental groups were subdivided into 3 subgroups (n=10), as follows: • exposure control: the cavities in this subgroup were immersed in 10 ml of distilled water at 37oc and were not subjected to any solution during execution of cycles of the other subgroups. • frequency of 9 cycles of des-re: each complete cycle consisted of immersion in 10 ml of hydrochloric acid solution (5% hcl ph=2.2) for 2 minutes at room temperature. after demineralization, the specimen was washed in 20 ml of distilled water in an ultrasonic bath for 2 minutes and immersed for 60 minutes in the remineralizing solution (1.5 mmol/l ca, 0.9 mmoll/l po4, 0.15 mol/l kcl, and 20 mmol/l tris buffer at ph 7.0)14. then, the specimen was washed in 20 ml of 120 resin composite restorations 30 control method (absence off surface protection) 30 topical application of fluoride (taf) 30 resinous sealant (fortfy) 30 resin-modified glass ionomer varnish (rmgi varnish) 10 control method (absence off surface protection) 10 9 cycles of des-re 10 18 cycles of des-re figure 1. distribution of the control method and levels of erosive challenge. 5 dias et al. distilled water in an ultrasonic washer (unique industria e comercio ltda, são paulo – sp – brazil) for 2 minutes. between the cycles, the units were stored in relative humidity, at 37oc. • frequency of 18 cycles of des-re: the specimens of this subgroup were subjected to double the frequency of cycles in order to promote a more aggressive challenge. each cycle was performed as previously described. the methodology about simulation of erosion the use in present study was based on the work of de queiroz et al.15. evaluation of marginal adaption examination of the specimens was mediated by a calibrated examiner, who recorded the percentage of continuous margins using a stereoscopic microscope, which captured the images at 15x magnification. the margin was first analyzed with the aid of coreldraw, an image processing program. the method of this evaluation was adapted from the work of bortolotto et al.16, and the captured images were classified in percentages of “continuous margins” absence of gaps, interruptions and total continuity of margin and percentage of “non-continuous margins” presence of gaps by adhesive and cohesive failure, fracture of restorative material or dental substrate. after the analysis of the external margins, the specimens were adapted to an acrylic plate with the aid of sticky wax (newwax, technew com. ind. ltda, rio de janeiro – rj – brazil) and sectioned in two parts, dividing the center of the cavity with the aid of a diamond cutting disc adapted to a precision cutter (extec corp. ®, enfield ct usa), with a diamond disk (extec corp. ®, enfield ct usa). this procedure was performed to reveal internal margins for the purpose of analyzing them, using the same image-processing program previously described. statistical analysis initially the exploratory analysis of the data was performed to verify the homogeneity of the variances and to determine if the experimental errors presented a normal distribution (parameters analysis of variance). inferential statistical analysis was performed by 2-way anova and tukey’s test for multiple comparisons. this analysis was done using sas statistical software, version 9.1, with significance level of 5%. results external margin table 1 shows the average and standard deviation of the % of external continuous margin found in the experimental groups. according to the statistical analysis of the data, a significant interaction was observed between the factors “prevention method” and “erosive challenge” (p=0.04), indicating dependence between levels of one factor on the results of the other. this statistical interaction was deployed by the tukey’s test. 6 dias et al. when erosive challenges were analyzed within each level of preventive methods, it was verified that in the groups exposed to the negative control (absence of protection) and to topical application of fluoride, there was a significant reduction in the percentage of continuous margins after 9 and 18 des-re cycles, compared to the absence of challenge. in the groups treated with the resin-modified glass ionomer varnish and resin sealants, no differences were observed in the percentage of continuous margins between the three challenges. in relation to the preventive methods at each level of erosive challenge, the percentage of continuous margins in the absence of an erosive challenge and after 9 cycles was similar between the methods of surface protection. however, after 18 cycles, the number of continuous margins protected with resin-modified glass ionomer varnish were higher than those of the negative control. the groups protected with topical application of fluoride and resin sealant presented intermediate values, with no significant differences among the groups. internal margin the % of internal data of continuous margins found in the experimental groups are shown in table 2. no significant effects were observed internally; neither between the statistical interaction among factors under study (p = 0.97), nor between the levels of each factor (preventive method p = 0.86, erosive challenge p = 0.85). table 1. mean (standard deviation) of experimental groups – external margin erosive challenge control methods erosive challenge negative control 9 cycles 18 cycles negative control 99.2 (1.7)aa 90.8 (5.1)ab 84.1 (6.7)bb topical application of fluoride 97.8 (3.3)aa 89.9 (5.4)ab 89.8 (8.8)abb resin-modified glass ionomer varnish 99.0 (3.0)aa 95.8 (2.8)aa 93.1 (7.8)aa resin sealant 94.6 (5.2)aa 92.3 (2.3)aa 89.9 (5.4)aba mediums followed by letters representing their statistical significance (anova 2-criteria/tukey, alfa=5%). capital letters compare control methods in the challenge factor’s levels and lowercase letters compare the erosive challenges in the control method’s level. table 2. mean (standard deviation) of experimental groups – internal margin erosive challenge control methods erosive challenge negative control 9 cycles 18 cycles negative control 99.4 (1.2) 99.3 (1.5) 99.4 (1.1) topical application of fluoride 99.8 (0.7) 99.4 (1.2) 99.5 (1.0) resin-modified glass ionomer varnish 99.8 (0.6) 99.6 (0.8) 99.4 (1.2) resin sealant 99.4 (1.2) 99.7 (0.8) 99.5 (1.0) mediums (standard deviation) of continuous internal margins percentage in experimental groups. 7 dias et al. discussion a higher frequency of regurgitation, which consequently increases the frequency of a lowering in the ph of the oral cavity over a period of time, may be present in individuals with gastroesophageal tract disorders. in these patients, the des-re mechanism is initiated at each episode of regurgitation, which can last between 1 and 2 minutes in the awake patient and between 15 and 20 minutes during sleeping. saliva helps to buffer the ph9. in the present work, the des-re mechanism, adapted from the methodology of austin et al.14, was used with the addition of 18 cycles of des-re, simulating moments of gastroesophageal crisis. the evaluation of the restored margins through a stereoscopic microscope can portray possible maladaptation of the composite resin17,18. this technique allows possible faults in the tooth-restoration interface to be determined and measured. however, the use of a stereoscopic microscope has limitations. the technique only demonstrates that there is maladaptation, but not the origin of the failure and whether it was due to an operative error, adhesive failure or the erosive challenge that caused material wear, as in the present study. the present study revealed signs of external degradation at the interface between tooth and restoration, even if minimal, in all the groups. the obtained images were enlarged to visualize the defects produced by the acids17, which gave confirmation that exposure to hydrochloric acid at ph=2.2 promotes irreversible changes in the external restoration margins. when teeth affected by dental erosion are restored, it is important that the adhesive system exhibits good bonding ability to enhance the longevity of the restoration. a three-step etch & rinse adhesive system was used in the present study. in previous studies, this material was more effective when compared to other systems19,20. the defects found on the eroded margins portray the hydrochloric acid’s ability to penetrate the dentin, dissolving the calcium crystals. the hybrid layer formed by the adhesive system seemed to be incapable of containing such damage19-21. thus, it is noted that even with the adhesive system of proven performance, marginal maladaptation is likely to occur due to factors such as morphology and composition of the dental tissue, the intensity of acid challenge episodes and lack of surface protection11,21. in the present study, the amount of external marginal defects increased significantly after erosive challenges in the groups without surface protection (negative control) and in those exposed to topical application of fluoride. however, there was no significant difference in the number of marginal defects when the erosive challenge was increased. therefore, it was not possible to determine whether the defects existing after 9 des-re cycles become more intense after 18 des-re cycles, since the methodology only measured the length of the defects, and not in other dimensions. further studies are therefore indicated to categorize the amplitude of the damage with more precision. the degradation of the composite subjected to acid exposure can result in softening the monomer, such as bis-gma and udma22. these compounds constitute the 8 dias et al. organic matrix of the composite resins, for example the nanofilled material tested in the present investigation. the softening of the monomers, degradation of the silane interface and the loss of fillers due to the irreversible damage caused by the acid can explain the failures found in the margins of restorations of the present study23. in the present investigation, the hypothesis that methods of surface protection could minimize and prevent damage at the adhesive interface was partially accepted. in the groups where the surface covering was a mechanical barrier using the resin and resin-modified glass ionomer varnish, no significant decrease in the percentage of continuous external margins was observed with the exposure to des-re cycles. unfortunately, the same result was not found using a topical fluoride application. there are reports that a topical application of fluoride performed with neutral sodium fluoride gel can promote the control and minimization of dental demineralization24,25. the fluoride ion, in contact with saliva, forms crystals of calcium fluoride, which can act as a physical barrier to prevent penetration of the acid in the exposed dentin25-27. some studies report that high amounts of fluoride can be retained in dentin, depending on their porosity and water content27,29,30. however, the findings of the restored outer margins protected using topical fluoride in the present investigation contradict those of previous studies. the fluoride ion did not prevent the increase of the marginal defects after 9 cycles of des-re, although it gave intermediate values after 18 cycles. it is therefore suggested that the frequency of the challenges, the dissolving ability of naf and the low ph of the gastric acid tested may be related to this result25,26. moreover, it should also be considered that clinically there would be the additional, effect of mechanical brushing forces, which would make a continual presence of fluoride deposits even more difficult to maintain29. also, the hypothesis that the acidity of some fluoride gels may influence the degradation of the surface of the restorative material should be taken into account, especially in patients with aesthetic restorations of composite resin18. in the present investigation, the percentage of continuous margins in the group with a physical barrier provided by the resin sealant did not significantly alter even after erosive challenges. this result might be related to the ability of the sealant to penetrate into irregularities present in the tooth-restoration interface, thus delaying the degradation of the margin and preventing possible marginal infiltration9,10. the use of a resin sealant could have decreased the progression of surface wear and acted as a physical barrier against acid attacks8,28. however, there are conflicting results in the literature regarding the effectiveness of resin sealants. it has been reported that sealants cannot minimize marginal infiltration because of their relatively high viscosity and incomplete wetting at the composite resin restoration interface31. the thickness of the applied material will also be low, which may also facilitate its’ vulnerability to removal because only superficial defects are filled32. it is important to note that although the percentage of continuous margins for the resin sealant remained unchanged throughout the different challenges, after 18 cycles of des-re, the result was intermediate. due to the monomeric nature and absence of filler particles in the resin sealant, it is possible that a greater absorption 9 dias et al. of water triggered the hydrolysis of the polymers7. this effect has already been documented previously, being associated with staining and pigmentation of the composite resin restoration7. the resin-modified glass ionomer varnish was also able to preserve the adhesive margins, even after the erosive challenges. however, an interesting result with this material was that it presented a significantly higher number of continuous margins after 18 des-re cycles. in dental enamel, a resin-modified glass ionomer varnish exhibits good remineralization and demineralization control11,13,25,28, probably due to the mechanism of long-term release of calcium, phosphorus and fluoride, adhesion to enamel and dentine and consequent sealing ability6,11,25. the glass-ionomer sealant modified by resin has important properties that enable maintenance of the dental organic matrix and conservation of the remaining substrate after the erosion process11,25,28. the manufacturer of clinpro xt varnish gives its composition as; polyalkenoic acid patented and modified by methacrylate, 2-hydroxyethyl methacrylate (hema), water, calcium glycerophosphate, bis-gma and fluoraluminiosilicate glass. the instant release of fluoride from the glass-ionomer modified by composite resin is triggered by chemical bonds carried out on the surface of the particle of a compound called fluoroaminosilicate, also described by the manufacturer. calcium glycerophosphate is present throughout the lifespan of the material, forming a reservoir until its’ complete disappearance from the surfaces of composite resin and tooth28,33,34. thus, there is a need for more in vivo studies on its behavior in clinical settings. the polyalkenoic acid present in the varnish can also occlude dentin tubules by the formation of an amorphous mineral similar to apatite, providing less exposure of the organic demineralized organic matrix (dom) to acid degradation13. therefore a sealant provides a physical and mechanical reduction in the diameter of the canaliculi that protect the dentin from future acid attacks, through this acid-resistant layer formed within the substrate12,34. there is an important relationship between the preservation of the demineralized organic matrix layer and the progression of dental erosion. the proteolytic degradation of the dom results in an increase in the rate of erosion, since this layer has the capacity of to buffer acids, which prevents the aggravation of the demineralization29. the resin-modified glass ionomer varnish component of the barrier material may also have the ability to penetrate into the organic matrix of the dental substrate, promoting stabilization of the collagen against the acidic environment25,35. when analyzing the condition of the margin of the restorations, the loss of structure from the composite resin and the dentin must be taken into account, since they maybe affected differently by the exposure to acid, as they react differently to the acid18. in this sense, it is believed that, in the present study, the good results obtained with the glass-ionomer sealant after 18 cycles of des-re are due, mainly to the preservation of the dental structure in the eroded margin. however, further studies regarding the use of this material should be performed. considering the limitations of the present in vitro study, it can be concluded that, in the face of simulated endogenous erosion, resin and resin-modified glass ionomer var10 dias et al. nish have the potential to prevent damage to the margins of composite resin adhesive restorations. however, the glass ionomer varnish material seems to be less affected by the erosive challenge intensity, which is relatively important in the control of dental erosion in patients with gastroesophageal disorders. acknowledgments authors thank fapesb, grant red 019/2013 and 3m-espe for material support for this investigation. conflicts of interest statement the authors report no conflicts of interest in this work. references 1. schwendicke f, felstehausen g, carey c, dörfer c. comparison of four methods to assess erosive substance loss of dentin. plos one. 2014 sep 17;9(9):e108064. doi: 10.1371/journal.pone.0108064. 2. caneppele tmf, jeronymo rdi, di nicoló, r, araújo mamd, soares les. in vitro assessment of dentin erosion after immersion in acidic beverages: surface profile analysis and energy-dispersive x-ray fluorescence spectrometry study. braz dent j. 2012;23(4):373-8. 3. scaramucci t, borges ab, lippert f, zero dt, aoki iv, hara at. anti-erosive properties of solutions containing fluoride and different film forming agents. j dent. 2015 apr;43(4):458-65. doi: 10.1016/j.jdent.2015.01.007. 4. cramer nb, stansbury jw, bowman, cn. recent advances and developments in composite dental restorative materials. j dent res. 2011 apr;90(4):402-16. doi: 10.1177/0022034510381263. 5. bartlett d, sundaram g, moazzez r. trial of protective effect of fissure sealants, in vivo, on the palatal surfaces of anterior teeth, in patients suffering from erosion. j dent. 2011 jan;39(1):26-9. doi: 10.1016/j.jdent.2010.09.007. 6. yamazaki h, litman a, margolis hc. effect of fluoride on artificial caries lesion progression and repair in human enamel: regulation of mineral deposition and dissolution under in vivo-like conditions. arch oral biol. 2007 feb;52(2):110-20. 7. kina m, lopes gc, monteiro junior s. [influence of surface sealant on microleakage and staining of composite resin restorations]. rpg rev pos-grad. 2011;18(3):148-53. portuguese. 8. oliveira gc, boteon ap, ionta fq, moretto mj, honório hm, wang l, et al. in vitro effects of resin infiltration on enamel erosion inhibition oper dent. oper dent. 2015 sep-oct;40(5):492-502. doi: 10.2341/14-162-l. 9. ranjitkar s, kaidonis ja, smales rj. gastroesophageal reflux disease and tooth erosion. int j dent. 2012;2012:479850. doi: 10.1155/2012/479850. 10. prakki a, ribeiro lwj, cilli r, mondelli rfl. assessing the tooth-restoration interface wear resistance of two cementation techniques: effect of a surface sealant. oper dent. 2005 nov-dec;30(6):739-46. 11. sohn s, yi k, son hh, chang j. caries-preventive activity of fluoride-containing resin-based desensitizers. oper dent. 2012 may-jun;37(3):306-15. doi: 10.2341/11-007-l. 12. suge t, ishikawa k, kawasaki a, suzuki k, matsuo t, noiri y et al. calcium phosphate precipitation method for the treatment of dentin hypersensitivity. am j dent. 2002 aug;15(4):220-6. 11 dias et al. 13. zhou sl, zhou j, watanabe s, watanabe k, wen ly, xuan k. in vitro study of the effects of fluoride-releasing dental materials on remineralization in an enamel erosion model. j dent. 2012 mar;40(3):255-63. doi: 10.1016/j.jdent.2011.12.016. 14. austin rs, stenhagen ks, hove lh, dunne s, moazzez r, bartelett dw, et al. a qualitative and quantitative investigation into the effect of fluoride formulations on enamel erosion and erosion– abrasion in vitro. j dent. 2011 oct;39(10):648-55. doi: 10.1016/j.jdent.2011.07.006. 15. queiroz mmv, shibasaki pan, lima mjp, araújo rpc, foxton rm, cavalcanti an. effect of erosion and methods for its control on the surface roughness of composite resin. rev odonto cienc 2017;32(2):88-93. 16. bortolotto t, doudou w, kunzelmann kh, krejci i. the competition between enamel and dentin adhesion within a cavity: an in vitro evaluation of class v restorations. clin oral investig. 2012 aug;16(4):1125-35. doi: 10.1007/s00784-011-0623-y. 17. soares le, de oliveira r, nahórny s, santo am, martin aa. micro energy-dispersive x-ray fluoresence mapping of enamel and dental materials after chemical erosion. microsc microanal. 2012 oct;18(5):1112-7. doi: 10.1017/s1431927612001535. 18. yu h, buchalla w, cheng h, wiegand a, attin t. topical fluoride application is able to reduce acid susceptibility of restorative material. dent mat j. 2012;31(3):433-42. 19. lenzi tl, raggio dp, soares fzm, rocha ro. bonding performance of a multimode adhesive to artificially-induced caries-affected primary dentin. j adhes dent. 2015 apr;17(2):125-31. doi: 10.3290/j.jad.a34058. 20. muñoz ma, luque i, hass v, reis a, loguercio ad, bombarda nhc. immediate bonding properties of universal adhesives to dentine. j dent. 2013 may;41(5):404-11. doi: 10.1016/j.jdent.2013.03.001. 21. flury s, koch t, peutzfeldt a, lussi a, ganss c. the effect of a tin-containing fluoride mouth rinse on the bond between resin composite and erosively demineralised dentin. clin oral investig. 2013 jan;17(1):217-25. doi: 10.1007/s00784-012-0697-1. 22. honório hm, rios d, francisconi lf, magalhães ac, machado ma, buzalaf ma. effect of prolonged erosive ph cycling on different restorative materials. j oral rehabil. 2008 dec;35(12):947-53. doi: 10.1111/j.1365-2842.2008.01856.x. 23. kaur s, makkar s, kumar r, pasricha s, gupta p. comparative evaluation of surface properties of enamel and different esthetic restorative materials under erosive and abrasive challenges: an in vitro study. indian j dent. 2015 oct-dec;6(4):172-80. doi: 10.4103/0975-962x.165047. 24. elkassas d, arafa a. remineralizing efficacy of different calcium-phosphate and fluoride based delivery vehicles on artificial caries like enamel lesions. j dent. 2014 apr;42(4):466-74. doi: 10.1016/j.jdent.2013.12.017. 25. martins, vl; ramos, rvc; lima, mjp; de araújo, rpc; cavalcanti, an. effect of surface protection on the permeability of eroded dentin. j conserv dent. 2018 jan-feb;21(1):16-20. doi: 10.4103/jcd.jcd_281_16. 26. algarni aa, lippert f, hara at. efficacy of stannous, fluoride and their their combination in dentin erosion prevention in vitro. braz oral res. 2015;29. pii: s1806-83242015000100276. doi: 10.1590/1807-3107bor-2015.vol29.0081. 27. lussi a, carvalho ts. the future of fluorides and other protective agents in erosion prevention. caries res. 2015;49 suppl 1:18-29. doi: 10.1159/000380886. 28. owens bm, johnson ww. effect of new generation surface sealants on the marginal permeability of class v resin composite restorations. oper dent. 2006 jul-aug;31(4):481-8. 29. ganss c ,klimek v,.schürmann a. effects of two fluoridation measures on erosion progression in human enamel and dentine in situ. caries res. 2004 nov-dec;38(6):561-6. 12 dias et al. 30. ganss c, klimek j, schäffer u, spall t. effectiveness of two fluoridation measures on erosion progression in human enamel and dentine in vitro. caries res. 2001 sep-oct;35(5):325-30. 31. takeuchi cy, flores vo, dibb rp, panzeri h, lara eh, dinelli w. assessing the surface roughness of a posterior resin composite: effect of surfasse sealing. oper dent. 2003 may-jun;28(3):281-6. 32. bertrand mf, leforestier e, muller m, lupi-pégurier l, bolla m. effect of surface penetrating sealant on surface texture and microhardness of composite resins. j biomed mater res. 2000;53(6):658-63. 33. orchardson r, gillam d. managing dentiny hypersensitivy. j am dent assoc. 2006 jul;137(7):990-8; quiz 1028-9. 34. chiga s, toro cv, lepri tp, turssi cp, colucci v, corona sa, et al. combined effect of fluoride varnish to er:yag or nd:yag laser on permeability of eroded root dentine. arch oral biol. 2016 apr;64:24-7. doi: 10.1016/j.archoralbio.2015.12.006. 35. zimmerli b, munck j, lussi a, lambrechts p, meerbeek bv. long-term bonding to eroded dentin requires superficial bur preparation. clin oral investig. 2012 oct;16(5):1451-61. doi: 10.1007/s00784-011-0650-8. 1 volume 16 2017 e17069 original article 1 dds, master student, school of dentistry and research institute, são leopoldo mandic, campinas, são paulo, brazil – email: ferfernandes89@hotmail.com 2 phd, professor, school of dentistry and research institute, são leopoldo mandic, campinas, são paulo, brazil – email: cecilia.turssi@ gmail.com; biagomes@yahoo.com; rbasting@yahoo.com 3 phd, professor, school of dentistry and research institute, são leopoldo mandic, campinas, são paulo, brazil – flbamaral@gmail.com corresponding author: prof. dr. flávia lucisano botelho do amaral rua josé rocha junqueira, 13, sala 24 ponte preta, campinas, sp – brazil zipcode 13045-775 tel (fax): 55 (19) 32113600 tel: 55 (19) 32113649 received: june 26, 2017 accepted: august 22, 2017 whitening mouthwash containing hydrogen peroxide decreases enamel microhardness in vitro fernando pelegrim fernandes1, cecília pedroso turssi2, fabiana mantovani gomes frança2, roberta tarkany basting2, flávia lucisano botelho do amaral3* aim: to assess the effect of a mouthwash containing hydrogen peroxide (hp) on knoop microhardness (kmh) of bovine enamel. methods: fifty-one enamel slabs were polished and divided into groups (n=17), according to the product used during 28 days: hp – mouthwash containing 1,5% of hp (4 min, once/day); cp 10% carbamide peroxide gel (2 hours/day); as no treatment (kept in artificial saliva (as). each fragment was submitted to kmh test (three indentations/fragment, with a 50 g load for 5 sec) four times: before (baseline); during (14 and 28 days) and after (7 days immerged in as) the bleaching treatment. the data were submitted to repeated-measures two-way anova (α=0.05). results: there was no effect of the interaction between the time and treatment factors (p=0.327). no significant effect was observed from the time factor (p = 0.054). the factor treatment showed significant effect (p =0.002). regardless of time, the kmh of the enamel submitted to hp was lower than the value observed with the use of cp, which did not differ significantly from the control group (as). conclusion: although there was a trend of decreasing enamel microhardness over time, only the mouthwash containing hydrogen peroxide had a significant effect. keywords: tooth bleaching; hydrogen peroxide; hardness tests. http://dx.doi.org/10.20396/bjos.v16i0.8650504 mailto:ferfernandes89@hotmail.com mailto:cecilia.turssi@gmail.com mailto:cecilia.turssi@gmail.com mailto:biagomes@yahoo.com mailto:rbasting@yahoo.com mailto:flbamaral@gmail.com 2 fernandes et al. introduction one of the most important factors for the aesthetic balance of the smile in our society is the color of our teeth, for being quickly noticed even before many other aesthetic anomalies. tooth discoloration or stains can be located on the surface of dental enamel (extrinsic stains) or in dental structure (intrinsic stains)1,2. extrinsic stains are obtained after the dental eruption in the oral cavity, from food or oral use products with great staining potential such as tea, coffee, tobacco, red wine, cola based beverages, etc. intrinsic stains have a multifactorial etiology, both pre-and post-eruptive3. in vital teeth, discoloration can be natural (teeth with yellow or gray shades), caused by the ingestion of tetracycline or fluoride, or defects of congenital formations (amelogenesis and dentinogenesis imperfecta)4,5. dental bleaching is being a highly requested procedure by patients seeking cosmetic dentistry resources as a result of a poor aesthetic, caused by dental discoloration6,7. hydrogen peroxide has been used for dental bleaching in the late 1800s, firstly with the aim of removing pigments from non vital and later from vital teeth8. hydrogen peroxide is a vehicle of oxygen radical, which promotes oxidation and reduction of pigments. these pigments are fractionated in to smaller molecular chains, being entirely or partially removed from the dental structure by diffusion9. for vital teeth, the techniques are the in-office technique, the over-the-counter bleaching, and the home-use nightguard vital bleaching technique, being each technique driven by different peroxide concentrations, time and mode of whitening product application. the home-use technique consists in applying a 10 or 16% carbamide peroxide gel that can be applied every night for 6 to 8 hours, or during the day, for one or 2 hours, with less tooth sensitivity within the decreased application time10. in the in-office technique the soft tissues are protected, and a hydrogen peroxide gel in a higher concentration is applied several times in a short period of time over the dental surface8. the over-the-counter technique uses products that are bought in supermarkets or drugstores, such as bleaching toothpastes and mouthwashes, aimed to whiten teeth in short periods of time with a low cost. these mouthwashes have in addition to water, antimicrobials, salts, and sometimes alcohol, and also contain hydrogen peroxide in its composition11, but in lower peroxide concentrations (around 1,5%) than those observed in in-office and home-use gels. although the effectiveness of over-the-counter products in dental bleaching has been reposted, it is less than the one obtained from home-use carbamide peroxide at 10%12. besides this, controversies are known about the effects of bleaching agents on the microhardness of dental structures, especially the enamel13. the over-the-counter products has been known to reduce the hardness of dental enamel14 and resin composites15 when used for 14 days, depending on the trade mark being used, and whether or not the product contains hydrogen peroxide (active component). the literature is still scarce to affirm the relation of mouthwashes recently launched on the market, which contains the bleaching agent (hydrogen peroxide) with the possible loss of minerals, and consequently a decrease of enamel microhardness. 3 fernandes et al. this issue is especially important if one considers that patients can use this product without the supervision of a professional and that manufacturer indicate that they can be used for 28 consecutive days, a relatively long time. considering that peroxide-containing mouthwashes are available for purchase without the need of a dentist recommendation and that they can affect enamel structure in someway, it is necessary to conduct experimental studies to verify this possible association. thus, the aim of this study was to monitor the effect of a mouthwash containing 1.5% of hydrogen peroxide, compared to 10% carbamide peroxide gel, by means of knoop microhardness test in bovine enamel, in the treatment periods (for 28 days) and post-bleaching (7 days). the null hypothesis was that neither bleaching agents nor treatment periods would affect enamel knoop microhardness. materials and methods ethical aspects this study was approved by the animal research ethical committee (protocol #2013/0143). experimental design the factors under study were the bleaching agents, at three levels, and times of treatment, at four levels: 1. treatment agents: cp – 10% carbamide peroxide. hp – mouthwash containing 1.5% hydrogen peroxide. as artificial saliva 2. evaluation times: before (baseline), during (14 days) and after (28 days) the bleaching treatment, 7 days of posttreatment. the experimental units were composed of 51 enamel slabs randomly distributed into the three levels of treatment agents (n=17). the response variables were knoop microhardness (kmh). analysis in each period was performed in the same specimen, which consisted of a block. the bleaching agents, as well as the artificial saliva, are shown in table 1. preparation of dental slabs in this experiment, 51 bovine incisors stored in thymol (0.1%, ph 7.0) were used. the teeth were debrided with scalpel blades and periodontal curettes and had their roots separate from the dental crowns using a diamond disc of high concentration (extec corp, enfield, ct, eua) in a precision cutter (isomet 1000, buehler lake bluff, illinois, eua). longitudinal sections were cut to obtain 3mm x 3mm enamel slabs, and those with stains or cracks were excluded after visual observation under a stereomicroscope loupe (ek3s3, são paulo, são paulo, brazil) at 30x magnification. the enamel slabs were embedded in polyester resin (maxi rubber ind quím ltda. diadema/sp/brazil) in 2.0-cm diameter polyvinyl chloride (pvc) molds, leaving the exter4 fernandes et al. nal surface of dental uncovered by resin. after 24 hours, the specimens were removed from the molds and flattened in a pneumatic polishing machine (ecomet/automet 250, buehler, lake bluff, ii, eua) with decreasing granulations (400, 600 and 1.200) (arotec s/a ind e comércio são paulo/sp/brazil) of abrasive paper under water cooling, and cleaned in a ultrasonic washer (unique ind. e com, de prod. elet. ltda model: usc 1400 são paulo/sp/brazil) after each granulation and polished with diamond paste (arotec granulation: 6µm / 3µm /1µm / ¹/4µm são paulo/sp/brazil) on felt discs (arotec são paulo/sp/brazil). before (baseline), during (14 and 28 days) and 7 days after the treatment, all specimens were submitted to the microhardness tests, using a microhardness tester (pantec digital microhardness tester hvs-1000/panambra, são paulo, são paulo, brazil) with a knoop penetrator, performing three indentations with a 50-g load for 5 seconds. as kmh is a non-destructive testing, analysis in each period was performed in the same specimen. treatment agent procedures the treatment agents used in this study were described in table 1, according to composition, manufacturer and ph values. group cp: 10% carbamide peroxide gel (opalescense pf, brazil´s ultradent, indaiatuba, são paulo, brazil), simulating the home-use bleaching, applying 0.02 ml of the product over each specimen for two hours/day12. then they were rinsed with distilled water and stored in 5 ml of artificial saliva, and kept in a bacteriological stove (odontobrás ind. e com. equip. med. odont. ltda model: ecb 1.3 digital, ribeirão preto/sp/brazil), at 37°c. this procedure was repeated for four weeks. hypodermic syringes were used for the accuracy of quantity of gel over each fragment. group hp: the specimens were immersed in the mouthwash containing 1.5% hydrogen peroxide (colgate plax whitening, colgate palmolive ind e comércio ltda. s.b. campo/sp, brazil), for four minutes, once a day, each specimen were submerged in table 1. products used in the experiment. materials manufacturer batch number composition ph opalescence pf 10% cp ultradent dental products ltda. indaiatuba/spbrazil b95zn 10% carbamide peroxide, 0.5% potassium nitrate and 0.11% fluoride ions 6.8 colgate plax whitening hp colgate palmolive s.b. campo/sp, brazil br122a br1210 br121a water, sorbitol, ethyl alcohol, 1.5% hydrogen peroxide, poloxamer 338, polysorbate 20, methyl silicate, menthol, sodium saccharin, cl 42090 3.4* artificial saliva described by featherstone et al.18 and modified by serra & cury19 as --1.5mm ca, 0.9 mm p, buffer tris 20 mm, 150 mm kci 7.0 * measured in laboratory, in triplicate. 5 fernandes et al. 4 ml of the solution and subjected to agitation on an agitator platform (cientec®, ct 158) simulating a mouth rinse, and rinsed with distilled water after the procedure and immersed in 5 ml of artificial saliva, kept in a bacteriological stove (odontobrás ind. e com. equip. med. odont. ltda model: ecb 1.3 digital, ribeirão preto/sp/brazil), at 37°c. this procedure was repeated for four weeks. group as: no bleaching agent was applied, the specimens were kept in 5 ml of artificial saliva that were renewed in every two days and the specimens were rinsed with distilled water and kept in a bacteriological stove (odontobrás ind. e com. equip. med. odont. ltda model: ecb 1.3 digital, ribeirão preto, sp, brazil), at 37°c. there was no evident calculus or material over specimens stored in saliva for 28 days. post-treatment period after the treatment, the fragments were kept in their individual receptacles with 5 ml of artificial saliva, in a bacteriological stove (odontobrás ind. e com. equip. med. odont. ltda model: ecb 1.3 digital, ribeirão preto/sp/brazil), at 37°c for 7 days to evaluate the posttreatment period and a possible remineralizing effect of this solution. the solution was changed every two days. knoop microhardness was also measured after 7 days of post-treatment. statistical analysis after checking normality (kolmogorov-smirnov; p>0.05) and homoscedasticity (levene; p>0.05) of data, the repeated measures two-way analysis of variance was performed, followed by tukey’s test for multiple comparisons. statistical calculations were performed with spss 20 (spss inc., chicago, il, usa). the significance level was set at 5%. results table 2 shows the averages and standard deviations of knoop microhardness for the tested groups, in different times. the two-way analysis of variance for repeated measurements showed no significant interaction between the factors under study (p =0.327). no significant effect was observed from the time factor (p = 0.054). the treatment factor showed significant effect (p =0.002). tukey’s test revealed that regardless of time, the microhardness mean of the enamel submitted to hp treatment was lower than that observed with the use of cp, which did not differ significantly from table 2. averages and standard deviations of knoop microhardness for the groups tested, in different times. baseline 14 days of treatment 28 days of treatment 07 days of post-treatment grand mean cp 351(43) 331(28) 333(26) 335(41) 337(35)a hp 317(55) 279(50) 251(46) 293(41) 285(55)b saliva 338(57) 329(46) 316(44) 325(42) 327(47)a grand mean 335(53)a 313(48)a 300(52)a 318(47)a grand means followed by distinct letters statistically differ between each other (lowercase compare times and uppercase compare treatments) 6 fernandes et al. the control group (artificial saliva). figure 1 illustrates a line chart of knoop microhardness of experimental groups over time. it can be observed that although there was a trend of decrease in microhardness within time, no statistical difference was observed among the different periods of evaluation. discussion the present in vitro study assessed the effect of a mouthwash containing 1.5% of hydrogen peroxide as an active component in its composition on bovine enamel surface microhardness and compared with 10% carbamide peroxide. the results obtained by microhardness analysis showed that the specimens of bovine enamel submitted to whitening mouthwash had smaller microhardness values compared to those submitted to 10% carbamide peroxide gel. therefore, the null hypothesis was rejected. the ph of the products used were measured and showed the result of 6.8 to the whitening gel and 3.4 for the whitening mouthwash. it can be assumed that the low ph of the whitening mouthwash may have acted on the demineralization of enamel surface favoring the decrease of microhardness, since the ph value is less than the enamel critical ph (5.5). in fact, lima et al.16 (2013) demonstrated, by 3d scanning eletronic microscopy, the erosive potential of plax whitening associated with toothbrushing. these results are similar to previous studies which also verified reduction of dental enamel microhardness14 and resin composite15 with the use of whitening mouthwashes. it would be interesting to investigate the effect of such products on the morphology of dental enamel by means of scanning electron microscopy and atomic force microscopy, however, literature is still scarce in these evaluations and should be considered in further studies. also, the manufacturer of the mouthwash recommends that the product should be used for 1 minute before and 1 minute after brushing the teeth, twice a day, totaling 4 minutes a day. in the present research, brushing was not considered so that only k no op m ic ro ha rd ne ss baseline 14 days 28 days 7 days post-treatment cp hp saliva 400 350 300 250 200 0 150 100 50 figure 1. chart representing knoop microhardness of experimental groups over time. 7 fernandes et al. the whitening mouthwash effect was observed. it is speculated that the application of mouthwashes in association with brushing could have brought different results, as the literature has demonstrated that this association may cause enamel loss16. on the other hand, the prolonged immersion time (4 consecutives minutes) can also have exacerbated the results in a more significative manner than fractioning the immersions two times a day, with storage of specimens in saliva between cycles. having this in mind, potgieter et al. (2014)17 demonstrated no statistical reduction in enamel microhardness when plax whitening was applied twice a day, for 1 minute in each immersion, although they did not perform toothbrushing. on the other hand, the use of the whitening gel containing 10% of carbamide peroxide did not influence significantly on the bovine enamel microhardness, a fact that may be related to the presence of fluoride ions in its composition, in addition to the ph value, greater than the critical ph for demineralization of dental enamel. in fact, cavalli et al.18 (2010) showed that the presence of fluoride in the composition of the carbamide peroxide based whitening gel minimized the loss of ions of the dental surface. in a literature review of attin et al.19 (2009) it was observed that 49% of the in vitro studies reviewed did not show microhardness reduction over time with the use of home-use bleaching agents. previous experimental studies had similar results to those found in the present study20,21. perhaps, the effects of carbamide peroxide gel on the dental structures may also be related to the time of gel in contact with dental structure. the manufacturer indicates the use of 10% carbamide peroxide gel for 8 hours, but, in the present study, the protocol of 2 hours of application was choosed because this time prooved to sufficiently bleach enamel12, reduce tooth sensitivity10, while does not affect the concentration of enamel minerals, assessed with the technique of enamel microbiopsy22. thus, if the manufacturer’s instructions were strictly followed, different results could be achieved. the results of this study also demonstrated that, although there was a tendency of decrease in microhardness values for the time of 28 days, there was no significant difference in the evaluated times, when considered in conjunction with the bleaching treatments. this may have been attributed due to the presence of artificial saliva in all groups, which may have leveled the results of microhardness for being a ion supersaturated solution23 that favors the absorption and precipitation of salivary components, such as calcium and phosphate24. although it has been reported the effectiveness of otc products to whiten dental enamel12,25 the present study demonstrated reduction in bovine dental enamel microhardness after using the mouthwash containing 1.5% of hydrogen peroxide, showing that such product should be used with caution, especially if considered the carcinogenic potential that such products may have26. still, literature is scarce regarding the effects of such products on the mineral loss of tooth structure, requiring, therefore, more studies on this subject, mainly in situ studies and clinical trials. also, the use of otc whitening mouthwashes in conjunction with toothbrushing should be evaluated in further studies. this study concluded that although there was a trend of decreasing microhardness values over time, only the whitening mouthwash containing hydrogen peroxide had a significant effect. 8 fernandes et al. references 1. alqahtani mq. tooth-bleaching procedures and their controversial effects: a literature review. saudi dent j. 2014 apr;26(2):33-46. doi: 10.1016/j.sdentj.2014.02.002. 2. nathoo as. the chemistry and mechanisms of extrinsic and intrinsic discoloration. j am dent assoc. 1997 apr;128 suppl:6s-10s. 3. price rb, loney rw, doyle mg, moulding mb. an evaluation of a technique to remove stains from teeth using microabrasion. j am dent assoc. 2003 aug;134(8):1066-71. 4. watts a, addy m. tooth discolouration and staining: a review of the literature. br dent j. 2001 mar 24;190(6):309-16. 5. sundfeld rh, croll tp, briso al, alexandre rs, sundfeld neto d. considerations about enamel microabrasion after 18 years. am j dent. 2007 apr;20(2):67-72. 6. sheoran n, garg s, damle sg, dhindsa a, opal s, gupta s. esthetic management of developmental enamel opacities in young permanent maxillary incisors with two microabrasion techniques a split mouth study. j esthet restor dent. 2014 sep-oct;26(5):345-52. doi: 10.1111/jerd.12096. 7. parreiras so, vianna p, kossatz s, loguercio ad, reis a. effects of light activated in-office bleaching on permeability, microhardness, and mineral content of enamel. oper dent. 2014 sep-oct;39(5):e225-30. doi: 10.2341/13-031-l. 8. haywood vb. history, safety, and effectiveness of current bleaching techniques and applications of the nightguard vital bleaching technique. quintessence int. 1992 jul;23(7):471-88. 9. eimar h, siciliano r, abdallah mn, nader sa, amin wm, martinez pp, et al. hydrogen peroxide whitens teeth by oxidizing the organic structure. j dent. 2012 dec;40 suppl 2:e25-33. doi: 10.1016/j.jdent.2012.08.008. 10. cardoso pc, reis a, loguercio a, vieira lc, baratieri ln. clinical effectiveness and tooth sensitivity associated with different bleaching times for a 10 percent carbamide peroxide gel. j am dent assoc. 2010 oct;141(10):1213-20. 11. moran jm. chemical plaque control-prevention for the masses. periodontol 2000. 1997 oct;15:109-17. 12. jaime im, frança fm, basting rt, turssi cp, amaral fl. efficacy of hydrogen-peroxide-based mouthwash in altering enamel color. am j dent. 2014 feb;27(1):47-50. 13. borges bc, borges js, de melo cd, pinheiro iv, santos aj, braz r et al. efficacy of a novel at-home bleaching technique with carbamide peroxides modified by cpp-acp and its effect on the microhardness of bleached enamel. oper dent. 2011 sep-oct;36(5):521-8. doi: 10.2341/11-013-l. 14. majeed a, grobler sr, moola mh, oberholzer tg. effect of four over-the-counter tooth-whitening products on enamel microhardness. sadj. 2011 oct;66(9):412-5. 15. miranda dde a, bertoldo ce, aguiar fh, lima da, lovadino jr. effects of mouthwashes on knoop hardness and surface roughness of dental composites after different immersion times. braz oral res. 2011 mar-apr;25(2):168-73.. 16. lima jp, melo ma, passos vf, braga cl, rodrigues lk, santiago sl. dentin erosion by whitening mouthwash associated to toothbrushing abrasion: a focus variation 3d scanning microscopy study. microsc res tech. 2013 sep;76(9):904-8. doi: 10.1002/jemt.22246. 17. potgieter e, osman y, grobler sr. the effect of three whitening oral rinses on enamel micro-hardness. sadj. 2014 may;69(4):152, 154-6. 18. cavalli v, rodrigues lk, paes-leme af, brancalion ml, arruda ma, berger sb et al. effects of bleaching agents containing fluoride and calcium on human enamel. quintessence int. 2010 sep;41(8):e157-65. 19. attin t, schmidlin pr, wegehaupt f, wiegand a. influence of study design on the impact of bleaching agents on dental enamel microhardness: a review. dent mater. 2009 feb;25(2):143-57. doi: 10.1016/j.dental.2008.05.010. http://www.ncbi.nlm.nih.gov/pubmed?term=watts a%5bauthor%5d&cauthor=true&cauthor_uid=11325156 http://www.ncbi.nlm.nih.gov/pubmed?term=addy m%5bauthor%5d&cauthor=true&cauthor_uid=11325156 http://www.ncbi.nlm.nih.gov/pubmed?term=parreiras so%5bauthor%5d&cauthor=true&cauthor_uid=24815914 http://www.ncbi.nlm.nih.gov/pubmed?term=vianna p%5bauthor%5d&cauthor=true&cauthor_uid=24815914 http://www.ncbi.nlm.nih.gov/pubmed?term=kossatz s%5bauthor%5d&cauthor=true&cauthor_uid=24815914 http://www.ncbi.nlm.nih.gov/pubmed?term=loguercio ad%5bauthor%5d&cauthor=true&cauthor_uid=24815914 http://www.ncbi.nlm.nih.gov/pubmed?term=reis a%5bauthor%5d&cauthor=true&cauthor_uid=24815914 http://www.ncbi.nlm.nih.gov/pubmed?term=jaime im%5bauthor%5d&cauthor=true&cauthor_uid=24902405 http://www.ncbi.nlm.nih.gov/pubmed?term=fran%c3%a7a fm%5bauthor%5d&cauthor=true&cauthor_uid=24902405 http://www.ncbi.nlm.nih.gov/pubmed?term=basting rt%5bauthor%5d&cauthor=true&cauthor_uid=24902405 http://www.ncbi.nlm.nih.gov/pubmed?term=turssi cp%5bauthor%5d&cauthor=true&cauthor_uid=24902405 http://www.ncbi.nlm.nih.gov/pubmed?term=amaral fl%5bauthor%5d&cauthor=true&cauthor_uid=24902405 http://www.ncbi.nlm.nih.gov/pubmed?term=oberholzer tg%5bauthor%5d&cauthor=true&cauthor_uid=23193870 http://www.ncbi.nlm.nih.gov/pubmed?term=miranda dde a%5bauthor%5d&cauthor=true&cauthor_uid=21537643 http://www.ncbi.nlm.nih.gov/pubmed?term=lima da%5bauthor%5d&cauthor=true&cauthor_uid=21537643 http://www.ncbi.nlm.nih.gov/pubmed?term=lovadino jr%5bauthor%5d&cauthor=true&cauthor_uid=21537643 http://www.ncbi.nlm.nih.gov/pubmed/20657849 http://www.ncbi.nlm.nih.gov/pubmed/20657849 9 fernandes et al. 20. maia e, baratieri ln, caldeira de andrada ma, monteiro s jr, vieira lc. the influence of two home-applied bleaching agents on enamel microhardness: an in situ study. j dent. 2008 jan;36(1):2-7. 21. abouassi t, wolkewitz m, hahn p. effect of carbamide peroxide and hydrogen peroxide on enamel surface: an in vitro study. clin oral investig. 2011 oct;15(5):673-80. doi: 10.1007/s00784-010-0439-1. 22. amaral fl, sasaki rt, da silva tc, frança fm, flório fm, basting rt. the effects of home-use and in-office bleaching treatments on calcium and phosphorus concentrations in tooth enamel: an in vivo study. j am dent assoc. 2012 jun;143(6):580-6. 23. freitas pm, turssi cp, hara at, serra mc. dentin microhardness during and after whitening treatments. quintessence int. 2004 may;35(5):411-7. 24. araujo fde o, baratieri ln, araújo e. in situ study of in-office bleaching procedures using light sources on human enamel microhardness. oper dent. 2010 mar-apr;35(2):139-46. doi: 10.2341/08-033-c. 25. oliveira j, sarlo rs, bresciani e, caneppele t. whitening efficacy of whitening mouth rinses used alone or in conjunction with carbamide peroxide home whitening. oper dent. 2017 may/jun;42(3):319-326. doi: 10.2341/15-361-l. 26. carlin v, matsumoto ma, saraiva pp, artioli a, oshima ct, ribeiro da. cytogenetic damage induced by mouthrinses formulations in vivo and in vitro. clin oral investig. 2012 jun;16(3):813-20. doi: 10.1007/s00784-011-0559-2. braz j oral sci. 15(4):287-292 family structure and oral habits among children age 1 to 12 years resident in ile-ife, nigeria oyedele ta1, 2, 3, kolawole ka1, 4, 5, folayan mo1, 4, 5, agbaje ho1, 5, oziegbe eo1, 4, 5, onyejaka n1, 6, chukwumah nm1, 7 1oral habit study group, ile-ife, nigeria 2department of surgery, benjamin carson, snr, school of medicine, babcock university, ilisan-remo, ogun state 3dental department, babcock university teaching hospial, ilisan-remo, ogun state 4department of child dental health, obafemi awolowo university teaching hospitals’ complex, ile-ife, nigeria 5department of child dental health, obafemi awolowo university, ile-ife, nigeria 6university of nigeria, enugu 7university of benin teaching hospital, benin-city edo state correspondence to: titus ayodeji oyedele; e-mail: ayotitus4christ@gmail. com; +234 706 2398941 e-mail addresses: kak: kikelomokolawole@gmail.com mof: toyinukpong@yahoo.co.uk hao: kimex10@yahoo.com eoo: elioziegbe@yahoo.com nko: nnekaonyejaka@yahoo.com nmc: nnechukwumah@yahoo.com abstract oral habits are repetitive actions that are done automatically. these behaviours are started and stopped spontaneously with or without deleterious effect on the developing occlusion. aim: to explore the family related factors associated with oral habits in children resident in sub-urban nigeria. methods: a cross sectional study utilizing a household survey to recruit 992 1year to 12-year-olds. information collected using a structured questionnaire included gender, family structure (parenting structure, birth rank, number of siblings, socioeconomic status) and types of non-nutritive habits. the association between family structures related variables and presence of non-nutritive oral habits was determined using chi square. logistic regression was used to determine the predictors of presence of oral habits. results: there was no significant association between the prevalence of oral habits and parenting structure (p=0.52), birth rank (p=0.50) and socioeconomic status (p=0.14). however, the association between oral habits prevalence and number of siblings the child had was significant (p=0.03). the odds of having a non-nutritive oral habit reduced insignificantly for those from middle (aor: 0.67; 95% ci: 0.42-1.08) and low (aor: 0.96; 95% ci: 0.59-1.55) socioeconomic class when compared with those with high socioeconomic status; and for last born and only children (aor: 0.94; 95% ci: 0.56-1.60) and children with 2-4 siblings (aor: 0.62; 95% ci: 0.36-1.09) when compared with those that have more than 4 siblings. the odds were higher for children who were living with single parents or guardians (aor: 1.41; 95% ci: 0.76-2.59; p=0.27) and for males (aor: 1.21; 95% ci: 0.82-1.78). conclusion: the study was unable to identify a significant family related predictor of presence of non-nutritive oral habits in the study population though a number of these factors increased the odds of having the habits. there is need to explore if specific family factors are associated with the presence of specific non-nutritive habits in this group of children.received for publication: january 18, 2017 accepted: july 23, 2017 original article braz j oral sci. october | december 2016 volume 15, number 4 http://dx.doi.org/10.20396/bjos.v15i4.8650041 introduction an oral habit is a repetitive action done automatically1. these repetitive behaviours are common in the infantile period. most of them are started and finished spontaneously2. oral habits are often associated with the stimulation of the mouth that is also a source of relief in passion and anxiety for both children and adults3. stimulation of this region with the tongue, finger or nail can be a palliative action for anxiety, hunger fear and stress3. the study was conducted in ife central local government area (lga) of osun state, a sub-urban population in the southwest region of nigeria. the estimated population of the lga is put at 96,580 based on 2006 census. the primary study generated a cross sectional data utilizing a household survey over a period of six months – august 2013 to february 2014. a household survey was conducted in view of the fact that not all children in nigeria are in school. it was estimated that 40% of primary school aged children and 60% of secondary school aged children were out of school22. it was more apt for recruiting a representative sample of the community. parts of the primary data had been published23,24. study population the participants were recruited from the national population enumeration sites in the lga. the enumeration sites were the same used for the 2010 national anc sero-sentinel survey and the 2012 national adolescent reproductive health survey. the study recruited children 6 months to 12-year-old whose legal guardians gave consent for study participation and who were present at home during the time of collecting this data. sample size sample size was calculated using leslie fischer’s formula25 for study population greater than10, 000. the sample size was determined using a prevalence of 34.1% based on the outcome of the study by quashie-williams et al.26 on the prevalence of oral habits among a group of children in nigeria. it would be necessary to examine 1,011 children to be able to identify 345 children with oral habits. the sample size was rounded to 1,200 to include a 10% study participation refusal rate. however, for this study, the data of 992 children age 1 year to 12 years was extracted for analysis. sampling technique participants were recruited used a multistage sampling procedure. stage 1 involved the random selection of 12 out of the 25 enumeration areas within the ife central lga. stage 2 involved the selection of eligible household within the enumeration sites for the survey. at each of the enumeration sites, every third household on each street was considered eligible for study participant recruitment. stage 3 involved the selection of actual respondent for interview and oral examination. only one eligible child in each household participated in the study. alternative sexes and age range identified for study recruitment were selected to participate in each consecutive household. study participant recruitment continued in the enumeration site until the study sample per each data collector was reached. more details of this sampling technique has been reported by folayan et al.23. data collection tool the data was collected using interviewer administered structured questionnaire which were administered by experienced field workers who were trained on this study protocol. the field supervisor reviewed the questionnaires within 24 hours of data collection and any problems encountered were addressed in the field. 288 a few non-nutritive habits have been associated with psychosocial disorders. bruxism, also known as tooth grinding, has been associated with psychosocial distress4 although bruxism in children below 6 years and sleep bruxism may not be related to any significant psychological traits5,6. wake clenching has however been associated with a number of psychopathological symptoms6. one of the most common repetitive oral behaviours in infantile period is digit sucking1. sucking reflex is one of the first complex patterns of behaviour in infant and appears around the 29 weeks of age in the utero7. digit sucking is naturally developed in 89% of infants in the second month of life and in 100% of them by the first year of age1,7. the frequency of engaging in this habit decreases as age increases, and most of the time; it is stopped by the age of 4 years1,8. however, a few children persist with the habit well into adolescence1. various factors have been associated with thumb sucking. parental educational status is one of such factors with a greater association demonstrated between thumb sucking and children whose fathers had a low level of schooling9. children with working mothers also have greater tendencies to develop sucking habits [digit, lip and tongue sucking] since the children must compete with other siblings for the mother’s limited time and attention10-13. the birth position is another with the youngest child in the family reported as having greater prevalence of thumb sucking14. very little is known about the effect of family various structures and the reason why non-nutritive oral habits persist in children beyond the accepted periods. prior studies had suggested that the duration of breastfeeding was inversely related to the age of pacifier use persistence15 and jahanbin et al.16 also showed that factors associated with family structure were associated with prolonged persistence of a non-nutritive oral habit. the family may be a significant influencing factor knowing that families influence the socialisation process of children17. family related factors could also produce continuous psychosocial distress that may inform the acquisition of non-nutritive oral habits after the preschool years or persistence of these oral habits. family related factors that could cause psychological stress to the child include the socioeconomic status18, the birth rank19, living with both parents, single parents or guardians20 and the size of the family21. this study was designed to generate information on family related factors that are associated with presence of non-nutritive oral habits in children 1 year and 12-year resident in a sub-urban region in nigeria. specifically, it explored the association between the child’s family socio-economic status, number of siblings, birth rank, the parental status (living with single parent, step parents, guardian or both parents) and the presence of non-nutritive sucking habits (finger sucking, tongue sucking, tongue thrusting, lip sucking, nail biting, object biting and bruxism). materials and methods study design this study is part of a larger study conducted to determine the relationship between non-nutritive oral habits and dental caries. family structure and oral habits among children age 1 to 12 years resident in ile-ife, nigeria braz j oral sci. 15(4):287-292 289family structure and oral habits among children age 1 to 12 years resident in ile-ife, nigeria table 1 profile of children resident in sub-urban nigeria with non-nutritive oral habits (n=992). variable oral habit age (years) absent n =863 n (%) one habit present n=117 n (%) two habits present n=11 n (%) three habits present n=1 n (%) total n=992 n (%) 1 55(6.4) 14(12.0) 3(27.3) 72 (7.3) 2 83(9.6) 12(10.3) 95 (9.6) 3 108(12.5) 13(10.2) 1(9.1) 122 (12.3) 4 86(10.0) 15(12.8) 1(9.1) 102 (10.3) 5 96(11.1) 9(7.7) 1(9.1) 106 (10.7) 6 90(10.4) 10(8.5) 100 (10.1) 7 61(7.1) 9(7.7) 2(18.2) 72 (7.3) 8 72(8.3) 11(9.4) 2(18.2) 85 (8.6) 9 64(7.4) 4(3.4) 68 (6.9) 10 70(8.1) 10(8.5) 1(9.1) 1(100.0) 82 (8.3) 11 46(5.3) 5(4.3) 51 (5.1) 12 32(3.7) 5(4.3) 37 (3.7) socioeconomic status high 203(23.5) 33(28.2) 3(27.3) 239 (24.1) middle 382(44.3) 43(36.8) 4(36.4) 1(100.0) 430 (43.3) low 277(32.1) 41(35.0) 4(36.4) 322 (32.5) missing 1(0.1) 1(0.1) braz j oral sci. 15(4):287-292 the questionnaire asked details on the family structure of the child. these include socioeconomic status, birth rank, number of siblings, parenting structure (living with single parent, step parents, both parents, guardian), and types of oral habit(s) child engaged in if any (digit sucking, tongue sucking, tongue thrusting, lip sucking, nail biting, object biting, bruxism). socio-economic status for the purpose of this study was obtained through a scoring index combining the mother’s level of education with the occupation of the father. it used an adapted version of the socio-economic status index developed by olusanya et al.27 and previously used by folayan et al.28,29 for studies assessing impact of socioeconomic status on oral health of children in nigeria. the mother’s level of education was classified as ‘no formal education, quranic and primary school education’ were scored 2; secondary school education scored as 1; and tertiary education scored as 0. the father’s occupation was also categorised into three: those who were civil servants or skilled professional with tertiary level of education were scored 1; those who were civil servants or skilled professional with secondary level of education were scored 2; and unskilled, unemployed individuals, students, and civil servants or skilled professional with primary and or quranic level of education were scored 3. the social class was obtained through the addition of the score of the mother’s level of education with that of the father’s occupation. each child was therefore allocated into social class iv where class i referred to upper class, class ii referred to upper middle class, class iii referred to middle class, class iv referred to lower middle class and class v referred to lower class. the socioeconomic status was regrouped into three: (i) class i and ii were the high socioeconomic class, class iii was middle socioeconomic class and class iv and v were the low socioeconomic class as described. data analysis statistical analysis was performed using stata 12. for this study, the dependent variables were non-nutritive oral habits (digit sucking, tongue sucking, tongue thrusting, lip sucking, nail biting, object biting, and bruxism) and the independent variables were family structures (birth rank, number of siblings and parenting structure). descriptive analysis was conducted for all the quantitative variables. associations between the dependent and independent variables were determined using chi-square. inferential analysis was conducted to determine the significant predictors of non-nutritive oral habits using logistic regression. adjustment was made for socioeconomic status as a possible confounder based on results of past studies showed socioeconomic status was associated with prevalence of oral habits in children30. statistical significance was defined as p<0.05. ethical consideration: approval for this study was obtained from the ethics and research committee of the obafemi awolowo university teaching hospital ile-ife, nigeria (erc/2013/07/14). permission for the conduct of the study was sought from the ife central local government authority. written informed consent was obtained from the legal guardians of children who participated in the study. results the mean age of the 992 study participants was 5.83 ± (3.15) years. table 1 highlights the profile of the study participants. children between the ages of 3 years and 6 years accounted for 43.3% of the study participants. also, 41.5% of the respondents were from the middle socioeconomic class. of the 992 study participants, 129 (13.0%) had one or more oral habits and 59 (45.7%) of those with oral habits were between the ages of 1 year and 4 years. in addition, 117 (11.8%) children had one oral habit, 11 (1.1%) had two oral habits and 1 (0.1%) had three oral habits. table 1 shows the socioeconomic status of the children with and without oral habit: 48 (37.2%) of children with oral habit were from the middle socioeconomic class, 45 (34.9%) were from low socioeconomic class and 36 (27.9%) were from high socioeconomic status. there was no statistically significant association between the prevalence of oral habits and socioeconomic status (p=0.29). 290 table 2 shows the distribution of study participants by family structure and oral habit. of the 129 study participants with oral habits, 62 (48.1%) were first born, 89 (69.0%) had 2-4 siblings and 111 (86.0%) lived with both parents. there was no significant association between the prevalence of oral habit and parenting structure (x2=1.78, p=0.18) and birth rank (x2=1.61, p=0.45). however, there was a significant association found between oral habit and number of siblings (x2=4.77, p=0.03), children with 2-4 sibling have higher probability of developing oral habits. family structure and oral habits among children age 1 to 12 years resident in ile-ife, nigeria table 2 distribution of children resident in sub-urban nigeria with non-nutritive oral habits by family structure and oral habit (n=129). table 3 logistic regression to determine predictors of presence of oral habits (n=992). variables oral habit present n=129 n(%) absent n=863 n(%) total n=992 n(%) x2 p value parenting structure both parents 111 (86.0%) 773 (89.6%) 884(89.1%) other parenting structure 15(11.6%) 70(8.1%) 85(8.6%) number of siblings 1 29(22.5%) 128(14.8%) 157(15.8%) 2-4 89(69.0%) 663(76.8%) 752(75.8%) 4.77 0.03 >4 11(8.5%) 66(7.6%) 77(7.8%) birth rank first born 62 (48.1%) 369 (42.8%) 431(43.4%%) last born and the only child 32(28.8%) 240 (27.8%) 272(27.4%%) 1.61 0.45 others 32(28.8%) 245(28.4%) 277(27.9%) variables oral habit adjusted or 95% c.i p value absent n=863 n % present n=129 n % sex female 425(49.2) 59(45.7) 1 male 438(50.8) 70(54.3) 1.21 0.82-1.78 0.33 socioeconomic status high 203(23.5) 36(27.9) 1 middle 382(44.3) 48(37.2) 0.67 0.42-1.08 0.10 low 277(32.1) 45(34.9) 0.96 0.59-1.55 0.86 birth rank first born 369 (42.8) 62(49.2) 1 last born and only child 240 (27.8) 32(25.4) 0.94 0.56-1.60 0.83 others 245(28.4) 32(25.5) 0.89 0.51-1.56 0.70 number of siblings 0-1 128(14.9) 29(22.5) 1 2-4 663(77.4) 89(69.0) 0.62 0.36-1.09 0.10 >4 66(7.7) 11(8.5) 0.79 0.31-2.00 0.62 parenting structure both parents 773(91.7) 111 (86.4) 1 other parenting structure 70(8.3) 15 (11.6) 1.41 0.76-2.59 0.27 table 3 shows the outcome of the multivariate logistic regression to determine the predictors of presence of oral habits. parenting structure, number of sibling and birth rank was not significant predictor of presence of non-nutritive oral habits. children who lived with other parents (single parents, step parent or guardians) had insignificantly increased odds of having an oral habit (aor: 1.41; 95% ci: 0.76-2.59; p=0.27) when compared with children who were living with both parents. also, children who were last born and only child (aor: 0.94; 95% ci: 0.561.60; p=0.83) and who had other birth ranks (aor: 0.89; 95% ci: 0.51-1.56; p=0.70) had insignificantly reduced odds of having oral habits when compared with children who were first born. in addition, children with 2 to 4 siblings (aor: 0.62; 95% ci: 0.361.09; p=0.10) and greater than 4 siblings (aor: 0.79; 95% ci: 0.31-2.00; p=0.62) had insignificant increased odds of having an oral habits when compared with children who had one or no siblings. braz j oral sci. 15(4):287-292 discussion this study was unable to find any significant association between presence of non-nutritive oral habits, parenting structure, birth rank, number of siblings and the socioeconomic status of children resident in ile-ife. however, the odds of having a non-nutritive oral habit reduced for males when compared to female, for those in the middle and low socioeconomic class when compared to those in the high socioeconomic class, for last born when compared with first born children and for children who were living with single parents or guardians when compared with those living with both parents. we also found that children with other birth ranks (not last borns) and those with more than one sibling were more likely to have a non-nutritive oral habit. the findings of this study differed from findings of some prior studies. for example, jahanbin et al.16 showed that the child’s birth rank, number of siblings and parents’ education level were significant factors associated with presence of non-nutritive oral habits in 7-year-old iranian children unlike what we found in this study. fernandes et al.31 on the other hand, showed that children of mothers with lower educational status were less likely to have non-nutritive oral habits similar to our observation that the prevalence of non-nutritive oral habits reduced with lowering socio-economic status. our observation however, did not reach statistical significance. there are a few studies that have identified family structure related factors associated with presence of non-nutritive oral habits in older children. williams et al.32 showed that more undergraduates not living with both parents bite their nails. also, murrieta et al.33 noted that primary school children who lived with both parents were more likely to be emotionally stable and therefore adopt less non-nutritive oral habits. quashie-williams et al.34 who studied the prevalence of non-nutritive oral habits in children 4-15 years, found that significantly more children from higher socioeconomic status had non-nutritive oral habits. they however did not study the relationship of other family related factors with the presence of non-nutritive oral habits. our study evaluated the role of multiple family related factors on the presence of non-nutritive sucking habits in children and we found no significantly associated family factor. this study was however limited in its ability to assess the effect of family related factors on the presence of specific nonnutritive oral habits because the small number of children with specific non-nutritive oral habits made it difficult to do these subanalyses. it would have been useful to identify if there are specific family related variables that are associated with the presence of specific non-nutritive oral habits. despite this limitation, the study was able to identify that parenting structure, birth rank, number of siblings and the socioeconomic status did not increase the chances of children in the study environment indulging in non-nutritive oral habits. also, the use of a household survey for study participants’ recruitment in a country where a significant number of children are out of school22 makes the finding of this study generalisable to the study population. further studies are needed to explore how family related factors are associated with presence of specific non-nutritive oral habits in children. conclusion the sex, socioeconomic status, birth rank, number of siblings and the parenting structure of children resident in ile-ife, nigeria were not significant predictors of presence of non-nutritive oral habits in children. there is a need to further explore if specific family factors are associated with the presence of specific nonnutritive habits in children 1 year old and older. authors’ contributions tao conceived the idea, designed and made substantial contributions to acquisition, analysis, and interpretation of the result and manuscript right up. mof, kak, eoo, nmc, no made substantial contribution to the design, acquisition, analysis and interpretation and manuscript right up. all authors agreed to the final version of the manuscript. competing interest the authors declare that there are no competing interests. acknowledgement the authors acknowledge the parents and children that participated in the study. references 1. maguire ja. the evaluation and treatment of pediatric oral habits. dental clin north am. 2000 jul;44(3):659-69, vii. 2. shahraki n, yassaei s, moghadam mg. abnormal oral habits: a review. j dent oral hyg. 2012 may;4(2):12-5. doi: 10.5897/jdoh12.001. 3. bear pn, lestor m. the thumb, the pacifier, the erupting tooth and a beautiful smile. j pedod. 1987 whinter;11(2):115-9. 4. funch dp, gale en. factors associated with nocturnal bruxism and its treatment. j behav med. 1980 dec;3(4):385-7. 5. kuch ev, till mj, messer lb. bruxing and non-bruxing children: a comparison of their personality traits. pediatr dent. 1979 sep;1(3):182-7. 6. manfredini d, lobbezoo f. role of psychosocial factors in the etiology of bruxism. j orofac pain. 2009 spring;23(2):153-66. 7. rani ms. synopsis of orthodontics. 2nd ed. delhi: all india publisher and distributors; 1998. p. 179-200. 8. larson ef. the prevalence and aetiology of prolonged dummy and finger-sucking habits. eur j orthod. 1985 aug; 7(3):172-6. 9. gupta bi, bhavna g, indushekar kr. childhood thumb sucking habit: the burden of a preventable problem! j dent med sci. 2012 jun;2(1):1-4. 10. adair sm. pacifier use in children: a review of recent literature. pediatr dent. 2003 sep-oct;25(5):449-58. 11. farsi nm, salama fs. sucking habits in saudi children: prevalence, contributing factors and effects on the primary dentition. pediatr dent. 1997 jan-feb;19(1):28-33. 12. turgeon-o'brien h, lachapelle d, gagnon pf, larocque i, maheurobert lf. nutritive and non-nutritive sucking habits: a review. asdc j dent child. 1996 sep-oct;63(5):321-7. 13. warren jj, bishara se. duration of nutritive and non-nutritive sucking behaviors and their effects on the dental arches in the primary dentition. am j orthod dento-facial orthop. 2002 apr;121(4):347-56. 14. rajchanovska d, zafirova-ivanovska b. oral habits among preelementary children in bitola. prilozi. 2012;33(1):157-69. 15. telles fb, ferreira ri, magalhãesldo n, scavone-junior h. effect 291family structure and oral habits among children age 1 to 12 years resident in ile-ife, nigeria braz j oral sci. 15(4):287-292 292 of breastand bottle-feeding duration on the age of pacifier use persistence. braz oral res. 2009 oct-dec;23(4):432-8. 16. jahanbin a, mokhber n, jabbarimani a. association between sociodemographic factors and nutritive and non-nutritive sucking habits among iranian girls. east mediterr health j. 2010 nov;16(11):1143-7. 17. richards mh, gitelson ib, peterson ac, hartig al. adolescent personality in girls and boys: the role of mothers and fathers. psychol women quart. 1991 mar;15(1):65-81. 18. chen e. why socioeconomic status affects the health of children: a psychosocial perspective. curr direct psychol sci. 2004 jun; 13(3):1125. 19. dohrenwend bs, dohrenwend bp. stress situations, birth order, and psychological symptoms. j abnormal psychology. 1966 jun;71(3):21523. 20. falana ba, bada fo, ayodele cj. single-parent family structure, psychological, social and cognitive development of children in ekiti state. j educ develop psychol. 2012 aug;2(2):158-64. doi: 10.5539/ jedp.v2n2p158. 21. oyerinde oo. the impact of family structure, parental practices and family size on children’s academic performance. nig school health j. 2001;13(2):160-8. 22. unicef. nigeria-the children education. 2005 [cited 2014 dec 20]. available from: www.unicef/org/nigeria/children. 23. folayan mo, kolawole ka, oyedele ta, chukwumah nm, onyejaka n, agbaje h, et al. erratum: association between knowledge of caries preventive practices, preventive oral health habits of parents and children and caries experience in children resident in sub-urban nigeria. bmc oral health. 2015 may 20;15:62. doi: 10.1186/s12903-015-0044-4. 24. folayan mo, kolawole ka, oziegbe eo, oyedele ta, oshomoji ov, chukwumah et al. prevalence and early childhood caries risk indicators in preschool children in suburban nigeria. bmc oral health. 2015 jun 30;15:72. doi: 10.1186/s12903-015-0058-y. 25. araoye mo. research methodology with statistics for health and social science. ilorin: nathadex publisher; 2003. p.115-9. 26. quashie-williams r, dacosta oo, isiekwe mc. oral habits, prevalence and effects on occlusion of 4-15 year old school children in lagos, nigeria. niger postgrad med j. 2010 jun;17(2):113-7. 27. olusanya o, okpere o, ezimokhai m. the importance of social class in voluntary fertility control in developing country. west afr j med. 1985;4(4):205-12 28. folayan mo, otuyemi od. reliability and validity of a short form of the dental subscale of the child fear survey schedule used in nigerian children population. niger j med. 2002 oct-dec;11(4):161-3. 29. folayan mo, idehen ee, ufomata d. the effect of sociodemographic factors on dental anxiety in children seen in a suburban nigerian hospital. int j peadiatr dent. 2003 jan;13(1):20-6. 30. taylor vj, cook pa. the prevalence of dummy sucking in two socioeconomic groups in leeds [abstract 72]. j dent res. 1989; 68:567. 31. fernandes ib, pereira ts, figueiredo de carvalho mf, ramos-jorge j, marques ls, ramos-jorge lm. non-nutritive sucking habits after three years of age: a case-control study. j indian soc pedod prev dent. 2015 jan-mar;33(1):19-24. doi: 10.4103/0970-4388.148965. 32. williams ti, rose r, chisholm s. what is the function of nail biting: an analog assessment study. behav res ther. 2007 may;45(5):989-95. 33. murrieta jf, hernández d, linares c, gonzáles m, juárez l, montaño v. parafunctional oral habits and its relationship with family structure in a mexican preschoolers group. j oral res. 2014;3(1):29-35. 34. quashie-williams r, dacosta oo, isiekwe mc. the prevalence of oral habits among 4 to 15 year old school children in lagos. nig j health biomed sci. 2007;6(1):78-82. family structure and oral habits among children age 1 to 12 years resident in ile-ife, nigeria braz j oral sci. 15(4):287-292 1http://dx.doi.org/10.20396/bjos.v18i0.8657254 volume 18 2019 e191573 original article 1 school of dentistry, university of ribeirão preto, ribeirão preto, sp, brazil. 2 school of dentistry, meridional faculty (imed), passo fundo, rs, brazil. 3 department of dental materials and prosthodontics, ribeirão preto school of dentistry, university of são paulo, ribeirão preto, sp, brazil. corresponding author: dr erica alves gomes university of ribeirão preto, school of dentistry av costábile romano, 2.201 ribeirão preto, são paulo 14096-900, brazil email: ericaagomes@yahoo.com.br received: february 13, 2019 accepted: september 10, 2019 different fabrication techniques of implant-supported prostheses: microhardness and fracture strength silas monteiro borges1, stephanie francoi poole1, izabela c. m. moris1, aloísio oro spazzin2, adriana cláudia lapria faria3, erica a. gomes1,* aim: this study evaluated the mechanical behavior of implant-supported crowns obtained by different fabrication technique after thermomechanical cycling. methods: thirtytwo external hexagon dental implants were divided into four groups (n=10): cc – conventional casting with torch; ei – electromagnetic induction casting; pl – plasma casting; and cad-cam – milling through computer-aided design and computer-aided manufacturing. vickers microhardness of the specimens were made before and after the thermomechanical cycling, and then subjected to fracture load. fracture pattern was evaluated. results: no significant difference was observed comparing the microhardness before and after thermomechanical cycling. cad-cam group presented significant lower microhardness than the other groups. no significant statistical difference was showed on fracture load between the groups. the cad-cam and pl presented lower number of failure by plastic deformation. conclusion: the manufacturing techniques affected the mechanical behavior and the failure pattern of implant-supported crowns tested. keywords: dental implants. prosthodontics. computer-aided design. flexural strength. 2 borges et al. introduction metal-free restorations have been used to obtain improved aesthetics for dental prostheses. however, metal–ceramic restorations remain widely and the most used because of their excellent mechanical properties and clinical performance1-2. the framework and aesthetic veneer must have sufficient mechanical properties to support masticatory loading and possible injuries to the oral environment3. the framework of metal–ceramic prostheses can be fabricated using casting or milling process, and some key factors should be considered for the long-term success of metal–ceramic prostheses, such as alloy composition, casting technique, workability, surface roughness, framework shape, and microhardness4-5. computer-aided design and computer-aided manufacturing (cad-cam) systems have been widely used for machining the framework of implant-supported prostheses. however, techniques using heating devices, such as torches, induction arcs, or electric arcs are still commonly used for manufacturing. the physical–chemical properties of the alloy can be altered according to the ability and knowledge of the operator, the equipment used, and the product quality, since the selected technique to obtain the framework should be considered5-7. structural failures of implant-supported prosthesis may be related to the material hardness and also associated to material wear resistance in the oral environment. and, the hardness of material is an important variable to be analyze, due the possibility of evaluate the resistance of material to the localized plastic deformation. therefore, the microhardness is an important method to predict the clinical success of the long-term treatments8. structural failures described in the literature refer to plastic deformations, such as the displacement and damage of the components without a fragments rupture or fractures, such as the fragments rupture and detachment of the structure of prosthetic crowns. both conditions compromise the mechanical functioning of the crown/screw/implant set and results in the failure of implant rehabilitation treatment9-10. the aim of this study was to evaluate the mechanical behavior (microhardness, fracture load and failure pattern) of implant-supported crowns obtained by different fabrication technique (conventional casting, electromagnetic induction casting, plasma casting or cad-cam) after thermomechanical cycling. materials and methods study design fourty external hexagon dental implants (4.1-mm in diameter × 13-mm in length) (pross; dabi atlante, ribeirão preto, sp, brazil) were divided into four groups considering on manufacturing of the implant-supported crown (n=10): cc – conventional casting using torch; ei – electromagnetic induction casting; pl – plasma casting; and cad-cam – milling through computer-aided design and manufacturing. vickers microhardness of the specimens were made before and after the thermomechanical cycling, and then they submitted to fracture load. fracture pattern was also evaluated. 3 borges et al. specimen preparations dental implants were embedded in polyurethane (f16 fastcast polyurethane, axson, cergy, france) using pvc tubes in a long axis assisted by a delineator (bio-art, são carlos, sp, brazil). a progressive waxing (kota, são paulo, sp, brazil) was initially made on a 4.1-mm anti-rotational castable cylinder (pross; dabi atlante, ribeirão preto, sp, brazil) on the anatomy of the maxillary canine. two-piece matrix was made in condensation silicone (zetaplus, zhermack, badia polesine, rovigo, italy) to standard the crown waxing maintaining the anatomical pattern. thirty waxes were obtained and randomly divided into three groups according to the manufacturing technique of the implant crowns (n=10): cc – conventional casting using torch; ei – electromagnetic induction casting; and pl – plasma casting. after casting in cobalt−chromium (co–cr) alloy (fit cast cobalto; talmax, curitiba, pr, brazil), the crowns were divested and sandblasted with 100-μm aluminum oxide particles (polidental, cotia, sp, brazil), under 80psi (5.51 bars), and separated of the sprue using carborundum disks (schelble, petópolis, rj, brazil). for the cad-cam group, the crowns were made using scanning and milling techniques in the cad-cam system (amangirgach, koblach, austria). a crown was waxed as priorly described and screwed to each implant (n=10), scanned, digitized (ceramill mind, amann girrbach, koblach, austria), and machined in pre-sintered co–cr alloy (ceramill sintron, amann girrbach, koblach, austria). all crowns were sintered under argon gas at 1300oc for six hours in a special furnace (ceramill argotherm, amann girrbach), following the manufacture instructions. the finishing and polishing were performed for all groups using specific burs and pastes for metals (exa-cerapol, edenta, au/sg, switzerland)11. vickers microhardness the vickers microhardness (hv) of the co–cr alloys obtained using different techniques (castings or milling) was measured with a load of 19.614 n for 20 s (hmv-2 microhardness tester; shimadzu, kyoto, japan)12. five measurements were performed for each specimen, before and after thermomechanical cycling. thermomechanical cycling the accelerated aging was performed using thermomechanical cycling in a pneumatic mastigation simulator (biopdi, são carlos, sp, brazil). six specimens were simultaneously submitted to the thermomechanical cycling, with a loading of 120n applied through a metallic tip with a flat surface, during 1×106 mechanical cycles with a frequency of 3 hz with temperatures ranging of 5°c to 55°c in distilled water with a dwell time of 40 s in each bath. fracture load the specimens were submitted to fracture loading in a universal testing machine (biopdi, são carlos, sp, brazil). each specimen (implant/screw/crown) was positioned in a 30° metal device (iso 14801) and compressive load was applied with crosshead speed of 1mm/min until specimen failure. the loading point was located 11.5mm 4 borges et al. from the surface of the implant platform. during the test, the load was applied on the specimens until plastic deformations occurred in some components of the set (implant/screw/crown). statistical analysis the analyses were performed using statistical software (20.0 spss statistics, ibm, chicago, il, usa). linear mixed-effects model and bonferroni complementary test were performed to analyze microhardness data (p<0.05). data of fracture load were submitted to one-way anova followed by tukey’s post-hoc test (p<0.05). results the results of microhardness are presented in the table 1. in intra-groups comparation (before and after thermomechanical cycling), the statistical analysis showed no significant difference (p>0.05). therefore, the thermomechanical cycling presented no effect in the microhardness. in relation to different manufacturing technique, cad-cam group presented significant lower microhardness than the other groups (p<0.05). in addition, the ei group after thermomechanical cycling presented higher microhardness values compared with cc and pl (p<0.05). the results of fracture load are shown in table 2. the statistical analysis showed no significant statistical difference on fracture load between the groups (p>0.05). after compression resistance test, the qualitative evaluation using micro ct images showed differences in the failure mode. in both cc and ei groups, six specimens failed with plastic deformation and two with fracture; whereas in the pl group, two specimens table 1. comparison of vickers microhardness (hv) before and after thermomechanical cycling. comparison thermomechanical cyclic loading mean difference p-valor confidence interval upper limit lower limit gc × gi before 154.025 0.001 57.376 250.674 after 117.212 0.000 63.541 170.884 gc × gp before -29.000 1.000 -125.649 67.649 after -61.650 0.018 -115.321 -7.979 gc × gcad before 10.350 1.000 -86.299 106.999 after 9.975 1.000 -43.696 63.646 gi × gp before 39.350 1.000 -57.299 135.999 after 71.625 0.004 17.954 125.296 gi × gcad before 183.025 0.000 86.376 279.674 after 178.863 0.000 125.191 232.534 gp × gcad before 143.675 0.001 47.026 240.324 after 107.237 0.000 53.566 160.909 5 borges et al. failed with plastic deformation and six with fracture. the cad-cam group presented one specimen with plastic deformation and seven with fracture. specimens with plastic deformation showed failure due to misfit to crown/screw/ implant sets during loading and, consequently, displacement of the prosthetic screw. the plastic deformation of the prosthetic screw and coronal third of the implant, including the platform, was observed in all groups (fig. 1). discussion the manufacturing methods affected the vickers microhardness results in the present study. the cad-cam group presented the lowest values, which were closest to those reported in the literature for co–cr alloy13. a previous study4 reported that casting procedures change the surface composition, but after polishing as-cast surface, the alloy composition is similar those obtained by the manufacturer previously the manipulation. the different microhardness found for the cad-cam group can be justified by the cast surfaces of the ei, cc, and pl groups that, probably presents a diftable 2. mean and standard deviation of compression strength (n). groups compression strength gc 884.00 (70.11) a gi 865.53 (157.32) a gp 930.32 (136.94) a gcad 912.22 (78.78) a values with the same letters in the same column present no statistically significant difference at 5% significance level. figure 1. micro-ct images representing the failure mode: (a) plastic deformation, and (b) plastic deformation and fracture. a b 6 borges et al. ferent surface composition and have a additional surface layer obtained because of the reaction between investment and alloy. although, casted crowns were polished for evaluating the microhardness, the depth of this surface layer remained unknown, and the composition of casted crowns can be different from cad-cam crowns. in addition, it is important to consider the difference of the microstructure of co–cr alloys obtained by casting and cad/cam. the microstructure of the cast co–cr alloys contain large grains, whereas co–cr alloys produced by cad/cam present finer grains, thereby improving the mechanical properties14. nevertheless, thermomechanical cycling did not affect the microhardness results. in the present study, specimens failures were not found during and after thermomechanical cycling. however, the plastic deformation failure of the specimen or implant fracture occurred when higher load was applied on the compressive strength test. the mean of values obtained for the compressive strength test (cc=884.00n, ei=865.53n, pl=930.32n, and cad-cam=912.22n) was within the acceptable limits of the masticatory load to the anterior region. the physiologic forces in this region ranged between 132–231n and the magnitude of masticatory load in patients with parafunctional habits presented a mean of 812.2n15-16. some studies have indicated that the frequency of mechanical complications (component fractures and screw loosening) is greater in hexagonal external connection than in internal connection9,17-18. it is believed that this phenomenon occurs because static and dynamic masticatory loads incident on specimens (crown/screw/ implant) are distributed along the prosthetic screw surface, which can lead to loosening or fracture. in the present study, the oblique load applied on specimens during the compressive loading test caused the prosthetic screw to exert a force against the lateral wall of the implant opposite to the loading point, leading to fracture because of excessive stress on the site. this characteristic of the stress concentration in the region contralateral to the load point is significantly increased when an angular load is applied, which ca be explained by the rigidity and flexion of the sample19. therefore, additional care should be taken during oral rehabilitation with external hexagonal connection, particularly in cases where intermediate abutments is not used. however, when there is a prosthetic abutment against high masticatory forces, the implant seems to be protected, but with failures in the abutment (abutment body or retaining-screw) and no implant fracture20-22, as related in this current study. thus, the use of intermediate abutment would be a favorable option because the clinician can reverse the situation using specific drills depending on the type of failure present in these abutments and replacing only the abutment and the crown, without damaging to the implant and, consequently, the osseointegration previously achieved. fracture load showed similar results among milled and casted crowns. however, a higher number of implant fracture was observed in the cad-cam group compared with the casting groups, which presented higher number of plastic deformations. we believe that these results were observed because of a difference in the alloy composition and surface oxidation due to different casting processes23. when the casting process ocurr in an open environment, the base alloys expose metals to deleterious 7 borges et al. gases and elements, such as nitrogen and oxygen, which can be absorbed during the heat produced, and negatively influencing the final cast, increasing the porosity of the alloy24, and inducing elastic instability25. thus, the cad/cam system may allow a greater appartness of the metallic alloy from the external environment protecting the material of the gas interaction, and probably offering a better prognosis of the proposed rehabilitation treatment. overall, the manufacturing techniques affected the mechanical behavior of implant-supported crowns being that the cad-cam system presented lowest microhardness. the fracture load was similar, while the failure pattern was different between the manufacturing techniques tested. acknowledgments the authors would like to thank the coordination for the improvement of higher eduaction personnel (capes) for the scholarship awarded, pross (dabi atlante, ribeirão preto, sp) 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exhibiting periapical pathology. clin oral implants res. 2011 jan;22(1):20-7. doi: 10.1111/j.1600-0501.2010.01973.x. 22. sghaireen mg. fracture resistance and mode of failure of ceramic versus titanium implant abutments and single implant-supported restorations. clin implant dent relat res. 2015 jun;17(3):554-61. doi: 10.1111/cid.12160. 23. serra-prat j, cano-batalla j, cabratosa-termes j, figueras-àlvarez o. adhesion of dental porcelain to cast, milled, and laser-sintered cobalt-chromium alloys: shear bond strength and sensitivity to thermocycling. j prosthet dent. 2014 sep;112(3):600-5. doi: 10.1016/j.prosdent.2014.01.004. 24. bauer jr, grande rh, rodrigues-filho le, pinto mm, loguercio ad. does the casting mode influence microstructure, fracture and properties of different metal ceramic alloys?. braz oral res. 2012 may-jun;26(3):190-6. 25. chen wc, teng fy, hung cc.characterization of ni–cr alloys using different casting techniques and molds. mater sci eng c mater. biol. appl. 2014 feb;35:231-8. doi: 10.1016/j.msec.2013.11.014. 1http://dx.doi.org/10.20396/bjos.v19i0.8660126 volume 19 2020 e200126 original article 1 federal university of pelotas, pelotas, rs, brazil. corresponding author: mateus bertolini fernandes dos santos graduate program in dentistry, federal university of pelotas, gonçalves chaves st, 457, 96015-560, pelotas, rs, brazil email: mateusbertolini@yahoo.com.br telephone number: +55 53 9.99349134 received: june 19, 2020 accepted: september 29, 2020 dental implant therapy in the brazilian public system: an overview of the last decade mateus de azevedo kinalski1 , ana luiza cardoso pires1 , júlia machado saporiti1 , mateus bertolini fernandes dos santos1,* aim: this retrospective study aims to describe and analyze the number of dental implants and implant-retained prostheses performed by the unified health system (sus) in the last decade. methods: this study is based on secondary data from the official government database (datasus) performed from january 2010 to december 2019 and is reported following the strobe. a descriptive analysis was performed of the total sample and the stratified sample divided by brazilian states. results: a total of 143,037 dental implants and 93,325 implant-retained prostheses were provided by sus. it is possible to observe that some states played a massive role on the provision of dental implants and implant-retained prostheses (parana state: 58.4% and 55.9% and paraiba state: 21.1% and 25.2% of the total amount of dental implants and implant-retained prostheses, respectively) while some states did not provide a single implant-retained prosthesis. also, inland cities were mostly responsible for the number of procedures compared to the state capitals. conclusion: although dental implant therapy is available in sus across the country, the number of treatments provided in the last decade is still very limited and is also mainly concentrated in the southeastern region of brazil. keywords: dental health services. dental implants. health policies. health services administration. https://orcid.org/0000-0002-8224-4740 https://orcid.org/0000-0002-8471-6422 https://orcid.org/0000-0001-5917-699x https://orcid.org/0000-0001-5477-4077 2 kinalski et al. introduction brazil is one of the few countries in the world to have a public health system that provides entirely free of cost services for any person, including foreigners. the unified health system (sistema unico de saude – sus) was instituted by the 1988constitution and is based on the principle that citizen’s health is a constitutional right and state’s duty1. regarding to numbers, sus is the largest public health system in the world considering the number of users, geographical extension, and size of the affiliated network2, whereas services are financed and provided at federal, state, or municipal levels. in 2004, a nationwide program called “smiling brazil” included oral health as one of the priority areas of the sus1. to do so, epidemiological census was conducted in the whole country and investments were made both in human resources (professional development) and infrastructure. the main focus of sus and its “smiling brazil” program is related to primary care including oral hygiene instructions, dental restorations, root and scaling, and tooth extractions. this program also focus on the expansion and qualification of specialized treatment, which is also covered by sus, including medium-complexity and tertiary care2. the last brazilian census on oral health have shown a high prevalence of edentulism in the elderly population (53.7%), while 17.4% of the brazilian adults have at least one tooth loss3. also, projections based on the population growth indicates that until 2040, 85.9% of the elderly population will have edentulous jaws4. in this perspective, oral rehabilitations with dental implants and implant-retained prostheses are considered the best treatment option to rehabilitate missing teeth, presenting high success and survival rates, as well as patients’ satisfaction5-8. dental implants and implant-retained prostheses were introduced in sus in 2010, through the ministry of health ordinances no 718/sas/ms and no 398/sas/ms9,10. to the best of our knowledge, the sus is one of the few public health systems that offer dental implants in the public service. however, it seems that the provision of dental implant rehabilitation in brazil is still mostly made by private practices. for this reason, this survey becomes important to assess the last decade of implants placement in the brazilian public service, whilst the results could represent a tool for the policy-makers, aiming to reducing inequalities and improving the coverage of these treatments. thus, the present study aims to describe and analyze the official government databank (datasus) regarding to dental implants, considering both the number of placed implants and implant-retained rehabilitations, made by sus since the inclusion of these treatments in it. materials and methods this retrospective study was designed as an ecologic study and is reported in accordance with the strengthening the reporting of observational studies in epidemiology (strobe) statement and is based on secondary data from datasus (brazilian health information databank)11. in accordance to a national resolution (cns, nº 510), the ethics committee approval was not mandatory12. 3 kinalski et al. source data was acquired from datasus (brazilian health information databank) using the tabnet tool, which provides information to support objective analyzes of the health situation, evidence-based decision making and the development of health action programs13. data acquisition a search comprising keywords and sus codes related to dental implants (osseointegrated dental implant – code 0414020421; and implant-retained prosthesis – code 0701070153) was performed considering all procedures performed in sus from january 2010 to the end of december 2019. data was collected on march 17th, 2020. all inputs were analyzed and categorized into: a) dental implants placement; b) implant-retained prostheses. the distribution of the number of each procedure/treatment was also distributed according to brazil’s socio-demographic regions (south, southeast, northeast, north, and central-west) and states. a descriptive analysis of the total sample and the stratified sample divided by state was performed using stata software 14.0 (stata corporation, college station, tx, usa). results table 1 summarizes the distribution of implant-related treatments provided by sus considering all brazilian states. in the last decade (january 2010 to december 2019), a total of 143,037 dental implants and 93,325 implant-retained prostheses were provided by sus. table 1. descriptive analysis stratified by state, considering the number of installed dental implants and number of installed dental prosthesis: datasus* (january 2010 to december 2019). national region state dental implants implant-retained prostheses n % in the region % in the country n % in the region % in the country south rio grande do sul (rs) 381 0.45 0.27 216 0.41 0.23 santa catarina (sc) 820 0.97 0.57 87 0.17 0.09 paraná (pr) 83,572 98.58 58.43 52,238 99.42 55.97 southeast são paulo (sp) 8,873 84.22 6.20 7,349 93.93 7.87 minas gerais (mg) 972 9.23 0.68 65 0.83 0.07 rio de janeiro (rj) 413 3.92 0.29 410 5.24 0.44 espírito santo (es) 277 2.63 0.19 0 central-west mato grosso do sul (ms) 1,132 7.05 0.79 73 0.84 0.08 goiás (go) 6,701 41.71 4.68 1,717 19.85 1.84 distrito federal (df) 0 0 mato grosso (mt) 8,234 51.25 5.76 6,862 79.31 7.35 continue... 4 kinalski et al. in figures 1 and 2, it is possible to observe that some states played a massive role on the provision of dental implants and implant-retained prostheses (parana state: 58.4% and 55.9% of the total amount of dental implants and implant-retained prostheses, respectively; paraiba state: 21.1% and 25.2% of the total amount of dental implants and implant-retained prostheses, respectively) while some states did not provide a single implant-retained prosthesis. figure 3 presents the number of dental implants and implant-retained prostheses provided by sus from january 2010 to december 2019. it is possible to observe a peak between the years of 2017, while a notable decrease was observed in the following years (2018-2019). in the table 2, a comparison whether the treatments were made in the capital region of each state or inland is presented. considering both treatments, inland cities were mostly responsible for the number of procedures compared to the state capitals. national region state dental implants implant-retained prostheses n % in the region % in the country n % in the region % in the country northeast bahia (ba) 70 0.23 0.05 102 0.43 0.11 sergipe (se) 0 0 alagoas (al) 42 0.14 0.03 42 0.18 0.05 pernambuco (pe) 175 0.57 0.12 0 paraíba (pb) 30,154 98.90 21.08 23,521 99.37 25.20 rio grande do norte (rn) 0 0 ceará (ce) 6 0.02 0.00 0 piauí (pi) 42 0.14 0.03 5 0.02 0.01 maranhão (ma) 0 0 north tocantins (to) 0 0 pará (pa) 0 0 amapá (ap) 1,173 100.00 0.82 638 100 0.68 roraima (rr) 0 0 amazonas (am) 0 0 acre (ac) 0 0 rondônia (ro) 0 0 total 143,037 100 93,325 100 *data extracted on march 17th, 2020 continuation 5 kinalski et al. figure 1. heat-map of the distribution of dental implants provided by sus for each brazilian state. implants <100 100–1,000 1,000–10,000 >10,000 figure 2. heat-map of the distribution of implant-retained prostheses provided by sus for each brazilian state. implants prostheses >10,000 1,000–10,000 100–1,000 <100 0 6 kinalski et al. table 2. comparison of implant-related treatments provided by sus in capitals or inland cities of brazil (number of installed dental implants and number of installed dental prosthesis: datasus*; january 2010 to december 2019) national region capital (state) capital inland total implants prostheses implants prostheses implants prostheses south porto alegre (rs)# 381 216 381 216 florianopolis (sc) 820 87 820 87 curitiba (pr) 611 461 83572 52238 83572 52238 southeast são paulo (sp)# 8873 7349 8873 7349 belo horizonte (mg) 28 20 972 65 972 65 rio de janeiro (rj) 6 5 413 410 413 410 vitória (es) 277 277 0 277 0 central-west campo grande (ms) 44 73 1132 73 1132 73 goiania (go)# 6701 1717 6701 1717 brasilia (df)# 0 0 0 0 cuiaba (mt)# 8234 6862 8234 6862 northeast salvador (ba)# 70 102 70 102 aracaju (se)# 0 0 0 0 maceió (al)# 42 42 42 42 recife (pe)# 175 0 175 0 joão pessoa (pb)# 30154 23521 30154 23521 natal (rn)# 0 0 0 0 fortaleza (ce)# 6 0 6 0 continue... figure 3. number of dental implants (red line) and implant-retained prostheses (green line) provided by sus from january 2010 to december 2019. 2019201820172016201520142013201220112010 prosthesesimplants 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 50000 7 kinalski et al. discussion the present study provides an insight of the sus role in regard to oral rehabilitations using dental implants in the last decade (2010-2019). although the incidence of tooth loss is decreasing worldwide in the last decades14 and, according with the last epidemiological brazilian census (2003 and 2010) it was observed a decline of tooth loss in teenagers and young adults; however, edentulism rates were still raising in the elderly as a result of the increase in life expectancy4,15,16. likewise, it is projected that tooth loss will continue to be a major dental problem in the next decades, which might impair patients general health and cause disabilities17. not only complete edentulism but a reduced number of teeth is directly related to quality of life worsening, since it can compromise daily activities, such as chewing, nutrition, phonation, social life, and self-esteem18,19. although dental implants are recognized as the gold standard approach to replace missing teeth5,6, brazil is one of the few countries in the world that provides free dental implant rehabilitations in the public health system while some countries with higher human development indexes (hdi) do not fully cover such treatments. the costs involved in implant rehabilitations are among the key factors that are considered by patients when choosing their therapy. it is well-known that the vast majority of edentulous persons usually belong to the poorest population stratum and have difficult access to treatment with dental implants20. in brazil, a single dental implant costs on average about 1,000-1,500 brazilian reais (brl), which is approximately 250 us dollars (usd). considering that brazil’s minimum wage is about to 245 usd, the low-income population has no option than to rely on the public health system to obtain their rehabilitations. in a recent review, a single implant rehabilitation was a more cost-effective option compared to a three-unit fixed dental prosthesis to replace a single tooth21. considering the rehabilitation of multiple teeth, dental implants were initially associated with higher initial costs; however, the patient-centered outcomes national region capital (state) capital inland total implants prostheses implants prostheses implants prostheses northeast teresina (pi) 42 5 42 5 42 5 são luis (ma)# 0 0 0 0 north palmas (to)# 0 0 0 0 belém (pa)# 0 0 0 0 macapa (ap)# 1173 638 1173 638 boa vista (rr)# 0 0 0 0 manaus (am)# 0 0 0 0 rio branco (ac)# 0 0 0 0 porto velho (ro)# 0 0 0 0 total 1008 564 141836 93022 143037 93022 #capital without data registered *data extracted on march 17, 2020 continuation 8 kinalski et al. were improved during life-course compared to other treatment options. in this perspective, the present study presents important results about the coverage of implants placement in the national health system. as above stated, although the costs could impact the coverage, the enhancement of dental implants in the sus could represent a key factor to improve the oral health-related quality of life in the brazilian population. in 2016, a previous study showed that the access to dental implants at sus was increasing until that moment, but the distribution was very unequal throughout the country22. our findings update their data, and it is clear that there was an important increase from 2015 to 2016 and from 2016 to 2017 (figure 3). however, the following years showed a clear reduction in the total number of procedures of 30.7% in 2018 and 29.6% in 2019 compared to 2017. it is important to highlight that since 2014 brazil is facing a serious economic crisis and that in 2016 the brazilian government changed its priorities, with a cascade of budget cuts that surrogated the health investments in the following years which could explain the massive reduction of procedures during these years23. bueno et al.24 evaluated the correlation between social and oral health determinants represented by the indicators of the national oral health policy (smiling brazil) and found that, hierarchically, clusters with the best performance in social determinants and oral health outcomes were composed by the brazilian capitals, presenting the highest values of notification of procedures. regions that presented high indexes of social determinants in the years 2000-2010 might have pioneered in the organization and availability of these procedures of medium complexity that, until then, were not offered by the public health system24. the organization and provision of health services are related to the human development of the macro-regions25 and with this, the south and southeast regions present higher rates of use of dental services, with a high number of specialized dental procedures. these findings are also observed in our study, where the south and southeast regions were the regions that provided more dental implant treatments in the last decade while the north region, which presents the lowest social and economic indexes in brazil, has also presented the lowest number of treatments regarding dental implants. it is important to highlight that the brazilian government has public policies to reduce health inequities for the north and northeast regions23,25,26; however, those inequities are evident in our findings. in order to provide information to support objective analyzes, evidence-based decision making, and the development of health action programs, sus have created the brazilian health information databank (datasus). this databank also allowed for decentralization and an improvement in the management of sus activities, contributions, viability, usage of available resources, and it is constantly updated and discloses the information needed for health actions13. as funding is only available after the execution of the procedures and the corresponding input in the databank, the datasus tool can be considered a reliable tool for accounting and analysis of the services provided by sus. however, according to a technical note from the brazilian ministry of health27, non-conformities in outpatient production reported by some municipalities were identified, which would explain the high number of procedures performed in some locations. the processing of outpatient production in the sus outpatient information system (sia/sus) is performed by the local manager, even when 9 kinalski et al. the services were provided by non-governmental companies, and therefore, those responsible were informed of the need to reimburse overpriced amounts. when data are not presented by the cities, it is assumed that the procedures were not carried out since the government’s funding occurs only after the registration and reporting of the performed procedures. consequently, eventual failures in the registration of procedures could underestimate the implants and prosthetic procedures. thus, the misregistration and lack of data of some locations constitutes a limitation of the present study. however, it is our understanding that such limitation impact also the sus management and planning and, therefore, specific actions should be made by sus in order to secure that all cities provide the information to the database. another important limitation of this study is the lack of sociodemographic data available in datasus, since the database does not provide access to patients’ medical records, preventing access to data that would be important to our study, such as gender, age range of individuals, type of rehabilitation (unitary, partial or total), number of implants placed in each patient, and clinical aspects, such as the need of reintervention or clinical success. regarding the type of the rehabilitation, it is important to highlight that sus have only one general code for implant rehabilitations that does not define whether a single crown or a full-mouth rehabilitation was made. thus, it is highly recommended that such code must be revised by sus since the costs of each type of treatment present very different costs and specificities. in this way, we suggest that codes for implant-retained single crowns, implant-retained partial fixed dentures, overdentures and full-arch fixed rehabilitations could be adopted by the sus. the findings of this study suggest that the public policies adopted by sus in the last decade are still far from providing the best treatment option to the population9,10. recent papers by hartmann et al.28,29 have found that the incremental costs for fullarch fixed prosthesis compared to overdentures retained by a single implant is not proportional to the respective gain in effectiveness, and that simplified implant treatments for edentulous patients result in favourable outcomes. considering that, we also suggest the standardization of overdentures retained by a single implant by sus, considering that it would reduce the costs and provide high-quality services to the population. finally, new policies and public actions should be made in order to provide this type of treatment for the brazilian population. in conclusion, although dental implant therapy is available in sus across the country, the number of treatments provided in the last decade is still very limited and is also mainly concentrated in the southeastern region of brazil. acknowledgements this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior – brasil (capes) – finance code 001. references 1. paim j, travassos c, almeida c, bahia l, macinko j. the brazilian health system: history, advances, and challenges. lancet. 2011 may 21;377(9779):1778-97. doi: 10.1016/s0140-6736(11)60054-8. 10 kinalski et al. 2. pucca ga jr, gabriel m, de araujo me, de almeida fc. ten years of a national oral health policy in brazil: innovation, boldness, and numerous challenges. j dent res. 2015 oct;94(10):1333-7. doi: 10.1177/0022034515599979. 3. peres ma, barbato pr, reis sc, freitas ch, antunes jl. [tooth loss in brazil: analysis of the 2010 brazilian oral health survey]. rev saude publica. 2013 dec;47 suppl 3:78-89. portuguese. doi: 10.1590/s0034-8910.2013047004226. 4. cardoso m, balducci i, telles dde m, lourenço ej, nogueira júnior l. edentulism in brazil: trends, projections and expectations until 2040. cien saude colet. 2016 apr;21(4):1239-46. doi: 10.1590/1413-81232015214.13672015. 5. dos santos mbf, agostini ba, de moraes rr, schwendicke f, sarkis-onofre r. industry sponsorship bias in clinical trials in implant dentistry: systematic review and meta-regression. j clin periodontol. 2019 apr;46(4):510-9. doi: 10.1111/jcpe.13100. 6. sarkis-onofre r, marchini l, spazzin ao, santos mbfd. randomized controlled trials in implant dentistry: assessment of the last 20 years of contribution and research network analysis. j oral implantol. 2019 aug;45(4):327-33. doi: 10.1563/aaid-joi-d-18-00276. 7. da cunha mc, santos jf, santos mb, marchini l. patients’ expectation before and satisfaction after full-arch fixed implant-prosthesis rehabilitation. j oral implantol. 2015 jun;41(3):235-9. doi: 10.1563/aaid-joi-d-12-00134. 8. de lima ea, dos santos mb, marchini l. patients’ expectations of and satisfaction with implant-supported fixed partial dentures and single crowns. int j prosthodont. 2012 sep-oct;25(5):484-90. 9. ministry of health of brazil. [health care secretariat ordinance n.718, from dec-12-2010]. brasilia: ministry of health; 2010. portuguese. 10. ministry of health of brazil. [health care secretariat ordinance n.398, from jul-28-2011]. brasilia: ministry of health; 2011. portuguese. 11. von elm e, altman dg, egger m, pocock sj, gotzsche pc, vandenbroucke jp, et al. the strengthening the reporting of observational studies in epidemiology (strobe) statement: guidelines for reporting observational studies. lancet. 2007 oct 20;370(9596):1453-7. doi: 10.1016/s0140-6736(07)61602-x. 12. ministry of health of brazil. [resolution nº 510, from apr-07-2016]. brasilia: ministry of health; 2016. 13. ministry of health of brazil. health unic system. [datasus health information (tabnet)]. [cited 2020 mar 19]. available from: tabnet.datasus.gov.br. portuguese. 14. kassebaum nj, bernabé e, dahiya m, bhandari b, murray cj, marcenes w. global burden of severe tooth loss: a systematic review and meta-analysis. j dent res. 2014 jul;93(7 suppl):20s-28s. doi: 10.1177/0022034514537828. 15. hammerle ch, chen st, wilson tg jr. consensus statements and recommended clinical procedures regarding the placement of implants in extraction sockets. int j oral maxillofac implants. 2004;19 suppl:26-8. 16. mello cc, lemos caa, verri fr, dos santos dm, goiato mc, pellizzer ep. immediate implant placement into fresh extraction sockets versus delayed implants into healed sockets: a systematic review and meta-analysis. int j oral maxillofac surg. 2017 sep;46(9):1162-1177. doi: 10.1016/j.ijom.2017.03.016. 17. tyrovolas s, koyanagi a, panagiotakos db, haro jm, kassebaum nj, chrepa v, et al. population prevalence of edentulism and its association with depression and self-rated health. sci rep. 2016 nov 17;6:37083. doi: 10.1038/srep37083. 18. haag dg, peres kg, brennan ds. tooth loss and general quality of life in dentate adults from southern brazil. qual life res. 2017 oct;26(10):2647-57. doi: 10.1007/s11136-017-1587-4. 11 kinalski et al. 19. rosing k, christensen lb, øzhayat eb. associations between tooth loss, prostheses and self-reported oral health, general health, socioeconomic position and satisfaction with life. j oral rehabil. 2019 nov;46(11):1047-54. doi: 10.1111/joor.12836. 20. de siqueira gp, dos santos mb, dos santos jf, marchini l. patients’ expectation and satisfaction with removable dental prosthesis therapy and correlation with patients’ evaluation of the dentists. acta odontol scand. 2013 jan;71(1):210-4. doi: 10.3109/00016357.2012.654612. 21. vogel r, smith-palmer j, valentine w. evaluating the health economic implications and cost-effectiveness of dental implants: a literature review. int j oral maxillofac implants. 2013 mar-apr;28(2):343-56. doi: 10.11607/jomi.2921. 22. almeida amr, gurgel gsca, campos cg, azevedo guimarães ea. [access to dental implant osseointegrated in the unified health system (sus): description of the national panorama]. arq odontol. 2016;52(3):145-53. portuguese. doi: 10.7308/aodontol/2016.52.3.03. 23. chaves scl, almeida amfl, reis cs, rossi tra, barros sg. oral health policy in brazil: transformations in the period 2015-2017. saude debate. 2018;42:76-91. doi: 10.1590/0103-11042018s206 24. bueno re, moysés st, bueno pa, moysés sj. [social determinants and adult oral health in brazilian state capitals]. rev panam salud publica. 2014 jul;36(1):17-23. portuguese. 25. neves m, giordani jmda, hugo fn. [primary dental healthcare in brazil: the work process of oral health teams]. cien saude colet. 2019 may 30;24(5):1809-1820. portuguese. doi: 10.1590/1413-81232018245.08892017. 26. fernandes jde k, pinho jr, queiroz rc, thomaz eb. avaliação dos indicadores de saúde bucal no brasil: tendência evolutiva pró-equidade? [evaluation of oral health indicators in brazil: a trend towards equity in dental care?]. cad saude publica. 2016 feb;32(2):e00021115. 27. ministry of health of brazil. [technical note/cgsb/dab/sas/ms: nº 23/ 2017. information on implantology procedures in sus]. brasilia: ministry of health; 2017 [cited 2020 sep 11]. available from: http://189.28.128.100/dab/docs/portaldab/documentos/nt_cgsb_23_2017.pdf. portuguese. 28. hartmann r, bandeira acfm, araújo sc, brägger u, schimmel m, leles cr. a parallel 3-group randomised clinical trial comparing different implant treatment options for the edentulous mandible: 1-year effects on dental patient-reported outcomes and chewing function. j oral rehabil. 2020 aug 9. doi: 10.1111/joor.13070. 29. hartmann r, de menezes bandeira acf, de araújo sc, mckenna g, brägger u, schimmel m, et al. cost-effectiveness of three different concepts for the rehabilitation of edentulous mandibles: overdentures with 1 or 2 implant attachments and hybrid prosthesis on four implants. j oral rehabil. 2020 aug 9. doi: 10.1111/joor.13071. 1http://dx.doi.org/10.20396/bjos.v20i00.8661654 volume 20 2021 e211654 original article 1 department of prosthodontics and periodontics, periodontics division, piracicaba dental school, university of campinas, piracicaba, sp, brazil. 2 department of dentistry, university of araras, araras, sp, brazil 3 department of periodontics, paulista university, são paulo, sp, brazil. corresponding author: renato corrêa viana casarin department of prosthodontic and periodontics avenida limeira, 901 – areião, piracicaba, zc 13414-903 email: rcasarin@unicamp.br phone: (19) 2106-5301 received: october 15, 2020 accepted: december 30, 2021 influence of rs6667202 snp on interleukin-10 levels in the gingival fluid of patients with periodontitis grade c camila schmidt stolf1 , tiago taiete1,2 , márcio zaffalon casati1,3 , enílson antônio sallum1 , francisco humberto nociti júnior1 , karina gonzales silvério ruiz1 , renato corrêa viana casarin1,* grade c periodontitis in youngers is characterized by a severe form of periodontitis, and il10 rs6667202 single nucleotide polymorphism (snp) has been described as an important feature in this disease etiology. aim: this study aimed to evaluate, in vivo, the functionality of il10 rs6667202 snp on il-10 gingival fluid levels. methods: thirty patients with perio4c were selected, 15 with the il10 aa genotype (rs6667202) and 15 with ac/cc genotypes. the gingival fluid was collected from two sites with probing depth ≥ 7 mm and bleeding on probing, and two healthy sites. the il-10 concentration was determined by luminex/magpix platform. results: in deep pockets, the il10 aa genotype presented a lower concentration of il-10 when compared with ac or cc genotypes (p<0.05). in shallow pockets, no difference between groups was seen (p>0.05). conclusion: il10 rs6667202 snp decreases the production of il-10 in crevicular fluid, potentially affecting this disease progression. keywords: aggressive periodontitis. interleukin-10. polymorphism, single nucleotide. https://orcid.org/0000-0002-5125-2326 https://orcid.org/0000-0003-2173-4620 https://orcid.org/0000-0001-9234-0536 https://orcid.org/0000-0003-3827-7091 https://orcid.org/0000-0001-6809-8866 https://orcid.org/0000-0001-5879-9095 https://orcid.org/0000-0003-1743-5855 2 stolf et al. introduction periodontitis is an inflammatory disease of multifactorial etiology, triggered by the host’s immune-inflammatory response to periodontopathogens present in the biofilm. it is clinically characterized by bone destruction and loss of conjunctive insertion, and if untreated, lead to tooth loss1. periodontitis stage 3 or 4, grade c (perio4c), previously named aggressive periodontitis2,3, is a severe form of periodontitis, affecting young systemically healthy individuals4. as additional characteristics, it presents a rapid rate of progression, familial aggregation of cases, and poor response to traditional therapeutic approaches4-8, leading to difficulties in the clinical management and early edentulism9. these patients’ host response presents a hyperinflammatory profile, with an imbalance between the release of pro and anti-inflammatory cytokines after the activation of inflammatory cells by periodontopathogens10-12. the host’s immune-inflammatory response is influenced by genetic factors, which determine different susceptibility profiles to the diseases’ development. the different allelic variants, such as the presence of a single nucleotide polymorphism (snp), may predispose the development of periodontitis by impacting the structure, function, or production of proteins13-15. previous studies demonstrated an association between the il10 rs6667202 snp (c>a) located in the il10 gene and the perio4c in a brazilian and a pooled german-austrian sample16-17. the ancestral c allele was detected in a lower frequency in perio4c patients when compared to individuals with periodontal health and chronic periodontitis, making it a potential protector against the occurrence of this disease16-17. this protective character could be explained by interleukin-10 (il-10) action, once this cytokine presents regulatory and anti-inflammatory properties, reducing the expression of pro-inflammatory cytokines such as interleukin 1β (il-1β) and tumor necrosis factor α (tnf-α). given its function, its greater or lesser expression is capable of modulating disease severity18-19. however, up to date, none functional analysis has been done. since rs6667202 snp is in the upstream genic region of the il10 gene (1.3 kb region), a regulatory region, it can be speculated that it may impact the expression of il-10 cytokine. this possibility is indirectly supported by other previous studies, which demonstrated that perio4c patients had reduced levels of il-10 in gingival crevicular fluid (gcf) and serum when compared to other periodontal profiles11,20-22. casarin et al.20 (2010) demonstrated a lower production of il-10 in the gingival crevicular fluid (gcf) in moderate and deep pockets of patients diagnosed with perio4c when compared with similar sites of individuals with chronic periodontitis. however, these findings were not based on genetic characteristics. it is impossible to determine whether there is a direct relationship between the presence of the polymorphism and differences in cytokine production. this type of relationship has been demonstrated for a pro-inflammatory cytokine, interleukin 6 (il-6), by nibali et al.15 (2013). in this study, the authors reported that perio4c patients with rs2069827 and rs2069825 il6 snps had higher levels of il-6 when compared with perio4c negative for both snps patients15. 3 stolf et al. therefore, this study aimed to evaluate, in vivo, the functionality of il10 rs6667202 snp on gcf levels, that is, assess whether il10 rs6667202 snp aa genotype may predispose an altered production of this anti-inflammatory marker in patients with perio4c when compared to those with ac or cc genotypes. material and methods patient selection this study was approved by the piracicaba dental school, university of campinas research ethics committee (number 58679416.4.0000.5418). for this cross-sectional study, 30 subjects were selected from taiete’s et al.16 (2019) study population, which determined the il-10 genotyping for 200 patients diagnosed with perio4c and 200 healthy patients. all selected patients signed an informed consent form to participate in this research, and during its execution, the principles of the declaration of helsinki were followed. the 30 selected subjects were allocated to: snp + group (n=15): perio4c with aa genotype (rs6667202). snp group (n=15): perio4c with ac or cc genotypes (rs6667202). the inclusion criteria for the perio4c group were the presence of true periodontal pockets and radiographic bone loss in patients up to 35 years of age; the presence of at least eight teeth with probing depth (ps) ≥ 5 mm (of which two teeth must have ps ≥ 7 mm) and bleeding on probing in 3 teeth not contiguous to the first molars and incisors; the presence of at least 20 teeth in the oral cavity. the exclusion criteria were the presence of systemic changes (diabetes, heart disease, hepatitis) or use of medications (such as antibiotics, anti-inflammatory drugs, phenytoin, cyclosporine) that may influence the response to periodontal treatment in the six months before the study; periodontal treatment including subgingival instrumentation in the six months before the study; smoking habit, pregnancy, or lactation period. collect of oral epithelial cell samples the patients rinsed with 5 ml of 3% dextrose for 1 minute. afterward, the solution was centrifuged for 10 min at 3000 rpm to sediment oral epithelial cells, as described by trevilatto and line23, 2000. dna extraction and genotyping for rs6667202 snp detection the oral epithelial cell samples were incubated at 55oc overnight in 1ml of lysis solution [10mm tris (ph 8.0), 0.5% sds, 5mm edta] with 10µl proteinase k (20mg/ml) (sigma chemical co., st. louis, mo, usa). after incubation, proteins and contaminants were removed by adding 500μl of 8m ammonium acetate solution and 1mm edta. the genomic dna was precipitated in 540μl of isopropanol. the dna was washed in 70% ethanol and resuspended in 50μl te ph 8.0 (10mm tris and 1mm edta). in the end, the amount of purified dna and its concentration were measured using a nanodrop spectrophotometer (thermo scientific). 4 stolf et al. the genotype for the rs6667202 variation was determined through dna polymerase chain reaction (pcr) in real-time, using the taqman® system for allele discrimination (applied biosystems, carlsbad, ca, usa). primers and probes were designed using the primer 3 program, allowing amplification of the regions where the variation is located. the taqman pcr was performed in a volume of 12µl (3ng of dna, 1x taqman master mix, 1 x assay mix, 900nm of each primer, and 200nm of each probe) and distributed in 96 wells. the fluorescence of pcr amplification was detected using stepone plus (applied biosystems, carlsbad, ca, usa) and analyzed with the manufacturer’s software. collect of gcf after removing the supragingival biofilm, the teeth were washed, and the area was isolated with cotton rollers and dried with air jets. gcf was collected through the insertion of filter paper strips (periopaper, oraflow) into the periodontal pocket or gingival sulcus for 15 seconds. samples were collected from four sites in each patient, two sites with pockets of pd ≥ 7mm and bleeding probing, two healthy sites with pd ≤ 3mm and no bleeding probing. the collected fluid volume was measured with a calibrated device for measurement of gingival fluid (periotron 8000, oraflow). the strips were stored in 400μl of phosphate-buffered saline (pbs) with 0.05% tween-20. strips contaminated with visible blood were discarded, and a new sample was collected after 30 seconds. all samples were stored at -80oc and remained under these conditions until the time of analysis. immunoenzymatic analysis aliquots of each gcf sample were analyzed for il-10 by luminex/magpix technology. for this, the analyzes were performed in 96-well plates with the aid of high-sensitivity panels (hscytomag 60k, millipore corporation, billerica, ma, usa), following the manufacturer’s instructions. briefly, the wells were washed with wash buffer, and aspirated and exclusive microspheres conjugated to monoclonal antibodies against the analyte (il-10) were added. samples and reagents for the standard curve were pipetted into the wells and incubated overnight at 4oc. then, the wells were washed, and a mixture of secondary antibodies was added. after incubation for 1 hour, the final detection was done through a third fluorescent marker, streptavidin-phycoerythrin, bound to the detection antibody. the concentration of il-10 was expressed in pg/ml. samples with quantification below the detection limit of the analysis were recorded as “zero”, and samples above the limit of quantification of the standard curve were recorded with a value equal to the curve’s highest value. statistical analyses the data from the immunoenzymatic assay were analyzed for normality by the shapiro-wilk test, and a comparison of il-10 levels between groups was performed using the t-student test. all tests were performed considering the significance level of 5%. results table 1 shows the clinical and demographic data of perio4c patients who participated in the study, indicating a similarity between the groups (p>0.05). 5 stolf et al. figure 1 shows the mean and standard deviation for shallow (1.a) and deep (1.b) pockets in the snp + and snp groups. regarding il-10 levels, in shallow pockets, there was no significant difference in il-10 gcf concentration between snp + and snp groups (0.05±0.04 and 0.15±0.32, respectively, p>0.05 figure 1.a). however, in deep pockets, the presence of rs6667202 snp (snp + group) was associated with lower levels of il-10 levels in the gcf when compared to the individuals with ac or cc genotypes (snp group) (0.005±0.003 and 0.01±0.01, respectively, p=0.03 figure 1.b). thus, based on the fact that in the deep pockets, the ac or cc genotypes had a higher level of il-10 when compared to aa genotype perio4c individuals, the protective character of the ancestral c allele for this disease could be firstly explained. discussion although the prevalence of perio4c appears to be exceptionally low in developed countries24, less developed regions show a higher frequency of this disease, where up to 5% of the population could be affected25,26. epidemiological studies indicate that the prevalence of perio4c can reach 5.5% of young individuals in the brazilian population, and among young people aged 29-34 years, this prevalence can reach 9.9%25. these percentages show that, especially in brazil, there is a greater urgency to meet the table 1. demographic and clinical full mouth data of study participants. characteristics aa group ac group age (years) 34.6 ± 4.5 34.4 ± 4.4 gender (m/f) 5/10 4/11 plaque index (%) 21.3 ± 6.5 24.4 ± 6.4 bleeding index (%) 26.8 ± 9.0 22.4 ± 7.9 probing depth (mm) 2.5 ± 0.5 2.2 ± 0.2 probing attachment loss (mm) 5.1 ± 1.1 5.6 ± 0.9 there was no significant difference between groups (student’s t and chi-square tests, p>0.05). * indicates the statistical difference between groups. figure 1. levels of il-10 in the gcf in shallow pockets (pd ≤ 3 mm) (a) and deep pockets (pd ≥ 7 mm) (b) of perio4c aa genotype (rs6667202) patients (snp + group) and perio4c ac or cc genotype (rs6667202) patients (snp group). pg /m l shallow pockets 0.5 a 0.4 0.3 0.2 0.1 0 snp + group snp group pg /m l shallow pockets 0.020 b 0.015 0.010 0.005 0 snp + group snp group * 6 stolf et al. demands caused by perio4c. however, with this higher proportion of affected subjects, achieving a large population is always a difficult deal in research, especially with rigid inclusion criteria. some previous studies have been done to identify one or more snps associated with this condition, and only a few in brazilians17,27-30. traditionally, genetic research in periodontics has focused on identifying specific genetic changes, mainly from snps, presented as risk factors for periodontitis14,15,30. despite these efforts, the genetic factors contributing to periodontal diseases’ pathogenesis, especially perio4c, are not yet fully defined29. in this sense, the current evidence suggests that perio4c is polygenic, like other complex diseases, with the involvement of multiple genes with little effect (usually more than 100 genes for complex diseases)27. some studies have recently used a broader or large-scale assessment approach, the genome-wide association studies (gwas), reporting the association of polymorphisms in genes hitherto not associated with perio4c but which need their validation in independent populations14,17,28. using this approach, schaefer et al.28 (2010) conducted the first gwas study in perio4c, involving german individuals. it was observed that the genetic variant rs1537415 in the glt6d1 gene, which indirectly controls the differentiation of th2 cells, could be related to perio4c. in the same context, rs1333048 snp, in the cdkn2b antisense rna 1 (anril) gene, regulates mechanisms associated with colonization of the subgingival biofilm by periodontopathogens, also were associated with perio4c in a european population30. in 2013, schaefer et al.17 (2013) showed an association between rs6667202 il10 gene snp and perio4c in a german-austrian cohort. however, this association was not significant in some geographically close populations, as in the dutch cohort. these results indicate the importance of validating snps for specific populations, once different regions could present a different genetic background. the brazilian population alone is highly miscegenated, resulting in genetic heterogeneity compared to other countries. thus, our group conducted a study aiming to identify if there could be any association between the snps previously reported and the perio4c in brazilians. we found that the only snp previously associated with perio4c that showed to be significant to this population was the il10 snp, rs6667202, and not glt6d1 rs1537415 and anril rs1333048 snps16, unlike what was found in other populations17,28,30. meanwhile, the populational association between snp and any disease relies on other aspects – how the snp could modify etiologic patterns and clinically impact disease occurrence. in this effort, this study assessed the local il-10 levels in perio4c affected-subjects to confirm rs6667202 functionality. genetic changes may have a direct influence on the immune response of patients affected by perio4c13. these statements can be observed in studies that seek to understand the functionality of genetic variants, that is, how these variations can influence cellular functioning and/or alter microbiological colonization. no functional analysis had been performed to understand better the relationship between the snp rs6667202 and the production of interleukin-10. since this snp is in a regulatory region of the il-10 gene, we can presume an altered production of this cytokine when this snp is present. when we analyze the results of this study, we notice an altered production of il-10 in the snp + group, which presents the aa genotype compared to 7 stolf et al. the snp – group, presenting ac or cc genotype (0.005±0.003 and 0.01±0.01, respectively, p=0.03 figure 1.b). this fact reinforces the protective character of the ancestral c allele. it corroborates with the previous findings made by taiete et al.16 (2019), where this allele was detected less frequently in perio4c patients since, in this study, the aa group had a lower dosage of this anti-inflammatory cytokine. however, this result was dependent on pocket strata, i.e., il-10 levels in gcf were differentially modulated by rs6667202 snp in deep and shallow pockets. in shallow pockets, there was no significant difference in il-10 production between snp + and snp groups (0.05±0.04 and 0.15±0.32, respectively, p>0.05 figure 1.a). it could be related to the fact that a symbiotic community is colonizing the subgingival environment in healthy sites and does not pose a challenge to the host immune system20,31,32. when we analyze the shallow pockets in figure 1, we see a significant difference between the mean values of il-10 in both groups. however, the standard deviation was high, especially for the snp group; that is, there was a significant impact of the data variability on the results. it may be related to the presence of pathogens and the onset of a dysbiotic environment at the gcf collected sites of some patients, who have not yet shown clinical changes at these sites despite already having an altered inflammatory response. the same thought could be attributed to deep pockets, where a mature and dysbiotic biofilm instigates the host response, but now leading to clinical alterations. it is well known that il-10 is highly expressed in inflamed periodontal tissues and acts as the main inhibitor of pro-inflammatory and harmful cytokines in the context of periodontal destruction. it can downregulate the synthesis of pro-inflammatory cytokines and chemokines, such as interleukin 1 (il‐1), il‐6, tnf‐α, nitric oxide, gelatinase, and collagenase33, and upregulate the synthesis of il‐1 and tnf‐α when specifically neutralized34. in addition, il-10 has a major impact on the adaptive immune response. it led b cells to differentiate and proliferate, reducing apoptosis of immune cells and stimulating cd8+ and natural killer cells18,19. thus, it could be suggested that when the subject presents the aa genotype, il-10 production is reduced, altering the immune response against microbial aggression in periodontal pockets, and inducing more severe destruction. corroborating to this pathological pathway, several immunological and microbiological factors have been strongly associated with perio4c, which can be associated with the occurrence and the low response of conventional periodontal treatment. in addition to the study of casarin et al.20 (2010) that showed reduced il‐10 levels in gcf associated with low production of immunoglobulin g (igg) against pg and aa in perio4c patients, other studies have also suggested a reduction in il‐10 expression both in gcf, blood serum, and gingival tissue21,22 in these individuals. these findings can probably be related to polymorphisms associated with perio4c, some of them described in previous studies15,35. for example, the haplotype ata of il‐10, as a “low interleukin‐10 producer”, was proved to be a risk indicator for perio4c35, emphasizing the importance of il‐10 in perio4c etiology. at the moment, this discovery indicates a better understanding of the pathogenesis of this condition. it soon will instigate other in vitro analysis to better comprehends it cell behavior and its relationship with the presence of pathogens in perio4c, and with a 8 stolf et al. larger population, to widely confirm its role on disease etiopathogenic aspects, allowing the identification of patients with greater susceptibility and establishing an individualized periodontal support therapy according to patient’s biological characteristics. we conclude that il10 rs6667202 snp is associated with lower production of il-10 in crevicular fluid, potentially affecting this disease progression and reinforcing the protective character of the ancestral c allele evaluated before. references 1. armitage gc. development of a classification system for periodontal diseases and conditions. ann periodontol. 1999 dec;4(1):1-6. doi: 10.1902/annals.1999.4.1.1. 2. albandar jm. aggressive and acute periodontal diseases. periodontol 2000. 2014 jun;65(1):7-12. doi: 10.1111/prd.12013. 3. caton jg, armitage g, berglundh t, chapple ilc, jepsen s, kornman ks, et al. a new classification scheme for periodontal and peri-implant diseases and conditions introduction and key changes from the 1999 classification. j 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zimmermann u, reichert y, gläser ch, et al. the interleukin-10 promoter haplotype ata is a putative risk factor for aggressive periodontitis. j periodontal res. 2008 feb;43(1):40-7. doi: 10.1111/j.1600-0765.2007.00992.x. untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8652648 volume 17 2018 e18127 original article 1 dds. department of dental materials and prosthodontics, araçatuba school of dentistry, são paulo state university – unesp, araçatuba, são paulo, brazil; 2 dds, ms, phd student. department of restorative dentistry, araçatuba school of dentistry, são paulo state university – unesp, araçatuba, são paulo, brazil; 3 dds, ms, phd, post-doctoral student. department of dental materials and prosthodontics, araçatuba school of dentistry, são paulo state university – unesp, araçatuba, são paulo, brazil; 4. associate professor, department of dental materials and prosthodontics, araçatuba school of dentistry, são paulo state university – unesp, araçatuba, são paulo, brazil. 5. associate professor, department of restorative dentistry, araçatuba school of dentistry, são paulo state university – unesp, araçatuba, são paulo, brazil. corresponding author: paulo henrique dos santos address: josé bonifácio st, 1193. araçatuba-sp, brazil. zip code: 16015-050 phone number: 5518 3636-2802 facsimile number: 5518 3636-3245 e-mail: paulosantos@foa.unesp.br received: november 17, 2017 accepted: february 15, 2018 microshear bond strength of conventional and self-adhesive resin cements to feldsphatic ceramic estéfani maminis soares couto1, bruna de oliveira reis2, thaís yumi umeda suzuki3, wirley gonçalves assunção4, andré luiz fraga briso5, paulo henrique dos santos4. aim: the aim of this study was to verify the microshear bond strength of conventional and self-adhesive resin cements to feldsphatic ceramic. methods: twenty discs of starlight ceramic (degudent) were made (15mm x  2mm). the bonding procedure was accomplished by insertion of resin cements into tubes  of 0.7mm internal diameter in contact with the ceramic. the resin cements used were: relyx arc (3m espe), panavia f (kuraray), relyx unicem (3m espe) and set (sdi). total of six tubes of each material on each ceramic disc.  the specimens were tested for microshear in a universal testing machine, 24hours and 6months after bonding procedure.  values of microshear bonding strength (mpa)  were subjected to anova and fisher plsd test (p<0.05). results: for 24hours analysis, relyx arc  showed the highest microshear bonding strength without statistical difference to relyx unicem and set.  panavia f showed the lowest values of microshear bonding strength in both periods. in the analysis after 6months there was a significant increase in the microshear bonding strength values for all cements compared to 24hours. only for relyx arc, the increase was not statistically significant. conclusion: in conclusion, the bond strength depends mainly on the type of resin cement used, and the self-adhesive cements behave similarly to conventional resin cement. keywords: resin cements. ceramics. dentin-bonding agents. shear strength. 2 santos et al. introduction the restorative dentistry has been changed in the last years, with significant improvement in the esthetic issue, biocompatibility and strength of direct and indirect restorative materials1. dental ceramics has been the choice material for aesthetic restorations  because of their optical properties.  the chemical stability and resistance to wear make the ceramic the indicated for both anterior and posterior areas, in which aesthetic demand  and strength are necessary2. traditionally, dental porcelain is composed by two distinct phases: crystalline phase, responsible for strength, and a glass phase, primarily responsible for the translucency of the material. polycrystalline ceramics are basically characterized by having a larger amount of crystalline phase as alumina, leucite, lithium disilicate and zirconia3. improvements in the clinical performance of ceramic restorations are closely related with the use of adhesive resin materials for luting procedures, including a photoactivation method and efficiency of the adhesive system4. the use of resin cements is preferred because they offer many advantages, as the ability to micromechanical bond to tooth structure, low solubility and greater strength to wear compared to conventional dental cements5. the resin cements are divided into conventional and self-adhesive materials and involve two bonding interfaces: tooth structure/resin cement and resin cement/restorative material6. the conventional resin cements require prior treatments of the tooth surface. the bonding of these materials to the tooth is dependent of an adhesive system, following the same principles of direct composite resin restorations. the bonding of the resin cement to the restoration depends on the type of ceramic material and the treatments performed on the internal surface which include roughness with a diamond bur, sandblasting with aluminum oxide, etching with hydrofluoric acid, ammonia bifluoride or acidulated phosphate fluoride, silanization and ultrasonic cleaning7. this total-etch adhesive protocol of conventional resin cements is complex, require multiple steps and may make the effectiveness of bonding vulnerable8. the self-adhesive resin cements were developed to simply the bonding procedures, once do not require any pre-treatment of dental substrate, reduces clinical steps and the “window of contamination”8,9. the cementation can be accomplished in a single step8. the organic matrix of these materials consists of a multifunctional monomer (generally a phosphoric acid ester methacrylate). this can react with the filler of the resin cement as well as hydroxyapatite of dentin and enamel10.  following the trend to simplify the technique of luting, many manufacturers launched their versions for the self-adhesive cements. usually most of scientific papers that involve the study of indirect restorative materials such as dental ceramics and their adhesive procedure focus in the investigation of the effectiveness of these adhesive materials to bond in tooth substrates, with few studies focusing the other bonding interface between resin materials and dental ceramics. thus, the present study aimed to evaluate the microshear bonding strength between conventional and self-adhesive resin cements to dental ceramics, 24 hours and 6 months after the bonding procedure. two null hypotheses were tested: (1) different resin cements did not have difference in the bonding strength to dental ceramics and (2) the bonding strength of resin cements to dental ceramics measured after 6 month of bonding procedure did not have difference for the measurements done after 24 hours. 3 santos et al. materials and methods twenty discs of starlight feldspathic ceramic (degudent gmbh, hanau-wolfgang, germany)  were made using a silicone matrix, measuring 15mm in diameter and 2mm in thickness. a centurion furnace (degussa-ney dental, yucaipa, ca, usa) was used for the ceramics sintering, following the time-temperature scale recommended by the manufacturer. after sintering, the samples were finished and polished with ninja points (talladium do brasil, curitiba, pr, brazil), #104023 diamond points flame-shaped and #35 inverted cone-shape. the measurements of the samples were checked with a digital caliper (mitutoyo, suzano, sp, brazil). one of the ceramic surfaces was etched with 10% hydrofluoric acid (porcelain conditioner, angelus, londrina, pr, brazil)  during 2 minutes. after the acid etching, the specimens were washed with air-water spray during 60 seconds and dried with compressed air for 30 seconds, and then submitted to the silanizing agent application (ceramic primer, 3m espe, st. paul, mn, usa) during 1 minute. the discs were divided into four groups, according to the resin cement tested (n=5). the bonding process of resin cements to ceramic  was performed by the insertion of resin cements in tygon®-microbore (tgy-030, small parts inc., miami lakes, fl, usa) tubes with 0.7mm of internal diameter, 2.1mm of external diameter and 1.0mm of height, located on the prepared surface of the ceramic. four resin cements were used: relyx arc (3m espe) and panavia f (kuraray, kurashiki, japan) conventional resin cements, relyx unicem (3m espe) and set (sdi, bayswater victoria, australia) self-adhesives resin cements. six tubes of each cement were placed in each ceramic specimen. the photoactivation of cement was performed using ultraled ii light-curing unit (dabi atlante, ribeirão preto, sp, brazil), with 700 mw/cm2 light output intensity for 40 seconds, through the ceramic surface. the photoactivation process was conducted in a darkened environment, so the external ambient light has not influenced the degree of conversion of the resin cement. the specimens were stored in deionized water at 37°c for 24 hours. the specimens  were submitted to microshear bonding test in a universal testing machine (dl 3000, emic, são josé dos pinhais, sp, brazil) at a crosshead speed of 1.0 mm/min. for the test, each specimen was horizontally positioned in a metal glove and an orthodontic wire with 0.3mm forming a loop surrounding the cylinder of resin cement composite has performed the tensile stress resulting in a microshear bonding test. the microshear bonding strength (mpa) was calculated by: f/a where: f was the force applied (n); and a, the area of bonding (mm²). to perform the microshear bonding strength after 6 months, the same ceramic discs were worn with no. 80-, 320and 600-grit silicon carbide paper (extec corp, enfield, ct, usa) and then submitted to same surface treatment  with hydrofluoric acid and silane application. resin cements were positioned in tygon®-microbore tubes as the same manner as above described. the specimens were stored in deionized water at 37ºc for 6 months, changed weekly, until the microshear bonding test. data were submitted to the kolmorov-smirnov normality test and the averages compared by anova and fisher´s plsd test (p=0.05). two factors were studied: material (relyx arc, panavia f, relyx unicem and set) and time (24 hours and 6 months).  4 santos et al. results anova (table 1) showed that there was a statistically significant difference between the cements studied (p<0.001) as well as between the evaluated times (p<0.001). the interaction between the groups was not statistically significant (p=0.94). at 24 hours, relyx arc showed the highest microshear bonding strength without statistical difference to relyx unicem and set (p>0.05). the lowest values were obtained for panavia f with significant difference for the other cements (p<0.001). these data are shown in table 2. the same phenomenon occurred to the specimens analyzed 6 months after the bonding procedure, in which panavia f showed the lowest value of microshear bonding strength, compared to the other cements studied. a significant increase in the values of microshear bonding strength was found 6 months after the bonding procedure compared to the 24 hour for all resin cements studied (table 2). just for relyx arc, this increase was not statistically significant (p=0.134).  discussion the success of the luting procedure  is dependent of a strong and durable bonding strength in both interfaces between resin cement to ceramics and between resin cement to tooth11. in this study, the focus was to evaluate the bonding strength in the resin cement-ceramic interface. currently, the bonding process between feldspathic ceramic to resin cements is provided by etching the ceramic surface with hydrofluoric acid, followed by silane agent application. both have the property to increase the wetting of the cement on the surface, facilitating the contact between the materials. furtable 2.  means of microshear bonding strength (mpa) between the resin cements and feldspathic ceramic, 24 hours and 6 months after the bonding procedure. microshear bonding strength (mpa) relyx arc panavia relyx unicem set 24 hours 21.13 ± 4.77 a a 8.40 ± 3.75 a b 20.86 ± 0.43 a a 19.12 ± 1.98 a a 6 months 26.54 ± 5.45 a a 13.18 ± 2.92 b b 27.11 ± 2.81 b a 25.60 ± 2.06 b a distinct letters, uppercase in the column and lowercase letter in the line, show a statistically significant difference between them (p<0.05). table 1.  anova results for microshear bonding strength (mpa). df sum of squares mean square f-value p-value lambda power groups 3 1191.924 397.308 34.491 <.0001 103.472 1.000 time 1 318.310 318.310 27.633 <.0001 27.633 1.000 groups * time 3 4.499 1.500 .130 .9414 .391 .071 residual 31 357.099 5 santos et al. thermore, the silane performs a chemical bonding between the silica contained in ceramic and the organic matrix of resin cements,  through siloxane bonding12,13.  for this reason, all ceramic specimens received the same surface treatment,  based on application of hydrofluoric acid followed by silanization. based on the results of this study, it was found that panavia f showed the lowest value of microshear bond strength in both periods studied (table 2). in the other hand, relyx arc showed the highest value of microshear bond strength after 24 hours and 6 months, without significant difference to relyx unicem and set cements. the difference in the behavior of conventional cements could be related to the formulation and their viscosity.  the difference in composition is inherent to the materials and could not be compensated by the polymerization, although an increased viscosity of the material could restrict the photoactivation process14. the conventional cements contain in their basic composition,  resin monomers as bis-gma (bisphenol a glycidyl methacrylate) or udma (urethane dimethacrylate). self-adhesive resin cements have in their composition phosphoric acid monomers as mdp or meta15. kern and thompson16  (1995) related in their studies that mdp-modified resin cements, as panavia f, has better adhesion to aluminized surfaces than conventional resin cements based on bis-gma (as relyx arc), because of the existence of chemical bonding between mdp and alumina. furthermore,  phosphate ester groups of mpd-modified resin cement have been described in the literature as able to bond directly to metal oxides17. however, this benefit may be limited when it bonds to feldspathic ceramics, as used in this study. the high viscosity shown by panavia f could not allow sufficient flow, negatively affecting the bonding to ceramics, as shown also in dentin interface18.  this could probably explain the lower values found for panavia f in this study. regarding the self-adhesive resin cements, relyx unicem and set did not show significant differences between them, but showed higher values of bonding strength than panavia f in both analyzed periods (table 2). one of the factors that could explain the performance of the self-adhesive is the way of commercial presentation.  they are available in the market in capsules, which require pre-activation, and need a mechanical device for mixing procedure,  with no manual mixing.  the mechanical mixing enables greater effectiveness for the cement, once the manual manipulation  could lead the formation and entrapment of air bubbles creating voids in the adhesive interface and therefore, interference in the performance of the material19. the analysis of the microshear bonding strength after 6 months showed a statistically significant increase in the values compared to the values after 24 hours for all cements studied, except for relyx arc (table 2). the late polymerization may be the main responsible for the increase of the bonding strength over time,  once the photoactivation provides the generation of free radicals responsible  for the induction of chemical polymerization20. according to arrais et al.21 (2009), the initial exposure to light may cause a change  in the viscosity of dual-cured materials,  making difficult the migration of active radicals, which could lead to a delay in the process of polymerization. this study has showed some limitations  as the difficulty of load standardization applied during the resin cement insertion inside the microtubes representing the pressure during the luting procedure,  the fixation of the tube so the cement could 6 santos et al. be adequately inserted and the removal of the tygon®-microbore  tubes  before the microshear bonding test. other aging process as thermal and mechanical cycling, are also necessary to guarantee the longevity of the bonded interface studied 22, since in the present study, only the storage in water was realized. however, it could be seen that the inherent properties of the resin cement affects the final performance of the bonding interface. conclusion based on the results, it can be observed that time had a directly influence in the bonding strength between resin cement and feldsphatic ceramic.  for all resin cements, except for relyx arc,  there was an increase of microshear bonding strength over time. panavia f resin cement showed the lowest value of microshear bonding strength compared to the other resin cements in both times analyzed. the characteristics of these materials have influenced its short and long-term performance. acknowledgements this study was supported by fapesp (2009/17826-3). conflicts of interest the authors declare no conflict of interest. references 1. seemann r, flury s, pfefferkornb f, lussia a, noackc mj. restorative dentistry and restorative materials over the next 20 years: a delphi survey. dent mater. 2014 apr; 30(4):442-8. doi: 10.1016/j.dental.2014.01.013. 2. nayar s, aruna u, bhat wm. enhanced aesthetics with all ceramics restoration. j pharm bioallied sci. 2015 apr;7(suppl 1):s282–4. doi: 10.4103/0975-7406.155957. 3. kim rj, woo js, lee ib, yi ya, hwang jy, seo dg. performance of universal adhesives on bonding to leucite-reinforced ceramic. biomater res. 2015 may;19:11. doi: 10.1186/s40824-015-0035-1. 4. kuguimiya rn, rode km, carneiro pm, aranha ac, turbino ml. influence of curing units and indirect restorative materials on the hardness of two dual-curing resin cements evaluated by the nanoindentation test. j adhes dent. 2015 jun;17(3):243-8. doi: 10.3290/j.jad.a34399. 5. tian t, tsoi jk, matinlinna jp, burrow mf. aspects of bonding between resin luting cements and glass ceramic materials. dent mater. 2014 jul;30(7):e147-62. doi: 10.1016/j.dental.2014.01.017. 6. pavan s, dos santos ph, berger s, bedran-russo ak. the effect of dentin pretreatment on the microtensile bond strength of self-adhesive resin cements. j prosthet dent. 2010 oct;104(4):258-64. doi: 10.1016/s0022-3913(10)60134-5. 7. leite fp, özcan m, valandro lf, moreira ch, amaral r, bottino ma et al. effect of the etching duration and ultrasonic cleaning on microtensile bond strength between feldspathic ceramic and resin cement. j adhes. 2013:89(3):159-73. 8. bellan mc, cunha pfjsd, tavares jg, 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resin cements to caries-affected dentin. j prosthet dent. 2013 jul; 110(1):47-55. doi: 10.1016/s0022-3913(13)60339-x. 19. aksornmuang j, nakajima m, foxton r, tagami j. mechanical properties and bond strength of dualcure resin composites to root canal dentin. dent mater. 2007 feb;23(2):226-34. 20. moraes rr, brandt wc, naves lz, sobrinho lc, piva e. light-and time-dependent polymerization of dual-cured resin luting agent beneath ceramic. acta odontol scand. 2008 oct;66(5):257-61. doi: 10.1080/00016350802241563. 21. arrais cag, giannini m, rueggeberg fa. kinetic analysis of monomer conversion in autoand dualpolymerizing modes of commercial resin luting cements. j prosthet dent. 2009 feb;101(2):128-36. doi: 10.1016/s0022-3913(09)60008-1. 22. blumer l, schmidli f, weiger r, fischer j. a systematic approach to standardize artificialaging of resin composite cements. dent mater. 2015 jul;31(7):855-63. doi: 10.1016/j.dental.2015.04.015. untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8652886 volume 17 2018 e18082 original article 1 piauí state university – uespi, school of dentistry, department of clinical dentistry, area of integrated clinic, parnaíba, pi, brazil corresponding author: ana de lourdes sá de lira universidade estadual do piauí, faculdade de odontologia rua senador joaquim pires 2076 ininga. fone (86) 999595004 cep: 64049-590 teresina-pi-brasil email: anadelourdessl@hotmail.com received: november 12, 2017 accepted: june 24, 2018 prevalence and etiology of dental trauma in schoolchildren aged 6 to 12 years ana de lourdes sá de lira1, luís paulo da silva dias1, cassius wander coelho martins1, tito cacau sousa santos1 aim: to investigate the prevalence and etiological factors of dental trauma in school-age children aged 6 to 12 years. methods: a study was developed in children and adolescents between the ages of 6 and 12 of both genders enrolled in 3 municipal public schools. an oral examination of the permanent or deciduous incisors was performed, if they were still present in the oral cavity, to evaluate the presence of dental trauma, need for treatment and sequels, and the method of examination and classification of dental trauma of o’brien. results: the prevalence of dental trauma was 4.03% (n=29). the most verified traumas were enamel crack / enamel fracture and enamel / dentin fracture without pulp exposure. it was observed that the most affected teeth were the permanent upper central incisors and the age at which trauma was most observed was 11 years, the main etiological factor was fall from a height, in 65.3% of schoolchildren (n=19). conclusion: the prevalence of dental trauma was 4.03%, with no statistically significant difference between genders. the permanent right upper central incisor was the most affected, occurring predominantly at home and at school. the dissemination of information about dental trauma and emergency protocols to parents and teachers need to be encouraged. keywords: tooth injuries. dentition, mixed. pediatric dentistry. http://dx.doi.org/10.20396/bjos.v17i0.8652886 mailto:anadelourdessl@hotmail.com 2 lira et al. introduction traumatic dental injury (tdi) is a common oral disorder in school-age children. it is caused by an external impact on the tooth and surrounding tissues, it constitutes a public health problem in our society, in increasing proportions of the population being affected, with attendant aesthetic, psychological, social and therapeutic damages, besides the high costs of oral rehabilitation, when accidents with dental losses occur1,2. due to the evident decay of caries in brazil and the low prevalence of periodontal disease in younger populations, traumatic lesions are documented as one of the most serious problems associated with oral health, becoming a public health problem among children and adolescents. its prevalence rate is high, and immediate care is necessary for a good prognosis3,4. although guidelines of the international association of dental traumatology (iadt) recommend focusing on the treatment of acute dental injuries, other sequelae of trauma such as crown discoloration should be considered1. the treatment for dental trauma varies with each patient, degree of severity, or duration of trauma5. the high rates of auto accidents, extreme sports, urban violence with firearms, falls and wrestling have favored the occurrence of this type of trauma in dental emergencies. in the vast majority of accidents children and adolescents are involved5,6. recent studies have shown an association between illicit drug use and tdi, considering this association as a worrying risk factor for fracture or tooth loss7. the upper central incisors are the teeth most prone to trauma, the most common fractures being those involving only enamel or enamel and dentin without pulp exposure8. obtaining epidemiological data from tdi is extremely important for the planning, execution and evaluation of actions in oral health, since variations in prevalence evidenced the need for successive studies and identification of factors associated with it9,10. it was desired to perform this research in schoolchildren, between 6 and 12 years of age, for the following reasons; because it is the period of mixed dentition, children at this age practice various sports activities and are very rxplorative, the permanent teeth i some may still be undergoing their eruptive process thus making management a bit more complicated, their chances of being exposed to dental traumas may be a bit increased as the tend to express some degree of malocclusion at this stage, since the permanent dentition has not yet been fully established. the objective of this research was to investigate the prevalence and etiological factors of dental trauma in children of school age from 6 to 12 years. materials and methods a cross-sectional study was developed and the reference population consisted of children and adolescents between the ages of 6 and 12 of both genders enrolled in the municipal public schools network in the city of parnaíba-piaui. the descriptors used were dental trauma, mixed dentition, pediatric dentistry. the researchers obtained a letter of consent from the directors of the school units são francisco dos capuchinhos school, rev. erasmo martins ferreira presbyterian 3 lira et al. school and caio passos municipal school, in the municipality of parnaíba, piauí. the research protocol was approved by the research ethics committee of the state university of piauí cep / uespi, under opinion 1665.758. according to the sample calculation 550 children should be examined, but this was exceeded during data collection to make the study more robust and all inclusive. the inclusion criteria were all children aged between 6 and 12 years, since it corresponded to the period of the mixed dentition, children whose parents or guardians gave consent and the children who accepted to participate in the research. as exclusion criteria, children under 6 years of age and over 12 years of age, due to permanent dentition, or the second erupting permanent molars and those with more than 12 years of age, due to permanent dentition, or the second erupting permanent molars, and those whose parents or guardians did not authorize the research. epidemiological questionnaire was applied to the participants and caregivers to make a survey about the circumstances that led to the event of the trauma. the children were examined by two researchers who were trained at the clinical school of dentistry (ceo) of the state university of piauí to identify dental traumas. subsequently, calibration exercises were performed with 20 children not participating in the sample plan, in a school in the municipality, according to the methodology described in another publication11. a pilot study was conducted involving 45 schoolchildren. as a result, its validity was observed, without adjustments. in order to measure intra and inter-examiner diagnostic reproducibility, 10% of the total sample was double-checked by each of the examiners, with the kappa coefficient for intraand inter-examiner agreement being 0.99 and 0.98, respectively. the oral examination of the permanent or deciduous incisors was performed at the school, if they were still present in the oral cavity, to evaluate the presence of tdi and the need for treatment, being adopted the method of examination and classification of the dental trauma of o’brien12, with the use of natural light and light of a led flashlight for better visualization, gloves for procedures and wooden spatula. for clinical examination, the teeth were dried with sterile gauze on all their surfaces13. when there was history and evidence of tdi, records were made in the odontogram. the diagnostic criteria adopted to investigate the occurrence of tdi included, according to o´brien12: enamel crack and fracture, enamel / dentin fracture without and with pulp exposure, change in crown color, presence of aesthetic restorations, bonding of coronary fragment, total restoration of the crown of the permanent tooth, fistula or abscess and dental absence due to tdi. a descriptive analysis of the data was carried out using frequency and number tables using excel windows 2013 software microsoft®. the possible association between the variables was verified by the chi-square test and the non-parametric mann-whitney test. the significance level adopted was 5% and the statistical package spss for windows 2010 (social package statistical science), version 20, was used for analysis. results in this cross-sectional study, there were a total of 719 children, 65.5% were males (n = 378) and 34.5% females (n = 341), the prevalence of tdi in this study population was 4.03% 4 lira et al. (n = 29). from the chi-square statistical calculation, it was verified that there was no statistically significant difference in the association between the genders of the participants χ² (1) = 2.80, p = 0.09, considering the level of significance of random error of 5 %. from the total prevalence of 4.03%, the traumas were distributed according to the classification of andersson et al.14, 2012: enamel crack / enamel fracture: 20.6% (n = 6); enamel / dentin fracture without pulp exposure: 65.5% (n = 19); enamel / dentin fracture with pulp exposure: 3.4% (n = 1); lateral dislocation 3.4% (n = 1); intrusive dislocation 3.4% (n = 1); avulsion: 3.4% (n = 1), (table 1). next, it was investigated if there was difference between the genders in the type of trauma of occurrence. for that, a non-parametric test was performed, mann-whitney. which indicated that they did not differ statistically as to the level of occurrence of different types of trauma [u = 73.80 (z = 1.17); p = 0.24]. the number and the traumatized teeth were shown in figure 1. the most affected were the permanent upper central incisors (n = 12 cases), followed by the permanent upper right lateral incisor (n = 5) and by the primary teeth 51, 52, 62, 81 and 82 (one case for each tooth). table 1. frequency of distribution by gender n (%) of the types of injuries observed (classification according to andersson et al14, 2012). dental trauma female n (%) male n (%) total n (%) enamel crack 1 (9.09) 5 (27.7) 6 (20.6) enamel and dentin fracture/without pulp exposure 7 (63.6) 12 (66.6) 19 (65.5) enamel and dentin fracture/with pulp exposure 0 (0.0) 1 (5.5) 1 (3.4) lateral dislocation 1 (9.09) 0 (0.0) 1 (3.4) iintrusive dislocation 0 (0.0) 1 (5.5) 1 (3.4) avulsion 1 (9.09) 0 (0.0) 1 (3.4) figure 1. distribution of affected teeth and their respective amounts. 14 12 10 8 6 4 2 0 ics dec 51 ils dec 52 ics dec 62 ils dec 62 ics dec 81 ili dec 82 ics 11 ils 12 ics 21 1 1 1 1 1 1 12 12 5 5 lira et al. as to age, it was observed that the age at which most trauma occurred was 11 years with 20.68% (n = 6). followed by the age of 7, 8, 9 and 10 years with 13.79% (n = 4) respectively, 12 years with 10.34% (n = 3) and finally the age of 6 years with 6.89% (n = 2), figure 2. according to the questionnaire given to the participants and parents/legal guardians, the main etiological factor was falling from a height, in 65.3% of the students (n = 19), followed by a collision with 10.2% (n = 3), a bicycle crash with 10.2% (n = 3), a sports accident 10.2% (n = 3). another factor was an automobile accident with 3.4% (n = 1) (table 2). the majority of schoolchildren who presented td reported that the home and school were the places where the accident occurred, corresponding to 37.9% (n = 11). then, the most cited site was the street, involving 17.1% (n = 5), and lastly, occurrences in leisure time were reported with 6.8% (n = 2). (table 2). discussion the prevalence of tdi found was 4.03%, with no statistically significant difference between genders. similar results were observed in studies by campos et al.4 and paiva et al.13. rodrigues et al.9 found a prevalence in the deciduous dentition between table 2. distribution of schoolchildren according to etiology and place of dental injury etiology home n (%) school n (%) recreation n (%) street n (%) total n (%) fall 11 (37.9) 6 (20.6) 1 (3.4) 1 (3.4) 19 (65.3) collision with people /objects 0 (0.0) 2 (6.8) 1 (3.4) 0 (0.0) 3 (10.2) bycicle drop 0 (0.0) 0 (0.0) 0 (0.0) 3 (10.3) 3 (10.2) sports accident 0 (0.0) 3 (10.2) 0 (0.0) 0 (0.0) 3 (10.2) car accident 0 (0.0) 0 (0.0) 0 (0.0) 1 (3.4) 1 (3.4) figure 2. number of schoolchildren affected by traumatic events by age. 7 6 5 4 3 2 1 0 6 years 7 years 8 years 9 years 10 years 11 years 12 years 6 4444 2 3 6 lira et al. 9.4% and 62.1%, while in the permanent dentition ranging from 8% to 58.6%, suggesting that there is a need for a methodological evaluation in the study designs because there are large variations in prevalence. the dissemination in high-access media of educational campaigns in schools in the city may have contributed to the low prevalence reported in this study, together with the fact that the unified health system (sus) has reorganized its priorities regarding oral health, adopting the model of health promotion with interventions based on risk factors15. the most frequent trauma was enamel and dentin without pulp exposure: 65.5%, followed by enamel crack: 30.6%, in agreement with the findings of gonçales et al.1 and goettems et al.8, but contrary to jung et al.16 who found that the most identified forms of trauma were fractures involving only enamel and fractures involving enamel and dentin. andersson et al.14,17 recommended that the fractured coronary fragment be stored in physiological saline for bonding (because it is a low-cost technique and satisfactory aesthetic results), or restoration with resin if collage was not feasible. it was observed that the most affected teeth were the permanent upper central incisors. similar results were observed by goettems et al.8, reis et al.18 and carvalho et al.19, who justified this fact due to eruption before the upper lateral incisors and their position in the dental arch, being subject to a longer period of exposure to risk factors. as for age, it was observed that the age at which most trauma occurred was 11 years with 20.68%, according to studies8-10,13. this fact, which can be explained by the greater participation of this age group, in activities, mainly sports that require greater physical effort, with greater risks to dental trauma. fall accident was presented as the main cause related to tdi, a correlation also found by marchiori et al.5 and paiva et al.13. the majority of schoolchildren who presented with dental trauma reported that the home and school were the places where the accident occurred, corresponding to 37.9%. levin et al.20 considered the school as the most prone place for the occurrence of trauma. the etiology is correlated to cultural factors and to the types of activities practiced by each community. from the reported cases of traumatic events, it was observed that of the total number of traumatized teeth found 17% were treated, with composite resin restorations, with no need to be redone. of these cases, 0.83% were in the upper left central incisor, one in the tooth in the upper right lateral incisor and one in the lower right lateral incisor. the low number of previously traumatized teeth that were treated suggests the parents’ lack of information about the adequate dental treatment, the difficulty of access to it, and the low socioeconomic status of the population involved in the research. bonding of coronary fragment in some traumatized tooth was not observed during the examinations. although the prevalence of dental trauma is considered low compared to other studies13,18-20, it is still a very worrying finding, strategies that guarantee access to health involving preventive and intervention measures, through the dissemination of information on dental trauma and emergency protocols to parents and teachers need to be encouraged. 7 lira et al. it was concluded that the prevalence of dental trauma was 4.03%, with no statistically significant difference between genders. the permanent right upper right central incisor was more affected. the interpretation of the results should be considered as an inherent limitation of the study. it is important that further studies are conducted on the prevalence of dental trauma and its associated etiological factors. references 1. goncalves bm, dias lf, pereira cs, ponte filho mx, konrath ac, bolan ms, et al. the impact of dental traumatism and aesthetical empairment on the quality of life of pre-schools children. rev paul pediatr. 2017;35(4):448-55. doi: 10.1590/1984-0462/;2017;35;4;00011. 2. traebert j, claudino d. epidemiology of dental trauma in children: brazilian scientific production. pesq bras odontop clin integr. 2012 abr-jun;12(2):263-72. doi: 10.4034/pboci.2012.122.17. portuguese. 3. martins vm, sousa rv, rocha es, leite rb, gomes mc, granville-garcia af. assessment of the association between overweight/obesity and traumatic dental injury among brazilian schoolchildren. acta odontol. latinoam. 2014;27(1):26-32. 4. campos dmks, almeida er, miotto mhmb, barcellos la. campos fs. dental trauma: prevalence among adolescents aged 15 to 19 years in the city of santa teresa/es, brazil. rev bras pesq saude. 2016 jul-sep;18(3):65-73. portuguese. 5. marchiori ec, santos se, asprino l, moraes m, moreira rw. occurrence of dental avulsion and associated injuries in patients with facial trauma over a 9-year period. oral maxillofac surg. 2013 jun;17(2):119-26. doi: 10.1007/s10006-012-0354-5. 6. pithon mm, santos rl, magalhães phb, coqueiro rs. brazilian primary school teachers’ knowledge about immediate management of dental trauma. dental press j orthod. 2014 sept-oct;19(5):110-5. doi: 10.1590/2176-9451.19.5.110-115. 7. paiva hn, paiva pcp, silva cjp, lamounier ja, ferreira ef, zarzar pm. use of illicit drugs as a risk factor for dental trauma in adolescents cad saude colet. 2016;24 (3):17-322. doi: 10.1590/1414-462x201600030083. portuguese. 8. goettems ml, torriani dd, hallal pc, correa mb, demarco ff. dental trauma: prevalence and risk factors in schoolchildren. community dent oral epidemiol. 2014 dec;42(6):581-90. doi: 10.1111/cdoe.12113. 9. rodrigues as, castilho t, antunes laa, antunes ls. epidemiological profile of dental traumatisms in children and adolescents in brazil. unopar cient cienc biol saude. 2015;17(4):267-78. portuguese. 10. soriano ep, caldas jr af, góes ps. risk factors related to traumatic dental injuries in brazilian schoolchildren. dent traumatol. 2004 oct;20(5):246-50. doi: 10.1111/j.1600-9657.2004.00246.x. 11. peres ma, traebert j, marcenes w. calibration of examiners for dental caries epidemiology studies. cad saude publica. 2001 jan-feb;17(1):153-9. doi: 10.1590/s0102-311x2001000100016. portuguese. 12. o’brien m. children’s dental health in the united kingdom 1993. in report of dental survey, office of population censuses and survey. london: her majesty´s stationery office; 1994. 13. paiva pcp, paiva hn, filho pmo, cortês mis. prevalence and risk factors associated with traumatic dental injury among 12-year-old schoolchildren in montes claros, mg, brazil. cienc saude colet. 2015 apr;20(4):1225-33. doi: 10.1590/1413-81232015204.00752014. 14. andersson l, andreasen jo, day p, heithersay g, trope m, diangelis aj, et al. international association of dental traumatology guidelines for the management of traumatic dental injuries: 2. avulsion of permanent teeth. dent traumatol. 2012; 28(2):88-96. doi: 10.1111/j.1600-9657.2012.01125.x. 8 lira et al. 15. frujeri mlv, frujeri ja, bezerra ac, cortes mi, costa ed jr. socio-economic indicators and predisposing factors associated with traumatic dental injuries in schoolchildren at brasília, brazil: a cross-sectional, population-based study. bmc oral health. 2014 july;14(91):1-7. doi:10.1186/1472-6831-14-91. 16. jung cp, tsai ai, chen cm. a 2-year retrospective study of pediatric dental emergency visits at a hospital emergency center in taiwan. biomed j. 2016 jun;39(3):207-13. doi: 10.1016/j.bj.2016.06.004. 17. andersson l, andreasen jo, day p, heithersay g, trope m, diangelis aj, et al. international association of dental traumatology guidelines for the management of traumatic dental injuries: 3. injuries in the primary dentition. dent traumatol. 2012 jun;28(3):174-82. doi: 10.1111/j.1600-9657.2012.01146.x. 18. reis ag, paiva pcp, oliveira filho pm. prevalence of dental trauma and associated factors in 11 to 19-year-old students in the rural areas of the town of diamantina, mg, brazil. arq cent estud curso odontol univ fed minas gerais. 2014 jan/mar; 50(1):42-8. portuguese. 19. carvalho b, brito as, heimer m, vieira s, colares v. dental injury in adolescents between 15 and 19 years in recifepe, brazil: preliminary study. pesq bras odontoped clin integr. 2013;13(1):95-100. doi: 10.4034/pboci.2013.131.14. portuguese. 20. levin l, samorodnitzky gr, schwartz-arad d, geiger sb. dental and oral trauma during childhood and adolescence in israel: occurrence, causes, and outcomes. dent traumatol. 2007 dec;23(9):356–9. doi: 10.1111/j.1600-9657.2006.00473.x. http://dx.doi.org/10.1590/1413-81232015204.00752014 1http://dx.doi.org/10.20396/bjos.v19i0.8656537 volume 19 2020 e206537 original article 1 graduate program of orthodontics, araras dental school, university center of hermínio ometto foundation-fho, araras, são paulo, brazil. 2 department of community dentistry, piracicaba dental school, university of campinas, piracicaba, sp, brazil. corresponding author: silvia a s vedovello graduate program of orthodontics-fho dr. maximiliano baruto av, 500 jardim universitário. araras, sp – brazil. 13607-339 +55 19 3543-1423 e-mail: silviavedovello@gmail.com received: september 03, 2019 accepted: april 05, 2020 influence of social capital on self-perception related to orthodontic treatment need sandra d f sedrez1, ana paula terossi de godoi1 , marcelo de c meneghim2 , silvia a s vedovello1 , giovana c venezian1 , carolina c de menezes1 aim: to evaluate the influence of social capital on self-perception related to orthodontic treatment need. methods: a cross-sectional study was conducted with a sample of 578 11-16 years-old adolescents from a city in southern brazil. social capital was evaluated using the social capital questionnaire for adolescent students (scq-as). index of orthodontic treatment need (iotn) assessed malocclusion and self-perception related to orthodontic treatment need. sociodemographic aspects of adolescents were also evaluated. individual analyses were performed, relating the study variables to the outcome, estimating the odds ratio with the respective confidence intervals of 95%. the variables with p<0.20 in the individual analyses were tested in the multiple logistic regression models, and those with p<0.10 remained in the model. results: social capital did not influence the self-perception related to orthodontic treatment need. adolescents with high orthodontic needs were 5.35 (ci 95%: 2.68 to 10.65) times more likely to perceived orthodontic treatment need (p<0.05). crowding and dental absence were associated with self-perception related to orthodontic treatment need (p<0.05). conclusions: social capital did not influence the self-perception related to orthodontic treatment need. keywords: index of orthodontic treatment need. orthodontics. self-concept. social capital. mailto:silviavedovello@gmail.com https://orcid.org/0000-0003-4631-2563 https://orcid.org/0000-0003-2673-3627 https://orcid.org/0000-0002-7203-2867 https://orcid.org/0000-0003-4643-7964 https://orcid.org/0000-0002-8875-8611 2 sedrez sdf et al. introduction oral health is a fundamental component of physical health and mental well-being. the values and attitudes of individuals and communities can influence oral health, as well as their experiences, perceptions, expectations and adaptability, reflecting on the physiological, social and psychological aspects that are essential for life quality1. considering the assessment of oral health, malocclusion is a condition of high prevalence2, with direct influence on quality of life. in addition has the potential to affect biological functions, appearance, interpersonal relationships, socialization, self-esteem and psychosocial well-being3,4. on the other hand, there is no consensus if the orthodontic treatment need is perceived by adolescents5-7. adolescents are a group exposed to situations of physical, emotional and social risks due to the environmental context8. thus, the change from the traditional orthodontic model to a more biopsychosocial model increased interest of the literature9. in the broader context of health, social capital can be defined as the interpersonal relationship networks that occur at different levels and positions of power and which have trust and reciprocity as foundations10,11. the possible influence of social capital on oral health has been highlighted from the social support12, bond and mutual understanding which contribute to the self-esteem, population well-being, quality of life and health11-17. recent studies reported that the social capital can reduce the negative effects of social inequality, it seems to be related to the political interest of the parents of adolescents12 and it can be seen as a protective factor, in socially disadvantaged locations8,11,16,18. this concept has been used increasingly in several aspects of mental health, self-assessment in health and, especially, oral health8,11-17,19,20. although the evidences in oral health are at an early stage of development, social capital is defined as a potential social determinant of oral health12. literature still lacks answers about the relationship between oral health and social capital13,14,16,17,21. according to a recent study, the association between income inequality and the oral health-related quality of life can be attenuated by contextual social capital21. on the other hand, did not affect the relationship between income inequality and oral disease, identified by professional21. in the same context of social capital, no relationship was found with dental trauma and alcohol consumption in adolescents17; however, the socioeconomic status and features of social capital may be associated with the levels of gingival bleeding among20. in addition, there was an effect of support and social cohesion on the caries index14, in adolescents and adults16, in a contextual level13,16. there is still limited and inconsistent evidence on the association between individual social capital and oral health in in the population of children and adolescents20. likewise, it is important in the oral health context to include the study of the impact of orthodontic treatment need on social capital. therefore, the aim of this study was to evaluate the influence of social capital on self-perception related to orthodontic treatment need in adolescents. the hypothesis was that the perceived orthodontic treatment need has an impact on social capital. 3 sedrez sdf et al. materials and methods study design and study population this cross-sectional study received ethical approval from the research ethics committee (#65187817.8.0000.5385), and was carried out in south brazil, in a city with 61,198 inhabitants, an 96.5% literacy rate and a human development index (hdi) of 0.802, between march and may 2017. all adolescents consented to participate, and their parents or guardians signed an informed consent form. the sample size was calculated considering a 5% significance level, test power greater than 80%, and 1.7 effect size, obtaining a minimum of 495 individuals. adolescents enrolled in public and private schools were invited to participate in the study, and only adolescents in permanent dentition were included. exclusion criteria were current or previous orthodontic treatment and systemic diseases, such as cerebral palsy or down syndrome. the final sample included 578 students (314 girls and 264 boys), aged 11-16-years-old. parents answered a sociodemographic questionnaire with information regarding family income, number of people living in the house, and level of parental education. family income was determined based on the sum of all salaries received by active residents in the home and categorized based on the current brazilian minimum salary; the threshold was the median response (r$ 3.000). mother and father’s schooling were defined as the number of years of study, with eight years used as the cut-off point; the threshold was the median response. social capital the self-administered instrument used to evaluate the adolescents social capital was the social capital questionnaire for adolescent students (scq-as), developed and validated in brazil22. this questionnaire is composed of items selected from the national and international literature and has been submitted to face validation, content analysis and analyses of internal consistency, reliability and reproducibility. the scq-as questionnaire is composed of 12 items grouped into four subscales: social cohesion at school; network of friends at school; social cohesion in the community/neighborhood; and trust at school and in the community/neighborhood. the answers are given in a three-point likert scale and scores range from 12 to 36 points, with a higher score denoting greater social capital. for this study, the median was adopted to dichotomize the value in high and low social capital17,22. self-perception related to orthodontic treatment need the self-perception related to orthodontic treatment need was determined by the aesthetic component (ac) of the index of orthodontic treatment need (iotn)12. this index consists of a visual scale of 10 photographs of different dental arrangements, which allows adolescents to identify which photograph most closely resembles the appearance of their teeth23. the photographs from 1 to 4 are classified as no or slight need for treatment, from 5 to 7 as moderate and from 8 to 10 as severe6,7,23. in this study, responses from 1 to 4 were considered without orthodontic treatment need, and equal to or greater than 5, with perceived orthodontic treatment need24. 4 sedrez sdf et al. normative orthodontic treatment need the dental health component (dhc) of iotn was used for a clinical assessment of orthodontic treatment need. the data were collected with the adolescents seated, under natural light, using a wooden spatula and a millimeter probe. the iotn-dhc categorizes the unfavorable effects of different deviant occlusal traits into five grades scale of orthodontic treatment priority, and the most severe change is used for index determination. the following features are evaluated: missing teeth, overjet, crossbite, displacement of contact points (also called crowding), and overbite (including deep bite and open bite). the sample was classified as having no need for treatment when iotn-dhc was 1 and 2, moderate need when it was 3 and high need for normative orthodontic treatment when it was 4 and 56,25. in addition, individual aspects of malocclusion were analyzed from the iotn-dhc components: overjet, negative overjet, overbite, open bite, crowding, posterior crossbite, dental absence and special cases. any of the conditions mentioned are considered special cases: cleft lip; ankylosed primary tooth; dental rash prevented by crowding, incorrect dental positioning, supernumerary tooth, retained primary tooth or pathology; partially erupted; inclined or impacted tooth; and, supernumerary tooth23. training and calibration a previously trained examiner performed the exams under artificial light at the schools. before the study began, a calibration process was conducted to obtain acceptable consistency for the malocclusion. the training stage consisted of a theoretical discussion followed by a practical stage. during training and calibration, the inter and intra-examiner agreement was estimated with the intraclass correlation coefficient (icc) for the components of the iotn-dhc, with an acceptable limit value greater than 0.97. statistical analysis data analysis was adjusted in simple logistic regression models, estimating the odds ratios (or) with their respective confidence intervals of 95%. the variables with p≤0.20 in the individual analyzes, for each outcome variable, were tested in multiple logistic regression models, with the remaining variables being p≤0.10. from the models, the adjusted odds ratios were estimated with the respective confidence intervals of 95%. the analyses were performed in the sas (release 9.2, sas institute inc., cary, nc, usa). the association of clinical evaluation and self-perception related to orthodontic treatment need was performed by the chi-square test in the epi info (version 7.02, centers for disease control and prevention, atlanta, georgia, usa). results the sample consisted of 578 adolescents, 314 girls and 264 boys aged 11-16-yearsold. a total of 74% of adolescents were from families that earned up to three times the brazilian monthly minimum wage, 47.9% of the fathers and 50.7% of the mothers had more than eight years of schooling, and 63.5% with more than four people living in the house. the adolescents presented a high social capital, with a median of 32. 5 sedrez sdf et al. according to table 1, adolescents with high treatment needs showed 5.35 (95% ci: 2.68 to 10.65) times more likely to perceive the need for orthodontic treatment (p<0.05). there was no  significant association between  sociodemographic variables,  social capital and self-perception related to orthodontic treatment need (p> 0.05). table 2 showed that in 11.1% of adolescents who perceived the orthodontic treatment need, the presence of crowding or the absence of some dental element were the components associated with the treatment need perception, and crossbite obtained a score very close to significance. discussion the social context can exert influence on health-related behavior10,17. however, the majority of the studies was carried out with populations of adults14,15,18,19,21 and little is known regarding the relationship between social capital and health outcomes among adolescents8,16,17,20, particularly on the impact of malocclusion. this study will contribute to the literature with an understanding of how the capital social influence the self-perception related to orthodontic treatment need. the study findings shows that social capital was not associated with the self-perception related to orthodontic treatment need. there is a growing application of the concept of social capital in various aspects of mental health, self-evaluation in health and, particularly, in oral health8,11-17,19. in addition, the self-perception of orthodontic treatment need (iont-ac) is influenced by several aspects, including self-esteem26-29. this relationship may have affected the results of this study, since the average social capital was high and the need for perceived treatment was low, possibly modulated by self-esteem. there are no reports in the literature of studies associating malocclusion and social capital. the evaluation of social capital can be performed at different levels, both individual and contextual10,11. regardless of this assessment, there is a diversity of results when associated with other aspects of oral health. there are reports that the individual social capital was relevant in the quality of life related to the oral health of pregnant women30 and influenced the report of toothache15. neighborhood and family social capital also has influenced oral health reported by parents of children31 and dental losses in adults are more frequent when there is less social capital18. however, studies did not find an association between social capital and regular dental brushing, visits to the dentist, time of the last visit15 and dental trauma17. therefore, there is no consensus about the influence of social capital on oral health and when present, whether it is on an individual or collective level. the diversity of results may be related to the sociocultural environment8, as well as the variety of tools and methodologies making it difficult to compare studies19. in this study, individual social capital evaluated characteristics such as trust11 and cohesion28 of adolescents, through the subscales of the social capital questionnaire for adolescent students22. it is important to highlight that the population studied belongs to a city in the south of brazil with a high human development index, compared to other cities in the country. the present study showed that 11.11% of adolescent’s perceived orthodontic treatment need (iotn-ac); such prevalence in the literature ranges from 10 to 40%5,6. 6 sedrez sdf et al. ta bl e 1. a ss oc ia tio ns b et w ee n th e se lfpe rc ep tio n re la te d to o rt ho do nt ic tr ea tm en t n ee d (i o tn -a c ), no rm at iv e or th od on tic tr ea tm en t n ee d (i o tn -d h c ), so ci od em og ra ph ic va ria bl es a nd s oc ia l c ap ita l. v ar ia bl es c at eg or y n( % ) s el fpe rc ep tio n re la te d to o rt ho do nt ic tr ea tm en t n ee d * g ro ss o r ( $ i c 95 % ) pva lu e o r – fi na l m od el (i c 95 % ) pva lu e       n o ne ed n ee d &         g en de r fe m al e 31 4 (5 4. 3% ) 28 3 (9 0. 1% ) 31 (9 .9 % ) r ef   m al e 26 4 (4 5. 7% ) 23 1 (8 7. 5% ) 33 (1 2. 5% ) 1. 30 (0 .7 8 2 .1 9)    0 .3 17     a ge ≤ 12 y ea rs o ld # 30 7 (5 3. 1% ) 27 1 (8 8. 3% ) 36 (1 1. 7% ) 1. 15 (0 .6 81. 95 ) 0. 59 4   > 12 y ea rs o ld 27 1 (4 6. 9% ) 24 3 (8 9. 7% ) 28 (1 0. 3% ) r ef         in co m e ≤ r $ 3. 00 0# 42 8 (7 4. 0% ) 38 3 (8 9. 5% ) 45 (1 0. 5% ) r ef > r $ 3. 00 0 13 7 (2 3. 7% ) 11 9 (8 6. 9% ) 18 (1 3. 1% ) 1. 29 (0 .7 22. 31 ) 0. 39 6   n o an sw er 13 (2 .2 % ) 12 (9 2. 3% ) 1 (7 .7 % )       sc ho ol p ub lic 47 3 (8 1. 8% ) 41 9 (8 8. 6% ) 54 (1 1. 4% ) 1. 22 (0 .6 02. 49 ) 0. 57 7   p riv at e 10 5 (1 8. 2% ) 95 (9 0. 5% ) 10 (9 .5 % ) r ef fa th er ’s ed uc at io n le ve l u p to 8 th g ra de # 27 7 (4 7. 9% ) 25 2 (9 1. 0% ) 25 (9 .0 % ) r ef a bo ve 8 th g ra de 21 3 (3 6. 9% ) 18 4 (8 6. 4% ) 29 (1 3. 6% ) 1. 59 (0 .9 02. 80 ) 0. 10 9 n o an sw er 88 (1 5. 2% ) 78 (8 8. 6% ) 10 (1 1. 4% )        m ot he r’s le ve l o f ed uc at io n u p to 8 th g ra de # 29 3 (5 0. 7% ) 26 2 (8 9. 4% ) 31 (1 0. 6% ) r ef a bo ve 8 th g ra de 27 4 (4 7. 4% ) 24 3 (8 8. 7% ) 31 (1 1. 3% ) 1. 08 (0 .6 41. 83 ) 0. 77 9 n o an sw er 11 (1 .9 % ) 9 (8 1. 8% ) 2 (1 8. 2% ) -        p eo pl e liv in g in th e sa m e ho us e u p to 4 # 36 7 (6 3. 5% ) 32 3 (8 8. 0% ) 44 (1 2. 0% ) 1. 36 (0 .7 72. 39 ) 0. 29 3 m or e th an 4 20 8 (3 6. 0% ) 18 9 (9 0. 9% ) 19 (9 .1 % ) r ef n o an sw er 3 (0 .5 % ) 2 (6 6. 7% ) 1 (3 3. 3% ) -        o rt ho do nt ic s ne ed (i o tn -d h c ) n o/ lig ht n ee d 24 5 (4 2. 4% ) 23 4 (9 5. 5% ) 11 (4 .5 % ) r ef r ef m od er at e ne ed 11 4 (1 9. 7% ) 10 5 (9 2. 1% ) 9 (7 .9 % ) 1. 82 (0 .7 34. 53 ) 0. 23 7 1. 82 (0 .7 34. 53 ) 0. 23 73 h ig h ne ed 21 9 (3 7. 9% ) 17 5 (7 9. 9% ) 44 (2 0. 1% ) 5. 35 (2 .6 810 .6 5) <0 .0 01 5. 35 (2 .6 810 .6 5) <0 .0 00 1 so ci al c ap ita l lo w (≤ 3 2# ) 35 8 (6 1. 9% ) 31 3 (8 7. 4% ) 45 (1 2. 6% ) 1. 52 (0 .8 62. 67 ) 0. 14 5 h ig h (> 3 2) 22 0 (3 8. 1% ) 20 1 (9 1. 4% ) 19 (8 .6 % ) r ef     * o dd s ra tio ; $ 95 % c on fid en ce in te rv al ; & r ef er en ce le ve l; # m ed ia n. 7 sedrez sdf et al. higher values are expected since some studies have their samples composed by patients seeking orthodontic treatment5, suggesting a greater perception of malocclusion. apart from that, cultural differences can influence the aesthetic perception of different societies32. recently, a study suggested that the greater the normative need for orthodontic treatment, the greater the need perceived by the patient5. similar results were observed in this study, in which adolescents with a high need for normative treatment (iotn-dhc) were more likely to need perceived treatment (iotn-ac). thus, although the need for perceived orthodontic treatment is low compared to normative, when it is high, it is suggested that it is more easily identified by adolescents, because the literature states that the psychosocial impact and self-perception of malocclusion increases with the increase in severity25. the presence of crowding and the absence of any dental elements were the normative components responsible for this perception; the crossbite obtained a score very close to significance suggesting that these components are more easily identified by lay people. in this study there was no association between sociodemographic factors and need for treatment, agreeing with the previous study6. however, an association between normative, perceived need and age was found in the literature and the greater the age the greater the normative and perceived need5, and there are also reports that increased age and low socioeconomic status were associated with the presence of malocclusion4. table 2. association of self-perception related to orthodontic treatment need (iotn-ac) and occlusal aspects, by iotn-dhc components. iotn-dhc components evaluation self-perception related to orthodontic treatment need p-valueno need need# n % n % overjet altered 250 87.1% 37 12.9% 0.210 normal 264 90.7% 27 9.3% negative overjet altered 26 81.2% 6 18.8% 0.256 normal 488 89.3% 58 10.7% overbite altered 258 88.3% 34 11.7% 0.756 normal 256 89.5% 30 1.04% open bite altered 48 87.3% 7 12.7% 0.609 normal 472 89.2% 57 10.7% crowding altered 280 86.1% 45 13.9% 0.022* normal 234 92.3% 19 7.5% posterior crossbite altered 92 83.6% 18 16.4% 0.072 normal 422 90.2% 46 9.8% dental absence altered 36 78.3% 10 21.7% 0.031* normal 478 89.8% 54 10.2% special cases& altered 70 86.4% 11 13.6% 0.558 normal 444 89.3% 53 10.6% chi-square; # median; *p<0.05; &cleft lip; ankylosed primary tooth; dental rash prevented by crowding, incorrect dental positioning, supernumerary tooth, retained primary tooth or pathology; partially erupted; inclined or impacted tooth; supernumerary tooth. 8 sedrez sdf et al. the differentials of our study was to evaluate the influence of social capital on the orthodontic treatment need. in addition, we included the normative need, assessed clinically, with that perceived by the adolescent. as a limitation, it is worth noting the cross-sectional study design, which evaluated the impact of social capital at a given period of time. a longitudinal design is suggested in order to improve the understanding this social influence over time. finally, considering the results, the evaluation of the influence of social capital on the perception of the orthodontic treatment need should consider different amplitudes and forms of evaluation, sociodemographic diversities, and that aspects such as self-esteem can be related in this association. in conclusion, social capital did not influence the self-perception of the orthodontic treatment need. references 1. glick m, williams dm, kleinman dv, vujicic m, watt rg, weiyant rj. a new definition for oral health developed by the fdi world dental federation opens the door to a universal definition of oral health. am j orthod dentofacial orthop. 2017 feb;151(2):229-31. doi: 10.1016/j.ajodo.2016.11.010. 2. brazil. health ministry. health attention general office basic attention department. [sb brazil 2003 project: oral health conditions of brazilian population 2002–2003: main results]. brasilia: health ministry; 2004. portuguese. 3. masood y, massoud m, zainul nnb, araby nbaa, hussain sf, newton t. impact of malocclusion on oral health related quality of live in young people. health qual life outcomes 2013 feb;11:25. doi: 10.1186/1477-7525-11-25. 4. vedovello sas, ambrosano gmb, pereira ac, valdrighi hc, vedovello filho m, meneghim mc. association between malocclusion and the contextual factors of quality of life and socioeconomic status. am j orthod dentofacial orthop. 2016 jul;150(1):58-63. doi: 10.1016/j.ajodo.2015.12.022. 5. omer yt, bouserhai j, hawas n, sayed aame. association between normative and self-perceived orthodontic treatment need in a lebanese population. int orthod. 2016 sep;14(3):386-98. doi: 10.1016/j.ortho.2016.07.002. 6. eslamipour f, riahi ft, etemadi m, riahi a. correlation coefficients of three self-perceived orthodontic treatment need indices. dent res j (isfahan). 2017 jan-feb;14(1):37-42. doi: 10.4103/1735-3327.201131. 7. santos pr, meneghim mc, ambrosano gmb, vedovello filho m, vedovello sas. influence of quality of life, self-perception, and self-esteem on orthodontic treatment need. am j orthod dentofacial orthop. 2017 jan;151(1):143-7. doi: 10.1016/j.ajodo.2016.06.028. 8. furuta m, ekudi d, suzuki e, morita m, kawachi i. social capital and self-rated oral health among young people. 2012 apr;40(2):97-104. doi: 10.1111/j.1600-0528.2011.00642.x. 9. agou s, locker d, strainer dl, tompson b. impact of self-esteem on the oral health-related quality of life of children with malocclusion. am j orthod dentofacial orthop. 2008 oct;134(4):484-9. doi: 10.1016/j.ajodo.2006.11.021. 10. bordieu p. the forms of capital. in: richardson jg, editor. handbook of theory and research for the sociology of education. new york, ny: greenwoodpress; 1986. 11. putnam rd. bowling alone: the collapse and revival of american community. new york: simon & schuster; 2000. 9 sedrez sdf et al. 12. rouxel pl, hilmann a, aida j, tsakos g, watt rg. social capital: theory, evidence, and implications for oral health. community dent oral epidemiol. 2015 apr;43(2):97-105. doi: 10.1111/cdoe.12141. 13. aida j, ando y, oosaka m, niimi k, morita m. contributions of social context inequality in dental caries: a multilevel analysis of japanese 3-year-old children. community dent oral epidemiol. 2008 apr;36(2):149-56. doi: 10.1111/j.1600-0528.2007.00380.x. 14. aida j, kuriyama s, ohmori-matsuda k, hozawa a, osaka k, tsuji i. the association between neighborhood social capital and self-reported dentate status in elderly japanese – the ohsaki cohort 2006 study. community dent oral epidemiol. 2011 jun;39(3):239-49. doi: 10.1111/j.1600-0528.2010.00590.x. 15. bezerra ia, goes psa. [association between social capital and oral health conditions and behavior]. cien saúde colet. 2014 jun;19(6):1943-50. doi: 10.1590/1413-81232014196.06242013. portuguese. 16. santiago bm, valença amg, vettore mv. the relationship between neighborhood empowerment and dental caries experience: a multilevel study in adolescents and adults. rev bras epidemiol. 2014;(suppl 2):15-28. doi: 10.1590/1809-4503201400060002. 17. paiva hn, paiva pcp, silva cjp, lamounier ja, ferreira e ferreira e, ferreira rc, et al. is there an association between traumatic dental injury and social capital, binge drinking and socioeconomic indicators among schoolchildren? plos one. 2015 feb;10(2):e0118484. doi: 10.1371/journal.pone.0118484. 18. borges cm, campos acv, vargas amd, ferreira e ferreira e. [adult tooth loss profile in accordance with social capital and demographic and socioeconomic characteristics]. cien saude colet. 2014 jun;19(6):1849-58. doi: 10.1590/1413-81232014196.02332013. 19. agampodi t c, agampodi s b, glozier n, siribaddana s. measurement the social capital in relations to health in low e middle income countries (lmic): systematic review. soc sci med. 2015 mar;128:95104. doi: 10.1016/j.socscimed.2015.01.005. 20. tomazoni f, vettore mv, zanatta fb, tuchtenhagen s, moreira ch, ardenghi tm. the associations of socioeconomic status and social capital with gingival bleeding among schoolchildren. j public health dent. 2017 dec;77(1):21-29. doi: 10.1111/jphd.12166. 21. aida j, kondo k, kondo n, watt r, sheiham a, tsakos g. income inequality, social capital and self-rated health and dental status in older japanese. 2011 nov;73(10):1561-8. doi: 10.1016/j.socscimed.2011.09.005. 22. paiva pcp, paiva hn, de filho pm de o, lamounier ja, ferreira ef, kawachi i, zarzar pm. development and validation of a social capital questionnaire for adolescent students (scq-as). plos one. 2014 aug;9(8):e103785. doi: 10.1371/journal.pone.0103785. 23. brook p h, shaw w c. the development of an index of orthodontic treatment priority. eur j orthod. 1989 aug;11(3):309-20. doi: 10.1093/oxfordjournals.ejo.a035999. 24. garcia g, brandão g, ferreira l, meneghim mc, pereira ac, vazquez f, et al. [influence of subjective determinants on the decision for orthodontic treatment in different dental caries prevalence]. rfo. 2012;17(3):303-8. doi: 10.5335/rfo.v17i3.2694. portuguese. 25. yi s, zhang c, ni c, qian y, zhang j. psychosocial impact of dental aesthetics and desire for orthodontic treatment among chinese undergraduate students. patient prefer adherence. 2016 jun;10:1037-42. doi: 10.2147/ppa.s105260. 26. boyce wf, davies d, gallupe o, shelley d. adolescent risk taking, neighborhood social capital, and health. j adolesc health. 2008 sep;43(3):246-52. doi: 10.1016/j.jadohealth.2008.01.014. 27. gohn mg. [empowerment and community participation in social policies]. saude soc. 2004;13(2):20-31. doi: 10.1590/s0104-12902004000200003. portuguese. 28. elgar fj, trites sj, boyce w. [social capital reduces socioeconomic differences in child health: evidence from the canadian health behavior in school-aged children study]. can j public health. 2010 nov-dec;101 suppl 3:s23-7. france. 10 sedrez sdf et al. 29. badran sa. the effect of malocclusion and self-perceived aesthetics on the self-esteem of a sample of jordanian adolescents. eur j orthod. 2010 dec;32(6):638-44. doi: 10.1093/ejo/cjq014. 30. lamarca ga, leal mc, leaos att, sheihams a, vettore mv. the difference roles of neighborhood and individual social capital an oral health-related quality of life during pregnancy and postpartum a multilevel analysis. community dent oral epidemiol. 2014 apr;42(2):139-50. doi: 10.1111/cdoe.12062. 31. reynolds jc, damiano pc, glanville jl, oleson j, macquistan mr. neighborhood and family social capital and parent-report oral health of children in iowa. community dent oral epidemiol. 2015 dec;43(6):569-77. doi: 10.1111/cdoe.12182. 32. heravi f, farzanegan f, tabatabaee m, sadeghi m. do malocclusions affect the oral health-related quality of life? oral health prev dent. 2011;9(3):229-33. 1http://dx.doi.org/10.20396/bjos.v21i00.8665385 volume 21 2022 e225385 letter to the editor braz j oral sci. 2022;21:e225385 1 department of pediatric dentistry, othodontics and public health. bauru school of dentistry, university of são paulo, bauru, são paulo, brazil. corresponding author: gerson aparecido foratori-junior department of pediatric dentistry, othodontics and public health. bauru school of dentistry, university of são paulo, bauru, são paulo, brazil. e-mail: gerson.foratori@usp.br phone: +551199766-7202 editor: dr altair a. del bel cury received: april 20, 2021 accepted: june 14, 2021 obesity during pregnancy and its oral repercussions: what is the current evidence? gerson aparecido foratori-junior1 , silvia helena de carvalho sales-peres1,* dear editor, periodontal medicine is a field that has been in evidence in recent years due to the increased number of diseases and conditions that have been linked to periodontitis1. a systematic review on periodontal medicine indicated 57 different conditions that may be associated with periodontal disease, among them, the pregnancy2. during pregnancy, physiological, immunological and hormonal changes occur in the women’s body. considering the high levels of estrogen and progesterone and the reduced antimicrobial activity of peripheral neutrophils, pregnant women are more prone to acute periodontal inflammation even with a small amount of dental plaque3. obesity is a chronic and inflammatory disease that has been considered one of the biggest public health problems4. in brazil, 63.3% and 30.2% of women are considered with overweight and obesity, respectively5. there is strong evidence to associate obesity with periodontitis6-8. this association is explained by the adipose tissue of overweight patients secreting inflammatory mediators, such as tnf-α, il-6, adiponectin, leptin and resistin, reducing the host’s immune response and causing a generalized inflammatory state of the body6-8. therefore, in the presence https://orcid.org/0000-0003-4760-8948 https://orcid.org/0000-0003-3811-7899 2 foratori-junior et al. braz j oral sci. 2022;21:e225385 of dental plaque, obese patients present an exacerbated inflammatory response of the periodontal tissues. considering the synergistic effect of pregnancy and obesity on periodontal tissues, previous studies have investigated the association of these outcomes in pregnant women9-20. most of them showed a positive association between maternal excessive weight and periodontitis9-18,20, and also associated maternal overweight with other systemic disorders, such as arterial hypertension and gestational diabetes mellitus, which in turn may further damage periodontal tissues. foratori-junior et al.17 (2020) conducted a longitudinal study in which they evaluated overweight/obese women during the 2nd and 3rd trimesters of pregnancy and also after delivery. the authors pointed out that overweight/obese women had a higher prevalence of periodontitis during pregnancy and this condition remained even with the reduction of hormone levels after childbirth. it is important to highlight that some limitations were found among those studies that sought to assess the association of these outcomes during pregnancy. most of them had a cross-sectional design, which makes it impossible to understand the cause-and-effects relationship between the outcomes. in addition, they differ in relation to the sample size, cutoff points of the bmi to classify excessive weight and, mainly, to the classification for periodontitis. these divergences make it difficult to compare studies. it is known that maternal periodontitis may result in negative perinatal outcomes, being associated with a doubled risk of prematurity21 and with low birth weight22. therefore, the holistic care of women during pregnancy is necessary, considering their systemic involvement. as future perspectives, it is expected that population-based longitudinal studies will be conducted in order to better understand the association of obesity and periodontitis during pregnancy. in addition, studies at biological levels are also important, aiming to identify the plausible pathophysiological mechanisms of the association of these outcomes and, possibly, to identify biomarkers that serve as instruments for us to adopt adequate protocols in the management of periodontitis during pregnancy in women affected by obesity acknowledgements the authors would like to thank the coordination for the improvement of higher education personnel (capes finance code 001) and the são paulo research foundation (fapesp; #2015/25421-4; #2018/13990-2; # 18/25934-0; #2018/20626-5) for the financial support to some of the studies mentioned in this letter. references 1. beck jd, papapanou pn, philips kh, offenbacher s. periodontal medicine: 100 years of progress. j dent res. 2019 sep;98(10):1053-62. doi: 10.1177/0022034519846113. 3 foratori-junior et al. braz j oral sci. 2022;21:e225385 2. monsarrat p, blaizot a, kémoun p, ravaud p, nabet c, sixou m, et al. clinical research activity in periodontal medicine: a systematic mapping of trial registers. j clin periodontol. 2016 may;43(5):390-400. doi: 10.1111/jcpe.12534. 3. silva de araujo figueiredo c, gonçalves carvalho rosalem c, costa cantanhede al, abreu fonseca thomaz éb, fontoura nogueira da cruz mc. systemic alterations and their oral manifestations in pregnant women. j obstet gynaecol res. 2017 jan;43(1):16-22. doi: 10.1111/jog.13150. 4. foratori-junior ga, andrade fjp de, mosquim v, peres m de cs, chaim ea, sales-peres sh de c. association of metabolic syndrome with oral and systemic conditions in morbidly obese patients. braz j oral sci. 2019 apr;18:e191484. doi: 10.20396/bjos.v18i0.8655299. 5. brazilian institute of geography and statistics. [national health survey 2019: primary health care and anthropometric information]. rio de janeiro: brazilian institute of geography and statistics; 2020. 57p. portuguese. available from: https://biblioteca.ibge.gov.br/index.php/biblioteca-catalogo? view=detalhes&id=2101758. 6. moura-grec pg, marsicano ja, carvalho ca, sales-peres sh. obesity and periodontitis: systematic review and meta-analysis. cien saude colet. 2014 jun;19(6):1763-72. doi: 10.1590/141381232014196.13482013. 7. keller a, rohde jf, raymond k, heitmann bl. association between periodontal disease and overweight and obesity: a systematic review. j periodontol. 2015 jun;86(6):766-76. doi: 10.1902/jop.2015.140589. 8. khan s, barrington g, bettiol s, barnett t, crocombe l. is overweight/obesity a risk factor for periodontitis in young adults and adolescents?: a systematic review. obes rev. 2018 jun;19(6):85283. doi: 10.1111/obr.12668. 9. chapper a, munch a, schermann c, piacentini cc, fasolo mt. obesity and periodontal disease in diabetic pregnant women. braz oral res. 2005 apr-jun;19(2):83-7. doi: 10.1590/s1806-83242005000200002. 10. piscoya md, ximenes ra, silva gm, jamelli sr, coutinho sb. periodontitis-associated risk factors in pregnant women. clinics (sao paulo). 2012;67(1):27-33. doi: 10.6061/clinics/2012(01)05. 11. vogt m, sallum aw, cecatti jg, morais ss. factors associated with the prevalence of periodontal disease in low-risk pregnant women. reprod health. 2012 jan 24;9:3. doi: 10.1186/1742-4755-9-3. 12. lee hj, jun jk, lee sm, ha je, paik di, bae kh. association between obesity and periodontitis in pregnant females. j periodontol. 2014 jul;85(7):e224-31. doi: 10.1902/jop.2014.130578. 13. xie y, xiong x, elkind-hirsch ke, pridjian g, maney p, delarosa rl, et al. prepregnancy obesity and periodontitis among pregnant females with and without gestational diabetes mellitus. j periodontol. 2014 jul;85(7):890-8. doi: 10.1902/jop.2013.130502. 14. zambon m, mandò c, lissoni a, anelli gm, novielli c, cardellicchio m, et al. inflammatory and oxidative responses in pregnancies with obesity and periodontal disease. reprod sci. 2018 oct;25(10):1474-84. doi: 10.1177/1933719117749758. 15. fusco nds, foratori-junior ga, missio alt, jesuino bg, sales-peres shc. systemic and oral conditions of pregnant women with excessive weight assisted in a private health system. int dent j. 2019 dec;69(6):472-9. doi: 10.1111/idj.12507. 16. caracho ra, foratori-junior ga, fusco nds, jesuino bg, missio alt, sales-peres shc. systemic conditions and oral health-related quality of life of pregnant women of normal weight and who are overweight. int dent j. 2020 aug;70(4):287-95. doi: 10.1111/idj.12547. 17. foratori-junior ga, da silva bm, da silva pinto ac, honório hm, groppo fc, de carvalho sales-peres sh. systemic and periodontal conditions of overweight/obese patients during pregnancy and after delivery: a prospective cohort. clin oral investig. 2020 jan;24(1):157-65. doi: 10.1007/s00784-019-02932-x. https://biblioteca.ibge.gov.br/index.php/biblioteca-catalogo?view=detalhes&id=2101758 https://biblioteca.ibge.gov.br/index.php/biblioteca-catalogo?view=detalhes&id=2101758 4 foratori-junior et al. braz j oral sci. 2022;21:e225385 18. foratori-junior ga, jesuino bg, caracho ra, orenha es, groppo fc, sales-peres shc. association between excessive maternal weight, periodontitis during the third trimester of pregnancy, and infants’ health at birth. j appl oral sci. 2020 mar;28:e20190351. doi: 10.1590/1678-7757-2019-0351. 19. gomes-filho is, batista jet, trindade sc, passos-soares js, cerqueira emm, costa tsd, et al. obesity and periodontitis are not associated in pregnant women. j periodontal res. 2020 jan;55(1):77-84. doi: 10.1111/jre.12690. 20. foratori-junior ga, missio alt, orenha es, de carvalho sales-peres sh. systemic condition, periodontal status, and quality of life in obese women during pregnancy and after delivery. int dent j. 2021 jan:s0020-6539(20)36543-6. doi: 10.1016/j.identj.2020.12.012. 21. manrique-corredor ej, orozco-beltran d, lopez-pineda a, quesada ja, gil-guillen vf, carratala-munuera c. maternal periodontitis and preterm birth: systematic review and meta-analysis. community dent oral epidemiol. 2019 jun;47(3):243-51. doi: 10.1111/cdoe.12450. 22. figuero e, han yw, furuichi y. periodontal diseases and adverse pregnancy outcomes: mechanisms. periodontol 2000. 2020 jun;83(1):175-88. doi: 10.1111/prd.12295. 1 volume 16 2017 e17011 original articlebjos 1 state university of são paulo (unesp), school of dentistry, department of pediatric dentistry and public health, araçatuba, sp, brazil. 2 federal fluminense university (uff), institute of health, department of dentistry, nova friburgo, rj, brazil. corresponding author: cristiane duque department of pediatric dentistry and public health state university of são paulo (unesp) school of dentistry address: r josé bonifácio, 1193, cep. 16015050, araçatuba, sp, brazil. tel: (+55) 1836363315. e-mail: cristianeduque@yahoo.com. br, cduque@foa.unesp.br received: january 31, 2017 accepted: august 02, 2017 effect of storage time and chlorhexidine addition on the mechanical properties of glass ionomer cements juliana de carvalho machado1, josânia pitzer de oliveira2, cristiane duque1*, angela scarparo2 aims: to evaluate the effect of the chlorhexidine (chx) incorporation and the storage time on the mechanical properties of glass ionomer cements (gics). methods: the following gics were evaluated: ketac molar easymix (km), vidrion r (vr) and vitromolar (vm), containing or not chx. gic liquid was modified by adding 1.25 % chx digluconate and then manipulated with the power and placed into the stainless steel cylindrical or bar-shaped molds. gics specimens were stored into water for 1, 7 and 28 days. after these periods, specimens were submitted to flexural, diametral tensile and compressive strength tests, according to iso standards. data from mechanical tests were statistically analyzed using 2-way anova and tukey tests. results: overall, the storage time did not influence any of the mechanical properties of the gics tested. in contrast, the inclusion of chx reduced significantly these properties for all gics tested. km presented the highest values of compressive strength for all storage times. km + 1.25% chx had lower compressive strength results than km, however, it showed similar results when compared to another gics without chx. conclusions: the presence of chlorhexidine, independent of the storage time, interfered on the mechanical characteristics of gic. keywords: glass ionomer cements, chlorhexidine, dental caries, water storage, mechanical properties http://dx.doi.org/10.20396/bjos.v16i0.8650457 2 machado et al. introduction the atraumatic restorative treatment (art) approach consisted in the removal of infected dentin using cutting hand tools under relative isolation followed by the restoration of the cavities with conventional glass ionomer cements (gics) as the material of choice1-6. this procedure is considered of low cost, easy handling and high applicability1-3,5-7, which causes minimal discomfort to the patient by eliminating the need for local anesthesia2,3,5. the world health organization (who) recognized art in 1994 as a new approach for dental treatment in regions economically less favored8. it has been stated in the minimum intervention philosophy for several reasons, such as the maintenance of healthy tooth structure, early intervention in the progression of caries lesions3,7, preservation of decayed teeth without endodontic involvement, promotion of oral health education concomitant with the restorative treatment9. art has contributed to the management of pediatric patients behavior mainly in the cases of non-cooperative patients and patients with special needs3. the use of gics for art approach is due to their intrinsic properties, such as adhesion to the dental structure, biocompatibility, coefficient of thermal expansion similar to tooth structure and fluoride release1,3-5,7,10. gics more indicated for art are those with a high proportion powder/liquid11, mainly ketac molar and fuji ix, due to improved mechanical properties for restorations in posterior teeth. previous studies showed that the restorations longevity rates are higher when the art of high viscosity cements are used, both in the primary dentition and in permanent11,12. however, high aspect ratio powder/liquid have resulted in decreased solubility and fluoride release13. studies have shown that gics have a variable antimicrobial activity, possibly related to different initial ph values, the amount of fluoride released as well as the chemical components present in each cement powder. the gics indicated for art have shown reduced antimicrobial activity5,6,14,15, and for this reason, the incorporation of antimicrobial agents (chlorhexidine and antibiotics) to the cement has been suggested in several investigations1,6,14-18, in order to increase the effect of these materials against residual microorganisms on cavities after partial caries removal. chlorhexidine is the antimicrobial agent commonly used in dentistry due its safety and broad-spectrum bactericidal effect, affecting the growth of gram-positive bacteria especially oral streptococci19, gram-negative bacteria, fungi and yeasts, facultative aerobic and anaerobic species20. conflicting results about the influence of the incorporation of chlorhexidine on the physical-mechanical properties of gics are still present in the literature. some studies have shown that the addition of chlorhexidine decreased the mechanical properties of gics, such as the compressive strength1,14-16, and others showed no negative effects on these properties, except for concentrations above 2% (8,17). however, the influence of the storage time on the mechanical properties of gics containing chlorhexidine has been not studied yet. thus, the aim of this study was to evaluate the effect of the chlorhexidine incorporation and the storage time on the mechanical properties of glass ionomer cements. the study’s hypotheses were that 1) chlorhexidine incorporation affects mechanical properties 3 machado et al. of glass ionomer cements along the time and 2) water storage affects mechanical properties of glass ionomer cements containing or not chlorhexidine along the time. material and methods dental materials three glass ionomer cements (gics) were evaluated: ketac molar easymix (3m/ espe, st. paul, usa), vidrion r (ss white dental products ltd., rio de janeiro – rj, brazil) and vitromolar (dfl, rio de janeiro – rj, brazil). the composition of each gic is presented in table 1 and the distribution of the groups according to the factors studied (gic, presence or not of chx, storage time and mechanical test) and the number of samples per group (n) is shown in table 2. gic liquid was modified by adding 1.25 % chlorhexidine digluconate (chx – c9394 sigma–aldrich, steinheim, germany), as proposed by türkün et al.15 and then manipulated with the power, according to each gic manufacturer’s instructions without altering liquid/powder ratio. the scoop of powder and the droop of liquid were previously weighted in an analytical balance in order to standardize the recommend powder/liquid ratio for each gic (bioprecisa, são paulo – sp, brazil). each gic containing or not chx were mixed and placed into the stainless steel cylindrical or bar-shaped molds using a restorative dispenser (and tip, slightly overfilled and compressed with polyethylene sheets and glass slabs. after 5 minutes of the initial setting of the materials, specimens were removed from the mold and excess were removed manually using a 600-grit sic paper. the dimensions of specimens were checked using a digital caliper (digimatic caliper, mitutoyo corp., tokyo, japan). a colorless glaze protection (nail enamel, revlon inc, ny, usa) was applied on all surfaces of gic samples, in order to inhibit the syneresis and imbibition processes. the specimens were then stored inside plates containing gauze soaked with water (relative humidity) at 37oc for 1 h. after this period, gic samples were immersed in 10ml of deionized water in individual plastic containers and stored for 1, 7 and 28 days. control group comprises specimens without chx incorporation. after the storage times, samples were submitted to mechanical tests described below. table 1. composition of the conventional glass ionomer cements used in this study cement code composition manufacturer liquid powder vidrion r vr sulphate na-ca-ba-alfluorosilicate, acrylic acid tartaric acid and water ss white dental products ltda, rio de janeiro, rj, brazil vitromolar vm ba-al silicate, polyacrylic acid and dehydrated zinc oxide polyacrylic acid, tartaric acid and distilled water dfl. rio de janeiro, rj, brazil ketac molar easymix km polialquenoic acid, tartaric acid and water fluorosilicate glass al-ca-la copolymer (5% acrylic acid and maleic acid 3m/espe, st. paul, usa 4 machado et al. mechanical tests flexural strength the flexural strength (fs) was measured according to the iso standard iso9917-2 using 25mm length x 2mm width x 2mm height bar-shaped specimens (n=10). after storage times, the specimens were submitted to a 3-point bending test (the distance between the two supports is 20 mm) on a universal testing machine (instron no. 4442, instron corp, canton, ma, usa) at a crosshead speed of 1mm/min until the fracture. the fs was calculated with the following formula: fs=3r/2wh2, where r is the load required to fracture in mpa; l is the distance between the supports (20.0 mm); w is the specimen width and h is the specimen height. diametral tensile strength the specimens (n=10) for diametral tensile strength (dts) were made using cylindrical metal molds with 4mm diameter x 6mm thickness, according to the specifications of iso standard iso9917-1. after the storage periods, were subjected to the dts test on a universal testing machine (instron), at a speed of 0.5 mm/min in horizontal position until the fracture. the dts was calculated with the following formula: dts =2f /pdt, where f is the load required to fracture; p is 3.1416; d is the diameter and t the thickness). compressive strength the compressive strength (cs) was measured under the same conditions (storage conditions, testing machine and number of specimens) with specimens with 4mm diameter and 6 mm thickness (n=10), at a speed of 1mm/min in a vertical position until the fracture, according to the specifications of iso9917-1. the values obtained were converted into mpa using the following formula: cs =f/1/4pd2, where f is the load at fracture, p is 3.1416 and d2 the diameter in mm2. statistical analysis two-way anova and tukey post hoc tests (p = 0.05) were used to test the influence of chx and storage time on the mechanical properties of the gics, considering p<0.05. the software used was spss version 17.1 (spss inc., chicago, usa). results the mean and standard deviation values (mpa) for flexural, diametral tensile and compressive strength are summarized in tables 2, 3 and 4, respectively. overall, the storage time did not influence any of the mechanical properties of the gic tested. in contrast, the inclusion of chx reduced significantly the mechanical properties of all gics tested. gic without chx did not differ from each other for flexural and diametral tensile strength tests (tables 2 and 3), independent on the storage time evaluated. the same was observed among gics containing chx. km presented the highest values of compressive strength for all storage times. km + 1.25% chx had lower compressive strength results than km, however, it showed similar results when compared to another gics without chx (table 4). 5 machado et al. discussion high viscosity gics for art technique have been introduced in the market promising better mechanical properties than conventional gics. however, the gics indicated for art have shown reduced antimicrobial activity5,6,14,15, and the incorporation of table 3. comparison of means (standard deviations) in mpa of diametral tensile strength for the glass ionomer cements containing or not 1.25% chx at different storage times. material 1 day 7 days 28 days vr 7.60 (2.80)aa 7.42 (0.74)aa 7.11 (0.97)aa vr + 1.25% chx 3.31 (1.86)ba 3.50 (1.19)ba 3.82 (1.93)ba vm 7.09 (2.68)aa 6.83(3.66)aa 6.64 (2.72)aa vm+ 1.25% chx 4.12 (2.08)ba 3.94(2.49)ba 4.08 (2.56)ba km 8.25 (3.99)aa 8.31(2.37)aa 7.54 (1.90)aa km + 1.25% chx 4.48 (1.03)ba 4.39(0.63)ba 4.37 (2.09)ba a different upper case letters showed statistical difference between gics and gics containing chx, according to anova and tukey tests. a different lower case letters showed statistical difference comparing the storage time for the same gic, according to anova and tukey tests. table 4. comparison of means (standard deviations) in mpa of compressive strength for the glass ionomer cements containing or not 1.25% chx at different storage times. material 1 day 7 days 28 days vr 10.60 (3.00)aa 10.98(3.79)aa 11.16 (2.64)aa vr + 1.25% chx 6.46 (2.22)ba 6.61(2.86)ba 6.71(2.21)ba vm 10.10(2.26)aa 13.96(5.09)a,cb 11.55 (3.18)aa vm+ 1.25% chx 8.15(3.40)ba 8.28(3.20)ba 8.36 (3.35)ba km 16.76 (5.04)ca 16.36(2.96)ca 16.62 (2.68)ca km + 1.25% chx 10.71 (2.48)aa 10.63(3.36)aa 10.33 (3.56)aa a different upper case letters showed statistical difference between gics and gics containing chx, according to anova and tukey tests. a different lower case letters showed statistical difference comparing the storage time for the same gic, according to anova and tukey tests. table 2. comparison of means (sd standard deviations) in mpa of flexural strength for the glass ionomer cements containing or not 1.25% chx at different storage times. material 1 day 7 days 28 days vr 17.08(5.62)aa 19.54(3.67)aa 19.31 (3.77)aa vr + 1.25% chx 5.00(2.61)ba 4.56(2.70)ba 3.95(1.85)ba.b vm 20.01 (6.78)aa 16.88(10.01)aa 18.21 (7.85)aa vm+ 1.25% chx 6.35 (2.09)ba 6.07(2.30)ba 6.13 (2.92)ba km 20.79(5.81)aa 16.82(9.97)aa 19.63 (6.18)aa km + 1.25% chx 5.18(2.50)ba 5.67(1.80)ba 4.49 (2.40)ba a different upper case letters showed statistical difference between gics containing or not chx, according to anova and tukey tests. a different lower case letters showed statistical difference comparing the storage time for the same gic, according to anova and tukey tests. 6 machado et al. chlorhexidine could be an alternative to improve their action against residual bacteria from cavities. in addition, restorations are constantly bathed with saliva, influencing the syneresis and imbibition processes of gics. however, the effect of water storage on the mechanical properties of these high viscosity gics containing chlorhexidine have been not studied yet. deionized water, artificial or human saliva have been frequently chosen as storage media simulating intraoral conditions. in this present study, gics were immersed in water as storage media. this choice was based in the study of mckenzie et al.21. these authors reported that physical-mechanical properties of conventional and resin-modified glass ionomer cements were not significantly different comparing storage in water or saliva up to 1 year. therefore, water was considered acceptable as the storage medium for in vitro analysis of gic properties. in this present study, the storage time did not influence any of the mechanical properties of gics evaluated. our results are according with zoergiebel and ilie22 that evaluated three conventional gics in comparison with newly developed zinc-containing gic and demonstrated low impact of storage agent (water and artificial saliva) and storage duration (7 and 30 days) on the flexural strength, modulus of elasticity and hardness properties. other studies reported a tendency for increasing the compressive strength after 1-week storage and remaining unchanged up to 1-year storage21,23. the early moisture contamination could decreases mechanical properties of gics favoring surface erosion and abrasion. in the present study, gic specimens were protected from the influence of water by a thin colorless glaze preventing water contamination on the initial phase of the setting to endure 24h up to 2 weeks24. the incorporation of chlorhexidine reduced the mechanical properties of gics studied in the current study. our results confirmed the findings obtained by takahashi et al.8, turkun et al.15, palmer et al.16, marti et al.25, mittal et al.26. the addition of chx may alter the powder/liquid ratio and consequently the mechanical strength of the material6,15,17. the form of chx salt could also influence in the gics properties. chx digluconate (liquid form) is solubilized faster into oral environment than the chx diacetate (powder form), however, both forms could hamper the chemical reaction of gics by neutralization of polyacids to release ions from glass particles or by the formation of base/polyacid complexes that block reactions between cationic ions and polyacrylic chains, consequently increasing the setting time27-29. marti et al.25 showed that the addition of chx at concentration of 2% resulted in significant increase on setting time and decrease on the surface hardness of high viscosity gics. in addition, the tensile bond strength of these materials also decreased significantly after adding 2% chx. although study of mittal et al.26 observed that the incorporation of 1.5% chx did not affect 24-h compressive strength of a high viscosity gic, the majority of the studies are in agreement that the addition of up to 1% chx in high viscosity gics did not change their physico-mechanical properties25,28,30,31. in the present study, ketac molar presented the best results for compressive strength. in addition, ketac molar with 1.25% chx showed similar results of compressive strength compared to another gics without chx. similar results were obtained by bonifácio et al.1 and algera et al.32. the strength of gics is influenced by their composition. high content of fluoride or zinc increases the ability of gic form a network with acrylic acid and 7 machado et al. decreased the setting time, induced a higher compressive and flexural strength33. superior flexural and compressive strengths have been reported for ketac molar in comparison to vitromolar1, similar to those found in the present study. these gics have been exhibited high concentration of fluoride in their composition similar to resin-modified gics and their high viscosity allows a faster setting reaction resulting on fluoride lockup within the matrix forming a reservoir to be released later. there was no relationship between the amount of fluoride in the composition of gic and the amount of fluoride release to the oral environment. the amount of fluoride release did not affect the changes of the compressive strength or the surface hardness up to 1-year water storage34. in conclusion, one of the hypotheses of this present study was rejected, because neither gic nor gic containing chx reduced their properties up to 30 days of water storage. however, the presence of chlorhexidine interfered on the mechanical characteristics of gic. among the gic tested, ketac molar, containing or not 1.25% chx, presented the highest values of compressive strength. acknowledgments the authors would like to thank to faperj (fundação de amparo à pesquisa do estado do rio de janeiro) for the financial support (process number #e-26/ 101.830/2010). references 1. bonifácio cc, kleverlaan cj, raggio dp, werner a, de carvalho rcr, van amerongen we. physical-mechanical properties of glass ionomer cements indicated for atraumatic restorative treatment. aust dent j. 2009 sep;54(3):233-7. doi: 10.1111/j.1834-7819.2009.01125.x. 2. davidovich e, weiss e, fuks ab, beyth n. surface antibacterial properties of glass ionomer cements used in atraumatic restorative treatment. j am dent assoc. 2007 oct;138(10):1347-52. 3. franca c, 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roughness, and anti-biofilm activity. am j dent. 2014 dec;27(6):318-22. 26. mittal s, soni h, sharma dk, mittal k, pathania v, sharma s. comparative evaluation of the antibacterial and physical properties of conventional glass ionomer cement containing chlorhexidine and antibiotics. j int soc prev community dent. 2015 jul-aug;5(4):268-75. doi: 10.4103/2231-0762.161754. 27. prosser hj, jerome sm, wilson ad. the effect of additives on the setting properties of a glass-ionomer cement. j dent res. 1982 oct;61(10):1195-8. 28. deepalakshmi m, poorni s, miglani r, rajamani i, ramachandran s. evaluation of the antibacterial and physical properties of glass ionomer cements containing chlorhexidine and cetrimide: an in-vitro study. indian j dent res. 2010 oct-dec;21(4):552-6. doi: 10.4103/0970-9290.74217. http://www.ncbi.nlm.nih.gov/pubmed/17518967 http://www.ncbi.nlm.nih.gov/pubmed/17518967 http://www.ncbi.nlm.nih.gov/pubmed/17518967 http://www.ncbi.nlm.nih.gov/pubmed/?term=mittal s%5bauthor%5d&cauthor=true&cauthor_uid=26310885 http://www.ncbi.nlm.nih.gov/pubmed/?term=soni h%5bauthor%5d&cauthor=true&cauthor_uid=26310885 http://www.ncbi.nlm.nih.gov/pubmed/?term=sharma dk%5bauthor%5d&cauthor=true&cauthor_uid=26310885 http://www.ncbi.nlm.nih.gov/pubmed/?term=mittal k%5bauthor%5d&cauthor=true&cauthor_uid=26310885 http://www.ncbi.nlm.nih.gov/pubmed/?term=pathania v%5bauthor%5d&cauthor=true&cauthor_uid=26310885 http://www.ncbi.nlm.nih.gov/pubmed/?term=sharma s%5bauthor%5d&cauthor=true&cauthor_uid=26310885 http://www.ncbi.nlm.nih.gov/pubmed/?term=deepalakshmi m%5bauthor%5d&cauthor=true&cauthor_uid=21187624 http://www.ncbi.nlm.nih.gov/pubmed/?term=poorni s%5bauthor%5d&cauthor=true&cauthor_uid=21187624 http://www.ncbi.nlm.nih.gov/pubmed/?term=miglani r%5bauthor%5d&cauthor=true&cauthor_uid=21187624 http://www.ncbi.nlm.nih.gov/pubmed/?term=rajamani i%5bauthor%5d&cauthor=true&cauthor_uid=21187624 http://www.ncbi.nlm.nih.gov/pubmed/?term=ramachandran 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properties of setting glass-ionomer cements. dent mater. 2006 sep; 22(9):852-6. 33. de barra e, hill rg. influence of glass composition on the properties of glass polyalkenoate cements. part iii: influence of fluorite content. biomaterials. 2000 mar; 21(6):563-9. 34. shiozawa m, takahashi h, iwasaki n. fluoride release and mechanical properties after 1-year water storage of recent restorative glass ionomer cements. clin oral investig. 2014 may;18(4):1053-60. doi: 10.1007/s00784-013-1074-4. http://www.ncbi.nlm.nih.gov/pubmed/?term=marti lm%5bauthor%5d&cauthor=true&cauthor_uid=24789289 http://www.ncbi.nlm.nih.gov/pubmed/?term=mata md%5bauthor%5d&cauthor=true&cauthor_uid=24789289 http://www.ncbi.nlm.nih.gov/pubmed/?term=ferraz-santos b%5bauthor%5d&cauthor=true&cauthor_uid=24789289 http://www.ncbi.nlm.nih.gov/pubmed/?term=azevedo er%5bauthor%5d&cauthor=true&cauthor_uid=24789289 http://www.ncbi.nlm.nih.gov/pubmed/?term=giro em%5bauthor%5d&cauthor=true&cauthor_uid=24789289 http://www.ncbi.nlm.nih.gov/pubmed/?term=zuanon ac%5bauthor%5d&cauthor=true&cauthor_uid=24789289 http://www.ncbi.nlm.nih.gov/pubmed/24789289 http://www.ncbi.nlm.nih.gov/pubmed/?term=ahluwalia p%5bauthor%5d&cauthor=true&cauthor_uid=22565516 http://www.ncbi.nlm.nih.gov/pubmed/?term=chopra s%5bauthor%5d&cauthor=true&cauthor_uid=22565516 http://www.ncbi.nlm.nih.gov/pubmed/?term=thomas am%5bauthor%5d&cauthor=true&cauthor_uid=22565516 http://www.ncbi.nlm.nih.gov/pubmed/?term=jaidka s%5bauthor%5d&cauthor=true&cauthor_uid=27195231 http://www.ncbi.nlm.nih.gov/pubmed/?term=somani r%5bauthor%5d&cauthor=true&cauthor_uid=27195231 http://www.ncbi.nlm.nih.gov/pubmed/?term=singh dj%5bauthor%5d&cauthor=true&cauthor_uid=27195231 http://www.ncbi.nlm.nih.gov/pubmed/?term=shafat s%5bauthor%5d&cauthor=true&cauthor_uid=27195231 http://www.ncbi.nlm.nih.gov/pubmed/?term=shiozawa m%5bauthor%5d&cauthor=true&cauthor_uid=23974799 http://www.ncbi.nlm.nih.gov/pubmed/?term=takahashi h%5bauthor%5d&cauthor=true&cauthor_uid=23974799 http://www.ncbi.nlm.nih.gov/pubmed/?term=iwasaki n%5bauthor%5d&cauthor=true&cauthor_uid=23974799 1http://dx.doi.org/10.20396/bjos.v19i0.8658221 volume 19 2020 e201669 original article 1 department of stomatology, division of periodontics, school of dentistry, university of são paulo (fo-usp), são paulo, brazil. corresponding author: claudio mendes pannuti address: av. professor lineu prestes, 2227, zip code 05508-000 – department of stomatology, division of periodontics, university of são paulo – fo-usp, são paulo, sp, brazil. telephone: +55 (11) 30917833. e-mail: pannuti@usp.br received: may 24, 2019 accepted: september 19, 2019 effect of magnification on root coverage surgery: a systematic review marcella goetz moro1, maria luisa silveira souto1, emanuel silva rovai1, joão batista cesar neto1, marinella holzhausen1, claudio mendes pannuti1,* root coverage surgery can be performed in patients with gingival recession to cover the exposed root aiming to control hypersensitivity and promotes better aesthetic. optical magnification has been proposed as a refinement in this surgical technique to increase root coverage. this approach may lead to enhanced soft tissue stability, less post-operative discomfort, better predictability and esthetic appearance. aim: this systematic review aimed to evaluate the effectiveness of magnification on root coverage surgery when compared to procedures performed without magnification. methods: randomized controlled trials with a follow-up of at least 6 months that compared surgeries for root coverage performed under optic magnification versus conventional (macro) root coverage surgery were screened. the primary outcome was mean root coverage (mm) (mrc) and secondary outcomes were percentage of root coverage (prc) and complete root coverage (crc). results: of 569 papers relevant to this review, seven were included. meta-analysis showed that the use of magnification may favor greater prc (7.38%, 95% ci 3.66-11.09). conclusion: magnification can increase prc in root coverage surgeries. more randomized trials with the use of magnification may be necessary to verify if this benefit is clinically relevant, in order to justify the use of this device. keywords: gingival recession. microsurgery. periodontitis. review. 2 moro et al. introduction gingival recession (gr) is the apical displacement of the gingival margin, which results in the exposure of the root surface1,2. it is a frequent condition, which affects a significant percentage of subjects and teeth3. it has been associated with older age, male gender4, smoking exposure5,6, higher education3,5,7,8, poor self-reported oral hygiene5,6,9,10, higher percentage of sites with gingivitis6, regular dental visits, history of periodontal treatment and presence of calculus3,5,7,8. exposed root surfaces present an increased risk for caries, abrasion and erosion1,11,12. furthermore, gr is related with hypersensitivity and poor esthetics, which has an impact on oral health-related quality of life13. root coverage surgery can be performed in these patients, aiming to cover the exposed root14,15. the main objective of root coverage surgery is to achieve clinically relevant root coverage (rc). several techniques have been proposed as root coverage procedures11,16,17, which result in correction of gingival deformities, position and/ or amount of keratinized tissue14,15. currently, optical magnification has been proposed as a refinement in mucogingival surgical techniques, aiming to increase rc. magnification of the operative field can be obtained by the use of loupes or microscope during the surgical procedure to amplify visual acuity and enhance illumination. as a consequence, magnification may minimize surgical invasiveness, enables more precise incisions and suture co-adaptation of wound edges18. this approach may lead to enhanced soft tissue stability, less post-operative discomfort, better predictability and esthetic appearance2,19. some clinical trials have observed that the use of optical magnification in root coverage procedures may enhance clinical outcomes and patient related outcomes, as aesthetic condition, when compared to conventional surgical procedures20-22, however, there is still lack of evidence in this field. a comprehensive evaluation, combining similar studies may contribute to understanding the impact of magnification on root coverage surgery. therefore, the present systematic review and meta-analyses aims to evaluate whether the use of magnification provides better clinical and aesthetic results when compared to conventional treatment in root coverage surgery. the following focused question was addressed: “in systemically healthy patients with miller class i and/or ii gingival recession, does magnification favor better clinical outcomes when compared to procedures without magnification?” materials and methods the protocol of this systematic review (sr) was registered at the national institute for health research prospero, international prospective register of systematic reviews (http://www.crd.york.ac.uk/prospero, registration number crd42017064682). the review text was structured according to prisma’s guidelines (preferred reporting items for systematic reviews and meta-analyses)23, cochrane handbook of systematic reviews of interventions24 and check review checklist25. 3 moro et al. eligibility criteria inclusion criteria randomized controlled trials, with follow-up of at least 6 months, that compared surgeries for root coverage performed under optic magnification versus conventional (macro) surgery in patients with miller class i and/ or ii gingival recessions were selected. only studies that mentioned the use of microscope or loupe in the surgical procedure were included. exclusion criteria trials that included patients with systemic disease (e.g., diabetes). non-randomized trials, studies that did not have a control group without magnification, animal studies, in vitro studies, reviews and letters. primary outcome mean root coverage (mrc), expressed in millimeters. secondary outcomes percentage of root coverage (prc), complete root coverage (crc), keratinized tissue width (ktw) change, keratinized tissue thickness (ktt) change, clinical attachment level (cal) change, probing pocket depth (ppd) change, aesthetic condition change, surgical operation time (min) and adverse effects. information source and search strategy medline via pubmed, embase and lilacs databases were used to search publications up to may 2019. mesh terms and keywords were combined with boolean operators (or; and) and used to search the databases. there was no restriction regarding language or publication year. search strategies were: 1# root coverage or gingival recession (mesh terms) or coronal advanced flap or connective tissue graft or periodontal plastic surgery or mucogingival surgery and; 2# microsurgery (mesh terms) or microscope or microsurgical or magnification or loupe. in addition, reference lists of the selected studies were hand-searched, and unpublished studies were searched at open grey. study selection study selection was completed in two phases, as follows: 1) titles and abstracts; 2) full text screening. in the first phase, two reviewers (m.g.m. and m.l.s.s.) independently screened titles and abstracts. in the second phase, the same reviewers independently read the full text of the selected articles. in both phases, any disagreement was resolved by a third reviewer (c.m.p.). data extraction and validity assessment were performed for publications that met the inclusion criteria and reasons for excluding publications were recorded. data collection two reviewers (m.g.m. and m.l.s.s.) collected data from the selected articles using extraction forms. any disagreements in the data extraction were discussed with a 4 moro et al. third reviewer (c.m.p.). also, if needed, the authors of the included studies were contacted to elucidate questions or provide missing data. the following data were recorded from the eligible studies: 1) citation, 2) country of the study, 3) characteristics of trial participants (age, gender and other trial´s eligibility criteria), 4) miller’s classification of the recession defect26, 5) length of follow-up, 6) intervention’s characteristics (type of surgery, type of microscope/ loupe, magnification and microsurgical instruments), 7) sample size, 8) outcome measures, 9) conclusions, and 10) financial support and conflict of interest. risk of bias of the included studies risk of bias was ascertained according to the cochrane collaboration’s tool for assessing risk of bias. two reviewers (m.g.m. and m.l.s.s.) independently evaluated quality of randomization and allocation concealment (selection bias); completeness of follow-up period/ incomplete outcome data (attrition bias); selective reporting (reporting bias); blinding of examiners (detection bias) and other forms of bias. performance bias was not evaluated since it is not possible to mask patients and operators in studies that use microscopes or loupes. each domain was classified as adequate (+), inadequate (-) or unclear (?). overall risk of bias was categorized as: 1) low risk of bias if all criteria were met; 2) unclear risk of bias if one or more criteria were partly met; or 3) high risk of bias if one or more criteria were not met. any disagreement was solved by a third investigator (c.m.p.). quality of evidence (grade) grade (grades of recommendation, assessment, development, and evaluation) guidelines were used to assess the strength of evidence across rcts for each outcome. the quality of evidence was classified into four categories: high quality, moderate quality, low quality, and very low quality, based on risk of bias, consistency, directness and precision27. summary measures and synthesis of results summary measures were calculated as difference in means for mrc and prc, gain of ktw and cal change, and as risk ratio for crc, using random-effects models. all meta-analyses were conducted with a software package (review manager software, version 5.3, the nordic cochrane centre, the cochrane collaboration, copenhagen, denmark). moreover, heterogeneity among the included studies was assessed with cochran q statistic and i2 28. results a total of 569 potentially relevant papers were identified. after screening of titles and abstracts, 558 were excluded, leaving 11 articles. after complete reading of full text, 4 papers were considered not eligible for inclusion. at the end of the process, 7 papers were included in the review, as shown in the flowchart (figure 1). 5 moro et al. included studies initially, 135 subjects with gingival recession were enrolled, and 128 (94.8%) completed the follow up period. the main characteristics of the included studies are shown in table 1. the age of the included patients ranged from 18 to 67 years old, and most of them were female. when miller’s classification of the recession defect was analyzed, class i was predominant. a total of 255 miller class i and ii gingival recessions were treated. four studies used a split mouth design20,22,29,30, and the other three used parallel groups19,21,31. the follow-up period of the trials were 629,30, 1219,20,22,31 and 24 months21. seven participants drop out the respective studies20,29 either because of relocation or refusal to complete the research. most of the selected studies excluded smokers20-22,29,31. however, two papers did not mention the smoking habits of the included participants19,30. risk of bias two studies were considered of low bias risk22,31 and the other five were considered of unclear risk of bias (figure 2)19-21,29,30. records identified through database searching (n = 569) additional records identified through other sources (n = 0) records after duplicates removed (n = 569) records screened (n = 569) records excluded (n = 558) full-text articles assessed for eligibility (n = 11) studies included in qualitative synthesis (n = 07) studies included in quantitative synthesis (meta-analysis) (n = 07) studies excluded, with reasons (n = 0) agarwal et al. (2016): comparison between microsurgical techniques. francetti et al. (2004): surgical technique around implant. thankkappan et al. (2015): comparison between microsurgical techniques. andrade et al. (2010): miller class iii gingival recessions id en ti fi ca ti on s cr ee ni ng el ig ib ili ty in cl ud ed full-text articles excluded, with reasons (n = 04) figure 1. flowchart. 6 moro et al. table 1. characteristics of the studies. study/ country study design follow-up sample size (baseline) participants recession areas (miller’s classification)/ number of recessions source of funding azaripour et al., 2016/ germany parallel rct 12 months n= 40 (15 male and 25 female) age range: 19-64 years (38.6 ± 12.8 years) test group: n baseline = 15 n end of trial = 15 control group: n baseline = 15 n end of trial = 15 at least one miller class i or ii buccal gingival recession defect ≥ 1 and < 6 mm in depth. n = 71 (42 test; 29 control) department of operative dentistry and periodontology of the university medical central, mainz. bittencourt et al., 2012/ brazil splitmouth rct 12 months n= 24 (13 male and 11 female) age range: 18-55 years (34 years) test group: n baseline = 24 n end of trial = 24 control group: n baseline = 24 n end of trial = 24 presence of bilateral miller class i or ii gingival recessions (> 2 mm) in maxillary canines or premolars. n = 48 (24 test; 24 control) research funding agency of bahia state, brazil. burkhardt et al., 2005/ switzerland splitmouth rct 12 months n= 10 (4 male and 6 female) mean age: 32-44 years (mean not mentioned) test group: n baseline = 10 n end of trial = 8 control group: n baseline = 10 n end of trial = 8 presence of bilateral canine root denudations of class i or ii. n = 20 (10 test; 10 control) no francetti et al., 2005/ italy parallel rct 12 months n= 24 (male and female not mentioned) age: not mentioned test group: n baseline = 12 n end of trial = 12 control group: n baseline = 12 n end of trial = 12 buccal recession at least 2 mm deep; no loss of interdental bone or soft tissue (class i or ii miller’s). n = 24 (12 test; 12 control) no jindal et al., 2015/ india splitmouth rct 6 months n= 7 (6 male and 1 female) mean age: 18-67 years (mean not mentioned) test group: n baseline = 7 n end of trial = 7 control group: n baseline = 7 n end of trial = 7 bilateral isolated or multiple miller’s class i or class ii gingival recession ≥2 mm when measured from cement enamel junction (cej) on anterior teeth or premolar. n = 30 (15 test; 15 control) no nizam et al., 2015/ turkey parallel rct 24 months n= 24 (11 male and 13 female) age range: 19-41 years (mean not mentioned) test group: n baseline = 15 n end of trial = 13 control group: n baseline = 15 n end of trial = 12 presence of miller class i or class ii gingival recession >2 mm in at least one canine or premolar tooth. n = 42 (21 test; 21 control) no pandey and mehta, 2013/ india splitmouth rct 6 months n= 10 (male and female not mentioned) age range: 20-45 years (mean not mentioned) test group: n baseline = 10 n end of trial = 10 control group: n baseline = 10 n end of trial = 10 at least two sites of miller’s class i or class ii gingival recession labially in different quadrants with thick and wide interproximal papilla not smaller than the recession defect. n = 20 (10 test; 10 control) no 7 moro et al. in four studies, treatment was randomly assigned by coin toss20-22,29 and two studies used computer-generated random sequence19,31. one publication did not report how random sequence was generated30. four studies reported that allocation concealment was made properly21,22,29,31 and three studies did not report this information19,20,30. effects of interventions individual outcomes of studies the individual outcomes of studies are present in table 2. five of the included trials used mrc as primary outcome19,21,22,30,31. as secondary outcomes, six trials used prc and crc19-22,29,31. although pandey and mehta30 (2013) did not use prc and crc as outcome, they used mrc, cal gain and ktt (in mm). the use of magnification promoted significantly greater mrc in bittencourt et al.22 (2012) and nizam et al.21 (2015) studies. these two trials and the study of burkhardt and lang20 (2005) showed that figure 2. risk of bias. + ++++++ + ????? ? ? ? ? ? ? ? ? ??? ? ? ? ++++++ + ++ + + +++ +++ +++++ azaripour et al. 2016 bittencourt et al. 2012 burkhardt et al. 2005 francetti et al. 2005 jindal et al. 2010 nizam et al. 2015 pandey & mehta et al. 2013 r an do m s eq ue nc e ge ne ra tio n (s el ec tio n bi as ) a llo ca tio n co nc ea lm en t ( se le ct io n bi as ) b lin di ng o f o ut co m e as se ss m en t ( de te ct io n bi as ) in co m pl et e ou tc om e da ta (a tt rit io n bi as ) se le ct iv e re po rt in g (r ep or tin g bi as ) sa m pl e si ze c al cu la tio n o ve ra ll ris k of b ia s 8 moro et al. ta bl e 2. p ar tic ip an ts , i nt er ve nt io ns , o ut co m es a nd re su lts . s tu dy in te rv en tio ns m ic ro su rg ic al eq ui pm en t a es th et ic c on di tio n p r c / m c r m m c r c si te s/ % c a l m m k t w m m a za rip ou r e t a l., 20 16 te st g ro up : m m t t + c tg c on tr ol g ro up : c a f + c tg ze is s m ic ro sc op e “p ic o ”, se tt in g va rie s be tw ee n 0. 4 to 0 .6 (x 47 m ag ni fic at io n) , m ic ro su rg ic al in st ru m en ts . p at ie nt ’s o pi ni on : s at is fa ct or y (t es t a nd c on tr ol ); p ro fe ss io na l’s o pi ni on (r es ): te st : 9 .2 ± 1 .1 c on tr ol : 9 .2 ±1 .3 te st : 9 7. 3 ± 7. 6% / 2. 1 ± 1. 1 c on tro l: 98 .3 ± 9. 2% / 2. 3 ± 1. 2 te st : 3 7/ 88 .1 % c on tro l: 28 / 96 .6 % n ot re po rt ed te st : 0. 48 ± 0 .6 c on tro l: 0. 36 ± 0. 6 bi tte nc ou rt e t a l., 20 12 te st g ro up : t ec hn iq ue pr op os ed by t ib be tt s an d sh an el ec a nd m od ifi ed b y c am po s et a l. + c tg (m ic ro su rg er y) c on tr ol g ro up : t ec hn iq ue pr op os ed by t ib be tt s an d sh an el ec a nd m od ifi ed b y c am po s et a l. + c tg (c on ve nt io na l t ec hn iq ue ) m ic ro sc op e at x 8 to x1 2 m ag ni fic at io n (s m p lu s, o pt o el et rô ni ca , sã o p au lo , s p, b ra zi l); m ic ro su rg ic al in st ru m en ts . p at ie nt ’s o pi ni on : t es t: 10 0% c on tr ol : 7 9. 1% p ro fe ss io na l’s o pi ni on : n ot re po rt ed te st : 9 8% */ 2 .4 6 ± 0. 38 * c on tro l: 88 .3 % / 2. 24 ± 0 .6 4 te st : 2 1/ 87 .5 % * c on tro l: 14 / 58 .3 % te st : 1. 96 ± 0 .8 2 c on tro l: 1. 99 ± 0. 69 te st : 1. 51 ± 1 .0 1 c on tro l: 1. 37 ± 1, 18 b ur kh ar dt e t a l., 20 05 te st g ro up : m ic ro su rg er y c on tr ol g ro up : m ac ro su rg er y th e su rg ic al p ro ce du re w as pe rf or m ed a cc or di ng to th e te ch ni qu e de sc rib ed b y h ar ris (1 99 2) u si ng fr ee c on ne ct iv e tis su e gr af ts c ov er ed b y a do ub le -p ed ic le p ap ill a fla p. o p m i® p ro m ag is a t 15 m ag ni fic at io n (c ar l ze is s) , m ic ro su rg ic al in st ru m en ts . n ot re po rt ed te st : 9 8. 0 ± 3. 4% * c on tro l: 89 .9 ± 8. 5% te st : 5 / 62 .5 % * c on tro l: 2/ 25 % n ot re po rt ed n ot re po rt ed fr an ce tt i e t a l., 20 05 te st g ro up : 6 c a f + c tg , 1 c a f + g tr , 4 c a f + c tg + em d , 1 s em ilu na r fl ap (m ic ro su rg er y) c on tr ol g ro up : 9 c a f + c tg , 1 c a f + g tr , 2 c a f (m ac ro su rg er y) th e ty pe o f s ur gi ca l t ec hn iq ue w as c ho se n in re la tio n to th e an at om ic fe at ur es o f t he s ite . th e m ic ro sc op e us ed ha d a fib er -o pt ic ill um in at io n sy st em , an d th e m ag ni fic at io n va rie d be tw ee n 5x a nd 30 x (c ar l z ei ss o m ni p ro 55 ), m ic ro su rg ic al in st ru m en ts . p at ie nt ’s o pi ni on : n ot re po rt ed p ro fe ss io na l’s o pi ni on : q ua lit at iv e (s ca rr in g, g in gi va l m ar gi n, a nd pa pi lla e ap pe ar an ce ). te st : b et te r r es ul ts o f s ca rr in g an d gi ng iv al m ar gi n* te st : 8 6% / 2. 67 ± 0. 87 c on tro l: 78 % / 2. 63 ± 0 .9 1 te st : 5 8. 3% c on tro l: 33 .3 % te st : 2. 63 ± 0 .8 6 c on tro l: 2. 38 ± 1. 15 te st : 1. 79 ± 0 .6 9 c on tro l: 1. 7 ± 1. 51 co nt in ue 9 moro et al. s tu dy in te rv en tio ns m ic ro su rg ic al eq ui pm en t a es th et ic c on di tio n p r c / m c r m m c r c si te s/ % c a l m m k t w m m ji nd al et a l., 2 01 5 te st g ro up : m ic ro su rg er y c o nt ro l g ro up : m ac ro su rg er y a p ar ti al t hi ck ne ss f la p, w it h tw o v er ti ca l i nc is io ns pl ac ed a t le as t o ne -h al f to o ne t o o th w id er m es io di st al ly t ha n th e ar ea o f gi ng iv al r ec es si o n an d pl ac em en t o f co nn ec ti ve ti ss ue g ra ft , r et ri ev ed f ro m pa la te t o r ec ip ie nt w as d o ne ac co rd in g to t he l an ge r an d la ng er t ec hn iq ue . su rg ic al m ic ro sc op e (e nf or te ) a t a m ag ni fic at io n of 1 0x , 6 -0 vi cr yl s ut ur es . p at ie nt ’s o pi ni on : n ot re po rt ed p ro fe ss io na l’s o pi ni on : q ua lit at iv e (s ca rr in g, g in gi va l m ar gi n, a nd pa pi lla e ap pe ar an ce ). b et te r e st he tic o ut co m es in te st , w he n co m pa re d to c on tr ol (n o st at is tic al ly s ig ni fic an t d iff er en ce ). te st : 6 7. 58 % c on tro l: 61 .7 8% te st : 4 / 26 .6 7% c on tro l: 3/ 20 % te st : 3 .1 3 c on tro l: 2. 43 n ot re po rt ed n iz am et a l., 2 01 5 te st g ro up : c p f + c tg (m ic ro su rg er y) c on tr ol g ro up : c p f + c tg (m ac ro su rg er y) m ic ro sc op e un de r x 3. 5 m ag ni fic at io n, u si ng th e eq ui pm en t d es ig ne d fo r m ic ro su rg er y (b la de s, ne ed le h ol de r, sc is so rs , an d tis su e fo rc ep s) . p at ie nt ’s o pi ni on ( v a s s ca le ): t he a es th et ic s co re s of t he in te rv en ti on s w er e si gn if ic an tl y an d si m ila rl y im pr ov ed d ur in g al l ev al ua ti on t im e po in ts c om pa re d w it h ba se lin e (s co re s be tw ee n 8 an d 9) . p ro fe ss io na l’s o pi ni on : n ot re po rt ed . te st : 9 5. 82 ± 8. 41 % */ 3 .6 2 ± 0. 85 * c on tro l: 83 .4 6 ± 16 .2 1% / 2. 96 ± 0. 69 te st : 1 5 c on tro l: 9 te st : 3 .4 4 ± 0. 97 * c on tro l: 2. 80 ± 0. 74 te st : 2 .2 4 ± 1. 17 c on tro l: 2. 09 ± 0. 84 p an de y an d m eh ta , 2 01 3 te st g ro up : f re e ro ta te d pa pi lla a ut og ra ft + c a f (m ic ro su rg er y) c on tr ol g ro up : f re e ro ta te d pa pi lla a ut og ra ft + c a f (m ac ro su rg er y) m ic ro sc op e (s er w el l c om pa ny , c he nn ai ) un de r x 10 m ag ni fic at io n, m ic ro su rg ic al in st ru m en ts . n ot re po rt ed te st : 2 .0 5 c on tro l: 2. 13 n ot re po rt ed te st : 0 .7 c on tro l: 0. 5 n ot re po rt ed k t w : k er at in iz ed ti ss ue w id th ; c p f: c or on al ly p os iti on ed fl ap ; c r c : c om pl et e ro ot c ov er ag e; m r c : m ea n ro ot c ov er ag e; e m d : e na m el m at rix d er iv at e; c a f: c or on al ly a dv an ce d fla p; m m t t: m od ifi ed m ic ro su rg ic al tu nn el te ch ni qu e; c tg : s ub ep ith el ia l c on ne ct iv e tis su e gr af t; r es : r oo t c ov er ag e ae st he tic s co re ; g tr : g ui de d tis su e re ge ne ra tio n; v a s: v is ua l a na lo g sc al e; c a l: c lin ic al a tt ac hm en t l ev el ; l m c a f: la te ra lly m ov ed c or on al ly a dv an ce d fla p. co nt in ua tio n 10 moro et al. the intervention with magnification promoted significantly more prc than conventional surgery. in addition, more sites with crc were found in the test group in the studies of bittencourt et al.22 (2012) and burkhardt and lang20 (2005). moreover, when cal gain was analyzed, two papers were not included20,31. just one study found out that magnification promotes significantly more cal gain when compared to control group21. on the other hand, four papers analyzed ktw change and none of them showed significant differences between groups19,21,22,31. four studies evaluated ppd change and no differences were found between control and test groups19,21,22,31. one study evaluated ktt change, and no differences were detected between groups22. two trials observed that the length of surgery was greater using microscopes, when compared to conventional technique (72 ± 8 min versus 51 ± 5 min20; 73 ± 12 min versus 55 ± 8 min)21 and one study did not found differences between groups (test: 60 min versus control: 54 min)22. divergences among studies were observed as regards to aesthetic condition change. as regards professional`s opinion, in one parallel study, using the root coverage aesthetic score (res), both conventional surgery and surgery under magnification were related with acceptable esthetic (test: 9.2 ± 1.1/ control: 9.2 ± 1.3)31. in another split-mouth study, the use of magnification resulted in 100% aesthetic satisfaction, while conventional surgery was associated with 79.1% satisfaction22. the parallel study of nizam et al.21 (2015) also used visual analog score (vas) to obtain patient’s opinion. the aesthetic scores of conventional surgeries and the technique with magnification were significantly improved, with no differences between groups. two trials used a qualitative scale to obtain professional’s opinion using pictures of treated sites, as follows: scarring, gingival margin, and papillae appearance19,29. although francetti et al.19 (2005) found better results for scarring and gingival margin in the magnification group, jindal et al.29 (2015) observed no difference between groups regarding esthetic outcomes. pooled outcomes pooled estimates of mrc (in mm) were available in 5 studies19,21,22,30,31 and showed no difference between the use of magnification and conventional treatment (mean difference = 0.20 mm, 95% ci -0.10-0.50; i2 = 35%, p = 0.18; low quality) (figure 3 and table 3). six studies were summarized in the meta-analysis of prc19-22,29,31, and indicated that magnification resulted in greater prc than conventional technique (mean difference = 7.38%, 95% ci 3.66-11.09; i2 = 0%, p < 0.0001; low quality) (figure 3 and table 3). crc data was available for 6 studies19-22,29,31. results indicated that magnification did not increased the chance of crc (rr = 1.35, 95% ci 0.94-1.92; i2 = 62%, p = 0.10; very low quality) (figure 3 and table 3). meta-analysis of cal gain19,21,22,30 showed no difference between the use of microscope and conventional technique (mean difference = 0.25 mm, 95% ci -0.11-0.61; i2 = 21%, p = 0.17; low quality) (supplementary material 1 and table 3). moreover, similar results were found when pooled outcomes were calculated in 5 studies that evaluated ktw (mean difference: 0.08 mm, 95% ci -0.10-0.27; i2 = 0%, p = 0.39; very low quality) (supplementary material 1 and table 3)19,21,22,30,31. 11 moro et al. meta-analysis for ppd and ktt were not conducted since few studies presented these variables. adverse effects one study reported absence of complications associated with conventional surgery and the use of magnification19. one study reported that in the conventional technique groups, three subjects had dentin hypersensitivity and 10 had postoperative pain, figure 3. forest plot of random effects meta-analysis evaluating mrc, prc and crc on magnification. 12 moro et al. ta bl e 3. g r a d e su m m ar y of fi nd in gs ta bl e fo r r oo t c ov er ag e su rg er y un de r m ag ni fic at io n ve rs us c on ve nt io na l r oo t c ov er ag e su rg er y. c er ta in ty a ss es sm en t № o f pa tie nt s ef fe ct q ua lit y of ev id en ce im po rt an ce № o f st ud ie s s tu dy de si gn r is k of bi as in co ns is te nc y in di re ct ne ss im pr ec is io n o th er co ns id er at io ns r oo t c ov er ag e su rg er y w ith m ag ni fic at io n c om ve nt io na l ro ot c ov er ag e su rg er y r el at iv e (9 5% c i) a bs ol ut e (9 5% c i) m ea n ro ot c ov er ag e (f ol lo w u p: ra ng e 6 m on th s to 2 4 m on th s; a ss es se d w ith : m m ; s ca le fr om : 2 .0 to 4 .4 ) 5 ra nd om iz ed tr ia ls se rio us a no t s er io us no t s er io us se rio us b no ne 11 4 11 3 m ea n 0. 26 m m m or e (0 .0 7 m or e to 0 .4 6 m or e) lo w c r it ic a l p er ce nt ag e of ro ot c ov er ag e (f ol lo w u p: ra ng e 6 m on th s to 2 4 m on th s; a ss es se d w ith : % ; s ca le fr om : 4 to 2 3) 6 ra nd om iz ed tr ia ls se rio us a no t s er io us no t s er io us se rio us b no ne 11 9 11 8 m ea n 7. 41 h ig he r (4 .2 6 hi gh er to 1 0. 57 hi gh er ) lo w c r it ic a l c om pl et e ro ot c ov er ag e (f ol lo w u p: ra ng e 6 m on th s to 2 4 m on th s; a ss es se d w ith : n um be r o f e ve nt s; s ca le fr om : 2 to 3 7) 6 ra nd om iz ed tr ia ls se rio us a se rio us c no t s er io us se rio us b no ne 11 9 11 8 m ea n 1. 35 m or e (1 .0 4 m or e to 1 .7 5 m or e) v er y lo w c r it ic a l k er at in iz ed ti ss ue w id th (f ol lo w u p: ra ng e 6 m on th s to 2 4 m on th s; a ss es se d w ith : m m ; s ca le fr om : 0 .0 to 5 .0 ) 4 ra nd om iz ed tr ia ls se rio us a no t s er io us no t s er io us ve ry se rio us b no ne 11 4 11 3 m ea n 0. 15 m m m or e (0 .0 2 fe w er to 0 .3 2 m or e) v er y lo w im p o rt a n t c lin ic al a tt ac hm en t l ev el (f ol lo w u p: ra ng e 6 m on th s to 2 4 m on th s; a ss es se d w ith : m m ; s ca le fr om : 0 .5 to 4 .3 ) 5 ra nd om iz ed tr ia ls se rio us a no t s er io us no t s er io us se rio us b no ne 99 98 m ea n 0. 33 m m m or e (0 .1 m or e to 0 .5 7 m or e) lo w im p o rt a n t a ut ho r( s) : m or o m g , s ou to m ls , r ov ai e s, c es ar n et o jb , h ol zh au se n m , p an nu ti c m . q ue st io n: r oo t c ov er ag e su rg er y un de r m ag ni fic at io n co m pa re d to c on ve nt io na l r oo t c ov er ag e su rg er y in h ea lth y pa tie nt s w ith g in gi va l r ec es si on . se tt in g: b ra zi l, g er m an y, s w itz er la nd , i ta ly , i nd ia , t ur ke y b ib lio gr ap hy : a za rip ou r e t a l. (2 01 6) ; b itt en co ur t e t a l. (2 01 2) ; b ur kh ar dt e t a l. (2 00 5) ; f ra nc et ti et a l. (2 00 5) ; j in da l e t a l. (2 01 5) ; n iz am e t a l. (2 01 5) ; p an de y an d m eh ta (2 01 3) . c i: c on fid en ce in te rv al ex pl an at io ns a. a cc or di ng to th e ta bl e of ri sk o f b ia s, th e m os t o f s tu di es w er e cl as si fie d as u nc le ar ri sk o f b ia s du e to th e fa ils o f r an do m iz at io n an d al lo ca tio n, b lin d of e xa m in er s, a s w el l, sa m pl e si ze c al cu la tio n. b. t he s am pl e si ze o f s tu di es is s m al l a nd ju st tw o st ud ie s re po rt ed h ow it w as c al cu la te d. a ls o, th e co nfi de nc e in te rv al d oe s no t r ul e ou t a n ul l e ff ec t o r h ar m . c. t he m od er at e he te ro ge ne ity c ou ld b e ex pl ai ne d by th e us e of d iff er en t p er io do nt al s ur gi ca l t ec hn iq ue s an d re ga rd in g th e ra ng e of m ag ni fic at io n. 13 moro et al. while 10 participants had postoperative pain in the magnification group22. three trials did not report information about the presence of postoperative complications20,29,31. in another study, one subject in each group had postoperative hemorrhage and one participant in the test group had partial necrosis and swelling in the donor area21. moreover, one trial showed that less subjects in test group (20%) had postoperative pain when compared to control group (60%)30. discussion the findings of this review suggest that magnification has a controversial influence on clinical outcomes in root coverage procedures. surgeries performed under magnification may result in higher prc than the ones performed without magnification. on the other hand, when analyzing the other outcomes, including the primary outcome, magnification did not promote additional benefit. magnification was associated with approximately 7% more root coverage than the conventional technique. although this percentage was considered statistically significant, the clinical relevance of this improvement must be discussed. the clinician should analyses if it is worth invest on magnification for gain more 7% of root coverage than conventional technique. moderate heterogeneity between studies was detected in meta-analysis of crc (62%), whereas pooled estimates of mrc and prc showed low heterogeneity (35% and 0%, respectively), what may reinforce the reliability of such findings. supplementary material 1. forest plot of random effects meta-analysis evaluating cal gain and ktw change on magnification. 14 moro et al. the rationale use of magnification in periodontal surgery involves a combination of practical considerations associated with scientific evidences that indicates, in some clinical situations, that magnification may be an advantage for both the practitioner and the patient. however, it is difficult to directly compare the available devices and to identify which magnification yielded the best results. loupe was defined as a double monocular telescope with converging lenses side by side to focus on the operative field. the range of magnification varies between 1.5 and 6 x32. the microscope provides a greater range of magnification (4-45 x). it incorporates an optical system coated with achromatic lenses and has a high optical resolution due to the enhanced depth of focus and field of view33. microscope allows the adjustment of magnification according to the preference of the user in each step of the procedure. the microscope magnification of the trials selected for this review ranged from 3 to 30 x. further, the values of magnification also varied within the same study. azaripour et al.31 (2016) used a magnification that varied between 4 and 7x, and the magnification of francetti et al.19 (2005) and bittencourt et al.22 (2012) studies varied between 5 and 30 x; and 8 and 12 x, respectively. the studies of burkhardt and lang20 (2005), jindal et al.29 (2015), nizam et al.21 (2015), and pandey and mehta30 (2013) applied just one value of magnification (15 x, 10 x, 3.5 x and 10 x, respectively). the use of different surgical techniques also difficult comparisons. the majority of the studies used coronally positioned flap (cpf) in association with subepithelial connective tissue graft (ctg)21,22,29. other studies used double-pedicle papilla flap20, and free rotated papilla autograft + coronally advanced flap (caf)30. azaripour et al.31 (2016) compared different techniques (modified microsurgical tunnel technique + ctg versus cpf + ctg), while francetti et al.19 (2005) used different techniques, according to the patient’s need. azaripour et al.31 (2016) was the only study that included upper first molars. the others included incisors, canines and premolars (maxilla and mandible)29; anterior area from maxilla and mandible19; canines and premolars from maxilla21,22; or upper canines20. despite the present interesting findings, it should be considered that rc may vary according to tooth types due to the anatomic characteristics as recession width, frenum attachments and lip muscles34,35. another point is about the operators. the use of magnification is associated with a well-trained and experience operator, while the conventional surgery can be performed by a less trained operator. the use of magnification is associated with an additional financial investment, training time and potential longer surgical time36,37. these factors induced the operator to get better and promoted more precise surgeries. the precision and refinement promoted by magnification may result in better final visual analyze38. esthetic evaluation was conducted by francetti et al.19 (2005) and bittencourt et al.22 (2012) that found superior results for surgery with magnification. two studies followed patients for 6 months29,30, the majority of the investigations followed the subjects for 12 months19,20,22,31 and nizam et al.21 monitored the subjects for 24 months. although some studies claim that the longer the follow-up time, the changes are more stable39, other studies have reported that results obtained after 6 15 moro et al. months are stable over time up to 12 months40,41 or even after 3 years of follow-up42. tissue stability is also associated with other aspects, including surgical technique, tissue thickness and mainly oral hygiene habits of the patients. within the limits of our knowledge, this is the second systematic review investigating the influence of magnification on root coverage procedures and some important differences have to be highlighted. while the present review included seven trials, the previous review was limited to the inclusion of four studies2. this difference could be explained due to the publication of recent papers addressing magnification and also, no restrictions for surgical technique. another difference is that meta-analysis in the kang et al.2 (2015) review included only two studies that used ctg in the surgical procedure20,22. despite our interesting findings, some limitations must be addressed. five studies were classified as unclear risk of bias19-21,29,30. studies that present unclear or high risk of bias tend to overestimate the effect of treatment and decrease the reliability of the trials’ conclusions. moreover, according to grade, three outcomes (mrc, prc and cal change) were related to low quality and two outcomes (crc and ktw change) were related to very low quality, which indicated that further research is recommended to confirm whether the estimates are close to real values. still, when patient related outcomes are analyzed, the use of microscope did not interfere positively on discomfort, postoperative pain and esthetic evaluation22,31. in this sense, well-conducted studies are needed, in order to focus not only in clinical aspects, but also evaluating the perspective of the practitioner. data regarding physical lesions caused by work, fatigue after working hours and frequency of pain in neck and column could bring interesting information for the field. these data could contribute not only to the understanding of the potential benefits of magnification on clinical results, but also whether the use of magnification devices could favor the quality of life of a practitioner during his career and after retirement. in conclusion, there is low evidence that magnification can increase prc in root coverage surgeries. however, more randomized trials with the use of magnification are necessary, in order to prove that this benefit is clinically relevant, in order to justify the use of this device. acknowledgements this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. references 1. kassab mm, cohen re. the etiology and prevalence of gingival recession. j am dent assoc. 2003 feb;134(2):220-5. 2. kang j, meng s, li c, luo z, guo s, wu y. microsurgery for root coverage: a systematic review. pak j med sci. 2015 sep-oct;31(5):1263-8. doi: 10.12669/pjms.315.7782. 3. rios fs, costa rs, moura ms, jardim jj, maltz m, haas an. estimates and multivariable risk assessment of gingival recession in the population of 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combination with a tunnel technique for the treatment of miller class ii and iii gingival recessions in mandibular incisors: clinical and esthetic results. int j periodont rest dent. 2016 jul-aug;36(4):591-8. doi: 10.11607/prd.2748. 36. tibbetts ls, shanelec da. a review of the principles and practice of periodontal microsurgery. tex dent j. 2007 feb; 124(2):188-204. 37. sitbon y, attathom t. minimal intervention dentistry ii: part 6. microscope and microsurgical techniques in periodontics. brit dent j. 2014 may;216(9):503-9. doi: 10.1038/sj.bdj.2014.356. 18 moro et al. 38. chambrone l and tatakis dn. periodontal soft tissue root coverage procedures: a systematic review from the aap regeneration workshop. j periodontol. 2015 feb;86(2 suppl):s8-51. doi: 10.1902/jop.2015.130674. 39. chambrone l, sukekava f, araujo mg, pustiglioni fe, chambrone la, lima la. root-coverage procedures for the treatment of localized recession-type defects: a cochrane systematic review. j periodontol. 2010 apr;81(4):452-78. doi: 10.1902/jop.2010.090540. 40. stefanini m, jepsen k, de sanctis m, baldini n, greven b, heinz b, et al. patient-reported outcomes and aesthetic evaluation of root coverage procedures: a 12-month follow-up of a randomized controlled clinical trial. j clin periodontol. 2016 dec;43(12):1132-1141. doi: 10.1111/jcpe.12626. 41. zucchelli g, mounssif i, mazzotti c, stefanini m, marzadori m, petracci e, et al. coronally advanced flap with and without connective tissue graft for the treatment of multiple gingival recessions: a comparative shortand long-term controlled randomized clinical trial. j clin periodontol. 2014 apr;41(4):396-403. doi: 10.1111/jcpe.12224. 42. jepsen k, stefanini m, sanz m, zucchelli g, jepsen s. long-term stability of root coverage by coronally advanced flap procedures. j periodontol. 2017 jul;88(7):626-633. doi: 10.1902/jop.2017.160767. 1http://dx.doi.org/110.20396/bjos.v20i00.8660699 volume 20 2021 e210699 original article 1 departament of orthodontics, university center of hermínio ometto foundation – fho, araras, são paulo, brazil. *corresponding author: silvia a. s. vedovello araras dental school, university center of hermínio ometto foundation-fho dr. maximiliano baruto av, 500 jardim universitário. araras, sp, brazil, 13607-339 +55 19 3543-1423 silviavedovello@gmail.com received for publication: july 30, 2020 accepted: december 30, 2020 knowledge and clinical practices of orthodontists regarding the treatment of patients with aids. a nationwide study elisabete s. rocha1, mario vedovello filho1 , giovana cherubini venezian1 , carolina carmo de menezes1 , silvia amélia scudeler vedovello1,* aim: to evaluate orthodontists’ knowledge and clinical practices regarding the treatment of patients with hiv/aids. methods: cross-sectional study performed with 655 brazilian orthodontists based on a previously calculated sample size. self-administered questionnaires were sent to orthodontists to collect information on knowledge and clinical conduct regarding the care of patients with hiv/aids. the study evaluated the awareness of possible risk factors for contamination, oral manifestations of hiv, need for more information on the care of hiv-positive patients, whether orthodontic treatment is indicated in hiv-positive patients, and whether they had knowingly performed orthodontic treatment in hiv-positive patients. simple regression models were adjusted, and crude odds ratios estimated the associations with 95% confidence intervals. the variables with p < 0.20 in the crude analysis were tested in multiple logistic regression models, and those with p ≤ 0.05 were maintained in the final model. magnitudes were estimated by adjusted odds ratios values, with 95% confidence intervals. results: orthodontists who were aware of the oral manifestations of hiv/aids, those having work experience of more than 20 years, and those who believed that orthodontic treatment could be indicated for these patients were 3.30 (1.79-6.10), 2.74 (1.36-5.52) and 1.92 (1.13-3.24) times more likely to perform orthodontic treatment in hiv-positive patients, respectively. most orthodontists (92.9%) reported they needed to obtain more information about orthodontic care in patients with hiv/aids. conclusion: although orthodontists reported feeling able and qualified to provide dental care to patients with  hiv/aids, gaps in their knowledge need to be addressed with further training. keywords: hiv. orthodontics. acquired immunodeficiency syndrome. immunologic deficiency syndromes. mailto:silviavedovello@gmail.com https://orcid.org/0000-0002-5944-7937 https://orcid.org/0000-0003-4643-7964 https://orcid.org/0000-0002-8875-8611 https://orcid.org/0000-0002-7203-2867 2 rocha et al. introduction human immunodeficiency virus (hiv) infection remains among the top ten major health issues worldwide. according to estimates of the world health organization (who), approximately 1 million people are infected with hiv every year1-4. official data compiled by the unaids (a joint united nations program on hiv/ aids) indicated that approximately 830,000 individuals were living with hiv/aids (acquired immunodeficiency syndrome) in brazil in 2018, most of them aged 20 to 34 years5,6. the perception of dentists, dental students, and academic scholars about the treatment of hiv-positive patients has been studied over the last years7-9. while these groups have the knowledge and/or willingness to treat hiv/aids individuals, they are usually faced with numerous queries10-12. based on typical oral manifestations resulting from hiv infection, the dentist can be the first health professional to suspect hiv positivity. despite that, all patients should be treated equally as if they were potentially infectious, mainly because it is known that most seropositive patients fail to report their infectious state to the oral health team4,13,14. awareness about the patient’s condition has increased the professional’s willingness and self-confidence during dental care11,12. among other oral health issues, malocclusion has a direct impact on one’s social interaction and self-esteem. overall, patients with malocclusion may significantly benefit from orthodontic therapy15,16, particularly hiv-positive patients who commonly wish to maintain a healthy appearance17. orthodontists should know the implications of hiv infection, considering that the presence of orthodontic appliances can directly dysregulate the oral microbiome18,19 and that hiv-positive patients are more prone to develop severe periodontitis14. due to frequent contact with hiv-positive patients, the orthodontist can identify the manifestations of hiv, diagnose and refer the patient for appropriate treatment. however, the orthodontist’s perception about the implications of dental care of hiv-positive patients is still poorly known compared to other dental specialties13,20. our study hypothesis was that orthodontists were unaware of the implications of orthodontic treatment in hiv-positive patients. thus, this study aimed to evaluate orthodontists’ knowledge and clinical practices regarding the treatment of patients with hiv/aids. materials and methods a nationwide cross-sectional study was carried out with brazilian orthodontists following the strobe guide21. the study included only orthodontists registered in the federal council of dentistry (cfo) of brazil, regardless of graduation time, sex, or age group. the sample size was determined in the epiinfo program (centers for disease control and prevention, atlanta, u.s.a.) based on a previous pilot study (n = 20). the following parameters were used for sample size calculation: significance level of 5%, test power of 80%, and an effect size of 1.8 totaling a sample of 524 orthodontists. an additional 30% was included to compensate for sample loss. a total of 5% of the sample was excluded due to incomplete responses. 3 rocha et al. a total of 655 orthodontists participated in the study, which was carried out between april and august 2018. electronic questionnaires were sent to orthodontists’ e-mail with the assistance of the regional dentistry councils of all states in brazil. all study volunteers signed an informed consent form to authorize their participation and provided information about their attitude, knowledge, and professional conduct regarding treating patients with hiv/aids. this study was previously approved by a research ethics committee (caae #83148618.1.0000.5385). study questionnaire a specific questionnaire addressing the orthodontists’ knowledge, attitude, and clinical practice was developed for this study on the google forms platform and  contained 16 closed items. the questions were formulated based on the previous studies7,22 and addressed demographic data, orthodontist’s basic knowledge about hiv/aids, and previous clinical experiences. the following variables were considered: • demographic data: gender, work experience and training (in years); • basic knowledge on possible risk factors for contamination; oral manifestations of hiv infection; the need for more information to assist hiv-positive patients; • attitudes: ability to treat a patient with hiv/aids; indication or contraindication of orthodontic treatment for this group of patients. • clinical practices on whether hiv/aids testing can be requested and whether orthodontic treatment can be performed in hiv-positive patients. each item had three answer options: “yes”, “no,” and “do not know”; the (1) attitudes and knowledge and (2) clinical conduct of orthodontists towards the treatment of patients with hiv/aids was considered the outcome variables. statistical analysis initially, a qualitative descriptive analysis was performed, expressing the data in tables using percentages. simple regression models were then adjusted, and crude odds ratios estimated the associations with 95% confidence intervals. the variables with p < 0.20 in the crude analysis were tested in multiple logistic regression models, and those with p ≤ 0.05 were maintained in the final model. magnitudes were estimated by adjusted odds ratios values, with 95% confidence intervals. the data were analyzed in the r program (r foundation for statistical computing, vienna, austria). results a total of 655 orthodontists completed the study, of which 481 (72.3%) were females, and 184 (27.7%) were males; 37.1% of them finished dental school between 2000 and 2008, 68.7% had obtained specialist certification less than ten years before. the characteristics of the study sample are shown in table 1. 4 rocha et al. table 1. characteristics of the study sample (n = 655). variable frequency, n (%) gender male 184 (27.7) female 481 (72.3) time since graduation 0-10 years 210 (31.6) 11-20 years 247 (37.1) 21-30 years 161 (24.2) over 30 years 47 (7.1) time since specialist certification 0-10 years 457 (68.7) 11-20 years 157 (23.6) 21-30 years 49 (7.4) over 30 years 2 (0.3) table 2 shows the analysis of orthodontists’ attitude towards the treatment of patients with hiv/aids. the findings show that 78.9% of the orthodontists considered themselves able to treat patients with hiv/aids, and 79.8% of them were aware of the oral manifestations of hiv infection. orthodontists who assumed there are risks of contamination in the treatment of hiv-positive patients were 2.53 (1.12-5.75) times more likely to consider themselves able to treat these patients as compared to 3.10 (1.25-7.69) for those who reported no risk of contamination. orthodontists who knew the oral manifestations of hiv infection were 3.30 (1.79-6.10) times more likely to consider themselves able to perform orthodontic treatment in these patients. those who considered that orthodontic treatment could be indicated for hiv-positive patients were 4.66 (2.84-7.66) times more likely to consider themselves able to treat these patients (p < 0.05). in contrast, orthodontists, who reported that orthodontic treatment is contraindicated for hiv-positive patients were 2.01 (1.08-3.78) times more likely to consider themselves able to treat them. when asked, the vast majority of orthodontists (92.9%) reported the need to obtain more information about orthodontic care for hiv/ aids patients. table 3 shows the associations between the study variables and the orthodontists’ clinical conduct towards the treatment of hiv-positive patients. few orthodontists (29.8%) had knowingly performed orthodontic treatment in patients with hiv/aids. also, a more extended training period was associated with a greater likelihood of treating hiv-positive patients (p < 0.05). orthodontists with work experience of more than 20 years were 2.74 (1.36-5.52) times more likely to treat hiv/aids patients than 1.96 (1.23-3.13) for those with over ten years of work experience. according to the results, orthodontists who believed that orthodontic treatment could be indicated for patients with hiv/aids and those who considered themselves able to treat these patients were 1.92 (1.13-3.24) and 5.39 (2.26-12.86) times more likely to have knowingly performed orthodontic treatment in patients with hiv/aids (p < 0.05), respectively. 5 rocha et al. table 2. association between the study variables and orthodontists’ attitude and knowledge regarding the care of patients with hiv / aids. variable category feels able to treat hiv+ patients $crude or (#95% ci) p-value $adjusted or ajustado(#ic95%) p-valueno / do not know yes* n (%) n (%) sex female 109 (22.7) 372 (77.3) ref male 31 (16.8) 153 (85.2) 1.45 (0.93-2.25) 0.1013 time since graduation 0-10 years 37 (17.62) 173 (82.4) 1.98 (0.97-4.07) 0.0619 11-20 years 58 (23.5) 189 (76.5) 1.38 (0.69-2.76) 0.3582 21-30 years 31 (19.2) 130 (80.8) 1.78 (0.85-3.72) 0.1259 over 30 years 14 (29.8) 33 (70.2) ref time since specialist certification 0-10 years 83 (18.2) 374 (81.8) 1.99 (1.04-3.82) 0.0390 11-20 years 40 (25.5) 117 (74.5) 1.29 (0.64-2.61) 0.4788 21-30 years 15 (30.6) 34 (69.4) ref over 30 years 2 (100.0) 0 (0.0) orthodontists may be at risk for hiv contamination no 24 (14.2) 145 (85.8) 8.63 (3.85-19.36) <0.0001 3.10 (1.25-7.69) 0.0145 do not know 20 (58.8) 14 (41.2) ref yes 96 (20.8) 366 (79.2) 5.44 (2.65-11.17) <0.0001 2.53 (1.12-5.75) 0.0261 has performed orthodontic treatment in hiv-positive patients no 123 (26.3) 344 (73.7) ref ref yes 17 (8.6) 181 (91.4) 3.80 (2.22-6.52) <0.0001 3.74 (2.06-6.81) <0.0001 orthodontists may no 12 (32.4) 25 (67.6) 1.05 (0.49-2.24) 0.8981 request hiv test do not know 58 (33.5) 115 (66.5) ref orthodontic treatment is contraindicated in hiv-positive patients yes 70 (15.4) 385 (84.6) 2.77 (1.85-4.16) <0.0001 no 50 (11.9) 371 (88.1) 3.89 (4.40-10.79) <0.0001 4.66 (2.84-7.66) <0.0001 do not know 65 (48.2) 70 (51.8) ref ref yes 25 (22.9) 84 (77.1) 3.12 (1.78-5.46) <0.0001 2.01 (1.08-3.78) 0.0289 knows the oral manifestations of hiv infection no 27 (40.3) 40 (59.7) 1.20 (0.60-2.38) 0.6003 1.04 (0.48-2.28) 0.9169 do not know 30 (44.8) 37 (55.2) ref ref yes 83 (15.6) 448 (84.4) 4.38 (2.56-7.48) <0.0001 3.30 (1.79-6.10) 0.0001 considers saliva as a means of hiv transmission no 94 (18.7) 408 (81.3) 4.61 (2.25-9.46) <0.0001 do not know 17 (51.5) 16 (48.5) ref yes 29 (22.3) 101 (77.7) 3.70 (1.67-8.22) 0.0013 needs to obtain more information on the topic no 2 (4.3) 45 (95.7) 6.47 (1.55-27.00) 0.0104 yes 138 (22.3) 480 (77.7) ref *reference category for the outcome variable; $odds ratio; #confidence interval. 6 rocha et al. table 3. association between the study variables and orthodontists’ clinical conduct regarding the care of patients with hiv / aids. variable category has performed orthodontic treatment in hivpositive patients $crude or (#95% ci) p-value $adjusted or (#95% ci) p-value no yes* n (%) n (%) sex female 350 (72.8) 131 (27.2) ref male 117 (63.6) 67 (36.4) 1.53 (1.07-2.20) 0.0210 time since graduation 0-10 years 162 (77.1) 48 (22.9) ref ref 11-20 years 174 (70.4) 73 (29.6) 1.42 (0.93-2.16) 0.1034 1.53 (0.99-2.36) 0.0550 21-30 years 103 (64.0) 58 (36.0) 1.90 (1.20-3.00) 0.0076 1.96 (1.23-3.13) 0.0047 over 30 years 28 (59.6) 19 (40.4) 2.29 (1.18-4.46) 0.0401 2.74 (1.36-5.52) 0.0049 time since specialist certification 0-10 years 334 (73.1) 123 (26.9) ref 11-20 years 102 (65.0) 55 (35.0) 1.46 (0.99-2.16) 0.0670 21-30 years 30 (61.2) 19 (38.8) 1.72 (0.93-3.17) 0.1307 over 30 years 1 (50.0) 1 (50.0) 2.72 (0.17-43.74) 0.5917 orthodontists may be at risk for hiv contamination no 113 (66.9) 56 (28.3) 2.31 (0.90-5.91) 0.1129 do not know 28 (82.4) 6 (17.6) ref yes 326 (70.6) 136 (29.4) 1.95 (0.79-4.81) 0.2036 feels able to treat hiv-positive patients no 54 (83.1) 11 (16.1) 2.34 (0.81-6.74) 0.1143 2.45 (0.84-7.18) 0.1013 do not know 69 (92.0) 6 (8.0) ref ref yes 344 (65.5) 181 (34.5) 6.05 (2.58-14.20) <0.0001 5.39 (2.26-12.86) 0.0001 orthodontists may request hiv test no 24 (64.9) 13 (35.1) 1.50 (0.70-3.18) 0.2958 do not know 127 (73.4) 46 (26.6) ref yes 316 (69.4) 139 (30.6) 1.21 (0.82-1.80) 0.3312 orthodontic treatment is contraindicated in hiv-positive patients no 275 (65.3) 146 (34.7) 2.58 (1.58-4.23) 0.0002 1.92 (1.13-3.24) 0.0155 do not know 112 (83.0) 23 (17.0) ref ref yes 80 (73.4) 29 (26.6) 1.76 (0.95-3.28) 0.0715 1.40 (0.74-2.67) 0.3030 knows the oral manifestations of hiv infection no 50 (74.6) 17 (25.4) 1.00 (0.46-2.18) 1.000 do not know 50 (74.6) 17 (25.4) ref yes 367 (69.1) 164 (30.9) 1.31 (0.74-2.35) 0.3559 *reference category for the outcome variable; $odds ratio; #confidence interval. discussion this study surveyed the attitude, knowledge, and clinical conduct of brazilian orthodontists regarding treating hiv/aids patients. our findings confirmed the hypothesis that orthodontists need further training on the implications of treating hiv-positive patients. our findings indicated that more extended work experience and knowledge about the risks of contamination by hiv, oral manifestations of the infection, and means of viral transmission, were associated with a greater likelihood of orthodontists feeling able 7 rocha et al. to perform orthodontic treatment in patients with hiv/aids or have knowingly done so. these findings are consistent with other studies conducted with different oral health-related populations, such as dentists and dental students7,11,12,22. hence, more extended work experience and more knowledge on the topic seem to increase self-confidence for the oral care of hiv-positive patients. the presence of orthodontic devices may directly affect the composition and quantification of the oral microbiome, particularly increasing the prevalence of microbial species such as streptococcus mutans, candida albicans, among others18,19. knowing the oral manifestations of hiv infection is an important predictive strategy for detecting and tracking the evolution of the infectious condition14,23-25. for instance, the presence of oral candidiasis and severe periodontitis, among other manifestations of hiv infection, may raise doubts as to whether orthodontic therapy should be contraindicated for patients with hiv/aids. to date, there are no studies in the literature that provide evidence to contraindicate orthodontic therapy for patients with hiv/ aids. nevertheless, the indication of orthodontic treatment in these cases requires an individualized analysis of the patient’s systemic health, especially with regard to the cd4 count, an indicative of the patient’s immune status. individuals  with  low cd4 counts (severe immunosuppression) are more likely to experience oral manifestations of hiv infection than those with higher cd4 counts23,24. the orthodontists who reportedly indicated that patients with hiv/aids might undergo orthodontic therapy were more likely to have already treated hiv-positive patients. in contrast, orthodontists who either indicated or contraindicated orthodontic therapy for hiv-positive patients showed a greater chance of considering themselves able to perform orthodontic treatment in these patients. in our study, 75.5% of the orthodontists believed that saliva is not a means of hiv transmission. although there is no evidence that saliva alone may transmit hiv – as salivary glands inhibit the virus’s infectivity, it can be contaminated with hiv-infected blood and therefore contain biological hazard7. therefore, it is recommended to comply with universal precautions concerning exposure to saliva in dental offices. according  to the literature, dentists and dental students are aware of contamination risks when treating patients with hiv/aids, especially concerning contact with infected blood22. our findings showed that orthodontists who were aware of the risk of contamination when treating patients with hiv/aids had a greater probability of considering themselves able to treat them. this result is in line with previous literature reports7,22, indicating that dentists and dental students who were aware of the risks of contamination when treating hiv/aids patients were more willing to treat these patients. however,  our study also revealed that orthodontists who reported having no risk of contamination when treating hiv-positive patients were more likely to feel able to treat these patients, which may lead us to reason that they need more information concerning hiv/aids. this point may be explained by the fact that being more knowledgeable about the condition is associated with having greater availability and increased ability to treat hiv-positive patients. among the orthodontists who answered the questionnaire, 68.4% stated that they could request an hiv test. according to the previous studies9,26,27, many hiv-in8 rocha et al. fected patients are unaware of their infectious state. however, recent studies have highlighted a need for cultural change among dentists regarding their responsibility towards the patient’s systemic health9,27. raising awareness through the integration of medical and dental workforces may be necessary for dentists to be held responsible for preliminary medical examinations. while 78.9% of the participants considered themselves able to perform orthodontic treatment in patients with hiv/aids, less than 30% (29.8%) of them had knowingly done so. the inclusion of additional information on orthodontic treatment in patients with hiv/ aids to the program content of dental schools could help minimize the lack of knowledge while improving professional attitude and clinical conduct and increasing orthodontists’ willingness to treat these patients. our study revealed that 92.9% of the orthodontists reported feeling a need to obtain more information related to hiv/aids care. especially at the present moment, in the face of the covid-19 epidemic, knowledge about biosafety methods and prevention of dentist activities is essential. the study has limitations. the cross-sectional design shows information about the knowledge of orthodontists only at a specific period. also, the data were collected by a structured electronic questionnaire, with the possibility of subjectivity. thus, qualitative clinical studies are essential to expand the analysis of the results reported here. however, we highlight that our findings contribute to determining training strategies for orthodontists concerning hiv infection. in addition to bringing a critical reflection on biosafety methods and prevention of clinical activities in orthodontics. in conclusion, although orthodontists reported feeling able and qualified to provide dental care to patients with hiv/aids, gaps in their knowledge need to be addressed with further training. references 1. verma m, erwin s, abedi v, hontecillas r, hoops s, leber a, et al. modeling the mechanisms by which hiv-associated immunosuppression influences hpv persistence at the oral mucosa. plos one. 2017;12(1):e0168133. doi: 10.1371/journal.pone.0168133. 2. rostamzadeh m, afkhamzadeh a, afrooz s, mohamadi k, rasouli ma. dentists’ knowledge, attitudes and 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[oral manifestations and dental caries in children exposed to human immunodeficiency virus]. rev salud publica (bogota) 2011;13(5):833-43. doi: 10.1590/s012400642011000500012. spanish. 10 rocha et al. 24. thanyasrisung p, kesakomol p, pipattanagovit p, youngnak-piboonratanakit p, pitiphat w, matangkasombut o. oral candida carriage and immune status in thai human immunodeficiency virus-infected individuals. j med microbiol. 2014;63(5):753-9. doi: 10.1099/jmm.0.069773-0. 25. gruffaz m, zhang t, marshall v, gonçalves p, ramaswami r, labo n, et al. signatures of oral microbiome in hiv-infected individuals with oral kaposi’s sarcoma and cell-associated kshv dna. plos pathog. 2020 jan 17;16(1):e1008114. doi: 10.1371/journal.ppat.1008114. 26. hutchinson mk, van devanter n, phelan j, malamud d, vernillo a, combellick j, et al. feasibility of implementing rapid oral fluid hiv testing in an urban university dental clinic: a qualitative study. bmc oral health. 2012; 12:11. doi: 10.1186/1472-6831-12-11. 27. santella aj, schlub te, schifter m, tolani m, hillman rj. australian dentists’ perspectives on rapid hiv testing. aust dent j. 2016;61(3):270-6. doi: 10.1111/adj.12371. https://doi.org/10.1111/adj.12371 1http://dx.doi.org/10.20396/bjos.v19i0.8658647 volume 19 2020 e208647 original article 1 sociedad científica de estudiantes de medicina los andes (sociemla). universidad peruana los andes (upla), huancayo, perú. 2 sociedad científica de estudiantes de medicina de la universidad privada san juan bautista (sociem upsjb); universidad privada san juan bautista. lima, perú. 3 instituto de investigación de interculturalidad, universidad privada san juan bautista, perú. corresponding author: aquino canchari christian renzo cc.hh juan parra del riego ii etapa, block 2, dpto: 101, el tambo, huancayo, peru email address: christian.aquino. canchari@gmail.com phone: 051 – 934 824 051 received: march 08, 2020 accepted: september 08, 2020 body mass index and dental caries in native peruvian communities christian renzo aquino-canchari1* , diego andre crisol-deza2,3 , joselyn linda zurita-borja2,3 oral disorders and eating disorders affect everyone, however, these will be more frequent in vulnerable populations such as native communities. aim: determining the body mass index and the prevalence of dental caries and its clinical consequences in native peruvian communities. methods: observational, correlational, cross-sectional study. the sample consisted of 169 adults from the native communities selected for convenience, meeting inclusion and exclusion criteria. nutritional status was evaluated through the body mass index, to quantify the experience of dental caries, the dmtf index was used, the severity was quantified by the significance index of dental caries, and its clinical consequences when not being treated by the pufa index. the evaluation was carried out in natural light by calibrated observers. the data were analyzed in the stata v 14 program using frequency distribution tables and figures, to determine the association, the pearson’s correlation coefficient was used. results: the majority of residents had an adequate weight for their height 71 (41.01%), followed by low weight 64 (37.87%), overweight 29 (17.16%) and obesity 5 (2.96% ). the prevalence of dental caries was 100% (dmtf = 13.23; sic = 19.01), of which 68.04% had clinical consequences, no association was found between: bmi and dmtf (p = 0.557) bmi and pufa-index (0.485). conclusions: no association was found between the body mass index and dental caries and its clinical consequences. keywords: nutritional status. oral health. population groups. epidemiology. dental caries. peru. https://orcid.org/0000-0002-7718-5598 https://orcid.org/0000-0001-7872-3901 https://orcid.org/0000-0003-4030-348x 2 aquino-canchari et al. introduction indigenous communities are culturally different societies and communities, presenting diverse religions, traditions, languages and histories, being a vulnerable population, in addition, it represents around 5% of the world population, of which 15% live in poverty1,2; in addition to having the worst health indicators compared to urban populations3.these inequalities and inequities may stem from globalization, poverty and marginalization, as well as barriers to accessing medical care4. the diverse native communities constitute a high percentage of the population with food insecurity. malnutrition, overweight or obesity has increased in this type of population, which can be related to the high consumption of carbohydrates, especially those in the form of sugars, increasing the risk of developing dental caries, this is caused by limited access to quality food, or due to the unavailability of rural stores where healthy products are sold5,6. in peru, the fight against malnutrition, anemia and obesity has improved, but there is still a large gap7. decades ago it was recognized worldwide that oral diseases are a public health problem, the most frequent being dental caries. peru is no stranger to this reality, according to the peruvian ministry of health (minsa) 90.4% of peruvians have dental caries and 85% suffer from periodontal diseases8,9, being this reality more frequent and acute in vulnerable populations such as indigenous communities10. the present study was carried out in the ashanincas indigenous communities of potsoteni, union puerto ashaninca and boca sanibeni, located in the ene river basin, belonging to the mazamari district, satipo province; junin department, peru, its economy is based on agriculture, hunting and fishing. its main access routes are river and road transport11. the objective of the present study was to determine the body mass index and dental caries in peruvian indigenous communities. materials and methods observational, correlational, cross-sectional study. the sample was made up of 169 residents of both sexes, selected for convenience, who attended the dental care module installed in each community during the days of intervention (04/08/2018 to 08/11/2020), in the xii camp multidisciplinary university of research and service (cumis) of the peruvian student medical scientific society (socimep), organized by the scientific society of medical students of the center (sociemc), belonging to the indigenous communities of potsoteni, boca sanibeni and union puerto ashaninka of mazamari district, satipo province, junin department, peru, the inclusion criteria were: belonging to the selected indigenous communities, over 18 years of age, signing the informed consent, the exclusion criteria were: presence of any impairment (physical, psychological or social). for the evaluation of nutritional status, the body mass index was used using the formula bmi = weight (kg) / height2 (m2). adults were evaluated barefoot and with a minimum of clothing, a calibrated seca brand scale was used. the weight was recorded in 3 aquino-canchari et al. kilograms (kg). to measure height, the adult stood up straight, barefoot on the height meter. height was recorded in centimeters (cm), down to the nearest 0.5. once the measurements were obtained, they were extrapolated to the anthropometric calculator, provided by the who anthro plus 1.0.4 software. bmi was classified as low weight (bmi <18.5), normal (bmi 18.5 24.9), overweight (bmi 25-30), and obese (bmi> 30)12. the experience of dental caries was evaluated using the dmft index (permanent decayed, lost and filled teeth)13, for the diagnosis of dental caries, explorers, oral mirrors, with natural light were used, fulfilling the biosecurity criteria. he inspected the occlusal, vestibular, palatal, lingual, mesial, and distal faces of all permanent teeth present except for the third molars. the presence of caries was diagnosed if there was a visible loss of continuity of the dental surface, without the characteristics of a developmental defect and in pits and fissures when the end of the explorer “hooked” under light pressure14, to the degree of severity the dental caries significance index (sic) was used15. to determine the clinical consequences of untreated dental caries, the pufa index was used, which quantifies the number of teeth with presence of pulp exposure (p / p), ulceration of the oral mucosa due to root fragments (u / u), fistula (f / f) and abscess (a / a)16. the clinical examination procedure was standardized, the research team was divided into three pairs: the first consisting of an examiner and an annotator, the second focused on the evaluation of weight and height, and a third focused on the collection of demographic data of the natives. the evaluators were subjected to calibration and concordance tests, the results were subjected to the cohen’s kappa index to establish the agreement between the observers, obtaining the value 0.87 (p <0.005). the evaluations were made in the local school, under natural light, adapting the resources according to ergonomics and biosafety. to avoid fatigue bias, breaks were taken every ten evaluations per pair. in order to collect the information, we had the support of bilingual translators from the community, who knew the research and the meaning of the questions to adapt the terms at the time of translation. after the inspection, each participant was given a hygiene kit consisting of a face soap, brush and toothpaste. this study complies with the ethical principles of the declaration of helsinki, has the approval of the research ethics committee of the national child teaching hospital san bartolome, and the patients were previously consulted about their willingness to participate in the research by signing informed consent, explained what it consisted of and the possible benefit it would bring them. the project had the approval of the chiefs and residents of the selected native communities. the data was analyzed in the microsoft excel 2016 program and subsequently statistical quantification was performed using the stata v.14.0 statistical package, for descriptive analysis, percentages and frequency measurements, mean scores and standard deviations of the variables were obtained. the pearson’s correlation coefficient was used to determine the association. results the sample was made up of 169 adults, the mean age was 29.13 ± 11.05 years, it was grouped by age group according to minsa: young adult (18-29 years), adult 4 aquino-canchari et al. (30-59 years) and adult older (60 years and older). the distribution of the selected sample was 99 women (58.57%) and 70 men (41.43%). (table 1) table 1. sample characteristics, by community, age group and sex. age group indigenous communities sex total potsoteni boca sanibeni unión puerto ashaninka male female n (%) n (%) n (%) n % n % n % young adult 31(18.34%) 30(17.75%) 43(25.44%) 36 21.30% 68 40.24% 104 61.54% adult 24(14.20%) 20(11.85%) 19(11.24%) 33 19.53% 30 17.75% 63 37;27% elderly 0 1(0.59%) 1(0.59%) 1 0.61% 1 0.61% 2 1.19% total 55(32.54%) 51(30.19%) 63(37.27%) 70 41.44% 99 58.6% 169 100% regarding the nutritional status of the native population, it was found that the majority presented an adequate weight for their height 71 (41.01%), low weight 64 (37.87%), overweight 29 (17.16%) and obesity 5 (2.96%). it should be noted that the female sex presented more cases of overweight and obesity. (table 2) table 2. body mass index of the inhabitants of native communities, according to sex. bmi sex total male female n % n % n % underweight 27 15.98 37 21.89 64 37.87 normal weight 34 20.12 37 21.89 71 42.01 overweight 9 5.33 20 11.83 29 17.16 obese 0 0 5 2.96 5 2.96 regarding the prevalence of dental caries, this was 100%, a population dmft of 13.23 (4.91) was found, being slightly higher in the female sex, and with a severity of dental caries of 19.01, according to the group age. (table 3) table 3. prevalence, experience and significance of dental caries of the inhabitants of native communities, according to age group. prevalence c x– (sd) p x– (sd) o x– (sd) cpod x– (sd) sic age group young adult 104 (100) 10.47 (2.83) 1.10 (1.46) 0 (0) 11.56 (2.63) 14.02 adult 63 (100) 13.87 ( 5.06) 2.05 (2.49) 0 (0) 15.34 (4.69) 16.66 continue... 5 aquino-canchari et al. prevalence c x– (sd) p x– (sd) o x– (sd) cpod x– (sd) sic elderly 2 (100) 15 (1.41) 0 (0) 0 (0) 15 (1.15) 0 (0) total 169 (100) 11.79 (4.15) 1.44 (1.96) 0 (0) 13.23 (4.91) 15.34 sexo male 70 (100) 11.21 (3.96) 1.14 (1.58) 0 (0) 12.36 (4.49) 15.65 female 99 (100) 12.20 (4.25) 1.65 (2.17) 0 (0) 13.85 (5.13) 16.81 total 169 (100) 3.77 (4.56) 0.99 (1.32) 0 (0) 13.23 (4.91) 16.23 in relation to the prevalence of the clinical consequences of untreated dental caries, 115 (68.04%) were residents, with the p component (pulp involvement) being the most frequent 112 (66.27%). (table 4) table 4. frequency of components of the pufa index of the inhabitants of native communities, according to age group and sex. index of clinical consequences of untreated tooth decay prevalence p u f a n % n % n % n % n % age group young adult 59 56.70 5 4.80 0 0 9 8.70 62 59.62 adul 51 81.10 16 25.4 3 4.80 9 14.3 51 80.95 elderly 2 100.0 0 0 1 50.0 1 50.0 2 100.0 total 112 66.27 21 12.43 4 2.36 19 11.24 115 68.04 sexo male 45 64.30 8 11.40 0 0 5 7.10 47 67.14 female 67 67.70 13 13.10 4 4.40 14 14.10 68 68.68 total 112 66.27 21 12.43 4 2.36 19 11.24 115 68.04 regarding the pearson’s correlation analysis between the bmi and the experience of dental caries and its clinical consequences, a very low correlation was found. furthermore, there was no statistically significant association (p> 0.05) between the variables studied. (table 5) table 5. correlation between bmi and caries experience and its clinical consequences when not treated. dmtf pufa index bmi rho p rho p 0.039 0.616 0.069 0.370 pearson correlation (significance p<0.05) continuation 6 aquino-canchari et al. discussion nutritional disorders such as low weight, overweight and obesity are of growing concern not only in developed countries, but also in low-income countries such as peru17. our study found that most of the inhabitants had an adequate body mass index, in agreement with what was reported by de souza-filho et al.18 [bmi (mean = 26.65)] in mura indigenous people of the amazon region of brazil, romero et al.19 [bmi (mean = 24.20)] in residents of five native ashaninka peruvian communities and with davison et al.20 [bmi (mean = 21.70)] in young indigenous adults from the north from australia21. however, our study evidenced that a large number of underweight and overweight residents coexist, known as the “double burden of malnutrition”, this is particularly noticeable in low-income countries such as peru, this can manifest itself within a community, in the home or even in the same individual. according to the food and agriculture organization of the united nations (fao), indigenous communities are at greater risk of food insecurity and malnutrition than other population groups22. another aspect to consider is that the majority of overweight and obese indigenous residents were female, in agreement with what was reported by romero and others19, boaretto and others23, this may be because women have a higher percentage of body fat, serotonin regulation, leptin levels, in addition to the progressive weight gain in pregnancy and menopause24. dental caries affects without distinction of social class, however, it affects more low-income people, our study showed a prevalence of 100%, being higher than that reported by soares and others (91.6%)25 and aamodt and others (99%)26, in adult indigenous residents of kaingang-brazil and chiapas-mexico; respectively. in relation to the dental caries experience, our study found average scores lower than those reported in adult guaraní indigenous settlers (dmtf = 13.9)27, xavantes-brasil (dmtf = 14.25)28 and from the park. national xingu-brazil (dmtf = 20.2)29 and higher than that reported by jayashantah and johnson (dmtf = 0.98) in indigenous people of sri lanka30. in relation to the age group, older adults presented a higher average number of carious lesions compared to the rest, this could be explained by the fact that the enamel of the older adult undergoes natural wear31, in addition to the fact that many of them continue to accept that oral deterioration is normal and inevitable in old age. regarding the clinical consequences of untreated dental caries, the majority presented teeth with the presence of pulp exposure, the prevalence of pufa was 68.04%, being more frequent in the female sex, however, this is relatively high compared to infants32-34. something important to mention is that no inhabitant had any dental restoration (amalgam and / or resin), this can be explained by the fact that the indigenous communities studied have a minor oral health service quality (limited to the topical application of fluoride), in addition, it is a challenge to have dental personnel who speak their indigenous language, which also compromises the quality of the service provided, this added to the distance from health institutions that have dental equipment35. our study found a very low correlation, and there was no statistically significant association between bmi and dental caries and its clinical consequences when not treated, 7 aquino-canchari et al. being similar to what was reported by adriano et al.36, in mexican adults and contrary to what reported by shestha and others in nepalese teachers37,bthese discrepancies in the literature may be due to the multifactorial etiology of dental caries. the change in the prevalence, experience of dental caries and its clinical consequences of not being treated and nutritional disorders in indigenous communities have increased mainly due to changes in diet, with the progressive inclusion of industrialized and sugar-rich products in their diet. in food, this can be explained by sociocultural, economic and environmental changes, as a result of their interaction with society38.39. limitations: 1. variability that exists in the different ethnic groups existing in peru, will not allow these results to be extrapolated to other countries in the region. 2. the cross-sectional design does not allow evaluating the causality of dental caries, and other variables such as food diet were not considered. 3. the sample size was not significant and the fact of evaluating the bmi values and not having considered other indicators to evaluate the nutritional status. our results show the need for the implementation of policies in oral health and food safety with an intercultural approach, both in prevention, as well as in the treatment of pathologies and their rehabilitation, guaranteeing accessible, quality care appropriate to the culture of these populations. references 1. world bank group. indigenous village. washington, dc: world bank; 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[high blood pressure and obesity in indigenous ashaninkas of junin region, peru]. rev peru med exp salud publica. 2014;31(1):78-83. spanish. 20. davison b, goodall j, whalan s, montgomery-quin k, howarth t, singh g. nutritional dual burden in indigenous young adults: the geographical differential. aust j rural health. 2019 feb;27(1):14-21. doi: 10.1111/ajr.12439. 21. monteban m, yucra velasquez v, yucra velasquez b. comparing indigenous and public health infant feeding recommendations in peru: opportunities for optimizing intercultural health policies. j ethnobiol ethnomed. 2018 nov 20;14(1):69. doi: 10.1186/s13002-018-0271-2. 22. arza insfran ea, collante lavand v, sanabria m, acosta j, morínigo martínez m. [double burden of malnutrition in mothers and children under five years of age in two indugenous communities of the central departament]. an fac cienc med (asunción). 2018;51(3):53-60. spanish. doi: 10.18004/anales/2018.051(03)53-060. 23. boaretto jd, molena-fernandes ca, pimentel gg. [the nutritional status of kaingang and guarani indigenous peoples in the state of paraná, brazil]. cien saude colet. 2015 aug;20(8):2323-8. portuguese. doi: 10.1590/1413-81232015208.14462014 24. pizzi r, fung l. [obesity and woman]. rev obstet ginecol venez. 2015;75(4):221-4. spanish. 25. soares gh, aragão as, frias ac, werneck ri, biazevic mgh, michel-crosato e. epidemiological profile of caries and need for dental extraction in a kaingang adult indigenous population. rev bras epidemiol. 2019 aug 19;22:e190042. doi: 10.1590/1980-549720190042. 9 aquino-canchari et al. 26. aamodt k, reyna-blanco o, sosa r, hsieh r, de la garza ramos m, garcia martinez m, et al. prevalence of caries and malocclusion in an indigenous population in chiapas, mexico. int dent j. 2015 oct;65(5):249-55. doi: 10.1111/idj.12177. 27. alves filho p, santos rv, vettore mv. [oral health of guaraní indians in the state of rio de janeiro, brazil]. cad saude publica. 2009 jan;25(1):37-46. portuguese. doi: 10.1590/s0102-311x2009000100004.  28. arantes r, santos rv, coimbra ce jr. [oral health among the xavánte indians in pimentel barbosa, mato grosso, brazil]. cad saude publica. 2001 mar-apr;17(2):375-84. portuguese. doi: 10.1590/s0102-311x2001000200012. 29. hirooka lb, mestriner-junior w, mestriner sf, nunes sac, lemos pn, franco lj. dental caries dental in mother-child couples of xingu. braz j oral sci [internet]. 2014;13(1):43-6. doi: 10.1590/1677-3225v13n1a09.  30. jayashantha p, johnson nw. oral health status of the veddas--sri lankan indigenous people. j health care poor underserved. 2016 feb;27(1 suppl):139-47. doi: 10.1353/hpu.2016.0039. 31. chávez-reátegui b, manrique-chávez j, manrique-guzmán j. [geriatric dentistry and gerodontology: aging and oral characteristics of elderly patients. review]. rev estomatol herediana. 2014;24(3):199-207. spanish. 32. tsai c, blinkhorn a, irving m. oral health programmes in indigenous communities worldwide-lessons learned from the field: a qualitative systematic review. community dent oral epidemiol. 2017 oct;45(5):389-97. doi: 10.1111/cdoe.12302. 33. carrasco-loyola m, orejuela-ramirez f. [clinical consequences of untreated dental caries in preschool and school children of public educational institutions]. rev estomatol. herediana. 2018;28(4):223-8. spanish. doi: 10.20453/reh.v28i4.3425. 34. rogers hj, tariq u, olsson l, riaz sa, miah mr. caries prevalence, clinical consequences and self-reported pain experienced by children living in the west bank. eur arch paediatr dent. 2019 aug;20(4):333-8. doi: 10.1007/s40368-018-00412-6. 35. aliaga-del castillo a, mattos-vela ma, aliaga-del castillo r, del castillo-mendoza c. [malocclusions in children and adolescents from villages and native communities in the ucayali amazon region in peru]. rev peru med exp salud publica. 2011 mar;28(1):87-91. spanish. doi: 10.1590/s1726-46342011000100014. 36. adriano-anaya mp, caudillo-joya t, caudillo-adriano pa. [dental caries its association with body mass index in a young adult population]. int j odontostomat. 2017;11(4):437-42. apanish. doi: 10.4067/s0718-381x2017000400437. 37. shrestha s, shrestha rm. correlation between oral health and body mass index among nepalese teachers. kathmandu univ med j (kumj). 2016 jul-sept.;14(55):231-4. 38. restrepo-arango m, gutiérrez-builes la, ríos-osorio la. food security in indigenous and peasant populations: a systematic review. cien saude colet. 2018 apr;23(4):1169-81. doi: 10.1590/1413-81232018234.13882016. retraction in: cien saude colet. 2019 jan;24(1):343. 39. hita sr. [health, globalization and interculturalism: an anthropological approach to the situation of indigenous peoples in south america]. cien saude colet. 2014 oct;19(10):4061-9. spanish. doi: 10.1590/1413-812320141910.09372014. revista fop n 13 evaluation of root structure loss using autocad assisted histomorphometry prohic samir1; nakas enita2 1dds,msc, phd, assistant professor, department of oral surgery and implantology 2dds, msc, senior research assistant, department of orthodontics, school of dentistry, university of sarajevo, sarajevo, bosnia and herzegovina received for publication: september 11, 2008 accepeted: october 14, 2008 correspondence to: prohic samir department of oral surgery and implantology school of dentistry, university of sarajevo bolnièka 4°, 71 000 sarajevo bosnia and herzegovina e-mail: samir@drprohic.ba; sprohic@gmail.com phone: ++ 387 61 17 00 71 a b s t r a c t aims: the quantitative measurement and characterization of microscopical images using a computer is histomorphometry. the purpose of this experimental research is to evaluate the validity of two methods of histomorphometry of root structure loss measuring antiresorptive efficacy of topical application of alendronate in delayed tooth replantation on a canine model. methods: we used fortyeight premolar mature roots from five dogs in the study. roots were soaked in: 1 mmol of aln, 3 mmol of aln and physiological saline. after 4 months, animals were sacrificed and a bone block was prepared for analysis. histological slides were photographed with digital camera (olympus c 5060 5.1 megapixel) which is directly attached to microscope (carl zeiss, axiolab,jena, germany) connected to a pc. the images obtained were stored as figures (tiff) for further interpretation. root structure loss due to resorption was measured in two ways. results: the t-test for two independent samples for evaluating root mass loss between different methods of measurement was used. (t=4.497; df=17; p<0.05). conclusion: the obtained results showed additional accuracy and precision by computer method that enables future researchers in the area of traumatic injuries of dento-alveolar system, for applying in pathohistology scientific approach, and for measuring the resorption changes on the roots of replanted teeth. key words: dental trauma; autocad; alendronate i n t r o d u c t i o n the most frequent therapy used on patients with avulsed teeth is the method of replantation. after replantation, there may be ankylosis and root resorption, which frequently lead to tooth loss in 4–6 years.1 clinical and histological studies have shown that the main factors that have impact on replanted teeth are: the length of the extra-alveolar period, storage condition and maturation of the root. many studies have tried to identify a medicament that would act in the manner of inhibiting root resorption of replanted teeth. these treatments have focused on the use of various forms of fluoride and antibiotics.25 aforementioned studies do not take into account the active role of osteoclast in root resorption. the possibility of osteoclast inhibition could increase the likelihood of pdl cell proliferation, differentiation, and maintenance of the cemental layer.6 because of the similar morphology, enzymatic properties and function of the cells leading to resorption of dentin, cement and bone, the processes of root and bone resorption may be considered similar7. thus, drugs that may inhibit bone resorption may also be effective for the treatment of tooth resorption. alendronate (aln) is a third-generation bisphosphonate, which demonstrates the osteoclast inhibitory activity that can slow down the resorptive process. solutions containing different concentrations of alendronate may inhibit the bone resorption; moreover, the higher the concentration, the higher the effect8,9. measurement of root structure loss as indicator of antiresorptive efficacy is important factor in estimation of drug effectiveness. in order to get precise data we measure root structure loss by standard method6,10. nowadays autocad as well as other computer programs are used in medicine for image analysis and they can be used for precise measurements.11-17 the purpose of this experimental research is to evaluate the validity of two methods of histomorphometry of root braz j oral sci. july/september 2008 vol. 7 number 26 1620 structure loss measuring antiresorptive efficacy of topical application of alendronate in different concentrations of 3 mmol and 1 mmol in delayed tooth replantation on a canine model. materials and methods we used five mongrel dogs skeletally mature with a mean weight of 13.9 ±1.5 kg. the animals were handled according to international standards of animal welfare that are accepted by the research and bioethics committee of the dentistry school, of the university of sarajevo (issue number 09/133-3/04). all experimental procedures were performed with the animals under general anaesthesia. this was accomplished by preaneasthetic sedation with acepromazin maleate (vetranquil, sanofi, france) at a dose of 0.05 mg/kg im, and anaesthetic induction with propofol (propofol abbott,pakistan) at the dose of 4 mg/kg iv, followed by intubations and maintenance with 2,5% halothane (fluothane, zeneca, uk) with the oxygen flow of 2 liters/ minute. immediately after intubations, the animals were given a one-time dose of tramadol hydrochloride (lumidol, belupo, croatia ) at a amount of 7 mg/kg sc. forty-eight mandibular premolar mature roots of five dogs were used in the study, each dog served as its own control. it was used two rooted mandibular premolar teeth (p2, p3, and p4). p1 premolar was excluded due to size of the root and impossibility of immobilization. also 12 roots (6 teeth) were excluded due to root or bone damage. the two-rooted premolars were hemisected and accessed, instrumented with stainless steel k-type files to the apical delta, and irrigated with sterile saline. canals were dried with paper points, obturated with laterally condensed gutta-percha (vdw, gmbh, germany) and roth’s 801 sealers. teeth were extracted as atraumatically as possible, and dried for 45 minutes at room temperature in sterile petri dishes. furthermore, roots were soaked in: 1 mmol of aln, 3 mmol of aln, physiological saline, for 5 minutes and replanted. alendronat solutions were prepared by dissolving alendronat (fosamax® 70mg alendronat sodium oral solution, merck &co inc. whitehouse station nj, 08889, usa) in distilled water. due to the stability of the replanted teeth, splinting of teeth was determined to be unnecessary. because of divergence of the roots of mandibular premolar teeth in canine model only sutures for fixation were performed (mersilk 3-0, 26 mm ½ c, round bodied, ethicon inc.usa). the dogs were maintained on a soft diet for 2 days. they tolerated the treatment procedures well, their food intake and behaviour did not change following the treatment. all procedures were done based on double blind investigation principle and were performed by experienced specialist. after four months, the animals were sacrificed by an overdose of a 6 % solution of sodium pentobarbital administered iv, and the bone blocks were prepared for analysis. block specimens containing the teeth and surrounding alveolar bone were dissected using diamond separators, and fixed in 10% neutral formalin. following this, the bone blocks were placed in 5% nitric acid hno3 for seven days and then in 5% sodium sulphate na2so4 for 24 hours. bone blocks were placed into a tissue processor (microm stp 120, microm international gmbh, germany) for the process of dehydration through various percentages of alcohol, from 70% to an absolute alcohol, after in organic solvent xylol (changing the solution every 60 minutes for two times i, ii). for tissue impregnation the blocks were immersed in liquid paraffin at the temperature of 56°c. after the process of impregnation was completed, the specimens were embedded in paraffin blocks sized 2x2x2 cm and sectioned on rotary microtome (leica rm 2145, leica microsystems, swiss). the bone blocks were sectioned horizontally in reference to the longitudinal axis of the teeth to the thickness of 5 µm, at 140 µm intervals. the process of staining was than completed by haematoxylin-eosin method in digester. histological slides were photographed with digital camera (olympus c 5060 5.1 megapixel) which is directly attached to microscope (carl zeiss, axiolab,jena, germany) connected to a pc. the images obtained were stored as figures (tiff) for further interpretation. root structure loss due to resorption was measured. histomorphometry methods to evaluate the extent of root structure loss due to resorption, the original circumference of the root was determined. the radius of the remaining root structure was ranked on a linear, integer 0–6 scale at each of the eight points of the superimposed grid, with the value of 6 given to an unaffected radius and the value of 0 given to a point without any remaining root structure.6 (fig. 1.) our original method of measuring the root structure was fig. 1 method of measuring root structure loss with superimposed eight point grid on histologic section hematoxiylin and eosin (x10) 1621 braz j oral sci. 7(26):1620-1623 evaluation of root structure loss using autocad assisted histomorphometry used, with further assistance from the computer-assisted program, auto-cad 2002 for windows op. the program imported the images and, by means of multiple points (polyline tool), it outlined the image of the original circumference of the root (p1), later we outlined the image of the remaining root structure (p2). then, by calculating differences between area p1 and area p2, we obtained the value of the loss of the root structure (rp). measurements were made in a manner as to evaluate the circular area. (fig. 2) root structure loss was assessed by the difference between the p1 and p2 areas. all measurement was performed by a blinded investigator in three different time periods. for statistical analysis we used mean value. fig.2 method of the measuring the root structure with auto-cad program. hematoxiylin and eosin (x10) statistical analysis the root mass loss data were subjected to scheffe f multiple comparison tests to identify differences between groups. the t-test for two independent samples was used to evaluate root mass loss calculation between different means of measurement. statistical calculations were carried out with statistic software programme spss® for windows®. r e s u l t s in order to evaluate root structure loss we compared previously published method of determining root structure loss and our method 15 slides were used and the histomorphometry was made using both methods. there was evident more root structure loss when measured with autocad (p<0.05). average root structure loss in different groups in the group of 1 mmol, the root structure loss was registered at a value of 17.21%. in the group of physiological saline, root structure loss was 34.28%. the result difference satisfies statistical significance (p<0.005). n mean sd sem t-test p value standard method 15 11.467 8.254 2.131 0.860 <0.0001 auto-cad method 15 16.800 8.914 2.302 table 1 – descriptive data of measurement methods on comparing the group of 1 mmol and the group of 3 mmol, it was observed 17.21% of root structure loss in the first group and 12.74% in the second group, with statistical significance (p<0.005). d i s c u s s i o n to date research indicates that the main mode of action of bisphosphonates in the prevention of bone structure loss is inhibition of osteoclast function through the break-down of enzyme passages, which are very important for osteoclast activity and survival7,11,18. results of root structure loss in our study are 17.21% root structure loss in the 1mmol group, while a loss of 34.28% was found in the group with the physiology solution (p<0.005). furthermore, in support of a more potent anti-resorption action with regards to the concentration, when we statistically compared 1 mmol and 3 mmol groups in our research, we found out that the former registered a 17.21% mass loss, and the latter a loss of 12.74%, (p<0.005). this certainly affirms the fact that locally applied alendronate is a strong inhibitor of resorption processes, regardless of the concentration. it proves unable to stop, but can only potently slow down resorptive processes in the root of the replanted tooth. the doses of systemic application of alendronate in the work of el-shinnawi et al.18, which proved a positive effect of bisphosphonate on bone density in patients affected by periodontal disorder, were undoubtedly selected on the basis of abundant clinical experiences in the per oral ordination of fosamax®; however, local doses have no such kind of support and they are not sufficiently examined, which is an additional reason and justification for our study. digital image quantification (morphometry and image analysis) involves the computer-assisted quantification of various measurement parameters performed on digital photographs of histologic sections. typical measurement parameters include object counts, linear measurements, area measurements and relative color intensities. digital image quantification has proven valuable in studies that concern the identification and documentation of subtle induced changes in tissues.19 in this study it was used autocad software program for histomorphometry. the accuracy of the results obtained in calculations of root mass loss was tested both through mathematical calculations of percentages expressing the root mass loss, and using measuring methods published in earlier works. it can easily be noticed that, from a geometric aspect, 1622 braz j oral sci. 7(26):1620-1623 evaluation of root structure loss using autocad assisted histomorphometry computer method displays a considerably higher precision. the obtained results showed additional accuracy and precision by computer method that enables future researchers in the area of traumatic injuries of dento-alveolar system, for applying in patho-histology scientific approach, and for measuring the resorption changes on the roots of replanted teeth. r e f e r e n c e s 1. andreasen jo, borum mk, jacobsen hl, andreasen fm. replantation of 400 avulsed permanent incisors. 1. diagnosis of healing complications. endod dent traumatol. 1995; 11:51–8. 2. shulman lb, et al. fluoride inhibition of tooth-replant root resorption in cebus monkeys. j oral ther pharm. 1968; 4:331–7. 3. coccia ct. a clinical investigation of root resorption rates in reimplanted young permanent incisors: a five year study. j endod 1980;6:413–20. 4. selvig ka, bjorvatn k, claffey n. effect of stannous fluoride and tetracycline on repair after delayed replantation of rootplaned teeth in dogs. acta odont scand 1990; 48: 107–12. 5. selvig ka, bjorvatn k, bogle gc, wikesjo um. effect of stannous fluoride and tetracycline on periodontal repair after delayed tooth replantation in dogs. scand j dent res. 1992; 100: 200–3. 6. levin l, bryson ec et al. effect of topical alendronate on root resorption of dried replanted dog teeth. dent traumatol. 2001:120-6. 7. ne rf, witherspoon de, gutmann ji. tooth resorption. quintessence int. 1999; 30: 9–25. 8. hughes de, macdonald br, russell rgg, gowen m. inhibition of osteoclast-like cell formation by bisphosphonates in long-term cultures of human bone marrow. j clin invest. 1989; 83: 1930–5. 9. ito m, chokki m, ogino y, satomi y, azuma y, ohta t, et al. comparison of catatonic effects of bisphosphonates in vitro and in vivo. calcif tissue int. 1998; 63: 143–7. 10. sae-lim v, metzger z,trope m. local dexamethasone improves periodontal healing of replanted dog s teeth. endod dent traumatol. 1998; 14:232-6. 11. eslaminejad mr, valojerdi mr, yazdi pe.computerized threedimensional reconstruction of cartilage canals in chick tibial chondroepiphysis. anat histol embryol. 2006; 35:247-52. 12. iqbal mk, rafailov h, kratchman si, karabucak b.a comparison of three methods for preparing centered platforms around separated instruments in curved canals.j endod. 2006; 32: 48-51. 13. ciobanu o. the use of a computer aided design (cad) environment in 3d reconstruction of anatomic surfaces.stud health technol inform. 2006; 119:102-4. 14. lemaire e. a cad analysis programme for prosthetics and orthotics.prosthet orthot int. 1994 aug;18(2):112-7 15. palaisa j, ngan p, martin c, razmus t.use of conventional tomography to evaluate changes in the nasal cavity with rapid palatal expansion. am j orthod dentofacial orthop. 2007; 132: 458-66. 16. gergi r, sabbagh c. effectiveness of two nickel-titanium rotary instruments and a hand file for removing gutta-percha in severely curved root canals during retreatment: an ex vivo study. int endod j. 2007; 40: 532-7. 17. biz mt, figueiredo ja. morphometric analysis of shank-toflute ratio in rotary nickel-titanium files. int endod j. 2004; 37:353-8. 18. el-shinnawi um, el.tatnawy si. the effect of alendronate sodium on alveolar bone loss in periodontitis (clinical trial). j int acad periodontal. 2003; 5: 5-10. 19. histomorphometry/image analysis. available in: http:// www.epl-inc.com/s-sp01.html. accessed on november 10, 2008 1623 braz j oral sci. 7(26):1620-1623 evaluation of root structure loss using autocad assisted histomorphometry 1http://dx.doi.org/10.20396/bjos.v19i0.8659197 volume 19 2020 e209197 original article 1 herminio ometto university center, school of dentistry, araras, sao paulo, brazil. 2 department of post-graduation in implantology, university of santo amaro, school of dentistry. são paulo, sp, brazil. corresponding author: renata siqueira scatolin herminio ometto university center, school of dentistry, araras-sp, brazil dr. maximiliano baruto avenue, 500 – university garden, araras sp, 13607-339. fone fax: +55 (19) 3543-1400 e-mail: re_scatolin@hotmail.com received: april 17, 2020 accepted: september 23, 2020 effect of violet led associated with low-concentration hydrogen peroxide on enamel surface roughness natalia eugenio1, innocenzo scandiffio1, marcia hiromi tanaka2, ana luisa botta martins de oliveira1, renata siqueira scatolin1,* aim: the present study aimed to assess in vitro the effect of violet led in tooth bleaching techniques associated or not with low-concentration hydrogen peroxide gel on enamel surface roughness. methods: fifty-two enamel fragments of bovine teeth were flattened and polished (4x4x3 mm) and divided into four groups according to bleaching treatment: vlviolet led; hp7.5% hydrogen peroxide; hp+vl7.5% hydrogen peroxide + violet led; cno bleaching (control). before the treatments, all specimens were immersed in 20 ml of black tea for six days, changing solutions every 24 h to simulate the staining of specimens. forty fragments were used to analyze surface roughness (n=10) and 12 fragments were used for the morphological analysis (sem) (n=3). results: the data were submitted to one-way anova and a post-hoc tukey test. the lower roughness values was observed for the group that did not receive bleaching treatment (c), differing significantly only from the group bleached with 7.5% hydrogen peroxide + violet led (hp+vl) (p=0.0077). the remaining groups did not show significant differences in roughness values (p>0.05). the scanning electron microscopy analysis showed irregularities on the enamel surface regardless of the treatment received. conclusion: the results showed that bleaching treatments with violet led associated with low-concentration hydrogen peroxide gels (7.5%) increase the surface roughness of tooth enamel. keywords: dental enamel. hydrogen peroxide. tooth bleaching. https://orcid.org/0000-0003-4890-8096 2 eugenio et al. introduction the most common bleaching process is the application of carbamide peroxide or hydrogen peroxide gel on tooth enamel1,2, using techniques performed in-office or at home by the patient. in the initial phases, after the application of gels, a chemical reaction of oxide reduction occurs, in which the pigmented carbon molecules are broken and converted into smaller molecules with a lighter color, promoting the bleaching effect3. light sources are often used in association with gels to accelerate the decomposition of peroxides, but they may also increase the temperature of the tooth structure depending on the light source used4. these tooth bleaching techniques show to be effective as a color change, but in many cases, it can cause tooth sensitivity2. they are also seen in the literature some unwanted effects on the enamel structure related to peroxide gels and some associated light sources, among which are roughness increase, hardness decrease, and surface morphology changes5,6. such changes may be harmful to patients because of the increase in enamel porosity, facilitating the adhesion of microorganisms7. in this light, the search for procedures that are less harmful to the dental structure has also led to the development of different tooth bleaching products and techniques8. violet led devices can supposedly fragment the pigmented molecules through a physical medium, in which the absorption peak of these molecules matches the violet light emission range (405-410 nm), transforming them into smaller and less pigmented molecules9 that can bleach even without the gel association, decreasing the risk of the harmful effects of bleaching related to peroxide, providing safety and comfort for patients10,11. thus, this study aimed to assess in vitro the enamel surface roughness after tooth bleaching techniques using violet led associated or not with low-concentration hydrogen peroxide gels, considering the literature is scarce of such information. materials and methods experimental design fifty-two fragments of bovine teeth were included, 40 of them were used for the surface roughness analysis (n = 10) and 12 fragments were used for the morphological analysis (sem) (n = 3). the experimental groups were divided according to the bleaching procedure proposed: vlviolet led; hp7.5% hydrogen peroxide; hp+vl7.5% hydrogen peroxide + violet led; cno bleaching (control). the outcome variables were subjected to surface roughness analysis (quantitative analysis) and scanning electron microscopy (qualitative analysis). sample collection the sample collection included bovine teeth without cracks or hypoplastic stains. the crowns of the teeth were sectioned in the middle third with an electric precision cutter (isomet 1000; buehler, lake bluff, il, usa) aided by a diamond disc, to obtain 40 fragments with dimensions of 4x4x3 mm. 3 eugenio et al. the enamel surface was then flattened in a water-cooled rotary polisher (dp-9u2; struers s/a, copenhagen, denmark) with #600 and #1200 abrasive files and polished with 0.3-μm alumina paste and polishing felt. after polishing, the specimens were cleaned in ultrasound for five minutes. staining of fragments for staining the enamel fragments, they were immersed individually in 20 ml of black tea (leão júnior s.a., curitiba, brazil) in the ratio of 1.6 g of black tea leaves to 100 ml of distilled water for five minutes, with posterior filtration to remove the tea leaves12. this solution was replaced every 24 h for six days8,12, ph 5,1 and room temperature (±22°c). at the end of the staining, the samples were washed with deionized water for one minute and dried with absorbent paper to receive the treatments. bleaching of enamel fragments the applications and number of bleaching sessions were performed according to the manufacturer’s instructions, to the following protocols: vlbleached with violet led (bright max whitening, mmo, são carlos, sp, brazil): seven sessions of 30 minutes were performed, in which the light was applied at 8 mm of distance from the tooth surface, 20 times for 60 seconds, and turned off between each application for 30 seconds. each session had a 7-day interval in between. hpbleached with 7.5% hydrogen peroxide (whiteness class, fgm, joinville, sc, brazil): fifteen 7.5% hydrogen peroxide gel applications were performed for one hour on a layer of 0.5 to 1 mm of thickness on the enamel surface. each application had a 24-hour interval in between. hp+vlbleached with 7.5% hydrogen peroxide (whiteness hp blue, fgm, joinville, sc, brazil) + violet led (bright max whitening, mmo, são carlos, sp, brazil): the 7.5% hydrogen peroxide was applied on the surface of the fragments as described for group hp, added by the association of violet led application as described for group vl. three sessions were performed with a 7-day interval in between. cno bleaching (control): during the bleaching period of groups vl, hp e hp+vl the specimens of group c remained in deionized water. during the intervals of treatments, the samples were kept in relative humidity with deionized water in an oven at 37 ° c. surface roughness analysis the roughness of the specimens was analyzed with a digital roughness meter (model sj 301, mitutoyo corporation inc., kanagawa, japan). roughness was assessed at two times: before and after the treatment with bleaching agents. the parameter for measuring roughness was ra (average roughness) and the cut-off used was 0.25 mm. the ra values represents the arithmetic mean of the size of peaks and valleys found during surface scanning. 4 eugenio et al. scanning electron microscopy three specimens from each group were put in distilled water in the ultrasound (10 minutes) and later they received dehydration in ascending levels of ethanol (labsynth ltda., diadema, sp, brazil): 25% (20 min), 50% (20 min), 75% (20 min), 95% (30 min), and 100% (60 min). as dehydration was concluded, the specimens were fixed in stubs and metalized with a layer of gold-palladium alloy in a vacuum metallization apparatus (sdc 050, bal-tec ag, fl9496, balzers, liechtenstein). the specimens were examined in a scanning electron microscope (zeiss, evo 50, cambridge, england) for the qualitative analysis of the morphological change of enamel caused by the treatments. the area that most represented each group was photographed with (x3000) magnification. statistical analysis after being tested for normality and homoscedasticity, using shapiro-wilk’s and levene’s tests, respectively, the data were submitted to one-way anova and a post-hoc tukey test. for all tests, the significance level of 5% was considered. the data were analyzed using the graphpad prism (graphpad software, la jolla, ca, usa) and jamovi (the jamovi project (2020). jamovi. (version 1.2) retrieved from https://www.jamovi.org). results comparing the roughness values before and after the treatments, there was a significant increase in the roughness values for all groups, with no differences between them. lower roughness values for the group that did not receive bleaching treatment (c) was observed, differing significantly only from the group bleached with 7.5% hydrogen peroxide + violet led (hp+vl) (p=0.0077). among the remaining groups, there were no significant differences in roughness values (p>0.05). (table 1) the images obtained with scanning electron microscopy (figure 1) showed irregularities on the enamel surface regardless of the bleaching treatment performed (vl, hp e hp+vl). changes in the enamel surface morphology are also present for the group that received only the process of staining with black tea and no bleaching (c). table 1. mean (±sd) of ra values (μm) of the specimens that were stained and later received bleaching treatments groups vl (violet led) hp (7.5% hydrogen peroxide) hp+vl (7.5% hydrogen peroxide + violet led) c (no bleaching) p value ra before (μm) 0.0633 (±0.0212)ab 0.0622 (±0.0210)ab 0.0588 (±0.0226)ab 0.0600 (±0.01187)ab 0.96 ra after (μm) 0.3778 (±0.2154) aba 0.3378 (±0.1258) aba 0.4422 (±0.1838) aa 0.2433 (±0.0678) ba 0.0077 p value 0.0022 0.0022 0.0003 <0.0001 *means followed by different uppercase letters indicate statistically significant difference between groups (lines) and different lowercase letters mean statistically significant difference between intragroups (collums). https://www.jamovi.org 5 eugenio et al. discussion the evaluation of the enamel changes after different bleaching techniques needs to be studied, because factors such a surface roughness can lead to the accumulation of biofilm with a consequent increase in the risk of developing caries injury and periodontal disease7. in this study, all groups showed an increase in average surface roughness values (ra) when compared to the initial surface roughness values. even though group c received no treatment, it was subjected to staining with black tea as all the other groups, which would explain the increase in roughness values, considering this beverage shows an erosive potential13. dental exposure to drinks with ph values below 5.5, considered critical for the dissolution of enamel prisms, with long duration and the absence of intrinsic buffering systems of saliva, may have contributed to the changes in the enamel microstructure14. it can also be identified by the scanning electron microscopy images. according to kury et al.15 (2020), the bleaching treatment with violet led (vl) showed similar enamel morphology to the untreated group (c), which shows the safety of its use for dental enamel. these similarities were also observed in this study. the roughness tests showed no significant differences between them too. when the violet led was applied associated to the 7.5% hydrogen peroxide (hp + vl), the surface roughness increased when compared with untreated group (c), but there was no difference in the roughness values when the treatments were applied alone. figure 1: a. violet led; b. 7.5% hydrogen peroxide; c. 7.5% hydrogen peroxide + violet led; d. no bleaching a b c d 10 µm eht = 20.00 kv mag = 3.00 k x detector = se1 10 µm eht = 20.00 kv mag = 3.00 k x detector = se1 10 µm eht = 20.00 kv mag = 3.00 k x detector = se1 10 µm eht = 20.00 kv mag = 3.00 k x detector = se1 6 eugenio et al. ergin et al.16 (2018) observed an increased enamel roughness by associating 35% hydrogen peroxide with different types of light (diode laser, er:yag laser, or blue led). light sources accelerate the bleaching procedure by heating the bleaching gels to increase the decomposition rate of oxygen to oxygen-free radicals and raise the release of stained molecules17. the free radicals produced during bleaching penetrate within the interprismatic regions and react with pigmented molecules and the organic matrix of the enamel, increasing the surface irregularity of this enamel18. the increased surface roughness after bleaching facilitates the staining of teeth and restorative materials by the adhesion of dye pigments and microorganisms19,20, which causes great concern to both professionals and patients. studies have shown that bleaching with hydrogen peroxide promotes structural changes in enamel, characterized by superficial depressions21,22, greater porosity21, exposure of enamel prisms21, increased surface roughness22, decreased hardness, and loss of mineral content23. these changes occur due to enamel demineralization and they may facilitate the accumulation of plaque24. the adverse effects of bleaching on enamel morphology depend on the concentration of hydrogen peroxide, its ph, and exposure time25. despite the increased values of tooth enamel roughness observed in the bleaching technique with gel associated with the violet led, it is difficult to affirm whether such changes are clinically reversible or not. this study was performed in vitro and perhaps the presence of saliva, fluorides, or other remineralizing solutions would have maintained a balance between the processes of demineralization and remineralization26, minimizing the adverse effects of tooth bleaching19,27. thus, in vivo studies are required to understand the effects of bleaching agents associated with a violet led on tooth enamel. acknowledgments the authors would like to thank the institutional scholarship program for scientific initiation and 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topography of enamel afther bleaching: tracking channels of penetration with silver nitrate. braz j oral sci. 2011;10(1):1-6. doi: 10.20396/bjos.v10i1.8641669. 19. hauss monteiro dd, valentim pt, elias dc, moreira an, machado cornacchia tp, et al. effect of surface treatments on staining and roughness of bleached enamel. indian j dent res. 2019;30(3):393-8. doi: 10.4103/ijdr.ijdr_233_16. 20. wongpraparatana i, matangkasombut o, thanyasrisung p, panich m. effect of vital tooth bleaching on surface roughness and streptococcal biofilm formation on direct tooth-colored restorative materials. oper dent. 2018;43(1):51-9. doi: 10.2341/16-366-l. 8 eugenio et al. 21. miranda cb, pagani c, benetti ar, matuda fda s. evaluation of the bleached human enamel by scanning electron microscopy. j appl oral sci. 2005;13(2):204-11. doi: 10.1590/s1678-77572005000200021. 22. pinto cf, oliveira rd, cavalli v, giannini m. peroxide bleaching agent effects on enamel surface microhardness, roughness and morphology. braz oral res. 2004;18(4):306-11. doi: 10.1590/s1806-83242004000400006. 23. ferreira sda s, araújo jl, morhy on, tapety cm, youssef mn, sobral ma. the effect of fluoride therapies on the morphology of bleached human dental enamel. microsc res tech. 2011;74(6):512-6. doi: 10.1002/jemt.20939. 24. hegedüs c, bistey t, flóra-nagy e, keszthelyi g, jenei a. an atomic force microscopy study on the effect of bleaching agents on enamel surface. j dent. 1999;27(7):509-15. doi: 10.1016/s0300-5712(99)00006-8. 25. ushigome t, takemoto s, hattori m, yoshinari m, kawada e, oda y. influence of peroxide treatment on bovine enamel surface--cross-sectional analysis. dent mater j. 2009;28(3):315-23. doi: 10.4012/dmj.28.315. 26. de abreu dr, sasaki rt, amaral fl, flório fm, basting rt. effect of home-use and in-office bleaching agents containing hydrogen peroxide associated with amorphous calcium phosphate on enamel microhardness and surface roughness. j esthet restor dent. 2011;23(3):158-68. doi: 10.1111/j.1708-8240.2010.00394.x. 27. sa y, chen d, liu y, wen w, xu m, jiang t, et al. effects of two in-office bleaching agents with different ph values on enamel surface structure and color: an in situ vs. in vitro study. j dent. 2012;40 suppl 1:e26-e34. doi: 10.1016/j.jdent.2012.02.010. 1http://dx.doi.org/10.20396/bjos.v20i00.8663736 volume 20 2021 e213736 original article 1 departamento of dentistry, federal university of rio grande do norte (ufrn), natal/rn, brazil. 2 university of west paulista (unoeste), são paulo/sp, brazil. corresponding author: ruthineia diógenes alves uchôa lins federal university of rio grande do norte – departamento of dentistry av. sen. salgado filho, 1787 – lagoa nova, 59056-000 – natal – brazil. phone: +55 84 32154100 – mobile: +55 84 994013056 email: aruthineia@gmail.com editor: dr altair a. del bel cury received: december 24, 2020 accepted: march 22, 2021 effect of spondias mombin l. extract on the wettability, roughness, color and morphology of bovine enamel franciara maria gomes alves1 , thaís oliveira cordeiro1 , ana margarida dos santos melo1 , larissa sgarbosa de araújo matuda2 , daniela lopes da silva amorieli2 , joselucia da nóbrega dias1 , boniek castillo dutra borges1 , ruthineia diógenes alves uchôa lins1,* although spondias mombin l. extract has an excellent antimicrobial effect against oral microorganisms, it should be clarified how it affects enamel surface properties. aim: to evaluate the color change, wettability/contact angle, surface roughness and morphology of bovine enamel submitted to the spondias mombin l. extract. methods: thirty bovine teeth were distributed into the following groups: 0.12% chlorhexidine digluconate, 1:32 spondias mombin l. extract and distilled water. color change (cc) was evaluated after immerging specimens into the solutions for 14 days. surface roughness (ra) was measured using a roughness meter; wettability/contact angles (ca) were determined by the sessile drop method, and scanning electron microscopy images were obtained to characterize the morphology (sma). the ph of the solutions was evaluated using a phmeter. the ra, ca, and cc data were parametric (kolmogorov-smirnov; p>0.05). two-way anova (for ra and ca) and one-way anova (for cc) with tukey’s posthoc tests at a significance level of 5% were used. sma was analyzed descriptively. results: the spondias mombin l. extract revealed an acidic ph, and when in contact with the bovine teeth, it increased the wettability, but it did not cause statistically significant differences in the ra. spondias mombin l. extract caused the highest color change. the sem images showed differences in the specimens’ surface submitted to the extract compared to the other groups. conclusion: spondias mombin l. extract provided negative effects on bovine enamel’s surface, including a high color change and a more wettable substrate. keywords: phytotherapy. anacardiaceae. mouthwashes. dental enamel. surface properties. https://orcid.org/0000-0001-9529-4694 https://orcid.org/0000-0003-0078-715x https://orcid.org/0000-0001-7234-226x https://orcid.org/0000-0002-9033-9029 https://orcid.org/0000-0001-9755-0807 https://orcid.org/0000-0002-0435-2869 https://orcid.org/0000-0003-4313-5776 https://orcid.org/0000-0002-0047-5976 2 alves et al. introduction clinical evidence shows that dental biofilm control is essential in preventive dentistry and directly reflects individuals’ oral health. the materials used for this purpose in dentistry attempt to maintain the dental surface’s natural properties, including mineral composition, hardness, smoothness, translucency and low surface free energy/wettability1-3. the use of mouthwashes, along with the mechanical control of the biofilm, has also shown substantial ability to prevent biofilm from growing. although 0.12% chlorhexidine digluconate is considered the gold standard in mouthwash, it promotes adverse effects such as staining of teeth, changes in gustation, and irritation of the mucosa1,4,5. therefore, there is a need for scientific studies that evaluate herbal medicines’ potential as an alternative of interest for future use in the control of dental biofilm, preventing and treating oral biofilm diseases6. the species spondias mombin has been gaining notoriety among the currently studied plant extracts. in brazil, this plant is mainly found in the north and northeast regions. however, this species is also found in several parts of the world, such as peru, venezuela, bolivia, mexico, and western india7. its leaves present, among other characteristics, components with antimicrobial and antioxidant properties, such as flavonoids, saponins, tannins, and phenolic compounds those last ones are also associated with antiviral and antitumor activities8. those phytochemicals have been constantly associated with effects against oral microorganisms, including streptococcus mutans. a recent study showed that the hydroethanolic extract of spondias mombin l. has an antimicrobial activity similar to chlorhexidine 0.12% against oral bacteria of the genus streptococcus, also showing an anti-adherent effect on streptococcus mutans9. besides, anti-inflammatory effects and properties arising from the extract of spondias mombin l., associated mainly with the tannin components, have also been observed6,10-16. in light of this knowledge, this in vitro study aimed to evaluate the effect of the spondias mombin l. extract on the wettability, color change, surface roughness and morphology of bovine enamel compared with 0.12% chlorhexidine digluconate and distilled water. the null hypothesis tested was that there would be no differences among the solutions concerning all properties analyzed. methods specimens’ preparation specimens of bovine teeth (n=30) collected from the nellore animal breed with a mean age of 36 months were used from the meat industry. fractured and irregular teeth were excluded after visual inspection. initially, the bovine teeth were cleaned with pumice and water, with the aid of a rubber cup in a slow-speed handpiece and stored in distilled water. subsequently, a transversal section was made, dividing the root and coronal portions, 2mm below the cementoenamel junction, having as reference the labial surface. the crowns were separated from the roots using a diamond disc (kg sorensen, cotia, sp, brazil) in a straight handpiece. it was then made 3 alves et al. longitudinal sections in the mesial, distal, and incisal surfaces, obtaining specimens (10mm x 10mm) from the labial enamel’s flatter part. specimens with cracks were excluded17. finally, the specimens were stored in distilled water at room temperature until the moment of the immersion protocol. obtaining the spondias mombin l. extract the sample of the collected raw vegetable material was deposited in the ufrn herbarium and identified by a botanist, with the number of exsiccate 12252. spondias mombin’s collected leaves were dried at room temperature for two weeks. from 100 g of the spondias mombin l., an extract was prepared by maceration, leaving it in contact with ethanol and distilled water (80:20, v/v) for seven days and subsequent lyophilization. after this period, the hydroethanolic extract was filtered, and the organic solvent was eliminated in a rotatory evaporator (te210, tecnal, piracicaba, brazil) under a vacuum at 45° c. the hydroethanolic extract of spondias mombin l. used in this research had a 1:32 dilution = 31.25 (mg/ml). this concentration was defined based on lima et al.8 (2017), which found that spondias mombin l. extract showed antimicrobial activity superior to 0.12% chlorhexidine digluconate only from that concentration. determination of ph ten ml of the tested solutions were analyzed in a ph meter (luca-210-e, lucadema, são josé do rio preto, brazil), calibrated with phosphate and acetate buffer solutions, ph 7.0 and 4.0, respectively, at 25° c. after the extract preparation, the solutions were performed in triplicate. the ph of distilled water, 0.12% chlorhexidine digluconate, and spondias mombin l. extract were, respectively, 7.0, 6.5, and 2.96. immersion protocol and analyzed variables the specimens (n=30) were randomly allocated into 3 groups: clx 0.12% chlorhexidine digluconate (n=10); dw distilled water (n=10); and de diluted spondias mombin l. extract (n=10). the following dependent variables were tested: wettability/contact angles (ca), surface roughness (ra), color change (cc) and surface micromorphology (smf). for ca and ra, solution (clx, dw, and de) and time (24 h and 14 days) were the independent variables. for cc and smf, solution was the independent variable. figure 1 presents a schematic representation of the methods. the immersion protocol used for all solutions followed the clx manufacturer’s (periomax©, iodontosul, porto alegre, rx, brazil). each specimen was positioned individually inside a flask containing 10ml of the specific solution, which was disturbed for 1min twice a day, with 12 hours intervals, for 14 days. in the interval between immersions, the specimens were stored in 1.5ml of artificial saliva (farmafórmula, natal, brazil. composition: single syrup [nipagin, nipazol, sugar, water] 20%; glycerin 10%; carboxymethyl cellulose gel [cmc] 2.5 to 8%; cherry flavorant 0.1%. ph = 7.0)17,18. the solutions and artificial saliva were renewed after each sample submission period. then, the specimens were dried at room temperature for 24 hours before the analyses4. 4 alves et al. 10 mm 10 mm dw group (n=10) clx group (n=10) de group (n=10) measurement of the ph of the solutions 14 days drying in oven at 37° 24 hours final analysis: surface roughness wettability colour stability sem initial analysis: surface roughness wettability colour stability distilled water, room temperature immersion in dw, clx, de 10ml, 1 min, 2x/day storage in artificial saliva 15ml, 12 hours figure 1. schematic representation of the methods. wettability/contact angles adapting the protocol described by costa et al.19 (2018), the wettability was evaluated using the sessile drop method. the contact angle between the dental surface and the liquid was determined. a drop of distilled water (5 μl) was released on the specimens’ surface with the aid of an automatic micropipette over a distance of 20mm, in front of a photographic camera. the surface was positioned in the central and perpendicular part of the lens. the images were captured at a distance of 30cm, 5s after the drop was dumped. subsequently, the contact angle was measured using the surftens 4.7 automatic software (oeg gmbh, frankfurt, oder, germany), adapting its settings for “distilled water” and “4 (four) point analysis”. each image was analyzed in triplicate, and an average was obtained. surface roughness assessment (ra) for ra analysis, the hommel etamic w10 roughness meter (jenoptik industrial metrology germany gmbh, germany) was used, equipped with a diamond needle with a radius of 2 mm. the needle was moved at a constant speed of 0.5 mm/s with a load of 0.7 mn. the cut-off value was set at 0.25 mm. the average surface roughness (ra) of different locations (parallel, oblique and perpendicular) was obtained20,21. color change analysis (cc) to determinate the cc, the specimens’ color was assessed at baseline (t0) and after 14 days (t14) of immersion in each solution. four measurements were performed on enamel using a digital spectrophotometer (easyshade, vitazahnfabrik, bad säckingen, germany), and an average value was obtained. the data recorded by the colorimeter were used to calculate the ciede2000 color change (δe00) according to the following equation: 5 alves et al. δe00 = + + rt+ 1/2 δl’ klsl δc’ kcsc δh’ khsh δc’ kcsc δh’ khsh the values of δl’, δc’, and δh’ are the differences in lightness, chroma, and hue between t14 and t0. sl, sc, and sh are the weighting functions for the lightness, chroma, and hue components, respectively. kl, kc, and kh are the parametric factors to be adjusted according to different viewing parameters. in this study, kl, kc, and kh were set to 1. color change considering t14-t0 was obtained with 50:50% perceptibility (pt = 0.81 δe00 units) and 50:50% acceptability (at = 1.77 δe00 units) thresholds22,23. surface microorphology analysis (sma) for the sms, three specimens of each group were randomly selected (n=3) at t14. after 24h dry, the specimens were gold-sputtered, and images were recorded through a scanning electron microscope (sem-feg, zeiss gemini, germany). images with 2000x magnification in the center of the sample were obtained and descriptively analyzed19. statistical analysis data analyses were performed through the statistical package for social sciences software (spss ibm spss statistics subscription, version 25). the normality of the data was verified using the kolmogorov-smirnov test (p>0.05). the descriptive analysis presented the mean and standard deviation. the ca and ra were analyzed using two-way anova/tukey posthoc tests (solution versus time). for cc, one-way anova/tukey posthoc tests were used. the level of significance was set at 95% (p<0.05). the photomicrographs of the surface morphology were evaluated qualitatively. results wettability/contact angles (ca) there were statistically significant differences among solutions (p>0.01), times (p<0.01) and in the interaction of solutions versus times (p<0.01). multiple comparisons are shown in table 1. regardless of the time, de showed statistically decreased contact angles than dw and clx. only de provided statistically lower contact angles at t14 compared to t0. table 1. mean (standard deviation) of the contact angles according to the solution and time tested in this study. solution time baseline (t0) 14 days (t14) distilled water (dw) 53.81 (3.46) aa* 48.36 (3.17) aa 0.12% chlorhexidine digloconate (clx) 56.86 (4.39) aa 54.95 (4.78) aa spondias mombin l. extract (de) 51.36 (3.68) ba 39.26 (4.90) bb *different capital letters reveal statistically significant differences among the solutions for the same time (p<0.05). different lowercase letters reveal statistically significant differences between times for the same solution (p<0.05). 6 alves et al. surface roughness (ra) there were no statistically significant differences among solutions neither between times (p>0.05). table 2 presents detailed ra values. table 2. mean (standard deviation) of surface roughness (μm) according to the solution and time tested. solution time baseline (t0) 14 days (t14) distilled water (dw) 2.12 (0.91) aa* 1.55 (0.87) aa 0.12% chlorhexidine digloconate (clx) 2.04 (1.04) aa 2.23 (0.91) aa spondias mombin l. extract (de) 2.36 (0.82) aa 1.65 (0.41) aa *different capital letters reveal statistically significant differences among the solutions for the same time (p<0.05). different lowercase letters reveal statistically significant differences between times for the same solution (p<0.05). color change (cc) there were statistically significant differences among the solutions (p<0.01). multiple comparisons are shown in table 3. de showed a statistically higher cc than dw and clx. only de presented δe00 units higher than the perceptibility (0.81) and acceptability (1.77) thresholds. table 4. mean (standard deviation) of the color change (δe00) according to the solution tested. solution distilled water (dw) 0.12% chlorhexidine digluconate (clx) spondias mombin l. extract (de) 0.74 (0.14) b* 0.79 (0.38) b (1.89) a *different lowercase letters reveal statistically significant differences among the solutions (p <0.05). surface micromorphology (sma) the images obtained (figure 2) point that exposition to clx promoted mineral-like precipitation compared with dw. however, specimens exposed to the de showed characteristics of enamel dissolution. dw clx de 2 µm eht = 3.00 kv wd = 5.1 mm mag = 2.00 k x pixel size = 57.29 nm signal a = inlens photo no. = 3307 date: 23 apr 2019 time: 20:37:52 2 µm eht = 5.00 kv wd = 7.4 mm mag = 2.00 k x pixel size = 57.29 nm signal a = inlens photo no. = 3200 date: 17 apr 2019 time: 15:06:55 2 µm eht = 5.00 kv wd = 9.5 mm mag = 2.00 k x pixel size = 57.29 nm signal a = inlens photo no. = 3224 date: 17 apr 2019 time: 16:22:38 figure 2. scanning electron microscopy images of specimens exposed to distilled water (dw), 0.12% chlorhexidine digluconate (clx), and spondias mombin l. extract (de). mineral-like precipitation was observed in specimens exposed to clx (arrows), while enamel dissolution was perceived in specimens exposed to de (arrows). 7 alves et al. discussion the null hypothesis tested in this experiment that there would be no differences among the solutions concerning all properties analyzed was rejected since exposition to the spondias mombin l. extract promoted enamel color and micromorphology changes. although the sem images demonstrated possible changes in the specimens’ surface from the spondias mombin l. extract group, the surface roughness before and after immersion protocol did not indicate statistically significant changes in any of the groups. according to field et al.24 (2013), the assessment of surface roughness may not provide details on the surface texture, wear resistance and the ability to retain liquids, limiting this type of analysis. however, the analysis of surface roughness can indicate possible changes in the dental structure, especially when associated with other variables’ analysis25. it is likely that an acidic ph presented by the spondias mombin l. extract caused enamel dissolution and changes on enamel topography (figure 2de), which were not detected employing the roughness test. dantas et al.26 (2015) defined wettability as the liquid’s ability to wet a solid, exemplifying it as a drop of liquid resting on a solid surface which the liquid may or may not spread. water is a polar liquid that tends to spread over a surface with high surface energy and form a drop in areas with low energy. regarding surface wettability, the contact angle data indicate that the dental enamel has undergone significant changes when exposed to the extract of spondias mombin l. a decrease in the contact angle was observed, which suggests an increase in the free surface energy and, consequently, in wettability. according to luz et al.27 (2008), the two main factors that can affect the wetting behavior of a solid by a liquid are: topographic inhomogeneity, caused by surface roughness or porosity and chemical inhomogeneity, caused by the presence of contaminants on the solid surface. likely, the acidic ph of the spondias mombin l. extract promoted enamel dissolution, increasing porosity (figure 2) and wettability. regarding color change, the specimens submitted to the spondias mombin l. extract presented the highest color change compared to distilled water and 0.12% chlorhexidine digluconate. since this extract has a brown color, an acidic ph, and contains alcohol, it presents greater potential to cause specimens darkness23,28. comparatively, the clx group showed no clinically noticeable color changes. however, chromatic changes on the dental surface exposed to clx are related to its ability to precipitate pigments on the dental surface, whether from drinks or food1,29,30. the fact that the present study did not establish a contact of specimens with any drink or food may explain this result. therefore, before clinical use of the spondias mombin leaf extract, their dark color and acidic ph should be modified to avoid enamel damage and darkening. as this work involved bovine enamel as the first substitute from human enamel, further investigations should perform similar evaluations using human enamel. finally, it is worth mentioning that one of the limitations of in vitro studies is the reproduction of real conditions. even when it is conducted as close as possible to a clinical 8 alves et al. situation, laboratory conditions do not reproduce exact oral conditions, known for their extreme complexity. thus, further clinical trials should be designed to investigate the interaction between spondias mombin l. extract and oral tissues. conclusion the spondias mombin l. extract altered the micromorphology, promoted color change and a more wettable bovine enamel surface. acknowledgements we are grateful to the departments of materials engineering and pharmacy at the universidade federal do rio grande do norte to support the production of extract and sem analysis, respectively. references 1. zanatta fb, antoniazzi rp, rösing ck. staining and calculus formation after 0.12% chlorhexidine rinses in plaque-free and plaque covered surfaces: a randomized trial. j appl oral sci. 2010 sepoct;18(5):515-21. doi: 10.1590/s1678-77572010000500015. 2. rodrigues ja, lussi a, seemann r, neuhaus kw. prevention of crown and root caries in adults. periodontol 2000. 2011 feb;55(1):231-49. doi: 10.1111/j.1600-0757.2010.00381.x. 3. kumar s, patel s, tadakamadla j, tibdewal h, duraiswamy p, kulkarni 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[effect of argon plasma therapy on teeth exposed to sodium hypochlorite: analysis of hydrophilicity]. rfo upf. 2015 jan-apr;20(1):34-8. portuguese. doi: 10.5335/rfo.v20i1.4440. 10 alves et al. 27. luz ap, ribeiro s, pandolfelli vc. [review article: use of the wettability in the investigation of the corrosion behaviour of the refractory materials]. cerâmica. 2008 jun;54(330):174-83. portuguese. doi: 10.1590/s0366-69132008000200007. 28. pereira cal, lima rbw, andrade akm, duarte rm. [diet children’s influence in net roughness of cattle teeth enamel]. arq odontol. 2016 set;52(3);154-9. portuguese. doi: 10.7308/aodontol/2016.52.3.04. 29. friedrich re, kristen u. toxicity assessment of mouthwashes in the pollen tube growth test. anticancer res. 2003 mar-apr;23(2a):941-7. 30. pegoraro j, silvestre l, cara g, stefenon l, mozzini cb. [adverse effects of 0.12% chlorhexidine gluconate]. j oral invest. 2014;3(1):33-7. portuguese. doi: 10.18256/2238-510x/j.oralinvestigations.v3n1p33-37. 1http://dx.doi.org/10.20396/bjos.v18i0.8657327 volume 18 2019 e191601 original article 1 department of restorative dentistry, school of dentistry of ribeirão preto, university of são paulo (usp), ribeirão preto, sp, brazil. corresponding author: juliana jendiroba faraoni avenida do café, s/n°, ribeirão preto, brazil phone +55(16)3315-4016 jujfaraoni@forp.usp.br https://orcid.org/0000-0003-0945-4028 received: march 22, 2019 accepted: october 01, 2019 color stability of nanohybrid composite resins in drinks juliana jendiroba faraoni1,*, isabela barbosa quero1, lívia semedo schiavuzzo1, regina guenka palma – dibb1 aim: the objective of this study was to evaluate the effects of solutions on the color stability of nanohybrid composite resins. methods: the experimental sample consisted of 90 composite specimens (beautifil ii; z350xt; premisa), divided into three subgroups (n=10) according to the solutions (matte tea; lemon flavor isotonic drink; artificial saliva). the specimens were immersed in the solutions (5 ml/specimen) while stirring for 5 minutes, four times a day, with 1-hour intervals, repeated for 15 days. the color of the specimens was analyzed before (baseline) and after the 15th day of cycling using the cielab system. data were analyzed using the two-way analysis of variance (anova) and tukey’s test (α=5%). results: different behaviors were observed among resins. beautifil ii presented the highest color change (∆e=4.18) and less color stability, statistically different from the others (p<.05). the solutions also presented different behaviors. the lemon flavor isotonic drink (∆e=3.95) promoted the highest color change, statistically different from saliva (∆e=2.75; p<.05). the interaction between beautifil ii and isotonic drink became even more evident and significant (p<.05). conclusion: the isotonic drink was the solution that most affected the resins, and beautifil ii presented the worst color stability. keywords: composite resins. nanocomposites. color. beverages. https://orcid.org/0000-0003-0945-4028 2 faraoni et al. introduction the demand and advances in the aesthetic area have grown, primarily with an evolution of composite resins in dental practice1,2,3. alteration of color is one of the many reasons for replacement of a composite resin restoration. however, this procedure costs time and money and increases the size of the cavity4. color alteration is attributed to intrinsic discolorations due to physicochemical reactions, such as the quality of the polymer matrix of the resin or the quality of photopolymerization5,6. there are also extrinsic discolorations, which are related to biofilm accumulation and staining by adsorption or absorption of pigments, mostly present in drinks and food7,8. in addition, other properties should be considered, such as the surface texture, staining agent, exposure time to the pigment, and characteristics of the restoration material4. some studies3,8-12 demonstrated that some drinks, such as tea, red wine, coffee, juice, and soft drinks, are responsible for staining the composite resins to various degrees. besides these, sports drinks can also influence the stability of color of composite resins13. the consumption of this kind of beverage is elevated14 due to a new beauty standard that promotes a “new” modern lifestyle with a “healthy diet” and regular exercise, and the researches are not involving this category14. many modifications have been taking place regarding composite resin to produce a material that is more aesthetic with better polish and good mechanical properties. this change has been occurring in the size of particles, which produce a functional material with nanosized phases called nanocomposites15. this change in charged particles and the monomers of the matrix results in a lower polymerization shrinkage, better retention, and better aesthetics1,16 without compromising mechanical strength17. despite this, there are few studies regarding the color stability of these materials. therefore, the aim of this study was to evaluate the color stability of nanohybrid composite resins in the immersion of different commercial drinks. materials and methods experimental design the experimental sample consisted of 90 resin specimens of composite resin. the specimens were divided into nine groups/subgroups (n=10), considering three composite resins: beautifil  ii (shofu, kyoto city, kyoto prefecture, japan), z350xt (3m espe, st. paul, minnesota, usa), and premisa (kerr, orange, california, usa) and three solutions: matte tea (leão alimentos e bebidas, fazenda rio grande, paraná, brazil), lemon flavor isotonic drink (gatorade ambev., jaguariúna, são paulo, brazil; glucose, water, sucrose, sodium chloride, sodium citrate, potassium phosphate, citric acid, and flavoring), and artificial saliva. this study was conducted using a randomized complete block design. the quantitative response was the color stability (∆e, ∆l, ∆a, and ∆b). 3 faraoni et al. preparation of specimens the composite resins (table 1) were manipulated according to the manufacturer’s instructions and were inserted into cylindrical metal molds of stainless steel (4  mm diameter x 2 mm thickness/height). this insertion was performed in a single increment. table 1. composite resins tested in this study. composite resin matrix size of fillers percentage of fillers filler manufacturer beuatifil ii bis-gma, tegdma 10nm 20nm 54% (l/v) 74% (l/wt) s-prg shofu inc filtek z350xt bis-gma, udma, tegdma, pegdma, bis-ema 4–11 nm, 20 nm 63.3% (v)/78.5% weight zirconia, silica cluster (20 nm) 3m espe primesa bis-gma, bisema, tegdma 0.02 μm 84% (wt) ppf filler, point 4 filler, 0.02 μm kerr corp after the insertion, the matrices were covered with a glass slide. then, an axial load of 500  g was applied on each specimen for 1 min. this compress created a flat surface and standardized the thickness. after 1 min, the load was removed, and the material was photopolymerized using kavo poly wireless (kavo do brasil, joinville, santa catarina, brazil) through glass with visible light for 20 seconds. the intensity of the visible light was monitored by a radiometer and was maintained at around 1100 mw/cm². after preparation, the specimens were held and stored in artificial saliva in the oven at 37ºc (+/1ºc). after 24 h, the specimens were submitted to finishing and polishing phases in a polish machine (arotec, são paulo, são paulo, brazil) with water sandpaper 600 and 1200 and with 0.3and 0.05-µm alumina suspensions. by the end of these procedures, the specimens were washed with distilled water for 30 s, submerged into distilled water at the ultrasound for 5 min, dried with paper towels, and then immersed in artificial saliva for 24 h at 37ºc. baseline color analysis before the cycling, the original color of each specimen was analyzed with a spectrophotometer (color guide 45/0, pcb 6807 byk-gardner gmbh, geretsried, bavaria, germany) on a white background. this handheld portable equipment measures color and gloss attributes simultaneously. the spectro-guide spectrophotometer allows repeatable results using color guide 45/0 and a 4-mm aperture and circumferential illumination. the standard of observation simulated by spectrophotometer follows the cielab system, recommended by the commission internationale de l’éclairage (cie). this consists of two axes: a* and b*. they have right angles, representing the size of the shade or color. the third axis is the brightness: l*. it is perpendicular to the 4 faraoni et al. plane with axes a* and b*. with this system, any color can be specified by the coordinates l*, a*, and b*. we activated the spectrophotometer (30 led lamps) with 10 different colors, arranged in a circular shape, and focused the light beam at 45º with the material surface. this beam is reflected back at 0º to the apparatus, and it captures and records the values l*, a*, and b* of each sample. cycling of samples specimens of each composite resin were randomly divided into three subgroups. the control group was kept in artificial saliva and the other two experimental groups were submitted into cycling with the selected drink (matte tea or lemon flavor isotonic drink). the drinks were used in their consumption temperature, with matte tea at 40ºc and the isotonic drink at 4ºc. temperatures were measured with a digital thermometer. for 5 min, specimens were immersed in the drinks (5  ml/specimen) under agitation (orbital shaker table ct-155, cientec laboratories equipment, piracicaba, são paulo, brazil), 4 times a day, with 1-h intervals. among the cycles, the specimens were immersed in artificial saliva at 37ºc (+/1ºc). for the control group, the specimens were kept in an oven at 37ºc (+/-1ºc) changing the solution daily. these procedures were repeated for 15 d. final color analysis after the cycling period, the color was measured again. the difference between the color results was obtained by calculating ∆e* = [(∆l*)2+(∆a*)2+(∆b*)2]12. the brightness differences of ∆l, ∆a, and ∆b were also calculated by the formulas ∆l* = l*(t)-l*(0), ∆a* = a*(t)-a*(0), and ∆b* = b*(t)-b*(0), where (t) is the time and (0) is the baseline. the color changes were obtained by the values ∆e, ∆l, ∆a, and ∆b. data were analyzed based on distribution and homogeneity, showing normal (shapiro-wilks) and homogeneous (levene’s) results. the analysis of variance (anova) used two criteria (two-way anova: resin and solution) and the tukey test (p < .05) to distinguish the means. results change in brightness (∆l) for factor composite resins, similar results (p > .05) were observed for the three nanohybrid composite resins. however, for the solutions, the lemon flavor isotonic drink was the solution that most affected the specimens, making them clearer, which was a statistically significant difference from the other solutions studied (p < .05). the other two solutions presented similar results (p > .05). in the interaction, the composite premisa showed no significant difference for the solutions (p > .05; table 2). the tea solution darkened the z350 and beautifil ii, and the isotonic drink samples were lighter. 5 faraoni et al. table 2. mean and standard deviation of δl for the different resins and solutions. composite resin saliva tea isotonic drink premisa 1.58±2.40 a a -0.04±3.22 a a 0.44±1.94 b a z350 xt 1.12±3.48 a a -1.92±2.49 a b 0.27±1.27 b ab beautifil ii -0.51±2.04 a b -0.97±2.41 a b 6.25±4.78 a a *capital letter indicates statistical difference among columns. lowercase indicates statistical difference among lines. change in color (∆e) in the color analysis for the composite resin factor, beautifil ii showed the greatest change and presented a statistical difference from the other composites studied (p < .05). in turn, premisa and z350 were similar (p > .05). comparing the solutions, the lemon flavor sports drink caused changes in the composite resin, with a statistically significant difference compared with the other solutions (p < .05). the other two solutions were similar (p > .05). considering the interaction, only beautifil ii showed significant changes for the isotonic drink (p < .05; table 3). table 3. mean and standard deviation of δe for the different resins and solutions. composite resin saliva tea isotonic drink premisa 2.57±1.43 a a 3.04±1.84 a a 2.53±1.27 b a z350 xt 3.34±2.09 a a 3.32±0.93 a a 1.98±0.82 b a beautifil ii 2.32±1.05 a b 2.88±1.65 a b 7.35±3.71 a a *capital letter indicates statistical difference among columns. lowercase indicates statistical difference among lines. changes in a* and b* regarding ∆a, the three resins presented different behaviors (p < .05), and the composite resin z350 demonstrated the most variance. however, statistically, the solutions did not affect the samples (p > .05). in the interaction of the factors, only beautifil ii showed significant changes for the isotonic drink (p < .05; table 4). table 4. mean and standard deviation of δa for the different resins and solutions. composite resin saliva tea isotonic drink premisa 0.28±0.44 a a 0.05±0.54 b a 0.29±0.38 a a z350 xt 0.50±0.82 a a 1.07±0.47 a a 0.80±0.63 a a beautifil ii 0.02±0.67 a a -0.05±0.70 b ab -0.72±3.71 b b *capital letter indicates statistical difference among columns. lowercase indicates statistical difference among lines. 6 faraoni et al. in the analysis, the three composites showed similar results for ∆b (p > .05). however, the lemon flavor isotonic drink affected the specimens and demonstrated a distinct behavior from the artificial saliva and matte tea (p < .05). in the interaction, only beautifil ii showed significant changes with the lemon flavor sports drink (p < .05; table 5). table 5. mean and standard deviation of δb for the different resins and solutions. composite resin saliva tea isotonic drink premisa 0.43±0.68 a a -0.33±1.70 a a -1.38±1.54 a a z350 xt -0.22±1.51 a a -0.34±1.09 a a -1.22±0.79 a a beautifil ii -1.00±1.4 a a -0.43±2.10 a a -2.12±1.27 b b *capital letter indicates statistical difference among columns. lowercase indicates statistical difference among lines. discussion consuming sports drinks and teas has increased considerably due to habit changes for a healthier lifestyle13. however, these solutions provide erosion and staining in tooth structure and esthetic restorative materials14,18,19, and this effect can directly affect patient satisfaction with the color of the restoration20. also, at long term, the association of the consumption of these kind of beverages with toothbrushing can influence on the material’s longevity in relation to the contour and coloration3. the perception of color is related to psychological aspects and can be interpreted based on different factors according to the observer’s skills. because of these errors, devices that assist in the evaluation of color were used, and data were obtained using the cielab system21,22. in several studies23,24, the color change is deemed acceptable for values up to ∆e = 3.3, determining a threshold for visual perception. in the present study, the three resins showed color alteration after immersion into the tested solutions, including artificial saliva (control). this might have occurred because of the period to which the samples were immersed, since the artificial saliva significantly influences the color stability of restorative materials because of its components and water sorption by the resin matrix3. the color alteration can be attributed to intrinsic discolorations due to physicochemical reactions and to the quality of the polymer matrix of the resin or the quality of photopolymerization6. it can also be attributed to the extrinsic discolorations, which are related to biofilm accumulation and staining by adsorption or absorption of pigments that are present in food and drinks20. different drinks, such as tea, soda, beer, coffee, and orange juice, can affect the physical and chemical structures of restorative materials17. in addition, the oral environment associated with the characteristics of the beverage can influence the discoloration of restorative materials and affect the surface integrity25. results shows that, among the drinks, isotonic drinks presented the most distinguished results affecting the brightness for axis a* and axis b* of the resins, leading to a significant color change in the composite beautifil ii. of the solutions, the lemon 7 faraoni et al. flavor isotonic drink most altered the stability of the tested composites, that can be explained because of the acid ph of these beverage19. although tea is considered one of the most decolorizing beverages26, its consumption is still very high in the population. in our study, it did not cause a major alteration of color in the composite resins tested, presenting similar behavior to saliva. in this study, artificial saliva did not provoke major color alteration either20. in the analysis of ∆e values, premisa presented better stability to immersion in various solutions, and any solution promoted color alteration up to 3.3, since ∆e values up to 3.3 is considered clinically accepted3. filtek z350 xt and beautifil ii showed a higher color change when compared to premisa. beautifil ii showed higher values (∆e = 7.35) when immersed into the sports drink (isotonic drink), indicating clinically visible change in color. one hypothesis for these results is that the acid solution may have degraded resin surface, interfering with the light reflection. this fact can be observed by the brightness in the analysis of the ∆l27; the values showed that the premisa resin was more stable and that beautifil ii had also suffered major changes in both drinks. the different results observed among the composites is due to its composition, which differ from resin matrix composition, particle size and conversion after polymerization3. the resin matrix, which is responsible for the stability of color, can influence a higher staining25,28. depending on the composition, it can absorb more or less water (and other substances), which leads to discoloration19. another possibility is the presence of triethylene glycol dimethacrylate (tegdma), which can increase the hydrophilicity compared to urethane dimethacrylate (udma), and is more resistant to staining than bisphenol a glycidyl ether dimethacrylate (bis-gma)29. thus, the color stability of premisa resin can be justified by its complex composition of the resin matrix: bis-gma, bisema, and tegdma. therefore, the unstable behavior of the beautifil ii can also be justified by its simplicity because of the presence of two resin monomers (bis-gma and tegdma). our results corroborates with taşkinsel et al.19 (2014), which demonstrated a considerable color alteration of nano and micro hybrids composites resins when frequently immersed in sports drinks. in agreement with mara da silva et al.3 (2019), in this present study beautifil ii had the highest value of ∆e as well as the highest variation compared to filtek z350 xt. it was demonstrated that the consumption of beverages able to stain associated with brushing challenges leads to a decrease in microhardness, which was severer in beautifil ii; therefore, the surface treatments reduced the properties of filtek z350 xt and beautifil ii. in another study30 was observed that beautifil ii had the highest values for surface roughness after some superficial treatments. this fact can explain the considerable color alteration in the present study, since the surface roughness can influence the esthetic and biological outcomes of the composites30. this study demonstrated the color alteration of composite resins induced by different solutions. this data complements the existing studies in the literature3,8,10,11,30. providing subsidies to conclude the different compositions of the resinous matrix can promote different results with natural or synthetic pigmentation drinks. additionally, the drink’s acidity can significantly alter the stability of the color of the composites, generating aesthetic disadvantages and disturbing clinical practice25. thus, professionals 8 faraoni et al. should focus more attention to the different characteristics of each patient, such as their habits and customs, and then select the best restorative material. more in vitro, ex vivo, and in vivo studies are needed for a greater understanding of the behavior of nanohybrid composite resins in the oral environment when in contact with solutions and commercially consumed drinks. considering the limitations of this study, it can be concluded that premisa resin showed less change, while beautifil ii was more susceptible to staining. among the beverages, the lemon flavor isotonic drink promoted major alterations. acknowledgment this study was supported by cnpq through grant 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light-induced yellowing of dental restorative resins. j prosthet dent. 1985 oct;54(4):483-7. 25. batra r, katarina p, kapoor s. effect of salivary ph on color stability of different flowable composites – a prospective in-vitro study. j clin diagn res. 2016 oct;10(10):zc43-6. 26. abed kahnamouei m, gholizadeh s, rikhtegaran s, daneshpooy m, kimyai s, alizadeh oskoee p, et al. effect of preheat repetition on color stability of methacrylateand silorane-based composite resins. j dent res dent clin dent prospects. 2017 fall;11(4):222-228. doi: 10.15171/joddd.2017.039. 27. kotanidis a, kontonasaki e, koidis p. color alterations of a pmma resin for fixed interim prostheses reinforced with silica nanoparticles. j adv prosthodont. 2019 aug;11(4):193-201. doi: 10.4047/jap.2019.11.4.193. 28. dietschi d, campanile g, holz j, meyer jm. comparison of the color stability of ten new-generation composites: an in vitro study. dent mater. 1994 nov;10(6):353-62. 10 faraoni et al. 29. bagheri r, burrow mf, tyas m. influence of food-simulating solutions and surface finish on susceptibility to staining of aesthetic restorative materials. j dent. 2005 may;33(5):389-98. 30. ruivo ma, pacheco rr, sebold m, giannini m. surface roughness and filler particles characterization of resin-based composites. microsc res tech. 2019 oct;82(10):1756-1767. doi: 10.1002/jemt.23342. 1http://dx.doi.org/10.20396/bjos.v20i00.8661168 volume 20 2021 e211168 original article 1 department of morphology, genetics, orthodontic and pediatric dentistry, araraquara school of dentistry, são paulo state university (unesp), araraquara, sp, brazil. 2 ces university, faculty of dentistry, medelin, colômbia. corresponding author: vinicius krieger costa nogueira department of morphology, genetics, orthodontic and pediatric dentistry, araraquara school of dentistry, (unesp) rua humaitá, 1680, 14801-903 araraquara, sp, brazil. fone/fax: +55 (16) 3301-6334. e-mail: vinicius.nogueira@unesp.br editor: dr altair a. del bel cury received: september 8, 2020 accepted: march 10, 2021 impact of the undergraduate clinical teaching-learning process on caries detection and treatment decision-making silas alves costa1 , vinicius krieger costa nogueira1,* , diego girotto bussanelli1 , manuel restrepo2 , alfonso escobar2 , rita de cássia loiola cordeiro1 aim: the aim of this study was to evaluate caries diagnosis and treatment decisions made by undergraduate dental students based on icdas or nyvad criteria. methods: twelve students analyzed 90 digital photographs of permanent teeth at different clinical stages of carious lesion development and chose among different treatments in three different assessments: when there was no knowledge of the criteria (described as “no knowledge (n)”; when there was theoretical knowledge of the criteria (described as “theoretical knowledge (t)” and when there was theoretical knowledge, clinical experience about dental caries and the criteria (described as “theoretical and clinical knowledge (tc)”. for “t” and “tc” the students were randomized into two experimental groups – icdas or nyvad (experimental units: 6 students/group). the reference standard was established cooperatively by two experienced researchers. criteria performance was evaluated by sensitivity, specificity, auc, and kappa statistics. treatment decision was described in percentage by contingency tables and spearman’s correlation with the reference standard. results: the first assessment demonstrated a high percentage of operative treatment even for initial enamel lesions based on icdas criteria and treatment was proposed for both active and inactive lesions, according to the nyvad criteria. in the second assessment, the students continued recommending treatments for initial or inactive lesions, but less frequently. in the third assessment, treatment decisions presented greater cohesion in relation to the assigned classification criterion. the criteria presented no differences between them in terms of diagnostic in the third assessment. conclusion: clinical experience may improve caries detection and treatment decisions with the use of icdas and nyvad criteria. keywords: dental caries. education. diagnosis. https://orcid.org/0000-0001-7982-5301 https://orcid.org/0000-0002-0371-6421 https://orcid.org/0000-0001-9078-7385 https://orcid.org/0000-0003-2621-2231 https://orcid.org/0000-0001-5779-3484 https://orcid.org/0000-0002-5644-2807 2 costa et al. introduction decision-making for the establishment of appropriate clinical treatment is a complex process that presents as a task of solving a particular problem1. most dentists examine their patients with the aim of determining a treatment plan without giving careful attention to establishing a correct diagnosis. thus, important information may be lost and some treatment options may be underestimated2,3. many factors may influence undergraduate students’ clinical management decisions during their education, such as theoretical knowledge and clinical training. by analyzing the curriculum content of european dental schools, bottenberg et al.4 found that the teaching content of cariology was unequal among the universities and was fragmented into many other subjects. nonetheless, knowledge of cariology is fundamental for the construction of critical thinking, both during undergraduate studies and in one’s career5,6. in addition, ferreira-nóbilo et al.7 observed that although most brazilian schools contemplating the theme in their disciplines, the little emphasis on specific clinical practice may be contributing to the inequality distribution of the disease in the country. visual inspection is a method commonly taught at dental schools for caries detection and allows having a good performance even if it is not associated with other methods8,9. in order to facilitate the detection of carious lesions, reduce subjective decision-making, and improve the standardization of studies and epidemiological surveys, different indices with well-defined criteria have been developed10,11. the international caries detection and assessment system (icdas) classifies lesions according to signs such as spots, pigmentations, cavities, and the association among these signs can suggest the extent of the carious lesion, with application of scores (1-6) for staging of the lesions10,12. icdas has shown good validity and reproducibility in determining the presence or absence of caries in both primary and permanent teeth. on the other hand, the criteria developed by nyvad are used as a scoring system to identify the physical traits for caries activity based on visual-tactile aspects, detecting and distinguishing clinical signs, such as translucency or opacity, surface texture, and plaque stagnation area13. theoretical knowledge and expertise seem to have some importance for the application of criteria for the detection of signs that indicate the presence or absence of carious lesions and, consequently, for the decisions about the treatment to be offered to the patient14-16. according to a recent systematic review8, undergraduate students demonstrate good performance in caries detection and staging using visual inspection, but their performance in the assessment of caries activity still needs improvement. bussaneli et al.16 pointed out poor clinical experience as a possible influence on the choices of undergraduate students for more invasive dental caries treatments. the few studies on this subject1,16-19 assessed a single moment of the students’ experience during their undergraduate program, mostly in their senior year. despite the considerable number of studies on the performance of visual criteria, the relationship between students’ experience, visual criteria, and treatment deci3 costa et al. sions is still unclear. therefore, the aim of this study was to evaluate dental caries diagnosis and treatment decisions made by undergraduate students at three different assessments: when there is no knowledge of the criteria (described as “no knowledge (n)”; when there is theoretical knowledge of the criteria (described as “theoretical knowledge (t)” and when there is theoretical knowledge, clinical experience about dental caries and the criteria (described as “theoretical and clinical knowledge (tc)”. materials and methods this study is part of a macro project, and its data have been partially published by nogueira et al.14 the data obtained and analyzed here are unique; however, the methodology is similar to that described in that other article. study participants after approval of the study protocol by the research ethics committee of the araraquara dental school (foar), araraquara, brazil (process 701/14), 75 undergraduate students in the second year/fourth semester were directly invited to participate. twelve students (response rate: 16%) agreed to participate. this means that the sample was selected by convenience sampling. they were all in the same academic semester, with the same level of knowledge and experience in all phases of this study. in the second year, students take basic courses in health sciences. this means they are taught about dental caries development; however, at that stage, they have not learned about the clinical specificities of carious lesions, have had no previous experience with icdas or nyvad criteria, and have not yet had clinical experience. we emphasize that this dentistry course lasts five years and does not have a specific discipline of cariology. the content on this topic is covered in the disciplines of histology, restorative dentistry and pediatric dentistry, without minimum workload. study design ninety digital photographs of permanent teeth at different clinical stages of carious lesion development were used. the photographs were taken during the selection of patients by the researchers in charge of establishing the reference standard. the photographed tooth should allow direct visualization of the carious lesion, regardless of whether the lesion was located on the proximal, occlusal, or buccolingual surface. the photographs were assessed at the informatics teaching laboratory of foar using a 21.5-inch led screen with 1920x1080 pixel resolution and the site to be analyzed was indicated precisely by an arrow. the design of the methodology described below is illustrated in our study flow-chart (fig. 1). initially, the undergraduate students analyzed the photographs by looking at the carious lesion site and assigning a treatment decision score according to their own theoretical knowledge about dental caries, without any training or calibration about the criteria. at this time, the students have not been evaluated about the diagnosis using icdas and nyvad criteria because this assignment is only possible after some knowledge about the codifications, for this reason this assessment 4 costa et al. was described as “no knowledge (n)”. for treatment decisions, students should assign a score among the options: (0) no treatment; (1) non-operative treatment: oral hygiene instruction and application of 5% fluoride varnish; (2) operative treatment: sealant or restoration with composite resin or restoration with conventional glass ionomer cement. after this first assessment, the students were then randomized through an online randomization tool (http://www.random.org) into two experimental groups – icdas or nyvad (experimental units: 6 students/group) criteria – and received the original article that described each criterion8,9. after reading the article, each group attended an expository lesson about the etiology, pathogenesis, and diagnosis of dental caries based on the assigned criteria (either icdas or nyvad) and treatment decisions. figure 1. study flowchart + no knowledge visual examination assigned the treatment decision theoretical knowledge interval: one week icdas criteria (n=6) nyvad’s criteria (n=6) original article reading according to the group theoretical class same 90 photographs visual examination (nyvad) + treatment decision theoretical and clinical knowledge interval: two years 2nd year / 4th semester of the course 4th year / 8th semester of the course 12 undergraduate students 90 photographs randomization visual examination (icdas) + treatment decision original article reading according to the group same 90 photographs visual examination (icdas) + treatment decision visual examination (nyvad) + treatment decision 5 costa et al. in the following week, the students performed the second assessment of the same photographs. the difference was that, before choosing the treatment score, students classified and scored the selected sites according to the icdas or nyvad criteria, depending on the group to which they belonged (fig. 2). the carious lesion classification criteria were provided on a printout. here, we reinforce that treatment decisions nyvad 0: sound normal enamel translucency and texture (slight staining allowed in otherwise sound fissure). 1: active caries (intact surface) surface of enamel is whitish/yellowish opaque with loss of luster; feels rough when the tip of the probe is moved gently across the surface; generally covered with plaque. no clinically detectable loss of substance. intact fissure morphology; lesion extending along the walls of the fissure. 2: active caries (surface discontinuity) same criteria as score 1. localized surface defect (microcavity) in enamel only. no undermined enamel or softened floor detectable with the explorer. 3: active caries (cavity) enamel/dentine cavity easily visible with the naked eye; surface of the cavity feels soft or leathery on gentle probing. there may or may not be pulpal involvement. 4: inactive caries (intact surface) surface of enamel is whitish, brownish or black. enamel may be shiny and feels hard and smooth when the tip of the probe is moved gently across the surface. no clinically detectable loss of substance. intact fissure morphology; lesion extending along the walls of the fissure. 5: inactive caries (surface discontinuity) same criteria as score 4. localized surface defect (microcavity) in enamel only. no undermined enamel or softened floor detectable with the explorer. 6: inactive caries (cavity) enamel/dentine cavity easily visible with the naked eye; surface of the cavity feels shiny and feels hard on gentle probing. no pulpal involvement. icdas 0: no or slight change in enamel translucency after prolonged air drying (5 s). 1: first visual change in enamel (seen only after prolonged air drying or restricted to within the confines of a pit or fissure). 2: distinct visual changes in enamel. 3: localized enamel breakdown in opaque or discolored enamel (without visual signs of dentinal involvement). 4: underlying dark shadow from dentine. 5: distinct cavity with visible dentine (involving less than half of the surface). 6: extensive distinct cavity with visible dentine (involving more than half of the surface). figure 2. criteria used in the visual assessment. 6 costa et al. were followed exclusively based on their theoretical knowledge, however, taken after the specific criterion training. for this reason, this assessment was described as “theoretical knowledge (t)”. the third assessment was carried out two years after the first one using the same photographs. the undergraduate students were now enrolled in the fourth year/eighth semester of the course. the original articles were handed out again and a new analysis was performed one week after the students read them. in this assessment, the students scored the selected sites according to the criterion belonged and also with a treatment score. at this moment, the treatment decisions were based on their theoretical knowledge enhanced with clinical experience about dental caries. for this reason, this assessment was described as “theoretical and clinical knowledge (tc)”. this methodology aimed to evaluate the different factors related to this study: the purely theoretical knowledge about dental caries; the theoretical knowledge about dental caries after specific training for each criterion; and knowledge about dental caries when theoretical and clinical practice are combined. for this, we analyze these factors through the performance of the students based on their treatment decisions supported or not by the criteria. reference standard the reference standard used for encoding criteria and for treatment decisions was established by two experienced researchers. initially, these researchers cooperatively analyzed 50 photographs and discussed the classification of the lesions according to the different criteria and possible treatments. after one week, they independently performed a new assessment and the reproducibility analyses resulted in good interrater reliability (kappa icdas: 0.84; kappa nyvad: 0.82; and kappa treatment decisions: 0.87). after the calibration process, the same researchers were responsible for selecting the teeth that were really part of the present study. this occurred in the clinical assessments during the selection of patients in the department of clinical pediatrics of foar during the obligatory inclusion check for regular treatment. the included teeth were classified according to the icdas and nyvad criteria, to the need for treatment and photographed. treatment decisions were chosen according to the depth/extent of the lesion associated with its activity. all different caries scores were included, but the ratio it was not necessary the same. statistical analysis medcalc software (mariakerke – be. version 9.3 for windows) was used for the statistical analysis. as response variables, it was adopted that both treatment and criteria scores represent the dependent variables, while the different assessments represent the independent variables. sensitivity, specificity, and area under roc curve (auc) determine different cutoff points according to the presence of cavitation (icdas) and in relation to disease activity (nyvad). cutoff points were established according to icdas for sound teeth and enamel-dentin lesions (considering icdas scores from 4 to 6 as disease) and accord7 costa et al. ing to nyvad criteria for sound teeth, inactive lesions, and active lesions (considering nyvad scores from 1 to 3 as disease). the groups were intra and inter-compared by means of a mcnemar’s test and the significance level was set at p < 0.05. kappa statistics (cohen’s kappa) were used to evaluate the diagnostic criteria performance in the second and third assessments of each group compared to the gold standard, represented by an interrater. for treatment decisions, the percentage distribution within icdas and nyvad score criteria was described in contingency tables and the correlation with the reference standard was determined using spearman’s correlation coefficient. results according to the reference standard established in the clinical evaluations, the sample consisted of photographs of 21 (23%) sound teeth, 30 (34%) teeth with enamel lesions, and 39 (43%) teeth with dentin lesions. considering the activity status, 50 (73%) were scored as active lesions and 19 (27%) as inactive lesions. the table 1 presents the results for sensitivity, specificity, area under the roc curve, and interrater reliability regarding the icdas and nyvad criteria used by students in two moments: when the students were just with theoretical knowledge about the criterion; and when they were with theoretical knowledge and clinical experience, after two years. these analyses were performed in order to clarify whether the students were able to base their treatment decisions on the different signs of carious lesions. the icdas criteria showed significant difference in sensitivity, specificity and auc when the students were with theoretical knowledge compared to nyvad criteria (mcnemar’s test, p < 0.05). however, after the students acquired theoretical knowledge and clinical experience, there was no difference in the performance of the two criteria. in fact, nyvad criteria presented higher specificity and auc values than did icdas criteria, but this difference was not significant. table 1. sensitivity, specificity, area under the roc curve (auc), and interrater reliability shown by dental students. threshold* sensitivity specificity auc interrater t tc t tc t tc t tc icdas 0.82 a,a (0.79 – 0.84) 0.94a,a (0.89 – 0.99) 0.79a,a (0.74 – 0.83) 0.93a,a (0.88 – 0.98) 0.80a,a (0.75 – 0.84) 0.85a,a (0.79 – 0.89) 0.65 (0.57 – 0.74) 0.82 (0.78 – 0.93) nyvad 0.50 a,b (0.42 – 0.61) 0.85b,a (0.82 – 0.87) 0.65a,b (0.57 – 0.77) 0.97b,a (0.95 – 0.99) 0.58a,b (0.52 – 0.60) 0.91b,a (0.90 – 0.94) 0.41 (0.34 – 0.47) 0.81 (0.77 – 0.86) assessment: n – no knowledge about dental caries; t theoretical knowledge; and tc theoretical and clinical knowledge, after two years. icdas: sound and enamel lesions = 0-3, dentin lesion = 4-6. nyvad: sound and inactive lesions = 0, 4-6, active lesions = 1-3. a,b difference between groups for the same criteria; a,b difference between groups for different criteria (mcnemar’s test, p < 0.05). regarding treatment decisions, table 2 presents the percentages for the different treatments suggested for each criterion score and also the correlation between students and the reference standard. the first assessment performed by the students 8 costa et al. when they were in the second year of dental school, without any training or calibration about dental caries or support for criteria-based evaluations, showed the lowest correlation with the reference standard for both icdas (rs = 0.43) and nyvad (rs = 0.40). for icdas criteria, operative treatment of initial lesions exhibited a high percentage (score 1 = 73%; score 2 = 49%; and score 3 = 76%), whereas non-operative treatments showed a lower percentage (score 1 = 4%; score 2 = 44%; and score 3 = 11%). using nyvad criteria, the students recommended non-operative treatments for inactive lesions (score 4 = 37%; score 5 = 43%) and non-treatment of active lesions (score 1 = 47%; score 2 = 55%). the second assessment, the students were just with theoretical knowledge about the criterion. when they were introduced to the criteria and went through the training process, the correlation with the reference standard showed a considerable leap of improvement both for icdas (rs = 0.75) and for nyvad (rs = 0.64). for icdas criteria, the second assessment revealed operative treatments were indicated less frequently for initial lesions (score 1 = 0; score 2 = 3%; and score 3 = 57%) and showed non-operative treatments were highly recommended for the same lesions (score 1 = 64%; score 2 = 73%; and score 3 = 43%). nonetheless, despite improved consensus after the introduction of nyvad criteria, preventive treatments continued to be recommended for inactive lesions (score 4 = 61%; score 5 = 73%). the third assessment was performed after two years, when the students were with theoretical knowledge and clinical experience about criteria and dental caries. this experience, combined with the repeated training process, might explain the good correlation values obtained by both criteria (icdas = 0.89; nyvad = 0.87). in addition, treatment decisions also presented greater cohesion in relation to the assigned classification criterion. for example, most of the students in the icdas group recommended preventive treatment for scores 1 (80%) and 2 (100%) and none of the students in the nyvad group assigned preventive treatments for any lesion classified as inactive. table 2. percentages of treatment decisions according to icdas and nyvad criteria used by dental students in three assessments. criterion assessment* td** criteria score rs*** 0 1 2 3 4 5 6 icdas n 0 99 23 7 13 9 4 0.431 1 4 44 11 11 2 73 49 76 80 96 100 t 0 93 36 24 0.751 7 64 73 43 11 1 2 3 57 89 99 100 tc 0 100 16 6 0.891 80 100 22 29 2 2 4 72 71 98 100 continue 9 costa et al. continuation nyvad n 0 100 47 55 2 42 8 2 0.401 31 37 4 37 43 13 2 22 8 94 21 49 85 t 0 100 25 31 1 5 0.641 70 39 17 61 73 25 2 5 61 83 8 26 70 tc 0 100 8 80 45 56 0.871 48 48 18 2 52 44 82 20 55 44 * assessment: n – no knowledge about dental caries; t theoretical knowledge; and tc theoretical and clinical knowledge, after two years. ** td (treatment decision) 0: no treatment; 1: non-operative treatment: oral hygiene instruction and application of 5% fluoride varnish; 2: operative treatment: sealant or restoration with composite resin or restoration with conventional glass ionomer cement. *** spearman’s correlation for treatment decisions proposed by students vs. standard treatment decisions. discussion in an attempt to investigate dental caries diagnosis and treatment decisions made by undergraduate students, this study used a longitudinal methodology to cover three different moments that could influence our results. such influence may be related to the students’ experience measured by caries diagnosis and treatment decision using icdas and nyvad’s system. photographs were used to depict clinical situations20,21 that allowed reproducing the same lesions in the two-year period that involved this study, which would not have been possible in a clinical study. the use of extracted teeth is more common in studies that evaluate visual diagnostic criteria performance, enabling the study of reproducibility and avoiding patient exposure to multiple examinations14,20,21. however, this study protocol is not ideal for the evaluation of diagnostic criteria involving lesion activity signs, since extracted teeth do not allow visualization of indicators such as plaque stagnation area, roughness, quality of the marginal gingiva and lesions, translucency, or opacity. moreover, the pre-specified surface to be evaluated also does not represent the clinical setting, where patient should be considered individually and the decisions should not be based specifically on a tooth22. the increase in the correlation coefficient between undergraduate students and the gold standard regarding criteria-based treatment decisions suggests that the knowledge obtained during undergraduate years may improve the caries decision-making process, as suggested by some authors4,5,14. however, foley23 did not observe improvement in the diagnosis of caries among students in the 2nd, 3rd, and 4th undergraduate years when criteria were used as an auxiliary tool. the differences in curricular components among undergraduate courses at different universities may affect the clinical approach of professionals in training4, and we believe these curricular disparities might explain the differences observed between our results and those obtained by the authors mentioned above. 10 costa et al. the visual examination commonly used for carious lesions detection in clinical practice has resulted in a lack of consensus on the proposed treatments because of subjective interpretation by the examiner5,24. regarding the first evaluation based on icdas criteria, operative treatments were highly recommended for initial lesions, defined as “first visual changes in enamel” (score 1), “distinct visual changes in enamel” (score 2), and “localized enamel breakdown in opaque or discolored enamel” (score 3), indicating that the proposed treatments were not compatible with the visual diagnosis, which could be due to lack of knowledge about the pathogenesis and management of dental caries. bussaneli et al.16 also observed this more invasive pattern for initial lesions when the examiner had limited clinical experience; however, those studies evaluated treatment decisions when radiographic examination supported the visual examination. in our study, after the introduction of icdas criteria to the students and the training process, treatment decisions were less invasive for this same type of carious lesions. according to the literature, visual-tactile diagnostic systems can minimize subjective interpretation and can help the understanding and recognition of different caries signs by dental students14. the correct recommendation of preventive and minimally invasive treatments23 may be better understood when considering the activity status of carious lesions because while active lesions need preventive measures for their inactivation, preventive treatment is dispensable13 for inactive lesions. regarding the first assessment made by undergraduate students using nyvad criteria, inactive lesions, presenting spots to large cavities (scores 4-6), received a high percentage of preventive treatments, and active lesions, especially with “intact surface” (score 1) and lesions with “surface discontinuity” (score 2) received a high percentage of non-treatment decisions. after the introduction of nyvad criteria and the training process, the recommendation of these treatments showed an inconsistent pattern. when evaluating criteria for the activity of carious lesions, parviainen et al.18 and gimenez et al.11 showed that undergraduate students were able to classify the signs of lesion activity when they had clinical/theoretical experience in cariology. we also observed a better application of nyvad criteria by undergraduate students two years after the first assessment, when they had already had some clinical experience. after some clinical experience, treatment decisions showed correct indications of preventive treatments for lesions classified as active and of non-treatment for lesions classified as inactive. for both icdas and nyvad criteria, most lesions with dentin involvement received indications for operative treatment, whereas those classified as sound lesions received indication of treatment in both assessments. the correct identification of sound surfaces is extremely important to avoid false-positive results that may lead to unnecessary interventions25. the literature considers the indication of operative treatment as appropriate for dentin lesions, mainly because the procedure should be limited to lesions with signs of activity and proven demineralization on the tooth surface26. among the limitations of this study, convenience sampling might have limited the extrapolation of our results. a more or less interventional approach may affect treatment decisions established by the reference standard. in this way, we believe discussing the conciseness of the treatments in relation to the proposed classification and the improvement of knowledge throughout the course would be more important than 11 costa et al. the correlation between the groups and the reference standard. we also understand that visualization of lesion activity signs in photographs can be difficult and that the lack of tactile perception, with probing of the classified sites. besides that, the fact that the criteria for the diagnosis of caries (icdas and nyvad’s) were used by different examiners is also a limitation of our study. we suggest that future studies assess other teaching-learning methods/tools for caries detection as expository lectures might not have been very significant for dental education. overall, the educational system does not focus on evaluating knowledge to determine the quality of student learning27,28. the learning score is a complex process because it encompasses factors that may not be measured in specific assessments; hence, the learning curve and academic experiences, either theoretical or practical, are just some aspects to be considered28. in conclusion, our results showed that clinical experience could improve the detection of carious lesion and treatment decisions, regardless of the criteria used (icdas or nyvad). these results are important to guide learning strategies in cariology, providing theoretical training and emphasizing the importance of clinical experience. conflicts of interest: none. acknowledgments this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior – brazil (capes) – finance code 001. references 1. maupomé g, schrader s, mannan s, garetto l, eggertsson h. diagnostic thinking and information used in clinical decision-making: a qualitative study of expert and student dental clinicians. bmc oral health. 2010 may 13;10:11. doi: 10.1186/1472-6831-10-11. 2. braga mm, martignon s, ekstrand kr, ricketts dnj, imparato jcp, mendes fm. parameters associated with active caries lesions assessed by two different visual scoring systems on occlusal surfaces of primary molars-a multilevel approach. community dent oral epidemiol. 2010 dec;38(6):549-58. doi: 10.1111/j.1600-0528.2010.00567.x. 3. thammasitboon s, cutrer wb. diagnostic decision-making and strategies to improve diagnosis. curr probl pediatr adolesc health care. 2013 oct;43(9):232-41. doi: 10.1016/j.cppeds.2013.07.003. 4. bottenberg p, ricketts dnj, van loveren c, rahiotis c, schulte ag. decision-making and preventive non-surgical therapy in the context of a european core curriculum in cariology. eur j dent educ. 2011 nov;15 suppl 1:32-9. doi: 10.1111/j.1600-0579.2011.00712.x. 5. zandona agf, al-shiha s, eggertsson h, eckert g. student versus faculty performance using a new visual criteria for the detection of caries on occlusal surfaces: an in vitro examination with histological validation. oper dent. 2009;34(5):598-604. doi: 10.2341/08-082-l. 6. el-damanhoury hm, fakhruddin ks, awad ma. effectiveness of teaching international caries detection and assessment system ii and its e-learning program to freshman dental students on occlusal caries detection. eur j dent. 2014 oct;8(4):493-7. doi: 10.4103/1305-7456.143631. 12 costa et al. 7. ferreira-nóbilo np, de sousa m da lr, cury ja. cariology in curriculum of brazilian dental schools. braz dent j. 2014;25(4):265-70. doi: 10.1590/0103-6440201300149. 8. turchiello rz, pedrotti d, braga mm, rocha ro, rodrigues ja, lenzi tl. do undergraduate dental students perform well detecting and staging caries and assessing activity by visual examination? a systematic review and meta-analysis. int j paediatr dent. 2019 may;29(3):281-93. doi: 10.1111/ipd.12463. 9. zain e, chew hp. update on clinical detection methods for noncavitated fissure caries. world j dent. 2020 feb;11(1):81-8. doi: 10.5005/jp-journals-10015-1687 10. ai i, sohn w, tellez m, amaya a, sen a, hasson h, et al. the international caries detection and assessment system (icdas): an integrated system for measuring dental caries. comunity dent oral epidemiol. 2007 jun;35(3):170-8. doi: 10.1111/j.1600-0528.2007.00347.x. 11. gimenez t, piovesan c, braga mm, raggio dp, deery c, ricketts dn, et al. visual inspection for caries detection: a systematic review and meta-analysis. j dent res. 2015 jul;94(7):895-904. doi: 10.1177/0022034515586763. 12. diniz mb, campos ph, wilde s, cordeiro rcl, zandona agf. performance of light-emitting diode device in detecting occlusal caries in the primary molars. lasers med sci. 2019 aug;34(6):1235-41. doi: 10.1007/s10103-019-02717-4. 13. nyvad b, machiulskiene v, baelum v. reliability of a new caries diagnostic system differentiating between active and inactive caries lesions. caries res. 1999 jul-aug;33(4):252-60. doi: 10.1159/000016526. 14. nogueira vkc, bussaneli dg, tagliaferro eps, spin-neto r, escobar a, cordeiro rdcl. examiner’s experience and the outcome interpretation of icdas and nyvad’s system–a prospective in vitro study. acta odontol scand. 2017 apr;75(3):186-90. doi: 10.1080/00016357.2016.1277260. 15. nogueira vkc, bussaneli dg, restrepo mr, spin-neto r, dos santos-pinto lam, boldieri t, et al. caries treatment decisions among undergraduate and postgraduate students supported by visual detection systems. int j paediatr dent. 2018 jan;28(1):92-101. doi: 10.1111/ipd.12312. 16. bussaneli dg, boldieri t, diniz mb, lima rivera lm, santos-pinto l, cordeiro rcl. influence of professional experience on detection and treatment decision of occlusal caries lesions in primary teeth. int j paediatr dent. 2015 nov;25(6):418-27. doi: 10.1111/ipd.12148. 17. luz pb, stringhini ch, otto br, port alf, zaleski v, oliveira rs, et al. performance of undergraduate dental students on icdas clinical caries detection after different learning strategies. eur j dent educ. 2015 nov;19(4):235-41. doi: 10.1111/eje.12131. 18. parviainen h, vähänikkilä h, laitala ml, tjäderhane l, anttonen v. evaluating performance of dental caries detection methods among third-year dental students. bmc oral health. 2013 dec 6;13:70. doi: 10.1186/1472-6831-13-70. 19. silva pf, forte fds, chaves ambp, farias iap, castro ks. reproducibility of caries diagnosis in permanent teeth according to who, icdas-ii and nyvad criteria. brazilian j oral sci. 2012;11(1):25-9. doi: 10.20396/bjos.v11i1.8641537. 20. heaven tj, gordan vv, litaker ms, fellows jl, brad rindal d, firestone ar, et al. agreement among dentists’ restorative treatment planning thresholds for primary occlusal caries, primary proximal caries, and existing restorations: findings from the national dental practice-based research network. j dent. 2013 aug;41(8):718-25. doi: 10.1016/j.jdent.2013.05.014. 21. carvalho jc, qvist v, aimée nr, mestrinho hd, bakhshandeh a. diagnosis, risk assessment, and treatment decisions for occlusal caries: a survey from the danish public dental health service. caries res. 2018;52(1-2):58-70. doi: 10.1159/000484987. 13 costa et al. 22. uhlen mm, valen h, karlsen ls, skaare ab, bletsa a, ansteinsson v, et al. treatment decisions regarding caries and dental developmental defects in children a questionnaire-based study among norwegian dentists. bmc oral health. 2019 may;19(1):80. doi: 10.1186/s12903-019-0744-2. 23. foley ji. dental students consistency in applying the icdas system within paediatric dentistry. eur arch paediatr dent. 2012 dec;13(6):319-22. doi: 10.1007/bf03320834. 24. gordan v v., bader jd, garvan cw, richman js, qvist v, fellows jl, et al. restorative treatment thresholds for occlusal primary caries among dentists in the dental practice-based research network. j am dent assoc [internet]. 2010 feb;141(2):171-84. doi: 10.14219/jada.archive.2010.0136. 25. baelum v, hintze h, wenzel a, danielsen b, nyvad b. implications of caries diagnostic strategies for clinical management decisions. community dent oral epidemiol. 2012 jun;40(3):257-66. doi: 10.1111/j.1600-0528.2011.00655.x. 26. de carvalho p, bönecker m, tello g, abanto j, oliveira lb, braga mm. inclusion of initial caries lesions in a population-based sample of brazilian preschool children: impact on estimates and treatment needs. plos one. 2020 jun;15(6):e0234122. doi: 10.1371/journal.pone.0234122. 27. hoffmann jml. [metering assessment a practice in construction from preschool to university]. porto alegre: mediação; 2018. 192 p. portuguese. 28. luckesi cc. [assessment of learning and ethics]. abc educ. 2006;7(54):20-1. portuguese. 1http://dx.doi.org/10.20396/bjos.v19i0.8659398 volume 19 2020 e209398 original article 1 department of prosthodontics, dental school, meridional faculty – imed, passo fundo, rio grande do sul, brazil. 2 department of dentistry, state university of ponta grossa, ponta grossa, paraná, brazil. corresponding author: lara dotto 304, senador pinheiro street, passo fundo, brazil graduate program in dentistry – meriodional faculty/imed, passo fundo, brazil laradotto@hotmail.com received: april 30, 2020 accepted: august 26, 2020 patients’ perception about the outcomes of prosthetic treatment in southern brazil a cross sectional study giseli sauer benetti1, lara dotto1,* , angélica maroli1 , fabíola barbon1, alfonso sanchez-ayala2 , atais bacchi1 aim: this study assessed the patients’ perception of the outcomes of prosthetic treatment in a university of southern brazil. methods: patients seeking for prosthetic treatment were invited to answer a questionnaire with 41 items about the potential risks, benefits, and consequences of no treatment. answers were obtained according to a five-item likert-scale varying from 1 (completely disagree) to 5 (completely agree). sociodemographic data was included in the comparison. mannwhitney or kruskal-wallis (95%) compared data depending on the variable. results: two-hundred twenty-five patients answered the questionnaire. median age of participants was 45-54 years. the potential benefits were similar among participants irrespective of the variable adopted. the perception of risks was significant influenced by variables gender, age, education level, type of edentulous space, prior treatment with prosthodontics, type of prosthodontics, and prosthodontics usage in years. consequences of no treatment were affected by age, prior treatment with prosthodontics, and prosthodontics usage in years. conclusions: the study showed the fundamental importance of correct elucidation about the potential risks (negative perception) in the initial appointment for the studied population. elucidation about the consequences of no treatment are relevant because some differences were seen into the variables. prosthodontics patients from the city of passo fundo seem well informed about the benefits of the prosthetic treatment, besides the significant influence of some sociodemographic and prosthetic conditions. keywords: dental prosthesis. education, dental. self concept. https://orcid.org/0000-0003-1535-4736 https://orcid.org/0000-0002-4063-8653 https://orcid.org/0000-0003-3426-0997 https://orcid.org/0000-0002-9913-8290 2 benetti et al. introduction in brazil, through an epidemiological survey of oral health, there are about 30 million edentulous individuals1. among these, about 97% of the elderly subjects need prosthetic rehabilitation of an average of 25.4 teeth. individuals between 35 and 44 years of age have an average of 7.4 teeth lost1. partial or total edentulism may result in a significant deterioration of the health of the stomatognathic system and may result in structural and pathological changes in the temporomandibular joint, which may be symptomatic or asymptomatic2,3. the loss of teeth limits the functions directly linked to the maintenance of quality of life4-6. their impact may still result in decreased chewing and phonation capacity, as well as nutritional, aesthetic and psychological losses, with reductions in self-esteem and social integration7. in view of the impact that dental prostheses can have on rehabilitated patients, there has been a growing increase in researches focusing on the effects of different perceptions of patients on prosthetic treatments8-11. this is also due to the subjectivity that the expectations can present, due to the lack of knowledge of the patient about the proposed treatment, being thus considered an unrealistic expectation12. with this, treatment decision-making should be cautious, shared and discussed with patients. this culture of treatment discussion must be initiated in an academic context13, with the guidance of professors to students. therefore, care becomes an important learning experience since the undergraduate period14. concepts, concerns, beliefs and attitudes regarding dental condition and prostheses are important variables that influence oral health satisfaction and the search for treatment12. in this sense, the questions about satisfaction15 and the perception on oral health4,10 of the rehabilitated patients were taken into account in order to better understand the effects of prosthetic treatment on patients’ lives4,5,10,15. given that there is a need for greater understanding of the subject matter, the aim of this study was to assess the patients’ perception of the outcomes of prosthetic treatment in a university of southern brazil, with individuals’ residents in the city of passo fundo, rs. the null hypotheses tested was that sociodemographic and prosthetic conditions would not affect the patient’s perception about the dental prosthetic treatment. materials and methods study design and location this cross-sectional study was performed in a university in southern brazil (meridional faculty imed), located in the city of passo fundo. the population of passo fundo is approximately 200,000 inhabitants16. passo fundo is a health reference in the state, and there are three dental schools with different types of treatment available for the population. this study was approved by the ethical committee of the imed (protocol number: 1.625.668/2016). 3 benetti et al. inclusion criteria and data collection patients’ perceptions about dental prosthesis treatment was assessed by applying a previously validated questionnaire10 as presented in table 1. the protocol for patients who sought treatments at imed is go through a screening and then the patient is forwarded to the specific clinic which capable to meet the patient’s demands. the participants who were scheduled at the prosthetic dental clinic and met the inclusion criteria were invited to answer the questionnaire and signed an informed consent form. the inclusion criteria involved patients who sought partially or full edentulous prosthetic treatment, and wearing (or not) any type of dental prosthesis. patients without cognitive capacity to understand the questions or patients who refused answer the questions were excluded. the demographic data of patients was obtained according to gender, age, and educational level. prosthetic-related data was collected according to the type of edentulous spaces, prior treatment with prosthodontics, type of prosthodontic treatment, and prosthodontic usage in years. the questionnaire contains 41 items10 (table 1) involving questions about: (1) the perceived potential benefits or positive consequences of the prosthetic treatment (positive perceptions); (2) the risks or negative consequences of the prosthetic treatment (negative perceptions); and (3) the consequences of no treatment with dental prosthesis. the possible answers for each item were presented in a 5-point likert-type scale: 1, strongly disagree; 2, disagree; 3, neutral; 4, agree; 5, strongly agree. one interviewer helped the patients to interpret the questions when they had any doubts. table 1. perceive potential outcomes investigated. benefits or positive perceptions risks or negative perceptions consequences of no treatment 1 – better chewing 18high cost 32digestive problems 2improve eating foods 19risk of rejection 33makes someone uglier 3better smile 20difficult to chew 34causes headache 4better appearance 21risk of cancer 35makes someone feel introverted and decrease self-esteem 5improve quality of life 22can cause harm to the bone and gingival tissues 36worsen personal relationship 6improve general health 23treatment is stressful 37may cause general health problems 7better speech 24prosthodontics need periodic recall 38food avoidance 8improve oral communication 25injury to the remaining teeth 39makes someone feel older 9better digestion 26prolonged treatment may cause anxiety 40may cause negative thoughts 10alleviate headaches or facial pain 27access to treatment is restricted by costs 41intervention is imperative 11improve bite 28treatment results can be disappointed continue 4 benetti et al. continuation 12improve professional opportunities 29demand more patient care than natural teeth 13help to protect remaining teeth 30will never be like natural dentition 14benefits overcome costs 31quality of service is professional-dependent 15when properly executed long-term outcomes are complementary 16feel younger 17feel pleased and confident data analysis data were explored using spss® software (version 20; ibm, armonk, ny, usa), and all inferences were performed with two-tailed trials using a significance level of 95% and statistical power of 80%. the mann-whitney test was used to compare data for the variables of gender and prior treatment with prosthesis. the kruskal-wallis test was used to compare the outcomes of the other variables. results two-hundred twenty-five subjects answered the questionnaire. median age of participants was 45-54 years. all demographic data is presented in table 2. considering all items (table 3), lower scores were observed for subjects aged 65-74 years old, with educational level ≥ 12 years, presenting single or total edentulous spaces, those prior treated with prosthodontics, and wearing partial fixed, complete denture or implant supported during 0-5 or 21 or more years when compared to subjects with 55-64 years old, educational level of 8-12 years, partial edentulous spaces, without prior prosthodontics treatment, and not wearing prosthodontics any time, respectively (p<0.05). the benefits or positive perceptions (table 3) did not differ among subjects (p>0.05). in this category, high values were observed within all variables (above 4.3), perceiving the benefits provided at least somehow by the prosthetic treatment. females and also subjects aged 65-74 years old (when compared to those with 55-64 years old), presenting educational level ≥ 12 years (vs 8-12 years), single or total edentulous spaces (different to partial spaces), prior treated with prosthodontics, and wearing partial fixed, complete denture or implant supported (vs not wearing and partial removable) during 0-5 years (in contrast to not use and use for 11-20 years) (p<0.05), showed the lower values to the potential risks or negative views (table 3). the patients in general agreed with the consequences of the no treatment. it is worth to mention that mean values were high in all the variables in this category (above 4.47). subjects with 35-44 and 65-74 years old, prior treated with prosthodontics, and using their dentures by 11-20 years presented lower scores than those with 45-54 years old, not previously treated, and not wearing prosthodontics or wearing during 0-5 years, respectively (p<0.05) (table 3). 5 benetti et al. table 2. demographic and clinical data of participants. n % sociodemographic aspects gender female 149 66.2 male 76 33.8 age 25-34 11 4.8 35-44 37 16.4 45-54 68 30.2 55-64 54 24.0 65-74 40 17.8 75-85 15 6.8 education level <8 years 55 24.4 8-11 years 71 31.6 ≥12 years 99 44.0 clinical aspects type of edentulous space single 57 25.3 partial 80 35.6 total 88 39.1 prior treatment with prosthodontics yes 183 81.3 no 42 18.7 type of prosthodontics do not use 38 16.9 partial removable 33 14.7 partial fixed 37 16.4 complete denture 72 32.0 implant-supported 45 20.0 prosthodontic usage (years) do not use 42 18.7 0-5 55 24.4 6-10 23 10.2 11-20 26 11.6 21 or more 79 35.1 6 benetti et al. table 3. comparison of clinical and demographic data. different uppercase letters denote significant difference within the same column for each variable. variable category all items benefits or positive risks or negative no treatment mean (sd) mean (sd) mean (sd) mean (sd) gender female 4.05 (0.3) a 4.49 (0.4) a 3.11 (0.6) a 4.61 (0.4) a male 3.98 (0.2) a 4.45 (0.4) a 2.92 (0.6) b 4.65 (0.4) a age 25-34 4.02 (0.3) ab 4.51 (0.5) a 3.00 (0.4) ab 4.59 (0.5) ab 35-44 3.95 (0.2) ab 4.44 (0.3) a 2.91 (0.5) ab 4.59 (0.2) b 45-54 4.05 (0.3) ab 4.49 (0.4) a 3.01 (0.6) ab 4.73 (0.4) a 55-64 4.09 (0.3) a 4.43 (0.4) a 3.24 (0.6) a 4.71 (0.3) ab 65-74 3.90 (0.2) b 4.48 (0.4) a 2.76 (0.6) b 4.49 (0.4) b 75-85 3.85 (0.3) ab 4.39 (0.5) a 2.73 (0.5) ab 4.49 (0.5) ab education level <8 years 4.02 (0.3) ab 4.50 (0.3) a 3.05 (0.6) a 4.57 (0.5) a 8-11 years 4.08 (0.2) a 4.50 (0.3) a 3.18 (0.5) a 4.64 (0.3) a ≥12 years 3.93 (0.2) b 4.42 (0.5) a 2.81 (0.6) b 4.67 (0.4) a type of edentulous space single 3.95 (0.2) b 4.43 (0.4) a 2.86 (0.5) b 4.66 (0.4) a partial 4.09 (0.2) a 4.46 (0.4) a 3.26 (0.6) a 4.63 (0.4) a total 3.95 (0.3) b 4.48 (0.4) a 2.82 (0.6) b 4.63 (0.4) a prior treatment with prosthodontics yes 3.97 (0.2) b 4.45 (0.4) a 2.92 (0.6) b 4.61 (0.4) b no 4.15 (0.3) a 4.49 (0.5) a 3.29 (0.5) a 4.77 (0.3) a type of prosthodontics do not use 4.13 (0.3) a 4.49 (0.6) a 3.27 (0.5) a 4.74 (0.3) a partial removable 4.09 (0.3) ab 4.33 (0.5) a 3.54 (0.6) a 4.47 (0.6) a partial fixed 3.94 (0.2) b 4.44 (0.2) a 2.81 (0.4) b 4.69 (0.2) a complete denture 3.95 (0.3) b 4.50 (0.4) a 2.77 (0.6) b 4.65 (0.4) a implant supported 3.95 (0.2) b 4.48 (0.3) a 2.83 (0.5) b 4.61 (0.4) a prosthodontic usage (years) do not use 4.15 (0.3) a 4.49 (0.5) a 3.29 (0.5) a 4.77 (0.3) a 0-5 3.96 (0.2) b 4.51 (0.3) a 2.78 (0.5) c 4.68 (0.4) a 6-10 4.01 (0.2) ab 4.56 (0.2) a 2.89 (0.6) abc 4.64 (0.2) ab 11-20 4.02 (0.2) ab 4.36 (0.4) a 3.23 (0.6) ab 4.55 (0.2) b 21 or more 3.94 (0.3) b 4.41 (0.4) a 2.92 (0.7) bc 4.57 (0.5) ab discussion the null hypothesis was rejected because the sociodemographic aspects showed to affect patients’ perceptions about dental prosthetic treatment (female and lower education showed more risk or a negative view). moreover, the prosthodontic condition such as partial edentulous space and removable partial prosthesis wearers also showed more risk or a negative view. not only the questionnaire used in this study10 but self-reporting in general may reflect in a validated form of the clinical oral condition in a brazilian context, since people without cognitive deficit have accurately identified conditions such as edentulism and denture use16. 7 benetti et al. the majority of the participants were female (66.2%), who presented a more negative perception than males. this result could be related to the fact that women have more concern for health and aesthetics, and these aspects are reflected in the search for procedures which improve these needs such as the use of prosthesis and more demanding treatments. these findings corroborate other studies where females participated the most, i.e. being the gender who most demanded care in prosthetic clinics10,17,18. this might be explained by the fact that women have greater aesthetic expectations, and their concern about health is greater than in men10 or which may be related to women’s negative self-perception of oral health17. most of the participants’ age ranged between 45-64 (54.2%) years. this finding may be related because normally people tend to lose more teeth over the years, or if the loss is of a unique dental element, especially in the posterior part of the dental arch, the patients tend to neglect seeking treatment. however, as the number of missing teeth increase, patients are more likely to demand treatment10, but it may also be too late for a simple rehabilitation. for this reason, this study found more partial (35.6%) and total (39.1%) edentulous spaces than single spaces (25.3%). one of reasons why the percentage of the partial and total edentulous in the present study was lower (74.7%) than that found by leles et al.10 (92.8%) is probably that some clinics at the faculty are separated by area and only patients treated with fixed partial prosthesis (single tooth missing) were interviewed on some days. another interesting finding is in relation to more negative perception for patients with partial edentulous spaces and removable partial prosthodontic rehabilitation. this might be explained due to the fact that removable partial dentures could be difficult for the patient to adapt to10, or because females most demanded treatment and this type of rehabilitation often fails in relation to aesthetics10. still, this type of treatment might only have been chosen for financial reasons19 and it could not be the desired rehabilitation, leading to a negative perception of treatment. an absence of differences was observed among partial fixed, complete dentures, and implant-supported prosthesis for the means of all items. however, the risks or negative consequences scores were higher for the do not use and partial removable denture categories. this might be related to the fact that fixed treatments favor a patient’s adaptation, while complete dentures may be easier to adapt to when compared to removable partial dentures, as observed in a previous study20. adaptation by neuroplasticity in oral rehabilitation procedures might explain the improved oral stereognostic ability, defined as the neurosensorial ability of the oral mucosa to recognize and discriminate the object forms in the oral cavity, sensorimotor activity of periodontal receptor, mainly encoding from anterior teeth, and masticatory function21. patients with less education reported to be more afraid, as well patients who never used any type of prosthodontics. these findings were also discussed in previous studies19 and may be due to the fact that patients are ashamed of or fear dental treatment, with these being some of the reasons for avoiding dental care19. furthermore, maybe their last experience in a dental clinical was bad or the patient had experienced an uncourteous reception by dentists and therefore have avoided facing this situation again19. however, leles et al.10 (2008) found that positive expectations were higher in 8 benetti et al. patients with low educational level, perhaps because these patients are not as critical of healthcare in relation to other social and cultural groups. meeting patients’ expectations is often complex because the majority of them already had previous experiences and have expectations to be fulfilled. in this sense, part of a clinician’s job is to clarify doubts and work on accurate prognosis for the proposed treatment. anxieties such as adaptation to removable or total prosthodontics is one of the points raised by patients after the first contact. moreover, there are concerns if the rehabilitation will be well adapted or at least will not disturb their phonetics or harm the aesthetics. after the rehabilitation, patients who had already used some type of prosthodontics may feel strange about the new rehabilitation and they do not adapt well to the new treatment. with the intuition to reduce the negative perceptions, leles et al.12 (2009) suggested that the active role of patients in making decisions about the proposed prosthodontic treatment is important to obtain positive results, and this makes the patient’s expectations more realistic and reduces anxiety and disappointment with the new treatment. our results agree with lemos et al.22 (2013), as their findings suggested that being completely edentulous or wearing non-fixed complete dentures, and regular, poor or extremely bad oral health were important features for negative self-perception of oral health with impact on the volunteer’s quality of life22. moreover, the oral health appreciation can be affected by general conditions such as rheumatoid arthritis23 or local commodities, such as stomatitis24. since this study was carried out in a prosthodontic clinic at a university where the operators were students, good interaction between teacher and student is necessary. consequently, this good relation should be transmitted to the patient to quell patients’ doubts, ambitions and expectations. thus, the rehabilitation should be well accepted by the patient, aiming to reduce negative perceptions as well as the patients’ fear of seeking prosthodontic treatment. this study has shown that in general, the studied population is aware of the benefits that the prosthetic treatment and of potential negative effects of the no treatment. however, some sociodemographic aspects as female and lower education showed to affect the patient’s perception about the dental prosthetic treatment (more risk or negative view). adequate information about risks of the treatment (negative perception) and consequences of the no treatment are relevant to provide to patients in the first appointment, because variation in the perception was identified in most variables. moreover, patients’ answers about risks ranged closer to the mean values representing a neutral perception (around 3 points), which means not able to inform about the risks or negative perceptions of treatment. acknowledgements this work is part of the fulfillment of the requirements for the conclusion of the undergraduate course in dentistry at faculdade meridional imed, passo fundo, brazil. the authors emphasize that this study was funded by the meridional foundation. however, these supporters had no role in the design of the study, in the collection or analysis of data, in the decision to publish or in preparing the manuscript. 9 benetti et al. references 1. peres ma, barbato pr, reis sc, freitas ch, antunes jl. 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mar/apr;34(2):481-8. doi: 10.11607/jomi.6980. 10. leles cr, morandini wj, da silva et, de f nunes m, freire mc. assessing perceived potential outcomes of prosthodontic treatment in partial and fully edentulous patients. j oral rehabil. 2008 sep;35(9):682-9. doi: 10.1111/j.1365-2842.2007.01839.x. 11. wolfart s, müller f, gerß j, heyedcke g, marré b, böning k, et al. the randomized shortened dental arch study: oral health-related quality of life. clin oral investig. 2014;18(2):525-33. doi: 10.1007/s00784-013-0991-6.. 12. leles cr, martins rr, silva et, nunes mf. discriminant analysis of patients’ reasons for choosing or refusing treatments for partial edentulism. j oral rehabil. 2009 dec;36(12):909-15. doi: 10.1111/j.1365-2842.2009.02018.x. 13. divaris k, polychronopoulou a, mattheos n. an investigation of computer literacy and attitudes amongst greek post-graduate dental students. eur j dent educ. 2007 aug;11(3):144-7. doi: 10.1111/j.1600-0579.2007.00437.x. 14. masella rs. the hidden curriculum: value added in dental education. j dent educ. 2006 mar;70(3):279-83. 15. steele jg, ayatollahi sm, walls aw, murray jj. clinical factors related to reported satisfaction with oral function amongst dentate older adults in england. community dent oral epidemiol. 1997 apr;25(2):143-9. doi: 10.1111/j.1600-0528.1997.tb00912.x. 10 benetti et al. 16. arenas-márquez mj, tôrres lhn, da silva dd, hilgert jb, hugo fn, neri al, et al. validity of self-report of oral conditions in older people. braz j oral sci. 2019;18:e191670. dx.doi.org/10.20396/bjos.v18i0.8657271. 17. gaspar mg, dos santos mb, dos santos jf, marchini l. correlation of previous experience, patient expectation and the number of post-delivery adjustments of complete dentures with patient satisfaction in a brazilian population. j oral rehabil. 2013 aug;40(8):590-4. doi: 10.1111/joor.12070. 18. miranda bb, dos santos mb, marchini l. patients’ perceptions of benefits and risks of complete denture therapy. j prosthodont. 2014 oct;23(7):515-20. doi: 10.1111/jopr.12164. 19. nordenram g, davidson t, gynther g, helgesson g, hultin m, jemt t, et al. qualitative studies of patients’ perceptions of loss of teeth, the edentulous state and prosthetic rehabilitation: a systematic review with meta-synthesis. acta odontol scand. 2013 may-jul;71(3-4):937-51. doi: 10.3109/00016357.2012.734421. 20. john mt, koepsell td, hujoel p, miglioretti dl, leresche l, micheelis w. demographic factors, denture status and oral health-related quality of life. community dent oral epidemiol. 2004 apr;32(2):125-32. doi: 10.1111/j.0301-5661.2004.00144.x. 21. kumar a, kothari m, grigoriadis a, trulsson m, svensson p. bite or brain: implication of sensorimotor regulation and neuroplasticity in oral rehabilitation procedures. j oral rehabil. 2018 apr;45(4):323-33. doi: 10.1111/joor.12603. 22. lemos mmc, zanin l, jorge mlr, flório fm. oral health conditions and self-perception among edentulous individuals with different prosthetic status. braz j oral sci. 2013 jan/mar;12(1):5-10. doi: 10.20396/bjos.v12i1.8641111. 23. alfenas bfm, de andrade km, carletti tm, cunha r, garcia mr. removable prostheses improve oral health-related quality of life and satisfaction of elderly people with rheumatoid arthritis. bra j oral sci. 2020;19:e206652. doi: 10.20396/bjos.v19i0.8656652. 24. morel ll, possebon apr, faot f, pinto lr. prevalence of risk factors for denture stomatitis in complete denture wearers. braz j oral sci. 2019;18:e191414. doi: 10.20396/bjos.v18i0.8655147 oral sciences n3 braz j oral sci. 11(3):362-367 original article braz j oral sci. july | september 2012 volume 11, number 3 satisfaction of patients and students at the integrated clinics of a dental school joão paulo de carli1, maria salete sandini linden2, micheline sandini trentin2, soluete da silva oliveira2, marcos eugênio de bittencourt3, luiz renato paranhos4 1dds, msc, professor, department of oral medicine, school of dentistry, university of passo fundo (upf-rs), passo fundo, rs, brazil 2dds, msc, phd, professor, department of oral medicine, school of dentistry, university of passo fundo (upf-rs), passo fundo, rs, brazil 3dds, msc, phd, professor, department of dentistry, school of dentistry, university of passo fundo (upf-rs), passo fundo, rs, brazil 4dds, msc, phd, professor, department of oral biology, school of dentistry, bauru, sp, brazil correspondence to: joão paulo de carli department of oral medicine, school of dentistry, university of passo fundo rua bento gonçalves, 967/204, centro, cep: 99010-010 passo fundo, rs, brasil e-mail: joaoestomatologia@yahoo.com.br abstract aim: the objective of this questionnaire-based research was to evaluate the level of satisfaction of students and patients at the school of dentistry of the university of passo fundo (foupf). methods: the questionnaire was administered after the patient’s care, as follows: first, the student explained the study purposes and methodology to the patient, the option of not participating and the full confidentiality of the information obtained. patients were selected among those undergoing treatment at the integrated clinic i and ii disciplines (5th year of undergraduate dental course) between march and november 2010. results: satisfactory results were obtained, as most interviewed patients (49; 98%) considered the care given by foupf students as positive. another important factor mentioned by patients was the use of individual protection equipment and biosafety measures adopted by the students; 98% (n = 49) of the patients affirmed that the students were properly wearing individual protection equipment. with regard to aspects that could be improved, 36.7% (n = 18) of interviewed patients mentioned the need to schedule appointments more than once a week, 12.2% (n = 6) felt the need to reduce waiting times for consultations, and 10.2% (n = 5) wanted to reduce appointment duration. as much as 98% of students (n = 49) declared being attentive to dentistry situations, 56% (n = 28) were satisfied with their technical aptitudes, and 96% (n = 48) mentioned enjoying helping patients. conclusions: the care given to patients by foupf students is done well, not requiring significant changes. keywords: oral health, dental health services, dental clinics. introduction dental schools play an important role in improving access to dental healthcare. studies on patient satisfaction at healthcare teaching facilities can contribute to the creation of planning measures to improve the quality of service. it is also important to evaluate the social-economic-cultural level of students and patients, as well as the reason for choosing the school for learning/undergoing dental treatment. from these data, it is possible analyze the negative and positive points of assistance for students and patients, aiming to overcome possible deficiencies and improve existing qualities. received for publication: march 09, 2012 accepted: may 17, 2012 363363363363363 research studies on the quality of dental services at integrated clinics and patient satisfaction with them start from the premise that there are certain needs established for users. patient satisfaction represents valuable feedback that contributes to considerations on the continuous improvement of teamwork. therefore, constant investigations are necessary to identify the factors that promote the satisfaction of patients assisted by healthcare services. mendes1 (2003) described that the concept of user satisfaction is part of a sociocultural and political context in a given society. the author also affirms that the quality of healthcare services depends on the subjects who practice the action, and particularly those who undergo the action the citizenry. garcia and almeida2 (2005), in a study involving the quality of dental healthcare services offered at a public dental care center (ceo rodolfo teófilo), concluded that the technical-scientific-assistance characteristics of health professions, in particular oral health, should be balanced within teaching practice. as such, the curriculum should promote integration between basic scientific concepts and clinical practice – with the latter valuing the human relationship between the dental surgeon and patient, within a realistic perspective. according to nobre et al.3 (2005), assessing client satisfaction in different teaching institutions is the best indicator to evaluate which standard of dental service is being offered to users. in a study conducted at the university of fortaleza/ce (unifor), these authors concluded that a large part of users are satisfied with the quality of the dentistry service provided by unifor. nevertheless, some flaws were found in the student-patient relationship with regard to communication and user autonomy, requiring reinforcement to some students on the importance of sharing the treatment plan and procedure to be performed. in view of the theoretical reference exposed herein, the aim of the present work was to evaluate the quality of the dental services and education provided at the upf school of dentistry (foupf), by applying questionnaires directed at patients and students at the institution. material and methods the present work was approved by the ethics committee of the university of passo fundo, rs, brazil (process #162/ 2009). it was regarded as a research field, performed by applying open-ended questionnaires to patients and students of the integrated clinical practice i and ii at foupf disciplines attending the 9th and 10th semesters of the undergraduate dental course. in such clinics are offered dental procedures in periodontics, surgery, endodontics, dentistry, occlusion and preventive orthodontics (approximately 350 weekly visits). the present study was divided into two different stages: a) application of the questionnaire to patients: performed at the foupf waiting room by a researcher (was not self-administered) who gave users an informed consent form and the questionnaire containing the open-ended questions (panel 1). the questionnaire was administered after the patient’s care, as follows: first, the student explained the objective and method of the research to the patient, the option of not filling it out, as well as the full confidentiality of the information obtained. it was also made clear that the answers would not influence the dental service provided at the clinics, and that the patient had the right, if so desired, to withdraw from the research at any time (free and informed consent form). the sample was randomly chosen, constituted approximately for 15% of the total number of patients treated during the study period (340 patients). patients were selected among those undergoing treatment in the integrated clinic i and ii disciplines (5th year of the undergraduate dental course), between march and november 2010. to take part in the research, users had to have been assisted at least once at the foupf integrated clinic and should be willing to answer the questionnaire. it is noteworthy that most (70%, n = 35) of users surveyed had been treated 3 to 4 times at foupf. the questionnaires given to patients and students were previously applied by means of a pilot study and was developed based on work done by other authors3-5. (panel 1) b) application of the questionnaire to students: performed at the foupf integrated clinics by a student participating in the research (was not self-administered), who gave students an informed consent form and the sheet containing the open-ended questions (panel 2). foupf has approximately 500 students, of which 100 (20%) are enrolled at the disciplines of integrated clinic i and ii. the questionnaire was administered to 50 enrolled students in such disciplines (50%), corresponding to the 9th and 10th levels, chosen by lot (100 names of students placed on strips of paper stored in an urn, of which 50 names were removed). the questionnaire was administered after the clinical care, as follows: first, the student explained to the participant the objective and method of the research, the option of not filling it out, as well as the full confidentiality of the information obtained. it was also made clear that the answers would not influence the student’s clinical performance/grade, and that the student had the right, if so desired, to withdraw from the research at any moment. next, a significant sample was selected, by random draw, of students enrolled in the integrated clinic i and ii disciplines at foupf between march and november 2010, who were willing to fill out the questionnaire. (panel2) the response rate of questionnaires to students (n = 50) and patients (n = 50) was 100%, since all subjects responded. the variables to be analyzed are specified in the questionnaires above; the main focal point of the work is the satisfaction level of patients with regard to the services rendered by the institution. data were arranged in an excel spreadsheet and analyzed using frequency descriptive statistics. results fifty patients were interviewed, mostly female (54%, n = 27), at the fourth decade of life (34.6%, n = 17), and satisfaction of patients and students at the integrated clinics of a dental school braz j oral sci. 11(3):362-367 364364364364364 age:__________ m ( ) f ( ) city of residence:___________________________________________________ clinic where treated: ( ) integrated i ( ) integrated ii 1. profession:______________________________________________________ 2. are you currently employed? ( ) yes ( ) no, i am unemployed ( ) yes, and i am also retired ( ) no, i am retired 3. what is your monthly family income? (minimum wage r$ 545.00) ( ) none ( ) 1 to 2 times the minimum wage ( ) 2 to 3 times the minimum wage ( ) 3 to 5 times the minimum wage ( ) 5 to 10 times the minimum wage ( ) over 10 times the minimum wage 4. what is your complete educational level? ( ) none (cannot read or write) ( ) some middle school ( ) middle school ( ) some high school ( ) high school degree ( ) some college ( ) college degree ( ) graduate school 5. does your family own its home? ( ) yes ( ) no 6. why did you seek treatment at the foupf clinic? ( ) recommended colleagues, relatives or friends ( ) already knew the student and was promised treatment by him/her ( ) was treated previously and did not conclude treatment ( ) could not currently afford private treatment ( ) referred by a public or private dentist for specialized treatment ( ) to get prosthetics ( ) because students have more patience with patients ( ) because the quality of service is good ( ) other reasons:__________________________________________________ 7. did you feel any pain during the treatment? ( ) yes ( ) no 8. at what time and why?____________________________________________ 9. was this solved by the student? how?________________________________ 10. what were you most afraid of during your dental treatment? ( ) was not afraid ( ) the instruments used by the students ( ) anesthesia ( ) the drill ( ) everything ( ) the student’s heavy hand ( ) other 11. description of the service given by the student yes no somewhat were you well treated? ( ) ( ) ( ) did the student portray confidence and security during the procedures? ( ) ( ) ( ) was the student properly attired? ( ) ( ) ( ) did the student go over the treatment plan with you? ( ) ( ) ( ) did the student explain to you the procedures being performed? ( ) ( ) ( ) were the explanations clear? did you understand them? ( ) ( ) ( ) 12. in general, how do you rate the service given by the student? ( ) excellent ( ) good ( ) average ( ) terrible 13. how do you rate the service given by the dental school at the clinic? ( ) excellent ( ) good ( ) average ( ) terrible 14. what suggestions would you have to improve service at the clinic where you are being treated? ( ) nothing, it is fine as is ( ) reduce wait time in the waiting room ( ) more frequent appointments during the week ( ) reduce wait time on the dentist’s chair ( ) the student should be more experienced 15. how did you perceive the environment during your dental treatment? excellent good regular terrible organization ( ) ( ) ( ) ( ) comfort ( ) ( ) ( ) ( ) cleanliness ( ) ( ) ( ) ( ) lighting ( ) ( ) ( ) ( ) student punctuality ( ) ( ) ( ) ( ) 16. how do you rate the clinic’s screening service? ( ) excellent ( ) good ( ) regular ( ) terrible 17. what was your impression of the clinic where you received treatment? ( ) clean ( ) organized ( ) quiet ( ) dirty ( ) disorganized ( ) noisy 18. were you well serviced by the person who made your appointment? ( ) yes ( ) no ( ) average 19. how do you rate the service fee? ( ) high ( ) satisfactory ( ) low 20. have you or would you recommend the university’s service to anyone? ( ) yes ( ) no 21. give a score from 0 to 10 to the dental service given by the university:_________ 22. suggestions to improve service at foupf___________________________ panel 1 questionnaire answered by patients satisfaction of patients and students at the integrated clinics of a dental school braz j oral sci. 11(3):362-367 age:_______ m( ) f( ) ( ) single ( ) married semester: ( ) ix ( )x 1. what is your monthly family income? (minimum wage r$ 545.00) ( ) none ( ) 1 to 2 times the minimum wage ( ) 2 to 3 times the minimum wage ( ) 3 to 5 times the minimum wage ( ) 5 to 10 times the minimum wage ( ) over 10 times the minimum wage 2. what is the educational level of your parents? (you can mark more than one) ( ) none (cannot read or write) ( ) some middle school ( ) middle school ( ) some high school ( ) high school degree ( ) some college ( ) college degree ( ) graduate school 3. does your family own its home? ( ) yes ( ) no 4. are you attentive when dealing with the dental problems of your patients? ( ) yes ( ) no ( ) sometimes 5. do you feel prepared to perform actions as planned, and are able to do so? ( ) yes ( ) no ( ) somewhat 6. are you satisfied with the technical quality of your work? ( ) yes ( ) no ( ) somewhat 7. are your relationships with your patients satisfactory? ( ) yes ( ) no ( ) somewhat 8. in your opinion, has your service promoted a high level of interpersonal relationship? ( ) yes ( ) no ( ) somewhat 9. do you enjoy helping patients? ( ) yes ( ) no ( ) somewhat 10. have you enjoyed working at the integrated clinic? ( ) yes ( ) no ( ) somewhat 11. with regard to the faculty at the integrated clinic, are they available when you need help? ( ) yes ( ) no ( ) not always 12. suggestions to improve patient service and your learning experience: panel 2 questionnaire answered by students times at which pain was felt n % unspecified situations 12 24 extraction 2 4 root canal treatment 4 8 prosthetic fitting 2 4 anesthesia 2 4 orthodontic appliance 1 2 table 3 – situations reported by patients with pain. residing in the city of passo fundo/rs (51.1%, n = 26). the professions declared by the subjects were predominantly retirees (16.3%, n = 8), housewives (12.2%, n = 6) and farmers (12.2%, n = 6). the most frequently reported reasons by patients for seeking dental care at foupf were recommendation by friends and colleagues (38%, n = 19), quality of service rendered (42%, n = 21), and lack of financial resources to pay for private dental treatment (26%, n = 13) – (table 1). according to users, the students portrayed confidence and security during treatment (87.3%, n = 44), explaining procedures before performing them (100%, n = 50) and properly wearing individual protection equipment (ipe) in 97.8% of cases (n = 49) (table 2). a total of 24% of patients (n = 12) reported feeling pain during dental treatment in unspecified situations (table 3). to improve the quality of dental service given by foupf, 36.7% (n = 18) of patients suggest that appointments be made more than once a week (table 4). finally, it was observed that 49 (98%) of users do or would recommend the foupf dental service to someone. fifty students were interviewed (25 male and 25 female), 365365365365365 table 1reasons for patients to seek dental care at foupf reason that led the user to seek dental care at foupf n % good quality of treatment 21 42 recommendation of colleagues, relatives or friends 19 38 financial reasons 13 26 to get prosthetics 9 18 unfinished previous treatment 7 14 dentist referral for specialized treatment 4 8 students have patience with patients 4 8 other reasons 1 2 knows the student, who promised to treat him/her 0 0 characteristics of service (students) yes n o somewhat % (n) % (n) % (n) treated well 97.9% (49) 2.1%(1) 0% (0) portrayed confidence and security 87.3% (44) 2.1% (1) 10.6% (5) wore adequate individual protection equipment 97.8% (49) 2.2% (1) 0% (0) discussed treatment plan 82.2% (41) 17.8% (9) 0% (0) explained procedures beforehand 100% (50) 0% (0) 0% (0) gave clear explanations 89.1% (45) 0% (0) 10.9% (5) table 2 – characteristics of service given by students, according to the patients. a total of 24% of patients (n = 12) reported feeling pain during dental treatment in unspecified situations (table 3). satisfaction of patients and students at the integrated clinics of a dental school braz j oral sci. 11(3):362-367 366366366366366 with mean age of 22.6 years. with regard to the integrated clinics, most students feel prepared to perform any procedure (58%, n = 29), are attentive when dealing with clinical dentistry situations (98%, n = 49), and enjoy working at the integrated clinics (70%, n = 35) (table 5). discussion patient satisfaction represents valuable feedback that contributes towards assessing the constant improvement required in teamwork. thus, constant investigation is required in order to seek out the factors that promote the satisfaction of patients assisted at health services. according to brazilian curricular guidelines for dental school courses, the profile of professionals to be formed is to be generalist. dental surgeons should act at all levels of healthcare, based on technical and scientific rigor. they are qualified to practice activities related to oral health of the general population, focusing their actions to transform reality for the benefit of society at large. health professionals, within the scope of their profession, must be prepared to develop health prevention, protection and rehabilitation actions, at both individual and collective levels. mendes1 (2003) described that the concept of user satisfaction is part of the sociocultural and political context of a given society. also, the quality of healthcare services depends on the subjects who practice the action and especially those who undergo it – that is, the citizen. these aspects could be observed in the present research, as most patients rated positively the service provided by foupf students (97.9%, n = 49). it should be reminded that most patients seen at foupf are mostly farmers, retirees and stayat-home individuals, having an education level probably lower than that of the average population. it is noteworthy that the level of education generally follows the income level of individuals. thus, individuals with less education and less sophisticated ordering treatments whose cost is generally lower. in the present study, this fact should be the subject of extensive analysis in the industry screening of college in order to perform a more careful selection of patients, primarily aimed at direct care to individuals with less education and lower income bracket. according to ferreira et al.6 (2004), anxiety is a phenomenon that can be characterized by subjective feelings of tension, apprehension, nervousness and preoccupation, experienced by an individual at a given time, such as during a dental appointment. studies have shown that a large share of the adult population regards dental treatment as very uncomfortable and associated with stress. with regard to dental fear and anxiety, the present study diverged from the literature, as 87.3% (n = 44) of interviewed patients reported that the students portrayed confidence and security during treatment performed at foupf, and 48.9% (n = 24) of individuals were not afraid during the procedures. according to barbisan et al.7 (1995), most patients assisted at the ufrgs dental school were satisfied with the service provided, and more than half of them believe that students can perform at the same level of service as a more experienced professional. they further reported that the physical environment of the school is clean and organized, and that they had already recommended or would recommend the service to others, giving the treatment a score of 10. the findings of the present study agrees with the results reported in the literature, as 97.9% (n = 49) of patients declared they had been well treated and only 4.2% (n = 2) mentioned that students should be more experienced. ramos8 (1997) analyzed the opinion of patients at the integrated clinic of the federal dental school of diamantina/ mg on what they most feared in a dental treatment. the results showed that 47.97% of patients were not afraid of anything and 52.03% had several causes for fear; moreover, 97.92% rated the service given students to users as excellent or good. those results agree with the present study, as 48.9% (n = 24) of individuals were not afraid during the performed procedures, and 97.9% (n = 49) declared having been well treated by students and staff at foupf. pinheiro et al.9 (2002), based on data obtained in their work, reported that 94.09% of patients subjected to a research informed their preference for being served by dental surgeons wearing glasses, masks, gloves and hairnet. those authors thereby concluded that most interviewed subjects were aware of the importance of individual protective equipment worn table 5 – profile of foupf students student profile (soon to graduate) yes % (n) no % (n) somewhat % (n) attentive with regard to dentistry situations 98% (49) 0% (0) 2% (1) prepared to perform dental work (skilled) 58% (29) 0% (0) 42% (21) satisfied with own technique 56% (28) 0% (0) 44% (22) satisfactory relationship with patients 88% (44) 2% (1) 10% (5) service provided with high level of interpersonal relationship 70% (35) 0% (0) 30% (15) enjoy helping patients 96% (48) 0% (0) 4% (2) enjoy working at the integrated clinics 70% (35) 4% (2) 26% (13) user suggestions % (n) none, it is fine as is 36.7% (18) more frequent appointments during the week 36.7% (18) reduce waiting time 12.2% (6) reduce appointment duration 10.2% (5) the student should be more experienced 4.2% (2) table 4 – patient suggestions to improve dental service at foupf. satisfaction of patients and students at the integrated clinics of a dental school braz j oral sci. 11(3):362-367 367367367367367 to prevent infectious and contagious diseases. the present study revealed that 97.8% (n=49) of patients observed that the student assisting them was properly dressed and protected. according to nobre et al.3 (2005), assessing client satisfaction at various learning institutions is the best indicator to evaluate what standard of dental care is being offered to users. in a research study conducted at the university of fortaleza/ce (unifor), those authors concluded that the service process should be improved, especially with regard to the excessive wait until users are seen and unnecessary visits to the services. in that sense, it should be mentioned that 36.7% (n = 18) of patients interviewed at foupf mentioned the need to be seen more than once a week, 12.2% (n = 6) felt a need to reduce the waiting time, and 10.2% (n = 5) wanted to reduce appointment duration. on that same topic, bottan et al.10 (2006) reported that the most prevalent negative feedback by patients concerned the waiting time until being called to begin treatment, waiting time in the waiting room and duration of the treatment at univali. mialhe et al.5 (2008), while evaluating the quality of dental service provided by graduates of the fop/unicamp dental school, observed that 92.5% of interviewed subjects declared not feeling pain during clinical care. with regard to the characteristics of the service provided by students, most did so portraying security and confidence to the interview subjects, discussing and clearly explaining the proposed treatment plan. in the present study, 82.2% (n = 41) of patients mentioned that foupf students explained the treatment plan in detail prior to assisting the patients. santos11 (2005) performed a research study in campos dos goytacazes/rj among professionals and users of public dental service units, who filled out self-applied questio-nnaires. the author observed that patients were satisfied with the service, professionals were interested in solving patients’ problems, and patients received care according to the explanations given for treatment. in the present study, 98% (n = 49) of students declared being attentive to dentistry situations, 56% (n = 28) claimed to be satisfied with their own technical skills, and 96% (n = 48) mentioned enjoying helping patients. in general, it could be observed that the service offered to patients by foupf students was adequate, not requiring significant changes. among the points to be modified with regard to satisfaction, patients emphasized scheduling visits more than once a week so that treatment could be completed sooner and in a more efficient manner. references 1. mendes vlps. evaluation of health services by users: a matter of citizenship. rev baiana enferm. 2003, 18: 97-110. 2. garcia eg, almeida mi. perceptions and expectations by health service users on the surgeon-dentist-patient relationships. rev odontol univ são paulo. 2005; 17: 29-39. 3. nobre es, câmara gp, silva kp, nuto sas. quality assessment of dental service provided by private university: user view. rev bras prom saude. 2005, 18: 171-6. 4. leão att, dias k. evaluation of health services provided by schools of dentistry: the user view. rev bras odontol saude colet. 2001; 2: 40-6. 5. mialhe fl, gonçalo cs, carvalho lms. evaluation by users about the dental service quality provided by students from the dentistry course at fop/unicamp. rev fac odontol univ passo fundo. 2008, 13: 19-24. 6. ferreira cm, gurgel-filho ed, valverde gb, moura eh, deus g, coutinho filho t. dental anxiety: level, prevalence and behavior. rev bras odontol. 2004, 17: 51-5. 7. barbisan ao, moschen az, weissheimer ap, cauduro ff, castro gd, muller s et al. opinion of patients on the efficacy in the attendance received at the school of dentistry of ufrgs. rev fac odontol porto alegre. 1995, 36: 34-6. 8. ramos fb. efficacy in the attendance provided to patients at the integrated clinic of the federal dentistry school of diamantina. rev cromg. 1997, 3: 56-63. 9. pinheiro jt, aguiar cm, santos vf. behavioral study of patients before the dental surgeon costume. rev bras odontol. 2002, 59: 57-60. 10. bottan er, sperb ral, telles ps, uriarte neto m. evaluation of dental services: the view of patients. rev abeno. 2006, 6: 128-33. 11. santos sas. organizational analysis of the demand and degree of satisfaction of professional and user at the dental public service unities in the municipality of campos dos goytacazes. [dissertation]. piracicaba: faculdade de odontologia de piracicaba. universidade estadual de campinas; 2005. satisfaction of patients and students at the integrated clinics of a dental school braz j oral sci. 11(3):362-367 1 volume 16 e17039 original article http://dx.doi.org/10.20396/bjos.v16i0.8651186 corresponding author: dr. abiodun-solanke iyabode m.funmilayo department of restorative dentistry, college of medicine, university of ibadan, nigeria phone no : +234-802-375-4987 e-mail address: abisolimf@yahoo.ca received: april 21, 2017 accepted: november 23, 2017 audit of crowns and fixed partial dentures in a nigerian teaching hospital abiodun-solanke iyabode m.funmilayo1, ajayi deborah mojirade2, sulaiman amidu omotayo3 1 bds, mmp, fmcds. department of restorative dentistry, college of medicine, university of ibadan. oyo state, nigeria 2 bds, m.sc (epid &med stat.), fwacs. department of restorative dentistry, college of medicine, university of ibadan. oyo state, nigeria 3 bds,fmcds. department of restorative dentistry, college of medicine, university of ibadan. oyo state, nigeria aim: the purpose of this investigation was to document the reasons for placement, and replacement of crowns and fixed partial denture in a nigerian teaching hospital. methods: a retrospective review of patients that had advanced conservative procedures. a data collection form was used to gather the relevant information from the patients’ case notes. section a sought information on age , gender, educational level, patients’ occupation etc. section b recorded information on tooth/teeth involved, reason for fabrication of prosthesis and material used. results: three hundred and twenty six had 398 crowns while 23 patients received bridges. patients’ ages ranged from 16-85 years with a mean of 47.7 ± 17.2 years and a male to female ratio of 1:1.3. thirty five percent of the crowned teeth were in the upper right quadrant, followed by the upper left quadrant with 29.4%. upper right central incisors were the most frequently (15.6%) crowned teeth. endodontics and esthetics were the most common reasons for initial crown placement and replacement respectively. about 60% of bridges fabricated were new, while 22.2% of replaced bridges were due to fracture of porcelain and unacceptable marginal adaptation. conclusion: endodontics and esthetics were the most common reasons for initial crown placement and replacement  keywords: prosthodontics. denture. partial, fixed. mailto:abisolimf@yahoo.ca https://www.ncbi.nlm.nih.gov/mesh/68003830 2 funmilayo et al. introduction the placement and replacement of crowns forms an important part of regular dental care which is provided in the general dental practice1. it covers half of the general practitioners time and it is usually quite expensive2. the main goal of crown placement is to provide patients with a long–lasting tooth–like restoration that is strong, easy to maintain, biologically compatible, and esthetically similar to natural teeth or improve the strength or appearance of natural teeth3. dental restorations do not last forever. studies4,5 have shown that restorations have a limited lifespan and once a tooth is restored, ‘the restorative cycle’ commences where the restoration will likely be replaced many times throughout the lifetime of the patients5. it has been shown that over 60% of all restorative dentistry involves the replacement of restorations6. for intracoronal restorations, reasons for placement and replacement include primary caries, secondary caries, unacceptable marginal adaptation, bulk fracture of tooth, unsightliness, non–carious tooth wear and pain/sensitivity7. deligeorgi et al. reported primary caries to be the main reason for the placement of initial restoration and secondary caries being the most frequent reason for the replacement of existing restorations8. ajayi et al., however, found fracture of restorations to be the commonest reason for replacement9. indirect restorations (crown and fixed partial dentures) can be fabricated using different materials such as metals, ceramics or the combination of the two materials. however, gold still remains the gold standard against which all other restorations are measured in terms of longevity. secondary caries and retention loss are the commonest causes of failure, it hasgold restorations have a survival rate of 96% and a failure rate of 1.4% in the posterior permanent dentition10, but secondary caries and retention loss are the commonest causes of failure. porcelain fused to metal and all ceramic restorations have a survival rate of 90% and 75-80% respectively over 10 years. studies11,12 conducted among americans revealed that secondary caries account for 22% and 37% of failure of crown and fixed partial denture and the mean service life of single crown was 8-9.4 years11,12. technical failure was reported to be the most prevalent (8%) cause of failure of crowns in a study by cheung13 in hongkong. other studies14,15 however, reported lack of retention to be the most common cause of failure of crown and bridges. oginni3 in his study in a suburban population in southwestern part of nigerian however reported poor esthetics to be the most frequent cause of failure and the overall mean years of service for replaced restorations were 5.6 years there is paucity of information on reasons for placement and replacement of crowns and fixed partial dentures especially in this environment. the purpose of this investigation was to document the reasons for placement, and replacement of crowns and fixed partial denture, as well as to assess the tooth/teeth involved and the different materials used in the fabrication of the restorations in a nigerian teaching hospital. materials and methods this is a retrospective review of all patients aged sixteen and above that had advanced restorative procedures done at the conservation clinic of the dental centre, university college hospital between august 2011and july 2016. a data collection form 3 funmilayo et al. was used to gather the relevant information from the patients’ case notes. section a sought information on sociodemographic characteristics of patients such as age, gender, educational level, patients’ occupation etc. section b recorded information on tooth/teeth involved, reasons for initial placement and replacement of crown and fixed partial denture, type of material used in fabrication of prosthesis. one of the authors (asimf) searched through the case notes for the relevant information required. patients case notes in which all the required information could not be found were excluded from the study. ethical approval was sought and obtained from the university of ibadan /university college hospital ethical review committee. there has not been a consensus on various socio-economic classifications in nigeria. therefore, for the purpose of this study, a modified version of standard occupational classification system designed by the office of population census and surveys, london (opcs 1991)16 was employed as given below; • class 1 = skilled worker e.g professionals and managerial officers and retirees of this cadre • class 2 = unskilled workers e.g artisan and traders • class 3 = dependants e.g housewives, students, non-pesionable retirees data were collected on the designed collection form, one sheet was used for each crown or bridge placed and the forms were bound together and then entered into the computer. data analysis was carried out with spss version 22.0 (spss inc, chicago, illinois, usa) using descriptive statistics. association between categorical variables was determined using chi – square with a level of statistical significance placed at p≤ 0.05 results a total of 415 patients received crowns and bridges during the study period out of which only 349 with complete records were included and those with incomplete data were excluded giving a ‘drop out’ rate of sixteen percent. three hundred and twenty six had 398 crowns while 23 patients received bridges. patients’ ages ranged from 16-85 years with a mean of 47.7± 17.2years. more than half (195, 55.9%) were females and the remaining were males giving a male to female ratio of 1:1.3. the highest proportion (37.8%) of the patients were in the age range of 41-60years and the least (1.1%) were those older than 80 years. a high majority (88.5%) of the patients had university education or its equivalent. (table 1). more than half (57.9%) of the patients were skilled workers ( class1) and the least (10.6%) were the unskilled (class 2).(table 1) three hundred and ninety eight teeth were crowned, out of which 140 (35.2%) were in the upper right quadrant, 117 (29.4%) were in the upper left quadrant and the least (67, 16.8%) were in the lower right quadrant. one hundred and seventy five (44%) of the total teeth crowned were incisors out of which 112 ( 28.1%) were central incisors and the highest proportion (15.6%) were the upper right central incisors. a total of 92 premolars were crowned and the upper right second premolar constituted the highest(6.3%) proportion, while out of the 106 crowned molars, the lower left second molars had the highest (23, 5.8%) proportion and the least were the third (upper 4 funmilayo et al. and lower) molars with only 4 crowned teeth. the maxilla had 258 (64.6%) of the crowned teeth while the remaining were in the mandible. slightly over half (54.5%) of all the crowned teeth were placed in female patients. (table 2). the average number of crowns placed in the patients ranged from one to twelve. the commonest reason for initial crown placement was following endodontics (194, 59.5%) which is statistically significant across gender with more female crowning table1. socio demographic status of patients age (years) 16 – 85 mean 47.7 ± 17.16 age group ≤ 20 years 20(5.7%) 21 – 40 years 106(30.4%) 41 – 60 years 132(37.8%) 6180 years 87 (24.9%) > 80 year 4(1.1%) total 349(100%) sex male 154 (44.1%) female 195 (55.9%) occupation class 1 202 (57.9.0%) class 2 37 (10.6%) class 3 110 (31.5%) educational level primary/arabic 17 (4.9%) secondary 23 (6.6%) tertiary 309 (88.9%) the highest proportion of patients were in the age group 41-60years. majority (88.5%) had university education and slightly over half (57.9%) were skilled workers table 2. distribution of crowned teeth according to gender ul1 ul2 ul3 ul4 ul5 ul6 ul7 ul8 50 17 6 9 19 12 4 0 117 m 25 11 2 4 6 3 1 52 f 25 6 4 5 13 9 3 65 ur1 ur2 ur3 ur4 ur5 ur6 ur7 ur8 62 17 9 17 25 6 4 0 140 m 36 4 6 8 5 3 1 63 f 26 13 3 9 20 3 3 77 ll1 ll2 ll3 ll4 ll5 ll6 ll7 ll8 11 6 3 3 7 20 23 1 74 m 10 3 1 2 3 6 5 1 31 f 1 3 2 1 4 14 18 0 43 lr1 lr2 lr3 lr4 lr5 lr6 lr7 lr8 7 5 3 1 11 17 20 3 67 m 6 4 1 8 6 8 2 35 f 1 1 2 1 3 11 12 1 32 a total of 398 teeth were crowned; 175 (44%)were incisors, 92 (23%) were premolars and the remaining 106 (27%) were molars. the upper right central incisors constitute the highest proportion (15.6%) of all the crowned teeth and the least were the third molars. about sixty four percent of the crowned teeth were in the maxilla and slightly over half (54.5%) were in females 5 funmilayo et al. their teeth. other reasons include previous failed crown, fracture, failed extensive restoration (table 3) . the most common reason for crown replacement was esthetics (23.1%), followed by secondary caries (13.5%), pulpal necrosis (10) (resulting in the need for endodontics) and unacceptable marginal adaptation (10%) (fig 1). table 3. reason for crown placement and replacement according to sociodemographs sociodemograph endodontic fracture aesthetic reasons failed extensive restoration tooth wear 10 caries failed crown total p value sex 0.007male 73 29 5 9 1 1 27 145 female 121 15 3 8 6 3 25 181 age group 0.72 ≤20years 12 4 0 1 2 19 21 40 63 16 5 7 1 1 10 103 41 – 60 69 16 3 4 4 3 23 122 61 – 80 48 8 5 2 17 80 >80 2 2 total 194 44 8 17 7 4 52 326 occupation 0.7 class 1 109 24 6 10 4 4 34 191 class 2 18 6 1 0 1 0 5 31 class 3 66 14 1 7 3 0 13 104 the commonest reason for initial crown placement was following endodontics (194, 59.5%) which is statistically significant across gender with more female crowning their teeth. other reasons include previous failed crown, fracture, failed extensive restoration. 0 12 7 2 10 2 3 0 0 12 1 3 1 10 1 2 5 crowns bridges2 10 8 6 4 2 se co nd ar y ca rie s u na cc ep ta bl e m ar gi na l ad ap ta tio n lo st c ro w n a es th et ic s w ea r en do do nt ic re as on s p er fo ra te d cr ow n p or ce la in fr ac tu re /d eb on di ng figure 1. reasons for replacement of crowns and bridges among patients seen. 6 funmilayo et al. twenty three bridges were used in replacing 44 missing teeth in different parts of the arch. the bridge designs used were fixed fixed (91.3%) and cantilever (8.7%). the number of units per bridge varied from 3 to 8, with the 3unit bridges constituting the highest (34.8%).(table 4 ). fourteen (60.9%) of the bridges fabricated were new while 9 (39.1%) were replacements. the reasons for replaced bridges include fracture of porcelain facing (22.2%), unacceptable marginal adaptation with resultant cementation failure (22.2%), and others.(fig 1). a high majority (88%) of all the prostheses (crowns and bridges) were fabricated using porcelain fused to metal. discussion in a developing country like ours, it is usually quite difficult to assess or evaluate restorations on long term basis because patient compliant to follow up is usually poor and most patients will only present when there is a fresh complaint, or a need for repair or replacement of the previous restoration. this, therefore, accounts for the retrospective evaluation of patients records seen during the study period. a lot of data is usually lost when evaluation like this is employed, more so, different criterion may be used by clinicians in recording their findings. the age range of patients seen in the present study of 16 – 85 years with a mean of 47±17.2 years is close to what was reported by akbar et al.17 in pakistan but slightly higher than that documented by oginni3 in the same region in nigeria while it is lower than that reported by akar et al.18 in turkey. this can be due to the fact that older age group now pays more attention to their dental care. epidemiological studies have shown that as life expectancy gradually increases, so does the percentages of elderly individuals in the population19. the tendency therefore is for this group of patients to retain more teeth in their late years and a desire towards fixed rather than removable prosthetic rehabilitation20,21. table 4. distribution of bridges fabricated n percentage(%) mode of bridge fabrication new 14 60.9 replacements 9 39.1 types of bridges cantilever 2 8.7 fixed fixed 21 91.3 no of units/bridge 3 units 8 34.8 4 units 7 30.4 5 units 2 8.7 6 units 4 17.4 8 units 2 8.7 a total of 23 bridges were fabricated out of which fourteen (60.9%) were new, 21 (91.3%) were fixed fixed. the number of units in the bridge vary from 3 to 8 with the 3 unit bridge been the most prevalent (34.8%) 7 funmilayo et al. this study reported a higher frequency (55%) of female with fixed prostheses which is corroborated by 51.7% and 66.67% reported by akbar et al and valderhaug respectively17,22 while on the contrary, a higher male preponderance of 57% was reported in another study23.the higher number of females with fixed prostheses in this study could be attributed to the fact that they tend to be more concerned with their esthetics and general appearance than male as earlier reported by ogunrinde et al.24. the fact that the highest proportion of patients that received crowns and bridges were in the age group 41 – 60 years may be an indication that this age group are those who are comfortable in their chosen profession and economically stable and can readily afford the cost of a fixed prosthesis more so that payments are done out of pocket3. in the present study, the upper central incisors were the most commonly (28.1%) crowned teeth. this is slightly lower than 33% reported by wilson et al.1 for the upper central incisors which were frequently crowned than lateral incisors. overall, the upper right central incisors were the most commonly crowned (15.6%) unlike in a previous study1 where the upper left central incisors were the most commonly (10%) crowned teeth. these incisors either the left or right are usually the most frequently involved in trauma thereby indicating them for root canal therapy followed by crowning. furthermore, the esthetic demand to restore these teeth to form and function is usually high because of the location. the upper second premolars were also found to be more frequently crowned than upper first premolar. this is similar to what has been previously reported1. this observation may not be unconnected with the positioning of the second premolar which is next to the first molar which takes active role in masticaion. furthermore, composite materials for the restoration of access cavity have been found to be less successful on posterior than on anterior teeth25. practitioners may therefore believe that the placement of crowns on posterior teeth with premolar inclusive remains a more predictable approach than placing large tooth-colored restorations. in addition, caries removal, access cavity preparation during endodontic treatment may also contribute to the fragility of the tooth26 and hence the need for full coronal coverage. in this study, almost 60% of teeth were crowned following endodontic treatment and 13.5% due to fracture. this is contrary to report by wilson et al.1 in which 26% of initial crown placement was due to intracoronal restoration failure, 38% to teeth fracture and esthetic reasons accounting for 15%. this therefore, suggests that contrary to certain perceptions, cosmetic considerations may not be a principal driver for resorting to the initial provision of crowns among practitioners in this environment. moreso, late presentation with the attendant extensive coronal destruction will necessitate crowning of most endodontically treated teeth in this environment. walton et al.11 and schwartz et al.12found caries to be the most common reason for crown failure accounting for 22% and 37% of cases seen respectively. loss of retention was also reported as the commonest reason for crown and fixed partial denture failure accounting for 28%15 and 45%14 in studies in cameroun15 and india14 respectively. however, in the study by oginni3, poor esthetics was the commonest reason for 40.5% of crown failure, followed by dental caries accounting for 15.4%. this is similar to present study where the most common reason for crown replacement were due to loss of esthetics (23.1%), and secondary caries accounted for 13.5% . this loss of 8 funmilayo et al. aesthetic may not be unconnected to the fact that the majority of these restorations were fabricated in porcelain-fused-to-metal and debonding of a large amount of the porcelain veneer will automatically lead to aesthetic failure. thus, esthetic demands may be seen to be on increase as patients’ expectations continue to rise, however further investigation is required. the ultimate goal of single crown replacement is to treat at first crown complication and secondly to improve esthetic and restore function26. the very few cases of fixed partial denture seen in this study does not allow for favorable comparison with other similar studies. the metal ceramic restoration remains the restoration of choice both in this study and previous studies1,15,18 because it combines both the esthetic and strength. in addition, this type of restoration can withstand high masticatory forces generated during chewing associated with the more fibrous nigerian diet3. though the strength of zirconia is comparable with metal, the lack of adequate facilities in our center makes the fabrication impossible. in conclusion, the most commonly crowned teeth in this study were the upper right central incisors. definitive restoration of endodontically treated teeth and esthetics were the commonest reasons for initial crown placement and replacement respectively. thus, esthetic demand may be on the increase as patients expectation continue to rise, however further investigation is required. references 1. wilson na, whitehead sa, mjor ia, wilson nhf. reasons for the placement and replacement of crowns in general dental practice. prim dent care. 2003 apr;10(2):53-9. 2. kim kl, namgung c, cho bh. the effect of clinical performance on the survival estimates of direct restorations. restor dent endod. 2013 feb;38(1):11-20. doi: 10.5395/rde.2013.38.1.11. 3. oginni ao. failures related to crowns and fixed partial dentures fabricated in a nigerian dental school. j contemp dent pract. 2005 nov 15;6(4):136-43. 4. fernandes na, vally zi, sykes lm. the longevity of restorations. a literature review. s.afr dent j. 2015 oct;70(9):410-3. 5. chadwick b, treasure e, dummer p, dunstan f, gilmour a., jones r, et al. challenges with studies investigating longevity of dental restorations – a critique of a sysytematic review. j dent 2001 mar;29(3):155-61. 6. sheldon t, treasure e. dental restoration, what type of filling. eff health care. 1999;5(2):1-12. 7. mjor ia. placement and replacement of amalgam restorations in italy. oper dent. 1992 mar-apr;17(2):70-3. 8. deligeorgi v, mjor ia, wilson nhf. an overview of the reasons for the placement and replacement of restorations. prim dent care. 2001 jan;8(1):5-11. 9. ajayi dm, abiodun-solanke, arigbede ao. evaluation and treatment of failed amalgam restorations in ibadan. west afr j med. 2013 oct-dec;32(4):248-53. 10. goldstein gr. the longevity of direct and indirect posterior restorations is uncertain and may be affected by a number of dentist, patient and material related factors. j evid based dent pract. 2010 mar;10(1):30-1. doi: 10.1016/j.jebdp.2009.11.015. 11. walton jn, gardner fm, agar jr. a survey of crown and fixed partial denture, failures, length of service and reasons for replacement. j prosthet dent. 1986 oct;56(4):416-21. 9 funmilayo et al. 12. schwartz nl, whitslf ld, berry tg, stewart jl. unserviceable crowns and fixed partial denture, the lifespan and causes for loss of serviceability. j am dent assoc. 1970 dec;81(6):1395-401. 13. cheung gsp. a preliminary investigation into the longevity and causes of failure of single unit extracoronal restorations. j dent. 1991 jun;19(3):160-3. 14. sudihr p. failures of crown and fixed partial dentures. a clinical survey. int j contemp dent. 2011jan;2(11):120-1. 15. elage ke. failure of crowns and fixed partial dentures in yaounde [thesis]. faculty of medicine and biomedical sciences, the university of yaounde i; 2015. 16. office of population census and surveys opcs. standard occupational classification. london: hmso; 1991. v. 3. 17. akbar k, fahad a, azmat ak. complaints among patients wearing metal ceramic fixed partial dentures. j kyber col dent. 2014 dec;5(1): 1-5. 18. akar gc, ozdemir n, uluer h, aksoy g. a clinical evaluation of fixed partial dentures. acta stomatol croat 2009 jan;43(2):99-109. 19. kanstrom l, zamaro g, sjosted c, green g, editors. healthy ageing profiles: guidance for producing local health profiles of older people. geneva: who; 2008 [2017 jan 16]. available from: http://www.euro.who.int/__data/assets/pdf_file/0011/98399/e91887.pdf. 20. zitzmann nu, staehelin k, walls aw, menghini g, weiger r, zemp stutz e. changes in oral health over a 10 year period in switzerland. eur j oral sci. 2008 feb;116(1):52-9. doi: 10.1111/j.1600-0722.2007.00512.x. 21. begwitz ic, soderfeldt b, palmqvst s, nilner k. oral prostheses and oral health –related quality of life: a survey of study of an adult swedish population. int j prosthodont. 2007 mar-apr;20(2):132-42. 22. valderhaug j, karlsen k. frequency and location of artificial crowns and fixed partial dentures constructed at a dental school. j oral rehabil. 1976 jan;3(1):75-81. 23. nandhini ga, sangeetha s. evaluation of post-operative complaints in fixed partial denture wearers and those with crowns: a questionnaire based study. int j cur res rev. 2016 aug;8(16):30-4. 24. ogunrinde tj, dosumu oo. the influence of sociodemographic factors and medical conditions on patients compliants with complete dentures. annals ib pg med 2002;10(2):16-21. 25. frieedl k-h, hiller ka, schmalz g. placement and replacement of composite restorations in germany. oper dent. 1995 jan-feb;20(1):34-8. 26. khiari a, hadyaoui d, saafi j, harzallah h, cherif m. clinical attitude for failed fixed restorations: an overview. dent open j 2015;2(4):100-4. doi :10.17140/doj-2-119. 1http://dx.doi.org/10.20396/bjos.v20i00.8664995 volume 20 2021 e214995 original article 1 pdm dental college & research institute, bahādurgarh, india. 2 jamia millia islamia, faculty of dentistry, delhi, india. corresponding author: abhishek mehta jamia millia islamia, faculty of dentistry, delhi, india e-mail: amehta@jmi.ac.in editor: dr altair a. del bel cury received: march 03, 2021 accepted: june 06, 2021 knowledge and practices of recording and maintaining patients ‘dental records among private dental practitioners of delhi, india gurkiran kaur1, abhishek mehta2,* , aastha sahani1, shradha malik1 proper recording and keeping dental records are an important part of any dental practice. it helps in improving patient care, has medico-legal importance and play significant role in human identification during mass disasters or criminal offences. aim: to assess the knowledge and practices of recording and maintaining patients ‘records among private dental practitioners of delhi, india. methods: data for this cross-sectional study was collected from 160 dentists of delhi using a self-administered questionnaire. the face and content validity as well as reliability of questionnaire was tested before the final data collection. a single trained examiner collected all the necessary information via personal visits or google forms. chi-square test was applied to check the statistically significant difference between the dichotomous independent variables with respect to study participants’ responses to the questionnaire. results: the mean age of the study participants was 34.5 (sd 7.2) years.digital method of recording patient’s data and x-ray storage was more prevalent than manual method among the study participants. slightly more than 40% of the dentists were keeping patient’s records safe for a period of 6 to 10 years. younger dentists with lesser years of practice were more explicit in recording and correcting patient records. conclusion: results of this study shows that private dental practitioners of delhi are aware of medico-legal importance of dental records. most of them were recording important findings and history of their patients. dentists must be educated in two aspects namely correct method of recording and the ideal duration of storing their patients’ dental records. keywords: dental records. dentists. forensic dentistry. https://orcid.org/0000-0002-9192-7615 2 kaur et al. introduction the dental record is an official document that has all the diagnostic information (odontogram, radiographs and casts), clinical notes, treatment performed and patient-related communications that occur in the dental office, including instructions for home care and consent to treatment1. recording the patient –related data in an accurate, comprehensive and efficient manner is an important part of clinical practice. dental records are useful in cases of malpractice suits, insurance claims and in the field of forensic dentistry. a complete dental record of the patient is a valuable commodity in human identification in cases of bite assaults, child abuse or age disagreements as in child marriages and child labour or criminal proceedings2,3. these records can aid in the identification of a dead or missing person, criminal investigations and in cases of mass disasters. numerous instances are on record wherein dental records have been used to identify the victims of mass disasters such as 2004 tsunami and in criminal investigations as in the 2012 nirbhaya rape case in delhi4,5. in a systematic review of 20 mass disasters, in 17 of them the use of forensic odontology for identification of the victims was found in about 14.70% cases. in kentucky air crash 100% of the identification was done by dental remains, 76% in newark air crash and 71.42% in nepal air crash6. dental records can play an important role in identification of a dead body which has been grossly decomposed and is difficult to identify visually. this is due to the capability of dental tissues to withstand high temperature, humidity and pressure. but the critical feature of dental identification lies in being able to obtain accurate ante-mortem records that can be matched with the post-mortem dental findings7,8. developed countries have a well-established system of maintaining patients’ dental record. this is unlike what we commonly find in developing countries like india wherein in spite of extensive dental treatment a person’s complete dental chart is usually not available. this acts as a hindrance in cases relating to mass disasters or crime involving decomposed unidentified remains as there is no ante-mortem data to match the available post-mortem findings. studies conducted in india have shown the lack of awareness and practice of dental record maintenance especially by private dental practitioners9-12. these practices make the dentist prone to being guilty of negligence under consumer protection law and also reduces the wider role for the dentists in the field of forensic science. delhi is the capital of india and lies in a zone-4 of earthquake prone seismic areas13. it is important that every dental health care professional in delhi should maintain proper records that can be obtained in case of mass disasters so as to supplement forensic medicine teams for victim identification. therefore, this study was planned with the objective to assess the knowledge and practices of dentists working in private clinics across delhi regarding the writing and maintenance of patients’ dental records. materials and methods study design, sampling frame and sample size this was a questionnaire-based cross-sectional study conducted among private dental practitioners in delhi state. based on the similar studies conducted in india, 3 kaur et al. it was observed that a sample size of around hundred-fifty dentists is appropriate for present study9,11. as there is no directory of registered dentists in delhi, we contacted local indian dental association branches for the list of their member dentists. we shortlisted 180 dentists via convenience sampling method from this list, in case few dentists refused to participate. participants and ethical clearance dentist practicing within delhi state limits and were willing to participate after signing written consent form were eligible for this study. before starting the study a research proposal was submitted to the ethical research committee of the prabhu dayal memorial dental college and research institute and ethical clearance was obtained on 27-04-2019 (letter no. pdm/iec/02/2019). setting and data collection the student investigator (as) collected the data under the supervision of the guide (gk). the selected dentists were contacted telephonically and explained the objectives of conducting this study. those willing to participate were requested for appointment and the student investigator personally went to their clinic to get the questionnaire filled. written informed consent was obtained before participants started to respond to the questionnaire. dentists were also given the option of filling online questionnaire through google form. twenty five dentists opted for online submission. data collection was done within two months (july and august 2019). questionnaire performa a self-designed questionnaire comprising of 24 close-ended questions was used to collect the data from the participating dentists. the questionnaire was divided into four blocks, namely, baseline characteristics of study participants, importance and method of recording case history and dental findings, technical considerations of record keeping and final block of question focused on radiographic records and their preservation. the questionnaire was sent to five experts in the field of forensic odontology. their inputs and suggestions were used to make changes and finalize the questionnaire (face and content validity). most of the suggestions were on changing the way questions are framed so that study participants are able to understand them. the reliability was tested by repeating the questionnaire to ten previously participating dentists. the kappa value was 0.8, hence found to be good. statistical analysis spss (statistical package for social sciences) version21 was utilized for statistical analysis of the recorded data. one-sample binomial test was applied to check statistical differences in frequency distribution of independent variables such as gender and highest qualification. statistical difference between the responses of the study participants according to dichotomous groups such as age and years of practices was assessed using chi-square test. p value was set at less than 0.05. 4 kaur et al. results among the 180 dentists contacted for this study, only 160 gave consent and completed the questionnaire. hence response rate was 88.8%. the main reason for refusing to participate in the study was lack of time to complete the questionnaire. there were significantly more males than females in the final sample. also, significantly more participating dentists had completed only bachelor’s degree (66.3%) when compared to those (33.7%) who had done post-graduation as well (one sample binomial test, p<0.05). therefore, we didn’t do further comparison of study participants based on these two variables. we divided study participants into approximately equal size dichotomous groups based on age i.e upto 32 and above 32 years and number of years of practice i.e upto 8 and above 8 years for further comparative analysis. these groups were created in such a way that approximately equal number of dentists are there in each group. most of the dentists agreed that not maintaining dental records is a medical negligence (n=156, 97.5%). slightly more than half (n=93, 58.1%) of the participating dentists felt dental records must be kept safe for medico legal purpose. forty-seven (29.4%) dentists felt good record keeping helps in providing better services to their patients (table 1). table 1. baseline characteristics of the study participants characteristics n % p value overall 160 100 age group <= 32 years 76 47.5 >32 years 84 52.5 gender* male 93 58.1 0.04 female 67 48.9 highest qualification** bds 106 66.3 0.0001 mds 54 33.7 number of years of practice <=8 years 88 55.0 >8 years 72 45.0 do you know not keeping dental records is a medical negligence? yes 156 97.5 no 04 2.5 most important reason for record keeping? medico legal purpose 93 58.1 better patient service 47 29.4 helps in transferring patient to other dentist 02 1.3 all of the above 18 11.3 *significant difference between the groups p<0.05, one sample binomial test ** highly significant difference, p<0.0001, one sample binomial test 5 kaur et al. more dentists were recording case history performa digitally (n= 74, 46.2%) when compared to writing on a printed form (n= 44, 27.5%) or on a blank page (n=42. 26.3%), although, the difference between them was not statistically significant. almost all the dentists (n= 157, 98.1%) were asking and recording medical history from the patients visiting their clinic. only two-third of the dentists were recording number of teeth present (n= 101, 63.1%), teeth with restorations (n= 107, 66.9%) or caries (n=139,86.9%) or any abnormalities in dentition or jaws (n= 107, 66.9%).around 3/4th of the participating dentists (n= 123, 76.9%) were recording the type of prosthesis their patient was wearing (table 2). table 2. study participants’ response on practice of recording case history and dental findings questions age upto 32 years age above 32 years p value years of practice upto 8 years years of practice above 8 years p value overall n (%) do you start a record file for all new patients? yes 73(96.1) 79(94) 0.41 83(94.3) 69(95.8) 0.47 152(95) no 03(1.9) 05(3.1) 05(5.7) 03(1.9) 08(5) how do you record case history? manually on a printed forms 17(22.4) 27(32.1) 22(25) 22(30.6) 44(27.5) manually on a blank page 22(28.9) 20(23.8) 0.37 22(25) 20(27.8) 0.56 42(26.3) digital 37(48.7) 37(44) 44(50) 30(41.7) 74(46.2) do you record medical history of the patients? yes 73(96.1) 84(100) 0.10 85(96.6) 72(100) 0.25 157(98.1) no 3(3.9) 0 3(3.4) 0 3(1.9) do you document teeth which are present and sound? yes 52(68.4) 49(58.3) 0.19 60(68.2) 41(56.9) 0.18 101(63.1) no 24(31.6) 35(41.7) 28(31.8) 31(43.1) 59(36.9) do you record all carious teeth? yes 68(89.5) 71(84.5) 0.48 79(89.8) 60(83.3) 0.24 139(86.9) no 08(10.5) 13(15.5) 09(10.2) 12(16.7) 21(13.1) do you record teeth with restorations? yes 56(73.7) 51(60.7)* 0.09 64(72.7) 43(59.7)* 0.09 107(66.9) no 20(26.3) 33(39.3) 24(27.3) 29(40.3) 53(33.1) do you record type of prosthesis? yes 63(82.9) 60(71.4)* 0.09 75(85.2) 48(66.7)* 0.008 123(76.9) no 13(17.1) 24(28.6) 13(14.8) 24(33.3) 37(23.1) do you note any additional abnormalities in the dentition and jaws? yes 60(78.9) 47(56)** 0.002 73(83) 34(47.2)** 0.0001 107(66.9) no 16(21.1) 37(44) 15(17) 38(52.8) 53(33.1) continue 6 kaur et al. significantly more number of younger dentists (up to 32 years) and those with less than 8 years of practice were recording teeth with restorations (n= 107, 66.9%) or any abnormalities in dentition or jaws, presence of any prosthesis as compared to their elder counterpart. they were also mentioning the name of medication prescribed by them and treatment done on the particular appointment in significantly higher proportion as compared to the corresponding group. most of the dentists were keeping a record of every treatment done (n=145, 90.6%) on their patients with younger dentists recording this finding in higher frequency as compared to older ones (table 2). slightly more than half (n=85, 53.1%) of the participating dentists were obtaining written informed consent from all of their patients whereas 43.1% (n=69) were obtaining it for selected cases only. when asked regarding method of making a correction in dental record sheet, 71.3% (n=114) dentists were using a single line to cross out and very few reported they cut the written matter in such a way that nobody can read it (n=21, 13.1%). slightly more than half (n=86, 53.8%) of the participating dentists were writing an explanation for the corrections they do in dental record sheet, this practice was followed in significantly higher frequency by younger group of dentists. there was variability in response to question related to duration of retaining patient records, 44.4% (n=71) of the dentists were retaining from 6 to 10 years but 43.2% (n=69) were retaining patients records for less than one year. sixty percent (n=96) were preserving patient record file with themselves, whereas 23.8% (n=38) were giving it to the patient and 16.3% (n=26) were just handing over a copy of the records sheet. dentists in the age group of upto 32 years and those with less than 8 years of practice reported in statistically significantly higher numbers that they are recording date and time of their patient’s every visit to the dental office (table 3). continuation do you mention prescribed medication in the record file? yes 57(75) 49(58.3)* 0.03 65(73.9) 41(56.9)* 0.02 106(66.3) no 19(25) 35(41.7) 23(26.1) 31(43.1) 54(33.8) do you keep record of every treatment done? yes 73(96.1) 72(85.7)* 0.02 85(96.6) 60(83.3)* 0.004 145(90.6) no 03(3.9) 12(14.3) 03(3.4) 12(16.7) 15(9.4) *statistically significant difference, p<0.05; chi-square test ** highly significant difference, p<0.0001 table 3. practice of study participants regarding technical considerations of record keeping questions age upto 32 years age above 32 years p value years of practice upto 8 years years of practice above 8 years p value overall n (%) do you record date and time of patient’s every visit to the clinic? yes 70(92.1) 68(81)* 0.06 82(93.2) 56(77.8)* 0.006 138(86.3) no 06(7.9) 16(19) 06(6.8) 16(22.2) 22(13.8) continue 7 kaur et al. continuation do you obtain written informed consent from your patients? yes for all patients 43(56.6) 42(50) 53(60.2) 32(44.4)* 85(53.1) only for few selected patients 29(38.2) 40(47.6) 0.36 31(35.2) 38(52.8) 0.08 69(43.1) only for child patients 04(5.3) 02(2.4) 04(4.5) 02(2.8) 06(3.8) what do you use for writing records? ball or ink pen 63(82.9) 66(78.6) 67(76.1) 62(86.1) 129(80.6) pencil 0 01(1.2) 0.54 1(1.1) 0 0.22 01(0.6) phone or tab 13(17.1) 17(20.2) 20(22.7) 10(13.9) 30(18.8) do you maintain a chronological order of the recorded data? yes 54(71.1) 57(67.9) 0.39 66(75) 45(62.5)* 0.06 111(69.4) no 22(28.9) 27(32.1) 22(25) 27(37.5) 49(30.6) how do you make a correction in dental records sheet? single line cross out 57(75) 57(35.6) 64(72.7) 50(69.4) 114(71.3) double line cross out 03(3.9) 07(8.3) 0.63 03(3.4) 07(4.4) 0.36 10(6.3) cut in a way that nobody can read it 10(13.2) 11(13.1) 12(13.6) 09(5.6) 21(13.1) correction fluid 06(7.9) 08(9.5) 09(10.2) 05(6.9) 14(8.8) with eraser 0 01(1.2) 0 01(1.4) 01(0.6) do you preserve all correspondence related to the patients? yes 53(69.7) 41(48.8)* 0.006 64(72.7) 30(41.7)** 0.0001 94(58.8) no 23(30.3) 43(51.2) 24(27.3) 42(58.3) 66(41.3) do you write any explanations for corrections in the dental record? yes 47(61.8) 39(46.4)* 0.03 57(64.8) 29(40.3)* 0.002 86(53.8) no 29(38.2) 45(53.6) 31(35.2) 43(59.7) 74(46.2) duration of retaining the dental records less than one year 36(47.4) 33(39.2) 40(44.9) 29(39.5) 69(43.2) 1 to 5 years 08(10.5) 10(11.9) 0.59 05(5.7) 13(18.1) 0.18 18(11.3) 6 to 10 years 31(40.8) 40(47.6) 42(47.7) 29(40.3) 71(44.4) more than 10 years 01(0.6) 01(0.6) 01(0.6) 01(0.6) 02(1.3) what do you do with the patient record file after treatment? preserve the file with myself 43(56.6) 53(63.1) 54(61.4) 42(58.3) 96(60) give file to the patient 21(27.6) 17(20.2) 0.54 22(25) 16(22) 0.60 38(23.8) handover a copy of record to the patient 12(15.8) 14(16.7) 12(13.6) 14(19.4) 26(16.3) *statistically significant difference, p<0.05; chi-square test 8 kaur et al. on the questions related to radiographs, 70% (n=112) of the dentists reported that they were taking digital x-ray exclusively. slightly more than half (n=84, 52.5%) of them were retaining digital copy of the patient’s radiograph, whereas some were retaining hard copy (n=19, 11.9%) or keeping one copy with them and providing other to patient (n=27, 16.9%). most of the dentists in the study participating reported that they mention radiographic findings in dental record sheet (n=131, 81.9%). no significant difference was observed for the responses to these questions based on age group or years of practice (table 4). discussion as dental patients are becoming more aware of their rights and protecting consumer laws, there has been an increase in identification of cases of dental malpractice. during any legal procedure a written document or the past radiograph of the patient has more weightage than verbal statement of the patient or the health care professional, hence, it has become crucial for dental professionals to maintain a comprehensive patient record. apart from medico-legal aspect, dental records are useful in forensic dentistry, as a mean of communication with other dentist, patient’s follow-up visit, providing information to third – party insurer and research purposes3. this questionnaire survey tried to probe on the awareness and practices of recording and maintenance of dental records by private dental practitioners of delhi. keeping the dental records of the patients in a safe, retrievable manner for a specified period is an important part of clinical dental care. various countries and associations have specified the minimum period of the maintenance of patient’s records. this duration usually ranges from 6 to 10 years1,14. in india, physicians are required to store their patient records for 3 years and clinical trials participants’ data upto 5 years15. but unfortunately, the laws in india are not practiced as strictly as they should be. table 4. responses of participants to questions related to radiographic records and their preservation questions frequency percentage which type of radiographs do you take in your clinic? conventional 25 15.6 digital 112 70 both 23 14.4 do you mention radiographic findings in case sheet? yes 131 81.9 no 29 18.1 what do you do with patient radiograph after treatment? retain x ray as a hard copy 19 11.9 retain x ray as a soft copy 84 52.5 handover x ray to the patient 28 17.5 one copy with you and other to patient 27 16.9 do not retain x ray 02 1.3 9 kaur et al. in the present study, only 44.4% (n=71) participating dentists were maintaining dental records for 6 to 10 years and 43.2% (n=69) for less than a year. in comparison, studies done in other states of india had reported a skewed picture of record maintenance where of the total sample only 7%16, 18%12, 38%10, 43%17to 50%11 dentists were maintaining dental records for the desired period. there are debates as to whom the dental records belong to, dentist or patient, therefore it is safe that both keep them safely. patient can be provided a copy of their original dental record sheet. around 16% of the dentists in our study were following this practice whereas majority of them (n=96, 60%) were keeping it with them only. recording the date and time of appointment of the patient has a medico-legal significance. it can be used as an evidence for time and location of the person. in our survey, 86.3% (n=138) dentists were recording date and time of their patient’s every visit to their clinic. these numbers are similar to those reported in a study done in sudan18. an encouraging finding of our survey was significantly more number of younger dentists and those with fewer years of practice were recording date and time of their patient’s every visit as compared to their comparative groups. another aspect of recording proper method of correcting the errors. if the dentist wishes to do any correction in the patient’s dental record it should be a single cross cut with a line. use of correction ink is not recommended nor should it be cut in a way that nobody can read it. around 30% of the participating dentists were not following the correct method of error correction in our study. these mistakes can go against the dental professional in the court of law during malpractice litigations. dental record keeping should not be a time-consuming exercise otherwise it will discourage the practicing dentists to follow its correct and systematic method. specifically designed computer software can save the time and energy of the dentist in record-keeping and should be encouraged in dental practice. in our study, only 46.2 % (n=74) of the participating dentists reported that they are keeping dental record digitally. this figure is higher than reported among dentists of mangalore (2%)12, pune (11%)11, madhya pradesh (24%)17and rajasthan (26%)10. dentists must be made aware of these soft wares. record keeping of additional investigations advised during the course of diagnosis and treatment is equally important. most common of these investigations is the radiographs. dentists must keep record of the radiograph/s taken and mention its findings in the patient record file. more than 80% of the participating dentists (n=130, 81.2%) were safe-keeping patient’s radiograph either as hard or digital copy with themselves. this figure is higher than reported in dentists of mangalore (59%)12, punjab and uttar pradesh (47%)9, and pune (77%)11. asking and recording medical history is an important and mandatory aspect of case history. failure to record proper medical history can have medico-legal consequences in case of any medical emergencies arising after dental treatment or medications prescribed by the dentist. majority of the participating dentists in our study had reported to record medical history (n=157, 98.1%) in patient case sheet. similar studies conducted in other parts of india had reported less figures ranging from 22%9, 31%12, 38% 10 to 44.6%11. a study in sudan reported that 57.1% of the surveyed dentists were recording medical history18. 10 kaur et al. few limitations of this study are, firstly, as the sampling frame for this study is limited to private dental practitioners of delhi state therefore, these results don’t reflect the knowledge and practices of indian dentists on dental record writing and keeping. second, as this is a self –reporting study it is prone to recall bias. third, there may be issue of selection bias as dentists are selected from a list of members of an association hence we may have missed interviewing non-member dentists. in conclusion, overall the participating dentists had fair knowledge of importance of patients ‘ record but still some of them were making mistakes while writing and maintaining them safely for recommended period of time. lack of knowledge and poor implementation of good practices were common among dentists of higher age group and more years of dental practice. this could be due to absence of regular reinforcement for good dental practices among indian dental fraternity. we suggest a two prong strategy to overcome this shortcoming by means of regulatory approach in form of stricter laws and second, an educational approach for correct information dissemination aimed at increasing awareness of dentists towards standard rules of recording and maintaining dental records. future research on this subject is required with a larger sample size and more questions pertaining to forensic dentistry. references 1. american dental association. council on dental practice. division of legal affairs. dental records. ada; 2010 [cited 2021 feb 25]. available from: https://www.aapd.org/globalassets/media/safetytoolkit/dental-records-ada.pdf. 2. avon sl. forensic odontology: the roles and responsibilities of the dentist. j can dent assoc. 2004 jul-aug;70(7):453-8. 3. gooneratne i. dental records: medico-legal and clinical significance. sri lanka dent j. 2015;45(2):63-8. 4. morgan ow, sribanditmongkol p, perera c, sulasmi y, van alphen d, sondorp e. mass fatality management following the south asian tsunami disaster: case studies in thailand, indonesia, and sri lanka. plos med. 2006 jun;3(6):e195. doi: 10.1371/journal.pmed.0030195. 5. kattiman bf. nirbhaya case: dharwad college helped with forensic analysis. the times of india. 2013 sep 13 [cited 2021 feb 24]. available from: https://timesofindia.indiatimes.com/city/hubballi/ nirbhaya-case-dharwad-college-helped-with-forensic-analysis/articleshow/22528229.cms. 6. prajapati g, sarode sc, sarode gs, shelke p, awan kh, patil s. role of forensic odontology in the identification of victims of major mass disasters across the world: a systematic review. plos one. 2018 jun;13(6):e0199791. doi: 10.1371/journal.pone.0199791. 7. chandra shekar br, reddy cv. role of dentist in person identification. indian j dent res. 2009 jul-sep;20(3):356-60. doi: 10.4103/0970-9290.57377. 8. devadiga a. what’s the deal with dental records for practicing dentists? importance in general and forensic dentistry. j forensic dent sci. 2014 jan;6(1):9-15. doi: 10.4103/0975-1475.127764. 9. gupta a, mishra g, bhutani h, hoshing c, bhalla a. forensic revolution need maintenance of dental records of patients by the dentists: a descriptive study. j int soc prev community dent. 2016 julaug;6(4):316-20. doi: 10.4103/2231-0762.186799. 10. astekar m, saawarn s, ramesh g, saawarn n. maintaining dental records: are we ready for forensic needs? j forensic dent sci. 2011 jul;3(2):52-7. doi: 10.4103/0975-1475.92143. 11 kaur et al. 11. sarode gs, sarode sc, choudhary s, patil s, anand r, vyas h. dental records of forensic odontological importance: maintenance pattern among dental practitioners of pune city. j forensic dent sci. 2017 jan-apr;9(1):48. doi: 10.4103/jfo.jfds_1_16. 12. wadhwani s, shetty p, sreelatha sv. maintenance of antemortem dental records in private dental clinics: knowledge, attitude, and practice among the practitioners of mangalore and surrounding areas. j forensic dent sci. 2017 may-aug;9(2):78-82. doi: 10.4103/jfo.jfds_64_15. 13. wikipedia. earthquake zones of india [cited 2019 jul 18]. availible from: https://en.wikipedia.org/ wiki/earthquake_zones_of_india. 14. college of dental surgeons of british columbia. dental records management. vancouver: clds; 1996 [cited 2020 may 3]. available from: https://www.academia.edu/6307817/dental_records_mgt. 15. indian council of medical research. national ethical guidelines for biomedical and health research involving human participants. new delhi: icmr; 2017 [cited 2020 sep 16]. available from: https://main.icmr.nic.in/sites/default/files/guidelines/icmr_ethical_guidelines_2017.pdf.  16. preethi s, einstein a, sivapathasundharam b. awareness of forensic odontology among dental practitioners in chennai: a knowledge, attitude, practice study. j forensic dent sci. 2011 jul;3(2):63-6. doi: 10.4103/0975-1475.92145. 17. tomar u, airen b, sarkar pa, singh h, bishen ka. a vigilance alert for forensic odontology: preservation and maintenance of dental records in central india. indian j dent sci. 2020;12(1):16-20. doi: 10.4103/ijds.ijds_61_19. 18. waleed p, baba f, alsulami s, tarakji b. improtance of dental records in foresic dental identification. acta inform med. 2015;23(1):49-52. doi: 10.5455/aim.2015.23.49-52. 1http://dx.doi.org/10.20396/bjos.v20i00.8661359 volume 20 2021 e211359 original article 1 department of health sciences and child dentistry, piracicaba dental school, university of campinas (unicamp), piracicaba, são paulo, brazil. 2 department of community health jundiai medical school jundiaí, são paulo, brazil. 3 department of physiatry and nursing, faculty of health sciences, university of zaragoza, spain. *corresponding author: maria da luz rosário de sousa av limeira 901 piracicaba, são paulo, brazil email: luzsousa@unicamp.br received for publication: october 9, 2020 accepted: december 21, 2020 effects of the intervention of the multicenter study idefics on the prevalence of caries in spanish children vinícius aguiar lages1 , maria paula rando meirelles1 , marília jesus batista2 , carolina matteussi lino1 , andréa moscardini da costa1 , luis alberto moreno aznar3 , maria da luz rosário de sousa1,* aim: to evaluate the effects of an intervention for the prevention of obesity on the prevalence of dental caries disease in spanish children. methods: two cities participated intervention study nested in a cohort idefics (identification and prevention of dietary and lifestyle induced health effects in children and infants): huesca, where there was a 2-year intervention, which encouraged less sugar consumption; and zaragoza (control). the prevalence of caries was evaluated by examining the 1st permanent molars in the 7-11 age range, using the icdas (international caries detection and assessment system). these teeth erupt at 6 years of age and at the baseline (2007-2008) were free of caries because they were not present in the oral cavity. as outcomes, white spots were selected, combining the icdas criteria 1 and 2, and untreated caries, combining criteria 4, 5 and 6. their  association with socioeconomic variables, bmi (body mass index), frequency of sugar intake, sex and parents’ perceptions of their children, was investigated. to  do so, the chi-square test was applied (p<0.05). results: the  sample consisted of 281 children. the prevalence of white spots and untreated caries was higher in huesca, despite the intervention. there was no association between the outcomes and the variables studied (p>0.05). conclusion: the intervention for the prevention of obesity did not exert any association with the prevalence of caries in spanish children. keywords: oral health. dental caries. obesity. http://dx.doi.org/10.20396/bjos.v20i00.8661359 mailto:luzsousa@unicamp.br https://orcid.org/0000-0003-0110-3987 https://orcid.org/0000-0001-6197-1757 https://orcid.org/0000-0002-0379-3742 https://orcid.org/0000-0001-6686-3296 https://orcid.org/0000-0003-1734-1779 https://orcid.org/0000-0003-0454-653x https://orcid.org/0000-0002-0346-5060 2 lages et al. introduction the prevalence of childhood obesity has increased rapidly in europe, and in other continents, and has become a serious global public health problem1. this problem tends to persist into adult life, and is a risk factor in the occurrence of various chronic diseases, such as cardiovascular disease and diabetes1. the fact that eating habits are a key common etiological component of both obesity and dental caries2 stimulates interest as the latter is also a serious public health problem in several countries3 and the prevalence of both diseases can be reduced with healthy diets and low sugar consumption2. studies on the relationship between obesity and dental caries present inconclusive results. some have shown a positive association between them2, while others4 show no relationship between these factors in schoolchildren, but found that the higher the parents’ educational level, the lower the prevalence of obesity and dental caries among their children. socioeconomic inequalities can create unequal opportunities among people5 and have different effects on these diseases, as they are influenced by factors such as education, income and saccharose-rich diets2,5. thus, new methodologies are necessary to verify this association. studies of schoolbased interventions6 have been effective in improving the health and behavioral conditions of adolescents, considering that the school period is an essential phase for acquiring knowledge, modifying and creating healthier lifestyle habits. different methods of interventions6  have managed to cause positive changes in lifestyle through increased consumption of fruits and vegetables and increased levels of physical activity. however, the scientific literature lacks studies of this type focused on oral health. to face this growth of obesity in the population, the multicentric idefics (identification and prevention of dietaryand lifestyle-induced health effects in children and infants) was set up, with intervention modules which addressed diet, physical activity, and stress management7. it was a prospective cohort study in a large diverse sample of children in europe, which started at the beginning in 2007. it investigated the causes of dietary and lifestyle-related diseases and disorders, with a main focus on overweight and obesity8. this cohort involved the drawing up, implementation and assessment of a community-oriented intervention program for the primary prevention of obesity in a controlled study project7. spain was the focus of this study and it took place in the cities of zaragoza and huesca. the i.family study, which continued from the idefics, verified determinants of eating behavior in children, adolescents and their parents of european origin9. based on data collected from more than 10,000 children who were under 10 years old in the idefics study, the i.family reevaluated these children and their families and identified those who maintained healthy diets and eating habits from the intervention program and compared them to children who had not received the intervention9. this phase, which began in 2013, included the oral health assessment of spanish children participating, thereby providing a pioneering dentistry study within a cohort with a focus on behaviors, lifestyles and eating habits. the purpose of this study was to evaluate the effects of an intervention for the prevention of obesity on the prevalence of dental caries disease in spanish children. 3 lages et al. materials and methods the idefics and ifamily studies a cohort of 16224 children between 2 to 9-year-olds from eight european countries (sweden, germany, hungary, italy, cyprus, spain, belgium and estonia) participated in the first population survey of the idefics study. defined as t0, it took place between september 2007 and may 2008 was the first stage (baseline) of the prospective cohort study and was the initial stage for the idefics intervention (figure a). all children in the defined age group residing in the regions studied and attending public primary schools (grades 1 and 2), preschools or kindergartens were eligible to participate. in addition to the signed informed consent provided by parents, each child was invited to give their verbal approval. the t0 exams included anthropometric data, lifestyle, biological markers, behavioral and sociodemographic characteristics and were based on a highly standardized protocol, as set out by ahrens et al.8 (2011). the idefics study was not designed with population representativeness of each country. in most countries, the regions selected were individual cities or communities, most of which were located in the same geographical area. the intervention and control regions in each country were selected for convenience, such as the distance between the research teams involved, in order to reduce costs. the t0 occurred in the intervention (where education actions would be carried out) and control (without health education actions) regions selected in these countries, allowing researchers to assess and describe health conditions, eating habits and lifestyle of children in europe, taking regional, social, biological and sex aspects into account. 2007 2008 2009 2010 2013 2014 t0 and intervention t1 and t2 idefics t3 and assessment of oral health ifamily figure a. schedule of exams of the idefics cohort and the ifamily study. t0, initial research phase (baseline); t1, first exam after intervention; t2, questionnaires sent to participants via postal service to assess the intervention; t3, second exam after intervention, with assessment of oral health in spain. after this phase, the primary prevention program was implemented. the intervention was designed to address key behaviors in relation to obesity (diet, physical activity and stress) at four levels: individual (children), family (parents), school and community. at these levels, six messages related to these behaviors were worked on, through 10 separate modules with intervention measures targeting each level. the six messages were “increase water consumption”; “increase consumption of fruit and vegetables”; “reduce daily screen time (television and computer)”; “increase daily physical activity”; “improve the quality of family life”; “ensure adequate sleeping hours”. the  interven4 lages et al. tion design and details of the different intervention modules of the idefics study are described, according to their level of intervention7,10. the effects of the intervention were evaluated during the t1, 2 years after t0, repeating the same exam modules with children from both control and intervention areas, to compare both regions of each country. at the end of t1, 69% (n = 11,189) of the children who had participated were reevaluated. dropout between the exams was higher among those with overweight, low schooling, children of single parents and low scores for well-being11. the prevalence of overweight and obesity stratified by sex and country were recorded. in 2010, a second follow-up, defined as t2, was conducted using a questionnaire sent through the postal service to all intervention participants to evaluate sustainability of behavioral change. as a starting point for the i.family study described in ahrens et al.9 (2017), another follow-up exam (t3) was carried out between 2013 and 2014, when those children had 7 to 15-year-olds, with an almost equal proportion of boys and girls. this stage included an assessment of the oral health conditions of the children who had participated in the study in spain, the only country where this initiative occurred. as the aim was to investigate entire families, all siblings in the same age range as these children were invited. the role of family characteristics, family structure and family life in relation to child development is one of the focuses of i.family9, in which at least one parent of each index child participated and provided information about their family. in spain, it is estimated that 1,591 children participated in the baseline and 445 of these had their oral health assessed in t3, 281 (59.2%) aged from seven to 11 and 194 (40.8%) from 12 to 15 years. this study was approved by the ethics committee (protocol p113/2012 spain). no child underwent any procedure without their parents’ prior consent for exam, sample collection, subsequent analysis and storage of personal data and samples collected. population and study design the present study included 281 children from the spanish cohort in the idefics study, participating from baseline (t0) and aged from 7 to 11 in t3 (i.family), when they participated in the oral health assessment. the spanish city of zaragoza was selected as a control and the intervention occurred in huesca. the aim of the dietary intervention, considered the most important for this study because of the common risk factor for caries and obesity, set out to improve the children’s eating habits, by increasing their daily consumption of water, fruit and vegetables7,10 and, thus reduce their intake of sugars. there was no intervention in oral hygiene habits, such as frequency of brushing or use of dental floss, for example. data on sex frequency of sugar intake and children’s anthropometrics were obtained at t0. from the questionnaires applied to parents also at t0, information on family income and parents’ levels of education were collected. another questionnaire answered by parents at t3 described their perception of their children’s weight. assessment of oral health occurred only at t3. frequency of sugar intake the frequency of sugar intake was estimated using a suitable appropriately reproduced12 and validated13 children’s eating habits questionnaire (cehq). this tool 5 lages et al. investigated the frequency of food consumption and behavior associated with overweight, obesity and children’s general health. the cehq includes a food frequency section (ffq) in which parents or other caregivers living with the child reported the frequency of their child’s consumption of sugar foods over a typical week in the previous 4 weeks. this allowed for the reduction of likelihood of a “special week”, for example due to holidays or illness. consequently, week-to-week variability was reduced. to facilitate completion of the questionnaire, the same response scale was used for all dietary items of the cehq-ffq. the response options were as follows: never/less than once a week, 1-3 times a week, 4-6 times a week, 1 time a day, 2 times a day, 3 times a day, 4 or more times a day, and no idea. this scale was adopted using the eating habits questionnaire of the early childhood longitudinal survey of the united states department of agriculture14 as a basis. intake frequencies were evaluated without trying to quantify portion sizes. for the present study, this variable was divided into “up to 3 times a day” and “4 or more times a day”, as a sugar consumption frequency greater than 4 times a day is directly related to the development of caries disease15. anthropometry the children’s weight was measured by tanita bc 420 sma scale (tanita europe gmbh, sindelfingen, germany) while their height was measured using a seca 225 stadiometer (seca gmbh & co. kg., hamburg, germany). the measurements were taken in the morning, with the children in fasting and wearing underwear only. body mass index (bmi) was calculated according to the international obesity task force (iotf)16, where low weight = bmi<17kg/m2, normal weight = 17kg/m2≤ bmi<25kg/m2, overweight = 25kg/m2 ≤ bmi<30kg/m2 and obesity = bmi ≥30kg/m2. questionnaire applied to parents parents answered questionnaires on the socioeconomic data of the family and their perceptions of their children’s body weight12. data on the parents’ education levels were based on the international standard classification of education (isced)17, which levels 0-2 are classified as low, while levels 3-4 are medium and 5-6 are high levels of education. we have summed low and medium categories, versus high level of education. family income was evaluated by the question “what is your monthly family income?” using specific spanish categories based on people’s average net income18. the answers were grouped into low to medium or high incomes. parents’ perceptions of their child’s weight were classified as “underweight, adequate or overweight”. oral health assessment the selection of the sample for the assessment of oral health was performed based on the absence/presence of the first molar in the baseline period (t0). as the first molars erupt around the 6 years old, in the baseline these teeth were free of caries, as they were not in the oral cavity. as no oral health assessment was performed previously, we opted to exclude children who, in the t0, had the first molar erupted, remaining in the sample only children from 7 to 11 years old who presented the eruption of these teeth after baseline (t0). the children’s oral health was assessed at t3 on the basis of exams for the prevalence of dental caries and periodontal disease, and observation of the amount of accu6 lages et al. mulated bacterial plaque in the first permanent molars (teeth 16, 26, 36 and 46). for all of these exams, a dental diagnostic plane mouth mirror nº5 and a spherical calibrated periodontal probe were used, under artificial light, with both examiner and child seated. caries disease was evaluated by the international caries detection and assessment system (icdas)19, which combines the use of visual and tactile signs of caries lesions and allows for detection of the disease in its early stages. the examination was performed after prophylaxis, relative isolation and drying of the teeth with a portable air jet. the clinical stages of the caries lesions were established according to the histological classification proposed by ekstrand et al.20 (1995), ranging from the identification of a white spot on a dental surface, which could need drying to be visualized (code 1) or not (code 2), lesions on dental enamel (code 3), to a cavity visible in dentin (codes 4, 5 and 6). codes 1 and 2 were considered for white spots, and codes 4, 5 and 6 were used for untreated caries, and these lesions were summed up for each outcome. the existence of periodontal disease was evaluated by the community periodontal index (cpi), according to the methodology recommended by the world health organization (who)21 for epidemiological surveys in oral health. the codes are 0 healthy sextant, 1 gingival bleeding, 2 dental calculus, 3 shallow periodontal pocket, between 3.5 and 5.5mm, and 4 deep periodontal pocket, greater than 5.5mm. the amount of plaque was observed according to the plaque index (pi) proposed by silness and loe22 (1964), by means of the following codes: 0 absence of biofilm, 1 – tooth clean with biofilm detectable only with probe, 2 – moderate deposit of biofilm visible to the naked eye, and 3 – large accumulation of biofilm filling the entire gingival margin. data on periodontal conditions were analyzed in a descriptive manner, with the prevalence of periodontal disease by sextant, with a view to describing the oral health of the patients, without any relation to the outcomes used. examiner calibration was carried out through theoretical discussions and practical activities, simulating the different conditions and situations that the professionals would encounter during the practical work. the examiners were calibrated for icdas caries criteria with a standard examiner, who also assisted in data collection first, they did an on-line calibration (https://www.icdas.org/icdas-e-learning-course), and then an exercise with 20 children. the kappa values weighted for inter-examiner and intra-examiner agreement exceeded 0.85 for the icdas and cpi indices. statistical analysis descriptive statistics were used to define sociodemographic and oral health characteristics. pearson’s chi-square test was used to compare categorical variables (gender, educational level, income, frequency of sugar intake and bmi, with dental caries and white spots as outcomes). data were analyzed using ibm spss statistics, version 20. the significance level was set at 0.05. results the socioeconomic characterization of the t3 sample was similar to that at the baseline. there was a slight reduction in the percentage of low-income children and an increase in the number of middle-income children in both cities (table 1). https://www.icdas.org/icdas-e-learning-course 7 lages et al. table 1. socioeconomic data of the total sample at t0 (2007) and of the sample of 7 to 11-year-olds at t3 (2013), per spanish city. variable per city t0 t3 total sample n=1,509 (100.0%) n=281 (100.0%) zaragoza (control) sample n=799 (52.9%) n=175 (62.3%) level of education low 76 (9.77%) 6 (5.6%) medium 281(36.1%) 40 (37.4%) high 421 (54.11%) 61 (57.0%) total 778 (100.0%) 107 (100.0%) family income low 125 (17.0%) 9 (7.4%) medium 409 (55.95%) 76 (62.8%) high 197 (26.94%) 36 (29.8%) total 731 (100.0%) 121 (100.0%) huesca (intervention) sample n=710 (47.1%) n=106 (37.7%) level of education low 39 (5.55%) 3 (4.1%) medium 316 (45.0%) 39 (53.4%) high 347 (49.4%) 31 (42.5%) total 702 (100.0%) 73 (100.0%) family income low 84 (13.0%) 3 (4.1%) medium 457 (70.96%) 59 (78.6%) high 103 (16.0%) 13 (17.3%) total 644 (100.0%) 75 (100.0%) note: between t0 and t3, the variables were not significant and, therefore, the characteristics of the sample remained the same. the prevalence of children with dentin carious lesions (p=0.015) and with white spots (p=0.016) in the first molars was higher in huesca, despite the intervention (figure b). 60 50 40 30 20 10 0 zaragoza (control) huesca (intervention) caries white spots 37.1% 52.8% 33.1% 40.6% p er ce nt ag e of c ar io us le si on s (% ) figure b. prevalence of caries and white spots assessed in the first permanent molars of 7 to 11-yearolds in the post-intervention period (t3), in zaragoza and huesca, spain, 2013-2014. 8 lages et al. the analysis of first molars with carious lesions in zaragoza shows that 54.2% of them were at the initial caries stage, while in huesca this rises to 59.6%. the zaragoza children presented a lower percentage of visible plaque, but presented a higher percentage of sextants with bleeding. in huesca, there were more children with localized visible plaque and plaque in the whole gingival margin, and although they presented a lower percentage of sextants with bleeding, 5% presented calculus, a more serious condition (table 2). table 2. oral health data for 7 to 11-year-olds per city, at t3, in terms of the i.family study, spain, 2013 oral health data zaragoza (control) huesca (intervention) n % n % first molars with carious lesions (icdas) white spots after drying 49 32.8 33 28.9 white spots without drying 21 14.0 18 15.8 enamel cavitation 11 7.4 17 14.9 darkened dentin without visible cavitation in enamel 7 4.7 2 1.38 dentin cavitation less than ½ the surface 3 2.0 1 0.9 dentin cavitation more than ½ the surface 58 38.9 43 37.7 total 149 100.0 114 100.0 bacterial plaque index (silness pi) no plaque 54 30.85 23 21.69 plaque visible with probe 68 38.85 41 38.67 localized plaque visible 24 13.7 21 19.81 plaque visible in whole gengival margin 29 16.57 20 18.86 total 175 100.0 106 100.0 periodonal condition per sextant examined (cpi) healthy 643 65.88 419 69.6 bleeding 319 32.68 152 25.25 calculus 14 1.43 31 5.14 total 976 100.0 602 100.0 there was no association between caries and white spot disease with the socioeconomic, sex, parents’ perception of their children’s weight, sugar consumption or body weight variables in any of the cities. the results of parents’ perception of their children’s weight showed responses which were inconsistent with their children’s actual weight for those considered underweight, despite having normal weight (tables 3 and 4). table 3. bivariate analysis for 7 to 11-year-olds per city in the idefics study in spain, for untreated caries variables untreated caries huesca (intervention) zaragoza (control) no yes total p value no yes total p value leval of education high 16(51.6%) 15(48.4%) 31(100.0%) 0.459 36(59.0%) 25(41.0%) 61(100.0%) 0.514 medium-low 18(42.9%) 24(57.1%) 41(100.0%) 30(65.2%) 16(34.8%) 46(100.0%) family income high 7 (53.9%) 6(46.1%) 13(100.0%) 0.432 21(58.3%) 15(41.7%) 36(100.0%) 0.590 medium_low 26(41.9%) 36(58.1%) 62(100.0%) 54(63.5%) 31(36.4%) 85 (100.0%) continue 9 lages et al. frequency of sugar intake up to 3 times a day 9 (47.3%) 10(52.6%) 19(100.0%) 0.305 25(64.1%) 14(35.9%) 39(100.0%) 0.167 4 or more times a day 1(100.0%) 0 (0.0%) 1 (100.0%) 5 (41.7%) 7 (58.3%) 12 (100.%) bmi underweight 1 (25.0%) 3 (75.0%) 4 (100.0%) 0.620* 4 (80.0%) 1 (20.0%) 5 (100.0%) 0.990*normal 37(47.4%) 41(52.6%) 78(100.0%) 72(59.0%) 50 (41.0% 122(100.0%) overweight 0(0.0%) 0 (0.0%) 0 (0.0%) 1 (50.0%) 1 (50.0%) 2 (100.0%) parents’ perception of their children’s weight underweight 9 (37.5%) 15(62.5%) 24(100.0%) 0.353* 14(45.1%) 17(54.9%) 31 (100.0%) 0.099*adequate 28(51.0%) 27(49.0%) 55(100.0%) 62(66.7%) 31(33.3%) 93 (100.0%) overweight 0 (0.0%) 1(100.0%) 1 (100.0%) 1(50.0%) 1 (50.0%) 2 (100.0%) sex boys 27(53.0%) 24(47.0%) 51(100.0%) 0.252 52(66.7%) 26(33.3%) 78 (100.0%) 0.350 girls 23(41.8%) 32(58.2%) 55(100.0%) 58(59.8%) 39(40.2%) 97 (100.0%) nota: * fisher’s exact test table 4. bivariate analysis for 7 to 11-year-olds per city in the idefics study in spain, for white spots variables white spots huesca (intervention) zaragoza (control) no yes total p value no yes total p value leval of education high 19(61.3%) 12(38.7%) 31(100.0%) 0.722 39(64.0%) 22(36.0%) 61(100.0%) 0.542 medium-low 24(57.1%) 18(42.9%) 42(100.0%) 32(69.6%) 14(30.4%) 46(100.0%) family income high 9 (69.2%) 4(30.8%) 13(100.0%) 0.395 23(63.9%) 13(36.1%) 36(100.0%) 0.676 medium-low 35(56.5%) 26(43.5%) 62(100.0%) 58(68.2%) 27(31.8%) 85(100.0%) frequency of sugar intake up to 3 times a day 15(79.0%) 4(21.0%) 19(100.0%) 0.608 26(66.7%) 13(33.3%) 39(100.0%) 0.597 4 or more times a day 1(100.0%) 0 (0.0%) 1 (100.0%) 7 (58.3%) 5 (41.7%) 12 (100.%) bmi underweight 1 (25.0%) 3 (75.0%) 4 (100.0%) 0.297* 4 (80.0%) 1 (20.0%) 5 (100.0%) 0.583*normal 48(61.5%) 30(38.5%) 78(100.0%) 78(66.1%) 40(33.9%) 118(100.0%) overweight 0 (0.0%) 0 (0.0%) 0 (0.0%) 1 (50.0%) 1 (50.0%) 2 (100.0%) parents’ perception of their children’s weight underweight 15(62.5%) 9 (37.5%) 24(100.0%) 0.885* 15(48.4%) 16(51.6%) 31 (100.0%) 0.266*adequate 33(60.0%) 22(40.0%) 55(100.0%) 67(72.0%) 26(28.0%) 93 (100.0%) overweight 0 (0.0%) 1(100.0%) 1 (100.0%) 1(50.0%) 1 (50.0%) 2 (100.0%) sex boys 31(60.8%) 20(39.2%) 51(100.0%) 0.785 53(68.0%) 25(32.0%) 78 (100.0%) 0.783 girls 32(58.2%) 23(41.8%) 55(100.0%) 64(66.0%) 33(34.0%) 97 (100.0%) nota: * fisher’s exact test continuation 10 lages et al. discussion the highlight of this study is the inclusion of oral health assessment in a multicentric european cohort project which investigates chronic diseases with common lifestyle and dietary risk factors such as obesity, diabetes and cardiovascular diseases. despite the fact that the dental exams took place in spain only, although eight countries participated in the study, it was innovative, as the idefics was the european multicentric cohort study involving the largest sample of children among studies undertaken to date, none of which included oral health among their analyses, though caries is also diet-related. as it was a longitudinal study, an analysis of the same population in early childhood and, subsequently, their health outcomes in early adolescence, also contributed to the significance of the study. in addition, the diagnosis of caries with the icdas index19, which considers initial stages of caries, such as white spots and enamel lesions during oral exams, has a great advantage over the decayed, missing and filled teeth (dmft)21, which only considers cavitations at the dentin level. among the first molars with carious lesions, more than half were at the initial stage, and would not have been noticed if the dmft had been used. the prevalence of untreated caries and white spots were higher in huesca, despite the intervention. de bourdeaudhuij et al.23 (2015) found that the intervention had no positive effects on diet or lifestyle, such as the consumption of water, soft drinks and fruit juice; fruit and vegetable intake; daily tv viewing time and computer use; and daily levels of physical activity (sports and outdoor games) for the total sample of 2 to 9-year-olds in the eight european countries. families with lower incomes, a poorer quality of life or were migrants presented the worst results, with lower adherence to the intervention measures11. however, we did not find this association in this study. possible limitations of the idefics intervention were mentioned: dependence on parents’ answering of questionnaires on child behavior; interference from local, regional or national governments in the fight against obesity in the different countries; differences in the penetration of the idefics intervention in each country, given the different cultural contexts, for which there were no adaptations in strategies; and differences in the measure of intervention at child and parent level23. it should be emphasized that there was no intervention in oral hygiene behavior in spain. there was no association between untreated caries and white spots with body weight. the fact that a child was underweight, of normal weight or overweight/obese did not associated with their oral health or vice versa. this result is in agreement with studies which also did not find this relation with the same age group in the netherlands, brazil and spain4, but found a relationship between social determinants and dental caries disease and obesity, such as lower family income and parents’ lower educational levels, although this was also not shown in our study. however, hayden et al.2 (2013), in a systematic review study with meta-analysis, argue that there is an association between these diseases, while dimaisip-nabuab et al. 24 (2018) found that underweight children are more likely to experience caries. through a systematic review of the literature, hooley et al.25 (2012) stated that there is no evidence to suggest that dental caries is associated with either high or low bmi. although the exact nature of such associations is not clear, it is possible that there are different factors involved in the development of caries in children with high or low 11 lages et al. bmi or with different socioeconomic profiles. according to costa  et  al.4 (2013), the conflicting results of the studies could be related to the research design, variations in the environment in which the data were collected, socioeconomic status of the sample, nutritional status measures used, caries assessment indexes used and age differences in the children examined. nevertheless, it was suggested that combined strategies be implemented simultaneously to control caries and obesity, as they present diet as a common risk factor26. the frequency of sugar consumption was not associated with caries and white spots in this study either, but that was probably due to the fact that only 71 parents answered this particular question, of whom only 12 reported consumption more than 4 times a day, as it has already been proven that sugar consumption is directly involved in these diseases3,27. most interventions in the prevention of obesity and dental caries focus on modifying health behavior. these policies and strategies must be accompanied by other government efforts to improve public education, and deterioration of the state of health and nutrition of the population. traditional preventive and curative approaches, based solely on education and health care are considered unsustainable and ineffective. such strategies should target the broader upward flow of social factors which affect sugar consumption. people’s  decisions to consume sugars is deeply rooted in the social, economic and environmental conditions in which they grow up, live, work and age. thus, sustainable environments for health promotion should be created so that there is a behavior of commitment to health, where the consumption of sugar is the difficult choice and the consumption of vegetables and fruit is the easy choice in terms of availability and accessibility5,27. political and economic changes at local, state and national levels could promote and encourage healthier food choices26,27. the united kingdom and mexico, for example, have successfully passed legislation on drinks containing sugar using successful taxation prototypes adapted from alcohol and tobacco28,29. some experiments already reported suggest that a 20% tax on sugar-added beverages could reduce the prevalence of caries, obesity, diabetes and other comorbidities, and reduce treatment costs and lead to behavioral changes in food choice29. at the same time, sugar-free food, drinks and medicines should not be taxed. fiscal returns could be used to subsidize the price of vegetables, fruits and sugar-free medicines29. for grinsberg30 (2017), the ideal would be to establish both shortand long-term goals for reducing people’s sugar consumption, until the who recommended rate of a maximum of 5% of daily total calorie intake is reached, as the rewards arising out of reduced mortality, morbidity and health expenditures would gradually be enormous, as is happening in israel. the who has recognized that dental diseases are the most common non-communicable diseases worldwide and that the treatment of dental disease is costly. governments must pressure the food and drug industries, with the backing of appropriate legislation, to reduce the sugar content of their products and offer a wide variety of sugar-free alternatives. meals provided in schools and other public establishments, such as hospitals, should be sugar free or at least with reduced sugar. all of the aforementioned policies should be supported by initiatives to increase public awareness of the need to reduce the intake of sugary foods and beverages from childhood through life, using national and mother and child nutrition programs27. 12 lages et al. all such changes occur amidst the complexities of interactive social, cultural, political, and financial forces which rarely align themselves to promote healthy eating. as in the case of public health efforts to reduce tobacco use, there are keen corporate interests with powerful incentives to oppose or undermine these efforts. this has long been recognized in the case of the tobacco industry and is becoming better recognized in the case of the food and drinks industry30. one limitation to this study was the fact that the oral health exam was undertaken only after the intervention, during t3, which meant it was possible to verify this condition only in the group 7 to 11 years old. another limitation was that the icdas ii index was used only in the first permanent molars, which was necessary in the circumstances. for future multicentric cohort studies focusing on diet, obesity and lifestyle, it is suggested that children’s oral health be assessed from the baseline, that all teeth be examined, and that macroeconomic policies of wide social reach, such as the taxation of foods rich in sugar and advertising restrictions be included in the intervention measures of the study. future investigations involving parents’ perception of their children’s weight would also be important, as a perception incompatible with their  children’s weight could stimulate diets which lead to weight gain and the development of dental caries. in conclusion, the intervention for the prevention of obesity in the idefics study did not shown an association with the prevalence of dental caries disease in the children participating in the spanish cohort. references 1. world health organization. global strategy on diet, physical activity and health: childhood overweight and obesity. geneva: who; 2004 [cited 2020 aug 20]. available from: https://www.who.int/ dietphysicalactivity/strategy/eb11344/strategy_english_web.pdf. 2. hayden c, bowler jo, chambers s, freeman r, humphris g, richards d, et al. obesity and dental caries in children: a systematic review and metaanalysis. community dent oral epidemiol. 2013 aug;41(4):289-308. doi: 10.1111/cdoe.12014. 3. gbd 2017 oral disorders collaborators, bernabe e, marcenes w, hernandez cr, bailey j, abreu lg, et al. global, regional, and national levels and trends in burden of oral 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https://www.cedefop.europa.eu/files/international_standard_classification_for_education_%28isced%29.pdf https://apps.who.int/iris/handle/10665/97035 14 lages et al. 24. dimaisip-nabuab j, duijster d, benzian h, heinrich-weltzien r, homsavath a, monse b, et al. nutritional status, dental caries and tooth eruption in children: a longitudinal study in cambodia, indonesia and lao pdr. bmc pediatr. 2018 sep 14;18(1):300. doi: 10.1186/s12887-018-1277-6.  25. hooley m, skouteris h, boganin c, satur j, kilpatrick n. body mass index and dental caries in children and adolescents: a systematic review of literature published 2004 to 2011. syst rev. 2012 nov 21;1:57. doi: 10.1186/2046-4053-1-57. 26. joury e, al-kaabi r, tappuni ar. constructing public health policies in post crisis countries: lessons to learn from the associations between free-sugars consumption and diabetes, obesity and dental caries before, during and after sanctions in iraq. z gesundh wiss. 2016;24(6):563-9. doi: 10.1007/ 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endodontics, and dental materials, university of são paulo, bauru, brazil. corresponding author: mariana maciel batista borges department of operative dentistry, endodontics, and dental materials, university of são paulo, bauru, brazil. mmbborges@usp.br editor: dr altair a. del bel cury received: november 16, 2021 accepted: june 04, 2021 physicochemical properties and antimicrobial activity of calcium hydroxide pastes in association with other compounds mariana maciel batista borges1,* , mayara conde frank1 , rafaela fernandes zancan1 , talita tartari1 , rodrigo ricci vivan1 , marco antonio hungaro duarte1 aim: this study aimed to evaluate the ph, ca2+ release, solubility, and antimicrobial activity of calcium hydroxide (ca(oh)2) pastes in association with different substances. methods: sixty acrylic teeth (n=10) were filled with pastes that associated ca(oh)2 with the following substances: benzalkonium chloride 5% (g1) and 50% (g2) both in propylene glycol, arnica glycolic extract (g3), green tea glycolic extract (g4), calen/pmcc™ (g5), and calen™ (g6). in the group g1 to g4 were used 1g of ca(oh)2 powder with 0,8g of vehicle. ph and ca 2+ release was measured after 7, 15, and 30 days. for solubility, micro-ct was used immediately and at the periods of 7, 15, and 30 days. for the antimicrobial analysis, a biofilm of e. faecalis was induced in vitro on bovine dentin discs. live/dead viability dye and confocal scanning microscopy were used. results: the highest ph values occurred on the first 7 days, and the g6, g1, g3, and g5 presented the highest ph values at this period (p <0.05). ca2+ release was higher in all groups at 7 days, with the highest values observed in g1, g5, and g6. the volume of all pastes showed no significant difference in the intragroup analysis at 7 and 15 days (p <0.05). g1 and g2 showed the highest antimicrobial action (p <0.05). for the biovolume, there was difference between the g6 and the other groups (p >0.05) with g1 presenting the lowest values. conclusion: benzalkonium chloride 5% increases the antimicrobial action of the ca(oh2), without impairs physicochemical properties. keywords: anti-infective agents. biofilms. calcium hydroxide. root canal filling materials. chemical phenomena. https://orcid.org/0000-0003-0049-8657 https://orcid.org/0000-0003-0997-7072 https://orcid.org/0000-0001-5040-5776 https://orcid.org/0000-0003-4372-0158 https://orcid.org/0000-0002-0419-5699 https://orcid.org/0000-0003-3051-737x 2 borges et al. introduction the aim of endodontic treatment of teeth with apical periodontitis is to eliminate microorganisms from the root canal system1. however, root canal preparation, due to its anatomical complexity, is not totally effective in complete disinfection, requiring the use of intracanal dressing2. calcium hydroxide (ca(oh)2) paste has been the most widely used intracanal medication for this purpose3. its mechanism of action involves the dissociation in calcium (ca²+) and hydroxyl (oh-) ions, showing biological properties such as antimicrobial activity, tissue dissolution capacity and inhibition of osteoclast activity4-5. the association of ca(oh)2 with inert vehicles has demonstrated limited antimicrobial effectiveness, especially against enterococcus faecalis3. therefore, the association with antiseptic substances to potentiate the antimicrobial activity of the paste has been recommended6. different substances and vehicles have been proposed to be associated with ca(oh)2 for improvement of its antimicrobial properties7. camphorated paramonochlorophenol (cpmc) is a phenolic compound used as root canal disinfectant and is one of these substances6. cpmc breaks the bacterial cytoplasmic membrane, denatures proteins, and inactivates enzymes such as oxidases and dehydrogenases, having a bactericidal effect, in addition to the release of chlorine, which is an antibacterial agent. the literature suggests that the association of ca(oh)2 / cpmc increases the spectrum of action and antimicrobial efficacy, in addition to greater dentinal penetration, when compared to the association of ca(oh)2 with inert vehicles 6-8. phytotherapy has been gaining popularity nowadays in the prevention and treatment of diseases because it causes few adverse effects and is inexpensive8. green tea (camelia sinensis) is a widely consumed beverage in many parts of the world8. it is rich in flavonoids, polyphenols, and catechins, which have potent antioxidant properties and eliminate free radicals8. furthermore, it presents favorable results in the antimicrobial action against e. faecalis in biofilm and planktonic forms, which can be justified by the generation of hydroxyl radical9. arnica is another vegetable substance with active ingredients such as sesquiterpene lactones, helenalin 11-alpha, 13-dihydroelenanine, and esters, as well as acetic, isobutyric, methacrylic, and other carboxylic acids, with anti-inflammatory properties, and also containing potent immunostimulants10,11. however, it has a low antimicrobial efficacy when used in isolation on endodontic microorganisms12. its root extract is used externally to treat bruises, sprains, rheumatic pain, phlebitis, and skin inflammation, and as homeopathic preparations for the stimulation of the immune system10. another vehicle that might be associated with ca(oh)2, because of its antimicrobial activity, is benzalkonium chloride (bc), which is a surfactant that presents a quaternary ammonium compound that can change cell penetrability, altering cytoplasmic components and leading to the death of bacteria, besides showing activity against viruses, fungi, and protozoa12-15. arias-moliz et al.14 (2015) demonstrated that the addition of this compound to endodontic cements, promoted significant improvements in antimicrobial and antibiofilm activity without affecting its properties, eliminating e. faecalis. despite the existing literature on the characteristics and proper3 borges et al. ties of these substances, little is known about the impact of the association of other substances, such as herbal compounds and benzalkonium chloride, on the physicochemical properties and antimicrobial activity of ca(oh)2 pastes. therefore, the aim of this study was to evaluate the ph, calcium ion release, solubility, and antimicrobial activity against enterococcus faecalis biofilm, of pastes that associate benzalkonium chloride, arnica glycolic extract, green tea glycolic extract and cpmc with ca(oh)2. the null hypothesis tested is that these substances do not influence in the antimicrobial activity and physicochemical properties of ca(oh)2 paste. materials and methods experimental pastes six ca(oh)2 pastes were evaluated and their compositions are shown in table 1. in the group g1 to g4 were used 1g of calcium hydroxide powder with 0,8g of vehicle and the final consistency of the pastes was similar to that of toothpaste. in the groups g5 and g6 were used 1,8g of the paste to fill the canal. analyses of ph, calcium ion release, and solubility for the ph and calcium ion release analyses, 60 artificial acrylic resin maxillary central incisors (n = 10) with an artificial foramen standardized to a diameter of 400 µm were filled, using a lentulo drills #30 (dentsply/ maillefer, ballaigues, switzerland). after that, the roots were analyzed using a micro-ct camera to guarantee the complete filling. then, the crowns were sealed with a composite resin and the teeth were individually immersed in tubes containing 10 ml of deionized water (ph 7.15). the tubes were hermetically sealed and taken to the oven at 37 °c. after 7, 15, and 30 days, the specimens were transferred to a new tube with the same volume of deionized water. the ph and calcium ion released were measured in each time interval immediately after the removal of the teeth from the tubes. table 1. composition of the ca(oh)2 pastes used. group composition g1 ca(oh)2 p.a (biodinâmica, londrina, brazil) + benzalkonium chloride 5% (bauru fórmulas, são paulo, brazil) in propylene glycol (ch/cb5) g2 ca(oh)2 p.a (biodinâmica, londrina, brazil) + benzalkonium chloride 50% (bauru fórmulas, são paulo, brazil) in propylene glycol (ch/cb50) g3 ca(oh)2 p.a (biodinâmica, londrina, brazil) + arnica glycolic extract (ch/arn) g4 ca(oh)2 p.a (biodinâmica, londrina, brazil) + green tea glycolic extract (camellia sinensis leaf extract) (ch/gt) g5 calen paste cpmc (ch/cpmc) (ss white artigos dentarios ltd, rio de janeiro, brazil) – composed by calcium hydroxide (2.5 g), zinc oxide (0.5 g), hydrogenized colophony (0.05 g), polyethyleneglycol 400 (1.75 ml), and camphorated paramonochlorophenol (0.15 ml). g6 calen paste (ch) (ss white artigos dentarios ltd, rio de janeiro, brazil) – calcium hydroxide (2.5 g), zinc oxide (0.5 g), hydrogenized colophony (0.05 g) and polyethyleneglycol 400 (12.75 ml) 4 borges et al. a ph meter (model 371; micronal, são paulo, sp, brazil) was used for the determination of the ph. to assure the accuracy of the device, constant measurements were performed with known buffers at ph of 4, 7, and 14. after the specimen removal, the tube was taken to a shaker and agitated for 5 seconds. after agitation, the liquid was poured into a beaker and then put in contact with the device’s electrode. calcium ion release was measured using an atomic absorption spectrophotometer (aa6800; schimadzu, tokyo, japan) equipped with a calcium-specific hollow cathode lamp. calcium release and ph were performed in triplicate. the solubility analyses were performed with the same teeth used for the determination of the ph and calcium ion release. for this experiment, immediately after the acrylic teeth were filled with the experimental pastes and sealed with the composite resin they were scanned with a microcomputed tomography (skyscan 1174v2; skyscan, kontich, belgium) to obtain the baseline values. the imaging parameters used were: voxel size of 19.70 μm and 360° at a rotation step of 0.5°. then, the teeth were immersed in plastic bottles and stored as previously described. at the time intervals of 7, 15, and 30 days, the teeth were removed from the bottles and scanned again before they were inserted in the new tubes. the images obtained were reconstructed and the volume (mm³) was measured with the aid of a software (ctan v1.11.10.0, sky-scan). solubility values at baseline and after the tests were recorded. the percentage of solubility in each period was also determined. analyses of antimicrobial activity to test the antimicrobial activity of the pastes, dentin discs were removed from bovine teeth with fully developed roots. for this, trephine burs (4 x 4 mm) were used coupled to a handpiece device and placed perpendicular to bovine roots. the trephine was forced from the mesial to the distal portion of cervical and middle thirds, under abundant irrigation, so that were obtained four dentin discs per tooth. then, the discs were polished with silicon carbide abrasive papers (buehler ltd, lake bluff, illinois) of 600, 400, and 200 granulations, providing a mean thickness of 0.8-1.0 mm of dentin disks. afterwards, the specimens were submitted to an ultrasonic bath of 5 min each, with 1% sodium hypochlorite and 17% edta to remove smear layer, and were subsequently sterilized in an autoclave. after confirmation of the strain purity by gram staining and colony morphology, 15 ml enterococcus faecalis standard strain (american type culture collection [atcc] 29212) were put into 3 ml sterile brain-heart infusion (bhi; oxoid, basingstoke, uk) at 37ºc in air for growth overnight. following, bacterial density was adjusted at 108 cells/ml with a spectrophotometer (uv-visibli, shimadzu, japan) at an optical density of 1 at 600 nm according to the 0.5 macfarland standard. the dentin surfaces were then infected for biofilm grown: 1 dentin block + 100 µl e. faecalis + 900 µl bhi were inserted into each well of a 24-well multiwell plate. the latter was kept under agitation in an oven at 37 °c (q816m20; composed of scientific chemis ltda, diadema, sp, brazil) for 21 days. to avoid nutrient deficiency, the culture medium was completely replaced every 48 hours, without addition of new microorganisms. for the antimicrobial test, dentin samples were immersed in experimental pastes and incubated at 37 °c for 7 days. in the control group, the biofilm received no treatment. 5 borges et al. after this period, the pastes were washed out inserting the disc in a recipient with distilled water until the complete removal of the paste under the disc, after that samples were stained with 15 μm of syto 8/propidium iodide (live/dead technique-in vitro gen) for 15 minutes. after staining, the samples were taken to a confocal laser scanning microscope (leica, mannheim, germany) to obtain images from the surface with a 40x magnification. the biovolume and percentage of live cells were calculated with the aid of the bioimage_l software (www.bioimagel.com). the data obtained in the ph analysis, calcium ion release, solubility, and antimicrobial activity were analyzed for normality by the shapiro-wilk test. as the values ph, calcium ion release, and solubility presented a normal distribution the analysis of variance anova with a post-hoc tukey’s test was applied for statistical comparison of the data. because of the absence of a normal distribution, the kruskal-wallis and dunn’s post-hoc tests were used to compare the antimicrobial activity. the level of significance was set at 5%. results table 2 shows the median (med), minimum and maximum (min max) values for the calcium and hydroxyl ions release (mg/l) by the pastes in the periods analyzed. in this period the higher alkalinity values were achieved by the viscous pastes ch/cb5 (g1), ch/cpmc (g5) and ch (g6) with exception of ch/cb50 (g2). (p < 0.05). the ch/gt (g4) presented the lowest values for calcium ion release in all periods. (p<0.05). for the groups ch/cb5 (g1), ch/cb50 (g2), ch/cpmc (g5), and ch (g6) the intragroup analysis revealed a statistically significant reduction in calcium ion table 2. median (med), minimum and maximum (min max) values for the calcium and hydroxyl ions release (mg/l) from the pastes in the different periods of time. groups 7 days 15 days 30 days ph med (min – max) ca2+ med (min – max) ph med (min – max) ca2+ med (min – max) ph med (min – max) ca2+ med (min – max) g1 (ch/cb5) 11.45 (10.93–11.78)ab 129.3 (82.12–355.7)ab.a 8.48 (7.9–9.6)d 55.07 (50.92–57.86) a.b 7.9 (7.74–8.06)cd 29.36 (15.26–57.84)ab.b g2 (ch/cb50) 10.67 (10.39–11.29)cd 64.03 (38.76–77.99)bc.a 9.37 (8.4–10.64)abc 18.72 (13.06–49.65) ab.b 8.06 (7.88–8.33)bcd 18.33 (0–49.05) ab.b g3 (ch/arn) 10.93 (10.36–11.26)bc 18.77 (8.71–33.48)c.a 10.54 (9.33–10.85)a 9.93 (3.59–30.84) bc.a 8.52 (8.32–8.65)a 21.53 (9.82–29.43) b.a g4 (ch/gt) 10.27 (9.5–10.82)d 2.92 (0.11–168)c.a 8.73 (8.3–9.33)cd 1.2 (0.15–9.87) c.ab 8.14 (8–8.4)bc 0.475 (0.13–4.76)c.b g5 (ch/cpmc) 11.63 (11.32–11.94)a 219.6 (150.5–446.6)ab.a 9.57 (9–10.78)ab 22.77 (3.28–24.16) bc.b 7.88 (7.82–7.96)d 14.02 (6.8–20.7)bc.b g6 (ch) 11.64 (9.2–12.25)ab 340.3 (69.93–899.3)a.a 8.98 (8.43–10.46)bcd 24.93 (10–33.74) ab.b 8.32 (7.8–9.35)ab 45.42 (36.88–51.36)a.b *repeated measures anova with tukey’s multiple comparison test, p-value < 0.05; different lowercase letters in rows indicate statistically significant intragroup differences; anova with tukey’s multiple comparison test, p-value < 0.05; different uppercase letters in columns indicate statistically significant intergroup differences in the same time period. ch/cb5 calcium hydroxide + benzalkonium chloride 5%; ch/cb50 calcium hydroxide + benzalkonium chloride 50%; ch/arn calcium hydroxide + arnica glycolic extract; ch/gt calcium hydroxide + green tea glycolic extract; ch/cpmc calen paste + camphorated paramonochlorophenol; ch calen paste http://www.bioimagel.com 6 borges et al. release between 7 and 15 days (p<0.05), however, between 15 and 30 days, no difference was observed (p>0.05). the median (med), minimum and maximum (min max) for the volume (mm³) of the different experimental pastes over time is presented in the table 3. in the intragroup analysis, in all groups, no statistical significant difference was seen between initial volume and the volume after 7 days. ch/cb5 (g1) presented the lowest solubility, showing statistical reduction of the initial volume just in 30 days. ch/cb50 (g2), ch/arn (g3) and ch/gt (g4) revealed similar solubility over time, with significant volume reduction after 15 days. ch/cpmc (g5) and ch (g6) presented no statistical significant difference in the volume between 15 and 30 days. table 4 shows the median (med), minimum and maximum (min max) values for the biovolume and the percentage of living bacteria in the biofilm after contact with table 3. median (med), minimum and maximum (min max) values of the experimental pastes volumes (mm3) in the different periods of time groups initial volume 7 days 15 days 30 days med (min – max) med (min – max) med (min – max) med (min – max) g1 (ch/cb5) 1.63 (1.22-2.24)a 1.6 (0.99 – 2.23)a 1.42 (0.79 – 2.06)ab 1.36 (0.57 – 1.94)b g2 (ch/cb50) 2.26 (3.44-2.01)a 2.13 (2.64-1.72)ab 1.99 (2.46-1.35)b 1.99 (2.47-1.45)b g3 (ch/arn) 1.92 (2.30-1.41)a 1.840 (2.18-1.17)ab 1.67 (2.18-1.04)b 1.67 (2.18-0.98)b g4 (ch/gt) 3.35 (4.02-1.24)a 2.53 (3.00-1.24)ab 2.37 (2.53-1.24)b 2.37 (2.53-1.24)b g5 (ch/cpmc) 2.37 (2.18-2.73) a 2.21 (2.09 – 2.38) ab 0.40 (0.19 – 0.67)bc 0.37 (0.07 – 0.62)c g6 (c) 1.64 (1.15 – 2.59)a 1.58 (1.08 – 2.50)ab 1.45 (0.93 – 2.18)bc 1.39 (0.71 – 1.95)c *repeated measures with friedman and dunn’s multiple comparison test, p-value < 0.05; different lowercase letters in rows indicate statistically significant intragroup differences; ch/cb5 calcium hydroxide + benzalkonium chloride 5%; ch/cb50 calcium hydroxide + benzalkonium chloride 50%;ch/arn calcium hydroxide + arnica glycolic extract; ch/gt calcium hydroxide + green tea glycolic extract; ch/cpmc calen paste + camphorated paramonochlorophenol; ch calen paste table 4. median (med), minimum and maximum (min max) values of the percentage of live cells and biovolume of the biofilm after the contact with the experimental pastes for 7 days. groups % of live cells biovolume med (min – max) med (min – max) g1 (ch/cb5) 13.39 (5.75 – 21.57)b 56.673 (5.182 – 451.825)b g2 (ch/cb50) 6.4 (2.59 – 16.64)b 67.114 (22.511-577.933)ab g3 (ch/arn) 70.48 (54.15 – 76.16)a 65.277 (12.295 – 460.183)b g4 (ch/gt) 81.43 (72.38 – 92.63)a 167.706 (16.582 – 1270.006)ab g5 (ch/cpmc) 58.73 (47.34 – 77.1)a 146.641 (6.947 – 550.611)ab g6 (c) 54.81 (34.13 – 87.86)a 133.222 (5.605 – 800.540)ab control 79.14 (68.30 – 86.56)a 255. 94 (12.882 – 1222.006)a *kruskal-wallis with dunn’s post-hoc, p-value <0.05; different uppercase letters in columns indicate statistically significant intergroup differences. ch/cb5 calcium hydroxide + benzalkonium chloride 5%; ch/cb50 calcium hydroxide + benzalkonium chloride 50%;ch/arn calcium hydroxide + arnica glycolic extract; ch/gt calcium hydroxide + green tea glycolic extract; ch/cpmc calen paste + camphorated paramonochlorophenol; ch calen paste 7 borges et al. the experimental pastes for 7 days (figure 1). the pastes with 5% benzalkonium chloride ch/cb5 (g1) and ch/cb50 (g2) were similar (p >0.05) and showed the lowest percentage of living bacteria, with statistically significant differences in comparison with all other groups (p < 0.05). a reduction on the biovolume of the biofilm was seen in all groups when compared to the control, with the best results for ch/cb5 and ch/arn (p < 0.05). discussion in the present study, different substances with phytotherapeutic and antiseptic properties were associated with the ca(oh)2 paste to evaluate their effects against e. faecalis biofilms. the results obtained with regard to ph, after 7 days, reinforce that the association with viscous vehicles, such as propylene glycol, is related to a greater diffusion of hydroxyl and calcium ions through dentinal tubules, and the penetration into the bacterial cell membrane16-18. except for g2, where the high concentration of the additive may have interfered with the action of ca(oh)2. similar results were observed in the released ca² +. hydroxyl ions are able to diffuse rapidly in the first 24 hours and reach a plate after 2 weeks in the apical region3. in our results, the ph peak was in the first week, and its decrease over the time could be explained by the absence of dentinal permeability, once in artificial teeth the ions were able to leave the canal space only through the foramen. however, even decreasing the ions release all the pastes maintained an alkaline ph in the external environment, which is important for the biological and antimicrobial activities of the medication2,3,16,18. estrela et al.2 showed that the ph necessary for activation of alkaline phosphatase ranges from 8.6 to 10.3. figure 1. confocal laser scanning microscopy of biofilms treated with benzalkonium chloride 5% (g1) and 50% (g2), arnica glycolic extract (g3), green tea glycolic extract (g4), camphorated paramonochlorophenol (cmcp) (g5), and calen (g6) (control). live cells are indicated in green, and dead cells are indicated in red. each picture represents an area of 275 x 275 mm. g1 (ch/cb5) g2 (ch/cb50) g3 (ch/arn) g4 (ch/gt) g5 (ch/cpmc) g6 (c) control 8 borges et al. regarding solubility, most studies use analytical methods based on ansi / ada no. 57 or iso 687619,20. however, this type of methodology was not applicable to pastes due to the absence of setting. thus, in this study, the solubility was measured by inserting the pastes in simulated canals. after, they were digitized in micro-ct to determine the volume of the paste in different periods of time, enabling the comparison of the volume lost over time3,21. high solubility is undesirable, as it interferes with the paste’s direct contact with microorganisms22,23. on the other hand, solubility is related to the ion release and consequently the alkalization of the environment16, in this sense there must be a balance. there was a decrease in the volume of pastes over time (15 and 30 days), indicating solubility that resulted from their immersion in the liquid, as it occurs in clinical scene, where fluids gain the root canal through the foramen and promote the solubility of the medication17,18. e. faecalis, mainly in the form of biofilm, can survive in alkaline ph and be resistant to ca (oh) 2 paste with an inert vehicle24, reaffirming the results of the present study. studies have shown that calcium hydroxide may need 10 days to disinfect dentinal tubules with facultative bacteria25,26, suggesting that e. faecalis exhibits the ability to maintain the ph homeostasis of the medium17. therefore, the association of ca(oh)2 with antiseptic vehicles is recommended to enhance its antimicrobial effect. although cpmc mixed with calcium hydroxide appear to increase the antimicrobial action4,17,27 in our results it was not evident. lima et al.19 (2012) showed that cpmc / calen maintained for 7 days did not show an effective antimicrobial action, but after 14 days, it resulted in 100% elimination of the bacteria. the authors suggested that the relation between the phenol and the peg could decrease the release of the group phenolic28, resulting in a less active paste against biofilm in the initial periods. studies show the inhibitory effect of green tea on the growth of s. mutans29-31, suggesting that its local application can reduce the acidity of saliva and bacterial plaque, acting as a preventive measure against caries29. however, the association with ca(oh)2 did not improve the antimicrobial activity against e. faecalis, compared to the positive control. a similar result was observed in the arnica group (g3), another phytotherapeutic substance rich in flavonoids and derived from thymol10. the addition of benzalkonium chloride to ca(oh)2 potentiated the release of calcium and hydroxyl ions, favoring their antimicrobial activity, reaffirming the results obtained by jaramillo et al.32 (2012), where approximately 70% of the bacterial biofilm was reduced with the use of benzalkonium chloride, suggesting that this surfactant interferes with the cellular mechanism of bacterial adhesion. the association of this substance with irrigating solutions or sealers presented good antimicrobial activity12-14,32. thus, the association of ca(oh)2 with the suggested compounds does not seem to significantly alter its ph, volume, and solubility, based on this, benzalkonium chloride in high concentration is not recommended, in spite of bc is recognized by the united states pharmacopoeia and it is used in numerous toothpastes and mouthrinses33. clinically, a lower concentration may be used in association with ca(oh)2, promoting greater decontamination in a safe manner by reducing any possibility of side effects if extruded into the periapical tissue. 9 borges et al. in conclusion, the results of this study showed that the type of vehicle and substances associated with ca(oh)2 powder can affect the antimicrobial effects, solubility, and ion release of the pastes obtained. after a comparison of all physicochemical and antimicrobial properties of ca(oh)2 pastes tested, it was possible to conclude that association with 5% benzalkonium chloride seems to offer the greatest advantages since it presented a high ion release, low solubility, and significant antimicrobial activity. acknowledgements the work was supported by state of sao paulo research foundation, fapesp (2018/26299-6). the authors deny any conflicts of interest related to this study. references 1. gründling gl, melo ta, montagner f, scarparo rk, vier-pelisser fv. qmix® irrigant reduces lipopolysacharide (lps) levels in an in vitro model. j appl oral sci. 2015 jul-aug;23(4):431-5. doi: 10.1590/1678-775720140488. 2. estrela c, pécora jd, souza-neto md, estrela cr, bammann ll. effect of vehicle on antimicrobial properties of calcium hydroxide pastes. braz dent j. 1999;10(2):63-72. 3. zancan rf, vivan rr, milanda lopes mr, weckwerth ph, de andrade fb, ponce jb, et al. antimicrobial activity and physicochemical properties of calcium hydroxide pastes used as intracanal medication. j endod. 2016 dec;42(12):1822-8. doi: 10.1016/j.joen.2016.08.017. 4. siqueira jf jr, lopes hp. mechanisms of antimicrobial activity of calcium hydroxide: a critical review. int endod j. 1999 sep;32(5):361-9. doi: 10.1046/j.1365-2591.1999.00275.x. 5. siqueira jf jr, de uzeda m. influence of different vehicles on the antibacterial effects of calcium hydroxide. j endod. 1998 oct;24(10):663-5. doi: 10.1016/s0099-2399(98)80151-9. 6. gomes bp, ferraz cc, vianna me, rosalen pl, zaia aa, teixeira fb, et al. in vitro antimicrobial activity of calcium hydroxide pastes and their vehicles against selected microorganisms. braz dent j. 2002;13(3):155-61. doi: 10.1590/s0103-64402002000300002. 7. lima ra, carvalho cb, ribeiro tr, fonteles cs. antimicrobial efficacy of chlorhexidine and calcium hydroxide/camphorated paramonochlorophenol on infected primary molars: a split-mouth randomized clinical trial. quintessence int. 2013 feb;44(2):113-22. doi: 10.3290/j.qi.a28932. 8. george de, shetty r, shetty pj, gomes la. an in vitro study to compare the effect of different types of tea with chlorhexidine on streptococcusmutans. j clin diagn res. 2017 sep;11(9):zc05-7. doi: 10.7860/jcdr/2017/26581.10538. 9. lee p, tan ks. effects of epigallocatechin gallate against enterococcus faecalis biofilm and virulence. arch oral biol. 2015 mar;60(3):393-9. doi: 10.1016/j.archoralbio.2014.11.014. 10. adkison jd, bauer dw, chang t. the effect of topical arnica on muscle pain. ann pharmacother. 2010 oct;44(10):1579-84. doi: 10.1345/aph.1p071. 11. šutovská m, capek p, kočmalová m, pawlaczyk i, zaczyńska e, czarny a, et al. characterization and pharmacodynamic properties of arnica montana complex. int j biol macromol. 2014 aug;69:214-21. doi: 10.1016/j.ijbiomac.2014.05.051. 12. koo h, gomes bp, rosalen pl, ambrosano gm, park yk, cury ja. in vitro antimicrobial activity of propolis and arnica montana against oral pathogens. arch oral biol. 2000 feb;45(2):141-8. doi: 10.1016/s0003-9969(99)00117-x.  10 borges et al. 13. oguz ahmet s, mutluay mm, seyfioglu polat z, seseogullari dirihan r, bek b, tezvergil-mutluay a. addition of benzalkonium chloride to self-adhesive resin-cements: some clinically relevant properties. acta odontol scand. 2014 nov;72(8):831-8. doi: 10.3109/00016357.2014.913307. 14. arias-moliz mt, ruiz-linares m, cassar g, ferrer-luque cm, baca p, ordinola-zapata r, et al. the effect of benzalkonium chloride additions to ah plus sealer. antimicrobial, physical and chemical properties. j dent. 2015 jul;43(7):846-54. doi: 10.1016/j.jdent.2015.05.003. 15. gjorgievska es, nicholson jw, coleman nj, booth s, dimkov a, hurt a. component release and mechanical properties of endodontic sealers following incorporation of antimicrobial agents. biomed res int. 2017;2017:2129807. doi: 10.1155/2017/2129807. 16. camões ic, salles mr, chevitarese o, gomes gc. influence on ph of vehicle containing glycerin used with calcium hydroxide. dent traumatol. 2003 jun;19(3):132-8. doi: 10.1034/j.1600-9657.2003.00114.x. 17. chávez de paz le, dahlén g, molander a, möller a, bergenholtz g. bacteria recovered from teeth with apical periodontitis after antimicrobial endodontic treatment. int endod j. 2003 jul;36(7):500-8. doi: 10.1046/j.1365-2591.2003.00686.x. 18. duarte ma, midena rz, zeferino ma, vivan rr, weckwerth ph, dos santos f, et al. evaluation of ph and calcium ion release of calcium hydroxide pastes containing different substances. j endod. 2009 sep;35(9):1274-7. doi: 10.1016/j.joen.2009.05.009. 19. lima rk, guerreiro-tanomaru jm, faria-júnior nb, tanomaru-filho m. effectiveness of calcium hydroxide-based intracanal medicaments against enterococcus faecalis. int endod j. 2012 apr;45(4):311-6. doi: 10.1111/j.1365-2591.2011.01976.x. 20. pereira tc, da silva munhoz vasconcelos lr, graeff msz, ribeiro mcm, duarte mah, de andrade fb. intratubular decontamination ability and physicochemical properties of calcium hydroxide pastes. clin oral investig. 2019 mar;23(3):1253-62. doi: 10.1007/s00784-018-2549-0. 21. gasperi tl, silveira jacd, schmidt tf, teixeira cds, garcia ldfr, bortoluzzi ea. physical-mechanical properties of a resin-modified calcium silicate material for pulp capping. braz dent j. 2020 jun;31(3):252-6. doi: 10.1590/0103-6440202003079. 22. hungaro duarte ma, minotti pg, rodrigues ct, zapata ro, bramante cm, tanomaru filho m, et al. effect of different radiopacifying agents on the physicochemical properties of white portland cement and white mineral trioxide aggregate. j endod. 2012 mar;38(3):394-7. doi: 10.1016/j.joen.2011.11.005. 23. cavenago bc, pereira tc, duarte ma, ordinola-zapata r, marciano ma, bramante cm, et al. influence of powder-to-water ratio on radiopacity, setting time, ph, calcium ion release and a micro-ct volumetric solubility of white mineral trioxide aggregate. int endod j. 2014 feb;47(2):120-6. doi: 10.1111/iej.12120. 24. rehman k, saunders wp, foye rh, sharkey sw. calcium ion diffusion from calcium hydroxide-containing materials in endodontically-treated teeth: an in vitro study. int endod j. 1996 jul;29(4):271-9. doi: 10.1111/j.1365-2591.1996.tb01381.x. 25. de andrade ferreira fb, silva e souza pde a, do vale ms, de moraes ig, granjeiro jm. evaluation of ph levels and calcium ion release in various calcium hydroxide endodontic dressings. oral surg oral med oral pathol oral radiol endod. 2004 mar;97(3):388-92. doi: 10.1016/j.tripleo.2003.08.020. 26. verma r, sharma ds, pathak ak. antibacterial efficacy of pastes against e faecalis in primary root dentin: a confocal microscope study. j clin pediatr dent. 2015 spring;39(3):247-54. doi: 10.17796/1053-4628-39.3.247. 27. stuart ch, schwartz sa, beeson tj, owatz cb. enterococcus faecalis: its role in root canal treatment failure and current concepts in retreatment. j endod. 2006 feb;32(2):93-8. doi: 10.1016/j.joen.2005.10.049. 11 borges et al. 28. orstavik d, haapasalo m. disinfection by endodontic irrigants and dressings of experimentally infected dentinal tubules. endod dent traumatol. 1990 aug;6(4):142-9. doi: 10.1111/j.1600-9657.1990.tb00409.x. 29. zancan rf, calefi phs, borges mmb, lopes mrm, de andrade fb, vivan rr, et al. antimicrobial activity of intracanal medications against both enterococcus faecalis and candida albicans biofilm. microsc res tech. 2019 may;82(5):494-500. doi: 10.1002/jemt.23192. 30. awadalla hi, ragab mh, bassuoni mw, fayed mt, abbas mo. a pilot study of the role of green tea use on oral health. int j dent hyg. 2011 may;9(2):110-6. doi: 10.1111/j.1601-5037.2009.00440.x. 31. gok b, kirzioglu z, kivanc m. green tea extract solutions can control bacterial biofilms formed on space maintainers. niger j clin pract. 2020 jun;23(6):783-91. doi: 10.4103/njcp.njcp_246_19. 32. jaramillo de, arriola a, safavi k, chávez de paz le. decreased bacterial adherence and biofilm growth on surfaces coated with a solution of benzalkonium chloride. j endod. 2012 jun;38(6):821-5. doi: 10.1016/j.joen.2012.03.012. 33. baron a, lindsey k, sidow sj, dickinson d, chuang a, mcpherson jc 3rd. effect of a benzalkonium chloride surfactant-sodium hypochlorite combination on elimination of enterococcus faecalis. j endod. 2016 jan;42(1):145-9. doi: 10.1016/j.joen.2015.08.035. 1http://dx.doi.org/10.20396/bjos.v18i0.8657247 volume 18 2019 e191406 original article 1 institute and research center são leopoldo mandic, campinas, brazil. 2 department of restorative dentistry, piracicaba dental school, university of campinas (unicamp), piracicaba, brazil. corresponding author: vanessa cavalli university of campinas, piracicaba dental school av. limeira, 901, 13414-018 piracicaba, sp, brazil email: vcavalli@yahoo.com received: september 13, 2018 accepted: september 11, 2019 do metal alloy primers increase the bond strength of orthodontic tubes? keity cristina moreira de oliveira1, daylana pacheco da silva2, cecília pedroso turssi1, flávia lucisano botelho do amaral1, vanessa cavalli2,* aim: to evaluate the bond strength (bs) and failure mode of orthodontic tubes treated with different alloy primers at the interface among enamel, resin and orthodontic tubes. methods: orthodontic tubes were bonded to the enamel of 80 bovine incisors with the orthodontic resin (transbond xt, 3m unitek). prior to bonding, the tubes were chemically treated with (n=20) metal/zirconia primer (mz, ivoclar), scothbond universal (sb, 3m espe); orthoprimer (op, morelli) or left untreated (control c). specimens were submitted to 5,000 thermal cycles (5 and 55o c) to age the bonded interface. a shear bs test and failure modes were conducted, and the results were analyzed using one-way analysis of variance and fisher’s exact test, respectively. results: no differences were observed among groups regardless of the type of alloy primer used (p = 0.254). however, no differences were observed among the failure modes of the groups tested (p=0.694). the adhesive failure mode between the resin and enamel was the most prevalent failure (45%) for groups op and c, whereas cohesive failure in the orthodontic resin was the most prevalent failure (40%) for groups sb and mz. conclusion: alloy primers were unable to increase the bs of the orthodontic tubes to enamel. keywords: shear strength. dental bonding. materials testing. alloys. orthodontics. 2 oliveira et al. introduction the use of tubes as an option for orthodontic treatment was responsible for a major advancement in orthodontics, and it was possible due to improvements in adhesive systems and orthodontic composite resins, which provide reliable and long-term enamel adhesion1. the orthodontic tubes offer advantages over traditional systems, such as orthodontic bands, as they facilitate hygiene, eliminate the need for spacers, and decrease the possibility of periodontal disease and secondary carious lesions2. to be effective, tubes must be able to withstand the forces that orthodontic mechanics generate, as well as masticatory forces. in addition, they should present a shear bond strength (bs) greater than 8 mpa, which is the minimum value necessary to maintain enamel adhesion under such circumstances3. however, under clinical conditions, enamel adhesive failures leading to the debonding of orthodontic tubes can occur4-6. in fact, debonding is the reason for many failures, causing 66% of total failures in molars7. adhesive failures are frequently associated with complications with the bonding technique, moisture contamination, occlusal contacts, the presence of aprismatic enamel, and changes in the enamel-etching pattern8. although the tubes can be replaced or submitted to a new bonding procedure, these procedures involve additional chairside appointments, which compromises the treatment progress. the interface between the orthodontic tube and enamel is not the only site susceptible to failure. debonding can also occur between the tube and the orthodontic resin, and although a number of treatments are described to improve adhesion to enamel4-6, only a few are proposed to increase bonding on the metal surface. mechanical treatments designed to increase roughness, such as aluminum oxide blasting, the use of a diamond drill9, and chemical treatment with the use of silane10, are amid the reported alternative treatments for the metal surface. base metal alloys containing chrome in their composition (ni-cr, ni-cr-be, co-cr) can oxidize when in contact with atmospheric air, forming a passivation layer11. some adhesive monomers that can chemically bond to the oxide film on the metal surface have been incorporated into resin cements with the goal of improving bonding across the interface. therefore, bonding between adhesive monomers and alloys is the result of a micromechanical interocking promoted by surface roughness, and a chemical interaction between the oxide film of the metal surface and the carboxylic of phosphate acidic monomer of the resin cements12. with the aim of overcoming the absence of adhesive monomers in the composition of resin cements, metal alloy primers were developed to be applied on the surfaces of alloys prior to cementation13. previous, studies have demonstrated alloy primers’ ability to improve bonding between polymer-based materials and metallic surfaces14,15. the first monomer to be used for adhesion to metal was 4-meta (methacryloyloxyethy trimellitate anhydride), which was designed to eliminate the need for mechanical retention and increase adhesion16. later, monomers containing either phosphoric or carboxylic acid groups, such as 11-methacryloxyunden, decarboxylic acid (mac10), 10 methacryloxydecyl dihydrogen phosphate (10 mdp), vinylbenzyl-n-propylamino-triazine-dithiol (vbatdt), and 6-methacryloxyhexyl-2-thiouracil-5-caboxylate (mtu-6), 3 oliveira et al. were synthesized14-16. the primers applied on the surface of the metal may contain mdp, vbtdt, mtu-6, mac-10, or the combination of these monomers. the functional monomer of 10-mdp is able to react chemically with the chromium oxide of the casting surface to promote adhesion14,16. mdp has two functional groups: one is a di-valent phosphoryl group that chemically bonds to the metal atoms of the metal surface, and the other is a methacryloyl group that copolymerizes with resin monomers either in the adhesive or in the resin cement composition14,16. recently, “universal “or ”multimold” adhesives containing both silane and a functional monomer have been developed and are indicated to bond to metal surfaces. however, these universal adhesives’ ability to bond to metal alloys is still being debated16. since alloy primers are indicated for metal surfaces, the potential of increasing the bs of orthodontic tubes seems to be promising17. however, the literature is scarce in studies evaluating the use of primers in metal orthodontic devices. therefore, the aim of this study was to evaluate the bs and failure mode at the interface among enamel, resin, and orthodontic tubes treated with various metal primers. the null hypothesis was that no differences would be found in the bs and failure mode among groups. materials and methods experimental design the experimental units consisted of 80 bovine incisors, and orthodontic tubes were bonded to the enamel buccal surfaces. before bonding, the orthodontic tubes received (n=20) two alloy primers (metal/zirconia primer – mz and orthoprimer op) and one multi-mode adhesive (scothbond universal – sb). the control group was left untreated (n = 20). the response variables were shear bs (in mpa) and the failure mode of the deboned area. the commercial name, manufacturer, and composition of the alloy primers are shown in table 1. table 1. composition of the metal primers and orthodontic composite. commercial name and manufacutrer composition metal/zirconia primer (mz, ivoclar vivadent, schaan, liechtenstein) methacrylate of phosphonic acid and methacrylate cross-linked in organic solvent. scotchbond universal (sb, 3m/espe, st. paul, mn, usa) bis-gma, hema, silane treated silica, water, ethanol, decamethylenedimethacrylate,10-decanediol phosphate methacrylate, acrylic copolymer and itaconic acid, camphorquinone, n, n-dimethylbenzocaine, 2-dimethylamonoethyl methacrylate, methyl ethyl ketone. orthoprimer (op, morelli, sorocaba, brazil) bis-gma, teg-dma, hema, dmpt, camphorquinone, hydroxytoluenebutylated, dimethyl aminoethyl methacrylate. transbond xt (3m-unitek, monrovia, usa) primer: bisphenol a diglycidyl ether dimethacrylate, teg-dma, triethylene glycol dimethacrylate, triphenylantimonium, 4(dimethalamino) -benzethanol, d-1-camphorquinone, hydroquinone. resin: bis-gma, bisphenol a bis (2-hydroxyethyl ether) dimethacrylate, quartz treated silane, silane treated silane, silanodimethacrylate, diphenyliodonium hexafluorophosphate bis-gmabisphenolglicedyl methacrylate; teg-dma-triethylene glycol dimethacrylate; hema-hydroxyethyl methacrylate; dmpt-dimethyl-p-toluidine. 4 oliveira et al. specimen preparation eighty bovine incisors crowns with enamel free of defects or cracks were selected and were stored in a 0.1% thymol solution for 24 hours. after debridement and pumicing, the teeth were embedded in polystyrene resin with the buccal surfaces facing up and sonicated. enamel bonding was performed according to the manufacturer’s instructions for the orthodontic composite (transbond xt). teeth were cleaned with water spray (15 s), air dried (15 s), and acid etched with 37% phosphoric acid gel for 30 s. the surface was rinsed with air-water spray for 20 s and air dried for 10 s. the primer of the transbond xt system was applied on the enamel surface, then sprayed with a mild air spray for 5 s and light cured for 20 s. the orthodontic composite (transbond xt) was applied on the inner surface of the tube and fixed on the enamel. light curing was performed for 40 s (20 s in the mesial and 20 s in the distal sites) using a led light-curing unit (bluephase, ivoclar, liechtenstein, with irradiance of 1200 mw/cm2). before the tube was bonded to the enamel surface, the base of the orthodontic tube was treated according to each experimental group: • mz: the primer was applied on the base of the tube (180 s) and air dried (5 s). • op: the primer was applied on the base of the tube (180 s) and air dried (5 s). • sb: the universal adhesive was applied on the base of the tube (20 s) and air-dried (5 s). • control: no treatment was performed on the tube. thermal cycling the samples were stored for 24 h in distilled water at 37 ºc, and for the purpose of aging the bonding interface, 5,000 thermal cycles were performed (msct-3, marcelo nucci me, são carlos, brazil) at 5 and 55 °c (± 1°c) with a dwell time of one minute each. shear bond strength test forty-eight hours later, specimens were submitted to a shear test in the occlusal-cervical direction, with the blade placed at the enamel and resin/tube interface. the tests were performed in a universal testing machine (emicdl 2000, instron brasil scientific equipment ltda, são josé dos pinhais, brazil) with a load cell of 1kn at a crosshead speed of 0.5 mm/min. the maximum force (n) up to failure was recorded. the shear bs (in mpa) was calculated from the force and the bonded area of the tube to the enamel surface. failure mode the failure mode of the debonded interface was observed under a stereomicroscope at 40x magnification. debonding was classified (table 2) based on a previous report18. 5 oliveira et al. statistical analysis shear bs data were submitted to exploratory analysis to verify normality and homoscedasticity, and they were also submitted to parametric one-way analysis of variance (anova). the fracture mode was analyzed using fisher’s exact test. in all analyses, sas software (sas institute inc., cary, nc, usa, release 9.2, 2010) was used considering the level of significance of 5%. results no significant difference was found among groups in terms of shear bs values (p = 0.254) as observed in table 3. additionally, no significant difference (p = 0.694) was observed in the failure mode distribution of the adhesive interface as a function of the treatments. the most common failure mode was type 3 (adhesive failure between the orthodontic composite and enamel), in which no orthodontic composite remnant was found in the enamel (45% of the experimental units). the least prevalent failure was type 1 (only in the sb group), in which the base of the orthodontic tube did not exhibit a resin remnant and all of the resin remained on the enamel (1.2% of the experimental units) (table 3). table 3. mean and standard deviation of shear bond strength and distribution of the failure mode according to treatments. group shear bs failure mode type 1 (adhesive orthodontic composite/tube) type 2 (cohesive in orthodontic composite) type 3 (adhesive orthodontic composite/enamel) type 4 (cohesive in enamel) mz 13.64 (6.24) a 0 (0%) 11 (55%) 5 (25%) 4 (20%) sb 12.26 (5.38) a 1 (1.25%) 11 (55%) 6 (30%) 2 (10%) op 13.15 (4.18) a 0 (0%) 4 (20%) 13 (65%) 3 (15%) control 10.40 (5.70) a 0 (0%) 6 (30%) 12 (60%) 2 (10%) total 1 (1.25%) 32 (40%) 36 (45%) 11 (13.75%) mz metal/zirconia primer; sb scotchbond universal; op – orthoprimer; nnumber of samples per group. means followed by the same letter indicate no statistical differences. no differences were observed in failure modes among experimental groups, according to fisher’s test (p = 0.1119). table 2. failure mode classification standard type of failure 1 adhesive failure between orthodontic composite and the base of the orthodontic tube (100% of the composite remains on enamel surface) 2 cohesive failure in the orthodontic composite (50% of the composite remains at the base of the orthodontic tube and 50% bonded on enamel) 3 adhesive failure between orthodontic composite and enamel (100% of the composite remains on the tube) 4 cohesive enamel fracture 6 oliveira et al. discussion the bs results showed no difference among the orthodontic tubes that were treated with various primers, even those containing acidic phosphate monomers (sb and mz). dias et al.19 observed that the application of an alloy primer (kuraray) did not increase the bs of the resin cement to zirconia, and the primer did not prevent bs decrease after six months of water storage. the alloy primer contains two functional acidic monomers vbatdt (vinylbenzyl-n-propylamino-triazine-dithiol) and 10-mdp in an acetone-based solution, and it is indicated to cement metal fixed prosthodontics structures. similarly, it was previously reported that an orthoprimer application to polycarbonate-based brackets did not influence the bs results15, which is comparable to the findings of our study. the orthoprimer agent does not contain acidic phosphate monomers except for methacrylate monomers and non-phosphoric hydrophilic monomers (tegdma e hema) (table 1). therefore, it is speculated that this primer is responsible for increasing wettability and improving the resin permeation of the composite in the irregularities of the base of the orthodontic tube. however, it does not chemically bond to the alloy surface as the function monomer does. contrary to the present results, cal neto et al.20 observed that the application of an alloy primer with the acidic monomer 4-meta (4-methacryloxyethyl trimellitic anhydride) increased the bs between the composite and the metallic brackets 48% compared with the control group, whereas in our research, alloy primers were able to increase the bs 17.9 to 31.2%. it should be noticed that these authors used a different primer (4-meta) and that this agent could have been more effective in increasing bs compared with the primers selected in this research. the absence of thermal cycles to age the bonded interface, and the fact that the authors used human pre-molars instead of bovine incisors cannot be ruled out. in a previous study20, it was observed that treating the surface of zirconia with alloy primers increased the bs of the zirconia to the metallic bracket. that study used z-prime plus (10-mdp and carboxylic acid) and the zirconia liner premium (silane with mdp)20. the authors credited the good performance of z-prime plus to the presence of mdp and the ability of the primer to co-polymerize with the resin monomers, as the functional group binds to the metallic oxide of the substrate via the phosphoric group21. it is important to notice that mz, sb, and z-prime plus exhibit different acidic phosphate monomers: the phosphonic acid methacrylate, 1,10-decanediol phosphate methacrylate, and 10-methacryloyloxydecyl dihydrogen phosphate, respectively. thus, different monomer compositions and application modes may influence bs results22. furthermore, the presence of organophosphate monomers in the universal adhesive sb could promote the instability of the silane component22. therefore, it is possible that the presence of silane hampered the monomer performance and that the adhesive presented similar bs results compared with the other primers. thermal cycling (5,000 cycles) was performed for all groups after bonding to age the interface. previous studies observed that mdp-based primers were effective in preserving the bonding stability to zirconia even after thermal cycling22-24. on the other hand, imai et al.25 observed that the bs between composite and alloy surfaces 7 oliveira et al. decreased after 20,000 thermal cycles. the authors suggested that the lower bs could have been the result of water infiltration in microgaps between the composite and the alloy25. as no differences were observed in our study, it is possible that the number of cycles performed could have been insufficient for influencing the bs. no significant differences were found among the failure modes. in the current study, the shear bs test evaluated multiple interfaces—enamel, orthodontic resin, and the base of the orthodontic tube. numerically, the most predominant failure was the adhesive type, between the enamel and orthodontic resin (45%). this may be an indication that the bs at the base of the orthodontic tube where the primer was applied was acceptable. in addition, it should be kept in mind that in shear bond tests, the knife of the apparatus slides down the bordering enamel, stressing the orthodontic resin more than the orthodontic tube. therefore, as expected, the second more prevalent failure was cohesive failure in the orthodontic composite (40%). the least prevalent failure was the adhesive type between the orthodontic composite and the base of the orthodontic tube (1.25%), which might indicate that although the primers did not improve the shear bs, they did not compromise it. in addition, the fact that no differences were found among the bs and failure modes implies that the irregularities at the base of the orthodontic tubes were sufficient for promoting a reasonable bs regardless of the presence of primers. mz, op, and sb exhibited 15%, 20% and 10% respectively, of enamel cohesive defects. although this type of failure indicates an acceptable performance of the primers applied to the metal, enamel fracture is undesirable when the tubes are removed at the end of orthodontic treatment. therefore, because no differences were observed among groups, we believe that the decision to apply primers to metallic surfaces should be reexamined, as it could cause enamel fracture during debonding. based on the above, the null hypothesis could be accepted, as the application of alloy primers to the base of the orthodontic tubes did not increase the bs to the enamel surface and did not influence the failure modes of the tubes bonded to the enamel surface. moreover, based on the failure modes and the existence of enamel cohesive failures when primers were applied, we endorse that no need exists to apply alloy primers to orthodontic tubes. considering that the application of alloy primers to orthodontic tubes can be dismissed and that debonding occurs due to adhesives (enamel/orthodontic resin) at the orthodontic resin, future research should focus on developing an orthodontic resin that will not fail cohesively and that possesses optimal enamel adhesion that will resist shear forces without compromising enamel integrity when the tube is removed at the end of the orthodontic treatment. in conclusion, the application of alloy primers did not increase the bs of the orthodontic tube to the enamel. in addition, its application should be better evaluated, as enamel fracture could occur during debonding. conflict of interest statement the authors do not have any financial interest in the companies whose materials are included in this article. 8 oliveira et al. references 1. murray pg, millett dt, cronin m. bonded molar tubes: a survey of their use by specialist orthodontists. j orthod. 2012 jun;39(2):129-35. doi: 10.1179/1465312512z.00000000016. 2. al-anezi sa. the effect of orthodontic bands or tubes upon periodontal status during the initial phase of orthodontic treatment. saudi dent j. 2015 jul;27(3):120-4. doi: 10.1016/j.sdentj.2014.11.010. 3. pickett kl, sadowsky pl, jacobson a, lacefield w. orthodontic in vivo bond strength: comparison with in vitro results. angle orthod. 2001 apr;71(2):141-8. 4. fleming ps, johal a, pandis n. self-etch primers and conventional acid-etch technique for orthodontic bonding: a systematic review and meta-analysis. am j orthod dentofacial orthop. 2012 jul;142(1):83-94. doi: 10.1016/j.ajodo.2012.02.023. 5. brunharo ihvp, fernandes dj, miranda ms, artese f. influence of surface treatment on shear bond strength of orthodontic brackets. dental press j orthod. 2013 may-jun;18(3):54-62. 6. bakhadher w, halawany h, talic n, abraham n, jacob v. factors affecting the shear bond strength of orthodontic brackets a review of in vitro studies. acta medica (hradec kralove). 2015;58(2):43-8. doi: 10.14712/18059694.2015.92. 7. millett, dt, mandall, na, mattick rcr, hickman j, glenny am. adhesives for bonded molar tubes during fixed brace treatment. cochrane database syst rev. 2017 feb 23;2:cd008236. doi: 10.1002/14651858.cd008236.pub3. 8. pandis n, christensen l, eliades t. long-term clinical failure rate of molar tubes bonded with a selfetching primer. angle orthod. 2005 nov;75(6):1000-2. 9. ribeiro aa, de morais av, brunetto dp, ruellas ac, de araujo mt. comparison of shear bond strength of orthodontics brackets on composite resin restorations with different surface treatments. dental press j orthod. 2013 jul-aug;18(4):98-103. 10. zhang y, sun mj, zhang d. designing functionally graded materials with superior load-bearing properties. acta biomater. 2012 mar;8(3):1101-8. doi: 10.1016/j.actbio.2011.11.033. 11. thery s, jaquet d, mantel m. a study of chemical interactions at the stainless steel/polymer interface by infrared spectroscopy. part 2: mechanical properties and study of the interface. j adhes. 1996;56:15-25. 12. matsumura h, kamada k, tanoue n, atsuta m. effect of thione primers on bonding of noble metal alloys with an adhesive resin. j dent. 2000 may;28(4):287-93. 13. pulido lg, powers jm. bond strength of orthodontic direct-bonding cement-plastic bracket systems in vitro. am j orthod. 1983 feb;83(2):124-30. 14. bulbul, m; kesim b. the effect of primers on shear bond strength of acrylic resins to different types of metals. j prosthet dent. 2010 may;103(5):303-8. doi: 10.1016/s0022-3913(10)60063-7. 15. souza nf, vilar rv, almeida ma, miguel ja, cal-neto jp. primer on the base of polycarbonate brackets: should we apply it or not? prog orthod. 2010;11(1):37-40. doi: 10.1016/j.pio.2010.04.006. 16. nima g, ferreira pvc, paula ab, consani s, giannini m. effect of metal primers on bond strength of a composite resin to nickel-chrome metal alloy. braz dent j. 2017 jan-apr;28(2):210-215. doi: 10.1590/0103-6440201701288. 17. pithon mm, oliveira mv, ruellas aco, bolognese am, romano fl. shear bond strength of orthodontic brackets to enamel under different surface treatment conditions. j appl oral sci. 2007 apr;15(2):127-30. 18. wang wn, lu tc. bond strengths with various etching times on young permanent teeth. am j orthod dentofacial orthop. 1991 jul;100(1):72-9. http://www.ncbi.nlm.nih.gov/pubmed/?term=bakhadher w%5bauthor%5d&cauthor=true&cauthor_uid=26455565 http://www.ncbi.nlm.nih.gov/pubmed/?term=halawany h%5bauthor%5d&cauthor=true&cauthor_uid=26455565 http://www.ncbi.nlm.nih.gov/pubmed/?term=talic n%5bauthor%5d&cauthor=true&cauthor_uid=26455565 http://www.ncbi.nlm.nih.gov/pubmed/?term=abraham n%5bauthor%5d&cauthor=true&cauthor_uid=26455565 http://www.ncbi.nlm.nih.gov/pubmed/26455565 http://www.ncbi.nlm.nih.gov/pubmed/26455565 http://www.ncbi.nlm.nih.gov/pubmed/?term=pandis n%5bauthor%5d&cauthor=true&cauthor_uid=16448245 http://www.ncbi.nlm.nih.gov/pubmed/?term=christensen l%5bauthor%5d&cauthor=true&cauthor_uid=16448245 http://www.ncbi.nlm.nih.gov/pubmed/?term=eliades t%5bauthor%5d&cauthor=true&cauthor_uid=16448245 https://www.ncbi.nlm.nih.gov/pubmed/28492752 https://www.ncbi.nlm.nih.gov/pubmed/28492752 9 oliveira et al. 19. dias tm, pacheco rr, de sá rb, francescantonio md, berger sb, giannini m. [evaluation of the effect of metal primers and type of resin cement on the bond strength to zirconia]. rev bras odontol. 2012 jan-jun;69(1):15-20. portuguese. 20. cal neto jp, calasans-maia ja, de almeida nv, rohen h, freire ma. effect of a metal primer on the adhesive interface between composite and lingual brackets. j contemp dent pract. 2013 nov 1;14(6):1106-8. 21. lee jy, kim js, hwang cj. comparison of shear bond strength of orthodontic brackets using various zirconia primers. korean j orthod. 2015 jul;45(4):164-70. doi: 10.4041/kjod.2015.45.4.164. 22. grifin jd, suh bi, chen l, brown dj. surface treatments for zirconia bonding: a clinical perspective. can j rest dent prosthodont. 2010 jan.3:23-9. 23. koizumi h, nakayama d, komine f, blatz mb, matsumura h. bonding of resin-based luting cements to zirconia with and without the use of ceramic priming agents. j adhes dent. 2012 aug;14(4):385-92. doi: 10.3290/j.jad.a22711. 24. nakayama d, koizumi h, komine f, blatz mb, tanoue n, matsumura h. adhesive bonding of zirconia with single liquid acidic primers and a tri-n-butylborane initiated acrylic resin. j adhes dent. 2010 aug;12(4):305-10. doi: 10.3290/j.jad.a17549. 25. imai h, koizumi h, shimoe s, hirata i, matsumura h, nikawa h. effect of thione primers on adhesive bonding between an indirect composite material and ag-pd-cu-au alloy. dent mater j. 2014;33(5):681-8. http://www.ncbi.nlm.nih.gov/pubmed/?term=cal neto jp%5bauthor%5d&cauthor=true&cauthor_uid=24858759 http://www.ncbi.nlm.nih.gov/pubmed/?term=calasans-maia jde a%5bauthor%5d&cauthor=true&cauthor_uid=24858759 http://www.ncbi.nlm.nih.gov/pubmed/?term=de almeida nv%5bauthor%5d&cauthor=true&cauthor_uid=24858759 http://www.ncbi.nlm.nih.gov/pubmed/?term=rohen h%5bauthor%5d&cauthor=true&cauthor_uid=24858759 http://www.ncbi.nlm.nih.gov/pubmed/?term=freire ma%5bauthor%5d&cauthor=true&cauthor_uid=24858759 1http://dx.doi.org/10.20396/bjos.v21i00.8664013 volume 21 2022 e224013 original article 1 school of health sciences, positivo university, curitiba, brazil. 2 piracicaba dental school, university of campinas, piracicaba, são paulo, brazil. 3 department of operative dentistry, school of dentistry, university of joinville, joinville, santa catarina, brazil. corresponding author: marilisa carneiro leão gabardo rua prof. pedro viriato parigot de souza, 5300 81280-330 curitiba, paraná, brazil. tel: +55 41 3526-5121 e-mail: marilisagabardo@gmail.com editor: dr altair a. del bel cury received: january 19, 2021 accepted: june 30, 2021 cleaning of long oval canals with waveone gold system associated with different irrigant agitation protocols prescila mota de oliveira kublitski1 , flávia sens fagundes tomazinho1 , bruno marques-da-silva1 , vinícius rodrigues dos santos2 , wander josé da silva2 , flares baratto-filho3 , marilisa carneiro leão gabardo1,* aim: the aim of the study was to evaluate the cleaning of mandibular incisors with waveone gold® (wo) under different preparation techniques. methods: a total of 210 human mandibular incisors were selected and divided into seven groups (n = 30), prepared by wo single-files (small 20/.07 – wos; primary 25/.07 – wop; medium 35/.06 – wom; or large 45/.05 wol) and sequential-file techniques (wos to wop; wos to wom; and wos to wol). further subdivision was made according to irrigation protocol: control group (manual irrigation con), e1 irrisonic® eir, and eddy® edd. debris removal and the smear layer were evaluated by scanning electron microscopy. data were analyzed by using spearman’s correlation test. the significance level was set at 5%. results: for debris and smear layer removal, wos and wop, eir differed from con and edd (p <0.05). conclusion: regardless of the instrumentation used, the agitation of the irrigant solution provided better cleanability. these findings reinforce the need for agitation techniques as adjuvants in cleaning root canal systems in mandibular incisors. keywords: endodontics. root canal irrigants. root canal preparation. microscopy, electron, scanning. https://orcid.org/0000-0002-3828-2328 https://orcid.org/0000-0001-5553-6943 https://orcid.org/0000-0002-6227-4125 https://orcid.org/0000-0002-0369-3485 https://orcid.org/0000-0002-5613-5081 https://orcid.org/ 0000-0002-5649-7234 https://orcid.org/0000-0001-6832-8158 2 kublitski et al. introduction endodontic therapy aims to promote the shaping of the root canal system, eliminating microorganisms, tissue remains and debris, promoting a sanitized place for obturation1. in all dental groups cleaning of the apical region is a challenge, as reported by many authors2,3, but some teeth have anatomical characteristics that make this process more difficult, such as the mandibular incisors. these teeth are especially flatted and narrow, and it is known that the excessive dentin wall wear due to the use of large instruments can lead to root weakening, while the use of less tapered instruments can lead to non-instrumented areas4. in the literature, findings indicate significant results regarding the reduction of root canal microorganisms using the combination of chemical and mechanical preparation5. better results in terms of disinfection are found proportionally with the root canal enlargement, especially in the apical region6, when the benefits of the action of sodium hypochlorite solution (naocl) appear to be significantly increased7. new systems and techniques aimed at improving the root canal preparation have been commercially introduced regularly. currently, most are presented as single-use instruments with the proposal of cost and operating time reduction8. the waveone gold® (wo) system was presented in this context and featured in small (20/.07), primary (25/.07), medium (35/.06), and large (45/.05) (dentsply sirona endodontics, ballaigues, switzerland). despite technological advances related to endodontic instruments, tips to agitate the irrigant solution have been proposed to enhance the disinfection process9. these techniques are detrimental to the conventional methods performed with a syringe and fine-caliber needles10. thus, the eddy® sonic tip (vdw gmbh, munich, germany) as well as the e1 irrisonic® (20/.01) (helse ultrasonic, santa rosa de viterbo, brasil) are aimed at agitating the irrigant solutions11. regarding the cleaning capacity, scanning electron microscopy (sem) has been used to analyze the presence of debris and smear layer adhered to the inner walls of the root canal after preparation12,13. therefore, the present study aimed to analyze the cleaning of mandibular incisors with the wo system under different preparation protocols. the null hypothesis tested was there is no difference between the preparation techniques. material and methods this study was approved by the institutional ethics committee of universidade positivo (approval number 2,420,082). only one trained operator performed the experimental procedures. for the analysis of the images, two researchers were trained and calibrated (kappa = 0.83). sample size estimation and characterization initially, sample data from previous studies were used as reference14,15. subsequently, the power observed in the sample was calculated, considering the value of α = 5% and 3 kublitski et al. the rejection of the null hypothesis (existence of difference between treatments) by the nonparametric anova test, which resulted in a power value > 99%. a total of 210 freshly extracted human mandibular incisors were selected, which at the digital radiographic examination towards bucco-lingual and mesio-distal directions, presented a single root canal, complete rhizogenesis, and had no endodontic treatment, calcifications, internal and external resorption, lacerations, or fractures. these teeth remained in 0.9% sodium chloride (nacl) solution (eurofarma, são paulo, brazil) until the beginning of the experiment, with the solution being replaced every seven days. the crowns were sectioned with a double-sided diamond disc (kg sorensen, cotia, brasil) with the remaining standardized at a length of 15 mm. the patency of all root canals was established with a 10 k-file (dentsply sirona endodontics, ballaigues, switzerland), at a working length (wl) of 14 mm. initially the specimens were divided into four groups according to the instrument used for the preparation (n = 30): wo small (20/.07) – wos; wo primary (25/.07) wop; wo medium (35/.06) wom; wo large (45/.05) wol. this allocation was based on the manual files used to negotiate the root canals, according to van der vyver et al.16. the surpluses of this division were distributed in the other groups, until each reached n = 30. specimen preparation then the root apexes were isolated with composite resin (filtek® z350; 3m espe saint paul, eua). the specimens were divided as follows (n = 30): wos, wop, wom, wol, wos to wop, wos to wom, wos to wol. in this study, the wo system, which has different conicities, was tested in a single-file technique (each specimen was prepared with only one instrument according to the initial apical diameter) and sequential-file techniques (specimen preparation was performed sequentially using the entire system). the instruments were coupled to the x-smart plus® electric motor (dentsply sirona endodontics, ballaigues, switzerland) and were used according to the manufacturer’s instructions. regarding the use of instruments reproducing clinical reality, in this study each instrument was used three times (three specimens) with the consideration that the instrumentation of molar teeth (with three to four root canals) with a single instrument is safe. irrigation protocols and solution agitation irrigation during preparation for all groups was performed with a 5 ml luer lock syringe (bd®, curitiba, brasil) coupled to a 30 g needle irrigation tip (navitip; ultradent, south jordan, eua), calibrated at 11 mm. at each 3 mm advance of the instrument inside the root canal, 2 ml 2.5% naocl was used. between advances, the wl was verified with a 10 k-file. after preparation, the specimens were divided into three subgroups (n = 10) according to the irrigation protocol (figure 1). the control group (con) received conventional irrigation, as described above. the e1 irrisonic® (20/.01) tip (eir) (helse ultrasonic, santa rosa de viterbo, brasil), and the eddy® tip (edd) (vdw gmbh, munich, germany) were used for agitation. the eir tip was coupled to the piezon® master 200 ultrasound 4 kublitski et al. device (ems, nyon, switzerland) power 1; edd was activated in a handpiece (sonic borden 2000n, kavo dental ltda., joinville, brazil) at a power of 5000 hz to 6000 hz. both instruments were calibrated at 1 mm from the wl (13 mm). in all specimens the methodology presented by plotino et al.13 was followed to both, eir and edd. the irrigation was performed at 1 mm from the wl with 2.5 ml of 2.5% naocl, activated for 20 s three times, renewed with 1 ml of fresh 2.5% naocl between them. again, the root canals were irrigated with 2.5 ml of 2.5% naocl. final irrigation was performed with 17% edta for a total of 2 min as follows: the activation occurred during 20 s three times, with solution renovation (1 ml of fresh 17% edta), and then another irrigation with 2 ml of 17% edta continuously for 1 min. for the con group, the irrigation was performed, without agitation, with 8 ml of naocl and 10 ml of edta. a final flush was performed with 2.5 ml of sterile saline solution in all canals. the final aspiration was done with 0.36 mm capillary tip tips (ultradent, south jordan, eua). preparation of specimens for sem exam for the cleavage of all of the specimens into two halves, two longitudinal grooves were made along the entire root length in the buccal and lingual walls with double-sided diamond discs (kg sorensen). an ochsenbein no. 1 micro chisel (quinelato®; schobell industrial ltda., rio claro, brazil) was introduced into the grooves, and lever movements were made throughout the root extension to avoid an uneven fracture of the apical third, which was the focus of the of analysis. for dehydration, the sample was placed in petri dishes, stored in a sterilization and drying oven, for 48 h at 36 ºc. prior to image acquisition, the specimens were subjected to gold sputtering. figure 1. flowchart of the division of experimental groups. wos, waveone gold® small; wop, waveone gold® primary; wom, waveone gold® medium; wol, waveone gold® large; con, control group; eir, e1 irrisonic®; edd, eddy®. (n = 210) wos (n = 30) con (n = 10) eir (n = 10) edd (n = 10) con (n = 10) eir (n = 10) edd (n = 10) con (n = 10) eir (n = 10) edd (n = 10) con (n = 10) eir (n = 10) edd (n = 10) wos to wop (n = 30) con (n = 10) eir (n = 10) edd (n = 10) wos to wom (n = 30) con (n = 10) eir (n = 10) edd (n = 10) wos to wol (n = 30) con (n = 10) eir (n = 10) edd (n = 10) wop (n = 30) wom (n = 30) wol (n = 30) single-files technique (n = 120) sequential-files technique (n = 90) 5 kublitski et al. analysis of dental wall cleaning by sem the root walls of the apical third were analyzed at 3 mm from the apex using a scanning electron microscope (jsm 6010; jeol, peabody, usa) at a power of 20 kv. magnifications of 100× and 1000× allowed evaluation of debris and smear layer, respectively13. for the acquisition of the images, the microscope’s own software measurement application was used. the wall submitted to analysis was the one with the highest thickness. the criteria used to quantify these variables followed the classification of gutmann et al.17: score 1, absence or little debris covering up to the 25% of the surface; score 2, little to moderate debris covering from 25% to 50% of the surface; score 3, moderate to high presence of debris covering from 50% to 75% of the surface; score 4, high amount of aggregated or scattered debris covering over 75% of the surface. for smear layer the criteria was: score 1, absence or little smear layer, covering less than 25% of the specimen with tubules visible and patent; score 2, little to moderate or patchy amounts of smear layer, covering from 25% to 50% of the specimen with many tubules visible and patent; score 3, moderate amounts of scattered or aggregated smear layer, covering from 50% to 75% of the specimen with minimal tubules visible or patent; score 4, high amount of smear layer covering over 75% of the specimen with no tubule orifices visible or patent. thus, debris and smear layer were considered criteria to evaluate the cleaning capacity in the different forms of irrigant solution agitation, compared to the manual irrigation technique in the apical region only. all analyses were performed using spss (ibm® statistics v. 25.0, spss inc, chicago, usa) with a significance level of 5%. the spearman correlation test was used for debris to test the correlation of the different scores between groups. results the sequential use of wos to wop instruments resulted in greater removal of debris and smear layer, with a statistically significant difference when compared to wop (p <0.05). differences were also observed in smear removal in single use between wom and wos (p = 0.025), and sequentially between wos to wom and wos to wop (p = 0.035) (table 1). table 1. results of debris and smear layer assessment, according to group and irrigation/agitation (median: minimum-maximum). group debris smear layer con eir edd con eir edd wos 1.50 (1-4)a,b,c 1.00 (1-4)a,b 1.00 (1-3)a,b 3.00 (1-4)b,b 1.00 (1-4)a,a 1.00 (1-3)a,a wop 2.50 (1-4)b,c,b 1.00 (1-1)a,a 2.50 (1-4)c,d,b 3.00 (1-4)b,c,b 1.00 (1-3)a,a 3.00 (1-4)b,b wom 1.50 (1-4)a,b,c 1.00 (1-2)b 1.00 (1-2)a 1.50 (1-4)a,c 1.00 (1-3)a 1.00 (1-3)a wol 3.00 (1-4)b 1.00 (1-4)a,b,c 2.00 (1-4)c 3.00 (1-4)b,c 2.00 (1-3)a,b 3.00 (1-4)a,b wos to wop 1.00 (1-4)a 1.00 (1-3)a,b 1.00 (1-4)a,c 1.00 (1-4)a 1.00 (1-4)a 1.50 (1-4)a,b wos to wom 2.00 (1-4)a,b 1.50 (1-4)a,b,c 1.00 (1-4)a,b,d 3.50 (1-4)b,c 1.50 (1-3)a 1.00 (1-4)a wos to wol 1.50 (1-4)a,b 2.00 (1-3)c 2.50 (1-4)b,c 1.00 (1-4)a,c 3.00 (2-4)b 2.00 (1-4)a,b note: capital letters indicate statistically significant differences in the column. lower case letters indicate statistically significant differences in the line. kruskal-wallis followed by dunn’s post hoc test (p <0.05). 6 kublitski et al. concerning agitation, for eir differences were observed in the comparisons between groups for wos a wop and wos a wol (p = 0.012); in the intra-group comparison, for wop, edd and con were the same (p = 0.682), but different from eir (p <0.05). these differences occurred for both debris and smear layer. edd only showed significant differences when dealing with a single-use instrument. for debris, wos and wom were the same (p = 0.654), but differed from wop and wol (p <0.05). for the removal of smear layer, there was a difference between wop and wos (p = 0.017), and wop and wom (p = 0.049) (table 1). the assessment of debris and smear layer scores is shown in figure 2. of a total of 210 specimens analyzed, 114 had a score of 1, i.e., more than 50% of the sample obtained satisfactory cleaning. this image also reveals that, in absolute numbers, edd performed at or above eir, based on the number of specimens recorded as score 1. figure 2. (a) number of specimens recorded for each score in relation to the apical third of root canals in the evaluation of residual debris of the different treatments (100×), and (b) smear layer (1000×). wos, waveone gold® small; wop, waveone gold® primary; wom, waveone gold® medium; wol, waveone gold® large; con, control group; eir, e1 irrisonic®; edd, eddy®. score 1 score 2 score 3 score 4 wos 10 9 8 7 6 5 4 3 2 1 5 1 1 3 8 2 1 2 1 3 3 1 3 2 4 1 1 1 1 7 1 1 3 5 1 4 4 1 1 3 6 4 5 1 3 3 2 4 3 6 1 1 1 8 1 1 8 1 3 6 7 2 3 2 2 3 3 2 5 3 2 8 2 8 2 10 3 0 con eir edd wos con eir edd wos con eir edd wos con eir edd wos to wop con eir edd wos to wom con eir edd wos to wol con eir edd score 1 a b score 2 score 3 score 4 wos 10 9 8 7 6 5 4 3 2 1 2 3 1 4 6 2 2 1 2 1 2 5 3 2 4 2 2 8 2 2 6 4 5 3 2 3 3 3 5 1 5 2 5 3 3 4 1 2 7 1 1 1 7 1 4 5 7 2 1 5 2 1 3 4 5 3 1 1 7 2 1 6 2 2 9 1 2 0 con eir edd wos con eir edd wos con eir edd wos con eir edd wos to wop con eir edd wos to wom con eir edd wos to wol con eir edd 7 kublitski et al. discussion this study aimed to evaluate the cleaning of mandibular incisors with wo under different preparation techniques. the results found in the present study revealed that even with the technological advances related to endodontic instruments, their use alone does not have efficient root canal cleaning capacity, and the combination of the use of irrigation solutions concomitantly with their agitation is necessary3,9. the failure of endodontic therapy is related to the maintenance of pathogens into the root canal system18, thus the chemical and mechanical combination in preparation is so important5. in this context, the apical region is a challenge, being the place with the largest amount of debris2,3. the mechanical debridement of the last millimeters of the root canals is not as effective as cervical region, and some authors attribute this fact due to caliber of the dentinal tubules or the narrower diameter of the canal in this region19, therefore less penetration and contact occurs between the canal walls and the irrigators2. once the literature reveals that limitations in endodontic treatment are also related to the variation of internal root anatomy20, the study of the apical region of mandibular incisors is opportune, because this dental group has root canals especially flatted and narrowed. care during preparation of these teeth is necessary, because the greater enlargement of the apical third promotes more cleaning6, but it know that excessive instrumentation can lead the root weak4. other justification to the method adopted here is due to the fact that protocols with an association of different instruments and irrigants are necessary to compare the performance in removing debris and smear layer21. the techniques (single or sequential instruments) used in this study to assess the cleaning capacity of the root canals with wo instruments showed differences between wop versus wos to wop. thus, the null hypothesis was rejected, with a significant difference regarding the removal of debris and smear layer on the root canal walls. with the same purpose in mandibular incisors, de-deus et al.22, revealed that better cleaning was observed when preparation was performed with sequential instruments. similar results were also highlighted in another study, although performed on molars, the differences occurred when the canals were also prepared in sequential mode23. considering the presence of regions in mandibular incisors that can provide the accumulation of debris and microorganisms24, the role of irrigant solutions comes to the fore25. given the ability to dissolve, neutralize and remove organic and inorganic matter26. among the most used are naocl, due to its bactericidal power, low surface tension, and antibacterial action, spreading throughout the deproteinization canals27. as for the concentration of naocl, a 2.5% liquid was adopted, based on the study of duque et al.14. another irrigant used in this study, 17% edta, aimed to dissolve inorganic material28. based on the chemical action of the aforementioned, an association of these irrigants was adopted, justified by the presence of organic and inorganic tissues within the root canal28. in addition to the chemical properties of these solutions, it is known that the way they are applied in difficult regions influences their effectiveness, due to the trapping of air such occurs in the apical region29, which impairs or prevents the flow of irrigants. 8 kublitski et al. despite the advances in the field of instruments, and the persistence of non-instrumented areas after preparation30, it was proposed to use ultrasonic inserts to enhance the cleaning of root canals, under agitation of the irrigant solution14. in the present study differences occurred. corroborating these findings, a recent systematic review also indicated an association, with ultrasonic activation being more effective than syringe irrigation in removing tissue debris and fragments of hard tissue31. regarding the removal of remnants, eir showed a lower score when compared to con and edd. the same tip was also evaluated by duque et al.14. the authors demonstrated that the agitation of the irrigant solution promoted a better cleaning of the canal and isthmus areas when compared to the syringe irrigation technique. evaluating the reduction of microorganisms within the root canals, the effectiveness of eir was tested against irrigation with syringe. although the naocl used was 6%, the clinical trial revealed a greater reduction in the amount of microorganisms the group where ultrasonic activation was instituted32. both mentioned studies corroborate the results of the present study, where eir was superior to con in removing debris and smear layer. the edd tip was commercially introduced as a sonic system for agitating irrigant solutions. here, this tip not showed differences in comparison to con and eir. according to the manufacturer it is made of polyamide and has a flexible tip, giving it the same effectiveness as ultrasonic. its vibration, with great amplitude, are due to the high flexibility, which promotes a three-dimensional movement that generates the same physical effects of cavitation and acoustic streaming achieved by ultrasonic. in comparison, syringe irrigation and edd, decontaminating root canals infected with enterococcus faecalis, the first one was significantly less efficient in eliminating microorganisms in single canals33. zeng et al.34 also evaluated the reduction in bacterial load, and identified that edd was superior to manual irrigation in relation to bacterial death in the intratubular region in the cervical and middle thirds. with regard to the use of edd, the present results differed from those mentioned above, since statistical differences were not identified in comparison to con. based on the results of this research, a satisfactory cleaning regarding the removal of debris and smear layer can be observed. even though there were no significant differences, between and within groups for some associations. such observation is due to the greater number of specimens registered for score 1, in both quantifications. a possible justification for this is associated with the homogeneity of the initial anatomy achieved in the specimens during the allocation between groups. for that, manual files were used to negotiate the canals and consequently indicated the preparation instrument, according to van der vyver et al.16. as a result, canals with smaller diameter were prepared with smaller taper instruments, as were the larger ones. critically, it was still possible to observe that in the edd groups, the number of specimens registered with score 1 was higher than the con group, and equally or higher than eir. therefore, it is noteworthy that in the quantification of debris and smear layer, score 1 is classified, with no or slight presence of surface debris, covering up to 25% of the dentin surface, and for smear layer, little or no smear layer, covering less than 25% of the specimen with visible tubules, respectively17. 9 kublitski et al. currently, this quantification has been proposed by the sem, through the presence of debris and smear layer, in different increases12. in this study, magnifications of 100× and 1000× were used, respectively, based on the study by plotino et al.13. it is known that this methodology presents restrictions, as it evaluates a limited area of the canal; therefore, the standardization of the studied region was adopted in an attempt to always analyze the same region, in relation to the root apex. thus, image acquisition was performed at a distance of 3 mm from the root apex, and defined at the central point between the dentin walls in the mesio-distal direction. the chosen face was always the one with the least irregularities resulting from the neckline, as the teeth were thin. a total of 420 images (210 samples versus two enlargements, versus one third) were analyzed by two blind, trained and calibrated observers. in reference to the enlargement of the preparation of the canals for the institution of the agitation protocols, considering that the taper of edd (25/.04) and eir (20/.01), there would be a need for preparation with a 60 taper instrument to allow the movement the tip of the instrument. according to ahmad et al.35 for ultrasonic irrigation to be effective and for acoustic streaming to occur, it must operate within a space three times larger than the diameter of the tip used. however, the anatomical characteristics of the dental group used, the apical preparation was performed with a maximum 45 taper instrument, which may have had an impact on the conduction of ultrasonic energy. within the limitations of this study with respect to cleaning, none of the preparation techniques associated with tips produced a debris-free dentin surface and smear layer. however, it is indicated that for the preparation of mandibular incisors with manual irrigation, a protocol with sequential file, with the association of the wos and wop instruments. in the presence of tips of the irrigant solutions, eir can be used after wop and wom instruments as well as wos to wop association. while the use of edd is indicated, it can be used after wom and wos to wom instruments. in conclusion, the agitation of the irrigant solution provided better cleaning capacity, with better results for the eir. these findings reinforce the need for agitation techniques as aids in cleaning the root canal system, especially in cases of canals with a tendency to flatten. acknowledgements the authors deny any conflicts of interest related to this study. the authors would like to thank professor sérgio aparecido ignácio, statistician that performed the data analysis. references 1. gonçalves lc, sponchiado junior ec, da frota mf, marques aa, garcia lf. morphometrical analysis of cleaning capacity of a hybrid instrumentation in mesial flattened root canals. aust endod j. 2011 dec;37(3):99-104. doi: 10.1111/j.1747-4477.2010.00255.x. 2. baumgartner jc, mader cl. a scanning electron microscopic evaluation of four root canal irrigation regimens. j endod. 1987 apr;13(4):147-57. doi: 10.1016/s0099-2399(87)80132-2. 3. urban k, donnermeyer d, schäfer e, bürklein s. canal cleanliness using different irrigation activation systems: a sem evaluation. clin oral investig. 2017 dec;21(9):2681-7. doi: 10.1007/s00784-017-2070-x. 10 kublitski et al. 4. wu mk, r’oris a, barkis d, wesselink pr. prevalence and extent of long oval canals in the apical third. oral surg oral med oral pathol oral radiol endod. 2000 jun;89(6):739-43. doi: 10.1067/moe.2000.106344. 5. zhao d, shen y, peng b, haapasalo m. micro-computed tomography evaluation of the preparation of mesiobuccal root canals in maxillary first molars with hyflex cm, twisted files, and k3 instruments. j endod. 2013 mar;39(3):385-8. doi: 10.1016/j.joen.2012.11.030. 6. lorencetti kt, silva-sousa ytc, 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irrigant agitating devices for debris 2017 feb;43(2):326-31. doi: 10.1016/j.joen.2016.10.009. 15. versiani ma, carvalho kkt, mazzi-chaves jf, sousa-neto md.. micro-computed tomographic evaluation of the shaping ability of xp-endo shaper, irace, and edgefile systems in long oval-shaped canals. j endod. 2018 mar;44(3):489-95. doi: 10.1016/j.joen.2017.09.008. 16. van der vyver pj, vorster m. waveone® gold reciprocating instruments: clinical application in the private practice: part 1. int dent – afr ed. 2017;7(4):6-19. 17. gutmann j, saunders w, nguyen l, guo i, saunders e. ultrasonic root‐end preparation part 1. sem analysis. int endod j. 1994 nov;27(6):318-24. doi: 10.1111/j.1365-2591.1994.tb00276.x. 18. prada i, micó-muñoz p, giner-lluesma t, micó-martínez p, collado-castellano n, manzano-saiz a. influence of microbiology on endodontic failure. literature review. med oral patol oral cir bucal. 2019 may 1;24(3):e364-72. doi: 10.4317/medoral.22907. 19. khalap nd, kokate s, hegde v. ultrasonic versus sonic activation of the final irrigant in root canals instrumented with rotary/reciprocating files: an in-vitro scanning electron microscopy analysis. j conserv dent. 2016;19(4):368-72. doi: 10.4103/0972-0707.186451. 20. sousa-neto md, silva-sousa yc, mazzi-chaves jf, carvalho kkt, barbosa afs, versiani ma, et al. root canal preparation using micro-computed tomography analysis: a literature review. braz oral res. 2018 oct 18;32(suppl 1):e66. doi: 10.1590/1807-3107bor-2018.vol32.0066. 11 kublitski et al. 21. alakshar a, saleh arm, gorduysus mo. debris and smear layer removal from oval root canals comparing xp-endo finisher, endoactivator, and manual irrigation: a sem evaluation. eur j dent. 2020 oct;14(4):626-33. doi: 10.1055/s-0040-1714762. 22. de-deus g, barino b, zamolyi rq, souza e, fonseca jr a, fidel s, et al. suboptimal debridement quality produced by the single-file f2 protaper technique in oval-shaped canals. j endod. 2010 nov;36(11):1897-900. doi: 10.1016/j.joen.2010.08.009. 23. saraf aa, patil ac, mangala tm, mahaparale r, mali s, pawar s. comparison of cleaning effectiveness of single rotary file oneshape and reciprocating f2 protaper with protaper universal sequence: a sem analysis. j oral biol craniofac res. 2020;10(4):337-42. doi: 10.1016/j. jobcr.2020.06.011. 24. hülsmann m, rümmelin c, schäfers f. root canal cleanliness after preparation with different endodontic handpieces and hand instruments: a comparative sem investigation. j endod. 1997 may;23(5):301-6. doi: 10.1016/s0099-2399(97)80410-4. 25. de gregorio c, arias a, navarette n, del rio v, oltra e, cohenca n. effect of apical size and taper on the volume of irrigant delivered at working length with apical pressure at different root curvatures. j endod. 2013 jan;39(1):119-24. doi: 10.1016/j.joen.2012.10.008. 26. van der sluis lw, gambarini g, wu mk, wesselink pr. the influence of volume, type of irrigant and flushing method on removing artificially placed dentine debris from the apical root canal during passive ultrasonic irrigation. int endod j. 2006 jun;39(6):472-6. doi: 10.1111/j.1365-2591.2006.01108.x. 27. rath pp, yiu cky, matinlinna jp, kishen a, neelakantan p. the effect of root canal irrigants on dentin: a focused review. restor dent endod. 2020 jun 30;45(3):e39. doi: 10.5395/rde.2020.45.e39. 28. garberoglio r, becce c. smear layer removal by root canal irrigants. a comparative scanning electron microscopic study. oral surg oral med oral pathol. 1994 sep;78(3):359-67. doi: 10.1016/0030-4220(94)90069-8. 29. tay fr, gu ls, schoeffel gj, wimmer c, susin l, zhang k, et al. effect of vapor lock on root canal debridement by using a side-vented needle for positive-pressure irrigant delivery. j endod. 2010 apr;36(4):745-50. doi: 10.1016/j.joen.2009.11.022. 30. espir cg, nascimento-mendes ca, guerreiro-tanomaru jm, cavenago bc, hungaro duarte ma, tanomaru-filho m. shaping ability of rotary or reciprocating systems for oval root canal preparation: a micro-computed tomography study. clin oral investig. 2018 dec;22(9):3189-94. doi: 10.1007/s00784-018-2411-4. 31. căpută pe, retsas a, kuijk l, de paz lec, boutsioukis c. ultrasonic irrigant activation during root canal treatment: a systematic review. j endod. 2019 jan;45(1):31-44.e13. doi: 10.1016/j.joen.2018.09.010. 32. aveiro e, chiarelli-neto vm, de-jesus-soares a, zaia aa, ferraz ccr, almeida jfa, et al. efficacy of reciprocating and ultrasonic activation of 6% sodium hypochlorite in the reduction of microbial content and virulence factors in teeth with primary endodontic infection. int endod j. 2020 may;53(5):604-18. doi: 10.1111/iej.13261. 33. hage w, de moor rjg, hajj d, sfeir g, sarkis dk, zogheib c. impact of different irrigant agitation methods on bacterial elimination from infected root canals. dent j (basel). 2019 jun 27;7(3):64. doi: 10.3390/dj7030064. 34. zeng c, willison j, meghil mm, bergeron be, cutler cw, tay fr, et al. antibacterial efficacy of an endodontic sonic-powered irrigation system: an in vitro study. j dent. 2018 aug;75:105-12. doi: 10.1016/j.jdent.2018.06.003. 35. ahmad m, pitt ford tj, crum la. ultrasonic debridement of root canals: acoustic streaming and its possible role. j endod. 1987 oct;13(10):490-9. doi: 10.1016/s0099-2399(87)80016-x. revista fop n 13 1624 gingival peripheral odontoma in a child: case report of an uncommon lesion vanessa de fátima bernardes1; luis otávio de miranda cota1; fernando de oliveira costa2; ricardo alves mesquita2; ricardo santiago gomez2; maria cássia ferreira aguiar2 1dds, ms, graduate student, department of oral surgery and pathology, school of dentistry, federal university of minas gerais, brazil 2dds, ms, phd, adjunct professor, department of oral surgery and pathology, school of dentistry, federal university of minas gerais, brazil received for publication: july 30, 2008 accepted: september 09, 2008 correspondence to: maria cássia ferreira de aguiar av. antônio carlos, 6627 faculdade de odontologia da ufmg cep: 31270-901 pampulha, belo horizonte, mg, brazil phone: +55-31-3409-2476. fax: +55-31-3409-2472 e-mail: cassiafa@odonto.ufmg.br, cassiafaster@gmail.com a b s t r a c t odontoma in an extraosseous location represents a challenge for diagnosis. this article reports a case of peripheral odontoma and its clinical presentation, histological evaluation and treatment. a 12-year-old boy reported a firm asymptomatic gingival mass in the anterior maxilla with two years of evolution. the procedures for diagnosis included intraoral examination, excisional biopsy and histological analysis. the diagnosis was peripheral odontoma. the follow-up revealed no sign of recurrence. peripheral odontoma is rare and the differential diagnosis with other gingival masses is rather difficult and must include inflammatory and reactive processes. the definitive diagnosis is based on microscopic features. key words: extraosseous odontoma, diagnosis, gingival lesion. i n t r o d u c t i o n odontomas are considered hamartomatous malformations of odontogenic origin in which all dental tissues are represented, occurring in a more or less disorderly pattern1. these lesions are usually diagnosed in the second decade of life and have no predilection for sex2. the intraosseous (central) odontomas represent the odontogenic tumors of greatest incidence 3. otherwise, odontomas arising in the extraosseous soft tissue, also known as peripheral odontoma (pos), are extremely uncommon4. po shows the histological characteristics of an intraosseous odontoma, but occurs only in the soft tissue covering the tooth-bearing portion of the mandible4. to the best of our knowledge only eight cases have been previously reported2,5-10. this article presents a case of po in a child referred to our dental clinic for treatment. case report a 12-year-old white boy was referred to dental clinic of the school of dentistry, federal university of minas gerais, with a single, asymptomatic nodule on the gingiva in the region of the left lateral incisor. the patient reported that the mass had a slow growth and two years of evolution. physical and intraoral examination showed a healthy boy with a slightly reddish, circumscribed, firm 4-mm-diameter nodule. the overlying gingiva had normal texture (figure 1a). neither signs nor history of trauma or infection were detected. teeth were visually free of caries and adjacent tissues were clinically normal. the hypotheses of diagnosis included pyogenic granuloma, periodontal abscess, and peripheral ossifying fibroma, and an excisional biopsy was performed for confirmation. no bone involvement was observed during the surgery. the specimen was immediately placed in 10% neutral buffered formalin, processed in the usual manner and submitted for histopathological analysis. histologically, a developing rudimentary tooth was observed in the submucosal tissue. enamel matrix, dentin, pulp tissue and ameloblasts were present (figure 1b). ghost cells were also noted (not shown). the lesion was completely separated from the overlying epithelium by a capsule composed of connective tissue with fibroblasts, blood vessels and islands of odontogenic epithelium. on the basis of the clinical and histopathological findings, the lesion was diagnosed as a developing peripheral compound odontoma. the two-week postoperative follow-up showed that the area had already healed. two years later, there is still no clinical sign of recurrence. d i s c u s s i o n two types of odontoma are histologically recognized: compound and complex lesions. the compound lesion is braz j oral sci. july/september 2008 vol. 7 number 26 1625 comprised of tooth-like structures while the complex lesion is comprised of a mixture of odontogenic tissues without dental organization2. clinically, three types of odontoma are described in literature: central (intraosseous), erupted, and peripheral odontoma (extraosseous or soft tissue)6. intraosseous odontomas represent the most common type. these extraosseous lesions are quite rare and the reports in the literature are limited7. the lesions are usually diagnosed between the first and the second decade of life but may be found at any age. the most common location for this type of lesion is the maxillary anterior region8, and most odontomas are relatively small, rarely exceeding 1 cm in diameter9. histological examination shows rudimentary denticles or tooth-like structures and epithelial elements may be absent10. in the present case, the relationship among the structures suggested a compound odontoma. the presence of ghost cells, as noted in this case, is not a pathognomonic sign. it has been described in odontoma as well as in other lesions, such as calcifying odontogenic cyst, fig.1. clinical and histopathological features of the gingival peripheral odontoma. a: clinical aspect of the lesion. b: mucosal fragment showing the lesion beneath the surface epithelium. h&e. (original magnification ×100). rudimentary tooth containing ameloblasts (arrow), enamel matrix (arrowhead) and primitive mesenchymal tissue (pm). h&e. (original magnification ×400) ameloblastoma, ameloblastic fibroma and ameloblastic fibroodontoma11-13. the radiographic examination may show three different developmental stages based on the amount of calcification present at the time of discovery. unfortunately, no radiographic exam was performed in this case. the histogenesis of po is controversial. pos are speculated to arise from the soft tissue remnants of the dental lamina. gingival rests of serres seems to retain the ability to pursue necessary epithelial-mesenchymal interactions, thus leading to an odontoma formation 10. the specific etiological factors responsible for their development have not been determined. theories have included physical trauma, infection and hereditary influence5,9. po occurs in gingival tissue, appearing as an exophytic mass that can be mistakenly diagnosed as other more common exophytic gingival lesions. the differential diagnosis includes lesions of inflammatory origin (e.g., periodontal abscess) and reactive proliferations (e.g., pyogenic granuloma, peripheral ossifying fibroma, peripheral giant cell granuloma)2,5,8. because of their rarity, peripheral odontogenic tumors are usually not included in differential diagnosis. when considered, the first suggestion is the peripheral odontogenic fibroma. peripheral ameloblastoma, peripheral calcifying odontogenic cyst and peripheral calcifying epithelial odontogenic tumor can also be suspected. however, they are extremely rare and usually occur in the third and sixth decade of life5,12. since these lesions are considered hamartomatous with a very limited growth potential, surgical removal should be conservative with no real expectation of recurrence10. the histological examination is mandatory for an accurate diagnosis9. in the present case, two years of follow-up indicate no sign of recurrence. a c k n o w l e d g e m e n t s the authors thank the brazilian national council for scientific and technological development (cnpq) and the the minas gerais state research support foundation (fapemig) for assistance and financial support. mesquita ra, gomez rs, and aguiar mcf are research fellows of the cnpq. r e f e r e n c e s 1. praetorius f, piatelli a, odontoma, complex type / odontoma, compound type. in: l. barnes, j.w. eveson, p. reichart, d. sidransky. world health organization classification of tumours. pathology and genetics of head and neck tumours. lyon: iarc press; 2005. p. 310-1 2. ledesma-montes c, perez-bache a, garces-ortiz m. gingival compound odontoma, int j oral maxillofac surg. 1996; 25: 296-7. 3. kaugars ge, miller me, abbey lm., odontomas. oral surg oral med oral pathol. 1989; 67: 172-6. 4. buchner a, sciubba jj. peripheral epithelial odontogenic tumors: a review. oral surg oral med oral pathol. 1987; 63: 688-97. 5. manor y, mardinger o, katz j, taicher s, hirshberg a. peripheral odontogenic tumours—differential diagnosis in gingival lesions. int j oral maxillofac surg. 2004; 33: 268-73. braz j oral sci. 7(26):1624-1626 gingival peripheral odontoma in a child: case report of an uncommon lesion 1626 6. junquera l, de vicente jc, roig p, olay s, rodriguez-recio o. intraosseous odontoma erupted into the oral cavity: an unusual pathology. med oral patol oral cir bucal. 2005; 10: 248-51. 7. castro gw, houston g, weyrauch c. peripheral odontoma: report of case and review of literature, asdc j dent child. 1994; 61: 209-13. 8. giunta jl, kaplan ma, peripheral, soft tissue odontomas. two case reports. oral surg oral med oral pathol. 1990; 69: 406-11. 9. swan rh. odontomas. a review, case presentation and periodontal considerations in treatment. j periodontol. 1987; 58: 856-60. 10. ide f, shimoyama t, horie n. gingival peripheral odontoma in an adult: case report. j periodontol. 2000; 71: 830-2. 11. vuletin jc, solomon mp, pertschuk lp. peripheral odontogenic tumor with ghost-cell keratinization. a histologic, fluorescent microscopic, and ultrastructural study. oral surg oral med oral pathol. 1978; 45: 406-15. 12. orsini g, fioroni m, rubini c, piattelli a. peripheral calcifying odontogenic cyst. j clin periodontol. 2002; 29: 83-6. 13. tanaka a, okamoto m, yoshizawa d, ito s, alva pg, ide f, kusama k. presence of ghost cells and the wnt signaling pathway in odontomas, j oral pathol med. 2007; 36: 400-4. braz j oral sci. 7(26):1624-1626 gingival peripheral odontoma in a child: case report of an uncommon lesion 1http://dx.doi.org/10.20396/bjos.v20i00.8660432 volume 20 2021 e210432 original article 1 federal university of rio de janeiro, school of dentistry, department of dental clinic, rio de janeiro, rio de janeiro, brazil. 2 federal university of rio de janeiro, technology center, department of nuclear instrumentation laboratory, rio de janeiro, rio de janeiro, brazil. corresponding author: heloisa gusman rua prof. rodolpho paulo rocco 325 / 2° andar, ilha da cidade universitária, rio de janeiro. zip code: 21941-913. brazil. e-mail: heloisa.gusman@odonto.ufrj.br phone: 55 21 996312426 received: july 9, 2020 accepted: january 21, 2021 retreatment of mesial roots of mandibular molars filled with resin-based and bioceramic sealers marina de almeida salim1, fabiola ormiga1 , ricardo tadeu lopes2 , heloisa gusman1,* aim: the aim of this study was to evaluate, by micro-computed tomography (micro-ct) analysis, the remaining filling material during endodontic retreatment performed with protaper retreatment without solvent. methods: forty mandibular molars were divided into two groups (n = 20) according to the sealer used in the obturation: the bioceramic totalfill bc (tf) or the resin-based ah plus (ahp). the specimens were scanned before instrumentation, after obturation and after filling removal. only the mesial roots were analysed. the filling volumes and the remaining filling material were calculated in the entire root canal and in the cervical, middle and apical thirds. results: the volume of obturation and the volume of remaining filling material in the entire root canal and in the cervical, middle and apical thirds of the canal between the groups were not statistically different (independent t-test, p > 0.05). in the ahp group, there was a higher percentage of remaining filling material in the middle third than in the cervical third (p < 0.05). conclusion: the filling material could not be entirely removed from any specimen. key words: root canal filling materials. retreatment. molar. x-ray microtomography. https://orcid.org/0000-0002-3764-944x https://orcid.org/0000-0001-6535-7236 https://orcid.org/0000-0002-7470-6650 2 salim et al. introduction endodontic retreatment is indicated when the treatment fails, in the presence of pain, edema, fistula and when the periodontal ligament space is radiographically abnormal1. removing the filling material during retreatment is essential for adequate cleaning, disinfection and shaping of the root canal system (rcs); therefore, this procedure plays an important role in the success of endodontic therapy2. during root canal filling, gutta-percha is the main constituent of the obturation material; however, because it does not adhere to dentin walls, it must be used in conjunction with endodontic sealers, which can adhere to canal walls and provide a more homogeneous obturation3. the composition of bioceramic sealers includes calcium silicate, and the sealers are biocompatible, have antimicrobial properties, are radiopaque, chemically stable and do not shrink after setting4-7. due to their chemical composition, they form an interfacial layer with tag-like structures at the cement–dentin interface, which increases the push-out bond strength of those materials8,9. however, there is no consensus about the difficulty in removing these sealers after setting10-15. kakoura and pantelidou13 (2018) observed by scanning electron microscopy (sem) no significant differences in the percentage of the remaining filling material after retreatment of single-rotted teeth filled with gutta-percha and bioroot, totalfill  bc or ah26. the disadvantages of the methods such as scanning electron microscopy, stereomicroscopy, optical microscopy and digital radiography, that have been used to evaluate the removal of bioceramic sealers from the rcs, include loss of sample, qualitative analysis, and two-dimensional evaluation of images12,16-19. in this context, micro-computed tomography (micro-ct) is a method that has been widely used in recent years to evaluate rcs obturation and retreatment through precise three-dimensional analysis of the root canal and the filling and remaining material volumes without destroying the sample10,11,14,15. ma et al.11 (2012) used micro-ct to evaluate the removal of the bioceramic iroot sp with or without solvent from incisors filled by lateral condensation and continuous wave condensation techniques. they found more remaining filling material in the continuous wave condensation group and in the no-solvent groups. recent studies, also using micro-ct, have shown more remaining filling material in uniradicular teeth obturated with bioceramic sealer, removed with solvent, than that of other sealers10,15. according to agrafioti et al.12 (2015), the calcium silicate-based sealers totalfill bc and mta fillapex are negotiable under simple root canal anatomy, but the conventional retreatment techniques are not able to fully remove them. all these studies used single-rooted teeth that present lower anatomical complexity when compared with maxillary and mandibular molars, where clinicians tend to find more difficulty during the retreatment. therefore, the objective of the present study was to evaluate, using micro-ct, the removal of the bioceramic totalfill bc from the mesial roots of mandibular molars using the protaper retreatment instrument without using gutta-percha solvent. the hypothesis is that the bioceramic sealer is harder to remove than the resin-based ah plus. 3 salim et al. material and methods specimen selection and initial preparation this study was approved by the research ethics committee of of clementino fraga filho hospital (protocol number 02235718.0.0000.5257). a sample calculation was performed using a test power of 0.8 and an alpha value fixed at 0.05, which resulted in a minimum of nine samples per group to show statistical significance. forty mandibular molars with complete root formation, a median length of 19.5 mm and a root curvature angle of up to 25°, extracted for clinical reasons, were used in this study. the teeth were stored in 0.1% thymol solution at 4°c until used. access cavities were created with round diamond and endo-z burs (dentsply maillefer, ballaigues, switzerland) in a water-cooled high-speed handpiece. no file was inserted through the mesiobuccal, mesiolingual or distal canals to prevent changes in the original anatomy of the apical region. micro-ct scanning micro-ct images were acquired according to the methodology of almeida et al.20 (2015); a custom-made mould of self-polymerizing resin was created for each tooth to standardize the specimen mounting. the teeth were placed in a micro-ct scanner (skyscan 1173; bruker microct, kontich, belgium) on a custom aluminium attachment. this attachment ensured high similarity between the images obtained before and after canal preparation because it enabled precise specimen repositioning inside the scanner. scanning was performed through 360° rotation with a rotation step of 0.30 using a 1.0-mm thick aluminium filter, 70 kv power, 114 ma current, 14.8 mm pixel size and 21.39 mm resolution. root canal instrumentation and obturation coronal third enlargement was performed with la axxess stainless steel burs (sybronendo, orange, ca, usa), and size 10 k-files (dentsply maillefer) were used during instrumentation to establish apical patency and to determine the canal lengths. after radiographic evaluation, the working length was established 1 mm short of the radiographic apex. the teeth were instrumented to the working length with k3 files (sybronendo) using the crown-down technique at 350 rpm with a torque control endodontic motor (easy, belo horizonte, brazil) using the sequence recommended by the manufacturer as follows: #25/.08, #25/.06 and #25/.04. all files were used passively, and apical enlargement was performed using #25.06 and #30.04 files. the pulp chamber was filled with 5.25% sodium hypochlorite (naocl) throughout the instrumentation. the canal was irrigated with 3 ml 5.25% naocl after each instrument. after instrumentation, all canals were irrigated with 3 ml 17% edta for 3 minutes (1 ml/min) and 3 ml 5.25% naocl, and dried with fm-size paper points (sybronendo). next, the teeth underwent postoperative micro-ct scanning with the parameter settings described above. upon acquisition of the initial images, the specimens were divided into two groups based on their anatomical equivalence according to the number of canal entrances, number of canals, ramifications and isthmus. 4 salim et al. the teeth were divided into two groups of 20 teeth each according to the sealer and technique used: the totalfill (tf) group used totalfill bc (fkg dentaire, la chauxde-fonds, switzerland), #30 or #35 gutta-percha cones and totalfill bc sealer (fkg dentaire). the ah plus (ahp) group used size fm or m gutta-percha cones (dentsply-maillefer) and ah plus sealer (dentsply-maillefer). in both groups, the cone was placed in the working length. after radiographic certification of the obturation limit, the canals were filled with 5.25% naocl and underwent passive ultrasonic irrigation (pui) with a delsonic 2000 (deldent, petah tikva, israel) with a #20 ultrasonic tip and 30-khz potency for 1 minute. the canals were irrigated with 5 ml distilled water, dried with #35 paper cones (dentsply-maillefer), filled with edta for 3 minutes (1 ml/min), irrigated with 5 ml 5.25% naocl, washed again with 5 ml distilled water and dried. the obturation technique used was the continuous heat wave. the sealer was introduced into the canal using a #15 k-file. the gutta-percha cone was covered with sealer and introduced to the working length. system b was used, with the medium tip inserted into the canal 5 mm short of the working length to remove the gutta-percha from the cervical and middle thirds. the obtura ii system (obtura, fenton, mo, usa) was used to fill the middle and cervical thirds with 4-mm increments of gutta-percha at 200°c. after filling, the pulp chamber of all teeth was sealed with cotton and zinc oxide–based temporary material, and stored for 7 days in an oven at 37°c and 100% moisture for the sealers’ total setting times. then, the dental elements underwent micro-ct image acquisition again. removal of filling material the filling material was removed using a #3 gates glidden bur (dentsply-maillefer) in the cervical 3 mm of the canal, followed by the rotary niti protaper universal retreatment (ptur) sequence (dentsply-maillefer), as per the manufacturer instructions: d1, d2 and d3 were used in the cervical, middle and apical thirds of the canal until the working length, respectively, without solvent. filling material removal was considered finished when no gutta-percha or sealer residue was observed in the instrument and when the working length was achieved. after that, a niti protaper next x4 (#40.06, dentsply-maillefer) was used until the working length, followed by irrigation with 5 ml naocl 5.25%. then, the dental elements underwent micro-ct image acquisition again. micro-ct scanning measurements only the mesial roots of the mandibular molars were evaluated. the images were analysed using ctan software (bruker microct). nrecon software (v1.6.1.0, bruker microct) was used for the image reconstruction with a 25% beam hardening correction, ring artifact correction of 2 and smoothing of 2, resulting in the acquisition of 700 to 800 axial sections per sample. the slices of the samples after obturation were recorded with their respective slices after endodontic retreatment by the affine plugin of the 3d slicer 4.4.0 program. after the images were recorded, the gray scale range required to recognize the dentin (range 60-255) and the remaining filling material (range 210-255) was determined on a density histogram using a threshold method. comparisons between acquisitions before and after root canal preparation were made to ensure the accuracy of the segmentation. all image analysis proce5 salim et al. dures were performed using the imagej 1.50d program (national institutes of health, bethesda, md, usa). after the reconstruction, the images were analyzed in the ctan software (v. 1.16.4.1), where the selection of the region of interest (roi), binarization and image segmentation for analysis is made. this program provides the necessary morphometric parameters for the proposed study, which was the volume of filling material per third (cervical, middle and apical) and total. the dataviewer (v. 1.5.2.4) and ctvox (v. 3.2.0.0) software were also used for visualization and qualitative analysis of 2d and 3d images of the anatomy of the teeth, respectively. the images obtained before the chemical–mechanical preparation were used to verify the anatomical equivalence of the groups. the filling volume was determined from the image obtained after obturation, and the total volume of remaining filling material was obtained after retreatment. the difference between these two values was calculated and resulted in δv, which represents the volume of material removed from the rcs. the volumes of filling and remaining material were also determined in the cervical, middle and apical thirds of the canal. the percentage of remaining filling material was calculated based on the formula: remaining volume × 100 ÷ filling volume. statistical analysis the volume values underwent normality analysis by the shapiro-wilk test, and showed normal distribution. the independent t-test was used to compare the groups for initial canal volume, volume of obturation, volume of remaining filling and δv. the paired t-test was used to compare the volume of obturation and the remaining filling volume within the same group. analysis of variance (anova) was used to compare the thirds of the canal in terms of the obturation volume and the remaining filling volume within the same group. an alpha value of 0.05 was established as the level of statistical significance (spss v20.0, chicago, il, usa). results table 1 shows the volume of obturation, the volume of remaining filling material, total δv and δv in the cervical, middle and apical thirds of the canal. there was no statistical difference between the groups for the volume of obturation, volume of remaining filling material and δv both in the entire root canal and in the cervical, middle and table 1. mean ± standard deviation (mm3) of obturation volume, remaining filling volume and δv in the cervical, middle, apical and entire root canal ah plus total fill bc sealer obturation volume remaining filling volume ∆v obturation volume remaining filling volume ∆v cervical 5.85 ± 2.16a 0.58 ± 0.46e 5.28 ± 2.06h 6.39 ± 2.16a 0.99 ± 0.85e 5.39 ± 2.13h middle 3.25 ± 1.19b 1.06 ± 0.77f 2.18 ± 0.95i 3.65 ± 1.57b 1.40 ± 1.45e,f 2.25 ± 1.70i apical 1.36 ± 0.89c 0.80 ± 0.78e,f 0.55 ± 0.46j 1.47 ± 0.75c 0.80 ± 0.88e,f 0.66 ± 0.76j total 10.46 ± 3.79d 2.44 ± 1.65g 8.01 ± 3.01k 11.51 ± 4.01d 3.19 ± 2.78g 8.30 ± 3.78k * different letters indicate statistical difference (p<0.05) 6 salim et al. apical thirds of the canal (independent t-test, p > 0.05). in both groups, the volume of obturation and volume of remaining filling material were significantly different in the entire root canal and in the three thirds of the canal (paired t-test, p < 0.05). however, the filling material was not completely removed in all specimens. in the ahp group, the remaining filling volume in the cervical third was lower than that of the middle third, without significant differences with the apical third (anova, p < 0.05). the tf group showed no significant differences for remaining filling volume in the three thirds of the canal. patency was not obtained in 35% and 25% of the samples in the ahp group and tf group, respectively (anova, p > 0.05). figure 1 shows the representative reconstructed micro-ct images of the root canal obturated (fig. 1a, f), with the remaining filling material (fig. 1b, g) and transversal sections of the cervical (fig. 1c, h), middle (fig. 1d, i) and apical (fig. 1e, j) thirds of the ahp and tf groups. we noted that the filling material was not completely removed in the groups analysed. in both groups, the presence of remaining filling material was visible in the canal walls and isthmus (fig. 1c–e, h–j). figure 1. three-dimensional images of the obturated root canal (a, f), remaining filling material (b, g) and transversal section images of the cervical (c, h), middle (d, i) and apical (e, j) thirds of the ahp and tf groups. (a) (b) (c) (d) (e) (f) (g) (i) (j) (h) ahp tf 7 salim et al. discussion the present study used micro-ct to evaluate the removal of filling material from the mesial roots of mandibular molars obturated with totalfill bc or ah plus sealers using ptur without gutta-percha solvent. the initial hypothesis has to be rejected since there was no difference between the analysed parameters of both groups. although the removal of filling material was significant when compared to the volume of obturation, complete removal of filling material from the rcs was not observed in any specimen. the sealers used in the present study have similar physicochemical characteristics, such as high flow, biocompatibility and radiopacity7,21, although their compositions differ and also their adhesion mechanism to dentin. ah plus is an epoxy resin sealer that interacts chemically with the root dentin collagen with covalent bonds between its epoxy rings and collagen amine groups22. total fill bc presents chemical bonding to mineralized tissues and therefore, the use of edta as an irrigating agent to remove smear-layer prior to obturation could decrease the interaction with the dentin wall, since edta removes calcium from dentin22,23. this could possibly explain the similar results obtained with the sealers used. micro-ct is a method that has been widely used in recent years to evaluate rcs anatomy, obturation and retreatment through three-dimensional analysis10,11,15,20,21. in the present study, micro-ct showed 23% remaining filling material in the ahp group, whereas that in the tf group was 28%; the two groups were not statistically significantly different. these results corroborate with other studies that also used micro-ct to evaluate the retreatability of bioceramic sealers, albeit with different methodologies15,17,18. ersev et al.17 (2012) used digital radiography to analyse the removal of ah plus, hybrid root seal, activ gp sealers and the endosequence bc bioceramic sealer using manual and rotary (protaper-r) instruments, while kim et al.18 (2015) used the profile rotary instrument to evaluate, by means of mev, the cleaning of root canals obturated with ah plus or endosequence bc sealers. oltra et al.15 (2017) used micro-ct to evaluate the removal of ah plus and endosequence bc sealers with the profile and vortex blue rotary instruments, with and without solvent, and observed no difference in the percentage of remaining material in the no-solvent groups. these authors also observed that the bioceramic sealer group had more remaining filling material in the apical third of the canal than the ahp group. furthermore, patency was not established in 86% of specimens in the bioceramic group, whereas the ahp group achieved 100% patency. in the present study, patency was not obtained in 35% and 25% of specimens from the ahp and tf groups, respectively. this difference in achieving patency can be related to the anatomic differences of the teeth used in the respective studies. oltra et al.15 (2017) utilized uniradicular teeth with a final apical diameter of #45.04, whereas we used the mesial canals of mandibular molars with a lower final diameter (#30.4). the diameter and more circular anatomy of the uniradicular teeth could have favoured more compact filling in the apical third with sealer, making it difficult to remove. on the other hand, in molar teeth, the presence of irregularities and re-entrances in the apical third can lead to empty spaces in the filling mass, which can allow the passage of small-diameter files. in fact, a study that compared root canals filled with bioceramic sealer showed that patency was estab8 salim et al. lished in 20% of the cases obturated in the working length and in only 70% of cases obturated 2 mm before the working length16. our results are not in agreement with other studies that also evaluated the retreatability of bioceramic sealers. uzunoglu et al.19 (2015) used stereomicroscopy and observed more remaining filling material in the group obturated with the bioceramic iroot sp sealer as compared with the resin-based ah 26 sealer. agrafioti et al.12 (2015) used a clinical microscope to compare the removal of ah plus, mta fillapex and totalfill bc sealers, and found more remaining filling material in the ah plus group. zuolo et al.10 (2016) used micro-ct and found more remaining filling material in teeth obturated with endosequence bc sealer when compared with a zinc oxide–based sealer. differences in the methodology and sample used may explain the divergent results. rotary instruments are recommended for removing filling material during root canal retreatment because they are fast, safe and efficient. in the present study, ptur was selected for its proven effectiveness and safety in the extravasation of debris24. in fact, our results demonstrate that the volume of remaining filling material was significantly lower than the obturation volume, which agrees with other studies that used the same instrument11,17,19. furthermore, no gutta-percha solvent was used during retreatment, as its use is still controversial in the literature. although the use of solvent favours the penetration of instruments in the obturation mass, it has been shown by mev that the dentinal tubules of teeth re-treated with solvents can be obliterated by residues of gutta-percha and sealer, thus making rcs cleaning difficult. studies that evaluated the influence of the solvent during the retreatment of teeth obturated with bioceramics have shown that filling material removal was either easier or harder when solvent was used11,15. according to the literature, the present study also demonstrates that filling material removal was not complete in any specimen analysed10-12,15,16-19. furthermore, the remaining filling material in the entire root canal and in the three thirds of the canal was higher when compared to other studies that also used micro-ct10,11,15. these results may be explained by the anatomical complexity of the mesial canal of the mandibular molars, with high prevalence of isthmus and ramifications, when compared with the uniradicular teeth used in the other studies. indeed, the ahp group had a higher percentage of remaining filling material in the middle rather than the cervical third of the canal, probably due to the presence of isthmus in that region. our findings based on the micro-ct reconstruction images showed the presence of filling material in the re-entrances, isthmus and canal walls, which demonstrates not only how challenging the retreatment of these high anatomical complexity teeth is, but also that the routine procedures during retreatment are ineffective, regardless of the type of filling material used. therefore, is imperative the need for an additional step of cleaning, as these anatomical areas cannot be reached by the routine procedures that aim at disinfecting the rcs. references 1. consensus report of the european society of endodontology on quality 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a, zuolo ml, da silveira bueno ce, chu r, cunha rs. evaluation of the efficacy of trushape and reciproc file systems in the removal of root filling material: an ex vivo micro-computed tomographic study. j endod. 2016 feb;42(2):315-9. doi: 10.1016/j.joen.2015.11.005. 11. ma j, al-ashaw aj, shen y, gao y, yang y, zhang c, et al. efficacy of protaper universal rotary retreatment system for gutta-percha removal from oval root canals: a micro-computed tomography study. j endod. 2012 nov;38(11):1516-20. doi: 10.1016/j.joen.2012.08.001. 12. agrafioti a, koursoumis ad, kontakiotis eg. re-establishing apical patency after obturation with gutta-percha and two novel calcium silicate-based sealers. eur j dent. 2015;9(4):457-61. doi: 10.4103/1305-7456.172625. 13. kakoura f, pantelidou o. retreatability of root canals filled with gutta percha and a novel bioceramic sealer: a scanning electron microscopy study. j conserv dent. 2018;21(6):632-6. doi: 10.4103/jcd.jcd_228_18. 14. wolf m, küpper k, reimann s, bourauel c, frentzen m. 3d analyses of interface voids in root canals filled with different sealer materials in combination with warm gutta-percha technique. clin oral investig. 2014 jan;18(1):155-61. doi: 10.1007/s00784-013-0970-y. 15. oltra e, cox tc, lacourse mr, johnson jd, paranjpe a. retreatability of two endodontic sealers, endosequence bc sealer and ah plus: a microcomputed tomographic comparison. restor dent endod. 2017 feb;42(1):19-26. doi: 10.5395/rde.2017.42.1.19. 16. hess d, solomon e, spears r, he j. retreatability of a bioceramic root canal sealing material. j endod. 2011 nov;37(11):1547-9. doi: 10.1016/j.joen.2011.08.016. 17. ersev h., yilmaz b., dinçol m.e., daglaroglu r. the efficacy of protaper universal rotatory retreatment instrumentation to remove single gutta-percha cones cemented with several endodontic sealers. int endod j. 2012 aug;45(8):756-62. doi: 10.1111/j.1365-2591.2012.02032.x. 10 salim et al. 18. kim h., kim e., lee sj, shin sj. comparisons of the retreatment efficacy of calcium silicate and epoxy resin based sealers and residual sealer in dentinal tubules. j endod. 2015; 41(12): 2025-30. 19. uzunoglu e, yilmaz z, sungur dd, altundasar e. retreatability of root canals obturated using gutta-percha with bioceramic, mta and resin-based sealers. iran endod j. 2015 dec;41(12):2025-30. doi: 10.1016/j.joen.2015.08.030. 20. almeida bc, ormiga f, de araújo mc, lopes rt, lima ic, dos santos bc, et al. influence of heat treatment of nickel-titanium rotary endodontic instruments on apical preparation: a micro-computed tomographic study. j endod. 2015 dec;41(12):2031-5. doi: 10.1016/j.joen.2015.09.001. 21. roizenblit rn, soares fo, lopes rt, santos bc, gusman h. root canal filling quality of mandibular molars with endosequence bc and ah plus sealers: a micro-ct study. aust endod j. 2020 apr;46(1):82-7. doi: 10.1111/aej.12373. 22. crozeta bm, lopes fc, menezes silva r, silva-sousa ytc, moretti lf, sousa-neto md. retreatability of bc sealer and ah plus root canal sealers using new supplementary instrumentation protocol during non-surgical endodontic retreatment. clin oral investig. 2020 jun 6. doi: 10.1007/s00784-020-03376-4. 23. soares imv, crozeta bm, pereira rd, silva rg, da cruz-filho am. influence of endodontic sealers with different chemical compositions on bond strength of the resin cement/glass fiber post junction to root dentin. clin oral investig. 2020 oct;24(10):3417-23. doi: 10.1007/s00784-020-03212-9. 24. çanakçi bc, ustun y, er o, genc sen o. evaluation of apically extruded debris from curved root canal filling removal using 5 nickel-titanium systems. j endod. 2016 jul;42(7):1101-4. doi: 10.1016/j.joen.2016.03.012. 1 volume 21 2022 e226611 original research braz j oral sci. 2022;21:e226611http://dx.doi.org/10.20396/bjos.v21i00.8666611 1 faculty of dentistry, tabriz university of medical sciences, daneshgah street, tabriz, iran. 2 department of oral and maxillofacial radiology, faculty of dentistry, tabriz university of medical sciences, daneshgah street, tabriz, iran. corresponding author: dr. shiva daneshmehr, d.d.s. address: faculty of dentistry, tabriz university of medical sciences, daneshgah street, tabriz, iran office phone: +98 (41) 328 86333 phone number: +98935 212 2461 e-mail: shivadaneshmehr@yahoo.com editor: altair a. del bel cury received: august 6, 2021 accepted: may 25, 2022 relationship between the condyle morphology and clinical findings in terms of gender, age, and remaining teeth on cone beam computed tomography images shiva daneshmehr1,* , tahmineh razi2 , sedigheh razi2 aim: this study aimed to evaluate the relationship between clinical findings and some factors such as age, gender, and remaining teeth on the anatomy of the temporomandibular joint in order to diagnose normal variations from abnormal cases. methods: in this cross-sectional study, cone-beam computed tomography (cbct) images of 144 patients referring to tabriz dental school for various reasons were selected and evaluated. the different aspects of the clinical parameters and the morphology of the condyle were evaluated on coronal, axial, and sagittal views. the cbct prepared using the axial cross-sections had been 0.5 mm in thickness. the sagittal cross-sections had been evaluated perpendicular to the lengthy axis of the condyle at a thickness of 1 mm and the coronal cross-sections had been evaluated parallel to the lengthy axis of the condyle at a thickness of 1 mm. data were analyzed with descriptive statistical methods and t-test, chi-squared test, using spss 20. the significance level of the study was p < 0.05. results: there was a significant relationship between the condyle morphology, number of the teeth, and mastication side (p = 0.040). there were significant relationships between the condyle morphology, age between 20-40, and occlusion class i on the all the three views (coronal, axial, sagittal) (p = 0.04), (p = 0.006), (p = 0.006). also, significant relationships were found in the condyle morphology and location of pain according to age, the number of remaining teeth, and gender. (p = 0.046) (p = 0.027) (p = 0.035). conclusion: there are significant relationships between the clinical symptoms and condyle morphology based on age, gender, and the number of remaining teeth. the clinical finding that has the most significant relationship between the condyle morphology, remaining teeth (9-16 teeth), all of the age range (20-80 year), and gender was mastication side. keywords: cone beam computed tomography. mandibular condyle. temporomandibular joint. https://orcid.org/0000-0002-4320-5327 https://orcid.org/0000-0001-7471-2180 https://orcid.org/0000-0003-1328-0083 2 daneshmehr et al. braz j oral sci. 2022;21:e226611 introduction the temporomandibular joint (tmj) is one of the sophisticated structures in the body that allows the mandible to move in several directions1. mastication patterns have a crucial role in preserving the functional balance of the tmj2. forces exerted on the craniomandibular joint might affect the bony components, which might change the thickness and shape of these parts. excessive loads are considered abnormal, thus some patients require to eliminate the causative agent3-5. the degenerative disorders have a great importance in temporomandibular joint remodeling. in the tmj, degenerative joint disease might be defined as a local condition or be a part of a systemic disease6. one of the other factors that affect mandibular condyle structure is tooth loss. posterior crossbite and tooth loss might be associated with mandibular condyle structural alterations7. the other factor is age rising. radiographic changes in the condylar morphology have increased with age. in addition, gender is one of the factors that will help to reproduce the appropriate tmj structure8. all in all, several factors, including age, gender, number of remaining teeth, mastication patterns, etc, were found to affect tmj morphology9,10. therefore, we need paraclinical examinations to evaluate the variation of condylar morphology. it is difficult to carry out thorough radiographic evaluations of the maxillofacial region due to its complex anatomy and superimposition of the images of adjacent structures11. because of the complexity of maxillofacial anatomy and superimposition of the images of adjacent structures, radiographic images do not precisely replicate the anatomy that is being assessed. therefore, different imaging techniques are used to evaluate the craniofacial defects. recent research has indicated that cone-beam computed tomography (cbct) is an efficient imaging technique for determining fractures in the facial and skull bones12,13. cbct imaging technique has been introduced for imaging of the hard tissues of the maxillofacial region. the technique evaluates the structure of the bones, the articular spaces, and the dynamic performance in all three spatial dimensions without superimposition and deformity. although cbct is a suitable technique for the evaluation of osteoarthritic changes of the temporomandibular joint (tmj), it is not a proper tool for the evaluation of articular disk displacement. in addition, soft tissue tumors cannot be properly evaluated on cbct images. one of the disadvantages of the cbct technique, compared to the mri technique, is the poor contrast of soft tissues14,15. although clinical examinations usually are adequate to reach an accurate diagnosis of tmj disorders, supplementary imaging examinations should be considered to diagnose and determine the origin of tmj disorders and structural alterations and functional disorders. thus, it is recommendable to use a combination of clinical and radiographic examinations to reach a definite diagnosis16,17. in recent years, several studies have evaluated the morphology of the tmj and condyle. these studies have investigated the morphology of the tmj in subjects with malocclusion, the overall shape of the condyle head in different individuals, the relationship between age and morphologic changes in the condyle bone, the prevalence of tmj changes in asymptomatic cases, etc18-20. considering the paucity of studies 3 daneshmehr et al. braz j oral sci. 2022;21:e226611 on the morphology of the condyle with the cbct technique, the purpose of this study was to investigate the relationship between the condyle morphology and clinical findings in terms of gender, age, and remaining teeth on cbct images. materials and methods study type and the subjects a total of 144 patients referring to the department of maxillofacial radiology, faculty of dentistry, tabriz university of medical sciences (iran) between 2019 and 2020, were included in the present cross-sectional study. the ethics committee of tabriz university of medical sciences approved the protocol of the study under the code ir.tbzmed.rec.1396.310. the patients had been referred to the radiology department for cbct examinations for various reasons and were 20–80 years of age. all the radiographic examinations were carried out for other diagnostic purposes, including implant placement. the inclusion criteria consisted of no history of surgery, fracture and congenital anomalies of the tmj, and no pathologic lesions of the jaws. subjects with faulty restorations, complete and partial dentures, edentulous patients, patients with a history of systemic diseases or use of medications affecting the joints, and subjects with a history of trauma, surgery, and jaw lesions were excluded from the study. procedural steps the cbct images were taken with a newtom vgi cbct unit (newtom, verona, italy) in the department of oral and maxillofacial radiology. this cbct unit has a cone-shaped x-ray beam, a flat panel detector, a pixel of 1536×1920, a pixel size of 127×127 µm2, a pixel depth of 14 bits, a rotation of 360º, a scan time of 18 s, and kvp of 110. nnt viewer (version 2.17) software was used for the initial and final reconstruction of images. the exposure conditions were set to “automatic.” the cbct images were viewed by two oral and maxillofacial radiologists and a dental student. the kappa correlation coefficient for the inter-examiner agreement was excellent for the two radiologists in 20 samples; therefore, only one radiologist proceeded with the evaluations. the results of all the clinical and radiographic examinations were recorded on a research-made checklist, which consisted of three sections: clinical findings, radiographic findings, and possible findings. the different aspects of the clinical parameters consisted of the following: parafunctional habits, mastication side, deviation of the jaw during mouth opening and closing, the type of occlusion, and the history of pain and tenderness to palpation. the radiographic parameters consisted of the shape of the condyle on coronal, sagittal, and axial views. any possible findings were recorded in the three categories of joint erosion, osteophytes, and articular mouse. the remaining teeth were reconstructed by panoramic radiographic images. to prepare the cbct images, 0.5 mm axial sections were used. based on our previous study, the morphology of the condyle is classified into six groups on the coronal view: convex, round, flat with on effect on the glenoid fossa, flat with no effect on the glenoid fossa, angled, and heart-shaped (figure 1)21. 4 daneshmehr et al. braz j oral sci. 2022;21:e226611 l b l b b l l l b b b a b c d e f figure 1. different shapes of the condyle on the coronal view from the left to right: convex, round, flat with on effect on the glenoid fossa, flat with no effect on the glenoid fossa, angled, and heart-shaped. on the sagittal view, the morphology of the condyle was classified into four groups: round, intermediate (between round and flat), flat with an effect on the glenoid fossa, and flat with no effect on the glenoid fossa (figure 2)21. l b l b l b figure 2. different shapes of the condyle on the sagittal view from the right to left: round, intermediate (between round and flat), flat with an effect on the glenoid fossa, and flat with no effect on the glenoid fossa. 5 daneshmehr et al. braz j oral sci. 2022;21:e226611 on the axial view, the morphology of the condyle was classified into three groups: oval, bean-shaped, and conical (figure 3)21. figure 3. different shapes of the condyle on the axial view from the left to night: oval, bean-shaped and conical. statistical analysis data were analyzed with descriptive statistics, and t-test, chi-squared test, correlation test, one-way anova, and multi-variate anova, using spss 20. the spss statistics package program (ibm spss for windows, ver.25, armonk, ny) was used for statistical analysis. statistical significance was set at p < 0.05. results in the present study, 144 subjects were evaluated, with 71 males (49.3%) and 73 females (50.7%); respectively, with an age range of 20–80 years. table 1 presents the correlation between the condyle morphology and mastication side, based on age and the number of remaining teeth and gender. table 1. correlation among the condyle morphology and mastication side, based on age and the number of remaining teeth and gender. variable cross-section condyle shape mastication side p-value 9-16 remaining teeth axial bean-shaped right side 0.040 age range: 20-40 axial bean-shaped anterior 0.044 age range: 20-40 sagittal flat with no effect on the glenoid fossa bilateral 0.013 age range: 41-60 sagittal flat with no effect on the glenoid fossa bilateral 0.045 age range: 61-80 sagittal flat affecting the glenoid fossa bilateral 0.002 male coronal heart-shaped posterior 0.023 female axial bean-shaped anterior 0.037 6 daneshmehr et al. braz j oral sci. 2022;21:e226611 there was a significant relationship between the condyle morphology, 9-16 remaining teeth, and mastication side in the axial view on the right side (p = 0.040). also, there was a significant relationship between the condyle morphology, age range 20-40, and mastication side in the axial view on the anterior side (p = 0.044). there was a significant relationship among the condyle morphology, age range 20-40, 41-60, 61-80, and mastication side in the sagittal view on the bilateral side (p = 0.013), (p = 0.045), (p = 0.002). in addition, a significant relationship was found between the condyle morphology, female, and mastication side in the axial view on the anterior side (p = 0.037). in males, there was a significant relationship between the condyle morphology and mastication side in the coronal view on the posterior side (p = 0.023). table 2 presents the relationships among the condyle morphology and occlusion, based on age and the number of remaining teeth and gender. table 2. relationships among the condyle morphology and occlusion, based on age and the number of remaining teeth and gender. variable cross-section condyle shape occlusion p-value age range: 20-40 coronal convex class i 0.04 age range: 20-40 sagittal flat with no effect on the glenoid fossa class i 0.006 age range: 20-40 axial oval class i 0.006 9-16 remaining teeth coronal convex class i <0.001 25-32 remaining teeth axial oval class i 0.031 male axial oval class i 0.034 there were significant relationships among the condyle morphology, age between 20-40, and occlusion class 1 in the axial, sagittal and coronal view (p = 0.006), (p = 0.006), (p = 0.04). in addition, there was a significant relationship between the condylar morphology, 9-16 remaining teeth, and occlusion class 1 in the coronal view. both variation include of male and 25-32 remaining teeth have significant relationship with occlusion class 1 in the axial view (p = 0.031), (p = 0.034). table 3 presents the correlation among the condyle morphology and location of pain, based on age and the number of remaining teeth and gender. table 3. correlation among the condyle morphology and location of pain, based on age and the number of remaining teeth and gender. variable cross-section condyle shape location of pain p-value 9-16 remaining teeth coronal flat with an effect on the glenoid fossa the right side 0.027 age range: 61-80 coronal flat with no effect on the glenoid fossa the left side 0.046 male coronal heart-shaped the right side 0.035 7 daneshmehr et al. braz j oral sci. 2022;21:e226611 all of the variables in table 3 have significant relationships between the condyle morphology and the location of pain in the coronal view. there was a significant relationship between the condyle morphology, location of pain, and 9-16 remaining teeth and male in the coronal view on the right side (p = 0.027), (p = 0.035). also, a significant relationship was found between the condyle morphology, location of pain, and age range 61-80 in the coronal view on the left side. (p = 0.046). in our study, there was no significant relationship between the condyle morphology, parafunctional habits, the number of remaining teeth, age, and gender (p > 0.05). in addition, no significant relationship was found between the mandibular deviation during mouth opening and closing and the condyle morphology based on the number of remaining teeth, age and gender (p > 0.05). discussion morphologic changes because of the effects of estrogen, testosterone, and metabolic activity during adulthood result in differences between male and female22. tecco et al.23 and al-koshab et al.24 evaluated the morphology of the mandibular condyle and reported that the condyle is larger in men compared to women, irrespective of the clinical signs and symptoms. tabatabaian evaluated the prevalence of temporomandibular disorders and did not report any significant relationship between temporomandibular disorders and gender25. in addition, razi did not report a significant relationship between the thickness of the glenoid fossa roof and gender on the left and right sides which is similar to the present study21. in the present study, the morphology of the condyle was evaluated solely by classification of the condyle shapes at different cross-sections. the findings in this study suggest that the males might be associated with the morphological alterations of the mandibular condyle and clinical findings more than females. shahidi did not report any significant relationships between the articular eminence slope and joint disorders26, which is different from the results of the present study. this discrepancy between the results might be attributed to the smaller sample size in the study above and the differences in research methodologies. few studies have assessed the relationship between tooth loss and condyle morphology. people with single missing teeth might have a decreased articular eminence inclination angle and the unipartite missing teeth may reduce the craniocervical angle9,27. ejima et al.28 evaluated the relationship between the thickness of the glenoid fossa and the shape of the condyle and did not report any significant relationship between the two variables in terms of the number of remaining teeth in asymptomatic patients. zabarović et al.29 showed that loss of teeth has no statistically significant correlation with the eminence inclination. in the present study, 9-16 remaining teeth seems to be associated with condyle morphology and clinical findings more than the categories with a higher number of remaining teeth. a study was conducted by alhammadi et al.30 in which the tmj joint of 90 patients aged 18 to 25 years with class i occlusion was measured and analyzed by using a 8 daneshmehr et al. braz j oral sci. 2022;21:e226611 three-dimensional cbct image. the position, slope, and width of the glenoid fossa and the articular height of the eminence, the position and the slope and dimensions of the condyle and the measurement of the joint space and how the joint was placed in the glenoid fossa were evaluated and finally the left and right joints of each patient were compared. in this study, there was no difference in the depth of the left and right glenoid fossa30. the results of this study are different from the results of the present study. this discrepancy between the results might be attributed to the smaller sample size in the study above and the differences in research methodologies. in addition, one of the factors affecting the morphology of the condyle is aging; articular degenerative changes have been recorded in older subjects31. katsavrias32 evaluated the condyle morphology at a young age with a small sample size, similar to the study by merigue et al.2, and reported that the size of the condyle reaches its maximum at a young age, with the oval shape being the most prevalent shape in the anteroposterior dimension32. in the present study, we found out that the patients in the age range 20-40 have the most meaningful relationship with condylar morphology and clinical findings. nevertheless, this study had certain limitations. therefore, additional studies are needed to investigate more detail in this regard. based on the results, there were significant relationships between the condyle morphology and the clinical findings based on age, gender and the number of remaining teeth. of all the results concerning the relationship between the clinical symptoms and condyle morphology based on age, gender, and the number of remaining teeth, the most significant relationship was detected on the mastication side especially on the bilateral side. there were no significant relationships between parafunctional habits and the condyle morphology based on number of remaining teeth, age, and gender. data availability datasets related to this article will be available upon request to the corresponding author. conflict of interests none. author contribution conceptualization: shiva daneshmehr. methodology: sedigheh razi, formal analysis: tahmineh razi. investigation: shiva daneshmehr. resources: shiva daneshmehr. data curation: tahmineh razi. writing—original draft preparation: shiva daneshmehr. writing—review and editing: shiva daneshmehr. visualization: shiva daneshmehr. supervision: tahmineh razi. project administration: tahmineh razi. funding acquisition: none. all authors have read and agreed to the published version of the manuscript. references 1. bordoni b, varacallo m. anatomy, head and neck, temporomandibular joint. in: statpearls. treasure island (fl): statpearls publishing; 2021 jul 26 [cited 2021 nov 5]. available from: https://www.ncbi.nlm.nih.gov/books/nbk538486. 9 daneshmehr et al. braz j oral sci. 2022;21:e226611 2. merigue lf, conti ac, oltramari-navarro pv, navarro rl, almeida mr. tomographic evaluation of the temporomandibular joint in malocclusion subjects: condylar morphology and position. braz oral res. 2016;30:s1806-83242016000100222. doi: 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temporomandibular joint measurements in normal occlusion: a three-dimensional cone beam computed tomography analysis. j world fed orthod. 2014;3(4):155-62. doi: 10.1016/j.ejwf.2014.08.005. 31. siriwat pp, jarabak jr. malocclusion and facial morphology is there a relationship? an epidemiologic study. angle orthod. 1985 apr;55(2):127-38. doi: 10.1043/0003-3219(1985)055<0127:mafmit>2.0.co;2. 32. katsavrias eg. morphology of the temporomandibular joint in subjects with class ii division 2 malocclusions. am j orthod dentofacial orthop. 2006 apr;129(4):470-8. doi: 10.1016/j.ajodo.2005.01.018. 1http://dx.doi.org/10.20396/bjos.v19i0.8657508 volume 19 2020 e207508 original article 1 department of restorative dentistry, piracicaba dental school, university of campinas (unicamp), piracicaba, sao paulo, brazil. corresponding author: renata pereira department of restorative dentistry, piracicaba dental school, university of campinas, limeira avenue, 901, piracicaba 13414-903, sp, brazil. +55 19 98214-3025 fax: +55 19 3421-0144. e-mail: re_pe@hotmail.com / r106926@dac.unicamp.br received: november 17, 2019 accepted: april 07, 2020 glass fiber posts: influence of cementation techniques on push-out bond strength renata pereira1,* , rodrigo barros esteves lins1 , victória castelan rodrigues1, débora alves nunes leite lima1 , luís roberto marcondes martins1 , flávio henrique baggio aguiar1 aim: glass fiber posts are indicated in the rehabilitation of extensively damaged teeth; their cementation represents a critical step in restorative dentistry. the aim of this study was to quantify and compare the push-out bond strength of glass fiber posts cemented by conventional technique, two-step technique with luting agent and two-step technique associating bulk-fill composite and luting agent. methods: eighty maxillary bovine incisors were endodontically treated and divided into eight groups (n = 10) according to the luting agent (rely x arc and duo-link) and cementation technique (conventional technique; two-step technique with luting agent; and two-step technique associating bulk-fill composite – filtek bulk-fill flow or surefil sdr flow – and luting agent). samples were submitted to pushout bond strength test, and the fracture pattern was evaluated through scanning electron microscope. data were submitted to two-way anova and tukey’s test (α = 0.05). results: when rely x arc was used, the conventional cementation technique obtained higher bond strength values than the twostep technique associated with filtek bulk-fill flow. when duolink was used, the two-step technique associated with filtek bulk-fill flow presented higher bond strength values than the conventional technique. the most prevalent fracture patterns were adhesive between luting agent and dentin, and adhesive between bulk-fill composite and dentin. conclusion: two-step cementation technique associated with bulk-fill composite may be promising depending on the luting agent used. keywords: cementation. composite resins. dental cements. https://orcid.org/0000-0002-7865-3365 https://orcid.org/0000-0002-8224-6578 https://orcid.org/0000-0001-5457-3347 https://orcid.org/0000-0001-6376-4540 https://orcid.org/0000-0003-3389-5536 2 pereira r et al. introduction glass fiber posts are indicated for the rehabilitation of extensively damaged teeth in order to ensure higher retention and support to restorative material, as well as better distribution of masticatory stresses1-4. among the advantages of glass fiber posts, one may cite their esthetic appearance, high tensile strength and modulus of elasticity similar to dentin; which provides uniform stress distribution along the post length3,5,6. if, on the one hand, glass fiber posts may be promising, on the other hand, their cementation represents a critical step and can be influenced by the post type and shape, dental geometry and luting agent5,7. furthermore, the cavity configuration factor (c-factor) should be considered. defined first by feilzer et al.8 (1987) as the ratio between bonded and unbounded surfaces, the higher the c-factor value, the greater the stress at the adhesive interface. cementation of fiber posts has been described as the worst possible scenario in relation to c-factor because of the geometric characteristics of the root canal. the root canal is figuratively a very deep class i cavity9. namely, the surface to be cured is deep, reducing the stress relief capacity and increasing the challenge for adhesion. post cementation through a two-step technique has been proposed in order to make the c-factor more favorable to adhesion10,11. namely, c-factor reduction should be achieved through layered application of luting agents, instead of the traditional single increment application. jogsma et al.10 (2010) explain that in the two-step technique, the unbounded surface is higher than in case of the one-step cementation technique, which could reduce the c-factor from 229 to 1.8. also, polymerization shrinkage stress should be reduced, generating less microleakage and, thus, increasing the restoration longevity10,11. the use of two layers of luting agent in post cementation has not been studied enough. the number of in-vitro studies related to the topic is low10,11, and to the best of our knowledge a single case report was published so far12. however, recently, bakaus et al.13 (2018) verified a high bond strength when the root canal was reinforced with bulk-fill composite before cementing the fiber post with traditional luting agent, similarly to the two-step cementation technique. bulk-fill composites emerged in 2010, when dentsply produced surefil sdr flow; the first composite able to be cured in 4 mm increments14. bulk-fill flowable composites, specifically, should represent a promising alternative to reinforce root canals, since they can be cured at depths of up to 4  mm, without the need to extend the light curing period15,16. additionally, they present reduced polymerization shrinkage and lower modulus of elasticity in deeper layers15. taking into account the lack of studies in respect to the two-step cementation technique, and considering a single study was reported on the use of bulk-fill composite to reinforce root canals before fiber post cementation, further studies are needed to clarify the efficiency of these protocols. the aim of this study was to quantify and compare the push-out bond strength of glass fiber posts cemented by a conventional technique, a two-step technique with luting agent and a two-step technique associating bulk-fill composite and luting agent. the working hypotheses tested were: (1) there would be significant differences in the bond strength of glass fiber posts cemented 3 pereira r et al. by different techniques; (2) two-step technique, either solely with luting agent or with bulk-fill composite and luting agent, would generate higher bond strength when compared to the conventional cementation technique. material and methods eighty freshly extracted maxillary bovine incisors teeth of similar shapes and sizes, with 18 cm straight root, closed apex and free of cracks were selected and kept in 0.1% thymol for up to two months. cleaning of the outer surfaces of the teeth was performed by root scaling, followed by blasting with sodium bicarbonate and water. after cleaning, the crown was separated from the root with the aid of a double-sided diamond disc (kg sorensen, barueri, sp, brazil) under water cooling. for confection of the samples, the pulp was removed with hand k-files (dentsply maillefer, tulsa, ok, usa) and irrigation with 1% naocl solution was performed to suspend any organic matter. endodontic treatment was manually performed using the crown-down technique with k-files at 17 mm working length, and an apical stop with a #40 file. during instrumentation, the root canal was abundantly irrigated with distilled water so that the irrigation solution did not cause bias to the study17. roots were dried with paper points (dentsply maillefer, ballaigues, switzerland) and filled by vertical compaction of warm gutta-percha points and endodontic cement sealer 26 (dentsply, york, pa, usa). conventional glass ionomer cement (vitro fil, nova dfl, rio de janeiro, rj, brazil) was used to temporarily seal the root access, and the roots were stored at 37ºc for ten days. gates glidden drills and peeso reamers #5 and #6 (dentsply maillefer, ballaigues, switzerland) were used to remove the filling from the root canal at a depth of 12 mm. intraradicular preparation was performed according to the recommendations of whitepost dc post manufacturer (fgm, joinville, sc, brazil), considering drill #3 for the final calibration. the roots were then randomly divided into eight groups (n = 10) according to experimental factorial design with independent variables: luting agent (two levels) and cementation technique (four levels), as follows: group 1 – conventional technique with rely x arc (3m oral care, st. paul, mn, usa); group 2 – two-step technique with rely x arc; group 3 – two-step technique with filtek bulk-fill flow (3m oral care, st. paul, mn, usa) and rely x arc; group 4 – two-step technique with surefil sdr flow (dentsply maillefer, ballaigues, switzerland) and rely x arc; group 5 – conventional technique with duo-link (bisco, schaumburg, il, usa); group 6 – two-step technique with duolink; group 7 – two-step technique with filtek bulk-fill flow and duo-link; group 8 – two-step technique with surefil sdr flow and duo-link. the details of the luting agents and composites used in this experiment are presented in table 1. for pre-treating the glass fiber posts, initially, #3 glass fiber posts (whitepost dc, 3m oral care, st. paul, mn, usa) were selected, cleaned with alcohol and air-dried. then, a coat of silane-based primer (relyx ceramic primer, 3m oral care, st. paul, mn, usa) was applied for one minute. finally, a layer of either adpter scotchbond multipurpose plus adhesive (3m oral care, st. paul, mn, usa) (groups 1-4) or all-bond 3 adhesive (bisco, schaumburg, il, usa) (groups 5-8) was applied for 20 seconds and air-dried. 4 pereira r et al. the treatment of the root canal was performed as follows: groups 1-4 root canal was etched with 35% phosphoric acid (ultra-echt, ultradent products inc., south jordan, ut, usa) for 15 seconds, rinsed for 30 seconds and dried with absorbent paper points. a coat of adper scotchbond multipurpose plus activator, followed by a catalyst, was applied. excess was removed with absorbent paper points. groups 5-8 etching, rinsing and drying were performed as previously described. a drop of part a and a drop of part b of all-bond 3 adhesive system were mixed into a mixing well. a coat was applied into the canal and excess was removed with paper points. the cementation of the fiber posts was then performed, as described: conventional technique luting agent was mixed in 1:1 ratio according to the manufacturer’s instructions, and was inserted into the root canal with the aid of a needle tube (centrix, shelton, ct, usa). the glass fiber post was then positioned. light curing was performed by a poly-wave light-emitting diode curing unit (valo, ultradent products inc., south jordan, ut, usa) in the high power mode: 1400 mw/cm² for 40 seconds. twostep technique with luting agent – the luting agent was mixed in 1:1 ratio according to the manufacturer’s instructions and was applied to the root canal with a needle tube (figure 1a). after, a non-stick simulated post, made of polyether-based impression material (impregum soft, 3m oral care), was placed inside the root canal in order to establish space for the definitive post cementation. the simulated post dimensions were standardized as follows: polyether-based material was manipulated according to the manufacturer’s instructions and applied into an s4 nylon plug fixing (fischer, são paulo, sp, brazil) with the aid of a needle tube, resulting in a post slightly larger than that of #3 glass fiber post (figure 1b). after the setting time, the simulated post was removed from the plug fixing and placed into the root canal along with the first layer of luting agent (figure 1c), which was light-cured for 20 seconds. the simulated post was then removed from the root canal, and the first layer of luting agent was lightcured for 40 seconds more (figure 1d). finally, the second layer of luting agent was manipulated, applied to the root canal (figure 1e) and the glass fiber post was positioned (figure 1f). light curing was performed for 40 seconds (figure 1g). two-step table 1. evaluated luting agents and composites and respective manufacturer information. material brand name (classification) manufacturer composition shade rely x arc (luting agent) 3m oral care, dental products, st. paul, mn, usa paste a: silane-treated ceramic, tegdma, bis-gma, silane-treated silica, functionalized dimethacrylate polymer. paste b: silane-treated ceramic, tegdma, bis-gma, silane treated silica, functionalized dimethacrylate polymer (eyfh) a1 duo-link (luting agent) bisco, schaumburg, il, usa base: bis-gma; tegdma; udma; glass filler. catalyst: bis-gma; tegdma; glass filler trans filtek bulk-fill flow (bulk-fill composite) 3m oral care, dental products, st. paul, mn, usa bis-gma, bis-ema, udma, tegdma, substituted dimethacrylate, edmab, benzotriazol, silate treated ceramic, ytterbium trifluoride a2 surefil sdr flow (bulk-fill composite) dentsply caulk, milford, de, usa modified udma, tegdma, ebpdma, bariumaluminofluoroborosilicate glass, strontiumaluminofluoroborosilicate glass universal abbreviations: bis-ema: ethoxylated bisphenol-a dimethacrylate; bis-gma: bisphenol-a diglycidyl ether dimethacrylate; edmab: ethyl 4-dimethylaminobenzoate; ebpdma: ethoxylated bisphenol-a dimethacrylate; tegdma: triethyleneglycol dimethacrylate; udma: urethane dimethacrylate. data were provided by manufacturers. 5 pereira r et al. technique associating bulk-fill composite and luting agent the same procedures of the previous technique were performed. nevertheless, the first layer of luting agent was replaced by bulk-fill composites, which were applied by their own dispensing tips. after seven days of storage in distilled water at 37ºc, the roots were fixed to acrylic plates with sticky wax (asfer ind., são caetano do sul, sp, brazil). the set was stabilized to a metallographic precision cutter (isomet 1000, buehler, lake bluff, il, usa) in which a diamond blade (isomet diamond wafering blades, buehler ltd., lake buff, il, usa) performed serial sections with water-cooling at 250 rpm, from the cervical to apical direction, to obtain three slices of 1  mm thick from cervical, middle and apical third. the samples were then submitted to push-out bond strength test, conducted at a crosshead speed of 1 mm/min, with the load applied to the apical-cervical direction using a metal tip of 1.2mm diameter, until failure (universal testing machine, ez test l, shimadzu, japan). the maximum failure load was recorded in newtons (n) and converted into mpa by dividing the load by the root canal area (a). the area was calculated through the formula: a = 2πr.h, where π is the constant 3.14; r the radius of the post #3; and h the root slice thickness. measurements of r and h were performed using a digital calliper (mitutoyo corporation, tokyo, japan). * a – application of first layer of luting agent to the root canal; b – application of polyether-based material to nylon plug fixing and obtainment of simulated post; c – placement of simulated post into root canal along with luting agent; d – light curing of first layer of luting agent; e – application of second layer of luting agent to root canal; f – placement of glass fiber post; g – light curing of second layer of luting agent and glass fiber post. figure 1. illustrative scheme of the two-step technique for glass fiber post cementation. a b e f g c d 6 pereira r et al. a mean push-out bond strength, in mpa, was calculated for each root from the values obtained by each slice. sample patterns of fractures were evaluated using scanning electron microscopy (sem) (jeol-jsm 5600lv, tokyo, japan) in 1) adhesive fracture between glass fiber post and luting agent; 2) adhesive fracture between first and second layers of luting agent; 3) adhesive fracture between luting agent and composite; 4) adhesive fracture between luting agent and dentin; 5) adhesive fracture between composite and dentin; 6) cohesive fracture in glass fiber post; 7) cohesive fracture in luting agent; 8) cohesive fracture in composite; 9) mixed fracture. cohesive and adhesive fractures were considered when at least 70% of the total area was composed of the same pattern. mixed fracture was stated when there was more than one pattern, and none prevailed. data were tabulated and statistically analyzed using spss 21.0 software (spss inc., chicago, il, usa). the results were submitted to normality and equality of variances tests (shapiro-wilk and kolmogorov-smirnov, p > 0.05), followed by parametric twoway anova and tukey’s post-hoc test (α = 0.05). results the results of push-out bond strength (mpa) for both variables, luting agent (p = 0.932), cementation technique (p = 0.744) and their interaction (p < 0.001) are presented in table 2. rely x arc obtained higher bond strength than duo-link when a conventional cementation technique was performed (p = 0.002). conversely, when associated with filtek bulk-fill, duo-link obtained higher bond strength than rely x arc (p = 0.005). both luting agents, associated with surefil sdr flow, obtained similar bond strength values (p > 0.05). also, bond strength values presented by the two-step technique with rely x arc and the two-step technique with duo-link were not statistically different (p > 0.05). when rely x arc was used, conventional cementation technique obtained higher bond strength values than two-step techniques associated with filtek bulk-fill flow (p = 0.027). two-step techniques with rely x arc and two-step techniques with surefil sdr flow did not differ statistically from other groups (p > 0.05). when duo-link was used, in turn, the two-step technique associated with filtek bulk-fill flow presented higher bond strength values than the conventional technique and the table 2. mean (standard deviation) push-out bond strength (mpa) of glass fiber posts cemented by different techniques. cementation technique luting agent rely x arc duo-link conventional 4.55 (1.56) aa 3.23 (1.16) bc luting agent + luting agent 4.15 (1.33) aab 3.86 (1.12) abc filtek bulk-fill flow + luting agent 3.62 (1.44) bb 4.81 (1.43) aa surefil sdr flow + luting agent 3.78 (1.40) aab 4.29 (1.53) aab mean values followed by distinct letters (uppercase in horizontal and lowercase in vertical) differ from each other (p≤0.05). n=10 specimens / group. 7 pereira r et al. two-step technique with duo-link (p = 0.0001 and p = 0.025, respectively). the two-step technique associated with surefil sdr flow presented higher bond strength than the conventional technique (p = 0.012). however, the same technique did not differ neither from the two-step technique associated with filtek bulk-fill flow nor from the two-step technique with duo-link. the values obtained by the two-step technique with duo-link did not differ from the values obtained by the conventional technique (p > 0.05). figure 2 shows the fracture patterns obtained by each group. the most prevalent fracture patterns were adhesive between the luting agent and dentin (groups 1, 2, 5, 6) and the adhesive between the composite and dentin (groups 3, 4, 7, 8), although mixed fractures also stood out. discussion in order to increase the bond strength of glass fiber posts to radicular dentin, several strategies have been proposed by dental material manufacturers and researchers to reduce luting agent thickness: posts individualization, roots reinforcement with restorative materials and layered application of luting agents by a two-step cementation technique10-13,17-19. the aim of this study was to quantify and compare the push-out bond strength of glass fiber posts cemented by a conventional technique, a two-step technique with luting agent and a two-step technique associating bulk-fill composite and luting agent. the luting agents tested in this study were rely x arc and duo-link, whereas the bulk-fill composites used in this study were filtek bulk-fill flow and surefil sdr flow. the first hypothesis, that there would be significant differences in the bond strength of glass fiber posts cemented by different techniques, was accepted. nevertheless, the second hypothesis, that the two-step technique would generate higher bond strength * 1 adhesive fracture between glass fiber post and luting agent; 2 adhesive fracture between first and second layers of luting agent; 3 adhesive fracture between luting agent and composite; 4 adhesive fracture between luting agent and dentin; 5 adhesive fracture between composite and dentin; 6 cohesive fracture of glass fiber post; 7 cohesive fracture of luting agent; 8 cohesive fracture of composite; 9 mixed fracture. figure 2. failure pattern (%) of glass fiber posts cemented by different cementation techniques. 1 100% 80% 60% 40% 20% 90% 70% 50% 30% 10% 0% g1 g2 g3 g4 g5 g6 g7 g8 2 3 4 5 6 7 8 9 8 pereira r et al. compared to the conventional cementation technique was rejected. interestingly, rely x arc and duo-link obtained opposite performance results. when rely x arc was used, the conventional cementation technique obtained higher bond strength values than two-step techniques with filtek bulk-fill flow. conversely, when duo-link was used, the two-step technique with filtek bulk-fill flow presented higher bond strength values than the conventional technique. although both luting agents are dual-cured, the amount of autoand light-polymerizing components varies between the products. such variation may result in differences in the polymerization characteristics20. rely x arc is a luting agent with a rapid response to light exposure, but low potential of cure when chemically activated20. namely, in the conventional cementation technique, despite the increase of luting agent volume, there is a single compound of cement, so light is able to be transmitted through the whole thickness and activate the high content of photo-initiators, which yield high degree of conversion and consequently high bond strength. conversely, when the two-step technique is applied, especially with bulk-fill composite, luting agent may depend more on chemical activation, since light may be attenuated by the different constituents of the root canal filling. arrais et al.20 (2009) found that in the auto-polymerizing mode, rely x arc takes longer than duo-link to initiate polymerization and its maximum rate of polymerization is lower. this explains the low bond strength values obtained by rely x arc when it was associated with filtek bulk-fill flow. when duo-link was used, in turn, the two-step technique associated with filtek bulk-fill flow presented higher bond strength values than the conventional technique. several points may be considered to explain the result. the two-step technique enables a thinner cementation layer, which provides: first, reduction of the polymerization shrinkage, generating less stress at the adhesive interface. second, decrease of incorporation of failures such as voids. third, increase of frictional retention, through the intimate contact between post and dentin. all these features should increase the adhesion21-23. the results may also demonstrate the influence of bulk-fill associated with a luting agent on fiber post cementation. according to the manufacturer’s information, the resin system of filtek bulk-fill flow produces low polymerization shrinkage associated with a low modulus that results in low shrinkage stress. low shrinkage stress provided by the composite should reinforce even more the bond to dentin and create a more uniform structure at the dentinal walls10. also, the semi-translucence of the composite enables light transmission and complete curing through the whole layer16,24. lastly, it is relevant to consider that the bond between filtek bulk-fill flow and duo-link might be uniform and high enough to provide high bond strength results. such speculation may be confirmed by the pattern of fracture analyses, which show that in the two-step technique associated with filtek bulk-fill flow and duo-link, no fracture between the luting agent and composite was observed. the results of this study are somewhat in accordance with the reports of bakaus et al.13 (2018), who found out that the bond strength of fiber posts cemented into roots reinforced by bulk-fill composite was not the highest in their study, but was constant. namely, bulk-fill composite was the only material able to maintain high bond strength values from the cervical to apical root third13. the two-step technique associating surefil sdr flow and duo-link presented similar bond strength values to two-step techniques associating filtek bulk-fill flow and duo-link, which point out once again the promising influence of bulk-fill composites 9 pereira r et al. associated with a luting agent on fiber post cementation. giovannetti et al. (2012) tested surefil sdr flow as a luting agent to cement fiber posts25. the authors found out that surefil sdr flow yielded post retentive strengths similar to those of the luting agent tested as the control. although the present study did not test bulk-fill composites exactly as luting agents, high bond strength results might be due to the same reasoning. surefil sdr flow is a flowable bulk-fill composite of high translucency. a high translucency associated with low filler volume, typical of flowable composites, enhance light transmission, enabling complete curing and increasing bond strength24. additionally, surefil sdr flow features a photo-initiator group, which is a modulator of polymerization reaction in urethane dimethacrylate (udma). the polymerization modulator reacts with camphorquinone, leading to the formation of polymers with low elastic modulus and decreased polymerization stress25-27. indeed, low polymerization stress should enhance the bond to dentin10,22. unlike the two-step technique associating bulk-fill composite and luting agent, the two-step technique solely with luting agent presented overall intermediate bond strength values. the inherent shrinkage of luting agents after the setting reaction may justify the results since gaps at the interface between the layers of luting agent could be developed. the findings are not in accordance with the reports of jongsma et al.10 (2010). yet, it should be highlighted that their study did not compare two-step technique solely with luting agent to two-step technique with bulk-fill composite and luting agent. comparing both techniques, the use of a bulk-fill composite in a twostep cementation procedure should yield higher bond strength of the fiber posts since bulk-fill composites exhibit singular composition that reduces polymerization shrinkage. assuming, however, that the layered application of both luting agents by the twostep technique yielded a statistically similar bond strength than those applied by the conventional technique, the use of such a protocol should be pondered. analyzing the pattern of fracture, although mixed fracture pattern was prominent among the groups, indicating homogeneity between the composite and/or luting agentpost-dentin composition, it is worth noting that failures occurred mainly between the luting agent and dentin or bulk-fill composite and dentin. based on these findings, it may be assumed that the bond between the fiber post and luting agent was higher than the bond between the luting agent or bulk-fill composite and dentin. this result is in accordance with previous studies28. shrinkage is inherent in resin-based cements, and such shrinkage may pull the resin cement away from dentin, resulting in weaker bond10. also, several factors may affect the luting agent-dentin bond strength: the presence of moisture in the root canal to allow the penetration of adhesive monomers, the number and diameter of dentin tubules relative to the portion of the root canal, and certainly the procedures related to endodontic treatment, post space preparation and post cementation9,23. the association between inherent shrinkage of the luting agent and the unfavourable features of the root canal may lead to failures at the luting agent/ composite–dentin interface9,28,29. pre-treatment of the post may have also played a role in the pattern of the fracture results, as the bond strength between the glass fiber post and luting agent was maximised. according to machado et al.30 (2015), the application of both silane and adhesive improve post retention since the adhesive allows a compatible and strong chemical interaction between the silanized post and luting agent. 10 pereira r et al. it is important to emphasise that, although the two-step cementation technique associating bulk-fill composites and a luting agent seems promising to enhance the bond strength of glass fiber posts, additional studies that evaluate a greater variety of bulkfill composite and luting agent combinations are necessary to consider this protocol superior in relation to a conventional cementation technique. also, the present study was performed under ideal laboratory conditions. thus, further studies may contribute to making the protocol feasible, so that the several steps involved in the technique can be clinically practicable. finally, glass fiber post cementation is considered a complex technique, usually performed in clinical environment, whose increased protocol steps appears to have limited benefits. within the limitations imposed by this in vitro study, it can be concluded that rely x arc performance was better when using a conventional cementation technique. conversely, duo-link bond strength was higher when it was associated with bulk-fill composites. findings suggest that two-step cementation technique associated with bulk-fill composite may be promising depending on the luting agent used. acknowledgments this work was supported by the brazilian 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freitas tl, vitti rp, miranda me, brandt wc. effect of glass fiber post adaptation on push-out bond strength to root dentin. braz dent j. 2019;30(4):350-5. doi: 10.1590/0103-6440201902491. 23. walcher jg, leitune vcb, collares fm, de souza balbinot g, samuel smw. physical and mechanical properties of dual functional cements-an in vitro study. clin oral investig. 2019;23(4):1715-21. doi: 10.1007/s00784-018-2598-4. 24. son sa, park jk, seo dg, ko cc, kwon yh. how light attenuation and filler content affect the microhardness and polymerization shrinkage and translucency of bulk-fill composites? clin oral investig. 2017;21(2):559-65. doi: 10.1007/s00784-016-1920-2. 25. giovannetti a, goracci c, vichi a, chieffi n, polimeni a, ferrari m. post retentive ability of a new resin composite with low stress behaviour. j dent. 2012;40(4):322-8. doi: 10.1016/j.jdent.2012.01.007. 12 pereira r et al. 26. fronza bm, ayres a, pacheco rr, rueggeberg fa, dias c, giannini m. characterization of inorganic filler content, mechanical properties, and light transmission of bulk-fill resin composites. oper dent. 2017;42(4):445-55. doi: 10.2341/16-024-l. 27. hernandes nm, catelan a, soares gp, ambrosano gm, lima da, marchi gm, et al. influence of flowable composite and restorative technique on microleakage of class ii restorations. j investig clin dent. 2014;5(4):283-8. doi: 10.1111/jicd.12058. 28. pereira jr, da rosa ra, do valle al, ghizoni js, só mv, shiratori fk. the influence of different cements on the pull-out bond strength of fiber posts. j prosthet dent. 2014;112(1):59-63. doi: 10.1016/j.prosdent.2013.10.009. 29. pereira jr, lins do valle a, ghizoni js, lorenzoni fc, ramos mb, dos reis só m. push-out bond strengths of different dental cements used to cement glass fiber posts. j prosthet dent. 2013;110(2):134-40. doi: 10.1016/s0022-3913(13)60353-4. 30. machado fw, bossardi m, ramos ts, valente ll, münchow ea, piva e. application of resin adhesive on the surface of a silanized glass-fiber reinforced post and its effect on the retention to root dentin. j endod. 2015;41(1):106-10. doi: 10.1016/j.joen.2014.09.014. 1http://dx.doi.org/10.20396/bjos.v18i0.8657256 volume 18 2019 e191591 original article 1 superior school of health sciences, state university of amazonas, manaus, amazonas, brazil 2 department of restorative dentistry, piracicaba dental school, state university of campinas, piracicaba, são paulo, brazil. corresponding author: caroline mathias department of restorative dentistry, piracicaba dental school, state university of campinas, limeira avenue, 901, mail box 52, piracicaba, são paulo, brazil, zip code 13414-903. phone number: +55 19 2106-5337. email: caroline.mathias@hotmail.com https://orcid.org/0000-0002-0718-1137 received: february 26, 2019 accepted: september 16, 2019 effect of diphenyliodonium hexafluorphosphate in the yellowing and properties of experimental resin cements kamila menezes guedes de andrade1, caroline mathias2*, hugo felipe do vale1, flávio henrique baggio aguiar2, giselle maria marchi2 aim: the yellowing effect is undesirable and can occur in the dental restoration margins when light-cured resin cements containing camphorquinone as photoinitiator is used. this study aimed to evaluate the effect of diphenyliodonium hexafluorphosphate (dpi) in photoinitiator systems that contained different concentrations of camphorquinone (cq) and dimethylamino ethyl benzoate (edab) on resin cements. methods: a base (1:1) of bisphenol a diglycidyl methacrylate (bisgma) and triethylene glycol dimethacrylate (tegdma) was obtained, and 60wt% of glass fillers was added. eight formulations were obtained: rc1 (0.5mol% cq / 1mol% edab), rc2 (0.5mol% cq / 2mol% edab), rc3 (1mol% cq / 1mol% edab), rc4 (1mol% cq / 2mol% edab), and rc5 to rc8, which contained the same bases plus 0.5mol% dpi. experimental resin cements were evaluated by the degree of conversion (dc), l*a*b* color analysis, water sorption (ws) and solubility (s), flexural strength, and elastic modulus. the data were analyzed by three-way anova, tukey’s and mann-whitney tests (α = 0.05). results: the combination of dpi and 0.5mol% cq increased dc in rc6 and l* in rc5 without increasing the ws and a* b* values. cq at 1mol% showed higher values of b* and lower values of a*, except for rc3. groups with 2mol% edab showed higher dc. conclusion: the addition of dpi reduces cq, generating a decreased yellowing effect, while maintaining adequate properties in the resin cements, especially with 2 mol% edab. keywords: resin cements. physical phenomena. photoinitiators, dental. polymers. https://orcid.org/0000-0002-0718-1137 2 andrade et al. introduction resin cements are widely used in luting procedures, such as laminate veneers, crowns, and posts. however, the choice of a light-cured or dual-cured fixation agent depends on the intended use1. the thickness of larger indirect restorations and the depth in root post fixations are factors that usually suggest the choice of dual-cured resin cements because of their distance from the light source. in these cases, dual-cured resin cements may perform better when the light power is reduced2. despite the advantage of chemical phase polymerization, the amount of tertiary amines in these materials is a potential factor for yellowing, because of the possibility of oxidation of these molecules. light-cured resin cements are more stable with a reduced tertiary amine concentration, which also provides a longer working time, leading to easier removal of excess resin during procedures3. improving the photoinitiator system can be an alternative to these materials that have limited indications. camphorquinone (cq) is the most common photoinitiator that is used for composites4,5. recently, total or partial replacement of this component was described as a possibility for providing better properties, such as a higher degree of conversion (dc). cq is an intense yellow powder with a conjugated diketone chromophore. because of this coloration, greater amounts of cq in a resin cement may impact the optical properties of restorations over time, marking the margins of restoration in esthetic areas and altering the color aspect of thin laminates because of the resin cement color change6. because of this characteristic, reducing this component in initiator systems may decrease these undesirable effects on the composite color6,7. polymerization is generally initiated using cq/amine systems, because, although reactions can occur using only the cq, they are more effective with a co-initiator as a reducing agent. dimethylamino ethyl benzoate (edab) is described as a tertiary aromatic amine in two-component photoinitiator systems that interacts with cq to form exciplexes that lead to efficient free radical formation8. an iodonium salt, diphenyliodonium hexafluorphosphate (dpi), has been investigated to improve polymerization in metacrylate-based resins and cements as the third molecule in a three-component photoinitiator system9. during the interaction with an excited photosensitive initiator, such as the cq, dpi can be decomposed into diphenyliodine. diphenyliodine generates phenyliodine and phenyl free radicals, which initiate polymerization in metacrylates. another mechanism can occur when amino radicals produced from cq/amine reduce dpi, also generating new free radicals. phenyl radicals generated in these ways can still react with residual amines and form new free amine radicals, which may be associated with better polymerization10. the c-i bond in iodonium salts has a low energy, which can explain its decomposition in these ways during the reactions11. however, the adequate concentration of each component of photoinitiator system that provides the least amount of yellowing of resin cements using cq as photoinitiator remains unclear. thus, it is important to investigate ternary systems using iodonium salt and the influence of each component and concentration on chemical and 3 andrade et al. physical properties of resin cements; previous studies did not change the concentrations of cq and edab associated with dpi in resin cements, to achieve the best combination9. adding dpi may allow cq reduction, which may improve color and provide better esthetic results. the objective of the present study was to evaluate the influence of adding dpi to different concentrations of cq/edab in ternary systems on the dc, color analysis (y), water sorption (ws), solubility (s), flexural strength (fs), and elastic modulus (e). the hypotheses tested were as follows: (1) as the concentration of cq/edab combined with dpi increases, the cement’s properties will also improve; (2) dpi does not affect color; and (3) the lower the cq concentration, the lower the yellowing effect. materials and methods preparation of the model cements experimental resin cements (rc) were prepared at a 1:1 mass ratio of bisphenol a diglycidyl methacrylate (bisgma, esstech inc., essington, pa, usa) and triethyleneglycol dimethacrylate (tegdma, esstech inc., essington, pa, usa). butylated hydroxytoluene (bht, esstech inc., essington, pa, usa) was added as an inhibitor at 0.1 mol%. four resin cements were tested using binary systems with 0.5 mol% or 1 mol% camphorquinone (cq, sigma–aldrich, st. louis, mo, usa) and 1 mol% or 2 mol% edab (sigma–aldrich), totaling eight formulations. diphenyliodonium hexafluorphosphate (dpi, sigma–aldrich, milwaukee, wi, usa) at 0.5 mol% was added in the other four resin cements, forming ternary systems according to the following proportion in mol%: rc1: cq + edab (0.5/1) rc2: cq + edab (0.5/2) rc3: cq + edab (1/1) rc4: cq + edab (1/2) rc5: cq + edab + dpi (0.5/1/0.5) rc6: cq + edab + dpi (0.5/2/0.5) rc7: cq + edab + dpi (1/1/ 0.5) rc8: cq + edab + dpi (1/2/0.5) the resins were blended and homogenized for 1 h at room temperature with a magnetic stirrer. each formulation was loaded with 60 wt% of 0.7 µm average size silanated barium borosilicate glass fillers (esstech inc., essington, pa, usa). all chemicals were used without further purification. degree of conversion for the dc assessment, five specimens from each group were prepared directly on the horizontal face of the attenuated total reflectance (atr) cell using a diamond crystal (pike technologies, madison, wi, usa) in fourier transform infrared spec4 andrade et al. troscopy (ftir, spectrum 100 optica, perkinelmer, billerica, ma, usa). a silicon bar mold (7 mm long, 2 mm wide and 1 mm high) was filled with material directly on the crystal, and a mylar strip was placed over the mold for polymerization with a light-emitting diode (led, bluephase g2, ivoclar-vivadent, schaan, liechtenstein) at an irradiance of 1.200 mw/cm2 for 20 s. the assay was performed at the bottom surface immediately after the light-curing protocol. before polymerization, the monomers’ baseline was recorded with a wave-number range of 1.665–1.580 cm−1, resolution of 4 cm−1, and happ-genzel apodization in absorbance mode (12). the dc was calculated according to a baseline technique based on band ratios of 1.638 cm−1 (aliphatic carbon-to-carbon double bond) and 1.608 cm−1 (aromatic component group) as an internal standard between the polymerized and unpolymerized samples, based on the following expression: dc=100 × [1 − (polymerized cement/ unpolymerized cement)]. color analysis silicon molds (5 mm in diameter and 1 mm thick) were filled with resin cements and irradiated for 20 s using light-emitting diodes (led, bluephase g2, ivoclar-vivadent) through a mylar strip. ten specimens of each group were stored dry in a dark container for 24 h at room temperature (25±1°c). the color reading was performed using a reflectance spectrophotometer (konica minolta cm-700d, shanghai, china) with a teflon device as a sample holder. a light cabin (gti mini matcher mm1e; gti graphic technology, newburgh, ny, usa) was used during measurements to standardize the ambient light. cie lab system was used to quantify the values considering the coordinates l* (luminosity, from 0=black to 100=white), a* (from negative values=green to positive values=red), and b* (from negative values=blue to positive values=yellow). water sorption and solubility five specimens of each group were prepared in a silicon mold (7 mm long, 2 mm wide, and 1 mm high). the reduced specimen dimensions were adapted to enable a single-step light-curing of resin cements for 20 s with light-emitting diodes (led, bluephase g2, ivoclar-vivadent). the specimens were individually stored in microtubes at 37°c, in a desiccator with silica gel. they were weighed daily in an analytical balance (discovery dv215cd, ohaus corporation, pine brook, nj, usa) with an accuracy of 0.01 mg. this procedure was performed three consecutive times until the loss of mass in each specimen was no higher than 0.1 mg each. these values were recorded as m1. the length, width, and height of the specimens were measured using a digital caliper (mitutuyo, tokyo, japan) with 0.01 mm accuracy for calculation of the volume (v) of each bar, in mm3. the specimens were then individually immersed in 3 ml of distilled water for 7 days. after immersion, the excess water was removed using absorbent papers and the specimens were gently dried with air for 10 s and weighed again to record m2 values. for recording of m3 values, specimens were stored again in a desiccator containing silica gel and weighed as described in the process for m1. the values of ws and s were calculated according to the formula: ws=(m2–m3)/v and s=(m1–m3)/v. 5 andrade et al. flexural strength and elastic modulus for three-point bending measurements (n=10), specimens were made in a similar way to that used for the ws and s, in bar-shaped specimens (7 mm long, 2 mm wide and 1 mm high). the specimens were irradiated for 20 s by light-emitting diodes (led, bluephase g2, ivoclar-vivadent). after storage for 24 h, their dimensions were measured using an electronic digital caliper (mitutuyo, tokyo, japan), and the specimens were tested on a universal testing machine (instron 4411, canton, ma, usa) with a span width of 5 mm at a crosshead speed of 0.5 mm/min with 50n. fs and e were monitored by blue hill 2 software (instron). fs (mpa) was calculated according to the formula: fs=3fl/2bh² where: f is the maximum load in newtons that is exerted on the specimen, l is the distance between the supports; b is the width of the specimen immediately before testing (mm); and h is the height of the specimen immediately before testing (mm). the values of e were obtained from the linear portion of load-displacement traces generated by the software, according to the formula: e=(∆f/∆y) x (3l/4bh³), where ∆f/∆y is the force variation (∆f) divided by unit change in deflection at the center of the specimen (∆y), l is the distance between the supports; b is the width of the specimen immediately prior to testing (mm); and h is the height of specimen immediately prior to testing (mm). statistical analysis the normality and variance homogeneity of the data were analyzed. the results of dc, ws/s, fs/e, and coordinates a*b* of the color analysis were analyzed using a threeway anova and tukey’s post-hoc test (α=0.05). the results of l* were analyzed using the non-parametric mann-whitney u test (α=0.05). results degree of conversion table 1 shows that when edab was used at 1 mol%, there was no difference between the groups regardless of the concentration of cq and the presence of dpi. adding dpi increased the dc for 0.5 mol% cq and 2 mol% edab cement. however, without salt, the cement containing 1 mol% cq and 2 mol% edab had higher results. for amines, the 2 mol% edab cements promoted better results than the 1 mol% cements. table 1. means (standard deviations) for dc (%) dpi (mol%) cq (mol%) edab (1 mol%) edab (2 mol%) 0 0.5 51.17 (0.22) ab 53.09 (0.37) ba# 1 50.68 (0.33) ab 56.05 (0.61) aa# 0.5 0.5 51.51 (0.44) ab 55.54 (0.78) aa* 1 51.52 (0.80) ab 54.12 (0.44) ba* values followed by the different upper case letter in the same column or by different lower case letter in the same row are statistically different (p < 0.05). * and # represents statistical difference for dpi. 6 andrade et al. color analysis color analysis considered the results of coordinates l*a*b* separately. table 2 shows that adding dpi increased the l* when combined with 0.5 mol% cq and 1 mol% edab. for coordinate a*, table 3 shows the influence of dpi when combined with 1 mol% cq and 1 mol% edab, which caused decreasing values. table 4 shows that dpi also affected b* values when added into cements containing 1 mol% cq and 1 mol% edab, which caused increasing results. the edab concentration of 2 mol% showed higher values than 1 mol% edab when combined with 1 mol% cq or 0.5 mol% cq with 0.5 mol% dpi. however, in all experimental resin cements tested, 1 mol% cq produced a higher yellowing effect than 0.5 mol%. table 4. means (standard deviation) of coordinate b* dpi (mol%) cq (mol%) edab (1 mol%) edab (2 mol%) 0 0.5 7.75 (0.68) ba 8.24 (0.50) ba 1 9.49 (1.74) ab* 11.96 (1.00) aa 0.5 0.5 7.33 (0.52) bb 8.91 (0.99) ba 1 11.05 (1.09) aa# 11.78 (1.21) aa values followed by the different upper case letter in the same column or by different lower case letter in the same row are statistically different (p < 0.05). * and # represents statistical difference for dpi. table 3. means (standard deviation) of coordinate a* dpi (mol%) cq (mol%) edab (1 mol%) edab (2 mol%) 0 0.5 -1.55 (0.15) ab -1.89 (0.23) ba 1 -1.69 (0.45) ab* -2.52 (0.36) aa 0.5 0.5 -1.72 (0.11) ba -1.84 (0.21) ba 1 -2.29 (0.20) aa# -2.43 (0.26) aa values followed by the different upper case letter in the same column or by different lower case letter in the same row are statistically different (p < 0.05). * and # represents statistical difference for dpi. table 2. median (minimum maximum) of coordinate l* dpi (mol%) cq (mol%) edab (1 mol%) edab (2 mol%) 0 0.5 82.27 (79.34 83.60) aa* 81.94 (80.53 83.26) aa 1 82.85 (81.97 83.24) aa 82.27 (81.65 82.86) ab 0.5 0.5 83.11 (82.40 83.53) aa# 82.45 (80.96 83.27) ab 1 82.75 (81.68 83.00) aa 82.70 (81.56 -83.28) aa values followed by the different upper case letter in the same column or by different lower case letter in the same row are statistically different (p < 0.05). * and # represents statistical difference for dpi. 7 andrade et al. water sorption and solubility table 5 shows the results of ws and s in µg/mm³. the addition of dpi had higher ws values in cements containing 1 mol% cq with 1 or 2 mol% edab. the edab concentration of 1 mol% produced lower values than 2 mol% edab when combined with 1 mol% cq with or without dpi. solubility did not differ statistically (p> 0.05). flexural strength and elastic modulus table 6 shows results for fs and e. the fs (mpa) and e (gpa) of the experimental resin cements tested did not differ statistically (p>0.05). discussion the present study investigated properties of experimental resin cements with different concentrations of edab, an aromatic tertiary amine, and cq, a photosensitizer molecule. although tertiary amines are prone to oxidation and can cause color changes, the yellowing effect can be also related to higher cq concentrations in photoactivated materials7,12. thus, ideal luting materials must have as low concentrations as possible of these molecules without compromising properties in the polymer network. table 6. means (standard deviations) of flexural strenght ( mpa) and elastic modulus (gpa). dpi (mol%) cq (mol%) flexural strenght elastic modulus edab (1 mol%) edab (2 mol%) edab (1 mol%) edab (2 mol%) 0 0.5 125.69 (17.98) 120.82 (23.21) 3.39 (0.33) 3.40 (0.28) 1 122.20 (9.88) 127.03 (22.98) 3.12 (0.28) 3.20 (0.27) 0.5 0.5 116.35 (17.26) 121.39 (18.36) 3.05 (0.35) 3.37 (0.24) 1 109.96 (18.97) 121.11 (15.12) 3.24 (0.40) 3.32 (0.25) values followed by the different upper case letter in the same column or by different lower case letter in the same row are statistically different (p < 0.05). * and # represents statistical difference for dpi. table 5. means (standard deviation) of water sorption and solubility (µg/mm³) dpi (mol%) cq (mol%) water sorption solubility edab (1 mol%) edab (2 mol%) edab (1 mol%) edab (2 mol%) 0 0.5 21.23 (4.70) aa 23.81 (4.12) aa -7.62 (2.61) aa -7.58 (2.32) aa 1 14.01 (2.21) bb* 20.95 (4.83) aa* -3.33 (3.19) aa -5.71 (6.21) aa 0.5 0.5 26.67 (3.10) aa 22.38 (4.32) ba -2.86 (3.10) aa -2.38 (4.76) aa 1 22.86 (3.19) ab# 29.39 (1.15) aa# -5.71 (3.19) aa -6.67 (2.61) aa values followed by the different upper case letter in the same column or by different lower case letter in the same row are statistically different (p < 0.05). * and # represents statistical difference for dpi. 8 andrade et al. dpi was added to this two-component system as a co-initiator forming a three-component photoinitiator system9. for dc, when the cq concentration was reduced from 1 to 0.5 mol%, it was expected that properties would be negatively affected. instead, in the cement containing 2 mol% edab, this was not observed on the dc when dpi was added, showing a positive effect of the salt even at lower cq concentrations. thus, the first hypothesis was partially rejected, because even with a higher concentration of components, not all properties were improved. because of the known effectiveness of generating free radicals, dpi must contribute to more reactive bonds in the polymer formation, resulting in a high value of conversion10,13. therefore, even at low concentrations, the initiator systems containing dpi were capable of efficiently polymerizing the experimental resins9,10. however, when dpi was added to 1 mol% cq, a decrease in dc was observed. generally, the necessary concentration of amine:cq is 2:1 or more in photoinitiator systems14. in the present study, the salt added to this system may have hindered the collision of molecules during the reaction because of its ionic nature and hydrophilic characteristics, which is in contrast to hydrophobic cq and edab. a lower amine concentration can also make the exciplex formation more difficult to form, which explains why cements containing 1 mol% edab showed lower values than 2 mol% edab, indicating that the tertiary amine concentration directly affects the dc13. adding dpi as a third component may enable a small amount of cq to be added into the model resin cement, and for this reason, the present study shows systems with 1 or 0.5 mol% cq. this partial removal of cq was intended to reduce the yellowing effect on coordinate b*. the coordinates l*a*b* were individually analyzed and the second hypothesis was rejected, because adding the salt influenced the values. adding dpi increased the l* values in cement with 0.5 mol% cq and 1 mol% edab, and this can be correlated to the color of the salt, which is a white powder. l* values (luminosity, from 0=black to 100=white) of around 80 indicate that all cements tested have a light color. dpi influenced coordinate a* in cement with 1 mol% cq and 1 mol% edab, decreasing the values. this influence was also demonstrated in another study that analyzed the composition with 0.5 mol% cq/1 mol% edab/0.5 mol% dpi, where values around −2 were also found15. considering the b* axis, all cements with 0.5 mol% cq were less yellow than 1 mol% cq. thus, the third hypothesis was accepted, because all formulations with a lower concentration of cq presented lower values of b* and, consequently, showed less yellowing effect. as is well known, cq is a yellow powder and its potential to yellow the material must be considered when it is added7. adding dpi was intended to compensate for the decrease in cq concentration. in the present study, dpi increased b* values only when combined with a 1:1 ratio of amine:cq. because of their double bonds, aromatic amines can react, creating higher energy states that could react with even oxygen or other aromatic groups, such as the salt radicals. these reactions may form color centers, as bigger conjugated systems in the polymer that lead to more visible light absorption in the blue region16. the dpi and edab reactions in these ways, or unreacted radicals, may be the cause of more yellowing in the cement containing the salt, which can also be related to the lower dc value 9 andrade et al. because of the 1:1 amine:cq ratio. however, in 2 mol% edab, this influence was not observed because higher amounts of amine may have contributed to a higher dc, which can be correlated to the photodecomposition of cq into colorless products, namely photobleaching17. bisgma/tegdma comonomer blends are capable of forming a heterogeneous polymeric structure with high cross-link density areas18. because of its structural characteristics, tegdma causes formation of less homogeneous copolymers. even in cross-link bond areas, microgel domains may be the result of free radical polymerization of dimethacrylates. these areas with unreacted monomers can present hydrogen bonds between >c=o and –o– of tegdma with –oh– groups of bisgma in the spaces between the chains formed19. these polar groups can explain how the process of sorption in the present study occurred, considering the water molecule as a polar structure that could easily be diffused in a polymer. another factor is that the ionic nature of dpi as a salt can lead the polymer to absorb more water13. the hydrophilic character of dpi molecules and the polar groups of the base comonomer blend may have contributed to allowing more water into the tested resin cements that contained dpi. the lower ws values in cement with 1 mol% edab was obtained with 1 mol% cq. despite the lower dc for 1 mol% edab, the combination with 1 mol% cq may have formed a more stable polymer cross-link structure, with less pendant polar groups, especially without the addition of hydrophilic dpi. ws is a poor predictor of dc because even in high cross-linked polymers, the process of water diffusion is highly dependent upon the chemistry of monomers20. in this study, all ws and s results were acceptable, considering that iso establishes ≤40 µg/mm3 and ≤7.5 µg/mm3, respectively, as the maximum values to accomplish the requirements. the present results are also consistent with those of previous studies demonstrating similar values for ws in other resin cements tested21. negative values in solubility may suggest an increase in the polymer mass because of the difficulty of removing water in the final process of the test22. in addition to the solubility test, flexural strength and elastic modulus showed that even with a variation in components, the polymer network achieved a similar behavior, showing no statistical differences in the results. the 60% filler mass fraction may have contributed to values that were higher than the 50 mpa reported in other studies23,24. this value is established as a minimum acceptable value for fs in type 1, class 2 materials, iso 4049/2009 requirements. the present study showed the influence of adding dpi and reducing cq in ternary initiator systems and its effects on the yellowing reduction and adequate property maintenance. thus, future investigations are needed to determine suitable photoactivation times and their relationship to the indirect restoration’s thickness, as well as the behavior of color properties as a function of time. within the limitations of this study, it can be concluded that the best formulation tested in this study was the cement with 0.5 mol% cq / 2 mol% edab / 0.5 mol% dpi, which provided an adequate dc and sorption with a reduced yellowing effect. additionally, the compensatory effect of dpi for reducing the cq concentration is more effective with 2 mol% edab. 10 andrade et al. acknowledgments this work was supported by amazonas research foundation (fapeam) – grant number 005/2012 – decision 269/2012. references 1. hoorizad ganjkar m, heshmat h, hassan ahangari r. evaluation of the effect of porcelain laminate thickness on degree of conversion of light cure and dual cure resin cements using ftir. j dent (shiraz). 2017 mar;18(1):30-6. 2. jang y, ferracane jl, pfeifer cs, park jw, shin y, roh bd. effect of insufficient light exposure on polymerization kinetics of conventional and self-adhesive dual-cure resin cements. oper dent. 2017 jan/feb;42(1):e1-e9. doi: 10.2341/15-278-l. 3. archegas lr, freire a, vieira s, caldas db, souza em. colour stability and opacity of resin cements and flowable composites for ceramic veneer luting after accelerated ageing. j dent. 2011 nov;39(11):804-10. doi: 10.1016/j.jdent.2011.08.013. 4. stansbury jw. curing dental resins and composites by photopolymerization. j esthet dent. 2000;12(6):300-8. 5. ilie n, hickel r. can cq be completely replaced by alternative initiators in dental adhesives? dent mater j. 2008 mar;27(2):221-8. 6. brandt wc, tomaselli l de o, correr-sobrinho l, sinhoreti ma. can phenyl-propanedione influence knoop hardness, rate of polymerization and bond strength of resin composite restorations? j dent. 2011 jun;39(6):438-47. doi: 10.1016/j.jdent.2011.03.009. 7. de oliveira dc, rocha mg, gatti a, correr ab, ferracane jl, sinhoreti ma. effect of different photoinitiators and reducing agents on cure efficiency and color stability of resin-based composites using different led wavelengths. j dent. 2015 dec;43(12):1565-72. doi: 10.1016/j.jdent.2015.08.015. 8. emami n, söderholm kj. influence of light-curing procedures and photo-initiator/co-initiator composition on the degree of conversion of light-curing resins. j mater sci mater med. 2005 jan;16(1):47-52. 9. gonçalves ls, morães rr, ogliari fa, boaro l, braga rr, consani s. improved polymerization efficiency of methacrylate-based cements containing an iodonium salt. dent mater. 2013 dec;29(12):1251-5. doi: 10.1016/j.dental.2013.09.010. 10. ogliari fa, ely c, petzhold cl, demarco ff, piva e. onium salt improves the polymerization kinetics in an experimental dental adhesive resin. j dent. 2007 jul;35(7):583-7. 11. crivello jv, lam jh. diaryliodonium salts – new class of photo-initiators for cationic polymerization. macromolecules. 1977 nov;10(6):1307-15. 12. schneider lf, cavalcante lm, consani s, ferracane jl. effect of co-initiator ratio on the polymer properties of experimental resin composites formulated with camphorquinone and phenyl-propanedione. dent mater. 2009 mar;25(3):369-75. doi: 10.1016/j.dental.2008.08.003. 13. guo x, wang y, spencer p, ye q, yao x. effects of water content and initiator composition on photopolymerization of a model bisgma/hema resin. dent mater. 2008 jun; 24(6):824-31. 14. andrade km, palialol ar, lancellotti ac, aguiar fh, watts dc, gonçalves ls, et al. effect of diphenyliodonium hexafluorphosphate on resin cements containing different concentrations of ethyl 4-(dimethylamino)benzoate and 2-(dimethylamino)ethyl methacrylate as co-initiators. dent mater. 2016 jun;32(6):749-55. doi: 10.1016/j.dental.2016.03.014. 11 andrade et al. 15. albuquerque pp, bertolo ml, cavalcante lm, pfeifer c, schneider lf. degree of conversion, depth of cure, and color stability of experimental dental composite formulated with camphorquinone and phenanthrenequinone photoinitiators. j esthet restor dent. 2015 mar-apr;27 suppl 1:s49-57. doi: 10.1111/jerd.12131. 16. furuse ay, mondelli j, watts dc. network structures of bis-ma/tegdma resins differ in dc, shrinkage-strain, hardness and optical properties as a function of reducing agent. dent mater. 2011 may;27(5):497-506. doi: 10.1016/j.dental.2011.02.007. 17. asmusen s, arenas g, cook wd, vallo c. photobleaching of camphorquinone during polymerization of dimethacrylate-based resins. dent mater. 2009 dec;25(12):1603-11. doi: 10.1016/j. dental.2009.08.010. 18. ferracane jl. resin composite--state of the art. dent mater. 2011 jan;27(1):29-38. doi: 10.1016/j.dental.2010.10.020. 19. sideridou i, tserki v, papanastasiou g. study of water sorption, solubility and modulus of elasticity of light-cured dimethacrylate-based dental resins. biomaterials. 2003 feb;24(4):655-65. 20. ferracane jl. hygroscopic and hydrolytic effects in dental polymer networks. dent mater. 2006 mar;22(3):211-22. 21. mese a, burrow mf, tyas mj. sorption and solubility of luting cements in different solutions. dent mater j. 2008 sep;27(5):702-9. 22. janda r, roulet jf, latta m, rüttermann s. water sorption and solubility of contemporary resin-based filling materials. j biomed mater res b appl biomater. 2007 aug;82(2):545-51. 23. kim kh, ong jl, okuno o. the effect of filler loading and morphology on the mechanical properties of contemporary composites. j prosthet dent. 2002 jun;87(6):642-9. 24. gonçalves f, azevedo cl, ferracane jl, braga rr. bisgma/tegdma ratio and filler content effects on shrinkage stress. dent mater. 2011 jun;27(6):520-6. doi: 10.1016/j.dental.2011.01.007. untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8651900 volume 17 2018 e18019 original article 1 community dentistry department, school of dentistry, university of são paulo, avenida professor lineu prestes, 2227, são paulo 05508-000, brazil. 2 center of anatomy and human identification, university of dundee, dow street, dundee 05508-000, scotland. 3 liverpool school of art and design, liverpool john moores university, 2 duckinfield st, liverpool l3 5rd, united kingdom. 4 institute of teaching and research in forensic sciences, rua maria de castro mesquita, 203 sala 03, guarulhos 07110-040, brazil. 5 community dentistry department, school of dentistry, university of são paulo, avenida professor lineu prestes, 2227, são paulo 05508-000, brazil. 6 community dentistry department, school of dentistry, university of são paulo avenida professor lineu prestes, 2227, são paulo 05508-000, brazil. corresponding author: maria gabriela haye biazevic, phd av. professor lineu prestes, 2227 são paulo-sp, brazil phone: +55 11 3091 7891 e-mail:biazevic@usp.br received: september 05, 2017 accepted: january 25, 2018 craniometric variation among brazilian and scottish populations: a physical anthropology approach thais torralbo lopez-capp1, christopher rynn2, caroline wilkinson3, luiz airton saavedra de paiva4, edgard michel-crosato5, maria gabriela haye biazevic6* aim: the present investigation intended to compare the craniometric variations of two samples of different nationalities (brazilian and scottish). materials and methods: the brazilian sample consisted of 100 modern complete skulls, including 53 female skulls and 47 male skulls, and the scottish sample consisted of 100 historical skulls (61 males, 39 females) and 36 mandibles (24 males, 12 females). the cranial measurement protocol was composed of 40 measurements, 11 bilateral and 29 unilateral, and the measurement protocol of the mandible was composed of 15 measurements, with six that were bilateral and nine that were unique. the comparative analysis of the metric variability between the two samples was performed using the means and medians analysis, the t-test, the wilcoxon test, and the coefficient of variance, with a significance level of 5%. results: the results showed that, among the 72 analysed variables, 44 measurements (61.11%) presented statistical differences between the samples. the scottish skull tends to have a cranial length (gol diff=5.53), breadth (xcb diff=3.78) and height (nph diff=5.33) greater than the brazilian skulls, and the scottish mandibles tend to show a higher mandibular ramus height (mrh diff=9.25), a higher mandibular body height (hmb diff=6.37) and a larger bigonial breadth (bgb diff=5.29) than the brazilians. the discriminant analysis of the 51 cranial measurements and 21 mandibular measurements showed a variation of the percentage of accuracy between 46.383.8%. conclusion: the metric analysis demonstrated that there is variability between the two samples studied (61.11%), but a concrete cause cannot be determined considering the multifactorial aspects of the variations of form and size. keywords: forensic sciences. craniometry. forensic anthropology. skull. forensic dentistry. 2 lopez-capp et al. introduction the existence of craniometric variability among the most diverse populations is well established in the scientific literature1-7, and the development of analytical standards specific to each geographic location is of paramount importance. the factors associated with changes in the shape and size of the skull can be categorized as intrinsic (genetic factors) or extrinsic (factors related to environment) factors3. inter-population and intra-population craniometric variations can be estimated based on the proportion of these two types of factors. in other words, the morphological variations of the human skull are the result not of the influence of a single factor, but rather of an association among factors. therefore, studies of human craniometric variability should be performed using a multifactorial approach. among the various factors that are associated with craniometric variability, age, climate and the human evolutionary process should be highlighted. bone remodelling is a continuous process throughout human life. certain bone alterations can be observed in the fundamental planes, such as increases in the maximum length of the skull, the bizygomatic width, and the maximum width of the skull. these changes occur during adulthood, which is between 20 and 80 years of age8. climate can be considered as a factor of great impact in regional and global craniometric variations, mainly in extreme climatic regions, including regions with extremely low or high temperatures9. the cranial modifications associated with temperature are the result of adaptive characteristics that human beings possess. the skulls of individuals living in cold and dry climates have a tendency to be wider compared to skulls from regions with high temperatures, which tend to be narrower and elongated9-11. the cranial structure that has the greatest climate-related plasticity is the nasal region. this fact can be explained by the adaptation of nasal structures over time relative to survival according to local climatic changes9,12. the morphology of the nasal cavity is extremely important to establishing the dynamics of airflow, so the dimensions of this structure are directly related to the airflow during the inspiratory and expiratory processes. in addition, the variability of the nasal region is associated with humidity and latitude. in analyses of the morphology of the nasal cavities, because of the adaptive processes relative to the climate, individuals who live in cold and dry regions tend to have higher and narrower cavities compared to individuals who live in hot and humid regions. in addition, in cold and dry regions, the nasal cavity tends to be deeper, increasing the contact area with the mucosa to optimize the air-heating process12-14. in brazil, beyond the diversity of demographic and ethnological conformations, the climate is also considered heterogeneous. this climatic variability may be attributed to several factors: the great territorial extension, the geographical physiognomy, and the relief, and the dynamics of air masses can characterize this diversity. in contrast, the scottish climate tends to offer smaller variations, and, similar to its demographic characteristics, it is more homogenous in all its territorial extension. when analysing the climatic characteristics of the two countries, the variation is evident. brazil is characterized by higher average temperatures compared to scotland. the brazilian demographic, ethnological and climate scenarios are more heterogeneous than scotland’s, which tend towards homogeneity. 3 lopez-capp et al. human evolution can also be considered a factor of great impact in the morphometric alterations of the skull. the craniometric variabilities occurring over time between generations can be categorized into two classes: short-term changes and long-term changes3. short-term changes, or secular trends, are variations occurring between two or three generations due to several environmental factors, such as industrialization, urbanization, migratory processes, nutritional factors and socioeconomic level. thus, the variations that have occurred and the intensity of these variations are specific for each population and do not follow a universal parameter. studies around the world have shown that several modifications of the skull have occurred over time1-5,15-23. the human skull and mandible plasticity are correlated with the processes of structural and functional adaptations occurring over time, resulting in the heterogeneity of cranial and mandibular morphology among populations around the world. consequently, the levels of accuracy and reliability of anthropological techniques are highly sensitive to the population type because the level of biological data varies significantly between populations. therefore, changes that seem to be appropriate for one population may not be appropriate for another. cranial plasticity and mandibular plasticity are factors of great importance to forensic anthropology. the study of diverse populations is essential for the development of references to help in the establishment of biological profiles. cranial and mandibular variability exists in intraand inter-populational spheres as a result of multifactorial causes that should be studied together. several studies have been published with the common objectives of explaining the morphological and metric variations of the human skull and mandible that occur in the interand intra-populational spheres and correlating these variations with regional demographic characteristics24-26. the present study aimed to evaluate the craniometric variations between two samples of different nationalities (brazilian and scottish). materials and methods the convenience sample consisted of 200 skulls and 136 mandibles of 114 males and 86 females, all catalogued with records regarding age, ancestry and sex. the brazilian sample consisted of 100 complete skulls (skulls and mandibles from the same skeleton), with 53 female and 47 male, from a 20th century collection that belongs to the institute of teaching and research in forensic sciences (instituto de ensino e pesquisa em ciências forenses, iepcf). the scottish sample consisted of 100 skulls (61 males, 39 females) and 36 mandibles (24 males, 12 females) from the anatomical museum of the university of edinburgh and from the center for anatomy and human identification at the university of dundee. the scottish skulls and mandibles were collected by anatomy professors during the 18th and 19th centuries to teach anatomy, anthropology, and comparative anatomy, and they represent a comprehensive range of human structure from across the world. the inclusion criteria adopted for this research were the absence of extensive fractures and skulls and mandibles belonging to individuals older than 18 years. the exclusion criteria were trauma and extensive fractures (table 1). the availability of osteological documented materials is limited, because of that the convenience sampling was the method of choice in the present study. the sample power were calculated using the g*power 3.1.9.2 software. the cranium sample power obtained was 0.96, with an effect size mean of 0.50, a significance level of 5%, a critical t of 1.65 and using post hoc analysis. the mandible sample power obtained was 0.99, with an effect size mean of 0.84, a significance level of 5%, a critical t of 1.97 and using post hoc analysis. 4 lopez-capp et al. the application of the measurement protocol had, as a reference, 34 craniometric landmarks, 15 odd points in the median sagittal plane and 19 even points (pl) located in the lateral planes (table 2). the cranial measurement protocol was composed of 40 measurements, including 11 bilateral and 29 unique variables (table 3). the measurements were grouped into five categories according to their anatomical location: superior cranial measurements, anterior cranial measurements, lateral cranial measurements, posterior cranial measurements and inferior cranial measurements. the measurement protocol for the mandible was composed of 15 measurements: six that were bilateral and nine that were unique (table 4). the measurement protocol was applied to the two samples following the same parameters. two fundamental plans were used as a reference for the standardization and alignment of the skulls: the median sagittal plane and the frankfurt horizontal plane. for the mandible analysis, the mandibular plane and the median sagittal plane were adopted as fundamental plans for the standardization of the protocol. measurements were performed with a digital calliper (lee tools, houston, texas, usa) with a minimum measurement of 0.01 mm, a maximum measurement of 150 mm, and a resolution of 0.01 mm. measurements that were not measurable with the digital calliper were taken using a curved compass, a protractor, and a compass (tables 3 and 4). in addition to the metrology equipment described above, two stabilizing devices, namely, a skull stabilizer (fig. 1) and a mandible stabilizer (fig. 2), were used in this research to standardize the measurement protocol by aligning the skull and mandible relative to the fundamental planes of the human body. the skull stabilizer patent was registered in 2012 under number p.i. 1,103,246-4, and the mandible stabilizer patent was requested from the national institute of industrial property in brazil (inpi), br 10 2013 003270-0. the data obtained in the craniometric analysis were registered using the excel program (microsoft office®) to generate an organized spreadsheet of values. statistical analysis was performed with spss 22.0, stata 13.0 and medcalc, with a significance level of 5%. initially, it was performed the skewness/kurtosis normality test to verify the distribution of the data, referring to the measurements made in the brazilian and scottish samples. among the analysed variables, the brazilian sample presented 59 variables within the normality curve and 13 variables with a non-normal distribution. the scottish sample presented 50 variables with a normal distribution and 22 variables with a non-normal distribution. therefore, a parametric test (t-test) and a non-parametric test (mann-whitney) test were implemented for all variables in addition to analyses of means, medians and the coefficient of variation. in order to verify the difference between both skull sample, it was applied discriminant analysis. this investigation was conducted in accordance with the international and national parameters for ethical investigations involving human beings, and the investigation protocol was submitted to and approved by the ethics committee of the university of são paulo’s school of dentistry (fousp), process number 1.556.080. table 1. description of the brazilian and scottish samples. colection skulls mandibles male female total male female total brazil 53 47 100 53 47 100 scotland 61 39 100 24 12 36 5 lopez-capp et al. table 2. definition of the landmarks. landmark abbreviation definition alare al instrumentally determined as the most lateral points on the nasal aperture in a transverse plane. alveolon alv the point where the mid-sagittal plane of the palate is intersected by a line connecting the posterior borders of the alveolar crests. asterion ast the point where the temporal, parietal, and occipital bones meet. bregma b the posterior border of the frontal bone in the midsagittal plane. basion ba the point at which the anterior border of the foramen magnum is intersected by the mid-sagittal plane. condylion laterale cdl the most lateral points of the mandibular condyles. dacryon d the point on the frontal bone where the frontal, lacrimal and maxillary sutures meet. ectoconchion ec the intersection of the anterior edge of the lateral orbital border and a line parallel to the superior orbital border that bisects the orbit into two equal halves. ectomolare ecm the most lateral point on the buccal surface of the alveolar process at the level of the second molar. endomolare enm the point on the lingual surface of the alveolar process at the level of the second molar. euryon eu the most laterally positioned point on the side of the braincase. frontomalare temporale fmt the most laterally positioned point on the fronto-malar suture. frontotemporale ft the point located generally forward and inward on the superior temporal line directly above the zygomatic process of the frontal bone. glabella g the most anteriorly projecting point in the mid-sagittal plane at the lower margin of the frontal bone, which lies above the nasal root and between the superciliary arches. gnathion gn the lowest point on the inferior margin of the mandibular body in the midsagittal plane. gonion go the point on the mandible where the inferior margin of the mandibular corpus and the posterior margin of the ramus meet. infradentale id the point between the lower incisor teeth where the anterior margins of the alveolar processes are intersected by the mid-sagittal plane. inion i the point at the junction of the upper nuchal lines with the mid-sagittal plane. lambda l the apex of the occipital bone at its junction with the parietals, in the midline. mastoideale ms the most inferior point on the tip of the mastoid process. maxillofrontale mf the point where the anterior lacrimal crest (on the medial border of the orbit) and frontolacrimal suture intersect. mentale ml the inferior point of the mental foramen. nasion n the point of intersection of the naso-frontal suture and the midsagittal plane. nasospinale ns the point where a line drawn between the inferiormost points of the nasal aperture crosses the midsagittal plane. opisthocranion op the most distant point posteriorly from glabella on the occipital bone, located in the mid-sagittal plane. opisthion o the point on the inner border of the posterior margin of the foramen magnum in the mid-sagittal plane. orale ol the most anterior point of hard palate where a line drawn lingual to the central incisors intersects the palatal suture. pogonion pg the most prominent point in the mental protuberance at the mandibular symphysis. porion po the most superior point along the upper margin of the external acoustic meatus. prosthion pr the most anterior point on the alveolar border of the maxilla between the central incisors in the mid-sagittal plane. radiculare ra the point located in the deepest curvature of the root of the zygomatic process at the temporal bone in a lateral view. staphylion sta the midpoint on the tangent line to the posterior concavities of the hard palate. zygion zy the most laterally positioned point on the zygomatic arches. zygomaxillare anterior zma the intersection of the zygomaxillary suture and the limit of the attachment of the masseter muscle. zygoorbitale zo the intersection of the orbital margin and the zygomaxillary suture. 6 lopez-capp et al. table 3. definition of the cranial measurements. measure abbreviation definition superior cranial measures frontal anglec fra the angle formed by underlying the frontal curvature at its maximum height and above the front cord at mid sagittal plane. maximum cranial lengthb gol linear distance from glabella (g) to opisthocranion(op) in the mid-sagittal plane. maximum cranial breadthb xcb linear distance between right and left euryon (eu). basion-bregma heightb bbh linear distance from basion (ba) to bregma (b). cranial base lengthb bnl linear distance from basion (ba) to nasion (n). basion-prosthion lengthb bpl linear distance from basion (ba) to prosthion (pr). frontal chorda frc linear distance from nasion (n) to bregma (b). parietal chorda pac linear distance from bregma (b) to lambda (l). anterior cranial measures upper facial breadtha ufb linear distance between right and left frontomalare temporale (fmt). upper facial heighta nph linear distance from nasion (n) to prosthion (pr). minimun frontal breadtha wfb linear distance between right and left frontotemporale (ft). orbital breadtha obb linear distance from dacryon (d) to ectoconchion (ec). orbital heighta obh linear distance from the superior orbital border to the inferior orbital border while perpendicular to the natural horizontal axis of the orbit. zygoorbitale breadtha zob linear distance between right and left zygoorbitale (zo). interorbital breadtha dkb linear distance between right and left dacryon (d). biorbital breadtha ekb linear distance between right and left ectoconchion (ec). frontal interorbital breadtha iob linear distance between right and left maxillofrontale (mf). nasal heighta nlh linear disatnce from nasion (n) to nasospinale (ns). nasal breadtha nlb linear distance between right and left alare (al). bizygomatica breadtha zyb linear distance between right and left zygion (zy). bimaxillary breadtha zmb linear distance between right and left zygomaxillare anterior (zma). lateral cranial measures minimum vertical archa iml linear distance from frontomalare temporale (fmt) to zygomaxillare anterior (zma). malar length, maximuma xml linear distance from zygoorbitale (zo) to the most inferior lateral point of the zygomaticotemporal suture. zygoorbitale-porion lengtha zpl linear distance from zygoorbitale (zo) to porion (po). asterion-porion lengtha apl linear distance from asterion (ast) to porion (po). porion-mastoidale lengtha pml linear distance from porion (po) to mastoideale (ms). asterion-mastoidale lengtha aml linear distance from mastoideale (ms) to asterion (ast). mastoid lengtha mdh vertical projection of the mastoid process below and perpendicular to the frankfurt plane posterior cranial measures biauricular breadtha aub linear distance between right and left radiculare (ra). biasterion breadtha asb linear distance between right and left asterion (ast). occiptal chorda occ linear disnatce from lambda (l) to opisthion (o). lambda-inion chorda lic linear distance from lambda (l) to inion (i). inferior cranial measures maximum length of occipital condylea mlc maximum linear distance from the length of the occipital condyle. maximun width of occipital condylea mwc maximum linear distance from the width of the occipital condyle. foramen magnum lengtha fol linear distance from basion (ba) to opisthion (o). foramen magnum breadtha fob distance between the lateral margins of the foramen magnum at the point of greatest lateral curvature. palatal breadtha pab linear distance between right and left endomolare (enm). palatal lengtha pal linear distance from orale(ol) to staphylion (sta). maxillo-alveolar breadtha mab linear distance between right and left ectomolare (ecm). maxillo-alveolar lengtha mal linear distance from prosthion (pr) to alveolon (alv). a-digital caliper b-curved compass c-protractor d-compass 7 lopez-capp et al. table 4. definition of the mandibular measurements. measure abbreviation definition mandibular cranial measures chin heighta chh linear distance from infradentale (id) to gnathion (gn). body height at mental foramena hmb distance from the alveolar process to the inferior border of the mandible at the level of the mental foramen. body thickness at mental foramena bmb maximum breadth at the level of the mental foramen and perpendicular to the long axis of the mandibular body. bimentale lengtha bml linear distance between right and left mentale (ml). bicoronoid breadtha bcb distance between the highest points of the mandibular coronoid processes. bicondylar breadtha cdb linear distance between right and left condylion laterale (cdl). mandibular notch breadtha mnb distance between the superior point of the condylar process and the superior point of the coronoid process. minimum ramus breadtha mrb the minimum breadth of the mandibular ramus measured perpendicular to the height of the ramus. maximum ramus breadtha marb the maximum breadth of the mandibular ramus measured perpendicular to the height of the ramus. maximum ramus heightc mrh the distance from gonion (go) to the highest point on the mandibular condyle. maximum mandibular lengtha mlt the distance from the anterior margin of the chin to the midpoint of a straight line extending from right gonion (go) and left gonion (go). bigonial breadtha bgb linear distance between right and left gonion (go). mandibular length (projection)a mlp distance between pogonion (pg) and the perpendicular line that tangent the posterior part of the condylar processes. mandibular angleb ma the angle formed by inferior border of the body and the posterior border of the ramus. mandibular notch deptha mnd distance between the inferior point of the mandibular notch and the midpoint of a straight line extending from the superior point of the condylar process and the superior point of the coronoid process. a digital caliper b protractor c compass figure 1. skull stabilizer 8 lopez-capp et al. results table 5 shows the results of the comparative analysis of the metric variability between the two (brazilian and scottish) samples. among the superior cranial measurements, only the cranial base length (bnl), the basion-prosthion length (bpl) and the frontal cord (frc) showed no significant differences between the two samples. variations of the standard deviation relative to the mean correlating the two samples ranged from 4.02% to 7.55%. among the anterior cranial measurements, seven variables showed differences between the two samples: upper facial height (nph), right and left orbital breadths (obbd and obbe), zygoorbital breadth (zob), interorbital breadth (dkb), nasal height (nlh) and nasal breadth (nlb). the measurements that showed the greatest variation among the samples were the frontal interorbital breadth (iob) (16.57%), interorbital breadth (dkb) (13.21%), upper facial height (nph) (12.31%) and zygoorbital breadth (zob) (12.24%). among the lateral cranial measurements, only the left asterion-porion length (aple) did not present a difference between the groups. all measurements showed between-sample variations greater than 8%. none of the variables related to the posterior cranial measurements presented a significant difference between the two samples. the lambda-inion chord (lic) presented a coefficient of variation of 12.38%. among the inferior cranial measurements, only two measurements had no significant differences, that is, the palatal length (pal) and maximum alveolar breadth (mab). all variables presented a coefficient of variation greater than 10%. among the mandibular measurements, 11 variables showed metric variability between the two samples: the right and left body heights (hmbd and hmbe), right and left body thicknesses (bmbd and bmbe), maximum ramus breadth (marb), right and left maximum ramus heights (mrhd), bigonial breadth width (bgb) and right mandibular angle (mad). among the 21 mandibular measurements analysed, five presented variations greater than 20%, nine had variations between 10% and 20%, and seven showed variations less than 10% (table 6). figure 2. mandible stabilizer 9 lopez-capp et al. table 5. the skewness/kurtosis normality test results. measurea brazil scotland measurea brazil scotland p-value p-value p-value p-value superior cranial measures posterior cranial measures fra 0.0046* 0.2352 aub 0.5402 0.0014* gol 0.8136 0.0000* asb 0.0144* 0.0017* xcb 0.7888 0.2164 occ 0.7738 0.9024 bbh 0.2885 0.0000* lic 0.2434 0.0016* bnl 0.7538 0.3458 inferior cranial measures bpl 0.0743 0.3378 mlcd 0.4055 0.4620 frc 0.3680 0.0000* mlce 0.6565 0.0143* pac 0.0970 0.0003* mwcd 0.3146 0.0000* anterior cranial measures mwce 0.0000* 0.0215 ufb 0.9925 0.2507 fol 0.2402 0.0000* nph 0.6302 0.0003* fob 0.9731 0.5406 wfb 0.9404 0.0000* pab 0.0000* 0.0000* obbd 0.4075 0.0000* pal 0.0602 0.9097 obbe 0.1552 0.0205* mab 0.3913 0.0717 obhd 0.1876 0.0000* mal 0.0492 0.6620 obhe 0.1093 0.0133* mandibular measures zob 0.0000* 0.2906 chh 0.0415* 0.0826 dkb 0.7499 0.0000* hmbd 0.0602 0.7354 ekb 0.3861 0.1126 hmbe 0.0659 0.7576 iob 0.9027 0.4545 bmbd 0.0055* 0.5928 nlh 0.4226 0.4533 bmbe 0.0144* 0.1217 nlb 0.9659 0.2684 bml 0.7473 0.457 zyb 0.8849 0.2346 bcb 0.1793 0.9521 zmb 0.0000* 0.1148 cdb 0.8366 0.936 lateral cranial measures mnbd 0.9996 0.1302 imld 0.0003* 0.0050* mnbe 0.0000* 0.0466* imle 0.0067* 0.1215 mrb 0.3452 0.0652 xmld 0.1744 0.3591 marb 0.3468 0.7064 xmle 0.5320 0.2040 mrhd 0.9736 0.9189 zpld 0.3208 0.8227 mrhe 0.2615 0.4299 zple 0.1526 0.8246 mal 0.7017 0.2948 apld 0.0001* 0.1933 bgb 0.417 0.6962 aple 0.6170 0.3122 mlp 0.1982 0.0000* pmld 0.9409 0.1200 mad 0.4003 0.0525 pmle 0.5500 0.1280 mae 0.6839 0.1464 amld 0.5801 0.0020* mndd 0.9123 0.7644 amle 0.1491 0.1401 mnde 0.3465 0.1915 mdhd 0.6471 0.9907       mdhe 0.3776 0.4371       * non-normal distribution 10 lopez-capp et al. ta b le 6 . c o m p a ra ti v e a n a ly s is o f th e m e tr ic v a ri a b il it y b e tw e e n t h e b ra z il ia n a n d s c o tt is h s a m p le s . m ea su re a b ra zi l s co tl an d t t es t t p -v al ue w ilc o xo n p -v al ue v c b m ed ia n m ea n s d c 9 5 % c if m ed ia n m ea n s d c 9 5 % c if u pp er c ra ni al m ea su re s f r a 9 6 .0 0 9 6 .4 3 2 .6 7 9 5 .9 0 9 6 .9 6 1 0 0 .0 0 9 9 .8 8 3 .7 9 9 9 .1 2 1 0 0 .6 4 -7 .7 6 0 .0 0 0 0 * 0 .0 0 0 0 * 4 .0 2 g o l 1 7 6 .0 0 1 7 6 .6 3 8 .0 7 1 7 5 .0 2 1 7 8 .2 4 1 8 4 .0 0 1 8 2 .1 6 1 0 .5 8 1 8 0 .0 5 1 8 4 .2 7 -4 .1 1 0 .0 0 0 1 * 0 .0 0 0 0 * 5 .6 8 x c b 1 3 3 .0 0 1 3 3 .5 6 7 .7 1 1 3 2 .0 2 1 3 5 .0 9 1 3 7 .0 0 1 3 7 .3 4 6 .3 1 1 3 6 .0 8 1 3 8 .6 -3 .8 1 0 .0 0 0 2 * 0 .0 0 0 4 * 5 .5 0 b b h 1 3 1 .0 0 1 3 1 .3 8 8 .0 3 1 2 9 .7 5 1 3 3 .0 0 1 2 9 .0 0 1 2 8 .5 6 8 .1 2 1 2 6 .9 1 1 3 0 .2 2 .5 4 0 .0 1 2 6 * 0 .0 1 1 4 * 6 .0 7 b n l 9 8 .0 0 9 7 .9 7 5 .6 7 9 6 .8 2 9 9 .1 2 9 9 .0 0 9 8 .3 4 5 .5 5 9 7 .2 2 9 9 .4 7 -0 .4 9 0 .6 2 4 3 0 .6 2 2 9 5 .3 6 b p l 9 1 .2 9 9 3 .1 6 6 .4 9 9 1 .6 0 9 4 .7 2 9 3 .6 9 9 2 .8 4 6 .6 2 9 1 .4 6 9 4 .2 2 0 .3 0 .7 6 2 5 0 .9 8 7 4 7 .5 5 f r c 1 1 0 .2 4 1 1 0 .3 7 5 .1 4 1 0 9 .3 2 1 1 1 .4 1 1 1 1 .5 3 1 1 1 .3 2 6 .4 1 1 1 0 .0 1 1 1 2 .6 2 -1 .1 2 0 .2 6 2 7 0 .2 9 5 2 5 .2 5 p a c 1 1 0 .8 8 1 1 0 .7 3 7 .1 5 1 0 9 .3 1 1 1 2 .1 5 1 1 2 .5 9 1 1 3 .0 3 7 .0 7 1 1 1 .6 2 1 1 4 .4 3 -2 .4 7 0 .0 1 5 0 * 0 .0 1 5 5 * 6 .0 0 fr on ta l c ra ni al m ea su re s u f b 1 0 2 .6 3 1 0 2 .8 1 4 .5 7 1 0 1 .8 9 1 0 3 .7 3 1 0 3 .0 2 1 0 2 .9 6 4 .4 3 1 0 2 .0 7 1 0 3 .8 5 -0 .2 2 0 .8 2 0 4 0 .9 6 4 7 4 .4 3 n p h 6 3 .4 0 6 3 .2 6 6 .3 8 6 1 .9 6 6 4 .5 6 6 9 .7 4 6 8 .5 9 7 .6 7 6 7 .0 3 7 0 .1 6 -5 .0 8 0 .0 0 0 0 * 0 .0 0 0 0 * 1 2 .3 w f b 9 7 .3 3 9 7 .4 1 4 .6 7 9 6 .4 8 9 8 .3 3 9 6 .2 5 9 5 .3 5 9 .7 4 9 3 .4 1 9 7 .2 8 1 .9 2 0 .0 5 7 0 0 .0 9 4 7 7 .9 5 o b b d 3 9 .2 4 3 9 .3 3 2 .1 4 3 8 .8 9 3 9 .7 0 4 0 .6 6 4 0 .6 8 2 .7 8 4 0 .1 2 4 1 .2 4 -4 .0 5 0 .0 0 0 1 * 0 .0 0 0 0 * 6 .2 2 o b b e 3 8 .9 4 3 8 .9 8 1 .9 0 3 8 .6 0 3 9 .3 7 4 0 .6 2 4 0 .5 5 2 .2 5 4 0 .0 9 4 1 .0 0 -5 .1 8 0 .0 0 0 0 * 0 .0 0 0 0 * 5 .9 3 o b h d 3 4 .2 5 3 4 .1 0 2 .5 7 3 3 .5 8 3 4 .6 2 3 3 .9 5 3 4 .3 8 3 .4 2 3 3 .6 9 3 5 .0 6 -0 .6 4 0 .5 2 0 0 0 .5 0 4 2 8 .7 7 o b h e 3 4 .0 3 3 4 .5 0 2 .5 1 3 3 .9 9 3 5 .0 1 3 4 .7 1 3 4 .7 5 2 .6 5 3 4 .2 1 3 5 .2 8 -0 .6 4 0 .5 1 9 0 0 .3 8 4 9 7 .6 3 z o b 5 6 .6 0 5 6 .7 9 6 .5 5 5 5 .4 4 5 8 .1 4 5 1 .6 8 5 1 .9 9 5 .0 9 5 0 .9 4 5 3 .0 4 5 .6 8 0 .0 0 0 0 * 0 .0 0 0 0 * 1 2 .2 a se e ta bl e 3 fo r m ea su re m en ts d efi ni tio n b s d = s ta n d a rd d e v ia ti o n c c i= co nfi de nc e in te rv al o f 9 5% d c v =c oe ffi ci en t o f v ar ia tio n 11 lopez-capp et al. ta b le 6 . c o m p a ra ti v e a n a ly s is o f th e m e tr ic v a ri a b il it y b e tw e e n t h e b ra z il ia n a n d s c o tt is h s a m p le s . c o n ti n u a ti o n d k b 2 0 .8 6 2 0 .9 4 2 .5 7 2 0 .4 3 2 1 .4 5 2 2 .1 7 2 2 .5 1 2 .5 8 2 2 .0 0 2 3 .0 3 -4 .1 5 0 .0 0 0 1 * 0 .0 0 0 1 * 1 3 .2 e k b 9 7 .0 9 9 6 .7 9 4 .1 4 9 5 .9 4 9 7 .6 4 9 7 .1 4 9 6 .8 5 4 .3 4 9 5 .9 6 9 7 .7 4 -0 .0 9 0 .9 2 3 8 0 .7 2 0 2 4 .5 6 io b 1 2 .4 7 1 2 .5 6 2 .3 9 1 2 .0 7 1 3 .0 4 1 2 .6 6 1 2 .7 9 2 .0 0 1 2 .3 8 1 3 .1 9 -0 .7 6 0 .4 4 3 5 0 .4 0 1 8 1 6 .6 n l h 4 9 .3 3 4 9 .2 9 3 .6 2 4 8 .5 5 5 0 .0 3 5 1 .5 0 5 1 .7 3 3 .7 0 5 0 .9 8 5 2 .4 9 -4 .8 4 0 .0 0 0 0 * 0 .0 0 0 0 * 7 .6 4 n l b 2 4 .3 1 2 4 .4 2 2 .1 5 2 3 .9 8 2 4 .8 6 2 3 .2 8 2 3 .5 1 1 .9 5 2 3 .1 1 2 3 .9 1 3 .4 0 .0 0 1 0 * 0 .0 0 1 3 * 8 .1 1 z y b 1 2 4 .0 0 1 2 4 .4 1 6 .3 0 1 2 2 .9 1 1 2 5 .9 1 1 2 7 .5 5 1 2 6 .5 7 7 .5 0 1 2 4 .7 8 1 2 8 .3 6 -1 .8 4 0 .0 6 8 6 0 .0 6 0 7 5 .6 z m b 8 9 .4 0 8 9 .4 0 6 .0 6 8 8 .1 6 9 0 .6 4 9 0 .8 0 9 0 .1 7 5 .5 1 8 9 .0 4 9 1 .3 0 -0 .9 1 0 .3 6 1 1 0 .3 8 1 7 6 .4 3 la te ra l c ra ni al m ea su re s im l d 4 4 .8 5 4 4 .9 8 4 .2 3 4 4 .1 1 4 5 .8 4 4 7 .7 4 4 7 .7 7 3 .5 3 4 7 .0 5 4 8 .4 9 -4 .6 2 0 .0 0 0 0 * 0 .0 0 0 0 * 9 .9 im l e 4 5 .0 2 4 4 .5 8 3 .7 9 4 3 .8 1 4 5 .3 4 4 7 .4 4 4 7 .5 2 3 .3 9 4 6 .8 3 4 8 .2 0 -5 .4 4 0 .0 0 0 0 * 0 .0 0 0 0 * 9 .3 x m l d 4 9 .3 8 4 9 .3 8 6 .0 2 4 8 .0 3 5 0 .7 3 5 4 .1 9 5 3 .4 2 4 .1 2 5 2 .5 0 5 4 .3 5 -4 .5 9 0 .0 0 0 0 * 0 .0 0 0 0 * 1 2 .0 x m l e 5 0 .6 0 4 9 .9 6 5 .4 5 4 8 .7 3 5 1 .1 9 5 3 .5 7 5 3 .1 9 3 .8 8 5 2 .3 1 5 4 .0 6 -4 .0 3 0 .0 0 0 1 * 0 .0 0 0 1 * 1 0 .6 z p l d 8 1 .4 0 8 2 .0 5 4 .7 4 8 1 .0 8 8 3 .0 2 8 4 .5 7 8 4 .4 3 4 .1 5 8 3 .5 7 8 5 .2 8 -3 .6 8 0 .0 0 0 4 * 0 .0 0 0 3 * 5 .6 5 z p l e 8 2 .0 2 8 2 .0 8 5 .0 0 8 1 .0 6 8 3 .1 0 8 4 .2 5 8 4 .1 8 3 .7 5 8 3 .4 1 8 4 .9 4 -3 .2 4 0 .0 0 1 6 * 0 .0 0 1 0 * 5 .6 2 a p l d 4 8 .2 6 4 7 .9 9 3 .9 7 4 7 .2 1 4 8 .7 8 4 6 .9 4 4 6 .8 7 4 .2 7 4 6 .0 2 4 7 .7 2 2 .0 8 0 .0 3 9 4 * 0 .0 3 2 0 * 8 .1 5 a p l e 4 8 .2 3 4 8 .0 8 4 .2 5 4 7 .2 2 4 8 .9 3 4 6 .8 1 4 7 .2 5 3 .7 7 4 6 .4 9 4 8 .0 1 1 .5 1 0 .1 3 1 9 0 .0 9 3 4 8 .0 3 p m l d 2 9 .8 8 2 9 .9 6 3 .4 7 2 9 .2 7 3 0 .6 5 3 1 .6 6 3 1 .2 3 3 .8 7 3 0 .4 6 3 2 .0 0 -2 .3 5 0 .0 2 0 6 * 0 .0 3 1 3 * 1 2 .7 p m l e 3 1 .1 3 3 1 .1 2 3 .4 8 3 0 .4 2 3 1 .8 2 3 2 .6 9 3 2 .2 0 3 .4 7 3 1 .5 0 3 2 .9 0 -2 .2 1 0 .0 2 9 0 * 0 .0 3 8 4 * 1 1 .0 a m l d 4 9 .8 4 5 0 .3 0 5 .3 8 4 9 .2 3 5 1 .3 8 4 8 .8 0 4 8 .4 5 5 .2 6 4 7 .4 0 4 9 .5 0 2 .4 7 0 .0 1 4 9 * 0 .0 5 5 3 * 1 0 .9 a m l e 5 0 .2 6 5 0 .6 3 5 .3 1 4 9 .5 6 5 1 .7 0 4 8 .2 6 4 8 .6 4 4 .6 2 4 7 .7 0 4 9 .5 7 2 .8 4 0 .0 0 5 5 * 0 .0 1 0 4 * 1 0 .2 a se e ta bl e 3 fo r m ea su re m en ts d efi ni tio n b s d = s ta n d a rd d e v ia ti o n c c i= co nfi de nc e in te rv al o f 9 5% d c v =c oe ffi ci en t o f v ar ia tio n 12 lopez-capp et al. ta b le 6 . c o m p a ra ti v e a n a ly s is o f th e m e tr ic v a ri a b il it y b e tw e e n t h e b ra z il ia n a n d s c o tt is h s a m p le s . c o n ti n u a ti o n m d h d 2 8 .2 2 2 7 .8 4 3 .6 5 2 7 .1 1 2 8 .5 7 3 0 .1 2 2 9 .9 7 3 .8 9 2 9 .1 9 3 0 .7 4 -4 .1 1 0 .0 0 0 1 * 0 .0 0 0 1 * 1 3 .6 m d h e 2 9 .5 2 2 8 .6 3 3 .5 8 2 7 .9 1 2 9 .3 5 3 0 .9 2 3 0 .9 2 3 .7 8 3 0 .1 6 3 1 .6 8 -4 .7 1 0 .0 0 0 0 * 0 .0 0 0 0 * 1 2 .5 p os te ri or c ra ni al m ea su re s a u b 1 1 9 .7 8 1 2 0 .2 7 5 .0 3 1 1 9 .2 7 1 2 1 .2 8 1 2 1 .3 3 1 2 1 .0 2 6 .6 7 1 1 9 .6 9 1 2 2 .3 5 -0 .8 6 0 .3 9 0 1 0 .1 9 2 4 5 .0 3 a s b 1 0 9 .3 6 1 1 0 .5 4 6 .1 9 1 0 9 .3 1 1 1 1 .7 7 1 0 9 .6 3 1 0 9 .8 1 6 .9 4 1 0 8 .4 3 1 1 1 .1 9 0 .7 7 0 .4 4 2 5 0 .6 8 7 5 6 .0 5 o c c 9 5 .6 2 9 5 .5 5 5 .8 0 9 4 .4 0 9 6 .7 0 9 6 .1 6 9 5 .9 8 5 .1 5 9 4 .9 6 9 7 .0 0 -0 .5 3 0 .5 9 3 9 0 .7 1 2 9 5 .9 1 l ic 6 4 .9 8 6 6 .3 1 8 .2 5 6 4 .6 4 6 7 .9 8 6 6 .9 3 6 6 .9 1 7 .3 8 6 5 .4 2 6 8 .4 1 -0 .5 0 .6 1 6 1 0 .4 6 3 7 1 2 .4 in fe ri or c ra ni al m ea su re s m l c d 2 1 .9 3 2 2 .3 7 2 .7 2 2 1 .7 9 2 2 .9 6 2 3 .9 8 2 4 .2 1 2 .3 9 2 3 .6 9 2 4 .7 2 -4 .6 9 0 .0 0 0 0 * 0 .0 0 0 0 * 1 2 .3 m l c e 2 1 .8 9 2 2 .1 2 2 .8 0 2 1 .5 3 2 2 .7 2 2 4 .1 5 2 3 .9 6 2 .6 4 2 3 .4 0 2 4 .5 2 -4 .7 7 0 .0 0 0 0 * 0 .0 0 0 0 * 1 2 .4 m w c d 1 0 .9 0 1 1 .0 8 1 .7 6 1 0 .7 1 1 1 .4 6 1 1 .8 8 1 2 .1 3 1 .6 8 1 1 .7 7 1 2 .4 9 -4 .5 7 0 .0 0 0 0 * 0 .0 0 0 0 * 1 4 .3 m w c e 1 1 .1 0 1 1 .0 9 1 .6 7 1 0 .7 4 1 1 .4 5 1 2 .1 0 1 2 .2 5 1 .7 9 1 1 .8 7 1 2 .6 3 -5 .1 6 0 .0 0 0 1 * 0 .0 0 0 0 * 1 5 .3 f o l 3 1 .5 0 3 1 .5 2 3 .3 9 3 0 .8 3 3 2 .2 1 3 5 .8 0 3 5 .9 1 3 .8 2 3 5 .1 3 3 6 .6 9 -8 .6 4 0 .0 0 0 0 * 0 .0 0 0 0 * 1 3 .8 f o b 3 3 .0 9 3 3 .0 7 3 .4 4 3 2 .3 7 3 3 .7 7 3 0 .3 8 3 0 .6 7 2 .3 9 3 0 .1 8 3 1 .1 5 5 .5 1 0 .0 0 0 0 * 0 .0 0 0 0 * 1 0 .8 p a b 3 4 .6 6 3 4 .9 7 3 .6 3 3 4 .2 1 3 5 .7 3 3 2 .9 0 3 3 .1 2 4 .0 3 3 2 .2 8 3 3 .9 6 3 .2 7 0 .0 0 1 5 * 0 .0 0 0 4 * 1 1 .8 p a l 4 7 .8 3 4 7 .1 9 6 .0 5 4 5 .8 5 4 8 .5 3 4 9 .0 3 4 8 .9 6 4 .9 4 4 7 .8 7 5 0 .0 6 -1 .8 7 0 .0 6 4 5 0 .0 9 7 9 1 2 .7 m a b 5 6 .9 4 5 6 .8 5 5 .7 4 5 5 .6 3 5 8 .0 8 5 7 .2 1 5 6 .8 5 4 .9 4 5 5 .8 0 5 7 .9 0 0 .0 0 0 .9 9 9 1 0 .5 3 2 4 1 0 .2 m a l 4 9 .8 5 5 0 .3 4 5 .4 4 4 9 .1 4 5 1 .5 4 4 8 .3 9 4 8 .0 8 4 .3 3 4 7 .1 3 4 9 .0 4 2 .7 7 0 .0 0 6 8 * 0 .0 1 6 0 * 1 0 .9 a se e ta bl e 3 fo r m ea su re m en ts d efi ni tio n b s d = s ta n d a rd d e v ia ti o n c c i= co nfi de nc e in te rv al o f 9 5% d c v =c oe ffi ci en t o f v ar ia tio n 13 lopez-capp et al. in the discriminant analysis of the 51 cranial measurements, the percentage of accuracy varied between 45-74.9%. since nine cranial measurements demonstrated an average percentage of classify correctly between 65-70% and two cranial variables showed a percentage accuracy higher than 70%. in the univariate discriminant analysis of the 21 mandibular measurements, four variables showed an average percentage of classify correctly between 65-70% and four showed a percentage accuracy higher than 70%, the percentage of accuracy varied between 46.3-83.8%. wilks’ lambda (λ) ranged from 0.681 to 1.0, the variables that showed the greatest difference between the two samples were the right and left body thicknesses (bmbd λ=0.680 and bmbe λ=0.714) and right and left maximum ramus heights (mrhd λ=0.726) (table 7). discussion the metric variability analysis of the samples showed that of the 72 variables, 44 measurements presented significant differences between the samples (61.11%). the scottish sample had a higher mean compared to the brazilian sample for 54 variables among the 72. considering only those measurements that showed significant differences, the scottish sample presented higher averages for 33 variables. however, among the measurements that showed differences between the samples, only seven variables had a mean difference greater than 5 mm: maximum cranial length (gol) (diff=5.53 mm), nasal height (nlh) (diff=5.33 mm), right body height (hmbd) (diff=5.2 mm), left body height (hmbe) (diff=6.52 mm), right (mrhd) (diff=8.93 mm) and left (mrhe) (diff=9.57 mm) maximum ramus height, and bigonial breadth (bgb) (diff=5.29 mm). in this study, the scottish sample was considered historical because the skulls came from the 18th and 19th centuries. in contrast, the brazilian sample was a contemporary, or modern, sample, with skulls and mandibles belonging to a collection originating from the 20th century. considering the plasticity of the skull over time, anatomical evaluations indicate a decrease in cranial measurements7, including a reduction of the facial breadth that results in narrower and elongated faces1,15,18 and mandibles5. it is not possible to confirm that the results found in the present study are related to inter-populational variation or cranial plasticity due to the temporal differences between the samples. this factor can be considered as a limitation of the study. the results show that the scottish skulls tended to have a greater cranial length (gol), breadth (xcb) and height (nph) compared to the brazilian skulls. these factors may be associated with the climate of a region with colder temperatures compared to the average temperatures in brazil. the skulls from regions with predominantly cold climates tend to be wider compared to those from hot and humid regions9,11. the nasal cavity also shows changes due to temperature, humidity and latitude. in hot and humid regions, this cavity tends to be lower and wider, but in cold and dry regions, it tends to be higher and narrower12-14. in the current study, the scottish sample had a mean nasal height (nlh) of 51.73 mm and an average nasal breadth (nlb) of 23.51 mm, and the brazilian sample values were 49.29 mm and 24.42 mm, respectively. the results of this study showed that the nasal cavities of the scottish skulls tended to be higher and narrower compared to those of the brazilian skulls, a feature that may be associated with variations in temperature, humidity and latitude, as described in the literature. 14 lopez-capp et al. ta b le 7 . c o m p a ra ti v e a n a ly s is o f th e m a n d ib u la r m e tr ic v a ri a b il it y b e tw e e n t h e b ra z il ia n a n d s c o tt is h s a m p le s . m ea su re a b ra zi l s co tl an d t t es t t p -v al ue w ilc o xo n p -v al ue v c b m ed ia n m ea n s d c 9 5 % c if m ed ia n m ea n s d c 9 5 % c if m an d ib ul ar m ea su re s c h h 2 7 .4 9 2 7 .0 5 6 .7 2 2 4 .7 8 2 9 .3 3 3 1 .6 3 3 0 .7 8 3 .8 6 2 9 .4 7 3 2 .0 9 -2 .9 9 0 .0 0 5 0 * 0 .0 0 8 1 * 2 0 .2 h m b d 2 5 .8 0 2 3 .1 4 5 .5 1 2 1 .2 7 2 5 .0 1 2 9 .4 9 2 9 .3 6 4 .2 5 2 7 .9 2 3 0 .8 0 -5 .1 3 0 .0 0 0 0 * 0 .0 0 0 0 * 2 5 .6 h m b e 2 5 .9 5 2 3 .1 6 5 .9 5 2 1 .1 4 2 5 .1 7 2 9 .2 7 2 9 .6 8 3 .6 3 2 8 .4 5 3 0 .9 1 -5 .2 4 0 .0 0 0 0 * 0 .1 7 6 7 2 6 .3 b m b d 1 0 .3 8 1 0 .0 8 1 .6 8 9 .5 1 1 0 .6 5 1 3 .0 7 1 3 .1 2 1 .5 5 1 2 .6 0 1 3 .6 5 -7 .8 8 0 .0 0 0 0 * 0 .0 0 0 0 * 2 3 .2 b m b e 1 0 .0 6 9 .8 6 1 .7 4 9 .2 6 1 0 .4 5 1 2 .4 6 1 2 .9 1 1 .5 3 1 2 .3 9 1 3 .4 3 -8 .2 9 0 .0 0 0 0 * 0 .0 0 0 0 * 2 3 .3 b m l 4 4 .1 9 4 4 .5 0 2 .8 4 4 3 .5 4 4 5 .4 6 4 5 .1 6 4 4 .9 4 3 .1 7 4 3 .8 6 4 6 .0 1 -0 .6 6 0 .5 1 2 3 0 .3 6 2 2 6 .1 8 b c b 9 4 .0 9 9 3 .3 8 6 .4 1 9 1 .2 1 9 5 .5 6 9 4 .2 5 9 6 .9 5 6 .1 1 9 1 .8 8 9 6 .0 2 -0 .3 5 0 .7 2 1 5 0 .6 9 4 5 7 .0 6 c d b 1 1 4 .5 9 1 1 4 .4 6 6 .7 6 1 1 2 .1 3 1 1 6 .7 8 1 1 5 .7 8 1 1 5 .0 2 7 .3 6 1 1 2 .4 9 1 1 7 .5 5 -0 .3 1 0 .7 5 6 1 0 .9 7 3 9 6 .4 5 m n b d 3 2 .5 2 3 1 .5 6 3 .2 6 3 0 .4 6 3 2 .6 7 3 2 .2 8 3 2 .4 9 3 .5 8 3 1 .2 8 3 3 .7 0 -1 .1 3 0 .2 6 6 1 0 .1 7 6 7 1 0 .9 m n b e 3 2 .4 4 3 2 .5 2 3 .5 5 3 1 .3 3 3 .7 4 3 1 .9 7 3 2 .1 4 3 .9 2 3 0 .7 9 3 3 .4 9 0 .3 9 0 .6 9 2 2 0 .8 3 1 4 1 2 .1 m r b 2 9 .8 0 2 8 .7 9 3 .4 2 2 7 .6 3 2 9 .6 4 2 8 .9 7 3 0 .1 5 4 .1 3 2 8 .7 5 3 1 .5 5 -1 .5 5 0 .1 2 7 8 0 .0 6 6 0 1 2 .8 m a r b 3 1 .3 2 3 0 .7 6 3 .4 3 2 9 .6 3 1 .9 2 3 4 .6 4 3 4 .9 6 3 .7 5 3 3 .6 9 3 6 .2 3 -5 .0 3 0 .0 0 0 0 * 0 .0 0 0 1 * 1 3 .9 m r h d 5 7 .8 1 5 6 .8 2 6 .2 5 4 .7 2 5 8 .9 2 6 5 .2 5 6 5 .7 5 6 .2 0 6 3 .6 5 6 7 .8 5 -6 .3 4 0 .0 0 0 0 * 0 .0 0 0 0 * 1 4 .1 m r h e 5 6 .8 6 5 5 .9 9 6 .1 8 5 3 .8 6 5 8 .1 1 6 5 .3 2 6 5 .5 6 6 .9 7 6 3 .1 6 6 7 .9 5 -6 .5 4 0 .0 0 0 0 * 0 .0 0 0 0 * 1 4 .9 m l t 6 7 .6 0 6 9 .7 5 5 .5 6 7 .8 8 7 1 .6 1 6 8 .2 6 6 8 .4 5 .1 0 6 6 .7 0 7 0 .1 2 1 .2 3 0 .2 2 4 4 0 .1 8 1 7 6 .7 5 b g b 8 9 .3 9 9 1 .1 5 7 .2 1 8 8 .7 1 9 3 .5 9 9 7 .1 5 9 6 .4 4 8 .6 8 9 3 .5 0 9 9 .3 8 -2 .6 5 0 .0 1 1 8 * 0 .0 1 0 9 * 9 .7 4 m l p 1 0 1 .8 7 1 0 1 .0 8 7 .6 1 9 8 .5 1 1 0 3 .6 6 1 0 4 .0 5 1 0 1 .9 2 1 6 .7 4 9 6 .2 6 1 0 6 .5 9 -0 .2 7 0 .7 8 2 3 0 .2 0 6 0 1 2 .5 m a d 1 2 2 .0 0 1 2 1 .0 8 7 .0 7 1 1 8 .6 8 1 2 3 .4 7 1 1 6 .5 1 1 7 .7 7 6 .2 6 1 1 5 .6 5 1 1 9 .8 9 2 .5 3 0 .0 1 6 1 * 0 .0 1 5 9 * 2 .3 9 m a e 1 2 3 .0 0 1 2 3 .1 6 8 .0 5 1 2 0 .4 3 1 2 5 .8 9 1 1 8 .0 0 1 1 8 .8 8 5 .2 1 1 1 7 .1 2 1 2 0 .6 5 3 .3 7 0 .0 0 1 8 * 0 .0 0 2 3 * 5 .0 4 m n d d 1 3 .1 9 1 3 .3 0 2 .0 5 1 2 .6 0 1 3 .9 9 1 3 .9 6 1 3 .9 9 2 .1 0 1 3 .2 7 1 4 .7 0 -1 .3 9 0 .1 7 2 6 0 .2 2 9 4 1 4 .2 m n d e 1 3 .3 4 1 3 .4 9 2 .2 7 1 2 .6 9 1 4 .2 8 1 3 .4 8 1 3 .8 6 2 .8 0 1 3 .1 3 1 4 .5 8 -0 .7 0 .4 8 7 9 0 .7 2 6 0 1 5 .8 a se e ta bl e 4 fo r m ea su re m en ts d efi ni tio ns b s d = s ta n d a rd d e v ia ti o n c c i= co nfi de nc e in te rv al o f 9 5% d c v =c oe ffi ci en t o f v ar ia tio n 15 lopez-capp et al. according to the results obtained in the present study, the scottish mandibles tended to have a greater mandibular ramus height (mrh), mandibular body height (hmb), and bigonial breadth (bgb) than the brazilian mandibles. martin and danforth5 concluded that the jaw tends to become longer and narrower and that these secular changes are the result of dietary changes and improved medical and dental care. the measurement of mandibular body height (hmb) is directly related to tooth loss. after dental extraction, the alveolar processes are reabsorbed, resulting in a reduction of the height of the mandibular body. furthermore, this variable is closely related to the age factor. however, the association with the age factor could not be determined in the present study due to the absence of documentation related to the scottish sample27. the discriminant analyses confirmed the differences found in the descriptive analyses. most of the variables showed a lower percentage of accuracy (<55%), which means that this variables did not discriminate the samples. on the other hand, six variables showed acceptable accuracy (>70%). the present study found metric differences between the two analysed samples. this variability can be considered as multifactorial because factors such as the temporal differences between the samples, age, temperature, humidity, latitude, diet, and ethnographic and demographic profiles, among other factors, may influence the variability. as a result, the present study affirms that certain variables presented statistically significant differences between the samples, but a concrete cause for this variability could not be determined. the study of diverse populations is important to understand the craniometric variations around the world and the factors that affect this variability. future studies performed using a multifactorial approach are required to understand the variations of the human skull. in conclusion, the variability analysis showed that metric variability exists between the two studied populations. scottish skulls tend to have a cranial length (gol), breadth (xcb) and height (nph) greater than those of brazilian skulls, and scottish mandibles tend to present a greater mandibular ramus height (mrh), mandibular body height (hmb) and bigonial breadth (bgb) than brazilian mandibles. acknowledgements this investigation was funded by the state of são paulo research foundation (fapesp, process numbers 2014/13340-7, 2014/23727-6 and 2011/18577-7). capes-ciências forenses (process 25/2014). references 1. buretić-tomljanović a, ostojić s, kapović m. secular change of craniofacial measures in croatian younger adults. am j hum biol. 2006 sep-oct;18(5):668-75. 2. jantz rl. cranial change in americans: 1850-1975. j forensic sci. 2001 jul;46(4):784-7. 3. jantz rl, meadows jantz l. secular change in craniofacial morphology. am j hum biol. 2000 may;12(3):327-38. 4. manthey l, jantz rl, bohnert m, jellinghaus k. secular change of sexually dimorphic cranial variables in euro-americans and germans. int j legal med. 2017 jul;131(4):1113-1118. doi: 10.1007/s00414-016-1469-2. 16 lopez-capp et al. 5. martin dc, danforth me. an analysis of secular change in the human mandible over the last century. am j hum biol. 2009 sep-oct;21(5):704-6. doi: 10.1002/ajhb.20866. 6. ousley s, jantz r, freid d. understanding race and human variation: why forensic anthropologists are good at identifying race. am j phys anthropol. 2009 may;139(1):68-76. doi: 10.1002/ajpa.21006. 7. spradley k, stull ke, hefner jt. craniofacial secular change in recent mexican migrants. hum biol. 2016 jan;88(1):15-29. 8. israel h. the dichotomous pattern of craniofacial expansion during aging. american journal of physical anthropology. 1977;47(1):47-51. doi: 10.1002/ajpa.1330470110. 9. hubbe m, hanihara t, harvati k. climate signatures in the morphological differentiation of worldwide modern human populations. anat rec (hoboken). 2009 nov;292(11):1720-33. doi: 10.1002/ar.20976. 10. harvati k, weaver td. human cranial anatomy and the differential preservation of population history and climate signatures. anat rec a discov mol cell evol biol. 2006 dec;288(12):1225-33. 11. roseman cc. detecting interregionally diversifying natural selection on modern human cranial form by using matched molecular and morphometric data. proc natl acad sci u s a. 2004 aug 31;101(35):12824-9. doi: 10.1073/pnas.0402637101. 12. noback ml, harvati k, spoor f. climate-related variation of the human nasal cavity. am j phys anthropol. 2011 aug;145(4):599-614. doi: 10.1002/ajpa.21523. 13. evteev a, cardini al, morozova i, o’higgins p. extreme climate, rather than population history, explains mid-facial morphology of northern asians. am j phys anthropol. 2014 mar;153(3):449-62. doi: 10.1002/ajpa.22444. 14. holton ne, yokley tr, franciscus rg. climatic adaptation and neandertal facial evolution: a comment on rae et al. (2011). j hum evol. 2011 nov;61(5):624-7; author reply 628-9. doi: 10.1016/j.jhevol.2011.08.001. 15. buretić-tomljanović a, ristić s, brajenović-milić b, ostojić s, gombač e, kapović m. secular change in body height and cephalic index of croatian medical students (university of rijeka). am j phys anthropol. 2004 jan;123(1):91-6. doi: 10.1002/ajpa.10306. 16. kouchi m. brachycephalization in japan has ceased. am j phys anthropol. 2000 jul;112(3):339-47. 17. kouchi m. secular changes in the japanese head form viewed from somatometric data. anthropol sci. 2004;112(1):41-52. doi: 10.1537/ase.00071. 18. weisensee ke, jantz rl. secular changes in craniofacial morphology of the portuguese using geometric morphometrics. am j phys anthropol. 2011 aug;145(4):548-59. doi: 10.1002/ajpa.21531. 19. hubbe m, strauss a, hubbe a, neves wa. early south americans cranial morphological variation and the origin of american biological diversity. plos one. 2015 oct 14;10(10):e0138090. doi: 10.1371/journal.pone.0138090. 20. pena sdj, di pietro g, fuchshuber-moraes m, genro jp, hutz mh, kehdy fdsg, et al. the genomic ancestry of individuals from different geographical regions of brazil is more uniform than expected. plos one. 2011 feb 16;6(2):e17063. doi: 10.1371/journal.pone.0017063. 21. perez si, bernal v, gonzalez pn, sardi m, politis gg. discrepancy between cranial and dna data of early americans: implications for american peopling. plos one. 2009 may 29;4(5):e5746. doi: 10.1371/journal.pone.0005746. 22. hens sm, ross ah. cranial variation and biodistance in three imperial roman cemeteries. int j osteoarchaeol. 2017 jun;27(5):880-7. doi: 10.1002/oa.2602. 23. nikita e. αge-associated variation and sexual dimorphism in adult cranial morphology: implications in anthropological studies. int j osteoarchaeol. 2014 sep-oct;24(5):557-69. doi: 10.1002/oa.2241. 24. relethford jh. apportionment of global human genetic diversity based on craniometrics and skin color. am j phys anthropol. 2002 aug;118(4):393-8. doi: 10.1002/ajpa.10079. 17 lopez-capp et al. 25. relethford jh. population-specific deviations of global human craniometric variation from a neutral model. am j phys anthropol. 2010 may;142(1):105-11. doi: 10.1002/ajpa.21207. 26. roseman cc, weaver td. multivariate apportionment of global human craniometric diversity. am j phys anthropol. 2004 nov;125(3):257-63. doi: 10.1002/ajpa.10424. 27. albert am, ricanek k jr., patterson e. a review of the literature on the aging adult skull and face: implications for forensic science research and applications. forensic sci int. 2007 oct 2;172(1):1-9. 1 volume 21 2022 e225967 original article braz j oral sci. 2022;21:e225967http://dx.doi.org/10.20396/bjos.v21i00.8665967 1 department of pedodontics and preventive dentistry, ame’s dental college and hospital, raichur 584103, karnataka, india corresponding author: dr. raghavendra havale department of pedodontics and preventive dentistry ame’s dental college and hospital raichur – 584103, karnataka, india email: raghavendrahavale@yahoo.co.in editor: dr. altair a. del bel cury received: april 10, 2021 accepted: june 8, 2021 pediatric dentist attire and relationship with anxiety in children and parents during covid-19 pandemic raghavendra havale1,* , dhanu g rao1 , s p shrutha1 , irin mathew1 , namratha tharay1 , kausar-e-taj1 , kanchan m tuppadmath1 aim: the study aimed to evaluate children’s and parent’s preferences of dentist’s attire during covid-19 pandemic and their relationship with dental anxiety. methods: a total of 139 children(71 boys, 68 girls) aged 6-12 years were shown videos of a pediatric dentist working with different attire such as personal protective equipment (ppe) and pedoscrub, and they were asked to express the way they preferred their dentist to be dressed. children’s anxiety levels with different attire of paediatric dentists were assessed in different age groups and for boys and girls separately and recorded it using the facial image scale. a questionnaire regarding dental anxiety was created online and completed by 139 parents (76 females, 63 males) of various ages and different educational backgrounds who were asked to choose between two outfits. results were tabulated and statistically analysed using chi-square test.  results: children aged 10-12 years preferred ppe by 50.6%, whereas 48.1% of children aged 6-9 years least preferred ppe (<0.05). about 46 (33%) were scored as anxious children and they had a preference for pedoscrub. also, nonanxious children 43(31%) preferred ppe. all educated parents (100%) selected ppe over pedoscrub and the result were shown to be statistically significant. (<0.05). conclusion: ultimately, the majority of the anxious children chosen pedoscrub, whereas non anxious children have chosen ppe. furthermore, the data reveals that both educated parents and older children preferred ppe as their attire for paediatric dentists. keywords: surgical attire. covid-19. parents. dentists. dental anxiety. https://orcid.org/0000-0001-8811-3445 https://orcid.org/0000-0002-4032-4582 https://orcid.org/0000-0002-9426-2719 https://orcid.org/0000-0002-0781-3835 https://orcid.org/0000-0002-2380-8868 https://orcid.org/0000-0001-6920-6486 https://orcid.org/0000-0002-9485-5493 2 havale et al. braz j oral sci. 2022;21:e225967 introduction the global outbreak of the covid-19 pandemic has had an impact on every aspect of the human life1. fear and anxiousness are strong emotions that could be linked to the covid-19 pandemic. dental anxiety is thought to be the most common cause for problematic behaviour in children2. the attitude of the parents has an impact on the children’s view. children are affected by parents who do have a high level of anxiety. anxiety in youngsters increases when they are surrounded by anxious parents3. the appearance of a paediatric dentist has been demonstrated to elicit a variety of behavioural patterns in both children and parents4. the enhanced pedoscrub not only made the environment more child-friendly, but also made it easier to converse with the child in the first place5. the highly contagious nature of the disease necessitates changes in paediatric dentists’ standard dress, such as the use of personal protective equipment (ppe). for children, seeing someone wearing ppe can be intimidating6. recognizing and determining dental anxiety in children and parents regarding paediatric dentist attire is essential for providing successful dental care7. although there are countless reports5,6,8-10, on perception towards attire of paediatric dentist, since the covid-19 pandemic, no studies have assessed about the anxiety levels of children and parents towards ppe and pedoscrub. the study was aimed to interpret the paediatric dentist’s attire by children and their parents and their relationship with dental anxiety while functioning during the outbreak of covid-19. materials and methods study design this was a questionnaire-based cross-sectional study. children and their parents who visited the department of paediatric and preventive dentistry ame’s dental college, raichur during august and september were included in the study. the study was approved by the institutional ethical committee (380/2019-20). participants were approached while waiting for their appointment, the parents and children gave their informed consent. the following were the conditions for inclusion criteria; • parents and children who could communicate in english as well as in kannada. • children between the ages of 6 and 12 years old (inclusive). • children with a physical status of asa 1 or 2 (american society of anaesthesiologists 2014). • children & parents who are willing to participate in the study • there should be no systemic disorder in children. • children who are visiting a dental clinic for the first time. the following were the conditions for exclusion criteria; 3 havale et al. braz j oral sci. 2022;21:e225967 • children and parents who were unable to complete the survey on their own due to cognitive disabilities. • children with a physical status of asa 3 (american society of anaesthesiologists) and above. • children who did not have their parents with them. • parents who didn’t give consent to take part in the study. sample size a convenience sampling was performed consisting of 145 children aged 6 to 12 years who visited dental clinics, and one of their parents who accompanied them from out patient department of pediatric and preventive dentistry during the covid-19 pandemic. out of this, 6 parent-child pairs who didn’t meet the inclusion criteria were excluded from the study and the study size was dropped to 139. (fig 1) sample size estimation: this was done with the formula n = (z1-α)2 {p(1-p))/d2 where: zα/2  is the critical value of the normal distribution at α/21.64 (at 90% confidence level);d is the margin of error3%;p is the sample proportion50%= 0.50 (assuming the anxiety levels of children);n is the population size. substituting the above values in the formula, sample size obtained is 139. data collection participants were identified and screened for any potential covid-19 symptoms (temperature, pulse rate and oxygen saturation were measured) in order to prevent cross infection and were confronted while they were waiting for their appointment. a total figure 1. recruitment flowchart total nº of parents-child pairs approached (n = 145) total nº of parents-child pairs recruited (n = 139) nº of parents-child pairs that decline to participate (n = 6) reasons given: no interest in participating in study (n = 3) did not have time to participate for the full length of their study (n = 3) 4 havale et al. braz j oral sci. 2022;21:e225967 of 139 parents were surveyed using an online interviewer-administered questionnaire and checked the anxiety levels in children using facial image scale (fig 2). validity and reliability of questionnaire before its administration to the study participants, reliability and content validity of questionnaire were tested. questionnaire was validated by 5 experts prior to the study. the questionnaire was pretested (reliability) on ten parents to ensure that the questions were appropriate, to see if they were easy to understand, and to evaluate the data collection workflow. reliability was assessed using test-retest method11. a total of 10 participants were given the same questionnaire one week apart. data from 9 participants were included for analysis. due to missing data, one participant was removed from the study. for each question, cohen’s kappa was used to assess the agreement at two time-points. it was observed that among the 13 questions, 12 questions had a kappa value ranging from fair (0.40) to perfect agreement (1) and hence were retained in the questionnaire. one question had a kappa value of 0.17 was removed from the questionnaire. parents were interviewed separately according to the questionnaire to assess their own level of anxiety. the parent’s questionnaire consisted of two sections, section one involved the consent and section two had the questionnaire. questionnaire survey for parents questionnaire was given after obtaining informed consent from the parents. a self-administered questionnaire was prepared and translated to local language kannada by the language experts. an online semi-structured questionnaire was developed by using google forms. the link of questionnaire was circulated through whatsapp, email and other social media and to the parents and received the response through an online survey submission. each participant was asked to complete a set of questionnaire while waiting for their appointment. clear instructions were given to avoid confusion. participants were automatically led to details about the study and informed consent after obtaining and clicking the connection. after agreeing to participate in the survey, they completed the demographic information. then a series of 12 questions related to dental anxiety emerged, which the figure 2. depicts faces which represents facial image scale with scores: (1) very happy, (2) happy, (3) moderate, (4) unhappy, (5) very unhappy 5 havale et al. braz j oral sci. 2022;21:e225967 participants were to answer in order. participants were only permitted to choose one answer per question and only make one request. child’s perception towards pediatric dentist’s attire all 139 children were asked to rate their preferred attire from the videos shown with different attire (ppe and pedoscrub) of pediatric dentist. the attires were as follows: (figure 3) a) pedoscrubcolourful uniform with cartoon images in order to reduce the white coat phobia in children. b) ppe – equipment that will protect the user against health or safety risks at work which includes goggles, face shield, mask, gloves, gowns, head and shoe cover. and their anxiety levels were assessed using facial image scale. the facial image scale consists of one item with a five-faces (ranging from a very sad to a very smiley face). it is a ‘state’ indicator of anxiety because children were asked to show which of the faces they feel most likely at that time. anxiety level of child for attire according to facial image scale in (fig 2) as follows. (1) very happy, (2) happy, (3) moderate, (4) unhappy, (5) very unhappy. children with score 4 & 5 as highly anxious12. statistical analysis observed data was coded, tabulated and analysed using ibm spss statistics for windows (version 20.0. armonk, ny: ibm corp.). descriptive data were reported as frequency and percentages for categorical variables and mean and standard deviation for continuous variables. comparison between study groups (genfigure 3. xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx. personal protective equipment pedo scrub 6 havale et al. braz j oral sci. 2022;21:e225967 der, age group and education level of parent) for categorical dependant variables (questions) and (gender, age) of child for child’s perception of ppe and scrub was done using chi-square test. a p-value of less than 0.05 was considered statistically significant. results of all 139 children 46 (33%) were scored as anxious children and had preference of pedoscrub and 42 (30%) preferred ppe. of all non-anxious children, 33(24%) had preference of pedoscrub and 43 (31%) preferred ppe. (fig 4). the comparison of perception of ppe and pedoscrub by gender of child using facial image scale showed that there is no statistical significance in anxiety scores between boys and girls. (p=0.097) (p=0.431)(table 1). out of 139 children, approximately 50.6 % of children aged 10-12 years preferred ppe and 48.1% of children aged 6-9 years least preferred it. it has also shown that elder figure 4. child’s perception of ppe and scrub using facial image scale 43 20 4 30 42 33 41 4 15 46 0 10 20 30 40 50 very happy happy moderate unhappy very unhappy child's perception of ppe and scrub ppe scrub table 1. comparison of perception of ppe and pedoscrub by gender of child (facial image scale) using chi square test perception score ppe score p value pedo scrub score p value male female male female very happy 26 (40%) 17 (23%) 0.097 17 (26.2%) 16 (21.6%) 0.431 happy 5 (7.7%) 15 (20.3%) 16 (24.6%) 25 (33.8%) moderate 2 (3.1%) 2 (2.7%) 3 (4.6%) 1 (1.4%) unhappy 15 (23.1%) 15 (20.3%) 9 (13.8%) 6 (8.1%) very unhappy 17 (26.2%) 25 (33.8%) 20 (30.8%) 26 (35.1%) * statistically significant at p < 0.05 7 havale et al. braz j oral sci. 2022;21:e225967 children were less anxious towards ppe when compared to younger ones. preference was statistically significant. (p<0.05) (table 2). about 50 % children aged 6-9 years were less anxious towards pedoscrub whereas 50.6% of children aged 10-12 years preferred their dentist not to wear them and the result was significant at (p<0.05) (table 2). the result from the questionnaire study showed that there were no statistical significant difference between the two age groups and different gender groups towards the attire of pediatric dentist. parent’s education showed significant relation in the preference of dentist attire during covid-19 pandemic. that is all the educated parents (100%) selected ppe over pedoscrub in reducing the droplet transmission. (p=0.001) (question 10) (table 3). discussion the impact of covid-19 pandemic led to an overwhelming feeling of fear and anxiety among parents and children. the need for ppe for paediatric dentists has caused increased anxiety in both parents and children, posing challenges in oral health management. several studies in the literature addressed preferences of children and parents attitude towards attire of the pediatric dentist and association between dental anxiety5,8-10,. since paediatric dentists began wearing ppe at the clinic during the covid-19 outbreak, the acceptability of this attire was evaluated along with anxiety level of parents and children. the findings of the current study showed that anxious children preferred pedoscrub which is similar to the findings from previous studies which concluded that anxious children preferred colourful attires5,13. in our opinion the reason behind this may be due to the increased access to the internet and social media which made children more aware about the use of protective wears in dental operatory. we also found that most of the non-anxious children preferred ppe as their attire of choice. in current study we also found that there is no significant difference in anxiety scores between boys and girls for ppe and pedoscrub. table 2. comparison of perception of ppe and pedoscrub by age group of children (facial image scale) using chi square test perception score ppe score p value pedo scrub score p value 6 9 years 10 12 years 6 9 years 10 12 years very happy 0 (0.0%) 43 (50.6%) < 0.001 27 (50.0%) 6 (7.1%) < 0.001 happy 5 (9.3%) 15 (17.6%) 21 (38.9%) 20 (23.5%) moderate 2 (3.7%) 2 (2.4%) 1 (1.9%) 3 (3.5%) unhappy 21 (38.9%) 9 (10.6%) 2 (3.7%) 13 (15.3%) very unhappy 26 (48.1%) 16 (18.8%) 3 (5.6%) 43 (50.6%) * statistically significant at p < 0.05 8 havale et al. braz j oral sci. 2022;21:e225967 ta bl e 3. r es po ns es b as ed o n a ge , g en de r a nd e du ca tio n le ve l o f p ar en ts u si ng c hi -s qu ar e te st q ue st io n r es po ns e to ta l a ge g en de r ed uc at io n of p ar en ts le ss th an or e qu al to 40 y ea rs m or e th an 40 y ea rs p va lu e m al e fe m al e p va lu e h ig h sc ho ol a nd be lo w in te rm ed ia te / d ip lo m a an d ab ov e p va lu e 1) h av e yo u ev er ta ke n yo ur c hi ld to a pe di at ric d en tis t ( c hi ld ’s d en tis t) ? n o 89 (6 4. 0% ) 64 (7 1. 9% ) 25 (5 0. 0% ) 0. 00 8* 39 (6 1. 9% ) 50 (6 5. 8% ) 0. 38 3 61 (6 8. 5% ) 28 (5 6. 0% ) 0. 09 8 ye s 50 (3 6. 0% ) 25 (2 8. 1% ) 25 (5 0. 0% ) 24 (3 8. 1% ) 26 (3 4. 2% ) 28 (3 1. 5% ) 22 (4 4. 0% ) 2) a re y ou a nx io us a bo ut v is iti ng a pe di at ric d en tis t f or th e tr ea tm en t o f yo ur c hi ld ? n o 74 (5 3. 2% ) 43 (4 8. 3% ) 31 (6 2. 0% ) 0. 08 4 36 (5 7. 1% ) 38 (5 0. 0% ) 0. 25 2 41 (4 6. 1% ) 33 (6 6. 0% ) 0. 01 8* ye s 65 (4 6. 8% ) 46 (5 1. 7% ) 19 (3 8. 0% ) 27 (4 2. 9% ) 38 (5 0. 0% ) 48 (5 3. 9% ) 17 (3 4. 0% ) 3) d o yo u kn ow p eo pl e in in di a ar e af fe ct ed b y c ov id -1 9? n o 4 (2 .9 % ) 4 (4 .5 % ) 0 (0 .0 % ) 0. 16 4 2 (3 .2 % ) 2 (2 .6 % ) 0. 61 6 1 (1 .1 % ) 3 (6 .0 % ) 0. 13 3 ye s 13 5 (9 7. 1% ) 85 (9 5. 5% ) 50 (1 00 .0 % ) 61 (9 6. 8% ) 74 (9 7. 4% ) 88 (9 8. 9% ) 47 (9 4. 0% ) 4) a re y ou a w ar e of th e m od e of sp re ad o f c ov id -1 9? n o 2 (1 .4 % ) 2 (2 .2 % ) 0 (0 .0 % ) 0. 40 8 2 (3 .2 % ) 0 (0 .0 % ) 0. 20 4 2 (2 .2 % ) 0 (0 .0 % ) 0. 40 8 ye s 13 7 (9 8. 6% ) 87 (9 7. 8% ) 50 (1 00 .0 % ) 61 (9 6. 8% ) 76 (1 00 .0 % ) 87 (9 7. 8% ) 50 (1 00 .0 % ) 5) d id y ou ta ke y ou r c hi ld to d en ta l cl in ic d ur in g th e c ov id -1 9 pa nd em ic ? n o 85 (6 1. 2% ) 55 (6 1. 8% ) 30 (6 0. 0% ) 0. 48 8 40 (6 3. 5% ) 45 (5 9. 2% ) 0. 36 7 64 (7 1. 9% ) 21 (4 2. 0% ) 0. 00 1* ye s 54 (3 8. 8% ) 34 (3 8. 2% ) 20 (4 0. 0% ) 23 (3 6. 5% ) 31 (4 0. 8% ) 25 (2 8. 1% ) 29 (5 8. 0% ) 6) a re y ou a w ar e th at p er so nn el pr ot ec tiv e eq ui pm en t ( p p e) w or n by pe di at ric d en tis t r ed uc es th e dr op le t tr an sm is si on fr om d en tis t t o ch ild an d vi ce v er sa ? n o / d on ’t kn ow 20 (1 4. 4% ) 16 (1 8. 0% ) 4 (8 .0 % ) 0. 08 4 8 (1 2. 7% ) 12 (1 5. 8% ) 0. 39 4 18 (2 0. 2% ) 2 (4 .0 % ) 0. 00 6* ye s 11 9 (8 5. 6% ) 73 (8 2. 0% ) 46 (9 2. 0% ) 55 (8 7. 3% ) 64 (8 4. 2% ) 71 (7 9. 8% ) 48 (9 6. 0% ) c on tin ua 9 havale et al. braz j oral sci. 2022;21:e225967 c on tin ua çã o 7) d o yo u th in k yo ur c hi ld w ill fe el an xi ou s se ei ng th e pe di at ric d en tis t in fu ll co ve re d p p e? n o / d on ’t kn ow 57 (4 1. 0% ) 36 (4 0. 4% ) 21 (4 2. 0% ) 0. 49 9 24 (3 8. 1% ) 33 (4 3. 4% ) 0. 32 2 39 (4 3. 8% ) 18 (3 6. 0% ) 0. 23 6 ye s 82 (5 9. 0% ) 53 (5 9. 6% ) 29 (5 8. 0% ) 39 (6 1. 9% ) 43 (5 6. 6% ) 50 (5 6. 2% ) 32 (6 4. 0% ) 8) d o yo u fe el th e co lo ur fu l d re ss w or n by th e pe di at ric d en tis t r ed uc es th e an xi et y in c hi ld re n? n o / d on ’t kn ow 23 (1 6. 5% ) 14 (1 5. 7% ) 9 (1 8. 0% ) 0. 45 1 12 (1 9. 0% ) 11 (1 4. 5% ) 0. 31 0 22 (2 4. 7% ) 1 (2 .0 % ) < 0. 00 1* ye s 11 6 (8 3. 5% ) 75 (8 4. 3% ) 41 (8 2. 0% ) 51 (8 1. 0% ) 65 (8 5. 5% ) 67 (7 5. 3% ) 49 (9 8. 0% ) 9) d o yo u th in k th at th e pl ay a re a fil le d w ith to ys re du ce s yo ur c hi ld ’s an xi et y le ve l ? n o / d on ’t kn ow 29 (2 0. 9% ) 15 (1 6. 9% ) 14 (2 8. 0% ) 0. 09 2 15 (2 3. 8% ) 14 (1 8. 4% ) 0. 28 4 23 (2 5. 8% ) 6 (1 2. 0% ) 0. 04 1* ye s 11 0 (7 9. 1% ) 74 (8 3. 1% ) 36 (7 2. 0% ) 48 (7 6. 2% ) 62 (8 1. 6% ) 66 (7 4. 2% ) 44 (8 8. 0% ) 10 )i s p p e m or e ef fe ct iv e th an co lo ur fu l d re ss o f p ed ia tr ic d en tis t i n re du ci ng d ro pl et tr an sm is si on d ur in g th is c ov id -1 9 pa nd em ic ? n o / d on ’t kn ow 15 (1 0. 8% ) 11 (1 2. 4% ) 4 (8 .0 % ) 0. 31 1 8 (1 2. 7% ) 7 (9 .2 % ) 0. 34 8 15 (1 6. 9% ) 0 (0 .0 % ) 0. 00 1* ye s 12 4 (8 9. 2% ) 78 (8 7. 6% ) 46 (9 2. 0% ) 55 (8 7. 3% ) 69 (9 0. 8% ) 74 (8 3. 1% ) 50 (1 00 .0 % ) 11 )d o yo u th in k pr ev en tio n of d ro pl et tr an sm is si on is m or e im po rt an t t ha n ha nd lin g ch ild ’s a nx ie ty ? n o / d on ’t kn ow 15 (1 0. 8% ) 10 (1 1. 2% ) 5 (1 0. 0% ) 0. 53 2 7 (1 1. 1% ) 8 (1 0. 5% ) 0. 56 2 15 (1 6. 9% ) 0 (0 .0 % ) 0. 00 1* ye s 12 4 (8 9. 2% ) 79 (8 8. 8% ) 45 (9 0. 0% ) 56 (8 8. 9% ) 68 (8 9. 5% ) 74 (8 3. 1% ) 50 (1 00 .0 % ) 12 )h as y ou r p er ce pt io n ch an ge d to w ar ds th e at tir e of p ed ia tr ic d en tis t af te r a ns w er in g th is q ue st io nn ai re ? n o 19 (1 3. 7% ) 13 (1 4. 6% ) 6 (1 2. 0% ) 0. 43 9 10 (1 5. 9% ) 9 (1 1. 8% ) 0. 32 9 18 (2 0. 2% ) 1 (2 .0 % ) 0. 00 1* ye s 12 0 (8 6. 3% ) 76 (8 5. 4% ) 44 (8 8. 0% ) 53 (8 4. 1% ) 67 (8 8. 2% ) 71 (7 9. 8% ) 49 (9 8. 0% ) 10 havale et al. braz j oral sci. 2022;21:e225967 in the present study we have found that elder children were less anxious towards ppe and younger ones preferred pedoscrub. this suggests that dental anxiety decrease with increasing age. our study revealed that elder children were less anxious towards ppe is due to the fact that they were better instructed about the transmission of covid-19 and they are aware about the better aspects of ppe whereas younger age group children had the preference of coloured attire to trust their pediatric dentist. this result effectively replicates the findings of babaji p et al (2017) where younger age group children had the preference of coloured attire of dentists2. this study also compared parents of different age group, and found that majority of the elder age group parents were ready to bring their child for dental treatment whereas younger age groups were less aware about the importance and benefits provided by the paediatric dentist and the result was statistically significant. (p =0.084). in the present study there was no statistically significant difference observed in between the gender of parents about the perception towards the paediatric dentist attire. the findings of the questionnaire study showed that parents who have only primary level education were not ready to take their children to clinic during covid-19 pandemic indicating that they are more scared about the transmission of the disease. in our study we also found that parents with higher education preferred ppe over pedoscrub. the level of education of parents was found to have a significant effect on their pediatric dentist attire preferences. result from our finding showed that all educated parents preferred ppe in reducing the droplet transmission which support the view of kiranmayi et al, (2021)14. from our point of view, people with better education have received better knowledge about ppe during this pandemic. some of the limitations of the present study are larger samples could not be covered due to cross sectional nature of the study and language barrier as it was conducted in only two languages (english and kannada). moreover, it could not be carried out in people who do not have smart phone to access the questionnaire. lastly, main drawback of the study was general anxiety was not assessed before the start of study which could have made our results more precise. in conclusion, most of the anxious children chose pedoscrub and non-anxious children have chosen ppe. and the study also suggests that both educated parents and elder children chose personal protective equipment as the attire for paediatric dentists. acknowledgements we would like to thank all the participants for giving their valuable time for the study. funding this research received no external funding. data availability datasets related to this article will be available upon request to the corresponding author. 11 havale et al. braz j oral sci. 2022;21:e225967 conflict of interest none author contribution raghavendra havale: design of the work, interpretation of data and revision of manuscript dhanu g rao: drafting the work and revision of manuscript. shrutha s p: manuscript editing irin mathew: data collection, design of the work, analysis of data and drafting the work namratha tharay: designing of the study. kausar-e-taj: acquisition of the data kanchan m tuppadmath: acquisition of the data all authors actively participated in  the manuscript’s findings, and have revised and approved the final version of the manuscript. references 1. ahmed ma, jouhar r, ahmed n, adnan s, aftab m, zafar ms, et al. fear and practice modifications among dentists to combat novel coronavirus disease (covid-19) outbreak. int j environ res public health. 2020 apr 19;17(8):2821. doi: 10.3390/ijerph17082821. 2. babaji p, chauhan pp, rathod v, mhatre s, paul u, guram g. a cross sectional evaluation of children preference for dentist attire and usage of camouflage syringe in reduction of anxiety. eur j dent. 2017 oct-dec;11(4):531-6. doi: 10.4103/ejd.ejd_223_17. 3. dikshit p, limbu s, bhattarai k. evaluation of dental anxiety in parents accompanying their children for dental treatment. orthod j nepal. 2013 dec;3(1):47-52. doi:10.3126/ojn.v3i1.9282. 4. panda a, garg i, bhobe ap. children’s perspective on the dentist’s attire. int j paediatr dent. 2014 mar;24(2):98-103. doi: 10.1111/ipd.12032. 5. kuscu oo, caglar e, kayabasoglu n, sandalli n. short communication: preferences of dentist’s attire in a group of istanbul school children related with dental anxiety. eur arch paediatr dent. 2009 jan;10(1):38-41. doi: 10.1007/bf03262666. 6. alsarheed m. children’s perception of their dentists. eur j dent. 2011 apr;5(2):186-90. 7. yon mjy, chen kj, gao ss, duangthip d, lo ecm, chu ch. an introduction to assessing dental fear and anxiety in children. healthcare (basel). 2020 apr 4;8(2):86. doi: 10.3390/healthcare8020086. 8. kamavaram ellore vp, mohammed m, taranath m, ramagoni nk, kumar v, gunjalli g. children and parent’s attitude and preferences of dentist’s attire in pediatric dental practice. int j clin pediatr dent. 2015 may-aug;8(2):102-7. doi: 10.5005/jp-journals-10005-1293. 9. ravikumar d, gurunathan d, karthikeyan s. children’s perception towards pediatric dentist attire: an observational study. int j pedod rehabil 2016 dec;1(2):49-51. doi: 10.4103/2468-8932.196479. 10. tong hj, khong j, ong c, ng a, lin y, ng jj, et al. children’s and parents’ attitudes towards dentists’ appearance, child dental experience and their relationship with dental anxiety. eur arch paediatr dent. 2014 dec;15(6):377-84. doi: 10.1007/s40368-014-0126-z. 12 havale et al. braz j oral sci. 2022;21:e225967 11. howard ke, freeman r. reliability and validity of a faces version of the modified child dental anxiety scale. int j paediatr dent. 2007 jul;17(4):281-8. doi: 10.1111/j.1365-263x.2007.00830.x. 12. prasad mg, nasreen a, radha krishna an, devi gp. novel animated visual facial anxiety/pain rating scale-its reliability and validity in assessing dental pain/anxiety in children. pediatr dent. j. 2020 aug;30(2):64-71. doi: 10.1016/j.pdj.2020.05.003. 13. asokan a, kambalimath hv, patil ru, maran s, bharath kp. a survey of the dentist attire and gender preferences in dentally anxious children. j indian soc pedod prev dent. 2016 jan-mar;34(1):30-5. doi: 10.4103/0970-4388.175507. 14. kiranmayi m, raju ss, reddy er, pm srujana, snehitha p, divya ss. parental attitude towards their child’s dental treatment during covid19 pandemic a questionnaire study ind j res. 2021 feb;10(2):61-3. doi: 10.36106/paripex/7004664. 1 volume 22 2023 e239087 letter to the editor braz j oral sci. 2023;22:e239087http://dx.doi.org/10.20396/bjos.v22i00.8669087 1 professor-in-residence oral and maxillofacial surgery, ucla dental school, los angeles california, usa. 2 public health nurse (retired) county of los angeles department of health, usa 3 associate professor, department of dentistry, school of medicine and life sciences, pontifical catholic university of paraná, curitiba, brazil. corresponding author: paulo couto-souza department of dentistry, school of medicine and life sciences, pontifical catholic university of paraná, curitiba, brazil. email: couto.s@pucpr.br editor: altair a. del bel cury received: apr 24, 2022 accepted: jun 10, 2022 analgesia for anticoagulated patients requires substituting gabapentin for ibuprofen arthur h friedlander1, ida k friedlander2, soraya de azambuja berti-couto3 , paulo couto-souza3* to the editor older brazilians with various cardiovascular disorders (e.g., atrial fibrillation, venous thrombosis, pulmonary embolia) are often provided long-term treatment with a vitamin k antagonist (i.e., warfarin) or direct acting oral anticoagulants (daoas) such as dabigatran, apixaban1. many members of this patient cohort often require complex dental implant procedures with anticipated moderate to severe post-operative pain. this scenario heralds a number of clinical dilemmas. non-steroidal anti-inflammatory medications (nsaids) and specifically ibuprofen are most commonly prescribed for their analgesic effects but may not provide adequate pain relief2,3. secondly, nsaids adversely effect platelet function and when concurrently administrated with an anticoagulant medication be it a vitamin k antagonist or a daoa there are enhanced risks of significantly bleeding. thirdly, there are both societal concerns regarding the addictive properties and diversion of opioid medications as well as the need for brazilian dentists to adhere to a set of stringent recommendations as to how the medications are to be administered as well as legal regulations (regulamento técnico sobre substâncias e medicamentos sujeitos a controle especial) set in place limiting their prescribing4-6. https://orcid.org/0000-0001-5189-6300 https://orcid.org/0000-0003-3655-397x 2 friedlander et al. braz j oral sci. 2023;22:e239087 thus, our interest was peaked when coming upon two prospective, double-blinded, placebo-controlled studies. the first demonstrating that an orally administered preoperative dose of gabapentin (600mg.) significantly (p=0.004) decreased the need for post-operative “narcotic rescue” pain medication administration among patients undergoing rhinoplasty and endoscopic sinus surgery7. the second demonstrating that the perioperative administration of gabapentin (1,200mg. preoperatively and 600 mg. 3 times a day postoperatively) to patients having total hip arthroplasty increased by 24% (h.r. 1.24; 95% ci, 1.20-1.54) the rate of opioid cessation after surgery8. gabapentin’s perioperative anti-inflammatory effects result from its ability to reduce pro-inflammatory mediators (e.g., tnf-α, il β, and il-6) and up-regulate anti-inflammatory cytokine il-10). its acute (nociceptive) pain analgesic effect, by binding to calcium channels thereby inhibiting the influx of calcium into nerve endings thus decreasing excitatory neurotransmitter release in the central and peripheral nervous systems. concomitantly, analgesia is also believed garnered from gabapentin’s activation of the descending noradrenergic pain inhibitory system9,10. in summary, our review of the medical literature suggests that patients concurrently receiving anticoagulant medications and presenting for dental implant surgery be administered oral gabapentin 600mg. one hour prior to surgery in order to decrease the inflammatory (painful) surgical insult (i.e., pre-emptive analgesic effect) and that the post-operative regimen consist of gabapentin 600mg combined with acetaminophen 500mg. every 6 hours as needed for pain control. the institution of our suggested regimen however should be held in abeyance until consultation with the patient’s physician for patients having renal impairment or those having chronic obstructive pulmonary disease (copd). these admonitions specifically because gabapentin is not metabolized in the body and is eliminated solely by renal clearance, therefore, toxic levels may arise in those with chronic kidney disease and because respiratory depression may arise in those with copd because gabapentin acts centrally. furthermore, patients need to be advised that gabapentin administration has been associated with somnolence resulting in impaired driving capabilities as well as dizziness increasing the propensity of falling. conflict of interest no potential conflict of interest relevant to this article was reported. data availability datasets related to this article will be available upon request from the corresponding author. references 1. marcolino ms, polanczyk ca, bovendorp ac, marques ns, silva la, turquia cp, et al. economic evaluation of the new oral anticoagulants for the prevention of thromboembolic events: a cost-minimization analysis. são paulo med j. 2016 jul-sep;134(4):322-9. doi: 10.1590/1516-3180.2016.0019260216. 3 friedlander et al. braz j oral sci. 2023;22:e239087 2. pereira gm, cota lo, lima rp, costa fo. effect of preemptive analgesia with ibuprofen in the control of postoperative pain in dental implant surgeries: a randomized, triple-blind controlled clinical trial. j clin exp dent. 2020 jan;12(1):e71-e78. doi: 10.4317/medoral.56171. 3. cruz ajsd, santos js, pereira júnior ea, ruas cm, mattos ff, castilho ls, et al. prescriptions of analgesics and anti-inflammatory drugs in municipalities from a brazilian southeast state. braz oral res. 2020 dec;35:e011. doi: 10.1590/1807-3107bor-2021.vol35.0011. 4. maia lo, daldegan-bueno d, fischer b. opioid use, regulation, and harms in brazil: a comprehensive narrative overview of available data and indicators. subst abuse treat prev policy. 2021 jan;16(1):12. doi: 10.1186/s13011-021-00348-z. 5. brazilian ministry of health. ordinance no. 834, of may 14, 2013. redefines the national committee for the promotion of rational use of medicines within the scope of the ministry of health. brasília: brazilian ministry of health; 2013 [cited 2022 jan 27]. available from: https://bvsms.saude.gov.br/ bvs/saudelegis/gm/2013/prt0834_14_05_2013.html. portuguese. 6. brazilian ministry of health. ordinance no 344, of may 12, 1998. approves the technical regulation on substances and medicines subject to special control. brasília: brazilian ministry of health; 2013 [cited 2022 jan 27]. available from: https://bvsms.saude.gov.br/bvs/saudelegis/svs/1998/ prt0344_12_05_1998_rep.html. portuguese. 7. kazak z, meltem mortimer n, sekerci s. single dose of preoperative analgesia with gabapentin (600 mg) is safe and effective in monitored anesthesia care for nasal surgery. eur arch otorhinolaryngol. 2010 may;267(5):731-6. doi: 10.1007/s00405-009-1175-5. 8. hah j, mackey sc, schmidt p, mccue r, humphreys k, trafton j, et al. effect of perioperative gabapentin on postoperative pain resolution and opioid cessation in a mixed surgical cohort: a randomized clinical trial. jama surg. 2018 apr;153(4):303-11. doi: 10.1001/jamasurg.2017.4915. erratum in: jama surg. 2018 apr 1;153(4):396. 9. chincholkar m. analgesic mechanisms of gabapentinoids and effects in experimental pain models: a narrative review. br j anaesth. 2018 jun;120(6):1315-34. doi: 10.1016/j.bja.2018.02.066. 10. anfuso cd, olivieri m, fidilio a, lupo g, rusciano d, pezzino s, et al. gabapentin attenuates ocular inflammation: in vitro and in vivo studies. front pharmacol. 2017 apr;8:173. doi: 10.3389/fphar.2017.00173. 1 volume 22 2023 e238076 original article braz j oral sci. 2023;22:e238076http://dx.doi.org/10.20396/bjos.v22i00.8668076 1 postgraduate in health sciences, faculty of health sciences, university of brasília, campus darcy ribeiro, asa norte, brasília, df, brazil. 2 dentistry course, catholic university of brasilia, brasília. df, brazil. 3 postgraduate in genomic sciences and biotechnology, catholic university of brasília, asa norte, brasília, df. brazil. corresponding author: taia maria berto rezende universidade católica de brasília e-mail: taiambr@gmail.com, taia@p.ucb.br editor: dr. altair a. del bel cury received: january 9, 2022 accepted: may 25, 2022 triclosan antimicrobial activity against dental-caries-related bacteria jade ormondes de farias1 , jamilca de almeida do espírito santo2 , ingrid aquino amorim3 , taia maria berto rezende1,2,3* triclosan (tcs) is a chlorinated diphenyl ether and a possible active agent against microorganisms. due to its probability of reducing dental plaque accumulation, tcs can be added as a substance for oral hygiene. aim: to evaluate the efficacy and antimicrobial capacity of tcs against pseudomonas aeruginosa and streptococcus mutans. methods: this work evaluates the percentage of bacteria inhibition of p. aeruginosa (atcc 27853) and s. mutans (atcc 25175). tcs concentrations between 2 and 128 µg.ml-1 were tested. results: an inhibitory potential of tcs was found against s. mutans. no percentage of inhibition was detected against p. aeruginosa (technical and biological triplicate). conclusion: tcs, an antimicrobial agent used in dentifrices, can reduce s. mutans levels therefore these dentifrices should be indicated for patients with a high risk of caries. however, further study is needed, including antimicrobial analyses against other microbial conditions. keywords: dental caries. triclosan. streptococcus mutans. https://orcid.org/0000-0001-9347-3330 https://orcid.org/0000-0002-9471-2038 https://orcid.org/0000-0002-1340-7091 https://orcid.org/0000-0002-4148-0659 2 farias et al. braz j oral sci. 2022;21:e238076 introduction tooth decay happens as a consequence of enamel and dentin tissue degradation, resulting from bacteria-produced acids. its consequences may include pulp inflammation, pain, infection, edema, and tooth loss1. different bacterial species cause dental caries, and many bacterial strains were already characterized as an etiological factor1. streptococcus mutans is described as one of the main etiological factors of dental caries2. this microorganism is capable of colonizing the oral cavity and forming bacterial biofilm3. in addition to s. mutans, several other bacteria are also present in dental biofilm. studies demonstrate p. aeruginosa in saliva, and the subgingival and supragingival biofilm of subjects with chronic periodontal infection4,5. also, p. aeruginosa is related to the failure of periodontal treatment6, and the development of aggressive periodontitis6. prevention of dental caries is directly related to biofilm remotion by flossing and brushing teeth6. in addition to the mechanical removal of dental biofilm, chemical agents are also good coadjuvants for oral health care promotion6. fluoride, the most used substance to prevent tooth decay, is found in toothpaste and water6. chlorhexidine  gluconate  (chx) is found in mouthwashes and is the most indicated antimicrobial for patients with periodontal diseases7. in addition to these substances, other agents are already being used to increase the preventive effect of toothpaste. triclosan (tcs) is a chlorinated diphenyl ether or bisphenol of broad-spectrum against gram-positive and negative bacteria and fungi and is characterized as a non-ionic molecule8. evidence indicates that tcs has antimicrobial capacity against aggregatibacter actinomycetemcomitans and porphyromonas gingivalis besides having anti-inflammatory properties that reduce bacterial biofilm9 and contribute to decreased bacterial load and a reduction in pathogenicity10,11. in addition, a study has demonstrated that tcs has an anti-inflammatory action, a long-lasting effect, and high substantivity, resulting in an active agent to reduce dental plaque accumulation12. due to these properties, this antimicrobial is part of the composition of some toothpaste and mouthwashes13. furthermore, the study suggests tcs could be incorporated as a component of glass ionomer cement. in this context, tcs has been employed as an adjunct to fluoride. a study indicated that a dentifrice containing 0.3% of tcs was highly effective in preventing and enhancing demineralization compared to a positive control sodium fluoride dentifrice14. therefore, it is essential to know the antimicrobial capacity of tcs against each oral bacterium, contributing to the correct indication of oral hygiene agents containing tcs. our study hypothesized that tcs has an antimicrobial potential against p. aeruginosa and s. mutans, and this analysis may help indicate oral hygiene agents containing tcs for specific conditions. thus, further studies should be performed to assess tcs activity on other microorganisms related to oral diseases. 3 farias et al. braz j oral sci. 2022;21:e238076 materials and methods triclosan and ampicillin preparation tcs (via magistral, df, brazil) was dissolved in 20% absolute ethanol and 80% sterile distilled water and used at different concentrations (serial dilutions from 2 to 128 µg.ml-1). gentamicin and chloramphenicol (sigma aldrich, ma, usa) were diluted in sterile distilled water and brain heart infusion (bhi) broth (thermo fisher, ma, usa) in 10 mg.ml-115. microorganisms’ preparation p. aeruginosa (atcc27853) and s. mutans (atcc 25175) were cultured in a petri dish containing muller hinton (mh) agar (sigma aldrich, ma, usa) for p. aeruginosa and bhi agar (kasvi, usa) for  s. mutans. the pre-inoculum preparation of each bacterium was carried out in a flow chamber, where three colonies of bacteria from each microorganism (biological replication) were selected and inoculated in 5 ml of mh broth and bhi broth. this culture was maintained under shaker conditions (200 rpm) at 37 °c, overnight. inoculum of bacteria was obtained through 100 µl of the pre-inoculum of each bacteria and added to 4.9 ml of mh and bhi under agitation (200 rpm) at 37 ºc, for 1 hour. optical density (o.d.) were performed until reaching 0.3 (p. aeruginosa) and 0.25 (s. mutans) at an absorbance (abs) of 600 nm. in this abs, 5.02x1011 cfu of p. aeruginosa  and  1x105 cfu of s. mutans  were being considered. technical and biological replicates were performed in a 96-well plate (tpp, usa)15. triclosan’s antimicrobial capacity tcs (0.004 g) was weighed in eppendorf (1.5 ml). tcs stock was obtained by diluting 200 µg.ml-1 of absolute alcohol and 800 µg.ml-1 of sterile distilled water. the antibiotic controls for each bacterium were diluted according to these antibiotics’ concentrations in their respective media. all samples were separated in each group: 1) medium, 2) medium and alcohol, 3) medium and bacteria, 4) bacteria and antibiotics, 5) bacteria and alcohol, and 6) bacteria and different dilutions of triclosan (128 to 2 µg.ml-1). soon after, plates were stored in an incubator at 37 ºc for 18 hours. the plates were read with an absorbance of 600 nm. the minimum inhibitory concentration (mic) and the minimum bactericidal concentration (mbc) were obtained following the standards of a previous protocol. percentage of bacteria inhibition was calculated from the absorbances of microdilution 15. statistical analysis technical and biological replicates were performed for all analyses. mean of absorbances were calculated on excel (microsoft software, san diego, ca, usa). and antimicrobial analyses were determined by comparing samples to the controls referring to 100% and 0% of microbial growth. the graphics were made in graphpad prism (graphpad software, san diego, ca, usa). 4 farias et al. braz j oral sci. 2022;21:e238076 results gentamicin mic against p. aeruginosa was 10 µg.ml-1 while chloramphenicol mic was also 10 µg.ml-1 against s. mutans (table 1). table 1. minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) in μg/ml-1 of triclosan and control (gentamicin and chloramphenicol) against pseudomonas aeruginosa and streptococcus mutans. nd: non detected until 128 µg.ml-1. microorganism tcs control mic mbc mic mbc s. mutans nd nd 10 µg.ml-1 10 µg.ml-1 p. aeruginosa nd nd 10 µg.ml-1 10 µg.ml-1 none of the tcs tested concentrations inhibited s. mutans and p. aeruginosa growth. also, no tcs percentage of inhibition was detected against p. aeruginosa in technical and biological triplicates. tcs showed around 80% of inhibitory activity at 128 to 8 μg.ml−1 against s. mutans (figure 1). 100 80 60 40 20 0 128 64 32 16 8 4 2 triclosan (μg•ml-1) s . m ut an s in hi bi tio n (% ) figure 1. percentage of s. mutans inhibition by various concentrations of triclosan in μg/ml -1. discussion the tcs inhibitory results against the bacteria represented in this work are consistent with the data already reported in the literature.  s. mutans  is one of the primary causative caries, so it is essential to assess how this bacterium is affected by tcs12. possibly, tcs is a multi-target inhibitor for s. mutans, which lack a triclosan-sensitive fabi enoylacp reductase, and that inhibition of glycolysis in dental plaque biofilms, in which tcs is retained after initial or repeated exposure, would reduce cariogenicity16. besides, other studies report the relationship of tcs and s. mutans, where possibly the gene called fabk, an isoenzyme present in the bacterial cell membrane, may be the target of the tcs17,18. 5 farias et al. braz j oral sci. 2022;21:e238076 p. aeruginosa has already been reported to be highly resistant to tcs and most conventional antibiotics, such as carbapenems19. in the present study, tcs was tested at concentrations from 2 to 128 µg.ml-1. however, none of these concentrations was able to inhibit the growth of p. aeruginosa. even though the tcs has a protein carrying enoyl acyl reductase of the fabi type, an isoenzyme present in the bacterial cell membrane, such protein is considered a target of triclosan20. however, it is believed that the fabv type enoyl acyl reductase carrier protein, which is also an isoenzyme present in the cell membrane of p. aeruginosa, confers resistance to tcs20. thus, tcs can possibly be indicated for patients at high risk for caries due to its antimicrobial action against s. mutans observed in this study and its effectiveness as an adjuvant to fluoride and remineralization capacity already reported14. although studies observed a decrease in dental plaque using toothpaste with triclosan9,11,12, no study has compared the caries incidence of patients using tcs with other agents. thus, further study is needed to confirm this indication. despite the resistance of p. aeruginosa to tcs, other studies demonstrate the indication of this antimicrobial in cases of gingivitis due to the antimicrobial action against a. actinomycetemcomitans and p. gingivalis8. another study has already reported a decrease in periodontitis markers, such as bleeding on probing and probing depth, in children from parents with aggressive periodontitis who used toothpaste with triclosan21. even though chx is the most used antimicrobial for periodontal diseases, tcs is more compatible with typical toothpaste ingredients, unlike chx which is mostly found in mouthwashes7,22. also, chx showed some disadvantages: staining the enamel surface21 and a higher cost compared to tcs. therefore, further study is needed, including testing different species of microorganisms related to periodontitis and gingivitis. our results reinforce the use of tcs as part of the composition of toothpaste, mouthwashes, and even dental materials such as glass ionomer cement, especially for patients with a high risk of caries21. further study is needed to make tcs more present in these products. in conclusion, it is estimated that the use of tcs against p. aeruginosa may be unfeasible due to its resistance, but other concentrations could be tested due to the clinical results already reported. however, this agent has an inhibitory potential against s. mutans, one of the main bacteria related to caries. thus, these results suggest that it is highly recommended to use tcs as an oral hygiene agent in toothpaste and mouthwashes, as well as in other materials such as glass ionomer, due to its antimicrobial capacity. also, it is possible to indicate toothpaste and mouthwashes with tcs for patients at high risk of caries. however, further work is needed to test the tcs in other microbial conditions and applications in these products. acknowledgments this study was supported by conselho nacional de desenvolvimento tecnológico (cnpq); coordenação de aperfeiçoamento de pessoal de nível superior (capes – grant 409196/2018-5) and fundação de amparo do distrito federal (fapdf – grant nº00193-00000782/2021-63). 6 farias et al. braz j oral sci. 2022;21:e238076 author contribution jade ormondes de farias: roles/writing original draft, writing review & editing. jamilca de almeida do espírito santo: methodology, investigation, data curation. ingrid aquino amorim: methodology, investigation, data curation. taia maria berto rezende: conceptualization resources, funding acquisition, supervision, writing review & editing. all authors actively participated in  the manuscript’s findings and have revised and approved the final version of the manuscript. data availability datasets related to this article will be available upon request to the corresponding author. references 1. paster bj, boches sk, galvin jl, ericson re, lau cn, levanos va, et al. bacterial diversity in human subgingival plaque. j bacteriol. 2001 jun;183(12):3770-83. doi: 10.1128/jb.183.12.3770-3783.2001. 2. forssten sd, björklund m, ouwehand ac. streptococcus mutans, caries and simulation models. nutrients. 2010 mar;2(3):290-8. doi: 10.3390/nu2030290. 3. krzyściak w, jurczak a, kościelniak d, bystrowska b, skalniak a. the virulence of streptococcus mutans and the ability to form biofilms. eur j clin microbiol infect dis. 2014 apr;33(4):499-515. doi: 10.1007/s10096-013-1993-7. 4. souto r, silva-boghossian cm, colombo ap. prevalence of pseudomonas aeruginosa and acinetobacter spp. in subgingival biofilm and saliva of subjects with chronic periodontal infection. braz j microbiol. 2014 aug;45(2):495-501. doi: 10.1590/s1517-83822014000200017. 5. da silva-boghossian cm, do souto rm, luiz rr, colombo ap. association of red complex, a. actinomycetemcomitans and non-oral bacteria with periodontal diseases. arch oral biol. 2011 sep;56(9):899-906. doi: 10.1016/j.archoralbio.2011.02.009. 6. lister pd, wolter dj, hanson nd. antibacterial-resistant pseudomonas aeruginosa: clinical impact and complex regulation of chromosomally encoded resistance mechanisms. clin microbiol rev. 2009 oct;22(4):582-610. doi: 10.1128/cmr.00040-09. 7. yates r, jenkins s, newcombe r, wade w, moran j, addy m. a 6-month home usage trial of a 1% chlorhexidine toothpaste (1). effects on plaque, gingivitis, calculus and toothstaining. j clin periodontol. 1993 feb;20(2):130-8. doi: 10.1111/j.1600-051x.1993.tb00327.x. 8. escalada mg, russell ad, maillard jy, ochs d. triclosan-bacteria interactions: single or multiple target sites? lett appl microbiol. 2005;41(6):476-81. doi: 10.1111/j.1472-765x.2005.01790.x. 9. pancer ba, kott d, sugai jv, panagakos fs, braun tm, teles rp, et al. effects of triclosan on host response and microbial biomarkers during experimental gingivitis. j clin periodontol. 2016 may;43(5):435-44. doi: 10.1111/jcpe.12519. 10. blinkhorn a, bartold pm, cullinan mp, madden te, marshall ri, raphael sl, et al. is there a role for triclosan/copolymer toothpaste in the management of periodontal disease? br dent j. 2009 aug;207(3):117-25. doi: 10.1038/sj.bdj.2009.669. 11. wallet ma, calderon nl, alonso tr, choe cs, catalfamo dl, lalane cj, et al. triclosan alters antimicrobial and inflammatory responses of epithelial cells. oral dis. 2013 apr;19(3):296-302. doi: 10.1111/odi.12001. 12. bedran tb, grignon l, spolidorio dp, grenier d. subinhibitory concentrations of triclosan promote streptococcus mutans biofilm formation and adherence to oral epithelial cells. plos one. 2014 feb;9(2):e89059. doi: 10.1371/journal.pone.0089059. 7 farias et al. braz j oral sci. 2022;21:e238076 13. macisaac jk, gerona rr, blanc pd, apatira l, friesen mw, coppolino m, et al. health care worker exposures to the antibacterial agent triclosan. j occup environ med. 2014 aug;56(8):834-9. doi: 10.1097/jom.0000000000000183. 14. mellberg jr, blake-haskins j, petrou id, grote ne. remineralization and demineralization in situ from a triclosan/co-polymer/fluoride dentifrice. j dent res. 1991 nov;70(11):1441-3. doi: 10.1177/00220345910700110901. 15. wiegand i, hilpert k, hancock re. agar and broth dilution methods to determine the minimal inhibitory concentration (mic) of antimicrobial substances. nat protoc. 2008;3(2):163-75. doi: 10.1038/nprot.2007.521. 16. phan tn, marquis re. triclosan inhibition of membrane enzymes and glycolysis of streptococcus mutans in suspensions and biofilms. can j microbiol. 2006 oct;52(10):977-83. doi: 10.1139/w06-055. 17. cullinan mp, bird ps, heng nc, west mj, seymour gj. no evidence of triclosan-resistant bacteria following long-term use of triclosan-containing toothpaste. j periodontal res. 2014 apr;49(2):220-5. doi: 10.1111/jre.12098. 18. kim to, im dw, jung hy, kwon sj, heo ys. purification, crystallization and preliminary x-ray diffraction analysis of enoyl-acyl carrier protein reductase (fabk) from streptococcus mutans strain ua159. acta crystallogr sect f struct biol cryst commun. 2012 mar;68(pt 3):292-4. doi: 10.1107/s1744309112000115. 19. huang yh, lin js, ma jc, wang hh. functional characterization of triclosan-resistant enoyl-acyl-carrier protein reductase (fabv) in pseudomonas aeruginosa. front microbiol. 2016 nov;7:1903. doi: 10.3389/fmicb.2016.01903. 20. zhu l, lin j, ma j, cronan je, wang h. triclosan resistance of pseudomonas aeruginosa pao1 is due to fabv, a triclosan-resistant enoyl-acyl carrier protein reductase. antimicrob agents chemother. 2010 feb;54(2):689-98. doi: 10.1128/aac.01152-09. 21. monteiro mf, tonelli h, reis aa, casati mz, silvério kg, nociti junior fh, et al. triclosan toothpaste as an adjunct therapy to plaque control in children from periodontitis families: a crossover clinical trial. clin oral investig. 2020 apr;24(4):1421-30. doi: 10.1007/s00784-019-03121-6. 22. pihlstrom bl, michalowicz bs, johnson nw. periodontal diseases. lancet. 2005 nov;366(9499):1809-20. doi: 10.1016/s0140-6736(05)67728-8. 1 volume 22 2023 e237216 original article braz j oral sci. 2023;22:e237216http://dx.doi.org/10.20396/bjos.v22i00.8667216 1 private dental practitioner, porto alegre, rs, brazil. 2 post-graduate program in dentistry, federal university of pelotas corresponding author: luísa quevedo grave ph.d. student at post-graduate program in dentistry, federal university of pelotas. address: 457, gonçalves chaves st. pelotas rs brazil zip: 96015-360 e-mail: luisagrave@gmail.com editor: dr. altair a. del bel cury received: october 5, 2022 accepted: february 4, 2023 dental students’ self-perception of security and biosafety measures in times of pandemic by covid-19: a cross-sectional study in private universities in southern brazil luísa quevedo grave1,2,* ; francine dos santos costa2 ; luiz alexandre chisini2 ; marcus cristian muniz conde1 the study investigated the factors associated with the self-perception safety of dental students in clinical activities during the covid-19 pandemic. this cross-sectional study was based on a semi-structured online (google forms) self-applied questionnaire, sent by e-mail to three private dental schools in brazil. the variables were: 1) sociodemographic information; 2) questions about the measures adopted by dental schools before returning to clinical activities; 3) dental students’ self-perception of security; 4) the general health questionnaire. data were submitted to fisher’s exact test (p<0.05). of the 294 eligible students, 97% were evaluated and 100% received previous specific biosafety training predominantly theoretical (72.16%) longer than one hour (51.55%). most students (81.44%) felt secure performing clinical care during the covid-19 pandemic. students undergoing specific biosafety training for longer than one hour felt safer than those perfoming training for up to 1 hour (p=0.004). students from schools where the aerosol-producing restriction was applied felt safer than those without restrictions (p=0.016). women reported feeling less secure than men (p=0.046), and students who submitted to covid-19 specific biosafety training felt safer in clinical activities than those submitted to theoretical training only (p=0.011). students from private universities presenting psychosomatic changes felt less secure in practicing clinical dental care activities (p=0.006). in conclusion, time-spent training in biosafety, restriction of the use of aerosol-producing procedures, and the gender of students were associated with the self-perception safety of students. students with practical training felt safer in clinical activities for patients with covid-19 than those who had only theoretical training. keywords: covid-19. students, dental. pandemics. https://orcid.org/0000-0002-8527-6473 https://orcid.org/0000-0001-9558-937x https://orcid.org/0000-0002-3695-0361 https://orcid.org/0000-0003-2662-3305 2 grave et al. braz j oral sci. 2023;22:e237216 introduction since december 2019, the world has been experiencing an unprecedented situation in public health due to the pandemic caused by the new coronavirus, the sars-cov-2. the disease was termed coronavirus disease 2019 (covid-19)1. covid-19 is an ss-rna-enveloped virus with an average incubation period varying from 4 to 14 days and with the potential to lead to severe acute respiratory tract infection2. the virus typically transmits from person to person via saliva and nasal droplets (>5-10 µm in diameter)3,4. thus, in february 2020, the first case of covid-19 disease was confirmed in brazil, and since then, the transmission and spread of sars-cov-2 have increased. with the introduction of vaccines, people who have completed the vaccination cycle have been at lower risk of developing complications compared to unvaccinated individuals5. however, dentists are exposed to occupational risk related to infections in the respiratory tract (such as covid-19), due to constantly exposed to aerosols-generating procedures6. this condition worsens during pandemics, which are disposable increasing significantly the costs related to dental care7 and affecting patients’ behavior related to the demand for dental services7-9. therefore, dental care in educational institutions was initially suspended10 and, subsequently, readjusted to meet the necessary biosafety measures, keeping the minimum of damage to the development of activities11. individuals involved with dental care adapted their clinical practices by including new personal protective equipment (ppe), such as n95/pff2 masks and face shields12. social distancing to protect students, staff, and patients were important strategies to ensure the continuity of dental education11. however, due to the contagious nature of covid-19 and the high risk of dental professionals to be contaminated, students have low self-perception of safety showing frequently fear and anxiety, which are observed also in dental assistants and professors13,14. corroborating, during the first months of pandemic, it has been shown that dental students reported several mental health issues including stress and depression15. a recent study observed that brazilian undergraduate students presented elevated symptoms of anxiety being associated with alcohol abuse16. although several biosafety measures have been adopted to protect students and professionals involved in dental care, no known study has investigated a possible relationship between the adoption of biosafety measures and the perception of safety by dental students in southern brazil. therefore, bearing in mind that dental students present as many risks as the professionals trained in the area to contract covid-19 disease, the study aimed to investigate the factors associated with the safety self-perception of dental students from private universities in clinical activities during the covid-19 pandemic. materials and methods the present study complies with the strobe statement for observational studies17. 3 grave et al. braz j oral sci. 2023;22:e237216 ethical issues the project was approved by the research ethics committee of blinded for peer review (caae: 44461221.9.0000.5310). study design and setting this cross-sectional study was conducted with students from three private universities of rio grande do sul state, in southern brazil. of the three private universities evaluated, one presents a community character. data collection was performed for three months (1st february to 30th april 2021). the study was a census comprising all students from dentistry courses performing clinical dental activities during the period investigated at the respective universities. thus, was applied an online self-administered survey consisting of questions regarding sociodemographic issues and biosafety measures adopted by dental schools before the return of students to practical activities during the covid-19 pandemic. besides, the student’s self-perception of safe when performing clinical care in the context of a pandemic was evaluated, even in the general health questionnaire 12 (ghq12). a questionnaire pre-test was performed aim to evaluate the understanding and clarity of the questions. thus, the questionnaire was randomicity sent to 10 students from institutions not eligible for the present study (public institutions). the questions were presented as well as a 5-score likert scale [a) not at all clear b) difficult to understand c) understandable but confusing d) clear e) fully understandable where students should mark the clarity of the questions. the official questionnaire was improved after the pre-test. participants were considered eligible students who resumed clinical activities during the covid-19 pandemic. thus, before the beginning of the questionnaire, the students were asked if they were in clinical activities. if they were not, the questionnaire was closed and the student would not be part of the study sample. such students were from three (blinded to peer review) private universities in the rio grande do sul. the institutions were selected by convenience. thus, the population universe of the present study was 294 (n=294) and it was obtained through contact with the coordination of each course/institution. students were contacted three times by institutional e-mail and by social media (instagram, whatsapp, and facebook). data were collected through a semi-structured self-applied questionnaire, formulated using the google forms platform. students who were not involved in academic clinical activities were not considered eligible for the study. variables the dependent variables provided data about the dental students’ self-perception of security related to dental clinical care. thus, the students were asked: “did you feel safe to perform clinical care during pandemics? (yes/no)” and “did you felt prepared to provide clinical care for a patient with covid-19 symptoms?” (yes/no). 4 grave et al. braz j oral sci. 2023;22:e237216 independent variables related to students comprised age, sex, undergraduate semester, and covid-19 diagnoses (appendix 1). additional biosafety measures adopted by dental schools were also investigated. in such a context, the students were asked whether the educational institution offered any type of covid-19 specific biosafety training (csbt) before resuming clinical activities. besides, the nature of received csbt, theoretical or practical was collected. in the theoretical csbt category, were included, those activities carried out in person and those virtualized; the category “practical” included any training performed in the clinical environment. in the same way, the time spent during the received csbt has been collected continuously and then categorized into “up to one hour of training” and “more than one hour of training”. regarding personal protective equipment, students were asked about the use of n95/ppf2 masks, both when acting as operators and assistants. in addition, they were also asked whether the aforementioned equipment was being subsidized by dental schools. the last part of the questionnaire contained the general health questionnaire 12 (ghq12). the instrument investigates whether the respondent has experienced a particular symptom or behavior and is composed of 12 questions presented on a 4-point likert-type scale18. each item is accompanied by four possible responses, typically being “not at all”, “no more than usual”, “rather more than usual” and “much more than usual”, scoring from 0 to 3, respectively. the scores are summed and the greater the score value, the greater the level of psychological disturbances19. to carry out the analysis of possible associations, the binary scoring method was applied, being the two least symptomatic (0 and 1) responses scoring “0” and the two most symptomatic (2 and 3) responses scoring “1”. any score that exceeded the threshold value of 3 was considered symptomatic for common mental disorders (cmd)19. such scores were categorized as follows: 0 3: no psychosomatic changes (asymptomatic). 4 12: the presence of psychosomatic changes (symptomatic) statistical methods records were tabulated in an excel spreadsheet and then uploaded into stata 14.0 (stata corp, college station, tx, usa) software package to perform analysis. data were submitted to fisher”s exact test considering a level of significance of p≤0.05 and a confidence interval of 95%. results from 294 eligible students, ninety-seven (33.0%) signed the consent term and participated in the study. the interviewed students were mostly female (83.5%), aging on average 23.1(±5.16) years old. most respondents attended the seventh (23.7%), eighth (19.6%), and ninth (35.1%) undergraduate semesters. besides, 72.2% responded not have received a positive diagnosis for covid until the date of this research (table 1). before the clinic started, 100% of students reported having received previous csbt, which was predominantly theoretical (72.2%) and longer than one hour (51.5%). more5 grave et al. braz j oral sci. 2023;22:e237216 over, most students (81.4%), reported to felt secure performing clinical care during the covid-19 pandemic. on the other hand, 61.9% of students reported not feeling secure to provide dental care for patients with covid-19 symptoms. most students (55.7%) reported their professors’ restricted aerosol-producing procedures. concerning the ghq-12 instrument, 48.4% of students were classified as symptomatic, presenting some symptoms of common mental disorders. table 1. number of observations and frequencies for independent and dependent variables regarding students’ self-perception of safety related to biosafety measures adopted by dental schools (n=97). variables n* (%) age 19 5 5.2 20 14 14.4 21 25 25.8 22 19 19.6 23 15 15.5 24 4 4.1 25 5 5.2 26 2 2.1 27 1 1.0 30 1 1.0 32 1 1.0 33 1 1.0 37 1 1.0 43 1 1.0 49 2 2.1 sex male 16 16.5 female 81 83.5 undergraduate semester fifth 12 12.4 sixth 4 4.1 seventh 23 23.7 eighth 19 19.6 ninth 34 35.1 tenth 5 5.1 students diagnosed with covid-19 yes 27 27.8 no 50 72.2 continue 6 grave et al. braz j oral sci. 2023;22:e237216 continuation did you receive some type of covid-19-specific biosafety training before returning? yes 97 100.0 no 0 0.0 how long was the covid-19 specific biosafety training offered? up to 1 hour 50 51.5 > 1 hour 47 48.5 how was the covid-19 specific biosafety training provided? theoretical 70 72.2 practical 27 27.8 was there a restriction on the use of aerosol generating-procedures? yes 54 55.7 no 43 44.3 did you use n-95 mask as operator? yes 97 100.0 no 0 0.0 did you use n-95 mask as auxiliar? yes 92 94.8 no 5 5.2 n95 mask was provided by the university? yes 74 76.3 no 23 32.7 did you felt prepared to provide clinical care? yes 79 81.4 no 18 18.6 did you felt prepared to provide clinical care for a patient with covid-19 symptoms? yes 37 38.1 no 60 61.9 ghq-12 symptomatic 47 48.4 asymptomatic 50 51.6 * n may vary in different questions and it is related to the number of each individual that have answered it students undergoing csbt for longer than one hour reported to felt safe performing clinical care during pandemics than those with training for up to 1 hour (p=0.004). similarly, students from schools where the aerosol-producing restriction was applied felt safer than without restrictions (p=0.016). in such a context, women self-reporting to felt less secure than the man to perform clinical care for patients with covid-19 (p=0.046). students that performed practical 7 grave et al. braz j oral sci. 2023;22:e237216 training in csbt felt safer performing clinical activities for patients with covid-19 than those who had only theoretical training (p=0.011) (table 2). table 3 displays the factor associated with symptoms of common mental disorders assessed by the ghq-12 instrument. although no associations were observed between mental disorders and sex (p=0.680) or the time spent in the covid-19 training (0.927), was found an association with the nature of csbt training (p=0.007). students who received practical activities showed fewer mental disorders symptoms. students in places without restrictions on the use of aerosol-generating procedures were associated with mental disorders symptoms (p=0.035). those students classified as symptomatic in ghq-12 reported feeling less secure in resuming practical activities related to clinical dental care (p=0.006). table 2. factors associated with safety’s self-perception to resume clinical care. did you feel safe to perform clinical care during pandemics? n (%) pvalue yes no how long did the training last?     0.004* up to 1 hour 35 (70.0) 15 (30.0)   more than 1 hour 44 (93.6) 3 (6.4)   schools adopting protocol to restrict the use of aerosol-producing procedures     0.016* yes 46 (92.0) 4 (8.0)   no 33 (70.2) 14 (29.8)   did you felt prepared to provide clinical care for a patient with covid-19 symptoms? n (%) yes no sex 0.046* male 10 (62.5) 6 (37.5) female 27 (33.3) 54 (66.7) nature of received csbt 0.011* theoretical 21 (30.0) 49 (70.0) practical 16 (59.3) 11 (40.7) * fischer’s exact test p < 0.05 table 3. factors associated with ghq-12 ghq-12 asymptomatic n (%) symptomatic n (%) 47 p-value sex 0.680male 9 (56.2) 7 (43.8) female 41 (50.6) 40 (49.4) continue 8 grave et al. braz j oral sci. 2023;22:e237216 continuation how long did the training last? 0.927up to 1 hour 26 (52.0) 24 (48.0) more than 1 hour 24 (51.1) 23 (48.9) nature of received csbt 0.007*theoretical 21 (30.0) 49 (70.0) practical 16 (59.3) 11 (40.7) did you felt prepared to provide clinical care? 0.006*yes 46 (58.2) 33 (41.8) no 4 (22.2) 14 (77.8) was there a restriction on the use of aerosol generating-procedures? 0.035*yes 33 (61.1) 21 (28.9) no 17 (39.5) 26 (60.5) * fischer’s exact test p < 0.05 discussion the new world scenario and the new services routine have also impacted practical activities20,21 and clinical care in dental schools22,23. in the present findings, it was observed that students who underwent training with a higher workload felt more secure to perform clinical care as well as students in places where aerosol use was restricted. in addition, female students were less secure to provide clinical care for patients with covid-19 as well it was also found that the type of activity was an important factor in the students’ feeling of security. thus, students exposed to training who performed practical activities showed a tendency to present greater confidence to perform consultations with patients with potential infection of covid-19 and tend to present fewer mental disorders symptoms. in the present study, 100.00% of respondents reported having received some kind of csbt, provided by dental schools before the return of clinical activities in that context (first semester of 2021). students, who received csbt for longer than 1 hour reported felt safe performing dental clinical care. a possible justification for the present result is that a longer discussion on the subject increases knowledge and understanding about the transmission of the disease and the respective prevention methods. thus, students with more training would be more empowered and able to carry out all prevention strategies more safely. in addition, in-depth knowledge of the disease and its symptoms is essential in carrying out pre-treatment screening, which aims to identify possible patients with the disease. in this way, dental care for these individuals can be performed at a more opportune time. according to meng et al.24 (2020) the biological risk of sars-cov 2 transmission is extremely high when performing dental procedures due to the use of handpieces under irrigation, which produces and diffuses significant amounts of aerosol par9 grave et al. braz j oral sci. 2023;22:e237216 ticles, containing saliva, blood, and secretions, contaminating the environment3,24. this increases the fear of performing these services, once the patient can carry the disease and does not know about it (asymptomatic), evidence confirmed that more than 50% of the virus transmission is asymptomatic25. the risk of contamination of any family member and the need for enough information about the virus have been associated with greater burden and suffering psychological aspect of dentistry student26. pre-clinical care training can be an alternative to reduce this risk since dentistry students during its formation may present higher stress levels than the general population27. despite available guidelines for the management of dental patients and to make dentists (and students) safe from risks27-29, the severity of the covid19 pandemic presents clear challenges to dental educational institutions worldwide, since it is necessary to think about the health of students, teachers, employees, and patients who frequent the environment23,30. appointments with patients with respiratory disease should be scheduled at the end of the day to minimize the risk of nosocomial infection6,31. isolated rooms with good ventilation or negatively pressurized rooms would be more appropriate for patients with suspected cases of covid-19. the brazilian association of dental education (abeno), has positioned itself, through the abeno consensus, which ensures that each dentistry course can build its own possible adjustments, between the ideal and the minimum necessary for the future resumption of teaching activities in classrooms, laboratories and clinics32. from the collected data, most students reported their professors restricted the use of aerosol-producing procedures between the months of february and april of the year 2021. in such cases, students reported more frequently feeling safe to provide clinical care than students in places without aerosol-producing procedures restrictions. in such context, it is important to highlight that there was an additional challenge for the dental profession during pandemics in the evaluated period33. as dentists work in close contact with patients’ oropharyngeal region, they are exposed to an additional risk for nosocomial infection34. besides, dental practice is strongly associated with aerosol-generating procedures potentially able to spread infections among dental professionals and their patients24 by creating a virus-laden aerosolized environment. thus, it is important to expose that the sars-cov-2 can survive in aerosols for hours and on surfaces for up to days33. such risk, related to dental practice, for transmitting respiratory infectious diseases due to aerosol‐producing procedures33,35 could explain why the students reported to felt safe preferably when performing clinical care in dental schools where the use of aerosol-producing procedures was restricted30. the production of aerosols is the main self-perceived contributor to covid-19 cross-infection in teaching clinics by students and staff30. in such a context, 93% of students perceived their health to be at risk while they were at the dental teaching clinics during the covid-19 pandemic30. in the same study, 87% of students reported a significant increase in their stress levels30. therefore, dental school clinics must perform and prioritize measures to reduce contaminated aerosol-producing during dental procedures, such as the use of rubber dams to minimize contamination34. 10 grave et al. braz j oral sci. 2023;22:e237216 this work showed that the majority of interviewed students reported it unsafe to provide dental clinical care to a person with covid-19 symptoms. there were statistical differences in self-reported by gender, with women more frequently reported to feel insecure to attend a person with covid-19 symptoms than men. previous studies evaluating dental students’ stressors during the covid-19 pandemic and, in agreement with our results, identified women being significantly more anxious most of the time about themselves or a family member contracting covid-1926. besides, depression and anxiety are more common symptoms among women, and during the covid-19 pandemic anxiety’s prevalence can be three times greater in women36. in literature, recent studies showed that male dentistry students felt more confident in their competence during clinical procedures37. the greatest ease female to articulate their emotions besides the biggest feminine propensity for the development of posttraumatic stress in a pandemic situation are some possible explanations for these results38. most of patients with covid-19 are asymptomatic or only mildly symptomatic but discharge large amounts of infectious viral particles in the early phase of infection. this poses an enormous challenge for containing the spread of the infection39. standard precautions are not enough to prevent the spread of the coronavirus, especially during the incubation phase of covid-1928, the whole dental team should be vigilant and keep patients and themselves in a safe environment. presented findings highlight that a high number of students presented a propensity to develop psychological disorders and the adoption of safety and training measures increased the students’ perception of safety. covid-19 has challenged the higher education sector worldwide, strongly affecting healthcare professionals and students. dentists, as well as dental students, are at the top of the pyramid of healthcare professionals at risk for contracting covid-1924. this fact affects the continuity of activities in dental schools during the pandemic outbreak, as they need to concern with the safety of all students, patients, staff, and professors22,40. the present study evaluated the self-perception of safety by dental students performing dental clinical care during the covid-19 pandemic related to biosafety measures adopted by dental schools in the south of brazil. we know that dental students are likely to develop stress-related disorders during their undergraduate courses, even more during a pandemic period. previous study24 proved the increase in stress levels, especially when related to some health risk, has a strongly negative impact on students’ clinical performance. precisely due to this affirmation, it is necessary to investigate students’ stress levels during the pandemic. the ghq12 is a tool used to identify the severity of disorders in non-psychotic psychiatric patients of the non-clinical population. it works as an identifier of potential causes of these disorders19,32. the questionnaire was chosen for this research due to the advantage of recognized validity in the literature studies carried out with samples composed of students. increased stress, anxiety, and fear are expected to happen during a pandemic period and dental schools need to be prepared to provide mental health support for students30. some limitations of the present study need to be discussed and considered when interpreting the results. a low response rate was achieved, and this is mainly because 11 grave et al. braz j oral sci. 2023;22:e237216 participants were invited by email. complementary strategies for forwarding e-mails and invitations via social networks were implemented but with little success. studies using electronic tools for data collection tend to have lower response rates when compared to studies where the invitation is made in person. thus, considering the low response rate affects the statistical power in the statistical analysis and additional associations may not have been found due to the limited statistical power. in addition, it is important to note that the sample was composed of students from private institutions. thus, the present data should only be extrapolated to populations with similar characteristics. in conclusion, students from private universities who underwent training with a higher workload and in places where aerosol use was restricted felt more secure to perform dental clinical care. moreover, students classified as symptomatic in ghq-12 reported feeling less secure in resuming practical activities related to clinical dental care. female students and students who performed only theoretical biosafety training were less secure to provide clinical care for the patient with covid-19. thus, students exposed to practical training showed a tendency to present greater confidence to perform the consultations with patients with potential infection of covid-19. data availability datasets related to this article will be available upon request to the corresponding author. declarations of interest none. funding none. author contribution lqg and mcmc conceived the ideas, analyzed the data, collected the data, and wrote the paper. lac and fsc rewired the paper. all authors actively participated in  the manuscript’s findings, and have revised and approved the final version of 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10.1007/s12630-020-01729-x. 40. ghai s. are dental schools adequately preparing dental students to face outbreaks of infectious diseases such as covid-19? j dent educ. 2020 jun;84(6):631-3. doi: 10.1002/jdd.12174. 1http://dx.doi.org/10.20396/bjos.v19i0.8656579 volume 19 2020 e206579 original article 1 phd of health education and health promotion, shemiranat health network, health deputy department, shahid beheshti university, tehran, iran. 2 department of public health, school of health, torbat heydariyeh university of medical sciences, torbat heydariyeh, iran. 3 health sciences research center, torbat heydariyeh university of medical sciences, torbat heydariyeh, iran. 4 health education and health promotion, faculty of medical sciences, tarbiat modares university, tehran, iran. corresponding author: dr. mohammad hossein delshad 7th floor, bldg no.2 sbums, arabi ave, daneshjoo blvd, velenjak, tehran, iran. (delshad@sbmu.ac.ir) p.o. box: 19839-63113 tel:+98 (21) 22211882 fax: +98 (21) 22211882 department of health education and health promotion, faculty of medical sciences, tarbiat modares university, tehran, iran. no 213, department of health education and health promotion, faculty of medical sciences, tarbiat modares university, ghisa st., jalae ale ahmd ave, tehran, iran. p.o. box: 14115-111 tel:+98 (21) 82880000 fax: +98 (21) 82880000 mail:delshad@sbmu.ac.ir received: september 07, 2019 accepted: april 07, 2020 factors predicting the oral health behaviors of the iranian students in the district 1 tehran, iran fatemeh pourhaji1,2,3 , mohammad hossein delshad1,2,3* , sedigheh sadat tavafian4 , alireza hidarnia4 , shamsodin niknami4 aim: the purpose of this examination is determining the predictors of oral health behaviors among iranian students in district 1 tehran based on the health belief model with added commitment to plan construct. methods: this cross-sectional study was conducted on 351 four grade female students in the first district of tehran, iran in 2017. the multi‑stage random cluster sampling method was used to recruit students. the inclusion criteria were being in four – graded level of elementary schools of the 1st district in tehran, being female students aged between 9-11 years and being physically and psychologically healthy student. logistic regression analysis was used to identify the variables that predict oral health behaviors. results: totally, (n= 31.8%) students reported that they were brushing less than twice a day and (n= 55.2%) students claimed using of dental floss once a week or less than once a day. the results indicated that perceived self‑efficacy (or=1.46, 95% ci=0.57‑3.78, p<0.001), commitment to plan (or=1.13, 95% ci=1.04‑1.23, p<0.001) and cues to action (or=1.42, 95% ci=1.14–1.76, p=0.002) were the significant predicting variables of brushing twice a day, and use of dental floss once a day or more (or=1.02, 95% ci=0.23‑3.53, p=0.003). conclusion: this study has shown the effectiveness of the health belief model with added commitment to plan construct to predict oral health behavior in female students. thus, it seems that the model as a acceptable framework for designing training programs to improve oral health behavior in students. keywords: health behavior. health education, dental. iran. oral health. students. tel:+98 (21) 82884547 tel:+98 (21) 82884547 tel:+98 (21) 82884547 https://orcid.org/0000-0001-6075-5307 https://orcid.org/0000-0002-3512-9010 https://orcid.org/0000-0003-2842-7172 https://orcid.org/0000-0003-1534-4757 https://orcid.org/0000-0002-8179-5719 2 pourhaji f et al. introduction oral disorders are the most common health problems. studies have shown that one of the commonest problems of early life is dental caries and oral diseases. oral health is a part of the public health and essential issue to enhancing the quality of life1. de faria campestrini et al.2 study shows that it is not enough to merely convey information about the functions of the oral cavity and describe the characteristics of diseases that affect it when attempting to develop healthy public attitudes toward health habits and it is needed educational preventive programs2.primarily based on this fact that prevention and training are the satisfactory manners of promoting oral health collectively, it has been argued that extra prematurely preventive measures and interruption on disease evolution could be more effective3. distribution and severity of oral conditions vary in different parts of the world and this is also real for specific geographic conditions within the equal country or area3. according to a countrywide oral health survey which performed in 2012, indicated a high level of carries inside the primary dentition and the mean dmft (full) index rated as 5.16/0.38 in 6-year-old children4. behavioral factors are shown as the best care in early childhood period..brushing and flossing are the very best methods to reduce the incidence of plaque5. in addition, health education is considered a critical method for health promotionthrough voluntary wonderful adjustments of individuals within healthy life. additionally health education can improve familiar and community behavior, producing political behaviors that allow the development of new strategies to promote health and enhance the quality of lifestyles of the population6-7.the implementation and effectiveness of educational preventive programs have become important because of the perception of risk factors for oral diseases, knowledge acquisition and consequently behavioral changes7. in health education; the use of models and theories of health behavior to designing interventions is recommended because they can cause powerful health education programs. in fact, the models provide a framework for expertise regarding how people analyze healthy messages and the way they behave and why humans behave as they distinguish8.the health belief model(hbm) is a comprehensive model that can be used for organizing educations. the hbm is one of the first models which were advanced for regulating health-related behaviors9. on this version there are specific patterns of social‑cognitive predictors can also appear (figure 1) the construct of “commitment to plan of action” from “health promotion model added to hbm model. the model assumes that different factors, consisting of the perceived severity of health trouble, perceived benefits, and perceived barriers preventing people from assignment preventive behaviors, affect health related beliefs and behaviors10.the purpose of this examination is determining predictors of oral health behaviors like teeth brushing and dental floss rate in iranian students in district 1 tehran based on hbm with added commitment to plan construct. 3 pourhaji f et al. materials and methods study design and participants this was a cross-sectional study which was conducted on the grade four female students (9‑11 years) of schools in the first district of tehran on april 21, 2017, for 2 months. to obtain samples from 33,179 female students (grade four) studying in this urban-rural, a multi-stage random cluster sampling method was used. in the first stage, out of 162 schools (145 urban schools and 17 rural schools), 10 schools [urban schools (n=6) and rural schools (n=4)] were randomly selected. in the second stage, from351 students based on the population rate of each school in the sample of each school were randomly selected. according to dropping 43 students totally 308 eligible students were selected (table 1). the inclusion criteria were being in four – graded level of elementary schools of the 1st district in tehran, being female students aged between 9-11 yearsand being physically and psychologically healthy student. the exclusion criterion was student or parent’s disagreement to be studied or not to responding to the study questionnaire (figure 2). the researcher was available while completing the questionnaire to help the students. the students were educated to answer truly. to assess the predictors of brushing, and use of dental floss, all the health promotion model added to hbm model constructs (figure 1) were examined as risk factors which could influence the probability of occurrence brushing, and use of dental floss and were interpreted through odds ratio (or). the odds ratio was used to determine whether particular exposures like hpm added to hbm model constructs could be risk factors for occurrence of the outcome like behaviors. logistic regression analysis was used to identify the variables that predict oral health behaviors. to determine the relationship between different hpm added hbm model constructs with each other and with brushing, and use of dental floss behavior, r spearman was used because k‑s test showed the data were non‑parametric. to predict the factors influencing brushing, and use of dental floss behavior logistic regression analysis was applied. figure 1. flow diagram of the expanded health belief model with the construct of “commitment to plan of action” from “health promotion model. age gender ethnicity personality socieconomics knowledge perceived threat perceived benefits perceived barriers perceived self-efficacy modifying factors perceived susceptibility to and severity of disease individual beliefs individual behaviors cues to action commiment to plan of action action 4 pourhaji f et al. results totally, 308 students took part in the study. the mean age of the subjects was 9.32 ± 0.8 years. the demographic variables of the study population are shown in table 1 and table 2. about 31.8% of the students (n =98) reported that they were brushing behavior less than twice a day, and 170 students (55.2%) reported that they brushed their teeth once a week or after using dental floss or less than once a day. while 210 students (68.2%) reported that they brushed at least twice a day, 138 students (44.8%) reported that they were using dental floss at least once a day. the results indicated that perceived self‑efficacy (or=1.46, 95% ci=0.57‑3.78, p<0.001), commitment to plan (or=1.13, 95% ci=1.04‑1.23, p<0.001) and cues to action (or=1.42, 95% ci=1.14–1.76, p=0.002) were the significant predicting variables which is the key predictor of brushing twice a day, and use of dental floss once a day or more (or=1.02, 95% ci=0.23-3.53, p=0.003). first stage the recognition of effective demographic variables on oral health behaviors chi-square statistics was used. the related data are shown in tables 1 and 2. based on the results given in table 1, the father’s educational level (p=0.03), and income (p = 0.04) had a significant relationship with the students’ brushing behavior. figure 2. flow diagram of student’s recruitment. 10 schools, out of 162 schools (145 urban schools and 17 rural schools) randomized multi stage cluster sampling out of 162 schools with 33,179 female students (grade four) total students (n=351) from 10 schools [urban schools (n=248) rural schools (n=103)] assessed for eligibility and analyzed (n=308) urban schools (n=6) rural schools (n=4) urban schools (n=215) rural schools (n=93) declined to participate (n=43) 5 pourhaji f et al. the children’s use of dental floss was significantly related to the father’s job (p = 0.04), father’s educational level (p = 0.03) (table 2). using a logistic model for testing, the effect of six structures of hbm and demographic variables had a significant relationship with oral health behaviors. tables 3& 4 show the data used in the model. in order to find out the relationship between oral health behavior and independent variables, simple and multiple logistic regression analyses were carried out with structures of hbm and demographic variables that were significant. mother’s education (p =0.005), income (p =0.007), self efficacy, commitment to plan (p <0.001) and cues to action (p =0.003) predicted the students’ behavior of dental floss using at least twice a day (tables 3). however, after adjustment, only perceived self‑efficacy, commitment to plan, cues to action remained significant, so that one unit increase in perceived self efficacy increased the possibility of teeth brushing behavior at least twice a day by 1.42 times, commitment to plan by 1.02 times cues to action by times. table 1. demographic characteristics affecting of the students brushing behavior demographic variables brushing frequency less than twice a day twice a day or more n (%) n (%) 98(31.8) 210(68.2) father’s educational level primary 20(20.4) 54(25.7) high school 35(35.7) 66(31.4) higher educational 43(43.9) 90(42.9) p-value 0.03 mother’s educational level primary 23(23.5) 43(20.5) high school 31(31.6) 80(38.1) higher educational 44(44.9) 87(41.4) p-value 0.07 father’s job private 75(76.6) 147(70) employee 23(23.4) 63(30) p-value 0.08 mother’s job un employed 50(51) 110(52.4) employed 48(49) 100(47.6) p-value 0.1 income low 10(10.2) 16(7.6) appropriate 13(13.3) 17(8.1) well 16(16.3) 87(41.4) excellent 59(60.2) 90(42.9) p-value 0.04 6 pourhaji f et al. the results showed that the students’ use of dental floss behavior was significantly related to the mother’s job (p = 0.006), father’s educational level (p = 0.004), income (p = 0.007) perceived self efficacy (p < 0.001), commitment to plan (p < 0.001), and cues to action (p = 0.003). when they were separately entered into the model (table 4) nevertheless, after adjustment, mother’s job (p = 0.012) and self efficacy (p = 0.016) and cues to action (p = 0.002) were found to be significantly related to the use of dental floss once a day or more. the increase of perceived self efficacy by one unit, the possibility of using dental floss at least once a day would increase by 1.30 times (or = 1.30, 95% ci = 0.99-2.34, p = 0.016). discussion the current survey was designed to investigate the predictors to oral health behaviors in iranian students in district 1 tehran based on the health belief model with added commitment to plan construct. consistent with this examine findings, other research table 2. demographic characteristics affecting of the students dental floss using demographic variables dental floss frequency once a week or less than once a day once a day or more n (%) n (%) 170(55.2) 138(44.8) father’s educational level primary 38(22.4) 34(24.6) high school 65(38.2) 48(34.8) higher educational 67(39.4) 56(40.6) p-value 0.03 mother’s educational level primary 33(19.4) 33(23.9) high school 67(39.4) 47(34) higher educational 70(41.2) 58(42.1) p-value 0.5 father’s job private 164(96) 89(64.5) employee 126(74) 49(35.5) p-value 0.04 mother’s job un employed 115(67.6) 73(52.9) employed 55(32.4) 65(47.1) p-value 0.8 income low 30(17.7) 24(17.4) appropriate 32(18.8) 22(15.9) well 31(18.2) 24(17.4) excellent 77(45.3) 68(49.3) p-value 0.2 7 pourhaji f et al. has mentioned a significant relationship between the education level of mother and father as aggarwal et al.11 study. contrary to the pourhaji et al.8 study that showed there was no significant relationship between education level and oral health behaviors1, a significant relationship between income, father’s job, dental floss behavior and brushing behavior in students same as phanthavong et al.12 study. this study indicated that perceived self‑efficacy, cues to action, and commitment to plan were the significant predictors which is the key factor of teeth brushing and brushing behavior at least twice a day, use of dental floss and brushing behavior once a day or more. according to the data, respectively the study carried out by rahnama et al.13 study and hazavei et al.14 study showed that self‑efficacy, cues to action had the highest percent of total variance observed in dental health behaviors. table 3. factors predicting brushing behavior at least twice a day among of students brushing behavior b simple or (95% ci) p-value b multiple or (95% ci) p-value mother’s educational level 0.005 0.108 primary 0.16 1 (0.40-2.51) 1.32 0.19 1.14(0.54-2.65) 0.26 high school 0.47 1.60(0.92-2.78) 0.63 0.38 1.46(0.57-3.78) 0.02 higher educational 0.57 1.78(0.66-4.74) 0.01 0.52 1.65(0.97-2.83) 0.01 income 0.008 0.123 low 0.18 1.12(0.52-2.63) 0.12 0.15 1.01(0.53-1.90) 0.24 appropriate 0.23 1.24(1.14-1.38) 0.18 0.20 1.13(0.53-2.64) 0.18 well 0.28 1.36(0.47-3.68) 0.02 0.25 1.18(0.41-2.59) 0.01 self-efficacy 0.38 1.46(0.57-3.78) <0.001 0.35 1.42(1.14-1.76) 0.012 commitment to plan 0.18 1.13(1.04-1.23) <0.001 0.15 1.02(0.36-2.52) 0.014 cues to action 0.16 1.02(0.23-3.53) 0.003 0.12 1 (0.87-1.26) 0.023 or = odds ratio, ci = confidence interval table 4. factors predicting use dental floss behavior at least once a day among of students dental floss behavior b simple or (95% ci) p-value b multiple or (95% ci) p-value mother’s job 0.006 0.012 father’s educational level 0.004 0.113 primary 0.18 1.20(0.54-2.70) 0.61 1.19 0.78(0.37-1.69) 0.23 high school 0.47 1.60(0.92-2.78) 0.01 0.28 1.36(0.47-2.68) 0.01 higher educational 2.61 0.74(0.33-1.65) 0.03 0.52 1.65(0.97-2.83) 0.01 income 0.007 0.104 low -0.56 0.56(0.18-1.72) 0.31 0.45 1.31(0.83-2.43) 0.28 appropriate -0.034 0.96(0.31-3.01) 0.95 0.20 1.15(0.55-2.66) 0.23 well 0.13 1.14(0.35-3.65) 0.81 0.21 1.12(0.35-2.53) 0.01 self-efficacy 0.53 1.78(0.66-4.74) <0.001 0.36 1.30(0.99-2.34) 0.016 commitment to plan 0.18 1.13(1.043-1.23) <0.001 0.15 0.89(0.38-1.54) 0.21 cues to action 0.16 1.02(0.23-3.53) 0.003 0.14 1.02(0.89-3.44) 0.002 or = odds ratio, ci = confidence interval 8 pourhaji f et al. however, there was a constrained correlation between oral health perceptions and elevated perceived benefits in solhi et al.15 study. buglar et al. study on the role of self efficacy in dental patients’ brushing and flossing, found that, barriers emerging, and self efficacy significantly predicted brushing and flossing behaviors16. however, like the current study it had no significant relation with perceived benefits and in contrast to current study with no relation to cues to action17. theses differences might be due to different gender and age rangeof the participants. within the reisi et al. study, besides to perceived barriers (with negative correlation), all constructs of hbm were definitely associated with oral health behaviors. self‑efficacy was the most powerful predictor of oral health behavior18. the kasmaei et al. findings recommend that perceived objective severity and perceived psychological barriers play an important position in adopting acceptable health behavior among younger young people19. moreover, according to the present study, numerous researches have revealed that commitment to plan has been as the best predictor variable for actual oral health behaviors19-20.therefore, strategies for enhancing commitment to plan in practice, such as strengthening self-extinguishing techniques, enhance commitment, pursuit of commitment and focus groups discussion could lead to more effective oral health behaviors programs for iranian students and should be considered in future intervention20-21.these programs could propose that highly commitment to plan individuals exert greater efforts to empowering individuals to prevent them from returning to unhealthy behavior22. pender stated that more commitment to plan could have a much impact on continuing health promotion behaviors23. in this study, the variables of cues to action with a positive relationship were demonstrated to be significant predictors for oral health behaviors among the iranian students. this finding is supported by many previous studies which found that cues to action are stimuli that trigger appropriate health behaviors. cues to action can be either internal, that is, the perception of bodily states, or external, that is, stimuli from the environment, such as interpersonal interactions or the mass media24-25.in the current study, there was also a relationship between self‑efficacy and oral health behaviors. similar to the present study, self‑efficacy was the most predictive factors of oral health behaviors. these results are consistent with previous studies26-28. there are several limitations to this study. first, this study was a cross-sectional design in addition to assessing oral health behaviors as self-report, in which humans typically might record the behavior better than the real amount. furthermore, the sample of this study were selected from volunteered individuals, so that it’s results might not be generalized to all iranian student groups. in this study, psychological tests for the studied participants were not done. therefore, it is suggested to consider this assessment in future studies to see if there would be some correlations with the prediction of the behavior. this study has shown the effectiveness of the health belief model with added commitment to plan construct to predict oral health behavior in female students. herefore, it seems that the model as a framework for designing training programs to improve oral health behavior can be used. the finding of this study provides needed data assisting the development of model-based behavioral prevention interventions to encourage students’ oral health behavior. 9 pourhaji f et al. acknowledgement the authors would like to thank all the participants who took part in the study. the authors also thank research deputy of shahid beheshti university for its financial support for this study (ir.sbmu.retech.rec.1396.625). finance/disclosure none declared. conflict of interest “the authors acclaimed that they have no rivaling interests”. references 1. peyman n, pourhaji f. the effects of educational program based on the health belief model on the oral health behaviors of elementary school students. mod carev j. 2015;12(2):74-8. 2. de faria campestrini nt, da cunha bm, de oliveira kublitski pm, kriger l, caldarelli pg, gabardo mcl. 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campinas, piracicaba, sp, brazil. 2 department of orthodontics, araras dental school, university center of hermínio ometto foundation-fho, araras, sp, brazil. corresponding author: patrícia rafaela dos santos av. limeira, 901 vila rezende. piracicaba, sp – brazil 13414-903. +55 19 999439853 e-mail: patriciarasantos@gmail.com received: september 12, 2019 accepted: january 27, 2020 exploring the impact of oral health-related quality of life on the child’s family structure patrícia rafaela dos santos1,*, felipe alexandre de souza2, diego patrik alves carneiro2, marcelo de castro meneghim1, silvia amélia scudeler vedovello2 aim: the aim of this study was to explore the impact of oral health-related quality of life (ohrqol) on the child’s family structure. methods: a representative sample of 613 children from public preschools in a city in southeastern brazil was included in this cross-sectional study. the sample was determined through probabilistic sampling in two stages (preschools and children). the outcome variable (early childhood oral health impact scale [ecohis]) was multi-categorized in children and family. independent individual variables were sex, race, family income, parents’/ caregivers’ level of education, family income, dental caries, and malocclusion. initially, individual analyses were performed, relating the study variables to the outcome variables, estimating the raw odds ratio with the respective confidence intervals of 95%. the variables with p < 0.20 in the individual analyses were tested in the multiple logistic regression models, and those with p < 0.10 remained in the model. results: impact on ohrqol was reported by 40.9% of the children and 17% of their families. children with low family income and caries experience had, respectively, 1.53 (95% ci: 1.00-2.32) (p = 0.0465) and 2.96 (95% ci: 1.81-4.84) (p < 0.0001) more chance of presenting negative impact on ohrqol. conclusion: the aspects that most affected the ohrqol of child’s family structure were low income and dental caries experience. keywords: child, preschool. dental caries. malocclusion. oral health. child, preschool. dental caries. malocclusion. oral health. 2 santos et al. introduction oral health-related problems can play an important role in social acceptance, resulting in functional limitations, mainly in more severe cases1-4. currently, it is known that assessment of oral health-related quality of life (ohrqol) goes beyond traditional clinical measures to provide insight into the social and emotional experiences of individuals and their family and social context5. dental caries and malocclusion are the most common oral health-related problems in preschoolers and have been associated with a negative impact on ohrqol2,6,7. despite a significant reduction in the prevalence and severity of dental caries in brazil in other age groups, 80% of affected teeth remain untreated in the primary dentition7. in this sense, caries disease, when left untreated, might lead to tooth loss, imposing functional limitations that have a negative impact on quality of life6,8,9. in addition, malocclusion is highly prevalent in populations at different stages of occlusal development10,11, varying according to ethnic group, age, and recording methods. however, severe malocclusion can affect the negative manner in which a person is perceived throughout his or her entire life. in this sense, studies have demonstrated the importance of detecting malocclusion in preschoolers as a prognostic action that allows for early treatment planning6. assuming that the clinical conditions do not exist in isolation, understanding the perception of family structure and exploring its impact on oral health aspects can help to assess treatment needs, prioritizing care and strategies2. parental socioeconomic status and family structure can be significant predictors of children’s ohrqol because a positive family environment is known to be associated with better oral health behavior12. on the other hand, these factors need to be better understood9 to know how the individual and socioeconomic characteristics influence ohrqol. a recent study13 also suggested that contextual socioeconomic factors are more important than individual socioeconomic factors with regard to this outcome. individual health promotion strategies can be strengthened if also expanded to the community level13, which implies knowing the impact of oral conditions on the family structure. it is known that the family is fundamental for the maintenance of oral health, in an important age group for the future development of the individual. thus, it is necessary to investigate how the clinical factors related to caries disease and occlusion, as well as contextual and behavioral factors, influence ohrqol. the aim of this cross-sectional study was to explore the impact of ohrqol on the child’s family structure. materials and methods this study received approval from the human research ethics committee of brazil (caae: 24905113.0.0000.5385) and the department of education of the municipality. parents/caregivers signed a statement of informed consent authorizing the participation of their children. 3 santos et al. a population-based cross-sectional study was conducted involving children aged 3-5 years enrolled at all public schools in the city of araras, located in the state of são paulo in southeast brazil, from march to april 2017. araras has an estimated population of 188,843 and a human development index of 0.78. all public schools (n = 14) with children in the target age range were included in the study, and probabilistic sampling was performed by conglomerates. the distribution of 3to 5-year-old children in each region of araras was determined from information provided by the municipal secretary of education. the sample was stratified according to administrative district and, in the first phase, schools were randomly selected. in the second phase, children were selected for the sample using a simple randomization procedure. classrooms were randomly selected at the schools, and children were randomly selected from the classes. the sample size was calculated considering a confidence interval of 95%, a test power of 80%, and an odds ratio of 1.6. the minimum sample size was defined as 604 children. the inclusion criteria were children in primary dentition with no systemic problems (based on the reports of parents/caregivers) and no history of orthodontic treatment. moreover, the parents/caregivers to be included in the study needed to have adequate reading and writing skills to answer the questionnaires. thus, a total of 613 schoolchildren and their families participated in the study. data were collected through clinical oral examinations and interviews. one calibrated examiner carried out clinical examinations and recorded dental caries and malocclusion. before the survey, the calibration process was performed in a group of 30 children who were 5 years old. theoretical and clinical training and calibration exercises were arranged for a total of 36 hours under the supervision of one benchmark examiner14. the mean kappa values were above 0.81 and 0.92 for caries and malocclusion, respectively. a blinded dentist conducted the interviews. the outcome variable was the early childhood oral health impact scale (ecohis), which was used to evaluate the impact of oral conditions on the ohrqol of the child’s family structure15. this questionnaire has been translated into portuguese and validated for use on brazilian populations (b-ecohis)16. parents/caregivers were previously contacted and asked to attend a meeting at the preschool, during which they were informed about the objectives of the study. the b-ecohis consists of 13 questions, divided into two sections: a child impact section with 4 domains (child symptoms, function, psychology, and self-image/social interaction domains) and a family impact section with 2 domains (parental distress and family function). parents/caregivers answer the questions using a rating scale from 0 to 5, where 0 = never, 1 = hardly ever, 2 = occasionally, 3 = often, 4 = very often, and 5 = do not know. total scores are calculated as the sum of the response codes, and the “do not know” answers are counted but excluded from the total ecohis score. higher scores denote a greater oral health impact and poorer ohrqol. sociodemographic data were collected to obtain an individual profile of the child’s family structure (age, gender, parental education, and household income). parental education was categorized considering the educational level of the mother and father 4 santos et al. and was dichotomized as ≤ 8 years of study or > 8 years of study. household income was dichotomized based on the median (us$350). caries were recorded using the dmf-t (mean number of decayed, missing, and filled primary teeth) in accordance with the who codes and criteria17. caries experience was dichotomized as absent (dmf-t = zero) or present (dmf-t > zero). malocclusion was classified according to the specific instrument for primary dentition, as recommended by the who17. the primary occlusion was examined according to the criteria of foster and hamilton18,19. overjet was considered the relationship of incisors in the horizontal direction. no distance between maxillary and mandibular incisors was defined as normal overjet (0 mm). increased overjet was recorded when the distance was > 2 mm, and anterior crossbite was recorded when the distance was < 0 mm. normal overbite was defined when maxillary incisors overlapped mandibular incisors by 2 mm. overbite greater than 2 mm was designated deep overbite. anterior crossbite was recorded when the mandibular incisors were observed in front of the maxillary incisors. anterior open bite was recorded in the absence of contact between anterior teeth when posterior teeth were in occlusion. posterior crossbite was recorded when maxillary primary molars were occluded in lingual relationship to mandibular primary molars in centric occlusion. the schoolchildren were diagnosed with absence of malocclusion when all the conditions were normal. when exhibiting at least one of the aforementioned conditions, they were classified as having absence of malocclusion6. the study methodology is presented in figure 1. figure 1. methodological flowchart. 613 schoolchildren and their families outcome variable 14 public schools age independent variables gender parental education household income caries experience – dmf-t malocclusion – who inclusion and exclusion criteria so ci od em og ra ph ic d at a c lin ic al da ta ecohis – child impact section ecohis – family impact section ecohis – total score o h r q ol 5 santos et al. statistical analysis initially, individual analyses were performed relating the study variables (sex, race, income, father’s and mother’s educational level) as outcome variables (impact of oral health on quality of life-ecohis of the child, family, and total scores), estimating the raw odds ratios with the respective confidence intervals of 95%. the variables with p < 0.20 in the individual analyses were tested in the multiple logistic regression models, and those with p < 0.10 remained in the model, estimating the adjusted odds ratio with the respective confidence intervals of 95%. analyses were performed in the r (r foundation for statistical computing, vienna, austria) program. results according to table 1, of the schoolchildren evaluated, 40.9% presented impact of oral health on quality of life (ecohis > 0). furthermore, children of families with income ≤ r$2000 were observed to have 1.39 (95% ci: 0.97-2.00) (p = 0.0696) times more chance of presenting impact of oral health on quality of life. regarding the clinical conditions, 39.2% presented malocclusion, and those with caries experience had 3.37 (95% ci: 2.29-4.97) (p < 0.0001) times more chance of presenting impact of oral health on quality of life. according to table 2, 17% of the families analyzed presented impact of oral health on quality of life (ecohis > 0). families with income ≤ r$2000 were observed to have 2.56 (95% ci: 1.47-4.46) (p = 0.0009) times more chance of presenting impact of oral health on quality of life. families with children with caries experience had 4.46 (95% ci: 2.84-6.99) (p < 0.0001) times more chance of presenting impact of oral health on quality of life. when the instrument was evaluated by means of the total scores, the ecohis of the child and family (table 3), it was observed that 44.7% of the children and/or families presented impact of oral health on quality of life. furthermore, families with income ≤ r$2000 were observed to have 1.43 (95% ci: 1.02-2.08) (p = 0.0359) times more chance of (income) causing impact on the quality of life of children and families. families with children with caries experience had 3.43 (95% ci: 2.31-5.08) (p < 0.0001) times more chance of presenting impact of oral health on quality of life. discussion the aim of this study was to contextualize the association of socioeconomic and clinical factors with the impact of ohrqol, from the perspective of family structure, because the clinical factors in isolation did not reflect the real impact of oral conditions on the individual’s well-being. clearly, despite the growing number of research studies on children’s ohrqol, there is a lack of studies related to family structure and support in the context of health20,21. 6 santos et al. ta bl e 1. u na dj us te d as se ss m en t o f t he a ss oc ia tio n of in di vi du al a nd c on te xt ua l v ar ia bl es o n th e o h r q ol o n th e ch ild ’s fa m ily s tr uc tu re c hi ld ’s li fe , b y ch ild im pa ct s ec tio n. v ar ia bl e c at eg or y n (% ) ec o h is ch ild im pa ct s ec tio n r aw o r ( $c i9 5% ) pva lu e a dj us te d o r ( ic 95 % ) pva lu e w ith ou t im pa ct w ith im pa ct n ( % ) n (% ) g en de r m al e 30 0 (4 8. 9) 17 7 (5 9. 0) 12 3 (4 1. 0) r ef fe m al e 31 3 (5 1. 1) 18 5 (5 9. 1) 12 8 (4 0. 9) 1. 00 (0 .7 21. 37 ) 0. 97 88 r ac e w hi te 40 6 (6 9. 3) 24 7 (6 0. 8) 15 9 (3 9. 2) r ef n on -w hi te 48 ( 8. 2) 24 ( 50 .0 ) 24 ( 50 .0 ) 1. 55 (0 .8 52. 83 ) 0. 15 01 m ul at to 13 2 (2 2. 5) 75 ( 56 .8 ) 57 ( 43 .2 ) 1. 18 (0 .7 91. 76 ) 0. 41 34 h ou se ho ld in co m e ≤m ed ia n (u p to u s$ 3 50 ) 40 6 (6 6. 2) 22 6 (5 5. 7) 18 0 (4 4. 3) 1. 53 (1 .0 82. 16 ) 0. 01 72 1. 39 (0 .9 72. 00 ) 0. 06 96 >m ed ia n (> u s$ 3 50 ) 20 7 (3 3. 8) 13 6 (6 5. 7) 71 ( 34 .3 ) r ef r ef fa th er ’s sc ho ol in g ≤8 y r o f s tu dy 50 1 (8 1. 7) 29 6 (5 9. 1) 20 5 (4 0. 9) 0. 99 (0 .6 61. 51 ) 0. 97 62 >8 y r o f s tu dy 11 2 (1 8. 3) 66 ( 58 .9 ) 46 ( 41 .1 ) r ef m ot he r’s sc ho ol in g ≤8 y r o f s tu dy 46 5 (7 5. 9) 27 1 (5 8. 3) 19 4 (4 1. 7) 1. 14 (0 .7 81. 67 ) 0. 48 97 >8 y r o f s tu dy 14 8 (2 4. 1) 91 ( 61 .5 ) 57 ( 38 .5 ) r ef c ar ie s ex pe rie nc e n o 46 4 (7 5. 7) 30 8 (6 6. 4) 15 3 (3 3. 6) r ef r ef ye s 14 9 (2 4. 3) 54 ( 36 .2 ) 95 ( 63 .8 ) 3. 47 (2 .3 65. 11 ) <. 0. 00 01 3. 37 (2 .2 94. 97 ) <. 0. 00 01 m al oc cl us io n n o 37 3 (6 0. 8) 22 5 (6 0. 3) 14 8 (3 9. 7) r ef ye s 24 0 (3 9. 2) 13 7 (5 7. 1) 10 3 (4 2. 9) 1. 14 (0 .8 21. 59 ) 0. 42 62 *o dd s ra tio ; $ c on fid en ce in te rv a 7 santos et al. ta bl e 2. u na dj us te d as se ss m en t o f t he a ss oc ia tio n of in di vi du al a nd c on te xt ua l v ar ia bl es o n th e o h r q ol o n th e ch ild ’s fa m ily s tr uc tu re c hi ld ’s li fe , b y fa m ily im pa ct s ec tio n. v ar ia bl e c at eg or y n (% ) ec o h is fa m ily im pa ct s ec tio n r aw o r ( $c i9 5% ) pva lu e a dj us te d o r ( ic 95 % ) pva lu e w ith ou t im pa ct w ith im pa ct n (% ) n (% ) g en de r m al e 30 0 (4 8. 9) 25 3 (8 4. 3) 47 ( 15 .7 ) r ef fe m al e 31 3 (5 1. 1) 25 6 (8 1. 8) 57 ( 18 .2 ) 1. 20 (0 .7 81. 83 ) 0. 40 19 r ac e w hi te 40 6 (6 9. 3) 34 6 (8 2. 2) 60 ( 14 .8 ) r ef n on -w hi te 48 ( 8. 2) 41 ( 85 .4 ) 7 (1 4. 6) 0. 98 (0 .4 22. 30 ) 0. 97 13 m ul at to 13 2 (2 2. 5) 10 3 (7 8. 0) 29 ( 22 .0 ) 1. 62 (0 .9 92. 66 ) 0. 05 49 h ou se ho ld in co m e ≤m ed ia n (u p to u s$ 3 50 ) 40 6 (6 6. 2) 32 0 (7 8. 8) 86 ( 21 .2 ) 2. 82 (1 .6 54. 84 ) 0. 00 02 2. 56 (1 .4 74. 46 ) 0. 00 09 >m ed ia n (> u s$ 3 50 ) 20 7 (3 3. 8) 18 9 (9 1. 3) 18 ( 8. 7) r ef r ef fa th er ’s sc ho ol in g ≤8 y r o f s tu dy 50 1 (8 1. 7) 41 5 (8 2. 8) 86 ( 17 .2 ) 1. 08 (0 .6 21. 88 ) 0. 78 03 >8 y r o f s tu dy 11 2 (1 8. 3) 94 ( 83 .9 ) 18 ( 16 .1 ) r ef m ot he r’s sc ho ol in g ≤8 y r o f s tu dy 46 5 (7 5. 9) 38 0 (8 1. 7) 85 ( 18 .3 ) 1. 52 (0 .8 92. 60 ) 0. 12 65 >8 y r o f s tu dy 14 8 (2 4. 1) 12 9 (8 7. 2) 19 ( 12 .8 ) r ef c ar ie s ex pe rie nc e n o 46 4 (7 5. 7) 41 4 (8 9. 2) 50 ( 10 .8 ) r ef r ef ye s 14 9 (2 4. 3) 95 ( 63 .8 ) 54 ( 36 .2 ) 4. 71 (3 .0 27. 34 ) <. 0. 00 01 4. 46 (2 .8 46. 99 ) <. 0. 00 01 m al oc cl us io n n o 37 3 (6 0. 8) 30 5 (8 1. 8) 68 ( 18 .2 ) r ef ye s 24 0 (3 9. 2) 20 4 (8 5. 0) 36 ( 15 .0 ) 0. 79 (0 .5 11. 23 ) 0. 29 90 *o dd s ra tio ; $ c on fid en ce in te rv al . 8 santos et al. ta bl e 3. u na dj us te d as se ss m en t o f t he a ss oc ia tio n of in di vi du al a nd c on te xt ua l v ar ia bl es o n th e o h r q ol o n th e ch ild ’s fa m ily s tr uc tu re c hi ld ’s li fe , b y to ta l s co re o f e c o h is . v ar ia bl e c at eg or y n (% ) ec o h is – to ta l s co re r aw o r ( $c i9 5% ) pva lu e a dj us te d o r ( ic 95 % ) pva lu e w ith ou t im pa ct w ith im pa ct n (% ) n (% ) g en de r m al e 30 0 (4 8. 9) 16 5 (5 5. 5) 13 5 (4 5. 0) r ef fe m al e 31 3 (5 1. 1) 17 4 (5 5. 6) 13 9 (4 4. 4) 0. 98 (0 .7 11. 34 ) 0. 88 30 r ac e w hi te 40 6 (6 9. 3) 23 3 (5 7. 4) 17 3 (4 2. 6) r ef n on -w hi te 48 ( 8. 2) 23 ( 47 .9 ) 25 ( 52 .1 ) 1. 46 (0 .8 02. 66 ) 0. 21 27 m ul at to 13 2 (2 2. 5) 67 ( 50 .8 ) 65 ( 49 .2 ) 1. 31 (0 .8 81. 94 ) 0. 18 32 h ou se ho ld in co m e ≤m ed ia n (u p to u s$ 3 50 ) 40 6 (6 6. 2) 20 9 (5 1. 5) 19 7 (4 8. 5) 1. 59 (1 .1 32. 24 ) 0. 00 78 1. 43 (1 .0 22. 08 ) 0. 03 59 >m ed ia n (> u s$ 3 50 ) 20 7 (3 3. 8) 13 0 (6 2. 8) 77 ( 37 .2 ) r ef r ef fa th er ’s sc ho ol in g ≤8 y r o f s tu dy 50 1 (8 1. 7) 27 7 (5 5. 3) 22 4 (4 4. 7) 1. 00 (0 .6 61. 51 ) 0. 98 96 >8 y r o f s tu dy 11 2 (1 8. 3) 62 ( 55 .4 ) 50 ( 44 .6 ) r ef m ot he r’s sc ho ol in g ≤8 y r o f s tu dy 46 5 (7 5. 9) 25 4 (5 4. 6) 21 1 (4 5. 4) 1. 12 (0 .7 71. 63 ) 0. 54 95 >8 y r o f s tu dy 14 8 (2 4. 1) 85 ( 57 .4 ) 63 ( 42 .6 ) r ef c ar ie s ex pe rie nc e n o 46 4 (7 5. 7) 29 1 (6 2. 7) 17 3 (3 7. 3) r ef r ef ye s 14 9 (2 4. 3) 48 ( 32 .2 ) 10 1 (6 7. 8) 3. 54 (2 .3 95. 24 ) <. 0. 00 01 3. 43 (2 .3 15. 08 ) <. 0. 00 01 m al oc cl us io n n o 37 3 (6 0. 8) 21 0 (5 6. 3) 16 3 (4 3. 7) r ef ye s 24 0 (3 9. 2) 12 9 (5 3. 8) 11 1 (4 6. 2) 1. 10 (0 .8 01. 54 ) 0. 53 50 *o dd s ra tio ; $ c on fid en ce in te rv al . 9 santos et al. it should be noted that, as an evaluation tool, the ecohis allowed for broadening this analysis, leaving the binomial disease individual and including the family. family structure is often referred to as the type of family in which a child is residing22, which justifies the choice of the ecohis as the outcome. detecting the impact of social and economic factors in the extended family perspective, as commonly employed, allows us to discuss the impact that oral problems have on this nucleus23. understanding this impact in the family nucleus allows us to suppose a greater adhesion of the families in the care of their children and to employ better directed strategies. the socioeconomic condition affected the ohrqol in all the contexts, in an increasing manner: of the child and, in a larger proportion, of the family. of the aspects evaluated, income was the factor that negatively impacted the family, leading the authors to conclude that low income has more chance of influencing quality of life. thus, the results corroborate the findings in the literature3-6,8,23, reaffirming that the child’s oral health status is frequently associated with social dimensions. the discrepancies of the results observed in the literature were related to the methodological differences. in some studies, the impact of socioeconomic conditions on ohrqol was evaluated only by the general score of the instrument, without determining the perspective of the child’s family structure. even when considering a sample with a low prevalence of dental caries, this experience had a negative impact on the quality of life of the child and the family, detected by the instrument of analysis. the importance of this perception can be felt by the fact that the parents of schoolchildren with a caries experience feel guilty because, at this age, children are dependent for the parents daily activities of oral hygiene. it is clear, therefore, that socioeconomic status and family structure may be significant predictors of children’s ohrqol12. of the schoolchildren evaluated in the present study, 63.7% presented some occlusal change. this prevalence was considered high when compared with the findings of other studies12,24 conducted in the same age group. however, malocclusion caused no impact on the quality of life of the child or his/her family, corroborating the results of previous studies24. the probable explanation for this is related to the stage of development and age group studied. in the stage of deciduous dentition, children still have no perception of the appearance of their teeth, which begins to be more important in the mixed dentition state, when the greater occlusal changes that affect the individual’s self-image begin25-27. this did not exclude the importance of evaluating orthodontic treatment need at school-going age or of acting in a preventive manner in deciduous dentition. although this study had a cross-sectional design, which does not allow a causal relationship to be established, it was possible to understand the influence of contextual and clinical factors in the children’s daily lives from different perspectives. moreover, the present study reinforces the need for using subjective measures associated with clinical criteria, considering the opinions of children and their families in the elaboration of treatment strategies. therefore, future studies of longitudinal designs are desirable to evaluate these effects over time. another important aspect to consider is the extent to which public policies directed toward oral health are indispensable, as far as the promotion of better quality of life for the population is concerned. 10 santos et al. in conclusion, the child’s family structure is affected by socioeconomic and clinical factors. low income and children with dental caries experience were negative aspects associated with ohrqol in the children’s family structure. references 1. feldens ca, dos santos dullius ai, kramer pf, scapini a, busato al, vargas-ferreira f. impact of malocclusion and dentofacial anomalies on the prevalence and severity 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19. grabowski r, stahl f, gaebel m, kundt g. relationship between occlusal findings and orofacial myofunctional status in primary and mixed dentition. part i: prevalence of malocclusions. j orofac orthop. 2007 jan;68(1):26-37. 20. kumar s, tadakamadla j, zimmer-gembeck mj, kroon j, lalloo r, johnson nw. parenting practices and children’s dental caries experience: a structural equation modelling approach. community dent oral epidemiol. 2017 dec;45(6):552-8. doi: 10.1111/cdoe.12321. 21. lai shf, wong mlw, wong hm, mcgrath cpj, yiu cky. factors influencing the oral health-related quality of life among children with severe early childhood caries in hong kong. int j dent hyg. 2019 nov;17(4):350-8. doi: 10.1111/idh.12414. 22. chan kl, chen m, chen q, ip p. can family structure and social support reduce the impact of child victimization on health-related quality of life? child abuse negl. 2017 oct;72:66-74. doi: 10.1016/j.chiabu.2017.07.014. 23. nkansah-amankra s, luchok kj, hussey jr, watkins k, liu x. effects of maternal stress on low birth weight and preterm birth outcomes across neighborhoods of south carolina, 2000–2003. matern child health j. 2010 mar;14(2):215-26. doi: 10.1007/s10995-009-0447-4. 24. perazzo mf, gomes mc, neves et, martins cc, paiva sm, costa emmb, et al. oral problems and quality of life of preschoold children: self-reports of children and perception of parents/caregivers. eur j oral sci. 2017 aug;125(4):272-9. doi: 10.1111/eos.12359. 25. gomes mc, clementino ma, pinto-sarmento tc, costa em, martins cc, granville-garcia af, et al. parental perceptions of oral health status in preschool children and associated factors. braz dent j. 2015 jul-aug;26(4):428-3 doi: 10.1590/0103-6440201300245. 26. gomes mc, perazzo mf, neves ét, martins cc, paiva sm, granville-garcia af. oral problems and self-confidence in preschool children. braz dent j. 2017 jul-aug;28(4):523-30. doi: 10.1590/0103-6440201601295. 27. gomes mc, neves étb, perazzo mf, paiva sm, ferreira fm, granville-garcia af. contextual and individual determinants of oral health-related quality of life among five-year-old children: a multilevel analysis. peer j. 2018 aug;29:e5451. doi: 10.7717/peerj.54. https://www.ncbi.nlm.nih.gov/pubmed/28653417 https://www.ncbi.nlm.nih.gov/pubmed/?term=gomes mc%5bauthor%5d&cauthor=true&cauthor_uid=29160407 https://www.ncbi.nlm.nih.gov/pubmed/?term=perazzo mf%5bauthor%5d&cauthor=true&cauthor_uid=29160407 https://www.ncbi.nlm.nih.gov/pubmed/?term=martins cc%5bauthor%5d&cauthor=true&cauthor_uid=29160407 https://www.ncbi.nlm.nih.gov/pubmed/?term=paiva sm%5bauthor%5d&cauthor=true&cauthor_uid=29160407 https://www.ncbi.nlm.nih.gov/pubmed/?term=granville-garcia af%5bauthor%5d&cauthor=true&cauthor_uid=29160407 https://www.ncbi.nlm.nih.gov/pubmed/29160407 https://www.ncbi.nlm.nih.gov/pubmed/?term=gomes mc%5bauthor%5d&cauthor=true&cauthor_uid=30186681 https://www.ncbi.nlm.nih.gov/pubmed/?term=neves %c3%89tb%5bauthor%5d&cauthor=true&cauthor_uid=30186681 https://www.ncbi.nlm.nih.gov/pubmed/?term=perazzo mf%5bauthor%5d&cauthor=true&cauthor_uid=30186681 https://www.ncbi.nlm.nih.gov/pubmed/?term=paiva sm%5bauthor%5d&cauthor=true&cauthor_uid=30186681 https://www.ncbi.nlm.nih.gov/pubmed/?term=ferreira fm%5bauthor%5d&cauthor=true&cauthor_uid=30186681 https://www.ncbi.nlm.nih.gov/pubmed/?term=granville-garcia af%5bauthor%5d&cauthor=true&cauthor_uid=30186681 https://www.ncbi.nlm.nih.gov/pubmed/30186681 1http://dx.doi.org/10.20396/bjos.v21i00.8666341 volume 21 2022 e226341 original article 1 department of child dental health, lagos state university teaching hospital, lagos, nigeria. 2 department of child dental health, college of medicine, university of lagos, lagos, nigeria. 3 department of preventive dentistry, faculty of dentistry, university of medical sciences, ondo state, nigeria. 4 department of preventive dentistry, faculty of dentistry, lagos state university college of medicine, lagos, nigeria. 5 faculty of family dentistry, national postgraduate medical college of nigeria, lagos, nigeria. 6 department of child dental health, faculty of dentistry, lagos state university college of medicine, lagos, nigeria. corresponding author: dr afolabi oyapero senior lecturer, department of preventive dentistry lagos state university college of medicine, lagos, nigeria. email: fola_ba@yahoo.com editor: dr altair a. del bel cury received: july 14, 2021 accepted: september 6, 2021 randomized controlled trial on the effectiveness of silver diamine fluoride in arresting caries in lagos, nigeria moses okechukwu azuoru1 , modupe olufunmilayo ashiwaju2 , augustine edomwonyi3 , afolabi oyapero4 , bola obisesan5 , aderinsola omotuyole6 treatment of dental caries in children still remains challenging due to lack of cooperation with conventional treatment modalities. recently, the use of silver diamine fluoride (sdf) has proved useful in addressing this challenge. aim: this clinical trial aimed to evaluate the effectiveness of silver diamine fluoride (sdf) in arresting caries in children in lagos, nigeria. methods: this was a phase iii balanced randomized controlled school based interventional study on 240 children. the study group was treated with sdf while gic was used in the control group. follow up visits in 2 weeks, 1 month, and 3 months were carried out to assess the treatment outcome. inferential statistics with the use of pearson chi-square test and independent student t-test were used at 5% level of significance. results: there was significant relationship between sdf and caries arrest in 2 weeks, 1 month and 3 months’ assessment period (p = 0.001). the control group showed continuous decline (71.7%, 54.3% and 50.9%) in restorative success from 2 weeks to 3 months respectively. the mean ± sd and confidence interval (ci) of arrested caries in the sdf group were 113± 1.24 and 113.1 – 113.5 respectively. in the control group the mean ± sd and ci of restorative success were 69.3±11.8 and 67.2 – 71.4. the effect size was 5.24. conclusion: the result of the study showed that sdf was effective in arresting caries in children without any harm and there was statistically significant difference in the use of 38% sdf in arresting caries in children. keywords: dental caries. glass ionomer cement. fluorides, topical. https://orcid.org/0000-0002-7613-8773 https://orcid.org/0000-0001-6767-2344 https://orcid.org/0000-0002-1531-610x https://orcid.org/0000-0003-4433-8276 https://orcid.org/0000-0001-5222-5453 https://orcid.org/0000-0003-0324-1144 2 azouru et al. introduction according to the global burden of disease study, an estimated 3.8 billion people are affected by dental caries1. in nigeria, the prevalence of caries among children is still high despite current preventive and control strategies2,3. recently, adeniyi et al.4 reported 14.8% prevalence of dental caries among children aged 5 – 10 years in lagos, nigeria. though this figure is still within the who millennium development goals target for dental caries, its impacts on children’s health cannot be over emphasized as it is estimated to be the most prevalent chronic childhood disease worldwide5. these impacts include pain and discomfort, difficulty in masticating, sleep and speech disturbance, poor self-esteem and social isolation among others. it can also negatively affect body weight, growth, school attendance, and school performance if left untreated5. the commonest treatment protocol for dental caries is traditional surgical intervention but prevention of caries is more cost effective and less invasive6. with the recent change from the surgical model, which places emphasis on restorative treatment, to a medical model which focuses on disease prevention and conservation of tooth structure, caries management in the present decade is fast becoming more patient friendly, effective and efficient7. despite recent advances, the management of dental caries still remains challenging particularly with children, the aged, vulnerable populations and special health care needs patients, where gaining cooperation is still a significant problem in traditional restorative treatment of dental caries8. furthermore, among those from low economic class, access to care and cost are hindrances to conventional dental caries treatment8,9. thus, innovative treatment approach that reduces the burden of care on patients and the need for more comfortable, effective and efficient treatment protocols that would promote patient cooperation have continued to engage dental researchers and clinicians in recent times. the primary focus in these researches has been the use of chemotherapeutic agents in the prevention and arrest of caries particularly in children10. consequently, a variety of chemotherapeutic agents such as metal ions, antibiotics and various types of fluoride containing agents have been developed, tested and used for preventing and arresting caries11. a notable innovative therapy among these is the use of silver diamine fluoride (sdf)10. silver diamine fluoride has clinical usefulness in children when patient cooperation for restorative dentistry is difficult due to situational anxiety, young age or intellectual and developmental disabilities12,13. sdf is also very useful where the restoration of primary tooth that is about to exfoliate is not an option. since this process requires non-invasive procedures, the risk of cross-infection is significantly reduced. clinical studies have shown that sdf prevents and arrests caries in children13. a review on sdf concluded that it is a safe, effective and efficient caries control agent that can be employed to meet the who millennium development goals for 21st-century12-15. randomized clinical trials on the effectiveness of sdf have been carried out in united states, europe and asia15-17. in an ex-vivo study, mei et al.18 reported a highly remineralized rich zone in calcium and phosphate on arrested caries lesion of primary teeth with sdf application. in a related study, milgrom et al.19 also reported that application of 38% sdf arrested caries and was effective for the short-term treatment of caries 3 azouru et al. in pre-school age children. there is however no available published or unpublished research in nigeria nor africa on the effectiveness of sdf in arresting caries after meticulous search of relevant literature. there is also no research evidence to support the use of sdf in africa despite the growing interest in this agent. thus, the aim of this study was to evaluate the effectiveness of 38% silver diamine fluoride in arresting caries in children in lagos, nigeria. materials and methods description of study area: the study was conducted in lagos, nigeria from october 2019 to march 2020. lagos lies between latitude 6.465422 and longitude 3.40644820. it is bordered essentially in the south by atlantic ocean and hence it is a coastal city. as epicenter of commerce and industry, it is a heterogeneous state with mixed proportion of different ethno-religious groups and socioeconomic class. more significantly, because it is a commercial city the production and consumption of refined sugars is very high21. this is a favorable predisposing factor for the development of caries. study population: children aged 3 – 10 years living in lagos, nigeria made up the study population. according to the national population commission the population of children aged 0 – 9 years in lagos state in 2006 population census was 2,109,86222. the study age group ranks highest in the prevalence of dental caries in lagos state estimated at 14.8%4. it is also the group with high incidence of early childhood caries4. more importantly, it is the age group that poses difficulty in tolerating traditional restorative technique due to situational anxiety, low emotional stability and low intellectual understanding23. study design: the study design was a phase iii balanced randomized controlled trial; parallel groups, multicentre school based interventional study conducted in lagos nigeria. ethical considerations: approval for the study was obtained from the lagos state university teaching hospital (lasuth) health research and ethics committee with reference number lrec/06/10/1221 dated 31 july 2019. approval for the study was also obtained from the lagos state government with reference no: ls/c-530/t.3/755-756 dated 28 oct 19 with respect to the use of schools and primary school children for the study. approval was also obtained from various school authorities where the study was conducted through the education secretaries of various districts used for the study. informed and signed consents were obtained from parents, guardians and caregivers of children who participated in the study. informed assent was obtained from the children during the study. participants with increased caries activities during the course of the study were planned to be withdrawn from the study and given conventional treatment. registration of trial: the clinical trial was registered at pan african clinical trials registry (pactr) with trial no: pactr201908699150281 dated 16/04/2019. trial was registered in accordance with who and icmje standards. sampling technique: a multistage sampling technique was utilized. eight (8) public primary schools in lagos state were selected in the first stage using computer gen4 azouru et al. erated random numbers, with the list of schools serving as the sampling frame. the second stage involved selection of classes in eligible classes in the schools by simple random sampling using the nominal rolls as a sampling frame. children who fulfilled the eligibility criteria for the study were eventually enlisted. eventual allocation of the recruited sample population into 2 equal groups was also done by simple randomization. sample size estimation: this was done with the formula: (zα + zβ ) 2 x 2p(1-p) d2 n = where: n = sample size required in each group; zα = value for α at desired confidence level of 95%; α = 0.05 (two-tailed test) = 1.96; zβ = value of β error, which is 1β (statistical power). at statistical power of 95% and β error of 5%, zβ = 1.94; p = proportion of dental caries in children aged 5 – 10 years in a reference study (prevalence is 14.8%; 0.148)4; and d = the minimum difference in the clinical performance between the two study groups. the minimum difference for this study was set at 20% in order to increase power of the study and give more validity to the study. hence d = 20%. thus, n = 96.7 with 10% attrition or follow up losses (9.67), minimum sample for each group was 107 or 214 for the two groups. however, 240 subjects were recruited in all. eligibility criteria inclusive criteria: children aged 3 -10 years, with dental caries icdas 5 or 6, with signed consent form from parents/guardians and signed or thumb printed the assent form that allowed for oral examination and application of intervention medicament and control treatment were included. exclusion criteria: children with symptomatic carious tooth (toothache or sensitivity), mobile carious tooth, with dental caries icdas 0-4; those who were exposed to fluoride from other sources apart from a dentifrice; those that were allergic to silver or heavy metals; with amelogenesis or dentinogenesis imperfecta; with oral ulceration, stomatitis, swelling or abscess; with cooperation challenge; obviously mentally retarded; systemic illness like asthma, epilepsy, leukaemia, kidney disease and those whose parent or guardian could not understand the consent documentation were excluded from the study. research questionnaire: an interviewer administered close ended structured questionnaire developed from previously validated questionnaires was used. it comprised sections a to e. section a to d captured a participant’s biodata, history, examination, and intervention and control data while section e documented the study outcomes. dental caries examination tools and treatment set: the caries examination tools and treatment set comprised a comfortable field examination mobile dental chair unit which was set up in a well-lit and airy room in the schools used for the study. the examination instrument comprised standard who periodontal probe, wooden spatula, mouth mirror, 5ml hypodermic syringes with water, cotton gauze and rolls, meth5 azouru et al. ylated spirit and dental tray. the treatment set for sdf comprised dispensing dish, microbrushes, guage, cotton wool/rolls and dental tray. the treatment set for gic restoration comprised atraumatic hand instruments (excavators, condensers, explorer, tweezers, dental hatchet, mouth mirrors, carvers, examination probes and periodontal probes), mixing pads, mixing spatula, plastic applicators, gauge, wedges, cotton wool/rolls/pellets, petroleum jelly and dental tray. the researchers were trained and calibrated to use these tools and instrument for diagnosis of icdas 5 or 624, treatment of caries and assessing the study outcome which is arrested caries using icdas ii criteria by a consultant paediatric dentist. intervention medicament and control medicament: the intervention or experimental medicament for the study was 38% silver diamine fluoride: tedequim s.r.l b.25 the control medicament was glass ionomer cement (gic): prevestdenpro.26 details and sequence of data collection phase i recruitment and intervention phase. day 1: on the first day in each school, school children aged 310 which cut across nursery 1-2 and primary 1-6 were examined for dental caries icdas 5 and 6. the examination was done in a well-lit open hall or school clinic provided by the school. the children were made to sit comfortably in a mobile dental chair unit. examination of icdas 5 and 6 caries was done by direct visual examination using wooden spatula by two calibrated examiners. records of the children with icdas 5 and 6 were taken and documented. the documented children with icdas 5 and 6 were given consent document and forms to give to their parents to authorize intervention. day 2 interventions: the children that were given the consent document and forms were recalled on day 2. the children with signed consent forms were randomized into experimental group and control group by randomization. the children in the two groups were counseled and then signed or thumb print the assent form. the children in group 1 received sdf while the children in group 2 received gic. administration of sdf: the children in group 1 thereafter were treated with topical sdf. using a comfortable mobile chair unit, section a c of the questionnaire were administered to the children by the investigators. thereafter, the affected carious tooth with icdas 5 or 6 was gently cleaned, dried and isolated with cotton rolls. two drops of sdf solution was then applied with a disposable microbrush on the carious tooth. the treated participants were instructed not to rinse for the next 10 minutes. subjects were also advised to continue their normal oral hygiene care. the intervention received with the treatment date as well as the tooth treated (icdas 5 or 6) and the caries class was all documented on section d (intervention section) of the questionnaire. the date of the treatment and the school name were also recorded in the study book to calculate subsequent time of follow up visits. administration of gic: the children in group 2 received gic intervention. like in group 1, the children in group 2 were administered section a c of the questionnaire by the investigator and the research assistant (house officer). thereafter the researcher administered conventional gic. the carious lesion of the affected tooth 6 azouru et al. with icdas 5 or 6 was excavated with disposable plastic excavator. the tooth was cleaned, dried and isolated with cotton rolls. conventional gic was then mixed in a high viscosity consistency and condensed on the tooth cavity and allowed to set. petroleum jelly as a separating medium was then used to seal the gic surface from saliva. the participants were instructed not to rinse for the next 10 minutes. participants were also advised to continue their normal oral hygiene care. the intervention received with the treatment date as well as the tooth treated (icdas 5 or 6) and the caries class was all documented on section d (intervention section) of the questionnaire. the dates of the treatment intervention and the school name were also recorded in the study book to calculate subsequent time of follow up visits. phase ii outcome phase: since it was a prospective study the second phase assessed the treatment outcomes. the outcomes were assessed in 2 weeks, 1 month and 3 months respectively. the outcomes that were assessed were as follows: primary outcome: the expected primary outcome for the experimental group was arrested caries while that for the control group was restorative success. arrested caries for sdf group (experimental group): arrested caries was assessed using the icdas ii criteria and findings were recorded in the questionnaire. the researcher examined the treated teeth for arrested and active caries. the assessment was done using visual tactile examination with aid of who probe and mouth mirror using the icdas ii criteria24 to classify active and arrested caries. restorative success for gic group (control group): the restorative success of glass ionomer cement was determined when signs of restorative failure were absent. signs of gic restorative failure included restoration losses (full or partial loss), fractures and wear. assessment of restorative failure was done using both visual and tactile examination with the aid of examination probe and mouth mirror. the restoration was gently probed to assess for any sign of failure such as fracture, wear, partial loss or total loss. findings were recorded accordingly in the participant’s questionnaire. the type of restorative failure was also noted in each case. the follow-up outcome assessments were carried out in 2 weeks, 1 month and 3 months respectively after the intervention. secondary outcomes: the secondary outcomes that were assessed in both the experimental group and control group included toothache, sensitivity, stain, nausea, vomiting, silver allergy, oral soft tissue ulceration, rashes and any other noted complications. the assessments for secondary outcomes were done concurrently with the assessment of primary outcome. there was no case that necessitated hospital referral. data analysis: the obtained data was analyzed using statistical package for social sciences (spss ibm new york, usa) windows version 23. data were coded and entered into microsoft excel spreadsheet and later imported into spss for cleaning and analysis. descriptive statistics with the use of frequencies, percentage/proportion, mean and standard deviation were used to summarize data. inferential statistics with the use of pearson chi-square test, fisher exact test and independent student t-test were used to test for association between bivariate at 5% level of significance (95% confidence interval). a value of p < 0.05 was considered statistically significant. 7 azouru et al. results a total of 240 school children aged 4 – 10 years participated in the study. out of these, 124(51.7%) were male while 116(48.3%) were female, giving a male-to-female ratio of 1.1:1. the sdf group had 64(53.3%) males and 56(46.7%) females giving male to female ratio of 1.1:1 the control group had 60 (50.0%) male and 60(50.0%) female giving male to female ratio of 1:1. enrollment assessed for eligibility (2,168) male (1,014) female (1,154) randomized (240) allocation (baseline) follow-up analysis lost to follow-up due to tooth exfoliation (2) lost to follow-up due to tooth exfoliation (4) • @ 2 weeks (120) • @ 1month & 3 months (118) analyzed • @ 2 weeks (120) • @ 1month & 3 months (116) analyzed • received intervention (120) • declined intervention (0) control group (120) • received intervention (120) • declined intervention (0) intervention group (120) • not meeting inclusion criteria (1,869) • parents declined consent (47) • declined assent (12) excluded (1,928) figure 1. participants flowchart the mean dmft in sdf group was 1.87±0.9 while the mean of control group was 1.75 ± 0.9. the total mean dmft was 1.81 ± 0.9. overall, dmft score1 had the highest percentage of 47.9% closely followed by dmft score 2 with 32.8%. dmft score 5 had the lowest percentage of 0.04%. there was no significant difference between the dmft scores in the two groups (p>0.05). table 1 8 azouru et al. table 1. distribution of dmft scores of participants variables sdf group (n=120) n (%) control (n=120) n (%) total (n=240) n (%) t-test* p dmft scores 1 52 (43.3) 63(52.5) 115(47.9) 1.032 0.302 2 42(35.0) 35(29.2) 77(32.8) 3 17(14.2) 14(11.6) 31(12.9) 4 8(6.6) 6 (5.0) 14( 5.8) 5 0( 0.0) 1 (0.8) 1 (0.04) 6 1 (0.8) 1 (0.8) 2 (0.1) mean±sd 1.87±0.9 1.75 ± 0.9 1.81 ± 0.9 *independent t-test table 2 shows the arrested/restorative success at different intervals of assessment. at 2 weeks’ outcome assessment, 94.2% of the treated carious teeth in the sdf group showed hard arrested dentine, while 71.7% of the restored carious teeth in the control group showed restorative success. at the 1-month outcome assessment, the sdf group had 97.5% of the teeth showing arrested caries, while the control group had 54.3% showing restorative success. at 3 months’ outcome assessment, 94.9% of the carious lesions in teeth treated with sdf were still arrested, while the control group showed 50.9% of teeth with restorative success. chi-square test of independence showed there was significant relationship between sdf and caries arrest at 2 weeks, 1 month and 3 months’ assessment period respectively (p = 0.001). there was a marginal increase of 3.3% in caries arrest in the sdf group between 2 weeks and 1-month outcome assessment. however, there was a slight decline in caries arrest of 2.6% between 1 month and 3 months of outcome assessment. the control group showed continuous decline (71.7%, 54.3% and 50.9%) in restorative success from 2 weeks to 3 months respectively. table 2. distribution of arrested caries /restorative success at different intervals of assessment variables sdf group (n=120) n(%) control (n=120) n(%) total x2 p-value 2 weeks’ assessment success 113(94.2) 86(71.7) 199(82.9) 21.444 <0.001* failure 7(5.8) 34(238.3) 41(17.1) 1 month’s assessment success 115(97.5) 63(54.3) 178(79.1) 60.031** <0.001* failure 3(2.5) 53(45.7) 56(23.9) 3 months’ assessment success 112(94.9) 59(50.9) 171(73.1) 57.700 <0.001* failure 6(5.1) 57(49.1) 63(26.9) two (2) and 4 subjects lost to follow up at experimental group and control group respectively for one month and 3 months’ assessments **fischer exact test 9 azouru et al. table 3 shows the intervention failure at different intervals of assessment. in the sdf group, observed failure signs ranged from presence of dull enamel/dentine to soft dentine on examination. in the control group observed signs of failure ranged from partial loss of restoration to total loss and wear/fracture of restoration. at 2 weeks’ outcome assessment, there were 7 non arrested carious teeth in the sdf group: 3(42.9%) were as a result of soft dentine while 4(57.1%) were as a result of dull enamel/dentine. at 1-month assessment, sdf group showed 3 non arrested cavities evidenced by dull enamel/ dentine. at three (3) months assessment. 5 treated teeth had dull dentine while one (1) had soft dentine. the control group recorded a greater number of restorative failures. there was a progressive failure rate of restoration. the restorative failure rate increased with time of assessment. a total of 57 restorative failures were observed out of 120 restored teeth giving a restorative failure rate of 47.5%. at 2 weeks’ assessment there were a total of 34 restorative failures; 13(38.2%) due to partial loss of restoration, 15(44.1%) due to total loss and 6(17.6%) due to wear of restoration. one-month assessment showed a total of 50 restorative failures; 9(16.1%) was due to partial loss, 40(75.5%) as a result of total loss and one (1.9%) due to wear of restoration. at 3 months’ assessment, there were 57 restorative failures: 7(12.3%) due to partial loss, 49(86.0%) as a result of total loss and one (1.8%) due to wear of restoration. table 3. distribution of intervention failure at different intervals of assessment sdf group n(%) control n(%) total x2 p-value 2 weeks 41.000 <0.001* dull enamel/dentine 4(57.1) 0(0.0) 4(9.8) partial loss of filling 0(0.0) 13(38.2) 17(31.7) soft dentine 3(42.9) 0(0.0) 3(7.3) total loss of filling 0(0.0) 15(44.1) 15(36.6) wear of filling 0(0.0) 6(17.6) 6(14.6) 1 month 26.415 <0.001* dull enamel/dentine 3(100.0) 3(5.7) 6(10.7) partial loss of filling 0(0.0) 9(17.0) 9(16.1) total loss of filling 0(0.0) 40(75.5) 40(71.4) wear of filling 0(0.0) 1(1.9) 1(1.8) 3 months 63.000 <0.001* dull enamel/dentine 5(83.3) 0(0.0) 5(7.9) partial loss of filling 0(0.0) 7(12.3) 7(11.1) soft dentine 1(16.7) 0(0.0) 1(1.6) total loss of filling 0(0.0) 49(86.0) 49(77.8) wear of restoration 0(0.0 1(1.8) 1(1.6) *fisher exact test 10 azouru et al. the mean ± sd and confidence interval (ci) of arrested caries in the sdf group were 113± 1.24 and 113.1 – 113.5 respectively. in the control group the mean ± sd and ci of restorative success were 69.3±11.8 and 67.2 – 71.4. the effect size ‘d’ of the study was 5.24. table 4 table 4. mean outcome of the two groups at different intervals of assessment and the effect size variables sdf n =120 control n =120 mean ± sd sdf mean ± sd control d arrested/rs 2 weeks 113 86 113.3±1.24 69.3±11.8 5.24 1 month 115 63 3 months 112 59 variance 1.5* 141.5* 95% ci 113.1113.5 67.2 -71.4** d = effect size, * variance, **ci rs restorative success at two weeks’ secondary outcome assessment, 6.7% of participants reported slight tooth sensitivity in the sdf group while 5.0% of participants reported tooth sensitivity in the control group. at 1-month, tooth sensitivity reduced in the sdf group to 0.8% while there was an increase in the control group to 5.2%. at 3 months the percentages of tooth sensitivity remained same for both the sdf group and control group at 0.8% and 5.2% respectively. there was significant relationship between tooth sensitivity and treatment modality at 1 month and 3 months’ assessment periods (p < 0.05). -table 5 table 5. tooth sensitivity at different intervals of assessment study group (n=120) control (n=120) total x2 p-value 2 weeks’ assessment yes 8(6.7) 6(5.0) 14(5.8) 0.303 0.582 no 112(93.3) 114(95.0) 226(94.2) 1-month assessment yes 1(0.8) 6(5.2) 7(3.0) 3.770** 0.041* no 117(99.2) 110(94.8) 227(97.0) 3 months’ assessment yes 1(0.8) 6(5.2) 7(3.0) 3.770** 0.041* no 117(99.2) 110(94.8) 227(97.0) two (2) and 4 subjects lost to follow up at experimental group and control group for one and 3 months’ assessments **fischer exact test 11 azouru et al. twenty-four (24) hours after the interventions, there was no adverse effect in both the sdf group and control group. at 2 weeks follow up 99.2% of the participants in the sdf group had black stain without pain on the treated tooth while only one (0.8%) participant reported slight pain in the control group. at 1 month, 98.3% of the treated teeth in the sdf group showed black stain while none in the control group had any adverse effect. at 3 months follow up the black stain on treated teeth declined to 97.4% while there was no adverse effect in the control group. no allergic reaction or any other adverse effects was observed in both sdf group and control group at different intervals of assessment. black teeth stains in the sdf group were statistically significant at 2 weeks, 1 month and 3 months’ assessment period (p <0.001). -table 6 table 6. distribution of adverse effect of intervention at different intervals of assessment sdf group (n=120) (%) control (n=120) (%) total fischer exacts p-value 24-hour assessment nil adverse effect 120(100.0) 120(100.0) 240(100.0) 0.000 1.000 2 weeks’ assessment black stain 119(99.2) 0(0.0) 119(49.6) slight pain 0(0.0) 1(0.8) 1(0.4) 236.033 <0.001* nil black stain/pain 1(0.8) 119(99.2) 120(50.0) 1-month assessment black stain 116(98.3) 0(0.0) 116(48.3) 226.802 <0.001* nil black stain/pain 2(0.02) 116(100) 118(49.2) 3-month assessment black stain 115(97.4) 0(0.0) 116(48.3) 226.802 <0.001* nil black stain/pain 3(0.06) 116(100) 118(49.2) two (2) and 4 subjects lost to follow up at experimental group and control group respectively for one and 3 months’ assessments discussion the intervention teeth used in the study were posterior primary teeth. most studies on the effectiveness of sdf in arresting caries in children also used posterior primary molars23,27,28. in this study, the second primary molars constituted the highest percentage (57.1%) of teeth used while the central incisors were least (0.4%). the high involvement of primary molars especially the second molar in caries formation could be due to the fact they are big, have broad surface areas with pits, grooves and fissures and are regularly used for chewing. secondly, within the age bracket of the study, the primary molars experience more time exposure to caries than even the first permanent molars. with respect to icdas class, this study observed high icdas 5 icdas 6 ratio of 1.5:1. this ratio is in congruence with rosenblatt et al.14 12 azouru et al. and zhi et al.17 in their separate studies using the icdas system on the effectiveness of sdf in arresting caries in children. icdas 5 caries was more in this study because cavitation is minimal compared to icdas 6 and most often asymptomatic. hence, it is unnoticed or ignored. the clinical effectiveness of 38% sdf in arresting dental caries among children has been extensively researched and well documented11,14,16,29-35. both rcts and systematic reviews have been carried out16,29,32-40 to evaluate its short and long-term effectiveness. in this study, the caries-arresting rate of 38% sdf was found to be 94.2% in 2 weeks, 97.5% in 1 month and 94.9% in 3 months. these findings agree comparably with recent clinical trials. santos jr et al.37 reported 81% caries arrest in 1 week and 72.7% in 5 months. in a short-term clinical trial, milgrom et al.19 demonstrated 77.6% caries arrest in 2 weeks with 38% sdf while clemens et al.32 reported 100% caries arrest after 3 months. furthermore, zhi et al.17 reported 91% caries arrest in 6 months and 79% arrest in 12 months while llorda et al.33 reported 77% caries arrest in 6 months with 38% sdf. a systematic review conducted on 8 studies concluded an overall proportion of 81% caries arrest after sdf treatment38 while another systematic review in 2016 documented a 65% caries arrest with sdf29. findings from this present study are in agreement with the reports from these systematic reviews of the effectiveness of sdf. both this present study and all the compared recent studies adhered strictly to consort guidelines for conducting rct; had similar methodology, though the different brands of 38% sdf used could be responsible for the varying rates of success observed. on the other hand, no recent study has reported low effectiveness. therefore, this study further validates the effectiveness of sdf in arresting caries in children. in addition, this study compared the effectiveness of sdf and restorative success rate of art (gic) at different intervals in the treatment of caries. compared to the restorative success in the control group, sdf with statistically significant higher rates of treatment success at 2 weeks, 1 month and 3 months’ treatment intervals over the control (p< 0.001). this also is in line with the reports of studies that compared the effectiveness of sdf and gic at different intervals. zhi et al.17, monse et al.39, dos santos et al.40 and braga et al.41 who all reported statistically significantly higher success rates (p < 0.05). additionally, in order to appreciate the clinical importance of sdf in arresting caries, the effect size was also evaluated. the effect size of this study was 5.24 (p<0.001). most studies on sdf failed to report effect size. however, castillo et al.42 in a study comparing the effectiveness of sdf and placebo in arresting caries reported a higher effect size of 12.4 compared to this study. the difference in effect size could be due to the fact that higher effect size is expected to be produced with placebo as against an active treatment. nonetheless, this high effect size underlines the invaluable clinical importance of sdf in arresting caries. this underscores the fact that sdf is a reliable protocol in treatment of caries not only in community-based programs but also in the clinic. with respect to secondary outcome assessment, this study observed slight tooth sensitivity in both the sdf group and the control group. however, tooth sensitivity was found to be statistically significant in the control group at 1 month and 3 months’ 13 azouru et al. periods of assessment (p < 0.05). the significant tooth sensitivity in the control group could likely be due to the fact that the study was field-based as against clinic based and caries excavation before placement of gic may not have been very thorough. secondly radiographic evaluation of carious teeth was not done in the field. nevertheless, milgrom et al.19 in a placebo controlled trial reported 16.0% slight tooth sensitivity in the sdf group even though it was not significant. this study observed more intervention failures in the control group compared to the study group. the study recorded 47.5% failure rate in the control group and total loss of restoration accounted for the highest proportion (40.8%). the treatment of multiple participants on the same day on the field rather than in the clinic where improved moisture control, superior lighting source and radiographic evaluation is more feasible may likely have contributed to this high failure rate. more importantly however, it has been documented that the drawbacks of gic include inadequate flexural strength, little toughness and low abrasive resistance leading to wear and loss of restoration thus necessitating frequent recall visits and follow up34. nevertheless similar studies have also recorded this proportion of failure in atraumatic restorative treatment (art) control group treatments17,38. many studies have documented and reported black staining of carious lesions as significant adverse effect associated with sdf treatment14,16,29,32,33. in this study, black staining of carious lesions was also observed to be associated with sdf treatment, which was significant at different assessment intervals. some studies recommend the use of potassium iodide along with sdf to keep staining to a minimum. no other adverse effect was observed to be significant in the sdf group. according to fda, tooth discoloration is not considered as harm that causes damage to health. however, this effect cannot be totally ignored as this drawback may cause dissatisfaction for children and parents, especially when treating anterior teeth. in addition, it can result in skin and mucosal stains which disappears after 2 days, thus, caution should be taken when applying this medicament. this study however has some limitations. paucity of published work on this subject in this part of the world made it difficult to have a sufficiently extensive local comparison for the findings from the study. furthermore, the assessment outcome of the study, which was proposed to be in 2 weeks, 1 month, 3 months and 6 months respectively, was limited to 2 weeks, 1 month and 3 months due to the shutdown of schools for many months in 2020 occasioned by the covid 19 pandemic. however, being the first study in nigeria and possibly the whole of africa to evaluate the effectiveness of 38% silver diamine fluoride in arresting caries, it provides vital reference data for further studies and also a possible template for policy proposals for implementing field based secondary preventive initiatives among nigeria’s large population of indigent children who have limited access to dental care. in conclusion, the result of the study showed that sdf was effective in arresting caries in children without any harm and there was statistically significant difference in the use of 38% sdf in arresting caries in children. the mean ± sd and confidence interval (ci) of arrested caries in the sdf group were 113± 1.24 and 113.1 – 113.5 respectively. in the control group the mean ± sd and ci of restor14 azouru et al. ative success were 69.3±11.8 and 67.2 – 71.4. the effect size ‘d’ of the study was 5.24. sdf was demonstrated to be effective and safe for short-term treatment of dental caries in children. source of funding this research did not receive any specific grant from funding agencies in the public, commercial, or not forprofit sectors. conflict of interest there are no conflicts of interest with respect to the study. dataset availability datasets related to this article will be available upon request to the corresponding author. references 1. gbd 2016 disease and injury incidence and prevalence collaborators. global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990-2016: a systematic analysis for the global burden of disease study 2016. lancet. 2017 sep;390(10100):1211-59. doi: 10.1016/s0140-6736(17)32154-2. 2. adeniyi aa, oyapero oa, ekekezie oo, braimoh mo. dental caries and nutritional status of school children in lagos, nigeria – a preliminary survey. j west afr coll surg. 2016 jul-sep;6(3):15-38. 3. oyapero a, bakare ti, goncalves tf. advancing oral health policy for mandatory dental screening before admission into public primary and secondary schools in lagos, nigeria. j family med 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tc, mancl ll, milgrom p. the short-term effects of diammine silver fluoride on tooth sensitivity: a randomized controlled trial. j dent res. 2011 feb;90(2):203-8. doi: 10.1177/0022034510388516. 1http://dx.doi.org/10.20396/bjos.v20i00.8660967 volume 20 2021 e210967 original article 1 faculty of medicine, department of pediatrics,college of medicine, gadarif university, gadarif, sudan. 2 departments of pediatrics and medical education, college of medicine, university of bisha, saudi arabia. 3 unaizah college of medicine and medical sciences, department of obstetrics and gynecology, qassim university, unaizah, kingdom of saudi arabia. *4 king khalid university, college of medicine, department of obstetrics and gynecology, abha, saudi arabia. * corresponding author: bahaeldin a. hassan king khalid university, college of medicine, department of obstetrics and gynecology, abha, saudi arabia. email: bahasuikt@hotmail.com received for publication: august 21, 2020 accepted: december 21, 2020 misconceptions and traditional practices toward infant teething symptoms among mothers in eastern sudan: a cross-sectional study mohammed ahmed a. ahmed1*,karimeldin m. salih2 , abdullah al-nafeesah 3, ishag adam3, bahaeldin a. hassan4,* there is no much published data on the mothers’ false beliefs about signs and symptoms associated with teething in sudan. aim: this cross-sectional hospital-based study was conducted to assess mothers` knowledge about infant teething process and to evaluate mothers’ practices used to alleviate teething disturbances in gadarif city, eastern sudan. methods: questionnaires were used to collect data. multivariate logistics regression models were performed and adjusted odds ratios (aor) and 95% confidence intervals (ci) were calculated. results/conclusion: of a total of 384 participating mothers, 126 (32.8%) had good knowledge about infant teething. the  mothers’ knowledge was associated with a higher number of children in the family (adjusted odds ratio [aor] = 1.14) and with having a job (aor = 2.22). mothers residing in rural areas (aor = 0.40) and mothers with lower than secondary education (aor = 0.43) were less likely to have good knowledge about teething. diarrhea (88.5%), fever (86.5%), an urge to bite (76.6%), and poor appetite (71.9%) were the signs and symptoms most attributed to teething by mothers. only  the mother’s knowledge about teething was associated with reporting fever as a sign. a considerable number (317; 82.6%) of mothers reported performing “dokhan” (acacia wood smoke), 313 (81.5%) preferred to administer paracetamol or other systemic analgesics, 262 (68.2%) agreed that a child with tooth eruption should be taken to a hospital or health center, and 216 (56.3%) believed that antibiotics relieved symptoms related to teething. keywords: tooth eruption. knowledge. signs and symptoms. mothers. child. http://dx.doi.org/10.20396/bjos.v20i00.8660967 mailto:bahasuikt@hotmail.com https://pubmed.ncbi.nlm.nih.gov/?sort=date&size=100&term=ahmed+maa&cauthor_id=31794569 https://orcid.org/0000-0002-0681-0827 https://pubmed.ncbi.nlm.nih.gov/?sort=date&size=100&term=al-nafeesah+a&cauthor_id=31794569 https://pubmed.ncbi.nlm.nih.gov/?sort=date&size=100&term=adam+i&cauthor_id=31794569 https://orcid.org/0000-0003-0528-9723 2 ahmed et al. introduction tooth eruption (teething) is a normal physiological process whereby a tooth moves from within the jaw (intraosseous position) to the oral cavity1. teething usually starts at around six months and continues up to around three years of age2. symptoms such as fever, diarrhea, dermatitis, constipation, irritability, respiratory diseases, repeated finger sucking, rashes, facial flushing, and poor appetite were believed to be associated with teething3,4. traditionally, “blistering, placing leeches on the gums, and cauterization of the back of the head were the treatment options reported by the parents for teething”5. mothers usually use systemic and topical analgesics to relieve teething pain and antibiotics to treat associated symptoms6. moreover, they have their children bite objects to alleviate symptoms7. many medical and non-medical (traditional or other) practices are used as teething treatments without consultation of a dentist or pediatrician8. mothers’ misconceptions about teething might lead to misdiagnosis and mismanagement of potentially serious childhood diseases9. therefore, ideas about teething must be assessed and changed if they are not based on evidence. although several relevant studies have been conducted in african and other neighboring countries6,10,11, little research has been conducted in sudan and none in eastern sudan8. therefore, the aim of this study was to assess mothers’ knowledge about teething and to evaluate their practices for alleviating teething symptoms in eastern sudan. materials and methods a cross-sectional hospital-based study was conducted between may 1st and may 31st, 2019 in gadarif pediatric teaching hospital in eastern sudan. gadarif is situated at a mean altitude of 496 m above sea level, has a population of 1,727,401 residents, covers an area of 75,000 km2, and lies between latitudes 14 and 16 north and longitudes 33 and 36 east. it is 400 km from the capital khartoum, on the ethiopian border. gadarif pediatric teaching hospital is a 170-bed tertiary care facility that serves as a referral center in gadarif state. it is a public hospital with dental unit which providing a free health services. between 150 and 200 patients refer to the pediatric outpatient clinic every day. between 40 and 80 patients are discharged within 24 hours. the pediatric hospital is staffed with 8 consultants, 10 specialists, and 25  medical doctors (registrars and residents). inclusion and exclusion criteria mothers with children between six months to three years of age referring to the hospital who agreed to participate were included in the study. mothers with only one child younger than six months or older that three years of age and mothers with more than one child whose youngest child was over three years old were excluded. mothers who did not agree to participate and mothers who were mentally unable to communicate were also excluded. questionnaire a questionnaire composed of four parts was used to collect data. 3 ahmed et al. the first part regarded sociodemographic information about the mothers and their families. the second part regarded mothers’ knowledge about teething. the third part concerned symptoms attributed by mothers to teething. the fourth part concerned treatments used by mothers for teething. responses to the second to the fourth sections were structured using “agree,” “disagree,” and “don’t know” options sample size a sample size of 384 mothers was calculated based on the expected knowledge rate of maximum 50%. the sampling was conducted at a 95% confidence level with a 5% margin of error and 80% power. statistics ibm spss statistics version 22.0 for windows was used for the statistical analysis. absolute numbers and frequencies were used to express mothers’ teething-related knowledge, experiences, and practices. multivariate logistics regression models (using backward likelihood ratios) were performed with mothers’ knowledge as a dependent variable (other models were performed for symptoms) and the mothers’ age, residence, and education, the children’s age and sex, and the number of children in the family as independent variables. adjusted odds ratios (aor) and 95% confidence intervals (ci) were computed. a two-sided p value less than 0.05 was considered statistically significant. results general characteristics of the study population a total of 384 mothers were enrolled in the study. the ranges and means ± standard deviations (sd) of the mothers’ age, number of children in the family, and age of the youngest child were 16–48 and 29.2 ± 6.4 years, 1–12 and 3.7 ± 2.4, and 6–36 and 14.7 ± 8.4 months, respectively. a total of 294 (76.6%) mothers resided in rural areas. the education level of 203 (52.9%) mothers was secondary or higher. of the 384 children involved in the study, 197 (51.3%) were male. mothers’ knowledge about tooth eruption four-fifths (80.5%) of the mothers knew that the first primary teeth erupt at 6–7 months, and 89.6% knew that the lower central incisors are the first to erupt. however, less than half (167; 43.5%) were aware that delayed eruption could be associated with the presence of a systemic disease. a total of 126 (32.8%) mothers answered correctly all the questions related to knowledge about teething (table 1). 4 ahmed et al. table 1. mothers’ knowledge about teething yes n (%) no n (%) i don’t know n (%) teeth start to erupt at 6–7 months. 309 (80.5) 45 (11.7) 30 (7.8) the lower central incisors are the first teeth to erupt. 344 (89.6) 26 (6.8) 14 (3.6) tooth eruption is complete at 2–3 years of age. 268 (69.8) 36 (9.4) 80 (20.8) delayed tooth eruption may indicate the presence of a systemic disease. 167 (43.5) 111 (28.9) 106 (27.6) the associations between demographic variables and mothers’ knowledge about teething are shown in table 2. logistic regression showed that knowledge about teething was associated with a higher number of children in the family (aor = 1.14, 95% ci = 1.4–1.27; p = 0.007) and with having a job (aor = 2.22, 95% ci = 1.21–4.01; p = 0.009). mothers residing in rural areas (aor = 0.40, 95% ci = 0.21–0.77; p = 0.006) and mothers with lower than secondary education (aor = 0.43, 95% ci = 0.25–0.73; p = 0.002) were less likely to have good knowledge about teething. the mother’s age and the age and sex of the youngest child were not significantly related to knowledge about teething (table 3). table 2. comparing the variables between women with poor and good knowledge variables total (n = 384) poor knowledge (n = 258) good knowledge (n = 126) p mean (sd) mother’s age (years) 29.2 (6.4) 29.0 (6.5) 29.6 (6.1) 0.377 number of children in the family 3.7 (2.4) 3.6 (2.3) 3.9 (2.6) 0.284 age of the youngest child (months) 14.7 (8.8) 14.2 (7.9) 15.8 (10.0) 0.092 number (%) residence urban 294 (76.6) 182 (70.5) 112 (88.9) <0.001 rural 94 (23.4) 76 (29.5) 14 (11.1) mother’s education level secondary or higher 203 (52.9) 118 (45.7) 85 (67.5) <0.001 below secondary 181 (47.1) 140 (54.3) 41 (32.5) mother’s occupation housewife 326 (84.9) 232 (89.9) 94 (74.6) <0.001 employee 58 (15.1) 26 (10.1) 32 (25.4) child’s sex male 197 (51.3) 132 (51.2) 65 (51.6) 1.000 female 187 (48.7) 126 (48.8) 61 (48.4) sd standard deviation 5 ahmed et al. table 3. multivariate logistic regressions analysis for the factors associated with good knowledge unadjusted adjusted variables or ci p or ci p mother’s age 0.95 0.90–1.01 0.150 residence urban reference rural 0.39 0.20–0.76 0.005 0.40 0.21–0.77 0.006 mother’s education level secondary or higher reference below secondary 0.40 0.23–0.70 0.001 0.43 0.25–0.73 0.002 mother’s occupation housewife reference employee 2.39 1.30–4.40 0.035 2.22 1.21–4.01 0.009 number of children in the family 1.283 1.109–1.48 0.001 1.14 1.4–1.27 0.007 age of the youngest child 1.250 0.912–1.70 0.156 child’s sex male reference female 0.97 0.61–1.53 0.887 or odds ratio, ci confidence interval perception of symptoms associated with teething diarrhea (88.5%), fever (86.5%), an urge to bite (76.6%), and poor appetite (71.9%) were the most common signs and symptoms attributed to teething by mothers (table 4). logistic regression showed that among all the examined factors (mother’s knowledge about teething, age, residence, education, and job status, age and sex of the youngest child, and number of children in the family) only the mother’s knowledge about teething was associated with reporting fever as a sign. none of the investigated factors were associated with reporting diarrhea as a sign (table 5). table 4. symptoms attributed to teething by mothers yes n (%) no n (%) i don’t know n (%) fever 332 (86.5) 46 (12.0) 6 (1.6) diarrhea 340 (88.5) 42 (10.9) 2 (0.5) vomiting 260 (67.7) 112 (29.2) 12 (3.1) irritability 284 (74.0) 82 (21.4) 18 (4.7) poor appetite 276 (71.9) 94 (24.5) 14 (3.6) excessive salivation 266 (69.3) 104 (27.1) 14 (3.6) sleep disturbance 206 (53.6) 156 (40.6) 22 (5.7) ear problems 110 (28.6) 233 (60.7) 41 (10.7) inflammation of the oral mucosa 153 (39.8) 189 (49.2) 42 (10.9) continue 6 ahmed et al. pain 198 (51.6) 165 (43.0) 21 (5.5) facial flushing 91 (23.7) 232 (60.4) 61 (15.9) gum irritation 233 (60.7) 118 (30.7) 33 (8.6) finger sucking 236 (61.5) 127 (33.1) 21 (5.5) constipation 85 (22.1) 264 (68.8) 35 (9.1) urge to bite 294 (76.6) 72 (18.8) 18 (4.7) rhinorrhea 215 (56.0) 139 (36.2) 30 (7.8) respiratory problems 177 (46.1) 155 (40.4) 52 (13.5) skin rash 59 (15.4) 260 (67.7) 65 (16.9) convulsion 50 (13.0) 272 (70.8) 62 (16.1) increased susceptibility to diseases 118 (30.7) 164 (42.7) 102 (26.6) table 5. multivariate logistic regressions analysis for the factors associated with fever and diarrhea as signs of infant teething fever diarrhea variables or ci p or ci p mother’s age 1.02 0.96–1.09 0.539 1.06 0.98–1.14 0.144 residence urban reference rural 1.98 0.87–4.48 0.102 0.52 0.25–1.12 0.097 mother’s education level secondary or higher reference below secondary 0.93 0.47–1.84 0.845 1.71 0.78–3.70 0.179 mother’s occupation housewife reference employee 1.61 0.57–4.50 0.365 0.62 0.25–1.54 0.310 number of children in the family 1.11 0.91–1.37 0.227 0.96 0.77–1.19 0.685 age of the youngest child 1.02 0.98–1.07 0.235 0.99 0.96–1.03 0.716 child’s sex male reference female 1.29 0.70–2.38 0.407 0.95 0.50–1.82 0.881 good knowledge reference poor knowledge 2.50 1.13–5.52 0.023 1.88 0.85–4.18 0.118 or odds ratio, ci confidence interval table 6 displays the mothers’ reported practices for alleviating pain and other teething symptoms. a considerable number of mothers (317; 82.6%) reported performing “dokhan” (acacia wood smoke), 313 (81.5%) preferred to administer paracetamol or other systemic analgesics, 262 (68.2%) agreed that a child with tooth eruption should be taken to a hospital or health center, and 216 (56.3%) believed that antibiotics relieved symptoms related to teething. continuation 7 ahmed et al. table 6. treatments provided by mothers for teething yes n (%) no n (%) i don’t know n (%) taking the child to a hospital or health center 262 (68.2) 109 (28.4) 13 (3.4) performing “dokhan” (acacia wood smoke) 317 (82.6) 62 (16.1) 5 (1.3) taking the child to grandmother 177 (46.1) 199 (51.8) 8 (2.1) taking the child to a more experienced mother in the neighborhood 113 (29.4) 261 (68.0) 10 (2.6) administering paracetamol or another systemic pain killer 313 (81.5) 66 (17.2) 5 (1.3) administering extra fluids to prevent dehydration (ors or other) 280 (72.9) 91 (23.7) 13 (3.4) administering antibiotics 216 (56.3) 153 (39.8) 15 (3.9) applying topical analgesia 99 (25.8) 252 (65.6) 33 (8.6) rubbing the gums with carrots 169 (44.0) 201 (52.3) 14 (3.6) using sesame oil 246 (64.1) 115 (29.9) 23 (6.0) giving a pacifier 95 (24.7) 239 (62.2) 50 (13.0) giving herbs 144 (37.5) 199 (51.8) 41 (10.7) extracting the teeth 74 (19.3) 283 (73.7) 27 (7.0) bottle feeding 98 (25.5) 273 (71.1) 13 (3.4) nothing 7 (1.8) 352 (91.7) 25 (6.5) discussion this study found that 32.8% of the participating mothers had good knowledge about teething. knowledge was associated with the number of children in the family, job status, residence, and education level. a study conducted in neighboring ethiopia reported that 65.4% of mothers knew that teeth start to erupt at 6–7 months of age, and 74.8% knew that the lower central incisors are the first to erupt. their knowledge was associated with their age and place of residence11. in a study conducted in saudi arabia, over three-quarters (87.5%) of participants had poor knowledge about teething, and none of the investigated factors were associated with knowledge6. another study similarly found that 60.5% of parents in saudi arabia had poor knowledge about teething and reported that knowledge was significantly associated with a higher number of children in the family9. a study conducted in jordan reported that 65.4% of mothers knew that tooth eruption normally starts at 6–7  months of age and found that the mothers’ knowledge was associated with their age9. a considerably higher knowledge rate (71.4%) about teething was reported in india12. we observed that diarrhea (88.5%), fever (86.5%), and an urge to bite (76.6%) were the signs and symptoms most commonly attributed to teething by mothers and that citing fever as a symptom was associated with the mother’s knowledge. this is in line with a study conducted in khartoum, sudan, which reported that fever (86.6%) and diarrhea (80.3%) were the symptoms most commonly attributed to teething8. however, in that study, the mothers’ age and educational status were not associated perceived8. in our study, there was no association between the child’s sex and systemic signs and symptoms reported by the mother. this is consistent with the findings of oziegbe et al.10, who also found no significant relationship between the child’s sex and signs and symptoms noticed by the mother9. also, a study in ethio8 ahmed et al. pia reported that 91.6% of the mothers believed that teething was associated with various symptoms, such as diarrhea (90.7%)11. owais  et  al. reported that 75% of the parents incorrectly associated fever and diarrhea with teething9. an urge to bite (93.1%), fever (87.0%), and diarrhea (83%) were the most common signs and symptoms reported by saudi parents6. contrary to our findings, fever (51.8%), diarrhea (12.5%), and vomiting (2.9%) were the most commonly reported signs and symptoms by nigerian mothers10. this and other studies’ findings show that although teething does not cause fever, diarrhea, or any respiratory illness, these are common misconceptions related to teething. unfortunately, such misconceptions may be obstacles to proper diagnosis and effective treatment of many illnesses. a high fever (>39°c) should not be considered a teething symptom and needs to be investigated. attention should be paid to detecting or excluding coincidental infections, such as upper respiratory and gastrointestinal infections. a plausible explanation for the presence of coincidental but unrelated systematic diseases during the teething period is a decrease in passive immunity/antibodies13. in our study, 81.5% of the mothers preferred to administer paracetamol or other systemic analgesics. in contrast, in a study in ethiopia, only one mother stated that the child should be given paracetamol to relieve symptoms, while 12.1% reported rubbing children’s gums with garlic, and 6.5% reported rubbing them with herbs to relieve teething pain11. in saudi arabia, 76.1% of parents used systemic analgesics, and 65.6% applied topical analgesics to children’s gums9. in our study, 56.3% of the mothers believed that antibiotics relieved teething-related symptoms. in saudi arabia, 45% of parents believed that antibiotics were indicated for teething6. antibiotic overuse or misuse is a harmful practice for both the individual and the community, as it can lead to bacterial resistance. unfortunately, in sudan, antibiotics are sold without restrictions. this study had certain limitations. as it was a single-center study, its results cannot be generalized to the rest of the population. moreover, as the data were obtained from mothers’ responses regarding teething, they may be subject to recall bias. in conclusion, this study documents a poor level of knowledge about teething, especially among mothers residing in rural areas and those with a low education level. ethics approval ethics approval was obtained from the ethics committee of the faculty of medicine of gadarif university (reference number: 2019/012), sudan. written informed consent was obtained from each participant. competing interests the authors declare that they have no competing interests. funding none received. 9 ahmed et al. acknowledgment the authors would like to thank all the mothers who participated in the study. references 1. cunha rf, pugliesi dm, garcia ld, murata ss. systemic and local teething disturbances: prevalence in a clinic for infants. j dent child (chic). 2004 jan-apr;71(1):24-6. 2. sahin f, camurdan ad, camurdan mo, olmez a, oznurhan f, beyazova u. factors affecting the timing of teething in healthy turkish infants: a prospective cohort study. int j paediatr dent. 2008 jul;18(4):262-6. doi: 10.1111/j.1365-263x.2007.00893.x. 3. massignan c, cardoso m, porporatti al, aydinoz s, canto gde l, mezzomo la, bolan m. signs and symptoms of primary tooth eruption: a meta-analysis. pediatrics. 2016 mar;137(3):e20153501. doi: 10.1542/peds.2015-3501. 4. tighe m, roe mf. does a teething child need serious illness excluding? arch dis child. 2007 mar;92(3):266-8. doi: 10.1136/adc.2006.110114. 5. hatibovic-kofman s, ari t. managing discomfort caused by teething. j can dent assoc. 2013;79:d141. 6. elbur ai, yousif ma, albarraq aa, abdallah ma. parental knowledge and practices on infant teething, taif, saudi arabia. bmc res notes. 2015 nov;8:699. doi: 10.1186/s13104-015-1690-y. 7. kumar s, tadakamadla j, idris a, busaily ia, alibrahim ay. knowledge of teething and prevalence of teething myths in mothers of saudi arabia. j clin pediatr dent. 2016;40(1):44-8. doi: 10.17796/10534628-40.1.44. 8. awadkamil m. mothers’ misconception and traditional practises towards infant teething’ symptoms in khartoum. iosr j pharm. 2012 jan;2(3):448-51. doi: 10.9790/3013-0230448451. 9. owais ai, zawaideh f, al-batayneh ob. challenging parents’ myths regarding their children’s teething. int j dent hyg. 2010 feb;8(1):28-34. doi: 10.1111/j.1601-5037.2009.00412.x. erratum in: int j dent hyg. 2010 nov;8(4):324. erratum in: int j dent hyg. 2017 aug;15(3):258. 10. oziegbe eo, folayan mo, adekoya-sofowora ca, esan ta, owotade fj. teething problems and parental beliefs in nigeria. j contemp dent pract. 2009 jul;10(4):75-82. doi: 10.5005/jcdp-10-4-75. 11. getaneh a, derseh f, abreha m, yirtaw t. misconceptions and traditional practices towards infant teething symptoms among mothers in southwest ethiopia. bmc oral health. 2018 sep;18(1):159. doi: 10.1186/s12903-018-0619-y. 12. kakatkar g, nagarajappa r, bhat n, prasad v, sharda a, asawa k. parental beliefs about children’s teething in udaipur, india: a preliminary study. braz oral res. 2012 mar-apr;26(2):151-7. doi: 10.1590/ s1806-83242012000200011. 13. jones m. teething in children and the alleviation of symptoms. j fam health care. 2002;12(1):12–3. 1http://dx.doi.org/10.20396/bjos.v19i0.8660181 volume 19 2020 e200181 original article 1 chair of dental materials, faculty of dentistry, university of the republic, montevideo, department of montevideo, uruguai. 2 department of dental materials, school of dentistry, federal university of rio grande do sul, porto alegre, rs, brazil. corresponding author: fabrício mezzomo collares department of dental materials, school of dentistry, federal university of rio grande do sul. ramiro barcelos street, 2492, rio branco, 90035-003, porto alegre, rs, brazil. fabricio.collares@ufrgs.br phone number: +55 51 33085198 received: june 22, 2020 accepted: september 25, 2020 surface and mechanical properties of adhesives with calcium phosphates challenged to different storage media marcelo matias mederos gómez1 , isadora martini garcia2 , vicente castelo branco leitune2 , fabrício mezzomo collares2,* aim: to evaluate the behavior of experimental dental adhesives with hydroxyapatite (hap), alpha-tricalcium phosphate (α-tcp) or octacalcium phosphate (ocp) after storing them in three different media: dry storage, distilled water, or lactic acid. methods: an experimental adhesive resin was formulated with bisphenol a glycol dimethacrylate, 2-hydroxyethyl methacrylate, and photoiniciator/co-initiator system. hap (ghap), α-tcp (gα-tcp), or ocp (gocp) were added to the adhesive resin at 2 wt.%, and one group remained without calcium phosphates to be used as a control (gctrl). the adhesives were evaluated for surface roughness, scanning electron microscopy (sem), and ultimate tensile strength (uts) after storing in distilled water (ph=5.8), lactic acid (ph=4) or dry medium. results: the initial surface roughness was not different among groups (p>0.05). ghap showed increased values after immersion in water (p<0.05) or lactic acid (p<0.05). sem analysis showed a surface variation of the filled adhesives, mainly for gα-tcp and ghap. ghap showed the highest uts in dry medium (p<0.05), and its value decreased after lactic acid storage (p<0.05). conclusions: the findings of this study showed that hap, ocp, and α-tcp affected the physical behavior of the experimental adhesive resins in different ways. hap was the calcium phosphate that most adversely affected the surface roughness and the mechanical property of the material, mainly when exposed to an acid medium. keywords: dentin-bonding agents. calcium phosphates. acids. polymers. tensile strength. http://orcid.org/0000-0002-1561-2283 https://orcid.org/0000-0002-7388-0200 https://orcid.org/0000-0002-5415-1731 https://orcid.org/0000-0002-1382-0150 2 gómez et al. introduction recurrent caries at the tooth-restoration interface is one of the major causes of restoration replacement over time1. this outcome is related to materials’ hydrolytic and enzymatic degradation when in contact with the oral environment, leading to higher surface roughness, biofilm accumulation at the margin, and caries development2. moreover, restorative materials may not completely seal the tooth interface. mainly over time, the sealing ability still is a concern since gaps are prone to caries development3. restorative resin-based materials have been modified to decrease polymerization shrinkage, hydrolysis degradation, and to decrease gaps formation at the interface via a biomimetic remineralization approach4. bioactive fillers have been added to resins to provide them bioactivity with the ultimate purpose of reducing the incidence of caries around the restoration’s margin. calcium orthophosphates (cap) are the most representative fillers able to release calcium and phosphate ions, which are retained in the oral biofilm and induce dental remineralization5-8. cap present different molecular forms, crystalline structures, and solubility values9,10. previous studies evaluated cap as fillers in experimental adhesive resins showing promising results such as increased bond strength11,12 and mineral deposition at the tooth6,8,11. currently, studies that evaluate ion-releasing bioactive materials, such as those filled with bioglasses and cap, stored them in water13,14, ethanol13, artificial saliva15 or simulated body fluid5. however, the release of ions may increase materials’ roughness over time16, especially when they are exposed to acid medium, which could occur in the presence of an acidogenic biofilm17. the results of a recent in vitro study suggest that low ph increases the surface roughness and alter the superficial topography of resin-based orthodontic adhesives18. the change in the roughness may indicate modifications not only in the morphology of the material but also in its chemical and physical stability19,20. the bioactive material must maintain its mechanical and chemical properties to seal the cavity adequately21. in a previous study, the authors formulated an experimental adhesive resin composed of bisphenol a glycol dimethacrylate, 2-hydroxyethyl methacrylate, and a photoinitiator/co-initiator system11. the material was filled with different calcium orthophosphates at 2 wt.%: hydroxyapatite (hap; ca5(po4)3(oh)), alpha-tricalcium phosphate (α-tcp; ca3(po4)2), or octacalcium phosphate (ocp; ca8h2(po4)65h2o) 11. the filled adhesives were compared to the base resin without cap (control group), and they showed a higher degree of conversion. furthermore, the α-tcp group showed a high microshear bond strength compared with the other groups. the α-tcp and hap groups induced mineral deposition at the tooth-resin interface, suggesting that these fillers could be an alternative to formulate bioactive dental resins. despite these findings, the effect of different storage media on the behavior of adhesives composed of α-tcp, hap, or ocp was not investigated so far. the aim of this study was to evaluate the behavior of experimental adhesive resins with hap, α-tcp, or ocp after storing them in three different storage media. the null hypotheses to be tested are: (1) there are no differences among the adhesives formulated with different cap regarding their surface and mechanical properties; (2) different storage media do not influence the surface and mechanical properties of the adhesives. 3 gómez et al. materials and methods in this study, the dependent variables analyzed are surface roughness, surface morphology, and ultimate tensile strength. two independent variables were analyzed: (1) the variable “addition of cap”, with four different adhesives composed solely by the base resin or with 2 wt.% of hydroxyapatite (hap), alpha-tricalcium phosphate (α-tcp), or octacalcium phosphate (ocp); (2) the variable “storage medium”, in which the adhesives were immersed: dry storage, distilled water, or acidic solution (figure 1). ocp ca8h2(po4)65h2o α-tcp ca3(po4)2 hap ca5(po4)3(oh) bis-gma hema photoinitiator system dry (no treatment) distilled water (ph 5.8) lactic acid (ph 4.0) adhesive resin storage media surface roughness scanning electron microscopy ultimate tensile strenght figure 1. representative illustration that summarizes the materials and methods of the study. experimental adhesive resin formulation the experimental adhesive resin was formulated mixing 66.66 wt.% bisphenol a glycol dimethacrylate and 33.33 wt.% 2-hydroxyethyl methacrylate. camphorquinone and ethyl 4-(dimethylamino) benzoate were added as a photoinitiator system at 1 mol%. butylated hydroxytoluene was added at 0.01 wt.% as polymerization inhibitor. these reagents were purchased from aldrich chemical, st louis, mo, usa. three cap were previously synthesized, and they were added in this base resin at 2 wt.%: α-tcp (gα-tcp, 6.03 µm) 22, ocp (gocp, 4.94 nm) 23 and hap (ghap, 26.7 nm) 24. a group without cap was used as control (gctrl), totaling four groups. the mixture (resin/particles) was hand-mixed for 5 min, sonicated for 180 s, and hand-mixed again for 5 min. surface roughness (ra) five samples per group were prepared (10 mm diameter x 1 mm thickness) using a polyvinylsiloxane mold. the uncured adhesive resins were inserted in the molds between two transparent mylar strips. the samples were light-cured for 30 s on each side (radii cal, sdi; bayswater, victoria, australia, 1200 mw/cm2). the top of each 4 gómez et al. sample was marked and divided in half. four measurements of surface roughness were performed immediately on one half using a profilometer (mitutoyo, surftest sj–201p, chicago, usa) with a tracing length of 2 mm and 0.25 mm cut-off. then, each sample was submerged and stored in individual-hermetic containers for 3 days at 37 °c17, in 10 ml25,26 of different media as distilled water or lactic acid with ph at 5.8 and 417, respectively. the samples were placed vertically so that the surfaces to be tested were kept exposed to the different media. the ph of these media was evaluated along with the study via a digital ph meter (dm-22, digimed, são paulo, sp, brazil). after 3 days of storage, four new measurements were performed on the other half on the top of each sample. the initial roughness (ra1), final roughness (ra2), and roughness variation (δra) were recorded for each group. surface morphology via scanning electron microscopy (sem) the surface morphology of three samples per group used in the roughness assessment (stored in distilled water and lactic acid) was evaluated via sem. other three samples per group were prepared and stored in a dry environment inside a desiccator with silicon dioxide at 37 °c for 3 days to be also analyzed via sem. the samples were placed on metallic stubs and gold-sputter coated (15–25 nm) (sdc 050, baltec, vaduz, liechtenstein). sem analysis (sem, jsm 6060, jeol, tokyo, japan) was performed under 7 kv, at 5,000´ and 8,000´ magnification. ultimate tensile strength (uts) thirty samples per group were prepared in a metallic matrix with an hourglass shape (8 mm long, 2 mm wide, 1 mm thickness, and 1 mm2 at constriction area) after photoactivation for 30 s on each side27. after photoactivation, the samples were measured with a digital caliper (mitutoyo, kawasaki, kanagawa, japan; accuracy of 10 µm) to obtain the constriction area of each one. then, the thirty samples from each group were divided and submerged into 1 ml of the three media of storage (n = 10, dry environment in a desiccator, distilled water, or lactic acid) to be sat for 3 days at 37 °c. the specimens were fixed in metallic jigs with cyanoacrylate resin to be tested for tensile strength. the tests were performed in a universal testing machine (ez test ez-sx, shimadzu, japan) at a crosshead speed of 1 mm/min. the values were obtained in newtons, and the final uts was expressed in megapascals (mpa) using the constriction area of each sample. statistical analysis the data were analyzed using the software sigmaplot®, version 12.0 (systat software, inc., san jose, ca, usa). data distribution was evaluated using the shapiro–wilk test. one-way analysis of variance (anova) was used to compare groups for initial surface roughness. paired t test was used for each group to evaluate the difference between immediate and final surface roughness. kruskal-wallis was used to compare δra among groups in both media, and dunn’s was used as post-hoc after immersion in lactic acid. two-way anova was used to compare groups for uts dry medium, distilled water or lactic acid. a significance level of 0.05 was considered. 5 gómez et al. results the results of surface roughness before (ra1) and after (ra2) immersion of the experimental adhesive resins, as well as the δra, in distilled water and in lactic acid are shown in table 1. the authors did not perform other statistical analysis such as threeway anova, split-plot two-way anova, or two-way anova with repeated measures to analyze this data because there is a dependency within the same group (the same group was tested before and after the storage in the liquids). moreover, the samples tested for immersion in water or lactic acid are not the same since this is a destructive method and the same sample could not be immersed in both liquids one after the other. statistical analysis with repeated measures considering different immersions (in water or lactic acid) should not be applied. in this context, the one-way anova revealed no statistically significant differences among groups for ra1 (p>0.05). after immersion in distilled water, ghap roughness increased (p<0.05), while the other groups showed no statistically significant differences (p>0.05). moreover, there was no difference among groups for δra after immersion in water (p>0.05). on the other hand, the materials presented different behavior after immersion in lactic acid solution. while there was no difference among groups for ra1 (p>0.05), the roughness of gα-tcp and ghap increased after immersion in lactic acid (p<0.05) and gctrl and gocp had no differences between ra1 and ra2 (p>0.05). when comparing δra after immersion in lactic acid, ghap showed the highest variation among groups, with statistical difference in comparison to gctrl (p<0.05). table 1. results of surface roughness of the experimental adhesive resins with different calcium phosphates before and after the immersion in distilled water or lactic acid. group storage in distilled water storage in lactic acid roughness before immersion roughness after immersion δra (%) roughness before immersion roughness after immersion δra (%) (ra1, μm) (ra2, μm) (ra1, μm) (ra2, μm) gctrl 0.09 (±0.01) aa 0.12 (±0.05) a 47.93 (±70.28) a 0.11 (±0.04) aa 0.13 (±0.03) a 24.74 (±49.88) b gocp 0.10 (±0.03) aa 0.15 (±0.07) a 59.35 (±76.50) a 0.10 (±0.04) aa 0.16 (±0.10) a 59.07 (±75.10) ab gα-tcp 0.08 (±0.03) aa 0.13 (±0.03) a 78.85 (±97.40) a 0.10 (±0.03) aa 0.21 (±0.06) b 116.94 (±83.58) ab ghap 0.07 (±0.01) aa 0.21 (±0.08) b 173.91 (±96.28) a 0.08 (±0.02) aa 0.30 (±0.06) b 285.64 (±110.34) a different capital letters indicate statistically significant difference in the same column (p<0.05). different small letters indicate statistically significant difference in the same row within the same medium of storage (distilled water or lactic acid) (p<0.05). the images from sem analyses corroborate the findings of surface roughness measurement. few differences can be observed in the surface of gctrl between dry storage and distilled water storage (figure 2 a–d). higher irregularities are identified for gctrl when it was stored in lactic acid (figure 2 e, f). as well as observed for gctrl, almost no differences are observed among images of dry storage and distilled water for gocp (figure 3 a–d). when exposed to lactic acid, more irregularities are observed (figure 3 e, f). compared with gctrl, gocp showed higher defects when exposed to 6 gómez et al. lactic acid. the surface of gα-tcp stored in distilled water, and lactic acid (figure 4 c–f) showed larger irregularities than gctrl. ghap showed the highest difference on the surface between dry storage and distilled water storage (figure 5 a–d) compared with gctrl, gocp, and gα-tcp. in addition, after storing in lactic acid (figure 5 e, f), ghap presented higher irregularities, with larger cracks and cavities with irregular borders distributed on an irregular surface. figure 2. scanning electron microscopy of gctrl at dry (no treatment), distilled water, or lactic acid storage. few differences are observed for this group without calcium phosphates addition when the surface is exposed to dry storage (a and b) compared to that after water storage (c and d). after the exposition to lactic acid, gctrl presents higher irregularities (e and f). n o tr ea tm en t control group a ft er 3 -d ay s to ra ge in di st ill ed w at er ( ph =5 .8 ) a ft er 3 -d ay s to ra ge in la ct ic a ci d (p h =4 .0 ) a c e b d f 7kv 7kv 7kv 7kv 7kv 7kv 5μm 5μm 5μm 2μm 2μm 2μm x5,000 x5,000 x5,000 x8,000 x8,000 x8,000 7 gómez et al. figure 3. scanning electron microscopy of gocp at dry (no treatment), distilled water, or lactic acid storage. few differences are observed for gocp when the surface is exposed to dry storage (a and b) compared to that after water storage (c and d). after the exposition to lactic acid, this group showed higher irregularities (e and f). n o tr ea tm en t octacalcium phosphate group a ft er 3 -d ay s to ra ge in di st ill ed w at er ( ph =5 .8 ) a ft er 3 -d ay s to ra ge in la ct ic a ci d (p h =4 .0 ) a c e b d f 7kv 7kv 7kv 7kv 7kv 7kv 5μm 5μm 5μm 2μm 2μm 2μm x5,000 x5,000 x5,000 x8,000 x8,000 x8,000 8 gómez et al. figure 4. scanning electron microscopy of gα-tcp at dry (no treatment), distilled water, or lactic acid storage. high differences are observed within this group when “no treatment” (a and b) is compared to the surfaces after water (c and d) or lactic acid (e and f) exposition. observe that gα-tcp shows a much more irregular surface after water or lactic acid storage in comparison to gctrl and gocp. n o tr ea tm en t alpha-tricalcium phosphate group a ft er 3 -d ay s to ra ge in di st ill ed w at er ( ph =5 .8 ) a ft er 3 -d ay s to ra ge in la ct ic a ci d (p h =4 .0 ) a c e b d f 7kv 7kv 7kv 7kv 7kv 7kv 5μm 5μm 5μm 2μm 2μm 2μm x5,000 x5,000 x5,000 x8,000 x8,000 x8,000 9 gómez et al. the results of the uts of the experimental adhesive resins exposed to different media are shown in table 2. after dry storage, the values ranged from 28.69 (±10.93) mpa for gctrl to 46.34 (±10.72) mpa for ghap, with a statistically significant difference between gctrl and ghap (p<0.05). the values of uts after distilled water storage ranged from 30.53 (±6.07) mpa for gocp to 39.26 (±9.44) mpa for gctrl, without differences among groups (p>0.05). after lactic acid storage, the values of uts ranged from 29.56 (±6.43) mpa for ghap to 33.87 (±11.93) mpa for gctrl, also without differences among groups (p>0.05). ghap was the only group that presented a statistically significant difference among the different storage media, with lower uts values after lactic acid storage (29.56 ± 6.43 mpa) than dry medium (46.34 ± 10.72 mpa) (p<0.05). figure 5. scanning electron microscopy of ghap at dry (no treatment), distilled water, or lactic acid storage. high differences are observed within this group when “no treatment” (a and b) is compared to the surfaces after water (c and d) or lactic acid (e and f) exposition. observe that, mainly after the lactic acid exposition, ghap shows more irregularities than the other adhesive resins, areas with large cracks and cavities surrounded by irregular borders. n o tr ea tm en t hydroxiapatite group a ft er 3 -d ay s to ra ge in di st ill ed w at er ( ph =5 .8 ) a ft er 3 -d ay s to ra ge in la ct ic a ci d (p h =4 .0 ) a c e b d f 7kv 7kv 7kv 7kv 7kv 7kv 5μm 5μm 5μm 2μm 2μm 2μm x5,000 x5,000 x5,000 x8,000 x8,000 x8,000 10 gómez et al. table 2. results of ultimate tensile strength of the experimental adhesive resins with different calcium phosphates after their storage in different media: dry, distilled water or lactic acid. group dry (mpa) water (mpa) lactic acid (mpa) gctrl 28.69 (±10.93) ba 39.26 (±9.44) aa 33.87 (±11.93) aa gocp 35.96 (±17.19) aba 30.53 (±6.07) aa 32.61 (±6.93) aa gα-tcp 34.43 (±11.48) aba 38.14 (±7.88) aa 30.36 (±13.22) aa ghap 46.34 (±10.72) aa 34.60 (±9.89) aab 29.56 (±6.43) ab different capital letters indicate statistically significant difference in the same column (p<0.05). different small letters indicate statistically significant difference in the same row (p<0.05). discussion bioactive materials with ion-releasing fillers such as cap have been investigated to induce the remineralization process of dental tissues7. studying the behavior of bioactive materials when exposed to different media could assist in understanding their physical properties. in this study, adhesive resins with hap, α-tcp, or ocp were tested regarding their physical properties after storing in distilled water, lactic acid, or dry medium. there were significant differences among the adhesives with different cap, leading to the rejection of the first null hypothesis. furthermore, the storage media influenced the behavior of the adhesives, which led us also to reject the second null hypothesis. dental materials are susceptible to suffering chemical and physical modifications in the oral environment due to hydrolysis and to bacterial enzymes, leading to their degradation over time28. high surface roughness contributes to the attachment of microorganisms and biofilm development28, besides making it more difficult to maintain hygiene28. in 1990, an in vivo study using fluorethylenepropylene or cellulose acetate strips suggested that the surface roughness of ra = 0.2 μm was a threshold value for bacterial retention in intraoral surfaces29. moreover, it is suggested that when the values are lower than 0.2 μm, the materials’ chemical properties may be more important for biofilm formation than the surface roughness. currently, lower values up to 0.1 µm are recommended for polishing resins with inorganic particles to reduce biofilm accumulation2. gα-tcp and ghap presented ra higher than 0.2 µm after immersing in lactic acid, and ghap showed values above 0.2 µm even after distilled water storage. in addition to inducing remineralization, ion-releasing materials have been suggested to inhibit biofilm formation by increasing the ph around them and delaying bacterial colonization30. however, the exposed cap on the materials’ surface, accompanied by the increase of surface roughness, was shown not to decrease bacterial adhesion17. in this study, as well as in the previous report17, the samples were not subject to ph cycles, which could lead to different results and, maybe, lower surface roughness differences. however, this method is a way to evaluate the material over an extreme situation. besides the surface roughness measurement, the surface morphology of the experimental adhesive resins was evaluated via sem, which supported the results observed for ra. in distilled water, gα-tcp and ghap showed larger holes interspersed with small prominences on an irregular surface compared with α-tcp or hap in a dry medium. a uniform pattern over the entire surface of gα-tcp and ghap was observed, probably due to a slight hydrolytic effect on the resin matrix31. after lactic acid storage, the variation 11 gómez et al. of surface integrity was more pronounced for cap groups than for gctrl. this result corroborated the values found for δra, mainly for ghap, which should statistically significant difference for gctrl after immersion in acid. we could also observe that the gocp showed small grooves and holes scattered on the surface after lactic acid storage, while gα-tcp presented a similar pattern to gα-tcp immersed in distilled water but with cracks in greater quantity. the group containing hap showed larger cavities with irregular limits distributed over a slightly smooth surface. it is possible that these cavities were created due to the release of hap agglomerates because low values of surface area were found for hap previously synthesized by the same method24. nanoparticles are prone to agglomeration due to their high surface energy. in composite resins, agglomerates of nanoparticles presented lower adhesion to the organic matrix compared with microparticles, detaching over time32. these agglomerates jeopardize the composite resins compared to microparticles, making the material more susceptible to mechanical failure and surface wear33. this process could occur with hap in the experimental adhesive resin because the small molecules of lactic acid could diffuse through pores among hap agglomerates and produce faster dissolution34. in the mechanical analysis, the immediate uts increased with hap incorporation compared with gctrl, without differences for gα-tcp and gocp. in distilled water, there were no differences in uts, neither among groups nor between the same group comparing dry storage and distilled water storage. on the other hand, the mechanical performance was different after immersion in lactic acid solution, with ghap showing reduced uts compared with ghap in dry storage. this group also presented the highest surface roughness variation after exposure to the lactic acid solution. these results suggest that, even without statistically significant differences among gα-tcp, ghap, and gocp after storing in lactic acid, the uts could be jeopardized for ghap over time in acid conditions. the ph of the medium and the type of filler determine the release rate of the ions7, altering materials’ mechanical properties. hap is soluble in acid solutions9, insoluble in alkaline solutions, and distilled water, while α-tcp and ocp are more soluble than hap at neutral ph9. even so, there were no differences for gctrl, gocp, and gα-tcp, depending on the storage media. the rationale for that may be a better distribution of ocp and α-tcp within the polymer, leading to lower cap–resin interfaces to be exposed and to react with lactic acid. another important factor related to the solubility of cap is the size of the particles, in which the decrease to a nanoscale level can increase their dissolution35. furthermore, cap stability decreases with the increase of impurities’ presence9 and the method used to synthesize the hap24 leads to the presence of carbonates in the final powder, which may have favored its dissolution9. here we observed the different behavior of bioactive resin-based restorative materials depending on the type of cap incorporated into them. interestingly, the physical response of the materials when facing various storing media depended on the cap added. therefore, further evaluations are encouraged in situ and in vivo to deeply understand the biological effects of these bioactive materials in patients with different risks of caries. 12 gómez et al. in conclusion, the findings of this study showed that hap, ocp, and α-tcp affected the physical behavior of the experimental adhesive resins in different ways. hap was the cap that most adversely affected the surface roughness and the mechanical property of the material, mainly when exposed to an acid medium. acknowledgments the authors gratefully acknowledge microscopy and microanalysis center (federal university of rio grande do sul) for the transmission electron microscopy analysis. this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior brasil (capes) finance code 001 – scholarship. conflicts of interest: none. references 1. demarco ff, corrêa mb, cenci ms, moraes rr, opdam njm. longevity of posterior composite restorations: not only a matter of materials. dent mater. 2012 jan;28(1):87-101. doi: 10.1016/j.dental.2011.09.003. 2. glauser s, astasov-frauenhoffer m, müller ja, fischer j, waltimo t, rohr n. bacterial colonization of resin composite cements: influence of material composition and surface roughness. eur j oral sci. 2017 aug;125(4):294-302. doi: 10.1111/eos.12355. 3. ranjkesh b, ding m, dalstra m, nyengaard jr, chevallier j, isidor f, et al. calcium phosphate precipitation in experimental gaps between fluoride-containing fast-setting calcium silicate cement and dentin. eur j oral sci. 2018 apr;126(2):118-25. doi: 10.1111/eos.12399. 4. jefferies sr, fuller ae, boston dw. preliminary evidence 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1;255(12):6087-91. doi: 10.1016/j.apsusc.2009.01.083. 1http://dx.doi.org/10.20396/bjos.v19i0.8658729 volume 19 2020 e208729 original article 1 center of biological sciences and of the health, school of dentistry, state university of western paraná, cascavel, pr, brazil. 2 department of oral diagnosis, piracicaba dental school, university of campinas, piracicaba, sp, brazil. 3 post-graduation program in rehabilitation sciences, hospital for rehabilitation of craniofacial anomalies, university of são paulo, bauru, sp, brazil. corresponding author: renato assis machado, department of oral diagnosis, school of dentistry, university of campinas (fop/unicamp), limeira avenue, 901, areião, 13414-018, piracicaba, sp, brazil, tel.: +55 35 991792635, renatoassismachado@yahoo.com.br received: march 15, 2020 accepted: august 02, 2020 patterns of dental anomalies in patients with nonsyndromic oral cleft natália ventura da cas1, renato assis machado2,3,* , ricardo della coletta2 , ana lúcia carrinho ayroza rangel1 aim: to characterize the patterns of dental anomalies (da) in the mixed and permanent dentitions of patients with nonsyndromic oral cleft (nsoc). methods: this cross-sectional, observational, case-control study included 173 patients, 61 with mixed dentition (nsoc=29 and control=32) and 112 with permanent dentition (nsoc=57 and control=55). all subjected were submitted to clinical and radiographic examination. dental anomalies of eruption, number, size and shape outside the cleft area were considered. results: although there was no statistical significance among patients with mixed dentition, dental agenesis was the anomaly more common in this group. in patients with permanent dentition, a higher prevalence of da in nsoc group compared to control group was observed (p=0.02). gyroversion and dental agenesis were the da more frequently observed in the permanent dentition and the second premolar was the tooth more affected (p=0.003). mandible and the left side were more involved, and dental agenesis was more frequently found in patients with unilateral cleft lip with or without cleft palate (nscl±p). conclusion: our findings show a higher frequency of da in nsocs than in the control group in patients with permanent dentition, mainly due to a higher occurrence of agenesis of second premolars in patients with unilateral nscl±p. keywords: anadontia. cleft lip. cleft palate. dentition, mixed. dentition, permanent. tooth abnormalities. https://orcid.org/0000-0002-1697-3662 https://orcid.org/0000-0001-5285-3046 https://orcid.org/0000-0003-1080-358x 2 cas et al. introduction oral clefts (oc) and dental anomalies (da) are common congenital alterations that may occur isolatedly or in combination1. the nonsyndromic oral clefts (nsoc) are traditionally separated in cleft lip only (nsclo), cleft lip and palate (nsclp) and cleft palate only (nscpo), and show a prevalence varying according to geographic location, ethnicity and socioeconomic status2. due to similarities in both epidemiologic features and embryologic timing for nsclo and nsclp, they are considered variants of the same defect and grouped together to form the group of nonsyndromic cleft lip with or without cleft palate (nscl±p). da refer to color, eruption, number, size and shape alterations of the teeth and its prevalence is also variable, depending on anomaly type, dentition and the population3. nsoc and da etiologies are attributed to complex interactions between genetic, epigenetic and environmental factors during the process of development2,4-6. the association between these congenital anomalies has been initially proposed because individuals with nsoc have a higher prevalence of da than the general population7,8, and later studies demonstrated that genes and pathways related to tooth development are also involved with nsoc, reinforcing the putative link between da and oral clefting9,10. dental agenesis, supernumerary teeth, microdontia, fused teeth, ectopic eruption, gyroversion, taurodontism and enamel hypoplasia are considerably the more prevalent da in individuals with nsoc1,8,11-19. in addition, the severity these da seems to be directly related to the severity of the nsoc11-13,20,21. supporting this hypothesis is the fact that the development of teeth, lips and palate occur almost concomitantly and are anatomically related22. thus, the aim of the current study was to characterize the patterns of da in a group of patients with nsoc. material and methods the sample consisted of 86 unrelated patients with nsoc (26 nsclo, 42 nsclp and 18 nscpo) and 87 healthy control individuals from the same geographic region. nsoc records were obtained from the association of patients with cleft lip and palate in cascavel apofilab and the control group from the state university of western paraná, cascavel, brazil. to estimate the sample size of this study, the frequencies of da reported in the study of letra and collaborators7, which was realized with 500 patients with nsoc and 500 health controls, were used. assuming that approximately 9% of the subjects in the general population show at least one da and this frequency is approximately 49% in nsoc patients, to achieve a power of 80%, at a two-sided p-value of 0.05, with a design effect of 423, the required sample size to each group was 72. all nsoc patients in treatment at the apofilab during 2016 were included. the study was approved by the ethics committee in research of the university (#1.741.771) and written informed consent was obtained from the parents or guardians and/or the participants. all patients were subjected to clinical examination, which included intraoral photos and dental casts, and panoramic radiography. patients with history of permanent tooth extraction and with previous orthodontic treatment were not included. in order 3 cas et al. to make sure that the agenesis of second premolars was not mistakenly noted due to individual variation, only patients older than 6 years were included. the alterations in the primary teeth of the mixed dentition and in the third molars were excluded. the patients with mixed dentition, 29 with nsoc and 32 controls, showed age ranging from 6 to 12 years, whereas patients with permanent dentition, 57 with nsoc and 55 controls, ranged from 12 to 39 years. the data collected were analysed using graphpad prism® software version 5.0 (san diego, usa). chi-square and fisher’s exact tests were applied to assess comparisons between groups. the significance level was set at 95% (p<0.05). results the main clinical characteristics of subjects included in this study are depicted in table 1. in the mixed dentition, 7 (24.1%) patients with nsoc showed 18 (62.1%) da and 6 (18.7%) patients had 11 (37.9%) da in the control group, whereas, in the permanent dentition, 65 (67.7%) da in 32 patients with nsoc (56.1%) and 31 (32.3%) da in 19 (34.5%) patients of control group were identified (table 1). the comparison between the groups shows that the occurrence of da was significantly more frequent in the permanent dentition of patients with nsoc than in healthy controls, yielding an or of 2.42 (95% ci: 1.61-5.36, p=0.02) (table 1). in the mixed dentition, the difference between groups was not significant (p=0.61) (table 1). table 1. distribution of the groups by gender, age, clinical extent of the clefts and dental anomalies. mixed dentition permanent dentition nsoc n=29 control n=32 nsoc n=57 control n=55 gender male 15 (51.7%) 18 (56.2%) 34 (59.6%) 32 (58.2%) female 14 (48.3%) 14 (43.8%) 23 (40.4%) 23 (41.8%) age (mean ± standard derivation) 12.0 (±2.0) 11.0 (±2.0) 17.0 (±5.0) 23.0 (±5.0) oral cleft subtype cleft lip 8 (27.6%) 18 (31.6%) cleft lip and palate 17 (58.6%) 25 (43.9%) cleft palate 4 (13.8%) 14 (24.5%) dental anomaly yes 7 (24.1%) 6 (18.7 %) 32 (56.1%)* 19 (34.5%) no 22 (75.9%) 26 (81.3%) 25 (43.9%) 36 (65.5%) *p value <0.05. in relation to da type, dental agenesis was the most frequently found in the mixed dentition, but the difference between groups was not statically significant (p=0.64, table 2). gyroversion showed the highest prevalence in the permanent dentition in both nsoc (n=31, 47.7%) and control (n=16, 51.6%) groups (p=0.32), followed by den4 cas et al. tal agenesis (table 2). the frequency of dental agenesis was almost twice higher in nsoc group than that observed in the control group (p=0.01), indicating high odds to dental agenesis for patients with nsoc (or: 2.14, 95% ci: 1.19-3.82). table 3 depicts the teeth most commonly affected by dental anomalies. the canines and second premolars were the most affected teeth, and the second premolars were significantly more affected in the nsoc group than in the control group (p=0.003). table 2. distribution of dental anomalies in the mixed and permanent dentitions in the nonsyndromic oral cleft and control groups. mixed dentition permanent dentition nsoc n=29 control n=32 nsoc n=57 control n=55 dental agenesis 15 (83.3%) 8 (72.7%) 28 (43.1%)* 8 (25.8%) gyroversion 2 (11.1%) 0 (0.0%) 31 (47.7%) 16 (51.6%) supernumerary tooth 1 (5.6%) 3 (27.3%) 1 (1.5%) 0 (0.0%) impacted tooth 0 (0.0%) 0 (0.0%) 1 (1.5%) 2 (6.5%) supernumerary root 0 (0.0%) 0 (0.0%) 2 (3.1%) 0 (0.0%) root dilaceration 0 (0.0%) 0 (0.0%) 2 (3.1%) 5 (16.1%) *p value <0.05. table 3. number of dental anomalies in mixed and permanent dentition in the nonsyndromic oral cleft and control groups. mixed dentition permanent dentition nsoc n=29 control n=32 nsoc n=57 control n=55 central incisor 2 (11.1%) 4 (36.4%) 2 (3.1%) 2 (6.4%) lateral incisor 1 (5.6%) 1 (9.0%) 5 (7.7%) 0 (0.0%) canine 4 (22.2%) 2 (18.2%) 21 (32.3%) 18 (58.1%) 1st pre molar 0 (0.0%) 0 (0.0%) 10 (15.4%) 5 (16.1%) 2nd pre molar 7 (38.9%) 0 (0.0%) 25 (38.4%)* 3 (9.7%) 1st molar 2 (11.1%) 0 (0.0%) 2 (3.1%) 0 (0.0%) 2nd molar 2 (11.1%) 4 (36.4%) 0 (0.0%) 3 (9.7%) *p value <0.05. regarding the distribution of da, the highest frequency of da in the mixed dentition was observed among nsclp patients (n=10, 55.6%), followed nsclo (n=7, 38.9%) and nscpo (n=1, 5.5%) (table 4). in the permanent dentition, patients with nsclp (n=35, 53.9%) were the most frequently affected, following by nscpo (n=19, 29.2%) and nsclo (n=11, 16.9%) (table 4). in both dentitions, dental agenesis was more frequent in patients with unilateral nscl±p, while cases of gyroversion were more prevalent among unilateral nscl±p and nscpo patients. out of 83 da in nsoc patients, 35 (42.2%) occurred in the maxilla and 48 (57.8%) in the mandible. among control 5 cas et al. individuals, 28 (66.7%) da occurred in mandibula and 14 (33.3%) in maxilla. the side more commonly affected by the oral cleft and da was the left in both groups, with exception of control group with mixed dentition. in addition, 1 mesiodent was found in the nsoc group with permanent dentition and 2 in the control group with mixed dentition. no size anomaly was found in this. table 4. number of dental anomalies by subtype of nonsyndromic oral cleft in the mixed and permanent dentitions. mixed dentition agenesis gyroversion supernumerary impacted supernumerary root root dilaceration cleft lip bilateral (n=1) 0 0 0 0 0 0 unilateral (n=7) 7 0 0 0 0 0 cleft lip and palate bilateral (n=7) 1 0 0 0 0 0 unilateral (n=10) 6 2 1 0 0 0 cleft palate (n=4) 1 0 0 0 0 0 permanent dentition agenesis gyroversion supernumerary impacted supernumerary root root dilaceration cleft lip bilateral (n=2) 0 0 0 0 2 0 unilateral (n=16) 7 2 0 0 0 0 cleft lip and palate bilateral (n=7) 2 3 1 0 0 0 unilateral (n=18) 13 14 0 0 0 2 cleft palate (n=14) 6 12 0 1 0 0 discussion considering that individuals with nsoc have a higher prevalence of congenital da compared to the general population and previous studies demonstrate that da outside the cleft area can serve as clinical markers for the definition of nsoc subphenotypes1,8,11-19, this study aimed to evaluate the occurrence of da in individuals with nsoc in a southern region of brazil. in addition, data on da are important for anthropological and clinical management of patients24,25. however, differences in da frequencies are observed among the dentitions26, and therefore, we evaluated two case-control groups, a group with mixed dentition and other with permanent dentition. the anomalies in deciduous teeth were not included because the patients had more than 6 years and their tooth were in the process of rhizolysis. although no significant differences were found in the mixed dentition, individuals with nsoc showed a significantly higher frequency of da in the permanent dentition than the control group. 6 cas et al. this lack of association with the mixed dentition may be due to smaller sample size compared to the group with permanent dentition. when the mixed and permanent dentitions were compared separately by type of da, no significant differences were found. however, a high frequency of dental agenesis was found among patients with nsoc from both dentitions. the most affected teeth were the second premolars, being that the dental agenesis was most frequent in patients with nsoc and only the group with permanent dentition showed significant difference between patients with nsoc and controls. this high occurrence of agenesis of second premolars in nsoc patients has been extensively verified in different populations and although some studies showed no significance, the agenesis of second premolars was very frequent1,11-13,15,17,27-38. the second premolar agenesis occurred more on the left side of the mandible in nscl±p in both dentitions. this association have been possibly confirmed because of the pattern of tooth agenesis in nscl±p mainly defined by variants in the genes msx1 and pax910. msx1-associated dental agenesis typically includes missing maxillary and mandibular second premolars and maxillary first premolars. however, the most distinguishing feature of pax9-associated tooth agenesis is the frequent absence of maxillary and mandibular second molars39. although all teeth were equally likely to be missing from the left and right sides in this study, the nscl±p of left side have more dental agenesis that the others nsoc subtypes. in relation to oral cleft subtype, da were most frequently found in nsclp than nscpo and nsclo in both dentitions. these results are in agreement with previous reports1,11,15,17,34,36,38. in addition, a higher prevalence of gyroversion was found in the permanent dentition of patients with nscl±p, which may be related to the malocclusion in patients with nsoc regardless of the cleft area40. in summary, since our findings showed a higher prevalence of dental agenesis in the permanent dentition outside the cleft region, mainly of second premolars in patients with unilateral nscl±p, compared to the healthy control group, a more complex treatment is expected in these patients. acknowledgments ram is supported by the national postdoctoral program of the coordination of training of higher education 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congenitally missing second premolars in cleft lip and cleft palate children. am j orthod dentofacial orthop. 1999;115(4):396-400. doi: 10.1016/s0889-5406(99)70258-1. 28. shapira y, lubit e, kuftinec mm. hypodontia in children with various types of clefts. angle orthod. 2000;70(1):16-21. doi: 10.1043/0003-3219(2000)070<0016:hicwvt>2.0.co;2. 29. karsten a, larson m. the relationship between hypodontia in the second premolar region and heredity of cleft, lip and palate in children with isolated cleft palate. swed dent j. 2004;28(1):47-52. 30. tortora c, meazzini mc, garattini g, brusati r. prevalence of abnormalities in dental structure, position, and eruption pattern in a population of unilateral and bilateral cleft lip and palate patients. cleft palate craniofac j. 2008;45(2):154-62. doi: 10.1597/06-218.1. 31. halpern rm, noble j. location and presence of permanent teeth in a complete bilateral cleft lip and palate population. angle orthod. 2010;80(3):591-6. doi: 10.2319/051009-250.1. 32. bartzela tn, carels ce, bronkhorst em, rønning e, rizell s, kuijpers-jagtman am. tooth agenesis patterns in bilateral cleft lip and palate. eur j oral sci. 2010;118(1):47-52. doi: 10.1111/j.1600-0722.2009.00698.x. 33. camporesi m, baccetti t, marinelli a, defraia e, franchi l. maxillary dental anomalies in children with cleft lip and palate: a controlled study. int j paediatr dent. 2010;20(6):442-50. doi: 10.1111/j.1365-263x.2010.01063.x. 34. bartzela tn, carels ce, bronkhorst em, kuijpers-jagtman am. tooth agenesis patterns in unilateral cleft lip and palate in humans. arch oral biol. 2013;58(6):596-602. doi: 10.1016/j.archoralbio.2012.12.007. 35. mikulewicz m, ogiński t, gedrange t, berniczei-royko a, prussak e. prevalence of second premolar hypodontia in the polish cleft lip and palate population. med sci monit. 2014 mar;20:355-60. doi: 10.12659/msm.890386. 36. berniczei-roykó á, tappe jh, krinner a, gredes t, végh a, gábor k, et al. radiographic study of the prevalence and distribution of hypodontia associated with unilateral and bilateral clef lip and palate in a hungarian population. med sci monit. 2016 oct;22:3868-85. doi: 10.12659/msm.897957. 37. schwartz jp, lauris rc, dalbén g, garib dg. second premolar agenesis as a subclinical phenotype of isolated cleft palate. orthod craniofac res. 2017;20(1):1-7. doi: 10.1111/ocr.12134. 9 cas et al. 38. de stefani a, bruno g, balasso p, mazzoleni s, baciliero u, gracco a. teeth agenesis evaluation in an italian sample of complete unilateral and bilateral cleft lip and palate patients. minerva stomatol. 2018;67(4):156-64. doi: 10.23736/s0026-4970.18.04114-6. 39. kim jw, simmer jp, lin bp, hu jc. novel msx1 frameshift causes autosomaldominant oligodontia. j dent res. 2006;85(3):267-71. doi: 10.1177/154405910608500312. 40. vettore mv, sousa campos ae. malocclusion characteristics of patients with cleft lip and/or palate. eur j orthod. 2011;33(3):311-7. doi: 10.1093/ejo/cjq078. 1http://dx.doi.org/10.20396/bjos.v20i00.8661181 volume 20 2021 e211181 original article 1 department of oral diagnosis, piracicaba dental school, university of campinas; piracicaba, são paulo, brazil. 2 resident of oral and maxillofacial surgery, federal university of piaui; teresina, piaui, brazil. 3 department of postgraduate program in dentistry, ceuma university, são luis, maranhão, brazil. 4 departament of dentistry, school of dentristy, federal university of maranhão, são luís, brasil. corresponding author: raísa sales de sá, departament of oral diagnosis, piracicaba dental school, university of campinas; piracicaba, são paulo, brazil. av. limeira, 901, areião, piracicaba, são paulo 13414-903, brazil. + 55 19 2106 5213. e-mail: raisadesa@hotmail.com received: september 9, 2020 accepted: december 9, 2020 dental developmental anomalies and post-eruption dental disorder: a series of panoramic radiographs raísa sales de sá1,* , lais inês silva cardoso2, jordana almeida brito3, alina nunes braga4, laíse nascimento correia lima4 , melaine mont’alverne lawall-silva4 , erika martins pereira4 panoramic radiographs are complementary exams to evaluate oral alterations in an early manner, these changes can be dental developmental anomalies, and post-eruption dental disorder. aim: this study evaluated the findings in panoramic radiographs and correlated the variables of gender and dental location. methods: a retrospective study was through the observation of 1.111 panoramic radiographs from the radiology department in brazil. it was included patients from 5 to 79 years of age of both gender, and it classified the anomalies in shape, size, and number and post-eruption dental changes in and correlated with gender and location. patients with syndromes were excluded from the sample. results: the majority of the sample was composed of fameles 752 (67.7%), as to the frequency of dental developmental anomalies related lesions 684 cases (61.6%) and post-eruption dental disorder 567 (51.8%), in the radiographs. the most prevalent change was endodontic treatment (32.6%), followed by root dilaceration (25.9%), and included tooth (19.5%). the most prevailing alteration when correlated with the gender variables was the cyst root (p<0.01) in females, and orthodontic treatment (p=0.02) in males and the variable location in the mandible was root dilaceration, giroversion, impacted tooth, taurodontia, microdontia, and endodontic treatment (p<0.01). conclusion: our findings provide evidence that dental developmental anomalies e post-eruption dental disorder are frequent alterations in the population with particular characteristics of distribution by sex and location. keywords: radiography, panoramic. tooth abnomalities. diagnosis, oral. https://orcid.org/0000-0003-4563-6531 https://orcid.org/0000-0002-1773-847x https://orcid.org/0000-0003-4773-1612 https://orcid.org/0000-0002-2247-0017 2 sales de sá et al. introduction panoramic radiography is an extraoral procedure of simple execution, in which it visualizes the whole maxillo-mandibular complex1-3. this technique uses an external image receptor that moves in synchronism around the head of the individual, significantly reducing the discomfort of the radiographic examination to the patient, in addition to subjecting the patient to minimal ionizing exposure4. panoramic radiography is the complementary examination of routine, used both for diagnosis and treatment planning, moreover, this exam may reveal some radiographic findings, which are relevant for patient prevention3. extensive research has provided shown that panoramic radiographs are useful for the discovery of numerous pathological changes, such as ectopic tooth germs, calcification in root canals, retained teeth, supernumeraries, root dilacerations, odontomas, periapical lesions, among others3,5,6. dental developmental anomalies (dda) and post-eruption dental disorder (pedd) are the radiographic findings that the early diagnosis that can occur with the use of this examination3,7. dda is a multifactorial disease change caused by genetic, epigenetic, and environmental factors. these can include changes in number, shape, dimensions, and structure of teeth5,7,8. pedd was associated environmental factors, and without associated with dental development. currently, clinical dentists are working to prevent dental and oral diseases that can lead to tooth anomalies or more severe changes within the oral mucosa, where panoramic radiographs are presented as the main exam. however, most epidemiological studies with large cohort have been demonstrated mainly in syndromic patients, children patients or before orthodontic treatment3,4,9,10. thus, the objective of our study was to characterize the epidemiological profile through a large cohort of non-syndromic patients and in a wide range of ages, through a survey of panoramic radiographs correlating developmental dental anomalies and post-eruption changes and associating them with gender and location. materials and methods a retrospective study was performed based on radiographic findings of 1.111 digital panoramic radiographs from a radiology clinic in the brazilian northeast, from january 2013 to december 2014. this research study was approved by the human research ethics committee of federal university of maranhão (620.034/2014), the free and informed conscience term was waived. the eligibility criteria required that the individuals be of both sexes, with ages over 5 years, that the radiographs present high contrast and minimal distortion, and patients with syndromes were excluded from the sample. the radiographs analyzed were taken in patients of both genders, ages ranging from 5 to 79 years, divided into 4 groups; group 1: from 5 to 13 years (children), group 2: 14 to 18 years (adolescents) (statute of the child and adolescentbrazil), group 3: 19 to 59 years (adults) and group 4: 60 to 79 years (aged). 3 sales de sá et al. this evaluation was carried out by two appropriately calibrated examiners. this calibration was performed by the analysis of 30 pairs of panoramic radiographs, from the same examiners randomly selected, twice, with an interval of 15 days between the analyses, supervised by a radiology specialist. the digital radiographs were saved in digital files in an appropriate quality for peer review of all cases, as seen in (figure 1), where they were then checked, and in case of disagreement, a third examiner would perform the evaluation. a b c d figure 1. radiographic findings of dental changes. a) microdontia in the upper second molars. b) upper third molar microdontia and orthodontic treatment. c) presence of taurodontia on the lower second molars. d) residual roots in the left upper second premolar and right lower second premolar, gyroscopic rotation in the left upper first molar, impacted right lower third molar and orthodontic treatment in the upper incisors. the changes for analysis were divided into two major groups: 1) dental developmental anomalies (dda), and 2) post-eruption dental disorder (pedd). in the dda we sought: a) changes in number (hypodontia and hyperdontia; b) changes in size (microdontia and macrodontia, transposition, included teeth, impacted teeth,); c) changes in shape (twinning, fusion, concrescence, hypercementosis, taurodontia, dilaceration, and supranumerary roots), demonstrated. the pedd was also analyzed for the occurrence of types of dental intervention, including the presence of periapical lesions, radiolucent lesions (suggestive of cysts) and odontomas, residuais roots, the presence of anterior endodontic treatment, and orthodontic treatment (figure 2). then all changes were tabulated in the excel spreadsheet and later analyzed by the spss statistics program (version 17.0). the variables collected included gender, den4 sales de sá et al. tal arch of the evaluated changes and detection of dental development anomalies, and post-eruption dental disorder. initially, descriptive statistics of the data were performed using absolute frequency and percentage. categorical variables were compared between genders and dental arches using the chi-square or fisher’s exact test. the significance level adopted was 5% (p<0.05). results the clinical features are present, it observed that most samples were composed of female individuals (719 cases, 64.7%,), and males (392 cases; 35.3%). regarding the age group, we noticed that the majority of the group was formed by adults (592 cases; 53.3%), followed by adolescents 284 cases (25.5%), children 184 cases (16.6%), and elderly 51 cases (4.6%), representing 25.5%, 16.6% and 4.6% respectivelyof the sample. most radiographs panoramics were characterized dda was related in 684 cases (61.6%), reveled for analysis of dilacerations (288 cases; 25.9%), followed by included teeth (217 cases; 19.5%), gyroversion (195 cases; 17.6%), impacted teeth (156 cases; 14.0%), taurodontia (88 cases; 7.9%) and hypercementosis (76 cases; 6.8%). otherwise, when the less frequent dda, reveled for analysis of dens invaginatus and tooth transposition, which were report with only (1 case; 0.1%), macrodontia had (6 cases; 0.5%), supernumerary (22 cases; 2.0%), microdontia (59 cases; 5.3%) and agenesis (70 cases; 6.3%). subsequently, dda was correlating with gender, where we observed that there was no statistically significant difference (table 1). table 1. incidental findings prevalence of dda their predilection for female or male patients gender p value female (n = 719) male (n = 392) n (%) n (%) dilaceration root 187 (26.0) 101 (25.8) 0.986 included tooth 137 (19.1) 80 (20.4) 0.642 dental gyroversão 125 (17.4) 70 (17.9) 0.908 impacted tooth 97 (13.5) 59 (15.1) 0.532 taurodontia 57 (7.9) 31 (7.9) 0.916 hypercementose 46 (6.4) 30 (7.7) 0.504 agenesis 49 (6.8) 21 (5.4) 0.408 microdontia 41 (5.7) 18 (4.6) 0.516 supernumerary 13 (1.8) 9 (2.3) 0.739 macrodontia 6 (0.8) 0 (0) 0.072 transposition 1 (0.1) 0 (0) 0.647 dens in dente 1 (0.1) 0 (0) 0.647 dda were compared it with the location in the maxillo-mabibular complex (table 2). among the most frequent disorders correlated with the mandible and we find dilaceration (p<0.01), dental gyroversion (p<0.01), impacted tooth (p<0.01), taurodontia 5 sales de sá et al. (p<0.01), hypercementosis (p=0.03). otherwise, when assessed maxilla the most frequentely lesion was microdontia (p<0.01). tabela 2. incidental findings prevalence of dda their predilection for maxila or mandible maxilla mandible p value n (%) n (%) dilaceration root 138 12.4 220 19.8 <0.001 included tooth 163 14.7 166 14.9 0.904 dental gyroversão 73 6.6 150 13.5 <0.001 impacted tooth 74 6.7 128 11.5 <0.001 taurodontia 39 3.5 75 6.8 <0.001 hypercementose 30 2.7 50 4.5 0.030 agenesis 39 3.5 38 3.4 1.000 microdontia 43 3.9 17 1.5 <0.001 supernumerary 15 1.4 9 0.8 0.305 macrodontia 2 0.2 5 0.5 0.226 transposition 1 0.1 0 0 0.5 dens in dente 1 0.1 1 0.1 0.75 pedd was related in 567 cases (51.8%), revealed in the analysis that most patients had orthodontic treatment (362 cases; 32.6%), followed by periapical lesions (183 cases; 16.5%), orthodontic treatment (175 cases; 15.8%), presence of residual roots (108 cases; 9.7%). radiolucent lesions and odontomas were less frequent (0.6% and 0.2%, respectively). pedd were correlation between with sex (table 3), where the presence of endodontic treatment might have been more frequent in famele (p=0.02), while radiolucent lesions exclusive to male (p=0.01). the odontoma was a lesion exclusively to famele, although it did not reach a statistically significant difference. tabela 3. comparative analysis between genders of the prevalence of pped and occurrence of types of dental intervention. gender p value female (n = 719) male (n = 392) n (%) n (%) periapical lesion 117 (16.3) 66 (16.8) 0.874 radiolucent lesions 0 (0) 5 (1.5) 0.001 odontoma 2 (0.3) 0 (0) 0.418 residual root 65 (9.0) 43 (11.0) 0.351 endodontic treatment 231 (32.1) 131 (33.4) 0.708 orthodontic treatment 127 (17.7) 48 (12.2) 0,022 6 sales de sá et al. comparative analysis of pped between locations showed that the maxilla was often more affected compared to mandibular (p=0.01) (table 4). periapical lesions were more frequent in the mandible, as well as the presence of residual roots, although these data did not reach statistical significance. some lesions were evaluated by this study, but we could not find any cases in this sample, such as germination and dental fusion lesions. tabela 4. comparative arch analysis of the prevalence of pped and occurrence of types of dental intervention. location p value maxilla mandible (n = 452 ) (n = 403) n (%) n (%) periapical lesion 1 0.2 1 0.2 na radiolucent lesions 94 20.8 119 29.5 0.103 odontoma 63 13.9 69 17.1 0.668 residual root 2 0.4 3 0.7 na endodontic treatment 292 64.6 211 52.4 0.0001 discussion dental anomalies are multifactorial and complex changes involving genetic, epigenetic and environmental factors throughout the dental development process6,8. these anomalies can develop from simple changes of shape or position to changes so complex that they lead to the disorganization of structures such as dentin and enamel8,11. in this study, were characterized the large cohort of non-syndromic patients and in a wide of ages, through a survey of panoramic radiographs correlating developmental dental anomalies and post-eruption changes and associating them with gender and location. among the developmental disorders that had the greatest expression was root dilaceration with a total of 25.9% of the cases, being more prevalent in females, despite not having a statistically significant difference, these data were similar to those found by ledesma-montes et al., where they also correlated these alterations with other dda12. regarding the location 19.8% of the cases were in the mandible, a systematic review showed that the most affected teeth are the lower third and second molars, followed by upper premolars and upper incisors respectively, thus showing the highest frequency in the lower arch7,13,14. taurondontia is a morphological anomaly in which the pulp cavity is vertically elongated, so clinical diagnosis becomes impossible, and radiographic diagnosis is essential15. in our study, it was noted the presence of taurodontia in only 7.9% of cases, where it presented with a predominance in the mandible. although this anomaly is related mainly as a marker of orofacial anomalies, it was noted that even in a normal population it can be a complication mainly for endodontic treatment, due the taurodontia was decreased the root surface area and for prosthetic treatments that require an adequate occlusal load15,16. 7 sales de sá et al. hypercementosis is described as enlargement or increase in the deposition of cementum around part or all of the root17. in our study, hypercementosis when observed showed that the mandible was more affected than the maxilla, these data corroborate with reports in the literature that demonstrate the mandible as the most affected region7,17. impacted tooth refers to a tooth that cannot erupt within the expected time until its normal position3. our sample observed that 14% had this change and was more frequent in the jaw region. this predilection for the mandible is mainly caused by lower third molars, which may encounter physical barriers and changes in tooth position for the eruption3. microdontia was observed with a most frequency in the maxilla. this alteration was unique dda correlated with the maxilla, mainly because it is an area more susceptible to the size/form anomaly in the upper anterior region, such as the conoid teeth and a high prevalence of upper third molar with microndontia. given the smaller diameter of the upper region in some patients, as in the case of patients with an ogival palate18. pped most commonly found were endodontic treatments where we found a percentage of 32.6% and is more correlated to the maxilla. although the lower molars appear like the teeth most prone to endodontic treatment, when it shows an overview the maxilla is more stricken19. a possible explanation for this might be that region is more prone to trauma which would cause pulp necrosis and extensive carious lesions leading to endodontic treatment. radiolucent lesions showed low incidence reported in the literature and this result is similar to the results3. radiolucid lesions were associated with the root apex are caused by reactions of periapical tissue to inflammatory stimulus, but may mimic more severe lesions such as lymphomas and oral metastasis, as described in other studies20,21. our study has some limitations, digital panoramic radiography which is not as clear as those of intraoral radiographs, distortion in the upper and lower anterior region, and limitation of three-dimensional visualization of structures, especially in cases where there is a need for vestibule-lingual location. however, to provide an initial diagnosis it becomes of great relevance. in summary, our study provide evidence of panoramic radiographic exams at the initial moment of the consultation, proving that dda related lesions (61.6%), and pedd (51.8%) are very frequent changes in the brazilian population with particular characteristics of distribution by sex and localization. in addition, the most frequent alterations were endodontic treatment, followed by root dilaceration, and included tooth. conflict of interest the authors state that they have no potential conflict of interest that could bias the results obtained in the current study. acknowledgment this work was supported by the e foundation of amparo to the research of the state of maranhão (fapema). 8 sales de sá et al. reference 1. santos kc p, oliveira as, hesse d, buscatti my, oliveira jx. 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[the importance of dental developmental anomalies]. acta pediatr port. 2008;39(5):195-200. 12. ledesma-montes c, jiménez-farfán md, hernández-guerrero jc. dental developmental alterations in patients with dilacerated teeth. med oral patol oral cir bucal. 2019 jan 1;24(1):e8-e11. doi: 10.4317/medoral.22698. 13. topouzelis n, tsaousoglou p, pisoka v, zouloumis l. dilaceration of maxillary central incisor: a literature review. dent traumatol. 2010 oct;26(5):427-33. doi: 10.1111/j.1600-9657.2010.00915.x. 14. jafarzadeh h, abbott pv. dilaceration: review of an endodontic challenge. j endod. 2007 sep;33(9):1025-30. doi: 10.1016/j.joen.2007.04.013. 15. macdonald d. taurodontism. oral radiol. 2020 apr;36(2):129-132. doi: 10.1007/s11282-019-00386-1. 16. weckwerth gm, santos cf, brozoski dt, centurion bs, pagin o, lauris jr, et al. taurodontism, root dilaceration, and tooth transposition: a radiographic study of a population with nonsyndromic cleft lip and/or palate. cleft palate craniofac j. 2016 jul;53(4):404-12. doi: 10.1597/14-299. 17. bürklein s, jansen s, schäfer e. occurrence of hypercementosis in a german population. j endod. 2012 dec;38(12):1610-2. doi: 10.1016/j.joen.2012.08.012. 18. d’la torre ochoa c, gurrola martínez b, casasa araujo a. multidisciplinary approach in patient with upper lateral incisor microdontia. case report. rev mex ortod. 2016;4(2):132-7. doi: 10.1016/j.rmo.2016.10.018. 9 sales de sá et al. 19. wigsten e, jonasson p; endoreco, kvist t. indications for root canal treatment in a swedish county dental service: patientand tooth-specific characteristics. int endod j. 2019 feb;52(2):158-68. doi: 10.1111/iej.12998. 20. torregrossa vr, faria km, bicudo mm, vargas pa, almeida op, lopes ma, et al. metastatic cervical carcinoma of the jaw presenting as periapical disease. int endod j. 2016 feb;49(2):203-11. doi: 10.1111/iej.12442. 21. hopp rn, marchi mt, kellermann mg, rizo vh, lopes ma, jorge j. lymphoma mimicking a dental periapical lesion. leuk lymphoma. 2012 may;53(5):1008-10. doi: 10.3109/10428194.2011.631161 1 volume 22 2023 e237543 original article braz j oral sci. 2023;22:e237543http://dx.doi.org/10.20396/bjos.v22i00.8667543 1 dentistry school, atitus educação, passo fundo, rs, brazil. corresponding author: lilian rigo dentistry school, atitus educação, passo fundo, rs, brazil. major joão schell, 1121, annes, passo fundo, rs, brazil; zip code: 99020-020 e-mail: lilian.rigo@atitus.edu.br editor: dr. altair a. del bel cury received: november 13, 2021 accepted: september 29, 2022 impact of xerostomia and the use of dental prosthesis on the quality of life of elderly: a cross-sectional study larissa steilmann demarchi1 , mayara trapp vogel1 , gabrielle haubert1 , lilian rigo1,* aim: to evaluate the impact of xerostomia, edentulism, use of dental prosthesis, and presence of chronic diseases on quality of life in relation to oral health in institutionalized elderly individuals. methods: this is a cross-sectional study. a questionnaire was administered containing the following instruments: oral health impact profile (ohip-14), which measures the quality of life related to oral health; the summated xerostomia inventory questionnaire (sxi-pl) for evaluation of xerostomia, sociodemographic data, clinical description, and patient-reported factors was assessed (edentulism, use of dental prostheses, and chronic diseases). results: most elderly individuals did not have any teeth in their mouths and used dental prosthesis. the impact on quality of life, considering the mean of the ohip-14 scores, was positive in 58.3% of the elderly. those who used a dental prosthesis were three times more likely to have their oral health negatively impacted (or=3.09; 95%ci =1.17 8.11), compared to those who did not use, and individuals with xerostomia were more likely to have their oral health negatively impacted (or=1.57; 95%ci=1.25-1.98) compared to those without xerostomia. there was no difference in the quality of life of individuals with and without chronic diseases. conclusions: the feeling of dry mouth and use of dental prostheses negatively impacted the quality of life in relation to oral health of the elderly. keywords: xerostomia. quality of life. oral health. aged. chronic diseases. aging. dental prosthesis. drug utilization. mouth, edentulous. https://orcid.org/0000-0002-9090-6214 https://orcid.org/0000-0003-0370-0048 https://orcid.org/0000-0001-5375-8446 https://orcid.org/0000-0003-3725-3047 2 demarchi et al. braz j oral sci. 2023;22:e237543 introduction aging of the global population is becoming increasingly evident. the increase in people’s life expectancy can be attributed to socioeconomic development, advances in science, improvements in healthcare, and a greater focus on health promotion1,2. thus, with an increase in life expectancy, there is also an increase in the number of institutionalized elderly people3. however, the aging process brings with it the issue of frailty in the elderly, a complex condition that affects their social, psychological, physical, and cognitive domains, increasing society’s concern regarding the health of this rising population3. aging has some consequences, such as an increase in chronic non-communicable diseases (ncd’s), which can negatively impact the quality of life of these individuals4,5. in addition to ncd’s, there are more chances of appearance of several lesions in the oral mucosa, which can arise from the absence of natural teeth, or can be manifestations of chronic oral diseases, oral infections, or other factor6. some oral manifestations affect the elderly and generate feelings of discomfort. xerostomia, a prevalent condition, is defined as a subjective sensation of dry mouth and is often associated with hypofunction of the salivary gland7. most of the time, xerostomia causes discomfort in the oral mucosa and lesions in hard and soft tissues of the mouth, leading to inflammation, such as stomatitis, fissured tongue, glossitis, angular cheilitis, mucositis, stomatodynia (burning sensation), aphthous and ulcerative lesions, traumatic ulcerations, chapped lips, tongue without papillae, and difficulty in using prostheses8-10. it is unquestionable that these comorbidities represent a problem for global public health, reflecting on the quality of life and general health of the population4. there is, hence, a growing concern regarding the quality of life across various dimensions. limitations of life concerning age are part of the physiological process, among which changes in the oral cavity stand out, which can cause poorer quality of food, social isolation, and dissatisfaction with life, among others11. some studies have reported a direct relationship between oral health and quality of life; for example, when an individual’s oral health is impaired, the quality of life will often also be affected12-14. subjective perceptions of the amount of saliva in the mouth and the experience of speaking difficulty affected the quality of life in patients with xerostomia13. dental prosthesis and edentulism negatively impacted the oral health of the elderly5,12. the hypothesis of this research is that edentulism, the use of dental prosthesis, and the presence of chronic diseases and xerostomia have a negative impact on the quality of life of the elderly. therefore, this study aimed to assess the impact of xerostomia, edentulism, use of dental prosthesis, and presence of chronic diseases on the quality of life about oral health in institutionalized elderly aged 70 years or older. 3 demarchi et al. braz j oral sci. 2023;22:e237543 materials and methods study design and sample our research work was previously submitted to the research ethics committee of faculty imed and approved under number 2.711.544, caae 90966718.0.0000.5319. this scientific article was written in accordance with the report of strobe (strengthening the reporting of observational studies in epidemiology)15. this cross-sectional study was conducted in 2020. the sampling was of the non-probabilistic type, consisting of interviews with the institutionalized elderly living in the seven nursing homes in the southern brazil municipality (passo fundo, rio grande do sul). the following inclusion criteria were employed: elderly aged 70 years or older, absence of neurological disease, and the possibility of answering the research questionnaire (not be illiterate). the strategy to select the sample was based on the total number of elderly residents of the twelve long-stay institutions for the elderly in the municipality. the total number of institutionalized elderlies in these institutions was 300, however, only 202 elderly people met the inclusion criteria of this study. of these, 46 refused to participate in the research, and the final sample consisted of 156 individuals. data collection instruments for data collection, a questionnaire was used to address the following parameters: sex, age, presence of diabetes, depression or anxiety, hypertension, rheumatoid arthritis, or other chronic diseases; edentulism; use of dental prostheses; self-reported xerostomia; and impact of quality of life-related to oral health. for the geriatric quality of life associated with oral health, the oral health impact profile instrument, in its reduced version (ohip-14)16,17, was used, containing questions related to the last four weeks, divided into seven dimensions: 1. functional limitations (have you had trouble pronouncing any words because of problems with your teeth, mouth or dentures?; have you felt that your sense of taste has worsened because of problems with your teeth, mouth or dentures?); 2. physical pain (have you had painful aching in your mouth?; have you found it uncomfortable to eat any foods because of problems with your teeth, mouth, or dentures?); 3. psychological discomfort (have you felt self-conscious because of problems with your teeth, mouth or dentures?; have you felt tense because of problems with your teeth, mouth, or dentures?); 4. physical disability (has your diet been unsatisfactory because of problems with your teeth, mouth or dentures?; have you had to interrupt meals because of problems with your teeth, mouth, or dentures?); 5. psychological disability (have you found it difficult to relax because of problems with your teeth, mouth or dentures?; have you been a bit embarrassed because of problems with your teeth, mouth, or dentures?); 6. social disability (have you been a bit irritable with other people because of problems with your teeth, 4 demarchi et al. braz j oral sci. 2023;22:e237543 mouth or dentures?; have you had difficulty doing your usual jobs because of problems with your teeth, mouth, or dentures?); and 7. social handicap (have you been a bit irritable with other people because of problems with your teeth, mouth, or dentures?; have you had difficulty doing your usual jobs because of problems with your teeth, mouth or dentures? the answer options were never, hardly ever, occasionally, fairly often, or very often. subsequently, scores were assigned to each answer: never = 0, hardly ever = 1, occasionally = 2, fairly often = 3, and very often = 416,17. to check the self-reported xerostomia, a short version of the xerostomia inventory (xerostomia inventory) was used as designed by thomson et al.18. the summated xerostomia inventory questionnaire (sxi-pl) was validated in the portuguese version and was used to check for dry mouth sensation in the brazilian population19. it was composed of five questions, with each item having four possible answers (never, occasionally, frequently, always): “do you feel dry mouth during meals?”, “do you feel dry mouth?”, “do you have difficulty eating dry foods?” , “do you have difficulty swallowing certain foods?”, “do your lips feel dry”? the responses had values of 1, 2, 3, and 4, respectively, which when added together, generated a score ranging from 5 to 20; the higher the value, greater the severity of xerostomia. first, a pilot test was carried out with 10 participants, similar to definitive research, making it possible to train the researcher in data -collection and guide the application of the questionnaire to the elderly, thus minimizing bias. variables in the study for this research, the outcome variable was “impact of oral health on quality of life”, according to ohip-14 values16,17, which was dichotomized in the presence and absence of impact on quality of life, with at least one answer meaning presence, such as “occasionally”, “fairly often”, or “very often”, and the answers “never” and “hardly ever” in the two items corresponded to the absence of impact on the quality of life of each the exposure variables analysed were: sex (male/female), age group (70-80 years/8190 years), edentulism (yes/no), use of dental prosthesis (yes/no), self-reported xerostomia (values quantitative responses with values of 1, 2, 3 and 4 were added together, generating a total score continuous variable), and presence of chronic diseases (yes/no) all comorbidities: the presence of diabetes, depression or anxiety, high blood pressure, rheumatoid arthritis and other chronic diseases were combined in this variable. data analysis for data analysis, all variables were descriptively analyzed accordingly. the pearson’s chi-square test (p <0.05), a univariate analysis, was performed between the outcome and exposure variables. logistic models were also used for bivariate and multivariate regression tests with the associated variables (p <0.20). in the multivariate analysis, odds ratios (or) and their respective 95% confidence intervals 5 demarchi et al. braz j oral sci. 2023;22:e237543 were estimated, both crude and adjusted for exposure variables in a binary logistic regression model (p-value <0.05) and were reported accordingly. the data were analyzed using the statistical program ibm spss® software (statistical package for the social sciences, version 20.0, armonk, ny, usa). results the study included 156 participants who completed the questionnaires. table 1 shows the descriptive results of the elderly, most of whom were women (64.1%), with a mean age of 80 years old (± 10.5). regarding chronic diseases, most of the elderly reported having depression and/or anxiety (45.5%), 41% hypertension, 22.4% diabetes, 21.2% heumatoid arthritis, and 46.2% reported having another disease(s), except those mentioned. regarding chronic diseases, 86.5% of the elderly had at least one comorbidity. more than half (51.3%) used some type of dental prosthesis, (complete dental prostheses to fixed rehabilitation) and 25.6% did not have any teeth in their mouth (edentulism). table 1. description of demographic variables, diseases chronic, and oral conditions, southern brazil municipality, 2020. (n = 156). variables n % sex feminine 100 64.1 male 56 35.9 age 70-80 years 88 56.4 81-97 years 68 43.6 diabetes no 121 77.6 yes 35 22.4 depression / anxiety no 85 54.5 yes 71 45.5 hypertension no 92 59.0 yes 64 41.0 rheumatoid arthritis no 123 78.8 yes 33 21.2 other diseases no 84 53.8 yes 72 46.2 continue 6 demarchi et al. braz j oral sci. 2023;22:e237543 continuation edentulism yes 40 25.6 no 116 74.4 dental prosthesis yes 80 51.3 no 76 48.7 table 2, which refers to the questions of the xerostomia inventory, shows that 56.4% of the elderly did not report dry mouth when eating a meal; however, 37.8% reported that their mouth frequently felt dry. when asked about the difficulty when eating dry foods, 55.1% reported not having any difficulties, and 9% reported frequently encountering difficulties while eating dry foods. regarding dry lips, a significant number (23.1%) of the elderly reported that their lips were always dry. for the prevalence of self-reported xerostomia, all answers were either “frequently” or “always” for the five questions in the questionnaire, resulting in a prevalence of 18.5%. table 2. xerostomia inventory data summated xerostomia inventory-5 (sxi-pl) of institutionalized elderly, southern brazil municipality, 2020. (n = 156). variables n % 1. my mouth feels dry when eating a meal never 88 56.4 occasionally 43 27.6 frequently 9 5.8 always 16 10.3 2. my mouth feels dry never 58 37.2 occasionally 59 37.8 frequently 13 8.3 always 26 16.7 3. i find it difficult to eat dry food never 86 55.1 occasionally 48 30.8 frequently 7 4.5 always 15 9.6 4. i have difficulty swallowing certain foods never 101 64.7 occasionally 41 26.3 frequently 6 3.8 always 8 5.1 continue 7 demarchi et al. braz j oral sci. 2023;22:e237543 continuation 5. i feel my lips dry never 50 32.1 occasionally 63 40.4 frequently 7 4.5 always 36 23.1 when analysing the measures of central tendency of the scores of the reduced xerostomia inventory-5 (sxi-pl), an overall average of self-reported xerostomia of 8.5 was observed. regarding the means, standard deviation, minimum and maximum quality of life, and the seven dimensions of the ohip-14 questionnaire, the average quality of life was 1.26 (± 1.13), whose domain was physical pain, which most negatively impacted the quality of life, having a value of 2.21 (± 2.08) (table 3). table 3. descriptive statistics of the scores of the five dimensions and self-reported total xerostomia summated xerostomia inventory-5 (sxi-pl) and the negative impact of oral health on quality of life related to oral health and the seven domains (ohip-14) of the elderly, southern brazil municipality, 2020 (n = 156). minimum maximum average standard deviation 1. dry mouth when eating meal 1 4 1.35 0.78 2. dry mouth 1 4 1.58 1.00 3. difficulty eating dry foods 1 4 1.65 1.05 4. difficulty swallowing food 1 4 2.04 1.19 5. dry lips 1 4 1.82 1.15 total xerostomia scores 5 20 8.5 3.80 1. functional limitation 0 8 1.10 1.65 2. physical pain 0 8 2.21 2.08 3. psychological discomfort 0 8 1.77 2.00 4. physical disability 0 8 1.18 1.70 5. psychological disability 0 7 1.27 1.73 6. social disability 0 7 0.63 1.20 7. social handicap 0 8 0.65 1.35 ohip-14 scores 0 7 1.26 1.13 however, in reaction to the impact of oral health on the quality of life of the elderly, it was observed that it was positive in 58.3% of the elderly and negative in 41.7%. to perform the binary logistic regression, all variables that were associated with pearson’s chi-square test with p <0.20 were entered in the crude model: sex, age, use of dental prosthesis, edentulism, self-reported xerostomia, and disease chronicles with variable oral health outcomes in quality of life. after multivariate adjustment, the vari8 demarchi et al. braz j oral sci. 2023;22:e237543 ables used for dental prosthesis and self-reported xerostomia remained significant (p<0.05), with the other variables losing their association in the final adjusted model of the multivariate regression analysis (table 4). elderly people who use a dental prosthesis are 3.09 times (or = 3.09; 95% ci 1.17-8.11) more likely to have a negative impact on oral health, and those with self-reported xerostomia were 1.57 times, more likely to have a negative impact on oral health (or = 1.57; 95% ci 1.25-1.98). table 4. bivariate (crude) and multivariate (adjusted) binary logistic regression model for the impact of oral health on oral health-related quality of life (ohip-14) of institutionalized elderly, southern brazil municipality, 2020. crude or (95% ci) p-value * adjusted or (95% ci) p-value ** age 70 to 80 1 0.029 1 81 to 97 2.22 (1.08-4.55) 0.73 (0.24-2.25) 0.596 sex male 1 0.116 feminine 1.63 (0.88-2.99) use of dental prosthesis no 1 0.001 1 <0.001 yes 3.50 (1.70-7.21) 3.09 (1.17-8.11) edentulism no 1 0.036 1 0.967 yes 2.09 (1.05-4.17) 1.02 (0.34-3.01) chronic diseases no 1 0.091 1 yes 2.23 (0.88-5.69) 2.92 (0.62-5.88) 0.252 self-reported xerostomia 1.60 (1.29-1.99) <0.001 1.57 (1.25-1.98) 0.022 * chi-square test; ** wald test (p <0.05 statistically significant) or chance ratio; 95% ci 95% confidence interval % frequency-percentage adjusted for the variables: age group, sex, use of dental prosthesis, edentulism, chronic diseases, and selfreported xerostomia (p <0.05). discussion this study evaluated the impact of certain conditions on the quality of life of institutionalized elderly people, and it was found that xerostomia and usage of dental prosthesis had a negative impact on their quality of life in relation to oral health. this study revealed that individuals who have self-reported xerostomia are 1.57 times more likely to have a negative impact on their oral health. in a study of 566 patients from a dental clinic, patients with xerostomia had worse quality of life 9 demarchi et al. braz j oral sci. 2023;22:e237543 scores than those without xerostomia19. in another study of 2,209 new zealanders aged 75 and over, quality of life was worse in individuals with xerostomia20. studies report problems that individuals affected by xertostomia may have: dysgeusia, dysphagia, dysphonia, masticatory efficiency reduction, nutritional inadequacy, candidiasis, oral lesions and ulcerations, atrophic tongue, dental caries, periodontal diseases, halitosis, loss of denture retention21-26. although the purpose of this study was not to verify the association between xerostomia and oral problems, there is much evidence to support this relationship. lesions in the hard and soft tissues of the mouth, which many patients report as causing discomfort and pain, are common in patients with xerostomia, due to dryness on the surface of the tongue, palate, oral floor, and mucosa15,21,27. the xerostomia scores in this study were high, consistent with other studies13,19,24,28-31. in the present study, 45.5% of the elderly have depression or anxiety. with the growth of the elderly population, the number of chronic physical and behavioural diseases increases and, consequently, the use of continuous medication. thus, it may be that the use of continuous medication for anxiety/depression or other diseases by the elderly has contributed to the prevalence of xerostomia. in the present study, having one or more chronic diseases and quality of life were not found to be associated with oral health in the elderly. it is emphasized that, often, an increase in life expectancy and longevity occurs with the increase in the prevalence of chronic diseases32. currently, longevity is an achievement of human beings; however, the high prevalence of chronic diseases is associated with the ability to live longer33. statistics show that between 80 to 85% of the elderly aged 65 years or older have at least one chronic medical condition34. the prevalence of comorbidities and multimorbidity, especially in the elderly, is high and may lead to, above all, a poor quality of life30,35,36. regarding the use of dental prostheses, it was found in the present study that the elderly are three times more likely to experience a negative impact on their oral health. this result corroborates that of a study by masood et al.37, conducted in 1,277 elderly people in the united kingdom, in which the use of total prosthesis negatively impacted oral health, and prosthetic wearers were twice as likely to have functional limitations, physical discomfort, and psychological discomfort than non-users. other studies have also reported an association between the use of dental prostheses and worse quality of life5,38,39. one of the ziggest consequences of poor oral health is edentulism12,40. data from the national oral health survey (sb brazil)41 showed that the characteristic of not having any teeth was common in many elderly people in the country, with a rate of 53.7%. in the present study, even though the prevalence of total edentulism patients is lower (25.6%) than that in the national survey, it is still quite high. in interpreting the results, it should be taken into account that the quality of the dental prosthesis and the masticatory efficiency were not evaluated in this study, which could influence the impact on quality of life. dental prostheses are not always ideal, so many individuals are not satisfied with the clinical effects of their dentures, due to deficiencies in feeding and speech, discomfort, poor retention, and stability42. in addition, dental care is not offered to institutionalized elderlies in the municipality investigated in the present research. 10 demarchi et al. braz j oral sci. 2023;22:e237543 it is important to highlight and recognise the limitations of the present study. one of the limitations is the fact that the study was not population-based, and therefore, the results cannot be generalised for the population of this age in the city. however, this limitation does not invalidate the present study. the study raises unique questions because the institutionalised population represents an often-forgotten contingent. besides, another limitation was the lack of information about the quality of the prosthesis and the evaluation of masticatory efficiency. we also point out as limitation, the absence of data on income, education, and knowledge of oral health by the elderly. increasing age, low education, ethnicity, low income, lack of knowledge about oral health has a negative impact on oral health-related quality of life among the elderly43. thus, the relevance of the quality of life of institutionalized elderlies is clear. it is also essential to carry out further studies on this condition, as it frequently presents itself in the elderlies, since there is a general increase in life expectancy and the geriatric contingent is increasingly on the rise. future research is encouraged, with a larger sample and from other locations, aiming at a greater understanding of the factors that contribute to the quality of life in relation to oral health. in conclusion, self-reported xerostomia and the use of dental prostheses negatively affected the quality of life with the oral health of the elderly, and people who have these conditions are more likely to have a worse quality of life. author contribution larissa steilmann demarchi: conceptualization, methodology, formal analysis, investigation, resources, data curation, writing original draft. mayara trapp vogel: writing review & editing, visualization. gabrielle haubert: supervision, project administration. lilian rigo: investigation, resources, formal analysis, data curation, writing original draft, writing review & editing, visualization, supervision, project administration. all authors actively participated, revised and approved the final version of the manuscript. references 1. kyu hh, abate d, abate kh, abay sm, abbafati c, abbasi n, et al. global, regional, and national disability-adjusted life-years (dalys) for 359 diseases and injuries and healthy life expectancy (hale) for 195 countries and territories, 1990-2017: a systematic analysis for the global burden of disease study 2017. lancet. 2018 nov;392(10159):1859-922. doi: 10.1016/s0140-6736(18)32335-3. 2. makovski tt, schmitz s, zeegers mp, stranges s, van den akker m. multimorbidity and quality of life: systematic literature review 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findings from a prospective cohort study in an urbanized low-income community. bmc geriatr. 2020 feb;20(1):78. doi: 10.1186/s12877-020-1480-9. 35. lee h-j, yun j. health-related quality of life in south korean community-dwelling older adults with multimorbidity: a convergent parallel mixed-methods approach. qual life res. 2020 mar;29(3):721-32. doi: 10.1007/s11136-019-02360-0. 36. zheng dd, mccollister ke, christ sl, lam bl, feaster dj, lee dj. chronic condition patterns in the us population and their association with health related quality of life. prev med. 2020 jul;136:106102. doi: 10.1016/j.ypmed.2020.106102. 37. masood m, newton t, bakri nn, khalid t, masood y. the relationship between oral health and oral health related quality of life among elderly people in united kingdom. j dent. 2017 jan;56:78-83. doi: 10.1016/j.jdent.2016.11.002. 13 demarchi et al. braz j oral sci. 2023;22:e237543 38. yen yy, lee he, wu ym, lan sj, wang wc, du jk, et al. impact of removable dentures on oral health-related quality of life among elderly adults in taiwan. bmc oral health. 2015 jan;15:1. doi: 10.1186/1472-6831-15-1. 39. paredes-rodríguez vm, torrijos-gómez g, gonzález-serrano j, lópez-pintor-muñoz rm, lópez-bermejo ma, hernández-vallejo g. quality of life and oral health in elderly. j clin exp dent. 2016 dec;8(5):e590-6. doi:10.4317/jced.53317. 40. felton da. edentulism and comorbid factors. j prosthodont. 2009 feb;18(2):88-96. doi: 10.1111/j.1532-849x.2009.00437.x. 41. brazilian ministry of health. [sb brazil 2010: national research on oral health: main results]. brasília: ministry of health; 2012 [cited 2022 jan 10]. available from: http://189.28.128.100/dab/docs/ portaldab/publicacoes/pesquisa_saude_bucal.pdf. portuguese. 42. sun x, zhai jj, liao j, teng mh, tian a, liang x. masticatory efficiency and oral health-related quality of life with implant-retained mandibular overdentures. saudi med j. 2014 oct;35(10):1195-202. 43. kwon sr, lee s, oyoyo u, wiafe s, de guia s, pedersen c, et al. oral health knowledge and oral health related quality of life of older adults. clin exp dent res. 2021 apr;7(2):211-8. doi: 10.1002/cre2.350. 1 volume 21 2022 e225388 original article braz j oral sci. 2022;21:e225388http://dx.doi.org/10.20396/bjos.v21i00.8665388 1 department of dentistry, hassan institute of medical sciences, hassan-573201, india. 2 department of orthodontics and dento-facial orthopaedics, dr syamala reddy dental college, hospital and research centre, bangalore, india. corresponding author: dr girish babu kl bds, mds professor and head, department of dentistry, hassan institute of medical sciences, hassan-573201, india e-mail:docgirish77@gmail.com editor: altair a. del bel cury received: april 21, 2021 accepted: december 12, 2021 dental arch characteristics among south indian twins – a morphometric study kl girish babu1 , geeta maruti doddamani2,* , gururaj hebbar kavyashree1 genetic and environmental factors are essential in occlusal variations and malocclusion and have been of considerable interest to orthodontists. studies on twin pairs are one of the most effective methods for investigating genetically determined occlusal variables. many studies have focused on distances between first molars or between canines but malocclusions can also occur in other regions of the dental arch. aim: to evaluate the characteristics of the dental arch between pairs of monozygotic (mz) and dizygotic (dz) twins from southern india. methods: a random sample of 51 twin pairs (12–18years old) participated in this study. the zygosity of twin pairs was recorded by facial appearance. the occlusion of the first permanent molars was recorded according to angle’s classification. study models were prepared to assess dental arch characteristics (i.e., arch form, arch perimeter, arch length; intercanine, intermolar width, and teeth size discrepancy). the obtained data was statistically analyzed using spss software 19.0. the student’s t-test (two-tailed, independent) and chi-square test was used to determine the significance of studied parameters. results: angle’s class i molar relation was more commonly observed followed by the class ii molar relationship among twins. the measured dental arch dimensions did not show a statistically significant difference among twin pairs. the ovoid arch form was commonly observed among monozygotic and dizygotic twins. there was a similarity among mz and dz twins in the anterior and overall bolton’s ratio. conclusion: there were similar occurrences of measured parameters among twins, which showed genetic predominance in the expression of measured dental arch traits. keywords: dental arch. twins. india. https://orcid.org/0000-0002-1449-3729 https://orcid.org/0000-0001-6315-9252 https://orcid.org/0000-0001-7243-1748 2 babu et al. braz j oral sci. 2022;21:e225388 introduction twin research has made important contributions to understanding normal and abnormal dentofacial development. some researchers believed that the value of twin studies would diminish owing to the revolution in molecular biology, which has occurred over the past 10–15 years. however, the data derived from twins and their families have allowed the researchers to adopt newer models and approaches that take advantage of the unique features of twins and the twinning process and combined these with advances in genotyping and phenotyping1. twins and the twinning process have been a continual source of fascination for human populations over the centuries. building on the pioneering work of sir francis galton in the 19th century, studies on twins became more common and sophisticated throughout the 20th century, which provided important insights into the relative contributions of genetic factors (nature) and environmental factors (nurture) to variation in many behavioral and physical features including some dental traits1. in dentistry, numerous differences in the dentofacial characteristics of individuals are seen even among family members. some children have large teeth, high prevalence of dental caries, while only some have good occlusion and low dental caries. therefore, the question arises as to whether the dental traits are inherited. twin research allows establishing detectable genetic variability and study genetic environment interactions in dental development2. othman et al. studied teenage twins and reported high genetic contribution to variation in dental arch dimensions3. cassidy et al., suggested that arch size and shape are mainly determined by environmental effects4. twin studies have demonstrated that, while genetic variance can be discerned for different occlusal variables, heritability tends to be low, which emphasizes the importance of environmental effects on occlusal variation5. harris and johnson obtained similar results in their longitudinal study on siblings6. the above mentioned researchers concluded that most observed variation in occlusion in permanent dentition was acquired rather than inherited6. some studies have implicated hereditary contributions to tooth size, dental malalignment, occlusion, and tooth morphology7-9. therefore, it is not clear whether dental arch characteristics are determined by genetic or environmental factors. thus, the aim of the study was to evaluate the characteristics of the dental arch between pairs of monozygotic (mz) and dizygotic (dz) twins from southern india. materials and methods before commencing this cross-sectional study, ethical clearance was obtained from the institutional review board. (hims/irc/126/20-21) the study protocol was as per the guidelines provided by the world medical association declaration of helsinki on ethical principles for medical research. the sample size calculation was done based on the previous studies1,9,10 on the sample size chart with the power of 0.09. twins aged 12–18-year-old studying in the schools participated in this study. permission from the schools was obtained, and preliminary identification of twins was done from the school admission register. each child was given an information sheet, 3 babu et al. braz j oral sci. 2022;21:e225388 which explained the purpose and nature of the study. children, who were willing to participate in the study, were given a consent form to obtain written informed consent from the parents. only the children who obtained informed consent were included in the study. strict confidentiality was assured for both parents and children. identification of twins was done by facial appearance and history10. the parents of each twin was met by an investigator, and a history of medical conditions, details of chorionicity, and the number of placental cords were obtained. twin pairs with (1) permanent occlusion including second molar teeth, (2) no history of dental extraction, prosthesis, or filling in specific contact or occlusal surfaces, (3) no history of diseases, injuries, or surgical intervention in the craniofacial region were included9,10. the following twin pairs were excluded9,10: (1) children with handicapping conditions including medically compromised individuals, (2) children on long-term medication, (3) children with not completely occluded second permanent molars, (4) children undergoing orthodontic treatment, (5) children with mixed dentition, (6) children with periodontal problems, and (7) children with developmental anomalies such as cleft lip and cleft palate, (7) missing teeth, (8) dental anomalies, (9) children with retained deciduous teeth, and (10) children with severe crowding (11) children with genetic syndromes. thus, a total of 51 pairs of twins were included, i.e., 27 pairs of mz and 24 pairs of dz twins. pro-forma was used to record the date of birth, gender, demographic details, and oral findings. the children were seated upright on a chair and examined in adequate natural daylight to receive maximum illumination. oral examination of children was performed by only one examiner to avoid inter-examiner variability. the kappa value for the intra-examiner agreement of the tooth status was 0.88. the examination was performed using a disposable sterile mouth mirror and probe. recording of data was done by a single trained assistant throughout the study. an oral examination was performed on a specific day and time scheduled as per the convenience of the children. the molar relation was recorded according to angle’s classification11. then; the impression of maxillary and mandibular teeth was made using a regular-setting very high viscosity impression material (aquasil® soft putty regular set, dentsply). the impression was analyzed for the presence of bubbles, accurate record of all teeth, and vestibular depth. any impression lacking these characteristics was repeated. the impression was dis infected with 0.5% sodium hypochlorite. the dental cast was prepared with a die stone.the dental cast was carefully separated from the impres sions, and its quality was analyzed. the dental cast with broken teeth, extended restorations to interdental contact points, broken cusp tips and incisal edges, increased number of porosity and presence of tooth wear were excluded12. if required a repeat cast was prepared. inter-molar width, inter-canine width, arch length,arch perimeter, and maxillary arch depth were measured using vernier’s caliper (calibrated with an error of ±0.01 mm) on the dental cast as described by moyer’s11. arch parameters for maxillary and mandibular jaws were separately measured. the dental arch form was assessed using arch form templates (orthoform; 3m unitek).the template was overlaid on the dental cast, and the best template was noted. the arch form was grouped into ovoid, tapered, and square according to chuck13. bolton14 analysis was used to 4 babu et al. braz j oral sci. 2022;21:e225388 determine tooth size discrepancy in maxillary and mandibular teeth.the mean was calculated for both the overall ‘12’ ratio and anterior ‘6’ ratio. all the dental arch measurements were recorded thrice and the mean of the three values was taken as the corresponding measurement. the obtained data were entered into a microsoft excel sheet and statistically analyzed using spss software 19.0 (ibm corp, released 2010, ibm spss statistics for windows, and version 19.0. armonk, ny: ibm corp.).in this study, descriptive statistical analysis was performed. significance was assessed at the 5% level of significance. the student’s t-test (two-tailed, independent) was used to determine the significance of study parameters on a continuous scale between two groups. intergroup analysis of metric parameters and chi-square/fisher exact test was used to determine the significance of study parameters on a categorical scale between two or more groups. the kappa statistic for the agreement was the inter-rater agreement statistic (kappa), which was used to evaluate the agreement between two classifications on ordinal or nominal scales. the agreement is quantified by kappa (κ) or weighted kappa (κw) statistics. results two independent examiners blinded to the type of twins measured the inter-molar width, inter-canine width, arch length, arch perimeter, and maxillary arch depth. they also determined the type of dental arch form and tooth size discrepancy in maxillary and mandibular teeth. kappa test was performed for the examiners and the score obtained was 0.87 and 0.90 for inter-examiner and intra-examiner, respectively. if there was any disagreement with the measurement, the examiners jointly reviewed and discussed and reached an agreement. if there was a disagreement between the examiners a lower measurement was considered. a total of 51 pairs of twins participated in this study. among 51 pairs of twins, there were 27 pairs of mz twins (14 male pairsand13 female pairs) and 24 pairs of dz twins (11 male pairs and 13 female pairs). angle’s class i molar relation was observed in 88.88% of mz twins and 95.33% of dz twins. angle’s class ii molar relation was seen in 14.81% of mz twins and 12.5% of dz twins (table1). figures 1 to 4 shows the mean value of inter-canine, inter-molar, arch length, arch perimeter, table 1. type of molar occlusal relationship among twins zygosity twin type of molar occlusal relationship class i class ii class iii right side n (%) p value left side n (%) p value right side n (%) p value left side n (%) p value right side n (%) left side n (%) mz (n=27) twin a 24 (88.88) 0.7 24 (88.88) 0.3 3 (11.11) 0.7 3 (11.11) 0.3 0 0 twin b 23 (85.18) 23 (85.18) 4 (14.81) 4 (14.81) 0 0 dz (n=24) twin a 21 (87.5) 0.7 21 (87.5) 0.4 3 (12.5) 0.7 3 (12.5) 0.4 0 0 twin b 23 (95.33) 23 (95.33) 1 (4.16) 1 (4.16) 0 0 5 babu et al. braz j oral sci. 2022;21:e225388 and arch depth of mz and dz twins both in maxillary and mandibular dental arches. on comparison of the mean values for all four parameters, there was no significant difference between mz and dz twins. the most common arch form was ovoid, which was seen in 70–81% of mz twins and 83–91% of dz twins. the tapered arch form was seen in 14–25% of twins. the square arch form was the least common (table 2). the tooth size discrepancies (i.e., mean of overall and anterior ratio among mz and dz twins) were similar (table 3). figure 1. maxillary dental arch characteristics among mz twins 0 10 20 30 40 50 60 70 80 intercanine width intermolar width arch length arch perimeter maxillary depth twin a twin b 31 .4 5 46 .5 8 28 .4 3 77 .9 6 10 .8 7 28 .4 5 44 .6 6 27 .0 8 76 .6 1 10 .4 5 table 2. type of dental arch form among twins zygosity twin arch dental arch form ovoid n (%) tapered n (%) square n (%) mz (n=27) twin a maxilla 19 (70.37) 7 (25.92) 1 (3.70) mandible 21 (77.77) 6 (22.22) 1(3.70) twin b maxilla 20(74.7) 5(18.51) 2(7.4) mandible 22 (81.48) 4 (14.81) 1 (3.70) dz (n=24) twin a maxilla 20 (83.3) 3 (12.5) 1 (4.1) mandible 21 (87.5) 3 (12.5) 0 twin b maxilla 21 (87.5) 2 (8.3) 1(4.1) mandible 22 (91.66) 1(4.1) 1(4.1) table 3. comparison of tooth size discrepancy between mz and dz twins twin monozygotic twins dizygotic twins overall ratio anterior ratio overall ratio anterior ratio twin a 91.3 ± 0.14 77.26 ± 0.162 91.34 ± 0.134 77.3 ± 0.128 twin b 91.4 ± 0.13 77.36 ± 0.173 91.27 ± 0.147 77.6 ± 0.133 p value 0.115 0.888 0.187 0.762 6 babu et al. braz j oral sci. 2022;21:e225388 figure 2. mandibular dental arch characteristics among mz twins 0 10 20 30 40 50 60 70 intecanine width intermolar width arch length arch perimeter 26 .4 4 41 .1 9 29 .9 4 67 .7 5 24 .2 1 42 .1 6 24 .4 3 67 .1 5 twin a twin b figure 3. maxillary dental arch characteristics among dz twins 0 10 20 30 40 50 60 70 80 intercanine width intermolar width arch length arch perimeter maxillary depth 31 .5 8 45 .9 2 32 .1 78 .3 10 .8 6 33 .4 6 4 5. 84 31 .6 2 77 .7 7 10 .6 3 twin a twin b figure 4. mandibular dental arch characteristics among dz twins 0 10 20 30 40 50 60 70 intercanine width intermolar width arch length arch perimeter 25 .1 8 43 .5 4 33 .3 3 66 .7 9 26 .8 3 40 .1 6 32 .9 3 66 .0 5 twin a twin b 7 babu et al. braz j oral sci. 2022;21:e225388 discussion genetic and environmental factors are essential in occlusal variations and malocclusion and have been of considerable interest to dentist. studies on twin pairs are one of the most effective methods for investigating genetically determined occlusal variables. however, studies on genetic effects on the dental arch have reported varying results3-9. zygosity can be determined using physical features, examining the placenta, or dna testing using cells from inside the cheek. in this study, twins were initially segregated as mz (identical) and dz (non-identical) based on general facial appearance. this method of zygosity recording is easier, non-invasive, and requires little cooperation from the twin pairs. comparison of facial appearance is a reasonably accurate approach for distinguishing between mz and dz twin pairs15-17. using this method, determination of zygosity is straight forward if the children are of different sexes (i.e., dz). among twins of the same sex, by the time the children are approximately two years old, their zygosity may be clear from their physical features. color of hair and eyes, the shape of ears, teeth eruption and formation, the shape of hands and feet, and pattern of growth provide a good indication as to whether or not the twins are identical18. genetic and environmental factors affect the occlusion and dental arch parameters. there is indisputable evidence for significant influence by many dental and occlusal variables19. in this study, most mz and dz twins showed angle’s class i molar relation followed by class ii molar relation, and none of them had class iii molar relation. there was concordance among mz and dz twin pairs in the first molar relation. similarly, lundstrom reported angle’s class i molar relationship in most of the studied twins followed by class ii molar relation20. among indian twins, the molar relation was highly significant within mz twin pairs and had a 100% concordance rate. however, there was a decrease in the correlation rate for mz twins compared to dz twins, which suggested that the environment also affected the development of malocclusion10. significant genetic variance of occlusal parameters (e.g., arch size and shape, overjet, overbite, and rotated teeth) have been reported9. however, an increased environmental component of variance in occlusion has also been reported21. eguchi et al.2 stated that occlusal variables are associated with low estimates of hereditability. corrruccini and potter21 and townsend et al.5 also noted that heritabilities for occlusal variables (e.g., overbite and overjet) were relatively low. orthodontists need to know how much the dental arch dimensions depend on genetics and how much on the environment because larger effects of genetics suggest less effective orthodontic treatment outcomes. the stability of such outcomes depends on a certain balance between genetic and environmental factors22. in this study, the measured dental arch parameters (i.e., intercanine width, intermolar width, arch length,arch perimeter, and maxillary depth) were not significantly different between mz or dz twin pairs. the similarities in mz twin pairs suggest a high genetic predisposition. a high intra pair concordance for dental size traits among indian mz twins has been reported, which suggests a significant genetic influence23. boraas et al.24 reported that arch width is under significant genetic influence. othman et al.3 observed 8 babu et al. braz j oral sci. 2022;21:e225388 a high genetic contribution in dental arch dimensions. among lithuanian twins, the largest genetic effect was observed on the upper dental arch breadth between lateral incisors9. a similar but lower heritability was inherent for canines and first premolars of the upper jaw and first premolars of the lower jaw. among twins, the arch breadths between posterior teeth showed lower heritability estimates than between anterior teeth on both jaws. the dental arch in the upper jaw has a more expressed genetic component than that in the lower jaw9. data among australian twins revealed that arch length was mainly determined by genetics than arch breadth parameters2. the same study also determined that in the upper jaw, the breadths had increasing heritability with increasing distality of teeth, while in the lower jaw this was less expressed2. dempsey et al.25 studied the width of teeth and revealed that genetic factors affected all incisors. townsend et al.26 stated that the heritability of lower arch length in australian twins can be as high as 0.92 and is mainly determined by genetics than arch breadth (0.82). less consistent differences between upper and lower arch parameters were also observed by eguchi et al.2, with more prevailing heritability in the upper jaw than in the lower jaw. shapiro27 reported that genetic factors more strongly contributed to variation in palatal height than either arch breadth or length. similarly, eguchi et al.2 reported that variation in the vertical dimension (height) of the maxillary dental arch is under considerable genetic control. a preliminary comparison of arch dimensions among australian twins failed to disclose any significant differences2. among them, both in maxilla and mandible, males exhibited a significant difference in arch dimensions than females but there was no significant difference in arch length and palatal heights displayed the greatest variation2. authors from india reported a considerable resemblance in mandibular irregularity, maxillary intercanine distance, mandibular intercanine distance, and open bite among twins and concluded that there is a significant genetic predisposition for the studied dental parameters10. all the above mentioned studies suggest that dental arch parameters are mainly determined by genetics. however, other researchers concluded that arch size is mainly determined by environmental than heredity factors; however, these researchers also stated that arch widths has the highest heritability estimates in adolescents4. they also suggested that arch length and width growth factors were mainly independent. harris and johnson6 studied the arch breadth and length in 4–20-year-old subjects and reported that heritability estimates of traits related to tooth position gradually decreased, whereas the heritability estimates of craniofacial variables increased with age. further, they highlighted the dynamic nature of the masticatory system and associated habitual activities and indicated that the relative contribution of genetic and environmental factors to occlusal variation was likely to change over time and also differed in different regions of dental arches6. kawamura et al.28 have proposed that dental arch breadth is related to buccolingual tooth inclination and, in turn, is affected by masticatory function, and tooth size is related to arch length. they stated that the three arch dimensions (i.e., arch length, breadth, and height, each occurring in a different plane) appeared to be independent of one another28. šidlauskas29 stated that total mandibular and corpus lengths are more heritable than maxillary. this result suggest that the effect of heritability on total jaw parameters does not apply to dental arch indicators. the difference in obtained results 9 babu et al. braz j oral sci. 2022;21:e225388 may be due to differences in the studied population, specific sample differences, or methodology that was used for the determination of zygosity. the dental arch form has a wide individual variation in humans. the establishment of dental arch form aids manufacturers in designing arch wires. the most common arch form observed among both mz and dz was the ovoid followed by tapered and square arch form. similar observations was reported in population of saudi arabian, iran, caucasia, malaysia and india, but among singletons3,12,30-32. sahoo et al. observed a narrow arch form to be most common among indians than among bhutanese33. kook et al.34 observed the square and tapered form of the dental arch were most common among the korean and united states population, respectively. to ensure proper inter-digitations, overbite, and overjet specific dimensional relationships must exist between maxillary and mandibular teeth. proportionality of the size of maxillary and mandibular teeth affects the establishment of good stable functional occlusion. in this study, mz and dz twins showed values that were similar to those in bolton’s analysis for both anterior and overall ratios. there was concordance among mz and dz twins when anterior and overall ratios were compared. studies on singletons conducted by smith et al. using bolton’s inter-arch ratios in three populations (i.e., black, hispanic, and white) identified significantly different relationships between lower and upper teeth35. these authors have also observed sexual dimorphism in tooth dimensions and the ratio of upper and lower arch tooth sizes35,36. lavelle reported that blacks have larger overall and anterior ratios than whites and asians36. stifter37 replicated bolton’s study in non-twins with class i dentitions and reported similar results, which match the results of our study. the results of our study shows a genetic predominance in determining dental arch characteristics among twins. however, because the data on the genetic component may be country or region-specific or have an ethnic or racial background, it is essential to estimate the genetics across different populations and countries. it is also necessary to analyze environmental factors because they are essential for the development and determining the shape of dental structures. the determination of twins using facial photographs and history. the cross-sectional nature and small sample size may have impacted the results of this study. future longitudinal studies involving a larger sample size and determining zygosity using dna of buccal cells should be performed to further validate our results. multidisciplinary studies of twins with input from dentists, molecular geneticists, and twin researchers need to be performed to clarify the role of genetic factors in contributing to dental characteristics. in conclusion, the similar occurrence of measured characteristics of dental arch between pairs of monozygotic (mz) and dizygotic (dz) twins suggest that there is genetic predominance in the expression of dental arch traits. acknowledgment the authors report that we do not have a financial affiliation or any conflicts of interest for this paper. 10 babu et al. braz j oral sci. 2022;21:e225388 conflict of interest none. funding agency none. author contribution dr girish babu kl: conceived the idea, concept and research design, literature search, data analysis and preparation of manuscript dr geeta maruti doddamani: conceived the idea, concept and research design, collected the data; conducted the experiment dr kavyashree g: conceived the idea, concept and research design, literature search, data analysis and preparation of manuscript all authors actively participated in the discussion of the manuscript’s findings, and have revised and approved the final version of the manuscript. references 1. hughes te, townsend gc, pinkerton sk, bockmann mr, seow wk, brook ah, et al. the teeth and faces of twins: providing insights into dentofacial development and oral health for practising oral health professionals. aust dent j. 2014 jun;59 suppl 1:101-16. doi: 10.1111/adj.12101. 2. eguchi s, townsend gc, richards lc, hughes t, kasai k. genetic contribution to dental arch size variation in australian twins. arch oral biol. 2004 dec;49(12):1015-24. doi: 10.1016/j.archoralbio.2004.07.006. 3. othman sa, xinwei es, lim sy, jamaludin m, mohamed nh, yusof zy, et al. comparison of arch form between ethnic malays and malaysian aborigines in peninsular malaysia. korean j orthod. 2012 feb;42(1):47-54. doi: 10.4041/kjod.2012.42.1.47. 4. cassidy km, harris ef, tolley ea, keim rg. genetic influence on dental arch form in orthodontic patients. angle orthod. 1998 oct;68(5):445-54. doi: 10.1043/0003-3219(1998)06. 5. townsend gc, corruccini rs, richards lc, brown t. genetic and environmental determinants of dental occlusal variation in south australian twins. aust orthod j. 1988 oct;10(4):231-5. 6. harris ef, johnson mg. heritability of craniometric and occlusal variables: a longitudinal sib analysis. am j orthod dentofacial orthop. 1991 mar;99(3):258-68. doi: 10.1016/0889-5406(91)70007-j. 7. goodman ho, luke je, rosen s, hackel e. heritability in dental caries, certain oral microflora and salivary components. am j hum genet. 1959 sep;11(3):263-73.  8. boraas jc, messer lb, till mj. a genetic contribution to dental caries, occlusion, and morphology as demonstrated by twins reared apart. j dent res. 1988 sep;67(9):1150-5. doi: 10.1177/00220345880670090201. 9. švalkauskienė v, šmigelskas k, šalomskienė l, andriuškevičiūtė i, šalomskienė a, vasiliauskas a, et al. heritability estimates of dental arch parameters in lithuanian twins. stomatologija. 2015;17(1):3-8. 11 babu et al. braz j oral sci. 2022;21:e225388 10. anu v, arsheya gs, anjana v, annison gk, lakshmi aruna mr, alice ap, et al. dental caries experience, dental anomalies, and morphometric analysis of canine among monozygotic and dizygotic twins. contemp clin dent. 2018 sep;9(suppl 2):s314-s317. doi: 10.4103/ccd.ccd_345_18. 11. moyers re. handbook of orthodontics. 4th ed. chicago: year book medical publishers; 1998. 12. toodehzaeim mh, mostafav sms. dental arch morphology in iranian population. iran j orthod. 2016 sep;11(2):e5863. 13. chuck gc. ideal arch form. angle orthod. 1934;4:312-27. 14. bolton wa. disharmony in tooth size and its relation to the analysis and treatment of malocclusion. angle orthod. 1958;28(3):113-30. 15. nylander pp. fingerprints and the determination of zygosity in twins. am j phys anthropol. 1971 jul;35(1):101-8. doi: 10.1002/ajpa.1330350112. 16. hamilton d, boyle ja, greig wr, jasani mk, buchanan ww. dermatoglyphic differences in determination of dizygosity diagnosis. j forensic sci soc. 1969 dec;9(3):141-6. doi: 10.1016/s0015-7368(69)70525-4. 17. parisi p, di bacco m. fingerprints and the diagnosis of zygosity in twins. acta genet med gemellol (roma). 1968 apr;17(2):333-58. doi: 10.1017/s1120962300012750.  18. subramaniam p, babu kl, vardhana b. assessment of dental caries and oral hygiene status among twins. j forensic sci med 2018;4:18-22. 19. tyagi r, khuller n, sharma a, khatri a. genetic basis of dental disorders: a review. j oral health comm dent 2008; 2(3):55-61. 20. lundström a. tooth size and occlusion in twins. basel: karger; 1948. p.71-5. 21. corruccini rs, potter rh. genetic analysis of occlusal variation in twins. am j orthod. 1980 aug;78(2):140-54. doi: 10.1016/0002-9416(80)90056-1.  22. van der linden fp. genetic and environmental factors in dentofacial morphology. am j orthod. 1966 aug;52(8):576-83. doi: 10.1016/0002-9416(66)90138-2. 23. sharma k, corruccini rs, henderson am. genetic variance in dental dimensions of punjabi twins. j dent res. 1985 dec;64(12):1389-91. doi: 10.1177/00220345850640121301. 24. boraas jc, messer lb, till mj. a genetic contribution to dental caries, occlusion, and morphology as demonstrated by twins reared apart. j dent res. 1988 sep;67(9):1150-5. doi: 10.1177/00220345880670090201. 25. dempsey pj, townsend gc, martin ng, neale mc. genetic covariance structure of incisor crown size in twins. j dent res. 1995 jul;74(7):1389-98. doi: 10.1177/00220345950740071101. 26. townsend g, richards l, messer lb, hughes t, pinkerton s, seow k, et al. genetic and environmental influences on dentofacial structures and oral health: studies of australian twins and their families. twin res hum genet. 2006 dec;9(6):727-32. doi: 10.1375/183242706779462516. 27. shapiro bl. a twin study of palatal dimensions partitioning genetic and environmental contributions to variability. angle orthod. 1969 jul;39(3):139-51. doi: 10.1043/0003-3219(1969)039<0139:atsopd>2.0.co;2.  28. kawamura a, kanazawa e, kasai k. relationship between teeth positions and morphological characteristics of vertical sections of the mandible obtained by ct scanning. orthod waves 1998;57:299-306. 29. šidlauskas m, šalomskienė l, andriuškevičiūtė i, šidlauskienė m, labanauskas ž, šidlauskas a. mandibular morphology in monozygotic twins: a cephalometric study. stomatologija. 2014;16(4):137-43.  12 babu et al. braz j oral sci. 2022;21:e225388 30. murshid z. patterns of dental arch forms in the different classes of malocclusion. j am sci. 2012;8:308-12. 31. paranhos lr, andrews wa, jóias rp, bérzin f, daruge junior e, triviño t. dental arch morphology in normal occlusions. braz j oral sci. 9(4):475-480. doi: 10.20396/bjos.v10i1.8641705. 32. patel vj, bhatia af, mahadevia sm, italia s, vaghamsi m. dental arch form analysis in gujarati males and females having normal occlusion. j ind orthod soc 2012;46(4):295-99. 33. sahoo n, mohanty r, mohanty p, sonal. comparison of arch forms between indian and bhutanese populations. j res adv dent 2016; 5:1:75-82. 34. kook ya, nojima k, moon hb, mclaughlin rp, sinclair pm. comparison of arch forms between korean and north american white populations. am j orthod dentofacial orthop. 2004 dec;126(6):680-6. doi: 10.1016/j.ajodo.2003.10.038. 35. smith ss, buschang ph, watanabe e. interarch tooth size relationships of 3 populations: “does bolton’s analysis apply?” am j orthod dentofacial orthop. 2000 feb;117(2):169-74. doi: 10.1016/s0889-5406(00)70228-9.  36. lavelle cl. maxillary and mandibular tooth size in different racial groups and in different occlusal categories. am j orthod. 1972 jan;61(1):29-37. doi: 10.1016/0002-9416(72)90173-x. 37. stifter j. a study of pont’s, howes’, rees’, neffs and bolton’s analyses on class 1 adult dentitions. angle orthod. 1958;28:215-25. 1 volume 22 2023 e238358 original article braz j oral sci. 2023;22:e238358http://dx.doi.org/10.20396/bjos.v22i00.8668358 1 graduate program in physical therapy, state university of northern paraná – uenp – jacarezinho, pr, brazil. 2 post graduate program in oral and maxillofacial surgery, piracicaba dental school, university of campinas – unicamp – piracicaba, sp, brazil. 3 post graduate program in human movement sciences, methodist university of piracicaba unimep – piracicaba, sp, brazil. 4 post graduate program in oral and dental biology, piracicaba dental school, university of campinas – unicamp – piracicaba, sp, brazil. corresponding author: elisa bizetti pelai post graduate program in oral and maxillofacial surgery, piracicaba dental school, state university of campinas – unicamp – av. limeira, 901 areião, piracicaba sp, 13414-903. phone number +55 19 998052628. e-mail: elisabpelai@gmail.com editor: dr. altair a. del bel cury received: july 3, 2022 accepted: february 8, 2022 maximum bilateral bite strength and rms emg for the diagnosis of myogenic tmd paulo fernandes pires1 , elisa bizetti pelai*2 , marcio de moraes2 , ester moreira de castro carletti3 , fabiana foltran mescollotto3 , fausto berzin4 , delaine rodrigues bigaton2 aim: the study aimed to evaluate the accuracy of the maximum bilateral molar bite force and the root mean square (rms) electromyography (emg) index of the masticatory muscles in the maximum bilateral molar bite (mmbmax) of women with myogenic temporomandibular disorder (tmd) and asymptomatic. methods: this is a cross-sectional study, composed of 86 women allocated to the tmd group (n=43) and control group (n=43) diagnosis through the diagnostic criteria for temporomandibular disorders. the maximum bilateral molar bite force was evaluated using a bite dynamometer and the rms emg index of the masticatory muscles (anterior temporalis, masseter) during 5 seconds of the mmbmax task. student t-test was used for data comparison between accuracy of the bite force and rms emg of masticatory muscles during the mmbmax. results: the maximum bilateral molar bite force showed high accuracy (auc=0.99) for the diagnosis of women with myogenic tmd and asymptomatic women, and the rms emg index evaluated during the mmbmax showed a moderate level of accuracy for all masticatory muscles (auc=0.70 to 0.75). conclusion: the bilateral bite dynamometer with a surface emg during bilateral bite can be used to diagnose tmd in young women. keywords: diagnosis. electromyography. temporomandibular joint disorders. https://orcid.org/0000-0003-3191-4772 https://orcid.org/0000-0003-0826-9744 https://orcid.org/0000-0002-5229-5723 https://orcid.org/0000-0002-0688-165x https://orcid.org/0000-0002-6145-7448 https://orcid.org/0000-0002-9179-1893 https://orcid.org/0000-0002-3423-5575 2 pires et al. braz j oral sci. 2023;22:e238358 introduction temporomandibular disorder (tmd), considered the main cause of pain in the orofacial region1, is characterized by joint and/or muscular pain, limited or irregular mandibular function, and noises in the temporomandibular joints (tmj)2,3. tmd presents a multifactorial etiology4 making the diagnosis complex5.the diagnostic criteria for temporomandibular disorders (dc/tmd)1 is the gold standard tool for tmd diagnosis. individuals with tmd exhibit many changes in the electrical activity of masticatory muscles due to their dysfunction or through a compensatory mechanism associated with symptoms6. therefore, surface electromyography (emg) emerges as a bioelectric and non-invasive instrument that allows the assessment of muscle electrical activity7, which can be used in the clinical environment to assist in the diagnosis of myogenic tmd8,9. the electrical activity of the masticatory muscles during the sustained submaximal molar bite task has been evaluated using a bite dynamometer or small thickness tension measurement sensors10,11. xu, et al.11 evaluated the submaximal unilateral molar bite sustained at 30% of the maximum bite force (mbf), using a small thickness unilateral bite dynamometer, for the maximum time tolerated by the volunteers, and found significantly higher values of normalized root mean square (rms) emg in the tmd group compared to the control group in masticatory muscles. mbf is one of the indicators of the functional status of the masticatory system12 and can be affected by some factors: craniofacial morphology, sex, age, and occlusal status13-15. todic et. al.12 indicated that the tmd significantly affects the potential of masticatory muscle action which is confirmed by the analysis of mbf with significantly lower values in patients with tmd16 and with the fact that mbf values decrease with the increase in the severity of tmd. one possibility is that the presence of masticatory muscle pain and/or tmj inflammation can play a role in the mbf17. however, it is currently unclear how tmd affects mbf. based on the capacity of emg to evaluate the electrical activity of the masticatory muscles and the dynamometer to measure force between individuals with tmd and asymptomatic individuals, it becomes important to analyze the instruments in question, by measuring their accuracy, since they present good applicability, easy access, and non-invasive assessments, which could assist health professionals in the diagnosis and treatment of tmd18,19. accuracy analysis is defined as the amount of agreement between the measurement results of an instrument studied with the measurement results of another instrument already established and used as the gold standard, which can be calculated using the receiver-operating characteristic curve (roc curve)20,21. berni et al.9 demonstrated that the rms emg index of the masticatory muscles has a moderate level of accuracy for discrimination between individuals with myogenic tmd and asymptomatic individuals during the tmj rest task and inadequate levels 3 pires et al. braz j oral sci. 2023;22:e238358 of accuracy during the maximum isometric bilateral molar bite task. in contrast, manfredini et al.8 demonstrated that the rms emg index of the masticatory muscles shows moderate to high accuracy for the diagnosis of myogenic tmd during the maximal isometric bilateral molar bite task, but low accuracy levels in the tmj resting task. based on the cited literature, data on the accuracy of the rms emg index of masticatory muscles during specific tasks are still conflicting. furthermore, no information was found on the electrical activity of the anterior masseter and temporal muscles considering the use of a bilateral molar bite dynamometer. we hypothesized that there is a significant difference between women with tmd and asymptomatic women for values of maximum force of bilateral molar bite using the bite dynamometer, in the rms emg index of the masticatory muscles during the maximum bilateral molar bite and that these are accurate for the diagnosis of women with muscular tmd and asymptomatic women. the present study aimed to evaluate the accuracy of the maximum bilateral molar bite force and the rms emg index of the masticatory muscles during the maximum bilateral molar bite in women with myogenic and asymptomatic tmd. methods study design this is a cross-sectional study, approved by the research ethics committee of the university, protocol no. 25/2015. the volunteers who agreed to participate in this research signed a consent form. subjects a sample size calculation was performed based on a pilot study. the outcome used was the surface emg (masticatory pattern). the mean and standard deviation normalized rms values of the anterior temporal muscle during the biting phase of the control (n=10) and the tmd group (n=10) were, respectively, 89.06±8.21% and 83.44±6.64%, and, an effect size of 0.34 was found. for a power of 95% and a 5% alpha, n was determined as 43 volunteers per group. the calculation was performed using gpower® software, version 3.1.9.2. (uiversität kiel kiel, schleswig-holstein, germany). the volunteers were recruited, from may 2016 to april 2017, from the surgery sector of a school of dentistry (sao paulo state, brazil). inclusion and exclusion criteria women aged between 18 and 45 years and body mass index (bmi)<25 kg/m²7,22 were selected. for the tmd group, the volunteers were required to be diagnosed with myogenic tmd (dc/tmd)1 with the presence of present pain and/or fatigue in the masticatory muscles for at least 6 months. women who received physical or pharmacological treatment (eg. analgesic), with dental losses, who used total or partial dentures, with a history of facial and tmj 4 pires et al. braz j oral sci. 2023;22:e238358 trauma, a history of subluxation or dislocation of the tmj, and who were diagnosed with degenerative joint diseases through the dc/tmd were excluded from the study. for the control group, the volunteers were required not to present pain and any diagnosis of tmd (dc/tmd). it is important to highlight that both groups contain only women for convenience, according to epidemiological studies, tmd is more prevalent in women than in men23. materials diagnostic criteria of temporomandibular disorder (dc/tmd) the dc/tmd is a validated questionnaire to diagnose tmd. axis i of the dc/tmd contains a physical evaluation and considers recurrent factors of the patient’s daily life; axis ii considers the previous history, beginning, and perpetuating factors of the dysfunction1. visual analogue scale (vas) the vas was used to measure orofacial pain. it is a linear scale, 10 cm in length, labeled at the two extremes with the boundaries of pain sensation: “no pain” at one end, and “worst pain imaginable” at the other end24,25. maximum bilateral molar bite force (mmbmax) the mmbmax force was assessed using a bite dynamometer dfm021115/200 (emg system do brasil, são josé dos campos, brazil) with iron rods designed for the oral bite, protected by silicone material 15 mm thick. the device has a kgf scale with a reading capacity from 0 to 200 kgf and was connected directly to one of the channels of the electromyographic acquisition module. the bite dynamometer was used to assess strength during mmbmax, maintaining a sampling frequency of 2000 hz. before the tasks, all volunteers were trained on the day of collection to use the instrument and were asked to bite the instrument stem with upper and lower molar teeth bilaterally. verbal encouragement was given during the bite tasks. electromyography the emg 830c signal acquisition module (emg system do brasil, são josé dos campos, brazil) was used for reading the semg signals, with an impedance of >10  mω, analog/digital converter, 16-bit resolution, a sampling frequency of 2000 hz, and fourth-order butterworth filter. four differential surface electrodes (self-adhesive, ag/agcl, conductive gel) were used, and the distance inter-electrode was 10  mm. a reference electrode (30×40 mm) was positioned on the manubrium of the sternum. the electrodes were positioned following the criteria proposed by cram26. the gain was 20×, a common mode rejection >130  db, an input impedance of 10  gω, and signal-to-noise ratio <3 μv rms.  5 pires et al. braz j oral sci. 2023;22:e238358 procedures on the same day, after recruiting volunteers according to screening via the dc/tmd, anthropometric data, pain, emg assessment of masticatory muscles, and mmbmax were collected in each volunteer for 5 seconds. for the emg evaluation procedure and mbf, the volunteers stayed seated in a chair respecting the frankfurt parallel plan. for the mmbmax task, the volunteers were asked to perform the bilateral molar bite on the dynamometer with the maximum strength possible, even if they felt pain in the tmj, to obtain the maximum bite force value and evaluation of the electrical activity of the masticatory muscles. emg signal processing and maximum bite force the matlab® software 8.5.0.1976.13 (r2015a, mathworks inc., natick, massachusetts, usa) was used to process the emg, and bite force data. a 4th order digital butterworth filter was applied to the emg signal, with zero phase delay (high pass of 10 hz, low pass of 400 hz). the first and second emg signals were always eliminated to avoid interferences that occurred at the beginning and end of each collection. the emg indices were processed in the amplitude domain to determine the rms values, through the evaluation of the magnitude of the electrical activity of the masticatory muscles during the mmbmax task. statistical analysis data were submitted to the normality test (kolmogorov-smirnov) and described as mean, standard deviation, and 95% confidence interval. the student t-test was used for intergroup comparisons of the rms emg index in the mmbmax task, as well as for anthropometric data, tmj rom movement, and maximum bilateral bite force. the roc curve was analyzed to determine the diagnostic accuracy (area under the curve auc), cut-off point, sensitivity and specificity of the maximum bilateral molar bite force, and rms emg index of the masticatory muscles referring to the mmbmax task. the values used for the auc classification followed the recommendations of greiner et al.27 and akobeng et al.20: excellent discrimination (0.90 to 1.0); good discrimination (0.80 to 0.90); moderate discrimination (0.70 to 0.80); poor discrimination (0.60 to 0.70); and discrimination no better than chance (≤0.50). the youden index (yi) was also calculated by the formula: yi = ([sensitivity + specificity]–1)28. based on the study by akobeng, et al.20, to identify the best cut-off point, the point with the lowest resultant value for the expression was selected: (1-sensitivity)2 + (1-specificity)2. to guarantee the methodological quality of this study, intra-rater reliability was also analyzed for each muscle on the rms emg index and the maximum bilateral bite force considering the two repetitions collected in the mmbmax. for this purpose, the intraclass correlation coefficients (icc), model: two-way mixed; type: absolute agreement; calculated reliability: single measurement. the icc values were classified according to weir29: icc<0.40 (low reliability), icc≥0.40to≤0.75 (good reliability), 6 pires et al. braz j oral sci. 2023;22:e238358 and icc>0.75 (excellent reliability). the following formula was used to calculate the sem: sem=standard deviationx√(1-icc). data processing was performed using spss® software, version 17.0 (chicago, il, usa). significance was set at 5% (p<0.05). results the final sample was composed of 86 women, divided into two groups: tmd group (n=43) and control group (n=43). concerning the level of overall intra-rater reliability of the data, between the 2 repetitions in the mmbmax, excellent reliability was observed for the maximum bite force assessed through the dynamometer (icc=0.97; epm=6.67 kgf). for the rms emg index, excellent levels of reliability were found for the right anterior temporal muscle (icc=0.94; epm:51.38 µv), left anterior temporal muscle (icc=0.95; epm:42.27 µv), right masseter (icc=0.85; epm:100.7 µv), and left masseter (icc=0.86; epm:101.39 µv). table 1 shows the tmd classifications of all volunteers, diagnosed using the dc/tmd. table 1. diagnosis of volunteers according to the dc/tmd (n=86). diagnosis control group 43 myalgia 22 myofascial pain 15 myofascial pain with referral 6 disc displacement with reduction (r/l) 14 (10/9) disc displacement without reduction, with limited opening (r/l) 4 (4/4) disc displacement without reduction, without limited opening (r/l) 6 (5/6) arthralgia 34 (25/28) r/l= right/left table 2 shows that the groups were homogeneous in terms of age and bmi and presented a significant difference in rom of mouth opening without pain. the groups also demonstrated homogeneity regarding the preference of the chewing side (x2=2.90; p=.14), since in the tmd group 15 volunteers had a preference for chewing on the left side and 28 on the right side and, in the control group, 8 volunteers had a preference for chewing on the left side and 35 on the right side. table 2. intergroup comparison of anthropometric data and rom of the tmj. mean±standard deviation significance age (years) tmd group 28.72±8.04 t=1.25; p=0.21 control group 22.69±6.94 continue 7 pires et al. braz j oral sci. 2023;22:e238358 continuation bmi (kg/m2) tmd group 22.43±2.51 t=-0.61; p=0.54 control group 22.74±2.13 orofacial pain intensity in rest (cm) tmd group 3.13±2.68 na control group 0.0±0.0 rom of active mouth opening without pain (mm) tmd group 33.04±11.73 t=-6.91; p<0.001* control group 46.85±5.51 rom of active mouth opening with pain (mm) tmd group 41.16±11.33 na control group na right lateralization rom of the atm (mm) tmd group 9.27±5.95 t=-1.63; p=0.10 control group 10.85±1.93 rom of lateralization to the left of the atm (mm) tmd group 10.81±8.06 t=-0.73; p=0.46 control group 11.76±2.29 tmj protrusion rom (mm) tmd group 6.32±4.80 t=-1.15; p=0.25 control group 7.23±1.8 *significant intergroup difference (t-student test). na: not applicable. table 3 expresses the values of bite force and rms emg in the mmbmax task, in which a significant intergroup difference was found in the mmbmax and the electrical activity of the temporal and masseter muscles, with greater strength and activity in the control group. table 3. comparison of maximum bite force (kgf), orofacial pain (cm), and rms emg index (µv) in the mmbmax task.   mean±standard deviation mean difference (95%ci) significance maximum bilateral molar bite force tmd group 30.85±9.69 -39.67 (-44.04\-35.30) t=-18.03; p<0.001* control group 70.52±11.13 orofacial pain intensity after mmbmax tmd group 5.76±2.68 na control group 0±0 continue 8 pires et al. braz j oral sci. 2023;22:e238358 continuation rms emg anterior temporalis left muscle tmd group 114.90±57.60 -63.46 (-100.71\-26.22) t=-3.40; p<0.001* control group 178.36±111.19 rms emg masseter left muscle tmd group 192.91±148.79 -69.50 (-125.34\-13.66) t=-2.48; p=0.02* control group 262.40±115.56 rms emg anterior temporalis right muscle tmd group 125.28±62.46 -78.89 (-120.41\-37.37) t=-3.80; p<0.001* control group 204.17±124.66 rms emg masseter right muscle tmd group 192.68±126.65 -89.15 (-140.18\-38.11) t=-3.47; p=0.00* control group 281.83±117.53 *significant intergroup difference (t-student test). 95% ci: 95% confidence interval. na: not applicable. figure 1 shows the roc curves, respectively, of the mmbmax and the rms emg values of the masticatory muscles in the mmbmax. maximal bite force reference line left temporal right temporal reference line left masseter right masseter 0.0 0.2 0.4 0.6 0.8 1.0 0.8 0.6 0.4 0.2 0.0 s en si tiv ity 1 – specificity 1.0 a 0.0 0.2 0.4 0.6 0.8 1.0 0.8 0.6 0.4 0.2 0.0 s en si tiv ity 1 – specificity 1.0 b figure 1. roc curve a of the maximum bilateral molar bite force; b emg rms of masticatory muscles in the mmbmax. table 4 shows the levels of accuracy, sensitivity, specificity, and best cut-off point of the data illustrated in figure 2. it was observed that the maximum force of the bilateral molar bite showed a high level of accuracy and the values of rms emg 9 pires et al. braz j oral sci. 2023;22:e238358 during the mmbmax showed a moderate level of accuracy for the masseter and temporal muscles. table 4. accuracy level of maximum bite force (kgf) and rms emg values (µv) of masticatory muscles in mmbmax. auc (ic95%) best cut point sensitivity (%) specificity (%) j ([sensitivity+specificity] – 1) maximum bilateral molar bite force 0.99(0.00 1.00) 52.41 97.78 97.78 0.95 rms emg anterior temporalis left muscle 0.74(0.64 0.85) 107.4 62.22 86.67 0.49 rms emg masseter left muscle 0.70(0.60 0.81) 173.03 62.22 75.56 0.38 rms emg anterior temporalis right muscle 0.73(0.63 0.83) 136.9 60 75.56 0.35 rms emg masseter right muscle 0.75(0.65 0.86) 208.59 68.89 75.56 0.44 auc: area under the roc curve; j: youden index discussion force the results demonstrated a high level of accuracy in the maximum bilateral molar bite strength assessed using a bite dynamometer for the diagnosis of women with myogenic tmd. it is important to emphasize that this research is pioneering since it defines the best cut-off point in the use of the instrument in question for the diagnosis of myogenic dysfunction. these findings need to be interpreted with caution, since they should be used in line with the criteria employed in this study, that is, in young women, using a 15 mm thick bilateral molar bite dynamometer. considering the methodology used in this research, the value of 52.41 kgf was established as the best cut-off point for the diagnosis of myogenic tmd; positive cases should be diagnosed when the mmbmax is less than the cut-off point, and negative cases when the maximum force is equal to or greater than the cut-off point. this research also found a significantly greater difference in mbf for the control group compared to the tmd group. this fact is in agreement with previous research, such as the study by kroon and naeije30, who evaluated maximum strength with a force transducer for an incisive bite; castroflorio et al.10 who portrayed the maximum force of the bilateral molar bite with an intraoral force transducer; and xu et al.11 who observed maximum strength with a unilateral molar bite dynamometer. on the other hand, koyano, kim and clark31, analyzed maximum strength with a bilateral molar bite dynamometer and found no significant difference between the group with dysfunction of the masticatory muscles and the control group. 10 pires et al. braz j oral sci. 2023;22:e238358 these disagreements are due to the influence of anatomical and physiological factors of each individual evaluated, which directly influences the measurement of the bite force. it is also known that the mbf varies according to the location of the force transducer or bite dynamometer in the oral cavity; the more posterior, the greater the maximum bite force record14. for the most adequate record of maximum bite force, an interocclusal distance of 9 to 20 mm should be used, with a load applied on several teeth for larger support area32. in the present study, the greater magnitude of maximum bite force found in the control group may be justified by the 15 mm thickness of the bite dynamometer, which in turn may have favored the optimization of the length-tension relationship of masseter muscles during the evaluation in the masticatory muscles or tmj in this group. the myogenic tmd group, due to the chronic installed dysfunction and pain during the bite task, may have presented inhibitory mechanisms of the maximum bite force17,33. electromyography this study showed a moderate level of accuracy in the rms emg values of the masticatory muscles during the mmbmax for the diagnosis of women with myogenic tmd. considering the masticatory muscles, the following values are established as the best cut-off points for the diagnosis of myogenic tmd, in which positive cases should be diagnosed when the rms emg value is less than the cut-off point, and negative cases when the maximum force is equal to or greater than the cut-off point: left anterior temporal muscle (107.4 µv), right anterior temporal muscle (136.9 µv), left masseter (173.3 µv), and right masseter (208.59 µv). manfredini et al.8 and berni et al.9 assessed the accuracy levels of the rms emg parameter in a bite task and reported inconsistent findings. in general berni, et al.9 demonstrated that the rms emg parameter of the masticatory muscles showed a moderate level of accuracy for the discrimination between individuals with myogenic tmd and asymptomatic individuals during the tmj rest task and inadequate levels of accuracy during the maximum isometric bilateral molar bite. conversely, manfredini, et al.8 demonstrated that the rms emg parameter of the masticatory muscles demonstrated a high level of accuracy for the diagnosis of myogenic tmd during the maximal isometric bilateral molar bite task, but low levels of accuracy in the tmj rest task. strengths and limitations this study presents as strengths a large sample size, robust statistical analysis, the cut-off of mmbmax and the rms emg index of the masticatory muscles during the maximum bilateral molar bite, and a new perspective of tools already used in research and clinical practice. the limitations were: absence of an evaluation of the facial pattern of the volunteers; the need for caution when interpreting and using the accuracy cut-off point for mmbmax for discrimination of women with myogenic and asymptomatic tmd, as these 11 pires et al. braz j oral sci. 2023;22:e238358 values should be used only for young women and with the use of a 15 mm thick bilateral molar dynamometer; the presence of some cases with other joint symptoms, such as disk displacement and arthralgia, in the sample. future studies the evaluation of the volunteers’ menstrual cycle phase is suggested. the literature portrays a greater capacity for modulating pain in the ovulatory phase of the cycle, which may complement the findings of this research. considering the ability of surface emg to assess myogenic tmd, non-invasively and painlessly, providing quantitative and reliable information both in the clinical and research environment10, its use is suggested for monitoring and directing the treatment of myogenic tmd, as already used in research over the years. in conclusion, the research hypothesis was confirmed, the maximum bilateral bite force demonstrated high precision for myogenic tmd diagnosis women, and the rms emg index of the masticatory muscles in the mmbmax was able to discriminate between groups. the bilateral bite dynamometer with a surface emg during bilateral bite can be used to diagnose tmd in young women. data availability datasets related to this article will be available upon request to the corresponding author. acknowledgments this study was financed by the coordenação de aperfeiçoamento de pessoal de nível superior – brazil (capes). finance code 001. paulo fernandes pires: conceptualization, methodology, data curation, data analysis, writing; 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india 4 sribalaji vidyapeeth university, puducherry union territory, india corresponding author: dr swetha paulose department of oral medicine and radiology, indira gandhi institute of dental sciences, sri balaji vidyapeeth university, puducherry union territory-607402, india. email: swethathomas1981@gmail. com phone: 9176070430 received: june 24, 2019 accepted: december 02, 2019 prevalence of oral potentially malignant disorders associated with habits in puducherry – a cross-sectional study swetha paulose1, vishwanath rangdhol2, lakshminarayanan kavya3, govindasamy ezhumalai4 tobacco and betel quid are the most common cause of oral cancer in india. very often oral cancers are preceded by a visible oral precursor lesion called as potentially malignant disorder (pmd). aim: the aim of this study was to assess the prevalence of oral pmds associated with habits in urban and rural areas of puducherry union territory, india. methods: a cross-sectional descriptive study in urban and rural areas of puducherry was conducted. the study group comprised of 450 patients with positive history of oral habits. a standard structured questionnaire was designed to record information about demographic details, socioeconomic status, type, duration and frequency of habits followed by clinical oral examination by single trained and calibrated examiner to detect the presence of pmd. statistical analysis used: epidata software (version 3.1). descriptive statistics were presented for all variables. pearson’s chi-square test and adjusted odds ratio (ors) with 95% confidence interval (ci) were calculated to estimate the suspected risk factors for pmd by using multivariate logistic regression analysis. p-value of ≤ 0.05 was considered to be statistically significant. results: prevalence of habit associated oral pmd was 64.2%. females were more prone to develop pmds (68.3%) as compared to males (62.8%). pmd was more common in the age group of 51-60 years (69.2%). smoking with alcohol consumption was the most common oral habit. habits, socio-economic status and diet were significantly associated with development of oral pmds in our study population. multivariate logistic regression analysis showed that chronic betel quid chewing and smoking were significant risk factors for pmd. increased frequency of vegetable consumption reduced the risk of pmd. conclusions: oral pmd were observed in more than half of the subjects with oral habits in puducherry. clearly, there is an increased risk of pmd with increased duration of tobacco and betel quid use in this region. patients and public need to be educated regarding pmd and encouraged to quit habits so as to prevent high risk population from developing cancer. keywords: arecanut. oral cancer. potentially malignant disorder mailto:swethathomas1981@gmail.com mailto:swethathomas1981@gmail.com 2 paulose et al. introduction among the modern epidemics, cancer is one of the major causes of mortality in developing countries1. india has one of the highest rates of oral cancer due to life style related habits such as smoking, betel quid chewing and alcohol. oral cancers are often preceded by clinically evident precursor lesions called as potentially malignant disorders (pmd). the term pmd introduced by who in 2007, conveys that not all lesions and conditions described under this term may transform to cancer, instead there is a family of morphological alterations among which some may have an increased potential for malignant transformation2. the overall prevalence of pmd worldwide is reported as 4.47%3. the most important pmds are erythroplakia, oral leukoplakia (ol), oral lichen planus (olp) and oral submucous fibrosis (osmf). besides oral habits, sun exposure, micronutrient deficiency and socio-economic status are independent risk factors for development of oral pmds. unlike oral cancer, public awareness regarding pmd is very poor in india. union territory of puducherry is one among the five districts in india which has recorded a much higher mouth cancer incidence as compared to the global scenario4. pmds offers a good platform to identify the high-risk population. identifying the risk factors and educating the patients about the ill effects of betel quid, tobacco, alcohol and various other risk factors will help in preventing the development of malignancy in these patients. there is lack of population-based studies in puducherry, regarding the prevalence of pmds and its association with various risk factors. thus, the present study was undertaken to assess the prevalence of oral pmds in relation to habits in urban and rural population of puducherry and to understand the clinical profile of these patients. materials and methods study design a cross-sectional descriptive study was conducted in puducherry during a 1-year period from 2017-2018 after getting approval from institutional ethical committee (igidsrid2017 nrp06faspomr). the district was divided into two zones for the study purpose and a stratified cluster sampling design was employed in which individuals were randomly selected from each zone (villages in rural area and wards in urban areas). with a relative precision of 10% and design effect of 1.5, the required sample size was estimated at 274. the study however included a larger sample in order to improve its precision. a total of 450 eligible participants who met the inclusion criteria were enrolled in the study. the inclusion criteria was that the individual was 15 years or older with history of oral habits and willingness to participate in the study. the study objectives were explained to the participants and written  informed  consent was taken from every patient in their own language regarding the questionnaire and intraoral examination. patients below 15 years and without oral habits were excluded from the study. 3 paulose et al. data collection data was collected by a trained dental surgeon using a standardized interviewer-based questionnaire. all the participants were enquired about past or present history of oral habits. a specially designed proforma was used to record the demographic data which included age, gender, occupation, monthly income, marital status (single, married, other), place of residence (rural or urban). the second part of the proforma recorded data on following factors. oral habits habits were classified according to number, type, frequency and duration. type of areca quid (areca nut, mawa, pan masala, gutkha) along with or without tobacco and lime, location of quid placement etc were recorded. tobacco was used to refer to the use of dried tobacco leaves. smoking included cigarette/beedi/pipe smoking. betel quid was used to refer to the use of betel nut and lime. socioeconomic status socioeconomic status was assessed using b.g. prasad’s socio-economic status scale (2015). the ses index was categorized into 5 index quartiles based on their per capita monthly income. the minimum value of the ses index was 0-869 and maximum value was 5798 and above. dietary intake the dietary risk factors were evaluated on a six-point likert scale based on the frequency of vegetable and fruit intake per day. the subjects were further classified into specific groups based on the frequency of vegetable and fruit intake. the interview was followed by a thorough clinical examination of the oral cavity which was performed by trained dental professional to identify the pmds based on well-established clinical features of the lesions. subjects were asked to rinse their mouth prior to the examination. all the study participants were explained about the adverse effects of oral habits and counselling was given regarding habit cessation. subjects diagnosed with pmd were referred to our institution for further investigations and management. statistical analysis the recorded data from the 450 cases and controls was entered and analysed using epidata software (version 3.1) descriptive statistics were presented for all variables. pearson’s chi-square test and adjusted odds ratio (ors) with 95% confidence interval (ci) were calculated to estimate the suspected risk factors for pmd by using multivariate logistic regression analysis. p-value of ≤ 0.05 was considered to be statistically significant. results table 1 presents the demographic characteristics of study participants. among 450 participants with oral habits, there were 349 men (77.5%) and 101 women (22.4%). 4 paulose et al. mean age group of the study population was 51 years. overall prevalence of pmd was 64.2%. pmd was more common among females (68.3%) than males (62.8%). leukoplakia (54.2%) was the most common pmd noticed followed by osmf (8.4%) and lichen planus (0.6%). least common pmd identified was erythroplakia. some of them had more than one lesion. there was slightly more cases of pmd among the urban population compared to rural areas (64.7% vs 63.5%). however, there was no significant association between age, gender, area of residence and presence of pmds (p values=0.23, 0.30 and 0.80 respectively). table 2 presents the association between oral habits and pmd. highest number of pmds were observed in group 7 (smoking with alcohol habit) (n=88) followed by betel table 1. distribution of subjects according to age, gender, area of residence and pmd. variable pmd chi-square value df p-value yes (%) no (%) total 288(64) 162(36) age(years) ≤ 30 19 (51.4) 18 (48.6) 5.57 4 0.23 31-40 30 (60.0) 20 (40.0) 41-50 78 (60.9) 50 (39.1) 51-60 92 (69.2) 41 (30.8) ≥ 60 69 (67.6) 33 (32.4) gender male 219(62.8) 130(37.2) 1.05 1 0.30 female 69 (68.3) 32 (31.7) area urban 119 (64.7) 65 (35.3) 0.06 1 0.80 rural 169(63.5) 9 7(36.5) table 2. distribution of subjects according to habits and pmd sl no habits pmd chi-square value p-value yes (%) no(%) 1. betel nut + lime 20 (74.1%) 7(25.9%) 24.98 0.009 2. tobacco leaf 9 (100%) 0(0%) 3. smoking 35(53.8%) 30(46.2%) 4. alcohol 2 (28.6%) 5(71.4%) 5. betel quid + tobacco leaf 67(62.0%) 41 (38.0%) 6. betel quid+ alcohol 3(50.0%) 3 (50.0%) 7. smoking+ alcohol 88(64.2%) 49 (35.8%) 8. tobacco + alcohol 2 (100%) 0 (0%) 9. betel quid + tobacco + alcohol 31(60.8%) 20 (39.2%) 10. betel quid+ tobacco+ smoking 14 (82.4%) 3(17.6%) 11. betel quid + smoking + alcohol 2 (100.0%) 0(0%) 12. betel quid +tobacco+ smoking+alcohol 15(78.9%) 4 (21.1%) 5 paulose et al. quid chewing with tobacco (n=67).the types of betel quid chewing varied from traditional paan consisting of betel leaf wrapped around a mixture of areca or betel nut (nut of areca catechu), slaked lime, catechu (extract of acacia)with or without dried tobacco leaf to gutkha, hans, pan parag and other commercially available tobacco products. statistically significant difference was observed between various forms of oral habits and development of pmds. (p value =0.009). adjusted residual value for habit-2 (smokeless tobacco chewing) was 2.3 which implies that dried tobacco leaf is a major contributory factor for development of pmd. table 3, table 4 and table 5 presents the correlation of duration of habits and pmd. there was a significant association between chronic betel quid chewing and smoking with pmd. (p value =0.01 and 0.00 respectively). however there was no significant association between pmd and duration of alcohol consumption (p value= 0.20). table 6 presents the correlation of pmd with ses. majority of the patients belonged to the 0-869 ses index quartile. chi-square analysis showed statistically significant difference in ses index and pmds. (p value = 0.003) 78.6 % of the study population diagtable 3. distribution of subjects according to duration of betel quid chewing and pmd. sl no duration of betel quid habit (years) pmd chi-square value p-value yes(%) no(%) 1. ≤ 30 136(59.6%) 92(40.4%) 13.36 0.01 2. 31-40 41 (59.4%) 28(40.6%) 3. 41-50 32 (74.4%) 11(25.6%) 4. 51-60 27 (90.0%) 3 (10.0%) 5. ≥ 60 52 (65.0%) 28(35.0%) table 4. distribution of subjects according to duration of smoking and pmd sl no duration of smoking habit (years) pmd chi-square value p-value yes (%) no (%) 1. ≤ 30 136 (63.3%) 79(36.7%) 21.8 0.00 2. 31-40 22 (40.7%) 32(59.3%) 3. 41-50 48 (68.6%) 22 (31.4%) 4. 51-60 21(91.3%) 2 (8.7%) 5. ≥ 60 61 (69.3%) 27 (30.7%) table 5. distribution of subjects according to duration of alcohol drinking and pmd sl no duration of alcohol habit (years) pmd chi-square value p-value yes(%) no (%) 1. ≤ 30 146(62.9%) 86(37.1%) 5.97 0.20 2. 31-40 37 (66.1%) 19(33.9%) 3. 41-50 35 (55.6% 28(44.4%) 4. 51-60 22 (81.5%) 5 (18.5%) 5. ≥ 60 48 (66.7%) 24(33.3%) 6 paulose et al. nosed with pmds belonged to families in the higher ses group with monthly income 2899-5797 and least number was noticed in the lower ses group (53.2%). table 7 and table 8 presents the correlation of nutritional intake and pmd. pmd was more common in patients who had less vegetable intake compared to those who took vegetables several times a day and the association was statistically significant. (p value=0.001). there was higher prevalence of pmds among individuals with reduced fruit consumption. there was statistically significant association between frequency of fruit consumption and pmd (p value= 0.002) table 9 presents multivariate analysis of different variables in cases with pmd. the data were analysed by multiple regression using age, gender, place of residence (urban/rural), habit duration, ses, vegetable and fruit intake as regressors. duration of betel quid chewing and smoking, frequency of vegetable intake interacted, and table 6. distribution of subjects according to socioeconomic status and pmd per capita monthly income pmd chi-square value pvalue yes n (%) no n (%) 0-869 91(53.2) 80(46.8) 16.30 0.003 870-1738 80(66.1) 41(33.9) 1739-2898 58(72.5) 22(27.5) 2899-5797 33(78.6) 9(21.4) 5798-infinity 26(72.2) 10(27.8) table 7. distribution of subjects according to frequency of vegetable intake and pmd sl no frequency of vegetable intake pmd chi-square value p-value yes (%) no (%) 1. several times a day 30 (65.2%) 16 (34.8%) 17.6 0.001 2. everyday 180 (59.0) 125(41.0%) 3. several times week 65(83.3%) 13(16.7%) 4. once a week 3(42.9%) 4(57.1%) 5. several times a month 10(71.4%) 4 (28.6%) 6. seldom/never 0 0 table 8. distribution of subjects according to frequency of fruits intake and pmd sl no frequency of fruits intake pmd chi-square value p-value yes(%) no (%) 1. several times a day 0 (0%) 2 (100%) 18.46 0.002 2. everyday 11(37.9%) 18 (62.1%) 3. several times week 49(64.5%) 27 (35.5%) 4. once a week 20 (50.0%) 20 (50.0%) 5. several times a month 94 (69.6%) 41(30.4%) 6. seldom/never 114(67.9%) 54(32.1%) 7 paulose et al. table 9. multivariate analysis of different variables in cases with pmd variables b s.e. wald chisquare df p value or 95.0% ci for or lower upper age group(years) ≤ 30 2.240 4 .692 31-40 .371 .508 .532 1 .466 1.449 .535 3.926 41-50 .068 .457 .022 1 .882 1.070 .437 2.620 51-60 .465 .472 .972 1 .324 1.593 .631 4.018 ≥ 60 .312 .497 .395 1 .530 1.367 .516 3.620 gender .490 .362 1.836 1 .175 1.632 .804 3.315 area of residence -.279 .247 1.271 1 .260 .757 .466 1.228 duration of habits (years) alcohol ≤ 30 4.807 4 .308 31-40 .400 .378 1.124 1 .289 1.492 .712 3.128 41-50 -.214 .350 .374 1 .541 .807 .407 1.602 51-60 .993 .601 2.735 1 .098 2.700 .832 8.761 ≥ 60 .171 .364 .221 1 .638 1.187 .582 2.421 betel quid ≤ 30 16.034 4 .003 31-40 .498 .376 1.753 1 .186 1.646 .787 3.442 41-50 1.272 .476 7.146 1 .008 3.568 1.404 9.067 51-60 2.587 .728 12.645 1 .000 13.292 3.194 55.319 ≥ 60 .685 .426 2.593 1 .107 1.984 .862 4.570 smoking ≤ 30 23.223 4 .000 31-40 -.151 .434 .122 1 .727 .860 .367 2.010 41-50 1.221 .421 8.423 1 .004 3.391 1.486 7.734 51-60 2.454 .833 8.676 1 .003 11.633 2.273 59.544 ≥ 60 years 1.085 .420 6.683 1 .010 2.960 1.300 6.737 frequency of vegetables and fruits consumption fruits several times a day 13.577 5 .019 once a day 20.753 2.799e4 .000 1 .999 1.030e9 .000 . several times a week 22.343 2.799e4 .000 1 .999 5.054e9 .000 . once a week 21.566 2.799e4 .000 1 .999 2.322e9 .000 . several times a month 22.438 2.799e4 .000 1 .999 5.554e9 .000 . seldom/never 22.139 2.799e4 .000 1 .999 4.118e9 .000 . veg several times a day 13.722 4 .008 once a day -.868 .425 4.160 1 .041 .420 .182 .967 several times a week .192 .536 .128 1 .721 1.211 .424 3.463 once a week -1.650 .920 3.214 1 .073 .192 .032 1.166 several times a month -.599 .783 .586 1 .444 .549 .118 2.547 constant -22.097 2.799e4 .000 1 .999 .000 b= parameter estimate β, se = standard error, df= degree of freedom, or= odd’s ratio, ci=confidence interval 8 paulose et al. showed strong association with development of pmd. patients with history of smoking for 50-60 years had 11 times more risk of developing pmds. similarly, those who had chronic betel quid chewing habit for 50-60 years were 13 times more prone to develop pmd. vegetable consumption everyday reduced the risk of pmd by 58%. discussion among 450 participants in our study, pmd was identified among 288 individuals (64.2%) which is consistent with the reports by kadashetti et al.1 in maharashtra (65%). our results were less compared to the corresponding values of 77.1% reported by gupta et al.5 in lucknow but distinctly higher than several other studies in india such as saraswathi et al.6 in chennai, tamil nadu(4.1%), vinay et al.7 telangana (4.2%), thada and pai8 manipal, (27.67%) and narasannavar and wantamuttet9 belgaum (karnataka) (51.21 %). the wide variation among the prevalence rates of pmd may be attributed to the changing trends in oral habits in various parts of the country and other associated risk factors like cultural, socioeconomic and environmental factors. we observed that highest number of pmd cases were in 50-60 yrs of age group (69.2%). this was comparable to the results from kadashhetti et al.1, saraswathi et al.6 and jagtap et al.10. several studies have reported equal prevalence of pmd among the middle aged and elderly subjects8 whereas few others have reported highest prevalence in 21–30 years of age group11 which was not in accordance with our study. this difference could be attributed to the study design and variation in habit pattern among different age groups with respect to different states. males have been reported to be more frequently affected by pmd due to higher prevalence of oral habits among men1,11,12. female predilection has been reported in few places such as manipal, southern karnataka (59.30%)8 and this finding was in concordance with our results. however, age, gender and area of residence were found to be neither a risk nor a protective factor for pmds in our population. smoking with alcohol was the most common oral habit (30.4%) prevalent in this area. among single habit, smoking was the most common habit (14.4%). similar to our findings, smoke form of tobacco was reported as the most common form of tobacco used in puducherry (64%) by aroquiadas et al.13. high prevalence of alcohol consumption has been noted among adult men (59.6%) in coastal areas of puducherry due to easy availability of alcohol at a subsidized rate14. habits were significantly associated with pmds (p value= 0.009). our study results were in agreement with the studies conducted at different parts of world1,8,15,16,17. maximum number of pmd cases were seen among smokers with alcohol consumption habit (64.2%) which was consistent with the study by marija et al.18 and baric et al.19. localized elevation in the temperature of the oral cavity following smoking, makes the epithelium more susceptible to genotoxic effect of tobacco products. ethanol as a solvent may damage the oral cells and increase the mucosal penetration of these toxic carcinogens. based on the adjusted residual values, it was found that tobacco leaf is a significant predictor for development of pmd. tobacco use in any form can trigger changes including inflammation, atrophy and hypertrophy of the mucosa, leading to development of pmd and oral cancer20. betel quid chewing with tobacco is said 9 paulose et al. to have an increased carcinogenic potential because it remains in contact with the oral mucosa for longer period of time compared to smoking/alcohol17. chronicity and increase in number of habits have been associated with increased risk of oral cancer and pmd8. in the current study, we noticed that duration of smoking and betel quid chewing were significant risk factor for pmd but not alcohol duration. it is possible that alcohol contributes to the development of pmds in the presence of other habits such as betel quid chewing /smoking. alcohol has been proved to be a risk factor for oral cancer21, but not for pmd5. some studies did not show any association between drinking and the risk of oral pmd6. while other studies that showed associations had not adjusted for potential confounders such as tobacco chewing and smoking22. the most common pmd noticed among the study population was leukoplakia (54.2%) followed by osmf and lichen planus. homogeneous leukoplakia was more frequently seen than non-homogeneous type. leukoplakia was identified as the most common pmd by hosagadde et al.11, in mumbai (45.71%) and aroquiadasse et al.15, in pondicherry (38.4%). there are contradictory results from other studies where osmf2,12,17,23 and lichen planus were found to be most prevalent pmd24. this alteration could be due to the differences in chronicity, composition, method of smoking/ chewing and combinations of habits which is prevalent in respective areas. in our study, buccal mucosa was the most common site affected which was comparable to the observations made by thada et al.8, zain et al.20 and mortazav et al.25. socioeconomic status showed strong association with pmd in our study population (p value= 0.003). changes in income are more likely to affect the tobacco, alcohol / betel quid usage26. most of our study subjects belonged to the lower socio-economic group with family income less than rs 869 per month. we believed that economically poor people are at an increased risk of developing oral cancer and pmd due to insufficient nutrition coupled with lack of oral health awareness27. however,  the  figures  were  very surprising as our data showed that most of the pmd lesions were observed in the middle and higher socioeconomic strata perhaps because of changing life style risk factors. this was matching with the observations made by gupta et al.5 in lucknow. the authors had reported that lower income group was at a lesser risk of developing osmf and leukoplakia. in our study, we had taken into consideration only per capita monthly income as a variable to assess ses. literacy rate/education, occupation etc may also affect the socioeconomic status. more longitudinal studies are recommended in this direction to determine the causal relationship to pmd. frequency of fruits and vegetable intake were significantly associated with pmd in our study population (p value = 0.002 and 0.001 respectively). majority of the subjects were frequent consumers of vegetables but not fruits. on multiple regression analysis we found that increased vegetable intake significantly reduces the risk of pmd. kumar et al.12 and pahwa et al.28 have reported that among patients with pmd, majority were infrequent consumers of fruits and vegetables.while the protective role of antioxidant rich diet against oral cancer and pmds is well established, data regarding the same in indian population is meagre. dietary habits of indians are in fact influenced by their socioeconomic status, culture and religious beliefs. antioxidant rich diet in sufficient 10 paulose et al. quantity is essential to protect against free radical induced oxidative damage which is precipitated in the presence of tobacco, betel nut and alcohol. our study results emphasize the importance of educating the patients regarding the importance of diet in maintaining oral health. results of logistic regression analysis showed that chronic betel quid chewing, smoking and reduced frequency of vegetable intake are the major risk factors for development of oral pmd. our study being a community-based survey provides the baseline data regarding the prevalence of pmd in this region. to our knowledge, this is the first study to investigate the prevalence of pmd in association with the habit trends and potential risk factors in puducherry population. however, there were some limitations. causal relationships between the risk factors and pmds were not completely clear due to the use of a cross-sectional design. diet pattern was not analysed with respect to quality of food. finally, there may have been some unrecognized confounding factors. the results of our study highlight the fact that individuals with oral habits in puducherry are at high risk of developing pmd. chronic smoking and betel quid chewing are the major risk factors for development of pmd. frequent intake of vegetables may serve as a protective factor in these individuals. socioeconomic status has an impact on individual oral health and development of pmd which could be related to lifestyle risk factors. oral health planners should address the socioeconomic status of the population while planning and evaluation of oral health care programs. as health professionals it is our duty to educate the public regarding the consequences of tobacco, alcohol and betel quid chewing habits. we suggest that every dental teaching institution should have a trained personal to operate a habit counselling clinic to promote and assist those who want to quit the habit. the knowledge regarding pmd, its associated risk factors and susceptibility to develop cancer must be imparted by awareness programs. early intervention in initial risk populations will help us to prevent irrevocable changes in oral mucosa. conflicts of interest nil. source of funding nil. references 1. kadashetti v, chaudhary m, patil s, gawande m, shivakumar km, patil s, et al. analysis of various risk factors affecting potentially malignant disorders and oral cancer patients of central india. j cancer res ther. 2015 apr-jun;11(2):280-6. doi: 10.4103/0973-1482.151417. 2. warnakulasuriya s. clinical features and presentation of oral potentially malignant disorders. oral surg oral med oral pathol oral radiol. 2018 jun;125(6):582-590. doi: 10.1016/j.oooo.2018.03.011. 11 paulose et al. 3. mello fw, miguel afp, dutra kl, porporatti al, warnakulasuriya s, guerra ens, et al. prevalence of oral potentially malignant disorders: a systematic review and meta-analysis. j oral pathol med. 2018 aug;47(7):633-640. doi: 10.1111/jop.12726. 4. bhardwaj n, daniel mj, srinivasan sv, jimsha vk. demographics, habits, and clinical presentation of oral cancer in puducherry’s population: an institutional experience. j indian acad dent spec res. 2015;2(2):64-9. doi: 10.4103/2229-3019.177926. 5. gupta s, singh r, gupta op, tripathi a. prevalence of oral cancer and pre-cancerous lesions and the association with 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india. asian pac j cancer prev. 2018 aug 24;19(8):2165-70. 1http://dx.doi.org/10.20396/bjos.v19i0.8659247 volume 19 2020 e209247 original article 1 faculty of dentistry, federal university of juiz de fora, juiz de fora, mg, brazil. 2 department of dental clinic, faculty of dentistry, federal university of juiz de fora, juiz de fora, mg, brazil. corresponding author: luan viana faria, fazenda da reforma, sn, araraí, alegre, es, 295350000, brazil. phone: +5528999523072; luanvfaria13@hotmail.com received: april 21, 2020 accepted: october 12, 2020 medical emergencies teaching in dentistry undergraduate courses in southeastern brazil luan viana faria1,* , yuri de lima medeiros1 , danielle fernandes lopes1 , eduardo machado vilela2 , neuza maria souza picorelli assis2 aim: the aim of this study is to offer an overview of the medical emergencies (me) discipline offer in dentistry graduations in southeastern brazil and to observe the curricular characteristics of the discipline when present. methods: this cross-sectional documentary study analyzed the available curricular frameworks in the official websites of higher education institutions (hei) in southeastern brazil registered on the ministry of education’s e-mec website. the data were analyzed and tabulated using the graphpad prism 8.1.2 software, being described by absolute and relative frequencies. fisher’s exact test was used to compare the proportions between public and private institutions. results: of the 176 courses in the southeast, 144 were included in the study for providing access to the curriculum, 19 (13.19%) were public and 125 (86.81%) were private. only 27 (18.75%) of the hei present the discipline of me, with a greater tendency of supply in private heis (20.80%) when compared to public heis (5.26%), but this difference was not statistically significant (p> 0.05). as a positive aspect, the discipline is predominantly mandatory (88.88%), and the with regard to the teaching methodology is predominantly theoretical (68.18%). the average workload is 50.14 hours (sd=19.54). conclusions: in only 18.75% of the dental institutions in southeast brazil, me discipline were offered. when offered, the discipline is predominantly theoretical and mandatory. this study raises an important discussion regarding the need to include specific and mandatory subjects on me in the dentistry curricula in brazil and reflects the need to update and standardize the national curricular guidelines for dentistry. keywords: education, dental. emergency service, hospital. first aid. dentistry. https://orcid.org/0000-0003-2336-9946 https://orcid.org/0000-0002-4013-7752 https://orcid.org/0000-0002-6737-9284 https://orcid.org/0000-0001-5634-9998 https://orcid.org/0000-0002-8612-5044 2 faria et al. introduction dental surgeons must be prepared to manage possible medical emergencies (me) that may arise in their daily professional practices1,2. in such situations, professionals and their teams need to be confident and up to date with their skills in order to provide initial treatment of quality3. despite the consensus on the importance of training dental surgeon in this subject, several national4-7 and international studies8-14 show unsatisfactory results by demonstrating low knowledge and confidence of professionals in managing me and first aid. based on these aspects, in recent years there has been a change in medical emergency management teaching methodologies for undergraduate students in many developed countries as an attempt to improve their management outcomes15-20. moreover, in some countries, emergency medical training is formally part of undergraduate dental programs, and the example is the united states, which has been offering the discipline since 198121. in brazil, the federal council of dentistry (cfo) determined that all specialization courses should include in their programs the discipline of medical emergency in dentistry, with a minimum workload of 15 hours22. arsati et al.4 (2010) point out that it is unacceptable that this discipline is compulsory only for postgraduate programs, since any practitioner may face a medical emergency during clinical practice, regardless of their level of education and specialty. a national study has shown that brazilian undergraduate dentistry students have only superficial knowledge about medical emergencies23. with this, the formation of a generalist dentist is compromised, bringing insecure and unprepared professionals to the brazilian job market. although several studies assess the knowledge of students and professionals in the management of me, few studies discuss the factors that contribute to the persistence of these unsatisfactory results. the poor approach of this content during graduation may be one of the reasons why dental surgeon dentists still demonstrate a low ability to manage me. in addition, this study is also useful because there are no reports in the literature regarding the comparative assessment between the provision of the me discipline from public and private brazilian institutions. therefore, the objective of this paper is to draw an overview of the insertion of the me discipline in the dentistry graduations of southeastern brazil, and observe the curricular characteristics of the discipline when present. materials and methods this is a cross-sectional and documentary evaluation study, in which the curricular matrices of the dentistry graduations were collected on the official institutional websites of the colleges, or via e-mail, through the coordinators of the dentistry courses. the search for discipline in the curricular matrices was carried out by two independent evaluators previously trained and calibrated. information collected that differed between the two researchers was checked by a third evaluator. the following variables were studied: location of institutions by states of the federation; hei administrative category: public or private; insertion and offer of the 3 faria et al. discipline: presence or absence; nature of the curricular component: mandatory or non-mandatory; teaching methodology: theoretical or theoretical-practical content and total workload. the sample of this research consists of all dentistry schools located in the southeast region of the country in active situation in march 2019, registered in the e-mec portal of the ministry of education and culture24 which is regulated by normative ordinance no. 21, of december 21, 2017.25 the collection period of the curricular grids was from march to june 2019. institutions were excluded from the sample according to the following criteria: did not have an official website and did not provide virtual media; did not present their complete curriculum available; did not open the dentistry course; and did not answer the e-mail after waiting for a deadline of two months, in case of absence of the curriculum on the institutional website. the collection period of the curricular grids was from march to june 2019. as it is a documentary evaluation study in the public domain, the approval of the ethics committee is waived. different poles of the same institution were considered in the composition of the final show, as well as institutions that offered the dentistry course in more than one modality, whether morning, afternoon, night or full, were treated as independent data, since the curricular matrix may vary between these situations, even within the same institution. the statistical software used to analyze the variables was graphpad prism 8.1.2 (graphpad software inc., la jolla, ca, usa), which was analyzed using descriptive statistics, absolute (n) and relative (%) frequency. the statistical test used for comparison was fisher’s exact test. the adopted significance level was 5%. results we identified 176 dental schools, of which 32 were excluded for not meeting the study inclusion criteria; one did not have an official website and did not provide virtual means of communication; six did not open the course and 25 did not respond to the email after the two-month period. it was necessary to consult 33 heis via e-mail, obtaining a response rate of 24.24%. the final research sample consisted of 144 institutions (81.82%), 10 (6.94%) located in the state of espírito santo (es), 51 (35.41%) in minas gerais (mg), 21 (14.58%) in rio de janeiro (rj) and 62 (43.05%) in são paulo (sp). according to the administrative category, brazilian dental schools may be public or private. the sample of this study consisted of 125 (86.80%) private and 19 (13.19%) public schools. regarding the offer and insertion of the discipline, other names were found in the curriculum as “basic life support” and “first aid”. in general, only 18.75% of the colleges offered the discipline in their pedagogical plans. for the variable nature of the discipline, out of the 27 institutions that provided this information, it was observed that discipline is compulsory in 88.88% of the courses, compared to 11.12% of institutions in which the discipline is optional. the prevalence of the discipline taking into account the nature of the institutions is described in table 1. 4 faria et al. table 1. prevalence and association test of the discipline of medical emergencies according to the administrative nature of dental schools in southeastern brazil, 2019. variables n (%) n (%) hie public + hie private p-value (hie public x hie private) administrative nature of dental schools hie public hie privete insertion and offer (n=144) 0.1264 present 1 (5.26%) 26 (20,.0%) 27 (18.75%) absent 18 (94.74%) 99 (79.20%) 117 (81.25%) nature of the discipline (n=27) >0.9999 compulsory 1 (100.00%) 23 (88.46%) 24 (88.89%) optional 0 (0.00%) 3 (11.54%) 3 (11.11%) teaching methodology (n=22) 0.3182 exclusively theoretical contente 0 (0.00%) 15 (71.43%) 15 (68.18%) conjugated practical methodologies 1 (100.00%) 6 (28.57%) 7 (31.82%) absolute (n) and relative (%) frequency of me subject to hei administrative category. fisher’s exact test (p<0.05). in private institutions, regarding teaching methodology, 22 institutions offered this information. in institutions which the discipline is only theoretical, four of them (26.66%) offer it in the distance learning modality (dl) and in 11 (73.33%), in the in-person modality. only seven dental schools offer practical me courses in their curriculum. regarding the workload, 21 courses offered this information in the curriculum matrices. the average workload was 50.14h (sd = 19.54). the workload per state of the federation is shown in figure 1.a. analyzing this variable according to the administrative category of the institutions, private schools had an average workload of 51.90h (sd = 18.27), while in public colleges the only school offering the subject had a workload of 15h. the minimum workload was 15h, and the maximum workload was 80h, with a median of 40h. figure 1. average workload of the discipline of medical emergencies offered by dental schools by states of the federation (es n = 3; mg n = 9; sp n = 6; rj n = 3) in southeastern brazil, 2019. 100 80 60 40 20 0 40.0 53.9 58.3 45.5 h ou rs es pír ito sa nt o m ina s g er ais ri o d e j an eir o sã o p au lo 5 faria et al. discussion me management is of crucial importance in dentistry practice and the starting point of training in this area is the undergraduate course in dentistry26. nevertheless, the results of this study show that brazilian dental schools are outdated in the teaching of me, as most of the institutions surveyed (86.81%) fail to offer a specific subject-oriented discipline in their curriculum. aggravating this picture, less than a third of institutions reserve practical content subjects in their curriculum matrices, although studies show that participation in hands-on training easily raises students’ awareness of the importance of me, as well as self-confidence in emergency management17,19. as a positive aspect, our study observed that in colleges that the discipline is offered, the tendency is to be mandatory, ensuring that all professionals trained in the institution had contact with the subject during graduation. according to our literature review, this is the first study to evaluate the supply of me disciplines in a sample of dentistry courses in brazil, as well as to compare how this discipline is distributed between public and private hei. in brazil, the opinion of national education council / higher education chamber nº 803/201827 that deals with the revision of the national curriculum guidelines of the undergraduate course in dentistry in the country, defines “the emergency approach and the basic life support in case of accidents that compromise the life and the individual’s health” among the essential curricular contents of the undergraduate course in dentistry, within the humanities and social sciences axis. in addition, law 5,081 of august 24, 196628, which regulates dental practice in brazil, states that brazilian dental surgeon can prescribe and apply emergency medication in the event of serious accidents that compromise the patient’s life and health. despite these guidelines and attributions, the discipline of me is not described as compulsory to be included in the pedagogical projects of undergraduate courses, and it is up to the institution to choose whether or not to offer it in the curriculum matrices, as proposed by law no. 9,39429, in its article 5329, which guarantees didactic autonomy to dentistry institutions. except in emergencies caused by trauma, dental surgeon can find almost all forms of me in their offices, such as fluctuating consciousness, unconsciousness, chest pain-related problems, hypoglycemia, generalized seizure, and adverse drug reactions30. therefore, dental professionals must be able to perform basic maneuvers in me situations, both general practitioners and specialists, to ensure the health quality and integrity of patients, until they are able to receive specialized care. however, in emergency situations, it is common for a rescuer to go through a situation of panic or indecision, resulting in increased morbidity and mortality for the patient, given the potential delay in response time to intensive care31 reflecting the need for good care subject training to assure that professionals are calm and secure to deal with the situation. an australian study32 evaluated patients’ perceptions of me in dentistry and their expectations for dental surgeon’ management, noting high patient expectations regarding dentists’ general medical knowledge. nevertheless, many studies support the lack of preparation of students23,26,33-35 and professionals4-14 in managing me, concluding that the subject should be addressed more deeply in dental education11,23, and for graduate dentists, refresher courses and workshops should be more frequently considered9. 6 faria et al. in this present study noted that few institutions offer practical content in me disciplines, which does not seem to follow the trend in developed countries, where courses are moving away from just didactic-theoretical instruction, and incorporating regular practical simulation training into the provision of education15,16,18,19. these exercises help dental professionals and staff to feel more confident in their duties during emergency situations, improving their level of knowledge and overall readiness15-17,19. the use of simulators can be expensive as it may require the purchase of various mannequins and advanced life support simulators, but the skill acquisition level and positive student feedback seem to justify this expense18. therefore, the existence of a specific theoretical-practical discipline in undergraduate studies is essential for the preparation of future professionals to lead with me. the course introduces the student to a weekly experience on the subject, allowing him to perform regular emergency exercises to reinforce learning. a us study published in 20182 assessed the knowledge and preparation of professionals, skilled residents, and faculty members. it has observed that participants who had recently graduated or were still in school were able to treat me cases more appropriately and often than the other participants. this finding positions the dental school as a valuable component in the preparation of clinicians. the authors also proposed the incorporation of periodic emergency exercises in dental settings, along with continuing education courses, to help dental surgeon manage me. a national study4 of 498 professionals found that brazilian dental surgeon are not fully prepared to manage me, as only 41% of dental surgeon thought they could diagnose the cause of me, and most felt unable to treat situations of anaphylaxis, myocardial infarction or cardiac arrest, or dealing with cardiopulmonary resuscitation procedures and intravenous injection. another important finding of the study is that the most common justifications for lack of knowledge and skills were lack of training and updating during and after the undergraduate course. despite geographical differences and variability in dental curricula around the world, studies show that students and practitioners have expressed their intention to improve their specific set of me management skills5,34. we believe that undergraduate dentistry courses should be reviewed to make it mandatory to include me in their curricula, and that this agenda should be raised by the brazilian association of dental education (abeno). likewise, upon graduation, all trained professionals, regardless of whether they are specialists, should be encouraged to take regular cfo-regulated theoretical and practical courses in the field, and more severely in the future become mandatory for record keeping from the board. a limitation of this study is the impossibility of identifying institutions that offer subject-oriented extracurricular projects related to the subject. another limitation is to have evaluated and included only disciplinary `by name`, but that the content could be approached in a theoretical or theoretical-practical way within another discipline, such as physiology, pharmacology and surgery, which can generate a possible analysis bias. however, it was not the objective at this time and due to the difficulty of accessing all menus for this type of evaluation. one suggestion is that future studies conduct an assessment based on semi-structured questionnaires sent to course coordinators to obtain additional information related to the prepa7 faria et al. ration of dental schools to respond to medical emergencies. the hei exclusion criteria adopted in this study culminated in an inclusion rate of 81.8% of institutions in southeastern brazil. therefore, the results discussed here reflect the reality of most heis in this region, but they should not be generalized as the real situation of teaching me. the evaluation of higher education curricula and discussions that lead to curricular changes are essential to achieve excellence in teaching35-38. it is important that the curricula of undergraduate courses in dentistry are reviewed to make it mandatory to include this discipline, in order to improve knowledge and practice in me, providing the skills that will allow a well trained professional to attend the needs of the patients. this need should be discussed by the abeno and by the cfo, in order to formulate a consensus document that guides the hei in future curricular reformulations, such as fundamental program content, minimum workload, offer period, nature of the discipline and methodologies of teaching ideas. in conclusion, this study observed the current panorama of the supply of the discipline of medical emergencies in dentistry courses, and observed that dental schools in southeastern brazil, both public and private, are outdated in the curriculum, since most heis do not offer the discipline (81.25%). in addition to the low supply of the discipline, 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[tmd and orofacial pain: curricular perspectives of dental schools in southeast brazil]. hu rev. 2020;46:1-7. portuguese. doi: 10.34019/1982-8047.2020.v46.30348. oral sciences n3 original article braz j oral sci. july | september 2012 volume 11, number 3 influence of wrong determination of occlusal plane in maxillary advancement: a model surgery study maximiana cristina de souza maliska1, robson garcia2, roger willian fernandes moreira3 1dds, master student, oral and maxillofacial surgery division, piracicaba dental school, university of campinas, piracicaba, sp, brazil 2dds, ms, phd, collaborative researcher, oral and maxillofacial surgery division, piracicaba dental school, university of campinas, piracicaba, sp, brazil 3dds, ms, phd, associate professor, oral and maxillofacial surgery division, piracicaba dental school, university of campinas, piracicaba, sp, brazil correspondence to: maximiana cristina de souza maliska departamento de cirurgia buco-maxilo-facial faculdade de odontologia de piracicaba, caixa postal 52, universidade estadual de campinas cep: 13414-903, piracicaba, sp, brasil phone: + 55 19 82221777 e-mail: maximaliska@yahoo.com.br abstract aim: this study investigated whether an occlusal plane error of acquisition can influence on the treatment planning of maxillary advancement in double-jaw surgeries. methods: advancement of 8 and 4 mm were studied in different groups. for each group, 20 maxillary models were mounted by a 13-degree platform with superior articulator arm (control group) and other 20 models mounted with 7 degrees (study group). intermediate splint was obtained by cast surgery performed in the control group. all the 40 maxillary models were remounted with this intermediate splint. measurements in vertical and anteroposterior planes were accomplished preand postoperatively by digital caliper rule and erickson’s platform. results: statistical analysis showed significant results in two planes. the alteration of occlusal plane from 13 degrees to 7 degrees modified the final result in vertical measurements: right molar from group of 4 mm advancement (p<0.0001) and left molar from two groups of advancement (p<0.0001); in anteroposterior measurements: maxillary incisor from 4 mm of advancement (p<0.005) and 8 mm of advancement (p<0.0001). conclusions: notwithstanding the importance of statistical findings, the result probably did not show clinical relevance in orthognathic surgery. clinical studies addressing these concerns must be supplied in scientific literature. keywords: occlusal plane, bimaxillary surgery, face bow, model surgery. introduction correction of dentoskeletal deformities of the jaws, where double-jaw surgery is necessary to establish optimal functional and esthetic relationships, requires accurate diagnoses and proper treatment planning. planning for orthognathic surgery involves integrating diagnostic information from patient examination, cephalometric radiograph, and dental models. the frankfort horizontal plane is a common reference plane accepted to link all this information and it is universally represented by the upper arm of the articulator1. it is imperative that the angle between the occlusal plane and the frankfort horizontal plane in the patient be the same as the angle between the occlusal plane of the maxillary model to the upper arm of the articulator 2. an important step to achieve excellence in orthognathic surgery concerns model surgery, which can provide a more accurate assessment of the surgical movements, as it allows assessment in all three dimensions3. face bow is the device that can transfer maxillary position in relation to frankfort plane from the patient to the received for publication: february 23, 2012 accepted: may 17, 2012 braz j oral sci. 11(3):368-372 semi-adjustable articulator. unfortunately, this face bow registration method often creates inaccuracies in the mounted maxillary model position. many authors1,4-5 studied different face bows recordings to investigate the inaccuracies presented in the maxillary mounted model in the articulator. ellis et al. 2 developed a simple technique of checking the angle between the occlusal plane and frankfort horizontal. with the use of a compass, recordings in the maxillary mounted model were transferred to cephalometric tracings. a line was drawn tangent to the compass recordings, and the angle between this line and the frankfort line was measured. an important average value of 7 degrees of inaccuracy was established with respect to the angulation of the occlusal plane. however, traditional face bow mounting of dental models remains with different and difficult inaccuracies to be corrected6. the three-dimensional position of patient’s external meatuses or condyles may be asymmetric from side to side; a shift could happen when tightening the bolts or screws of the face bow components, which show the sensibility of the device; some anatomic aberrations or absent structures may be present and are not reproducible on the articulator 6. in regard of face bow recording inaccuracies, the purpose of this study was to analyze the influence of a 6-degree hypothetical error of occlusal plane registration in model surgery with maxillary advancement of 4 and 8 mm. material and methods maxillary and mandibular plaster models were obtained from a dental manikin with corrected dental alignment. twenty models for each group, control and study, were necessary for this study. the models had a specific number to permit the use of the same model for maxillary advancement of 4 and 8 mm. the same articulator adjusted with the recommended arbitrary average values of 15° and 30°, respectively for bennett angle and eminence angle, was used for mounting all samples. maxillary models of the control and study groups were firstly mounted in 13and 7-degree planes retrospecti-vely from two splints with a flat inferior surface adapted on the superior surface of the planes to give a standardized position of the models (figures 1-3). this standardization was checked by measurements made in specific vertical and anteroposterior points: the tip of the upper incisor and the mesiobuccal cusps of the left and right first molars in the vertical plane, and the most convex point of the upper incisor in the anteroposterior plane (figure 4). measurements were performed with erickson’s platform and digital caliper similar to the ellis technique described5. models that could not achieve an error margin of 0.05 mm in the average of measurements of all mounting models were remounted. a malocclusion splint was obtained by an anteroposterior discrepancy simulation of about 4 mm of mandibular prognathism. the normal (control group) and altered (study group) maxillary plane angulation data was transferred to mandibular models: two mandibular models were mounted fig. 1. superior view of the splints with a flat inferior surface adapted with acrylic resin on the superior surface of the seven and thirteen degrees planes. fig. 2. lateral view of the splints with a flat inferior surface adapted with acrylic resin on the superior surface of the seven and thirteen degrees planes. fig. 3. lateral view of the maxillary model of the control group mounted in a 13 degrees plane. by the adapted malocclusion splint and according to each maxillary model angulation of 13 and 7 degrees. maxillary advancement of 4 and 8 mm were performed during model surgery from two extra models mounted in a 13-degree plane (control group) according to standardized procedures. a mandible with 13 degrees of mandibular occlusal angle and operated models as cited above was used to the confection of the intermediate splints (figure 5). 369369369369369influence of wrong determination of occlusal plane in maxillary advancement: a model surgery study braz j oral sci. 11(3):368-372 fig. 4. measurements realized in vertical and anteroposterior dimension with erickson platform and digital caliper. all the samples of the control and study groups, totalizing 40 maxillary models, were remounted according to intermediate splint from maxillary advancement of 4 mm. it is important to highlight that it was used the mandible of 13-degree mandibular plane angle to set the control group and the mandible of 7-degree mandibular plane angle to set the study group. the same measurements performed in vertical and anteroposterior plane for standardization was applied and registered as post-operatory values. forty maxillary models for advancement group of 8 mm were remounted, and measurements were registered in the same manner. data was submitted to double f test for homogeneity of variances, and to shapiro-wilk test for normality of variances followed by student’s t-test. the level of fig. 5. lateral view of the maxillary model advancement of 8 millimeters and mandible model with the intermediate splint made from acrylic resin. significance adopted in the study was 0.05, and calculations were accomplished by the sas system (sas institute inc. the sas system, release 9.2 – ts level 2mo. sas institute inc., cary, nc, usa). results vertical variable did not show statistical significance between control and study groups in the tip of upper incisor from advancement of 4 and 8 mm and mesiobuccal cusp of the right first molar from maxillary advancement of 8 mm. however, there were subsidies to affirm that the mean difference of the measures were different between control and study groups in mesiobuccal cusps of the right molar from maxillary advancement of 4 mm (p<0.0001) and the left molar from 4 and 8 mm (p<0.0001) of advancement, as seen in table 1. in the clinical practice, the flattening of 6 degrees of occlusal plane angle could downward the first molars cusps of 0.55 mm in right molar with advancement of 4 mm compared with upward movement showed with angulation of 13 degrees observed in all control groups from vertical variable. the left molar demonstrated a similar course: a downward movement of 0.52 and 0.62 mm in retrospectively 4 and 8 mm of advancement. anteroposterior variable showed statistically significant difference between control and study groups in both 4 and 8 mm advancements (p<0.005; p<0.0001), as seen in table 2. study groups in both maxillary advancements did not follow the predicted advancement in control groups. the study group advanced 0.23 mm less than the control group in 4 mm analysis and 0.5 mm less than control group in 8 mm analysis. discussion several studies have been designed to compare the occlusal plane angulation obtained in the articulator with the occlusal plane in the patient. results showed great variation about type or brand of the articulator and the face bow used. the differences in methodologies applied increase the variability of the results, even though all studies demonstrated different angulations between patient and position of transferred maxilla in the articulator. o’malley and milosevic5 found 1.9 degrees of difference between the occlusal plane of patients and whipmix articulator, 5.2 degrees of difference for denar articulator, and 6.5 for dentatus articulator. gateno et al.1 showed statistically difference to sam’s face bow: 7.8 degrees and 4.4 degrees to erickon’s face bow. this study demonstrated statistically significant values for both planes studied: vertical and anteroposterior in all measures analyzed, except for the right molar in 8 mm advancement, considering a hypothetic error of 6 degrees of occlusal plane transference. the choice of 6 degrees of inaccuracy is perfectly acceptable for analysis of the influence 370370370370370 influence of wrong determination of occlusal plane in maxillary advancement: a model surgery study braz j oral sci. 11(3):368-372 advancement group average standard deviation t value p-value 4 mm c -11.950 0.2658 -1.74 0.0898 upper incisor s -10.35 0.3503 8 mm c -0.181 0.1732 -0.39 0.7019 s -0.211 0.3076 t test calculated with satterthwaite method 4 mm c -10.610 0.3641 -5.31 0.0001 right molar s -0.517 0.2779 8 mm c 0.5895 0.2458 1.03 0.3112 s 0.4855 0.3807 t test calculated with pooled method 4 mm c -0.783 0.2961 -6.86 (s) 0.0001 left molar s -0.267 0.1596 8 mm c 0.231 0.2269 9.22 (p) 0.0001 s -0.399 0.2043 (s) satterthwaite method; (p) pooled method table 1. average, standard deviation, and student t test for comparison of vertical variable from analyzed groups in advancement of 4 and 8 millimeters advancement group average standard deviation t value p value 4 mm c 52.650 0.2151 2.93 (p) 0.0058 upper incisor s 50.385 0.2712 8 mm c 86.040 0.2567 5.21 (s) 0.0001 s 81.050 0.3428 (s) satterthwaite method; (p) pooled method table 2. average, standard deviation, and student t test for comparison of anteroposterior variable from analyzed groups in advancement of 4 and 8 millimeters in planning the orthognathic movements, as seen in previous studies, cited above. a variation of vertical movement was expected in this upper incisor variable because the condyle axis determines the aperture amplitude in articulator, notwithstanding the results were not statistically significant. during splint fabrication, special attention was dedicate to the incisor position, so their unaltered position can be explained by the axis formed by the tip of crowns in maxillary incisor that transfer the amplitude movement for this incisor axis, thus determining a variation in first molar’s vertical position as demonstrated in this study. the movement that first molars in study group did not exert to accompany the vertical movement in control group, or a flattening of occlusal plane, can be considered a counterclockwise rotation of occlusal plane. according to wolford et al.7, some important anatomic alterations were found in this type of movement such as anterior projection of chin, increase of maxillary incisor inclination and decrease of mandibular incisor inclination, increase in the posterior facial height, and flattening of mandibular occlusal plane. esthetic relationships of the jaws with other facial structures in counterclockwise rotation are also determined by the center of rotation adopted for the movement. this study adopted the center of rotation at the maxillary incisor edge, so the perinasal area, subnasale area, and the nasal tip could move posteriorly and the chin could come forward. on the other hand, if rotation is around point a or higher, then the perinasal area and the nose could be less affected, but the maxillary incisor edges could come forward, thus increasing the anteroposterior support to the upper lip 8. no study has yet demonstrated a relationship between the decrease of vertical height in first molars in a counterclockwise rotation and the influence in some soft tissue point. coleta et al.9 showed results with different values of hard and soft tissue after a counterclockwise movement adjunct with tmj reconstruction in vertical and horizontal planes, unfortunately, not applicable in this study. doubts about the clinical influence of 0.5 mm of vertical height decrease in 6 degrees of flattening occlusal plane founded in this study still persist. probably, this value, which is even statistically significant, could not represent the real problems in clinical esthetic planning and the results in orthognathic surgery. maxillary advancement induces some important changes in soft tissue, mainly, in the nasal area and upper lip. rotation and translation from the subnasale point are typical movements that the upper lip exerts when maxillary advancement is performed, followed by the advancement of the tip of the upper incisor in a soft-hard tissue proportion of 0.4:1 and 0.9:110-11. jensen et al.12 found 78% of soft-hard tissue alteration measured in the most projected point in the upper lip (labrale superius) in a 1.9 mm advancement from the tip of maxillary incisor. in addition to this tendency, stella et al.13 observed that postoperative upper lip had a pattern more than 25% 371371371371371influence of wrong determination of occlusal plane in maxillary advancement: a model surgery study braz j oral sci. 11(3):368-372 thinner than pre-operative upper lip in maxillary advancements13. anteroposterior upper incisor lack of projection induces errors of prediction. soft tissue never followed totally hard tissue in maxillary advancement, even with different points of reference and methods of investigation. if transference of occlusal plane has inaccuracies, probably the amount of advancement planned will not be the expected. the same doubt still remains about clinical influence of little lack of movements predicted in the orthognathic surgery results. developments in different three-dimensional technologies, such as multiplanar computed tomography and magnetic resonance imaging scanning and three-dimensional photography modalities, permit fusion of images that can provide an accurate and realistic prediction of model treatment planning in orthognathic surgery14. digital model surgery and the obtainment of prototyped wafers for orthognathic surgery became an accurate and effortless alternative to model surgery. however, these new technologies are not yet achievable for oral and maxillofacial surgeons from some developing countries. in these cases, manual model surgery still remains the only alternative to achieve excellent results in orthognathic surgery15. the authors believe that sensible errors from acquisition of occlusal plane with face bow and semi-adjustable articulator technique will not produce serious problems with facial esthetic and functional outcomes in bimaxillary surgery that requires pure advancement of maxilla. a limitation of this study is that only one movement was analyzed: maxillary advancement. perhaps, with the exception of non-asymmetric cases, or in patients with jaw aberrations, a camper table could be used as alternative mounting of maxillary model in the articulator. analysis of other orthognathic movements and clinical studies will be necessary to make it clearer and feasible the use of camper table as an alternative technique. references 1. gateno j, forrest kk,camp b. a comparison of 3 methods of face-bow transfer recording: implications for orthognathic surgery. 2001; 59: 635-40. 2. ellis e iii. accuracy of model surgery: evaluation of an old technique and introduction of a new one. j oral maxillofac surg. 1990; 48: 1161-7. 3. ellis e iii, tharanon w, gambrell k. accuracy of face-bow transfer: effect on surgical prediction and postsurgical result. j oral maxillofac surg. 1992; 50: 562-7. 4. bamber ma, firouzai r, harris m, linney a. a comparative study of two arbitrary face-bow transfer systems for orthognathic surgery planning. 1996; 25: 339-43. 5. o’malley am, milosevic a. comparison of three facebow/semi-adjustable articulator systems for planning orthognathic surgery. br j oral maxillofac surg. 2000; 38: 185-90. 6. wolford lm, galiano a. a simple and accurate method for mounting models in orthognathic surgery. j oral maxillofac surg. 2007; 65: 1406-9. 7. wolford lm, chemello pd, hilliard f. occlusal plane alteration in orthognathic surgery – part i: effects on function and esthetics. am j orthod dentofac orthop. 1994; 106: 304-16. 8. reyneke jp, evans wg. surgical manipulation of the occlusal plane. int j adult orthod orthognath surg. 1990; 5: 99. 9. coleta ked, wolford lm, gonçalves jr, dos santos pinto a, cassano ds, gonçalves dag. maxillo-mandibular counterclockwise rotation and mandibular advancement with tmj concepts total joint prostheses –part iv –soft tissue response. int j oral maxillofac surg. 2009; 38: 637-46. 10. carlotti ae, aschaffenburg ph, schendel sa. facial changes associated with surgical advancement of the lip and maxilla. j oral maxillofac surg. 1986; 44: 593-6. 11. dann jj, fonseca rj, bell wh. soft tissue changes associated with total maxillary advancement: a preliminary study. j oral surg. 1976; 34: 19-23. 12. jensen ac, sinclair pm, wolford lm. soft tissue changes associated with double jaw surgery. am j orthod dentofacial orthop. 1992; 101: 266-75. 13. stella jp, streater mr, epker bn, sinn dp. predictability of upper lip soft tissue changes with maxillary advancement. j oral maxillofac surg. 1989; 47: 697-703. 14. plooij jm, maal tj, haers p, borstlap wa, kuijpers-jaqtman am, bergé sj. digital three-dimensional image fusion processes for planning and evaluating orthodontics and orthognathic surgery. a systematic review. int j oral maxillofac surg. 2011; 40: 341-52. 15. mccormick su, drew sj. virtual model surgery for efficient planning and surgical performance. j oral maxillofac surg. 2011; 69:638-44. 372372372372372 influence of wrong determination of occlusal plane in maxillary advancement: a model surgery study braz j oral sci. 11(3):368-372 1http://dx.doi.org/10.20396/bjos.v19i0.8659594 volume 19 2020 e209594 original article 1 university of são paulo, hospital for rehabilitation of craniofacial anomalies, sector of endodontics, bauru, são paulo, brazil. 2 university of são paulo, bauru school of dentristy, department of dentistry, endodontics and dental materials, bauru, são paulo, brazil. corresponding author: mirela cesar de barros bauru school of dentristy university of são paulo al. octávio pinheiro brisolla, 9-75 17012-901 bauru sp brazil phone: +55 14 32358344 e-mail: mirela.barros@usp.br received: may 13, 2020 accepted: november 24, 2020 consequences of chemical residue formation during potentiation of final irrigation – in vitro study mirela cesar de barros1,* , jessica de almeida coelho2 , lidiane de castro pinto1 , marco antônio húngaro duarte2 , flaviana bombarda de andrade2 seeking to increase the efficiency of endodontic irrigation, the association of different solutions as final irrigant has been investigated, such as sodium hypochlorite with chlorhexidine. the literature shows that the combination of these substances leads to the formation of a brownish precipitate, but does not reveal measurements of the intensity of this precipitate and its consequences. aim: the present study aimed to evaluate the change in dentin color and the obliteration of the dentinal tubules after the association of sodium hypochlorite (naocl) with chlorhexidine (chx) in the final irrigation. methods: fifty sterile human lower premolars were prepared with a prodesign r 35.05 files and divided into 6 groups. four different naocl concentrations (0.5%; 1%, 2.5% and 5.25%) associated with 2% chx were tested, in addition to 2 control groups, using only 2.5% naocl and 2% chx, respectively. after the final irrigation protocol, the dentin color change was evaluated by spectrophotometry immediately and after 24 hours, and the dentinal tubule obliteration was assessed by scanning electron microscopy. results: it was possible to verify that regardless the naocl concentration used when associated with chx, a chemical residue was formed, with consequent dentin pigmentation and tubular obstruction. there was a trend towards increased dentin pigmentation and tubular obstruction due to the deposition of the chemical residue formed by this association. conclusion: it can be concluded that all concentrations of naocl associated with chx caused color changes and tubular obstruction, being proportional to the concentration of naocl used. keywords: sodium hypochlorite. chlorhexidine. root canal irrigants. endodontics. https://orcid.org/0000-0002-4711-3841 https://orcid.org/0000-0001-6642-9908 https://orcid.org/0000-0001-9764-0327 https://orcid.org/0000-0003-3051-737x https://orcid.org/0000-0002-1238-2160 2 barros et al. introduction the purpose of endodontic treatment is to clean and model the root canal system (rcs). however, due to the anatomical complexity presented by this three-dimensionally complex system, only mechanized instrumentation is not able to remove all pulp and bacteria content present in the isthmus and branches. therefore, it is necessary to use chemical agents during mechanized instrumentation for greater success rates in endodontic therapy1-3. sodium hypochlorite (naocl) is one of the irrigating solutions widely used in endodontics due to its excellent antimicrobial and tissue dissolution properties, being present in concentrations of 0.5% to 5.25%3,4. although sodium hypochlorite has advantages in its use, it still can not be considered the ideal solution. naocl does not have the full capacity for debridement of the dentinal tubules; it is irritating when in contact with periapical tissues, in addition to not having substantivity5,6. seeking to increase the efficiency of irrigation, the possibility of associating different irrigating solutions to naocl has been studied due to the positive synergistic effects on antimicrobial activity7. thus, other irrigating solutions are recommended for the association with sodium hypochlorite, such as chlorhexidine digluconate6,7. chlorhexidine (chx) in a concentration of 2% has a broad-spectrum antimicrobial effect used as an alternative to sodium hypochlorite7-10, and demonstrates similar antimicrobial activity. the literature shows that chx can be used as a final irrigant after naocl due to its residual antimicrobial action11. however, sodium hypochlorite must be removed from the rcs since the concomitant use of these two substances leads to the formation of a brown chemical residue, that is difficult to remove and can cause discoloration of dentinal structures12. it also promotes the obliteration of the dentinal tubules, compromising the filling of the canals. the literature shows that the precipitate formed may contain para-chloroaniline (pca), a compound formed through the hydrolysis of chx in a reaction dependent on time, alkaline ph, and temperature2,12,13. this precipitate is toxic and carcinogenic and may lead to methaemoglobinemia in humans. the international agency for research on cancer (international agency for research on cancer, 2006) classified pca as a carcinogen of the 2b group, with limited evidence about its carcinogenic potential in humans12,14,15. the objective of this study was to evaluate the consequences of the interaction of sodium hypochlorite and chlorhexidine on the obliteration of the dentinal tubules and the alteration of the dentin staining, after the final irrigation, immediately and after 24 hours. the null hypothesis tested is that there is a tendency for greater pigment deposition as sodium hypochlorite concentration is increased. materials and methods preparation of teeth the present study was approved by the local ethics committee (caae: 04269418500005421). fifty healthy human lower premolars were selected and 3 barros et al. radiographed to confirm the absence of internal calcifications, the presence of more than one canal, stones, or pulp nodules. all procedures were performed by a single operator. the external root surfaces of the specimens were cleaned of tissue remnants and stored in 0.9% saline solution until the moment of use16,17. the length was standardized at 15 mm using an isomet cutting machine (isomet 1000, buehler ltd, lake bluff, il, usa) with a diamond disc at 250 revolutions per minute (rpm), under irrigation, reducing the height of the tooth crown and gaining access to the canal18. the patency was performed by inserting a # 15 k file (dentsply, maillefer, ballaigues, switzerland) until the tip of the instrument was seen juxtaposed to the apical foramen19,20. soon after, 1mm was subtracted from this measurement, obtaining a working length of 14mm. subsequently, the teeth were immersed in 1% sodium hypochlorite for 12 hours for initial disinfection and dissolution of organic tissues. to open the dentinal tubules, the specimens received three 10-minute baths each in an ultrasonic tub (odontobras, ribeirão preto, sp, brazil) with 1% sodium hypochlorite (naocl) (formula e ação, são paulo, sp, brazil), 17% ethylenediaminetetraacetic acid (edta) (biodynamic chemistry and pharmaceuticals, ibiporã, pr, brazil) followed by distilled water to neutralize the previous substances. the specimens were dried for 24 hours before being autoclaved at 121 °c18. the root apex of each specimen was closed with pink wax # 7, avoiding the extrusion of irrigants20,21. the specimens were inserted into a sterile metallic device and adjusted until they were firm. all specimens were prepared with prodesign r 35.05 files (easy equipamentos odontológicos, belo horizonte, mg, brazil) following the speed and torque indications suggested by the manufacturer. during instrumentation, the canal was irrigated with distilled water, using a 5 milliliter (ml) syringe (ultradent products, south jordan, ut, usa) with a 30 gauge needle (ultradent products, south jordan, ut, usa) at 1 mm from the working length 3 times with 3 ml, totaling 9ml. distilled water was used because it is an inert solution, which did not influence the association of compounds in the final irrigation. with a # 15 k file (dentsply, maillefer, ballaigues, switzerland), the working length was recapitulated17,22. final irrigation protocol after instrumentation, the specimens were flooded with 3ml of 17% edta for three minutes to remove organic residues from the instrumentation, followed by irrigation with 5ml of distilled water to neutralize the substance previously used. next, they were randomly divided into 6 groups, 4 experimental groups with n = 10 and 2 control groups with n = 5, which received the following final irrigation protocol: group 1: (n = 10) 2ml of 0.5% naocl followed by 2ml of 2% chx; group 2: (n = 10) 2ml of 1% naocl followed by 2ml of 2% chx; group 3: (n = 10) 2ml of 2.5% naocl followed by 2ml of 2% chx; 4 barros et al. group 4: (n = 10) 2ml of 5.25% naocl followed by 2ml of 2% chx group 5: (n = 5) 2ml of 2.5% naocl group 6: (n = 5) 2ml of 2% chx sodium hypochlorite solutions in concentrations of 0.5%; 1%; 2.5% and 2% chlorhexidine were obtained through the manufacturer (formula e ação, são paulo, sp, brazil) and 5.25% by manipulation in a pharmacy (specifica ltda, bauru, sp, brazil). the canals were immediately dried with logic tanari 35.05 sterile absorbent paper tips (tanariman industrial ltda., manacapuru, am). analysis of color change in a spectrophotometer color measurements were performed using a spectrophotometer (vita easyshade® compact; vita zahnfabrik, bad saichen, germany) under standard conditions, calibrating the equipment before measuring each group. the following intervals were used for the measurement: t = 0 (before the specimens received the proposed final irrigation protocols); t = 1 (just after the proposed final irrigation protocols); t = 2 (24 hours after the proposed final irrigation protocols). to avoid optical changes caused by dehydration and to simulate the environment of the oral cavity, the specimens were stored in a humid environment and kept in an oven at 37ºc for the third reading and their water excess removed with a sterile paper filter (melitta, minden, germany). the readings were performed on the root vestibular surface of each specimen, in the region of the cervical and apical thirds23. the specimens were measured three times, with 5 seconds for each reading, so that the average of the measurements was used, avoiding possible bias in the reading. the measurement was standardized in the cervical and apical thirds. the values of (cie) (international lighting commission, 1913), l, a and b were noted, and the color change (δe) concerning the interval between before the final irrigation protocol (t0), immediately after the association of the substances in the final irrigation (t1) and 24 hours after the proposed final irrigation protocol (t2), was calculated using the following formula: δe = [(δl) 2 + (δa) 2 + (δb) 2] ½ where “l” represents the luminosity values of the color, “a” is the measurement along the red-green axis, “b” corresponds to the measurement along the yellow-blue axis and “δe” is the measure of the difference between the color in the initial reading and the final reading23,24. the values obtained from “δe” were statistically evaluated by the friedman test. scanning electron microscopy (sem) for scanning electron microscopy analysis (jeol, jsmtlloa, tokyo, japan), longitudinal sections of the specimens from each group were performed with a diamond disc and sterile saline solution on an isomet machine (isomet, buehler, il, usa) to obtain two halves. the sections obtained were dipped in absolute alcohol for 1 minute and allowed to dry at room temperature for 24 hours, and subsequently mounted on metal bases to receive the gold bath. they were sprayed with 200a 300a (angstroms) 5 barros et al. of gold (hammer vi sputtering system; anatech ltd, alexandria, va) to become electrically conductive. images of the cervical, middle, and apical thirds were obtained at 10-15kv with a standard increase of 750x25. scoring system the images obtained in sem were classified according to the presence of the precipitate observed in the thirds and punctuated by three calibrated examiners using a classification system described by pirani26 (2009) and prati27 (2004): 0 more than 75% of the tubules visibly exposed and free from the smear layer. 1 smear layer present and <75% of tubules visibly exposed. 2 smear layer visibly limited and <50% of tubules visibly exposed. 3 smear layer homogeneous in dentine and without exposed dentinal tubules. the term smear layer in the present work corresponds to the presence of the precipitate. intraand an inter-examiner agreement was assessed using the kappa test (p <0.05). statistical analysis in the analysis of the color change by spectrophotometry, the tabulated data showed non-normal distribution, being subjected to the kruskal-wallis test followed by the dunn’s test when all groups were compared. the evaluation of these data by time was performed using the friedman test. in the scanning electron microscopy, the analyses by scores were also submitted to the kruskal-wallis test, followed by the dunn’s test using the prism 6.0 software (graphpad software inc., la jolla, usa) as an analytical tool and the level of significance was established at p <0.05. results color change in all tested groups, the precipitate was formed with consequent dentin color change (δe) of the specimens. in figure 1, it is possible to observe the color and appearance of this precipitate right after the irrigators’ interaction. figure 1. precipitate formed after the association of the proposed irrigators. 6 barros et al. table 1 shows the median, maximum and minimum color change (δe) values for all groups before final irrigation (t0), after final irrigation (t1) and 24 hours after the proposed final irrigation protocols (t2). although without statistically significant differences when comparing δe between times t0 (before final irrigation) and t1 (after final irrigation); g4 showed higher values than the other groups (figure 2). table 1. values for color change (δe) in the different groups evaluated before the irrigation protocol, immediately after and 24h after. friedman test p <0.05. groups median (min-max) δe t0 t0 t0 g1 – naocl 0.5% + clx 2% 15.22 (4.80-32.25) 15.22 (4.80-32.25) 15.22 (4.80-32.25) g2 – naocl 1% + clx 2% 16.13 (11.00-24.00) 16.13 (11.00-24.00) 16.13 (11.00-24.00) g3 – naocl 2.5% + clx 2% 14.72 (5.90-26.30) 14.72 (5.90-26.30) 14.72 (5.90-26.30) g4 – naocl 5.25% + clx 2% 15.98 (13.20-26.50) 15.98 (13.20-26.50) 15.98 (13.20-26.50) g5 – naocl 2.5% 7.93 (4.20-11.90) 7.93 (4.20-11.90) 7.93 (4.20-11.90) g6 – clx 2% 7.26 (3.40-14.90) 7.26 (3.40-14.90) 7.26 (3.40-14.90) figure 2. mean and standard deviation of variation of the color (de) of the experimental and control groups measured at the moment after biomechanical preparation (t0), immediately after the final irrigation (t1) and 24 hours after (t2). g1 – 0.5% naocl + chx g4 – 5.25% naocl + chx g3 – 2.5% naocl + chx g6 – chxg5 – 2.5% naocl g2 – 1% naocl + chx t0 t1 t2 time 30 20 10 0 ∆ e t0 t1 t2 time 25 20 10 0 ∆ e t0 t1 t2 time 30 20 10 0 ∆ e t0 t1 t2 time 25 20 10 0 ∆ e t0 t1 t2 time 20 15 5 0 ∆ e t0 t1 t2 time 20 15 10 0 ∆ e 5 15 10 5 15 5 7 barros et al. when t1 (just after the final irrigation) and t2 (24 hours after the final irrigation) were compared, there was a reduction in δe in groups g2 and g4, but without statistically significant differences. as a result, it is suggested that pigmentation of the dental structure occurs immediately after the association of irrigants, but it is not maintained after 24 hours. groups g1 and g3, on the other hand, showed an increasing trend in their δe between t1 and t2, without statistically significant differences. the positive control groups, g5 and g6, showed small variations in δe between the proposed times, with no statistical differences (p> 0.05) (figure 3). scanning electron microscopy in all experimental groups, there was residue formation, regardless of the concentration of naocl associated with clx. in g2, this precipitate was more visible and obliterated most dentinal tubules, according to the classification proposed to blind examiners. the control groups showed fewer chemical residues deposited in the dental tubules (tubular obstruction) when compared to the experimental groups (figure 4). however, the results showed that there was no statistical difference between the groups regarding the presence of residue in the dentin. table 2 shows the median, maximum, and minimum values of the scoring system used to classify the images according to the presence of precipitate in the three thirds. figures 5 and 6 show some sem images related to the cervical, middle and apical thirds, respectively, of different groups, where the formation and deposition of residue were greater in the cervical and middle thirds when compared to the apical. figure 3. mean and standard deviation of variation of the color (de) between groups in the moments immediately after irrigation (t1) and 24 hours after the irrigation protocol (t2). 30 time 1 time 2 groups groups 20 10 0 ∆ e 0.5 % na oc l + c x 2.5 % na oc l + c x 2.5 % na oc l 2% c x 5.2 5% n ao cl + cx 1% n ao cl + cx 0.5 % na oc l + c x 2.5 % na oc l + c x 2.5 % na oc l 2% c x 5.2 5% n ao cl + cx 1% n ao cl + cx 30 20 10 0 ∆ e 8 barros et al. table 2. score values for the presence of precipitate on the dentin surface in the three thirds of the different groups. kruskal-wallis test p <0.05. groups median (min-max) score (sem) g1 – naocl 0.5% + clx 2% 2.13 (0.00-3.00) g2 – naocl 1% + clx 2% 2.20 (0.00-3.00) g3 – naocl 2.5% + clx 2% 1.86 (0.00-3.00) g4 – naocl 5.25% + clx 2% 2.10 (0.00-3.00) g5 – naocl 2.5% 1.46 (0.00-3.00) g6 – clx 2% 1.26 (0.00-3.00) figure 5. sem images (magnification 750x) of dentin surface after irrigants association showing precipitate formation in the cervical (a) and middle (b) thirds. a b c 10 kv x 750 10 µm 000026 10 kv x 750 10 µm 000067 10 kv x 750 10 µm 000043 figure 4. mean and standard deviation of residue formation based on the examiner’s analysis and score classification. 4 residues groups 3 1 0 sc or es 0.5 % na oc l + c x 2.5 % na oc l + c x 2.5 % na oc l 2% c x 5.2 5% n ao cl + cx 1% n ao cl + cx 2 9 barros et al. discussion the present study evaluated the chemical residue formed by the association of irrigants commonly used in endodontic therapy11. this knowledge is important because this association results in a smear layer with the potential to obliterate the dentinal tubules, compromising the sealing of the root canal during obturation14,16,17, in addition to aesthetically compromising some roots. the null hypothesis was partially accepted since there were no statistical differences between the groups in the analyses carried out, but there was a tendency for greater deposition of residues and pigmentation as the naocl concentration increased. in the spectrophotometric analysis, the values of l, a and b established by the ciee (international lighting commission, 1913) were observed to detect color changes (δe) using the formula δe = [(δl) 2 + (δa) 2 + (δb) 2] 1/2, concerning the established reading protocols. the findings of the present study are in agreement with other results presented in the literature14,19. when the association of naocl in concentrations of 0.5% to 5.25% with the 2% clx solution was carried out, it was possible to observe the formation of dense chemical residue, whose intensity of the staining varied according to the concentration of naocl used. when t0 (before final irrigation) and t1 (immediately after final irrigation) were compared, the results in the spectrophotometry showed an increase in δe in most of the tested groups, although without statistically significant differences. g1 showed the highest standard deviation when compared to the other groups, which can be associated with chemical instability of 0.5% naocl. however, values of greater variations of δe were detected for g4 where the association of 5.25% naocl + 2%chx was used. these results reinforce the findings of the literature6,19. in the results obtained in t1 (just after the final irrigation) and t2 (24 hours after the final irrigation), the g1, g2, g3, g5, and g6 groups showed an increase in δe, diverging from g4, which showed a reduction in δe between the times. the result obtained in g4 suggests that the color change did not last due to the low stability of sodium hypochlorite in high concentrations, which rapidly loses active chlorine, as stated in the study by clarkson and moule28 (1998). the control groups, g5 and g6, had their concentrations selected based on the concentrations of the solutions with the greatest scientific relevance29. in the evaluation of δe between the times, they presented lower values concerning the formation of residues, without statistical differences between them. figure 6. sem images (magnification 750x) of precipitate formation in the apical third of several specimens, but in smaller amounts. 10 kv x 750 10 µm 000001 10 kv x 750 10 µm 000054 10 kv x 750 10 µm 000088 10 barros et al. in the analysis by scanning electron microscopy, residue formation was found in all groups of the present study to a greater or lesser degree, however, there was no statistical difference between the groups25. in the cervical and middle thirds of g1, g2, g3, and g4, there was a greater amount of chemical residue formed (figure 5) when compared to the apical third of each group (figure 6), corroborating the findings in the literature6,19. g2 was the group that showed the greatest evidence of the chemical residue formed, a fact that may be associated with the use of stabilizers in the formulation of 1% naocl30 (figure 3). its presence is probably responsible for less loss of active chlorine and, therefore, there is a greater formation of final chemical residue. our findings are in line with other studies in the literature19,31,32, showing that this chemical residue from the association of these two different irrigants obstructs the dentinal tubules. although the association of sodium hypochlorite with chlorhexidine in the final irrigation may result in positive synergistic effects on antimicrobial activity11, it triggers the formation of a chemical precipitate capable of altering dentin staining and obstructing the dentinal tubules. according to the limitations of this in vitro study, it was concluded that there was the formation of a precipitate, in greater concentration in the cervical and middle thirds of the root canal, and pigmentation in all concentrations of naocl, when associated with 2% chlorhexidine, observing that the pigmentation in extracted teeth, remains for at least 24 hours. clinically, the use of sodium hypochlorite followed by chlorhexidine is not recommended without the use of an intermediate solution, such as saline or distilled water. references 1. irala led, melo taf, sores rg, louzada f, juchem j. [evaluation of the dark pigment formation when mixing the sodium hypochlorite solution in different concentrations, with chlorhexidine gluconate 0.2%]. rsbo. 2009;6(3):286-90. portuguese. 2. basrani br, manek s, mathers d, fillery e, sodhi rn. determination of 4-chloroaniline and its derivatives formed in the interaction of sodium hypochlorite and chlorhexidine by using gas chromatography. j endod. 2010 feb;36(2):312-4. doi: 10.1016/j.joen.2009.10.031. 3. kolosowski kp, sodhi rn, kishen a, basrani br. qualitative analysis of precipitate formation on the surface and in the tubules of dentin irrigated with sodium hypochlorite and a final rinse of chlorhexidine or qmix. j endod. 2014 dec;40(12):2036-40. doi: 10.1016/j.joen.2014.08.017. 4. estrela c, estrela cr, barbin el, spanó jc, marchesan ma, pécora jd. mechanism of action of sodium hypochlorite. braz dent j. 2002;13(2):113-7. doi: 10.1590/s0103-64402002000200007. 5. peters oa. current challenges and concepts in the preparation of root canal systems: a review. j endod. 2004 aug;30(8):559-67. doi: 10.1097/01.don.0000129039.59003.9d. 6. zehnder m. root canal irrigants. j endod. 2006 may;32(5):389-98. doi: 10.1016/j.joen.2005.09.014. 7. haapasalo m, shen y, wang z, gao y. irrigation in endodontics. br dent j. 2014 mar;216(6):299-303. doi: 10.1038/sj.bdj.2014.204. 8. cathro p et al. the importance of irrigation in endodontics. contemp endod. 2004;1:3-7. 9. okino la, siqueira el, santos m, bombana ac, figueiredo ja. dissolution of pulp tissue by aqueous solution of chlorhexidine digluconate and chlorhexidine digluconate gel. int endod j. 2004 jan;37(1):38-41. doi: 10.1111/j.1365-2591.2004.00749.x. 11 barros et al. 10. vianna me, horz hp, gomes bp, conrads g. in vivo evaluation of microbial reduction after chemo-mechanical preparation of human root canals containing necrotic pulp tissue. int endod j. 2006 jun;39(6):484-92. doi: 10.1111/j.1365-2591.2006.01121.x. 11. prado m, santos júnior hm, rezende cm, pinto ac, faria rb, simão ra, et al. interactions between irrigants commonly used in endodontic practice: a chemical analysis. j endod. 2013 apr;39(4):505-10. doi: 10.1016/j.joen.2012.11.050. 12. orhan eo, irmak ö, hür d, yaman bc, karabucak b. does para-chloroaniline really form after mixing sodium hypochlorite and chlorhexidine? j endod. 2016 mar;42(3):455-9. doi: 10.1016/j.joen.2015.12.024. 13. basrani br, manek s, sodhi rn, fillery e, manzur a. interaction between sodium hypochlorite and chlorhexidine gluconate. j endod. 2007 aug;33(8):966-9. doi: 10.1016/j.joen.2007.04.001. 14. stojicic s, shen y, qian w, johnson b, haapasalo m. antibacterial and smear layer removal ability of a novel irrigant, qmix. int endod j. 2012 apr;45(4):363-71. doi: 10.1111/j.1365-2591.2011.01985.x 15. siddique r, sureshbabu nm, somasundaram j, jacob b, selvam d. qualitative and quantitative analysis of precipitate formation following interaction of chlorhexidine with sodium hypochlorite, neem, and tulsi. j conserv dent. 2019;22(1):40-47. doi: 10.4103/jcd.jcd_284_18. 16. bui tb, baumgartner jc, mitchell jc. evaluation of the interaction between sodium hypochlorite and chlorhexidine gluconate and its effect on root dentin. j endod. 2008 feb;34(2):181-5. doi: 10.1016/j.joen.2007.11.006. 17. krishnamurthy s, sudhakaran s. evaluation and prevention of the precipitate formed on interaction between sodium hypochlorite and chlorhexidine. j endod. 2010 jul;36(7):1154-7. doi: 10.1016/j.joen.2010.01.012. 18. andrade fb, arias mp, maliza ag, duarte ma, graeff ms, amoroso-silva pa, et al. a new improved protocol for in vitro intratubular dentinal bacterial contamination for antimicrobial endodontic tests: standardization and validation by confocal laser scanning microscopy. j appl oral sci. 2015;23(6):591-8. doi: 10.1590/1678-775720140261. 19. akisue e, tomita vs, gavini g, poli de figueiredo ja. effect of the combination of sodium hypochlorite and chlorhexidine on dentinal permeability and scanning electron microscopy precipitate observation.  j endod. 2010 may;36(5):847-50. doi: 10.1016/j.joen.2009.11.019. 20. magro gm, kuga mc, victorino rk, vázquez-garcia fa, aranda-garcia aj, faria-junior nb, et al. evaluation of the interaction between sodium hypochlorite and several formulations containing chlorhexidine and its effect on the radicular dentin-sem and push-out bond strength analysis. microsc res tech. 2014 jan;77(1):17-22. doi: 10.1002/jemt.22307. 21. marchesan ma, pasternak jb, afonso mm, sousa-neto md, paschoalato c. chemical analysis of the flocculate formed by the association of sodium hypochlorite and chlorhexidine. oral surg oral med oral pathol oral radiol. 2007 may;103(5):e103-5. doi: 10.1016/j.tripleo.2006.11.008. 22. mortenson d, sadilek m, flake nm, paranjpe a, heling i, johnson jd, cohenca n. the effect of using an alternative irrigant between sodium hypochlorite and chlorhexidine to prevent the formation of para-chloroaniline within the root canal system. int endod j. 2012 sep;45(9):878-82. doi: 10.1111/j.1365-2591.2012.02048.x. 23. silva mam. [physico-chemical and biological properties of cement experiment with portland base associated with different radiopacities] [thesis]. bauru: university of são paulo, bauru dental school; 2011. portuguese. 24. lenherr p, allgayer n, weiger r, filippi a, attin t, krastl g. tooth discoloration induced by endodontic materials: a laboratory study. int endod j. 2012 oct;45(10):942-9. doi: 10.1111/j.1365-2591.2012.02053.x. 12 barros et al. 25. bernardes ra, duarte mah, vivan rr, alcalde mp, vasconcelos bc, bramante cm. comparison of three retreatment techniques with ultrasonic activation in flattened canals using micro-computed tomography and scanning electron microscopy. int endod j. 2016 sep;49(9):890-897. doi: 10.1111/iej.12522. 26. pirani c, pelliccioni ga, marchionni s, montebugnoli l, piana g, prati c. effectiveness of three different retreatment techniques in canals filled with compacted gutta-percha or thermafil: a scanning electron microscope study. j endod. 2009 oct;35(10):1433-40. doi: 10.1016/j.joen.2009.06.002.  27. prati c, foschi f, nucci c, montebugnoli l, marchionni s. appearance of the root canal walls after preparation with niti rotary instruments: a comparative sem investigation. clin oral investig. 2004 jun;8(2):102-10. doi: 10.1007/s00784-004-0253-8. 28. clarkson rm, moule aj. sodium hypochlorite and its use as an endodontic irrigant. aust dent j.1998;43(3):250-6. 29. cintra l, watanabe s, samuel ro, da silva facundo ac, de azevedo queiroz io, dezan-júnior e, et al. the use of naocl in combination with chx produces cytotoxic product. clin oral investig. 2014 apr;18(3):935-40. doi: 10.1007/s00784-013-1049-5. 30. brazil. national health surveillance agency. [technical regulation of sanitary products classified as sodium hypochlorite or calcium bleaches and other arrangements]. 2016 [cited 2019 oct 22]. available from: www.anvisa.gov.br. portuguese. 31. bernardi a, texeira cs. the properties of chlorhexidine and undesired effects of its use in endodontics. quintessence int. 2015;46(7):575-82. doi: 10.3290/j.qi.a33934. 32. nocca g, ahmed hma, martorana ge, callà c, gambarini g, rengo s, et al. chromographic analysis and cytotoxic effects of chlorhexidine and sodium hypochlorite reaction mixtures. j endod. 2017 sep;43(9):1545-1552. doi: 10.1016/j.joen.2017.04.025. 1http://dx.doi.org/10.20396/bjos.v18i0.8657328 volume 18 2019 e191627 original article 1 department of oral pathology and diagnosis, school of dentistry, universidade federal do rio de janeiro (ufrj), rio de janeiro, rj, brazil. 2 department of pediatric dentistry and orthodontics, school of dentistry, universidade federal do rio de janeiro (ufrj), rio de janeiro, rj, brazil. 3 laboratory for nuclear instrumentation, coppe, universidade federal do rio de janeiro (ufrj), rio de janeiro, rj, brazil. 4 laboratory for nuclear instrumentation, coppe, universidade federal do rio de janeiro (ufrj), rio de janeiro, rj, brazil. corresponding author: maria augusta portella guedes visconti, universidade federal do rio de janeiro, departamento de patologia e diagnóstico oral rua professor rodolpho paulo rocco, 325, cidade universitária, zip code 21941-617 rio de janeiro, rj, brazil, phones: +55 (21) 988899383; +55 (21) 39382045 e-mail: gutavisconti@odonto.ufrj.br received: april 16, 2019 accepted: october 06, 2019 root canal segmentation in cone-beam computed tomography: comparison with a micro-ct gold standard juliane freitas machado1, paula maciel pires2, thais maria pires dos santos3, aline de almeida neves2, ricardo tadeu lopes4, maria augusta portella guedes visconti1,* aim: the purpose of this study was to compare root canal volumes (rcvs) obtained by means of cone beam computed tomography (cbct) to those obtained by micro-computed tomography (micro-ct) after applying different segmentation algorithms. methods: eighteen extracted human teeth with sound root canals were individually scanned in cbct and micro-ct using specific acquisition parameters. two different images segmentation strategies were applied to both acquisition methods (a visual and an automatic threshold). from each segmented tooth, the root canal volume was obtained. a paired t-test was used to identify differences between mean values resulted from the experimental groups and the gold standard. in addition, pearson correlation coefficients and the agreement among the experimental groups with the gold standard were also calculated. the significance level adopted was 5%. results: no statistical differences between the segmentation methods (visual and automatic) were observed for micro-ct acquired images. however, significant differences for the two segmentation methods tested were seen when cbct acquired images were compared with the micro-ct automatic segmentation methods used. in general, an overestimation of the values in the visual method were observed while an underestimation was observed with the automatic segmentation algorithm. conclusion: cone beam computed tomography images acquired with parameters used in the present study resulted in low agreement with root canal volumes obtained with a microct tomography gold standard method of rcv calculation. keywords: root canal therapy. x-ray microtomography. conebeam computed tomography. imaging, three-dimensional. mailto:gutavisconti@odonto.ufrj.br 2 machado et al. introduction cone-beam computed tomography (cbct) is an important resource for examination of bone and dental structures in the maxillofacial region. the tridimensional nature of the obtained images is used in diagnosis, treatment planning and follow up of patients treated for diverse oral conditions1,2. in endodontics, cbct images enable determination of root canal morphology and length, as well as the presence of accessory canals, particularly in complex cases, in which periapical radiographs fail to reveal with precision, important anatomic features3,4. some cone-beam scanners are equipped with a small field of view (fov), allowing examination of specific areas of interest and, especially in endodontics, high resolution images are obtained with small fov equipments. this is important because it restricts the area of exposure, possibly reducing the radiation dose to the patient5,6. certain factors however, such as voxel size, acquisition parameters, and number of acquired images, can directly influence the quality of the produced tomographic images7. on the other hand, micro-ct has been recently suggested as a possible gold standard for a precise and non-destructive in vitro study of the 3d anatomy of the root canal system8,9 due to its high resolution, low noise and precise three-dimensional reproduction of the internal and external morphology of the tooth10,11. image segmentation is an important tool in digital image analysis, providing information on the volume and dimensions of a specific area of interest. selection of threshold values in micro-ct acquired images of root canals can be done visually, based on the operator’s ability to detect histogram peaks and valleys or automatically, by means of computer-based algorithms12. however, it is unclear whether differences among segmentation methods are indeed significant in the determination of root canal volume by cbct. thus, the purpose of the present study was to evaluate the accuracy of cbct examination in calculating the root canal volume after application of two segmentation methods (visual and automatic) compared to a gold standard micro-ct evaluation. materials and methods specimen screening and preparation this in vitro study protocol has been approved by the ethics in research committee of the host institution (registration number 1.884.298). in this work, 18 extracted human permanent teeth were used. single and multiradicular teeth were randomly included, provided they presented intact apical root thirds. all teeth were disinfected by immersion in 2% glutaraldehyde for two hours, after which they were kept in distilled water. in order to simulate the condition of the teeth being implanted in the alveoli, the roots were entirely covered in utility wax, and the teeth were individually placed in a custom-made transparent acrylic positioner. this device allowed a standardized placement of the sample to be scanned and simulated soft tissues, without interfering in the quality of the obtained images13. 3 machado et al. image acquisition and data preparation eighteen individual specimen acquisitions were obtained for each scanning method. for the cbct images, acquisitions were performed in a picasso trio 3d apparatus (vatech, hwaseong, republic of korea), using the following parameters: 85kv, 4.5ma, 8x8 cm fov, 0.2 mm isotropic voxel size, and exposure time of 15 seconds. for the micro-ct procedures, the skyscan 1173 system was used (bruker micro-ct, kontich, belgium) and acquisition parameters were 70kv, 114µa, isotropic voxel size of 14.25µm, 1.0mm al filter, exposure time of 250ms and step rotation of 0.5° under 360°. reconstruction was performed using the nrecon software (nrecon, version 1.51, skyscan, kontich, belgium) using a 50% beam hardening correction scheme, ring artefact correction of 5 and input of contrast limits between 0 and 0.1. the reconstruction parameters were specifically optimized for the characteristics of the specimens used in the present study. both cbct and micro-ct image stacks were visualized and prepared using the imagej/ fiji open-source software14 (fig. 1a and b). a volume of interest (voi) containing the root part of the tooth was selected from each image stack. the images were saved in optical media and imported in.tiff format into the software interface. image stacks from the cbct modality were resized to match the dimensions of the micro-ct images. root canal segmentation in cbct and micro-ct images imagej/fiji software was used to perform segmentation in both image modalities: cbct (n=18) and micro-ct (n=18). two segmentation methods were used for each image modality: a visual (n=36) and an automatic based algorithm (n=36), resulting in a total of 72 segmented images. first, the images were converted into 8-bit grayscale, and for the visual threshold method, a simple binary format was attributed (0 for background and 255 for the foreground) (fig. 2a, b, c and d). the visual threshold was applied at the lowest gray value representing dentin tissue, as judged by the operator. the automatic segmentation method was based on the application of a minimum algorithm15, incorporated into the imagej threshold menu, for both tomographic and micro-ct images. for both threshold methods, after binary format conversion, an image subtraction method was applied, in order to obtain the final root canal volume16. figure 1. image stacks visualized using imagej/fiji software. cone beam computed tomography (a); micro-computed tomography (b). a b 4 machado et al. the segmented root canals were then individually visualized, and its volume was obtained (fig. 3a and b). the precision of the root canal volume acquired by the cbct images and the degree of agreement between the tested segmentation methods were compared to the automatic segmentation method of the micro-ct images, which was considered as the gold standard for root canal volume evaluation. statistical analysis statistical analysis was performed using the spss® software (spss statistics for windows, version 13.0. chicago, usa). the variables were expressed by means, standard deviation, medians, interquartile range, minimum and maximum values. paired t-test was used to verify differences between root canal volumes obtained by each tested segmentation method and modality with the gold standard (automatic micro-ct threshold). pearson’s correlation coefficients were also obtained, to verify the degree of correlation between the tested variables and the gold standard. a correlation is considered strong whenever the high values of a given variable were related to the high values of another variable, but as this does not imply that variables are in agreement17, these were also calculated among the groups. results for micro-ct, automatic and visual segmentation methods resulted in similar mean root canal volumes (7621.8 and 7741.8 voxels, respectively; t= -1.621; df = 17; p=0.123). for cbct, automatic segmentation resulted in the lowest root canal volume (4144 voxels), while the visual method resulted in the largest volume (11572 voxels). differences between the gold standard and cbct automatic segmentation were positive (t=4.135; df=17; p≤0.001), showing that cbct with automatic segmentation resulted in underestimation of root canal volume. differences between the gold standard and cbct visual method, were negative (t=-3.950; df=17; p ≤0.001), showing that this method overestimated root canal volume. table 1 shows distribution of root canal volumes among the groups. figure 2. image segmentation process before and after a binarization. non-binarized micro-computed tomography image (a); binarized micro-computed tomography image (b); non-binarized cone beam computed tomography image (c); binarized cone beam computed tomography image (d). a b c d figure 3. root canal volume after segmentation. cone beam computed tomography (a); micro-computed tomography (b). a b 5 machado et al. pearson correlations and agreement between the volumes obtained for the tested acquisitions and thresholds compared to the gold standard are described in figure 4. although correlation coefficients were statistically significant and positive for all comparisons (figure 4 a-c), no agreement has been found between the gold standard and cbct segmentation methods (figure 4e and f). discussion optimal knowledge of the internal anatomy of the root canal, in addition to an accurate diagnosis and treatment planning, are essential pre-requisites for a successful endodontic treatment, since appropriate root canal cleaning and shaping procedures rely on this information18. in fact, imaging technology are currently being applied to clinical diagnosis of teeth in need of endodontic treatment to gain additional information regarding the root canal anatomy, in an attempt to help clinical decisions19. main drawbacks of tridimensional imaging as cbtc, as applied for the precise evaluation of root canal morphology include patient’s overexposure to radiation20. the need to acquire more detailed images of complex root canal structures has been combined with technological advances and development of imaging techniques, such as digital radiography, cbct and micro-ct4,21. in addition, many resources for image analysis using specific software have been nowadays applied to tomographic images9. in the present study, the accuracy of root canal segmentation obtained from tomographic images was compared to those obtained by micro-ct images, using an automatic micro-ct segmentation method as a gold standard. results showed no statistical differences when the volumes obtained by the “visual micro-ct” and the gold standard were compared. thus, for micro-ct images, both segmentation methods are reliable to calculate root canal volume, corroborating a previous study11. such findings may be attributed to the high resolution and low noise produced by micro-ct, table 1. descriptive data of root canal volume obtained for the tested groups. mean, median, minimum and maximum root canal volumes (in voxels) for all threshold and acquisition methods are shown. threshold mean (dp) median [q1 ; q3] minimum ; maximum micro-ct automatic (gold standard) 7621.8 (6585.1)a 5788 [2560 ; 10168] 872 ; 25072 micro-ct visual 7741.8 (6630.3)a 6196 [2384 ; 9848] 744 ; 25200 cbct automatic 4144 (3703.3)b 4040 [832 ; 5096] 208 ; 12944 cbct visual 11572 (7189.4)c 11936 [5680 ; 15488] 1504 ; 26520 difference (micro-ct automatic, micro–ct visual) -120 (314.1) -128 [-216 ; 120] -912 ; 320 difference (micro-ct automatic, cbct automatic) 3477.8 (3568.1) 2112 [1208 ; 4.680] 120 ; 14872 difference (micro-ct automatic, cbct visual) -3950.2 (2905.0) -3380 [-5512 ; -1472] -9624 ; 152 * different lowercase superscript letters indicate statistically significant differences. paired t-test, p<0.05 † micro-ct: micro-computed tomography; ‡ cbct: cone beam computed tomography. 6 machado et al. what makes identification of dentin borders accurate. in fact, it has been shown that micro-ct has a unique potential of showing detailed root canal morphological features in an accurate manner, without destruction of the tooth, while offering reproducible data in three-dimension10,12,18. figure 4. correlations and agreement between the root canal volumes for the tested acquisitions and thresholds and the gold standard. micro-ct automatic volume m ic ro -c t v is ua l v ol um e 30 00 0 25 00 0 20 00 0 15 00 0 10 00 0 50 00 50 00 0 10 00 0 15 00 0 20 00 0 25 00 0 30 00 0 0 micro-ct automatic volume m ic ro -c t v is ua l v ol um e a ut om at ic m ic ro -c t x v is ua l m ic ro -c t (r =0 ,9 99 ; p <0 ,0 01 ) micro-ct automatic volume m ea n (m ic ro -c t a ut o v ol ., m ic ro -c t v is ua l v ol .) 16 00 0 12 00 0 -1 20 00 80 00 -8 00 0 40 00 -4 00 00 50 00 0 10 00 0 15 00 0 20 00 0 25 00 0 30 00 0 -1 60 00 a gr ee m en t b et w ee n a ut om at ic m ic ro -c t x v is ua l m ic ro -c t (d if= -1 20 ; t =1, 62 1; g l= 17 ; p =0 ,1 23 ) c u l= 49 6; b ia s= -1 20 ; c ll =73 6 micro-ct automatic volume m ea n (m ic ro -c t a ut o v ol ., c b c t a ut o v ol .) 16 00 0 12 00 0 -1 20 00 80 00 -8 00 0 40 00 -4 00 00 50 00 0 10 00 0 15 00 0 20 00 0 25 00 0 30 00 0 -1 60 00 a gr ee m en t b et w ee n a ut om at ic m ic ro -c t x a ut om at ic c b c t (d if= 34 77 ; t =4 ,1 35 ; g l= 17 ; p =0 ,0 01 ) c u l= 10 47 1 c ll =35 16 b ia s= 34 78 micro-ct automatic volume m ea n (m ic ro -c t a ut o v ol ., c b c t a ut o v ol .) 16 00 0 12 00 0 -1 20 00 80 00 -8 00 0 40 00 -4 00 00 50 00 0 10 00 0 15 00 0 20 00 0 25 00 0 30 00 0 -1 60 00 a gr ee m en t b et w ee n a ut om at ic m ic ro -c t x v is ua l c b c t (d if= 39 50 ; t =5 ,7 69 ; g l= 17 ; p <0 ,0 01 ) c u l= 17 44 c ll =96 44 b ia s= -3 95 0 a ut om at ic m ic ro -c t x a ut om at ic c b c t (r =0 ,9 14 ; p <0 ,0 01 ) 30 00 0 25 00 0 20 00 0 15 00 0 10 00 0 50 00 50 00 0 10 00 0 15 00 0 20 00 0 25 00 0 30 00 0 0 micro-ct automatic volume m ic ro -c t v is ua l v ol um e a ut om at ic m ic ro -c t x a ut om at ic c b c t (r =0 ,9 14 ; p <0 ,0 01 ) 30 00 0 25 00 0 20 00 0 15 00 0 10 00 0 50 00 50 00 0 10 00 0 15 00 0 20 00 0 25 00 0 30 00 0 0 7 machado et al. regarding the segmentation performed on cbct images, the results showed that, when compared to the gold standard, both the “automatic cbct” and “visual cbct” were statistically different compared to the gold standard, revealing a limitation of the accurate determination of root canal volume using this image modality (with acquisition parameters used in this study). despite the accuracy of cbct in allowing a three-dimensional and detailed view of bone3,22, in this study, it did not allow a precise determination of root canal volume. this may be probably explained by the specific acquisition parameters and resolution of the cbct used. it is known that results of image segmentation in cbcts depend on the acquisition configuration, because they have a direct influence on the reconstructed image quality20. an increase in milliamperage leads to an increase in the signal-noise ratio but also increase the radiation dose. an increase in kilovoltage increases the mean photon energy and reduces the grayscale resolution. the present study used 85 kv and 4.5 ma as acquisition parameters in cbct, in other words, a low milliamperage, when compared to the gold standard (114 µa). in addition, the higher kilovoltage used in cbct compared to the micro-ct acquisition (70kv) may have led to the decrease of the image contrast. another study, using high spatial resolution cone beam tomography (76μm) showed very strong correlations between root canal areas obtained from selected slices in cbct and histologic sections23 or root canal volume obtained by micro-ct data24. in both cases, the automatic segmentation implemented resulted in cbct data which was slightly smaller than the gold standard (underestimation), corroborating results of the present study. on the other hand, the whole volume tends to be selected in the visual segmentation, rather than being restricted to the root canal area, due to the difficulty in perceiving the different attenuation coefficients of the dentin structure, explaining overestimation of cbct after visual threshold compared to the gold standard. in the present study, the correlation among the analyzed variables were high (r=0.99; r=0.914 and r=0.922), demonstrating that the grayscale values increased or decreased in a correlated manner, regardless of the method. however, there was only agreement when the automatic and visual micro-ct methods were compared (figure 4d), corroborating the other comparisons shown in the present study (table 1). unfortunately, micro-ct analysis is not a viable alternative for clinical practice. instead, cbct, the most common technique used for this, presents resolution limitations depending on the available system. the acquisition parameters used to obtain the tomographic images may significantly interfere with the results, especially the spatial resolution. therefore, the difference between the segmented volumes of the root canals obtained in both cbct methods, when compared to the gold standard, can be attributed to the high noise level and the used voxel size in the cbcts. although the segmentation methods were efficient, they depended directly on the acquisition parameters and the fact that the used voxel was rather large may have had a significant influence on the results. the visual and automatic segmentation methods performed on cbct images overestimated and underestimated, respectively, the volume of the root canals. they were therefore considered inconsistent with root canal volumes considered as gold standards. however, cbct is certainly an additional resource for treatments in dentistry, and is recognized as an accurate method for analysis of root canals25,26, however, volumetric 8 machado et al. analysis of the data obtained from cbct image stacks should be interpreted taking into account the acquisition parameters, including spatial resolution, especially for endodontic applications. new studies are needed to improve root canal segmentation methods by testing different tomographic scanners with varying acquisition parameters. in conclusion, volumetric analysis of root canals in single or multiradicular teeth obtained with cbct should not be used as absolute values, since no agreement with gold standard values were obtained. further studies are needed to elucidate optimized acquisition parameters of cbct scanners to ensure the best endodontic segmentation image processing protocol that can be applied in clinical situations. acknowledgments we would like to thank brazilian research funding agencies (faperj and capes), the school of dentistry of the universidade federal do rio de janeiro, and university hospital clementino fraga filho (hucff) for their support. references 1. nasseh i, al-rawi w. cone beam computed tomography. dent clin north am. 2018 jul;62(3):361-391. doi: 10.1016/j.cden.2018.03.002. 2. scarfe wc, li z, aboelmaaty w, scott sa, farman ag. maxillofacial cone beam computed tomography: essence, elements and steps to interpretation. aust dent j. 2012 mar;57 suppl 1:46-60. doi: 10.1111/j.1834-7819.2011.01657.x. 3. scarfe wc, levin md, gane d, farman ag. use of cone beam computed tomography in endodontics. int j dent. 2009;2009:634567. doi: 10.1155/2009/634567. 4. patel s, durack c, abella f, shemesh h, roig m, lemberg k. cone beam computed tomography in endodontics a review. int endod j. 2015 jan;48(1):3-15. doi: 10.1111/iej.12270. 5. iikubo m, nishioka t, okura s, kobayashi k, sano t, katsumata a et al. influence of voxel size and scan field of view on fracture-like artifacts from gutta-percha obturated endodontically treated teeth on cone-beam computed tomography images. oral surg oral med oral pathol oral radiol. 2016 nov;122(5):631-7. doi: 10.1016/j.oooo.2016.07.014. 6. nascimento ha, andrade me, frazão ma, nascimento eh, ramos-perez fm, freitas dq. dosimetry in cbct with different protocols: emphasis on small fovs including exams for tmj. braz dent j. 2017 jul-aug;28(4):511-6. doi: 10.1590/0103-6440201701525. 7. bechara b, mcmahan ca, moore ws, noujeim m, geha h, teixeira fb. contrast-to-noise ratio difference in small field of view cone beam computed tomography machines. j oral sci. 2012 sep;54(3):227-32. 8. swain mv, xue j. state of the art of micro-ct applications in dental research. int j oral sci. 2009 dec;1(4):177-88. doi: 10.4248/ijos09031. 9. nair mk, nair up. digital and advanced imaging in endodontics: a review. j endod. 2007 jan;33(1):1-6. 10. wolf tg, paque f, woop ac, willershausen b, briseno-marroquin b. root canal morphology and configuration of 123 maxillary second molars by means of micro-ct. int j oral sci. 2017 mar;9(1):33-7. doi: 10.1038/ijos.2016.53. 11. tomaszewska im, skinningsrud b, jarzebska a, pekala jr, tarasiuk j, iwanaga j. internal and external morphology of mandibular molars: an original micro-ct study and meta-analysis with review of implications for endodontic therapy. clin anat. 2018 sep;31(6):797-811. doi: 10.1002/ca.23080. 9 machado et al. 12. queiroz pm, rovaris k, santaella gm, haiter-neto f, freitas dq. comparison of automatic and visual methods used for image segmentation in endodontics: a microct study. j appl oral sci. 2017 nov-dec;25(6):674-9. doi: 10.1590/1678-7757-2017-0023. 13. visconti ma, verner fs, assis nm, devito kl. influence of maxillomandibular positioning in cone beam computed tomography for implant planning. int j oral maxillofac surg. 2013 jul;42(7):880-6. doi: 10.1016/j.ijom.2013.03.001. 14. schindelin j, arganda-carreras i, frise e, kaynig v, longair m, pietzsch t, et al. fiji: an open-source platform for biological-image analysis. nat methods. 2012 jun 28;9(7):676-82. doi: 10.1038/nmeth.2019. 15. prewitt jm, mendelson ml. the analysis of cell images. ann n y acad sci. 1966 jan;128(3):1035-53. 16. neves aa, silva ej, roter jm, belladona fg, alves hd, lopes rt, et al. exploiting the potential of free software to evaluate root canal biomechanical preparation outcomes through micro-ct images. int endod j. 2015 nov;48(11):1033-42. doi: 10.1111/iej.12399. 17. giavarina d. understanding bland altman analysis. bioch med. 2015 jun;25(2):141-51. doi: 10.11613/bm.2015.015. 18. fan b, yang j, gutmann jl, fan m. root canal systems in mandibular first premolars with c-shaped root configurations. part i: microcomputed tomography mapping of the radicular groove and associated root canal cross-sections. j endod. 2008 nov;34(11):1337-41. doi: 10.1016/j.joen.2008.08.006 19. cohenca n, shemesh h. clinical applications of cone beam computed tomography in endodontics: a comprehensive review. quintessence int. 2015 sep;46(8):657-68. doi: 10.3290/j.qi.a34396. 20. michetti j, georgelin-gurgel m, mallet jp, diemer f, boulanouar k. influence of cbct parameters on the output of an automatic edge-detection-based endodontic segmentation. dentomaxillofac radiol. 2015;44(8):20140413. doi: 10.1259/dmfr.20140413. 21. zhang r, wang h, tian yy, yu x, hu t, dummer pm. use of cone-beam computed tomography to evaluate root and canal morphology of mandibular molars in chinese individuals. int endod j. 2011 nov;44(11):990-9. doi: 10.1111/j.1365-2591.2011.01904.x. 22. elsherief sm, zayet mk, hamouda im. cone-beam computed tomography analysis of curved root canals after mechanical preparation with three nickel-titanium rotary instruments. j biomed res. 2013 jul;27(4):326-35. doi: 10.7555/jbr.27.20130008. 23. michetti j, maret d, mallet jp, diemer f. validation of cone beam computed tomography as a tool to explore root canal anatomy. j endod. 2010 jul;36(7):1187-90. doi: 10.1016/j.joen.2010.03.029. 24. michetti j, basarab a, diemer f, kouame d. comparison of an adaptive local thresholding method on cbct and microct endodontic images. phys med biol. 2017 dec 19;63(1):015020. doi: 10.1088/1361-6560/aa90ff. 25. venskutonis t, plotino g, juodzbalys g, mickeviciene l. the importance of cone-beam computed tomography in the management of endodontic problems: a review of the literature. j endod. 2014 dec;40(12):1895-901. doi: 10.1016/j.joen.2014.05.009. 26. de carlo bello m, tiburcio-machado c, dotto londero c, branco barletta f, cunha moreira ch, pagliarin cml. diagnostic efficacy of four methods for locating the second mesiobuccal canal in maxillary molars. iran endod j. 2018 spring;13(2):204-8. doi: 10.22037/iej.v13i2.16564. 1http://dx.doi.org/10.20396/bjos.v20i00.8661656 volume 20 2021 e211656 original article 1 departament of restorative dentistry, piracicaba dental school, university of campinas (unicamp), piracicaba, são paulo, brazil. 2 private clinic. 3 university center uniftc, salvador, bahia, brazil. 4 department of dental clinic, school of dentistry, federal university of bahia, salvador, bahia, brazil. *corresponding author: paula mathias email: pmathias@yahoo.com received: october 15, 2020 accepted: february 22, 2021 editor: dr altair a. del bel cury effect of ceramic thicknesses and opacities on water sorption and solubility of a light-curing resin cement by different units gabriela alves de cerqueira1 , lais sampaio souza2 , rafael soares gomes3 , giselle maria marchi1 , paula mathias4,* aim: this study evaluated the water sorption and solubility of a light-cured resin cement, under four thicknesses and four opacities of a lithium disilicate ceramic, also considering three light-emitting diode (led) units. methods: a total of 288 specimens of a resin cement (allcem veneer trans – fgm) were prepared, 96 samples were light-cured by each of the three light curing units (valo – ultradent / radii-cal – sdi  /  bluephase ii – ivoclar vivadent), divided into 16 experimental conditions, according to the opacities of the ceramic: high opacity (ho), medium opacity (mo), low translucency (lt), high translucency (ht), and thicknesses (0.3, 0.8, 1.5, and 2.0  mm) (n = 6). the specimens were weighed at three different times: mass m1 (after making the specimens), m2 (after 7 days of storage in water), and m3 (after dissection cycle), for calculating water sorption and solubility. results: the higher thickness of the ceramic (2.0  mm) significantly increased the values of water sorption (44.0± 4.0) and solubility (7.8±0.6), compared to lower thicknesses. also, the ceramic of higher opacity (ho) generated the highest values of sorption and solubility when compared to the other opacities, regardless of the thickness tested (anova-3 factors / tukey’s test, α = 0.05). there was no influence of light curing units. conclusion: higher thicknesses and opacities of the ceramic increased the water sorption and solubility of the tested light-cured resin cement. keywords: cementation. ceramics. light-curing of dental adhesives. resin cements. mailto:pmathias@yahoo.com https://orcid.org/0000-0002-4581-6797 https://orcid.org/0000-0001-7083-413x https://orcid.org/0000-0002-7989-0098 https://orcid.org/0000-0002-0945-1305 https://orcid.org/0000-0002-2589-3760 2 de cerqueira et al. introduction in recent years, minimal intervention dentistry has presented, as an aesthetic restorative alternative, the use of ceramic veneers, with reduced dental preparation1,2. among the ceramic options, lithium disilicate has been widely used because it allows adhesive cementation procedures3 and is highly aesthetic, reproducing optical effects similar to those of natural teeth and enabling efficient masking of teeth with chromatic changes4,5. ceramic laminates are fixed to the teeth using adhesive systems and resin cement, which is capable of generating a strong bond between the dental substrate and the laminate ceramic1,5. resin cement can be classified according to its polymerization: self-curing, light-curing, and dual5,6. for ceramic laminate veneers, light-cured cements are the most suitable, since self-curing and dual cements present, in their composition, tertiary amines, which, when reacted with benzoyl peroxide, become responsible for the yellowing of the material6. considering that ceramic laminates have a reduced thickness in many cases, this yellowing could compromise the aesthetic result of the treatment, especially in long term1,7. the ceramic material used in the restorative technique can present different thicknesses and opacities, which are associated with the depth of preparation and the darkness of the tooth2. the different thicknesses and opacities of the ceramics directly affect the amount of light available to light-cure the resin cement underlying the ceramic material4,6. this is because the light radiated by the light-curing unit must go through the ceramic and reach the cement, in a uniform, efficient, and satisfactory manner, to achieve a high degree of conversion of the resin monomers of the cement, ensuring its mechanical properties5,6. more translucent laminates, as well as less thick ones, tend to facilitate the passage of light through the restoration, allowing a greater amount of light to reach the resin cement below the restoration5,8. however, in some clinical situations, it is necessary to mask darkened teeth, using a more opaque and thicker ceramic restorations1,8,9. there are no conclusive studies in the literature associating different opacities of ceramics with different thicknesses to determine which situations could ensure the best passage of light through the ceramic restoration; or about which of the two variables – thickness or opacity – is able to compromise this passage of light more significantly1,5,6,10. in addition to the optical characteristics and thickness of the ceramic, the parameters of the light must also be considered11. a high intensity of power, along with a homogeneous and collimated light beam, are required so that the light is able to pass through the ceramic, without losing so much power and without dissipating too much into the material12. the delivery of this light energy to resin cement is also strongly influenced by the time of application of the light2,10. in addition, the light must have wavelengths in the absorbance range of the photoinitiators contained in the resinous material, such as camphorquinone, considered the main photoinitiator, which shows its absorption peak around 468-470 nm2,6,10. thus, the importance of light in the light-curing process of resin cements makes the analysis of the light-curing parameters essential for the safety of clinical procedures. 3 de cerqueira et al. when the light curing of resin cement is compromised, some negative effects can occur, such as a low degree of conversion and high water sorption and solubility of the material, which will generate poor mechanical properties, infiltration and detachment of the restoration5,6,11,13. in the oral environment, fluid sorption can occur, resulting in the swelling of the resinous material at the tooth interface/restoration, causing changes in its organic matrix and impairment in the structure of this material, because of the dissociation of the inorganic charge. this decreases the resistance and the solubility of the material, reducing its mass by leaching its components and, consequently, releasing unreacted monomers, which can even cause damage to the dental pulp1,13-16. thus, it is important to understand the influence of dental ceramic thickness and opacity both alone and in association on water sorption and solubility of resin cement, using different light-curing units. the hypothesis of this study was that the greater the thickness and opacity of the ceramic interposed, the higher the water sorption and solubility values of the resin cement, regardless of the light-curing unit used. therefore, this study aimed to evaluate the physical properties (water sorption and solubility) of a light-cured resin cement under four thicknesses and four opacities of a lithium disilicate-based ceramic, considering three different light-emitting diode (led) units. materials and methods preparation of specimens a total of 288 specimens of allcem veneer trans resin cement (fgm, dentscare ltda, joinville-sc, brazil) were prepared (table 1), being light-cured under four different thicknesses and opacities of a ceramic based on lithium disilicate (ips e.max press – ivoclar-vivadent), also considering three different light emitting diode (led) units. for each of the three light-curing units (valo – ultradent, radii-cal – sdi, and bluephase ii – ivoclar vivadent) tested, 96 specimens were obtained, divided into 16 experimental conditions: four opacities (ht – high translucency, lt – low translucency, mo – medium opacity, and ho – high opacity) and four thicknesses (0.3 / 0.8 / 1.5 / 2 mm) (n = 6). g1-ht/0.3mm; g2-ht/0.8mm; g3-ht/1.5 mm; g4-ht/2.0mm; g5-lt/0.3mm; g6-lt/0.8mm; g7-lt/1.5mm; g8-lt/2.0mm; g9-mo/0.3mm; g10mo/0.8mm; g11-mo/1.5mm; g12-mo/2.0mm; g13-ho/0.3  mm; g14-ho/0.8  mm; g15-ho/1,5 mm; g16-ho/2.0mm table 1. the composition of the light-curing resin cement allcem venner trans (fgm, dentscare ltda, joinville-sc, brazil) resin cement composition allcem venner trans light-curing methacrylate monomers (udma, bis-ema, bis-gma, tegdma) photoinitiators (camphorquinone, peak absorption between 400-500 nm) coinitiators stabilizers pigments silanized barium-aluminum-silicate glass particles and silicon dioxide 4 de cerqueira et al. all specimens were made using a split stainless steel matrix with 3-mm diameter and 1-mm thickness. on this metallic matrix, filled with a single increment of the resin cement, a polyester strip was placed, followed by a glass coverslip and a weight of 500 mg, left for 30 seconds to drain the excess material. then, the weight and the glass coverslip were removed and the specimen was light-cured for 40 s, through the polyester strip, for the control group. for the other groups, the resin cement was light-cured under a piece of ceramic material that varied its respective opacity and thickness, according to each experimental condition. this sequence was applied, in an identical manner, to the three light-curing units (valo, radii-cal and bluephase ii) (table 2). table 2. description of light-curing units (lcu) used in this study, considering their respective light parameters (power intensity and wavelength) operation conditions led devices valo – ultradent radii-cal – sdi bluephase ii – ivoclar vivadent light intensity 1400 mw/cm2 1200 mw/cm2 1200 mw/cm2 wavelength range 385-515 nm 440-480 nm 385-515 nm evaluation of water sorption and solubility after preparation, the specimens were measured using a digital caliper with 0.01 mm precision (mitutoyo, suzano – sp, brazil). means of diameter duplicates were calculated for each specimen, to determine the radius (r), height (h), and the individual volume. following the iso 4049 specifications17, the specimens were placed in a desiccator and transferred to an incubator at 37oc, for preconditioning. after 24hours, the specimens were weighed repeatedly, in an interval of 24  hours, until a constant mass (m1) was reached, on an analytical scale (shimadzu, mod. auw220d, barueri – sp, brazil), with an accuracy of 0.0001 of 1g. this stabilization was verified when the variation of the values of m1 was lower than 0.2mg, in a period of 24h, for each specimen. the specimens were individually identified and stored in an incubator at 37ºc (quimis, diadema – sp, brazil), being kept in a container with silica gel, for desiccation, for seven consecutive days. after stabilization of m1, the specimens were immersed in 2  ml of distilled water (ph 7.2) and again stored at 37 ºc, where they remained for seven consecutive days. afterwards, the specimens were removed from the distilled water, dried with absorbent paper (sorella, canoinhas-sc, brazil), and weighed again on an analytical scale to obtain the mass (m2). after m2 registration, the specimens were stored individually in the incubator at 37ºc, for desiccation. all specimens were weighed, repeatedly, at 24h intervals, until a constant mass (m3) was reached, considering a variation lower than 0.2mg for each specimen. after acquiring all the mass values of the specimens, water sorption (so) and solubility (sol) were calculated using the following formulas: 5 de cerqueira et al. so = m2 – m3/v (1) sol = m1 – m3/v (2) where m1 is the constant mass, in μg, found before immersion in water; m2 is the mass, in μg, after immersion in water for 7 days; m3 is the constant mass, in μg, after desiccation; and v is the volume of the specimens in mm3. statistical analysis the collected data were tabulated and evaluated for their homogeneity and normality, with levene and shapiro-wilk tests being applied, respectively, with a 5% significance level, for each of the variables (water sorption and solubility). considering the assumptions for the application of the parametric tests, an analysis of variance with 3 factors was applied: 1. light-curing unit in 3 levels (valo, radii-cal, and bluephase); 2. ceramic thickness in 4 levels (0.3, 0.8, 1.5, and 2.0 mm); and 3. opacity in 4 levels (ht – high translucency, lt – low translucency, mo – medium opacity, and ho – high opacity). tukey’s test was used as post hoc. results the statistical analysis showed that the thickness and the degree of translucency of the ceramic interfered in the water sorption and solubility values of the light-cured allcem resin cement, regardless of the light-curing unit used. as a result of water sorption (table 3), it was found that the greater thickness of the lithium disilicate ceramic (2.0 mm) increased the water sorption values when statistically compared to the thicknesses of 0.3 mm and 0.8 mm. similarly, medium and high opacity ceramics (mo and ho) contributed to higher values of water sorption compared to high translucency (ht) ceramics. the higher opacity lithium disilicate (ho) ceramics resulted in higher water sorption values of resin cement than the values showed by high and low translucency ceramics (ht and lt). the less thick ceramic (0.3 mm) with greater opacity (ho) showed increased water sorption values, regardless of the light-curing unit tested. the same behavior was found for high translucency (ht), thick (2.0 mm) ceramic. the type of the light-curing unit tested did not influence the water sorption values of the resin cement, with no statistically significant difference between them. table 4 shows the results obtained by analyzing the solubility data of the resin cement. it was found that, as with sorption, the greater thickness of the lithium disilicate ceramic (2 mm) increased the solubility values, compared to the values obtained with the thickness of 0.3 mm, regardless of the light-curing unit tested. likewise, the difference for the other thicknesses was statistically significant with the increase in the opacity of the ceramic. the more translucent the ceramics (ht and lt), the lower the solubility values of the resin cement compared to the values obtained with the higher opacity ceramic (ho), for the three light-curing units tested. similarly to water sorption, the type of light-curing unit, by itself, did not affect the solubility values, with no statistically significant difference between them. 6 de cerqueira et al. ta bl e 3. w at er s or pt io n va lu es o f th e lig ht -c ur ed r es in c em en t co ns id er in g fo ur d iff er en t th ic kn es se s (0 .3 / 0 .8 / 1 .5 / 2  m m ) an d op ac iti es ( h t – h ig h tr an sl uc en cy , l t – lo w t ra ns lu ce nc y, m o – m ed iu m o pa ci ty , a nd h o – h ig h o pa ci ty ) of t he c er am ic li th iu m d is ili ca te ( ip s e. m ax p re ss – iv oc la rv iv ad en t) a nd t hr ee li gh tcu rin g un its ( v al o; r ad iica l a nd b lu ep ha se ). thickness v a lo li g h tc u r in g u n it s r a d iic a l b lu ep h a s e c er a m ic o pa c it ie s h t lt m o h o h t lt m o h o h t lt m o h o 0. 3 †2 0. 6± 2. 6b c †2 2. 5± 2. 6b bc †2 5. 1± 3. 2c b †2 9. 0± 2. 5b a †2 1. 0± 1. 9b c †2 2. 9± 1. 9b bc †2 5. 0± 3. 1b b †2 9. 9± 2. 3b a †2 0. 3± 2. 5b c †2 2. 6± 2. 1b bc †2 5. 1± 3. 2b b †2 9. 7± 2. 9b a 0. 8 †2 1. 1± 2. 1b b †2 2. 3± 3. 0b b †2 6. 0± 3. 8b c a †2 9. 3± 3. 3b a †2 1. 5± 2. 6b c †2 2. 4± 2. 3b bc †2 5. 9± 2. 2b b †3 1. 0± 2. 5b a †2 0. 9± 2. 4b b †2 3. 0± 2. 5b b †2 7. 6± 3. 5b a †2 9. 0± 3. 4b a 1. 5 †2 2. 1± 1. 9b b †2 4. 0± 4. 5b b †2 8. 7± 3. 9b a †3 1. 5± 2. 2b a †2 2. 6± 3. 1b b †2 4. 6± 3. 6b b †2 9. 9± 3. 0a a †3 1. 8± 3. 1b a †2 2. 7± 2. 6b b †2 4. 6± 3. 3b b †2 9. 2± 4. 6b a †3 1. 8± 3. 1b a 2. 0 †2 7. 1± 3. 1a c †3 0. 3± 3. 9a bc †3 3. 6± 3. 1a b †4 1. 1± 3. 9a a †2 9. 0± 3. 2a c †3 1. 0± 2. 4a bc †3 2. 7± 2. 9a b †4 4. 0± 4. 0a a †2 6. 6± 3. 2a c †3 0. 6± 3. 6a b †3 3. 5± 3. 1a b †4 3. 1± 4. 2a a d iff er en t c ap ita l l et te rs in di ca te d iff er en ce b et w ee n th ic kn es se s in th e sa m e op ac ity a nd li gh t c ur in g (c ol um n) . d iff er en t l ow er ca se le tt er s in di ca te d iff er en ce b et w ee n op ac iti es in th e sa m e th ic kn es s an d lig ht c ur in g (r ow ). d iff er en t s ym bo ls († , * , ‡ ) i nd ic at e a di ff er en ce a m on g lig ht -c ur in g un its fo r t he s am e th ic kn es s an d op ac ity (a n o v a -3 fa ct or s / tu ke y’ s te st ; α = 0. 05 ). ta bl e 4 s ol ub ili ty v al ue s of t he li gh tcu re d re si n ce m en t co ns id er in g fo ur d if fe re nt t hi ck ne ss es ( 0. 3 / 0. 8 / 1. 5 / 2  m m ) an d op ac iti es ( h t – h ig h tr an sl uc en cy , l t – l ow tr an sl uc en cy , m o – m ed iu m o pa ci ty , a nd h o – h ig h o pa ci ty ) of t he c er am ic l ith iu m d is ili ca te ( ip s e. m ax p re ss – i vo cl ar -v iv ad en t) a nd t hr ee l ig ht -c ur in g un its ( v al o; r ad iica l a nd b lu ep ha se ). thickness v a lo li g h tc u r in g u n it s r a d iic a l b lu ep h a s e c er a m ic o pa c it ie s h t lt m o h o h t lt m o h o h t lt m o h o 0. 3 †3 .1 ±0 .4 b b †3 .3 ±0 .4 c b †4 .0 ±0 .4 b a †4 .1 ±0 .4 c a †3 .2 ±0 .5 b c †3 .3 ±0 .5 b bc †3 .9 ±0 .6 b ab †4 .2 ±0 .6 c a †3 .1 ±0 .4 b c †3 .3 ±0 .5 c bc †3 .9 ±0 .4 c ab †4 .4 ±0 .8 c a 0. 8 †3 .4 ±0 .4 a b c †3 .5 ±0 .3 b c bc †4 .0 ±0 .4 b ab †4 .3 ±0 .4 c a †3 .4 ±0 .5 b c †3 .6 ±0 .5 b bc †4 .1 ±0 .6 b ab †4 .5 ±0 .7 c a †3 .5 ±0 .3 a b b †3 .6 ±0 .6 b c b †4 .2 ±0 .5 b c a †4 .5 ±0 .6 c a 1. 5 †3 .4 ±0 .4 a b c †3 .9 ±0 .6 b bc †4 .4 ±0 .6 b b †5 .1 ±0 .4 b a †3 .6 ±0 .5 b c †3 .9 ±0 .6 b c †4 .5 ±0 .5 b b †5 .2 ±0 .6 b a †3 .5 ±0 .5 a b c †4 .0 ±0 .7 b bc †4 .5 ±0 .6 b b †5 .2 ±0 .6 b a 2. 0 †3 .9 ±0 .5 a d †5 .3 ±0 .5 a c †6 .1 ±0 .6 a b †7 .5 ±0 .5 a a †4 .4 ±0 .3 a d †5 .2 ±0 .5 a c †6 .0 ±0 .7 a b †7 .8 ±0 .6 a a †3 .8 ±0 .4 a d †5 .3 ±0 .7 a c †6 .0 ±0 .9 a b †7 .7 ±0 .4 a a d iff er en t c ap ita l l et te rs in di ca te d iff er en ce b et w ee n th ic kn es se s in th e sa m e op ac ity a nd li gh t c ur in g (c ol um n) . d iff er en t l ow er ca se le tt er s in di ca te d iff er en ce b et w ee n op ac iti es in th e sa m e th ic kn es s an d lig ht c ur in g (r ow ). d iff er en t s ym bo ls († , * , ‡ ) i nd ic at e a di ff er en ce a m on g lig ht -c ur in g un its fo r t he s am e th ic kn es s an d op ac ity (a n o v a -3 fa ct or s / tu ke y’ s te st ; α = 0. 05 ). 7 de cerqueira et al. discussion the mechanical properties of resin cement can be affected by the water sorption and solubility that occur in an aqueous environment, such as in the oral environment5. studies show that increasing the thickness and opacity of the ceramic restoration reduces the passage of light during the photoactivation of cement, compromising the performance of the material1,8,10,18. in this study, the experimental hypothesis was partially accepted, since the ceramics of greater thickness and opacity interposed during light-curing procedures increased the values of water sorption and solubility of the resin cement. however, this increase was not gradual as the opacity and/or thickness of the ceramic increased, with similar behaviors being observed among ceramics of different thicknesses. the tested light-curing units were not different. in our study, thicknesses of 0.3 and 0.8 mm resulted in similar values to each other and significantly lower for water sorption and solubility, compared to the thickness of 2  mm, for all three light curing units tested. these results corroborate the data reported by runnacles et al.8 (2014), who has shown that the effect of light attenuation by ceramic veneers is not significant in thicknesses up to 1.0 mm. previous studies have also shown that the thinner the ceramic material interposed between the resin cement and the light source, the greater the degree of conversion of the resin material18,19. according to calgaro  et  al.20 (2013), the increase in thickness is a key factor in attenuating the light emitted by the light-curing unit, since they observed that the polymerization decreases as the thickness increases. in this study, increasing the thickness of the ceramic to 2.0  mm also significantly increased the water sorption and the solubility of the resin cement under it. similar  observations were reported in a previous study, where the resin microhardness and roughness was reduced when the thickness of the ceramic increased from 1  mm to 2  mm21. the explanations for this change in properties of the light-cured resin cement under thicker ceramics may be related to the reduction of the light transmitted by the light-curing unit when crossing a 2 mm thick ceramic22. according to liebermann et al.23 (2018), light transmittance is inversely related to the thickness of the ceramic, which will be crossed by the light beam. that is, the thicker the material, the lower the transmittance of that light22,23. transmittance can be defined as the amount of light that passes through a material, part of which is reflected or absorbed23. if a small part of this light is scattered and most of it is transmitted through the material, higher transmittance values will be achieved22,23. in a ceramic material, the light ends up being too dispersed and diffusely reflected, generating an opaque appearance22,23. it should also be noted that more translucent materials show changes in light transmittance due to the variation in thickness, thus, even translucent materials, when thicker, reduce light transmittance22,23. this may explain the fact that the interposition of a translucent ceramic (ht), with a thickness of 2.0 mm, also results in an increase in the values of water sorption and solubility of the light-cured cement under it in this study. as well as the thickness, the degree of translucency of the ceramic laminate has a strong influence on the polymerization of the resin cement under the ceramic lami8 de cerqueira et al. nat1,10. in this study, when the ht and lt ceramics were used with the same thickness, the resin cement showed the lowest values of water sorption and solubility, for the three light units tested. this influence of the degree of translucency of the ceramic can be explained by the microstructure of the ceramic material, especially its crystalline phase, which tends to present differences in the transmittance and dispersion of light, affecting light transmission and, consequently, the light irradiance that reaches the resin cement underlying the laminate4. leal  et  al.1 (2016) observed that ceramics with lower translucency (more opaque) limit the passage of light emitted by the light-curing units. likewise, calgaro  et  al.20 (2013), when testing different types of ceramics, observed that, among the ht, lt, and mo ceramics, the best performance in the degree of conversion of the underlying resin cement was achieved by ht ceramics. the results found in our study corroborate these observations, since the resin cement under ceramics with a low level of translucency (mo and ho) showed greater water sorption and solubility. the less translucent ceramics are often used to mask teeth with severe stains or with significant color differences between substrate and final color of the restoration1,10. when correlating the thickness and translucency variables of the ceramics tested in this study, it can be seen that, regarding thickness for ceramics up to 1.5  mm, in ht and lt opacities, no significant differences were observed for water sorption and resin cement solubility. the increase in the thickness of the ceramic piece to 2.0  mm resulted in significant increases in sorption and solubility for all degrees of translucency tested, confirming the care with measuring ceramic thickness during adhesive cementation procedures with light-curing resin cements. de jesus  et  al.4 (2020) also showed that ceramics with thickness higher than 1.5  mm reduce the values of degree of conversion and microhardness of the resinous material below to ceramic restoration. the association of 2.0 mm thickness with higher opacities (mo and ho) increased the values of water sorption and solubility even more, probably due to the greater impairment of light transmission through the respective ceramic pieces. concerning the opacity variable, for the ho ceramic, all thicknesses resulted in higher values of water sorption and solubility, except the minimum thickness of 0.3 mm. ho ceramics with thicknesses of 1.5 and 2.0 mm make the tested diffusion dynamics values even more critical. higher values of water sorption and solubility of resin cements under thicker and more opaque ceramic laminates probably come from a lower degree of conversion and less microhardness of these cements, after polymerization4,9,22,23. with the increase in the thickness and opacity of the ceramic, there is a decrease in the passage of light to reach the resin cement, reducing its degree of conversion and its microhardness4,9. according to the american dental association specification, the sorption of resinous materials must be less than 40 lg/mm3 and the water solubility must be less than 7.5 lg/mm3, for a storage period of seven days24. in this study, when the resin cement was light-cured under the 2-mm thick ceramic piece with high opacity (ho), it exceeded this acceptable water sorption limit for the three light-curing units tested: valo (41.1  lg/mm3), radical (44.0 lg/mm3), and bluephase (43.1 lg/mm3). the same occurred for the solubility, with values above the limit for all led units: radical (7.8 lg/mm3), bluephase (7.7 lg/mm3), and valo (7.5 lg/mm3). therefore, the association of high 9 de cerqueira et al. opacity with a 2.0mm ceramic tile thickness demonstrates a detrimental effect on the physical properties of diffusion dynamics of resin cement, and should be considered in clinical cementation procedures. in their study, leal et al.1 (2016) identified mean values higher than the acceptable limit both for water sorption and solubility of light-cured resin cement, when ceramic pieces of medium and low translucency were positioned on the resin cement..on the other hand, when highly translucent surfaces are used, values similar to the control conditions were found, indicating no significant influence to light transmission, which allowed an adequate conversion of resin monomers25,26. when a light-curing material does not receive the appropriate amount of light energy, an impaired formation of free radicals that initiate polymerization and a lower degree of conversion of the polymer network are observed27. the resin cement used in this study has low viscosity, and, in its composition, high and low molecular weight monomers (table 1). it is known that the composition of the organic matrix of resin cements can influence water sorption and solubility5. lower  molecular weight monomers, such as tegdma (triethylene glycol dimethacrylate) or udma (urethane dimethacrylate), are mixed with higher molecular weight monomers, such as bis-gma (bisphenol a-diglycidyl dimethacrylate), to promote less viscosity, changing the material handling properties and collaborating with the technical cementation procedures5,13. however, monomers of lower molecular weight also have a more hydrophilic nature, which may allow greater diffusion dynamics of resin cements28-30. the hydrophilicity of these monomers can generate undesirable clinical consequences, such as microleakage, susceptibility to degradation, discoloration and decreased mechanical properties, postoperative sensitivity, and recurrent caries1,8,31. it is believed that water gain is related to the composition of the material, the content, concentration and type of inorganic fillers, as well as the size and nature of the particles1. therefore, the water sorption and solubility values of the resin cement used in this study may also have been influenced by the hydrophilicity characteristics of the monomers incorporated in its matrix. in our study, the behavior of this cement was not compared to that of other cements, so that a more accurate assessment of the influence of this composition could be considered. therefore, in future research, comparative investigation between different cements is recommended. in this study, three high power light-curing units were tested, with no significant difference between them. light intensity, irradiance, and light application time are factors that may be associated with the conversion of resin monomers from resinous material18. for some authors, variations in the power of the light emitting device used can directly affect the mechanical properties of the material, showing the need to work at maximum intensity1. it should be noted, however, that the use of high energy densities, per se, is not directly related to higher degrees of conversion32. other factors should be considered, such as the use of a light-curing unit with an appropriate wavelength, to sensitize photoinitiators to the resin material used33. in view of the need for the emergence of clearer resin materials for cosmetic procedures in dentistry, alternative photoinitiators were inserted in their composition, changing the peak wavelength absorption used for camphorquinone34. therefore, some led units were created with polywave technology, which allows them to emit a broader wavelength spectrum, photosensitizing all the photoinitiators present in the composition of the 10 de cerqueira et al. material, unlike monowave led units, which normally only reach camphorquinone, since they have a more limited spectrum34,35. in this study, two of the light-curing units tested are considered polywave (bluephase and valo). however, the absence of difference between them can be justified by the composition of the resin cement, which has a photoinitiator compatible with the wavelength spectrum emitted for all three light-curing units. this highlights the need for including different resinous materials, with different compositions and photoinitiators, in future studies. in addition, the presence of a collimated light beam seems to have a considerable influence on the light-curing of resinous materials, especially when a barrier is placed between the light source and the resinous material, as in the case of ceramic restorations12,33. the linear orientation of the light beams prevents this light from dispersing, which favors the delivery of energy density to the restorative material12,33. considering  the exposure time factor, archegas  et  al.25 (2012) observed that the polymerization of resin cements for 40s through an opaque ceramic resulted in a lower degree of conversion than through a translucent ceramic. also, a time of 120s resulted in similar degree of conversion values for opaque and more translucent  ceramics25. uctasli et al.10 (1994) found that the use of a thicker and more opaque ceramic requires greater exposure to irradiated light, but even so, there is a limit of thickness and opacity so that the appropriate polymerization is obtained at the best irradiation conditions. the authors emphasized that the photoactivation time should not be less than 40s. in this study, the exposure time used was standardized at 40s, for all three light-curing units tested, and there was no statistically significant difference among them. based on the results found in the present study, the factors opacity and thickness significantly affected the water sorption and solubility of the light-cured resin cement underlying the ceramics; therefore, they need to be evaluated by the professional. often, ceramic pieces have variable thicknesses and need to be measured and evaluated when opting for adhesive cementation with light-curing resin cements, since the use of a ceramic restoration may not ensure effective light transmission through it, if its thickness is equal or greater to 2.0mm. another factor that must be considered is the light-curing unit used, which must have adequate characteristics, especially regarding a high intensity of emitted power, for effective photopolymerization. in conclusion, the light-curing resin cement under lithium disilicate ceramics with a thickness equal to or greater than 2.0  mm and/or high opacity presents high water sorption and solubility. the greater degree of opacity (ho) increased values of water sorption and solubility of resin cement in all tested thicknesses, regardless of the high-power light curing units tested. acknowledgments the authors thank espaço da escrita – dean of research – unicamp for the language services provided. we also thank the institutional scientific initiation scholarship program (pibic), the bahia research foundation (fapesb), and the national council for scientific and technological development (cnpq) for the financial support given to this study. 11 de cerqueira et al. references 1. leal cl, queiroz apv, foxton rm, argolo s, mathias p, cavalcanti an. water sorption and solubility of luting agents used under ceramic laminates with different degrees of translucency. oper dent. 2016;41(5):e141-8. doi: 10.2341/15-201-l. 2. rasetto fh, driscoll cf, von fraunhofer ja. effect of light source and time on the polymerization of resin cement through ceramic veneers. j prosthodont. 2001 sep;10(3):133-9. doi: 10.1111/j.1532849x.2001.00133.x. 3. mobilio n, fasiol a, catapano s. qualitative evaluation of the adesive interface between lithium disilicate, luting composite and natural tooth. ann stomatol (roma). 2016 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https://www.sciencedirect.com/science/article/pii/s0109564116301014 https://www.sciencedirect.com/science/article/pii/s0109564116301014 https://www.sciencedirect.com/science/article/pii/s0109564116301014 1http://dx.doi.org/10.20396/bjos.v21i00.8665042 volume 21 2022 e225042 original article 1 state university of montes claros, montes claros, brazil. 2 dental school, pitágoras integrated colleges, montes claros, mg, brazil. 3 school of dentistry, federal university of minas gerais, belo horizonte, brazil. 4 dental school, united colleges of northern minas (funorte), brazil. 5 dental science school (fco), montes claros, brazil. 6 dental research division, guarulhos university, guarulhos, brazil. corresponding author: joão g. s. souza, dental research division, guarulhos university. praça tereza cristina, 88 centro – guarulhos, são paulo, brazil. 07023-070 e-mail: jgabriel.ssouza@yahoo.com.br editor: altair a. del bel cury received: march 21, 2021 accepted: september 13, 2021 short-term effect of adhesive system on clinical performance of bulk fill composite: randomized clinical trial vanessa cristiane araújo oliveira1 , fernanda piana santos lima de oliveira2 , claudia silami magalhães3 , fabíola belkiss santos de oliveira4 , isabella pereira marques4, mayara dos santos noronha5 , joão gabriel silva souza5,6* , daniela araújo veloso popoff1,2 aim: although bulk fill composites have been widely used as restorative material, there is no consensus regarding the best clinical protocol in terms of composite technique and adhesive system. therefore, this clinical trial evaluated the clinical performance of bulk fill composites for class i restorations under different protocols. methods: a randomized clinical trial including 155 class i restorations was conducted using different adhesive systems: conventional technique (phosphoric acid + conventional three-step adhesive system) (group 1, 2 and 3); or self-etching adhesive system (groups 4, 5 and 6). control groups 1 and 4 were restored with conventional composite; groups 2 and 5 with low viscosity bulk fill and conventional composite as occlusal coverage; groups 3 and 6 with high viscosity bulk fill. the fdi criteria was used for clinical evaluation at baseline and after 6 months. results: all groups showed good clinical performance. at baseline, the adhesive system did not affect postoperative hypersensitivity. after 6 months, group 5 showed a significant reduction in color and translucency; group 6 a reduction in terms of anatomical form and for postoperative sensitivity and an improvement in patient satisfaction (p<0.05). considering the same restorative technique, the use of the self-etching adhesive system showed a significant decrease in color and translucency (p<0.05). conclusion: all groups showed favorable clinical performance, and promising results were found for the conventional adhesive system and high viscosity bulk fill protocol. keywords: composite resins. phosphoric acids. adhesives. dental restoration, permanent. esthetics, dental. clinical studies as topic. https://orcid.org/0000-0002-4905-3985 https://orcid.org/0000-0002-8826-6852 https://orcid.org/0000-0002-5101-8089 https://orcid.org/0000-0003-1643-8819 https://orcid.org/0000-0003-3963-7503 https://orcid.org/0000-0001-5944-6953 https://orcid.org/0000-0001-8313-2495 2 oliveira et al. introduction the improvement of dental materials, as well the increased demand for esthetic treatments by patients, have improved the development of less invasive restoration techniques and the use of composites with enhanced biomechanical and esthetic properties1-3, even for posterior teeth4. however, the polymerization shrinkage and the development of stress in the tooth-restoration interface, still being an important clinical problem that can degrade the adhesive layer, create cracks, and, consequently lead to treatment failure5. in this sense, different incremental techniques have been developed to reduce the material shrinkage6,7. although an increment thickness of 2 mm of composite has been suggested for tooth restoration to reduce this effect4, this approach can increase the clinical time8. therefore, to reduce the polymerization shrinkage effect different materials and techniques have been suggested, among them, bulk fill composites4. bulk fill composites have been widely used for tooth restoration and shown enhanced translucence and polymerization properties compared to conventional composites, allowing the use of a single increment (4–5 mm). these properties reduce clinical time and the polymerization shrinkage stress9,10. modifications in the organic matrix of bulk fill composites, such as monomers with higher molecular weight and the size of particles, explain these advantages9,11. these composites are available in two forms: low (flow) and high viscosity, in terms of organic matrix composition12. overall, clinical trials have compared the use of bulk fill and conventional composites showing similar clinical performance for both materials13, but with a better marginal adaptation for bulk fill material4. however, different clinical protocols have been tested by these studies, and differences in the experimental designs make it hard to compare between the materials and techniques used. although bulk fill composites have been evaluated by long-term clinical studies14-16, there is no consensus regarding the optimal clinical protocol, as well as the effect of the adhesive system in the clinical performance of this material16. previous studies evaluated the use of high viscosity bulk fill restorations under different adhesive systems16-18. however, to the best of our knowledge, no study compared the short-term effect of different adhesive systems under different clinical protocols, considering the bulk fill form (low and high viscosity) and conventional composites under the same technique. therefore, the present randomized clinical trial evaluated the clinical performance of low and high viscosity bulk fill composites for class i restorations, compared to conventional composites, and under different adhesive systems. the hypothesis is that the adhesive does not affect the clinical performance. materials and methods ethical aspects this study was approved by the local ethics committee (caae 96708418.5.0000.5109) and registered and approved by the brazilian clinical trials registry (rebec) (protocol rbr-2h9qkd), being conducted according to consort guidelines. 3 oliveira et al. trial design this is a prospective, double-blind, controlled, and randomized clinical trial. participants the restoration was the experimental unit. for sample calculation, a 50% difference between the groups was considered, a power (1 β) = 0.90 and a type i error (α) = 0.05, totaling 155 experimental units (more than 22 units per group was considered), resulting in a minimum increase of 15% in the pre-defined n for each group. patients (35) were chosen from a dental clinic school and met the inclusion criteria described by marques et al.19 (2018), exhibiting the need for class i restorations and/ or restoration replacement considering the assessment in score 5 (clinically poor: fédération dentaire internationale fdi) in at least one of the fdi criteria20. the same patients received more than one treatment according to clinical necessity. patients with physiological limitations that contraindicated dental treatment were excluded from the study19. eligible patients were checked for visible plaque index (ipv) and submitted to oral hygiene instructions (iho) and diet. all the included teeth received a professional cleaning before the restoration protocol. randomization and intervention a sequence of random numbers was generated by ms excel software (microsoft corp, redmond, wash., usa) to assign each experimental unit to treatment groups according to the randomization list generated. the experimental units were randomized and submitted to the restorative procedures listed below: group 1 (control 1): 37% phosphoric acid etching + conventional adhesive system (adper scotchbond multipurpose adhesive) + restoration with conventional nanoparticulated composite resin by incremental technique (filtek z350 xt). group 2: 37% phosphoric acid etching (maquira®) + conventional adhesive system (adper scotchbond multipurpose adhesive – 3m) + restoration with low viscosity bulk fill composite resin (filtek bulk fill flow – 3m) as a single increment base + conventional nanoparticulate composite resin (filtek z350 xt – 3m) by incremental technique for occlusal coverage. group 3: 37% phosphoric acid etching (maquira®) + conventional adhesive system (adper scotchbond multipurpose adhesive – 3m) + restoration of high viscosity bulk fill composite resin by a single increment (filtek one bulk fill – 3m). group 4 (control 2): self-etching adhesive system (single bond universal – 3m; multi-mode) + restoration with conventional nanoparticulated composite resin by incremental technique (filtek z350 xt – 3m). group 5: self-etching adhesive system (single bond universal 3m) + restoration with low viscosity bulk fill composite resin (filtek bulk fill flow – 3m) as a single increment base + conventional nanoparticulate composite resin (filtek z350 xt – 3m) by incremental technique for occlusal coverage. 4 oliveira et al. group 6: self-etching adhesive system (single bond universal – 3m) + restoration of high viscosity bulk fill composite resin by a single increment (filtek one bulk fill – 3m). the cavity depth (≈3–5 mm) was measured using a millimeter probe. all restorations protocols were conducted according to the manufacturer’s recommendations for each restorative material and performed by three calibrated blinded dentists19. the dentists were calibrated by an expert in this field with more than 20 years of experience in operative dentistry and conducting clinical trials. for the clinical protocol, the composite increments were inserted in an oblique direction; the conventional technique used 2 mm increments; bulk fill composites only one increment. furthermore, a photoactivation (ec 450 ecel® ≥900 mw/cm2 or valo® fotopolimerizador curing light shield – ultradent 1000 mw/cm²) also followed each composite and adhesive manufacturer’s recommendation regarding the application and photoactivation time (in a range of 10–20 seconds). immediately after performing the restorations, occlusal adjustment was conducted and the finishing and polishing were performed with a 9714ff® carbide burr (kg sorensen) and enhance® system (dentsply) 7 days after the restorative procedure19. clinical evaluation clinical evaluations at baseline (after polishing) and after 6 months were performed by two calibrated dentists using items from the fdi criteria20. divergences between examiners about the assessments were reviewed and a consensus was reached through discussion among them. to assess postoperative hypersensitivity, a pain scale numbered from 0 to 10 was applied to the participants, using 0 for the absence of pain and 10 for unbearable pain. then, the vitality of the restored tooth was evaluated using the endo-ice cold vitality test (maquira). patient satisfaction with the treatment was also determined by a scale from 0 to 10, where 0 was totally dissatisfied and 10 was totally satisfied. statistical analysis the ibm spss 22.0 for windows software was used. treatment groups were described by the percentage of the best clinical condition for each criterion (clinically adequate excellent / good). the kruskal–wallis test was used to compare treatment groups within each clinical criterion and assessment time (baseline and 6 months). then, pair comparisons among the groups were performed using the mann–whitney test. wilcoxon’s test was used to assess changes in assessments over time. a significance level of 5% (p<0.05) was adopted. results 155 teeth were submitted to the restoration procedure at baseline according to the treatment groups. the loss to follow-up rate was approximately 23.5% (fig. 1). on average, each patient received 4.42 restorations. at baseline, all groups showed clinically satisfactory results with a positive assessment (excellent or good) greater than 70% for all criteria (table 1). however, a significant difference was identified between the groups for surface staining (p=0.012) and anatomical contour (p<0.001). at baseline, the results suggest no clinical effect of the adhesive system. 5 oliveira et al. figure 1. flowchart clinical trial (consort) 155 tooth (35 patients) baseline 6 months group 1 (33 tooth) group 2 (28 tooth) group 3 (23 tooth) group 4 (23 tooth) group 5 (22 tooth) group 6 (26 tooth) group 1 (18 tooth) group 2 (24 tooth) group 3 (15 tooth) group 4 (15 tooth) group 5 (19 tooth) group 6 (20 tooth) table 1. comparison among the treatment groups for each clinical criterion at baseline and after 6 months according to clinical condition evaluated. the percentage of best clinical outcome was reported (clinically adequate excellent / good). the average rating scale was used for patient satisfaction and postoperative sensitivity. comparison among all the groups at each time (baseline and 6 months) and clinical criterion was conducted by kruskal–wallis test (p (all groups)). comparison in the same group at different times (baseline x 6 months) by wilcoxon test (p (time)). mann–whitney test was used for paired comparisons at 6 months and different upper letters mean statistical difference (p<0.05). criterion group 1 group 2 group 3 group 4 group 5 group 6 p (all groups) surface brightness baseline 84.8 89.3 90.0 91.3 90.9 96.2 0.822 6 months 100.0 100.0 100.0 100.0 100.0 100.0 1.000 p (time) 0.317 0.157 1.000 1.000 0.180 1.000 surface staining baseline 87.9 100.0 100.0 100.0 100.0 100.0 0.012 6 months 100.0 100.0 100.0 100.0 100.0 100.0 1.000 p (time) 0.317 1.000 1.000 1.000 1.000 1.000 marginal staining baseline 90.9 96.4 90.0 100.0 95.5 100.0 0.388 6 months 100.0 100.0 100.0 100.0 100.0 100.0 1.000 p (time) 1.000 0.317 0.157 1.000 1.000 1.000 color and translucence baseline 97.0 96.4 100.0 100.0 100.0 100.0 0.693 6 months 72.7a 100.0b 100.0b 60.0a 73.7a 100.0b <0.001 p (time) 0.102 0.317 1.000 0.014 0.025 1.000 anatomical contour baseline 72.7 96.4 95.0 100.0 100.0 96.2 <0.001 6 months 94.4ab 91.7ab 100.0b 73.3a 94.7ab 75.0a 0.069 p (time) 0.285 1.000 1.000 0.046 0.317 0.025 fracture baseline 100.0 100.0 100.0 95.7 100.0 100.0 0.351 6 months 100.0 100.0 100.0 100.0 100.0 100.0 1.000 p (time) 1.000 1.000 1.000 1.000 1.000 1.000 marginal adaptation baseline 90.6 82.1 95.0 91.3 77.3 84.6 0.507 6 months 100.0 100.0 100.0 100.0 100.0 100.0 1.000 p (time) 0.083 0.066 0.317 0.317 0.025 0.157 continue 6 oliveira et al. after 6 months, all groups remained with a proper clinical performance (table 1). however, the groups were statistically different regarding color and translucency (p<0.001), with lower values of clinical performance for groups 1, 4 and 5. regarding the individual comparisons of the clinical performance of the treatment groups over time (baseline x after 6 months) in each criterion, there was a significant decrease for restorations using the self-etching adhesive system (table 1). moreover, there was a significant reduction (p<0.05) in the evaluation of color and translucency for groups 4 and 5; and anatomical contour for groups 4 and 6 (table 1). however, a significant improvement (p<0.05) in the satisfactory assessment was identified for marginal adaptation for group 5; and in postoperative sensitivity and patient satisfaction in group 6 (table 1). the use of the self-etching adhesive system led to a significant decrease of clinical performance after 6 months in terms of color/translucency for restorations using low viscosity bulk fill + conventional composite, and the anatomical contour for high viscosity bulk fill restorations (table 2). moreover, comparing the restorative techcontinuation patient satisfaction baseline 9.5 9.5 9.7 9.6 9.6 9.3 0.351 6 months 9.8 9.4 9.6 9.6 9.6 9.9 0.238 p (time) 0.317 0.713 0.317 0.655 0.655 0.034 postoperative hypersensitivity baseline 1.0 0.8 0.3 0.3 0.8 1.4 0.328 6 months 0.3 0.6 0.4 0.4 0.2 0.3 0.794 p (time) 0.067 0.573 0.655 1.000 0.102 0.010 group 1 – conventional adhesive + conventional composite resin, group 2 – conventional adhesive + low viscosity bulk fill + conventional composite resin, group 3 – conventional adhesive + high viscosity bulk fill composite resin, group 4 – self-etching adhesive + conventional composite resin, group 5– self-etching adhesive + low viscosity bulk fill + conventional composite resin, group 6 – self-etching adhesive + high viscosity bulk fill composite resin. table 2. pair comparison of the adhesive systems tested of treatment groups using the same restorative technique (same composite) after 6 months. mann–whitney test (p <0.05). p values. criterion group 1 x 4 group 2 x 5 group 3 x 6 surface brightness 1.000 1.000 1.000 surface staining 1.000 1.000 1.000 marginal staining 1.000 1.000 1.000 color and translucence 0.465 0.008 1.000 anatomical contour 0.097 0.698 0.039 fracture 1.000 1.000 1.000 marginal adaptation 1.000 1.000 1.000 patient satisfaction 0.417 0.313 0.804 postoperative hypersensitivity 0.717 0.272 0.836 group 1 – conventional adhesive + conventional composite resin, group 2 – conventional adhesive + low viscosity bulk fill + conventional composite resin, group 3 – conventional adhesive + high viscosity bulk fill composite resin, group 4 – self-etching adhesive + conventional composite resin, group 5– self-etching adhesive + low viscosity bulk fill + conventional composite resin, group 6 – self-etching adhesive + high viscosity bulk fill composite resin. 7 oliveira et al. nique (high viscosity bulk fill only or low viscosity bulk fill + conventional composite), a better performance was identified for group 6 in terms of color and translucency (p=0.015) compared to group 5. discussion bulk fill composites are a promising clinical approach for tooth restoration due to the enhanced polymerization process and reduced clinical time. thus, the clinical evaluation of this restorative material under different techniques allows the standardization of appropriate protocols4. the results of the present study showed similar clinical performance of restorations made under different adhesive systems, with slight differences that were not clinically relevant. moreover, we also showed a good clinical performance of bulk fill composites after 6 months, associated or not with conventional composite. however, a slight negative effect on the clinical performance was observed in terms of color, translucency and anatomical form in the groups treated with bulk fill composite. this result was found only for groups treated with the self-etching adhesive system, which, in general, showed worse performance after 6 months. restorations using only high viscosity bulk fill composite with the self-etching adhesive system showed less postoperative sensitivity and a higher level of patient satisfaction after 6 months. moreover, the use of this material associated with the conventional adhesive system showed a similar pattern of results, but without statistical differences. since patient satisfaction and lower postoperative sensitivity are important results to be achieved in esthetic restorative procedures, mainly at a short-term parameter, high viscosity bulk fill composites seem to be a promising strategy. the postoperative sensitivity for bulk fill and conventional composites using the single bond universal adhesive showed a similar response, as also demonstrated by a previous study5. in fact, the adhesive strategy may not play an important role in the intensity of postoperative sensitivity16, but the depth of cavity can affect this parameter directly. these results can also be explained by the composite composition, mainly in terms of monomers, which allow additional fragmentation and structural rearrangement of their bonds favoring the tension relief of the polymer chain. then, these properties may reduce the polymerization contraction process and, consequently, lower the postoperative sensitivity. sixyear follow-up clinical studies10 showed similar performances between combined or incremental techniques. it should be noted that there is a promising result from the use of only high viscosity bulk fill without coverage with conventional resin using conventional adhesive, with higher percentages of good/excellent evaluation in all criteria. after 6 months some restorations showed a decrease in terms of color/translucency when using the bulk fill resin associated with the conventional resin and universal adhesive system. in fact, bulk fill resins have greater translucency, which allows a greater depth of polymerization21. thus, the use of the universal adhesive system, also known as single bond universal, can affect color properties when associated with conventional composite22. this result for bulk fill composites is expected, since it was identified by a previous clinical study18. however, this effect was not considered 8 oliveira et al. to be clinically important. it suggests that the translucency present in low viscosity bulk fill composites, a property that allows a greater depth of polymerization of these materials, may have influenced the clinical evaluation. the comparison between the adhesive strategies showed a decrease in the clinical performance for restorations using self-etching adhesive. the universal adhesive tested resulted in greater color changes, a condition that appears to be associated with the oxidation of camphorquinone in this material22. additionally, there is no consensus on the influence of the adhesive system on the clinical performance of this restorative material. although some adhesive failures are to be expected in conventional and bulk fill restorations using the conventional three-step adhesive system, there is no clinical consensus on whether variations in adhesive techniques can interfere with the restorative clinical performance. clinical studies compared the high viscosity bulk fill composite using different adhesive systems16,18 but did not compare clinically with conventional composites. moreover, bulk fill and conventional composites restorations using different adhesive systems have been evaluated using limited clinical criteria17. therefore, clinical studies that also consider adhesive systems in the clinical performance of restorative techniques and materials are of great importance. clinical deficiency in anatomical form was identified after 6 months for the groups using conventional resin or bulk fill. a previous study showed that restorations with the low viscosity bulk fill composite provided clinically unacceptable scores after 1 year for anatomical form, showing a significant increase in this type of failure when compared to the other evaluation periods (one week and 6 months)23. however, it is noteworthy that studies whose restorative procedures involved more than one professional, as in the present study, revealed that some of the variables evaluated were more dependent on the operator than the tested material24. therefore, it is expected that the anatomical form is not affected by material properties. in conclusion, the clinical protocols evaluated showed a similar pattern of results and good clinical performance. although the results presented here are preliminary, they highlight important comparisons to determine restorative protocols using bulk fill material. at baseline, the adhesive systems and composite did not significantly affect the clinical performance of restorations. the use of high viscosity bulk fill resin with a conventional adhesive system seems to be a promising restorative strategy for class i restorations. longitudinal evaluation is necessary to assess the clinical performance of these restorations considering all the evaluated clinical parameters. data availability datasets related to this article will be available upon request to the corresponding author. conflicts of interest: none 9 oliveira et al. references 1. ayar mk, guven me, burduroglu hd, erdemir f. repair of aged bulk-fill composite with posterior composite: effect of different surface treatments. j esthet restor dent. 2019 may;31(3):246-52. doi: 10.1111/jerd.12391. 2. klapdohr s, moszner n. new inorganic components for dental filling composites. monatshefte chem. 2005;136:21-45. 3. ferracane jl. resin composite state of the art. 2011 jan;27(1):29-38. doi: 10.1016/j.dental.2010.10.020. 4. fahim se, mostafa ma, abi-elhassan mh, taher hm. clinical behaviour and marginal sealing of bulk-fill resin composite restorations using light amplified high-intensity leds curing: a randomized controlled clinical trial. open access maced j med sci. 2019 apr 29;7(8):1360-8. doi: 10.3889/oamjms.2019.216. 5. ayar mk. postoperative sensitivity after 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10.1007/s00784-018-2509-8. 24. colak h, tokay u, uzgur r, hamidi mm, ercan e. a prospective, randomized, double-blind clinical trial of one nano-hybrid and one high-viscosity bulk-fill composite restorative systems in class ii cavities: 12 months results. niger j clin pract. 2017 jul;20(7):822-31. doi: 10.4103/1119-3077.212449. 1http://dx.doi.org/10.20396/bjos.v21i00.8663759 volume 21 2022 e223759 original article 1 graduate program in dentistry, cruzeiro do sul university, são paulo, sp, brazil. 2 camilo castelo branco university, são paulo, sp, brazil. 3 department of biomaterials and oral biology, school of dentistry, university of são paulo (usp), são paulo, brazil. corresponding author: ângela toshie araki av robert kennedy 2126/sala 4, zip code 09861-080 são bernardo do campo, sp brazil tel.: +55-11-992534078 fax: +55-11 -43921575 a_araki@me.com editor: altair a. del bel cury received: december 29, 2020 accepted: august 12, 2021 effect of intracanal diode laser irradiation on fracture resistance of roots restored with cad/cam posts flavia florentino teixeira da silva1 , andré hayato saguchi1 , sidnea aparecida freitas paiva1 , guilherme espósito pires1 , mariana isidoro1 , aldo brugnera junior2 , paulo francisco cesar3 , ângela toshie araki1* aim: to evaluate the fracture resistance of roots restored with cad/cam-fabricated posts, receiving or not intracanal laser treatment, compared with glass fiber posts under mechanical cycling. methods: twenty-seven endodontically treated, single-rooted teeth were divided into 3 groups: group 1 (control), prefabricated glass fiber posts relined with resin composite; group 2, cad/cam-fabricated intraradicular posts using resin nano ceramic (rnc) blocks; and group 3, cad/cam-fabricated intraradicular posts using rnc blocks in canals irradiated with a 940-nm diode laser (100 mj, 300-um optic fiber, coronal-apical and apical-coronal helical movements, speed of 2 mm/second, 4 times each canal). after cementation of the coping, cyclic loading was applied at an angle of 135° to the long axis of the root, with a pulse load of 130 n, frequency of 2.2 hz, and 150,000 pulses on the crown at a point located 2 mm below the incisal edge on the lingual aspect of the specimen. every 50,000 cycles, the specimens were evaluated for root fracture occurring below or above the simulated bone crest. results were analyzed by one-way anova followed by tukey’s test (p<0.05). results: group 1 was the least resistant, while groups 2 and 3 were the most resistant. group 1 differed significantly from groups 2 and 3 (p<0.01), but there was no difference between groups 2 and 3 (p<0.01). conclusion: treatment of the intracanal surface with diode laser had no influence on fracture resistance of roots restored with cad/cam-fabricated posts, but a longer cycling time is required to evaluate the real benefits of diode laser irradiation. keywords: lasers. endodontics. dentistry. computer-aided design. https://orcid.org/0000-0001-7752-584x https://orcid.org/0000-0002-1903-4986 https://orcid.org/0000-0001-5882-8802 https://orcid.org/0000-0003-2721-6752 https://orcid.org/0000-0002-8929-5516 https://orcid.org/0000-0003-0023-191x https://orcid.org/0000-0001-5834-105x https://orcid.org/0000-0003-4402-7531 2 paiva et al. introduction endodontically treated teeth are at increased risk of fracture due to loss of healthy tooth structure, which may result from inadequate access cavity preparation, carious lesions, and/or extensive restorations1. in order to improve crown retention and stress distribution on the remaining tooth structure, the use of intraradicular posts fabricated from different materials is indicated2. metal alloys are the oldest materials, but their high modulus of elasticity (100-200gpa) is much higher than the dentin (18gpa), what can lead to root fracture. in contrast, prefabricated carbon and glass fiber posts have a lower modulus of elasticity-similar to dentinand are presented in different diameters and tapers. even when the most adjustable post to the prepared root canal is selected, the film thickness of the luting cement may be higher, which reduces fracture resistance3. with the development of the cad/cam system, intraradicular posts can replace the intracanal and coronal portions in one piece, thus reducing the resin-cement interface, shortening clinical time, and enhancing adaptation4. recent studies have shown that cad/cam-fabricated intraradicular posts increase root fracture resistance, providing an alternative to the use of prefabricated and cast metal posts5. an in vitro study6 compared the fracture resistance of flared canals restored with cad/cam postand -core with prefabricated glass fiber and cast gold alloy posts. radicular fractures occurred in all 3 groups. however, cad/cam post-and -core reduced the occurrence fractures in the middle and in apical 1/3 part of root. fractures in those regions are irreparable and, consequently, catastrophic to root survival6. debonding of posts to the dentin wall is a currently concern. despite the promising results obtained with cad/cam fabricated intraradicular posts regarding to fracture resistance of root canals, there is no protocol for post surface treatment that ensures better adhesion to the dentin. simple procedures are indicated, such as use of 70% alcohol during cleaning of the posts and use of an universal adhesive. different surface treatments of cad/cam glass fiber posts with 24% hydrogen peroxide and a moisture of silane and ethanol solution did not influence in bond strength values7,8. the bonding quality can be influenced by the type of dentin and the presence of smear layer produced during chemomechanical preparation9,10. smear layer can be removed by using suitable chemical substances and high-power lasers11-16. studies testing fracture resistance of roots after dentin pretreatment using diode lasers and luting a cad/cam post are scarce, especially using resin nanoceramic cad/cam blockslava ™ ultimate cad / cam restorative, 3m espe, usa which have, according to the manufacturer, a modulus of elasticity (12,8gpa) similar to the dentin (18 gpa). in view of this scenario, it seems pertinent to evaluate the fracture resistance of roots restored with cad/cam-fabricated intraradicular posts made from lava™ ultimate blocks, receiving or not intracanal treatment with high-power diode laser, compared with glass fiber posts relined with resin composite under mechanical cycling. 3 paiva et al. materials and methods specimen selection and preparation twenty-seven single-rooted human permanent teeth without fractures, cracks, or fissures were obtained by donation from the human tooth bank of apcd são caetano do sul, são paulo, brazil. the sample size was based on previous studies6,7 that used 10 samples per group. in our study, 9 specimens were used, as there was a loss of 3 samples in the preparation (considering a significance level of 5%, we had a power of 0.77 to detect differences between groups with specimen rates of 25% and 85%, respectively). the crowns were removed with a carborundum disc, leaving a root length of 14 mm. the root canals were prepared with r25 reciproc files (vdw, munich, germany) and irrigated with 1% sodium hypochlorite (naocl), and the cavities were lubricated with a water-soluble lubricating gel (endo-ptc leve; fórmula e ação, são paulo, sp, brazil). after chemomechanical preparation, final irrigation was performed with 10 ml of 1% naocl, 10 ml of 17% edta-t, and 10 ml of 1% naocl. the specimens were obturated with r25 reciproc gutta-percha cones (vdw, munich, germany) and ah plus sealer (dentsply, pennsylvania, usa). the apical 4 mm of gutta-percha were left in the canal, and the remaining 10 mm of the root canal were prepared with a #2 dcr drill of the white post dce glass fiber post kit (fgm, joinville, sc, brazil), leaving a 10-mm post space. randomization of specimens the teeth were numbered and randomly divided (www. randomizer.com) into 3 experimental groups of 9 teeth each. in group 1 (control), prefabricated glass fiber posts (fgm, joinville, sc, brazil) relined with resin composite (filtek z350 xt, 3m espe, brazil) were usedg1 gfrc. in group 2, root canals were scanned for fabrication of intraradicular posts with a cad/cam system (cerec inlab mc xl, sirona dental systems, inc., ny, usa) using resin nano ceramic (rnc) blocks (lava™ ultimate cad/cam restorative, 3m espe, usa)g2 cad. in group 3, root canals were scanned for fabrication of intraradicular posts with a cad/cam system (cerec inlab mc xl, sirona dental systems, inc., ny, usa) using rnc blocks (lava™ ultimate cad/cam restorative, 3m espe, usa) and then irradiated with a 940-nm diode laser (biolase) at 100 mj, with an optic fiber of 300 um in diameter and helical movements from the coronal to the apical part of the canal and from the apical to the coronal part of the canal at a speed of 2 mm/second. each canal was irradiated 4 timesg3 cad/laser. post preparation and cementation all posts were cleaned with alcohol and dried with air spray. silane was then applied for 1 minute, followed by spray drying and a thin layer of single bond universal adhesive (3m espe, brazil) was applied during 20 seconds without polymerization. the canals were irrigated with 1% naocl and dried with sterile paper points. a thin layer of single bond universal adhesive (3m espe, brazil) was applied, followed by application of relyx™ ultimate dual cure resin cement (3m espe, brazil) inside the canal. the prepared post was inserted into the root canal and light-cured for 3 seconds. after excess luting material was removed, a final light curing was performed for an additional 40 seconds. 4 paiva et al. mechanical cycling the #2 drill previously used for post space preparation was inserted into the root canal, and the set (root + drill) was placed in the surveyor so that it was tilted at an angle of 135°. the root was fixed inside an acrylic cylinder (diameter of 12 mm and height of 20 mm) by using a high-viscosity crystalline epoxy resin. for cycling, ceramic copings were manufactured with a cad/cam system (ips emax cad, ivoclar vivadent). they were cleaned with alcohol and dried with air spray, followed by application of silane (monobond n, ivoclar vivadent, barueri, sp, brazil) for 1 minute, followed by spray drying for 1 minute. a thin layer of adhesive was applied without polymerization, and the coping was secured on the already cemented post with dual cure resin cement (relyx™ ultimate, 3m espe, brazil). load was applied at an angle of 135° to the long axis of the root (figures 1 and 2), with a pulse load of 130 n and frequency of 2.2 hz, for a total of 150,000 pulses on the crown at a point located 2 mm below the incisal edge on the lingual aspect of the specimen. analysis of specimens every 50,000 cycles, the roots were evaluated by a calibrated observer at 4x magnification with prismatic loupes (zeiss eyemag pro s, carl zeiss). fractures that occurred in the cementation during the tests were classified as favorable (above 3 mm, which was the simulated bone crest) and unfavorable (below the simulated bone crest). the collected data was analyzed by one-way anova followed by tukey’s test (p<0.05). load acrylic cylinder 45° 135° figure 1. load was applied at an angle of 135° to the long axis of the root. figure 2. specimen being cycled in the biocycle piopdi cycler (são carlos, sp, brazil). 5 paiva et al. results table 1 shows the total number of cycles until fracture occurred. in group 1 (prefabricated glass fiber posts relined with resin composite), fractures occurred just above the simulated alveolar bone crest (favorable) – figure 3. figure 4 shows a boxplot of the total number of cycles until fracture occurred per experimental group. figure 3. two specimens above the simulated alveolar bone crest 0 50000 100000 150000 g1 – fr g2 – cad g3 – cad/laser experimental groups n u m b er o f c y c le s figure 4. boxplot of total number of cycles/ fracture. 93 x 67mm (600 x 600 dpi) table 1. total number of cycles until fracture occurred root fracture g1 fr g2 cad g3 cad/laser 1 20000 71434 2 17148 3 17853 4 24926 5 39779 6 54230 7 8 6 paiva et al. group 1 was the least resistant, while groups 2 (cad/cam-fabricated posts) and 3 (cad/cam-fabricated posts in laser-irradiated root canals) were the most resistant. one-way analysis of variance revealed statistically significant differences between the groups (p<0.01), and tukey’s test showed the following results: significant differences between groups 1 and 2 and between groups 1 and 3 (p<0.01), but no difference between groups 2 and 3. groups 2 and 3 showed virtually no fracture in the parameters analyzed. the specimen survival rate (non-fractured roots) was 25% in group 1 and 87.5% in groups 2 and 3 (table 2). the specimens number 1 in group 2 and number 4 in group 3 showed fracture of the coping, thereby adversely affecting the test results. discussion restoring endodontically treated teeth is challenging due to weakened roots and absence of dental crown or intraradicular dentin17,18. intraradicular posts are commonly indicated to aid in the reconstruction of these elements19, but even though irreversible root fracture may still occur, leading to tooth loss. metal intraradicular posts have a high modulus of elasticity, thus placing greater stress on the remaining root and increasing the risk of fracture20-27. for this reason, metal posts were not included in this study. in vitro23,28-35 and clinical21,22,36 studies have shown a better performance of prefabricated glass fiber posts when they are relined with resin composite, as this would reduce the amount of cement between the resin and intracanal wall. this justifies the relining of specimens in group 1. however, relining the prefabricated posts with resin composite did not ensure fracture resistance in this group compared to cad/cam groups (2 and 3). the rate of non-fractured roots (survival rate) was only 25%. in glass fiber posts, fracture occurs by shear stress when forces are loading at an angle of 45° to the long axis of the tooth31. table 2. specimen survival rate specimen group g1 fr g2 cad g3 cad/laser 1 20000 71434 150000 2 17148 150000 150000 3 17853 150000 150000 4 24926 150000 48654 5 39779 150000 150000 6 54230 150000 150000 7 150000 150000 150000 8 150000 150000 150000 survival rate 25% 87,5% 87,5% 7 paiva et al. for a long time, prefabricated glass fiber posts have been the posts of choice to restore endodontically treated teeth. however, with the development of the cad/cam system, it is possible to fabricate posts that better adapt to the intraradicular canal. these posts are indicated mainly for use in oval or wide canals, in addition to providing a better aesthetic outcome, increased retention, and lower film thickness of the luting cement6,37. it is imperative to highlight the increased fracture resistance that the cad/cam system provides to the root6,38, which supports the choice of this system in the present study. moreover, the specimen survival rate in groups 2 and 3 was higher than that in group 1, i.e., there was less root fracture – except for the fracture of copings in specimens number 1 and 4 in groups 2 and 3, respectively – which is consistent with the results reported in other studies5,6,39. several materials can be used in the cad/cam system. in the present study, lava™ ultimate rnc was chosen because of its good performance in root fracture resistance tests compared with other materials, according to the results reported by spina et al.37. in addition, according to the manufacturer (3m espe, brazil), this material has a nanoceramic content of 80%, thus providing the benefits of both resin (modulus of elasticity similar to that of dentin) and ceramic (color stability and hardness). bond strength is another extremely important factor when investigating intraradicular posts, as well as a determinant of treatment success40-45. dentin-post-cement interactions are influenced by some factors such as integrity of the root canal wall, polymerization of the resin cement, and contamination of the root canal, all of which can increase the risk of root fracture42. in the present study, before cementation, the canal walls were examined under an operating microscope and, if impurities were observed in the intracanal wall, they were removed with an xp endo finisher rotary file (fkg, switzerland) aiming to improve cement bonding to the root canal wall. the significantly superior results obtained with cad/cam-fabricated posts (groups 2 and 3) over prefabricated glass fiber posts (group 1) were expected by the authors of the present study based on the existing literature. however, a question arose as to whether diode laser treatment of root canals would have a beneficial effect on post bonding to the canal walls and, consequently, on root fracture resistance, since there is a vast literature on the action of diode laser on dentin permeability, smear layer removal, and structural changes in the dentin11,46-49, factors closely related to bond strength. this justifies the inclusion of group 3. studies with intraradicular surfaces irradiated with a 980-nm diode laser50 and er:yag, er,cr:ysgg51 presented an improvement in bond strength of glass fiber posts compared to non-irradiated groups. these findings are in agreement with this study, when group 3 is compared to group 1. however, because bond strength of groups 2 and 3 did not differ significantly, authors can not state that diode laser treatment improved fracture resistance. the present study performed 150,000 cycles. for more robust results, it will be necessary to manufacture new copings and to continue mechanical cyclic loading until all specimens have fractured, simulating longer time in the mouth, according to other studies37,39. clinical studies are important to validate these in vitro findings. in addition, studies applying different lasers and parameters are required. 8 paiva et al. based on the present results, it can be concluded that cad/cam-fabricated intraradicular posts made from lava™ ultimate blocks provided greater fracture resistance of roots than prefabricated glass fiber posts relined with resin composite. treatment of the intracanal surface with diode laser had no influence on fracture resistance of roots restored with cad/cam-fabricated posts, but a longer cycling time is required to evaluate the real benefits of diode laser irradiation. author disclosure statement financial disclosure: the authors have no financial relationships relevant to this article to disclose. conflict of interest: no competing financial interests exist. references 1. novais vr, rodrigues rb, simamoto junior pc, lourenco cs, soares cj. correlation between the mechanical properties and structural characteristics of different fiber posts systems. braz dent j. 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10.1007/s10103-013-1442-y. 49. da costa ribeiro a, nogueira ge, antoniazzi jh, moritz a, zezell dm. effects of diode laser (810 nm) irradiation on root canal walls: thermographic and morphological studies. j endod. 2007 mar;33(3):252-5. doi: 10.1016/j.joen.2006.09.002. 50. garcia lda f, naves lz, farina ap, walker cm, consani s, pires-de-souza fc. the effect of a 980 nm diode laser with different parameters of irradiation on the bond strength of fiberglass posts. gen dent. 2011;59(1):31-7; quiz 38-9, 80. 51. gomes kgf, faria ns, neto wr, colucci v, gomes ea. influence of laser irradiation on the push-out bond strength between a glass fiber post and root dentin. j prosthet dent. 2018 jan;119(1):97-102. doi: 10.1016/j.prosdent.2017.01.013. revista fop n 13 1627 halitosis: a review of basic principles naila aparecida de godoi machado1; paulinne junqueira silva andresen strini2; mario olavo pena carneiro1; roberto bernardino júnior3; alfredo júlio fernandes neto3 1 dds graduate student, dental school, federal university of uberlândia, brazil 2 dds master’s degree student, dental school, federal university of uberlândia, brazil 3 dds, msc, phd professor, dental school, federal university of uberlândia, brazil received for publication: november 11, 2007 accepted: june 12, 2008 correspondence to: alfredo júlio fernandes neto av. pará, 1720, campus umuarama, bl 2b, sala 2b01, cep: 38400-902 uberlândia, mg, brazil phone: +55-34-3218-2222 e-mail: alfredon@ufu.br a b s t r a c t halitosis, also known as bad breath or oral malodor, is the general term used to describe any disagreeable odor in expired air, regardless of whether the odorous substances originate from oral or non-oral sources. bad breath can be detrimental to one’s self-image and confidence, causing social, emotional and psychological anxiety. this paper provides a comprehensive review of the historical background, prevalence, social aspects and pathological causes of halitosis as well as the dental professional’s responsibility. key words halitosis, bad breath, oral malodor. i n t r o d u c t i o n halitosis is the general term used to describe any disagreeable odor in expired air, regardless of whether the odorous substances originate from oral or non-oral sources. other names used are fetor ex ore, fetor oris, bad or foul breath, breath malodor and oral malodor. the latter term is reserved for halitosis from the mouth1. although halitosis is one the most common medical conditions, relatively little is known about this embarrassing and sometimes debilitating problem2. this paper provides a comprehensive review of the historical background, prevalence, social aspects and pathological causes of halitosis as well as the dental professional’s responsibility. historical background the importance of oral malodor goes beyond the knowledge of its cause, diagnosis and therapy because this condition interacts with other sociological issues such as culture, religion, race, gender and social taboos. innumerous ancient folk remedies are still in use. in the bible, the book of genesis mentions ladanum (mastic), a resin derived from the pistacia lentiscus tree, which had been used in mediterranean countries for breath freshening for thousands of years3. chewing of natural products for breath freshening has long been practised worldwide, such as cloves (iraq), parsley (italy), anise seeds (far east), cinnamon (brazil), and guava peels (thailand)1. mouthwashes containing flavored elements like menthol, eucalyptol and methyl salicylate are also widely used4. knowledge and written reference to this condition date back to ancient cultures. references were found in papyrus manuscripts dating back to 1550 bc. the hebraic liturgics (the talmud), dating back more than two thousand years ago, clearly state that the terms of a marriage license (the ketuba) may be legally broken in case of malodor of one of the partners5. prevalence and social aspects halitosis affects a large proportion of the population and may cause significant social or psychological impairment to those suffering from this condition6. information regarding its prevalence is scarce. it is very difficult to determine the exact number or percentage of people who have bad oral breath because there is a lack of epidemiological studies addressing this issue7. malodor is a condition that has health and social implications rendering it an area of oral science that spans medical and psychological issues. current social guidelines emphasize the importance of personal image and interpersonal relationships. in this context, halitosis may be an important factor in social communication and hence the origin of concern due not only to a possible health condition, but also to psychological alterations leading to social and personal isolation8. pathological causes of halitosis the production of halitosis is multi-factorial and may involve both oral and non-oral sources. non-oral sources braz j oral sci. july/september 2008 vol. 7 number 26 1628 are generally related to systemic problems and/or medications. several extraoral diseases, including bronchial and lung infections, kidney failure, different types of carcinomas, metabolic dysfunctions and biochemical disorders, can cause halitosis. however, taken together, all these diseases affect only a very small percentage of people experiencing oral malodor9. some medications, especially those that reduce salivary flow such as antidepressants, antipsychotics, narcotics, decongestants, antihistamines, and antihypertensives10, may also be the source of this condition. certain foods, tobacco, alcohol and some prescription or over-the-counter drugs may cause halitosis11. an estimated 90% of halitosis cases originate within the oral cavity4. in most of these cases, halitosis occur primarily as a result of oral microbial metabolism, in which volatile sulfur compounds (vsc) and other volatile components are produced through deglycosylation, proteolysis and putrefaction of glycoproteins and proteins12. concentration of vsc can be measured using a portable sulphide monitor13. halitosis of oral origin is associated with poor oral hygiene, dental plaque, dental caries, gingivitis, stomatitis, periodontitis, tongue coating and oral carcinoma. dry mouth (xerostomy) might also be the cause, although a correlation is not always observed14. in addition, to the most common intraoral sites of bad breath production (tongue, interdental and subgingival areas), other foci may include faulty restorations (e.g.: as overhanging restorations and leaking crowns), sites of food impaction and abscesses. carious cavities are usually not necessarily malodorous, unless large enough to entrap food. dentures, particularly those are worn overnight, are another common cause of halitosis. dentures usually have a somewhat sweet but unpleasant and characteristic odor that is readily identifiable, especially if the dentures are placed in a plastic container and smelled following several minutes3,9. in people with rigorous oral hygiene, clean and sound dentition and a healthy periodontium, the source of bad breath is likely to be the back of the tongue. although the anterior part of the tongue usually smells (a simple test is to lick one’s wrist, left dry for a few seconds and then smell the area), the main source of odor is usually farther back in the posterior region. some studies have shown that simply brushing the tongue reduced bad breath measurements by 70%11,15. the posterior area of the dorsal surface can be readily assessed by a gentle but thorough scraping using a disposable plastic spoon. afterwards, the spoon can be smelled to compare with the overall mouth odor. the diagnosis of halitosis is established based on a detailed clinical interview that reviews the history of the disease, previous dental/medical treatment, severity and the impact of the disease on the patient’s everyday life, systemic changes and emotional issues and dietary habits. clinical examination should evaluate the patient’s oral hygiene, presence of caries, periodontal status and plaque retention sites. the presence of dental caries, alveolar bone defects and defective restorations should be assessed radiographically. special tests are performed to detect the foul-smelling vscs with the associated bacteria. different diagnostic techniques are currently available, including organoleptic measurement, halimeter examination and gas chromatography, which are described below7,16. organoleptic measurement is based on the subjective sensation of the examiner to the mouth odor. it is recorded on a point scale according to the examiner’s perception of the intensity of halitosis from the air expelled through a straw at a specific distance. the examination is simple to conduct and does not require any specific equipment. in some cases, organoleptic measurement accurately reflects the severity and intensity of halitosis, but it is considered a subjective technique because the smelling capacity of the evaluator may oscillate. a potential risk of the organoleptic measurement is the transmission of diseases via expelled air7. the halimeter is a portable instrument that measures the vsc concentration in the oral cavity. it is sensitive to volatile compounds and has to be calibrated to the background air prior to making a reading. the patient is instructed not to drink, smoke, eat, chew gum, suck confectionary, use mouthwash or breath fresheners, or perform oral hygiene for at least 4 hours before being tested. also, the use cosmetic products like perfumes, after-shave and scented lipstick should be avoided. just before the measurement, the patient is instructed to keep the mouth closed for 3 minutes. a straw connected to the halimeter is gently placed over the dorsum of the tongue without touching and the patient is asked to keep the mouth wide open. a measurement is taken once a peak reading has been reached. there may be falsepositive results due to othervolatile vapours, such as acetone, ethanol, and methanol, which do not contribute to oral halitosis7. gas chromatography is a method that uses a specific sulfur detector to identify the source of intraoral and extraoral halitosis. however, this method requires expensive equipment and trained personnel. a newly developed portable gas chromatograph (oralchroma™; abilit corporation, japan) has been developed for measuring vscs in the same way as standard gas chromatographs. this device can become the choice for diagnosis of oral breath and can perfectly differentiate between intraoral and extraoral bloodborne halitosis. the equipment is very sensitive and presents advantages in relation to the halimeter, which that detects only intraoral halitosis. its hardware, though, needs revision16. halitosis can also be assessed by the analysis of saliva viscosity, color and amount of secretion. in some cases, laboratory tests may be necessary to investigate possible systemic causes and microbiological tests can also be performed in outbreaks of disease7. the dental professional’s responsibility dental practitioners have traditionally neglected halitosis, is spite of its high priority for the public. however, interest in halitosis has recently increased. dentists are increasingly braz j oral sci. 7(26):1627-1630 halitosis: a review of basic principles 1629 being called on to help patients with complaints of bad breath and should consider psychological and physiological factors in diagnosing and treating such cases17. patients who do not visit a dentist regularly are at high risk of having halitosis from dental plaque, dental caries or periodontitis. it is important to ascertain whether it is subjective or objective halitosis. most cases are objective, but subjective halitosis may occur due to use of medications, such as lithium, zinc deficiency or in association with some psychoses18. the general dentist is frequently the first health professional to examine and treat patients with halitosis of extraoral etiology, and is their responsibility to refer these patients to an otorhinolaryngologist in order to rule out the presence of chronic tonsillitis or chronic sinusitis. if the otorhinolaryngologist does not detect alterations in the examined regions, the patient should be referred to other medical specialists to explore other organs and systems: the digestive system, to rule out gastric pathology, obstructions or inflammatory gastrointestinal processes; the liver, to rule out hepatic insufficiency or cirrhosis; the endocrine system, to exclude diabetes or trimethylaminuria; the airways, to rule out bronchiectasis or pulmonary abscesses; and the kidney, to exclude renal failure. finally, if no systemic pathology is detected, the possibility of halitosis of psychiatric etiology should be considered and requires evaluation of the patient’s psychological profile by an specialist19. one of the most intriguing issues regarding halitosis is the apparent incapacity to determine whether someone has this condition and to what extent. several people have bad breath for years without being at all aware of it. others overestimate their own oral malodor and are more prone to developing an obsessive behavior, avoiding social interactions2. people who complain about having halitosis may harbor a selfperception that does not reflect objective findings. to deal with these patients, clinicians should investigate not only the physiological causes of malodor and associated parameters, but also the nature of the subjective complaint. in addition, because of the multifactorial complexity of the problem, patients with halitosis should be treated individually, rather than categorized. instead of informing a patient that he/she does not have halitosis, clinicians can suggest that there might be an odor, but that it is barely detectable and is not evident at the time of consultation. the dentist can then recommend appropriate oral hygiene measures, again providing the patient with a sense of increased control over the problem17. in such cases, a condition that causes malodor can be originated from dry mouth (xerostomy) and associated with uncomfortable symptoms of altered taste, mucosal roughness and burning tongue, characterizing a sort of pseudo-halitosis. recognizing this condition is of importance in patient care because treatment of pseudo-halitosis as genuine halitosis, i.e., with oral products like alcohol-containing mouthwashes, can aggravate the case. xerostomia can be caused by a number of factors, including some medications and stress, which lead to salivary gland secretion and effects on the production of volatile sulfur compounds and malodor20. unfortunately, most halitophobic patients, those with imaginary halitosis, refuse to acknowledge that they may have a psychological problem, which prevents them from receiving adequate psychological treatment and prolongs their suffering and social isolation. additional guidance from mental health professionals will help dentists providing support to these patients18. it is important that dental professionals have in mind the treatment protocol and/or products to be prescribed for patients with halitosis should be based on an individualized evaluation rather than in remedies and standardized regimens available for sale to patients. dentists can play a major role in helping these patients by eliminating caries and periodontal diseases, with an approach based on counseling and clinical procedures that include4: 1. oral hygiene instructions to reinforce toothbrushing, flossing and denture hygiene; 2. scaling and root planing on regions of periodontal pockets, and tongue cleaning; 3. chemical control of biofilm with mouthwashes; 4. dietary advice to reinforce mouth cleaning after eating or drinking dairy products, fish, meat, garlic, onion, coffee, and after smoking. asking an experienced health professional is always the best way to confirm a breath odor problem. this, however, can be awkward and embarrassing for both the patient and the dentist, who has historically been hesitant to broach the subject. since the dental office is the most indicate place to investigate and treat halitosis, it is important that dentist develop their communication skills and knowledge in order to respond to patients that seek information and treatment for halitosis. because certain medical conditions or medications can be the source of this problem, making a comprehensive review of medical/dental history, including questions about breath complaints, can lay the groundwork for open dialogue about breath problems. when patients initiate a dialogue about their breath concerns, dental professionals should be comfortable with explaining the etiology of and treatments for oral malodor10. health-care professionals should be aware that the patient seeks not only treatment for his/her condition, but also support and understanding. it is the patient’s right to be offered all currently available treatments options and be given full information to understand his/her condition and make a decision in his/her best interest. r e f e r e n c e s 1. tangerman a. halitosis in medicine: a review. int dent j. 2002; 52: 201-6. 2. rosenberg m, kozlovsky a, gelernter i, cherniak o, gabbay j, baht r et al. self-estimation of oral malodor. j dent res. 1995; 74: 1577-82. 3. rosenberg m. clinical assessment of bad breath: current concepts. j am dent assoc. 1996; 127: 475-82. 4. lee pp, mak wy, newsome p. the aetiology and treatment of oral halitosis: an update. hong kong med j. 2004; 10: 414-8. braz j oral sci. 7(26):1627-1630 halitosis: a review of basic principles 1630 5. elias ms, ferriani mgc. historical and social aspects of halitosis. rev latino-am enfermagem. 2006; 14:821-3. 6. hine mk. halitosis. j am dent assoc. 1957; 55: 37-46. 7. ayers kms, colquhoun ank. halitosis: causes, diagnosis, and treatment. n z dent j. 1998; 94: 156-60. 8. sanz m, rolda s, herrera d. fundamentals of breath malodour. j contemp dent pract. 2001; 2: 1-17. 9. lee ss, zhang w, li y. halitosis update: a review of causes, diagnoses, and treatments. j calif dent assoc. 2007; 35: 258-60. 10. lenton p, majerus g, bakdash b. counseling and treating bad breath patients: a step-by-step approach. j contemp dent pract. 2001; 2: 46-61. 11. what you should know about bad breath. j am dent assoc. 2003; 134: 135. 12. tanaka m, anguri h, nishida n, ojima m, nagata h, shizukuishi s. reliability of clinical parameters for predicting the outcome of oral malodor treatment. j dent res. 2003; 82: 518-22. 13. rosenberg m, mcculloch ca. measurement of oral malodor: current methods and future prospects. j periodontol. 1992; 63: 776-82. 14. almas k, al-hawish a, al-khamis w. oral hygiene practices, smoking habits, and self-perceived oral malodor among dental students. j contemp dent pract. 2003; 4: 77-90. 15. rosenberg m, knaan t, cohen d. association among bad breath, body mass index, and alcohol intake. j dent res. 2007; 86: 9971000. 16. tangerman a, winkel eg. the portable gas chromatograph oralchromatm: a method of choice to detect oral and extra-oral halitosis. j. breath res. 2008; 2: 017010 (6 p.). 17. eli i, baht r, koriat h, rosenberg m.self-perception of breath odor. j am dent assoc. 2001; 132: 621-6. 18. tomás carmona i, limeres posse j, diz dios p, fernández feijoo j, vázquez garcía e. extraoral etiology of halitosis. med oral. 2001; 6: 40-7. 19. attia el, marshall kg. halitosis. can med assoc j. 1982; 126: 1281-5. 20. kleinberg i, wolff ms, codipilly dm. role of saliva in oral dryness, oral feel and oral malodour. int dent j. 2002; 52: 236-40. braz j oral sci. 7(26):1627-1630 halitosis: a review of basic principles 1http://dx.doi.org/10.20396/bjos.v18i0.8657248 volume 18 2019 e191417 original article 1 department of periodontology, school of dentistry, veiga de almeida university (uva), brazil. 2 department of periodontology, school of dentistry, rio de janeiro state university (uerj), brazil. corresponding author: antonio canabarro, dds, phd department of periodontology, veiga de almeida university (uva), rua ibituruna 108, casa 3, sala 201. cep 20271-020, tijuca, rio de janeiro, rj, brazil. fax: +55-21-25748871 e-mail: canabarro@uva.br conflict of interest: the authors declare that they have no conflict of interest received: september 27, 2018 accepted: june 14, 2019 putative periodontal bacteria in clinically healthy and diseased sites of periodontitis patients carlos eduardo barros1, vivian siqueira1, dennis carvalho1, antonio canabarro1,2,* aim: the aim of this study was to compare the microbial profile of subgingival sites in periodontitis (pd) patients and healthy ones. methods: eighteen patients with pd and 18 gender-matched healthy controls were selected. subgingival samples were collected from three types of sites: 1) healthy site of healthy subjects (probing pocket depth (ppd) ≤ 3mm, cg), 2) healthy site of pd patients (ppd ≤ 3mm, pg-c) and 3) diseased site (ppd > 3mm) of the same pd patients (pg-t). all sites were subjected to microbial analysis for the detection of 40 bacterial species by the “checkerboard dna-dna hybridization” technique. results: it was observed a great diversity of bacteria in all patients evaluated. the sites from the pd groups (pg-t and pg-c) showed a higher overall count of the studied bacteria than those of the cg group, especially from green, orange, and red complexes. also, pg-t showed a higher prevalence of red complex bacteria than cg. individual pathogens, such as porphyromonas gingivalis, treponema denticola and treponema socranskii were detected in higher levels and/or prevalence in pd than in control patients. however, it was not observed any difference between pg-t and pg-c. conclusion: pd patients showed higher prevalence and counts of some putative periodontal bacteria, especially from the red complex, than control ones, regardless of the severity of their sites. keywords: periodontitis. bacteria. molecular biology. 2 barros et al. introduction gingivitis and periodontitis are diseases that affect many people worldwide. while gingivitis is considered a reversible marginal inflammation, periodontitis causes irreversible destruction of the supporting tissues of the teeth, resulting in the formation of periodontal pockets and eventually in tooth loss1. in periodontal disease, microorganisms adhered to the tooth surface in the biofilm and can release a large number of inflammatory mediators in the adjacent periodontal tissues. these microorganisms cause inflammation and, in many cases destroy of these tissues2. periodontal pockets can harbor over 500 bacterial species that are mostly resident species. however, a part of these are potentially pathogenic3 and may result, under certain circumstances, in an infection due to the excessive release of inflammatory mediators4,5. in the 1990s, the theory that biofilm contained specific bacteria gained strength. six microbial complexes with distinct characteristics seem to be involved in the formation of subgingival biofilm in sequential phases. the red complex microorganisms, porphyromonas gingivalis, tannerella forsythia, and treponema denticola, are potential etiologic agents of periodontitis6-9. although these bacteria allegedly have an important role in the pathogenesis of periodontal disease, there does not seem to be a single causative agent in inflammatory periodontal diseases. gram-positive bacteria, anaerobes, and facultative organisms, as well as viruses and fungi, have also been associated with periodontitis10,11. in fact, periodontal pathogens can also be detected in healthy individuals although at low levels12. large communities of microorganisms, collectively called microbiomes, inhabit the surfaces of our body13, including teeth. the diversity and abundance of these communities are huge13, and this situation is no different in the mouth. recent studies indicate that the participation of specific pathogens is not as obvious as we previously thought when considering both periodontitis and dental caries. both diseases appear to result from an imbalance among the constituents of bacterial communities, resulting in dysbiosis14. therefore, identifying certain bacteria in individuals with and without periodontal disease in healthy and diseased sites using modern techniques based on dna identification may be an interesting way to understand the etiology of periodontitis in greater depth. the study aimed to assess the microbial profile of healthy and diseased sites of patients with periodontitis using the checkerboard dna-dna hybridization technique and to compare it to data from control subjects. materials and methods patients eighteen patients of both sexes were enrolled in this controlled cross-sectional study. the following inclusion criteria, based on the criteria of the 2017 world 3 barros et al. workshop on the classification of periodontal and peri-implant diseases and conditions15 were used: if interdental clinical attachment loss (cal) is detectable at ≥2 non-adjacent teeth, or buccal or oral cal ≥3 mm with probing pocket depth (ppd) >3 mm is detectable at ≥2 teeth (periodontitis group pg). they were compared to 18 gender-matched healthy control patients of both sexes without periodontitis (no cal, control group cg). all participants were recruited as they came to the clinic for treatment and met the inclusion criteria of the study. after anamnesis, all patients signed an informed consent form. the study was approved by the ethics committee on human research, under number 1397046. the exclusion criteria were: periodontal treatment for at least a year, pregnancy, lactation, use of prostheses, medical conditions that could affect the existence of bacteria in periodontal tissues (e.g., hiv, antibiotic or non-steroidal anti-inflammatory therapy for at least six months). sample collection after the clinical examination, cotton-rolls and saliva-ejector were used to keep teeth dry. subgingival samples were collected from three types of sites: 1) healthy site of healthy subjects (cg), 2) healthy site of subjects with periodontitis (pg-c) and 3) diseased site (ppd > 3mm) of periodontitis subjects (pg-t). pg-t and pg-c sites were selected from the same patients. subgingival biofilm samples were collected using one sterile paper point, size 45 (dentisply, petropolis, rio de janeiro, brazil) for each tooth. only one site per tooth was studied. the most severe site was chosen for analysis in pg-t, while healthy sites were randomly selected in pg-c and cg. paper points were introduced into periodontal pockets (diseased sites) or gingival sulcus (control sites) for at least 30 seconds. the samples were immediately placed into individual plastic tubes containing 150 μl of te buffer solution (10mm tris-hcl (invitrogen life technologies, carlsbad, ca, usa), 1 mm edta (labsynth products for laboratories ltd., diadema, sp brazil), ph 7.6), and then 100 ul of 0.5 m naoh (labsynth) was added so that the bacterial dna remained viable for a longer period of time. these plastic tubes were previously labeled with the individual’s name, date, and site; and, after collection, were stored under refrigeration at -20°c until the samples were analyzed by the dna-dna hybridization checkerboard technique for bacterial strains. checkerboard dna-dna hybridization counts of 40 bacterial species were determined using the checkerboard dnadna hybridization technique8. the analyses were performed at the microbiology laboratory of guarulhos university as previously described16,17. table 1 presents the 40 reference strains used to develop the dna probes according to the bacterial complexes6,9. the readings of the radiographic films were carried out by a single trained examiner, calibrated and blind to the objectives of the study. readings were performed on different days to verify the results. each signal produced by a given plaque sample probe 4 barros et al. was compared in intensity to the signal produced by the same probe in the two control lines containing 105 and 106 bacteria. the number 0 was recorded when no signal was detected; 1 was equivalent to a less intense signal than the control of 105 cells; 2 was equivalent to 105 cells; 3 was between 105 and 106 cells; 4 was equal to or approximately 106 cells, and 5 was more than 106 cells. these logs were used to determine the levels of different species investigated in the different samples under evaluation. the sensitivity of the assay allowed the detection of 10,000 units of each bacterial species studied by adjusting the concentration of each dna probe. the number of bacteria in each site was estimated according to the signal intensity number: 0 = 0, 1 = 10,000, 2 = 100,000 3 = 500,000, 4 = 1,000,000 and 5 = 10,000,000. table 1. list of the bacterial strains used for the preparation of dna probes. the species are grouped by bacterial complexes.6,17 species strain species strain blue complex orange complex (cont.) actinomyces gerencseriae 23860a fusobacterium nucleatum ssp nucleatum 25586a actinomyces israelii 12102a fusobacterium nucleatum ssp polymorphum 10953a actinomyces naeslundii 1 12104a fusobacterium nucleatum ssp vincentii 49256a actinomyces naeslundii 2 43146a fusobacterium periodonticum 33693a purple complex parvimonas micra 33270a actinomyces odontolyticus 17929a prevotella intermedia 25611a veillonella parvula 10790a prevotella nigrescens 33563a yellow complex streptococcus constellatus 27823a streptococcus gordonii 10558a red complex streptococcus intermedius 27335a tannerella forsythia 43037a streptococcus mitis 49456a porphyromonas gingivalis 33277a streptococcus oralis 35037a treponema denticola b1b streptococcus sanguinis 10556a other species green complex eubacterium saburreum 33271a aggregatibacter actinomycetemcomitans a + b 43718a 29523a gemella morbillorum 27824a leptotrichia buccalis 14201a capnocytophaga gingivalis 33624a neisseria mucosa 19696a capnocytophaga ochracea 33596a prevotella melaninogenica 25845a capnocytophaga sputigena 33612a propionibacterium acnes i + ii 11827a eikenella corrodens 23834a 11828a orange complex selenomonas noxia 43541a campylobacter gracilis 33236a streptococcus anginosus 33397a campylobacter rectus 33238a treponema socranskii s1b campylobacter showae 51146a eubacterium nodatum 33099a a atcc (american type culture collection); b forsyth institute 5 barros et al. statistical analysis statistical evaluation was performed using spss  statistics version 17 (ibm, armonk, ny,  usa). initially, the normal distribution of data was checked with the kolmogorov-smirnov test. subsequently, the non-parametric kruskal-wallis test and the chi-square test (χ2) were used to analyze the prevalence and proportion of the positive sites for different types of bacteria, i.e., sites with values ≥1, and the nonparametric mann-whitney test was used to evaluate the differences in the bacteria count among the three types of sites. the analysis unit was the patient. the significance level of 5% was established for the remaining analyzes. the sample size was calculated using the percentage of sites with p. gingivalis as the primary outcome variable. pg-t showed 80% and cg 30% of sites colonized by these bacteria based on a pilot study (data not shown). considering a statistical power of 85% and a 95% confidence level, a total of 18 individuals per group was needed. results eighteen patients, 12 female and 6 male, aged between 30 and 70 years old (mean age 51.50 ±14.24), with a mean cal of 4.72 (±1.40) mm (periodontitis group pg), and 18 gender-matched periodontally and systemically healthy control patients (control group cg), aged between 20 and 30 years old (mean age 24.65 ±3.12) were included in this study (table 2). diseased (pg-t) and healthy sites (pg-c) from the same periodontitis patients and healthy sites from control ones (cg) were analyzed using the checkerboard dna-dna hybridization technique. the overall prevalence of the bacterial species evaluated did not differ among groups (p= 0.131). pg-t, pg-c, and cg showed 72%, 64% and 49% of positive sites, respectively. the most prevalent bacteria in pg-t were t. socranskii (100%) and actinomyces naeslundii 1 (94%). in the pg-c, the most prevalent bacteria were actinomyces gerencseriae (100%) and t. denticola (94%). finally, in the cg the most prevalent were fusobacterium nucleatum ssp vincentii (84%) and a. naeslundii (77%) (figure 1). on evaluating each bacterium individual, pg-t and pg-c groups compared to cg showed higher prevalence of: a. gerencseriae (pg-t (94%)=pg-c (100%)>cg (28%), p< 0.001), table 2. characteristics of studied individuals. characteristics pg (n= 18) cg (n= 18) age (mean and sd) 51.5 (14.2) 24.7 (3.1)* gender: male / female 6/12 6/12 number of teeth (mean and sd) 21.4 (6.3) 24.5 (5.6)* ppd (mm, mean and sd) 4.5 (1.8) cal (mm, mean and sd)** 4.7 (1.4) *statistical difference between groups (p< 0.05). ppd = periodontal pocket depth; cla = clinical attachment loss. pg= periodontitis group; cg= control group. 6 barros et al. a. naeslundii 1 (pg-t (94%)=pg-c (72%)>cg (50%), p= 0.012), streptococcus sanguinis (pg-t (83%)=pg-c (78%)>cg (28%), p= 0.001), capnocytophaga sputigena (pg-t (83%)=pg-c (72%)>cg (33%), p= 0.005), streptococcus constellatus (pg-t (72%)=pg-c (67%)>cg (28%), p< 0.014) and t. socranskii (pg-t (100%)=pg-c (89%)>cg (50%), p< 0.001) (figure 1). f. nucleatum ssp vincentii was the only bacteria statistically more prevalent in cg and pg-t than in pg-c (cg (84%)= pg-t (61%)>pg-c (33%), p< 0.009) (figure 1). figure 1. prevalence of the 40 bacterial species evaluated in the three groups (healthy – control, cg; periodontitis – test, pg-t and periodontitis – control, pg-c). statistically significant differences among groups were evaluated by chi-square test (*). treponema socranskii streptococcus anginosus selenomonas noxia propionibacterium acnes i+ii prevotella melaninogenica neisseria mucosa leptotrichia buccalis gemella morbillorum eubacterium saburreum treponema denticola porphyromonas gingivalis tannerella forsythia streptococcus constellatus prevotella nigrescens prevotella intermedia parvimonas micra fusobacterium periodonticum fusobacterium nucleatum ssp vincentii fusobacterium nucleatum ssp polymorphum fusobacterium nucleatum ssp nucleatum eubacterium nodatum compylobacter showae compylobacter rectus compylobacter gracilis eikenella corrodens capnocytophaga sputigena capnocytophaga ochracea capnocytophaga gingivalis a. actinomycetemcomitans streptococcus sanguinis streptococcus oralis streptococcus mitis streptococcus intermedius streptococcus gordonii veilonella parvula actinomyces odontolyticus actinomyces naeslundii actinomyces naeslundii 1 actinomyces israelii actinomyces gerencseriae positive sites (%) * * * * * * * cg pg-t 0% 20% 40% 60% 80% 100% pg-c 7 barros et al. comparison of positive sites and bacterial complexes demonstrated a statistical difference in the red complex only (figure 2). a higher number of positive sites were found in diseased sites of pd patients compared to cg (pg-t (72% ±31)>cg (44 ±36), p= 0.027; pg-t (72% ±31)= pg-c (59% ±27), p= 0.171 and pg-c (59% ±27)=cg (44 ±36), p= 0.226). figure 2. proportion (mean % and dp) of bacterial complexes in different groups (healthy – control, cg; periodontitis – test, pg-t and periodontitis – control, pg-c). statistically significant differences among groups were evaluated by kruskal-wallis followed by mann-whitney test (*). treponema socranskii streptococcus anginosus selenomonas noxia propionibacterium acnes i+ii prevotella melaninogenica neisseria mucosa leptotrichia buccalis gemella morbillorum eubacterium saburreum treponema denticola porphyromonas gingivalis tannerella forsythia streptococcus constellatus prevotella nigrescens prevotella intermedia parvimonas micra fusobacterium periodonticum fusobacterium nucleatum ssp vincentii fusobacterium nucleatum ssp polymorphum fusobacterium nucleatum ssp nucleatum eubacterium nodatum compylobacter showae compylobacter rectus compylobacter gracilis eikenella corrodens capnocytophaga sputigena capnocytophaga ochracea capnocytophaga gingivalis a. actinomycetemcomitans streptococcus sanguinis streptococcus oralis streptococcus mitis streptococcus intermedius streptococcus gordonii veilonella parvula actinomyces odontolyticus actinomyces naeslundii actinomyces naeslundii 1 actinomyces israelii actinomyces gerencseriae positive sites (%) cg pg-t 0% 20% 40% 60% 80% 100% pg-c * * * * * * * 8 barros et al. the overall mean count of the bacterial species evaluated differed among groups (p= 0.002). pg-t, pg-c, and cg showed 2.35 x105 (±2.25), 1.85 x105 (±2.25) and 0.45 x105 (±0.50) of bacteria, respectively (pg-t=pg-c>cg). when evaluating the mean count of each bacteria, no significant difference was found between the pg-t and pg-c groups. however, at the pg-t group sites compared to the cg ones there were significantly more (x105 ±dp): a. gerencseriae (5.28 ±4.22 vs 0.18 ±0.38, p< 0.001), veillonella parvula (3.15 ±3.82 vs 0.06 ±0.05, p= 0.001), s. sanguinis (0.78 ±1.58 vs 0.03 ±0.05, p< 0.001), capnocytophaga gingivalis (2.24 ±2.87 vs 0.15 ±0.31, p= 0.008), c. sputigena (1.75 ±2.72 vs 0.18 ±0.38, p= 0.006), f. nucleatum ssp nucleatum (4.62 ±4.33 vs 0.19 ±0.38, p= 0.002), p. gingivalis (13.02 ±31.86 vs 0.08 ±0.23, p< 0.011), t. denticola (3.79 ±3.98 vs 0.41 ±0.48, p= 0.008), leptotrichia buccalis (9.62 ±22.99 vs 0.25 ±0.41, p= 0.008) and t. socranskii (3.91 ±3.48 vs 0.30 ±0.45, p< 0.001) (figure 3). all the bacteria above mentioned were also in higher number in the pg-c group compared to the cg sites, except p. gingivalis (0.86 ±2.32 vs. 0.08 ±0.23, pg-c=cg, p< 0.118) (figure 3). comparison of mean bacterial count and complexes demonstrated that there were more green (p= 0.012), orange (p= 0.001), red (p= 0.002) complexes and others bacteria (p= 0.010) in pg-t and pg-c than in cg (pg-t=pg-c>cg) (figure 4). discussion several studies have demonstrated the relationship between colonization of specific microorganisms and the presence and/or severity of periodontal disease. the bacteria involved in deep periodontal pockets are mainly of the red complex such as p. gingivalis, t. denticola and t. forsythia (formerly bacteroides forsythus)18. moore et al.19 observed, in patients with periodontal disease, species such as p. gingivalis, eubacterium nodatum, eubacterium timidum, eubacterium brachy, and peptostreptococcus anaerobius. corroborating with it, here, red complex, as well as green and orange ones, were detected in higher levels in pd patients than in control ones, including either diseased or healthy sites. also, pd patients harbored higher proportion and/or counts than cg of the following bacteria: a. gerencseriae, s. sanguinis, c. sputigena, p. gingivalis, t. denticola, and t. socranskii. however, we observed that both healthy and diseased sites of pd patients presented similar prevalence and counts of bacteria, contradicting findings of a former study which showed that levels of red complex bacteria seem to be related to periodontitis severity18. it is important to note that this study only assessed deep sites18. furthermore, red complex can be detected in both supraand subgingival samples as well as in healthy and diseased sites from periodontitis patients7,8. although the literature suggests that the  levels  of specific gram-negative organisms in subgingival plaque biofilm play a major role in the initiation and progression of the disease, there is little evidence in the literature on the correlation of the levels  of periodontal pathogens of sites with different pocket depth with periodontal disease activity20. 9 barros et al. periodontal pathogens are necessary but are not sufficient by themselves to provoke periodontal disease, and depend on risk factors, genetic factors and the immunological response of the host. so, as shown by our study, periodontitis cannot be strictly considered a site-specific infectious disease but the outcome of a polymicrobial dysbiosis12. the microbial ecology is the relationship between the microorfigure 3. mean levels (and sd) of the 40 bacterial species evaluated in the three groups (healthy – control, cg; periodontitis – test, pg-t and periodontitis – control, pg-c). statistically significant differences among groups were evaluated by kruskal-wallis followed by mann-whitney test (*). treponema socranskii streptococcus anginosus selenomonas noxia propionibacterium acnes i+ii prevotella melaninogenica neisseria mucosa leptotrichia buccalis gemella morbillorum eubacterium saburreum treponema denticola porphyromonas gingivalis tannerella forsythia streptococcus constellatus prevotella nigrescens prevotella intermedia parvimonas micra fusobacterium periodonticum fusobacterium nucleatum ssp vincentii fusobacterium nucleatum ssp polymorphum fusobacterium nucleatum ssp nucleatum eubacterium nodatum compylobacter showae compylobacter rectus compylobacter gracilis eikenella corrodens capnocytophaga sputigena capnocytophaga ochracea capnocytophaga gingivalis a. actinomycetemcomitans streptococcus sanguinis streptococcus oralis streptococcus mitis streptococcus intermedius streptococcus gordonii veilonella parvula actinomyces odontolyticus actinomyces naeslundii actinomyces naeslundii 1 actinomyces israelii actinomyces gerencseriae bacterial count (x105) cg pg-t 0 10 20 30 40 50 pg-c * * * * * * * * * 10 barros et al. ganisms and their habitat. microbial homeostasis is the result of the dynamic balance of microbial interactions, including synergism and antagonism21. according to oliveira et al.22 the mere presence of putative periodontal pathogens in the gingival sulcus is not enough to cause periodontal inflammation. the hypothesis that disease can be prevented not only by the inhibition of pathogens but also by interfering with the factors responsible for the transition of commensal biofilm microbiota to pathogenic microbiota, has been postulated23,24. thus, perturbations in the structure of commensal communities (dysbiosis) can lead to a host immune deficiency and the subsequent development of diseases mediated by the immune system. these changes in microbial composition are factors contributing to the initiation and/or persistence of many diseases25-27; they are characterized by the loss of beneficial organisms, expansion of potentially pathogenic microorganisms or by the loss of global microbial diversity28,29. the search for the etiological factors of periodontitis, as well as any disease, is related to a dynamic process in which several microbial species dominate the biofilm at different stages of the infection due to changes in nutrient availability, oxygen level, and local ph24. therefore, the knowledge of the ecological relationships between bacterial species in periodontitis should direct and focus the research to a critical bacterial interaction30, since polymicrobial infectious diseases such as periodontal diseases appear to be caused more by an imbalance of inter-microbial relationship in the subgingival site, as shown in this study, rather than by specific isolated bacteria. so, although periodontitis is considered a site-specific disease associated with red complex bacteria, the findings of this study seem to indicate a patient-associated microbial profile rather than a site-specific microbiota. based on it, the use of sysfigure 4. levels (mean and sd) of bacterial complexes in different groups (healthy – control, cg; periodontitis – test, pg-t and periodontitis – control, pg-c). statistically significant differences among groups were evaluated by kruskal-wallis followed by mann-whitney test (*). cg pg-t pg-c * * * * 20.00 18.00 0.00 2.00 4.00 blue purple yellow green orange red others 16.00 14.00 12.00 10.00 8.00 6.00 bacterial complex (count x 105) 11 barros et al. temic antibiotics in the treatment of periodontal disease may be considered interesting, since it would reduce the presence and number of putative bacteria in the whole oral cavity. indeed, adjunctive use of metronidazole showed a greater reduction in the levels of periodontal pathogens in pd patients compared to mechanical control alone31. however, it is important to note that good oral hygiene continues to be fundamental in the long-term control of the disease. poor oral hygiene in pd patients diminishes the beneficial effects of any treatment32. although pd and cg groups were matched by gender to avoid the influence of this factor on the results, this study has some limitations. it is important to highlight the relatively small sample size and the age of subjects. pd patients were older than clinically healthy ones due to the difficult to find elderly subjects with no signs of periodontitis. in conclusion, pd patients showed higher prevalence and counts of some putative periodontal bacteria, especially from the red complex, than control ones, regardless of the severity of their sites. acknowledgments we acknowledge the financial support of faperj and funadesp. references 1. philstrom bl, ammons wf. treatment of gingivitis and periodontitis. research, science and therapy committee of the american academy of periodontolgy. j periodontol. 1997 dec;68(12):1246-53. 2. hienz sa, paliwal s, ivanovski s. mechanisms of bone resorption in periodontitis. j immunol res. 2015;2015:615486. doi: 10.1155/2015/615486. 3. jarvensivu a, hietanen j, rautemaa r, sorsa t, richardson m. candida yeasts in chronic periodontitis tissues and subgingival microbial biofilms in vivo. oral dis. 2004 mar;10(2):106-12. 4. darby i, curtis m. microbiology of periodontal disease in children and young adults. periodontol 2000. 2001;26:33-53. 5. khan sa, kong ef, meiller tf, jabra-rizk ma. periodontal diseases: bug induced, host promoted. plos pathog. 2015 jul 30;11(7):e1004952. doi: 10.1371/journal.ppat.1004952. 6. socransky ss, haffajee ad. periodontal microbial ecology. periodontol 2000. 2005;38:135-87. 7. socransky ss, haffajee ad, smith c, dibart s. relation of counts of microbial species to clinical status at the sample sites. j clin periodontol. 1991 nov;18(10):766-75. 8. ximénez-fyvie la, haffajee ad, socransky ss. comparison of the microbiota of supraand subgingival plaque in health and periodontitis. j clin periodontol. 2000 sep;27(9):648-57. 9. socransky ss, haffajee ad, cugini ma, smith c, kent jr rl. microbial complexes in subgingival plaque. j clin periodontol. 1998 feb;25(2):134-44. 10. koshy g, corbet ef, ishikawa i. a full-mouth disinfection approach to nonsurgical periodontal therapy – prevention of reinfection from bacterial reservoirs. periodontol 2000. 2004;36:166-78. 11. canabarro a, valle c, farias mr, santos fb, lazera m, wanke b. association of subgingival colonization of candida albicans and other yeasts with severity of chronic periodontitis. j periodontal res. 2013 aug;48(4):428-32. doi: 10.1111/jre.12022. 12 barros et al. 12. camelo-castillo aj, mira a, pico a, nibali l, henderson b, donos n et al. subgingival microbiota in health compared to periodontitis and the influence of smoking. front microbiol. 2015 feb 24;6:119. doi: 10.3389/fmicb.2015.00119. 13. oever jt, netea mg. the bacteriome-mycobiome interaction and antifungal host defense. eur j immunol. 2014 nov;44(11):3182-91. doi: 10.1002/eji.201344405.. 14. costalonga m, herzberg mc. the oral microbiome and the immunobiology of periodontal disease and caries. immunol lett. 2014 dec;162(2 pt a):22-38. doi: 10.1016/j.imlet.2014.08.017. 15. papapanou pn, sanz m, buduneli n, dietrich t, feres m, fine dh, et al. periodontitis: consensus report of workgroup 2 of the 2017 world workshop on the classification of periodontal and peri-implant diseases and conditions. j periodontol. 2018 jun;89 suppl 1:s173-s182. doi: 10.1002/jper.17-0721. 16. matarazzo f, figueiredo lc, cruz se, faveri m, feres m. clinical and microbiological benefits of systemic metronidazole and amoxicillin in the treatment of smokers with chronic periodontitis: a randomized placebo-controlled study. j clin periodontol. 2008 oct;35(10):885-96. doi: 10.1111/j.1600-051x.2008.01304.x. 17. lima ja, santos vr, feres m, de figueiredo lc, duarte pm. changes in the subgingival biofilm composition after coronally positioned flap. j appl oral sci. 2011 jan-feb;19(1):68-73. 18. farias bc, souza pr, ferreira b, melo rs, machado fb, gusmão es et al. occurrence of periodontal pathogens among patients with chronic periodontitis. braz j microbiol. 2012 jul;43(3):909-16. doi: 10.1590/s1517-83822012000300009. 19. moore lv, moore we, cato ep, smibert rm, burmeister ja, best am et al. bacteriology of human gingivitis. j dent res. 1987 may;66(5):989-95. 20. dosseva-panova vt, popova cl, panov ve. subgingival microbial profile and production of proinflammatory cytokines in chronic periodontitis. folia med (plovdiv). 2014 jul-sep;56(3):152-60. 21. hajishengallis g. periodontitis: from microbial immune subversion to systemic inflammation. nat rev immunol. 2015 jan;15(1):30-44. doi: 10.1038/nri3785. 22. oliveira lf, jorge aoc, santos ss f. in vitro minocycline activity on superinfecting microorganisms isolated from chronic periodontitis patients. braz oral res. 2006 jul-sep;20(3):202-6. 23. takahashi n. oral microbiome metabolism: from “who are they?” to “what are they doing?”. j dent res. 2015 dec;94(12):1628-37. doi: 10.1177/0022034515606045. 24. kolenbrander pe, egland pg, diaz pi, palmer rj jr. genome interactions: bacterial communities in initial dental plaque. trends microbiol. 2005 jan;13(1):11-5. 25. frank dn, st amand al, feldman ra, boedeker ec, harpaz n, pace nr. molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel disease. proc natl acad sci u s a. 2007 aug 21;104(34):13780-5. 26. karlsson fh, tremaroli v, nookaew i, bergström g, behre cj, fagerberg b et al. gut metagenome in european women with normal, impaired and diabetic glucose control. nature. 2013 jun 6;498(7452):99-103. doi: 10.1038/nature12198. 27. abrahamsson tr, jakobsson he, andersson af, björkstén b, engstrand l, jenmalm mc. low gut microbiota diversity in early infancy precedes asthma at school age. clin exp allergy. 2014 jun;44(6):842-50. doi: 10.1111/cea.12253. 28. petersen c, round jl. defining dysbiosis and its influence on host immunity and disease. cell microbiol. 2014 jul;16(7):1024-33. doi: 10.1111/cmi.12308. 29. gootz td. the global problem of antibiotic resistance. crit rev immunol. 2010;30(1):79-93. 30. ng hm, kin lx, dashper sg, slakeski n, butler ca, reynolds ec. bacterial interactions in pathogenic subgingival plaque. microb pathog. 2016 may;94:60-9. doi: 10.1016/j.micpath.2015.10.022. 13 barros et al. 31. soares gm, mendes ja, silva mp, faveri m, teles r, socransky ss et al. metronidazole alone or with amoxicillin as adjuncts to non-surgical treatment of chronic periodontitis: a secondary analysis of microbiological results from a randomized clinical trial. j clin periodontol. 2014 apr;41(4):366-76. 32. mdala i, olsen i, haffajee ad, socransky ss, de blasio bf, thoresen m. multilevel analysis of bacterial counts from chronic periodontitis after root planing/scaling, surgery, and systemic and local antibiotics: 2-year results. j oral microbiol. 2013 jul 9;5. doi: 10.3402/jom.v5i0.20939. 1http://dx.doi.org/10.20396/bjos.v20i00.8662755 volume 20 2021 e212755 original article 1 department of conservative dentistry and prosthodontics, al-quds university, palestine. 2 department of conservative dentistry and prosthodontics, arab american university, jenin, palestine. corresponding author: dr. naji ziad arandi assistant professor (operative dentistry) department of conservative dentistry and prosthodontics faculty of dentistry, arab american university, jenin palestine p.o box 240 jenin,13 zababdeh. telephone: 00972598126111 editor: dr altair a. del bel cury received: november 21, 2020 accepted: march 9, 2021 restorative treatment decisions regarding approximal and occlusal carious lesions among general dental practitioners in palestine tarek rabi , naji ziad arandi aim: to investigate restorative decisions made by dentists and to examine what demographic characteristics are associated with the decisions for managing approximal and occlusal lesions. methods: a questionnaire was randomly sent to 900 palestinian dentists. it noted the demographic details of the dentists and the years of experience. the questionnaire evaluated the respondents for their treatment decisions regarding approximal and occlusal carious lesions. the data was analyzed using the ibm spss statistics for windows. the associations between gender and years of experience of the respondents and their restorative decisions were assessed. results: the response rate was 58.2%. for occlusal carious lesions, 93.9% of the respondents would postpone operative treatment until the lesion was in dentine (grade 3 to 5). for approximal lesions, intervention was deemed appropriate by 92.6% of the respondents when there was radiographic evidence of a carious lesion reaching the dej or deeper. around 53% preferred to prepare approximal lesions according to the traditional principles of cavity preparation. for both approximal and occlusal lesions, the participants opted for resin composites. statistically, there was a significant association between the restorative decisions with the years since graduation and gender. conclusion: the study showed variations between the treatment decisions of palestinian dentists. the subjects chose conservative treatment plans but still adhered to traditional learned practices especially when cavity preparation for approximal lesions was concerned. the years since graduation and gender played a significant role in the choice of treatment opted for. resin composites seemed to be a popular choice for treatment. keywords: dental caries. dental cavity preparation. practice patterns, dentists’. population characteristics. composite resins. https://orcid.org/0000-0003-0015-6382 https://orcid.org/0000-0001-5961-6975 2 rabi and arandi introduction dentists all over the world face challenges with cases that are at various stages of caries progression for which they have to decide the proper management strategy. the expansion of knowledge and understanding of the various stages of caries progression, along with developments in dental materials have encouraged a fundamental change in the management of dental caries. there is considerable evidence that discourages operative management of carious lesions confined to enamel and limits it to the management of cavitated lesions1,2. conversely, this evidence supports the management of non-cavitated early lesions by the provision of non-invasive and micro-invasive treatment strategies1,3,4. non-invasive strategies aim to modify the micro environment, shifting the dynamic process toward remineralisation. they do not remove dental hard tissue and involve, for example, topical fluorides and other chemical agents for controlling mineral balance, biofilm control measures and dietary control5. micro-invasive strategies predominantly act by sealing the lesion, depriving the bacteria within of fermentable carbohydrates required for acid production. they remove the dental hard tissue surface at the micron level, usually during an etching step, such as used in sealing or resin-infiltration techniques2. minimally-invasive operative strategies are recommended only to those lesions presenting with cavitation. cavitation indicates the irreversible clinical endpoint of continued mineral loss and requires a surgical approach to restore form, function and plaque control. it involves removing a limited amount of gross dental hard tissue, through the use of hand excavators, rotary instruments or other devices. in most cases, this process is associated with the subsequent placement of restorations6,7. selecting the management strategy lies completely in the practitioners’ hands. the decisions that they undertake will eventually affect the treatment costs and the tooth’s restorative cycle. the thresholds for restorative treatment among dentists in the clinical settings have been studied in many countries and a wide variation in treatment modalities between and within study populations have been shown. educational background and years of experience of the dental clinical practitioner seem to govern treatment decisions all over the world8–15. however, there have not been any studies of this nature conducted in palestine. therefore, this study aimed to examine restorative treatment decisions made by dental practitioners in palestine and to investigate what demographic characteristics are associated with restorative treatment decisions made by the respondents in their management of proximal and occlusal lesions. materials and methods the present study was a cross-sectional survey which was sent to 900 randomly selected general dentists from a list of those who were registered as members of the palestinian dental association before december 2019. the sample size was calculated using an online sample size calculator (raosoft.com) using a population of 4000, marginal error of 3%, a response distribution of 50% at a confidence interval of 95%. this yielded a proposed sample size n=843, thus a 900 sample size was used for convenience. the questionnaire was presented online (google forms) and its link was sent to the practitioners. the study started on december 12, 2019 and was completed in february 2020. a reminder 3 rabi and arandi was sent 3 and 6 weeks after the launch. the questionnaire included a brief introduction on the background, objectives, and voluntary nature of the study. declarations of confidentiality and anonymity, instructions for filling in the questionnaire, and statement of consent were included as well. accepting to proceed with survey was considered as an agreement to the statement of consent. ethical approval for the study was obtained from the research and ethics committee at al-quds university (9/rec/18). the questionnaire was adopted with modification from two previously published questionnaires16,17. before the formal survey, a pilot study was conducted among 20 participants, who were not included in the final survey. after evaluating the responses, the questionnaire was revised and considered appropriate and then used as the final version in this survey. through the questionnaire, socio-demographic details of the dentists were obtained and their years of experience were recorded too. in the questionnaire, the subjects were presented with an illustration of radiographic stages (1 to 6) of approximal carious lesions as shown in figure 1. the proximal staging used was defined by radiolucency depth. they were asked at which stage they think an immediate restorative (operative) treatment is required. the dentists were also asked which type of preparation they would prefer for the smallest of the lesions they decided to need immediate restorative treatment assuming that the lesion is present distally on the upper second premolar. three choices were given, namely traditional class ii preparation, tunnel preparation, or saucer-shaped preparation. then the respondents were asked which restorative material they would choose for the smallest approximal lesion they would restore. the options were amalgam, composite, packable glass ionomer cement (gic), a combination of composite and resin-modified glass ionomer cement (sandwich technique). figure 1. shows photographs of progressive occlusal carious lesions. grade 1 – white or brown discoloration in enamel, no clinical cavitation, no radiographic evidence of caries; grade 2 – small cavity formation, or discoloration of the fissure with a surrounding opaque or grey zone of enamel and/or radiolucency in enamel; grade 3 – moderately sized cavity and/or radiolucency in the outer 1/3 of the dentin; grade 4 – large cavity and/or radiolucency in the middle 1/3 of the dentin; grade 5 – extensive cavity and/ or radiolucency in the inner 1/3 of the dentin on the bitewing radiograph16 photographs illustrating the clinical presentation of occlusal carious lesions in a lower second molar (grade 1-5) with radiographic descriptions were also presented to the participants as shown in figure 2. the dental practitioners were asked to verify the ‘’grade’’ at which immediate restorative intervention was needed for a patient who was 20 years old, used fluoridated toothpaste, had good oral hygiene, low caries activity, and visited a dentist once a year. subsequently, the questionnaire asked the participants what restorative material they would choose to treat such teeth if the need for invasive clinical restorative treatment arises. the options were amalgam, composite, packable glass ionomer cement (gic), a combination of composite and resin-modified glass ionomer cement (sandwich technique). 4 rabi and arandi 1 2 3 4 5 6 outer half of the enamel outer third of the dentin outer half of the dentin inner half of the enamel inner half of the dentin enamel-dentin border figure 2. an illustration of the radiographic stages of approximal carious lesions17. statistical analysis the data was collected and analysed using the ibm spss statistics for windows. version 25.0 (ibm corp., armonk, ny, usa). the statistical associations between the demographic characteristics of the dentists and their restorative decisions for approximal and occlusal lesions were assessed using pearson’s χ2 test. p-value was considered significant when less than 0.05. fisher’s exact test was used to assess statistical associations wherever pearson’s χ2 test is not appropriate. results the response rate was 58.2%. a total of 524 dental practitioners completed the questionnaire, 451 (86.1%) were females and 73 (13.9%) were males. for the years since the graduation section of the questionnaire, 166 participants displayed 0-5 years, 190 ticked 6-10 years and 168 reported more than 10 years since graduation. occlusal carious lesions with radiographic description of the 524 participants, relatively few respondents (n = 32; 6.1%) chose to operatively intervene when the carious lesion is in enamel (grade ii). whereas 83 (15.8%) chose to intervene with restorative treatment until the lesion in the outer 1/3 of the dentin (grade iii). the majority of the participants (n= 409; 78.1%) chose not to intervene until the lesion was in the inner third of the dentin on the radiograph (grade iv). none of the respondents would wait until the carious lesion reaches the inner third of dentine (grade v). composite was said to be used as the restorative material of choice among 264 (50.4%) of the participants. preference for other restorative materials were amalgam 29.8%, gic 11.5% and 8.4% would use a combination of composite and rmgi (sandwich technique). there was significant association (p<0.05) between the gender and the restorative threshold and material of choice for restoring an occlusal lesion. there was significant association (p<0.05) between the years of experience and the restorative threshold and material of choice for a restoring a proximal lesion. 5 rabi and arandi ta bl e 1. a ss oc ia tio n be tw ee n ye ar s of e xp er ie nc e an d re st or at iv e va ria bl es <5 y ea rs 610 y ea rs > 10 y ea rs to ta l 16 6 19 0 16 8 n w ith in re st or at iv e va ria bl es % w ith in e xp er ie nc e le ve l % n w ith in re st or at iv e va ria bl es % w ith in e xp er ie nc e le ve l % n w ith in re st or at iv e va ria bl es % w ith in e xp er ie nc e le ve l % p -v al ue r es to ra tiv e th re sh ol d fo r a p ro xi m al le si on o ut er h al f o f t he e na m el 0 0 0% 0% 0 0% 0% 0 0% 0% in ne r h al f o f t he e na m el 39 0 0% 0% 7 18 % 3. 7% 32 82 % 19 .0 5% d ej 26 7 43 16 % 25 .9 % 12 0 45 % 63 .2 % 10 4 39 % 61 .9 % o ut er th ird o f t he d en tin 21 8 12 3 56 .4 % 74 .1 % 63 28 .9 % 33 .1 % 32 14 .6 % 19 .0 5% <0 .0 01 o ut er h al f o f t he d en tin 0 0 0% 0% 0 0% 0% 0 0% 0% in ne r h al f o f t he d en tin 0 0 0% 0% 0 0% 0% 0 0% 0% p re pa ra tio n te ch ni qu e fo r a pp ro xi m al c ar io us le si on s tu nn el p re pa ra tio n 40 29 72 .5 17 .5 % 11 27 .5 % 5. 79 % 0 0% 0% tr ad iti on al p re pa ra tio n 28 0 78 27 .9 47 % 66 23 .5 7% 34 .7 4% 13 6 48 .5 7% 80 .9 5% sa uc er -s ha pe d pr ep 20 4 59 28 .9 35 .5 % 11 3 55 .4 % 59 .4 7% 32 15 .6 9 19 .0 5% <0 .0 01 r es to ra tiv e m at er ia l f or a pp ro xi m al c ar io us le si on s a m al ga m 16 0 30 18 .7 5 18 .0 7 15 9. 37 7. 89 11 5 71 .8 7 68 .4 5% g ic 68 36 52 .9 21 .6 8 32 47 .1 % 16 .8 4 0 0% 0% c om po si te 27 2 88 32 .4 % 53 .0 1 13 6 50 % 71 .5 8 48 17 .6 % 28 .5 7% r m g i & c om po si te 24 12 50 % 7. 22 7 29 .2 % 3. 69 5 20 .8 % 2. 98 <0 .0 01 ea rli es t s ta ge o f o cc lu sa l c ar io us le si on s at in te rv en tio n g ra de 1 0 0 0 0 0 0 0 0 0 0 g ra de 2 32 0 0 0 12 37 .5 6. 32 20 62 .5 11 .9 % g ra de 3 83 33 39 .7 6 19 .8 8 18 21 .6 9 9. 47 32 38 .5 5 19 .0 % g ra de 4 40 9 13 3 32 .5 1 80 .1 2 16 0 39 .1 2 84 .2 1 11 6 28 .3 6 69 .0 % <0 .0 01 g ra de 5 0 0 0 0 0 0 0 0 0 0 r es to ra tiv e m at er ia l f or o cc lu sa l c ar io us le si on s a m al ga m 15 6 28 17 .9 5 16 .9 13 8. 33 6. 84 11 5 73 .7 2 68 .5 % g ic 60 34 56 .6 6 20 .5 26 43 .3 3 13 .6 8 0 0 0. 0% c om po si te 26 4 84 31 .8 50 .6 12 7 48 .1 66 .8 4 53 20 .1 31 .5 % r m g i & c om po si te 44 20 45 .5 12 24 54 .5 12 .6 3 0 0 <0 .0 01 6 rabi and arandi approximal carious lesions around half (n = 267; 50.9%) of the respondents would wait until caries reach the dej, while 218 respondents (41.6%) would wait until the carious lesion reaches the outer third of dentine, and 39 (7.4%) said they would restore a carious lesion confined to the inner half of the enamel. none would operatively restore an approximate lesion limited to the outer half of the enamel nor to the inner two thirds of dentine. the preferred preparation type for 280 (53.4%) of the participants was traditional class ii, followed by the saucer-shaped preparation (n=204; 38.9%) and the least preferred cavity design the tunnel preparation (n = 40; 7.6%). composite was the most preferred restorative material of 272 (51.9) of the respondents, followed by amalgam (n=160; 30.5%), and gic (n= 68; 13%). only 24 (4.6%) chose to restore carious lesions by using the sandwich technique (rmgi and composite). there was significant association (p<0.05) between the gender and the restorative threshold, preparation technique, and material of choice for restoring a proximal lesion (table 1). the statistical analysis also showed that there was significant association (p<0.05) between years of experience and the restorative threshold, preparation technique, and material of choice for a restoring a proximal lesion (table 2). table 2. association between years of experience and restorative variables males females total n = 73 n = 451 n within restorative variables % within gender % n within restorative variables % within gender % p-value restorative threshold for approximal lesions outer half of the enamel 0 0 0 0 0 0 0 inner half of the enamel 39 28 71.8 38.36 11 28.2 2.44 dej 267 31 11.6 42.46 236 88.4 52.33 outer third of the dentin 218 14 6.4 19.18 204 93.6 45.23 <0.001 outer half of the dentin 0 0 0 0 0 0 0 inner half of the dentin 0 0 0 0 0 0 0 preparation technique for approximal carious lesions tunnel preparation 40 12 30 16.44 28 70 6.2 traditional preparation 280 50 17.86 68.5 230 82.14 51 saucer-shaped preparation 204 11 5.4 15.06 193 94.6 42.8 <0.001 restorative material for approximal carious lesions amalgam 160 11 6.89 15.06 149 93.11 33.04 gic 68 13 19.12 17.8 55 80.88 12.2 composite 272 37 13.6 50.7 235 86.4 52.1 rmgi & composite 24 12 50 16.44 12 50 2.66 <0.001 earliest stage of occlusal carious lesions at intervention grade 1 0 0 0 0 0 0 0 continue 7 rabi and arandi continuation grade 2 32 16 50 21.92 16 50 3.56 grade 3 83 16 19.28 21.92 67 80.72 14.85 grade 4 409 41 10.02 56.16 368 89.92 81.6 <0.001 grade 5 0 0 0 0 0 0 0 restorative material for occlusal carious lesions amalgam 156 43 27.56 58.9 113 72.4 25.05 gic 60 0 0 0% 60 100 13.3 composite 264 23 8.71 31.5 241 91.29 53.44 rmgi & composite 44 7 15.9 9.59 37 84.1 8.2 <0.001 discussion questionnaire surveys help to understand the current status and decision-making process to facilitate translation of research evidence into clinical practice and reduce overtreatment. questionnaire surveys investigating the restorative treatment threshold and caries management strategies (from diagnosis to treatment) among general practitioners have been used in norway8, usa9, uk11, france10, croatia12, kuwait13, iran14, uae15. this study aimed to evaluate the restorative threshold, restorative treatment, and restoration materials that the general dental practitioners in palestine use for approximal and occlusal carious lesions. to the best of our knowledge, this is the first study exploring the restorative thresholds of any group of palestinian dentists regarding occlusal and proximal carious lesions. the majority of the respondents were females, which reflect the actual enrolment of females in dental schools. the present study reported significant difference between the gender and the restorative threshold, type of preparation and choice of restorative material used. female dentists took a more conservative approach to restoration on the case scenario that involved approximal and occlusal lesions. our findings are similar to those of previous studies13,18–20 where gender differences in the management of dental caries and restorative procedures were reported to be significant. studies from different countries have shown variations among dental practitioners in terms of restorative treatment thresholds. chana et al.11 reported that 84.8% (n=217) of the dentist who responded to a survey investigating the restorative treatment decisions in london preferred a conservative approach by only opting to operatively treat dentine lesions. in norway, out of 2375 respondents, only 12.3% would restore posterior occlusal enamel lesions (grades 1–2) compared with 87.7% who would tend to defer operative treatment until the lesion had manifested in the dentin (grade 3 to 5)8. around 95.7% of the 185 dentists who responded to a similar survey in kuwait opted to initiate operative treatment only when the occlusal lesions approached the dentine (grade 3 to 5)13. around 41% out of 1842 respondents to a survey in usa suggested an immediate restoration for an early stage of caries progression (grade 1 and 2), while 59.3% would restore an occlusal lesion that involved the dej or deeper (grade 3 to 5)9. the majority (60.7% out of 770) of the respondents to a survey in france would wait until the lesion was in dentine (grade 3 to 8 rabi and arandi 5) while 39.3% would restore a lesion confined to enamel (grades 1 and 2)10. the results of the present study are consistent with previous studies8–11,13, the majority of the respondents (93.9%) would postpone operative treatment until the lesion was in dentine (grade 3 to 5) and fewer (6.1%) would start operative treatment earlier for a lesion confined to enamel (grade 1 and 2). however, gordan et al.19 stated that not all of the thresholds are appropriate; some may be too conservative and some too aggressive, but no one has yet identified the “right” threshold. they added, that at present, the only thresholds that definitely can be identified as not appropriate are those that call for surgical treatment when caries is confined to enamel, owing to enamel lesions’ potential for arrest or reversal. in analysing the treatment threshold of approximal carious lesions, the participants in this study showed that intervention was deemed appropriate by the majority (92.6%) of the respondents when there was radiographic evidence of a carious lesion reaching the dej or deeper. in contrast to this conservative behaviour, almost 7.4% of the respondents would recommend restorative intervention at much earlier stages with lesions confined to the enamel. this attitude (intervening when lesions are confined to enamel) is inconsistent with the literature and potential remineralisation and reversal of these lesions21. similar to the results of the present study, the majority of dentists in kuwait13, uk11, iran14, croatia12, and usa9 opted to interfere operatively only when the approximal carious lesion had reached the dentine. although there have been great advances in the field of restorative dentistry, quite many participants (53.4 %) opted for traditional class ii preparations. similarly, most respondents in kuwait13, uk11, and usa9 chose the traditional class ii approach. the gv black preparation requires removal of a large amount of healthy tooth structure to gain access to very small areas of proximal caries so that a restoration can be placed.  this traditional preparation is unnecessarily invasive if restoring small approximal lesions with resin composite. an alternative tooth substance preserving cavity design is either the tunnel preparation or the minimal slot preparation. the tunnel preparation which leaves the marginal ridge unaffected during preparation is quite popular among dentists in croatia12. this preparation technique was the least preferred by the respondents to the present survey. only 7.6% of the respondents opted for this preparation technique. this might be due to the difficulty of completely removing the caries and the subsequent frequent collapse of the marginal ridge. in the slot preparation, access is achieved through the marginal ridge, but preserving this structure where ever possible. slot preparations aim to keep removal of healthy tooth structure to a minimum and therefore have been reported to perform significantly better than tunnel restorations22. a study among dental practitioners in the college of dentistry at ajman university – uae reported that the most preferred cavity design for aproximal lesions was the simple box preparation (72.8%), followed by tunnel preparation (20.6%) and the least preferred was the conventional class ii preparation (6.7%)15. the most preferred material to restore both occlusal and proximal cavities by the respondents was composite resins. the respondents’ preferences correspond to the trend towards the teaching of posterior composite restorations over amalgam restorations in dental schools provided that the majority of participants being recent graduates. the results of the present study follow along with those of similar studies10–13,15. 9 rabi and arandi the findings of this study support other researches where the treatment decisions regarding occlusal and approximal carious lesions and the demographic factors of the dentists were interlinked. the significant differences found could be due to the fact that there are various methods of teaching restorative dentistry during the learning phase of dentists. therefore, the diagnosis and treatment plan differs from one clinical practitioner to the other. the limitations of this study were that the data collected through the questionnaire was heavily influenced by self-reported clinical practices of the dentists and the participants were presented with hypothetical scenarios. therefore, the answers of the participants might not have accurately depicted their clinical and practical treatment plans. in addition, the present study does not highlight treatment modalities like selective removal of caries in deep dentin lesion to prevent pulp exposure and clinical judgement of the practitioners on the same. this can be added in the present questionnaire to explore more scope in attitude of the dentists in pulp preservation. another limitation of this is that the questionnaire is based on a given scenario and therefore the results of this study only reflect the treatment decisions of this scenario. it has been reported that the restorative decisions of dentists will change depending on the caries risk of the patient23,24. nevertheless, even if questionnaire surveys are not able to assess the dentists’ specific clinical decisions, they can still provide a good indication of their treatment modalities and knowledge. also, it could help in the development of guidelines for dentists’ education and promoting better practices. further studies should investigate the provision of non-invasive and micro-invasive treatment strategies in the management of early lesions. in conclusion, this study was successful in portraying that for approximal and occlusal lesions, palestinian dentists delayed invasive restorative intervention until the carious lesions progressed to the dentin. the subjects were conservative but still adhered to traditional learned practices especially when cavity preparation for approximal lesions were concerned. the years since graduation and gender played a significant role in the choice of treatment opted for in our study. also, resin materials seemed to be a popular choice for treatment and this shows the changing trends in dental education and research. conflict of interests all authors declare that there is no conflict of interests. ethics approval this study approved by the research and ethics committee at al-quds university (9/ rec/18). there is no conflict with ethical considerations. funding self-financed. acknowledgements none 10 rabi and arandi references 1. dorri m, sm d, walsh t, schwendicke f. micro-invasive interventions for managing proximal dental decay in primary and permanent teeth. cochrane database syst rev. 2015 nov 5;(11):cd010431. doi: 10.1002/14651858.cd010431.pub2. 2. splieth ch, 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sumida f, matsumoto y, manabe k, yokoyama y, gilbert gh, et al. restorative treatment thresholds for proximal caries in dental pbrn. j dent res. 2012 dec;91(12):1202-8. doi: 10.1177/0022034512464778. 1http://dx.doi.org/10.20396/bjos.v19i0.8660436 volume 19 2020 e200436 original article 1 dentistry department, university center christus (unichristus), fortaleza, ceará, brazil. 2 hospitalar health and management institute, fortaleza, ceará, brazil. 3 oral pathology department, federal university of ceará (unichristus), fortaleza, ceará, brazil. corresponding author: tácio pinheiro bezerra address: israel bezerra st, 1033, tauape, fortaleza/ce, zip 60.135-460. brazil email: taciopg@gmail.com phone number: +55 85 988995998 received: july 10, 2020 accepted: september 23, 2020 pneumonia and the role of dentistry on icu staff: 8-year analysis of health indicators tácio pinheiro bezerra1,2,* , clarissa sales de paula campêlo1 , francisco artur forte oliveira1 , clarissa pessoa fernandes forte1 , aghata kelma palácio gomes1 , paulo goberlânio de barros silva1 , bráulio matias de carvalho1,2 , ana paula negreiros nunes alves3 , fabrício bitu sousa1 abstract: ventilator-associated pneumonia (vap) is one of the most prevalent healthcare-associated infections (hai) and causes of death in intensive care units (icus), and studies have shown its relation to oral health. aim: to report the impact of the incorporation of dental professionals into multidisciplinary icu staff on the incidence of vap. methods: a retrospective observational study was carried out to collect and analyze health indicators of patients in the icus from 2011 to 2018 and to differentiate these indicators between the periods before and after the participation of dental staff in the icu. this study was approved by the research ethics committee. results: the average number of monthly icu admissions was 105.89 ± 169.72, and the discharge was 105.21 ± 168.96, with a monthly average number of deaths within 24 h of 38.61 ± 62.27. the average number of monthly hai-related deaths decreased from 2011 to 2018, followed by a reduction in cases of hai per month. the average monthly number of hais related to mechanical ventilation (mv) decreased, and the same was observed for the infection density of hais related to mv (p < 0.001). in multivariate analysis, there was a significant decrease in the number of hais related to mv (p = 0.005). conclusion: although a reduction in the number of admissions or complexity of cases was not observed in the study period, multidisciplinary staff practices were essential for controlling hais and the presence of dental professionals can assist in the control of hais related to mv. keywords: respiration, artificial. pneumonia, ventilatorassociated. dentistry. http://orcid.org/0000-0002-1058-4172 http://orcid.org/0000-0002-5622-7314 http://orcid.org/0000-0002-6913-3261 http://orcid.org/0000-0001-6032-8436 http://orcid.org/0000-0003-3825-3751 http://orcid.org/0000-0002-1513-9027 http://orcid.org/0000-0001-5848-5301 http://orcid.org/0000-0002-5090-6877 http://orcid.org/0000-0002-6430-9475 2 bezerra et al. introduction in brazil, intensive care was implemented in the 1970s. this measure brought about a substantial decrease in the number of deaths in intensive care units (icus)1 and reduced the number of healthcare-associated infections (hais), which are among the main causes of increased length of stay in icus and the incidence of death during hospitalization2. lack of attention to oral hygiene and reduced salivary flow in icu patients increases the complexity and amount of biofilm, which can favor the interaction between indigenous plaque bacteria and respiratory pathogens such as p. aeruginosa and enteric gram-negative bacilli. contamination of the lower respiratory tract by saliva can cause respiratory infections3. perhaps one of the most important contributions of dental professionals to the hospital environment is the prevention of ventilator-associated pneumonia (vap). vap remains a major cause of morbidity and mortality, and it also increases the cost of care for critically ill patients2,4-6. the incidence of vap ranges from 6% to 52%; in icus, it represents 25% of all infections. unquestionably, intubation is associated with an increased risk; approximately 90% of vap episodes in icus occur in patients on mechanical ventilation (mv)7. despite a study that showed mortality rates of 50% in patients who develop vap8; on average, death occurs in 10% of cases4. the native oral microbiota plays an important role in the risk of vap development. abnormal colonization and presence of potential respiratory pathogens in biofilms are among the main etiological causes9,10. pathogen migration occurs when patients aspirate contaminated oropharyngeal secretion, which disseminates through spaces between the trachea and tracheal tube cuff7. other variables affecting this outcome are possible systemic disabilities caused by antibiotic use, stress, chronic respiratory diseases, gastroesophageal reflux, and immunosuppression11. controlling the oral microbiota has a relevant effect on vap prevention4. among the applicable methodologies, chemical control with chlorhexidine gluconate (chx) associated with mechanical cleaning of mucous and dental surfaces has a significant impact on reducing these infectious conditions5. oral care in the icu is provided by nurses and deemed of moderate-to-high importance compared with other care activities12. dekeyser ganz et al.13 (2009) showed that no consistent practices related to oral care existed, and that most nurses had little or no knowledge of the current best evidence-based practice13. this paper aims to report the impact of the incorporation of dental professionals into multidisciplinary icu staff on the incidence of vap. this topic is significant for dentistry because it could offer subsidies for programs aimed at improving the multidisciplinary treatment offered to the patients. in addition, it could reduce treatment costs related to patients in critical care. materials and methods this was a retrospective and observational study of hospital indicator data on icu occupation and healthcare-associated infections from 2011 to 2018. the study was 3 bezerra et al. carried out in a general hospital with 3 icus (a total of 21 beds), and where the patients were treated and followed by a multidisciplinary staff that incorporated a team of dental professionals in 2014. this addition to the staff was the main subject of the present investigation. the icus receive adult patients with several medical conditions, such as community-acquired pneumonia, chronic obstructive pulmonary disease, stroke, exogenous intoxication, and pancreatitis, among others. every month, the committee on infectious disease control and prevention monitors and records infectious events and uploads the information to an electronic database. this information was consulted to gather the following data for the present study: number of icu admissions and discharges, number of patients discharged from the icus to regular nursing floors or transferred to other hospitals, number of icu deaths in the first 24 h, number of hai-related deaths, number of hai cases, number of hai patients, number of mvs/day (mv/d), number of hais related to mv, and infection density of hais related to mv. the infection density of hais related to mv was calculated using the ratio between the number of reported pulmonary infections and the number of ventilations/day ×1 000. as the primary hypothesis of the present study, it was supposed that the presence of an oral health team would reduce the number of hai related to mv in critical patients. this impact would be associated with the quality and frequency of oral hygiene performed. the vap prevention bundle is an institutional recommendation that has five steps to be performed in patients under mv. the recommendations are: peptic ulcer prevention, daily sedation interruption, elevated headboard, prevention of thromboembolic events, and oral hygiene. it was the same before and after 2014, but before 2014, the nursing team was the only one responsible for performing these procedures. with the multidisciplinary staff improvement, one dental hygienist and one dentist (qualified in hospital dentistry) were responsible for helping with the oral hygiene essential procedure. before and after 2014, the oral hygiene protocol was exactly the same; the only difference was the presence of dental specialists evaluating and training the nurses on oral hygiene, mainly for patients on mv. the dental staff also had as objective to perform oral examinations of all the patients referred to the icu, identifying oral lesions or treatment necessities. as an important landmark to evaluate this impact on the hais related to mv, the data from 2011-2014 were compared to that from 2015-2018. the collected data were tabulated in an excel spreadsheet and exported to the statistical package for the social sciences (spss) software for statistical analysis. monthly rates were compared by year using pearson’s correlation test. in addition, the multiple linear regression model was employed to assess the collinearity between the data and the temporal profile as well as the number of hais. all analyses were performed using the 95% confidence interval in the spss software. this study was submitted and approved by the research ethics committee under protocol 1,418,708. 4 bezerra et al. results to present the number of patients treated in the icus in this study, table 1 shows information about the number of icu admissions and discharges, and the main reasons for discharge (transfer to nursing floors and transfer to other hospitals). the average number of monthly admissions in the study period was 105.89 ± 169.72, and discharge was 105.21 ± 168.96, with the transfer to a regular nursing floor as the most frequent reason (60.98 ± 96.87). to show the severity level of the treated patients, table 1 shows the average number of deaths within the first 24 h and the hai-related deaths. it is important to highlight that the average number of monthly hai-related deaths decreased from 7.69 ± 20.05 to 7.35 ± 28.99 after 2015. the infection data collected (hai-related deaths; total hai; number of hai patients) showed that despite an increase in the average number of deaths between the periods, there was a reduction in the cases of hai per month from 22.08 ± 35.63 to 19.85 ± 34.13 (table 1). considering the information on hai of the respiratory tract (number of mv/day; number of hai related to mv; mv-related to hai density), table 1 also shows that the average monthly number of mv/d slightly reduced between the periods, the same as hais related to mv that decreased with a more expressive numeric change. however, only the reduction in the mv-related to hai density was statistically significant (p < 0.001). in multivariate analysis, it is important to highlight that there was a reduction in the number of hais related to mv (p = 0.005) from 2011 to 2018 (table 2). this analysis means that it was independent from the other data tabulated. table 1. general data from 2011-2014 and 2015-2018. period total p-valor 2011-2014 2015-2018 number of icu admissions 100.02±160.05 111.65±180.08 105.89±169.72 0.730 number of icu discharges 98.69±158.45 111.73±180.17 105.21±168.96 0.696 transfer to nursing floors 58.98±94.34 63.02±100.29 60.98±96.87 0.834 transfer to other hospitals 2.77±5.00 5.61±9.69 4.17±7.78 0.066 icu deaths (24h) 35.56±58.14 41.73±66.66 38.61±62.27 0.618 hai related deaths 7.69±20.05 7.35±28.99 7.52±24.80 0.944 total hai 22.08±35.63 19.85±34.13 20.96±34.73 0.745 number of hai patients 19.04±30.70 17.73±29.89 18.38±30.16 0.826 number of mechanical ventilation/day 872.43±1438.35 864.94±1383.03 868.69±1404.10 0.978 number of hai-related to mv 6.35±10.29 3.77±6.70 5.06±8.74 0.133 mv-related to hai density 7.15±3.52 4.41±3.64 5.78±3.82 *<0.001 *p<0.05, t de student test, data expressed as mean ± sd. mv – mechanical ventilation; hai healthcareassociated infection. 5 bezerra et al. discussion hospital infections are potential life-threatening conditions for patients in icus and are among the main causes of hospital morbidity and mortality14. throughout the study period, there was a reduction in the total number of nosocomial infections, which might result from an increase in quality of care, especially after the incorporation of dental professionals into the multidisciplinary staff. almost half of the microorganisms in the human body are found in the oral cavity15. these organized microorganisms form biofilms that can be colonized by potential respiratory pathogens9. therefore, it is important to pay attention to oral hygiene and to invest in professionals trained for this function. moreover, the acquisition of components related to mechanical and chemical control of dental biofilm appears to be a necessary measure for maintaining the health of hospitalized patients16. matos et al.16 (2013) conducted a national survey on physicians working in icus with questionnaires addressing the participation of dentists in the hospital environment. the results showed that 83.68% of the physicians did not supervise the oral hygiene of patients, 76.52% did not indicate the use of mouthwash in their patients, and most physicians were unable to diagnose oral conditions such as periodontal disease (93.88%) and dental caries (84.7%). the work by matos et al.16 (2013) highlights the importance of the participation of dentists in the hospital environment to minimize systemic problems resulting from oral conditions, mainly because this is not a specific concern of icu physicians. dekeyser ganz et al.13 (2009) conducted a survey with nurses working in icus asking about the current oral care practices performed for patients, including the type of equipment used, solutions used, technique, and the type and timing of oral assessment. nurses were also asked about their perceived level of priority for oral care on a scale from 0 to 100. the authors showed that nurses were often not adhering to the latest evidence-based practice on oral hygiene and therefore need to be educated and table 2. multivariate analysis year p-value β ajusted ci 95% number of icu admissions 0.659 -0.037 -0.205 0.130 number of icu discharges 0.650 -0.044 -0.235 0.148 transfer to nursing floors 0.563 0.030 -0.072 0.132 transfers to other hospitals *0.015 0.223 0.044 0.402 icu deaths within 24 hours *0.043 0.128 0.004 0.252 hai-related deaths 0.991 0.000 -0.036 0.035 total number of hai 0.981 0.004 -0.282 0.289 number of patients with hai 0.739 0.058 -0.285 0.400 number of mv/day 0.666 0,001 -0.003 0.005 number of hai associated with mv *0.005 -0.283 -0.479 -0.088 mv-related to hai density 0.500 -0.002 -0.006 0.003 *p<0.05, multiple linear regression. anova / bonferroni test; pearson’s correlation [p-value (pearson’s correlation coefficient)]. data expressed as mean ± sd. mv – mechanical vetilation; hai healthcare-associated infection 6 bezerra et al. encouraged to do so to improve patient care. only 44% of the sample brushed their patients’ teeth. this is a significant health care problem, and is not only present in israel as shown by dekeyser ganz et al.13 (2009). the data of the present study shows that without the participation and action of a dental staff, the icu patients did not have adequate oral hygiene, which had a positive impact on mv-related pneumonia. moreover, as cited by alhazzani et al.12 (2013), a high workload makes nurses give more attention or importance to other critical necessities of icu patients. the results of the cited literature agree with the present study, considering that the results presented here show the benefits of oral health care provided by a dental team. both the literature and the results presented highlight the necessity of having dental professionals included in the multidisciplinary staff of critical patients. the addition of dental professionals to the multidisciplinary staff leads to a significant reduction in the length of stay on mv17. this may be a consequence of the implementation of a daily oral hygiene routine, reducing oral contamination. however, there is still no consensus on an oral hygiene protocol that can be implemented to reduce infections related to mv. in general, chx, liquid or gel, 0.12% or 0.2%, three or four times a day9,18 is the most used method19. oral antisepsis demonstrates efficacy in controlling oral biofilms in patients exposed to long periods of mv20,21. a review published on the cochrane platform concluded that the use of chx, whether in liquid or gel form, is associated with a 40% reduction in the occurrence of vap in critically ill adult patients22. nonetheless, larger studies are still needed to verify the impact of these practices on hospital indicators23. however, as stated by prasad et al.24 (2009), it is difficult to ascertain whether the significant improvement in oral hygiene found in their study was due to chlorhexidine use or the improved technique of the ward staff or, indeed, both factors combined. the author also said that the training element should not be underestimated when having dental staff in the icu. this is one more reason to support that the better the staff the better the care provided, considering the stimulus of one person to another on the care provided to the patients. this study analyzed a vast database and demonstrated the importance of controlling infections caused by mv, demonstrating that the incorporation of dental professionals in high complexity hospitals can contribute to the control of hai. the results of the multivariate analysis showed that although there was an increase in the number of deaths (within 24 h) in the study period, a reduction of approximately 28% per year in the number of hais related to mv was achieved. in summary, despite the increase in complexity of critical patients, the actions implemented to improve care influenced the control of nosocomial infections, especially in relation to oral health. the dentist’s role in areas with a high prevalence of vap, such as in icus, is important to ensure adequate oral hygiene for patients and to help reduce intubation time, length of stay, and mortality21. the data from the present research highlight that after the incorporation of dental professionals in 2014, there was a significant decrease in the density of hais related to mv from 7.15 ± 3.52 between 2011 and 2014 to 4.41 ± 3.64 between 2015 and 2018. this reveals that the presence of dental professionals can improve oral health even when a nursing staff is already responsible for oral healthcare. 7 bezerra et al. it should also be noted that vap occurrences increase hospitalization costs; however, economic values have not yet been quantified because of the numerous variables involved in the process4. there are few available data on the cost reduction that hospital dentistry can generate in high complexity hospitals. nevertheless, the most significant reduction is probably the improvement of indicators related to mv17. this economic impact should be investigated in future studies. in conclusion, although a reduction in the number of admissions or complexity of cases was not observed in the study period, the data of the present study reinforce that the practices conducted by the multidisciplinary staff were essential for the control of hais and that the presence of dental professionals can assist in the control of hais related to mv. acknowledgments the authors would like to thank the staff that works in the icu of the general hospital dr. waldemar de alcântara, the hospital directors, and the hospital infection control service, who supports the work of the hospital dental service. references 1. do nascimento er, trentini m. [nursing care at the intensive care unit (icu): going beyond objectivity]. rev lat am enfermagem. 2004 mar-apr;12(2):250-7. portuguese. doi: 10.1590/s0104-11692004000200015. 2. munro cl, grap mj, sessler cn, elswick rk jr, mangar d, karlnoski-everall r, et al. preintubation application of oral chlorhexidine does not provide additional benefit in prevention of early-onset ventilator-associated pneumonia. chest. 2015 feb;147(2):328-34. doi: 10.1378/chest.14-0692. 3. gomes sf, esteves mcl. [role of the surgeon dentist in icu: a new paradigma]. rev bras odontol. 2012;69(1):67-70. portuguese. 4. klompas m, branson r, eichenwald ec, greene lr, howell md, lee g, et al. strategies to prevent ventilator-associated pneumonia in acute care hospitals: 2014 update. infect control hosp epidemiol. 2014 aug;35(8):915-36. doi: 10.1086/677144. 5. lorente l, lecuona m, jiménez a, palmero s, pastor e, lafuente n, et al. ventilator-associated pneumonia with or without toothbrushing: a randomized controlled trial. eur j clin microbiol infect dis. 2012 oct;31(10):2621-9. doi: 10.1007/s10096-012-1605-y. 6. koeman m, van der ven aj, hak e, joore hc, kaasjager k, de smet ag, ramsay g, dormans tp, aarts lp, de bel ee, hustinx wn, van der tweel i, hoepelman am, et al. oral decontamination with chlorhexidine reduces the incidence of ventilator-associated pneumonia. am j respir crit care med. 2006 jun 15;173(12):1348-55. doi: 10.1164/rccm.200505-820oc. 7. wang j, liu kx, ariani f, tao ll, zhang j, qu jm. probiotics for preventing ventilator-associated pneumonia: a systematic review and meta-analysis of high-quality randomized controlled trials. plos one. 2013 dec 18;8(12):e83934. doi: 10.1371/journal.pone.0083934. 8. needleman ig, hirsch np, leemans m, moles dr, wilson m, ready dr, et al. randomized controlled trial of toothbrushing to reduce ventilator-associated pneumonia pathogens and dental plaque in a critical care unit. j clin periodontol. 2011 mar;38(3):246-52. doi: 10.1111/j.1600-051x.2010.01688.x. 9. özçaka ö, başoğlu ok, buduneli n, taşbakan ms, bacakoğlu f, kinane df. chlorhexidine decreases the risk of ventilator-associated pneumonia in intensive care unit patients: a randomized clinical trial. j periodontal res. 2012 oct;47(5):584-92. doi: 10.1111/j.1600-0765.2012.01470.x. 8 bezerra et al. 10. charles mp, easow jm, joseph nm, ravishankar m, kumar s, sivaraman u. aetiological agents of ventilator-associated pneumonia and its resistance pattern a threat for treatment. australas med j. 2013 sep 30;6(9):430-4. doi: 10.4066/amj.2013.1710. 11. american thoracic society; infectious diseases society of america. guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia. am j respir crit care med. 2005 feb;171(4):388-416. doi: 10.1164/rccm.200405-644st. 12. alhazzani w, smith o, muscedere j, medd j, cook d. toothbrushing for critically ill mechanically ventilated patients: a systematic review and meta-analysis of randomized trials evaluating ventilator-associated pneumonia. crit care med. 2013 feb;41(2):646-55. doi: 10.1097/ccm.0b013e3182742d45. 13. dekeyser ganz f, fink nf, raanan o, asher m, bruttin m, nun mb, benbinishty j. icu nurses’ oral-care practices and the current best evidence. j nurs scholarsh. 2009;41(2):132-8. doi: 10.1111/j.1547-5069.2009.01264.x. 14. laupland kb, zygun da, doig cj, bagshaw sm, svenson lw, fick gh. one-year mortality of bloodstream infection-associated sepsis and septic shock among patients presenting to a regional critical care system. intensive care med. 2005 feb;31(2):213-9. doi: 10.1007/s00134-004-2544-6. 15. amaral sm, cortes aq, pires fr, pneumonia nosocomial: importância do microambiente oral. j bras pneumol. 2009;35(11)1116-24. doi: 10.1590/s1806-37132009001100010. 16. matos fz, porto na, caporossi ls. semenoff tadv, borges ah, segundo as. hospital physicians’ knowledge of oral hygiene and oral manifestations in hospitalized patients. pesq bras odontoped clin integr. 2013;13(3):239-43. doi: 10.4034/pboci.2013.133.03. 17. de souza is, santaella ng, da silva santos ps. the practice of hospital dentistry in brazil: an integrative literature review. rev bras odontol. 2017;74(3):232-9. doi: 10.18363/rbo.v74n3.p.232. 18. fourrier f, dubois d, pronnier p, herbecq p, leroy o, desmettre t, pottier-cau e, boutigny h, di pompéo c, durocher a, roussel-delvallez m; pirad study group. effect of gingival and dental plaque antiseptic decontamination on nosocomial infections acquired in the intensive care unit: a double-blind placebo-controlled multicenter study. crit care med. 2005 aug;33(8):1728-35. doi: 10.1097/01.ccm.0000171537.03493.b0. 19. bergan eh, tura br, lamas cc. impact of improvement in preoperative oral health on nosocomial pneumonia in a group of cardiac surgery patients: a single arm prospective intervention study. intensive care med. 2014 jan;40(1):23-31. doi: 10.1007/s00134-013-3049-y. 20. bellissimo-rodrigues wt, menegueti mg, gaspar gg, de souza hcc, auxiliadora-martins m, basile-filho a, et al. is it necessary to have a dentist within an intensive care unit team? report of a randomised clinical trial. int dent j. 2018 dec;68(6):420-7. doi: 10.1111/idj.12397. 21. tuon ff, gavrilko o, almeida s, sumi er, alberto t, rocha jl, et al. prospective, randomised, controlled study evaluating early modification of oral microbiota following admission to the intensive care unit and oral hygiene with chlorhexidine. j glob antimicrob resist. 2017 mar;8:159-63. doi: 10.1016/j.jgar.2016.12.007. 22. shi z, xie h, wang p, zhang q, wu y, chen e, ng l, worthington hv, needleman i, furness s. oral hygiene care for critically ill patients to prevent ventilator-associated pneumonia. cochrane database syst rev. 2013 aug 13;(8):cd008367. doi: 10.1002/14651858.cd008367.pub2. update in: cochrane database syst rev. 2016 oct 25;10 :cd008367. 23. scannapieco fa, yu j, raghavendran k, vacanti a, owens si, wood k, mylotte jm. a randomized trial of chlorhexidine gluconate on oral bacterial pathogens in mechanically ventilated patients. crit care. 2009;13(4):r117. doi: 10.1186/cc7967. 24. prasad r, daly b, manley g. the impact of 0.2% chlorhexidine gel on oral health and the incidence of pneumonia amongst adults with profound complex neurodisability. spec care dentist. 2019 sep;39(5):524-32. doi: 10.1111/scd.12414. 1http://dx.doi.org/10.20396/bjos.v20i00.8664034 volume 20 2021 e214034 original article 1 federal university of patos, patos, pb, brazil. 2 cruzeiro do sul university, são paulo, sp, brazil. 3 santa cecilia university, santos, sp, brazil. 4 camilo castelo branco university, são paulo, sp, brazil. 5 dentistry department at university of são paulo, sp, brazil. 6 federal university of paraná, curitiba, pr, brazil. corresponding author: ângela toshie araki av robert kennedy 2126/sala 4, zip code 09861-080 são bernardo do campo, sp brazil tel.: +55-11-992534078 fax: +55-11 -43921575 a_araki@me.com editor: dr altair a. del bel cury received: january 20, 2021 accepted: may 25, 2021 comparative in vitro study of intracanal enterococcus faecalis reduction using photosensitizers in apdt maria cleide azevedo braz1 , andré hayato saguchi2 , eduardo akisue3 , adriana de oliveira lira2 , sidnea aparecida freitas paiva2 , aldo brugnera junior4 , mary caroline skelton macedo5 , andré luiz da costa michelotto6 , ângela toshie araki2,* aim: to compare enterococcus faecalis reduction after antimicrobial photodynamic therapy (apdt) used with methylene blue, toluidine blue, tannin, and curcumin as photosensitizers, an adjunct to endodontic chemomechanical preparation (cmp) in root canals of human teeth. methods: a total of 120 single-rooted teeth were divided into 6 groups (n  =  20): g1cmp and 2.5% sodium hypochlorite (naocl); g2cmp and saline solution; g3cmp, 2.5% naocl, and apdt with 0.005% methylene blue; g4cmp, 2.5% naocl, and apdt with 0.005% toluidine blue; g5cmp, 2.5% naocl, and apdt with 0.005% tannin; and g6cmp, 2.5% naocl, and apdt with 0.005% curcumin. a portable semiconductor laser was used (660  nm, 100  mw, 1.8  j, 180s) in groups 1, 2, 3, 4, 5, and a blue led light-curing (420-480  nm, 1200 mv/cm2) in g6. for all groups, a 5 min pre-irradiation time was applied. samples were collected before (initial collection), immediately after (intermediate collection) and 7 days after cmp (final collection) for colony-forming unit (cfu) counting. the kruskal-wallis test and analysis of variance (anova) were performed (p < 0.05; 95% confidence interval). results: in between-group comparisons, there was no significant difference observed in the number of cfus at the initial (p < 0.001) and final collections (p < 0.001) for g2 and g3. in within-group comparisons, the number of cfus showed a decreasing trend in g4 (p = 0.007) and g5 (p = 0.001). conclusion: photosensitizers promoted e. faecalis reduction, with better results for tannin and curcumin. alternative photosensitizers should be the focus of further studies. keywords: photodynamic. endodontics. photosensitizing agents. curcumin. tannins. mailto:a_araki@me.com https://orcid.org/0000-0002-0290-6987 https://orcid.org/0000-0002-1903-4986 https://orcid.org/0000-0002-7934-7255 https://orcid.org/0000-0001-7295-803x https://orcid.org/0000-0001-5882-8802 https://orcid.org/0000-0003-0023-191x https://orcid.org/0000-0002-4189-5088 https://orcid.org/0000-0001-7855-4407 https://orcid.org/0000-0003-4402-7531 2 braz et al. introduction the main goal of endodontic treatment is to eliminate or reduce the intraradicular microbial load to a level that allows for the prevention or cure of apical periodontitis1. the development and/or persistence of apical periodontitis is closely related to the presence of microorganisms within the root canal, and insufficient root canal disinfection has been suggested as one of the main reasons for treatment failure2. in addition to auxiliary chemicals, intracanal medicaments, passive ultrasonic irrigation, magnification, and ultrasound, antimicrobial photodynamic therapy (apdt) has also been used as an adjunct to conventional endodontic treatment3-5. apdt requires a photosensitizer and a light source emitting at a wavelength close to its absorption peak that will absorb energy from light photons6. the photosensitizing agent binds to the microorganism membrane or cell wall or accumulates selectively in them, becoming target to the light irradiated. in the presence of oxygen, photosensitizers generate reactive oxygen species (ros) – singlet oxygen and free radicals – that, by oxidative reactions, can lead to death of various organismsfungi, viruses, protozoa and bacteria7. photosensitizers initially act on the cell membrane to increase cell permeability, and then penetrate the cell acting on other intracellular organelles8. methylene blue and toluidine blue, at low concentrations, have been widely used as photosensitizers for not exerting cytotoxic effects on keratinocytes and fibroblasts9. tannins and curcumin are also used in apdt. tannins are astringent, hemostatic, polyphenolic compounds that have been used as natural dyes and are major active constituents of plants, which justifies the traditional use of plants as anti-inflammatory and healing agents10. they have a strong absorption band at 672 nm and rapid skin clearance. tannins are non-toxic and have favorable photophysical properties, with high triplet-state yields. curcumin, a polyphenolic compound extracted from curcuma longa rhizomes, has been widely used in traditional chinese medicine and food industry, with anti-inflammatory, antitumor, antifungal, antibacterial, and anticarcinogenic properties11. in apdt, curcumin has high light absorption capacity at wavelengths in the blue spectral region, between 455 nm and 492 nm12 which is commonly used in dental offices for the light curing of resin-based composites by using a led or halogen light device. reduction of intracanal microorganisms promoted by apdt ranges from 65 to 99.6% depending on the photosensitizer used, and some studies have been conducted to compare the reducing ability of photosensitizers12,13. this way, in this study had compared methylene blue, toluidine blue, tannin, and curcumin for their ability to reduce intracanal enterococcus faecalis. the purpose of this study was to compare microbial reduction after apdt (660nm) used with methylene blue, toluidine blue, tannin, and curcumin as photosensitizers as an adjunct to chemomechanical preparation (cmp) in root canals of human teeth contaminated with e. faecalis. 3 braz et al. materials and methods study design and setting this in vitro study was conducted after approval by the institutional research ethics committee (approval number 2.332.759). the sample size was calculated using g*power, version 3.1, based on a pilot data set to obtain a medium to large effect size, with a significance level of 5%, 95% confidence interval (ci), statistical power of 80% (β = 0.20), and a 1:1 allocation ratio. a sample size of 20 specimens per group was required. specimen preparation a total 120 extracted single-rooted mandibular incisors with fully formed apices were selected for this study. all teeth had been donated to the human tooth bank of our institution, and written informed consent was obtained from all patients prior to tooth donation. after periodontal tissue removal and rubber cup and pumice prophylaxis, the crowns were removed with a diamond disc. the remaining 12-mm roots were autoclaved at 120ºc, 1 atm, for 20 min, and the apical third were sealed with araldite epoxy resin (brascola, são paulo, sp, brazil) to prevent leakage of bacterial cultures after contamination. microorganisms and culture medium the root canals were contaminated with a pure culture of e. faecalis (atcc 29212) in brain heart infusion (bhi) broth (bhi00oxoid; basinkstoke, uk). isolated colonies were suspended in screw-cap tubes containing 5  ml of bhi broth. the suspension was mechanically shaken and adjusted in a spectrophotometer with absorbance at 800 nm to match a 1.0 mcfarland standard (3.0 x 108 bacteria/ml). the specimens were transferred to flasks containing sterile bhi broth and remained incubated at 37°c for 24 h to confirm sterility. contamination with e. faecalis in a laminar flow hood, 5.0 ml of bhi broth were removed and replaced with 4.0 ml of e. faecalis suspension. the flasks containing the specimens were sealed and incubated at 37°c in a 10% co2 atmosphere for 7 days, with 2.0 ml of contaminated bhi broth being replaced with 2.0 ml of sterile bhi broth every 2 days to avoid medium saturation. bacterial growth during incubation was determined by turbidity of the culture medium. experimental groups the 120 specimens were divided into 6 groups (n = 20) according to the disinfection protocol used during cmp, as shown in table 1. 4 braz et al. chemomechanical preparation and photodynamic therapy the teeth were arranged in a table-top lathe machine and prepared with a size 35.06 waveone gold file (dentsply). all root canals were prepared and irrigated with 10 ml of 2.5% sodium hypochlorite (naocl), followed by irrigation with 10 ml of saline solution, except group 2 that irrigation was realized with saline solution. in groups submitted to apdt after instrumentation, the canals were irrigated with photosensitizers solution (3 ml) of 0.005% with a 5-min pre-irradiation period. portable semiconductor laser (laser duo®, algaas, ingaalp, λ880 nm and λ660 nm, mm optics ltda, são carlos, sp, brazil) was used at a wavelength of 660 nm, 100 mw output power, and laser beam area of 3 mm2, for a total energy of 1.8 j per spot area over an irradiation time of 180s. in final, the canals were irrigated with 10 ml of saline solution to remove the dyes according to the protocol of gomes et al.14 in group 6, apdt irradiation was performed with a blue led light-curing unit (gnatus), at a wavelength of 420-480 nm, with an intensity of 1200 mv/cm2. microbiologic collection samples were collected at 3 time points: before cmp (initial collection), immediately after cmp (intermediate collection), and 7 days after cmp (final collection). all procedures were performed in a laminar flow hood. initial collection before cmp, the specimens were irrigated with 10 ml of saline solution, dried with 15-mm diameter sterile paper points for 1 min (figure 1), and stored in eppendorf tubes (figure 2). table 1. groups distribution n disinfection protocol g1(control) 20 cmp + 2.5% sodium hypochlorite g2(control) 20 cmp + saline solution g3 20 cmp + methylene blue + apdt g4 20 cmp + toluidine blue + apdt g5 20 cmp + tannin + apdt g6 20 cmp + curcumin + apdt figure 1. microbiologic collection with a sterile paper point. 5 braz et al. intermediate collection cmp was performed after the initial collection, and the canals were dried with sterile paper points and stored individually in eppendorf tubes, following the same protocol used for the initial collection. the specimens were filled with bhi broth and sealed with a gutta-percha stick to prevent contamination. the specimens were placed in individual eppendorf tubes containing 60 µl of sterile bhi broth to maintain hydration, so that the bhi broth did not reach the middle third of the canal to prevent infiltration into the gutta-percha sealing. the tubes were incubated at 37°c in a 10% co2 atmosphere for 7 days. final collection after 7 days, the root canals were again irrigated with 10 ml of saline solution, dried with 35-mm diameter sterile paper points, and placed in individual eppendorf tubes containing 1 ml of sterile bhi broth. after collection, the eppendorf tubes were vortexed (biomixer-ql-901) for 60 s to homogenize the solution. dilution and plating after mechanical agitation, the collected samples were diluted 1:10 and 1:100 in sterile bhi broth. a 50-µl aliquot of the 10-2 dilution was seeded on plates containing bhi agar, in triplicate, and the plates were incubated at 37°c in a 10% co2 atmosphere. after 48 h, colonies on plates were counted to determine the colony-forming units (cfus). the number of cfus was multiplied by 2000 to account for dilutions during sample preparation (100 times) and 50-µl plating (20 times less than 1  ml), thus yielding the number of cfus per ml of sample (cfu/ml). the average of the 3 plates of each collection was taken as the final count (figure 3). figure 2. eppendorf tube with the paper point after collection 6 braz et al. statistical analysis data were analyzed using biostat 4.0. the shapiro-wilk test was used to assess the normality of data distribution. the kruskal-wallis test and analysis of variance (anova) were used for nonparametric data, at a significance level of p  <  0.05 and 95% ci. results table 2 shows the results of the comparative analysis of bacterial load (cfu/ml) in each group at the 3 assessment time points. in between-group comparisons, a significant difference was observed in the number of cfus at the initial (p < 0.001) and final collections (p < 0.001). in within-group comparisons, the number of cfus showed a decreasing trend in groups 4 (p = 0.007) and 5 (p = 0.001). when comparing initial and final values, percentage reductions in relation to mean cfus were 68.2% for group 4 and 69.9% for group 5. figure 3. schematic representation of microbiologic collection in the initial, intermediate, and final stages 100 µl 10-1 1,000 µl 9,000 µl 9,000 µl 30 µl 50 µl 50 µl sum of 3 plates 3 2,000 100 µl 10-2 10-2 table 2. comparison of bacterial load at 3 assessment time points time point group g1 g2 g3 g4 g5 g6 p-value(1) initial collection mean 116,36a,ba 8,37aa 225,12ba 269,66ba 272,30ba 267,39ba < 0,001* sd 207,46 9,46 215,97 283,75 259,81 286,61 intermediate collection mean 31,73aa 85,00aa 74,26ab 192,92aa,b 54,67ab 292,07aa < 0,078 sd 35,23 200,93 66,68 209,94 88,20 408,84 final collection mean 74,34a,ba 1856719,65bb 272,40ca,b 85,77a,bb 81,83a,bb 12,23aa < 0,001* sd 159,94 8303252,97 280,49 91,62 164,47 18,38 p-valor(2) 0,630 < 0,001* 0,007* 0,007* 0,001* 0,089 % inhibition (initial-final) 36,1% 68,2% 69,9% 95,4% sd = standard deviation; values with different superscript letters are significantly different (p < 0.05); (1) uppercase letters compare values in the same row (between-group comparisons); (2) lowercase letters compare values in the same column (within-group comparisons). 7 braz et al. the control group 2 (with saline) and group 3 (with methylene blue) showed no reduction in cfus. all other groups showed bacterial reduction between the initial and final collections, but group 6 (with curcumin) had a marked standard deviation, and the result was not statistically significant. discussion e. faecalis is a valuable microbiologic marker for in vitro studies because of its ability to colonize the root canal in a biofilm-like style2. in addition, e. faecalis does not require strict culture conditions: it grows in culture media supplemented with blood or serum15, justifying the use of bhi agar as a culture medium, as previously done by silva garcez et al.16 (2006). the incubation period was 7 days, according to dametto et al.17 (2005). the procedures for microbiologic collection vary between studies. bonsor et al.18 (2006) used an endodontic file to remove dentin debris from the root canal and this instrument together with the swarf sample (dentin debris) was analyzed, while soukos et al.19 (2006) and fimple et al.9 (2008) analyzed the solution leaking from the apical foramen. in the present study, microbiologic samples were collected from within the root canal by using sterile paper points, according to previous studies14,16,20,21. in all groups, the auxiliary chemical used was 2.5% naocl, because its bactericidal effect and ability to dissolve necrotic and living tissues22,23. in control group 1 (cmp with 2.5% naocl), there was only a 36.1% reduction in bacterial growth, which was not statistically significant (p  =  0.63). higher concentration of naocl would improve its properties24-26 but increase the risk of accidents27 because substances are less biocompatible with peri radicular tissues24,28. increasing the volume of the solution to compensate for the effects of concentration22,29-32 would improve its bactericidal properties. the present study compared the efficacy in reducing e. faecalis of 4 dyes used as photosensitizers in apdt: methylene blue, toluidine blue, tannin, and curcumin. methylene blue is the most commonly used dye in endodontics9,19, followed by toluidine blue14,18,20,21. they are known to be more effective against gram-positive than gram-negative species33. methylene blue is used at various concentrations, ranging from 1 μg/ml to 25 μg/ml, as is toluidine blue, ranging from 12.5 μg/ml to 100 μg/ ml. in accordance with the study by gomes et al.14 (2008) the 4 photosensitizers tested in this study were used at a concentration of 5 μg/ml (0.005%). an unexpected statistically significant (p<0.001) bacterial growth was observed between the initial and final collections using methylene blue. however, when a higher concentration was used there was a reduction of 80%9 and 97%19. besides to the concentration, better results could also be achieved using more energy density of light (j/cm²)9 and combining it with passive ultrasonic irrigation34,35. in contrast to methylene blue, toluidine blue at a concentration of 0.005% promoted a statistically significant reduction of 68.2%, in accordance with other studies12,18,36. extracted from guava leaves (psidium guajava l. myrtaceae), ‘espinheira-santa’ leaves (maytenus sp., celastraceae), and brazilian peppertree leaves (schinus terebinthifolius raddi), tannins are astringent, hemostatic substances37 commonly used in the leather tanning industry and paint industry. the hydrolyzable tannins have aroused the interest of researchers to be tested against microorganisms that infect the root canal sys8 braz et al. tem because they are non-toxic and have favorable photophysical properties with high triplet-state yields9. the use of tannin in group 5 promoted a statistically significant bacterial reduction of 69.9% (p = 0.001), better than groups 3 and 4 (methylene blue and toluidine blue, respectively) and group 1 (naocl alone). according to verza et al.38 (2012) hydrolyzable tannins have a stable binding to central glycose residues and consequently increased solubility and binding of its molecules to low-density lipoproteins, facilitating entry into the target cell. given the 69.9% bacterial load reduction and results superior to those of methylene blue and toluidine blue, the present authors hypothesize that tannin may be indicated in cases of persistent e. faecalis infection. tannins are easily extracted from plants such as guava tree, ‘espinheira-santa’ (maytenus sp., celastraceae), and brazilian peppertree at a low cost. so, they have become a highly promising dye for use in apdt. curcumin is a pigment extracted from curcuma longa, a plant used as a spice in cuisine that also has anti-inflammatory, antitumor, antifungal, antibacterial, and anticarcinogenic properties, in addition to antiviral effects against hepatitis b and h1n1 viruses. it has an antimicrobial effect against gram-negative and gram-positive bacteria when used in apdt and can be purchased at a compounding pharmacy. curcumin has the advantage of being activated by a blue spectral range between 455 nm and 492 nm, which allows for the use of led or halogen light devices12. in this study, the curcumin group (group 6) showed a marked standard deviation and the 95,4% bacteria reduction was not statistically significant. da frota et al.12 (2015) evaluated the efficacy of apdt using curcumin in the elimination of enterococcus faecalis from root canals, varying the time of led irradiation (5 or 10 min) after 5 min of pre-irradiation time. they observed that 5min reduced more bacterial viability than 10 min led irradiation. mahdi et al.39 (2015) investigated the effect of apdt using a combination of curcumin (60 μm), h2o2 (0.3 mm), and erythrosine (22 μm) in planktonic cultures of porphyromonas gingivalis and fusobacterium nucleatum and observed a 100% bacterial reduction after 5 min of blue light activation (450 nm). in sum, the control group 2 (cmp with saline) and group 3 (cmp + apdt with methylene blue) showed no reduction in cfus. all other groups showed bacterial reduction between the initial and final collections, but group 6 (cmp + apdt with curcumin) had a marked standard deviation and a not statistically significant reduction. group 5 (cmp + apdt with tannin) showed the best results, introducing tannin as an alternative dye with less toxicity and good antibacterial activity. despite the fact of all advances in rotary and reciprocating instruments, microbiology should always be the focus of endodontic treatment. it is wellknown these instruments become instrumentation of radicular canal faster, reducing clinical time. in addition, studies have shown effectiveness of these instruments systems on microbial reduction40. however, the present authors have a concern if clinicians and specialists are, in fact, irrigating successfully in such reduced clinical time. apdt emerges in this scenario improving and ensuring microbial load reducton inside radicular system canal. this study presents tannin and curcumin as alternatives to methylene blue and toluidine blue, showing promising results. however, randomized controlled trials are warranted to further test them in apdt given their scientific relevance. alternative photosensitizers and other concentrations in apdt should be the focus of further in vitro and in vivo studies. 9 braz et al. in conclusion, apdt using a wavelength of 660  nm, 100  mw as light source during 180s in association with toluidine blue or tannin at a 0.005% concentration used as an adjunct to cmp with 2.5% naocl reduced e. faecalis significantly. methylene blue at a 0.005% concentration and curcumin did not promote microbial reduction. author disclosure statement financial disclosure: the authors have no financial relationships relevant to this article to disclose conflict of interest: no competing financial interests exist. references 1. orstavik d. time-course and risk analyses of the development and healing of chronic apical periodontitis in man. 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effectiveness of rotary and reciprocating systems on microbial reduction: a systematic review. j conserv dent. 2019 mar-apr;22(2):114-22. doi: 10.4103/jcd.jcd_523_18. 1http://dx.doi.org/10.20396/bjos.v19i0.8657981 volume 19 2020 e207981 original article 1 postgraduate program in public health, rené rachou institute, oswaldo cruz foundation (fiocruz), brazil. 2 rené rachou institute, oswaldo cruz foundation (fiocruz), brazil. corresponding author: eduardo josé pereira oliveira, dds, msd, postgraduate program in public health, rené rachou institute, oswaldo cruz foundation (fiocruz). avenida augusto de lima, 1715 – barro preto. 30.190-002. belo horizonte, mg, brasil. email: eduardooliveiraacustico@ gmail.com; phone: +55 35 98868-8867 received: december 27, 2019 accepted: may 17, 2020 oral health-related quality of life among 12-year-olds: results from sb-minas gerais eduardo josé pereira oliveira1,* , fabíola bof de andrade2 aim: to assess oral health-related quality of life (ohrqol) and associated factors among the 12-year-old population of the state of minas gerais, brazil. methods: cross-sectional data from the sb-minas gerais 2012 study were used. the presence of poor ohrqol was assessed using the oral impact on daily performance (oidp) and its dimensions (physical, psychological and social domains). independent variables included sociodemographic factors and variables related to the use of dental care and oral health conditions. the association between the outcomes and the independent variables were tested using logistic regression and the results reported as odds ratio with 95% confidence interval. results: prevalence of poor ohrqol was 31.4%; the psychological domain was the most affected (22.6%). pain and dissatisfaction with oral health were associated with poor ohrqol on overall oidp and all its domains. non-whites had greater poor ohrqol than whites on overall oidp and physical domain. conclusion: self-perceived oral health and social inequalities were associated with poor ohrqol. keywords: dental health surveys. oral health. quality of life. socioeconomic factors. https://orcid.org/0000-0002-9043-5299 https://orcid.org/0000-0002-3467-3989 2 oliveira et al. introduction poor oral health-related quality of life (ohrqol) is reported by one-third of 12-yearolds1 and two-fifths of the 15–19-year olds in brazil2. its main determinants are socioeconomic characteristics, access to dental services2,3 and oral health impairments, such as untreated caries, malocclusion and tooth loss1,4-5. the negative impact of oral health on quality of life can be understood as the burden that oral disorders play in daily life, the system of values, and perception of life as a whole within a cultural context and in relation to personal objectives, standards and concerns6. these impairments occur hierarchically7; speech and chewing functions are the first dimensions to be affected1,8, next are psychological losses, such as restrictions on smiling9, sleep disorders and anxiety or irritability1. finally, there may be disadvantages to social life, including association with bullying10,11, disruption of study and restrictions on leisure among children and adolescents1. in the last decades, important advances have been made in the control of oral diseases in brazil, mainly among schoolchildren12,13. these advances may be attributed to the fluoridation of the public water supply14, dissemination of fluoride toothpaste, decrease in sugar consumption12, as well as improvements in living conditions, and the implementation of public policies featured by expansion in health promotion actions13. however, dental caries and incidence of other oral diseases have increased due to the weakening of successful programs and policies15, with those most harmed being the most socially vulnerable16. similarly, there are inequalities in distribution of poor ohrqol in brazil1-3,8,9 and worldwide17. thus, non-whites2, those in lower levels of income1 and schooling2, and people facing barriers to access dental services1 have been the most affected in their quality of life. although several studies have identified the determinants of ohrqol1-5, continuous monitoring of trends and patterns in different contexts shows they are relevant in addressing oral impairments. studies are lacking that describe ohrqol determinants among 12-year-olds in brazilian states3. we performed this study because we recognized the importance of these studies in identifying regional variations for planning and setting public health priorities. this study aimed to assess oral health-related quality of life and associated factors in a representative sample of 12-year olds from minas gerais state, brazil. materials and methods design, setting and participants a study analyzing observational, secondary data was conducted using data from the last oral health survey performed in minas gerais, brazil (sb-minas gerais 2012), including in its capital (belo horizonte) and in 60 cities within the state. in 2012, minas gerais was the second most populous state in brazil18. sb-minas gerais was conducted using probabilistic sampling by multi-stage conglomerates, with proportional probability of participation by size. the sample plan considered the participants’ region of residence, according to factors used for allocating financial resources pro3 oliveira et al. duced by the joão pinheiro foundation. the cities within the state were classified into quartiles according to economic size and health needs, and the two lower quartiles were grouped in the interior i domain, representing the municipalities with the lowest relative need for financial resources. the upper quartiles were grouped in interior ii—municipalities with the greatest relative need for financial resources. sb-minas gerais was designed to be representative of the state, its capital and the two interior domains at five age groups: 5, 12, 15–19, 35–44, and 65–74 years. clinical and self-perceived oral health measures, demographic and socioeconomic characteristics, access and use of dental services and ohrqol were investigated19. data were collected by trained and calibrated dentists, according to world health organization criteria20. the minimum level of agreement accepted intra and inter examiners was established with kappa equal to 0.65. more details about the design and sampling have been published elsewhere19,21,22. of the 1,217 participants aged 12 years, 996 presented complete data for the variables of interest and were included in the analysis, representing 208,763 adolescents from minas gerais, brazil. variables the dependent variable ohrqol was evaluated using the oral impacts on daily performances (oidp) survey. oidp has three dimensions with a total of nine questions related to daily activities that may be affected by oral conditions in the previous six months. the response options were “no” or “yes” for each of the following activities: 1) physical domain: eat and enjoy food, speak and pronounce clearly, brush teeth, and play sports; 2) psychological domain: sleep and relaxation, smile without embarrassment, and maintain the usual emotional state without anxiety or irritability; 3) social domain: study and attend school, and enjoy contact with people (going out, having fun, going to parties, outings)23. poor ohrqol was considered for participants who reported difficulty in performing one or more activities for overall oidp as well as the physical, psychological and social domains. the independent variables included in the analysis were divided into the following blocks: 1) demographic and socioeconomic characteristics: gender; self-declared race / skin color evaluated according to the brazilian institute of geography and statistics (instituto brasileiro de geografia e estatística–ibge) and categorized as white / non-white (black, brown, yellow [asian]), and indigenous; family income (≤r$500 / r$501–2500 / > r$2500); 2) dental services—time since the last dental appointment (≤1 year / >1 year); type of service used (public / private–including health insurance and covenants); reason for last dental appointment (prevention / other–including pain, extraction, treatment); 3) oral health: presence of untreated caries; dental pain in the last six months; satisfaction with oral health (satisfied–’very satisfied’ and ‘satisfied’) / dissatisfied (‘neither satisfied nor dissatisfied’, ‘dissatisfied’ and ‘very unsatisfied’). statistical analysis descriptive analysis was used to estimate relative frequencies, next were bivariate and multiple analyses. in the bivariate analysis, associations between the independent variables and the outcomes (overall oidp and its domains –physical, psychological and social) were tested using the chi-square test with rao and scott correction24. 4 oliveira et al. the association between the outcomes and the independent variables was tested by means of a logistic regression, and the results reported as odds ratio with 95% confidence interval (95% ci). all the variables with a p<0.2 in the bivariate analysis were included in the multiple logistic regression analyses, according to a hierarchical approach25, following the theoretical framework proposed in figure 1. first, demographic and socioeconomic characteristics (block 1) were included in the model and adjusted by themselves. the use of dental services (block 2) was adjusted for demographic and socioeconomic characteristics and for themselves. finally, the oral health variables (block 3) were adjusted for demographic and socioeconomic characteristics, for the use of dental services and for themselves. all analyses were performed in stata v. 14.0 software using the survey command, which allows to analyze data from complex samples, incorporating sample weights, stratification processes, conglomeration and study design26. ethics statement sbminas gerais was ethically conducted based on the helsinki declaration. this survey received approval from the ethics in research committee of the pontifical catholic university of minas gerais under protocol number 9,173. results the 12-year-old children from minas gerais were characterized by a predominance of non-whites (59%), and family income ranging from r$501 to r$2,500 (77%). most participants had their last dental appointment for reasons other than prevention (63.1%). the prevalence of untreated caries, dental pain in the last six months, and dissatisfigure 1. conceptual model used in hierarchical analysis. demographic & socioeconomic characteristics dental services oral health outcome poor oral health-related quality of life satisfaction with oral health dental paincaries last dental appointment reason for consultation dental service sex skin-color income 5 oliveira et al. faction with oral health were 35.3%, 18.1%, and 32.9%, respectively. about 31.4% of participants had at least one negative impact of oral health on quality of life, 22.6% had impacts on psychological domain, and 6.2% on the social domain. in the bivariate analysis, dental services and oral health were associated with poor ohrqol in overall oidp and all its domains. demographic and socioeconomic characteristics were associated with overall oidp, physical and psychological domains (table 1). table 1. sample characteristics and bivariate analysis of factors associated with poor oral-related quality of life (ohrqol) at 12 years. sb-minas gerais, 2012 (weighted estimates). total oidp overall physical psychological social % % orc % orc % orc % orc total 100 31.4 19.8 22.6 6.2 demographic and socioeconomic characteristics sex men 53.4 28.1 1 18.8 1 20.2 1 5.0 1 women 46.6 35.1 1.38 20.9 1.14 25.4 1.35 7.6 1.55 skin-color white 41.0 22.9 1 12.8 1 17.0 1 3.6 1 non-white 59.0 37.3 2.00** 24.6 2.23** 26.6 1.77* 8.0 2.30 income ≤r$500 8.4 44.5 1 32.3 1 32.2 1 9.1 1 r$501–r$2,500 77.0 32.0 0.59* 19.1 0.49* 23.5 0.65 7.1 0.76 >r$2,500 14.5 20.8 0.33** 16.3 0.41 12.4 0.30** 0.0 1.00 dental services last dental appointment ≤1 year 65.0 29.2 1 18.3 1 21.9 1 5.3 1 >1 year 35.0 35.2 1.33* 22.5 1.29 24.0 1.13 8.0 1.56 reason for consultation prevention 36.9 23.4 1 12.0 1 16.2 1 2.0 1 other 63.1 36.1 1.85** 24.3 2.35** 26.4 1.85** 8.7 4.75** dental service public 52.5 32.0 1 20.2 1 23.3 1 7.7 1 private 47.5 30.7 0.94 19.2 0.93 21.9 0.92 4.6 0.58 oral health caries no 64.7 28.3 1 15.8 1 19.8 1 3.5 1 yes 35.3 37.1 1.49** 27.0 1.97** 27.9 1.57* 11.2 3.46** dental pain no 81.9 22.1 1 11.4 1 15.3 1 1.3 1 yes 18.1 73.4 9.71** 58.0 10.80** 55.6 6.92** 28.3 29.52** satisfaction with oral health satisfied 67.1 18.9 1 12.3 1 10.9 1 3.0 1 dissatisfied 32.9 56.8 5.62** 35.1 3.86** 46.6 7.17** 12.7 4.66** source: sb-minas gerais. oidp: oral impacts on daily performance; orc: crude odds ratio. * p<0.05; ** p<0,01. 6 oliveira et al. table 2 shows the analysis of factors associated with overall oidp and its domains, after adjustment for demographic and socioeconomic characteristics, dental services and oral health. for overall oidp and physical domain, the following groups were more likely to have poor ohrqol: non-white, those with dental pain, and those dissatisfied with their oral health, independently of socioeconomic conditions, dental services and dental care. for the psychological and social domains, after adjustment for multiple variables, subjects with dental pain and dissatisfied with their oral health were more likely to have poor ohrqol. table 2. multiple analysis of factors associated with poor ohrqol at 12 years. sb-minas gerais, 2012 (weighted estimates). oidp overall physical psycological social ora (95%ci) ora (95%ci) ora (95%ci) ora (95%ci) demographic and socioeconomic characteristics sex men 1 1 1 women 1.32 (0.92-1.89) 1.25 (0.79-1.96) 1.35 (0.70-2.61) skin-color white 1 1 1 1 non-white 1.58 (1.06-2.37) 1.72 (1.04-2.84) 1.30 (0.85-1.99) 1.46 0.56-3.85) income ≤r$500 1 1 1 r$501–r$2,500 0.71 (0.38-1.32) 0.64 (0.30-1.34) 0.71 (0.44-1.16) >r$2,500 0.44 (0.18-1.07) 0.73 (0.28-1.94) 0.32 (0.10-1.01) dental services last dental appointment ≤1 year 1 1 >1 year 1.14 (0.81-1.59) 1.36 (0.70-2.64) reason for consultation prevention 1 1 1 1 other 1.14 (0.77-1.67) 1.39 (0.91-2.12) 1.09 (0.68-1.75) 2.30 (0.71-7.47) dental service public 1 private 0.88 (0.46-1.67) continue... 7 oliveira et al. discussion this was the first study to assess oral health-related quality of life and associated factors in a representative sample of the 12-year-old population of minas gerais state, brazil. one-third of participants at this age had poor ohrqol, being the psychological domain the most affected. pain and dissatisfaction with oral health were associated with poor ohrqol on overall oidp and all its domains. non-whites had greater odds of poor ohrqol for overall oidp and physical domain. the prevalence of poor ohrqol at 12 years in minas gerais is similar to that presented by other studies conducted with children and adolescents in brazil1,27 and worldwide28. however, unlike 12-year-old brazilians (for whom negative impacts on the physical domain prevail)1,8, the psychological domain was the most affected in minas gerais. according to locker and allen7, ohrqol is gradually and hierarchically impaired, with physical being the first and social being the last domains to be affected. there is also a gradient of severity ranging from discomfort, pain, disability, impairment and social disadvantage7. thus, the deterioration of ohrqol at 12 years in minas gerais would be at a more advanced stage than in brazil. furthermore, in brazil1,27, worldwide29,30, and for the state of minas gerais, social impacts are the least prevalent, as they represent the final and most severe stage of losses in ohrqol. in the adjusted models, pain and dissatisfaction with oral health were independently associated with the overall oidp and all its domains. similar associations have been reported in other studies for 12-years-olds1 as well as for other age groups2,3,5,6,31,32. besides being associated with physical losses (such as eating, brushing, speaking and playing sports), pain and dissatisfaction with oral health may trigger psychological problems such as sleep disorders, irritation and restrictions on smiling. this process may also be associated with bullying and restrict social life, reaching leisure activities and resulting in school absenteeism11. continuation... oral health caries no 1 1 1 1 yes 0.72 (0.48-1.06) 1.01 (0.69-1.46) 0.77 (0.48-1.25) 1.42 (0.67-3.02) dental pain no 1 1 1 1 yes 7.68 (4.97-11.86) 7.81 (4.77-12.79) 5.15 (3.15-8.39) 17.85 (7.99-39.91) satisfaction with oral health satisfied 1 1 1 1 dissatisfied 4.98 (3.16-7.85) 2.74 (1.66-4.52) 6.39 (4.14-9.86) 2.20 (1.04-4.67) source: sb-minas gerais. oidp: oral impacts on daily performance; ora: adjusted odds ratio; 95%ci: 95% confidence interval. 8 oliveira et al. this study showed no independent association between untreated caries and overall oidp as well as its domains. given the effects of dental pain and dissatisfaction with oral health on ohrqol1,3, one can state that not the cavity itself, but its severity (expressed by pain) and location (which may affect satisfaction, mostly in anterior tooth decay) would result in greater impacts on ohrqol. however, a study with the 12-year-old brazilians found an independent association of caries with oidp in psychological and social domains. cultural, socioeconomic aspects, provision of dental services, distribution and severity of oral diseases in different regions33 may influence the perception of ohrqol and, hence, the associations found. this reinforces the importance of regional representative studies in detecting different disease patterns and related inequalities. the association between satisfaction with oral health and ohrqol reported here was also observed for other authors6,31, especially regarding the psychosocial component33. indeed, even the prevalence of poor ohrqol and dissatisfaction with oral health were similar in the population studied. thus, dissatisfaction with oral health could partially represent poor ohrqol. on the demographic and socioeconomic characteristics, only skin color was associated with the overall oidp and physical domain. this means that non-whites had greater odds of poor ohrqol, as observed by colussi et al.27. on the other hand, in the studies performed by souza et al.1 and scapini et al.34, socioeconomic inequalities in ohrqol among adolescents were associated with family income, but not skin color. in another study conducted with adolescents, adults and elderly from the state of são paulo, brazil, skin color and income remained associated with poor ohrqol3. in some scenarios, skin color is able to identify vulnerable social contexts, as non-white individuals are in lower levels of schooling and income as well as have restricted access to dental services in brazil35. as a consequence, they also present worse oral35 and overall health32. this study has limitations as its cross-sectional design does not allow causal inferences. it also has some strengths: this is one of the few representative data of ohrqol for a brazilian state3, corroborating the understanding of its determinants and distribution in different regions. in conclusion, the prevalence of poor ohrqol at the age of 12 years in minas gerais is significant. recent dental pain and dissatisfaction with oral health were associated with overall oidp and all its domains, and there are inequalities regarding skin color for the physical domain and overall oidp. future studies should explore the origins of these inequalities. strengthening equity in access to dental services, taking into account socioeconomic conditions and the self-perception of individuals may contribute to improved ohrqol. acknowledgments: sb-minas gerais study was supported by state health secretariat of minas gerais, brazil. ejp oliveira received a doctoral scholarship from coordenação de aperfeicoamento de pessoal de nível superior (capes). conflict of interest statement: the authors declare no competing interests. 9 oliveira et al. references 1. souza jg, martins am, silveira mf, jones km, meirelles mp. impact of oral clinical problems on oral health-related quality of life in brazilian children: a hierarchical approach. int j paediatr dent. 2017 jan;27(1):66-78. doi: 10.1111/ipd.12229. 2. peres kg, cascaes am, leão att, côrtes mls, vettore mv. 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[racial inequity in oral health in brazil]. rev panam salud publica. 2012 feb;31(2):135-41. doi: 10.1590/s1020-49892012000200007. portuguese. 1 volume 22 2023 e239097 original article braz j oral sci. 2023;22:e239097http://dx.doi.org/10.20396/bjos.v22i00.8669097 1 dds, msc, endodontist and doctoral graduate student on sciences of rehabilitation, hospital for rehabilitation of craniofacial anomalies, university of são paulo, bauru, brazil. 2 dds, msc, phd, hospital for rehabilitation of craniofacial anomalies, university of são paulo, bauru, brazil. corresponding author: sávio brandelero junior rua silvio marchioni, 3-20 cep: 17012-230 bauru sp, brazil saviobjr@usp.br editor: altair a. del bel cury received: apr 25, 2022 accepted: jun 07, 2022 does the rehabilitation treatment predispose the patient with cleft to endodontic treatment? sávio brandelero junior1* , renata artioli moreira1 , lidiane de castro pinto2 , gisele da silva dalben2 aim: to verify the relation between endodontic treatment of teeth adjacent to the cleft area and the rehabilitation of patients with unilateral cleft lip and palate, at the hospital for rehabilitation of craniofacial anomalies. methods: the present split-mouth study was composed of 406 individuals with complete unilateral cleft lip and palate who had completed the rehabilitation process in a single tertiary cleft center (55.9% males). the information was collected from the dental history on the patients’ records and radiographs. the frequency of endodontic treatment in the upper incisors and canines was calculated for the cleft and non-cleft sides. the comparison between sides was performed by the chi-square test (p <0.05). results: endodontic treatment was more frequent in teeth adjacent to the cleft than in contralateral teeth. the frequency of endodontic treatment in at least one tooth adjacent to the cleft was 18.97%, and 11.6% on the contralateral side. it was observed that endodontic treatment was necessary in 63.5% of patients who had been submitted to orthodontic treatment, 42.4% of those rehabilitated with fixed partial dentures and 12.0% of patients who underwent dental reshaping of teeth adjacent to the cleft. the treatments performed included vital pulp therapy (46.1%), non-vital pulp therapy (46.8%) and endodontic retreatment (7.1%). conclusion: in individuals with complete cleft lip and palate, teeth close to the bone defect area and used for rehabilitation treatment presented greater need of endodontic intervention. keywords: cleft palate. cleft lip. endodontics. diagnosis. https://orcid.org/0000-0002-7695-3858 https://orcid.org/0000-0001-7976-2945 https://orcid.org/0000-0001-9764-0327 https://orcid.org/0000-0002-5203-796x 2 brandelero junior et al. braz j oral sci. 2023;22:e239097 introduction endodontic treatment is a relatively complex procedure. for this reason, knowledge on the anatomy of dental structures is essential, since many morphological variations can occur in patients with cleft lip and palate1. in individuals with cleft, the diagnosis of endodontic lesions becomes more challenging. depending on the extent, the bone defect can be seen as a radiolucent area surrounding the periapical portion of teeth adjacent to the cleft. this evidences the importance of performing adequate anamnesis, physical and complementary exams for an accurate diagnosis2,3. the need for endodontic intervention can have several etiologies. the incorrect hygiene4, due to poor positioning of teeth in the arch, can contribute to tooth decay. exacerbated orthodontic mechanical forces, tooth resorption and prosthetic rehabilitation in the cleft area may also be related to the need for endodontic treatment5. knowing the characteristics of individuals with clefts in all aspects is very important for endodontic treatment. dental changes in shape, size and position, difficult cleaning and a long period of rehabilitation are frequent6. therefore, the aim of this study was to investigate the relationship between endodontic intervention in teeth adjacent to the unilateral complete cleft lip and palate and dental rehabilitation treatment. the null hypothesis was that teeth adjacent to the cleft do not present a greater risk for endodontic treatment when compared to contralateral teeth. materials and methods this present split mouth study was approved by the institutional review board of the hospital for rehabilitation of craniofacial anomalies. a list of individuals with complete unilateral cleft lip and palate that had received hospital discharge was requested to the hospital informatics service. the search retrieved 2,521 patients. from sample calculation, 406 were randomly selected to be part of the study. the information was obtained based on the patient’s dental records. sample calculation considered an alpha of 5% and test power of 80%. the sample calculation indicated a minimum sample of 288. data collected were gender, side affected by the cleft, type of rehabilitation at the cleft area, presence of endodontic treatment and pulp diagnosis. the experimental group (cs) included the teeth adjacent to the cleft area (central incisor, lateral incisor or canine). the control group (ncs) was composed of analysis of contralateral teeth at the non-cleft side. after collection, data related to the individual, rehabilitation treatment and endodontic intervention were tabulated. descriptive analysis of data related to gender, side affected by the cleft, rehabilitation modalities, pulp diagnosis and type of endodontic treatment were performed. the chi-square test (x2) was applied to compare the number of endodontic treatments between sides with and without cleft, and endodontic treatments performed concurrently or outside the period of orthodontic intervention. a p-value smaller than 5% was considered significant. 3 brandelero junior et al. braz j oral sci. 2023;22:e239097 results there was predominance of males, and the occurrence of complete unilateral cleft lip and palate was greater on the left side (64.1%). as for the records, 37.9% of individuals in the study were submitted to at least one endodontic treatment. overall, 18.9% had undergone endodontic treatment in at least one tooth adjacent to the cleft, greater than on the non-cleft side (11.5%) (table 1). table 1. number and frequency of individuals undergoing endodontic treatment involving teeth adjacent to the cleft side compared to the non-cleft side (pearson’s chi-square test with yates correction). endodontic treatment cleft side % non-cleft side % absent 329 81.1 359 88.4 present 77 18.9 47 11.6* total 406 100.0 406 100.0 *represents statistically significant difference between groups (p<0.05) when the type of rehabilitation to which these individuals were submitted was analyzed, the most common was fixed partial dentures (30.4%), followed by dental reshaping with composite resin (30%). no information was found in 103 records (table 2). table 2. rehabilitation modalities in individuals with complete unilateral cleft lip and palate. type of rehabilitation n % fixed partial denture 92 30.4 reshaping 91 30.0 removable partial denture 60 19.8 implant 41 13.5 complete denture 19 6.3 total 303 100.0 among the 406 patients, 286 had undergone orthodontic treatment. the mean treatment time was 10.4 (+/4.7) years. among these patients, 104 underwent endodontic treatment, of which 66 were treated during the period of orthodontic treatment, while 38 underwent endodontic treatment outside the period of orthodontic intervention (table 3). table 3. endodontic treatment in individuals with complete unilateral cleft lip and palate performed during or outside the orthodontic intervention period (pearson’s chi-square test with yates correction). treatment n % endodontics with orthodontics 66 63.5* endodontics without orthodontics 38 36.5 total 104 100.0 *represents statistically significant differences between groups (p <0.05). 4 brandelero junior et al. braz j oral sci. 2023;22:e239097 table 4 shows the frequency of endodontic interventions associated or not with each rehabilitation modality. table 4. need for endodontic treatment for rehabilitation with fixed partial dentures (fpd) and dental reshaping in individuals with complete unilateral cleft lip and palate. treatment n % fpd + endodontics 39 42.4 fpd 53 57.6 reshaping + endodontics 11 12.0 reshaping 80 88.0 the most frequent pulp diagnosis was pulp necrosis (54.0%). the most frequent endodontic treatment was non-vital pulp therapy (46.8%), followed by vital pulp therapy (46.1%) (table 5). table 5. pulp diagnosis, number and frequency of endodontic treatments to which individuals with complete unilateral cleft lip and palate were submitted. pulp diagnosis n % healthy pulp 47 16.1 reversible stage 11 3.8 transition stage 11 3.8 irreversible stage 65 22.3 necrotic pulp 157 54.0 treatment n % vital pulp therapy 136 46.1 non-vital pulp therapy 138 46.8 retreatment 21 7.1 discussion this is a quantitative, descriptive, retrospective and documental study, with no reports of similar studies in individuals with cleft. there was predominance of complete unilateral cleft lip and palate in the male gender (55.9%). these data agree with studies by martelli júnior et al.7 (2006) and cymrot et al.8 (2010), who found, respectively, the frequencies in the male gender of 61%, 53.5%, 60% and 53%. concerning side, it was observed that the left side is significantly more affected than the right. this information agrees with the studies of carvalho and tavano9 (2008); cymrot et al.8 (2010). additionally, it was found that the probability of a tooth adjacent to the cleft area requiring endodontic treatment is almost two times higher when compared to contralateral teeth. teeth adjacent to the cleft area may present adverse conditions for 5 brandelero junior et al. braz j oral sci. 2023;22:e239097 the rehabilitation treatment, such as the presence of crowding, rotation, malformations of the dental structure10, partial eruption, absence of keratinized mucosa11, high prevalence of gingival recession12, shallow vestibule13 and hygiene difficulties14. the negligent hygiene can cause dental caries and, consequently, gingival, periodontal and endodontic changes15. this fact becomes worrying when it was found that 37.9% of individuals in the sample needed at least one endodontic intervention. fixed orthodontic devices such as bands, brackets, elastics, and arches are also complicating factors, since they influence the biofilm accumulation and can lead to enamel decalcification and caries16. among the individuals evaluated, 63.5% who used brackets required at least one endodontic intervention during orthodontic treatment. the mean duration of orthodontic treatment was 10 years and 4 months. according to ahluwalia et al.17 (2004), individuals with clefts need longer treatment than individuals without clefts. however, orthodontics cannot always solve more complex cases, such as those with marked discrepancies. in these situations, orthognathic surgery is indicated and often must be planned together with prosthetic rehabilitation, to establish a good prognosis18. the present study demonstrated that 63.7% needed some type of prosthesis during rehabilitation. this agrees with siqueira et al. (2021)5, who found that 30% of individuals with clefts need some type of prosthesis. therefore, the need of fixed partial dentures led to greater need of endodontic treatment for rehabilitation procedures at the cleft areas5. the esthetic function includes improving the individual’s profile, supporting the upper lip and aligning the teeth. individuals with cleft lip and palate usually have a low smile line, that is, most of these individuals do not have exposure of teeth and gingival tissues when smiling, which facilitates cosmetic prosthesis, even in the absence of bone or gingival tissue19,20. treatment with fixed partial dentures is still widely indicated, especially in case of failure or impossibility of performing the alveolar bone graft21. when the graft is performed satisfactorily and in the ideal period of rehabilitation, orthodontic treatment is only complemented with dental revitalization, mainly because these individuals frequently present changes related to shape, size, number and position. in some cases, the canine assumes the position of the lateral incisor in the arch and it is necessary to transform the shape of this tooth to maintain the function and esthetics2. this type of rehabilitation is generally less traumatic for the dental structure. this fact can be confirmed by the results obtained, in which only 12.0% of reshaped teeth required endodontic treatment22. the most frequent endodontic diagnosis was necrotic pulp (54.0%), followed by irreversible pulpitis (22.3%). regarding the type of endodontic treatment performed, it was found that non-vital pulp therapy was the most frequent with 46.8%, followed by vital pulp therapy (46.1%). in the study by hussne et al.6 (2009), 1377 teeth were evaluated. non-vital pulp therapy was the most frequently performed therapy (51.34%), followed by vital pulp therapy (36.60%) and endodontic retreatment (12.06%). the authors also found that the main reason for performing endodontic treatment in individuals with clefts was dental caries (56.14%), followed by prosthetic purposes (18.95%) the high prevalence of dental caries and the frequent need for prosthetic rehabilitation of these individuals increase the likelihood of the need for endodontic treatment. many individuals present themselves for reparative surgeries without ever having 6 brandelero junior et al. braz j oral sci. 2023;22:e239097 received any dental assistance, presenting decayed teeth with substantial loss of dental structure and the need for extensive oral rehabilitation. in addition to the peculiar characteristics of these individuals, the socioeconomic and geographic aspects can contribute to this situation. the difficult access to adequate treatment due to the distance from specialized centers and the refusal of many professionals to treat individuals with cleft, due to insecurity or lack of knowledge, end up restricting oral health care to many of these individuals23. however, rehabilitative procedures must be performed at a suitable period to not jeopardize the craniofacial growth and development. moreover, it is imperative to combine a specialized and qualified multidisciplinary team with clinical care and surgical experience24,25. in conclusion, teeth adjacent to the alveolar cleft are at greater risk for endodontic treatment when compared to the contralateral teeth, especially during the period of orthodontic treatment and when there is need to rehabilitate the cleft area with partial fixed denture. this shows the importance of dental support during the rehabilitation process in efforts aimed at preserving the teeth and maintaining the volume of alveolar bone adjacent to the cleft. data availability datasets related to this article will be available upon request to the corresponding author. conflict of interests none. author contribution sávio brandelero junior – contributed substantially to the conception and design of the study, the acquisition of data, the manuscript’s findings, and the analysis and interpretation, have revised and approved the final version of the manuscript. renata artioli moreira – contributed substantially to the conception and design of the study, the acquisition of data, the manuscript’s findings, and the analysis and interpretation, have revised and approved the final version of the manuscript. lidiane de castro pinto agree to be accountable for all aspects of the work as the manuscript’s findings, in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved, have revised, and approved the final version of the manuscript. gisele da silva dalben the manuscript’s findings, drafted or provided critical revision of the article. provided final approval of the version to publish. agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. all authors actively participated in the manuscript’s findings, and have revised and approved the final version of the manuscript. 7 brandelero junior et al. braz j oral sci. 2023;22:e239097 references 1. arora a, batra p, sood sc, raghavan s, sood a. comparison of dentofacial morphology between parents of children with and without craniofacial anomalies using cephalogram imaging. indian j dent res. 2021 oct-dec;32(4):472-9. doi: 10.4103/ijdr.ijdr_623_21. 2. pinheiro cr, de castro pinto l, mateo-castillo jf, siqueira v da s, garib d. response to pulp vitality tests in teeth submitted to orthodontic movement, adjacent to the cleft area. cleft palate craniofac j. 2022 jan;59(1):72-8. doi: 10.1177/1055665621996115. 3. khademi aa, shahtouri mm, attar bm, rikhtegaran n. pulp vitality of maxillary canines after alveolar cleft bone grafting: pulse oximetry versus electric pulp test versus cold test. j craniofac surg. 2021 may;32(3):e314-7. doi: 10.1097/scs.0000000000002544. 4. siqueira vds, castillo aes, mateo-castillo jf, pinto ldc, garib d, pinheiro cr. dental hypersensitivity in individuals with cleft lip and palate: origin and therapies. j dent res dent clin dent prospects. 2021 winter;15(1):42-6. doi: 10.34172/joddd.2021.008. 5. siqueira vs, mateo-castillo j-f, pinto lc, garib d, pinheiro c-r. etiological factors commonly related to the need of endodontic treatment in individuals with orofacial clefts. j clin exp dent. 2021 jun;13(6):e580-5. doi: 10.4317/jced.57980. 6. hussne rp, berbert fl, nishiyama ck, câmara as, pinheiro cr, leonardo rt. investigation of the endodontic needs and planning in patients with cleft lip and or palate submitted to surgical treatment. perspect oral sci. 2009;1(2):19-23. 7. martelli júnior h, orsi júnior j, chaves mr, barros lm, bonan prf, freitas jas. [epidemiologic study of cleft lip and palate in alfenas minas gerais from 1986 to 1998]. rpg rev pos-grad. 2006 jan-mar:13(1)31-5. portuguese. 8. cymrot m, sales fcd, teixeira faa, teixeira junior faa, teixeira gs, cunha filho jfo, et al. [prevalence of kinds of cleft lip and palate at a pediatric hospital in northeast of brazil]. rev bras cir plást (impr). 2010;25(4):648-51. portuguese. doi: 10.1590/s1983-51752010000400015. 9. carvalho lcf, tavano o. [dental agenesis in cleft lip and/or palate pf the centro pró-sorriso universidade josé do rosário vellano]. rgo. 2008;56(1):39-45. portuguese. 10. ruiz la, maya rr, d’alpino php, atta mt, da rocha svizero n. prevalence of enamel defects in permanent teeth of patients with complete cleft lip and palate. cleft palate craniofac j. 2013 jul;50(4):394-9. doi: 10.1597/11-200. 11. almeida alpf, esper la, kaizer rof, fernandes js, greghi sla, carrilho gpb. surgical treatment of mucogingival alterations in cleft lip and palate patients: a clinical report. periodontal pract today. 2006 jan;3(1):31–5. 12. de almeida alpf, esper la, pegoraro ta, valle ald. gingival recession in individuals with cleft lip and palate: prevalence and severity. cleft palate craniofac j. 2012 jan;49(1):92-5. doi: 10.1597/10-052. 13. de almeida al, pedro pf, kogawa em, pereira t, de barros carrilho gp, aiello ca, et al, comparative evaluation of two different vestibuloplasty surgical procedures in cleft patients: a pilot study. cleft palate craniofac j. 2005 jul;42(4):439-41. doi: 10.1597/04-052r.1.. 14. brasil jmp, pernambuco ra, dalben gs. suggestion of an oral hygiene program for orthodontic patients with cleft lip and palate: findings of a pilot study. cleft palate craniofac j. 2007 nov;44(6):595-7. doi: 10.1597/06-057.1. 15. al-dajani m. comparison of dental caries prevalence in patients with cleft lip and/or palate and their sibling controls. cleft palate craniofac j. 2009 sep;46(5):529-31. doi: 10.1597/08-003.1. 16. pilli ln, singaraju gs, nettam v, keerthipati t, mandava p, marya a. an extensive comparison of the clinical efficiency of acidulated phosphate fluoride (apf) and neutral sodium fluoride (naf) 8 brandelero junior et al. braz j oral sci. 2023;22:e239097 oral rinses in the prevention of white spot lesions during fixed orthodontic treatment: a randomized controlled trial. biomed res int. 2022 mar;2022:6828657. doi: 10.1155/2022/6828657. 17. ahluwalia m, brailsford sr, tarelli e, gilbert sc, clark dt, barnard k, et al. dental caries, oral hygiene, and oral clearance in children with craniofacial disorders. j dent res. 2004 feb;83(2):175-9. doi: 10.1177/154405910408300218. 18. fukuda m, takahashi t, yamaguchi t, kochi s, inai t, watanabe m, et al. dental rehabilitation using endosseous implants and orthognathic surgery in patients with cleft lip and palate: report of two cases. j oral rehabil. 2000 jun;27(6):546-51. doi: 10.1046/j.1365-2842.2000.00539.x. 19. esper la, sbrana mc, ribeiro iwj, siqueira de en, de almeida alpf. esthetic analysis of gingival components of smile and degree of satisfaction in individuals with cleft lip and palate. cleft palate craniofac j. 2009 jul;46(4):381-7. doi: 10.1597/07-189.1. 20. fiamengui filho jff, de almeida alpf. aesthetic analysis of an implant-supported denture at the cleft area. cleft palate craniofac j. 2013 sep;50(5):597-602. doi: 10.1597/11-193. 21. watanabe i, kurtz ks, watanabe e, yamada m, yoshida n, miller aw. multi-unit fixed partial denture for a bilateral cleft palate patient: a clinical report. j oral rehabil. 2005 aug;32(8):620-2. doi: 10.1111/j.1365-2842.2005.01468.x. 22. janiszewska j. wędrychowskaszulc b. [missing permanent tooth buds] magazyn stomatol. 2000;10(4):42-3. polish 23. freitas ja, almeida al, soares s, neves lt, garib dg, trindade-suedam ik, yaedú ry, et al. rehabilitative treatment of cleft lip and palate: experience of the hospital for rehabilitation of craniofacial anomalies/usp (hrac/usp) part 4: oral rehabilitation. j appl oral sci. 2013;21(3):284-92. doi: 10.1590/1679-775720130127. 24. global strategies to reduce the health care burden of craniofacial anomalies: report of who meetings on international collaborative research on craniofacial anomalies. cleft palate craniofac j. 2004 may;41(3):238-43. doi: 10.1597/03-214.1. 25. freitas j de s. neves lt das, almeida alpf de, garib dg, trindade-suedam ik, yaedú ryf, et al. rehabilitative treatment of cleft lip and palate: experience of the hospital for rehabilitation of craniofacial anomalies/usp (hrac/usp)-part 1: overall aspects. j appl oral sci. 2012 feb;20(1):9-15. doi: 10.1590/s1678-77572012000100003. 1http://dx.doi.org/10.20396/bjos.v19i0.8661431 volume 19 2020 e201431 original article 1 department of preventive dentistry, faculty of dentistry, lagos state university college of medicine, ikeja, lagos, nigeria. 2 department of child dental health, faculty of dentistry, lagos state university college of medicine, ikeja, lagos, nigeria. 3 department of preventive dentistry, faculty of dentistry, university of medical sciences, ondo, ondo state, nigeria. 4 department of child dental health, faculty of dental sciences, college of medicine university of lagos, nigeria. corresponding author: afolabi oyapero department of preventive dentistry, faculty of dentistry, lagos state university college of medicine, ikeja, lagos. email: fola_ba@yahoo.com received: september 29, 2020 accepted: november 26, 2020 association between dental caries, odontogenic infections, oral hygiene status and anthropometric measurements of children in lagos, nigeria afolabi oyapero1,* , aderinsola adenaike2 , augustine edomwonyi3, abiola adeniyi1, olubukola olatosi4 dental caries is a significant public health problem afflicting about a third of the world’s population which impacts nutrition, quality of life and systemic health. aim: we explored associations between dental caries, odontogenic infections, oral hygiene and anthropometric measurements of children in lagos, nigeria. methods: a pretested validated questionnaire was administered on 278 children who also received anthropometric assessment and dental examinations. caries was scored according to who criteria and untreated dental caries by the pulpal exposure, ulceration, fistula, abcess (pufa/pufa) index. the weight for age (waz), height for age (haz), and weight for height (whz) parameters evaluated nutritional status. categorical and continuous data were analysed by χ2-test and anova. regression analysis was done and statistical significance set at p ≤ 0.05. results: the prevalence of decayed, missing, and filled teeth (dmft + dmft) > 0 was 220 (79.1%) and the proportion of d+d teeth in dmft+dmft index was 194 (70.0%). the prevalence of odontogenic infections due to caries (pufa + pufa > 0) was 172 (61.8%). 74 (26.6%) children were stunted; 12 (4.3%) were underweight while 30 (10.8%) were wasted. children with pufa + pufa > 1 had increased risk of wasting (or: 2.45; 95% ci: 1.16-4.88). children with dmft+dmft >5 were also significantly underweight with odds ratios of 2.34 (95% ci 1.04-4.33). conclusions: there was significant association between untreated dental caries, odontogenic infections and stunting, wasting and being underweight among the children studied. policy makers should be aware of the additional burden that oral neglect has on anthropometric indices. key words: anthropometry. body mass index. dental caries. dental caries susceptibility. oral hygiene. http://orcid.org/0000-0003-4433-8276 https://orcid.org/0000-0003-0324-1144 http://orcid.org/0000-0003-1395-8261 2 oyapero et al. introduction dental caries is a significant public health problem afflicting about a third of the world’s population with wide ranging impacts on nutrition, quality of life and systemic health1. it is a lifelong progressive and cumulative disease, associated with pain and anxiety, time lost from work and school, and when untreated, can lead to hospitalization2. the greatest disease burden of dental caries is borne by children in developing countries where health promotion initiatives and access to dental treatment are limited, in an existing milieu of underlying poverty, malnutrition and communicable diseases3. in addition to negative impact of dental caries on quality of life, it is also costly to health care systems, accounting for ≤10% of health care budgets in industrialized countries, being the fourth most expensive disease to treat4. the cost of treating dental caries is estimated to be us $3513 per 1000 children in most low-income countries, exceeding the total health budget of these countries5. furthermore, in these low-income countries, oral health policies are usually of low priority, often being completely neglected due to limited resources. the carious process commences and progresses when oral bacteria metabolize fermentable carbohydrates, producing acids, which diffuse into hard dental tissue, and demineralize tooth enamel. some authors observed that the frequency of sugar consumption is a stronger predictor of caries risk than the total amount consumed, while others observed that the amount of sugar consumed is more important than consumption frequency6. oral hygiene is likewise crucial for caries prevention, and the likelihood of progression of the carious process is higher when oral hygiene is poor.  thus, the inadequate removal of dental plaque, the frequent intake of sugary foods and drinks and poor access to fluoride in susceptible individuals is invariably linked to caries development and progression7. consequently, several recent preventive interventions emphasize the maintenance of a favorable oral environment through oral hygiene, restriction of sugar consumption and access to adequate levels of fluoride8. the high prevalence of dental caries is however not only influenced by risk factors like fermentable carbohydrates and a susceptible tooth surface that interact with the causative microorganisms, but other risk indicators such as developmental characteristics at birth, socioeconomic background and access to oral health services9. socio-demographic and behavioral indicators that predispose an individual to increased caries experience include age, gender, educational status, social class, genetic factors and systemic health10. furthermore, certain common risk factors exist between caries and certain systemic condition, such as the frequent ingestion of fermentable sugars, which can also lead to obesity11. researchers have always suspected that a link exists between growth/development and dental caries presumably due to a shared common pathogenesis because nutrition, parenting, lifestyle, physical and social environments, as well as psychosocial factors can influence both conditions12. moreover, untreated dental decay is thought to be a neglected determinant of low body mass index (bmi)13. these research findings that link oral health to general health problems provide an opportunity to put oral health in the agenda of health care policies in low-income developing countries14. 3 oyapero et al. the decayed, missing, and filled teeth (dmft) index is a valid tool for assessing the extent and prevalence of dental caries as well as for assessing overall oral health15 but it has some limitations due to skewed distributions of caries prevalence in many countries and its inability to assess the consequences of untreated caries. the pulpal exposure, ulcers due to root fragments, fistula, abcess (pufa/pufa) index introduced by monse et al.16. in 2010 complements the dmft index by displaying the severity of dental decay and quantifying odontogenic infections of the pulp and surrounding tissues due to untreated caries. many researches that aimed to correlate dental caries with bmi did not account for oral hygiene and did not stratify caries based on the pufa index. it is necessary to do this because it is neglected and untreated dental caries that often results in pain and discomfort that may affect ability to chew and ultimately nutrition. although evidence on the probable effect of untreated dental caries is receiving attention in developed countries, in developing countries, the impact of untreated dental caries on malnutrition and well-being is not adequately researched, with no such study documented in nigeria. this study thus aimed to determine the relationship between caries risk, untreated dental caries (pufa), oral hygiene and the anthropometric measurements of children at a tertiary paediatric dental clinic in lagos, nigeria. materials and methods ethical considerations: ethical approval for the study was obtained from the health research and ethics committee of the lagos state university teaching hospital, ikeja (lrec/06/10/1415). the study was implemented in line with the declarations of helsinki and the confidentiality of all the participants was assured by the researchers. study design and study population: this was a descriptive study conducted among paediatric patients that presented for treatment at the child dental health clinic after they were referred for treatment from the oral diagnosis clinic of the lagos state university teaching hospital, ikeja, lagos, nigeria (lasuth). lasuth is a tertiary teaching hospital in the cosmopolitan city of lagos. lagos state university teaching hospital is an urban tertiary center located in the heart of lagos, nigeria. it serves as a referral center for public and private hospitals in lagos and her neighboring states.  sample size: a confidence interval of 95% was used for the sample size calculation, with an absolute precision of 0.05 and with a standard error of 2% or less; using a recent nigerian caries prevalence (p= 20.4%)17, a minimum sample size of 240 was required. we however 272 recruited for the study to make provision for incomplete data. inclusion and exclusion criteria: the inclusion criteria was children and adolescents aged 4-16 years of both genders whose parents agreed to participate in the study and who gave informed consent and assent where appropriate. children with physical or mental disability with such conditions that have difficulty in managing routine oral hygiene measures and hence are more prone to dental caries, those with any developmental dental anomaly and those with ongoing dental treatment were excluded. sampling technique: our study sample was obtained by a systematic random sampling technique. we randomly selected every third patient on the attendance register 4 oyapero et al. after balloting for the starting point. we planned to recruit 6 patients per clinic day, given that about 20 patients are booked for each session. they were recruited on each wednesday and friday paediatric dentistry clinic day until the required sample size was attained. study procedure study instrument: an interviewer administered questionnaire was utilized for data collection. data on the socio-demographic profile was obtained from the caregiver of each study participant. these included information on the age, gender, as well as maternal and paternal education and occupation. oral health-related behaviour related to dietary habits, oral hygiene practices and past dental visits was also obtained. anthropometric measures were then taken and clinical examinations were conducted to determine the dental caries status. standardisation of examiners: the intraand inter-examiner-reproducibility was assessed by the kappa (κ) statistics. calibration of examiners (two dental resident doctors) was done by a consultant community dentist. duplicate examinations were taken on 20 randomly selected children in the oral diagnosis unit to evaluate intra-examiner and inter-examiner reproducibility. the κ values for inter-examiner-reproducibility ranged from 0.88 (o.a.) to 0.90 (a.a.) for the pufa/pufa index, and between from 0.92 (o.a.) to 0.93 (a.a.) for the dmft/dmft index. intra-examiner reproducibility ranged from 0.91 to 1.00 (o.a.) and between 0.92 to 1.00 (a.a.) for both indices. anthropometric measures: the height of the children standing upright and without shoes was measured with a portable stadiometer (seca 216 height rod; seca gmbh & co. kg, hamburg, germany) to the nearest 0.5 cm. weight was measured with children wearing light outfit using an electronic digital scale (soehnle gala xl; leifheit ag, nassau, germany) to the nearest 0.5 kg. the measuring equipment was re-calibrated daily and all measurements were done by a well-trained nurse following standardized guidelines. the measuring equipment was re-calibrated daily. oral examinations: oral examinations were done using plane mouth mirrors and blunt dental probes oral examination. when an active or restored carious cavity or extracted tooth due to caries was detected on manual inspection, it was recorded as caries on the dental charts using the dmft+dmft index. pufa+pufa was used to record the presence of a visible pulp (p/p), ulceration of the oral mucosa due to root fragments (u/u), a fistula (f/f) or an abscess (a/a) resulting from untreated caries in the primary (pufa) and permanent (pufa) dentition for the presence of either the pufa+pufa and dmft+dmft score per child was calculated in the a cumulative way without any dichotomy. data entry and analysis: data was analysed with spss version 22.0 (ibm, armonk, ny). mean dmft+dmft score for both primary and permanent dentition was reported together. untreated dental caries severity was recorded using pufa+pufa index and reported together as mean scores. caries prevalence and overall pufa+pufa prevalence was calculated as a percentage.  the weight for age (waz), height for age (haz), and weight for height (whz) parameters were calculated using who epi 3.5 nutritstat software. using age and gender specific criteria, children were categorized as 5 oyapero et al. being at significant risk for either inadequate (< -2 sd) or excessive (> +2 sd) growth. nutritional status, an independent variable, was regrouped, stunting was defined as haz <-2.0, thinness as waz <-2.0, overweight as waz >1.0 and obesity as waz >2.0. frequency tables were generated for all variables and mean scores computed for numerical variables. the correlation between dmft and pufa scores was computed by the spearman’s rank correlation coefficient (ρ). categorical data were compared between groups using the chi-square test and continuous data were analyzed by t-test and anova. logistic regression model controlled for variables that showed statistical significance (p < 0.05) in bi-variate analysis with low bmi as dependent variable. the probability level of p<0.05 was considered statistically significant. results out of the 278 children seen, 146 (52.5%) were female, the highest proportion (116; 41.7%) were aged between 6-10 years and the mean age was 8.82 ±3.4 yrs. mean maternal age was 36.45 ±3.22; most (180; 64.7%) were of the middle social class and 212 (76.8%) of mothers had ≥12 years of formal education. for caries risk factors and indicators, the children consumed carbonated drinks and beverages an average of 2.14 ±2.26 times per week and cookies, biscuits or sweets 3.65 ±2.54 times per week. 74(26.6%) children has poor oral hygiene while 209(75.2%) had no dental home or previous dental visits. 48(17.3%) children had enamel hypoplasia. maternal educational level, social class, number of children in the family, consumption of cariogenic meals, poor oral hygiene and history of dental visits were all significantly associated with dmft+dmft and pufa+pufa values (p<0.05). (table 1) table 1. demographic characteristics, caries risk indicators and caries status of the sample. category mean ±sd or no. (%) dmft+dmft; χ2; p value pufa+pufa;  χ2; p value children’s demographics n=278 male 132 (47.5) dmft=dmft≥1: 99 (75.0) 25.20; 0.406 5.88; 0.437 female 146 (52.5) dmft=dmft≥1: 121 (82.9) age, y 8.82 ±3.4 8.45;0.207 6.25;0.396 1.0–5.0 52 (18.7) 6.0–10.0 116 (41.7) 11.0–15.0 110 (39.6) parents’ demographics age, y 36.45 ±3.22 length of formal education, y (mean) 13.35 ±3.49 <12 years of formal education (mum) 66 (23.2) 11.65; 0.039** 6.22; 0.027** ≥12 years of formal education (mum) 212 (76.8) survey respondent is the mother 268 (96.4) continue https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4638248/table/tbl1/ 6 oyapero et al. continuation social class lower 40 (14.4) 4.67; 0.014** 3.55; 0.012** middle 180 (64.7) upper 58 (20.9) number of children 4.45; 0.028** 6.70; 0.012** 1-4 226 (81.3) 5-8 52 (18.7) caries risk factors and indicators carbonated drinks and beverages times/wk 2.14 ±2.26 never or rarely 54 (19.5) 4.09; 0.000** 4.23; 0.000** weekly or more frequently 56 (20.1) daily or more frequently 168 (60.4) cookies, biscuits or sweets times/wk 3.65 ±2.54 never or rarely 56 (20.1) 2.88; 0.014** 1.72; 0.001** weekly or more frequently 84 (30.2) daily or more frequently 138 (50.8) use of fluoridated toothpaste (no) 98 (35.3) 2.52; 0.281 4.09; 0.129 child has poor oral hygiene; visible plaque 74 (26.6) 2.49; 0.012** 1.83; 0.016** child has enamel hypoplasia 48 (17.3) 1.032; 0.59 1.217; 0.54 no dental home or previous dental visits 209 (75.2) 2.75; 0.025** 4.23; 0.020** mother or sibling has un-restored cavities 42 (15.1) 1.62; 0.032** 1.02; 0.044** impaired salivary flow 8 (2.9) ** significant the overall prevalence of caries (dmft + dmft > 0) was 79.1%) while the overall prevalence of odontogenic infections due to caries (pufa + pufa > 0) was 61.8%. decayed teeth (d/d) accounted for 70% in dmft + dmft and the mean no. of decayed teeth (dt/dt x ± sd) was 3.01 ±2.34. mean dmft+dmft score was 3.63 ±3.18 and 112 (40.3%) of the children had dmft+dmft scores > 5. the mean pufa+pufa score was 2.42 ±1.95 and pulp exposure accounted for the highest proportion (mean p x ± sd=1.98±1.54). for child nutritional outcomes, 74 (26.6%) children were stunted, 12 (4.3) were underweight and 30 (10.8) were wasted. (table 2) table 2. child oral health outcomes and nutritional parameters. variables categories mean ±sd or no. (%) valid no. has any tooth decay (yes) 220 (79.1) 278 proportion of d teeth in dmft+dmft index (d/d) 194 (70.0) decayed teeth (dt/dt x ± sd) 3.01 ±2.34 missing teeth (mt/mt x ± sd) 0.55 ±1.13 continue 7 oyapero et al. continuation filled teeth (ft/ft x ± sd) 0.07±0.18 severity of dental caries dmft+dmft = 0 58 (20.9) dmft+dmft = 1–5 108 (38.8) dmft+dmft > 5 112 (40.3) average dmft+dmft score 3.63 ±3.18 care index (%) pulp involved decayed teeth yes (pufa+pufa prevalence) 172 (61.8) pufa+pufa (x ± sd) 2.42 ±1.95 pulp exposure (p x ± sd) 1.98±1.54 ulceration (u x ± sd) 0.0±0.00 fistula (f x ± sd) 0.11±0.12 abcess (a x ± sd) 0.33±0.46 child nutritional outcomes undernourished statusa height for age (stunted), no. 74 (26.6) height for age (stunted), z-score 1.81±0.6 weight for age (underweight), no. 12 (4.3) weight for age (underweight), z-score -0.19±1.4 bmi for age (wasted), no. 30 (10.8) bmi for age (wasted), z-score 0.2±2.8 overweight status no. 50 (19.9) z-score 0.2±2.8 there was a low positive correlation between poor oral hygiene and dental caries severity (ρ = 0.307;  p =0.005) and a moderate positive correlation between caries severity and odontogenic infection (ρ = 0.604; p <0 .001). we likewise found significant associations between the severity of the children’s carries experience and their anthropometric measurements. for children with the high-severity caries group, the mean height-for-age, weight for ageand bmi-for-age  z-scores were –0.36 (95% ci -1.12, -0.67); –0.53 (95% ci -0.62, -0.17) and –1.03 (95% ci -0.58, -0.16) lower, respectively, than those in the caries-free children. similarly, for children with >1 odontogenic infection, the mean height-for-age, weight for ageand bmi-for-age z-scores were –0.79 (-1.88, -1.17); –0.75 (-1.01, -0.62) and –0.65 (-0.62, 0.20) lower than in children with no odontogenic infection. (table 3) https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4638248/table/tbl1/?report=objectonly#tblfn1 8 oyapero et al. table 3. bivariate analysis between caries indices and nutritional parameters. predictive variable outcome variable correlation factor (ρ) or test statistic ( f) mean z-score mean difference from reference (95% ci) p poor oral hygiene (di score > 1.5) mean dmft+dmft ρ = 0.307 0.005** mean dmft+dmft mean pufa+pufa ρ = 0.645 <0 .001** dmft+dmft = 0 mean height for age (stunting) f = 1.88 -0.58±3.5 (ref) dmft+dmft = 1–5 -0.69±3.9 –0.11 (-0.83, -0.35) 0.042 dmft+dmft > 5 -0.94±2.7 –0.36 (-1.12, -0.67) 0.001 dmft+dmft = 0 mean weight for age (underweight) f = 6.38 -0.08±1.2 (ref) dmft+dmft = 1–5 -0.26±1.8 –0.34 (-0.41, -0.88) 0.031 dmft+dmft > 5 -0.45±1.4 –0.53 (-0.62, -0.17) 0.002 dmft+dmft = 0 mean bmi for age(wasting) f = 14.8 0.64±2.3 (ref) dmft+dmft = 1–5 0.23±3.2 –0.41 (0.09, 0.38) 0.003 dmft+dmft > 5 -0.39±2.6 –1.03 (-0.58, -0.16) 0.000 pufa+pufa=0 mean height for age (stunting) f= 0.745 -0.58±3.5 (ref) pufa+pufa= 0.1-1 -0.75±2.8 –0.17 (-0.96, -0.53) 0.478 pufa+pufa> 1 -1.37±1.2 –0.79 (-1.88, -1.17) 0.019 pufa+pufa=0 mean weight for age (underweight) f = 0.745 -0.08±1.2 (ref) pufa+pufa= 0.1-1 -0.28±1.6 –0.20 (-0.51, -0.91) 0.004** pufa+pufa 1 -0.83±1.1 –0.75 (-1.01, -0.62) <0 .001 pufa+pufa=0 mean bmi for age (wasting) f= 1.87 0.23±3.2 (ref) pufa+pufa= 0.1-1 0.67±2.3 –0.44 (0.51, 0.86) 0.018** pufa+pufa> 1 -0.42±4.3 –0.65 (-0.62, 0.20) < 0.001 f= anova ρ = spearman rank correlation. 9 oyapero et al. table 4 displays the odds ratio and confidence interval values obtained from the logistic regression model between significant predictor variables in the bivariate analysis and outcome variable (low bmi). maternal education of <12 years of formal education – or 0.52 (ci 0.03-1.21) and p = 0.015; social class: middle or 0.41 (ci 0.17-0.72) and p < 0.008 and upper or 0.38 (ci 0.13-0.69) and p = 0.001; dmft+dmft 1- 5 or 0.83 (ci 0.89-3.97) and p = 0.027 and pufa+pufa= 0.1-1 or 98 (ci 1.0-2-4.43) and p = 0.008 – also showed significantly more likelihood to be associated with low bmi. table 4. odds ratio from the logistic regression model between significant predictor variables in the bivariate analysis and outcome variable (low bmi). variables mean bmi for age (wasting) odds ratio confidence interval p-value maternal education <12 years of formal education 1 ≥12 years of formal education 0.52 0.03-1.21 0.015 social class lower 1 middle 0.41 0.17-0.72 0.008 upper 0.38 0.13-0.69 0.001 number of children 1-4 1 5-8 1.65 0.78-3.23 0.043 severity of dental caries (dmft+dmft) dmft+dmft = 0 1 dmft+dmft = 1–5 1.83 0.89-3.97 0.027 dmft+dmft > 5 2.34 1.04-4.33 0.005 pulp involved decayed teeth (pufa+pufa) pufa+pufa=0 1 pufa+pufa= 0.1-1 1.98 1.02-4.43 0.008 pufa+pufa> 1 2.45 1.16-4.88 0.001 oral hygiene index (ohi) poor 1 fair 0.87 0.41-1.63 0.453 good 0.71 0.33-1.45 0.074 discussion the development of dental caries in children encompasses a multifaceted interaction between infectious, genetic, biochemical, social and physical environmental, and behavioral factors which also have implications for systemic health. females constituted a higher proportion of children with caries in our study, confirming the observation by previous authors that the odds of being susceptible to caries is higher in girls18. 10 oyapero et al. this has been adduced to the early hormonal fluctuation in girls, compared with boys and the fact that the permanent dentition of girls erupts two to 10 months earlier than that of boys19. the children were observed to consume carbonated drinks and beverages an average of 2.14 ±2.26 times per week and cookies, biscuits or sweets 3.65 ±2.54 times per week. those who consumed more cariogenic meals had significantly more carious teeth and odontogenic infections. sheiham and james20  documented a dose-response relationship, between the caries process and sugar consumption. about 18% of our study subjects also had enamel hypoplasia. a defective tooth surface could provide a suitable site for the adhesion and colonisation of cariogenic bacteria, and the progress of caries can depend on the degree of surface defect or alterations. the ensuing cleaning difficulties may cause bacteria to be retained at the base of the defect resulting in a more rapidly developing carious lesion than otherwise would occur on a sound tooth surface. we also observed a low positive correlation between poor oral hygiene and dental caries severity which is in agreement with what was reported by other researchers2. a recent meta-analysis identified a 1.5-fold higher risk of dental caries among people brushing less than once daily compared to those brushing twice daily. (odds ratio (or) = 1.56; 95% ci 1.37–1.78)21 streptococcus mutans acting with a group of other microorganisms have been implicated in the initiation and progression of dental caries22. tooth brushing is the most effective method of mechanical plaque control which simultaneously delivers fluoride topically through toothpaste. oral health promotion programs constantly underscore the importance of tooth brushing while most evidence based preventive interventions emphasize plaque control, restriction of sugar consumption, and adequate exposure to topical fluoride delivery systems. we also observed a significant association between low maternal educational attainment, lower social class and many children in the family with high caries experience. this was similarly observed by other researchers who observed that unfavorable socioeconomic status, including lower parental educational level and household income, was demonstrated to affect children’s oral health23. dietary habits and conditions that are influenced by life style like dental caries, overweight, obesity, and malnutrition are shown to covary with socioeconomic status24. overall, caries prevalence has waned in most populations, but socioeconomic disparities increasingly define dissimilarities observed among these populations with caries transiting from a disease of affluence to a disease of deprivation25. it has been postulated that people from lower socio-economic backgrounds have poorer access oral health information and dental services. the overall prevalence of caries in our study population was high at 79.1% with decayed teeth accounting for 70% of dmft+dmft scores, though it was a hospital based study group. offering only dmft data to policy makers however leaves them unmindful of the severity and related consequences of untreated caries on oral and systemic health26. the overall prevalence of odontogenic infections due to caries (mainly pulp exposure) was also high at 61.8%. the pufa/pufa prevalence in different countries vary from 24 % in brazilian 6to 7-year-olds27 to 85 % in 6-year-old children from the philippines16, indicating a high occurrence of untreated dental caries and dental neglect. https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4700662/#bibr55-0022034515609034 11 oyapero et al. teeth with pulp exposure cause significant discomfort and pain which diminishes the ability of the child to take on the task of a full day of school. it also reduces concentration and energy, the ability to eat healthy food and leads to malnutrition. it is also associated with sleeping problems, learning disorders and absence from school as well as chewing difficulties potentially impacting on children’s, nutrition, growth and body weight. persistent pain also causes the child to lose school hours, and when in school, the time spent is markedly less productive28. this is because pain interferes with the ability of the child to concentrate. therefore, oral health fundamentally influences children’s general health and quality of life. additionally, odontogenic infections can progress to dentoalveolar abcess and ludwigs angina, a frequently cited reason for the hospitalisation of young children29. thus, children with pufa scores should be characterized to be at high caries risk for early caries onset in permanent teeth. we observed a moderate positive correlation between caries severity and odontogenic infection. regarding child nutritional outcomes, 26.6% of the children were stunted, 4.3% were underweight and 10.8% were wasted. we likewise found significant associations between the severity of the children’s carries experience and their anthropometric measurements. numerous researches with inconsistent findings have observed an association between caries experience and anthropometric measurements in children due to differences in study designs and internal or external validity influences30. some researchers observed a strong association between obesity and a high caries experience31 while sheller et al32. observed no association. others however reported an inverse relationship33. there is a complex interplay of many variables that can produce divergent and bi-directional outcomes depending on the population studied. overweight or obese children also have relatively high levels of dental caries due to the consumption of high levels of soda and other energy-dense foods, which are cariogenic and obesogenic. protein-energy malnutrition in children in indigent populations can also result in reduced salivary flow and a high count of lactobacilli and streptococcus mutans, as well as altered saliva composition and impaired secretion, predisposing to dental caries34. conversely, reduced chewing ability due to caries can negatively impact affect nutritional intake which further exacerbates susceptibility to dental caries. it should however be pointed out that in developing countries like ours, the high prevalence of dental caries is closely related to poverty and low socioeconomic status. malnutrition of children is highly prevalent in low-income and middle-income countries, resulting in substantial increases in mortality and overall disease burden. to establish internal validity, we controlled for socioeconomic and behavioural factors and still observed a significant association between being underweight and dental caries. the implication of our research is emphasizing the added burden that caries possess on the abysmal health indices in developing countries, aside the intense scourge of hiv/aids in addition to the rising prevalence of non-communicable diseases (ncds) like hypertension, diabetes mellitus, and other medical conditions. decision and policy makers should be made aware of the additional burden that oral neglect can have on the anthropometric indices of children. the identification of high-risk groups provides motivation to enhance community awareness and its 12 oyapero et al. involvement in preventive efforts; as well as re-orient oral health services towards oral health promotion and prevention. prevention strategies through early institution of dental home include dietary modifications to reduce high sweetener consumption, supervised tooth brushing, systemic fluoride supplements for children living in areas without public health fluoridation, and professional delivery of fluoride varnish and sealants35. thus, multiple strategies encompassing both upstream and downstream preventive approaches are now required to translate the recommendations into policy and practice. study limitations apart from being one of the only study that determined that correlated untreated dental caries using the pufa index with bmi in nigeria, the other strength of our study lies in our exploration of the impact of oral hygiene on caries experience. this investigation, however, had a number of limitations. one is non-use of radiographs in identification of caries in the schools, even though we adhered to the who protocol for clinical diagnosis. secondly, we used a hospital based cohort rather than obtaining our sample from the community. nevertheless, within the limits of our study design, the data provides useful information for further empirical studies on subsets of the population that still have a high caries experience. conclusions there was a significant association between untreated dental caries, odontogenic infections and stunting, wasting and being underweight among the children studied. we observed a high prevalence of decayed, missing, and filled teeth of which the proportion of decayed teeth was 70.0%. the overall prevalence of odontogenic infections due to caries was also high (61.8%). there was a significant correlation between poor oral hygiene and untreated caries. children with pufa + pufa > 1 had an increased risk of wasting while those with dmft+dmft >5 were significantly underweight. references 1. vos t, flaxman ad, naghavi m, lozano r, michaud c, ezzati m, et al. years lived with 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university of brasília. 3 department of cell biology, institute of biological sciences, university of brasília. 4 department of medicine, faculty of medicine of ribeirão preto, university of são paulo. 5 department of pharmacy, faculty of health sciences, university of brasília. corresponding author: danielly de mendonça guimarães cnd 4, lote 06, loja 01/02, taguatinga norte brasília/distrito federal 72120045 e-mail: daniellymendoncaguimaraes@ gmail.com editor: dr. altair a. del bel cury received: august 8, 2021 accepted: april 9, 2022 xerostomia and dysgeusia in the elderly: prevalence of and association with polypharmacy danielly de mendonça guimarães*1 , yeda maria parro2 , herick sampaio muller3 , eduardo barbosa coelho4 , vicente de paulo martins3 , rafael santana5 , érica negrini lia1,2 xerostomia is defined as the perception of dry mouth, and dysgeusia, as a change in taste. both are common complaints in the elderly, especially among those making use of polypharmacy drug combinations. aim: this study aimed to determine the prevalence of xerostomia and dysgeusia and to investigate their association with polypharmacy in the elderly. methods: older people under follow-up at the multidisciplinary elderly center of the university hospital of brasília were interviewed and asked about health problems, medications used, presence of xerostomia and dysgeusia. descriptive statistics were used to determine the prevalence of the symptoms surveyed. the chi-square test was used to investigate the relationship between xerostomia and dysgeusia and polypharmacy. secondary associations were performed using binomial logistic regression. results: ninety-six older people were evaluated and of these, 62.5% had xerostomia and 21.1%, had dysgeusia. the average number of medications used was 4±3 medications per individual. polypharmacy was associated with xerostomia but not dysgeusia. it was possible to associate xerostomia with the use of antihypertensive drugs. conclusion: xerostomia was a frequent complaint among elderly people making use of polypharmacy, especially those using antihypertensives. antihypertensives and antidepressants were used most drugs by the elderly and exhibited interactions with drugs most prescribed in dentistry. two contraindications were found between fluconazole and mirtazapine; and between erythromycin and simvastatin. keywords: xerostomia. dysgeusia. drug interactions. dental care for aged. https://orcid.org/0000-0002-9385-5184 https://orcid.org/0000-0001-8428-6226 https://orcid.org/0000-0003-2474-8294 https://orcid.org/0000-0003-3491-3396 https://orcid.org/0000-0001-7611-861x https://orcid.org/0000-0003-4481-210x https://orcid.org/0000-0001-5691-415x 2 guimarães et al. braz j oral sci. 2023;22:e236637 introduction elderly brazilians represent about 15% of the country’s population1, and the number is expected to increase due to the demographic transition2. in view of this reality, the aging process requires multidisciplinary attention, especially from health professionals. older people are often affected by mutimorbidies and are exposed to polypharmacy3, predisposing to drug interactions and adverse reactions4. the concept of polypharmacy is variable in the literature, and it can be considered to be the simultaneous continuous long-term use of 3 or more different drugs, in addition to those inappropriate for the clinical conditions5, such as medications that meet the beers criteria, in the case of the elderly5. among the common adverse effects of polypharmacy, xerostomia and dysgeusia have been found4,6. xerostomia is a symptom defined as a perception of dry mouth7 and may or may not be associated with hyposalivation, characterized by decreased salivary flow8. the sensation of dry mouth affects about 50% of the elderly over 60 years9, and it can occur due to salivary gland agenesis7, in patients undergoing radiotherapy treatment of the head and neck region, those with sjogren’s syndrome, and metabolic disorders such as diabetes mellitus and rheumatoid arthritis10. in addition, certain classes of drugs have been related to xerostomia, such as antidepressant, anxiolytic, opioid, antihypertensive, diuretic, and antihistamine drugs11. dysgeusia is characterized as a change in the sense of taste. it can be qualitative when the change occurs in the altered perception of the taste of food or quantitative when the change refers to the lack of taste in the food4. this condition can be caused by using some groups of drugs, such as antineoplastic agents, systemic antibiotics, and drugs indicated for the treatment of nervous system diseases and the result of drug interactions12. infection with the new coronavirus (sars-cov-2) also drew attention to dysgeusia, as it has been reported by approximately 43% of patients affected by covid-1913. both xerostomia and dysgeusia significantly impact the quality of life12,14. xerostomia affects the perception of oral health and is related to a burning sensation in the mouth and halitosis15. it can also induce caries lesions and periodontal disease, taste disorders, candidiasis, dysphagia, and speech difficulties14. dysgeusia can cause feeding difficulties, leading to malnutrition and sarcopenia in the elderly12. in addition, it reduces the ability to differentiate excessive concentrations of salt and sugar, which can worsen the clinical condition associated with chronic diseases such as diabetes and high blood pressure12. given the above, this study aimed to determine the prevalence of xerostomia and dysgeusia in the elderly and to associate it with polypharmacy. an additional purpose was to determine the prevalence of medications used by the participants and map possible interactions between the medications used with those most prescribed in dentistry. material and methods study design and location a cross-sectional study was conducted at the geriatric outpatient clinic of the multidisciplinary center of the elderly hospital universitário de brasília, from july to 3 guimarães et al. braz j oral sci. 2023;22:e236637 august 2018. the ethics committee approved the study for research with human beings of the faculty of sciences of the health of the university of brasília (opinion no. 3,033,121; caae 818897177.7.0000.0030). participants all the participants who attended the “centro multidisciplinar do idoso” (multidisciplinary center for the elderly) aged 60 years or over, between july and august 2018 were interviewed. the exclusion criterion was patients with cognitive deficit or dementia. the interview was conducted after the objective of the research had been explained to the participants and they had signed the term of free and informed consent (tfic). the sample calculation was based on the elderly population in brazil in 2017, with a 95% confidence level and a 10% margin of error. assessment the assessment consisted of data collection from the personal interview, such as age and gender, clinical history (comorbidities and continuous use of medications), and evaluation of the participants’ clinical records. participants were asked about the number of drugs they used continuously, and their generic or trade names. in case the elderly had difficulty in providing this information, the medical record was consulted. in addition, the participants were asked about their self-perception of dry mouth (xerostomia) and taste alteration (dysgeusia). mapping of drug interactions the drugs listed by the participants were grouped according to their drug class into hypoglycemic, antihypertensive, antiplatelet and anticoagulant, antilipidemic, analgesic, and non-steroidal anti-inflammatory (nsaid), muscle relaxant, benzodiazepine, and others. the medications most used by the participants (those mentioned more than five times in the formatted form) were selected, and their possible association with xerostomia and dysgeusia was verified. the micromedex drug interactions® and dynamed® database was used to verify the possibility of interaction with the drugs most frequently prescribed in dentistry, such as non-opioid analgesics and non-steroidal anti-inflammatory drugs (nsaids), antibiotics, steroidal anti-inflammatory drugs (aies), anxiolytic and antifungal agents, and local anesthetics16. drug interaction was selected and classified according to its severity in minor, moderate, major, and contraindicated use. statistical analysis general and sociodemographic data were provided in the form of descriptive statistics. the chi-square test with the calculation of the prevalence ratio (pr) was performed to assess an association between xerostomia and dysgeusia ( dependent variables) with polypharmacy (independent variable), defined as the use of 3 or more medications17. the binomial logistic regression was performed to verify the association between the medications most used by the participants and the presence of xerostomia and dysgeusia. the level of significance adopted was for p <0.05 4 guimarães et al. braz j oral sci. 2023;22:e236637 results a total of 96 older people were interviewed, all of whom were being monitored at the multidisciplinary center of the university hospital of brasília (hub), between july and august 2018. the characteristics of the research participants are shown in table 1. table 1. general characteristics and oral health conditions, and several medications used by elderly people followed-up at the geriatric outpatient clinic (university hospital of brasília) between july and august 2018. data expressed in the form of mean and standard deviation or absolute number and percentage (n = 96) age (years) 74 ± 8 gender m 17(17.7%) f 79 (82.3%) number of drugs used by participants 4.4 ± 3.1 xerostomia 60 (62.5%) dysgeusia 50 (21.1%) total 96 (100%) among the drug classes most used by the participants, antihypertensives (72.9%) and antidepressants (33%) were outstanding (graph 1). medicines included in “others” are food supplements like calcium, glucosamine, omega 3, cholecalciferol, melatonin, lactulose, lithium carbonate, and folic acid; thyroid treatment agents such as levothyroxine; hormone therapy medications such as tibolone; parkinson’s treatment agents such as levodopa and benserazide hydrochloride; gastric protectors such as omeprazole and pantoprazole; medications for cardiac arrhythmias such as propafenone, amiodarone; for treatment of benign prostatic hyperplasias such as tamsulosin and dutasteride; for treating glaucomas such as latanoprost and timolol maleate; nasal decongestant such as sodium chloride; and anti-vertigo drugs such as bestatin. graph 1. percentage distribution of drug classes used by older people followed-up at the geriatric outpatient clinic (university hospital of brasília) between july and august 2018 100.00% 75.00% 50.00% 25.00% 0.00% hypoglycemic antihypertensive anti-platelet and anticoagulant agents antidepressants antilipidemics analgesics and nsaids muscle relaxants benzodiazepines others 5 guimarães et al. braz j oral sci. 2023;22:e236637 the medications most used by research participants are listed in table 2. the use of losartan (51%), amlodipine (22.9%), and metformin (16.6%) were outstanding. medicines mentioned five times or more by the elderly were considered. table 2. prevalence of medication use by older people monitored at the geriatric clinic between july and august 2018 medication prevalence of use among participants losartan 51% amlodipine 22.9% metformin 16.6% hydrochlorothiazide (hctz) 15.6% simvastatin 13.5% acetylsalicylic acid(asa) 12.5% calcium 12.5% levothyroxine 11.4% dipyrone 11.4% atenolol 10.4% indapamide 10.4% omeprazole 10.4% acetaminophen 7.2% rosuvastatin 7.2% mirtazapine 6.2% gliclazide 5.2% duloxetine 5.2% the interactions between the drugs used with those most prescribed in dentistry16 were described in chart 1. no relevant drug interactions with local anesthetics used in dental practice were found. there was an association between polypharmacy and xerostomia (pr = 1.57, 95%ci, 1.10-2.23, p = 0.004), but there was no association between polypharmacy and dysgeusia (pr = 1.14, 95%ci, 0.862-1 .51, p=0.348). table 3 shows the result of the binomial logistic regression. the use of antihypertensive drugs was associated with the occurrence of xerostomia. there was no association between the use of the drugs listed and the presence of dysgeusia. 6 guimarães et al. braz j oral sci. 2023;22:e236637 c ha rt 1 . i nt er ac tio ns b et w ee n th e dr ug s m os t u se d by s tu dy p ar tic ip an ts a nd th os e fr eq ue nt ly p re sc rib ed in d en tis tr y, a cc or di ng to th e se ve rit y of th e in te ra ct io n de sc rib ed by m ic ro m ed ex d ru g in te ra ct io ns ® a nd d yn am ed ® d ru g c la ss es m ed ic in es o ft en pr es cr ib ed in de nt is tr y m ed ic in es m os t u se d by th e ol de r pe op le a s a a te no lo l d ul ox et in e d ip yr on e h c t z in da pa m id e lo sa rt an m irt az ap in e o m ep ra zo le a ce ta m in op he n p re dn is on e ro su va st at in si m va st at in n on -o pi oi d a na lg es ic s / n sa id s/ o pi oi d a na lg es ic s d ip yr on e ++ ++ + ++ + ++ + a sa ++ ++ + ++ + ++ d ic lo fe na c so di um ++ + ++ ++ + ++ + ++ + ++ ++ + ib up ro fe n ++ + ++ ++ + ++ + ++ + ++ ++ + c od ei ne ++ + ++ + a nt ib io tic s er yt hr om yc in x a zi th ro m yc in ++ + ei a d ex am et ha so ne ++ ++ + a nx io ly tic s (b en zo di az ep in es ) d ia ze pa m ++ + + a nt ifu ng al s fl uc on az ol e x ++ ++ ++ se ve rit y of d ru g in te ra ct io n: x : c on tr ai nd ic at ed ; + ++ : m aj or ; + +: m od er at e; + :m in or . 7 guimarães et al. braz j oral sci. 2023;22:e236637 chart 2. details about the interactions between the drugs most used by study participants and those frequently prescribed in dentistry described by micromedex drug interactions® and dynamed® medicines most used by the older people medicines often prescribed in dentistry interactions asa 1. diclofenac sodium 1. may result in an increased risk of bleeding and cardiovascular events 2. ibuprofen 2. may result in decreased antiplatelet effect of acetylsalicylic acid, an additive risk of bleeding, and risk of cardiovascular events 3. dexamethasone 3. may result in an increased risk of gastrointestinal ulceration and lower aspirin serum concentrations atenolol 1. dipyrone 1. may result in decreased antihypertensive activities 2. asa 2-4. concurrent use of them may result in an increased blood pressure 3. diclofenac sodium 4. ibuprofen duloxetine 1. dipyrone 1. may result in increased risk or severity of gastrointestinal bleeding 2. asa 2-4. may result in an increased risk of bleeding3. diclofenac sodium 4. ibuprofen 5. codeine 5. may result in an increased risk of serotonin syndrome increased codeine plasma concentrations and reduces plasma concentrations of the active metabolite dipyrone 1. dexamethasone 1. may result in increased risk or severity of gastrointestinal irritation hctz 1. dipyrone 1. may result in decreased hydrochlorothiazide therapeutic efficacy 2. asa 2-4. may result in reduced diuretic effectiveness and possible nephrotoxicity 3. diclofenac sodium 4. ibuprofen indapamide 1. dipyrone 1. may result in decreased indapamide therapeutic efficacy 2. asa 2-4. may result in reduced diuretic effectiveness and possible nephrotoxicity 3. diclofenac sodium 4. ibuprofen losartan 1. asa 1-3. may result in renal dysfunction and/or increased blood pressure 2. diclofenac sodium 3. ibuprofen mirtazapine 1. diazepam 1. may result in an increased risk of somnolence 2. fluconazole 2. may result in increased mirtazapine plasma concentrations and increased risk of qt-interval prolongation and ventricular arrhythmias omeprazole 1. diazepam 1. may result in enhanced and prolonged diazepam effects 2. fluconazole 2. may result in increased plasma concentrations of omeprazole acetaminophen 1. asa 1. may result in an increased risk of bleeding continue 8 guimarães et al. braz j oral sci. 2023;22:e236637 continuation prednisone 1. asa 1. may result in an increased risk of gastrointestinal ulceration and lower aspirin serum concentrations 2. diclofenac sodium 2-3. may result in an increased risk of a gastrointestinal ulcer or bleeding3. ibuprofen 4. fluconazole 4. may result in a decrease in the metabolic degradation of prednisone and an increase in prednisone efficacy rosuvastatin 1. fluconazole 1. may result in increased rosuvastatin exposure and an increased risk of myopathy or rhabdomyolysis simvastatin 1. codeine 1. may result in decreased simvastatin metabolism 2. erythromycin 2. may result in an increased risk of myopathy or rhabdomyolysis 3. azithromycin 3. may result in an increased risk of rhabdomyolysis table 3. binomial logistic regression model between xerostomia and the drugs most used by the participants. drugs classes estimate standard error z1 p value hypoglycemic 0.188 0.597 0.31484 0.753 antihypertensives 1.397 0.536 2.60448 0.009 platelet antiaggregants and anticoagulants -1.414 0.777 -1.82012 0.069 antidepressants 0.600 0.597 1.00482 0.315 antilipidemics -0.177 0.676 -0.26170 0.794 analgesics and nsaids 0.517 0.608 0.85171 0.394 benzodiazepines -18.991 2452.989 -0.00774 0.994 others 0.919 0.588 1.56239 0.118 1z-score discussion the majority of study participants reported having xerostomia, which was statistically associated with polypharmacy. therefore, the prevalence of xerostomia was high (62.5%) compared with that of another study, also conducted in brazil, in which it was prevalent in 49% of non-institutionalized elderly18. a systematic review with meta-analysis showed a prevalence of xerostomia of 22% in adults, and that this percentage was higher in the elderly19 due to the aging process predisposed to salivary gland agenesis, immunological disorders such as sjogren’s syndrome, metabolic disorders such as diabetes mellitus and rheumatoid arthritis, and use a lot of medications 7. xerostomia is a subjective measure, and its diagnosis is often the patient’s report10. methods to assess the quantity and quality of saliva can be used simultaneously, 9 guimarães et al. braz j oral sci. 2023;22:e236637 such as chewing gum test, paraffin, or saxon test7. in addition, scintigraphy, sialography, and minor salivary gland biopsy can help gland dysfunctions diagnosis7,8. it is essential to emphasize the importance of a multidisciplinary approach to the elderly with xerostomia with the purpose of verifying the etiology and implementing the most appropriate treatment for this situation10,11. the doctor and the pharmaceutical responsible can help in the alternative medications to improve the dry mouth sensation; use of chewing gums and substitutes of saliva are non-pharmacological alternatives that can relieve the xerostomia8. our study showed a low prevalence of dysgeusia (21.1%), similar to rates in another brazilian study, which found a prevalence of 19.4% in the elderly20. dysgeusia has a multifactorial etiology and may result from sensory and nutritional disorders, medications, and polypharmacy. furthermore, it may be related to infections since half of those infected with sars-cov-2 have experienced a loss of taste21. dysgeusia interferes with the quality of life of the elderly, as it can cause feeding difficulties related to the lack of perception and taste distinction and consequently lead to weight loss12. in both xerostomia and dysgeusia, polypharmacy and drug use are common causes4,6. in the present study, there was an association between xerostomia and polypharmacy (particularly the use of antihypertensive drugs), but not between dysgeusia and polypharmacy. previous studies have shown elevated rates of xerostomia associated with polypharmacy that included use of antihypertensives, anticholinergic, adrenergic, thyroid-stimulating hormones, sedative, hypoglycemic, nonsteroidal anti-inflammatory, corticosteroid, and antiulcerogenic hormones18. although the literature has previously included the association of dysgeusia with various medications, such as antimicrobials, angiotensin-converting enzyme inhibitors, chemotherapeutic agents, among others12, in this study, no drug classes were found to be associated with dysgeusia. the medications most used by the participants were antihypertensives (losartan and amlodipine) and hypoglycemic agents (metformin), followed by antidepressants. a cohort conducted in the united states showed that the drug classes most used among the population were antihypertensives, analgesics, statins, anticholinergics, psychiatric drugs, and antibiotics6. a brazilian study also highlighted antihypertensives, used by 70.9% of the elderly, followed by antilipemic agents, antacids, hypoglycemic agents, antiplatelet agents, thyroid hormone, antidepressants, and benzodiazepines22 . these data reflect the epidemiological transition experienced in brazil. there is an increase in the prevalence of chronic and mental diseases when compared with the high number of infectious diseases reported in the past23. drug therapy in dentistry includes infection, inflammation, pain, and anxiety24. therefore, it is necessary to use non-opioid and opioid analgesics, non-steroidal anti-inflammatory drugs (nsaids), antimicrobials, anxiolytics, in addition to local anesthetics16. we considered the medications used by the participants in our study, and drug interactions relevant to dental practice were found. the concomitant use of simvastatin and erythromycin is contraindicated, as it can reduce the effect of simvastatin. consequently, it can lead to myopathy, as erythromycin contains an inhibitory effect on a cyp3a4 enzyme, which metabolizes simvastatin25. another 10 guimarães et al. braz j oral sci. 2023;22:e236637 relevant contraindication is between fluconazole and mirtazapine because the cyp3a enzyme metabolizes mirtazapine, and fluconazole initiates its activity. the concomitant use of these two drugs increases the plasma concentration of mirtazapine, therefore, increases the risk of prolongation of the qt interval and episodes of ventricular arrhythmia26 . furthermore, healthcare professionals should be aware of the contraindication between nsaids and thiazide diuretics since this combination is associated with diuretic efficacy and can lead to nephrotoxicity26. increased risk of gastrointestinal ulcers can occur when there is concomitant use of nsaids and corticosteroids26. the use of duloxetine may cause a relevant interaction with nsaids, which may increase the risk of bleeding26. the use of fluconazole is also considered a risk for simultaneous use with simvastatin due to the possibility of [leading to] myopathy and rhabdomyolysis26. drug interactions may occur due to drugs that have a high rate of binding to plasma proteins, a long half-life, and a narrow therapeutic window. furthermore, they are more common in patients with chronic diseases, making use of polypharmacy and self-medication, including herbal medicines16. given the above, health professionals who prescribe medications, such as physicians and dentists, should know about possible drug interactions and adverse reactions27. therefore, it is necessary to carry out a detailed anamnesis that will allow the professional to recognize the possibility of drug interactions occurring, thus preventing adverse effects and even providing treatment when necessary28. among the study limitations, the use of a non-probabilistic sample, limited to a research center can be mentioned. the discrepancy between men and women is due to the study location and the trend towards greater self-care in women29. this fact may have influenced the prevalence of xerostomia and dysgeusia, as hormonal changes are frequent in elderly women and predispose to changes in taste and dry mouth4,11. furthermore, the dose and frequency of medications used by the participants were not evaluated. in conclusion, xerostomia is a frequent complaint among older people using polypharmacy, especially those using antihypertensives. antihypertensives and antidepressants were used most drugs by the elderly and exhibited interactions with drugs most prescribed in dentistry. two contraindications were found between fluconazole and mirtazapine; and between erythromycin and simvastatin. author contribution danielly de mendonça guimarães: assisted in collecting and tabulating data, carried out the article’s writing, and carried out the final review. yeda maria parro: responsible for collecting the data, helping with the study design and reviewing the final version of the article. herick sampaio muller: responsible for assisting in data interpretation and reviewing the final version of the article. 11 guimarães et al. braz j oral sci. 2023;22:e236637 eduardo barbosa coelho: responsible for assisting in the elaboration of the work methodology, statistical calculations, and reviewing the final version of the article. vicente de paulo martins: responsible for assisting in data interpretation and reviewing the end of the article. rafael santana: responsible for assisting in the interpretation and organization of data, guiding the discussion about pharmacology, and reviewing the final version of the article. érica negrini lia: responsible for outlining the study and guiding and reviewing the final version of the article. all authors actively participated and revised and approved the final version of the manuscript. references 1. brazilian institute of geography and statistics (ibge). national household sample survey (pnad) 2019 [cited 2020 oct 28]. available from: https://www.ibge.gov.br/busca.html?searchword=pnad. portuguese. 2. ervatti lr, borges gm, jardim ap, organizators. [demographic change in brazil at the beginning of the 21st century – subsidies for population projections]. rio de janeiro: ibge; 2015 [cited 2020 oct 28]. available from: https://biblioteca.ibge.gov.br/visualizacao/livros/liv93322.pdf. portuguese. 3. roughead ee, vitry ai, caughey ge, gilbert al. multimorbidity, care complexity and prescribing for the elderly. aging health. 2011;7(5):695-705. 4. rademacher wmh, aziz y, hielema a, cheung kc, de lange j, vissink a, et al. oral adverse effects of drugs: taste disorders. oral dis. 2020 jan;26(1):213-23. doi: 10.1111/odi.13199. 5. masnoon n, shakib s, kalisch-ellett l, caughey ge. what is polypharmacy? a systematic review of definitions. bmc geriatr. 2017 oct;17(1):230. doi: 10.1186/s12877-017-0621-2. 6. marcott s, dewan k, kwan m, baik f, lee yj, sirjani d. where dysphagia begins: polypharmacy and xerostomia. fed pract. 2020 may;37(5):234-41. 7. anil s, vellappally s, hashem m, preethanath rs, patil s, samaranayake lp. xerostomia in geriatric patients: a burgeoning global concern. j investig clin dent. 2016 feb;7(1):5-12. doi: 10.1111/jicd.12120. 8. guggenheimer j, moore pa. xerostomia. etiology, recognition and treatment. j am dent assoc. 2003 jan;134(1):61-9; quiz 118-9. doi: 10.14219/jada.archive.2003.0018. 9. rech ca, medeiros aw. 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[drug interactions: factors related to the patient (part i)]. rev cir traumatol bucomaxilo-fac. 2007;7(1):17-27. 25. simvastatin. [package insert for the drug simvastatin®]. américo brasiliense/sp. folk remedy foundation; 2017 [cited 2020 oct 28]. available from: https://consultas.anvisa.gov.br/#/bulario/deta lhe/1000858?nomeproduto=sinvastatina. portuguese. 26. dynamed [database online]. ipswich (ma): ebsco information services [cited 2021 jun 28]. available from: http://www.dynamed.com. 27. sousa itc, pestana am, araujo mar. [clinical implications of the use of nsaids in hypertensive patients: drug interactions in dentistry]. rev bras hipertens. 2019;26(3):91-6. portuguese. 28. ouanounou a, haas da. pharmacotherapy for the elderly dental patient. j can dent assoc. 2015;80:f18. 29. levorato cd, de mello lm, da silva as, nunes aa. [factors associated with the demand for health services from a gender-relational perspective]. cien saude colet. 2014 apr;19(4):1263-74. portuguese. doi: 10.1590/1413-81232014194.01242013. https://consultas.anvisa.gov.br/#/bulario/detalhe/1000858?nomeproduto=sinvastatina 1http://dx.doi.org/10.20396/bjos.v18i0.8657257 volume 18 2019 e191603 original article 1 federal university of bahia, department of clinical dentistry, salvador, bahia, brazil. 2 bahiana school of medicine and public health and federal university of bahia, department of clinical dentistry, salvador, bahia, brazil. corresponding author: emilena maria castor xisto lima address: rua waldemar falcão, n. 1906, torre paysage, apt. 1402 horto florestal salvador – bahia cep – 40295-010 brazil tel: +55 (71) 99194-6656 e-mail: emilenalima@gmail.com received: march 23, 2019 accepted: june 28, 2019 marginal adaptation of provisional crowns made of acrylic and bisacrylic resins using different impression materials camilla lopes cerqueira1, roniel kappler1, andrea araújo nobrega cavalcanti2, emilena maria castor xisto lima2,* aim: to evaluate the marginal adaptation of provisional crowns made of acrylic and bisacrylic resins using different impression materials. methods: a metal die and a matrix applied through a direct technique were used to fabricate the specimens. the impression materials used as a matrix were divided into four groups: irreversible hydrocolloid (ih), laboratory silicone (ls), condensation silicone (cs), and addition silicone (as). after the impression procedures, each matrix was loaded with the provisional prosthetic materials, alike, duralay, protemp 4, and structur 3 (n = 12). marginal discrepancy was evaluated using a stereomicroscope at  ×45 magnification. the images obtained were transferred to the corel draw x7 program, and the distances from the cervical margins of the specimen to the reference lines at the metal die were measured vertically. the data were analyzed by using 2-way anova followed by the tukey test (α=.05). results: the acrylic resins had higher values of marginal discrepancy compared to the bisacrylic resins. a statistically significant difference was found between all impression materials, and the irreversible hydrocolloid presented higher values of discrepancy (303.28–613.31 μm), whereas addition silicone had the lowest (48.61–190.06 μm). conclusions: the bisacrylic resins had a better marginal adaptation compared to the acrylic resins. the addition silicone promoted a better marginal adaptation of the provisional prosthetic materials tested, followed by condensation silicone, laboratory silicone, and irreversible hydrocolloid. keywords: dental marginal adaptation. dental restoration, temporary. dental impression materials. 2 cerqueira et al. introduction temporary prostheses are needed to maintain gingival health, prevent the migration of abutment teeth, establish oclusal contact, and provide esthetic value during treatment. they also provide professionals with a prognosis as to the success of the final restoration in terms of its aesthetic and functional aspects1,2. therefore, the exclusion or negligence of this step can lead to a failure of the final treatment3. a marginal adaptation is one of the fundamental requirements of a fixed prosthesis because a poor marginal fit allows the formation of a gap between the restorative material and the prepared tooth1. open marginal configurations encourage the microleakage of bacteria and their by-products owing to the dissolution of the luting agentes4, and may predispose the tooth to caries or pulpitis. in addition, poorly adapted provisional prosthetic materials cause mechanical irritation to the surrounding tissues and enhance biofilm accumulation with subsequent periodontal problems1,5. choosing the appropriate combination of materials and techniques for the fabrication of a high-quality provisional restoration is important because the only difference between this and the definitive restoration should be the material used6. interim restorations can be directly fabricated intraorally, indirectly in the laboratory, or with a direct-indirect technique when the restoration is formed extraorally and finalized intraorally7. although an indirect technique produces a restoration with a superior marginal fit7,8, most dentists use a direct method with satisfactory results. a matrix planned for provisional fabrication may copy existing tooth contours from the mouth with a diagnostic cast9, or reproduce customized contours created through diagnostic waxing10. it has been further suggested that, when possible, this matrix should include at least one adjacent tooth on each side11. the matrices applied for a direct technique are made of thermoplastic, vacuum-formed templates, irreversible hydrocolloid, or elastomeric impression materials12, although the matrix of choice depends upon the many variables of each particular situation. to select an appropriate material, it does help to have a feel for the classification of the impression materials, as well as concepts such as the working time, setting time, permanent deformation, and dimensional stability. there are certainly other important factors that influence the decision, such as ease of manipulation, taste, and tackiness, but they have thus far eluded quantitative measurement13. in fact, an accurate reproduction of the preparation margins in an impression is a necessary requirement for achieving a good marginal quality14. the materials available for fabricating provisionally fixed partial dentures include autopolymerizing polymethyl methacrylate, polyethylene methacrylate, polyvinyl methacrylate, urethane methacrylate, bis-acryl, and microfilled resins15. historically, autopolymerizing polymethyl methacrylate resin (pmma) has been the most popular material and is widely used by clinicians, although during the last several years, temporary bis-acryl composite materials have been introduced and are rapidly gaining acceptance15 owing to such benefits as ease of use3, better color stability16, resistance to wear17, and low exothermic heat18. 3 cerqueira et al. studies conducted to assess the degree of marginal gap formation of provisional materials have shown contradictory results. some studies have indicated that acrylic resins have lower marginal discrepancies compared to bisacrylic resins, some of which have shown a comparable fit between both types, whereas other studies have demonstrated bis-acryl composite resin to be superior to acrylic resin5,17,19. thus, the aim of this study was to evaluate the marginal adaptation of provisional crowns made of acrylic and bisacrylic resins using different impression materials (irreversible hydrocolloid, laboratory silicone, condensation silicone, and addition silicone). the null hypothesis was that there was no difference between marginal adaptations of these restorative materials and between these impression materials. materials and methods the materials used in this study are described in tables 1 and 2. specimen preparation an in vitro method was used to simulate a direct clinical technique in which a provisional crown was made directly on the prepared tooth using different impression materials as a matrix (table 1). mandibular left first molar prepared for a complete table 1. impression materials used in the present study. impression material product manufacturer irreversible hydrocolloid hydrogum 5 zhermack spa, germany laboratory silicone zetalabor zhermack spa, badia polesine, ro, italy condensation silicone zetaplus putty/oranwash zhermack spa, badia polesine, ro, italy addition silicone elite hd/putty regular zhermack spa, badia polesine, ro, italy table 2. provisional prosthetic materials used in the present study. product name material classification composition manufacturer alike polymethymethacrylate (pmma) barium silicate glass, benzoyl peroxide, dimethylp-toluidene, methacrylate polymer, methyl methacrylate, ethyl-2-cyano-3diphenylacrylate, methanol gc américa, alsip il, eua duralay polymethymethacrylate (pmma) copolymer of plasticizable methacrylate, monomer of methylmethacrylate, paraffin, mineral oil reliance, cotia, são paulo, brasil structur 3 bis-acryl composite bis-gma, bht, amines, benzoyl peroxide, dimethacrylates, glass particles voco smbh cuxhaven, alemanha protemp 4 bis-acryl composite dimethacrylate polymer, bis-gma, zirconium, silica and silane particles, pigments 3m-espe, seefeld, alemanha 4 cerqueira et al. metaloceramic crown with a 1-mm chamfer finish line and a taper of approximately 5 degrees was cast in a base metal alloy and adapted to the left mandibular hemi-arch (fig. 1). a standard crown was fabricated for the prepared die to represent the form of the tooth prior to preparation (fig. 2). a matrix planned for provisional fabrication was used to copy the contours from the standard crown adapted to the metal die. this matrix was extended onto at least one tooth adjacent to the teeth being restored. the impression materials were divided into four groups: irreversible hydrocolloid (ih), laboratory silicone (ls), condensation silicone (cs), and addition silicone (as). twelve impressions were taken for each impression material. this impression served as a matrix for making the provisional crowns for all materials applied: alike, duralay, protemp 4, and structur 3 (n=12). a verticulator (bio-art equipamentos odontológicos ltda., são carlos – sp, brazil) was used to standardize the path of insertion and removal of the partial stock tray and the applied force (fig. 2). figure 1. the metal die (copy of the mandibular left first molar prepared for a complete metaloceramic crown) adapted to the left mandibular hemi-arch. figure 2. the partial stock tray and standard crown on the metal die in the left mandibular hemi-arch. both were adapted to the upper and lower parts of the verticulator, respectively. 5 cerqueira et al. group ih: the irreversible hydrocolloid (ih), (hydrogum 5, zhermack, badiapolesine – ro, italy) was used at a ratio of 1:1; the powder was dispensed into water in a rubber vat and manipulated using a plastic spatula until homogenization. the irreversible hydrocolloid was loaded into the partial stock tray and seated on the standard crown with the aid of the verticulator, where it remained locked for 2 min until the gelation was complete. group ls: laboratorial silicone (zetalabor, zhermack, badiapolesine – ro, italy) was proportioned and manipulated following the manufacturer’s instructions. the material was then loaded into the stock tray and seated on the standard crown with the aid of the verticulator. it remained locked until complete polymerization of the laboratory silicone. group cs: condensation silicone (zetaplus, zhermack, badiapolesine – ro, italy) was used, and a putty material was proportioned and manipulated following the manufacturer’s instructions, and loaded into the stock tray. a light paste and catalyst were placed on a glass plate (at a 1:1 ratio) and handled using a #24 spatula. the light-body material was dispensed using an elastomer syringe on the standard crown. thereafter, the stock tray loaded with the putty material was seated on the standard crown, and the verticulator was locked until complete polymerization of the condensation silicone. group as: addition silicone (elite hd, zhermack, badiapolesine – ro, italy) was used at a 1:1 ratio. the putty material was provided and handled following the manufacturer’s instructions and loaded into the stock tray. the light-body material was dispensed using a dispenser gun and compatible mixing tip on the standard crown. the stock tray loaded with the material was seated on the standard crown and the verticulator was locked until complete polymerization of the addition silicone. next, the metal die (copy of the tooth prepared) was lubricated (vaseline; quimidrol) to prevent adhesion to the resin samples. the acrylic resins (duralay and alike) were dispensed and mixed according to the manufacturer’s instructions, and placed in the matrices with the aid of a manipulating spatula (#24). the bisacryl composites (protemp 4 and structur 3) were inserted into the matrices using a dispenser gun and compatible mixing tips. the resin-filled impression was seated on the metal die with aid of the verticulator, which remained locked during polymerization. before complete polymerization (plastic phase), any excess material was trimmed from the margins of the provisional restorations using a scalpel blade (15c). the test specimens were kept in a closed, dry container for 24 h until an analysis of the marginal discrepancy was conducted. analysis of marginal discrepancy the provisional crowns were adapted to the metal die with the aid of a “c” clamp (metasul, braço do norte sc, brazil) (fig. 4) and kept at a standardized position during the analysis in a stereomicroscope lupe (optima mdce-5ª, 2.0, hiperquímica, santo andré sp, brazil). photographs at 45x magnification were obtained from the buccal, lingual, mesial, and distal surfaces, and the images were transferred to corel draw x7 program. the mar6 cerqueira et al. ginal discrepancy of the provisional crowns was determined by measuring the space (marginal opening) between the margin of the provisional crowns and the finish line of the metal die. for each provisional crown, the measurements were made at four vertical reference lines previously marked at the midpoint of the metal die finish line at four locations to represent the buccal, lingual, mesial, and distal surfaces of the die. the measurements were made three times along the long axis of the die at each of the four reference points. all procedures were performed by one operator calibrated. statistical analysis the amounts of marginal discrepancy were compared among the 4 provisional restoration materials and impression materials with a 2way analysis of variance (anova) followed by the tukey test for multiple comparisons (a=.05). the analyses were conducted using the statistical program, sas, version 9.1 (sas institute, cary, nc, usa). results table 3 shows the mean and standard deviation of the marginal discrepancy values obtained. table 3. mean (standard deviation) of the marginal discrepancy (μm). impression materials / resins alike duralay protemp 4 structur 3 irreversible hydrocolloid 613.31 (19.53) aa 605.08 (25.97) aa 334.35 (24.99) ab 303.28 (16.76) ac laboratorial silicone 440.92 (18.61) ba 387.21 (24.69) bb 237.90 (11.82) bc 238.87 (15.16) bc condensation silicone 258.77 (19.48) ca 250.85 (16.71) ca 133.86 (12.49) cb 104.00 (4.79) cc addition silicone 190.06 (9.74) da 192.83 (6.01) da 73.58 (6.68) db 48.61 (7.08) dc mean followed by distinct letters represents a significant difference (anova 2-way/tukey, a=.05)). upper case letters compare the impression materials within each level of the resin factor. lower case letters compare resins within each level of the impression material factor. figure 3. temporary crown adapted to the metal die with a “c” clamp. 7 cerqueira et al. the statistical analysis showed the significance of the interaction between the main factors impression material x resin (p <0.0001), demonstrating dependence between the levels of one factor on the outcome of another. statistically significant differences among the impression materials were found at all resin factor levels. irreversible hydrocolloid was shown to have the highest discrepancy, followed by laboratory silicone, condensation silicone, and addition silicone. for the resins, it was observed that when irreversible hydrocolloid, condensation silicone, or addition silicone was applied as the impression material, the acrylic resins alike and duralay showed statistically similar and significantly higher values than the bisacrylic resins protemp 4 and structur 3, presenting statistical differences among them. for the laboratory silicone material, the acrylic resins alike and duralay presented statistically significant differences between them; however, both showed a superior discrepancy compared to the bisacryl resins protemp 4 and structur 3. discussion in this study, 4 provisional restorative materials and 4 impression materials were evaluated for marginal fit. the results indicate that the null hypothesis was rejected. the accuracy of fit has been extensively investigated in the dental literature. marginal openings of between 100 and 200 mm are considered clinically acceptable with regard to longevity20-22. however, some authors have considered a marginal discrepancy between 50 and 120 μm to be within the range of clinical acceptance23-25. holmes et al.26 (1989) described this divergence of values between various studies as resulting from the fact that, unlike the physical and mechanical properties of the materials applied, the fit of a restoration has never been strictly defined. the reference points for the measurements and the descriptive terminology defining a fit vary considerably among investigators. in the present study, some groups, namely, addition silicone for all resins, and condensation silicone for bisacryl composites (table 3) were within the clinically acceptable threshold established in the literature20-22. however, the other groups presented values above these limits. the variations in the results of this study as compared with other studies could be because of the differences in the materials and fabrication techniques applied. a direct relationship exists between an initially poor marginal adaptation and the dissolution of cement (with a resultant microleakage)27, and damage to the adjacent tissues28. the bisacrylic resins showed a significantly lower marginal discrepancy when compared with the acrylic resins. young et al.29 (2001) compared the quality of the provisional restorations fabricated by dental students using two materials (bis-acryl composite resin and pmma), and found that the bis-acryl composite resin (integrity) was significantly superior to pmma (c&b resin and snap) as a provisional restorative material, including marginal adaptation. tjan et al.5 (1997) evaluated the vertical discrepancies of the margins for complete crowns and indicated that provisional crowns fabricated using protemp garant (bys-acryl composite) and splintline (ema) recorded the least marginal discrepancies. these results corroborate with those of the present study despite the different materials used. 8 cerqueira et al. this difference in adaptation between acrylic and bisacrylic resins can be attributed to the higher polymerization shrinkage of the acrylic resins. deficiencies can occur when autopolymerizing acrylic resin is used owing to a dimensional contraction because of the difference in density between the polymer and monomer applied30. temporary crowns maintained in water at 20–30° c for 10 min help to obtain a better marginal fit31. this technique decreases the monomer content and reduces the chance of irritation from the free monomer content, which may directly influence the polymerization shrinkage, and consequently the marginal adaptation. moreover, a bis-acrylic or bis-acrylate composite differs from a methacrylate resin, but is similar to a composite restorative material because it is made of a bis-acryl resin and inorganic fillers, the latter of which reduces polymerization shrinkage32. it has been stated that volumetric polymerization shrinkage for pmma is 6%, compared with 1–4% for composite materials33. the most common materials used for the fabrication of provisionals are polymethylmethacrylate (pmma) and composite-based resin (cbr)34. the pmma comes in the form of a polymer, which is a powder and monomer in liquid form that has to be hand mixed. the cbr comes in the form of an auto-mixed paste or in paste tubes. pmma is chemically polymerized, whereas cbr is available as a chemically or light cured material34. one attractive aspect of a bisacryl composite is its convenient, cartridge-based dispensing system, which should result in a more accurately proportioned and consistent mix29. this innovative approach may have contributed to the superior results credited to protemp 4 and structur 3. however, wang et al.17 (1989) observed that acrylic resin and bisacrylic produce similar marginal gaps. they indicated that alike e protemp produced the best fitting crowns, exhibiting respective marginal gaps of 90.7 and 94.2 mm. few studies in the dental literature have compared the accuracy of the impression material used to fabricate a temporary crown. in this study, a direct technique was used with different impression materials, and a significant difference in marginal discrepancy was found among all of them. it was verified that the impression material influenced the accuracy of the temporary crowns. addition silicone promoted the smallest marginal discrepancy in the provisional crowns, followed by condensation silicone, laboratory silicone, and finally, irreversible hydrocolloid. addition-cured silicone rubber is considered the most dimensionally stable impression material, which is set using an addition-cured polymerization reaction, with no by-product produced during cross-linkage, resulting in an extremely stable impression13. irreversible hydrocolloid promoted the highest marginal discrepancy in the provisional crowns. this material has two major disadvantages, firstly, very poor dimensional stability because of the ready loss or imbibition of water on standing in dry or wet environments respectively. secondly, low tear resistance which can be a real problem when attempting to record the gingival sulcus13. faria et al.35 (2008) evaluated the the accuracy of different impression materials used for fixed partial dentures (irreversible hydrocolloid, condensation silicone, addition silicone, polyether, and polysulfide), and verified that different impression materials and techniques influence the accuracy of the stone casts in such a way 9 cerqueira et al. that polyether, polysulfide, and addition silicone, when following a single-phase technique, are more accurate than the other materials35. an accurate stone cast is indispensable for the fabrication of a crown or bridge, and the choice of the impression material is a vital step. vitti et al.36 (2013) compared the dimensional accuracy of casts made using three impression techniques with addition and condensation silicone, and concluded that stone casts made from addition silicone is also dimensionally more accurate, corroborating the results of the present study. however, an analysis of the accuracy of the impression material in a stone cast differs from that of a specimen36. as with the present study, the authors also used putty and a light-body material. the high filler loading of the putty was initially devised to reduce the effects of polymerization shrinkage. putty is commonly combined with a low viscosity silicone when recording an impression to produce more accurate results13. this study attempted to highlight some of the issues related to the marginal opening of the provisional materials available in the market associated to a direct tecnhnique used to fabricate provisional restorations with diferentt impression materials. however, further studies are needed for the development of more durable and accurate materials for provisional restorations. in conclusion, bisacrylic resins showed a better marginal adaptation than acrylic resins. addition silicone promoted a better marginal fit of the provisional prosthetic materials tested, followed by condensation silicone, laboratory silicone, and irreversible hydrocolloid. references 1. nejatidanesh f, lotfi hr, savabi o. marginal accuracy of interim restorations fabricated from four ínterim autopolymerizing resins. j prosthet dent. 2006 may;95(5):364-7. 2. haselton dr, diaz-arnold am, vargas ma. flexural strength of provisional crown and fixed partial denture resins. j prosthet dent. 2002 feb;87(2):225-8. 3. burns dr, beck da, nelson sk. a review of selected dental literature on contemporary provisional fixed prosthodontic treatment: report of the committee on research in fixed prosthodontics of the academy of fixed prosthodontics. j prosthet dent. 2003 nov;90(5):474-97. 4. rastogi a, kamble v. comparative analysis of the clinical techniques used in evaluation of marginal accuracy of cast restoration using stereomicroscopy as gold standard. j adv prosthodont. 2011 jun;3(2):69-75. doi: 10.4047/jap.2011.3.2.69. 5. tjan ahl, castelnuovo j, shiotzu g. marginal fidelity of crowns fabricated from six proprietary provisional materials. j prosthet dent. 1997 may;77(5):482-5. 6. reshad m, cascione d, kim t. anterior provisional restorations used to determine form, function, and esthetics for complex restorative situations, using all-ceramic restorative systems. j esthet restor dent. 2010 feb;22(1):7-16. doi: 10.1111/j.1708-8240.2009.00305.x. 7. dumbrigue hb. composite indirect-direct method for fabricating multiple-unit provisional restorations. j prosthet dent. 2003 jan;89(1):86-8. 8. regish km, sharma d, prithviraj dr. techniques of fabrication of provisional restoration: an overview. int j dent. 2011;2011:134659. doi: 10.1155/2011/134659. 10 cerqueira et al. 9. clements wg. predictable anterior determinants. j prosthet dent. 1983 jan;49(1):40-5. 10. amet em, phinney tl. fixed provisional restorations for extended prosthodontic treatment. j oral implantol. 1995;21(3):201-6. 11. konstantinidis i, kotsakis g, pallis k, walter mh. a novel technique for the direct fabrication of fixed interim restorations. j prosthet dent. 2013 mar;109(3):198-201. doi: 10.1016/s0022-3913(13)60044-x. 12. ayuso-montero r, martinez-gomis j, lujan-climent m, salsench j, peraire m. influence of matrix type on surface roughness of three resins for provisional crowns and fixed partial dentures. j prosthodont. 2009 feb;18(2):141-4. doi: 10.1111/j.1532-849x.2008.00392.x. 13. wassell rm, barker d, walls awg. crowns and other extra-coronal restorations: impression materials and technique. br dent j. 2002 jun 29;192(12):679-84, 687-90. 14. hamalian ta, nasr e, chidiac jj. impression material in fixed prosthodontics: influence of choice on clinical procedure. j prosthodont. 2011 feb;20(2):153-60. doi: 10.1111/j.1532-849x.2010.00673.x. 15. christensen gj. the fastest and best provisional restorations. j am dent assoc. 2003 may;134(5):637-9. 16. sham ask, chu fcs, chai j, chow tw. color stability of provisional prosthodontic material. j prosthet dent. 2004 may;91(5):447-52. 17. wang k, moore bk, coodacre c, sifdrfe ml, andres cj. a comparison of resins for fabricating provisional fixed restorations. int j prosthodont. 1989 mar-apr;2(2):173-84. 18. driscoll cf, woolsey g, ferguson wm. comparison of exothermic release during polymerization of four materials used to fabricate interim restorations. j prosthet dent. 1991 apr;65(4):504-6. 19. koumjian jh, holmes jb. marginal accuracy of provisional restorative materials. j prosthet dent. 1990 jun;63(6):639-42. 20. fransson b, oilo g, gjeitanger r. the fit of metal-ceramic crowns, a clinical study. dent mater. 1985 oct;1(5):197-9. 21. karlsson s. the fit of procera titanium crowns. an in vitro and clinical study. acta odontol scand. 1993 jun;51(3):129-34. 22. mclean jw, fraunhofer ja von. the estimation of cement film by an in vivo technique. br dent j. 1971 aug 3;131(3):107-11. 23. abduo j, lyons k, swain m. fit of zirconia fixed partial denture: a systematic review. j oral rehabil. 2010 nov;37(11):866-76. doi: 10.1111/j.1365-2842.2010.02113.x. 24. colpani jt, borba m, della bona a. evaluation of marginal and internal fit of ceramic crown copings. dent mater. 2013 feb;29(2):174-80. doi: 10.1016/j.dental.2012.10.012. 25. habib sr, asiri w, hefne mj. effect of anatomic, semi-anatomic and nonanatomic occlusal surface tooth preparations on the adaptation of zirconia copings. j adv prosthodont. 2014 dec;6(6):444-50. doi: 10.4047/jap.2014.6.6.444. 26. holmes jr, bayne sc, holland ga, sulik wd. considerations in measurement of marginal fit. j prosthet dent. 1989 oct;62(4):405-8. 27. meyer filho a, vieira lcc, araujo e, baratieri ln. ceramic inlavs and onlavs: clinical procedures for predictable results. j esthet restor dent. 2003;15(6):338-51; discussion 352. 28. karlsson s. a clinical evaluation of fixed bridges, 10 years following insertion. j oral rehabil. 1986 sep;13(5):423-32. 29. young hm, smith ct, morton d. comparative in vitro evaluation of two provisional restorative materials. j prosthet dent. 2001 feb;85(2):129-32. 11 cerqueira et al. 30. patras m, naka o, doukaudakis s, pissiots a. management of provisional restorations deficiencies: a literature review. j esthet restor dent. 2012 feb;24(1):26-38. doi: 10.1111/j.1708-8240.2011.00467.x. 31. dhillon n, kumar m, d’souza dsj. effect of water temperature and duration of immersion on the marginal accuracy of provisional crowns. med j armed forces india. 2011 jul;67(3):237-40. doi: 10.1016/s0377-1237(11)60049-x. 32. darvell, b.w. resin restorative materials. materials science for dentistry. 9th ed. cambridge: woodhead publishing; 2009. 103p. 33. lepe x, bales dj, johnson gh. retention of provisional crowns fabricated from two materials with the use of four temporary cements. j prosthet dent. 1999 apr;81(4):469-75. 34. al rifaiy mq. evaluation of vertical marginal adaptation of provisional crowns by digital microscope. niger j clin pract. 2017 dec;20(12):1610-1617. doi: 10.4103/1119-3077.196083. 35. faria ac, rodrigues rc, macedo ap, mattos mda g, ribeiro rf. accuracy of stone casts obtained by different impression materials. braz oral res. 2008 oct-dec;22(4):293-8. 36. vitti rp, da silva ma, consani rl, sinhoreti ma. dimensional accuracy of stone casts made from silicone-based impression materials and three impressions techniques. braz dent j. 2013 sep-oct;24(5):498-502. doi: 10.1590/0103-6440201302334. https://www.ncbi.nlm.nih.gov/pubmed/?term=macedo ap%5bauthor%5d&cauthor=true&cauthor_uid=19148382 https://www.ncbi.nlm.nih.gov/pubmed/?term=mattos mda g%5bauthor%5d&cauthor=true&cauthor_uid=19148382 https://www.ncbi.nlm.nih.gov/pubmed/?term=ribeiro rf%5bauthor%5d&cauthor=true&cauthor_uid=19148382 https://www.ncbi.nlm.nih.gov/pubmed/?term=sinhoreti ma%5bauthor%5d&cauthor=true&cauthor_uid=24474292 1 volume 21 2022 e226343 original article braz j oral sci. 2022;21:e226343http://dx.doi.org/10.20396/bjos.v21i00.8666343 1 department of pediatric dentistry, orthodontics and public health, bauru school of dentistry, university of são paulo, bauru, são paulo, brazil. 2 hospital for rehabilitation of craniofacial anomalies, university of são paulo, bauru, são paulo, brazil. 3 department of orthodontics, ingá university center, maringá, paraná, brazil. corresponding author: thais marchini oliveira bauru school of dentistry, university of são paulo alameda dr. octávio pinheiro brisolla, 9-75, bauru, são paulo, 17012-901brazil telephone: +55 14 3235-8224 e-mail: marchini@usp.br editor: altair a. del bel cury received: june 14, 2021 accepted: january 30, 2022 surgical effects of rehabilitation protocols on dental arch occlusion of children with cleft lip and palate paula karine jorge1 , níkolas val chagas2 , eloá cristina passucci ambrosio1 , cleide felício carvalho carrara2 , fabrício pinelli valarelli3 , maria aparecida andrade moreira machado1 , thais marchini oliveira1,* aim: to evaluate the surgical effects of two rehabilitation protocols on dental arch occlusion of 5-year-old children with or without cleft lip and palate. methods: this is a retrospective longitudinal study the sample comprised 45 digitized dental casts divided into followed groups: group 1 (g1) – children who underwent to cheiloplasty (millard technique) at 3 months and to one-stage palatoplasty (von langenbeck technique) at 12 months; group 2 (g2) – children who underwent to cheiloplasty (millard technique) and two-stage palatoplasty (hans pichler technique for hard palate closure) at 3 months and at 12 months to soft palate closure (sommerlad technique); and group 3 (g3) – children without craniofacial anomalies. linear measurements, area, and occlusion were evaluated by stereophotogrammetry software. shapiro-wilk test was used to verify normality. anova followed by posthoc tukey test and kruskal-wallis followed by posthoc dunn tests were used to compared groups. results: for the measures intercanine distance (c-c’), anterior length of dental arch (i-cc’), and total length of the dental arch (i–mm’), there were statistical differences between g1x g3 and g2xg3, the mean was smaller for g1 and g2. no statistically significant differences occurred in the intermolar distance and in the dental arch area among groups. the occlusion analysis revealed significant difference in the comparison of the three groups (p=0.0004). conclusion: the surgical effects of two rehabilitation protocols affected the occlusion and the development of the anterior region of the maxilla of children with oral clefts when compared to children without oral clefts. keywords: cleft lip. cleft palate. dental arch. imaging, threedimensional. dental occlusion. https://orcid.org/0000-0002-9221-8052 https://orcid.org/0000-0002-2783-8933 https://orcid.org/0000-0003-2322-3832 https://orcid.org/0000-0002-3219-5936 http://orcid.org/0000-0002-4285-486x https://orcid.org/0000-0003-3778-7444 https://orcid.org/0000-0003-3460-3144 2 jorge et al. braz j oral sci. 2022;21:e226343 introduction the individual with cleft lip and palate undergoes a complex rehabilitative treatment through primary plastic surgeries, namely cheiloplasty and palatoplasty1. these surgical procedures aim to rehabilitate and return the proper speech, hearing, and masticatory functions, directly influencing the self-esteem and social-affective integration of individuals with oral clefts2. different rehabilitation protocols have been used over the years to repair the lip and palate3. to understanding the outcomes of plastic surgery and searching for suitable technical approach to decrease the iatrogenic effects of the rehabilitative procedures are essential to the rehabilitation of individuals with oral clefts, and they provide more favorable results that would consequently improve the quality of life4. the main aspects of the clef lip and palate repair is to understand the outcomes of different rehabilitative procedures. the rehabilitation starts with the closure of the lip. one of the most techniques used is millard’s, which consists in incisions that allow the rotation of the flap for lip closure5. von langenbeck’s technique, a procedure for close palate, requires relaxing incisions to promote union of the muscles at the level of the septum6,7. another technique for hard palate repair that can be used is hans pichler, which consist in the closure using a vomer flap8. sommerlad’s technique, is a procedure to enhance velopharyngeal competence, the performance is to reposition and reinserted of the elevated muscle of the soft palate, in posterior edge of hard palate, in order to reestablish the muscle complex, contributed to the function of the soft palate9. the differences in protocols can improve maxillary growth in cleft patients, and its importance is related to an achievement of the best rehabilitative protocol, since there is no gold standard protocol for cleft patient. the protocols are performed by the experience of the surgeon or for the convenience of the type, extension of the cleft. this justifies the evaluation and comparison of the dental arch development and the impact of the different surgical protocols in 5-year-old children. this study null hypothesis is that the dental arch morphology of children undergoing different rehabilitation surgical protocols is not statistically different from that of children without oral clefts, thus, the dental arch with patients with cleft remain without any restriction after primary surgeries, being as the same pattern with match-control-peers. thus, this study aimed to evaluate the surgical outcomes of two rehabilitation protocols on dental arch occlusion of 5-year-old children with or without cleft lip and palate. materials and methods the approved protocol by the institutional review board is caae: 40034620. 6.0000.5441. this is a retrospective longitudinal study, in the period of 2010 to 2019. the study the sample comprised 45 digitized dental casts divided into the three different groups: group 1 (g1) – children submitted to cheiloplasty (millard technique) at 3 months and one-stage palatoplasty (von langenbeck technique) at 12 months; group 2 (g2) – children submitted to cheiloplasty (millard technique) and two-stage palatoplasty (hans pichler technique for hard palate closure) at 3 months and 12 3 jorge et al. braz j oral sci. 2022;21:e226343 months to soft palate closure (sommerlad technique); and group 3 (g3) – children without craniofacial anomalies (control group). sample size estimative was accomplished according to the study of maulina et al.10 (2007). we considered a standard deviation of 2.73 millimeter (mm) in the intercanine distance of children with unilateral cleft lip and palate, the level of significance used was 5%, where p≤0.05 was considered significant, power test of 80%, and the clinically minimum difference to be detected of 2.95 mm. the minimum sample size for each group was 14 children.  inclusion criteria (g1 and g2) comprised maxillary dental casts of children from 5 years old, with unilateral cleft lip and palate, and without other craniofacial anomalies, with complete primary dentition, operated by the same plastic surgeon during their first year of life, at the rehabilitative center. the exclusion criteria (g1 and g2) were syndrome or other associated malformations, uncooperative children, and absence of the maxillary primary canines and/or second molars. inclusion criteria (g3) comprised maxillary dental casts of children from 5 years old, without cleft lip and palate and with complete primary dentition, at a dental school university. the exclusion criteria (g3) were absence of the maxillary primary canines and/or second molars. the analyzed images was obtained from digitized dental casts by a three dimensional (3d)d scanner (scanner r700tm scanner; 3shape as, copenhagen, denmark), and the digitized images were analyzed by two examiners in the stereophotogrammetry software (mirror imaging software, canfield scientific, inc., fairfield, nj, usa)11–13. the linear measurements were evaluated: intercanine distance (c–c’) – transversal line between the cusps of the maxillary left and right primary canine; intermolar distance (m–m’) – transversal line between the distal points of the palatal surface of the primary second molars; anterior dental arch length (i–cc’) – straight line passing from the interincisive point (i) perpendicularly to the c–c’ distance; and total dental arch length (i–mm’) – straight line from the point (i) perpendicularly to the distance m–m’14. the linear measurements were quantified in mm, figure 1. the palate area figure 1. linear measurements. 4 jorge et al. braz j oral sci. 2022;21:e226343 was marked by points passing through the palatal surfaces of the teeth. the posterior limit of the dental arch was the distance m–m’10, figure 2. the area was quantified in square millimeters (mm2). the three-dimensional images of the models in occlusion were evaluated by the index of atack et al.15 (1997). this index defines the systematization criteria for quantifying the occlusion morphology in individuals with unilateral cleft lip and palate, ranging from 1 to 5. the greater the index, the greater is the severity of the occlusion considering the interarch relationship, the maxillary arch shape, and the tipping of the maxillary incisors (table 1). all the statistical analyses were performed by graphpad prism software (prism 5 for windows version 5.0 – graphpad software., inc. san diego, usa), with the level of significance used was 5%,  where p≤0.05 was considered significant. the normality of the samples were analyzed by shapiro-wilk test. to check the table 1. classification of the index of atack. index description prognosis 1 positive overjet. normal or palatal tipping of the maxillary incisors. lack of open or crossbite. excellent 2 positive overjet. normal or labial tipping of the maxillary incisors. tendency towards crossbite and unilateral crossbite. tendency towards open bite at the cleft side. good 3 anterior edge-to-edge bite. labial tipping of the maxillary incisors or overjet with palatal tipping of the incisors. tendency towards open bite at the cleft side. regular 4 negative overjet. normal or labial tipping of the maxillary incisors. tendency towards open bite at the cleft side. tendency towards posterior unilateral or bilateral crossbite. poor 5 negative overjet. labial tipping of the maxillary incisors. bilateral crossbite. very poor figure 2. palate area. 5 jorge et al. braz j oral sci. 2022;21:e226343 method reliability, 1/3 of the sample was evaluated twice with a 15-day interval12,13. wilcoxon test verified the intraexaminer analysis, while mann-whitney test verified the interexaminer analysis. dahlberg’s formula quantified the causal error. anova and posthoc tukey test, kruskal-wallis and posthoc dunn tests were used to compared all three groups. results the sample was comprised by 16 children in g1, 14 children in g2, and 15 children in g3, totalizing 45 evaluated dental casts. the study participants mean age was 6.08 (± 0.65) years (table 2).  both the linear measurements and the area revealed no statistically significance in intraexaminer (wilcoxon test, p= 0.114 and dahlberg’s formula = 0.829) and interexaminer analyses (mann-whitney test, p=0.579). the occlusion analysis revealed no statistically significance differences in intraexaminer (wilcoxon test, p = 0.423) and interexaminer analyses (mann-whitney test, p=0.983). for the measures intercanine distance (c-c’), anterior length of dental arch (i-cc’), and total length of the dental arch (i–mm’), there were statistical differences between g1x g3 and g2xg3, the mean was smaller for g1 and g2. there was no significant differences presented in the intermolar distance (m–m’) and in the dental arch area among groups (table 3). table 4 shows the occlusion analysis of g1, g2, and g3 according to the index of atack. the intergroup comparison showed no statistically significant differences for g1 vs. g2, but statistically significant differences for g3 vs. g1 and g3 vs. g2. table 2. statistical analysis of sample. parameters group 1 group 2 group 3 p-value (test) male / female (n) 9 / 7 11 / 3 10 / 5 0.433 (chi-square) age (years) 6.93 5.92 5.39 0.367 (kruskal-wallis) table 3. intergroup analysis of the anthropometry of the dental arches (anova post-hoc tukey test). analyses unit group 1 mean sd group 2 mean sd group 3 mean sd p c–c’ mm 25.29 a 3.90 25.04 a 3.01 29.90 b 1.56 <0.0001* i–cc’ mm 5.19 a 2.51 5.78 a 1.27 6.87 b 0.86 0.034* m–m’ mm 35.31 a 3.99 35.06 a 2.39 36.29 a 1.59 0.425 i–mm’ mm 24.04 a 3.84 23.96 a 1.36 26.95 b 1.29 0.002* area mm² 788.23 a 144.65 826.27 a 77.64 845.28 a 98.56 0.361 * statistically significant difference. sd: standard deviation. different capital letters in line means statistically significant difference. 6 jorge et al. braz j oral sci. 2022;21:e226343 discussion this present study justifies in the attempted to understand better the differences between two different rehabilitative protocol, highlighting all the children were operated by one surgeon. in this way, the outcomes can be more favorable for the comprehension of what is important in the rehabilitative process. this present study exhibited a greater measurement of the intercanine distance, anterior dental arch length, and total dental arch length for children without clefts. the rationale behind this finding would be the restriction initiated by primary surgeries in the anterior (canine area) and anterior-posterior transversal growth. there were no significant differences in the intermolar distance and area. thus, it can be affirmed that the primary surgeries did not change the posterior transversal growth and the dental arch area. by corroborating with the maxillary restriction caused by the primary surgeries, bruggink et al.16(2019), evaluated longitudinally individuals without oral clefts and followed the maxillary growth through the first year of life and remarkably found that the rate growth between the canines increased between 3 and 6 months of life. previous study analyzed the maxillary dimensions at the first six months of life and estimated that the relative transversal growth of the anterior portion of the maxilla is around three times quicker than that of the posterior portion (81.9% vs. 26.2%)17. this points out to an anterior widening of the maxilla during that period and highlights the impact of cheiloplasty performed at the first months of life. moreover, the treatment prognosis is categorized by the cleft amplitude severity, highlighting the width size. the bigger the cleft size, the greater is the probability of the healing tissue negatively impact on the maxillary growth18. the study of huang et al.19(2002), evaluated the maxilla of individuals with unilateral cleft lip and palate and found an increasing in the dental arch linear measurements after a period of 12 months, except for the anterior region that displaced towards palatine after the cheiloplasty. the method to obtain these measurements is very important. according to kongprasert et al.20(2019), three-dimensional evaluation has better accuracy and validity than two-dimensional evaluation, and it performances an important role in the follow-up of the change in dental arch dimensions towards all directions. digitized models have the advantages of construction and analysis, absence of damage, that table 4. classification of the index of atack by group. intergroup analysis of the index of atack (kruskal-wallis test post-hoc dunn test). index group 1 n (%) group 2 n (%) group 3 n (%) p 1 1 (6.25) 0 (0) 7 (46.67) 2 6 (37.50) 4 (28.75) 6 (40) 3 0 (0) 5 (35.71) 2 (13.33) 4 6 (37.50) 4 (28.57) 0 (0) 5 3 (18.75) 1 (7.14) 0 (0) total 16 (100) a 14 (100) a 15 (100) b 0.0004* * statistically significant difference. different capital letters in line means statistically significant difference. 7 jorge et al. braz j oral sci. 2022;21:e226343 is, preservation of the dental casts. thus, digitized dental cast has replaced dental casts as gold-standard20. previous studies reported the validity of the 3d stereophotogrammetry, including the clinical environment21. the occlusal analysis by the index of atack is performed at 5 years-old because this is the age children are at complete deciduous denture. this index is measured in a scale ranging from 1 to 5, seeing that the greater the index, the worst is the facial profile, oscillating from regular occlusion to anterior and/or posterior crossbite15. the study of dental casts plays a relevant therapeutic role in the treatment of individuals with oral clefts because it points out the dimensional alterations and enables the use of indexes regarding treatment22. this present study showed no statistically differences between the groups with clefts. this may suggest the interference of the different primary surgeries techniques on the occlusion development. the impact of the primary surgeries is still difficult to measure, which one is more suitable for the growth23. indeed, the literature lacks comparative studies on the occlusal analysis in children with and without cleft lip and palate. thus, the comparison with children without clefts revealed that different surgical techniques directly influenced on the occlusal outcome. this result may contribute with the elaboration of a satisfactory rehabilitation protocol. the potential strength of this study is the sample, because all the patient present in this study was operated by the same surgeon, so the sample have no operator bias, however this limits the sample number. a limitation issue that can be pointed is the size of the cleft before primary surgeries, this can be an important challenge for the surgeon, because wider is the cleft, more soft tissue is needed and more in the potential of scar and retraction. therefore, further studies can be delineated in relation of cleft width. in conclusion, the surgical effects of two rehabilitation protocols affected the occlusion and the development of the anterior region of the maxilla of children with oral clefts when compared to children without oral clefts. acknowledgments são paulo research foundation, grant/award number: 2017/02706-9. data availability datasets related to this article will be available upon request to the corresponding author. conflict of interests none. author contribution paula karine jorge – have made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data. and have been involved in drafting the manuscript or revising it critically for important intellectual content 8 jorge et al. braz j oral sci. 2022;21:e226343 níkolas val chagas – contributed substantially to the conception and design of the study, the acquisition of data, and the analysis and interpretation eloá cristina passucci ambrosio drafted or provided critical revision of the article, the analysis and interpretation, and provided final approval of the version to publish cleide felício carvalho carrara – have given final approval of the version to be published fabrício pinelli valarelli – have been involved in drafting the manuscript or revising it critically for important intellectual content maria aparecida andrade moreira machado agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. thais marchini oliveira drafted or provided critical revision of the article. provided final approval of the version to publish. agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. references 1. shi b, losee je. the impact of cleft lip and palate repair on maxillofacial growth. int j oral sci. 2015 mar;7(1):14-7. doi: 10.1038/ijos.2014.59. 2. pereira rmr, siqueira n, costa e, vale d do, alonso n. unilateral cleft lip and palate surgical protocols and facial growth outcomes. j craniofac surg. 2018 sep;29(6):1562-8. doi: 10.1097/scs.0000000000004810. 3. arosarena oa. cleft lip and palate. otolaryngol clin north am. 2007 feb;40(1):27-60, vi. doi: 10.1016/j.otc.2006.10.011. 4. sakoda kl, jorge pk, carrara cfc, machado maam, valarelli fp, pinzan a, et al. 3d analysis of effects of primary surgeries in cleft lip/palate children during the first two years of life. braz oral res. 2017 jun;31:e46. doi: 10.1590/1807-3107bor-2017.vol31.0046. 5. demke jc, tatum sa. analysis and evolution of rotation principles in unilateral cleft lip repair. j plast reconstr aesthet surg. 2011 mar;64(3):313-8. doi: 10.1016/j.bjps.2010.03.004. 6. von langenbeck b. operation der anageborene totalen spaltung des harten gauments nach einer methode. dtsch arch klin med. 1861;13:231. 7. silva filho og, freitas jas. caracterização morfológica e origem embriológica. fissuras labiopalatais: uma abordagem interdisciplinar. são paulo: santos; 2007. 8. bosi v, brandão g, yamashita r. speech resonance and surgical complications after primary palatoplasty with intravelar veloplasty in patients with cleft lip and palate. rev bras cir plast. 2016;31(1):43-52. doi: 10.5935/2177-1235.2016rbcp0007. 9. sommerlad bc, mehendale fv, birch mj, sell d, hattee c, harland k. palate re-repair revisited. cleft palate craniofac j. 2002 may;39(3):295-307. doi: 10.1597/1545-1569_2002_039_0295_prrr_2.0.co_2. 10. maulina i, priede d, linkeviciene l, akota i. the influence of early orthodontic treatment on the growth of craniofacial complex in deciduous occlusion of unilateral cleft lip and palate patients. stomatologija. 2007;9(3):91-6. 9 jorge et al. braz j oral sci. 2022;21:e226343 11. carrara cfc, ambrosio ecp, mello bzf, jorge pk, soares s, machado ma, et al. three-dimensional evaluation of surgical techniques in neonates with orofacial cleft. ann maxillofac surg. 2016 juldec;6(2):246-50. doi: 10.4103/2231-0746.200350. 12. ambrosio ecp, sforza c, de menezes m, gibelli d, codari m, carrara cfc, et al. longitudinal morphometric analysis of dental arch of children with cleft lip and palate: 3d stereophotogrammetry study. oral surg oral med oral pathol oral radiol. 2018 dec;126(6):463-8. doi: 10.1016/j.oooo.2018.08.012.. 13. ambrosio ecp, sforza c, de menezes m, carrara cfc, machado maam, oliveira tm. post-surgical effects on the maxillary segments of children with oral clefts: new threedimensional anthropometric analysis. j craniomaxillofac surg. 2018 sep;46(9):1511-4. doi: 10.1016/j.jcms.2018.06.017. 14. rando gm, jorge pk, vitor llr, carrara cfc, soares s, silva tc, et al. oral health-related quality of life of children with oral clefts and their families. j appl oral sci. 2018 feb;26:e20170106. doi: 10.1590/1678-7757-2017-0106. 15. atack n, hathorn i, mars m, sandy j. study models of 5 year old children as predictors of surgical outcome in unilateral cleft lip and palate. eur j orthod. 1997 apr;19(2):165-70. doi: 10.1093/ejo/19.2.165. 16. bruggink r, baan f, kramer g, maal tjj, kuijpers-jagtman am, bergé sj, et al. three dimensional maxillary growth modeling in newborns. clin oral investig. 2019 oct;23(10):3705-12. doi: 10.1007/s00784-018-2791-5. 17. zen i, soares m, pinto lmcp, ferelle a, pessan jp, dezan-garbelini cc. maxillary arch dimensions in the first 6 months of life and their relationship with pacifier use. eur arch paediatr dent. 2020 jun;21(3):313-9. doi: 10.1007/s40368-019-00487-9. 18. reiser e, skoog v, andlin-sobocki a. early dimensional changes in maxillary cleft size and arch dimensions of children with cleft lip and palate and cleft palate. cleft palate craniofac j. 2013 jul;50(4):481-90. doi: 10.1597/11-003. 19. huang c-s, wang w-i, liou ej-w, chen y-r, chen pk-t, noordhoff ms. effects of cheiloplasty on maxillary dental arch development in infants with unilateral complete cleft lip and palate. cleft palate craniofac j. 2002 sep;39(5):513-6. doi: 10.1597/1545-1569_2002_039_0513_eocomd_2.0.co_2. 20. kongprasert t, winaikosol k, pisek a, manosudprasit a, manosudprasit a, wangsrimongkol b, et al. evaluation of the effects of cheiloplasty on maxillary arch in uclp infants using three-dimensional digital models. cleft palate craniofac j. 2019 sep;56(8):1013-9. doi: 10.1177/1055665619835090. 21. othman sa, saffai l, wan hassan wn. validity and reproducibility of the 3d vectra photogrammetric surface imaging system for the maxillofacial anthropometric measurement on cleft patients. clin oral investig. 2020 aug;24(8):2853-66. doi: 10.1007/s00784-019-03150-1. 22. ozawa to, shaw wc, katsaros c, kuijpers-jagtman am, hagberg c, rønning e, et al. a new yardstick for rating dental arch relationship in patients with complete bilateral cleft lip and palate. cleft palate craniofac j. 2011 mar;48(2):167-72. doi: 10.1597/09-122. 23. shaye d, liu cc, tollefson tt. cleft lip and palate: an evidence-based review. facial plast surg clin north am. 2015 aug;23(3):357-72. doi: 10.1016/j.fsc.2015.04.008. 1 volume 22 2023 e238082 original article braz j oral sci. 2023;22:e238082http://dx.doi.org/10.20396/bjos.v22i00.8668082 1 department of restorative dentistry, piracicaba dental school, university of campinas. 2 department of restorative sciences, division of dental materials, college of dentistry, oklahoma university health sciences center. corresponding author: vanessa cavalli department of restorative dentistry, piracicaba dental school, university of campinas cavalli@unicamp.br editor: dr. altair a. del bel cury received: january 9, 2022 accepted: june 11, 2022 effects of black tea tooth staining previously to 35% hydrogen peroxide bleaching samuel da silva palandi1 , matheus kury1 , mayara zaghi dal picolo1 , fernando luís esteban florez2 , vanessa cavalli1 * aim: to determine if the artificial staining with black tea (bt) influences the enamel microhardness before in-office bleaching and if bt staining is necessary to evaluate the efficacy of bleaching with 35% hydrogen peroxide methods: enamel/dentin blocks were randomized into groups according to the staining protocol (n=5/group): (co) control – maintained in artificial saliva solution (as); (bt4) immersed in black tea solution for 4 h; (bt24) immersed in black tea solution for 24 h. after the staining protocols, all specimens were kept in as for one week, followed by bleaching (three sessions of hp application for 40 min). knoop surface microhardness (kgf/mm2) was determined at baseline (t0), after staining (t1), after 7 days of storage in as (t2), and after bleaching (t3). the color (∆e00) and coordinate changes (∆l, ∆a, ∆b) were measured using a digital spectrophotometer at t0 and t3. data were submitted to one-way (∆e00, ∆l, ∆a, ∆b) or two-way anova repeated measures (kgf/mm2) and tukey’s test (a=5%). results: the staining protocols (bt4 and bt24) promoted significantly lower microhardness (t1 and t2, p<0.05) than co, whereas co was the only group to maintain microhardness values over time. bleaching promoted perceptible ∆e00 without a significant difference among the groups regardless of the staining protocol (p=0.122). co and bt4 showed no differences in terms of ∆l and ∆a (p>0.05), but bt4 displayed a higher ∆b than co. conclusion: the artificial staining with bt negatively affected the enamel surface microhardness and was not essential to evaluate the efficacy of 35% hydrogen peroxide bleaching. keywords: bleaching agents. hydrogen peroxide. staining and labeling. https://orcid.org/0000-0001-7355-9954 https://orcid.org/0000-0002-9971-0568 https://orcid.org/0000-0002-4141-5283 https://orcid.org/0000-0002-8351-0721 https://orcid.org/0000-0002-9459-1926 2 palandi et al. braz j oral sci. 2023;22:e238082 introduction tooth discoloration is classified based upon its location1. intrinsic color is associated with structural and thickness alterations to dentin during tooth formation or after its eruption2, while extrinsic discoloration is determined by the adsorption of polyphenolic compounds onto the surface of dental enamel and their interaction with pellicle proteins3. most of these organic chromogens are present in the daily habits of the patients, i.e., intake of foods and smoking4. additionally, enamel defects, dental caries, or restorative materials may facilitate the incorporation of such compounds within the tooth structure5. however, it should be noted that the extrinsic staining deposited on the enamel is not resistant to removal with either regular toothbrushing or professional prophylaxis4,6. tooth bleaching remains the most ultraconservative and efficient approach to treat dental discoloration even after prophylaxis7. the technique consists of applying carbamide or hydrogen peroxide gels on the enamel surface8 in concentrations that vary according to the bleaching regimen. the chemical byproducts of the reaction, the reactive oxygen species (ros) resulting from peroxide decomposition, diffuse through the enamel into the dentin9, supposedly breaking down the intrinsic staining into smaller molecules. consequently, teeth look whiter8. several in vitro studies evaluated tooth bleaching protocols’ efficacy and adverse effects by using artificial staining on enamel before hp bleaching. one of the reasons for that approach is the necessity of the standardization of tooth color among groups before the bleaching treatments10,11. previous studies reported tooth staining with soft drinks12, tobacco smoke, coffee13, red wine14, and black tea11. there is evidence that black tea exhibits a higher staining effect than cola-based soft drinks and coffee15. in this regard, sulieman et al.3 (2003) validated an artificial staining protocol with black tea specifically for a dental bleaching evaluation. according to the authors, the 24-hour immersion of enamel/dentin specimens in black tea solution did not differ from a 6-day immersion3. in other words, an overnight staining protocol would suffice to assess the bleaching efficacy of peroxides. nevertheless, staining agents may influence composition and structure other than tooth optical properties. for instance, it has been reported that teeth stained with black tea exhibited lower ros penetration after highly concentrated hp bleaching than unstained teeth16. in addition, long-lasting enamel exposure to solutions or products with a ph lower than 5.5 can trigger enamel demineralization17. since evidence shows that black tea’s ph can range from 3.3 to 6.5 in commercial ready-to-drink beverages18, artificial staining before bleaching may compromise the enamel mineral composition and surface morphology. moreover, tooth bleaching may modify the enamel composition and structure by decreasing the enamel mineral content19 and surface microhardness20, increasing the surface roughness14, and changing the enamel morphology11. given these facts, artificial black tea staining may interfere with the outcomes and decrease the reliability of the results related to tooth bleaching itself. although several studies have already investigated the effects of tooth bleaching on enamel sur3 palandi et al. braz j oral sci. 2023;22:e238082 face properties11,14,19,20, there is no evidence showing if the step of the artificial tooth staining isolated would negatively affect the enamel microhardness and become a confounding variable. additionally, studies have not indicated whether the specimens’ darkening in different exposure times is necessary to determine enamel color change compatible with efficient tooth bleaching. hence, this study determined the effect of artificial staining with black tea, in short (4 h) or long (24 h) exposures, on enamel surface microhardness and the efficacy of tooth bleaching with 35% hydrogen peroxide (hp) on stained and nonstained teeth. the null hypotheses were that (a) artificial staining would not decrease the enamel surface microhardness and that (b) the staining protocols tested would not affect the color change after bleaching. materials and methods experiment design dental blocks were submitted to artificial staining protocols with or without black tea solution (bt): • co: control without black tea and stored in artificial saliva (as) • bt4: black tea immersion for 4 h (short exposure) • bt24: black tea immersion for 24 h (long exposure) after staining, all specimens were stored in as for 7 d and submitted to an in-office bleaching protocol (35% hp). the color and the surface microhardness of the specimens were assessed before (t0), after staining (t1), after storage in as (t2), and after bleaching treatment (t3). specimen preparation bovine incisors were extracted, cleaned with periodontal scrapers, and stored in 0.1% thymol solution at 4°c for no longer than 30 days. fifteen teeth without enamel cracks or defects were selected, and the roots were separated from the crowns using a low-speed diamond saw (isomet, buehler; lake bluff, illinois, usa) under refrigeration, 2 mm below the cementoenamel junction. blocks (6 mm × 6 mm and 3 mm thick) were obtained from the central portion of the crown. the dentin was flattened using a rotary polisher (arotec ind. com., são paulo, sp, brazil) with silicon carbide paper #600 under water-cooling. the enamel surface was finished with aluminum oxide grit #600 and #1200 and polished using diamond aqueous suspensions (6, 3, 1, and 0.25 μm, metaldi supreme, buehler, lake bluff, illinois, usa). the blocks were ultrasonically cleaned with distilled water for 5 min among each progression in grits. artificial staining and groups distribution the dentin of specimens was isolated using wax to allow the exposure of the enamel surface only. the blocks were randomly assigned to groups according to the black tea protocol (n=5/group): (co) without bt and stored in as [1,5 mm ca; 0,9 mm p; 150 mm kcl e 0,1 m tris; ph 7,0] 21, (bt4) immersion in bt for 4 h, (bt24) immersion for 24 h. 4 palandi et al. braz j oral sci. 2023;22:e238082 the sample size was calculated using the software g*power. the mean and standard deviation values of the enamel surface microhardness reported by costa et al.22 (2021) were used to detect the effect size since these authors also used artificially stained enamel to evaluate the action of hydrogen peroxide bleaching. the detected effect size (f = 1.5) was applied on g*power with a predetermined significance level of 0.05, and a power test of 80%. as a result, five specimens were required for each group. the black tea solution was prepared according to an adaptation of the protocol described by sulieman et al.3 (2003). two grams of black tea (dr. oetker, são paulo, sp, brazil) was diluted in 100 ml of boiling distilled water for 5 min, filtered, and cooled. the specimens were immersed in bt (20 ml) at a constant temperature of 37°c. the ph of the solution was checked before and after immersion (4 h: 4.7 – 4.9 and 24 h: 4.6 – 4.9). after staining, the specimens were washed in distilled water and stored in as for 7 d to allow color stabilization of the specimens. bleaching procedures in-office bleaching (35% hp, total blanc office h35, nova dfl, rio de janeiro, rj, brazil) was performed according to the manufacturer’s recommendation: three 40-min sessions at 72-h intervals. the specimens were stored in as at intervals for seven consecutive days after bleaching. surface microhardness the mean enamel surface microhardness was obtained by three impressions in the central area of the block, with a knoop diamond penetrator (future tech-fm-1e, tokyo, japan), under a static charge of 25 grams for 5 seconds and 100 µm of distance from each other. no differences in kgf/mm2 among groups were found at baseline (t0). the microhardness was evaluated immediately after staining (t1), after stabilization in as (t2), and seven days after bleaching (t3). colorimetric evaluation the colorimetric measurements on the enamel surface were performed using a digital spectrophotometer (easyshade, vita zahnfabrik, bad säckingen, germany). the equipment was fixed on a platform to allow the tip to face enamel as parallel as possible. the background in which the dental blocks were placed was standardized with an opaque tile. this apparatus was positioned inside a light-controlled chamber. the l*, c*, h*, a*, and b* coordinates were obtained from the average of three measurements. l* represents the luminosity of an object (black to white), while a* and b* represent + red/-green and + yellow/-blue, respectively. the c* parameter determines the chroma of the substrate, and h*, the tooth hue. because this study aimed to evaluate the effect of bleaching in stained teeth, colorimetric data were collected 7 days after staining and storage in as (t2) and 7 days after the last bleaching session (t3). the color variation was calculated according to ciede2000 (∆e00), with the color difference between t3 and t2. the 50:50% perceptibility threshold adopted was 1.2 ∆e00 units23. the variation in the l*, a*, and b* coordinates were also individually calculated, taking into consideration the same time points mentioned above. the groups exhibited no significant differences in the mean baseline l*, a*, and b* values. 5 palandi et al. braz j oral sci. 2023;22:e238082 statistical analysis the normal distribution (shapiro–wilk, p>0.05) and homoscedasticity (levene, p>0.05) of the data were confirmed. data were statistically analyzed by one-way (∆e00, ∆l, ∆a, ∆b) or two-way repeated-measures anova (microhardness), and tukey’s test detected differences among groups at a significance level of 5%. all analyses were performed by spss version 23 (ibm corp. released 2015. ibm spss statistics for windows, version 23.0. armonk, ny: ibm corp.). results microhardness evaluation. at baseline (t0), no differences in microhardness were detected among groups (table 1), but both staining protocols (bt4 and bt24) decreased enamel microhardness (t1) in comparison with co (p<0.001). at this time-point (t1), bt24 exhibited lower microhardness than bt4 (p<0.001). after storage in as for 7 d (t2), the results remained unaltered. seven days after bleaching (t3), co and bt4 displayed no differences (p=0.36) but exhibited higher knoop values than bt24 (p<0.05). co microhardness remained unaltered over time, while microhardness recovery was detected for bt24 at t3. table 1. mean values and standard deviation of microhardness values (kgf/mm2). groups t0 t1 t2 t3 co 307.1 (20.5) aa 288.5 (14.5) aa 286.5 (6.2) aa 285.89 (10.74) aa bt4 307.0 (16.3) aa 260.1 (16.9) bb 259.5 (17.7) bb 267.82 (25.35) ab bt24 310.3 (16.2) aa 189.0 (24.4) cc 204.6 (25.0) cc 224.60 (11.97) bb means followed by distinct letters are different according to two-way anova repeated measurements and tukey’s test at a significance level of 5%. capital letters compare staining protocols (columns), and lowercase letters compare time points (lines). t0: baseline; t1: after staining; t2: after 7 days of storage in as; t3: after bleaching. co: control; bt4: black tea immersion for 4 h; bt24: black tea immersion for 24 h. color evaluation. no differences in ∆e00 among groups were detected (p=0.122) after bleaching (t3 -t2). the mean color differences promoted by bleaching were above the perception threshold (∆e00>0.8), regardless of the staining protocol (table 2). bt24 showed higher ∆l, ∆a, and ∆b values than co but no difference from bt4 in ∆l and ∆b. bt4 and co displayed no differences in ∆l and ∆a (>0.05) but higher ∆b than co. table 2. mean values and standard deviation of the alteration in euclidian coordinates (∆l, ∆a, and ∆b) and color alteration between color stabilization in as and bleaching. groups ∆l ∆a ∆b ∆e00 co -2.8 (3.7) b -2.3 (1.4) a -15.1 (1.8) b 9.0 (1.1) a bt4 -1.0 (6.1) ab -4.2 (1.6) a -18.8 (2.1) a 10.7 (0.2) a bt24 6.4 (5.9) a -8.6 (1.4) b -17.2 (1.0) a 11.5 (2.9) a means followed by distinct letters are different according to one-way anova and tukey’s test at a significance level of 5%. capital letters compare staining protocols (columns). co: control; bt4: black tea immersion for 4 h; bt24: black tea immersion for 24 h. 6 palandi et al. braz j oral sci. 2023;22:e238082 discussion black tea staining reduced the enamel microhardness before the bleaching procedures, regardless of immersion time in bt. this outcome remained unchanged even after storage in artificial saliva for 7 days elapsed from the last bleaching session. therefore, the first null hypothesis was rejected because artificial staining with bt affected the enamel surface microhardness. artificial dental staining is a method developed to mimic the natural intrinsic staining that occurs with the deposition of secondary and tertiary dentin along the tooth´s lifetime3. the protocol described in 20033 has been extensively used by researchers as an attempt to simulate an ideal substrate to be bleached and as an approach to standardize the teeth color of groups24-26. nevertheless, that study3 failed to report the ph of black tea and its effects on enamel after 24 h of immersion. to the best of our knowledge, studies investigating the bleaching effect on enamel have not reported the surface microhardness immediately after immersion in the staining solution. for instance, a previous report displayed the microhardness values at baseline and after bleaching while evaluating the effects of light-activated 6% hp on the enamel surface22,27. in that study, black tea with an unknown ph and manufacturer was used to immerse dental blocks for 18 h. since a mean surface microhardness reduction was observed over time, the black tea immersion should also be considered in the interpretation of the results. bearing this in mind, the time points of the present study allowed a more comprehensive understanding of the real impact of bleaching on the enamel surface. the results showed that 35% hp was not responsible for reducing the enamel surface microhardness, contrary to the black tea solution. similar to the tea used in our study, with a ph ranging from 4.6 to 4.9, others have reported the use of bt with a ph lower than 5.528-30. the study of farawati et al.30 (2019) investigated the effect of black tea, wine, soda, coffee, and water on the surface properties of enamel submitted to bleaching with carbamide peroxide. the data from that study suggested that only black tea and wine changed the enamel mineral composition. one might say that the presence of fluoride in tea31 could uphold the level of enamel mineralization; nonetheless, it was demonstrated that the immersion of eroded enamel in a ready-to-drink black tea with 0.760 mg/l of fluoride, in a much lower immersion time than our study, decreases the enamel microhardness significantly more compared to other types of drinks32. additionally, jameel et al.29 (2016) showed that a black tea solution (ph 4.9, 0.938 mg/l) negatively impacted not only the surface microhardness but also the enamel roughness. hence, the existing evidence suggests that the ph of the solutions might play a more important role than the presence of fluoride in the maintenance of the surface properties. it is important to highlight that group bt4 was set as an alternative to diminishing the necessary time of staining. despite the lower immersion time, a significant decrease in khn was also observed for this group. however, it is noteworthy that this reduction in the microhardness was significantly lower than that of bt24, which may indicate a time-dependent effect of black tea on the enamel microhardness. even though other protocols could adjust the ph of the staining solution as an attempt to overcome 7 palandi et al. braz j oral sci. 2023;22:e238082 the low-ph value, raising the ph of this solution could impact the capability of the staining molecules to penetrate the dental structure. since no studies were found attempting this approach, future studies could investigate its application. the colorimetric data inferred that the staining protocols did not affect the color change promoted by bleaching. the group stored in artificial saliva showed a similar ∆e00 to the bt groups. thus, the second null hypothesis was accepted since the staining protocols did not affect the color change after bleaching. according to paravina et al.23 (2019), a color change above 5.4 units is compatible with the excellent effectiveness of tooth bleaching, thereby indicating a very highly perceptible outcome. therefore, the commercial bleaching gel used herein was very effective even without a previous staining step [∆e00 = 9.0 (1.1)]. additionally, in vitro studies using artificial staining may exhibit exacerbated ∆e00 results. for instance, different studies evaluating the same bleaching protocols presented higher ∆e00 values when teeth were artificially stained 11,13 compared to nonstained teeth13,33,34. differences in the l* and a* coordinate changes among the groups could be explained by the absence (co) or lower amount (bt4) of pigments, suggesting a minor challenge for the hydrogen peroxide gel. on the other hand, the high shift in the b* coordinate from b+ (yellow) to b(blue), independent of the staining protocol, might suggest that the bleaching gel could breakdown the chromogens even without previous artificial staining. these results indicate that artificial staining before bleaching to mimic and standardize the specimens for color evaluation may be discarded since bleaching therapy was effective in nonstained teeth. in this regard, an interesting research approach would be avoiding dental blocks with a high initial luminosity (l*) and low values of the b* coordinate to observe the bleaching effects. updated colorimetric systems, such as the color change calculated by ciede200035, could overcome the drawbacks of previous calculations (cielab or visual shade guides). moreover, ∆e00 adjusts the low influence that the a* coordinate plays on the color of teeth. the sulieman et al.3 (2003) study only applied a subjective color evaluation of the specimens while establishing the staining protocol. this fact emphasizes the need for further analysis using appropriate objective color measurements and corresponding updated color and whiteness indexes7. a limitation of the present research is the absence of topography analysis and groups with additional bt immersion. however, the purpose of this investigation was to stress the importance of carefully employing artificial staining for bleaching evaluation. artificial staining should be carefully designed since overexposure and ph might damage the enamel surface and lead to a mistaken interpretation of the hp bleaching effects and enamel mineral content. in addition, excluding artificial staining may not impact bleaching efficacy, and the results could be more reliable and translated to a clinical setting. in conclusion, artificial staining with black tea showed detrimental effects on enamel surface microhardness, wherein the higher the time of exposure to tea was, the lower the hardness. in addition, bleaching with 35% hp did not decrease surface microhardness in the stained or nonstained specimens, and the staining protocols (4 h or 8 palandi et al. braz j oral sci. 2023;22:e238082 24 h immersion in black tea) were not essential to detect the bleaching effects. all groups displayed similar color changes after bleaching. acknowledgments this study was supported by the são paulo state research foundation (fapesp #2020/08440-3 and #2020/06782-4). this study was also supported in part by the coordenação de apoio ao pessoal de nível superior (capes – financial code 001). conflict of interest the authors have no conflicts of interest to declare. data availability datasets related to this article will be available upon request to the corresponding author. author contribution • samuel da silva palandi: methodology, investigation, data curation, writing (original draft, review, and editing); • matheus kury: conceptualization, methodology, formal analysis, data curation, writing (review and editing); • mayara zaghi dal picolo: methodology, investigation, data curation, writing (review and editing); • fernando luis esteban florez: conceptualization, funding acquisition, writing (review and editing); • vanessa 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randomized clinical trial. clin oral investig. 2022 jan;26(1):837-47 16. doi: 10.1007/s00784-021-04062-9. 35. sharma g, wu w, dalal en. the ciede2000 color-difference formula: implementation notes, supplementary test data, and mathematical observations. color res appl. 2005 feb;30(1):21-30. doi: 10.1002/col.20070. 1http://dx.doi.org/10.20396/bjos.v21i00.8665991 volume 21 2022 e225991 original article 1 department of restorative dentistry, state university of londrina – uel, londrina, pr, brazil. 2 department of restorative dentistry, school of dentistry, araraquara, são paulo state university – unesp, araraquara, sp, brazil. 3 study group of tissue regeneration, adaptation and repair, center of biological sciences, state university of londrina – uel, londrina, pr, brazil. corresponding author: joão felipe besegato araraquara school of dentistry 1680 humaitá street – 3rd floor zip code: 14801-903 araraquara, são paulo, brazil. tel.: +55 16 3301-6393 e-mail: besegato@outlook.com editor: dr altair a. del bel cury received: june 6, 2021 accepted: november 11, 2021 clinical performance of resin composite restorations placed by dental students: a retrospective, cross-sectional, and observational study daiane cristina mendonça dos santos1 , joão felipe besegato2,* , joissi ferrari zaniboni2 , solange de paula ramos3 , sueli de almeida cardoso1 , márcio grama hoeppner1 aim: a retrospective, cross-sectional study was carried out to evaluate the performance of resin composite restorations placed by undergraduate dental students with 1 to 15 years of placement based on dental records. methods: four calibrated operators evaluated 498 restorations (anterior and posterior) of 120 patients according to ryge’s validated criteria (usphs). results: the criteria that showed the smallest changes between the types of failures were color matching, marginal discoloration and surface texture. regarding the longevity, the surface texture showed an increase in the frequency of failures from the second time interval (3.1 to 6 years). higher prevalence of failure was found in class ii and iii restorations, with secondary caries being the main reason. no significant differences were found for anatomic form, marginal adaptation, and color matching. class v restorations showed a higher fracture rate with total displacement of the restoration, with no increase in the frequency of fracture over time. conclusion: high rate of restoration failure was observed, possibly due to the lack of experience and skills of the students. this outcome highlight the need for continuous revision and improvements of teaching practice regarding the development of clinical competences and skills by dental students. keywords: dental restoration permanent. dental restoration failure. composite resins. https://orcid.org/0000-0003-1929-1680 https://orcid.org/0000-0001-8747-779x https://orcid.org/0000-0003-0172-9891 https://orcid.org/0000-0003-0107-1057 https://orcid.org/0000-0001-5238-9845 https://orcid.org/0000-0001-5075-3412 2 santos et al. introduction resin composite (rc) is the choice material for direct restoration of teeth with caries lesion, fracture, and/or unsatisfactory restoration due to its aesthetic and physical properties, adhesion to dental tissues, reinforcement of the remnant tissue, low-cost and conservative approach compared to indirect restorations1-3. although good clinical survival with an annual failure rate ranging from 0.3 to 4.1%2,4-6, the number of rc restorations has increased7,8 and dental clinicians (dc) have been replaced rc restorations more often6. rc restorations can fail due to several reasons. the number of surfaces affected, tooth position in the arch, and cavity size are tooth-related conditions impacting the longevity of restorations1,4-6,9,10. concomitantly, oral hygiene, deleterious habits4,6,11, and socioeconomic factors6,12 of patients directly affect the clinical performance of rc restorations. besides, dc can also interfere in survival rates of rc restorations since expertise and clinical skills are crucial to correctly develop the restorative technique6. in this way, rc restorations may be compromised when placed by dental students since their skills and knowledge are under progress through the course13,14. several studies clinically evaluating rc restorations performed by qualified and calibrated dc have been reported2,3-5,9,10. however, there is a lack of studies14-17 reporting clinical outcomes of rc restorations placed by dental students. these studies will certainly be useful to assess the quality of restorative techniques and guide some improvements in dental teaching and qualification. this retrospective, cross-sectional, and observational study aimed to clinically evaluate the performance of rc restorations placed by dental students from the college of dentistry of the state university of londrina (uel) based on ryge’s criteria (modified usphs)16. material and methods experimental design and ethical aspects in this retrospective, cross-sectional, and observational study, rc restorations (anterior and posterior) placed by dental students from the state university of londrina were clinically evaluated. the restorations were classified according to black’s classification in i, ii, iii, iv, and v. this study received ethical approval by the research ethics committee involving human beings from the state university of londrina (cep/uel 1.607.965/2016). all the volunteers were properly informed of the objective of this study. those who agreed to participate, a consent form was signed. none invasive procedure was performed in the participants. patients’ recruitment the patients were selected based on dental charts of the screening section of university dental clinic from the state university of londrina. we evaluated only rc restorations performed by dental students from the third, fourth, and fifth grades of 3 santos et al. the dentistry course. these restorations were placed during curricular dental practice activities under the supervision of qualified professors in patients older than 18 years. for inclusion in this study, rc restorations should be correctly recorded in the dental chart, which must contain: grade of the student, date of the procedure, cavity type, restorative material used, and finishing and polishing procedures. only rc restorations with a minimum of 12 months and a maximum of 15 years were included. restorations performed in private practice or any other dental care, by formed dental clinicians, and in patients with special needs were excluded. data acquisition clinical evaluation for data acquisition was uninterruptedly performed for 24 months by four dental clinicians from the residency program in restorative dentistry. the operators were calibrated in two time points with an interval of 30 days between them. the intraclass correlation coefficient (icc) obtained was 0.87. during the clinical evaluation, teeth were cleaned, dried with air spray, and a clinical probe (nº 5, golgran, são caetano do sul, brazil) was used to evaluate the surfaces. artificial spotlights directly illuminated the operatory field, and it was considered adequate by the operators. rc restorations were classified according to ryge’s criteria (modified usphs) in: alpha (a) – satisfactory; bravo (b) – regular, and charlie (c) – unsatisfactory (table 1)8. regarding the time of placement, the restorations were allocated in five intervals: 1º interval = 1 to 3 years; 2º interval = 3.1 to 6 years; 3º interval = 6.1 to 9 years; 4º interval = 9.1 to 12 years, and 5º interval = 12.1 to 15 years. table 1. ryge’s criteria modified united states public health service (usphs). ryge criteria alpha (a) bravo (b) charlie (c) color matching restoration matches with the color and translucence of the adjacent dental tissues. restoration does not match with the color and translucency of the adjacent dental tissues. however, the unmatched is clinically acceptable, i.e., less changed. restoration does not match the shade and translucency of the adjacent dental tissues. the unmatched is outside the normal range of tooth shade and translucency, i.e., very altered. marginal discoloration no visual evidence of marginal discoloration different from the restorative material shade and the adjacent dental tissue shade. there is visual evidence of marginal discoloration at the interface tooth/ restoration. but the discoloration has not penetrated along the restoration in a pulpal direction. staining at the cavosurface angle <1mm. there is visual evidence of marginal discoloration at the tooth structure junction and the restoration that has penetrated along the restoration in a pulpal direction. staining along the restoration interface. secondary caries the restoration is a continuation of the existing anatomical shape adjacent to the restoration or is slightly flattened (absence of secondary caries). there is visual evidence of discoloration remaining dark next to the restoration (presence of secondary caries). 4 santos et al. statistical analysis data normality was verified by shapiro-wilk test. frequency of failure in different classes of restorations and different time intervals were expressed in absolute numbers and relative frequencies. the frequency differences were determined by chi-square test with yates’s correction or fisher exact test at a significance level of 5%. graphpad prism statistical software was used. results 498 rc direct restorations (anterior and posterior) were evaluated in 120 patients with mean age of 54 (±13.6) years-old, being 81 (67.5%) females and 39 (32.5%) males. class iv restorations was the more prevalent type, representing 137 of the 498 restorations (27.5%) (table 2). continue continuation anatomic form the restoration is a continuation of the existing anatomical shape or is slightly flattened. it can be bypassed and when the exploratory probe side is placed tangentially through the restoration, it must touch both sides, i.e. the tooth surface and the restoration at the same time. the surface concavity is evident. when the exploratory probe side is placed tangentially on the entire restoration, it does not touch two opposite sides of the cavus angle at the same time, but the dentin or base is not exposed. poor contact point, cervical excess. there is a loss of restorative substance such that a concavity is evident and the base or dentin is exposed marginal adaptation the probe does not grip when drawn over the restoration surface toward the tooth, there is no visible slit, or that threads the exploratory probe, along the restoration periphery. there is visible evidence of a slit, which penetrates the exploratory probe, indicating that the restoration edge does not closely adapt to the tooth structure. the dentin and base are not exposed and the restoration is not movable. the probe penetrates an extended gap to the dentin-enamel junction. surface texture the surface texture similar to the polish enamel. surface with a sandy texture or similar to a white stone surface or similar to a macroparticulated resin composite. surface is thick enough to inhibit the continuous circulation of an exploratory probe over the entire surface. probe does not slide. fracture restoration is intact and fully placed. restoration is partially placed with some portion of the restoration still intact. restoration completely absent. 5 santos et al. ta bl e 2. f re qu en cy o f f ai lu re a cc or di ng to th e r yg e cr ite ria in d iff er en t t yp es o f c av iti es o f r c re st or at io ns . c la ss i (n =5 2) c la ss ii (n =1 09 ) c la ss ii i ( n= 10 4) c la ss iv (n = 13 7) c la ss v (n =9 6) to ta l (n =4 98 ) c ri te ri a s co re n (% ) n (% ) p* n (% ) p* n (% ) p* n (% ) p* n (% ) c ol or m at ch a 42 (8 0. 7) 68 (6 8. 0) >0 .0 5 72 (7 3. 5) >0 .0 5 96 (7 9. 3) >0 .0 5 46 (6 7. 6) >0 .0 5 32 4 (7 3. 8) b 09 (1 7. 3) 28 (2 8. 0) 22 (2 2. 4) 22 (1 8. 2) 20 (2 9. 4) 10 1 (2 3. 0) c 01 (0 1. 9) 04 (4 .0 ) 04 (0 4. 1) 03 (0 2. 5) 02 (0 2. 9) 14 (0 3. 2) m ar gi na l di sc ol or at io n a 25 (4 8. 1) 33 (3 3. 0) >0 .0 5 42 (4 2. 9) >0 .0 5 67 (5 5. 3) >0 .0 5 45 (6 5. 2) >0 .0 5 21 2 (4 8. 2) b 24 (4 6. 1) 63 (6 3. 0) 48 (4 8. 9) 51 (4 2. 1) 20 (2 8. 9) 20 6 (4 6. 8) c 03 (5 .7 ) 04 (4 .0 ) 08 (0 8. 2) 03 (0 2. 5) 04 (0 5. 8) 22 (0 5. 0) se co nd ar y ca rie s a 44 (8 4. 6) 53 (5 3. 0) <0 .0 00 1i * 66 (6 6. 7) <0 .0 2i * >0 .0 5i i 10 0 (8 2. 6) >0 .0 5i <0 .0 00 1i i * >0 .0 5i ii 57 (8 2. 6) >0 .0 5i <0 .0 00 1i i * <0 .0 2i ii * >0 .0 5i v 32 0 (7 2. 6) b 08 (1 5. 3) 47 (4 7. 0) 33 (3 3. 3) 21 (1 7. 4) 12 (1 7. 4) 12 1 (2 7. 4) a na to m ic fo rm a 27 (5 1. 9) 35 (3 5. 3) >0 .0 5i 55 (5 6. 1) >0 .0 5i <0 .0 2i i * 60 (4 9. 6) >0 .0 5i <0 .0 2i i * >0 .0 5i ii 32 (4 6. 4) >0 .0 5i >0 .0 5i i <0 .0 1i ii * >0 .0 5i v 20 9 (4 7. 6) b 24 (4 6. 1) 55 (5 5. 6) 43 (4 3. 9) 58 (4 7. 9) 32 (4 6. 4) 21 2 (4 8. 3) c 01 (0 1. 9) 09 (0 9. 1) 03 (0 2. 5) 05 (0 7. 2) 18 (0 4. 1) m ar gi na l ad ap ta tio n a 28 (5 3. 8) 32 (3 2. 0) <0 .0 2i * 51 (5 2. 0) >0 .0 5i <0 .0 1i i * 73 (6 0. 3) >0 .0 5i <0 .0 00 1i i * >0 .0 5i ii 40 (5 7. 9) >0 .0 5i <0 .0 03 ii * >0 .0 5i ii >0 .0 5i v 22 4 (5 0. 9) b 22 (4 2. 3) 58 (5 8. 0) 38 (3 8. 8) 40 (3 3. 1) 26 (3 7. 7) 18 4 (4 1. 8) c 02 (0 3. 8) 10 (1 0. 0) 09 (0 9. 2) 08 (6 .6 ) 03 (0 4. 3) 32 (0 7. 3) su rf ac e te xt ur e a 23 (4 4. 2) 29 (2 9. 0) >0 .0 5 34 (3 4. 7) >0 .0 5 45 (3 7. 2) >0 .0 5 27 (3 9. 1) >0 .0 5 15 8 (3 5. 9) b 24 (4 6. 1) 53 (5 3. 0) 53 (5 4. 1) 59 (4 8. 8) 33 (4 7. 8) 22 2 (5 0. 4) c 05 (0 9. 6) 18 (1 8. 0) 11 (1 1. 2) 17 (1 4. 0) 09 (1 3. 0) 60 (1 3. 6) fr ac tu re a 43 (8 2. 7) 87 (7 9. 8) >0 .0 5i 92 (8 8. 5) >0 .0 5i >0 .0 5i i 98 (7 1. 5) <0 .0 3i * >0 .0 5i i >0 .0 5i ii 61 (6 4. 2) <0 .0 00 1i * <0 .0 00 7i i * <0 .0 00 1i ii * 0. 00 1i v * 38 1 (7 6. 6) b 09 (1 7. 3) 13 (1 1. 9) 06 (5 .8 ) 23 (1 6. 8) 07 (0 7. 4) 58 (1 1. 7) c 09 (0 8. 0) 06 (0 5. 8) 16 (1 1. 7) 27 (2 8. 4) 58 (1 1. 7) to ta l 52 (1 0. 5) 10 9 (2 1. 9) 10 4 (2 0. 8) 13 7 (2 7. 5) 96 (1 9. 3) 49 8 (1 00 ) c hi -s qu ar e te st . d iff er en ce s w er e si gn ifi ca nt w he n p < 0 .0 5 (* ). i cl as s i; ii c la ss ii ; i ii c la ss ii i; iv c la ss iv ; v c la ss v . 6 santos et al. secondary caries was the main reason for failure in class ii (47%) and iii (33.3%) restorations. the same rc restorations (class ii and iii) exhibited marginal adaptation failure (10 and 9.2% respectively). regarding anatomic form, class ii failed more frequently (9.1%), while class iii did not exhibit any failure. color matching, marginal discoloration, and surface texture were the less affected criteria without differences between the cavity types (p > 0.05) (table 2). class v restorations showed the highest fracture rate (28.4%) (table 1), but without increase between the time intervals (table 3). table 3. frequency of failure of rc restorations according to the ryge criteria in relation to the time of placement. time intervals criteria score (1–3 years) (3.1–6 years) (6.1–9 years) (9.1–12 years) (12.1–15 years) n (%) p* n (%) p* n (%) p* n (%) p* n (%) p* color match a 106 (67.0) 0.05 151 (76.2) >0.05 35 (87.5) 16 (64.0) >0.05 10 (83.3) >0.05 b 45 (28.4) 41 (20.7) 03 (7.5) >0.05 09 (36.0) 02 (16.6) c 07 (04.4) 06 (03.0) 02 (5.0) total 158 (100) 198 (100) 40 (100) 25 (100) 12 (100) marginal discoloration a 78 (49.3) >0.05 93 (46.90 >0.05 20 (50.00 >0.05 07 (28.0) >0.05 10 (83.3) >0.05 b 70 (44.3) 96 (48.4) 18 (45.0) 18 (72.0) 02 (16.6) c 10 (06.3) 09 (04.5) 02 (05.0) total 158 (100) 198 (100) 40 (100) 25 (100) 12 (100) secondary caries a 122 (77.2) 131 (66.1) <0.02a * 30 (75.0) >0.05a >0.05b 20 (80.0) >0.05a >0.05b >0.05c 12 (100) >0.05a <0.01b* >0.05c >0.05d b 36 (22.7) 67 (33.8) 10 (25.0) 05 (20.0) total 158 (100) 198 (100) 40 (100) 25 (100) 12 (100) anatomic form a 80 (50.6) >0.05 92 (46.4) >0.05 18 (45.0) >0.05 09 (36.0) >0.05 09 (75.0) >0.05 b 69 (43.6) 97 (48.9) 20 (50.0) 16 (64.0) 03 (25.0) c 09 (05.7) 09 (04.5) 02 (05.0) total 158 (100) 198 (100) 40 (100) 25 (100) 12 (100) marginal adaptation a 81 (51.2) >0.05 96 (48.4) >0.05 24 (60.0) >0.05 11 (44.0) >0.05 09 (75.0) >0.05 b 62 (39.2) 89 (44.9) 13 (32.5) 12 (48.0) 03 (25.0) c 15 (09.4) 13 (06.5) 03 (07.5) 02 (08.0) total 158 (100) 198 (100) 40 (100) 25 (100) 12 (100) continue 7 santos et al. color matching and marginal discoloration criteria did not show significant changes on frequency over time. however, the surface texture experienced more failures after the second time interval (3.1 to 6 years) (table 3). higher occurrence of secondary caries was observed in the second time interval regardless of the cavity type (p < 0.02). for surface texture, more failures were observed in the second, thirdand fifth-time intervals (p < 0.01). no differences between the interval times were verified for color matching, marginal discoloration, anatomic form, marginal adaptation, and fracture (table 3). discussion to evaluate the clinical performance of rc restorations placed by dental students, retrospective studies can display the clinical reality more accurately than prospective cross-sectional studies conducted by calibrated operators with selected patients6,18. herein, the main outcome was that class ii was the most failed restoration, due to the number of restored surfaces1,4,10. furthermore, secondary caries was the most prevalent reason for failure, as opdam et al.5 (2014) reported. however, moura et al.15 (2011) evaluated rc restorations placed by dental students for no longer than 3 years and have found that marginal adaptation was the main reason for failure due to negligence during adhesive procedures. in clinical short-term evaluations with a limited number of rc restorations placed in patients with low risk of caries, it is quite difficult to observe differences in the type of restorative materials used6. however, our study showed a higher failure rate by secondary caries occurring after 3 years of placement. especially in class ii restorations, the lack of clinical skills by dental students can be highlighted in: the use of rc in extensive cavities with unfavorable cervical limit; the isolation of the operatory field; the application of adhesive systems to dentin; the use continuation surface texture a 38 (24.0) 84 (42.4) <0.001a * 19 (47.5) <0.01a * >0.05b 8 (32.0) >0.05a >0.05b >0.05c 08 (66.6) <0.005a* >0.05b >0.05c >0.05d b 96 (60.7) 90 (45.4) 17 (42.5) 13 (52.0) 03 (25.0) c 24 (15.1) 24 (12.1) 04 (10.0) 04 (16.0) 01 (08.3) total 158 (100) 198 (100) 40 (100) 25 (100) 12 (100) fracture a 141 (78.7) >0.05 167 (75.5) >0.05 33 (67.3) >0.05 21 (77.7) >0.05 12 (85.7) >0.05 b 16 (8.9) 31 (14.0) 07 (14.2) 04 (14.8) c 22 (12.2) 23 (10.4) 09 (18.3) 02 (07.4) 02 (14.2) total 179 (100) 221 (100) 49 (100) 27 (100) 14 (100) chi-square test. differences were significant when p<0.05 (*). a time interval of 1-3 years; b time interval of 3.1-6 years; c time interval of 6.1-9 years; d time interval of 9.1-12 years; e time interval of 12.1-15 years. 8 santos et al. of matrix band systems and wood wedges; and the correct insertion and light-curing of rc increments. failures during these clinical steps may result in reduced marginal adaptation and gap formation on the cervical interproximal region14,19. in this study, for anterior rc restorations, class iii and iv showed high rates of secondary caries, differently than heintze et al.2 (2015) has found. secondary caries occurs by the invasion of cariogenic microorganisms within the tooth/restoration interface due to adhesive failure20. taking into account that we evaluated rc restorations placed by dental students in different levels of knowledge and clinical skills, some factors may be attributed to the high prevalence of secondary caries, such as inadequate use of rc in extensive cavities with high occlusal loads and little or any enamel in the cervical cavo-superficial angle; negligence in adhesive system application, resulting in material’s degradation; inefficient light-curing by inadequate irradiance and time of light exposure; and the inherent polymerization shrinkage and stress15,21. adhesive failure can compromise the marginal adaptation and lead to total or partial fracture of the restoration without the occurrence of secondary caries15. the prevalence of restoration displacement was higher in class v cavities, followed by class iv. these results corroborate with previous studies2,5 and show similar failure rates compared to other rc restorations placed by dental students14,15. cervical lesions are normally non-carious and showed hypermineralized, less-permeable, and acid-resistant dentin22. these features associated with the cavity type, polymerization shrinkage, quality and amount of enamel in the cervical cavo-superficial angle, difficulty in achieving adequate isolation of the operatory field may be contributed to high rates of adhesive failure and displacement of class v restorations22. however, the fracture of class iv restorations can be mainly associated with failure or absence of occlusal adjustment since the incisal angle region suffers from tensions not observed in any other type of cavities15. color matching was the criteria that showed the less failure rate, followed by surface texture, which is in accordance with previous studies23,24. these results highlighted that in this clinical step of the restorative technique, the dental students perform the finishing and polishing procedures satisfactorily. clinically, adequate finishing and polishing procedures may reduce biofilm accumulation on the resin surface, ensure the maintenance of marginal adaptation, the control of marginal discoloration, and may reduce the occurrence of secondary caries consequently23. from the second interval time, there is an increase of failure due to surface texture. in this context, the oral environment provides critical and challenging conditions to resin-based materials, such as humidity and ph changes24. this scenario may result in degradation of the polymeric matrix25, which alters the surface texture of the rc and contributes to surface wear, plaque accumulation, and less survival rates of the restoration25. we emphasize that dental students are constantly improving their competencies and skills over the dentistry course6,10,14. thus, the relative lack of clinical skills and the subjectivity of diagnosis may be contributed to the results obtained. in this way, periodical evaluation of the teaching practice is crucial to discuss and establish novel 9 santos et al. improvement strategies. additionally, further clinical studies evaluating the performance of rc restorations placed by dental students are needed. conclusion within the limitations of this study, a high rate of failure in rc restorations placed by dental students was verified. this outcome can be attributed to their lack of expertise and clinical skills. we encourage the constant revision and updating of the teaching process in order to enhance the development of clinical skills by dental students. acknowledgment this study was financed in part by the coordination for the improvement of higher education personnel (capes) – finance code 001. conflict of interest none. data availability datasets related to this article will be available upon request to the corresponding author. references 1. da rosa rodolpho pa, cenci ms, donassollo ta, loguercio ad, demarco ff. a clinical evaluation of posterior composite restorations: 17-year findings. j dent. 2006 aug;34(7):427-35. doi: 10.1016/j.jdent.2005.09.006. 2. heintze sd, rousson v, hickel r. clinical effectiveness of direct anterior restorations a meta-analysis. dent mater. 2015 may;31(5):481-95. doi: 10.1016/j.dental.2015.01.015. 3. moraschini v, kafai c, monte alto r, santos go. amalgam and resin composite longevity of posterior restorations: a systematic review and meta-analysis. j dent. 2015 sep;43(9):1043-50. 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doi: 10.1016/s0142-9612(02)00617-8. untitled 1http://dx.doi.org/10.20396/bjos.v17i0.8651901 volume 17 2018 e18021 original article 1 phd, msc, dds, department of clinical dentistry, school of dentistry, federal university of bahia, salvador, bahia, brazil 2 dds, department of clinical dentistry, school of dentistry, federal university of bahia, salvador, bahia, brazil 3 msc, dds, department of dentistry and health, school of dentistry, federal university of bahia, salvador, bahia, brazil 4 msc, dds, department of restorative dentistry, piracicaba dental school, state university of campinas, piracicaba, são paulo, brazil corresponding author: paula mathias av. araújo pinho, 62 canela, salvador ba, 40100-150. phone +55 71 3283-8986 e-mail: pmathias@yahoo.com; caroline.mathias@hotmail.com received: september 11, 2017 accepted: november 27, 2017 effect of air-polishing on properties of nanocomposite submitted to coffee, red wine and cigarette smoke paula mathias1; thayz mota de souza cunha2; isadora almeida rios rocha2; lívia andrade vitória3; caroline mathias4; andrea nóbrega cavalcanti1. abstract aim: the aim of this study was to evaluate the surface roughness and the color stability of nanocomposite exposed to the sodium bicarbonate air-polishing (sbap) followed by red wine, coffee and cigarette smoke exposure. materials and methods: 64 nanocomposite specimens were prepared and allocated in 8 groups: g1 (sbap + distilled water), g2 (sbap + coffee), g3 (sbap + red wine), g4 (sbap + cigarette smoke), g5 (distilled water), g6 (coffee), g7 (red wine) and g8 (cigarette smoke). the surface roughness was evaluated in three periods: before and after sbap and after exposuring to agents tested. the color was evaluated according to ciel*a*b* parameters using reflection spectrophotometer in two moments: initial and 30 days after the exposure to staining agents. data were subjected to three-way repeated measures anova and tukey’s test (5%). results: the results showed a higher surface roughness of the nanocomposite submitted only to the sbap and those exposed to the sbap followed by exposure to the coffee or wine solution. the previous application of sbap followed by cigarette smoke exposure did not increase the roughness of nanocomposite. the sbap procedure just increased the staining for cigarette smoke group. conclusion: the sbap increases resin surface roughness, which worsens when there is exposure to coffee and red wine solution. in addition, sbap may also provide increased staining of nanocomposites exposed to cigarette smoke. keywords: composite resins. dental materials. beverages. smoke. mailto:pmathias@yahoo.com 2 mathias et al. introduction the composite resins are widely used in restorative dentistry due to the evolution of aesthetic and mechanical properties, as well as the simplified clinical protocols compared to ceramic restorations1–3. the development of the chemical composition, inorganic particles distribution and size have provided better physical and mechanical properties, resulting in less surface roughness and better color stability against staining1,4. the nanoparticulate resin composite is composed of nanomer and nanoclusters and it is used on posterior and anterior teeth as universal resin composite by clinicians due to the characteristics that achieve good properties and better aesthetic2,5. the wear resistance of nanocomposites is related as comparable to or superior to that of microfill and microhybrid resin composites1,5. overall, the surface quality of the restorative material influences the clinical performance and durability of restorations5, since the maintenance of flat surface decreases early alterations of color and shine, besides of reducing the biofilm growth on surface restoration, which decreases risk of secondary caries lesions and periodontal inflammation4–9. the roughness present on resin surface can be detected by the tip of the patient’s tongue since 0.3µm10. these irregularities are directly associated to the characteristics of the material, as well as the action of instruments used on it4, besides several kinds of finishing and polishing procedures and follow-up sessions of aesthetic restorations, that include the use of air-polishing powders3. a regular and efficient practice in professional dental prophylaxis is the use of sodium bicarbonate air-polishing (sbap)1,11. this system releases air jet, water and sodium bicarbonate particles that have the size by 250µm, and they may cause loss of resin surface smoothness, therefore they favor the staining and degradation of restorations10,12. on the other hand, studies have reported that the abrasiveness from air-polishing can be used as strategy to reduce dental and material surface staining11,12, due to the ability of sbap to remove waste substances containing dyes from food, drinks and cigarette smoke13,14. the advantages of air-polishing compared to the conventional rubber cup prophylaxis are the fast removal of tooth deposits, less hypersensitivity, lower operator fatigue and better access to pits and fissures3,11,15. furthermore, the sodium bicarbonate particles are less abrasive than particles contained in commercially available polishing pastes or pumice16. in spite of the air-polishing has not been developed to direct use on aesthetic restorative materials and it be even condemned by some researchers11,16, it is observed regularly its applications on clinical conditions. besides, resin composite is a restorative material mightily influenced by oral environment conditions, as moisture, the contact with low ph substances, contact with alcohol or other solvents and even changes of temperature17,18.these factors can be more harmful to the resin composite structure because it shows a more degraded or porous surface6. 3 mathias et al. the resin extrinsic staining occurs due to adsorption of dyes by the composite resin matrix, leading to changes on its surface17. therefore, the exposure of composite resin restorations to individuals habits such as the consumption of coffee, alcoholic beverages, such as red wine and the smoking are determinant for their color stability19. therefore, it becomes important to evaluate the effects of sbap on roughness surface and color stability of resin composite and if the air-polishing is able to intensify the resin surface roughness and staining, when associated to substances as coffee, red wine and cigarette smoke, agents often used by patients. thus, the null hypothesis tested was the sbap is not able to intensify the surface roughness and staining of nanocomposite not even when it is exposed to coffee, red wine and cigarette smoke. materials and methods specimen preparation sixty-four disc-shaped nanocomposite specimens (filtek z350 xt, 3m espe, st paul, mn, eua) were made on a metalic mold (6x1.5mm) in single increment and photocured using led (radii, sdi, 1200mw/cm2, bayswater, victoria, australia) through a polyester matrix strip, for 40 seconds. the light intensity of led was measured before the photoactivation using a radiometer (rd-7, ecel, ribeirão preto, são paulo, brazil). then, the specimens were identified and stored in 3ml of distilled water at 37ºc for 24h. after this period, the specimens were planned and sanded with sandpapers with granulation of 2000, 1200 and 600 on metallographic polymeter (aropol – 2v, arotec, cotia, são paulo, brazil), and they were submitted to ultrasonic bath with distilled water for 2 minutes. posteriorly, the specimens were randomly allocated in 8 groups (n=8) according to the surface treatment and the exposure to agents (table 1).the roughness analysis was performed in three different periods: initial records, after sodium bicarbonate air-polishing and after the exposure to staining agents (experimental groups) or distilled water (control group). moreover, the specimens were analyzed by spectrophotometer to measure color parameters, in two times: initial and after 30 days of exposure to staining agents. table 1. groups according to the surface treatment and the staining agents. sodium bicarbonate air polishing exposure agents groups yes distilled water (control) g1 coffee g2 red wine g3 cigarette smoke g4 no distilled water (control) g5 coffee g6 red wine g7 cigarette smoke g8 4 mathias et al. surface roughness measurements all the specimens were analyzed for surface roughness, for initial records (time 1) using a rugosimeter (surftest 3000, mitutoyo sul, americana, são paulo, brazil). the value considered was the arithmetic mean from peaks and valleys (ra), recorded at 1.25mm and 0.1mm/s. the mean value obtained was the result of 3 measurements for each specimen made in different positions to cover the analyzed surface. after the initial evaluation (1), the roughness was again measured after sodium bicarbonate air-polishing (2) and after the exposure to the staining agents (3). color measurements the color measurements were performed initially and after the period of exposure to the agent,using a reflection spectrophotometer (uv-2600; shimadzu, tokyo, japan) and the software uv probe, where reflectance spectra were obtained from the specimens. the spectral curves obtained from the reading of each test specimen were transported to the software color analysis for color evaluation following the parameters of the ciel * a * b * system (commission internationale de l’eclairage), with standardization of the illuminant d65. the coordinate values l* (lightness; 0 = black/100 = white), a* (green [negative]/red [positive]), and b* (blue [negative]/yellow [positive]) were measured at baseline and after 30 days of exposure to staining agents. the l * (brightness), a * (green-red variation) and b * (blue-yellow variation) parameters were analyzed separately and the respective values were used to calculate the total color variation (δe), applying the formula : δe = √ (l-l0) ² + (a-a0) ² + (b-b0) ². sodium bicarbonate air-polishing the sbap procedure was performed from g1 to g4 using a device for professional prophylaxis (profi ceramic ii, dabi atlante, ribeirão preto, sp, brazil) for 30 seconds, distance of 15mm of the specimen and pressure of 60psi. the sodium bicarbonate powder was composed by: nahco3 (99.35%)/ chloride (cl 0.003%)/ phosphate (po4 0.001%)/ sulfate (so4 – 0.003%)/ ammonium (nh4 – 0.001%)/ iron (fe – 0.001%)/ potassium (k – 0.02%)/ precipitate (ca/ mg/ p2o3 – 0.02%). after the air-polishing, the specimens were submitted to ultrasonic bath with distilled water for 2 minutes to remove particles that may have impregnated on resin surface. exposure to staining agents the composite resin specimens were immersed in red wine (santa ana seleccion – 12.5% alcoholic gradient, mendonza, argentina) and in coffee solution (maratá traditional, itaporanga d’ajuda, sergipe, brazil) for 3 minutes, twice a day for 30 days. during this period the specimens that were not exposed to the staining agents were stored on distilled water at 37ºc, as well as the specimens of control group. the cigarette used in the present study contained 10mg of tar (malboro, philip morris international, brazil) and the method used was a simulation of frequent cigarette smoking in an acrylic box contained 2 cameras interconnected by holes. the lit cigarettes were allocated in the first camera and the air was pumped up to the second camera, where the specimens were kept. the specimens were exposed to the smoke 5 mathias et al. of 20 cigarettes for day (10 cigarettes for 8 minutes, twice a day), during 30 days. in the intervals between cigarettes exposures the specimens were stored in distilled water at 37ºc. statistical analysis data were submitted to 3-way repeated measures anova and tukey test for surface roughness and 2-way repeated anova and tukey test for δe data. the statistical procedures were performed using sas 9.1 (sas institute, cary, nc, usa) at a significance level of 5%. the power obtained with the sample size in this analysis was higher than 80%. results surface roughness the mean values and statistical comparisons for surface roughness analysis are shown in table 2. according to the analysis, there was statistical significance in the triple interaction between the main factors (p< 0.0001). when comparing the exposure agents in time vs. air-polishing levels, the differences were observed only in the time 3 both in the presence and in absence of the sbap. in these experimental conditions, the control group presented mean values significantly different from experimental groups exposed to different agents, these values were higher in the presence of sodium bicarbonate air-polishing and lower in the absence of this one. regarding the differences of time in exposure agents vs. air-polishing levels, both in the presence and absence of air-polishing, higher means were found in the time 3 and lower means in the time 1, independent of exposure agent. however, in the absence of air-polishing and in the control group no differences between the times were found. table 2. surface roughness means (standard deviations) according to the groups and different times. sodium bicarbonate air polishing exposure agents time 1 2 3 yes control 0.362(0.058) ab 0.654 (0.418) ab 1.074 (0.373) aa coffee 0.402(0.105) ac 0.721 (0.373) ab 0.896 (0.337) ba red wine 0.432(0.107) ac 0.746 (0.174) ab 0.907 (0.144) ba cigarette smoke 0.339(0.060) ac 0.759 (0.150) ab 0.888 (0.147) ba no control 0.376(0.070) aa 0.358 (0.069) aa* 0.350 (0.121) ba* coffee 0.369(0.087) ab 0.382 (0.042) ab* 0.565 (0.231) aa* red wine 0.376(0.068) ab 0.384 (0.050) ab* 0.591 (0.150) aa* cigarette smoke 0.384(0.065) ab 0.391 (0.041) ab* 0.871 (0.103) aa means followed by different letters are statistical significance (3-way repeated measures anova/ tukey p<0.0001). uppercase letters compare staining agents in time vs. air polishing levels. lowercase letters compare times in air polishing vs. staining agents levels. asterisks represent differences between the use of air polishing in staining agents vs. time levels. 6 mathias et al. lastly, the differences between the use of air-polishing in exposure agents vs. time were found only in the times 2 and 3, for all exposure agents tested, except for cigarette smoke group in the time 3. color analysis the statistical analysis of the data obtained in the δe variable indicated a significant interaction between the main factors (p<0.0001). according to table 3, the only statistical difference between the use of sbap or no sbap was in cigarette groups, where the sbap increased the staining. for other solutions (red wine and coffee) and in the control group, no difference was observed between using or not sbap. for sbap groups, the exposure to colouring agents increased the staining if compared to control group, but there were no differences between the agents used. the same result could be seen in the groups without sbap, where the staining values were higher in the groups exposed to pigment agents than in the control group, but without differences between them. discussion according to this study, the use of sbap increased the composite resin surface roughness and also intensified the roughness when the resin was submitted to other substances as coffee and red wine. moreover, the sbap just increased staining for cigarette group. thus, the null hypothesis of this study was partially rejected. in agreement with other studies3,20–23, the sbap was able to increase the surface roughness even in a nanoparticulate composite.the abrasive jet action, which contains sodium bicarbonate particles, on the restorative material surface was demonstrated by comparison of the groups with and without sbap at time 2 (after the sbap jet application). the superficial abrasion of a composite resin leads to the loss of surface inorganic particles, increasing the roughness and it interferes in the reflective ability of the material20. thereby, the sbap is able to remove surface inorganic particles, due to the large particle size of sodium bicarbonate, exhibiting the organic matrix to degradation process, which increases the water sorption, solubility and hydrolysis22. this superficial degradation can result in microcracks that allow penetration of substances and dyes, further increasing the staining20. besides the sbap’s abrasive effect on composite resin surface, other substances are able to alter the resin matrix, elevating the superficial roughness and thus the degradation of the material24–26. the exposure to coffee increased the resin surface roughness and staintable 3. δe means (standard deviations) according to the groups. staining control coffee red wine cigarette smoke sbap 4.66 (1.24) ab 32.65 (3.19) aa 30.13 (2.69) aa 28.94 (1.1) aa no sbap 5.74 (0.63) ab 33.24 (3.88) aa 31.50 (2.80) aa 26.82 (1.21) ba means followed by different letters are statistical significance (2-way repeated measures anova/ tukey test, p<0.0001). uppercase letters compare the air polishing levels vs. each staining agents. lowercase letters compare the surface vs. staining agents levels. 7 mathias et al. ing if compared to control group, independently of the association to sbap. this fact can be due to coffee’s acid ph, around 5.0, as well as the presence of long-chain organic acids, that they can promote dissolution and corrosion on restorative materials6.it is reported that the coffee also has high temperature, as used in this experiment, that it can promote degradation of the resin matrix, since areas of material exposed to high temperatures exhibited considerable increased of roughness and superficial staining27. the prior use of sbap followed by immersion of the composite resin in coffee was able to significantly intensify surface roughness when compared to the roughness found in the resins only exposed to the coffee. this increasing can be due to the resin surface previously air polished has more pores and then, resulting in larger areas that remain in contact with coffee. therefore, this greater contact can intensify the harmful effects of coffee on organic and inorganic matrix of resin, affecting its surface roughness. the alcohol contained in the red wine can degrade chemically the resin composite surface, by softening of its organic matrix or by hydrolyzing the silane coupling agent, thus it being able to remove filler particles8,28,29. although the inorganic particles protect the resins from deeper decomposition, the surface of material is more exposed and thus, compromised by the presence of microcracks in the interface between the inorganic particles and organic matrix26. this micromorphological change of resin surface can explain the surface roughness and the greater staining presented by the nanocomposite in this study, when immersed in red wine, twice a day during 30 days. the sbap applied prior to immersion in the red wine further increased the surface roughness of the material tested. it can be explained by the creation of porosities by the sbap in the composite resin, increasing the surface area that remains in contact with the wine. this contact can intensify the deleterious potential of red wine on the resin, acting as a plasticizer of the polymer matrix29. in this study, the exposure to cigarette smoke was also able to increase the surface roughness and staining of composite resin, in agreement with other studies that have shown the smoke can influence on chemical and mechanical properties of resinous materials13,17,25,30.the surface roughness showed by the composite resin exposed to cigarette smoke did not differ from other groups, regardless of the presence of sbap. the roughness changes of resin exposed to smoke can be attributed to combustion process of organic matter present in tobacco, resulting in presence of carbon monoxide and carbon dioxide, among other harmful substances besides the increase of temperature13,31. furthermore, an acidic ph solution can be produced by mixing water contained in saliva and cigarette components as carbon dioxide, promoting damage to resin surface integrity32.these factors can compromise the organic matrix stability, causing superficial degradation of material. however, there was no statistical difference between the roughness values of resin exposed to cigarette smoke submitted or not to sbap. it can be explained by the excessive deposits of cigarette smoke components, that remained adhered to resinous surface17,25and it may have occluded the pores previously created by the sbap, contributing for a more regular final surface of composite resin. it can be emphasized by the cigarette group have been the only group that showed increased staining after sbap procedure. 8 mathias et al. the high roughness found in the sbap group followed by exposure to cigarette smoke can result in sites that favor the deposition of particles containing brown pigments, as nicotine and tobacco-specific nitrosamines ranging from 0.1 to 1.0 𝜇m in diameter, on resin surface, altering significantly the color of composite13,30. on the other hand, the previous use of sbap did not alter the color of other groups tested. the remaining of the sodium bicarbonate could be able to prevent the detection of a staining, maybe due to the white coloration of the sodium bicarbonate powder. the professional prophylaxis using sbap was able to increase the roughness surface of the nanoparticulate resin tested and it worsen the action of other agents, as coffee and red wine. although in the present study sbap did not raise the surface roughness values for composite resin exposed to cigarette smoke, the sbap was able to increase the staining potential of the cigarette smoke. this result may emphasize the recommendation to perform the re-polishing procedures in composite restorations after they are submitted to sbap procedure3.the sbap can compromise the aesthetic and longevity of restorations, since the color stability also depends of the surface roughness and it can allow greater biofilm retention on these surfaces9. considering that resins are in constant contact with several substances, ph and temperature variations in the oral environment, studies evaluating the effects of air-polishing in restorations for a long period should be performed. based on the results obtained in this study, it can be concluded that: 1. the sodium bicarbonate air-polishing is able to increase the nanoparticulate resin surface roughness; 2. the coffee, red wine and cigarette smoke increase the surface roughness and staining of the resin tested if compared to control group; 3. the previous use of sbap intensifies the effects of immersion in coffee and red wine increasing the surface roughness of nanoparticulate resin; 4. the previous use of sbap followed by cigarette smoke exposure does not affect the surface roughness of the material, when compared to the resin surface roughness exposed only to cigarette, but it increases the staining. acknowledgments the authors acknowledge the pibic-ufba program from national council for scientific and technological development (cnpq) and bahia research foundation (fapesb) agencies for its funding. references 1. arhun n, celik c, yamanel k. clinical evaluation of resin-based composites in posterior restorations: two-year results. oper dent. 2010 jul-aug;35(4):397-404. doi: 10.2341/09-345-c. 2. gönülol n, yilmaz f. the effects of finishing and polishing techniques on surface roughness and color stability of nanocomposites. j dent. 2012 dec;40 suppl 2:e64-70. doi: 10.1016/j. jdent.2012.07.005. 3. güler au, duran i, yücel aç, özkan p. effects of air polishing powders on the surface roughness of composite resins. j dent sci. 2010 sep;5(3):136-43. doi: 10.1016/s1991-7902(10)60020-7. 9 mathias et al. 4. da costa j, goncalves f, ferracane j. comparison of two-step versus four-step composite finishing/polishing disc systems: evaluation of a new two-step composite polishing disc system. 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falkensammer f, arnetzl gv, wildburger a, freudenthaler j. color stability of different composite resin materials. j prosthet dent. 2013 jun;109(6):378-83. doi: 10.1016/s0022-3913(13)60323-6. 20. arabaci t, ciçek y, ozgöz m, canakçi v, canakçi cf, eltas a. the comparison of the effects of three types of piezoelectric ultrasonic tips and air polishing system on the filling materials: an in vitro study. int j dent hyg. 2007 nov;5(4):205-10. 10 mathias et al. 21. salerno m, giacomelli l, derchi g, patra n, diaspro a. atomic force microscopy in vitro study of surface roughness and fractal character of a dental restoration composite after air-polishing. biomed eng online. 2010 oct 12;9:59. doi: 10.1186/1475-925x-9-59. 22. giacomelli l, salerno m, derchi g, genovesi a, paganin pp, covani u. effect of air polishing with glycine and bicarbonate powders on a nanocomposite used in dental restorations: an in vitro study. int j periodontics restorative dent. 2011 sep-oct;31(5):e51-6. 23. sturz 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of pyrolysis temperature on the mutagenicity of tobacco smoke condensate. food chem toxicol. 2001 may;39(5):499-505. 32. mahross hz, mohamed md, hassan am, baroudi k. effect of cigarette smoke on surface roughness of different denture base materials. j clin diagn res. 2015 sep;9(9):zc39-42. doi: 10.7860/jcdr/2015/14580.6488.   oral sciences n3 original article braz j oral sci. july | september 2012 volume 11, number 3 preliminary investigation to achieve patency of mb2 canal in maxillary molars tauby de souza coutinho-filho1, eduardo diogo gurgel-filho2, francisco josé souza-filho3, emmanuel joão nogueira leal da silva4 1dds, msc, phd, professor, state university of rio de janeiro, endodontics department, rio de janeiro, rj, brazil 2dds, msc, phd, professor, university of fortaleza, department of endodontics, fortaleza, ce, brazil 3dds, msc, phd, professor, state university of campinas, endodontics division, piracicaba, sp, brazil 4dds, msc, phd, student, state university of campinas, endodontics division, piracicaba, sp, brazil correspondence to: tauby de souza coutinho-filho rua santo afonso 110, sala 707, tijuca, rio de janeiro, rj, brasil cep: 20511-170 phone: + 55 21 22347197 e-mail: taubycf@hotmail.com abstract aim: to establish a preliminary investigation about the possibility to achieve patency of second mesiobuccal canal (mb2) in mesiobuccal roots of maxillary molars. methods: three hundred and five first maxillary molars were examined with direct vision and with magnification. the root canal configurations were classified according to the possibility to achieve patency in mb2 canals. clearing technique was also performed to illustrate root canal anatomy and verify the presence of extra canals not identified with magnification. results: the prevalence of mb2 canals detected only with direct vision was 53.4% and the use of the surgical operating microscope increased rate detection to 90.7%. the clearing technique revealed the presence of mb2 canal in 12 more teeth (94.7%). in 49.1% of the localized mb2 canals, it was not possible to achieve patency. conclusions: the findings of the presented study revealed that it was only possible to achieve patency in 50.9% of the mb2 canals, showing that achieve patency in the mb2 canal is much more challenging than locating them. keywords: maxillary first molar, mesiobuccal second canal, operating microscope. introduction the success of endodontic treatment depends on the location, cleaning, shaping and sealing of the root canal system1. the knowledge of root canal system anatomy is essential to determine the success of endodontic therapy. anatomical studies are of great importance to aid the understanding of the internal morphology and anatomical variations of the root canal system2-4. the permanent maxillary first molar has one of the most complex root and canal anatomies. this morphology has been studied and it is accepted that the mesial root often has more than one canal1-2,5-6. in the literature, the mesiobuccal (mb) root of the maxillary first molar has generated more research and clinical investigation than any other root7. the incidence of second mesiobuccal canals (mb2) has been reported in a range of between 33% and 96%1-2,5-10. however, the clinical detection of mb2 in maxillary molars has been lower than that of laboratorybased reports and this canal could be detected in less than 40% of maxillary first molars11-12. the use of the surgical operating microscope (som) facilitates detection received for publication: february 28, 2012 accepted: may 17, 2012 braz j oral sci. 11(3):373-376 374374374374374 and negotiation of more mb2 canals in both in vitro and clinical studies13-15. a high incidence of mb2 canals was reported in the mesiobuccal roots of the first maxillary molars ex vivo, and it was also demonstrated that the use of the operating microscope increased the ability of the dental clinician to locate the mb213. although the literature shows a high incidence of mb2 canal, sempira & hartwell 16 reported that only 33% of maxillary first molars had a negotiable mb2 canal, as determined by use of som in vivo. the most important causes of non-negotiable mb2 canal location are diffuse calcification, pulp stones and debris 17. given this low incidence of negotiable mb2 canals, the aim of this study was to establish a preliminary investigation about the possibility to achieve of mb2 canals in mesiobuccal roots. material and methods a total of 305 extracted human maxillary molars that had been stored in distilled water were used in this study. the floor of the pulp chamber was then explored in order to locate the mb1 and mb2 and to verify the possibility to achieve patency in mb2 canals. initially the canals were located with direct vision. the canal was negotiated and confirmed by insertion of a size 08 and 10 k-file (dentsply-maillefer, ballaigues, switzerland). further efforts to locate canals were carried out under magnification using the som (x20, dfv, mc-m 1232; são paulo, sp, brazil) and the canals were negotiated with a size 08 and 10 k-file. after instrumentation a radiograph was performed. the root canal configuration in each root was determined by using a simplified classification of root canal configurations. type i being two separate and distinct canals from pulp chamber to the apex, type ii being two canals leaving the pulp chamber and merging to form a single canal short to the apex, and type iii being interrupted access with no canal or roots with just one canal. type i and type ii were classified as possible to achieve patency and type iii was the canal that was not accessible to the apex. after this, to illustrate root canal configuration, teeth were prepared using a clearing technique. teeth were immersed in indian ink (pelikan, são paulo, sp, brazil) under 600-mmhg vacuums for 40 min and then kept for 10 days in an incubator at 37ºc. a final wash with tap water to remove excess ink was performed. the teeth were demineralized with a 5% hydrochloric acid solution for 3 days, dehydrated with ascending grades of alcohol, immersed in methyl salicylate and rendered transparent18. results under direct vision, the mb2 canal was located in 163 teeth (53.4%). with the use of the som, the mb2 canal was located in 114 additional teeth, totalizing 277 teeth (90.7%). the clearing technique disclosed the presence of a mb2 canal in 12 more teeth, totalizing 289 teeth (94.7%), while 16 teeth had a single canal in the mesiobuccal root (5.3%). this prevalence can be seen in figure 1. a total of 45 canals were classified as type i canals (14.8%), 110 classified as type ii canals (36.1%), 150 as type iii canals (49.1%). these results can be seen in figure 2 and the images and examples of the classification are found in figure 3. patency was achieved in only 50.9% of the localized mb2 canals. discussion successful endodontic treatment demands adequate cleaning, shaping and filling of the root canal system. for this, clinicians must have comprehensive knowledge about root canal morphology. anatomical variations may be present in teeth subjected to endodontic treatment. in that perspective, the incidence of the mb2 canal in the mesiobuccal root of the maxillary first molar is always a matter of interest to the endodontic community13. failure to detect and treat the second mb2 canal system will result in a decreased longterm prognosis2. previous studies showed that the use of som increased detection of mb2 canals from 51 to 81%13,19-20. a total mb2 detection rate of 93.3% was observed when cone bean computed tomography (cbct) was used in first molars21. fig. 2. simplified classification of mb2 canals fig. 1. mb2 location in teeth in different stages. preliminary investigation to achieve patency of mb2 canal in maxillary molars braz j oral sci. 11(3):373-376 braz j oral sci. 11(3):373-376 fig. 3. examples of simplified classification. (1a) laboratory radiograph of a type i tooth; (1b) clearing of a type i tooth; (1c) clinical radiograph of a type i tooth; (2a) laboratory radiograph of a type ii tooth; (2b) clearing of a type ii tooth; (2c) clinical radiograph of a type ii tooth; (3a) laboratory radiograph of a type iii tooth; (3b) clearing of a type iii tooth; (3c) clinical radiograph of a type iii tooth; the findings of the present study observed that the prevalence of mb2 canals detected with direct vision was 53.4% and the association with the som increased rate detection to 90.8%. this can be justified by the fact that the higher magnification and illumination can be useful for access cavity preparation, instrumentation and filling22-23. it can improve the clinician’s view of the complexity of the root canal anatomy and aid in the location of additional canals, fins or ribbons. thus, the use of som to detect the ml canal orifice of maxillary first and second molars may enhance the success of endodontic procedures13. although there are several reports demonstrating the efficacy of the som in locating mb2 canals, sempira and hartwell16 reported that 33% of maxillary first molars had a negotiable mb2 canal as determined by use of som in vivo. in this study, the accessibility of located mb2 canals was evaluated. only 14.8% of the tooth had two separate and distinct canals from pulp chamber to the apex. in the remaining cases occurs the intersection of mb2 with the mb canal (36.1%) or its unexpected ending at 3 or 4 mm of entry and roots with just one canal (49.1%). this unexpected ending may be because of calcified mb2 canals that prevented the full instrumentation of the canal. previous research suggests that non-negotiable factors of mb2 canal location were diffuse calcification, pulp stone debris and tortuous pathway17,24-25. the mb2 canal can be challenging to treat because it might share an orifice with mb1 or can be harbored within, or just apical to, that of the mb1 canal. in cases of union with the mb canal, treatment is possible since it passes the entrance angle to the mb. clinically, it can be seen the impossibility of instrument penetration on both canals simultaneously. it becomes evident when a file is introduced into one canal and the other does not go down and vice versa. the most complex cases are those where the penetration of the files is stopped suddenly, usually at 3 or 4 mm from its entrance. in the present study, this type of canal was observed in 49.1% of maxillary first molars in mb2 canals. the patency was achieved in only 50.9% of the localized mb2 canals. this prevalence is much lower than the total number of mb2 canal orifices (94.7%). previous reports, have also shown that the accessibility of located mb2 canals orifices is much lower than the total number of these canals16,26. neaverth et al.27 suggested that a modified access preparation and countersinking techniques may help in the mb2 canals treatment. they also reported that with these techniques they were able to treat 77.2% of mb2 canals in maxillary first molars. however, even with these highs rates of treated canals, several mb2 canals could not be instrumented. this large number of canals left untreated in several studies demonstrates the need to emphasize de frequency and clinical significance of these canals. achieving patency of mb2 canal is much more challenging than locating them. in the endodontic treatment of first molars, the mb2 canal occurs on an average frequency of 94.7%. it should be possible to perform its treatment at the same frequency, but this does not occur. in the present study, the cleaning technique revealed that the complex anatomy and the tortuous pathway of the root canal system justify the difficulties in penetrating the entire length of the mb canal. based on the findings of the present study, it can be concluded that it was only possible to achieve patency in 50.9% on mb2 canals, showing that achieve patency in the mb2 canal is much more challenging than locating them. references 1. pattanshetti n, gaidhene m, kandari am. root canal morphology of the mesiobuccal and distal roots of permanent first molars in a kuwait populationa clinical study. int endod j. 2008; 41: 755-62. 2. weine fs, healey hj, gerstein h, evanson l. canal configuration in the mesiobuccal root of the maxillary first molar and its endodontic significance. oral surg oral med oral pathol. 1969; 28: 419-25. 3. vertucci fj. root canal anatomy of the human permanent teeth. oral surg oral med oral pathol. 1984; 58: 589-99. 4. friedman s. prognosis of initial endodontic therapy. endod top. 2002; 2: 59-88. 5. weng xl, yu sb, zhao sl, wang hg, mu t, tang rt, zhou xd. root canal morphology of permanent maxillary teeth in the han nationality in chinese guanzhong area: e new modified root canal staining technique. j endod. 2009; 35: 651-6. 6. blattner tc, george n, lee cc, kumar v, yelton cdj. efficacy of cone375375375375375preliminary investigation to achieve patency of mb2 canal in maxillary molars 376376376376376 beam computed tomography as a modality to accurately identify the presence of second mesiobuccal canals in maxillary first and second molars: a pilot study. j endod. 2010; 36: 867-70. 7. cleghorn b, christie w, dong ccs. root and root canal morphology of the human permanent maxillary first molar: a literature review. j endod. 2006; 32: 813-21. 8. kobayashi c, sunada i. root canal morphology and its possibility for penetration. part 3.maxillary molar. jap j cons dent. 1987; 30: 1674-83. 9. carvalho mcc, zuolo ml. orifice location with a microscope. j endod. 2000; 26: 532-4. 10. imura n, hata g, toda t, otani sm, fagundes mirc. two canals in mesiobuccal roots of maxillary molars. j endod. 1998; 38: 410-4. 11. weller rn, hartwell gr. the impact of improved access and searching techniques on detection of the mesiolingual canal in maxillary molars. j endod. 1989; 15: 82-3. 12. wasti f, shearer ac, wilson nh. root canal systems of the mandibular and maxillary first permanent molar teeth of south asian pakistanis. int endod j. 2001; 34: 263-6. 13. coutinho-filho t, cerda rsl, gurgel-filho ed, de-deus g, magalhães km. the influence of the surgical operating microscope in locating the mesiolingual canal orifice: a laboratory analysis. braz oral res. 2006; 20: 59-63. 14. yoshioka t, kikuchi i, fukumoto y, kobayashi c, suda h. detection of the second mesiobuccal canal in mesiobuccal roots of maxillary molar teeth ex vivo. int endod j. 2005; 38: 124-8. 15. wolcott j, ishley d, kennedy w, johnson s, minnich s,meyers j. a 5 yr clinical investigation of second mesiobuccal canals in endodontically treated and retreated maxillary molars. j endod. 2005; 31: 262-4. 16. sempira hn, hartwell gr. frequency of second mesiobuccal canals in maxillary molars as determined by use of an operative microscope: a clinical study. j endod. 2000; 26: 673-4. 17. ibbarrola jl, knowles ki, ludlow mo, mckinley ib jr. factors affecting the negotiability of second mesiobuccal canals in maxillary molars. j endod. 1997; 38: 236-8. 18. almeida jf, gomes bp, ferraz cc, souza-filho fj, zaia aa. filling of artificial lateral canals and microleakage and flow of five endodontic sealers. int endod j 2007; 40: 692-9. 19. yoshioka t, kobayashi c, suda h. high detection rate of root canal orifices under microscope. j endod. 2002; 28: 452-3. 20. alaçam t, tinaz ac, genç o, kayaoglu g. second mesiobuccal canal in maxillary first molars using microscope and ultrasonics. aust endod j. 2008; 34: 106-9. 21. bauman r, scarfe w, clark s, morelli j, scheetz j, farman a. ex vivo detection of mesiobuccal canals in maxillary molars using cbct at four different isotropic voxel dimensions. int endod j. 2011; 44: 752-8. 22. görduysus mo, görduysus m, friedman s. operative microscope improves negotiation of second mesiobuccal canals in maxillary molars. j endod. 2001; 27: 683-6. 23. kontakiotis eg, palamidakis fd, farmakis et, tzanetakis gn. comparison of isthmus detection methods in the apical third of mesial roots of maxillary and mandibular firts molars: macroscopic observation versus operating microscope. braz dent j. 2010; 21: 428-31. 24. constante ig, davidowicz h, barletta fb, moura aa. location and angulation of curvatures of mesiobuccal canals of mandibular molars debrided by three endodontic techniques. braz oral res. 2007; 21: 22-8. 25. estrela c, bueno mr, sousa-neto md, pécora jd. method for determination of root curvature radius using cone-beam computed tomography images. braz dent j. 2008; 19: 114-8. 26. tuncer ak, haznedaroglu f, sert s. the location and accessibility of the second mesiobuccal canal in maxillary first molar. eur j dent. 2010; 4: 12-6. 27. neaverth ej, kotler lm, kaltenbach rf. clinical investigation of in vivo endodontically treated maxillary first molars. j endod. 1987; 13: 506-12. preliminary investigation to achieve patency of mb2 canal in maxillary molars braz j oral sci. 11(3):373-376 1 volume 22 2023 e230282 original article braz j oral sci. 2023;22:e230282http://dx.doi.org/10.20396/bjos.v22i00.8670282 1 department of dental materials and prosthodontics, ribeirão preto school of dentistry, university of são paulo, ribeirão preto, são paulo, brazil. 2 dds, ms, phd, department of oral rehabilitation, school of dentistry, national university of concepción, concepción, paraguay. corresponding author: tatiane cristina dotta department of dental materials and prosthodontics school of dentistry of ribeirão preto, university of são paulo av. do café, s/n, 14040-904, ribeirão preto – sp, brazil telephone: +55(16) 3315-4790 e-mail: tatianedotta@usp.br editor: dr. altair a. del bel cury received: june 28, 2022 accepted: december 8, 2022 changes in the properties of bulk-fill resins under conditions of gastroesophageal reflux and bulimia mayara manfrin arnez1 , tatiane cristina dotta1,* , leonardo de pádua andrade almeida1 , raisa castelo1 , david emanuel ugarte2 , andréa cândido dos reis1 , alma blasida concepcion elizaur benitez catirse1 aim: evaluate the roughness, microhardness and color change of different bulk fill resins when submitted to the condition of gastroesophageal reflux and bulimia. methods: 60 specimens (n = 10) of bulk-fill composite resins were made: m1 – filtek™; m2 – tetric n-ceram and m3 – opus, through a matrix 2x6 mm and light cured by the valo light source. after polishing, initial analyzes (48 hours t0) of surface roughness (ra), microhardness (vhn) and color change (δe) were performed. to simulate the oral condition of severe gastroesophageal reflux and bulimia, the specimens were immersed in hydrochloric acid (s1) (ph 1.7) 4 minutes a day, for 7 days. control group specimens were immersed in artificial saliva (s2). subsequently to immersions, mechanical brushing was performed for 3 minutes, three times a day, simulating 7 days of brushing. and again, the analyzes of ra, vhn and δe were performed (7 days t1). thus, hydrochloric acid immersion, mechanical brushing and ra analysis were repeated at 14 days (t2) and 21 days (t3); and t2, t3 and t4 (3 years) for vhn and δe. results: after shapiro-wilk statistical test, anova and tukey test with bonferroni adjustment (p>0.05), m3 showed the lowest ra at all times compared to the other resins, while the highest ra was at t0. m1 and t1 showed higher vhn. and m2 and t4 showed higher δe. conclusion: bulk fill resins can be indicated for patients with gastroesophageal reflux and bulimia, nonetheless, tetric n-ceram resin showed the worst results. keywords: composite resins. color. hardness. hydrochloric acid. https://orcid.org/0000-0001-6194-517x https://orcid.org/0000-0001-6376-679x https://orcid.org/0000-0002-9211-0826 https://orcid.org/0000-0002-4312-7170 https://orcid.org/0000-0001-5104-1986 https://orcid.org/0000-0002-2307-1720 https://orcid.org/0000-0001-5817-5768 2 arnez et al. braz j oral sci. 2023;22:e230282 introduction dental erosion and consequent loss and demineralization of mineralized tissues may be present in individuals with eating disorders, bulimia nervosa or gastroesophageal reflux disease, due to exposure to gastric acids1-4. acids of extrinsic or intrinsic origins in contact with the dental surface can promote the irreversible loss of this substrate, whose increase in incidence and prevalence has been documented5. the ph of pure hydrochloric acid varies between 0.9 to 1.5 and after episodes of vomiting in the oral cavity, this ph does not fall below 1.5 due to esophageal buffering and dilution of saliva, leading to a serious and high-risk condition for the formation and progression of erosive lesions6,7. the durability of restorations depends on some factors, such as the choice of a suitable restorative material. composite resins provide excellent restoration properties such as increased wear resistance8. however, acidic conditions can damage the physical and mechanical properties of these materials, leading to the degradation of the organic matrix and the exposure of inorganic filaments, changing the properties of resins reducing the durability of restorations9. with advances in the development of dental materials and clinical techniques, composite resins have become more widely used as direct restorative materials to satisfy patients with esthetic demands10. many changes in its composition have been carried out since its inception, as well as the use of low-shrink, high-molecular-weight monomers to overcome the effects of polymerization shrinkage, one of the main deficiencies in the mechanical and chemical properties of these materials11. bulk-fill resins were developed to simplify the time-consuming incremental technique, with 4-5mm depth increments, and featuring bisgma, udma, bisema and procrylate monomer composition, plus a combination of ytterbium trifluoride and zirconia/ silica, giving the material a lower polymerization shrinkage11-13. surface roughness, color stability and microhardness can affect the survival of restorations as well as the dentist’s decision to replace them14. faced with the development of materials with new chemical formulations, there is a need for new experimental studies that evaluate the physical and mechanical properties of these new composite resins, in the conditions of oral challenges of patients with gastroesophageal reflux and/or bulimia, in order to to propose a more specific observation in the manufacture of materials, so that the longevity of these resins can be guaranteed during aggressive and extreme oral situations. thus, this study aims to evaluate the effect of hydrochloric acid on the roughness, microhardness and color of different bulk resins over 48 hours, 7 days, 14 days, 21 days and 3 years. material and methods experimental design for the evaluation of the effect of the acid challenge associated with the mechanic on the variables surface roughness, microhardness and color change, the factors 3 arnez et al. braz j oral sci. 2023;22:e230282 for this study were: restorative material (3 levels: m1 – composite resin filtektm bulk fill (3m, ribeirão preto, são paulo, brazil); m2 – composite resin tetric n-ceram bulk fill (ivoclar vivavent, são paulo, brazil) and m3 – composite resin opus bulk fill (fgm, santa catarina, brazil)), solution (2 levels: s1 hydrochloric acid, s2 artificial saliva) and time (4 levels: t0 48 hours; t1 7 days; t2 14 days; t3 21 days; t4 3 years) (table 1). table 1. division of groups according to response variables, variation factors and different levels surface roughness microhardness color change restorative material m1 – composite resin filtektm bulk fill; m1 m1 m2 – composite resin tetric n-ceram bulk fill; m2 m2 m3 – composite resin opus bulk fil m3 m3 solution s1 – hydrochloric acid; s1 s1 s2 – artificial saliva s2 s2 time t0 – 48 hours; t0 t0 t1 – 7 days; t1 t1 t2 – 14 days; t2 t2 t3 – 21 days; t3 t3 t4 – 3 years t4 fabrication of test specimes 60 specimens (n=10) were made according to the manufacturer’s instructions using a teflox matrix measuring 6 mm in diameter and 2 mm in depth. with the aid of a resin spatula (duflex, são paulo, brazil), the material was inserted into the matrix in a single increment. a polyester matrix and a glass plate were put on top of the filled cavity. additionally, a weight of 1 kg was put on top to guarantee the complete filling of the matrix and to produce the overflowing of any excess material. next, the light activation was carried out on the specimens light-curable using a curing light valo (ultradent – são paulo, brazil), in accordance with instructions from the manufacturer. after the polymerization, samples were taken out of the matrix and kept in relative humidity for 24 hours, in the oven at 37±1 °c. afterwards, the specimens were polished with sof lex discs (3m, são paulo, brazil) in a decreasing sequence of granulation, and one of the faces was marked to serve as a positioning guide, to be used with the confocal laser microscope, microhardness and spectrophotometer. storage of specimens all specimens were kept in relative humidity of artificial saliva in an oven at 37 ±1°c throughout the experiment period, they were only removed from the oven to be submitted to the action of hydrochloric acid and to the tests at the proposed times. 4 arnez et al. braz j oral sci. 2023;22:e230282 gastroesophageal reflux condition and bulimia for the specimens of each material that were subjected to the acid challenge, each specimen was individually immersed in 15 ml of hydrochloric acid (ph = 1.7) for 4 min, once a day, for 21 days, under vibration. and for the time of 3 years, the specimens were immersed for 3 uninterrupted days15. mechanical challenge the brushing of the specimens was performed using the pepsodent brushing machine. this test was performed before the readings of the times of 7 days, 14 days, 21 days and 3 years. colgate total 12 toothpaste was used. the volume of 10 g suspended in 10 ml of distilled water (1:1 proportion) in the appliance vats on the specimens. to perform the brushing, the time of 3 minutes was used, corresponding to 1025 cycles of the machine to simulate 7 days of brushing, three times a day. and for the 3-year brushing time, 2 hours and 5 minutes of brushing was used. after brushing, the specimens were washed in running water for 30 seconds and inserted again in relative humidity with artificial saliva in the oven at 37±1 °c. surface roughness readouts surface roughness readouts were performed after polishing the specimens at 48 hours, 7 days, 14 days and 21 days using a confocal laser microscope (lext ols4000, olympus, japan). the device was calibrated to focus an image at 1500 μm through the 5x objective lens. the average roughness of the area (sa, µm) of the polished surface of the specimens was measured. data were obtained using ols4000 software version 2.0 (lext ols4000, olympus corporation, tokyo, japan). microhardness readouts the microhardness readouts were performed in the experimental time intervals of 48 hours, 7 days, 14 days, 21 days and 3 years. for this analysis, the microdurometer (hmv-2000 shimadzu corporation, japan) was used, with a pyramid-shaped diamond coated penetrator of the vickers type, with a load of 100 g, applied for 10 seconds. 3 readings were made in the upper surface region of each specimen at points equidistant from each other, and the average of the measurements was obtained. color change readouts the color change readouts were performed after polishing the specimens at 48 hours, 7 days, 14 days, 21 days and 3 years using the sp62s spectrophotometer with model qa master i software (x-riteincorporated neu-isenburg germany. each specimen was carefully manipulated using clinical forceps (millennium, golgran, sp, brazil), dried with absorbent paper, and kept in a device duly prepared with niches for placement of the specimens and standardization of the readouts against an opaque white background. color measurements were performed using the cie l* a* b* color system. the δe* value is the total difference between two color stimuli and was calculated using following formula: 5 arnez et al. braz j oral sci. 2023;22:e230282 δe* = (δl*)² + (δa*)² + (δb*)². statistical analysis the results obtained were submitted to the shapiro-wilk normality test and data were analyzed using anova test (p ≤ 0.05) and tukey test with bonferroni adjustment, using the assistat (7.7 beta) software package. results in the interaction of the material x time of surface roughness, it was found that t0 (48 hours) had higher averages than the other times and m3 had means statistically lower than m1 and m2 (p< 0,001) (table 2). table 2. mean values for roughness by interaction material (m) x time (t) m1 m2 m3 t0 2,39 ± 0,56 aa 2,80 ± 0,37 ba 2,48 ± 0,30 aba t1 2,43 ± 0,62 aa 2,63 ± 0,50 aa 1,78 ± 0,27 bb t2 2,37 ± 0,59 aa 2,36 ± 0,55 ab 1,89 ± 0,38 bb t3 2,28 ± 0,56 aa 2,77 ± 0,68 ba 1,77 ± 0,23 cb lowercase letter line sense capital letter column sense for microhardness, in the means for the interaction of the time x material (p< 0,001) and time x solution (p< 0,001), it was found that the t1 time presented averages statistically higher than the other times. the acid solution (s2) showed statistically higher averages than artificial saliva (s1). it is possible to verify that the material m2 showed statistically lower averages than m1 and m3 (table 3). table 3. mean values for microhardness by interactions time (t) x material (m) and time (t) x solution (s) t0 t1 t2 t3 t4 m1 49,15 ± 6,47 aa 52,53 ± 3,94 ca 53,03 ± 5,60 ba 47,23 ± 2,76 da 47,72 ± 6,27 da m2 40,09 ± 6,35 ab 44,32 ± 7,23 bb 38,99 ± 5,31 ab 40,60 ± 5,34 ab 41,72 ± 7,04 ab m3 43,74 ± 5,51 bb 51,26 ± 7,11 aa 46,31 ± 6,56 bc 47,42 ± 6,26 ca 52,58 ± 6,60 ac s1 45,46 ± 7,78 aa 48,06 ± 7,78 ba 42,88 ± 6,51 aa 43,54 ± 5,59 aa 44,75 ± 7,80 aa s2 43,20 ± 6,26 aa 50,67 ± 6,35 ba 49,33 ± 8,44 bb 46,63 ± 5,83 cb 49,96 ± 7,24 bb lowercase letter line sense capital letter column sense for color change, in the means for the interaction of the time x material (p< 0,001), it was found that m2 had higher averages than m1 and m3 and t4 had means statistically higher than the other times (table 4). 6 arnez et al. braz j oral sci. 2023;22:e230282 table 4. mean values for color change by time (t) x material (m) m1 m2 m3 t1 2,72 ± 0,93 aa 2,84 ± 1,42 aa 2,63 ± 1,33 aa t2 1,87± 0,87 ab 3,16 ± 1,59 ba 2,19 ± 1,76 aa t3 2,92 ± 0,99 ac 3,74 ± 1,81 bb 2,34 ± 1,45 ab t4 2,85 ± 1,48 ac 4,20 ± 1,65 bb 2,83 ± 1,19 aa lowercase letter line sense capital letter column sense discussion the null hypothesis is that the acid does not change the properties of bulk fill resins, although in this work, opus bulk fill resin presented less roughness than filtek bulk fill and tetric n ceram. this result may be related, mainly to the amount of inorganic components of bulk fill resins. the opus bulk fill resin (79% by weight) has more inorganic component than filtek bulk fill (76% by weight) and tetric n ceram (75% by weight), this composition probably favored its lower roughness and greater hardness. the roughness was higher in the 48 hours when compared to the times of 7 days, 14 days, 21 days and 3 years. these results are not in accordance with several studies16 where they stated that, over time, there is a degradation of the organic matrix of resins, which provides an increase in surface roughness as a function of the time of restorative materials. according to ishii et al.17 (2020), the polishing technique can cause the release of the particles of charge, which generate voids on the resin surface and thus collaborate to increase the roughness. on the other hand, the effect of daily brushing, may favor the smoothing of the surfaces of the specimens over time, due to the abrasion process, which is in accordance with somacal et al.18 (2020). somacal and collaborators18 (2020) evaluated the effect of ph cycling and simulated brushing on the surface roughness of bulk fill resins, and although the ph cycle caused changes in the surface of the studied resins, it was not enough to generate changes in surface roughness. this result corroborates with the present study, since the low ph acid solution did not negatively influence the roughness of bulk fill resins. in the literature, acidic solutions can result in damage to the surface and reduce the microhardness of restorative materials, as they cause the material to dissolve, soften the polymeric matrices and detach the filler particles(11,14). tanthanuch et al.16 (2018) also reported that the immersion of bulk fill resins in liquids and acid-simulating food drinks can negatively influence the surface properties of restorative materials. the lower microhardness of the tetric n-ceram bulk fill (m2) resin is probably related to the type of photoinitiator that this material presents in its composition, ivocerin. this germanium-based initiator system has a high light-curing activity and an absorption spectrum that extends below 380 nm to 460 nm, with an absorption peak close to 408 nm19. when materials with this photoinitiator are photopoly7 arnez et al. braz j oral sci. 2023;22:e230282 merized with polywave light sources, they may present polymerization impairment, since the light source with this characteristic may present a problem of homogeneous light emission and thus interfere with the material’s microhardness20,21. in addition, the lower microhardness of the tetric n-ceram bulk fill (m2) resin may be related to the reduction in the percentage of inorganic components when compared to the other resins. this lower inorganic amount in the composition could have influenced the lower microhardness of the restorative material16. alencar and collaborators22 (2020), reported that after 7 days of immersion, the restorative materials used in the study (filtek z350xt, grandioso, filtek bulk fill, x-tra fil) showed less microhardness in different solutions (deionized water, acid citric 5% and hydrochloric acid 0.1%). these previous results corroborate with the data of the present study, where the microhardness was reduced over the different analyzed times. acidic solutions can degrade the monomeric matrix of restorative materials, impairing hardness, roughness and increasing water sorption. thus, the importance of the correct choice of restorative material in patients with severe dental erosion is evidenced23. it was found that the tetric n-ceram bulk resin (m2) under the action of acid (s1) was more sensitive, as it presented a greater color change than the m1 and m2 resins after 14 days, a result that showed that there was time addiction. when the color change occurs, this change may be related to the composition of the material, such as the type of photoinitiator system, type of monomer, percentage by weight and volume; and size of the charge particles, which can influence the stain susceptibility24-26. the color stability of composite resins can be mainly caused by water absorption and the hydrophilicity of the matrix. most resin matrix compositions such as bisphenolglycidyl methacrylate (bis-gma) and urethane dimethacrylate (udma) present hydrophilic molecules in their formulation, that is, with the ability to attract water with bis-gma, a slightly more hydrophilic component when compared to udma. such a situation will have a direct impact on the detection of stains found when resins with this monomeric composition are immersed in solutions. in the present study, tetric n-ceram resin was the compound that showed the greatest color change, and this fact is probably due to the presence of bis-gma in its monomeric composition14,27,28. according to rüttermann et al.29 (2010), color stability is related to the conversion of the photoinitiator system. this system can form by-products that fade thermally or under ultraviolet light and shift the color of the resin to a more red or yellow color. tetric n-ceram bulk fill resin has its own photoinitiator, ivocerin. this type of photoinitiator can influence the susceptibility of material stains30. in addition, a hydrophilic matrix contributes to the discoloration of the material. however, even if the matrix structure is not hydrophilic, water and coloring fluids can diffuse in the composite resin and cause susceptibility to discoloration. diffusion and discoloration occur when the inorganic and organic contents are not silanized correctly or when the integration in the resin matrix is not sufficient31. moreover, the tetric 8 arnez et al. braz j oral sci. 2023;22:e230282 n-ceram bulk fill resin compared to the other study resins is the material that has a lower percentage of charge, an amount that could contribute to a greater color change in this material. smaller particles affect the pigment adsorption on the material surface, affecting the overall color saturation after staining32. according to gönülol and yilmaz33 (2012), the monomer content and the surface roughness affect the color change of composite resins, more than the size of the filler particles. in the absence of pigments, the degree of conversion (proportion of remaining unreacted carbon-carbon bonds) and greater translucency of composite resins may be one of the other factors of color change, that is, color change is a multifactorial problem. acid solutions can promote the degeneration of resins leaving the surface rougher, which could allow greater pigment retention and thus influence the color change, justifying the results found when observing the significant averages according to the respective solution16,30. clinical experiments are necessary for the validation of the methods used in this study, since the evaluation made in the present research was an in vitro analysis. the results of this study show that the choice of material should be considered when planning restorations in patients with gastroesophageal reflux. furthermore, it can be observed that the composition of the restorative material (monomer, photoinitiator, particle size, and inorganic filler) and the presence of acid can have considerable effects on the properties of the different resins tested. in conclusion and according to the methodology used, it is possible to conclude that the roughness was higher at 48 hours and the composite resin opus bulk fill always showed lower roughness when compared to the other resins. in addition, it was possible to observe that the acid did not negatively influence this property. the composite resin filtek™ bulk fill showed the highest microhardness in 7 days. the acid negatively influenced the microhardness of the resins, however, tetric n-ceram bulk fill behaved better. also, color change has increased over time. at 3 years, composite resin tetric n-ceram bulk fill showed greater color change when associated with hydrochloric acid. acknowledgements the university of são paulo, ribeirão preto school of dentistry (department of materials and prosthodontics) and school of philosophy, sciences and letters (department of chemistry). declaration of interests the authors certify that they have no commercial or associative interest that represents a conflict of interest in connection with the manuscript. author contribution substantial contributions to the conception and design of the work: mma; tcd; lpaa; rc. substantial contributions to the analysis; mma; tcd; lpaa, rc, substan9 arnez et al. braz j oral sci. 2023;22:e230282 tial contributions to the interpretation of data for the work: mma; abcebc. drafting the work: mma; abcebc. reviewing it critically for important intellectual content: mma; acr; abcebc. manuscript findings: mma; tcd; lpaa; rc; deu; acr; abcebc. final review and approval of the version to be published: mma; tcd; lpaa; rc; deu; acr; abcebc. agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved: mma; tcd; acr; abcebc. all authors actively participated in the manuscript’s findings, revised and approved the final version of the manuscript. references 1. milani dc, borba m, farré r, grando lgr, bertol c, fornari f. gastroesophageal reflux disease and dental erosion: the role of bile acids. arch oral biol. 2022 jul;139:105429. doi: 10.1016/j.archoralbio.2022.105429. 2. davis ha, wildes je. eating disorders: bulimia nervosa. in: caballero b, editor. encyclopedia of human nutrition. 4.ed. academic press; 2023. p.304-13. doi: 10.1016/b978-0-12-821848-8.00074-3. 3. ranalli dn, studen-pavlovich d. eating nt. dent clin north am. 2021;65(4):689-703. doi: 10.1016/j.cden.2021.06.009. 4. li y, wang z, fang m, tay fr, chen x. association between gastro-oesophageal reflux disease and dental erosion in children: a systematic review and meta-analysis. j dent. 2022 oct;125:104247. doi: 10.1016/j.jdent.2022.104247. 5. marro f, o’toole s, bernabé 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for innovation and advanced specialties, calle de albarracín, 35, 28037 madrid, spain email: pcapdevicaz@gmail.com 0034628709408. received: march 18, 2020 accepted: october 06, 2020 attractive perception of profile class ii patients treated with extractions versus dentoalveolar mandibular advancement devices paul capdeville-cazenave1,*, iván nieto sánchez1, inés díaz renovales1, javier de la cruz pérez1 aim: this study aimed to compare the esthetic perception of profile silhouette of pictures of class ii patients before and after treatment (extractions or mandibular advancement), according to a visual analog scale (vas) among orthodontists, general dentists and lay people. methods: a presentation of 18 class ii adult patients silhouette was shown to three groups of participants (25 orthodontists, 25 general dentists, and 25 lay people) in two phases in an cross-sectional survey study. an initial presentation pre-treatment and a second one post-treatment, one month later. the presentation consisted of nine pictures of four extractions orthodontic cases (two maxillary premolars and two mandibular premolars): five males, four females, and other nine pictures with a dentoalveolar mandibular advance (forsus® and/or class ii elastics): four males, five females. to compare pre and post-treatment scores, an anova test was performed. independent variables studied were: sex, age, and previous or present orthodontic treatment of participants. results: a total of 75 of evaluators scored 18 patient profiles before and after treatment. in the three groups, the post treatment silhouette scored significantly higher. advancement treatment scored significantly higher than extractions, especially among lay people. orthodontist gave the lowers score regardless of treatment. no differences were found between male and female scoring (p>0.05). the participants between 30-39 years old gave higher scores than other age groups(p<0,05). conclusion: in our sample, class ii orthodontic treatments did always improve esthetic perception of patients profile. advancement treatment achieved a better esthetic perception than extractions, especially among lay people. keyword: orthodontics. malocclusion, angle class ii. esthetics. cross-sectional studies. 2 capdeville-cazenave et al. introduction the controversy remains in literature as to whether lay people or orthodontics, general dentist, and other dental professionals agree on their perception of facial attractiveness. according to peretta et al.1, a symmetrical face is more attractive. the skeletal class ii malocclusion is most often found in orthodontic practice after class i, according to profitt2 and simmons et al.3 with a prevalence of 15% in the population. in non-growing patients with skeletal class ii discrepancies, the orthodontist is often faced the choice of either carrying out surgery or camouflage treatment. in this scenario, orthodontists have two dentoalveolar treatment choices, class ii extractions or class ii advancement devices (forsus® device, class ii elastics). modern society places a strong emphasis on physical attractiveness and facial beauty. the face remains one of the key features for determining human physical attractiveness4. one reason that motivates patients to seek orthodontic treatment is to improve facial aesthetics. orthodontic treatment can influence facial aesthetics in a number of different ways, including well-aligned teeth5,6, an attractive smile6,7 and a pleasing facial profile7-9. subsequently, several studies have evaluated lip profile preferences8,9, where orthodontists and lay people have been requested to choose what they consider as most pleasant from groups of pictures or silhouettes profiles. peck and peck9 in 1970, found that public preferred profiles with lips that were consistently fuller and more prominent, which are considered as ideal by orthodontists. according to yehezkel and turley10 cephalometrics standards show that the public now prefers a more complete and more convex facial profile. foster et al.11 found that lay people chose profiles with fuller lips in children than in adults, what is consistent with the fact that lip profiles became more retruded with age, and were preferred over slightly fuller lips. czarnecki et al.12 also reported that subjects preferred fuller women lips, but said that lips were closer and linked to the positions of the nose and chin.subjects prefer a profile of protruded lips with a big nose and advanced chin. lai et al.13 supported the importance of balancing the relations of the nose and lips. abu arqoub and al-khateeb14 stated that images with increased lower facial proportions were considered less attractive than corresponding images with reduced lower facial proportions, and those with class ii profile features were considered to be less attractive than corresponding images with class iii profile features. lips and chin are paramount to orthodontic patients to decide whether extract or not. these principles have become commonly accepted and used in contemporary orthodontic treatment planning. the aim of this research was: firstly, to evaluate and score the differences, on a scale visual analog (vas), of the perceived attractiveness among orthodontists, general dentists, and those outside field of the dental sciences, in class ii patients, between the beginning and the end of treatment. 3 capdeville-cazenave et al. secondly, to compare how score changes a visual analog scale (vas) of perceived attractiveness was used to compare between treatment with extractions and treatment with mandibular advancement among class ii patients prior to the start and at the end of treatment time. materials and methods the present study was approved by the committee of ethics in research of alfonso x university (madrid, spain) (protocol approval no. 2015-44/011) , all participants signed an informed consent form. sample size was calculated based on a pilot study. a minimum of 25 subjects was required for each group of evaluators (orthodontists, dentists and laypeople) this cross sectional survey study consists of two populations: patients and reviewers. patients population: the study population consisted of photographs of patients who were treated with orthodontics and presented skeletal class ii malocclusion (according to cephalometric steiner anb). inclusion criteria were: • patients with skeletal class ii. • patients treated either with extraction or advancement mandibular devices (forsus® and or class ii elastics in adults). • patients between 13 and 35 years old. as they were only silhouette tracings, the age of the patients could not be known by the participants. • evaluations of facial profiles were made from tracings of the soft tissue contour obtained from the lateral cephalograms from the g´ point to the me point. tracings were scanned in jpeg format, at a 1:1 ratio, with 300 dpi, using an officejet 6500 scanner. the frankfurt plane was oriented parallel to a true horizontal plane, and tracings were filled with black color using photoshop© cs6 software (adobe systems). inclusion criteria were: • patients with skeletal class i or class iii. • patients not treated either with extraction or advancement mandibular devices (forsus® and or class ii elastics in adults) • patients younger than 13 or older than 35 years old ). • participants were divided into three groups: group i: orthodontists of master of orthodontics at alfonso x el sabio university (madrid, spain) group ii: dentists of the clinic of dental specialties at alfonso x el sabio university (madrid, spain). group iii: patients attending the clinic of dental specialties at alfonso x el sabio university (madrid, spain). 4 capdeville-cazenave et al. the dependent variable of the study was the aesthetic perception of the profile of patients with skeletal class ii malocclusion assessed by a vas scale of 0 to 10 (0 less attractive, 10 most attractive silhouette). independent variables the studied independents variables were: • sex. • facial biotype : according to ricketts cephalometric study. • the initial and final incisors inclination, given the involvement they have on the degree of protrusion or retraction of lips. • patient age in years. the independent variables were considered for the group of evaluators were: • sex. • age of the evaluators. • orthodontic treatment of the participant (previous, at the time of the survey or non-orthodontic treatment at all) : all the patients were treated with one of this two options: • patients treated with class ii advancement device. within this group patients treated with forsus® were inlcuded. • class ii skeletal patients treated with extractions of first upper and lower premolars (teeth 1.4, 2.4, 3.4, 4.4). and at the end of treatment which ended in molar and canine class i. patients’ pictures were obtained from the medical records owned by the master of orthodontics clinic at alfonso x el sabio university (madrid, spain) since 2005. all patients had previously signed their consent for using their medical records for educational purposes and for publishing in dental journals or congresses. profile photos collected from the patients were taken with a reflex device objective of 60 mm or more and ring flash described above. those photos were taken with a manual mode camera photo with an iso of 100 and f 10 or less inside a room with natural light without overexposure of light. once the photos were selected, they were cropped with adobe photoshop 6.0 version 13 software package (adobe systems, san jose, calif) among all the photos, only the outer contour of the profile photo, from the base of the bridge of the nose to the cervical region, was selected. photos were cut in a way that the profile end result was a black line representing these outer contours describing the face of the patient on a white background. a questionnaire was given initially to the three groups of examiners to score profile photos of the 18 patients pretreatment that afterward were treated class ii. nine before advancement devises and extractions (figure 1 and figure 2) and other nine 5 capdeville-cazenave et al. before (figure 3 and 4) and both, the questionnaire sheet to fill using a vas visual scale and informed consent, were given to the participants before starting the study in the three groups. figure 1. silhouette of advancement devices pre treatment. figure 2. silhouette of advancement devices post treatment. figure 3. silhouette of class ii premolars extractions pre treatment. 6 capdeville-cazenave et al. figure 4. silhouette of class ii premolars extractions post treatment. participants were asked to evaluate the attractiveness of the presented silhouettes before treatment. a month later, the same participants were required to evaluate the presented silhouettes from the same patients after orthodontic treatment (extractions or dentaolveolar mandibular advancement). esthetics was evaluated through a visual analog scale, where 0 was the lowest value (or the least aesthetic), and 10 was the highest (or the most aesthetic) after collecting the pre-treatment profiles questionnaires, a second one was given to the same three groups one month later for post-treatment profiles evaluation. analysis  was carried out  using the spss software (v. 16.0, spss inc., chicago, il, usa). descriptive statistics was used to describe the distribution of data, and means and standard deviations were calculated. the following comparisons were also made: comparison of scores before and after treatment were performed by t test of student for related samples, such as descriptive statistics mean and standard deviation (sd) of the difference of pre-post treatment is presented. these comparisons have been made for the total assessments and separately for pretreatment and post-treatment assessments. comparison of the score of both treatments (advancement vs extractions) by t test of student´s t-test for independent data. comparisons were made in both, pretreatment and the post-treatment and in the difference between them, descriptive statistics as the mean (sd) are presented. comparison between categories of each of the variables that describe the evaluators were carried out with t-test for independent data if the variable is dichotomous (sex), or by analysis of variance anova if the variable has tree or more categories (age, profession and background orthodontic). the calculation of the sample size has been designed to detect differences in the aesthetic evaluation between groups of at least 1.5 points, considering a standard deviation of 1.9 points. 7 capdeville-cazenave et al. the precision level (1-α) = 95%, and a statistical power (1-β) = 80%. a sample of 25 evaluators will be required in each group. this comparisons are both pre and post-treatment and in the difference between them. descriptive statistics such as the mean (sd) is presented and the adopted level of significance for this study was p<0,01. results differences between pre and post-treatment as well as differences between groups of evaluators (lay people, orthodontists and dentists) were evaluated. significant differences were observed in the three cases (figure 5) ie. example: the mean (sd) score prior to the treatment was 4.8 (sd ±2.55) in treatments with “advancement” and 3.3 (sd ± 2,17) in treatments with “extractions”, this difference was highly significant (p <0.001). orthodontists general dentists lay people pre advance post advance pre extractions post extractions 0,00 1,75 3,50 5,25 7,00 mean 0 to 10 p = 0,01 figure 5. esthetic scoring as per evaluator group. according to the sex of the evaluator. the differences after-before treatment are significantly higher in both, the total sample, and separately in both treatments, with the highest score post-treatment. advancement patient scores are considerably higher in advance treatment, even before any intervention. in the case of male, the difference pre-post in extraction treatment is significantly higher, while this difference has not been appreciated in the case of female patients. 8 capdeville-cazenave et al. only in pre-treatment of the total sample extraction and treatment of case, there are significant differences in scores associated with the sex of the evaluators, being male evaluators the ones who scored highest. depending on the age of the evaluator. in all age groups, differences after-before are very significant in both, the total sample, and separately in both treatments, with the highest score after appropriate intervention. in all age groups are scored higher in patients treated with advance, even before the intervention. although there are some differences in scoring related to age, there is no clear trend. according to the evaluator profession. in all profession groups, differences after-before are (<0.0001) in both, the total sample, and separately in both treatments, with the highest score after appropriate intervention. there are significant differences in scoring related to professional groups among patients treated with advance. these one got higher scoring than extraction treatment, even before the intervention. except in the case of “lay people” there is not relationship found with treatment in post-pre differences (figure 6). in most of the other comparisons, there are significant differences associated with the score given by profession group, being generally orthodontists. this group was the one giving the lowest scores. mean 0,0 1,5 3,0 4,5 5,0 p = 0,01 both pre treatments both post treatment advance pre treatment extraction pre treatment advance post treatment extraction post treatment figure 6. esthetic scoring as per treatments. according to personal orthodontic experience of the evaluator. regardless of the personal dental knowledge, differences between after-before are very significant in both, the total sample, and separately in both treatments, with the highest score after treatment. 9 capdeville-cazenave et al. in all groups, patients treated with advance scored higher. only in the case of those who never had orthodontic treatment there is not direct link with the post-pre differences, being greater the difference in the case of extractions. but for post-treatment evaluations in all other comparisons no significant differences in scores associated with the group of personal orthodontic experience were found. discussion mergen et al.15 found, as in our study, that most attractive profiles occurred post-treatment but they did not find any remarkable differences depending on the type of treatment. also according to nomura et al.16, an attractive soft tissue profile greatly varies among ethnicities and continents. in europe, the preferred profile is usually flat or slightly protrusive. therefore, extractions and advancement treatment could be both solutions that are socially accepted. nevertheless, in our sample, although both treatments are considered esthetically accepted, advancement profiles were scored higher. peck and peck9, a protruded lower lip was found more attractive in the case of female. however, the opposite happens for women´s upper lip, that feature is considered unattractive, being attractive among men. cassia molina de paula et al.17 explain that the treatment of class ii patients performed with a combination of mandibular protraction appliance and fixed corrective orthodontics, had a positive effect on how orthodontists and laypeople were perceiving facial silhouettes, being this effect more frequently identified by laypeople. moresca et al.18 focused on class ii malocclusion treatment using the herbst appliance, concluding that may produce a more esthetically improved facial profile silhouette compared with the one using the forsus appliance. our results did not compare skeletal but only dentoalveolar advancement devices, though. for rocha et al.19 the orthodontic treatment of class ii division 1 malocclusion both with extraction of maxillary first premolars and with the forsus appliance had positive effects on facial profile esthetics. this was evaluated by both lay examiners and orthodontists, who assigned greater scores to the posttreatment profile. both treatment protocols produced similar results on the facial profile esthetics. the group of orthodontists assigned higher scores to both preand posttreatment profiles than lay individuals, which is slightly different to our results. in both studies18,19 results are similar to ours: class ii treatment had positive effect in both cases. in this same way, mandible advancement profiles produced more esthetic results than upper and lower extractions in our sample. conversely, it has been reported that the great benefit of treatment by extraction is achieved in cases with a greater dental protrusion, marked crowding, and thick and protruded lips. however, in individuals presenting a thin upper lip and an increased 10 capdeville-cazenave et al. nasolabial angle at pretreatment, extraction may affect the upper lip positioning negatively and increase the nasolabial angle20. orthodontists used to measure changes in lateral cephalometrics and to evaluate the sagittal plane of patients is skeletal class, molar or canine class. however, in real life and in everyday social interactions, orthodontic result is rated from a face to face interaction rather than from a lateral view. one of the improvements that should be developed in the future would be assessing changes of patients profile treated with extractions or with mandibular advancement through cbct. it would also be interesting to evaluate the atractiveness of a 3d profile patient before and after treatment with different treatment modalities trough a stereophotogrammetry system. it would also be a good option to compare the profiles treated with molar distalization in class ii or the differences between female and male patients, in order to know what treatment achieves the most attractive profiles among the three groups of examiners. another study line to develop in the future could be applying this type of study in class iii, class i with open bite or excessive overbite patients. this study has some limitations. this study included patients with 13-year-old or older. however, orthognathic surgery is usually performed after the patient’s skeletal maturation period, which occurs in individuals aged 18 years. thus, the perception of patients below this age by dental professionals may generates a bias assessment on the volume and position of the lip and chin. also the extracted tooth may be an issued as differences in profile between first and second premolar extraction cases. in our study, participants were asked to sit down and to fill in a paper form to carry out the study. this request may limit the motivation of certain evaluators that could have been reduced in case of using computerized questionnaire. the fact that the study was only made up of 2d profile photos, could lead to think that a study with 3d photos could have been more suitable. also the mood and state of mind of those evaluating at the time of giving scores could also have an effect on the results. in conclusion, the difference scoring after-before treatment are significant, all pictures systematically scored higher after treatment. dentoalveolar advancement treatment is clearly preferred to extractions both in case of posttreatment. however, the advantage of the advance against extractions as to the difference in posterior and anterior to the intervention score, only becomes significant in all evaluations in of the male group, laypeople and those who they never underwent orthodontic treatment. orthodontists treating class ii patients should consider not only the esthetic but also the inclination of lower incisor, crowding, and the ability to cooperate during treatment among other concerns and have a thorough chat with the patients explaining in full detail the advantages and disadvantages of extractions and dentoalveolar advancement. having this said, both treatments offer an improvement in esthetics, that is slightly higher for non-extraction treatment. 11 capdeville-cazenave et al. references 1. perretta di, burt md, penton-voak is, lee kj, rowland da, edwards r. symmetry and human facial attractiveness. evol hum behav.1999; 20:295-307. 2. proffit wr. contemporary orthodontics. madrid: elsevier; 2005. 3. simmons hc 3rd, oxford de, hill md. the prevalence of skeletal class ii patients found in a consecutive population presenting for tmd treatment compared to the national average. j tenn dent assoc. 2008 fall;88(4):16-8; quiz 18-9. 4. riggio re, widaman, kf, tucker js, salinas c. beauty is more than skin deep: components of attractiveness. basic appl soc psych.1991;12(4):423-39. doi: 10.1207/s15324834basp1204_4. 5. giddon db. orthodontic applications of psychological and perceptual studies of facial esthetics. 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10.1016/j.ajodo.2003.05.007. 11. foster la, thomson wm, locker d. assessing the responsiveness of the cpq11-14 in new zealand adolescents. soc sci dent. 2010;1:48-53. 12. czarnecki st, nanda rs, currier gf. perceptions of a balanced facial profile. am j orthod dentofacial orthop. 1993 aug;104(2):180-7. doi: 10.1016/s0889-5406(05)81008-x. 13. lai j, ghosh j, nanda rs. effect of orthodontic therapy on the facial profile in long and short vertical facial patterns. am j orthod dentofacial orthop. 2000 nov;118(5):505-13. doi: 10.1067/mod.2000.110331. 14. abu arqoub sh, al-khateeb sn. perception of facial profile attractiveness of different anteroposterior and vertical proportions. eur j orthod. 2011 feb;33(1):103-11. doi: 10.1093/ejo/cjq028. 15. nomura m, motegi e, hatch jp, gakunga pt, ng’ang’a pm, rugh jd, et al. esthetic preferences of european american, hispanic american, japanese, and african judges for soft-tissue profiles. am j orthod dentofacial orthop. 2009 apr;135(4 suppl):s87-95. doi: 10.1016/j.ajodo.2008.02.019. 16. mergen jl, southard ka, dawson dv, fogle ll, casko js, southard te. treatment outcomes of growing class ii division 1 patients with varying degrees of anteroposterior and vertical dysplasias, part 2. profile silhouette evaluation. am j orthod dentofacial orthop. 2004 apr;125(4):457-62. doi: 10.1016/j.ajodo.2003.06.004. 17. molina de paula ec, de castro ferreira conti ac, siqueira df, valarelli dp, de almeida-pedrin rr. esthetic perceptions of facial silhouettes after treatment with a mandibular protraction appliance. am j orthod dentofacial orthop. 2017 feb;151(2):311-6. doi: 10.1016/j.ajodo.2016.06.038. 18. moresca ahk, de moraes nd, topolski f, flores-mir c, moro a, moresca rc, et al. esthetic perception of facial profile changes in class ii patients treated with herbst or forsus appliances. angle orthod. 2020 feb 24. doi: 10.2319/052719-362.1. 12 capdeville-cazenave et al. 19. rocha ad, casteluci cevf, ferreira fpc, conti ac, almeida mr, almeida-pedrin rr. esthetic perception of facial profile changes after extraction and nonextraction class ii treatment. braz oral res. 2020 jan 31;34:e003. doi: 10.1590/1807-3107bor-2020.vol34.0003. 20. tadic n, woods mg. incisal and soft tissue effects of maxillary premolar extraction in class ii treatment. angle orthod. 2007 sep;77(5):808-16. doi: 10.2319/081706-336. 1http://dx.doi.org/10.20396/bjos.v20i00.8660971 volume 20 2021 e210971 original article 1 state university of feira de santana, department of health, feira de santana, bahia, brazil. 2 são judas tadeu university, department of operative dentistry, sao paulo, sao paulo, brazil. 3 state university of feira de santana, department of biological sciences, feira de santana, bahia, brazil. * corresponding author: ynara bosco de oliveira lima-arsati uefs – av. transnordestina, s/n. bairro novo horizonte, feira de santana, bahia, brasil. zip code: 44036-900. phone/fax: (+55) 75 3161-8019. email: ynaralima76@gmail.com received: august 22, 2020 accepted: december 14, 2020 in vitro determination of potentially bioavailable fluoride in diet and toothpaste after ingestion cristiane brandão santos almeida1 , josé augusto rodrigues2 , valéria souza freitas1 , ynara bosco de oliveira lima-arsati3,* aim: to propose a new method to determine in vitro potentially bioavailable fluoride (f) in diet and toothpaste after ingestion. methods: diet samples (d) were obtained from 15 portions of a meal served to children in a day care centre. to simulate the ingestion of toothpaste during brushing after meals, a specific amount of toothpaste was added to the diet samples (d + t). f was determined in d and d + t after incubation in a solution that simulated “gastric juice” (0.01 m hydrochloric acid) at 37oc for 30, 60 and 120  min. microdiffusion facilitated by hmds was used to determine the total f concentrations in samples d and d + t. the analyses were performed using an ion specific electrode. results: for d samples, incubation in “gastric juice” for 30, 60 and 120 min resulted in f concentrations (μg f/ml) of 0.75 ± 0.06c, 0.77 ± 0.07c and 0.91 ± 0.09b, corresponding to 75.3, 77.3 and 90.7% of the total f (1.02 ± 0.12a), respectively (p = 0.0001; anova + tukey). for d + t samples, these values of f concentrations (μg f/ml) were 2.55 ± 0.46b, 2.83 ± 0.44ab and 3.15 ± 0.37a, corresponding to 86.9, 94.8 and 106.7% of the total f (2.99 ± 0.34a), respectively (p = 0.0023; anova + tukey). conclusion: then, it can be concluded that the proposed method of “gastric juice” is a promising protocol for determining potentially bioavailable fluoride in the diet and toothpaste after ingestion. however, additional studies are desirable. keywords: fluoride. dentifrice. toothpaste. diet. dental fluorosis. http://dx.doi.org/10.20396/bjos.v20i00.8660971 mailto:ynaralima76@gmail.com https://orcid.org/0000-0002-7424-1160 https://orcid.org/0000-0003-4172-1499 https://orcid.org/0000-0002-7259-4827 https://orcid.org/0000-0002-1059-2797 2 almeida et al. introduction several studies were developed to estimate the effect of fluoride (f) intake and risk of dental fluorosis development. the mean intake of f from diet and toothpaste observed in scientific studies ranged from 0.036 to 0.090 mg f/kg/day1-7. however, the correlation between high doses of f exposure and high prevalence of dental fluorosis has not been established yet8-9. this is probably because the doses determined may be overestimated for two reasons: the daily brushing frequency is not as high as that reported3 and the fact that the dose is based on the amount of ingested f, rather than absorbed. in vivo studies demonstrate that the presence of food in the stomach decreases the absorption of f from the toothpaste ingested during brushing after regular meals10. the reason is probably the increase in the ph of the stomach or the formation of low solubility salts between f and cations calcium (ca2+), aluminum (al3+) and magnesium (mg2+) from food. another aspect to consider is the type of abrasive present in the toothpaste. toothpastes with calcium-based abrasives, such as calcium carbonate (caco3) have part of the insoluble f, because it is bounded to calcium. the silica-based abrasive toothpastes present the entire f in the soluble form, being bioavailable for absorption11. thus, the reported dose values may be overestimated, as discussed in some of these studies3,8. when f is needed to be quantified in food samples using ion-specific electrode, it is necessary to extract the f ion (f-), since the electrode can only detect f ion in solution12-13. the recommended method to determine f in food samples is microdiffusion facilitated by hmds14-15, originally described by taves14 (1968), and manages to extract total f of the samples, even solid ones, besides concentrating them approximately 6.7 times, which increases the sensitivity of the method. however, it is a laborious, expensive, and time-consuming technique. considering the use of different protocols for microdiffusion, martínez-mier  et  al.15 (2011) demonstrated that a standardization of techniques increased the recovery of f and resulted in very precise and exact values between different laboratories. for food samples, they advocated the use of microdiffusion in a protocol which has some differences from the original technique, in addition to having the same critical step of completing the final volume of the drops with deionized water to compensate for evaporation. although the protocols for total f extraction are established, not all f is soluble in food and cannot be absorbed. therefore, methods that can determine potentially bioavailable fluoride are desirable to improve the discussion about f intake and the risk of dental fluorosis16. it was reported that the use of 0.01 m hcl to dissolve samples of prenatal supplements resulted in the recovery of 38.3% of the total f17. the authors suggested that body temperature and peristaltic movements should be simulated to provide more realistic results for potentially bioavailable f during digestion. considering that the main sources of systemic f for children at risk for dental fluorosis are diet (food and beverages) and toothpaste 1-7, the aim of the present study was to propose a new method to determine in vitro the concentration of potentially bioavailable f in samples of diet and toothpaste. 3 almeida et al. methodology experimental design this was an in vitro study. the experimental units were 15 diet samples, which were analyzed pure (d) or with toothpaste (d + t); there were four experimental groups, corresponding to the method used to extract fluoride from the samples: “gastric juice” 30 min, “gastric juice” 60 min, “gastric juice” 120 min and microdiffusion facilitated by hmds. the response variable was the f concentration, expressed in μg / ml. obtaining diet samples (d) the sample size was statistically determined using the bioestat software, based on data from previous studies, as follows: minimum difference between treatments = 0.15; standard deviation = 0.1; study power = 0.8; level of significance = 0.05. the  result was 11, but we used 15 just in case. after authorization from the municipal education secretary of feira de santana, bahia, diet samples were collected at a daycare center. the meals were prepared with optimally fluoridated water (0.76 ± 0.01 μg f/ml) and consisted of rice, beans, pasta and meat. the cook placed the meals on dishes as usual, reproducing the amount usually consumed by children aged 2 to 3 years during lunch. fifteen dishes were selected at random and each meal was collected separately in plastic containers. in the laboratory, samples of the diet were weighed, 100 ml of distilled and deionized water were added to each sample and homogenized with a mixer, without a filtration method. the final volume was determined, and each sample was frozen (18oc) until analysis. knowing its volume before and after adding water, the dilution factor was calculated for each one. obtaining diet + toothpaste samples (d+t) the used toothpaste was tandy® (colgate, strawberry flavor, lot 6289br121k, validity 10/19), containing 942.8 ± 3.8 µg f/g as total soluble fluoride (tsf), from sodium fluoride. the tsf concentration was determined by the direct method using an ion-specific electrode18. according to a previous study7, children residing in feira de santana, bahia, aged between 15 and 30  months, used an average of 0.47  g of toothpaste for brushing their teeth, and ingested 70.5% of this, resulting in 0.33 g of toothpaste ingested. in the present study, the average volume of the diet collected at the daycare center, simulating lunch, was 250 ml. hypothetically considering that children would brush their teeth right after lunch, the gastric content of these children would be 250 ml of diet and 0.33 g of toothpaste. we used this proportion to determine the amount of toothpaste to be added to the diet + toothpaste samples (d+t), considering the volume of diet used in each analytic method: ta = da * ti/ di where: • ta: weight of toothpaste to be added to sample in laboratory analysis (g) • da: volume of diet used in laboratory analysis (ml) • ti: estimated weight of toothpaste ingested during toothbrushing (g) • di: estimated volume of diet ingested per meal (ml) 4 almeida et al. so, for microdiffusion facilitated by hmds, ta = 3 * 0.33/ 250 = 0.004 g. and for the “gastric juice”, ta = 7 * 0.33/ 250 = 0.009 g. f determination using the “gastric juice” method (shown in fig. 1) this simulation was performed by incubation of the samples in a solution that simulated gastric juice (0.01 m hydrochloric acid) at 37oc for 30, 60 and 120 min. this solution was called “gastric juice”. this protocol was based on the reported by fernandes and cury17 (1993), and in a preliminary study of our group19, which showed that the 7:1 proportion of sample to “gastric juice” resulted in a ph value corresponding to the gastric content during digestion (ph = 4.7612). in addition, the preliminary study showed that the incubation in “gastric juice” for 120 min resulted in a recovery of 95.51% of total f concentrations in samples. so, we decided to test this incubation time (120 min) against lower times (60 and 30 min). d samples were prepared adding 1 ml of 0.01 m hcl to 7 ml of diet. d+t samples were prepared adding 1 ml of 0.01 m hcl to 7 ml of diet and 0.009 g of toothpaste (based on the formulae previously demonstrated: ta = da * ti/ di). plastic tubes containing the samples were incubated for 30, 60 or 120 min in an oven (sterilifer, sx 300) at 37°c. then centrifugation (thermo scientific) was performed for 5 min at 10,000 rpm. fat was cautiously removed from the surface using an absorbent paper, and 0.4 ml of supernatant was buffered with 0.4 ml of tisab ii for f analysis. the f concentration was determined by means of an ion-specific electrode (ise; orion model 96-09, orion research incorporated, cambridge, ma, usa) and an ion analyzer (orion star a214, orion research incorporated), previously calibrated with standards solutions (0.4, 0.8, 1.6, 3.2 and 6.4  μg f/ ml) in triplicate, prepared in the same conditions of the samples. the calibration and concentrations determined were tested in the linear regression curve, using excel software, where a calculation program transformed the values of mv provided by the electrode in f concentration. blank correction was done. d and d+t samples were analyzed in separate days. for d samples, the mean variation between obtained and expected values for calibration curve was 0.0085% and r2 = 0.9998. for d+t samples, the mean variation for calibration curve was 0.001% and r2 = 0.9999. read in f-specific electrode under stirring, and calculate [f] from mv readings using a spreadsheet d samples: 1 ml of 0.01 m hcl + 7 ml of diet or d + t samples: 0.009 g of toothpaste + 1 ml of 0.01 m hcl + 7 ml of diet mix vigorously ("vortex") 30, 60 or 120 min in an oven at 37o c centrifugate 5 min at 10,000 rpm remove fat from the surface with absorbent paper add 0.4 ml of supernatant + 0.4 ml of tisab ii figure 1. f determination using the “gastric juice” method. 5 almeida et al. f determination using microdiffusion facilitated by hmds (shown in fig. 2) it was based on the method described by taves14 (1968). a cap from a plastic tube was placed, using vaseline, in the center of a plastic petri dish. for d samples, 3 ml of homogenized diet was added to the petri dish. for d+t samples, before the diet, 0.004 g of toothpaste was added to the petri dish (based on the formulae previously demonstrated: ta = da * ti/ di). for calibration curve, 1 ml of standard solution and 2 ml of distilled and deionized water were added to the petri dish. then, 0.10 ml of 1.65 m sodium hydroxide was placed in the cap. the petri dish was closed, sealed with vaseline and 1.0 ml of 6 m hydrochloric acid saturated with hmds added to the sample through a hole made in the petri dish cap. the hole was sealed with vaseline and the petri dish was shaken at room temperature for 14 h in an orbital shaker (kline, nt-150). then, the plastic cap containing f diffused from the sample was dried at 60°c for 2 h in an oven (sterilifer, sx 300). this cap was then used to close a plastic tube containing 0.40 ml of 0.66 m acetic acid. the tube was inverted and vigorously shaken to dissolve the fluoride crystals present in the cap. the f concentration was determined by means of ise, previously calibrated with standards solutions (0.4, 0.8, 1.6, 3.2 and 6.4 μg f/ ml) in triplicate, prepared in the same conditions of the samples. blank correction was done. d and d+t samples were analyzed in separate days. for d samples, the mean variation between obtained and expected values for calibration curve was 0.054% and r2 = 0.9989. for d+t samples, the mean variation for calibration curve was 1.423% and r2 = 0.9527. read in f-specific electrode and calculate [f] from mv readings using a spreadsheet fix a cap, using vaseline, in the center of a plastic petri dish add 3 ml of sample or 1 ml standard solution + 2 ml distilled and deionized water in the petri dish add 0.1 ml of 1.65 m naoh in the central cap close the petri dish, sealing with vaseline add 1 ml of 6 m hcl/ hmds by the hole of petri´s dish, sealing imediatelly with vaseline leave 12-14h under agitation (orbital shaker) at room temperature remove the central cap and dry in an oven at 60o c for 2 h use the cap to close a plastic tube containing 0.40 ml of 0.66 m acetic acid, invert and shake vigorously figure 2. f determination using microdiffusion facilitated by hmds. statistical analysis a descriptive statistical analysis was performed to obtain values of central tendency and dispersion. then, analysis of variance was used to compare the results of f concentration obtained for the different methods (one-way anova + tukey test). the coefficient of variation (cv%) and the intraclass correlation coefficient (icc) were determined for all samples, analyzed in triplicate, to evaluate reproducibility. the software bioestat 5.0 and spss were used; the level of significance was 5%. 6 almeida et al. results results are expressed in table 1. regarding diet (d) samples, “gastric juice” resulted in lower f concentrations than the microdiffusion facilitated by hmds. comparison among incubation times in “gastric juice” showed that 120  min resulted in higher f concentration than 30 and 60 min. both the “gastric juice” method (cv% = 4.79% and icc = 0.86) and the hmds-facilitated microdiffusion (cv% = 6.58% and icc = 0.89) showed high reproducibility20. the diet + toothpaste (d+t) samples comparison showed that the “gastric juice” with 60 and 120 min of incubation did not differ from hmds-facilitated microdiffusion. the “gastric juice” method (cv% = 6.68% and icc = 0.88) showed high reproducibility, but the hmds-facilitated microdiffusion (cv% = 10.45% and icc = 0.42) showed low reproducibility. table 1. mean, standard deviation and range of f concentration (μg f/ml) in diet samples (d) and diet+toothpaste samples (d + t), using different methods of f extractiona. method diet (d) (n = 15) diet+toothpaste(d+t); (n = 15) microdiffusion facilitated by hmds 1.02 ± 0.12 a (0.77 1.25) 2.99 ± 0.34 a (2.70 4.06) “gastric juice” 30 min 0.75 ± 0.06 c (0.63 0.88) 2.55 ± 0.46 b (1.25 – 3.16) 60 min 0.77 ± 0.07 c (0.69 0.92) 2.83 ± 0.44 ab (2.24 – 3.85) 120 min 0.91 ± 0.09 b (0.78 1.09) 3.15 ± 0.37 a (2.49 3.96) a values followed by distinct letters indicate a statistically significant difference between the analytical techniques within each group (d or d+t) (p < 0.05; one-way anova and tukey’s test). considering the results of the microdiffusion method as total f concentration, it was found that, using “gastric juice” for 30 min, 75.25% (± 10.77) of the total f were extracted from the samples of the diet. these values for 60 and 120 min were 77.33% (± 12.80) and 90.70% (± 9.69), respectively. for the samples of diet + toothpaste, using “gastric juice” for 30, 60 and 120 min extracted 86.90% (± 18.39), 94.83% (± 10.06) and 106.65% (± 15.96) of total f, respectively. discussion after ingestion, the potentially bioavailable fluoride is represented by its soluble and in ionic form (f-), which can be converted into hydrofluoric acid (hf) and absorbed, having some systemic effect. the formation of hf depends on the ph of the medium. gastric ph is acidic due to the presence of hydrochloric acid (hcl) in gastric juice. in  addition to hcl, gastric juice is composed of pepsinogen, intrinsic factor and mucus. the rate of hcl secretion varies depending on the stimuli. when the stomach is “empty”, it contains about 50  ml of gastric juice and its ph is approximately 221. after eating, the ph increases to 4.1 6.322. therefore, it is necessary to assess the presence of potentially bioavailable fluoride in these conditions. in laboratory analysis, the electrode can detect f-. tisab ii is generally used as a buffer, in order to regulate the ph about 5.5, in which most of the soluble f will be 7 almeida et al. in ionic form (f-)23. it must be considered that not all the f detected by the electrode represents the absorbed f, as there are different conditions for it to be detected in the laboratory (to be in the fform)23 and to be absorbed in vivo (to be in the hf form)24. in an in vivo situation, perhaps the main reason for the difference between ingested and absorbed f is related to gastric ph. the f will only be absorbed in the form of hydrofluoric acid (hf)24, which is predominant when the ph of the gastrointestinal tract is less than 3.2 (pka value of hf). therefore, depending on the gastric content, the ph might be different and therefore the absorption of f might also be different. considering the gastric ph between 4.1 and 6.3, the predominant f will be in the ionic form (f-), therefore, it will not be absorbed24.the “gastric juice” method was proposed to simulate gastric conditions during digestion (ph and temperature). thus, when the samples were subjected to these conditions, it was assumed that f would be solubilized in the same way that it occurs in real conditions. the results showed that there was no difference in f concentration obtained by using microdiffusion ou “gastric juice” for 120 min to extract f from samples of diet. it means that “gastric juice” method, with 120 min of incubation, can extract total f from diet samples. this is an important result, considering the advantages of the “gastric juice” method (simplicity and cost). the amount of f detected by the electrode analysis represents the ionic f, which might not be totally absorbed, since not all fwill form hf. therefore, despite being potentially bioavailable, most of the f in the diet will not be absorbed. this could also justify the lack of correlation between f intake and dental fluorosis reported in the literature8-9. however, when the diet samples were incubated in “gastric juice” for shorter periods, less potentially bioavailable f was detected, showing that in the “gastric juice” method, the time of incubation affected the extraction of f from the diet samples. in fact, this method actually extracts fluoride from the samples, instead of just detecting the one already soluble; it is only a matter of time. then additional studies are needed, to correlate shorter and longer incubation periods to in vivo conditions and assess the relationship between incubation time and potentially bioavailable f extraction from diet samples. considering the samples of diet + toothpaste, it is possible that the duration of the incubation was important only for f in the diet, since the f concentrations were lower than the total f concentration only for the group incubated for 30 min. perhaps the f in toothpaste, which contained sodium fluoride (naf) and was already in ionic form, remained so, without being inactivated by cations present in food. it is also necessary to note that d + t analyzes using microdiffusion showed low reproducibility (icc = 0.42). this was not expected, since microdiffusion is a well-established methodology for food15, and the toothpaste used contained sodium fluoride. our hypothesis is that the toothpaste samples dried out because they were the first to be added (weighed). only after toothpaste was added to all petri dishes, the diet (homogenized food) was added. although the petri dish remained under agitation (orbital shaker) overnight, the toothpaste may not have adequately solubilized in food. to avoid this, it is suggested to add toothpaste after the diet. among the limitations of the “gastric juice” method, is the fact that it is not as dynamic as the digestion process, where hcl secretions are not constant, so neither is the 8 almeida et al. ph. in addition, gastric juice is not exclusively composed of hcl. another limitation was that the samples were not under agitation while incubated at 37oc, therefore, peristaltic movements were not simulated, as suggested by fernandes and cury17 (1993). for the “gastric juice” method to be considered adequate to determine potentially bioavailable f, additional studies must be conducted, evaluating the effect of the following factors: absence of incubation time; repetition of analyzes over time (short periods) in the same sample until [f] reaches a plateau; addition and recovery of known amounts of f to samples. in addition, it must be considered that not all potentially bioavailable f will be absorbed, the “gastric juice” technique would also overestimate the dose of systemic exposure to fluoride and, consequently, the risk of dental fluorosis. therefore, clinical studies, using validated biological samples that reflect fluoride metabolism are essential to complement in vitro studies16. then, it can be concluded that the proposed method of “gastric juice” is a promising protocol for determining potentially bioavailable fluoride in the diet and toothpaste after ingestion. however, additional studies are desirable. acknowledgements we are grateful to fapesb (bahia research foundation) for the master’s degree granting (process n. bol 93/2016) and financial support (fapesb/cnpq, n. 485/2011). references 1. guha-chowdhury n, drummond bk, smillie ac. total fluoride intake in children aged 3 to 4 years a longitudinal study. j dent res. 1996 jul;75(7):1451-7. doi: 10.1177/00220345960750070401. 2. rojas-sanchez f, kelly sa, drake km, eckert gj, stookey gk, dunipace aj. fluoride intake from foods, beverages and dentifrice by young children in communities with negligibly and optimally fluoridated water: a pilot study. community dent oral epidemiol. 1999 aug;27(4):288-97. doi: 10.1111/j.1600-0528.1998.tb02023.x. 3. lima ybo, cury ja. [fluoride intake by children from water and dentifrice]. rev saude publica. 2001;35(6):576-81. doi: 10.1590/s0034-89102001000600012. portuguese. 4. paiva sm, lima ybo, cury ja. fluoride intake by brazilian children from two communities with fluoridated water. comm dent oral epidemiol. 2003 jun;31(3):184-91. doi: 10.1034/j.16000528.2003.00035.x. 5. omena lmf, silva mf, pinheiro cc, cavalcante jc, sampaio fc. fluoride intake from drinking water and dentifrice by children living in a tropical area of brazil. j appl oral sci. 2006 oct;14(5):382-7. doi: 10.1590/s1678-77572006000500015. 6. lima cv, cury ja, vale gc, lima md, moura lf, moura ms. total fluoride intake by children frmo a tropical brazilian city. caries res. 2015;49(6):640-6. doi: 10.1159/000442029. 7. lima-arsati ybo, gomes arlf, santos hka, arsati f, oliveira mc, freitas vs. exposure to fluoride of children during the critical age for dental fluorosis, in the semiarid region of brazil. cien saude colet. 2018 apr;23(4):1045-1054. doi: 10.1590/1413-81232018234.07952016. portuguese. 8. martins cc, paiva sm, lima-arsati ybo, ramos-jorge ml, cury ja. prospective study of the association between fluoride intake and dental fluorosis in permanent teeth. caries res. 2008;42(2):125-33. doi: 10.1159/000119520. https://doi.org/10.1590/s0034-89102001000600012 9 almeida et al. 9. warren jj, levy sm, broffitt b, cavanaugh je, kanellis mj, weber-gasparoni k. considerations on optimal fluoride intake using dental fluorosis and dental caries outcomes a longitudinal study. j public health dent. 2009;69(2):111-5. doi: 10.1111/j.1752-7325.2008.00108.x. 10. cury ja, del fiol fs, tenuta lma. low-fluoride dentifrice and gastrointestinal fluoride absorption after meals. j dent res. 2005 dec;84(12):1133-7. doi: 10.1177/154405910508401208. 11. falcão a, tenuta lma, cury ja. fluoride gastrointestinal absorption from na2fpo3 /caco3 and naf/sio2 – based toothpastes. caries res. 2013;47(3):226-33. doi: 10.1159/000346006. 12. martinez-mier ea, tenuta lma, carey cm, cury ja, van loveren c, ekstrand kr, et al. orca fluoride in toothpaste analysis work group. european organization for caries research workshop: methodology for determination of potentially available fluoride in toothpastes. caries res. 2019;53(2):119-36. doi: 10.1159/000490196. 13. reshetnyak vy, nesterova ov, admakin oi, dobrokhotov da, avertseva in, dostdar sa, et al. evaluation of free and total fluoride concentration in mouthwashes via measurement with ionselective electrode. bmc oral health. 2019 nov 20;19(1):251. doi: 10.1186/s12903-019-0908-0. 14. taves dr. separation of fluoride by rapid diffusion using hexamethyldisiloxane. talanta. 1968 sep;15(9):969-74. doi: 10.1016/0039-9140(68)80097-9. 15. martínez-mier ea, cury ja, heilman jr, katz bp, levy sm, li y, et al. development of gold standard ion-selective electrode-based methods for fluoride analysis. caries res. 2011;45(1):3-12. doi: 10.1159/000321657. 16. idowu os, azevedo lb, valentine ra, swan j, vasantavada pv, maguire a, et al. the use of urinary fluoride excretion to facilitate monitoring fluoride intake: a systematic scoping review. plos one. 2019 sep;14(9):e0222260. doi: 10.1371/journal.pone.0222260. 17. fernandes lmag, cury ja. [prenatal fluoride metabolic evaluation]. rbm rev bras med. 1993 nov;50(11):1546-52. portuguese. 18. cury ja, oliveira mjl, martins cc, tenuta lm, paiva sm. available fluoride in toothpastes used by brazilian children. braz. dent. j. 2010;21(5):396-400. doi: 10.1590/s0103-64402010000500003. 19. lima-arsati ybo, santos hka, gomes arlf. evaluation of a protocol to simulate in vitro the decreased absorption of fluoride from toothpaste due to gastric content. in: xix aboprev meeting. braz j oral sci. 2015;15(1):87. doi: 10.20396/bjos.v14i1.8641314. 20. munro bh. statistical methods for health care research. 3rd ed. new york: lippincott williams & wilkins; 1997. 21. hall je. guyton and hall textbook of medical physiology. 13. ed. philadelphia, pa: elsevier; 2016. 22. mclauchlan g, fullarton gm, crean gp, mccoll ke. comparison of gastric body and antral ph: a 24 hour ambulatory study in healthy volunteers. gut. 1989 may;30(5):573-8. doi: 10.1136/ gut.30.5.573. 23. thermo fisher scientific. thermo scientific orion fluoride ion selective electrode user guide. 254792-001, revision b. 2016 sep [cited 2020 jul 23]. available from: https://www.thermofisher.com/document-connect/document-connect. html?url=https%3a%2f%2fassets.thermofisher.com%2ftfs-assets%2flsg%2fmanuals% 2fd15872~.pdf&title=rmx1b3jpzgugsw9uifnlbgvjdgl2zsbfbgvjdhjvzguglsbvc2vyied1awrl. 24. whitford gm. absorption and plasma concentrations of fluoride. in: whitford gm. the metabolism and toxicity of fluoride. 2. ed. basel: karger; 1996. chapter 2, p.10-29. doi: 10.1159/ isbn.978-3-318-04022-7. 1http://dx.doi.org/10.20396/bjos.v19i0.8656652 volume 19 2020 e206652 original article 1 department of prosthodontics and periodontology, piracicaba dental school, university of campinas (unicamp), piracicaba, são paulo, brazil. corresponding author: renata cunha matheus rodrigues garcia https://orcid.org/0000-0001-8486-3388 department of prosthodontics and periodontology, piracicaba dental school, university of campinas, av. limeira, no 901, bairro areião, piracicaba, sp, brazil, cep: 13414-903, phone number: +55 19 2106-5240 / fax number: +55 19 2106-5211 e-mail: regarcia@fop.unicamp.br received: september 14, 2019 accepted: december 02, 2019 removable prostheses improve oral health-related quality of life and satisfaction of elderly people with rheumatoid arthritis bruna fernandes moreira alfenas1, kelly machado de andrade1, talita malini carletti1, renata cunha matheus rodrigues garcia1,* rheumatoid arthritis (ra) is an autoimmune disease that affects joint tissues and causes severe physical and functional impairments on quality of life due to muscular and articular pain. the involvement of temporomandibular joint in ra interferes with mouth opening and masticatory process. however, no studies addressed the impact of ra on oral health-related quality of life (ohrqol) and satisfaction with prostheses use in elderly people. aim: this study assessed the impact of oral rehabilitation with conventional dentures on the ohrqol and prostheses satisfaction in elderly patients with ra, associated or not with temporomandibular disorder (tmd). methods: forty-five elderly were enrolled and divided into three groups: (1) ra and tmd (n=15, experimental), (2) ra without tmd (n=15, experimental), and (3) without ra and without tmd (n=15, control). the ohrqol and the prostheses satisfaction were evaluated before and after new oral rehabilitation with partial and/or complete dentures. the ohrqol and prosthesis satisfaction were assessed and verified through ohip-14 questionnaire and visual analogue scale, respectively. results: tmd group exhibited the worst mean values (p<0.05) for all ohip-14 domains before insertion of new dentures. group 2 showed worst means (p<0.05) compared to controls for functional limitation and physical pain domains of the ohip-14, but not in the general score. patients showed better outcomes of satisfaction with prostheses use only after the new rehabilitation. conclusion: the use of new and well-fitted dentures improves all domains of ohrqol in patients with ra and tmd and all groups were satisfied with prostheses use after the new rehabilitation with conventional dentures. keywords: arthritis rheumatoid. dental prosthesis. quality of life. oral health. patient satisfaction. https://orcid.org/0000-0001-8486-3388 2 alfenas et al. introduction rheumatoid arthritis (ra) is a chronic auto-inflammatory disease that affects joint tissues and causes severe physical and functional impairments on quality of life (qol) in the disease bearers1-4. the occurrence of ra is over the middle-aged people, between the fourth and sixth decades. it is an autoimmune disease probably caused by environmental factors, such as drugs and diet, in association with regulatory genes responsible to express the immune disease5,6. the american college of rheumatology has updated the guidelines on ra, so that approaches to manage the disease can be applied to prevent pain, loss of function and joint disturbances7. to diagnose ra, in most cases the synovitis is identified through clinical exam in phalangeal joints, in combination with large or other small joints, with swelling and pain episodes1. once temporomandibular joint (tmj) is the unique synovial orofacial joint, it can be frequently affected in ra, including condylar and disc alterations8. however, literature is variable in reporting the frequency of temporomandibular disorder (tmd) due to ra8-10. some authors found the presence of limited mouth opening11, tmj pain10,11, muscular hyperactivity, and masticatory damage12. still, some risk factors have been associated with the involvement of tmj in patients with ra, as being of female gender, presence of mental disorders, insomnia or stroke13. concerning facial joints involvement and the consequences of ra, few reports14,15 verified the impact of oral rehabilitation with conventional dentures in patients with the disease. improvement of masticatory efficiency, bite force and mandibular movements after new prostheses use in individuals with ra has been reported14,15. it is also known that ra is associated with a decreased qol16 due to pain, ageing process17 and, functional limitation18. however, the authors are unaware of studies on ra and oral health-related quality of life (ohrqol) in elderly patients wearing complete or partial conventional dentures. for this reason, considering completely or partially edentulous people wearing old and misadjusted prostheses, the replacement of missing teeth could bring positive results on oral and general health. therefore, the present study aimed to evaluate the impact of oral rehabilitation with conventional dentures on the ohrqol and satisfaction with prostheses use in elderly patients with ra, associated or not with tmd. materials and methods study design this cross-sectional study performed subjective assessments concerning the ohrqol and prosthesis satisfaction among elderly individuals with or without ra and/or tmd, before and after a 2-month prosthetic rehabilitation with new removable dentures. the participants of this clinical research had already participated in previous study14, in which masticatory function and mandibular movements were observed. all participants were first evaluated with their old prosthesis in the mouth, and after receiving new removable dentures. ohrqol was assessed by ohip-14 questionnaire19-21, and prosthesis satisfaction by a visual analogue scale (vas)22. 3 alfenas et al. subjects according to sample size calculation (test power of 80% and α value of 0.05.), a total of 45 participants were considered to be enrolled in the study (figure 1). subjects were selected without restriction of gender and race and were divided into 3 groups: (1) elderly with ra and tmd (n = 15, experimental), (2) elderly with ra without tmd (n = 15, experimental), and (3) elderly without ra and without tmd (n = 15, control). following the inclusion criteria, elderly volunteers must present: 60 years or older; be partially or completely edentulous, and using inadequate removable partial and/ or complete dentures, according to the criteria of vigild23; and present diagnosis of ra. besides these criteria, group 1 participants must present tmd. all volunteers agreed with voluntary participation and signed the informed consent form, approved by the ethics committee of piracicaba dental school, university of campinas (#068/2012). this study was also registered in the brazilian registry of clinical trials (rebec #rbr-6 qkjzy). volunteers with ra were selected from those attended in the medical specialties ambulatory, department of rheumatology (limeira, são paulo, brazil), managed by university of campinas. controls were selected from patients who sought prosthetic treatment at the piracicaba dental school, university of campinas and also met the same inclusion criteria described above, but with absence of both ra and tmd. patients with the presence of severe malocclusions, craniofacial injuries or recent orofacial surgeries, intake of muscle-related medication, or with degenerative diseases, including parkinson’s or alzheimer’s disease, were excluded of this study. the diagnosis of ra was performed by a rheumatologist, according to clinical and laboratory criteria, based on the american college of rheumatology classification: (1) morning joint stiffness (up to 1 hour); (2) arthritis in three or more joints with soft tissue edema or joint effusion; (3) arthritis in the hand joints (wrist, proximal interphalangeal and metacarpophalangeal joints); (4) symmetric arthritis; (5) rheufigure 1. flowchart for selection of volunteers. experimental groups ra medical specialties ambulatory/ unicamp control group without ra piracicaba dental school/ unicamp clinical exam and radiographs rdc/tmd – axis i group 1 (n=15) ra and tmd group 2 (n=15) ra without tmd group 3 (n=15) without both ra and tmd 4 alfenas et al. matoid nodules; (6) high levels of serum rheumatoid factor (destructive antibodies); and (7) radiographic changes (erosions or decalcification located in hands and wrists). in the presence of four of the seven criteria, for at least 6 weeks, (american college of rheumatology subcommittee on rheumatoid arthritis guidelines) it is confirmed the ra diagnosis. meanwhile, for the tmd diagnosis, all volunteers underwent a clinical examination by means of the axis i of research diagnostic criteria (rdc/tmd)24. in the axis i, clinical aspects of tmds are involved and divided into three aspects: (1) muscle disorders, (2) joint disorders and (3) arthralgia, arthrosis, and arthritis. the last one was used to characterize and select volunteers of groups 1 and 2, in the presence of tmd or not, respectively. after clinical examination of muscle insertions, mucosa-bearing area and remaining teeth, vigild´s criteria23 were applied to assess prosthesis conditions with regards to stability, retention, occlusion, vertical dimension and defects. to participate in this study, at least one of these criteria should be unsatisfactory. prosthesis new prosthetic rehabilitation with removable complete and/or partial dentures had been provided to the volunteers. the prosthetic devices were made following conventional techniques25,26. firstly, dental impressions were taken from both arches (hydrogum, zhermack, rovigo, italy) so that stone casts were made for all the volunteers. after that, elderly who received complete dentures had custom trays manufactured with acrylic resin (vipiflash, vipi, são paulo, brazil), while those in need for a removable partial denture had their metallic framework made by a dental technician, following the previous surveying analysis of the casts. for the completely edentulous elderly, functional impressions of both arches were taken. in the partially edentulous arches, the frameworks were proved in the mouth. following these steps, maxillomandibular relationships of all patients were obtained through the occlusal vertical dimension, and dental casts were positioned in a semi-adjustable articulator (a7 plus, bioart, são paulo, brazil), with the help of the facial bow. artificial teeth (biotone, dentsply, ny, usa) were mounted over the wax rims, following the bilateral balanced occlusion. after the prostheses being clinically evaluated by means of aesthetic features, they were finished, polished and delivered to patients. all patients were instructed to keep hygiene and care of the prostheses. prosthetic adjustments on occlusion and acrylic base of the dentures were performed for 2 months to allow patient’s adaptation with no complaints. oral-health related quality of life (ohrqol) to evaluate the ohrqol, the simplified portuguese version of the oral health impact profile (ohip-14) was used20,21. this is a questionnaire comprised of 14 questions, in seven conceptual dimensions of oral health: (1) functional limitation, (2) physical pain, (3) psychological discomfort, (4) physical incapacity, (5) psychological incapacity, (6) social incapacity, and (7) social disadvantages. for each of the domains, volunteers could answer “often”, “sometimes”, “rarely”, “never” and “do not know”; whose weights/ scores were 4, 3, 2, 1 and 0, respectively. the final index could 5 alfenas et al. vary between 0 and 28 points for each volunteer and it was classified as weak (0-9 points), average (10-18 points) and strong (19-28 points). thus, the lower the score, the better the ohrqol. patient satisfaction with the prosthetic treatment volunteer’s satisfaction with their prostheses was measured by a visual analogue scale (vas)22. it consists on a 10 cm ruler, whose extremities indicate the minimum (zero or no satisfaction) and the maximum value (10 or the highest satisfaction), concerning domains of retention, comfort, mastication, speaking, hygiene, aesthetics, and general satisfaction, of both old and new, upper and lower prostheses. the volunteers were instructed to indicate, with a vertical mark over the ruler, the level of satisfaction with prosthesis use before and after the rehabilitation treatment. the greater the score (ranging from 0 to 10), the better the subjective perception with the prosthetic treatment. statistical analysis data were first submitted to the shapiro-wilk test and evaluations of asymmetry coefficient and kurtosis. anova was used when the residues adhered to the gaussian distribution; otherwise, the analysis of variance on ranks (anova-r) was adopted. a generalized linear mixed model with repeated measures was applied to test the effect of prosthetic treatment (before versus after) in each group and for the effect of group (experimental groups versus control) at each time point. tukey-kramer for multiple comparisons was applied as a post-hoc test. all statistical analyses were performed on sas system (release 9.3; sas institute inc., cary, usa) with a significance level of 5%. results sociodemographic data were published in previous studies14. volunteers were aged 60 to 80 years old. a total of 21 subjects received complete dentures in both jaws, while 12 were rehabilitated by upper and lower removable partial dentures. the remaining participants received upper complete dentures and lower removable partial dentures. most of them were married, lived in the urban area, had completed the elementary school and were retired. groups 1 and 2 were diagnosed with ra for, approximately, 10.9 years14. table 1 shows the anova f and p values for main effects of group and time point, and the interaction. the following ohip-14 domains: psychological discomfort, physical incapacity, psychological incapacity, social incapacity, and social disadvantages showed significant group-time point interaction. in contrast, functional limitation and physical pain domains, as well as the general score of the ohip-14 showed significant main effects for both, group and time point factors, while the patient satisfaction with prosthesis revealed significant effect only of time point. 6 alfenas et al. mean comparisons between before and after prosthetic treatment showed that ra and tmd group exhibited the highest means values (p < 0.05) for psychological discomfort, physical incapacity, psychological incapacity, social incapacity, and social disadvantages domains scores on the ohip-14 before insertion of new dentures (table 2). a similar trend was observed for the patients with ra without tmd, however only for psychological discomfort, physical incapacity, and psychological incapacity domains. table 1. f and p values from anova to test ohip-14 domains and prosthesis satisfaction scores. variable group time point group × tp interaction f p f p f p ohip-14 general score 7.87 0.0013 74.93 0.0001 2.07 0.1392 functional limitation 8.64 0.0007 18.78 0.0001 1.52 0.2298 physical pain 12.06 0.0001 31.71 0.0001 2.31 0.1117 psychological discomfort 9.17 0.0005 59.66 0.0001 5.39 0.0082 physical incapacity 3.66 0.0342 51.23 0.0001 4.29 0.0202 psychological incapacity 0.0004 0.0001 0.0172 social incapacity 4.50 0.0170 28.27 0.0001 4.50 0.0170 social disadvantages 0.0043 0.0001 0.0043 prostheses satisfaction 0.3713 0.0001 0.2989 p values < 0.05 indicate significant evidence of the main effects (group and time point) and interaction. table 2. mean (standard deviation) of ohip-14 domains with significant interaction between group and time points. ohip-14 domain groups time point before pt after pt psychological discomfort ra + tmd 1.92 (1.12) a 0.27 (0.69) b ra – tmd 1.49 (1.21) a 0.23 (0.41) b control 0.29 (0.44) b 0.00 (0.00) b physical incapacity ra + tmd 1.74 (1.10) a 0.20 (0.56) c ra – tmd 1.05 (1.10) a 0.07 (0.18) c control 0.50 (0.70) bc 0.10 (0.20) c psychological incapacity ra + tmd 1.71 (0.97) a 0.31 (1.03) bc ra – tmd 0.89 (1.37) b 0.00 (0.00) c control 0.33 (0.64) bc 0.00 (0.00) c social incapacity ra + tmd 1.04 (0.99) a 0.00 (0.00) b ra – tmd 0.28 (0.75) b 0.00 (0.00) b control 0.39 (0.86) ab 0.00 (0.00) b social disadvantages ra + tmd 0.95 (0.90) a 0.00 (0.00) b ra – tmd 0.39 (0.73) b 0.00 (0.00) b control 0.21 (0.58) b 0.00 (0,00) b mean values followed by the same letter do not differ significantly from each other by the tukey-kramer test, with a significance level of 5%. pt, prosthesis treatment. 7 alfenas et al. prior to prosthetic treatment, the control group showed significantly lower mean values (p <0.05) than the ar + tmd group for all the ohip-14 domains, except for social incapacity. besides, controls also presented significantly lower means (p < 0.05) than the ar-tmd group in the psychological discomfort and physical incapacity domains. after the insertion of the new prostheses, no significant differences among the groups were detected (table 2). concerning those ohip-14 domains which showed significant main effects for group and time point (but not for the interaction), their mean values (sd) are shown in table 3. there was a significant reduction in the mean values (p < 0.05) for the general ohip-14 score, as well as for the functional limitation and physical pain domains after the prosthetic treatment for all studied groups, which means that ohrqol was greatly improved after rehabilitation with new dentures. in addition, irrespectively of the prosthetic treatment, patients with ra and tmd presented significant (p < 0.05) higher values than control group. in addition, patients with ra without tmd group showed increased means (p < 0.05) compared to controls for functional limitation and physical pain domains of the ohip-14, but not in the general score. finally, figure 2 illustrates the analysis of patient satisfaction with prostheses use, showing a significant main effect exclusively for the time point factor. thus, regardless of the ra and/or tmd, all patients were greatly satisfied with the new dentures. figure 2. mean values (sd) of patient satisfaction with the prosthetic treatment. p ro st he se s sa tis fa ct io n before time point after 9,40 (0,81) b 4,18 (2,28) a 10,00 9,00 8,00 7,00 6,00 5,00 4,00 3,00 2,00 1,00 0,00 table 3. mean (standard deviation) of ohip-14 domains with significant main ohip-14 domains time point group before pt after pt ra+tmd ra–tmd control general 6.65 (6.04) a 1.23 (2.23) b 6.34 (6.17) a 3.81 (5.15) ab 1.67 (3.12) b functional limitation 0.85 (0.99) a 0.31 (0.58) b 0.91 (0.91) a 0.71 (0.98) a 0.12 (0.26) b physical pain 1.21 (1.06) a 0.44 (0.65) b 1.34 (1.09) a 0.88 (0.89) a 0.27 (0.45) b effects for group and time points. mean values followed by the same letter do not differ significantly from each other by the tukey-kramer test, with a significance level of 5%. pt, prosthesis treatment 8 alfenas et al. discussion this clinical trial assessed the ohrqol and satisfaction with prosthetic treatment between patients with ra and tmd or not, before and after 2 months of new removable dentures (cd or rpd) insertion. most of ohip-14 domains exhibited significant interactions among groups and time points with increased values in elderly with ra and tmd before new dentures installation, meaning that such elderly had worse ohrqol. the use of old and worn out dentures contributes to the worse perception that elderly may have about their qol27. a recent report showed a correlation between denture wearing and better outcomes of ohrqol4. even though the last authors4 did not mention whether prostheses conditions were satisfactory or not, other reports showed that the use of well-fitted dentures improves masticatory performance, bite force and mandibular movements of patients with ra and tmd14,15. thus, it can be suggested that improvements in such objective variables, could positively influence the subjective measurements, like those of ohip-14 questionnaire. still on groups comparisons before new dentures insertion, the oral implications of tmd in ra11, such as pronounced muscular symptoms, soreness, pain during movement of masticatory muscles, arthralgia of tmj and tinnitus, associated with the absence of teeth and use of misfit dentures may impact the subjective perception of elderly ohrqol. thus, we can suppose that prosthetic problems, resembling the use of misfit dentures, negatively influence ohrqol when tmj are affected by the ra. similarly, patients with ra but without tmd showed worse outcomes of qol before prosthetic treatment, apart from social incapacity and social disadvantages domains, standing for impaired ohrqol in this group. hence, these outcomes possibly indicate that ra does not influence the social life aspects of elderly, but uniquely the presence of tmd and the use of misfit dentures. in this sense, a systematic review exhibited that the use of new and comfortable dentures provided better masticatory efficiency and fitting over the soft tissues, contributing to the higher values of ohip functional and physical domains28. indeed, given the worries concerning the old prostheses conditions, the lack of retention, support, and stability, may bring a psychological discomfort to make social contacts, keep interpersonal interaction, rather than strictly because of an autoimmune disease. preceding prosthetic treatment, controls also presented better values of ohip-14 domains than those with ar and tmd, except for social incapacity. it is reported that personal behavior concerning the social disability present a low impact on ohrqol, once patients with absence of teeth do not avoid social interaction29. thus, our outcome is confirmed by previous data29, which emphasizes that the presence of old dentures does not avoid the social welfare of patients, even in the presence of ra. furthermore, even with the use of old dentures, elderly volunteers without the autoimmune disease (control group) showed better perception of ohrqol in psychological discomfort and physical incapacity domains, compared to patients without tmd. previous studies reported that both domains are of greater impact over elderly patients qol29,30. for this reason, regardless of tmd, ra is a chronic and limiting health condition, due to the severe functional disability on body joints, affecting individuals qol1,2,4. therefore, the psychological concern about teeth, mouth or dentures are mainly related to damage 9 alfenas et al. on masticatory function and changes in diet habits30. a systematic review showed a greater commitment of ra on physical domain than mental health16, since bodily pain and decreased physical functioning is commonly found in ra patients. accordingly, our findings possibly reinforce the idea of a physical-limited life, as well as the psychological processes to identify and deal with daily pain, signs, and symptoms of the disease, which do not occur in a patient without the ra diagnosis. following prosthetic rehabilitation with new conventional dentures, no differences were noticed among the three groups, with respect to the ohip-14 domains. the multidimensional concept of qol involves the individual perception of social, physical, psychological, environmental, spiritual and level of independence31. thereby, since elderly wearing dental prostheses presented prominent expectations regarding the study treatment, our experimental groups reached the same qol values of control group. irrespective of ra and tmd presence, insertion of new dentures improved ohip-14 general, functional limitation and physical pain domains. our outcomes corroborate with those from alves et al.32 (2018), which also showed enhancement of ohip-edent domains after complete dentures rehabilitation. contrastingly, bonnet et al.3 (2016) assessed the qol of patients in need of new prosthetic rehabilitation, however, regardless of prostheses type, whether complete or partial removable dentures, there was not a considerable impact on ohrqol before and after rehabilitation3. despite the fact that implant prostheses were freely offered in the study, these authors adopted gohai questionnaire to assess the qol and, either, did not evaluate elderly with an autoimmune disease3. in addition, irrespective of the prosthetic treatment, controls exhibited greater ohip-14 scores with regards to functional limitation and physical pain. again, this finding confirms the limitations of ra, which causes polyarticular inflammatory alterations, damaging and limiting the physical integrity and the performance of daily activities16-18,33. our results also showed that regardless of groups, patient satisfaction was greater after the installation of the new dentures. these results corroborate with medeiros et al. (2019)28, who also reported the recovery of functional and aesthetical parameters with new rpd and/or cd. in the presence of a damaged health condition, it is reasonable to think that after a deficient dental prostheses use, delivery new prosthetic devices may increase the level of satisfaction of the autoimmune disease bearers’ patients. moreover, all prosthetic devices were made following the conventional technique by experienced professionals, which might have contributed to the positive results. although the literature contains a distinct tool to evaluate the consequences of ra in qol34, the use of the ohip-14 in the present study can figure as a limitation. however, different from a specific instrument to detect the impact of an autoimmune disease on qol, the ohip-14 allowed the perception of questions concerning the mouth, dentures, and teeth deterioration. in addition, a two-month follow-up period is limited and might not reflect the long-term ohrqol or satisfaction with prostheses use of ra patients, thus longer periods can be further assessed. finally, the diagnosis of an autoimmune disease is of great relevance to clinicians and associated professionals. 10 alfenas et al. this way, identifying the patient’s perception about their qol is extremely important to define clinical strategies and organize treatment proposals according to their needs. in conclusion, the use of new and well-fitted dentures improves all domains of ohrqol in patients with ra and tmd. meanwhile, better results of qol after prosthetic rehabilitation were observed for the psychological discomfort, psychological incapacity, and physical incapacity domains in patients with ra without tmd group. all groups were satisfied with prostheses use after the new rehabilitation with conventional dentures. acknowledgments the authors acknowledge the support of the são paulo research foundation (grant number 12/08374-4). references 1. aletaha d, neogi t, silman aj, funovits j, felson dt, bingham co 3rd,et al. 2010 rheumatoid arthritis classification criteria: an american college of rheumatology/european league against rheumatism collaborative initiative. arthritis rheum. 2010 sep;62(9):2569-81. doi: 10.1002/art.27584. 2. hoyuela cp, furtado rn, chiari a, natour j. oro-facial evaluation of women with rheumatoid arthritis. j oral rehabil. 2015 may;42(5):370-7. doi: 10.1111/joor.12255. 3. bonnet g, batisse c, segyo jw, veyrune jl, nicolas e, bessadet m. influence of the renewal of removable dentures on oral health related quality of life. springerplus. 2016 nov 28;5(1):2019. doi: 10.1186/s40064-016-3699-7. 4. chamani g, shakibi mr, zarei mr, rad m, pouyafard a, parhizkar a, et al. assessment of relationship between xerostomia and oral health-related quality of life in patients with rheumatoid arthritis. oral dis. 2017 nov;23(8):1162-1167. doi: 10.1111/odi.12721. 5. scott dl, wolfe f, huizinga tw. rheumatoid arthritis. lancet. 2010 sep 25;376(9746):1094-108. doi: 10.1016/s0140-6736(10)60826-4. 6. nemtsova mv, zaletaev dv, bure iv, mikhaylenko ds, kuznetsova eb, alekseeva ea, et al. epigenetic changes in the pathogenesis of rheumatoid arthritis. front genet. 2019 jun 14;10:570. doi: 10.3389/fgene.2019.00570. 7. american college of rheumatology subcommittee on rheumatoid arthritis guidelines. guidelines for the management of rheumatoid arthritis: 2002 update. arthritis rheum. 2002 feb;46(2):328-46. 8. helenius lm, hallikainen d, helenius i, meurman jh, könönen m, leirisalo-repo m, et al. clinical and radiographic findings of the temporomandibular joint in patients with various rheumatic diseases. a case-control study. oral surg oral med oral pathol oral radiol endod. 2005 apr;99(4):455-63. 9. larheim ta, smith hj, aspestrand f. temporomandibular joint abnormalities associated with rheumatic disease: comparison between mr imaging and arthrotomography. radiology. 1992 apr;183(1):221-6. 10. mortazavi n, babaei m, babaee n, kazemi hh, mortazavi r, mostafazadeh a. evaluation of the prevalence of temporomandibular joint involvement in rheumatoid arthritis using research diagnostic criteria for temporomandibular disorders. j dent (tehran). 2018 nov;15(6):332-8. 11 alfenas et al. 11. crincoli v, anelli mg, quercia e, piancino mg, di comite m. temporomandibular disorders and oral features in early rheumatoid arthritis patients: an observational study. int j med sci. 2019 jan 1;16(2):253-263. doi: 10.7150/ijms.28361. 12. marim gc, machado bcz, trawitzki lvv, de felício cm. tongue strength, masticatory and swallowing dysfunction in patients with chronic temporomandibular disorder. physiol behav. 2019 oct 15;210:112616. doi: 10.1016/j.physbeh.2019.112616. 13. lin cy, chung ch, chu hy, chen lc, tu kh, tsao ch, et al. prevalence of temporomandibular disorders in rheumatoid arthritis and associated risk factors: a nationwide study in taiwan. j oral facial pain headache. 2017 fall;31(4):e29-e36. doi: 10.11607/ofph.1917. 14. andrade km, alfenas bfm, rodrigues garcia rcm. influence of removable prostheses on mastication in elderly subjects with rheumatoid arthritis. j oral rehabil. 2018 apr;45(4):295-300. doi: 10.1111/joor.12592. 15. andrade km, alfenas bf, campos ch, rodrigues garcia rc. mandibular movements in older people with rheumatoid arthritis. oral surg oral med oral pathol oral radiol. 2017 may;123(5):e153-e159. doi: 10.1016/j.oooo.2017.01.014. 16. matcham f, scott ic, rayner l, hotopf m, kingsley gh, norton s, et al. the impact of rheumatoid arthritis on quality-of-life assessed using the sf-36 : a systematic review and meta-analysis. semin arthritis rheum. 2014 oct;44(2):123-30. doi: 10.1016/j.semarthrit.2014.05.001. 17. hillsdon mm, brunner ej, guralnik jm, marmot mg. prospective study of physical activity and physical function in early old age. am j prev med. 2005;28(3):245-50. 18. jakobsson u, hallberg ir. review pain and quality of life among older people with rheumatoid arthritis and/or osteoarthritis: a literature review. j clin nurs. 2002;11(4):430-43. 19. slade gd, spencer aj. development and evaluation of the oral health impact profile. community dent health. 1994 mar;11(1):3-11. 20. oliveira bh, nadanovsky p. psychometric properties of the brazilian version of the oral health impact profile-short form. community dentistry oral epidemiology. 2005 aug;33(4):307-14. 21. pistorius j, horn jg, pistorius a, kraft j. oral health-related quality of life in patients with with removable dentures. schweiz monatsschr zahnmed. 2013;123(11):964-71; 955. 22. awad ma., feine js. measuring patient satisfaction with mandibular prostheses. community dent oral epidemiol. 1998 dec;26(6):400–5. 23. vigild m. denture status and need for prosthodontics treatment among institutionalized elderly in denamark. community dent oral epidemiol. 1987 jun;15(3):128-33. 24. dworkin sf, leresche l. research diagnostic criteria for temporomandibular disorders: review, criteria, examinations and specifications, critique. j craniomandib disord.1992 fall;6(4):301-55. 25. telles d. [total prosthesis conventional and on implants]. são paulo: santos; 2009. portuguese. 26. phoenix rd; cagna dr, defreest cf. stewart’s clinical removable partial prosthodontics. 4. ed. quintessence; 2008. 518 p. 27. hadzipasic-nazdrajic a. quality of life with removable dentures. mater sociomed. 2011;23(4):214-20. doi: 10.5455/msm.2011.23.214-220. 28. de medeiros akb, campos mftp, da silva costa rsg, de melo la, barbosa gas, carreiro adfp. improvement in quality of life of elderly edentulous patients with new complete dentures: a systematic review. int j prosthodont. 2019 may/jun;32(3):272-277. doi: 10.11607/ijp.6075. 29. sáez-prado b, haya-fernández mc, sanz-garcía mt. oral health and quality of life in the municipal senior citizen’s social clubs for people over 65 of valencia, spain. med oral patol oral cir bucal. 2016 nov;21(6):e672-8. 12 alfenas et al. 30. echeverria ms, wünsch is, langlois co, cascaes am, ribeiro silva ae. oral health-related quality of life in older adults — longitudinal study. gerodontology. 2019 jun;36(2):118-124. doi: 10.1111/ger.12387. 31. the world health organization quality of life assessment (whoqol): position paper from the world health organization. soc sci med. 1995 nov;41(10):1403-9. 32. alves ac., cavalcanti rva., calderon ps., pernambuco l, alchieri jc. quality of life related to complete denture. acta odontol latinoam. 2018 aug;31(2):91-96. 33. hodkinson b., musenge e., ally m., meyer pw, anderson r, tikly m. functional disability and health-related quality of life in south africans with early rheumatoid arthritis. scand j rheumatol. 2012 oct;41(5):366-74. doi: 10.3109/03009742.2012.676065. 34. de jong z, van der heijde d, mckenna sp, whalley d. the reliability and construct validity of the raqol: a rheumatoid arthritisspecific quality of life instrument. br j rheumatol. 1997 aug;36(8):878-83. 1http://dx.doi.org/10.20396/bjos.v18i0.8657330 volume 18 2019 e191663 original article 1 department of restorative dentistry, piracicaba dental school, state university of campinas unicamp, piracicaba, sp, brazil. corresponding author: débora alves nunes leite lima, (dds, msc, phd, associate professor), e-mail: dalima@ unicamp.br; department of restorative dentistry operative dentistry division, piracicaba dental school university of campinas, p.o. box 52, 13414-903, piracicaba, sp, brazil. received: may 16, 2019 accepted: october 01, 2019 resolution of a fluorosis case through the association of minimally invasive techniques: microabrasion and tooth bleaching danielle ferreira sobral-souza1, josué junior araújo pierote1, flávio henrique baggio aguiar1, luís alexandre maffei sartini paulillo1, débora alves nunes leite lima1,* color changes may interfere with smile balance and they represent a clinical challenge to dentists. dental fluorosis originates from intrinsic factors and it is a disorder of enamel formation during the phase of teeth development, resulting in the change of enamel color. this clinical case report aimed to present the resolution of a case of dental fluorosis through the association of minimally invasive techniques, namely microabrasion and tooth bleaching. a 27-year-old male patient sought the dental clinic of the school of dentistry of piracicaba (fop unicamp, brazil) presenting striped and symmetrical white stains and generalized chromogenic biofilm. after anamnesis and clinical examination, the patient was diagnosed with fluorosis stains. initially, adequacy of the oral environment was performed with prophylaxis and supragingival scraping. then, the enamel microabrasion technique was performed with 6% hydrochloric acid associated with silicon carbide (whiteness rm fgm) and supervised at-home bleaching was performed with 16% carbamide peroxide (whiteness simple 16% fgm). in conclusion, the treatment performed reestablished the aesthetics and harmony of smile color with minimally invasive procedures without causing tooth sensitivity. keywords: tooth bleaching. enamel microabrasion. fluorosis, dental. mailto:dalima@unicamp.br mailto:dalima@unicamp.br 2 sobral-souza et al. introduction dental fluorosis occurs due to the excessive intake of fluoride above the adequate limit and for a long period during the phase of teeth development, resulting in the malformation of tooth enamel. clinically, fluorosis is presented as opaque stains that go from small transverse streaks up to large opaque areas in homologous teeth, with white, yellowish, or brownish colors, and even surface erosion, varying according to the degree of severity1,2. several techniques are indicated for the treatment of fluorosis, from conservative methods such as microabrasion associated or not with tooth bleaching to more invasive methods such as restorations with composite resin, infiltrating resin, veneers, laminates, and ceramic crowns. for the correct treatment indication, a precise diagnosis of the type and depth of the stain is required, which varies according to the severity of fluorosis2-4. the microabrasive agents used for the microabrasion procedure are 6% hydrochloric acid associated with silicon carbide, which is found commercially, or a combination of pumice stone with 37% phosphoric acid, performed in-office5,6. the minimally invasive treatments have been the first option in the scope of aesthetic dentistry, as long as the clinical case allows their indication. thus, this study aims to report the association of two techniques based on such principle for the resolution of a dental fluorosis case. case report a 27-year-old male patient sought assistance at the dental clinic of the school of dentistry of piracicaba (fop unicamp, brazil) because he was unsatisfied with the striped white stains associated with yellowish areas in the buccal aspect of his teeth (figure 1a). after anamnesis and clinical examination, the patient was diagnosed with stains due to dental fluorosis and generalized chromogenic biofilm (figure 1b). initially, the adequacy of the patient’s oral environment was performed with supragingival scraping aided by duflex periodontal curettes (ss white, rio de janeiro, rj, brazil) and prophylaxis was performed with pumice stone (ss white, rio de janeiro, rj, brazil), water at the ratio of (2:1), and rubber cup (kg sorensen, barueri, sp, brazil) coupled with a low-rotation contra-angle handpiece (kavo do brasil, joinville, sc, brazil). after these procedures, the patient was instructed on the required oral hygiene care, toothbrushing, and use of dental floss. next, the transillumination technique was performed to assess the depth of superficial and deep stains (figures 1c and 1d) using a led light-curing unit of 5 w with blue light emission, wavelength of 470 nm, and power of 1200 mw/cm2. hence, considering the age of the patient and the depth of the stain, the more conservative treatment was selected based on a combined approach of enamel microabrasion with at-home bleaching using 16% carbamide peroxide. prior to treatment, the initial color of the patient was recorded, but determining the color tends to be harder in teeth with fluorosis due to the polychrome of the dental 3 sobral-souza et al. elements. tooth color was chosen with the help of a vitapan™ classical visual color scale (vitazähnfabrik, bad säckingen, germany). the upper teeth presented color a1 for incisors and a2 for canines, while the lower teeth presented color a3 for incisors and a3.5 for canines (figures 2a, 2b, 2c, and 2d). figure 1. a: initial smile, front view; b: patient presented with white enamel stain on the maxillary and mandibular teeth; c and d: transillumination to evaluate the staining. after this process it was observed that the upper central incisors, showed superficial stains and tooth 33, demonstrated deep stain. a c b d figure 2. a, b, c, and d: initial color registration with the vitapan® classical visual vision. a c b d 4 sobral-souza et al. microabrasion was performed before tooth bleaching for better controlling stain removal, due to the higher contrast of the stain with the healthy tooth. the following materials were used for this procedure: young metal archwire (golgran, são caetano do sul, sp, brazil), rubber mat (madeitex, são josé dos campos, sp, brazil), 14a and 26 duflex clamps (ss white, rio de janeiro, rj, brazil), and dental floss (hillo, nilópolis, rj, brazil), for the absolute isolation of the surgical area and the protection of soft tissues (figures 3a and 3b). the abrasive agent (whiteness rm, fgm, joinville, sc, brazil) was handled according to manufacturer’s instructions, applied (figure 3c) on the stained enamel surface and rubbed from 5 to 10 seconds aided by a rubber cup (kg sorensen, barueri, sp, brazil) coupled with a low-rotation contra-angle handpiece (kavo, joinville, sc, brazil) (figure 3d). the microabrasive agent was applied 10 times in only one session. immediately after this, the product was removed from the tooth surface with abundant wash and disposable suction device (ss plus do brasil ltda, maringá, pr, brazil) coupled to a high-power compressor (figure 3e). the need for either applying more of the product or not with the wet tooth surface was assessed. at the end of the microabrasion procedure, the tooth surface was polished with diamond paste (diamond excel, fgm, joinville, sc, brazil) in a felt disc (diamond flex, fgm, joinville, sc, brazil) (figure 3f) and then it was washed to remove the product. later, a colorless neutral fluoride gel (2% naf, nova dfl, jacarepaguá, rj, brazil) was applied for four minutes (figures 3g and 3h). figure 3. a: absolute isolation (front view); b: absolute isolation (palatine view); c and d: application of whiteness rm abrasive agent with rubber cup in enamel stained surfaces; e: cleaning of the abrasive agent; f: surface polishing with diamond paste and felt disc; g and h: application of neutral fluorine gel (2% naf) for 4 minutes. a c e g b d f h 5 sobral-souza et al. the second aesthetic treatment performed for resolving this case was at-home tooth bleaching, which was performed one week after the microabrasion session. this technique was performed through alginate molding of the patient (hydrogum, zhermack clinical, italy) and after obtaining the mold, it was poured with type iii plaster stone (herodent tipo iii, coltene, rio de janeiro, brazil), thus originating the models of the upper and lower arches (figure 4a). after cutting out the models, an eva plate (whiteness placas para moldeiras, fgm, joinville, sc, brazil) and a vacuum plasticizer (plastvac p7, bioart, são carlos, sp, brazil) (figure 4b) were used to produce the individualized impression trays. after producing and cutting out the impression trays (figure 4c), their adaptation and comfort in the mouth of the patient were verified (figure 4d). the bleaching agent used was 16% carbamide peroxide (whiteness simple, fgm, joinville, sc, brazil), which was applied in an impression tray (figure 4e) for four hours daily according to the manufacturer’s protocol, for four weeks. after finishing the bleaching procedure, two weeks were waited to reassess the color obtained. the upper incisors and canines presented color b1 (figures 4f and 4g), the lower incisors presented color b1 (figure 4h), and the lower canines presented color a2 (figure 4i), providing uniformity in tooth color when compared to the smile before the treatments performed (figures 5a, 5b, and 5c). the patient received and signed an informed consent form so his images and other clinical information were reported in the journal. figure 4. a: plaster model; b: preparation of individualized trays and vacuum plasticizer; c: tray cutting; d: checking the adaptation of the tray in oral cavity; e: application of the bleaching agent in tray; f, g, h, and i: final color registration with the vitapan® classical visual vision. a c e f h b d g i 6 sobral-souza et al. discussion enamel microabrasion is a conservative technique for removing stains or surface defects in tooth enamel7. the abrasive products associated with acids allow, among other possibilities, performing conservative clinical procedures and achieving immediate and lasting results, with limited loss of tooth enamel8. the microabrasion technique is indicated for the aesthetic treatment of fluorotic white stains, mineralized white stains caused by demineralization from caries or post orthodontic treatment, localized hypoplasia from dental trauma or infections, and idiopathic hypoplasia in which discoloration is limited to the most superficial layer of tooth enamel8,9. clinically, it is difficult to diagnose with precision the actual depth of the intrinsic stain in tooth enamel9,10. therefore, this case report presented a method for analysis of enamel hypoplasia that uses transillumination technique with a led light-curing followed by a combination of enamel microabrasion with carbamide peroxide at-home tooth bleaching. these association of techniques showed good results. transillumination procedure is simple and easy to perform and it helps to differentiate deep from superficial stains, as well as guiding the professional to choose the most adequate treatment for the stained tooth surface. however, the location of the stain in the middle and/or cervical third of the tooth leads to a better indication for the abrasive technique, considering that the incisal third is usually more translucent and that any enamel surface removal in this area may become apparent. besides stain location, another factor that aids the indication of such technique is the transillumination procedure, which is characterized by showing, with light reflection through teeth, the changes in enamel and dentin, favoring the interpretation process11-13. this can be used to estimate the lesion depth, as a darker color indicates deeper staining14. park et al.4 described the utility quantitative light-induced fluorescence (qlf) to determine the depth of the stains. the qlf is used be to analyze the enamel fluorescence through a computer program (qa2 v1.18, inspektor research systems bv) that measures the white stain. however, qlf also have limitation as it is not indicated in the analysis of for interproximal lesion. in the clinical case presented, enamel microabrasion was indicated successfully, because it allowed removing the changed and/or stained tissue with minimal abrasion of the stained surface. this technique used hydrochloric acid at low concentration (6%) associated with particles of silicon carbide, which is commercially available9,14,15. the enamel microabrasion involves minimal enamel loss (25 a to 200 micrometer) when performed correctly, being considered a safe, conservative and atraumatic method9. however, the thickness of the cervical enamel region is smaller. therefore, figure 5. a: initial smile; b: smile after microabrasion treatment; c: final smile after bleaching treatment. a b c 7 sobral-souza et al. care should be taken with the pressure used during the microabrasion procedure in these regions so that the higher the pressure, the greater the amount of enamel removed. in addition, microabrasion enamel wear depends on the time, the number of applications and acids concentration9,14. in case the stains may not be fully removed, other clinical procedures may be applied, among which are direct restorations and bleaching procedures16. in this context, tooth bleaching is considered the most conservative aesthetic treatment option17; however, peroxide cannot remove white stains in enamel, but only reduce the contrast of the stain with the healthy tooth, making the stain any less apparent. a rather common adverse effect of the tooth bleaching technique is tooth sensitivity. although sensitivity is slightly observed in at-home treatments with carbamide peroxide at low concentration after microabrasion, such technique is the most indicated, because it provides a more conservative treatment to an enamel that has already been minimally abraded18,19. thus, at-home bleaching with 16% carbamide peroxide was indicated in this case. either during or after microabrasion and tooth bleaching, the patient did not report the occurrence of tooth sensitivity. based on the results obtained, it may be concluded that the association of microabrasion and at-home tooth bleaching techniques was effective for clinical resolution and aesthetic reestablishment, without causing tooth sensitivity. conflict of interest no potential conflict of interest relevant to this article was reported. references 1. wang y, sa y, liang s, jiang t. minimally invasive treatment for esthetic management of severe dental fluorosis: a case report. oper dent. 2013 jul-aug;38(4):358-62. doi: 10.2341/12-238-s. 2. joiner a. tooth colour: a review of the literature. j dent. 2004;32 suppl:3-12. doi:10.1016/j.jdent.2003.10.013. 3. gugnani n, pandit ik, gupta m, gugnani s, soni s, goyal v. comparative evaluation of esthetic changes in 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[removal of tooth-enamel white stains: clinical and microscopical study]. rev bras odontol. 1990 may-jun;47(3):29-34. 11. litzenburger f, heck k, pitchika v, neuhaus kw, jost fn, hickel r, et al. interand intraexaminer reliability of bitewing radiography and near-infrared light transillumination for proximal caries detection and assessment. dentomaxillofac radiol. 2018 feb 7;47(3):20170292. doi: 10.1259/dmfr.20170292. 12. laitala m-l, piipari l, sämpi n, korhonen m, pesonen p, joensuu t, et al. validity of digital imaging of fiber-optic transillumination in caries detection on proximal tooth surfaces. int j dent. 2017;2017:1-6. doi: 10.1155/2017/8289636. 13. miguéns-vila r, martín-biedma b, varela-patiño p, ruíz-piñón m, castelo-baz p. vertical root fracture initiation in curved roots after root canal preparation: a dentinal micro-crack analysis with led transillumination. j clin exp dent. 2017 oct;9(10):e1218-23. doi: 10.4317/jced.54227. 14. sundfeld rh, sundfeld-neto d, machado ls, franco lm, fagundes tc, briso alf. microabrasion in tooth enamel discoloration defects: three cases with long-term follow-ups. j appl oral sci. 2014 jul;22(4):347-54. doi: 10.1590/1678-775720130672. 15. sundfeld r, franco l, gonçalves r, de alexandre r, machado l, neto d. accomplishing esthetics using enamel microabrasion and bleaching — a case report. oper dent. 2014 apr;39(3):223-7. doi: 10.2341/13-002-s. 16. sundfeld rh, rahal v, croll tp, de alexandre rs, briso alf. enamel microabrasion followed by dental bleaching for patients after orthodontic treatment case reports. j esthet restor dent. 2007;19(2):71-7; discussion 78. doi: 10.1111/j.1708-8240.2007.00069.x 17. kihn pw. vital tooth whitening. dent clin north am. 2007 apr;51(2):319–31, viii. doi: 10.1016/j.cden.2006.12.001. 18. basting r, amaral f, frança f, flório f. clinical comparative study of the effectiveness of and tooth sensitivity to 10% and 20% carbamide peroxide home-use and 35% and 38% hydrogen peroxide in-office bleaching materials containing desensitizing agents. oper dent. 2012 sep;37(5):464-73. doi: 10.2341/11-337-c. 19. moghadam fv, majidinia s, chasteen j, ghavamnasiri m. the degree of color change, rebound effect and sensitivity of bleached teeth associated with at-home and power bleaching techniques: a randomized clinical trial. eur j dent. 2013 oct;7(4):405-11. doi: 10.4103/1305-7456.120655. 1http://dx.doi.org/10.20396/bjos.v19i0.8661689 volume 19 2020 e201689 original article 1 academic department of dentistry for children and adolescents, faculty of stomatology, universidad peruana cayetano heredia, lima, peru. 2 department of biosciences, piracicaba dental school, university of campinas, piracicaba, sp, brazil. * corresponding author: jaime aparecido cury department of biosciences, piracicaba dental school, university of campinas av limeira, 901, piracicaba, sp, brazil, 13414-903 email: jcury@unicamp.br received: october 18, 2020 accepted: november 23, 2020 a simplified protocol to determine total fluoride concentration in naf/ silica-based toothpastes jenniffer quiroz-torres1 , cinthia pereira machado tabchoury2 , carlos liñán-durán1 , antonio pedro ricomini-filho2 , ailin cabrera-matta1 , jaime aparecido cury2,* aim: to determine total fluoride (tf) concentration in na2fpo3/ca-based toothpastes, using fluoride ion selective electrode (f-ise) by the direct technique, it is necessary to use acid (ac+) to hydrolyze the fpo3 2ion and to dissolve insoluble fluoride salts bound to the abrasive. for naf/silicabased toothpastes, the use of acid is not necessary (ac-) and a simplified protocol could be followed. methods: thus, we evaluated tf concentration in seven brands of naf/silicabased toothpastes, following the validated conventional cury’s protocol (ac+) or a simplification of this protocol (ac-). fluoride was analyzed with ise calibrated with fluoride standard solutions prepared in the same conditions as the samples (ac+ or ac-). results: the mean (±sd; n=21) of tf concentrations (µg f/g) found by ac+ (971.3±191.2) and ac(982.4±201.3) protocols were not statistically different (t test, p=0.22). the tf concentrations found agree with those declared by the manufacturers, except for one toothpaste imported from china. conclusion: the findings suggest that the determination of fluoride in naf/silica-based toothpastes can be accurately made using a simplified protocol of analysis. key words: fluorides. toothpastes. silicic acid. dentifrices. ion-selective electrodes. http://dx.doi.org/10.20396/bjos.v19i0.8661689 mailto:jcury@unicamp.br https://orcid.org/0000-0001-7957-8662 https://orcid.org/0000-0002-7660-5685 https://orcid.org/0000-0003-2669-842x https://orcid.org/0000-0002-6593-6040 https://orcid.org/0000-0002-4493-7036 https://orcid.org/0000-0003-1046-5605 2 quiroz-torres et al. introduction toothpaste is considered the most rational way of fluoride use to control caries and the total fluoride concentration in toothpastes is under legislation control worldwide. thus, different methods are used to determine fluoride concentration in toothpastes1 and fluoride ion specific electrode (f-ise) has been used for a long time2,3. the choice of the method for analysis mainly depends on the type of fluoride salt present in the formulation and the abrasive system of the toothpaste. in toothpastes containing calcium-based abrasives, such as calcium carbonate (caco3) or calcium phosphate dihydrate (cahpo4.2h2o), sodium monofluorophosphate (na2fpo3) is the source of fluoride salt used due to the chemical incompatibility of sodium fluoride (naf), stannous fluoride (snf2) or amine fluoride (amf) with calcium (ca++). in formulations na2fpo3/ca-based, part of the total fluoride (tf) is soluble (tsf) as po3f 2ion plus fluoride ion (f-), but part is insoluble (fins.) bound to the abrasive4. naf, snf2 and amf toothpastes are formulated with hydrated silica (sio2), in which tf is soluble as f -. fluoride ion specific electrode (f-ise) has been successfully used to determine the different forms of fluoride in na2fpo3/ca-based and in naf/sio2-based toothpastes following the same protocol of analysis5. to determine total fluoride in na2fpo3/ca-based toothpastes with f-ise, acid is used to hydrolyze fpo3 2ion and to dissolve insoluble fluoride salts bound to the abrasive5. for the determination of tf in ca-free naf toothpastes with f-ise, it is not necessary to use acid because all tf is soluble as fto be promptly analyzed with the electrode. although the use of acid is not theoretically necessary and the protocol used since 1980 by cury  et  al. (1981)3 is valid because there is a high correlation (p=0.996) between tf found and that present in 27 naf/silica-based toothpastes6, the protocol could be simplified. therefore, we evaluated if the step of acid hydrolysis used in the validated protocol of cury et al. (2010)5, could be simplified with accuracy for the determination of tf in naf/silica-based toothpastes. material and methods sampling seven brands (n=3/brand) of fluoride toothpastes marketed for children were purchased; six in lima, peru, and one in piracicaba, brazil. each tube from each brand was purchased in different supermarkets and drugstores and were from different fabrication batches, except for peppa pig brand. table 1 shows information about the toothpastes used. complying with the purpose of this study, all toothpastes chosen were naf/silica-based and were analyzed within their expiration time. 3 quiroz-torres et al. table 1. information on the toothpastes analyzed brand code country of purchase abrasive agent fluoridated agent declared [f] (ppm f) expiration date colgate 6+ a1 peru silica naf 1100 01/2020 colgate 6+ a2 peru silica naf 1100 02/2020 colgate 6+ a3 peru silica naf 1100 10/2019 aqua fresh my big b1 peru silica naf 1150 02/2019 aqua fresh my big b2 peru silica naf 1150 09/2018 aqua fresh my big b3 peru silica naf 1150 06/2018 vitis jr c1 peru silica naf 1000 05/2019 vitis jr c2 peru silica naf 1000 12/2019 vitis jr c3 peru silica naf 1000 04/2019 colgate kids d1 peru silica naf 1100 02/2020 colgate kids d2 peru silica naf 1100 02/2020 colgate kids d3 peru silica naf 1100 02/2020 dento jr e1 peru silica naf 1100 04/2020 dento jr e2 peru silica naf 1100 10/2019 dento jr e3 peru silica naf 1100 peppa pig f1 peru silica naf 1450 06/2021 peppa pig f2 peru silica naf 1450 peppa pig f3 peru silica naf 1450 tandy g1 brazil silica naf 1100 02/2020 tandy g2 brazil silica naf 1100 02/2020 tandy g3 brazil silica naf 1100 02/2020 toothpaste preparation for fluoride analysis toothpastes samples were prepared (figure 1) for analysis of total fluoride (tf) with f-ise by the protocol described by cury et al. (2010)5. an amount of 90 to 110 mg of toothpaste was weighed (± 0.01 mg) and vortexed in 10.0 ml of purified water. duplicate volumes of 0.25 and 1.0 ml of the suspension were transferred to tubes codified ac+ and ac-, respectively conventional (figure 1a) and simplified protocols (figure 1b). to the tubes ac+, 0.25 ml of 2.0 m hcl was added and after 1 h at 45 oc the extracts were buffered with 0.50 ml of 1.0 m naoh plus 1.0 ml of tisab ii. to the tubes ac-, just 1.0 ml of tisab ii was added. 4 quiroz-torres et al. two calibration curves were made (figure 2). for samples prepared according to the conventional protocol (ac+; figure 1a), standards ranging from 0.25 to 4.0  µg f/ml prepared in 0.25 m hcl, 0.25 m naoh and tisab ii 50% (v/v) were used. for samples evaluated by the simplified protocol (ac-; figure 1b), fluoride standards ranging from 4.0 to 32.0 µg f/ml prepared in tisab ii 50% (v/v) were used. all fluoride standards were prepared from naf 99.99% (sigma-aldrich, st louis, mo, usa). the accuracy of the analysis was checked with a standard fluoride solution (orion 940907, thermo scientific, boston, ma, usa) and the average coefficient of variation from triplicates was 1.0 and 2.4%, respectively for the conventional and the simplified protocol. for the analysis, a f-ise (orion 96-06; orion research inc., boston, ma, usa) coupled to an ion analyzer (orion star a214; orion research inc., boston, ma, usa) was used. fluoride concentration in the samples was determined from linear regression of the logarithm of fluoride concentrations of the standards with the respective mv values (r2=0.999 for both calibration curves), using excel spreadsheet (microsoft®). the results were expressed in ppm f (µg f/g; mg f/kg). calibration curve (a) mv 40.0 50.0 60.0 70.0 80.0 90.0 100.0 110.0 120.0 lo g µg f /m l y = -0.0171x + 1.4244 r2 = 1 0.8000 0.6000 0.4000 0.2000 0.0000 -0.2000 -0.4000 -0.6000 -0.8000 calibration curve (b) mv -10.0 0.0 10.0 20.0 30.0 40.0 50.0 lo g µg f /m l y = -0.0174x + 1.4022 r2 = 0.9999 1.5000 1.3000 1.1000 0.7000 0.9000 0.5000 figure 2. calibration curves (n=3) and data of the correlation between the logarithm of fluoride concentrations in standards and the relative mv generated for the conventional (a) and simplified (b) protocols of analysis. toothpaste (90-110 mg) 1.0 ml suspension + 1.0 ml tisab ii accalibration with fluoride standards ranging from 4.0 to 32.0 µg f/ml, prepared in tisab ii 50% (v/v) calibration with fluoride standards ranging from 0.25 to 4.0 µg f/ml, prepared in 0.25 m hcl, 0.25 m naoh and tisab ii 50% (v/v) 0.5 ml 1 m naoh + 1.0 ml tisab ii 0.25 ml suspension + 0.25 ml 2 m hcl 1h 45°c ise analysis ac+ (b)(a) suspension 10 ml h2o ise analysis figure 1. toothpaste sample preparation for total fluoride determination by the conventional (a) and the simplified protocol (b) 5 quiroz-torres et al. figure 4 shows means (sd; n=21) of fluoride concentration found in the toothpaste tubes by the conventional protocol and by the simplified one. the difference between the protocols was not statistically significant (p=0.22). the variation coefficient in percentage was 19.8 and 20.5%, respectively by the conventional and simplified protocol. 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 pp m f (µ g f/ g) toothpastes brand colgate 6+ aqua fresh my big vitis jr colgate kids dento jr peppa pig tandy tf declared tf found conventional tf found simplified figure 3. total fluoride (tf) declared (ppm f) by the manufacturer and concentrations found using the conventional (ac+) and the simplified (ac-) protocol (mean;sd;n=3) statistical analysis the data of tf found in the 21 samples, analyzed according to the conventional protocol using acid (ac+) and those found by the simplified protocol (ac-), were compared by paired t test at 5%. results figure 3 shows total fluoride (tf) concentration declared by the manufacturer and the mean (sd; n=3) concentrations found by the conventional protocol (ac+; acid treatment) and by the simplified one (ac-; without acid treatment) of the seven toothpastes evaluated. excluding the unusual finding for peppa pig toothpaste, the difference (%) between tf found and that declared for the other six toothpastes was -4.1 and -2.8%, respectively for the conventional and simplified protocol. 6 quiroz-torres et al. discussion the protocol of fluoride determination in toothpastes with f-ise by the direct technique and used since 1980 in laboratory of oral biochemistry from fop-unicamp3,5,7-20 is considered chemically valid, reliable, and feasible1. in addition, it is able to estimate how much of the total fluoride presents in na2fpo3/caco3-based toothpaste is bioavailable to be released in the oral cavity during toothbrushing21. however, this protocol could be simplified to determine fluoride in ca-free toothpaste formulations, which contains ionic fluoride salts, such as naf. the findings clearly showed that the simplified protocol used is valid, reliable, and feasible when compared with the results found for the conventional protocol. as shown in figure 4, the mean of total fluoride found in the 21 samples of toothpastes analyzed with the simplified protocol did not statistically differ from the conventional protocol. the results found with the simplified protocol were as reproducible as those found with the conventional protocol, because the variation coefficient (%) of duplicates of analyses (n=21) was 1.3% compared with 1.5% for the conventional. the simplified protocol is cheaper, consumes less time of work and is easier than the conventional one, because the number of laboratorial steps is reduced from the calibration curve up until the sample determination. therefore, it can be used instead of the conventional protocol not only for naf/ca-free-based toothpastes as well as for snf2 or amf formulations. however, the analyst should check if in fact the commercial toothpaste was not formulated with na2fpo3 or the abrasive is ca-free. this warning is important because we have previously found toothpastes containing caco3 as abrasive but according to the manufacturer it was silica19. 0 100 200 300 400 500 600 700 800 900 1000 conventional protocol simplified protocol pp m f ; µ g f/ g sample treatment figure 4. fluoride concentration (ppm f) found in the toothpastes by the conventional (ac+) and the simplified (ac-) protocol; (mean;sd;n=7;p=0.22) 7 quiroz-torres et al. in all toothpastes, except in peppa pig, tf concentration found agreed with the declared by the manufacturer. according to the label of peppa pig’s toothpaste, it should contain 1450 ppm f but we found 512.0 and 520.7 ppm f, respectively for the conventional and the simplified protocol. this result confirmed our previous fluoride determination in other batches of this same toothpaste19, where we found 515.1 ppm f using our conventional protocol5. in addition to the development of this simplified protocol for fluoride determination in toothpastes, the present data confirm the necessity of sanitary vigilance of imported toothpastes from china because if there are approximately 3 times lower tf than the declared in the peppa pig brand analyzed, in previous analyses we found the opposite14. also, we have observed in the present study problems with the type of abrasive declared and that found, as it is already described by chávez et al. (2019)19. furthermore, the present study confirms that when the use of acid is necessary to hydrolyze po3f 2ion and to dissolve insoluble f bound to abrasive, hcl as used in cury’s protocol (2010)5 does not produce artifacts in the analysis because the tf concentration found did not statistically differ comparing the results found with the simplified protocol (figure 3). in conclusion, the findings suggest that the determination of fluoride in naf/silica-based toothpastes can be accurately made using a simplified protocol of analysis. acknowledgments the manuscript was based on the master’s degree in dentistry with mention in pediatric dentistry thesis of the first author at the universidad peruana cayetano heredia. a preliminary report of this study was presented at the 65th orca congress, copenhagen, denmark, july 4-7, 2018. references 1. martinez-mier ea, tenuta lma, carey cm, cury ja, van loveren c, ekstrand kr, et al. european organization for caries research workshop: methodology for determination of potentially available fluoride in toothpastes. caries res. 2019;53(2):119-36. doi: 10.1159/000490196. 2. pearce eif. a laboratory evaluation of new zealand fluoride toothpastes. n z dent j. 1974 apr;70(320):98-108. 3. cury ja, guimarães loc, arbex st, moreira bw. [analysis of fluoride dentifrices: concentration and chemical formula of the fluorides encountered in brazilian products]. rev assoc paul cir dent. 1981 mar-apr;35(2):142-7. portuguese. 4. tenuta lma, cury ja. laboratory and human studies to estimate anticaries efficacy of fluoride toothpastes. monogr oral sci. 2013;23:108-24. doi: 10.1159/000350479. 5. cury j, oliveira m, martins c, tenuta l, paiva s. available fluoride in toothpastes used by brazilian children. braz dent j. 2010;21(5):396-400. doi: 10.1590/s0103-64402010000500003. 6. marin lm, vieira filho w, tenuta lma, tabchoury cpm, cury ja. [reproducibility and validity of fluoride dentermination in toothpastes with ion specific electrode by direct analysis]. braz oral res 2016;30(suppl 1):68. abstract ao0090. portuguese. 8 quiroz-torres et al. 7. sarmiento rv, issao m, cury ja. [study of the availability and stability of fluoride in dentifrices sold in peru]. rev stomatol hered 1994(1-2):12-20. spanish. 8. conde nc, rebelo ma, cury ja. evaluation of the fluoride stability of dentifrices sold in manaus, am, brazil. pesq odontol bras. 2003;17(3):247-53. doi: 10.1590/s1517-74912003000300009. 9. hashizume ln, lima yb, kawaguchi y, cury j. fluoride availability and stability of japanese dentifrices. j oral sci. 2003 dec;45(4):193-9. doi: 10.2334/josnusd.45.193. 10. cury ja, tabchoury cpm, piovano s. [fluoride concentration and stability in dentifrices sold in the autonomous city of buenos aires]. bol ass argent odontol ninos. 2006;35:4-8. spanish. 11. cury ja, tenuta lm, ribeiro cc, paes leme af. the importance of fluoride dentifrices to the current dental caries prevalence in brazil. braz dent j 2004;15(3):167-74. doi: 10.1590/s0103-64402004000300001. 12. carrera ca, giacaman ra, muñoz-sandoval c, cury ja. total and soluble fluoride content in commercial dentifrices in chile. acta odontol scand 2012 dec;70(6):583-8. doi: 10.3109/00016357.2011.640287. 13. ricomini filho ap, tenuta lm, fernandes fs, calvo af, kusano sc, cury ja. fluoride concentration in the top-selling brazilian toothpastes purchased at different regions. braz dent j. 2012;23(1):45-8. doi: 10.1590/s0103-64402012000100008. 14. giacaman ra, carrera ca, muñoz-sandoval c, fernandez c, cury ja. fluoride content in toothpastes commercialized for children in chile and discussion on professional recommendations of use. int j paediatr dent 2013 mar;23(2):77-83. doi: 10.1111/j.1365-263x.2012.01226.x. 15. cury ja, vieira-dantas ed, tenuta lma, romão da, tabchoury cpm, nóbrega df, et al. [fluoride concentration in the most sold mfp/caco3-based brazilian toothpastes at the expiration time]. rev assc paul cir dent. 2015;69:248-51. portuguese. 16. fernández ce, carrera ca, muñoz-sandoval c, cury ja, giacaman ra. stability of chemically available fluoride in chilean toothpastes. int j paediatr dent. 2017 nov;27(6):496-505. doi: 10.1111/ipd.12288. 17. marín lm, vieira w, tenuta lma, tabchoury, cpm, cury ja. [available fluoride concentration in local dentifrices from brazilian regions]. rev assoc paul cir dent. 2017;71(1):60-5. portuguese. 18. soysa ns, cury ja, alles cnra. fluoride concentration and stability in commonly used dentifrices in sri lanka. braz j oral sci. 2018;17:e181244. doi: 10.20396/bjos.v17i0.8654067. 19. chávez ba, vergel gb, cáceres cp, perazzo mf, vieira-andrade rg, cury ja. fluoride content in children’s dentifrices marketed in lima, peru. braz oral res. 2019 jul 1;33:e051. doi: 10.1590/18073107bor-2019.vol33.0051. 20. marin lm, castiblanco ga, usuga-vacca m, cury ja, martignon s. fluoride chemically soluble in toothpastes sold in colombia. ces odontologia 2020. forthcoming. 21. coelho css, cury ja, tabchoury cpm. chemically soluble fluoride in na2fpo3/caco3-based toothpaste as an indicator of fluoride bioavailability in saliva during and after toothbrushing. caries res. 2020;54(2):185-93. doi: 10.1159/000506439. 1 volume 21 2022 e227228 original research braz j oral sci. 2022;21: e227228http://dx.doi.org/10.20396/bjos.v21i00.8667228 1 departament of dentistry school, meridional faculty/imed, rs, brazil. 2 department of business school, meridional faculty/imed, rs, brazil. 3 meridional faculty/imed, brazil corresponding author: lilian rigo meridional faculty/imed, passo fundo, rio grande do sul, brazil senador pinheiro street, 304 passo fundo (rs) – brazil 99070-220 (+55 54) 99927-0441 e-mail: lilian.rigo@imed.edu.br editor: altair a. del bel cury received: october 7, 2021 accepted: april 16, 2022 affect and cognition as antecedents of patients’ trust in the dentist: cross-sectional study lilian rigo1* , kenny basso2 , jandir pauli2 , michele natara portilio3 aim: to evaluate the association of the patients’ perception about dentist’s affect and cognition on trust and, consequently, on intention to return and patient satisfaction with life. methods: analytical cross-sectional study conducted in patients’ adults and elderly at of two dentistry clinics in the south of brazil. patients had to have a previous relationship with the dentist (at least one previous consult) and 18 years of age or older. the data were collected through self-administrated questionnaire using measures adapted from other research, using structural equation modeling. we test using a chi-square difference test (p-value<0,05). results: the mean age of the 197 patients was 37.0 years (σ = 15.5). the affect perceived by the patient at the dentist had a positive effect (β = .53) on the trust that the patient develops in relation to the dentist. the same is true for the effect of the rational or cognitive aspects perceived by the patient at the dentist (β = .41). the trust positively influences the patient’s intention to return to that dentist (β = .82). in addition, the intention to return to the dentist positively influences the patient’s satisfaction with life (β = .49). conclusions: affective and cognitive aspects positively influenced the patient’s trust in the dentist. the greater the patient’s trust in the dentist, the greater the intention to return to that dentist. furthermore, a good relationship with the dentist improve the patient’s satisfaction with life. keywords: affect. cognition. patient satisfaction. trust. https://orcid.org/0000-0003-3725-3047 https://orcid.org/0000-0003-2853-0903 https://orcid.org/0000-0003-4618-6958 https://orcid.org/0000-0002-3169-9470 2 rigo et al. braz j oral sci. 2022;21: e227228 introduction although recent decades in the field of dentistry have been characterized by an evolution of the technologies and procedures of intervention, the interactional aspects of the patient-dentist relationship remain a central aspect of the trust perception in the work developed. in this sense, themes such as trust, affect and cognition begin to cross the provision of this service, and it cannot be disregarded that the quality of this relationship can also significantly impact on the patient’s satisfaction with life1. in medical service research, trust is considered to be a vital element of the physician-patient relationship2. trust is an acceptance of vulnerability by the patient and a belief that the service provider will take care of their interests3,4. corroborating with these authors, maynard and bloor2 (2003) and hupcey and miller5 (2006) argue that despite the various definitions of trust that are proposed, a central element would be the acceptance of vulnerability and belief that the doctor will do the best for the patient. the authors add that the doctor will be the “guardian” of the patient and will ensure that the best treatment is provided5. trust is maintained if expectations are confirmed; however, if they are disconfirmed, trust will likely be lost or minimized. the difference between satisfaction and trust would be that satisfaction refers to an evaluation of an experience already lived and trust refers to a future-oriented vulnerability provision3. mcallister6 (1995) states that interpersonal trust has two main forms: interpersonal trust based on affect and interpersonal trust based on cognition. johnson and grayson7 (2005) argue that cognitive trust would be based on competence and accumulated knowledge, and affective trust based on the feelings generated by the level of care, attention and concern shown by the partner. levels of trust between patient and professional can reveal systematic failures or possible individual communication obstacles. thom et al.8 (2004) emphasized that low levels of trust can be changed and, consequently, improved trust can reduce some disparities, increase access and notably improve health outcomes. in addition, jacquot9 (2005) reported that trust helps significantly in reducing patient anxiety and even reducing fear of dental procedures. therefore, patient trust in the dentist is a key predictor of continuity of treatment, and of whether the patient will believe in the dentist’s assessments and follow the dentist’s guidelines and prescriptions, returning for other consults10. several studies have placed trust as a determinant of relational commitment11-13. authors have sought to relate trust to intention to return and focus on the consequences of development and conquest of trust in relationships; the main consequences studied are loyalty (intention to return) and cooperation12. in the field of dentistry, patient-centered care (pcc) requires assessment of the influence of subjective dimensions involving the dentist-patient interaction13. the relationship between communication skills and empathy of health professionals influences more effective treatment14, the perception of service value15 and customer loyalty16. however, a systematic review of the literature by mills et al.17 3 rigo et al. braz j oral sci. 2022;21: e227228 (2014) has shown that research is still needed to understand the most important features of pcc. the relationship between loyalty and satisfaction with health services is known in the literature18-20. a recent study by zhou et al.21 (2017) reaffirmed the idea that loyalty is the key to the business success of professionals in this area. the research developed a conceptual model integrating the different determinants of loyalty from the literature on the subject, emphasizing the role of organizational citizenship satisfaction and behaviors21. the studies presented show, therefore, the influence of subjective aspects on the perception of satisfaction with the services provided by the dentist, as well as their contribution to generating trust for continuity of treatment (intention to return and loyalty). although satisfaction is a multidimensional concept22, the literature demonstrates the psychometric properties of the measure of satisfaction with life23, the relationship between oral health and life satisfaction24,25, and satisfaction with health services and life satisfaction26. on the other hand, there is a gap between the formation of trust in the dentist, the intention to return (loyalty) and the perception of satisfaction with life. in our research, the problem is approached and presented whit a quantitative analytical method, correlating the various variables, which differs from other studies. the theoretical framework of this study adopted 4 hypotheses: h1: there will be a positive effect of affect shown by the dentist on patient trust. h2: there will be a positive effect of cognition shown by the dentist on patient trust. h3: there will be a positive effect of patient trust on intention to return to that dentist. h4: there will be a positive effect of intention to return to that dentist on the patient’s satisfaction with life. the objective of the present study was to evaluate the association of the patients’ perception about dentist’s affect and cognition on trust and, consequently, on intention to return and patient satisfaction with life. methods ethical aspects this research was approved by the ethics research committee under the number caae 50601915.9.0000.5319 and approval number 1.372.040 (december 16, 2015). design and setting analytical cross-sectional study conducted at of two dentistry clinics in the south of brazil, located in the city center and serve only private patients and do not have health insurance. the sample was not probabilistic containing all patients who were present on the days of the research during the months of august to november 2016 (four months) 4 rigo et al. braz j oral sci. 2022;21: e227228 the participants were patients’ adults and elderly of two dentistry clinics private in the city of passo fundo, rio grande do sul, brazil. participants and data collection to participate in the study, the patient had to have a previous relationship with the dentist (at least one previous consult). as criteria of this study, all patients who were being seen by the dentist for the first time were excluded from the study during the four months of data collection. patients 18 years of age or older were invited to participate in the study, and those who agreed to participate signed the informed consent form (icf). an icf copy was delivered to the respondent. we obtained written consent from the parents/guardians of all participants involved in the study under 18 years of age, which was approved by the ethics research committee. data were collected through a self-administered questionnaire applied by researchers in the dentistry clinics. before data collection, a pretest was done in order to test the methodology in the application of the research instrument to ten (10) patients. the pretest subjects were not included in the final sample, as the purpose of the pilot test was only to verify the participants’ understanding. patients filled out the questionnaires while waiting for service in the waiting room of the dentistry clinics. in this room, there was a box in which the patient anonymously, deposited the completed questionnaire. there were no problems in filling out the instrument by the patients, so it was approved for its application in the sample of this research. measures the affect and cognition scales were adapted from thom27 (2001); the trust scale was adapted from dagger et al.28 (2009), the scale of intention to return was adapted from balkrishnan et al.29 (2003), and satisfaction-with-life scale of diener et al.1 (1985). we based the affection and cognition scales on thom27 (2001), having been adapted and published in a study carried out with the population of a hospital in brazil by silva et al.30 (2015). regarding affection, the patients were asked how much they agree with the following statements: “the dentist tells me everything, being truthful and honest”, “the dentist comforts and reassures me, making me feel cared for”, “the dentist is someone i can count on”. the cognition scale requested the participant to evaluate the following statements: “the dentist is one of the best in his/her area”, “the dentist has good experience in his/her area of expertise”, “the dentist demonstrates up-to-date knowledge in his/her area of expertise”. the trust scale was adapted from dagger et al.28 (2009) adapted and published in brazil by silva et al.30 (2015) and requested the patient to evaluate the following statements: “this doctor can be trusted”, “this doctor can be counted on to do what is right”, “this doctor has integrity” and “this doctor is trustworthy”. the scale of the intention to seek a second opinion included the following items, which were based on our in-depth interviews and balkrishnan et al.29 (2003), adapted and published in brazil by silva et al.30 (2015): “i would consider this dentist as my first 5 rigo et al. braz j oral sci. 2022;21: e227228 choice to treat this type of problem”, “if necessary, i would make further consults with this dentist in the future”, “i would return to this dentist if i had health problems similar to what led me to look for him or her”. all scales were measured using the 7-point likert scale and are presented in table 1 and a higher score represents better results. table 1. confirmatory factor analysis constructs / indicators average variance extracted composed reliability factor loadings mean (sd) trust .88 .97 6.54 (.94) this doctor can be trusted. .93 this doctor can be counted on to do what is right. .94 this doctor has integrity. .95 this doctor is trustworthy. .93 intention to return .88 .96 6.45 (1.04) i would consider this dentist as my first choice to treat this type of problem. .90 if necessary, i would make further consults with this dentist in the future. .96 i would return to this dentist if i had health problems similar to what led me to look for him or her. .95 satisfaction with life .59 .87 5.56 (1.12) in most ways my life is close to my ideal. .72 the conditions of my life are excellent. .82 i am satisfied with my life. .90 so far i have gotten the important things i want in my life. .77 if i could live my life over, i would change almost nothing. .58 affect .70 .87 6.36 (.95) the dentist tells me everything, being truthful and honest. .84 the dentist comforts and reassures me, making me feel cared for. .89 the dentist is someone i can count on. .77 cognition .78 .91 6.28 (.97) the dentist is one of the best in his/her area. .88 the dentist has good experience in his/her area of expertise. .93 the dentist demonstrates up-to-date knowledge in his/her area of expertise. .84 sd = standard deviation. the scale of positive satisfaction with life was adapted from diener et al.1 (1985). in the brazilian validation, the scale presented a cronbach’s α value of 0.8931, and contained the following items: “in most ways my life is close to my ideal”, “the con6 rigo et al. braz j oral sci. 2022;21: e227228 ditions of my life are excellent”, “i am satisfied with my life”, “so far i have gotten the important things i want in my life”, “if i could live my life over, i would change almost nothing”. participants answer it based on a 7-point scale, ranging from 1 (totally disagree) to 7 (totally agree). data analyses once collected, data were processed using the spss® software, version 20.0 (armonk, new york). the missing values were replaced by the maximum expectancy in each variable. the normality of the data was verified through a kolmogorov-smirnov test; multicollinearity was verified by the bivariate correlation and by the variance inflation factor (vif), and the homoscedasticity was verified by levene’s test32. no cases of non-normality, multicollinearity or homoscedasticity were found. the data were analyzed using structural equation modeling with amos® software, version 20.0. to the matrix of data entry, a maximum likelihood estimation model was used. we test for the common method bias using a chi-square difference test between one factor and the multiple factor solution. to analyze the measurement model according to anderson and gerbing33 (1988), reliability and validity were measured using a confirmatory factor analysis. the goodness-of-fit indexes found for the model (χ ² = 340.37, df = 125, p < .001, gfi = .84, nfi = .91, cfi = .94, rmsea = .09) indicated appropriate adjustment. all constructs showed satisfactory levels of composed reliability (> .70). regarding average variances extracted, all the measures showed levels above .50, as indicated by the literature33. we test using a chi-square difference test (p-value< .05). to test the study hypotheses, we used structural equation modeling using maximum likelihood (ml) estimation with the amos (v. 20) software. the model adjustment was appropriate, following the indications of hair et al.32 (2005) (χ ² = 275.33, df = 124, p < .001, gfi = .87, nfi = .93, cfi = .96, rmsea = .07). this adjustment of the model indicated that the solution of the structural equation model has quality and enable us to follow with the test of the hypotheses. results sample profile the mean age of 197 respondents was 37.0 years old (σ = 15.5 years old). more than half (58.9 %) were female. educationally, almost half (48%) of respondents had completed or were currently enrolled in high school; 42% were single. regarding monthly household income, 67.0% of individuals reported an income of us $1,000 at maximum. related to dentistry care, on average the patients had consulted with the same dentist during the last 13.9 months (σ = 24.9 months). most patients informed that their dentist was a woman (65.5%) of less than 30 years old (65.0%). most patients indicated that they frequently visited the dentist, more than one time per month (39.6%) or monthly (37.1%). 7 rigo et al. braz j oral sci. 2022;21: e227228 measurement model along the average variance extracted and composed reliability, all the factor loadings were significant and higher than .50, which indicate that all variables are significantly linked to the respective construct, evidencing convergent validity. further detail of the scales can be observed in table 1. based on the correlation analysis, we verify the discriminant validity. for this, we compared the extracted and the shared variance (square of the correlation) between constructs34. the correlation and the extracted variance are presented in table 2. table 2. square of the correlation constructs trust intention to return satisfaction with life affect cognition trust .88 intention to return .72 .88 satisfaction with life .52 .50 .59 affect .81 .80 .42 .70 cognition .78 .75 .42 .70 .78 from the comparison between the average extracted variance and the shared variance, all constructs present evidence of discriminant validity. specifically, the higher shared variance is between trust and affect (.65), which is lower than the average extracted variance of both trust and affect scales. test of hypotheses to verify the hypotheses, figure 1 presents the coefficient of each structural path, along with the t-value and the r2 (explication coefficient). it is important to note that all coefficients were significant at the level of p < .01. affection cognition patient trust r2 = .76 h1 β = .53 h2 β = .41 r2 = .85h3 β = .82 r2 = .24h4 β = .49intention to return satisfaction with life figure 1. test of the structural model the affect perceived by the patient at the dentist had a positive effect (β = .53) on the trust that the patient develops in relation to the dentist. the same is true for the effect of the rational or cognitive aspects perceived by the patient at the dentist (β = .41). both affective and cognitive aspects positively influenced the formation of patient 8 rigo et al. braz j oral sci. 2022;21: e227228 trust in the dentist, which offers support to hypotheses h1 and h2. moreover, these two aspects explicate 76% of the variances identified in patient trust. consequently, to patient trust, in the structural model, the patient develops an intention to return to that dentist. our model identifies a stronger and positive effect of the trust in the dentist on the intention to return (β = .82). this implies that the more trust the patient has in that dentist, the higher will be his or her intention to return to that dentist. this finding supports h3. it is important to also highlight that 85% of the variance of the intention to return is directly explained by the perceived affect, cognition and trust in the dentist. finally, in the model, the relationship between patient and dentist can have a broad influence on satisfaction with life. specifically, the intention to return to the dentist positively influences the patient’s satisfaction with life (β = .49). this finding supports h4. the complete model can explain 24% of the satisfaction with the life of the patients, evidencing that, despite the results of the treatment, the relationship between dentist and patient is important to increase the patient’s satisfaction with his or her life. the following is a summary of the testing of the four hypotheses. h1: there is a positive effect of affect shown by the dentist on patient trust – corroborated. h2: there is a positive effect of cognition shown by the dentist on patient trust corroborated h3: there is a positive effect of patient trust on intention to return to that dentist – corroborated. h4: there is a positive effect of intention to return to that dentist on the patient’s satisfaction with life corroborated discussion the present study identified that the affect perceived by the patient from the professional assumes a positive effect on the trust developed by the patient and, in addition, the same goes for the effect of the cognitive aspects. thus, these two aspects positively influence the formation of trust that the patient develops in the dentist. in addition, we observed in the research that there was a positive effect of the patient’s trust on the intention to return to that dentist, corroborating thom et al.8 (2004), who reported that the healthcare professional who establishes trust with the patient ensures, in this way, a greater likelihood of the patient seeking the care offered again. thus, the greater and more solid the trust established between the patient and the professional, the greater the chances that the patient will return in search of other treatments. patients who trust their health care providers report better health outcomes. regarding this, trust in the health professional has been currently suggested as the basis for effective treatments35. in different studies, health outcomes encompass different dimensions, such as objectively measured indicators, clinical observations 9 rigo et al. braz j oral sci. 2022;21: e227228 (e.g., clinical diagnoses) and subjective self-assessments of patients (e.g., patient satisfaction)35. from the clinical point of view, patients reported more beneficial health behaviors, fewer symptoms and higher quality of life, and were more satisfied with treatment when they trusted their health care provider36. in the present study, the intention of the patient to return to the dentist positively influences satisfaction with life, or else, the established relationship between the patient and the professional influences broad aspects of life, as satisfaction with life. that is, the relationship between dentist and patient is important to increase the patient’s satisfaction with his or her life. moreover, hurst et al.37 (2004) revealed that the interpersonal skills present in the health professional, including recommendations and searches for health care, can influence the aspects related to the behavior of the patient. coulter38 (2002) stated that sick people need to establish relationships with practitioners who offer empathy, support and honesty about their health condition and treatment options, as well as being open to listening to their concerns and preferences. research by hall et al.3 (2002) and hupcey and miller5 (2006) emphasized the importance of elements such as care, concern, attention and interest in building patients’ trust in doctors and nurses. trust is related to the support of the professionals, the importance of the communication of support in terms of emotional and informative support. rempel et al.39(1985) argue that trust in interpersonal relationships has a fundamental element of faith, which promotes a sense of emotional security that allows one to go beyond the physical evidence and feel that the partner takes care of and responsibility for it40. in addition, trust can be considered as a collective good, similar to “social capital,” that is necessary for a health care system. there is evidence that patient trust is linked to desired or reported adherence to treatment recommendations8. with this, interpersonal competence involving caring, concern and compassion were the most commonly reported aspects of trust in the research of mechanic and meyer41 (2000), with individual listening as the central focus. this research analyzed the patient-dentist relationship under the prism of the social and behavioral issues involved. thus, trust as a central mechanism for the maintenance of longstanding relationships is a key element for evaluating the durability of the patient’s relationship with his or her dentist. based on this, it is important verify how cognitive and affective cues from the dentist influence patient trust and, consequently, how this trust creates intentions to return and greater satisfaction with life in the patients. therefore, this research has important findings for both dentists and patients. for dentists, this paper presents evidence for how a patient’s intention to return is created, developing cognitive (e.g., competence) and affective aspects (e.g., attention) that influence trust and consequently create an intention to return. for patients, this research examines how the characteristics of dentists influence their satisfaction with life. the limitations of this study are that we conducted a survey in private clinics. the sample convenience was restricted to patients from two dentistry clinics private in in the south of brazil. therefore, the results cannot be generalized to all patients. future 10 rigo et al. braz j oral sci. 2022;21: e227228 research could extend this sample to other health-care settings, including public health-care services. the present study has a cross-sectional design, which does not allow us to establish the temporal relation between the observed correlations to assess the continuing trend of dental care. thus, the interpretation of the findings is limited. however, since this is an unexplored topic, the study has the role of contributing to the construction of knowledge regarding aspects of the patient-dentist relationship. despite the limitations, we do not know of any other study that has examined the influence of the two dimensions of trust, cognitive and affective, which makes our findings suggest that the role of the professional dental surgeon in establishing trust is a determinant for the return of the patient and their satisfaction with life. in addition, our study provides important findings for future longitudinal research needed to better follow the causal factors directly related to the aspects investigated here in this study, and to better understand the complex interaction between trust and health outcomes. with the confirmation of the four hypotheses proposed, the study allows dentists to establish a comprehensive and integrative model to correlate different dimensions that involve the provision of dental services. this study promotes an advance in the literature on the subject because it relates elements related to satisfaction, such as affectivity and cognition, with loyalty generation and satisfaction with life. in other words, the study articulates, in a model, a link between subjective factors of the dentist-patient relationship, with an objective indicator of this satisfaction: the intention to return to the clinic. in this sense, a second contribution of the study is to show that loyalty is strongly related to subjective aspects. this study therefore corroborates the findings of previous studies on the importance of good communication and empathy by the dentist for satisfaction with the service provided, while incorporating the dimension of satisfaction with life as a factor related to the intention to return (loyalty). conclusions it can be concluded that: a. affective and cognitive aspects positively influenced the patient’s trust in the dentist; b. the greater the patient’s trust in the dentist, the greater the intention to return to that dentist. affect, cognition and trust in the dentist are important factors for the intention to return to the dentist; and c. the relationship between the patient and the dentist influenced the patient’s satisfaction with life, highlighting the importance of a good relationship with the dentist in satisfaction as a broad aspect of the individual’s life. data availability datasets related to this article will be available upon request to the corresponding author. conflict of interest authors do not have any conflicts to declare. 11 rigo et al. braz j oral sci. 2022;21: e227228 author’s participation basso k worked in structuring the article, designed the method, preparation of database and analysis of results. pauli j worked in project design, in data collection and entering the database. portilio mn worked in discussion of the methodology, in the statistical analysis. rigo l worked in review of the english language and the final wording of article. references 1. diener e, emmons ra, larsen rj, griffin s. the satisfaction with life scale. j pers assess. 1985;49(1):71-5. doi: 10.1207/s15327752jpa4901_13. 2. maynard a, bloor k. trust and performance management in the medical marketplace. j r soc med. 2003;96(11):532-9. doi: 10.1258/jirsm.96.11.532. 3. hall ma, zheng b, dugan e, camacho f, kidd ke, mishra a, et al. measuring patients’ trust in their primary care providers. med care res rev. 2002;59(3):292-318. doi: 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oral sci. 2022;21: e227228 reply to reviewers id – 8667228 title: affect and cognition as antecedents of patients’ trust in the dentist: cross-sectional study dear altair a del bel cury editor-in-chief, brazilian journal of oral science corrections are highlighted in the text of the manuscript and answers on a point-bypoint basis are provided below. reviewer a authors this original investigation is in accordance with focus and scope of the brazilian journal of oral sciences (bjos). the manuscript is related to an original theme and relevant to the area. thank you very much for the corrections to our manuscript! abstract: the abstract is intelligible and accurately describes the objective and results obtained. it is a sufficient summary of the contents of the paper keywords: the keywords are available and in accordance with the study. the authors indicated why the study was undertaken and the background information was provided adequate to understand the aims of the study. the authors important references about the research problem. “some studies”, but the authors cite only one reference: “12”. the text has been adjusted citing the reference number 12. “...authors have sought to relate trust to intention to return and focus ....12” i suggest presenting the theoretical framework of this study before the objectives in text form. the purpose of the study is consistently stated throughout the paper. the text has been adjusted! the methods were described and organized in sufficient detail i suggest that the authors present a better characterization of the “two dentistry clinics in the south of brazil”. the text has been adjusted! “dentistry clinics are located in the city center and serve only private patients and do not have health insurance.” … in the same dentistry clinic? i suggest describing the pretest succinctly. the text has been adjusted! “data were collected through a self-administered questionnaire applied by researchers in the dentistry clinics. before data collection, a pretest was done in order to test the methodology in the application of the research instrument. the pretest subjects were not included in the final sample, as the purpose of the pilot test was only to verify the participants’ understanding. patients filled out the questionnaires while waiting for service in the waiting room of the dentistry clinics. in this room, there was a box in which the patient anonymously, deposited the completed questionnaire. the authors specified the statistical procedures used to analysis of the data. .15 rigo et al. braz j oral sci. 2022;21: e227228 the results are interesting. the authors clearly highlighted the information collected using the methods described to meet the study objectives. the tables and the figure with the presentation of the results are organized and clear. the discussion is adequate.the authors cite appropriate and actual papers in the discussion. the authors identify the limitations of the study in the end of the manuscript and make suggestions for future research. the conclusion is clear and adequate. the authors use appropriate english grammar, but it is important a final review to check. authors should review “author guidelines”: https://periodicos.sbu. unicamp.br/ojs/index.php/bjos/about/submissionsthe conclusion is clear and adequate. include the year of publication. the year of publications were included in the citations. english should be improved because there are many type errors and grammatical problems. we send the certificate of translation of the manuscript into the english language carried out by the company “wiley editing services”. reviewer b authors corrections made in the topic abstract (aim, methods, results and conclusions) all requests to change the abstract have been made. page 1 corrections made in the topic introduction: … remains to be exposed what the chosen method can differentiate in approaching the problem presented. ok, all requests to change the introduction have been made. page 3 corrections made in the topic methods: ethical aspects design and setting participants and data collection data analyses and measures ok, all requests to change the methods have been made. pages 4-6 corrections made in the topic results all requests to change the results have been made and references have been revised. pages 6-7 corrections made in the topic discussion ok, all requests to change the discussion have been made. pages 8-10 corrections made in the topic conclusions ok! the conclusions were reduced, according to the objectives. page .10 corrections made in the topic references ok, references have been corrected! page 11-15 1http://dx.doi.org/10.20396/bjos.v20i00.8661194 volume 20 2021 e211194 original article 1 department of restorative dentistry, endodontic division, piracicaba dental school, university of campinas, piracicaba, sp, brazil. corresponding author: josé flávio affonso de almeida assistant professor, department of restorative dentistry, endodontic division address: piracicaba dental school university of campinas p.o. box 52 13414-903, piracicaba, sp, brazil phone: 55 19 21065344 e-mail: jfa.almeida@gmail.com editor: dr altair a. del bel cury received: september 10, 2020 accepted: march 9, 2021 effect of ethanol-conditioned dentine on sealer penetration into dentinal tubules: a confocal microscopy study carlos augusto de morais souto pantoja¹ , diogo henrique da silva¹ , adriana de jesus soares¹ , caio cezar randi ferraz¹ , brenda paula figueiredo de almeida gomes¹ , alexandre augusto zaia¹ , josé flávio affonso de almeida¹,* aim: the aim of this study was to evaluate the effect of ethanol-conditioned dentin on endodontic sealer penetration into dentinal tubules by confocal laser scanning microscopy (clsm). methods: forty human maxillary anterior teeth were instrumented and divided into four groups (n = 10) according to the drying methods: 1) wet: vacuum only, 2) paper points: vacuum + absorbent paper points, (3) 70% ethanol: 70% ethanol (1 min) + vacuum + absorbent paper points, and (4) 100% ethanol: 100% ethanol (1 min) + vacuum + absorbent paper points. all root canals were filled with resin-based endodontic sealer. four sections from each third (cervical, middle, and apical) were examined by clsm. root canal wall perimeter infiltrated by sealer, maximum depth of sealer penetration, percentage of penetrated area, and fluorescence intensity of rhodamine b were measured. statistical analysis was performed by analysis of variance and tukey’s tests (α = 0.05). results: no statistical difference was found when percentage of root canal wall coverage infiltrated by sealer were compared. the groups in which ethanol solutions were used presented greater depth of sealer penetration, higher percentage of penetrated area, and higher fluorescence intensity of rhodamine b (p< 0.05) when compared with the wet and paper point groups. overall, 100% ethanol produced better results than 70% ethanol, except for rhodamine b intensity (cervical third). in addition, the absorbent paper points drying method behaved better than did vacuum only group, except for rhodamine b intensity (apical third). conclusion: ethanol-conditioned dentin improved the penetration of resin-based sealer into dentinal tubules, especially at the concentration of 100%. keywords: microscopy, confocal. dentin. endodontics. ethanol. wettability. resin cements. https://orcid.org/0000-0002-7041-9179 https://orcid.org/0000-0001-8874-5522 https://orcid.org/0000-0002-8078-1606 https://orcid.org/0000-0002-1513-682x https://orcid.org/0000-0002-8449-0646 https://orcid.org/0000-0003-1354-1466 https://orcid.org/0000-0002-5874-9247 2 pantoja et al. introduction bacteria causing persistent endodontic infections are usually located in areas unaffected by instruments and antimicrobial agents, including anatomically complex areas of the root canal system, especially dentinal tubules1,2. sealer penetration into these spaces is considered a desirable outcome. in addition to their antibacterial effect, sealers can penetrate into dentinal tubules, entombing residual bacteria3,4. however, many factors may influence the penetration of the filling material, such as the physicochemical properties of the sealer, filling techniques, and different levels of residual moisture in the root canal5. clinicians should be aware of these characteristics and try to establish a protocol that can promote greater percentage and maximum depth of sealer penetration into dentinal tubules6. final irrigation of root canals with different chemical solutions has been proposed5,7-9 to improve sealer penetration into dentinal tubules. for many years, drying the dentine walls prior to filling procedures has been carried out with vacuum cleaner tips and absorbent paper points2,3,7. ethanol solution is a dehydrating agent10 that has been tested to improve dentine wettability and to increase sealer penetration along the root canal walls8. some authors suggest there is a higher compatibility between ethanol-saturated dentine and hydrophobic resin monomers, preventing collagen shrinkage and allowing for higher impregnation11, infiltrating hydrophobic monomers into the demineralised dentine collagen matrix12-14. the use of 70% or 100% ethanol increased dentine wettability and improved the interaction between resin-based endodontic sealer and dentine root canal walls15. these findings suggest that different ethanol concentrations could enhance sealer penetration into dentinal tubules and corroborate the better endodontic treatment outcomes. hence, the aim of this study was to evaluate the effect of ethanol-conditioned dentine on endodontic sealer penetration into dentinal tubules by confocal laser scanning microscopy (clsm). materials and methods specimen preparation forty recently extracted human maxillary anterior teeth were selected. the teeth were cleaned with an ultrasonic scaler and washed with saline solution. preoperative mesiodistal and buccolingual radiographs were taken to ensure the presence of a single root canal. the teeth were stored in 0.2% thymol solution at 4 °c. the crowns were removed using a 0.3-mm saw microtome (isomet 1000; buehler, lake bluff, il, usa) to standardise root length at 18 mm. the canals were accessed and the working length established. all canals were instrumented with the crown-down technique using rotary nickel-titanium k3 files (sybronendo, glendora, ca, usa) according to the “procedure pack” guidelines for instrumentation. the canals were prepared up to apical size 25/06. after each instrument change, the canals were irrigated with 5 ml of saline solution and filled with 2% chlorhexidine gel, an auxiliary chemical substance (endogel, essencial farma – itapetininga, sp, brazil). apical patency was maintained by passing # 15 k-file (dentsply maillefer, ballaigues, switzerland) through the apical foramen between files. the canals were then irri3 pantoja et al. gated with 3 ml of 17% ethylenediaminetetracetic acid for 3 min, followed by a final rinse with 5 ml of saline solution. all irrigation solutions and auxiliary chemical substances were introduced into the root canal using a 5-ml disposable plastic syringe (ultradent products inc, south jordan, ut, usa) and a 20 x 0.55 mm (24 g) needle (becton dickinson (bd) – curitiba, brazil) inserted 2 mm short of the working length. the samples were randomly divided into four groups (n=10) according to the drying methods: g1 – wet: the root canals were dried for only 5 s using a capillary tip 0.014 attached to the vacuum adapter (ultradent products inc, south jordan, ut, usa); g2 – paper points: the root canals were dried with a capillary tip 0.014 attached to the vacuum adapter dried for 5 seconds, followed by the use of two absorbent paper points (endo points industrial amazônica, manacapuru, am, brazil); g3 – 70% ethanol: the canals were filled with 70% ethanol using a 5-ml disposable plastic syringe (ultradent products inc, south jordan, ut, usa) with 20 x 0.55 mm (24 g) needle inserted 2 mm short of the working length. after 1 min, the ethanol solution was removed with a 0.014 capillary tip attached to the vacuum adapter dried for 5 s then with the use of two paper points; and g4 – 100% ethanol: the same procedure applied in g3 was performed using 100% ethanol. the canals were then obturated with ah plus (dentsply – konstanz, germany) and autofit gutta-percha cones (analytic endodontics, orange, ca, usa) using schilder’s technique. the sealer was labelled with 0.1% fluorescent rhodamine b (sigma-aldrich, st. louis, mo, usa) for clsm9,15,16. in endodontics, clsm is used to determine the degree of adaptation and penetration of the root canal filling into dentine walls and into dentinal tubules, respectively17. rhodamine must be incorporated with cement at a ratio of 0.1%. it did not interfere with the physicochemical properties of endodontic sealers13,16. autofit gutta-percha cones were coated with sealer and placed 2 mm short of the working length. radiographs were exposed from facial and proximal surfaces to make sure no voids were present. the access cavities were sealed with intermediate restorative material (coltosol, vigodent / coltene – rj, brazil). the teeth were stored in an incubator at 37 °c and 100% relative humidity for 24 h to allow sealers to set. the roots were sectioned perpendicularly (1 mm thick) with a slow-speed, watercooled 0.3-mm saw microtome (buehler isomet lake bluff, il, usa). four sections from each third were obtained at distances of 2, 6, and 10 mm from the apex. after sectioning, gutta-percha was gently removed by a probe, without touching in sealer or dentine areas, removed from the root canal and the sections were polished manually with silicone carbide abrasive papers. the specimens were mounted onto glass slides and examined with a leica tcs-spe confocal microscope (leica, mannheim, germany) and leica microsystems software (las-af). clsm analysis six images from each section were obtained (fig 1a), merged, and exported to image j software (fig 1b). the image of the final section was evaluated by the depth of sealer penetration into dentinal tubules at four different points. percentage of root canal wall coverage in each image of the sections, the circumference of the root canal wall was outlined and the perimeter was measured with the image j software measuring tool (fig 1c). 4 pantoja et al. then, the areas along the canal walls in which the sealer had penetrated into dentinal tubules were outlined and measured by the same method (fig 1d). the outlined distances were divided by the circumference of the canal to calculate the percentage of each canal wall area covered by sealer in that section. the depth of sealer penetration did not matter in this evaluation. maximum depth of sealer penetration the depth of sealer penetration into dentinal tubules was measured at four standardised points in each image (fig 1e). they were 2 pairs of points, each one located on left and right, up and down sides of each root third slice, centralizing the sample. the canal wall was the starting point, and the final point of each pair marked the maximum depth of sealer penetration into dentinal tubules (fig. 1f) which was measured on each slice of each third root section for each section, so an average was obtained and used. percentage of penetrated area the limits of each section and root canal wall were outlined and the area was measured with the image j software measuring tool (fig 1g). thus, it was possible to calculate only the dentine wall area, by subtracting the root canal space. then, the areas along the dentine in which the sealer had penetrated into dentinal tubules were outlined and measured using the same method (fig 1h). the total area into which the sealer had penetrated was divided by the dentine wall area to calculate the percentage of penetrated area. figure 1. design evaluation parameters of each radicular section. (a) captured images in clsm; (b) merged image at image j software; (c) root canal wall perimeter; (d) root canal wall perimeter covered by sealer; (e) four standardized points on each image; (f) maximum depth sealer penetration; (g) area of each section; (h) area penetrated with sealer; (i) intensity values of rhodamine b and (j) dentin area outlined and rhodamine b intensity. a b c d e f g h i j percentage of sealer penetration maximum depth of sealer penetration percentage of area penetrated intensity rhodamine b 5 pantoja et al. rhodamine b intensity in dentinal tubules in each section of the images, the dentin area was outlined and rhodamine b intensity (fig. 1i-j) was determined. then, the intensity of rhodamine b in each section was summed, according to each root third, and the overall value was calculated. statistical analysis statistical analysis was performed with sas software. data normality evaluation was performed by shapiro-wilk test and one-way anova and tukey’s tests were carried out to compare each treatment at a specific root canal third. the level of significance was set at p < 0.05. results sealer penetration occurred on each side of the root canal wall, resulting in statistically similar perimeter percentages in all groups and at all root canal thirds (fig 2a). figure 2. representative graphics of evaluation parameters. a. percentage of root canal wall covered; b. maximum depth of sealer penetration; c. percentage of penetrated area; d. intensity of rhodamine b in dentinal tubules. a b c d wet paper point 70% alcohol 100% alcohol ns cervical middle apical 150 100 50 0 0.0 0.5 1.0 1.5 d d dc c cb b b a d d d d c c c c c c c b b b b b b a a a a a a a a a 100 80 60 40 20 0 100 80 60 40 20 0 m ax im um d ep th o f s ea le r p en et ra tio n (m m ) p er ce nt ag e of ro ot c an al w al l c ov er ag e (% ) p er ce nt ag e of p en et ra te d ar ea (% ) in te ns ity o f r ho da m in e b in d en tin al tu bu le s cervical middle apical cervical middle apical cervical middle apical 6 pantoja et al. on the other hand, 100% ethanol-conditioned dentine promoted deeper sealer penetration (fig 2b), higher percentage of penetrated area (fig 2c), and higher rhodamine b intensity (fig 2d), followed by the 70% ethanol, paper point, and wet groups (p<0.05). the only two exceptions were the non-significant differences in rhodamine b intensity (p>0.05) between the 100% and 70% ethanol groups (cervical third) and the absorbent paper point and wet groups (apical third). figure 3 shows the representative patterns of sealer penetration into dentinal tubules in all experimental groups and at all root canal thirds. discussion scanning electron microscopy (sem) has been widely used to evaluate sealer penetration into dentinal tubules3,7,18-20. sem micrographs allow observation of the dentinal tubules and accurate measurement of sealer penetration depth at high magnification21. although this technique may be advantageous, some requirements need to be analysed such as artefacts during specimen preparation and inability to obtain a detailed overall view at low magnification20. benefits of clsm have been shown in the assessment of sealer penetration into dentinal tubules15,17,21,22. background inforfigure 3. clsm images according to drying methods and root canal thirds. a – c: wet group images showed lower penetration of sealer into dentinal tubules at all thirds; d – f: paper points group images with more penetration of sealer into dentinal tubules, than wet groups; g – i: 70% ethanol group images showed better sealer penetration, when compared to wet and paper points group and j – l: 100% ethanol group images presented a greater amount of sealer penetrated into dentinal tubules, especially at middle third. apicalmiddlecervical wet paper points 70% ethanol 100% ethanol a b c d e f g h i j k l 7 pantoja et al. mation away from the focal plane enables the acquisition of images with fewer artefacts11. another advantage of clsm is the control over the depth of the field, which allows obtaining excellent images in different planes. in this study, clsm images allowed detailed visualisation of sealer penetration into dentinal tubules and measurement of the percentage of sealer penetration area at each root canal third. also, clsm provided the rhodamine b intensity values in each dentine sample, which could suggest that higher rhodamine b intensity is related to greater sealer volume in dentinal tubules. the ethanol solutions used for the final rinse demonstrated that the root canal cervical third presented a significantly higher perimeter percentage and maximum depth of sealer penetration when compared to the middle and apical thirds5,7,22. however, the quantity, volume, and orientation of dentinal tubules at each root third are different23. moreover, root dentine is not uniformly mineralised, and the density or number of dentinal tubules increases from the apical-coronal direction to the root surface23. apical dentine is more frequently sclerosed and the tubules are irregular in number and cannot be observed in some areas18,23. therefore, root canal thirds were not compared in this study because establishing comparisons among them could not be reliable and would not provide any important information about sealer penetration. the root canal perimeter in which the endodontic sealer could penetrate into dentinal tubules has been analysed and differences were found after several final rinse regimens, sealer applications, or filling techniques8,24-27. our results did not show any difference among groups when root canal wall coverages were compared. despite the different evaluation techniques, the same root canal coverage results were achieved after a final rinse with 95% ethanol prior to obturation25. although our previous study showed that ethanol could improve root canal surface wettability24, an adequate sealer placement and/or a good filling technique could overcome any difference in surface substrates. a uniform sealer distribution can be obtained even when different sealer penetration into dentinal tubules is observed. this emphasises that sealer penetration into dentinal tubules would be better evaluated by other methods such as maximum depth of sealer penetration, percentage of sealer penetration area, or intensity of fluorescent dye using clsm. penetration of sealers into dentinal tubules can form a physical barrier to prevent bacterial microleakage and recontamination of the root canal system24, maintaining their bactericidal effect24,28, which favours the healing of periapical lesions. besides, the intensity of rhodamine b in dentinal tubules shows a higher sealer volume, as well as better filling and sealer performance, leading to periapical repair. these findings have been poorly discussed in previous studies and are probably more meaningful and more clinically relevant than those of other evaluations. final rinse with 95% ethyl ethanol presented significantly deeper sealer penetration than the rinse with naocl solution8, as in this study 100% ethanol-conditioned dentine promoted deeper sealer penetration. ethanol is generally considered a dehydrating medium10, which removes the water from among the collagen fibrils29 and dentinal tubules30. the reduction of root dentine wetness improved the wettability of the dentine surface in our previous study24, which could let into more penetration and flow of sealer upon dentin, corroborating with this study and others8,30. this could also occur 8 pantoja et al. on the inner surfaces of dentinal tubules, favouring diffusion of sealer into deeper regions, as founded. therefore, the decreased wetness on the dentin wall or tubules may improve adhesion31 or penetration of hydrophobic materials such as ah plus. moreover, the ethanol solution was used to condition the root canal prior to filling8 or bonding14, instead of dehydrating the dentine using a technique known as ‘ethanol wet-bonding’. conditioning of the dentine promotes a surface with hydrophobic characteristics. an interaction with a hydrophobic sealer – such as “ah plus” would provide a low contact angle between sealer and surface, higher sealer penetration, greater mechanical interlock into the tubules, retention, sealing ability and, consequently, in vivo antibacterial effectiveness6,32. this technique combines the reduction of polarity of the collagen network with the low polarity of highly hydrophobic resins, since hydrophobic monomers can better infiltrate into the ethanol-saturated demineralised dentine. therefore, as epoxy resin binds to collagen14, especially in the demineralised dentine, it is suggested that in the presence of ethanol instead of water, some hydrophobic materials, such as ah plus, have improved flow into dentinal tubules and infiltration into collagen interfibrillar spaces. in conclusion, a 1-minute final rinse with 100% ethanol seems to be a good dentine conditioning method before root canal drying procedures, as it can improve sealer penetration into dentinal tubules, which clinically could led into entombing more bacteria and enhancing endodontic treatment outcomes. acknowledgements the authors deny any conflicts of interest in this study. this study was supported by capes (coordination for the improvement of higher education personnel) finance code 001. references 1. ricucci d, siqueira jr jf. biofilms and apical periodontitis: study of prevalence and association with clinical and histopathologic findings. j endod. 2010 aug;36(8):1277-88. doi: 10.1016/j.joen.2010.04.007. 2. rosales ji, marshall gw, marshall sj, watanabe lg, toledano m, cabrerizo ma, et al. acid-etching and hydration influence on dentin roughness and wettability. j dent res. 1999 sep;78(9):1554-9. doi: 10.1177/0022034599078009100. 3. mamootil k, messer hh. penetration of dentinal tubules by endodontic sealer cements in extracted teeth and in vivo. int endod j. 2007 nov;40(11):873-81. doi: 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26. generali l, cavani f, serena v, pettenati c, righi e, bertoldi c. effect of different irrigation systems on sealer penetration into dentinal tubules. j endod. 2017 apr;43(4):652-656. doi: 10.1016/j.joen.2016.12.004. 27. wilcox lr, wiemann ah. effect of a final ethanol rinse on sealer coverage of obturated root canals. j endod. 1995 may;21(5):256-8. doi: 10.1016/s0099-2399(06)80992-1. 28. almeida jf, gomes bp, ferraz cc, souza-filho fj, zaia aa. filling of artificial lateral canals and microleakage and flow of five endodontic sealers. int endod j. 2007 sep;40(9):692-9. doi: 10.1111/j.1365-2591.2007.01268.x. 29. picoh t, stotz s, buff e, duschner h, staehle hj. influence of different etching times on hybrid layer formation and tensile bond strength. american j dent. 1998 oct;11(5):202-6. 30. zhang h, shen y, ruse nd, haapasalo m. antibacterial activity of endodontic sealers by modified direct contact test against enterococcus faecalis. j endod. 2009 jul;35(7):1051-5. doi: 10.1016/j.joen.2009.04.022. 31. heling i, chandler np. the antimicrobial effect within dentinal tubules of four root canal sealers. j endod. 1996 may;22(5):257-9. doi: 10.1016/s0099-2399(06)80144-5. 32. ordinola-zapata r, bramante cm, cavenago b, graeff ms, gomes de moraes i, marciano m, et al. antimicrobial effect of endodontic solutions used as final irrigants on a dentine biofilm model. int endod j. 2012 feb;45(2):162-8. doi: 10.1111/j.1365-2591.2011.01959.x. 1 volume 22 2023 e238670 original article braz j oral sci. 2023;22:e238670http://dx.doi.org/10.20396/bjos.v22i00.8668670 1 department of dentistry, tuiuti university of paraná, curitiba, paraná, brazil. 2 healthcare division, joinville municipal authority, joinville, santa catarina, brazil. 3 school of health sciences, positive university, curitiba, paraná, brazil. 4 department of dentistry, joinville region university, joinville, santa catarina, brazil. corresponding author: marilisa carneiro leão gabardo universidade positivo r. prof. pedro viriato parigot de souza, 5300 cep 81.280-330 – curitiba – paraná brazil e-mail: marilisagabardo@gmail.com editor: altair a. del bel cury received: march 11, 2022 accepted: jul 13, 2022 calcium hydroxide diffusion after agitation of endodontic irrigants: an ex vivo study lucas takeo wakasugui1 , camila paiva perin1 , allan abuabara2 , marilisa carneiro leão gabardo3* , liliane roskamp1 , flares baratto-filho4 , natanael henrique ribeiro mattos1 aim: to compare the influence of two methods of agitation of endodontics irrigants, by diffusion of calcium hydroxide [ca(oh)2] through the dentinal tubules, measuring the ph of the medium where they were kept. methods: twenty mandibular incisors were prepared using a waveone gold large file, in a reciprocating movement, and then divided into (n = 10): gutta-percha cone (gpc) or easy clean system (ecs) agitation of 1% sodium hypochlorite and 17% ethylenediaminetetraacetic acid. the specimens were filled with ca(oh)2 paste, placed in flasks with 4 ml of deionized water, and stored in an incubator. the ph was read using a digital ph meter immediately after storage (t0), after 7 (t1), 14 (t2), 21 (t3), and 35 (t4) days. results: statistical difference between groups was observed regardless of the day ph was measured (p < 0.01). from t2 on, ecs presented higher ph values in comparison with gpc, with significant difference (p < 0.01). conclusion: agitation of endodontic irrigants with ecs enhances the ca(oh)2 diffusion, providing higher ph values, from the 14th day on, when compared with gpc. keywords: calcium hydroxide. endodontics. root canal irrigants. sodium hypochlorite. https://orcid.org/0000-0002-7628-1350 https://orcid.org/0000-0002-4646-8851 https://orcid.org/0000-0003-2454-3360 https://orcid.org/0000-0001-6832-8158 https://orcid.org/0000-0002-2331-0090 https://orcid.org/0000-0002-5649-7234 https://orcid.org/0000-0003-2755-2270 2 wakasugui et al. braz j oral sci. 2023;22:e238670 introduction cleaning and shaping can be considered the most important step of endodontic treatment, because, in addition to preparing the site for three-dimensional obturation, it eliminates bacterial infection of the root canal system1. root canal anatomical complexities, including curvatures, isthmuses, lateral canals, apical ramifications, and recesses of oval-shaped, c-shaped, or flattened canals, may be challenging for endodontic therapy2. in these cases, the chemical effects of irrigants can take on their main role, especially in antisepsis in necrotic teeth and teeth with failed root canal treatments3. moreover, irrigants as sodium hypochlorite (naocl) and ethylenediamine tetraacetic acid (edta) can penetrate mechanically inaccessible areas, dissolving organic tissues and removing smear layers, being essential in endodontic treatment. naocl is an acid solution (ph ≅ 5,0) commonly used in endodontics, because it has a wide antimicrobial spectrum and it is capable of inhibiting or deactivating some bacterial enzymes through the formation of reactive chlorine, which causes irreversible oxidation of the sulfhydryl group of the bacterial enzymes4,5. although naocl eliminates microorganisms6, ethylenediaminetetraacetic acid (edta – ph ≅ 12) is used because of its capacity to dissolve the inorganic component of the smear layer5,7. the antibacterial effect, achieved by endodontic treatment, is more likely affected by the degree of the penetration of irrigants to scavenge bacteria residing deeply inside infected dentinal tubules than by the instrumentation of the root canal system, once the shaping protocol revealed deficient debridement and areas untouched by both, the manual files and rotary or reciprocating instruments8. due to these limitations, in recent years, research on root canal irrigation quality and efficiency has focused on irrigants with better cleaning and antibacterial activity as a necessary complement to mechanical preparation9. also, it is important to mention the influence of endodontic irrigants on sealer penetration into the dentinal tubules. naocl, when compared to other irrigant solutions, promoted more uniform sealer penetration10. root canal irrigants reduce the number of microorganisms within superficial layers of root dentine, however, bacteria more deeply embedded within tubules often remain unaffected11 and may contribute to persistent periradicular disease12. this barrier may be overcome through the use of irrigant activation techniques, allowing deeper areas of the dentine tubules to be reached5,13. the main techniques for this purpose are basically divided between manual techniques or use of a device14, indicating that the latter has superior results15,16. in this context, the easy clean system ecs (easy equipamentos odontológicos, belo horizonte, mg, brazil) is a device similar to a rotary endodontic instrument; its active part is in the shape of an “aircraft wing” and it is recommended for promoting endodontic irrigant agitation17,18. thus, for eliminating microorganisms, the use of calcium hydroxide [ca(oh)2] paste is established as a form of intracanal medication, complementary to irriga3 wakasugui et al. braz j oral sci. 2023;22:e238670 tion. with a ph of 12.5 to 12.8, its main antibacterial actions, tissue dissolution, inhibition of tooth resorption and induction of tissue deposition, result from the dissociation of calcium (ca2+) and hydroxyl (oh-) ions19. this dissociation allows the ions to diffuse through the dentinal tubules20. for antibacterial action to occur within the dentinal tubules, the ionic diffusion of ca(oh)2 must be greater than the buffering capacity of the dentine, and the ph levels must be enough to eliminate microorganisms19. for this reason adequate prior cleaning with irrigants is essential. another important factor refers to the vehicles used to manipulate ca(oh)2 paste. the vehicles for ca(oh)2 powder aggregate are primarily of three types: aqueous, viscous, and oily. the high molecular weights of the vehicles minimize the dispersion of the materials in the tissues and retain the paste in the desired area for a longer duration. with respect to viscosity, the lower the viscosity, the higher the rate of ion dissociation, as evident with aqueous vehicles21, that generate a higher ionic dissociation speed, while the opposite occurs with viscous vehicles22. based on the presented, given that irrigant agitation techniques may have some impact on ca(oh)2 penetration through the dentinal tubules, this study aimed to compare manual use of gpc with ecs with regard to irrigant activation, measured by the ph values in the deionized water. the null hypothesis was that there would be no significant difference in ph values. material and methods this ex vivo study was approved by the human research ethics committee (no. 4.260.956). sample size calculation used data from previous studies as a reference11. the power observed in the sample was calculated considering α = 5% and the rejection of the null hypothesis through the anova test, which resulted in a power value greater than 99%. twenty mandibular incisors, with a single root canal, were selected from the dental school’s tooth bank. all procedures were performed by the same operator, an endodontist, with 20 years of experience. the inclusion criteria were: teeth with almost straight roots with similar size and shape (standardized using cone beam computed tomography – cbct), completely closed apices, and absence of previous root canal treatment. the exclusion criteria were: presence of caries in the root and presence of visible fracture lines in the root. calcified canals were replaced to maintain the sample (n = 20). the method for ion diffusion analysis was adapted from batista et al.23. teeth were sectioned with rotating carborundum discs (dentorium international, new york, ny, usa) using a low-speed handpiece at 14 mm from the apex. thereafter, the specimens were stored in 0.9% saline solution (eurofarma, são paulo, sp, brazil) at 9 oc for further procedures. initial canal exploration was performed with manual #10 k-files (dentsply sirona, ballaigues, switzerland), proglider files (dentsply sirona, ballaigues, switzerland), syringe, and navitip fx irrigation needle (ultradent products inc., south jordan, ut, 4 wakasugui et al. braz j oral sci. 2023;22:e238670 usa) containing 0.9% saline solution to verify the presence of a single root canal and to achieve apical patency. working length (wl) was set at 13 mm for all specimens. root canal preparation was performed using a waveone gold large file (dentsply sirona, ballaigues, switzerland) fitted to a vdw silver motor (vdw, munich, germany) in a reciprocating movement. the specimens were washed in deionized water. then, the apices were closed using composite resin (vh opallis; fgm, joinville, sc, brazil). the specimens were then divided into two groups (n = 10): • gpc: 1 ml of 1% naocl was applied at the entry of the canal using a syringe and navitip fx irrigation needle (ultradent products inc., south jordan, ut, usa). the solution was manually activated in an up-and-down motion for 30 seconds using a #25 gpc (dentsply sirona, ballaigues, switzerland) at the wl. subsequently, the canals were irrigated with 17% edta (biodinâmica, ibiporã, pr, brazil) and agitated as described above for 30 seconds. 1 ml of 1% naocl was replaced at the entry of the canal and activated manually for a further 30 seconds. • ecs: the procedure was similar to that used in the previous group, but activation was performed with ecs in continuous motion using an electric device at approximately 900 rpm and 2-newton centimeters (ncm) of torque (dforce 1000 endo, dentflex, ribeirão preto, sp, brazil), at 2 mm from the wl. after preparation the canals were dried with #25 paper points (tanariman industrial ltda., manacapuru, am, brazil), and filled with ultracal using a syringe and a navitip fx needle (utradent products inc., south jordan, ut, usa). the access cavities were sealed with composite resin. the specimens were placed in flasks with 4 ml of deiozined water, and stored in an incubator with 100% humidity at 36 oc for up to 35 days. the ph of the deionized water was read using a digital ph meter device (quimis, diadema, sp, brazil) immediately after storage (t0), after 7 (t1), 14 (t2), 21 (t3), and 35 (t4) days. prior calibration was performed using buffer solutions with acetic acid (ph 4.0) and sodium acetate (ph 7.0). for each measurement, the ph meter electrode was rinsed with deionized water and dried with absorbent paper to eliminate residues that might cause interference. the data were analyzed using stata/se v.14.1 statistical software (statacorp llc., college station, tx, usa). the kolmogorov-smirnov test at 0.05 level showed that the samples were normally distributed (p > 0.05), allowing differences in mean ph values to be assessed. two-way analysis of variance (two-way anova) tested the difference between the mean ph values of gpc and ecc, and also between the days the ph readings were taken. when two-way anova indicated a difference between the variables (p < 0.05), the tukey hsd (honestly significant difference) test was performed for homogeneous variances, or the games-howell test was used for heterogeneous variances, both for multiple comparisons. levene’s test was used to compare the homogeneity of variance. the level of all tests was set at 0.05. 5 wakasugui et al. braz j oral sci. 2023;22:e238670 results regardless of the day on which ph was measured, a statistically significant difference (p < 0.01) occured between the mean ph values of the two groups (table 1). table 2 shows the statistical difference between each method at different observation periods (t). the highest ph value was observed at t4, with means and standard deviations for gpc and ecs of 7.28 (± 0.09) and 7.65 (± 0.08), respectively. from t2 on, gpc and ecs presented an increase in mean ph values, but the ecs mean (7.29 ± 0.16) differed statistically from the gps mean (p < 0.01). this difference was also found at t3 (7.47 ± 0.13) and t4 (7.65 ± 0.08) (table 2). table 1. mean ph values following each agitation technique, regardless of the observation periods. group n mean (sd) 95%ci lower limit upper limit gpc 50 7.05 (0.24)a 6.98 7.12 ecs 50 7.29 (0.30)b 7.20 7.38 note: gpc, manual activation using gutta-percha cones; ecs, easy clean activation; sd, standard deviation; 95%ci, 95% confidence interval. mean values followed by different letters indicate significant difference (p <0.05). figure 1 shows the increase in mean ph values according to the technique and time. higher mean ph values were found for ecs. the difference increased and was statistically significant (p < 0.01) from t2 onwards (table 2). 7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 t0 gpc ecs t1 t2 t3 t4 note: gpc, manual activation using gutta-percha cones; ecs, easy clean activation; t0, immediately after storage; t1, after 7 days; t2, after 14 days; t3 after 21 days; t4 after 35 days. figure 1. mean ph values according to technique and time. 6 wakasugui et al. braz j oral sci. 2023;22:e238670 table 2. mean ph values in different observation periods (t) and agitation technique. group x t n mean (sd) 95%ci lower limit upper limit gpc x t0 10 6.95 (0.19)a,b 6.81 7.09 gpc x t1 10 6.81 (0.33)a 6.57 7.05 gpc x t2 10 7.07 (0.13)a,b 6.98 7.16 gpc x t3 10 7.17 (0.08)b,c 7.11 7.23 gpc x t4 10 7.28 (0.09)c,d 7.20 7.35 ecs x t0 10 6.98 (0.27)a,b 6.78 7.17 ecs x t1 10 7.06 (0.19)a,b,c 6.92 7.19 ecs x t2 10 7.29 (0.16)c,d 7.17 7.40 ecs x t3 10 7.47 (0.13)d 7.38 7.57 ecs x t4 10 7.65 (0.08)e 7.59 7.71 note: gpc, manual activation using gutta-percha cones; ecs, easy clean activation; t0, immediately after storage; t1, after 7 days; t2, after 14 days; t3 after 21 days; t4 after 35 days; sd, standard deviation; 95%ci, 95% confidence interval. mean values followed by different letters indicate significant difference (p <0.05). discussion this study aimed to compare the different results for ph values of the deionized water, when gpc or ecs were used to agitate irrigants before ca(oh)2 paste filling. the null hypothesis tested was rejected because there was a significant difference between the techniques. from the 14th (t2) day on, ecs demonstrated a significant difference and higher mean ph values. conventional irrigation, such as manual techniques, have reduced ability to remove dentine debris, especially in anatomically complex areas7,14. the use of apically fitted gpc seems limited, since the volume of fresh solution in the apical region remains small, although some authors have revealed good results with this technique24. these characteristics may have influenced the better results achieved by ecs compared to gpc in this study. some authors advocate the use of devices the use of irrigant activation techniques, allowing deeper areas of the dentine tubules to be reached5,13, which is clinically relevant, resulting in a more favorable endodontic treatment25. it is known that the instrumentation of the root canal system, with manual or rotary instruments has its limitations, with the maintenance of untouched areas with microorganisms not reached and, thus, lead to endodontic treatment failure8. thus, authors focused their investigations on irrigants with better cleaning and antibacterial activity, as a necessary complement to mechanical preparation9. naocl and edta are extensively studied irrigants, with physical actions that include smear layer and debris removal from the root canal walls17,26, removal of root canal dressing27, flow of irrigant into the lateral canals28 and ability to penetrate dentinal tubules29. the ecs manufacturer suggests its use in reciprocation motion, but its use with continuous rotary movement at low speed has demonstrated more efficacy in cleaning the isthmus area as well as the root canal walls18,26. ecs presents a minimum risk of 7 wakasugui et al. braz j oral sci. 2023;22:e238670 deforming the canal walls because it uses an acrylonitrile butadiene styrene (abs) plastic instrument. it is therefore possible to introduce it up to the wl18. the technique is based on the premise that energy released by the instrument enhances the properties of the irrigants, promoting more effective debris and smear layer removal when compared with ultrasonic irrigation17. regarding the use of naocl, beyond the physical aspects, the solution was chosen because of its recognized tissue-dissolving and antimicrobial capabilities6. at a concentration of 1% its effectiveness is recognized, which justifies having been elected in the present research6. also, naocl seems to promote more uniform sealer penetration when compared to other irrigants10. on the other hand, edta solution, which has a basic ph, is also susceptible to agitation and this improves its results30. as the purpose of this study was to evaluate the agitation of the solutions prior to filling with ca(oh)2 paste, it is important to discuss this material. its penetration in the dentinal tubules is essential for it to act31 as an ally in combating microorganisms that remain in the root canal system even after cleaning and shaping. the literature points out that the action of ca(oh)2 is associated with its ph and, depending on the vehicle used, this can occur faster (aqueous vehicle) or slower (viscous vehicle) due to ionic dissociation21,22. in this study it was opted for ultracal paste (utradent products inc., south jordan, ut, usa), the vehicle of which is aqueous. data highlight that the action of ca(oh)2 ranges from 7 to 45 days 32, but the greatest antimicrobial effect occurs in 14 days33, which was corroborated by the results found here by analyzing the ph of the deionized water. considering the differences in the methods adopted, plataniotis and abbott34 compared ohion diffusion through root dentine taking various ca(oh)2 preparations, but they did not find significant differences. eftekhar et al.35 identified differences between three ca(oh)2 pastes regarding ion diffusion. the study conducted by batista et al.23, using a similar method to that adopted here, revealed that high ph values were maintained, even after 30 days of filling with ca(oh)2 pastes manipulated with different vehicles: saline, propylene glycol, and aloe vera gel. thus, it is evident that the results of this study depend on the technique used, and limitations may be related to this. the standardization of specimens by cbct is a positive aspect. likewise, the possibility of bias in the results was minimized by the fact that only one experienced operator conducted all the experiment. although this was an ex vivo study, the use of ecs for 30 seconds in continuous motion to agitate the naccl and edta solutions enhances the ca(oh)2 diffusion, providing higher ph values, from the 14th day on, when compared with manual agitation (gpc). such results imply the recommendation of the technique that promotes better results, favoring the endodontic treatment with greater chances of success. acknowledgements the authors declare that they have no conflicts of interests. 8 wakasugui et al. braz j oral sci. 2023;22:e238670 data availability datasets related to this article will be available upon request from the corresponding author. conflicts of interest none authors contribution conceptualization: lucas takeo wakasugui, liliane roskamp, natanael henrique ribeiro mattos. methodology: camila paiva perin, marilisa carneiro leão gabardo, flares baratto-filho, natanael henrique ribeiro mattos. formal analysis and investigation: lucas takeo wakasugui, camila paiva perin, allan abuabara, marilisa carneiro leão gabardo. writing original draft preparation: lucas takeo wakasugui, camila paiva perin, liliane roskamp. writing review and editing: allan abuabara, marilisa carneiro leão gabardo, flares baratto-filho. resources: lucas takeo wakasugui, camila paiva perin, natanael henrique ribeiro mattos. supervision: flares baratto-filho, liliane roskamp, natanael henrique ribeiro mattos. all authors actively participated in the manuscript’s findings have revised and approved the final version of the manuscript. references 1. european society of endodontology. quality guidelines for endodontic treatment: consensus report of the european society of endodontology. int endod j. 2006 dec;39(12):921-30. doi: 10.1111/j.1365-2591.2006.01180.x. 2. vertucci fj. root canal morphology and its relationship to endodontic procedures. endod topics. 2005 aug;10(1):3-29. doi: 10.1111/j.1601-1546.2005.00129.x. 3. zehnder m. root canal irrigants. j endod. 2006 may;32(5):389-98. doi: 10.1016/j.joen.2005.09.014. 4. estrela c, estrela cr, barbin el, spanó jc, marchesan ma, pécora jd. mechanism of action of sodium hypochlorite. braz dent j. 2002;13(2):113-7. doi: 10.1590/s0103-64402002000200007. 5. dioguardi m, gioia gd, illuzzi g, laneve e, cocco a, troiano g. 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antibiotics and biocides as antimicrobial medicaments in endodontics. aust dent j. 2007 mar;52(1 suppl):s64-82. doi: 10.1111/j.1834-7819.2007.tb00527.x. 23. batista ves, olian dd, mori gg. diffusion of hydroxyl ions from calcium hydroxide and aloe vera pastes. braz dent j. 2014;25(3):212-6. doi: 10.1590/0103-6440201300021. 24. huang ty, gulabivala k, ng yl. a bio-molecular film ex-vivo model to evaluate the influence of canal dimensions and irrigation variables on the efficacy of irrigation. int endod j. 2008 jan;41(1):60-71. doi: 10.1111/j.1365-2591.2007.01317.x. 25. oliveira kv, silva bmd, leonardi dp, crozeta bm, sousa-neto md, baratto-filho f, et al. effectiveness of different final irrigation techniques and placement of endodontic sealer into dentinal tubules. braz oral res. 2017 dec;31:e114. doi: 10.1590/1807-3107bor-2017.vol31.0114. 10 wakasugui et al. braz j oral sci. 2023;22:e238670 26. cesario f, hungaro duarte ma, duque ja, alcalde mp, de andrade fb, reis so mv, et al. 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mar-apr;31(2):109-15. doi: 10.1590/0103-6440202002829. 30. walsh lj, george r. activation of alkaline irrigation fluids in endodontics. materials (basel). 2017 oct;10(10):1214. doi: 10.3390/ma10101214. 31. cai m, abbott p, castro salgado j. hydroxyl ion diffusion through radicular dentine when calcium hydroxide is used under different conditions. materials (basel). 2018 jan;11(1):152. doi: 10.3390/ma11010152. 32. sharma g, ahmed hma, zilm ps, rossi-fedele g. antimicrobial properties of calcium hydroxide dressing when used for long-term application: a systematic review. aust endod j. 2018 apr;44(1):60-5. doi: 10.1111/aej.12216. 33. nascimento gg, rabello dgd, corazza bjm, gomes apm, silva eg, martinho fc. comparison of the effectiveness of singleand multiple-sessions disinfection protocols against endotoxins in root canal infections: systematic review and meta-analysis. sci rep. 2021 jan;11(1):1226. doi: 10.1038/s41598-020-79300-3. 34. plataniotis e, abbott p. a comparison of hydroxyl ion diffusion through root dentine from various calcium hydroxide preparations. aust endod j. 2018 may 28. doi: 10.1111/aej.12281. 35. eftekhar b, moghimipour e, eini e, jafarzadeh m, behrooz n. evaluation of hydroxyl ion diffusion in dentin and injectable forms and a simple powder-water calcium hydroxide paste: an in vitro study. jundishapur j nat pharm prod. 2014 jun;9(3):e14029. doi: 10.17795/jjnpp-14029. 1 volume 22 2023 e237544 original article braz j oral sci. 2023;22:e237544http://dx.doi.org/10.20396/bjos.v22i00.8667544 1 department of dentistry, school of dentistry, catholic university of minas gerais (puc minas), belo horizonte, brazil. corresponding author: soraya de mattos camargo grossmann department of dentistry – icbs/puc minas rua dom josé gaspar, 500 – prédio 45 – sala 111, oral biology laboratory, coração eucarístico belo horizonte mg, brazil. 30535-610 voice: +55-31-33194341 e-mail: sorayagrossmann@gmail.com editor: dr. altair a. del bel cury received: november 13, 2021 accepted: march 12, 2022 a survey on brazilian dentists’ awareness, perception, and knowledge of bisphosphonates raquel laís ottoni nunes1 , nicole ribeiro dos anjos1 , luciano henrique ferreira lima1 , ana paula cota viana1 , larissa de ávila pereira1 , fábio fernandes borém bruzinga1 , soraya de mattos camargo grossmann*1 aim: the purpose of the study was to analyze the knowledge of dentists in belo horizonte, brazil, about bisphosphonates and their clinical implications. methods: a cross-sectional questionnaire-based study was conducted with a convenience sample of dentists in belo horizonte, in a period of 8 weeks. the questionnaire was self-applied and was structured with 10 items about the dentists’ demographic characteristics, professional profile, and knowledge about bisphosphonates. associations in the data were analyzed by with the fischer’s exact test with a significance level of 5%. results: of the 214 participating dentists, 163 (76.17%) were women, and 51 (23.83%) were men, with age ranged for 21 to 73 years (mean of 30 years) and mean of 6 years of professional activity. nearly half (106/49.53%) reported having knowledge about bisphosphonates, and undergraduate courses were the primary source of such information (73/34.11%). osteoporosis was the most identified indication for use (75/35.04%), although no participants correctly identified all indications. regarding the drugs’ side effects, only three dentists (1.40%) could correctly identify all responses, with bone necrosis being recognized by the majority (88/41.12%). sodium alendronate (54/25.23%) and sodium ibandronate (15/7.01%) were the most identified examples of bisphosphonates. last, only nine dentists (4.20%) could identify all examples of the drugs, and their capacity was associated with self-reported knowledge (p<0.05). conclusions: more information about bisphosphonates should be disseminated in belo horizonte, ideally via better approaches in local undergraduate and postgraduate courses. until then, knowledge of the basic aspects of bisphosphonates will remain limited. keywords: diphosphonates. knowledge. awareness. drug utilization. dentists. brazil. https://orcid.org/0000-0003-2952-8178 https://orcid.org/0000-0002-4214-5529 https://orcid.org/0000-0003-0118-0958 https://orcid.org/0000-0002-5648-8565 https://orcid.org/0000-0003-0935-2574 https://orcid.org/0000-0002-0197-139x https://orcid.org/0000-0002-8920-3853 2 nunes et al. braz j oral sci. 2023;22:e237544 introduction bisphosphonates are chemical compounds analogous to pyrophosphoric acid that are found in the form of pyrophosphate in the human body. they act on osteoclasts and osteoblasts in the process of bone resorption and are used in treating diseases that alter bone metabolism, including osteoporosis, paget’s disease, malignant hypercalcemia, bone metastases, breast cancer, prostate cancer, osteogenesis imperfecta, and lung cancer1–3. with oral or intravenous application, bisphosphonates are generally well tolerated by the body systems. however, some adverse effects may be observed, including low and transient fever, fatigue, arthralgia, nausea, esophagitis, renal failure, hypocalcemia, bone pain, and osteonecrosis of the jaws3–5. bisphosphonate-related osteonecrosis of the jaw (bronj) is considered to be the most relevant adverse event for dentistry, characterized as an area of bone exposure in the mandible and/or maxilla without remission in 8 weeks and affecting patients who use or have used systemic bisphosphonates and have not received irradiation on the head and neck region5,6. in the field of dentistry, bronj may develop after dental procedures such as extractions, implant placement, and periodontal therapy or arise either spontaneously or by prosthetic trauma1. a recent systematic review showed a prevalence of 2.7% of bronj in patients using bisphosphonates and undergoing dental treatment, with a specific prevalence of 6.9% for intravenous use7. although bisphosphonates are not new in the market, with the first report of bronj in 20038, the literature shows low knowledge by most dentists, in brazil9 and worldwide10-13. to minimize the risks of developing bronj, preventive dental treatment should be performed before bisphosphonate therapy begins8. likewise, the dentist’s knowledge of bisphosphonates is important to assess the patient’s risk and advise him on the need to maintain good oral hygiene to reduce the need for dental procedures7. in view of the low knowledge about bisphosphonates reported in the literature and the importance of the dentist in the prevention and treatment of bronj, associated with little data on the subject in brazil and the absence of information about local dentists, the objective of the present study was to analyze the knowledge of dentists in belo horizonte brazil about bisphosphonates and their possible clinical implications. materials and methods study design, local, population, and sample following approval by the local ethics and research committee (protocol no. 1,961,961), a cross-sectional questionnaire-based study was conducted for eight consecutive weeks. local, population and sample participated in this study dentists working in public service and private practice across belo horizonte, brazil. participants were recruited by convenience sampling through 3 nunes et al. braz j oral sci. 2023;22:e237544 data obtained from the regional council of dentistry and signed a consent form to participate in this study. the sample was categorized according to age group and duration of professional activity. data collection a questionnaire with specific questions to assess the participants’ level of knowledge about bisphosphonates was developed by the researchers. a pilot study was conducted to verify the applicability of the questionnaire and make adjustments. the questionnaire had ten items, addressing participants’ demographics (age and gender), professional profile (duration of professional activity and dental specialty), and knowledge about bisphosphonates (self-declared knowledge, source of information, indications, drugs’ side effects, examples of drugs, and screening of use during anamnesis). questions 1 and 3 requested information about age and time of clinical practice. questions 2, 4, 5, and 6 presented a single option, whereas questions 7–10 were multiple-choice. the questionnaire was self-applied without the intervention of the researchers. when necessary, trained dentistry students answered questions regarding the completion of the questionnaire without influence or prejudice in the responses. statistical analysis for the data analysis, single option questions were treated as dichotomous variables and presented as absolute and relative frequencies. multiple-choice questions were categorized into correct answer (when all possible answers were marked) and wrong answer (when any correct answer was not identified). also, for multiple-choice questions, the correct answers were also analyzed separately in the form of relative and absolute frequency. the fischer’s exact test were used to analyze the associations between self-reported knowledge about bisphosphonates and specific questions about the drug class (i.e., indication, examples of drugs, and side effects). statistical analysis was performed with epi info™ (version 7.2; centers for disease control and prevention, atlanta, ga, usa). the level of significance was set at 0.05. results of the 214 participating dentists, 163 were women (76.17%), and 51 were men (23.83%) with age ranged from 21 and 73 years and mean of 30 years (table 1). their professional activity ranged from 0 to 43 years (mean of 6 years), and most had up to 6 years of dental education (57.01%). in terms of dental specialty, 106 dentists were specialists (49.53%), thirty-six were orthodontists (16.82%), seventeen were endodontists (7.94%), and fourteen were implantodontists (6.54%). regarding knowledge about bisphosphonates, 106 dentists (49.53%) reported having knowledge about the drugs (table 1). undergraduate courses were the primary source of knowledge about bisphosphonates for 73 dentists (34.11%), whereas 19 (8.88%) obtained such information in postgraduate courses, and 83 (38.78%) reported 4 nunes et al. braz j oral sci. 2023;22:e237544 “i don’t know much about it.” almost all, 201 dentists (93.92%) reported questioning patients during anamnesis about the use of systemic medications. table 1. gender, duration of professional activity, and knowledge about bisphosphonates by decade of life and gender, according to 214 dentists from belo horizonte. brazil, 2017. gender decade of life total female male 3rd 4th 5th 6th 7th 8th gender female n (%) 163 (76.71) 95 (44.39) 49 (22.89) 10 (4.67) 8 (3.73) 1 (0.46) 0 (0) 163 (76.71) male n (%) 51 (23.83) 16 (7.47) 18 (8.41) 6 (2.80) 4 (1.86) 6 (2.80) 1 (0.46) 51 (23.83) duration of professional activity ≤ 6 years n (%) 105 (49.41) 17 (7.94) 105 (49.05) 15 (7.00) 0 (0) 1 (0.46) 0 (0) 1 (0.46) 122 (56.97) > 6 years n (%) 58 (27.30) 34 (15.89) 6 (2.81) 52 (24.3) 16 (7.47) 11 (5.13) 7 (3.26) 0 (0) 92 (42.97) self-report knowledge about bisphosphonates yes n (%) 77 (47.23) 29 (56.86) 66 (30.83) 26 (12.14) 7 (3.26) 3 (1.39) 3 (1.39) 1 0.46) 106 (49.47) no n (%) 86 (52.76) 22 (43.13) 45 (21.03) 41 (19.16) 9 (4.21) 9 (4.21) 4 (1.87) 0 (0) 108 (50.48) total n (%) 163 (76.71) 51 (23.83) 111 (51.86) 67 (31.30) 16 (7.47) 12 (5.59) 7 (3.26) 1 (0.46) 214 (100) note: table made by the authors. none of the participants correctly identified all the indications of the bisphosphonates, nine dentists (4.20%) correctly identified all examples of drugs, and three (1.40%) identified all correct clinical implications present in the questionnaire. table 2 presents the number of dentists who identified each correct alternative separately in the multiple-choice questions about indications, drug examples and possible clinical dental implications in the use of bisphosphonates. concerning examples of bisphosphonate-based drugs, 94 (43.92%) did not answer the question. as for possible clinical implications of bisphosphonates, 68 dentists (31.77%) marked “none of the alternatives” as the correct answer. table 2. correct alternatives of the multiple-choice questions and the number of dentists who ticked these alternatives, according to 214 dentists from belo horizonte. brazil, 2017. correct answer n % question 7 – tick the indications to use bisphosphonates osteoporosis 75 35.04 bone metastasis 34 15.88 breast cancer 24 11.21 continue 5 nunes et al. braz j oral sci. 2023;22:e237544 continuation hypercalcemia 4 1.87 question 9 – mark examples of bisphosphonates-based drugs alendronate sodium 54 25.23 risedronate sodium 13 3.27 ibandronate sodium 15 7.01 zoledronic acid 7 3.27 question 10 – mark the possible dental clinical implications of the use of the bisphosphonates bone necrosis 88 41.12 osteomyelitis 18 8.41 decreased of the blood supply 13 6.07 bone exposure 8 3.73 presence of ulcerations 3 1.40 note: table made by the authors. responses related to indications for bisphosphonates and possible adverse events of drug use were not related to self-reported knowledge, whereas the ability to correctly identify all drugs exemplified in the questionnaire was (p<0.05), as shown in table 3. table 3. self-reported knowledge about bisphosphonates of 214 dentists from belo horizonte, brazil, 2017. self-reported knowledge about bisphosphonates p valueyes n (%) no n (%) total n (%) q7 correct respond 0 (0.0) 0 (0.0) 0 (0.0) ns wrong or incomplete 106 (49.53) 108 (50.47) 214 (100.00) q9 correct respond 8 (3.74) 1 (0.47) 9 (4.21) <0.05 wrong or incomplete 98 (45.80) 107 (49.99) 205 (95.79) q10 correct respond 2 (0.94) 1 (0.47) 3 (1.41) ns wrong or incomplete 104 (48.59) 107 (50.00) 211 (98.59) note: table made by the authors. q7 – question seven (tick the indications to use bisphosphonates); q9 – question nine (mark examples of bisphosphonates-based drugs); q10 – question 10 (mark the possible dental clinical implications of the use of the bisphosphonates); ns not significant discussion the present study identified low self-reported knowledge among dentists in belo horizonte, with slightly less than half of participants reporting knowledge about bis6 nunes et al. braz j oral sci. 2023;22:e237544 phosphonates. similarly, another study from brazil reported knowledge about bisphosphonates among “over half” of participants9. in different locations worldwide, the knowledge reported ranged from 40.1% to 71%10-13. of the retrieved publications, dentists in saudi arabia presented the lowest level of knowledge: 40.1% considered “reasonable to good” and only 2.4% considered “good”13. this data is important given recent findings that only 12.4% of jordan patients using bisphosphonates were aware of the risk of bronj due to medication14 and that only 16.7% israel patients knew about the need to discontinue the medication before the installation of dental implants15. thus, patients using bisphosphonates are unaware of the drug’s peculiarities regarding dental care. in this sense, the low knowledge of dentists could increase the risk of complications due to inadvertently performing dental procedures without specific care. in addition, it is the dentist’s role to inform the patient about the dental risks relevant to the health condition. a study conducted in canada revealed that dentists had satisfactory knowledge about bisphosphonates regarding their indications, route of administration, and treatment of bronj. for canadian dentists, the primary sources of information on the subject were scientific journals16. in this study, by contrast, undergraduate courses were the primary sources of knowledge about bisphosphonates. comparative studies of the knowledge of dentists versus dental students about bisphosphonates have shown that dentists had superior knowledge, although both groups had low knowledge on the subject9. when only undergraduate students were evaluated, knowledge about specific issues (i.e., indications, examples of drugs, and routes of administration) was high; however, knowledge about clinical decisions to establish appropriate dental treatment plans for patients using the medication and the ability to recognize and treat the stages of osteonecrosis were extremely low17. this shows that the information acquired during graduation is not enough to cover all specific knowledge in the area, requiring updates and other sources of knowledge, such as scientific journals or postgraduate courses. additionally, the low knowledge of dentists in the present study can be attributed, in part, to the insufficiency in the undergraduate curriculum, given that most of them present the undergraduate degree as the main source of information. analysis of the curriculum of the undergraduate course in dentistry at the university with greater local importance and national and international recognition did not find any specific mention of bisphosphonates18. considering that this curriculum serves as the basis for several other local courses, the absence of this specific topic may represent a possible omission of this information during graduation, representing a possible justification for the low knowledge. corroborating the hypothesis raised, a brazilian study shows the dentists with specialties focused on oral medicine, pathology, and palliative care had greater knowledge about risk factors, treatment planning for patients using bisphosphonates, preventive measures, and the treatment of osteonecrosis19. in this study, orthodontics and implantodontics were among the three most frequent specialties, and because they are areas that deal directly with bone metabolism, greater knowledge about bisphosphonates was expected. however, that expectation could not be confirmed because the data were not sufficient to verify statistical significance. 7 nunes et al. braz j oral sci. 2023;22:e237544 the sample results of this study show that, although participants reported knowledge about bisphosphonates, there is a conflict between low knowledge about indications, examples of medications, and adverse events, which suggests that such knowledge has not been consolidated. similar findings have also been evidenced by other studies in brazil and various locations around the world9-11,20, indicating that almost 20 years after the first report of bronj8, bisphosphonates are still a topic that many dentists have not mastered and that they may not be prepared for provide clinical care of patients using the drug. a study conducted with patients in use of bisphosphonate for osteoporosis and osteopenia undergoing dental treatment revealed that almost halt dentists were unaware of the patients’ health condition and that majority did not question patients about the type of medication used or the duration of treatment for osteoporosis15. although this study showed that almost all participants questioned their patients about the use of systemic medications, the limited knowledge evidenced and the failure to pinpoint the indications, types, and adverse effects of bisphosphonates may prompt the underestimation of the drug and increase patients’ exposure to the risk of osteonecrosis. a study conducted in south korea showed similar results, with one also almost all of dentists questioned about the use of systemic medications but only a little more than half wanting to know about the type and duration of the medication21. although dentists question their patients about the use of systemic medications, many ignore the information collected or do not understand its clinical significance. thus, the simple fact of questioning patients about medications in use does not guarantee that the dentist really knows the subject and applies this finding in his/her practice. knowledge about adverse events from medications in use is important both to establish a correct treatment plan and to predict possible surgical complications. although bronj is considered to be an uncommon adverse event5 with low incidence (i.e., 1.04–69.0 per 100,000 patients/year for oral bisphosphonates and 0.0–12.22 per 100,000 patients/year for intravenous bisphosphonates)6, it represents a significant finding for dental practice. a few of participants in this study could correctly identify all of the adverse events resulting from the use of bisphosphonates on the questionnaire and almost half could identify osteonecrosis was clinical implication. although most participants identified bronj as an adverse event, the relative frequency was quite low, which suggests the risk of performing procedures without proper care or based on any protocols. sodium alendronate was the most recognized drug by the participants in this study, possibly because it is the most commonly used oral bisphosphonate22. however, a systematic review has classified it as the third drug most related to adverse reactions of dental interest1. in the same review, zoledronate was identified as the most frequent in adverse situations and was the fourth most identified by participants in this study1. in other study, sodium alendronate was also the most-cited type of bisphosphonate, followed by zoledronate11. although the number of participants in this study able to correctly indicate all types of bisphosphonates on the questionnaire was small, the capacity significantly related to self-reported knowledge (p<0.05). in turn, knowledge about indications and adverse reactions were not associated 8 nunes et al. braz j oral sci. 2023;22:e237544 with self-reported knowledge (p>0.05). this may indicate that the knowledge presented by dentists is limited to the identification of the types of bisphosphonates and the existence of this pharmacological class. more specific topics whose clinical implications are more significant are neglected, increasing the risk of complications and a negative impact on patients’ quality of life. although this study has limitations in terms of sample size, the results are important to start the discussion on the need to improve the knowledge of bisphosphonates by dentists. thus, the data can be treated as a pilot study, serving as a basis for future studies with a representative sample of the dentist population in belo horizonte. the results of this study reveal that the dentists interviewed have poor knowledge about bisphosphonates. although they reported knowing the type of drug, they could not correctly identify the examples of the drug, indications for use, or adverse reactions, thereby making their clinical practice and planning susceptible to errors and complications associated with the use of the drug. information on bisphosphonates therefore needs to be better disseminated among dentists in belo horizonte, and undergraduate courses, as a primary source of information, and postgraduate courses need better approaches to teaching the subject. other studies focused on the curriculum of local undergraduate and postgraduate courses may help to identify the causes associated with the low knowledge found. furthermore, studies associating the level of knowledge about bisphosphonates with the occurrence of adverse reactions in the dental clinic due to the use of the drug may provide information on the impact of low knowledge on clinical practice. as a suggestion, didactic videos addressing primary information on bisphosphonates and deficiencies identified in this study, should also be broadcast on digital platforms with free access and shared with dentists. conflict of interest the authors have no conflict of interest statement of compliance with ethical standards of research this study was approval by the local ethics and research committee (protocol no. 1,961,961). all procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. informed consent was obtained from all individual participants involved in the study. funding this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. data statement author elects to not share data. 9 nunes et al. braz j oral sci. 2023;22:e237544 author contribution study concepts and study design: soraya mc grossmann data acquisition: raquel lo nunes, nicole r dos anjos, luciano hf lima, ana pc viana, larissa a pereira quality control of data and algorithms: raquel lo nunes, nicole r dos anjos data analysis and interpretation: raquel lo nunes, nicole r dos anjos, luciano hf lima statistical analysis and manuscript editing: soraya mc grossmann, fábio fb bruzinga, manuscript preparation: soraya mc gossmann, fábio fb bruzinga, luciano hf lima manuscript review: soraya mc gossmann, fábio fb bruzinga. all authors actively participated in the manuscript and approved the final version of the manuscript. references 1. fliefel r, tröltzsch m, kühnisch j, ehrenfeld m, otto s. treatment strategies and outcomes of bisphosphonate-related osteonecrosis of the jaw (bronj) with characterization of patients: a systematic review. int j oral maxillofac surg. 2015 may;44(5):568-85. doi: 10.1016/j.ijom.2015.01.026. 2. billington eo, reid ir. benefits of bisphosphonate therapy: beyond the skeleton. curr osteoporos rep. 2020 oct;18(5):587-96. doi: 10.1007/s11914-020-00612-4. 3. barbosa js, almeida paz fa, braga ss. bisphosphonates, old friends of bones and new trends in clinics. j med chem. 2021 feb;64(3):1260-82. doi: 10.1021/acs.jmedchem.0c01292. 4. vannala v, palaian s, shankar pr. therapeutic dimensions of bisphosphonates: a clinical update. int j prev med. 2020 oct;11:166. doi: 10.4103/ijpvm.ijpvm_33_19. 5. adler ra. update on rare adverse events from osteoporosis therapy and bisphosphonate drug holidays. endocrinol metab clin north 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( ) yes ( ) no 6. do you know the bisphosphonate based drugs? ( ) yes ( ) no 7. tick the indications to use bisphosphonates? *multiple-choice ( ) breast cancer ( ) bone metastasis ( ) rheumatological diseases ( ) hypercalcemia ( ) osteoporosis ( ) i don’t know about it 8. by what means did you gain knowledge about bisphosphonates? *multiple-choice ( ) during the graduation ( ) during the specialization 12 nunes et al. braz j oral sci. 2023;22:e237544 ( ) during the postgraduation ( ) reading articles and books ( ) search sites ( ) i don’t know much about it 9. mark examples of bisphosphonates-based drugs: *multiple-choice ( ) alendronate sodium ( ) risedronate sodium ( ) ibandronate sodium ( ) zoledronic acid ( ) prednisone ( ) clavulanic acid 10. mark the possible dental clinical implications of the use of the bisphosphonates: *multiple-choice ( ) decreased of the blood supply ( ) bone necrosis ( ) osteomyelitis ( ) presence of the ulcerations ( ) bone exposure ( ) none of the alternatives 1http://dx.doi.org/10.20396/bjos.v19i0.8660201 volume 19 2020 e200201 original article 1 são leopoldo mandic school, campinas, brazil. 2 department of stomatology, school of dentistry, university of são paulo, são paulo, brazil. 3 gobeyond institution, curitiba, brazil. department of dentistry, universidade positivo. curitiba, brazil. 4 department of oral rehabilitation and community care, faculty of dental surgery, university of malta, msida, malta. 5 department of dental surgery, faculty of dental surgery, university of malta, msida, malta. *corresponding author: dr. arthur rodriguez gonzalez cortes department of dental surgery – faculty of dental surgery university of malta block a, level 0 mater dei hospital, msida msd 2080 malta phone/fax: +356 99729634 email: arthur.nogueira@um.edu.mt received: june 25, 2020 accepted: november 14, 2020 clinical relevance of digital dentistry during covid-19 outbreak: a scoped review roberto adrian markarian1 , renan lucio berbel da silva2 , shaban burgoa3 , otavio henrique pinhata-baptista2 , juliana no-cortes4 , arthur rodriguez gonzalez cortes5 ,* aim: to perform a scoped literature review on advantages of digital workflows in dentistry that could be widely adopted to address safety issues raised during the coronavirus (covid-19) pandemic. methods: recent studies on any advantages of digital dentistry – as compared to conventional methods – that could help addressing the new safety demands for dental treatments that emerged due to the current pandemic were included. pubmed, embase, and web of knowledge databases were searched for eligible articles published in the last five years. the guidelines of prisma statement were followed during data extraction and evaluation. results: the present search strategy yielded 181 publications. after application of exclusion criteria, a total of 34 studies were finally considered eligible to be discussed. among the most important advantages of digital dentistry that contribute to safety during the current pandemic are: reduced number of clinical appointments required, shorter chairside time, less invasive surgeries and safer procedures. conclusion: within the limitations of this study, the findings observed herein suggest that the use of digital workflows in dentistry could lead to increased safety and reduced transmission of covid-19 during the current pandemic. key words: technology, dental. dentistry. workflow. covid-19. http://dx.doi.org/10.20396/bjos.v19i0.8660201 mailto:arthur.nogueira@um.edu.mt https://orcid.org/0000-0003-3281-6891 https://orcid.org/0000-0002-5841-0892 https://orcid.org/0000-0003-2014-9729 https://orcid.org/0000-0003-3197-9576 https://orcid.org/0000-0003-1991-5465 https://orcid.org/0000-0001-6591-7256 2 markarian et al. introduction recently, the world health organization (who) declared covid-19 a current pandemic related to a disease caused by the new coronavirus (sars-cov-2/covid-19). who suggested several actions to be taken by health-related institutions. as a result, healthcare facilities considered to be essential had to adapt to this new reality by taking measures of biosafety and social isolation to actively break the covid-19 chain of transmission. it is known that covid-19 can be transmitted directly from person to person by means of respiratory droplets and contact routes/fomites1. in this context, virus carrying saliva droplets may lead to covid-19 infection by means of inhalation, ingestion, and direct mucous contact2-4. the first contamination of dental professionals during the first covid-19 outbreak revealed that its transmission can also occur from asymptomatic patients5. in addition, a previous study suggested that saliva should be considered a major transmitting agent of covid-19 in asymptomatic cases6. while droplets of saliva discharged by people sneezing or coughing generally varies in size from 1 to 5  mm, particles in aerosol are considerably smaller, varying from 0.3  mm to 100  μm in diameter. this emphasizes the high contamination risk that clinicians are submitted during dental procedures7, which in turn require appropriate disinfection of devices and facilities offering dental treatments8-10. to facilitate disinfection of dental clinics, it is recommended that disposable guards are placed on surfaces and devices to avoid direct contamination. such disposable guards can be easily removed after each clinical procedure, which in turn should be followed by proper chemical disinfection11. furthermore, dental professionals should be adequately informed on possible covid-19 transmission routines12-13. there is agreement among studies that all dental elective treatment should be avoided during the current pandemic and quarantine periods4-14, whereas only emergency dental procedures should still be performed15. for this purpose, a screening process would be advisable prior to the patient appointment in dental clinics to avoid excessive exposure of the dental team and patients themselves16. as an essential area of healthcare, dentistry should adapt to the above mentioned changes during and after the current pandemic. in this context, relatively recent studies on digital dentistry have presented methods and technological improvements that offer advantages in the different fields of study in dentistry to treat patients during the current pandemic. nevertheless, little is known on how digital workflow tools and methods could be useful to address the safety concerns related to the current covid19 pandemic. thus, the aim of this study is to perform a literature review to identify advantages of digital workflows in dentistry that could be widely adopted to address safety issues raised during the covid-19 pandemic. material and methods a literature review was carried-out to describe the advantages of digital dentistry – as compared to conventional methods – that could help addressing the new safety 3 markarian et al. demands during dental treatments due to the current pandemic. such advantages were addressed by area of knowledge. included areas were: oral and maxillofacial radiology, esthetic dentistry, dental emergency, prosthodontics, orthodontics, periodontics, endodontics, oral and maxillofacial surgery and implant dentistry. pubmed and embase databases were searched for articles published in the last five years (i.e. from april of 2015 to april of 2020), with restriction to english language publications according to keywords following the search strategy: (digital dentistry or digital workflow) and (conventional or analog) and (advantages or comparison) and (oral radiology or endodontics or periodontology or dental implants or prosthodontics or aesthetic or restorative dentistry or dental emergency or oral surgery). reference lists of any potential articles and opengrey12 database were screened for relevant unpublished studies or papers not identified by electronic searching. the inclusion criteria considered original articles, systematic reviews, technique reports and clinical reports for the analyses. conference abstracts and book chapters were excluded from the study. to be considered eligible for inclusion, studies should have reported at least one advantage of any digital workflow in dentistry that could be useful to improve safety against the covid-19 pandemic. the guidelines of prisma statement were followed during data assessment and evaluation17. data extraction two independent reviewers with expertise in digital dentistry screened the titles, abstracts and full texts of articles identified. when considered required, attempts to contact study authors were performed. data extracted from the studies included: year of publication; location of the study, sample and group characteristics; methodologic characteristics; outcome measures and conclusions. results a total of 181 potentially eligible papers were screened. of these, 122 were excluded after title, keywords and/or abstract assessment, yielding 59 papers that potentially met the inclusion criteria. then, a total of 25 studies were also excluded, since their full texts did not present any direct evidence of advantages of digital dentistry, as compared to conventional methods. as a result, a total of 34 studies were finally identified as eligible for inclusion in this literature review. characteristics of advantages raised by the studies included are summarized in table 1. of the studies analyzed, 21% were published in 2020, 39% in the 2019, 18% in 2018, and 22% between 2015 and 2017. furthermore, 36% of the studies were performed in the american continent (8% from north america), 31% in asia, and 33% in european countries. regarding the study design, 89% of the studies were observational, descriptive and analytical (of these, 48% were case reports), whereas 11% were experimental studies. characteristics of clinical relevance concluded by the studies are also summarized in table 1. 4 markarian et al. oral and maxillofacial radiology one of the most important aspects raised by the literature that could be important in the current pandemic is the establishment of faster, safer and optimal imaging diagnostic protocols18. for this purpose, imaging diagnostic centers should have adequate equipment hardware to perform faster scans, as well as to store and send images digitally to the responsible clinician, avoiding generation of printed exams. however, one of the main concerns raised by the literature, is the fact that most dental clinicians seem to be using printouts or static image files sent by email, instead of receiving original digital communication in medicine (dicom) files and performing digital analyses on software programs19. with the advent of cone beam computed tomography (cbct) technology, significant improvements in hardware and software components have reduced radiation doses for patients. these enhancements include changes in sensor technology, smaller fields of view, and pulsed radiation technique following the alara (as low as reasonably achievable) radiation guidelines. furthermore, several optimized exposure protocols (e.g. child mode, adult mode, high-resolution mode, high definition mode) have constantly been developed by manufacturers20. for this reason, modern and faster dental cbct devices with smaller field of views have replaced medical ct scans of the craniofacial region, thus decreasing the total radiation dose for the patient21. in addition to cbct, some non-invasive methods of diagnosis have become increasingly implemented in the clinical routine. intraoral scanners are fast and prevent manipulation of impression trays with saliva. evaluation of carious lesions using near-infrared light transillumination (nilt) is also feasible and has been shown to be as accurate as interproximal radiographs22. some intraoral scanners have implemented this technology, such as the itero 5d (align technologies, san jose, california, usa). table 1. summary of advantages of digital dentistry with relevance for the current pandemic raised by the studies assessed field of study studies assessed relevance for the covid-19 pandemic oral and maxillofacial radiology references 18-26 scans are faster and safer. digital 3d data storage decreases number of appointments. aesthetic dentistry references 27-29 facially driven digital wax-up decreases number of clinical appointments. orthodontics reference 30 orthodontic aligners require less clinical appointments. prosthodontics references 31-38 cad-cam procedures lead to less chairside time and reduced number of appointments. implant dentistry references 23, 39-42 prosthetically-driven guided surgery decreases surgical time and number of appointments. oral and maxillofacial surgery references 43-46 cad-cam surgical guides lead to faster and less invasive procedures. endodontics references 47-50 endodontic guides lead to faster procedures while decreasing the risk of iatrogenesis. periodontology reference 51 surgical guides for crown lengthening decrease chairside time and number of appointments. 5 markarian et al. with the appropriate computer-aided design and computer-aided manufacturing (cad-cam) techniques, fully digital workflow methodologies can be performed from stored digital imaging files of the exams, leading to a reduced number of clinical appointments to finalize oral rehabilitation treatments23. for this purpose, several types of files such as photographs, videos, intraand extraoral scans and cbct can be imported and combined in the same software to orientate treatment planning and execution by means of cad-cam technologies24,25. while cbct should not be performed routinely without proper clinical indications, intraand extraoral scans could be routinely stored, in order for the professional to have a backup of a patient’s dental arch. this would allow the professional to analyze such dental arch without requiring a clinical appointment solely for this reason26. aesthetic and restorative dentistry in aesthetic dentistry, the use of facially driven digital wax-up tools such as the digital smile design (dsd), combined with the traditional mock-up technique allows for predictability of treatments, leading to higher success rates27. digital imaging allows patients to visualize the expected final result, as compared to their current oral health condition27,28. in this context, conventional mockups still used in some digital workflows can be replaced by photos, videos, and 3d models. dsd programs incorporate digital technology to the smile design process and can be used as tools for diagnosis, treatment plan visualization, and communication with the patient and technician that can increase treatment outcome predictability29. therefore, treatment plan and details can be discussed with no need of a physical appointment. orthodontics in the case of orthodontic patients, one of the most important preventive measures to prevent the spread and control the infection by covid-19 is to perform an adequate screening of patients who should not attend the dental clinic16. in this context, digital workflow with orthodontic aligners requires less clinical appointments at the chairside, as compared with other orthodontic therapies. according to recent evidences, the use of orthodontic aligners is considered an aesthetic technique recommended to solve simple to moderate alignment cases30. on the other hand, an orthodontic aligner can be broken or lost by the patient. in this case, patients are oriented to continue to use a previous aligner in the sequence, depending on how long the patient had already used the broken / lost aligner. this decision would avoid the need of the patient to immediately schedule a physical appointment, which can be therefore postponed to a more adequate occasion, when the clinician is able to perform new digital impressions. moreover, any treatment planning changes could still be performed digitally, which also avoids unnecessary physical appointments with the patient16. prosthodontics in conventional prosthetic dentistry, a number of clinical steps are usually necessary for try-in and adjustments of prostheses coming from the laboratory. however, digital workflow techniques can optimize prosthetic planning and fabrication, which is espe6 markarian et al. cially beneficial for patients and professionals during the current pandemic due to several reasons. firstly, cad/cam procedures have the potential to produce reliable prostheses with a reduced number of appointments, with shorter chairside times, as well as higher precision and accuracy, as compared with conventional methods31,32. furthermore, optimal occlusal and proximal contacts can be forecasted and designed in software, especially when making monolithic complete anatomy prostheses, without the need of further ceramic layering. therefore, an anatomic analysis using full digital workflow is encouraged to obtain optimized results for digital crowns, fixed bridges, and full arch implant rehabilitations, leading to improved adaptation and reduced number of laboratory adjustments required33. selection of materials for cad-cam restorations depends on the extension of the prosthetic span and the expected duration of the prosthesis (e.g. pmma can be used for provisionalization, vitroceramics are indicated for crowns, liquid resin is indicated for provisionalization or prototypes, translucent zirconia can be used from crowns to full-arch prostheses)34. similarly, cad-cam technology can be also applied to fabricate removable dental devices, such as complete dentures, removable partial dentures and splints with a reduced number of clinical steps, shorter chairside time and better adaptation results, as compared with the conventional workflow35,36. despite such evidences, dentists must be aware that the use of high speed handpieces generate spray and airborne saliva particles9. therefore, procedures involving such handpieces should be avoided during the current pandemic. nevertheless, in cases that tooth preparation is mandatory, finishing line of preparations may be left at gingival level to avoid bleeding, and to facilitate digital impressions without the presence of blood37,38. implant dentistry digital technology was introduced in implant rehabilitations, leading to new different workflows to enable easier and faster ways to resolve cases. digital implant-prosthetic planning and image-guided surgery techniques decrease the number of clinical appointments, since there is no need for clinically trying-in a conventional diagnostic wax-up in the mouth. therefore, it is possible to perform an image-guided implant surgery after performing imaging examinations, virtual planning and surgical guide 3d-printing. use of surgical guide developed from full digital workflow increasing predictability of implant positioning23,39. digital workflow can also be used in cases of immediate implants. the advantages of immediately placed implants include reduced treatment time, fewer surgical procedures, better aesthetics and preservation of the alveolar bone in height and thickness40. similarly, image-guided immediate implant surgery is performed less invasively, with less bleeding, often being a flapless surgery, and thus diminishing the dental cabinet potential contamination. finally, since digital prosthetic planning is carried-out before surgical planning to allow for a prosthetically-driven implant placement, infrastructures and temporary prostheses can be manufactured even before implant placement surgery. such advantages also apply for cases of bone grafting surgeries related to implant dentistry. in this context, two studies have reported the usefulness of cadcam digital workflow to virtually plan the shape of block bone grafts41, and to fabri7 markarian et al. cate surgical guides to remove fixation screws of block grafts allowing for subsequent flapless implant placement that leads to reduced surgical time and enhanced postsurgical healing42. oral and maxillofacial surgery during the current pandemic, oral and maxillofacial surgical procedures should allow for faster procedures with reduced amount of aerosol produced in the environment15. in this context, digital simulation of third-molar extraction surgeries based on cbct scans has proved valuable for surgical guidance43. this leads procedures to be faster and less invasive and therefore safer for the patient, while requiring a lower amount of aerosol from clinical procedures during surgeries. models and surgical guides digitally designed from cbct scans in cad software and manufactured with 3d printing facilitate various procedures related to oral surgery, such as cases of periapical surgeries44, surgeries for excision of large oral lesions45, and surgeries to treat maxillofacial fractures46. among the advantages of imageguided surgery described by the aforementioned studies are fast procedures and shorter chairside time required to perform surgeries. endodontics treatment planning using digital workflow and the use of endodontic guides simplified root channel treatment in obliterated teeth while decreasing the risk of iatrogenesis at the same time47,48. in addition, scientific evidences in endodontics suggest that treatment of compromised cases can be safely performed with digital workflow by less specifically experienced or skilled clinicians47. among the advantages described in the literature discussed herein are: reduced treatment time for the clinician while offering higher predictability and success rates. surgical endodontic treatment can also benefit from digital dentistry methods. with freehand osteotomy and resection, clinicians make sequential adjustments to correct for errors in perforation site, angulation, and depth, possibly resulting in overextended osteotomy and nonideal resection, prolonging the surgical time49. on the other hand, guided surgery allows for a more efficient osteotomy and resection ideal of the surgical site and reduced surgical time potentially augmenting the healing process49. surgical endodontic treatment aided by a 3d printed surgical guide provides an accurate osteotomy and root-end resection procedures50. periodontology in the field of periodontology, the most common treatment performed with digital workflow is the crown lengthening procedure, in which virtual planning allows for creation of cad-cam surgical guides to orientate gingivectomy and osteoplasty procedures in the aesthetic region. in this context, periodontal surgical outcomes can be predicted after the assembly of the digital project in a cad-cam software to carry out the aesthetic planning51. such digital planning may include a face-guided wax pattern, a cbct scan and intraoral scans of both dental arches51. the aforementioned digital workflow has been found to decrease the number of necessary clinical appointments when compared with the conventional methodology, while also improving treatment predictability. 8 markarian et al. discussion digital dentistry is based on the use of cad-cam methodologies. as described by the articles discussed herein, the initial project phase is characterized by transfer of patient’s data to the digital world by using cad methodologies to enable virtual treatment planning. when the latter is ready, one or more dental treatment devices can be fabricated with a cam technique (e.g. 3d printing or milling). in this context, acquisition of intraoral scans is the first step of a digital workflow, and lead to greater comfort and less direct contact of the operator with the infected oral tissues, blood and saliva, as compared with conventional impressions. nevertheless, all removable parts from intraoral scanners, such as tips, must undergo steam heat sterilization after each use. similarly, fixed parts of the hardware such as screens and computers must be disinfected with chemical solutions with a single-use disposable wipe52,53. despite its high cost, intraoral scanners can be used to accurately collect the data required to fabricate fixed or removable prosthetic devices54. such data may include: implant tridimensional locations, details on gingival contours; prosthetic implant emergence profiles; previous prosthesis gingival volume; previous tooth shapes and tridimensional positions, vertical dimension, the opposing arch anatomy and dynamic occlusal contacts33. after data acquisition, digital workflows can be performed usually requiring less clinical appointments and shorter chairside time during treatment, reducing inaccuracies, and decreasing the number of steps and chances of occurrence of human errors54. among the general advantages of digital over conventional workflows for the covid19 pandemic discussed herein are: decreased number of steps, higher productivity and possibility to perform several of the treatment steps directly in software, with no need of human contact22,23. as a result, planners and cad professionals do not necessarily need to perform some of these tasks at laboratories or clinical facilities. instead, in situations where quarantine is indicated, tasks could be performed remotely from the professionals’ homes, following the recommendations to lessen the impact of the current pandemic. for dental technicians working in laboratories, there are also several safety advantages provided by digital dentistry, such as avoiding conventional impressions that may carry biologic materials that are risk factors for the transmission of any type of microbial agents, including the coronavirus12,15. another intrinsic advantage of digital workflow is the decreased working time from dental technicians during fabrication of devices with cam, in which a milling machine and/or digital 3d printer can produce several pieces for hours simultaneously. as a result, digital laboratory workflows can be performed faster and with a reduced number of workers inside the laboratory environment, as compared to conventional workflows that require the physical presence of more dental technicians on benches to zmanually produce prosthetic devices and components55. the above-mentioned observations indicate that the advent of digital dentistry by dental laboratories could encourage dentists to work with and send preferably digital impressions in order to reduce human contact to biologic material and to benefit from the usefulness of digital workflows. on the other hand, in situations where the use of regular impressions is unavoidable, dentists could decontaminate the material prior 9 markarian et al. to sending it to laboratories by using 0.1% sodium hypochlorite or 70% ethanol or 0.5% hydrogen peroxide, which are disinfectants already available in dental clinics56. it is advisable to re-decontaminate all material when arriving, while changing laboratory sector, and when leaving the dental laboratory. in addition to the new measures in dentistry discussed herein, dental clinicians could also benefit from cad-cam methodologies to participate in projects developing novel tools to improve biosafety during the current pandemic. in this context, the use of rubber dam and additional suction devices could prevent contamination from airborne particles during dental procedures4. moreover, cad-cam knowledge could also be applied for the fabrication of customized equipment such as 3d-printed face masks and face shields57. among the limitations of this scoped literature review is the limited number of experimental studies on digital dentistry. this occurs due to the fact that literature on digital workflows are very recent, including several case reports and description of techniques. such techniques, in turn, have been developed along with the current fast technological improvements, and still need to be assessed in larger clinical and experimental studies. another limitation is that, as expected, the relationship between clinically relevant advantages of digital workflows and the current pandemic is not necessarily explicit in articles on digital dentistry. therefore, future public health studies would still be recommended to address the actual impact of digital workflows on the reduction covid-19 transmission in dental clinics. within the limitations of this study, the findings observed herein suggest that the use of digital dentistry tools can lead to increased safety and reduced transmission of covid-19 during the current pandemic. acknowledgments we wish to thank capes (coordination of the advancement of higher education, brasília, brazil) for its financial support. the authors deny any conflicts of interest related to this study. references 1. liu j, liao x, qian s, yuan j, wang f, liu y, et al. community transmission of severe acute respiratory syndrome coronavirus 2, shenzhen, china, 2020. emerg infect dis. 2020 jun;26(6):13203. doi: 10.3201/eid2606.200239. 2. khurshid z, asiri fyi, al wadaani h. human saliva: non-invasive fluid for detecting novel coronavirus (2019-ncov). int j environ res public health. 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int. 2019;50(1):58-65. doi: 10.3290/j.qi.a41337. 53. boyce jm. alcohols as surface disinfectants in healthcare settings. infect control hosp epidemiol. 2018 mar;39(3):323-8. doi: 10.1017/ice.2017.301. 54. mangano c, luongo f, migliario m, mortellaro c, mangano fg. combining intraoral scans, cone beam computed tomography and face scans: the virtual patient. j craniofac surg. 2018 nov;29(8):2241-6. doi: 10.1097/scs.0000000000004485. 55. piedra cascón w, parra nuñez a, charlén díez i, revilla-león m. laboratory workflow to obtain long-term injected resin composite interim restorations from an additive manufactured esthetic diagnostic template. j esthet restor dent. 2019 jan;31(1):13-19. doi: 10.1111/jerd.12419. http://j.qi 13 markarian et al. 56. kampf g, todt d, pfaender s, steinmann e. persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents. j hosp infect. 2020 jun:s0195-6701(20)30285-1. doi: 10.1016/j.jhin.2020.06.001. 57. cortes ar, galea k, no-cortes j, sammut ej, alzoubi ee, attard nj. use of free cad software for 3d printing individualized face masks based on face scans. int j comput dent. 2020;23(2):183-9. 1http://dx.doi.org/10.20396/bjos.v20i00.8663781 volume 20 2021 e213781 original article 1 school of health sciences, positivo university up, curitiba, paraná, brazil. 2 department of dentistry, university of joinville region – univille, joinville, santa catarina, brazil. 3 department of dentistry, tuiuti university of paraná – utp, curitiba, paraná, brazil. *corresponding author: flares baratto-filho 10 paulo malschitzki, zona industrial norte, joinville, santa catarina, 89219-710, brazil. phone: +55 41 991042685 e-mail: fbaratto1@gmail.com editor: dr altair a. del bel cury received for publication: december 30, 2020 accepted: march 29, 2021 influence of different reciprocating systems on the apical extrusion of debris in flat-oval canals monique marchiori1 , arissa michelle yamada corrêa2 , flávia sens fagundes tomazinho1 , marilisa carneiro leão gabardo1 , natanael henrique ribeiro mattos3 , luiz fernando fariniuk3 , flares baratto-filho2,3,* aim: to evaluate the apical extrusion of debris in flat-oval canals, using three reciprocating systems at two different working lengths (wl), 0 mm and 1 mm from the apical foramen. methods: ninety mandibular incisors were randomly divided into three groups based on the systems: waveone gold #25.07 (wog), prodesign r #25.06 (pdr), and x1 blue #25.06 (x1b). extruded debris were collected and dried in pre-weighed eppendorf tubes. three consecutive weighings were performed for each tube, and the mean was calculated. if the assumptions of normality and homogeneity of variance were not met, the kruskal-wallis test was used to analyze the amount of extruded debris between groups with the same wl, and the mann-whitney u test was used for comparison within groups for each wl. results: all groups had extruded debris, with higher median values occurring at 1 mm. no significant difference regarding the amount of debris extrusion was observed at 0 mm (p>0.05) and 1 mm (p>0.05) between groups. however, within the groups, at different wl, there was greater extrusion at 1 mm (p<0.05), with pdr differing significantly from the other systems (p<0.05). conclusion: the extrusion of debris occurred regardless of the group, with higher values at 1 mm. however, using pdr at 1 mm from the apical foramen showed the highest values of extrusion. keywords: dental pulp cavity. root canal preparation. tooth apex. https://www.univille.edu.br/ https://www.univille.edu.br/ https://www.univille.edu.br/ https://orcid.org/0000-0001-6693-0663 https://orcid.org/0000-0002-2771-4287 http://orcid.org/0000-0001-5553-6943 http://orcid.org/0000-0001-6832-8158 http://orcid.org/0000-0003-2755-2270 http://orcid.org/0000-0003-0731-9893 http://orcid.org/0000-0002-5649-7234 2 marchiori et al. introduction root canal preparation is an essential step of endodontic treatment that includes the cleaning, shaping, and disinfection of root canals1. the irrigation procedure assists in the lubrication of the root canal walls, favoring the cutting action of endodontic instruments and the removal of tissue remnants, such as pulp and dentin1. the quality of root canal preparation depends on the design, kinematics, and cutting ability of instruments. in this context, the nickel-titanium (niti) alloy increases the flexibility and cyclic fatigue strength of instruments, making them resistant to torsional failure2,3. reciprocating systems with this alloy allow faster and more efficient preparation of the root canal with a single instrument4. waveone gold (wog) (dentsply sirona, ballaigues, switzerland) is a reciprocating system represented by a parallelogram cross-section with a variable taper. this system is manufactured with niti gold alloy technology that increases flexibility, providing better resistance to cyclic fatigue3,4. the prodesign r (pdr) instrument (easy equipamentos odontológicos, belo horizonte, brazil) is a reciprocating system with an s-shaped cross-section. it is produced with niti cm control memory alloy, making it extremely flexible, but without shape memory5,6. the x1-blue (x1b) file (mk life, porto alegre, brazil) features single-use reciprocating kinematics with a triangular cross-section and is manufactured with blue heat treatment technology7. thermomechanically treated niti alloys have been reported to be more flexible with improved cyclic fatigue resistance and greater angle of deflection at failure than conventional niti alloy8. the design of reciprocating instruments is such that debris removal is in the coronary direction and its apical extrusion is decreased7,9. debris can be released in the periapical region during root canal preparation, causing damage to peri-radicular structures and delayed healing10,11, with probable causes related to root canal anatomy, instrument design, and technique9,10. several studies have aimed to evaluate this extrusion9-18, including analysis with flat-oval canals13,18, and regardless of the instrumentation used, extrusion does occur10,13. another relevant issue concerning the preparation is the working length (wl) determination, which must be adjusted in the apical constriction associated with the main foramen, thereby allowing a better mechanical debridement of the apical area, better irrigation, disinfection, and reduction of the amount of extruded debris9. to date, no endodontic instrument/system has completely eliminated the possibility of apical extrusion of debris. this study aimed to evaluate this occurrence with the reciprocating instruments wog, pdr, and x1b at two different wl. the null hypothesis tested was that there are no differences between the amount of debris extruded by the different instruments used, even when comparing the two wl, 0 mm and 1 mm. material and methods sample selection the local research ethics committee approved the research project (nº 2.716.775). a priori sample calculation was not performed. however, for the variable debris weight 3 marchiori et al. (mg), which was different at different wl (mm) using the mann-whitney non-parametric test, the debris weight rank (mg) and the test power was calculated, which was 70%. thus, 90 recently extracted mandibular central incisors were selected, cleaned, and stored in distilled water. the inclusion criteria were single root canal, complete rhizogenesis, at least 19 mm of length, canal with single foramen and patency, absence of internal/external root resorption, no root fractures, and no previous endodontic treatment. digital radiographs in the mesiodistal and buccolingual directions were taken to select the flat-oval canals (buccolingual diameter four times greater than mesiodistal diameter). to standardize the dental groups and each system, the classification by wu et al.18 was chosen for selecting the flat-oval canals. sample preparation the specimens (n = 90) were individually stored and distributed randomly into two experimental groups (n = 45) with different wl: 0 mm and 1 mm of the apical foramen. subsequently, the teeth crowns were removed with 3080 diamond bur (kg sorensen, são paulo, brazil), and the lengths were standardized to 19 mm, measured using a digital pachymeter (mtx 316119, guarulhos, brazil). the foramen patency was then determined with a size 10 k-file (dentsply sirona, ballaigues, switzerland) till its tip was visible, and the wl was established according to the pre-established groups. evaluation of apical extrusion of debris for debris evaluation, the experimental model adapted from myers and montgomery19 was used. an orifice was created in the cap of an eppendorf tube, and a specimen was inserted until the cement-enamel junction remained 1-2 mm above, fixed with a cyanoacrylate-based glue (ic-gel ethyl cyanoacrylate gel, insta-curebsi, odeme dental research, luzerna, brazil). a 27-g needle was placed beside the root to balance the air pressure inside and outside the tube. thus, the specimen was fixed in the eppendorf tube to collect the debris extruded from the foramen. after root canal preparation, the tube was placed in an oven for evaporation. an aluminum foil covered the tube, thus blinding the operator to the root apex and process. eppendorf tube pre-weighing the tubes were weighed (in mg) three times using a precision analytical balance (m214ai, bel engineering, piracicaba, brazil) with an accuracy of 10-4 mg, and the mean value of each weighing was obtained. root canal preparation a single operator, a specialist in endodontics with five years of experience, performed all stages of the experiment. irrigation was carried out with a total of 5 ml distilled water (ssplus do brasil ltda, maringá, brazil) using a 27-g gauge needle (endo-eze irrigator tip – ultradent, salt lake city, usa) with a plastic syringe (bd plastipak 10 ml bd, curitiba, brazil), with its tip, inserted in the wl at 3 mm20. the instruments were driven with the x-smart plus motor (dentsply sirona, ballaigues, switzerland) using the “wave one” settings. after three pecking motions, the instrument was removed from the root canal, cleaned, and inspected before being 4 marchiori et al. reused. the canal was irrigated with 1 ml distilled water, and a size 10 k-file was used to check the patency. the selected group (0 mm or 1 mm) repeated this procedure till the instrument reached the pre-established wl. at the end of the root canal preparation, the specimens were submitted to a final irrigation with 1 ml distilled water with the irrigation needle positioned at 2 mm from the wl of each group. the same instrument was used to prepare three specimens and was then discarded. extruded debris quantification after complete instrumentation, the cap, cannula, teeth, and aluminum foil were separated from the eppendorf tube. the tubes with debris were then stored in an incubator (incubator inc.410, new ethics and scientific equipment products ltd., são paulo, brazil) at 37 °c for seven days for the evaporation of distilled water. after this period, the tubes were weighed using the same precision analytical balance described above to quantify only extruded debris. the mean was calculated after weighing each tube three times, in which the final weight was subtracted from the initial weight. statistical analysis statistical analysis was performed using the software spss v. 25 (ibm spss inc., armonk, usa). the shapiro-wilk and levene tests were used to verify the normality and homogeneity of data, respectively. to analyze the amount of debris extrusion between groups with the same wl, a non-parametric test (kruskal-wallis test) was used. the mann-whiney u test was used to analyze this amount within the same group, considering the two wl. the level of significance was set at 5%. results the data obtained are shown in table 1. regardless of the instrument used, higher median values of extrusion were observed at 1 mm. regarding the wl, no significant difference in the amount of debris extrusion was observed at 0 mm (p>0.05) and 1 mm (p>0.05) between the instruments. in contrast, taking into consideration the wl, there was a difference in the amount of extruded debris at 1 mm (p<0.05), with pdr differing significantly from the others (p<0.05). table 1. median (minimum-maximum) of extruded debris (mg) after root canal preparation working length group median (min.-max.) of debris weights regardless of instrument wog pdr x1b 0 mm 0.0023 (0.0000-0.0300)aa 0.0020 (0.0010-0.0200)aa 0.0023 (0.0007-0.0123)aa 0.0023 (0.0000-0.0300)a 1 mm 0.0037 (0.0020-0.0063)aa 0.0040 (0.0020-0.0090)ab 0.0033 (0.0003-0.0553)aa 0.0037 (0.0003-0.0553)b note: lower case letters indicate a significant statistical difference between the groups of instruments in each wl (kruskal-wallis test, p<0.05). capital case letters indicate a significant statistical difference between each group of instrument in different wl (mann-whitney u test, p<0.05). 5 marchiori et al. discussion this study was conducted to analyze the amount of apical debris extrusion in mandibular incisors with flat-oval-shaped root canals, using reciprocating kinematics at two different wl. the reciprocating systems wog, pdr, and x1b were chosen because they have the same kinematics but with different tapers, cross-sections, and heat treatments. in the present study, the null hypothesis was rejected since significant differences were found in the analysis of debris extrusion when comparing the instruments and wl, where higher values of extrusion were observed at 1 mm. all instruments used in this study had the same kinematics. the literature comparing manual, rotary, and reciprocating instruments has shown that greater apical extrusion of debris was obtained with manual instruments12,15,19. the study and development of new instrument designs and different niti alloys with various heat treatments allow an efficient, faster and safer root canal preparation associated with the reciprocating movement21. according to varela-patiño et al.22, the reciprocating kinematics relieves tension in the instrument due to special counter-clockwise (cutting action) and clockwise (instrument release) movements, providing a balanced force technique that causes less extrusion of debris. dincer et al.14 compared the reciprocating, rotary, and adaptive kinematics and showed the lowest apical extrusion values for the reciprocating and twisted file adaptive (tfa) system. boijink et al.15, analyzing the wog, tfa (sybronendo, orange, usa), and manual instruments, observed that reciprocating instruments extruded the least amount of debris in curved root canals. in a comparison between single-file systems, wog and reciproc (vdw gmbh, munich, germany), authors found no significant statistical difference in the amount of the debris extruded12. using a similar dental group as that of this study (flat-oval root canals), kirchhoff et al.13 evaluated the amount of apically extruded debris with four different instrumentation systems: protaper next (ptn; dentsply tulsa dental, tulsa, usa), wog, tfa, and self-adjusting file (saf) (redent-nova, ra’anana, israel). the authors revealed fewer values with ptn, wog, and tfa than saf. however, farmakis et al.16, in an ex vivo study with mandibular premolars with oval-shaped canals showed that wog had a higher mean mass of apically extruded debris compared to saf, with a significant difference. frota et al.9, using manual and reciprocating systems such as reciproc, wog and pdr found lower extrusion results with pdr, different from the results obtained in the present study in which the highest debris extrusion value was found for the pdr group. this can be possibly explained by the s-shaped cross-sectional characteristic of the pdr, which provides a greater cutting ability of the root canal walls5, when compared to the design of the cross-section of the wog, a parallelogram with only one cutting edge in contact with the canal wall, and x1b which presents a convex triangular cross section. regarding the diameter of the instruments, bürklein et al.23 and tinaz et al.24 confirmed that the larger the diameter of the instrument’s tip, the greater the apical patency, and consequently the amount of extruded debris. the differences in diameters between the instruments, that 6 marchiori et al. could be an interference factor in the results, were excluded in this study since a diameter size 25 was used. even though the same diameter with different tapers .06 and .07 was used in this study, the results showed no significant differences due to diameter variability. concerning the wl, the present results showed greater extrusion of debris at 1 mm of the apical foramen than at the limit of the apical foramen (0 mm), with a statistically significant difference. this may have occurred because the final patency made with a size 10 k-file may have pushed these debris toward the apex instead of being moved in the coronal direction. the apical plug is formed when the instrument works in1 mm at the apical foramen with an over instrumentation of the root canal compacting the debris near the apex19. confirming this possibility, other studies have observed that during endodontic treatment, the movements of the k-file instruments toward the apex resemble that of a piston, with push and pull kinematics, and together with irrigation can cause greater debris compaction, tissue remnants, dentin scrapings, and irrigants in periapical tissues with a blockage of the apical foramen24,25. in a recent study to evaluate the extrusion of debris associated with the use of the reciproc and wog in curved canals instrumented up to different wl (at the apical foramen and 1 mm short of the foramen). the authors concluded that regardless of the wl, both reciprocating systems were associated with similar results26. another factor that may be correlated with the results of this study is the diameter of the irrigation needle and the depth at which it is positioned during the final irrigation process. this may have influenced the higher debris extrusion values at 1 mm of the apical foramen. the irrigation needle used in this study was 27-g, and the irrigation length during the root canal preparation was pre-established at 2 mm below the wl of each group, which corroborates the methodology described in the studies of uzunoglu-özyürek et al.26 and boutsioukis et al.20. in these studies, it was established that the needle should be positioned 2-3 mm at the wl, and the space available around and below the needle is necessary for the irrigating solution to have a reverse flow towards the apex. based on these results, it was suggested that needles with a diameter below the apical diameter of the root canal after preparation, positioned at 3 mm of the wl, could improve the penetration of the apical third preventing the extrusion of debris. to avoid the influence of different irrigating solutions, distilled water instead of sodium hypochlorite (naocl) was chosen as observed in several studies11,16,23. naocl, when subjected to high temperatures and consequent evaporation, results in the formation of crystals that mix with the debris, interfering with the results of the quantification of extruded debris27. thus, the amount of irrigation is directly associated with greater cleaning of the root canals. it is important to emphasize that this study used extracted teeth, and there are some limitations since the presence of periapical tissues around the foramen, in vivo, can avoid the extrusion of debris and irrigating solutions through the apical foramen17. in this study, mandibular incisors with flat-oval canals were used, in which it may be difficult to remove all intracanal residues properly13,18. therefore, new instruments have been developed and tested that contemplate the difficulties in cleaning and shaping root canals. additional studies are needed in the face of these challenges to compare the results found. 7 marchiori et al. in conclusion, extrusion of debris 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in flat-oval root canals after using different instrumentation systems. j endod. 2015 feb;41(2):237-41. doi: 10.1016/j.joen.2014.09.023. 14. dincer an, guneser mb, arslan d. apical extrusion of debris during root canal preparation using a novel nickel-titanium file system: waveone gold. j conserv dent. 2017 sep-oct;20(5):322-325. doi: 10.4103/jcd.jcd_407_16. https://pubmed.ncbi.nlm.nih.gov/31340226/ https://pubmed.ncbi.nlm.nih.gov/31340226/ 8 marchiori et al. 15. boijink d, costa dd, hoppe cb, kopper pmp, grecca fs. apically extruded debris in curved root canals using the waveone gold reciprocating and twisted file adaptive systems. j endod. 2018 aug;44(8):1289-92. doi: 10.1016/j.joen.2018.04.011. 16. farmakis et, sotiropoulos gg, abràmovitz i, solomonov m. apical debris extrusion associated with oval shaped canals: a comparative study of waveone vs self-adjusting file. clin oral investig. 2016 nov;20(8):2131-8. doi: 10.1007/s00784-016-1709-3. 17. uslu g, özyürek t, yılmaz k, gündoğar m, plotino g. apically extruded debris during root canal instrumentation with reciproc blue, hyflex edm, and xp-endo shaper nickel-titanium files. j endod. 2018 may;44(5):856-9. doi: 10.1016/j.joen.2018.01.018. 18. wu mk, r’oris a, barkis d, wesselink pr. prevalence and extent of long oval canals in the apical third. oral surg oral med oral pathol oral radiol endod. 2000 jun;89(6):739-43. doi: 10.1067/ moe.2000.106344. 19. myers gl, montgomery s. a comparison of weights of debris extruded apically by conventional filing and canal master techniques. j endod. 1991 jun;17(6):275-9. doi: 10.1016/s0099-2399(06)81866-2. 20. boutsioukis c, lambrianidis t, verhaagen b, versluis m, kastrinakis e, wesselink pr, et al. the effect of needle-insertion depth on the irrigant flow in the root canal: evaluation using an unsteady computational fluid dynamics model. j endod. 2010 oct;36(10):1664-8. doi: 10.1016/j. joen.2010.06.023. 21. yared g. canal preparation using only one ni-ti rotatory instrument: preliminary observations. int endod j. 2008 apr;41(4):339-44. doi: 10.1111/j.1365-2591.2007.01351.x. 22. varela-patiño p, ibañez-párraga a, rivas-mundiña b, cantatore g, otero xl, martin-biedma b. alternating versus continuous rotation: a comparative study of the effect on instrument life. j endod. 2010 jan;36(1):157-9. doi: 10.1016/j.joen.2009.09.023. 23. bürklein s, benten s, schäfer e. quantitative evaluation of apically extruded debris with different single file systems: reciproc, f360 and one shape versus mtwo. int endod j. 2014 may;47(5):405-9. doi: 10.1111/iej.12161. 24. tinaz ac, alacam t, uzun o, maden o, kayoglu g. the effect of disruption of apical constriction on periapical extrusion. j endod. 2005 jul;31(7):533-5. doi: 10.1097/01.don.0000152294.35507.35. 25. mendonça de moura jd, bueno ceds, fontana ce, pelegrine ra. extrusion of debris from curved root canals instrumented up to different working lengths using different reciprocating systems. j endod. 2019 jul;45(7):930-4. doi: 10.1016/j.joen.2019.03.002. 26. uzunoglu-özyürek e, karaaslan h, türker sa, özçelik b. influence of size and insertion depth of irrigation needle on debris extrusion and sealer penetration. restor dent endod. 2017 dec;43(1):e2. doi: 10.5395/rde.2018.43.e2. 27. tanalp j, güngör t. apical extrusion of debris: a literature review of an inherent occurrence during root canal treatment. int endod j. 2014 mar;47(3):211-21. doi: 10.1111/iej.12137. 1http://dx.doi.org/10.20396/bjos.v21i00.8665442 volume 21 2022 e225442 original article 1 department of oral and maxillofacial surgery, school of dentistry, university of são paulo, são paulo, brazil. 2 department of stomatology, division of radiology, school of dentistry, university of são paulo, são paulo, brazil. corresponding author: joão gualberto c. luz, department of oral and maxillofacial surgery, school of dentistry, university of são paulo – usp. av. prof. lineu prestes, 2227 cidade universitária, 05508–900, são paulo – sp, brazil. phone: 5511 3091-7887, fax: 5511 3091-7879. e-mail address: jgcluz@usp.br editor: altair a. del bel cury received: april 25, 2021 accepted: september 02, 2021 cone-beam computed tomography analysis of degenerative changes, condylar excursions and positioning and possible correlations with temporomandibular disorder signs and symptoms nayara stefany leite-de-lima1 , eduardo felippe duailibi-neto2 , israel chilvarquer 2 , joão gualberto cerqueira luz 1* aim: to describe cone-beam computed tomography (cbct) features in patients with temporomandibular disorders (tmds), in terms of degenerative changes, condylar excursions and positioning as well as their possible correlations with signs and symptoms. methods: clinical records of patients diagnosed with tmd who were seen between january 2018 and december 2019 were retrospectively evaluated. these patients were divided into the following groups based on the diagnostic criteria for temporomandibular disorders (dc/tmd): arthralgia, myalgia, and arthralgia and myalgia groups. the cbct examination findings of the patients were evaluated in relation to degenerative changes, estimates of condylar excursion, and condylar positioning. the likelihood ratio test was used to verify the possible differences among the three groups, whereas the chi-square test was used to verify the possible differences among the signs and symptoms for the tomographic findings (p ≤ 0.050). results: in this study, 65 patients with tmd were included. these patients were predominantly female (84.6%) with a mean age of 40.6 years. tomographic findings of flattening, hyperexcursion and posterior condylar positioning were frequent. a significant correlation was noted between osteophyte and lateral capsule pain (p = 0.027), erosion and posterior capsule pain (p = 0.026), and flattening, pseudocysts (p < 0.050) and condylar excursion (p < 0.001) with mouth opening. conclusion: few correlations were noted between degenerative changes and signs of joint pain as well as degenerative changes and condylar hypoexcursion with mouth opening. these correlations were likely associated with division by diagnosis, whereas condylar positioning did not correlate with signs and symptoms. keywords: cone-beam computed tomography. mandibular condyle. signs and symptoms. temporomandibular joint disorders. https://orcid.org/0000-0002-6968-5082 https://orcid.org/0000-0002-1226-0366 https://orcid.org/0000-0003-3967-0143 https://orcid.org/0000-0002-7686-7829 2 leite-de-lima et al. introduction temporomandibular disorders (tmds) are diagnosed on the basis of a combination of clinical features and diagnostic imaging findings1. when indicated, diagnostic imaging is an important part of the examination process for patients with tmd. diagnostic imaging is used to confirm suspected disease, rule out disease, or obtain additional information2. computed tomography (ct) is considered valuable for evaluating tissues3. however, the identification of pathologies on the basis of imaging findings can be difficult4. in recent years, cone-beam computed tomography (cbct) has been the method of choice for assessing the bone morphology of the temporomandibular joint (tmj)5,6. cbct provides high-resolution multiplanar images with a lower radiation dose than multislice ct. this imaging technique allows examination of the tmj without superimposition or distortion, facilitating the analysis of bone morphology, joint spaces and dynamic function5,6. the signs and symptoms of patients with tmd include localized or diffuse pain in the tmj and masticatory muscles, articular sounds and functional disorders, which can occur in isolation or in association7,8. pain-related tmd can impact the individual’s daily activities, psychosocial functioning, and quality of life9. the main functional disorder is partial limitation of mouth opening. this condition occurs predominantly in females, representing from 67 to 82.2% of cases7,8. myalgia (m) is classified into three types: local myalgia, which is defined as pain localized to the site of palpation; myofascial pain, which is defined as pain spreading beyond the site of palpation but within the boundary of the muscle being palpated; and myofascial pain with referral, which is defined as pain at a site beyond the boundary of the muscle being palpated. arthralgia with disc displacement (add) represents three main types: disc displacement with reduction, disc displacement without reduction and degenerative joint disease. finally, myopain with arthralgia and disc displacement (maad) represents the association of the two main previous diagnoses9. many types of degenerative bone changes identified by cbct, such as flattening, erosion, sclerosis and osteophytes, have been described in individuals with tmd, and the changes reported vary in different studies10,11. however, degenerative alterations are common in asymptomatic individuals, representing up to 40% of cases12. in addition, there are reports of some correlations between hypermobility and joint symptoms13,14. on the other hand, correlations as well as an absence of correlations between condylar positioning and certain symptoms of tmd have been described15,16. thus, it is important to describe these features of cbct in patients with tmd due to the controversy regarding the presence or absence of correlations between tomographic findings and signs and symptoms of tmd. the objective of this study was to describe cbct features in patients with tmd in terms of degenerative changes, condylar excursions and positioning as well as their possible correlations with signs and symptoms. materials and methods a retrospective study was conducted with information collected from the medical charts of patients with tmd seen between january 2018 and december 2019 and 3 leite-de-lima et al. aged 18 years or older regardless of their gender, race and social status. patients with dental absences of up to three elements were admitted provided that they were isolated and included two posterior and one anterior teeth and did not include central incisors. patients with a history of parafunctional habits (e.g., bruxism) were admitted. patients who presented a history of previous orthodontic treatment, maxillofacial trauma, orthognathic or tmj surgery or neurological disorders were excluded from the study. tmd was diagnosed on the basis of the chief complaint and the findings from a clinical examination conducted according to the diagnostic criteria for temporomandibular disorders (dc/tmd)9. tmd was diagnosed using the dc/tmd diagnostic decision tree, the completed clinical examination form, and the symptom questionnaire. the patients were divided into groups based on their tmd diagnosis to assess for possible correlations between cbct findings and dysfunction groups or specific symptoms. the cases were divided into 3 groups according to the tmd diagnosis: m comprising localized myalgia, myofascial pain, and myofascial pain with referral; add comprising disc displacement with reduction, disc displacement without reduction and degenerative joint disease; and madd comprising the association of the two main above diagnoses9. prior to the initiation of the clinical examination, the first author (research fellow) underwent calibration sessions with a specialist trained in the use of the dc/tmd protocol9. ethical approval for this study was provided by the human research ethics committee of the school of dentistry, university of são paulo, brazil (protocol caae 09536918.5.0000.0075). all of the tomographic images were obtained at the same radiological facility using a carestream dental cs 9600 scanner (carestream dental llc, atlanta, ga, usa). the sagittal and coronal tomographic views were analyzed under standard conditions separately by the authors. the second author, who specializes in dentomaxillofacial radiology, analyzed the cbct findings. no tomographic examinations were performed for the purpose of this study. the occurrence of degenerative bony changes was defined as the presence of flattening of the condylar head due to loss of condylar convexity; sclerosis due to increased bone density; osteophyte formation as the result of a bony protrusion on the condylar margins; erosion, which represents a decrease in cortical and subcortical bone densities; and single or multiple subchondral cysts (scs) or pseudocysts, which represent pyriform-shaped subchondral lesions with sclerotic margins (figure 1)1,2. figure 1. examples of degenerative bone changes. (a) erosion; (b) sclerosis; (c) flattening; (d) osteophytes; (e) subchondral cysts. a b c d e 4 leite-de-lima et al. to obtain condylar excursion estimations, sagittal images that were taken while the patient was in maximum opening were used. the type of condylar excursion was classified as: normal excursion when top-to-top positioning of the apex of the articular eminence and the condyle was present, hyperexcursion when the condylar location was in front of the apex of the articular eminence, and hypoexcursion when the condyle was positioned below the apex of the articular eminence (figure 2)14,17. linear measurements of the superior, anterior, and posterior joint spaces were made using the following formula, and the images of the patient were obtained in maximum intercuspation to evaluate the positioning of the condyle in the mandibular fossa: (posterior − anterior)/(posterior + anterior). a zero value was classified as the equidistant position, a positive value was classified as the anteriorized position, and a negative value was classified as the posteriorized position (figure 3)18,19. figure 2. examples of condylar excursion estimates. (a) hypoexcursion; (b) normal excursion; (c) hyperexcursion. a b c figure 3. example positions of the condyle in the mandibular fossa. (a) equidistant; (b) anteriorized; (c) posteriorized. a b c 5 leite-de-lima et al. the data were submitted for statistical analysis. the likelihood ratio test was used to verify the possible differences among the three groups studied in the variables of interest. the chi-square test was used to verify the possible differences among the categories of signs and symptoms for the tomographic findings of interest. the statistical package for social sciences (spss) version 25.0 (ibm software group, chicago, usa) was used for the analysis. the level of significance adopted was p ≤ 0.050. results in this study, 78 cases of tmd were identified and 65 cases were included. the mean age of the patients was 40.6 years, with an age range of 18 to 74 years, and a predominance of females (84.6%). the main diagnoses were myopain with arthralgia and disc displacement (maad) in 32 cases (49.2%), myopain (m) in 26 cases (40.0%) and arthralgia with disc displacement (add) in 7 cases (10.8%). there was a predominance of normal amplitude of mouth opening (73.1% of cases). the most frequent degenerative change was flattening. in the add group, flattening (55.6%) was followed by erosion (27.8%). in the madd group, flattening (60.2%) was followed by osteophytes (22.7%). finally, in the m group, flattening (64.6%) was followed by osteophytes (21.5%) (table 1). no significant differences were noted among groups. the most common condylar excursion estimate was hyperexcursion. in the add group, hyperexcursion (64.3%) was followed by hypoexcursion (21.4%). in the madd group, hyperexcursion (67.2%) was followed by normoexcursion (18.8%). finally, in the m group, hyperexcursion (59.6%) was followed by hypoexcursion (21.2%) (table 1). no significant differences were noted among groups. table 1. cross tabulation of the occurrence of degenerative changes, estimates of condylar excursion and positions, the dc/tmd and the significance of the likelihood ratio test. variable category dc/tmd classification* p valueadd madd m n % n % n % degenerative changes  flattening  p  10  71.4  53  82.8  42  80.8  0.619  a  4  28.6  11  17.2  10  19.2  sclerosis  p  0  0.0  8  12.5  5  9.6  0.366  a  14  100.0  56  87.5  47  90.4  osteophyte  p  2  14.3  20  31.3  14  26.9  0.432  a  12  85.7  44  68.8  38  73.1  erosion  p  5  35.7  3  4.7  0 0.0  0.109  a  9  64.3  61  95.3  52  100.0  scs p  1  7.1  4  6.3  3  5..8  0.981  a  13  92.9  60  93.8  49  94.2  condylar excusions  hyperexcursion  9  64.3  43  67.2  31  59.6  0.853 hypoexcursion  3  21.4  9  14.1  11  21.2  normoexcursion  2  14.3  12  18.8  10  19.2  continue 6 leite-de-lima et al. no predominant condylar position was common to the three groups. in the add group, there was a predominance of the equidistant position (50.0%) followed by the posteriorized (28.6%) position. in the madd group, there was a predominance of the posteriorized position (43.8%) followed by the anteriorized (31.3%) position. finally, in the m group, there was a predominance of the anteriorized (46.2%) position followed by the posteriorized (32.7%) position (table 1). no significant differences were noted among groups. the distribution of the occurrence of muscle signs regarding degenerative changes is shown in table 2. in the category of masseter muscle pain, flattening (82.7%) was more frequent, followed by osteophytes (31.6%). in the temporal muscle, there was a predominance of flattening (82.7%) followed by osteophytes (28.6%). finally, in the medial pterygoid muscle, there was a predominance of flattening (83.8%) followed by osteophytes (33.8%) (table 2). no significant differences were noted among groups. continuation condylar position  anterior  3  21.4  20  31.3  24  46.2  0.109 equidistant  7  50.0  16  25.0  11  21.2  posterior  4  28.6  28  43.8  17  32.7  *according to the diagnostic criteria for temporomandibular disorders (dc/tmd).15 m=myopain, add=arthralgia with disc displacement, madd= myopain with arthralgia and disc displacement. p=present, a=absent. total degenerative changes exceed 100%, as many patients had more than one finding. table 2. cross tabulation of the occurrence of degenerative changes and muscle signs and the significance of the chi-square test. degenerative changes category muscle signs masseter temporal medial pterygoid y n y n y n n % n % n % n % n % n % flattening p 81 82.7 24 75.0 81 82.7 24 75.0 62 83.8 43 76.8 a 17 17.3 8 25.0 17 17.3 8 25.0 12 16.2 13 23,2 p value 0.340 0.340 0.316 sclerosis p 10 10.2 3 9.4 11 11.2 2 6.3 7 9.5 6 10.7 a 88 89.8 29 90.6 87 88.8 30 93.8 67 90.5 50 89.3 p value 0.892 0.415 0.813 osteophyte p 31 31.6 5 15.6 28 28.6 8 25.0 25 33.8 11 19.6 a 67 68.4 27 84.4 70 71.4 24 75.0 49 66.2 45 80.4 p value 0.079 0.695 0.074 erosion p 0 0.0 8 25.0 0 0.0 8 25.0 0 0.0 8 14.3 a 96 100.0 24 75.0 98 100.0 24 75.0 74 100.0 48 85.7 p value 0.072 0.068 0.069 scs p 7 7.1 1 3.1 6 6.1 2 6.3 5 6.8 3 5.4 a 91 92.9 31 96.9 92 93.9 30 93.8 69 93.2 53 94.6 p value   0.412 0.979 0.742 y=yes, n=no, p=present, a=absent. 7 leite-de-lima et al. the distribution of articular signs in relation to the occurrence of degenerative changes is shown in table 3. regarding the presence of lateral pain to the capsule, there was a predominance of flattening (83.7%) followed by osteophytes (32.7%). regarding the presence of posterior pain to the capsule, flattening (82.7%) was most frequent followed by osteophytes (27.1%). in the presence of clicking, there was a predominance of flattening (81.3%) followed by osteophytes (27.1%). finally, when crepitation was present, there was a predominance of flattening (75.0%) followed by osteophytes (50.0%). there was a significant difference in osteophyte findings in the group with lateral pain to the capsule, and in erosion findings in the group with posterior pain to the capsule. the distribution of muscle signs according to the excursion estimates and condylar positioning is shown in table 4. in the masseter muscle, the most frequent type was hyperexcursion (65.3%) followed by hypoexcursion (20.4%). in the temporal muscle, the most frequent type was hyperexcursion (64.3%) followed by normoexcursion (20.4%). finally, in the medial pterygoid muscle, the most frequent type was hyperexcursion (60.8%) followed by normoexcursion (21.6%). no significant differences were noted among groups. table 3. cross tabulation of the occurrence of degenerative changes and articular signs and the significance of the chi-square test. articular signs degenerative changes category lateral pain to the capsule posterior pain to the capsule clicking crepitation y n y n y n y n n % n % n % n % n % n % n % n % flattening p 82 83.7 23 71.9 81 82.7 24 75.0 39 81.3 66 80.5 6 75.0 99 81.1 a 16 16.3 9 28.1 17 17.3 8 25.0 9 18.8 16 19.5 2 25.0 23 18.9 p value 0.141 0.340 0.915 0.669 sclerosis p 8 8.2 5 15.6 11 11.2 2 6.3 5 10.4 8 9.8 0 0.0 13 10.7 a 90 91.8 27 84.4 87 88.8 30 93.8 43 89.6 74 90.2 8 100.0 109 89.3 p value 0.222 0.415 0.904 0.330 osteophyte p 32 32.7 4 12.5 29 29.6 7 21.9 13 27.1 23 28.0 4 50.0 32 26.2 a 66 67.3 28 87.5 69 70.4 25 78.1 35 72.9 59 72.0 4 50.0 90 73.8 p value 0.027 0.397 0.905 0.146 erosion p 6 6.1 3 9.4 4 4.1 5 15.6 3 6.3 6 7.3 1 12.5 8 6.6 a 92 93.9 29 90.6 94 95.9 27 84.4 45 93.8 76 92.7 7 87.5 114 93.4 p value 0.529 0.026 0.817 0.521 scs p 7 7.1 1 3.1 8 8.2 0 0.0 2 4.2 6 7.3 0 0.0 8 6.6 a 91 92.9 31 96.9 90 91.8 32 100.0 46 95.8 76 92.7 8 100.0 114 93.4 p value   0.412 0.095 0.471 0.455 y=yes, n=no, p=present, a=absent. 8 leite-de-lima et al. for condylar positioning, when pain in the masseter muscle was present, there was a predominance of the anteriorized condylar position (38.8%) followed by the posteriorized (35.7%) position. in the temporal muscle, there was a predomince of the anteriorized position (43.9%) followed by the posteriorized (34.7%) position. finally, in the medial pterygoid muscle, there was a predominance of the anteriorized position (43.2%) followed by the posteriorized position (35.1%). no significant differences were noted among groups. the distribution of articular signs according to the excursion estimates and condylar positioning is shown in table 5. in the presence of pain lateral to the capsule, there was a predominance of hyperexcursion (64.3%) followed by normoexcursion (18.4%). in the presence of posterior pain to the capsule, there was a predominance of table 4. cross tabulation of the excursion estimates, condylar positions, muscular signs and the significance of the chi-square test. variable category masseter temporal medial pterygoid y n y n y n  n % n % n % n % n % n % hiperexcursion 64 65.0 19 59.4 63 64.3 20 62.5 45 60.8 38 67.9 condilar excursion hipoexcursion 20 20.4 3 9.4 15 15.3 8 25.0 13 17.6 10 17.9 normoexcursion 14 14.3 10 31.3 20 20.4 4 12.5 16 21.6 8 14.3 p value 0.062 0.348 0.555 condilar position anteriorized 38 38.8 9 28.1 43 43.9 4 12.5 32 43.2 15 26.8 equidistant 25 25.5 9 28.1 21 21.4 13 40.6 16 21.6 18 32.1 posteriorized 35 35.7 14 43.8 34 34.7 15 46.9 26 35.1 23 41.1 p value   0.540 0.140 0.133 y=yes, n=no. table 5. cross tabulation of the excursion estimates, condylar positions, articular signs and the significance of the chi-square test. variable category lateral pain to the capsule posterior pain to the capsule clicking crepitation y n y n y n y n n % n % n % n % n % n % n % n % condilar excursion hiperexcursion 63 64.3 20 62.5 63 64.3 20 62.5 33 68.8 50 61.0 4 50.0 79 64.8 hipoexcursion 17 17.3 6 18.8 19 19.4 4 12.5 8 16.7 15 18.3 3 37.5 20 16.4 normoexcursion 18 18.4 6 18.8 16 16.3 8 25.0 7 14.6 17 20.7 1 12.5 23 18.9 p value 0.980 0.440 0.621 0.315 anteriorized 37 37.8 10 31.3 36 36.7 11 34.4 14 29.2 33 40.2 1 12.5 46 37.7 condilar position equidistant 24 24.5 10 31.3 25 25.5 9 28.1 14 29.2 20 24.4 2 25.0 32 26.2 posteriorized 37 37.8 12 37.5 37 37.8 12 37.5 20 41.7 29 35.4 5 62.5 44 36.1 p value   0.703 0.951 0.447 0.257 y=yes, n=no. 9 leite-de-lima et al. hyperexcursion (68.8%) followed by hypoexcursion (19.4%). when clicking was present, there was a predominance of hyperexcursion (68.8%) followed by hypoexcursion (16.7%). finally, in the presence of crepitation, there was a predominance of hyperexcursion (50.0%) followed by hypoexcursion (37.5%). no significant differences were noted among groups. for condylar positioning, when pain lateral to the capsule was present, the anteriorized and posteriorized positions were the most frequent (37.8%). in the presence of pain posterior to the capsule, the posterior position was most frequent (37.8%), followed by the anterior position (36.7%). in cases with clicking, the posteriorized position was more frequent (41.7%) followed by the anteriorized and equidistant positions (29.2%). finally, when crepitation was present, there was a predominance of the posterior position (62.5%) followed by the equidistant position (25.0%). no significant differences were noted among groups. the distribution of the amplitudes of mouth opening in relation to the occurrences of degenerative changes is shown in table 6. in patients with decreased mouth opening, flattening (92.9%) was predominant followed by osteophytes (32.1%). in patients with normal opening, flattening (75.0%) was the most frequent condition followed by osteophytes (25.0%). significant differences in the degenerative changes regarding flattening and the formation of subchondral cysts were noted. the distribution of the excursion estimates and condylar positioning in relation to the mouth opening classifications is shown in table 7. in cases of hyperexcursion, there was a predominance of normal mouth opening (71.1%) and in cases of hypoexcursion and normoexcursion, decreased mouth opening predominated (42.9% and 28.6%, respectively). a significant difference was noted. in cases with the anteriorized table 6. cross tabulation of the occurrence of degenerative changes, the mouth opening and the significance of the chi-square test. degenerative change category mouth opening p valuedecreased normal n % n % flattening p 26 92.9 57 75.0 0.044 a 2 7.1 19 25.0 sclerosis p 2 7.1 6 7.9 0.898 a 26 92.9 70 92.1 osteophyte p 9 32.1 19 25.0 0.466 a 19 67.9 57 75.0 erosion p 3 10.7 4 5.3 0.325 a 25 89.3 72 94.7 scs p 5 17.9 1 1.3 0.001 a 23 82.1 75 98.7 p=present, a=absent. 10 leite-de-lima et al. position, decreased mouth opening predominated (42.9%); in cases with the equidistant position, normal opening predominated (26.3%); and in cases with the posteriorized position, decreased opening predominated (42.9%). no significant differences among groups were noted. discussion the present study revealed few correlations between degenerative changes and signs of joint pain as well as degenerative changes and condylar hypoexcursion with mouth opening, whereas condylar positioning did not correlate with signs and symptoms. there was a predominance of females, and the mean age was 40.6 years. these findings are consistent with the characteristics reported in the literature7,8,20,21. however, given the wide age range, age-related degenerative changes could be present in this sample22,23. regarding degenerative changes, most patients had flattening, many exhibited osteophytes, and few showed sclerosis, erosion and scs. the predominance of flattening and osteophytes has been reported8,10,11. other studies have suggested that the prevalence of erosion or sclerosis represents the condition with the greatest prevalence24,25. studies with cbct in tmj osteoathritis showed common flattening, erosion and osteophytes26,27. a correlation between disc displacement and condylar degenerative changes has been demonstrated28. however, asymptomatic individuals can also present degenerative changes on cbct, and such findings should be exclusively used with care12. in older age groups, tmd patients are expected to exhibit more degenerative bony changes22,23. a study reported that no significant correlation was found between degenerative changes verified in cbct and clinical symptoms of tmd29. another study evaluated whether a relationship existed between degenerative changes and bone quality of the mandibular condyle and articular eminence in patients with tmd, and no causality relationship between these factors was found30. it should be considered that these table 7. cross tabulation of the condylar excursion estimates, condylar positions, the mouth opening and the significance of the chi-square test variable category mouth opening p valuedecreased normal n % n % condylar excursion hiperexcursion 8 28.6 54 71.1 < 0.001hipoexcursion 12 42.9 9 11.8 normoexcursion 8 28.6 13 17.1 condylar position anteriorized 12 42.9 25 32.9 0.392equidistant 4 14.3 20 2.3 posteriorized 12 42.9 31 40.8 11 leite-de-lima et al. studies did not classify the cases according to groups of diagnoses in contrast to our study, which may have provided some correlations between degenerative alterations and signs and symptoms of tmd. there was a predominance of hyperexcursion in the three groups based on the condylar excursion estimates. patients with intra-articular dysfunctions are more likely to have joint hypermobility13,17. hyperexcursion can lead to internal derangement, which can damage articular tissues. condylar excursion can significantly influence pain perception in patients with tmd15. it has been noted that pain in the tmj is correlated with a large amplitude of maximal mouth opening14. the predominant condylar position varied among the diagnostic groups, but without significant differences. our findings are not consistent with those in previous studies. the condyle is more commonly positioned posteriorly in patients with tmd, and anterior and equidistant positions are more common in asymptomatic patients15,18. a relationship was found between the condylar position and tenderness of a specific muscle group16. the posterior condylar position is associated with anterior disc displacement18. a study that evaluated the bone components of the tmj in asymptomatic individuals and patients with tmd using cbct demonstrated that the presence of tmd was associated with the condylar position with the anterior joint space being larger31. again, this study did not divide the cases according to groups of diagnoses, unlike our study, which may have led to no correlations between condylar positions and signs and symptoms of tmd. there was a predominance of amplitudes of mouth opening that were considered normal. however, degenerative changes, especially flattening and scs, were more prevalent in the group with decreased mouth opening compared with the other groups. cbct studies have revealed a weak correlation with reduced maximum mouth opening26. no significant difference was noted between the categories of tomographic findings and the groups of diagnoses according to dc/tmd9. additionally, no significant differences were noted between the tomographic findings and the signs of muscular pain. few significant differences were noted between degenerative tomographic findings and signs of articular pain. the probability of these signs of articular pain being associated with intra-articular dysfunctions with articular disc displacement and degenerative joint disease must be considered7,9. additionally, few significant differences were noted between degenerative changes and condylar hipoexcursion with mouth opening. the probability of these mouth opening limitations being associated with intra-articular dysfunctions with articular disc displacement must be considered7,9. one of the limitations of this study could be the sample size. although 65 cases of tmd were included, the sample size was not calculated. another limitation is the wide age range, which would lead to the inclusion of age-related degenerative changes. this study confirmed that cbct can reveal degenerative changes with high precision and detail. a previous study that used the same criteria for diagnosing dysfunctions and degenerative findings but used conventional ct reported a lower incidence of degenerative changes, such as osteophytes and erosion, and no cases of scs8. most likely, due to the use of cbct, these findings were more frequent in this study. 12 leite-de-lima et al. based on the data collected in this study, it was concluded that few correlations exist between degenerative changes and signs of joint pain as well as degenerative changes and condylar hypoexcursion with mouth opening. these correlations are likely associated with division by diagnosis. in contrast, condylar positioning exhibited no correlations with signs and symptoms. statement of ethics: this study was approved by the research ethics committee of the school of dentistry, university of são paulo, brazil. disclosure statement: the authors declare that there are no conflicts of interest regarding the publication of this paper. funding sources: this research was funded by a grant from ffo-fundecto (fundação faculdade de odontologia), são paulo, brazil (first author). references 1. suenaga s, nagayama k, nagasawa t, indo h, majima hj. the usefulness of diagnostic imaging for the assessment of pain symptoms in temporomandibular disorders. jpn dent sci rev. 2016 nov;52(4):93-106. doi: 10.1016/j.jdsr.2016.04.004. 2. hunter a, kalathingal s. diagnostic imaging for temporomandibular disorders and orofacial pain. dent clin north am. 2013 jul;57(3):405-18. doi: 10.1016/j.cden.2013.04.008. 3. hussain am, packota g, major pw, flores-mir c. role of different imaging modalities in assessment of temporomandibular joint erosions and osteophytes: a systematic review. dentomaxillofac radiol. 2008 feb;37(2):63-71. doi: 10.1259/dmfr/16932758. 4. larheim ta, hol c, ottersen mk, mork-knutsen bb, arvidsson lz. the role of imaging in the diagnosis of temporomandibular joint pathology. oral maxillofac surg clin north am. 2018 aug;30(3):239-49. doi: 10.1016/j.coms.2018.04.001. 5. alkhader m, kuribayashi a, 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kiliç n, sümbüllü ma. temporomandibular joint osteoarthritis: cone beam computed tomography findings, clinical features, and correlations. int j oral maxillofac surg. 2015 oct;44(10):1268-74. doi: 10.1016/j.ijom.2015.06.023. 14 leite-de-lima et al. 27. al-ekrish aa, al-juhani ho, alhaidari ri, alfaleh wm. comparative study of the prevalence of temporomandibular joint osteoarthritic changes in cone beam computed tomograms of patients with or without temporomandibular disorder. oral surg oral med oral pathol oral radiol. 2015 jul;120(1):78-85. doi: 10.1016/j.oooo.2015.04.008. 28. dias im, coelho pr, assis nm, leite fp, devito kl. evaluation of the correlation between disc displacements and degenerative bone changes of the temporomandibular joint by means of magnetic resonance images. int j oral maxillofac surg. 2012 sep;41(9):1051-7. doi: 10.1016/j.ijom.2012.03.005. 29. abdel-alim hm, abdel-salam z, ouda s, jadu fm, jan am. validity of cone-beam computed tomography in assessment of morphological bony changes of temporomandibular joints. j contemp dent pract. 2020 feb 1;21(2):133-9. doi: 10.5005/jp-journals-10024-2732. 30. ulay g, pekiner fn, orhan k. evaluation of the relationship between the degenerative changes and bone quality of mandibular condyle and articular eminence in temporomandibular disorders by cone beam computed tomography. cranio. 2020 dec 3;1-12. doi: 10.1080/08869634.2020.1853307. 31. yasa y, akgül hm. comparative cone-beam computed tomography evaluation of the osseous morphology of the temporomandibular joint in temporomandibular dysfunction patients and asymptomatic individuals. oral radiol 2018 jan;34(1):31-9. doi: 10.1007/s11282-017-0279-7. 1http://dx.doi.org/10.20396/bjos.v19i0.8658224 volume 19 2020 e201704 original article 1 dentistry course, school of medicine and public health of bahia (bahiana). dentistry course school of medicine and public health of bahia (bahiana), salvador, bahia, brazil. 2 department of restorative dentistry, school of dentistry, northeast independent school (fainor), vitoria da conquista, bahia, brazil. 3 kings college london dental institute, king’s college london, se1 9rt, london, uk. 4 department of restorative dentistry, school of dentistry, federal university of bahia (ufba), salvador, bahia, brazil. 5 dentistry course, school of medicine and public health of bahia (bahiana), salvador, bahia, brazil, and school of dentistry, federal university of bahia (foufba), salvador, bahia, brazil. corresponding author: ana paula menezes vaz queiroz https://orcid.org/0000-0002-0329-3078 dentistry course school of medicine and public health of bahia. telephone: (71) 99210-4297. e-mail: anapaulavaz.odonto@outlook.com received: july 11, 2019 accepted: december 02, 2019 effect of the ceramic translucency on the long-term water sorption and solubility of resin cements ana paula menezes vaz queiroz1,*, mariana menezes vaz queiroz1, saryta argolo2, richard mark foxton3, paula mathias4, andrea nóbrega cavalcanti5 aim: the aim of this study was to evaluate the influence of the translucency of ceramic on water sorption and solubility of resin cements over time. methods: lithium disilicate ceramic slides (15x15x1 mm), a1 in color and with different translucencies (high-medium-low) were manufactured; and a glass slide with similar dimension was used as control. under every slide, 15 specimens (8x0.5 mm) from each resin cement were prepared: light-cured (relyx veneer); conventional dual-cured (relyx arc); and self-adhesive dual-cured (rely-x u200). the specimens were then sub-divided according to the period of evaluation (immediately, after 6 and 12 months of storage) (n=5). to evaluate the loss or gain of mass, the specimens were dried until a constant mass was reached. subsequently, they were divided according to the respective period of storage at 37°c in distilled water and weighed immediately following removal from water. after each period, specimens underwent a new dehydration. values from water sorption and solubility were calculated and statistically analyzed (anova 3-way/tukey test). results: the lower translucency resulted in greater water sorption and solubility for all cements, regardless of the experimental period. the self-adhesive dual-cured cement showed higher water sorption under all experimental periods and had worse values after 1 year aging. differences among solubility could only be detected after aging, and the light-cured material had higher values after 6 and 12 months. conclusions: it could be concluded that the low degree of translucency can negatively influence the passage of light and interfere on the durability of the resin cement. the conventional dual-cured resinous agent seemed to be less affected by such condition. keywords: ceramics. dental restoration, permanent. resin cements. solubility. https://orcid.org/0000-0002-0329-3078 2 queiroz et al. introduction lithium disilicate glass ceramics are highly esthetic materials that have different levels of translucency for reproducing the optical effects from natural teeth and for masking possible discolorations of the substrate1. however, for attaining successful and long-lasting restorations, such materials rely on a strong and durable cementation protocol2,3. in adhesive cementation of glass ceramic surfaces, light-activated or conventional dualcured resin cements are frequently used. particularly, by their capacity for adhesion to dental tissues, and by their improved mechanical properties, when compared with other agents4,5. in addition to these, self-adhesive dual-cured cements have been introduced on the market, in an endeavor to simplify the clinical steps and minimize the working time4. the polymerization process from dual-cured resin cements occurs by two means: physical, through the action of the light on the photoinitiators; and chemical, through the reaction of benzoyl hydrogen with the tertiary amines6,7. whereas light activated resin cements depend on the visible light of an efficient photoinitiator system so that polymerization of the material occurs effectively, and the properties of the material can be attained to the maximum extent5. inadequate polymerization of resin cements may be related to an insufficient quantity of light radiation that passes through the restorative material and activates the monomers, as a result of the thickness and opacity of the ceramic restoration5,8,9. therefore, the optical characteristics of materials, such as the refractive index and translucency, may determine the quantity of light transmitted, and consequently, the degree of conversion of resin cements5,10. long term permanence in a humid environment such as the oral cavity may also have an influence on the dynamics of diffusion of resin cements9,10. this may be proved by measuring the water sorption and solubility of materials, which are important tools for predicting the clinical behavior, and particularly the stability of adhesively cemented restorations11-14. however, it is important to consider that the durability and stability of the material are primordial characteristics, therefore more in depth information is obtained by means of long term studies. in view of the foregoing, the experimental hypothesis tested by the present study was that the translucency of the ceramic could interfere in the polymerization of cements with different forms of activation (light or dual) changing their loss and gain of mass in the long term. therefore, the aim of the present study was to evaluate the water sorption and solubility of light activated or dual-cured resin cements used under the surfaces of ceramics with different levels of translucency, in different time intervals (immediate and after 6 and 12 months of storage in distilled water). materials and methods experimental design main factors • ceramic translucency (4 levels): high (ht), low (lt), medium (mo) and glass slide (negative control) 3 queiroz et al. • resin cement (3 levels): light-cured (relyx veneer); conventional dual-cured (relyx arc); and self-adhesive dual-cured (rely-x u200) • time interval (3 levels): immediate; 6 months and 12 months sample: 180 resin cement specimens (n=5) dependent variables: water sorption and solubility test specimen preparation in accordance with the manufacturer’s recommendations, lithium disilicate ceramic (ips e-max press, ivoclar-vivadent, schaan, liechtenstein) slides were fabricated. briefly, this process included waxing-up the plates, sprueing, investing, pressing the respective ingots, divesting and finishing. ceramic slides measured 1.0 mm thick, 15 mm high and 15 mm long, were a1 in shade, and had variation in translucency: high (ht), low (lt) and medium (mo). a glass slide, without any opacity, and of similar dimensions was used as negative control. for preparation of resin cement specimens, a polyvinyl siloxane mold (ma) (elite h-d+ puttysoft normal setting, badia polesine, rovigo, italy) was made. the mold had an internal orifice measuring 0.5 mm thick and 8 mm in diameter and was used for accommodating the cement. another polyvinyl siloxane mold (mb) was fabricated for embracing the ceramic slide, leaving just an upper orifice of 8-mm diameter. the function of this device was to prevent both the dissipation of light at the time of polymerization and the interference of external light8. materials with similar color (a1/light yellow) were selected. all the cementation agents were investigated in association with the different levels of translucency, in three experimental time intervals (n=5), as described in figure 1. the cementation agents and their formulations are described in table 1. to prepare each specimen, the cementation agent was inserted into the first mold (ma), and a polyester strip was placed over it to accommodate the material and maintain a smooth and uniform surface. after this, a ceramic slide was placed on the above-mentioned assemble, with the second mold (mb) on top of it. a glass slide was used to exert pres• control (c) • high translucency (ht) • medium translucency (mt) • low translucency (lt) water sorption solubility • light-cured • conventional dual-cured • self-adhesive dual-cured storage time immediate (n = 5) 6 months 12 months (n = 5) cementing agentsurface translucency figure 1. distribution of groups for water sorption and solubility tests. 4 queiroz et al. sure on this to force out the excess material. after removing the glass slide, polymerization was performed with a light emitting diode (led) unit (radii plus, sdi, victoria, australia) for an exposure time of 60 seconds, with an intensity of 1,500 mw/cm2 wavelength of 470 nm (figure 2). water sorption and solubility evaluation after been removed from the mold, the specimens were individually placed in a dark environment to prevent additional polymerization until they were ready for the water sorption and solubility test, based on the iso 4049 specifications: 20007. immediately 1 mm 0.5 mm 8 mm matrix (ma) internal orifice/specimen polyester strip ceramic slide matrix (mb) light-curing device figure 2. illustrative diagram of test specimen fabrication. table 1. materials used and their compositions material composition emax (ips e-max, ivoclar/ vivadente r, schaan, principality of liechtenstein) lithium dioxide, phosphorous oxide, aluminum, potassium oxide, and other components, that are combined to produce cast glass. dual-cured resin cement, relyx arc (3m espe dental products. st. paul, mn, usa). bis-gma and tegdma monomers, zirconium/silica particle with mean particle size of 1.5µm, paste a: pigments and tertiary amine. paste b: benzyol peroxide. filler percentage of 68% by weight. light polymerized resin cement, relyx veneer (3m espe dental products. st. paul – usa). bis-gma and tegdma monomers zirconium/silica particles and colloidal silica mean particle size of 0.6 mm filler percentage of 66% by weight. self-adhesive dual-cured resin cement, relyx u200 (3m espe dental products. st. paul, mn, usa). bis-gma and tegdma monomers inorganic particles with 70% by weight and particle size of approximately 12.5 µm. base paste: glass fiber, methacrylate phosphoric acid esters, triethyleneglycol dimethacrylate, silica treated with silane and sodium persulphate. catalyzer paste: glass fiber, substitutive dimethacrylate, silica treated with silane, sodium p-toluenosulphate and calcium hydroxide. 5 queiroz et al. after polymerization of the test specimens, the thickness of the resin cement cylinders was measured with a digital caliper, with precision of 0.01 mm, and these values were used to calculate the volume (v) of each unit (mm3). later, these specimens were individually placed in eppendorf flasks, in a desiccator containing silica gel and then transferred for preconditioning in an oven at 37ºc. the specimens were repeatedly weighed at intervals of 24 hours until the constant mass (m1) was obtained (variation of less than 0.2 mg over a period of 24 hours). the weighing procedures were performed on an analytical balance (analytical plus, ohaus® corporation, florham park, switzerland), with precision of one hundredth of a thousandth of a gram. after stabilization of the mass in m1, the groups were subdivided (n=5) according to storage time in 2 ml of distilled water (ph 7.2) at 37ºc: immediate analysis (7 days) or storage (6 and 12 months). after the respective time interval, the test specimens were removed from storage; washed in running water; excess was removed with absorbent paper until water was no longer visualized, and the weight was noted (m2). after weighing, the test specimens were put back into dry eppendorf vials and put into a desiccator containing silica gel in an oven at 37ºc to eliminate absorbed water. the specimens were weighed daily until the constant mass (m3) was obtained (variation of less than 0.2 mg over a period of 24 hours). water sorption (so) and solubility (sol) in 7 days, 6 months and 12 months of storage in water were calculated using the following formula: so = (m2 – m3)/v sol = (m1 – m3)/v where m1 was the mass of the sample in μg before immersion in distilled water; m2 was the mass of the sample in μg after immersion in distilled water for 7 days; 6months and 12 months; m3 was the mass of the sample in μg after being conditioned in a desiccator with silica gel and v is the volume of the sample in mm3. statistical analysis exploratory analysis of the water sorption and solubility data was performed to verify the homogeneity of the variances and determine whether the experimental errors presented normal distribution. inferential statistical analysis was performed by using 3-way anova and the tukey post-hoc test for multiple comparisons of the measures, by means of the statistical program sas, version 9.1, with a level of significance of 5%. the measurement of water sorption and solubility occurred in an independent sample over time. results statistical analysis of the data pointed out significant double interaction between the main factors “resin cement” and “time”, both in water sorption (p=0.00015) and in solubility (p=0.00019). the other statistical interactions between main factors were not considered significant (p>0.05). furthermore, statistically significant differences were observed among the levels of the factor “ceramic translucency” in both variables (water sorption – p=0.00001 / solubility – p=0.0005). therefore, the effect of the main factor “ceramic translucency” was tested irrespective from the other ones. 6 queiroz et al. table 2 presents the result of statistical analysis of the water sorption data. according to this analysis, it was verified that regardless of the ceramic level of translucency, in all the storage time intervals, the resins cements presented statistical differences among them, with the highest values being presented by the self-adhesive dual-cured cement, followed by the light activated, and conventional dual-cured cement. furthermore, all the cements tested presented statistically higher mean values after 6 months. however, the difference in water sorption obtained between the time interval of 6 months and 1 year was not statistically significant for both the conventional dualcured resin cement and the light activated type. on the other hand, the self-adhesive dual-cured resin cement showed statistically higher water sorption values after 1 year. relative to the effect of ceramic translucency, it was observed that irrespective of the cement tested and time interval, the highest water sorption values were noted when the low translucency ceramic was used. the results obtained with the medium and high translucency level ceramics and the control group were statistically similar among them. table 3 shows the results obtained by analysis of the solubility data. the differences between the resin agents was only noted after storage in water for 6 or 12 months, because in the initial period, all the resin agents presented statistically similar mean values. however, after 6 months and 1 year of storage, the light activated resin cement table 2. mean (standard deviation) of water sorption data in experimental groups ceramic resinous cement time initial 6 months 1 year control (c) conventional dual 20.8 (1.0) cb 28.1 (1.3) ca 30.5 (4.0) ca *light activated 27.6 (1.4) bb 34.2 (1.9) ba 34.2 (6.7) ba self-adhesive dual 40.2 (1.8) ac 54.0 (1.2) ab 58.9 (6.0) aa high translucency (ht) conventional dual 21.7 (3.6) cb 27.6 (4.4) ca 29.0 (3.2) ca *light activated 22.3 (3.0) bb 30.1 (5.6) ba 32.8 (4.7) ba self-adhesive dual 42.3 (3.0) ac 50.2 (0.3) ab 52.8 (4.7) aa medium translucency (mo) conventional dual 20.5 (1.1) cb 27.0 (1.7) ca 25.8 (1.2) ca *light activated 26.4 (1.3) bb 28.0 (0.2) ba 30.5 (3.6) ba self-adhesive dual 41.4 (1.4) ac 51.2 (3.4) ab 54.4 (3.5) aa low translucency (lt) conventional dual 23.0 (2.8) cb 29.3 (1.8) ca 30.2 (2.2) ca #light activated 32.3 (4.2) bb 47.3 (6.5) ba 45.9 (5.6) ba self-adhesive dual 43.9 (4.6) ac 59.6 (5.2) ab 61.2 (3.9) aa different letters or symbols represent statistically significant differences (3-way anova / tukey, alpha=5%). lower case letters used to compare levels of factor time for each ceramic/cement. capital letters were used to compare cements at each level of ceramic/time. symbols used to compare differences among ceramics. variation coefficient = 9.5% 7 queiroz et al. presented higher solubility values compared with those of the other materials. the effect of storage in water on the solubility of the three materials was similar; all presented statistically different mean values in the time intervals evaluated, with the highest values verified after 1 year of immersion. similarly to the water sorption data, the highest solubility values were associated with the surface that had low translucency. however, the surface with a mean level of opacity could also interfere in the solubility results, by presenting an intermediate effect when compared with the values verified between the surfaces with low translucency, control and high translucency. discussion inadequate polymerization of the resin cement may be related to decreased longevity of ceramic restorations, greater stress at the interface with the dental structure; incomplete conversion of monomers into polymers, and impaired properties of the material9. ceramic restorations in the oral medium are in constant contact with humidity, and in many cases, close to the region of the gingival sulcus. in these cases, water can act as a plasticizer, decreasing the mechanical strength and dimensional stability of the resin material, which could result in the degradation of polymers in the long term12. the experimental hypothesis of the present study was verified, since it proved that the resin cements polymerized under the ceramics with a low level of translucency presented higher levels of loss and gain of water, and that this effect was directly related to the type of material and time of storage. table 3. mean (standard deviation) of solubility data in experimental groups ceramic resinous cement time initial 6 months 1 year control (c) conventional dual 2.9 (0.1) ac 4.8 (1.3) bb 14.9 (2.9) ba *light activated 6.2 (0.1) ac 10.2 (5.3) ab 15.2 (4.2) aa self-adhesive dual 3.2 (0.1) ac 8.3 (1.3) bb 8.1 (3.4) ba high translucency (ht) conventional dual 2.7 (0.2) ac 6.1 (1.2) bb 12.6 (2.3) ba *light activated 4.7 (2.6) ac 14.0 (0.9) ab 21.0 (5.0) aa self-adhesive dual 2.8 (0.1) ac 7.3 (3.0) bb 10.4 (4.2) ba medium translucency (mo) conventional dual 3.4 (1.2) ac 7.0 (1.0) bb 13.8 (3.5) ba *#light activated 3.4 (0.1) ac 16.4 (1.5)ab 23.6 (3.2) aa self-adhesive dual 5.0 (1.2) ac 9.4 (3.7) bb 15.7 (7.3) ba low translucency (lt) conventional dual 6.0 (0.5) ac 10.9 (2.5) bb 16.0 (5.6) ba #light activated 8.1 (1.1) ac 16.8 (3.3)ab 20.1 (1.7) aa self-adhesive dual 6.0 (1.2) ac 11.9 (6.4) bb 15.5 (10.0) ba different letters or symbols represent statistically significant differences (3-way anova / tukey, alpha=5%). lower case letters used to compare factor time for each ceramic/cement. capital letters used to compare levels of cements at each level of ceramic/time. symbols used to compare differences among ceramics variation coefficient = 31.9% 8 queiroz et al. the time of immersion in the aqueous medium may affect the absorption of water and solubility of the resin materials13,14. in time-following, all the cementation agents tested presented higher values of water sorption and solubility. the composition of the organic matrix of the resin cements was based on a bis-gma (bisphenol a-diglycidyl dimethacrylate) or udma (urethane dimethacrylate) matrix in combination with other monomers with lower molecular weight, such as tegma (triethyleneglycol dimethacrylate). the latter promotes the formation of more hydrophilic matrices in comparison with other monomers such as bis-ema (bisphenol a-ethoxylate dimethacrylate), which may have allowed a higher level of diffusion dynamics of the cements tested13,15. in a similar manner, aguiar et al in 201414, conducted a study comparing the water sorption and solubility of five resin cements of different commercial brands, in two experimental time intervals (24 hours and 7 days). the authors concluded that time has an influence on the durability of the cements, because the presence of hydrophilic monomers could compromise the durability of the bond, whereas hydrophilicity and hydrolytic stability are considered antagonistic properties13,14. there are theories explaining how water diffuses into a resin-based material after its application in an aqueous environment. an explanation of free volume theory reports that water can diffuse through polymer voids caused by resin-filler interfaces and morphological defects. another possibility is that water molecules form hydrogen bonds with specific ionic groups in the polymer chain, causing water diffusion according to affinity16. fabre et al.16 in 2007, compared the water sorption and solubility of different dentin bonding agents with respect to classification and light activation system, and the authors concluded that simplified systems were more susceptible to adverse effects of water and may become more prone to degradation over time independent of the type of light activation source. accordingly, wei et al in 2011 investigated the kinetic process of water diffusion and mass change in five new resin matrix composites, and the samples were prepared following iso 4049. each resin matrix composite varied in water sorption and solubility cycles, which may affect clinical behavior. therefore, this study reinforces the hypothesis that water sorption in resin composites depends on polymer (monomer, degree of conversion, polar interaction), charge (fraction, generic type, morphology, particle size, matrix dispersion), properties of the resin-filler interface, concentration of catalysts and initiators in the system and surface exposed to water. thus, the polymer plays a dominant role in the water sorption in composite resin17. soares et al.9 studied the microhardness of a dual-cured resin cement under the influence of thickness and color of a feldspathic ceramic. the authors concluded that good conversion of monomers depended on the quantity of light radiation that attained them. in spite of the present study having studied ceramics of the same color, different levels of translucency were tested, thus, the principle of influence on polymerization of the resin material could have been similar; that is, acting on the formation deficiently converted polymer networks, whose weak bonds favored water sorption and solubility of the material. runnacles et al.8 evaluated the influence of the thickness of ceramic restorations with different levels of translucency on the degree of conversion of a light activated resin 9 queiroz et al. cement, similar to the type used in the present investigation. the cement was light activated after interposition of ceramic restorations at low and high levels of translucency, of four thicknesses (0.5 mm; 1.0 mm; 1.5 mm and 2.0 mm). the authors concluded that the light activated cements must be used with caution under ceramics thicker than 1.5 mm, due to the possibility of reduction in the degree of conversion with larger thicknesses. in this study, a thickness of 1 mm was selected because it is commonly used when masking a darkend substrate is necessary. faria and silva in 2017 carried out an in vitro study to evaluate the light transmission of light curing units through ceramic cylinders and their effect on the polymerization kinetics of resin cement. in this study the ceramic ingots (ips empress esthetic, shadow et1) were sectioned to produce 0.5, 1.0 and 2.0 mm thick cylinders that were activated by two light curing units: smartlite focus and valo cordless. the authors concluded that total energy and irradiance decreased with increasing ceramic thickness in all cases18. another study by martins et al in 2019 through a meta-analysis evaluated how the thickness variations in lithium disilicate ceramic restorations and the use of different light curing agents influence the degree of conversion of resin cements. the authors concluded that the thinner the ceramic material, the greater the degree of conversion. a thickness greater than 1.0 mm dramatically reduces the degree of conversion of double cured or photoactivated resins19. the self-adhesive dual-cured cement exceeded iso 4049 minimum requirements for cementing agents, in the three-time intervals (water sorption 40 um/mm3; solubility 7.5 um/mm3) according to the iso 4049 standards for water sorption and water solubility of polymer-based materials, water-sorption values under the limit of 40 mg mm and water-solubility values lower than 7.5 mg mm are considered acceptable20. the water sorption and solubility of self-adhesive dual cements was compared with those of other resin and ionomer cements in a previous study21, and the conclusion was that the they were more susceptible to water sorption. this finding was associated to their higher degree of acidity and hydrophilia, because these are the characteristics required in its process of bonding to dental structures21. also, the acidic monomers from self-etching dual cements might negatively affect their degree of conversion through chemical interactions with the amine initiator. the solubility of cements exceeded the iso requirements in most conditions tested. furthermore, after 6 and 12 months, the light activated cement was found to present statistically higher solubility values than the others at all the levels of translucency. in the study of jung et al.22, different sources of polymerization and modes of exposure were compared in the activation of a dual-cured resin cement through ceramic discs of different thicknesses: 1.0 and 2.0mm. the thickness of the restoration was found to act as a negative effect on the depth of conversion of the monomers, caused by the exponential reduction in light energy transmitted, called underpolymerization. this event could also have occurred in the light activate cement tested in this study, whereas, it depended exclusively on light to attain a good degree of conversion. 10 queiroz et al. the dual-cured resin cement presented statistically lower water sorption values in comparison with the other cementation agents, and low solubility values in comparison with the light activated cement in the storage time intervals tested. these results found could be justified due to the characteristic of addition of catalyzers in the chemical setting, which increased the degree of polymerization, because the conversion of monomers occurs even after use of the light source22. braga et al.5 investigated the flexural strength; flexural modulus, and the hardness of four resin cements: enforce (light activated), variolink ii (dual), relyx arc (dual) and c&b (chemically activated), and verified the influence of the method of polymerization on the different properties of the material, and observed that dual-cured cements depend on light activation to reach higher degree of conversion values. according to the limitations of the present in vitro study, the authors verified that the ceramic with the low degree of translucency had a negative influence on the passage of light, thereby elevating the loss and gain of water. however, the conventional dual cement possibly due to the presence of the chemical setting propagated activation of the monomers even after polymerization and demonstrated more stable results when compared with the other cements, even after a long time of storage in water. in a preliminary study, leal et al.23 in 2016 evaluated the effect of the same levels of translucency of a laminated ceramic on the water sorption and solubility of three different adhesive cements (relyx arc, relyx veneer and z350xt flow). the authors observed that there was an inverse relationship between translucency of the ceramic and water sorption and solubility of the cementation agent in the immediate period (7 days of storage). ceramics with higher levels of translucency produced acceptable water sorption and solubility values for the cementation agents studied. on the other hand, for ceramics with lower levels of translucency, the dual-cured resin cements or flowable resin composites must be preferred. in view of these results, it would be necessary to seek an efficient way to compensate the attenuation of energy influenced by the translucency of the restorative material or for accentuating the activation of monomers. a prolonged period of activation by light could be used, as well as higher irradiation, up to 3500 mw/cm2, or an increase in temperature of the agent. however, further studies are necessary to confirm the suitability, feasibility and clinical protocol of these procedures. in conclusion, ceramic restorations with a low level of translucency may have a negative influence on the passage of light. the light activated resin, or conventional dual cementation agents are better indicated in these situations, because the chemical setting propagates activation of the monomers, thereby promoting good polymerization of the cement, acting on the longevity of the restoration even under the influence of time and humid environment of the oral cavity. conflict of interests the authors declare no potential conflict of interests with respect to the authorship and/or publication of this paper. 11 queiroz et al. references 1. guess pc, schultheis s, bonfante ea, coelho pg, ferencz jl, silva, nr. all-ceramic systems: laboratory and clinical performance. dent clin north am. 2011 apr;55(2):333-52, ix. doi: 10.1016/j.cden.2011.01.005. 2. uctasli s, hasanreisoglu u, wilson hj. the attenuation of radiation by porcelain and its effect on polymerization of resin cements j oral rehabil. 1994 sep;21(5):565-75. 3. ilie 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filho f, gonzaga cc, furuse ay. degree of conversion of a resin cement light-cured through ceramic veneers of different thicknesses and types. braz dent j. 2014 jan-feb;25(1):38-42. 9. soares cj, silva nr, fonseca rb. influence of the feldspathic ceramic thickness and shade on the microhardness of dual resin cement. oper dent. 2006 may-jun;31(3):384-9. 10. rasetto fh, driscoll cf, von fraunhofer ja. effect of light source and time on the polymerization of resin cement through ceramic veneers. j prosthodont. 2001 sep;10(3):133-9. 11. gerdolle da, mortier e, jacquot b, panighi mm. water sorption and water solubility of current luting cements: an in vitro study. quintessence int. 2008 mar;39(3):e107-14. 12. mese a, burrow mf, tyas m. sorption and solubility of luting cements in different solutions dent mater j. 2008 sep;27(5):702-9. 13. reis af, giannini m, pereira pnr. influence of water-storage time on the sorption and solubility behavior of current adhesives and primer/adhesive 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jung h, friedl kh, hiller ka, furch h, bernhart s, schmalz g. polymerization efficiency of different photocuring units through ceramic discs. oper dent. 2006 jan-feb;31(1):68-77. 23. leal cv, queiroz apv, foxton rm, argolo s, mathias p, cavalcanti an. water sorption and solubility of luting agents used under ceramic laminates with different degrees of translucency. oper dent. 2016 sep-oct;41(5):e141-8. 1http://dx.doi.org/10.20396/bjos.v20i00.8659638 volume 20 2021 e219638 original article 1 university of são paulo, faculty of dentistry, department of social dentistry, são paulo, são paulo, brasil. 2 federal university of juiz de fora, campus governador valadares, department of nutrition, governador valadares, minas gerais, brasil 3 federal university of juiz de fora, campus governador valadares, department of dentistry, oral diagnosis, governador valadares, minas gerais, brasil corresponding author: rebeca cardoso pedra, federal university of juiz de fora, department of dentistry, campus governador valadares. raimundo monteiro rezende avenue, 330, governador valadares mg, 35010173, brazil. e-mail: rebecapedra@yahoo.com.br. telefone: +5533011000. editor: dr altair a. del bel cury received: may 16, 2020 accepted: february 2, 2021 factors associated with advanced-stage oral and oropharyngeal squamous cell carcinoma in a brazilian population rebeca cardoso pedra1,* , clarice lima álvares da silva2 , ismênia edwirges bernardes3 , francielle silvestre verner3 , karla machado andrade3 , rafael de sousa santos3 , sibele nascimento de aquino3 diagnosis of oral and oropharyngeal cancer in advanced stages may be associated with social nature factors, access to health care, education, occupation, and behavioral/ cultural factors. aim: to determine the factors related to high clinical-staging in patients diagnosed with squamous cell carcinoma in the oral and oropharyngeal region in a cancer center in brazil between 2009 and 2015. methods: it is an epidemiological, retrospective, and exploratory study. patients diagnosed with squamous cell carcinoma had their medical records analyzed. the variables considered were sociodemographic, lifestyle, and disease characteristics. descriptive and exploratory tests (pearson’s, chi-square test and, student’s t-test) were realized. results: we analyzed 365 patient records, among which 289 (79.17%) were male, and 73 (20.0%) were female. age ranged from 16 to 101 years, with a mean of 61.13. regarding education, 157 (43.01%) studied < 8 years, 103 (28.21%) were illiterate and 102 (27.94%) studied > 8 years. 305 (83.56%) patients live in urban areas. there was an association between high clinical-staging and low educational level. for high clinical-staging, symptomatology, tobacco, and alcohol intake as well. conclusion: patients with low educational levels tend to report the disease later, and their diagnostics occurred in advanced stages. thus, specific public health policies for this population, including access to dental care to recognize the clinical signs and early diagnosis, are necessary. keywords: education status. mouth neoplasms. oropharyngeal neoplasms. socioeconomic factors. https://orcid.org/0000-0001-5769-5913 https://orcid.org/0000-0002-1257-8964 https://orcid.org/0000-0003-3827-8182 https://orcid.org/0000-0001-5770-316x https://orcid.org/0000-0002-4746-0579 https://orcid.org/0000-0002-0345-787x https://orcid.org/0000-0003-3843-3517 2 pedra et al. introduction about 40 to 60% of patients diagnosed with oral and oropharyngeal squamous cell carcinoma (opscc) start the treatment in the advanced-stages of the disease1. several factors of social nature, such as the time between the disease perception, diagnosis, and treatment, are responsible for the disease identification in advanced-stages. also, access to health care services, education, occupation, behavioral/cultural factors, exposure to risk factors such as tobacco, topographical distribution of the disease, and the tumor stage may affect the disease perception as well2. individuals in an underprivileged socioeconomic situation usually present a higher prevalence of head and neck cancer and inadequate medical access. some authors identify socioeconomic status as the fundamental cause of inequalities in mortality and, therefore, relevant to health protection3,4. socio-demographic characteristics are related to the advanced clinical staging of oral cancer and its diagnosis delay5. the delay in diagnosis compromises the treatment, its prognosis, and survival6. lack of health insurance affects access to the clinical examination of the oral and oropharyngeal region. it is a highly effective prevention strategy to detect cancerous lesions in their early stages, allowing a better prognosis and effective therapeutic intervention5. it is relevant to delineate the profile of individuals affected by opscc and identify the main risk factors associated with the disease’s appearance, evolution, and survival to develop and implement primary and secondary public health measures. also, dentists and physicians could be more accurate in oral examinations and population screening by being aware of this information. it may result in the improvement of public health measures for the prevention and control of opscc1,6. knowing the profile of these patients and identifying the factors associated with the late diagnosis will be useful for public policy formulation for the prevention and early diagnosis of opscc. thus, this study aims to describe the clinical and socio-demographic profile of patients who received treatment for opscc to evaluate the association between the socio-demographic and the disease characteristics with the clinical-staging at the diagnosis moment as well as to analyze possible associations between the tumoral subsite, socio-demographic characteristics, and disease evolution. materials and methods it is a retrospective and exploratory study about the epidemiological profile of opscc cases treated in a cancer center in governador valadares, minas gerais state, between 2009 and 2015. four hundred and five patients’ medical records who had been diagnosed with head and neck cancer were analyzed. the inclusion criteria for medical records selection were to have an anatomopathological report description confirming the diagnosis during the period proposed in this study. the exclusion criteria were medical records without information or incomplete, for not allowing a proper data analysis. 3 pedra et al. the dependent variables of this study were disease characteristics, including the histological type, tumor location, clinical-staging (subdivided in 1, 2, 3, 4 according to the degree of severity), treatment (chemotherapy, radiotherapy, surgery), clinical follow-up, and closure. the variables considered as independent for analyses were the socio-demographics: sex (male and female), age (years), marital status (single, married, divorced, widowed), educational level (<8 years of study, ≥8 years, illiterate), occupation/employment classified according to brazilian occupational code, residence location (urban or countryside), family history of cancer, tobacco (smoker and never smoker) and alcohol consumption (alcoholic and never drank). data collection in the medical records was monitored and verified by the coordinators of this research. stata® version 13.0 (stata corp., college station, united states) was the software applied for data tabulation and statistical analysis, with double data entry to control potential tabulation errors. the statistic tests selected were descriptive statistics (prevalence, absolute and relative frequency, means, and standard deviation) and exploratory tests (pearson’s chi-square test and student’s t-test), which adopted statistical significance of 5% (p<0.05). the study was approved by the ethics in research with human beings committee of the federal university of juiz de fora (#1.300.203) and with the 1964 helsinki declaration and its later amendments. results among the four hundred and five head and neck cases of cancer analyzed, we selected three hundred and sixty-five medical records of patients diagnosed with opscc. age at diagnosis ranged between sixteen and one hundred and one years, and the mean age was 61,13 years. the majority of the patients were male. they studied for less than eight years, were married, and lived in the urban area (table 1). regarding occupation, one hundred and nine (30.28%) patients were multipurpose agricultural workers and similar, followed by workers that cannot be classified by profession 65 (18.6%), masons and plasterers 25 (6.94%), and specialized agricultural workers not classified under other headings 22 (6.11%), among other less frequent occupations as well. regarding habits, 84.65% were smokers, and 67.4% used to consume alcohol regularly. the health care used for 92.31% of the patients was the brazilian public health system, sistema único de saúde or health unic system (sus), and 24 (6.59%) used private health care. disease identification occurred predominantly by medical professionals (84.65%) (table1). the most affected anatomic sites were oropharynx 151 (48.55%), tongue 114 (36.65%), palate 34 (10.93%), and floor of the mouth 12 (3.85%). as diagnostic methods, we identified clinical examination and biopsy used in 223 (61.10%) cases and the combination of clinical examination, biopsy, and imaging exam in 124 (33.97%) patients. 4 pedra et al. table 1. sociodemographic and clinical characteristics of patients with squamous cell carcinoma. sociodemographic data n % gender male 289 79.17% female 73 20.0% no information 03 0.82% education no 103 28.21% <8 years 157 43.01% >8years 102 27.94% no information 03 0.82% marital status single 78 21.49% married 201 55.06% divorced 30 8.21% widowed 54 17.79% no information 02 0.54% residence urban 305 83.56% countryside 58 15.89% no information 02 0.54% smoke no 56 15.34% yes 309 84.65% alcohol no 119 32.60% yes 246 67.4% referral professional dentist 32 8.76% physician 309 84.65% other 6 1.64% no information 18 4.93% family history of cancer no 273 74.79% yes 92 25.21% the majority of patients reported symptoms (80.38%). the most common were pain reported by 75.50% of patients, feed struggles by 74.17%, speech difficulties by 24.17%, trismus by 18.54%, and 32.78% felt other symptoms. when comparing the symptomatology reports between the same cancer subsites, some locations were more symptomatic: in the tongue (96 of 114), mouth floor (09 of 12), ridge (04 of 05), buccal mucosa (05 of 07), oropharynx (121 of 151), retromolar area (07 of 07), and soft palate (27 of 33). there was no report of symptoms in the lips. the most common clinical-staging (cs) among patients at the beginning of the treatment was cs 4, with 55.68% of patients, followed by cs 3 with 26.99%. cervical lymphadenopathy was present in 60.44% of the patients. among them, 68.66% had unilateral lymphadenopathy, and 31.34% had it bilaterally. forty (11.02%) patients had metastasis. twenty of them were in the lungs, six in the brain, and 5 in the liver and bones. the treatments available were surgery, chemotherapy, and radiotherapy. a large number of patients received treatment but remained with the disease in progress. death was a frequent outcome (table 2). 5 pedra et al. analyzing clinical-staging in opscc according to sociodemographic and clinical characteristics, we found association between worse cs and educational level, symptoms, smoking habit, and alcohol consumption. however, we did not find a significant association with gender (table 3). there was no significant association between alcohol and tobacco consumption or education level and death. table 2. patient treatment data and outcomes of patients with squamous cell carcinoma. treatment n (363) % no 74 20.39% yes 289 79.61% treatment received n (289) % surgery no 263 91.00% yes 26 9.00% chemotherapy no 72 24.91% yes 217 75.09% radiotherapy no 71 24.57% yes 218 75.43% disease follow up disease progressing 153 42.15% complete remission 80 22,04% no information 50 13.77% stable disease 30 8.26% no therapeutic possibility 18 4.96% partial remission 17 4.68% abandoned the treatment 15 4.13% death no 175 48.21% yes 188 51.79% table 3. analysis of clinical-staging in oral and oropharyngeal squamous cell carcinoma according to sociodemographic and clinical characteristics. patients per stage 18 41 95 195 p-value clinical-staging 1 2 3 4 alcoholism no 11 (61.11%) 19 (44.19%) 34 (35.79%) 49 (25.00%) 0.002 yes 7 (38.89%) 24 (55.81%) 61 (64.21%) 147 (75.00%) smoking no 8 (44.44%) 8 (18.60%) 12 (12.63%) 23 (11.73%) 0.002 yes 10 (55.56%) 35 (81.40%) 83 (87.37%) 173 (88.27%) symptoms no 11 (64.71%) 8 (19.51%) 16 (16.84%) 21 (10.77%) 0.000 yes 6 (35.29%) 33 (80.49%) 79 (83.16%) 174 (89.23%) educational level >8 years 11 (61.11%) 16 (38.10%) 27 (28.72%) 43 (22.05%) 0.015<8 years 5 (27.78%) 15 (35.71%) 39 (41.49%) 92 (47.18%) no 2 (11.11%) 11 (26.19%) 28 (29.79%) 60 (30.77%) gender female 6 (33.33%) 10 (24.39%) 23 (24.21%) 32 (16.41%) 0.170 male 12 (66.67%) 31 (75.61%) 72 (75.79%) 163 (83.59%) 6 pedra et al. discussion this study describes the clinical and sociodemographic aspects of 365 cases of opscc, and the results show that educational level, consumption of tobacco, and alcohol are associated with high cs. the patients in our study were predominantly low educated men who lived in urban areas. they were rural workers, and their opscc diagnosis occurred in advanced cs. patients with oral and oropharyngeal cancer might face many social nature factors that could affect their health condition. most of them have a low educational level and low financial income, which usually are associated with a high risk for cancer due to late diagnosis and high morbidity rates7,8. our findings are characteristic from developing countries where the diagnosis commonly occurs at advanced cs. otherwise, in developed countries, the cs most common at diagnosis are i and ii. probably due to the highest educational levels and better health care access7,9. low educational level and socioeconomic status are also considered risk factors to develop and die from other types of cancer. in lung cancer, the prevalence and intensity of smoking were associated with educational level and mortality among men10,11. the highest educational inequalities are associated with mortality from lung, esophagus head and neck cancer in european studies12,13. compared to highly educated men, low educated men were about 2.2 (95% ci: 2.1–2.3) and 2.0 (95% ci: 1.7–2.2) times more likely to die from lung and head and neck cancer, respectively12. the association between education level, risk factors, and advanced cs observed in our study is similar to the results observed in the literature7. low-educated men are more inclined to smoke, which is a high associated risk factor with the development of opscc, especially if combined with alcohol consumption 9,12,14,15. the educational level influence the presence of the risk factors in these patients’ lives. the ideal solution to prevent the disease from reaching this stage is early diagnosis. although the public absence of knowledge about the opscc compared to other cancers is a challenge to the auto-perception of disease signs and symptoms. the lack of dental examinations and the patient’s fear of them also contribute to the diagnostic delay16. diagnosis in advanced cs brings a treatment challenge insofar as, in these stages, the prognosis worsens. in our findings, there was a positive association between a symptomatology increase and advanced cs, few patients have achieved complete remission in the disease follow up, and more than 50% died. patients classified in high stages present patterns of bigger and invasive lesions17. these tumor standards, when associated with the anatomical characteristics of the subsites mainly affected, explains the high symptomatology presence and its cs associations. also, the low number of patients submitted to surgeries, which might result in poor functional outcome18. a study with 646 patients found that localized swelling, pain, and alterations of the mucosa were the predominant signs and symptoms of the disease in advanced cs in most cases. the hospitalization rate was high, corresponding to 66.9% of patients after four months of the first symptoms notification. in our study, we found similar findings regarding symptomatology (75.50% pain) and clinical staging. patients clas7 pedra et al. sified in high stages presented patterns of bigger and invasive lesions17, which are related to symptomatology. physicians were responsible for most patient’s referral and the diagnosis. the low participation of dentists in the diagnosis is a critical finding. dentists should be responsible for the primary prevention of oral cancer by identifying precancerous lesions, advising on smoking cessation, alcohol diminution, and sun protection19. opscc were diagnosed in the early stages by dentists than to physicians20,21. regular dental visits were also associated with diagnosis at early stages21. dentists are essential in the early diagnosis of oral cancer since they have access to all oral and oropharyngeal regions during clinical examinations22. the preponderant part of the patients in our study worked in the countryside. rural areas usually are characterized by higher rates of poverty and tobacco use. generally, they have worse education levels, lower incomes, and socioeconomic status. rural residents may also indirectly have limited access to care, such as low availability of specialty health facilities and long travel times. consequently, patients from these areas have higher incidence and mortality rates for tobacco-related cancer23,24. besides that, in brazil, rural populations have poor access to oral public health services in comparison to those from urban areas25. tobacco and alcohol consumption are the etiological factors most associated with the development of oral squamous cell carcinoma9,14,15. similarly to our findings, a study has associated alcohol and tobacco consumption with advanced clinical staging7. these risk factors contribute to the disease worsening, and the diagnosis delay results in high morbidity rates1. in our study, most patients died because of opscc, during or after treatment. these are facts that urge attention to the need to implement more effective public policies that strongly encourage early diagnosis enabling these patients to have more effective treatment, better prognosis, and survival rates6. in our study, we observed that educational level deepens cultural limits. it brings harmful habits to population health, to those who have less access to education. the low education level might influence consumption patterns affecting health negatively with risky habits. public policies that encourage schooling are also relevant for preventing opscc not only to provide economic growth12. social inequality goes even further, transcending cultural issues. the at-risk population, being rural, face difficulties accessing health services. health policies in sus that guide active searches on the communities who live or work in rural areas and are smokers are needed to promote health education and opscc early detection. besides, encourage greater participation by dental surgeons in these actions, especially in rural areas13. opscc is a health problem that, to be combated effectively, poses a massive challenge for public policy elaboration. it demands that the policies developed should involve the citizen needs globally. for effective prevention, it is crucial to improve educational levels and access to health care. besides, it is also necessary to approach the rural population and health services. promote a better involvement of dental professionals in this issue as well. these actions could culminate in the achievement of better results as an early disease diagnosis25,26. 8 pedra et al. the present study demonstrates that low educational level, smoking, and symptoms may be related to the diagnosis of oral and oropharyngeal cancer at advanced clinical-staging. therefore, it is relevant to implement specific public policies to improve this population’s health. including the information about this disease to the inhabitants with low educational levels and improve their access to dental care in order to recognize the clinical signs and early diagnosis. references 1. gigliotti j, madathil s, makhoul n. delays in oral cavity cancer. int j oral maxillofac surg. 2019; 48(9):1131-7. doi: 10.1016/j.ijom.2019.02.015. 2. dantas ts, de barros silva pg, sousa ef, da cunha mdp, de aguiar as, costa fw, et al. influence of educational level, stage, and histological type on survival of oral cancer in a brazilian population. medicine (baltimore). 2016; 95(3):e2314. doi: 10.1097/md.0000000000002314. 3. phelan j, link b, 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10.4103/0973-1482.180687. 15. peltanova b, raudenska m, masarik m. effect of tumor microenvironment on pathogenesis of the head and neck squamous cell carcinoma: a systematic review. mol cancer. 2019;18(1):63. doi: 10.1186/s12943-019-0983-5. 16. logan hl, guo y, marks j. disparities in survival patterns for oral and pharyngeal cancer in florida: can we do anything about it? today’s fda. 2015;27(4):58–61. 17. crescenzi d, laus m, radici m, croce a. tnm classification of the oral cavity carcinomas: some suggested modifications. otolaryngol pol. 2015;69(4):21-30. doi: 10.5604/00306657.1160919. 18. alzahrani r, obaid a, al-hakami h, alshehri a, al-assaf h, adas r, et al. locally advanced oral cavity cancers: what is the optimal care?. cancer control. 2020;27(1):1073274820920727. doi: 10.1177/1073274820920727. 19. gigliotti j, madathil s, makhoul n. delays in oral cavity cancer. int j oral maxillofac surg. 2019;48(9):1131-7. doi: 10.1016/j.ijom.2019.02.015. 20. langton s, cousin gcs, 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[oral cavity cancer: epidemiology and early diagnosis]. refuat hapeh vehashinayim (1993). 2015;32(3):55-63, 71. hebrew. 22. abadeh a, ali a, bradley g, magalhaes m. increase in detection of oral cancer and precursor lesions by dentists. j am dent assoc. 2019;150(6):531-9. doi: 10.1016/j.adaj.2019.01.026. 23. zuniga s, lango m. effect of rural and urban geography on larynx cancer incidence and survival. laryngoscope, 2018;128(8):1874-80. doi: 10.1002/lary.27042. 24. roberts me,  doogan nj, kurti an, redner r, gaalema de, stanton ca, et al. rural tobacco use across the united states: how rural and urban areas differ, broken down by census regions and divisions. health place. 2016;39:153-9. doi: 10.1016/j.healthplace.2016.04.001. 25. arruda nm, maia ag, alves lc. inequality in access to health services between urban and rural areas in brazil: a disaggregation of factors from 1998 to 2008. cad saude publica. 2018;34(6):e00213816. doi: 10.1590/0102-311x00213816. 26. cunha a, prass t, hugo f. mortality from oral and oropharyngeal cancer in brazil: impact of the national oral health policy. cad saude publica. 2019;35(12):e00014319. doi: 10.1590/0102-311x00014319. https://www.ncbi.nlm.nih.gov/pubmed/?term=doogan nj%5bauthor%5d&cauthor=true&cauthor_uid=27107746 https://www.ncbi.nlm.nih.gov/pubmed/?term=kurti an%5bauthor%5d&cauthor=true&cauthor_uid=27107746 https://www.ncbi.nlm.nih.gov/pubmed/?term=redner r%5bauthor%5d&cauthor=true&cauthor_uid=27107746 https://www.ncbi.nlm.nih.gov/pubmed/?term=gaalema de%5bauthor%5d&cauthor=true&cauthor_uid=27107746 https://www.ncbi.nlm.nih.gov/pubmed/?term=stanton ca%5bauthor%5d&cauthor=true&cauthor_uid=27107746 1http://dx.doi.org/10.20396/bjos.v19i0.8659272 volume 19 2020 e209272 original article 1 school of dentistry, university of passo fundo, passo fundo, rio grande do sul, brazil. 2 federal university of pelotas, pelotas, rio grande do sul, brazil. 3 federal university of rio grande do sul. porto alegre, rio grande do sul, brazil. corresponding author: francisco wilker mustafa gomes muniz. federal university of pelotas rua gonçalves chaves, 457, pelotas/rs, brazil, zip code: 960105-560. phone/ fax: +55 53 991253611. email: wilkermustafa@gmail.com received: april 23, 2020 accepted: august 02, 2020 concern with dental appearance and associated factors among the elderly of two southern brazilian cities: a cross-sectional study diandra genoveva sachetti1 , fernanda pretto zatt1, thais carleso trevizan1, caroline fernandes e silva2, francisco wilker mustafa gomes muniz2,* , cassiano kuchenbecker rösing3 , paulo roberto grafitti colussi1 aim: this study aimed to evaluate the prevalence of concern with dental appearance (da) and associated factors among the elderly of two southern brazilian cities. methods: a cross-sectional study was conducted in the cities of cruz alta and veranópolis, brazil. oral health examination and a structured questionnaire were applied. questions from the pcatool-sb brasil tool and the questionnaire about teeth appearance, both validated to brazilian samples, were used. the collected independent variables were: sex, age, ethnicity, education level, marital status, retirement, health problem, use of medication, smoking exposure, alcohol exposure, access to the dentist, toothbrushing frequency, use of dental floss, edentulism, use of and need for dental prosthesis. concern with da was dichotomized into yes/no. associations between dependent and independent variables were assessed by chi-square or mannwhitney tests. moreover, uniand multivariate analyses were conducted by poisson regression with robust variance. level of significance was established as p<0.05. results: the prevalence of concern with da was 18.8% (n=107). the prevalence ratio (pr) of concern with da decreased 5.8% for each year increase (pr:0.942; 95% confidence interval [95%ci]:0.911–0.973). elderly without access to the dentist in the last 12 months presented 62.5% (p=0.006) higher pr of concern with da when compared to those with access to dental care. dentate elderly showed 219% higher pr for concern with da (pr:2.197; 95%ci:1.364–3.539) in comparison to edentulous individuals. conclusion: prevalence of concern with da was low and associated with demographics, access to dental care and edentulism. keywords: dental care for aged. epidemiology. esthetic, dental. jaw, edentulous. self concept. https://orcid.org/0000-0002-7581-2762 https://orcid.org/0000-0002-3945-1752 https://orcid.org/0000-0002-8499-5759 2 sachetti et al. introduction the phenomenon of human aging is a reality, and reflects an intense demographic challenge, probably one of the largest in recent decades. according to this reality, there is need to provide a higher quality of life for these individuals, which includes better physical, social, and psychological aspects1. regarding oral health in the elderly, the literature is consistent, demonstrating that these individuals have high rates of periodontal disease and tooth loss2,3. historically, dental services and public policies did not prioritize oral health care in the elderly4. this may be one of the explanations for the worse oral health condition in this population5. those aspects are more evident in underdeveloped countries, such as brazil, where mean number of lost teeth is higher and there is a high prevalence of edentulism6. moreover, data from the latest national survey showed that most brazilian elderly need prosthetic rehabilitation7. this impairs functional aspects, such as chewing and speaking, influencing aesthetics. however, issues involving dental aesthetics still seem to be of minor importance to the elderly population. studies demonstrate that less than 20% of the elderly report to be unsatisfied with dental appearance8,9. however, the literature indicates that greater satisfaction with oral appearance is a protective factor for the decline in some domains of quality of life10. additionally, there is a high perception of dental treatment needs and rehabilitation among the elderly11. nevertheless, questions involving human aging and oral health, as well as their psychosocial consequences, need further understanding in the literature. currently, aspects related to elderly aesthetics are receiving more attention, mainly related to oral rehabilitation needs12. in contrast, knowledge about the elderly oral aesthetic self-perception is scarce in the literature. the present study aimed to evaluate the prevalence and associated factors of concern with dental appearance among the elderly, in two cities of the state of rio grande do sul, brazil. the null hypothesis of this study is that there are no significant associations between concern with dental appearance and demographic, socioeconomic and dental variables. material and methods study design and location this study followed is reported according to the strobe checklist guidelines. elderly aged ≥60 years from two cities in the state of rio grande do sul, brazil were interviewed and examined in this cross-sectional study. the cities of cruz alta and veranópolis are located in the north of rio grande do sul state, approximately 350 and 160 km from the capital porto alegre, respectively. the population of cruz alta is approximately 62,821 inhabitants, while the population of veranópolis is approximately 22,810 inhabitants13. about 95% and 87% of the population live in the urban area, respectively, and, in both cities, around 60% of the elderly are female. the municipal human development index of veranópolis, in 2010, was 0.7514, and the gini index, in the same year, was 0.4836, whereas these data, in cruz alta, were 0.75 and 0.5419, 3 sachetti et al. respectively15. for each city, two distinct research protocols were developed, reviewed and approved by the ethical committee of the university of passo fundo (protocol numbers 1,531,862 and 2,990,088). all participants read and signed the informed consent form before their inclusion in the study. sample size calculation sample calculation was based on the elderly oral health self-perception prevalence reported in another study16. when assuming a prevalence of 31.92% of poor oral health self-perception, an alpha error of 5%, a confidence interval of 99% and a population size of 7,284, a sample of 535 elderly people was considered necessary. an attrition rate of 5% was added. sampling strategy to be representative of both cities, different sampling strategies for the cities of cruz alta and veranópolis were developed. in cruz alta, a per cluster probabilistic sample was conducted. due to the high number of individuals in the urban area, only the elderly living in this area were included. the city of cruz alta has 68 district or neighborhoods, which were listed and received a number. of these, seventeen were randomly selected (25%), through the website www.random.org, considering the proportion of elderly residents in the area. more information about the sampling strategy in cruz alta city can be found elsewhere17. for the city of veranópolis, another probabilistic sample by cluster was performed to visit 300 residences, respecting the proportion of urban and rural areas. based on the city urban area map, all blocks were numbered. a total of 82 blocks (20% of the total blocks) were randomly selected in this area, using the website www.random. org. additionally, the blocks corners were numbered from one to four, and a new draw was conducted to determine the first interview. in each block, three households with at least one resident elderly were visited. after the first interview, the visits continued in a clockwise direction until complete the planned work. whenever necessary, new blocks were selected in order to include the necessary number of households. in veranópolis, a total of three rural communities were part of the study, randomly selected using the website www.random.org, among all 21 rural communities in the city. households were visited until reaching at least 12 elderly people interviewed and examined in each community. rural area households involved residences in the central nucleus of the community and others located along the several community side roads. inclusion and exclusion criteria individuals to be included in the present study should be at least 60 years old and living in the selected households in the sampling strategy. participants that answered all questions and allowed the clinical oral examinations were included in the present study. if more than one elderly person met the eligibility criteria, in each residence, all of them were included. in the case of absence on the day of data collection, two more attempts were made before excluding the household. homes for the aged (i.e. geriatric long-term care facilities), stores, elderly who were visiting the selected area and uninhabited homes were not included. 4 sachetti et al. clinical examination and interview elderly were interviewed between july and august 2016, in cruz alta, and between december 2018 and january 2019, in veranópolis. in both cities, the same structured questionnaire, which included socio-demographic, behavioral, medical and dental history data, was applied. these variables were obtained by the question blocks from the pcatool-sb brasil tool18. the complete questionnaire may be found elsewhere19. the questions related to concern with dental appearance and dental aesthetic perception were obtained by the questionnaire about teeth appearance, a validated questionnaire for the brazilian population20. oral health was assessed by counting teeth and verifying use and need for dental prosthesis, according to the world health organization criteria21. oral examinations were performed with a wooden spatula, without artificial lighting or odontoscope. individuals were examined and interviewed by teams composed of an interviewer and an oral health examiner, who were previously trained by the study coordinators to ensure uniformity of data. training consisted of lectures, discussion of all the questions in the questionnaire and explanations about oral health examination. the same coordinator was responsible for all teams in both cities. before the study, training was conducted with the questionnaire application and with the oral health exam in elderly patients undergoing treatment in the school of dentistry of the university of passo fundo clinics. the intraand inter-examiner reproducibility of the clinical oral health examination was verified in 5% of the total population. after 14 days, the examination was repeated. when considering all the examiners, the interand intra-examiner reproducibility demonstrated a kappa index of at least 0.70 for all variables. statistical analysis the dependent variable of the present study was concern with dental appearance determined by a validated instrument20. the question used was: “during the past two months, how much did your teeth appearance concern you”? the options of answers were: a lot, a little, very little, nothing and i do not know. the sample was categorized into two groups, those who answered “very little”, “nothing” or “i don’t know” were included in the group that was not concerned about their teeth appearance. those who answered “a lot” or “a little” were included in the group that was concerned about their teeth appearance. gender, age, ethnicity/skin color, educational level, marital status, retirement, health problems, use of medication, exposure to smoking and alcohol, access to the dentist in the last 12 months, frequency of toothbrushing, flossing, edentulism, use of prosthesis, need for prosthesis were considered independent variables in the present study. age was recorded in years, and sex, as male or female. ethnicity/skin color was classified as white or non-white (the non-white classification included elderly people who referred themselves as black, yellow, brown or indigenous). level of education was classified as low, which included illiterate participants and those with incomplete or complete elementary school; medium, for those with incomplete or complete high school; and high level, for those with at least incomplete college/university degrees. marital status was classified as married or not married. the not married group was composed of those who reported being single, divorced or widowed. regarding retirement, the elderly were dichotomized into retired and not retired. 5 sachetti et al. regarding general health conditions, the elderly were classified into two groups: one that involved individuals with no health problems or who were not aware of having them, and those who reported having some type of health problem. use of medication was classified into two groups: one with participants who reported using one or more medications, and another group who did not reported use of medication. smoking exposure was classified into three groups: no history of smoking, current smokers and former smokers. alcohol exposure was classified into yes or not. those who reported consuming alcohol frequently or moderately were considered as alcohol users, and the other individuals were considered as non-users. access to the dentist in the last 12 months and the use of dental floss use were dichotomized as yes or not. toothbrushing frequency was classified into two groups: at least once a day and twice or more a day. elderly that used any dental prosthesis in at least one arch were included in the group that uses dental prosthesis. similarly, the elderly that needed oral rehabilitation in at least one arch were included the in the group that needs dental prosthesis. these criteria were used based on the world health organization for epidemiologic studies on oral health20. tooth loss was categorized as edentulism, for those without any teeth, and non-edentulism, for those with one or more teeth. associations between dependent and independent variables were assessed through chi-square or mann-whitney tests. additionally, uniand multivariate analyses were performed by poisson regression with robust variance to verify associations between independent variables and concern with dental appearance. in the multivariate model, the variables that presented p <0.20 in univariate analysis were included. the maintenance of these variables, in the final multivariate model, was determined by the combination of p <0.05 and changing effect models analysis. moreover, multicollinearity analyses were performed and none was observed. results a total of 569 individuals were interviewed and examined, of which 287 were from cruz alta and 282 were from veranópolis. in the present sample, mean (± standard deviation) age was 70.35±6.18, with 67.8% (n=386) of female individuals. the majority of the included elderly considered themselves to be white (78.7%, n=448), had a low educational level (72.9%, n=415), were retired (82.2%, n=468), and 55.4% (n=315) of them were married. the prevalence of concern with dental appearance was 18.8% (n = 107). age, edentulism and need for dental prosthesis were significantly associated with concern with dental appearance (table 1). in table 2, univariate analysis of the association between concern with dental appearance and the exploratory variables is shown. age, smoking exposure, edentulism, use of dental prosthesis and need for dental prosthesis were associated with concern with dental appearance. in this model, by each additional year of age, there was a 7% decrease (p<0.001) in the prevalence ratio (pr) of the elderly being concerned with their dental appearance. elderly with no history of smoking had 42.7% lower pr of concern with dental appearance when compared to elderly that currently smoke. dentate elderly had approximately 2.5 times higher pr of concern about their teeth appearance when compared with edentulous elderly (p <0.001). those that did not 6 sachetti et al. use dental prosthesis had 51.6% higher pr of concern with dental appearance when compared to those that use. the elderly without need of oral rehabilitation with prosthesis had 47.6% (p <0.001) lower pr of concern about their teeth appearance when compared to elderly that needed dental prosthesis. in addition to the already mentioned variables, ethnicity/skin color, education, access to the dentist, and use of dental floss were included in the initial multivariate model. table 3 shows the final multivariate analysis associating concern with dental appearance and the independent variables. age, access to the dentist in the last 12 months and edentulism remained associated. by each additional year of age, there was a 5.8% decrease in the pr of the elderly being concerned with their dental appearance (pr: 0.942; 95% confidence interval; 95% ci: 0.911 – 0.973). the elderly lacking access to the dentist in the last 12 months had 62.5% (p = 0.006) higher pr of concern with table 1. demographic, behavioral, medical, and dental variables according to concern with dental appearance. variables n – (%) or mean±sd concerned (n=462; 81.2%) not concerned (n=107; 18.8%) p-value sex male female 150 (32.5) 312 (67.5) 33 (30.8) 74 (69.2) 0.819* age 70.87±6.39 68.09±4.57 <0.001# ethnicity/skin color white non-white 369 (79.9) 93 (20.1) 79 (73.8) 28 (26.2) 0.190* education level low medium high 341 (73.8) 65 (14.1) 56 (12.1) 74 (69.2) 22 (20.6) 11 (10.3) 0.234* marital status married not married 253 (54.8) 209 (45.2) 62 (57.9) 45 (42.1) 0.590* retirement yes no 380 (82.3) 82 (17.7) 88 (82.2) 19 (17.8) 1.000* health problem yes no 404 (87.4) 58 (12.6) 92 (86.0) 15 (14.0) 0.748* use of medication yes no 392 (84.8) 70 (15.2) 90 (84.1) 17 (15.9) 0.882* smoking exposure smokers ex-smokers never smokers 39 (8.4) 123 (26.6) 300 (64.9) 16 (15.0) 31 (29.0) 60 (56.1) 0.079* alcohol exposure yes no 220 (47.6) 242 (52.4) 46 (43.0) 61 (57.0) 0.392* access to the dentist yes no 222 (48.1) 240 (51.9) 44 (41.1) 63 (58.9) 0.199* tooth brushing frequency <2/day ≥2/day 44 (9.5) 418 (90.5) 12 (11.2) 95 (88.8) 0.591* dental floss use yes no 129 (27.9) 333 (72.1) 37 (34.6) 70 (65.4) 0.194* edentulism yes no 201 (43.5) 261 (56.5) 22 (20.6) 85 (79.4) <0.001* use of dental prosthesis yes no 401 (86.8) 61 (13.2) 85 (79.4) 22 (20.6) 0.067* need for dental prosthesis yes no 144 (31.2) 318 (68.8) 54 (50.5) 53 (49.5) <0.001* legend: *qui-square; #mann-whitney 7 sachetti et al. table 2. univariate analysis of the association of concern with dental appearance and sociodemographic variables, medical and dental histories. variables prevalence ratio (ci 95%) p-value sex male female ref. 1.063 (0.734 – 1.540) 0.746 age 0.930 (0.901 – 0.959) <0.001 ethnicity/skin color white non-white ref. 1.312 (0.896 – 1.922) 0163 education level low medium high ref. 1.418 (0.935 – 2.150) 0.921 (0.516 – 1.642) 0.100 0.780 marital status married not married ref. 0.900 (0.637 – 1.273) 0.552 retirement yes no ref. 1.000 (0.639 – 1.563) 0.998 health problem yes no ref. 1.108 (0.680 – 1.803) 0.681 use of medication yes no ref. 1.046 (0.657 – 1.667) 0.848 smoking exposure smokers ex-smokers never smokers ref. 0.692 (0.412 – 1.163) 0.573 (0.357 – 0.919) 0.164 0.021 alcohol exposure yes no ref. 1.164 (0.824 – 1.645) 0.389 access to the dentist yes no ref. 1.257 (0.887 – 1.780) 0.198 tooth brushing frequency <2/day ≥2/day ref. 0.864 (0.507 – 1.473) 0.592 use of dental floss yes no ref. 0.779 (0.546 – 1.111) 0.169 edentulism yes no ref. 2.490 (1.608 – 3.857) <0.001 use of dental prosthesis yes no ref. 1.516 (1.009 – 2.277) 0.045 need for dental prosthesis yes no ref. 0.524 (0.374 – 0.734) <0.001 table 3. multivariate analysis of the association of concern with dental appearance and sociodemographic variables, medical and dental histories. variables prevalence ratio (ci 95%) p-value age 0.942 (0.911 – 0.973) 0.017 smoking exposure smokers ex-smokers non smokers ref. 0.748 (0.447 – 1.251) 0.657 (0.409 – 1.055) 0.268 0.082 access to the dentist yes no ref. 1.625 (1.150 – 2.297) 0.006 edentulism yes no ref. 2.197 (1.364 – 3.539) 0.001 use of dental prosthesis yes no ref. 1.000 (0.656 – 1.523) 0.999 need for dental prosthesis yes no ref. 0.779 (0.544 – 1.116) 0.173 8 sachetti et al. dental appearance when compared to ones with access to dental care. moreover, dentate had approximately 2.2 times higher pr of concern with dental appearance (pr: 2.197; 95% ci: 1.364 – 3.539) when compared with edentulous individuals. discussion the present study aimed to assess the prevalence of concern with dental appearance and associated factors in the elderly of two cities in south brazil. the analysis merged data from two similar cities in order to increase observations and better support the analytical approach. in this particular population, concern with dental appearance was low and associated with age, access to the dentist and edentulism. studies focusing on dental aesthetics among the elderly have increased, as their interaction with society has increased in recent decades, as well as due to aging of the populations22. several aspects, such as greater social interactions, professional activities and the desire to enjoy life, require better health conditions of these individuals, including oral health. oral aesthetics at this age group should receive attention especially due to the fact that satisfaction with dental appearance is often related to the general appearance, well-being, social interaction, self-esteem, and ultimately, quality of life23,24. in the present study, the prevalence of concern with dental appearance was 18.8%. this demonstrates that the percentage of elderly dissatisfied with their dental appearance is somewhat low. however, the percentage of concerns with dental appearance in the elderly seems to be slightly higher than those found in other studies, as published studies show estimates of dissatisfaction of 11%25, 12%9 and 13%8. it is important to highlight that some differences between the findings of the present study and the other studies reported in the literature may be detected, which include the country in which they were performed. most of them were performed in european countries and did not evaluate the percentage of edentulism and its impact on appearance perception. moreover, the presence of edentulism and use of dental prosthesis were not assessed in those studies, which is a strength of the present study. also, another possible explanation for such difference is the fact that those studies were performed more than five years ago and, with time, there is change in health tendencies, probably including oral health. in this sense, it is speculated that concern with dental appearance in the elderly will increase in the coming years. the percentage of concern with dental appearance in the present study can be justified by the appearance acceptance process that seems to occur when one gets older. however, appearance investments should be considered more and more to maintain successful aging26. therefore, the relationship between human aging and aesthetics may increase in the coming years, which warrants epidemiological studies and the development of appropriate assessment instruments for this age group24. elderly in the work forces, the greater social interaction and use of social media can be situations involved in this process. important factors have been identified as potential influencers in body image construction and acceptance, including age. in the present study, age was associated with lower concern with oral aesthetics. in fact, by each additional age year, there was a 5.8% decrease in the pr of the elderly being concerned about their smile appearance. it should 9 sachetti et al. be put into perspective that this decrease with aging is among elderly and not related to whole adulthood. these results corroborate with studies that demonstrate that aesthetics concern tends to decrease as one gets older12,22. age is not necessarily associated with an oral aesthetics negative perception, but with cognitive factors. it must be considered that the human aging process is generally followed by a greater susceptibility to chronic degenerative diseases. in this scenario, it is expected that oral health issues are still seen as less important or secondary25. it is also important to recognize that other aspects have been reported in the literature as associated with image self-perception. older adults exposed to higher social interaction was associated with better self-esteem when compared to those with limited or reduced social activities27,28. when considering that 82.2% of our sample consisted of retired elderly, participation in social activities may have a beneficial influence on the participants’ perception of self-image. however, this variable was not assessed, which might be a limitation of the present study. in the present study, lack of access to professional oral care was associated with greater concern with dental aesthetics. there seems to be a lower predisposition to dental treatments among the elderly, especially regarding preventive strategies29,30. in addition, the cost of dental treatment should be considered when observing this association31. it is particularly true for the majority of the elderly from brazil, in which need of prosthesis reaches almost 70% of this population7. access to dental treatment is directly related to educational level and income. a better educational level is usually associated with greater purchasing power, which results in more resources for investments in aesthetic treatments32. unfortunately, the majority of the brazilian elderly have a low level of education and income13. it should be emphasized that the majority of the population of the present study is from underprivileged strata in terms of education and is retired, which per se reduces income. moreover, oral health-related issues were also associated with concerns with appearance. the effect of most chronic oral diseases makes tooth extraction often the main treatment option, especially for the population from lower socioeconomical levels33. in the present study, elderly with at least one tooth present (dentate) were more concerned about their dental aesthetics. these individuals are probably more predisposed to dental treatments. in this sense, studies showed that edentulous elderly perceive less the need for dental treatment34 and present a better oral health-related quality of life35 in comparison to elderly with teeth. this is especially true due to the presence of dentures, that, in one way or another, solve the aesthetic concern. in addition, the process of adaptation, that comes with age should be taken into consideration and could be part of the explanation for such findings. also, it should be emphasized that having natural teeth, as assessed in the present study, does not mean having healthy dentition or having a satisfactory oral rehabilitation. in the present study, sex was not associated with greater concern with dental appearance. although similar results can be observed in the literature32, female gender is generally more associated with oral aesthetics concerns. several studies demonstrate that women are less satisfied with their smile and express a greater desire for dental aesthetic treatments12,24,36. for a long time, having certain appearance standards were the only possibility of social advancement for women in many cultures and, therefore, they were resigned to aesthetic impositions still perceived today37. 10 sachetti et al. this study is representative of the two cities from the rio grande do sul state regarding socioeconomic status and sex, taking into consideration the probabilistic sample per cluster conducted in both cities. in this sense, educational level and gender distribution of participants were similar to those observed in the last national census13. additionally, examiners were trained and calibrated to data collection by the same researcher, which increases its internal validity. on the other hand, this study has some limitations: the cross-sectional design, which does not allow the temporality assessment of concern with dental appearance and the exploratory variables. additionally, the quality of prosthetic rehabilitation was not evaluated. the time between evaluations for both cities (from july/august 2016 to december 2018/january 2019) may also be faced as another limitation, and must be considered when interpreting the results of the present study. despite the limitations, the study design allows data generalization for comparisons with other home-based studies with a representative sample. it was concluded that concern with dental appearance was low among the elderly. higher concern with dental appearance was observed at younger ages, in those without access to the dentist, and who presented at least one natural tooth. however, smoking exposure was not associated with concern with dental appearance. the use of and need for dental prosthesis was not associated with concern with dental appearance among the elderly. acknowledgements this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior brasil (capes) finance code 001. all other funding was self-supported by the authors. the authors report no conflict of interest related to this study. references 1. tourani s, behzadifar m, martini m, aryankhesal a, mirghaed mt, salemiet m, et al. health-related quality of life among healthy elderly iranians: a systematic review and meta-analysis of the literature. health qual life outcomes. 2018 jan;16(1):18. doi: 10.1186/s12955-018-0845-7. 2. kassebaum nj, bernabé e, dahiya m, bhandari b, murray cj, marcenes w. global burden of severe tooth loss: a systematic review and meta-analysis. j dent res. 2014 jul;93(7 suppl):20s-28s. doi: 10.1177/0022034514537828. 3. van der putten gj, de baat c, de visschere l, schols j. poor oral health, a potential new geriatric syndrome. 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october university of modern sciences and arts, egypt. 2 lecturer in department of endodontics, faculty of dentistry, october university of modern sciences and arts, egypt. corresponding author: mai h. abdelrahman department of dental biomaterials, faculty of dentistry, october university of modern sciences and arts, egypt. e-mail: mhesham@msa.eun.eg editor: dr altair a. del bel cury received: january 24, 2021 accepted: may 5, 2021 a comparative evaluation of the sealing ability of two calcium silicate based sealers and a resin epoxy-based sealer through scanning electron microscopy and bond strength: an in vitro study mai h. abdelrahman1,*, mohamed y. hassan2 aim: this study aimed to compare the sealing ability of two types of commercially available calcium silicate bioceramic based root canal sealers and a resin based root canal sealer. methods: twenty one single-rooted teeth were used, samples (n= 21) were randomly divided into three groups according to the sealer used (group a; adseal, group b; wellroot, group c; ceraseal). roots were then cleaved longitudinally in the labiolingual direction; all samples were then sectioned at three, six, and nine mm from the root tip. the penetration of sealers into the dentinal tubules was examined at 1000x with a scanning electron microscope. data were tested for normality using shapiro wilk test. anova test was used for analyzing normally distributed data followed by bonferroni post hoc test for pair-wise comparison. significance level p≤0.001. results: groups b and c showed better sealing ability than group a in all the three sections. the coronal section showed higher sealing ability than the middle section followed by the apical section in the three tested groups. conclusion: it can be concluded that both calcium silicate-based sealers had better sealing ability and higher bond strength than the resin epoxybased sealer. keywords: calcium compounds. silicates. epoxy resins. root canal filling materials. electron microscope tomography. https://orcid.org/0000-0002-6056-3417 2 abdelrahman et al. introduction efficient filling and complete sealing of the previously cleaned and shaped root canal system are crucial steps that have an impact on the long-term success of the treatment1,2. incorporation of sealers is mandatory as gutta-percha does not adhere to the dentinal walls and thus cannot prevent leakage by it-self, accordingly sealers are used to fill the irregularities and to penetrate into dentinal tubules attempting to achieve a hermetic seal of the root canal system2. therefore, root canal sealers should attain strong adherence between gutta-percha and the dentinal walls preventing gap existence at the sealer-dentine interface2,3. thus achieving a three-dimensional seal of root canal which is essential to ensure complete prevention of reinjection of the canal and for preserving the health of the periapical tissues, consequently ensuring successful treatment1,4. there is no real chemical bond between root canal sealers and the dentinal wall of root canals; however, tubular penetration of root canal sealers may enhance the micromechanical bonding of sealers and subsequently their sealing properties5. adaptation of a sealer to the dentinal wall is evaluated using different ways; stereo-microscope, confocal laser microscopy, scanning electron microscopy (sem), leakage tests and digital imaging3. currently, there is a variety of commercially available sealers. however, none of the existing sealers satisfies all the required idealistic properties3. calcium silicate based sealers have attracted clinicians due to their excellent biocompatibility and bioactivity as claimed by their manufacturers, combined with their ability to bond to the tooth structure2,6. however, there is scarce literature on their properties and performance in vitro and in vivo. accordingly, this in-vitro study aimed to compare the sealing ability of two types of commercially available calcium silicate based root canal sealers and a resin based root canal sealer. the study was performed under the null hypothesis that no differences in the ability of sealing the dental tubules would be observed between the three tested root canal sealants. materials and methods materials used in this study were; adseal, well-root st and ceraseal. table 1. materials used in this study, their manufacturer, lot number and composition. materials manufacturer lot number composition adseal meta biomed korea ads1406171 a two paste system; base: • epoxy oligomer resin. • ethylene glygol salicylate. • calcium phosphate. • bismuth subcarbonate. • zirconium oxide. continue 3 abdelrahman et al. continuation adseal meta biomed korea ads1406171 catalyst: • poly aminobenzoate. • triethanolamine. • calcium phosphate. • bismuth subcarbonate. • zirconium oxide. • calcium oxide well-root st vericom, gangwon-do, korea wr8n0200 calcium aluminosilicate compound. zirconium oxide filler and thickening agent. ceraseal meta biomed korea csl1912161 2g pre-mixed syringe × 1ea intra canal tips × 15ea methods twenty-one recently extracted human upper incisors with straight fully formed roots were selected. all teeth were inspected under stereomicroscope (image j, earl f, glynn ii, and over park, usa) at a magnification of (10x). incisors with more than one canal, open apex, endodontically treated, internal or external resorption, caries, cracks or fractures on the root surfaces were excluded. five from each group were used for push out bond strength testing. a total sample size of 15(5 sample in each group) will be sufficient to detect an effect size of 2.18, a power (1-β error) of 0.85, using a two-sided hypothesis test, significance level (α error) 0.05 for data. the remaining two samples were used for scaning. teeth were cleaned using ultrasonic scalar, and then placed in 2.5%naocl for 30 minutes for surface disinfection then stored in distilled water until use. crowns were removed at the level of cement-enamel junction by the use of micro-saw under water cooling (isomet 4000 micro saw, buehler,usa), leaving averagely 15 mm long root segments. working lengths were recognized for all canals by a # k file (mani, tochigi, japan). cleaning and shaping were conducted by the use of protaper system (dentsply maillefer, ballaigues, switzerland) starting by; sx, followed by s1, s2 in a brushing motion, followed by f1,f2 f3,f4 and f5 in a non brushing motion. all root canals were irrigated during cleaning and shaping with 5ml of 25% naocl solution using end-perforated 27 gauge needle sung shim, seoul, korea) to ensure efficient cleanliness of the canal. after complete instrumentation, all samples we placed in a glass box (n= 21), and were randomly divided by a blind technician in to three groups (n=7) according to the type of sealer used. samples were obturated with protaper universal gutta percha points and the type of sealer was used according to its group (group a: adseal, group b: well-root and group c: ceraseal) using lateral condensation technique. roots were then coded according to the type of sealer used and stored in a moist environment for 1 week to ensure complete setting of the sealers before testing. 4 abdelrahman et al. 1. scanning electron microscopy: two roots from each group were cleaved longitudinally in the labio-lingual direction using a hammer and chisel, all samples were then sectioned at three, six, and nine mm from the root tip using a 0.3 mm disk thickness7,8. the penetration of sealers into the dentinal tubules and adaptation of each sealer to the canal wall were examined at 1000x magnification with a scanning electron microscope (quanta 250 feg (field emission gun) attached with edx unit (energy dispersive x-ray analyses), with accelerating voltage 30 k.v (netherland). 2. push out bond strength: five roots from each group were tested. each root was embedded in a centralized manner in acrylic resin using a transparent plastic mold (diameter 10 mm, length 16mm) so that the tooth surface flushes with the upper acrylic surface. after setting of the acrylic resin, coronal, middle and apical thirds were defined and a section of 1mm thickness was cut from the center of each third using water-cooled precision microsaw9. test was conducted using computer-controlled universal testing machine (instron universal testing machine model 3354 instron instruments england) with a load cell of 5-kn. a plunger with diameter of (0.9mm or 0.7mm or 0.5mm) acted as a force probe to apply a push out load at a crosshead speed of 0.5mm/min in an apical coronal direction. the selected diameter of the plunger was chosen so that it only contacts the filling to displace it downwards9. the maximum failure load was recorded in newtons (n) and was used to calculate the push-out bond strength in mega pascals (mpa) according to the following formula. shear bond strength (mpa) = maximum load in (n) / adhesion area of root canal filling (mm2) data were presented as mean and standard deviation. data were tested for normality using shapiro wilk test. anova test was used for analyzing normally distributed data followed by bonferroni post hoc test for pair-wise comparison. analysis was performed using ibm spss statistics for windows, version 25.0. armonk, ny: ibm corp. figure 1. sections in the coronal, middle and apical thirds 5 abdelrahman et al. results 1. scanning electron microscopy: the morphology of sealer/ dentin interface of the three tested sealers using a scanning electron microscope were evaluated, they showed a true hybridization and sealers tags formation inside the dentinal tubules. group a revealed few numbers of tags having small diameters and a clear interfacial gap. while groups b and c showed numerous number of tags with large diameters protruding into the dentinal tubules with a gap at the sealer/dentin interface as shown in figure 1. a b c acc v 30.0 kv spot 6.0 maq 1500x det bse wd 15.4 e.m.r.a-xl30 20 µm acc v 30.0 kv spot 6.0 maq 1500x det bse wd 15.4 e.m.r.a-xl30 20 µm acc v 30.0 kv spot 6.0 maq 1500x det bse wd 15.4 e.m.r.a-xl30 20 µm figure 2. scanning electron micrograph (1000x) of the interface at sealer/ dentin using a:adseal, b: well-root and c: ceraseal. 2. push-out comparison between the three groups: 2a. in the coronal, middle and apical thirds between the three groups: in the coronal section; the mean and standard deviation values of group a were (57.93 ± 1.21), while in group b were (68.49 ± 1.62) and in group c were (73.5 ± 1.88). there was no statistically significant difference between the three groups. in the middle section; the mean and standard deviation values of group a were (44.46 ± 2.16), while in group b were (63.4 ± 1.41) and in group c were (67.59 ± 1.18). there was no statistically significant difference between the three groups. in the apical section; the mean and standard deviation values of group a were (4.13 ± 0.71), while in group b were (10.74 ± 0.62) and in group c were (11.6 ± 0.51). there was no statistically significant difference between the three groups. table 2. mean and standard deviation (sd) and the results of anova test for comparison of push-out (mpa) in the coronal, middle and apical thirds between the three groups: coronal group a group b group c p value mean 57.93 68.49 73.50 <0.001 sd 1.21 1.62 1.88 middle continue 6 abdelrahman et al. continuation mean 44.46 63.4 67.79 <0.001 sd 2.16 1.41 1.18 apical mean 4.13 10.74 11.6 <0.001 sd 0.71 0.62 0.51 significance level p≤0.001 2b. pair wise comparison: the push-out in the coronal (figure 3) and middle thirds (figure 4) exhibited a statistically significant difference between all group pairs. in the apical third (figure 5), a statistically significant difference was found between groups a and b and between groups a and c, while no statistically significant difference was found between groups b and c. table 3. results of bonferroni post hoc test for pair-wise comparison of push-out (mpa) in the coronal, middle and apical thirds between the three groups: coronal p-value group a – group b <0.001* group a – group c <0.001* group b – group c <0.001* middle group a – group b <0.001* group a – group c <0.001* group b – group c <0.001* apical group a – group b <0.001* group a – group c <0.001* group b – group c <0.055 significance level p≤0.001 u ni t group a group b group c mean push-out 80.00 70.00 60.00 50.00 40.00 30.00 20.00 10.00 0.00 figure 3. bar chart representing the mean push-out in the coronal third in the three groups. 7 abdelrahman et al. u ni t group a group b group c mean push-out 80.00 70.00 60.00 50.00 40.00 30.00 20.00 10.00 0.00 figure 4. bar chart representing the mean push-out in the middle third in the three groups. u ni t group a group b group c mean push-out 14.00 12.00 10.00 8.00 6.00 4.00 2.00 0.00 figure 5. bar chart representing the mean push-out in the apical third in the three groups. discussion in order to achieve a successful endodontic treatment and good prognosis, it is crucial to have a tight apical seal which is dependent on proper instrumentation and cleaning of the coot canal system in conjunction to adequate obturation10. penetration of root canal sealers into dentinal tubules allows a better sealing ability thus preventing residual bacteria from re-growing within the tubular space11. accordingly, there is a continuous improvement in the field of root canal filling materials and recently bioactive materials are becoming of high demand7. calcium silicate-based sealers are hydrophilic in nature, possess an alkaline ph, insoluble in tissue fluids and they don’t shrink on setting. moreover, the moisture environment of the tooth has an influence on the adhesion between the bioceramic sealers and root canal dentin. these sealers are characterized by their potential bio8 abdelrahman et al. active properties, where calcium hydroxide and hydroxyapatite are formed once the sealer contacts water, resulting in a high alkaline ph that activates and initiates the expression of alkaline phosphatase, favoring the formation of mineralized tissue and possessing an antibacterial effect12-14. an resin epoxy-based sealer was used in this study because it is characterized by its good adherence to the root canal dentin, outstanding biocompatibility and low risk of unfavorable postoperative inflammatory reactions12. several methods have been postulated for evaluating the sealing ability of obturation materials; dye penetration, fluid filtration techniques, radioisotopes, scanning electronic microscopic analysis, electrochemical leakage tests, glucose penetration and bacterial penetration test13,15. scanning electronic microscope was utilized in this study as it allows proper evaluation of the sealing ability and adhesiveness of the sealer to dentin walls or sealer-gutta-percha interface on the various levels of root sections15. moreover it provides high magnification thus allows better observation of surface topography11. improvement in the sealing ability of the sealers is achieved through the mechanical interlocking of the sealer plug inside the dentinal tubules (push-out). a strong bond is critical in order to maintain the integrity of the sealer-dentin interface16. therefore, this study aimed to evaluate and compare the sealing ability of two types of calcium silicate-based sealers and a resin epoxy-based root canal sealer by the use of scanning electron microscopy (sem) and bond strength. results of the current study revealed that groups b and c had higher push-out bond strength than group a in the three root sections namely; coronal, middle and apical. this can be attributed to the excellent physical properties of the calcium silicate-based sealers such as flow, low film thickness and dimensional stability. in addition to the alkaline nature of the byproducts produced by the calcium silicate-based sealers that might have denaturized the dentin collagen fibers thus facilitated the sealers penetration. this was in accordance with baruah et al.13 in 2018. while, the lower bond strength exhibited by group c could be due to the incomplete polymerization and the setting shrinkage of its resinous components resulting in formation of poor microtags which consequently exhibits low adhesion properties. this was also suggested by huang et al.7 in 2018 and baruah et al.13 in 2018. in the present study, results of the sealers push-out strength revealed that coronal section exhibited the highest mean value followed by the middle section while, the apical section had the lowest mean value in the three tested groups. this might result from presence of dentinal tubules with larger diameter at both coronal and middle parts when tested against the apical part16. moreover wang et al.3 in 2018, stated that despite of the kind of sealers or obturation techniques used, the percentages of penetrated dentinal tubules of root canal increased from apical to coronal part due to the increased amount of eliminated smear layer in the upper-middle section of the root canal. 9 abdelrahman et al. the fact that the degree of adhesion of the sealers to the dentin wall depends on the surface energy of the dentin, surface tension and wettability of the sealer in addition to the cleanliness of the dentin surface7. dentin in the coronal, middle, and apical sections has different surface energies, in conjunction with obstacles faced during complete removal of the smear layer from the apical region might be the cause of its lower sealer penetration this was in accordance with huang et al.7 in 2018 and eid et al.16 in 2019. these results were confirmed by the descriptive characteristics of the tags revealed by the scanning electron microscopic study as shown in figure (2). where groups b and c demonstrated a clearly recognizable thin hybrid layer, including numerous numbers of tags with a large diameter protruding into the dentinal tubules however, group a showed a thin hybrid layer with few numbers of resin tags having a small diameter. the images obtained through scanning electron microscopy have some limitations; are only representative of sectioned canal levels examined. further studies are required to evaluate the mineralogical characteristics of both well root and ceraseal when it is in contact with different solutions, in addition to their physiochemical properties. in conclusion, with the limitations of this study, it can be concluded that both calcium silicate-based sealers had sufficient sealing qualities and higher bond strength than the resin based sealer. the higher bond strength of the two tested calcium silicate-based sealers in conjunction with their bioactivity might help in improvement of the root canal system sealing. this conclusion needs further investigations. conflicts of interest there are no conflicts of interest. references 1. al-haddad a, che ab aziz za. bioceramic-based root canal sealers: a review. int j biomater. 2016;2016:9753210. doi: 10.1155/2016/9753210. 2. reszka p, nowicka a, lipski m, dura w, droździk a, woźniak k. a comparative chemical study of calcium silicate-containing and epoxy resin-based root canal sealers. biomed res int. 2016;2016:9808432. doi: 10.1155/2016/9808432. 3. wang y, liu s, dong y. in vitro study of dentinal tubule penetration and filling quality of bioceramic sealer. plos one. 2018 feb 1;13(2):e0192248. doi: 10.1371/journal.pone.0192248.. 4. ha jh, kim hc, kim yk, kwon ty. an evaluation of wetting and adhesion of three bioceramic root canal sealers to intraradicular human dentin. materials (basel). 2018 jul;11(8):1286. doi: 10.3390/ma11081286. 5. abdul khader m. an in vitro scanning electron microscopy study to evaluate the dentinal tubular penetration depth of three root canal sealers. j int. oral health. 2016;8(2):191-4. 6. gad ra, farag am, el-hediny ha, darrag am. sealing ability and obturation quality of root canals filled with gutta-percha and two different sealers. tanta dent. j. 2016;13(4):165-70. doi: 10.4103/1687-8574.195703. 10 abdelrahman et al. 7. huang y, orhan k, celikten b, orhan ai, tufenkci p, sevimay s. evaluation of the sealing ability of different root canal sealers: a combined sem and micro-ct study. j appl oral sci. 2018 jan;26:e20160584. doi: 10.1590/1678-7757-2016-0584. 8. kim hj, baek sh, bae ks. sealing ability of root canals obturated with gutta-percha, epoxy resin-based sealer, and dentin adhesives. j korean acad conserv dent. 2004;29(1):51-7. doi: 10.5395/jkacd.2004.29.1.051. 9. vemisetty h, ravichandra pv, reddy s j, ramkiran d, krishna m jn, sayini r, et al. comparative evaluation of push-out bond strength of three endodontic sealers with and without amoxicillin-an invitro study. j clin diagn res. 2014 jan;8(1):228-31. doi: 10.7860/jcdr/2014/7180.3919. 10. kapasi a, bishnoi a, meena ss, singh p, patodia a. sealability of bioceramic cements on root ends prepared using a hard tissue laser evaluated by stereomicroscope an in vitro study. acta scient dent sci. 2018;2(8):9-18. 11. toursavadkohi s, zameni f, afkar m. comparison of tubular penetration of ah26, easyseal, and sure-seal root canal sealers in single-rooted teeth using scanning electron microscopy. j res dent maxillofac sci. 2018;3(3):27-32. doi: 10.29252/jrdms.3.3.27. 12. gyulbenkiyan e, gusiyska a, vassileva r, dyulgerova e. scanning electron microscopic evaluation of the sealer/dentine interface of two sealers using two protocols of irrigation. j imab. 2020;26(1):2887-91. doi: 10.5272/jimab.2020261.2887. 13. baruah k, mirdha n, gill b, bishnoi n, gupta t, baruah q. comparative study of the effect on apical sealability with different levels of remaining gutta-percha in teeth prepared to receive posts: an in vitro study. contemp clin dent. 2018 sep;9(suppl 2):s261-5. doi: 10.4103/ccd.ccd_196_18. 14. abdelrahman mh, hassan my. comparison of root canals walls cleanliness filled with two different calcium silicate bioceramic based sealers and a resin-based sealer after retreatment. int. j dentistry res. 2020;5(1):20-3. 15. vujasković m, teodorović n. analysis of sealing ability of root canal sealers using scanning electronic microscopy technique. srp arh celok lek. 2010 nov-dec;138(11-12):694-8. doi: 10.2298/sarh1012694v. 16. eid bm, waly as, princy p, venkatesan r. scanning electron microscope evaluation of dentinal tubules penetration of three different root canal sealers. ec dent. sci. 2019;18(6):1121-7. 1http://dx.doi.org/10.20396/bjos.v21i00.8665074 volume 21 2022 e225074 original article 1 usp – university of são paulo, ribeirão preto medical school, department of pathology and legal medicine. ribeirão preto, são paulo, brazil. 2 usp university of são paulo, school of dentistry of ribeirão preto. ribeirão preto, são paulo, brazil. 3 unicamp – university of campinas, piracicaba dental school, department of health sciences and pediatric dentistry. campinas, são paulo, brazil. 4 usp university of são paulo, school of dentistry of ribeirão preto, department of stomatology, public health and forensic dentistry. ribeirão preto, são paulo, brazil. corresponding author: ricardo henrique alves da silva usp – faculdade de odontologia de ribeirão preto. departamento de estomatologia, saúde coletiva e odontologia legal. área de odontologia legal. avenida do café, s/n, bairro monte alegre, 14040-904, ribeirão preto, sp, brasil. e-mail: ricardohenrique@usp.br editor: dr altair a. del bel cury received: march 24, 2021 accepted: august 28, 2021 dental mapractice litigance in the city of são paulo (sp), brazil silmara pereira de sousa1,2 , bruna saud borges1,2 , ana luisa rezende machado1,2 , giovanna teixeira matteussi1,2 , paulo henrique viana pinto1,2 , larissa dutra bittencourt de oliveira1 , rienne assis matos2 , marcos vinicius coltri3 , ricardo henrique alves da silva4 the relationship between patients and dentists today is more worn and less based on trust, which can lead to high rates of lawsuits related to civil liability and dental malpractice. aim: verify if there has been an increase in the number of lawsuits related to questioned dental treatments, and against dentists registered in the city of são paulo (sp), brazil, from 2012 to 2017. methods: we outlined an overview based on the list from the são paulo council of dentistry containing 30,238 registered dentists in the city of são paulo, and searched for lawsuits on the public base of the são paulo state court’s. results: the search, after the inclusion and exclusion criteria, found 247 lawsuits, with dental implants as the most involved specialty. the total indemnities requested ranged from r$ 227.42 to r$ 937,000.00, but no indemnity granted exceeded the amount of r$ 100,000.00. conclusion: according to the analysis of cases, there is a progressive increase in the number of civil liability lawsuits against dentists involving dental malpractice litigance. keywords: insurance, liability. forensic dentistry. patient harm. jurisprudence. https://orcid.org/0000-0002-8124-9611 https://orcid.org/0000-0002-8622-0068 https://orcid.org/0000-0001-8169-0063 https://orcid.org/0000-0002-6294-3704 https://orcid.org/0000-0003-0945-9566 https://orcid.org/0000-0002-4221-5275 https://orcid.org/0000-0002-8183-4001 https://orcid.org/0000-0002-3614-6437 https://orcid.org/0000-0002-1532-1670 2 sousa et al. introduction with the advent of the brazilian code of consumer protection, which has provided patients greater knowledge in regards to their rights and easier access to justice, dentists have been increasingly faced with complaints about treatment and/or lack of information in lawsuits1. the dentist’s civil liability is defined as the duty to repair damage caused to the patient2. the brazilian civil code characterizes an unlawful act and its obligation of reparation can be found in articles 186, “that who, by voluntary action or omission, negligence or recklessness, violates law and causes harm to others, even if exclusively moral, commits an unlawful act”; and 927, “that who, by an unlawful act, causes harm to another, is obliged to repair it”3. therefore, a civil liability is characterized by the guilty conduct of the professional, damage suffered by the patient, and a causal link between them, thus generating the obligation of pecuniary reparation (material, moral, and/or esthetic damage)4. once the lawsuit initiates, an impartial professional acting as an expert witness can be appointed by the judge to carry out an examination and prepare a report, which aims to clarify the technical and scientific issues of the case. moreover, a lawsuit in brazil can rely on the presence of a technical assistant who can be appointed by any of the parties involved, and who can assist during the lawsuit5. the patient/dentist relationship is classified as a consumption relationship, with the consumer here interpreted as the patient and author of the lawsuit. in these cases, there is a mechanism used to facilitate consumer protection, known as the inversion of the proof onus. thus, it is not the patient/author who must prove that the dentist is culpable; it is the professional/defendant who is responsible for proving that there was no fault on his/her part during the professional act. in this regard, the brazilian code of consumer protection article 6 states in relation to consumer rights: the facilitation of the defense of your rights, including the inversion of the proof onus in your favor in civil lawsuits, when at the discretion of a judge, the claim is credible or when the consumer is dependent, in accordance with the ordinary rules of experience6. currently, there is an increase in the number of lawsuits against dentists in brazil, and the most valuable proof for the defense of the professional are the patient’s dental records. among other functions, they may show that the dentist’s conduct was based on technical and scientific literature, the patient did not suffer any damage, or that the eventual damage presented by the patient was not related to the dentist’s actions or performance7. therefore, this research aimed to conduct an analysis of civil liability lawsuits against dentists related to dental treatments in the biggest city in brazil, the city of são paulo. materials and methods this research was approved by the ethics commission under caae (registration number 03381318.1.0000.5419). data were collected from the list provided by the https://dictionary.cambridge.org/pt/dicionario/ingles-portugues/the https://dictionary.cambridge.org/pt/dicionario/ingles-portugues/patient https://dictionary.cambridge.org/pt/dicionario/ingles-portugues/did https://dictionary.cambridge.org/pt/dicionario/ingles-portugues/not https://dictionary.cambridge.org/pt/dicionario/ingles-portugues/suffer https://dictionary.cambridge.org/pt/dicionario/ingles-portugues/damage 3 sousa et al. são paulo council of dentistry of são paulo (cro-sp) containing the names of 30,238 dentists registered in the city of são paulo. in addition, the são paulo state court’s website (www.tjsp.jus.br) was searched for all available court cases in the city of são paulo. the lawsuit inclusion criteria adopted were: available as full electronic records; involving, as one of the requested parties, the name of at least one dentist present in the mentioned list; related to civil liability in dentistry regarding the questioning of dental treatments; and started between 2012 and 2017. the year 2012 was selected as the starting point because it was the year that the system of electronic records was implemented in the são paulo state court. for the exclusion criteria, the following were listed: lawsuits available only physically (paper documents) or partially electronically; lawsuits that involved only the company providing dental care as a required part; lawsuits that cited names of dentists from the aforementioned list, but were not related to the object of the research (dental treatments); lawsuits for which electronic records were unavailable; lawsuits that did not start within the period between 2012 and 2017. first of all, it’s important to mention that the research couldn’t work on more recent years due to the magnitude of the study. the first part was a manual search, including each name on the são paulo state court public basis, resulting in 30,238 different quests, and it take around six months, and reached 1325 lawsuits. then, the second phase was to check each one of the 1325 lawsuits and apply the inclusion and exclusion criteria, and it took more eight months. finally, with 247 lawsuits selected, it was necessary to read and study the whole documents (in average with more than 300 pages each) to collect the data of this study, and it took more ten months. after the selection, a full verification of the records was performed, with the following data collected: (1) year the lawsuit began; (2) dental specialty involved; (3) qualification(s) of the prosecuted dentist; (4) whether or not an expert witness was appointed; (5) the expert witness’ specialty; (6) whether or not technical assistants for the parties were indicated, and their specialties; (7) amount of compensation requested; and (8) data related to the sentence. in line with the analysis of these data, the specialties of the professionals (dentists as defendants, expert witnesses, and technical assistants) were checked through the public database of cro-sp and the brazilian federal council of dentistry (cfo), and it included more than 300 quests in two different data basis. results from the 247 lawsuits involving dental malpractice litigance that corresponded to the inclusion criteria of the study, it was observed an increase over the years (figure 1). http://www.tjsp.jus.br 4 sousa et al. 100 90 80 70 60 50 40 30 20 10 0 2 14 36 44 60 91 2012 2013 2014 2015 2016 2017 figure 1. number of lawsuits against dentists involving dental malpractice litigance, 2012-2017, são paulo, sp, brazil. figure 2 shows the five most cited dental specialties in the analyzed lawsuits. it is important to note that these specialties refer to the procedures claimed by the patient’s request within the lawsuits. thus, we have related the procedures to existing specialties within dentistry to better illustrate the results. some of the lawsuits mentioned more than one procedure. additionally, while 60% (n=149) of the lawsuits mentioned referred to procedures related to a single specialty, 27% (n=67) mentioned two distinct specialties, 11% (n=27) cited three specialties, and 2% (n=4) linked to four specialties. thus, the number of specialties cited exceeds the number of cases found. 120 100 80 60 40 20 0 103 89 52 26 16 im pl an to lo gy p ro st hd on tic s o ra l a nd m ax ill of ac ia l s ur ge ry en do do nt ic s o rt ho do nt ic s figure 2. dental specialties in lawsuits against dentists involving dental malpractice litigance, 2012-2017, são paulo, sp, brazil. the qualification of the dentists involved in the lawsuits were also investigated; 60% (n=149) of the cases dealt with professionals who had no registered specialty at the time of data collection, while 40% (n=98) dealt with dentists with a registered specialist title, 31 of whom claimed more than one specialty. the registered specialties are detailed in figure 3. 5 sousa et al. 40 30 20 15 10 5 0 38 31 19 14 9 im pl an to lo gy o ra l a nd m ax ill of ac ia l s ur ge ry p ro st hd on tic s p er io do nt ic s o rt ho do nt ic s 25 35 figure 3. dentists’ specialties in lawsuits against dentists involving the questioning of dental treatments, 2012-2017, são paulo, sp, brazil. regarding the data collected on expert witnesses, in 12% (n=29) of the lawsuits, no expert witness was appointed. in 65% (n=161) of the lawsuits, an expert witness was appointed. in the remaining 23% of lawsuits, it was not possible to determine expert witness appointment, as cases had not reached this legal phase at time of the data collection. in the lawsuits where an expert witness was appointed, 71% (n=115) were specialized dentists, of which 61% (n=70) had one specialty, 37% (n=43) had two specialties, and 2% (n=2) of the expert examinations were performed by medical doctors. in addition, among the appointed expert witness, 63% (n=72) were registered as forensic dentistry specialists (figure 4). 40 30 20 10 0 72 27 12 11 11 fo re ns ic d en tis tr y o ra l a nd m ax ill of ac ia l s ur ge ry r ad io lo gy p er io do nt ic s im pl an to lo gy 80 70 60 50 figure 4. specialties of expert witnesses appointed in lawsuits against dentists involving dental malpractice litigance, 2012-2017, são paulo, sp, brazil. regarding the 161 lawsuits that appointed an expert witness, 68% (n=110) of these included indications of technical assistants, with 9% (n=10) of the indications made only by the requesting part, 63% (n=69) only by the requested part, and 28% (n=31) by both parties. regarding question formulations, they were present in 84% (n=136), with 7% (n=10) being performed only by the requesting part, 21% (n=29) only by the requested part and 71% (n=97) by both parties. 6 sousa et al. regarding the 161 cases that appointed an expert witness, two of them experienced conciliation of the parties before the expert examination, and two other cases did not present the report for different reasons. one lawsuit was suspended before they could give the report, and in three other lawsuits, it was not possible to identify the reason for the absence of the report. additionally, in 51 lawsuits, the reports had not been presented yet due to the phase of the lawsuit at the time of this research. this resulted in 102 cases in which there was presentation of the expert report. of these, 48% (n=49) indicated the presence of causal link and 52% (n=53) indicated the absence of a causal link. regarding sentencing data from the 247 included lawsuits, 64% (n=158) had not yet reached the sentence stage, 0.4% (n=1) were suspended and 36% (n=88) had sentences. from the 88 lawsuits that already had a sentence, 52% (n=46) were valid and 25% (n=22) unfounded; for 23% (n=20), were extinguished. thus, regarding indemnities clamed, total amounts ranged between r$ 227.42 (usd 40.74) and r$ 937,000.00 (usd 167,860.98)8; however, the sentences did not bring amounts higher than r$ 100,000.00 (usd 17,914.72)8. the study demonstrated that in many lawsuits, there was a distinction between material, moral, and esthetic damages (table 1). table 1. damages requested and sentenced in lawsuits against dentists involving dental malpractice litigance, 2012-2017, são paulo, sp, brazil. damage requested indemnities sentenced indemnities min. average max. min. average max. material r$200.00 (usd 35.82) r$16,862.90 (usd 3,020.94) r$386,120.00 (usd 69,172.33) r$200.00 (usd 35.82) r$9,860.48 (usd 1,766.47) r$48,266.00 (usd 8,646.72) moral r$1,000.00 (usd 179.14) r$46,030.51 (usd 8,246.23) r$500,000.00 (usd 89,573.62) r$1,000.00 (usd 179.14) r$15,323.76 (usd 2,745.20) r$100,000.00 (usd 17,914.72) esthetic r$1,350.00 (usd 241.84) r$44,932.52 (usd 8,049.53) r$210,000.00 (usd 37,620.92) r$5,000.00 (usd 37,620.92) r$18,500.00 (usd 3,314.22) r$40,000.00 (usd 7,165.89) total r$227.42 (usd 40.74) r$67,636.71 (usd 12,116.93) r$937,000.00 (usd 167,860.98) r$850.00 (usd 152.27) r$20,861.18 (usd 3,737.22) r$100,000.00 (usd 17,914.72) discussion no professional is exempt from making mistakes, and this awareness is the first step to combat possible professional weaknesses in the face of processes related to civil liability. therefore, certain conduct is essential in preventing or minimizing the chances of possible lawsuits, such as maintaining a harmonious relationship with the patient, producing well-organized dental records, providing all the information to the patient as often as possible, and continuous monitoring of the patient9. in brazil, the dentist’s civil liability is guided by the civil code and the code of consumer protection. these codes assure the patient that it is the professional’s duty to act with caution in the practice of dentistry1. 7 sousa et al. to begin discussion of the analysis of this research we will highlight the limitations inherent to the data collection. the analyzed period was decided based on the beginning of the implementation of the electronic lawsuit records by the são paulo state court, since these can be fully accessed, unlike “paper” lawsuits that only have some or no information available online. it would be logistically impossible to analyze physical records one by one, justifying the methodological option of considering only electronic lawsuits in order to obtain more complete and real data. because of this, it is possible that the low number of lawsuits in the initial years was due to the ongoing implementation of this new form of producing and filing of court records. another limitation is the dependence on a list of registered professionals, which does not follow a historical series, meaning if any professional requested a transference or cancellation of their registration before the time of this research, it is not possible to have information on this record, which them limits the search for their respective lawsuits. finally, there is a time lag between the data presented in the study and its publication, as the achievement of these results required the nominal and individual search of each of the 30,238 names on the list available manually, that is, in 2018 each of the full names of registered dentists were inserted one by one on the platform of the são paulo state court’s website, followed by a full reading of the records of the cases found, and tabulation of data, one must take into account the great time demand of this process since it does not allow automation. in the following year, the detailed data analysis of each civil liability lawsuit was carried out, configuring a cross-sectional analysis. however, despite these limitations, this study was able for the first time to draw a design of civil lawsuits against dentists in the city of são paulo in the first instance court, regarding questioning the dental treatments. this followed an increasing curve over the years, which reaffirms the trend observed in other cities and regions throughout brazil1,10,11. the analysis found a continuous annual increase in lawsuits, with the final year, 2017, responsible for 36.8% of the total litigations analyzed. such an increase can be due to a number of factors, such as a constant increase in the number of dentists, the rise in law professionals specializing in health, competition among dentists and dental companies, and changes in the professional-patient relationship over the years10,12. this relationship was previously based solely on trust, with the professional being the one who was knowledgeable and made decisions; today, however, the patient has easier access to knowledge about his contractual situation, and has become more demanding of information regarding the services provided10. melani et al.13 (2010) demonstrated this problem by showing that about 29% of the lawsuits analyzed in their study resulted from unwanted care in the face of dissatisfaction or doubt, concluding that the lack of communication and clarification resulted in a serious problem in the professional-patient relationship, often resulting in a breach of trust. additionally, there is a social appeal in searching for monetary compensation from dental damages, often encouraging the patient to resort to the judicial system5. 8 sousa et al. another point to discuss is that health care practitioners who experience fatigue, overwork, and stress may be more likely to commit an error14-16, which increases the susceptibility of being involved in a civil liability lawsuit. nowadays, several reports maintain that dentistry is an exceedingly stressful profession where distress and eventual burnout are distinct possibilities17. yansane et al.18 (2020), evaluated the relationship between burnout, work engagement, and self-reported dental errors among northamerican dentists and of the 391 responding dentists, 46.1% reported concern that they had made a dental error in the last 6 months, 12.1% of the dentists were informed by dental staff that they may have committed an error in the last six months, 16% were concerned that a malpractice lawsuit would be filed against them, and 3.6% were actively involved in a malpractice lawsuit. dental treatments often seemed related to the specialties of dental implants, prosthodontics, oral and maxillofacial surgery, endodontics, and orthodontics, confirming the results obtained in the londrina population1 and, previously, in general research conducted in the national territory10. some of these specialties seem to be common, even when evaluated in different regions or time frames, as in the studies by magalhães et al.19 (2019), where the most involved specialties were, in decreasing order, prosthodontics, orthodontics, and dental implants, and rosa et al.20 (2012), where the specialties remained the same, but in reverse order. this scenario is repeated even internationally, where thavajanah et al.21 (2019) found that the treatments that led to a lawsuit most often in india were tooth extraction, root canal treatment and implants, and in rome, the authors found a similar perspective where the most litigious dental activities was prosthetic, implantology and endodontia22. this phenomenon can be explained by the consideration that these procedures take a longer time to complete, are usually more costly, and bring a greater esthetic appeal and, therefore, create a greater expectation by the patient. this, many times, can be encouraged by the dentist’s advertisements, which assigns to the dentist an obligation of result, where they must reach a certain goal desired by the patient5,7. as previously mentioned, we observed that 60% of the professionals involved in litigation did not have a registered specialty. according to brazil law number 5.081/1966, dentists are allowed to “perform all acts related to the dentistry field, resulting from knowledge acquired from undergraduate and graduate courses”23. however, having the necessary professional legal qualification does not mean having the ability to perform all the procedures the degree confers, and it is necessary to be aware of the limits of one’s competence, always bearing in mind the responsibility and legal consequences regarding all professional acts performed1,24. comparing the research by lino junior. et al.1 (2017), where only 7.18% of individuals had a registered specialty, to our study, where 40% of the professionals had such qualifications, we can consider the difference to be related to distinct geographic location and population demands that required a more generalist approach by the professionals, as in the case of the first study mentioned, and a more specialized approach, as in the case of the present study. therefore, despite the high number of dentists without a specialist title registered, it is not possible to state that they do not have the capacity to carry out the dental treatments. 9 sousa et al. we found that in 65% of the cases an expert witness was appointed, and meaningful results were found by zanin et al.12 (2015), where 84.2% of cases involved an expert witness, and montagna et al.25 (2008), where 73.1% of cases involved an expert witness. this highlights the importance of the expert as a clarifying agent for technical and scientific issues, who will produce impartial evidence to the judge and the related parties13. on the other hand, the study by magalhães et al.19 (2019) observed that expert reports were only used in 3.9% of the assessed lawsuits20. of the appointed expert witnesses, 115 had some registered specialty, and we observed that forensic dentistry as a specialty that goes side-by-side with law. for silva9 (2010), the expert examination cannot be performed by just any professional in the field (here referring to dental treatment), since it is not enough just to be his or her profession, but requires a professional qualified and specialized in a specific area, especially with the expert routine that is part of the scope of the forensic dentistry specialty. in this research, 63% of expert witnesses were registered as forensic dentistry specialists. in the lawsuits with an appointed expert witness, we found that in 68% of these cases, a technical assistant (often referred to as “part’s expert witness”) was hired. compare this to the data found by zanin et al.12 (2015), in which only 10.3% of the parts involved in the lawsuits indicated hiring these professionals, even though more than 80% of the lawsuits involved expertise examination. this increase in hiring of technical assistants over the years denotes a greater awareness on the importance of these professionals in the lawsuit. the importance of such a service during a lawsuit is justified by the guarantee of the technical adversary, that is, the partiality to the part that hired the service. the technical assistant can assist the lawyer in formulating questions, focusing on technical matters, limiting and guiding the parameters followed by the expert examination, and participate in due diligence regarding the expert examination. technical assistants can also prepare an independent opinion that may agree with, complement, or even refute the report issued by the appointed expert witness.(5,12). when comparing the number of lawsuits in which at least one of the parts indicated a professional as a technical assistant and those in which questions to expert witness were submitted, it is clear that there are more lawsuits with questions presented than those with designated technical assistants. this is due to the fact that the elaboration of the questions is not strictly linked to the presence of technical assistants, and it can be done by the part itself, by the lawyer, or even by the court. the questions work as a device to try to prove the allegations made in the initial petition (by the author/patient) or in the defense (by the required/dentist)5,26. for this reason, it is worth reflecting the usefulness of a professional, who has not only the technical and scientific knowledge of the areas involved, but also the expertise in knowing which points are worth highlighting, with ethics, in favor of the client. when instituted, an indemnity action aims to make reparation to the victim to compensate any damage suffered by the patient when the causal nexus inherent to the dental treatment and the professional conduct are proven. often, indemnity is subdivided into material and moral damage, and more recently, esthetic damage. when it comes to valuing the material damage, there are not many difficulties, considering 10 sousa et al. that it is enough to have proof of the equity losses by the requesting part1,27. with regard to moral damage, subjectivity and psychological effects make determination complex, and the judge must use subjective criteria for its definition, using proportionality and reasonableness, as the objective of the indemnity is not enrichment, but the reparation of unfair suffering28. the same reasoning applies to the arbitration of any compensation for esthetic damage. in this study, the most varied claims were collected, ranging from r$ 227.42 (usd 40.74) to r$ 937,000.00 (usd 167,860.98)8. however, there was a tendency for the sentence, when the lawsuit was deemed valid, to impose condemnatory amounts lower than that requested in the initial petition, not exceeding r$ 100,000.00 (usd 17,914.72)8. the same seens to occours in india, where thavarajah et al.21 (2019) found the average compensation claimed as inr 5,772.87 (usd 78,36)8 ± 9,058.98 (usd 122.96)8 while the average compensation awarded was 1,039.98 (usd 14.11)8. the data obtained in this research are in line with information provided in other studies, given that the research points to a discrepancy between the values pleaded and the condemnations set out in the sentences1,29,30. the number of lawsuits involving dental malpractice litigance in the city of são paulo (sp), brazil, has grown progressively over the years, and the dental treatments claimed often seemed related to the specialties of dental implants, prosthodontics, oral and maxillofacial surgery, endodontics, and orthodontics. this rases an alert for dentists to use their technical and scientific knowledge in conjunction with current ethics and legislation. acknowledgements own funding. references 1. lino-junior hl, terada assd, silva rha, soltoski mpc. [civil liability lawstuits in dentistry, londrina, brazil]. rev jurid. 2017;1(46):515-31. portuguese. doi: 10.21902/revistajur.2316-753x.v1i46.2261. 2. figueira junior e, trindade go. [responsibility of the surgeon dentist accord to the code of defense of the consumer]. cad unifoa. 2010 mar;5(12):63-70. portuguese. doi: 10.47385/cadunifoa.v5i12.1006. 3. presidency of the republic of brazil. [law n. 10,406, 2002 january 10. establishes the civil]. diario oficial união. 2002 [cited 2020 feb 30] available from: http://www.planalto.gov.br/ccivil_03/leis/2002/l10406.htm. portuguese. 4. medeiros uv, coltri ar. [civil responsibility of the dentist]. rev bras odontol, 2014;71(1):10-6. portuguese. 5. silva rha, musse jo, melani rfh, oliveira rn. [surgeon dentist’s civil liability: the technical assistant’s importance]. rev dent press ortod ortop facial. 2009;14(6):65-71. portuguese. doi: 10.1590/s1415-54192009000600009. 6. presidency of the republic of brazil. [law n. 8,078, 1990 september 11. 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[scenario of civil liability actions against dentists in brazilian courts]. rev paul odontol. 2010;32(4):22-8. portuguese. 12. zanin aa, strapsson rap, melani rfh. [jurisprudential study: evidences in dental civil liability lawsuit]. rev assoc paul cir dent. 2015;69(2):119-27. portuguese. 13. melani rfh; oliveira rn, tedeshi-oliveira sv, juhás r. legal devices and arguments mostly used in civil lawsuits: casuistry analysis in dentistry. rpg rev pós-grad. 2010;17(1):46-53. portuguese. 14. shanafelt td, balch cm, bechamps g, russell t, dyrbye l, satele d, et al. burnout and medical errors among american surgeons. ann surg. 2010 jun;251(6):995-1000. doi: 10.1097/sla.0b013e3181bfdab3. 15. leape ll. error in medicine. jama. 1994 dec;272(23):1851-7. doi: 10.1001/jama.1994.03520230061039. 16. leape ll, berwick dm, bates dw. what practices will most improve safety? evidence-based medicine meets patient safety. jama. 2002 jul;288(4):501-7. doi: 10.1001/jama.288.4.501. 17. gorter rc, eijkman ma, hoogstraten j. a career counseling program for dentists: effects on burnout. patient educ couns. 2001 apr;43(1):23-30. doi: 10.1016/s0738-3991(00)00141-5. 18. yansane a, tokede o, walji m, obadan-udoh e, riedy c, white j, et al. burnout, engagement, and dental errors among u.s. dentists. j patient saf. 2020 apr 3. doi: 10.1097/pts.0000000000000673. 19. magalhães lv, costa b, silva rha. [analysis of the lawsutisinvolving dentistry in metropolitan vitória, espírito santostate, brazil]. rev bras odontol leg rbol. 2019;6(2):13-20. portuguese. doi: 10.21117/rbol.v6i2.232. 20. rosa fm, fernandes mm, daruge-júnior e, paranhos lr. [material and moral damages in cases involving dentists in the state of são paulo]. rfo. 2012;17(1):26-30. portuguese. doi: 10.5335/rfo.v17i1.2537. 21. thavarajah r, saranya v, priya b. the indian dental litigation landscape: an analysis of judgments on dental negligence claims in indian consumer redressal forums. j forensic leg med. 2019 nov;68:101863. doi: 10.1016/j.jflm.2019.101863. 22. manca r, bruti v, napoletano s, marinelli e. a 15 years survey for dental malpractice claims in rome, italy. j forensic leg med. 2018 aug;58:74-7. doi: 10.1016/j.jflm.2018.05.005. 23. presidency of the republic of brazil. [law n. 5,081, 1966 august 24. [regulates the practice of dentistry.]. diario oficial união. 1966 [cited 2020 feb 30] available from: https://presrepublica.jusbrasil.com.br/legislacao/128600/lei-5081-66. portuguese. 24. lino-júnior hl, gabriel m, daruge-júnior e, silva rha. [forensic dentistry education in brazil: an invitation to reflection]. rev abeno. 2015;15(2):38-46. portuguese. 25. montagna f, manfredini d, nuzzolese e. professional liability and structure of litigation in dentistry. minerva stomatol. 2008 jul-aug;57(7-8):349-57. 26. sarmento ms, dezem tu, medeiros uv. [the importance of the dentistry expert in lawsuits]. rev bras crimin. 2018;7(3):44-52. portuguese. doi: 10.15260/rbc.v7i3.231. 12 sousa et al. 27. giostri ht. [ethical civil liability of dentists: a new vision]. curitiba: juruá; 2012. portuguese. 28. presidency of the republic of brazil. [constitution of the federative republic of brazil, 1988]. 1988 [cited 2020 feb 30] available from: http://www.planalto.gov.br/ccivil_03/constituicao/constituicao.htm. portuguese. 29. bouchardet fch, vieira slg, miranda ge, fernandes mm, vieira dnp, silva rf. [assessment of the aesthetic damage in the jurisprudence of the court of justice, minas gerais]. robrac. 2013;22(63):116-9. portuguese. doi: 10.36065/robrac.v22i63.820. 30. terada assd, araujo lg, flores mrp, silva rha. [civil liability of dental surgeon: legal proceedings analysis in the city of ribeirao preto, são paulo state, brazil]. int j odontostomat. 2014; 8(3), 365-369. spanish. doi: 10.4067/s0718-381x2014000300008. 1http://dx.doi.org/10.20396/bjos.v21i00.8662812 volume 21 2022 e222812 original article 1 program of interactive process of organs and systems, federal university of bahia. 2 institute of the science of health, federal university of bahia. corresponding author: natália nascimento odilon. av. reitor miguel calmon, s/n, canela, salvadorba, cep: 40110-902., 4° andar, sala 413. e-mail:natalia.odi@gmail.com editor: dr altair a. del bel cury received: november 23, 2020 accepted: june 11, 2021 the influence of the cielab parameters on the perception of color after the use of whitening toothpastes natália nascimento odilon1,* , rafaela silva oliveira1, max josé pimenta lima2 , elisângela de jesus campos2 aim: to evaluate the influence of the parameters l* a* b* on the variation of the color of bovine tooth enamel submitted to artificial darkening, after simulated brushing, with whitening toothpastes containing blue covarine. methods: to undertake this study in vitro, 60 specimens (sp) were divided into 6 groups (n=10): control group (cgwater) and 5 test groups (gt1-colgate total 12, gt2-oral-b 3d white perfection, gt3colgate luminous instant white, gt4-closeup white diamond attraction, gt5-sorriso xtreme white). the specimens were darkened with coffee and submitted to simulated brushing for 6, 12, and 24 months. the alteration in the color was evaluated using cielab parameters and the ∆l, ∆a, ∆b and ∆e were calculated. the data was analyzed through generalized linear models using the r program and considering a level of significance of 5%. results: the parameters l*, a* and the ∆l, ∆a obtained better results in the test group than in the control group. there were no statistical differences between cg and the test groups for the evaluation of the b* parameter. in the evaluation of the ∆b, the gt3 differed statistically from the cg. in relation to the ∆e, all the group tests showed a variation in color statistically greater than that of the cg and the gt4 showed the greatest variation, not differing from the gt3 during the periods studied. conclusion: the mechanical and optical whitening agents positively influenced the values l*a* and b*, as well as in its variations and in the ∆e. it is important to emphasize, however, that to analyze tooth whitening it is necessary to evaluate their parameters together. keywords: color. tooth. toothpastes. bleaching agents. https://orcid.org/0000-0001-7797-9569 https://orcid.org/0000-0002-7017-1185 https://orcid.org/0000-0002-7751-0493 2 odilon et al. introduction color is a perceptual phenomenon and, for that reason, subjective which depends on the observer, lights and object. thus, with the purpose of establishing colorimetric criteria to quantify and describe this human perception, the comission internationale de l’eclairage (cie) defined color in three dimensions: luminosity or value, which is the luminosity relative to the color and encompasses the quantity of gray; the shade, the real color of the object such as green, red, blue and yellow; and chroma, which is the concentration or intensity of the shade1. cie also instituted in 1976, the tridimensional space of color (cielab) in order to provide representation to perceptions of colored stimulate2. cielab is defined numerically by three measurements: luminosity (l*) which varies between 0 to 100; the a* coordinate, the red-green axis, which varies between -80 to +80, and the b* coordinate, the yellow-blue axis, which varies between -80 to +802. the l* parameter corresponds to the luminosity, the closer it is to zero, the darker the object is and the closer to 100 the lighter it will be. positive values of b* indicate yellow tones and negative b* values indicate blue tones. and negative values of a* indicate green tones, whereas positive values indicate red tones. thus, the higher the value of l* and the lower the values of a* and b*, the tooth will be lighter and whiter1,3. since the tooth whitening occurs through the reduction of yellowing tones of the teeth, the alteration in the b* value seems to be the most important parameter to perceive the whitening, followed by the increase in the l* value and with less impact through the reduction of the value of a*. further, through the cielab system, it is possible to determine the difference between two colors through the calculation of the numerical distance between the l*a*b* coordinates, denominated as ∆e, when evaluated together1,3,4. toothpastes with a whitening function have been developed to service the aesthetic demands of having whiter teeth. the most used whitening agents in toothpaste are mechanical agents (abrasives), chemical (peroxides) and optical (blue covarine). the abrasives, such as hydrated silica, calcium carbonate and mica, mechanically remove the teeth’s extrinsic stains and are the most commonly used agents in toothpaste5. the chemical agents, such as hydrogen peroxide, oxidize the organic pigments located within the structure of the tooth, however their effects in toothpaste are considered limited due to their low concentration level and more importantly, hydrogen peroxide’s instability in aqueous environments5,6. the use of the optical agent blue covarine, on the other hand, aims to modify the spectrum of absorption and reflection of light, altering the color of the shaft of the tooth from yellow to blue, decreasing the value of b*7. blue covarine-based toothpaste has been recommended for daily use, and produces an immediate whitening action after using it, fostering a gradual effect1,8. together with the whitening agents, other agents that help to remove extrinsic stains such as anti-plaque and anti-calculus agents, surfactants and enzymes, as well as activated carbon, are also found in toothpaste5,9. since the expected tooth whitening results from the removal of extrinsic pigments and stains, as well as the removal of optical agents, the changes in the teeth’s coloration do not always correspond to the patient’s expectations. thus, it is important to 3 odilon et al. perform studies that investigate the role of each of the parameters within the cielab system in the alteration of the teeth’s color. given what has been outlined here, the objective of this study was to evaluate the influence of the l* a* b* parameters in the color variation of bovine dental enamel after simulated brushing with whitening toothpaste. the tested hypothesis evaluated was there are no difference among the groups regarding the l* a* b* parameters. materials and methods selection and preparation of the specimens to perform this study in vitro, 60 units of bovine incisors composed the specimens. initially, the teeth were affixed and adapted in a precision cut-off machine (model elsaw, elquip®, são paulo, sp, brazil) and, with the aid of a diamond wheel (model er04003 hc 4 x 0.012 x 1/2, erios® equipment, são paulo, sp, brazil) were sectioned, separating the crown from the root of the dental units. from the crowns, cuts in the oral-lingual direction were made to obtain 60 fragments (8mm x 8mm x 2 mm) which were then flattened in a milling machine (politriz metalográfica pl vo60 biopdi®, são carlos, sp, brazil) on the oral surface, with the aid of water sanding discs in a sequence of granulation (320, 400, 600, 1200), in order to make the surfaces uniform and without the exposure of the enamel. preparation of the specimens was finalized after they were fixed in centerglass® orthophtalic resin. after polishing, the specimens were put into the l-200 ultrasonic cube (schuster® ltda.) for 10 minutes to remove any possible residues of polishing. experimental groups the 60 specimens were randomized and divided into 6 groups (n=10), the control group (cg – water) and the test groups (gt1, gt2, gt3, gt4 e gt5) (table 1). the main whitening agents present in the toothpastes are described in table 2. table 1. description of the test groups with the toothpastes. groups toothpastes composition manufacturer gt1 colgate total 12 sodium fluoride 0,32%, triclosan 0,3%, sorbitol, hydrated silica, sodium laurylsulphatepvm/ma, carrageenan, sodium hydroxide, sodium saccharin, titanium dioxide, limonene. colgate-palmolive gt2 oral-b 3d white perfection sodium fluoride (1100 ppm de flúor), glycerine, hydrated silica, sodium hexametaphosphate, peg-6, tisodium phosphate, carrageenan, sodium lauryl sulphate, cocamidopropil betaine, mica (cl 77019), sodium saccharin, peg-20m, xanthum gum, titanium dioxide (cl 77891), sucralose, limonene, ci74160. procter & gamble (p&g) gt3 colgate luminous instant white hydrated silica, sorbitol, glycerine, peg-12, pentasodium triphophate, tetrapotassium pyrophosphate, sodium laurysuphate, aroma, taste, cellulose gum, cocamidopropil betaine, sodium saccharin, xanthum gum, sodium fluoride (1100 ppm of flurine), sodium hydroxide, hidroxipropilmetil cellulos, propilenglicol, polysorbate 80, mica, cl 74160 (pigmento azul),ci77891, titanium dioxide, ci 73360, cl 17200, cl 42051, eugenol. colgate-palmolive continue 4 odilon et al. darkening of the specimens after randomization, the specimens are kept immersed in soluble coffee solution (nescafé, nestlé®, brazil) at 37 °c, for 7 days, with daily changes according to the methodology proposed by munchow et al.10 (2016). to prepare the coffee, the manufacturer’s instructions were followed. abrasion test simulated brushing was performed at three intervals of time: 6 months (25,000 cycles), 12 months (50,000 cycles) and 24 months (100,000 cycles) with a brushing machine (elquip®, são paulo, sp, brazil). the “heads” of the dental brushes with soft bristles (classic clean/colgate-palmolive company® são paulo, brazil) were used for the brushing of the specimens. toothpaste solutions were prepared (1:2) of slurry of toothpaste: water according to the tao et al.11 (2017) and and submitted to ph verification (phmetro modelo 2000 quimis® aparelhos científicos ltda., são paulo, diadema, brazil). color evaluation color alteration was evaluated by the spectrophotometer easyshade vita®, which provides the readings for the cie l* a* b*. this system allows for the measurement of the difference in color between the two samples and shows the quantity of color alteration between the two readings (∆e). parameters for colors were obtained: before the beginning of the experiment, after the process of darkening and after the simulated brushing over the periods of 6, 12 and 24 months. groups toothpastes composition manufacturer gt4 close up diamond attraction sorbitol, water, hydrated silica, sodium layryl sulphate, peg-32, aroma, sodium fluoride, cellulose gum, mica, sodium saccharin, trisodium phosphate, copolymen pvm/ma. unilever gt5 sorriso xtreme white evolution hydrated silica, sorbitol, glycerine, peg-12, pentasodium triphosphate, tetrapotássio pirophosphate tetrapottasium, sodium lauryl sulphate, aroma, carboximetilcellulose, sodium fluoride, xanthum gum, sodium hydroxide, ci 74160, ci 42090. colgate-palmolive continuation table 2. principal whitening agents present in the toothpastes tested. groups toothpastes mechanical optical gti colgate total 12 hydrated silica gt2 oral-b 3d white perfection hydrated silica mica blue covarine (ci: 74160) gt3 colgate luminous instant white hydrated silica mica blue covarine (ci: 74160) gt4 close up diamond attractionpower white hydrated silica mica blue covarine (ci: 74160) gt5 sorriso xtreme white evolution hydrated silica blue covarine (ci: 74160) 5 odilon et al. from the determinations of the cielab parameters, the ∆l, ∆a, ∆b and ∆e were calculated according to the following equation12 : ∆l: (l1-l0) ∆a: (a1-a0) ∆b: (b1-b0) ∆e=[(∆l*)2+(∆a*)2+(∆b*)2]1/2 statistical analysis exploratory analysis indicated that the data did not meet the presuppositions of a parametric analysis and was analyzed through generalized linear models. the parameters l*a*b* followed the model of repeated measurements in time, but as significant differences between the groups appeared at the beginning, this time was considered as a co-variable in these models. the ∆l, ∆a, ∆b and ∆e were also calculated at different intervals of time and generalized linear models were applied, since the data did not meet the presuppositions of anova. analyses were performed in the r program, considering the level of significance of 5%. results the results were analyzed using the cielab parameters and those of ∆l, ∆a, ∆b and ∆e. in relation to the l* parameter, all the test groups showed a value of l* higher than that of the control group in all periods of time studied. after 06 and 12 months of simulated bushing, the group gt2 showed the highest values of l*, however it did not differ from gt1. after 24 months of simulated brushing, it was observed that the groups gt1 and gt2 showed higher values of l*, not differing significantly from gt3 and gt5 (table 3). table 3. median (minimum; maximum) of the variable relative to color (values l, a and b) in function of group and time. variable group time 1initial (covariable) 26 months 312 months 424 months l cg 55.15 (39.40; 69.00) 54.70 (39.50; 69.70) ad 56.60 (38.20; 70.40) ac 55.75 (37.80; 70.40) ad gt1 64.50 (32.20; 71.10) 74.95 (42.90; 76.90) ca 75.40 (45.10; 79.70) ba 78.35 (51.90; 81.90) aa gt2 66.30 (55.00; 74.70) 75.20 (60.90; 85.40) ba 77.25 (63.00; 86.30) ba 78.15 (64.60; 87.00) aa gt3 60.75 (50.90; 74.70) 74.00 (50.60; 82.80) cc 75.40 (55.60; 86.40) bab 76.90 (69.50; 86.40) aab gt4 49.05 (25.80; 70.20) 74.30 (43.70; 87.90) ab 71.00 (62.20; 79.30) ab 72.40 (68.30; 79.30) abc gt5 56.50 (44.80; 67.40) 71.45 (53.50; 75.90) cc 74.45 (58.90; 79.00) bb 75.40 (61.00; 81.50) aab a* cg 19.60 (11.40; 25.80) 17.35 (11.16; 25.80) aa 19.25 (11.20; 26.40) aa 19.40 (11.10; 26.00) aa gt1 12.15 (7.40; 24.60) 8.00 (4.10; 21.40) ae 7.05 (3.30; 24.10) ad 5.50 (2.50; 21.70) acd gt2 11.15 (4.50; 14.30) 6.20 (0.30; 9.80) aef 5.10 (-0.10; 9.80) abd 5.25 (-0.40; 8.60) bcd gt3 13.00 (8.00; 20.60) 5.95 (3.70; 13.30) af 5.80 (3.50; 13.20) bd 4.90 (1.50; 9.00) cd gt4 21.65 (9.00; 29.90) 9.00 (2.80; 24.20) ad 9.25 (5.70; 15.30) ab 7.95 (5.70; 11.20) ab gt5 16.90 (7.50; 23.20) 10.45 (4.80; 13.50) ac 8.95 (4.20; 12.70) bbc 7.45 (3.70; 11.80) ccd continue 6 odilon et al. analyzing the ∆l after 06, 12 and 24 months of simulated brushing, all the test groups showed higher ∆l than the cg. however there was no statistical difference in the three time periods studies between the test groups (table 4). variable group time 1initial (covariable) 26 months 312 months 424 months b* cg 43.10 (35.60; 48.20) 42.30 (35.30; 47.40) aa 43.15 (32.60; 47.30) aa 43.10 (33.20; 47.60) aa gt1 43.85 (29.30; 47.10) 41.05 (34.60; 44.60) aa 39.80 (34.90; 44.90) aab 38.55 (34.20; 44.60) aab gt2 43.70 (37.70; 45.60) 39.65 (30.80; 43.00) aa 38.25 (31.50; 43.10) aab 37.50 (32.50; 42.40) aab gt3 43.65 (40.50; 46.80) 38.70 (33.50; 43.90) aa 38.55 (33.00; 44.00) aab 37.00 (34.30; 41.90) aab gt4 40.10 (17.40; 44.90) 37.95 (33.60; 46.10) ba 43.50 (37.60; 51.00) aa 42.90 (36.00; 49.10) aba gt5 43.05 (40.00; 46.40) 41.20 (34.10; 46.70) aa 41.40 (34.70; 45.50) aa 41.25 (35.40; 45.10) aab 1before brushing, 2 25,000 cycles, 350,000 cycles and 4100,000 cycles. distinct letters (capital letters on the horizontal small letters on the vertical show the statistically significant differences (p≤0.05). valor l*: p(grupo)<0,0001; p(tempo)=0,0002; p(interação)=0,7009 valor a*: p(grupo)=0,0031; p(tempo)<0,0001; p(interação)=0,0049 valor b*: p(grupo)=0,041; p(tempo)=0,0004; p(interação)<0,0001 continuation table 4. median (minimum; maximum) of the variables relative to color (δl, δa and δb) in function of group for each time interval. variable group time 0 to 6 months 0 to 12 months 0 to 24 months δl cg 0.40 (-0.60; 5.50)c 1.55 (-1.20; 2.60)b 0.50 (-3.40; 2.80)b gt1 10.00 (5.80; 12.20)ab 11.05 (8.40; 14.00)a 14.90 (10.30; 19.70)a gt2 10.10 (5.10; 13.50)ab 11.55 (4.40; 14.50)a 11.65 (9.60; 17.00)a gt3 13.20 (-8.40; 20.60)ab 13.85 (-3.40; 23.90)a 16.10 (10.50; 24.00)a gt4 19.95 (8.80; 35.10)a 24.00 (0.80; 49.40)a 25.15 (1.30; 50.90)a gt5 12.65 (0.10; 24.00)ab 14.10 (5.50; 30.90)a 15.00 (7.60; 29.80)a p-value <0.0001 0.0009 0.0003 δa cg -0.07 (-7.30; 1.23)a -0.20 (-1.50; 0.90)a 0.10 (-2.50; 1.60)a gt1 -3.90 (-6.00; -3.00)ab -5.20 (-6.70; -0.50)ab -6.05 (-8.60; -2.90)ab gt2 -4.60 (-5.60; -3.00)b -4.55 (-7.50; -2.80)ab -5.30 (-7.50; -2.40)ab gt3 -5.70 (-11.00; -0.10)b -5.80 (-11.90; -1.80)b -6.90 (-12.60; -5.60)b gt4 -7.90 (-17.40; 1.20)b -10.55 (-22.20; 3.10)b -12.65 (-22.60; 2.10)b gt5 -4.55 (-11.70; -1.90)b -5.95 (-13.80; -2.90)b -6.60 (-14.90; -3.80)b p-value 0.0004 0.0007 0.0003 δb cg -0.55 (-1.70; 2.00)ab -0.10 (-3.00; 1.40)ab -0.60 (-3.50; 1.30)ab gt1 -2.80 (-3.80; 5.30)abc -3.95 (-5.20; 9.60)abc -5.80 (-7.80; 12.10)bc gt2 -3.95 (-6.90; -2.10)bc -4.65 (-7.30; -1.50)bc -4.75 (-7.10; -1.70)bc gt3 -5.05 (-7.10; -1.70)c -5.35 (-9.40; -1.60)c -6.25 (-9.00; -0.70)c gt4 0.35 (-11.30; 20.60)abc 2.95 (-2.20; 28.10)a 3.15 (-1.80; 26.80)a gt5 -1.55 (-17.80; 15.70)abc -2.20 (-18.90; 15.90)abc -1.95 (-19.00; 16.90)abc p-value 0.0004 <0.0001 <0.0001 *distinct letters on the vertical side indicate statistically significant differences (p≤0.05). 7 odilon et al. in relation to the a* parameter, all the test groups showed lower a* values than that of cg in the three periods studied. comparing the groups, after 06 months of simulated brushing, the gt3 group showed a lower value of a*, not differing significantly from gt2. after a period of 12 months, the gt2 group showed a lower value of a*, not differing statistically from gt1 and gt3. and after 24 months of simulated brushing, the gt3 showed a lower value of a*, not differing statistically from gt1, gt2 and gt5 (table 3). in relation to ∆a, over the interval of 0-6 months, all the groups showed a ∆a statistically greater than that of the control group except gt1. over the period of 0-12 months and 0-24 months, the groups gt3, gt4 and gt5 showed a ∆a statistically higher than that of cg (table 4). in relation to the b* parameter, after the periods of 06, 12, and 24 months of simulated brushing, there were no statistical differences between the cg and test groups (table 3). as to the ∆b, in the three periods of time studied, it was observed that the gt3 showed a higher ∆b and was the only group which differed statistically from the cg (table 4). in the time intervals of 0-6, 0-12 and 0-24 months, all the groups presented a ∆e significantly higher than that of the cg. the gt4 group showed a higher ∆e, not differing statistically from gt3 in the three periods of times studied and from the gt5 at the interval of 0-6 months (table 5). discussion the literature relative to the evaluation of color through the individual parameters of cielab demonstrates that the perception of dental whitening occurs with the increase of l*, decrease of b* and to a lesser degree the decrease in the a* parameter1,8. the whitening agents can be incorporated into toothpastes to be used for daily oral hygiene. in general, whitening toothpastes promote teeth whitening through the abrasive removal of the extrinsic stains on the enamel13. the abrasives have the capacity to polish the surface of the enamel, according to the format and size of the abrasive particles, resulting in surface smoothness and an increase in the luminosity of the surface of the tooth14, directly influencing the value of the l* parameter of the cielab system, which represents the luminosity of the surface. table 5. median (minimum; maximum) of δe. group time 0 to 6 months 0 to 12 months 0 to 24 months cg 1.38 (0.33; 8.19) c 2.29 (0.86; 3.31) c 2.08 (1.21; 4.89) c gt1 11.40 (7.45; 13.61) b 13.02 (10.22; 16.09) b 17.33 (12.54; 23.30) b gt2 11.82 (7.71; 15.21) b 13.43 (7.21; 15.84) b 14.05 (11.11; 19.05) b gt3 15.00 (9.14; 23.72) ab 16.06 (8.43; 27.09) ab 18.74 (13.82; 27.85) ab gt4 22.37 (11.19; 39.18) a 26.83 (7.16; 61.01) a 27.84 (5.58; 61.80) a gt5 14.91 (3.34; 26.88) ab 15.99 (8.41; 33.77) ab 16.38 (11.44; 33.16) b p-value <0.0001 0.0001 0.0001 * distinct letters on the vertical side show the statistically significant differences (p≤0.05) 8 odilon et al. in the present study, all the whitening toothpastes and for conventional use have the mechanical agent hydrated silica isolated or associated with mica, in their formulation. this suggests that the presence of abrasives results in the polishing of the surface of the specimens, increases shine, and thus justifies the greater ∆l observed in the test groups, even those brushed with conventional use toothpaste. however, different concentrations, shapes and sizes of the abrasive particles present in the toothpastes can explain the different values of luminosity in each group when evaluating the l* parameter in an isolated manner. the parameter a* considers the pivot of the red-green color, so after dental whitening there is a reduction in the value of a*. however, according to the literature, this parameter has less influence on the perception of tooth whitening3,15. in the current study, all the test groups showed lower values for a* than the control group. this finding can also be justified by the presence of mechanical agents in all the toothpastes evaluated which resulted in the removal of extrinsic stains on the enamel. studies affirm that the reduction in the b* parameter is the most important factor for perception of tooth whitening, principally when the optical whitening agent blue covarine is used1,3. in the current study, when the b* parameter was evaluated in an isolated manner, there was no difference observed between the test groups and the control group. however, in ∆b, only the gt3 differed statistically from the control group in all the intervals of time studied. analysis of the formulation of gt3’s toothpaste showed the presence of two mechanical whitening agents, hydrated silica and mica, as well as blue covarine and the blue pigment evident, which when associated, seems to result in a whitening effect3. in addition to the abrasive agents, surfactant active and anti-plaque/anti-calculus agents help in tooth whitening. the surface-active agents act by removing the hydrophobic compounds from the surface of the tooth, while the anti-plaque/anti-calculus agents prevent deposit of the chromophores on the surface of the enamel and inhibit the formation of calculus, where the formation of extrinsic stains could occur5. the gt3 group was the only one to show two surface active agents and three anti-calculus agents simultaneously in its composition. according to the literature, sodium laurylsulphate5,16 and cocamidopropil betaine16 are the surface active and the anti-calculus agents are pentasodium triphosphate5,16, tetrapotassium pyrophosphate5,16 and sodium hydroxide16.thus, the composition of the mechanical and optical whitening agents and agents which aid in the whitening present in the formulation of toothpastes of the gt3 seems to explain the results observed in this group. the ∆e represents the variation of color of the same object from those of ∆l, ∆a, ∆b calculated together12. in the current study, all the test groups showed significantly higher ∆e than the control group, and this finding could be justified by the presence of the mechanical agent hydrated silica which is present in all the whitening toothpastes and for conventional use. however, in the comparison between the test groups, only gt4 differed from gt1, the group brushed with all the whitening toothpastes and for conventional use toothpaste. such a finding can be justified by the different concentrations of whitening agents present in their formulations. however, due to the patent rights of the manufacturers, access to the detailed information about the composition of the toothpastes is limited. thus, conclusive comparisons could not be undertaken17. 9 odilon et al. the literature is controversial in relation to the influence of the cielab parameters on the perception of the color alteration when evaluated separately. some authors report that the b* parameter is the most important for tooth whitening7,18, while other authors observe greater alteration in the l* parameter after whitening19,20. further, there are studies which point out that the results of the three parameters influence in the same way for whitening21,22. however, when analyzing the results of the studies, the fact that the research was performed using different methodologies, different types of whitening materials, agents and methods should be taken into consideration, and therefore there was no consensus in the literature. in the current study, it was observed that in the gt4 group, which had the greatest variation in color, the parameters which were most prominent were the ∆l and ∆a, suggesting that these parameters had more influence on the result than ∆e. in light of this, it is possible to conclude that the simulated brushing of the bovine enamel with the whitening and conventional toothpastes promoted a whitening effect, mainly due to the presence of abrasive and mechanical agents, and that the presence of other components in the formulation seems to help in the removal of extrinsic stains. in conclusion, mechanical whitening and optical agents influence the values of l*a* and b*, as well as their variations in ∆e, after simulated brushing, resulting in improvement in the color of bovine enamel. however, it is important to emphasize that to analyze dental whitening through the cielab system, it is necessary to evaluate the parameters together. conflicts of interest there are no conflicts of interest references 1. joiner a, luo w. tooth colour and whiteness: a review. j dent. 2017 dec;67s:s3-10. doi: 10.1016/j.jdent.2017.09.006. 2. international commission on illumination. colorimetry. 4rd ed. vienna: cie; 2004. 3. tao d, sun jn, wang x, zhang q, naeeni ma, philpotts cj, et al. in vitro and clinical evaluation of optical tooth whitening toothpastes. j dent. 2017 dec;67s:s25-8. doi: 10.1016/j.jdent.2017.08.014. 4. sullivan c, pan q, westland s, ellwood r. a yellowness index for use in dentistry. j dent. 2019 dec;91:103244. doi: 10.1016/j.jdent.2019.103244.  5. epple m, meyer f, enax j. a critical review of modern concepts for teeth whitening. dent j (basel). 2019 aug 1;7(3):79. doi: 10.3390/dj7030079. 6. jurema al, claudino es, torres cr, bresciani e, caneppele tm. effect of over-the-counter whitening products associated or not with 10% carbamide peroxide on color change and microhardness: in vitro study. j contemp dent pract. 2018 apr;19(4):359-66. 7. joiner a, philpotts cj, ashcroft at, laucello m, salvaderi a. in vitro cleaning, abrasion and fluoride efficacy of a new silica based whitening toothpaste 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perez mm, pecho oe, ghinea r, pulgar r, della bona a. recent advances in color and whiteness evaluations in dentistry. cdent. 2019;1:23-9. doi: 10.2174/2542579x01666180719125137. 13. wang c, lucas r, smith aj, cooper pr. an in vitro screening assay for dental stain cleaning. bmc oral health. 2017 jan;17(1):37. doi: 10.1186/s12903-016-0328-3. 14. lippert f. an introduction to toothpaste its purpose, history and ingredients. monogr oral sci. 2013;23:1-14. doi: 10.1159/000350456. 15. shamel m, al-ankily mm, bakr mm. influence of different types of whitening tooth pastes on the tooth color, enamel surface roughness and enamel morphology of human teeth. f1000res. 2019 oct 16;8:1764. doi: 10.12688/f1000research.20811.1. 16. vertuan m, de souza bm, machado pf, mosquim v, magalhães ac. the effect of commercial whitening toothpastes on erosive dentin wear in vitro. arch oral biol. 2020 jan;109:104580. doi: 10.1016/j.archoralbio.2019.104580. 17. odilon nn, lima mjp, ribeiro pl, araújo rpc, 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[in vitro evaluation of the effect of bleaching dentifrices containing blue covarine on bovine dental enamel]. rev odontol unesp. 2018;47(6):388-94. portuguese. doi:10.1590/1807-2577.1211. 18. chang jy, chen wc, huang tk, wang jc, fu ps, chen jh, et al. evaluating the accuracy of tooth color measurement by combining the munsell color system and dental colorimeter. kaohsiung j med sci. 2012 sep;28(9):490-4. doi: 10.1016/j.kjms.2012.04.006. 19. carlos nr, pinto a, do amaral f, frança f, turssi cp, basting rt. influence of staining solutions on color change and enamel surface properties during at-home and in-office dental bleaching: an in situ study. oper dent. 2019 nov/dec;44(6):595-608. doi: 10.2341/18-236-c. 20. bergesch v, henrique f, aguiar b, turssi cp, mantovani f, frança g, et al. shade changing effectiveness of plasdone and blue covarine based whitening toothpaste on teeth stained with chlorhexidine and black tea. eur j dent. 2017;11(4):432-7. doi: 10.4103/ejd.ejd_97_17. 21. malekipour m, norouzi z, shahlaei s. effect of remineralizing agents on tooth color after home bleaching. front dent. 2019 may-jun;16(3):158-65. doi: 10.18502/fid.v16i3.1586. 22. pirolo r, mondelli rf, correr gm, gonzaga cc, furuse ay. effect of coffee and a cola-based soft drink on the color stability of bleached bovine incisors considering the time elapsed after bleaching. j appl oral sci. 2014 nov-dec;22(6):534-40. doi: 10.1590/1678-775720130578. https://www.researchgate.net/deref/http%3a%2f%2fdx.doi.org%2f10.2174%2f2542579x01666180719125137 https://doi.org/10.1016/j.kjms.2012.04.006 1http://dx.doi.org/10.20396/bjos.v18i0.8657249 volume 18 2019 e191430 original article 1 dharamshila narayana hospital, new delhi 2 department of oral & maxillofacial pathology, maulana azad institute of dental sciences, new delhi, india 3 department of microbiology, maulana azad medical college, new delhi, india corresponding author: dr. garima rawat, m.d.s. oral & maxillofacial pathology, dharamshila narayana hospital, new delhi email idgarima3103@gmail.com phone number-+91-9873776634 received: october 22, 2018 accepted: june 13, 2019 dna damage in buccal cells in oral pmds and malignant disorders by comet assay: a comparison with blood leukocytes garima rawat1, aadithya b urs2, anita chakravarti3, priya kumar2,* aim: dna damage associated with oral squamous cell carcinoma (oscc) and potentially malignant disorders (pmds) is produced due to carcinogenic agents or increased oxidative stress. comet assay can assist in early detection and evaluation of the amount of dna damage; lymphocytesare the most commonly used cells for performing comet assay. utilisation of buccal epithelial cells in comet assay can be a minimally invasive and rapid method. the present study compared the efficacy of comet assay in assessing dna damage in buccal cells over peripheral blood leucocytes (pbls) in oral potentially malignant and malignant disorders. methods: the study included fifty five patients each of leukoplakia, oral submucous fibrosis (osmf) and oscc along with fifty five healthy individuals as control. buccal epithelial cells were collected from all the selected subjects. dna damage was evaluated bymeasuring the mean tail length (µm). results: a significantly increased mean tail length (µm) and higher dna damage were found in oscc (26.1096 + 1.84355) and there was a progressive stepwise increase in mean tail length from control (8.4982 + 0.93307) to pmd [leukoplakia (14.6105 + 0.71857); osmf (12.5009 + 1.12694)] to oscc.the mean tail length in different habit groups was greater than controls, though no significant difference was noted between habit groups. the mean tail length of buccal cells was significantly greater than the mean tail length of pbls in all study groups and controls. conclusion: hence, use of comet assay on buccal epithelial cells can prove to be beneficiary for evaluation of dna damage. keywords: comet assay. dna damage. epithelial cells. leukocytes. mouth neoplasms. 2 rawat et al. introduction the most common potentially malignant disorders (pmds) affecting the oral cavity include leukoplakia, erythroplakia and oral submucous fibrosis (osmf)1,2. 30-80% of the oral malignancies arise from pmds like leukoplakia and osmf. malignant transformation rates for leukoplakia and osmf range from 0.13-17.5% and 2.3-7.6% respectively3. oral squamous cell carcinoma (oscc) is the most common malignant disorder of the oral cavity, accounting for over 90% of all malignant neoplasms4-6. etiopathogenesis of oscc is multifactorial including tobacco either smokeless or smoked with or without alcohol which has synergistic role, some viruses, genetic and epigenetic factors and also, gene-environmentinteractions7. the oral deleterious habits stated earlier lead to oxidative stress or reactive oxygen species (ros) generation which in turn produces damage of dna at cellular level. these factors cause base alteration promoting break in the dna helix, these double-strand breaks (dsbs) are lethal to cells and lead to loss of genetic information. dna damage incurred may promote genomic instability and aid the development of disease, including cancer8-10. for evaluation of dna damage various methods known as genotoxicity assays are known such as single cell gel electrophoresis (scge) or comet assay and micronucleus assay (mn assay). comet assay (scge) is most widely used as an in vitro and in vivo genotoxicity test9,11. comet assay was first developed in 1984 by ostling and johansson and later in 1988, it was modified by singh et al. it is commonly utilised for quantification and analysis of dna damage in individual cells and depends on partial unwinding of the supercoiled dna inagarose-coatedslides. this allows the dna to be attracted towards the anode underelectrophoresis, forming ‘comet-like’ images as seen underfluorescence microscopy. the relative amount of dna inthe comet tail indicates dna break frequency12-15. a cell with dna damage appears as a ‘comet’ and undamaged cell appears as a ‘halo’. the head of the comet is composed of intact dna while the tail is composed of damaged dna. the length of the comet tail formed is directly proportional to the amount of dna damage present in that cell16. any eukaryotic cell type that can be obtained as a single cell or nuclear suspension assay can be used to perform this assay13. amongst eukaryotic cells, lymphocytes are the most commonly used cells. using buccal cells in comet assay to assess dna damage can be a minimally invasive and rapid method11. the aim of the present study was to evaluate the efficacy of comet assay in assessing dna damage in buccal epithelial cells in oral potentially malignant disorders and oscc and comparing the findings with those of peripheral blood leucocytes (pbls). materials and methods ethics this study was conducted in the department of oral pathology and microbiology, maulana azad institute of dental sciences, new delhi, india. the study was approved by the institutional ethical committee board. 3 rawat et al. study design this prospective study was designed to evaluate the efficacy of comet assay in buccal mucosal cells over pbls in assessing dna damage in oral potentially malignant and malignant disorders. part i of this project evaluated the efficacy of comet assay in assessing dna damage in peripheral blood leukocytes (pbls) in oral potentially malignant and malignant disorders. subjects of study clinically and histopathologically confirmed patients with leukoplakia, osmf and oscc in the age range from 18 years to 80 years were included in the study. the study sample comprised of 220 patients including leukoplakia(group b; n=55), oral submucous fibrosis (group c; n=55), oral squamous cell carcinoma (group d; n=55) along with healthy age and sex matched individuals without any habit history (control, group a; n=55) were included in the study. the patients with habits were categorised into three habit groups: • smokeless group (usage of tobacco, gutkha, pan or supari) • smoked group (usage cigarette or bidi) • mixed group(usage of both smoked and smokeless forms) patients not willing to participate, suffering from any infectious or contagious disease, with any other white patch such as candidiasis, oral lichen planus and lichenoid reaction and previous history of surgery, radiotherapy or chemotherapy, or any vitamin, or dietary supplement use were not included in the study. sample collection buccal mucosal cellsexfoliated buccal cells were collected using cytobrush from patients (site of lesion) and controls (buccal mucosa). buccal cells were stored in rpmi-1640 medium at -80º c untilprocessed. preparation of buffers lysis buffer: ingredients per 1000 ml were 2.5 m nacl 146.1 gm, 100 mm edta 37.2 gm, 10 mm trizma base -1.2 gm, 1% triton x100 10 ml, 10% dmso 100 ml. ingredients were added to 700 ml dh2o, and the mixture was stirred to dissolve the constituents. the ph was adjusted to 10.0 using concentrated (1n) hcl or naoh. thetriton x-100 and dmso were added and the final volume was made to 1 litre with dh2o. the buffer was stored at room temperature. alkaline buffer: ph > 13: ingredients per 1000 ml were 300 mm naoh 12 gm and 01 mm edta – 0.3 gm neutralising buffer: ph=7.5: ingredients per 1000 ml were 0.4 m tris 48.45 gm. this was added to ~800 ml dh2o, ph was adjusted to 7.5 with concentrated 4 rawat et al. (1n) hcl and the final volume was made to 1 litre. the buffer was stored at room temperature. trypsin solution: ingredients per 1000 ml were 0.25% trypsin and 1 mm edta in pbs 0.03 gm. staining solution: ethidium bromide dye (sigma aldrich): 2 mg is dissolved in 100 ml of distilled water and used for staining. procedure for comet assay in buccal cells buccal epithelial cells were collected from patients using a cytobrush gently from the buccal mucosa or the lesion (in study groups). the brush was then swirled into an eppendorf tube containing rpmi-1640 medium and centrifuged at 2000 rpm for 10 min. the supernatant was removed and 300µl of trypsin solution was added to the buccal cells and incubated for 30 min at 37°c. then the cells werecentrifuged and the supernatant was discarded. the cells werethen washed thrice by centrifugation at 2000 rpm for 10 min in cold pbs. about 40µl of cell suspension and 60µl of 0.5% lmpa were mixed and placed on frosted slides previously coated with 1% nmpa. to the solidified agarose, a third layer of 1% lmpa was applied and the slides were dipped in cold lysis buffer and refrigerated for 24 hours. following lysis, the slides were placed in the cold alkaline buffer (ph > 13) for 20 min to unwind dna strands and expose the alkali labile sites als (alkali unwinding). then the slides were subjected to electrophoresis under alkaline conditions (ph >13) at 300ma and 15v for 25 min. after this, the same procedureas used for pbls was followed. statistical analysis the mean values, standard deviation and ranges (maximum and minimum) were calculated for each variable. the resulting data was analyzed using spss software, version 20 (armonk, ny: ibm corp). data was expressed as mean ± standard deviation. differences between different variables were analyzed using parametric student t-test and analysis of variance (anova). correlation was calculated using the pearson’s correlation. a p value ≤ 0.05 was considered to be statistically significant. results the results for mean tail length of pbls in different study groups and habit groups were obtained from the previous part of this study and then these were compared with the results of buccal epithelial cells. distribution of patients and controls according to age, gender and habit was studied. [table 1] comet assay was performed on peripheral blood leucocytes of all the subjects. the dna damage (mean tail length) in the buccal cells in leukoplakia, osmf, oscc and control groups were assessed and results of each are illustrated in figures 1, 2, 3 and 4 respectively. the mean tail length (µm) buccal cells in leukoplakia, osmf and oscc were compared with controls. [table 2] 5 rawat et al. comparison of mean tail length of pbls and buccal cells in different study groups was done using anova. [table 3] the mean tail length (µm) of buccal cells was significantly more than that of pbls in all study groups compared to controls. comparison of mean tail length of buccal cells between different habit groups and control was done using student ttest. [table 4] though the mean tail length was table 1. table showing demographic data of patients included in the study group no of patients age gender (m:f) sample collected habit group a (control) 55 15-76 41:14 both buccal cells and pbls group b (leukoplakia) 55 20-70 50:5 both buccal cells and pbls mixed 14 smoked 14 smokeless 27 group c(osmf) 55 18-73 39:16 both buccal cells and pbls mixed 10 smokeless 45 group d(oscc) 55 28-80 44:11 both buccal cells and pbls mixed 16 smoked 5 smokeless 34 figure 1. non-fragmented and undamaged dna in buccal cell samples of control. (insetmagnified view of single cell without dna damage) 1 6 rawat et al. figure 2. dna damage in the form of comet in buccal cell samples of leukoplakia. (insetmagnified view of single comet) 2 figure 3. dna damage in buccal cell samples of osmf. (insetmagnified view of single cell with dna damage) 3 7 rawat et al. figure 4. dna damage in the form of comet in buccal cell samples of oscc. (insetmagnified view of single comet with tail) 4 table 2. comparison of mean tail length of buccal cells between different study groups control study groups mean tail length (µm) (mean + standard deviation) p-value group a (8.4982 + 0.93307 ) group b (14.6105 + 0.71857) 0.000 p value < 0.05 is considered as statistically significant group c (12.5009 + 1.12694) 0.000 group d (26.1096 + 1.84355) 0.000 on comparing the mean tail length of buccal cells by student t-test between various study groups, it was found to be highest for oscc followed by leukoplakia and osmf compared to the control. highly significant difference was obtained between the study groups and control. table 3. table showing p-values obtained by comparison of mean tail length of pbls and buccal cells in different study groups study groups buccal cells p-value pbls group a 0.000 p value < 0.05 is considered as statistically significant group b 0.000 group c 0.000 group d 0.000 on comparing the mean tail length of peripheral blood leucocytes and buccal cells of various study groups, the mean tail length of buccal cells was significantly more than that of pbls in every study group. 8 rawat et al. increased in the patients with habits, there was no significant difference between the various habit groups. discussion prolonged exposure to carcinogenic agents induces oxidative stress or reactive oxygen species (ros) generation that isgenotoxic and cytotoxic to human cells and causes damage leading to genetic alterations. accumulation of these genetic alterations may initiate development of premalignant disorders and subsequently oscc8. when the amount of ros generated in the cells is significantlyhigh, it leads to cellular damage, as well as dnadamage8,9. the ros also affect the dna repair mechanisms essential for maintenance of dna integrity and prevention of cancer8. thus, the progression of oscc from potentially malignant disorders is a multistep process3,5. the common potentially malignant disorders like leukoplakia and oral submucous fibrosis (osmf) have malignant transformation rates in the range of 0.13-17.5% and 2.3-7.6% respectively3. the comet assay or single cell gel electrophoresis (scge) is a well known assay in assessing the dna damage attributable to its rapidity, sensitivity, inexpensiveness and requirementof little biologicalmaterial17-20. the most commonly used cells in such assays are peripheral blood lymphocytes (pbls) and there is a need to replace or complement lymphocytes with some other cell type. the age of patients in the study ranged from 18 years to 80 years with the mean age for controls, leukoplakia, osmf and oscc being 36.33 years, 43.54 years, 38.95 years and 49.05 years respectively. 79 % of the patients included in the study were males, these findings were comparable to other studies3,7. amongst the patients, apredominance of smokeless tobacco usage (48.2%) was observed, while mixed tobacco (both smokeless and smoked) and smoked form accounting for 18.2% and 8.6% of the patients respectively. in the buccal cells, the mean tail length (µm) was found to be significantly increased in oscc (26.1096 + 1.84355μm), leukoplakia (14.6105+ 0.71857μm) and osmf (12.5009 + 1.12694μm) compared to controls (8.4982 + 0.93307μm). the comet tail lengths were found to be greater in all study groups compared to controls, which symbolises the presence of increased dna damage in the buccal cells of these table 4. comparison of mean tail length of buccal cells between different habit groups and control control habit groups mean tail lengths (µm) (mean + standard deviation) p-value group a (8.4982 + 0.93307) smokeless (17.3530 + 6.05758) 0.000 p value < 0.05 is considered as statistically significant smoked (17.6421 + 5.55230) 0.000 mixed (18.8135 + 6.62379) 0.000 on comparing the mean tail lengths of buccal cells by student t-test between various habit groups, the tail lengths were significantly higher in the habit groups compared to control. 9 rawat et al. patients. the amount of dna damage was most in oscc patients as this group had the greatest mean tail length followed by leukoplakia and osmf. in malignancies, there is loss of genomic stability or increased genomic instability, which can be either inherent or induced by external agents. the basal dna damage that is observed in normal healthy individuals due to exposure to risk factors also plays an important role in carcinogenesis as well as in progression of the disease. the level of dna instability of an individual at baseline level is critical in cancer predisposition and progression. thus, an increase in mean tail length which is observed from healthy individuals to precancer to cancer may be due to progressive increase in genomic instability21,22. udupa et al.23 reported that the mean tail length of buccal cells in osmf group (12.92 ± 0.90 μm) was significantly higher (p< 0.05) compared to the healthy group (8.34 ± 0.36 μm), indicating osmf patients had increased dna damage. in osmf patients, the buccal epithelial cells are in direct contact with the deleterious effect of betel quid and gutkha containing areca nut in comparison to other surrogate cells. the generation of ros by the aqueous extract of arecanut leads to the genotoxic damage in buccal epithelial cells, therefore, accounting for increased tail length and dna damage24. studies using alternative epithelial cells to assess dna damage in other types of cancers of the body have been published in literature. increased mean basal dna damage was discerned by udumudi et al.25 in epithelial cells of patients with cervical cancer and cervical dysplasia compared to controls. the authors of the present study in part 1 of this project had evaluated dna damage in the peripheral blood leucocytes (pbls) using comet assay in the same group of patients. these authors found that the mean tail length (µm) was significantly increased in oscc (22.4335 + 1.52341), leukoplakia (13.0022 + 0.74316) and osmf (10.6085 + 0.88140) compared to controls (6.8307 + 0.84261 µm). the increased comet tail lengths in all study groups compared to controls depicts presence of dna damage in the pbls of these patients26,27. the generation of ros and exposure to genotoxins causes dna breaks, reduced dna repair capacity and oxidation of purines or pyrimidines. these genotoxins attack different sites on the dna leading to the accumulation of dna damage which increases the risk of cancer18,28-29. they also observed that the amount of dna damage was greatest in oscc patients as this group had the maximum mean tail length followed by leukoplakia and osmf. there was significant stepwise increase in dna damage in the pbls from control to pre-cancer patients and from pre-cancer to oral cancer patients26. cancer patients have maximum dna damage as depicted by greatest mean tail length of comet in lymphocytes. this has been observed in cancers other than those of the oral cavity25,29-32. on comparing the results of the present study and the study done using pbls by the current authors, it was found that the mean tail length of buccal cells was significantly more than pbls in all study groups. the tail length of leukoplakia was closer to oscc compared to osmf suggesting that leukoplakia has severe dna damage, which can be directly correlated with its high possibility to undergo malignant transformation3. 10 rawat et al. katarkar et al.3 and mukherjee et al.7 in their respective studies also obtained significantly higher tail length in oscc and leukoplakia compared to osmf. a thorough search of literature revealed that there is no study published till date comparing the mean tail length of pbls and buccal cells using only comet assay in oral pmds and oscc. we hypothesize that the buccal epithelial cells come into direct contact with the mutagenic agents for prolonged duration as all the lesions of the study groups are habit associated. these cells are sensitive to dna damage because of direct exposure to the carcinogen. also, the buccal cells are short-lived cells (with renewal of 10–14 days) due to their continued renewal as compared to pbls which are considered to be longer living cells. hence, the presence of buccal cells with comet-like appearances is indicative of recent exposure to genotoxic agents. this may explain the increased mean tail length and higher levels of dna damage that were observed in buccal cells compared to pbls. thus, comet assay of buccal cells can be a more sensitive biomarker to assess early damage in target tissue in comparison to pbls. tobacco smoking and smokeless tobacco are important etiologic factors leading to oral cancer. these products are composed of carcinogens such as polyaromatic hydrocarbons nitrosamines and aromatic amines. these carcinogenic agents after deactivation in the liver are converted into electrophilic intermediates which in turn react with dna to form covalently bound adducts. the formation of dna adducts and the resulting mutations are responsible for oncogene activation and inactivation of tumor suppressor genes, leading to cancer. few authors have reported presence of these dna adducts in smokers28,33-34. in the current study, the mean tail length of buccal cells in different habit groups when compared with the control group of no habit showed highly significant results. the results of our study are comparable to those of jyoti et al.8 wherein gutkha chewers, gutkha chewers along with smoking, pan masala chewers, pan masala chewers along with smoking, and smokers had significantly increased (p < 0.05) tail length in buccal epithelial cells compared to the control group. in the current study, no statistically significant difference was obtained in the tail length between different habit groups in both pbls and buccal cells. udupa et al. also observed no statistically significant difference between the different types of habits and tail length of buccal cells23, though the tail lengths were increased compared to no habit group as seen in our study. thus, the increased tail length is associated with deleterious oral habits. jyoti et al.8 observed that highest tail length of buccal cells was among gutkha chewers along with smoking (36.9 ± 3.60 µm) amongst all groups which was not observed in our study. these contrasting results may be due to non standardisation of duration and frequency of the habit in our study. katarkar et al.3 also showed that the patients with multiple habits had significantly increased (p< 0.0001) tail length compared to patients with single habit. similar results were obtained by rawat et al.26 on comparing the mean tail length of pbls in different habit groups with the control group with no habit and obtained 11 rawat et al. highly significant results. similar results have been stated by other authors also28. many other investigators have investigated the effect of habit on mean tail length and have obtained comparable results11,35. however, hoffmann et al.36 showed no significant difference in dna damage between smokers and non-smokers. these authors concluded that cigarette smoking had no effect on the amount of dna damage in peripheral blood cells. no correlation between the length of thecomet and the number of cigarettes or the frequency of smoking was detected by frenzilli et al.37 and mohankumar et al.33. these findings can be explained by assuming that the single dna strand breaks can be induced in leucocytes also by free radicals generated due to the inflammation normally present in smokers. this reaction is independent of the amount of cigarette smoked and is related to individual susceptibility. the dna single strand breaks (ssb) induced by agents like hydrogen peroxide are quickly repaired16,34. in the current study, we obtained a positive correlation between the mean tail length of pbls and buccal cells in controls and the study groups as well as in the habit groups. also, oscc and other pmds are epithelial in origin and hence, detection of dna damage in this cell type can prove to be beneficial. this could be because the buccal epithelial cells are the first cells that come into direct contact with the carcinogenic agents and hence display maximum dna damage. thus, reflecting that buccal cells are more sensitive, non-invasive and effective indicators for evaluating dna damage in potentially malignant and malignant disorders when compared to pbls. hence, with the present study we advocate the utilization of buccal cells for evaluation of genotoxicity during the earlier stages of carcinogenesis and should be considered as a replacement or alternative to lymphocytes. compliance with ethical standards: no funding conflict of interest: no conflict exists ethical approval: all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 helsinki declaration and its later amendments or 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post labeling. cancer res. 1993 apr 1;53(7):1522-8. 35. dhawan a, mathur n, seth pk. the effect of smoking and eating habits on dna damage in indian population as measured in the comet assay. mutat res. 2001 mar 1;474(1-2):121-8. 36. hoffmann h, speit g. assessment of dna damage in peripheral blood of heavy smokers with the comet assay and the micronucleus test. mutat res. 2005 mar 7;581(1-2):105-14. 37. frenzilli g, betti c, davini t, desideri m, fornai e, giannessi l et al. evaluation of dna damage in leukocytes of ex-smokers by single cell gel electrophoresis. mutat res. 1997 apr 29;375(2):117-23. 1http://dx.doi.org/10.20396/bjos.v20i00.8661443 volume 20 2021 e211443 original article 1 associate prof, omfs, dow international dental college, dow university of health sciences, karachi pakistan. 2 mds trainee, dr ishrat ul ebad khan institute of oral and health sciences, dow university of health sciences, karachi pakistan. 3 associate professor omfs, dow international dental college, dow university of health sciences, karachi pakistan. 4 mds trainee, dr ishrat ul ebad khan institute of oral and health science, dow university of health sciences, karachi pakistan. 5 assistant prof, dr ishrat ul ebad khan institute of oral and health science, dow university of health sciences, karachi pakistan. 6 assistant prof, baqai medical university karachi. 7 fdsrcs, principal dikiohs, duhs dr ishrat ul ebad khan institute of oral and health science, dow university of health sciences, karachi pakistan. corresponding author: shaheen ahmed drshaheenahm@gmail.com address: dow international dental college, chanesar, goth mehmoodabad defence campus, dow university of health sciences received: september 30, 2020 accepted: december 9, 2020 the retromandibular transparotid approach vs. retromandibular retroparotid approach for the mandibular condyle: our clinical experience shaheen ahmed1,*, reema usmani2, abdul hafeez shaikh3, usman ashraf4, syeda noureen iqbal5, abdullah salman6, anwar ali7 aim: the mandible is regarded as a frequently fractured bone in patients who present with maxillofacial trauma accounting for almost 15.5% to 59% of all facial fractures. managing condylar trauma has remained to be a point of contention amongst experts, regardless of the advances in surgical modalities and methodologies, and the treatment plan is often determined by the preference and the experience of the surgeon. there exist various approaches in the literature, each with its own specific benefits and drawbacks. with this study, we aimed to evaluate the prevalence of post-operative complications in patients who experienced orif by means of the retromandibular approach, by comparing the outcomes of one group having undergone transparotid surgery, with another that underwent retroparotid surgery. methods: an experimental trial was undertaken. convenience sampling was done from among the cases of condylar neck and base fracture visiting the department of omfs, dow university of health sciences from january 2017 to december 2019. an overall 26 patients were divided into 2 groups of 13 members each; one was managed using open reduction internal fixation (orif) by means of a retromandibular transparotid approach while the other group was treated with orif by means of a retromandibular retroparotid approach. a 6 month follow-up was done to assess range of active motion, occlusion, and complications such as deviation/deflection, neural injury, infections, sialocele, salivary fistulae and frey’s syndrome in both groups. results: there was no statistically significant difference between the two groups in terms of inter-incisal opening, right and left lateral movements, or protrusion. one patient in the retroparotid group had deviation on mouth opening (7.69%), while one in the transparotid group reported with infection (7.69%), and 2 developed post operative seromas (15.38%). none had persisting facial nerve palsy at 6 months. conclusion: we find no significant disparity between the 2 approaches at a follow-up of 6 months; therefore, the primary determining factor for selection of either technique is surgeon preference and appropriate case selection. keywords: mandibular fractures. mandibular condyle. oral surgical procedures. mailto:drshaheenahm@gmail.com 2 ahmed et al. introduction the mandible is a frequently fractured bone in maxillofacial trauma, accounting for almost 15.5% to 59% of all facial fractures. the condyle accounts for 20% to 43% of all mandibular fractures. condylar fractures are intricate due to the articulation of the mandible with the temporal bone (squamous part). fractured condyles as well as the protracted immobilization employed as the conventional management of the aforementioned fractures, may result in disturbed occlusion, internal derangement, ankylosis, and alterations in the growth pattern of the mandible. patient may present with a myriad of complaints, ranging from chronic pain, restricted jaw mobility and compromised functionality to facial asymmetry, and traumatic occlusion. in fact, 13 – 100% of all tmj ankylosis cases are a direct sequelae of condylar trauma1, specially trauma that results in medially displaced condylar fractures2. the management of condylar trauma has remained a point of contention amongst experts, regardless of the advances in surgical modalities and methodologies, and the treatment plan is often determined by the surgeon’s preference and experience3. owing to the composite anatomical site, surgical approaches in this area should provide good visualization and allow the surgeon to precisely reduce the fracture and provide stable internal fixation4. various approaches have been employed for this purpose in the past, each with its own specific benefits and drawbacks. the retromandibular approach was initially defined by hinds and girotti in 1967; its outstanding benefit is the provision of forthright access to low condylar fractures5. however, certain complications are linked to hinds’ approach as well: infection, neurological deficits, seromas, sialoceles, salivary fistulae, dupuy’s syndrome etc.6 several modifications of the retromandibular approach have emerged over time to counteract these complications, ranging from anteroparotid to a high cervical transmasseteric approach7. with this study, we aimed to evaluate the prevalence of post-operative complications in patients who underwent open reduction and internal fixation (orif) of condylar neck and base fractures by means of the retromandibular approach, by comparing the outcomes of one group having undergone transparotid surgery, with another that underwent retroparotid surgery. materials and methods an experimental trial was undertaken; ethics approval was acquired from the university institutional review board (ref: irb-1130/duhs/approval/2019). the study sample was acquired according to convenience sampling from among the cases visiting the department of oral and maxillofacial surgery (omfs), dow university of health sciences, from january 2017 to december 2019. after obtaining informed consent, patients were counselled regarding the management options available, as well as the disadvantages and advantages associated with each option. this communication was done in a clear and coherent manner. an overall 26 patients were selected for orif, of whom 22 were males and 4 were females. patients were placed into 2 groups of 13 members each; one group was managed using orif by means of a retromandibular transparotid approach which has recently been well described by parihar et al.3, while the other group was treated with orif by means of the retromandibular retroparotid approach described by ebenezer et al in 20148. 3 ahmed et al. after inclusion into our trial, all of the patients had a standard panoramic radiograph (opg), a plain lateral and posteroanterior radiograph, or computed tomography (ct) of the face done in order to classify the condylar fracture according to the arbeitsgemeinschaft für osteosynthese cranio-maxillo-facial (aocmf) classification into head, neck and base, as well as to assess its severity, and the amount of dislocation and displacement of the condylar segment9. pre operatively, the nerve function was also assessed in accordance with house & brackmann’s grading system. the inter-incisal mouth opening and maximum lateral excursion was noted in millimeters (mm). the preoperative evaluation was done by the same examiner for all patients. all patients were operated by the same surgical team which included two professors, who were the primary surgeons, assisted by two residents. functionally stable osteosynthesis was applied via two miniplates in accordance with meyer’s tensile strain lines of the mandibular condyle in all cases10. inclusion criteria 1. age above 18 years 2. condylar neck and base fractures (unilateral/bilateral both) 3. no fragmentation/ minor fragmentation 4. all amounts and directions of displacement 5. 0-45 degrees of angulation, medially or laterally, as seen on a pa view or anterioposteriorly, as seen on a lateral view. 6. undisplaced or displaced fractures (condylar head in relation to fossa) exclusion criteria 1. inadequate dentition for reproduction of occlusion 2. condylar head fractures 3. major fragmentation 4. >45 degrees of angulation in any direction 5. dislocated fractures (condylar head in relation to fossa) 6. existing facial nerve deficits 7. previous tmj surgeries 8. sjogren syndrome or other coexisting autoimmune disorder 9. preexisting diseases of the parotid follow up protocol post operatively, the patients were assessed by one resident and one professor from the surgical team at one week, one month, three months and six months to assess: 4 ahmed et al. 1. range of active motion by measuring the maximum inter-incisal distance, maximum protrusion, and lateral movements. 2. deviation/deflection on opening the mouth (clinical assessment) 3. imperfect occlusion (clinical assessment) 4. presence of neural injury (cn vii) using house-brackmann facial nerve grading system 5. infections 6. seromas 7. sialocele and salivary fistulae 8. frey’s syndrome 9. unaesthetic scars statistical analysis all statistical analyses were done using spss statistics for windows, version 23 (spss inc.). probabilities of <0.05 were considered to be significant. independent sample t test was applied to compare the means of the two groups and a consort 2010 flow chart of the trial is also shown in figure 1. results a total of 10 males and 3 females were included in the transparotid (tp) group and 12 males and 1 female were included in the retroparotid (rp) group. the mean age of the tp group was 32.6 while that of the rp group was 37.5 years. the tp group included 10 fractures of the right condyle, 5 of the left (2 were bilateral cases); of these 7 patients had neck fractures and 4 had condylar base fractures and the bilateral cases had a neck and base, and neck and neck fractures on the right and left side respectively. 11 fractures were displaced in relation to the fossa, 8 were nonangulated and 7 were anteromedially angulated. in the rp group, 9 fractures were found on the right condyle, 5 on the left (1 was a bilateral case); of these 6 patients had neck fractures while 6 had condylar base fractures and the bilateral case had a condylar neck fracture on the right and base fracture on the left. 10 fractures were displaced in relation to the fossa, 9 were nonangulated and 5 were anteromedially angulated. there were no laterally or posteriorly angulated fractures in our study, incidentally. the mean operative time in the tp group was 29.33 minutes while that of the rp group was 30.13 minutes. no significant intraoperative complication was encountered in any case of either group. facial nerve dysfunction at one-week post op was encountered in 3 cases from the tp group; 2 involving the buccal branch (house brackmann grade iii and iv), and 1 involving the marginal mandibular branch (house brackmann grade iii). 2 cases of nerve dysfunction at one week post op were also found in the rp group; 1 involving the buccal branch (house brackmann grade iii) and 1 involving the marginal mandibular branch (house brackmann grade iv). all of these dysfunctions related to the nerve were transient and had resolved by the 6 month follow-up appointment. 5 ahmed et al. no evident malocclusion was found in patients in either group. however, one patient from the rp group complained of premature contacts on the ipsilateral side that were resolved by selective occlusal adjustment. no patient had any malocclusion at 6 months post op. of the overall complications seen, the cases in the tp group had a higher incidence of infection (7.69%) and seroma (15.38%) while those in the rp group had more deviation on mouth opening (7.69%). the 6 month post-operative mobility was similar in both groups; there was no statistically significant difference in the interincisal opening, right and left lateral and protrusive movements in either group (table 1). figure 1. consort flow diagram of the study assessed for eligibility (n=30) randomized (n=26) allocation follow up analysis allocated to intervention (n=13) lost to follow up (n=0) analyzed (n=13) allocated to intervention (n=13) lost to follow up (n=0) analyzed (n=13) excluded (n=4) • not meeting inclusion criteria (n=1) • declined to participate (n=3) • other reasons (n=0) enrollment table 1. 6 month postoperative mobility in both study groups (in millimeters) variables study groups mean p value inter incisal opening transparotid 34.02 0.928 retroparotid 35.6 left lateral movement transparotid 5.17 0.427 retroparotid 5.04 right lateral movement transparotid 5.38 0.47 retroparotid 5.36 protrusion transparotid 5.33 0.577 retroparotid 6.0 6 ahmed et al. discussion the clinical relevance of condylar fractures is well known in terms of their effect on the temporomandibular joint mobility, occlusion, facial symmetry, function and esthetics, and a long debate existed concerning the pros and cons of open versus closed management of the condyle. traditionally, fractures of the mandibular condyle, ranging in frequency from 14% to 30%9,11 have almost wholly been addressed by closed or “conservative approaches” such as mmf, due to complications attributed to open approaches such as limited access, danger to the facial nerve branches, and to anatomic structures such as the parotid12. lately however, there has been a paradigm shift towards the consideration of open approaches to condylar fractures as the gold standard of treatment13, owing in part to the advent of functionally stable osteosynthesis in the 1980s and to the multitude of benefits associated with early mobilization and loading of the temporomandibular joint. while open reduction and internal fixation of condylar fractures is gaining traction in the omfs world, there is yet to be a standardized protocol of approaches to the condyle14. this study was undertaken in order to examine two different approaches to the condylar neck and base, the transparotid and retroparotid modifications of the retromandibular approach, in order to establish which of the two had a more favorable outcome in terms of function, aesthetics and lower post-surgical complications. the majority of patients in our study were male, with a male to female ratio of 5.5:1, a circumstance reported by other studies as well15,16, owing largely to the higher incidence of rtas and interpersonal violence in pakistan. the right condyle was more frequently fractured than the left side, and the majority of fractures in our study were either not angulated or anteromedially angulated, a substantiation of findings by lindahl17 who noted that adults with condylar fractures tend to have medially angulated proximal segments. all the participants of our study were in the 4th decade of life. no incidence of permanent 7th cranial nerve paresis was seen in either group in our study; our results reflect those of other studies that state the transient palsy of the facial nerve in the range of 13 to 22% in both approaches collectively18, with permanent paralysis limited to 5% or less19. some studies, however, have reported a higher incidence of transient nerve dysfunction after the transparotid approach as compared to a non-transparotid approach, with the nontransparotid approach leading to a more permanent paralysis12,20. rozeboom et al.19 (2018) have proposed excessive stretching and traction on the nerve to be a contributing factor to permanent dysfunction. a mean difference of 1.58 mm in the interincisal opening was found between the two approaches in our study, which is not statistically significant; this finding also corroborates reports by rozeboom et al who found no difference in mouth opening, lateral deviations or pain across different transcutaneous approaches to the condyle, including retromandibular transparotid and non transparotid20. post-operative occlusal discrepancies though rare, are still encountered after orif of condylar fractures. no patient in either of our groups had occlusal disturbance at 6 months post op, a finding reflected by other studies as well19. 7 ahmed et al. development of seroma is a relatively rare complication; however, 2 cases of seroma developed in the tp group in our study. there were no cases of sialocele or salivary fistula formation in the tp group, though different studies report a 2.6% incidence of sialocele and a ≥4.8% incidence of salivary fistula with the transparotid approach19. appropriate layered closure is required to prevent both sequelae. frey’s syndrome occurs as a result of condylar fracture management due to the intimate relationship of the capsule of the condyle with the auriculotemporal nerve. in our study, none of the patients reported with frey’s syndrome21,22, in contrast to different studies quoting a 1.1% incidence after the retromandibular approach19. in conclusion, we find no statistically significant disparity between the 2 approaches at a follow-up of 6 months; therefore, the primary determining factor for selection of either technique is surgeon preference and appropriate case selection. strengths our study contributes significantly to the existing literature regarding the pros versus cons of retroparotid and transparotid approaches to the mandibular condyle. limitations our study was limited to one university setting only, and the sampling technique was convenience sampling. conflicts of interest the authors state no conflicts of interest. funding no external sources of funding were employed in this study. acknowledgements we acknowledge the contribution of all surgeons in the preparation of this manuscript, as well as dow university of health sciences for granting ethics approval for the study. references 1. long x. the relationship between temporomandibular joint ankylosis and condylar fractures. chin j dent res. 2012;15(1):17-20. 2. ferretti c, bryant r, becker p, lawrence c. temporomandibular joint morphology following post-traumatic ankylosis in 26 patients. nt j oral maxillofac surg. 2005 jun;34(4):376-81. doi: 10.1016/j.ijom.2004.09.003. 3. parihar v, bandyopadhyay t, chattopadhyay p, jacob s. retromandibular transparotid approach compared with transmasseteric anterior parotid approach for the management of fractures of the mandibular condylar process: a prospective randomised study. br j oral maxillofac surg. 2019 nov;57(9):880-5. doi: 10.1016/j.bjoms.2019.07.010. 8 ahmed et al. 4. tang w, gao c, long j, lin y, wang h, liu l, et al. application of modified retromandibular approach indirectly from the anterior edge of the parotid gland in the surgical treatment of condylar fracture. j oral maxillofac surg. 2009 mar;67(3):552-8. doi: 10.1016/j.joms.2008.06.066. 5. tomar k. efficacy of the retroparotid trans-masseteric approach via retromandibular incision in orif of subcondylar fractures: our institution experience. j maxillofac oral surg. 2018 sep;17(3):332-8. doi: 10.1007/s12663-017-1022-y. 6. bhutia o, kumar l, jose a, roychoudhury a, trikha a. evaluation of facial nerve following open reduction and internal fixation of subcondylar fracture through retromandibular transparotid approach. br j oral maxillofac surg. 2014 mar;52(3):236-40. doi: 10.1016/j.bjoms.2013.12.002. 7. trost o, trouilloud p, malka g. open reduction and internal fixation of low subcondylar fractures of mandible through high cervical transmasseteric anteroparotid approach. j oral maxillofac surg. 2009 nov;67(11):2446-51. doi: 10.1016/j.joms.2009.04.109. 8. ebenezer v, ramalingam b, sivakumar m. trans parotid and retroparotid approach for the management of condylar fracture (case study). world j med sci. 2014;10(2):229-32. doi: 10.5829/idosi.wjms.2014.10.2.82244. 9. neff a, cornelius cp, rasse m, torre dd, audigé l. the comprehensive aocmf classification system: condylar process fractures level 3 tutorial. craniomaxillofac trauma reconstr. 2014 dec;7(suppl 1):s044-58. doi: 10.1055/s-0034-1389559. 10. meyer c, kahn j-l, boutemi p, wilk a. photoelastic analysis of bone deformation in the region of the mandibular condyle during mastication. j craniomaxillofac surg. 2002 jun;30(3):160-9. doi: 10.1054/jcms.2002.0297. 11. ahmed s, usmani rv, shaikh ah, iqbal n, hassan smu, ali a. mandibular fractures; pattern and presentation of mandibular fractures in dow international dental college: five year review. professional med j. 2018;25(10):1596-9. doi 10.29309/tpmj/18.4574. 12. al-moraissi ea, louvrier a, colletti g, wolford lm, biglioli f, ragaey m, et al. does the surgical approach for treating mandibular condylar fractures affect the rate of seventh cranial nerve injuries? a systematic review and meta-analysis based on a new classification for surgical approaches. j craniomaxillofac surg. 2018 mar;46(3):398-412. doi: 10.1016/j.jcms.2017.10.024. 13. al-moraissi ea, ellis e 3rd. surgical treatment of adult mandibular condylar fractures provides better outcomes than closed treatment: a systematic review and meta-analysis. j oral maxillofac surg. 2015 mar;73(3):482-93. doi: 10.1016/j.joms.2014.09.027. 14. vincent ag, ducic y, kellman r. fractures of the mandibular condyle. facial plast surg. 2019 dec;35(6):623-6. doi: 10.1055/s-0039-1700888. 15. ajmal s, khan ma, jadoon h, malik sa. management protocol of mandibular fractures at pakistan institute of medical sciences, islamabad, pakistan. j ayub med coll abbottabad. 2007;19(3):51-5. 16. cheema sa, amin f. incidence and causes of maxillofacial skeletal injuries at the mayo hospital in lahore, pakistan. br j oral maxillofac surg. 2006 jun;44(3):232-4. doi: 10.1016/j.bjoms.2005.05.017. 17. lindahl l. condylar fractures of the mandible: i. classification and relation to age, occlusion, and concomitant injuries of teeth and teeth-supporting structures, and fractures of the mandibular body. int j oral surg. 1977 feb;6(1):12-21. doi: 10.1016/s0300-9785(77)80067-7. 18. bouchard c, perreault m-h. postoperative complications associated with the retromandibular approach: a retrospective analysis of 118 subcondylar fractures. j oral maxillofac surg. 2014 feb;72(2):370-5. doi: 10.1016/j.joms.2013.08.014. 19. rozeboom avj, dubois l, bos rrm, spijker r, de lange j. open treatment of condylar fractures via extraoral approaches: a review of complications. j craniomaxillofac surg. 2018 aug;46(8):1232-40. doi: 10.1016/j.jcms.2018.04.020. 9 ahmed et al. 20. rozeboom a, dubois l, bos r, spijker r, de lange j. open treatment of unilateral mandibular condyle fractures in adults: a systematic review. int j oral maxillofac surg. 2017 oct;46(10):1257-66. doi: 10.1016/j.ijom.2017.06.018. 21. mellor t, shaw r. frey’s syndrome following fracture of the mandibular condyle: case report and literature review. injury. 1996 jun;27(5):359-60. doi: 10.1016/0020-1383(96)00010-1. 22. sverzut ce, trivellato ae, serra ecs, ferraz ep, sverzut at. frey’s syndrome after condylar fracture: case report. braz dent j. 2004;15(2):159-62. doi: 10.1590/s0103-64402004000200014. 1 volume 22 2023 e231336 original article braz j oral sci. 2023;22:e231336http://dx.doi.org/10.20396/bjos.v22i00.8671336 1 department of conservative dentistry, college of dentistry, university of mosul, iraq. corresponding author: raghad s. jamel department of conservative dentistry, college of dentistry, university of mosul, iraq raghadsabah@uomosul.edu.iq tel 009647740895662 editor: dr. altair a. del bel cury received: oct 28, 2022 accepted: feb 3, 2023 influence of medium-translucency monolithic zirconia thicknesses and light-curing time on the polymerization of dual-cure resin cements raghad s jamel1* aim: to investigate and compare the effects of different thicknesses of medium-translucency monolithic zirconia and light curing times on the polymerization of two types of dual-cured resin cement. methods: a total of 200 cement specimens were prepared from theracem and relyx u200 cement. the specimens were divided into 5 groups: group i, without interposing zirconia; group ii, 0.50 mm thickness; group iii, 1.00 mm; group iv, 1.50 mm; and group v, 2.00 mm thickness. each group was subdivided into (1) relyx u200 and (2) theracem. each subgroup was subdivided according to the light-curing time into (a) 20 s and (b) 40 s (n =5). the polymerization was tested using fourier-transform infrared (ftir) spectroscopy and a vickers microhardness tester. the data were statistically analyzed using anova, an independent sample t-test, and tukey’s test at a significance level of 0.05. results: the control group had the highest values of dc and vmh, followed by 0.50, 1.00, and 1.50 mm, respectively, while the 2.00 mm group showed the lowest values. the specimens irradiated for 40 s had greater dc and vmh than those irradiated for 20 s. relyx u200 revealed higher values for both parameters compared to theracem cement. conclusion: the polymerization of selfadhesive cement depends on the thickness of the monolithic zirconia, the light curing time, and the composition of the cement. the cement should be irradiated for a longer period than recommended to overcome the light attenuation of zirconia. tegdma-based self-adhesive cement showed a higher dc and vmh than bisgma-based cement. keywords: zirconium. resin cements. polymerization. https://orcid.org/0000-0002-7842-7149 2 jamel rs braz j oral sci. 2023;22:e231336 introduction all-ceramic restorations have been used for a long time because they meet the demands of both dentists and patients, including superior aesthetics and a natural appearance. conventional zirconia (first generation) is a partially stabilized zirconia that was introduced over fifteen years ago. it was distinguished by its opaque appearance and poor aesthetics. as a result, it served as a framework for more aesthetic veneered materials1. cracking or chipping of the veneer ceramic materials was the main reason for the clinical failure of veneered zirconia restorations2. to overcome the issue of ceramic veneer chipping, monolithic zirconia (second generation) was introduced as a full-contour zirconia restoration for single or multiple units. several in vitro studies on the second generation have revealed increased strength in addition to higher translucency. monolithic zirconia is an excellent choice for all-ceramic restorations with limited occlusal space and high occlusal loads owing to its good fracture resistance, mechanical properties, and biocompatibility3-5. the third generation of monolithic zirconia was introduced in 2015 as a fully stabilized zirconia. the translucency of these materials was compared to glass ceramics. recent investigations have found that the monolithic zirconia of this generation can be effectively employed for restorations with less occlusal reduction and lower occlusal strength due to the lower fracture toughness of these materials5,6. the clinical performance of a zirconia fixed prosthesis is determined not only by the material’s strength but also by the choice of an appropriate luting agent used to form a good bond between the dental structure and the restoration7. the success of the luting agent depends on polymerization’s efficiency, which is determined by the degree of monomer conversion (dc) and the number of free radicals generated. inadequate polymerization compromises the hardening of the resin cement, affecting its mechanical properties. moreover, it leads to negative consequences, including increased water absorption, microleakage, secondary caries, and postoperative sensitivity3,8,9. various methods have been used to evaluate the dc. the direct method (fourier -transform infrared spectroscopy) is commonly used for dc analysis and provides reliable results10,11 and the indirect method (vickers microhardness test)12. several factors may influence polymerization, including ceramic thickness, translucency, type of luting agent, and light-curing time13-15. this in-vitro study aimed to investigate and compare the effects of different thicknesses of medium-translucency monolithic zirconia and light-curing times on the polymerization efficiency of two newly generated dual-cure, self-adhesive resin cement. the null hypothesis was that the different thicknesses of medium-translucency monolithic zirconia and the light-curing times had no effects on the vmh and dc of dual-cure resin cement (dcrc). materials and methods zirconia specimens’ preparation the materials and their composition tested in the current study are recorded in table 1. the monolithic zirconia specimens were prepared from partially sintered yttrium 3 jamel rs braz j oral sci. 2023;22:e231336 oxide–stabilized zirconium blocks (ips e. max zircad mt blank disc, ivoclar vivadent schaan, liechtenstein). cubic specimens (10×10 mm) of different thicknesses (0.50, 1.00, 1.50, and. 2.00 mm, n = 8 for each thickness) were fabricated using a cad/cam system (hint-els, griesheim, germany). after that, the zirconia specimens were separated from the blank disc and carefully finished using a fine fissure diamond bur to remove any excess. the specimens were sintered using a special furnace, following the manufacturer’s recommendations (programat s1 1600, ivoclar vivadent, schaan, liechtenstein). sintering was performed for 2.5 h at 1500 °c with heating and cooling rates of 10 °c /min, then specimens were carefully polished with 600, 800, and 1200 grit silicon carbide polishing papers (sailbrand, china) with a water-cooling system, cleaned for 15 min with an ultrasonic cleaner (shenzhen langee ultrasonic electric co., china), and air dried for the 20s. a digital caliper (bosch, germany) was used to standardize the total thickness of the zirconia specimens (± 0.01 mm). finally, the external specimen surfaces were glazed to remove any surface defects. table 1. the composition of the tested materials materials type manufacturer composition ips e.max zircad (mt) monolithic zirconia ivoclar vivadent schaan, liechtenstein 86.0 – 93.5% zirconium oxide (zro2), > 6.5 % – ≤ 8.0 % yttrium oxide (y2o3 ), ≤ 5.0% hafnium oxide (hfo2 ), ≤ 1.0% aluminium oxide (al2o3 ), ≤ 1.0% other oxides. relyx u200 dual-cured self-adhesive resin cement 3m espe; seefeld, germany base: methacrylate monomers with the phosphoric acid group, triethylene glycol dimethacrylate (tegdma), silanated filler, initiators, and stabilizers. catalyst: methacrylate monomers, silanated fillers, alkaline fillers, initiators, pigments. filler content: 72 wt.%. theracem dual-cured, self-adhesive resin cement bisco, schaumburg, u.s.a. base: calcium base filler, glass filler, bisphenol glycidyl dimethacrylate (bisgma), fluoride components, amorphous silica, and initiators catalyst: methacryloyloxydecyl dihydrogen phosphate (mdp), glass fillers. filler content: 60-65 wt.%. cement specimens’ preparation two hundred specimens were prepared from the two newly generated dual-cure self-adhesive resin cement: theracem (bisco, schaumburg, usa) and relyx u200 (3m espe, seefeld, germany) to test dc and vmh (100 specimens for each test, n =5). five experimental groups were formed: group i: control group, cement specimens were irradiated without interposing monolithic zirconia specimens. group ii: cement specimens were irradiated through 0.50 mm-thick monolithic zirconia specimens. group iii: cement specimens were irradiated through 1.00 mm-thick monolithic zirconia specimens. group iv: cement specimens were irradiated through 1.50 mm-thick monolithic zirconia specimens. group v: cement specimens were irradiated through 2.00 mm-thick monolithic zirconia specimens. 4 jamel rs braz j oral sci. 2023;22:e231336 each main group was randomly subdivided according to the type of resin cement tested: (1) relyx u200 cement and (2) theracem cement. each subgroup was further divided according to the light-curing time as follows: (a) 20 s and (b) 40 s. degree of conversion (dc) one hundred specimens were made from two types of dual-cured resin cement using a teflon mold with standard dimensions (2 mm inner diameter and 1 mm thickness, n = 5) to test the degree of conversion8. the molds were positioned on glass slides which were covered with transparent strips to prevent material bonding. each cement was mixed following the manufacturer’s recommendations and packed into molds. one more transparent strip and a glass slide were placed at the top and pressed lightly to remove excess material. after removing the glass slide, one of each thickness of the zirconia specimens was positioned on top, and then the tip of the curing device was carefully placed on the upper surface of the zirconia specimens to permit light to pass through the zirconia toward the cement (fig.1a). the light-curing device (led, guilin woodpecker, medical instrument co., ltd., germany) with a light intensity output of 1600 mw/cm2 was figure 1. schematic diagram showing the preparation of the tested resin cement specimens (a, with interposing of different thicknesses of monolithic zirconia. b, direct activation without interposing of monolithic zirconia). a light curing device light curing device transparent strip transparent strip teflon mold teflon mold glass slides glass slides monolithic zirconia specimen 0.50, 1.00, 1.50, 2.00 mm resin cements, diameter = 2–5 mm thickness = 1 mm resin cements, diameter = 2–5 mm thickness = 1 mm b 5 jamel rs braz j oral sci. 2023;22:e231336 used to irradiate cement specimens with an exposure time of the 20s or 40s. light intensity was checked using a radiometer (bluephase meter ii, ivoclar vivadent). the control group was prepared by direct activation without interposing zirconia specimens (fig.1b). the specimens were left for 5 min to self-cure and carefully polished with 600 and 800-grit silicon carbide polishing paper. finally, the specimens were kept at 37°c for 24 h in completely dark containers to prevent further exposure to light before testing. fourier-transform infrared (ftir) spectroscopy (bruker atr-diamond ft-ir, germany) was used to evaluate the dc. the spectrophotometer system operated in the range of 500–3500 cm-1 with a resolution of 4 cm-1. a small amount of uncured resin cement was evaluated using a spectrophotometer, and the resulting spectrum was considered an unpolymerized reference. dc was calculated using a method that evaluated the changes in the ratio of aliphatic c=c (1638 cm-1) to the aromatic c-c bond (1608 cm-1) for the uncured and cured conditions based on the following equation8. dc% = 1 – x 100 1638 cm-1 / 1608cm-1 cured 1638 cm-1 / 1608cm-1 uncured vickers microhardness test (vmh) one hundred specimens were made from two types of cement using a mold with standard dimensions (5 mm diameter and 1 mm thickness, n =5) to test the vmh9. the specimens were prepared using the same procedure mentioned above. the polymerized specimens were evaluated using a vickers microhardness tester (otto wolper-werke, germany). a load of 0.5 kg was applied with a dwell time of 30 s. four indentations were made on the surface of the specimen. the four values were averaged to obtain the vickers hardness number (vhn) of each specimen. the vhn was calculated in kg/mm2 using this equation: vhn=1.8544*l /d2 where l = applied load recorded in (kg), d = mean diagonal length recorded in (mm). statistical analysis spss (version 25, ibm corp., armonk, usa) was used to analyze the data statistically. an anova was applied to determine the significant differences between all different thicknesses of zirconia. tukey’s post hoc test was used to compare the significant groups. to investigate the differences between the two tested groups of resin cement and light-curing times, an independent samples t-test was used. p ≤ 0.05 was considered statistically significant. results the mean values and standard deviation (sd) of the dc and vmh tests for relyx u200 and theracem resin cement for different monolithic zirconia thicknesses in the 20s and 40s are recorded in tables 2 and 3, respectively. a one-way anova for both resin 6 jamel rs braz j oral sci. 2023;22:e231336 cement revealed significant changes in the mean values of the dc and vmh tests (p ≤ 0.05) between different monolithic zirconia thicknesses at both light-curing times. table 2. mean, standard deviation (sd) of the degree of conversion for different thicknesses of monolithic zirconia and light curing time for both resin cements. resin cement zirconia thickness (mm) time 20 s mean ± sd 40 s mean ± sd relyx u200 0.00 mm 68.8 a ± 0.45 73.4 a ± 0.44 0.50 mm 65.5 b ± 0.85 70.6 b ± 1.24 1.00 mm 62.4 c ± 1.41 67.9 c ± 2.18 1.50 mm 58.6 d ± 0.50 63.1 d ± 1.59 2.00 mm 54.0 e ± 2.27 58.6 e ± 0.82 theracem 0.00 mm 65.0 a ± 1.98 68.7 a ± 0.70 0.50 mm 62.1 b ± 0.97 66.3 b ± 1.61 1.00 mm 59.1 c ± 1.57 62.7 c ± 2.37 1.50 mm 53.6 d ± 0.80 58.2 d ± 0.97 2.00 mm 50.1 e ± 2.41 55.7 e ± 1.06 different letters are significantly different depending on tukey’s test. number of specimens = 5 table 3. mean, standard deviation (sd) of the vickers microhardness test (kg/mm2) for different thicknesses of monolithic zirconia and light curing-time for both resin cements. resin cement zirconia thickness (mm) time 20 s mean ± sd 40 s mean ± sd relyx u200 0.00 mm 70.7 a ± 1.05 75.0 a ± 1.34 0.50 mm 65.2 b ± 1.08 69.7 b ± 0.90 1.00 mm 62.1 c ± 1.31 65.5 c ± 1.32 1.50 mm 59.1 d ± 1.10 64.8 d ± 1.08 2.00 mm 53.5 e ± 1.34 60.7 e ± 2.87 theracem 0.00 mm 52.1 a ± 1.22 57.3 a ± 0.83 0.50 mm 48.6 b ± 1.55 54.3 b ±1.21 1.00 mm 44.7 c ±1.14 51.2 c ±1.06 1.50 mm 41.7 d ± 1.13 46.0 d ± 0.99 2.00 mm 38.4 e ± 0.99 42.3 e ± 1.12 different letters are significantly different depending on tukey’s test. number of specimens = 5 tukey’s post hoc test exhibited that the specimens irradiated without zirconia interpose (control group) had the highest mean values of dc and vmh, followed by the specimens irradiated with 0.50, 1.00, and 1.50-mm thick zirconia, respec7 jamel rs braz j oral sci. 2023;22:e231336 tively while specimens interposing with 2.00 mm had the lowest values compared to all groups, as shown in tables 2 and 3. based on these results, as the thickness of monolithic zirconia restoration increased, the vmh and dc values of resin cement decreased. an independent t-test exhibited that there were significant changes between the two light-curing times for all groups. the specimens irradiated for the 40s had greater dc and vmh mean values than those irradiated for the 20s as shown in tables 4 and 5. in addition, there were significant changes between relyx u200 and theracem resin cement, as shown in tables 6 and 7. relyx u200 revealed significantly higher mean values for both parameters compared to theracem resin cement in all groups. table 4. independent samples t-test of the degree of conversion for the different thicknesses of monolithic zirconia between two light-curing times. resin cement zirconia thickness (mm) t-test df se p-value relyx u200 0.00 mm 16.016 8 0.28 0.000** 0.50 mm 7.575 8 0.67 0.000** 1.00 mm 4.721 8 1.16 0.002** 1.50 mm 5.897 8 0.74 0.002** 2.00 mm 4.285 8 1.08 0.008** theracem 0.00 mm 4.025 8 0.94 0.010** 0.50 mm 4.996 8 0.84 0.002** 1.00 mm 2.834 8 1.27 0.025* 1.50 mm 8.193 8 0.56 0.000** 2.00 mm 4.766 8 1.17 0.004** se = standard error, df = degree of freedom, * normal significant at p ≤ 0.05, ** highly significant at p ≤ 0.01 table 5. independent samples t-test of the vickers microhardness test for the different thicknesses of monolithic zirconia between two light-curing times. resin cement zirconia thickness (mm) t-test df se p-value relyx u200 0.00 mm 5.684 8 0.76 0.001** 0.50 mm 7.188 8 0.63 0.000** 1.00 mm 4.097 8 0.83 0.003** 1.50 mm 8.217 8 0.69 0.000** 2.00 mm 5.054 8 1.42 0.003** theracem 0.00 mm 7.919 8 0.66 0.000** 0.50 mm 6.425 8 0.88 0.000** 1.00 mm 9.315 8 0.69 0.000** 1.50 mm 6.279 8 0.67 0000** 2.00 mm 5.859 8 0.67 0.001** se = standard error, df = degree of freedom, ** highly significant at p ≤ 0.01 8 jamel rs braz j oral sci. 2023;22:e231336 table 6. independent samples t-test of degree of conversion for different thicknesses of monolithic zirconia between two resin cements. time zirconia thickness (mm) t-test df se p-value 20 s 0.00 mm 4.19 8 0.91 0.011** 0.50 mm 5.83 8 0.57 0.000** 1.00 mm 3.47 8 0.95 0.008** 1.50 mm 11.87 8 0.42 0.000** 2.00 mm 2.64 8 1.48 0.029* 40 s 0.00 mm 12.39 8 0.37 0.000** 0.50 mm 4.71 8 0.91 0.002** 1.00 mm 3.59 8 1.44 0.007** 1.50 mm 5.73 8 0.83 0.000** 2.00 mm 4.85 8 0.60 0.002** se = standard error, df = degree of freedom, * normal significant at p ≤ 0.05, ** highly significant at p ≤ 0.01 table 7. independent samples t-test of vickers microhardness test for different thicknesses of monolithic zirconia between two resin cement. time zirconia thickness (mm) t-test df se p-value 20 s 0.00 mm 25.76 8 0.72 0.000** 0.50 mm 19.62 8 0.84 0.000** 1.00 mm 22.24 8 0.78 0.000** 1.50 mm 24.48 8 0.71 0.000** 2.00 mm 20.31 8 0.74 0.000** 40 s 0.00 mm 25.03 8 0.70 0.000** 0.50 mm 22.75 8 0.67 0.000** 1.00 mm 18.80 8 0.75 0.000** 1.50 mm 28.61 8 0.65 0.000** 2.00 mm 13.34 8 1.37 0.000** se = standard error, df = degree of freedom, ** highly significant at p ≤ 0.01 discussion the basic factors for the success of indirect ceramic restorations are the stable and strong bond between the luting agent and ceramic restoration and between the luting agent and dental tissues. the bond strength of resin-based luting agents is determined by satisfactory polymerization of the resin matrix16. optimal polymerization of the cement is important for obtaining appropriate mechanical and physical properties and is considered a fundamental target for the success of ceramic-based restorations13,17. clinically, the minimum thickness for monolithic zirconia restorations is 0.50 mm. however, to fabricate monolithic zirconia anatomical posterior restorations, the thickness can reach 2.00 mm, which may be critical to the polymerization 9 jamel rs braz j oral sci. 2023;22:e231336 of the cement18. to simulate a clinical condition of monolithic zirconia cementation, the dcrc was irradiated from the top of the zirconia specimens with different thicknesses (0.50-2.00 mm). the statistical analysis revealed that the mean dc and vmh values decreased significantly with increasing the thickness of the monolithic zirconia compared to the control group that was directly exposed to light (tables 2 and 3). based on this result, the first null hypothesis was rejected. the current study agrees with many previous studies19-21. a possible explanation for this result is that increasing the thickness of zirconia causes the light to be attenuated by the zirconia material. the combination of absorption, scattering, and reflection explains the attenuation of the light passing through the restoration, which negatively affects the polymerization reaction of the cement10,15,22-25. it has been reported that approximately 25% of the light energy reaching the cement is available at 1 mm thickness, and the polymerization efficiency of the dual-polymerized resin cement decreases by approximately 70% with the increasing thickness of indirect restoration26. bragança et al.27 2020 showed that a 0.50 mm thickness of ceramic reduced the light reaching the resin cement by about 50%. on the other hand, some studies concluded that the dc of the resin cement irradiated under 0.50 and 1.00 mm thick ceramic specimens was similar to that of the control groups, but there was a decrease in the dc at thicknesses of 1.50 mm and above28,29. this disagreement may be due to differences in the methodology and materials used. regarding the light curing time, the specimens irradiated for 40 s showed higher values for both parameters than those irradiated for 20 s. hence, the second null hypothesis was rejected. it appears that extending the exposure time is necessary to enhance the polymerization efficiency of the resin-based cement. these results agree with several authors who explained that the curing time used by dental clinicians is too short and may not be sufficient to achieve optimum polymerization. they suggested extending the curing time further than recommended by the manufacturer to compensate for the deficiency of light intensity and achieve proper chemical polymerization9,22,27-31. other factors may influence the polymerization of dcrc, including the type of luting agent, light irradiance, and post-curing times13,14,22. according to sulaiman et al.22 2015, the amount of light-curing irradiance decreased as the thickness of the zirconia increased. therefore, a high light irradiance device is preferred to increase the quantity of light reaching the cement, particularly for those under thick monolithic zirconia restorations. moreover, light irradiance decreases with aging. thus, clinicians should frequently check the conditions of light-curing devices to avoid inadequate polymerization9. several authors confirmed that dc and vhn values significantly improved within the first 24 hours. this is clarified by the presence of unpolymerized monomers with the potential mobility to permit low-rate reactions. the ‟dark polymerization” of unpolymerized monomers may continue after the cessation of photoactivation32. “dark polymerization” can adequately compensate for the deficiency in initial polymerization and reach the ultimate polymerization to obtain the best clinical performance15. 10 jamel rs braz j oral sci. 2023;22:e231336 relyx u200 and theracem resin cement exhibited different behaviors when irradiated under the same conditions. this may be related to the variances in the cement compositions, including the amount and type of monomer structure, inorganic filler content, concentration, and quality of chemical and photoinitiators33-35. the results obtained from (ftir) spectroscopy showed that the dc of theracem and relyx u200 ranged from 50% to 74% as shown in table 2. it has been proved that resins have a degree of monomer conversion (50-75%), which is clinically acceptable36,37. in all groups, relyx u200 had significantly higher mean dc values than theracem resin cement; this could be attributed to the fact that relyx u200 is a triethylene glycol dimethacrylate (tegdma)-based resin cement38. tegdma is a low-molecular-weight and high-mobility monomer that allows the formation of more cross-linking between polymeric chains, resulting in a high degree of polymerization37,39. long-chain tegdma molecules are believed to improve the polymerization reaction, which continues for 24h after light activation. the polymerization efficacy increases with an increase in tegdma percentage owing to its reactivity and greater mobility40, while theracem is bisphenol glycidyl dimethacrylate (bis-gma)-based resin cement41 which is a high-molecular-weight monomer that enhances the mechanical properties of the resins but decreases the dc8,42. because of the higher degree of polymerization compared to bis-gma-based cement, it is recommended to use a self-adhesive resin cement with a high tegdma content43. furthermore, theracem contains a methacryloyloxydecyl dihydrogen phosphate (mdp) functional group, which has negative effects on the polymerization reaction because it chemically interacts with the tertiary amine (co-initiator) and camphorquinone44. the high percentage of inorganic fillers incorporated into relyx u200 may increase the degree of monomer conversion compared to theracem due to the lower amount of residual monomers. relyx u200 had higher mean vhn values than theracem in all groups. this result could be clarified by the fact that the relyx u200 contains more inorganic filler (72 by weight) than theracem (60-65 by weight)41. the results of the current study are in agreement with other authors who stated that the surface hardness of resin cement depends on the proportion, type, and distribution of the inorganic filler content and that increasing the proportion of inorganic fillers enhances the surface hardness of the cement14,15. the limitation of this in vitro study was that the test conditions did not fully mimic the oral environment. additional evaluations will be required to test the polymerization efficiency of dcrc under monolithic zirconia in vivo. further research will also be needed  to evaluate the impact of zirconia thickness on cement’s optical characteristics. within the limits of the current study, it can be concluded that the polymerization efficiency of dual-cured self-adhesive cement depends on the thickness of the medium-transparency monolithic zirconia, light curing time, the monomer type, and the concentration of filler contents. increasing the thickness of monolithic zirconia from 0.50 to 2.00 mm exhibited a negative effect on the polymerization efficiency of the underlying cement. the dual-cured resin cement should be clinically irradiated for a longer period than recommended by the manufacturer to overcome the light atten11 jamel rs braz j oral sci. 2023;22:e231336 uation of zirconia material. tegdma-based self-adhesive cement showed statistically higher dc and vmh values than bisgma-based cement. the dc values for both types of cement ranged within the clinically accepted limit of 50–75%. thus, both relyx u200 and theracem can be proposed as luting agents for monolithic zirconia restorations. acknowledgments the author thanks the university of mosul and the college of dentistry for their assistance and the availability of the laboratories and equipment that enabled the development of the current study. conflict of interest none to declare. references 1. piconi c, maccauro g. zirconia as a ceramic biomaterial. biomaterials. 1999 jan;20(1):1-25. doi: 10.1016/s0142-9612(98)00010-6. 2. silva lhd, lima e, miranda rbp, favero ss, lohbauer u, cesar pf. dental ceramics: a review of new materials and processing methods. braz oral res. 2017 aug;31(suppl 1):e58. 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cements. dent clin n am. 2017 oct;61(4):821-34. doi: 10.1016/j.cden.2017.06.006. 1http://dx.doi.org/10.20396/bjos.v18i0.8657258 volume 18 2019 e191605 original article 1 department of orthodontics, araras dental school, uniararas, araras, sp, brazil. 2 department of restorative dentistry, dental materials division, piracicaba dental school, unicamp – universidade estadual de campinas, piracicaba, sp, brazil. corresponding author: heloisa cristina valdrighi av. dr. maximiliano baruto, 500 jardim universitário. araras, sp – brazil. 13607-339 +55 19 35431423 e-mail: heloisavaldrighi@gmail.com https://orcid.org/0000-0001-7567-1990 received: march 29, 2019 accepted: august 23, 2019 evaluation of friction on self-ligating and conventional brackets associated with different types of archwires submitted to sliding mechanics william carlos silva barbosa1, américo bortolazzo correr2, diego patrik alves carneiro1, mário vedovello filho1, ana paula terossi de godoi1, heloísa cristina valdrighi1,* aim: the aim of this study was to verify the frictional force during sliding mechanics in orthodontic tooth movement, using conventional metal brackets of the active and passive selfligating types with stainless steel and copper nickel titanium archwires. methods: this experimental in vitro study was conducted with conventional metal (morelli, sorocaba, sp, brazil) brackets, active self-ligated (sli morelli, sorocaba, sp, brazil) and passive self-ligated (slp morelli, sorocaba, sp, brazil), with slot 0.022 x 0.028 inches and roth prescription. the brackets were tested with rectangular section 0.019 x 0.025 inch copper nickel titanium and stainless steel archwires. for each type of bracket, 10 sets of plate/bracket/archwire segment (n=10) were fabricated. non-parametric kruskal wallis and dunn tests were used for comparison between types of brackets and wilcoxon tests for comparison between types of archwires. results: the results showed that the frictional force values were higher with copper nickel titanium than with stainless steel archwires (p<0.05). when copper nickel titanium archwires were used, the active self-ligating brackets showed higher frictional force values than the other types, followed by the conventional brackets. lower frictional force values were observed with passive selfligating brackets. for stainless steel archwires, no difference was observed between conventional and active self-ligating brackets, the passive self-ligating type presented lower frictional force values than the others. conclusion: it was concluded that the higher frictional force was observed when active self-ligating brackets were associated with copper nickel titanium archwires. lower frictional force was verified between passive self-ligating brackets combined with stainless steel archwires. keywords: orthodontic brackets. friction. orthodontic appliance design. https://orcid.org/0000-0001-7567-1990 2 barbosa et al. introduction the frictional resistance present when performing orthodontic sliding mechanics results from interactions between the bracket, arch and method of ligation1-9. a high frictional coefficient may reduce the force used for orthodontic movement by half, diminishing the speed of tooth movement and making it difficult to control anchorage. the frictional force should be as low as possible with the goal of achieving greater mechanical efficiency; that is, the force applied must be sufficient to break the static friction and enable tooth movement2,6,9,10-13. the frictional force may vary according to the materials used, and whether the environment is wet or dry, type of brackets, archwires and ligations. self-ligating brackets may be divided into active and passive types; the active types have a spring clip that invades the bracket slot putting pressure on the archwire, while in the passive system, the clip does not invade the slot, it only covers the slot without putting pressure on the archwire1-5,7,10,12,14-19. among the various wires used for making orthodontic arches, stainless steel wires are outstanding as they have a polished surface14,20. however, the technological evolution has led to new archwires being used, among them the nickel titanium type with the addition of copper (cuniti)17,21. the incorporation of copper has resulted in these archwires having more defined thermoactive properties than the superelastic niti archwires, therefore, they exert more homogeneous forces throughout the arch, providing faster, more effective movement, in an optimal system of forces, with greater control of tooth movement; they may be used in different orthodontic treatment protocols, as they achieve more biologically compatible results by releasing more physiological force and shortening the time of treatment2,4,10,21-23. the frictional forces between conventional and (passive and active) self-ligating brackets, associated with archwires that have different section and compositions have been studied2,6,12,24. however, no reports were found of studies comparing the frictional force between active and passive self-ligating brackets with rectangular 0.019 x 0.025 inch sections of copper nickel titanium and stainless steel, in a wet environment at a temperature of 36.50 c. the hypothesis under study was that metal self-ligating brackets would produce lower frictional force than that of conventional metal brackets; and copper nickel titanium archwires would produce higher frictional forces than those of stainless steel archwires. thus the aim of this study was to verify the frictional force during sliding mechanics in orthodontic tooth movement, using conventional metal brackets of the active and passive self-ligating types in combination with stainless steel and copper nickel titanium archwires. materials and methods: this experimental in vitro study was conducted with 30 sets of plate/bracket/archwire segments that were divided into three groups according to the brackets used, i.e., 3 barbosa et al. conventional metal (ref. 10.10.901, morelli, sorocaba, sp, brazil), active self-ligating metal (ref. 10.14.900, sli/ morelli, sorocaba, sp, brazil) and passive self-ligating metal brackets (ref. 10.13.900, slp/ morelli, sorocaba, sp, brazil). for each bracket type, 10 sets of plate/bracket/segment (n=10) were fabricated6,16,25. the brackets were tested with rectangular section 0.019 x 0.025 inch copper nickel titanium (ref. 50.62.154, morelli, sorocaba, sp, brazil), and stainless steel archwires (ref. 50.62.004, morelli, sorocaba, sp, brazil)2,3,11. the test specimens were made up of a rectangular acrylic plate3,5,10,14,25, measuring 8.5 cm long, 4 cm wide and 0.5 cm thick with metal brackets (conventional), slot 0.022 x 0.028 inches, from the maxillary right 2nd premolar to the maxillary right central incisor5,14, combined with a segment of rectangular section 0.019 x 0.025 inches of copper nickel titanium and stainless steel (morelli, sorocaba, sp, brazil)3,5,25, as shown in figure 1. 1 cm 1 cm 2 cm 2 cm2 cm 5 cm 2 cm figure. 1 schematic drawing of the acrylic plate (8.5 cm long, 4 cm wide and 0.5 cm thick). metal brackets were positioned 0.5 mm distant from each other. the archwire was fixed on the brackets to frictional force evaluation. the position of each bracket was demarcated on the plate and abraded with a spherical bur at low speed, cleaned with gauze imbibed in 70% alcohol, and dried with absorbent paper towels to prevent the presence of substances or dirt that could compromise the results obtained, thereby increasing the relationship of brackets and preventing 4 barbosa et al. these from debonding from the plate during the tests11,13. the brackets were aligned in parallel on in the most central region of the plate, so that the center of each bracket remained at a distance of 2 cm from the lateral borders and at a distance of 0.5 cm from each other. the brackets localized in the upper and lower extremities remained at a distance of 2 cm from the top and bottom edges of the acrylic plate3,5,14,25. after this, the brackets were fixed with cyanoacrylate adhesive (super bonder, loctite henkel, sp, brazil), before a device was bonded on a 0.021 x 0.025 inch thick “u”-shaped11 stainless steel archwire (ref. 55.03.015, morelli, sorocaba, sp, brazil), which was fitted into the channels of the brackets, and its extremities were fixed in holes made in the plate at a distance of 1 cm from its top and bottom edges. this was done to obtain the maximum level of standardization among the groups, to prevent any poorly positioned bracket from affecting the reliability of the results11,14. after the brackets were fixed, the rectangular archwire segments of copper nickel titanium and stainless steel, measuring 0.019 x 0.025 inches and 20 cm long were positioned3,10,23,24. the arch segments of 0.019 x 0.025 inches were fixed to the conventional metal brackets by conventional elastomeric ligatures (ref. 60.06.101, morelli, sorocaba, sp, brazil), in accordance with previously used methodology5,14,25. to simulate the conditions of the oral cavity, the tests were performed in a wet environment. the test specimens remained in a glass receptacle submersed in 12 liters of water at a temperature of 36.5 oc, because activation of the copper nickel titanium archwire occurs at 35 oc. the temperature of the water was controlled by two mercury thermometers2,14,15,20. assay to determine the frictional force to evaluate the frictional force, a universal test machine (instron model 4411, buckinghamshire, england) with a 50 n load cell and 5 mm/min crosshead speed was used25. the archwire was moved 5 mm on the brackets and the friction evaluated. the results corresponding to the static frictional force were transmitted to the bluehill 2.0 materials testing software (instron, norwood, ma 020622643, u.s.a.), coupled to the testing machine. the tests were repeated five times in each plate/ bracket/archwire set up and the mean obtained. in the conventional metal brackets, the elastomeric ligatures were removed and replaced with new elastomeric ligatures in each test. for removal and insertion of the elastomeric ligatures in the conventional brackets, an elastic tie applicator was used (ref. 75.01.001, morelli, sorocaba, sp, brazil)5,10,15.24. the data did not comply with the presuppositions of a normal variance analysis. therefore, the non-parametric kruskal wallis and dunn tests were used for comparison between types of brackets and wilcoxon tests for comparison between types of archwires. the wilcoxon test was used because the same 30 test specimens were analyzed with the stainless steel archwires and those made of copper nickel titanium. the analyses were performed in the r program (r foundation for statistical computing, vienna, austria) considering the level of significance of 5%1,11,13,14. 5 barbosa et al. results in table 1, it was possible to observe that the frictional force values were higher with copper nickel titanium than they were with stainless steel archwires, for the same type of bracket (p<0.05). when copper nickel titanium archwires were used, the active self-ligating brackets showed higher frictional force values than the other types (p<0.05), followed by the conventional brackets. lower frictional force values were observed with passive self-ligating brackets (p<0.05). for stainless steel archwires, no significant difference was observed between conventional and active self-ligating brackets (p>0.05), but the passive self-ligating type presented significantly lower frictional force values than the others (p<0.05). discussion the hypothesis that self-ligated metal brackets would produce lower frictional force than conventional metal brackets was rejected. the hypothesis that the titanium nickel copper archwires would produce higher frictional force than the stainless steel wires was accepted. the questions raised in our study reinforced the affirmations of other authors that the supposed advantage of lower friction in self-ligating brackets was still controversial, when compared with conventional brackets associated with archwires with rectangular sections, particularly when comparisons were made between active self-ligating brackets and the conventional types4,11,14,16,26,27. our findings showed that the association of conventional and self-ligating active and passive brackets with copper nickel titanium archwires with rectangular 0.019 x 0.025 inch sections presented higher friction values in the active self-ligating brackets, followed by the conventional types. the lowest frictional force values were observed for the passive self-ligating brackets; these results corroborated the findings of previous studies that did not find lower frictional force with the use of self-ligating brackets. the elasticity of the copper nickel titanium archwire, with a rougher and more irregular surface associated with the pressure exerted by the clip of the self-ligating brackets could increase the surface of contact between the wire and the internal part of the slot. consequently this would increase the frictional force, in agreement with previtable 1. median (minimum and maximum values) of frictional force (n) considering bracket and type of archwire bracket archwire copper nickel titanium stainless steel conventional 6.18 (4.38-6.86) ab 4.96 (4.13-6.26)ba active self-ligating 13.21 (10.58-15.35) aa 9.56 (4.80-12.78)ba passive self-ligating 0.52 (0.46-0.96) ac 0.01 (0.00-0.02) bb medians followed by different letters (capitals on horizontal lines and lower-case in vertical position) differ between them (p≤0.05) 6 barbosa et al. ous studies in which the composition of the copper nickel titanium archwire could increase the frictional force17,22,23,28. in conventional brackets associated with copper nickel titanium archwires, the friction would be lower due to the smaller area of contact of the ligature with the archwire, and also due to the lower pressure exerted by the elastic ligature on the archwire. these findings corroborated those of studies in which lower pressure exerted by the elastic ligatures were found, making the pressure smoother and diminishing the points of contact of the wire with the internal part of the slot21. the difference in composition between the material of the ligature and that of the clip could also have an influence on the friction. the lower friction values observed in the presence of passive self-ligating brackets would result from the smaller surface of contact between the archwire and internal part of the slot, resulting from the absence of pressure exerted by the clip of this bracket. the findings of this study corroborated those of previous studies in which it was proved that the increase in contact surface increased the friction1,2,5,7,11,13,14,16,18,24. the result of the present research showed that the combination between copper nickel titanium archwires associated with active self-ligating brackets generated higher frictional forces than those generated by the combination of this archwire with conventional brackets, disagreement with some reports in the literature, in which the low friction observed in self-ligating brackets was considered an advantage3,5. our findings corroborated the results found by researchers when they made a comparison between self-ligating and conventional brackets, in which the lower frictional resistance would only be observed when these brackets were combined with wires with smaller diameters. these results would be justified by the reduction in the surface of contact between the slot and archwire1,2,5,11,13,14,24,26,29. the results of the present study revealed that active self-ligating brackets produced similar friction values when compared with conventional brackets with the use of stainless steel archwires with rectangular sections. this fact may be explained by the more polished, smoother surface and greater rigidity of this wire, so that in spite of the pressure exerted by the clips of the active self-ligating brackets, the contact surface of this wire would not be increased. in the case of conventional brackets, the elastic ligature would not produce sufficient force to increase the surface of contact at the bracket/archwire interface, corroborating the findings of previous studies in which the composition of the archwire and clip of the bracket were reported2,11,16,18,19,21. the lowest friction values in this study were observed in passive self-ligating brackets, irrespective of the archwire used. this result corroborated the findings in the literature14,18. the lower friction in these brackets would be explained because of the smaller number of contacts between the archwire and bracket slot, since this system ends up creating a tunnel in which the archwire remains relatively free, thus transforming the bracket into a tube. this also explains why lower frictional values were observed with archwires of smaller calibers; that is, the smaller the surface of contact, the lower would be the friction, reinforcing the results of previous researches1,2,11,13,18,24. 7 barbosa et al. the results of the present study contribute to the orthodontic practice of sliding mechanics, since the current literature does not show unanimity regarding the friction produced by wires of rectangular section of different compositions in self ligating brackets. however, the results of this study have limitations because it is an in vitro study, suggesting that clinical studies are performed. in conclusion, higher frictional forces were observed between copper and nickel titanium arches associated with active self-ligating brackets, while lower frictional forces were observed with the use of stainless steel arches associated with passive self-ligating brackets. references 1. muguruma t, iijima m, brantley wa, ahluwalia ks, kohda n, mizoguchi i. effects of third-order torque on frictional force of self-ligating brackets. 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88(1):67-74. doi: 10.2319/030117-159.1. 19. kim ks, han sj, lee th, park tj, choi s, kang yg, park kh. surface analysis of metal clips of ceramic self-ligating brackets. korean j orthod. 2019 jan;49(1):12-20. doi: 10.4041/kjod.2019.49.1.12. 20. leal rc, amaral flb, frança fmg, basting rt, turssi cp. role of lubricants on friction between self-ligating brackets and archwires. angle ortho. 2014 nov; 84(6):1049-53. doi: 10.2319/110513-805.1. 21. henriques jfc, higa rh, semenara nt, janson g, fernandes tmf, sathler r. evaluation of deflection forces of orthodontic wires with different ligation types. braz oral res. 2017 jul; 31:49. doi: 10.1590/1807-3107bor-2017.vol31.0049. 22. gravina ma, canavarro c, elias cn, chaves mgam, brunharo ih, quintão cc. mechanical properties of niti and cuniti wires used in orthodontic treatment part 2: microscopic appraisal and metallurgical characteristics. dental press j orthod. 2014 jan-fev;19(1):69-76. doi: 10.1590/2176-9451.19.1.069-076.oar. 23. aydin b, semisk ne, koskan o. evaluation of the alignment efficiency of nickel-titanium and copper-nickel-titanium archwires in patients undergoing orthodontic treatment over a 12-week period: a single-center, randomized controlled clinical trial. korean j orthod 2018;48(3):153-62. doi: 10.4041/kjod.2018.48.3.153. 24. barbosa ja, elias cn, basting r. evaluation of friction produced by self-ligating, conventional and barbosa versatile brackets. rev odontol unesp. 2016 mar-apr;45(2):71-7. doi: 10.1590/1807-2577.09515. 25. venâncio fr, vedovello sas, tubel cam, degan vv, lucato as, lealdim ln. effect of elastomeric ligatures on frictional forces between the archwire and orthodontic bracket. braz j oral sci. 2013 jan-mar;12(1):41-5. doi: 10.1590/s1677-32252013000100009. 26. castro r. ]self-ligating brackets: efficiency versus scientific evidence]. rev dent press ortod ortop facial. 2009 jul-ago;14(4):20-4. portuguese. doi: 10.1590/s1415-54192009000400002. 27. carneiro gkm, roque ja, garcez segundo as, suzuki h. evaluation of stiffness and plastic deformation of active ceramic selfligating bracket clips after repetitive opening and closure movements. dental press j orthod. 2015 jul-aug;20(4):45-50. doi: 10.1590/2176-9451.20.4.045-050.oar. https://www.ncbi.nlm.nih.gov/pubmed/27220902 https://www.ncbi.nlm.nih.gov/pubmed/27220902 9 barbosa et al. 28. leite vv, lopes mb, gonini júnior a, almeida mr, moura sk, almeida rr. comparison of frictional resistance between self-ligating and conventional brackets tied with elastomeric and metal ligature in orthodontic archwires. dental press j orthod. 2014 may-jun;19(3): 114-9. 29. ehsania s, mandichb ma, el-bialy th, mirc cf. frictional resistance in self-ligating orthodontic brackets and conventionally ligated brackets. a systematic review. angle orthod. 2009 may;79(3):592-601. doi: 10.2319/060208-288.1. https://www.ncbi.nlm.nih.gov/pubmed/?term=10.2319/060208-288.1 1http://dx.doi.org/10.20396/bjos.v20i00.8660445 volume 20 2021 e210445 original article 1 instructor of dental biomaterials, biomaterials department, faculty of dentistry, ain-shams university, organization of african unity st, elqobba bridge, al waili, cairo, egypt. 2 professor of dental biomaterials, biomaterials department, faculty of dentistry, ain-shams university, organization of african unity st, elqobba bridge, al waili, cairo, egypt. 3 professor and head of biomaterials department, faculty of dentistry, ain-shams university, organization of african unity st, elqobba bridge, al waili, cairo, egypt. *corresponding author: amany a. salem bds, instructor of dental biomaterials, biomaterials department, faculty of dentistry, ain-shams university, organization of african unity st, el-qobba bridge, al waili, cairo, egypt. email: dr.amany@dent.asu.edu.eg address: 500 el narges buildings, 5th settlement, cairo governorate, egypt. phone: 00201090555433 received: july 10, 2020 accepted: january 16, 2021 durability of bond strength to dentin using two universal adhesives in different etching modes amany a. salem1,* , mohamed s. nassif2, dalia i. el-korashy3 aim. to evaluate the bond durability of two universal adhesives; mild and ultra-mild in both etch-and-rinse and self-etch modes after simulated in-vitro degradation by long-term water storage or thermocycling. methods. a total of 144 specimens were used in this study; 120 specimens (prepared from 30 teeth) for microshear bond strength testing and fracture mode assessment, and 24 specimens for scanning electron microscopic evaluation (prepared from 24 teeth). specimens  were prepared from 54 recently extracted caries free third molars and randomly divided into 12  groups, according to the adhesive treatment (all bond or scotchbond universal), etching mode (etch-andrinse or self-etch) and aging method (thermocycling or water storage). each  tooth was sectioned mesio-distally into two halves exposing free dentin surface for bonding where dentin substrate 1  mm below the dentino-enamel junction was used. after adhesive application and composite build up, specimens were tested in shear mode after storing in distilled water at 37˚c for 24 hours or 1 year, or after being thermocycled between 5 and 55 °c for 10,000 cycles (n=10). microshear bond strength (µsbs) was tested using a universal testing machine. adhesive-dentin interface was examined using scanning electron microscope (sem) (n=2). data were analyzed using 3 way an0va and pairwise comparisons was performed using bonferroni correction at significance level of α ≤0.05. results. statistical analysis revealed non-significant difference for etching mode in both adhesives (p=0.596). after aging, the bond strength was only reduced in scotchbond universal after one year of water storage in both modes (p<0.001). sem evaluation revealed intact hybrid layer and longer resin tags for etch-and-rinse mode than selfetch mode of both adhesives. conclusion. the bond strength of universal adhesives is not affected by the etching mode, however its durability was shown to be material dependent. keywords: dental cements. dentin. dental bonding. https://orcid.org/0000-0002-9471-6274 let12 highlight i recommend omitting the keyword (dental stress analysis) and adding other keywords like (universal adhesives, aging, durability) as they are more relevant to the study. let12 sticky note can you specify what keywords should we use? 2 salem et al. introduction the success of most of the current esthetic restorations depends greatly on the quality of the bond between the tooth structure and the restorative material1. in this context, a great effort has been directed towards the development of dental adhesives that can achieve a strong bond to the tooth structure by relatively simple adhesive procedures2. recently, manufacturers have introduced a single product for all situations, referred to as ‘universal’ or ‘multi-mode’ adhesives3. universal adhesives represent the last generation of adhesives in the market 4. they are “universal” in two main ways: first, they are recommended by dental manufacturers for use both in etch-and-rinse and selfetch modes with claims by manufacturers that there is no compromise on bonding effectiveness when either bonding strategy is employed5. second, they can be used on a wide range of substrates; they can be used to bond to dentin and enamel, for the placement of both direct and indirect restorations. moreover, some universal adhesives can be used as adhesive primers on substrates such as zirconia, noble and non-precious metals, composites and various silica-based ceramics without the need for dedicated and separately placed primers6. the immediate bond strength of contemporary adhesives are quite satisfactory, however the long term durability upon aging is more critical and more clinically relevant2. the ph of universal adhesives greatly influences the long term stability and durability of the bond strength to dentin. universal adhesives can be classified according to the ph into “ultra-mild (ph > 2.5) or mild (ph > 2) or intermediately strong (ph approximately equal 1.5). generally, lower stability of bonding to dentin has been reported to intermediately strong universal adhesives after aging due to the high acidity of residual monomers that continue to demineralize the dentin and further weaken the adhesive interface4. resin-dentin bonds are more challenging and less durable than resin-enamel bonds. the limited durability of resin-dentin bonds severely compromises the lifetime of tooth-colored restorations7. this may be attributed to several factors including the heterogeneity of dentin composition (high organic and water content), the variation in the density of dentinal tubules with dentinal depth and the structural changes as in carious and sclerotic dentin which are usually accompanied with decrease in the dentin permeability8. in contrast to clinical trials, simulated oral environment testing can be used to rapidly determine the relative bonding durability of materials. in-vitro degradation of restored teeth can be simulated allowing standardization of conditions before and after storage, and assessing degradation using various tests allows easy comparison9. several methodologies of aging have been reported in literature such as water storage, thermocycling, mechanical loading as well as degradation by enzymes and various chemical substances among which water storage and thermocycling represent the most popular artificial aging methods10. water is thought to play a major role in degradation of dentin-resin bond. in long-term water storage experiments, degradation is accelerated by hydrolysis of hydrophilic resin components and by host-derived proteases with collagenolytic activity. in addition, the restorations are clinically subjected to repetitive expansion and contraction stresses caused by temperature fluctu3 salem et al. ations within the oral cavity. these stresses have been proposed to affect the bonded interface. in thermocycling, the bonded specimens are subjected to cyclic temperature changes through water immersion9. there are insufficient data in the literature regarding the effect of different aging methods on the performance of universal adhesives. moreover, there is heterogeneity in the results of studies testing the long term durability of universal adhesives9,11-16 hence, this study was designed to evaluate the effect of the application mode and the simulated in vitro degradation method on the micro-shear bond strength of two different universal adhesives. the null hypotheses were that (i) the application mode will not affect the microshear bond strength of an ultra-mild or mild universal adhesives, (ii) the bond strength is not affected by the simulated in vitro degradation methods. material and methods materials: adhesive systems, their description, composition and application procedures according to the manufacturer’s instructions are shown in table 1. table 1. adhesive systems, their description, composition and application procedures according to the manufacturer’s instructions. brand name and manufacturer description composition application procedures scotchbond universal (3m espe, neuss, germany) mild universal adhesive. (ph=2.7) mdp monomer, dimethacrylate resins, hema, vitrebond™ copolymer, fillers, ethanol, water, initiators, silane. self-etching mode: 1. the adhesive was applied to the exposed dentin surface with a micro brush and rubbed for 20 seconds. 2. a gentle stream of air was directed over the adhesive for 5 seconds until the adhesive no longer moved indicating that the solvent has completely evaporated. 3. the adhesive was then light cured for 10 seconds. etch-and-rinse mode: 1. the etchant (meta etchant 37% phosphoric acid semi gel) was applied on dentin surface for 15 seconds. 2. the etchant was thoroughly rinsed under running water for 15 seconds. 3. excess water was removed by blotting the surface with a wet cotton pellet leaving the surface visibly moist. 4. the adhesive was then applied as for the self-etch mode. all bond universal (bisco, schaumburg, illinois, usa) ultra-mild universal adhesive. (ph = 3.2) mdp, bis-gma, hema, ethanol, water, initiators. self-etching mode: 1. two separate coats of adhesive were applied by scrubbing the dentin surface with a microbrush for 10 s per coat (no light polymerization was performed between coats). 2. excess solvent was evaporated by thoroughly airdrying wi th an air syringe for 10 s until the adhesive no longer moved, leaving the surface with a uniform glossy appearance. 4. the adhesive was then light cured for 10 s. etch-and-rinse mode: 1. the etchant (meta etchant 37% phosphoric acid semi gel) was applied on the dentin surface for 15 seconds. 2. the etchant was thoroughly rinsed under running water for 15 seconds. 3. excess water was removed by blotting the surface with a wet cotton pellet leaving the surface visibly moist. 4. the adhesive was then applied as for the self-etch mode. mdp: methacryloyloxydecyl dihydrogen phosphate, hema: hydroxyethylmethacrylate, bis-gma: bisphenol a glycidyl methacrylate. 4 salem et al. methods microshear bond strength testing specimens grouping a total of 144 specimens were used in this study; 120 specimens for microshear bond strength testing and fracture mode assessment, and 24 specimens for scanning electron microscope evaluation. the specimens were prepared from 54  recently extracted caries free third molars (30 teeth for microshear bond strength testing with 4 specimens prepared on each tooth and 24 for scanning electron microscopic evaluation). molars were washed, cleaned from debris, stored in distilled water for 1  month. then, they were randomly divided into twelve groups according to the aging method (24  hours of water storage, one year of water storage or thermocycling), the adhesive used (all bond universal or single bond universal) and etching mode (etch-and-rinse or self-etch) where n=10 for micro-shear and n=2 for sem evaluation. specimens preparation each tooth was sectioned mesiodistally into two halves under copious air-water coolant spray by diamond disc (honeycomb design 6924, komet, usa). the roots of the teeth were then removed17. each sectioned half was embedded in chemically cured acrylic resin placed in a polyvinyl ring with enamel surface facing the acrylic and the cut surface facing upward exposing a free dentin surface for bonding. in each sectioned half, the portion of dentin substrate 1 mm below the dentino-enamel junction was used. the exposed dentin surfaces were manually polished in a circular motion with a wet 600-grit sic paper for 60 s in order to standardize the smear layer3. the specimens were then cleaned ultrasonically to ensure removal of any debris from the surface18 and were randomly divided into twelve groups for microshear bond strength test (n=10) according to the adhesive used (all bond universal or scotchbond universal), etching mode (etch-and-rinse or self-etch) and aging method (24 hours of water storage, one year of water storage or thermocycling). each adhesive was strictly applied according to manufacturers’ instructions as shown in table 1. two rubber microtubes (tygon, norton performance plastic co., akron, oh, usa) of 0.8 mm diameter and 1 mm height were then placed at different positions 1mm below the dentinoenamel junction on the treated dentin surfaces. the adhesive was then light cured with the led light curing device (elipar s10 free light, 3m espe, usa) with an output intensity 1200 mw/cm2 with the light curing device resting directly on the tubes. the a2 shade flowable nano-hybrid composite (tetric n flow) was carefully inserted into the tubes, covered by a celluloid strip and light-irradiated with the led light curing device. then the tubes were carefully removed leaving the resin-bonded composite cylinders18. aging method and testing procedures for the control group, specimens were stored in distilled water at 37 ˚c for 24 hours19. 5 salem et al. for the water storage group, specimens were stored in distilled water at 37 ̊ c for 1 year9. for the thermocycling group, specimens were stored in distilled water at 37 ˚c for 24  hours and were then subjected to 10,000 thermal cycles (tcs) between 5 and 55 ˚c with a dwell time of 30 s and a transfer time of 5 s in a thermocycler (sd mechatronic, feldkirchen-westerham germany)20. microshear bond strength was tested by a universal testing machine (lloyd lr 5k, lloyd instruments ltd., hampshire, uk). a thin steel wire (0.18mm diameter) was looped around the resin composite cylinder touching the tooth surface and attached to upper compartment parallel to the load cell movement direction and to the bonded surface21 22. force was applied at a cross head speed of 1.0 mm/min until failure23. bond strength values were calculated by dividing the maximum debonding forces by the surface area automatically using (nexygen software). fracture mode analysis after debonding of each sample, the fractured interface was assessed using a stereomicroscope (olympus stereozoom sz 40 microscope, tokyo, japan) at magnification of 40x to determine the mode of failure which was classified as ‘cohesive’ (entirely within dentine substrate or resin composite), ‘adhesive’ (at the dentine-resin interface) or ‘mixed’ (at dentine-resin interface including failure into one of the substrates)19. scanning electron microscopic evaluation of the tooth-restoration interface scanning electron microscope analysis was carried out for assessment of the tooth restoration interface of the different experimental groups. standard class v cavities were prepared in 24 recently extracted caries free third molars and randomly divided into the twelve groups where the number of specimens (n=2) for each experimental condition as described for microshear bond strength test. the adhesive system and resin composite were then applied to the exposed dentin of class v. the teeth were stored in distilled water for 24 hours then sectioned bucco-lingually using a diamond disc under water coolant. the sections were flattened and smoothed using silicon carbide papers with sequential grit of 400, 600, and 1000 under water. the specimens were acid etched using a 37% phosphoric acid gel for 5 seconds and rinsed for another 30 seconds. the specimens were then immersed in a 3% naocl for five minutes. then the specimens were placed in 70%, 80%, 90%, and 99% alcohol to eliminate all the water present24. the specimens were gold sputtered (emitech k550x sputter coater, east sussex, england) and the adhesive/dentin interface was evaluated using scanning electron microscope (quanta 250 feg field emission gun), with an accelerating voltage 30 k.v. at magnification of 3000x. statistical analysis numerical data were explored for normality using kolmogorov-smirnov and shapiro-wilk tests. data showed parametric distribution so; it was represented by 6 salem et al. mean and standard deviation (sd) values. levene’s test was performed and the result was not significant so there was homogeneity of variances. three-way anova was then used to study the effect of different tested variables (adhesive, etching mode and aging method) and their interaction. comparison of main and simple effects was done utilizing bonferroni correction. the significance level was set at α≤0.05 within all tests. statistical analysis was performed with ibm (ibm corporation, ny, usa.) spss (spss, inc., an ibm company). statistics version 25 for windows. results micro-shear bond strength results mean and standard deviation (sd) values of micro-shear bond strength (mpa) for different aging methods (p<0.001), adhesives (p=0.001) and etching modes (p=0.596) are presented in table 2. in the control group, scotchbond universal adhesive showed significantly higher bond strength than all bond universal in both etching modes. after one year of water storage, all bond universal adhesive retained its bond strength for both modes. however, the bond strength of scotchbond universal was significantly reduced when compared with immediate bond strength. after thermocycling, statistical analysis revealed no significant difference between the immediate and thermocycled groups for both all bond universal adhesive and scotchbond universal as shown in table 2. regarding the effect of the etching mode, both adhesives showed no significant difference in bond strength either in the self-etch or the etch-and-rinse mode under all aging methods (p=0.596). table 2. mean ± standard deviation (sd) values of micro-shear bond strength (mpa) for the effect of different aging methods, adhesives and etching modes. etching mode adhesive aging method (mean±sd) p-value control one year water storage thermo cycling selfetch scotchbond universal 20.55±3.19aa 12.78±1.96ba 17.75±3.43aa <0.001* all bond universal 15.94±2.64ab 14.67±2.05aa 14.05±2.03ab 0.268ns p-value <0.001* 0.112ns 0.003* etchandrinse scotchbond universal 19.11±2.39aa 11.18±3.65bb 18.83±1.85aa <0.001* all bond universal 15.40±3.38ab 14.90±2.80aa 14.77±1.70ab 0.864ns p-value 0.004* 0.004* 0.002* different upper and lowercase superscript letters indicate a statistically significant difference within the same row or column respectively*; significant (p ≤ 0.05) ns; non-significant (p>0.05) failure mode analysis in immediate groups, the predominant failure mode was adhesive failure followed by mixed failure with remnants of adhesive and composite on the dentin surface, a similar tendency was found for thermocycled specimens. in water storage groups, the predominant mode of failure was adhesive failure as shown in table 3. 7 salem et al. table 3. bond failure mode for different groups. mode of failure aging methods control one-year water storage thermocycling sbse sber abse aber sbse sber abse aber sbse sber abse aber adhesive failure 70% 80% 70% 60% 100% 100% 80% 90% 90% 100% 80% 80% cohesive failure 0 0 0 0 0 0 0 0 0 0 0 0 mixed failure 30% 20% 30% 40% 0 0 20% 10% 10% 0 20% 20% sbse: scotchbond universal self etch, sber: scotchbond universal etch and rinse. abse: all bond universal self etch, aber: all bond universal etch and rinse. scanning electron microscope evaluation results in the control and thermocycling groups, both adhesives showed good integrity at the adhesivedentin interface with intact hybrid layer formation and numerous long intact resin tags formation in the etch-and-rinse mode with mean length of resin tags of 21.13 µm, 23.1 µm, 21.7 µm,24.5 µm in sber control, aber control, sber thermocycling and aber thermocycling groups respectively. however, in the self-etch mode a less distinct hybrid layer is seen with very few resin tags in case of scotchbond universal adhesive and absence of resin tags in case of all bond universal adhesive as shown in figures 1 and 2. a 21.13 ± 1.84 23.1 ± 2.1 c b d c d d d c c c d figure 1. sem image 3000x showing tooth /restoration interface in the control group, (a) scotchbond universal etch-and-rinse mode; (b) all bond universal etch-and-rinse mode; (c) scotchbond universal selfetch mode; (d) all bond universal self-etch mode (d) dentin; (c) composite; red arrows marking the hybrid layer; white arrows marking the resin tags, rectangle: mean length(µm) of resin tags ± standard deviation 8 salem et al. a 21.7 ± 2.14 23.5 ± 2.8 c b d c d d d c c c d figure 2. sem image 3000x showing tooth restoration interface after thermocycling, (a) scotchbond universal etch-and-rinse mode; (b) all bond universal etch-and-rinse mode; (c) scotchbond universal self-etch mode; (d) all bond universal self-etch mode; (d) dentin; (c) composite; red arrows marking the hybrid layer; white arrows marking the resin tags, rectangle: mean length(µm) of resin tags ± standard deviation. after one year of water storage, in scotchbond universal adhesive there was a clear deterioration in the hybrid layer with gap formation in both modes. on the other hand, in case of all bond universal adhesive a continuous hybrid layer is retained in both modes, with long resin tags in the etch-and-rinse mode as shown in figure 3. 9 salem et al. a 22.3 ± 3.1 19.9 ± 1.64 c b d c d d d cc c d figure 3. sem image 3000x showing tooth/restoration interface after one year water storage, (a) scotchbond universal etch-and-rinse mode; (b) all bond universal etch-and-rinse mode; (c) scotchbond universal self-etch mode; (d) all bond universal self-etch mode (d) dentin; (c) composite; red arrows marking the hybrid layer; white arrows marking the resin tags; yellow arrows marking gaps, rectangle: mean length(µm) of resin tags ± standard deviation. discussion adhesive technology has been well developed over the past few decades25. universal adhesives are claimed to offer the versatility of being used in either etch-and-rinse or self-etch modes to bond direct and indirect restorations6. in the present study, the two adhesives (scotchbond universal and all bond universal adhesives) have been evaluated to determine their dentin bonding ability under different etching modes and after aging. although both adhesives are marketed as universal adhesives, they differ from each other in the composition26. in the control group, the bond strength of scotchbond universal didn’t change significantly regardless the etching mode. this was in agreement with the studies carried out by marchesi et al.14 and munoz et al.3. similarly, the bond strength of all bond universal adhesive didn’t change significantly regardless the etching mode. this was in agreement with a study carried out by wanger et al.19. yet other studies3,27 showed that there was a drop in the bond strength of all bond universal in the self-etch mode when compared to the etch-and-rinse mode. the application of all bond universal without active brushing, was considered the main reason for the decrease in the bond strength in the self-etch mode. it has been demonstrated that active application of adhesives improves their bonding performance in the selfetch mode28. in the present study, all bond universal was applied by scrubbing the preparation surface according to the manufacturer’s instructions, which could explain the similar bond strength results in both modes19. 10 salem et al. however, the bond strength of scotchbond universal was higher than all bond universal. this may be attributed to the presence of fillers in the composition of scotchbond universal. many authors29-32 have reported higher bond strength for adhesive systems with incorporated filler particles than for unfilled products. this  may be because the addition of fillers increases the cohesive strength of the adhesive itself and plays a role in increasing the fracture resistance of the dentin-adhesive interface33. additionally, these nano-sized fillers may infiltrate into the demineralized tubules and intertubular dentin stabilizing the hybrid layer9,34. in the present study, the results of long-term water storage showed that responses to one year of water storage differ between the two adhesives. all bond universal adhesive retained its bond strength after one year of water storage. on the contrary, the bond strength of the scotchbond universal dropped for both bonding techniques. this was in agreement with study conducted by sai et al.9 so, the second null hypothesis is rejected. the bond stability of all bond universal adhesives after water storage, may be explained by the presence hydrophobic mdp functional monomers in their composition. these monomers are capable of bonding chemically to dentin, since mdp contains a polymerizable methacrylate group and a phosphate group capable of forming a stable salt with the calcium in hydroxyapatite35. in addition, the mdp monomers self assemble in hydrophobic nanolayers, which increases the strength of the adhesive interface3. it has been found that these water insolouble 10-mdp calcium salts have no effect on the immediate bond strength but contribute to the bond stability through protecting the hybrid layer from hydrolytic degradation5. although scotchbond universal adhesive also contains mdp monomers, the bond strength dropped in both modes after water storage. this might be attributed to the presence of poly-alkenoic acid copolymer (vitrebondtm) in its composition. poly-alkenoic acid copolymer bonds chemically and spontaneously to hydroxyapatite in dentin, yoshida  et  al.36 postulated that poly-alkenoic acid copolymer may compete with the mdp present in scotchbond universal reducing 10-mdp-calcium salts within the resin dentine interface5. moreover, it was postulated that the presence of polyalkenoic acid copolymer could have prevented the monomer approximation during polymerization resulting in a lower degree of conversion. in one study3, where the degree of conversion of universal adhesives was measured, a decreased degree of conversion was found for scotchbond universal when compared to other adhesives including allbond universal. the decreased degree of conversion and presence of unreacted monomers within the hybrid layer would increase the permeability of the adhesive layer, and thus decreasing the hydrolytic stability of the dental adhesive consequently reducing the interfacial strength of the adhesive interface37. another study38  measuring the extent of oxygen inhibition on free radical polymerization of several universal adhesives, found that scotchbond universal adhesives exhibited higher inhibition. moreover, the oxygen inhibition was not limited to the layer exposed to atmospheric oxygen but air bubbles were also incorporated in the adhesive film. these structural defects associated with oxygen inhibition were assumed to affect the long term stability and durability of the adhesive39. 11 salem et al. additionally, in contrast to all bond universal, scotchbond universal contains silane in its composition. it was postulated that silane could increase the hydrophilicity of scotchbond universal, thereby increasing the susceptibility of the adhesive layer to hydrolytic degradation40. all the abovementioned reasons may account for the decrease in the bond strength of scotchbond universal adhesive upon water storage. the second in-vitro degradation method used in the present study was the effect of thermocycling on the bond strength of universal adhesives. during thermocycling, specimens were subjected to thermal stresses in addition to the exposure to water. so thermocycling is expected to affect the bond strength in two ways; first through the stresses generated at the tooth-restoration interface due to the mismatch in the coefficient of thermal expansion and contraction between the restorative material and the tooth tissues19. second, the hot water may accelerate hydrolysis of non-protected collagen and extract poorly polymerized resin oligomers41. it has been postulated that 10,0000 cycles correspond to 1 year of clinical service, based on the hypothesis that around 20 – 30 cycles might occur intraorally per day42. another variable in the thermocycling protocol is the dwell time. it is the period of time that the specimen is immersed in a bath of a particular temperature. it represents the latency period that the oral capacity needs to reach its normal temperature again, after consuming hot or cold food and drink. a short dwell time of 10-15 s was suggested by several authors relying on the limited tolerance of patients to direct contact between vital tooth and extremely hot or cold substances43. in literature, there is controversy on the effect of thermocycling on the bond strength. some studies reported that thermocycling had no effect on bond strength to dentin, while others showed that it decreased the bond strength significantly19,44-46. in the present study, thermocycling had no effect on the bond strength of both adhesives. this may be justified by suggesting that higher temperatures during thermocycling induce secondary curing of unreacted monomers9. this secondary curing may result in higher resistance to the generated stresses and may overcome the hydrolytic effect of thermocycling since the susceptibility of resin to hydrolysis may result from a low degree of conversion47. the higher bond strength of single bond universal than all bond universal after thermocycling can be due the presence of fillers in its composition. it has been postulated that the addition of fillers not only reinforces the hybrid layer but also make the coefficient of thermal expansion of the adhesive resin closer to those of the dentin and resin based composites48. from the results of this study, the application mode didn’t affect the bond strength results in both adhesives in different groups. so the first null hypothesis is accepted. the bond strength results were further justified by sem findings. upon evaluation of the generated interface by sem, the adhesive interface morphology of the control groups revealed close morphological appearance to that of the thermocycling groups. in the self-etch mode of scotchbond universal, the hybrid layer was very thin with very few resin tags. in case of all bond universal, there was absence of hybrid layer and resin tags which may be explained by its ultramild acidity49. in case of etch-andrinse mode, acid etching resulted in removal of smear layer and smear plugs, deeper 12 salem et al. penetration of both universal adhesives into dentin with formation of long resin tags and thicker hybrid layers when compared to the self-etch mode. although there was a better interface morphology after acid etching, no relationship was observed between the quality of the hybrid layer (i.e., percentage of adhesive penetration)and bond strength50. in this study, there was no difference in the immediate bond strength of etch-and-rinse as well as self-etched dentin in both adhesives. in case of water storage, the sem analysis showed deterioration in the hybrid layer with gap formation after one year of water storage in either modes in case of scotchbond universal adhesive. however, in case of all bond universal adhesive, the sem micrographs showed that a continuous hybrid layer was retained in both modes, with long resin tags in the etch-and-rinse mode. this supports the bond strength results in the water storage. regarding the failure mode, it did not seem to follow any pattern in relation to treatment type. the predominant failure mode was adhesive failure regardless the degradation condition or etching mode. from this study, it can be concluded that universal adhesives represent a new class of adhesives that can be used in self-etch or etch-and-rinse mode without altering the bond strength to dentin. however, the durability of these adhesives depends greatly on the composition of these adhesives. more studies with different degradation methodologies are required to ensure the long term durability of universal adhesives. funding this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. references 1. vaidyanathan tk, vaidyanathan j. recent advances in the theory and mechanism of adhesive resin bonding to dentin: a critical review. j biomed mater res b appl biomater. 2009 feb;88(2):558-78. doi: 10.1002/jbm.b.31253. 2. nagarkar s, theis-mahon n, perdigão j. universal dental adhesives: current status, laboratory testing, and clinical performance. j biomed mater res b appl biomater. 2019 aug;107(6):2121-31. doi: 10.1002/jbm.b.34305. 3. muñoz ma, luque i, hass v, reis a, loguercio ad, bombarda nhc. immediate bonding properties of universal adhesives to dentine. j dent. 2013 may;41(5):404-11. doi: 10.1016/j.jdent.2013.03.001. 4. cuevas-suarez ce, da rosa wlo, lund rg, da silva af, piva e. bonding performance of universal adhesives: an updated systematic review and meta-analysis. j adhes dent. 2019;21(1):7-26. doi: 10.3290/j.jad.a41975. 5. chen c, niu l-n, xie h, zhang z-y, zhou l-q, jiao k, et al. bonding of universal adhesives to dentine–old wine in new bottles? j dent. 2015 may;43(5):525-36. doi: 10.1016/j.jdent.2015.03.004. 6. alex g. universal adhesives: the next evolution in adhesive dentistry. compend contin educ dent. 2015 jan;36(1):15-26; quiz 28, 40. 7. liu y, tjäderhane l, breschi l, mazzoni a, li n, mao j, et al. limitations in bonding to dentin and experimental strategies to prevent bond degradation. j. dent. res. 2011 aug;90(8):953-68. doi: 10.1177/0022034510391799. 13 salem et al. 8. sofan e, sofan a, palaia g, tenore g, romeo u, migliau g. classification review of dental adhesive systems: from the iv generation to the universal type. ann stomatol. 2017 jul 3;8(1):1-17. doi: 10.11138/ads/2017.8.1.001. 9. sai k, shimamura y, takamizawa t, tsujimoto a, imai a, endo h, et al. influence of degradation conditions on dentin bonding durability of three universal adhesives. j dent. 2016 nov;54:56-61. doi: 10.1016/j.jdent.2016.09.004. 10. van meerbeek b, peumans m, poitevin a, mine a, van ende a, neves a, et al. relationship between bond-strength tests and clinical outcomes. dent mater j. 2010 feb;26(2):e100-21. doi: 10.1016/j. dental.2009.11.148. 11. zhang z-y, tian f-c, niu l-n, ochala k, chen c, fu b-p, et al. defying ageing: an expectation for dentine bonding with universal adhesives? j dent. 2016 feb;45:43-52. doi: 10.1016/j. jdent.2015.11.008. 12. kaczor k, gerula-szymanska a, smektala t, safranow k, lewusz k, nowicka a. effects of different etching modes on the nanoleakage of universal adhesives: a systematic review and meta-analysis. j esthet restor dent. 2018 jul;30(4):287-98. doi: 10.1111/jerd.12375. 13. sezinando a, perdigao j, ceballos l. long-term in vitro adhesion of polyalkenoate-based adhesives to dentin. j adhes dent. 2017;19(4):305-16. doi: 10.3290/j.jad.a38895. 14. marchesi g, frassetto a, mazzoni a, apolonio f, diolosa m, cadenaro m, et al. adhesive performance of a multi-mode adhesive system: 1-year in vitro study. j dent. 2014 may;42(5):603-12. doi: 10.1016/j.jdent.2013.12.008. 15. lezaja zebic m, dzeletovic b, miletic v. microtensile bond strength of universal adhesives to flat versus class i cavity dentin with pulpal pressure simulation. j esthet restor dent. 2018 may;30(3):240-8. doi: 10.1111/jerd.12363. 16. manfroi fb, marcondes ml, somacal dc, borges ga, junior lh, spohr am. bond strength of a novel one bottle multi-mode adhesive to human dentin after six months of storage. open dent. 2016 jun;10:268-77. doi: 10.2174/1874210601610010268. 17. perdigao j, swift jr e, denehy g, wefel j, donly k. in vitro bond strengths and sem evaluation of dentin bonding systems to different dentin substrates. j. dent. res. 1994 jan;73(1):44-55. doi: 10.1177/00220345940730010601. 18. zhang l, wang d-y, fan j, li f, chen y-j, chen j-h. stability of bonds made to superficial vs. deep dentin, before and after thermocycling. dent mater j. 2014 nov;30(11):1245-51. doi: 10.1016/j. dental.2014.08.362. 19. wagner a, wendler m, petschelt a, belli r, lohbauer u. bonding performance of universal adhesives in different etching modes. j dent. 2014 jul;42(7):800-7. doi: 10.1016/j.jdent.2014.04.012. 20. tsujimoto a, barkmeier ww, takamizawa t, wilwerding t, latta ma, miyazaki m. interfacial characteristics and bond durability of universal adhesive to various substrates. oper. dent. 2017;42(2):e59-e70. doi: 10.2341/15-353-l. 21. sadr a, ghasemi a, shimada y, tagami j. effects of storage time and temperature on the properties of two self-etching systems. j dent. 2007 mar;35(3):218-25. doi: 10.1016/j.jdent.2006.08.004. 22. harnirattisai c, roengrungreang p, rangsisiripaiboon u, senawongse p. shear and micro-shear bond strengths of four self-etching adhesives measured immediately and 24 hours after application. dent mater j. 2012;31(5):779-87. doi: 10.4012/dmj.2012-013. 23. cheetham jj, palamara je, tyas mj, burrow mf. a comparison of the micro-shear bond strength and failure mode of non-enclosed and mold-enclosed luting cements bonded to metal. dent mater j. 2013;32(6):896-905. doi: 10.4012/dmj.2013-065. 24. lafuente d. sem analysis of hybrid layer and bonding interface after chlorhexidine use. oper. dent. 2012;37(2):172-80. doi: 10.2341/10-251-l. 14 salem et al. 25. van meerbeek b, yoshihara k, yoshida y, mine a, de munck j, van landuyt k. state of the art of selfetch adhesives. dent mater j. 2011 jan;27(1):17-28. doi: 10.1016/j.dental.2010.10.023. 26. costa dm, somacal dc, borges ga, spohr am. bond capability of universal adhesive systems to dentin in self-etch mode after short-term storage and cyclic loading. open dent j. 2017 jun 30;11:276-83. doi: 10.2174/1874210601711010276. 27. lee i-s, son s, hur b, kwon y-h, park j-k. the effect of additional etching and curing mechanism of composite resin on the dentin bond strength. j adv prosthodont. 2013 nov;5(4):479-84. doi: 10.4047/jap.2013.5.4.479. 28. pleffken pr, de almeida lourenco ap, torres c, buhler borges a. influence of application methods of self-etching adhesive systems on adhesive bond strength to dentin. j adhes dent. 2011 dec;13(6):517-25. doi: 10.3290/j.jad.a21417. 29. fortin d, swift jr ej, denehy ge, reinhardt jw. bond strength and microleakage of current dentin adhesives. dent mater j. 1994 jul;10(4):253-8. doi: 10.1016/0109-5641(94)90070-1. 30. wakefield cw, draughn ra, sneed wd, davis tn. shear bond strengths of six bonding systems using the pushout method of in vitro testing. oper. dent. 1998;23(2):69-76. 31. haller b. recent developments in dentin bonding. am j dent. 2000 feb;13(1):44-50. 32. frankenberger r, perdigao j, rosa bt, lopes m. “no-bottle” vs “multi-bottle” dentin adhesives--a microtensile bond strength and morphological study. dent mater j. 2001 sep;17(5):373-80. doi: 10.1016/s0109-5641(00)00084-1. 33. baracco b, fuentes mv, garrido ma, gonzález-lópez s, ceballos l. effect of thermal aging on the tensile bond strength at reduced areas of seven current adhesives. odontology. 2013 jul;101(2):17785. doi: 10.1007/s10266-012-0073-2. 34. carvalho cn, francci ce, costa jf, bauer j. effect of filler and application mode on micro-shear bond strength of etch-and-rinse adhesive systems. rev port estomatol cir maxilofac. 2015;56(2):89-94. doi: 10.1016/j.rpemd.2015.05.002. 35. lawson nc, robles a, fu cc, lin cp, sawlani k, burgess jo. two-year clinical trial of a universal adhesive in total-etch and self-etch mode in non-carious cervical lesions. j dent. 2015 oct;43(10):1229-34. doi: 10.1016/j.jdent.2015.07.009. 36. yoshida y, yoshihara k, nagaoka n, hayakawa s, torii y, ogawa t, et al. self-assembled nano-layering at the adhesive interface. j. dent. res. 2012 apr;91(4):376-81. doi: 10.1177/0022034512437375. 37. hass v, dobrovolski m, zander-grande c, martins gc, gordillo la, rodrigues accorinte mde l, et al. correlation between degree of conversion, resin-dentin bond strength and nanoleakage of simplified etch-and-rinse adhesives. dent mater j. 2013 sep;29(9):921-8. doi: 10.1016/j.dental.2013.05.001. 38. papadogiannis d, dimitriadi m, zafiropoulou m, gaintantzopoulou m-d, eliades g. universal adhesives: setting characteristics and reactivity with dentin. materials (basel). 2019 may 27;12(10):1720. doi: 10.3390/ma12101720. 39. hashimoto m, fujita s, endo k, ohno h. in vitro degradation of resin–dentin bonds with onebottle self-etching adhesives. eur j oral sci. 2009 oct;117(5):611-7. doi: 10.1111/j.16000722.2009.00664.x. 40. kim j, chae s, lee y, han g, cho b. effects of multipurpose, universal adhesives on resin bonding to zirconia ceramic. oper dent. 2015;40(1):55-62. doi: 10.2341/13-303-l. 41. de munck j, van landuyt k, coutinho e, poitevin a, peumans m, lambrechts p, et al. micro-tensile bond strength of adhesives bonded to class-i cavity-bottom dentin after thermo-cycling. dent mater j. 2005 nov;21(11):999-1007. doi: 10.1016/j.dental.2004.11.005. 15 salem et al. 42. morresi al, d’amario m, capogreco m, gatto r, marzo g, d’arcangelo c, et al. thermal cycling for restorative materials: does a standardized protocol exist in laboratory testing? a literature review. j mech behav biomed mater. 2014 jan;29:295-308. doi: 10.1016/j.jmbbm.2013.09.013. 43. amaral fl, colucci v, palma-dibb rg, corona sa. assessment of in vitro methods used to promote adhesive interface degradation: a critical review. j esthet restor dent. 2007;19(6):340-53; discussion 354. doi: 10.1111/j.1708-8240.2007.00134.x. 44. gale ms, darvell bw. thermal cycling procedures for laboratory testing of dental restorations. j dent. 1999 feb;27(2):89-99. doi: 10.1016/s0300-5712(98)00037-2. 45. abo-hamar se, hiller ka, jung h, federlin m, friedl kh, schmalz g. bond strength of a new universal self-adhesive resin luting cement to dentin and enamel. clin oral investig. 2005 sep;9(3):161-7. doi: 10.1007/s00784-005-0308-5. 46. shirai k, de munck j, yoshida y, inoue s, lambrechts p, suzuki k, et al. effect of cavity configuration and aging on the bonding effectiveness of six adhesives to dentin. dent mater j, 2005 feb;21(2):11024. doi: 10.1016/j.dental.2004.01.003. 47. helvatjoglu-antoniades m, koliniotou-kubia e, dionyssopoulos p. the effect of thermal cycling on the bovine dentine shear bond strength of current adhesive systems. j. oral rehabil. 2004 sep;31(9):911-7. doi: 10.1111/j.1365-2842.2004.01318.x. 48. fanning de, wakefield cw, robbins jw, bagley al. effect of a filled adhesive on bond strength in three dentinal bonding systems. gen dent. 1995;43(3):256-62. 49. ermis rb, de munck j, cardoso mv, coutinho e, van landuyt kl, poitevin a, et al. bond strength of self-etch adhesives to dentin prepared with three different diamond burs. dent mater j. 2008 jul;24(7):978-85. doi: 10.1016/j.dental.2007.11.019. 50. perdigao j, sezinando a, monteiro pc. laboratory bonding ability of a multi-purpose dentin adhesive. am j dent. 2012;25(3):153-8. 1 volume 21 2022 e226666 original article braz j oral sci. 2022;21: e226666http://dx.doi.org/10.20396/bjos.v21i00.8666666 1 dental materials department, school of dentistry, federal university of rio grande do sul, porto alegre, brazil. 2 department of preventive and social dentistry, school of dentistry, federal university of rio grande do sul, porto alegre, brazil. 3 post-graduate program in dentistry, federal university of rio grande do sul, porto alegre, brazil. corresponding author: fabricio mezzomo collares +555133085198. fabricio.collares@ufrgs.br rua ramiro barcelos, 2492. porto alegre, rio grande do sul. brazil. editor: altair a. del bel cury received: august 13, 2021 accepted: april 2, 2022 cost-related variables in the public purchase of dental materials for endodontic application: 10-year analysis gabriela de souza balbinot1 , roger keller celeste2 , francisco montagner3 , fabricio mezzomo collares1,* aim: this study analyzed public procurements for different endodontic materials used in the brazilian public health system and evaluated the variables related to their cost. methods: a time-series study was performed by screening materials for endodontic application in the public brazilian databank of healthcare prices from 2010 to 2019. data were categorized according to material composition and clinical application. the collated variables were used in a multiple linear regression model to predict the impact of unit price in procurement processes. results: a total of 5,973 procurement processes (1,524,693 items) were evaluated. calcium hydroxides were found in 79% of the observations (4,669 processes). prices drop each year by us$1.87 while mtas and epoxy resins are increasingly purchased at higher prices (us$50.87; us$67.69, respectively). the microregion, the procurement modality, and the type of institution had no influence on unit prices in the adjusted model (p > 0.05). conclusions: calcium hydroxide-based materials were the cheapest and most frequently purchased endodontic materials in the public health care system. novel formulations are being implemented into clinical practice over time and their cost may be a barrier to the broad application of materials such as mtas, despite their effectiveness. keywords: dental materials. endodontics. public health dentistry. translational science, biomedical. costs and cost analysis. https://orcid.org/0000-0001-9076-2460 https://orcid.org/0000-0002-2468-6655 https://orcid.org/0000-0002-7850-0107 https://orcid.org/0000-0002-1382-0150 2 balbinot et al. braz j oral sci. 2022;21: e226666 introduction oral diseases are prevalent conditions that impact public health1 and an individual’s quality of life2. dental caries and trauma frequently lead to damage of dental pulp tissue, contributing to pain and infection, which may progress to tooth loss. in these cases, endodontic treatments aim to reestablish periapical tissues by controlling bacterial infection in the root canal system, contributing to the maintenance of the tooth structure3,4. access to these treatments is known to impact patients’ pain and comfort levels and represents an essential factor in the maintenance of oral health5. the prevalence of endodontic-related conditions varies among populations, and the need for intervention ranges from 4.8% to 6.3% for different populations5,6. the need for technology and a specialized dental workforce limits the implementation of endodontic care in health care systems, especially considering the cost of these procedures and the impact they have on the public budget7,8. endodontic interventions range from pulp capping to endodontic surgeries that require specific materials that modulate the healing process in most cases. these materials aim to stimulate new tissue formation using minimally invasive approaches for small pulp injuries and to seal the root canal space after tissue removal in cases where root canal treatment is needed9. the selection of materials is related to the type of intervention, professional experience or clinical skills, and the available evidence about the material’s performance. these factors should guide the dentist’s and the public system manager’s decision-making process in purchasing materials for clinical practice10. the procurement of materials in the public systems is a tool to understand the current clinical practice and the adherence to evidence-based knowledge in the endodontic field11. the brazilian public health system includes dental treatment from primary to advanced care, including endodontic interventions12. the application of materials must consider both the effectiveness of treatments as well as the cost of these materials and their impact on the public budget. the screening for public expenditure in the biomedical fields helps to design cost-effective strategies for the public healthcare system and is observed currently for medicines and health care materials13,14, including dental adhesives11, but no information regarding the endodontic materials is known. this study aims to collate and analyze endodontic materials’ public procurements from 2010 to 2019, investigating the purchase frequency of different materials and the variables related to their cost. material and methods a time-series study was performed by screening the brazilian databank of healthcare prices (bdhp), a public database that collates secondary data of dental materials for endodontic application from january 1, 2010, to december 31, 2019. data were organized in a single databank, and all analyses were conducted using stata 14 (statacorp llc, tx, usa). the material description, the date of purchase, the institution that purchased the item, the product manufacturer, the number of purchased items, and the unit price were collated. materials were 3 balbinot et al. braz j oral sci. 2022;21: e226666 divided into four different categories according to their composition: 1) calcium hydroxide-based (ch) materials, 2) zinc oxide-eugenol (zoe), 3) mineral trioxide aggregates (mtas), and 4) epoxy resins (ers). as secondary data was assessed, the classification took the materials manufacturer into consideration, and inconsistencies in the relationship between these two variables were adjusted to avoid misinterpretation. observations were further divided within each composition by considering their commercial presentation and, consequently, their application. the procurement processes were divided according to the type of institution that purchased the products and their location. the unit price was adjusted according to the national wide consumer price index (índice nacional de preços ao consumidor amplo or ipca) for each month/year over time. the adjusted brazilian reais (brl) were converted to united states dollars (us$) using the daily exchange rate of the central bank of brazil. the frequency in the procurement process and the number of purchased items were descriptively analyzed, and the average adjusted us$ prices were used in a multiple linear regression model using ordinary least squares. the categories that most frequently appear in the databank were used as a reference in the multiple linear regression analysis. data were analyzed with bivariate tables and statistical significance was tested with a one-way analysis of variance. as part of regression diagnostic, studentized residuals larger than 3 were investigated for their impact on the assumption. then, 17 (0.28% of the total number of observations) values larger than 12.37 were excluded. the multiple regression linear model was cluster adjusted, as an interclass correlation coefficient showed that 10.4% of data variability may have been related to the institution that purchased the material. this adjustment considered 789 institutions that registered processes in the databank. results a total of 5,973 procurement processes were evaluated, resulting in 1,524,693 purchased items. figure 1 shows the descriptive analysis of purchased items in each composition and for different commercial presentations. chs-based corresponded to 79% of the procurement process (fig.1a) and 64% (fig.1c) of the overall number of purchased endodontic materials in the databank. the frequency of purchase for each composition over the years is found in figure 2. the number of procurement processes increase over the years for all analyzed materials. 4 balbinot et al. braz j oral sci. 2022;21: e226666 a b c d ch zoe mta er ch zoe mta er paste/paste powder syringepowder/liquid number of procurement process number of procurement process in each composition for different commercial presentation number of purchased itens number of purchased itens in each composition for different commercial presentation ch zoe mta er ch zoe mta er paste 4% 6% 12% 79% 5% 6% 25% 64% figure 1. descriptive analysis for the number of the procurement process and the overall number of purchased materials from 2010 to 2019. the screening in public procurement described the number of the procurement process that were conducted to purchase different dental materials (a) and their different commercial presentations (b). the number of purchased items in each process was used to calculate the overall number of purchased items in each composition (c) and their commercial presentations (d). a b b c 1.000 800 600 400 200 0 year calcium hydroxides zinc oxide-eugenol mta epoxi resin n um be r o f p ro cu re m en t p ro ce ss 2012 2013 2014 2015 2016 2017 2018 2019 1.000 1.200 1.400 800 600 400 200 0 macroregionn um be r o f p ro cu re m en t p ro ce ss south southeast mid-west northeast north 5.000 4.000 3.000 2.000 1.000 0 procurement modality n um be r o f p ro cu re m en t p ro ce ss auction bid-waiver other 2.500 3.000 4.000 3.500 2.000 1.500 1.000 500 0 types of institution n um be r o f p ro cu re m en t p ro ce ss universities other federal institutions municipalities figure 2. the procurement process for each composition, according to the analyzed categories. 5 balbinot et al. braz j oral sci. 2022;21: e226666 as shown in table 1, all variables were statistically significant and were thus included in the adjusted model in table 2. the adjusted model for the regression analysis is shown in table 2. the prices among different material compositions show that ch (reference category) was the cheapest material in the analyzed data. zoe sealers were purchased for us$6.19 more than the reference (p < 0.01) while ers were shown to cost us$44.76 more than ch-based materials (p < 0.01). year predicted a reduction of us$1.87 in endodontic material unit prices (p < 0.01). institution location did not modify the price of the materials, and the procurement processes that were conducted by bid-waiver were us$10.50 more expensive than the ones performed by auction. municipalities paid less for the products than did other federal institutions (us$2.25; p < 0.01). the differences between manufacturer prices showed high variation among the categories. table 1. the average price and standard deviation were calculated for each category on the variables. the analysis was conducted considering the different compositions. univariate analysis (us$) st.dev frequency p-value material composition ch 12.41 19.20 4,669 zoe 19.20 24.15 768 mta 50.86 25.12 323 er 67.69 24.46 308 <0.01 year 2012 21.38 30.49 168 2013 22.13 33.63 635 2014 18.39 24.09 591 2015 13.96 20.04 718 2016 16.92 22.48 856 2017 19.73 27.31 905 2018 14.83 20.71 974 2019 15.95 19.84 1,121 <0.01 macroregion south 18.46 29.48 1,315 southeast 18.66 25.32 1,557 mid-west 18.59 24.59 795 northeast 15.14 19.37 1,519 north 15.46 19.50 782 <0.01 procurement modality auction 16.65 27.12 5,159 bid waiver 21.82 23.74 774 other 11.58 14.96 35 <0.01 continue 6 balbinot et al. braz j oral sci. 2022;21: e226666 continuation type of institution universities 15.35 25.06 1,239 other federal institutions 18.65 24.80 4,202 municipalities 11.09 14.24 527 <0.01 manufacturer aaf 5.38 6.13 83 angelus 51.32 25.18 330 biodinâmica 4.60 7.79 830 caitech 8.23 5.28 4 coltene 10.39 6.16 53 dentsply 35.33 30.83 903 iodontosul 2.95 2.95 94 kavokerr 67.44 28.96 21 maquira 2.45 3.56 366 septodont 45.99 0 2 ss white 16.32 6.20 661 technew 7.54 6.43 1,559 ultradent 12.21 5.71 81 other 23.76 33.44 981 <0.01 table 2. adjusted price differences (coeff) from multiple linear regression of the procurement processes (n=5,968). price coeff (us$) 95% conf. interval material composition (reference category: calcium hydroxides) zinc oxide eugenol 6.19 3.31 9.06 mta 16.99 11.05 22.93 epoxy resin 44.76 38.24 51.29 year -1.87 -2.51 -1.22 macroregion (reference category: southeast) south 1.85 -2.94 6.64 mid-west 0.82 -1.95 3.59 northeast 0.03 -2.16 2.22 north -0.98 -3.28 1.30 procurement modality (reference category: auction)  bid waiver 10.15 -34.68 14.30 other 0.908 -4.70 6.51 continue 7 balbinot et al. braz j oral sci. 2022;21: e226666 continuation type of institution (reference category: other federal institutions) universities 0.17 -4.01 4.37 municipalities -2.25 -5.17 0.67 manufacturer (reference category: technew) aaf -6.84 -9.11 -4.57 angelus 31.44 25.53 37.34 biodinâmica -3.10 -4.44 -1.76 caitech -5.69 -10.10 -1.29 coltene 1.37 -1.33 4.07 dentsply 19.91 14.72 25.11 iodontosul -5.64 -7.16 -4.12 kavokerr 53.00 34.18 72.61 maquira -2.50 -3.75 -1.25 septodont 31.13 27.43 34.83 sswhite 10.09 8.23 11.25 ultradent 8.23 5.75 10.71 other 20.85 -3.40 45.10 cons 3782 2485 5079 note: r2= 45.41%. discussion access to endodontic treatment in a public health system depends on different health care levels, as the complexity of treatments may vary according to the diagnosis and available resources for assistance15. most endodontic therapies involve the application of the materials screened in the present analysis. four material compositions were used to address the public purchases of endodontic materials between 2010 and 2019. the registered procurement process showed that ch-based products were the most purchased and cheapest materials during the analyzed period. increasing purchases of mtas and er are observed despite their higher prices compared to ch-based materials. the linear model showed that a reduction in prices is expected. procurement processes that were conducted with auction modality presented a reduced cost for the health care system. the selected materials were divided by composition based on well-known formulations commonly used for endodontic procedures in clinical practice (fig.1). ch-based materials are indicated for several procedures with different commercial presentations as seen in figure 1. this may be one reason why these are the most frequently purchased materials in the analyzed period. the purchase of ch-based materials, regardless of their presentation, was consistent over the years as the number of registered processes increased (fig.2). their application as an intracanal medication in the paste presentation justifies the high number of the purchased product while the most purchased presentation is the paste/paste indicated for 8 balbinot et al. braz j oral sci. 2022;21: e226666 pulp-capping procedures16. the high number of procurement processes is maintained in this case despite the recent clinical evidence that shows mtas’ superior performance in pulp-capping treatments17. recently, mtas have been compared to ch and pulpotomy dressing agents for pulpotomy treatment in immature permanent teeth18, with a slightly greater benefit found for mtas in a 12-month analysis. in addition, mtas had overall better clinical and radiographic success rates for endodontic treatment in immature necrotic permanent teeth when compared to ch replacement or revascularization strategies19. although figure 2 shows an increase in mta purchases from 2015 to 2019, these purchases represent only 6% of the total purchased materials (fig. 1). as the mta materials were more recently developed, it was expected that they would occupy a smaller portion of the market share while their increasing applications and clinical effectivity were reported. the extensive clinical use of ch-based products and their low costs may be important factors in the decision-making of dentists and managers. the procurement processes for ch-based materials were conducted with lower average prices while mtas areus$16.99 is more expensive than chs (table 2). although these two compositions are not necessarily indicated for the same treatment in all clinical conditions, treatments with mtas could represent a higher cost for the system. mtas’ cost is known to be a drawback for its application in clinical practice, and it may be especially critical for a large-scale public system where this could affect the public budget and access to this treatment and where budget cuts are common20. still, it is essential to consider the clinical and laboratory data that has recently shown better outcomes with mtas than ch-based materials16-18,21. besides clinical success, mtas’ use may increase patient compliance and reduce the number of dental appointments, impacting the overall cost of treatment22. a prospective randomized clinical trial reported that 4 out of 15 teeth treated with ch as a material for inducing root apex closure in immature necrotic teeth exhibited coronal or radicular fractures after 12 months; no tooth loss was observed in mta-treated teeth21. it must be considered that premature tooth loss may have negative psychological effects on the patient, on the patient’s quality of life, and the costs for the health system8. although the implementation of novel technologies in health care may take time and there are still clinical situations that may benefit from ch, novel materials are constantly under development and investigation. the generation of novel evidence may shift the standard of care when new and effective products are available. while mtas show promising results in regenerative therapies, the need for translation of current evidence and the impact on the public budget may limit its use in the public health care system7. root canal sealers were also found in the databank, and as observed in figure 1, the zoe sealers were purchased in 710 procurement processes, representing 25% of the total number of purchased materials. the application of these sealers for root canal filling is well established and is observed in the purchases made for the powder and liquid commercial presentation (fig. 1b and 1d). the high number of procurement processes and the consistency of these numbers over the years may be due to these materials’ long clinical use9. besides the popularity of zoe, its price may play an important role in the procurement of these materials, as observed in 9 balbinot et al. braz j oral sci. 2022;21: e226666 the linear model (table 2). the unit price for these sealers is reduced compared to the reference, but when zoe coefficients are compared to that of er and mtas, it is possible to observe decreasing values in the adjusted model (us$6.19; p < 0.05). er was the most expensive material (fig.1) and, although it represents a small fraction of the total amount of endodontic materials (4%), an increase in the number of procurements was observed throughout 2016. although evident physicochemical and biological differences are observed between root canal sealers’ compositions9, there is still no evidence for superior clinical performance for any of these materials in root-filling procedures9. the establishment of evidence-based protocols, in this case, requires further high-quality analysis to provide information for practitioners and managers in the decision-making process. the implementation of guidelines and innovative materials requires solid scientific evidence and the diffusion of knowledge for clinical practice. the absence of synthesized evidence, in this case, may contribute to the broad utilization of well-known zoe sealers. the increase in ers and mtas purchases may represent modifications of clinical practice that must consider the cost-effectiveness of the treatments in the decision-making process. it should be noted that besides direct and indirect costs related to treatment and a material’s cost on the public expenditure, more effective and lasting therapies directly impact the need for reinterventions and may be beneficial for impacting the dental health in the population23. public procurement processes were analyzed considering different variables that may affect dentists’ and managers’ choices. the year of purchase was shown to significantly predict a reduction in unit prices (table 2). a drop of us$1.87 was predicted for each year (table 2; p < 0.01), and this may be related to the increased number of available materials on the market24. the procurement modality predicts a reduction, and these findings follow the recommendations of regulatory agencies that classify auctions as the procurement modality for public purchases due to a reduction in costs and an increase in transparency25. the manufacturer also modifies the unit price. although a high variation is observed in the linear coefficients, it is possible to observe that differences in prices are driven by material compositions (table 2). the er and mta manufacturers presented higher coefficients than the reference category known to produce mainly ch-based materials. comparisons within each composition were not possible in the current model due to the variations in the commercial presentations. the follow-up in the analysis of the databank in the next years may provide enough data for further analysis. the information provided by this analysis may contribute to the formulation of public policies to the application of endodontic materials considering the laboratory and clinical evidence and the need for rationality in public purchases. despite a large number of laboratory studies on dental materials for endodontic purposes, a robust synthesis of knowledge with high-quality clinical studies is required to guarantee clinically and cost-effective treatments26 and to reduce the influence of the personal preferences of professionals. variations in recommended protocols and the lack of a conclusive synthesis may jeopardize scientific-knowledge diffusion and the establishment of standardized clinical practice. the continuous analysis of 10 balbinot et al. braz j oral sci. 2022;21: e226666 the purchased products could provide insights into the current standard of care in endodontics. with the present findings, it is possible to understand the modifications in clinical behavior over the system and to assess their influence on the public budget and the quality of treatments provided in the field. acknowledgements g.s.b would like to thank capes “coordenação de aperfeiçoamento de pessoal de nível superior” brazil finance code 001-schoolarship. data availability datasets related to this article will be available upon request to the corresponding author. conflict of interest none author contribution g.s.b: conception and design of the work; analysis and interpretation of data; drafting. r.k.c: analysis and interpretation of data; work revision for important intellectual 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public health dent. 2019 dec;79(4):273-4. doi: 10.1111/jphd.12344. 1http://dx.doi.org/10.20396/bjos.v19i0.8658127 volume 19 2020 e208127 original article 1 school of dentistry, yasuj university of medical sciences, iran. 2 social determinants of health research center, yasuj university of medical sciences, iran. (*corresponding author) 3 department of operative dentistry, school of dentistry, yasuj university of medical sciences, iran. *corresponding author: mohammad malekzadeh social determinants of health research center, yasuj university of medical sciences, iran email: mzh541@yahoo.com mobile number: 00989171454340 received: january 18, 2020 accepted: july 12, 2020 dental anxiety and the effectiveness of local anesthesia hadi esmaeili1 , mohammad malekzadeh2,* , davood esmaeili1 , farid nikeghbal3 aim:  the successful anesthesia is an essential factor for dental treatment. this study aimed at determining the effectiveness of local anesthesia and it’s relationship with dental anxiety. methods:  this cross-sectional study was carried out on 256 dental patients, in gachsaran, iran in 2017. dental anesthesia was administered after completing the modified dental anxiety scale by the patients. the level of anesthesia was recorded in one of three states (successful, difficult, and failed). collected data was analyzed using spss version 22 and tests of, chi-square, independent t-test, analysis of variance, and logistic regression model.  results:  about 60.5% subjects had moderate-severe dental anxiety. the mean of dental anxiety significantly was lower in the successful anesthesia group (p<0.01). patients with elementary education had a significantly higher level of dental anxiety (p<0.01). dental anxiety was significantly higher in the age group of ≥59 years, compared to the other age groups, except for 49-58 years (p<0.05). subjects with a significantly higher level of dental anxiety more delayed their visits to the dentist. the logistic regression model showed that the dental anxiety (high anxiety) and literacy level (elementary) were the most important predictors of failed or difficult anesthesia.  conclusion:  informing patients about dental treatment procedures, regular and periodic visits to the dentist, using psychotherapeutic techniques to reduce dental anxiety before anesthesia, could play an important role in the success of anesthesia. keywords: anesthesia, local dental anxiety, pain. https://orcid.org/0000-0003-3400-263x https://orcid.org/0000-0002-0451-0097 https://orcid.org/0000-0001-9493-5904 https://orcid.org/0000-0001-9147-8744 2 esmaeili et al. introduction dental anxiety is a patients’ response to stress in a dental setting1. medical procedures cause a feeling of fear, inability, as well as anxiety2. it can be provoked due to multiple factors, such as previous negative or traumatic experience, sensory triggers such as sights of needles and air-turbine drills, sounds of drilling and screaming, vicarious learning from anxious people, patients’ personality characteristics and their coping strategies3-8. several studies have reported high dental anxiety levels in approximately 10–20% of participants9,10. pain perception during the administration of local anesthetics is an essential reason for anxiety, and it may be caused by tissue puncture, pressure and velocity of fluid injection, the temperature of the anesthetic, and operator’s skills11. this problem can affect various treatment stages and complicate the situation for patients and dentists12. pain seems to be multifactorial and is influenced by psychological factors such as catastrophizing and anxiety13,14. people with a high score on pain catastrophizing reported more severe pain and anxiety, and they consume more analgesic medication15-17. anxious patients experience more negative and irrational thoughts related to dental treatment. they usually consider the worst-case scenario in their treatment. avoiding dental treatment due to dental anxiety is related to more missing and decayed teeth18. poor oral and dental health can lead to dental diseases, which reduces the patients’ quality of life and creates a vicious cycle, where patient’s anxiety level increases and their health level decreases19,20. one of the most important factors in patient satisfaction is pain control techniques. canakci has stated that measuring pain is difficult because it has physical and psychological aspects. it is subjective and depends on the patient’s perception21. local anesthesia has enabled the profession to make tremendous therapeutic advances. patients experience severe pain in case of failed anesthesia, which prevents many dental treatments, including root canal surgery, periodontal surgery, and tooth extraction22. although dental anesthesia is an essential aspect of treatment for patients, an injection can induce anxiety or fear and maybe a reason for patients to avoid dental treatment23. patients with high levels of anxiety usually exhibit lower pain thresholds24, and therefore, there is a decreased anesthesia success rate among these patients, and complementary methods are required in this regard. notably, dentists’ efforts and motivation to prevent pain in patients play an essential role in keeping them calm and relax25. dental anxiety can affect a patient’s life. physiological effects include signs and symptoms of fear and fatigue appears after a dentist’s appointment, whereas cognitive impacts are a set of negative beliefs and thoughts. behavioral results show itself as eating, lack of oral hygiene, self-treatment, and aggression. moreover, dental anxiety can have adverse effects on general health due to its association with sleeping disorhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmc4790493/#b10-ccide-8-035 3 esmaeili et al. ders. furthermore, the social interactions and performance of these individuals at the workplace may decrease due to a lack of self-esteem and self-confidence26. medical evidence shows a strong relationship between oral and general health. periodontal diseases and systemic diseases have a bidirectional relationship, and there are more than 100 systematic diseases with oral manifestations, such as cardiovascular diseases, stroke, respiratory infections (e.g., aspiration pneumonia), pancreas cancer, diabetes, and nutritional problems. therefore, it can be stated that the treatment of oral diseases plays a vital role in the general health27,28. given the importance of successful anesthesia for dental care treatments, recognition, and control of factors involved in its effectiveness can help increase the possibility of successful anesthesia. this study differs from other studies in two ways. in most studies, patients ‘anxiety levels were measured at the end of treatment. in contrast, in the present study, patients’ anxiety was measured before starting treatment, indicating dental anxiety related to the patient’s previous experiences. besides, most studies have measured patients’ pain at the end of treatment with methods such as visual analog scale (vas), while in this study, the success rate of anesthesia has been considered. given the fact that the majority of studies have assessed the relationship between anxiety and pain level, and little attention has been paid to the success of anesthesia, the present study aimed to determine the relationship between dental anxiety and the success rate of anesthesia in dental patients. materials and methods this cross-sectional study was conducted between february and december 2016. after receiving permission from the research council and the ethics committee of yasuj university of medical sciences. from 15 dental clinics in gachsaran city in the south of iran, five clinics were randomly selected by cluster sampling. among the patients referred to these clinics, 256 patients who met inclusion criteria were randomly selected by simple random sampling. the inclusion criteria for participation in the study were adults aged between 18 and 70 years, no cognitive impairment, and obtaining informed consent to participate in the study. in this study, all patients approached by the same processes. at each dental clinic, patients were given the necessary explanations about the purpose of the research and how to fill out the questionnaires by a person with a bachelor’s degree in psychology who had received the necessary training. also, five dental surgeons with more than ten years of experience performed local anesthesia, who had already received the required training about the study, method of local anesthesia, and its success rate. at first, research objectives were explained to the patients waiting for their dental treatment that required local anesthesia. in order to assess the anxiety level of patients before anesthesia, the modified dental anxiety scale (mdas), demographic characteristics (age, gender, level of education, and the last dental visit) and informed 4 esmaeili et al. consent form were completed by patients. after local anesthesia it’s success rate was recorded by the dentist.  research tools modified dental anxiety scale (mdas): the mdas was applied to assess the anxiety of dental patients. this five-items questionnaire is scored based on a five-point likert scale (from no anxiety=1 to extremely anxious=5). besides, the score range of the scale is 5-25; a higher score indicates a higher anxiety level. this total score can be classified, as follows: minimum anxiety (5-9), moderate anxiety (10-12), high anxiety (13-17), and extremely anxious (18-25) who need special care29. in the present study, we applied 1.8 ml 2% lidocaine and 1:10000 epinephrine as local anesthesia. after 10 minutes of administration, the dentist evaluated the soft tissue (e.g., lips and gum) and hard tissue anesthesia in the mouth and related teeth using pinprick and cavity tests, respectively. in the pinprick test, a relatively sharp tool (e.g., a probe) is used to assess soft tissue anesthesia. in this context, the probe is entered into the desired tissue, and the level of pain perceived was compared to the soft tissues on the other side of the jaw. in case of soft tissue anesthesia, the cavity test was performed with a drill to determine whether hard tissue anesthesia was achieved or not. in addition, the effect of dental anesthesia was recorded in one of the following three items: 1. successful anesthesia: this item is selected if soft and hard tissue anesthesia is achieved in both tests, and the patient has no irregular pain during the treatment process.  2. difficult anesthesia: anesthesia is re-administered by the dentist if the pain is perceived in a probe or drill test. in case of complete soft and hard tissue anesthesia after re-administration of anesthesia and the use of complementary injection techniques, this item is selected by the dentist.  3. failed anesthesia: in case of lack of anesthesia in lips and mouth after re-administration of anesthesia and the use of complementary techniques, treatment is postponed to another day. in fact, the dentist announces anesthesia failure and selects this item. data analysis data analysis was performed in spss version 22 using chi-square, independent t-test, analysis of variance (anova) tests and logistic regression model.  results from 256 subjects, 144 were female (56.3%), and 112 were male (43.8%). in addition, the mean age and standard deviation of the participants were 35.28±12.56 years. moreover, the mean and standard deviation of dental anxiety of the subjects was 14.08±6.56. furthermore, the mean and standard deviation of the last visit to the dentist was 33.06±35.87 months. other research variables are presented in table 1.  5 esmaeili et al. table 1. the number and percent of research variables percentnvariable age 28.97418-28 29.37529-38 11.73039-48 3.5949-58 6.61759 and higher gender 43.8112male 56.3144female education level 28.573elementary school 41.4106high school 28.974university local analgesia 4.311failure 40.6104difficult 54.7140successful dental anxiety 10.2026no anxiety 29.3075low 11.3029moderate 12.5032high 36.7094extreme according to table 1, most participants were in the age group of 29-38 years (29.3%). in terms of literacy, most subjects were in the high school group (41.4%). furthermore, regarding dental anesthesia, the majority of participants were in the successful anesthesia group (57.4%), followed by the group of difficult anesthesia group (40.6%). in terms of dental anxiety, most subjects had mild anxiety (39.5%), while 31.6% of the participants had very high dental anxiety. in general, 39.5% of the participants had mild anxiety, and 60.5% had anxiety ranging from moderate to extreme.  in this study, we applied the analysis of variance (anova) to determine whether there was a difference among three groups of successful, difficult and failed anesthesia in the mean of anxiety (table 2). table 2. anova test to compare dental anxiety in three groups of successful, difficult and failed anesthesia local anesthesia n anxiety f sig mean st.d failed 11 17.9091 7.31375 29.95 0.001difficult 104 17.1923 6.44781 successful 140 11.4857 5.40515 st.d: standard deviation 6 esmaeili et al. according to the mentioned table, there was a significant difference among the groups in the mean of anxiety (p=0.001, f=29.95). also, the tukey post hoc test was exploited to determine the substantial difference in groups regarding the mean of anxiety. in this regard, there was a significant difference between the group of successful anesthesia and the two groups of difficult anesthesia and failed anesthesia (p<0.01). the results were indicative of a significantly lower dental anxiety in the subjects of the successful anesthesia group, compared to the other groups. according to anova results (table 3), there was a significant difference between the three groups of elementary education, high school, and university degrees in the mean of dental anxiety (p>0.01). table 3. anova test to compare dental anxiety in three groups of elementary, high school and university level educational level n mean st.d f sig elementary school 73 17.08 6.45 11.22 0.001high school 106 12.77 6.33 university 74 13.14 6.23 st.d: standard deviation according to table 3, the mean dental anxiety was significantly different in three groups of elementary education, high school, and university degrees (p<0.01). the tukey post hoc test showed a significant difference between the group of elementary education with groups of high school and university degrees regarding mean dental anxiety (p<0.01). the level of dental anxiety was higher in subjects with elementary education, compared to the other groups. however, no significant difference was found between the groups of high school and university degrees in this respect (p>0.05). besides, anova was indicative of a significant difference among various age groups regarding the mean anxiety level (p<0.01). according to the results, there was a considerable difference between the age group of ≥59 years and the age groups of 18-28 years (p<0.05), 29-38 years (p<0.01), and 39-48 years (p<0.01) in terms of dental anxiety. however, no significant difference was observed between the age groups of ≥59 years and 49-58 years (p>0.05). the findings were also indicative of a higher dental anxiety level in the age group of ≥59 years. according to the chi-square results, no significant relationship was observed between the level of anesthesia and variables of age and level of education (p>0.05). table 4 shows the mean and standard deviation of the last visit (month) to the dentist at various anxiety levels. 7 esmaeili et al. table 4. the mean and standard deviation of the last visit to the dentist (month) at several levels of anxiety anxiety n last visit mean st.d no anxiety 17 12.02 12.28 low 50 17.12 26.34 moderate 20 14.91 11.75 high 28 25.66 30.72 extremely 91 52.01 39.20 st.d: standard deviation according to this table, the highest mean of last visits to the dentist (52.01±39.20) was related to subjects with a high anxiety level. also, anova showed that the mean of the last visit to the dentist had a significant difference at various anxiety levels (p=0.001, f=15.12).  according to the tukey test, the group of high anxiety levels had a significant difference with other groups (no anxiety, low anxiety, moderate anxiety, and high anxiety) regarding the mean last visit to the dentist (p<0.01). in addition, the mean and standard deviation of the last visit to the dentist was significantly lower in female participants (22.74±27.71) compared to male subjects (45.12±40.44) (p<0.001). furthermore, we applied the logistic regression test to determine the most important predictors of the level of anesthesia. level of anesthesia at two levels (successful anesthesia and difficult and failed anesthesia) was considered as the dependent variable while the variables of age, gender, literacy, and dental anxiety level were entered into the equation as independent variables. finally, the variables of literacy level and anxiety were significant and remained in the regression equation. since the r square of the equation was equal to 0.39, the independent variables (literacy level and dental anxiety level) predicted 39% of changes in the dependent variable (level of anesthesia). according to results, the most changes in the dependent variable (anesthesia) were related to dental anxiety (moderate anxiety), in a way that changes dental anxiety from moderate to very severe increased the risk of difficult or failed anesthesia by 30.90 times. moreover, regarding the literacy level, the change of level of education from elementary education to university increased the chance of successful anesthesia by 6.99 times. discussion according to the results of the present study, 60.5% of the participants had significant dental anxiety. in this regard, our findings are in line with the results obtained by dou et al. they resulted that the majority of participants (83.1%) had dental anxiety30. one of the most important factors associated with dental anxiety is the experience of pain in previous visits to the dentist. according to the results of the present study, there was a significant difference among the three groups of successful, difficult, and failed anesthesia in terms of the level of dental anxiety. in this regard, the anxiety level 8 esmaeili et al. was lower in the group of successful anesthesia, compared to the other two groups. besides, the anxiety level was recognized as one of the most important predictors of dental anesthesia level.  pain is as much a cognitive and emotional construct as it is a physiological experience31. thus, emotional states, such as anxiety and fear, can affect the severity of pain32. some studies have demonstrated that patients with a high anxiety level experience difficult anesthesia and more pain during different dental treatments23,33,34. dental patients usually expect more pain than their previous experiences, and perception of pain is an essential factor in this regard. according to the literature, people with a high anxiety score tend to exaggerate their level of pain and fear23,35. this exaggeration leads to recording a higher score for pain, which is subjective and self-assessed by patients. however, the level of exaggeration in pain was somehow reduced in the present study due to assessing the effect of anesthesia using the pinprick and cavity tests. increased dental anxiety results in a higher expectation of pain, which itself increases anxiety in patients. this vicious cycle between pain and anxiety can be partially improved by providing the patient with positive, useful information about the anesthetic process and treatment36. moreover, people with a high level of anxiety are often hypervigilant37. in other words, people with a high level of anxiety is always in an increased state of awareness, and they notice the slightest change in their body. therefore, the use of distraction techniques can somehow reduce dental anxiety in these patients38. since dental anxiety is influenced by various factors such as psychosocial factors, solving these problems can have a significant impact on reducing dental anxiety39. patient response is an essential factor in sensibility tests. patients with a high level of anxiety may have a premature or false-positive response because they expect to feel an unpleasant sensation40. individuals’ cognitive and affective processing, mood, emotions, coping strategies can influence pain perception. at the same time, people with a high level of anxiety, they are more inclined to catastrophizing the pain41,42. according to the results of the present study, there was a significant difference among the three groups of elementary education, high school, and university degrees in terms of mean dental anxiety. in this regard, subjects with primary education experienced a higher level of anxiety, compared to the other two groups, which is congruent with the results obtained by saeed, saatchi, et al., and firat43-45. according to these studies, the level of dental anxiety decreased with increased literacy level. it may be due to the inability of illiterate patients to communicate with dentists. also, people with a higher knowledge level have more information about the importance of dental and oral health. moreover, people with a higher level of education are also generally well-positioned, which would enable them to pay the dental treatment costs. according to the results of the present study, the mean dental anxiety was significantly higher in the age group of ≥59 years, compared to the other age groups (18-28, 29-38, and 39-48 years). in this regard, our findings are in accordance with the results obtained by nair et al.46 and thomson et al.47. the results of humphris’s study were inconsistent with our findings9. according to these results, the level of dental 9 esmaeili et al. anxiety decreases with aging. most subjects in the age group of ≥50 years are more involved in issues related to their health and disease. in this age group, in addition to chronic disease, root canal problems, and periodontal diseases are somewhat prevalent; therefore, it can be said that these problems may cause more anxiety in this age group. according to the results of the present study, those who had not seen a dentist for a long time experienced higher dental anxiety. this result is in line with the results obtained by quteish taani and appukuttan et al. they concluded that people with a higher anxiety level more delay their visit to the dentist12,48. in other words, people who regularly visit for dental examinations have less anxiety compared to those who visit the dentist at longer periods and irregularly. anxious people usually tend to avoid dental treatment, which is a major problem and affects their quality of life. notably, various factors, such as lack of time and expensive dental costs, can be related to delayed visits to the dentist. like all studies, this study has its limitations, including that dental clinics, did not have access to an electrical pulp tester to confirm local dental anesthesia. for this purpose, they used the pinprick test for soft oral tissue and cavity test for hard tissue. although, according to the study of lin and chandler, the use of electrical pulp testers is known as a subjective method which, based on stimulation of sensory nerves, and requires and relies on subjective assessments and comments from the patient49. also, agbaje and de laat, concluded that there is a high correlation (89% to 94%) between quantitative and qualitative sensory testing. this result showed that qualitative somatosensory testing could be used as a screening tool in the clinical setting50. in conclusion, although a high level of anxiety is an indication of sedation in the patients, proper interventions, such as informing patients about anesthesia, dental treatment procedures, regular and periodic visits to the dentist, as well as using psychotherapeutic techniques to reduce dental anxiety before anesthesia, could play an essential role in the success of anesthesia.  acknowledgments authors would like to thank yasuj university of medical sciences, research and technology deputy for their financial support of this study. disclosure of interest the authors report no conflicts of interest. ethics approval and consent to participate the research proposal approved in research ethics committee of yasuj university of medical sciences and in accordance with the ethical standards of the 1964 helsinki declaration. the ethical registration code is ir.yums.rec.1395.211 10 esmaeili et al. references 1. corah nl, gale en, illig sj. assessment of a dental anxiety scale. j am dent assoc. 1978 nov;97(5):816-9. doi: 10.14219/jada.archive.1978.0394. 2. newton jt, buck dj. anxiety and pain measures in dentistry: a guide to their quality and application. j am dent assoc. 2000 oct;131(10):1449-57. doi: 10.14219/jada.archive.2000.0056. 3. locker d, shapiro d, liddell a. overlap between dental anxiety and blood-injury fears: psychological characteristics and response to dental treatment. behav res ther. 1997 jul;35(7):583-90. doi: 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2017 jan;44(1):30-42. doi: 10.1111/joor.12455. 1 volume 22 2023 e230302 original article braz j oral sci. 2023;22:e230302http://dx.doi.org/10.20396/bjos.v22i00.8670302 1 federal university of amazonas (ufam), school of dentistry, manaus, am, brazil. corresponding author: andressa coelho gomes federal university of amazonas (ufam), school of dentistry, manaus, am, brazil. avenida waldemar pedrosa, 1539, praça 14 de janeiro, manaus, am, 69025-050. fone: (92) 991658611 email: andressa.coelho.gomes@ gmail.com editor: altair a. del bel cury received: jun 30, 2022 accepted: oct 10, 2022 parents’ sense of coherence in the use of dental services by children aged 12 years old in the city manaus/am andressa coelho gomes1* , vivian casanova da silva1 , larissa neves quadros1 , maria augusta bessa rebelo1 , janete maria rebelo vieira1 aim: the aim of this study was to evaluate the association between the sense of coherence (soc) of parents/guardians and the use of dental services by children who are 12 years old. methods: this is a cross-sectional study conducted in the eastern zone of the city of manaus, with 358 children enrolled in the 7th grade in the municipal public school system. data collection was performed through self-administered questionnaires sent to parents/guardians, which contained questions about socioeconomic conditions, use of dental services by children and antonovsky’s soc-13 scale. data were examined by descriptive and bivariate analysis, using the chi-square test, with a 5% significance level. results: of the 358 children, 58.4% were female; in addition, 75.4% of the parents/guardians self-reported to be brown and 39.9% of them had a family income of ½ to 1 minimum wage. soc was categorized as strong soc and weak soc by the median. the association between strong or weak soc and the use of dental services in terms of frequency (p= 0.839) and reason for last visit (p= 0.384), was not significant. conclusion: it was concluded that soc of parents/guardians and utilization of dental services by children were not associated. keywords: sense of coherence. dental health services. child health. https://orcid.org/0000-0001-9306-8474 https://orcid.org/0000-0001-8672-3936 https://orcid.org/0000-0003-2828-5450 https://orcid.org/0000-0002-0099-9868 https://orcid.org/0000-0002-7125-9082 2 gomes et al. braz j oral sci. 2023;22:e230302 introduction the utilization of health services represents the center of health systems operation, comprising every direct or indirect contact with the services, being considered the result of the interaction between the individual who is seeking care and the professional who will perform it and guide the patient within the system1. it is also considered a complex health behavior, determined by a wide variety of factors, predisposing or restricting the use of these services by users2,3. access to and use of dental services are considered essential factors for prevention and management of oral diseases4, and it has been demonstrated that regular use of these services results in less tooth loss and dental caries5. on the other hand, obtaining access to dental care is often challenging. this situation represents a public health problem in countries where the use of dental services is observed to be unequal among different social and demographic groups4,6. childhood and adolescence are particularly relevant times for studying the use of oral health services. in a way, the ages of growth reflect the family context in the search for such services7. thus, understanding the use of dental services by children becomes even more complex, as it reflects the interaction between psychosocial, material, cultural, and behavioral factors, as well as the perception of the individual or of their parents/guardians about their health condition and the need for care8,9. among the psychosocial factors that may influence the use of dental services, the sense of coherence (soc) has been shown to be a protective factor for the adoption of more favorable oral health behaviors10. soc is the basis of the salutogenic theory developed by antonovsky, which measures the individual’s ability to deal with adverse situations without influencing his/her quality of life11. the soc is composed of the following attributes: understandability, manageability, and meaningfulness. that is, an individual’s soc shows the degree to which he/she sees life as comprehensive, manageable, and meaningful12. according to the salutogenic model, it is important for people to focus on their resources and their ability to generate health rather than on the causes of their illness. a stronger soc leads a person to perceive the environment as less stressful, disruptive, and chaotic. it also facilitates the selection of more effective health behaviors13. several studies that have worked with soc have shown that people with lower levels of education, low income, and weak soc tend to take their children to dental care less frequently14. on the other hand, parents or caregivers with a strong soc showed association with higher frequency and more favorable dental services utilization pattern, reflecting directly in the oral health of their children15,16. such findings show that the level of parent/guardian soc can be considered an important psychosocial determinant of children’s oral health status and practices10,17. added to this is the fact that children are dependent on their parents to have good general and oral health, which points to the relevance of considering not only individual factors, but also the psychological and sociodemographic factors of parents/caregivers in order to ensure comprehensive health in childhood. 3 gomes et al. braz j oral sci. 2023;22:e230302 therefore, the aim of this study was to evaluate the association between soc of parents/guardians and the use of dental services by 12-year-old children in the city of manaus/am. materials and methods study design and population as part of a larger research project entitled: “socioenvironmental determinants, clinical oral conditions, health-related behaviors, and psychosocial factors psychosocial factors of quality of life in children: a longitudinal study” (process no. 423309/2016-1), the present study was a cross-sectional, conducted in the eastern zone of the city of manaus between september and december 2016 with 12-year-old school-age children and their parents and/or guardians. the eastern zone, which is composed of 11 neighborhoods, is the second most populous zone of the city, characterized by being a socially deprived urban area. children enrolled in the municipal public school network who were 12 years old at the initial research period were included in the study, and those using orthodontic appliances, and those with any syndrome and/or requiring special care were excluded. a representative sample of 12-year-old children enrolled in the 7th grade in municipal public schools was then selected by stratified random sampling according to the size of the school population in the 11 neighborhoods of eastern manaus. thus, 25 schools were randomly selected, proportionally to the number of schools per neighborhood. the sample size was estimated at 528 children. of these, 86 did not return the consent form or their parents did not agree with their participation. of the remaining 442 adolescents, 27 were excluded due to the use of orthodontic appliances. pilot study and instrument reliability prior to the main study, a pilot study was carried out involving parents/guardians of 10 schoolchildren children who were not selected for the main study sample. parents/ guardians were interviewed to verify the understanding of the questionnaire items on the sense of coherence. data collection data collection was performed using self-administered questionnaires sent to parents and/or guardians, containing questions about socioeconomic characteristics, use of dental services, and soc. the socioeconomic characteristics included questions to the parents and/or guardians about number of people in the household (total number of people making up the family unit in the household); number of rooms in the house (total number of rooms in the dwelling, used by residents to meet basic needs such as rest, food preparation, hygiene, and others); number of assets (measured by the continuous variable from 0 to 11 assets, where 0 meant no assets in the household and 11 meant the maximum number of assets); family income (sum of monthly 4 gomes et al. braz j oral sci. 2023;22:e230302 incomes of the components of the family unit, in minimum wages in brazil, in effect in 2016, which was classified into three categories: less than or equal to brl 440.00 – up to half a minimum wage, between brl 441.00 to brl 880.00 – between half and one minimum wage, and brl 881.00 or more – more than one minimum wage). regarding the use of dental services, we asked whether the child had dental insurance, how many times they had visited the dentist during their life, how long it had been since their last visit, and for what reasons their parents/guardians had taken them to the dentist. the soc of parents/guardians was measured using the antonovsky’s soc-13 scale, cross-culturally adapted for the portuguese language18. each question has five response options that follow a likert-type psychometric pattern, always from the worst to the best condition. the scores of the questions that are negative to the sense of coherence were inverted for the final composition of the scale score. the minimum value of the scale is 13 and the maximum, 6518. the higher the sum of the items, the higher the sense of coherence. data analysis the data were analyzed by descriptive analysis, including the distribution of variables by medians, means, and standard deviations. the chi-square (χ) test was performed, with a significance level of 5%, to evaluate the association between the dependent variable (use of dental services) and the independent variable (soc of parents/guardians). all analyses were performed in spss (statistical package for social sciences) version 22.0 software. ethical aspects the study project was submitted to the municipal education secretary of the city of manaus (semed) and to the research ethics committee of the federal university of amazonas (caae: 57273316.1.0000.5020). all eligible students, including their parents, signed the free and informed consent form and the minor’s consent form. results the final sample consisted of 358 children, due to the exclusion of 57 of them for lack of complete data in the questionnaire. of these, 149 children (41.6%) were male and 209 (58.4%) were female. of the parents/guardians, 75.4% declared themselves as brown; 13.1% as white; 5.9% as black; 4.2% as indigenous, and 1.4% as yellow. regarding the number of years of schooling, most parents/caregivers declared to have eight to eleven years of schooling (72.9%), equivalent to complete elementary school (8 years) and complete high school (11 years). most of the population studied (41.6%) had a family income of ½ to 1 minimum wage, which at the time of data collection was brl 441.00 to brl 880.00. children with their respective parents/guardians lived predominantly in households with less than two residents per room (70.7%), and on average each family owned 6.62 assets in the household (table 1). 5 gomes et al. braz j oral sci. 2023;22:e230302 table 1. sociodemographic characteristics of the study population (n=358), manaus 2016. variable n % mean ± sd variance sex (children) female 209 58.4 male 149 41.6 race/skin color (parents/caregivers) yellow 5 1.4 white 47 13.1 indigenous 15 4.2 brown 270 75.4 black 21 5.9 years of schooling (parents/caregivers) 1-7 55 15.4 8-11 261 72.9 ≥12 years 42 11.7 monthly family income ≤ brl 440,00 91 25.4 brl 441,00-880,00 149 41.6 brl > 881,00 118 33.0 n. of residents per room < 2 residents 253 70.7 2 residents 71 19.8 3 residents or more 34 9.5 n. of goods in the household 6.65 ± 2.55 0-11 the median and mean scores for parent/guardian sense of coherence were 47.00 and 46.27 (sd=6.68), respectively; 196 (53.9%) of the parents/guardians had scores less than or equal to 47 and 165 (46.1%) had scores greater than 47. when stratifying the soc 13 questions, the question 6 (“do you think the things you do in your life have little meaning?”) had the lowest mean (1.99) and question 3 (“are you interested in what is going on around you?”) had the highest (3.90). regarding the use of dental services by children, 46.1% of parents/guardians said they took their children to the dental office less than 1 year ago, and 15.9% answered that the children never had a dental appointment. it was also evidenced that the main reasons for the last visit were: revision/prevention (31%), followed by treatment (26.8%), and pain or extraction (26.2%). the public service was the most used (47.8%,) and 89.4% of the children had no dental care plan. when asked why the child had never been to the cd or had not been in the last year, 81 answered there was no need. on the other hand, 68 children needed it, were referred to the dental office, but did not attended (table 2). 6 gomes et al. braz j oral sci. 2023;22:e230302 table 2. frequency and percentage of questions about use of dental services (n=358) manaus, 2016. questions frequency (%) when was the last time your child visited a dentist? never 57 (15.9) less than 1 year 165 (46.1) 1-2 years 84 (23.5) 3 years and more 52 (14.5) what was the reason for the last visit? checkup / prevention 111 (31.0) pain 46 (12.8) extraction 48 (13.4) treatment 96 (26.8) not applicable 57 (15.9) if your child has been to the dentist at least once in his or her life, where was the last visit? public service 171 (47.8) private service 82 (22.9) health insurance or health plan 41 (11.5) other 7 (2.0) why has your child never been to the dentist or did not go to the dentist last year? he/she didn’t have to 81 (22.6) needed it, but didn’t use it for whatever reason 38 (10.6) needed, sought, but did not go to 68 (19.0) used in the last year 171 (47.8) does your child have a dental plan? yes 38 (10.6) no 320 (89.4) table 3 presents the chi-square (χ) test to analyze the association between the soc of parents/guardians, categorized into weak soc (score below or equal to the median) and strong soc (score above the median) and the utilization of dental services. no significant difference was found between strong and weak soc regarding the frequency in the use of dental services (p = 0.839). both groups showed a higher frequency for using dental services for less than 1 year, followed by 1 to 2 years and 3 years or more. similarly, no significant difference was found between soc and the reason for the last dental visit (p = 0.384) (table 4). 7 gomes et al. braz j oral sci. 2023;22:e230302 table 3. association between poor and strong parent/guardian soc and utilization of dental services (n=358) manaus, 2016. soc use of dental health services total p never less than 1 year 1-2 years 3 years and more poor 33 (17.1%) 85 (44.0%) 46 (23.8%) 29 (15.0%) 193 (100.0%) 0.839strong 24 (14.4%) 80 (48.5%) 38 (23.0%) 23 (13.9%) 165 (100.0%) total 57 (15.9%) 165 (46.1%) 84 (23.4%) 52 (14.5%) 358 (100.0%) p>0,05 table 4. association between poor and strong parent/guardian soc and reason for using dental services (n=358) manaus, 2016. soc reason for using dental services total pcheck-up / prevention treatment extraction pain never been poor 51 (26.4%) 55 (28.5%) 27 (14.0%) 27 (14.0%) 33 (17.1%) 193 (100.0%) 0.384strong 60 (36.4%) 41 (24.8%) 21 (12.7%) 19 (11.5%) 24 (14.5%) 165 (100.0%) total 111 (31.0%) 96 (26.8%) 48 (13.4%) 46 (12.8%) 57 (15.9%) 358 (100.0%) p>0,05 discussion the present study investigated the possible role of parent/guardian soc and the use of dental services by 12-year-old children in the city of manaus/am. the results showed that the frequency of use of dental services by children, as well as their pattern of use (prevention, treatment, or pain) showed no significant association with the soc score of parents/guardians in families with low socioeconomic status. such results indicate that not all child oral health-related behaviors are related to parents/guardians soc, corroborating the findings of qiu et al.19 (2013) and differing from those found in national studies14-16. comparable results were also found in studies with adults20 and adolescents21. the reason for soc not being associated with all oral health-related behaviors has not yet been clarified. a possible explanation for this would be that children’s oral health behaviors are mainly influenced by the oral health behaviors of parents/guardians and not necessarily by their soc, or the analysis used was not able to detect the differences. the mean score obtained by the responses of parents/guardians on the soc 13 scale was 46.27, a mean similar to the studies of bonanato et al.17 (2009) and fernandes et al. (2017)14, which obtained 47.50 and 47.2 points, respectively. the total score in the study ranged from 22 to 62 points, similar to the study of bonanato et al.17 (2009), which showed a range of 23 to 62. 8 gomes et al. braz j oral sci. 2023;22:e230302 a significant percentage (84.1%) of children participating in this study used the dental service at least once in their lives and about 16% had never been to the dentist, findings that are relatively lower (18.9%) than those reported by da silva et al.22 (2011) and those presented in the last national survey on oral health (18.1%)23, which may express the increased access to and use of dental services by children, as also identified in a previous study24. the frequency of use of dental services proved to be regular, since more than half of the children had an appointed with the dentist within 2 years. despite this, the use of dental services among children was more related to treatment than to prevention, suggesting a need for change in the work process of oral health teams, especially regarding the expansion of prevention and health education, for a model of integral care to child and adolescent health7. another worrying fact is the non-use of dental services, represented by children who sought care but were not seen; this suggests failures in the organization of the local health system. on the other hand, a recent study conducted in brazil showed an association between greater coverage of oral health teams in the family health strategy (esf-sb), greater use of dental services in the public network, and the use of services for treatment by 12-year-old children, revealing the influence that contextual factors have on individual choices7. in addition, a very small proportion of children had dental insurance, indicating that these families rely exclusively on the public system to ensure basic oral health care for their children. this is particularly relevant for the use of dental services, since brazilian individuals with private health insurance tend to use more dental services than those who do not have health insurance25. these aspects are paramount to address inequalities in the use of dental services by underprivileged children15. thus, it is evident that, considering the soc in isolation may not be enough to clarify the use of dental services, in view of other factors that are involved in the complexity of the behavior or attitude of using or not using such services. some limitations of the present study should be considered. the cross-sectional study design restricts the causal relationships between the variables. it should also be recognized that the use of self-completed questionnaires may result in measurement bias to some extent. however, the use of validated questionnaires for the brazilian population potentially reduces reporting error. finally, only 12-year-old children residing in socially disadvantaged areas were investigated. therefore, our findings should not be attributed to other age groups and children of other socioeconomic levels. conclusion the results showed that the soc of parents/guardians and the use of dental services, both in terms of frequency and pattern of use, were not correlated, suggesting that the soc of parents/guardians, analyzed in isolation, cannot explain the use of dental services for children. data availability datasets related to this article will be available upon request to the corresponding author. 9 gomes et al. braz j oral sci. 2023;22:e230302 conflict of interest none. author contribution jmrv and mabr designed the study. acg, lnq, vcs contributed to data collection and analysis. acg, vcs wrote the first draft of the article. all authors read and approved the final manuscript and actively actively participated in the discussion of the manuscript’s findings. references 1. travassos c, martins m. [a review of concepts in health services access and utilization]. cad saude publica. 2004;20 suppl 2:s190-8. portuguese. doi: 10.1590/s0102-311x2004000800014. 2. andersen rm, davidson pl. improving access to care in america: individual and contextual indicators. in: andersen rm, rice t h, kominski gf, editors. changing the u.s. health care system: key issues in health services policy and management. 3rd. san francisco, ca: jossey-bass, 3–31; 2007. 3. andersen rm, davidson p, baumeister se. improving access to care. in: kominski gf, editors. changing the u.s. health care system: key issues in health services policy and management. 4th. san francisco, ca: jossey-bass; 2013. p.33-69. 4. reda sf, reda sm, thomson wm, schwendicke f. inequality in utilization of dental services: a systematic review and meta-analysis. am j public health. 2018 feb;108(2):e1-e7. doi: 10.2105/ajph.2017.304180. 5. thomson wm, williams sm, broadbent jm, poulton r, locker d. long-term dental visiting patterns and adult oral health. j dent res. 2010 mar;89(3):307-11. doi: 10.1177/0022034509356779. 6. peres ma, macpherson lmd, weyant rj, daly b, venturelli r, mathur mr. lislt s, celeste rk, guarnizo-herreño, kearns c, benzian h, allison p, watt rg. oral diseases: a global public health challenge. lancet. 2019 jul;394(10194):249-60. doi: 10.1016/s0140-6736(19)31146-8. 7. martinelli dlf, cascaes am, frias ac, souza lb, bomfim ra. oral health coverage in the family health strategy and use of dental services in adolescents in mato grosso do sul, brazil, 2019: cross-sectional study. epidemiol serv saude. 2021 nov;30(4):e20201140. doi: 10.1590/s1679-49742021000400010. 8. silva bdm, forte fds. [access to dental treatment, mother’s perception of oral health and intervention strategies in the city of mogeiro, pb, brazil]. pesq bras odontoped clin integr. 2009;9(3):313-19. portuguese. doi: 10.4034/1519.0501.2009.0093.0011. 9. jönsson b, holde ge, baker sr. the role of psychosocial factors and treatment need in dental service use and oral health among adults in norway. community dent oral epidemiol. 2020 jun;48(3):215-4. doi: 10.1111/cdoe.12518. 10. elyasi m, lucas abreu lg, badri p, saltaji h, flores-mir c, amin m. impact of sense of coherence on oral health behaviors: a systematic review. plos one. 2015 aug;10(8):e0133918. doi: 10.1371/journal.pone.0133918. 11. antonovsky a. the structure and properties of the coherence sense scale. soc sci med. 1993 mar;36(6):725-33. doi: 10.1016/0277-9536(93)90033-z. 12. antonovsky a. unraveling the mystery of health. how people manage stress and stay well. san francisco: jossey-bass publishers; 1987. 10 gomes et al. braz j oral sci. 2023;22:e230302 13. marçal ccb, heidemann itsb, fernandes gcm, rumor pcf, oliveira so. the salutogenesis in health research: an integrative review. uerj nursing j. 2018;26:1-6. portuguese. doi: 10.12957/reuerj.2018.37954. 14. fernandes ib, costa dc, coelho vs, sá-pinto ac, ramos-jorge j, ramos-jorge ml. association between sense of coherence and oral health-related quality of life among toddlers. community dent health. 2017 mar;34(1):37-40. doi: 10.1922/cdh_3960fernandes04. 15. maffioletti f, vettore mv, rebelo mab, herkrath fj, queiroz a, herkrath ap, et al. predisposing, enabling, and need characteristics of dental services utilization among socially deprived schoolchildren. j saúde pública dent. 2020 jun;80(2):97-106. doi: 10.1111/jphd.12349. 16. neves étb, perazzo mf, gomes mc, ribeiro ila, paiva sm, granville-garcia af. association between sense of coherence and untreated dental caries in preschoolers: a cross-sectional study. int dent j. 2019 apr;69(2):141-9. doi: 10.1111/idj.12439. 17. bonanato k, paiva sm, pordeus ia, ramos-jorge ml, barbabela d, allison pj. relationship between mothers’ sense of coherence and oral health status of preschool children. caries res. 2009;43(2):103-9. doi: 10.1159/000209342. 18. bonanato k, branco dbt, mota jpt, ramos-jorge ml, paiva sm, pordeus ia. trans-cultural adaptation and psychometric properties of the ‘sense of coherence scale’ in mothers of preschool children. int j psychol. 2009;43(1);144-53. 19. qiu rm, wong mc, lo ec, lin hc. relationship between children’s oral health-related behaviors and their caregiver’s sense of coherence. bmc public health. 2013 mar;13:239. doi: 10.1186/1471-2458-13-239. 20. lindmark u, hakeberg m, hugoson a. sense of coherence and its relationship with oral health-related behaviour and knowledge of and attitudes towards oral health. community dent oral epidemiol. 2011;39(6):542-53. doi: 10.1111/j.1600-0528.2011.00627.x 21. freire mc, sheiham a, hardy r. adolescents’ sense of coherence, oral health status, and oral health-related behaviours. community dent oral epidemiol. 2001;29(3):204-12. doi: 10.1034/j.1600-0528.2001.290306.x. 22. da silva an, mendonca mh, vettore mv. the association between low-socioeconomic status mother’s sense of coherence and their child’s utilization of dental care. community dent oral epidemiol. 2011;39:115-26. doi: 10.1111/j.1600-0528.2010.00576.x. 23. ministry of health of brazil. [sb brazil 2010: national research on oral health: main results]. brasília: ministry of health; 2012. 116p. portuguese. 24. barasuol jc, garcia lp, freitas rc, dalpian dm, menezes jvnb, santos bz. dental care utilization among children in brazil: an exploratory study based on data from national household surveys. cien saude colet. 2019 feb;24(2):649-57. doi: 10.1590/1413-81232018242.03232017. 25. pilotto lm, celeste rk. the relationship between private health plans and use of medical and dental health services in the brazilian health system. cien saude colet. 2019 jul;24(7):2727-36. doi: 10.1590/1413-81232018247.24112017. 1http://dx.doi.org/10.20396/bjos.v20i00.8658796 volume 20 2021 e218796 original article 1 department of social odontology, legal odontology division, piracicaba dental school, university of campinas, são paulo, brazil. 2 biostatistic of the post-graduation course from paulista university, brazil. 3 forensic odontology service, afrânio peixoto legal medicine institute, rio de janeiro, brazil. corresponding author: luiz francesquini júnior department of social odontology, legal odontology division, piracicaba dental school, university of campinas, avenida limeira, nº 901, bairro areião, piracicaba, sp, brazil, cep: 13414-903, phone number: +55 19 21065281, e-mail: francesq@unicamp.br editor: dr altair a. del bel cury received: march 20, 2020 accepted: march 6, 2021 evaluation of effectiveness of cranial morphological characteristics for sex estimation in a brazilian sample larissa stasievski1 , viviane ulbricht1 , vanessa gallego arias pecorari2 , vanessa moreira andrade1,3 , luiz francesquini júnior1,* forensic physical anthropometry allows the determination of animal species and estimates sex, ancestry, age and height. aim: to analyze the effectiveness of a cranioscopic/ morphological evaluation for sex estimation with a sample of the brazilian mixed-race population by conducting a qualitative visual assessment without prior knowledge of sex. methods: this is a blind cross-sectional study that evaluated 30 cranial characteristics of 192 skulls with mandible, 108 male and 84 female individuals, aged 22 to 97 years, from the osteological and tomographic biobank. the qualitative characteristics were classified and compared to the actual sex information of the biobank database. the statistical analysis was used to calculate de cohen’s kappa coefficient, total percentage of agreement, sensitivity and specificity of visual sex classification. results: of the 30 cranial variables analyzed, 15 presented moderate degree of agreement, achieving value of kappa test between 0.41–0.60: glabella (gl), angle and lines (at), mental eminence (em), mandible size (tm), cranial base (bc), mouth depth (pb), nasal aperture (anl), supraorbital region (rs), orbits (orb), mastoid processes (pm), alveolar arches (aa), zygomatic arch (az), orbital edge (bo), supraorbital protuberances (pts), and supramastoid crests and rugosity (crsm). the facial physiognomy (ff) presented substantial reliability (0.61-0.80) with 89.8% sensitivity for male sex and 70.2% specificity. conclusions: cranial morphological characteristics present sexual dimorphism; however, in this study only 15 variables showed moderate degree of agreement and can be used in sex estimation. only one variable (ff) 81.2% total agreement with substantial reliability. quantitative methods can be associated for safe sex estimation. keywords: sex characteristics. forensic anthropology. skull. mandible. https://orcid.org/0000-0003-4516-5493 https://orcid.org/0000-0001-7441-7667 https://orcid.org/0000-0002-0300-5697 https://orcid.org/0000-0002-1291-9642 https://orcid.org/0000-0002-6344-3488 2 stasievski et al. introduction in an anthropological examination for forensic purposes, the determination of sex, species, ethnic group and estimated age and height are essential as such information help build an individual’s biological profile1,2 and subsequent identification. musilová et al.2 (2016) e durić et al.3 (2005) portrayed that the pelvis is the structure of the human skeleton that presents the highest degree of sexual dimorphism, being the most reliable bone for sex estimation. according to musilová et al.2 (2016) the pelvis responds with evolutionary adaptation to bipedal locomotion and birth mechanisms, enabling parturition of children with relatively big brains. but, in situations where the skeleton is not complete4 or when the pelvis is not fully recovered, sex estimation can be achieved by performing a cranial analysis. as mentioned by spradley and jantz4 (2011), the skull has a high correct classification of sex, of 90-91%. in a skull examination, an anthropologist may use quantitative (metric) and/or qualitative (non-metric) methods2,5,6. lewis and garvin (2016)7, biancalana et al. (2015)8, godde (2015)9, tallman and go (2018)10, walker (2008)11 and langley et al. (2018)12 are some authors which estimated the sex based on the skull morphology. this qualitative analysis is based on visual examination of the presence or degree of expression of morphological characteristics7, but despite its subjectivity8-11,13, may be the only possible method in cases of bone fragmentation7. an examination of non-metric traits also ensures an easy and fast analysis, without requiring any devices7,10,12. in general, bone aspects such as prominences, crests and apophyses are more notable in men, while women have more delicate and less pronounced characteristics14. walker (2008)11 stated that the accuracy of sex determinations based on visual inspection depends on the osteologist’s familiarity with the population being studied. and franklin et al. (2013)15 mentioned that the forensic practitioner should access an osteological database for their specific geographic jurisdiction. this changes in skull shape and size are population-specific11,15, and can be explained because each population is submitted to its own forces of evolution1,9,10. environmental interventions8,10,15, nutritional status8,9, temporal changes8-12 and biomechanical processes related to neck, face and head movement9 can alter cranial morphological aspects, smoothing or enhancing the robustness of some characteristics. in view of these situations, this study aimed to analyze the effectiveness of a cranioscopic/morphological evaluation (a qualitative visual assessment without prior knowledge of sex) with a sample of the brazilian mixed-race population, for sex estimation. materials and methods this study was approved by the research ethics committee (caae nº 38522714.6.0000.5418). the main sample consisted of 192 human skulls without alterations that impaired the analysis of morphological characteristics, 108 were male and 84 were female skulls, 3 stasievski et al. aged between 22 and 97 years, with median age of 57 years, from the osteological and tomographic biobank. the year of death of this sample varied from 2006 to 2010. the skulls of individuals who were 22 years or older at the time of their death were analyzed, excluding skulls of individuals who had not reached puberty as they show slightly pronounced qualitative characteristics, providing little information for sex estimation12,14. all analyses were performed by a single rater. the researcher was previous calibrated analysing all the dichotomous variables (male or female) in 10 skulls, obtaining 100% of consensual agreement between itself and a gold rater. the calibration has not been made through a statistical test. and the sample used for calibration was not included in the main sample. then the main sample of 192 skulls was evaluated, of which 30 cranial anatomical structures that were analyzed by this rater through visual inspection, using a non-metric method without prior knowledge of sex. table 1 shows the morphological characteristics analyzed in this study. table 1. morphological characteristics of skulls according to sex. acronym description female male pe weight less heavy heavier at angle and lines less angled, round and thin more angled and pronounced lines iof frontal bone inclination vertical inclined pts supraorbital protuberances level pronounced rs supraorbital region none to moderate medium to excessive gl glabella flat and not very delimited prominent bo orbital edge thin and sharp thick fc canine fossa not very deep deep pm mastoid processes small, little protruding in lower plane robust, protruding in lower plane rcrm condyle protuberance in relation to the mastoid with greater protuberance without greater protuberance mcsp skull movement on a flat surface does not move when supported moves when supported sd digastric groove not very deep and narrow deep and wide az zygomatic arch thinner and shorter more robust and wide enl nasal spine less prominent more prominent anl nasal aperture less tall and wide, with rounded edges taller and wider, with sharp edges ff facial physiognomy indicates female indicates male orb orbits tall and round low and angular ct temporal crests slightly marked marked lns superior nuchal lines slightly marked marked rpn nuchal plane surface slightly marked and smooth rough continue 4 stasievski et al. the visual analysis of all 30 variables generated a subjective differentiation between female and male skulls (nominal qualitative variable). based on the knowledge of actual sex of the individuals, the degree of agreement was measured using kappa test, considering the significance level α=0.05, the levels of strength of agreement measure by cohen’s kappa are interpreted as proposed by landis and koch16 (1977): almost perfect (0.81–1.00), substantial (0.61–0.80), moderate (0.41–0.60), fair (0.21–0.40), slight (0.00–0.20), or poor (<0.00). the percentage of correct sex was calculated using frequency tables (crosstab) and the cohen’s kappa coefficient, total percentage of agreement, sensitivity, specificity visual sex classification were calculated. it was used spss statistics version 25 statistical package (ibm corporation, chicago, usa) for data processing. results the frequency and percentage of skull’s real sex are described in table 2. the sample was relativily balanced with 56.3% of male and 43.8% female. 32.8% of the sample consisted of skulls which age range was between 26–50 years old, 33.3% between 51–70 years and 30.7% over 70 years old. and 96.5% of the skulls analysed presented the year of death 2010. table 2. frequency and percentage of actual sex classification, range age and death’s date of the skulls. sample frequency n % actual sex female 84 43.8 male 108 56.3 age range ≤ 25 6 3.1 26 a 50 63 32.8 51 a 70 64 33.3 over 70 59 30.7 death date 2006 1 0.5 2009 6 3.1 2010 185 96.4 crsm supramastoid crests and rugosity just perceptible marked aa alveolar arches small raised td tooth size smaller larger tm mandible size smaller larger emd mandible thickness smaller larger cm mandibular condyles smaller larger am mandibular angle more obtuse straighter em mental eminence pointed, rounded square pb mouth depth narrow and not very deep larger and deeper bc skull base level and delicate rough and strong continuation 5 stasievski et al. due to the sample consisted of skulls aged older than 22 years, the authors performed the skull concordance tests without separating them by age. table 3 shows the frequency and percentage of sex classification based on a subjective analysis of different variables. through kappa test, variables pe (k=0.08) and ct (k=-0.04), should not be used to determine sex, as they do not present statistical significance in the agreement test (p>0.05). table 3. frequency and percentage of sex classification based on a subjective rater analysis of different variables. sex male n (%) female n (%) pe 111 (57.8) 81 (42.2) at 117 (60.9) 75 (39.1) iof 135 (70.3) 57 (29.7) pts 141 (73.4) 51 (26.6) rs 136 (70.8) 56 (29.2) gl 123 (64.1) 69 (35.9) bo 108 (56.3) 84 (43.8) fc 99 (51.6) 93 (48.4) pm 127 (66.1) 65 (33.9) rcrm 108 (56.3) 84 (43.8) mcsp 77 (40.1) 115 (59.9) sd 104 (54.2) 88 (45.8) az 88 (45.8) 104 (54.2) enl 106 (55.2) 86 (44.8) anl 125 (65.1) 67 (34.9) ff 122 (63.5) 70 (36.5) orb 141 (73.4) 51 (26.6) ct 148 (77.1) 44 (22.9) lns 152 (79.2) 40 (20.8) rpn 151 (78.6) 41 (21.4) crsm 144 (75.0) 48 (25.0) aa 96 (50.0) 96 (50.0) td 69 (35.9) 32 (16.7)* tm 121 (63.0) 71 (37.0) emd 130 (67.7) 24 (32.3) cm 138 (71.9) 54 (28.1) am 106 (55.2) 86 (44.8) em 98 (51.0) 94 (49.0 pb 132 (68.7) 60 (31.3) bc 135 (70.3) 57 (29.7) actual sex 108 (56.3) 84 (43.8) *absent structures did not allow sex classification. 6 stasievski et al. although the variable tooth size (td) has been visually analyzed, its statistical analysis was not made due to several skulls had no tooth. the other variables showed statistically significant agreement from slight to fair level. among these variables, lns and am had a slight level of agreement (0.00–0.20); emd, iof, cm, mcsp, enl, fc, rpn, sd and rcrm showed a fair level of agreement (0.21–0.40); gl, at, em, tm, bc, pb, anl, rs, orb, pm, aa, az, bo, pts and crsm presented a moderate level of agreement (0.41–0.60). and only ff presented substantial level of agreement (0.61–0.80). table 4 shows the degree of agreement according to kappa test and the p-value of all morphological variables, and the 16 variables with the best agreement degrees are highlighted with †symbol. table 4. percentage of total agreement, cohen’s kappa coefficient and 95% ci kappa of variables in relation to actual sex. total agreement (%) cohen’s kappa coefficient ic 95% kappa pe 54.7 0.08 -0.06 – 0.217 at 81.7 0.60*† 0.49 – 0.72 iof 71.4 0.40* 0.27 – 0.52 pts 73.5 0.44*† 0.31 – 0.56 rs 76.1 0.49*† 0.37 – 0.61 gl 80.7 0.60*† 0.49 – 0.71 bo 72.9 0.43*† 0.32 – 0.58 fc 72.4 0.34* 0.21 – 0.47 pm 73.4 0.45*† 0.32 – 0.58 rcrm 62.5 0.24* 0.10 – 0.37 mcsp 66.2 0.34* 0.21 – 0.46 sd 65.6 0.30* 0.17 – 0.44 az 72.9 0.46*† 0.34 – 0.58 enl 66.7 0.32* 0.20 – 0.46 anl 75.5 0.49*† 0.37 – 0.61 ff 81.2 0.61*† 0.50 – 0.72 orb 74.5 0.46*† 0.34 – 0.58 ct 51.1 -0.04 -0.12 – 0.12 lns 62.5 0.19* 0.07 – 0.31 rpn 68.3 0.32* 0.19 – 0.44 crsm 72.9 0.42*† 0.30 – 0.54 aa 74.0 0.48*† 0.36 – 0.60 tm 77.6 0.54*† 0.42 – 0.66 emd 70.8 0.39* 0.26 – 0.52 cm 70.9 0.38* 0.26 – 0.51 am 59.4 0.18* 0.04 – 0.32 em 79.1 0.58*† 0.47 – 0.69 pb 77.0 0.52*† 0.40 – 0.64 bc 76.6 0.51*† 0.40 – 0.62 *indicates significance in the kappa’s test (p<0.05); † represent the variables with the best agreement degrees (k>0.40). 7 stasievski et al. table 5 shows the percentage of correct sex through crosstab of 16 (sixteen) variables that presented statistically significant moderate and substantial agreement (k>0.40; p<0.05). the visual sex classification of ff, at and gl variables presented more than 80% of total agreement. the visual sex classification of ff variable showed 89.8% sensitivity and 70.2% specificity for male sex classification. the at showed 87% sensitivity and 72.6% for male sex specificity and gl showed 89.8% sensitivity and 69.0% specificity. the visual classification was more sensitivity for male sex, whereas for the female classification the sensitivity was lower. table 5. the sensitivity, specificity for visual sex classification of the variables that presented moderate and substantial reliability in cohen’s kappa coefficient (k>0.40; p<0.05). male n (%) female n (%) total gl# actual sex male 97 (89.8)* 11 (10.2) 108 (100.0) female 26 (31.0) 58 (69.0)†‡ 84 (100.0) at# actual sex male 94 (87.0)* 14 (13.0) 108 (100.0) female 23 (27.4) 61 (72.6)†‡ 84 (100.0) ff# actual sex male 97 (89.8)* 11 (10.2) 108 (100.0) female 25 (29.8) 59 (70.2)†‡ 84 (100.0) em# actual sex male 91(84.3)* 17 (15.7) 108 (100.0) female 39 (46.4) 45 (53.6)†‡ 84 (100.0) tm actual sex male 93 (86.1)* 15 (13.9) 108 (100.0) female 28 (33.3) 56 (66.7)†‡ 84 (100.0) bc actual sex male 99 (91.7)* 9 (8.3) 108 (100.0) female 36 (42.9) 48 (57.1)†‡ 84 (100.0) pb# actual sex male 98 (90.7)* 10 (9.3) 108 (100.0) female 34 (40.5) 50 (59.5)†‡ 84 (100.0) anl actual sex male 93 (86.1)* 15 (13.9) 108 (100.0) female 32 (38.1) 52 (61.9)†‡ 84 (100.0) rs# actual sex male 99 (91.7)* 9 (8.3) 108 (100.0) female 37 (44.0) 47 (56.0)†‡ 84 (100.0) orb# actual sex male 100 (92.6)* 8 (7.4) 108 (100.0) female 41 (48.8) 43 (51.2)†‡ 84 (100.0) pm # actual sex male 92 (85.2)* 16 (14.8) 108 (100.0) female 35 (41.7) 49(58.3)†‡ 84 (100.0) aa actual sex male 77 (71.3)* 31 (28.7) 108 (100.0) female 19 (22.6) 65 (77.4)†‡ 84 (100.0) az actual sex male 72 (66.7)* 36 (33.3) 108 (100.0) female 16 (19.0) 68 (81.0)†‡ 84 (100.0) bo actual sex male 82 (75.9)* 26 (24.1) 108 (100.0) female 26 (31.0) 58 (69.0)†‡ 84 (100.0) pts# actual sex male 99 (91.7)* 9 (8.3) 108 (100.0) female 42 (50.0) 42 (50.0)†‡ 84 (100.0) continue 8 stasievski et al. the variable orb showed 92.6% sensitivity for male sex, however, 51.2% of specificity. the ff, gl and at were the variables with the highest index of sensitivity and specificity. the percentages of correct sex for the 10 morphological variables with the best agreement degrees are highlighted with a hash in the same table 5 and are presented in the figure 1; all of them had higher percentages in the determination of male in relation to female sex, that is, such characteristics are more evident and facilitate identification in male sex. crsm# actual sex male 100 (92.6)* 8 (7.4) 108 (100.0) female 44 (52.4) 40 (47.6)†‡ 84 (100.0) # represent the variables with the best results; * sensitivity to male sex classification; † specificity to male sex classification; ‡ sensitivity to female sex classification. continuation a b c d e f g h figure 1. a: glabella (gl); b: supraorbital region (rs) and supraorbital protuberances (pts); c: orbits (orb); d: mastoid processes (pm); e: angle and lines (at) and facial physiognomy (ff); f: mental eminence (em); g: mouth depth (pb); h: supramastoid crests and rugosity (crsm). 9 stasievski et al. discussion in our study, using a balanced sample, although 30 cranial characteristics were analysed, only 15 variables achieved moderate agreement and one substantial agreement, but according to cicchetti and feinstein17 (1990) a low kappa can occur at a high agreement. it is remarkable that age is a variable that can influence the quantitative measurement of bone size. however, the present study classified each skull qualitatively and the sample consisted of skulls aged older than 22 years, not dealing with the stage before puberty, between 10 and 21 yearsold which can be a confounding bias in the identification of the gender. for this reason, the skulls concordance tests were performed without separating them by age. as the sex of all skulls was indexed in the biobank, it was possible to estimate the percentage of correct sex for female and male population, which was not obtained in the study by biancalana et al.8 (2015). correct sex percentages for the 10 characteristics presenting the best agreement results were high, ranging from 47.6% to 92.6%. being observed that this qualitative classification is more sensitivity for male sex. as observed in other studies2,7,13,18,19, glabella presented a high sexual dimorphism index, reaching 89.8% sensitivity and 69.0% of specificity for male sex classification, and a total agreement of 80.7%. in a qualitative analysis of the glabella region similar to ours, abdel fatah et al.13 (2014) and walker11 (2008) found correct sex classification of 82% and 82.6%, respectively. langley et al.12 (2018), when analyzing non-metric cranial traits, observed that mental eminence was the only variable that did not present a reasonable to moderate agreement for sex estimation, and should be avoided for such purpose. low accuracy of 45.03% was also obtained in the study by durić et al.3 (2005) for the size of mental eminence. in contrast, in our study, mental eminence was among the 10 best variables, with total agreement of 79.1%, having 84.3% of sensitivity for male sex and 53.6% for female sex. similarly, lewis and garvin7 (2016) and walker11 (2008) found correct sex classification for this variable of 75.0% and 76.6%, respectively. in our study, regarding supraorbital protuberances (pts), moderate agreement was observed (73.5%), while lewis and garvin7 (2016), when evaluating the eyebrow region, obtained 96.7% of correct sex classification. nikita and michopoulou19 (2018) also analyzed the mastoid process profile and found for this variable, up to 75.2% and 74.5% correct classification for both sexes, similar to the percentages found by walker11 (2008), while our study found for this characteristic, 85.2% of sensitivity for male sex and 58.3% for female sex. in a study conducted by graw et al.20 (1999), the analysis of the supraorbital margin shape allowed correct identification of sex, with about 70% accuracy, an index that is similar to what was observed by walker11 (2008), while durić et al.3 (2005) reported sharpness of the supraorbital margins as the least reliable indicator with 28.75% accuracy only. regarding orbits (orb), our study showed the best percentage (92.6%) of sensitivity for male sex. 10 stasievski et al. in a qualitative study conducted with european skulls, williams and rogers21 (2006) obtained high accuracy value for gonial angle (80.0%), which do not agree with the results of our study, as the strength of agreement for that variable was slight with a cohen’s kappa coefficient of 0.18. in addition, the authors mentioned above20 reported orbit shape and position, and forehead inclination should not be considered as reliable variables for sex determination. in contrast, in our study, the orbits showed moderate strength of agreement with 74.5% of total agreement and frontal bone inclination presenting a fair level of strength of agreement with 71.4% of total agreement. when analyzing jaw robustness, durić et al.3 (2005) found high accuracy (70.93%) for sex determination. in our study, mandible variables reached different agreements, such as mandible size (presenting moderate agreement), mandible thickness and mandibular condyles (fair agreement), and mandibular angle (slight agreement). similarly, keen22 (1950) reports that the angle of the mandible did not present a high index in sex differentiation. in 2018, tallman and go10 evaluated asian skulls, qualitatively analyzing nuchal crest, mastoid process, supraorbital margin, glabella and mental eminence, and obtaining 57.9% correct classification for female and 92.4% for male sex. similarly, was observed in our study that the visual classification of the cranial characteristics was more sensitivity for male sex. and to increase the probability of correct sex determination, we agree with loth and henneberg23 (1996) when they advise that a complete examination should be made of all available bones known to belong to an individual, combining qualitative and quantitative methodologies, to ensure improved certainty and reliability of forensic anthropological reports. the percentages showed here are helpful for forensic practioners according to which preserved cranial trait is available in the skull that they are working on, and according to the population the skull is originated. however, this study presents some limitations, as the analyses were performed by only one examiner, and although calibrated, by being a qualitative analysis it can be influenced by subjectivity, so the authors suggest that future studies use some examiners. another limitation was common lighting and tables to analyze the skulls, being evaluated with the naked eye. in conclusion, the visual classification of ff variable presented the best sensitivity and specificity to male sex with substantial reliability. next in decreasing order for the best qualitative evaluation of sex were the variables gl, at, em, tm, bc, pb, anl, rs, orb pm, aa, az, bo, pts and crsm which presented moderate agreement (41% to 60%). the visual classification was more sensitivity for male sex. however, for improved certainty and reliability in sex estimation, quantitative methods are recommended. acknowledgments the authors thank espaço da escrita/unicamp’s general coordination for the translation services provided. 11 stasievski et al. references 1. krüger gc, l’abbé en, stull ke, kenyhercz mw. sexual dimorphism in cranial morphology among modern south africans. int j legal med. 2015 jul;129(4):869-75. doi: 10.1007/s00414-014-1111-0. 2. musilová b, dupej j, velemínská j, chaumoitre k, bruzek j. exocranial surfaces for sex assessment of the human cranium. forensic sci int. 2016;269:70-7. doi: 10.1016/j.forsciint.2016.11.006. 3. durić m, rakocević z, donić d. the reliability of sex determination of skeletons from forensic context in the balkans. forensic sci int. 2005;147(2-3):159-64. 4. spradley mk, jantz rl. sex estimation in forensic anthropology: skull versus postcranial elements. j forensic sci. 2011;56(2):289-96. doi:10.1111/j.1556-4029.2010.01635.x. 5. celbis o, iscan my, soysal z, cagdir s. sexual diagnosis of the glabellar region. leg med (tokyo). 2001;3(3):162-70. 6. ulbricht v, schmidt cm, groppo fc, júnior ed, queluz dp, júnior lf. sex estimation in brazilian sample: qualitative or quantitative methodology? braz j oral sci. 2017;16:1-9. doi:10.20396/bjos.v16io.8650495. 7. lewis cj, garvin hm. reliability of the walker cranial nonmetric method and implications for sex estimation. j forensic sci. 2016;61(3):743-51. doi:10.1111/1556-4029.13013. 8. biancalana, rc, ortiz ag, de araújo lg, semprini m, galo r, silva rha. determinação do sexo pelo crânio: etapa fundamental para a identificação humana. rev. bras. crimin. 2015;4(3): 38-43. 9. godde k. secular trends in cranial morphological traits: a socioeconomic perspective of change and sexual dimorphism in north americans 1849-1960. ann hum biol. 2015;42(3):253-9. doi:10.3109/03014460.2014.941399. 10. tallman sd, go mc. application of the optimized summed scored attributes method to sex estimation in asian crania. j forensic sci. 2018;63(3):809-14. doi:10.1111/1556-4029.13644. 11. walker pl. sexing skulls using discriminant function analysis of visually assessed traits. am j phys anthropol. 2008;136(1):39-50. doi:10.1002/ajpa.20776. 12. langley nr, dudzik b, cloutier a. a decision tree for nonmetric sex assessment from the skull. j forensic sci. 2018;63(1):31-7. doi:10.1111/1556-4029.13534. 13. abdel fatah ee, shirley nr, jantz rl, mahfouz mr. improving sex estimation from crania using a novel three-dimensional quantitative method. j forensic sci. 2014;59(3):590-600. doi:10.1111/1556-4029.12379. 14. francesquini júnior l, francesquini ma, de la cruz bm, pereira sd, ambrosano gm, barbosa cm, et al. identification of sex using cranial base measurements. j forensic odontostomatol. 2007;25(1):7-11. 15. franklin d, cardini a, flavel a, kuliukas a. estimation of sex from cranial measurements in a western australian population. forensic sci int. 2013;229(1-3):158.e1-8. doi:10.1016/j.forsciint.2013.03.005. 16. landis jr, koch gg. the measurement of observer agreement for categorical data. biometrics. 1977 mar;33(1):159-74. 17. cicchetti dv, feinstein ar. high agreement but low kappa: ii. resolving the paradoxes. j clin epidemiol. 1990;43(6):551-8. 18. garvin hm, ruff cb. sexual dimorphism in skeletal browridge and chinmorphologies determined using a new quantitative method. am j phys anthropol. 2012;147(4):661-70. doi: 10.1002/ajpa.22036. 19. nikita e, michopoulou e. a quantitative approach for sex estimation based on cranial morphology. am j phys anthropol. 2018;165(3):507-17. doi: 10.1002/ajpa.23376. http://dx.doi.org/10.21117/rbol.v3i2.7 http://dx.doi.org/10.21117/rbol.v3i2.7 http://dx.doi.org/10.21117/rbol.v3i2.7 http://dx.doi.org/10.21117/rbol.v3i2.7 http://dx.doi.org/10.21117/rbol.v3i2.7 http://dx.doi.org/10.21117/rbol.v3i2.7 http://dx.doi.org/10.21117/rbol.v3i2.7 http://dx.doi.org/10.21117/rbol.v3i2.7 http://dx.doi.org/10.21117/rbol.v3i2.7 http://dx.doi.org/10.21117/rbol.v3i2.7 http://dx.doi.org/10.21117/rbol.v3i2.7 12 stasievski et al. 20. graw m, czarnetzki a, haffner ht. the form of the supraorbital margin as a criterion in identification of sex from the skull: investigations based on modern human skulls. am j phys anthropol. 1999 jan;108(1):91-6. doi: 10.1002/(sici)1096-8644(199901)108:1<91::aid-ajpa5>3.0.co;2-x. 21. williams ba, rogers t. evaluating the accuracy and precision of cranial morphological traits for sex determination. j forensic sci. 2006;51(4):729-35. 22. keen ja. a study of the differences between male and female skulls. am j phys anthropol. 1950;8(1):65-79. 23. loth sr, henneberg m. mandibular ramus flexure: a new morphologic indicator of sexual dimorphism in the human skeleton. am j phys anthropol. 1996;99(3):473-85. 1 volume 21 2022 e226202 original article braz j oral sci. 2022;21:e226202http://dx.doi.org/10.20396/bjos.v21i00.8666202 1 department of restorative dentistry, piracicaba dental school, university of campinas, piracicaba, sp, brazil. 2 department of dentistry, pontifical catholic university of minas gerais, belo horizonte, minas gerais, brazil. corresponding author: flávio henrique baggio aguiar dds, msc, phd, associate professor department of restorative dentistry, piracicaba dental school, university of campinas. av. limeira, 901, zip code 13414-903, piracicaba, sp, brazil. phone: 55 19 2106-5337 / fax: +55 19 3421-0144 e-mail address: baguiar@unicamp.br editor: altair a. del bel cury received: june 28, 2021 accepted: january 31, 2022 assessment of influence of led curing units used on microhardness of resin-modified glass ionomer sealants joyce figueiredo de lima marques1 , laura nobre ferraz1 , beatriz kelly barros lopes2 , tamires aparecida borges vasconcelos2 , thiely roberts teixeira2 , débora alves nunes leite lima1 , flávio henrique baggio aguiar1,* , diogo de azevedo miranda2 aim: resin modified glass ionomer (rmgi) is class of material that can be used as sealant for preventing and arresting the progression of caries in pits and fissures. as these are hybrid materials, their properties can be affected by factors related to the polymerization process. therefore, this study aimed to evaluate the influence of different generations of led curing units (elipar deepcure-l and valo grand) on knoop microhardness values (khn) of rmgi sealants (clinpro xt and vitremer). methods: forty cylindrical specimens (6mm ø x 1 mm high) were prepared according to the manufacturer’s instructions and divided into four groups (n=10) according to the type of rmgi and led used. the khn of the top surface of each sample was calculated 7 days after light-curing. data were submitted to two-way anova (α = 0.05). results: vitremer had higher khn values than clinpro xt after using both leds (p<0.0001), but especially when light-cured with the use of valo grand (p<0.0001). whereas the khn value of clinpro was not influenced by the led device (p>0.05). conclusions: top surface microhardness values of rgmi sealants were affected by both material composition and generations of led curing units used. third generation led curing units seemed to be more efficient for the polymerization of rmgi-based sealants. keywords: hardness. curing lights, dental. pit and fissure sealants. http://orcid.org/0000-0001-7800-1255 http://orcid.org/0000-0002-6234-8913 http://orcid.org/0000-0002-8598-7777 http://orcid.org/0000-0002-4407-5632 http://orcid.org/0000-0001-9185-0868 http://orcid.org/0000-0001-5457-3347 http://orcid.org/0000-0003-3389-5536 http://orcid.org/0000-0003-1035-7129 2 marques et al. braz j oral sci. 2022;21:e226202 introduction dental caries is a multifactorial disease caused by changes in the composition of bacterial biofilm, leading to an imbalance between the demineralization and remineralization processes and manifested by the formation of caries lesions in primary and permanent teeth1. pit and fissure caries accounts for around 44% of caries in the primary posterior teeth in children and adolescents, and 90% of the caries of permanent posterior teeth1. this happens because the complex morphology of the occlusal surface makes it difficult to perform mechanical cleaning and reduces the effects of preventive measures2. therefore, sealants that are capable of providing a physical barrier that prevents the retention of microorganism and food particles in pits and fissure have been introduced as one of many minimally invasive approaches in dentistry3,4. their clinical efficiency has been well documented in the literature and reviews have demonstrated that they are effective for preventing both pits and fissure caries, and for minimizing the progression of non-cavitated occlusal carious lesions5,6. according to the american academy of pediatric dentistry and american dental association, pit and fissure sealants can be classified into two broad categories: glass ionomer (gi) sealants and resin-based sealants7. when resin is incorporated into glass ionomer, it is classified as a subcategory of material known is resin-modified glass ionomer (rmgi)1,7. the development of rmgi was proposed to improve the mechanical properties and reduce the early sensitivity to moisture of gi sealants, while preserving their clinical advantages such as esthetics, self-adhesion to dental tissue, fluoride release and thermal insulation8. as this is a hybrid material, the setting reaction of rmgi sealant is initiated by light activation of the resin component, followed by the acid-base reaction of the ionomer component1. although it has been suggested that the latter reaction can compensate the light attenuation that occurs in deeper areas to increase the depth of cure of rmgi9, the main mechanism responsible for the curing process of this type of material is light activation8,10. therefore, their physical and mechanical properties can be greatly affected by factors related to the curing process11,12. problems associated with inadequate polymerization of rmgi sealants include solubility in the oral environment, and partial or complete loss of the material resulting in recurrent caries13,14. considering that the clinical efficiency of fissure sealants depends on their retention14,15, the relevance of the curing-process for achieving a successful outcome after sealing teeth cannot be neglected16,17. rmgi sealants can be polymerized by using many different light sources (e.g., quartz tungsten-halogen, plasma arc, leds)2. however, at present led curing units have dominated the market for many reasons since they eliminate the need for filters, weigh less and are smaller than other appliances used for light curing technologies. they also offer a more consistent radiant energy density, generate minimal heat and are long-lasting9,12,13. according to their stage of development, led curing units can be classified into the first, second and third generations9,12. different methods have been used to evaluate the quality of rmgi sealant polymerization. among them, the microhardness test has been used in many studies for 3 marques et al. braz j oral sci. 2022;21:e226202 indirect assessment of rgmi polymerization and evaluation of the light sources efficiency2,8,11,18, but there is little information available about different the generations of led curing units9. it is well known that for effective light-curing of a resin-based material, sufficient radiant exposure at the correct wavelengths of light of the photoinitiators is required12. however, the spectral radiant power derived from led curing units varies greatly due to unique optical characteristics used within a given design16,17,19. therefore, the influence of factors related to led curing units on the rmgi sealants properties should also be investigated. in view of the significant role of effective polymerization in the long-term clinical success of rgmi sealants used for caries prevention, and the recent advances in the area of light curing, the aim of this study was to evaluate the effect of different generations of led curing units on knoop microhardness values of rmgi. the null hypothesis tested were: (1) there would be no difference in khn values between the rmgi sealants being evaluated; (2) there would be no difference in the khn values of rmgi sealants cured using a second-generation led curing unit and a third-generation led curing unit. material and methods two rmgi sealants (clinpro xt and vitremer) and two led curing units of different generations (elipar deepcure-l and valo grand) were used in this study. table 1 provides details about them. table 1. details of rgmi sealants and led curing units used in this study light-curing unit manufacturer type emission spectrum (nm) irradiance (mw/cm2) elipar deepcure-l 3m espe, st. paul, mn, usa 2nd generation of led monowave 430-480 continuous mode: 1470 (-10%/+20%) valo grand ultradent products inc, south jordan, ut, usa 3rd generation of led polywave 385-515 standard mode: 1000 (±10%) rmgi sealants manufacturer composition manufacturer’s instructionsa vitremer 3m espe, st. paul, mn, usa powder: silane treated glass, potassium persulfate. liquid: copolymer of acrylic and itaconic acids, water, hema, ethyl acetate and diphenyliodonium hexafluorophosphate. place an equal number of level powder scoops and liquid drops. mix the powder into the liquid within 45 seconds. lightcure for 40 s. clinpro xt varnish 3m espe, st. paul, mn, usa part a: silane treated glass, hema, water, bis-gma, silane treated silica, edmab. part b: copolymer of acrylic and itaconic acids, water, hema, calcium glycerophosphate. dispense 1 click onto mixing pad. mix for 15 seconds. light-cure for 20s. notes: ain the present study, all materials were light-cured for 20 s. abbreviations: monomer abbreviations: hema: 2-hydroxyethyl methacrylate; bis-gma: bisphenol a glycidyl methacrylate; edmab: ethyl 4-dimethyl aminobenzoate. 4 marques et al. braz j oral sci. 2022;21:e226202 specimen preparation forty specimens were prepared and divided into four groups (n=10) according to material/light-curing unit combination, as shown in the experimental design (figure 1). the rgmi sealants were manipulated according to the respective manufacturers’ instructions (table 1) and inserted into a cylindrical teflon matrix (6 mm x 1 mm thick) placed on a glass plate. after insertion, a mylar strip was placed on the surface of the unpolymerized material, and another glass plate was pressed over the strip to adapt the material completely and produce a flat surface. the tip of the led was then placed in contact with the mylar strip on the matrix top surface, and specimens were light-cured for 20 s using one of the led curing units according to material/light-curing unit combination (fig.1). the radiant emittance of the led curing units was periodically assessed using a properly calibrated radiometer (rd-7, ecel, ribeirão preto, brazil). immediately after exposure to light, the specimens were removed from the matrix and stored in dry, lightproof receptacles until they were tested. microhardness measurements the microhardness test was performed 7 days after storage of the specimens, with the use of a digital knoop hardness measuring instrument (hmv-2t e, shimadzu corporation, tokyo, japan). three indentations were made on the top surface of all specimens: one central (defined by the location of light application) and the other two at approximately 200 µm from the central location, under 50 kgf load for 10 s. the khn values for each sample was recorded as the average of the three readings. statistical analysis after descriptive and exploratory data analysis, two-way anova was used to evaluate the influence of the two variables tested (rgmi sealants and led curing units) on khn values. the software r core team 2019 was used (r: a language and envifigure 1. experimental design of this study. vitremer clinpro 20 s (n = 10) vitremer clinpro 20 s (n = 10) led curing unit materials exposure time khn measurements elipar deepcure-l 2nd generation valo grand 3rd generation 5 marques et al. braz j oral sci. 2022;21:e226202 ronment for statistical computing. r foundation for statistical computing, vienna, austria) at a significance level of 0.05. results as may be visualized in table 2, the microhardness (khn) values were significantly higher when the vitremer (p < 0.0001) sealer was used. for the clinpro sealer, there was no significant difference irrespective of the led curing unit tested (p > 0.05). whereas the vitremer sealer showed higher khn values when the valo grand was used (p < 0.0001). discussion the dental light-curing unit (lcu) is an essential part of the process of light-curing a resin-based material, yet the relevance of the lcu and how it is used to achieve a successful restoration outcome is often underestimated16,17. due to the advances in led technology, this study aimed to evaluate the effect of different generations of led curing units on khn values of rgmi sealants. only the top surface of the specimens was tested since materials used as pit and fissure sealants are applied in a thin layer on the occlusal surface3. vitremer exhibited higher khn values than clinpro, irrespective of the led curing unit used (p < 0.05) and, therefore, the first null hypothesis was rejected. according to the manufacturers, vitremer contains glass filler particles of a relatively large average size (~3µm)20 corresponding to 65% by weight (according to the material safety data sheet). whereas clinpro is considered an unfilled rmgi1. as the physical and mechanical properties of dental resin-based materials depend on the concentration and size of filler particles19,21,22, this characteristic of vitremer in comparison with clinpro might have contributed to the result. moreover, vitremer is considered a “tri-cure” restorative material, which means that its setting reaction depends on three mechanisms: (1) the acid-base reaction between the fluoroaluminosilicate glass and the polycarboxylic acid (the same reaction as in a conventional glass ionomer), (2) a light-activated free radical polymerization of methacrylate groups of the polymer and hema (2-hydroxyethylmethacrylate), and (3) a chemically-initiated reaction between remaining methacrylate groups of the polymer system and hema (technical profile vitremer). the latter is possible because a potassium persulfate/ascorbic acid redox initiation system was incorporated to its composition23. table 2. means (standard deviation) of microhardness (khn) values in function of sealant and light-curing unit led curing units sealants clinpro vitremer valo grand 35,31 (2,26) ba 60,92 (3,86) aa elipar deepcure-l 32,30 (2,13) ba 50,65 (1,57) ab means followed by different letters (uppercase in the horizontal and lowercase in the vertical) differ from each other (p ≤ 0.05). p (sealant) < 0.0001; p (light-curing unit) < 0.0001; p (interaction) < 0.0001. 6 marques et al. braz j oral sci. 2022;21:e226202 when powder and liquid are mixed, the reaction is initiated and proceeds independent of light. higher values of microhardness24 and dc13 that have been relatively stable over time have been demonstrated for vitremer in other studies and were attributed to the enhanced physical and mechanical properties due to the complementary mechanism of cure. whereas clinpro has only two mechanisms of cure (acid-base, light-activated). furthermore, rafeek et al.25 (2008) showed that the setting process of rmgi can be benefited by heat. this may result in the accelerated maturity and improved mechanical properties of rgmi sealants26,27 in addition to their enhanced adhesion to the tooth tissue of the cavity walls28. the increase in temperature of a resin-based material has been attributed to light emitted by the light source, heat released in an exothermic reaction of material hardening and its rate of polymerization16,29. considering that vitremer has three mechanisms of cure, one may suggest that its rate of polymerization and internal heating produced during curing could also be higher than those of clinpro, which could also contribute to its higher khn values, irrespective of the led curing unit used (p<0.05). another characteristic of vitremer that might have contributed for its results is the presence of diphenyliodonium hexafluorphosphate (dpi) in its composition. dpi is an important onium salt catalyst used to improve the reactivity of dental materials30. although it cannot absorb light in the blue wavelength range31, in the presence of the excitatory state of camphorquinone, this co-initiator is decomposed in phenyliodonium and free phenyl radicals that can improve the photopolymerization kinetics of methacrylates, especially in ternary systems32,33. it has been demonstrated that even low concentrations of dpi participate efficiently in the monomer polymerization reducing the photo-activation time required to reach higher conversion when compared to systems without the co-initiator31. the benefit of improved mechanical properties due to increased degree of conversion by the incorporation of dpi have been demonstrated for many resin-based materials30-33 and agrees with the results of this current study, even though vitremer was light-cured for half the time recommended by the manufacturer. since vitremer showed higher khn values when valo grand was used (p <0.05), but there was no significant difference irrespective of the led curing unit (p> 0.05) used for clinpro, the second null hypothesis was partially rejected. the explanation may depend on the characteristic of both the led curing units tested and composition of the materials. shimokawa et al.34 (2018) evaluated the potential effect of four different lcus, including valo grand and elipar deepcure-s, on the curing profile of two bulk fill resin-based composites (rbcs). both the tip diameter and the homogeneity of the light emitted from the lcus affected their results. they found that valo grand produced the most homogeneous microhardness values across top and bottom surfaces of all the rbcs tested (p > 0.05). whereas when elipar deepcure-s was used, the hardness values obtained in the central, middle and outer regions across the rbc specimens differed significantly (p < 0.05). moreover, they demonstrated that the light distribution of valo grand was more homogeneous than that of elipar deepcure-s. it should be noted that according to manufacturers, elipar deepcure-s and l offers an identical technical performance (technical pro7 marques et al. braz j oral sci. 2022;21:e226202 file elipar deep-cure). the main differences between the two versions are the housing and how the units are charged. therefore, the association of a wide tip (valo grand: 11.5mm, elipar deepcure-l: 10mm) and a more homogenous light distribution may explain why vitremer showed higher values of khn when light-cured by valo grand than when using elipar deepcure-l (p<0.05). these features are especially important when light-curing a rgmi sealant because the material is applied on the total extension of pit and fissures of the occlusal surface, and it is necessary to completely cure the material to ensure long-term retention. there is concern that high-power led curing units, such as those used in this study, could be capable of harming the pulp and oral tissues12. nonetheless, pit and fissure sealants are applied on the occlusal surface more thinly than a resin composite in a cavity and, therefore, the pulp is protected by the overlying dentin. furthermore, considering that the tip of the light device cannot be placed directly on top of the sealant surface due to the morphology of fissures and cusps, this type of material could benefit from the use of high-power led curing units, since higher irradiances can compensate the distance between the material and the light tip8,19. third-generation of led curing units have further advantages over other light sources and their previous generations. time-saving procedures are an ongoing demand for restorative application, especially in pediatric dentistry, and the development of third-generation of led curing units has resulted from recent research focused on achieving shorter curing times without adverse consequences9,12. in this study, the fact that the vitremer showed higher khn values when cured with valo grand in standard mode for only 20 s, appears to have demonstrated the benefit of using a high-power curing unit. this could have resulted from the very thin layer of sealant and low light attenuation that tended to provide high levels of light energy within the sealant8,13,18. however, care should be taken since incident irradiance has only limited ability to compensate for the reduction in polymerization time and increase in efficiency9,19. gonulol et al.9 (2016) compared the polymerization of many tooth-colored restorative materials using three different modes of valo and elipar s10 as controls and showed that when valo was used in extra power mode (3200 mw/cm2) for 6 s, insufficient polymerization was achieved in all of the tested materials. according to the cited authors, this was especially noted for rmgi sealants containing fluoride particles, because they might produce light attenuation in thicker increments that could have a negative effect on monomer conversion in deep layers. another advantage of the third-generation led curing units refers to their wider spectral range of light emission. in order to be effective, it is well known that sufficient spectral radiant power must fall within the spectral range, as this is required to activate the photoinitiator(s) present in the material being used16. considering that manufacturers rarely reveal the proprietary constituents their products contain, and that alternative phoinitiators requiring activation by lower wavelength of light have been developed and introduced in resin-based materials34, the use of broad-spectrum light sources that deliver both violet and blue light, are preferable12. nonetheless, the characteristics of lcus are not the only factors that affect the quality of polymerization of resin-based sealants2,22. different compositions, as well as 8 marques et al. braz j oral sci. 2022;21:e226202 differences in the refractive indices of the organic matrix and inorganic filler components of the materials influence the transmission of visible light through them3,19. in this study, between the two rgmi sealants tested, clinpro has the less heterogenous mixture, mainly composed of organic matrix. this could be the reason why the differences between the led curing units tested were less evident when based on the khn values of this material. the continual development of led technology and composition of materials should be borne in mind. moreover, microhardness cannot be the final indicator for evaluating the setting of rmgi sealants. further studies using different mechanical and physical tests applied individually or in combination are needed in order to understand the complex relationship between polymerization efficiency and the use of different generations of led devices, and their effect on the properties of resin-based materials. within the limitations of this study, it could be concluded that surface microhardness of rgmi sealants was affected by both material composition and generations of led curing units used. third-generation of led curing units seemed to be more effective in relation to the polymerization efficiency of rgmi sealants than their previous generation. more information about led curing units and composition of materials should be provided by manufacturers to enable clinicians to determine proper protocols for their particular rgmi/led curing unit combinations. acknowledgments the authors are grateful to the department of restorative dentistry at fop-unicamp for to provide the use of the digital knoop hardness measuring instrument. conflicts of interest the authors have no proprietary, financial, or other conflict of interest of any nature or kind in any product, service, and/or company that is presented in this article. data availability datasets related to this article will be available upon request to the corresponding author. author contribution joyce figueiredo de lima marques: investigation, formal analysis, writing – review & editing, final approval of the version to be published. laura nobre ferraz: methodology, data acquisition, project administration, final approval of the version to be published. beatriz kelly barros lopes: investigation, writing – original draft, final approval of the version to be published. tamires aparecida borges vasconcelos: investigation, writing – original draft, final approval of the version to be published. thiely roberts teixeira: investigation, writing – original draft, final approval of the version to be published. débora alves nunes leite lima: resources, review of the manuscript critically for important intellectual content, final approval of the version to be published. flávio henrique baggio aguiar: conceptualization, methodology, 9 marques et al. braz j oral sci. 2022;21:e226202 resources, review of the manuscript critically for important intellectual content, final approval of the version to be published. diogo de azevedo miranda: conceptualization, project administration, review of the manuscript critically for important intellectual content, supervision, final approval of the version to be published. references 1. naaman r, el-housseiny aa, alamoudi n. the use of pit and fissure sealants-a literature review. dent j (basel). 2017;5(4):34. doi: 10.3390/dj5040034. 2. bani m, tirali re. effect of new light curing units on microleakage and microhardness of resin sealants. dent mater j. 2016;35(3):517-22. doi: 10.4012/dmj.2015-357. 3. borges bcd, souza-junior ej, catelan a, ambrosano gm, paulillo la, aguiar fh. impact of extended radiant exposure time on polymerization depth of fluoride-containing fissure sealer materials. acta odontol latinoam. 2011;24(1):47-51. 4. seixas g, guiraldo sb, lemos lvfm, myaki si, balducci i, moura s. clinpro ™ xt sealant adhesion to the occlusal surface of primary molars: longitudinal evaluation. j health sci. 2018;20(2):112-8. doi: 10.17921/2447-8938.2018v20n2p112-118 5. wright jt, crall jj, fontana m, gillette ej, nov´y bb, dhar v, et al. evidence-based clinical practice guideline for the use of pit-and-fissure sealants: a report of the american dental association and the american academy of pediatric dentistry. j am dent assoc. 2016;147(8):672-82.e12. doi: 10.1016/j.adaj.2016.06.001. 6. ahovuo-saloranta a, forss h, walsh t, nordblad 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treatment on selected properties of a conventional and a resin-modified glass ionomer cement. j mater sci mater med. 2008;19:1913-20. doi: 10.1007/s10856-007-3270-8. 26. kleverlaan cj, van duinen rn, feilzer aj. mechanical properties of glass ionomer cements affected by curing methods. dent mater. 2004;20(1):45-50. doi: 10.1016/s0109-5641(03)00067-8. 27. tolidis k, dionysopoulos d, gerasimou p, sfeikos t. effect of radiant heat and ultrasound on fluoride release and surface hardness of glass ionomer cements. j appl biomater funct mater. 2016;14(4):e463-9. doi: 10.5301/jabfm.5000292. 28. gorseta k, skrinjarić t, glavina d. the effect of heating and ultrasound on the shear bond strength of glass ionomer cement. coll antropol. 2012;36(4):1307-12. 29. dobrzynski m, herman k, bryla e, fita k, dudek k, kowalczyk-zajac m, et al. the heat risk during hardening of dental glass-ionomer cements using a light-curing. j therm anal calorim. 2019;135(6):3123-8. doi: 10.1007/s10973-018-7504-4. 30. mathias c, gomes rs, dressano d, braga rr, aguiar fhb, marchi gm. effect of diphenyliodonium hexafluorophosphate salt on experimental infiltrants containing different diluents. odontology. 2019;107(2):202-8. doi: 10.1007/s10266-018-0391-0. 31. ogliari fa, ely c, petzhold cl, demarco ff, piva e. onium salt improves the polymerization kinetics in an experimental dental adhesive resin. j dent. 2007;35(7):583-7. doi: 10.1016/j.jdent.2007.04.001. 32. gonçalves ls, moraes rr, ogliari fa, boaro l, braga rr, consani s. improved polymerization efficiency of methacrylate-based cements containing an iodonium salt. dent mater. 2013;29(12):1251-5. doi: 10.1016/j.dental.2013.09.010. 11 marques et al. braz j oral sci. 2022;21:e226202 33. augusto cr, leitune vcb, ogliari fa, collares fm. influence of an iodonium salt on the properties of dual-polymerizing self-adhesive resin cements. j prosthet dent. 2017 aug;118(2):228-34. doi: 10.1016/j.prosdent.2016.10.013. 34. shimokawa cak, turbino ml, giannini m, braga rr, price rb. effect of light curing units on the polymerization of bulk fill resin-based composites. dent mater. 2018;34(8):1211-21. doi: 10.1016/j.dental.2018.05.002. 1 volume 22 2023 e239237 original article braz j oral sci. 2023;22:e239237http://dx.doi.org/10.20396/bjos.v22i00.8669237 1 school of dentistry, federal university of pelotas, pelotas, brazil. 2 department of periodontology, graduate program in dentistry, federal university of pelotas, pelotas, brazil. 3 graduate program in medical sciences neuroscience and behavior laboratory, federal university of ceará, fortaleza, brazil. 4 department of oral pathology, graduate program in dentistry, federal university of pelotas, pelotas, brazil. corresponding author: francisco wilker mustafa gomes muniz rua gonçalves chaves, 457, pelotas/rs, brazil, zip code: 96015-560. telephone: +5553991253611 wilkermustafa@gmail.com editor: dr. altair a. del bel cury received: may 10, 2022 accepted: august 08, 2022 prevalence of depressive symptoms among dental students is influenced by sex, academic performance, smoking exposure, and sexual orientation: cross-sectional study bruna oliveira de freitas1 , maísa casarin2 , rafaela zazyki de almeida1 , jessica maria pessoa gomes3 , isadora vilas boas cepeda4 , francisco wilker mustafa gomes muniz2,* aim: to estimate the prevalence and associated factors of self-reported depressive symptoms in undergraduate and graduate dental students. methods: the depression, anxiety and stress scale (dass-21) was applied, and only the depression domain was verified. a structured questionnaire was used to collect sociodemographic, behavioral, and covid-19 pandemic-related fear variables. academic performance was assessed based on academic records, ranging from 0 (worst possible grade) to 10 (best possible grade). respondents included 408 regularly enrolled dental students. biand multivariate analyses were performed using poisson regression with robust variance to verify the association between at least moderate depressive symptoms and independent variables. results: the prevalence of at least moderate depression was 40.5% among undergraduate students and 26% among graduate students. the prevalence of fear and anxiety due to the covid-19 pandemic was 96.1% among undergraduate students and 93.5% among graduate students. in the final multivariate analysis, being female (prevalence ratio [pr]:2.01; 95% confidence interval [95%ci]:1.36–2.96) was associated with a higher pr for depression. conversely, no exposure to smoking (pr:0.54; 95%ci:0.36–0.82) and a final academic performance average ≥7.0 (pr:0.56; 95%ci:0.41–0.76) was associated with a lower pr for depression. finally, among graduate students, a non-heterosexual orientation was associated with a higher pr for depression (pr:6.70; 95%ci:2.21–20.29). conclusion: higher rates of depression symptoms were observed in female undergraduates, students with lower academic performance and smoking exposure, and graduate dental students with a non-heterosexual orientation. keywords: academic performance. students, dental. depression. mental health. https://orcid.org/0000-0001-6032-9076 https://orcid.org/0000-0002-3750-5091 https://orcid.org/0000-0003-2284-432x https://orcid.org/0000-0003-3345-6576 https://orcid.org/0000-0001-6608-1358 https://orcid.org/0000-0002-3945-1752 2 freitas et al. braz j oral sci. 2023;22:e239237 introduction depression is part of a group of mental disorders called minor psychiatric disorders1. such disorders are characterized by a combination of thoughts, perceptions, and emotions that may include physical symptoms with no apparent cause. more than 300 million people have been diagnosed with depression worldwide, generating high costs and strong socioeconomic impacts2. as a chronic and recurrent mental disorder, depression can significantly impact an individual’s personal and professional performance, ability to relate to others, and ability to address and solve everyday problems3,4. when evaluating the presence of a depressive disorder, it is necessary to consider not only dimensional characteristics but also factors such as intensity, duration, persistence, physiological and psychological interference, and disproportionate reactions to triggering factors. other critical considerations include environmental circumstances and an individual’s life history, as these can be triggers for depressive symptoms. these factors together determine the limit of normality; based on the disorder’s characteristics, depression can vary from normal (when it is considered a human reaction to a difficulty) to severe (when the patient is at risk of suicide and/or psychotic symptoms). in brazil, recent data from the brazilian institute of geography and statistics (instituto brasileiro de geografia e estatística [ibge]) (2020)5 showed that cases of depression have grown by 34.2%. a high prevalence of mental disorders has also been reported among college-educated students and, reflecting data from various parts of the world, the number of depression cases is even higher among students in the health field. in 2019, one study6 reported a prevalence of 83% for mild to moderate depression among health science students. in a study performed among final-year undergraduate dentistry students in colombia, an overall prevalence of 30.3% for common mental disorders was found7. another study showed that depression affects three out of ten dental students at a private university in central-western brazil8. crucially, the high variability for the prevalence of depression among students, variability which is dependent on the instruments used and the categorizations established. the beginning of academic life is a period full of changes for undergraduate students, who must adjust their routines and lifestyles to a new environment9. new students experience an adaptive physiological response to these changes, generating temporary anxiety and stress, which is expected during this phase10. however, when individuals have recurrent feelings of sadness and depression, these feelings may interfere with their professional training11. factors such as moving to another city, reduction or absence of family support, lack of leisure time, deadlines, professional expectations, and personal demands have been associated with mental strain for students6,12. the consumption of mind-altering substances (legal or illegal) typically also increases during the period of transition from adolescence to adulthood13. in addition, the covid-19 pandemic—with its attendant lockdowns, online classes, and changes in routine—has significantly affected students, and may have increased symptoms of anxiety14 and depression15. further3 freitas et al. braz j oral sci. 2023;22:e239237 more, the psycho-pedagogical impact of mental disorders on students has been associated with poor academic performance16. although several studies have evaluated the prevalence of depression among dental students, investigation of the factors associated with depression, especially through adjusted analytical strategies, remains scarce in the literature. therefore, this study aimed to assess the prevalence and associated factors of self-reported depressive symptoms among undergraduate and graduate dental students during the covid-19 pandemic. material and methods study design, location, and ethical aspects a cross-sectional study of undergraduate and graduate dental students at the local university was performed from june 2020 to august 2020. the present study was written following the recommendations of the strobe reporting checklist. the study protocol was reviewed and approved by the university ethics committee (n° 3.910.723). all participants electronically signed an informed consent form prior to inclusion in the study. population, inclusion and exclusion criteria all regularly enrolled dental students were invited to participate in the study. exceptions included students who had cancelled the course, those under 18, and graduate students without a degree in dentistry. data collection invitations to participate in this study were distributed via email, sent to all class representatives, and made available on the university’s digital platform and social networks. data were collected online through an electronic, self-applied questionnaire available on google forms. the questionnaire consisted of both structured and semi-structured questions. in this questionnaire, participants were asked if they consented to releasing access to their academic records. authorization to access academic records was electronically obtained. participants who denied access to their academic records did not have their data included in this study. participants who authorized access to their academic records were responsible for attaching the relevant academic record document to the final question of the questionnaire; in case of refusal, this field remained blank. all academic records were automatically sent only to the researcher, and remained confidential. the instrument was divided into several sections, addressing questions about socioeconomic, demographic, behavioral, and health data. no attempt was made at validation for these participant profile questions. the depression, anxiety and stress scale (dass-21), validated for brazil17, was then applied. in addition, three questions from a prior questionnaire18 about fear and anxiety related to the covid-19 pandemic were applied: 1) “do you want to close your dental practice until the number of covid-19 4 freitas et al. braz j oral sci. 2023;22:e239237 cases starts declining?” 2) “are you afraid of getting quarantined if you get infected?” and 3) “are you anxious about the cost of treatment if you get infected?” outcome definition self-reported depression symptoms were defined as the primary outcome of the present study. the dass-21 scale, with 21 questions, was used in its translated and validated version for portuguese17 to assess symptoms of depression in dentistry students in a context unrelated to their daily dental work routine for the previous week. the scale includes questions that measure depression, anxiety, and stress, with seven questions corresponding to each of the three domains. for each question, there were four possible answers according to the likert scale. score 0: not applied at all; score 1: applied to some degree, or for a short time; score 2: applied to a considerable degree, or for a significant amount of time; score 3: applied to an overwhelming degree, or most of the time. for the present study, only the seven questions related to the depression domain were considered. thus, sample data were categorized into the following depression symptom severities: normal (0 to 9 points), mild (10 to 13 points), moderate (14 to 20 points), severe (21 to 27 points), or extremely severe (≥28 points) self-reported depressive symptoms. independent variables the independent variables included the following: age (in years); sex (female, male, or other); skin color or race according to the ibge classification (white, brown, black, yellow, or indigenous); monthly family income (average value in brazilian reais); physical activity (yes or no); sexual orientation (heterosexual, homosexual, bisexual, or others); dentistry as a first choice of undergraduate course (yes or no); current semester for undergraduate students (1st to 10th semester); program level for graduate students (master’s or doctorate); academic performance (final average for undergraduate students, or frequency of the concepts achieved in the curriculum subjects for graduate students); receives salary/scholarship (yes or no); alcohol use (yes or no); cigarette use (yes or no); marijuana use (yes or no); and questions related to fear and anxiety within the context of the covid-19 pandemic. for the present analysis, sex was dichotomized into “male” and “female,” as no participant answered “other.” regarding skin color, the sample was categorized into “white” and “non-white.” sexual orientation was dichotomized into “heterosexual” and “non-heterosexual.” years of undergraduate study were categorized into 1st year, 2nd year, 3rd year, 4th year, and 5th year. academic performance at the university is measured in different ways for undergraduate and graduate students. undergraduate students receive grades ranging from 0 (worst possible grade) to 10 (best possible grade). graduate students, on the other hand, receive concepts ranging from a (best possible concept) to e (worst possible concept). thus, for the academic performance of undergraduate students, the sample was dichotomized into those who presented a final average of ≥7.0 or <7.0. for graduate students, the percentage of “a” grades was calculated. 5 freitas et al. braz j oral sci. 2023;22:e239237 for the presence of salary/scholarship, participants were categorized as “yes” if they reported being scholarship holders, being self-employed, being civil servants, or having some other type of employment relationship. regarding the questionnaire related to covid-19 pandemic fear and anxiety, the sample was divided into two groups: participants who answered “yes” to any question related to the covid-19 pandemic, and participants who answered “no or do not know” to all questions. statistical analysis only the depression domain of the dass-21 tool was considered in this study. therefore, mean scores in this domain were compared between sexes for undergraduate and graduate students, and between different years of study (undergraduate) or level of education (graduate). for this variable, normality was tested using the shapiro-wilk test, and no symmetric distribution was detected. therefore, the non-parametric mann-whitney and kruskal-wallis tests were used. bivariate and multivariate analyses using poisson regression with robust variance were employed to verify the association between self-reported depressive symptoms and exploratory variables. for these analyses, the sample was dichotomized into “normal or mild depressive symptoms” and “moderate, severe, or extremely severe depressive symptoms.” independent analyses were performed for undergraduate and graduate students. four variables were included in the final multivariate model based on theoretical and biological plausibility, regardless of the observed p-value. these variables were sex, years of education or education level, academic performance, and anxiety and fear related to the covid-19 pandemic. academic performance was defined as the main exploratory variable in this study. in addition, data collection was performed during the period of restricted face-to-face activities at the university, prompting the inclusion of the variable related to fear and anxiety in the face of the covid-19 pandemic. for all other variables, only those that presented a p-value <0.25 in the bivariate analysis were included in the initial multivariate model. the final multivariate model was built using the “backwards” strategy, i.e., observing the statistical significance and the effect of the modifications on the model. statistical significance was defined as p<0.05. all analyses were performed using spss software (version 21.0, statistics ibm, college station, tx, usa). results in the first semester of 2020, the school of dentistry enrolled 570 students, of which nine undergraduate students were excluded from this study for not being regularly enrolled. from the remaining 561, 409 students initially responded, with one respondent refusing to participate further after reading the informed consent form. thus, 408 students ultimately responded to the survey, including 331 undergraduate (response rate: 71.18%) and 77 graduate (response rate: 74.29%) students. no significant difference was found between participants and non-participants in terms of the overall population sex for undergraduate (p=0.550) and graduate (p=0.273) students. 6 freitas et al. braz j oral sci. 2023;22:e239237 the majority of the sample consisted of female students (68.6%) and white students (81.9%). the occurrence of cigarette use was higher among undergraduate students (8.5%) compared to graduate students (6.5%); the same result was observed for marijuana use, with an occurrence higher in undergraduate (10.3%) than in graduate (6.5%) students. the prevalence of fear and anxiety due to the covid-19 pandemic was 96.1% in undergraduate students and 93.5% in graduate students (table 1). table 1. descriptive characteristics of the sample regarding sociodemographic and behavioural variables for both undergraduate and graduate dental students (n=408) from the local university, 2020. variables undergraduate (n=331; 81.1%) graduate (n=77; 18.9%) total (n=408) age mean±sd 22.70±3.56 30.29±6.97 24.13±5.31 sex male – n (%) 107 (32.3) 21 (27.3) 128 (31.4) female – n (%) 224 (67.7) 56 (72.7) 280 (68.6) skin colour white – n (%) 266 (80.4) 68 (88.3) 334 (81.9) non-white – n (%) 65 (19.6) 9 (11.7) 74 (18.1) monthly family income (in thousand brazilian reais) mean±sd 8.37±30.79 8.50±6.14 8.40±27.68 absent 26 1 27 practice of physical activity yes – n (%) 159 (48.0) 44 (57.1) 203 (49.8) no – n (%) 172 (52.0) 33 (42.9) 205 (50.2) sexual orientation heterosexual – n (%) 291 (87.9) 73 (94.8) 364 (89.2) non-heterosexual – n (%) 40 (12.1) 4 (5.2) 44 (10.8) dentistry as the first choice yes – n (%) 225 (68.0) 57 (74.0) 282 (69.1) no – n (%) 106 (32.0) 20 (26.0) 126 (30.9) year 1st year – n (%) 55 (16.6) 55 (16.6) 2nd year – n (%) 72 (21.8) 72 (21.8) 3rd year – n (%) 60 (18.1) 60 (18.1) 4th year – n (%) 67 (20.2) 67 (20.2) 5th year – n (%) 77 (23.3) 77 (23.3) mean final grade <7.0 – n (%) 46 (18.8) 46 (18.8) ≥7.0 – n (%) 199 (81.2) 199 (81.2) absent 86 86 continue 7 freitas et al. braz j oral sci. 2023;22:e239237 continuation graduate level master’s – n (%) 32 (41.6) 32 (41.6) doctorate – n (%) 45 (58.4) 45 (58.4) percentage of grades a mean±sd 89,44±9.05 89.44±9.05 absent 18 18 is a scholarship holder or has some paid activity yes – n (%) 75 (22.7) 74 (96.1) 149 (36.5) no – n (%) 256 (77.3) 3 (3.9) 259 (63.5) alcohol use in the last 30 days yes – n (%) 229 (69.2) 54 (70.1) 283 (69.4) no – n (%) 102 (30.8) 23 (29.9) 125 (30.6) cigarette use in the last 30 days yes – n (%) 28 (8.5) 5 (6.5) 33 (8.1) no – n (%) 303 (91.5) 72 (93.5) 375 (91.9) marijuana use in the last 30 days yes – n (%) 34 (10.3) 5 (6.5) 39 (9.6) no – n (%) 297 (89.7) 72 (93.5) 369 (90.4) answer yes to any covid-19 question? yes – n (%) 318 (96.1) 72 (93.5) 390 (95.6) no/do not know – n (%) 13 (3.9) 5 (6.5) 18 (4.4) covid-19: coronavirus disease 2019; sd: standard deviation. as shown in table 2, a comparison of the mean scores on the dass-21 tool reveals a significantly higher depression score among female undergraduate students when compared to male undergraduate students (p<0.001). it was also observed that 40.5% (n=134) of undergraduate students had scores indicating moderate, severe, or extremely severe depression symptoms. however, when considering only graduate students, no significant differences between sexes (p=0.154) or education levels (p=0.481) were identified for the dass-21 scores. table 2. description of the depression, anxiety and stress scale (dass-21) tool, according to sex, years of study for undergraduation and graduate level (n=408) from the local university, 2020. variables undergraduate (n=331; 81.1%) graduate (n=77;18.9%) total (n=408) depression domain mean±sd 12.74±10.97 8.16±9.13 11.88±10.79 depression domain normal 156 (47.1) 48 (62.3) 204 (50.0) mild 41 (12.4) 9 (11.7) 50 (12.2) continue 8 freitas et al. braz j oral sci. 2023;22:e239237 continuation moderate 64 (19.3) 14 (18.2) 78 (19.1) severe 26 (7.9) 2 (2.6) 28 (6.9) extremely severe 44 (13.3) 4 (5.2) 48 (11.8) p-value p-value depression domain male 9.48±8.96 0.001* 5.33±7.91 0.154* female 14.30±11.51 9.21±9.39 depression domain 1st year 12.58±10.89 0.786# 2nd year 13.17±10.87 3rd year 12.50±12.24 4th year 13.73±11.10 5th year 11.79±10.13 depression domain master’s 7.00±7.92 0.481* doctorate 8.98±9.90 sd: standard deviation; *mann-whitney test; #kruskal-wallis test. the prevalence of at least moderate depression symptoms was 40.5% (n=134) and 26% (n=20) for undergraduate and graduate students, respectively. in the univariate analysis (table 3), when considering only undergraduate students, the variables of sex (p<0.001) and final course average (p=0.001) were significantly associated with the occurrence of at least moderate depression symptoms. when considering only graduate students, sexual orientation was the only variable significantly associated with the presence of depression symptoms (p=0.001). table 3. bivariate analysis for the presence of self-reported symptoms of at least moderate depression among undergraduate and graduate dental students (n=408) at the local university, 2020. undergraduate pr (95%ci) p-value graduate pr (95%ci) p-value age 1.00 (0.96 – 1.04) 0.973 0.99 (0.94 – 1.04) 0.617 sex male 1 <0.001 1 0.415 female 1.89 (1.33 – 2.70) 1.50 (0.57 – 3.97) skin colour white 1 0.282 1 0.577 non-white 1.18 (0.87 – 1.60) 1.33 (0.49 – 3.67) monthly family income 1.00 (0.99 – 1.01) 0.304 0.97 (0.92 – 1.03) 0.307 continue 9 freitas et al. braz j oral sci. 2023;22:e239237 continuation practice of physical activity yes 1 0.231 1 0.079 no 1.18 (0.90 – 1.53) 2.00 (0.92 – 4.33) sexual orientation heterosexual 1 0.072 1 0.001 non-heterosexual 1.35 (0.97 – 1.88) 3.22 (1.60 – 6.50) dentistry as the first choice yes 1 0.613 1 0.087 no 1.07 (0.82 – 1.41) 1.90 (0.91 – 3.96) year 1st year 1 2nd year 1.18 (0.79 – 1.77) 0.422 3rd year 0.83 (0.51 – 1.35) 0.459 4th year 1.05 (0.68 – 1.61) 0.842 5th year 0.97 (0.64 – 1.49) 0.904 mean final grade <7.0 1 0.001 ≥7.0 0.61 (0.45 – 0.83) graduate level master’s 1 0.495 doctorate 1.321 (0.59 – 2.94) percentage of grades a 1.01 (0.97 – 1.06) 0.627 is a scholarship holder or has some paid activity yes 1 0.319 1 0.756 no 1.19 (0.85 – 1.67) 1.30 (0.25 – 6.74) alcohol use in the last 30 days yes 1 0.212 1 0.988 no 0.83 (0.61 – 1.12) 1.01 (0.44 – 2.29) cigarette use in the last 30 days yes 1 0.248 1 0.421 no 0.79 (0.53 – 1.18) 0.63 (0.20 – 1.97) marijuana use in the last 30 days yes 1 0.640 1 0.762 no 0.91 (0.61 – 1.36) 1.32 (0,22 – 7.94) answer yes to any covid-19 question? yes 1 0.129 1 0.762 no/do not know 0.37 (0.10 – 1.34) 0.76 (0.13 – 4.56) 95%ci: 95% confidence interval; covid-19: coronavirus disease 2019; pr: prevalence ratio. 10 freitas et al. braz j oral sci. 2023;22:e239237 in the final multivariate model (table 4), female undergraduate students showed significantly higher levels of depression symptoms. the prevalence ratio (pr) was approximately twice as high (p<0.001) for the occurrence of at least moderate depression symptoms in female students than in male students. however, in graduate students, no significant association was identified for sex (p=0.099). non-heterosexual graduate students presented a 6.7 times higher pr (p=0.001) for at least moderate depression symptoms than heterosexual students. undergraduate students with a final average ≥7.0 showed a 44% lower pr (p<0.001) for the presence of depression symptoms when compared to students with a final average <7.0. furthermore, smoking was associated with the occurrence of depression symptoms among undergraduate students; those not having used a cigarette in the last 30 days had a 46% lower pr for at least moderate depression symptoms when compared to those having used one recently (p=0.004) (table 4). table 4. multivariate analysis for the presence of at least moderate depression among undergraduate and graduate dental students (n=408) from the local university, 2020. undergraduate pr (95% ci) p-value graduate pr (95% ci) p-value sex male 1 <0.001 1 0.099 female 2.01 (1.36 – 2.96) 3.47 (0.79 – 15.16) sexual orientation heterosexual 1 0.001 non-heterosexual 6.70 (2.21 – 20.29) year 1st year 1 2nd year 0.89 (0.51 – 1.54) 0.677 3rd year 0.85 (0.48 – 1.52) 0.580 4th year 0.93 (0.54 – 1.59) 0.786 5th year 0.65 (0.35 – 1.18) 0.155 mean final grade <7.0 1 <0.001 0.99 (0.94 – 1.04) 0.675 ≥7.0 0.56 (0.41 – 0.76) graduate level master’s 1 0.057 doctorate 2.99 (0.97 – 9.23) cigarette use in the last 30 days yes 1 0.004 no 0.54 (0.36 – 0.82) answer yes to any covid-19 question? yes 1 0.798 1 0.322 no/do not know 0.87 (0.29 – 2.61) 2.98 (0.34 – 25.95) 95%ci: 95% confidence interval; covid-19: coronavirus disease 2019; pr: prevalence ratio. 11 freitas et al. braz j oral sci. 2023;22:e239237 discussion this study assessed the prevalence of self-reported depressive symptoms among undergraduate and graduate dental students. a significant prevalence of at least moderate depression symptoms was found, mainly in undergraduate dental students. higher rates of depression symptoms were demonstrated in female undergraduates, undergraduates with lower academic performance, undergraduates with smoking exposure, and graduate dental students with a sexual orientation other than heterosexual. to understand these findings, it is important to highlight that higher rates of regular exercise and smoking exposure were observed in undergraduate male dental students (57% and 14%, respectively) when compared to female students (43.8% and 5.8%, respectively). however, 83.2% of female undergraduate students had an overall mean ≥7.0, while this rate was 76.9% among male students. sexual orientations other than heterosexual were reported among 14.3% and 1.8% of male and female graduate students, respectively. although these rates may partially explain the current results, they also emphasize the necessity of conducting an adjusted analysis to control possible confounders associated with depression symptoms. studies involving academics in the health sciences have suggested that higher rates of mental disorders are most strongly related to demanding clinical routines, extensive scheduling, career doubts, and the highly competitive environment. these factors are responsible for creating an exhausting environment that can trigger stress events and psychological illnesses such as burnout syndrome and impostor syndrome19,20. due to the characteristics of the program, dental students are constantly exposed to risky or unhealthy environments, serious and complicated pathological processes, and the fear of making mistakes during educational training, all of which can negatively impact a student’s physical and psychological health19. one systematic review showed an overall prevalence of 29% (95%ci: 26–34) for depression among dental students21. similarly, 37.8% of the dental students included in this study reported at least moderate depression symptoms. the higher prevalence of depressive symptoms detected among female undergraduate students highlights the impact of several sociocultural factors in contemporary society. these include the oppression experienced by women throughout their lives, the history of demands on women, and the greater ease women typically have with reporting and admitting feelings and seeking medical treatment, in addition to low self-esteem and biological factors such as sex hormones, endocrine reactivity to stress, and lower levels of vitamin b1222,23. such concerns expose women to risk factors for developing depression, increasing their vulnerability to the illness24. all these factors contribute to the higher occurrence of self-reported depression in women, which has already been demonstrated in the dental literature21. in addition to sex, tobacco consumption among the participants was significantly associated with the occurrence of depressive symptoms. however, the direction of this relationship is still not well understood, since nicotine acts as a self-medication to relieve feelings of sadness or negative mood25,26. smoking can also momentarily distract the individual from stressful situations and problems27. another hypothesis 12 freitas et al. braz j oral sci. 2023;22:e239237 for this association is that smoking and depressive symptoms have a bidirectional relationship, wherein smoking is positively reinforced when depressed smokers use tobacco to alleviate their negative feelings, obtaining the desired effect from nicotine28. it must also be noted that the present study did not assess the frequency of consumption of cigarettes, alcohol, or illicit drugs. thus, assuming a dose-response gradient is not possible given the results found. in the present study, poorer academic performance was associated with a higher occurrence of at least moderate self-reported symptoms of depression. stress and depression have the potential to impact reasoning, memory, and motivation, which may reflect on the learning process. furthermore, stress is one of the symptoms of depressive disorder, causing damage to an individual’s performance and a further decrease in productivity29. thus, the lower academic performance observed among undergraduate students with depression symptoms can be justified. the literature indicates that young people who belong to sexual minorities are significantly more likely to present depressive symptoms and face the double stigma of belonging to this marginalized group and experiencing mental health problems30,31. this disparity in the prevalence of depressive symptoms among non-heterosexual individuals has been associated with stressors such as discrimination, structural prejudices, concealment of sexual orientation, and isolation among heterosexual individuals32,33. in addition to the many stressors discussed above, the interruption of teaching activities due to the covid-19 pandemic has induced fear of losing manual skills, anxiety related to the consequences for long-term plans, and concerns regarding new dental protocols during the resumption of normal activities34,35. for these reasons, all multivariate analyses performed in the present study were adjusted for fear and anxiety specifically related to the covid-19 pandemic. however, no significant associations were found between this variable and the occurrence of depressive symptoms. it is critical for health education institutions—which are responsible for training future professionals who will provide assistance to the community—to adopt preventive measures of psychological support for students facing academic and social challenges. measures that promote awareness and inclusion of stigmatized groups are also necessary in order to reduce requirements for supportive and psychological treatment. higher education should thus aim to promote total academic success, embracing the individual and their anxieties. therefore, it is important to highlight that different academic levels and circumstances are related to different factors that might trigger depression in different ways. the limitations of this study were due primarily to the covid-19 pandemic, which made it difficult to contact participants and significantly changed their routines and activities. in addition, the aim of this study was restricted to evaluating responses to self-reported symptoms of depression, and was not concerned with any formal diagnosis of this condition. as a result, further studies that include an interview or psychiatric evaluation are needed to provide a more nuanced understanding. furthermore, the cross-sectional nature of this study did not allow for the establishment of causality between the detected associations. in this context, the reverse causal13 freitas et al. braz j oral sci. 2023;22:e239237 ity hypothesis cannot be ruled out. although the present study did not find significant associations between depressive symptoms and fear and anxiety regarding the covid-19 pandemic, it must be noted that the pandemic might have had a serious impact on all students, which may have contributed to the mental health burdens faced by these particular students. moreover, only one public higher education institution was included in the present study, which could be seen as a limiting factor. however, this also means that a higher internal validity can be expected, and thus that a high external validity may be applied for other public brazilian institutions with similar socioeconomic characteristics. this study ultimately concluded that female undergraduate dental students exhibit higher levels of depression symptoms when compared to male students. poorer academic performance was also associated with the occurrence of depression symptoms in undergraduate students, as was cigarette consumption within the last 30 days. finally, graduate dental students belonging to sexual minorities reported greater depressive symptoms when compared to heterosexual students. acknowledgments this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior brasil (capes) finance code 001. all other funding was self-supported by the authors. the authors report no conflict of interest. author contribution bruna oliveira de freitas: methodology, validation, investigation, data curation, writing – original draft preparation. maísa casarin: conceptualization, methodology, investigation, resources, writing – review and editing. rafaela zazyki de almeida: validation, investigation, writing – review and editing. jessica maria pessoa gomes: validation, writing – review and editing, visualization. isadora vilas boas cepeda: writing – original draft preparation, visualization. francisco wilker mustafa gomes muniz: conceptualization, methodology, formal analysis, investigation, resources, writing – review and editing, supervision. all authors actively participated in the discussion of the manuscript’s findings, revised, and approved the final version of the manuscript. references 1. world health organization, pan american health organization. transtornos mentais. genebra: who; 2022 june 8 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lira universidade estadual do piauí, faculdade de odontologia rua senador joaquim pires 2076 ininga. fone (86) 999595004 cep: 64049-590 teresina-pi-brasil email: anadelourdessl@hotmail.com received: august 25, 2020 accepted: october 29, 2020 prevalence of sleep bruxism in children in primary dentition ana de lourdes sá de lira1,* , francisco dário carvalho de sousa1, francisca janiele de sousa1, maria karen vasconcelos fontenele1, carlos kelvin campos ribeiro1, luiz eduardo gomes ferreira1 aim: to evaluate the prevalence of sleep bruxism in children between 2 to 6 years old in primary dentition in a public school (a1) and a private school (a2) in the state of piauí. method: it was cross-sectional and quantitative study in 370 participants, 180 in a1 and 190 in a2. the questionnaire to help diagnose bruxism was applied to parents. in the clinical examination, dental wear was evaluated, checking for the presence of shiny and polished facets on deciduous incisors and / or molars (palatal surface, incisal edges and working cusps) and the results were noted in the odontogram. students who had bruxism participated in group 1 (g1) and those who did not have it were in the control group (cg) in both a1 and a2. results: one hundred and five children had sleep bruxism. tooth wear was more prevalent in the male gender. both in a1 and in a2 and in both genders, wear on primary molars was more frequent than in canines. conclusion: the prevalence of bruxism in children between 2 and 6 years of age was 28.3%, with a predominance in males. there was an association between sleep bruxism and the habit of sucking a finger, pacifier, bottle use and the habit of awake bruxism. in 98.09% of the children who had sleep bruxism, teeth pain was found. keywords: sleep bruxism. epidemiology. child. https://orcid.org/0000-0002-9299-1416 2 lira et al. introduction bruxism is a repetitive jaw-muscle activity characterized by clenching or grinding the teeth and/or by bracing or thrusting the mandible that occurs in adults and children, characterized by clenching or clenching teeth. it can occur during the day, daytime bruxism or at night, sleep bruxism (sb)1. a study carried out in a brazilian city observed the prevalence of sleep bruxism in the preschool children approximately 14 percent. with no difference between genders1. the etiology of bruxism is still undefined, but studies suggest several predisposing factors, including systemic and hereditary factors, of psychological and functional origin, such as: strong emotional tension, family problems, existential crises, state of anxiety, depression and children in the self-affirmation phase2,3. the most common signs and symptoms are: dental wear, marks on the cheek mucosa, fractures of restorations, temporomandibular pain and dysfunction, muscle hypertrophy and headache. in children they can be related to headaches and cause extensive damage to primary teeth, such as pain and tooth wear. as primary dentition has an influence on the development of permanent dentition, regular examination of children is an opportunity to intercept the appearance of disorders of the stomatognathic system4. although different methods of diagnosis are used to sleep bruxism including the assessment of masseter muscle contraction through bite strip electrode and polysomnography, it is noteworthy that the american academy of sleep medicine (aasm) considers the reports of parents/caregivers to be quite reliable and sufficiently objective for use in epidemiological studies3,5. the diagnosis is based on the reports of family members who describe characteristic sounds generated by the grinding of teeth during sleep, when the questionnaire associated with the clinical examination to identify tooth wear is applied6,7. preschoolers can develop habits that upset the balance between function and growth. in recent years, bruxism in children has become a growing concern, since more often parents seek out a pediatric dentist with this complaint, in addition, this parafunctional habit has a direct impact on quality of life, as it compromises the sleep period, in addition to be considered a risk factor for temporomandibular disorder and damage to the stomatognathic system8-11. as bruxism is a subconscious reflex that is uncontrolled and mild, it is most often unknown or unnoticed by patients and their families. thus, it is necessary to establish an early diagnosis of possible changes that may occur, before the vicious cycle results in serious and permanent damage12. socioeconomic and cultural characteristics may be associated with the prevalence of sleep bruxism. the literature has reported that the prevalence is higher in children of greater socioeconomic power, probably due to the stress they are subjected to with numerous daily educational activities and poor sleep quality of the child, causing stress and anxiety in children3,12. 3 lira et al. it is assumed that there is a low prevalence of bruxism in children in primary dentition and ignorance of parents and / or guardians of children with bruxism regarding the damage caused by this harmful oral habit. knowledge of the distribution and factors associated with the manifestation of sleep bruxism in children in the primary dentition is of considerable importance to be able to draft treatment strategies and public health policies directed at oral health. since children at an early age can develop habits that impair the balance between function and growth and there are few studies conducted on children on the topic addressed, it was desired to investigate the prevalence of sleep bruxism in children preschoolers in primary dentition. the aim of this research was to evaluate the prevalence of sleep bruxism in children between 2 to 6 years old in primary dentition in a public school (a1) and a private school (a2) in the state of piauí. materials and methods it was a cross-sectional study, whose descriptors used were: sleep bruxism, epidemiology, children, prevalence. the sample calculation was based on the target population: children between 2 and 6 years old enrolled, specifically in the preschool phase, from the city of parnaíba in 2018, totaling 4.087 students and the required sample size was 365 participants. thus, in order to successfully achieve the objective of the present study, it was based on the survey by the brazilian institute of geography and statistics, which estimates an estimated 15.000 people in the target population: children living in the city of parnaíba-pi aged 2 to 6 years. from this, the sample size formula was performed, resulting in a number of 365.the survey was carried out with 10 more children, totaling 370, to compensate for the withdrawal of 5, whose parents did not answer the questionnaire. this minimum number of participants is considered sufficient considering the proposed analyzes, the 5% sampling error, and 95% confidence level, indicating that the probability of the mistake made by the research does not exceed 5%13. the researchers were provided with a letter of consent from the principals of two public schools (a1) and two private schools (a2) chosen by lot in the municipality of parnaíba-piauí, which authorized the development of the research after the ethical approval of the research committee. research ethics at the state university of piauí cep / uespi number: 3.289.714. this research was guided by the obedience of all the ethical principles that guide the research involving human beings, as foreseen in resolution nº 466/12 (cns / ms), having the parents or guardians signed the informed consent form (icf) and the children signed the term of assent, after having been advised that the child’s participation was not mandatory, and could withdraw at any time, without any harm to them. inclusion criteria were children in primary dentition, between 2 and 6 years of age, with no motor disturbances or psychiatric disorders or no systematic diseases (based on parents’/caregivers’ reports), whose parents or guardians and children accepted the research. as exclusion criteria, children under 2 years old, as the primary dentition would not yet be fully formed, and those over 6 years old, because they are already in 4 lira et al. mixed dentition(presence of one or more erupted permanent teeth) and a history of orthodontic treatment. to standardize the diagnosis of tooth wear, clinical training of examiners was carried out by the professor of the children’s clinic at clinic school of dentistry (csd) of the universidade estadual do piauí (uespi), experienced in epidemiological studies. as a pilot study, 20 children who did not participate in the research were examined to test the methodology and 10% of the sample was examined twice, with an interval of two weeks, to determine the intra-examiner and inter-examiner agreement, with the kappa coefficient for inter and intraexaminer agreement being 0.81 and 0.85, respectively. the diagnosis of sb was based on the reports of tooth grinding during sleep, as proposed by the aasm for preschool children that include: parents who report an occurrence of audible tooth grinding at night, but no other medical or mental disorders (e.g., sleep-related epilepsy, accounts of abnormal movements during sleep), and no other sleep disorders (e.g., obstructive sleep apnea syndrome)5. the children were also submitted to a clinical examination to determine tooth wear, and evaluate its possible association with sleep bruxism. this oral condition was diagnosed in the presence of wear on the incisal surfaces of the anterior teeth, and/or occlusal surfaces of the posterior teeth2. the parents attended a lecture on sb and its clinical manifestations and symptoms, before answering the questionnaire validated applied by the researchers (figure 1)14. using an age-compatible language, the children received an explanation of how the research would be carried out. figure 1. questionnaire for evaluation of bruxism. 5 lira et al. in a second moment in the school environment, the children were examined under artificial light by two examiners, who used sterilized cotton rollers, a flat mouth mirror and an explorer probe. the children were seated in a school chair, with their head positioned in the examiner’s lap. in the clinical examination, dental wear was evaluated, checking for the presence of shiny and polished facets on deciduous incisors and / or molars (palatal surface, incisal edges and working cusps) and the results were noted in the odontogram. children who had sb participated in group 1 (g1) and those who did not have it were in the control group (cg) in both a1 and a2. with spss, in its version 25, it was possible to perform descriptive statistics, with percentages and frequencies, association analyzes from the chi-square and mean comparisons from the t test, all with the level of significance measured by p value> 0.05. results based on the data collected from 370 children, it was possible to observe that the sb rate was 28.3%, that is, 105 children showed the behavior of grinding and / or clenching their teeth during sleep. specifically regarding the distribution of the sample in relation to gender and school, its prevalence was observed in table 1 and figure 2. table 1. characteristics and dental status of subjects. variable frequency total g1 cg p value n % n % n % public (a1) 180 48.6 41 22.8 139 77.2 0.05* private a2) 190 51.4 64 33.7 126 66.3 0.05* male 209 56.5 63 30.1 146 69.9 female 161 43.5 42 26.1 119 73.9 normal occlusion 320 86.5 70 21.8 250 78.2 0.01* malocclusion 50 13.5 35 70.0 15 30.0 absence dental carie 335 90.6 88 26.2 247 73.8 0.01* dental carie 35 9.4 17 48.5 18 51.5 note: chi-square test; * p<0.05. 160 140 120 100 80 60 40 20 0 41 64 63 42 139 126 146 119 with bruxism (g1) 41 64 63 42 no bruxismo (cg) 139 126 146 119 public (a1) private (a2) male female public (a1) private (a2) male female figure 2. prevalence of bruxism regarding gender and school. 6 lira et al. based on these values, complemented with statistical chi-square calculations, it was found that in the children of the cg there were no statistically significant differences (p> 0.05). however in the children of g1 there was a difference in terms of gender (x2 = 4.20; p <0.05), with boys having a higher prevalence and regarding the type of school (x2 = 5.04; p <0.05) and school a2 showed a higher prevalence of children with bruxism. thirty-five children in g1 presented anterior open bite, 25 of them due to the habit of sucking a finger or pacifier and 10 due to mouth breathing. in the cg, all 15 children with this type of malocclusion were due to the finger sucking habit. only 35 children had caries on the proximal surfaces of the upper incisors (17 in g1 and 18 in g2) (table 1). the t test was applied to compare the mean age between g1 and cg, and it was possible to verify that there was a statistically significant difference (t = 3.47; p = 0.01), with higher mean ages for g1 (4.81 years; sd = 0.96) when compared to the cg (4.38; sd = 1.27). table 2 showed the possible etiological factors and side effects and their association with sb. table 2. distribution of harmful habits and symptoms associated with sleep bruxism harmful habits and symptoms (g1) (cg) total x2 p valor suck finger/pacifier yes 25 92 117 22.42 no 90 163 253 0.01* use bottle yes 26 109 135 10.78 no 79 156 235 0.01* bite nail yes 61 125 186 2.45 no 44 144 184 0.12 biting lips yes 72 171 243 0.08 no 33 94 127 0.78 awake bruxism yes 75 66 141 64.76 no 30 199 229 0.01* chewing gum yes 102 228 330 5.82 no 3 37 40 0.01* biting objects yes 63 174 237 2.24 no 42 91 133 0.13 snoring at night yes 18 42 60 0.24 no 87 223 310 0.88 drooling asleep yes 18 27 45 2.92 no 87 238 325 0.09 talking asleep yes 9 18 27 0.25 no 96 247 343 0.62 waking up while sleeping yes 18 60 78 1.79 no 97 195 292 0.18 continue 7 lira et al. continuation breathe through your mouth yes 25 38 63 1.85 no 82 225 307 0.17 headache yes 48 99 147 1.46 no 57 166 223 0.23 neck and shoulder pain yes 31 68 99 0.31 no 74 197 271 0.58 toothache yes 103 93 196 113.88 no 2 172 174 0.01* note: chi-square test; * p<0.05 based on table 2, with the frequency data, followed by the chi-square test, it was possible to observe an association of sb with the habit of sucking a finger and / or pacifier (p = 0.01), with 15 children showing such habit. there was also an association between the fact that the child uses a bottle (p = 0.01) showing 26 children with sb. there was an association with awake bruxism and sb (p = 0.01). this fact was also observed with the majority of children who chew gum (p = 0.01). finally, there was a prevalence of almost all children who have sb, feel pain in the teeth (p = 0.01). according to table 3, tooth wear was more prevalent in the male gender. both in a1 and in a2 and in both genders, wear on primary molars was more frequent than in canines. table 3. distribution of dental wear in relation to schools and gender variable total p value molars canines p value n % n % n % a1 51 48.6 5.3 35 68.6 16 31.4 0.05* a2 54 51.4 39 72.2 15 27.8 0.05* male 65 61.9 0.05* 45 69.2 20 30.8 0.05* female 40 38.1 30 75 10 25 0.05* note: chi-square test; * p<0.05. discussion based on the data collected with 370 children, it was possible to observe that the bs rate was 28.3%, corroborating the study by junqueira et al.15, with preschool children, from 2 to 6 years old, whose prevalence was 29.3%. approximate values were found by clementino et al.8 and alves et al.16 who found a prevalence of 32.4% and 25.2% respectively. insana et al.17 studying preschool children in kentucky-usa, they found a high prevalence of 36.8%, with greater frequency in the male gender. in brazilian researches including older children in mixed dentition, drumond et al.9 and feitosa et al.10 found a higher prevalence, 40% and 53.2%, respectively. the difference 8 lira et al. between these studies may be explained by the application of different methodologies and/or the differences between the age groups of the patients. the clinical implication of knowing the prevalence of bs is the possibility of informing parents about the importance of early diagnosis and immediate treatment, since parents’ knowledge about the subject is fundamental. in the present study, there was a difference regarding gender, with boys having a higher prevalence of sb, corroborating the study by sousa et al.11. this fact may be related to the fact that boys are more agitated and, in general, motivated to contain their emotions, which would favor the occurrence of involuntary movements18. however, in the study by clementino et al.8 there was a higher prevalence in girls. in this research, it was found that sb was more frequent in children studying in private schools. it is assumed that socioeconomic factors may be associated with the prevalence of sb. although this association has not yet been clarified, it may be related to the number of daily activities children perform. on the other hand, serra-negra et al.7 did not detected no significant association between social vulnerability and sleep bruxism, but found that most children without this parafunctional activity belonged to more privileged social classes. in this study, it was possible to observe an association between sb and the habit of sucking a pacifier / finger. similar results were observed by simões-zenari and bitar19, when they stated that children with a habit of biting their lips present a five-fold increased risk for bruxism, just as the use of a pacifier increases the risk by approximately seven times. miamoto et al.20 found that individuals with sucking habits were four times more likely to develop sb. there was an association between the fact that the child uses a bottle and presents bruxism. rodrigues et al.12 in a systematic review stated that bruxism in children is associated with the use of a pacifier. lamenha et al.21 identified the connection between digital sucking habits, nail biting, pacifier use and sb. in this research, there was also an association between the fact that the child uses a bottle and presents bruxism. on the other hand, orengul et al.22 did not find an association between the use of a bottle and the presence of sb. lamenha et al.21 also found no relationship of sb in children with the use of bottles. in this research, attention is drawn to the association of awake bruxism (n = 75) and sb (n = 105). it was observed that the prevalence of almost all children with sb (n = 105) experience pain in the teeth (n = 103). probably the painful symptoms are the result of loss of tooth structure (enamel and dentin) during bruxism, whereas tooth wear is more severe in the primary dentition due to a lower degree of mineralization than in the permanent dentition. in addition, only 35 children out of a total of 370 had enamel caries on the proximal surfaces of the upper incisors (17 in g1 and 18 in g2) (table 1). goldstein and auclair23 concluded that awake bruxism is associated with tooth clenching instead of grinding teeth awake, suggesting that awake bruxism is more associated with emotional stress than with sb. countless times it goes unnoticed until a dental emergency occurs, such as pain or fracture. although in this research there was no significant correlation between chewing gum and sb, lamenha et al.21 and orengul et al.22 observed that most children who chew 9 lira et al. gum have this habit, especially the female gender. it is noteworthy that there was a statistical correlation between bruxism and oral habits of digital sucking and pacifier. however, castelo et al.4 in their research found a correlation between sb and chewing gum, mouth breathing, nail biting and lip biting habits. although polysomnography is the best test for the diagnosis of sb, its high cost makes its use unfeasible, especially in epidemiological investigations. thus, the present study has limitations that should be addressed. the cross-sectional nature of the design allows the demonstration of associations but not causality. longitudinal studies should be carried out to gain a better understanding of the factors that influence the occurrence of bruxism in children. in conclusion, the prevalence of bruxism in children between 2 and 6 years of age was 28.3%, with a predominance in males. there was an association between sleep bruxism and the habit of sucking a finger, pacifier, bottle use and the habit of awake bruxism. in 98.09% of the children who had sleep bruxism, teeth pain was found. references 1. vieira-andrade rg, drumond cl, martins-junior pa, correa-faria p, gonzaga gc, marques ls, et al. prevalence of sleep bruxism and associated factors in preschool children. pediatr dent. 2014; 36(1):46-50. 2. lobbezoo f, ahlberg j, raphael kg, wetselaar p, glaros ag, kato t, et al. international consensus on the assessment of bruxism: report of a work in progress. j oral rehabil. 2018 nov;45(11):837-844. doi: 10.1111/joor.12663. 3. gomes mc, neves ét, perazzo mf, souza egc, serra-negra jm, paiva sm, et al. evaluation of the association of bruxism, psychosocial and sociodemographic factors in preschoolers. braz oral res 2018 feb;32(1):9-16. doi: 10.1590/1807-3107bor-2018.vol32.0009. 4. castelo pm, gavião mb, pereira jl, bonjardim lr. relationship between oral parafunctional/nutritive sucking habits and temporomandibular joint dysfuction in primary dentition. int j paediatr dent. 2005;15(1):29-36. doi: 10.1111/j.1365-263x.2005.00608.x. 5. american academy of sleep medicine. international classification of sleep disorders. diagnostic and coding manual. westchester, il: aasm; 2005. vol. 2. 208p. 6. lam mh, zhang j, li am, wing yk. a community study of sleep bruxism in hong kong children: association with comorbid sleep disorders and neurobehavioral consequences. sleep med. 2011 aug. 12(7):641-5. doi: 10.1016/j.sleep.2010.11.013. 7. serra-negra j m, ramos-jorge ml, flores-mendoza ce, paiva sm, pordeus ia. influence of psychosocial factors on the development of sleep bruxism among children. int j paediatr dent. 2009 sep;19(5):309-17. doi: 10.1111/j.1365-263x.2009.00973.x. 8. clementino ma, siqueira mb, serranegra jm, paiva sm, granville-garcia af. the prevalence of sleep bruxism and associated factors in children: a report by parents. eur arch paediatr dent. 2017 dec;18(6):399-404. doi:10.1007/s40368-017-0312-x. 9. drumond cl, ramos-jorge j, vieira-andrade rg, paiva sm, serra-negra jmc, ramos-jorge ml. prevalence of probable sleep bruxism and associated factors in brazilian schoolchildren. int j paediatr dent. 2018.29(2):221-7. doi:10.1111/ipd.12443. 10. feitosa gma, félix r c r, sampaio dc, vieira-andrade rg, santos cco, fonseca-silva t. bruxism during childhood behavior profile, features of sleep and symptomatology. rev bahiana odonto. 2016;7(2):94-104. doi: 10.17267/2238-2720. https://doi.org/10.1007/s40368-017-0312-x 10 lira et al. 11. sousa hcs, lima mdm, dantas neta nb, tobias rq, moura ms, moura lfad. [prevalence and associated factors to sleep bruxism in adolescents from teresina, piauí]. rev bras epidemiol. 2018;21:e180002. doi: 10.1590/1980-549720180002. portuguese. 12. rodrigues ja, azevedo cb, chami vo, solano mp, lenzi tl. sleep bruxism and oral health‐related quality of life in children: a systematic review. int j paediatr dent. 2019 sep;30(2):1-8. doi:10.1111/ipd.12586. 13. luchesa cj, chaves neto a. [calculation of sample size in administration surveys]. curitiba: edição do autor; 2011. 27p. portuguese. 14. owens ja, spirito a, mcguinn m. the children´s sleep habits questionnaire (cshq): psychometric properties of a survey instrument for school-aged children. sleep. 2000;23(8):1043-51. doi: 10.1037/t33022-000. 15. junqueira th, nahás-scocate anr, valle-corott jm, conti accf, trevisan s. association of infantile bruxism and the terminal relationships of the primary second molars. braz oral res. 2013 jan;27(1):42-7. doi:10.1590/s1806-83242013000100008. 16. alves cl, fagundes dm, soares pbf, ferreira mc. knowledge of parents/caregivers about bruxism in children treated at the pediatric dentistry clinic. sleep sci. 2019;12(3):185-9. doi: 10.5935/1984-0063.20190083. 17. insana s, gozal d, mcneil dw, montgomery-downs he. community based study of sleep bruxism during early childhood. sleep med. 2012;14(2):183-8. doi:10.1016/j.sleep.2012.09.027. 18. renner ac, silva aa, rodriguez jd, simões vm, barbieri ma, bettiol h. are mental health problems and depression associated with bruxism in children? community dent oral epidemiol. 2012 nov;40(3):277-87. doi:  10.1111/j.1600-0528.2011.00644.x. 19. simões-zenari m, bitar m l. [factors associated to bruxism in children from 4-6 years]. pro fono. 2010;22(4):465-72. doi:10.1590/s0104-56872010000400018. portuguese. 20. miamoto cb, pereira lj, ramos-jorge ml, marques ls. prevalence and predictive factors of sleep bruxism in children with and without cognitive impairment. braz oral res. 2011 sep-oct;25(5):439-45. doi:10.1590/s1806-83242011000500011.  21. lamelha rml, cavalcanti mcc, mello lf, vilela mh, santos-junior ve. probable sleep bruxism in children and its relationship with harmful oral habits, type of crossbite and oral breathing. j clin pediatr dent. 2020;44(1):66-9. doi:10.17796/1053-4625-44.1.12. 22. orengul ac, tarakcioglu mc, gormez v, akkoyun s, zorlu a, aliyeva n, et al. duration of breastfeeding, bottle-feeding, and parafunctional oral habits in relation to anxiety disorders among children. breastfeed med. 2018 nov;14(1):57-62. doi:10.1089/bfm.2018.0013. 23. goldstein re, auclair wc. the clinical management of awake bruxism. j am dent assoc. 2017 may;148(6):387-91. doi:10.1016/j.adaj.2017.03.005. https://www.researchgate.net/deref/http%3a%2f%2fdx.doi.org%2f10.1037%2ft33022-000?_sg%5b0%5d=g4trygdv8490ju_uxfx73wycze-rkhtmf7y9k1fidoogououyco6atfyvotwk8jjal5o913r3zymmrswmiqdvwjbaa.pqcexd28v7nooydxpvaix7pkm78bv2loy1ihhv9vqk2-wqt3nb7gwi7vkpwilekxr4rycayp7yngk3nia3nrhq https://www.researchgate.net/deref/http%3a%2f%2fdx.doi.org%2f10.1590%2fs1806-83242013000100008?_sg%5b0%5d=lgmygdukjjsko-jqyasqa6otljyahwiiov6ossqniz6ir0jsh0e8d0fvvgsglnbmq442uf95ukxtmm_iojeeh-p0eq.s7rocfeix5dtvtkjjnr7d2owu_fdtntkcgdojl5dwxbnfwo5metr0ywemphovjbrieo8lxpq63z_zp4hskdmoq https://www.researchgate.net/deref/http%3a%2f%2fdx.doi.org%2f10.1016%2fj.sleep.2012.09.027?_sg%5b0%5d=d5uwprbzddrvwd50lwzssufvvqrb5z_vqndxtudndbolzwh6zp7dlceyy2kbicledvwnfdrjtihxuz9rnf9h7kncoa.7cose64_fqoflfscdexghuyki_smynxvhewbn0upt2legezw-htifufg5hdoufhcenwdrlcclkygqnoqzw7uaq https://www.researchgate.net/deref/http%3a%2f%2fdx.doi.org%2f10.1111%2fj.1600-0528.2011.00644.x?_sg%5b0%5d=nm8jpnwm-ct9bi_npzlr8rmx9oed254if4z1oyz9iixuv1czuuev8agyonrd9it9bx2dcsxuunqfa5c5up5z_0pujg.wnicf7mdhe8cfo6knczakvpodqfsuy0rnqbynmj_ghewutqvlr3xij1xxic_n6nt-dgbah9bq4kglewi0zgvrg 1 volume 22 2023 e238152 original article braz j oral sci. 2023;22:e238152http://dx.doi.org/10.20396/bjos.v22i00.8668152 1 phd student, department of prosthodontics and periodontology, university of campinas piracicaba dental school, piracicaba, sp, brazil. 2 professor, department of prosthodontics, faculty of technology and sciences (uniftc), salvador, ba, brazil. 3 professor, department of prosthodontics and periodontology, university of campinas piracicaba dental school, piracicaba, sp, brazil. 4 postdoctoral research fellow, department of prosthodontics and periodontology, university of campinas piracicaba dental school, piracicaba, sp, brazil. corresponding author: dra. raissa micaella marcello-machado department of prosthodontics and periodontology piracicaba dental school, university of campinas limeira avenue, 901 piracicaba, sp, brazil. phone: +55 (019) 21065211 e-mail: raissammm@gmail.com editor: valentim a. r. barão received: november 30, 2021 accepted: february 5, 2022 influence of diameter in the stress distribution of extra-short dental implants under axial and oblique load: a finite element analysis vanessa felipe vargas-moreno1 , rafael soares gomes2 , michele costa de oliveira ribeiro1 , mariana itaborai moreira freitas1 , altair antoninha del bel cury3 , raissa micaella marcello-machado4* aim: this study evaluated the influence of a wide diameter on extra-short dental implant stress distribution as a retainer for single implant-supported crowns in the atrophic mandible posterior region under axial and oblique load. methods: four 3d digital casts of an atrophic mandible, with a single implant-retained crown with a 3:1 crown-to-implant ratio, were created for finite element analysis. the implant diameter used was either 4 mm (regular) or 6 mm (wide), both with 5 mm length. a 200 n axial or 30º oblique load was applied to the mandibular right first molar occlusal surface. the equivalent von mises stress was recorded for the abutment and implant, minimum principal stress, and maximum shear stress for cortical and cancellous bone. results: oblique load increased the stress in all components when compared to axial load. wide diameter implants showed a decrease of von mises stress around 40% in both load directions at the implant, and an increase of at least 3.6% at the abutment. wide diameter implants exhibited better results for cancellous bone in both angulations. however, in the cortical bone, the minimum principal stress was at least 66% greater for wide than regular diameter implants, and the maximum shear stress was more than 100% greater. conclusion: extra-short dental implants with wide diameter result in better biomechanical behavior for the implant, but the implications of a potential risk of overloading the cortical bone and bone loss over time, mainly under oblique load, should be investigated. keywords: jaw, edentulous, partially. dental implants. dental prosthesis, implant-supported. finite element analysis. https://orcid.org/0000-0003-3334-6297 https://orcid.org/0000-0002-7989-0098 https://orcid.org/0000-0001-7679-0502 https://orcid.org/0000-0001-7729-8536 https://orcid.org/0000-0002-4329-0437 https://orcid.org/0000-0001-7661-703x 2 vargas-moreno et al. braz j oral sci. 2023;22:e238152 introduction implant-supported rehabilitation of the mandibular posterior region is challenging when severe mandibular bone resorption is present. the poor bone availability above the mandibular canal difficult the insertion of regular length implants1,2. there are different treatments for this clinical situation, including short dental implants (sdi), >6 to <10 mm in length, extra-short dental implants (esdi), ≤6 mm in length3, or surgeries for vertical bone augmentation2,4. a recent systematic review showed at 1-year follow-up that sdis have less morbidity, rehabilitation cost, and better survival rate (97%) than regular implants (92.6%) installed in a grafted bone area5. besides, in this same study, the proportion of patients with biological and mechanical complications was lower for sdis, with an incidence of 6%, while 39% of complications were reported for regular implants in grafted areas5. meanwhile, over a 5-year follow-up period, it was shown that there was no statistically significant difference in implant survival and success rates between sdis and regular implants in the grafted area4. also, esdis compared to regular implants have similar survival rates, 96.2%, and 99%, respectively, as well as the technical complications incidence, 14.14%, and 18.36%, respectively, after 3-years of follow-up6. in addition, a study that evaluated the long-term effectiveness of esdi reported a survival rate of 94.1% at a five-year follow-up1. this slightly lower survival rate, when compared to regular implants, can be explained by its unfavorable biomechanics7, due to an increased crown-to-implant ratio (c:i) that creates a more significant vertical lever arm and a disadvantageous stress distribution2. these implants have a smaller bone/ implant contact surface, which leads to increased stresses at the bone and prosthetic components8. therefore, sdi and esdi generally have a wide diameter (wd) compensating the limitation in high, increasing the surface and its bulk, which improves the stress dissipation9, leading to better biomechanical behavior10. the treatment plan also requires checking the patients’ occlusion and the antagonistic type affecting the implant success10. in a physiological occlusion predominantly occur axial loads (al), in the mandibular posterior region, transmitted by the long implant axis to the bone, resulting in an adequate stress dissipation11,12. however, when a non-physiological occlusion is present, the resultant occlusal force is an oblique load (ol), creating an unbalanced stress distribution8. therefore, when the high c:i anchored by esdi is used, the incidence of ol increases the bending moment of the vertical lever arm, causing a non-homogeneous force dissipation, leading to poor prognosis, which may contribute to peri-implant bone loss8,12. clinical and in vitro studies showed that the increased c:i only negatively influences the stress distribution when an ol is present8,13. previous systematic reviews focused on c:i evaluation have shown no significant differences in biological complications and peri-implant health results14,15, being 2.36:1 the higher c:i evaluated15. meanwhile, a recent four-year retrospective clinical trial concluded that the higher the c:i ratio (0.47 to 3.01), the less the marginal bone loss16. however, the biomechanical behavior of a challenging scenario where a 3:1 c:i crown supported by an esdi, with 5 mm in length, at the severe 3 vargas-moreno et al. braz j oral sci. 2023;22:e238152 reabsorbed mandibular posterior region, in the presence of ol, has not yet been investigated. that is critical since it can make the long-term success of this type of rehabilitation uncertain. besides, the benefits of using wd in esdi have not reached a consensus in the literature since clinical and laboratory studies have not found differences in survival rates when assessing different diameters and lengths2,17. this fact contradicts the prerogative of better biomechanics due to its larger contact surface10. therefore, there is a need for further studies to evaluate the rehabilitation mechanical behavior12 before future prospective clinical studies. thus, by using finite element analysis (fea), the present study evaluated the influence of wd on the stress distribution of esdi as support for single implant-supported crowns in the posterior region of the atrophic mandible, with a 3:1 c:i ratio, under al or ol. for then, to verify if the wd is relevant enough to justify the insertion of an implant that will wear out more bone. the tested null hypothesis stated that wd would have no difference from the rd regarding the stress distribution. materials and methods through the computer-aided design (cad) software (solidworks; dassault-systemes solidworks corp; waltham, massachusetts, usa) were created the 3d virtual models of a single crown, cement layer, cortical and cancellous bone. also, cad models of a universal abutment (4.5 x 2 x 6 mm) and two morse-taper implants of 4 x 5 mm (28.274 mm3, bone/implant contact surface: 101.39 mm2) and 6 x 5 mm (75.75 mm3, bone/implant contact surface: 155.36 mm2) were assessed virtually, and were left 2 mm submerged into the bone, which were obtained by the manufacturer (s.i.n implant system, são paulo, sp, brazil). two study factors were evaluated: i) implant diameter: 4 mm (rd: regular diameter, being this the control group) or 6 mm (wd) (fig. 1); ii) load angulation: al or ol (30° off-axis) being applied at the mesiobuccal cusp (fig. 2)18. the bone model had a 12.94 mm height and 16.11 mm of thickness, and a 2 mm layer of cortical bone surrounding the cancellous bone (fig. 1)19. the crown of a mandibular right first molar, 13 mm in height with a 3:1 c:i15 (fig. 1), was virtually cemented on the abutment (70-μm layer), and four groups were created: rdal (regular diameter implant under al); wdal (wide diameter implant under al); rdol (regular diameter implant under ol); wdol (wide diameter implant under ol). the fea models validation20 was performed by past literature for the location of force application and bone layers dimensions and by past in vivo study for crown and c:i. 4 vargas-moreno et al. braz j oral sci. 2023;22:e238152 13.47 mm 12.80 mm 13 mm a b figure 1. sagittal view: (a) regular implant diameter, 4mm; (b) wide implant diameter, 6 mm. dimensions of bone and crown (red) used are equal in all groups. axial load oblique load figure 2. load angulation applied at the mesiobuccal cusp for different groups, axial load and 30º oblique load. after assembly, the virtual models were exported to finite element software (ansys workbench 15.0; ansys inc; canonsburg, pennsylvania, usa) for a mathematical solution. a tetrahedral mesh was generated with an element size of 0.6 mm after convergence analysis with 5% tolerance. the young modulus (gpa) and poisson ratio (δ) of each material were set in the software according to table 1. the number of elements and nodes of each element is described on table 2. all components were considered homogenous, isotropic, and linearly elastic. also, the contact conditions between implant/abutment were assumed as no separation, and the contacts crown/ abutment and implant/bone were assumed as bonded. 5 vargas-moreno et al. braz j oral sci. 2023;22:e238152 table 1. mechanical properties of materials. material young modulus (gpa) poisson ratio (d) titanium grade iv21,22 110 0.33 co-cr alloy23 220 0.3 cortical bone24 13.7 0.3 cancellous bone24 1.370 0.3 resin cement 25 18.3 0.33 table 2. numbers of nodes and elements of each component. components nodes elements crown 16769 9940 abutment 11121 6327 cement layer 7862 3966 cortical bone (rd) 30221 17527 cortical bone (wd) 30757 17910 cancellous bone (rd) 28762 17244 cancellous bone (wd) 30325 18004 implant (rd) 106384 61350 implant (wd) 142268 82199 rd, regular diameter groups; wd, wide diameter groups. then, the models were fixed in two lateral portions of the bone segment and were submitted to a 200n load on the occlusal surface of the mandibular right first molar (fig. 2)8. the equivalent von mises stress (σvm) was used for the implant and the abutment8,10. minimum principal stress (σmin), and maximum shear stress (τmax)8,26, were used for both cortical and cancellous bone. a qualitative analysis was performed for the implants, abutment, and bone, using the colors of the resulting fea images. the colors varied from warmer (red) to cooler (blue) tones, with the peak stress represented by the warmest tone. results results for the fea assessment are presented in table 3. regardless of diameter, there was a significant increase in stress in all components, over 200%, under ol compared to al results. also, the stress was greater on the abutment and cortical bone and less on the cancellous bone and implant for wd groups. a substantial increase in stress was observed in cortical bone for wd groups compared to rd groups, being higher 66.3% for σmin and 99.8% for τmax under al and higher 125.7% for σmin and 201.7% for τmax under ol (table 3). for the al groups, the peak stress concentration was in the area in contact with the apical region of the implant, being the maximum values found at σmin of 72.34 mpa (wdal) (fig. 3) and τmax of 42.02 mpa (wdal) (fig. 4). meanwhile, in the ol groups, the highest stress concentration was in the cervical third of the bone, and the maximum values were at σmin 266.7 mpa (wdol) (fig. 3) and τmax 130.88 mpa (wdol) (fig. 4). the analysis of σmin and τmax showed decrease stress in the cancellous bone for wd groups, about 44.9% for σmin and 55.9% for τmax under al and 73.2% for σmin and 6 vargas-moreno et al. braz j oral sci. 2023;22:e238152 71.9% for τmax under ol (table 3). also, the images showed a peak stress concentration in the cervical third of the bone in all groups, and the minimum value of the σmin was 9.79 mpa (wdal) and of the τmax 7.32 mpa (wdal) (fig. 5 and fig. 6). besides, the σvm evaluation images showed that in all groups, the peak stress area was at the abutment collar level (fig.7) and in the corresponding region of the implant (fig. 8). the analysis demonstrated that with the wd, a low increase occurred in the abutment stress of 3.6% under al (wdal: 202.94 mpa) and 12.7% under ol (wdol: 1157.4 mpa) (table 3). however, a decrease in the implant of 38.7% was observed under al (wdal: 185.98 mpa) and 38.2% under ol (wdol: 873 mpa) (table 3). table 3. von-mises criteria (mpa) for implants and abutment, minimum principal stress and shear stress for cortical and cancellous bone (mpa), and the differences between the groups and direction of the load. axial load oblique load of 30º axial load x oblique load of 30º rdal wdal % stress rdol wdol % stress % rdal/ rdol % wdal/ wdol abutment (σvm) 200.97 202.94 *3.6% 1026.9 1157.4 *12.7% *511.0% *570.3% implant (σvm) 303.48 185.98 #38.7% 1414.4 873.4 #38.2% *466.1% *469.6% cortical bone (τmax) 21.02 42.02 *99.8% 43.37 130.88 *201.7% *206.3% *311.5% cortical bone (σmin) 43.53 72.34 *66.3% 118.19 266.7 *125.7% *271.5% *368.7% cancellous bone (τmax) 16.62 7.324 #55.9% 73.5 20.66 #71.9% *442.2% *282.1% cancellous bone (σmin) 17.78 9.795 #44.9% 94.19 25.23 #73.2% *529.8% *257.6% rdal, regular diameter implant under axial load; wdal, wide diameter implant under axial load; rdol, regular diameter implant under oblique load; wdol, wide diameter implant under oblique load; *, increased stress; #, stress decreased. rdal 20,119 max -43,535 min rdal -72,342 min wdal -118,19 min rdol -266,7 min wdol wdal rdol wdol figure 3. minimum principal stress peak concentration for cortical bone (mpa) for all groups. blue to red color represents stress values from higher to lower, respectively. 7 vargas-moreno et al. braz j oral sci. 2023;22:e238152 rdal 130,88 max wdol 21,024 max rdal 42,016 max wdal rdol 43,374 max 0,13952 min wdal rdol wdol figure 4. maximum shear stress peak concentration for cortical bone (mpa) for all groups. blue to red color represents stress values from lower to higher, respectively. rdal -25,228 min wdol -17,778 min rdal -9,7946 min wdal -94,192 min rdol 29,32 max wdal rdol wdol figure 5. minimum principal stress peak concentration for cancellous bone (mpa). blue to red color represents stress values from higher to lower, respectively. 8 vargas-moreno et al. braz j oral sci. 2023;22:e238152 rdal wdol 20,658 max 16,622 max rdal 7,324 max wdal 73,503 max rdol 0,0094777 min wdal rdol wdol figure 6. maximum shear stress peak concentration for cancellous bone (mpa). blue to red color represents stress values from lower to higher, respectively. rdal wdal rdol wdol 1157,4 max wdol rdal 200,97 max 202,94 max wdal 1026,9 max rdol 0,34574 min figure 7. von-mises stress peak concentration (mpa) in abutment. blue to red color represents stress values from lower to higher, respectively. 9 vargas-moreno et al. braz j oral sci. 2023;22:e238152 rdal wdal rdol wdol 873,4 max wdol rdal 303,48 max 185,98 max wdal 1414,4 max rdol 0,45843 min figure 8. von-mises stress peak concentration (mpa) in implant. blue to red color represents stress values from lower to higher, respectively. discussion there is no consensus in the literature about the benefits of using wd in esdi in the treatment of severe mandibular bone resorption in the posterior region12. also, recent studies showed that a high c:i ratio only increases the stress concentration when ol is present8,27, being traumatic occlusion the primary cause of biomechanical complications8,13,27. thus, by fea, the present study evaluated the influence of wd on esdis stress distribution as support for single implant-supported crowns in the posterior region of the atrophic mandible, under al and ol. the hypothesis that wd would have no difference from the rd regarding the stress distribution, had to be rejected. it was observed that wd in esdi, under both load directions, showed a decrease of stress at the implant and the cancellous bone (wdal: τmax=7.324 mpa, σmin=9.795; wdol: τmax=20.66 mpa, σmin=25.23 mpa), a relevant increase in the cortical bone, and a possible slight increase in the abutment. besides, when submitted to ol, there was an increase in stress in all components and groups by more than 200%, corroborating with previous studies8,13,27. in this study, the stress distribution on the peri-implant bone was different when a wd was used. a relevant increase (up to 66%) in the stress can be observed in the cortical bone when τmax and σmin were evaluated independently of load angulation. this is important since some studies have reported, without a consensus, a critical threshold of compressive (ranging from 50 mpa to 170 mpa) and tensile stress (ranging from 34.72 mpa to 100 mpa) of the bone28-31, and in the wdal, rdol, and 10 vargas-moreno et al. braz j oral sci. 2023;22:e238152 wdol these values were overtaken. what shows the need for more studies and other methods of evaluation of bone impact when wd is used. also, the figures in wd groups shows a stress peak in the cervical third of the bone of at least 311.5% under ol higher than the findings of the al groups, which could be explained by the use of the wd implant providing a 34.73% higher bone/implant contact and wear on the cortical bone. these results corroborate with elias et al.27, which evaluated the influence of the prosthetic crown height in sdi and found a higher stress concentration in the ol groups. meanwhile, in the wd groups, a decrease in the stress was observed in the cancellous bone, bringing the mpa values found within the limits of compressive and tensile stress at wdol28-31. this may be related to its young modulus, since its value is lower than that of the cortical bone. the greater the young modulus the stiffer the material, the greater the stress accumulation10, and more resistance to deformation32. in the present study, when the wd implant was evaluated the contact between the implant and cortical bone was increased, leading to higher stress on the cortical bone and a reduction on the cancellous bone, which can explain the results10. this enhanced contact with the cortical bone may negatively influence the bone remodeling around the implants since the cortical bone is less vascularized than the cancellous bone, which leads to interference of blood supply that directly affects the bone resorption response33. according to the results of this study, this would only be a problem in the presence of oblique load. considering that in the posterior region the pattern of forces is axial, perhaps it would not be a clinical problem, as long as the patient has a favorable occlusal pattern. the consequences of higher stress concentration on the cortical bone associated with its decrease on the cancellous bone remain uncertain since low-stress values around the implant resulting in a bone loss due to disuse atrophy, while high-stress cause microfracture at the bone resulting either in bone loss or fatigue failure of the implant32,33. also, since wd in esdi increases the stress at the implant/cortical bone interface, being mpa values over the compressive and tensile limits of the bone28-31, it represents a potential biological risk for marginal bone loss that might be even higher under ol. besides, the mechanical loading conditions regulate the morphology of the bone34, and it is still unknown how much bone/implant contact is necessary for the success of esdis27. the results of von mises stress showed, in all groups, a higher stress concentration at the surface of the abutment collar level and at the implant platform where it touches the abutment collar. in both loads, the wd showed an increase of 12.7% in stress at the abutment and a reduction of at least 38.2% in the implant. despite this percentage difference, the color pattern exhibits a great similarity in the stress distribution in general for the abutment, and under axial load for the implant. this substantial stress reduction at wd implants might be explained by its structure 62% bulkier than rd implants. since the stress increased over 400% at implant and abutment at the ol groups, clinically, would increase the risk of the implant, and abutment failure once was exceeded the limits of tensile yield strength 0.2% (483 mpa) and ultimate tensile strength (550 mpa) of the titanium grade iv35. suggesting that 11 vargas-moreno et al. braz j oral sci. 2023;22:e238152 should be avoided the use of esdi when it is impossible to eliminate ol during mandibular excursive movements, for example, in a parafunction scenario. another important point to be highlighted is that a wd implant might reduce the bone mechanical resistance, since the remaining bone around it is reduced when compared to a rd implant. there is a literature gap regarding the effects generated by an overload on the cortical bone, when a mandibular implant-retained crown is evaluated under different load directions. also, the maximum stress values of fea studies strongly depend on the size of the mesh used. so, even with this study results being encouraging, showing that the wd esdi can be a reliable option as shown in the al groups, it also shows the necessity to perform further studies on this behalf. clinically the masticatory forces are not acting in just one way, and it is impossible to isolate the force direction. so, it is essential to perform in silico studies, which allow the researcher to evaluate and study every direction of occlusal forces like was performed in this study. besides, the present study is a numerical theoretical analysis, and its results should be validated with an in vitro study assessing implant failure mode in the same conditions of this study. in addition, other simulations could be performed to estimate possible statistical differences, for example, by using different prostheses, abutments, and materials with different elastic modulus since they could reach a different result because of its dampers chewing loads10. finally, a reliable way to effectively assess the influence on the bone would be performing randomized controlled trials. these studies must include patients with severe bone atrophy in the posterior region of the mandible with different types of occlusal patterns and a minimum of 1 mm cortical bone wall to surround the implant. therefore, extra-short implants with wide diameter result in better biomechanical behavior for the implant, but the implications of a potential risk of overloading the cortical bone and bone loss over time, mainly under oblique load, should be investigated. acknowledgements this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior brasil (capes) finance code 001. the authors thank s.i.n. implant system for their support with the cads used in this study. data availability datasets related to this article will be available upon request to the corresponding author. conflicts of interest none. 12 vargas-moreno et al. braz j oral sci. 2023;22:e238152 author contribution vanessa felipe vargas-moreno: design of the work; acquisition and interpretation of data for the work; drafting the work; revising it critically for important intellectual contente; final approval of the version to be published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. rafael soares gomes: drafting the work; interpretation of data for the work; revising it critically for important intellectual contente; final approval of the version to be published. michele costa de oliveira ribeiro: drafting the work; interpretation of data for the work; revising it critically for important intellectual contente; final approval of the version to be published. mariana itaborai moreira freitas: drafting the work; revising it critically for important intellectual contente; final approval of the version to be published. altair antoninha del bel cury: drafting the work; revising it critically for important intellectual contente; final approval of the version to be published. raissa micaella marcello-machado: design of the work; drafting the work; revising it critically for important intellectual contente; final approval of the version to be published; agreement to be accountable for all aspects of the work in ensuring that 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volume 20 2021 e211701 original article 1 graduate program in dentistry, meridional college/imed, passo fundo, brazil. 2 mscid and phd graduate program in oral science, faculty of dentistry, federal university of santa maria (ufsm), santa maria, rio grande do sul state, brazil. corresponding author: rafael sarkis-onofre graduate program in dentistry – meridional faculty/imed, 304, senador pinheiro street passo fundo – brazil 99070-220 e-mail: rafael.onofre@imed.edu.br received: october 26, 2020 accepted: december 16, 2021 how are meta-analyses being conducted and reported in dentistry? a meta-research study letícia tainá de oliveira lemes1 , lara dotto1 , bernardo antonio agostini1 , gabriel kalil rocha pereira2 , rafael sarkis-onofre1,* aim: this study aimed to evaluate how meta-analyses are conducted and reported in dentistry. methods: we conducted a search to identify dentistry-related systematic reviews (srs) indexed in pubmed in 2017 (from january 01 until december 31) and published in the english language. we included only srs reporting at least one meta-analysis. the study selection followed the 4-phase flow set forth in the preferred reporting items for systematic reviews and meta-analyses statement (prisma), and it was independently conducted by two researchers. data extraction was performed by one of three reviewers, and data related to conducting and reporting of the meta-analysis were collected. descriptive data analysis was performed summarizing frequencies for categorical items or median and interquartile range for continuous data. results: we included 214 srs with meta-analyses. most of the studies reported in the title that a meta-analysis was conducted. we identified three critical flaws in the included studies: ninety (90) meta-analyses (43.1%) did not specify the primary outcome; most of the meta-analyses reported that a measure of statistical heterogeneity was used to justify the use of a fixed-effect or random-effects meta-analysis model (n=114, 58.5%); and a great part did not assess publication bias (n=106, 49.5%). conclusion: we identified deficiencies in the reporting and conduct of meta-analysis in dentistry, suggesting that there is room for improvement. educational approaches are necessary to improve the quality of such analyses and to avoid biased and imprecise results. keywords: oral health. systematic reviews as topic. research report. 2 lemes et al. introduction meta-analyses are an important component of systematic reviews and are a statistical method to combine results from two or more independent studies1. this method enables improving the precision of estimates and answering conflicting questions or questions not discussed in individual studies2. however, meta-analyses might be misused and biased in a similar way to other research methods. ioannidis3 (2016) demonstrated that 9,135 meta-analyses were published in pubmed in 2014, corresponding to an increase in the publication rate of 2,635% between 1991 and 2014. the author highlighted that many meta-analyses are redundant, unnecessary, or have methodological flaws. page et al.4 investigated flaws in the application and interpretation in a sample of meta-analyses of therapeutic interventions. the findings demonstrated problems in aspects such as interpretation of the model used, subgroup analyses, and the minimum number of studies recommended to test funnel plot asymmetry. in dentistry, saltaji et al.5 assessed 1,118 systematic reviews (srs) published between 1991-2012; the findings demonstrated that almost 50% performed a meta-analysis, and the median of included studies in the largest meta-analysis was 9. moreover, several articles assessed the quality of meta-analyses performed in different oral health specializations, demonstrating that the overall quality varies from low to medium6. however, there are no studies in dentistry evaluating reporting and conducting meta-analysis characteristics, which would enable highlighting areas for future improvement. thus, this study aimed to evaluate how meta-analyses are conducted and reported in dentistry. materials and methods we used a dataset of srs in dentistry indexed in pubmed in 2017. the reporting and conduct characteristics of these srs were previously published7. we have only evaluated data from srs with meta-analysis and assessed how meta-analyses are conducted and reported. search and eligibility criteria a full description of the search strategy and eligibility criteria is available in the study of bassani et al. (2019)7. first, we conducted a search in pubmed to identify dentistry-related srs published in the english language indexed in 2017 (from january 01 until december 31). we considered the article as a sr based on the preferred reporting items for systematic reviews and meta-analysis protocols (prisma-p) definition8. in this article, we have only included srs reporting at least one meta-analysis. in addition, we included articles regardless of questions answered and study designs included (clinical studies, in vitro, in situ, etc.). the search strategy used is presented in table 1 and was based on mesh terms of pubmed and a specific filter (u.s. national library of medicine). screening we selected the studies using a reference manager software (endnote x7, thomson reuters, new york, usa). details about the pilot test screening and study selection are 3 lemes et al. available in the study by bassani et al.7. the study selection followed the 4-phase flow set forth in the preferred reporting items for systematic reviews and meta-analyses statement9. two researchers independently identified articles by reviewing titles and abstracts, and then screening the full text in a second phase. data extraction a standardized form was created using microsoft excel based on the study by page et al. 10 (2016). a pilot data extraction was performed to ensure consistency, and the details are available in the study by bassani et al.7. data related to the conduct and reporting of meta-analyses were extracted by one of three reviewers. data regarding the journal category (general or specialized journal), number of authors, sr focus (epidemiology, diagnosis, prevention, prognosis, treatment/ therapeutic, other, unclear, or mixed), dental specialization, funding (reported or not reported), and data related to conducting and reporting of meta-analysis such as details about the primary outcome, the model used, number of studies in the largest meta-analysis, statistical heterogeneity, publication bias, additional analysis and software used to conduct the meta-analyses were extracted. the form used is available in the supplementary material. one author subsequently verified the data consistency, and the data were extracted again in the case of any doubt or inconsistency. data analysis descriptive analysis of the data was performed by summarizing frequencies for categorical items and calculating median and interquartile range for continuous data. all analyses were performed using stata 14.2 software. characteristics of the meta-analyses were assessed considering all included srs and meta-analyses. table 1. search strategy “oral health”[mesh] or “oral health” or “health, oral” or “dentistry”[mesh] or “dentistry” or “dental research”[mesh] or “dental research” and (((systematic review [ti] or meta-analysis [pt] or meta-analysis [ti] or systematic literature review [ti] or this systematic review [tw] or pooling project [tw] or (systematic review [tiab] and review [pt]) or meta synthesis [ti] or meta synthesis [ti] or integrative review [tw] or integrative research review [tw] or rapid review [tw] or consensus development conference [pt] or practice guideline [pt] or drug class reviews [ti] or cochrane database syst rev [ta] or acp journal club [ta] or health technol assess [ta] or evid rep technol assess summ [ta] or jbi database system rev implement rep [ta]) or (clinical guideline [tw] and management [tw]) or ((evidence based[ti] or evidence-based medicine [mh] or best practice* [ti] or evidence synthesis [tiab]) and (review [pt] or diseases category[mh] or behavior and behavior mechanisms [mh] or therapeutics [mh] or evaluation studies[pt] or validation studies[pt] or guideline [pt] or pmcbook)) or ((systematic [tw] or systematically [tw] or critical [tiab] or (study selection [tw]) or (predetermined [tw] or inclusion [tw] and criteri* [tw]) or exclusion criteri* [tw] or main outcome measures [tw] or standard of care [tw] or standards of care [tw]) and (survey [tiab] or surveys [tiab] or overview* [tw] or review [tiab] or reviews [tiab] or search* [tw] or handsearch [tw] or analysis [ti] or critique [tiab] or appraisal [tw] or (reduction [tw]and (risk [mh] or risk [tw]) and (death or recurrence))) and (literature [tiab] or articles [tiab] or publications [tiab] or publication [tiab] or bibliography [tiab] or bibliographies [tiab] or published [tiab] or pooled data [tw] or unpublished [tw] or citation [tw] or citations [tw] ,or database [tiab] or internet [tiab] or textbooks [tiab] or references [tw] or scales [tw] or papers [tw] or datasets [tw] or trials [tiab] or meta-analy* [tw] or (clinical [tiab] and studies [tiab]) or treatment outcome [mh] or treatment outcome [tw] or pmcbook)) not (letter [pt] or newspaper article [pt]))) 4 lemes et al. results figure 1 presents a flow diagram outlining the study selection process. the initial search in pubmed yielded 1375 records, and we included 214 srs with meta-analysis after study screening of the title/abstract and full-text analysis. table 2 presents the epidemiological characteristics of the srs with meta-analyses included in the study. most of the meta-analyses were published in specialized journals (n=159, 74,3%). considering the country where those meta-analyses were produced, 4 countries produced 64.3% of sr with meta-analysis published, in which brazil had the greatest contribution with 61 (28.5%). the main specialization was oral and maxillofacial surgery (n=32, 14.9%), followed by implantology (n=31, 14.5%) and periodontics (n=29, 13.5%). the main focus of most of the srs with meta-analyses was treatment/therapeutic (n=101, 47.2%), and a great number of the included meta-analyses reported no funding (n=80, 37.4%). the median number of authors was 5 (iqr 4-6). figure 1. flow diagram of study selection prisma 2009 flow diagram records identified through database searching (n = 1375) additional records identified through other sources (n = 0) id en ti fi ca ti on s cr ee ni ng el ig ib ili ty in cl ud ed meta-analyses (n = 214) srs included (n = 495) full-text articles assessed for eligibility (n = 616) records screened (n = 1375) records excluded (n = 759) full-text articles excluded, with reasons (n = 121) see bassani et al., 2019 records after duplicates removed (n = 1375) 5 lemes et al. table 2. epidemiological characteristics of meta-analyses included in the study journal n % general 55 25.7% specialty 159 74.3% country brazil 61 28.5% usa 24 11.2% china 18 8.4% united kingdon 11 5.1% switzerland 9 4.2% germany 8 3.7% sweden 7 3.2% other 30 countries and unclear 76 35.7% dental specialties oral and maxillofacial surgery 32 14.9% implantology 31 14.5% periodontics 29 13.5% orthodontics 26 12.1% oral and maxillofacial pathology 24 11.2% restorative and esthetic dentistry 19 8.8% pediatric dentistry 17 7.9% endodontics 13 6.1% public health 12 5.6% prosthodontics 5 2.3% radiology 5 2.3% others 1 0.5% focus treatment/therapeutic 101 47.20% diagnosis 41 19.2% epidemiology 29 13.5% prognosis 22 10.3% prevention 9 4.2% other 6 2.8% unclear 6 2.8% funding no funding 80 37.4% not reported 77 36% non-profit sponsor 53 24.8% unclear 3 1.4% mixed 1 0.5% number of authors median 5 (iqr = 4-6) iqr – interquartile range 6 lemes et al. table 3 presents the conduct and reporting characteristics of meta-analyses. most of the srs reported that a meta-analysis was conducted in the title (n=187, 87.4%). the “continuous variable” was the type of outcome most used (n=41, 34.4%), and the unit of measure of the first reported result of the primary outcome was “mean difference” (n=45, 38.5%). most of the meta-analyses reported the statistical significance of the first reported result of the primary outcome as “favorable, statistically significant” (n=60, 50.8%), and the majority of studies used a random effect model for all meta-analyses (n=130, 62.5%). regarding the statistical heterogeneity of included studies in the meta-analysis, the majority of meta-analyses described some method to formally evaluate the statistical heterogeneity of included studies (n=180, 87.0%), and the method most used was the i2 test (n=161, 75.2%). when additional analyses were conducted, the most used was subgroup (n=65, 30.4%); however, most of the meta-analyses did not conduct additional analyses (n=121, 56.6%). the software most used to conduct meta-analyses was revman (n=73, 34.1%), followed by stata (n=34, 15.9%). the median of studies included in the largest meta-analyses was 9 (iqr 5-16). forty (40) meta-analyses (18.7%) presented a funnel plot graph; however, 10 did not report that the analysis was performed, and a further 3 did not report that publication bias was assessed. table 3. characteristics of conduct and reporting of meta-analyses terms in the title n % systematic review and meta-analysis 160 74.8% only meta-analysis 27 12.6% only systematic review 17 7.9% neither 10 4.7% did the review authors specify one or more primary outcome(s)? yes 106 50.7% no 90 43.1% no but only one outcome reported 13 6.2% what type of outcome is the primary outcome? continuous 41 34.4% dichotomous 25 21% both dichotomous and continuous 23 19.3% not reported 15 12.6% rate 12 10.1% unclear 3 2.5% what is the unit of measure of the first reported result (effect estimate) of the primary outcome? mean difference 45 38.5% risk ratio 24 20.5% odds ratio 18 15.4% standardized mean difference 12 10.3% prevalence 6 5.1% continue 7 lemes et al. continuation not reported or unclear 6 5.1% likelihood ratios 5 4.3% event rate 1 0.8% what is the statistical significance of the first reported result (effect estimate) of the primary outcome? favorable, statistically significant 60 50.8% favorable, non-statistically significant 19 16.1% unfavorable, non-statistically significant 14 11.9% unfavorable, statistically significant 12 10.2% non comparative 12 10.2% not reported 1 0.8% which meta-analysis model was used in the meta-analyses? random-effects model for all meta-analyses 130 62.5% varied 47 22.6% fixed-effect model for all meta-analyses 21 10.1% other 3 1.4% not reported 7 3.4% was the risk of bias (or quality) assessment incorporated into any meta-analyses in the review? no 119 59.9% yes 79 40.1% was any method described to formally evaluate statistical heterogeneity of included studies? no 25 12.1% yes 180 87% statistical heterogeneity was not taken into account using formal statistical evaluation, but heterogeneity of the studies was qualitatively assessed 2 0.9% which methods were used to formally evaluate statistical heterogeneity of included studies? (*considering 214 studies) chi-square or cochran’s q 109 50.9%* i^2 (i-square) 161 75.2%* tau^2 (tau-square) 17 7.9% other 6 2.8%* did the authors report that a measure of statistical heterogeneity was used to justify use of a fixed-effect or random-effects meta-analysis model? no 81 41.5% yes 114 58.5% did the authors report assessing (or an intent to assess) publication bias? no, publication bias was not assessed, and the authors did not report an intention to assess it 106 49.5% no, publication bias was not assessed, but the authors reported that they intended to assess it if they identified a sufficient number of studies 29 13.5% yes, publication bias was assessed 79 36.9% continue 8 lemes et al. continuation which methods did the authors report using (or intending to use) to assess publication bias? (*considering the number total of studies) begg’s test 6 2.8%* egger’s test 25 11.7%* funnel plot 64 30%* sensitivity analysis comparing fixed-effect to randomeffects model 3 1.4%* subgroup analyses by sample size 2 0.9%* other 2 0.9%* funnel plot presented (*considering the number total of studies) 40 18.7% which of the following additional analyses did the authors conduct? meta-regression 21 9.8% network meta-analysis 2 0.9% sensitive 41 19.2% subgroup 65 30.4% no additional analyses 121 56.6% what statistical software was used to perform meta-analyses? revman 73 34.1% stata 34 15.9% comprehensive meta-analysis 31 14.5% r 18 8.4% not reported 18 8.4% others 13 6.1% two or more different programs 13 6.1% medcalc 8 3.7% openmeta 6 2.8% median of studies included in the largest meta-analysis 9 (iqr =16-5) for the primary outcome, 90 meta-analyses (43.1%) did not specify the primary outcome, and most of the meta-analyses did not incorporate the risk of bias assessment into them (n=119, 59.9%). most of the meta-analyses reported that a measure of statistical heterogeneity was used to justify the use of a fixed-effect or random-effects meta-analysis model (n=114, 58.5%) and did not assess publication bias (n=106, 49.5%). discussion our study is the first in dentistry to assess how meta-analyses are conducted and reported. we identified that the reporting and conduct characteristics of meta-analyses are varied. most of the studies reported that a meta-analysis was conducted in the title and formally evaluated the statistical heterogeneity of included studies. however, most of the meta-analyses reported that a measure of statistical heterogeneity was 9 lemes et al. used to justify the use of a fixed-effect or random-effects meta-analysis model and did not assess publication bias, thus demonstrating that there is room for improvement in conducting meta-analyses in dentistry. in addition, our estimates showed an increase rate of srs with meta-analyses published in recent years compared to a previous study5. several studies in dentistry assessed the quality of meta-analysis published in different dental specializations11,12. both publications demonstrated that the meta-analysis quality can vary from medium to low and highlighted that important aspects such as publication bias assessment were not conducted in most meta-analyses; these findings were also corroborated by our study. page et al.4 demonstrated that flaws in meta-analyses are not restricted to dentistry. the authors evaluated 110 statistical analyses in systematic reviews of therapeutic interventions in the biomedical field and pointed out that the involvement of statisticians in conducting systematic reviews could improve the quality of meta-analyses. three important flaws in the conduct/report of meta-analyses were identified. first, almost 50% of the included studies did not assess the impact of publication bias. publication bias occurs when there is a predilection for publishing particular results, such as in studies reporting high effect sizes which are more likely to be published than studies reporting lower effect sizes, and this type of bias can affect systematic reviews. failure to assess the possible impact of publication bias on systematic reviews results can generate an invalid estimate, which could guide interventions or actions based on biased conclusions1,13. koletsi et al. 14 (2016) assessed 162 systematic reviews in dentistry, with the findings demonstrating a low rate of publication bias analysis performed and a high rate of improperly conducted publication bias assessment. the second important flaw is the use of a statistical heterogeneity measure to justify the use of a fixedor random-effects meta-analysis model. this could be considered a shortcoming since the choice of each effect only based on a statistical measure could exacerbate the effect of bias, thus producing a spurious estimate and an inappropriate conclusion. recent recommendations clearly affirm that the decision between the use of the model is a topic of much debate. an alternative to the topic is a pragmatic approach planned to perform both analyses (fixed and random models), while the random effect model can be present in cases of not identifying asymmetry in funnel plot, even knowing that funnel plot asymmetry suggests that both methods are problematic. in addition, the author pointed out that “the choice between a fixed-effect and a random-effects meta-analysis should never be made based on a statistical test for heterogeneity”1. lastly, almost 50% of the meta-analyses included did not specify the primary outcome. tricoo et al. 15 (2016) evaluated a sample of 96 systematic reviews and demonstrated that one-third of the studies did not specify or change the primary outcome. we did not assess if the studies included in our study were registered or did not compare the information reported in protocols and in the final publication, however it could be possible that study results were related to outcome reporting bias, generating imprecise results. 10 lemes et al. there are some undeniable limitations of our study. we only considered one database and only studies published in english, and we performed a cross-sectional analysis only considering 2017. also, our data extraction was based on the study report, and it is possible that some srs were conducted more rigorously than was specified in the report. additionally, we did not include meta-analyses without a systematic review, which could have led to selecting studies with high methodological quality, and we did not judge the study quality and if a correct methodology was used to perform the meta-analyses because this was not the aim of this study. various approaches are encouraged to minimize the flaws identified in our analysis: 1) graduate program students should be receiving adequate training to perform and report meta-analyses; 2) involvement of statisticians on the systematic review team could improve analysis quality; 3) dissemination of prisma statement extensions such as prisma for individual patient data and network meta-analyses could help in reporting adequate meta-analyses. in conclusion, we identified deficiencies in the reporting and conduct of meta-analyses in dentistry, suggesting that there is room for improvement. educational approaches are necessary to improve the quality of such analyses and to avoid unbiased and imprecise results. acknowledgements baa, and rso are funded in part by meridional foundation (passo fundo – brazil). ll and ld are supported by capes, brazil. this study was conducted in a graduate program supported by capes, brazil (finance code 001). the funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. references 1. page mj, higgins jpt, sterne jac. assessing risk of bias due to missing results in a synthesis. in: higgins j, thomas j. cochrane handbook for systematic reviews of interventions. cochrane; 2019. version 6.0. chapter 13. 2. mckenzie je, beller em, forbes ab. introduction to systematic reviews and meta-analysis. respirology. 2016 may;21(4):626-37. doi: 10.1111/resp.12783. 3. ioannidis jp. the mass production of redundant, misleading, and conflicted systematic reviews and meta-analyses. milbank q. 2016 sep;94(3):485-514. doi: 10.1111/1468-0009.12210. 4. page mj, altman dg, mckenzie je, shamseer l, ahmadzai n, wolfe d, et al. flaws in the application and interpretation of statistical analyses in systematic reviews of therapeutic interventions were common: a cross-sectional analysis. j clin epidemiol. 2018 mar;95:7-18. doi: 10.1016/j.jclinepi.2017.11.022. 5. saltaji h, cummings gg, armijo-olivo s, major mp, amin m, major pw, et al. a descriptive analysis of oral health systematic reviews published 1991-2012: cross sectional study. plos one. 2013 sep;8(9):e74545. doi: 10.1371/journal.pone.0074545. 6. jayaraman j, nagendrababu v, pulikkotil sj, innes np. critical appraisal of methodological quality of systematic reviews and meta-analysis in paediatric dentistry journals. int j paediatr dent. 2018 nov;28(6):548-560. doi: 10.1111/ipd.12414. 11 lemes et al. 7. bassani r, pereira gkr, page mj, tricco ac, moher d, sarkis-onofre r. systematic reviews in dentistry: current status, epidemiological and reporting characteristics. j dent. 2019 mar;82:71-84. doi: 10.1016/j.jdent.2019.01.014. 8. moher d, shamseer l, clarke m, ghersi d, liberati a, petticrew m, et al. preferred reporting items for systematic review and meta-analysis protocols (prisma-p) 2015 statement. syst rev. 2015 jan;4(1):1. doi: 10.1186/2046-4053-4-1. 9. liberati a, altman dg, tetzlaff j, mulrow c, gøtzsche pc, ioannidis jp, et al. the prisma statement for reporting systematic reviews and meta-analyses of studies that evaluate healthcare interventions: explanation and elaboration. bmj. 2009 jul;339:b2700. doi: 10.1136/bmj.b2700. 10. page mj, shamseer l, altman dg, tetzlaff j, sampson m, tricco ac, et al. epidemiology and reporting characteristics of systematic reviews of biomedical research: a cross-sectional study. plos med. 2016 may;13(5):e1002028. doi: 10.1371/journal.pmed.1002028. 11. kattan s, lee sm, kohli mr, setzer fc, karabucak b. methodological quality assessment of meta-analyses in endodontics. j endod. 2018 jan;44(1):22-31. doi: 10.1016/j.joen.2017.07.019. 12. el-rabbany m, li s, bui s, muir jm, bhandari m, azarpazhooh a. a quality analysis of systematic reviews in dentistry, part 1: meta-analyses of randomized controlled trials. j evid based dent pract. 2017 dec;17(4):389-98. doi: 10.1016/j.jebdp.2017.06.004. 13. thornton a, lee p. publication bias in meta-analysis: its causes and consequences. j clin epidemiol. 2000 feb;53(2):207-16. doi: 10.1016/s0895-4356(99)00161-4. 14. koletsi d, valla k, fleming ps, chaimani a, pandis n. assessment of publication bias required improvement in oral health systematic reviews. j clin epidemiol. 2016 aug;76:118-24. doi: 10.1016/j.jclinepi.2016.02.019. 15. tricco ac, cogo e, page mj, polisena j, booth a, dwan k, et al. a third of systematic reviews changed or did not specify the primary outcome: a prospero register study. j clin epidemiol. 2016 nov;79:46-54. doi: 10.1016/j.jclinepi.2016.03.025. 1 volume 22 2023 e237545 original article braz j oral sci. 2023;22:e237545http://dx.doi.org/10.20396/bjos.v22i00.8667545 1 faculdade são leopoldo mandic. corresponding author: profa. dra. roberta tarkany basting faculdade são leopoldo mandic departamento de dentística e materiais dentários rua josé rocha junqueira, 13. bairro swift, campinas – sp cep: 13045-755 brazil telephone/fax: 55-19-3211-3600 e-mail: rbasting@yahoo.com editor: dr. altair a. del bel cury received: november 11, 2021 accepted: april 2, 2022 dentin permeability after pretreatment with titanium tetrafluoride and self-etching or universal adhesive systems gabriel greco franco1 , ruthinea faria de moraes cardoso1 , natália russo carlos1 , cecilia pedroso turssi1 , flávia lucisano botelho do amaral1 , fabiana mantovani gomes frança1 , roberta tarkany basting1 aim: to evaluate dentin permeability after pretreatment with 2.5% aqueous solution of titanium tetrafluoride (tif4), followed by a self-etching universal adhesive system. methods: forty dentin discs (1.5 mm thick) were randomly divided into groups according to the application or non-application of a pretreatment, and the type of adhesive system to be tested (two-step self-etching/ clearfil se bond/ kuraray medical, or universal adhesive system/ single bond universal/ 3m espe). both sides of the discs were conditioned with 37% phosphoric acid to remove the smear layer. the first hydraulic conductivity measurement (l1) was performed in a permeability machine, under 5 psi pressure. the samples were sanded again to form a standardized smear layer. the teeth designated for pre-treatment with tif4 received the tif4 solutions applied actively for 60 seconds, and the adhesive systems were applied according to the manufacturer’s instructions. then, a new hydraulic conductivity measurement (l2) was performed for the purpose of calculating the hydraulic conductance at a later time, considering the water viscosity and the thickness of the specimen. the percentage (l) of dentin permeability after application of the adhesive system was obtained (l (%) = [(l1-l2) x100] / l1). the mann-whitney non-parametric test was applied. results: there was no difference between the two adhesive systems, or between the groups with or without pretreatment, as regards dentin permeability (p>0.05). conclusion: pretreatment with 2.5% tif4 did not influence dentin permeability, irrespective of the adhesive system used. keywords: dentin permeability. dentin-bonding agents. titanium. fluorides. https://orcid.org/0000-0001-7869-803x https://orcid.org/0000-0002-9035-0236 https://orcid.org/0000-0003-3229-4333 https://orcid.org/0000-0002-0078-9895 https://orcid.org/0000-0002-8934-6678 https://orcid.org/0000-0002-2877-6797 http://orcid.org/0000-0002-5345-5776 2 franco et al. braz j oral sci. 2023;22:e237545 introduction advancements in the development of products for adhesive restorative systems has allowed direct restorative procedures to be performed with minimally invasive cavity preparations1. although the methods for bonding contemporary adhesive systems to dental substrates are easy to perform, there is always room for improvement in their composition, with the aim of increasing their bond strength to different substrates, and thus increasing the longevity of the restorations2. dentin has dental tubules filled with circulating dentinal fluid, under constant pulp pressure, which keeps the surface of this substrate permanently moist and makes adhesion difficult3, especially when there are active carious lesions in deep dentin4. in self-etching adhesive systems, the tubules remain partially occluded, because of incomplete smear layer removal; hence, the dentin surface is less susceptible to the effects of pulp pressure5. since universal adhesive systems work by way of chemical bonding to the tooth structure, it is questionable whether the tooth permeability resulting from these adhesive systems is lower than it would be with older generation adhesives. the suggestion has been that some products can be used together with adhesive systems to increase the longevity of the bond to dental tissues, especially dentin, among them chlorhexidine6, and some phenolic compounds present in green tea7. the aforementioned products have been evaluated for their potential of inhibiting endogenous enzymes, preventing proteolytic activity in collagen fibers, and maintaining the hybrid layer for a longer time. their ability to increase cross-links between collagen fibrils has also been assessed with a view to promoting increased resistance to degradation by endogenous proteases8. titanium tetrafluoride (tif4) is an inorganic fluoride compound that has also been researched as a dentin pretreatment to increase the longevity of the hybrid layer9-11. tif4 is usually used in the form of gel, varnish or aqueous solution, applied to the enamel surface to prevent caries lesions, reduce in vitro demineralization12, and inhibit the progression of caries lesions in situ and in vivo13. it has the ability to prevent erosion and abrasion injuries14, and to reduce the risk of dentin hypersensitivity15. when tif4 is hydrolyzed, it acquires a low ph rate, at which titanium tends to bind to an oxygen atom of a phosphate group on the tooth surface16. this leads to the formation of a solid element composed of titanium oxides or organometallic complexes, and a stable, modified, acid-resistant smear layer17,18. in this case, occlusion of the dentinal tubules occurs18, which is capable of reducing dentin permeability19. when dentin pretreatment with 2.5% tif4 was applied, before or after acid etching, followed by the application of a conventional adhesive system, there were no changes in the bond strength to dentin20, and similar results were reported with self-etching adhesives9-11. however, the influence of dentin pretreatment with 2.5% tif4 associated with self-etching and universal adhesive systems on dentin permeability have not yet been evaluated. therefore, the aim of this study was to evaluate dentin permeability after pretreatment with a 2.5% aqueous solution of titanium tetrafluoride (tif4), followed by the use of a self-etching of universal adhesive system. 3 franco et al. braz j oral sci. 2023;22:e237545 materials and methods sample preparation forty healthy human third molars (approved by the ethics committee caae 16187519.6.0000.5374) were used, cleaned with scalpel blades, washed with water, and stored frozen until ready to use. the crowns were sectioned perpendicular to the long axis of the tooth, 1.5 mm above the enamel-cement junction, using a precision saw (isomet 1000, buehler, springfield, va, usa) under cooling, to obtain the dentin discs21. the samples were sanded on both sides with 600 grit sandpaper to ensure uniformity and smoothness of the surfaces22. the occlusal surface of the sample was sanded until it was completely free of enamel. a diamond tip was used to mark the occlusal face that was used to adjust the slice of the permeability machine. the thickness of each sample was checked with a digital caliper (mitutoyo sul americana, mip/e, suzano, sp, brazil). the final thickness of the dentin discs was 1.5 mm. the discs were rinsed with water and stored in a flask with 5 ml distilled water in an incubator for 24 h. after this period, the slices were removed from the flask and first dried with paper for 15 s, and then with air for 5 s. after this, the smear layer was created by removing the polish with 37% phosphoric acid for 15 s21 and washing with water, on both sides for 30 s. acid etching was performed23 to open the dentinal tubules and increase dentin permeability, as a way to ensure standardization of the first hydraulic conductivity measurement (l1) (considered 100%), corresponding to the maximum filtration. afterwards, the occlusal surfaces were polished with 300 grit sandpaper for 30 s to form a standardized smear layer22. a total of 40 samples were prepared and randomly distributed into four groups (n=10). then they were dried with absorbent paper for 15 s, and received adhesive treatment on the occlusal surface, as specified for the particular group, according to the manufacturer’s recommendations. dentin treatments the 40 dentin specimens were separated into groups (n = 10): pretreatment + clearfil se bond, pretreatment + single bond universal, clearfil se bond, single bond universal. the composition of the adhesive systems is described in table 1. table1. adhesive systems evaluated in this study materials composition ph manufacturer (city, state, country) clearfil se bond primer: hema, 10-mdp, hydrophilic aliphatic dimethacrylate, dlcamphorquinone, water, accelerators, dyes bond: bis-gma, hema, 10-mdp, hydrophobic aliphatic dimethacrylate, silica, dl-camphorquinone, initiators, accelerators bond 1.55 primer 1.55 kuraray medical inc. (1621 sakazu, kurashiki, okayama, japan) single bond universal hema, bis-gma, ethanol, water, 10-mdp, silica, copolymer of acrylic and itaconic acid, silane, camphorquinone, dimethylaminobenzoate 2.82 3m espe (sumaré, são paulo, brazil) bis-gma: bisphenol a diglycidyl methacrylate; hema: 2-hydroxyethyl methacrylate; 10-mdp: 10-methacryloyloxydecyl dihydrogen phosphate 4 franco et al. braz j oral sci. 2023;22:e237545 the teeth assigned to receive the dentin pre-treatment were brushed with tif4 p.a. (sigma aldrich, saint louis, mo, usa) dissolved in distilled water to a final concentration of 2.5% (w/v; ph 1.0). the solution was actively applied to the dentin surface using a disposable brush (microbrush corporation, grafton, wi, usa) for 60 s. after this, the adhesive systems for each group were applied according to the respective manufacturer’s instructions. the primer assigned for use with the clearfil se bond adhesive system was actively applied with a microbrush for 20 s, and then air dried for 5 s at a distance of 10 cm. another microbrush was used to actively apply the adhesive over the primer for 20 s, and air was applied for 5 s immediately afterwards. the single bond universal adhesive system was actively applied for 20 s and received an air spray for 5 s. the adhesive system was light-cured with a photoactivation device (valo, ultradent, ut, usa) for 20 s with a light intensity of 1000 mw/cm2. then, a new hydraulic conductivity measurement was performed (l2). permeability test a permeability machine (thd, odeme dental research, luzerna, sc, brazil) was used under 5 psi pressure, equivalent to 351.54 cmh2o 23. the dentin disc was attached to the filtration chamber device, and the machine system was adjusted. the water entered the dentinal tubules and exerted pressure toward the surface. detachment of the liquid was marked by the difference in location of the air bubble inside the glass microtube of the equipment three measurements were performed during continuous movement of the liquid inside the glass microtube, to ensure that the amount of fluid that passed through the sample could be calculate using the following mathematical formula: q=(ri2l)/ t, where q (μl / min-1) was the amount of liquid passing through the sample, l(cm) was the linear displacement in the glass capillary, t (min) was the time, and ri (cm2) was the internal measurement of the glass microtube. the hydraulic conductivity (l) was obtained considering the viscosity of the water and thickness of the constant specimen, l = q / (ap), where l is the hydraulic conductivity (μl cm-2 min-1 cmh2o -1), a (cm2) was the dentin surface area, and p (cmh2o) was the pressure imposed. the hydraulic conductivity of each dentin disc was evaluated at two time points: initially, after acid etching (l1), and after applying the adhesive system protocol with or without tif4 pretreatment (l2). the percentage of dentin permeability after application of the adhesive system was obtained using the equation below, with each tooth being its own control: l (%) = [(l1-l2) x100] / l124, where l was the percentage of permeability, l1 was the hydraulic conductance after removal of the smear layer, and l2 was the hydraulic conductivity after application of the adhesive system. statistical analysis data distribution was evaluated by the shapiro-wilk test. exploratory analysis indicated that the data did not meet the assumptions of analysis of variance (anova). the mann whitney non-parametric test was then applied. the results were summa5 franco et al. braz j oral sci. 2023;22:e237545 rized and presented with median, minimum and maximum values. all the analyses were performed using the r program, with a significance level of 5%. results there was wide variability (%) in permeability among the dentin discs in the same group (table 2). no significant differences were found between the two groups studied, or between the groups with or without dentin pretreatment, as regards permeability (p>0.05). table 2. median (%) (minimum and maximum value) dentin permeability according to application of dentin pretreatment and type of adhesive system adhesive system dentin pretreatment p-value none 2.5% tif4 aqueous solution single bond universal 43.84 (17.68; 81.59) 47.76 (-64.42; 77.32) 0.5967 clearfil se bond 40.26 (-7.57; 71.01) 53.67 (-73.23; 70.84) 0.6501 p-value 0.3258 0.8206 discussion the formation of a hybrid layer with greater permeability causes greater degradation that compromises the tooth-restoration interface and dentinal sealing25. instead, lower permeability of the hybrid layer is desirable because it leads to less elution of resin components26, and minimum sorption and solubility rates?/levels?/. nevertheless, dentin pretreatment with 2.5% tif4 did not influence dentin permeability, irrespective of the association with either of the two adhesive systems. use of tif4 in an aqueous solution has been shown to form a layer with a vitreous aspect and promote a reduction in dentin permeability when used in concentrations of 0.1, 0.5, 1.027 and 4%28. this mechanism has been explained by the occlusion of the dentinal tubules18, which reduced dentin permeability19, especially when the smear layer was removed before the application of tif4 28. in contrast, this procedure differed from that of the present study, in which the smear layer was preserved. however, it should be noted that the adhesives used in this study had a mild ph (about 1.55 for clearfil se bond), and ultra-mild (about 2.82 for single bond universal)29. it could be suggested that these ph values caused the adhesives to dilute the vitreous layer, especially considering that the adhesive systems were actively applied (by brushing the dentin)9. although sen and büyükyilmaz17(1998) showed that the smear layer treated with tif4 was resistant to treatments with 17% edta and 5.25% sodium hypochlorite, their results cannot be compared with those of the present study, since the concentration of tif4 was 4%, whereas a 2.5% solution was used in the present study. the ability of self-etching adhesives to establish a mechanically resistant bond to dentin surfaces treated with an aqueous solution of 2.5% tif4 has been reported in 6 franco et al. braz j oral sci. 2023;22:e237545 previous studies9-11,30, despite this pretreatment solution tending to flocculate, and having a higher-than-average particle size30. these adhesive systems have been observed to be capable of penetrating the vitreous layer formed by the application of an aqueous solution of tif4. the acidity of the self-etching primer probably caused demineralization of the modified dentin surface layer, leading to the formation of a hybrid layer9-11,30. furthermore, the hydrophilic functional monomer 10-mdp most likely contributed significantly to the chemical bonding to dentin31. single bond universal is a single-bottle adhesive, containing hydrophilic components believed to promote greater permeability, by contributing to the formation of a semi-permeable hybrid layer25. whereas clearfil se bond adhesive has fewer hydrophilic components when compared with single bond. however, both adhesives are self-etching, a type of system that does not require etching with 37% phosphoric acid. cruz et al.32 (2021) also compared etch-and-rinse vs. self-etch adhesive system application modalities and observed similar results to those found in the present study, with no reduction in dentin permeability for single bond universal or clearfil se adhesives, irrespective of the application modality. in the present study, both adhesive systems were used without removing the smear layer, hence favoring a decrease in permeability5,33. both adhesives also promoted a chemical interaction with hydroxyapatite through the 10-methacryloxidecyl phosphate monomer (10-mdp), thereby favoring a more stable adhesive interface34,35. thus, the self-etching application mode and the presence of 10-mdp in both adhesive systems may have contributed to their similar permeability level. the results of the present study suggested that dentin pretreatment with 2.5% tif4 did not reduce dentin permeability. however, future studies must be conducted to assess whether this permeability is affected over time, especially in relation to reducing hybrid layer degradation. in conclusion, the use of 2.5% tif4 as a dentin pretreatment, combined with self-etching and universal adhesive systems, did not influence dentin permeability. acknowledgements the authors would like to thank the cnpq (conselho nacional de desenvolvimento científico e tecnológico) for providing grants (registration number 800280/2018-0). data availability datasets related to this article are available from the corresponding author upon request. conflict of interest the authors declare no conflict of interest author contribution gg franco: methodology, data acquisition and interpretation, wrote the draft; rfm cardoso: methodology, data acquisition, and data interpretation, wrote the draft; 7 franco et al. braz j oral sci. 2023;22:e237545 nr carlos: data interpretation, revised the draft; cp turssi: data interpretation, revised the draft; flb amaral: data interpretation, revised the draft; fmg frança: data interpretation, revised the draft; rt basting: responsible for conception and design, interpretation of the data, wrote and revised the draft. all authors actively participated in the manuscript’s findings have revised 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dent mater. 2021 oct;37(10):1463-85. doi: 10.1016/j.dental.2021.08.014. 1http://dx.doi.org/10.20396/bjos.v21i00.8666351 volume 21 2022 e226351 original article 1 graduate program in dentistry, meridional faculty/imed, passo fundo, brazil. 2 graduate program in dentistry, pontifical catholic university of rio grande do sul – pucrs, porto alegre, brazil. corresponding author: rafael sarkis onofre graduate program in dentistry, meridional faculty/imed, passo fundo, brazil. email: rafael.onofre@imed.edu.br editor: dr altair a. del bel cury received: july 14, 2021 accepted: october 27, 2021 reporting characteristics of systematic review abstracts published in the proceedings of the sbpqo meeting william vinícius de oliveira santos1 , lara dotto2 , rafael sarkis-onofre1,* aim: this study aimed to assess the reporting characteristics of systematic review abstracts published in the proceedings of the sociedade brasileira de pesquisa odontológica (sbpqo) meeting. methods: we selected abstracts published in the sbpqo meeting proceedings of 2019 and 2020, mentioning that a systematic review was conducted in the title, objective or methods sections. one researcher performed the screening and the data extraction after a pilot test training. the following data were extracted: affiliation of the primary author, dental specialization, the term “systematic review” mentioned in the title, reporting of the objective, reporting of eligibility criteria, reporting of information sources, reporting of the number of included studies and if a meta-analysis was performed. a descriptive analysis of the data was performed with data summarized as frequencies. results: we included 235 abstracts. a total of 20 studies were from the universidade de uberlândia (8.5%), and the main specialization was restorative and esthetic dentistry, with 47 studies (20%). most of the studies mentioned the term “systematic review” in the title (n=219; 93.2%) and reported the objective (n=231; 98.3%). a great majority of studies did not report the eligibility criteria (n=97; 41.3%) or it was classified as unclear (n=96; 40.8%). the great majority of studies only reported the databases searched (n=103; 43.8%) or databases and date of search (n=74; 31.5%). most of the studies reported the number of included studies (n=204; 86.8%). conclusion: based on this study, the reporting characteristics of systematic review abstracts published in the proceedings of the sbpqo meeting are satisfactory. however, there is room for improvement. keywords: dental research. research report. systematic reviews of topic. https://orcid.org/0000-0002-8643-5026 https://orcid.org/0000-0003-1535-4736 https://orcid.org/0000-0002-1514-7879 2 santos et al. introduction a systematic review is an important tool in health, and it is used for identifying, appraising, and integrating the results of a specific field1,2. the number of systematic reviews in dentistry has been increasing in recent years, and the reporting quality is highly variable3-5. much health research is presented at conferences and is publicly available as abstracts in the proceedings. the reporting quality of these abstracts is important because systematic reviewers will in some situations decide to include a study (or not) based on the conference abstract because the full article is not available. the reporting quality of conference abstracts was assessed in different topics in health, including sports injury prevention, oncology, urology, psychiatry, surgery, and oral health6-13. however, there are no studies assessing abstracts of systematic reviews in dentistry published in the proceedings of conferences. the sociedade brasileira de pesquisa odontológica (sbpqo) meeting is the most important conference in oral health research in brazil, and it is the brazilian division of the international association for dental research. since 2019, the sbpqo meeting has presented a special section dedicated to systematic review presentation, and all studies are published in the proceedings of the sbpqo meeting14,15. therefore, this study aimed to assess the reporting characteristics of systematic review abstracts published in the proceedings of the sbpqo meeting. material and methods eligibility criteria and search we included abstracts mentioning that a systematic review was conducted in the title, objective, or methods sections, regardless of the dental specialization discussed. we excluded studies which cited performing scoping reviews, overviews or assessing reporting quality of studies, or other methodological aspects characterizing the study as a meta-research. we performed a search in the proceedings of 201914 and 202015 to identify abstracts based on the eligibility criteria cited above, examining only the systematic reviews section. screening two researchers initially performed a pilot screening test discussing the inclusion criteria using the 2018 proceedings of the sbpqo meeting. one of the researchers subsequently identified studies by reviewing the titles and abstracts through the pdf versions of the 2019 and 2020 proceedings available at www.sbpqo.org.br. in case of any doubts, the opinion of a second researcher was requested. data extraction we created a standardized form using the excel program (microsoft excel 2020). we initially performed a pilot data extraction through a discussion between two reviewers to consider all data for extraction. data from each systematic review were 3 santos et al. subsequently extracted by one reviewer. the following data were collected: affiliation of primary author, dental specialization (public health, endodontics, oral and maxillofacial pathology/stomatology, radiology, oral and maxillofacial surgery, restorative and esthetic dentistry, pediatric dentistry, periodontics, orthodontics/orthopedics, implantology, and others), the term “systematic review” mentioned in the title (yes or no), reporting of the objective (yes or no), reporting of eligibility criteria (only inclusion criteria, only exclusion criteria, inclusion and exclusion criteria, unclear, not reported), reporting of information sources (only databases, only date of search, databases and date of search, unclear, not reported), reporting of the number of included studies (yes, no, unclear) and if a meta-analysis was performed (yes or no). data analysis a descriptive analysis of the data was performed with the data summarized as frequencies using the excel program (microsoft excel 2020). results we identified 262 abstracts published in the proceedings of the sbpqo meeting and classified as “systematic review”. we included 235 abstracts after the screening based on the eligibility criteria (see supplemental material). table 1 presents the data related to the affiliation of the primary author and the dental specialization of the abstract. as a result, 20 studies were from the universidade de uberlândia (8.5%), followed by the universidade de santa catarina (n=16;6.8%), while the universidade federal do pará, universidade federal do rio de janeiro, faculdade de odontologia de piracicaba (unicamp), and the universidade estadual paulista (araçatuba) presented 15 studies each (6.4%). the main specialization was restorative and esthetic dentistry, numbering 47 studies (20%), followed by oral and maxillofacial pathology/stomatology (n=41; 17.4%). table 1. characteristics of included studies filiation of main author n % universidade federal de uberlândia 20 8.5% universidade federal de santa catarina 16 6.8% faculdade de odontologia de piracicaba (unicamp) 15 6.4% universidade federal do pará 15 6.4% universidade federal do rio de janeiro 15 6.4% universidade estadual paulista araçatuba 15 6.4% universidade federal de minas gerais 10 4.3% faculdade de odontologia de são leopoldo mandic 9 3.8% universidade federal do rio grande do sul 8 3.4% universidade federal fluminense 7 3.0% universidade estadual da paraíba 7 3.0% universidade do estado do rio de janeiro 6 2.55% universidade de pernambuco 5 2.13% continue 4 santos et al. continuation universidade de são paulo são paulo 5 2.13% universidade federal fluminensepólo nova friburgo 4 1.7% universidade positivo 4 1.7% universidade federal da paraíba 4 1.7% universidade de são paulobauru 4 1.7% pontifícia universidade católica de minas gerais 4 1.7% universidade de são pauloribeirão preto 4 1.7% centro universitário christus 3 1.3% universidade federal do ceará 3 1.3% universidade de cuiába 3 1.3% universidade federal de juiz de fora 3 1.3% universidade estadual de ponta grossa 3 1.3% universidade de brasília 3 1.3% universidade estadual do pará 2 0.8% universidade do oeste paulista 2 0.8% instituto de ciências e tecnologia / ict-unesp-sjc 2 0.8% centro de estudos superiores de maceió 2 0.8% universidade federal de pelotas 2 0.8% universidade santo amaro 2 0.8% faculdade paulo picanço 2 0.8% universidade federal do amazonas 2 0.8% universidade ibirapuera 2 0.8% universidade luterana do brasil 1 0.4% universidade cruzeiro do sul 1 0.4% universidade federal de goiás 1 0.4% faculdade federal da paraíba 1 0.4% universidade federal do paraná 1 0.4% universidade norte do paraná 1 0.4% faculdade de odontologia de nova friburgo 1 0.4% pontifícia universidade católica do rio grande do sul 1 0.4% faculdade meridional 1 0.4% universidade federal de pernambuco 1 0.4% universidade federal do maranhão 1 0.4% centro universitário santo agostinho 1 0.4% universidade iguaçu 1 0.4% universidade estadual paulista araraquara 1 0.4% universidade federal do espírito santo 1 0.4% centro universitário da fundação hermínio ometto 1 0.4% escola bahiana de medicina e saúde pública 1 0.4% universidade estadual de maringá 1 0.4% universidade nove de julho 1 0.4% pontifícia universidade católica do paraná 1 0.4% universidade de taubaté 1 0.4% continue 5 santos et al. figure 1 presents the reporting characteristics of the included abstracts. most of studies mentioned the term “systematic review” in the title (n=219; 93.2%) and reported the objective (n=231; 98.3%). a great majority of studies did not report the eligibility criteria (n=97; 41.3%) or it was classified as unclear (n=96; 40.8%). in addicontinuation universidade guarulhos 1 0.4% dental specialties     restorative and esthetic dentistry 47 20.00% oral and maxillofacial pathology/stomatology 41 17.4% orthodontics/orthopedics 28 11.9% periodontics 26 11.1% endodontics 25 10.6% pediatric dentistry 18 7.7% public health 16 6.8% oral and maxillofacial surgery 15 6.4% implantology 11 4.7% radiology 6 2.5% others 2 0.8% figure 1. reporting characteristics of included abstracts. systematic review mentioned in the title objective reported 219 16 250 200 150 100 50 0n um be r o f a bs tr ac ts yes no answers elegibility criteria reported 33 96 120 100 80 60 40 0n um be r o f a bs tr ac ts only inclusion criteria inclusion and exclusion criteria only exclusion criteria unclear not reported answers answers 231 40 250 200 150 100 50 0n um be r o f a bs tr ac ts yes no answers unclear 97 63 20 reporting of information sources 103 8 120 100 80 60 40 0n um be r o f a bs tr ac ts only databases databases and date of search only date of search unclear not reported 49 74 120 number of included studies n um be r o f a bs tr ac ts 204 2110 250 200 150 100 50 0 yes no answers unclear meta-analysis performed 131 104 140 120 80 40 20 0n um be r o f a bs tr ac ts yes no answers 100 60 6 santos et al. tion, the great majority of studies only reported the databases searched (n=103; 43.8%) or databases and date of search (n=74; 31.5%). most of the studies reported the number of included studies (n=204; 86.8%), and most of the studies performed a meta-analysis (n=131; 55.7%). discussion this is the first study in dentistry to assess the reporting characteristics of systematic review abstracts published in proceedings of conferences. our results demonstrated that most of the aspects evaluated are well-reported, and we believe that results could be related to the fact that the conference abstracts included were peer-reviewed by experienced reviewers before the publication of conference proceedings. also, our results are significant because the abstract could become a pivotal element to support clinical decision-making in some situations, as highlighted by johnson et al. (2013)16. however, most studies did not report the eligibility criteria, or it was classified as unclear. this fact could be related to the limited number of words to write the abstract. details about what evidence was eligible or ineligible are important to the readers to comprehend the review scope. one of the possibilities to report the eligibility criteria is to use the pico framework highlighting the population, intervention, comparator, and outcome included in the review17. a significant number of systematic review abstracts (n=235) were presented in the sbpqo meeting since establishing a special section for this type of study. three aspects could be involved in this result: 1) bassani et al. (2019)3 demonstrated that brazil is one of the countries that publish the most systematic reviews in dentistry; 2) dotto et al. (2020)18 showed that systematic reviews are well accepted as a master’s or phd thesis by brazilian graduate programs in dentistry; and 3) brazil has an upper-middle-income economy and systematic reviews are cheaper than other methodologies such as randomized controlled trials resulting in a research methodology appropriate for this setting18. when analyzing the primary authors’ affiliation, we can observe universities from different regions of brazil which presented systematic reviews in the sbpqo meeting. the top university contributors could be the institutions where systematic reviews are well accepted in graduate programs, reflecting in their students presenting systematic reviews in that meeting. the main specializations reported were restorative and esthetic dentistry (20%) and oral and maxillofacial pathology/stomatology (17.4%). bassani et al. (2019)3 found that most specialization reporting in systematic reviews in dentistry indexed within pubmed during 2017 was oral and maxillofacial surgery, followed by oral and maxillofacial pathology/stomatology. the prisma 2020 statement17 was recently published, providing an updated reporting guidance for systematic review abstracts. the guidance includes a 12-item checklist specifying details about the systematic review title, background, methods, results, discussion, funding and registration. however, the number of words in abstracts could be limited in some conferences, jeopardizing the completeness of information reported. for example, the sbpqo recommendations about the number of words in 7 santos et al. abstracts allow 1,470 characters, 120 in the title and 1,350 in the body of the text19. also, the recommendations of the international association for dental research20 general session for abstracts allow 300 words or less, which is better than sbpqo, but it is still limited. we believe that it would be better to expand the number of words accepted in abstracts, but there are costs involved in this process. also, one crucial aspect is including the use of reporting guidelines in the instructions to authors to help in the abstract writing. the most important limitation of our study is that we did not assess the abstracts published before the establishment of a systematic review section, and it is impossible to evaluate the impact of this establishment in terms of the number of systematic reviews abstracts and reporting quality, and we did not assess all items recommended by prisma for abstracts. we believe that future assessments should focus on spin strategies and the extent and level of spin involved in systematic reviews abstracts. thus, in light of the existence of a specific guideline for systematic review abstracts (prisma 2020)17, the sbpqo, which is the most important conference in oral health research in brazil, should endorse the use of this statement to improve the reporting of abstracts and encourage students and researchers to use it. in conclusion, based on this study, the reporting characteristics of systematic review abstracts published in the proceedings of the sbpqo meeting are satisfactory. however, there is room for improvement. acknowledgements this study was conducted in a graduate program supported by capes, brazil (finance code 001). rso is funded in part by meridional foundation (passo fundo, brazil), and wvos is funded by the national council for scientific and technological development (cnpq, brazil). however, these supporters had no role in the design of the study, in the collection or analysis of data, in the decision to publish or in preparing the manuscript. data availability datasets related to this article will be available upon request to the corresponding author. references 1. cook dj, mulrow cd, haynes rb. systematic reviews: synthesis of best evidence for clinical decisions. ann. intern. med. 1997;126(5):376-80. doi: 10.7326/0003-4819-126-5-199703010-00006. 2. murad mh, montori vm. synthesizing evidence: shifting the focus from individual studies to the body of evidence. 2013; 309(21):2217-8. doi: 10.1001/jama.2013.5616. 3. bassani r, pereira gkr, page mj, tricco ac, moher d, sarkis-onofre r. systematic reviews in dentistry: current status, epidemiological and reporting characteristics. j dent. 2019;82:71-84. doi: 10.1016/j.jdent.2019.01.014. 4. saltaji h, cummings gg, armijo-olivo s, major mp, amin m, major pw, et al. a descriptive analysis of oral health systematic reviews published 1991-2012: cross sectional study. plos one. 2013 sep;8(9):e74545. doi: 10.1371/journal.pone.0074545. 8 santos et al. 5. t o lemes l, dotto l, agostini ba, pereira gkr, sarkis-onofre r. how are meta-analyses being conducted and reported in dentistry?: a meta-research study. braz j oral sci. 2021;20:e211701. doi: 10.20396/bjos.v20i00.8661701. 6. hopewell s, clarke m, askie l. reporting of trials presented in conference abstracts needs to be improved. j clin epidemiol. 2006 jul;59(7):681-4. doi: 10.1016/j.jclinepi.2005.09.016. 7. duan y, li j, ai c, chen y, chen p, zhang m, et al. quality of trials reported as conference abstracts in china: how well are they reported? int j technol assess health care. 2009 oct;25(4):479-84. doi: 10.1017/s0266462309990365. 8. turpen rm, fesperman sf, smith wa, vieweg j, dahm p. reporting quality and information consistency of randomized, controlled trials presented as abstracts at the american urological association annual meetings. j urol. 2010 jul;184(1):249-53. doi: 10.1016/j.juro.2010.03.045. 9. yoon u, knobloch k. assessment of reporting quality of conference abstracts in sports injury prevention according to consort and strobe criteria and their subsequent publication rate as full papers. bmc med res methodol. 2012 apr;12:47. doi: 10.1186/1471-2288-12-47. 10. yoon u, knobloch k. quality of reporting in sports injury prevention abstracts according to the consort and strobe criteria: an analysis of the world congress of sports injury prevention in 2005 and 2008. br j sports med. 2012 mar;46(3):202-6. doi: 10.1136/bjsm.2008.053876. 11. speich b, mc cord ka, agarwal a, gloy v, gryaznov d, moffa g, et al. reporting quality of journal abstracts for surgical randomized controlled trials before and after the implementation of the consort extension for abstracts. world j surg. 2019 jun;43(10):2371-8. doi: 10.1007/s00268-019-05064-1. 12. narayan vm, cone eb, smith d, scales cd jr, dahm p. improved reporting of randomized controlled trials in the urologic literature. eur urol. 2016 dec;70(6):1044-9. doi: 10.1016/j.eururo.2016.07.042. 13. kumar s, mohammad h, vora h, kar k. reporting quality of randomized controlled trials of periodontal diseases in journal abstracts-a cross-sectional survey and bibliometric analysis. j evid based dent pract. 2018 jun;18(2):130-141.e22. doi: 10.1016/j.jebdp.2017.08.005. 14. sociedade brasileira de pesquisa odontológica. proceedings of the 36th sbpqo annual meeting. braz oral res. 2019;33(suppl 2). 15. sociedade brasileira de pesquisa odontológica. proceedings of the 37th sbpqo virtual annual meeting. braz oral res. 2020;34(suppl 2). 16. johnson hl, fontelo p, olsen ch, jones kd 2nd, gimbel rw. family nurse practitioner student perception of journal abstract usefulness in clinical decision making: a randomized controlled trial. j am assoc nurse pract. 2013 nov;25(11):597-603. doi: 10.1111/1745-7599.12013. 17. page mj, mckenzie je, bossuyt pm, boutron i, hoffmann tc, mulrow cd, et al. the prisma 2020 statement: an updated guideline for reporting systematic reviews. bmj. 2021 mar;372:n71. doi: 10.1136/bmj.n71. 18. dotto l, t o lemes l, o spazzin a, sousa ytcs, pereira gkr, bacchi a, et al. acceptance of systematic reviews as master/phd theses in brazilian graduate programs in dentistry. j evid based med. 2020 may;13(2):125-9. doi: 10.1111/jebm.12382. 19. sociedade brasileira de pesquisa odontológica. instruções para envio de resumos. sbpqo; 2020 [cited 2021 mar 5]. available from: https://www.sbpqo.org.br/hotsite2020/conteudo.asp?id=7. 20. international association for dental research. call for abstracts. [cited 2021 mar 5]. available from: https://www.iadr.org/portals/69/docs/meetings/iags/2021/2021ia_callforabstracts.pdf. 9 santos et al. supplemental material list of numbers of included abstracts (2019). 1-rs001 42-rs045 83-rs097 2-rs002 43-rs046 84-rs098 3-rs003 44-rs048 85-rs099 4-rs004 45-rs049 86-rs100 5-rs005 46-rs050 87-rs101 6-rs006 47-rs051 88-rs102 7-rs007 48-rs052 89-rs103 8-rs008 49-rs054 90-rs104 9-rs010 50-rs055 91-rs105 10-rs011 51-rs057 92-rs106 11-rs012 52-rs058 93-rs107 12-rs013 53-rs059 94-rs109 13-rs014 54-rs060 95-rs110 14-rs016 55-rs061 96-rs111 15-rs017 56-rs062 97-rs113 16-rs018 57-rs064 98-rs114 17-rs019 58-rs065 99-rs115 18-rs020 59-rs068 100-rs116 19-rs021 60-rs069 101-rs117 20-rs022 61-rs070 102-rs118 21-rs023 62-rs071 103-rs119 22-rs024 63-rs072 104-rs121 23-rs025 64-rs073 105-rs122 24-rs026 65-rs075 106-rs123 25-rs027 66-rs076 107-rs124 26-rs028 67-rs077 108-rs126 27-rs029 68-rs078 109-rs128 28-rs030 69-rs079 110-rs129 29-rs031 70-rs082 111-rs130 30-rs032 71-rs083 112-rs131 31-rs033 72-rs084 113-rs132 32-rs034 73-rs085 114-rs134 33-rs035 74-rs086 115-rs135 34-rs036 75-rs087 116-rs136 35-rs037 76-rs088 117-rs137 36-rs038 77-rs090 118-rs138 37-rs040 78-rs091 119-rs139 38-rs041 79-rs092 120-rs140 39-rs042 80-rs093 121-rs142 40-rs043 81-rs094 41-rs044 82-rs096 10 santos et al. list of numbers of included abstracts (2020). 122-rs001 160-rs041 198-rs081 123-rs002 161-rs043 199-rs082 124-rs003 162-rs044 200-rs083 125-rs004 163-rs045 201-rs084 126-rs005 164-rs046 202-rs085 127-rs006 165-rs048 203-rs086 128-rs007 166-rs049 204-rs087 129-rs008 167-rs050 205-rs088 130-rs009 168-rs051 206-rs089 131-rs010 169-rs052 207-rs090 132-rs012 170-rs053 208-rs091 133-rs013 171-rs054 209-rs092 134-rs014 172-rs055 210-rs093 135-rs015 173-rs056 211-rs094 136-rs016 174-rs057 212-rs095 137-rs017 175-rs058 213-rs096 138-rs018 176-rs059 214-rs097 139-rs019 177-rs060 215-rs098 140-rs020 178-rs061 216-rs099 141-rs021 179-rs062 217-rs100 142-rs023 180-rs063 218-rs101 143-rs024 181-rs064 219-rs102 144-rs025 182-rs065 220-rs103 145-rs026 183-rs066 221-rs104 146-rs027 184-rs067 222-rs105 147-rs028 185-rs068 223-rs107 148-rs029 186-rs069 224-rs108 149-rs030 187-rs070 225-rs109 150-rs031 188-rs071 226-rs111 151-rs032 189-rs072 227-rs112 152-rs033 190-rs073 228-rs113 153-rs034 191-rs074 229-rs114 154-rs035 192-rs075 230-rs115 155-rs036 193-rs076 231-rs116 156-rs037 194-rs077 232-rs117 157-rs038 195-rs078 233-rs118 158-rs039 196-rs079 234-rs119 159-rs040 197-rs080 235-rs120 11 santos et al. characteristics of each abstract included. author/year systematic review mentioned in the title objective reported eligibility criteria reported reporting of information sources number of included studies meta-analysis conducted nunes et al., 2019 yes yes not reported only databases yes yes reis et al., 2019 yes yes only inclusion only databases yes no frazão et al., 2019 yes yes only inclusion only databases yes yes granja et al., 2019 yes yes unclear unclear yes yes lacerda-santos et al., 2019 yes yes unclear unclear yes yes paulo et al., 2019 yes yes not reported only databases yes no pereira et al., 2019 yes yes not reported only databases yes yes cetira-filho yes yes unclear only databases unclear yes novais et al., 2019 yes yes unclear unclear yes yes amaral et al., 2019 yes yes not reported only databases yes no thuller et al., 2019 yes yes only exclusion only databases yes yes falcão et al., 2019 yes yes unclear both yes yes barbosa et al., 2019 yes yes only inclusion only databases yes no stringhini-junior et al., 2019 yes yes unclear both yes yes moraes et al., 2019 yes yes only inclusion both yes yes alves et al., 2019 yes yes only inclusion both yes yes basso et al., 2019 yes yes unclear only databases yes yes wembier et al., 2019 yes yes unclear only databases yes yes silva et al., 2019 yes yes unclear both yes yes bronzato et al., 2019 yes yes unclear only databases yes yes albuquerque et al., 2019 yes yes not reported both yes no castro et al., 2019 yes yes unclear only databases yes yes pinto et al., 2019 yes yes unclear both yes yes scarsi et al., 2019 yes yes only inclusion only databases yes yes campos et al., 2019 yes yes unclear only databases yes yes spinola et al., 2019 yes yes not reported only databases yes no oliveira et al., 2019 yes yes not reported only databases yes yes terto et al., 2019 yes yes only exclusion both yes yes castilho et al., 2019 yes yes unclear only databases yes yes lima et al., 2019 yes yes unclear unclear no yes andrade et al., 2019 yes yes unclear only databases yes yes baroni et al., 2019 yes yes unclear only databases yes no rende et al., 2019 no yes unclear unclear yes yes guimaraes et al., 2019 no yes not reported only databases yes yes fontes et al., 2019 yes yes unclear only databases yes no dantas et al., 2019 yes yes only inclusion both yes yes continue 12 santos et al. continuation milani et al., 2019 yes yes unclear only databases unclear yes bedran et al., 2019 yes yes not reported only databases yes yes cruz et al., 2019 yes yes not reported unclear yes yes ribeiro-lages et al., 2019 yes yes not reported only databases yes yes mocchelini et al., 2019 yes yes not reported only databases yes yes masson et al., 2019 yes yes unclear both yes yes duarte et al., 2019 yes yes not reported only databases yes yes seabra et al., 2019 yes yes not reported both yes yes nadelman et al., 2019 yes yes only inclusion only databases yes no messignan et al., 2019 yes yes not reported only databases yes yes dantas et al., 2019 yes yes not reported unclear no no gonçalves et al., 2019 no yes not reported only databases yes yes oliveira et al., 2019 yes yes both both yes no bellini-pereira et al., 2019 yes yes only inclusion both yes yes prado et al., 2019 yes yes only inclusion only databases yes yes pinheiro et al., 2019 yes yes unclear only databases yes no cardoso et al., 2019 yes yes not reported both yes yes farias junior et al.,2019 yes yes unclear both yes no silva et al., 2019 yes yes only inclusion both yes no silveira et al;. 2019 yes yes unclear unclear yes no nascimento et al., 2019 yes yes unclear both yes no neves et al., 2019 yes yes unclear not reported unclear yes nunes et al., 2019 yes yes unclear only databases unclear no lins et al., 2019 no yes not reported only databases yes yes farias et al., 2019 yes yes unclear only databases yes no dietrich et al., 2019 yes yes unclear both yes no polmann et al., 2019 yes yes not reported only databases yes no maran et al., 2019 yes yes not reported only databases yes yes lima et al., 2019 no yes unclear both yes no martini et al., 2019 yes yes not reported only databases yes no antonio et al., 2019 yes yes only inclusion only databases yes no né et al., 2019 yes yes unclear unclear yes no fidalgo et al., 2019 yes yes unclear only databases yes no nascimento et al., 2019 no yes not reported only databases yes yes bacchin et al., 2019 yes yes not reported only databases yes no roithmann et al., 2019 yes yes unclear both yes yes maroli et al., 2019 yes yes both only databases yes no carneiro-campos et al., 2019 yes yes only inclusion both unclear yes brunetto et al., 2019 yes yes unclear both yes yes continue 13 santos et al. continuation minatel et al., 2019 yes yes not reported only databases yes no lemos et al., 2019 yes yes not reported unclear yes yes silva et al., 2019 yes yes unclear both yes yes barcelos et al.,2019 yes yes only exclusion both yes no gomez et al., 2019 yes yes not reported both yes no magalhães et al., 2019 yes yes not reported only databases yes no sarmento et al., 2019 yes yes unclear both yes no barbosa et al., 2019 yes yes not reported unclear yes yes lhano et al., 2019 yes yes unclear only databases unclear yes oliveira et al., 2019 yes yes not reported both yes yes nascimento et al., 2019 no yes not reported only databases yes yes oliveira et al., 2019 yes yes unclear only databases yes yes silva et al., 2019 yes yes both only databases yes yes dutra et al., 2019 yes yes not reported only databases unclear yes macedo-filho et al., 2019 yes yes unclear both yes yes oliveira et al., 2019 yes yes not reported unclear yes yes kammer et al.,2019 yes yes not reported unclear yes yes berretta et al., 2019 yes yes only inclusion unclear yes no diniz et al., 2019 yes yes unclear unclear yes no reis et al., 2019 yes yes unclear unclear yes no silveira et al;. 2019 yes yes only inclusion unclear yes yes martins et al., 2019 yes yes not reported unclear yes yes jerônimo et al., 2019 yes yes unclear unclear yes yes rosa et al., 2019 yes yes unclear both yes yes lago et al., 2019 yes yes unclear both yes yes haas et al., 2019 no unclear unclear only databases yes yes melo et al., 2019 yes yes unclear unclear yes no lavôr et al., 2019 yes yes unclear unclear yes no oliveira et al., 2019 yes yes unclear both yes no paulo et al., 2019 yes yes unclear unclear yes yes lisboa et al., 2019 yes yes not reported only databases yes no martins et al., 2019 yes yes only inclusion only databases yes no campos et al., 2019 yes yes not reported only databases no no rolim et al., 2019 yes yes unclear both yes yes silva et al., 2019 yes yes unclear only databases yes yes campos et al., 2019 yes yes not reported unclear unclear no souza et al., 2019 yes yes unclear only databases yes yes miranda et al., 2019 yes yes not reported only databases yes yes raymundo et al., 2019 yes yes not reported only databases no yes caldas et al., 2019 yes yes not reported both yes no continue 14 santos et al. continuation miranda et al., 2019 yes yes unclear only databases yes no alvarenga et al., 2019 yes yes not reported unclear yes yes santos et al., 2019 no yes not reported only databases yes no rendohl et al., 2019 yes yes only inclusion both no yes limírio et al., 2019 yes yes not reported both yes yes borges et al., 2019 yes yes not reported not reported yes yes reis et al., 2020 yes yes unclear only databases yes no gonçalves et al., 2020 yes yes unclear both yes yes vidigal et al., 2020 yes yes not reported unclear yes yes leal et al., 2020 yes yes not reported both yes no silva et al., 2020 no yes unclear both yes no rosatto et al., 2020 yes yes not reported unclear yes no sarmento et al., 2020 yes yes not reported unclear yes no matos et al., 2020 yes yes not reported unclear yes no gabriel et al., 2020 yes yes not reported unclear yes yes dias-junior et al., 2020 yes yes unclear only databases yes yes nóbrega et al., 2020 yes yes unclear only databases unclear yes sponchiado-júnior et al., 2020 yes yes unclear only databases yes no martins et al., 2020 yes yes unclear only databases yes no pirovani et al., 2020 yes yes unclear both yes no feitosa et al., 2020 yes yes only inclusion only databases yes no salomão et al., 2020 yes yes only inclusion only databases yes yes kwiatkowski et al., 2020 yes yes unclear both yes no lopes et al., 2020 yes yes unclear only databases yes no soares et al., 2020 yes yes not reported only databases yes no né et al., 2020 yes yes unclear only databases yes yes carvalho et al., 2020 yes yes unclear only databases yes no araujo et al., 2020 yes yes not reported only databases yes no silver et al., 2020 yes yes only inclusion only databases yes yes schoeffel et al., 2020 yes yes only inclusion unclear yes yes muknickas et al., 2020 yes yes unclear only databases unclear no rezende et al., 2020 yes yes unclear both yes no oliveira et al., 2020 yes yes only inclusion both yes yes cruz et al., 2020 yes yes not reported unclear yes yes oliveira et al., 2020 yes yes both both yes no martins et al., 2020 yes yes not reported only databases yes yes mocchelini et al., 2020 yes yes not reported only databases yes yes miyahira et al., 2020 yes yes unclear only databases yes yes machado et al., 2020 yes yes only inclusion both yes no miranda et al., 2020 yes yes only inclusion both yes no continue 15 santos et al. continuation soares et al., 2020 yes yes unclear both yes no pintor et al., 2020 yes yes only inclusion both yes no camatta et al., 2020 yes yes unclear only databases no yes sanglard et al., 2020 yes yes not reported both yes no haibara et al., 2020 yes yes unclear both yes no fontes et al., 2020 yes yes unclear only databases yes no latieri et al., 2020 yes yes unclear both yes no bonzanini et al., 2020 yes yes not reported both yes yes soares et al., 2020 yes yes not reported only databases yes yes neves et al., 2020 yes yes not reported only databases yes no inocêncio et al., 2020 no yes unclear unclear unclear yes figueiredo et al., 2020 yes unclear unclear both unclear no rodrigues et al., 202 yes yes unclear both unclear no jácome-santos et al., 2020 yes yes not reported both yes no sant ´anna et al., 2020 yes yes not reported only databases yes yes sant’anna et al., 2020 yes yes unclear unclear yes yes mattos et al., 2020 yes yes unclear both yes yes silveira-júnior et al., 2020 yes yes not reported only databases yes no caetano et al., 2020 yes yes unclear only databases yes no figueiredo et al., 2020 yes yes only inclusion only databases no yes naal et al., 2020 yes yes unclear both yes no tardelli et al., 2020 yes yes only inclusion both unclear no lopes et al., 2020 yes yes only inclusion both unclear yes kunz et al., 2020 no yes unclear only databases yes yes uehara et al., 2020 yes yes both only databases yes no limírio et al., 2020 yes yes not reported both yes yes sanches et al., 2020 yes yes not reported both yes yes veloso et al., 2020 yes yes only inclusion only databases yes yes oliveira et al., 2020 yes yes only inclusion unclear yes yes ortiz et al., 2020 yes yes only inclusion unclear yes no scherer et al., 2020 yes yes not reported both yes yes andrade et al., 2020 yes yes unclear only databases yes no martini et al., 2020 yes yes not reported only databases unclear yes valesan et al., 2020 yes yes unclear unclear yes yes oliveira et al., 2020 yes yes not reported unclear yes yes santos et al., 2020 yes yes not reported only databases yes yes corso et al., 2020 yes yes not reported only databases yes yes gama et al., 2020 yes yes unclear unclear yes no pinto et al., 2020 yes yes unclear both yes no parize et al., 2020 yes yes not reported unclear yes no continue 16 santos et al. continuation sabatini et al., 2020 yes yes unclear unclear yes no camarini et al., 2020 yes yes not reported not reported no no sakuma et al., 2020 yes yes not reported unclear yes yes silva et al., 2020 yes yes not reported only databases no yes santaella et al., 2020 yes yes not reported only databases yes no guerra et al., 2020 yes yes not reported not reported yes yes munhoz et al., 2020 no yes not reported only date yes no ibarra et al., 2020 yes yes not reported not reported yes no ribeiro et al., 2020 yes yes not reported both yes no galdino et al., 2020 yes yes not reported only databases yes yes magrin et al., 2020 yes yes unclear unclear yes no souza et al., 2020 yes yes not reported both yes yes borges et al., 2020 yes yes only inclusion both yes yes magalhães et al., 2020 yes yes both only databases yes no martinho et al., 2020 no yes not reported unclear unclear yes souza et al., 2020 yes yes unclear both yes yes resende et al., 2020 yes yes unclear only databases yes yes langa et al., 2020 yes yes unclear unclear yes yes basso et al., 2020 no yes unclear only databases yes no oliveira et al., 2020 yes unclear not reported unclear yes yes campos et al., 2020 yes yes not reported both unclear yes albuini et al., 2020 yes yes unclear not reported yes no koch et al., 2020 yes yes unclear only databases yes no soares et al., 2020 yes yes unclear unclear unclear yes jakymiu et al., 2020 yes unclear unclear both yes no peinado et al., 2020 yes yes not reported only databases yes no domingues et al., 2020 yes yes unclear only databases yes no barbosa et al., 2020 yes yes unclear both yes no vieira et al., 2020 yes yes not reported unclear yes yes santos et al., 2020 yes yes not reported only databases yes no macedo et al., 2020 yes yes not reported unclear yes yes dini et al., 2020 yes yes not reported not reported unclear yes macena et al., 2020 yes yes not reported only databases yes yes spessato et al., 2020 yes yes not reported both yes yes baccaro et al., 2020 yes yes only inclusion both unclear no silva et al., 2020 yes yes not reported both yes yes linhares et al., 2020 yes yes only inclusion only databases no no ribeiro et al., 2020 yes yes not reported only databases yes no bezerra et al., 2020 yes yes not reported unclear yes yes dias et al., 2020 no yes not reported both yes yes 1http://dx.doi.org/10.20396/bjos.v20i00.8661202 volume 20 2021 e211202 original article 1 state university of piauí – uespi, school of dentistry, department of clinical dentistry, area of integrated clinic, parnaíba, pi, brazil. corresponding author: ana de lourdes sá de lira universidade estadual do piauí, faculdade de odontologia rua senador joaquim pires 2076 ininga. fone (86) 999595004 cep: 64049-590 teresina-pi-brasil email: anadelourdessl@hotmail.com editor: dr altair a. del bel cury received: september 12, 2020 accepted: february 17, 2021 prevalence and predisponent factors of molar-incisor hypomineralization in primary dentition ana de lourdes sá de lira1,* , francisca janiele de sousa1, francisco dário carvalho de sousa1, maria karen vasconcelos fontenele1, carlos kelvin campos ribeiro1, luiz eduardo gomes ferreira1 aim: to evaluate the prevalence and predisposing factors for hypomineralization of second molars in children in primary dentition. methods: a questionnaire was applied to parents to analyze predisposing factors and to assist in the diagnosis of hypomineralization in children between 2 and 6 years old, followed by an intraoral examination based on indices of non-fluorotic enamel defects in the primary dentition, according to the “modified index dde” to determine demarcated opacity and hspm presence / severity index to assess hypomineralization. children from public and private schools were dived into two groups: if they presented hspm-group 1 (g1) and if they did not have hspm-control group (cg). results: the most frequent predisposing factors associated with the child were illness in the first year of life (x2= 6.49; p=0.01) and antibiotic use in the first year of life (x2= 41.82; p= 0.01). the factors associated with the mother were hypertension (x2= 9.36; p=0.01), infections during pregnancy (x2=14.80; p=0.01) and alcohol consumption during pregnancy (x2=97.33; p=0.01). there was a prevalence of 3.9% of hspm in 14 children, with statistical difference regarding gender (x2 = 4.57; p <0.05), with boys presenting a higher frequency. in g1 hypomineralization was of the type with demarcated opacity, with more prevalent characteristics the yellowish spot, with moderate post-eruptive fracture and acceptable atypical restorations. all lesions were located in the labial region with 1/3 of extension. conclusion: the prevalence of hspm in children between 2 and 6 years old was 3.9%, with a predominance in males, with tooth 65 being the most affected. there was an association between hspm and infection in the first year of life, as well as the use of antibiotics and sensitivity in the teeth affected by the lesion. there was an association between hspm and hypertension, infection and mothers’ alcohol use during pregnancy. keywords: tooth demineralization. dental enamel. amelogenesis. 2 lira et al. introduction molar-incisor hypomineralization (mih) is a defect of systemic origin in the dental enamel that affects the first permanent molars and is often associated with the incisors. it is characterized by qualitative defects in dental tissues and is visually identified by the change in enamel translucency1. it is a clinical condition that appears in the permanent dentition, but that can be related to hypomineralization in the primary dentition. several studies attempt to investigate factors potentially involved with the occurrence of mih, however the findings are variable and inconclusive2,3. the etiology remains unknown, although it is related to environmental factors that are associated with systemic conditions during the prenatal period (last three months of pregnancy) and during the perinatal and postnatal periods. these factors may include low birth weight, congenital heart disease and use of continuous medication to treat systemic disease. common conditions in the first 3 years of age, such as diseases of the upper respiratory tract, asthma, otitis, tonsillitis, gastrointestinal diseases, malnutrition, chickenpox, measles and rubella, also seem to be associated with the occurrence of mih4-6. due to the temporal association between the mineralization of the crowns of the first permanent molars and the second deciduous molars, they may also be affected by hypomineralization if any etiological factor, such as some systemic disorder, acts during this stage of development, especially during the period prenatal and perinatal7. the hypomineralization of second primary molars (hspm) is similar to mih with respect to both asymmetric distribution and post-eruptive structural losses and atypical restorations. the researchers suggest that the presence of hspm in the primary dentition poses a risk for the presence of mih. the explanation for this association is probably related to the etiological factors of developmental defects in the enamel, which are the same in both dentitions. the intensity and duration of injuries that occur in late pregnancy and in the early perinatal period can affect both dentitions8. the characteristics of hspm are the same as those of mih: opaque spots that vary in tone between white, yellow and brown, break post-eruptive enamel, atypical restorations and / or extensive caries with opacities in the margins, sensitivity, which can affect one to four second molars7. in the same patient it can be presented in a different way, being possible to observe a slight opacity in one second molar, and great structural losses in another, mainly the contralateral one, with the occurrence of fractures soon after the tooth eruption8,9. because the characteristics and clinical consequences are similar and the intention to identify the etiological factors involved in this enamel alteration, the epidemiological knowledge of the distribution of this defect is necessary. this research is justified because there is no study that reports the prevalence of hspm in the city where the search was conducted. it is believed that there is a low prevalence of hspm in the primary dentition and ignorance of parents and / or guardians about the damage of the injury in the child’s oral 3 lira et al. health quality. in addition, the presence of hspm can be considered a predictive factor for mih, although the absence of this defect in the primary dentition does not exclude the future appearance of mih. the aim of this research was to evaluate the prevalence and predisposing factors for hypomineralization of second molars in children in primary dentition. material and methods this study was approved by the research ethics committee of the state university of piauí cep / uespi under the number: 3,289,708. it was a cross-sectional study, whose sample calculation was based on the target audience: preschool students from the city of parnaíba-pi, attending in public and private schools in 2019. the sample size calculation was according to a survey conducted by the brazilian institute of geography and statistics, which showed an estimated 15.000 children living in the city of parnaíba-pi aged 2 to 6 years. thus, the necessary sample size was 360 participants. it was decided to increase the exhibition to 365, assuming the possibility of giving up participation in the research. in fact, 5 children were excluded from the survey because their parents answered the questionnaire incompletely, making a total of 360 participants. this minimum number of participants was considered sufficient considering the proposed analyzes, the sample error of 5%, in addition to the 95% confidence level, indicating that the probability of the error made by the research did not exceed 5%10. the researchers were provided with a letter of consent from the principals of the schools, chosen by lot, being a private and a public institution, in the municipality of parnaíba-piauí, which authorized the development of the research after the ethical approval of the research ethics committee of the state university of piauí cep / uespi, from august to december 2019. inclusion criteria were children in primary dentition, randomly included, between 2 and 6 years of age, who had four primary second molars fully erupted at the time of examination whose parents or guardians and children accepted the research. as exclusion criteria, children under 2 years old, as the primary dentition would not yet be fully formed, and those over 6 years old, because they would already be in mixed dentition, those pre‐schoolers with a syndrome or special need, who did not tolerate the dental examination or who had fixed orthodontic appliance. in order to standardize the diagnosis of hspm, clinical training of examiners was carried out in the children’s clinic of clinic school of dentistry (csd) of of the state university of piauí (uespi) campus of parnaíba. to measure the intra and inter-examiner diagnostic reproducibility, 10% of the total sample was doubly verified by two examiners, with the kappa coefficient for intra and inter-examiner agreement being 0.87 and 0.84, respectively. before data collection, a pilot study was carried out with 50 children from municipal schools who did not participate in the sample, to evaluate the methods and it was found that there was no need to make changes to the methodology initially proposed. 4 lira et al. the parents / guardian of the children answered the questionnaire to help diagnose hypomineraization (figure 1), after having attended a lecture at the school on the topic. using an age-compatible language, the children received an explanation of how the research would be conducted. in a second moment in the school environment, the children were examined. the children were seated in a school chair, with their head positioned on the examiner’s lap. the clinical examination was carried out under natural light, in a school environment, by two specially calibrated examiners, previously calibrated, with the aid of a wooden spatula, mouth mirror and explorer probe. after cleaning and drying the teeth with sterile gauze, a complete inspection was performed, based on the following indices: non-fluorotic enamel defects in the primary dentition, according to the “modified index dde”, assessing the coloring of the demarcated opacity ( white, yellow or brown), extension of defect (less than 1/3 of a tooth surface; at least 1/3 but less than 2/3; and at least 2/3 of the tooth surface), location (labial/buccal, lingual/palatal or occlusal), figure 1. evaluation of hypomineralization of second primary molars (hspm). 5 lira et al. if there is presence of post-eruptive fracture (light, moderate or severe) and atypical restoration (acceptable/no acceptable) (figure 2)11. according to the characteristics of the enamel defects, children were classified as having or not hypomineralization. those who presented hspm were named group 1 (g1) and those who did not present the lesion constituted the control group (cg) in both public (a1) and private schools (a2). the observations were transcribed to a standardized clinical record according to the established codifications. during the examination, it was possible to identify the need to apply fluoride to minimize tooth sensitivity or restorative treatment in the presence of irregularities in the enamel structure. in such situations the children were referred to the csd of uespi for assistance and follow-up at the pediatric dentistry clinic. the results were stored in the excel windows 2007 microsoft® database and displayed in graphs and tables for better interpretation and discussion. with spss, in its version 25, descriptive and association statistical tests were performed, with chi square, in addition to comparison of means using the t test. all considering the significance level p value of 0.05. results at first, we sought to know the prevalence of hspm. based on the total sample of 360 children, a prevalence of 3.9% was observed, that is, 14 children, 8 of whom were brown, 5 white and 1 black. of this total, 11 had lesions on two teeth, with 43% (n=6) of children having teeth 55 and 65 (upper deciduous second molars) affected, followed by those with teeth 75 and 85 (lower deciduous second molars) (36%; n = 5), and finally, those with only tooth 65 affected (21%; n = 3). of the children in g1, 11 were male and 3 were female, with 6 studying in public schools (a1) and 8 in private schools (a2). on the other hand, those in cg, 190 were male and 156 female, with 171 studying in a1 and 175 in a2 (figure 3). according to table 1, 9 children presented tooth 65 as the most affected by hypomineralization, followed by 55 in 6 children. hspm presented as demarcated opacity, with the most prevalent characteristics being the type of yellowish spot, with moderate post-eruptive fracture, but with atypical restorations acceptable. dental characteristic code code code demarcated opacity white stain (a) yellow stain (b) brown stain (c) post-eruptive fracture light (d) moderate (e) severe (f) atypical restoration acceptable (g) no aceptable (h) figure 2. judgment used in the diagnosis of hspm based on severity. 6 lira et al. table 1. teeth affected and characteristics of hspm in children. tooth number of children type of stain post –eruptive fracture atypical restoration 55 6 yellowish moderate acceptable 65 3 yellowish moderate acceptable 75 2 yellowish moderate acceptable 85 4 yellowish moderate acceptable 65 5 brown moderate not acceptable 65 1 white light acceptable 75 3 brown light acceptable 85 1 brown moderate not acceptable according to the frequency distribution of the affected teeth, there was no statistically significant difference between them, based on the chi-square test (p> 0.05). the t test was performed to compare the mean breastfeeding time of g1 and cg, and it was possible to verify that there was a statistically significant difference (t = 2.66; p = 0.01), with higher averages of time for the cg (13.3 months) when compared to g1 (10.5 months). as for age, there was no difference between groups (p> 0.05). finally, it is highlighted that 100% of children in g1 have tooth sensitivity. in sequence, it was possible to observe, from figure 4, the difference in the frequency of hspm between gender and children’s school. from the data and the statistical calculation of the chi-square, it was observed that in the children of the cg there were no differences, however in the children of g1 there was a difference regarding gender (x2 = 4.57; p <0.05), having boys showed a higher prevalence. table 2 shows the distribution of the predisposing factors for children. figure 3. comparison between g1 and gc regarding gender and school. 55 e 65 75 e 85 65 43% 21% 36% 7 lira et al. table 2. distribution of the predisposing factors for children from cg and g1. predisposing factors/child cg g1 total x2 p valor breathing problem at birth no 319 14 333 1.18 yes 27 0 27 0.28 neonatal hospitalization no 280 11 291 0.05 yes 66 3 69 0.83 premature baby no 334 14 348 0.51 yes 12 0 12 0.48 birth weight < 2.5kg 102 244 346 0.42 > 2.5kg 3 11 14 0.52 need for incubator no 313 14 327 1.47 yes 33 0 33 0.23 born with jaundice/bluish no 268 11 279 0.10 yes 78 3 81 0.92 illness in the first year of life no 111 0 111 6.49 yes 235 14 249 0.01* high fever often in the first year of life no 331 14 345 0.63 yes 15 0 15 0.42 antibiotic use in the first year of life no 267 0 267 41.82 yes 79 14 93 0.01* tooth sensitivity no 345 0 345 335.03* yes 1 14 15 0.01* foot note: *p < 0,05 figure 4. difference in the frequency of hspm between gender and children’s school. 200 180 160 140 120 100 80 60 40 20 0 6 8 11 3 171 175 190 156 g1 6 8 11 3 cg 171 175 190 156 a1 a2 male female 8 lira et al. based on table 2, with the frequency data, followed by the chi-square test, it was observed in g1 that there was an association between the presence of hspm and the fact that the child had some disease in the first year of life (p = 0 , 01), as well as, with the use of some antibiotic (p = 0.01) and sensitivity in the teeth affected by the lesion (p = 0.01). table 3 shows the predisposing factors for hspm for mothers. table 3. distribution of predisposing factors for mothers of children in g1 and cg. predisposing factors/mother cg g1 total x2 p valor hypertension no 312 9 321 9.36 yes 34 5 39 0.01* diabetes no 319 14 333 1.18 yes 27 0 27 0.57 infections during pregnancy no 337 11 348 14.80 yes 9 3 12 0.01* smoking during pregnancy no 337 14 351 0.37 yes 9 0 9 0.54 alcohol consumption during pregnancy no 343 8 351 97.33 yes 3 6 9 0.01* use of medication during pregnancy no 313 11 324 2.11 yes 33 3 36 0.15 type of childbirth normal 277 14 291 3.54 cesarean 69 0 69 0.06 complications during childbirth no 322 14 326 1.04 yes 24 0 24 0.31 premature childbirth no 334 14 348 0.50 yes 12 0 12 0.48 foot note: *p < 0,05 from table 3, with the frequency data, followed by the chi-square test, it was possible to observe an association between the presence of hspm in g1 and the fact that the mother was hypertensive (p = 0.01). that is, in five of the 14 children in g1, the mothers were hypertensive. in the same line of analysis, an association was observed between g1 and mothers who were affected by some infection during pregnancy (n = 3) (p = 0.01) and those who consumed alcohol during pregnancy (n = 6) (p = 0.01). discussion regarding the prevalence of 3.9% of hspm in the present study, similar results were observed by some authors8,12 whose prevalence was 2.9%. other authors13-16 found varying prevalences, since they are in accordance with population characteristics, methodological differences between studies, sample size and children’s age range. 9 lira et al. according to elfrink et al.12 (2012) and aine et al.17 (2000) there is a strong correlation between hspm and mih. in this research, most of the children (n=6) had their upper deciduous second molars affected. one study evaluated the prevalence and association between hmi and hspm and primary canines in brazilian schoolchildren. it was found a prevalence for second molars of 6.48% and for deciduous canines 2.22%. it is worth mentioning that according to sé et al.18 (2017), children with hypomineralized second molars and primary canines are six times more likely to develop imh. in view of this fact, the 14 children who presented hspm should be monitored until the eruption of the first molars and permanent incisors because they are highly likely to present hmi, since the enamel mineralization phase of these teeth occurs in the same period15,19. in this study, it was observed that in children in g1 there was a significant difference in terms of gender (x2 = 4.57; p <0.05), with boys having a higher prevalence of hspm. disagreeing with the studies by kemoli20 (2008) and jeremias et al.21 (2013) whose frequency was higher in females, 76% and 62% respectively, and by subramaniam et al.22 (2016) who did not find a gender predilection. the t test was performed to compare the average breastfeeding time between g1 and cg, verifying that there was a statistically significant difference (t = 2.66; p = 0.01), with higher averages of time for cg (13,28 months) when compared to g1 (10.50 months). paradoxically, research carried out by some authors23,24 suggested that children who had been exposed to dioxin through prolonged breastfeeding had a higher risk of developing hypomineralization. in the survey, it was highlighted that 100% of children with hypomineralization had stained teeth with demarcated opacity, tooth wear with loss of enamel, increased susceptibility to caries, since all teeth were restored and tooth hypersensitivity. according to the literature, hypomineralization has a greater impact on the quality of life of affected children, whether due to pain, sensitivity to thermal stimuli or the aesthetic appearance of opacities in teeth25-27. in this research it was possible to observe that the presence of hypomineralization was associated with the fact that the child had had a disease in the first year of life. according to elfrik et al.8(2015), the first year of life is a critical period for the formation of the crown of permanent molars and incisors, and thus for the development of hmi. infectious conditions with repeated episodes of high fever can be considered as risk factors for the development of hypomineralization, as well as prenatal complications, premature birth, childhood illnesses accompanied by high fever in the first three years of life, as shown by our search. the current study showed that all children in g1 used some antibiotic, the most frequent of which were amoxicillin and azithromycin. following the same line, faustino-silva et al.28(2020) in their research showed that there was an association between children with dental enamel defects (ded) and those who used antibiotics, with amoxicillin being the most commonly prescribed medication. a similar fact was observed by wuollet et al.27 (2016) and lopes-fatturi et al.29 (2019) when they found that children with hypomineralization sought more care for infectious dis10 lira et al. eases, received more penicillin in the first year of life, or amoxicillin until the 3 years, than children without hypomineralization. it was observed in this research the association between hypomineralization and the fact that the mother had hypertension, some infection, or consumed alcohol during pregnancy. corroborating the study by lopes-fatturi et al.29 (2019) when they observed that smoking use, presence of hypertension, complications during childbirth are associated with a higher prevalence of hypomineralized deciduous second molars (hspm). the causes of hypomineralization in teeth have not been fully clarified since there are several hypotheses such as diseases of the mother during pregnancy, premature birth, systemic diseases (mainly respiratory), exposure to products and medications that are considered to be risk factors7,30. however, according to the literature, hypomineralization is defined as a qualitative enamel defect caused by a disturbance during initial calcification and / or during maturation, being an important risk factor for caries in hypomineralized deciduous and permanent molars and that the presence of hspm can be a precursor to mih, and the need for monitoring and controlling injuries is essential. it is worth mentioning that the absence of this defect in the primary molars does not exclude the appearance of mih7,31. in conclusion, the prevalence of hspm in children between 2 and 6 years old was 3.9%, with predominance in males, with tooth 65 being the most affected. hspm presented as demarcated opacity, with the most prevalent characteristics being the type of yellowish spot, with moderate post-eruptive fracture and acceptable atypical restorations. there was an association between hspm and infection in the first year of life, as well as the use of antibiotics and sensitivity in the teeth affected by the lesion. there was an association between hspm and hypertension, infection and mothers’ alcohol use during pregnancy. references 1. weerheijm kl, duggal m, mejare i, papagiannoulis l, koch g, martens lc, et al. judgement criteria for molar incisor hypomineralisation (mih) in epidemiologic studies: a summary of the european meeting on mih held in athens, 2003. eur j paediatr dent. 2003;4(3)110-3. 2. jalevik b. 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teeth. environ toxicol pharmacol. 1996 may;1(3):193-7. doi:10.1016/1382-6689(96)00007-5. 12 lira et al. 24. garot e, rouas p, d’incau e, lenoir n, manton d, couture c. mineral dentisty of hypomineralized and sound enamel. bull group int sci stomatol odontol. 2016;53(1):33-6 25. oyedele ta, folayan mo, oziegbe eo. hypomineralized second primary molars: prevalence, pattern and associated co-morbidities in 8to 10-year-old children in ile-ife, nigeria. bmc oral health. 2016;16(1):65. doi:10.1186/s12903-016-0225-9. 26. buchgraber b, kqiku l, ebeleseder ka. molar incisor hypomineralization: proportion and severity in primary public school children in graz, austria. clin. oral investig. berlin. 2017 mar;22(2):757-62. doi: 10.1007/s00784-017-2150-y. 27. wuollet e, laisi s, salmela e, ess a, alaluusua s. molar-incisor hypomineralization and the association with childhood illnesses and antibiotics in a group of finnish children. acta odontol scand. 2016 may;74(5):1-7. doi: 10.3109/00016357.2016.1172342. 28. faustino-silva dd, rocha af, da rocha bs, stein c. use of antibiotics in early childhood and dental enamel defects in 6to 12-year-old children in primary health care. acta odontol latinoam. 2020;33(1):6-13. 29. lopes-fatturi a, menezes jvnb, fraiz fc, assunção lrds, de souza jf. systemic exposures associated with hypomineralized primary second molars. pediatr dent. 2019;41(5):364-70. 30. bullio fragelli cm, jeremias f, feltrin de souza j, paschoal ma, de cássia loiola cordeiro r, santos-pinto l. longitudinal evaluation of the structural integrity of teeth affected by molar incisor hypomineralization. caries res. 2015;49(4):378-83. doi: 10.1159/000380858. 31. temilola od, folayan mo, oyedele t. the prevalence and pattern of deciduous molar hypomineralization and molar-incisor hypomineralization in children from a suburban population in nigeria. bmc oral health. 2015 jun;15(1):73-9. doi: 10.1186/s12903-015-0059-x. 1 volume 22 2023 e238727 original article braz j oral sci. 2023;22:e238727http://dx.doi.org/10.20396/bjos.v22i00.8668727 1 herminio ometto university center, school of dentistry, ararassp, brazil. 2 university of campinas, piracicaba school of dentistry, graduate program in clinical dentistry, piracicaba-sp, brazil. corresponding author: renata siqueira scatolin herminio ometto university center, school of dentistry, araras sp, brazil dr. maximiliano baruto avenue, 500 – university garden, araras sp, 13607-339. re_scatolin@hotmail.com editor: dr. altair a. del bel cury received: march 24, 2022 accepted: september 6, 2022 bond strength of bulk fill composite to teeth prepared with er :yag laser marcos roberto de lima benati1 , jean carlos baioni1 , amanda guerra cavalcante de souza2 , laura nobre ferraz1 , ana luisa botta martins de oliveira1 , rafael pino vitti1 , renata siqueira scatolin1,* aim: the present in vitro study aimed to evaluate the bond strength of a bulk fill composite on dentin surfaces prepared with the er: yag laser. methods: twenty-four permanent third molars were selected and divided into 2 groups: cp conventional preparation with high-speed handpiece (control) and la (laser) preparation with er: yag laser. the occlusal surface was removed to expose coronal dentin, which was subsequently prepared with a high-speed handpiece or er: yag laser (350mj, 4hz, 1.5 ml/min water flow). both groups were restored with filtek one bulk fill (3m espe) composite resin. after 24 hours, the samples were evaluated for microtensile bond strength (µtbs), fracture pattern, and scanning electron microscopy (sem). results: the data obtained in the µtbs test were submitted to t-test (α=0.05). the results showed no difference in µtbs when the different types of cavity preparation were compared (ρ=0.091). fracture patterns revealed the prevalence of cohesive fracture in composite resin in cp (83.3%) and adhesive fracture in la (92.1%). in the sem analysis, the la group demonstrated the presence of gaps between the composite resin and the irradiated dentin surface. the hybrid layer exhibited more regularity with the presence of longer and uniform resin tags in the cp group. conclusion: the type of cavity preparation did not influence the values of bulk fill composite resin µtbs to dentin. fracture patterns and scanning electron microscopy analyses suggested less interference at the adhesive interface in preparations performed using cp. keywords: composite resins. dental materials. lasers. adhesiveness. https://orcid.org/0000-0002-6289-8114 https://orcid.org/0000-0003-2294-1883 https://orcid.org/0000-0002-9374-8346 https://orcid.org/0000-0002-6234-8913 https://orcid.org/0000-0001-8412-5588 https://orcid.org/0000-0001-6366-5868 https://orcid.org/0000-0003-4890-8096 2 benati et al. braz j oral sci. 2023;22:e238727 introduction composite resins are popular in dental clinics, due to their characteristics of mimicking dental structures, and their high physical1 and mechanical2 properties. however, despite all the technology and research applied to the development of composites, the knowledge about the tissues to which they must adhere, and improvement in restorative materials and techniques must be constantly evaluated3. the search for faster and simpler clinical procedures, along with the attempt to reduce polymerization shrinkage, led to a new class of resin composites, known as bulk fill, which have gained increasing visibility in the market4. whereas conventional composites are typically inserted in increments with a maximum thickness of two millimeters5, the bulk fill resins allow a reduction in working time by decreasing the number of increments in the cavity to be restored, as it allows layers of up to four millimeters to be effectively polymerized4,6. bulk fill composite can be inserted in a single increment since changes have been made either in the filler content or in their organic matrix. these approaches can result in lower viscosity monomers by the substitution or reduction of bis-gma and/or monomers with higher molecular weight, commonly based on tegdma, ebpdma, bis-ema, and udma monomers. as a result, a more translucent material, with improved healing capacity and consequent decrease in polymerization shrinkage was obtained6,7. the type of composite resin and the filling technique can have a great impact on the resin composite bond to the tooth structure8. however, these are not the only factors to be considered. preparation of the surface to be bonded is important for attaining clinical success and restoration durability9. the adhesion mechanism of the restorative material and dentin surface occurs through the interlock between the polymerized monomers and partially demineralized collagen fibrils10. the treatment of the dentin surface with er: yag laser is among the new methods studied. this is an alternative to the use of conventional rotary instruments, making it possible to eliminate noise during cavity preparation, decrease the pain sensation and perform a more conservative preparation11-14. er: yag laser is effective in ablating mineralized tissues because it emits a wavelength of 2.94 µm, which coincides with the absorption peaks of water and hydroxyapatite present in dental tissues. with the vaporization of water, there is an increase in the internal pressure of the molecules, generating micro explosions that lead to the eruption of the substrate in the form of microscopic particles, resulting in a micro-retentive pattern15,16. thus, the changes in dentin morphology, resulting from the use of the er: yag laser, can promote a larger adhesive area because of surface micro retentions, opening of dentinal tubules, and absence of smear layer. this can influence the bonding quality of restorative materials when compared to cavity preparations performed with diamond burs used in high-speed handpiece17-20. thus, this study aimed to evaluate the microtensile bond strength (µtbs) of an adhesive protocol associating the characteristics of an er: yag laser-prepared den3 benati et al. braz j oral sci. 2023;22:e238727 tin surface and the advantages of bulk fill composite and the develop a faster restorative procedure with less polymerization shrinkage. the null hypothesis would be that the er: yag laser does not promote differences in µtbs than the conventional protocol using diamond bur. materials and methods experimental design this is a randomized study and the sample consisted of 30 dentin blocks, of which 24 blocks were used in the bond strength test (n= 12) and 6 in the morphological analysis (sem) of the adhesive interface between the bulk fill composite and the dentin surface (n= 3). the groups were determined as follows: cpconventional preparation using high-speed handpiece (control) and la preparation with er: yag laser. the quantitative variable response was µtbs (mpa). qualitative analysis of fracture patterns (adhesive, cohesive or mixed) and scanning electron microscopy (sem) were also performed to assess the adhesive interface. specimens preparation after approval by the ethics committee (caae: 25790619.7.0000.5385), 30 healthy third molars (absence of carious lesions or fractures) were selected and cleaned with periodontal curettes (millenium, golgran, são caetano do sul, sp, brazil), pumice paste/water (sswhite produtos odontológico, são cristovão, rj, brazil) and rubber cups (soft, american burrs, palhoça, sc, brazil). the teeth were stored in distilled water and kept in an oven at 37°c until the beginning of the experiment. the clinical crown was horizontally sectioned with the aid of a diamond-cutting disc mounted on a cooled cutting machine (isomet 1000, model 11-2180) to remove 1/3 of the occlusal surface, thereby obtaining exposure to all the dentin. subsequently, the coronal dentin was flattened with abrasive papers #600 and #1200, using a metallographic polisher (dp-9u2; struers s/a, copenhagen, denmark)21. preparation technique for samples that received er: yag laser preparation (kavo key laser ii – kavocorp. biberach, germany) the non-contact mode was used, perpendicularly to the surface and focused at a distance of 12 mm from the sample22 with scanning of the entire surface. this laser has a fiber diameter of 0.63mm; irradiation was performed using the energy of 350mj and frequency of 4hz17, with a constant flow of 1.5 ml/min of water23. for the samples that received cavity preparation with a high-speed handpiece (dabi atlante, ribeirão preto, brazil), a diamond tip #2096 (kg sorensen, alphaville, sp, brazil) was used, perpendicularly to the surface, under constant cooling with distilled water23 and treating the entire dentin surface. restorative procedure immediately after performing the cavity preparation (conventional with high-speed handpiece or er: yag laser), the samples in all groups received the restorative material. 4 benati et al. braz j oral sci. 2023;22:e238727 35% phosphoric acid was applied (3m, espe st. paul, mn, usa) for 15 seconds on the dentin surface, followed by washing with distilled water for 30 seconds and drying with absorbent paper. subsequently, with the aid of a disposable brush, two layers of the single bond 2 adhesive system (3m, espe st. paul, mn, usa) were applied with gentle jets of air between them to allow volatilization of the solvent. the adhesive system was light-cured for 10 seconds as recommended by the manufacturer (radii-cal sdi, victoria, australia). dentin surfaces were restored with filtek one bulk fill resin (3m espe st. paul, mn, usa), by the single increment technique, in a single layer 4mm high. the polymerization was carried out for 40 seconds, at a distance of 1 cm. the samples were kept in distilled water and stored in an oven at 37°c for 24 hours for performing the µtbs test. microtensile test after storage, the specimens (n=12) were placed in the water-cooled diamond saw and sectioned to obtain four sticks of each tooth, measuring approximately 1.0mm2 (+-0.2mm2). the sticks were measured, identified and fixe,d in the device used for the µtbs test by using cyanoacrylate gel glue (super bonder gel, henkel ltda., são paulo, sp, brazil). then, the µtbs test was performed in a universal testing machine (ez test shimadzu, tokyo, japan) at a speed of 0.5 mm/min until failure occurred. the values obtained were recorded in newton (n). the average of each tooth (four sticks) was calculated and, finally, the mean values were from each tooth were obtained. the dimension of the fractured area was recorded with a digital caliper (king tools 150mm/6”, são paulo, sp, brazil) and subsequently, the microtensile bond strength values were converted into megapascal (mpa)21. fracture pattern analysis after the specimen ruptured, the surfaces were evaluated with the aid of a clinical microscope (model all 03 el. commercial alliance of são carlos ltda. me, são carlos, sp, brazil) to identify the type of fracture. specimens were evaluated at 16x magnification. failures were classified as adhesive (fracture between the substrate/restorative material interface), cohesive in dentin (fracture in dentin), cohesive in resin (fracture in the restorative material) or mixed (combined adhesive and cohesive fracture). scanning electron microscopy of bond interface three specimens were prepared for each group, following the same preparation and restoration protocols performed for the microtensile strength test. after the restoration, the specimens were sectioned longitudinally with a double-faced diamond disk, finished with water abrasives paper with decreasing grain (#600 and 1200) on the inner portion of the adhesive interface and polished with pastes containing aluminum in suspension, with a granulation of 0.3 µm (arotec, cotia, sp, brazil). the specimens were washed in an ultrasound bath for 10 minutes to remove possible residues on the surface. 5 benati et al. braz j oral sci. 2023;22:e238727 after obtaining the specimens, they were immersed for 12 hours, in a glutaraldehyde solution (2.5%) in 0.1m sodium cacodylate buffer, ph7.4. after this period, the specimens were washed with distilled water. the sections were then dehydrated with ascending grades of ethanol: 25% (20 min), 50% (20 min), 75% (20 min), 95% (30 min) and 100% (60 min). later, they were fixed on metal stubs, sputter coated with gold and analyzed by scanning electron microscopy (evo 50; carl zeiss, cambridge, england). representative areas were photographed at 1500x, aiming to verify the quality of hybrid layer restoration, the presence of irregularities and gaps21. data analysis the data were assessed for normality with the shapiro-wilk test. the results were submitted to t-test at the significance level of α=0.05. all statistical analyses were carried out using spss for windows software (ibm spss statistics 21). results the results showed no difference in µtbs between the different types of cavity preparation (ρ=0.091) (table 1). table 1. mean (±sd) of µtbs values (mpa) of the specimens that received preparations performed with high-speed handpiece or er: yag laser groups µtbs cp (conventional preparation) 27.81 (7.68) la (er:yag laser) 34.68 (12.66) relative to fracture patterns, the prevalence of cohesive fracture in composite resin in cp (83.3%) and adhesive fracture in la (92.1%) were observed (graph 1). 0% 20% 40% 60% 80% 100% 120% adhesive cohesive in resin group cp group la graph 1. percentage of fracture patterns for each group studied 6 benati et al. braz j oral sci. 2023;22:e238727 scanning electron microscopy analysis of the bond interface of group la demonstrated the formation of irregular hybrid layer, with presence of cracks between the resin composite and irradiated dentin surface, with accumulation of adhesive in the regions of valleys and a thin layer in the regions of peaks. short and small quantity of resin tags were observed. in group cp, formation of regular hybrid layer was observed, with presence of numerous, longer and uniform resin tags (figure 1). a b c d 10 µm eht = 20.00 kv mag = 1.50 kx detector = se1 10 µm eht = 20.00 kv mag = 1.50 kx detector = se1 10 µm eht = 20.00 kv mag = 1.50 kx detector = se1 10 µm eht = 20.00 kv mag = 1.50 kx detector = se1 figure 1. images representative of scanning electron microscopy in the different groups. a and b represent samples of group cp (conventional preparation). c and d represent samples of group la (er: yag laser). discussion an effective bond between the restorative material and the tooth structure is an essential factor for successful procedures in restorative dentistry. a failure in the adhesive protocol can damage tooth/restoration interface, which may lead to marginal discolorations, recurrence of caries lesions, postoperative hypersensitivity and other harmful impacts on the pulp24. the null hypothesis was accepted since the results showed that there was no significant difference in the µtbs of bulk fill composite to surfaces conventionally prepared 7 benati et al. braz j oral sci. 2023;22:e238727 with high-speed handpiece or with er: yag laser. the results obtained in this in vitro study suggest that the adhesive protocol is efficient and can be a good alternative for restorative procedures cases like in pediatric dentistry, geriatric dentistry, and special patients by reducing clinical time and facilitating care. bulk fill composite resin is still a new material on the market and is being scientifically tested6,25,26. as for the adhesion to dentin substrate, regardless of the adhesive used25 and the type of cavity27,28, its bond strength showed high µtbs values, validating the results found in the cp group. no studies in the literature have evaluated the bond strength between bulk fill composite and er: yag laser-treated dentin. however, a study reported by tekce et al.29 (2018) evaluated, qualitatively and quantitatively, the microleakage of bulk fill in enamel prepared with diamond bur or laser through sem analysis and like in the la group of this study, also found irregular hybrid layer and gaps at the adhesive interface, even though it was statistically similar to the conventional preparation. the adhesive system used in this study was the conventional two-step system (single bond 2 3m) and the µtbs results found did not corroborate the study by ramos et al.30 (2014), which states that the self-etching adhesive system showed better results than the etch-and-rinse for er: yag laser preparation. future studies can be developed to evaluate different adhesives systems under these same conditions, however the results found in a literature review by lopes et al.31 (2015) are contradictory, because there is no defined standard protocol and laser data vary from the type of adhesive, parameters, restorative material and even the bond strength methodology used. the laser parameters used in this study were based on the study by corona et al.17 (2007) who used 350 mj energy and a pulse repetition rate of 4 hz, thereby achieving greater depth of the ablation and slight dentinal tubules enlargement when compared with smaller parameters. as regards water flow, the samples were irradiated with 1.5 ml/min of water. this factor is extremely important during surface preparation, as the laser produces less thermal injury to the pulp when it is associated with water-cooling32,33. in this study, sem analysis of the bond interface showed an irregular hybrid layer, with the presence of cracks and gap formation between the composite resin and the dentin irradiated with er: yag laser, suggesting changes in collagen fibrils and reaffirming the studies by he et al.34 (2017) and aranha et al.35 (2007). the irregular hybrid layer could have occurred due to the micro retentive surface created by laser irradiation, with the accumulation of adhesive in regions called valleys, where higher pulse energy was used, and a thinner layer of adhesive filling the regions called peaks, as reported in the literature21,36. short and small amounts of resin tags were observed in the group irradiated with the laser, differing from the studies by aranha et al.35 (2007) and galafassi et al.21 (2014). as the parameters they used were lower, there may have been less potential for ablation of the dentin structure and less influence on the collagen content. as regards the preparations performed with high-speed handpiece, sem revealed the formation of a regular hybrid layer, with the presence of numerous, longer and more even resin tags. 8 benati et al. braz j oral sci. 2023;22:e238727 although no statistically significant difference values were observed in the microtensile bond strength test, when the fracture patterns were evaluated, it was possible to note a difference between the groups. the prevalence of adhesive fractures found in the la group corroborated the findings in the study by comba et al.37 (2019) who used a conventional resin composite. irrespective of the adhesive protocol, they observed the same fracture pattern predominance, suggesting that failure in hybrid layer formation might have negatively affected bonding. a possible explanation for these adhesive bond failures was reported in a study that evaluated the chemical and mechanical modifications of dentin irradiated with er: yag laser. he et al.34 (2017) observed that irradiation negatively affected the nanomechanical properties in the subsurface layer of dentin (< 15 µm in depth), with a decrease in organic and mineral components and a higher degree of crystallinity, due to phosphate and carbonate ions being recrystallized during irradiation with the laser. in addition, they observed changes in collagen content, with denaturation of fibrils and a consequent reduction in the interfibrillar space, which limits composite resin diffusion, causing poor hybridization and affecting its bond to the dentin structure. despite the limitations of this study, the restorative technique and surface preparation method chosen can directly influence the bond quality9,19. further research showing the longevity of these restorations is needed, in order to find out whether the interferences found in this study will harm long-term bond strength. furthermore, investigations into the interaction of bulk fill resin composites on dentin surfaces prepared with different parameters of er: yag laser are also of fundamental importance, with the aim of suggesting an effective protocol for performing direct restorations in posterior teeth, with less invasive preparations and use of restorative materials in procedures that require less time to perform. in conclusion, the type of cavity preparation did not influence the values of bulk fill composite µtbs to dentin, but fracture patterns and scanning electron microscopy analysis suggested less interference at the adhesive interface in preparations performed at high-speed handpiece. acknowledgments this work was supported by the são paulo research foundation 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10.1038/sj.bdj.2008.491. 34. he z, chen l, hu x, shimada y, otsuki m, tagami j, et al. mechanical properties and molecular structure analysis of subsurface dentin after er:yag laser irradiation. j mech behav biomed mater. 2017 oct;74:274-82. doi: 10.1016/j.jmbbm.2017.05.036. 11 benati et al. braz j oral sci. 2023;22:e238727 35. aranha ac, de paula eduardo c, gutknecht n, marques mm, ramalho km, apel c. analysis of the interfacial micromorphology of adhesive systems in cavities prepared with er,cr:ysgg, er:yag laser and bur. microsc res tech. 2007 aug;70(8):745-51. doi: 10.1002/jemt.20459. 36. moretto sg, azambuja n jr, arana-chavez ve, reis af, giannini m, eduardo cde p, et al. effects of ultramorphological changes on adhesion to lased dentin-scanning electron microscopy and transmission electron microscopy analysis. microsc res tech. 2011 aug;74(8):720-6. doi: 10.1002/jemt.20949. 37. comba a, baldi a, michelotto tempesta r, cedrone a, carpegna g, et al. effect of er:yag and burs on coronal dentin bond strength stability. j adhes dent. 2019;21(4):329-35. doi: 10.3290/j.jad.a42932. 1http://dx.doi.org/10.20396/bjos.v20i00.8659912 volume 20 2021 e219912 original article 1 department of oral and maxillofacial surgery, school of dentistry, university of pernambuco, recife, pernambuco, brazil. *corresponding author: ricardo josé de holanda vasconcellos email: ricardo.holanda@upe.br received for publication: june 9, 2020 accepted: december 18, 2020 feasibility of three-dimensional ct reconstruction in assessing mandibular fractures: a survey among oral surgeons and dental maxillofacial radiologists victor nogueira moura1 , emerson nogueira1 , ewerton daniel rocha rodrigues1,* , caio césar gonçalves silva1 , ricardo josé de holanda vasconcellos1 aim: evaluation of the reliability of 3d computed tomography (3d-ct) in the diagnosis of mandibular fractures. methods: a cross-sectional, quantitative and qualitative study was carried out, through the application of a questionnaire for 70 professionals in the area of oral and maxillofacial surgery and radiology. 3d-ct images of mandibular fractures were delivered to the interviewees along with a questionnaire. participants answered about the number of traces, the region and the type of fracture. the correct diagnosis, that is, the expected answer, was based on the reports of a specialist in oral and maxillofacial radiology after viewing the images in the axial, sagittal and coronal sections. the resulting data from the interviewees was compared with the expected answer and then, the data was analyzed statistically. results: in the sample 56.9% were between 22 and 30 years old, 52.8% were oral and maxillofacial surgeons (omf), 34.7% were residents in omf surgery and 12.5% omf radiologists. each professional answered 15 questions (related to five patients) and 50.8% of the total of these was answered correctly. specialists in oral and maxillofacial surgery and traumatology correctly answered 53.9%. interviewees with experience between 6 and 10 years correctly answered 58.2%. in identifying fracture traces, 46.1% of the questions were answered correctly. in terms of location, 5.6% of interviewees answered wrongly while 14.2% answered wrongly regarding classification. conclusion: 3d computed tomography did http://dx.doi.org/10.20396/bjos.v20i00.8659912 https://orcid.org/0000-0001-7569-7955 https://orcid.org/0000-0002-4560-7733 https://orcid.org/0000-0003-1969-8288 https://orcid.org/0000-0002-7519-7894 https://orcid.org/0000-0002-7934-5743 2 moura et al. not prove to be a reliable image for diagnosing mandibular fractures when used alone. this made necessary an association with axial, sagittal and coronal tomographic sections. keywords: tomography, x-ray computed. imaging, three-dimensional. diagnostic imaging. mandibular fractures. introduction in recent years, computed tomography (ct) has enabled better accuracy assessment of face fractures. it boosted and made imaging examination to present the best details, enabling the surgeon to view structures in three dimensions1,2. fracture traces, location, extension and displacement of fragments are evaluated in sections without image overlap1. two-dimensionally (2d) or three-dimensional (3d) reconstructed images can be obtained from original ct data, which allow indirect reconstructions in any desired plane3. 3d tomographic reconstructions have the advantage of helping communication between professionals, and between professionals and patients, however, according to some authors, they are not reliable in the diagnosis of facial fractures, especially those involving the middle third of the face, due to overlapping of images, artifacts, and the limitation in viewing details4. some studies3–5 suggest the utility of three-dimensional computed tomography technologies for preoperative and intraoperative decision-making, but it has not yet been determined to which level three-dimensional computed tomography (3d-ct) is a useful tool for diagnosing the maxillofacial trauma. although some professionals have high confidence in 3d-ct in the diagnosis of some facial fractures. therefore, the objective of this research was to assess the feasibility of 3d-ct reconstructed images to diagnose mandibular fractures and related factors. materials and methods a cross-sectional, quantitative and qualitative study was carried out through the application of a printed questionnaire. this questionnaire was developed specifically for this study by 2 maxillofacial surgeons professors of the discipline of surgery at the university of pernambuco. the questions asked were reviewed by the radiologist responsible for preparing the answers. all interviewees were informed about the content of the research and signed an informed consent form. the research started after approval by the research ethics committee of universidade de pernambuco (upe), with the caae registration: 91652217.3.0000.5207. residents, oral and maxilofacial surgeons, as well as oral and maxillofacial radiologists working in the state of pernambuco (brazil) participated in the research. corresponding to a total of 70 participants. the inclusion criteria were: 1) be oral and maxillofacial (omf) surgeon, omf resident, omf radiologist and; 2) be active in their professional área. participants who did not answer the questionnaire completely were excluded. sampling was through spontaneous demand from professionals who agreed to participate in this study. 3 moura et al. the examinations were performed on young patients (ages ranging from 20 to 36  years) who were victims of facial trauma (motorcycle accident, car accident and physical aggression) treated at a trauma hospital. computed tomography (ct) was performed using he somatom definition as, siemens (34 chanels). the slice thickness used in this study was 3 mm, the pitch was 1.2 and scan area diameter was 30 cm. ten clinical cases of mandible fracture were randomly selected the excell’s randbetween function. from these cases, the printing of 6 images in 3d reconstruction was standardized (frontal, axial, ¾ right, ¾ left, right profile, left profile) (figure 1). the images were printed on photographic paper at 29.7 x 42.2 cm. a c e b d f figure 1. tomography of a patient in 3d reconstruction in: a) frontal view, b) axial, c) ¾ right, d) ¾ left, e) right profile and f) left profile of one of the evaluated clinical cases. each participant received a questionnaire containing 3d reconstructed tomographic images representative of five random clinical cases. for each case, the volunteer should have answered the following questions. the questionnaires were randomized using the random.org program, and applied in hospitals and radiological clinics, where each interviewee had 30 minutes to answer the questions. 4 moura et al. the questions were: a. how many fracture traces can you see in the image? (1, 2, 3, 4, 5 or more than 5) b. at which region the fractures are located? (condyle, branch, angle, body, parasymphysis, symphysis, dentoalveolar) c. what is the type of fracture? (simple, complex, incomplete, comminutive, favorable, unfavorable) in each case the second and third questions could have more than one correct answer. a specialist in oral and maxillofacial radiology gave the correct answer to these questions after analyzing the cases, but with the images in coronal, axial and sagittal section. the radiologist answered the same questions ten days ago to assess the intra-examiner efficiency. it was considered as correct the one answered correctly in its entirety; partially correct when the question was answered incompletely correct; incorrect when neither of the alternatives had been filled out correctly or the candidate did not answer. in addition, demographic data such as age, education and time since graduation were collected from the interviewees. residents were asked about time since undergraduation. for specialists, the time since graduation. data was analyzed descriptively using absolute and percentage frequencies for categorical variables and measures: average, standard deviation, median, minimum and maximum values for numerical variables (age and number of questions: correct, partially correct and incorrect). to assess the association between two categorical variables, the pearson’s chi-squared test was used, or the fisher’s exact test when the condition for using the chi-squared test was not verified. mann-whitney test was used to compare the groups for the numerical variables. the choice for the mann-whitney test was due to the absence of normality of data, a condition verified through the shapiro-wilk test. the margin of error used in deciding the statistical tests was 5%. the data was entered into excel spreadsheet and the program used to perform the statistical analysis was ibm spss version 23.0 (ibm corporation, sommers, ny, usa) for windows (microsoft corporation, redmond, wa, usa). results seventy professionals were interviewed and table 1 presents the results of the sample’s demographic profile, 44 male and 26 female. this table highlights that: the average age in the total group was 31.86 years and the median 30.00 years; the age group of 22 to 30 years old was the most prevalent with 56.9% and the remaining 43.1% were 31 years old or more. a little more than half (52.8%) of the participants were omf surgeons, followed by 34.7% who were omf residents and the remaining 12.5% were radiologists. the most prevalent “time since graduation” time corresponded to those who were up to 2 years after graduation (41.4%) and the other time ranges had percentages ranging from 18.6% to 21.4%. 5 moura et al. table 1. demographic profile evaluation. variable total group age: average ± dp (median) 31.86 ± 9.10 (30.00) age range: n (%) (1) 22 to 30 33 (56.9) 31 or more 25 (43.1) total 58 (100.0) specialty: n (%) omf surgeons 38 (52.8) omf residents 25 (34.7) omf radiologists 9 (12.5) formation time (in years): n (%) (2) until 2 29 (41.4) 3 to 5 13 (18.6) 6 to 10 15 (21.4) more than 10 13 (18.6) total 70 (100.0) (1) for 14 respondents age was not informed. (2) for two respondents the time since graduation was not informed. general assessments (that is, regardless of formation) corresponding to the answers given to all questions are shown in table 2. there was an average of 7.63 questions answered in their entirety. for questions with more than one answer, an average of 3.69 partial hits was observed. and there was an average of 3.68 errors in total. table 2. statistics on the number of correct, partially correct and incorrect answers from the 15 questions presented. variable statistics total group correct answers in total average ± dp 7.63 ± 2.80 median 7.00 minimum 3.00 maximum 14.00 partially correct answers in total average ± dp 3.69 ± 1.42 median 4.00 minimum 0.00 maximum 7.00 incorrect answers in total average ± dp 3.68 ± 1.90 median 4.00 minimum 0.00 maximum 8.00 table 3 shows the answers to each question, together with the number of correct answers according to each specialty and formation, while table 4 shows the results based on formation time. 6 moura et al. table 3. evaluation of the total of 15 questions and blocks according to specialty speciality variable specialist resident radiologist total group p value n % n % n % n % total block correct 307 53.9 181 48.3 61 45.2 549 50.8 p(1) = 0.290partially correct 134 23.5 95 25.3 37 27.4 266 24.6 incorrect 129 22.6 99 26.4 37 27.4 265 24.5 questions about fracture trace correct 92 48.4 53 42.4 21 46.7 166 46.1 p(1) = 0.575 incorrect 98 51.6 72 57.6 24 53.3 194 53.9 diagnosis of the fractured region correct 93 48.9 52 41.6 17 37.8 162 45.0 p(1) = 0.541partially correct 87 45.8 65 52.0 26 57.8 178 49.4 incorrect 10 5.3 8 6.4 2 4.4 20 5.6 fracture classification correct 122 64.2 76 60.8 23 51.1 221 61.4 p(1) = 0.215partially correct 47 24.7 30 24.0 11 24.4 88 24.4 incorrect 21 11.1 19 15.2 11 24.4 51 14.2 (*) significant difference at the level of 5.0% (1) through pearson’s chi-square test. table 4. evaluation of the total of 15 questions and blocks according to the time since graduation variable time since graduation p valueuntil 2 3 to 5 6 to 10 more than 10 total group n % n % n % n % n % total block correct 218 50.1 107 54.9 131 58.2 83 42.6 539 51.3 p(1) = 0.048*partially correct 107 24.6 48 24.6 50 22.2 54 27.7 259 24.7 incorrect 110 25.3 40 20.5 44 19.6 58 29.7 252 24.0 questions about fracture trace correct 63 43.4 33 50.8 42 56.0 25 38.5 163 46.6 p(1) = 0.143 incorrect 82 56.6 32 49.2 33 44.0 40 61.5 187 53.4 diagnosis of the fractured region correct 64 44.1 30 46.2 39 52.0 26 40.0 159 45.4 p(2) = 0.971partially correct 73 50.3 32 49.2 32 42.7 36 55.4 173 49.4 incorrect 8 5.5 3 4.6 4 5.3 3 4.6 18 5.1 fracture classification correct 91 62.8 44 67.7 50 66.7 32 49.2 217 62.0 p(1) = 0.137partially correct 34 23.4 16 24.6 18 24.0 18 27.7 86 24.6 incorrect 20 13.8 5 7.7 7 9.3 15 23.1 47 13.4 (*) significant difference at the level of 5.0% (1) through pearson’s chi-square test. in the general assessment, professionals who had between 6 to 10 years since graduation, obtained the highest rate of correct answers with 58.2%, while professionals with more than 10 years of experience obtained the lowest rate, 42.6%. in questions 7 moura et al. about fracture traces, the highest rate of correct answer was in the range of 6 to 10 years since graduation and the lowest was with the group that was over 10 years since graduation, with 56.0% and 38.5% respectively. in the diagnosis of the fractured region, these groups again obtained the highest and lowest rate of correct answers, with 52.0% and 40.0% respectively, however the interviewees with more than 10 years of formation, obtained the highest rate of partial correct answers, with 55.4% against 42.5% of candidates with experience between 6 and 10 years. the lowest error rate for this assessment was among respondents with experience between 3 and 5 years and with those with more than 10 years, with 4.5%. in the classification of fractures, the group with experience between 3 and 5 years was the one that got the highest rate of correct answers (67.7%) while the group with more than 10 years of experience was the one that obtained the lowest rate (49.2%). discussion despite the clinical examination being the most important procedure for the correct diagnosis, radiographic investigation is of great importance, and is consequently important in the elaboration of the treatment plan and in the postoperative follow-up of patients with fractures of the facial bones3,6. the management of facial fractures is based on accurate clinical and radiographic diagnosis3,7. of the 15 questions in our study, an average of 7.63 questions was found to be correct answers in their entirety. that is, there may be a difficulty in interpreting 3d images, leading to possible flaws in the diagnosis and consequently in the surgical planning. the literature says that two-dimensional computed tomography with axial, coronal and sagittal projections is a standard criterion for evaluating facial fractures through images, especially when dealing with orbital fractures8. however, 3d tomography has become a useful tool in the diagnosis of facial fractures5,8. in conventional radiography, anatomy is represented in two dimensions. facial injuries usually produce significant edema, which can make the diagnosis of underlying bone lesions difficult9. fox et al.10 reported in their study that 3d-ct scans were interpreted in shorter time and with greater precision when compared to conventional ct scans, in addition, these scans were more accurate when assessing zygomatic fractures. however, they also noted that these exams were less effective than 2d exam in the evaluation of orbital fractures. these findings are in line with what was observed in our study, in which 3d-ct scans were not interpreted correctly, with a percentage of 53.9% of errors in relation to the number of fractures in the mandible. in our findings, surgery specialists were the group that obtained the highest percentage of correct answers, when compared to residents and radiologists. these findings can be attributed to the fact that specialists usually have more experience in interpreting these exams than the other two groups, since oral radiologists do not use medical tomography frequently. however, it is important to point out the fact that three interviewees (2 surgery specialists and 1 radiologist) were unable to identify any fractures in one of the patients, which could prevent that patient of being treated. this reinforces the importance and sovereignty of the clinical exam in relation to the complementary exams. 8 moura et al. saigal  et  al.4 drew attention to the usefulness of 3d imaging in cases of complex facial trauma. according to these authors, visualization in three dimensions helps the complex process in which the surgeon visualizes operational planning4. this advantage is also pointed out in the clinical trial by shah  et  al.11. perandini,  et  al.12 who reinforce in their articles the importance of 3d-ct in the diagnosis of relatively common conditions such as thrombosis, stenosis of the airways, exophytic cancer and trauma. wolf et al.13 evaluated whether the diagnostic information by 3d imaging had a significant impact on the decision process in six different classes of surgical indications. these authors observed that tomographic images resulted in significantly more relevant surgical information in dental implant and in cases of maxillofacial surgery, however, the 3d image information did not significantly change the surgical plan based on the 2d examination wolf et al.13. in the study by kaeppler et al.14, the authors sought to determine whether 3d tomography would lead to a change in the treatment of patients with suspected jaw fracture with ambiguous conventional radiographic and clinical signs. it was observed that in 63.2% of cases, the suspected diagnosis was confirmed and additional fractures were identified in 17.75% of the evaluated patients14. regarding the location of fractures, interviewees in this study were able to correctly observe most cases and obtained few errors. fox  et  al.10 observed that there is a better understanding of the spatial relationships of fractured elements with 3d, as evidenced by their ability to locate the traces. in our study, radiologists obtained the lowest error rate in the location of the fractures when compared to specialists and residents in the current research. the combination of section thickness, gap between sections and pipe current can influence the quality of the 3d reconstruction. sales  et  al.15 reported that 3d reconstructions are accurate for the detection of injuries and destruction of bone marrow. however, these authors drew attention to the fact that the quality of the ct scan can be affected by several digitalization configurations, they also reported that axial sections of 0.5 mm in width and 0.3 mm reconstruction interval were used to optimize the results16. in compliance with the report by kim et al.17, who observed that thinner sections helped to establish more accurate 3d cranial measurements. during the development of the research, we can observe some limitations. infinite forms of fracture can exist, and we select only a few cases. another difficulty observed was that the interviewees analyzed the printed exams, which makes it difficult to manipulate and observe other tomographic sections. for a better analysis, a larger sample is recommended, and perhaps to analyze the mandibular regions in isolation (only condylar fractures, only symphysis fractures ...). sensitivity and specificity testing would bring a stronger scientific result. in conclusion, imaging exam is an essential part of diagnosis and treatment planning for maxillofacial trauma. although 3d reconstruction provides an important global view of the investigated area, in this research, 3d-ct did not prove to be a reliable image in the visualization of the traces of mandibular fractures when used alone, since only half of the interviewees were able to diagnose them correctly. 9 moura et al. references 1. shintaku wh, venturin js, azevedo b, noujeim m. applications of cone-beam computed tomography in fractures of the maxillofacial complex. dent traumatol. 2009;25(4):358-66. doi: 10.1111/j.16009657.2009.00795.x. 2. bai l, li l, su k, bleyer a, zhang y, ji p. 3d reconstruction images of cone beam computed tomography applied to maxillofacial fractures: a case study and mini review. j xray sci technol. 2018;26(1):115-23. doi: 10.3233/xst-17342. 3. aydin u, gormez o, yildirim d. cone-beam computed tomography imaging of dentoalveolar and mandibular fractures. oral radiol. 2019;(0123456789):23-8. doi: 10.1007/s11282-019-00390-5. 4. saigal k, winokur rs, finden s, taub d, pribitkin e. use of three-dimensional computerized tomography reconstruction in complex facial trauma. facial plast surg. 2005;21(3):214-9. doi: 10.1055/s-2005-922862. 5. jarrahy r, vo v, goenjian ha, tabit cj, katchikian h v, kumar a, et al. diagnostic accuracy of maxillofacial trauma two-dimensional and three-dimensional computed tomographic scans: comparison of oral surgeons, head and neck surgeons, plastic surgeons, and neuroradiologists. plast reconstr surg. 2011;127(6):2432-40. doi: 10.1097/prs.0b013e318213a1fe. 6. nardi c, vignoli c, pietragalla m, tonelli p, calistri l, franchi l, et al. imaging of mandibular fractures: a pictorial review. insights imaging. 2020;11(1). doi: 10.1186/s13244-020-0837-0. 7. manson pn, markowitz b, mirvis s, dunham m, yaremchuk m. toward ct-based facial fracture treatment (letter). plast reconstr surg. 1990 oct;86(4):806. 8. dediol e. the role of three-dimensional computed tomography in evaluating facial trauma. plast reconstr surg 2012;129(2):354-5. doi: 10.1097/prs.0b013e31823aee2f. 9. kaur j, chopra r. three dimensional ct reconstruction for the evaluation and surgical planning of mid face fractures: a 100 case study. j maxillofac oral surg. 2010;9(4):323-8. doi: 10.1007/s12663-010-0137-1. 10. fox la, vannier mw, west oc, wilson aj, baran ga, pilgram tk. diagnostic performance of ct, mpr and 3dct imaging in maxillofacial trauma. comput med imaging graph. 1995;19(5):385-95. doi: 10.1016/0895-6111(95)00022-4. 11. shah s, uppal sk, mittal rk, garg r, saggar k, dhawan r. diagnostic tools in maxillofacial fractures: is there really a need of three-dimensional computed tomography? indian j plast surg. 2016;49(2):225-33. doi: 10.4103/0970-0358.191320. 12. perandini s, faccioli n, zaccarella a, re t, mucelli r. the diagnostic contribution of ct volumetric rendering techniques in routine practice. indian j radiol imaging. 2010;20(2):92-7. doi: 10.4103/0971-3026.63043. 13. wolff c, mücke t, wagenpfeil s, kanatas a, bissinger o, deppe h. do cbct scans alter surgical treatment plans? comparison of preoperative surgical diagnosis using panoramic versus cone-beam ct images. j cranio-maxillofacial surg. 2016;44(10):1700-5. doi: 10.1016/j.jcms.2016.07.025. 14. kaeppler g, cornelius cp, ehrenfeld m, mast g. diagnostic efficacy of cone-beam computed tomography for mandibular fractures. oral surg oral med oral pathol oral radiol. 2013;116(1):98104. doi: 10.1016/j.oooo.2013.04.004. 15. sales mao, gaia bf, perrela a, cavalcanti mgp. comparison between multislice and cone-beam computed tomography for the identification of simulated bone lesions using 3d reconstruction. rev odonto cienc. 2013;28(2):47-52. 16. severo fc, barbosa gf. risk factors and success rates associated with orthodontic mini-implants: a literature review. rev odonto cienc. 2015;30(4):200-4. doi: 10.15448/1980-6523.2015.4.15801. 17. kim do, kim hj, jung h, jeong hk, hong s il, kim kd. quantitative evaluation of acquisition parameters in three-dimensional imaging with multidetector computed tomography using human skull phantom. j digit imaging. 2002;15 suppl 1(4):254-7. doi: 10.1007/s10278-002-5054-5. 1http://dx.doi.org/10.20396/bjos.v20i00.8663400 volume 20 2021 e213400 original article 1 department of dentistry, ponta grossa state university, pr, brazil. 2 private practice, ponta grossa, pr, brazil. 3 department of dentistry, campos gerais higher education center (cescage), ponta grossa – pr, brazil. 4 department of dentistry, ponta grossa state university, pr, brazil. corresponding author: fábio andré santos department of dentistry, ponta grossa state university, pr, brazil; ave. carlos cavalcanti, n.4748, zipcode: 84030-900 ponta grossa, pr, brazil e-mail: fasantos@uepg.br received: november 27, 2020 accepted: january 17, 2021 clinical and behavioral conditions in the oral health of volleyball and soccer athletes: a cross-sectional study jullian josnei de souza1 , juliana squizatto leite1 ,ricardo bahls2 ,rodrigo stanislawczuk grande3,4 ,fabio andre santos4 aim: in this cross-sectional study, we evaluated the oral hygiene habits, oral health conditions, and the perception about the influence of oral health conditions on the physical performance of youth and professional volleyball and soccer athletes. methods: a total of 96 male athletes participated: 48 volleyball players (25 youth and 23 professional players); and 48 soccer players, of whom 22 were youth, and 26 were professional players. we analyzed the oral hygiene and oral health condition (daily toothbrush, flossing, mouthwash, dental plaque, orthodontic treatment, dental/facial trauma, temporomandibular dysfunction, malocclusion, and the athletes’ perception about the influence of oral health conditions on the physical performance (yes or no)). comparisons were made between the youth and professional athletes for each sport (volleyball and soccer). according to each variable, we applied the chi-square, fisher’s exact, and mann-whitney tests. results: for soccer athletes, we found significant differences between youth and professionals for: flossing (p=0.014), orthodontic treatment (p=0.028), dental/facial trauma (p=0.041), and the athletes’ perception about oral health and physical performance (p<0.001). considering the category (youth and professional) regardless of the type of sport, we found significant differences for dental plaque (p=0.024) and dental/facial trauma (p=0.005). according to the sport (volleyball and soccer), independent of the category, we found significant differences for daily brushing, dental/facial trauma (p=0.005), and the athletes’ perception about oral health and physical performance (p=0.006). conclusion: we concluded that the surveyed athletes had good oral health and believed that oral health can influence sports performance. keywords: oral health. athletic performance. sports. mailto:fasantos@uepg.br https://orcid.org/0000-0003-2547-5961 https://orcid.org/0000-0001-7084-7467 https://orcid.org/0000-0003-4874-7157 https://orcid.org/0000-0003-0600-753x https://orcid.org/0000-0003-0347-0270 2 souza et al. introduction oral health has an impact on the general health, well-being, and quality of life of athletes. most of the studies that have evaluated the relationship between oral health and sports have focused on the risk of orofacial trauma and its prevention1-4. however, poor oral health can contribute negatively to the quality of life, increase systemic inflammation, and result in psychosocial impacts such as difficulties with eating, sleeping, and socialization5-7. self-reported evidence from athletes suggests that poor oral health negatively affects their training and performance8. factors that can compromise an athlete’s oral health include orofacial trauma, infections resulting from poor oral hygiene, including caries, and periodontal disease; dental erosion; malocclusion and temporomandibular dysfunction6,9,10. poor oral health affects professional athletes from both developed and developing countries. the causes are related to nutritional challenges (intake of carbohydrates and acid sports drinks); the impairment of host immune response due to dehydration, xerostomia, intensive training; poor health behaviors and knowledge about oral health; and the lack of effective, preventive health promotion/support5,6. the objectives of this study were to evaluate oral hygiene habits, oral health conditions, and the perception about the influence of oral health conditions on the physical performance of youth and professional volleyball and soccer athletes. materials and methods in this cross-sectional study, the total number was 117 male athletes (66 professionals and 51 young athletes) from volleyball (caramuru vôlei, brazilian men’s volleyball super league – season 2020/21) and soccer (operário ferroviário esporte clube, brazilian soccer league serie b – season 2020/21). this study was approved by the research ethics committee (protocol #2.888.375). the athletes were evaluated over a period of 18 months (september 2018 to march 2020); the oral conditions were assessed by anamnesis (main complaint and previous medical and dental history) and extraand intraoral physical examination. two trained examiners (jjs and jsl) performed all anamnesis and the oral physical examinations. the training exercises involved two sections (theoretical and practical training) discussing the parameters with another investigator (fas). the anamnesis consisted of the main complaint and current medical and dental history. we also inquired about the frequency of daily toothbrushing, the use of dental floss and mouthwash, and trauma (dental/facial), as well as the athletes’ perception about the influence of oral health conditions on physical performance, which was evaluated in a dichotomous way (yes or no). dental plaque was assessed dichotomously, considering whether plaque was present (or not) on four tooth sites; the percentage of the positive site was calculated per subject11. we analyzed changes in the temporomandibular joint (tmj), considering the onset, intensity, duration, location of pain and changes over time, relief factors, and treatment 3 souza et al. reports. patients with previous reports of chronic temporomandibular dysfunction (tmd) also underwent behavioral and psychosocial developmental assessment12,13. malocclusion was determined using angle’s molar classification and was classified as class i; class ii, division 1; class ii, division 2; and class iii9. class i athletes did not present malocclusion and class ii and iii athletes were grouped into athletes with malocclusion. the athletes were evaluated if they needed orthodontic treatment or were already being treated. statistical analysis we initially performed a descriptive analysis of the data, presenting the absolute and relative values of the evaluated parameters. comparisons were made between each sport (volleyball and soccer) for youth and professional athletes (independent variables). we applied the chi-square test for the qualitative nominal dependent variables (toothbrushing, flossing, mouthwash, trauma, oral health conditions and sports performance, tmd, malocclusion, and orthodontic treatment). if the lowest expected frequency in any cell was less than 5, we applied the fisher’s exact test. the percentages of dental plaque and age (dependent variables) did not show normal distribution (kolmogorov-smirnov test, p > 0.05), and therefore the statistical analysis was performed using the mann-whitney test. the level of significance was 5% (ibm® spss® 21.0 statistics, ibm corp., armonk, ny, usa). results however, 96 athletes participated in this study (82% of the population): youth volleyball players, n = 25; professional volleyball players, n = 23; youth soccer players, n = 22; and professional soccer players, n = 26. the average age of the athletes was 16.7 ± 0.7 and 25.3 ± 3.3 years for the youth and professional athletes respectively. a number of 21 athletes (17 professionals and 4 young athletes) were not included in the study. the main reasons for the non-inclusion in the study were the non-attendance to dental appointments due to training routine, participation in official competitions, and change of sports team. considering the soccer players (youth and professional), the result for flossing, orthodontic treatment, dental/facial trauma, and athletes’ perception about the influence of oral health on the sports performance showed significant differences (table 1). table 1. oral hygiene habits, oral health conditions and athletes’ perception about the influence of oral health conditions on the physical performance of youth (y) and professional (p) volleyball and soccer athletes (ponta grossa, paraná, brazil, 2018 to 2020). parameters volleyball athletes p value soccer athletes p valuey (n=25) p (n=23) y (n=22) p (n=26) daily toothbrush (%) 0.224ns (††) 0.307ns (††)≤2x/day 0 (0) 2 (9) 3 (14) 7 (27) ≥3x/day 25 (100) 21 (91) 19 (86) 19 (73) flossing (%) 0.157ns (†) 0.014s (†)no 8 (32) 12 (52) 17 (77) 11 (42) yes 17 (68) 11 (48) 5 (23) 15 (58) continue 4 souza et al. according to category (youth and professional), we found significant differences for dental plaque and dental/facial trauma. when we compare the two sports independent of the category, we found significant differences for daily brushing, dental/facial trauma, and the athletes’ perception about the influence of oral health on the sports performance (table 2). mouthwash (%) 0.214ns (†) 0.316ns (†)no 13 (52) 16 (70) 18 (82) 18 (69) yes 12 (48) 7 (30) 4 (18) 8 (31) dental plaque (%) 0.158ns (‡) 0.101ns (‡) mean ± sd 30 ± 23 35 ± 20 26 ± 19 37 ± 23 median (iqr) 21 (11–48) 25 (18–50) 18 (11–43) 42 (14–58) orthodontic treatment (%) 0.102ns (†) 0.028s (†)no 14 (56) 18 (78) 17 (77) 12 (46) yes 11 (44) 5 (22) 5 (23) 14 (54) trauma dental/facial (%) 0.091ns (††) 0.041s (†)no 18 (78) 24 (96) 18 (82) 14 (54) yes 5 (22) 1 (4) 4 (18) 12 (46) tmd (%) 0.487ns (††) 0.106ns (††)no 21 (84) 17 (74) 21 (96) 20 (77) yes 4 (16) 6 (26) 1 (4) 6 (23) malocclusion (%) 0.719ns (††) 0.159ns (†)no 21 (84) 18 (78) 19 (86) 18 (69) yes 4 (16) 5 (22) 3 (14) 8 (31) athletes’ perception about the influence of oral health on the sports performance (%) 0.133ns (†) <0.001s (†)no 4 (16) 8 (35) 18 (82) 7 (27) yes 21 (84) 15 (65) 4 (18) 19 (73) †chi-squared test ††fisher’s exact test ‡mann-whitney test sd. standard deviation iqr. interquartile range s significant ns not significant continuation table 2. frequency of toothbrushing and oral health conditions category: youth (y) and professional (p) athletes, and sport: volleyball (v) and soccer (s). (ponta grossa, paraná, brazil, 2018 to 2020). parameters category p value sport p valuey (n=47) p (n=49) v (n=48) s (n=48) daily toothbrush (%) 0.076ns (†) 0.014s (†)≤2x/day 3 (6) 9 (18) 2 (4) 10 (21) ≥3x/day 44(94) 40 (82) 46 (96) 38 (79) continue 5 souza et al. discussion the daily frequency of toothbrushing was higher for volleyball athletes compared to soccer athletes. youth soccer athletes had the lowest percentage of individuals who reported using dental floss in comparison with professional soccer players. this result may have been due to the fact that becoming a professional soccer player in brazil is often a project which involves the whole of the athlete’s family, possibly to the detriment of the athlete’s formal education14. brazil still has a high level of social inequity; in 2014 the gini index for brazil was 0.518 (0 = no inequality, and 1 = maximum inequality), indicating a high degree of social inequality (ipea, http://www.ipeadata. gov.br). similar situations have been observed in other developing countries, where parents see sports as the best option for young people to escape from poverty15. we should consider that poor socioeconomic conditions such as limited income and lower education levels are associated with poor oral health16. we should consider that flossing (%) 0.540ns (†) 0.102ns (†)no 25 (53) 23 (47) 20 (42) 28 (58) yes 22 (47) 26 (53) 28 (58) 20 (42) mouthwash (%) 0.719ns (†) 0.127ns (†)no 31 (66) 34 (69) 29 (60) 36 (75) yes 16 (34) 15 (31) 19 (40) 12 (25) dental plaque (%) 0.024s (‡) 0.977ns (‡) mean ± sd 28 ± 21 36 ± 22 32 ± 21 32 ± 22 median (iqr) 18 (11–46) 25 (18–57) 23 (14–50) 25 (14–55) orthodontic treatment (%) 0.630ns (†) 0.525ns (†)no 31 (66) 30 (61) 32 (67) 29 (60) yes 16 (34) 19 (39) 16 (33) 19 (40) trauma dental/facial (%) 0.005s (†) 0.015s (†)no 42 (89) 32 (65) 42 (88) 32 (67) yes 5 (11) 17 (35) 6 (12) 16 (33) tmd (%) 0.076ns (†) 0.423ns (†)no 42 (89) 37 (75) 38 (79) 41 (85) yes 5 (11) 12 (25) 10 (21) 7 (15) malocclusion (%) 0.160ns (†) 0.615ns (†)no 40 (85) 36 (73) 39 (81) 37 (77) yes 7 (15) 13 (27) 9 (19) 11 (23) athletes’ perception about the influence of oral health on the sports performance (%) 0.103ns (†) 0.006s (†)no 22 (47) 15 (31) 12 (25) 25 (52) yes 25 (53) 34 (69) 36 (75) 23 (48) †chi-squared test ‡mann-whitney test sd. standard deviation iqr. interquartile range s significant ns not significant continuation 6 souza et al. soccer is a very popular sport in brazil and becomes more attractive for adolescents from low social class14. regarding the issue of flossing, we did not find differences between youth and professional athletes (category) and sport (volleyball and soccer). we observed the same results considering the mouthwash use. these results could be explained by the fact that the athletes are continually monitored by their team’s medical staff. our results showed that professional athletes had a higher percentage (significant difference) of visible dental plaque in comparison with youth athletes. literature shows males aged 15-18 have the worst patterns of oral hygiene, probably due a greater tendency to neglect oral hygiene during the teenage years17. our results can be explained by the intense training routine and psychophysical stress during professional athletes’ competitions7. orthodontic treatment was reported more frequently by the youth volleyball and professional soccer athletes. we found statistical difference considering orthodontic treatment between youth and professional soccer players. the majority of the soccer athletes came from low-income families; consequently, it was only when they reached a professional level that they were able to pay for dental treatment. occlusal problems may be associated with mouth breathing, tmd, digestive problems, as well as contributing to the occurrence of dental trauma3,9, therefore negatively impacting on sport performance10. consequently, orthodontic treatment can contribute to an improvement in sport performance. we observed a normal pattern in relation to tmj, and there was no significant difference between the groups regarding malocclusion. the clinical features of tmd are found in about 25% of the population; they include symptoms such as crackling, muscle and tmj pain, muscle fatigue, opening limitations, and headaches12. stress can be a factor that influences tmj; however, there was no significant difference between the youth athletes and the professional athletes. stress can accentuate and perpetuate pre-existing dysfunction, due to excessive tension in the joints or muscles involved in biting or the grinding of teeth during sports13. the causes of this dysfunction are multifactorial; however, direct trauma to the jaw due to sports is one of the main factors. severe mandibular shock caused during sports may also result in macro-trauma, causing disc deformities and dislocations, as well as ligament distension13. in less aggressive shocks, symptoms may disappear quickly, while more severe injuries can cause permanent changes in function2,8. for this reason, athletes from so-called contact sports, such as hockey, soccer, basketball, and rugby, who commonly suffer shocks and impacts, have a higher incidence of the signs and symptoms of tmd1. our study showed that the professional athletes reported having had more orofacial trauma than the youth athletes. orofacial trauma was most frequent for the soccer players. in addition, professional soccer players presented more dental/facial trauma than youth athletes (significant difference). soccer is a sport in which there is direct physical contact between athletes; consequently, there may be a higher incidence of trauma when compared to volleyball2-4. differences in the reported athletes’ perception about the influence of oral health conditions on the physical performance were detected between the groups, especially 7 souza et al. between the sports (volleyball and soccer), and youth and professional soccer athletes. the differences in the perceptions of the volleyball and soccer players may have been related to socio-educational differences between the two groups. youth soccer athletes generally seek to invest in their careers to become professionals; however, they tend to put educational priorities in second place once they have difficulties in reconciling the daily routines of athletes with school activities14. in our study, 69% of the professional athletes reported that oral health can interfere with sports performance. in a study of elite olympic athletes, 33-66% reported that oral health could interfere with sports performance8. professional athletes demonstrate a better understanding of the relationship between oral diseases and sports performance. professional athletes often have dentists on their medical staff who are responsible for orientation and the promotion of oral health. we should also consider that professional athletes always seek to improve their sports performance, which is why they value oral health and its impact on such performance. our study presents some limitations, such as the population involved in which all the soccer athletes came from one team (brazilian soccer league serie b) and one volleyball club (brazilian men’s volleyball super league). both clubs are located in the southern region of brazil, involving only male athletes. considering these characteristics of the population included, the results need to be interpreted with caution, since there may be differences considering first division clubs and volleyball teams with higher investment in basic categories and salary income. as future perspectives, we recommend additional studies including other sports from different regions of the country. we concluded that most of the surveyed athletes had good oral health and believe that oral health can influence sports performance. the use of mouthguards should be recommended in physical contact sports to reduce the risk of orofacial trauma. in order to improve oral hygiene conditions, and knowledge about health, sports teams should include dentists in their medical staff for both youth and professional athletes. acknowledgments the authors wish to thank dr. sean stroud for reading this manuscript and offering his valuable comments. this study was financed in part by the coordination of higher education and graduate training (capes) – finance code 001. we would also like to thank all the professionals of the caramuru vôlei and operário ferroviário esporte clube (brazil) who participated in this study. conflict of interest the authors have no conflict of interest to declare. consent for publication all authors have approved the final version and its publication. 8 souza et al. references 1. ashley p, di iorio a, cole e, tanday a, needleman i. oral health of elite athletes and association with performance: a systematic review. br j sports med. 2015;49(1):14-9. doi: 10.1136/bjsports-2014-093617. 2. knapik jj, hoedebecke bl, rogers gg, sharp ma, marshall sw. effectiveness of mouthguards for the prevention of orofacial injuries and concussions in sports: systematic review and meta-analysis. sports med. 2019;49(8):1217-32. doi: 10.1007/s40279-019-01121-w. 3. gay-escoda c, vieira-duarte-pereira dm, ardevol j, pruna r, fernandez j, valmaseda-castellon e. study of the effect of oral health on physical condition of professional soccer players of the football club barcelona. med oral patol oral cir bucal. 2011;16(3):e436-9. doi: 10.4317/medoral.16.e436. 4. rodd hd, chesham dj. sports-related oral injury and mouthguard use among sheffield school children. community dent health. 1997;14(1):25-30. 5. gallagher j, ashley p, petrie a, needleman i. oral health and performance impacts in elite and professional athletes. community dent oral epidemiol. 2018;46(6):563-8. doi: 10.1111/cdoe.12392. 6. needleman i, ashley p, petrie a, fortune f, turner w, jones j, et al. oral health and impact on performance of athletes participating in the london 2012 olympic games: a cross-sectional study. br j sports med. 2013;47(16):1054-8. doi: 10.1136/bjsports-2013-092891. 7. márquez-hidalgo j, zamora-campos d, acurio-benavente p, kinoshita-rivas h, lópez-rodriguez g, moreno-sekula k, et al. relationship between the quality of life and oral health in athletes at a peruvian university. gen dent. 2020;68(5):73-7. 8. needleman i, ashley p, fine p, haddad f, loosemore m, de medici a, et al. oral health and elite sport performance. br j sports med. 2015;49(1):3-6. doi: 10.1136/bjsports-2014-093804. 9. de souza al, elmadjian t, brito e dias r, coto n. prevalence of malocclusions in the 13-20-year-old categories of football athletes. braz oral res. 2011;25:19-22. doi: 10.1590/s1806-83242011000100004. 10. leroux e, leroux s, maton f, ravalec x, sorel o. influence of dental occlusion on the athletic performance of young elite rowers: a pilot study. clinics (sao paulo). 2018;73:e453. doi: 10.6061/clinics/2017/e453. 11. ainamo j, bay i. problems and proposals for recording gingivitis and plaque. int dent j. 1975;25(4):229-35. 12. durham j, newton-john tr, zakrzewska jm. temporomandibular disorders. bmj. 2015 mar;350:h1154. doi: 10.1136/bmj.h1154. 13. weiler rm, vitalle ms, mori m, kulik ma, ide l, pardini sr, et al. prevalence of signs and symptoms of temporomandibular dysfunction in male adolescent athletes and non-athletes. int j pediatr otorhinolaryngol. 2010;74(8):896-900. doi: 10.1016/j.ijporl.2010.05.007. 14. rocha hpad, bartholo tl, melo lbsd, soares ajg. [young sportsmen: professionalization in soccer and formation in school]. motriz: j phys ed. 2011;17(2):252-63. portuguese. doi: 10.5016/1980-6574.2011v17n2p252. 15. franz ce, cook k. utilisation of social determinants of health to improve education among youth in dominican baseball academies. health soc care community. 2020;28(2):423-30. doi: 10.1111/hsc.12874. 16. bastos jl, boing af, peres kg, antunes jl, peres ma. periodontal outcomes and social, racial and gender inequalities in brazil: a systematic review of the literature between 1999 and 2008. cad saude publica. 2011;27 suppl 2:s141-53. doi: 10.1590/s0102-311x2011001400003. 17. broadbent jm, thomson wm, boyens jv, poulton r. dental plaque and oral health during the first 32 years of life. j am dent assoc. 2011;142(4):415-26. doi: 10.14219/jada.archive.2011.0197. 1http://dx.doi.org/10.20396/bjos.v20i00.8663795 volume 20 2021 e213795 original article 1 department of orthodontics, college of stomatology, the first affiliated stomatological hospital, xi’an jiaotong university, xi’an 710004, pr china 2 department of orthodontics, school of dentistry, shahed university, tehran, iran 3 school of dentistry, guilan university of medical sciences, rasht, iran 4 school of dentistry, islamic azad university, tabriz branch, tabriz, iran corresponding author: ali amiri department of orthodontics, college of stomatology, the first affiliated stomatological hospital, xi’an jiaotong university, xi’an 710004, pr china tell: +86-2982655450 email: draliamiri2020@gmail.com editor: dr altair a. del bel cury received: january 1, 2021 accepted: april 5, 2021 evaluation of the clinical effectiveness of miniscrews in class i and ii malocclusion patients: a systematic review and meta-analysis ali amiri1,* , setareh khosravi2 , abolfazl habibi arbastan3 , sara jafarizadeh4 aim: the present systematic review and meta-analysis aimed to evaluate the clinical effectiveness of miniscrews in class i and ii malocclusion patients. methods: from electronic databases, between 2010 and 2020, pubmed, embase, cochrane library, isi were used to conduct systematic literature. two reviewers extracted data blindly and independently from the abstract and full text of the studies they used for data extraction. the mean differences between the two groups (miniscrews vs. conventional anchorage) with a 95 % confidence interval (ci), the inverse-variance method, and the fixed-effect model were calculated. the meta-analysis was evaluated using the statistical software stata/mp v.16 (the fastest version of stata). results: a total of 186 potentially relevant titles and abstracts were found during the electronic and manual search. finally, the inclusion criteria required for this systematic review were met by a total of seven publications. the mean difference of molar mesiodistal movement among seven studies and heterogeneity was -0.53 mm (md, -0.53 95 % ci -0.69, -0.38. p= 0.00) (i2 = 96.52 %). this result showed maximum reinforcement in miniscrews with fewer mesial movements. conclusion: the result of the current systematic review and meta-analysis shows that miniscrews in patients with class ii and i malocclusion help maintain better anchorage preservation than traditional anchorage devices. keywords: orthodontic anchorage procedures. malocclusion, angle class ii. https://orcid.org/0000-0001-9416-808x https://orcid.org/0000-0002-7360-269x https://orcid.org/0000-0001-7611-4133 https://orcid.org/0000-0001-5196-0296 2 amiri et al. introduction malocclusion was first introduced by edward angel, the father of modern orthodontics. malocclusion is a misalignment between the two dental arches’ teeth when they approach each other as the jaws close with a bite1. it is also a growing problem in public health due to its high prevalence2. malocclusions feature the third-highest prevalence among oral pathologies, second only to tooth decay and periodontal disease, and therefore rank third among worldwide dental public health priorities3-5. one of the skeletal classes’ treatment methods is to limit the decreased arch length due to mesial movement6. in class i and ii malocclusions, traditional methods such as trans-palatal arches and multi-tooth differential moments in the anchorage segment are used7. however, traditional methods are not recommended, because in some cases, anchorage loss has been observed. miniscrews are used for maximum anchorage8. the survival rate in studies reported between 80 and 90%. the difference between this method and other methods is that they are not directly connected to the teeth9-12. it is important to note that miniscrews do not allow any unnecessary movement after placement13,14. recent studies show that anchorage losses are observed after the use of miniscrews15-17. as a result, more studies are needed to be able to compare new and traditional methods. over the past few years, differences between study results have left little evidence for the exact effects of miniscrews. lack of studies showing significant anchorage losses and movements of miniscrews. in previous studies, insufficient evidence has been provided, the sample size is low, and the quality of studies is very low, so the present study was conducted to provide stronger evidence. however, previous studies have been written as literature; the present study is a meta-analysis. also, for successful treatment results, a comparison of miniscrews effectiveness in malocclusion class i or ii is required. the aim of the systematic review and meta-analysis study is to evaluate the miniscrew outcomes in patients with class i and ii malocclusion, given the importance of the subject and the gap between the studies’ results. materials and methods search strategy pubmed, embase, cochrane library were used from electronic databases to conduct systematic literature between 2010 and 2020. therefore, to manage the electronic titles, a software program (endnote x8) was used. searches have been performed with mesh terms: (“orthodontic anchorage procedures”[mesh] or “dental abutments”[mesh]) and “orthodontic brackets”[mesh]) or (“malocclusion”[mesh] or “malocclusion, angle class ii”[mesh] or “malocclusion, angle class i”[mesh] ), and keywords orthodontic anchorage procedures, dental abutments, skeletal anchorage, temporary anchorage devices, orthodontic brackets, miniscrew implant, micro-implant, malocclusion, angle class ii, angle class i were used for other databases. on prisma guidelines, this systematic review and meta-analysis were conducted18 and pico or peco strategy (table1). 3 amiri et al. table 1. pico or peco strategy. pico or peco strategy description p population/ patient: patients with class i and ii malocclusion e exposure/ intervention: miniscrews c comparison: miniscrews vs. traditional anchorage o outcome: mesiodistal and vertical movement of incisors and molars selection criteria inclusion criteria 1. randomized controlled trial studies, controlled clinical trials, prospective and retrospective cohort studies. 2. patients treated with fixed orthodontic treatment 3. only patients with class i and ii malocclusion 4. maxillary or bimaxillary protrusion 5. efficiency outcomes of buccal inserted maxillary miniscrews 6. intervention group: miniscrews/mini-implants temporary anchorage devices (tad) 7. control group: traditional anchorage 8. mesiodistal movement of the maxillary first molars, vertical movement of the molars 9. english language exclusion criteria 1. in vitro studies, reviews, case-control studies, case report, and animal studies 2. incomplete or inconsistent data for the present study. 3. onplant, orthosystem, mini-plates 4. patients with class iii malocclusion 5. miniscrews placed in palatal or zygomatic areas data extraction and analysis method the data were extracted from the research included years, study design, malocclusion type, duration of space closure, traditional anchorage group, sample size, mean/range of age, group of miniscrews, measurement techniques. using the cochrane collaboration tool, the quality of the randomized clinical trials the studies included was analyzed19. the scale scores for low risk were one and for high and unclear risk was 0. scale scores range from 0 to 6. a higher score means higher 4 amiri et al. quality. also nonrandomized clinical trial studies were evaluated using the newcastle-ottawa scale (nos)20; the scale scores range from 0 (lowest grade) to 8 (highest grade). two reviewers extracted data blindly and independently from the abstract and full text of the studies they used for data extraction. the mean differences of mesiodistal dental movement, vertical dental movement between the two groups (miniscrews vs. conventional anchorage) with a 95 % confidence interval (ci), the inverse-variance method, and the fixed-effect model were calculated. to deal with potential heterogeneity, random effects were used, and i2 showed heterogeneity. for the meta-analysis, stata v16 software was used. results according to the purpose of the study, in the initial search with keywords, 186 articles were found. in the first step of selecting studies, 184 studies were selected to review the abstracts. then, studies that did not meet the inclusion criteria were excluded from the study. in the second step, the full text of 43 studies was reviewed. finally, seven studies were selected (figure1). studies identified (n = 186) pubmed (61) embase (76), cochrane library (49) studies after copies expelled (n = 184) studies screened (n = 184) studies excluded (n = 141) in vitro studies, reviews, case-control studies, case report and animal studies, incomplete or inconsistent data for the present study, onplant, orthosystem, mini-plates, patients with class iii malocclusion, miniscrews placed in palatal or zygomatic areas. not meet eligibility criteria. full content article surveyed for eligibility (n = 43) full content article excluded (n = 36) not meet eligibility criteria the included studies (n = 7) id en ti fi ca ti on s cr ee ni n el ig ib ili ty in cl ud ed figure 1. study attrition bias assessment according to cochrane collaboration’s tool, two studies, 23, 24 had a total score of 5/6 with high quality and a low risk of bias (table 3). according to nos, three studies 19,22,21 had a total score of 6/8, two studies 25, 27 had a total score of 7/8. this outcome showed scores ranged from 6 to 8 and low risk of bias or high quality of all studies (table 4). 5 amiri et al. ta bl e 2. s tu di es w er e se le ct ed fo r s ys te m at ic re vi ew a nd m et aan al ys is . s tu di es . y ea rs s tu dy de si gn n um be r of p at ie nt s m ea n/ r an ge o f a ge (y ea rs ) ty pe o f m al oc cl us io n m et ho ds o f an ch or ag e m ea su re m en t te ch ni qu es o rt ho do nt ic s pa ce c lo su re (m on th ) m s c a m s c a m al e fe m al e m al e fe m al e m s c a c ho pr a et a l. 20 17 21 p 12 13 12 13 15 .1 2 15 .0 8 cl as s i o r cl as s ii n an ce b ut to n; lin gu al a rc h lc a 21 .1 6 21 .7 6 c he n et a l. 20 15 22 p 6 9 7 9 26 .5 3 25 .2 5 cl as s i o r cl as s ii h ea dg ea r lc a 21 .9 3 23 .8 8 sa nd le r e t a l. 20 14 23 r c t 11 16 19 7 14 .1 5 14 .2 6 cl as s i o r cl as s ii h ea dg ea r an d n an ce b ut to n 3d 26 .8 3 27 .7 2 a l-s ib ai e an d h aj ee r 2 01 42 4 r c t 12 16 9 19 23 .0 2 20 .4 6 cl as s i o r cl as s ii tr an sp al at al ar ch lc a 12 .9 0 16 .9 7 p ar k et a l. 20 12 25 p 4 8 1 11 18 .8 25 .4 cl as s i o r cl as s ii tr an sp al at al ar ch , h ea dg ea r 3d 8. 6 9. 8 k oy am a et a l. 20 11 26 p 1 13 2 12 25 24 .8 cl as s i h ea dg ea r lc a n r le e an d k im 20 11 27 p 0 20 0 20 24 .6 4 22 .1 6 cl as s i tr an sp al at al ar ch , h ea dg ea r lc a n r p : p ro sp ec tiv e st ud y; r c t: ra nd om iz ed c lin ic al tr ia ls ; m s: m in is cr ew s; c a : t ra di tio na l a nc ho ra ge ; l c a : l at er al c ep ha lo m et ric a na ly si s; 3 d :3 d s tu dy m od el a na ly si s; n r : n ot re po rt ed ; 6 amiri et al. table 3. risk of bias assessment (randomized clinical trials). study r an do m g en er at io n of s eq ue nc es c on ce al m en t o f a llo ca ti on b lin di ng o f pa rt ic ip an ts a nd pe rs on ne l b lin di ng o f o ut co m e as se ss m en t in co m pl et e da ta o n ou tc om es s el ec ti ve re po rt in g to ta l s co re sandler et al. 201423 + + + + + 5 al-sibaie and hajeer 201424 + + + + + 5 low (+), unclear (?), high (-) table 4. risk of bias assessment (non-randomized clinical trials). study s el ec t t he m ai n gr ou p s el ec t t he g ro up o f c on tr ol d et er m in at io n of th e m ai n g ro up d em on st ra ti on th at th e ou tc om e of in te re st a t t he b eg in ni ng o f th e st ud y w as n ot p re se nt c om pa ra bi lit y of b ot h gr ou p pa rt ic ip an ts th e in de pe nd en t b lin dn es s p ou tc om es e va lu at io n s ui ta bi lit y of fo llo w -u p fo r ou tc om es to o cc ur lo st to fo llo w -u p ac ce pt ab le to ta l s co re chopra et al. 201721 + + + + + + 6 chen et al. 201522 + + + + + + 6 park et al., 201225 + + + + ++ + 7 koyama et al., 201126 + + + + + + 6 lee and kim, 201127 + + + + ++ + 7 +=1, +=2, -=0 mesiodistal dental movement molars seven studies (two randomized controlled trials and five prospective studies) have been included. there were 46 and 95 male and female patients, with a mean age of 21.03 years, respectively, in the miniscrews group. the number of male and female patients was 50 and 91, with a mean of 21.05 years, respectively, in the traditional anchorage group. measurement techniques in five studies were lateral cephalometric analysis, and in two studies17, 22 were 3d study model analysis. the mean of orthodontic space closure in the miniscrews and traditional anchorage groups was 18.24 and 20.02 months, respectively (table 2). the mean difference of molar mesiodistal movement among seven studies and heterogeneity was -0.53 mm (md, -0.53 95 % ci -0.69, -0.38. p= 0.00) (i2 = 96.52 %). this result shows no sig7 amiri et al. nificant statistical difference between the traditional anchorage and miniscrews (p=000) (figure 2). this result showed maximum reinforcement with fewer mesial movements in miniscrews. incisors six studies (one randomized controlled trial and five prospective studies) have been included. there were 35 and 79 male and female patients, with a mean age of 22.18 years, respectively, in the miniscrews group. the number of male and female patients was 31 and 84, 22.19 years, respectively, in the traditional anchorage group. measurement techniques in five studies were lateral cephalometric analysis and, in one study, 22 were 3d study model analysis. the mean of orthodontic space closure in the miniscrews and traditional anchorage groups was 16.04 and 18.10 months, respectively (table 2). the mean difference in mesiodistal incisor movement among seven studies and heterogeneity found was -0.66 mm (md, -0.66 95 % ci -0.94, -0.37. p= 0.00) (i2 = 73.76 %). no statistically significant difference between miniscrews and traditional anchorage groups (p=0.00) is shown in this result (figure 2). in the miniscrew group, this result showed more retraction than in the traditional anchorage group. figure 2. the forest plot showed the mesiodistal movement of incisors between miniscrews vs. traditional anchorage. vertical dental movement molars three studies (prospective study) have been included. there were 5 and 41 male and female patients, with a mean age of 22.81 years, respectively, in the miniscrews group. the number of male and female patients was 3 and 42, with a mean of age 24.21 years, respectively, in the traditional anchorage group. measurement techniques in three studies were lateral cephalometric analysis, and in one study were 3d study model analysis. the mean of orthodontic space closure in the miniscrews and traditional anchorage groups was 8.6 and 9.8 months, respectively (table 2). mean difference of vertical movement of molars was -0.5 mm (md, -0.5 95% ci -1.11, 0.1. 8 amiri et al. p= 0.1) among three studies and heterogeneity found (i2 = 92.91%). this result shows no statistically significant difference between miniscrews and traditional anchorage groups (p=0.1) (figure 3). this result showed in the miniscrews group, maxillary molars have a higher intrusion. figure 3. the forest plot showed the vertical movement of molars between miniscrews vs. traditional anchorage. incisors four studies (prospective study) have been included. there were 11 and 50 male and female patients, with a mean age of 23.74 years, respectively, in the miniscrews group. the number of male and female patients was 10 and 52, with a mean of age 24.4 years, respectively, in the traditional anchorage group. measurement techniques in five studies were lateral cephalometric analysis and in one study were 3d study model analysis. the mean of orthodontic space closure in the miniscrews and traditional anchorage groups was 15.26 and 16.84 months, respectively (table 2). mean difference of vertical movement of incisors was -0.19 mm (md, -0.19 95% ci -0.5, 0.13. p= 0.25) among four studies and heterogeneity found (i2 = 87.36%). this result shows no statistically significant difference between miniscrews and traditional anchorage groups (p=0.25) (figure 4). this result showed better intrusion in the miniscrews group than the traditional anchorage group. figure 4. the forest plot showed the vertical movement of incisors between miniscrews vs. traditional anchorage. 9 amiri et al. discussion many anchorage reinforcing appliances are available, but achieving the desired result and controlling the absolute anchorage during treatment bimaxillary is very important and challenging. several factors must be considered to select a suitable anchor booster. miniscrews can enhance orthodontic anchors and have received a great deal of attention recently because they attached to the bony appendages and provided the ideal movement of only the targeted teeth20. the first part of the meta-analysis findings showed maximum reinforcement in miniscrews with the fewer mesial movement of molars vs. traditional anchorage. clinically, a reduction of 2 mm on each side can show better results. sandler et al. study showed miniscrews were better than headgear and nance groups23. meta-analysis findings showed that incisors’ mesial movement was more retraction in the miniscrews group than the traditional anchorage group. in molars’ vertical movement, more retraction in miniscrews than traditional anchorage and vertical movement of incisors was a better intrusion in the miniscrews group than the traditional anchorage group. papadopoulos et al.3 evaluate the clinical effectiveness of miniscrew implants used for anchorage reinforcement compared with conventional anchorage. the result showed the mean difference of anchorage loss between the two groups was −2.4 mm (95% ci = −2.9 mm to −1.8 mm, p = 0), miniscrew implants significantly decreased or negated loss of anchorage3. yao et al. showed only in the miniscrews group molar intrusion observed, thereby improving class ii malocclusion. although there are advantages to using miniscrews, some studies have shown that miniscrews cannot achieve absolute anchorage28. horiuchi et al. suggest that the movement of the anterior teeth using conventional anchoring devices depends more on the forces acting on the posterior teeth and the patient’s adaptation and is therefore, less than miniscrews29. although numerous benefits are available for miniscrew implants, some studies have shown that miniscrew implants can not achieve absolute anchorage compared to miniplates. however, it is better than conventional anchorage, and no side effects have been reported in the included studies28. clinically, the traditional anchorage is more suitable in some cases that require a maximum anchorage. in any case, no side effects have been reported in the included studies. a patient-reported outcome measures should be obtained from the patient better to determine treatment outcomes30,31. the limitations of the present study are low sample size, low rct studies, high heterogeneity in the selected study. large sample size, follow-up period, rct, and prospective and retrospective cohort studies are required in this field. it is suggested that more studies be performed for the present study. due to the high heterogeneity between the studies and the working method, a similar way of evaluating the data is required. in the present study, patients’ opinions about comfort and quality of life satisfaction with traditional anchorage and miniscrews were not reported because a study that addressed all these dimensions was not found, so the patient’s perceived benefit is not recognizable. however, most traditional anchorages are extraoral; they are uncomfortable for patients, which can negatively affect21. further studies are needed to address these parameters. 10 amiri et al. in conclusion, the present systematic review and meta-analysis show that miniscrews in patients with class ii and i malocclusion help maintain better anchorage preservation than traditional anchorage devices. miniscrews can also reduce anchorage loss by minimization of molar mesial movement. references 1. batista kb, thiruvenkatachari b, harrison je, o’brien kd. orthodontic treatment for prominent upper front teeth (class ii malocclusion) in children and adolescents. cochrane database syst rev. 2018 mar;3(3):cd003452. doi: 10.1002/14651858.cd003452.pub4. 2. khela s, newton jt, jeremiah hg. the effect of malocclusion on dating prospects. j orthod. 2020 mar;47(1):30-7. doi: 10.1177/1465312519888926. 3. papadopoulos ma, papageorgiou sn, zogakis ip. clinical effectiveness of orthodontic miniscrew implants: a meta-analysis. j dent res. 2011 aug;90(8):969-76. doi: 10.1177/0022034511409236. 4. tak m, nagarajappa r, sharda aj, asawa k, tak a, jalihal s, et al. prevalence of malocclusion and orthodontic 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10.1016/j.ajodo.2014.03.020. 24. al-sibaie s, hajeer my. assessment of changes following en-masse retraction with mini-implants anchorage compared to two-step retraction with conventional anchorage in patients with class ii division 1 malocclusion: a randomized controlled trial. eur j orthod. 2014 jun;36(3):275-83. doi: 10.1093/ejo/cjt046. 25. park hm, kim bh, yang ih, baek sh. preliminary three-dimensional analysis of tooth movement and arch dimension change of the maxillary dentition in class ii division 1 malocclusion treated with first premolar extraction: conventional anchorage vs. mini-implant anchorage. korean j orthod. 2012 dec;42(6):280-90. doi: 10.4041/kjod.2012.42.6.280. 26. koyama i, iino s, abe y, takano-yamamoto t, miyawaki s. differences between sliding mechanics with implant anchorage and straight-pull headgear and intermaxillary elastics in adults with bimaxillary protrusion. eur j orthod. 2011 apr;33(2):126-31. doi: 10.1093/ejo/cjq047. 27. lee ay, kim yh. comparison of movement of the upper dentition according to anchorage method: orthodontic mini-implant versus conventional anchorage reinforcement in class i malocclusion. isrn dent. 2011;2011:321206. doi: 10.5402/2011/321206. 28. consolaro a. mini-implants and miniplates generate sub-absolute and absolute anchorage. dental press j orthod. 2014 may-jun;19(3):20-3. doi: 10.1590/2176-9451.19.3.020-023.oin. 12 amiri et al. 29. horiuchi a, hotokezaka h, kobayashi k. correlation between cortical plate proximity and apical root resorption. am j orthod dentofacial orthop. 1998 sep;114(3):311-8. doi: 10.1016/s0889-5406(98)70214-8. 30. hujoel pp. levels of clinical significance. j evid based dent pract. 2004;1(4):32-6. doi: 10.1016/j.jebdp.2004.02.012. 31. feu d, miguel ja, celeste rk, oliveira bh. effect of orthodontic treatment on oral health-related quality of life. angle orthod. 2013 sep;83(5):892-8. doi: 10.2319/100412-781.1 1 volume 21 2022 e227259 original article braz j oral sci. 2022;21: e227259http://dx.doi.org/10.20396/bjos.v21i00.8667259 1 department of health sciences and children’s dentistry, school of dentistry of piracicaba, university of campinas, piracicaba, são paulo, brazil. 2 teacher at the faculty of dentistry of the state university of ponta grossa, ponta grossa, paraná, brazil. 3 university of zaragoza, spain. 4 faculty of medicine of jundiaí, jundiaí, são paulo, brazil. corresponding author: dr marília jesus batista department of health sciences and children’s dentistry, piracicaba dental school – university of campinas. avenida limeira, 901 – piracicaba, sp – brazil. zip code: 13414-018. p.o. box 52. phone: 55 (19) 2106 5209; fax: 55 (19) 2106 5218. cell phone: 55 (11)94449 1191. e-mail: mariliajbatista@yahoo.com.br, mariliamota@g.fmj.br editor: altair a. del bel cury received: october 13, 2021 accepted: may 25, 2022 health literacy dimensions among public health service users with chronic diseases in piracicaba, brazil, 2019 carla fabiana tenani1 , manoelito ferreira silva junior2 , maria da luz rosário de sousa1,3 , marilia jesus batista1,4,* aim: this study analyzes factors associated with dimensions of health literacy (hl) functional, communicative and critical among public health service users with chronic non-communicable diseases. methods: a cross-sectional analytical research was carried out in piracicaba, são paulo, brazil, with adults and older adults attending family health units (fhu). data were collected by oral exam (cpod and cpi) and a questionnaire on systemic conditions, sociodemographic factors, health behaviors and hls (hls-14). the outcomes consisted of functional, communicative, and critical hl dimensions dichotomized by median (high and low), which were analyzed by chi-square test (p<0.05) to find associations with the variables studied. results: the study sample comprised 238 fhu users with 62.7 (± 10.55) mean age, of which 47.5% (n=113) showed high functional hl, 50.0% (n=119) high communicative hl, and 46.2% (n=110) high critical hl. high functional hl was associated with men (p<0.05). functional and communicative hl were associated with having higher education (p<0.001 and p=0.018, respectively). high communicative and critical hl were associated with regular use of dental and medical services (p<0.05). individuals with low functional hl were more likely to present poor tooth brushing (p=0.020). high hl (in all three dimensions) was associated with regular flossing and having more teeth (p<0.05). conclusion: functional, communicative and critical hl were associated with health behaviors and clinical outcomes, whereas the functional dimension was also associated with sociodemographic factors. hl dimensions allowed to differentiate health-related factors. keywords: health literacy. oral health. chronic disease. health policy. national health programs. public health. https://orcid.org/0000-0001-7203-2763 https://orcid.org/0000-0001-8837-5912 https://orcid.org/0000-0002-0346-5060 https://orcid.org/0000-0002-0379-3742 2 tenani et al. braz j oral sci. 2022;21: e227259 introduction the demographic and epidemiological transition has widened the age pyramid and increased the prevalence of chronic non-communicable diseases (ncd) in the world population and, subsequently, in the brazilian population1. of strong behavioral character, these morbidities require co-responsibility between health professionals and patients to control their consequences. in this regard, health literacy (hl) has been considered a key to health promotion and to improve health decision-making2. health literacy refers to personal knowledge, motivation, and skills to make health decisions throughout life2. according to nutbeam’s concept, hl comprises three dimensions: functional, communicative, and critical literacy3. functional hl consists of sufficient basic reading and writing skills to be used in everyday situations. in this dimension, one’s role is passive. in the communicative dimension, one seeks information through direct communication with reliable sources, such as health professionals, thus playing an active role. the critical dimension requires more advanced cognitive skills, such as critical analysis to judge whether a health information is appropriate and represents a greater control over one’s own health, requiring a proactive role3,4. a low hl can have an impact on people’s health5, representing difficulties in making health-related decisions. studies suggest that adults and older individuals with low hl have less access to and understanding of health information, use medications inappropriately, have less disease prevention and control, with higher rates of morbidity and hospitalization3. conversely, a high hl means being able to take responsibility for the collective health and one’s own6. measuring health literacy remains a challenge for health professionals and managers, especially regarding the elaboration of strategies for developing critical thinking6. most of the existing instruments for hl measurement target specific health conditions such as oral health7 or diabetes8, and few of them take on a multidimensional approach9,10. most measure only functional hl11 and one more dimension12. using an instrument able to assess the three hl dimensions, as described by nutbeam, would thus allow us to identify the different aspects that might interfere in how people manage their health. in a community approach, information on the associated factors of hl dimensions contribute to assist and to plan health strategies in different health contexts, health conditions and/or age groups13. the health literacy scale (hls-14)10, for example, is a validated instrument that presents three dimensions14. given this context, this study sought to analyze the socio-demographic variables, health behaviors and clinical factors associated with hl dimensions among public health service users with chronic non-communicable diseases. materials and methods study design and location a cross-sectional analytical study was carried out with users of the unified health system (sus), with follow up at family health units (fhu) in the municipality of pira3 tenani et al. braz j oral sci. 2022;21: e227259 cicaba, são paulo, brazil, using the strengthening the reporting of observational studies in epidemiology (strobe) guidelines15. location according to the last census (2010), piracicaba has a population of 364,571 inhabitants in the urban area, with an adult and older population of 261,56716. in 2018, the municipality’s health network had 71 basic health units, of which 51 were fhus. this study included only adult and older adult hypertensive and/or diabetic users. sample we performed a sample calculation considering the prevalence of low hl as 50%, based on puello (2018)17, with a margin of error of 0.1 and design effect (deff)=2. predicting probable losses, we added 20%, totaling 298 participants. sample selection selection took place in two stages: first, we chose the fhu and then the participants. we performed a probabilistic drawing of eight family health units and then four alternates, considering the number of hypertensive and/or diabetic users in the population registered at the fhu, according to a study by morgan (2013)18. after two fhu refused to participate, two of the alternates were included. we had to include the remaining two alternate fhu to reach the sample size, thus totaling a final sample of 10 participating fhu (figure 1). estimating possible losses and refusals, we added 10 participants for each selected fhu, with 40 users taken from the list of hypertensive and/or diabetic patients registered at each health unit. 8 fhu n = 240 c al cu la te d s am pl e usf 1 n = 40 usf 2 n = 40 usf 33 n = 40 usf 4 n = 40 usf 5 n = 40 usf 6 n = 40 usf alternate 1 usf alternate 2 usf alternate 3 usf alternate 4 usf 7 n = 40 usf 8 n = 40 alternates figure 1. distribution of the sample of ncd patients (type 2 diabetes and hypertension) and the fhus selected for the study, adapted from morgan (2013). 4 tenani et al. braz j oral sci. 2022;21: e227259 the health teams of each selected fhu randomly distributed 40 invitations to registered users with type 2 diabetes and/or sah in attendance on the scheduled dates to participate in the study. data collection took place at the fhu during its opening hours. inclusion criteria consisted of patients registered and monitored at the fhu in piracicaba for type 2 diabetes and/or sah, who attend the fhu on the scheduled day and time. exclusion criteria included presence of abscesses or emergency oral health care on the day of collection, refusal to undergo clinical dental examination, and being unable to answer the questionnaire due to physical and/or psychological status (informed by the respective fhu). data collection clinical data were collected by a dental surgeon (ds) after an 8-hour theoretical and practical training with an experienced examiner, with intra-examiner agreement which, considered within reliability standards, ranged from 90.6% to 100.0% for caries and periodontal disease19,20. clinical oral examinations were performed by the examining board, properly dressed, and under world health organization (who) criteria, using a sterile periodontal probe and a clinical mirror, with the participant sitting in a chair, under natural light21, at the fhu offices. the clinical conditions evaluated were visible dental biofilm22, index of decayed, lost and filled permanent teeth (dmft) and community periodontal index (cpi)21. interviews were conducted with the participants following a questionnaire with 66 objective questions about behaviors, oral and general health determinants20,23. subsequently, we applied the health literacy scale (hls-14)10 validated in brazilian portuguese14. this instrument presents 14 questions (5 for the functional and communicative dimensions and 4 for the critical dimension), answered by a 5-point likert-type scale, with the following categories: “strongly disagree,” “disagree,” “neither agree nor disagree,” “agree” and “strongly agree.” total score ranges from 14 to 70 points, with higher scores indicating better hl. in the functional dimension (questions 1 to 5) the score is reversed, where agreeing means having low hl, whereas the questions related to communicative (questions 6 to 10) and critical literacy (questions 11 to 14) refer to high hl10. data on blood pressure and glycemic indexes were collected from the current information in the medical records. application of the questionnaire and hl instrument and the clinical oral examination were performed on the same day. study variables our variable of interested was hl, presented, in each dimension, at two levels: low and high, dichotomized by the median. cutoff points for high and low levels were 11.0 for the functional dimension, 16.5 points for the communicative dimension, and 14.0 for critical literacy. figure 2 summarizes the three dimensions3 and roles4, namely: functional hl – passive role, communicative hl – active role, and critical hl – proactive role. 5 tenani et al. braz j oral sci. 2022;21: e227259 levels of health literacy functional hl communication hl critical hl passive active proactive basic reading and writing knowledge that allows undestanding everyday health situations empowerment and interaction: more advanced skills to actively participate in daily life, extract information and meaning from different forms of communication, and apply them to change circumstances advanced knowledge in health analysis and critical thinking. social and support networks, and skills to critically analyze information that allows for greater control over life events and situations figure 2. flowchart of the adapted health literacy dimensions (kickbusch, 2004; nutbeam, 2000). the study variables were grouped into sociodemographic, behavioral, and clinical data. sociodemographic data consisted of age (considered continuously), gender (man or woman), and schooling level (less than 4 years, 4 full years, or 5 years or more), the cutoff point being elementary school20,23. toothbrushing (up to 2 times/day, 3 or more times/day), flossing (daily use or no daily use), use of medical services (1 time/year [regular use], less than 1 time/year [irregular use]), and use of dental services (1 time/year [regular use], less than 1 time/year [irregular use]) were the health behaviors analyzed20,23. oral and systemic clinical conditions comprised: tooth loss, not considering third molars in calculation performed by codes 4 and 5 of the dmft index (has 20 teeth or more, or has between 1 and 19 teeth, or edentulous) based on the reduced dental arch theory24; presence of periodontal pocket with code 3 or 4 per sextant in the cpi index (> 4mm) (yes or no); blood glucose (up to 126mg/dl, 127mg/dl or more); blood pressure considered normal (systolic [<130mmhg] and diastolic [85-89]); and hypertension (systolic [≥140mmhg] and diastolic [90mmhg or more])25,26. data analysis we performed a descriptive analysis to obtain the frequency, mean, median and standard deviation, using the statistical package for the social sciences (spss) software version 20.0. chi-square tests were performed comparing the hl dimen6 tenani et al. braz j oral sci. 2022;21: e227259 sions with the variables studied (p<0.05). internal consistence was estimated by cronbach’s α (>0.70). ethical aspects study submitted and approved by the research ethics committee under caae 94104618.7.0000.5418. the research started after approval and signing of the informed consent form by the research participants. results a total of 238 users with chronic diseases participated in the six-month data collection period. two users refused to undergo clinical oral examination, and a sample loss characterized by the non-attendance of 162 invited users, which was expected and calculated in the sample size and selection method. mean age was 62.7 (±10.55) years old, and 78.5% (n=187) had lower schooling level. regarding health behaviors, 68.1% (n=162) of the patients flossed regularly, and 74.8% (n=172) made irregular use of dental services (+1 year). as for the oral clinical exams, 57.6% (n=147) of participants presented a periodontal pocket > 4mm (table 1). table 1. characteristics of sociodemographic variables, access, health behavior and health conditions among patients with chronic non-communicable diseases (n=238), users of primary health care in piracicaba, sp, brazil, 2019. variables sociodemographic n (%) age (years) mean 62.7(±10.55) gender women 165 (69.3) men 73 (30.7) schooling level 4 years 86 (36.1) 4 complete years 101 (42.4) 5 years or over 51 (21.4) health behaviors toothbrushing up to 2 times/day 103 (43.3) 3 or more times/day 135 (56.7) flossing daily use 76 (31.9) no daily use 162 (68.1) medical service frequency regular use (+ 1 time/year) 181 (76.1) irregular use (1 time/year) 57 (23;9) dental service frequency regular use (up to 1 time/year) 58 (25.2) irregular use (+ 1 time/ year) 172 (74.8) clinical conditions (1)dental loss have 20 teeth or more 75 (31.5) between 20 and 27 teeth 86 (36.1) edentulous 77 (32.4) continue 7 tenani et al. braz j oral sci. 2022;21: e227259 continuation periodontal pocket (> 4mm) yes 137 (57.6) no 101 (42.4) glycemia up to 126 mg/dl 113 (47.5) 127 mg/dl or more 125 (52.5) systolic blood pressure up to 139 mmhg 174 (73.1) 140 mmhg or over 64 (26.9) diastolic blood pressure up to 89 mmhg 210 (88.2) 90 mmhg or over 28 (11.8) source: prepared by the authors (2020). note: (1) reduced dental arch theory (armellini and fraunhofer, 2002). regarding health literacy (hl), total mean was 40.4 (± 9.3) points and the median 42.0 points. analyzed by dimensions, the mean and standard deviation found were 11.0 (±4.4) for functional hl, 16.5 (±4.5) for communicative hl, and 14.0 (±3.4) for critical literacy. among users, 47.5% (n=113) showed high functional hl, 50% (n=119) high communicative hl and 46.2% (n=110) high critical literacy. table 2 presents the distribution of the hl dimensions for each question of the hls-14 instrument. most patients showed low hl for all three dimensions: functional (questions 1 to 4) had a higher percentages of agreement; communicative (questions 7 to 10) and critical (questions 11 to 14) had higher percentages of disagreement. table 2. distribution of health literacy for each question of the hls-14 instrument among individuals with chronic non-communicable diseases (n=238), users of primary health care in piracicaba, sp, brazil, 2019. questions according to the health literacy dimensions* answers strongly agree agree neither agree nor disagree disagree strongly disagree n (%) n (%) n (%) n (%) n (%) functional 1. i find words i cannot read 88 (37.0) 82 (34.5) 39 (16.4) 23 (9.7) 6 (2.5) 2. the print is too small for me 82 (42.0) 90 (37.8) 22 (9.2) 22 (9.2) 4 (1.7) 3. the content is very difficult to understand 88 (37.0) 89 (37.4) 35 (14.7) 20 (8.4) 6 (2.5) 4. it takes me a long time to read (the instructions) 72 (30.3) 75 (31.5) 39 (16.4) 48 (20.2) 4 (1.7) 5. i need someone to help me read 65 (27.3) 45 (18.9) 27 (11.3) 91 (38.2) 10 (4.2) communicative 6. i look for information in several places 31 (13.0) 76 (31.9) 31 (13.0) 69 (29.0) 31 (13.0) 7. i find the information i need 28 (11.8) 70 (29.4) 36 (15.1) 83 (34.9) 21 (8.8) 8. i understand the information found 26 (10.9) 70 (29.4) 45 (18.9) 80 (33.6) 17 (7.1) 9. i tell my opinion about the disease to my doctor, family, or friends 10 (4.2) 52 (21.8) 37 (15.5) 115 (48.3) 24 (10.1) continue 8 tenani et al. braz j oral sci. 2022;21: e227259 continuation 10. i put the information found into practice in my daily life 7 (2.9) 19 (8.0) 47 (19.7) 139 (58.4) 26 (10.9) critical 11. i know when the information is good for my case 10 (4.2) 37 (15.5) 52 (28.1) 110 (46.2) 29 (12.2) 12. i consider whether the information is true 6 (2.5) 27 (11.3) 35 (14.7) 143 (60.1) 27 (11.3) 13. i have knowledge to judge whether the information is reliable 17 (7.1) 72 (30.3) 57 (23;9) 78 (32.8) 14 (5.9) 14. i get information that helps me make decisions about how to improve my health 10 (4.2) 61 (25.6) 29 (12.2) 112 (47.1) 26 (10.9) source: prepared by the authors (2020). note: *hls-14 instrument (suka et al., 2013), validated in brazil by batista et al. (2020). hl dimensions were associated with the sociodemographic, behavioral, and clinical variables. the bivariate analysis showed that having more than 20 teeth and regular flossing were associated with high hl in all three dimensions (table 3). confirmatory analysis obtained a cronbach’s α = 0.87. table 3. sociodemographic factors, access, health behaviors, and clinical conditions associated with health literacy dimensions among patients with chronic non-communicable diseases (n=238), users of primary health care in piracicaba, sp, brazil, 2019. variables health literacy dimensions (hl) functional hl communicative hl critical hl < hl > hl p-value < hl > hl p-value < hl > hl p-value n (%) n (%) n (%) n (%) n (%) n (%) sociodemographic gender women 95 (57.6) 70 (42.4) 0.019 79 (47.9) 86 (52.1) 0.325 86 (52.1) 79 (47.9) 0.440 men 30 (41.1) 43 (58.9) 40 (54.8) 33 (45.2) 42 (57.5) 31 (42.5) schooling level 4 years 54 (62.8) 32 (37.2) <0.001 50 (58.1) 36 (41.9) 0.018 48 (55.8) 38 (44.2) 0.056 4 complete years 57 (56.4) 44 (43.6) 52 (51.5) 49 (48.5) 60 (59.4) 41 (40.6) 5 years or over 14 (27.5) 37 (72.5) 17 (33.3) 34 (66.7) 20 (39.2) 31 (60.8) health behaviors toothbrushing up to 2 times/day 63 (61.2) 40 (38.8) 0.020 46 (44.7) 57 (55.3) 0.150 58 (56.3) 45 (43.7) 0.494 3 or more times/day 62 (45.9) 73 (54.1) 62 (45.9) 73 (54.1) 70 (51.9) 65 (48.1) flossing daily use 32 (42.1) 44 (57.9) 0.028 24 (31.6) 52 (68.4) <0.001 28 (36.8) 48 (63.2) <0.001 no daily use 93 (57.4) 69 (42.6) 95 (58.6) 67 (41.4) 100 (61.7) 62 (38.3) continue 9 tenani et al. braz j oral sci. 2022;21: e227259 continuation medical service frequency regular use (+ 1 time/year) 98 (54.1) 83 (45.9) 0.372 101 (55.8) 80 (44.2) <0.001 107 (59.1) 74 (40.9) 0.003 irregular use (1 time/year) 27 (47.4) 30 (52.6) 39 (68.4) 18 (31.6) 21 (36.8) 36 (63.2) dental service frequency regular (up to 1 year since last time) 25 (43.1) 33 (56.9) 0.094 15 (25.9) 43 (74.1) <0.001 20 (34.5) 38 (65.5) <0.001 irregular (+ than 1 year since last time) 96 (55.8) 76 (44.2) 102 (59.3) 70 (40.7) 105 (61.0) 67 (39.0) clinical conditions dental loss have 20 teeth or more 29 (38.7) 46 (61.3) 0.013 25 (33.3) 50 (66.7) 27 (36.0) 48 (64.0) <0.001 between 20 and 27 teeth 49 (57.0) 37 (43.0) 48 (55.8) 38 (44.2) 0.002 47 (54.7) 39 (45.3) edentulous 47 (61.0) 30 (39.0) 46 (59.7) 31 (40.3) 54 (70.1) 23 (29.9) periodontal pocket (> 4mm) yes 51 (50.5) 50 (49.5) 0.591 45 (44.6) 56 (55.4) 0.149 88 (64.2) 49 (35.8) <0.0001 no 74 (54.0) 63 (46.0) 74 (54.0) 63 (46.0) 40 (39.6) 61 (60.4) glycemia up to 126 mg/dl 61 (54,0) 52 (46,0) 0,668 50 (44,2) 63 (55,8) 0,092 60 (53,1) 53 (46,9) 0,840 127 mg/dl or over 64 (51,2) 61 (48,8) 69 (55,2) 56 (44,8) 68 (54,4) 57 (45,6) systolic blood pressure up to 139 mmhg 92 (52.9) 82 (47.1) 0.857 95 (54.6) 79 (45.4) 0.019 103 (59.2) 71 (40.8) 0.006 140 mmhg or over 33 (51.6) 31 (48.4) 24 (37.5) 40 (62.5) 25 (39.1) 39 (60.9) diastolic blood pressure up to 89 mmhg 112 (53.3) 98 (46.7) 0.492 105 (50.0) 105 (50.0) 1.000 112 (53.3) 98 (46.7) 0.704 90 mmhg or over 13 (46.4) 15 (53.6) 14 (50.0) 14 (50.0) 16 (57.1) 12 (42.9) source: prepared by the authors (2019). note: *reduced dental arch theory (armellini and fraunhofer, 2002) discussion our study highlighted different associations between the dimensions of health literacy (hl) and sociodemographic factors, health behaviors, and clinical outcomes. a multidimensional evaluation of hl provides a broader approach that can deepen our understanding regarding hl levels and enhance one’s health autonomy. hence, the differential of a multidimensional instrument used to increase measurement sensitivity is evident, allowing more variables associated with the construct to be identified. despite the research on validated hl tools, few studies have assessed hl dimensions and associated factors27. the health literacy scale (hls-14), validated in brazilian portuguese, showed good internal consistency, which is considered adequate when greater than or equal to 0.70. its psychometrics properties were satisfactory to evaluate health literacy, as showed by batista et al.14. 10 tenani et al. braz j oral sci. 2022;21: e227259 recent studies using hl instruments associated with ncds, including oral diseases27, have assessed mainly reading and writing skills28, that is, only the functional dimension, disregarding communication and/or broad interaction with health care systems. in our study, therefore, we chose to use the hls-14 instrument, a pioneering tool for measuring the three hl dimensions (functional, communicative, and critical), according to nutbeam (2000)3. rapidly applicable, with reliable psychometric indexes not restricted to a specific area or health condition10, it can serve both to define clinical protocols more consistent with reality, thus improving people’s level of understanding of health information, and to carry out interventions capable of improving health literacy29. the questions with the greatest impact on literacy inquired about the difficulty in reading and finding information when needed, and in communicating one’s opinion about a health condition and being able to judge whether the information is reliable. regarding sociodemographic factors, men showed greater functional literacy, result not found in other studies30. gender inequity is an important social marker in brazil, especially in a sample of predominantly older adults. this finding may indicate a lack of study opportunities in a generation where these opportunities, including decision-making, were restricted for women. today, as observed in the 2010 census, women have a high level of schooling, with female school attendance increasing 9.8% in high school compared to men16. studies also highlight that older adults may have limited understanding of health information31 and greater participation of women due to the feminization of the aging process32. but even with this limited functional literacy, the literature points to greater self-care among women, including regular use of health services33. consequently, hl needs to go beyond the functional level. our findings showed that high functional and communicative hl were associated with high schooling level. these hl dimensions are related to passive and more active attitudes, such as communication. however, we must consider the cognitive differences, skills, and roles between people with the same educational level34. as such, research that exclude illiterate individuals from its sample30 may lose heterogeneity of results and restrict the understanding of literacy dimensions after all, literacy is one of and not the only aspect analyzed by hl dimensions. studies show that functional literacy focuses on reading skills, in which the people act more passively in health-related issues4. better reading and comprehension skills are associated with better formative education, which is related to schooling level, a marker and social determinant of health35. hl is thus related to one’s schooling, reflecting on their health behaviors; consequently, developing health literacy can reduce health inequalities36. regarding oral health behaviors, our results revealed that regular flossing was associated with high levels of all hl dimensions. lower frequency of tooth brushing was associated with low functional hl, corroborating a recent study37. oral health care and use of dental services can have an impact on clinical health conditions13. in our study, regular use of dental services was also associated with communicative and critical hl. the literature points out that, besides greater use of services, individuals with higher hl seek preventive consultations, showing a more active role in 11 tenani et al. braz j oral sci. 2022;21: e227259 the pursuit of health4. an unexpected finding in the present research was the association between low communicative and high critical hl and irregular use of medical services. this result can be explained by the sample characteristic of patients with chronic disease, who need continuous medical follow-up. the presence of a periodontal pocket was associated with a low critical hl. oral hygiene is associated with hl and with the risk of developing periodontal disease38, which can lead to tooth loss. tooth loss the worst oral health outcome was associated with all hl dimensions, but remains inconclusive13. when associated with risk behaviors for oral diseases, hl becomes relevant as a measure to reduce and control tooth loss, as it can help promote oral and general health, and studies exploring this topic have been performed39. thus, an in-depth knowledge of one’s hl level can be an important differentiator in the health-disease process40. sah was associated with communicative and critical hl, as shown by borges et al. (2019)30. considering that such dimensions of hl are associated with people’s proactive abilities4, blood pressure indices may, in this case, be influenced by aspects that interfere with their discharge, such as: frequency, type, and access to health services, interaction with health professionals, and others37. since the outcomes of oral and general health diseases and aggravations, such as periodontal disease, tooth loss and sah, are associated with more advanced dimensions of literacy, such as communicative and critical hl, it becomes clear that inequality negatively impacts health. as for the limitations, we can cite the restricted sample of the study. nonetheless, it was representative of unified health system users with sah and diabetes, where important associations between the hl dimensions and aspects involving the integral health of these users, often neglected in research2, were contemplated. measuring health literacy by a self-report instrument is always challenging, but using a validated questionnaire and proper analysis can control bias, thus improving the quality of the study. despite the limitations, our exploratory study presents unprecedent results that show a new perspective regarding the application and analysis of health literacy dimensions, reaffirming the need for greater research interest in exploring and improving on this topic in future studies. improving population hl can reduce the prevalence of chronic health conditions and the individual and collective impacts of these morbidities. our results showed that using instruments that cover only functional literacy may be insufficient to assess health literacy, and that the analyzes need to incorporate all three dimensions to formulate safer and more accurate strategies for professionals, managers, and users. the present work contributes to greater attention to the complexity and challenges involved in advancing the topic, serving as a starting point for future studies and as an aid to evidence-based public health policies that seek to improve the health of sus users. thus, future studies should consider hl using a multidimensional approach for public health policies and health promotion strategies. 12 tenani et al. braz j oral sci. 2022;21: e227259 in conclusion, functional, communicative, and critical hl dimensions were associated with sociodemographic, behavioral and clinical factors among adults and older adults with ncds, users of public health services in a different way. declaration of conflicting interests the authors declare no potential conflicts of interest regarding the research, authorship, and publication of this manuscript. funding we thank the coordination for the improvement of higher education personnel – capes (code 01) for funding this research. the authors thank espaço da escrita – pró-reitoria de pesquisa – unicamp for the language services provided. data availability datasets related to this article will be available upon request to the corresponding author. authors contribution it is stated that, for the conception of the manuscript, the authors marilia jesus batista and carla fabiana tenani made substantial contributions, such as the design and elaboration of the work. the author carla fabiana tenani performed the data acquisition. the authors carla fabiana tenani, manoelito ferreira silva junior, maria da luz rosário de sousa and marilia jesus batista analyzed and interpreted the data for the study. all authors critically reviewed the intellectual content and final approval of the version to be published. all authors agreed to be responsible for all aspects of ensuring that issues relating to the accuracy or completeness of any part of the work were properly investigated and resolved. references 1. hazra nc, gulliford m. evolution of the “fourth stage” of epidemiologic transition in people aged 80 years and over: population-based cohort study using electronic health records. popul health metr. 2017 may;15(1):18. doi: 10.1186/s12963-017-0136-2. 2. who. health literacy: the solid facts. geneva: world health organization; 2013. 3. nutbeam d. health literacy as a public health goal: a challenge for contemporary health education and communication strategies into the 21st century. health promot int. 2000 sep;15(3):259-67. doi: 10.1093/heapro/15.3.259. 4. kickbusch, i. improving health literacy in the european union: towards a europe of informed and active health citizens. in: european health forum gastein. 2004 oct 8 [cited 2022 jan 5]. available from: https://www.infosihat.gov.my/images/bahan_rujukan/he_ict/improving_health_literacy.pdf. 5. antunes ml, lopes c. 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population health and public health practice; roundtable on health literacy. integrating oral and general health through health literacy practices: proceedings of a workshop. wojtowicz a, olson s, editors. washington (dc): national academies press (us); 2019 jul 31. 1http://dx.doi.org/10.20396/bjos.v21i00.8665136 volume 21 2022 e225136 original article 1 school of health sciences, graduate program in dentistry, positivo university, curitiba, brazil. 2 department of dentistry, school of health sciences, university of brasilia, brasilia, brazil. corresponding author: carla castiglia gonzaga universidade positivo rua prof. pedro viriato parigot de souza, 5300 – 81280-330 – curitiba – pr – brasil phone: (5541) 3317-3180 fax: (5541) 3317-3082 e-mail: carlacgonzaga2@gmail.com editor: dr altair a. del bel cury received: march 30, 2021 accepted: june 15, 2021 fit of metal-ceramic crowns: effect of coping fabrication method and veneering ceramic application patrícia valéria manozzo kunz1 , gabriela andrade serpa1 , leonardo fernandes da cunha2 , gisele maria correr1 , carla castiglia gonzaga1,* aim: to evaluate the influence of cobalt-chromium (co-cr) coping fabrication methods and ceramic application on the marginal and internal fit of metal-ceramic crowns. methods: co-cr copings for metal-ceramic crowns were prepared by lost wax casting or cad-cam machining of sintered blocks. the fit was analyzed using the silicone replica technique at four assessment points: marginal gap (mg), axial wall (aw), axio-occlusal (ao) angle, and central occlusal (co) wall. after the initial analysis, the copings were ceramic-veneered with the layering technique, and the fit was again determined. data were statistically analyzed by paired and unpaired student’s-t test (α=0.05). results: marginal and internal fit before ceramic application according to the coping manufacturing method showed significant differences only at co (p < 0.001), with milled copings (137.98±16.71 μm) showing higher gap values than cast copings (112.86±8.57 μm). for cast copings, there were significant differences at mg (before 109.13±8.79 μm; after 102.78±7.18 μm) and co (before 112.86±8.57 μm; after 104.07±10.63 μm) when comparing the fit before and after ceramic firing. for milled copings, there was significant difference only at ao (before 116.39±9.64 μm; after 108.54±9.26 μm). conclusion: this study demonstrated that the coping fabrication method influenced the internal fit. ceramic firing maintained or improved the fit of the metal-ceramic crowns. the marginal discrepancy of all restorations, before and after ceramic firing, can be considered clinically acceptable. keywords: dental marginal adaptation. crowns. metal ceramic alloys. ceramics. mailto:carlacgonzaga2@gmail.com https://orcid.org/0000-0002-2581-0929 https://orcid.org/0000-0001-7522-6846 https://orcid.org/0000-0002-2073-0954 https://orcid.org/0000-0002-5032-0948 https://orcid.org/0000-0001-6374-1605 2 kunz et al. introduction metal-ceramic crowns are still commonly used for indirect single-unit restorations and fixed partial dentures1,2, because they combine the strength of the metal infrastructure and the esthetics provided by the veneering ceramic3. even with the increase in the clinical indications of all-ceramic restorations, metal-ceramic crowns are the treatment of choice for patients with parafunctional oral habits, mainly for the posterior region2. it happens because of their high mechanical properties and predictability of long-term clinical outcomes4. marginal fit is a critical factor for the success of indirect restorations5. poorly fit crowns may cause hypersensitivity, biofilm accumulation, gingivitis, periodontitis, and alveolar bone loss, which could eventually lead to tooth loss6-8. indirect restorations with good marginal fit may reduce gingival inflammation and cement dissolution, which are among the most common causes for prosthetic failures, in addiction, excellent internal fit promotes proper seating of the crown, compromising neither the margins nor the preparation9. among the methods used for the fabrication of metal copings, lost wax casting is the most widely used technique, both for noble and basic alloys, but the several steps necessary and the dependence on the ability of the technician can cause discrepancies in the marginal and internal fit of the final restoration10. because of the challenges involved with the lost-wax technique, new technologies for the manufacture of metal copings have been introduced. the most commonly used methods include computer-aided design and computer-aided manufacturing (cad-cam) of pre-sintered or sintered metal blocks and additive techniques, such as selective laser melting10-13. these new technologies facilitate laboratory procedures, save time and may result in restorations with improved marginal and internal adaptation10,13. different methods can be used for application of the veneering ceramic over metal copings. conventional layering technique (also known as vibration-condensation) and heat-pressing are two of them14. in the layering technique, powder and modeling liquid are mixed and applied on the coping in a larger volume to compensate for the shrinkage of the ceramic material during sintering15. however, multiple applications and firing cycles until application of the ceramic is complete (at least three firing cycles, considering the dentin, enamel, and glaze) may interfere with marginal and internal fit. moreover, the ceramicist’s skills are important at this stage so that a consistent and uniform ceramic thickness is maintained during application16. the evaluation of the marginal and internal fit of metal-ceramic crowns fabricated through different methods and the effect of ceramic firings may help clinicians choose the most appropriate fabrication method for indirect metal-ceramic restorations, preventing failures related to poor-fitted crowns, increasing the clinical longevity of the restorative treatments. thus, the aim of this study was to assess the influence of co-cr coping fabrication methods and of ceramic veneer application on the marginal and internal fit of metal-ceramic crowns. the null hypotheses were 3 kunz et al. that: i) the co-cr coping fabrication method would not influence the marginal and internal fit of metal-ceramic crowns, and ii) the ceramic veneering application would not influence the marginal and internal fit of co-cr copings, regardless of coping fabrication method. materials and methods a model for the preparation of a metal-ceramic crown in a lower molar was milled using cad/cam acrylic resin block (vipi block, pirassununga, sp, brazil). the preparation was made by simulating supragingival circumferential chamfer finish line, 2.0-mm occlusal reduction, 1.5-mm axial reduction, axial convergence angle of 6o, and rounded internal line angles. the master model was digitally scanned (ceramill map400, amann girrbach, koblach, austria) for the fabrication of twenty co-cr copings, with thickness of 0.5 mm at the margins and 0.8 mm at the axial and occlusal walls. ten copings were obtained by lost wax casting fabricated with a co-cr alloy (fit cast cobalt, talmax, curitiba, brazil) with the following composition: 61% cobalt, 30% chromium, 5.9% molybdenum, <1% silicon, and <1% manganese other ten copings were subjected to cad-cam milling (ceramill motion, amann girrbach, koblach, austria) of sintered blocks (ceramill sintron, amann girrbach, koblach, austria) with the following composition: 66% cobalt, 30% chromium, 5% molybdenum, <1% silicon, <1% iron, and <1% manganese. sample size was determined based on previous studies that also evaluated the marginal fit of metal-ceramic restorations before and after ceramic firing17-19. the silicone replica technique was used to assess marginal and internal fit of the copings20,21. the replicas were obtained i) after the fabrication of the copings and ii) after ceramic veneering. to produce the replica, each coping was filled with a light-body pvs material (adsil light body, coltène/whaledent, altstätten, switzerland) and positioned on the master model. firm finger pressure simulating a definitive cementation was applied and after 5 minutes the coping was removed from the master model. this procedure was practiced in a pilot study and has been previously reported in the literature19-22. to take out the light-body material from the coping and determine its thickness, which corresponds to the marginal and internal gap, a regular-body pvs material with contrasting color (adsil regular body, coltène/whaledent, altstätten, switzerland) was placed into the coping to produce the replica. after polymerization, the replica was cut with a sharp scalpel blade in buccolingual and mesiodistal directions to obtain four cross-sections (figure 1). 4 kunz et al. the four cross-sections of each replica were placed on a scanner (c3180 hp photosmart; hp) for digitization. the high-resolution images (1200 dpi) were saved as jpg files and a single operator determined the thickness of the light-body pvs material using an image analyzing software (imagej, u. s. national institutes of health, bethesda, maryland, usa). the following assessment points were evaluated in each of the sections: marginal gap (mg), axial wall (aw), axio-occlusal (ao) angle, and central occlusal (co) wall20,21 (figure 2). four measurements of each of the points were made for each replica, and the mean was estimated for each point in each replica. figure 1. silicone replica technique: a – metal-ceramic crown; b – light-body pvs material (in green), which corresponds to the marginal and internal gap; c – regular-body pvs material with contrasting color placed into the coping; d – cross-sections obtained after cutting the replica in buccolingual and mesiodistal directions. a c b d figure 2. light-body pvs replica showing the four assessment points. co ao aw mg 5 kunz et al. after determining the marginal and internal fit of the copings (initial – before ceramic application), they were ceramic-veneered with the layering technique. the porcelain powder (heraceram, heraeus-kulzer, hanau, germany) was mixed with water and applied over the coping with a brush, and the excess water was wiped off with absorbent paper. porcelain was applied in excess to compensate for the shrinkage observed after the porcelain was fired. the application was standardized using four firings (opaque layer firing, first firing for dentin, second firing for dentin, and glaze). all firings were performed in the same furnace for porcelain sintering (dekema austromat 624, jensen dental, north haven, ct, usa). statistical analyses were performed individually for each assessment point. the fit before or after ceramic application, comparing cast versus milled copings, was assessed by student’s t-test. paired student’s t-tests were used to evaluate the marginal and internal fit for each coping manufacturing method before and after ceramic veneering. all analyses were carried out at a significance level of 0.05. results the means and standard deviations of the fit (before and after ceramic firing) according to the coping manufacturing method (casting or milling) for each assessment point are shown in table 1. considering the fit before ceramic firing, the statistical analysis revealed significant differences only at co (p < 0.001), with milled copings (137.98 ± 16.71 μm) showing higher gap values than cast copings (112.86 ± 8.57 μm). mg (p = 0.224), aw (p = 0.818) and ao (p = 0.112) showed statistically similar values. comparing the fit of cast versus milled copings after ceramic application, the statistical analysis revealed significant differences at aw (p = 0.019, higher gap for cast copings) and co (p < 0.001, higher gap for milled copings). mg (p = 0.225) and ao (p = 0.156) showed statistically similar values. for cast copings, there were statistically significant differences at mg (before 109.13 ± 8.79 μm; after 102.78 ± 7.18 μm; p = 0.048) and co (before 112.86 ± 8.57 μm; after 104.07 ± 10.63 μm; p = 0.016) when comparing the fit before and after ceramic firing. for aw and ao, no statistically significant difference was observed between the copings before and after ceramic veneering. for milled copings, there was statistically table 1. means and standard deviations of marginal and internal fit (in μm) at the assessed points for cast and milled copings before and after ceramic veneering. mg aw ao co ceramic firing ceramic firing ceramic firing ceramic firing before after before after before after before after cast copings 109.13 ± 8.79 ab 102.78 ± 7.18 aa 107.43 ± 10.63 aa 104.48 ± 7.31 ba 107.77 ± 13.15 aa 100.24 ± 15.11 aa 112.86 ± 8.57 ab 104.07 ± 10.63 aa milled copings 104.18 ± 8.80 aa 97.94 ± 9.78 aa 106.06 ± 14.90 aa 93.25 ± 11.70 aa 116.39 ± 9.64 ab 108.54 ± 9.26 aa 137.98 ± 16.71 ba 127.65 ± 9.82 ba for each point, in the columns, values followed by the same uppercase superscript letters are statistically similar when comparing cast x milled copings before or after ceramic application (p > 0.05). for each point and coping fabrication method, in the lines, values followed by the same lower case superscript letters are statistically similar when comparing before and after ceramic application (p > 0.05). 6 kunz et al. significant difference only at ao (before 116.39 ± 9.64 μm; after 108.54 ± 9.26 μm; p = 0.049). it is important to note that, when statistically significant differences were observed, lower gap values were obtained always after ceramic veneering. discussion in this study, the first null hypothesis, that there would be no difference in marginal and internal fit of co-cr copings for the different techniques used (casting and cad-cam milling of sintered blocks), was accepted for marginal fit and rejected for internal fit. co assessment point of cad-cam-milled copings showed poorer internal fit than cast copings. the findings of the present study are in accordance with previous ones in the literature. han et al.23 investigated the effect of the manufacturing method (casting or cad-cam milling) on the marginal and internal fit of titanium crowns, and showed that cast crowns had better marginal and internal fit both at the margin and in the occlusal area than did cad-cam-milled crowns. among the different methods for the fabrication of metal copings, casting is more likely to have poor fit because of the larger number of steps involved13, which was not confirmed in the present study. our results showed that the marginal fit of both copings was similar, but internal fit was better for cast copings. the higher values obtained for the occlusal surface when compared with the axial wall are also described in previous studies13,20,21,24, despite the fact that milled copings are believed to have lower marginal discrepancies, since their fabrication has fewer steps. according to the literature, cement film thickness varies considerably, but most authors describe 120 µm as the clinically acceptable maximum thickness for a long-term good prognosis25. in most studies, thickness is close to 120 µm, but there are some questions about whether this value is still valid given the new materials and fabrication methods now commercially available. at the axio-occlusal angle and on the occlusal surface, the poorer fit of milled copings as compared to cast copings can be explained by the quality of data acquisition and processing in cad-cam26. poorer internal fit may result from round edges produced by the finite resolution of the cad-cam imaging system and difficulty in scanning the axio-occlusal angle. fit at ao is influenced by the image captured by the scanner and reconstruction of this angle by the software program, as the light reflected during the reading procedure is stronger than in flat regions27. moreover, poor preparation makes this area more irregular, and could also lead to poor fit. overshoot, a phenomenon that simulates virtual peak values close to the edges, could also cause larger internal discrepancies28,29. similar findings were also previously reported30, showing significant lower gap values in axio-occlusal and occlusal regions for co-cr cast copings when compared to co-cr milled copings. it is also important to note that the compositions of the metal alloys used in the present study are very similar. the main difference between the alloys is in the percentage of cobalt (61% and 65%). in general, the composition of the alloys may vary from one commercial brand to another, and even more when different manufacturing methods (casting and cad/cam milling) are used. however, it has been reported that the weight percentage of cobalt for co-cr alloys usually ranges from 53 and 68%31. thus, the two alloys used in the present study have the percentage of cobalt within that range. 7 kunz et al. the second null hypothesis, that the ceramic veneering application would not influence the marginal and internal fit of co-cr copings, was accepted for cast and milled copings. with regard to marginal and internal fit before and after ceramic application, higher gap values were observed always in copings before ceramic firing. the changes in fit for cast copings corresponded to -5.82% at mg and to -7.78% at co. in milled copings, the change occurred only at ao, exhibiting a rate of -6.75% after ceramic application. in the present study, when significant differences were observed, lower gap values were obtained after ceramic veneering. for the marginal fit, this result was also reported by real-voltas et al. (2017), who attributed this result to the fact that the porcelain firing improved the mismatch of the metal coping32. several studies demonstrated that the porcelain veneering application could change the fit of crowns and cause distortion in the metal substructure17,33,34. one of the possible explanations for this effect is the development of compressive forces on metal coping as a consequence of the sintering contraction that occurs in the veneering porcelain during firing35. another study also indicated that the marginal fit of metal copings can be altered after porcelain veneering at different stages36. interestingly, the largest marginal discrepancies occurred in basic alloys and after opaque layer firing. the present study assessed marginal and internal fit using the silicone replica technique. this technique has produced reliable outcomes and has been reported to be as accurate as the cross-section of the die and restoration for later direct microscopic evaluation of the fit37. its major advantages include the ease with which it is performed and its low costs38, in addition to the simulation of finger pressure at the time of luting of the crowns over the tooth preparation. lately, micro-ct imaging has also been used to assess the marginal and internal fit of crowns and fixed partial dentures11,28,39. both non-destructive methods are considered effective to evaluate the adaptation of indirect restorations20,24. metal-ceramic restorations may show some distortions in the different stages of coping fabrication and of ceramic firing, resulting in marginal and internal discrepancies. the main factors could be the difference in the coefficient of thermal expansion between the metal coping and the porcelain, the coping fabrication method, and sintering shrinkage. therefore, studies that assess the change in the fit of copings by different methods and after application of veneering ceramic may be better at preventing remarkable and clinically unacceptable poor fit. large marginal discrepancies may be harmful to both abutment teeth and periodontal tissues. despite the significant increase in the number of all-ceramic crowns and fixed partial dentures, metal-ceramic crowns still play an important role in oral rehabilitation and may be indicated and used in clinical practice for a long time, as they have a high rate of clinical success and proven longevity. the limitations of this study include the use of only two co-cr alloys for metal-ceramic crowns. other base metal alloys, such as ni-cr and ti-based alloys were not investigated in the present study. also, the marginal and internal fit of single crowns was investigated and the results may not be valid for multiple fixed partial dentures. the application of finger pressure to obtain the silicone replica, despite clinically significant, could have affected the reproducibility of the measurements. finally, since the adaptation of the crowns was evaluated under in vitro conditions, the results may not be directly extrapolated to the clinical practice. 8 kunz et al. in conclusion, the coping fabrication method influenced internal fit. ceramic application maintained or improved the fit of the metal-ceramic crowns. the marginal discrepancy of all restorations, before and after ceramic firing, can be considered clinically acceptable. data availability datasets related to this article will be available upon request to the corresponding author. references 1. huang z, zhang l, zhu j, zhang x. clinical marginal and internal fit of metal ceramic crowns fabricated with a selective laser melting technology. j prosthet dent. 2015 jun;113(6):623-7. doi: 10.1016/j.prosdent.2014.10.012. 2. makhija sk, lawson nc, gilbert gh, litaker ms, mcclelland ja, louis dr, et al. dentist material selection for single-unit crowns: findings from the national dental practice-based research network. j dent. 2016 dec;55:40-7. doi: 10.1016/j.jdent.2016.09.010. 3. sola-ruiz mf, agustin-panadero r, campos-estelles 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10.1007/s00784-010-0414-x. 30. gurel k, toksavul s, toman m, tamac e. in vitro marginal and internal adaptation of metal-ceramic crowns with cobalt-chrome and titanium framework fabricated with cad/cam and casting technique. niger j clin pract. 2019 jun;22(6):812-6. doi: 10.4103/njcp.njcp_570_18. 10 kunz et al. 31. roberts hw, berzins dw, moore bk, charlton dg. metal-ceramic alloys in dentistry: a review. j prosthodont. 2009 feb;18(2):188-94. doi: 10.1111/j.1532-849x.2008.00377.x. 32. real-voltas f, romano-cardozo e, figueras-alvarez o, brufau-de barbera m, cabratosa-termes j. comparison of the marginal fit of cobalt-chromium metal-ceramic crowns fabricated by cad/cam techniques and conventional methods at three production stages. int j prosthodont. 2017;30(3):304-5. doi: 10.11607/ijp.5038. 33. gemalmaz d, alkumru hn. marginal fit changes during porcelain firing cycles. j prosthet dent. 1995 jan;73(1):49-54. doi: 10.1016/s0022-3913(05)80272-0. 34. regish km, sharma d, prithviraj dr, nair a, raghavan r. evaluation and comparison of the internal fit and marginal accuracy of base metal (nickelchromium) and zirconia copings before and after ceramic veneering: a sem study. eur j prosthodont restor dent. 2013 mar;21(1):44-8. 35. weaver jd, johnson gh, bales dj. marginal adaptation of castable ceramic crowns. j prosthet dent. 1991 dec;66(6):747-53. doi: 10.1016/0022-3913(91)90408-o. 36. shokry te, attia m, mosleh i, elhosary m, hamza t, shen c. effect of metal selection and porcelain firing on the marginal accuracy of titanium-based metal ceramic restorations. j prosthet dent. 2010 jan;103(1):45-52. doi: 10.1016/s0022-3913(09)60216-x. 37. rahme hy, tehini ge, adib sm, ardo as, rifai kt. in vitro evaluation of the “replica technique” in the measurement of the fit of procera crowns. j contemp dent pract. 2008 feb;9(2):25-32. 38. an s, kim s, choi h, lee jh, moon hs. evaluating the marginal fit of zirconia copings with digital impressions with an intraoral digital scanner. j prosthet dent. 2014 nov;112(5):1171-5. doi: 10.1016/j.prosdent.2013.12.024. 39. pimenta ma, frasca lc, lopes r, rivaldo e. evaluation of marginal and internal fit of ceramic and metallic crown copings using x-ray microtomography (micro-ct) technology. j prosthet dent. 2015 aug;114(2):223-8. doi: 10.1016/j.prosdent.2015.02.002. 1http://dx.doi.org/10.20396/bjos.v19i0.8658335 volume 19 2020 e201574 original article 1 prosthodontic unit, faculty of odontology, franciscan university, santa maria, rio grande do sul, brazil. 2 prosthodontic unit, faculty of odontology, cnec faculty of santo angelo, santo angelo, rio grande do sul, brazil. 3 department of biomedical sciences and neuromotor, division of prosthodontics, alma mater studiorum, university of bologna, bologna, italy. 4 department of restorative dentistry, faculty of odontology, federal university of santa maria, santa maria, rio grande do sul, brazil. corresponding author: vinícius felipe wandscher, d.d.s., m.sci.d., ph.d., adjunct professor, franciscan university faculty of odontology prosthodontics-biomaterials unit r. silva jardim, 1175, 97010-491, rio grande do sul state, santa maria, brazil. phone/fax: +55 30251202/30259002 e-mail: viniwan@hotmail.com (dr vinicius) received: february 13, 2019 accepted: january 22, 2020 grinding of composite cores using diamond burs with different grit sizes: the effects on the retentive strength of zirconia crowns vinicius felipe wandscher1,*, ana maria estivalete marchionatti2, damiano lodi giuliani3, roberto scotti3, paolo baldissara3, luiz felipe valandro4 aim: to evaluate the retention of y-tzp crowns cemented in aged composite cores ground with burs of different grit sizes. methods: sixty composite resin simplified full-crown preparations were scanned, while 60 y-tzp crowns with occlusal retentions were milled. the composite preparations were stored for 120 days (wet environment-37°c) and randomly distributed into three groups (n=20) according to the type of composite core surface treatment. the groups were defined as: ctrl (control: no treatment), efb (extra-fine diamond bur [25µm]), and cb (coarse diamond bur [107µm]). the grinding was performed with an adapted surveyor standardizing the speed and pressure of the grinding. the intaglio surfaces on the crowns were air-abraded with silica-coated alumina particles (30 µm) and then a silane was applied. the crowns were cemented with self-adhesive resin cement, thermocycled (12,000 cycles; 5/55°c), stored (120 days) and submitted to a retention test (0.5mm/min). the retentive strength data (mpa) were analyzed using one-way analysis of variance and tukey test, as well as weibull analysis. failures were classified as 50c (above 50% of cement in the crown), 50s (above 50% of cement in the substrate) and coe (composite core cohesive failure). results: no statistical difference was observed among the retention values (p=0.975). however, a higher weibull modulus was observed in the ctrl group. the predominant type of failure was 50s (above 50% of cement in the substrate composite). conclusion: the retention of zirconia crowns was not affected by grinding using diamond burs with different grit sizes (coarse/extra-fine) or when no grinding was performed. keywords: composite resin. dental bonding. dental retention. surface properties. zirconium. mailto:viniwan@hotmail.com 2 wandscher et al. introduction yttria-stabilized tetragonal zirconia polycrystal (y-tzp) ceramics have been increasingly used over the years because of their superior flexural strength, flexural toughness, and phase-transformation toughening mechanism compared with traditional materials1. however, in spite of its excellent mechanical properties, zirconia is resistant to acid etching because of its highly crystalline microstructure, hence limiting adhesion to resin materials1,2. to improve the bond strength of zirconia to resin cements, tribochemical air-abrasion is commonly employed. this technique uses alumina particles coated with silica to generate micromechanical retention and a reactive surface for silanization1. although many strategies have been proposed to improve the bond strength of y-tzp to resin cements, there has been very little focus on the substrate over which the restoration is cemented. significant loss of coronal tissue is commonly observed in endodontically treated teeth, resulting in the need for post-retained restorations for both aesthetic and functional rehabilitation3. these restorations can be performed with prefabricated fiber-post cementation followed by a core build-up with composite resin3. recently, amaral et al.4 showed that the retention of y-tzp crowns is higher when cemented to dentin in comparison with composite resin. preparation for restorations includes core build-up, prosthetic preparation and impression. following these procedures, composite resin cores present few unreacted methacrylate groups at their surface, which reduce the potential for their adhesion to the resin cement5. during the clinical treatment with prosthetics, dentists will often use provisional restorations in patients before permanent restoration can be performed. during this period, the composite core build-up can be exposed to moisture, variances in ph and temperature6 and temporary luting cements7. as a result, surface alterations of the composite core could be required to improve their adhesion to resin cements and for optimal crown retention6. when a composite core is built up, its external surface is completely cured7-10. the interaction with the surrounding environment may promote water absorption, leading to softening of the matrix, the formation of micro-cracks, resin degradation, debonding of the filler/matrix interface, and leaching of some constituents8-10. some in vitro methods, such as thermocycling and water storage for different periods, can be used to simulate the aging process of resin-based composites that occurs in vivo11-13. thus, some studies have proposed that the surface treatment of aged resins could increase their adhesions to fresh resins. some of these techniques include: grinding with a diamond bur14-16 or with a diamond bur followed by acid etch/adhesive11,17,18, lasers irradiation19-23, air abrasion with aluminum oxide particles and silanization15,16,18,24, air abrasion with silica-coated alumina particles and silanization14,15,24,25, and treatment with hydrofluoric acid18,26-28. however, some of these techniques (e.g., air-abrasion and laser) require extra armamentarium in the clinical setting. as a result, the cost of the treatment increases29, a rubber dam is required to avoid damage to the patient’s periodontium and inhalation of particles24, and materials that are potentially unsafe (e.g., silica particles and hydrofluoric acid can be corrosive and toxic) are used intra-orally. 3 wandscher et al. in terms of crown retention, the retentive strength of ceramic crowns is associated with tooth preparation, as well as the type of luting material and polymerization used30,31. factors such as temperature, exposure to saliva, and mechanical stresses during mastication can influence the longevity of bond stability of the zirconia crown-resin cement-dentin complex32. in previous studies, when castings were cemented onto the surfaces of teeth using conventional cements (e.g., zinc phosphate), the rough surfaces seemed to influence the retention of crowns33,34. indeed, the retention quality of conventional cements is associated with both their physical strength and the micromechanical retention of the filler particles on the rough surface of the prepared tooth (and not with adhesive quality)33. however, with advances in adhesive technology for promoting adhesion between different substrates, there could be an increase in interaction between the composites used for core build-up and resin cements7. until now, we unknown studies has evaluated the use of diamond burs with different grit sizes of composite cores for finishing (i.e., grinding) on the retention of y-tzp crowns cemented with resin-luting cements. thus, the aim of this study was to evaluate the effect of finishing (i.e., grinding by diamond burs with different grit sizes) of the prosthetic preparation made of composite resin on the retentive strength of zirconia crowns. we tested the hypothesis that grinding with a coarse diamond bur (i.e., rougher surface) would generate higher retentive strength than grinding with an extra-fine diamond bur and no treatment (i.e., smoother surface). materials and methods composite core prosthetic preparation, aging, and finishing method split transparent templates were used to produce sixty composite resin prosthetic preparations (tetric evoceram, ivoclar vivadent, schaan, liechtenstein) with identical, simplified full-crown preparations (16 mm in total height: 6 mm in preparation height with a total occlusal convergence angle of 12° with rounded corners + 10 mm in base height). small portions of composite resin (2 mm) were inserted incrementally into the templates, until they were filled completely. next, a screw of 25 mm in height was screwed into the center of the composite. this screw was used to help with the fixation of the composite preparation sample in the embedding resin for the purpose of a retention test (figure 1). each surface of composite resin preparation was photo-activated for 20 s with a high-power led (1200 mw/cm2, radiical, sdi, bayswater, vic, australia) and placed into a vacuum-mirrored polymerization chamber (visio™ beta vario light unit, 3m espe, seefeld, by, germany) using a specific protocol for photo curing materials (1 min of light followed by 1 min of vacuum and light) to increase the conversion degree. afterwards, the composite preparations were stored in a bacteriological furnace (wet environment, 37°c) for 120 days11-13. after aging, the composite preparations were assigned to three groups (n=20) according to the grit size of the diamond bur used to finish the surface: • ctrl (control): without roughening the surface, • extra-fine bur: roughening the surface with an extra-fine bur (878ef.314.014 – parallel-chamfer, torpedo – komet, gebr. brasseler, lemgo, nw, germany), 4 wandscher et al. • coarse bur: roughening the surface with a coarse bur (878.314.014 – parallel-chamfer, torpedo – komet, gebr. brasseler, lemgo, nw, germany). to perform the grinding procedures, the composite core preparations were placed in a rotatory mounting of a purposely-built device allowing the core to rotate counter-clockwise around its own axis at a speed of 30 rpm. the diamond burs (extra-fine and coarse bur) were installed on a handpiece (kavo dental gmbh/kaltenbach & voigt gmbh, biberach an der riß, bw, germany) oriented to steadily hold the bur axis parallel to the composite core surface. the bur rotated at 20.000 rpm in the opposite direction (clockwise) of that of the core. the whole rotatory mounting was positioned above a movable x-y micrometric table: this arrangement allowed a standardized core grinding by setting the cutting depth on a dial caliber (make, model, 0.001 micron resolution). a cutting depth pattern of 5.0 ± 1 µm with a total of three rounds (or revolutions) for each preparation was performed (figure 2a, 2b, 2c). the abrasion was carried-out under water cooling exclusively on the axial surfaces, preserving the preparation shoulder (chamfer) which remained intact. in the manner here described, it was possible to standardize the same geometry and surface type in each composite core specimen. figure 1. split transparent templates used to produce the composite cores (note the screw on the composite base center). figure 2. a. special device for grinding the composite preparations. b. composite core after grinding (bur positioned parallel to the composite surface). c. micrometer installed onto a movable x-y table to standardize the grinding pressure. a rotatory base movable x-y table b c 5 wandscher et al. zirconia crowns production, cementation, and aging each composite preparation was scanned (ineos x5, sirona dental systems, bensheim, germany) and the images were transferred to the inlab software (sirona sw 15.0, sirona). crowns with occlusal retentions were designed for each preparation and the y-tzp crowns were milled by a milling machine (cerec inlab mc xl4, sirona) (ips e.max zircad for inlab c-15, dimensions of 14.5 x 15.5 x 18.5 mm3, ivoclar vivadent) with a cement space of 80 µm. sintering was produced according to the manufacturer’s instructions (zircomat, vita zahnfabrik, bad säckingen, germany). the crowns of each preparation were checked for passive adaptation (carbono arti-spray, bausch, bausch articulating papers, inc., nashua, nh, usa) and cleaned with an ultrasonic device (1440 d – odontrobras, ind. & com. equip. méd. odonto. ltda, ribeirao preto, sp, brazil) and distilled water for 10 min. to standardize the procedure, the intaglio surface of y-tzp crowns were air-abraded with an adapted device using silica-coated aluminum oxide particles (30 µm) (cojet sand, 3m espe, seefeld, by, germany) at a distance of 15 mm for 10 s and a pressure of 2.8 bar35. a coupling agent based on methacryloxypropyltrimethoxysilane (relyx ceramic primer s, 3m espe) was applied with a microbrush and crowns were left untouched for 5 min to allow for evaporation of the solvent. self-adhesive resin cement (relyx u200, 3m espe) was manipulated according to the manufacturer’s instructions and applied to the intaglio surface of the crowns, which were positioned on the composite preparation. with an adapted surveyor (b2, bioart, sao carlos, sp, brazil), a load of 750 g was applied to the crown, the cement excess was removed, and photo-activation was performed for 20 s on each surface (1200 mw/cm2, radii-cal, sdi, bayswater, vic, australia). the specimens were stored in distilled water (37°c) for 24 h, submitted to thermocycling (12.000 cycles; 5°c-55°c; 30 s per bath and 5 s between baths; ethik technology, vargem grande paulista, sp, brazil), and then stored for 30 days in a wet environment at 37°c. embedding and retentive strength test before a retentive strength test, the specimens were partially embedded inside the acrylic resin to fix the zirconia crown and the composite preparations. the margins of the crown preparations were kept free for testing. first, the crown from the crown/ preparation assembly was fixed onto an adapted surveyor perpendicular to the x axis (b2, bioart) to keep the adequate orientation of the specimen. subsequently, this preparation allowed the base of the composite preparation to be embedded in self-curing resin (vipi flash, vipi, pirassununga, sp, brazil) until 2 mm above the marginal zone. after acrylic-resin polymerization, the previously embedded part was fixed onto the surveyor perpendicular to x axis (for the same aforementioned reason) and the zirconia crown was embedded until the occlusal retentions were covered. both parts were then embedded using metallic templates with transversal holes that allowed for the attaching of the superior part (crown) and inferior part (composite preparation) in the universal testing machine (dl-1000, emic, são josé dos pinhais, pr, brazil). the superior part was fixed to a load cell (1000 n) which was attached to movable axle of the testing machine, while the inferior part was fixed at the fixed base of the machine. next, a retention force (pull-out) was applied until failure (0.5 mm/min). 6 wandscher et al. adhesive area calculation the amount of adhered area (129 mm2) was calculated by solidworks software (ds solidworks corporation, waltham, ma, usa) according to the measures of the composite cores. the retentive strength (r) was calculated using the formula: r = fmax/a, where fmax = maximum force for failure (decementation) and a = adhered area. failure analysis to evaluate the type of fracture, the tested assemblies were analyzed under a stereomicroscope (discovery v20, carl-zeiss, gottingen, ni, germany), and the fractures were classified according to the localization of the largest portion of cement. these classifications are described as follows: 50c (more than 50% of the cement on the crown), 50s (more than 50% of the cement on the substratum (composite core preparation)), and coe (cohesive failure of composite preparation) (these data were not included in the statistical analysis). representative images were taken with a scanning electronic microscope (sem) (jsm-6360lv, jeol usa, inc., peabody, ma, usa). this classification was adapted from amaral et al.4 and rippe et al.6. data analysis the retentive strength data were statistically analyzed with the spss software (version 21, ibm, chicago, il, usa). both normality and homoscedasticity were verified, and the data were subjected to one-way analysis of variance (anova) and post-hoc tukey’s test. the reliability of the retentive strength values (m: weibull modulus) and the characteristic retentive strength (σ0: strength value at which 63.2% of the specimens survive) were performed by a weibull analysis. results a one-way anova showed no statistical difference among the retention values (p=0.975) (table 1). in addition, no difference in characteristic strength (σ0) was table 1. means (standard deviation) of the tensile strength (mpa), weibull analysis (m= modulus; σ0= characteristic tensile resistance (mpa); ic= confidence interval), and percentage of failure types. groups tensile strength* weibull parameters** failures*** m ic σ0 ic 50c (%) 50s (%) coe (%) no-treatment (control) 2.1 (0.41)a 5,9a 3.3 – 8.3 3.4a 3 – 3.8 5 (25) 9 (45) 6 (30) coarse diamond bur 2.03 (1.03)a 2,1b 1.2 – 2.9 3.3a 2.4 – 4.5 15 (75) 5 (25) extra-fine diamond bur 2.04 (0.97)a 2,2b 1.3 – 3 3.3a 2.5 – 4.5 1 (5) 16 (80) 3 (15) *different lowercase letters in the same column indicate a significant difference. ** different lowercase letters in the same column indicate a significant difference (no overlap of the confidence intervals) *** 50s: more than 50% of cement adhered on the substratum; 50c: more than 50% of cement adhered on the crown. coe: cohesive failure: composite die fracture. 7 wandscher et al. observed. however, the weibull modulus (m) was higher in the ctrl group compared with the cb and efb groups (overlap of confidence intervals). the most common type of failure was 50s (more than 50% of cement adhered to the substratum) (figure 3). figure 3. representative scanning electric microscopies. a. zirconia crown of the coarse bur group (white arrow: circular machining marks on the internal occlusal surface; black arrow: axial machining marks on the axial internal surface; red arrow: semicircular machining marks on crown shoulder). b. composite core of the coarse bur group (it is possible to note that the resin cement layer is adhered totally on the core and the machining marks are reproduced on the cement layer) – 50s failure. c. zirconia crown of the ctrl group (yz: zirconia and cem: cement) – cement partially adhered on crown. d. composite core of the ctrl group (major part of cement adhered on composite surface) and machining marks on the layer cement – 50s failure. e. zirconia crown of the extra fine bur group – cohesive failure (part of the core fracture into crown. f. composite core of the extra fine group (red circle: fractured occlusal third; cement adhered on the composite shoulder with semicircular machining marks). a c f comp comp comp comp cemcem cem cem cem cem cem cem eht = 20.00 kv wd = 29.2 mm signal a = vpse g4 mag = 45x eht = 20.00 kv wd = 39.4 mm signal a = vpse g4 mag = 33x eht = 20.00 kv wd = 31.7 mm signal a = vpse g4 mag = 40x eht = 20.00 kv wd = 34.2 mm signal a = vpse g4 mag = 38x eht = 20.00 kv wd = 38.4 mm signal a = vpse g4 mag = 33x eht = 20.00 kv wd = 32.6 mm signal a = vpse g4 mag = 38x 1 mm 1 mm 1 mm 1 mm1 mm 1 mm yz yz yz yzyz b d 8 wandscher et al. discussion the retentive strengths of the three groups were found to be statistically similar (table 1). therefore, our formulated research hypothesis was rejected. other studies have used bond strength tests with simplified geometry (shear or tensile bond strength on flat surfaces) to evaluate bonding to aged composites36-39. however, these studies have shown conflicting results. in relation to the surface treatment with burs, our results align with other studies that showed no effect of burs on composite-composite bonding36,37. in contrast, valente et al.38 and costa et al.16 showed that surface roughening with diamond burs improved the tensile bond strength to new composites. however, these studies used intermediate agents between the aged and fresh composite layers, which could have enhanced the adhesion. bonstein et al.39 suggested that surface treatment with only a diamond bur on aged composites is simple, efficient, and does not require additional dental materials or instrumentation. other methods of surface roughening were tested, including sandpapers7,24,40, abrasive stone11, and pumice41, but for these studies the increased bonding is associated with surface grinding, followed by the application of a primer/adhesive. we chose to test burs for their finishing abilities (i.e., surface treatments) because the method is simple, has low cost and is available in the dental office. notably, we did not apply any intermediate agent since a self-adhesive resin cement was chosen to lute the y-tzp crowns. this cement is easier and less technical to use, and promotes similar bond strength to conventional luting resin cements42-44. most failures were classified as 50s (more than 50% of cement adhered to substratum) (table 1). these findings agree with those of both amaral et al.4 and rippe et al.6, who also used composite cores finished with fine diamond burs and observed that failure occurred between the cement and zirconia (adhesive failure). a main explanation for a non-significant result could be the association of resin materials (e.g., composite resin and resin cement) with similar chemical compositions7, thus favoring a bond between them. it is possible that the rougher surface produced with an extra-fine bur and coarse bur had no effect on retentive strength because of the similar compositions of resin cement and composite resin. in contrast, other studies demonstrated that the majority of cement was adhered to the intaglios of zirconia crowns after thermocycling32,44,45. however, dentin substrate was used in these studies. in addition, failure analysis showed that the cement remained attached on the internal occlusal surface of the zirconia crown for some samples in the extra-fine and coarse bur groups (figure 3). this result possibly occurred because the occlusal surface of the composite core was not prepared and, therefore, the cement remained adhered on ground axial surfaces of the composite. notably, this result was also observed by palacios et al.44. amaral et al.4 and rippe et al.6 did not evaluate the roughness of the composite core on zirconia crown retention. however, both of these studies presented failure patterns similar to those observed in our study, which used composite cores and resin cements. hence, independently of surface treatment, factors including the type of substratum, resin cement, and taper preparation can be more important than surface roughness in influencing the retention of zirconia crowns. 9 wandscher et al. taper preparation is another factor that could have affected the retention values. kaufman et al.46 examined the effect of variation of the convergence angle (1°, 5°, 10°, 15° and 20°) on crown retention and showed that retention increases as the convergence angle decreases. in our study, we utilized the total convergence angle of 12° and, consequently, the retentive effect may have been higher than both bonding and roughness effects. this convergence angle could have contributed to cohesive failure as observed by this study, amaral et al.4, and rippe et al.6. despite the similar retention strengths depicted by our findings, the weibull modulus of control group (ctrl) was higher (higher reliability) than the efb and cb groups (table 1). ayad et al.47 stated that excessive roughness could lead to trapped air between the cement and tooth preparation, which could cause adhesive failure; this event could have occurred in the current study. furthermore, the standard deviation of the control group was lower than in the efb and cb groups, possibly due to the fact that the procedure could have promoted heterogeneous morphological surface patterns on the treated cores. there were some limitations of our study. first, unreal retention values were generated with cohesive failures of the composite cores. as a result, these data were removed from the statistical analysis to avoid overestimation or underestimation of the retention values. in a prior study, cohesive failure occurred before reaching the maximum load supported by adhesive interfaces44. in the current study, cohesive failures varied from 15% to 30%, depending on the group (table 1). it is important to emphasize that if this type of failure had not occurred, the retention values would probably be higher. these failures may be associated with taper preparation — if the convergence angle had been greater, maybe the cohesive failures would not have occurred. secondly, composite preparations were created using highly standardized procedures. in clinical practice, however, dental tissue will be present at the chamfer preparation when restoring endodontically treated teeth with posts and composite cores. therefore, it is difficult to compare our results with those in the current literature. indeed, most studies employ different methodologies including varying geometries of the preparation, as well as different taper preparations, resin cements, and substrate. further studies should be performed with other types of aging, composite-core surface treatments, luting cements (e.g., zinc phosphate, glass ionomer, resin modified glass ionomer, and resin cement of different compositions), adhesive techniques, and taper preparations. finally, tests applying intermittent loading and fatigue investigations should also be conducted. in conclusion, the retention of zirconia crowns cemented with self-adhesive resin cement was not affected by grinding using diamond burs with different grit sizes on composite resin preparations with a convergence angle of 12º. references 1. thompson jy, stoner br, piascik jr, smith r. adhesion/cementation to zirconia and other non-silicate ceramics: where are we now? dent mater. 2011 jan;27(1):71-82. doi: 10.1016/j.dental.2010.10.022. 2. aboushelib mn, feilzer aj, kleverlaan cj. bonding to zirconia using a new surface treatment. j prosthodont. 2010 jul;19(5):340-6. doi: 10.1111/j.1532-849x.2010.00575.x. 10 wandscher et al. 3. aurelio il, fraga s, rippe mp, valandro lf. are posts 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resin based composite restorations. am j dent. 2003 feb;16(1):17-22. 14. ozcan m, barbosa sh, melo rm, galhano ga, bottino ma. effect of surface conditioning methods on the microtensile bond strength of resin composite to composite after aging conditions. dent mater. 2007 oct;23(10):1276-82. 15. perriard j, lorente mc, scherrer s, belser uc, wiskott hw. the effect of water storage, elapsed time and contaminants on the bond strength and interfacial polymerization of a nanohybrid composite. j adhes dent. 2009 dec;11(6):469-78. doi: 10.3290/j.jad.a18141. 16. costa tr, ferreira sq, klein-júnior ca, loguercio ad, reis a. durability of surface treatments and intermediate agents used for repair of a polished composite. oper dent. 2010 mar-apr;35(2):231-7. doi: 10.2341/09-216-l. 17. papacchini f, dall’oca s, chieffi n, goracci c, sadek ft, suh bi, et al. composite-to-composite microtensile bond strength in the repair of a microfilled hybrid resin: effect of surface treatment and oxygen inhibition. j adhes dent. 2007 feb;9(1):25-31. 18. loomans ba, cardoso mv, roeters fj, opdam nj, de munck j, huysmans mc, et al. is there one optimal repair technique for all composites? dent mater. 2011 jul;27(7):701-9. doi: 10.1016/j.dental.2011.03.013. 19. polat s, cebe f, tunçdemir a, öztürk c, üşümez a. evaluation of the bond strength between aged composite cores and luting agent. j adv prosthodont. 2015 apr;7(2):108-14. doi: 10.4047/jap.2015.7.2.108. 20. burnett lh jr, shinkai rs, eduardo cde p. tensile bond strength of a one-bottle adhesive system to indirect composites treated with er:yag laser, air abrasion, or fluoridric acid. photomed laser surg. 2004 aug;22(4):351-6. 11 wandscher et al. 21. kimyai s, mohammadi n, navimipour ej, rikhtegaran s. comparison of the effect of three mechanical surface treatments on the repair bond strength of a laboratory composite. photomed laser surg. 2010 oct;28 suppl 2:s25-30. doi: 10.1089/pho.2009.2598. 22. lizarelli rde f1, moriyama lt, bagnato vs. ablation of composite resins using er:yag laser-comparison with enamel and dentin. lasers surg med. 2003;33(2):132-9. 23. correa-afonso am, pecora jd, palma-dibb rg. influence of pulse repetition rate on temperature rise and working time during composite filling removal with the er:yag laser. photomed laser surg. 2008 jun;26(3):221-5. doi: 10.1089/pho.2007.2120. 24. cotes c, cardoso m, melo rm, valandro lf, bottino ma. effect of composite surface treatment and aging on the bond strength between a core build-up composite and a luting agent. j appl oral sci. 2015 jan-feb;23(1):71-8. doi: 10.1590/1678-775720140113. 25. rinastiti m, özcan m, siswomihardjo w, busscher hj. effects of surface conditioning on repair bond strengths of non-aged and aged microhybrid, nanohybrid, and nanoflled composite resins. clin oral investig. 2011 oct;15(5):625-33. doi: 10.1007/s00784-010-0426-6. 26. loomans ba, cardoso mv, opdam nj, roeters fj, de munck j, huysmans mc, et al. surface roughness of etched composite resin in light of composite repair. j dent. 2011 jul;39(7):499-505. doi: 10.1016/j.jdent.2011.04.007. 27. lucena-martín c, gonzález-lópez s, navajas-rodriguez de mondelo jm. the effect of various surface treatments and bonding agents on the repair strength of heat-treated composites. j prosthet dent. 2001 nov;86(5):481-8. 28. passos sp, özcan m, vanderlei ad, leite fp, kimpara et, bottino ma. bond strength durability of direct and indirect composite systems following surface conditioning for repair. j adhes dent. 2007 oct;9(5):443-7. 29. özcan m, corazza ph, marocho sm, barbosa sh, bottino ma. repair bond strength of microhybrid, nanohybrid and nanofilled resin composites: effect of substrate resin type, surface conditioning and ageing. clin oral investig. 2013 sep;17(7):1751-8. doi: 10.1007/s00784-012-0863-5. 30. sun r, suansuwan n, kilpatrick n, swain m. characterisation of tribochemically assisted bonding of composite resin to porcelain and metal. j dent. 2000 aug;28(6):441-5. 31. stewart gp, jain p, hodges j. shear bond strength of resin cements to both ceramic and dentin. j prosthet dent. 2002 sep;88(3):277-84. 32. ehlers v, kampf g, stender e, willershausen b, ernst cp. effect of thermocycling with or without 1 year of water storage on retentive strengths of luting cements for zirconia crowns j prosthet dent. 2015 jun;113(6):609-15. doi: 10.1016/j.prosdent.2014.12.001. 33. oilo g, jørgensen kd. the influence of surface roughness on the retentive ability of two dental luting cements. j oral rehabil. 1978 oct;5(4):377-89. 34. felton da, kanoy be, white jt. (1987) the effect of surface roughness of crown preparations on retention of cemented castings. j prosthet dent. 1987 sep;58(3):292-6. 35. amaral r, ozcan m, valandro lf, bottino ma. effect of conditioning methods on the microtensile bond strength of phosphate monomer-based cement on zirconia ceramic in dry and aged conditions. j biomed mater res b appl biomater. 2008 apr;85(1):1-9. 36. bouschlicher mr, reinhardt jw, vargas ma. surface treatment techniques for resin composite repair. am j dent. 1997 dec;10(6):279-83. 37. shen c, mondragon e, gordan vv, mjör ia. the effect of mechanical undercuts on the strength of composite repair. j am dent assoc. 2004 oct;135(10):1406-12; quiz 1467-8. 38. valente ll, silva mf, fonseca as, münchow ea, isolan cp, moraes rr. effect of diamond bur grit size on composite repair. j adhes dent. 2015 jun;17(3):257-63. doi: 10.3290/j.jad.a34398. 12 wandscher et al. 39. bonstein t, garlapo d, donarummo j jr, bush pj. evaluation of varied repair protocols applied to aged composite resin. j adhes dent. 2005 spring;7(1):41-9. 40. staxrud f, dahl je. role of bonding agents in the repair of composite resin restorations. eur j oral sci. 2011 aug;119(4):316-22. doi: 10.1111/j.1600-0722.2011.00833.x. 41. padipatvuthikul p, mair lh. bonding of composite to water aged composite with surface treatments. dent mater. 2007 apr;23(4):519-25. 42. de munck j, vargas m, van landuyt k, hikita k, lambrechts p, meerbeek b. bonding of an auto-adhesive material to enamel and dentin. dent mater. 2004 dec;20(10):963-71. 43. piwowarczyk a, lauer hc, sorensen ja. in vitro shear bond strength of cementing agents to fixed prosthodontic restorative materials. j prosthet dent. 2004 sep;92(3):265-73. 44. palacios rp, johnson gh, philips km, raigrodski aj. retention of zirconium oxide ceramic crowns with three types of cement. j prosthet dent. 2006 aug;96(2):104-14. 45. ernst cp, aksoy e, stender e, willershausen b. influence of different luting concepts on long term retentive strength of zirconia crowns. am j dent. 2009 apr;22(2):122-8. 46. kaufman eg, coelho dh, colin j. factors influencing the retention of cemented gold castings. j prosthet dent. 1961 may-jun;11(3):487-502. doi: 10.1016/0022-3913(61)90232-3. 47. ayad mf, rosenstiel sf, hassan mm. surface roughness of dentin after tooth preparation with different rotary instrumentation. j prosthet dent. 1996 feb;75(2):122-8. 1 volume 21 2022 e225454 original article braz j oral sci. 2022;21:e225454http://dx.doi.org/10.20396/bjos.v21i00.8665454 1 department of restorative dentistry, university of pernambuco, recife, pe, brazil. 2 department of restorative dentistry, federal university of paraíba, joão pessoa, pb, brazil. 3 department of restorative dentistry, faculty of nova esperança , joão pessoa, pb, brazil. corresponding author: caroline de farias charamba street joaquim ferreira da costa, 40 58038-540, joão pessoa, pb, brazil phone: 55 83-996074931 e-mail: carolfariasch21@gmail.com editor: altair a. del bel cury received: april 26, 2021 accepted: december 12, 2021 bond strength of bulk-fill resin composites: the effect of cavity preparation and aging caroline de farias charamba1,* , larissa dias boson silva2 , renally bezerra wanderley lima3 , rosângela marques duarte2 , ana karina maciel andrade2 aim:  evaluating the resin-dentin bond strength of class ii conventional and bulk-fill composite restorations, using different cavity sizes before and after aging. methods: seventy-five human molars were distributed into groups according to the buccolingual width of the cavities, conservative (n=25) and extended (n=50). they were divided according to the restorative material: conventional (z100/control group) or bulk-fill resin composites (filtek bulk fill/fbf; tetric n ceram bulk fill/tncbf; filtek bulk fill flow/fbff; surefill sdr flow/sdr). the restored teeth were sectioned on sticks (n=50 per restorative materials + width cavities group), half were stored in water/ethanol 75% for 30 days and the other half were submitted to the immediate microtensile bond strength (μtbs) test. data were analyzed applying the three-way analysis of variance (anova), bonferroni test, test t, and weibull analyses (p<0.05). results: sdr and fbf presented lower μtbs values for extended preparation when compared to the conservative preparation, before aging. after aging, only for the fbff, a decrease in the μtbs values was observed. comparing the μtbs values, before and after aging, the sdr demonstrated lower μtbs values after aging when the conservative cavity was used.  a decrease in the μtbs values was observed for the z100, the fbf and, the fbff, after aging, when the extended cavity was used. conclusion: the effect of cavity preparation and aging on the resin-dentin of class ii is material dependent. most of the bulk-fill resin composites evaluated presented a similar performance to the conventional resin composites for all the conditions of this study. keywords: composite resins. tensile strength. aging. dental cavity preparation. https://orcid.org/0000-0002-5811-1796 https://orcid.org/0000-0002-6091-1063 https://orcid.org/0000-0003-4477-7850 https://orcid.org/0000-0003-4369-2951 https://orcid.org/0000-0003-4520-5176 2 charamba et al. braz j oral sci. 2022;21:e225454 introduction bulk-fill resin composites have been used by clinicians to simplify dental operative procedures. these resin composites were introduced to be inserted in a single increment of 4–5mm, being an attractive alternative for posterior restorations1. manufactures have applied different strategies to formulate a material presenting better light transmission and reduced polymerization stress. to improve the depth of polymerization, alternative and more reactive photoinitiators, as well as lower filler concentrations, are used2-5. modified monomers, such as novel stress-relieving monomers and methacrylate monomers, containing a third reactive site, have been incorporated into the bulk-fill resin composites to reduction of the polymerization stress2-5. two types of bulk-fill composites viscosity are available: low-viscosity and high-viscosity. low-viscosity bulk-fill resin composite is indicated to replace dentin, filling most of the cavity, followed by capping with the conventional resin composites. using high-viscosity bulk-fill resin composites, only one increment can be applied and sculpt the occlusal surface simultaneously6. some factors, including polymerization shrinkage of the resin composites, may negatively affected clinical durability of resin composite restorations7. polymerization shrinkage stress may create tensile stress on the adhesive tooth restoration interface, affecting the bond strength and the marginal integrity of restorations8. as a result, some clinical consequences such as post-operative hypersensitivity, marginal discoloration, cohesive tooth fractures at the margins, recurrent caries and pulpal inflammation can be observed9. to provide better sealing for the cavity margins, bulk-fill resin composites have been developed. these resin composites seem to be an interesting option to enhance the resin-adhesive bonding to the tooth structure in regions without adequate marginal integrity, such as the cervical margins of class ii cavities10. micro-leakage and bond strength tests, associated with artificial aging, have been used to investigate marginal integrity and bonding quality to tooth of resin composite-restorations11. also is suggested that artificial aging has influence on the integrity tooth-composite interface12. controversial results about the bulk-fill resin composites presenting better sealing of the cavity margins and adequate bond strength to the dental substrate have been reported in the literature5,12,13. consequently, clinicians are still insecure about the use of this new class of materials in the clinical practice11. therefore, this in vitro study aimed to evaluate the effect of the cavity size and artificial aging on the resin-dentin bond strength of class ii conventional and bulk-fill composite restorations. the following experimental hypotheses were tested: 1) conservative cavity size will have better resin-dentin bond strength of class ii conventional and bulk-fill composite restorations than the extended cavity; 2) artificial aging will have effect on the resin-dentin bond strength of class ii conventional and bulk-fill composite restorations; 3) the resin-dentin bond strength of the conventional and the bulk-fill composite restorations will be comparable. 3 charamba et al. braz j oral sci. 2022;21:e225454 materials and methods tooth selection and experimental groups seventy-five healthy human third molars were used in this study after the approval from the research ethics committee of the university of paraiba (protocol n. 2.048.942). the teeth inspection was performed using an optical microscopy to select only teeth free from caries and with no cracks or developmental defects. after the selection, the teeth were cleaned, stored in a 0.2% thymol solution and used within one month after extraction. all tooth roots were embedded in self-curing acrylic resin. initially, the teeth were randomly distributed into groups according to the combination of the buccolingual width, conservative (n=50) and extended (n=25). this difference in the number of teeth between the groups is because conservative preparations provide smaller number of toothpicks than extended group. this step is better described below. a second distribution was made according to the resin composite used. three types of bulk-fill resin composites were used: filtek bulk fill, 3m espe dental products (fbf), tetric n ceram bulk fill, ivoclar vivadent, (tncbf), filtek bulk fill flow 3m espe dental products (fbff), surefil sdr flow, dentsply (sdr) and a conventional resin composite z100, 3m espe dental products, (z100). tested materials are in the table 1. table 1. tested materials composite composition batch number z100 (3m) bis-gma, tegdma, zirconia/silica with 71% weight by volume). particle size: 0.01 to 3.5 µm (average: 0.6 µm). 1822500253 filtek bulk fill (3m) audma, udma and 1,12-dodecane-dma. zirconia (4-11 nm) and silica (20 nm) that can be aggregated and agglomerated or not. iterbium trifluoride from agglomerated particles (100 nm). 76,5% by weight (58.4% by volume). n920657 tetric n ceram bulk fill (ivoclar) bis-gma, bis-ema and udma (19-21% by weight) and 75-77% by weight (53-55% by volume) inorganic particles (average: 0.6 µm). the filler consists of barium glass, prepolymer, ytterbium trifluoride and mixed oxides. the particle size of the inorganic fillers is between 0.04 and 3 μm. w94624 filtek bulk fill flow (3m) bis-gma, udma, bis-ema 6 and procrylat. ytterbium trifluoride and zirconia/silica with 64.5% by weight (42.5% by volume). particle size, respectively: 0.1 to 5.0 microns and 0.01 to 3.5 μm. 1531700424 surifil sdr (dentsply) ebpadma, tegdma, camphoroquinone (cq) as photoinitiator; photoaccelerator; hydroxy toluene butylate (bht); uv stabilizer; titanium dioxide; fluorescent agents. particle size: 20nm to 10μm, and the charge content by volume is about 47.3%. 150827 4 charamba et al. braz j oral sci. 2022;21:e225454 specimen preparation and restorative procedure the cavities were prepared according to standardized dimensions: occlusal box deep was 3mm and mesiodistal length at the bottom of the proximal box was 5mm. the proximal box (mesially and distally) was 5mm deep with margins located 1mm below the cemento-enamel junction. each cavity had the inner walls perpendicular to the top and bottom surfaces, with round angles defined by the bur’s shape. teeth were distributed into two groups according to the buccolingual width: conservative cavity (2mm wide in the buccolingual direction) and extended cavity (4mm wide in the buccolingual direction). the cavities were prepared using a diamond bur under water cooling (#1150, kg soresen; barueri, sp, brazil).  the two-step etch and rise adhesive adper single bond 2 (3m espe, st. paul, mn, usa) was applied following the manufacturers’ instructions. after the adhesive application, a metal matrix band was placed, and the teeth were restored according to the restorative material: conventional or bulk-fill composite resin. the conventional composite (z100-3m espe st. paul, mn, usa) was placed in a 1−1.5mm thick horizontal layer, applying an incremental technique. each increment was separately light cured for 20 s (800 mw/cm2, emitter c, schuster, santa maria, rs, brazil). the bulk-fill resin composites were applied in a 3.5 to 4-mm layer and then, light cured, following the manufactures instructions. the restored teeth were stored at 37 °c (±1°c) in distilled water/ethanol 75% for 24 hours. a single operator performed all procedures. after storage time, the proximal box of restorations was longitudinally sectioned in the mesiodistal and buccolingual directions across the bonded interface. the sections were executed using a slow-speed with a diamond saw in a lab-cut 1010 machine (extec, enfield, ct, usa) underwater cooling to obtain resin-dentin sticks with a rectangular cross-sectional area of approximately 1mm2. for each group (conservative and extensive of each restorative material)., fifty sticks were obtained from proximal boxes. twenty-five sticks were submitted to microtensile bond strength testing and the other half was stored at 37 °c (±1°c) in distilled water/ethanol 75% for 30 days.  microtensile bond strength testing (μtbs) the μtbs testing was performed with a crosshead speed of 5 mm/min using a universal testing machine (odeme, luzerna, sc, brazil). the sticks were attached to a modified microtensile testing device with cyanoacrylate resin (super bonder, loctite; são paulo, sp, brazil). to obtain μtbs (mpa) values, the measured force (n) was divided by the individual bonded area (mm2). when sticks failed while being sectioned or attached to the tester, they were excluded from the study. the failure mode was evaluated at 200x using light stereo microscopy (hmv-2, shimadzu, kyoto, japan). the failure modes were categorized as follow: cohesive failure in the adhesive (type i), cohesive failure in the dentin (type ii), cohesive failure in the hybrid layer (type iii), mixed failure (cohesive failure in the adhesive and in the hybrid layertype iv), cohesive failure in the resin composite (type v). statistical analysis the μtbs data were subjected to the kolmogorov-smirnov test to verify the normality. then, the data were analyzed using a three-way analysis of variance (anova) and 5 charamba et al. braz j oral sci. 2022;21:e225454 post hoc bonferroni test, as well as the test t at 0.05 level of significance. to evaluate the reliability of the bond strength, the weibull analysis was applied for each group. the weibull moduli (shape parameter) (slope of the line relating applied stress and the probability of specimen failure, m) were calculated, applying maximum likelihood estimation. the 95% upper and lower confidence intervals were calculated using the likelihood ratio (minitab 17.0, state college, pennsylvania, usa). results comparing the μtbs values of the conservative and the extended cavities, the sdr bulk-fill composite (p=0,03) and the filtek bulk fill flow (p=0,04) presented lower μtbs values for the extended preparation before artificial aging. on the other hand, a decrease in the μtbs values was observed only for the filtek bulk fill flow (p=0,01) after aging (table 2).  table 2 shows the results of the μtbs values, comparing the values before and after artificial aging for conservative and extended cavities. regarding the conservative cavity, the sdr bulk-fill (p=0,01) composite demonstrated lower μtbs values after 30 daysstorage in distilled water/ethanol. a decrease in the μtbs values was observed for the z100 (p=0,01), the filtek bulk fill (p=0,03), and the filtek bulk fill flow (p=0,03) after artificial aging when the extended cavity was used. no significant difference between the resin composites in the μtbs values was noted, before or after the aging process. table 3 shows the results of the failure mode analysis after bond testing, revealing that most of the failures were mixed fractures for all experimental conditions. the results of the weibull analysis are showed in table 4 and figure 1. no difference in the m values was observed for all experimental groups and conditions. table 2. means and standard deviation of μtbs values for resin composites studied (mpa) storage composite conservative extended before z100 31,48 (13,29) aa 29,22 (9,61) aa fbf 30,13 (14,60) aa 32,36 (13,92) aa tncbf 29,48 (14,63) aa 28,90 (10,44) aa fbff 33,67 (16,11) aa 25,06 (11,31) ab sdr 35,36 (16,24) aa 27,24 (10,89) ab after z100 28,28 (12,34) aa 21,69 (10,83) bb fbf 23,04 (9,73) ba 24,68 (9,98) ba tncbf 24,39 (9,77) aa 26,19 (12,90) aa fbff 27,09 (13,77) aa 18,84 (8,73) bb sdr 24,77 (13,56) ba 22,52 (9,77) aa different letters represent significant differences (p < 0.05): uppercase within columns (for before and after storage independently); lowercase within rows. 6 charamba et al. braz j oral sci. 2022;21:e225454 table 3. classification of failure modes (%) before and after storage before storage extended conservative failure modes i ii iii iv v i ii iii iv v z100 0% 0% 0% 84% 16% 0% 0% 0% 84% 16% fbf 16% 0% 0% 48% 36% 16% 0% 0% 48% 36% tncbf 4% 4% 0% 80% 12% 4% 4% 0% 80% 12% fbff 12% 0% 0% 80% 8% 12% 0% 0% 80% 8% srd 4% 12% 0% 76% 8% 4% 12% 0% 76% 8% after storage extended conservative failure modes i ii iii iv v i ii iii iv v z100 8% 0% 0% 67% 16% 0% 0% 0% 80% 20% fbf 16% 0% 0% 48% 36% 4% 0% 0% 76% 20% tncbf 12% 0% 0% 76% 12% 8% 4% 0% 72% 16% fbff 4% 0% 0% 80% 16% 8% 0% 0% 82% 10% srd 9% 0% 0% 79% 12% 7% 0% 0% 83% 12% type i cohesive failure in adhesive; type ii cohesive failure in detin; type iii cohesive failure in hybrid layer; type iv mixed failure (cohesive failure in adhesive and hybrid layer);type v cohesive failure in resin composite. table 4. weibull moduli (m) values, among the experimental groups comparing the resin composites for conservative and extended cavity before (24 h) and after storage (30 days). storage time composite conservative extented 24 hours z100 2.63 (1.93-3.58)aa 2.85 (2.30-3.54)aa fbf 2.26 (1.65-3.08)aa 2.37 (1.90-2.96)aa tncbf 2.18 (1.60-2.96)aa 2.52 (2.04-3.13)aa fbff 2.27 (1.73-3.31)aa 2.16 (1.75-2.66)aa sdr 2.39 (1.68-3.06)aa 2.35 (1.88-2.93)aa 30 days z100 2.68 (1.98-3.64)aa 2.36 (1.90-2.94)aa fbf 2.63 (1.92-3.61)aa 2.76 (2.21-3.44)aa tncbf 2.73 (2.02-3.69)aa 2.35 (1.90-2.90)aa fbff 2.13 (1.56-1.91)aa 2.35 (1.90-2.90)aa sdr 1.99 (1.49-2.66)aa 2.28 (1.92-2.72)aa means followed by same uppercase letters in the same row and column indicate no statistically significant differences between the groups (p >0.05). 7 charamba et al. braz j oral sci. 2022;21:e225454 discussion bulk-fill composites have been developed to be inserted in increments of up to 4mm in thickness without compromising the mechanical properties and marginal quality of the restoration14. the performance of these resin composites in terms of bond strength to dentin is still unclear, mainly when those composites are used to restore large cavities. in this study, the resin-dentin bond strength of class ii high viscosity and flowable bulk-fill resin composites restorations was evaluated, using different cavity sizes. previous research studies verified that large cavities were not favorable for bonding composites to tooth material, being an incremental technique more effective in those cavities15. according to the current study, the sdr and the filtek bulk fill resin composites restorations demonstrated lower μsbs values in extended cavity preparation when compared to the conservative cavity before artificial aging. after artificial aging, a decrease in the μsbs values was observed only for the filtek bulk fill flow. thus, the first experimental hypothesis was rejected.  polymerization shrinkage stresses developed in the adhesive interface of restorations can affect resin-dentin bond strength when composites’ contraction is restricted by the cavity walls16,17. several factors, including material composition, composite resin placement technique, geometry, and cavity extension can influence the magnitude of the polymerization stress18-20. this study showed the negative influence of extended cavity size on the bond strength of some bulk-fill resin composites to the dentin figure 1. weibull distribution plots of microtensile bond strength data for the experimental groups comparing the resin composites. fbf-filtek bulk-fill; tncbftetric n ceram bulk-fill; fbff-filtek bulk-fill flow; sdrsdr flow. (a) conservative cavity group after 24 hours storage; (b) conservative group after 30 days storage; (c) extended cavity group after 24 hours storage; (d) extended cavity group after 30 days storage. a b c d 1 3 5 10 15 20 30 40 50 60 80 microtensile bond strength (mpa) 1 2 pr ob ab ili ty o f f ai lu re (% ) f (t) = 1 -r (t) variable z100 24h fb 24h tncb 24h fbf 24h sdr 24h 3 5 10 20 30 40 50 60 70 80 90 99 99.9 1 3 5 10 15 20 30 40 50 60 80 microtensile bond strength (mpa) 1 2 pr ob ab ili ty o f f ai lu re (% ) f (t) = 1 -r (t) variable z100 24h fb 24h tncb 24h fbf 24h sdr 24h 3 5 10 20 30 40 50 60 70 80 90 99 1 3 5 10 15 20 30 40 50 60 80 microtensile bond strength (mpa) 0.1 2 pr ob ab ili ty o f f ai lu re (% ) f (t) = 1 -r (t) variable z100 30d fb 30d tncb 30d fbf 30d sdr 30d 3 5 10 20 30 40 50 60 70 80 90 99 99.9 1 3 5 10 15 20 30 40 50 60 80 microtensile bond strength (mpa) 1 2 pr ob ab ili ty o f f ai lu re (% ) f (t) = 1 -r (t) variable z100 30d fb 30d tncb 30d fbf 30d sdr 30d 3 5 10 20 30 40 50 60 70 80 90 99 1 8 charamba et al. braz j oral sci. 2022;21:e225454 (table 2). these results are not following previous study8. this fact can be related to the difference in cavity configuration and testing methodology. regarding the influence of artificial aging in the bond strength to the dentin, results demonstrated a significant influence of artificial aging (distilled water/ethanol) on the resin-dentin strength of class ii bulk-fill composite restorations. hence, the results of this study lead to the rejection of the second experimental hypothesis. a decrease in the μsbs values for the sdr bulk-fill composite (conservative cavity), the z100, the filtek bulk fill and the filtek bulk fill flow (extended cavity) was observed after artificial aging. this may be attributed to hydrolytic action of distilled water/ethanol on resin composite and the adhesive interface between the adhesive system and the resin composite, yielding a degradation of polymeric matrix21.  modifications in the matrix and filler of bulk-fill resin composites were made to increase their translucency and decrease the shrinkage stress. an increase in the filler size and the addition of more reactive photoinitiators are strategies used to allow greater light transmission with depth)2-5. regarding shrinkage stress, the inclusion of proprietary stress reliever molecules and polymerization modulators seems to decrease the shrinkage stresses generated during resin polymerization22. probably, the strategies used by bulk-fill manufactures explain the results of this study, in which conventional and bulk-fill composite restorations showed similar µsbs values in all studied conditions, agreeing with other studies8. therefore, the third was rejected. additional studies showed that bulk-fill resin composites presented better results of bond strength to the dentin than conventional composites for class ii23,24. systematics reviews of laboratory studies have shown similar or better performance of bulk-fill materials compared to the traditional composite resins in terms of polymerization stress, cusp deflection, marginal gap, degree of conversion, flexural strength, and fracture strength25,26. furthermore, systematic review and meta-analysis of clinical trials have revealed no differences in the performance of bulk-fill and conventional materials after 01 to 10 years of follow up27,28. thus, it seems that bulk-fill resin composites seem to be a suitable alternative to conventional layered resin composites when used in a 4 -5mm single-increment (bulk-fill technique)29.  the bond strength values were analyzed using the weibull statistic30. the bonding effectiveness to dentin and ceramics can be assessed by weibull survival analysis31. probably, high values of modulus mean that the bonding procedure is more reliable32. the weibull analysis revealed that similar m values were obtained for all groups. this finding suggests that the bond strength between bulk-fill resin composite to dentin present equal reliability than conventional resin composites. considering the analysis of fracture mode, mixed failure was the predominant fracture pattern for all experimental groups. these results agree with other studies8,33, suggesting that the hybrid layer was formed, but was fractured due to concentrated tension at the adhesive interface34-37. the results of this research study indicate that the type of cavity size (conservative or extended) and artificial aging negatively influenced the bond strength of some bulk-fill resin composites to the dentin. moreover, the studied bulk-fill resin composites presented similar bonding effectiveness to the dentin than conventional resins for all experimental conditions. however, this in vitro study does not test all bulk-fill resin composites available in the market and does not reproduce intraoral conditions. 9 charamba et al. braz j oral sci. 2022;21:e225454 therefore, further investigations, using different materials and conditions to simulate the buccal environment, are necessary to validate these findings. within the limitations of the current study, the following was concluded: 1. the type of cavity preparation affected the bond strength of the filtek bulk fill flow and the srd flow restorations before artificial aging and filtek bulk fill flow restorations after artificial aging. 2. the bond strength of the z100, the filtek bulk fill and the filtek bulk fill flow restorations was influenced by artificial aging when an extensive preparation cavity was used. while the srd flow restorations bond strength was affected by the conservative preparation cavity. 3. the bond strength of most bulk-fill resin composite restorations was similar to conventional composite restorations regardless of the type of cavity preparation and artificial aging employed.  acknowledgments the authors would like to thank the financial support received from the national council for scientific and technological development (cnpq, brasil). data availability datasets related to this article will be available upon request to the corresponding author. author contribution caroline de farias charamba: substantial contributions to the conception and design of the work, acquisition, analysis and interpretation of data for the work, drafting the work and revising it critically for important intellectual content, final approval of the version to be published, agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. larissa dias boson: contributed with the design of the article, acquisition and interpretation of data, drafting the article, final approval of the version to be published, agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. renally bezerra wanderley lima: contributed in the acquisition of data, analysis and interpretation of data, drafting the article and revising critically for important intellectual content, final approval of the version to be published, agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. rosangela marques duarte: contributed to conception and design of the article, acquisition of data and drafting the paper, final approval of the version to be published, agreement to be accountable for all aspects of the work in ensuring that ques10 charamba et al. braz j oral sci. 2022;21:e225454 tions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. ana karina maciel de andrade: contributed to conception and design, acquisition of data, and analysis and interpretation of data. additionally, this author was important in drafting the article and revising it critically for important intellectual content. final approval of the version to be published, agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. all authors actively participated in the discussion of the manuscript’s findings, and have revised and approved the final version of the manuscript. references 1. yazici ar, kutuk zb, ergin e, karahan s, antonson sa. six-year clinical evaluation of bulk-fill and nanofill resin composite restorations. clin oral investig. 2021 jun 10. doi: 10.1007/s00784-021-04015-2. 2. 3m oral care. filtek bulk fill posterior restorative-technical product profile. ontario: 3m esp; 2021 [cited 2021 april 18]. avaliable from: https://multimedia.3m.com/mws/media/976634o/filtek-bulkfill-posterior-restorative-technical-product-profile.pdf. 3. loguercio ad, rezende m, gutierrez mf, costa tf, 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of a multi-mode adhesive system: 1-year in vitro study. j dent. 2014 may;42(5):603-12. doi: 10.1016/j.jdent.2013.12.008. 37. munoz ma, luque i, hass v, reis a, loguercio ad, bombarda nh. immediate bonding properties of universal adhesives to dentine. j dent. 2013 may;41(5):404-11. doi: 10.1016/j.jdent.2013.03.001. 1http://dx.doi.org/10.20396/bjos.v20i00.8659280 volume 20 2021 e219280 original article 1. department of diagnosis and surgery, são paulo state university – unesp, school of dentistry at araraquara, araraquara, sp, brazil. corresponding author: joni augusto cirelli rua humaitá, 1680. zip code 14801-930, araraquara, sp, brazil phone: +55(16) 3301-6375/ fax: +55(16) 3301-6359; e-mail: joni.cirelli@unesp.br received: april 24, 2020 accepted: january 12, 2021 effect of electric, ultrasonic and manual toothbrushes on biofilm removal and gingivitis control: in vitro and parallel randomized controlled clinical trial study thamiris cirelli1 , guilherme josé pimentel lopes de oliveira1 , andressa vilas boas nogueira1 , isis jordão pinheiro ribaldo1, emilly yukiko diz furuta1, joni augusto cirelli1,* aim: to evaluate the effect of manual (m), electric (e) and ultrasonic (us) toothbrushes on the removal of oral biofilm and control of gingivitis. also, the roughness and tooth wear production were evaluated in vitro. methods: for the in vitro analyses, thirty bovine dentin specimens were submitted to a 3-month brushing simulation (9  minutes) with the three types of toothbrushes (n = 10). subsequently, a randomized controlled clinical trial was performed with 36 patients divided into 3  groups according to the toothbrushes used (n = 12). gingival index, visible plaque index and the volume of crevicular fluid were evaluated at baseline and 3  months after the beginning of the toothbrush use. furthermore, the performance of the biofilm removal per brushing cycle of 1 and 3 minutes with each toothbrush was made monthly until the end of the experiment. results: the us group had the highest dentin wear. clinically, the us group had a lower plaque index at 3  months than the m group. the m group also showed less biofilm removal efficiency from the second month of follow-up and more worn bristles at the end of the 3 month period than the e and us groups. conclusion: the ultrasonic, electric and manual toothbrushes showed no differences in gingivitis control in the present study. the  ultrasonic and electric toothbrushes had a more significant effect on biofilm removal than a manual toothbrush, but the ultrasonic toothbrush promoted greater dentin tissue wear. key words: gingivitis. oral hygiene. toothbrushing. http://dx.doi.org/10.20396/bjos.v20i00.8659280 mailto:joni.cirelli@unesp.br https://orcid.org/0000-0002-0521-0205 https://orcid.org/0000-0001-8778-0115 https://orcid.org/0000-0002-2756-5947 https://orcid.org/0000-0002-7082-9290 2 cirelli et al. introduction the dental biofilm represents a true complex biofilm that forms on the non-descamative surface of the teeth1. it is considered the major etiological factor of the most prevalent human oral diseases: dental caries and periodontal disease. the periodic removal of dental biofilm plays a key role in the prevention of these diseases2. as such, personal daily oral hygiene by brushing and using other hygiene aids is an accessible, effective, and economical method to maintain oral health3. it has been extensively demonstrated in clinical trials conducted in different geographical regions that the effective removal of dental biofilm is essential to maintain dental and periodontal health4. there are currently several toothbrush options on the market to increase motivation and facilitate brushing techniques, such as electric and ultrasonic brushes that have emerged as an alternative to conventional ones5. the ideal brushing technique is the one that allows for complete plaque removal in a shorter time, without causing any damage to tissues. also, a safe toothbrush should not contribute to the formation of gingival recession and excessive tooth wear, which may lead to the formation of non-carious cervical lesions6. in this context, a comparison of the safety of manual and electric toothbrushes has been little explored. systematic reviews7,8 studies have demonstrated the superiority of the electric and ultrasonic brushes in biofilm removal and gingivitis control when compared to manual brushes. however, other studies did not confirm these results9. besides, the degree of heterogeneity of included studies in the systematic reviews makes these results have only moderate evidence, which indicates the need for further studies. therefore, the objective of this study was to compare the efficiency of manual (m), electric (e), and ultrasonic (us) toothbrushes in the control of dental biofilm and gingival inflammation after 3 months of use. also, an in vitro analysis evaluated the effect of these toothbrushes on the wear and roughness of dentin samples after a 3 month-brushing simulation test. the study hypothesis is that the us and e brushes have a greater effect on biofilm removal and gingivitis control and induce less wear and roughness on dentin samples than manual brushes. material and methods in vitro preparation of the samples thirty intact bovine incisor teeth were selected, cleaned with the aid of mccall curettes (nº 13/14, golgran, são paulo, brazil) to remove the remaining periodontal tissues and immersed in physiological saline until the samples preparation. the sectioning of the teeth was performed using a diamond disk mounted in a low rotation driller. two cuts were made: one transversally, for the exclusion of the crown, and one longitudinally, dividing the root into two equal parts. the samples were planned in a polishing machine to obtain 60 samples with 10x4 mm2 containing only dentin. with the aid of cylindrical diamond drills mounted in a high rotation driller, a groove was made in the center of each sample dividing the sample into two areas. one of the 3 cirelli et al. areas was enveloped by insulating tape, thus constituting the control region, which was not subjected to the brushing simulation. the other region was exposed to the brushing simulation procedure10. the samples were then placed in a metal matrix prepared for this study and embedded in self-polymerizable acrylic resin (vip cril, pirassununga, são paulo, brazil). a random distribution of these specimens was performed in three experimental groups (n=10/group), according to the toothbrush used for the brushing simulation: 1) ultrasonic toothbrush (us) (ultrasonex ultima toothbrush®, sonex international corp, brewster, new york, eua ); 2) electric toothbrush (e) (braun oral b 3d plaque remover, braun gmbh, kronberg, alemanha); 3) manual toothbrush (m) (oral b model 30, gilette do brasil, manaus, brazil). the same dentifrice was used for all groups during the simulation of brushing (colgate anticarie, colgate do brasil, são bernardo do campo, sp, brazil). brushing simulation the specimens of the m group were submitted to the brushing simulation test in a brushing machine designed in the department of prosthesis from the school of dentistry at araraquara unesp (araraquara, são paulo, brazil). in this device, the samples were arranged in horizontally framed metal bases, which provided, in addition to the fixation, the immersion of the specimens in distilled water or distilled water/ dentifrice solutions in a ratio of 1: 1. the active part of the toothbrushes was fixed in metallic arms, which made horizontal movements of constant amplitude over the sample. the simulation of three months of brushing was obtained with 300 cycles, with a vertical force of 200n and frequency of 8rpm11. for the simulation of brushing with the electric and ultrasonic brushes, a previously described12 device was adapted. the heads of these brushes were positioned directly under the specimens with a constant force of 200n. with a metal rod’s aid, the specimens were fixed and raised slightly to allow maximum contact with the brushes. the samples were brushed for 9 minutes to simulate 3 months of brushing, considering patients with an average of 20 teeth and the use 2 minutes per brushing section: 120/20=6 seconds per tooth. six seconds x 2 times a day x 90 days=18  minutes. as only one face was brushed, the brushing time was approximately 9 min. roughness analysis the surface roughness measurement (ra) was recorded on each sample’s surface with the aid of a profilometer (surftest sj-401, mitutoyo sul americana ltda, santo amaro, sp) with an accuracy of 0.01mm. three readings were performed per region of each specimen, at distinct locations within a predetermined area and similar for all specimens. for each reading, the needle of the device scanned 1.5 mm always in a single direction with a cutoff of 0.8 mm. after registering the roughness values, an average of the three readings of each area of the sample was determined. analysis of the tooth wear the specimens were decalcified in morse solution (formic acid and sodium citrate) for 30 days, followed by paraffin embedment. serial 5 μm-thick histological sections were obtained and stained by hematoxylin-eosin. the relative wear of the experimen4 cirelli et al. tal surfaces was analyzed in 3 equidistant sections (72μm) about a control surface10. histological images with 25x magnification were obtained using an optical microscope (leica-reichert diastar products & jung, wetzlar, germany). image j software (image j, jandel scientific, san rafael, usa) was used for image analysis performed by a blinded and trained examiner (ijp). in vivo patient selection thirty-six patients participated in this randomized clinical trial, after having read and signed the term of free and informed consent, approved by the human ethics committee of the school of dentistry at araraquara – unesp (cep: 03/11) and registered at the brazilian clinical trials records (u1111-1204-1231). this study was performed following the helsinki declaration of 1975, revised in 2015. the inclusion criteria for this study were: 1) presence of at least 20 teeth in the mouth; 2) clinical diagnosis of biofilm-induced gingivitis13; 3) presence of marginal bleeding and visible dental biofilm in more than 50% of the sites, 4)probing pocket depth (ppd) ≤ 3mm, 5) 10% or more of sites with bleeding on probing and 6) absence of loss of attachment. after this evaluation, the patients were not included in the study if they had the following exclusion criteria: 1) periodontal treatment in the last 12 months; 2) systemic diseases that may affect the treatment outcome; (3) pregnancy; 4) use of systemic antibiotics in the last 6  months; 5) use of anti-inflammatory drugs in the last 3 months; 6) smokers and ex-smokers; 7) use of oral contraceptives; 8) patients with orthodontic appliances; 9) patients with total dentures, partial removable or fixed prosthesis with more than 2 elements and implant prosthesis. sample size calculation the sample size calculation was based on data published in a previous clinical study comparing mechanical plaque control in patients using manual and ultrasonic brushes14. a relevant clinical difference was determined for the reduction in the variable plaque index before and after treatments of 0.5 with a standard deviation of 0.35. therefore, the standardized difference at 0.85 (1-β = 0.85) and α = 0.05 determined a sample size of at least 12 patients to perform the mechanical control with each brush. study design this study was a randomized, single-blind, controlled clinical trial with a parallel model in which each patient used one type of toothbrush. the 36 patients were randomly divided into 3 groups according to the type of toothbrush used for 3 months: 1) ultrasonic toothbrush (us); 2) electric toothbrush (e); 3) manual toothbrush (m). during  that period, patients were oriented to perform only mechanical oral hygiene with the selected toothbrush, avoiding other mechanical or chemical plaque control methods. patients receive guidance on adequate oral hygiene using the modified bass brushing technique15. at each return appointment, the brushing instructions were redone. besides, patients were asked about adverse effects of the use of toothbrushes, such as sensitivity, discomfort, or pain. the flow of the study is depicted in figure 1. the same dentifrice was used for all patients during all the study period (colgate anticarie, colgate do brasil, são bernardo do campo, sp, brazil). the demographic data of the patients are exposed in table 1. 5 cirelli et al. allocation follow-up analysis enrolment assessed for eligibility (n = 62) excluded (n = 26) not meeting inclusion criterial (n = 24) decline to participate (n = 2) others (n = 0) manual (n = 12)electric (n = 12)ultrassonic (n = 12) after 3 months withdraws (n = 0) analyzed (n = 12) analyzed (n = 12) analyzed (n = 12) after 3 months withdraws (n = 0) after 3 months withdraws (n = 0) randomized (n = 36) figure 1. study flowchart table 1. demographic characteristics of the patients at baseline. parameter/groups ultrasonic (n = 12) electric (n = 12) manual (n = 12) age(y) 38.83 ± 12.40 35.58 ± 13.51 36.08 ± 12.75 females (n) 8 7 6 males (n) 4 5 6 teeth 27.00 ± 3.27 27.42 ± 2.19 27.33 ± 3.33 clinical analysis the patients were analyzed at baseline and after 3 months for the following clinical parameters: 1) gingival index (gi)16; 2) visible plaque index16. these clinical parameters were analyzed by a blinded, trained, and calibrated examiner (tc). in addition, the following parameters were analyzed at baseline for patient selection: 1) marginal gingival bleeding; 2) probing depthmeasured from the gingival margin to the bottom of the gingival sulcus, 3) gingival margin levelmeasured from the cement-enamel junction to the gingival margin; 4) clinical attachment level – measured from the cement-enamel junction to the bottom of the gingival sulcus. analysis of biofilm removal efficiency per brushing cycle after the clinical analysis at the baseline, the patients were instructed about using the different toothbrushes. the bacterial biofilm was stained, and each patient underwent dental brushing for 1 and 3 minutes. during these periods, the quingley & hein modification of the turesky plaque index17 was evaluated to analyze plaque removal efficiency 6 cirelli et al. in each brushing cycle. this analysis was repeated at 1, 2, and 3 months after the baseline. these exams were performed by a blind, trained, and calibrated examiner (tc). analysis of the volume of the crevicular fluid to collect crevicular fluid, a strip of paper periopaper (periopaper oralflow inc. new york usa) was introduced to the base of the gingival sulcus at the mesiobuccal site of the first molars until some resistance was felt, remaining in place for 30 seconds. soon after, the crevicular fluid volume was measured through the periotron 6000 equipment (oraflow inc. new york-usa). in the absence of a first molar, the sample was collected at the mesiobuccal site of the 2nd premolar at the same quadrant. this analysis was performed by a blinded to group allocation and trained examiner (tc) at baseline and after 3 months. analysis of the toothbrushes bristles in the baseline period and 3 months after being used by the patients, the toothbrushes were evaluated for bristle wear and their tips’ morphology. to analyze the wear of the bristles, five measurements were recorded for each brush using a digital caliper (series 500-144b, mitutoyo, suzano, brazil), according to the methodology used by rawls et al. 198918: fll (free-longlength): corresponds to the length of the brush head at the top of the larger side; bll (base-longlength): corresponds to the length of the brush head at the bottom (base) of the larger side; ffl (front free length): corresponds to the length of the brush head measured at the top of the smaller side; bfl (base free length): corresponds to the length of the brush head measured at the bottom (base) of the smaller side and brl (bristles length): a measure of the height of the bristles. the wear index was calculated using the formula: wi = fll-bll + ffl-bfl / brl18. for the analysis of the bristle tips’ morphology, four images with 20x magnification were reproduced from each brush using an optical microscope (leica microsystems, wetzlar, germany). two of the images were taken from the top view to evaluate the central bristles, and two images were made in lateral view to evaluate lateral bristles. all the images were captured in aleatory fields. a blinded and trained examiner evaluated the images twice in different moments using the index proposed by silverstone and featherstone19 (1988) (figure 2). the analysis was performed by a blinded, trained, and calibrated examiner (ef). a1 n1a2 n2 n3 n4 n5 figure 2. classification of bristle tip morphology. group a represents the acceptable rounding of the tip of the bristles, and group n represents the non-acceptable rounding of bristles tips. statistical analysis data on tooth wear and roughness, clinical parameters, and brush bristles wear were submitted to the shapiro-wilk normality test. data from tooth wear and dentin rough7 cirelli et al. ness presented normal distribution, and the parametric test one-way anova complemented by the tukey’s test was applied for inferential data analysis. data from clinical analysis and the bristle wear were performed using the non-parametric tests of kruskall-wallis supplemented by the dunn for the comparison between groups in the same evaluation period. the comparison within each group at the baseline and 3-month periods was performed using the wilcoxon test. besides, the analysis within each group in the analysis of efficiency of removal of bacterial biofilm in different brushing cycles times (baseline, 1  minute and 3  minutes) was performed by the friedman test complemented by the dunn test. data from the analysis of the morphology of the bristle tips were analyzed using the chi-square test. all tests of this study were applied using the software graphpad prism 6 (san diego, ca, usa) at a significance level of 5%. results in vitro the in vitro analysis presented no differences among the groups concerning the degree of roughness obtained after the brushing simulation. however, it was observed that the us group presented higher dentin wear than the e and m groups (figure 3). ultrasonic r ou gh ne ss r a (μ m ) 1.5 1.0 0.5 0.0 a electric manual ultrasonic * d en tin w ea r ( m m ) 1.5 1.0 0.5 0.0 b electric manual figure 3. representative graphs of the dentin a) roughness and b) wear data. it´s possible to note that the us presented higher dentin wear than the other groups. *p<0.05-higher dentin wear than the other groupsone-way anova complemented by tukey. clinical trial there were no differences (p > 0.05) among the groups regarding the demographic data (table 1). no patient in this study had clinical attachment loss at baseline. none of the patients mentioned adverse effects. us and e groups had a reduction in the plaque index levels at 3 months, a fact not observed in the m group. in the intergroups comparison, the us group presented lower plaque index values than the m group at the end of the experiment (figure 4a). regarding gingival inflammation analysis, the e and m groups presented a reduction in the gingival index at 3 months, a fact not observed in the us group (figure 4b). however, no differences were identified between the groups at the end of the experiment about the gingival index (figure 3b) and in the volume of the crevicular gingival fluid (figure 4c). no clinical attachment level loss of enhance in probing depth was detected in this study. 8 cirelli et al. ultrasonic # # * v is ib le p la qu e in de x 1.5 2.0 2.5 baseline 3 months 1.0 0.5 0.0 a electric manual ultrasonic g in gi va l i nd ex 1.5 2.0 2.5 baseline 3 months 1.0 0.5 0.0 b electric ## # manual ultrasonic c re vi cu la r f lu id c ol um e 100 150 200 baseline 3 months 50 0 c electric manual δ figure 4. representative graphs of the clinical analysis of the a) visible plaque index, b) gingival index, and c) crevicular fluid volume evaluated at the baseline and after 3  months of the toothbrushes use. *p<0.05-higher visible plaque index than the us group at 3 months-kruskall-wallis complemented by dunn; #p < 0.05; ##p < 0.01lower visible plaque index and gingival index than at baseline-wilcoxon; δp < 0.05less crevicular fluid volume than the e group at baseline-kruskall-wallis complemented by dunn. the analysis of the brushing cycle performed monthly showed statistically significant removal of biofilm after 3 minutes of brushing in all groups. however, in the second month, the m group presented less biofilm removal after 1 minute of brushing than the us group. in the third month, less removal of biofilm than the e group at1 and 3 minutes of brushing (figure 5). ultrasonic c c b b b b a a a q ui gl ey & h ei n in de x 8 6 4 2 0 baseline 1 minute 3 minutes a electric brushing cycles after 1st month manual ultrasonic * b b b aa a a a,b c * q ui gl ey & h ei n in de x 4 3 2 1 0 baseline 1 minute 3 minutes b electric brushing cycles after 2nd month manual ultrasonic * * b b b a a a a,b a,b a,b q ui gl ey & h ei n in de x 8 6 4 2 0 baseline 1 minute 3 minutes c electric brushing cycles after 3rd month manual figure 5. representative graphs of the effect of plaque removal after the brushing cycles of 1 and 3 minutes performed at the a)1st month, b) 2nd month, and c)3rd month after the toothbrushes use. different letters represent statistical differences within each group-friedman complemented by dunn; *p<0.05-higher plaque index than the us group at the 2nd month-kruskall-wallis complemented by dunn; #p<0.05-higher plaque index than the e group at the 3rd month-kruskall-wallis complemented by dunn. 9 cirelli et al. the m and us toothbrushes were more worn at 3 months than in the baseline timepoint. also, the m toothbrushes presented more worn bristles than the other groups at 3  months. there were no differences between the groups concerning the bristle tips’ morphology at the baseline and after 3 months of brushing (figure 6). the bristle tip morphology showed an improvement in its pattern after the 3 months of use in the m and us groups (figure 6b). a b g h c d e f ultrasonic ## * ## *** r aw ls in de x bristle wear 1.5 baseline 3 months 1.0 0.5 0.0 electric manual acceptable not acceptable morphology of the bristle tip ultrasonic-baseline ultrasonic-3 months eletric-baseline eletric-3 months manual-baseline manual-3 months n um be r o f r ev ie w s 50 40 30 20 10 0 δ δδ figure 6. figures a-f show the representative images of the tip of the bristles of the different types of toothbrushes used in this study. the images a, c, and e represent the conditions of the tip of the bristles of  the us, e and m toothbrushes before the use, respectively. the images b, d and f represent the conditions of the tip of the bristles of the us, e and m toothbrushes after 3 months of use, respectively. representative graphs of the g) bristle wear index and h) morphology of bristle tips index. the m group presented more worn bristles than the us and e groups after the 3 months of use. *p<0.05; ***p<0.001 less bristle wear than the m group at 3 months-kruskall-wallis complemented by dunn ##p<0.01-more bristle wear than the baseline period-wilcoxon; δp<0.05; δδp<0.01morphological pattern more acceptable than the baseline periodchi-square test. discussion in the present study, in vitro and clinical analyses evaluated the performance of different toothbrushes in the removal of dental biofilm and the control of gingival inflammation in patients with gingivitis. the results showed that us and e toothbrushes had a greater effect on biofilm removal than the manual toothbrush. however, this observation was not followed by a significant reduction in gingival inflammation. clinically, the three evaluated toothbrushes showed no differences between them in gingivitis control in the investigated period. this fact was demonstrated by the non-significance in the gi and the crevicular fluid volume analysis studied. although the removal of the biofilm evaluated by the plaque index has shown a superior statistical result for the us compared to the m, these differences were not sufficient to result in a clinical improvement in gingival inflammation, as also observed in previous studies20,21. the greater variability of the results in the us group may explain, at least in part, the improvement in the results of gingivitis parameters without significant difference. 10 cirelli et al. regarding the plaque index, the present results corroborate with previous studies. a possible explanation for this result is that the ultrasonic waves could remove adhered bacteria and induce cell surface alterations, affecting biofilm attachment22. however, this is not a consensus in the literature since other short-term studies did not observe a significant difference between those toothbrushes23. in addition, it has been suggested that long-term studies provide a more accurate evaluation of the effect of brushing. this fact may explain why no differences were found in the studies mentioned above24 for plaque index and why we did not find differences among brushes in gingivitis control. another possible reason for this contrast is the hawthorne effect23, induced by the monthly appointments during the study period. in this kind of studies generally, we have a patient’s positive contribution. in other words, patients pay more attention to their oral hygiene when they know that this will be evaluated25. patients may have improved their brushing only previously to their visit to the clinic, which affected the biofilm index level but not the inflammatory parameters. the advantage of powered toothbrushes in removing dental biofilm was confirmed by the brush cycles analysis from the second month of follow up. the toothbrushes of the us and e groups also showed less bristle wear than the toothbrushes of the m group. in vivo26 and in vitro27 studies showed that worn toothbrushes lose their effect on biofilm removal, so it is recommended that the toothbrush should be replaced whenever any signs of bristle wear are identified. however, the literature has shown no statistically significant differences in biofilm removal between used and new toothbrushes28. these studies indicate that other factors, such as brushing time, brushing strength, and patient motivation, are as important as the bristles’ integrity for oral hygiene performance29. another aspect of powered toothbrushes is their greater cost in comparison to the manual toothbrush30. another effect observed in this kind of study is the novelty effect, which hinders the effect of mechanical devices for plaque control22. this effect relates to the fact that a new brush attracts more attention while it is a novelty, resulting in more collaboration from the patient in controlling plaque22. in our study, it can be suggested that patients from the us and e groups became more susceptible to the novelty effect than patients from the m group, affecting the patient motivation and the results of the study. besides, to analyze the effect on the removal of dental biofilm, another important aspect to be analyzed in toothbrushes is their risk of causing dental wear and gingival recession. according to a recent literature review, the factors most associated with oral injuries caused by brushing are the tooth brushing frequency, a horizontal or scrub tooth brushing method, bristle hardness, brushing duration, the morphology of the bristle tip, and the frequency of changing a toothbrush. the principal tooth brushing factors associated with non-carious cervical lesions were tooth brushing method and frequency6. despite the greater dentin wear caused by the us toothbrushes in the  present study, they were not associated with gingival recession, as confirmed by the literature31. it has been reported that us toothbrushes users apply less lateral force during the brushing procedures, which may explain the absence of side effects during the use of those toothbrushes, observed in this and other studies32. 11 cirelli et al. the different groups of toothbrushes presented similar morphology of bristle tips before the begging of the study. the majority of the bristles presented an inappropriate morphology, which reveals that this parameter does not present an adequate pattern in the studied brushes, as demonstrated in the literature33. the inadequate morphology of bristle tips has been related to the possibility of causing gingival lesions, which may induce gingival recessions34. the short-term follow-up of this study cannot provide if the use of these types of toothbrushes may induce a gingival recession, and longterm periods of evaluation will be necessary to test this hypothesis. another important finding was that the morphology of the bristle tips in the m and us groups became more acceptable after 3  months of use. one study demonstrated that toothbrushes with hard bristles, with greater strength, improved in the morphological pattern of the tips of the bristles in comparison to toothbrushes with soft and extra-soft bristles in a 2-year brushing simulation35. likely, the improvement observed in the morphological pattern of bristles tip of toothbrushes with hard bristles should be observed at earlier periods of use of toothbrushes with soft bristles. perhaps this explains the findings of this study and the non-observance of gingival lesions induced by brushing. an advantage of this study was that the monitoring during the brushing cycles provided a more realistic analysis of the potential for biofilm removal of each toothbrush since the supervision induces the maximum effect of the patient to perform oral hygiene14. therefore, the differences found between the toothbrushes in this analysis were more related to their cleaning potential than to the patients’ motivation. however, this study does not mimic what happens during the patients’ daily oral hygiene practices, which may not reproduce the good clinical outcomes verified in this study. besides, the toothbrushes here evaluated may have different ideal brushing times. another aspect that limits the extrapolation of this study’s findings was that the patients returned every month for the recall during three months, which is not a standard maintenance protocol for patients with gingivitis, usually called at longer intervals. in conclusion, the ultrasonic, electric and manual toothbrushes showed no differences between them in gingivitis control in the present study. the ultrasonic toothbrush had a greater effect on biofilm removal than a manual toothbrush and promoted greater dentin tissue wear. besides, the manual toothbrush presented greater bristle wear compared to the other toothbrushes. acknowledgement this study was financed by the brazilian agency cnpq (conselho nacional de desenvolvimento científico e tecnológico-pibic/cnpq/unesp). the authors declare no other conflict of interest regarding this study. references 1. chapple ilc, mealey bl, van dyke te, bartold pm, dommisch h, eickholz p, et al. periodontal health and gingival diseases and conditions on an intact and a reduced periodontium: consensus report of workgroup 1 of the 2017 world workshop on the classification of periodontal and peri-implant diseases and conditions. journal of periodontology. 2018 jun;89 suppl 1:s74-s84. doi: 10.1002/ jper.17-0719. file:///d:/trabalho/ingroup/bjos/bjosv020-9280/9280/consolidado/javascript:void(0) 12 cirelli et al. 2. loe h, theilade e, jensen sb. experimental gingivitis in man. j periodontol. 1965 mayjun;36:177-87. doi: 10.1902/jop.1965.36.3.177. 3. suomi jd, west jd, chang jj, mcclendon bj. the 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brackets. braz oral res. 2010 julsep;24(3):316-22. doi: 10.1590/s1806-83242010000300010. 1http://dx.doi.org/10.20396/bjos.v21i00.8664265 volume 21 2022 e224265 original article 1 graduate program in dental sciences, federal university of santa maria, santa maria, rs, brazil 2 school of dentistry, federal university of santa maria, santa maria, rs, brazil 3 department of restorative dentistry, federal university of santa maria, santa maria, rs, brazil 4 department of dental materials and prosthodontics, institute of science and technology, são paulo state university, são josé dos campos, sp, brazil corresponding author: camila da silva rodrigues, dds, ms, phd department of dental materials and prosthodontics, institute of science and technology, são paulo state university address: av. eng. francisco josé longo, 777, são josé dos campos, sp, brazil, 12245-000 phone: (55)999474213 e-mail: camilasrdg@gmail.com editor: altair a. del bel cury received: february 25, 2021 accepted: march 29, 2021 color and translucency stability of cad/cam restorative materials rafaelo fagundes dalforno1 , maria luíza auzani2 , camila pauleski zucuni3 , camila da silva rodrigues4* , liliana gressler may3 aim: this study assessed the color and translucency stability of a polymer infiltrated ceramic network (picn) and compared it with a resin composite (rc) and a feldspathic ceramic (fel). methods: disc-shaped samples of a picn (vita enamic), a feldspathic ceramic (vitablocks mark ii), and a resin composite (brava block) were prepared from cad/cam blocks. picn and rc surfaces were finished with a sequence of polishing discs and diamond paste. fel samples received a glaze layer. the samples were subjected to 30-min immersions in red wine twice a day for 30 days. ciel*a*b* coordinates were assessed with a spectrophotometer at baseline and after 15 and 30 days of immersion. color alteration (∆e00) and translucency parameter (tp00) were calculated with ciede2000. average roughness was measured before the staining procedures. color difference and translucency data were analyzed with repeated-measures anova and tukey’s tests. roughness was analyzed with the kruskal-wallis test. results: roughness was similar among the experimental groups. all materials had their color alteration significantly increased from 15 to 30 days of staining. picn reached an intermediate ∆e00 between fel and rc at 15 days. picn revealed a color alteration as high as the composite after 30 days. no statistical difference was observed regarding translucency. conclusion: picn was not as color stable as the feldspathic ceramic at the end of the study. its color alteration was comparable to the resin composite when exposed to red wine. however, the translucency of the tested materials was stable throughout the 30-day staining. key words: color. ceramics. composite resins. computeraided design. materials testing. surface properties. http://orcid.org/0000-0003-0388-9020 http://orcid.org/0000-0003-4906-8199 http://orcid.org/0000-0002-7966-9879 http://orcid.org/0000-0003-4162-3303 http://orcid.org/0000-0002-4572-6142 2 dalforno et al. introduction computer-aided design and computer-aided manufacturing (cad/cam) technology has simplified the workflow for indirect restorations processing and enabled fabricating strong polycrystalline and glass-ceramics for dental applications. in addition to ceramics, resin composite blocks are industrially polymerized under standardized temperature and pressure parameters, which ensure their mechanical properties for cad/cam systems usage1. dental ceramics are generally stronger and more wear-resistant than resin composites2,3. however, the brittleness of ceramics together with its susceptibility to slow crack growth4,5 might result in worse fatigue behavior compared to some composites6. in an attempt to combine characteristics such as the resilience from resin composites and the resistance to abrasion from ceramics, a hybrid material was developed and made available as milling blocks. vita enamic (vita zahnfabrik) is a polymer-infiltrated ceramic-network (picn) material which gathers a sintered feldspathic ceramic scaffold (86w%) filled with a polymeric network (14w%) in a fully integrated structure. this combination results in a material with elastic modulus in the range of human dentin (~30 gpa) and provides it with easy machinability. picn can be milled more quickly than ceramics, which gives it a great advantage for chairside usage7. ceramics are more esthetically stable than composites3,8,9. resin composites are more susceptible to water sorption, mainly facilitated by hydrophilic compounds of its organic matrix10. water sorption degrades the bonding between resin matrix and filler particles and pigments infiltrate easily in these interfaces11. in contrast, particle-filled glass-ceramics consists of a vitreous matrix filled with glass or crystalline particles12, which gives it a denser microstructure and results in less discoloration. picn is expected to present an intermediate optical behavior between composite and ceramic. however, studies comparing the color and translucency stability of picn with other cad/cam materials are required to confirm this information. studies have evaluated the color and/or translucency stability of picn after days of immersion in staining beverages (e.g., coffee, tea, red wine, cola, or juice) and compared it with other restorative materials9,13-16. these studies applied diverse staining protocols and methods for calculating the optical properties, such as cielab9,14, ciede200013,15,16, translucency parameter13,14, percentage of light transmission9, or contrast ratio13,16. in addition, most of the studies perform silicon carbide paper polishing for all materials. the surfaces of the samples are finished on different grits (i.e. p4000, p1200) in each study, which might influence color stability and hinder the comparison of results. composite, ceramics, and hybrid materials are clinically subjected to different finishing procedures prior to cementation. this should be considered for studying the optical behavior of restorative materials since it leads to more realistic results. moreover, there is a need for studies using translucency and color calculation methods and clinical thresholds comparisons that are described in the literature as the most accurate17,18. 3 dalforno et al. esthetic issues are one of the main cited reasons for substituting anterior composite restorations19 and feldspathic ceramic veneers20 in clinical follow-ups. as picn is still a new material, there is little evidence from clinical studies21,22 and no consensus about its clinical behavior regarding esthetic issues. furthermore, in vitro studies gives us an estimation of what to expect in vivo. hence, this study aimed to assess the color and translucency stability of picn and compare it with a resin composite (rc) and a feldspathic ceramic (fel) also available in cad/cam blocks. the tested hypotheses were that picn would: 1) show intermediate color stability between the ceramic and the composite; and 2) its translucency would keep stable throughout the 30-day staining challenge. materials and methods study design the factors analyzed in this in vitro study were material (hybrid ceramic, feldspathic ceramic, or resin composite) and exposure time in the staining media (15 or 30 days). measurements from the same sample were compared between the checkpoints (repeated measures approach). the outcomes studied were color difference (∆e00) and translucency (tp00). the commercial brands, shades, and composition of the restorative materials used in this study are described in table 1. sample preparation cad/cam blocks of a hybrid ceramic (picn – vita enamic, vita zahnfabrik, bad sackingen, germany), a feldspathic ceramic (fel – vita mark ii, vita zahnfabrik, bad sackingen, germany), and a resin composite (rc – brava block, fgm dental group, joinville, brazil) with initial dimensions of 12 mm × 14 mm × 18 mm were used to prepare disc-shaped samples (10 mm diameter × 1.2 mm thick, n = 12). the blocks were ground into cylinders using a 100-grit sic paper under water-cooling in a polishing machine (ecomet 250, buehler, lake bluff, usa). the cylinders were then sectioned into discs in a precision cutting machine (isomet 1000, buehler, lake bluff, usa) with a diamond blade. the picn and rc discs had both sides ground with a 100-grit sic paper to a thickness of 1.3 mm. the fel discs were ground until they were 1.2 mm thick. table 1. labels, material type, commercial brand, shades, and composition of each cad/cam material used in the study. label material commercial brand shade composition picn hybrid ceramic vita enamic (vita zahnfabrik) 1m2 t sio2-al2o3-na2o-k2o-br2o3-zro2-cao, udma tegdma fel feldspathic ceramic vita mark ii (vita zahnfabrik) a2c al2o3sio2na2o-k2o rc resin composite brava block (fgm dental group) a2 ht methacrylate monomers, initiator, co-initiator, stabilizers, silane, glass-ceramic particles, silica, and pigments. 4 dalforno et al. the top surface of picn and rc samples were subjected to a sequence of coarse, medium, fine, and superfine polishing discs (sof-lex, 3m-espe, st. paul, usa), and finished using a felt disc with aluminum-oxide extra fine (6 – 8 µm grit) polishing paste (diamond flex and diamond r, fgm dental group, joinvile, brasil). the polishing discs and felts were placed in parallel to the samples and the polishing was performed for 20 seconds for each disc. the samples were rinsed in water between discs. the polishing procedures were performed by a trained operator using a low-speed motor associated with a contra-angle handpiece (~10,000 rpm) and light pressure. new polishing discs were used for each sample. the final thickness was 1.2 mm (± 0.05 mm). the fel samples received a thin glaze layer on their top surfaces (akzent plus, vita zahnfabrik, bad sackingen, germany). the glaze powder was mixed with the building liquid to obtain a creamy consistency. the mix was applied over the ceramic top surface with a brush, and the samples were subsequently fired in a furnace (vacumat 600 mp, vita zahnfabrik, bad sackingen, germany). glaze firing was performed according to the manufacturer’s instructions (950°c, 1 min dwell time). all the samples had their thickness measured with a digital caliper after the firing process. the thicknesses ranged from 1.23 mm to 1.27 mm. the bottom surfaces (not glazed) of the ceramic samples were slightly ground with 100-grit sic paper until 1.2 mm thick discs were obtained. all samples (n = 12) were stored in distilled water at 37°c for 24h. then, baseline ciel*a*b* measurements were taken and the top surfaces of all samples had roughness measured to ensure standardization. roughness measurements the roughness measurements were taken in a contact roughness tester (mitutoyo sj-410, mitutoyo) according to the iso standard 4287-1997. the average roughness (ra) parameter of all samples was evaluated (n = 12). three measurements were obtained from the polished/glazed side of each sample in both the x and y-axis. a cut-off of lc 0.8 mm (n = 5) and a ripple filter of ls 2.5 mm was used. the mean ra values of each sample were used in the statistical analysis. staining procedures the samples were immersed in red wine (salton classic cabernet sauvignon, vinícola salton, bento gonçalves, brazil) for 30 min at 37°c twice a day with a dwell time of 12 h between the immersions. this procedure was carried out for 30 days, totaling 30 h of immersion. after each immersion, the samples were rinsed and stored in distilled water at 37°c until the next immersion. the wine was replaced after every immersion. red wine was chosen because it is acidic and rich in pigment, which has been demonstrated to result in high color alteration in ceramics, composites, and hybrid materials9,14. color and translucency stability analyses the ciel*a*b*(comission international l´eclairage) parameters were assessed with a spectrophotometer (sp60, x-rite, grand rapids, usa). the samples were placed 5 dalforno et al. over white, black, and gray backgrounds and the l*a*b* parameters were recorded. the lightness axis (l*) in this system ranges from 0 (black) to 100 (white), and a* and b* are the color coordinates on green-red and in blue-yellow axes, respectively. the spectrophotometer was calibrated prior to the measurements. the assessments were carried out using a d65 light source (6500 k), observer angle of 10º, and specular component excluded (spex). a drop of a coupling agent with a refractive index of 1.47 was used (glycerol c3h8o3) to avoid the light dispersion between the sample and the background. each sample was measured three times over each background and the average of these three measurements was used for color and translucency calculations. these measurements were taken at baseline and after 15 and 30 days of staining in red wine. the values obtained over the gray background were used for color difference calculations with the ciede2000 formula (equation 1). the color alteration was calculated using the ciel*a*b* measurements at 15 and 30 days compared to the baseline ciel*a*b* values (mutual comparison). the perceptibility (∆e00 > 0.8) and unacceptability (∆e00 > 1.8) thresholds were considered for clinical inference 17. δe00 = 2 2 2 + + + rt 2 1 ∆l’ kl sl ∆c’ kc sc ∆h’ kh sh ∆c’ kc sc ∆h’ kh sh (1) where ∆l′, ∆c′, and ∆h′ are the differences in lightness, chroma, and hue, respectively, for a pair of measurements (baseline and 15 or 30 days of staining). the rotation function rt accounts for the interaction between chroma and hue differences in the blue region. weighting functions sl, sh, and sc adjust the total color difference for variation in the location of the color difference pair in l′, a′, b′ coordinates. the parametric factors kl, kc, and kh are correction terms for deviation from reference experimental conditions. in this study, these parametric factors of the ciede2000 formula were set as 1. the translucency parameter (tp00) was also calculated with the ciede2000 formula (equation 1). however, the pair of measurements used were the ciel*a*b* parameters obtained from each sample over the white and black backgrounds, separately for baseline, and after 15 and 30 days of staining. statistical analysis the statistical analysis was carried out using the sigmaplot 12.0 (systat software inc, san jose, usa) software program. data were subjected to normality (shapiro-wilk test) and homoscedasticity (levene test) tests. next, average roughness data were analyzed with the kruskal-wallis test. color difference data were analyzed with two-way repeated-measures anova (material*staining time) and tukey’s test as post-hoc. translucency stability was analyzed separately for each material using the one-way repeated measures anova test. the cad/cam blocks chosen for this study are available in different color scales, so that translucency comparisons among materials would be biased. the significance level was set at 5%. 6 dalforno et al. results table 2 shows the roughness mean values for each material after the polishing procedures. no significant difference was observed in the ra parameter among the experimental groups (p > 0.05). the statistical analysis regarding color stability (described in table 3) showed a significant effect from material (p = 0.002) and staining time (p < 0.001) on the studied outcome, as well as a significant interaction between these factors (p = 0.004). all three materials significantly increased their color alteration from 15 to 30 days of staining. picn reached an intermediate color alteration between fel and rc after 15 days. however, picn revealed a color alteration as high as the resin composite after 30 days of staining, and the feldspathic ceramic was the most stable material. the restorative materials reached the color unacceptability threshold (∆e00 > 1.8) after 15 days of staining. on the other hand, all three materials had their translucencies stable over the 30-day staining since no statistically significant differences were observed (table 4). table 2. means (standard deviations) of average roughness (ra) of each experimental group after polishing procedures. materials ra (µm) picn 0.36 (0.08)a fel 0.36 (0.22)a rc 0.27 (0.18)a different lowercase letter within a column indicates statistical differences among groups (kruskal-wallis test, p < 0.05). table 3. means (standard deviations) of color difference (∆e00) of each material after 15 and 30 days of staining in red wine. materials ∆e00 15 days ∆e00 30 days picn 3.74 (0.35)b,ab 4.95 (0.80)a,a fel 3.22 (1.04)b,b 3.82 (1.24)a,b rc 4.22 (0.88)b,a 5.49 (0.73)a,a different uppercase letter within a row indicates significant statistical differences between immersion times of the same material. different lowercase letter within a column indicates statistical differences among materials in the same immersion time measurement (two-way rm anova, tukey’s test, p < 0.05) table 4. means (standard deviations) of translucency parameter (tp00 ) of each material at baseline and after 15 and 30 days of staining in red wine. materials tp00 baseline tp00 15 days tp00 30 days picn 13.02 (1.25)a 13.52 (1.34)a 12.77 (1.33)a fel 21.23 (3.13)a 21.11 (1.40)a 20.39 (1.49)a rc 24.44 (3.35)a 25.32 (1.28)a 23.34 (1.16)a distinct uppercase letter within a row indicates significant statistical differences among the immersion time measurements of the same material (one-way rm anova, tukey’s test, p < 0.05). 7 dalforno et al. discussion picn exhibited the same color alteration as a machinable resin composite, which was less stable than a glass-ceramic after the total exposure time to red wine. however, its translucency was maintained throughout the 30 days of staining. this made the first and second tested hypotheses to be rejected and accepted, respectively. all the materials reached the clinical unacceptability threshold at the first checkpoint (15 days). this observation was predictable since red wine has been described as the most pigmented beverage in in vitro studies9,15. the color stability of each material depends on the staining exposure time. picn reached ∆e00 values similar to both composite and ceramic after the first 15 days of exposure to red wine. in contrast, its color alteration was statistically similar to the composite and greater than the glass-ceramic after 30 days. picn has udma and tegdma monomers in their composition. great water sorption has been reported in composites containing high tegdma content compared to other methacrylate monomers23. this is explained by the hydrophilicity of tegdma. in this sense, hydrophilic monomers facilitate pigment infiltrations leading to easier discoloration. on the other hand, feldspathic ceramics consist of a vitreous matrix filled with silicon oxide and leucite crystals. since glasses do not suffer water sorption as polymers, glass-ceramics are more resistant to discoloration than resin composites. to date, the manufacturer of brava block does not disclose the main methacrylate monomers in the materials’ composition. however, it is well known that resin composites are more color unstable than dental ceramics8,15,24. picn has only 14 w% of udma and tegdma in its composition. still, this amount of composite was sufficient to decrease its color stability when compared to a feldspathic ceramic. a previous study proposed a new classification for ceramic and ceramic-like materials25. they classified polymer-matrices containing predominantly inorganic refractory compounds as resin-matrix ceramics. in this sense, vita enamic and brava block would be included in the same category, which also corroborates the similarity observed in our results. in contrast to our results, previous studies have found the highest discolorations in resin composites, followed by picn, and glass ceramics, which reach the lowest values after staining in red wine9,26. nonetheless, these studies used the cielab formula for color difference calculations and days straight of immersion in the beverages. one should note that the ciede2000 formula is a more sophisticated tool which better represents the color differences perceived by the human eye than cielab27,28. therefore, despite conflicting with previously published results, using ciede2000 for color and translucency calculations might bring more accuracy to the results of the present study. according to the values obtained, the initial translucency of picn would be lower than fel and rc (table 4). however, the cad/cam blocks used in this study are available in different color scales so that comparisons among the materials’ translucency would be biased. therefore, we evaluated the translucency stability of each material throughout the 30-day staining separately to avoid unfair comparisons. our results showed that all the materials maintained their translucency values over the staining process. previous studies have observed changes in translucency of picn after being subjected to red wine13,14. nevertheless, these studies were obtained from days straight of immersion 8 dalforno et al. in red wine, which might have overestimated the results. authors that implemented staining protocols similar to the one used in the present study found no differences in the translucency of glass-ceramics or resin composites8. previous studies have observed that surface finishing methods can increase roughness and consequently decrease the translucency29, or lead to color alteration of restorative materials8,30. different finishing approaches were performed in our study, since the fel samples received a glaze layer and the rc and picn samples were polished with sof-lex discs. this polishing sequence was chosen since diamond discs are frequently used in daily dental practice. furthermore, the diamond disc sequence is suggested by the manufacturers for repair, pre-polishing, and/or polishing of picn and rc31,32. even at the risk of influencing the results due to the different surfaces, it was decided to reproduce finishing procedures closer to the clinical conditions. on the other hand, the initial roughness was proven to be similar among the experimental groups (table 2). this evidences standardization of the samples regarding the surfaces subjected to the staining process. the described results are somehow clinically applicable to patients who have a pigment-rich diet. we employed a 30-min immersion in red wine twice a day with 12 h of dwell time. the staining protocol is plausible since the aforementioned patients might keep their restorations in contact with pigments for this amount of time a day. brushing is an important clinical factor and it was proven to reduce staining in resin composites33 and to cause color alteration in glass-ceramics34. nonetheless, brushing was not included in our study design. even so, our findings indicate that polished picn tends to behave as a composite regarding color stability when in contact with highly pigment beverages. moreover, picn has shown mechanical properties superior to composites35,36, which must also be considered when choosing the best restorative material for each clinical scenario. conclusion picn was not as color stable as the feldspathic ceramic at the end of the study. its color alteration was comparable to the resin composite when subjected to contact with red wine. all tested cad/cam materials reached the unacceptable threshold of discoloration already at 15 days of staining. however, the translucency of all restorative materials was stable throughout the 30-day staining protocol. acknowledgments the authors are thankful to the federal agency for support and evaluation of graduate education (capes) (finance code 001) for master and ph.d. scholarships. conflicts of interest: none references 1. stawarczyk 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j am dent assoc. 2008 sep;139 suppl:4s-7s. doi: 10.14219/jada.archive.2008.0359. 13. barutcugil c, bilgili d, barutcigil k, dündar a, büyükkaplan us, yilmaz b. discoloration and translucency changes of cad-cam materials after exposure to beverages. j prosthet dent. 2019 sep;122(3):325-31. doi: 10.1016/j.prosdent.2019.01.009. 14. quek shq, yap auj, rosa v, tan kbc, teoh kh. effect of staining beverages on color and translucency of cad/cam composites. j esthet restor dent. 2018 mar;30(2):e9-e17. doi: 10.1111/jerd.12359. 15. seyidaliyeva a, rues s, evagorou z, hassel aj, rammelsberg p, zenthöfer a. color stability of polymer-infiltrated-ceramics compared with lithium disilicate ceramics and composite. j esthet restor dent. 2020 jan;32(1):43-50. doi: 10.1111/jerd.12525. 16. sulaiman ta, suliman aa, mohamed ea, rodgers b, altak a, johnston wm. optical properties of bisacryl-, composite-, ceramicresin restorative materials: an aging simulation study. j esthet restor dent. 2021 sep;33(6):913-8. doi: 10.1111/jerd.12653. 17. paravina rd, ghinea r, herrera lj, bona ad, igiel c, linninger m, et al. color difference thresholds in dentistry. j esthet restor dent. 2015 mar-apr;27 suppl 1:s1-9. doi: 10.1111/jerd.12149. 18. salas m, lucena c, herrera lj, yebra a, della bona a, pérez mm. translucency thresholds for dental materials. dent mater. 2018 aug;34(8):1168-74. doi: 10.1016/j.dental.2018.05.001. 10 dalforno et al. 19. demarco ff, collares k, coelho-de-souza fh, correa mb, cenci ms, moraes rr, et al. anterior composite restorations: a systematic review on long-term survival and reasons for failure. dent mater. 2015 oct;31(10):1214-24. doi: 10.1016/j.dental.2015.07.005. 20. morimoto s, albanesi rb, sesma n, agra cm, braga mm. main clinical outcomes of feldspathic porcelain and glass-ceramic laminate veneers: a systematic review and meta-analysis of survival and complication rates. int j prosthodont. 2016 jan-feb;29(1):38-49. doi: 10.11607/ijp.4315. 21. chirumamilla g, goldstein ce, lawson nc. a 2-year retrospective clinical study of enamic crowns performed in a private practice setting. j esthet restor dent. 2016 jul;28(4):231-7. doi: 10.1111/jerd.12206. 22. spitznagel fa, scholz kj, vach k, gierthmuehlen pc. monolithic polymer-infiltrated ceramic network cad/cam single crowns: three-year mid-term results of a prospective clinical study. int j prosthodont. 2020 mar-apr;33(2):160-8. doi: 10.11607/ijp.6548. 23. gusmao gm, de queiroz tv, pompeu gf, menezes filho pf, da silva ch. the influence of storage time and ph variation on water sorption by different composite resins. indian j dent res 2013 jan-feb;24(1):60-5. doi: 10.4103/0970-9290.114954. 24. choi ys, kang kh, att w. evaluation of the response of esthetic restorative materials to ultraviolet aging. j prosthet dent. 2020 oct 8;s0022-3913(20)30471-6. doi: 10.1016/j.prosdent.2020.09.007. 25. gracis s, thompson vp, ferencz jl, silva nr, bonfante ea. a new classification system for all-ceramic and ceramic-like restorative materials. int j prosthodont. 2015 may-jun;28(3):227-35. doi: 10.11607/ijp.4244. 26. alharbi a, ardu s, bortolotto t, krejci i. stain susceptibility of composite and ceramic cad/ cam blocks versus direct resin composites with different resinous matrices. odontology 2017 apr;105(2):162-9. doi: 10.1007/s10266-016-0258-1. 27. gomez-polo c, portillo munoz m, lorenzo luengo mc, vicente p, galindo p, casado amm. comparison of two color-difference formulas using the bland-altman approach based on natural tooth color space. j prosthet dent. 2016 apr;115(4):482-8. doi: 10.1016/j.prosdent.2015.07.013. 28. ren j, lin h, huang q, zheng g. determining color difference thresholds in denture base acrylic resin. j prosthet dent. 2015 nov;114(5):702-8. doi: 10.1016/j.prosdent.2015.06.009. 29. akar gc, pekkan g, cal e, eskitascioglu g, ozcan m. effects of surface-finishing protocols on the roughness, color change, and translucency of different ceramic systems. j prosthet dent. 2014 aug;112(2):314-21. doi: 10.1016/j.prosdent.2013.09.033. 30. kanat-erturk b. color stability of cad/cam ceramics prepared with different surface finishing procedures. j prosthodont. 2020 feb;29(2):166-72. doi: 10.1111/jopr.13019. 31. vita enamic – working instructions. bad sackingen: vita zahnfabrik. [cited 2021 mar 10] available from: http://data.dt-shop.com/fileadmin/media/ga/1034_ga_enu.pdf. 32. fgm dental group. perfil técnico brava block. joinvile: fgm dental group; 2019 feb 1 [cited 2021 mar 10]. available from: https://issuu.com/fgmprodutosodontologicos/docs/perfil_brava-block. 33. mozzaquatro lr, rodrigues cs, kaizer mr, lago m, mallmann a, jacques lb. the effect of brushing and aging on the staining and smoothness of resin composites. j esthet restor dent. 2017 apr;29(2):e44-e55. doi: 10.1111/jerd.12293. 34. yuan jc, barao var, wee ag, alfaro mf, afshari fs, sukotjo c. effect of brushing and thermocycling on the shade and surface roughness of cad-cam ceramic restorations. j prosthet dent. 2018 jun;119(6):1000-6. doi: 10.1016/j.prosdent.2017.06.001. 11 dalforno et al. 35. porto ts, roperto rc, akkus a, akkus o, teich s, faddoul f, et al. effect of storage and aging conditions on the flexural strength and flexural modulus of cad/cam materials. dent mater j. 2019 mar;38(2):264-70. doi: 10.4012/dmj.2018-111. 36. facenda jc, borba m, benetti p, della bona a, corazza ph. effect of supporting substrate on the failure behavior of a polymer-infiltrated ceramic network material. j prosthet dent. 2019 jun;121(6):929-34. doi: 10.1016/j.prosdent.2018.08.008. 1http://dx.doi.org/10.20396/bjos.v20i00.8661512 volume 20 2021 e211512 original article 1 postgraduate program in health sciences, university of brasília, brasília, distrito federal, brazil. 2 proteomic and biochemical analysis center, postgraduate program in genomic sciences and biotechnology, catholic university of brasilia, brasília, distrito federal, brazil. 3 dentistry course, catholic university of brasília, brasília, federal district, brazil. 4 postgraduate program in biotechnology and biodivesity, brasilia university, brasilia, distrito federal, brazil. 5 graduate program in health and development in the midwest region. faculty of medicine. federal university of mato grosso do sul. campo grande. brazil. 6 s-inova biotech, graduate program in biotechnology, dom bosco catholic university, campo grande, mato grosso do sul, brazil. corresponding author: name: taia maria berto rezende full postal address: universidade católica de brasília pós-graduação em ciências genômicas e biotecnologia sgan 916n – av. w5 – campus ii – módulo c, room c-221 brasília-df, brazil fone: + 55-61-98134-9001 fax: + 55-61-3347-4797 e-mail: taiambr@gmail.com secondary e-mail: taia@ucb.br received: october 06, 2020 accepted: january 21, 2021 host defense peptides clavanins a and mo reduce in vitro osteoclastogenesis ingrid aquino amorim1,2 , stella maris de freitas lima2,3 , ana paula de castro cantuária1,2 , mirna de souza freire2,4, jeeser alves de almeida5 , octávio luiz franco2,6 , taia maria berto rezende1,2,3,* aim: several systemic diseases, such as periodontitis and apical periodontitis, can cause extensive bone resorption. host defense peptides may have the potential for the development of novel therapies for the bone resorption process. this study evaluated the potential of host defense peptides clavanins a, mo, and ll-37 in in vitro osteoclastogenesis. methods: raw 264.7 cultures were stimulated with recombinant of receptor activator of nuclear factor kappa b ligand in the presence of different tested concentrations of host defense peptides, besides calcium hydroxide and doxycycline. cellular viability, nitric oxide production, and a number of differentiated osteoclast-like cells were also evaluated. results: results showed that none of the substances were cytotoxic, except for 128 μg.ml-1 of doxycycline after 3 days. host defense peptides, calcium hydroxide, and doxycycline did not interfere in nitric oxide production or downregulated it. an exception was observed in the presence of 2 μg.ml-1 of doxycycline, in which nitric oxide production was up-regulated. all host defense peptides were capable of reducing osteoclast-like cell differentiation. conclusion: host defense peptides clavanins a and mo demonstrated to be potential suppressors of osteoclastogenesis in vitro without interfering in cellular viability and nitric oxide production. these promising results need to be further analyzed in in vivo models of bone resorption. keywords: bone resorption. antimicrobial cationic peptides. nitric oxide. osteogenesis. mailto:taia@ucb.br https://orcid.org/0000-0002-1340-7091 https://orcid.org/0000-0003-1359-8761 https://orcid.org/0000-0001-5828-450x https://orcid.org/0000-0002-3409-8005 https://orcid.org/0000-0001-9546-0525 http://orcid.org/0000-0002-4148-0659 2 amorim et al. introduction bone remodeling is a process balanced between osteoblast-mediated bone deposition and osteoclast-developed bone resorption. many oral diseases are mediated by an inflammatory process, increasing the recruitment of osteoclasts and enhancing bone erosion1. periodontal disease and apical periodontitis present bone resorption with high osteoclast formation or hyperactivation, overcoming bone formation, and decreasing osteoblast activity2. inflammatory conditions, such as local osteolysis, can be associated with inducible nitric oxide synthase (inos) activation3. no can also promote cytokine production and bone turnover besides indirect induction of bone resorption3. in this regard, periodontitis results in higher production of no compared to healthy gingiva4. periodontal treatment may involve the use of several systemic antibiotics such as tetracycline (minocycline and doxycycline) as adjuvants due to its local distribution. it was demonstrated that doxycycline hyclate gel (local therapy) could aid in scaling and root planning in patients with moderate to severe chronic periodontitis, but the benefit is still uncertain5. for the endodontic treatment, the use of calcium hydroxide (ca(oh)2) as a local antimicrobial is accepted worldwide as intracanal dressing6. despite the high success rate in existing periodontal and endodontic therapies, there are some limitations, mainly in tissue repair activity6. to improve bone repair in these diseases, it is essential to develop new substances. new therapies involving a direct effect on the bone can prolong the maintenance of the tooth in the oral cavity due to tissue support health. host defense peptides (hdps) are biomolecules from many organisms released in early defense response to infection and invasion by bacteria and other microorganisms7. hdps may possess antimicrobial and immunomodulatory properties besides tissue repair induction8. in this context, human cells can be potential sources of hdps7. clavanin a is a promising hdp due to its known antibacterial, immunomodulatory, antitumor, and antiviral activities9. besides, clavanin a was used as a model to create clavanin mo. five hydrophobic amino acid residues (flpii) were added to the n-terminus, being selected based on a computational search of the conserved region of other peptides with higher immunomodulatory activities10. it has been shown that different hdps could improve therapies in the dental field. some peptides have been reported as having the potential to inhibit osteoclastogenesis such as ll-3711, human beta-defensin-3 with c-terminal end contains a 15-amino acid polypeptide (hbd3 c15)12, synoeca-mp,13 and hhc-1013. a previous study demonstrated that ll-37 and clavanins a and mo can modulate the inflammatory response of active cytokines presented in the osteoclastogenesis process, such as tnf-α, while ca(oh)2 up-regulated the il-6 and il-1α production 14. this fact leads us to believe that clavanins a and mo may have the potential to inhibit osteoclastogenesis, a fact that has not yet been evaluated. thus, this study aims to evaluate the biotechnological potential of hdps clavanin a and mo in the oral osteoimmunological context and their capability to reduce in vitro osteoclastogenesis, 3 amorim et al. compared to ll-37 (hdp control), ca(oh)2 (used in the endodontic treatment) and doxycycline (used in the periodontal treatment). material and methods peptide synthesis clavanin a (vfqflgkiihhvgnfvhgfshvf-nh2), clavanin mo (flpiivfqflgkiihhvgnfvhgfshvf-nh2), and ll-37 (llgdffrkskekigkefkrivqrikdflrnlvprtes-nh2) were synthesized and purified (>95% purity) by peptide 2.0 inc. (usa). molecular mass and purity of all peptides were analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry on an auto-flex iii speed instrument (bruker daltonics, billerica, ma). peptides were diluted in ultrapure water and quantified by uv absorption at 205, 215, and 225 nm, according to murphy and kies15. doxycycline and calcium hydroxide preparation ca(oh)2 (iodontosul, porto alegre, brazil) was weighed and diluted in ultrapure water before each experiment. doxycycline (pharmac, brasilia, brazil) was handled in capsules (100 mg in each unit). the capsules were opened, and doxycycline was weighed and diluted in ultrapure water before each experiment. cell culture, experimental groups and osteoclasts osteoclast precursor raw 264.7 cell line (raw; bcrj code 0212; rrid cvcl_0493 – rio de janeiro, brazil) is composed of monocytes derived from tumors induced in male balb/c mice (mus musculus), infected with murine leukemia abelson virus16. raw cells were grown in high glucose dulbecco’s modified eagle’s medium (dmem; gibco, california, usa) supplemented with 10% fetal bovine serum (gibco, california, usa), 1% penicillin/streptomycin (1000 u.ml-1) (gibco, california, usa), 1% nonessential amino acid solution (gibco, california, usa), 1% l-glutamine (gibco, california, usa) and 0.1% gentamicin (gibco, california, usa). cell cultures were maintained in an incubator containing 5% co2 at 37°c and 95% humidity. experiments were conducted with 2.5x103 cells per wells in 96-well plates (kasvi, china), stimulated with or without rrankl 100 ng.ml-1 (peprotech, new jersey, usa) and hdps clavanin a, clavanin mo, and ll-37 (2, 8, 32, and 128 μg.ml-1). peptide stimulated cultures were compared to doxycycline and ca(oh)2 (2, 8, 32, and 128 μg.ml -1). the concentrations were based on a previously published result14. cell viability assay and no production were analyzed after 3 and 7 days of cell culture. half of the culture medium and stimuli were changed every 3 days. after 7 days, trap staining was performed, and the number of differentiated osteoclast-like cells was determined. cytotoxicity analyses peptides, ca(oh)2, and doxycycline cytotoxicity were analyzed by mtt colorimetric assay (sigma-aldrich, st. louis, usa), read in a microplate reader (bio-tek power wave ht, usa) at 570 nm17. cell viability was determined after 3 and 7 days of cul4 amorim et al. ture. all samples were compared to a positive control group (raw culture), considered as 100% cell viability. nitric oxide production analysis nitrite production was evaluated in supernatants of cell cultures by griess reaction, with adaptations18. briefly, 100 μl of cell culture supernatant was transferred to a new 96-well plate (kasvi, china). then, 100 μl of 1% sulfanilamide phosphoric acid solution and 2.5% of 1% naphthyl ethylenediamine phosphoric acid (1:1) was added. after 10 min, reading was performed in a microplate reader (bio-tek powerwave ht, usa) at 490 nm. the amount of nitrite was calculated based on a standard curve of sodium nitrite (1.5625 µm to 200 µm)18. tartrate-resistant acid phosphatase (trap) staining trap staining was performed after 7 days of incubation for the quantification of differentiated osteoclast-like cells. the tartrate-resistant acid phosphatase (trap) kit (sigma-aldrich, st. louis, usa) was used according to the manufacturer’s specifications. osteoclast-like cells were considered as trap-positive cells (with red/orange trap staining) with more than three nuclei. statistical analysis data obtained was analyzed by the standard error of the mean for each experiment. the normality was evaluated (kolmogorov-smirnov test), and subsequent parametric statistical analysis was carried out by two-way analysis of variance (two-way anova) for the data from mtt and no production and one-way anova for trap analyses. tukey’s posthoc test was applied to identify statistical differences. analyses were considered at the 95% significance level, and statistical differences were considered when p<0.05. statistical analysis was performed using graphpad prism 6.0 software (instat california, usa). results hdp cytotoxicity the cytotoxicity of substances was determined by cell viability assays after 3 and 7 days of cell culture in the presence of hdps clavanin a, clavanin mo, ll-37, ca(oh)2 and doxycycline. hdps and ca(oh)2 were not cytotoxic to pre-osteoclasts (data not shown). however, doxycycline, at the high concentration (128 μg.ml1) reduced cell viability by 48% (p<0.05), after 3 days of incubation, compared to the control group (data not shown). similar viability results were observed in osteoclast-like cells (raw cells with rrankl), and substances after 3 and 7 days incubation. hdps, ca(oh)2 and doxycycline were not cytotoxic to rrankl-stimulated cells (figure 1). however, 128 μg.ml-1 of doxycycline reduced cell viability by 42% after 3 days, compared to the control group (p<0.05). indeed, hdps were not cytotoxic and only doxycycline at 128 μg.ml-1 demonstrated a cytotoxic effect on osteoclast-like cells (rrankl-stimulated and raw cells). 5 amorim et al. nitric oxide production cell cultures with hdps, ca(oh)2, and doxycycline produced basal levels of no, compared to the control group (data not shown). the rrankl increased no production in figure 1. clavanin a, clavanin mo, ll-37, ca(oh)2 and doxycycline cytotoxicity at 2, 8, 32 and 128 μg.ml -1 on 2.5x103 raw cells, after 3 and 7 days, by mtt assay. cultures were stimulated with 100 ng.ml-1 of rrankl. cell viability was represented by percentage. control group was represented by 2.5x103 raw cells stimulated with 100ng.ml-1 of rrankl and considered 100% of cell viability. all experiments were done in technical and biological triplicates. statistical differences by two-way anova test and tukey’s post hoc were represented by *p<0.05, **p<0.005, ***p<0.0005 and ****p<0.0001 compared to each concentration and time tested conditions; dark green bars represent statistical differences observed on day 3; light green bars represent statistical differences observed on day 7. c el l v ia bi lit y (% ) clavanin a ** 250 200 150 100 50 0 co nt ro l 2 μ g.m l -1 8 μ g.m l -1 32 μg .m l -1 12 8 μ g.m l -1 c el l v ia bi lit y (% ) clavanin mo ** *** * 250 200 150 100 50 0 co nt ro l 2 μ g.m l -1 8 μ g.m l -1 32 μg .m l -1 12 8 μ g.m l -1 c el l v ia bi lit y (% ) ll-37 250 200 150 100 50 0 co nt ro l 2 μ g.m l -1 8 μ g.m l -1 32 μg .m l -1 12 8 μ g.m l -1 c el l v ia bi lit y (% ) doxycycline 3 days 7 days 250 200 150 100 50 0 co nt ro l 2 μ g.m l -1 8 μ g.m l -1 32 μg .m l -1 12 8 μ g.m l -1 c el l v ia bi lit y (% ) ca(oh)2 250 200 150 100 50 0 co nt ro l 2 μ g.m l -1 8 μ g.m l -1 32 μg .m l -1 12 8 μ g.m l -1 * ** ** **** *** **** *** * ** *** * ** ** **** ** **** **** * ** **** * ** 6 amorim et al. raw 264.7 cell cultures (figure 2). after 3 days, rrankl-stimulated cells with clavanin a downregulated no levels at all concentrations, compared to the control group, while on the seventh day, no levels were similar to the control group (p<0.0001). the downregulation of no production was also observed in cultures stimulated with clavanin figure 2. nitric oxide production in the presence of clavanin a, clavanin mo, ll-37, ca(oh)2 and doxycycline at 2, 8, 32 and 128 μg.ml-1 on rrankl-stimulated-raw cells, after 3 and 7 days, as described in the method of green et al., with adaptations. cultures were stimulated with 100 ng.ml-1 of rrankl. control group consisted of 2.5x103 raw cells stimulated with 100 ng.ml-1 of rrankl. bars represent the standard error of the mean of nitrite oxide production. all experiments were done in technical and biological triplicates. statistical differences by two-way anova test and tukey’s post hoc were represented by *p<0.05, **p<0.005, ***p<0.0005 and ****p<0.0001 compared to each concentration and time-tested conditions; dark green bars represent statistical differences observed on day 3; light green bars represent statistical differences observed on day 7. n itr ite (μ m ) clavanin a **** 5 4 3 2 1 0 co nt ro l 2 μ g.m l -1 8 μ g.m l -1 32 μg .m l -1 12 8 μ g.m l -1 n itr ite (μ m ) clavanin mo5 4 3 2 1 0 co nt ro l 2 μ g.m l -1 8 μ g.m l -1 32 μg .m l -1 12 8 μ g.m l -1 n itr ite (μ m ) ll-37 5 4 3 2 1 0 co nt ro l 2 μ g.m l -1 8 μ g.m l -1 32 μg .m l -1 12 8 μ g.m l -1 n itr ite (μ m ) doxycycline 3 days 7 days 5 4 3 2 1 0 co nt ro l 2 μ g.m l -1 8 μ g.m l -1 32 μg .m l -1 12 8 μ g.m l -1 n itr ite (μ m ) ca(oh)2 5 4 3 2 1 0 co nt ro l 2 μ g.m l -1 8 μ g.m l -1 32 μg .m l -1 12 8 μ g.m l -1 ************ **** ** *** ** *** * **** **** *** * ** ** **** **** ******** **** **** ** * * **** **** **** **** **** 7 amorim et al. mo at 128 μg.ml-1, after 7 days (p <0.05). however, the presence of 128 μg.ml-1 of ll-37 after 7 days, increased the no levels compared to the control group (p<0.0001), while the others concentrations at 3 and 7 days were similar to baseline levels. ca(oh)2 was able to upregulate no production at 8 (p<0.005), 32 (p<0.05) and 128 μg.ml-1 (p<0.005) after 7 days of cell incubation. no levels in the presence of ca(oh)2 at other different concentrations were similar to the control group (p<0.05), after 3 and 7 days. the lower concentration of doxycycline up-regulated no production after 7 days compared to all concentrations, including the control group (p<0.0001). overall, reduced levels of no were observed in some concentrations of all substances, except for doxycycline and ca(oh)2 7 days after the test. number of differentiated osteoclast-like cells raw cell cultures stimulated with hdps, ca(oh)2, doxycycline and rrankl were submitted to trap staining after 7 days of incubation (figure 3a-b), for quantification of differentiated osteoclast-like cells. all hdps, ca(oh)2, and doxycycline, at all tested concentrations, were capable of reducing the differentiation of osteoclast-like cells (figure 3b). indeed, clavanin a reduced osteoclastogenesis in an inverse dose-dependent concentration. clavanin a at 128 μg.ml-1 demonstrated the lowest number of differentiated osteoclast-like cells (p<0.05). osteoclastogenesis was similarly reduced by clavanin mo at all tested concentrations (p<0.05). likewise, ll-37 also downregulated osteoclastogenesis at 2, 4, 8 and 128 μg.ml-1, with the lowest number of osteoclast-like cells in 2 and 4 μg.ml-1 (p<0.05). ca(oh)2 was most effective in reducing osteoclast-like cells at 8 μg.ml-1 (p<0.05). however, 2 μg.ml-1 of ca(oh)2 showed the highest number of differentiated osteoclast-like cells (p <0.05). meanwhile, doxycycline exhibited a gradual reduction in osteoclastogenesis, and 128 μg.ml-1 stimulated cells demonstrated the lowest number of differentiated osteoclast-like cells. based on the number of osteoclast-like cells differentiated by rrankl-stimulated raw cell culture, the concentration of 8 μg.ml-1 was the lowest common concentration for hdps, ca(oh)2, and doxycycline, capable of reducing differentiation in osteoclast-like cells. therefore, the best results were exhibited by ll-37 and ca(oh)2. ll-37 showed approximately 67% fewer osteoclasts than clavanin a, clavanin mo and doxycycline, while ca(oh)2 showed 59% fewer osteoclasts compared to clavanins and doxycycline. ca(oh)2 and ll-37 demonstrated better osteoclastogenesis downregulation, compared to the same concentration of clavanin a, clavanin mo and doxycycline (p<0.0001). therefore, among the tested hdps, ll-37 presented the best ability to reduce the number of osteoclasts in vitro (p<0.05). 8 amorim et al. figure 3. representative photos of trap positive stained cells (a) and number of multinucleated osteoclastlike cells (b) induced by 2.5x103 raw cell and 2.5x103 raw cells stimulated with 100 ng.ml-1 rrankl (control). cultures were rrankl-stimulated and tested with 2, 8, 32, and 128 μg.ml-1 of clavanin a, clavanin mo, ll-37, ca(oh)2 and doxycycline, after 7 days. scale bar: 50 μm. each well was completely checked, and osteoclasts were counted at 20x magnification. black arrows show osteoclast-like cells with more than 3 nuclei. statistical differences by one-way anova test and tukey’s post hoc were represented by *p<0.001 compared to control in each tested condition. statistical differences of comparative analysis of the lowest common concentration, with greater reduction in osteoclast-like cell differentiation, of all tested materials: 8μg.ml-1 of clavanin a, clavanin mo, ll-37, ca(oh)2 and doxycycline (samples at 8μg.ml -1) were represented by *p<0.0001. number of osteoclast-like cells was represented as the standard error of the mean. all experiments were done in technical and biological triplicates. o st eo cl as ts clavanin ab a 50 40 30 20 10 0 co nt ro l 2 μ g.m l -1 8 μ g.m l -1 32 μg .m l -1 12 8 μ g.m l -1 * * * * o st eo cl as ts clavanin mo 50 40 30 20 10 0 co nt ro l 2 μ g.m l -1 8 μ g.m l -1 32 μg .m l -1 12 8 μ g.m l -1 * * * * o st eo cl as ts ll-37 clavanin acontrol 2 μg .m l1 8 μg .m l1 r a w c el ls r a w c el ls + rr a n k l 32 μ g. m l1 12 8 μg .m l1 clavanin mo ll-37 50 40 30 20 10 0 co nt ro l 2 μ g.m l -1 8 μ g.m l -1 32 μg .m l -1 12 8 μ g.m l -1 * * * * o st eo cl as ts ca(oh)2 50 40 30 20 10 0 co nt ro l 2 μ g.m l -1 8 μ g.m l -1 32 μg .m l -1 12 8 μ g.m l -1 * * * * o st eo cl as ts doxycycline 50 40 30 20 10 0 co nt ro l 2 μ g.m l -1 8 μ g.m l -1 32 μg .m l -1 12 8 μ g.m l -1 * * * * o st eo cl as ts samples at 8 μg.ml-1 50 40 30 20 10 0 cl av an in a cl av an in m o ll -37 ca (o h) 2 do xy cy cli ne ** * * * * * ca(oh)2 doxycycline 9 amorim et al. discussion although periodontal and endodontic therapies are highly effective, new substances can improve outcome expectations. antimicrobial, immunomodulatory, and reparative activity could be better achieved by new therapies and biologic substances19. indeed, antimicrobial resistance is also a current limitation for both therapies6. the present study demonstrated the in vitro potential of hdps clavanins a and mo in an osteoclastogenesis model. results of cellular no production, cytotoxicity, and the effects of hdps on rrankl-mediated osteoclastogenesis were compared to ca(oh)2 and doxycycline, widely used medications in endodontic 6 and periodontal5 areas, respectively. study related to clavanin a has demonstrated different activities regarding this peptide, including important points for dentistry, such as antibiofilm and antimicrobial activity. hdp clavanin a showed antibiofilm activity against fungal biofilms when used to coat an amniotic membrane, which is frequently used in ophthalmologic surgery for rapid ocular surface reconstruction20. hdps clavanin a, clavanin mo and ll-37 did not show any degree of cytotoxicity to raw cells. a previous study using clavanin a also showed no cytotoxicity against mammalian cells (l929) with low concentrations9. moreover, another study showed that 128 µg.ml-1 of clavanin mo did not demonstrate cytotoxicity compared to the other antimicrobial agents, with or without additional stimulation. ll-37 increased cell viability on raw cells, and ca(oh)2 did not interfere with cell viability at the same concentration. besides, after 6 h of incubation, clavanins alone reduced cell viability14. no regulates bone resorption through the regulation of the synthesis of opg/ rankl in bone marrow cells21, although other factors, including cytokines, are also involved. our results demonstrated that hdps downregulated no production with or without the stimulation of rrankl. accordingly, clavanin a demonstrated a significant reduction in the number of osteoclast-like cells in a dose-dependent manner. clavanin mo also reduced the number of differentiated osteoclast-like cells. similarly, ll-37 at 8 and 128 μg.ml-1 demonstrated the best inhibition activity. according to previous results, hdps can also modulate inflammatory mediators that contribute to the bone resorption activation process, such as tnf-α, il-6 and il-1α, and no production14. different substances already used in clinical practice were also evaluated in this study. ca(oh)2 did not show cytotoxicity, and doxycycline demonstrated a toxic effect on cells at high concentration and increased cell viability at low concentration. accordingly, a study evaluated the effects of a sub-antimicrobial dose of doxycycline (sdd) on ligature-induced periodontitis in spontaneously hypertensive rats. it concluded that sdd therapy exerted a systemic modulating effect on inflammation, with reduced periodontal tissue destruction in hypertensive rats22. ca(oh)2 presented similar no results compared to hdps, and doxycycline presented an increase in no levels, especially at 2 µg.ml-1. in the osteoclastogenesis process, both tested drugs decreased the number of osteoclast-like cells in the presence of all concentrations tested. these facts suggest that although no is strongly associated with osteoclast differentiation, this is not the only factor involved in the osteoclasto10 amorim et al. genesis process23. also, doxycycline might have another mechanism for downregulating the osteoclastogenesis pathway23. however, these results are in agreement with previous results that suggest the inhibition of osteoclastogenesis in raw cells in the presence of ca(oh)2 23. other studies suggest that the alkaline ph of ca(oh)2 can neutralize the lactic acid secreted by osteoclasts and may help prevent the destruction of mineralized tissue24. in summary, this study aims to initiate the assessment of the biotechnological potential of hdps clavanin a and mo in the oral osteoimmunological context and their capability to reduce in vitro osteoclastogenesis, compared to ll-37 (hdp control), ca(oh)2 (used in the endodontic treatment) and doxycycline (used in the periodontal treatment). we highlighted the results observed in the presence of 8 μg.ml-1 of ll-37 and ca(oh)2, thus considering the use of these peptides as a possible product for endodontic and periodontal applications, in order to reduce the osteoclastogenesis process. on the other hand, ca(oh)2 shows low production costs when compared to ll-37. this hdp presents a relatively long sequence of amino acids, which raises its cost for synthesis, and it would probably only be indicated for restricted cases. indeed, because of its immunomodulatory benefits and its biocompatibility, by being a peptide present in the oral cavity, ll-37 presents itself as a good candidate for dentistry use. when ll-37 results were compared to the doxycycline, the hdp demonstrated better efficiency in osteoclastogenesis downregulation at low concentrations, thus showing an even greater potential in the context of periodontal bone loss. despite the benefits highlighted in these data, in vitro results should be interpreted with caution and other in vivo studies are necessary to evaluate the potential of this biomolecule for clinical use. other important points for future investigations should be focused on the large-scale expression of this peptide (lowering its cost), and the analysis of its integrity through various oral conditions, such as temperature changes, ph, and presence of lytic enzymes. in addition, other parameters should be evaluated, such as the peptides’ mechanism of action in the osteoclastogenesis process, in order to enhance the knowledge on these potential products indicated for bone resorption processes, present in the periradicular area and periodontitis. acknowledgments this work was supported by the conselho nacional de desenvolvimento científico e tecnológico (cnpq) grant: 409196/2018-5, coordenação de aperfeiçoamento de pessoal de nível superior (capes), fundação de apoio à pesquisa do distrito federal (fapdf) grant: 0193.001702/2017 and fundação de apoio ao desenvolvimento do ensino, ciência e tecnologia do estado de mato grosso do sul (fundect). the authors deny any conflicts of interest related to this study. references 1. schett g, gravallese e. 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ll-37, in vitro: an endodontic perspective. peptides. sep 2017;95:16-24. doi:10.1016/j.peptides.2017.07.005. 15. murphy jb, kies mw. note on the spectrophotometric determination of proteins in dilute solutions. biochim biophys acta. 1960;45:382-4. doi: 10.1016/0006-3002(60)91464-5. 16. raschke wc, baird s, ralph p, nakoinz i. functional macrophage cell lines transformed by abelson leukemia virus. cell. 1978;15(1):261-7. doi:10.1016/0092-8674(78)90101-0. 17. van de loosdrecht aa, nennie e, ossenkoppele gj, beelen rh, langenhuijsen mm. cell mediated cytotoxicity against u 937 cells by human monocytes and macrophages in a modified colorimetric mtt assay. a methodological study. j immunol methods. 1991;141(1):15-22. doi:10.1016/0022-1759(91)90205-t. 18. green lc, wagner da, glogowski j, skipper pl, wishnok js, tannenbaum sr. analysis of nitrate, nitrite, and [15n]nitrate in biological fluids. anal biochem. oct 1982;126(1):131-8. doi:10.1016/0003-2697(82)90118-x. 12 amorim et al. 19. lima 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periodontitis in hypertensive rats. arch oral biol. 2019;101:77-84. doi:10.1016/j.archoralbio.2019.03.011. 23. guo j, yang d, okamura h, teramachi j, ochiai k, qiu l, et al. calcium hydroxide suppresses porphyromonas endodontalis lipopolysaccharide-induced bone destruction. j dent res. 2014;93(5):508-13. doi:10.1177/0022034514526886. 24. modena kc, casas-apayco lc, atta mt, costa ca, hebling j, sipert cr, et al. cytotoxicity and biocompatibility of direct and indirect pulp capping materials. j appl oral sci. 2009;17(6):544-54. doi:10.1590/s1678-77572009000600002. 1http://dx.doi.org/10.20396/bjos.v20i00.8660219 volume 20 2021 e210219 original article 1 department of periodontology, school of dentistry, university center of state of pará, belém, pará, brazil. 2 tropical medicine nucleus, federal university of pará, belém, pará, brazil. 3 laboratory of virology, institute of biological sciences, federal university of pará, belém, pará, brazil. 4 department of pharmaceutics, school of pharmacy, university center of state of pará, belém, pará, brazil. 5 program of post-graduation in chemistry, federal university of pará, belém, pará, brazil. 6 program of post-graduation in food science and technology, faculty of food engineering (labex/fea), federal university of pará, belém, pará, brazil. 7 adolpho ducke laboratory, botany coordination, museu paraense emílio goeldi, belém, pará, brazil. 8 study and research group on vulnerable populations, institute for coastal studies, federal university of pará, bragança, pará, brazil. corresponding author: silvio augusto fernandes de menezes university center of state of pará, school of dentistry. nine of january street; nº 927; neighborhood: são braz; zip code: 66037000; belém, pa – brazil; telephone: (55) 09198412623/ 09132662044; email: menezesperio@gmail.com received: june 25, 2020 accepted: december 9, 2020 chemical composition, antimicrobial and antifungal activity of lippia thymoide essential oil in oral pathogens tabata resque beckmann carvalho1 , erich brito tanaka1, amujacy tavares vilhena1, paula cristina rodrigues frade2 , ricardo roberto de souza fonseca3 , tânia maria de souza rodrigues1, mileide da paz brito4 , sebastião gomes silva5, raul nunes de carvalho junior6 , mozaniel santana de oliveira6 , eloisa helena de aguiar andrade7 , aldemir branco oliveira-filho8 , silvio augusto fernandes de menezes1,* aim: this study evaluated the chemical composition of lippia thymoides (lt) essential oil and its antimicrobial activity against fungal strains of candida albicans (ca) and gram-negative bacteria prevotella intermedia (pi) and fusobacterium nucleatum (fn). methods: lt essential oil was obtained by hydrodistillation apparatus with a modified clevenger extension. the chemical analysis was analyzed by gas phase chromatography and mass spectrometry on shimadzu qp 2010 plus. sample sensitivity evaluation was performed by abhb-inoculum and culture plates were developed with triphenyltetrazolium chloride, also fn and pi samples analysis were in anaerobic environment and ca sample analysis was performed in aerobic environment. the minimum inhibitory concentration (cim) was determinated by microdilution in eppendorfs tubes. results: the chemical analysis showed that thymol (59,91%) is the main compound found in lt essential oil, also other antifungal and antimicrobial agents were present γ-terpinene (8.16%), p-cymene (7.29%) and β-caryophyllene (4.49%), thymol is a central ingredient of many medicinal plants and has a potent fungicidal, bactericidal and antioxidant activity, it has been previously shown to have anti-inflammatory activity against periodontal disease (pd) cause can reduces prostanoids, interleukins, leukotrienes levels in periodontium. cim result pi was 6.5 μg/ml, fn was 1.5 μg/ml and ca was 0.19 μg/ml. conclusion: the antimicrobial activity of l. thymoides, through the compound thymol, has been shown promising potential against gram-negative periodontopathogenic bacteria and fungi whose therapeutic arsenal is still very restricted. keywords: microbiota. plant extracts. oils, volatile. lippia. antifungal agents. periodontitis. https://orcid.org/0000-0002-7046-5490 https://orcid.org/0000-0001-7455-3490 https://orcid.org/0000-0003-0312-0553 https://orcid.org/0000-0002-0065-4966 https://orcid.org/0000-0002-4433-6580 https://orcid.org/0000-0002-4076-2443 https://orcid.org/0000-0003-0640-7496 https://orcid.org/0000-0002-4888-3530 https://orcid.org/0000-0002-1679-9756 2 carvalho et al. introduction it is well documented that oral microbiome is formed about 700 different microbial species and these interactions might result in a distinct environmental and microbial communities, among the microorganisms we cite bacterias, fungus and viruses, which might coexist in specific and formed organization in oral cavity habitats1-5. which one of these species can colonize a different sub-habitat in our mouth, this discrepant preferences due to anatomy of the region, nutrients sources, oxygen availability, ph range and host immune cells activity, among the colonized sub-habitats we mention: tongue surface and/or dorsum, keratinized and non-keratinized mucosa, cheek, periodontium (supra and subgingival) and teeth5-7. in most cases, the organisms presented in microbial communities live in oral cavity normally, harmless and helpful, but under certain conditions such as8,9: inadequate oral hygiene, host immunosuppression, risk factors, oral dysbiosis and presence of pathogenic organisms may lead patient to infectious diseases caused by the organisms cited above and the three main oral diseases are: caries, periodontal diseases (pd) and oral candidiasis10-12. oral and periodontal medicine are two dentistry fields, where oral pathologies like pd and oral candidiasis are treated through reducing dental plaque levels, oral hygiene improvement, host immune balance, decrease of pathogenic organisms and risk factors13,14. pd is a worldwide oral health problem; it is a multifactorial and poly‐microbial disease with host‐specificity. pd can be classified mainly in gingivitis and periodontitis, the diagnosis is based upon disease’s severity stage and grade, clinical attachment loss, periodontal probe depth, bleeding and/or suppuration on probing, alveolar bone resorption and eventual tooth loss15,16. pd’s etiopathogenesis is inflammatory and it is resulted to complexes interactions between dental plaque accumulation based on the specific plaque hypothesis and according to this theory the oral microbiome dysbiosis and polymicrobial synergy is caused by specific gram-negative bacterias like porphyromonas gingivalis, aggregatibacter actinomycetemcomitans, treponema denticola, tannerella forsythia, prevotella intermedia (pi) and fusobacterium nucleatum (fn) and associated with host-immune response decrease, systemic and local risk factors such: hiv, tobacco, diabetes, dental braces and even alzheimer’s disease17,18. as the third most common oral disease, oral candidiasis is clinic diagnosed by the presence of a superficial inflammation on the oral mucosa due to the overgrowth of fungal agents such as candida albicans (ca), c. tropicalis, c. glabrata and c. parapsilosis4,11.oral candidiasis clinical presentations include white plaques on tongue, mouth pain and burning, fungal organisms like ca can develop in certain condition and might begin due to systemic or local impairments such as host immune response, organ transplantation, hiv infection, chemotherapy, radiotherapy, elder age, oral prosthesis, poor oral hygiene, tobacco and alcohol use, hyposalivation and sometimes antibiotic drug use12,19. pd is an infectious inflammatory disease manifested and aggravated by a subgingival bacterial dysbiosis in dental plaque accumulation14,15. pd’s treatment, like 3 carvalho et al. gingivitis and early stages of periodontitis, basically consists in supra/ subgingival dental plaque accumulation reduction through non-surgical and mechanical removal known as scaling and root planning (srp), srp is well documented as the standard periodontal treatment, although its clinical benefits srp alone does not always induce in periodontal ecological modification necessary to accomplish and sustain clinical improvements17. therefore, periodontal adjunctive therapies such as systemic antimicrobials and specific mouthwashes have been used to improve clinical and microbiological status and features specially in necrotizing periodontal diseases and severe periodontitis which presents severe clinical conditions and a subgingival microbial ecology bacterias from socransky’s and orange complex18,20. as mentioned above, mechanical therapy as srp besides periodontitis advanced stage and aggressive subgingival bacterias, other limitations might decrease srp effectiveness, such factors as deeper depths of the periodontal pocket, root anatomy, periodontal instrument design, and operator ability may influence negatively in pd’s treatment20. mouthwashes just as chlorhexidine gluconate, sodium fluoride, zinc chloride and saline solution may act as a periodontal adjunctive therapy to control and sometimes even to prevent infections to spread systemically21,22. mouthwashes like chlorhexidine due to great activity against a variety of gram-positive and gram-negative bacterias can be used by dental surgeons during srp sessions to treat severe pd, despite chlorhexidine well-documented and guaranteed effectiveness few complications might appeared during periodontal treatment as chlorhexidine cationic properties, bacterial resistance and strong reactivity21,22. systemic antimicrobial therapy is also used as periodontal adjunctive therapy, antibiotics like metronidazole can be used locally or systemically to treat infections such necrotizing periodontal diseases and severe periodontitis and its effectiveness is influenced by the agent and protocol used20,23. according to teughels et al. (2020)23 antibiotics associations like amoxicillin and metronidazole has been showed an effective effect in periodontitis therapy. however, despite its positive clinical effects, the enormous and inaccurate use of systemic antimicrobials contributes to bacteria specific-drug-resistant and multidrug resistant and still according to teughels et al. (2020)23 periodontopathogens specific-drug-resistant is more frequent due to populations who are more exposure to systemic antimicrobials, like brazilian population20,22. regarding oral candidiasis treatment when it is caused by ca, it can be carried out through the association between oral hygiene improvement with the use of azole drugs, nystatin and polyenic antibiotics8,9. nystatin is cited as the gold-standard to treat oral candidiasis, nystatin is a polyene antifungal agent and it is widely used due to its effective influence and wide spectrum of pathogenic fungi, especially against ca21. regarding nystatin although is a safe drug, because it is not easily absorbed through skin or mucosae, low toxicity and no reported drug interactions some effects of oral nystatin must be considered such as high risk of developing caries and resistance to polyene antifungals which will decrease oral candidiasis treatment19. medicinal plants (mp) or essential oils (eo) usage as treatment or adjunctive therapy for various pathologies has been a practice since antiquity folk medicine, 4 carvalho et al. although mp and eo have been used for several years by ancient societies24-28. in brazil the scientific and commercial interesting upon natural substances like eo increased in recent years, so naturally the number of studies trying to understand eo medicinal and dental properties such as analgesic, anti-inflammatory, antimicrobial and/or antifungal effects enlarged24-28. in dental medicine, oral hygiene products based on eo have been used and one of the substances mainly used is thymol, it has been shown that thymol is an alternative mouthwash due to its potential anti-inflammatory, fungicidal and bactericidal action may serve as medical coadjutant against pd and oral candidiasis27,28. the objective of this study was to evaluate the chemical composition of lippia thymoides (lt) essential oil and its antimicrobial and antifungal activities against the fungal strains of candida albicans (ca) and the gram-negative bacterias prevotella intermedia (pi) and fusobacterium nucleatum (fn). materials and methods plant material lippia thymoides was collected from abaetetuba, brazilian state of pará, amazon region, always at the same horary and geographical location 1° 46’15.9 “south latitude and 48° 47’02.2” west longitude, at the end of the first month of each season: april 2017 (autumn), july 2017 (winter) and october 2017 (spring). voucher specimens were deposited under the number 213373 at the herbarium of the museu paraense emílio goeldi, in belém, brazilian state of pará, amazon region. the specimen was identified and categorized by a single calibrated botanical researcher, who had previously experience in botanical classification and taxonomy29,30. leaves of a pool of individuals were separated from the stem and air-dried at room temperature, protected from light, until constant weight, and powdered in the cutting mill. the determination of the residual humidity was carried out in a humidity-determining balance of marte®, model id 50, with infrared at the time of extraction30. essential oils extraction the botanical material was collected manually at 6 o’clock in the morning, transported immediately to the laboratory, then placed on trays, in a greenhouse at 34oc, with ventilation, for drying for three days, after being crushed in a botanical processor30,31. the extraction of the essential oil from the dry leaves of lt was made by hydrodistillation using a modified clevenger type glass system coupled to a cooling system to maintain the water condensation at 10-15ºc for 3 hours. after extraction the oils were centrifuged for 5 min at 3000 rpm, dehydrated with anhydrous na2so4 and again centrifuged under the same conditions. the oils were stored in amber glass vials, flamesealed and conditioned in a refrigerated environment at 5ºc at a concentration of 31.25 μm to immediately use after collection32,33. the mass yield in % of the essential oil was on a dry basis according to the equation in figure 1. 5 carvalho et al. figure 1. equation used to determine the mass yield in % of the essential oil on a dry basis. t = v oil m sample x ( )100-u100 chemical analysis the chemical composition of the essential oils was analyzed by gas phase chromatography and mass spectrometry (gc-ms) on shimadzu qp 2010 plus, self-injector (figure 2): aoc-20i equipped with rtx-5ms silica capillary column (30m x 0,25 mm; 0,25 mm film thickness) under the following operating conditions: temperature program: 60ºc 240ºc, with a gradient of 3ºc/min; injector temperature was 250ºc; the drag gas was helium (linear velocity of 32 cm/s, measured at 100ºc); without flow division (0.1 ml of a 2: 1000 of n-hexane); temperature of the ions source and other parts was 200ºc. the quadrupole filter was used for scanning in the range of 39 to 500 daltons every second. ionization was obtained by the electronic impact technique at 70 ev34-36. the identification of the volatile components was based on the linear retention index, which was calculated in relation to the retention time of a homologous series of n-alkanes and on the fragmentation, pattern observed in the mass spectra, by comparing them with authentic samples in the libraries the data system and the literature34-36. microbial species and culture conditions the evaluation of antimicrobial and antifungal activities of lt essential oil against fn, pi and ca strains of were performed by the microdilution method that is based on successive dilutions of the sample in culture containing blood. the microbiological assays were used with 5 strains of ca-atcc 90028; 5 strains of pi-atcc 49046 and 5 strains of fn-atcc 25586 purchased from the osvaldo cruz foundation (fiocruz), brazil. each inoculum was made and developed indefigure 2. l. thymoides chromatographic analysis. 1.25 1.00 0.75 0.50 0.25 0.00 uv (x1,000,000) chromatogram 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 min 6 carvalho et al. pendently according the needs of each microorganism, the bacterial inoculums were obtained from a fresh culture suspension (maximum 48h at 72h) of fn and pi37. the fungal inoculum was obtained from a a fresh culture suspension (maximum 24h) of ca in 0.85% saline solution (m/v). the concentration of the microorganism was standardized by comparing turbidity of the inoculum with the macfarland scale equivalent to a 1.5x108 cfu/ml concentration in a turbidimeter (grant-bio-den-1, model v.1gw)38. for the antibiotic control, vancomycin (sigma aldrich®) was used as a positive control at the concentration of 16 μg/ml; as negative control the microorganism culture was evaluated in a sterile medium area. the culture media to be used for analysis were the agar, the brain and heart broth (abhb), respectively, containing polysorbate 80 at 0.5% (m/v) in sheep blood, which will allow bacterial and fungal by growth decreasing interfacial tension neutralizing any disinfectants agents in the agar and defibrinated was also used39. as fungal control, nystatin (nystatin®) n6261sigma-aldricht was used as a positive control at (1 mg/ml) concentration; as negative control the microorganism culture was evaluated in a sterile medium area. the culture media to be used for analysis were the abhb, containing polysorbate 80 at 0.5% (m/v) and defibrinated sheep blood40. sample sensitivity evaluation to prepare the plates, 11 ml of abhb were poured into 15x100 mm plates. on this layer of solidified medium was added 10 ml of abhb-inoculum containing 106 cfu/ml. with the two layers uniformly superimposed and already solidified, filter paper was added with approximately 6 and 8 mm diameters and impregnated with 10 μl of essential oil. the samples were placed on the culture and incubated at 35°c / 24h for fn and pi; the ca culture was incubated at 25°c/24h. fn and pi samples analysis were in anaerobic environment (n2: 80%, h2: 10%, co2: 10%) and ca sample analysis was performed in aerobic environment22. after the incubation period, both ca, fn and pi culture plates were developed with triphenyltetrazolium chloride (in the fungal culture plate, it was added to confirm that it was only colonized by ca and in the bacterial culture plate it was added to improve the determination of minimum inhibitory concentrations at 7 mg/ml in bacteriological agar at 1% (m/v) and the results of the halos (mm) were measured using a pachymeter37,38. determination of minimum inhibitory concentrations the essential oil minimum inhibitory concentrations (mic) was performed in eppendorfs tubes by microdilution, where a 50 μl aliquot of the essential oil was diluted 1:2 in abhb with 5% (v/v) and polysorbate 80 defibrinated blood containing 105 cfu/ml to 20 dilutions. then, as cultures, those aliquots were incubated in triplicate (to increase positive results and prevent experiments failures), fn and pi bacterial strains aliquots were grown in tryptic soy broth and supplemented with hemin (5 mg/ml) and menadione (0.5 mg/ml) 35°c/ 24 hours in anaerobic environment 7 carvalho et al. (n2: 80%, h2: 10%, co2: 10%) 37. ca aliquots was performed in a laboratory greenhouse to allow better fungal growth, it contained 105 cfu/ml up to 15 dilutions and incubated at a temperature of 25°c/ 24 hours38. after the incubation period the plates were seeded with 40μl of the culture of each dilution in petri dishes (5x50 mm) containing 5% (v/v) defibrinated blood soybean casein agar and then incubated for further 35°c/24h for fn and pi, for ca agar incubated for further 25°c/ 24 hours. the plaques were evaluated for the presence or absence of bacterial and fungal growth, when compared to the negative and positive control groups. mic were revealed at the last dilution where there was no microbial growth after incubation, the plates were developed with 1% (m/v) bacteriological broth containing 7mg/ml of triphenyltetrazolium solution. the maintenance of the red color in the medium was interpreted as presence of microbial growth. results chemical components the yield of lt oil obtained by hydrodistillation was 0.7%. the chemical composition was determined by gc-ms, where thirty-five volatile constituents were identified, representing 96.7% of the total compounds present in the oil. the main constituents found in the essential oil were thymol (59,91%) (figure 3); γ-terpinene (8,16%); p-cymene (7, 29%); thymol acetate (6, 26%) and β-caryophyllene (4, 49%) (table 1). figure 3. thymol mass spectrum in l. thymoides chromatogram. % 100 50 0 40 41 45 50 51 57 60 65 69 70 77 80 80 91 92 90 100 110 103 107 115 122 120 130 133 135 140 150 150 152 table 1. chemical composition of lippia thymoides essential oil. compounds % α-thujene 0.74 α-pinene 0.20 β-pinene 0.09 myrcene 2.06 α-phellandrene 0.23 δ-3-carene 0.12 α-terpinene 1.58 p-cymene 7.29 (e)-β-ocimene 0.14 continue 8 carvalho et al. continuation γ-terpinene 8.16 terpinolene 0.30 linalool 0.13 camphor 0.04 umbellulone 0.21 terpinen-4-ol 0.34 methylthymol 1.43 thymol 59.91 thymol acetate 6.26 α – copaene 0.02 methyl eugenol 0.05 β-caryophyllene 4.49 β-copaene 0.11 trans-α-bergamotene 0.14 γ-elemene 0.14 αhumulene 0.75 allo-aromadendrene 0.06 γ-muurolene 0.24 germacrene d 0.26 γ-amorphene 0.18 δ-amorphene 0.16 trans-β-guiaene 0.05 γ-cadinene 0.20 δ-cadinene 0.42 elemol 0.04 caryophyllene oxide 0.12 total 96.7 %percentage of each compound found in the gc-ms table 2. inhibition zone diameter, antimicrobial and antifungal activities of lippia thymoides essential oil. samples f. nucleatum p. intermedia c. albicans halo (mm) 15 19 38 mic (µl ml-1) 1.6 6.5 0.19 mbc (µl ml-1) 2.60 2.44 mfc (µl ml-1) 1.30 1vcm (µg ml-1) 34 28 2nys (µg ml-1) 32 1vancomycin; 2nystatin; minimum bacterial concentration (mbc) and minimum fungicidal concentration (mfc). 9 carvalho et al. antimicrobial and antifungal activities the results showed that lt had a satisfactory bactericidal and antifungal against the 15 strains of ca-atcc 90028, pi-atcc 49046 and fn-atcc 25586 with eo 2% concentration. the inhibition halos were measured and resulted in 15mm (fn), 19mm (pi) and 38mm (ca). as bacterial positive control vancomycin was used and as fungicidal positive control nystatin was used and the respective mic values were 6.5 μg/ml (pi), 1.5 μg/ml (fn) and 0.19 μg/ml (ca) and the minimum bacterial concentration (mbc) were 2.60 μg/ml (pi) and 2.44 μg/ml (fn) and minimum fungicidal concentration (mfc) was 1.30 μg/ml (ca) and the peak of bactericidal and antifungal action could be observed after 2 hours and maintain during 4 hours. discussion pd and oral candidiasis are two of the three most common oral pathologies in the world, as the aforementioned this pathology is the result of an interaction between microorganisms and an accumulation of dental biofilm and subgingival surface; and non-surgical periodontal mechanical therapy associated with systemic or local anaerobicides and antifungal has been the standard treatment established in the literature for these pathologies20-22,41,42. however, this standard protocol is not always effective due to variations of occurrences, being more common cases of bacterial and fungal resistance due to the unreasonable and unrestrained prescription use of antibiotics and fungicides, which leads the periodontist to use more potent antibiotics with bigger adverse effects. in an attempt to resolve such complications and promote more effective and less harmful treatment, different therapeutic alternatives may be tested such as the herbal medicine25-27,43-45. the use of phytotherapy is a part of popular medicine, mainly in the amazon region (northern brazil). several studies have shown that medicinal plants sources are abundant in active biological compounds25-27. in recent years, the number of studies about medicinal plants has increased and through the results of these studies, we verified that medicinal plants have anti-inflammatory, antimicrobial, fungicidal and adverse effects on its extracts and essential oil which shows the great potential for several therapeutic applications43-47. studies such as warad et al.25 (2013) and mandras et al.27 (2016) reported excellent therapeutic properties of essential oils in a variety of aerobic, anaerobic and fungal microorganisms. in our study we used lt as the herbal medicine of choice; lt is a native species of brazilian vegetation, popularly used to treat various pathologies. thus, due to its ethno-pharmacological potential and innovative; our research was dedicated to studying its chemical composition and the antimicrobial and antifungal properties of the essential oil of this species, looking for a possible auxiliary agent in the periodontal treatment48-50. in the present study, we demonstrated that the essential oils extracted from lt maintain an antimicrobial activity against two of the main gram-negative periodontal pathogens, such as fn and pi47,50. in addition, it presented satisfactory activity against ca 10 carvalho et al. strain, which we can observe to be the most susceptible strain at mic <0.19 μl/ml. in contrast, pi was the most resistant strain, with mic> 6.5 μl/ml. according to botelho et al.43 (2016) the use of oral antiseptic containing essential oils associated with non-surgical periodontal therapy can significantly inhibit the levels of fn and pi in the supragingival and subgingival biofilm. in the study of sharifzadeh et al.46 (2018), the authors confirmed in their in vitro study that thymol has a highly fungicidal action. in their study the authors used different concentrations of thymol in samples of seropositive patients with candidiasis in the most diverse forms, the authors had the same conclusions that our study obtained that thymol is a promising fungicide against ca. in relation to the antimicrobial activity, lt extract showed to be sensitive against both bacteria, being more effective against fn corroborating with the results of arbia et al.47 (2017). the results of this study are considered promising and innovative because juiz et al.45 (2015) analyzed the effects of lippia alba on fn and found that this essential oil had no effect on its samples. in our study, when we analyze lt extracted essential oil, thymol (59%) was presented as the main chemical constituent. in the literature thymol it is already well documented as a viable antifungal agent for oral candidiasis, according with ahmad et al.48 (2013) thymol has amphipathic characteristics which in oral ca can affect the cell membrane structure and its electrostatic surface generating asymmetric tension in the membrane then its rupture, furthermore thymol might also interfere in mitochondria activities and in the production of viable filamentous forms that are essential to c. albicans during oral biofilm formation28,48-53. regarding using thymol as an alternative to periodontal disease, in the last few years this compound has been used commercially in mouthwashes due to its antimicrobial activity. since 1879, listerine® is mouthwash used in dentistry as a surgical disinfectant and one of its ingredients is thymol. sköld et al.50 (1998), antunes et al.51 (2015) e vlachojannis et al.28 (2016) demonstrated thymol’s antimicrobial activity efficiency against aerobic bacterias, such as streptococcus mutans, and anaerobic bacterias, such as fusobacterium nucleatum and yet when associated with chlorhexidine it decreased prostaglandin e2 levels in gingival crevicular fluid. contrasting our results of chemical composition silva et al. (2016)36 evaluated in their extract of lt that thymol was not present and had as main constituent sesquiterpene β-caryophyllene; such chemical variation may have occurred due to environmental, climatic and regional factors28,48-53. although our study was unable to perform cytotoxic studies and studies about the effectiveness of antimicrobial and antifungal activities through the inhibition zone halo diameter, specifically, it was due to the activity of thymol or any other components of the sample and such deficiencies occurred due to laboratory limitations, sample and financial. we quote this limit because among the other components of lt we verified the presence of γ-terpinene (8.16%), p-cymene (7.29%) and β-caryophyllene (4.49%) which are also compounds with antimicrobial, antifungal and anti inflammatory effects in studies such as those by emiroğlu et al.52 (2010), li et al.53 (2020) and amankwaah et al.24 (2020). despite there were decreasing of fn, pi and ca in our in vitro study the oral environment is far more complex due to conditions like temperature, ph, oral hygiene quality, polymicrobial biofilms and mouth humid11 carvalho et al. ity so in vivo or randomized clinical trials using and testing different eo concentrations and posology is needed to guarantee lt essential oil viability against pd and oral candidiasis28,48-53. the antibacterial activity of thymol is related to its activity on the phospholipids present in the cell membrane, which will increase the permeability of the membrane, destruction of the cell membrane, cytoplasmic leakage, cell lysis, and consequently cell death that’s why we assumed by the higher percentage of thymol this was the main antimicrobial and antifungal agent. although our study has not been able to perform cytotoxic studies on our samples, some authors state that using essential oils with values above 4.3 ml of thymol can cause episodes of cytotoxicity. however, de la chapa et al.44 (2018) state that thymol has antimicrobial properties and can be considered as a natural product with low cytotoxicity, even though further studies should be carried out to evaluate the toxicity of the product28,48-53. conclusion in summary, this study demonstrated that lt has the ability to produce essential oils with biological components such as thymol, which have potential to antifungal and antimicrobial results. this in vitro test evidenced a possible coadjuvant agent for periodontal treatment and further studies are needed. so further research on new medicinal coadjuvants should be developed to better clarify the biological activities of l. thymoides. acknowledgments the authors contributed equally to this paper. conflict of interest the authors declare that there is no conflict of interest. author’s contribution tmsr and safm conceived the original idea and supervised the project and the manuscript; trbc, ebt and atv collected the data and wrote the manuscript; rncj and ehaa analyzed the statistical methods; mpb, sgs and 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10.1016/j.ijbiomac.2020.02.066. 1 volume 21 2022 e226694 original article braz j oral sci. 2022;21:e226694http://dx.doi.org/10.20396/bjos.v21i00.8666694 1 universidad peruana los andes (upla), facultad de medicina humana, sociedad científica de estudiantes de medicina los andes (sociemla), huancayo, perú. 2 universidad peruana los andes (upla), facultad de odontología. sociedad cientifica de estudiantes de odontología universidad peruana los andes (sceoupla), huancayo, perú. 3 universidad continental, facultad de medicina humana, huancayo, perú corresponding author: christian renzo aquino canchari e-mail: christian.aquino.canchari@ gmail.com direction: cc.hh. juan parra del riego, ii etapa, block 2, dpto. 101, el tambo, huancayo, junín, perú. postal code: 051 cell phone: 051-934824051 editor: altair a. del bel cury received: august 16, 2021 accepted: january 29, 2022 accessibility to student publication in dental journals in the world christian renzo aquino-canchari1 , anabel jimena osco-mueras2 , luis arturo santivañez-isla2 , katia medalith huamán castillón3 scientific research and publication play an important role during the training of dentists, but one of the most outstanding barriers is the authorship conditions of the journals. aim: the objective of the study was to determine the accessibility to student publication in dental journals in the world. methods: a retrospective study was carried out. 208 journals indexed in scimago journal & country rank that met inclusion and exclusion criteria were included. the instructions for the authors were reviewed, an email was subsequently sent to the journal contact and articles with student affiliation were searched in the database of each journal. for the analysis of the descriptive statistical data of frequencies and percentage, the ibm spps statistics standard edition 22 program was used. results: 208 journals were included, 77.67% accepted the student publication without condition. the united states, united kingdom and india were the countries with the highest number of journals with student participation. likewise, the journals of q4 (85.70%), q3 (85.40%) and basic sciences (100%), dental education (100%), endodontic (100%), geriatrics and gerontology (100%) and public dental health (100%), mostly accepted student authorship. conclusion: it is concluded that 167 (77.67%) of the dental journals accept the publication of dental students without condition, being more frequent in journals positioned in q4 (85.70%). also, journals with thematic areas on basic sciences, dental education, endodontic, geriatrics and gerontology and public dental health. keywords: dentistry. students, dental. research report. school dentistry. research. https://orcid.org/0000-0002-7718-5598 https://orcid.org/0000-0002-3218-4508 https://orcid.org/0000-0002-5337-0609 https://orcid.org/0000-0001-7771-099x 2 aquino-canchari et al. braz j oral sci. 2022;21:e226694 introduction research plays a crucial role in professional training in dentistry. it is clear that better patient care is the result of technical advances made possible by research1. there is a clear recommendation to incorporate biomedical research training into undergraduate studies. in addition to the primary focus of the research, it can also be a valuable tool in dental education to prepare the next generation of leaders in oral health care2. scientific publication is the main means of transmitting evidence-based information to dentists and health professionals, especially if they are carried out in journals indexed to prestigious databases3. it also constitutes the most significant contribution that can be made to the academic community from undergraduate, as well as allowing the visibility of research at this level. it is a necessary good to promote the publication from undergraduate dentistry, however, even student participation within them is low, and among the causes, we find lack of time, little information, lack of academic incentives, few opportunities to integrate a research team, greater encouragement to the training of professionals dedicated to care activities, among the main ones4,5. rejection of undergraduate authors may be due the lack of knowledge of the student’s editorial itinerary added the prejudice towards them, for this reason some journals consider it necessary to condition the co-authorship with a professional, in addition to this there is a low number of citations of articles executed by undergraduate students in high-impact journals, thus limiting its usefulness6. currently, there are no studies that evaluate dental student’s disqualification as authors of scientific publications. therefore, the objective of the study was to determine the accessibility of student publication in dental journals in the world, indexed to scopus through scimago journal & country rank. materials and methods study design an observational, descriptive, retrospective study was carried out. analysis unit the unit of analysis was the dental journals indexed to scopus, in its 2020 edition. process the scimago journal & country rank (sjr) website (https://www.scimagojr.com) was accessed to find out about the journals included in the subject area of «dentistry», under «all subject categories», in «all regions/countries» and type «journals». subsequently, a first selection was made where non-current (discontinued) journals, without access to their website, were excluded, journals not classified in a quartile. the journals were classified according to the following categories: accepts student publication, accepts student publication with conditions, accept with or without condition, https://www.scimagojr.com 3 aquino-canchari et al. braz j oral sci. 2022;21:e226694 does not accept student publications, does not specify7. the research had three phases: first the «instructions for authors» or «guidelines for authors» journal’s section was reviewed, then the collection form was completed according to the established categories, if the journal did not specified whether or not to accept manuscripts with students as authors, a second phase was accessed, where an email was sent to the journal’s contact, a period of one month for a response was stablished. the collection form was simultaneously completed, and last, in the third phase, the journals that did not respond to the email were included, in which a manual search was carried out in the «archives» section, data base revision, in order to find an article that includes a student as author. this search was carried out up to three years old (2018-2020), recording the findings in a separate category. the analysis variables were: country of the journal, quartile, subject area of the journal. statistic analysis an information collection template prepared for this purpose was used. the data obtained was collected in a database (ms excel, microsoft corp., usa, 2018). the statistical program ibm spps statistics standard edition 22 was used, with which the descriptive statistical analysis was performed through frequencies and percentages. results 208 journals were included, distributed in 33 countries that met the inclusion and exclusion criteria of the study. in the first phase of the study, no data was collected on student accessibility since none required it; in the second stage after sending the emails, a response was obtained from 62 journals, and in the third phase it was verified that 121 journals presented articles with student affiliation. (figure 1) 208 journals first phase second phase third phase revision of the “instructions for authors” the 208 journals do not require sending mailings database revision 7 do not accept student publication 48 accept student publication 7 accept student publication on condition 121 journals have students as authors 25 journals do not have students as authors figure 1. flowchart of the selection and evaluation process of selected journals. 4 aquino-canchari et al. braz j oral sci. 2022;21:e226694 the countries with the highest percentage of journals that accept the student as an author without any conditions were canada (100%) and chile (100%) in america, bulgaria (100%), croatia (100%), italy (100%), poland (100%), denmark (100%), sweden (100%), russia federation (100%) on the european continent, south korea (100%), china (100%), united arab emirates in asia, and australia in oceania. it should be noted that the united states, united kingdom and india were the countries with the highest number of journals with student participation. in contrast, japan (44.44%) and egypt (50.00%) are the countries with the highest percentage of journals that do not accept student affiliation. (table 1) table 1. accessibility of student publication in dental journals of the world indexed to sjr, according to the country of the journal. continent country of journal journals that accept journals that acccept conditional journals that accept with or without condition journals that do not accept journals that don’t need it n (%) n (%) n (%) n (%) n (%) a m er ic a (n =5 ) united states 36 (73.40) 2 (4.08) 38 (77.48) 0 11 (22.44) canada 2 (100) 0 2 (100) 0 0 brasil 7 (70.00) 0 7 (70.00) 2 (20.00) 1 (10.00) chile 1 (100) 0 1(100) 0 0 cuba 0 1 (100) 1 (100) 0 0 eu ro pe (n =1 4) united kingdom 34 (87.18) 1 (2.56) 35 (89.74) 0 4 (10.26) bulgaria 1 (100) 0 1 (100) 0 0 croatia 2 (100) 0 2 (100) 0 0 denmark 3 (100) 0 3 (100) 0 0 france 2 (66.60) 0 2 (66.60) 0 1 (33.40) germany 5 (55.50) 1 (11.10) 6 (66.60) 0 3 (33.40) italy 5 (100) 0 5 (100) 0 0 netherlands 8 (80.00) 1 (10.00) 9 (90.00) 0 1 (10.00) poland 4 (10.00) 0 4 (100) 0 0 rusian federation 1 (100) 0 1 (100) 0 0 serbia 0 0 0 0 1 (100) spain 5 (83.30) 1 (16.70) 6 (100) 0 0 sweden 1 (100) 0 1 (100) 0 0 switzerland 4 (66.60) 0 4 (66.60) 1 (16.70) 1 (16.70) continue 5 aquino-canchari et al. braz j oral sci. 2022;21:e226694 continuation a si a (n =1 1) turkey 4 (80.00) 1 (20.00) 5 (100) 0 0 india 15 (88.24) 0 15 (88.24) 0 2 (11.76) iran 4 (80.00) 0 4 (80.00) 0 1 (20.00) japan 8 (44.40) 0 8 (44.40) 1 (5.50) 9 (50.00) malasya 1 (100) 0 0 0 0 saudi arabia 1 (100) 0 1 (100) 0 0 singapoure 1 (100) 0 1 (100) 0 0 south korea 5 (100) 0 5 (100) 0 0 taiwan 1 (100) 0 1 (100) 0 0 united arab emirates 1 (100) 0 1 (100) 0 0 china 1 (100) 0 1 (100) 0 0 o ce an ia (n =2 ) australia 1 (100) 0 1 (100) 0 0 new zealand 2 (66.60) 0 2 (66.60) 1 (33.40) 0 a fr ic a (n =1 ) egypt 1 (50) 0 1 (50) 0 0 total 167 (77.67) 8 (3.72) 175 (81.39) 5 (2.32) 35 (16.28) regarding the quartile of the journals, it was evidenced that those classified in q1 were the ones that most frequently did not accept the student publication (24.10%), followed by q2 (16.70%), q4 (10.40%) and q3 (8.30%). (figure 2) 100 90 80 70 60 50 40 30 20 10 0 q1 (n = 54) q2 (n = 54) q3 (n = 48) q4 (n = 49) 85.7 4.1 10.28.3 6.3 85.4 quartile journals that accept 16.7 1.9 81.5 72.2 3.7 24.1 journals that accept conditional journals that do not accept % figure 2. accessibility of student publication, according to quartile in dental journals in the world indexed to sjr. 6 aquino-canchari et al. braz j oral sci. 2022;21:e226694 the subject areas of dental journals that fully accept the student as an author are basic sciences (100%), dental education (100%), endodontic (100%), geriatrics and gerontology (100%) and public dental health (100%), followed by areas such as oral rehabilitation (88.90%), implantology (87.50%), compared to journals that did not accept student publication such as oral radiology (33.30%), pediatric dentistry (28.60%). (figure 3) speciality accept accept conditional does not accept ba sic s cie nc es (n = 4) de nt al ed uc at ion (n = 2) en do do nt ic (n = 3) or al re ha bil ita tio n ( n = 9) pu bli c h ea lth (n = 6) im pla nt olo gy (n = 8) es th eti c d en tis try (n = 6) de nt ist ry (m isc ell an eo us )... pr th od on tic s ( n = 18 ) or al su rg er y ( n = 29 ) pe dia tri c d en tis try (n = 7) or al ra dio log y ( n = 3) ge ria tri cs an d g er on to log y ( n = 1) 100 90 80 70 60 50 40 30 20 10 0 % 11.1 11.1 11.1 100 100 100 100 100 88.9 87.5 83.3 79.4 77.8 77.5 71.4 12.5 16.7 24.5 28.6 33.3 33.3 33.3 16.7 3.9 figure 3. accessibility of student publication, according to subject area of dental journals in the world indexed to sjr. discussion the study of the forms of production and the channels of dissemination of scientific knowledge are important to understand the forms of socialization and the advancement of disciplines and their academic communities; as is also the case in dentistry. in this context, scientific journals play a very important role since they are directly related to the advancement of research and professional training; however, the global participation of student researchers and publications from regions such as latin america and their contribution to the advancement of science have traditionally been modest, justified by various factors in accordance with delgado’s research8. one of 7 aquino-canchari et al. braz j oral sci. 2022;21:e226694 these factors is the limitation in terms of authorship and affiliation, not allowing student publications in some journals, which means a barrier for the development of knowledge and research, triggering a greater number of rejections, frustration of the authors and overload in the editorial management of the journals, agreeing at the latin american level with aquino-canchari et al.9 and alzate-granados et al.10; which could be avoided if it were included in detail in the instructions for authors. from 208 journals distributed in 33 countries, the first phase of the study was not possible to collect data on student accessibility since none required this information, thus giving us an indicator of the lack of information and precision in the instructions and limitations for journal authors, being a limiting factor for the submission and publication of articles by the student group, a claim that is supported by toro-huamanchumo et al. 7. additionally, it was verified that only 121 journals presented articles with student affiliation indexed to sjr in the world; there being for 2010, only in the latindex directory around 17 600 journals, of which some 15 500 were active; from this point the question arises: and why is the accessibility of student publication in the journals worldwide so scarce?, and despite the fact that the journals have grown in number and quality in recent years since electronic journals were included, information that is corroborated by delgado8. in the systematic review by pulgar et al.11, worldwide, they state that the number of authors per publication increased and co-authorship was related to funding, author productivity, promotion requirements for university publications, and increased of the competition for scientific research grants which would demonstrate that student collaboration can improve clinical and administrative relationships with other specialties. in relation to the quartile of the journals, it was evidenced that those classified in q1 were the ones that most frequently did not accept the student publication (28.10%), followed by q2 (16.70%), q4 (10.40%) and q3 (8.30%), data that fall in contradiction with that reported by toro-huamanchumo et al.7 who state as interesting data that more than half of the journals allow student publication, whether conditioned or not; however, the data from our research show the opposite, leading us to analyze possible research biases. reconsidering that perhaps the real problem centers on the descriptions and indications of the journals which are not sufficiently clear, or the lack of research initiative on the part of the students, or the high standards for the positioning of the journal, among others; leading to a very low number of citations of scientific articles with student authorship in high impact journals, which limits their usefulness and impact; information that was described by aquino-canchari et al.9 dental journals that fully accept the students as author are basic sciences (100%), dental education (100%), endodontic (100%), geriatric and gerontology (100%) and public dental health (100%), followed by areas such as oral rehabilitation (88.9%), implantology (87.50%), data that were corroborated by alarcon et al.12 who highlight the scientific productivity measured according to specialties, having a multidisciplinary predominance in general dentistry and as it progresses in the specialization, fewer publications; that would explain the acceptance of student authorship; in comparison to journals that did not accept them such as oral radiology (33.30%), pediatric dentistry (28.60%), whose reason can be understood by the fact that they 8 aquino-canchari et al. braz j oral sci. 2022;21:e226694 are official publications of medical societies and that only accept contributions from specialists, however, toro-huamanchumo et al.7 affirm that it should not be ruled out allowing the publication of students as co-authors, since currently some medical schools really emphasize the incorporation of the scientific method in clinical practice and the basic education of their students, which provides a adequate level of knowledge that is positively reflected in the scientific research process. in addition, the student author must show greater interest in research and publication, looking for journals that enhance their initiatives and, on the other hand, the journals must evaluate the student contribution in the field of health sciences since in the near future they will be the professionals specialists who will contribute to the scientific production of the institutions and countries. this study only included dental journals indexed to sjr, not including other journals indexed to other prestigious bases. another limitation was the low response of the contact correspondence, which could modify the appreciation of the journal’s editorial team regarding the inclusion of the student as an author, in addition, limiting the results to a second source of information by reviewing the published articles in every journal. it is concluded that 167 (77.67%) of the dental journals accept the publication of dental students without condition, being more frequent in journals positioned in q4 (81.50%) and q3 (85.40%). also, the journals with thematic areas on basic sciences, dental education, geriatrics and gerontology and endodontic. for a conditional publication, the main requirement was that the research article have as one of the authors a doctor or graduate professional. interest conflict the authors declare no conflict of interest. funding the authors did not receive any sponsoring to carry out this article. data availability datasets related to this article will be available upon request to the corresponding author. authors contribution crac: conceptualization, methodology, formal analysis, funding acquisition, investigation, validation, visualization, writing original draft, review & editing. ajom: data curation, formal analysis, investigation, visualization, writing original draft, review & editing. lasi: data curation, investigation, visualization, writing original draft, review & editing. kmhc: investigation, validation, visualization, writing original draft, review & editing. 9 aquino-canchari et al. braz j oral sci. 2022;21:e226694 all authors actively participated in the discussion of the manuscript’s findings, and have revised and approved the final version of the manuscript. references 1. bertolami cn. the role and importance of research and scholarship in dental education and practice. j dent educ. 2002 aug;66(8):918-24. 2. elangovan s. importance and implications of research experience in undergraduate dental curriculum. indian j dent res. 2019 may-jun;30(3):329-30. doi: 10.4103/ijdr.ijdr_568_19. pmid: 31397401. 3. asnake m. the importance of scientific publication in the development of public health. cien saude colet. 2015 jul;20(7):1972-3. doi: 10.1590/1413-81232015207.08562015. 4. gutiérrez c, mayta p. [publication from undergraduate in latin america: importance, limitations and alternative solutions]. cimel. 2003 [cited 2021 nov 5];8(1):54-60. available from: https://www.redalyc.org/pdf/717/71780110.pdf. spanish. 5. sanchez-duque ja, gómez-gonzáles jf, rodríguez-morales aj. [undergraduate publication in latin america: difficulties and associated factors among medical students]. inv ed med. 2017;6(22):104-8. spanish. doi: 10.1016/j.riem.2016.07.003. 6. huamaní c, chávez-solis p, mayta-tristán p. [students contribution to scientific papers publication in scielo-peru´s indexed medical journals, 1997 – 2005]. an fac med. 2008;69(1):42-5. spanish. 7. toro-huamanchumo cj, landa-hernández f, arce-villalobos lr, ruiz-pingo d, díaz-vélez c. [accessibility of student publication in health science’s journals indexed in scielo]. fem. 2015;18(5):319-23. spanish. doi :10.4321/s2014-98322015000600005.  8. delgado je. [iberic-american and colombian open access scientific dental journals]. rev fac odontol univ antioq. 2014;26(1):126-51. spanish. 9. aquino-canchari c. villanueva-zuñiga lm, alvarez-vilchez ml, lópez-orihuela ke, chavez-bendezu c. [accessibility analysis of student publication in medical journals in latin america]. educ med. 2021 jul-aug;22(4):215-21. spanish. doi: 10.1016/j.edumed.2021.03.002. 10. alzate-granados jp, caicedo-roa m, saboya-romero dm, pulido jc, gaitán-duarte hg. [participation of undergraduate medicine students in medical and academic colombian journals indexed in publindex, categories a1 and a2, in the period 2009-2012: a systematic review of the literature]. rev fac med. 2014;62(1):9-15. spanish. doi: 10.15446/revfacmed.v62n1.43657. 11. pulgar r, jiménez-fernández i, jiménez-contreras e, torres-salinas d, lucena-martín c. trends in world dental research: an overview of the last three decades using the web of science. clin oral investig. 2013 sep;17(7):1773-83. doi: 10.1007/s00784-012-0862-6. 12. alarcón m, aquino c, quintanilla c, raymundo l, álvarez l. [evidence based dentistry: the 82 highest impact journals]. int j odontoestomatol. 2015;9(1):43-52. spanish. doi: 10.4067/s0718-381x2015000100007. https://doi.org/10.1016/j.riem.2016.07.003 1http://dx.doi.org/10.20396/bjos.v20i00.8660525 volume 20 2021 e210525 original article 1 department of restorative dentistry, dental school, shahid beheshti university of medical sciences, tehran, iran. 2 department of restorative dentistry, dental school, shahid beheshti university of medical sciences, tehran, iran. 3 dental clinic of amiralam hospital, tehran university of medical science, tehran, iran. 4 dental school, hamedan university of medical sciences, hamedan, iran. corresponding author: mahsa mohammadi email: mmahsa604@gmail.com received: july 17, 2020 accepted: january 16, 2021 influence of laser deproteinization of acid-etched dentin on marginal microleakage of class v composite restoration shahin kasraei1 , sogol nejadkarimi2 , mona malek3 , mahsa mohammadi4,* aim: recent reports indicate that deproteinization of acid-etched dentin surface can extend penetration depth of adhesive agents. the main goal of the present research was to investigate the deproteinization effect of nd:yag and diode 940 lasers on acid-etched dentin and microleakage grade in class v composite restorations. methods: 36 extracted human premolar teeth were selected to make standard buccal and lingual class v cavities. these samples were randomly split into three sub-groups: 1.control group, in which composite was applied for restoration after etch and bonding process without deproteinization; 2.nd:yag laser group, in which the teeth were deproteinized with nd:yag laser after etching and painting internal surfaces of cavities with van geison stain and then composite restorations applied just as control group; 3.diode laser group, in which the process was similar to nd:yag laser group, but instead, diode 940 laser was irradiated. the teeth were bisected into two equal longitudinal buccal and lingual halves. marginal microleakage of samples was scored by using a stereomicroscope. kruskalwallis, mann-whitney u and fisher’s statistical tests were employed for analysis of the obtained data. results: a significant reduction in marginal microleakage was observed for both groups treated with laser (nd:yag and diode 940)compared to control (p=0.001 & p=0.047). there was no significant difference in marginal microleakage between nd:yag laser and diode 940groups (p = 0.333). conclusion: nd:yag and diode 940 laser deproteinization of acid-etched dentin decreased the marginal microleakage of in-vitro class v resin composite restorations. key words: acid etching, dental. dentin-bonding agents. dental leakage. lasers. https://orcid.org/0000-0003-0167-4704 https://orcid.org/0000-0001-5226-2502 https://orcid.org/0000-0002-9926-2363 2 kasraei et al. introduction there have been many studies to improve the quality of composite restorations bonding and decrease microleakage until now. many techniques such as oblique layering technique, using lining materials, different curing modes and pre-polymerized composite inserts have been tried on adherend surface to reduce this microleakage, however, there exists no method to eliminate the microleakage of gingival margin completely yet1-4. one of the recommended method for adherent surface to reduce the marginal microleakage complication is to eliminate or decrease the collagen network in acid-etched dentin and composite interface by the use of different materials such as enzymes, proteolytic agents3-6 or lasers2,5,7. considering the recent progress in laser technology, laser irradiation is used to improve bonding quality in dental restorations by chemical modification of the substrate materials8,9. the collagen network can be ablated selectively by using special features of lasers. additionally collagen network has the potential to be painted by special dyes which can differentiate the collagen network and enhance the effectiveness of collagen removal from acid-etched dentin surface without any effect on mineralized tissue2,3,8,9. deproteinization of dentin surface creates an adequate mineral substrate for adhesion to resin, hence, decreases marginal microleakage in composite restorations significantly2,7. the decreased marginal microleakage causes more durability and success for restoration and decreases post operative sensitivity2,10,11. in this regard, few studies recommended nd:yag laser as a deproteinizing agent for acid-etched dentin2,12. notably, diode lasers as well, have a wavelength in about 800 to 1064 nm and hemoglobin and pigmented material have a high absorption affinity for these lasers11,13-15. in the previous studies it has shown that employing lasers after etching the dentin by acid would improve the strength of bonding, reduce the microleakage and enhance the marginal adaption between the tooth and the restorative materials12,16-19. in the present study, the effect of simultaneous utilization of two lasers, including nd:yag and diode 940, in addition to employing adper single bond 2 and led curing either using dyes to absorb the laser beams are considered as the main novelty. in this regard, the influence of deproteinization of acid-etched dentin by nd:yag and diode 940 laser on the marginal microleakage of in vitro class v composite restorations are assessed and consequently the effectiveness of the two lasers in reducing microleakage is compared. methods thirty six extracted human premolar teeth, without restorations, caries, anomalies, observable defect or crack, were scrubbed and stored in 10% buffered formalin. one week prior to the experiments, the teeth were placed in distilled water at 20-22°c 3 kasraei et al. room temperature. the teeth were randomly split into three sub-groups, each with twelve specimens. conventional bevel classv cavities (3x2x2 mm in height, width, and depth, respectively) were provided in the buccal and lingual surfaces of each tooth 1-mm above the cementoenamel junction (cej) area. therefore, there attained three groups, each group contains 12 teeth (24 samples). the classical classv cavities, in which 1 mm was above the cej in enamel, 1 mm in cementum or dentin under the cej. the cavities were prepared by one practitioner with grain diamond burs (835/010, diamant gmbh, d&z, berlin, germany, 1 mm-diameter), under water cooling, which were replaced after each 9cavities prepared. a bevel with 0.5 to 0.7 mm width was produced on enamel, only the incisal margins, with angel of 45 degree. specimens with exposed pulp were not considered in the current study. the classifications of the groups tested in the present study are as follows: control group: based on manufacturer’s instructions, the teeth were etched with a 35% phosphoric acid etchant (3m oral care, st. paul, mn, usa) for 15 seconds which was used in the dentin and enamel starting with the enamel margins. the samples completely rinsed off phosphoric acid gel with distilled water. the surface of the dentin was dried with an absorbing tissue paper butlet to retain a slight moist surface with no visible excess of water on the tooth surface (blot drying). immediately after blotting, 2 adhesive consecutive coats (adper single bond2, 3m oral care, st. paul,mn,usa) employed in acid-etched dentin for 15 seconds by applying gentle agitation utilizing a fully saturated applicator and carefully dried for five seconds with air spray to eliminate solvents. applied bonding agent was light cured (woodpecker led, guilin , china) for 10 seconds at 1000mw/cm2light intensity (to ensure accurate performance, radiometer was applied for assessment of the light-curing unit). finally, cavities were filled with composite (3m espe, filtek z250, st. paul, mn,usa) in three incremental oblique layers respectively cervical layer at first, occlusal layer second, and the last layer was complete composite restoration. to ensure complete polymerization, each layer light cured for 40 seconds separately. nd:yag laser group: the same strategy established for control group, except that after etching, nd:yag laser irradiated with van geison stain for removal of the exposed collagen network. van geison stain was provided by blending 1% aqueous fuchsine solution (9 ml) with 50 ml distilled water and 50 ml saturated aqueous picric acid solution. a fine layer of van geison stain was used on the internal surface of the cavities with a micro-brush applicator. the dye was carefully brushed into the dentin for 5 seconds in a fine motion. then the teeth were sprayed with air and rinsed with water and treated with nd:yag laser (fotona, fidelis3 plus, ljubljana, slovenia); the pulse width of 300µ second (sp mode) with 1 mm distance from the surface; with 1.5 w output power and a frequency of 15 hz for 10 seconds. the diameter of fiber optic was 300 µm and fiber tip adjusted perpendicular to the cavity surface by hand at an approximate distance of 1 mm in sweeping movement with the speed of 2mm/sec. since the hand piece is applied and controlled by the operator, the same approach is used to have similar output. cavities restored with composite and cured the same as control group. 4 kasraei et al. diode 940 laser group: the same procedure performed as for nd:yag laser group except that diode 940 nm (epic 10, biolase, inc.4 cromwell irvine, ca ,usa) was irradiated. the specifications of the used laser beam were as follows: 940 nm wavelength; fiber optic diameter of 300 µm without initiation, mode cp1, pulse length 100 µ second, an average output power of 1 w, duty cycle was 33% and pulse interval was 200 µ second. the distance of lasers tip was about 1mm vertically from cavity surface and all the surfaces lased for 10 seconds by the speed of 2mm/sec. finally, the procedure of restoration was terminated with fine grit diamond burs (chatsworth, ca, usa) and polishing disks (soft-lex, 3m espe, st. paul, usa) were used to polish the samples similar to the other groups. the samples were put in distilled water at 24°c for 24hr after restoration procedure. the specimens were subjected to thermocycling (nemo co., mashad, iran) for 3000 cycles at 5°c (±2) and 55°c (±2) with 30 sec and 20 sec dwell and transfer times, respectively. afterwards, distilled water at 24°c was used for storing the restored teeth for six months. subsequently, the apex of teeth sealed with wax and the surfaces of the samples, except the restoration surfaces and 1 mm around the restorations’ margins, were coated with nail varnish (fig1). figure 1. the teeth were coated with nail varnish up to 1mm of restoration border. afterwards, the samples were put in 1% fuchsin basic solution at 20-22°c for 72 hr, then rinsed and dried. a diamond disk (diamant gmbh, d&z, berlin, germany) was applied in a handpiece in low-speed and water spraying condition to divide the samples into two pieces, in order to make them appropriate for observation under stereomicroscope. the assessment of the microleakage was performed under a stereomicroscope (sz40, olympus, tokyo, japan) at ×40 by two blind observers. in cases of difference values, the higher one is selected as the final value. the dye penetration of two halves were examined separately and evaluated at interface of tooth–restoration and scored as follows: grade 0: without dye penetration. grade 1: dye penetration up to the half of the lateral cavity wall, either incisal or the cervical, of the tooth–restoration interface. 5 kasraei et al. grade 2: dye penetration to the wall of whole lateral cavity without involving the axial wall. grade 3: dye penetration in direction of the axial wall (fig 2). 1 12 23 3 axial wall dentin dentin enamel occlusal margin cervical margin composite filling figure 2. schematic figure of dye penetration in specimens. kruskal-wallis, mann-whitney u and fisher’s statistical tests were applied to analyze the data at a significant level of p<0.05. results microleakage scores at dentin and enamel margins of restoration are represented in table 1. the kruskal-wallis tests showed significant differences between cervical (dentin) margins (p=0.001) but there were no significant differences between groups in occlusal (enamel) margins (p=0.549). the microleakage score of occlusal margins of restorations were less than cervical margins (p=0.001) table1. comparison of the microleakage scores achieved in margins of classv composite restorations in studied groups scores microleakage score in dentin margins microleakage score in enamel margins groups 0 1 2 3 *p-value 0 1 2 3 *p-value control 0 10 (41.66%) 9 (37.5%) 5 (20.83%) 0.001 19 (79.16%) 5 (20.83%) 0 0 0.549nd:yag 8 (33.3%) 13 (54.16%) 3 (12.5%) 0 (0%) 21 (87.5%) 3 (12.5%) 0 0 diode 940 5 (20.8%) 11 (45.83%) 7 (29.16%) 1 (4.16%) 18 (75%) 6 (25%) 0 0 *kruskal wallis test at significant level of p<0.05 6 kasraei et al. pairwise comparison between groups in dentin margins of restorations by complementary mann-whitney u tests showed significant difference between dentin margins of restorations in control versus nd:yag groups (p=0.001) and between control versus diode laser groups (p=0.047); although there were no significant difference in microleakage of restorations between two laser groups (p=0.333). discussion microleakage is among the most important challenges in restorative dentistry20,21. uncompleted diffusion of adhesive resin component via the outermost demineralized dentin will lead to non-resin infiltrated collagen networks low hydrolysis, the process that weakens the resin–collagen network over time2. decreasing or removing the collagen network in resin-dentin interface can improve the strength of bond and reduce marginal microleakage2,22-24. the aim of the current research was to assess the effect of deproteinization of acid-etched dentin by nd:yag and diode 940nm laser on microleakage of in vitro class v composite restoration. the results revealed that deproteinization of dentin surface by irradiation of each nd:yag and diode 940 lasers significantly decrease microleakage of cervical margins in classv restorations. previous studies showed that applying nd:yag and diode lasers before etching in classv composite restorations have no impact on decreasing of dentin marginal microleakage of composite restoration25,26.it was reported that laser irradiation of cavity surface before etching with acid increases subsurface cracks and micro cracks on laser-irradiated surfaces27. these micro cracks increase microleakage in dentin-restoration interface26,27. however, other studies revealed that using lasers after etching the dentin by acid can increase the bonding strength, decrease microleakage and improve marginal adaptation between restoration and tooth12,16-19. they stated that etching the dentin with phosphoric acid decrease the stability of the collagen fibers and removal of the exposed collagen matrix from the etched dentin surface increase dentin wettability and bond strength of composite restorations2,19,28. the collagen network has potential to be stained by special dyes (e.g. van geison stain); etched dentin with acid, stain the collagen network, and expose the dentin to laser by modifying favorable parameters, the collagen network will be removed selectively and deproteinized the dentin without effecting the inorganic substance. the elimination of collagen from the etched dentin leads to reduction in organic content, improve the energy of the surface and change the dentin hydrophilic features; consequently, the adhesive monomers penetration into the dentin will be enhanced3. dayem12 showed that the nd:yag laser can remove the collagen network from acid etched dentin more thoroughly than can 10% naocl. incomplete diffusion of resin monomers into etched dentin, and hydrolysis of collagen fibrils that have not been covered with resin, lead to increasing microleakage and reducing bond strength over time. improving the penetration of resin monomer into demineralized dentin creates more reliable hybrid layer that increase bond quality, so marginal microleakage of class v composite restorations will be reduce by this deproteinization method2,28. 7 kasraei et al. it seems that nd:yag laser irradiation via fusion and re-crystallization of dentin apatite crystals, removing of smear layer and elimination of collagen fibril of the smear layer, leads to formation of stronger and more durable bond20,29. their results agreed with a significant reduction in cervical microleakage in the present study. however, we did not observe any significant differences between the two types of lasers in reducing cervical microleakage. despite the results of the present study, kawaguchi et al. reported that the use of lasers after etching has no effect on restorations marginal microleakage30. it should be noted that the lasers irradiated parameters used in our study were completely different from these researches. obeidi et al. showed decrease in marginal microleakage of composite depends on parameters of irradiated laser beam31. additionally we used dyes to absorb the laser beams. these differences may be impressive to explain the different results of current study. the use of van gieson dye increases the absorption of nd: yag laser energy by stained collagen fibers, so better removal of organic matter and etched dentin collagen occurs, without causing the dentin to crack or change the hydroxyapatite crystals by laser beam energy. some researchers have suggested that the negative effect of nd: yag laser beam radiation on the bond strength before applied adhesive agent is due to absorbed energy of the laser to mineral materials and changing the chemical structure of the dentin and causing cracks in it30. it should be noted that the results of the present study could not be obtained by reducing the nd:yag laser energy fluency without collagen staining, because by performing stained collagen, the ratio of laser absorption coefficient in organic to mineral materials has changed. by increasing this ratio, the energy required to degrade and eliminate collagen fibrils is obtained without structural changing the dentin minerals. there was no significant difference in microleakage of dentinal margins between the two lasers tested (p=0.102). these two lasers had similar wavelengths in infrared, both absorbed by melanin and other stains and both have almost the same effect on collagen fibers colored with stains. laser absorption by stained collagen fibers cause protein distortion and finally the deproteinization effect of them are similar to each other approximately32. more evaluation of the differences between these two lasers, are suggested by using different parameters such as irradiation time and pulse energy, using other etch and rinse bonding systems and laser employing without coloring, according to laser absorption in protein materials. future studies comparing this method with other techniques might further characterize the advantages and disadvantages of this procedure. fourier transform infrared spectrometry and near-infrared spectrometry for better evaluations was recommended10,11,15.  dentin in gingival margin has a wet surface refers to its nature. since, it is critical point for bonding mechanism of current dental bonding agents and can increase the microleakage therefore, like most previous studies we have observed that the microleakage on dentin margins of restorations is more than enamel margins20,33,34. in order to enhance the survival of composite restoration, it is necessary to ensure a durable bonding of resin composite to enamel and dentin margins35. under the conditions set for the current study, it can be concluded that nd:yag and diode 940 lasers deproteinization of acid-etched dentin can lead to less marginal microleakage of in-vitro classv composite restorations. 8 kasraei et al. conflict of interests all of the named authors have been involved in the work leading to the publication of the paper and have read the manuscript before its submission for publication. the authors declare that they have no conflict of interest. acknowledgments the authors thank the dental research center and the vice chancellor of research of hamadan university of medical sciences for supporting this study. the present article is in-vitro and does not require institutional ethics committee approval in our country, iran. references 1. gupta j, saraswathi v, ballal v, acharya s, gupta s. comparative evaluation of microleakage in class v cavities using various glass ionomer cements: an in vitro study. j interdiscip dent. 2012;2(3):164. doi:10.4103/2229-5194.113245. 2. dayem rn. assessment of the penetration depth of dental adhesives through deproteinized acid-etched dentin using neodymium: yttrium-aluminum-garnet laser and sodium hypochlorite. lasers med sci. 2010;25(1):17-24. doi:10.1007/s10103-008-0589-4. 3. dayem rn, tameesh m. a new concept in hybridization: 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pedro, juiz de fora, mg, brazil – 36.036-643 55 (32) 99128-5974 editor: dr altair a. del bel cury received: july 7, 2021 accepted: october 27, 2021 comparison of the reproducibility of two cervical vertebrae maturation methods paulo henrique da rocha duque1 , rodrigo césar santiago2 , rogério lacerda dos santos3 , fernanda ramos de faria3 , carolina de sá werneck3 , robert willer farinazzo vitral3 , marcio josé da silva campos3 aim: facial orthopaedic treatments based on the stimulation or restrictions of craniofacial bone growth are more effective when carried out during the pubertal growth spurt. the aim of this cross-sectional study was to evaluate the reproducibility of two cervical vertebrae methods (cvm) with manual tracing and direct visual inspection. methods: a sample of 60 lateral cephalometric radiographs (10 of each of the 6 cvm stages) was randomly selected from 171 records. 5 orthodontists classified these radiographs according to the skeletal maturation stage in 2002 and 2005, and the application of both methods was conducted by direct visual inspection and evaluation through manual tracing. results: the average reliability of the two methods determination and the two forms of evaluation was substantial. the direct visual inspection evaluation showed the highest reliability and agreement interexaminer values for both methods, as well as the intraexaminers evaluation. conclusion: the reproducibility of cvm method was substantial, indicating its clinical use to determine the skeletal maturity and the ideal moment for treatment execution. keywords: reproducibility of results. cervical vertebrae. bone development. http://orcid.org/0000-0002-7100-0556 http://orcid.org/0000-0001-5290-4878 http://orcid.org/0000-0002-6213-9206 http://orcid.org/0000-0002-6834-2862 http://orcid.org/0000-0002-1660-0428 http://orcid.org/0000-0003-0861-7243 http://orcid.org/0000-0003-3217-9001 2 duque et al. introduction facial orthopaedic treatments based on the stimulation and/or restriction of craniofacial bone growth are more effective when carried out during the pubertal growth spurt, as during this period, the facial bone structures perform at maximum capacity in response to stimuli offered by applied mechanics1,2. therefore, the determination of skeletal maturation is widely used, as the chronological age has been considered a parameter of little reliability to assess the craniofacial development stage of the subject1,3-5. the degree of skeletal maturation of craniofacial bones can be determined by the hand-wrist x-ray or by the evaluation of the cervical vertebrae maturation6-8. the hand-wrist x-ray evaluation is considered the gold standard method, since it allows the evaluation in a small area of the development of a great number of ossification centres, that present close relation with the whole skeletal bone development6. however, the evaluation of the cervical vertebrae maturation stage has the advantage of reducing costs and the patients’ exposure to x-rays9,10, as the cephalometric radiograph is part of the initial orthodontic documentation5,8,10,11. in 2002, bacetti et al.12 published a modified version of the method of determining skeleton maturity from the analysis of cervical vertebrae. with five stages of cvm, this method made determining skeletal maturation possible through the vertebrae c2, c3 and c4 using only one cephalometric radiograph. later, in 200513, the same authors presented an improved version of the method to evaluate cvm with six stages of mvc, which allowed the physician to identify the ideal moment for dental skeletal disharmony treatment. according to gabriel et al.7 (2009), the clinical usage of cvm analysis must be conditioned to its accuracy and reproducibility. although some studies have reported reproducibility levels over 90%6,14-16, they show some methodological flaws that interfere directly with these findings7. cunha et al.17 (2018) showed that there was no significant difference when comparing the reproducibility of the evaluation of skeletal maturity through hand-wrist to cervical vertebrae x-rays, and both methods were considered useful for clinical planning. a systematic review by santiago et al.18 (2012) showed that the levels of scientific evidence related to the reliability of cvm to foresee the pubertal bone growth spurt is low due to few amounts of studies on the subject, even though some studies report a good correlation between both methods and considerable levels of reproducibility. the aim of this study was to evaluate the reproducibility of two cervical vertebrae methods (cvm) with manual tracing and direct visual inspection. the study hypothesis is that the reproducibility of the two methods is sufficient to determine the skeletal maturity of young patients. materials and methods initially, in this cross-sectional study, pre-treatment records of 171 subjects treated on federal university of juiz de fora were selected between the ages of 7 and 3 duque et al. 18 years old, regardless of sex, and who did not present: 1) history of facial, hand or wrist trauma; 2) congenital malformations nor acquired ones affecting the cervical vertebrae, hand or wrist; or 3) any syndrome or hormonal alteration associated with development alterations. moreover, subjects would have to present good quality of both hand-wrist and lateral cephalometric x-rays that were taken on the same date. this study was approved by the ethics committee of federal university of juiz de fora (comment number: 2.634.344). in order to guarantee the homogeneity of the sample, the hand-wrist x-rays were evaluated (by a single researcher p.h.r.d.) and the subjects were classified in one of the 11 stages according to the fishman’s method19. subjects were also classified in cvm stages based on the correlation between this method with the stages established by fishman19, as suggested by hassel and farman14 (1995) (table 1). the sample was composed by 10 randomly selected subjects of each of the 6 cvm stages (table 1), with a total of 60 subjects. the lateral cephalometric x-rays were evaluated and subjects were classified according to skeletal maturation stages using the qualitative methods proposed by baccetti et al.12 in 2002 (method 1) and in 200513 (method 2). they were applied through direct visual inspection and manual tracing evaluation of c2, c3 and c4 vertebrae using 0.5mm mechanical pencil (faber castel®, stein, germany) on acetate sheet (orthometric, franklin, usa). the four evaluations (methods 1 and 2, with direct visual inspection and manual tracing) were carried out between june and august 2015, by 5 orthodontists without previous experience with the methods. manual tracing was carried out by the orthodontists at each evaluation stage. immediately before the evaluations, the examiners were trained on applying the methods through an expository lesson given by a dental surgeon, specialized in radiology and with methodological experience. all examiners were trained together. in a dark room, the lateral cephalometric x-rays were located on a constant source of white light (negatoscope) and covered with a black sheet of paper of 300g/m2 of table 1. distribution of the 171 subjects preselected accordingly to skeletal maturation stages through fishman19 and hassel and farman’s14 methods. maturation stages (hand-wrist) maturation stages (cvm) subjects preselected sample 1 and 2 1 48 10 3 and 4 2 38 10 5 and 6 3 22 10 7 and 8 4 31 10 9 and 10 5 21 10 11 6 11 10 total 171 60 4 duque et al. grammage and with a rectangular clipping in the centre, which only allowed the view of the cervical vertebrae. the positioning of the lateral cephalometric radiographs was performed by a single researcher (p.h.r.d.). during the evaluations, templates of methods 1 and 2 were provided to the examiners, and access to the information of the participants was not permitted (age, gender nor dentition images). the evaluation of the 60 lateral cephalometric radiographs by the 5 examiners was done at two different moments (t1 and t2), with a six-week interval, and the order of the lateral cephalometric radiographs was randomly modified and the training repeated by all examiners. therefore, 1200 evaluations were carried out at each moment, being 600 at each different evaluation moment. statistical analysis the degree of reliability of the methods herein evaluated by direct inspection and manual tracing was evaluated by weighted kappa test, determining intra and interexaminers coefficients. reliability was considered moderate when the values of kappa varied between 0.41 and 0.60; substantial, when they varied between 0.61 and 0.80 and excellent, when they were above 0.8120. intraexaminers disagreements were evaluated in accordance to the amount of cvm stages presented between the evaluations at t1 and t2 for each examiner. the analyses were done using the software spss statistics 23 (ibm, chicago, usa), significance level of 0.05. the sample power was determined using the statistical r pwr package. results the power of the sample (n=60) for this study was 73.5% (1-β = 0.735), with a type β error of 0.265. a minimum effect of 0.30 and β/α = 1 were considered. table 2 shows the intraexaminer reliability between t1 and t2 moments for both methods. reliability values showed small variation when comparing methods 1 and 2, considering each of the evaluation forms (manual tracing and direct visual inspection). considering each examiner, the method and the evaluation the findings are approximately 5% of excellent reliability, 75% of substantial and 20% of moderate reliability and all averages were substantial. table 2. intraexaminers reliability between t1 and t2 moments according to weighted kappa coefficient and the reliable interval of 95%. examiner method 1 method 2 manual tracing direct inspection manual tracing direct inspection 1 0.67 (0.55-0.79) 0.73 (0.60-0.85) 0.68 (0.56-0.80) 0.73 (0.60-0.86) 2 0.72 (0.60-0.84) 0.79 (0.67-0.91) 0.76 (0.65-0.86) 0.81 (0.71-0.90) 3 0.63 (0.50-0.77) 0.58 (0.44-0.71) 0.69 (0.58-0.80) 0.58 (0.44-0.71) 4 0.54 (0.40-0.67) 0.62 (0.48-0.76) 0.58 (0.45-0.71) 0.61 (0.47-0.74) 5 0.65 (0.52-0.78) 0.63 (0.51-0.76) 0.61 (0.48-0.73) 0.64 (0.52-0.73) average 0.64 (0.51-0.77) 0.67 (0.54-0.80) 0.66 (0.54-0.78) 0.67 (0.55-0.79) 5 duque et al. intraexaminers disagreements between t1 and t2 moments occurred, in its majority, due to a difference of one cvm stage in both evaluation forms of methods 1 and 2 (table 3). almost all cases (above 94%) occurred due to a variation of up to two cvm stages. despite the fact that the manual tracing evaluation of method 1 presented the minor average of intraexaminer reliability (table 2), it showed the highest disagreement occurrence (87.4%) for only 1 cvm stage (table 3). the comparison between classifications of cvm by the five examiners resulted in substantial reliability, varying between 0.62 and 0.70, as seen in table 4. the highest percentage of interexaminers agreement (67.50%) was observed in method 1 evaluation through direct visual inspection and the minor percentage (51.83%) was obtained at method 2 evaluation through manual tracing. the evaluation through direct visual inspection showed the highest values of interexaminer reliability and agreement for both methods (table 4), as well as in the intraexaminers evaluation. table 3. distribution of intraexaminers degree of disagreement in accordance to the amount of cvm stages in disagreement between t1 and t2 moments. degree of disagreement* method 1 method 2 manual tracing direct inspection manual tracing direct inspection 1 stage 87.4% 81.7% 83.2% 77.8% 2 stages 10.9% 15.5% 16.0% 17.1% 1 or 2 stages 98.3% 97.2% 99.2% 94.9% 3 stages 1.7% 1.9% 0.8% 3.4% 4 stages 0% 0.9% 0% 1.7% 5 stages – – 0% 0% * amount of cvm stages in disagreement between classifications in t1 and t2. table 4. interexaminers reliability and agreement and distribution of the disagreements in accordance to the number of cvm stages in disagreement. method 1 method 2 manual tracing visual inspection manual tracing visual inspection t1 t2 t1 t2 t1 t2 t1 t2 agreement number 340 356 405 397 311 328 367 360 agreement 56.6% 59.3% 67.5% 66.1% 51.83% 54.6% 61.1% 60.0 reliability* 0.62 0.63 0.70 0.68 0.63 0.64 0.68 0.66 degree of disagreement** 1 stage 85.0% 88.1% 75.4 87.7% 83.0% 83.4% 76.0% 80.0% 2 stages 13.1% 9.9% 23.1% 10.3% 14.9% 12.9% 19.0% 15.8% 1 or 2 stages 98.1% 98.0% 98.5% 98.0% 97.9% 96.3% 95.0% 95.8% 3 stages 1.9% 2.0% 1.5% 2.0% 2.1% 3.3% 5.0% 3.0% 4 stages 0% 0% 0% 0% 0% 0.4% 0% 1.2% 5 stages ----0% 0% 0% 0% * weighted kappa coefficient ** amount of cvm stages in disagreement between the examiners. 6 duque et al. the evaluation through manual tracing showed an increase of the reliability and agreement degree from t1 to t2, while the evaluation through visual inspection showed a reduction of the degree for methods 1 and 2 (table 4). the majority of the disagreements (above of 75%) between examiners occurred due to a difference of one cvm stage (table 4). in all the classification forms, there was an increase of one stage in disagreement percentage from t1 to t2. however, apart from classification of method 2 through visual inspection, all situations showed a reduction of the concentration from t1 to t2 considering the degree of disagreement in one or two stages what demonstrates an increase of the occurrence of the most discrepant disagreements (three or four cvm stages). discussion the application of the analysis of the cervical vertebrae maturation (cvm) as a method to determine the skeletal development stage must be conditioned to its accuracy and reproducibility, allowing identification of the period where the craniofacial bones respond more effectively to facial orthopaedic treatments1,2. however, low reliability of the cvm method in identifying the bone development stages5 and methodological flaws in some researches21 raise doubts concerning its clinical applicability. in order to reduce the possibility of the sample to contain a discrepant number of subjects at specific cvm stages and whose identification might have been either easier or more difficult, in the present research we initially determined the skeletal maturity through fishman method19 and its correlation with cvm method14, and only then select the participants in a random and homogeneous way for each of the stages. the segregation of the sample in cvm stages based on the hand-wrist radiograph determination of skeletal maturation was considered possible because high levels of correlation between the methods were reported15,16,21,22. in the present research, all examiners made use of templates for consultation during evaluations7 and they were presented to all lateral cephalometric radiographs at the same moment and immediately after training so as to prevent any possibility of examiners to be confused between methods or between stages. the clinical and scientific validity of the method of determining cvm is directly related to its reproducibility among different examiners. as for the clinical application of the method, it is necessary for professionals to have a consensus in its determination. however, according to cericato et al.23 (2015) the majority of studies which evaluate these methodologies does not address interexaminers tests, therefore compromising the level of scientific evidence. the values of inter (0.62-0.70) and intraexaminers (0.64-0.67) reliability obtained in the present research are inferior to other findings in literature, which report reliability coefficients between 0.85 and 0.9816,21,22. nevertheless, most of these studies did not use rigorous statistical evaluations specific for association with ordinal data7. considering method 2 through direct visual inspection, gabriel et al.7 (2009) reported an average value of intraexaminers agreement of 62.32% and reliability of 0.62, which are lower than the values obtained in the present research, while 7 duque et al. sohrabi et al.8 (2016) reported higher values of reliability (average of 0.74). an individual analysis of the 20 examiners of the present study and others7,8 showed that only four had results with reliability different from moderate or substantial, one had poor reliability7 and three with excellent values (obtained in the present research and reported by sohrabi et al.8 (2016). the percentage of disagreement in one cvm stage reported by gabriel et al.7 (2009) is also lower than the values obtained in the present research and other studies24,25, indicating that when their examiners disagreed, it was in a more discrepant way. gabriel et al.7 (2009) reported that agreement among their 10 examiners was lower than 50%, although with values of reliability between 0.72 and 0.76, determined using kendall’s w test. the lower interexaminer agreement presented by gabriel et al.7 (2009) can be related to the fact that the authors used a higher number of examiners, hampering conformity of classification among them, whereas the highest values of reliability may have occurred due to the reduced number of lateral cephalometric radiographs (30 radiographs) in comparison to the 60 used in the present research. moreover, the use of kendall’s w test to determine the reliability may have embodied some inaccuracies to the results, since this test is indicated for comparisons between up to 2 examiners8, so the weighted cohen’s kappa test is more indicated. although gabriel et al.7 (2009) have adopted a reduced interval between evaluations (2 weeks) and provided one template of the method to the examiners at the moment of the evaluation, which could increase the reproducibility of the method, its pointers of inter and intraexaminers agreement and reliability were lower than the ones in the present research. this can be associated with the lack of standardization of the intensity, clearness and contrast of the radiographic images and the luminosity of the environment for evaluating these images, as the images were available in digital format and not printed, as in this research, which may have influenced the examiners’ perception and interpretation. as well as in other studies24,25, in the present research the reliability pointer was determined through the weighted kappa coefficient for intra and interexaminers evaluation which takes into account not only the percentage of agreement between the evaluations but also the degree of inconsistency among disagreements7, widely characterizing the reproducibility degree of the method. this explains the difference between agreement percentages and reliability values (coefficient of kappa) obtained for each method12,13 and evaluation (direct visual inspection and manual tracing) used in the present research. another factor that may have contributed for the divergence of results obtained by the present research and gabriel et al.7 (2009) was the homogeneous distribution of the lateral cephalometric radiographs during the different cvm stages. when cephalometric radiographs of different cvm stages do not have equal chances of being selected for the sample or they are not homogeneously enclosed, a selection bias can be incorporated when the occurrence of a higher number of cvm stages with easier or more difficult identification is allowed. other studies described higher values of agreement among examiners using method 2. however, they used questionable means to evaluate the classification reproducibility. wiwatworakul et al.25 (2015) reported an average percentage of 96.6% of interex8 duque et al. aminers agreement although they used only two examiners, facilitating the equality of classification among them. perinetti et al.24 (2014) found interexaminers reliability between 0.81 and 0.82, although previously to cvm classification. their examiners had been trained in the method until reaching 75% of correct identification of cases, which calibrated them in advance. the results show that the analysis through direct visual inspection, even though presenting higher values of agreement and reliability, had greater prevalence of disagreement in two or more cvm stages in relation to manual tracing, possibly due to the fact that once the tracing is finished, the definition of the form of the vertebra is facilitated because its tracing is based on a defined contour and new mental delimitations of vertebrae limits based on radiographic image are no longer necessary. in accordance with the present research findings, method 1 performed better in terms of interexaminer agreement and reliability in direct visual inspection evaluation. this may have occurred due to the additional stage of method 2, which was identified by the presence of the concavity in the inferior edge of c2. this characteristic generated a great doubt during classification according to the examiners of the present research. on the contrary, sohrabi et al.8 (2016) and nestman et al.26 (2011) reported higher values of reproducibility when determining the concavity of the inferior edge of c2, c3 and c4 vertebrae than their general form. however, despite the evaluated characteristic of the cervical vertebrae, the fact is that a lower number of stages adopted by method 1 in comparison with method 2 reduces the possibility of disagreement among evaluations. we acknowledge a limitation of clinical application of the results in the present study regarding the training received by orthodontists prior to cvm definition. the training was needed for the results not to be affected by the levels of knowledge of the examiners, although we understand the difficulties regarding the access to specific training of cvm by orthodontists. in conclusion, the methods of determining cvm published by baccetti, franchi and mcnamara in 2002 and 2005 presented substantial reproducibility both for direct visual inspection and for manual tracing of the cervical vertebrae. the analysis through direct visual inspection presented higher values of reliability and agreement when compared with the manual tracing. references 1. caldas mp, ambrosano gm, haiter grandson f. computer-assisted analysis of cervical vertebral bone age using cephalometric radiographs in brazilian subjects. braz oral res. 2010;24(1):120-6. doi: 10.1590/s1806-83242010000100020. 2. fudalej p, bollen am. effectiveness of the cervical vertebral maturation method to predict post peak circumpubertal growth of craniofacial structures. am j orthod dentofacial orthop. 2010 jan;137(1):59-65. doi: 10.1016/j.ajodo.2008.01.018. 3. caldas mp, ambrosano gmb, haiter neto f. new formula to objectively evaluate skeletal maturation using lateral cephalometric radiographs. braz oral res. 2007 oct-dec;21(4):330-5. doi: 10.1590/s1806-83242007000400009. 9 duque et al. 4. mahajan s. evaluation of skeletal maturation by comparing the hand wrist radiograph and cervical vertebrae as seen in lateral cephalogram. indian j dent res. 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cervical vertebral maturation as a biologic indicator of skeletal maturity. angle orthod. 2012 nov;82(6):1123-31. doi: 10.2319/103111-673.1. 19. fishman ls. radiographic evaluation of skeletal maturation. a clinically oriented method based on hand-wrist films. angle orthod. 1982 apr;52(2):88-112. doi: 10.1043/0003-3219(1982)052<0088:reosm>2.0.co;2. 20. landis jr, koch gg. the measurement of observer agreement for categorical data. biometrics. 1977 mar;33(1):159-74. http://www.ncbi.nlm.nih.gov/pubmed/21180964 http://www.ncbi.nlm.nih.gov/pubmed/21180964 10 duque et al. 21. lai eh, liu jp, chang jz, tsai sj, yao cc, chen mh. radiographic assessment of skeletal maturation stages for orthodontic patients: hand-wrist bones or cervical vertebrae? j formos med assoc. 2008 apr;107(4):316-25. doi: 10.1016/s0929-6646(08)60093-5. 22. soegiharto bm, moles dr, cunningham sj. discriminatory ability of the skeletal maturation index and the cervical vertebrae maturation index in detecting peak pubertal growth in indonesian and white subjects with receiver operating characteristics analysis. am j orthod dentofacial orthop. 2008 aug;134(2):227-37. doi: 10.1016/j.ajodo.2006.09.062. 23. cericato go, bittencourt ma, paranhos lr. validity of the assessment method of skeletal maturation by cervical vertebrae: systematic review and meta-analysis. dentomaxillofac radiol. 2015;44(4):20140270. doi: 10.1259/dmfr.20140270. 24. perinetti g, caprioglio, contardo l. visual assessment of the cervical vertebral maturation stages: a study of diagnostic accuracy and repeatability. angle orthod. 2014 nov;84(6):951-6. doi: 10.2319/120913-906.1. 25. wiwatworakul, manosudprasit m, pisek p, chatrchaiwiwatana s, wangsrimongkol t. agreement of tracing and direct viewing techniques will be cervical vertebral maturation. j med assoc thai. 2015 aug;98 suppl 7:s77-83. 26. nestman ts, marshall sd, qian f, holton n, franciscus rg, southard te. cervical vertebrae maturation method morphologic criteria: poor reproducibility. am j orthod dentofacial orthop. 2011 aug;140(2):182-8. doi: 10.1016/j.ajodo.2011.04.013. 1 volume 22 2023 e230356 original article braz j oral sci. 2023;22:e0356http://dx.doi.org/10.20396/bjos.v22i00.8670356 1 assistant lecturer in operative dentistry department, faculty of dental medicine, al–azhar university (boys-cairo) 2 professor of operative dentistry and vice dean for post graduate affairs, faculty of dental medicine, al–azhar university (boys-cairo) 3 assistant professor in operative dentistry department, faculty of oral and dental medicine, al–azhar university corresponding author: nabil al aggan n.a.a.elagan@gmail.com editor: dr. altair a. del bel cury received: july 11, 2022 accepted: november 4, 2022 influence of the cervical margin relocation on stress distribution a finite element analysis on lower first molar restored by direct nano-ceramic composite nabil al aggan1,* , sameh mahmoud nabih2 , abd allah ahmed abd al hady3 aim: evaluate the influence of the cervical margin relocation (cmr) on stress distribution in the lower first molar restored with direct nano-ceramic composite (zenit). methods: a 3d model of the lower first molar was modeled and used. standardized mesio-occluso-distal (mod) preparation consisted in two models used in this study with mesial subgingival margin in model ii. (cmr) was applied in model ii using flowable composite or resin glass ionomer (riva). both models were restored with nanoceramic composite and then subjected to six runs (2 for the model i and 4 for model ii) with load (100n) as two load cases, one at (11º) and other at (45º) from the vertical axis. the stress distributions (fea) in the final restoration and (cmr) material were analyzed using 3d models. results: the two models recorded an equivalent von mises stress and total deformation in the final restoration, regardless of the difference in the oblique angle incidence from (11º to 45º) or the type of the material used for (cmr) there was no significant difference in the (fea) between the model with cmr (model ii) and the model without cmr (model i). conclusions: (cmr) technique seems to be biomechanically beneficial with high eccentric applied stress, (cmr) with resin glass ionomer or flowable composite resin in combination with nanoceramic composite improved the biomechanical behavior of (mod) cavities extended below cement enamel junction (cmr) with high modulus elasticity material like (riva) exhibits a more uniform stress distribution. keywords: finite element analysis. composite resins. glass ionomer cements. https://orcid.org/0000-0001-9635-7538 https://orcid.org/0000-0002-2371-698x https://orcid.org/0000-0002-8659-1010 2 al aggan et al. braz j oral sci. 2023;22:e0356 introduction long-term clinical observations showed that even large cavities encompassing three or more surfaces and cusps of load-bearing posterior teeth can be restored successfully with minimally invasive direct restoration techniques. however, direct restoration of deep proximal defects beyond the cemento enamel junction (cej) requires elaborate treatment techniques and considerable operator skill1,2. (cmr) technique has been proposed as a non-invasive pretreatment for the restoration of deep class ii cavities with proximal cervical margins extending below (cej)3,4. (cmr) is an alternative for performing surgical crown lengthening and offers the possibility of a stepwise relocation of deep proximal margins to uplift cavity outlines for direct or indirect restorations5,6. step one consists of placing a base of flowable or direct resin composite to elevate the margin above the (cej). step two allows the practitioner to decide on whether to place a direct or an indirect restoration according to the restoration of choice under improved clinical conditions5,6. with current adhesive technology and modern composite resin materials it has become possible to restore even severely damaged teeth and undermine tooth defects using direct composite resin materials such as nano-ceramic composite6,7. restorations should be strong enough to resist the intra-oral forces; in fact, as a result of bite forces, restored teeth are exposed to high mechanical stresses8. therefore, biomechanical principles have an important part in the clinical success of restorative materials8. classical methods of mathematical stress analysis are extremely limited in their scope and are inappropriate for dental structures that have an irregular structural form and complex loading9. currently, (fea) is a numerical method for stress analysis. it involves a set of computational procedures to calculate the stress and strain in each component, generating a model solution10. the development of technology enabled (fea) to evolve from two-dimensional to three-dimensional modeling. the difference between 2d and 3d modeling is that 3d models are more realistic and have a closer to reality representation of the biomechanical interactions in the human anatomy, restorations, and implant components10,11. stresses acting upon the materials during function in the oral cavity are normal or principal stress which acts perpendicular to the cross section and causes elongation or compression and shear stress which acts parallel to the cross section and causes distortion (changes in original shape)11. the main advantages of (fea) are the variables can be easily changed, simulation can be performed without the need of the patient, it offers maximum standardization, and it helps to visualize the point of maximum stress and displacement. however, it is not easy to predict failure in complex designs made of different materials and complex loading varying in relation to time and point of application12. it is now considered the most theoretically accurate method of solving equations involving compatibility and elasticity. finite elements are fundamental when analyzing bone and tooth failure as these 3 al aggan et al. braz j oral sci. 2023;22:e0356 are intimately connected with stress and strain behavior10,12. null hypothesis of the present study is that the (cmr) has an adverse effect on (fea) of restored teeth. the aim of this study was to evaluate the influence of the (cmr) on stress distribution (fea) on the lower first molar restored by direct nano-ceramic composite (zenit). materials and methods this in-vitro study was performed to evaluate and compare the influence of the (cmr) on (fea) on the lower first molar restored by direct nano-ceramic composite (zenit). two models were used in this study; standardized mod cavity preparations were performed in the two models where proximal margins were located 2 mm above (cej) in (moodle1) and in (model ii) the mesial proximal margin located 1 mm below (cej)10. the generalized steps to perform a finite element analysis can be summarized as follows 1. model scanning 2. geometric model preparation. 3. definition of the materials properties. 4. mesh generation (nodes and elements generation). 5. application of load, and boundary conditions. 6. obtaining the data of resultant stresses and comparing the results10,12,13. 1. model scanning a three dimensional (3d) finite element model was constructed by 3d scanning of a sample tooth (lower first molar). the teeth geometry was digitized with a laser scanner (geometric capture, 3d systems, cary, nc, usa). such a scanner produced a data file containing a cloud of points coordinates. an intermediate software was required (rhino 3.0 mcneel inc., seattle, wa, usa) to trim a newly created surface by the acquired points. then, the solid (closed) teeth geometry was exported to a finite element program as step file10,12,13. standardized mesio-occlusal-distal (mod) preparation consisted in two models used in this study with mesial subgingival margin in model ii. (cmr) was applied in model ii using flowable composite or resin glass ionomer (riva). 2. geometric model preparation first, we set up the directions (top, bottom, mesial, distal, anterior, posterior). then, we set up the mask thresholds to define the mask of enamel and the mask of dentin to define tooth tissues with its mechanical properties and finally, we calculate 3d objects10,12,13. we used a “cut orthogonal to screen” tool to cut through the tooth to reproduce the mod of the molar, then we formed the pulpal extension part by cutting in the facial aspect and proximal surface of the molar, then the two parts 4 al aggan et al. braz j oral sci. 2023;22:e0356 were merged to form the whole mod10,12,13. then all the dentin parts were merged then the enamel part was constructed to be applied in the finite element analysis test with its mechanical properties10,12,13. after applying a set of boolean operations (add, subtract, overlap, etc.) the two models’ parts were ready for material assignment and meshing. thus model i can be defined as no dentin removal under (cej) while model ii can be defined as 1mm dentin removal under c.e.j. the 1mm dentine removed from root geometry under (cej) was restored by dynamic flow flowable composite and riva light cure glass ionomer as two case studies10. 3. definition of the material properties for linear static stress analysis, there are two essential parameters that need to be defined; elastic (young’s) modulus and poisson’s ratio, which are enough for defining the linear part of the stress strain curve of any isotropic material. the properties of the materials used in the present study were listed in table 1 . table 1. material’s properties of models’ components. materials modulus of elasticity in mpa poisson’ s ratio enamel 80,350 0.33 ref 8 dentin 19,890 0.31 ref 8 zenit 18754 0.3 ref 14 dynamic flow 5,300 0.28 ref 15 riva glass ionomer 10,860 0.3 ref 8 4. mesh generation (nodes and elements generation) each component of the model was assigned to a material property on the finite element package ansys workbench version 16 (ansys inc., canonsburg, pa, usa). then a parabolic tetrahedral element was used for meshing the model, and adequate mesh density was selected to ensure results accuracy for the discrete model10,12,13. 5. application of load and boundary conditions after the models were meshed, two different oblique forces each of 100n14-16 were applied as two load cases, one at (11º)10 and the other at (45º)10,14-18 from the vertical long axis of the tooth. each load was equally divided on 15 points representing; outer, inner surface cusp tips of labial cusp, inner surface of lingual cusp, central and mesial triangular fossa distal and mesial marginal ridge18 as presented in (figure 1). thus, totally six runs were performed on the two models as following:-two runs on the model1 (one at 11º and other at 45º) from vertical axis and four runs on cmr materials of model ii (one run at 11º and other at 45º on dyract flow) & (one run at 11º and other at 45º on riva light cure glass ionomer) from the vertical axis10,13. 5 al aggan et al. braz j oral sci. 2023;22:e0356 figure 1. loading points 6. obtaining the data of resultant stresses the resultant von mises stresses and total deformations were calculated under both loading conditions and distributions as; maximal resultant values. data analysis was performed in several steps. initially, descriptive statistics for each group results. one-way anova followed by pairwise tukey’s post-hoc tests were performed to detect significance between subgroups. student t-test was done between paired groups. two-way anova was done to show the effect of each variable (main group and subgroup). statistical analysis was performed using graph-pad instat statistics software for windows (www.graphpad.com). p values ≤0.05 are statistically significant in all tests. hp z820, with dual intel xeon e5-2660, 2.2 ghz processors, 64gb ram . results the distribution and magnitude of von mises stresses and total deformation in each component of the model were calculated. in the present study table 4 & figure (3,5) revealed that an equivalent value of maximum von mises stress at 11degree (234.7), at 45 -degree (299.8) and equivalent value of total deformation at 11degree (0.0173), at 45 -degree (0.0526) were recorded on the final restoration of the two models. also, there was a positive correlation between increase in the oblique angle incidence from the long axis of the tooth from (11º to 45º) and the stress received by the restorations. 6 al aggan et al. braz j oral sci. 2023;22:e0356 the result of the present study table 4 & figure (7,9) revealed that both (cmr) materials showed nearly the same total deformation at 11-degree (0.0109) and at 45-degree (0.0338), while flowable composite received less von mises stresses at 11-degree (4.7) and at 45-degree (5) than riva at 11-degree (6.5) and at 45-degree (7.1) by about 40% in the model ii. regardless of the difference in the oblique angle incidence from (11º to 45º) or the type of the material used for cervical marginal relocation material there was no significant difference in the stress distribution (finite element analysis) between the two models where the (cmr) technique was used or not. the von mises stresses and total deformation results of the six runs applied on final restorative material & (cmr) materials for (fea) on the two models were illustrated in the table 2 and figures (2 9). table 2. the von mises and total deformation of the six runs on the two models. total deformationvon misesruns 0,0173234,71model i –ob 110 zenit 0,0526299,8 2model i -ob 450 zenit 0,01094,7 3model ii – ob 110 dyract 0,03385,0 4model ii – ob 450 dyract 0,01096,55model ii – ob 110 riva 0,03377,16model ii – ob 450 riva figure 2. model ii (final restoration) von mises stress under oblique load at 11º from vertical axis 7 al aggan et al. braz j oral sci. 2023;22:e0356 350.0 300.0 250.0 200.0 150.0 100.0 50.0 – 234.7 299.8 234.7 299.8 234.7 299.8 model #1 – ob11 model #1 – ob45 model #2 – d flow – ob11 model #2 – d flow – ob45 model #2 – riva light – ob11 model #2 – riva light – ob45 von mises stress – restoration figure 3. column chart showing comparison of von mises of final restoration between the two models. figure 4. model ii (final restoration) total deformation under oblique load at 11º from vertical axis 8 al aggan et al. braz j oral sci. 2023;22:e0356 0.0600 0.0500 0.0400 0.0300 0.0200 0.0100 – 0.0173 0.0526 0.0173 0.0527 0.0173 0.0526 model #1 – ob11 model #1 – ob45 model #2 – d flow – ob11 model #2 – d flow – ob45 model #2 – riva light – ob11 model #2 – riva light – ob45 total deformation – restoration figure 5. column chart showing comparison of total deformation of final restoration of the two models. figure 6. model ii (flowable resin relocation material) maximum von mises stress under oblique load at 11º from vertical axis 9 al aggan et al. braz j oral sci. 2023;22:e0356 8 7 6 5 4 3 2 1 0 4.7 5.0 6.5 7.1 model #1 – ob11 model #1 – ob45 model #2 – d flow – ob11 model #2 – d flow – ob45 model #2 – riva light – ob11 model #2 – riva light – ob45 von mises stress – cervical figure 7. column chart showing comparison of von mises of (cmr) materials in model ii. figure 8. model ii (flowable resin relocation material) maximum total deformation under oblique load at 11º from vertical axis 10 al aggan et al. braz j oral sci. 2023;22:e0356 0.0109 0.0338 0.0109 0.0337 model #1 – ob11 model #1 – ob45 model #2 – d flow – ob11 model #2 – d flow – ob45 model #2 – riva light – ob11 model #2 – riva light – ob45 total deformation – cervical 0.0400 0.0350 0.0300 0.0250 0.0200 0.0150 0.0100 0.0050 – figure 9. column chart showing comparison of total deformation of (cmr) materials in model ii. discussion (fea) has been widely employed as an effective tool to evaluate the stress-strain distribution. it could evaluate the biomechanical characteristics of both the restored teeth and the dental restorative system. further, the results carry significant clinical implications regarding the ability to withstand the masticatory forces in the oral cavity11. (fea) values are divided as von mises stress, maximum principle stress (tensile stress), minimum principle stress (compressive stress), and shear stress. however, in most finite element studies presented in the literature, the von mises criterion is a formula for combining three principal stresses into an equivalent stress, which is then compared to the yield stress of the material. if the “von mises stress exceeds the yield stress, then the material is at the failure condition9,10. the 100 n load used in this study was chosen as the average chewing force, which is supposed to be one third of the maximum biting force. a restoration must resist natural forces that occur in the mouth14-16. a 45-degree angle to the long axis of the tooth was chosen to match the lateral the force (eccentric force) applied on the teeth during mastication12,17-20 while an 11-degree angle to the long axis of the tooth was chosen to match the applied perpendicular force (centric force) on the teeth (90 degrees) during mastication10. null hypothesis that the (cmr) has an adverse effect on stress distribution (fea) on restored teeth was rejected because in this study (cmr) by resin glass ionomer or flowable composite resin in combination with nanoceramic composite improved the biomechanical behavior of mod cavities extended below (cej). the present study revealed that an increasing in the total deformation and von mises stresses applied on the final restoration under both models by increasing oblique angle from (110 to 450); this was in agreement with rodrigues10 (2016) who 11 al aggan et al. braz j oral sci. 2023;22:e0356 observed that the loads applied with a 45-degree angle incidence causes more stress accumulation on the final restoration than the load applied with an 11-degree angle incidence. in the present study, an equivalent von mises stress and total deformation on the final restoration of the two models can be explained by the ability of the two (cmr) materials in the model ii to support the deformations and stresses excreted on final restoration as in model i; this may be dependent on the fact that the elastic modulus of restorative materials plays an essential role in stress absorption and load transmission21. hence, (cmr) could work as an “absorber,” in which an intermediate layer of material with low elastic modulus reduces stress concentrations in the restoration and tooth structure22. such findings corroborate those found by other authors10,23-25 who found that there was no significant difference in the stress distribution between the two models, cmr was not negative for biomechanical behaviors and the use of glass ionomer cement or flowable composite resin in combination with a bulk-fill composite improved the biomechanical behavior of deep class ii mo cavities. however, diverging from the findings of ausiello et al.26 (2017) who found that the direct resin-based composite materials applied in multilayer techniques to large class ii cavities produced adverse fea stress distributions. in our study (cmr) materials showed nearly the same deformation at (110,450), while flowable composite received less stress than riva at (110,450).this finding may be correlated to the material elasticity modulus; using restoration material with high elasticity received higher stress without differences in deformation so riva absorbed more stresses than dynamic flow due to (dynamic flow) lower in modulus of elasticity (5.3) than riva (10.8) 15,29(2003). such findings corroborate those found by other authors8,27 who showed that a restorative material with appropriate elasticity module was able to absorb more stress. in the current study, regardless the difference in the oblique angle incidence from (11º to 45º) or the type of the material used for (cmr) material there was no significant difference in the (fea) between the two models where the (cmr) technique was used or not; this may be attributed to the (cmr) technique reduce the gingival extension of the restoration, placing it in a more coronal position, which may have reduced the lever arm and consequently the restoration deflexion28. also, the base under the resin composite restoration might have acted as a tampon layer reducing the effects of stress concentration and the modulus of elasticity of dynamic flow is close to dentin28. this finding was in agreement with rodrigues10 (2016) who observed that there was no significant difference in the stress distribution between the two models regardless the difference in the oblique angle incidence from (11º to 45º) or the type of the material used for (cmr) material. a limitation of the present research is that several assumptions were made during designing of the models since the stress distribution pattern directly depends on the model design and the materials’ properties assigned to each layer of the model, any inaccuracy may be directly reflected in the results. also, the magnitude and direction of the maximum bite force and masticatory bite force considered in this study are averaged values and may not match the in vivo conditions accurately. in addition, this study does not simulate the ideal structure of the tooth. further study is needed to 12 al aggan et al. braz j oral sci. 2023;22:e0356 allow the definition of the (enamel, dentin, periodontal ligament & cementum) in the model to mimic all dental structure related to the influence of (cmr) on (fea). in conclusion, within the limitations of the present study, the following conclusions were drawn: (cmr) technique seems to be biomechanically beneficial with high eccentric applied stress, (cmr) by resin glass ionomer or flowable composite resin in combination with nanoceramic composite improved the biomechanical behavior of mod cavities extended below (cej), (cmr) with high modulus elasticity material like (riva) exhibits a more uniform stress distribution. clinical significance (cmr) does not impair biomechanical behavior. acknowledgments this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. conflict of interest the authors have no conflicts of interest to declare. data availability datasets related to this article will be available upon request to the corresponding author. author contribution • nabil al aggan: methodology, validation, investigation, data curation, writing – original draft preparation and resources. • sameh nabih: conceptualization, methodology, investigation, writing – review and editing. • abd allah ahmed abd al hady: validation, investigation, writing – review, and editing. all authors actively participated in the manuscript’s findings and revised and approved the final version of the manuscript. reference 1. da rosa rodolpho pa, cenci ms, donassollo ta, loguercio ad, demarco ff. a clinical evaluation of posterior composite restorations: 17-year findings. j dent.2006 aug;34(7):427-35. doi: 10.1016/j.jdent.2005.09.006. 2. opdam nj, van 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10.1590/s1516-14392008000400014. 17. zhong q, huang y, zhang y, song y, wu y, qu f, et al. finite element analysis of maxillary first molar with a 4-wall defect and 1.5-mm-high ferrule restored with fiber-reinforced composite resin posts and resin core: the number and placement of the posts. j prosthet dent. 2022 mar:s0022-3913(22)00077-4. doi: 10.1016/j.prosdent.2022.01.029. 18. zhu j, luo d, rong q, wang x. effect of biomimetic material on stress distribution in mandibular molars restored with inlays: a three-dimensional finite element analysis. peerj. 2019 sep;7:e7694. doi: 10.7717/peerj.7694. 19. kumar p, rao rn. three-dimensional finite element analysis of stress distribution in a tooth restored with metal and fiber posts of varying diameters: an in-vitro study. j conserv dent. 2015 mar-apr;18(2):100-4. doi: 10.4103/0972-0707.153061. 20. agarwal sk, mittal r, singhal r, hasan s, chaukiyal k. stress evaluation of maxillary central incisor restored with different post materials: a finite element analysis. j clin adv dent. 2020;4:22-27. doi: 10.29328/journal.jcad.1001020. https://doi.org/10.1016/s0168-874x(00)00059-7 https://doi.org/10.29328/journal.jcad.1001020 14 al aggan et al. braz j oral sci. 2023;22:e0356 21. tribst jpm, dal piva amo, borges als, araújo rm, da silva jmf, bottino ma, et al. effect of different materials and undercut on the removal force and stress distribution in circumferential clasps during direct retainer action in removable partial dentures. dent mater. 2020 feb;36(2):179-86. doi: 10.1016/j.dental.2019.11.022. 22. friedl kh, schmalz g, hiller ka, mortazavi f. marginal adaptation of composite restorations versus hybrid ionomer/composite sandwich restorations. oper dent. 1997;22(1):21-9. 23. ausiello p, ciaramella s, de benedictis a, lanzotti a, tribst jpm, et al. the use of different adhesive filling material and mass combinations to restore class ii cavities under loading and shrinkage effects: a 3d-fea. comput methods biomech biomed engin. 2021 apr;24(5):485-95. doi: 10.1080/10255842.2020.1836168. 24. alp ş, gulec alagoz l, ulusoy n. effect of direct and indirect materials on stress distribution in class ii mod restorations: a 3d-finite element analysis study. biomed res int. 2020 dec;2020:7435054. doi: 10.1155/2020/7435054. 25. grassi eda, de andrade gs, tribst jpm, machry rv, valandro lf, ramos nc, et al. fatigue behavior and stress distribution of molars restored with mod inlays with and without deep margin elevation. clin oral investig. 2022 mar;26(3):2513-26. doi: 10.1007/s00784-021-04219-6. 26. ausiello p, ciaramella s, martorelli m, lanzotti a, gloria a, watts dc. cad-fe modeling and analysis of class ii restorations incorporating resin-composite, glass ionomer and glass ceramic materials. dent mater. 2017 dec;33(12):1456-65. doi: 10.1016/j.dental.2017.10.010. 27. yaman sd, sahin m, aydin c. finite element analysis of strength characteristics of various resin based restorative materials in class v cavities. j oral rehabil. 2003 jun;30(6):630-41. doi: 10.1046/j.1365-2842.2003.01028.x. 28. vertolli tj, martinsen bd, hanson cm, howard rs, kooistra s, ye l. effect of deep margin elevation on cad/cam-fabricated ceramic inlays. oper dent. 2020 nov;45(6):608-17. doi: 10.2341/18-315-l. 1http://dx.doi.org/10.20396/bjos.v19i0.8658189 volume 19 2020 e208189 original article 1 department of pedodontics, orthodontics and preventive dentistry, college of dentistry, university of mosul, mosul, iraq. 2 department of pedodontics, orthodontics and preventive dentistry, college of dentistry, university of mosul, mosul, iraq. 3 department of pedodontics, orthodontics and preventive dentistry, college of dentistry, university of mosul, mosul, iraq. corresponding author: ali r. al-khatib department of pedodontics, orthodontics and preventive dentistry, college of dentistry, university of mosul, mosul, iraq email: alirajih@uomosul.edu.iq received: january 24, 2020 accepted: june 29, 2020 the effect of different mouth washes and text messages reminder in the oral health of orthodontic patients saba n. yaseen1 , aisha a. qasim2 , ali r. al-khatib3,* aim: the aim of this study was to investigate the effect of different mouth washes and to study the effect of text message reminder on the oral health status of orthodontic patients treated with fixed appliances. methods: this study was a clinical trial with pre-test and post-test control group design, conducted on 24 patients undergoing fixed orthodontic treatment aged 15-30 years. the patients randomly divided into two groups; one received a weekly telephone text massage reminder for the instructions of brushing and rinsing while the second group did not receive any messages. ortho-plaque index and gingival index were used to evaluate the patient’s oral hygiene status. the reading indices after brushing only were regarded as a control, then the patients were instructed to use two mouth washes, one containing sodium fluoride with cetylpyridinium chloride, the second containing chlorhexidine digluconate with cetylpyridinium chloride and aloe vera. each one was used routinely for 4 weeks with the same amount and method. the washout period between the two types mouth washes was 4 weeks with a standardized toothbrush and paste. results: ortho-plaque index values decreased over the time between first, second and third visits. however, this index raised up at the fourth visit and again dropdown at the fifth visit. similar results were detected for gingival index. also, significant interaction between messages and the assessments was recorded for gingival index with a significant difference between the group of text messages and without text messages group (p<0.05). conclusions: the results of this study showed that the combination of sodium fluoride with cetylpyridinium chloride was more potent for plaque control, while chlorhexidine digluconate with cetylpyridinium chloride and aloe vera combination showed a better gingival improvement. moreover, the text messages reminder could enhance, but not replace direct oral hygiene instruction in orthodontic patients. keywords: aloe vera, cetylpyridinium chloride, chlorohexidine, text messages. https://orcid.org/0000-0002-1478-4649 https://orcid.org/0000-0002-6574-5076 https://orcid.org/0000-0003-1068-3127 2 yaseen et al. introduction malocclusion is considered as one of the important problems in dental health which is usually relieved by orthodontic treatment1. this procedure encourages the accumulation of dental plaque that increases the risk of dental caries and periodontal diseases2,3. during orthodontic treatment, these problems can be controlled via maintaining effective oral hygiene. however, proper mechanical control is not performed effectively by the majority of the population, mainly due to the lack of the motivation and manual skill. therefore, the use of chemical agents as an adjunct to mechanical tooth cleaning has been shown to be useful in plaque control for patients4. chemotherapeutic agents such as mouth washes and dentifrices can act as a clinical adjunct for reducing the dental health problems during the active phase of orthodontic treatment5,6. among available products, chlorhexidine (chx) is highly effective in the reduction of dental plaque and gingivitis7,8. however, chx mouth rinses have some drawbacks such as reversible discoloration of the teeth and tongue, desquamation of the oral mucosa, burning sensation and dryness of the mouth8. another product that usually recommended in dental practice is fluoridated mouth washes. over years, fluoride plays a significant role as an anti-cariogenic product, due to its inhibitory action against salivary streptococcus mutans. sodium fluoride (naf) rinses have been applied to reduce enamel decalcification, plaque formation and gingivitis9. combinations with other products including chx are now available for further improvement of oral health10. cetylpyridinium chloride (cpc) is an agent used to control formation of the plaque. authors indicated that the regular use of cpc mouth washes reduced oral bacteria, controlled biofilms, and gingivitis11,12. in another study, ghiraldini et al.13 examined the effects of 0.5 % cpc in combination with 12.5% xylitol on the formation of dental plaque and gingivitis. this combination recorded a positive effect on dental biofilm, but have limited action against the gingivitis. in another clinical trial, mouth washes containing 0.1% cpc showed an effective in preventing plaque formation, however, no statistically significant effects recorded against gingivitis14. additionally, aloe vera is used as an effective anti-inflammatory and anti-microbial agent15,16. vangipuram et al.17 compared the efficacy of aloe vera (99% aloe juice) and 0.12% chx mouth washes for 30 days. they found no significant difference in plaque and gingival indices between the two types of mouth washes. all previously mentioned chemical products represent a practical options in controlling oral hygiene. their effect can be increased by the combination of other substances that can contribute further improvement of oral status. for example, farid ayad et al.18 showed that a mouth wash with cpc and naf has a clinical superiority in reducing plaque and gingivitis in comparison to mouth wash containing only naf. also, cpc and hyaluronic acid mouth wash was proven to be more effective against plaque and gingivitis compared with 0.20% chx rinse19. in addition to chemotherapeutic agents, it is very important to emphasize the oral hygiene instructions for orthodontic patients treated with a fixed appliance20. an earlier study showed that oral hygiene can be improved with the reward system or active 3 yaseen et al. reminder therapy21. another study demonstrated the significant positive influence of text messages on behavioral changes22. nowadays, mobile usage has increased dramatically, automated text messages even help to keep the dentist in contact with patients during longer appointment intervals. so, the text message reminder method is effective for improving the oral hygiene of patients during dental practice23, 24. several types of mouth washes are available today, they may confuse both patient and dentist about the most useful and effective type to reduce or eliminate oral hygiene problems during orthodontic treatment. according to the best of our knowledge, limited information is available regarding the comparative efficacy of (naf and cpc) with (chx, cpc, and aloe vera) combinations on oral hygiene during orthodontic treatment. moreover, no studies have tested the effect of reminder text messages on dental health throughout the period of orthodontic therapy. thus, the aims of this study were to compare the effects of mouth wash that contains (naf and cpc) with others that contain (chx, cpc and aloe vera). also, study the effect of the text messages reminder regarding oral hygiene on the oral health status of orthodontic patients. the null hypotheses tested are that: 1. there is no difference between mouth wash that contains (naf and cpc) and that which contains (chx, cpc and aloe vera) regarding anti-plaque and anti-gingivitis activities in orthodontic patients. 2. there is no effect of text messages reminder regarding oral hygiene on the oral health status of orthodontic patients. materials and methods the ethical approval no. 5033 was released by the academic authorities at university of mosul in 2018. trial design and sample size: this is a randomized, controlled, crossover clinical trial conducted between december 2018 and may 2019. the study conducted on a total of 24 patients underwent fixed orthodontic appliance treatment (16 females, 8 males) aged 15-30 years (mean age 18 years) randomly selected from a private practice clinic at duhok city in the north of iraq. the sample size was calculated on the basis of single mean formula [n = (z r ⁄ d)2]. resulted number was adjusted, and the final sample size was = n + (n ×0.2). in this study, n was considered as the number of subjects, z = 1.96 for 95% confidence, r (standard deviation) = 0.32.25 and (d) precision = 0.2 unit accordingly, the estimated total sample size was 12 participants for each study group. eligibility criteria and randomization procedures: the inclusion criteria were as follows: 1. no significant medical/dental problems. 2. no history of antibiotic consumption for at least three months before the study. 3. no dental hygienic intervention during a month preceding the study, including mouth washes utilization. https://en.wikipedia.org/wiki/randomization 4 yaseen et al. 4. no smoking. 5. no mouth breathing. 6. no known hypersensitivity to chx and/ or any other mouth washes. in the present study, aim and procedures were explained to subjects who agreed to participate in this study. each eligible participant has received specific sequential number. such a number was written on precise sheet which was placed in a container by randomly chosen researcher (sny). thereafter, another randomly chosen researcher (ark) selected the participants for the study groups with an equal allocation ratio. all of study participants underwent fixed orthodontic therapy. the brackets used in this study were 0.022 stainless steel mini roth brackets of mesh (ortho technology inc., tampa, fl). interventions: 1. text messages: each participant has his / her own cellular telephone with text messaging services. they were randomly divided into two equal groups; the first group received a weekly telephone text message reminder for the instructions of brushing and rinsing and the second group did not receive any messages. messages group received one text message each friday morning during all the study period. the text message was: hello, i hope that you are well. this is a reminder messages to follow the oral hygiene instructions given to you. best regards. 2. assessments: total time of assessments was four months. assessment of otho-plaque (opi) and gingival (gi) indices was done on each monthly visit of orthodontic patients by the same examiner. in order to exclude the individual variations between the patients regarding their oral environments and their commitments to the instructions, the two tested mouth washes were conducted by the same participant in the study period. first assessment: this represents the (baseline assessment) that was taken before any interventions. at the beginning of the visit, (opi) and (gi) indices were recorded for the participants. all, participants received free standardized toothpaste (orthokin strawberry mint toothpaste, kin, barcelona spain) and a soft orthodontic toothbrush (kin, barcelona spain). oral hygiene instructions were given to the participants by the same examiner. participants were instructed to brush their teeth three times daily after meals with a constant amount of tooth paste using a colored marker on the tooth brush with horizontal brushing technique for two minutes. second assessment: this is a positive control that represents the effect of brushing only without any mouth wash. after four weeks of the brushing, (opi) and (gi) indices were recorded for the same participants in each group. at the end of the appointment, participants were given the first mouth wash (mw1) that contains 0.11 sodium fluoride, 0.05 cetylpyridinium chloride and 100ml excipients (orthokin strawberry mint mouth wash, kin, barcelona spain) with instructions of use twice a day for 5 yaseen et al. 4 weeks period, (10 ml for one minute) after lunch and before bedtime. subjects were instructed to avoid eating and drinking for 30 minutes after rinsing. third assessment: this represents the effects of (mw1). after four weeks of rinsing with (mw1), (opi) and (gi) indices were recorded again for the same participants. then, participants were instructed to use brushing only without any mouth wash. this step is considered as a washout period. fourth assessment: this represents the washout period where the participants use brushing only without any mouth wash. after four weeks of washing out, (opi) and (gi) indices were recorded for the same participants. then, they received the second mouth wash (mw2) that contains 0.05 chx digluconate, 0.05 cetylpyridinium chloride, 0.062 aloe vera and 100ml excipients (kin care mouth wash, kin, barcelona spain) with the same instructions of the (mw1). fifth assessment: this represents the effects of (mw2). (opi) and (gi) indices for all participants were recorded again after four weeks of (mw2) utilization. outcome: the main outcome of the study was ortho-plaque index and gingival index for the two main groups of orthodontic patients (with text massage and without text massage) groups. ortho-plaque index evaluation of plaque was done using ortho-plaque index (opi). it is a special index used for patients with fixed orthodontic appliances26. in this study, disclosing tablets (mgs disclosing tablets, gap research co., ui) for the index calculation was used. the participant was asked to chew one tablet for 30 seconds, sluice saliva over the teeth and through the inter dental gaps, finally spit the tablet and saliva directly into a spittoon. standardized digital images were taken to the participant’s teeth from frontal, right and left and lateral views (fig.1). the images were analyzed to measure the percentage of the area covered by plaque27. figure 1. coloured dental plague after application of disclosing tablet from frontal and lateral views 6 yaseen et al. photographic analysis photographs were analyzed to measure the percentage of the area covered by plaque using digital image analysis of these photographs27. the advantages of a photograph are that they can be assessed at free time, they are permanent record, can be viewed on numerous occasions and enable reproducibility of assessment28. yes-no system was used in the evaluation of the dyed plaque in three areas of the buccal surface of the tooth. depending on the accessibility for cleaning, each area has its own level of difficulty (fig. 2). i = occlusal area (easily accessible) = score 1, ii = cervical area (accessible with certain difficulty) = score 2, iii = central area (poorly accessible) = score 3. values found were entered into a work table (tab. 1). the following formula was used in calculating opi29. opi % = (total value from dyed areas / total number of teeth×6)100 % the condition of oral hygiene was evaluated according to the following scheme29: 0-30%= excellent hygiene, 30-50%= average hygiene, over 50% = insufficient hygiene. i = occlusal area (easily accessible) = score 1 ii = cervical area (accessible with certain difficulty) = score 2 iii = central area (poorly accessible) = score 3 figure 2. scheme distribution of the buccal surface of a tooth for evaluation of opi according to ticha et al.29 i iii ii 7 yaseen et al. table 1. orthodontic plaque index: recorded dental plaque in individual areas multiplied by the relevant factor of cleaning difficulty29 cervical x2 total central x3 total occlusal x1 total teeth 5 4 3 2 1 1 2 3 4 5 occlusal x1 total central x3 total cervical x2 total gingival index loe and silness’s gingival index (gi) was used to measure the gingival status30. the scored teeth:16,12,24,36,32 and 44. each of four surfaces of the tooth near the gingival margin was scored according to the following criteria: 0 = normal gingiva (absence of inflammation), 1 = mild inflammation (slight alteration in color, slight edema, no bleeding on probing), 2 = moderate inflammation (moderate glazing, edema, redness, hypertrophy and bleeding on probing), 3 = sever inflammation (obvious redness, hypertrophy, ulceration, and tendency to spontaneous bleeding). statistical analyses: in this study, the descriptive statistics including mean and standard deviation were used to present the data. whereas, the two way repeated measures anova was recorded to assess the difference between and within the study groups for opi and gi. the significance level was sited at p< 0.05. calibration procedures: before data collection, the calibration and intra-examiner reliability were assessed, an examinations of 10 subjects were conducted on two occasions by the same examiner (sny) using the study indices (opi and gi). intra-examiner reliability was tested using intra-class correlation (0.91 % and 0.93%) for opi and gi respectively. results in this study, 36 orthodontic patients with fixed appliances were examined, 12 subjects were excluded because 10 of them did not meet the inclusion criteria and 2 subjects refused to participate. twenty four orthodontic patients were included and distributed equally in the study groups as shown in the flow chart (fig. 3). descriptive statistics including mean values and standard deviations were determined for each index (tab. 2). the opi values showed a decrease over time among first, second and 8 yaseen et al. third readings. however, this index raised up at the fourth visit and again dropdown at the fifth visit and similar values were recorded for gi (figs. 4 and 5). also, higher mean values of opi and gi in with text messages group were recorded alone the study groups (tab. 2). figure 3. diagrammatic illustration for the study flow chart patients with different orthodontic problems (n = 36 ) study sample (n = 24) excluded (n = 12 ) • not meeting inclusion criteria (n = 10 ) • declined to participate (n = 2) randomization procedures of any mouth washes one week before the study (intraoral examination and scaling) of any mouth washes text massage group n = (12) without text massage group (n = 12) first assessment: baseline assessment (opi,gi) first assessment: baseline assessment (opi,gi) 4 weeks second assessment: effect of brushing only (opi,gi) second assessment: effect of brushing only (opi,gi) 4 weeks third assessment: effect of mw1 (opi,gi) third assessment: effect of mw1 (opi,gi) 4 weeks fourth assessment: washout period (opi,gi) fourth assessment: washout period (opi,gi) 4 weeks fifth assessment: effect of mw2 (opi,gi) fifth assessment: effect of mw2 (opi,gi) analyzed participants (n = 12) analyzed participants (n = 12) results conclusions 9 yaseen et al. figure 4. shows the means plot of ortho-plaque index change (total values) throughout study period without text message with text message 1 2 3 4 5 assessment 0.000 20.000 40.000 60.000 80.000 o rt ho do nt ic p la qu e in de x figure 5. shows the means plot of gingival index change (total values) throughout study period without text message with text message 1 2 3 4 5 assessment 0.00 0.25 0.75 1.00 1.25 g in gi va l i nd ex 0.50 table 2. mean values and standard deviations (sd) for study indices at each visit according to the text messages groups assessments groups opi gi mean sd mean sd 1st assessment (baseline) without text message (n=12) 69.33 20.96 0.90 0.49 with text message (n=12) 70.27 13.47 1.28 0.20 total (n=24) 69.80 17.24 1.09 0.41 continue... 10 yaseen et al. analysis of variance the two way repeated measures anova were recorded for the opi and gi. ortho-plaque index the results of opi showed that there was no significant interaction between messages and the assessments (f value = 0.85, p value = 0.434). also, results showed that there were no significant differences between the group of with text messages and without text messages group (f value = 1.83, p value = 0.203) . significant main effect for the assessments was recorded (f value = 40.76, p value = 0.001), since it has 5 levels, post hoc test was conducted (tab. 3). in terms of scheffe’s post hoc test, the difference in opi between 4th and 5th visits was significantly smaller than in the other comparisons (p<0.05) (tab. 3). however, the opi in 2nd visit did not differ significantly when compared to the 4th and 5th visits. assessments groups opi gi mean sd mean sd 2nd assessment (brushing only) without text message (n=12) 40.21 21.28 0.52 0.17 with text message (n=12) 45.75 16.73 0.67 0.17 total (n=24) 42.98 18.93 0.59 0.18 3rd assessment (mw1) without text message (n=12) 21.95 17.44 0.25 0.17 with text message (n=12) 33.01 15.42 0.33 0.11 total (n=24) 27.48 17.06 0.29 0.14 4th assessment (washout) without text massage (n=12) 50.25 13.27 0.55 0.25 with text massage (n=12) 56.01 16.62 0.52 0.25 total (n=24) 53.12 15.01 0.54 0.25 5th assessment (mw2) without text massage (n=12) 37.72 14.83 0.18 0.08 with text massage (n=12) 49.27 14.09 0.24 0.14 total (n=24) 43.50 15.33 0.21 0.12 orthodontic plaque index (opi), gingival index (gi), mouth wash 1 (mw1), mouth wash 2 (mw2) ...continuation. table 3. post hoc scheffe’s pairwise comparisons within study groups for opi comparison mean difference of opi 1st assessment (baseline) 2nd 26.81* 3rd 42.32* 4th 16.67* 5th 26.30* 2nd assessment (brushing) 1st 26.81* 3rd 15.50* 4th 10.14 5th 0.51 continue... 11 yaseen et al. gingival index the results of gi showed that there were significant differences between the group of with text messages and without text messages group (f value = 6.60, p value = 0.026). significant main effect for the assessments was recorded (f value = 13.65, p value = 0.001). the repeated measures analysis of variance for gi showed that there was significant interaction between messages and assessments (f value = 88.85, p value = 0.001). for that reason, the main effect was ignored and the simple main effects were examined. the difference in assessments within each study group was presented separately. the results for scheffe’s post hoc test recorded significant difference in gi values among the different study groups for both text and without text messages groups (tab. 4). discussion orthodontic treatment by fixed appliances may extend for a considerable period of time producing difficult conditions for maintaining oral hygiene. for that reason, patients must gain a full understanding of their responsibilities during this period and have to be involved in preventive programs to maintain proper oral hygiene. in this study, the clinical effect of two different mouth washes in addition to the motivation by text messages were assessed. the results of this study showed that the mouth wash with (naf and cpc) was more potent for plaque control, while mouth wash with (chx+cpc and aloe vera combination) showed better gingival improvement. so, the null hypothesis of the first objective was rejected. moreover, the result of the current study showed significant difference between text message and without text message groups regarding gi only. this indicate that the text messages reminder could enhance, but not replace direct oral hygiene instruction in orthodontic patients31. so, the null hypothesis of the second objective was also rejected. comparison mean difference of opi 3rd assessment (mw1) 1st 42.32* 2nd 15.50* 4th 25.64* 5th 16.01* 4th assessment (washout) 1st 16.67* 2nd 10.14 3rd 25.64* 5th 9.62* 5th assessment (mw2) 1st 26.30* 2nd 0.51 3rd 16.01* 4th 9.62* *significant at p<0.05 orthodontic plaque index (opi), mouth wash 1 (mw1), mouth wash 2 (mw2) ...continuation. 12 yaseen et al. table 4. post hoc scheffe’s pairwise comparisons within each study groups for gi group comparison mean difference of gi without text massage 1st assessment (baseline) 2nd 0.38* 3rd 0.64* 4th 0.34* 5th 0.71* 2nd assessment (brushing) 1st 0.38* 3rd 0.26* 4th 0.03 5th 0.33* 3rd assessment (mw1) 1st 0.64* 2nd 0.26* 4th 0.30* 5th 0.07 4th assessment (washout) 1st 0.34* 2nd 0.03 3rd 0.30* 5th 0.37* 5th assessment (mw2) 1st 0.71* 2nd 0.33* 3rd 0.07 4th 0.37* with text massage 1st assessment (baseline) 2nd 0.60* 3rd 0.95* 4th 0.75* 5th 1.04* 2nd assessment (brushing) 1st 0.60* 3rd 0.34* 4th 0.15 5th 0.43* 3rd assessment (mw1) 1st 0.95* 2nd 0.34* 4th 0.19 5th 0.09 4th assessment (washout) 1st 0.75* 2nd 0.15 3rd 0.19 5th 0.28* 5th assessment(mw2) 1st 1.04* 2nd 0.43* 3rd 0.09 4th 0.28* *significant at p<0.05 gingival index (gi), mouth wash 1 (mw1), mouth wash 2 (mw2) 13 yaseen et al. text messages can reduce plaque and gingival inflammation by reminding the patients at home to follow the instructions that were given to them at the clinic. while patients without text messages also could get a reduction in plaque index because they were already follow the instructions of brushing and rinsing that were given to them at the clinic, but may be in a less extend compared to text messages group. these patients might still have some plaque accumulation on their teeth that can lead to gingival inflammation. opi used in this study gave a percentage of plaque accumulation with ranging, for example: 0 30%= excellent hygiene but, there is difference between 0% plaque accumulation compared to 30% although they have the same score and this may be added to the limitations of using this index. previous studies found significantly lower scores for plaque, bleeding, and gingival indices in the text message group compared with control. they concluded that the text message reminder method is effective for improving oral hygiene and in maintaining communication with orthodontic patients23,24. kumar et al.31 stated that text message reminder helps in the improvement of oral hygiene of patients under orthodontic treatment this approach is economical, useful for mass communication, and not disturbing to the patient’s working conditions. whereas, li et al.32 found no difference in pi and modified gingivitis index between the two study groups in the pre and post-orthodontic treatment. however, they recorded that messaging helps in patient’s management and education. the diagnostic indices used in this study were opi and gi. orthodontic plaque index is a special index used for patients with fixed orthodontic appliances26. it has a higher diagnosis performance and accuracy compared to quigley and hein index, and modified navy plaque index. re‐measurement of the stored images can be done for reliability and for comparability between studies27. but this index has not have been used widely by many authors, possibly because of its relative complexity of calculation when compared with the modified silness and lo¨e index. the outcome of our study recorded a significant reduction in opi and gi among the 1st (baseline), 2nd (brushing only) and 3rd (naf and pcp). the reduction in the opi and gi between the 1st and 2nd assessment may be attributed patients’ following the instructions given to them especially when they knew that they are part of the study. they are also given dental brush and toothpaste, this may encourage them to maintain their oral health. this is in agreement with wang et al.33 who concluded that oral hygiene instructions can lead to the efficient control of dental plaque accumulation in patients with fixed orthodontic appliances. the difference between the 2nd and 3rd assessments may be due to the use of mouth wash with brushing compared with the use of brushing only. this is in agreement with pahwa et al.34 who concluded that cetylpyridinium mouth wash was found to be effective in reducing the bleeding and plaque index scores in orthodontic patients compared to the patients using brushing only. in contract to that, wiraja et al.35 have shown that plaque control by mechanical means is always the most influential way in reducing plaque on fixed orthodontic patients while mouth wash has just a chemical assistance of reducing dental plaque. 14 yaseen et al. in our study, the opi and gi index values raised up at the 4th assessment and again dropdown at the 5th assessment after rinsing with (chx, cpc, and aloe vera) combination. the raising up in the 4th assessment may be due to that this visit represents the washout period (between the two types of mouth washes) when the patients used brushing only without any mouth wash. these results are in agreement with other studies that concluded that tooth brushing alone is not enough in maintaining oral hygiene of orthodontic patients, they suggested that prescribing of mouth washes is necessary to maintain a good oral hygiene34,36. regarding the second mouth wash (chx, cpc and aloe vera), a dropdown of opi and gi index values were recorded at the fifth visit. this result was in agreement with previous studies17,37,38. also, these results were in accordance with karim et al.39 who found a significant reduction in plaque and gingival bleeding scores after using aloe vera mouth washes. dehghani et al.40 concluded that the patients who instructed to maintain regular oral hygiene, in addition to mouth rinsing with chx combined with other products can help in control plaque, decrease gingival inflammation and improve patients’ oral health status. comparing the results of first and second mouth washes showed that the reduction in opi was higher for (naf and cpc), whereas gi reduction was higher for the (chx, cpc and aloe vera combination). this indicated that (naf and cpc) are more beneficial for plaque control. this may be attributed to fluoride that has the ability to reduce supragingival plaque by accumulation in the plaque and decreasing the proportion of streptococcus mutans9. the results of this study were in agreement with wiraja et al.35 who compared the effects of 0.05% sodium fluoride and 0.2% chx mouth washes on plaque index in orthodontic patients. they concluded that naf mouth rinse significantly reduced plaque index compared to chx. however, they mentioned that the most effective way of reducing plaque is tooth brushing while mouth wash is just an additional way. chauhan et al.36 indicated that among the mouth washes, fluoridated mouth wash seems to be more effective as compared to the mouth wash containing cpc. in another study, an attempt has been made to distinguish between chx (0.06%), naf (0.05%) mouth washes and combined one with chx-naf. although, no significant difference was recorded between groups, chx-naf demonstrated a higher decrease in bleeding, modified gingival and plaque indices40. the results of this study indicated that the second mouth wash (chx, cpc and aloe vera) was better for gingival improvement. the reduction in gi scores can be attributed to aloe vera component of this mouth wash. aloe vera extracts can reduce gingival inflammation by inhibition of the cyclooxygenase pathway and decrease prostaglandin synthesis from arachidonic acid. some of the components of aloe vera like hyaluronic acid, vitamin c and dermatan sulfate are involved in collagen synthesis, increasing the concentration of oxygen at the wound site due to the dilation of blood vessels and therefore, provide relief in swelling and bleeding gums39,41. furthermore, (chx, cpc and aloe vera ) mouth wash contains chx that is considered as the “gold standard” due to its broad anti-microbial spectrum. it is efficient against streptococcus.mutans and lactobaci llus bacteria7,40. it can reduce gingival disease because of its anti-bacterial action against oral pathogens by increasing in cellular membrane permeability followed by the coagulation of cytoplasmic macromolecules7. so, it is advisable to use this mouth wash when orthodontic patients have 15 yaseen et al. gingival inflammation to get the benefits of it on gingiva and reduce the side effects of prolong use of chx at the same time. finally, cpc (which present in both mouth washes) had significant anti-plaque and anti-gingival effect and revealed a wide spectrum of anti-microbial activity12,37. cpc is a cationic surface-active agent that can rapidly kills gram-positive pathogens and yeasts by disrupting the membrane function, cell membrane damage causing leakage of cell components and finally cell death42. the main limitations of this study were: 1. no negative control availability, because it is difficult to instruct the orthodontic patients to avoid tooth brushing. 2. there may be an individual variations between the participants regarding their response to the instructions. the results of this study showed that: 1. the incorporation of mouth wash with standard tooth brushing procedures can significantly improve oral hygiene in orthodontic patients in comparison with brushing alone. 2. mouth wash with naf and cpc can be used as a daily mouth rinse in orthodontic patients to control plaque. while, the mouth wash with chx+cpc+aloe vera combination is advisable when the orthodontic patient has gingival inflammation. 3. repeating oral hygiene instructions is important for the control of oral health condition in orthodontic patient. this can be enhanced by the text messages reminder. recommendations further long term studies are recommended to confirm the efficacy of different mouth washes as an anti-plaque and anti-gingivitis agents in orthodontic practice. moreover, there is a need for new combinations of mouth washes for anti-plaque, with anti-carious and anti-inflammatory actions with least side effects. acknowledgement the authors wish to acknowledge college of dentistry, university of mosul for the support. also we wish to acknowledge dr. mohamad nihad form college of dentistry, university of mosul for his linguistic assessment and for all of the study participants for their patients and cooperation. conflict of interest the authors have no conflict of interest. references 1. alogaibi ya, murshid za, alsulimani ff, linjawi ai, almotairi m, alghamdi m, et al. prevalence of malocclusion and orthodontic treatment needs among young adults in jeddah city. j 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b%5bauthor%5d&cauthor=true&cauthor_uid=24603910 https://www.ncbi.nlm.nih.gov/pubmed/?term=bhaskar dj%5bauthor%5d&cauthor=true&cauthor_uid=24603910 https://www.ncbi.nlm.nih.gov/pubmed/?term=gupta d%5bauthor%5d&cauthor=true&cauthor_uid=24603910 https://www.ncbi.nlm.nih.gov/pubmed/?term=gupta rk%5bauthor%5d&cauthor=true&cauthor_uid=24603910 https://www.ncbi.nlm.nih.gov/pubmed/?term=jain a%5bauthor%5d&cauthor=true&cauthor_uid=24603910 1http://dx.doi.org/10.20396/bjos.v19i0.8658798 volume 19 2020 e208798 original article 1 department of surgical, oncological and oral sciences, university of palermo, palermo, italy. 2 prosthodontics unit, school of dentistry, cnec faculty of santo angelo, rs, brazil. 3 department of biomedical sciences and neuromotor, division of prosthodontics, alma mater studiorum university of bologna, bologna, italy. corresponding author: vinicius felipe wandscher franciscan university, brazil viniwan@hotmail.com received: march 20, 2020 accepted: september 26, 2020 retention strength of ball-attachment titanium post for removable partial denture or overdenture dario melilli1 , vinicius felipe wandscher2, leonardo ciocca3 , giuseppe currò1, candida parisi3, giuseppe gallina1, paolo baldissara3 aim: to evaluate the retention of an endodontic titanium post with a spherical head for removable partial denture or overdenture attachment according to surface treatment type. methods: sixty healthy single-rooted teeth, sectioned at the enamel/cementum junction, were treated endodontically and steadily fixed in the embedding acrylic resin. the titanium posts were subdivided into four groups: control, no surface treatment (ctrl); posts with macroretentive grooves (mr); air abrasion of the post surface (ab); and posts with macro-retentive grooves and air abrasion of the post surface (mr+ab). the posts were luted in the root canal using self-adhesive dual resin cement. pull-out testing was performed using a universal testing machine until complete detachment was achieved. after pull-out testing, the metallic posts were examined under an optical microscope and the failures were classified based on the cement distribution pattern on the extracted posts: 0, no cement left on the post (cement/post failure); 1, post surface partially covered by adhered cement (post/cement and dentin/cement mixed failure); 2, post surface completely covered by cement (dentin/cement failure). the retention data were analyzed by one-way anova, bonferroni–dunn test (p<0.05) and weibull analysis. results: ab showed the highest retention value (485.37±68.36), followed by mr+ab (355.80±118.47), mr (224.63±42.54) and ctrl (113.12 ± 51.32). ab and mr showed the highest weibull moduli. conclusions: the data indicated that air abrasion alone could significantly increase the retention of titanium posts/attachments for use with overdentures or removable partial denture. keywords: air abrasion, dental. cementation. denture retention. surface properties. https://orcid.org/0000-0002-7993-5073 https://orcid.org/0000-0002-2127-484x 2 melilli et al. introduction the classic tooth-supported overdenture consists of a complete denture anchored by single attachments to two or more natural teeth that act as bearing and retention points. the attachment, cemented in the root canals, can be used for retention of overdentures or removable partial dentures (rpd). rehabilitation therapy with these anchorage methods could increase retention and stability compared to conventional rpds (retained by clasps)1,2, and have aesthetic advantages due to the absence of any visible metallic clasps. this procedure is a simple, economic and conservative solution, which retains the principles and advantages of classic overdentures1,2. the choice and cementation of posts in root canals are important considerations for the tooth-supported overdenture. clinically, at least two options are available, i.e. prefabricated or cast posts. although prefabricated posts present limitations of inadequate intraradicular morphology and reduced post space, they also have a number of advantages, including low cost, removal of less dentin (thus reducing the risk of root fracture) and a rapid clinical procedure3. titanium is the metal of choice as it has excellent biocompatibility, good cement adhesion and high resistance to wear4. post retention depends on the morphology, size and surface texture of the posts5. in particular, parallel posts are more retentive than tapered posts, but they require more extensive and specific root canal preparation. while a longer post length ensures better retention, its diameter has little influence on the retention force5. moreover, it has been reported that retention is greater for threaded than smooth posts5-10. therefore, dentists should be aware of the risk of debonding of these posts, manifested as adhesive failure between intraradicular dentin, cement and/or post. historically, the gold standard for cementing posts was zinc oxyphosphate followed by glass ionomer cements. after the introduction of composite resin, it became possible to increase adhesion and retentive forces, to reduce solubility and minimise the risk of microfractures11-19. the best results have been obtained by using adhesive systems associated with resin cements. the clinical success of resin cements is due to the mechanical properties of these materials, such as high flexural strength, compression strength, and elastic modulus, and adhesion to dental tissues20,21. both post/cement and dentin/cement interfaces are critical for the cementation of posts, especially when the post surface is smooth. in this case, retention can be increased by surface treatment of the post. most studies have evaluated the retention or adhesive strength of fibre posts; there have been few studies regarding cementation techniques and surface treatment of metallic posts. therefore, this study was performed to evaluate the retention (pull-out) and interfacial adhesive failure of intraradicular posts with a spherical attachment (pivot block; rhein83, bologna, italy) and cemented with a self-adhesive resin cement, according to different surface treatments; the reliability of the treatments was also assessed. the null hypothesis was that there would be no difference in retention between the groups (α = 0.05) materials and methods all described procedures comply with the internal ethical committee guidelines, approved on 17/02/2016 (university hospital “p. giaccone” of palermo, approval no. 2/2016). sixty 3 melilli et al. healthy single-rooted human upper incisors and lower premolars, extracted for periodontal or orthodontic reasons, were selected according to the following inclusion criteria: monoradicular teeth without caries and without endodontic treatment; absence of fractures; radicular length of at least 12 mm; and radicular canal diameter inferior to the post size. after removing the residual soft and hard tissues using an ultrasonic scaler, the teeth were stored in 0.5% chloramine for 3 days, immersed in distilled water at 4°c for 2 days to prevent dehydration22, and then maintained in 0.02% thymol solution. the teeth were sectioned using diamond burs at the cervical level of the enamel/ cementum junction (ecj). mechanical chemical preparation of the radicular canal was performed using 10-15 proglider hand instruments (dentsply maillefer, ballaigues, switzerland) and 2.5% sodium hypochlorite. the post space was then standardised to a size of 10 mm using a customised drill (mooser, a01mog; rhein 83, bologna, italy). macro-retentions were performed on the external surface of the roots by using a diamond bur (881.141.014; komet dental, lemgo, germany) attached to a high-speed handpiece, to enhance retention in resin material for the retention test. the teeth were embedded in resin cylinders (tecnovit 4071, heraeus kulzer, hanau, germany) using a friction-grip mounting pin inserted in the root canal, then the pin head, along with the connected dental root, was mounted in a poly-vinylsiloxane cylindrical jig. the jig was securely placed in the ptfe mould until polymerization of the liquid resin around the root was reached. the goal was to maintain the axis of the root canal, then the axis of the luted titanium post, aligned with the direction of the pulling force, that actually was coaxial to the resin cylinder (fig. 1) the pivot block posts were divided into four groups according to surface treatment (fig. 2): control posts without surface treatment (ctrl); posts with semi-circular macro-retentive grooves 0.4 mm deep made using a diamond bur (diameter, 0.8 mm, 835.107.008; komet dental) at 3, 5 and 7 mm from the coronal portion of the post (mr); air abrasion of the post surface with alumina (al2o3) particles 50 μm in diameter, 2.8 bar at a distance of 10 mm (international dental supply, savona, italy) (ab); macro-retentive features associated with air abrasion of the post surface with al2o3 particles (same procedures as in the mr and ab groups) (mr+ab). figure 1. specimen prepared for to pull out test. it is clearly visible the slot-type engagement system to the ball-attchment sphere. the force is coaxially directed to both titanium post and embedding resin cylinder. 4 melilli et al. ctrl mr ab mr+ab figure 2. representative image of the study groups. ctrl: control group, surface without treatment; mr: surface with macro retentions; ab: air-abraded surface; mr+ab macro retentions + air-abraded surface. all posts were cemented using relyx unicem automix (3m espe, st. paul, mn, usa), a self-adhesive dual resin cement, and photoactivated for 20 × 2 s on opposite surfaces, following the manufacture instructions. an alphanumeric code with the letter of the group and the number of the specimen was written on the resin cylinder. the pull-out test was performed by attaching the post/root/resin assembly to the fixed axis of an universal testing machine (instron 4301; instron, norwood, ma, usa) and and a flexible clamp, connected to the machine traverse, was engaged in the ball-attachment of the post through a slot-type engagement (fig. 2). the specimens were tested at a crosshead speed of 0.5 mm/min until post detachment and the maximum force (n) was recorded (fig. 1). the data were analyzed for normality of distribution using the kolmogorov–smirnov test. as the data were normally distributed, one-way analysis of variance (anova) was performed to determine differences in pull-out force measurements. in the case of significant differences, pairwise comparisons were performed using the bonferroni–dunn test. in all analyses, p<0.05 was taken to indicate statistical significance. data analysis was performed using statview software (ver. 5.0.1; sas institute, cary, nc, usa). weibull analysis was also performed: m (weibull modulus) represents the reliability of the pull-out values expressing the variation in the retention data, and the size distribution of the flaw population within a structure; σ0 (characteristic strength), indicates the retention value at which 63.2% of the specimens survived. after pull-out testing, the metallic posts were examined under an optical microscope (×10) and the failures were classified into six categories (areas), i.e. apical, middle and cervical for both sides of the post, indicated by colouring half of the ball attach5 melilli et al. ment with a marking pen. for each of the six areas, a value was recorded according to the presence of cement on the post surface, as follows: 0, metallic surface of the post completely polished (principally cement/post failure); 1, metallic surface of the post with visible cement (post/cement and dentin/cement mixed failure); 2, metallic surface completely covered with cement (principally dentin/cement failure). for each group, the failure distribution is described by percentages. results statistical analyses by one-way anova and the bonferroni–dunn test showed that all groups were significantly different from each other when compared (table 1, p≤0.0002). the highest mean values (n) were found for ab (485.37 ± 68.36), followed by mr+ab (355.80 ± 118.47), mr (224.63 ± 42.54) and ctrl (113.12 ± 51.32) (table 1). two roots (one in the ab group and one in the mr+ab group) fractured during the test and were excluded from the analysis. table 1. means (newtons), standard deviations (sd) values of pull out test and bonferroni/dunn test; significance: alphα = 0.05. all the groups were significantly different from each other. groups mean ± sd* bonferroni/dunn test mean diff. crit. diff p-value significance control 113.12 ± 51.32 d ctrl-mr -111.513 75.552 .0002 y mr 224.63 ±42.54 c ctrl-ab -372.258 76.890 <.0001 y ab 485.37 ±68.36 a ctrl-mr+ab -242.758 76.890 <.0001 y mr + ab 355.88 ±118.47 b mr-ab -260.745 76.890 <.0001 y mr-mr+ab -131.245 76.890 <.0001 y ab-mr+ab 129.500 78.204 <.0001 y *different uppercase letters indicate a statistical difference between groups the weibull moduli of mr and ab were the highest, while the characteristic strength was highest for ab and mr+ab (table 2). table 2. weibull analysis of the pull out values. groupsa weibull analysisb m ci α0 ci control 2.25 1.29 – 3.14bc 128.16 95.87 – 171.16c mr 6.2 3.57 – 8.65a 242.72 218.45 – 269.59b ab 7.9 4.55 – 11a 510.67 470.1 – 554.59a mr + ab 2.95 1.66 – 4.15c 406.17 322.44 – 511.98a amr: macro retentions; ab: air-abrasion; mr + ab: macro retentions + air-abrasion. bweibull analysis for pull out values: (α0) characteristic resistance in mpa, and m = weibull modulus (95% confidence intervals [ci]). lowercase letters were used for α0 values and capital letters for m values. 6 melilli et al. figure 3 shows the failure distributions after stereomicroscope observation (x10) of the post surface, following the codification described in figure 4. 45,2 74.4 29.7 12.2 46.4 22.2 58.3 73.2 8.3 3.3 11.9 14.4 0 10 20 30 40 50 60 70 80 ctrl mr ab mr+ab % post-cement mixed dentin/cement figure 3. failure distribution: 90 portions of the control and mr groups, 84 portions of the ab and mr+ab groups were analyzed. a b c a: code 0 = metallic surface of the post completely polished (principally cement/post failure) b: code 1 = metallic surface of the post with visible cement (post/cement and dentin/cement mixed failure) c: code 2 = metallic surface completely covered with cement (principally dentin/cement failure). figure 4. some representative images of the types of failures. discussion this study was performed to evaluate the retention of spherical attachment titanium posts to dentin and the types of adhesive failure. the null hypothesis, i.e. that there would be no difference in retention value among groups, was rejected because the ab group showed higher values than the other groups. 7 melilli et al. the success of intraradicular posts depends on the presence of sufficient adhesive force at the dentin/cement and post/cement interfaces. resin cements show better performance than other types of cement, but their use is technique-sensitive and requires adequate skill. therefore, self-adhesive resin cements have been introduced to simplify the clinical procedures and reduce the operation time23. although the initial ph of composites is approximately 2 (ph of relyx unicem < 2 in the first minute), no demineralisation of the dentin or hybrid layer formation has been reported24. monomers show multiple mechanisms of action on dentin; for example, 10-mdp (10-methacryloyloxydecyl dihydrogen phosphate) widely used in dental cements and adhesives creates nanolayer precipitates that are responsible for the stability and adhesive strength. the infiltration of dentin could be hindered by the high viscosity of the cement. the polymerisation reaction gives rise to the formation of molecules with high molecular weight, while the acid–base reaction with hydroxyapatite increases the ph up to 725,26. acid neutralisation occurs due to the bond between phosphate and alkaline groups present in the fillers and hydroxyapatite crystals. this reaction produces water, which renders the cement more hydrophilic and better able to moisten the dentin. water is fundamental in this adhesive system, as it facilitates the release of hydrogen ions that interact with the hydroxyapatite27. the bond between resin cement and the titanium surface is based on two factors: chemical adhesion and micromechanical retention28. three types of pre-treatment on the titanium surface have been shown to increase this bond: promoters of chemical adhesion, promoters of micromechanical retention, and promoters of chemical and micromechanical adhesion. specific primers for metals (meps, 4-meta and later mdp), which react with the oxides present on the surface of titanium, have been used to improve chemical adhesion29. as alternatives, bifunctional silanes chemically bound to both the resin matrix and the metal have been employed30. however, there have been few studies regarding chemical treatment of intraradicular posts. the most common method used for post preparation is air abrasion with al2o3 particles (30 μm, 50 μm, 110 μm, 250 μm) to promote micromechanical retention. this treatment increases the adhesive area, decreases the surface tension and increases wettability of the surface31,32. schneider et al. reported that there was no statistically significant difference in retention between cementing posts with dual resin cements at 10 minutes versus 24 hours after air abrasion33. this could be due to the ability of the oxide layer to self-limit, acting as a barrier to the reaction between titanium and the environment. schmage et al. reported no difference between cementing posts with resin cements and applying air abrasion to their surfaces versus cementing posts with zinc phosphate cement34. a combination of chemical and micromechanical effects was obtained by air abrasion of the post surface with al2o3 particles coated with silica (sio2 tribochemical treatment); the particles hit the surface with high energy and develop temperature peaks that melt and deposit silica on the surface, rendering the surface rich in silica so that it reacts with the adhesive materials (silane and resin cements)35. however, a positive 8 melilli et al. effect on retention is not achieved by all resin cements because they contain different adhesives, e.g. 10-mdp shows reduced bonding on silica surfaces, whereas silanes promote adhesion34. contrasting results have been found in relation to al2o3 particle dimensions. in some studies, higher retention values were achieved using small air abrasion particles compared to larger particles, while other studies showed the opposite result36,37. larger particles likely lead to a rougher titanium surface, thereby increasing the contact area with the cement. on the other hand, when air abrasion is associated with silanisation, differences in retention due to the different sizes of the air abrasion particles become less significant38. in a similar study, nergiz et al. showed that air-abraded titanium posts with macro-retentions on the surface had higher retention values compared to the ctrl group, although these posts were cemented with zinc phosphate cement39. furthermore, groups wherein air abrasion was associated with macro-retentions showed a threefold greater mean retention value. few studies similar to ours have been reported in the literature and the tested treatments often require more complex or expensive procedures; more generally, the tested specimens were typically titanium plates rather than titanium posts38,40. regarding the weibull analysis, ab and mr showed the highest weibull modulus (m), indicating that the retention of titanium posts treated with air abrasion or macro-retention had higher reliability than the other treatments. however, in relation to the failure distribution data, mr showed a high percentage of cement/post failures with weaker adhesion at this interface than the dentin/cement interface, unlike the ab group. with regard to characteristic strength (σ0), ab and mr+ab showed the highest values, possibly because these treatments promoted major topographic changes (chemical and mechanical) on the titanium post surface and consequently produced a greater interaction with the resin cement. the failure distribution data confirmed this, as both groups (ab and mr+ab) showed a higher percentage of mixed failures. the mr+ab group showed a mean retention value threefold greater than the ctrl group, while the ab group had a mean value fourfold greater than the ctrl group. comparison of our results with those of nergiz et al. indicated differences between the air abrasion and air abrasion with macro-retention groups39. in the present study, the cement may not have been able to penetrate sufficiently into the macro-retentions of the posts, generating defects at the post/dentin interface or due to greater cement thickness. it will be necessary to evaluate the differences in dentin/cement interactions according to failure mode; nevertheless, even with detailed failure analysis, it is difficult to precisely determine which of the two interfaces failed first. the macro-retentions likely increased retention by strengthening the shear forces on the post/cement interface. however, a completely air-abraded post surface allows better retention at the cement/dentin interface and the presence of macro-retentions is insignificant because they may not overcome the retention threshold at the cement/ dentin interface. 9 melilli et al. in conclusion the results of the present study suggested that retention of the rhein 83 preformed titanium post can be improved by surface pre-treatment prior to cementing with self-adhesive resin cement. these procedures are recommended because the untreated smooth surface does not provide satisfactory results and there is a high risk of detachment. the results suggested that the posts should be 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10.1016/j.dental.2005.04.015. 11 melilli et al. 34. thompson jy, stoner br, piascik jr, smith r. adhesion/cementation to zirconia and other non-silicate ceramics: where are we now? dent mater. 2011 jan;27(1):71-82. doi: 10.1016/j.dental.2010.10.022. 35. papadopoulos t, tsetsekou a, eliades g. effect of aluminium oxide sandblasting on cast commercially pure titanium surfaces. eur j prosthodont restor dent. 1999 mar;7(1):15-21. 36. watanabe i, kurtz ks, kabcenell jl, okabe t. effect of sandblasting and silicoating on bond strength of polymer-glass composite to cast titanium. j prosthet dent. 1999 oct;82(4):462-7. doi: 10.1016/s0022-3913(99)70035-1. 37. de almeida-júnior aa, fonseca rg, haneda ig, abi-rached fde o, adabo gl. effect of surface treatments on the bond strength of a resin cement to commercially pure titanium. braz dent j. 2010;21(2):111-6. doi: 10.1590/s0103-64402010000200004. 38. nergiz i, schmage p, platzer u, mcmullan-vogel cg. effect of different surface textures on retentive strength of tapered posts. j prosthet dent 1997 vol 78;5:451-7. 39. elsaka se, swain mv. effect of surface treatments on the adhesion of self-adhesive resin cements to titanium. j adhes dent. 2013 feb;15(1):65-71. doi: 10.3290/j.jad.a27827. 1http://dx.doi.org/10.20396/bjos.v20i0.8661711 volume 20 2021 e211711 original article 1 graduate program in dentistry, school of dentistry, university of san carlos of guatemala, guatemala city, guatemala. 2 graduate program in dentistry, school of dentistry, federal university of pelotas, pelotas, brazil. 3 graduate program in dentistry, school of dentistry, university of vale do taquari (univates), lajeado, brazil. *corresponding author: luiz alexandre chisini university of vale do taquari avelino talini st. 171 lajeado, rio grande do sul, 95914014, brazil tel: + 55 53 981121141 e-mail: alexandrechisini@gmail. com received for publication: october 26, 2020 accepted: december 9, 2020 dentist’s preferences on vital and nonvital tooth bleaching: findings from a guatemalan survey víctor ernesto villagrán colón1, mirna oldemia calderón márquez1, ricardo alfredo carrillo-cotto1, flávio fernando demarco2 , luiz alexandre chisini2,3,* aim: the present study aimed to investigate if the guatemalan dentist’s options on tooth bleaching could be influenced by their time in clinical practice, the level of specialization or their working place. methods: a representative sample of dentists working in clinical practice in guatemala was selected. data  were collected using a self-administered questionnaire with information related to gender, professional characteristics (time since graduation in years and working place) and preferences regarding vital (at-home or in-office; type and concentration of bleaching agent) and the nonvital tooth bleaching (bleaching agent used). the analysis was performed and the association between preference for bleaching technique and independent variables were investigated using fisher’s exact test. results: 200 dentists were interviewed. more than half of dentists were male (57.0%) with time since graduation between 11 and 20 years (n= 64; 32.3%). dentists mostly (60.5%) preferred in-office technique for vital bleaching, with 10-20% carbamide peroxide (cp) as the preferred agent (50%). for nonvital teeth, the preferred agent (46.8%) was 37% hydrogen peroxide (hp). about the associations, younger dentists (< 20 years of graduation) selected mostly in-office technique, while those with more than 20 years indicated more the at-home technique. also,  the dentists working in private practice chose more frequently in-office technique. thus, the in-office technique was more popular among guatemalan dentists, with 10-20% cp and 37% hp selected as favorite bleaching agents for vital and nonvital techniques, respectively. conclusion: the time of clinical practice and working place influenced some choices. keywords: tooth bleaching agents. cross-sectional studies. practice patterns, dentist’s. surveys and questionnaires. http://dx.doi.org/10.20396/bjos.v20i0.8661711 mailto:alexandrechisini@gmail.com mailto:alexandrechisini@gmail.com https://orcid.org/0000-0003-2276-491x https://orcid.org/0000-0002-3695-0361 2 colón et al. introduction esthetics plays a pivotal role in modern society. discolored or darkened teeth could impair individuals´ social life and negatively impact the oral health-related quality of life (ohrqo)1. whiter and aligned teeth are considered central requirements for satisfaction with teeth appearance2-5. aesthetic dental treatments are highly desired for individuals in several studies6,7. a survey with university students observed that 74% of them desired to carry out an esthetic treatment in their teeth and 16% have already bleached their teeth at least once in their lives7. similar results were observed in a population study in adults, where 86% desired to bleach their teeth6. indeed, tooth bleaching is one of the treatments most popular in dental clinics. there  are different bleaching agents with variate concentrations (generally ranging from 10 to 37%) available for professional use. these products are mainly based on hydrogen peroxide (hp) or carbamide peroxide (cp), which breaks down into hp and urea. these oxidizing chemical agents can be used for vital or nonvital tooth bleaching; while sodium perborate (sp) is used exclusively for nonvital tooth bleaching8-10. moreover, low concentration agents [10-22% cp and 6% hp] are used at home by the patients under professional supervision, which is defined as an at-home technique; and high concentrations [>30% cp or hp] are applied only in the dental office by the dentist in the so-called in-office technique11. bleaching agents seem to be able to improve tooth color for vital and nonvital teeth12. the bleaching effect is also capable to positively impact in the ohrqol13. when comparing at-home and in-office vital bleaching protocols, a systematic review found similar results between both techniques14. these techniques have also been reported to exhibit similar side effects: tooth sensitivity and gingival irritation15. considering the large number of products available in the market and the different techniques, most of the time the choice of bleaching technique and agents relies on the professional opinion. in fact, a survey with brazilian dentists showed that the decision for vital or nonvital bleaching protocols was impacted by the post-graduation training, time since graduation and working place16. there is none study in central america evaluating the preference of dentists regarding the vital and nonvital bleaching techniques and materials. therefore, the present study aimed at the guatemalan dentist’s options for vital or nonvital tooth bleaching and additionally to investigate if their choices could be influenced by dentists characteristics (the time in clinical practice, the level of specialization or their working place). materials and methods the present study was reported following the strobe guideline (strengthening the reporting of observational studies in epidemiology). study design, setting and study size the present self-administered survey was carried out in guatemala, a country located in central america with a population of around 17.3 million. guatemalan dentists are registered in the “colegio estomatológico de guatemala”, which maintains actualized 3 colón et al. the respective dentist’s address and telephones. in 2015, 2,258 dentists were registered being distributed in all guatemalan territory. a representative sample of this population was calculated considering an α error of 5%, power of 80% and a prevalence of 50% (unknown) of the outcome. thus, a sample of 181 individuals was estimated. considering potential losses and refusals, we added around 20% to the initial sample, thus comprising 220 individuals. dentists were designated systematically, selecting at random the first position in the list. subsequent individuals were selected by calculating the sample interval. selected dentists were initially contacted by cellphone and invited to participate. individuals that agreed to participate scheduled a meeting to deliver and complete the self-administered questionnaire. the questionnaires were personally delivered and collected after filling in by the participant. they have included only active graduated dentists that carry out clinical practice in the republic of guatemala. data were collected using a self-administered questionnaire including 12 questions related to demographic information (sex), professional characteristics (time since graduation in years), and information regarding bleaching techniques. all participants that agreed to participate in the study signed an informed consent form. all data collection was performed during may of 2016. independent variables independent variables were collected regarding the gender of participants, time since graduation was continuously collected and then categorized in a) ≤10; b) 11 to 20; c) 21 to 30; d) >3016. dentists were also asked about their level of specialization and categorized in general practitioners (no post-graduation) and specialists (with post-graduation training)17. the place of work was also assessed by the question “where do you get your professional clinical practice from most of the time?” the answers were categorized as “private” and “public/university”. outcome the outcomes of present studies were the choice of the vital bleaching technique and the materials used in vital and nonvital tooth bleaching16. 1. considering vital teeth, “what is your favorite protocol to bleach vital teeth?”. two possible answers were possible: a) at-home; b) in-office 2. in cases of vital teeth “what is your first choice to bleach discolored vital teeth?”: a) 10% carbamide peroxide (cp); b) 15 to 22% cp; c) 37% of cp; d) 37% of hp or e) over-the-counter (otc) products; 3. in cases of nonvital teeth “what is your first choice to bleach discolored nonvital teeth?” a) 10% to 22% cp; b) 37% cp; c) 37% hp; d) sodium perborate (sp) + water/or hp. statistical methods data were tabulated in excel™ (microsoft corporation) software. descriptive analysis was performed and the association between outcome and independent variables was investigated using fisher’s exact test. all the analyses were done with 4 colón et al. stata 12.0 (statacorp, college station, tx, usa) software package. confidence intervals of 95% were calculated and a level of significance of a ≤0.05 was used for the analyses. results a total of 200 dentists participated of present study, which were mainly in the guatemala city (n=135; 67.5%) followed by sacatepéquez (n=18; 9.0%) and chimaltenango (n=16; 8.0%). while 20 (9.1 %) of dentists declined to participate. more  than half of dentists were male (n= 114; 57.0%) with time since graduation between 11 and 20 years (n= 64; 32.3%) followed by individual with less than ten years since graduation (n=48; 24.2%) (table 1). most of the dentists worked in private practice (n=170; 92.9%). table 1. descriptive analyses of the studied variables among guatemalan dentists (n=200). guatemala, 2016 variable/category n=200 % (ic 95%) gender male 114 57.0 (49.8 – 64.0) female 86 43.0 (36.0 – 50.2) time since graduation (years)* ≤ 10 59 29.8 (23.5 – 36.7) 11 to 20 64 32.3 (25.8 – 39.3) 21 to 30 48 24.2 (18.5 – 31.0) >30 27 13.6 (3.1 – 43.7) post-graduation training* yes 64 32.7 (26.2 – 39.7) no 135 67.8 (60.8 – 74.3) working place private 170 92.9 (88.2 – 96.2) public/university 13 7.1 (3.8 – 11.8) vital bleaching technique (vital teeth)*a at-home 62 39.5 (31.8 – 47.6) in-office 95 60.5 (52.4 – 68.2) vital bleaching*b cp 10-22% 88 50.0 (52.4 – 57.6) cp 37% 38 21.6 (15.7 – 28.4) hp 37% 50 28.4 (21.9 – 35.7) nonvital bleaching*c cp 10-22% 15 10.8 (6.2 – 17.2) cp 37% 38 27.3 (20.1 – 35.5) hp 37% 65 46.8 (38.3 – 55.4) sp + water or hp 21 15.1 (0.9 – 22.2) *number of valid answers.a 19 (10.8%) dentists replied that they do not perform tooth whitening on this issue. b18 (9.3%) dentists replied that they do not perform tooth whitening on this issue. c57 (29.1%) dentist replied that they do not perform tooth whitening on this issue 5 colón et al. regarding the technique for bleaching vital teeth, 10.8% of dentists reported not to perform vital bleaching. considering only those participants who performed tooth bleaching, most of the dentists (n=95; 60.5%) carried out the in-office technique. the bleaching agent of choice by most guatemalan dentists was the carbamide peroxide between 10-22% (n=88; 50.0%). regarding nonvital bleaching, hydrogen peroxide was the material used by 46.8% (n=65) of the surveyed dentists. table 2 shows the association between the choice of the vital bleaching technique and independent variables. an association was found between the time since graduation and the type of vital bleaching technique (p=0.017). younger dentists (<20 years) indicate more in-office technique while dentists with more than 30 years since graduation indicate more at-home. although post-graduation training did not was associated with vital bleaching therapy (p=0.194), the place of work was a factor that influenced the choice of professionals (p=0.021). dentists in public/university place work choose more at-home vital bleaching compared with individuals that work in private practice, which preferred mostly in-office. table 2. association between the choice of the vital bleaching technique (at-home or in-office) and the independent variables vital bleaching technique variable/category at-home in-office p-value n (%) n (%) time since graduation (years) ≤ 10 27 (45.8) 32 (54.2) 0.017* 11 to 20 28 (43.8) 36 (56.3) 21 to 30 31 (64.6) 17 (35.4) >30 19 (73.1) 7 (26.9) post-graduation training yes 76 (56.7) 58 (43.3) 0.194 no 30 (46.9) 34 (53.1) working place private 87 (51.5) 82 (48.5) 0.021* public/university 11 (84.6) 2 (15.4) regarding the association between the materials used for nonvital tooth bleaching (table 3), no association was observed with time since graduation (p=0.319), post-graduation training (p=0.714) and place of work (p=0.447). similarly, no association was observed between the choice of vital bleaching materials and time since graduation (p=0.271), post-graduation training (p=0.085) and place of work (p=0.911). 6 colón et al. table 3. association between materials used for vital and nonvital tooth bleaching therapies and the independent variables variable/category nonvital tooth bleaching p-value10-22%cp 37%cp 37% hp sp n (%) n (%) n (%) n(%) time since graduation (years) ≤ 10 5 (12.5) 7 (17.5) 26 (60.0) 4 (10.0) 0.319 11 to 20 4 (8.2) 13 (26.5) 24 (49.0) 8 (16.3) 21 to 30 3 (9.1) 11 (33.33) 11 (33.3) 8 (24.2) >30 3 (18.6) 6 (37.50) 6 (37.5) 1 (6.25) post-graduation training yes 5 (10.9) 13 (28.3) 19 (41.3) 9 (19.6) 0.714 no 10 (10.75) 25 (26.9) 46 (49.5) 12 (12.9) working place private 13 (10.7) 33 (27.0) 57 (46.7) 19 (15.6) 0.447 public/university 0 (0.0) 3 (50.0) 3 (50.0) 0 (0.0) vital tooth bleaching time since graduation (years) ≤ 10 23 (41.8) 18 (32.7) 14 (25.4) 0.271 11 to 20 27 (49.1) 12 (21.8) 16 (29.1) 21 to 30 25 (56.8) 6 (13.6) 13 (29.5) >30 12 (57.1) 2 (9.5) 7 (33.3) post-graduation training yes 21 (37.5) 15 (26.8) 20 (35.7) 0.085 no 66 (55.5) 23 (19.3) 30 (25.2) working place private 75 (48.7) 36 (23.4) 43 (27.9) 0.911 public/university 5 (55.6) 2 (22.2) 2 (22.2) discussion the overall results of the present questionnaire-based survey showed that professionals’ characteristics such as place of work and time since graduation influenced the dentist’s decision concerning indication of at-home or in-office vital tooth bleaching. to investigate the opinion of dentists is a useful tool to understand what is happening in the dental clinic in real life for different procedures, including the preferences for tooth bleaching17,18. in this study, in-office dental bleaching was largely preferred (60.5%) by guatemalan dentists over at-home (39.5%) for vital tooth bleaching. a systematic review showed that both techniques are effective in bleaching capacity14. although the literature showed that while different concentration agents exhibit similar efficacy12, the use of therapies with lower peroxide concentration has been recommended as the first choice because they usually are associated with less sensitivity during treatment19. moreover, an in vitro study showed that higher concentrations of peroxide in bleaching materials can degrade more easily than peroxides with lower concentrations and thus affect its effectiveness8. in fact, 60.5% of dentists choose the in-office protocol, 7 colón et al. of which 50% indicated cp 37% or hp 37% and 10.5% indicated the cp 10-22%, concentrations used to at-home. this result could be explained due to possible higher sensitivity indices observed at higher concentrations. however, it is important to note that the use of cp in low concentrations (10-22%) is indicated only in the at-home technique. the choice of approach to tooth vital bleaching of guatemalan dentists was different from those reported by demarco et al.16 (2013), which investigated dentists in the south of brazil. in this study, brazilian dentists preferred more (78.1%) at-home than in-office bleaching16. also, the main dentist characteristic associated with the indication of bleaching treatments was the post-graduation training and younger dentists16. dentists with post-graduation preferred to indicate at-home bleaching, like as younger dentists16. in our study, we have not observed any influence of post-graduation training in the choice of vital bleaching technique. however, the place of work was an important factor associated with dentist preferences. thus, a dentist that works in public or university prefers more an at-home approach compared that in-office. the choice for this kind of treatment in private practice could be related to the higher control of the treatment for the dentist or related to the belief that in-office could produce a faster or more strong bleaching effect, which is not demonstrated by a systematic review comparing at-home and in-office treatments14. moreover, we identify that time since graduation was another factor that influences the indication of vital bleaching technique. older dentists (between 21 and 30 and more than 30 years since graduation) prefer more at-home tooth bleaching to the vital tooth. these results do not corroborate with brazilian dentists, were younger dentists indicate more at-home than older dentists16. concerning the nonvital bleaching, 37% hydrogen peroxide was the favorite bleaching agent in this present study. indeed, this agent has been largely been used to carry out bleaching treatments in nonvital teeth for more than 70 years20. hydrogen peroxide is no longer indicated for application in nonvital bleathing into the pulp chamber due to the risk of external cervical resorption. therefore, sodium perborate can be a safer alternative to hydrogen peroxide as an intracoronal bleaching agent. some  studies have also suggested that 35% carbamide peroxide present similar efficacy of 35% hydrogen peroxide even as is more safety21,22. also, in the brazilian study, above 30% hydrogen  peroxide was the preferred bleaching agent for nonvital treatments15. considering the materials used by guatemalan dentists, we did not observe any association regarding time since graduation, post-graduation training or place of work, both to vital as nonvital tooth bleaching. these results corroborate with a study carried out in brazil, which did not found an association between materials used for nonvital tooth bleaching and dentist’s characteristics16. a possible explanation for differences between the studies that evaluated dentists’ preferences can be linked to differences in dental schools. a  study investigated the attitudes of the final-year students from three european dental schools towards bleaching23, and found important differences between these schools (concerning confidence, teaching received in bleaching and attitudes to provision of bleaching and recommendations to patients). students declared to feel more confident about providing nonvital bleaching than vital bleaching. besides, the complete understanding of mechanisms that professional decision making is a hard task and can englobe several levels of comprehension, beginning in the professional knowledge17,24, influenced by the school of dentistry 8 colón et al. and, also, can be influenced by patients’ characteristics25-27. some patient’s characteristics could influence the decision making of health professionals25-27. moreover, it was observed that competition affected the clinical decision-making of dentists in canada28. dentists located in regions with competitive pressure from other dentists presented an odds 63% higher to indicate a more aggressive approach than dentists  located in medium competitive regions. also, the dentist in very low competitive areas presented an odds 31% higher to indicate aggressive dentistry treatments when compared with medium density regions28. therefore, considering the brazilian and guatemalan distribution of dental schools and dentists we found some differences that could help us. the dentist labor market in brazilian is very competitive in capitals and this decreases considering interior country29; similarly, dentist guatemalan distribution is concentered (around 75%) in the capital. moreover, some straight points must be highlight. practice-based studies carried out in dentists’ clinical practice have paramount relevance to understanding factors influencing the clinician’s preference of a specific technique/material over another. yet, we included dentists from all regions of guatemala through a representative sample of all countries. moreover, some limitations could also be emphasized. there were more than 9.1 % of the 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influence the treatment decision-making of dentists? a randomized questionnaire-based study. clin oral investig. 2019 mar;23(3):1023-30. doi: 10.1007/s00784-0182526-7. 26. chisini la, collares k, bastos jld, peres kg, peres ma, horta bl, et al. skin color affect the replacement of amalgam for composite in posterior restorations: a birth-cohort study. braz oral res. 2019 jul 29;33:e54. doi: 10.1590/1807-3107bor-2019.vol33.0054. 27. cabral ed, caldas jr af, cabral ha. influence of the patient’s race on the dentist’s decision to extract or retain a decayed tooth. community dent oral epidemiol. 2005 dec;33(6):461-6. doi: 10.1111/j.1600-0528.2005.00255.x. 28. ghoneim a, yu b, lawrence hp, glogauer m, shankardass k, quinonez c. does competition affect the clinical decision-making of dentists? a geospatial analysis. community dent oral epidemiol. 2020 apr;48(2):152-62. doi: 10.1111/cdoe.12514. 29. san martin as, chisini la, martelli s, sartori lrm, ramos ec, demarco ff. distribution of dental schools and dentists in brazil: an overview of the labor market. rev abeno. 2018; 18(1):63-73. doi: 10.30979/rev.abeno.v18i1.399. 1 volume 22 2023 e237617 original article braz j oral sci. 2023;22:e237617http://dx.doi.org/10.20396/bjos.v22i00.8667617 1 departamento de odontologia, universidade federal do rio grande do norte (ufrn), natal, rn, brazil. corresponding author: av senador salgado filho, 1787, lagoa nova, natal-rn, brazil; zip-code: 59056-000; phone/fax: +55 84 32154101; e-mail: rodolfo_xsl@hotmail.com editor: dr. altair a. del bel cury received: november 20, 2021 accepted: february 3, 2023 bottom/top hardness ratio and dentin bonding stability of conventional and bulk-fill resin composites maria eduarda lima do nascimento marinho1 , rodolfo xavier de sousa-lima1* , letícia virgínia freitas chaves1 , boniek castillo dutra borges1 aim: to evaluate the bottom/top hardness ratio (b/t) and the dentin bonding stability of conventional and bulk-fill resin composites in high c-factor preparations. methods: regular conventional (tetric n-ceram – tnc, and polofil supra – pfs), regular bulk-fill (tetric n-ceram bulk fill – tbf, and admira fusion x-tra – afx), and low viscosity bulk-fill resin composites (tetric n-flow – tnf, and x-tra base – xtb) were used to restore 180 dentin conical preparations. the specimens were randomly distributed in 12 groups (n = 15) according to the resin composites and storage time-points (24 h and six months) tested. after 24 h storage, all specimens were subjected to the bottom/top hardness ratio analysis. then, the push-out bond strength test was performed in half of the specimens and the other half were maintained for six months on water storage before testing. the failure modes were analyzed in a stereomicroscopic. the data were analyzed statistically using oneand two-way anova and tukey post-test (p <0.05). results: there were no statistically significant differences for the bottom/top hardness ratio among the resin composites (p>0.05). regardless of the storage timepoint, regular bulk-fill resin composites showed the highest bond strength values statistically (p<0.05). only conventional resin composites showed statistically lower bond strength values at six-month storage (p<0.05). adhesive failures were more predominant for low-viscosity bulk-fill resin composites. conclusion: although the doc was not affected by different materials tested, only bulk-fill resin composites did not present dentin bond strength loss after six-month of water storage. keywords: dental bonding. longevity. composites resins. dental materials. https://orcid.org/0000-0002-3037-990x https://orcid.org/0000-0002-4968-1340 https://orcid.org/0000-0001-9089-341x https://orcid.org/0000-0003-4313-5776 2 marinho et al. braz j oral sci. 2023;22:e237617 introduction the stability of the adhesive interface is one of the primary factors for the success of restorations. the resin composite bonding to dental tissues must be stable to promote durability to the restoration. bonding to dentin is a challenge due to its tubular conformation, water content, and organic components1. thus, an effort has been made to find an adhesive protocol to promote greater dentin bonding stability to resin composites over time2. mechanical properties such as the depth of cure are related to resin composites’ dentin bonding performance3-5. an insufficient monomer conversion in the bottom of resin composite restorations can compromise their strength and durability due to the material’s hydrolytic degradation6,7.thus, regardless of the resin composite type used, a well-polymerized material is required, which can be accessed using the bottom/top hardness ratio3-5. low and regular viscosity bulk-fill resin composites were introduced in the market to become easier filling of high c-factor posterior tooth preparations with increments of up 4-5 mm8,-9. low viscosity bulk-fill resin composites polymerized in 4-mm increments had lower shrinkage stress, higher bond strength and lower hardness than conventional resins composites4. regular bulk-fill composite resins obtained similar or better results for bottom/top hardness ratio, marginal adaptation and interfacial nanoleakage compared to conventional composite resins3. however, the evaluation of dentin bonding stability to compare the performance of low and regular viscosity bulk-fill resin composites and its relation with bottom/top hardness ratio need further investigation. thus, this study aimed to evaluate the bottom/top hardness ratio (b/t) and the dentin bonding stability of conventional and bulk-fill resin composites with different viscosities. the null hypothesis tested in this study is that there will be no statistically significant differences between the materials for both properties analyzed. methods and materials experimental design this research was characterized as an experimental in vitro study, whose composites used are listed in table 1. table 1. materials used in this study. material manufacturer type/viscosity shade lot composition tetric n-ceram ivoclar (liechtenstein) conventional/ regular a2 w91364 urethane dimethacrylate (≥10 >25%), ytterbium trifluoride (≥10 >20%), bis-ema (2.5 – 10%), bis-gma (≥2.5-<10%) tetric n-ceram bulk fill ivoclar (liechtenstein) bulk-fill/regular iva w91962 bis-gma (3 <10%), urethane dimethacrylate (3 <10%), ytterbium trifluoride (3 <10%), bis-ema (3 <10%) continue 3 marinho et al. braz j oral sci. 2023;22:e237617 specimens’ preparation a schematic representation of specimens’ preparation and methods performed in this study is shown in figure 1. the technique described by sousa-lima et al.10 (2017) guided the methodological aspects of this research. one hundred eighty healthy bovine incisors with no enamel cracks or structural defects were selected and decontaminated in an aqueous solution of thymol (0.1%) at 4°c for one week. then, they were distributed in 12 groups (n = 15), according to the six resin composites (table 1) and the two storage time-points tested (24 hours and six months). the roots’ teeth were sectioned using a diamond flexible disc (kg, cotia, são paulo, sp, brazil) at the highest point of the cementitious junction and discarded. subsequently, a parallel cross-section was made 5 mm above the first cut (in the incisal direction), through which a 5-mm thick specimen was obtained with a central void referring to the pulp cavity. to obtain 4-mm thick flat dentin specimens, #400 and #600 grit sandpapers (labopol-21, struers, copenhagen, denmark) were used to ground the upper (incisal) and lower (cervical) specimens’ surfaces. the central space referring to the pulp cavity was used for the cavity preparation with a tungsten carbide burs (komet inc., lemgo, germany) coupled to a handpiece under air-water cooling (kavo, joinville, santa catarina, brazil), which was connected to in a standardizer device. the bur penetrated the center of the samcontinuation tetric n-flow ivoclar (liechtenstein) bulk-fill/low iva w41268 urethane dimethacrylate (≥10 <25%), ytterbium trifluoride (≥10 <20%), bis-gma (10 – 25%), triethylene glycol dimethacrylate (≥2.5 <10%) polofil supra voco (germany) conventional/ regular a2 1810034 bis-gma (10-25%), urethanedimethacrylate (5 – 10%), triethylene glycol dimethacrylate (2.5 – 5%) admira fusion x-tra voco (germany) bulk-fill/regular e1 1736584 organically modified silicic acid (10 – 25%) xtra base voco (germany) bulk-fill/low a2 1742724 bis-ema (10 – 25%), aliphatic dimethacrylate (10 – 25%) single bond universal 3m espe (usa) 1724700342 ethyl alcohol (25-35%*), bis-gma (10 – 20%*), silane treated silica (10 – 20%*), hema (5 – 15%*), copolymer of acrylic and itaconic acids (515%*), glycerol 1,3 dimethacrylate (5 – 15%*), udma (<5%*), water (<5%*), diphenyliodonium hexafluorophosphate (<0.5%) * trade secret source: material safety data sheet (msds). 4 marinho et al. braz j oral sci. 2023;22:e237617 figure 1. bovine incisors were used. sections at the highest point of the cementoenamel junction and 5 mm above were made (a). the specimens were ground with sandpapers to obtain 4 mm heigh (b). the cavity was prepared using a tungsten carbide bur (c), and a final preparation was obtained (d). the adhesive system was applied according to the manufacturer’s recommendations (e-g), and the preparation was filled according to the resin composite used (h-k). the restorations were finished and polished (l) before submitting to the hardness (m) and bond strength (n) analyses. the failure modes were then analyzed (o). #400 #600 5 mm 4 mm 5.5 mm 4.5 mm 2 mm + 2 mm 4 mm #600 #1200 4 mm a b c d e f g h i j k l m n o 5 marinho et al. braz j oral sci. 2023;22:e237617 ple, giving rise to an open, standardized conical cavity, with 5.5 mm upper diameter (incisal) x 4.5 mm lower diameter (cervical) and 4 mm thick. the bur was changed every 30 preparations. after all the cavity preparations, excess water was blotted with absorbent paper, leaving the dentin surface visibly moist (wet bonding). the single bond universal system (3m espe, st paul, mn, usa) was applied according to the manufacturer’s instructions, and its solvent was volatilized with an air spray for 5 s. the device tip was positioned on a glass slide to standardize the distance between the curing device and the upper specimen surface. the photoactivation was performed for 10 seconds with the coltolux led device (coltène / whaledent, altstätten, switzerland – 1200 mw/cm2). each specimen was placed over a glass slide (1 mm thick) with the largest diameter opening upwards and the smallest diameter supported on the glass plate. the traditional resin composites were placed in two 2-mm thick increments separately photoactivated according to instructions of the manufacturer’s with the coltolux led device (coltène / whaledent, altstätten, switzerland) during the time determined by the manufacturer (table 2). in contrast, the low and regular viscosity bulk-fill composites were dispensed in single 4 mm increments and photoactivated according to the manufacturer’s recommendations (table 2). the curing device tip was placed over a glass slide (1 mm thick) on the resin composite surface to standardize the photoactivation distance for all resin composites. the restorations were finished with #600 and #1200 abrasive sandpapers coupled to a polishing machine (labopol-21, struers, copenhagen, denmark). half of the samples were kept for 24 hours in distilled water at 37 ° c and the other half for six months. bottom/top hardness ratio the bottom-to-top hardness ratio was performed according to previous studies3,5,11. after 24 h water storage, the specimens were positioned on the base of a microhardness tester device (hmv-2t e, shimadzu corporation, tokyo, japan) and three vickers indentations were performed in the central region of the top and bottom table 2. operative protocol for each resin composite used in this study. resin composite number of increments increment thickness photoactivation* time per increment tnc 2 2 mm 10s tbf 1 4 mm 10s tnf 1 4 mm 10s pfs 2 2 mm 40s afx 1 4 mm 20s xtb 1 4 mm 10s *the device used in this research had a power > 1000 mw/cm² which was measured with a radiometer (model 100, kerr, orange, ca, usa) for every eight specimens. tnf: tetric n-ceram; tbf: tetric n-ceram bulk fill; tnf: tetric n-flow; pfs: polofil supra; afx: admira fusion x-tra; xtb: x-tra base. 6 marinho et al. braz j oral sci. 2023;22:e237617 surfaces of each specimen with a distance of 200 μm between them. a 50 g load was used for 30 s. the mean vickers hardness number was obtained per surface, and the bottom/top hardness ratio was calculated. push-out bond strength test and failure modes the bond strength was assessed after 24 h (n = 90) and six months (n = 90) of water storage through the push-out test in a universal testing machine (microtensille om150, odeme, joaçaba, santa catarina, brazil). the specimens were placed on the device with its larger diameter (incisal) surface facing the metal base. a cylindrical 2.25 mm diameter metal tip pushed the smaller diameter (cervical) surface. it touched only the composite that filled the cavity, connected to the equipment’s load cell (100 n) at a 0.5mm/min speed until the restoration rupture. the load required for the restoration failure was recorded in n and converted to mpa, according to the following equation: mpa = n π(r + r)√(h2 + (r – r)2 where ‘r’ is the radius of the larger base, ‘r’ is the radius of the smaller base, and ‘h’ is the thickness of the specimen. after the test, the failure mode was examined using a dissecting microscope (stereozoom; bausch & lomb, rochester, ny, usa), using the following classification: adhesive between adhesive and dentin, cohesive in resin composite/dentin, and mixed (adhesive/cohesive) represented in figure 2. statistical analysis after confirming the parametric distribution of the errors, one-way anova (for bottom/ top hardness ratio) and two-way anova (for bond strength) followed by tukey posthoc tests were used to analyze the data (p<0.05). all statistical tests were performed using the graphpad prism 8 (graphpad software inc, san diego, california, usa). results there were no statistically significant differences for the bottom/top hardness ratio (p>0.05). comparisons among the groups are shown in table 3. figure 2. failure modes obtained in this study: adhesive (a), cohesive (b), mixed (c). a b c 7 marinho et al. braz j oral sci. 2023;22:e237617 there were statistically significant differences among resin composites (p<0.05) and time-points (p<0.01) for bond strength. comparisons among the groups are shown in table 3. at 24h, the resin composites tnc, pfs, tbf and afx showed statistically higher bond strength than tnf and xtb. at six months, tbf and afx provided the highest bond strength statistically, while tnc and psf provided the lowest bond strength statistically. considering the comparison between time-points, tnc and pfs showed statistically lower bond strength at six months, while tnf and xtb showed statistically higher bond strength at six months. tbf and afx showed statistically similar bond strength between 24 h and six months. failure modes are shown in figure 3. while adhesive failures were predominant for low-viscosity bulk-fill resin composites, other regular viscosity conventional and bulkfill resin composites showed more mixed failures. discussion the null hypothesis tested in this study that there will be no statistically significant differences between the materials for both properties analyzed was rejected. although the b/t was not statistically affected by the different materials, statistically significant differences in bond strength were found among them. as bottom/top hardness ratio of resin composites above 80% are adequate 12-15 all materials used in this study showed comparable polymerization between the bottom and top surfaces. thus, even bulk-fill resin composites were inserted in the preparation in a single 4 mm thick increment, they were able to promote adequate polymerization in the depth region of the specimens. a factor that may have been crucial for this favorable result for bulk-fill composites is the quantity and type of monomers, their molecular weight, and the mobility of the tested resin composites. the greater translucency and similar refractive index of components of bulk-fill resin composites are often associated with increased light transmutation into the depth portion of the material, which might guarantee an adequate degree of conversion5,16. table 3. means ± deviation from the bottom/top hardness ratio (b/t) and bond strength (mpa) according to the resin composite and time-points tested. resin composites b/t bond strength 24 hours 6 months tnc 0.85 ± 0.16 a 10.57 ± 2.32 aa 8.19 ± 1.63 bb tbf 0.90 ± 0.11 a 9.82 ± 1.99 aa 10.38 ± 1.89 aa tnf 0.91 ± 0.24 a 2.15 ± 0.86 bb 4.64 ± 155 ac pfs 0.95 ± 0.15 a 9.92 ± 2.08 aa 6.92 ± 1.01 bb afx 0.88 ± 0.09 a 10.63 ± 2.17 aa 11.17 ± 2.65 aa xtb 0.90 ± .18 a 3.07 ± 0.83 bb 3.88 ± 0.95 ac b/t: bottom/top hardness ratio. tnf: tetric n-ceram; tbf: tetric n-ceram bulk fill; tnf: tetric n-flow; pfs: polofil supra; afx: admira fusion x-tra; xtb: x-tra base. different lowercase letters indicate statistically significant differences between the same time for different composites (p <0.05). different capital letters indicate statistically significant differences between the different times for the same composite. 8 marinho et al. braz j oral sci. 2023;22:e237617 thus, the increments with thicknesses of up to 4 mm used in this research did not compromise the performance of the bulk-fill resin composites in the depth of cure compared to the conventional composites studied. for bond strength, regular viscosity resin composites (either traditional or bulk-fill tnc, tbf, pfs, and afx) showed higher bond strength than low viscosity bulk-fill resin composites (tnf and xtb). likely, low-viscosity bulk-fill resin composites have fewer filler particles, so bond strength decreased compared with resin composites containing more filler particles. conversely, only low viscosity bulk-fill resin composites provided higher bond strength at six months than 24 h. less polymerization shrinkage stress was observed for a low viscosity bulk-fill resin composite than a traditional regular viscosity composite10. also, low viscosity resin composites can dissipate easier polymerization stress due to a low elastic modulus than regular viscosity resin composites5. these findings may justify why only the low viscosity bulk-fill resin composites tested increased bond strength at six months of water storage. the higher elastic modulus of regular viscosity resin composites4 may impair stress dissipation during polymerization. however, as a regular viscosity bulk-fill resin composite can show decreased polymerization contraction stress than a traditional resin composite5, figure 3. distribution of the failure modes according to the resin composite and aging time analyzed. column: aging time to perform the test (24 24h or 6 6 months) after specimen preparation. lines: % of the failure modes. tnf: tetric n-ceram; tbf: tetric n-ceram bulk fill; tnf: tetric n-flow; pfs: polofil supra; afx: admira fusion x-tra; xtb: x-tra base. xtb-24 xtb-6 afx-24 afx-6 pfs-24 pfs-6 tnf-24 tnf-6 tbf-24 tbf-6 tcn-24 tcn-6 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% cohesive adhesive mixed 9 marinho et al. braz j oral sci. 2023;22:e237617 only the dentin adhesive interface of preparations filled with traditional resin composites showed decreased bond strength at six months of water storage. the stress generated at the adhesive interface at the time of polymerization and aging can compromise the integrity of the dentin adhesive interface of preparations restored with conventional resin composites, resulting in loss of strength after six months17. this study used the push-out bond strength method to measure dentin bonding stability in high c-factor preparations. the bond strength of resin composites can also be analyzed using a microtensile test after filling class i and class ii preparation, which requires cutting beams with diamond saws. thus, external stress is transferred to the tooth/composite interface and may underestimate bond strength values. in contrast, the push-out method allows the measurement of bond strength without this external stress. in the push-out bond strength test, stress generated by polymerization is transferred directly to the adhesive interface, as the resin composite shrinks into the cavity10. thus, the results obtained in this study state that regular-viscosity bulk-fill composite, in comparison with regular-viscosity and low-viscosity bulk-fill composite resins, may provide better clinical performance in terms of stability. however, more clinical trials need to be carried out to confirm this assumption. therefore, the bottom/top hardness ratio was not affected by the different materials tested. only bulk-fill resin composites did not present dentin bond strength loss after six months of water storage. only the low-viscosity bulk-fill resin composites were able to improve bond strength after aging. acknowledgments this study was supported by the brazilian council for scientific and technological development/cnpq (grant number: 402132/2016-5) data of availability datasets related to this article will be available upon request to the corresponding author. author contribution maria eduarda lima do nascimento marinho: drafting the work or revising it critically for important intellectual content; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. rodolfo xavier de sousa lima: drafting the work or revising it critically for important intellectual content 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10.1016/s0109-5641(87)80085-4. 11 marinho et al. braz j oral sci. 2023;22:e237617 16. meereis ct, leal fb, lima gs, de carvalho rv, piva e, ogliari fa. bapo as an alternative photoinitiator for the radical polymerization of dental resins. dent mater. 2014 sep;30(9):945-53. doi: 10.1016/j.dental.2014.05.020. 17. masarwa n, mohamed a, abou-rabii i, abu zaghlan r, steier l. longevity of self-etch dentin bonding adhesives compared to etch-and-rinse dentin bonding adhesives: a systematic review. j evid based dent pract. 2016 jun;16(2):96-106. doi: 10.1016/j.jebdp.2016.03.003. 1http://dx.doi.org/10.20396/bjos.v20i00.8661060 volume 20 2021 e211060 original article 1 pontifical catholic university of paraná. curitiba, pr, brazil. 2 positivo university. curitiba, pr, brazil. corresponding author: prof. dr. rodrigo nunes rached pucpr (ppgo) rua imaculada conceição, 1155. curitiba, pr, brazil, 80215-901. e-mail: r.rached@pucpr.br; ronura@gmail.com phone: 55 41-3271-1637 editor: dr altair a. del bel cury received: september 1, 2020 accepted: april 5, 2021 randomized clinical trial of complete denture cleaning methods in residents of care institutions ana paula sponchiado1 , maria cecília carlos lopes giacomel2 , evelise machado de souza1 , rosimeire takaki rosa1 , edvaldo antonio ribeiro rosa1 , rodrigo nunes rached1,* aim: to evaluate the efficacy of ultrasonic bath and traditional brushing in the hygiene of complete dentures of dependent residents in long-term care institutions (ltcis). methods: a randomized cross-over clinical study was conducted in 17 maxillary complete denture wearers living in ltcis. cleaning protocols were brushing or ultrasonic bath, both applied with neutral liquid soap. biofilm biomass were estimated by mtt reduction assay and specific microbial load (cfu) of streptococcus spp., staphylococcus spp. and candida spp. were quantified by selective and differential culture media. results: ultrasound method showed higher percentage reduction of biofilm biomass and specific microbial loads of streptococcus spp. compared to brushing (p<0.01). reduction of microbial loads of the other microorganisms were not different between cleaning methods (p>0.05). conclusions: the ultrasonic bath proved to be a feasible alternative method for the mechanical cleaning of complete dentures in ltcis. keywords: dentures. hygiene. biofilms. health services for the aged. elderly. https://orcid.org/0000-0002-5035-0741 https://orcid.org/0000-0003-3659-0797 https://orcid.org/0000-0002-7490-4868 https://orcid.org/0000-0002-8352-9078 https://orcid.org/0000-0001-6087-4365 https://orcid.org/0000-0003-4667-6762 2 sponchiado et al. introduction denture stomatitis affects approximately 65% of patients with maxillary complete dentures and it is commonly associated to poor oral hygiene1. the condition may be aggravated by trauma, continuous use of the dentures, allergic reaction to the denture base and cleaning products, hyposalivation, inadequate diet and use of antibiotics2,3. candida albicans is the main etiological agent of this condition or associated to other pathogenic and opportunistic microorganisms, such as streptococcus spp., staphylococcus spp. and lactobacillus4–6. systemic dissemination of microorganisms from oral infections associated with biofilm accumulation in complete dentures may cause bacterial endocarditis, aspiration pneumonia and chronic obstructive pulmonary disease in dependent elderly patients7–9. denture cleaning for biofilm control is essential to reduce the risk of these diseases8. complete dentures can be cleaned using chemical and/or mechanical methods such as hypochlorites, peroxides, enzymes, mouthwashes, brushing, sonic and ultrasonic vibration10,11. complete dentures wearers in long-term care institutions (ltcis) may be unable to maintain hygiene of their dentures due to illness, dementia or low motor coordination, which makes them dependent on caregivers’ support12–15. although these professionals consider oral hygiene important, lack of time, work overload and refusal behavior by patients may difficult its adequate performance16. brushing alone or associated with soap, toothpaste or abrasives has been the most commonly reported method for cleaning complete dentures8,11,17. ultrasonic bath is an accessible and low-cost cleaning method, especially for old and physically compromised patients18–20. the aim of this study was to compare the efficacy of ultrasonic bath and brushing as cleaning methods of complete dentures in ltci dependent residents. the outcomes evaluated were the percentage changes of biofilm biomass and specific microbial loads after application of the cleaning methods. the work hypotheses were: 1. there would be no difference between cleaning methods for biofilm biomass; 2. there would be no difference between cleaning methods for specific microbial load; materials and methods the present study was registered in rebec (rbr-3jj84f) and approved by the institutional research ethics committee (protocol number 2,225,015). sample selection the sample was composed of individuals selected among residents of two ltcis institutions located in the metropolitan area of the city of curitiba (parana, brazil). all residents were dependent elderly individuals. inclusion criteria were 60 years of age or older, complete maxillary edentulism and wear of conventional maxillary complete denture for at least one year. 3 sponchiado et al. individuals were excluded if: they wore conventional maxillary dentures that were fractured, repaired or relined; were using or had used antibiotics, antifungal agents or corticosteroids for a period of three months prior to the beginning of the research; were using denture adhesives or dentures with excessive calculus; were smokers. a sample size calculation for a non-inferiority trial of no differences between one treatment (ultrasound) to another (brushing) indicated a sample size of 13 patients for a power of 80%21. the consort 2010 flowchart22 (figure 1) illustrates the selection of study participants, allocation, monitoring and analysis. the institutions and their residents were invited to participate in the research after presentation of the risks, benefits and objectives of the study. the final sample consisted of 17 patients (4 men and 13 women) with a mean age of 72.5 years, who provided written informed consent to participate in the study. for the incapable patients, the legally designated representative was contacted, given explanations about the research and asked to provide written informed consent. enrollment assessed for eligibility (n = 25) excluded (n = 8) • not meeting including criteria (n = 8) • declined to participate (n = 0) • other reasons (n = 0) randomized (n = 17) allocation follow-up analysis allocated to intervention (n = 17) cross-over study: all 17 participants underwent both interventions. the sequence of interventions was randomized. • received allocated intervention (n = 17) • did not receive allocated intervention (n = 0) lost to follow-up (n = 0) discontinued intervention (n = 0) samples analysed (n = (17 participants x 2 interventions) = 34) • excluded from analysis (n = 0) figure 1. consort 2010 flow diagram. study design and cleaning methods this clinical study was randomized, double-blind, controlled, crossover, clinical study. all dentures were exposed to the two interventions, for five days each step. 4 sponchiado et al. intervention 1) manual brushing with neutral dishwashing soap: brushing once a day, at a fixed time, for two minutes20 using a soft brush specifically for denture cleaning (twister, colgate, são bernardo do campo, sp, brazil) associated with neutral dishwashing soap without glycerin (ype, goiania, go, brazil). intervention 2) ultrasonic bath with neutral liquid soap solution: the dentures were individually placed into identified and closed glass jars containing 100 ml of tap water and one drop of neutral dishwashing soap (ype), which were subjected to ultrasonic vibration (cristofoli, campo mourao, pr, brazil) at 160 watts once daily, at a fixed time, for 15 minutes19,23. the dentures were rinsed in running water and returned to the patient. one of the researchers (rnr) through a lottery system using wrapped cards placed in a plastic jar containing one of two possible sequences of interventions to be tested performed randomization. during the experimental stage, a reminder poster display was fixed on the bedroom wall of each patient with a daily checklist, according to the randomized sequence of cleaning methods. on the bathroom walls, posters were also fixed to emphasize that denture cleaning was not to be performed by anyone other than the previously calibrated caregiver. before the research began, alginate impressions of each maxillary complete denture were taken (jeltrate, dentsply, petropolis, rj, brazil) and dentures replicated using acrylic resin (jet classic, sao paulo, sp, brazil). this procedure was performed so that the replicates could be digitized with a dental scanner (ceramill map400, amann girrbach, koblach, austria) and the images were then analyzed using the solid edge v20 software (siemens plm, plano, usa) to calculate the total area of the dentures (cm2), which comprised both the external surface of the prosthesis (palate and dental arch) and the internal and retention area. with the purpose of starting the research with all dentures at the same level of hygiene, they were immersed in 4.2% acetic acid solution for 10 min and then brushed with 2% chlorhexidine digluconate (fgm, joinville, sc, brazil) for two min24. the dentures were washed with running water and returned to the patients. they wore the dentures normally for two days, and these were cleaned by brushing once a day. on the third day of the research, possible dietary debris were eliminated by using an indirect vortex of tap water for 30 s, with each denture being placed in a plastic receptacle with a volume of 500 ml. subsequently, the dentures were immersed in 100 ml of sterile saline solution. the sets were taken to the ultrasonic vessel for 15 min and the suspensions obtained were considered baseline in the microbiological analyses. after this collection, the dentures were again disinfected by immersion in 0.12% chlorhexidine digluconate (periogard®, colgate-palmolive, sao paulo, sp, brazil) for 15 min, followed by brushing with 2% chlorhexidine gel (maquira, maringa, pr, brazil) for 2 min and returned to the patients, whose dentures were then exposed to the study procedures. each of the cleaning methods was implemented for five consecutive days, and after a washout period of one week between each stage, the steps previously mentioned were repeated for all patients prior to beginning with the second intervention, so that all subjects were submitted to both cleaning methods. after apply5 sponchiado et al. ing each of the methods, the dentures were immersed in identified sterilized glass jars containing 100 ml of saline solution. the sets were taken to the ultrasonic vessel for 15 min and the suspensions obtained were considered “treatment” in the microbiological analyses. the “baseline” and “treatment” suspensions were sonicated (2840d, odontobrás, ribeirao preto, sp, brazil) for 20 min for maximum biofilm disintegration. aliquots of 500 μl were collected for quantification of the specific microbial load. the remaining suspension was used for estimating biofilm biomass by the reduction test of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (mtt; sigma-aldrich m5655, st louis, mo, usa)25. specific microbial load quantification the following dilution protocol was determined after a pilot trial with a patient’s complete denture. five hundred microliters aliquots of the suspensions were combined with 500 μl of distilled water and vortexed (ap56, phoenix, araraquara, sp, brazil) at 1500 rpm for 30 s. aliquots of 100 μl were taken and combined with 900 μl of distilled water and again vortexed at 1500 rpm for 30 s. one hundred microliter-aliquots of the above-mentioned diluted suspensions were plated in selective and differential culture media chromocult (merck, germany), manitol salt agar (newprov, brazil) and sabouraud agar dextrose (newprov, brazil). these media allow the presumptive quantification of streptococcus spp. (after confirmation by gram staining), staphylococcus spp. and candida spp., respectively. plates containing media for bacterial quantification were incubated at 37°c and plates with yeast media were incubated at 28°c (orion-502, fanem, sp, brazil). all incubations were conducted for 72 h in a normoxic atmosphere. after the incubation periods, the specific microbial load of the dentures was determined by counting the number of colonies formed (cfu = colony forming units) that presented phenotypes indicating the studied genera, followed by correction for dilution. the specific microbial density was obtained by dividing the numerical cfu values by the area of the dentures (cm2). estimation of biofilm biomass by mtt reduction assay the sonicated suspensions were centrifuged at 4500×g (be-4004, bioeng, curitiba, pr, brazil) for 10 min. the supernatants were removed and 1.5 ml of sterile water was added. after resuspension and transfer to microtubes, the suspensions were centrifuged at 10,000×g (eppendorf, hamburg, germany) for 1 min. after removal of the supernatant, 500 μl of 1 mg/ml-1 mtt was added and the microtubes were incubated at 37°c for 3 h. after every one-hour time interval the microtubes were vortexed at 1500 rpm for 30 s. at the end of the incubation, 500 μl of distilled water was added to the microtubes, which were agitated and again centrifuged for 3 min. the supernatants were removed and 200 μl of isopropyl alcohol was added to the sediments. after agitating for 30 seconds, the contents were separated at 10,000×g for 1 min. one hundred microliter-aliquots of the supernatants were collected and transferred 6 sponchiado et al. to wells of a flat-bottom microplate reader. the optical densities of the formazan crystals formed were measured in a tp-reader (thermoplate, go, brazil) at 540 nm. the optical density values were divided by the area of the respective dentures. the final results were expressed as od540/cm 2. estimation of biofilm biomass by mtt reduction assay and specific microbial load were performed by a blinded investigator (aps). statistical analysis statistical analyses were made with the statistical package for social science 24.0 (spss inc, chicago, il, usa). data were tested for normality by the kolmogorov-smirnov test with lilliefors significance correlation, and shapiro-wilk test. comparisons between cleaning methods were made using the mann-whitney u-test. comparisons between the percentage change from baseline to treatment of biofilm biomass and specific microbial load count were done with wilcoxon signed test. all tests were performed at a significance level of 5%. results the means and standard deviation of percentage change of biofilm biomass for brushing and ultrasound methods are presented in table 1. ultrasound method showed a significantly higher percentage change of biofilm biomass compared to brushing (p<0.05) (table 1). means and 95% confidence intervals of biofilm biomass are shown in figure 2. table 1. means and standard deviations of percentage change of biofilm biomass.   mean (±standard deviation) brushing -23.62% (±51.12%) p=0.0004851 ultrasound -70.77% (±16.95%) mtt/cm2 -23.62% -70.77% 10% 0% -10% -20% -30% -40% -50% -60% -70% -80% -90% brushing ultrasound figure 2. means and 95% confidence intervals of percentage change of biofilm biomass. 7 sponchiado et al. the means and standard deviation of percentage change of specific microbial loads of streptococcus spp., staphylococcus spp. and candida spp. are shown in table 2. streptococcus spp. was the only group that showed significant reduction in the count of microorganisms in both methods. staphylococcus spp. and candida spp. showed significant reduction in the percentage of microorganisms only in ultrasound method. means and 95% confidence intervals of specific microbial loads are shown in figure 3. table 2. means and standard deviations of percentage change of specific microbial load for streptococcus spp., staphylococcus spp. and candida spp. according to cleaning method. cleaning method mean (±standard deviation) streptococcus spp. brushing -23.39% (±45.95%) p=0.009 ultrasound -42.51% (±37.70%) staphylococcus spp. brushing -18.99% (±42.81%) p=0.094 ultrasound -36.54% (±44.07%) candida spp. brushing -16.27% (±35.37%) p=0.239 ultrasound -24.07% (±37.05%) 0.00% -10.00% -20.00% -30.00% -40.00% -50.00% -60.00% streptococcus spp. ufc/cm2 staphylococcus spp. candida spp. brushing ultrasound figure 3. mean and 95% confidence intervals of percentage change of microbial loads. discussion the current study compared the efficacy of the mechanical cleaning methods brushing and ultrasonic bath, on denture biofilm mass and specific microbial loads. the first null hypothesis was rejected as the positive impact of ultrasonic cleaning on biofilm mass was superior to brushing. the second null hypothesis was confirmed for the streptococcus spp. group, which showed significant changes on the percentage of specific microbial load in both methods, whereas it was rejected for staphylococ8 sponchiado et al. cus spp. and candida spp. groups, which had significant change on the percentage of specific microbial load only in ultrasound method. the current study compared the efficacy of the mechanical brushing cleaning and ultrasonic bath methods on denture biofilm mass and specific microbial loads. the first work hypothesis was rejected since the positive effect of ultrasonic cleaning on biofilm mass was superior to brushing. the second work hypothesis was rejected since the streptococcus spp. group showed significant changes on the percentage of specific microbial load in both methods. the choice of estimating biofilm biomass by the mtt reduction essay was shown to be adequate, since it is a widely used method in different areas of study25,26, and is applicable for quantifying the microbial content covering dental materials27. however, mtt reduction essay in complete denture cleaning methods is innovative. the method of scores based on biofilm staining, although frequently cited in the literature, is limited and not very accurate20,24,28–30. the polymerase chain reaction (pcr), another widely used test for biofilm evaluation in complete dentures24,31,32, does not allow discrimination of living cells from dead cells, since it is based on the indistinct amplification of dna segments, and may promote erroneous estimation of the microbial load24. on the other hand, the estimation of microbial biomass by the quantification of formazan formed from the mtt reduction is more reliable for estimating the load of living cells26. in the present study, the ultrasound method showed to be more efficient in the reduction of the percentage compared to brushing. as for the effect of cleaning methods on the percentage of streptococcus ssp. microorganism count, a statistically significant reduction was shown for brushing and ultrasound groups. this reduction has also been previously demonstrated23, however, the ultrasonic bath was associated with effervescent tablets and applied only once at the end of the research. in the current study, the counts of staphylococcus spp. and candida spp. were similarly reduced by the two cleaning methods. another study also reported a decrease in the levels of candida spp. with ultrasound associated to effervescent tablets19. on the other hand, studies have not observed reduction in candida spp., neither with the method of brushing nor ultrasound, associated or not with effervescent tablets23,24. a previous study has suggested that s. mutans from the biofilm of complete dentures create a glucan barrier that could limit the exposure of other species to chemical cleaning methods, including candida spp33. reduced counts of candida spp. were found when bactericidal and/or fungicidal solutions were associated with ultrasonic bath34. based on these results, it is possible to propose that this association could be effective, since the mechanical cleaning with ultrasound is capable of destabilizing the superficial layers of biofilm on dentures, that contain glucans produced by streptococcus spp. this disruption would expose the candida spp. of deeper layers of the biofilm to the action of chemicals33–35. the results of the current study comply with these assumptions, since significant reduction in the microbial load was only shown for streptococcus spp., both in the ultrasound and brushing groups. these microorganisms are found more superficially in the biofilm layers and, thus, may be more easily removed. on the other hand, can9 sponchiado et al. dida spp. and staphylococcus spp., are found in deeper biofilm layers, being positively influenced by the ultrasound method. ubiquitous microorganisms, such as candida spp., are present in healthy patients or in those with prosthetic stomatitis. in addition, candida albicans is found in the prostheses of patients with pathological changes1,6. counts of candida spp. in multi-species biofilm of complete dentures are difficult to reduce even when mechanical cleaning and effervescent tablets are associated23,33,36. in the current study, only the ultrasound was able to reduce the microbial load of candida spp. staphylococcus spp. are related to pneumonia that affects many elderly residents in ltcis7. in the current study, they were significantly reduced in concentration when the ultrasound method was applied to the complete dentures. therefore, it is expected that the method will result in minimizing a possible etiological factor for respiratory diseases related to these microorganisms. although brushing with water and toothpaste is the most commonly cleaning method used by complete denture wearers8,11,17, elderly patients may experience a decline in hand dexterity that affects their capacity to brush the dentures effectively19. ultrasonic cleaning is not a commonly used technique for denture in nursing homes, probably due to lack of information of caregivers and residents, but could be effective in an institutional home that provides care for the elderly with physical or cognitive deficiencies19,37,38. the effectiveness of ultrasonic cleaning is not dependent on the number of caregivers of the institution, as opposed to the brushing method. the method makes it possible to clean more than one denture at the same time. other advantages are easy handling of the equipment and low risk of occupational infection to caregivers. in the current study, the ultrasonic bath proved to be efficient in all the analyzed parameters: capability of significantly reducing the biofilm biomass as shown in the mtt assay, as well as of reducing the microbial load for all microorganisms studied groups. chemical disinfection is effective when associated with biofilm on denture surfaces30,39. there is a considerable variety of chemicals available for denture cleaning; however, the difficulty in manipulating and the elevated cost of some of these products limits their adoption in ltcis. as an example, effervescent tablets have demonstrated to be effective for the cleaning of dentures, however, they have an elevated cost, making it difficult to establish their use in underprivileged institutions20,40,41. an ideal cleaning agent for complete dentures should remove organic and inorganic materials, be non-toxic, cost-effective, easy to handle, and reduce biofilm39. as an example, dishwashing liquid soap, a conventional cleaning product, has proven to present emulsifying action that degrades biofilms. nevertheless, ultrasonic cleaning has demonstrated to be more effective when associated to chemical agents with these characteristics23,35. the chemical action results from protein solubilization and consequent reduction in microbial adhesion on complete denture surfaces40,42. the advantage over effervescent tablets is centered on non-abrasiveness, low cost and accessibility43. finally, it is important to note that, as proposed herein, the prostheses should be somehow stored individually during ultrasonic bath conduction, avoiding possible cross-contamination between residents. 10 sponchiado et al. in conclusion, the ultrasound method was more effective than brushing in reducing the biofilm biomass and the count of streptococcus spp. the reduction percentage of the specific count of the microorganisms staphylococcus spp. and candida spp microorganisms did not differ between the two cleaning methods. the ultrasonic bath proved to be a feasible alternative method for mechanical cleaning of complete dentures in ltcis. acknowledgments the authors thank the elderlies of the long term care 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streptococcus mutans biofilms towards detergent-stimulated detachment. eur j oral sci. 1999 aug;107(4):236-43. doi: 10.1046/j.0909-8836.1999.eos107402.x. 43. salles aes, macedo ld, fernandes rag, silva-lovato ch, paranhos hfo. comparative analysis of biofilm levels in complete upper and lower dentures after brushing associated with specific denture paste and neutral soap. gerodontology. 2007 dec;24(4):217–23. 1http://dx.doi.org/10.20396/bjos.v19i0.8659930 volume 19 2020 e209930 original article 1 head of the department of surgical dentistry, maxillofacial surgery and oncostomatology, uzhhorod national university, ukraine 2 head of scientific and research centre of forensic odontology, department of prosthetic dentistry, uzhhorod national university, ukraine. corresponding author: myroslav goncharuk-khomyn uzhhorod national university universitetska 16/a st., uzhhorod, ukraine area code: 88000 phone number: 0991212813 e-mail address: myroslav. goncharuk-khomyn@uzhnu.edu.ua received: june 04, 2020 accepted: november 23, 2020 economical treatmentrelated burden assessment of maxillofacial trauma among ukrainians patients pavlo brekhlichuk1 , myroslav goncharuk-khomyn2,* aim: quantitative evaluation of prognostic correspondence between initial maxillofacial traumatic injury assessed by facial injury severity score and maxillofacial injury severity score, treatment cost and duration of hospitalization among ukrainian patients. methods: design of present study was retrospective and based on the medical data of patients hospitalized with signs of maxillofacial trauma. quantitative assessment of maxillofacial trauma was held with the use of facial injury severity score (fiss) and maxillofacial injury severity score (mfiss). average treatment cost and hospitalization duration were used as coordinative criteria for economical treatmentrelated burden verification. results: levels of correlation between fiss, treatment charges and hospitalization duration were r=0.69 (р<0.05) and r=0.67 (р<0.05) respectively, while analogical correlations for mfiss were 0.74 (р<0.05) and 0.69 respectively (р<0.05). statistical correspondence between fiss and mfiss scores among study sample reached r=0.71 (р<0.05). cases with milder maxillofacial trauma types, characterized with initial lower levels of fiss and mfiss scores, demonstrated greater degree of fiss-to-mfiss inter-relation compare to cases with severe maxillofacial trauma. conclusion: even though fiss and mfiss scores both demonstrated reliable levels of correlation with hospitalization duration and cost of dental rehabilitation after maxillofacial trauma injury, but mfiss approach characterized by prognostically greater level of statistical relationship with economically related treatment derivates. moreover, differentiation capabilities of mfiss is relative greater than fiss, since independent grading of separate functional disabilities becomes possible. keywords: maxillofacial injuries. treatment outcome. cost of illness. hospitalization. http://dx.doi.org/10.20396/bjos.v19i0.8659930 mailto:myroslav.goncharuk-khomyn@uzhnu.edu.ua mailto:myroslav.goncharuk-khomyn@uzhnu.edu.ua https://orcid.org/0000-0001-6754-5142 https://orcid.org/0000-0002-7482-3881 2 brekhlichuk et al. introduction relevant predictive models of dental care supply, especially under the conditions of different insurance policies, should be based on the valid quantitative approaches aimed at primary differentiation and categorization of maxillofacial injuries in terms of needed dental interventions, their cost and treatment efficiency1-3. nevertheless, most of the used insurance calculation protocols considering the fact of maxillofacial trauma and it’s situational parameters as main criteria for future prognosis regarding payments of claims2-5. argumentative choice of economically-available and predictively-effective treatment modality could help to optimize overall rehabilitation process among maxillofacial trauma patients considering initial injuries of anatomical structures and associated functional alterations2,6,7. rapid scoring protocol of maxillofacial trauma also could provide further perspectives for primary patients categorization within trauma center conditions1,7,8. due to the number of previously published studies it is argumentative to resume that maxillofacial injury severity scoring system (mfiss) and facial injury severity scoring system (fiss) are considered as ones of the most prevalent among researchers’ use1,6,7,9-12, while in one of the studies such conclusion even was solidly clarified2. taking into account original scoring methodologies of mfiss and fiss scores and available literature data on their use with research objective, the first one is considered to be more functionally-oriented, while second – anatomically-based1,2,6,7,9-11. on the other hand number of publications revealed that both of these scores demonstrated comparatively analogical statistical associations with treatment duration, rehabilitation charges, injury severity, complication rates and some other parameters1,2,7,10-12. in multicentered study of european maxillofacial trauma it was noted that even though fiss scores were relatively analogical by the mean values among different centers, the longest hospitalization durations were noted in kiev (ukraine)13. since treatment methods and primary post-traumatic care differ among different countries, even though such are following the same biological and medical principles, it is important to find out how the fiss and mfiss scores predictively relate with the regionally-specific economic burden parameters of hospitalization and rehabilitation, thus widening the perspective of their use with an aim of dental care and insurance support optimization. considering all above-mentioned facts, our research was aimed at the quantitative evaluation of prognostic correspondence between initial maxillofacial traumatic injury assessed by fiss and mfiss scores, treatment cost and duration of hospitalization among ukrainian patients. materials and methods design of present study was retrospective and based on the medical data of patients hospitalized to the uzhhorod city clinical hospital (uzhhorod, ukraine) with signs of maxillofacial trauma during 2015-2019. study sample among all received patients’ data sets was formed due to the next inclusion criteria: 1) preliminary diagnosis of maxillofacial trauma provided at the time of hospitalization; 2) accessibility of full trauma characteristics description in provided medical documentation; 3) presence of supplemental x-ray diagnostics results or their full interpretations inside medical 3 brekhlichuk et al. documentation; 4) availability of full description regarding provided treatment with exact postscript of hospitalization duration and cost of treatment. exclusion criteria presented by the next parameters: 1) absence of necessary information related to the diagnostic process, anamnesis, provided treatment, cost or hospitalization duration; 2) concomitant traumatic injury; 3) combined trauma with related head and neck, eye-ball injury or neurotrauma; 4) compromised anamnesis with allied somathopathologies that potentially could alter rehabilitation process14. due to the inclusion and exclusion criteria group of 65 patients with maxillofacial trauma was formed. all patients were treated at the same accident and emergency department with further admission to the in-patient facility. data extraction was provided considering anonymization and ethical principles with further analysis of only next parameters: age, gender, characteristics of trauma, duration of hospitalization, average treatment cost15. average treatment cost was accounted by provisional monetary units due to the provided diagnostics complex, dental surgical rehabilitation, hospitalization and in-patient care without considering cost of pharmacological support. hospitalization duration was defined by the period between primary patient’s admission to the hospital till the official discharge2. such criteria as average treatment cost and hospitalization duration were used as coordinative for economical treatment-related strain verification for each patient from study group2. quantitative assessment of maxillofacial trauma was held with the use of facial injury severity score (fiss) and maxillofacial injury severity score (mfiss). evaluation was provided due to the original protocols of such scoring systems proposed by bagheri et al. and zhang et al. respectively6,9. use of mfiss and fiss scores as comparable and referent for analytical prognosis considering average treatment cost and duration of hospitalization, was argumented by high level of such criteria correlation with expert maxillofacial injury evaluation, described in previous studies1,2,12. assessment of fiss and mfiss scores was provided by two independent investigators (members of scientific and research center of forensic odontology, uzhhorod national university), who were previously calibrated with the use of reference data and characterized with inter-observer agreement of k=0.81. design of provided study was previously approved by ethical committee of faculty of dentistry (uzhhorod national university) as a part of the complex research related to the clinical and laboratorial assessment of advanced dental technologies and expert evaluation of treatment methods (ethical approval № 25072017-13). exploratory data analysis principles were used for study sample characterization with the evaluation of above-mentioned criteria (age, gender, characteristics of trauma, duration of hospitalization, average treatment cost). univariative statistical analysis with estimation of mean, maximum and minimum was provided considering criteria of fiss and mfiss scores independently among study group patients. pearson’s r was used for the estimation of correlation between fiss and mfiss scores and such parameters as average cost of treatment and hospitalization duration, while probability value (p-value) lower than 0.05 was considered as statistically reliable. student’s t-test was used for assessment of statistical difference between mfiss and fiss criteria patterns among study group patients, and distinction trends of their relation to the average treatment cost and hospitalization duration16,17. bland and altman analysis was provided with xlstat 2020 software tool (xlstat.com)18,19. stratification of data and its’ further graphical representation were provided via microsoft excel software (microsoft office, 2019). http://xlstat.com 4 brekhlichuk et al. results distribution of maxillofacial trauma patients sample by the age and gender criterions was presented as following: out of 65 subjects 53 (81.54%) were males and 12 (18.46%) were females with ratio of 4.41:1; 18 persons (27.69%) were within age group of 20-30 years (mean age – 26.72 years), 37 (56.92%) – within age group of 30-40 years (mean age – 34.54 years), 10 persons (15.38%) – within age group of 40-50 years (mean age 46.31 years). obtained distribution tendencies partially promoted by used specific inclusion criteria. main causes of maxillofacial trauma among study sample were presented by interpersonal violence (assaults) – 29 patients (44.62%), road-traffic accidents – 22 patients (33.85%), falls – 9 patients (13.85%), work-associated and sport-related – 5 patients (7.69%). most cases of interpersonal violence, road-traffic accidents and work/ sport-related traumas as causes of maxillofacial trauma were registered among male patients (65.52%, 63.64% and 60.0% respectively), while most cases of falls (55.56%) were noted among females. among all 65 analyzed cases 47 (72.30%) of them were presented with bone fractures (mandible fractures – 14 cases (21.54%), le fort i fractures – 5 cases (7.69%), le fort ii fractures – 4 cases (6.15%), le fort iii – 4 cases (6.15%), zygoma complex fractures – 6 cases (9.23%), nasal fractures – 10 cases (15.39%), orbital fractures – 4 cases (6.15%)), while in 18 cases (27.69%) such were also associated with pronounced adjacent soft tissue injuries (projected mostly at the lower one third of the face in 7 cases (10.77%), at the mid-face area – in 6 cases (9.23%), and at the upper one third – in 5 cases (7.69%), while in most cases lacerations projected beyond restricted area of some one third part of the face). numerical results received during fiss and mfiss scoring were characterized with normal distribution pattern, which also was described in previous study1. mean fiss score for study sample was 3.70±1.06 (mode – 3.0), while mean mfiss score was 16.37±6.04 (mode – 20) (table 1). average period of hospitalization duration was equal to 8.9±2.4 days. table 1. fiss and mfiss scores statistical characteristic registered among study sample variable observations obs. with missing data obs. without missing data minimum maximum mean std. deviation fiss 65 0 65 1.000 6.000 3.708 1.057 mfiss 65 0 65 5.000 30.000 16.369 6.035 levels of correlation between fiss, average treatment charges and hospitalization duration were r=0.69 (р<0.05) and r=0.67 (р<0.05) respectively, while analogical correlations for mfiss were 0.74 (р<0.05) and 0.69 respectively (р<0.05) (table 2). table 2. correlation level between fiss, mfiss, hospitalization duration and cost of treatment criteria cost of treatment p-value hospitalization duration p-value fiss p-value fiss 0.69 р<0.05 0.67 р<0.05 1.0 р<0.05 mfiss 0.74 р<0.05 0.69 р<0.05 0.71 р<0.05 statistical correspondence between fiss and mfiss scores among study sample reached r=0.71 (р<0.05) (fig. 1). 5 brekhlichuk et al. without preliminary standardization of obtained data statistical difference was noted during pairwise comparison of fiss and mfiss results during analysis of 57 (87.69%) individual cases. patients with fiss scores greater than 3 and mfiss scores greater than 10 were characterized with statistically longer period of hospitalization compare to study subjects with lower obtained scores levels (р<0.05). results of bland-altman analysis considering relationship between fiss and mfiss scores represented on the figures 2-4, with primary received data presented in table 3. m fi s s fiss 35 30 25 20 15 10 5 0 0 21 3 4 65 7 figure 1. correlation between fiss and mfiss scores registered among study sample. 30 25 20 15 10 5 0 bias cl bias (95%) cl (95%) 0 62 4 8 10 12 16 1814 20 figure 2. bland-altman plot for fiss and mfiss scoring results 6 brekhlichuk et al. 30 25 20 15 10 5 0 d ife re nc e mean minimum/maximum figure 4. correspondence between mean and median of fiss/mfiss due to the bland-altman analysis 0,08 0,07 0,05 0,06 0,03 0,04 0,02 0,01 0 0 105 15 diference diference normal d en si ty 20 25 30 figure 3. distribution of differences in fiss/mfiss scores due to the normality assumption 7 brekhlichuk et al. table 3. data received during bland-altman analysis of mfiss and fiss score bias standard error ci bias (95%) confidence interval (differences): lower limit upper limit lower limit upper limit 12.66 5.33 11.33 13.98 2.19 23.12 obtained results shown that both scores are analogically effective from the evaluation point of view and could be used for maxillofacial trauma assessment considering treatment expenses and hospitalization duration as targeted research parameters. but specific pattern of fiss/mfiss relationship was noted, due to which cases with milder maxillofacial trauma types, characterized with initial lower levels of fiss and mfiss scores, demonstrated greater degree of fiss-to-mfiss statistical inter-relation compare to cases with severe maxillofacial trauma. discussion the variability of the maxillofacial traumatic lesions cases and the prevalence of such among able-bodied persons justify the need for investigation, development and improvement of expert evaluation approaches considering dental changes of primary traumatic and secondary iatrogenic origin13,20-22. in present research we have argument the correlational levels between obtained fiss/mfiss scores, which indirectly related to the anatomical and functional severity of maxillofacial trauma, and economical parameters of provided dental care in means of average treatment cost and hospitalization duration. the results of previous analytical studies indicated the presence of relationship between the necessary amount of dental rehabilitation interventions and initial characteristics (location, severity, spread) of obtained dental injuries1,2,13,20. due to the eurmat project data the most prevalent causes of maxillofacial trauma were assault and falls13, while in our study most of trauma injuries were caused by interpersonal violence and road-traffic accidents. falls and work-associated/sport-related injuries were third and fourth the most prevalent causes of patients’ hospitalization with maxillofacial trauma. analogically to eurmat project data related to ukraine, male/female ratio in our study also was characterized with predominant number of male patients compare to female (4.41:1), while overall european ratio was at level of 3.6 to 113. similarly, to the findings noted by siber et al. (2015)20, we have also registered the bone injuries as the most prevalent among study sample of patients with maxillofacial trauma. providing retrospective study bocchialini and castellani (2019) had found that increase of fiss score parameter by 1 point associated with the increase in hospitalization duration on 12% (1.44 days)23. more pronounced relationship between fiss score and length of hospital stay was described in siregar  et  al. study (2019)24. authors mentioned that increase of fiss at the level of more than 3 characterized with 14 times more chances of longer hospitalization24. under the conditions of retrospective study, it was found the fiss scores greater than 5 causing 18 times more chances to be hospitalized compare to the situations when fiss score was lower than 6, while fiss scores greater than 5 were also statistically associated with need of minimum 3 days hospitalization (p  <  0.01)25. other researchers highlighted that level of fiss≤3 was 8 brekhlichuk et al. relevant for cases of maxillofacial trauma hospitalization up to 6 days, while fiss level of greater than 12 in most cases caused hospitalization for more than 10 days23. bagheri himself as an author of fiss scoring system mentioned that even though such demonstrated statistical association with the hospitalization duration, but it could not be categorized as fully reliable predictor9. in his research 3 cases of death were highlighted, while non-survivors’ fiss scores were not statistically different from those registered among survivors9. nevertheless, in all above-mentioned lethal cases victims demonstrated fiss scores greater compare to average ones noted during analysis9. considering variability of fiss/mfiss scores and hospitalization duration, we could not register some specific pattern of correspondence between 1 fiss/ mfiss point increase and additional number of days with needed in-patient care, but it was found that fiss scores greater than 3 and mfiss scores greater than 10 associated with more prolonged period of hospitalization compare to situations with lower obtained scores levels, difference between which was statistically approved (р<0.05). analogical to our, study was provided also by ramalingam s. (2015), who have found out that both mfiss and fiss scores were characterized with relatively equal correlation due to the cost (r=0.862 and r=0.845 respectively) and duration of hospitalization (r=0.828 and r=0.819) among indian population2. considering such results author highlighted the role of mfiss and fiss as “economic burden” indices, while in our study fiss demonstrated lower correlation levels with duration of hospitalization and the cost of treatment2. the presence of analogical study gives us a unique possibility to analyze potential causes of obtained results dissimilarities. such could be provoked by the influence on next factors: different approaches of patients stratification in india and in ukraine; different calibration levels of dental experts, who provided the evaluation of patients regarding mfiss and fiss criteria; different distribution of costs for specific dental trauma treatment algorithm in india and ukraine; variances of national currency due to the standardized cost of dental treatment calculated by insurance companies in means of provisional monetary units; differences of samples sizes. analogical to our findings, such also were described in giriyan et al. (2019) study, in which authors had registered spearmen’s correlation levels of r=0.398 and r=0.429 between mfiss/fiss values and treatment cost respectively, and correlation levels of r=0.477 and r=0.433 between mfiss/fiss values and hospitalization time respectively12. while estimated levels of correlation were lower compare to those in ramalingam’s study2, they were statistically approved. similarly to previous study, it was found that in cases of mid-face fracture among chinese population mfiss and fiss scores represent statistically analogical interrelations with hospitalization duration – r=0.415 and r=0.464 respectively26. moreover, authors found out statistically reliable dependencies between fiss scores and gender, age, etiology and fracture type parameter, while mfiss scores demonstrated connection with gender at the p=0.201 and with age at p=0.052. nevertheless, mfiss and fiss scores correlated between themselves at r=0.0592 (p=0.01)26. in our study relationship between above mentioned parameters (gender, age, etiology and fracture type) were out of primary formulated objective, while they will be considered as a perspective for future research it is interesting to note that in the comparative study of different maxillofacial trauma grading approaches, fiss scores demonstrated the lowest level of correlation with 9 brekhlichuk et al. expert evaluation results (r=0.699), while mfiss demonstrated the greatest (r=0.801) among all studied scoring systems1. despite that fiss scores were characterized by statistically the highest interrelation pattern with the cost of operation (r=0.742), while correlation levels of fiss (r=0.620) and mfiss (r=0.636) scores were comparable due to the operation time parameter1. our results are partially consistent with those described by chen et al. (2014)1 in terms, that in our study mfiss scores also have shown the highest level of correlation not only with duration of hospitalization, but also with the average cost of treatment. originally fiss was described as anatomically-based by the methodology of calculation in many of previously published studies1,2,7,10-12, but considering specific categorization of such grading trauma system, we could resume that this criteria is also partially functionally-oriented, even if such traumatic functional association is not so directly represented, as in mfiss methodology. for example, le fort iii fracture gains greater score than le fort i or le fort ii, which is logical, because such fracture is causing greater anatomical disruption, but it should be noted that le fort iii fracture is  also associated with more pronounced functional alterations. so, we would propose to classify fiss scoring system as “predominantly anatomically-oriented”, rather than just “anatomically-based”. considering today’s progress in maxillofacial surgery and forensic dentistry new scoring systems for maxillofacial trauma evaluation still developing. canzi and colleagues described comprehensive facial injury  (cfi) score, which differs by high descriptive capacity and with this characteristic supports patient differentiations in trauma centers27,28. potentially cfi could be used as statistical tool for hospitalization duration prognosis. another perspective could be related to the use of novel cbct-superimposition principle, which is effective for objectification of all possible dental status changes29. based on the obtained results we can resume that both fiss and mfiss scores are reliable baseground parameters that could be effectively used for prediction of treatment cost and duration of hospitalization, as component parts included in non-linear insurance proceedings calculation. greater correlation level of mfiss compare to fiss could be argumented by the orientation of such grading approach not only on the anatomical, but also on the functional evaluation of maxillofacial alterations. limitations of provided study related to its retrospective design, considering the use of medical patients records as primary data source, which related with risk of possible documentational errors. but such limitation was partially overcome by inclusion into study sample only cases with available x-ray diagnostic results or at least with their complete description (interpretation). other limitation of the research is related to the relatively small study sample compare to the analogical studies provided previously. on the other hand, such situation could be argumented by the use of specific exclusion criteria, such as concomitant traumatic injury, combined trauma with related head and neck, eye-ball injury or neurotrauma and compromised anamnesis with allied somathopathologies. neglect of such criteria potentially could help to increase the primary size of study sample, but in such situation, we would be limited in possibility to make reliable conclusion considering connection between fiss and mfiss scores with specifically maxillofacial trauma characteristics. logically,  that inclusion of patients with combined or concomitant trauma or aggravated anam10 brekhlichuk et al. nesis would complicate statistical analytical approach, and deviates from originally formulated objective. nevertheless, even considering above-mentioned limitations, obtained results demonstrated analogical pattern of relationship between fiss/ mfiss scores and economically associated rehabilitation parameters. moreover, verified covariances could be categorized as quantitively specific for ukrainian patients. the perspective of future study includes the statistical representation of such indices in the form of correction coefficients incorporated in the equations of the insurance indemnity amount calculation, which could be used not with ad hoc aim, but with prospective objective. considering limitations of provided retrospective study it could be resumed that even though fiss and mfiss scores both demonstrate reliable levels of correlation with hospitalization duration and average cost of dental rehabilitation after maxillofacial trauma injury, but mfiss approach characterized by prognostically greater level of statistical relationship with economically related treatment derivates. moreover, differentiation capabilities of mfiss is relative greater than fiss, since independent grading of separate functional disabilities become possible. taking this into account it could be recommended to include mfiss score as correction subcomponent or predictive factor into insurance calculation protocols during conceptual foresight of insurance coverages, or during court cases related to the assessment of dental health loss with the need of further dental rehabilitation. financial support none. conflict of interest the authors declare no conflicts of interest. references 1. 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14. van spall hg, toren a, kiss a, fowler ra. eligibility criteria of randomized controlled trials published in high-impact general medical journals: a systematic sampling review. jama. 2007;297(11):1233-40. doi: 10.1001/jama.297.11.1233. 15. general assembly of the world medical association. world medical association declaration of helsinki: ethical principles for medical research involving human subjects. j am col dent. 2014;81(3):14. 16. hannigan a, lynch cd. statistical methodology in oral and dental research: pitfalls and recommendations. j dent. 2013;41(5):385-92. doi: 10.1016/j.jdent.2013.02.013. 17. shintani a. primer of statistics in dental research: part i. j prosthod res. 2014;58(1):11-6. doi: 10.1016/j.jpor.2013.12.006. 18. giavarina d. understanding bland altman analysis. biochemia med. 2015;25(2):141-51. doi: 10.11613/bm.2015.015 19. doğan nö. bland-altman analysis: a paradigm to understand correlation and agreement. turkish j emerg med. 2018;18(4):139-41. doi: 10.1016/j.tjem.2018.09.001 20. siber s, matijević m, sikora m, leović d, mumlek i, macan d. assessment of oro-maxillofacial trauma according to gender, age, cause and type of the injury. acta stomatol croatica. 2015;49(4):340-7. doi: 10.15644/asc49/4/10 21. x maliska mcds, borba m, asprino l, de moraes m, moreira rwf. oral and maxillofacial surgeryhelmet and maxillofacial trauma: a 10-year retrospective study. braz j oral sci. 2012;11(2):125-9. doi: 10.20396/bjos.v11i2.8641434 22. galvão-moreira lv, cantanhede alc, de sousa neto ac, da cruz mcfn. factors affecting hospital discharge in maxillofacial trauma patients: a retrospective study. braz j oral sci. 2017;16:1-10. doi: 10.20396/bjos.v16i0.8650491 23. bocchialini g, castellani a. facial trauma: a retrospective study of 1262 patients. annals mxillofac surg. 2019;9(1):135. doi: 10.4103/ams.ams_51_19 24. siregar df, buchari fb, tarigan ua, lelo a. correlation of facial injury severity scale (fiss) with length of stay, the need for surgery and the involvement of other specialists in maxillofacial trauma patients at h. adam malik general hospital medan. global j res anal. 2019;8(11):8-10. doi: 10.36106/gjra https://doi.org/10.12968/jowc.2001.10.3.26062 https://www.researchgate.net/deref/http%3a%2f%2fdx.doi.org%2f10.5958%2f0974-1283.2019.00016.1?_sg%5b0%5d=f8bdnqav1iykkdgyc87iskntaqbfntynwcn1bdv3gitsj74imb6-zmblppkpv4n8svurzfqbwburs8sf9hdkbjyhig.rhxz2psf6z2rlhn3ps4uq8ylqkavhdi7u4228xtgamnt5avtstyhkqo-vvchqn9yf9w0p648ymntovprtk-c7g https://www.researchgate.net/deref/http%3a%2f%2fdx.doi.org%2f10.5958%2f0974-1283.2019.00016.1?_sg%5b0%5d=f8bdnqav1iykkdgyc87iskntaqbfntynwcn1bdv3gitsj74imb6-zmblppkpv4n8svurzfqbwburs8sf9hdkbjyhig.rhxz2psf6z2rlhn3ps4uq8ylqkavhdi7u4228xtgamnt5avtstyhkqo-vvchqn9yf9w0p648ymntovprtk-c7g https://dx.doi.org/10.11613%2fbm.2015.015 https://www.researchgate.net/deref/http%3a%2f%2fdx.doi.org%2f10.1016%2fj.tjem.2018.09.001?_sg%5b0%5d=pwzy5womkbse1ku0hldcac__k3mbxnnczdfzwhppmpiu2cflqbkjfskdwhylalregs2prbh0dx7ure_ho1ko4ptk-g.bls5dl0jx32wiosnmzpsz3mvfgp1f8lugimw-khvcmhwmjtdkzyrdxmpfalnecchb5qxltddephmqdewntiegq https://dx.doi.org/10.15644%2fasc49%2f4%2f10 https://www.researchgate.net/deref/http%3a%2f%2fdx.doi.org%2f10.20396%2fbjos.v16i0.8650491?_sg%5b0%5d=3wyn_kw6ezgrnqdrstsel8zg-ptsg065efopz9erncalbyksy5wz53b_yb2uwxkllm1yre1fofe6hrf1uhsm4skmha.m00jbcxd5kvpkpqrpzkzaze6socdbuby8pibrrsrrbb3mpqdg1mqw5sk1gyalelyseulyp8_eamnowstql0thq https://dx.doi.org/10.4103%2fams.ams_51_19 12 brekhlichuk et al. 25. aita tg, stabile clp, garbelini ccd, stabile gav. can a facial injury severity scale be used to predict the need for surgical intervention and time of hospitalization? j oral maxillofac surg. 2018;76(6):1280-e1. doi: 10.1016/j.joms.2018.02.002. 26. suwal r. analysis of mid-face fractures using mfiss and fiss scoring systems. j nepal dent assoc. 2018;18(1):21-9. 27. canzi g, de ponti e, novelli g, mazzoleni f, chiara o, bozzetti a, et al. the cfi score: validation of a new comprehensive severity scoring system for facial injuries. j craniomaxillofac surg. 2019;47(3):377-82. doi: 10.1016/j.jcms.2019.01.004.  28. canzi g, de ponti e, fossati c, novelli g, cimbanassi s, bozzetti a, et al. understanding the relevance of comprehensive facial injury (cfi) score: statistical analysis of overall surgical time and length of stay outcomes. j craniomaxillofac surg. 2019;47(9):1456-63. doi: doi: 10.1016/j.jcms.2019.07.005. 29. goncharuk-khomyn m, andrii k. evaluation of peri-implant bone reduction levels from superimposition perspective: pilot study among ukrainian implantology practice. pesq bras odontoped clin integr. 2018;18(1):3856. doi: 10.4034/pboci.2018.181.10. https://doi.org/10.1016/j.jcms.2019.07.005 1 volume 22 2023 e237270 original article braz j oral sci. 2023;22:e237270http://dx.doi.org/10.20396/bjos.v22i00.8667270 1 oral and maxillofacial diseases research center, mashhad university of medical sciences, mashhad, iran. 2 surgical oncology research center, mashhad university of medical sciences, mashhad, iran.  3 faculty of health, mashhad university of medical sciences, mashhad, iran. corresponding author: maryam amirchaghmaghi, ala ghazi contact information: oral and maxillofacial diseases research center, department of oral medicine of mashhad faculty of dentistry, vakilabad blvd, postal code:9177948959, mashhad, iran. amirchakhmaghim@mums.ac.ir, ghazial@mums.ac.ir editor: dr. altair a. del bel cury received: october 14, 2021 accepted: july 30, 2022 assessment of salivary opiorphin in oral lichen planus zohreh dalirsani1 , maryam amirchaghmaghi1*≠ , ala ghazi1*≠ , seyed isaac hashemy2 , mahboobeh taherizadeh3 opiorphin is a pentapeptide, which could be isolated from human fluids and has a decreasing effect on pain. aim: since lichen planus is a chronic mucocutaneous disease, which causes pain or burning feeling in the oral mucosa, this study aimed to compare salivary opiorphin levels of oral lichen planus (olp) patients with healthy subjects. methods: this case-control study, was performed on 24 patients with olp lesions and 21 healthy subjects. after collecting unstimulated saliva, opiorphin levels were compared between two groups through statistical analyses. results: there was not any significant difference between olp patients and healthy subjects according to salivary opiorphin concentration (p=0.378). also, in the olp group, opiorphin concentration was not significantly different between males and females (p=0.601). analytical analysis could not show any remarkable difference between various severity of olp lesions regarding to salivary opiorphin levels (p=0.653). conclusion: in this study, salivary opiorphin levels was not significantly different between patients with olp and healthy subjects; however, more studies are suggested for better assessment of salivary opiorphin levels in various types of olp lesions and its correlation with pain severity. keywords: oligopeptides. lichen planus, oral. ≠ maryam amirchaghmaghi and ala ghazi contributed equally to this work. about:blank about:blank https://orcid.org/0000-0002-5898-3180 https://orcid.org/0000-0002-6504-2725 https://orcid.org/0000-0002-0064-1233 https://orcid.org/0000-0002-1323-5250 https://orcid.org/0000-0003-1061-3284 2 dalirsani et al. braz j oral sci. 2023;22:e237270 introduction opiorphin is a pentapeptide, which could be isolated from human fluids including saliva1. it seems that opiorphin inhibits encephalininactivating enzymes and increases half-life of encephalin2. moreover, opiorphin could inhibit nociception due to chronic and acute stimulation, and perception of pain, this effect is compatible with morphine1. since, this peptide has a preventive effect on pain, there is a hypothesis, which depict that every chronic disease imposes chronic pain or discomfort, might influence on opiorphin levels. lichen planus is a chronic mucocutaneous disease, in which, the patients mostly tolerate a long time burning and pain in their oral mucosa. despite huge progression about etiology of olp, the exact mechanism and definite treatment of this condition is unclear. the researchers have been trying to find some acceptable and noninvasive methods for diagnosis and assessment of efficacy of the treatments. up to now, several studies have been performed on some salivary biomarkers in olp patients; however, none of them evaluate salivary opiorphin, as an index of chronic pain. this study was conducted to investigate salivary opiorphin concentrations of olp patients and compare this marker with healthy subjects. methods and materials this case-control study was performed in the oral and maxillofacial medicine department of mashhad faculty of dentistry from september 2018 until june 2019. twenty-four patients with olp (both keratotic and non-keratotic), and 21 healthy subjects, after signing a written informed consent form, enrolled in this study. diagnosis of olp was confirmed through clinical and histopathological examinations, according to who modified criteria3. the healthy individuals were selected from the patients, who were referred to mashhad faculty of dentistry, who had no oral lesions or systemic diseases, or any pain; they had only been referred for common dentistry procedures. the inclusion criteria for the case group were: newly diagnosed oral lichen planus patients, as confirmed by clinical and histopathological examination; patients who had signed the informed consent form. the exclusion criteria for the case group were as bellow: the subjects, who had used any drugs interacting with opioids or analgesics; the patients, whose histopathological examination was reported as lichenoid reactions. the exclusion criteria for the control group was the usage any medicine for relief of the pain, in recent days. prepared checklists were completed for all subjects including demographic characteristics consisting of age, gender, and medical history; lesion properties including the site of lesion, clinical features (keratotic and non-keratotic), type and severity of 3 dalirsani et al. braz j oral sci. 2023;22:e237270 olp lesion, based on thongprasom criteria3. the patients were categorized into five categories 20-30, 30-40, 40-50, 50-60, and ≥60 years according to their age. the correlation of opiorphin levels, and sex and age groups was statistically analyzed. assessment of opiorphin all subjects were asked not to drink, eat, or smoke for 90 minutes before collecting unstimulated saliva. saliva samples were collected through spitting method between 9 to 12 a.m. the patients were asked to sit in a convenient position and after gathering the saliva in their mouth, spit into 50 ml falcon sterilized tube every 1 minute for 5-10 minutes4. the salivary samples were stored with ice and sent to the laboratory and kept at -20°c for 24 hours for mucolysis. in order to separate debris and mucous, the saliva samples were centrifuged for 15 minutes at 3000 rpm. until collecting all samples and analysis, the samples were kept at -20°c. after that, the watery parts of the salivary samples were transferred to microtubes with a volume of 1.5 cc. then opiorphin levels were measured using the kits of “human opiorphin elisa kit (catalog # mbs760008)” according to the protocol of the company. the bioten double antibody sandwich was used for the assessment of opiorphin. first, 40 µl of the salivary sample and 10 µl of opiorphin antibody were added to 50 µl of standard solution and 50 µl of streptavidin-hpr; after that, the mixed solution was incubated at 37ºc for 60 min. next, this solution was washed with 300 µl diluted buffer five times. then, 50 µl of chromogen a and 50 µl of chromogen b solutions were added to prepare the samples. after mixing them, the samples were incubated at 37ºc for 10 minutes. finally, 50 µl of stop solution was added to them, which change the yellow color to blue color. after 10 minutes, the optical density was measured at the wavelength of 450 nm to assess the opiorphin concentration. the opiorphin levels were compared between healthy subjects and olp patients through statistical analyses. statistical analysis the data were entered into spss software (version 24) and statistical analysis was performed. kolmogorov-smirnov test was employed for quantitative data with normal distribution. in order to assess the qualitative variables between the groups, chi-do and fisher’s exact test were used. for quantitative variables with normal distribution, parametric statistical methods including independent t-test and anova, and for nonparametric methods in case of abnormal distribution, mann-whitney test and kruskal-wallis were employed. the significance level for the statistical tests was considered as 0.05. the protocol of this study was approved by ethical committee of mashhad university of medical sciences (the ethical code: ir.mums.dentistry.rec.1397.045). results in this study, salivary opiorphin levels of 24 olp patients and 21 healthy subjects were assessed. 4 dalirsani et al. braz j oral sci. 2023;22:e237270 kolmogorov-smirnov test showed that the variables of age and opiorphin have normal distribution. therefore, independent ttest was used for assessment of these variables. the mean ages of olp patients and healthy persons were 46.41±11.89 and 46.09±9.12 years, respectively. statistical analysis revealed that there was no any significant variations between the two groups regarding the mean age (p=0.920). also, more number of olp patients belonged to 50-60-year group; however, chi square test showed that there was no remarkable correlation between development of olp and subjects’ age (p=0.389) (table 1). the mean of salivary opiorphin levels were 2.76±0.66 and 2.93±0.55 for the case and control groups, respectively (table 1 and figure 1). independent ttest did not show any significant difference between olp patients and healthy subjects according to salivary opiorphin levels (p=0.378) (table 1). pearson’s chi-square test did not reveal any significant correlation between olp patients’ age and salivary opiorphin levels (p=0.187). furthermore, this statistical analysis did not show any remarkable relationship between healthy subjects’ age and salivary opiorphin concentration (p=0.597). in the case group, the mean of salivary opiorphin levels were 2.72±0.69 and 2.89±0.58 for the females and males, respectively. statistical analysis did not show any remarkable difference between two genders in the olp patients according to salivary opiorphin levels (p=0.601) (table 2). also, in the control group, the mean of salivary opiorphin levels were 2.79±0.40 and 3.07±0.67 for the females and males, respectively, which were not significantly different (p=0.257) (table 2). thus, we concluded that subjects’ gender has no effect on salivary opiorphin level. table 1. demographic data and opiorphin levels in the case and control groups. variables number (%) case group control group p-value number (%) sex male 6(62.1) 10(37.5) p=0.114*female 18(37.9) 11(62.5) total 24(100) 21(100) age (years) 20-30 3(12.5) 0(0) p=0.389* 30-40 5(20.8) 8(38.1) 40-50 6(25) 6(28.6) 50-60 8(33.3) 6(28.6) ≥60 2(8.3) 1(4.8) total 24(100) 21(100) opiorphin 2.76±0.66 2.93±0.55 0.378** *chi square test **independent samples t-test 5 dalirsani et al. braz j oral sci. 2023;22:e237270 moreover, according to opiorphin levels, there was no remarkable difference between olp females and healthy females (p=0.758), as well as between olp males and healthy males (p=0.587). analytical analysis showed that opiorphin levels were not statistically different between various severity of olp lesions, based on tongprasom criteria (p=0.653); and there was no significant difference between keratotic, and non-keratotic types of olp lesions (p=0.453). we concluded that there is no association between salivary opiorphin levels, and severity of olp lesions. discussion some biomarkers, which are secreted into saliva, could indicate some physiologic and pathologic conditions. opiorphin is a pentapeptide, which might be isolated from human saliva1 and could alleviate pain due to inflammation and physical damage. also, this biomarker is a natural suppressor of enzymes, which protects enkephalins and endorphin from degradation. since enkephalin and endorphin are the native painkillers; this process could reduce the pain sensation. moreover, opiorphin has anti-depressant5, anti-inflammatory and anti-tumoral effects1,6. sobocińska et al.7 (2020) studied the effects of sialorphin and spinorphin on a mouse model of colitis and suggested these peptides as anti-inflammatory elements. nejad et al.6 (2020) study revealed that salivary opiorphin levels in the patients, who had painful oral soft tissue lesions including traumatic and inflammatory table 2. opiorphin levels based on the sex in the case and control groups. variables sex case group control group p-value* opiorphin female 2.72±0.69 2.79±0.4 0.758 male 2.89±0.58 3.07±0.67 0.587 p-value* 0.601 0.257 *independent samples t-test figure 1. box plot of opiorphin levels in the case and control groups. 5.00 * 18 4.00 3.00 2.00 1.00 olp healthy group o pi or ph in 6 dalirsani et al. braz j oral sci. 2023;22:e237270 conditions, as well as, in patients with oral pre-malignant or malignant lesions, were higher than in healthy subjects; their study confirmed that the severity and type of pain could effect on opiorphin levels. therefore, assessment of patients’ salivary opiorphin could be an indicator of the severity of pain. furthermore, these rising opiorphin levels in oral cancer patients could suggest an anti-tumoral role of this marker. an in-vivo study on a mouse model of melanoma showed that opiorphin and sialorphin conjugate to a proapoptotic and antimicrobial peptide (called klak) leading to the formation of compounds, sialo-klak and opio-klak. these compounds have positive cytotoxic effects on cancer cells8. in the present study, the opiorphin levels were assessed in the patients involved with olp, as potentially malignant lesions. although opiorphin levels of olp patients were higher than in healthy persons, the difference was not significant. concurrently, similar study on painful oral lesions, showed that the opiorphin levels in these patients were higher than the controls6. furthermore, ozdogan et al.9 (2019) study showed that rate of salivary opiorphin concentration has a positive correlation with the severity of tooth pain. they concluded that inflammation due to the pulp or periodontal diseases could raise opiorphin levels. in the present study, which was performed on 24 olp patients including 7 keratotic and 17 non-keratotic olp; there was not statistically significant difference between salivary opiorphin levels of patients with keratotic and non-keratotic lesions. also, this biomarker level did not noticeably rise in severe lesions based on tongprasom criteria. therefore, this study could not prove that inflammation due to an erosive form of olp increase opiorphin levels in these patients. the results of this study were contradictory with ozdogan et al.9 (2019) study, which observed that there was a direct correlation between opiorphin concentration and severity of inflammation. with respect to the fact that another study did not perform on various types of olp lesions and opiorphin levels, a comprehensive study with a greater sample size on different severity of olp is suggested for better judgment about the relationship of salivary opiorphin levels and severity of olp lesions. in this study, there was no significant difference between the salivary opiorphin concentration of olp patients and healthy subjects; as well as, various types of olp lesions. further studies are recommended to evaluate salivary opiorphin levels in different scores of olp lesions and its correlation with pain severity. author contribution zohreh dalirsani, maryam amirchaghmaghi, ala ghazi and seyed isaac hashemy contributed in designing and performing the project. mahboobeh taherizadeh contributed in analyzing the data. all authors actively participated in the manuscript’s findings and have revised and approved the final version of the manuscript. 7 dalirsani et al. braz j oral sci. 2023;22:e237270 conflict of interest the authors declare no conflict of interest in this study. references 1. wisner a, dufour e, messaoudi m, nejdi a, marcel a, ungeheuer mn, et al. human opiorphin, a natural antinociceptive modulator of opioid-dependent pathways. proc natl acad sci u s a. 2006 nov;103(47):17979-84. doi: 10.1073/pnas.0605865103. 2. sujatha s, priyadharshini r, niloofar khansari n, yashoda devi bk, shwetha v, pavan kumar t, et al. an analgesic to bridge the gap between narcotics and nsaids: opiorphin. int j basic clin pharmacol. 2018;7(7):1432-6. doi: 10.18203/2319-2003.ijbcp20182695. 3. thongprasom k, luangjarmekorn l, sererat t, taweesap w. relative efficacy of fluocinolone acetonide compared with triamcinolone acetonide in treatment of oral lichen planus. j oral pathol med. 1992 nov;21(10):456-8. doi: 10.1111/j.1600-0714.1992.tb00974.x. 4. shirzaiy m, dalirsani z. the effect of glycemic control on salivary lipid peroxidation in type ii diabetic patients. diabetes metab syndr. 2019 may-jun;13(3):1991-4. doi: 10.1016/j.dsx.2019.04.004. 5. javelot h, messaoudi m, garnier s, rougeot c. human opiorphin is a naturally occurring antidepressant acting selectively on enkephalin-dependent delta-opioid pathways. j physiol pharmacol. 2010 jun;61(3):355-62. 6. nejad nk, ramakrishna p, kar a, sujatha s. quantitative analysis and expression of salivary opiorphin in painful oral soft-tissue conditions: a descriptive study. j global oral health. 2020;3(2):123-7. doi: 10.25259/jgoh_41_2020. 7. sobocińska m, salaga m, fichna j, kamysz e. anti-inflammatory effect of homoand heterodimers of natural enkephalinase inhibitors in experimental colitis in mice. molecules. 2020 dec;25(24):5820. doi: 10.3390/molecules25245820. 8. kamysz e, smolarczyk r, cichoń t, jarosz-biej m, sikorska e, sobocińska m, et al. antitumor activity of opiorphin, sialorphin and their conjugates with a peptide klaklakklaklak. j pept sci. 2016 nov;22(11-12):723-30. doi: 10.1002/psc.2936. 9. ozdogan ms, gungormus m, ince yusufoglu s, ertem sy, sonmez c, orhan m. salivary opiorphin in dental pain: a potential biomarker for dental disease. arch oral biol. 2019 mar;99:15-21. doi: 10.1016/j.archoralbio.2018.12.006. 1 volume 22 2023 e238271 original article braz j oral sci. 2023;22:e238271http://dx.doi.org/10.20396/bjos.v22i00.8668271 ¹ department of prosthodontics and periodontology, piracicaba dental school, university of campinas, piracicaba, são paulo, brazil. ² department of clinical and social dentistry, federal university of paraíba, joão pessoa, paraíba, brazil. corresponding author: renata cunha matheus rodrigues garcia, dds, phd. department of prosthodontics and periodontology, piracicaba dental school, university of campinas, avenida limeira, no. 901, bairro areião, piracicaba, sp, brazil, cep: 13414-903. e-mail: regarcia@fop.unicamp.br editor: altair a. del bel cury received: january 31, 2022 accepted: may 5, 2022 association of sociodemographic characteristics, mental health, and sleep quality with covid-19 fear in an elderly brazilian population talita malini carletti1 , ingrid andrade meira1 , lorena tavares gama1 , mariana marinho davino de medeiros1 , yuri wanderley cavalcanti2 , renata cunha matheus rodrigues garcia1,* aim: this study determined whether covid-19 fear is correlated with sociodemographic characteristics, general health, mental health, and sleep quality in an elderly brazilians. methods: elderly people aged ≥ 60 years replied to an online survey containing questions about their sociodemographic characteristics; general health; levels of stress, anxiety, and depression; sleep quality; and covid fear. results: data were statistically analyzed using descriptive statistics (α = 5%). in total, 705 elderly people with mean age of 66 ± 5 years, and most (82.7%) respondents were women, graduated and from southeastern brazil. covid-19 fear correlated positively and moderately with sleep quality and symptoms of depression, anxiety, and stress (all p < 0.001). it was associated with females. elderly people from northern and northeastern brazil and diabetics had increased covid-19 fear (all p < 0.05). conclusion: the fear of covid-19 exists among brazilian female old people, diabetics, increases anxiety and stress symptoms, and worsen sleep quality in elderly people. keywords: coronavirus. covid-19. mental disorder. sleep. aged. https://orcid.org/0000-0002-1549-2621 https://orcid.org/0000-0002-3631-0030 https://orcid.org/0000-0002-6224-0072 https://orcid.org/0000-0002-2472-8747 https://orcid.org/0000-0002-3570-9904 https://orcid.org/0000-0001-8486-3388 2 carletti et al. braz j oral sci. 2023;22:e238271 introduction severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has spread worldwide, causing the novel 2019 coronavirus disease (covid-19), considered by the world health organization (who) to be a public health emergency of international concern. the rapid progression of covid-19, the fear of contracting the infection, shortages of essential survival elements (e.g., food, water, clothing), and the closure of educational and business institutions have had adverse effects on human health during the pandemic1. anxiety and fear associated with social distancing measures implemented during the covid-19 outbreak may lead to excessive mental overload, which is often harmful to individuals’ psychological health2-4. the adoption of these measures has resulted in the loss of daily routines, leading to sleep dysregulation and worsening insomnia symptoms in most of the population5. the measures’ adverse psychological effects can exacerbate pre-existing illnesses, increasing clinical and subclinical mental conditions such as anxiety, depression, acute stress syndrome, and post-traumatic stress disorder2. incidence of depressive and anxiety-, trauma-, and stress-related disorders are expected to increase worldwide during and after the covid-19 pandemic. depression and anxiety already affect 4.4% and 3.6% of the world’s population, respectively; in brazil, 5.8% and 9.3% of citizens (the largest proportion among latin american countries)6, have been affected by these disorders. according to the world mental health report7, depression is common among elderly people. however, depressive disorders are detected less in this population than in young adults, as the aging process is often neglected. with populational aging, the numbers of people with depression and anxiety are increasing, mainly in developing countries8. given the covid-19 pandemic, elderly people are at high risk of infection, and stricter social isolation measures (e.g., family confinement) may cause more significant mental health problems in this age group. mental disorders, in turn, can be related to psychosocial and physiological problems, such as metabolic diseases (e.g., diabetes and cardiovascular, autoimmune, neurocognitive, and neurobiological diseases) and other age-related health problems, in elderly people9. however, little is known about the effects of covid-19–related fear on the mental health of brazilian elderly people. thus, this study was conducted to assess associations of elderly brazilians’ sociodemographic characteristics and medical factors with covid-19 fear during social isolation. we also examined correlations of covid-19 fear with anxiety, stress, depression, and sleep quality in this population. material and methods study design, sample, and procedure in this cross-sectional observational study, the effects of the covid-19 pandemic on sociodemographic and psychological factors of elderly brazilian people were assessed using an online survey. 3 carletti et al. braz j oral sci. 2023;22:e238271 according to the continuous national household sample survey, the brazilian population contained an estimated 33,707,000 people aged ≥ 60 years, of whom 37.8% (12,590,000) had internet access (and thus could participate in an online survey), in 2018. sample calculation was based on the variable with the lowest prevalence (depression), which is a psychological factor accounting for about 20% of the elderly population10. to obtain power and response rate of 80%, and 20% of expected sample loss, a study design effect of 2.0 and a 95% confidence level were adopted. thus, the minimum sample needed to detect significant differences was determined to be 615 respondents. the sample yielded a final target of 705 participants. volunteers from all brazilian regions were selected to participate in this study through digital media (open facebook and instagram accounts, journalistic websites, and local radio stations). the inclusion criteria were: 1) age ≥ 60 years, 2) brazilian citizenship and residence, and 3) internet access. this study conformed the recognized standards required by declaration of helsinki and was approved by the local ethics committee. the researchers established a hyperlink that took eligible individuals to the survey site, hosted on a google forms platform (google llc, mountain view, ca, usa). those who accepted the invitation to participate digitally signed an informed consent form to gain access to the questionnaire. the questionnaire could be filled out using a cellphone, tablet, computer, or other electronic means. data collection took place from september to december 2020. incomplete questionnaires were excluded from the study automatically; data from these surveys could not be displayed and were not available for analysis. socioeconomic characteristics and general health data on respondents’ age, sex, civil status, city and state of residence, monthly income, and educational level were collected. general health data (weight, height, presence of disease, previous diagnoses, symptoms) and data on the receipt of covid-19 information were also collected. depression, anxiety, and stress the short (21-item) version of the depression, anxiety and stress scale (dass-21), adapted for brazil11, was used to assess participants’ depression, anxiety, and stress (with seven items each) in the past week, during the covid-19 pandemic. responses are structured by a likert scale (0, not applicable at all; 1, applicable to some degree or for a short time; 2, applicable to a considerable degree or for a good part of the time; 3, applicable much or most of the time)12. dass-21 scores are used to classify the three mental conditions as normal, mild, moderate, severe, and extremely severe, with higher scores reflecting greater severity11,12. sleep quality sleep quality was assessed using the pittsburgh sleep quality index (psqi), validated for brazilian population, which consists of 19 items in seven domains: subjective perception of sleep quality, latency, duration, efficiency, disorders, medications, and 4 carletti et al. braz j oral sci. 2023;22:e238271 daytime disorders13. responses covered the past month of sleep. component scores ranged from 0–3 and are summed to yield final scores (0–21); higher scores indicate worse sleep quality13. total scores > 5 indicate significant or moderate difficulties in at least two or three scale components. covid-19 fear the survey also included the brazilian version of the covid-19 fear scale (fcv19s), which consists of seven items assessing respondents’ fear of sars-cov-214,15. responses are structured by a likert scale (1, strongly disagree; 2, disagree; 3, neither agree nor disagree; 4, agree; 5, strongly agree), with total scores ranging from 7 to 35 and higher scores indicating greater fear15. data analysis the data were characterized using descriptive statistics [frequencies, dispersions, and central tendencies (absolute and relative frequencies, means and standard deviations, medians, and interquartile ranges)]. as the kolmogorov–smirnov test demonstrated that the dependent variable (covid-19 fear) was not distributed normally, non-parametric tests were used for inferential data analysis. all analyses were performed using spss software (version 20.0 for windows; spss inc, chicago, il, usa). spearman’s correlation test was used to evaluate correlations of covid-19 fear with mental health domains and sleep quality. poisson regression was used to analyze associations between covid-19 fear and the independent variables (sociodemographic characteristics, comorbidities, depression, anxiety, stress, and sleep quality). the independent variables were allocated hierarchically to distal and proximal blocks. the distal block included sociodemographic characteristics (age, sex, region of residence, educational level) and comorbidities (diabetes, hypertension, cardiomyopathy, respiratory disease, kidney disease, cancer, stroke, obesity). the proximal block included depression, anxiety, stress, and sleep quality. using the backward procedure, only variables with p values < 0.20 in the crude model (sex, region of residence, diabetes, obesity, depression, anxiety, and stress) were included in the adjusted model. measures of the regression coefficient (b), prevalence ratios, and 95% confidence intervals were obtained. the significance level was set to p < 0.05. results in total, 705 elderly people participated in the study. considering dropouts, for the psqi, only 600 completed the questionnaire; thus, only for this variable the reduced sample was considered. their sociodemographic characteristics are presented in table 1. most participants were women (82.7%) and from southeastern brazil (42.7%) and had complete higher education (44.5%). their mean age was 66 years (range, (61–71 years; table 2) and most elderly present only one comorbidity. 5 carletti et al. braz j oral sci. 2023;22:e238271 table 1. sample characterization according to the nominal variables: sociodemographic data and presence of comorbidities (n = 705). characteristics frequency n % sex female 583 82.7 male 122 17.3 brazilian regions northern 41 5.8 northeastern 144 20.4 southeastern 300 42.7 midwestern 51 7.3 southern 167 23.8 educational level elementary school 66 9.4 high school 162 23.0 graduate 314 44.5 postgraduate 163 23.1 diabetes yes 107 15.2 no 598 84.8 hypertension yes 333 47.2 no 372 52.8 cardiomyopathy yes 59 8.4 no 646 91.6 respiratory disease yes 61 8.7 no 644 91.3 kidney disease yes 12 1.7 no 644 91.3 cancer yes 25 3.5 no 680 96.5 stroke yes 3 0.4 no 702 99.6 obesity yes 294 41.7 no 411 58.3 total 705 100.0 6 carletti et al. braz j oral sci. 2023;22:e238271 table 2. sample characterization according to the quantitative variables: age, number of comorbidities, fear of covid-19 (fcv-19s), depression, anxiety, stress, and sleep quality. characteristics mean (sd) median percentile 25-75 age (years) 66 +/5 64 62-68 number of comorbidities 1 +/1 1 0-2 covid-19 fear scale (fcv-19s) 21+/6 20 17-25 depression (dass-21) 4 +/5 3 0-7 anxiety (dass-21) 3 +/4 2 0-5 stress (dass-21) 6 +/5 5 1-8 dass21 total score 13 +/14 10 2-20 sleep quality (psqi) 8 +/4 7 4-10 dass-21 = depression, anxiety and stress scale short version. psqi = pittsburgh sleep quality index. fcv-19s = fear of covid-19 scale. the total fcv-19s score was high (mean, 21 ± 6; table 2). dass-21 stress scores (mean, 6 ± 5; median, 5) were higher than anxiety and depression scores, although the dass-21 overall score was classified as normal for 97.2% of respondents (table 3). the median sleep quality score was 7 (mean, 8 ± 4), indicating major or moderate sleep difficulties (table 2). fcv-19s score was correlated positively and moderately with dass-21 scores for symptoms of depression (r2 = 0.518; p < 0.001), anxiety (r2 = 0.587; p < 0.001), and stress (r2 = 0.595; p < 0.001), and with sleep quality scores (psqi; r2 = 0.424; p < 0.001)) (p < 0.001; table 4). in the adjusted regression model, fcv-19s score was related to female (b = 0.061), residence in northern (b = 0.132) and northeastern (b = 0.067) brazil relative to residence in southern brazil, diabetes (b = 0.047; all p < 0.05), and the presence of more anxiety (b = 0.013) and stress (b = 0.026) symptoms on dass-21 (both p < 0.001; table 5). table 3. frequency (n) and percentage (%) of dass-21 score classification in the study volunteers. dass-21 classification n % normal 685 97.2% mild 2 3% moderate 7 1% severe 3 0.4% extremely severe 3 0.4% total 705 100% dass-21 = depression, anxiety and stress scale – short version. 7 carletti et al. braz j oral sci. 2023;22:e238271 table 4. correlation between fear of covid-19 (fcv-19s), mental health and sleep quality in brazilian elderly people. mental health variables fear of covid-19 r2 p-value depression (dass-21, n=705) 0.518 <0.001* anxiety (dass-21, n=705) 0.587 <0.001* stress (dass-21, n=705) 0.595 <0.001* sleep quality (psqi, n=600) 0.424 <0.001* * p-value < 0.05 was considered statistically significant. dass-21 = depression, anxiety and stress scale short version. psqi = pittsburgh sleep quality index. fcv-19s = fear of covid-19 scale. table 5. poisson regression for factors associated with fear of covid-19 (fcv-19s) in brazilian elderly people (n=597). variable adjusted model b p-value pr 95% ci lower upper sex female 0.061 0.039* 1.063 1.003 1.126 male ref brazilian regions northern 0.132 0.002* 1.141 1.051 1.238 northeastern 0.067 0.026* 1.069 1.008 1.134 southeastern 0.042 0.086 1.043 0.994 1.094 midwestern 0.057 0.117 1.059 0.986 1.138 southern ref diabetes yes 0.047 0.049* 1.048 1.000 1.098 no ref obesity yes 0.024 0.182 1.024 0.989 1.061 no ref dass-21 depression -0.005 0.179 0.995 0.989 1.002 anxiety 0.013 <0.001* 1.013 1.006 1.021 stress 0.026 <0.001* 1.027 1.019 1.034 b = regression coefficient pr = prevalence ratio; 95% ci = 95% confidence interval dass-21 = depression, anxiety and stress scale short version. psqi = pittsburgh sleep quality index. fcv-19s = fear of covid-19 scale. ref = reference category used in the poisson regression. * p-value < 0.05 was considered statistically significant. 8 carletti et al. braz j oral sci. 2023;22:e238271 discussion in this study, associations of covid-19 fear with mental health and sleep quality were evaluated in a nationwide elderly brazilian population. emerging mental health issues related to the covid-19 pandemic may evolve into long-lasting health problems and isolation in elderly people, who are at high risk of complications of this disease16,17. the literature on the impacts of covid-19 on people’s quality of life is diverse and accumulating rapidly, but it lacks information about elderly people’ mental health during the pandemic. the fear of covid-19 has also been found to be most pronounced in elderly participants18, which emphasizes the concern about the virus disease course. the effects of quarantine on the mental and physical health of aged people have been summarized in only one review to date19. the present study revealed that the covid-19 outbreak has had a negative impact on the mental health of the elderly population of brazil, with covid-19 fear associated with high stress and anxiety levels, especially among women, residents of northern and northeastern brazil, and diabetics. the ease of access to technology, digital media, and information likely contributed to our achievement of the target number of participants. the predominance of women in our sample corroborates previous findings, although most respondents in other recent studies about covid-19 have been young adults20-22. moreover, the association of covid-19 fear with females is noteworthy. women’s sense of care, consciousness, educational levels, and concerns about post-outbreak family well-being likely increase their fear of the disease23. consistent with our findings, other researchers have noted that covid-19 fear was pronounced in females, families with children, laid-off workers, and individuals with symptoms of depression and anxiety20,24. as expected, most of our elderly respondents had complete higher education and were from southeastern brazil, which has the greatest concentration of elderly people with the country’s highest social, educational, and economic levels25. however, elderly people from northern and northeastern brazil were more afraid of sars-cov-2 than were those residing in southern brazil. covid-19 fear is concentrated in densely populated urban areas, which have larger case numbers24. in march 2020, when brazil was ranked 50th in the world in the number of covid-19 cases, the greatest average daily percentage changes in confirmed cases had been reported in the northern and northeastern regions of the country26. these regional disparities highlight the non-egalitarian distribution of public health, education, and human development resources in brazil. poor people are more vulnerable to covid-19 given the lack of health insurance, as well as limited intensive care unit availability in low-income regions27. similar to our findings, demenech et al. observed the greatest economic inequity and mortality rates in the north rather than the lowest imbalance in the south of brazil27. to date, the poorest regions have had enlarged numbers of covid-19 deaths and, considering the insufficient sars-cov 2 surveillance in brazil, the adoption of measures to restrict virus spread has also been inadequate. hypertension and obesity were prevalent in our sample, although they did not affect the majority of respondents. the prevalence of these conditions is increasing in 9 carletti et al. braz j oral sci. 2023;22:e238271 latin america due to socioeconomic trends and increasingly sedentary lifestyles28. obesity and metabolic syndrome are potential risk factors for the development of hypertension28, cardiovascular disease, dyslipidemia, and cancer. in the elderly population, changes in metabolism also result in lean mass reduction and the accumulation of more body fat29. this study revealed a high level of covid-19 fear among people with diabetes, who made up a minority of our sample. many patients admitted to hospitals with diagnoses of covid-19 have severe chronic diseases, such as hypertension and diabetes30,31. in individuals of advanced age, such conditions are of great concern once no treatment has been identified to the various symptoms the virus disease may induce in organs besides the respiratory system32. more elderly people than expected have contracted sars-cov-2 infection, although studies have explored the impact of the pandemic in adults20,24,33. due to the limited understanding of the pathophysiology of covid-19 and the lack of therapeutic interventions32, disease severity may be greatest in infected elderly people with pre-existing chronic diseases. high covid-19 fatality rates have been associated with elderly age, cardiovascular and cerebrovascular diseases, and diabetes16,17. patients with these conditions may be frequent users of medications, such as angiotensin-converting enzyme inhibitors and other angiotensin blockers, that upregulate protein expression in the cells to which the virus attaches and into it penetrates34. thus, the severity of sars-cov-2 infection would be intensified in these patients34, requiring an awful lot of healthcare workers’ attention. in the context of isolation and social distancing, elderly people with chronic diseases are feeling a great deal of apprehension about covid-19 progression, treatments, and future protocols. dissemination of scientific arguments and information in the press and social media may have contributed to covid-19 fear among elderly diabetic individuals. other comorbidities requiring constant medical attention (i.e., hypertension and obesity) were more prevalent than diabetes in our sample but were not associated significantly with covid-19 fear, raising questions about how elderly people in brazil are dealing with their general healthcare in terms of medical treatment and follow-up assessments. not surprisingly, elderly people with symptoms of anxiety, stress, and depression had more covid-19 fear. most studies of the effects of isolation during the covid-19 pandemic have documented the high prevalence of these mental disorders in elderly people19. in contrast, a brazilian study20 revealed higher levels of sadness and anxiety often or all the time during the pandemic in young adults than in elderly people; in 70% of respondents, prior diagnoses of depression contributed to these symptoms. as we did not collect such data in the present study, we cannot comment on the causality of the relationships we identified. perrin et al.4(2009) reported that determinants of psychological outcomes included being elderly, female, in isolation or quarantine, having a low educational level, and having a weak or support network. duarte et al.21(2020) also reported that women are 2.73 times more likely than men to present minor mental disorders; as our sample was predominantly women, this confirms our results. the critical associations of stress and anxiety symptoms with covid-19 fear in this study agree with a previous report35. anxiety and fear are intimately related, but they are different concepts. the former is a state mood bearing 10 carletti et al. braz j oral sci. 2023;22:e238271 a possible negative situation, and the latter is an alarm response to a present or perceived hazard35. symptoms of both may range from subjective perceptions to motor acts or visceral activities, such as muscle tension, nausea, sweating, heart palpitation, avoidance, and worry35. as a consequence, stress disorders may also develop. thus, the imminent possibility of infection with a fatal disease such as covid-19 generated an alerted state of mind and emotional and behavioral reactions in many of our elderly respondents, although most respondents had normal mental health according to the dass-21 classification. elderly people with covid-19 fear are also more prone to have worse sleep quality, leading to moderate or major sleep difficulties. more than half of participants in an american study had worse sleep quality, high anxiety rates, and signs of insomnia caused by the pandemic23. the authors of that study23 did not identify sex differences in sleep disorders and anxiety, but they noted a major female predisposition for mental disorder development. in contrast, other authors have reported pronounced sleep difficulties among women20, supporting our findings. however, no conclusion about sleep quality during lockdown can be addressed once people have shown heterogeneous sleep results before the pandemic due to individual attitudes, and different experiences towards emotions during the pandemic36. the protective factor of the lack of lifestyle change during isolation has been observed in elderly samples, although most elderly people wake up early or during the night and have difficulties falling asleep37. quarantine provided better sleep opportunities and reduced responsibilities among respondents23. our findings suggest that the retirement condition did not affect the way elderly people dealt with their daily routines, but uncertainties about the future and new measures implemented to restrain disease spread may have impacted their quality of sleep. finally, elderly people in brazil appear to have socioeconomic and social vulnerability and fear of covid-19. the country failed to adopt public measures to restrain the spread of the disease in time, which has resulted in massive numbers of covid-19 infections and deaths since the beginning of the pandemic26,27. despite this, strategies have been proposed to the population to avoid psychological problems and maintain quality of life during the covid-19 outbreak; they include limiting the time spent receiving covid-19 updates and news, working, and exercising as much as possible, and seeking behavioral therapy to minimize the development of mental disorders5. limitations of this study include: 1) its online basis, as elderly people may have difficulty filling in online questionnaires; 2) the use of digital media for recruiting, which is challenging for the target elderly group to access it without help from a familiar member; 3) the possibility of bias common to all online surveys, as responses could be softened, misleading, or incomplete; 4) the self-reporting nature of the survey, with no clinical evaluation performed by appropriate professionals, which could probably be misunderstood and answered; and 5) the inability to make causal inferences concerning covid-19 fear and mental disorders, as we did not know respondents’ pre-pandemic mental health status. the findings suggest, however, that the development of healthcare strategies and mental health programs to control the onset of anxiety and stress symptoms, especially for those with chronic diseases, 11 carletti et al. braz j oral sci. 2023;22:e238271 will aid elderly people across brazil. further clinical research could explore the ability of mental health assistance and other proposed treatments to minimize post-outbreak side effects in the elderly population. conclusions the fear of covid-19 exists among brazilian female old people, diabetics and increases the symptoms of anxiety and stress and worsen sleep quality in the aged population. acknowledgments this work was supported by são paulo research foundation – fapesp under grant 2018/23013-4 (iam); national council for scientific and technological development (cnpq) under grants 140391/2020-7 (tmc) and 140396/2020-9 (ltg); and coordenação de aperfeiçoamento de pessoal de nível superior brasil (capes), finance code 001 (mmdm). authors acknowledge piracicaba dental school (fop/unicamp) marketing service; journal of piracicaba (piracicaba, são paulo); globonews (g1-piracicaba, são paulo), and newscast sp tv (sistema brasileiro de televisão sbt) for helping with the research questionnaire dissemination around brazil. data availability datasets related to this article will be available upon request to the corresponding author. conflict of interest none author contribution the participation of each author is described below, according to the authorship and co-authorship criteria: • substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work – talita, ingrid, lorena, mariana, yuri and renata; • drafting the work or revising it critically for important intellectual content – talita, ingrid, lorena, mariana, yuri and renata; • final approval of the version to be published – talita and renata; • agreement to be 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[prevalence of overweight, general and central obesity in elderly women from curitiba, paraná, brazil]. rev nutr. 2008;21(5):525-33. portuguese. doi: 10.1590/s1415-52732008000500005. 30. li q, guan x, wu p, wang x, zhou l, tong y, et al. early transmission dynamics in wuhan, china, of novel coronavirus–infected pneumonia. n engl j med. 2020 mar;382(13):1199-207. doi: 10.1056/nejmoa2001316. 31. zhang j, dong x, cao y, yuan y, yang y, yan y, et al. clinical characteristics of 140 patients infected with sars-cov-2 in wuhan, china. allergy. 2020 jul;75(7):1730-41. doi: 10.1111/all.14238. 32. yuki k, fujiogi m, koutsogiannaki s. covid-19 pathophysiology: a review. clin immunol. 2020 jun;215:108427. doi: 10.1016/j.clim.2020.108427. 33. wu b. social isolation and loneliness among older adults in the context of covid-19: a global challenge. glob heal res policy. 2020 jun 5;5:27. doi: 10.1186/s41256-020-00154-3. 14 carletti et al. braz j oral sci. 2023;22:e238271 34. wan y, shang j, graham r, baric rs, li f. receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus. j virol. 2020 mar 17;94(7):e00127-20. doi: 10.1128/jvi.00127-20. 35. craske mg, rauch sl, ursano r, prenoveau j, pine ds, zinbarg re. what is an anxiety disorder? depress anxiety. 2009;26(12):1066-85. doi: 10.1002/da.20633. 36. kocevska d, blanken tf, van someren ejw, rösler l. sleep quality during the covid-19 pandemic: not one size fits all. sleep med. 2020;76(december 2019):86–8. 37. pinto j, van zeller m, amorim p, pimentel a, dantas p, eusébio e, et al. sleep quality in times of covid-19 pandemic. sleep med. 2020 oct;74:81-5. doi: 10.1016/j.sleep.2020.07.012. 1http://dx.doi.org/10.20396/bjos.v20i00.8663555 volume 20 2021 e213555 original article 1 graduate program in dentistry, federal university of pelotas (ufpel), pelotas, rio grande do sul, brazil. 2 faculty of dentistry, university of fortaleza/unifor, fortaleza, ceará, brazil. corresponding author: fernanda geraldo pappen address: faculty of dentistry, federal university of pelotas rua gonçalves chaves 457, sala 507 pelotas, rs, brasil cep 96015-320 phone: 55 53 984033599 received: december 9, 2020 accepted: april 13, 2021 editor: dr altair a. del bel cury endodontic treatment during covid-19 pandemic – economic perception of dental professionals eduarda carrera malhão1 , fabio de almeida gomes2 , cláudio maniglia ferreira2 , danilo lopes ferreira lima2 , maísa casarin1 , fernanda geraldo pappen1* aim: this study evaluated, by the application of questionnaires, the impact of the covid-19 pandemic on the clinical routine and inspection by the competent authorities, on the flow of patients in the office, as well as on possible changes in endodontic treatment costs and the amounts charged to patients. methods: this cross-sectional study was conducted from may 2nd, 2020 to may 6th, 2020, using an online questionnaire with a convenience sample. the inclusion criterion was professionals who perform endodontic treatments in daily clinical practice and who professional setting is private practice. the questionnaire brought questions about the impact on costs and the amount charged to the patient. results: a total of 1042 questionnaires were answered from all the different states of brazil, by professional who usually perform endodontic treatment, and who is working in private practice. a total of 1010 (96.9%) respondents affirm it was necessary to modify the protective equipment in endodontic treatment due to pandemic and longer intervals between appointments was cited by 922 (88.5%), economically affecting the dental practice. there was no association between routine changes and economic impacts with gender, professional experience, area of residence or education level. conclusion: in conclusion, most dental professionals recognized changes in the routine of endodontic treatment during the covid-19 pandemic. they have a perception of increase in endodontic costs, and reduction in the volume of patients. keywords: coronavirus. dentistry. endodontics. economics. https://orcid.org/0000-0002-8259-6025 https://orcid.org/0000-0003-0066-6729 https://orcid.org/0000-0001-8072-9376 https://orcid.org/0000-0002-9916-013x https://orcid.org/0000-0002-3750-5091 https://orcid.org/0000-0002-8483-455x 2 malhão et al. introduction in march 2020, the world health organization (who) declared the outbreak of covid19 as a pandemic due to the severity of the disease and the global spread. a pandemic is not only a health problem; it is also an economic, social and political issue1. the physical distance measures needed to prevent the virus proliferation have unleashed the most severe global recession on record since the great depression, since people have been recommended to stay at home whenever possible, which damages the economy as a whole2. the demand for social distance resulted in closing of stores, restaurants, gyms, factories, etc3. currently, more than a billion people worldwide are at risk of losing their jobs or at least suffering a cut in wages2. with regard to dental services, this economic crisis can significantly affect both demand and supply. on one side, patients may face financial problems resulting from economic crisis, with a decrease in the financial resources available for spending on dental services. on the other side, the pandemic can affect the availability of labor and the supply of materials4. in any case, dentists cannot fail to offer their services to the population to avoid overburden on hospitals in cases of urgency and emergency. initially, elective procedures were suspended in many places, prioritizing essential urgent care. consequently, these professionals must be prepared to perform their procedures safely during this turbulent period. it has been recommended that dentists should avoid the production of aerosols to reduce the risk of contamination. professionals were also instructed on the use of specific personal protective equipment (ppe), increasing the costs of dental treatment5. past six months since the who had declared the covid-19 pandemic, most professionals are working as usual and performing the most varied procedures. however, besides the need of precautions to protect themselves and their patients, dentists also face the economic consequences of covid-19 pandemic. the generated changes in clinical practice and in the daily lives of these professionals caused possible changes in the inspection of dental offices and in the costs involved. thus, the objective of this study is to evaluate, by the application of questionnaires, the impact of the covid-19 pandemic on the clinical routine, on the flow of patients in the office, as well as on possible changes in endodontic treatment costs and the amounts charged to patients. materials and methods this study was approved by the institutional review board (caae: 31064820.6.0000.5052). participants signed an informed consent form (icf) after acceptance to be part of the survey. as inclusion criteria, the respondent should be a dentist who usually performs endodontic procedures in clinical practice, with no need to be a specialist in endodontics. the submission was considered only when the ‘submit’ button was clicked at the end of the questionnaire. this cross-sectional study obtained data by filling out questionnaires developed on the google forms platform, available at: https://docs.google.com/forms/d/10pkh60cwbptjtuxyoknm8rjsadowkisq8kljxkpl3e8/edit. the design, pilot study and https://docs.google.com/forms/d/10pkh60cwbptjtuxyoknm8rjsadowkisq8kljxkpl3e8/edit https://docs.google.com/forms/d/10pkh60cwbptjtuxyoknm8rjsadowkisq8kljxkpl3e8/edit 3 malhão et al. strategies to enroll dentists were previously discussed by the research team. the pilot study had 20 participants and their answers were excluded from the main sample. since this questionnaire includes many specific questions in the area of endodontics, aiming to assess the impact of the pandemic on this specialty, the inclusion criterion was professionals who perform endodontic treatments in daily clinical practice and whose professional setting is private practice. in the present study, the questionnaires were applied to a convenience sample, which is a type of non-probability sampling method where the sample is taken from a group of people that is easy to reach. this type of sampling is also known as grab sampling or availability sampling. there are no other criteria to the sampling method except that people be available and willing to participate. in addition, this type of sampling method does not require generation of a simple random sample, since the only criterion is whether the participants agree to participate. this study was conducted from may 2nd, 2020 to may 6th, 2020. the questionnaire was distributed by email as well as posted on several social media platforms, as facebook and whatsapp. this is not a representative sample, and the regional councils were not contacted since they would not be able to provide information about dentists who perform endodontic treatment routinely. it was not possible to calculate the number of professionals reached by the social media platforms, as well as the number of dentists in brazil who perform endodontic treatment; thus, all individuals who perform root canal treatment as a routine and agreed to participate were included in the research. the questionnaire consisted of 21 questions designed to collect information about socioeconomic data, professional experience, biosecurity measures (to protect both professionals and patients) and the impact on the economic costs for professionals. the survey was divided into two parts: the first part contained questions related to personal data such as sex, age, area of residence, years of experience in dental practice; the second part included questions about the impact on costs and the amount charged to the patient. after responding to the questionnaire, the subjects were requested to provide their registration number in the regional dental council (cro) to prevent duplication of data. data were collected and analyzed by spss 25.0 for windows (ibm corp., spss, inc., chicago, il, usa). descriptive statistics were performed. chi-square and fisher’s exact tests were used to test the significance of possible associations. the significance level was 5% (p < 0.05). results a total of 1042 questionnaires were answered from all different brazilian states, by professionals who usually perform endodontic treatment and are working in private practice. female respondents accounted for 65.5% (n=683) and males 34.5% (n=359) of the total. the mean age of respondents was 37.5±10.3 years. regarding the time elapsed since graduation, 44.9% (n=468) of respondents graduated in the last 10 years; while 29.9% (n=312) graduated between 11 and 20 years ago; and 25.1% (n=262) graduated more than 20 years ago (table 1). 4 malhão et al. the percentage of respondents by brazilian regions was 23.7% from the south, 29.6% from the southeast, 37.7% from the northeast, 4.8% from the midwest and 4.2% from the north (table 1). a total of 1010 (96.9%) respondents stated that it was necessary to modify the protective equipment in endodontic treatment due to the pandemic. major attention with biosecurity measures was cited by 1021 respondents (98.0%), and longer intervals between appointments was cited by 922 (88.5%), economically affecting the dental practice. most participants believe that the new habits necessary to dental practice during the pandemic will last even after pandemic. table 2 describes the routine changes in endodontic treatment, volume of patients and economic impact of the pandemic. most professionals reported changes in routine and economic impacts. there was no association between routine changes and economic impacts with gender, professional experience, area of residence or educational level (p>0.05). the perception of dental professionals from different brazilian regions regarding the impact of the covid-19 pandemic is reported in table 3. table 1. percent distribution of respondents by background characteristics n % gender female 683 65.5 male 359 34.5 professional experience up to 10 years 468 44.9 11 to 20 years 312 29.9 more than 20 years 262 25.1 area of residence south-east 308 29.6 south 247 23.7 north-east 393 37.7 mid-west 50 4.8 north 44 4.2 marital status single 341 32.7 divorced, separated or widowed 78 7.5 married 625 60.0 professional setting private only 801 76.9 public and private practice 241 23.1 highest education level phd/ms 259 24.9 specialist degree 646 62.0 dds 137 13.1 5 malhão et al. table 2. routine changes in endodontic treatment, volume of patients and economic impact of pandemic n % should the total cost of endodontic treatment change due to pandemic? yes 901 86.5 no 141 13.5 have work routine changes led to increased financial costs of endodontic treatment? no 77 7.4 yes, but prices were not adjusted 663 63.6 yes, and prices were adjusted for patients 302 29.0 number of patients compared with prepandemic period there was not a reduction in the volume of patients 54 5.2 there was a reduction in the volume of patients 988 94.8 expected time, pospandemic, until the number of patients return to previous state up to 1 year 535 51.3 1 year or more 507 48.7 table 3. perception of dental professionals regarding the impact of pandemic considering the different brazilian regions. total south-east south north-east mid-west north p value n (%) which changes in your practice routine were necessary during pandemic? due to pandemic? changes in the protective equipment in endodontic treatment 1010 (96.9) 305 (99.0) 243 (98.4) 384 (97.7) 48 (96.0) 43 (97.7) 0.328 major attention to biosafety measures 1021 (98.0) 302 (98.1) 243 (98.4) 384 (97.7) 48 (96.0) 44 (100) 0.689 longer intervals between dental appointments 922 (88.5) 272 (88.5) 211 (85.4) 359 (91.3) 43 (86.0) 37 (84.1) 0.160 should the total cost of endodontic treatment change due to pandemic? yes 901 (86.5) 266 (86.4) 209 (84.6) 345 (87.8) 44 (88.0) 37 (84.1) 0.804 no 141 (13.5) 42 (13.6) 38 (15.4) 48 (12.2) 6 (12.0) 7 (15.9) have work routine changes led to increased financial costs of endodontic treatment? no 77 (7.4) 23 (7.5) 15 (6.1) 28 (7.1) 4 (8.0) 7 (15.9) 0.546 yes, but prices were not adjusted 663 (63.6) 165 (63.3) 153 (61.9) 255 (64.9) 33 (66.0) 27 (61.4) yes, and prices were adjusted for patients 302 (29.0) 90 (29.2) 79 (32.0) 110 (28.0) 13 (26.0) 10 (22.7) patients flow in comparison with prepandemic period there was not a reduction in the volume of patients 54 (5.2) 16 (5.2) 13 (5.3) 17 (4.3) 3 (6.0) 5 (11.4) 0.546 there was a reduction in the volume of patients 988 (94.8) 292 (94.8) 234 (94.7) 376 (95.7) 47 (94.0) 39 (88.6) expected time, pospandemic, until the number of patients return to previous state up to 1 year 535 (51.3) 162 (52.6) 127 (51.4) 195 (49.6) 26 (52.0) 25 (56.8) 0.878 1 year or more 507(48.7) 146 (47.4) 120 (48.6) 198 (50.4) 24 (48.0) 19 (43.2) 6 malhão et al. discussion the present cross-sectional study reported the perception of brazilian dentists regarding endodontic treatment during the covid-19 pandemic, and its possible economic impact for these professionals, by a qualitative analysis. the study was conducted in the early period of the pandemic in brazil, from may 2nd, 2020 to may 6th, 2020 and at this time a quantitative analysis of losses or percentages was still not possible. the questionnaire was distributed by e-mail and also by several social media platforms (facebook, whatsapp, instagram). unfortunately, it was not possible to calculate the number of professionals reached, as well as the number of dentists in brazil who perform endodontic treatment. even though not knowing the exact number of professionals contacted could represent a methodological failure, it is well recognized that using social media platforms may optimize the number of reached answers in this kind of study. most professionals reported changes in the routine of endodontic treatment, mainly related to the volume of patients assisted and increased costs of dental treatment. despite the reduced number of patients in dental clinics, according to an infodemiological study between march and may in brazil, the volume of tweets related to dental treatment needs increased during the covid-19 pandemic. pain/urgencies and orthodontic treatment were the most common needs6. the routine of dental procedures generates aerosols that characterize a risk to dental care personnel. in this sense, a higher interval between dental appointments is mandatory7. given the high transmissibility of sars-cov-2, dental teams must be attentive to maintain a healthy care environment for patients and themselves8. hygiene and cleaning care need to be increased and controlled routinely; thus, dentists need to adapt their practice and take special precautions during this period9. moreover, in this study, 1021 (98.0%) of respondents reported the need of greater attention to biosecurity measures during dental practice and consequently the higher cost of dental procedures (table 2). as expected, the present results demonstrated that 965 respondents (92.6%) stated that changes in work routine have already increased the financial costs of endodontic treatment; however, surprisingly, the prices have not changed according to 663 participants (63.6%). dentists are spending more and charging the same amount from each patient. it is important to highlight that this whole situation can bring difficulty in managing the dental office in the medium and short term. according to the literature, reducing the number of patients assisted each day in the dental office can be extremely useful to avoid the risk of cross infection10. in this sense, there will be reduced number of people in the waiting room at the same time, enabling a distance of 2 meters between individuals, given that the distance of approximately 1 meter has been established as a risk area11. in addition, the reduction in the number of patients offers a longer time interval between consultations and can provide the team with the necessary time to properly disinfect the clinical area9. this information is recognized by most participants of this study, since more than 88% of respondents reported that the interval between consultations should be longer during the pandemic period. 7 malhão et al. conversely, the reduction in the volume of patients assisted has negative impacts for professionals, considering the reduction in the number of procedures performed per day. in this study, 94% of respondents reported a reduction in the number of patients compared to the pre-covid-19 pandemic period. besides the need of increasing the measures to avoid cross infection, the search and use of dental services has also decreased, due to the fear and anxiety demonstrated by the patients, as well as the financial crisis, with a tendency to migrate to the public service12, evidencing the importance to strengthen it as soon as possible13. there was no association between routine changes and economic impacts with gender, professional experience, area of residence or educational level. this result possibly occurred because the questionnaires were applied in the initial months of pandemic in brazil, thus the impact was similar across groups, since most brazilian areas were in quarantine, and the number of confirmed cases and deaths was also still low14. at that moment, probably a percentage of clinicians had not yet felt the effects of the pandemic in the dental practice. additionally, all brazilian regions were affected similarly along the pandemic period. governments around the world are not only aiming to reduce the virus spread, but also to ease the economic burden of covid-19. it is known that the codiv-19 pandemic has caused an unprecedented challenge for all economic sectors15. in brazil, more than 50% of the population is in the informal economy, according to government data16. this had not occurred since 2007, and consequently it is probable that several dental professionals will be experiencing a strong crisis in the financial sector. one limitation of the present study is the fact that the individual income or family income was not assessed, which would require a longitudinal study. besides, information was obtained only by questionnaires. however, questionnaires are good for gathering data about abstract ideas or concepts that are otherwise difficult to quantify, such as opinions, attitudes and beliefs17. in conclusion, most dental professionals recognized changes in the routine of endodontic treatment during the covid-19 pandemic. they have a perception of increase in endodontic costs, and reduction in the volume of patients. acknowledgements e. c. m.’s work was financed by coordenação de aperfeicoamento de pessoal de nivel superior (capes), brazil. the authors would like to thank all persons who helped to disseminate the campaign on social media and the dental professionals who volunteered to participate. references 1. açikgöz ö, günay a. the early impact of the covid-19 pandemic on the global and turkish economy. turk j med sci. 2020 apr;50(si-1):520-6. doi: 10.3906/sag-2004-6. 2. guerriero c, haines a, pagano m. health and sustainability in post-pandemic economic policies. nat sustain. 2020 jun;17:1. doi: 10.1038/s41893-020-0575-9. 8 malhão et al. 3. mckee m, stuckler d. if the world fails to protect the economy, covid-19 will damage health not just now but also in the future. nat med. 2020 may;26(5):640-2. doi: 10.1038/s41591-020-0863-y. 4. consolo u, bellini p, bencivenni d, iani c, checchi v. epidemiological aspects and psychological reactions to covid-19 of dental practitioners in the northern italy districts of modena and reggio emilia. int j environ res public 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https://pubmed.ncbi.nlm.nih.gov/?term=xia+jj&cauthor_id=32425001 https://pubmed.ncbi.nlm.nih.gov/?term=fu+xh&cauthor_id=32425001 https://pubmed.ncbi.nlm.nih.gov/?term=zhang+yz&cauthor_id=32425001 https://pubmed.ncbi.nlm.nih.gov/?term=schwendicke+f&cauthor_id=32473182 https://pubmed.ncbi.nlm.nih.gov/?term=krois+j&cauthor_id=32473182 https://pubmed.ncbi.nlm.nih.gov/?term=gomez+j&cauthor_id=32473182 https://doi.org/10.1590/0103-1104202012400 https://pubmed.ncbi.nlm.nih.gov/?term=la+rochelle+js&cauthor_id=24661014 https://pubmed.ncbi.nlm.nih.gov/?term=dezee+kj&cauthor_id=24661014 https://pubmed.ncbi.nlm.nih.gov/?term=gehlbach+h&cauthor_id=24661014 1http://dx.doi.org/10.20396/bjos.v20i00.8663859 volume 20 2021 e213859 original article 1 department of restorative dentistry, piracicaba dental school, university of campinas, piracicaba, sp, brazil. 2 general practioner, taubaté, sp, brazil. 3 department of dental clinic, dental school, federal university of bahia, salvador, ba, brazil. corresponding author: vanessa cavalli piracicaba dental school, university of campinas av. limeira, no. 901, areião, 13414-903 piracicaba, sp, brasil cavalli@unicamp.br editor: dr altair a. del bel cury received: january 6, 2021 accepted: march 29, 2021 bond strength of resin composite to enamel submitted to at-home desensitizer and bleaching agents matheus kury1 , bruna guerra silva1 , francine de almeida zandonadi2, leonardo gonçalves da cunha3, vanessa cavalli1,* aim: this study evaluated the effect of a desensitizer agent (des) during bleaching with 10% carbamide peroxide (cp) on enamel microshear bond strength (μsbs). methods: sixty bovine incisors were obtained and randomly distributed into groups (n=15): (c) control: no desensitizing or bleaching, (des) desensitizing gel application, (cp) bleaching with 10% cp and (cp/des) bleaching with 10% cp combined with des. bleaching was performed for 6 h/day for 14 consecutive days. des was applied for 8 h only on the 7th and 14th days of therapy. specimens were stored in artificial saliva among the cp or des applications and submitted to μsbs testing at three postrestoration times (n=5): 24 h, 7 days, and 14 days after bleaching using a universal testing machine. failure modes were observed under a stereomicroscope. data were analyzed by two-way anova and tukey’s test (α=5%). results: immediately after bleaching (24 h), cp promoted lower μsbs than the c and des groups (p<0.05) but with no differences from the cp/des. μsbs increased in the des, cp, and cp/des groups (p<0.05) when bonding was performed for 7 or 14 days elapsed from bleaching. cp/des exhibited the highest μsbs among the groups 14 days after bleaching (p<0.05). cohesive failure in enamel was predominant in the cp groups, while adhesive failure was mostly observed for the other groups. conclusion: the use of a desensitizer during at-home bleaching maintained the enamel immediate bond strength, and its application favored bonding when the restoration was delayed for 14 days. keywords: tooth bleaching. dental enamel. shear strength. http://orcid.org/0000-0002-9971-0568 http://orcid.org/0000-0002-2690-6906 http://orcid.org/0000-0002-9459-1926 2 kury et al. introduction tooth bleaching is a widespread technique commonly performed and desired by patients due to its conservative approach and efficacy in providing color alterations1,2. carbamide (cp) and hydrogen (hp) peroxides are active compounds of bleaching agents, and their concentrations vary according to the bleaching technique3. in-office bleaching uses high concentrations of cp or hp (higher than 25%), while the home-applied technique uses lower cp (up to 22%) or hp (up to 10%) concentrations. although a highly concentrated agent could accelerate the bleaching effect, both methods achieve acceptable outcomes1,3. studies have reported that at-home bleaching resulted in mild tooth sensitivity4, but a recent systematic study revealed that the high variation among bleaching protocols could not confirm that at-home bleaching yielded a lower risk or intensity of sensitivity5. therefore, the management of this clinical symptom still represents a challenge during tooth bleaching, even when performed at lower concentrations. in vitro  studies have reported an increase in enamel surface roughness6 and a decrease in microhardness7 and immediate enamel bond strength8 following at-home bleaching. these alterations to the enamel surface could result in pore formation and consequent entrapment of oxygen residuals released from the peroxide agents, which could, in turn, negatively interfere with the light curing of the adhesive agent. poor light curing could lead to a decreased number, dimension, and quality of the resin tags in the etched enamel9. however, despite the cause, enamel bond strength may recover by surface remineralization promoted by saliva or remineralizing solutions10,11. although several in vitro studies proposed applying antioxidant agents to reverse the immediate bond strength of bleached enamel12-14, no randomized clinical trials attest to the long-term performance of restorations placed immediately after bleaching with the antioxidant application. consequently, delaying the bonding procedures to up to 3 weeks still seems to be an effective method to guarantee satisfactory restorative results after tooth bleaching10. in this scenario, studies investigating the effect on the enamel bond strength of potassium nitrateand sodium fluoride–based desensitizer use before, during, or after tooth bleaching are scarce15. although desensitizer application could be a feasible solution to overcome tooth sensitivity16 and maintain the resin composite bond strength to enamel due to its composition, a study showed that the application of a desensitizer containing potassium nitrate and sodium fluoride reduced the enamel bond strength when the bonding procedure was performed immediately after bleaching17. the authors credited the bonding reduction to the fluoride deposition on the surface, thereby compromising the adhesion between resin and teeth17. however, it is necessary to determine if a desensitizer agent influences the enamel bond strength even in delayed bonding procedures. based on these facts, this study evaluated the enamel bond strength at different postrestorative times after home-applied bleaching combined or not with a desensitizer agent. the null hypothesis was that the use of home-applied bleaching combined with a desensitizer agent would not influence enamel bond strength immediately or after 7 and 14 days of bleaching treatment. 3 kury et al. material and methods experimental design. sixty bovine incisors were submitted to the factors (n=5): 1. treatment (four levels): • (c) control: no bleaching or desensitizing treatment; • (des) enamel application of a desensitizer agent containing 0.25% sodium fluoride and 3% potassium nitrate (ultra ez, ultradent products inc., south jordan, ut, usa); • (cp) enamel bleaching with 10% cp (opalescence 10%, ultradent products); • (cp/des) enamel bleaching with 10% cp combined with the application of des. 2. time elapsed after bleaching (three levels): • (24 h) immediately; • (7d) 7 days; • (14d) 14 days. the variable responses evaluated were microshear bond strength (μsbs, in mpa) and fracture mode analysis, observed at 40 χ magnification. table 1 displays the composition of the materials used. table 1. description of the bleaching and restorative products used in the study product and manufacturer composition application mode* (*according to manufacturer) ultra ez (ultradent products inc., south jordan, ut, usa) sodium hydroxide, sodium fluoride and potassium nitrate the recommended treatment times can range from 15 min to 1 h. the length and number of times depend on condition, patient and clinician. 10% opalescence (ultradent products) carbamide peroxide, sodium hydroxide and poly(acrylic acid) wear opalescence 10% gel 8-10 hours or overnight. alternatively, treatments can be from 15 minutes to several hours/day, depending on the patient’s needs, level of sensitivity and day-to-day activities. ultra etch (ultradent products) phosphoric acid, cobalt blue spinel aluminate and siloxane apply etchant to enamel and dentin (15 s). rinse thoroughly, dry and proceed per adhesive manufacturer’s instructions. adper single bond 2 adhesive (3m oral care, st paul, mn, usa) ethyl alcohol, bisgma, hema, udma, edmab, treated silica, glycerol 1,3-dimethacrylate, copolymer of acrylic acid and itaconic acid, water, diphenyliodonium hexafluorophosphate. immediately after blotting, apply 2-3 consecutive coats of adhesive to etched enamel for 15 s with gentle agitation using a fully saturated applicator. gently air thin for 5 s evaporate solvents. light cure for 10 s. filtek z350 xt flow (3m oral care) certified silanized ceramics, dimethacrylate, bisgma, edmab, tegdma, silane treated silica, yttrium fluoride (ybf3), polycaprolactone reacted polymer, benzotriazole, diphenyliodonium hexafluorophosphate. place and light cure restorative in increments (20 s for each 2.0 mm increment). bis-gma: bisphenol a diglycidyl ether dimethacrylate; hema: 2-hydroxyethyl methacrylate; udma: diurethane dimethacrylate; edmab: ethyl 4-dimethylaminobenzoate; tegdma: triethylene glycol dimethacrylate. 4 kury et al. specimen preparation. sixty extracted bovine incisors, stored in 0.1% thymol solution, were cleaned with periodontal scalers, and the remaining debris was removed with a bicarbonate jet. the roots were cut at the amelocement junction, and enamel/ dentin blocks (5 x 5 mm and 3 mm of thickness) were obtained from the central area of the crown using a double face diamond disc (kg sorensen, barueri, sp, brazil). the blocks were embedded in polystyrene resin, and the buccal surface was flattened with #600, 800, 1200-grit silica carbide papers (3m espe, sumaré, sp, brazil) and polished with 6, 3, 1, and ¼ μm diamond pastes in a polishing machine (arotec, cotia, sp, brazil). bleaching and desensitizing treatments. specimens were randomly distributed into four groups according to bleaching and desensitizing treatments as described previously. a 1-mm layer of the bleaching agent (10% cp) was applied on the exposed enamel surface of groups cp and cp/des for 6 h/day for 14 days. during bleaching, specimens were stored underneath moisture gauze at 370c and relative humidity. after bleaching, specimens were thoroughly rinsed with deionized water and stored in artificial saliva [1.5 mm calcium (cacl2), 0.9 mm phosphate (nah2po4), 0.15 mm potassium chloride (kcl)], adjusted to ph 7.0 and 3.125 ml of solution for each mm2 of exposed enamel until the next gel application18,19. the desensitizer agent in the cp/des group was applied over the enamel, similar to the bleaching gel, for 8 h. however, desensitizer application was only performed on the 7th and 14th days of treatment. specifically, in the cp/des group, des application was carried out after the bleaching procedure. after treatments, the specimens were thoroughly rinsed with deionized water and stored in artificial saliva. desensitizer gel was also applied on the 7th and 14th days in the des group, but no bleaching agent was applied to the specimens; instead, they were kept in artificial saliva in the remaining hours. in the c group, neither bleaching nor desensitizing treatment was performed; specimens were kept in artificial saliva throughout the 14 days of treatment. the artificial saliva was replaced daily. restorative procedure. the specimens were submitted to restorative procedures according to the posttreatment times (n=5): 24 h, 7 d, and 14 d. at each posttreatment time, three composite cylinders were built upon the enamel surface. enamel was etched with 37% phosphoric acid gel (ultra etch, ultradent products inc., south jordan, ut, usa) for 15 s, rinsed with distilled water and air-dried. one-bottle adhesive (adper single bond, 3m oral care, st paul, mn, usa) was applied according to the manufacturer’s instructions (table 1) and light-cured for 20 s. a silicone matrix mold with a cylindrical configuration (0.75 mm diameter by 2.0 mm high) was placed over the treated surfaces and filled with composite resin (filtek z350 flow – 3m oral care, st paul, mn, usa) using a composite instrument (# 1/2, duflex ss white, rio de janeiro, rj, brazil). the composite was light-cured for 20 s, and the matrix molds were removed to expose the resin composite cylinders bonded to the enamel surfaces. thus, three bonded small resin cylinders were obtained for each specimen. composite cylinders were checked under an optical microscope (40c) (emz-tr, meiji techno co., saitama, japan) to evaluate the composite and interface integrity. cylinders with no interfacial defects, bubble inclusion or leaking of the composite were tested. the restored specimens were stored in artificial saliva at 37 °c for 24 h. 5 kury et al. microshear bond strength testing. specimens were fixed to the tested device with cyanoacrylate glue, and the bond strength of each composite cylinder was evaluated in a universal testing machine (4411/instron corp, canton, ma, usa). the shear load (50 n) was applied to the base of the composite cylinder with a thin orthodontic wire (0.2 mm diameter) at a crosshead speed of 0.5 mm/min until failure. the μsbs was calculated and expressed in mpa. three bond strength measurements were recorded, and the μsbs mean was obtained for each specimen. failure mode. the enamel failure modes (%) were analyzed with a stereomicroscope at 40χ magnification (emz-tr, meiji techno co., saitama, japan) and classified as follows: adhesive in enamel (ad), cohesive in enamel (coe), cohesive in resin (cor) and mixed (mix) failures involving ad coe or ad cor. statistical analysis. the normal distribution of the values was verified by kolmogorov-smirnov and lilliefors tests (p>0.05), and a parametric analysis was performed. data were analyzed by two-way anova, according to the factors  treatment  and elapsed  time  following bleaching, and tukey’s test with the significance level set at 5%. sas 9.0 software was used for all tests (sas institute, cary, nc, usa). results table 2 displays the results of the μsbs test. immediately after bleaching (24 h), cp promoted lower μsbs than the c and des groups (p<0.05) but with no differences from cp/des. seven days after bleaching, the c group exhibited the lowest μsbs values (p<0.05), and des, cp, and cp/des displayed no significant differences (p>0.05). in addition, at this time point (7 d), the μsbs of the des, cp, and cp/des groups increased in comparison to that of the immediate (24 h) corresponding groups (p<0.05). fourteen days elapsed from bleaching, cp/des exhibited the highest μsbs among groups, with no significant differences between the 7th and 14th days elapsed from treatment (p>0.05). in addition, no differences were detected among c, des, and cp at this time point (14 d, p>0.05). c exhibited no bond strength differences, regardless of the evaluation time (p>0.05). the des and cp groups produced higher μsbs at the 7th and 14th days elapsed from bleaching than at the immediate time point. table 2. μsbs and standard deviation (mpa) values of bleached and/or desensitized teeth restored at three different elapsed times. groups 24 h 7d 14d c 11.10 (4.28) aa 10.53 (3.39) ba 11.29 (3.42) ba des 10.65 (3.10) ab 14.58 (2.48) aa 13.76 (3.70) ba cp 7.99 (3.93) bb 14.49 (4.78) aa 13.65 (3.69) ba cp/des 9.13 (3.16) abb 13.52 (4.94) aa 17.00 (3.61) aa according to 2-way anova and tukey’s test, means followed by different letters differ statistically at 5% uppercase letters compare the different bleaching protocols within the same evaluation time, and lowercase letters compare the same bleaching protocol within the evaluation times. 6 kury et al. figure 1 depicts the failure modes (in %) of the tested groups according to the posttreatment evaluation times. adhesive failures (ad) and cohesive failures at enamel (coe) were the most predominant failures found, regardless of the treatment or the evaluation time. adhesive failures (ad) were predominantly observed for the c (40 60%) and des (30 – 50%) groups, while cohesive failures (coe) were observed for the cp (60 70%) and cp/des (40 – 60%) groups, regardless of the postevaluation times. mixed failures (mix) were detected in all groups (10 30%), and cohesive failure at the resin was detected for des, cp (7 days), and cp/des (14 d) at 10%. immediate failure mode c des cp cp/de 100 90 80 70 60 50 40 30 20 10 0 failure mode after 7 days c des cp cp/de 100 90 80 70 60 50 40 30 20 10 0 failure mode after 14 days adhesive cohesive at enamel cohesive at resin mixed c des cp cp/de 100 90 80 70 60 50 40 30 20 10 0 figure 1. failure mode distribution (%) of the groups according to the postponement of bonding (24 h – immediate, 7 d and 14 d). c: control; des: desensitizer; cp: carbamide peroxide; cp/des: carbamide peroxide and desensitizer. 7 kury et al. discussion a potassiumand nitrate-based desensitizer combined with cp maintained the enamel μsbs and displayed values higher than those of the other groups when bonding was performed 14 days after bleaching. in addition, the desensitizer alone promoted higher μsbs than the control on the 7th day of bonding postponement. therefore, the results rejected the null hypothesis because this protocol influenced the enamel μsbs after bleaching. the fluoride effect in the remineralization process could explain this outcome20. the addition of ca and f to low-concentrated cp has been demonstrated to control enamel mineral loss during bleaching21,22. these findings support the theory that the fluoride in the desensitizer maintained enamel integrity and hence the bond strength. contrary to these findings, a report from amuk et al.17 (2018) showed that the application of a desensitizer (ultraez) during 22% cp bleaching decreased the bond strength. the authors stated that desensitizer deposition weakened the adhesive interface. the differences in both studies could rely on the different cp concentrations, the frequency of the desensitizer applications, and mainly by the time elapsed from the bleaching and bonding procedures. in that study, only immediate bond strength was carried out, and the desensitizer was applied daily for 14 days17. therefore, divergences in the protocols might impact the effects of desensitizers on enamel bonding. even though bleaching with low-concentrated cp decreased immediate enamel μsbs20, the postponement (7 or 14 days) of bonding reversed that result. the oxygen release and entrapment in the structures during bleaching may decrease enamel/dentin bond strength23. in addition, hp could interfere with enamel morphology due to the inorganic content changes produced during bleaching24. these have been the rationale for replacing restorations on bleached teeth only after a few weeks3. although the desensitizing gel did not increase the immediate bond strength, the results suggested that the combination of cp with a fluorideand potassium nitrate-containing desensitizer favored bonding in the case of the restoration’s postponement. thus, the 10% cp protocol combined with des would benefit the enamel μsbs 14 days elapsed from bleaching. fluoride interacts in the tooth demineralization process when chemically soluble, increasing remineralization and reducing mineral loss20. the presence of fluoride ions during an acidic challenge, promoted by a solution with ph 6.5, leads to the exchange of ca of hydroxyapatite (ha) for fluoride, thereby forming fluorapatite (fa). as fa has a lower solubility and dissociation constant than ha, the ph will have to drop more dramatically (approximately at 4.5) to demineralize enamel25. therefore, it is likely that the fluoride in the desensitizer gel controlled the μsbs of bleached enamel similarly to fluoride-containing bleaching agents controlling enamel mineral content22. it is worth mentioning that bleaching gels dispensed in syringes often display neutral or even acidic ph to extend the product’s shelf life1. moreover, ph may drop over the gel’s application26. therefore, contact of the enamel surface with a fluoride-containing desensitizer after acidic challenges may have reversed this condition. several studies have investigated enamel bond strength after at-home and in-office bleaching treatment10-13. cavalli et al.10 (2001) evaluated the enamel bond strength 8 kury et al. of an etch-and-rinse adhesive after bleaching with cp gels (10 to 20%) for ten days. the authors concluded that bond strength was recovered three weeks elapsed from bleaching and artificial saliva storage14. in contrast, others27 reported that enamel treated with 10% cp and restored with self-etching adhesive exhibited similar bond strength to the control group 24 h after bleaching. furthermore, bond strength after 35% hp bleaching presented similar values to untreated enamel after one week27. such discrepancies between studies14,27 could rely on differences in bonding and bleaching procedures. nevertheless, no consensus exists regarding shear bond strength for bleached teeth bonded with self-etching adhesive9. the predominance of cohesive failures in enamel (coe) in the cp groups at all times corroborates the action of hp byproducts on enamel. the decrease in enamel strength was previously reported even after cp treatment24. according to that study, cp produced morphological alterations, with loss of enamel prism core, decreasing enamel cohesive strength24. in addition, the predominance of adhesive failures of the cp/des group at 7 and 14 days after bleaching indicates an increase in enamel resistance to fracture, which was probably promoted by remineralization. conversely, faria de lacerda et al.28 (2016) observed that 0.05% sodium fluoride remineralized enamel exhibited mixed failure modes for etch-and-rinsed and self-etched restorations. that study observed that the μsbs of the f-treated group bonded with the self-etching system was lower than that of the control group28. this contrast may be explained by the 8-week fluoride daily application, which may have hypermineralized the enamel. in summary, the μsbs behavior of bleached enamel and its recovery or even increase within the following weeks reaffirms previous data10-14. due to fluoride incorporation in the tooth structure, the desensitizer may have enhanced enamel bond strengths. since esthetic dental treatments frequently begin by tooth bleaching, these findings suggest that desensitizer application during bleaching could favor enamel bond strength of restorative procedures when postponed for two weeks. the manufacturer recommends the desensitizer’s application from 15 min to 1 h, but the frequency depends on the tooth condition, patient sensitivity, and professional decision (table 1)29. because of that recommendation, several application times can be found in the literature (from 15 min30 to overnight use31). in this study, the desensitizer’s gel application occurred on the 7th and 14th days of bleaching for 8 h without intervals, which corresponds to 1 h 15 min of daily use. however, the prolonged application adopted is a limitation of this in vitro design and cannot be extrapolated to a clinical situation because of the risks of swallowing the desensitizer. furthermore, authors have attested32 that the shear bond test also holds limitations because shear stress concentrates where the load is applied, causing an uneven tension distribution and a higher % of cohesive failures within the resin composite (cor), underestimating the μsbs values. however, all groups exhibited a lower % of cor (10-30%), contradicting this idea and supporting the μsbs test performed. clinically, a desensitizer agent containing fluoride and potassium nitrate attenuates the risk and intensity of tooth sensitivity19 during bleaching by obstructing dentin tubules and preventing depolarization of nerve fibers33. even though this study presents the inherent limitation of an in vitro design, it demonstrated that the desensitizer 9 kury et al. might positively impact the bond strength of postponed procedures. further investigations could attempt different protocols to determine an acceptable effect on immediate enamel bonding. in conclusion, the combination of a desensitizer agent with bleaching gel (10% cp) upheld the enamel bond strength immediately after bleaching. the protocol combining low-concentrated cp with a desensitizer agent presented the highest bonding performance under a 14-day postponement. references 1. rodríguez-martínez j, valiente m, sánchez-martín mj. tooth whitening: from the established treatments to novel approaches to prevent side effects. j esthet restor dent. 2019 sep;31(5):431-40. doi: 10.1111/jerd.12519. 2. llena c, villanueva a, mejias e, forner l. bleaching efficacy of at-home 16% 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appl oral sci. 2018 oct; 4(26):e20170589. doi: 10.1590/1678-7757-2017-0589. 23. spyrides gm, perdigão j, pagani c, spyrides sm. effect of whitening agents on dentin bonding. j esthet dent. 2000;12(15):264-70. doi:10.1111/j.1708-8240.2000.tb00233.x 24. cavalli v, giannini m, carvalho rm. effect of carbamide peroxide bleaching agents on tensile strength of human enamel. dent mater. 2004 oct;20(8):733-9. doi: 10.1016/j.dental.2003.10.007. 25. pitts nb, zero dt, marsh pd, ekstrand k, weintraub ja, ramos-gomez f, et al. dental caries. nat rev dis prim. 2017 may;25;3:17030. doi: 10.1038/nrdp.2017.30. 26. cavalli v, silva bgd, berger sb, marson fc, tabchoury cpm, giannini m. decomposition rate, ph, and enamel color alteration of at-home and in-office bleaching agents. braz dent j. 2019 jul;22;30(4):385-96. doi: 10.1590/0103-6440201902484. 27. unlu n, cobankara fk, ozer f. effect of elapsed time following bleaching on the shear bond strength of composite resin to enamel. j biomed mater res b appl biomater. 2008 feb;84(2):363-8. doi: 10.1002/jbm.b.30879. 28. farias de lacerda aj,  ferreira zanatta r, crispim b, borges ab, torres crg, tay fr, et al. influence of de/remineralization of enamel on the tensile bond strength of etch-and-rinse and self-etching adhesives. am j dent. 2016 oct;29(5):289-93. 29. ultradent products. ultraez desensitizing gel instructions. south jordan: ultradent; 2011 [cited 2020 nov 16]. available from: https://assets.ctfassets.net/wfptrcrbtkd0/6jde30pbmbbqxnxnpokqzs/4f05 fccb574be7a2a0cf5089fde77de6/ultraez-syringe-kits-ifu-10121.15.pdf. 30. kyaw ky, otsuki m, hiraishi n, segarra ms, tagami j. effect of application of desensitizers before bleaching on change of tooth shade. dent mater j.  2019 oct;38(5):790-7. doi: 10.4012/dmj.2018-129. https://www.ncbi.nlm.nih.gov/pubmed/?term=zero dt%5bauthor%5d&cauthor=true&cauthor_uid=28540937 https://www.ncbi.nlm.nih.gov/pubmed/?term=marsh pd%5bauthor%5d&cauthor=true&cauthor_uid=28540937 https://www.ncbi.nlm.nih.gov/pubmed/?term=cobankara fk%5bauthor%5d&cauthor=true&cauthor_uid=17626292 https://www.ncbi.nlm.nih.gov/pubmed/?term=ozer f%5bauthor%5d&cauthor=true&cauthor_uid=17626292 https://www.ncbi.nlm.nih.gov/pubmed/?term=ferreira zanatta r%5bauthor%5d&cauthor=true&cauthor_uid=29178743 https://www.ncbi.nlm.nih.gov/pubmed/?term=crispim b%5bauthor%5d&cauthor=true&cauthor_uid=29178743 https://assets.ctfassets.net/wfptrcrbtkd0/6jde30pbmbbqxnxnpokqzs/4f05fccb574be7a2a0cf5089fde77de6/ultraez-syringe-kits-ifu-10121.15.pdf https://assets.ctfassets.net/wfptrcrbtkd0/6jde30pbmbbqxnxnpokqzs/4f05fccb574be7a2a0cf5089fde77de6/ultraez-syringe-kits-ifu-10121.15.pdf 11 kury et al. 31. türkkahraman h, adanir n. effects of potassium nitrate and oxalate desensitizer agents on shear bond strengths of orthodontic brackets.  angle orthod.  2007 nov;77(6):1096-100. doi: 10.2319/101906-431.1 32. tribst jpm, anami lc, özcan m, botino ma, melo rm, saavedra gsf. self-etching primers vs acid conditioning: impact on bond strength between ceramics and resin cement. oper dent. 2018 jul/ago;43(4):372-9. doi: 10.2341/16-348-l. 33. alencar cm, pedrinha vf, araújo jln, esteves ra, silva da silveira ad, silva cm. effect of 10% strontium chloride and 5% potassium nitrate with fluoride on bleached bovine enamel. open dent j. 2017 aug;31(11):476-84.doi: 10.2174/1874210601711010476. https://doi.org/10.2319/101906-431.1 https://www.ncbi.nlm.nih.gov/pubmed/?term=alencar cm%5bauthor%5d&cauthor=true&cauthor_uid=28979576 https://www.ncbi.nlm.nih.gov/pubmed/?term=pedrinha vf%5bauthor%5d&cauthor=true&cauthor_uid=28979576 https://www.ncbi.nlm.nih.gov/pubmed/?term=ara%c3%bajo jln%5bauthor%5d&cauthor=true&cauthor_uid=28979576 https://www.ncbi.nlm.nih.gov/pubmed/?term=effect+of+10%25+strontium+chloride+and+5%25+potassium+nitrate+with+fluoride+on+bleached+bovine+enamel https://www.ncbi.nlm.nih.gov/pubmed/?term=effect+of+10%25+strontium+chloride+and+5%25+potassium+nitrate+with+fluoride+on+bleached+bovine+enamel 1 volume 21 2022 e228852 original article braz j oral sci. 2022;21:e228852http://dx.doi.org/10.20396/bjos.v21i00.8668852 1 department of restorative dentistry, são leopoldo mandic college, são leopoldo mandic research institute, campinas, sp, brazil. corresponding author: fabiana mantovani gomes frança faculdade são leopoldo mandic, instituto de pesquisas são leopoldo mandic rua josé rocha junqueira, 13 – swift, campinas, sp, 13045-755, brazil telephone/fax: 55-19 3211-3604 phone: 55-19 9112-1764 e mail: biagomes@yahoo.com editor: dr. altair a. del bel cury received: march 31, 2022 accepted: june 9, 2022 influence of restorative materials on occlusal and internal adaptation of cad-cam inlays ana carolina rodrigues cabral1 , waldemir francisco vieira junior1 , roberta tarkany basting1 , cecília pedroso turssi1 , flávia lucisano botelho do amaral1 , fabiana mantovani gomes frança1,* aim: to evaluate the occlusal and internal marginal adaptation of inlay restorations made of different materials, using cad-cam. methods: preparations were made for mod inlays of one-third intercuspal width and 4 mm depth in 30 third human molars. the teeth were restored using cad-cam materials (n=10) of nanoceramic resin (lava ultimate), polymer-infiltrated ceramic network (vita enamic), or lithium disilicate glass-ceramic (ips e.max cad). the specimens were cemented with dual resin cement and sectioned at the center of the restoration, after which the two halves were evaluated, and photographed the occlusal and internal discrepancy (µm) was determined at five points: cavosurface angle of the occlusal-facial wall (ca-o); center of the facial wall (fw); faciopulpal angle (fpa); center of the pulpal wall (pw); and center of the lingual wall (lw). the data were submitted to the kruskal-wallis and the dunn tests (α=0.05). results: no difference was observed among the materials regarding the occlusal discrepancy at the ca-o, fpa, or pw internal points. the e.max cad measurement at fw showed larger internal discrepancy than that of lava (p=0.02). the internal discrepancy at lw was greater for e.max cad than vita enamic (p=0.02). conclusion: lithium disilicate glass-ceramic presented greater internal discrepancy in relation to the surrounding walls of the inlay preparations. keywords: dental marginal adaptation. ceramics. computeraided design. dental materials. inlays. https://orcid.org/0000-0002-8160-5452 https://orcid.org/0000-0001-8226-3100 https://orcid.org/0000-0002-5345-5776 https://orcid.org/0000-0002-0078-9895 https://orcid.org/0000-0002-8934-6678 https://orcid.org/0000-0002-2877-6797 2 cabral et al. braz j oral sci. 2022;21:e228852 introduction indirect restorations are indicated in clinical situations where there is loss of coronary dental structure, and difficulty in obtaining contour and an interproximal contact point, or where anatomical shape must be addressed. indirect restorations are durable, have adequate strength, and maintain their aesthetic quality1. direct restorative procedures are based on inserting composite resin directly into the cavity to reestablish the dental anatomic form; however, indirect restorations are manufactured out of the mouth, and the polymerization stress is restricted to the resin cement, used in a lower layer. this advantage can increase the survival rate of the restoration, and is especially important for posterior teeth, which must support chewing forces2. nevertheless, issues exist in conventional inlay manufacturing techniques, such as the delay in making the inlay, and the difficulty in molding the silicone and casting the models, factors which could cause greater distortion of the mold, and hinder the mounting of the articulator3. the development of computer-aided design and computer-aided manufacturing (cad-cam) technology makes it easier to perform indirect restorations, because the restoration can be designed and manufactured2. cad-cam systems can be used with several types of materials, including ceramics, resin composites and hybrids. resin-based materials have been developed for cad-cam technology with a high degree of homogeneity4. hybrid materials can consist of a combination of ceramics, polymers and lithium disilicate reinforced zirconia5. polymeric materials are optimally indicated for indirect intracoronary restorations, owing to their adhesion to the dental structure, mechanical characteristics, and elastic recovery6,7. the biomechanical behavior of teeth and their interface with cad-cam inlay restorations have not been fully investigated. the adaptation of unitary prostheses or partial restorations can affect their clinical success and survival rates8. the clinically acceptable marginal discrepancy ranges from 100 to 150  µm, whereas previous studies9,10 have suggested that the fit of cad-cam restorations may produce a marginal discrepancy of less than 80 µm. thus, cad-cam systems could improve the average fitting quality of prostheses more than conventional manufacturing methods, and investigations should be conducted to disseminate and popularize these findings among professionals worldwide. the marginal adaptation of materials impacts clinical outcomes and failure rates, considering that any spaces or gaps left in the adhesive or cement may promote biofilm accumulation and marginal pigmentation, and lead to long-term degradation9. the adaptation of indirect restorations is more commonly studied in the cervical region, and the occlusal and axial walls. it is known that the smaller the gap in the cervical region, the lower the risk of gum irritation, microleakage, and secondary caries lesions11. in addition, better internal fit of the prosthetic parts improves the mechanical performance of restorations, by imparting strength and retention12. considering the relevance of evaluating the marginal and internal adaptation of polymeric materials produced by the cad-cam method8,9, the objective of this study was to evaluate the occlusal and internal adaptation of intracoronary indirect restorations made from different materials, specifically lithium disilicate glass-ceramic, hybrid ceramic, and composite resin, all using cad-cam technology. the null hypothesis 3 cabral et al. braz j oral sci. 2022;21:e228852 tested was that the materials used for making inlays would not differ in terms of occlusal and internal fit. materials and methods experimental design this study was submitted to and approved by the institutional research ethics committee (caae: 69083117.5.0000.5374). the factors under study involved indirect restoration materials at three levels: nanoceramic resin (lava ultimate cad-cam restorative for cerec 3m espe, st. paul, mn, usa); polymer-infiltrated ceramic network (vita enamic cad-cam for cerec inlab, vita zahnfabrik, bad säckingen, germany); and lithium disilicate glass-ceramic (ips e.max cad, ivoclar vivadent, schaan, liechtenstein). the occlusal and internal marginal discrepancy was determined with images evaluated by image j software. the experimental units consisted of 30 third molars restored with mesial-occlusal-distal (mod) inlays produced by cad-cam. table 1 presents the composition of the materials studied, and figure 1 illustrates the study design and the sequence of the procedures. table 1. composition, manufacturer and lot of main materials used in the study materials, manufacturer, and lot composition lava ultimate 3m espe st. paul, mn, usa lot: 1727700558 & 1635400334 20 nm silica filler, 4–11 nm zirconia filler, 80 wt% bis-gma, udma, tegdma, bis-ema vita enamic vita zahnfabrik bad säckingen, germany lot: 40970 & 48001 sio2, al2o3, na2o, 86 wt% udma, tegdma ips e.max cad ivoclar vivadent schaan, liechtenstein lot: w12668 sio2 (57 80% by weight), li2o (11 to 19% by weight), k2o, mgo, al2o3, p2o5, and other components single bond universal 3m/espe st. paul, mn, usa lot: 1702700590 bis-gma, hema, sio2, and ethyl alcohol hydrofluoric acid maquira maringa, pr, brazil lot: 587417 10% hydrofluoric acid, thickener, dye, and purified water silane maquira maringa, pr, brazil lot: 7 898561 540287 silane and ethanol relyx ultimate adhesive resin cement 3m/espe st. paul, mn, usa lot: 652581 paste 1: methacrylate monomers, radiopaque silanated fillers, initiator components, stabilizers, rheological additives. paste 2: methacrylate monomers, radiopaque alkaline fillers, initiator components, stabilizers, pigments, rheological additives, fluorescence dye, dark polymerize activator for scotchbond universal adhesives. legend: sio2 (silicon dioxide); li2o (lithium oxide); k2o (potassium oxide); mgo (magnesium oxide); al2o3 (aluminum oxide); p2o5 (phosphorus pentoxide); bis-gma (bisphenol a-glycidyl methacrylate), na2o (sodium oxide); udma (urethane dimethacrylate); tegdma (triethylene glycol dimethacrylate); hema (2-hydroxyethyl methacrylate). https://multimedia.3m.com/mws/media/783784o/3m-relyx-ultimate-adhesive-resin-cement-technical-data-sheet.pdf https://pubchem.ncbi.nlm.nih.gov/compound/lithium-oxide 4 cabral et al. braz j oral sci. 2022;21:e228852 sample selection and cavity preparation thirty healthy third molars were obtained, scraped with periodontal curettes, and stored in 0.1% thymol solution. all the teeth were radiographed to establish the distance between the central groove and the pulp chamber ceiling. the total tooth length was recorded, as well as the crown length, the root length, the mesiodistal and buccolingual diameters of the crown, and the crown height. all these measurements were taken to make it easier to obtain equal distribution among the experimental groups, and ultimately ensure that all the groups would be composed of the same proportion of teeth of the same dimensions. the teeth were embedded in condensation silicone (zetaplus zhermack, badia polesine, italy) to allow adaptation to a cavity preparation machine. legend: a) class ii cavity preparation; b) scan of preparations; c) inlay milling; d) inlays made; e) polishing of inlays; f) tooth prepared with phosphoric acid, active application of universal adhesive and photoactivation; g) lava inlay prepared with aluminum oxide blasting followed by application of universal adhesive. vita enamic and e.max cad inlays prepared by conditioning with 10% hydrofluoric acid gel, and application of silane and universal adhesive; h) inlay ready for cementation; i) cementation of inlays. figure 1. study design. cad-cam inlay manufacturing and cementation. a b c d e gf h i lava ultimate vita enamic ips e.max 5 cabral et al. braz j oral sci. 2022;21:e228852 mod cavities (class ii) of one-third intercuspal width and 4 mm depth were made by a cavity preparation machine (elquip, são carlos, sp, brazil), without a proximal box, using high rotation under abundant water irrigation, and a tapered trunk diamond tip (#2096 kg sorensen, cotia, sp, brazil). each diamond tip was used for three preparations, and then discarded. the #3131 diamond tip (kg sorensen, cotia, sp, brazil) for intracoronary preparations was then used to determine the required cavity size. all the samples were stored individually in pots immersed in distilled water, and distributed randomly for restoration with the three materials studied (n=10). cad-cam inlay manufacturing all the preparations were scanned directly (cerec blue cam, sirona dental, benshein, hessen, germany), and both the designs and the drawings of each inlay were made separately, considering a cement space of 100 µm13. then, the inlays were milled according to the experimental group (nanoceramic composite resin, hybrid ceramic or lithium disilicate glass-ceramic). the inlays of all the materials were made by an experienced professional, after which the materials were polished with abrasive rubber of two granulations (vita enamic polishing set clinical, vita zahnfabrik, bad säckingen, germany) for 5 seconds on the occlusal face, 5 seconds on the mesial face, and 5 seconds on the distal face, after which a medium-grain, followed by a fine-grain, rubber tip was applied for 5 seconds respectively on each side. the restorations were submitted to ultrasound individually for 3 minutes. afterwards, they were polished with abrasive paste (diamond excel, fgm dental group, santa catarina, brazil) for 5 seconds on each outer face, and again submitted to ultrasound for 3 minutes. only lithium disilicate inlays were submitted to single-glaze firing and then oven-crystallized, according to the manufacturer’s parameters (820oc for 10 min, ivoclar programat ep 3000, ivoclar vivadent, schaan, liechtenstein). cementation of inlays the teeth were treated the same way in all the groups. the enamel was conditioned selectively for 15 seconds, then washed with water for 15 seconds, and dried gently, making sure to keep the dentin moist. universal adhesive was applied (single bond universal 3m espe, st. paul mn, usa) for 20 seconds, followed by light air-blasting for 5 seconds, and photoactivation for 10 seconds. the internal treatment of the inlays depended on each individual material, as described in table 2. all the inlays were cemented with dual resin cement (rely x ultimate 3m espe, st. paul, mn, usa) using finger pressure14 for 15 seconds. excess cement was removed, and photoactivation was performed with led light, at a minimum irradiance of 1000 mw/cm2 (bluephase ivoclar, vivadent, schaan, liechtenstein) for 1 minute. after cementation of the inlays, the margins were polished using the same polishing sequence and application time on each face, as previously described. 6 cabral et al. braz j oral sci. 2022;21:e228852 occlusal and internal adaptation at the tooth/restoration interface the specimens were placed on a precision cutter (isomet buhler) and sectioned in the central region toward the lingual vestibule. each half of the dental element was photographed with a digital camera (sony α-200, sony, japan), using a standardized procedure, and a 105  mm lens (sigma lens for sony, sigma corporation, japan). the camera was docked at a height of 10 cm between the lens and the sample. digital camera specifications were also standardized as follows: firing speed of 1/125, diaphragm opening at f16, iso 200 sensor sensitivity, manual function with the flash triggered. the images of the two halves of each tooth were introduced in the image j software (image processing and analysis in java, bethesda, md, usa)15, and the average cement space gap of each half was calculated per sample. the occlusal and internal discrepancy, or cement space (µm), was determined16 at five points: ca-o (occlusal fit); center of the facial wall (fw, internal fit); faciopulpal angle (fpa, internal fit); center of the pulpal wall (pw, internal fit); and center of the lingual wall (lw, internal fit) (figure 2). statistical analysis exploratory analysis indicated that the data did not meet the assumptions of parametric analysis, and kruskal wallis and dunn tests were performed considering a significance level of 5%. the analyses were performed using r* software (r core team, r foundation for statistical computing, vienna, austria). table 2. sequential steps of internal inlay treatments material internal treatment of inlays nanoceramic resin (lava ultimate) 1clean the restoration ultrasonically for 3 minutes and dry by air blast. 2blast with aluminum oxide (50 μm to 30 psi) until each inner surface is matte. 3apply alcohol to remove the excess from blasting. 4actively apply universal adhesive (single bond universal 3m espe, st. paul mn, usa) for 20 seconds. 5light air blasting. hybrid ceramic (vita enamic) 1condition with 10% hydrofluoric acid gel (maquira, maringa, pr, brazil) for 60 seconds. 2wash with water for 30 seconds. 3apply silane (maquira, maringa, pr, brazil). 4actively apply universal adhesive (single bond universal 3m espe st. paul mn, usa) for 20 seconds. 5light air blasting. lithium disilicate glass-ceramic (ips e.max cad) 1condition with 10% hydrofluoric acid gel (maquira, maringa, pr, brazil) for 20 seconds 2wash with water for 30 seconds. 3apply silane (maquira, maringa, pr, brazil). 4actively apply universal adhesive (single bond universal 3m espe st. paul mn, usa) for 20 seconds. 5light air blasting. 7 cabral et al. braz j oral sci. 2022;21:e228852 results table 3 shows that there was no significant difference among the materials, regarding marginal discrepancy (µm) at ca-o, fpa and pw (internal fit). the measurement at fw showed significantly higher marginal discrepancy (p=0.02) when lithium disilicate glass-ceramics (e.max cad) versus nanoceramic resin (lava) was used. in contrast, the marginal discrepancy at the lw, on the inner face of the restoration (lw), was significantly greater (p=0.0198) for e.max cad than vita enamic. legend: ca-o (cavosurface angle of occlusal-facial wall); fw (facial wall); fpa (faciopulpal angle); pw (pulpal wall); lw (lingual wall). figure 2. illustration of the site of the points where the measurements were performed. lw pw ca-o fw fpa table 3. median (minimum value maximum value) of the internal marginal adaptation in micrometers (µm) for the five points assessed, according to the material used (n=10). material point ca-o fw fpa pw lw lava1 98.5 (67-301) a 77 (58-243) b 310 (123-939) a 261 (89-861) a 111.5 (74-174) ab e.max cad2 138 (78-728) a 187.5 (80-281) a 354 (192-635) a 360.5 (188-826) a 177 (67-738) a vita enamic3 105.5 (56-424) a 127.5 (36-380) ab 296 (143-841) a 237 (131-700) a 100 (92-160) b p-value 0.3484 0.02 0.4818 0.3329 0.0198 legend: 1nanoceramic resin (lava ultimate cad-cam restorative for cerec 3m espe, st. paul, mn, usa); 2lithium disilicate glass-ceramic (ips e.max cad, ivoclar vivadent, schaan, liechtenstein); 3polymer-infiltrated ceramic network (vita enamic cad-cam for cerec inlab, vita zahnfabrik, bad säckingen, germany). points assessed: ca-o (cavosurface angle of occlusal-facial wall); fw (facial wall); fpa (faciopulpal angle); pw (pulpal wall); lw (lingual wall). median followed by different lowercase letters indicates a statistically significant difference between the materials at a set point. 8 cabral et al. braz j oral sci. 2022;21:e228852 discussion indirect restorations are used to restore large and deep cavities. however, conventional techniques have a limited run time, and require molding, plaster casts and mounting of the articulator, which may decrease technique accuracy17. cad-cam technology emerged to facilitate the planning and fabrication of prostheses and restorations performed by computer, and features a reading tool, which creates a virtual model for the preparation of prostheses1. the resulting information is then sent to manufacture the restoration of the model using a software process. this technology also allows the cementation time6 of the restoration to be reduced to a single session10, decreases the chances of error, and provides better marginal adaptation18. considering the results, the null hypothesis was partially rejected, because a difference was found in the adaptation at the interface of the surrounding wall with the cad-cam inlay. even when the parameters for adaptation are entered in the software, the program may not reproduce them when the part is milled19. considering the tooth preparation used in the present study, it should be borne in mind that non-retentive cavity preparations display better adaptation of milled parts than more retentive preparations, and that cementation may increase marginal discrepancy20. the evolution of cad-cam systems has led to the development of restorative materials that currently include aesthetic ceramics, high strength ceramics, and both definitive and temporary polymeric materials2,3. according to a previous study21, ceramics are the most widely studied materials of all those used in cad-cam technology, owing to their aesthetics, low thermal conductivity, and biocompatibility; however other materials have emerged to enable different treatments to be performed. commercially acquired lithium disilicate glass-ceramic blocks (ips e.max cad) consist of 70% lithium disilicate22. in fact, use of these ceramic blocks in the present study led to obtaining adequate values9,10 of discrepancy for the cavosurface angle of the occlusal-facial wall, faciopulpal angle and pulpal wall. unlike other materials, these blocks must then be submitted to firing to complete the crystallization process, at which point they reach their highest strength. awada and nathanson6 reported that lithium disilicate blocks had high fracture resistance and a low wear rate. these properties have promoted their widespread use in crown-making, especially because this material leaves the restorations with color and translucency similar to those of the tooth1,2. although there are other clinical parameters that come into play, lithium disilicate has greater antagonist enamel wear than nanoceramic resin (lava) and polymeric materials (vita enamic)23. nanoceramic resin (lava, 3m espe) resulted in lower discrepancy than lithium disilicate glass-ceramic in the facial wall. in addition to having the advantages inherent in ceramic, this material absorbs chewing forces, hence reducing restoration stress7,24. commercial nanoceramic resin blocks have high wear resistance because of their low elastic modulus, and because they are composed of about 80% nanoceramic particles, incorporated into a highly polymerized organic matrix6,7. thus, lava is indicated for making unitary adhesive restorations, such as crowns, inlays, onlays and laminates, inasmuch as it absorbs the chewing load and promotes less wear of the antagonist. 9 cabral et al. braz j oral sci. 2022;21:e228852 vita enamic is composed of two interconnected networks, that of a dominant ceramic, and that of a polymer. although it showed adaptation values similar to those of nanoceramic resin (lava), its marginal discrepancy values in the lingual wall were smaller than those of the lithium disilicate glass-ceramic (e.max cad). vita enamic networks consist of urethane dimethacrylate crosslinked (udma) with triethylene glycol dimethacrylate (tegdma)25. moreover, the differences of nanoceramic resin and polymer-infiltrated ceramic compared to lithium disilicate glass-ceramics may have occurred in the regions analyzed. this is because the lower modulus of elasticity (lava 16 gpa and enamic 21.5 gpa)26 of these two materials is similar to that of dentin (20 gpa)25, hence making them commonly indicated for inlays. in addition, these hybrid ceramics are more malleable and ductile, thus allowing thinner margins. elmougy et al.27 suggested that vita enamic was harder than lava, because of its higher ceramic content. intracoronary restorations do not cover the cusps; the materials employed should be able to bond to the walls of the preparation using resin cementation. thus, according to the present results, the differences in the composition of the materials studied explain why different adaptations were made to the inlays20. the results of this study indicated a discrepancy in the lw and fw for e.max cad. this could be related to the fact that lithium disilicate ceramics is harder23, and to the high crystalline phase content, which may make it more difficult to adapt the ceramic to the walls. although all the materials used (vita enamic, e.max cad and lava) were acquired using the same cad-cam method, they have different properties. thus, although cad-cam machinability was the same for all three materials, the block wear can be expected to be different for each of the materials manufactured. conversely, given that the process of obtaining the inlays by cad-cam manufacture was the same, a very similar fit could be achieved between the ceramic and the polymeric materials. however, when the machinability of the polymeric and lithium disilicate-based materials was compared, the former showed faster cad-cam-induced wear28, perhaps because the former is not as hard as the latter. in general, the machinability of a cad-cam material affects brittleness and marginal chipping, and ips e.max cad presents higher brittleness and marginal chipping values, compared to resin-based or hybrid indirect materials29. in addition, the edges of polymeric materials chip less, because these materials are less friable than lithium disilicate ceramics28. these characteristics of polymeric materials could be why they were able to adapt better to the pw cavity and angles. moreover, the polymer-based materials adapted better than the ceramic materials, specifically at the cervical margin20. microstructure is one of the critical factors that influence the adhesion of materials in the cavity30. materials with a polymeric mesh have better adhesion, and a lower chance of fracture, compared with materials with a scattered filler. the surface structure of the restoration is modified by blasting or etching with hydrofluoric acid to increase the micromechanical retention even further24. however, micromechanical bonds do not necessarily promote the best bond. the chemical bond between the materials and the cement11, promoted by silane, is also of great importance. roughness increases the contact surface, interlocking the cement with the poly10 cabral et al. braz j oral sci. 2022;21:e228852 meric ceramic31. comparatively, mechanical gripping is of great importance to ceramic-loaded polymers, while chemical bonding is more relevant to polymer-infiltrated ceramic material32. several factors may influence marginal and internal adaptation, such as preparation design, location of the margin, milling, milling drill size, milling machine calibration, scanner, restorative material, pressure applied during cementation and cement space thickness33. it stands to reason that any changes in these variables can interfere with the results, and should be investigated in future research. another form of standardizing pressure during cementation should be looked into, since it impacts cement thickness and the occurrence of bubbles. furthermore, any 2d assessments made using photographs should be complemented with 3d analysis, since the latter characterizes the margins more generally. in conclusion, polymeric materials resemble acid-sensitive ceramic in marginal adaptations and promote better internal adaptation when obtained by cad-cam. e.max cad presented a greater internal discrepancy in relation to the surrounding walls. however, there was no difference among the materials, regarding the pulpal wall, the cavosurface, or the faciopulpal angles of the inlays. data availability datasets related to this article are available upon request to the corresponding author. acknowledgments this study was funded in part by cnpq-brazil (#0022/2017-0). disclosure statement the authors declare no conflict of interest. author contribution a.c.r. cabral: conceptualization, data curation, formal analysis, funding acquisition, investigation, validation, visualization, writing original draft, review & editing. w.f. vieira jr.: data curation, formal analysis, investigation, validation, visualization, review & editing. r.t. basting: formal analysis, investigation, validation, visualization, review & editing. c.p.turssi: formal analysis, investigation, validation, visualization, review & editing. f.l.b. amaral: formal analysis, investigation, validation, visualization, review & editing. f.m.g. frança: conceptualization, data curation, formal analysis, funding acquisition, investigation, validation, visualization, project administration, supervision, writing original draft, review & editing. all authors actively participated in the discussion of the manuscript’s findings, and have revised and approved the final version of the manuscript. 11 cabral et al. braz j oral sci. 2022;21:e228852 references 1. 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novdec;26(6):382-93. doi: 10.1111/jerd.12100. 33. tribst jpm, dal piva amo, penteado mm, borges als, bottino ma. influence of ceramic material, thickness of restoration and cement layer on stress distribution of occlusal veneers. braz oral res. 2018 nov;32:e118. doi: 10.1590/1807-3107bor-2018.vol32.0118. 1 volume 22 2023 e239246 original article braz j oral sci. 2023;22:e239246http://dx.doi.org/10.20396/bjos.v22i00.8669246 1 mds, school of medicine, lutheran university of brazil, canoas, brazil. 2 professor of periodontology, department of periodontology, federal university of pelotas, pelotas, brazil. 3 professor of genetics, postgraduate program in cellular and molecular biology, lutheran university of brazil, canoas, brazil. 4 ph.d. student, postgraduate program in genetics and molecular biology, federal university of rio grande do sul, porto alegre, brazil. corresponding author: francisco wilker mustafa gomes muniz rua gonçalves chaves, 457. pelotas, rio grande do sul, brazil. zip code: 96015-560. telephone: +5553991253611. e-mail: wilkermustafa@gmail.com editor: dr. altair a. del bel cury received: may 5, 2022 accepted: august 06, 2022 higher maternal age is associated with higher occurrence of cleft lip/palate in neonates under intensive care luísa de souza maurique1 , francisco wilker mustafa gomes muniz2,* , nathalia preissler vaz silveira1 , melissa camassola3 , bibiana mello de oliveira4 aim: to assess the prevalence of cleft lip and/or cleft palate (cl/p) and associated variables in neonates admitted to neonatal intensive care units (icu). methods: medical charts for neonates born and admitted to the icu between 2012 and 2018 were reviewed. obstetric and neonatal variables were collected by a trained researcher. in the case group, all neonates with cl/p were included. the control group was formed by matching sex, prematurity and month of birth using random number generation. neonates with congenital malformations were excluded from the control group. adjusted logistic regression was used (p<0.05). results: the prevalence of cl/p was 0.43% (n=15). five cases were excluded, as pairing was not possible. twenty neonates were included in the control group. in the final multivariate model, cl/p was only associated with increased maternal age. for each year of increase in maternal age, neonates had a 35.2% higher chance of presenting cl/p (95% confidence interval: 1.021–1.792). conclusions: higher maternal age was associated with higher occurrence of cl/p in neonates admitted to the icu. no other neonatal or maternal independent variables were associated with cl/p. due to missing data, interpretation of study results must be approached with caution. keywords: cleft palate. congenital abnormalities. infant, newborn. intensive care, neonatal. https://orcid.org/0000-0003-3669-5679 https://orcid.org/0000-0002-3945-1752 https://orcid.org/0000-0002-8229-3198 https://orcid.org/0000-0003-2288-2131 https://orcid.org/0000-0002-2679-6858 2 maurique et al. braz j oral sci. 2023;22:e239246 introduction congenital malformation is defined as a change in the structure, metabolism or function present at birth which causes a physical–motor or mental limitation and, in severe cases, death1,2. many studies have reported that the overall prevalence of congenital malformation is 2.3% to 3.8%3,4. moreover, between 5% and 38% of neonatal deaths are related to malformations5. cleft lip (cl) is a congenital malformation that occurs due to non-fusion of the medial nasal prominence with the maxillary prominence and palate6. in contrast, cleft palate (cp) is a problem with the fusion of the primary and secondary palates that occurs during different weeks of pregnancy6. only 30% of patients with cp also present with other malformations or syndromes7. furthermore, literature demonstrated that the occurrence of cl is higher in comparison to cp8, and that a higher occurrence has been observed in male than in female individuals9. the same study demonstrated that the prevalence of cl with or without cp was 14.8 per 10,000 births, while the prevalence of cl with cp was 6.9 per 10,000 births9. neonates with cleft lip/palate (cl/p) may require multidisciplinary treatment starting at birth8. many such patients are also transferred to intensive care units (icu), as specialized feeding and respiratory support may be necessary10. a higher mortality rate is expected for neonates admitted to icus when compared to neonates not admitted to icus11. it has been suggested that the occurrence of cl/p is multifactorial, triggered by environmental, genetic and multifactorial causes12, with more than 400 associated genes with mendelian and non-mendelian inheritance13. when considering all factors, several variables may be associated with cl/p, including maternal smoking and use of alcohol during pregnancy14. furthermore, other malformations in different systems may be associated with cl/p, such as nervous, circulatory, respiratory, genitourinary system and poly-malformative chromosomal abnormalities (trisomy 13 and 18)15. however, it is important to highlight that most of the available data in this area originate with studies that involved cl/p neonates without intensive care needs. it remains necessary to determine what variables are associated with cl/p neonates admitted to icus. therefore, the present study aimed to assess the prevalence of cl/p and its associated variables in neonates admitted to icus when compared to neonates without any malformations. material and methods study design and ethical aspects this is a case-control study, nested to a retrospective cohort of live neonates admitted to a neonatal icu. this study was approved by the local ethics committee (under protocol #2.876.678). hospital administration approved the study. sample, inclusion and exclusion criteria medical charts were reviewed for all neonates born and admitted to the neonatal icu of hospital universitário de canoas, canoas, rio grande do sul, brazil, between 3 maurique et al. braz j oral sci. 2023;22:e239246 2012 and 2018. canoas is located in the metropolitan region of porto alegre, the state capital. the hospital universitário de canoas is a tertiary referral center to many surrounding cities for high-risk pregnancies and neonatal intensive care. the hospital primarily serves users of brazil’s public health system. both the case and control groups were composed of neonates admitted to the hospital’s icu. in the case group, only neonates with cl/p were included. as all neonates with cl/p born between 2012 and 2018 were included in the present study, no sample size calculation was performed. ten neonates were included in the case group. detailed information is provided in the results section. the control group was composed of neonates randomly chosen through use of a number generator. in order to be included in the control group, all neonates had to present no congenital malformations. all neonates selected were aged 0 to 28 days. neonates who were not born in the hospital universitário de canoas were excluded. sampling strategy in the control group for each neonate initially detected with cl/p, two control neonates without any malformations were included. therefore, 20 control neonates were included. the control group was randomly chosen and paired by month of birth (±1 month), sex and presence (<37 weeks) or not (≥37 weeks) of prematurity. a numbered list of all neonates admitted to the icu between 2012 and 2018 was obtained. overall, 3,463 newborns were admitted during this period. with the support of a website (www.randomizer.org), random numbers were selected between 1 and 3,463. a researcher otherwise uninvolved in data collection (fwmgm) performed this process. random numbers were selected until a sufficient number of control neonates were included. data collection and variables during the first phase of the present study, all medical charts were reviewed in order to detect all neonates with cl/p. these charts were reviewed by four medical students previously trained to detect information related to any malformation. students identified malformations using a latin american collaborative study of congenital malformations (eclamc) form16. based on this information, a previously trained researcher (lsm) collected all data from both the case and control groups. training consisted of lectures on how to manage the charts and how to locate the data in the system. lectures were given by a more experienced researcher, who was involved only in data collection for the prevalence of malformations. a microsoft excel spreadsheet was specifically developed in order to collect this data. the following variables were extracted from patient charts: sex (male or female), month and year of birth, gestational age (in weeks + days), delivery method (vaginal or caesarean), toxoplasmosis serologies (igg and igm+ status), maternal age (in years), number of previous pregnancies, number of previous vaginal deliveries, number of previous caesarean deliveries, apgar (5’), length (in cm), cephalic 4 maurique et al. braz j oral sci. 2023;22:e239246 perimeter (in cm), thoracic perimeter (in cm), weight at birth (in kg), length of stay in icu (in days), weight at discharge (in kg), death before discharge (yes or no) and syndromic diagnosis before discharge (yes or no). serologies for hiv, syphilis, hepatitis b and hepatitis c were collected. in addition, exposures to smoking, alcohol and illicit drugs were extracted from the charts. however, as few charts presented information for these variables, it was ultimately impossible to consider them in the statistical analysis. statistical analysis the main outcome of the present study was the presence of cl/p. categorical variables were expressed as observed and percent; continuous variables were expressed as mean and standard deviation. toxoplasma serologies were categorized into immune (igg+ and igm-), susceptible (iggand igm-) or active infection (iggand igm+, or igg+ and igm+). both cephalic perimeter and weight at birth were converted into percentiles. cephalic perimeter percentiles for all neonates, as well as weight at birth percentiles for premature neonates, were obtained using the intergrowth-21st newborn size application tool, adjusted to the gestational age. weight at birth percentiles for term newborns were obtained through the world health organization (who) anthroplus software17. the study groups were compared using chi-square and mann-whitney tests for categorical and continuous variables, respectively. moreover, biand multivariate analyses were performed using binary logistic regression. the initial multivariate model was formed with all independent variables that presented p<0.25 in the bivariate analysis. however, weight at birth was included in the final multivariate model regardless of the p-value detected in the univariate analysis, as the literature shows a strong association between weight at birth and occurrence of cl/p18. a backward strategy was used for the final multivariate model. the maintenance of independent variables was determined using a combination of statistical significance and changing effect model. a p-value of <0.05 was established for statistical significance. all analyses were performed using the software spss, version 21.0 (ibm corp., armonk, ny, usa). results between 2012 and 2018, the prevalence of cl/p was 0.43% (n=15), meaning that for every 10,000 neonates admitted to the icu, 43.32 of them presented with cl/p. among these, cl alone was detected in one neonate (6.67%) while cl + cp was detected in 3 of them (20%). more than one associated malformation was observed in 11 neonates (73.33%), of which six, four and one, respectively, presented with cl + cp, cp and cl. however, due to missing data (e.g., information on sex and gestational age), three of these neonates were excluded, as pairing was not possible. in two cases, ambiguous data were extracted from charts regarding sex. figure 1 presents a flowchart of patient inclusion in the present study. 5 maurique et al. braz j oral sci. 2023;22:e239246 neonates in nicu n = 3,463 excluded missing data n = 3 ambiguous data n = 2 evaluated (case group) n = 15 evaluated (control group) n = 545 included and analyzed n = 10 included and analyzed n = 20 excluded pairing not possible (sex) n = 95 pairing not possible (month of birth) n = 369 pairing not possible (prematurity) n = 9 other place of birth n = 28 presence of other malformation n = 24 figure. flowchart of the participants in the study. neonates in the control group were admitted to the icu due to respiratory distress (n=9; 45%), exposure to perinatal vertical infections (n=6; 30%), weight problems (n=5; 25%), hypoglycemia (n=5; 25%), prematurity (n=7; 35%) or other reasons (n=9; 45%). all patients in the case group were admitted due to malformation (n=10; 100%); however, it was unclear whether cl/p was the main cause of admission. thus, other causes of admission were detected in the case group as well, including weight problems (n=1; 10%), hypoglycemia (n=1; 10%) and prematurity (n=2; 20%). for both groups, more than one cause of admission was detected. all included neonates were admitted on the day of delivery. among the included patients, three (30%) had isolated cl + cp, one (10%) had cl associated with other malformations, two (20%) had cp and other malformations and four (40%) had cl + cp and other malformations. no patient had isolated cl or cp. other malformations were also observed in the case group, including gastrointestinal malformations (n=1), cardiovascular malformations (n=2), urogenital malformations (n=3), upper limb malformations (n=5) and other craniofacial malformations (n=6). overall, up to the date of discharge, no patient died in either group. furthermore, no patient was given a syndromic conclusive diagnosis before neonatal discharge or underwent surgical procedures to correct cl/p while in the icu. ultimately, seven male neonates and three female neonates were included in the case group. when considering patient weight according to gestational age, seven neonates were adequate for gestational age (aga) in the case group, while 11 were aga in the control group. conversely, five and two neonates small for gestational age (sga) were detected in the control and case groups, respectively. four and one neonates large for gestational age were included in the control and case groups, respectively. the majority of the included neonates presented a cephalic perimeter within the normal range (80% in the case group and 85% in the control group). 6 maurique et al. braz j oral sci. 2023;22:e239246 the frequency of distribution for independent variables between cases and controls is expressed in table 1. when considering the whole sample, five women gave birth with advanced maternal age (≥35 years of age), two of whom were from the case group. the minimum maternal ages were 14 years for the control group and 17 years for the case group. meanwhile, the maximum maternal ages were 39 years for the control group and 42 years for the case group. no statistically significant difference between cases and controls was observed for any obstetric variables. however, length of stay in icu was significantly higher among cases (25.89 days±19.82) when compared to controls (10.00±7.13) (p=0.011). table 1. frequency distribution of independent variables among cases and controls, canoas, 2012–2018. variables cases (n=10) controls (n=20) p-value sex male – n (%) 7 (70.0) 14 (70.0) female – n (%) 3 (30.0) 6 (30.0) delivery method normal birth – n (%) 3 (30.0) 9 (45.0) 0.694# caesarean – n (%) 7 (70.0) 11 (55.0) toxoplasmosis serologic status immune – n (%) 2 (25.0) 12 (70.6) 0.081# susceptible – n (%) 6 (75.0) 5 (29.4) absent (n) 2 3 maternal age (years) mean±sd 29.40±8.67 25.65±5.82 0.248β absent (n) 0 0 number of pregnancies mean±sd 2.6±2.12 2.55±1.46 0.713β absent (n) 0 0 number of vaginal deliveries mean±sd 1.40±1.90 1.05±1.39 0.681β absent (n) 0 0 number of caesarean deliveries mean±sd 1.20±1.48 1.25±1.41 0.983β absent (n) 0 0 apgar (5’) mean±sd 8.70±0.67 8.55±1.36 0.681β absent (n) 0 0 length (cm) mean±sd 47.07±3.48 46.95±3.47 0.962β absent (n) 1 1 continue 7 maurique et al. braz j oral sci. 2023;22:e239246 continuation cephalic perimeter (percentile) mean±sd 68.60±39.72 54.37±26.13 0.135β absent (n) 3 1 thoracic perimeter (cm) mean±sd 31.38±3.11 31.97±3.64 0.605β absent (n) 2 2 weight at birth (percentile) mean±sd 44.68±35.19 50.37±33.49 0.764β absent (n) 1 0 length of stay in icu (days) mean±sd 25.89±19.82 10.00±7.13 0.011β absent (n) 1 0 weight at discharge (kg) mean±sd 3.04±0.56 3.01±0.86 0.847β absent (n) 1 1 legend: #fisher’s exact test; *chi-square test; β mann-whitney test. the univariate analysis for the comparison between cl/p and both obstetric and neonatal variables is shown in table 2. women susceptible to toxoplasmosis presented a 7.2 times higher chance of having a child with oral facial cleft (ofc) (95% confidence interval [95%ci]: 1.066–48.639) in comparison to immune women. regarding the neonatal length of stay in icu, a significantly higher age was demonstrated in the case group (odds ratio [or]: 1.109; 95%ci: 1.013–1.214). no other variable was significantly associated with cl/p. both variables were included in the initial multivariate model, along with maternal age and weight percentile at birth. table 2. univariate analysis for the association between independent variables and cleft lip and/or palate. variables or (95% ci) p-value delivery method normal birth 1 0.432 caesarean 1.909 (0.380–9.590) toxoplasmosis serologic status immune 1 0.043 susceptible 7.200 (1.066–48.639) maternal age (years) 1.084 (0.966–1.218) 0.171 number of pregnancies 1.019 (0.644–1.612) 0.938 number of vaginal deliveries 1.155 (0.713–1.871) 0.559 number of caesarean deliveries 0.974 (0.560–1.694) 0.926 continue 8 maurique et al. braz j oral sci. 2023;22:e239246 continuation apgar (5’) 1.138 (0.532–2.435) 0.739 length (cm) 1.011 (0.797–1.282) 0.930 cephalic perimeter (percentile) 1.018 (0.985–1.052) 0.288 thoracic perimeter (cm) 0.949 (0.741–1.215) 0.677 weight at birth (percentile) 0.995 (0.971–1.019) 0.668 length of stay in icu (days) 1.109 (1.013–1.214) 0.026 weight at discharge (kg) 1.000 (0.999–1.001) 0.929 the final multivariate model for the association between outcome and independent variables is presented in table 3. analysis demonstrated that neither toxoplasmosis serologies nor percentile of weight at birth were significantly associated with cl/p (p>0.05). however, for each year of increase in maternal age, neonates had a 35.2% higher chance of presenting cl/p (95% ci: 1.021–1.792). table 3. multivariate analysis for the association between independent variables and cleft lip and/or palate. variables or (95% ci) p-value toxoplasmosis serologic status immune 1 0.093 susceptible 11.264 (0.670–189.493) maternal age (years) 1.352 (1.021–1.792) 0.036 weight at birth (percentile) 1.058 (0.999–1.120) 0.051 discussion the present study evaluated the prevalence of cl/p and compared maternal and neonatal variables between neonates with malformations and those without. analytical results demonstrated that higher maternal age is significantly associated with higher chances of cl/p. conversely, other collected variables, such as serologies for toxoplasmosis and weight at birth, were not significantly associated with cl/p. approximately 7 to 15 in every 10,000 live births present with cl/p, representing one of the most common craniofacial malformations in the world19. a population-based study conducted in foz do iguaçu, brazil demonstrated that the prevalence of cl/p was 9.5 in 10,000 live births20. a higher rate was observed in the present study (43.32 in 10,000 icu admissions), likely because the sample was composed of neonates requiring intensive care. in addition, the hospital universitário de canoas is a referral center for many high-risk pregnancies, including those involving congenital malformations. moreover, this hospital has a highly demanding neonatal intensive care facility, which partially explains the high rate observed in this study. 9 maurique et al. braz j oral sci. 2023;22:e239246 it was determined that 73.34% of the case group had cl/p and another malformation. two prior studies have identified that 39%15 to 41%20 of neonates with cl/p will present with at least one additional malformation20. however, different prevalence rates for multiple associated malformations in neonates with cl/p have been described in the literature, ranging from 25%19 to 29.9%21. a systematic review concluded that cardiac malformation was eight times more frequent in apparently non-syndromic patients with cl/p when compared to the general population22, demonstrating a strong association between cl/p and other malformations. although studies involving neonates admitted to icus are scarce in the literature, they are critical to current research, since a high mortality rate is detected in this group of individuals. one such study showed that the overall mortality rate was 20% (95%ci: 16.7–23.8%), of which 4.08% was related to congenital malformation, making congenital malformation the fifth leading cause of mortality23. as isolated cl was detected in only one patient and isolated cp was not detected, it can be hypothesized that this isolated malformation does not justify admission to a neonatal icu. higher mortality rates are also expected in neonates with cl/p when compared to neonates without this malformation (5.08 vs. 0.33 deaths in every 1,000 births per year, p<0.001)24. despite these previous findings, no neonate in the present study with cl/p died before icu discharge. regarding maternal age, only two mothers in the control group presented an advanced maternal age (age ≥35 years). the present study demonstrated that, for each year of increase in maternal age, there is a 35.2% increase in the or of having a child with cl/p. in a population-based, cross-sectional study which used data from all live births between 2012 and 2017, cl/p was found to be the most common malformation at 9.5 per 10,000 live births (lip, n=3; palate, n=15; lip and palate, n=1)20. among these patients, mean maternal age was 26.4±6.8, results which closely mirror those of the present study. in addition to maternal age, the literature reports that the age of both parents is important when considering the occurrence of cl/p. for example, one study demonstrated that when one of the parents is young (up to 30 years), there is no significant association with isolated cp, regardless of the age of the other parent25. however, if an advanced age was observed in at least one of the parents (mother aged >40 years and/or father aged >50 years), there is a higher risk of cl. furthermore, a significantly higher risk for cl (1.24 per 1,000) was detected in infants of mothers aged >40 years, even those with fathers aged <37 years. in this sense, it may be speculated that maternal age is crucial to the occurrence of cl/p, but that paternal age may not be disregarded. however, in this study, paternal age could not be extracted from the available charts, representing one of the limitations of the present study. furthermore, the literature reports that weight at birth is associated with cl/p18. for instance, one study showed that low birth weight children (<2,500g) presented a 2.5 higher chance of presenting cl/p when compared to those with normal weight at birth (≥2,500g)18. in consideration of this information, the percentile of weight at birth was included in the final multivariate model of the present study. however, no statistically significant association was detected between weight at birth and cl/p in neonates admitted to the icu. thus, it may be hypothesized that other vari10 maurique et al. braz j oral sci. 2023;22:e239246 ables are responsible for the similar lower weight at birth identified in the control group, including congenital infections, maternal use of alcohol or tobacco during pregnancy, placental insufficiency, previous maternal diseases and maternal nutrition. these health conditions are potential causes for admission to the icu in neonates without congenital anomalies. prior literature has also demonstrated that exposure to maternal smoking, abuse of alcohol and use of other illicit drugs is associated with cl/p13. however, this information could not be retrospectively extracted from the available charts in the present study. medical records were often found to be incomplete, possibly due to inadequate standardization of criteria. thus, several data categories remain unexplored, and this is another limitation of the present study. moreover, five neonates with cl/p had to be excluded from the analysis, as minimal data for pairing, such as missing sex or gestational age and ambiguous data, were available. in addition, many maternal infections may be associated with congenital malformations, including rubella, cytomegalovirus, syphilis and toxoplasmosis26,27. in the present study, only serologies for toxoplasmosis were analyzed due to missing data. conversely, no statistically significant association with cl/p was observed. the present study used international growth standard data, using percentiles of birth weight and cephalic perimeter according to sex (in all cases) and gestational age (in pre-term newborns). this approach was used in order to provide more accurate and adjusted data28. neonatal anthropometry, when compared to growth patterns, can be a tool for predicting early and long-term postnatal complications, dysmorphology assessment and body surface estimation29. public data from the sistema nacional de nascidos vivos (sinasc), a national system of population surveillance for live births, allowed for the recognition of cl/p at birth in brazil between 2012 and 2018. the state of rio grande do sul, where the present study was conducted, presented the highest incidence of orofacial clefts (0.072%) in brazil. the national mean was 0.053%. however, the city of canoas demonstrated a smaller incidence of cl/p (0.051%) during the same period as the present study (2012–2018)30. when considering all neonates admitted to the icu, the prevalence of orofacial cleft in the present study was 0.43% (n=15), which is higher than all other comparisons. it is important to highlight that these data are restricted to neonates admitted to the icu, and may not apply to all patients born in the referred hospital. some limitations of the present study must be disclosed. first, the sample was restricted to neonates admitted to the local icu. therefore, a lower external validity may be expected for neonates with cl/p but without intensive care needs. second, several different health care professionals filled in the collected medical reports, and the hospital does not use a standardized reporting method. thus, missing data were often detected, restricting data collection and analysis. third, no sample size calculation was performed, as all neonates with cl/p and admitted to the icu were included in the present study. however, several strengths must also be pointed out. only one trained researcher extracted all data for both the case and control groups. two control neonates were 11 maurique et al. braz j oral sci. 2023;22:e239246 included for each neonate with cl/p, which increases the power of the present study. moreover, three important variables were used to pair individuals from the case and control groups: sex, prematurity and month of birth. additionally, both groups were composed of individuals from the same icu, which increases the comparability between individuals, allowing for a higher internal validity. this is a case-control study nested to a retrospective cohort among a population admitted to an icu. these characteristics are strengths of the present study. based on the present study, higher maternal age may be associated with higher incidence of cl/p. this information should be discussed with couples when planning for pregnancy. further studies should rely on adjusted data using reliable medical records. these records should also be completed in a standardized manner to prevent missing data. ultimately, this study concluded that higher maternal age was associated with higher occurrence of cl/p in neonates admitted to the icu. no other neonatal or maternal independent variables were associated with cl/p. neonates with cl/p presented a significantly higher length of icu stay; however, no etiological diagnosis was provided for this group during the stay in icu. in addition, the lack of complete information in the studied medical records may impair the current results. acknowledgments this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior brasil (capes) finance code 001. all other funding was self-supported by the authors. the authors report no conflicts of interest. the authors are thankful for the assistance of rodolfo tomé soveral and caroline freiesleben cruz during data collection. author contribution luísa de souza maurique: protocol development, data collection, manuscript writing. francisco wilker mustafa gomes muniz: protocol development, data analysis, manuscript writing. nathalia preissler vaz silveira: data collection manuscript editing melissa camassola: protocol development, data analysis, manuscript writing. bibiana mello de oliveira: data analysis, manuscript writing. all authors actively participated in the discussion of the manuscript’s findings, revised, and approved the final version of the manuscript. references 1. candotto v, oberti l, gabrione f, greco g, rossi d, romano m, et al. current concepts on cleft lip and palate etiology. j biol regul homeost agents. 2019 may-jun;33(3 suppl 1):145-51. 12 maurique et al. braz j oral sci. 2023;22:e239246 2. kang sl, narayanan cs, kelsall w. mortality among infants born with orofacial clefts in a single cleft network. cleft palate craniofac j. 2012 jul;49(4):508-11. doi: 10.1597/10-179. 3. giang htn, bechtold-dalla pozza s, ulrich s, linh lk, tran ht. prevalence and pattern of congenital anomalies in a tertiary hospital in central vietnam. j trop pediatr. 2020 apr;66(2):187-93. doi: 10.1093/tropej/fmz050. 4. charan pal a, mukhopadhyay dk, deoghuria d, mandol sk, patra ac, murmu s. prevalence of congenital malformations in newborns delivered in a rural medical college hospital, west bengal. j dent med sci. 2015 dec;14(12):26-32. doi: 10.9790/0853-141222632. 5. lehtonen l, gimeno a, parra-llorca a, vento m. early neonatal death: a challenge worldwide. semin fetal neonatal med. 2017 jun;22(3):153-60. doi: 10.1016/j.siny.2017.02.006. 6. worley ml, patel kg, kilpatrick la. cleft lip and palate. clin perinatol. 2018 dec;45(4):661-78. doi: 10.1016/j.clp.2018.07.006. 7. dao am, goudy sl. cleft palate repair, gingivoperiosteoplasty, and alveolar bone grafting. facial plast surg clin north am. 2016 nov;24(4):467-76. doi: 10.1016/j.fsc.2016.06.005. 8. matute j, lydick ea, torres or, owen kk, jacobsen kh. prevalence of cleft lip and cleft palate in rural north-central guatemala. cleft palate craniofac j. 2015 may;52(3):377-80. doi: 10.1597/13-347. 9. paaske eb, garne e. epidemiology of orofacial clefts in a danish county over 35 years before and after implementation of a prenatal screening programme for congenital anomalies. eur j med genet. 2018 sep;61(9):489-92. doi: 10.1016/j.ejmg.2018.05.016. 10. hubbard ba, baker cl, muzaffar ar. prenatal counseling’s effect on rates of neonatal intensive care admission for feeding problems cleft lip/palate infants. mo med. 2012 mar-apr;109(2):153-6. 11. christensen k. the 20th century danish facial cleft population--epidemiological and genetic-epidemiological studies. cleft palate craniofac j. 1999 mar;36(2):96-104. doi: 10.1597/1545-1569_1999_036_0096_tcdfcp_2.3.co_2. 12. bille c, knudsen lb, christensen k. changing lifestyles and oral clefts occurrence in denmark. cleft palate craniofac j. 2005 may;42(3):255-9. doi: 10.1597/03-139.1. 13. schutte bc, murray jc. the many faces and factors of orofacial clefts. hum mol genet. 1999;8(10):1853-9. doi: 10.1093/hmg/8.10.1853. 14. mbuyi-musanzayi s, kayembe tj, kashal mk, lukusa pt, kalenga pm, tshilombo fk, et al. non-syndromic cleft lip and/or cleft palate: epidemiology and risk factors in lubumbashi (dr congo), a case-control study. j craniomaxillofac surg. 2018 jul;46(7):1051-8. doi: 10.1016/j.jcms.2018.05.006. 15. koga h, iida k, maeda t, takahashi m, fukushima n, goshi t. epidemiologic research on malformations associated with cleft lip and cleft palate in japan. plos one. 2016 feb 22;11(2):e0149773. doi: 10.1371/journal.pone.0149773. 16. eclamc. latin american collaborative study of congenital malformations. atlas of birth. 2020 [cited 2020 sep 21]. available fotmt: http://en.atlaseclamc.org. 17. world health organization. application tools. 2020 [cited 2020 sep 21]. available from: https://www.who.int/growthref/tools/en/. 18. shibukawa bmc, rissi gp, higarashi ih, oliveira rr. factors associated with the presence of cleft lip and/or cleft palate in brazilian newborns. rev bras saude mater infant. 2019 sep-dec;19(4):947-56. 19. cassinelli a, pauselli n, piola a, et al. national health care network for children with oral clefts: organization, functioning, and preliminary outcomes. arch argent pediatr 2018 feb;116(1):e26-33. doi: 10.5546/aap.2018.eng.e26. 13 maurique et al. braz j oral sci. 2023;22:e239246 20. de souza s, nampo fk, pestana cr. major birth defects in the brazilian side of the triple border: a population-based cross-sectional study. arch public health. 2020 jun 30;78:61. doi: 10.1186/s13690-020-00443-w. 21. de bérail a, lauwers f, noirrit esclassan e, woisard bassols v, 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[epidemiology of malformations associated with cleft lip and palate: a retrospective study of 324 cases]. arch pediatr. 2015 aug;22(8):816-21. french. doi: 10.1016/j.arcped.2015.05.005. 22. munabi nco, swanson j, auslander a, sanchez-lara pa, davidson ward sl, magee wp 3rd. the prevalence of congenital heart disease in nonsyndromic cleft lip and/or palate: a systematic review of the literature. ann plast surg. 2017 aug;79(2):214-20. doi: 10.1097/sap.0000000000001069. 23. desalew a, sintayehu y, teferi n, amare f, geda b, worku t, et al. cause and predictors of neonatal mortality among neonates admitted to neonatal intensive care units of public hospitals in eastern ethiopia: a facility-based prospective follow-up study. bmc pediatr. 2020 apr;20(1):160. doi: 10.1186/s12887-020-02051-7. 24. malic cc, lam m, donelle j, richard l, vigod sn, benchimol ei. incidence, risk factors, and mortality associated with orofacial cleft among children in ontario, canada. jama netw open. 2020 feb;3(2):e1921036. doi: 10.1001/jamanetworkopen.2019.21036. 25. berg e, lie rt, sivertsen å, haaland øa. parental age and the risk of isolated cleft lip: a registry-based study. ann epidemiol. 2015 dec;25(12):942-7.e1. doi: 10.1016/j.annepidem.2015.05.003. 26. davanzo r, antonio c, pulella a, lincetto o, schierano s. neonatal and post-neonatal onset of early congenital syphilis: a report from mozambique. ann trop paediatr. 1992;12(4):445-50. doi: 10.1080/02724936.1992.11747612. 27. voordouw b, rockx b, jaenisch t, fraaij p, mayaud p, vossen a, et al. performance of zika assays in the context of toxoplasma gondii, parvovirus b19, rubella virus, and cytomegalovirus (torch) diagnostic assays. clin microbiol rev. 2019 dec;33(1):e00130-18. doi: 10.1128/cmr.00130-18. 28. villar j, cheikh ismail l, victora cg, ohuma eo, bertino e, altman dg, et al. international standards for newborn weight, length, and head circumference by gestational age and sex: the newborn cross-sectional study of the intergrowth-21st project. lancet. 2014 sep;384(9946):857-68. doi: 10.1016/s0140-6736(14)60932-6. 29. pereira-da-silva l. neonatal anthropometry: a tool to evaluate the nutritional status and predict early and late risks. in: the handbook of anthropometry: physical measures of human form in health and disease. new york: springer; 2012. p.1079-104. 30. brazil. datasus; 2020 [cited 2020 sep 21]. available from: datasus.saude.gov.br. 1http://dx.doi.org/10.20396/bjos.v20i00.8660298 volume 20 2021 e210298 original article 1 department of oral pathology, saveetha dental college and hospitals, saveetha institute of medical and technical sciences, saveetha university. corresponding author: snega thamilselvan, saveetha dental college & hospitals, saveetha institute of medical and technical sciences, saveetha university, 162, poonamallee high road, velappanchavadi, chennai – 600077. tamilnadu, india. snegathamilselvann@gmail.com phone number: 7397458628 email id – 151907001.sdc@ saveetha.com editor: dr altair a. del bel cury received: july 1st, 2021 accepted: february 2, 2021 p53 & cyclin d1 expression in surgically resected clear margins of oral squamous cell carcinoma snega thamilselvan1,* , archana santhanam1 , herald j. sherlin1 , gifrina jayaraj1 , k. r. don1 oral squamous cell carcinoma (oscc) is one of the most well-known malignancies that affect the human population worldwide. the early diagnosis and early intervention of oscc help improve the survival rate of the patients. the tumour free surgical margins are a positive prognostic factor for recurrence-free survival. the molecular markers can be used to detect the tumour free surgical margins. aim: the aim of the study is to evaluate the expression of p53 & cyclin d1 marker in resected surgical apparently clear margins and to correlate the p53 & cyclin d1 expression with clinicopathological characteristics and patient outcome. methods: the study population included retrospective cases of oscc with apparently clear margins (2017-18) n=10 and clinicopathological variables relevant to survival analysis were recorded. finally, two margins were selected from each case, a total of 20 margins were included in this study. paraffin-embedded wax blocks retrieved and tissue sections were made. expression of cyclin d1 and p 53 was assessed by the immunohistochemical staining procedure results: positive expressions cyclin d1 in 40% of mild dysplasia margins and 60% in clearance adequate margins were present. p53 expression was seen in 16% of mild dysplasia margins and 84% in clearance adequate margins. the expression of p53 and cyclin d1 molecular markers are noted in the basal & parabasal layer of epithelium. conclusion: molecular markers could play a more reliable method for the assessment of dysplasia at the margins. keywords: tumor suppressor protein p53. cyclin d1. carcinoma, squamous cell. https://orcid.org/0000-0001-7901-5050 https://orcid.org/0000-0002-9143-5813 https://orcid.org/0000-0003-4177-1648 https://orcid.org/0000-0003-4194-1774 https://orcid.org/0000-0003-3110-8076 2 thamilselvan et al. introduction oral cancer makes up to 2% of all the cancer cases with the majority being oral squamous cell carcinoma (oscc) which accounts for 90% of all the oral malignancies1. oral cancer is the 8th most frequent cancer among males and the 14th most frequent cancer among females globally2. the current findings state the increasing prevalence of oral cancer in asia, especially in india has been documented1. surgical resection is the first-line management of oscc followed by adjuvant radiotherapy and chemotherapy when needed3. the primary goal of surgical resection is to obtain tumour-free margins. the tumour-free surgical margin is an important prognostic factor for recurrence free survival in oscc managed with primary surgery4. regardless of whether the histological status of surgical margins is apparently clear the local recurrence rate of oscc still ranges from 10% 30%4. the severe dysplasia margins are considered positive margin which requires a re-excision while mild/moderate dysplasia margins are being overseen leading to local recurrence. head and neck squamous cell carcinoma (hnscc) is a multistep process characterized by genetic and epigenetic alterations. these alterations in the tumour-free surgical margins that lead to recurrence may not be detected by conventional microscopic histological analysis but may be detected using immunohistochemical (ihc) staining5,6. tnm staging and histopathological grading are considered as the main prognostic factors in oscc. but patients with similar stages of disease treated in a uniform manner experience a wide range of outcomes. the biological behavior of cancer for each patient differs7, which necessitates assessing the molecular markers separately and according to which the treatment modalities can be tailor-made. oscc is characterized by imbalances in cell cycle control. the assessment of p53 & cyclin d1 molecular markers in surgical margins is more valuable in surgical margins for patients undergoing surgical treatment. p53 is a gene that codes for a protein that regulates cell growth and proliferation through its role in cell-cycle checkpoint control hence functions as a tumour suppressor5. the cyclin d1 is a proto-oncogene that encodes the cyclin d1 nuclear protein, a positive regulator of g1 cell-cycle checkpoint and may play an important role in tumorigenesis of oscc8. therefore, expression of p53 & overexpression of cyclin d1 in the resected surgical apparently clear margins is considered to have better prognostic value in oscc. this study evaluates the expression of p53 & cyclind1 markers in resected surgical apparently clear margins and to correlate the p53 & cyclind1 expression with clinicopathological characteristics and patient outcome. materials and methods sample selection a total of 40 retrospective cases of oscc patients who reported to saveetha dental college & hospitals from 2017-2018 were selected initially. clinic-pathological variables relevant to survival analysis were recorded. all the patients had been treated https://paperpile.com/c/ujmkrl/9qf1 https://paperpile.com/c/ujmkrl/9qf1 https://paperpile.com/c/ujmkrl/9qf1 https://paperpile.com/c/ujmkrl/9qf1 https://paperpile.com/c/ujmkrl/9qf1 https://paperpile.com/c/ujmkrl/9qf1 https://paperpile.com/c/ujmkrl/9qf1 https://paperpile.com/c/ujmkrl/9qf1 https://paperpile.com/c/ujmkrl/9qf1 https://paperpile.com/c/ujmkrl/9qf1 3 thamilselvan et al. surgically and the margins of the excised specimens were histopathologically evaluated for adequate clearance. 30 retrospective cases were excluded since the surgical margins had moderate to severe dysplasia histopathologically, inadequate thickness of the epithelium, fragmented tissue sections and also the patients who underwent adjuvant chemotherapy and radiotherapy in follow-up were excluded. a final of 10 cases that were reported with adequate epithelial thickness and apparently clear or mild dysplasia margins were included in the study. 2 margins from each case were included in the study. finally, the histological analysis for the respective slides was reviewed and with a total of 20 margins, the study was performed. approval for the study was obtained from the institutional review board srb/sdc/mds/002/03. immunohistochemistry the paraffin wax blocks were retrieved from the department of oral & maxillofacial pathology from saveetha dental college & hospital. 3 μm sections were cut from formalin-fixed paraffin-embedded blocks mounted on gelatin-coated slides. then sections were deparaffinized in xylene for 10 mins & followed by dehydration in 100% alcohol for 5 mins and rinsed in distilled water. following which heat mediated antigen retrieval with tris-edta buffer solution of 9.0 ph was done in a pressure cooker for 5 mins. depressurize the pressure cooker to 37 c under running tap water. endogenous peroxidase was blocked for 30 mins. sections were incubated with the primary antibody, p53 (dako, monoclonal mouse anti-human p53 protein, denmark) & cyclin d1(dako, monoclonal mouse anti-human cyclin d1, denmark) for 1 hour at room temperature. detection was performed using polyexcel hrp/dab detection system (pathnsitu, conjugated by goat anti-mouse/rabbit igg, usa). the sections were then counterstained with mayer’s hematoxylin and were then dehydrated and mounted using dibutyl phthalate in xylene mountant. negative and positive controls were used in each run. scoring criteria the presence of brown-coloured reactions at the site of the target antigen was indicative of positive reactivity. the parameters used for assessing the immunostaining was propensity index, which indicates the percentage of tumour cells which had taken up the stain and staining intensity, which indicates the amount of stain taken up by the tumour cells. immunostaining was assessed by the evaluation of a total score obtained by combining the staining intensity and staining proportion scores of p 53 and cyclin -d1 cells which were scored from 0 to 3+. the scores for evaluation of immunostaining are tabulated in table 1. there were 2 observers who assessed and evaluated the respective slides for immunohistochemical analysis. table 1. scoring criteria for evaluation of expression of p53 and cyclin d1. score proportionality index intensity of staining 0 no labelling or <10% of tumour cells negative 1+ 10 -24 % of tumour cells mild 2+ 25-49% of tumour cells moderate 3+ >50% of tumour cells severe 4 thamilselvan et al. statistical analysis all the results were tabulated and assessed for statistical analysis using spss (ibm spss statistics for mac version 20.0). the results of the two markers were compared using the chi‐square test, and p-value = 0.05 was statistically significant. the kaplan-meier method was used to estimate local recurrence-free survival and the statistical significance was determined by the log-rank test. results all the resected surgical margins of n=10 oscc cases included in the study were histopathologically clear/mild dysplasia margins. a total of n=10 cases with 2 margins for each were selected and analyzed for immunohistochemistry staining to evaluate the expression of p53 and cyclin d1. among 20 margins evaluated for ihc expression 6 (30%)were mild dysplasia margins and 14(70%) were apparently clear margins. table 2 shows patient characteristics. pathologically, 80% of the patients had metastasis of which 30% involved level i, 40% involved level ii & 10% involved level iv and 20% of the patients had no metastasis. out of 10 cases, 2 cases( 20%) had a recurrence and the survival rate was 90% among 10 patients. table 2. table depicting the demographics, tumour characteristics and the overall survival of the patients included in the study. factor group total sample n(%) gender male 8(80%) female 2(20%) age >50 5(50%) <50 5(50%) location lateral border of the tongue 3(30%) left buccal mucosa 3(30%) right buccal mucosa 1(10%) left maxilla 1(10%) gingivobuccal sulcus 1(10%) palate 1(10%) nodal status l i 3(30%) lii 4(40%) l iii 0 l iv 1(10%) no involvement 2(20%) histological grade microinvasion 1(10%) wdscc 7(70%) mdscc 1(10%) pdscc 1(10%) recurrence yes 2(20%) no 8(80%) survival alive 9(90%) expired 1(10%) 5 thamilselvan et al. cyclin d1 expression in resected surgical margins showed positive expression in 5 (25%) margins. out of the 5 margins, 2 (40%) margins were mild dysplasia margins and 3 (60%) were clearance adequate margins. there were negative expressions of cyclin d1 in 15 margins (75%) with 4 (27%) in mild dysplasia margins and 11 (73%) in clearance adequate margins. the level of expression of cyclin d1 was seen in the basal and parabasal layers of the epithelium (table 3). the positive margins of cyclin d1 in basal and parabasal layers are depicted in figure 1. among the 20 margins evaluated for p53 expression, positive expression was present in 12 (60%) margins. out of the 12 margins, 2 (16%) margins were mild dysplasia margins and 10 (84%) were clearance adequate margins. there were negative expressions of p53 in 8 (40%) margins with 4 (50%) in mild dysplasia margins and 4 (50%) in clearance adequate margins. the level of expression of p53 was seen in the basal and parabasal layers of the epithelium (table 4) (figure 2). table 3. expression of cyclin d1 in the resected surgical apparently clear margins. margins positive expression (n=5) negative expression (n=15) level of expression mild dysplasia 2(40%) 4(27%) basal & parabasal clearance adequate 3(60%) 11(73%) basal & parabasal figure 1. positive expression of cyclin d1 in the basal and parabasal layer of resected surgical margins. table 4. expression of p53 according in the resected surgical apparently clear margins. margins positive expression (n=12) negative expression (n=8) level of expression mild dysplasia 2(16%) 4(50%) basal & parabasal clearance adequate 10(84%) 4(50%) basal & parabasal 6 thamilselvan et al. among the 20 margins, 2 (10%) clearance adequate margins of one case showed positive expression for both p53 and cyclin d1. the level of expression of p53 and cyclin d1 was seen in basal and parabasal layers of the epithelium. the kaplan-meier survival local recurrence-free survival curve according to the ihc status in surgical margins showed patients with cyclin d1 positive expression in surgical margins are 100% alive, 80% of cyclin d1 negative expressions in surgical margins are alive and 20% with cyclin d1 negative expression in the surgical margin are deceased. the p-value = 0.373(p >0.050) (figure 3). figure 2. positive expression of p53 in the basal and parabasal layers of resected surgical margins. figure 3. cumulative local recurrence free survival curve for cyclin d1 expression in resected surgical margins c um s ur vi va l months survival functions cyclin d1 status cyclin d1 (-ve) cyclin d1 (+ve) 1.0 0.8 0.6 0.4 0.2 0.0 0 10 20 30 40 7 thamilselvan et al. the kaplan-meier survival local recurrence-free survival curve according to the ihc status in surgical margins showed patients with p53 negative expressions in surgical margins are 100% alive, 80% of p53 positive expressions in surgical margins are alive and 20% with positive expression in the surgical margin is deceased. the p-value = 0.665 (p >0.05) (figure 4). discussion immunohistochemistry (ihc) is an integration of histological and immunological techniques that mainly visualizes the distribution and localization of specific molecular biomarkers within a tissue9. ihc staining has an important role in the histopathological diagnosis of many tumours10. a local recurrence after resection indicates the presence of molecular alterations in cells. p53 is a classical tumour suppressor gene, its expression is related to the tumorigenesis and overexpression of cyclin d1 represents the same. the pathological evaluation of oral epithelial dysplasia is based on the epithelial architectural and cellular features and is graded accordingly4. severe dysplasia has been considered as positive margins that require re-excision after histopathological evaluation. the mild/ moderate dysplasia margins are usually overlooked which indicates the need for evaluation of molecular markers to avoid recurrence. to date, there have been several studies done with the primary tumour specimen or invasive tumour front for evaluating the expression of molecular biomarkers and prognosis in oral squamous cell carcinoma patients. however, the analysis of molecular markers in resected surgical margins would be more appropriate in determining the prognosis and survival of the patients. to the best of our knowledge, this is the first study to evaluate p53 and cyclin d1 expression in resected surgical margins. figure 4. cumulative local recurrence free survival curve for p53 expression in resected surgical margins. c um s ur vi va l months survival functions p53 expression p53 (-ve) p53 (+ve) 1.0 0.8 0.6 0.4 0.2 0.0 0 10 20 30 40 https://paperpile.com/c/ujmkrl/9qf1 https://paperpile.com/c/ujmkrl/9qf1 https://paperpile.com/c/ujmkrl/9qf1 8 thamilselvan et al. the positive expressions of p53 were seen in 12 margins out of the total 20 margins of which 10 margins were clearance adequate margins and 2 were mild dysplasia margins. interestingly, in patients who had clear margins, a majority of the patients who had p53 positive expression did not develop local recurrence. only one patient developed local recurrence at the end of five months and another patient with positive p53 expression expired after 8 months. also, the statistical significance of the overall survival rate with the positive expression of p53 was not significant (p-value = 0.665). since the level of expression for p53 was restricted to basal and parabasal layers, it cannot be accepted as a confirmatory prognostic indicator yet it adds value for existing cancer with respect to local recurrence. the previous literature suggests the presence of a strong impact of p53 on local recurrence. also, they have observed p53 expression in the late event of carcinogenesis11. use of immunohistochemistry alone to determine whether the positive p53 expression reflects the presence of stable mutant p53 protein or stabilization of normal p53 through its binding to certain cellular gene products is not sufficient. on the other hand, there will be false-negative staining for p53 when the nonsense and frame-shift mutations result in the absence of p53 in the tumour cells. therefore, p53 immunoexpression does not exactly correspond with the p53 gene status12. our study results showed positive expression of cyclin d1 in 5 margins of which 3 were clearance adequate margins and 2 were mild dysplasia margins. all the patients with positive cyclin d1 expression in surgical margins were alive and disease-free. the cyclin d1 positivity in surgical margins did not have any significance in our study because carcinogenesis is multifactorial with the involvement of numerous genes and pathways. the low-level expression of cyclin d1 is typical of epithelial cells in a normal state as a cell cycle regulator in the g1-s phase transition. cyclin d1 overexpression in surgical margins may cause future carcinogenesis13. overexpression of cyclin d1 has been previously reported in many malignancies such as breast cancer, colon cancer, prostate cancer, lymphomas, melanomas and carcinomas13,14. sakashita et al.5 identified cyclin d1-positive tumour specimens did not indicate a worse prognosis, but cyclin d1-positive margins could be a worse prognostic factor for local recurrence. the presence of cyclin d1-positive surgical margins did not have any statistical significance on overall survival (p=0.373). hence, cyclin d1-positive status in surgical margins can be considered as an unbiased prognostic indicator for local recurrence. in the present study, the positive expression of both the molecular markers, p53 and cyclin d1 was noted in one case in both the margins. both the margins were clear margins. unfortunately, the positive expression does not prove any correlation with the prognosis since the patient is alive and disease-free. the overall survival of oscc patients is determined by several factors such as age, gender, t and n stage, tumour differentiation, primary site, multiple nodal metastases, extracapsular spread, massive primary cancer and the presence of adjunctive treatment15. local recurrence is observed less frequently in patients with histopathologically tumour-free surgical margins. the retrospective analysis was done with a minimum sample size selected from paraffin-embedded wax blocks. hence, large scale retrospective studies are required to substantiate the results obtained. https://paperpile.com/c/ujmkrl/9qf1 https://paperpile.com/c/ujmkrl/9qf1 https://paperpile.com/c/ujmkrl/9qf1 https://paperpile.com/c/ujmkrl/9qf1 https://paperpile.com/c/ujmkrl/9qf1 https://paperpile.com/c/ujmkrl/9qf1 9 thamilselvan et al. a clear surgical margin is an important determinant of a good outcome. in the present study, some patients with clear margins developed local recurrence while some patients did not. ihc analysis of surgical margins can augment standard histopathological assessment and may improve the prediction of local recurrence. these data may have a major impact on future diagnostic workups for patients with oral carcinoma after surgical treatment. in conclusion, molecular markers could play a more reliable method for the assessment of dysplasia at the margins. further large scale studies to examine the association between p53 and cyclin d1 expression at the margin of oscc and the development of local recurrence are required. acknowledgements the authors would like to acknowledge the help and support rendered by the department of oral pathology of saveetha dental college and hospitals and the management for their constant assistance with the research. references 1. ghafari r, jalayer naderi n, emami razavi a. a retrospective institutional study of histopathologic pattern of oral squamous cell carcinoma (oscc) in tehran, iran during 2006-2015. j res med sci. 2019 jun;24:53. doi: 10.4103/jrms.jrms_882_18. 2. coelho kr. challenges of the oral cancer burden in india. j cancer epidemiol. 2012;2012:701932. doi: 10.1155/2012/701932. 3. kamat m, rai bd, puranik rs, datar uv. a comprehensive review of surgical margin in oral squamous cell carcinoma highlighting the significance of tumor-free surgical margins. j cancer res ther. 2019 jul-sep;15(3):449-54. doi: 10.4103/jcrt.jcrt_273_17. 4. yang xh, ding l, fu y, chen s, zhang l, zhang xx, et al. p53-positive expression in dysplastic surgical margins is a predictor of tumor recurrence in patients with early oral squamous cell carcinoma. cancer manag res. 2019 feb;11:1465-72. doi: 10.2147/cmar.s192500. 5. sakashita t, homma a, suzuki s, hatakeyama h, kano s, mizumachi t, et al. prognostic value of cyclin d1 expression in tumor-free surgical margins in head and neck squamous cell carcinomas. acta otolaryngol. 2013 sep;133(9):984-91. doi: 10.3109/00016489.2013.795287. 6. bilde a, von buchwald c, dabelsteen e, therkildsen mh, dabelsteen s. molecular markers in the surgical margin of oral carcinomas. j oral pathol med. 2009 jan;38(1):72-8. doi: 10.1111/j.1600-0714.2008.00715.x. 7. sheriff k, santhanam a. knowledge and awareness towards oral biopsy among students of saveetha dental college. res j pharm technol. 2018;11(2):543-6. doi: 10.5958/0974-360x.2018.00101.4. 8. zhong lp, zhu dw, william wn jr, liu y, ma j, yang cz, et al. elevated cyclin d1 expression is predictive for a benefit from tpf induction chemotherapy in oral squamous cell carcinoma patients with advanced nodal disease. mol cancer ther. 2013 jun;12(6):1112-21. doi: 10.1158/1535-7163.mct-12-1013. 9. duraiyan j, govindarajan r, kaliyappan k, palanisamy m. applications of immunohistochemistry. j pharm bioallied sci. 2012 aug;4(suppl 2):s307-9. doi: 10.4103/0975-7406.100281. 10. hornick jl. novel uses of immunohistochemistry in the diagnosis and classification of soft tissue tumors. mod pathol. 2014 jan;27 suppl 1:s47-63. doi: 10.1038/modpathol.2013.177. http://paperpile.com/b/ujmkrl/9zkd http://paperpile.com/b/ujmkrl/9zkd 10 thamilselvan et al. 11. singh j, jayaraj r, baxi s, mileva m, skinner j, dhand nk, et al. immunohistochemical expression levels of p53 and eif4e markers in histologically negative surgical margins, and their association with the clinical outcome of patients with head and neck squamous cell carcinoma. mol clin oncol. 2016 feb;4(2):166-72. doi: 10.3892/mco.2015.689. 12. sawair f, hassona y, irwin c, stephenson m, hamilton p, maxwell p, et al. p53, cyclin d1, p21 (waf1) and ki-67 (mib1) expression at invasive tumour fronts of oral squamous cell carcinomas and development of local recurrence. asian pac j cancer prev. 2016;17(3):1243-9. doi: 10.7314/apjcp.2016.17.3.1243. 13. fu m, wang c, li z, sakamaki t, pestell rg. minireview: cyclin d1: normal and abnormal functions. endocrinology. 2004 dec;145(12):5439-47. doi: 10.1210/en.2004-0959. 14. lukas j, bartkova j, rohde m, strauss m, bartek j. cyclin d1 is dispensable for g1 control in retinoblastoma gene-deficient cells independently of cdk4 activity. mol cell biol. 1995 may;15(5):2600-11. doi: 10.1128/mcb.15.5.2600. 15. shehenaz a, santhanam a, jayaraj g, ramani p. correlation of levels of lymph node, node involvement, gender predilection and histopathology in surgical pathology specimen of oral squamous cell carcinoma. int j res trends innov. 2017;2(8):149-53. http://paperpile.com/b/ujmkrl/zxld http://paperpile.com/b/ujmkrl/zxld http://paperpile.com/b/ujmkrl/zxld 1http://dx.doi.org/10.20396/bjos.v20i00.8664270 volume 20 2021 e214270 original article 1 faculty of health sciences, universidad autónoma de chile, temuco, chile. 2 faculty of dentistry, universidad de concepción, chile. 3 fundación kimntrum, chile corresponding author: valeria campos email: valeriacamposcannobbio@ gmail.com editor: dr altair a. del bel cury received: february 5, 2021 accepted: march 10, 2021 factor analysis of the cross-cultural adaptation of the multidimensional attitudes scale towards deaf persons in chilean dental students valeria campos1 , luis luengo2 , ricardo cartes-velásquez3,* there are various instruments to measure attitudes toward persons with disabilities (pwd). the multidimensional attitudes scale (mas) toward pwd is a three-dimension scale with good psychometric properties; the spanish version has been validated with a four-factor structure. aim: to examine the factor structure of a cross-cultural adapted version of the spanish mas towards deaf persons in a sample of chilean dental students. methods: this cross-sectional study involved five chilean public health experts that reviewed the scale for obtaining a preliminary version of a 30-item modified mas towards deaf persons; a pilot with 15 dental students was performed, and a final sample composed of 311 students was included. for the exploratory factor analysis (efa), maximum likelihood estimation (ml) for determining the number of factors and parallel analysis (pa) was used, with oblimin for the rotation method. cronbach’s alpha was used to assess reliability. the root mean square error of approximation (rmsea), comparative fit index (cfi), incremental fit index (ifi), goodness of fit index (gfi), tucker-lewis fit index (tli-nnf) and root mean square of residuals (rmsr) were used to assess model fit. results: all items had a normal distribution with the exception of items 7 and 10. the four-factor structure without item 10 in this efa presented an adequate cronbach’s alpha (>0.83), suggesting acceptable reliability. rmsea, tli-nnfi, rmsr, gfi and cfi indices suggested a good fit of the model and were consistent with the literature. conclusion: the spanish modified version of the mas towards deaf persons has a four-factor structure, which in consistent with a previous version of the mas. keywords: deafness. validation studies as topic. attitude. students, dental. hispanic americans. chile. https://orcid.org/0000-0003-0697-1345 https://orcid.org/0000-0002-9643-4334 https://orcid.org/0000-0001-5831-7324 2 campos et al. introduction one out of seven persons have some kind of disability, comprising one of the most marginalized groups in the world1,2. in the case of the deaf population, more than 460 million people have a hearing disability worldwide2. in chile, 20% of the population has a disability, from which 8.2% corresponds to a hearing disability3. a significant proportion of the obstacles that people with disabilities (pwd) face when entering society are determined by the attitudes of the rest of the population, since it has been described that negative attitudes hinder their inclusion in education, employment, health care and social participation4-6. unfavorable attitudes and feeling of discomfort regarding people with hearing disability also has been described7,8 hampering their inclusion. attitudes are usually defined as: “the disposition or tendency to respond positively or negatively about a certain idea, object, person or situation”, which is closely related to our opinions and beliefs, and based on our own experiences9. it has been proposed that attitudes involve three components: a cognitive component that refers to thoughts, beliefs, or perceptions about an object/person; an affective component is related to emotion, which can be positive or negative; and a behavioral component linked to a way of acting10,11. there are various instruments to measure attitudes toward pwd for general population. they can assess attitudes towards disability in general, as the attitude to disability scale for people with disabilities12 or the attitudes towards disabled persons scale13. also, they can assess attitudes towards a specific disability, such as attitudes towards deafness scale14-16, attitudes toward intellectual disability questionnaire17 and the multidimensional attitudes scale toward persons with disabilities (mas)18, among others. the mas, an originally three-dimension scale, with good psychometric properties18, has been cross-culturally adapted to different languages reporting four dimensions19-22. moreover, the french and chinese modified versions of the mas for autism spectrum have been validated as a four-dimension scale21,22. although the mas was created to measure attitudes in the general population, the samples used in its construction and cross-cultural adaptations were mostly college students18-20,22. moreover, as the attitudes towards deafness scale was created for health professionals, there are no instruments to measure attitudes towards the deaf population in college students, including dental students. this situation could be considered a long-standing oversight in dental education research, as inclusion must be a relevant part of the training of future dentists. based on the above, and considering that mas has not been used in chile, our aim was to examine the factor structure of a cross-cultural spanish modified version of the mas towards deaf persons using exploratory factor analyses in a sample of chilean dental students. materials and methods study type this cross-sectional study aimed to examine the factor structure of a cross-cultural spanish modified version of the mas towards deaf persons using exploratory factor analyses in a sample of chilean dental students. 3 campos et al. sample the target population was comprised of dental students from universidad de concepción, from first to fifth year, with a grand total of 350 students. students from sixth year were excluded as they participated in the pilot test. the final sample was composed of 311 participants; all were 18 years old or older. there is no exact number for validation studies of scales, but between 2 to 20 participants per item are recommended, with a minimum of 100-250 participants22. the mas scale is composed of 30 items, so the estimated sample size ranges from 60-600. instrument the spanish version of the mas is a 30-item scale that measures attitudes towards pwd by asking participants to react to a social scenario between ‘‘josé” or ‘‘claudia” and another individual who is using a wheelchair in a public space. thus, using a person in a wheelchair as prototypical for pwd. respondents are asked to mark their answers on a five-point likert scale, ranging from 1 (not at all) to 5 (very much). high scores indicate negative attitudes towards people with physical disabilities while lower scores indicate positive attitudes toward people with physical disabilities. the calm dimension items need to be inverted as they describe positive statements. in the present research, the stimulus ‘‘person in a wheelchair’’ was changed to “deaf person”. the application of the instrument takes around 5 minutes. the factor analysis, performed by the scale’s authors, revealed the four following factors or dimensions: negative affections (11 items), calm (3 items), cognitions (10 items) and behaviors (6 items). the confirmatory factor analysis, conducted in all the other participants, confirmed the validity and adequacy of the model to 4 factors (v2 = 2.15; rmsea =.068; cfi =.88; ifi =.88; tli =.87)19. validation assessment face and content validity was executed via a cross-cultural adaptation process which was carried out according to beaton et al.23. five chilean public health experts with different academic backgrounds were selected for the committee, which included a psychologist with expertise in disability research and validation studies, three health researchers with experience in disability and public health, and a statistician with experience in validating instruments in health sciences. the committee reviewed the process for obtaining a preliminary version of a modified chilean version of the mas towards deaf persons regarding feasibility, readability, consistency of style and formatting and the clarity of the language used. a test with the pre-final version was carried out with 15 dental students in sixth year at the university of concepción. the difficulties and problems of understanding the instrument were evaluated, and a few changes were made in the final version of the instrument regarding the clarity of the language used. cross-cultural adaptation: the original spanish version says (josé/claudia) with a person in a wheelchair (female/male). firstly, only the stimulus of “person in wheelchair” was replaced by “deaf person”. as students during the pilot reported getting confused whether to focus on josé or claudia and if the deaf person was female or male, after the pilot it was decided to develop four versions of the instrument, one that 4 campos et al. says “josé” and a “young deaf male”; “josé” and a “young deaf female”; “claudia” and a “young deaf male”; “claudia” and a “young deaf female”. data collection two researchers collected sociodemographic data and the application of the instrument through an auto applied survey. to ensure that the questions are not addressed to the respondent directly but based on a projection mechanism ensuring greater honesty of the answers, the four versions were randomly distributed to female/male participants. data were collected in the classroom at the beginning of theory or clinical activities, during the first two months of the second semester of 2018. each course was visited twice in case a student was absent the first time. there was no monetary nor academic compensation associated with participation. all students were invited to participate. each student was informed of the study aim and their voluntary participation was obtained through signed informed consent. participants answered the modified version of the mas towards deaf persons and provided sociodemographic data. ethics this study was approved by the research and bioethics committee of the universidad de concepción school of dentistry (c.i.y.b. n°053/17) and was conducted in full accordance with the world medical association declaration of helsinki. all respondents agreed to voluntary participate by providing signed informed consent. the survey was anonymous, and the information was used only for the purposes of this research. statistical analysis data were tabulated in an excel spreadsheet (ms corp., usa). to examine the adequacy of the data, a multivariate normal distribution was executed by examining the univariate symmetry coefficient and kurtosis of each item, the pearson correlation matrix with the bartlett test of sphericity, and kaiser-meyer-olkin (kmo). for the exploratory factor analysis (efa), maximum likelihood estimation (ml) for determining the number of factors parallel analysis (pa) was used with oblimin for rotation method, loadings lower than 0.3 were not considered. finally, to determine reliability, cronbach’s alpha was calculated for the total score and subscale scores. the indices used to determine the model fitness were root mean square error of approximation (rmsea), the comparative fit index (cfi), incremental fit index (ifi), goodness of fit index (gfi), tucker-lewis fit index (tli-nnf) and for the fitted residuals root mean square of residuals (rmsr). data analyses were conducted using stata 16 (stata corp., usa) and factor 10 (universitat rovira i virgili, spain). results the sample was composed of 311 participants. table 1 details the changes made on this adapted version from the original one. in general, all items had a normal distribution, with the exception of items 7 and 10 (table 2). different models were executed in order to determine the best fit. 5 campos et al. table 1. adaptations made from the original mas. original version: imagine la siguiente situación. josé/claudia fue a almorzar con un grupo de amigos a un restaurante. una persona en silla de ruedas, a quien josé/claudia no conoce, entra en la cafetería y se une al grupo. first version for pilot: imagine la siguiente situación. josé/claudia fue a almorzar con un grupo de amigos a un restaurante. una persona sorda, a quien mario/maría no conoce, entra en el restaurante y se une al grupo. final versions 1. imagine la siguiente situación. claudia fue a almorzar con un grupo de amigos a un restaurante. una joven sorda, a quien claudia no conoce, entra en el restaurante y se une al grupo. 2. imagine la siguiente situación. claudia fue a almorzar con un grupo de amigos a un restaurante. un joven sordo, a quien claudia no conoce, entra en el restaurante y se une al grupo. 3. imagine la siguiente situación. josé fue a almorzar con un grupo de amigos a un restaurante. una joven sorda, a quien josé no conoce, entra en el restaurante y se une al grupo. 4. imagine la siguiente situación. josé fue a almorzar con un grupo de amigos a un restaurante. un joven sordo, a quien josé no conoce, entra en el restaurante y se une al grupo. table 2. symmetry measures for each item. item asymmetry kurtosis p12 -,536 -,222 p13 -,421 -,412 p14 -,381 -,433 p21 -,201 -,849 p30 -,168 -,677 p4 -,164 -,738 p18 ,073 -,565 p22 ,109 -,878 p3 ,130 -,721 p9 ,155 -,906 p27 ,178 -1,081 p1 ,276 -,664 p15 ,312 -,627 p19 ,361 -,040 p17 ,363 -,338 p16 ,390 -,359 p20 ,420 -,315 p29 ,465 -1,025 p23 ,493 -,193 p24 ,624 -,304 p25 ,669 -,654 p8 ,745 -,779 p6 1,033 ,047 p5 1,108 ,197 p26 1,162 ,588 p28 1,391 1,170 p2 1,491 2,213 p11 1,604 1,662 p7 1,878 2,933 p10 2,730 7,582 6 campos et al. model 1 all 30 items were included. a significant bartlett test of sphericity was obtained (p<0.001) and a kmo of 0.855 and an overall 0.8779 cronbach’s alpha. the ml and pa analysis advised a four-dimension model (table 3a) the oblimin rotation for the advised model (table 4a) showed a good internal consistency (factor 1= 0.8918; factor 2= 0.9038; factor 3= 0.8332, factor 4= 0.8315). in table 5, indices of the fitted model can be observed. the dimensions were: negative effects (11 items), calm (3 items), cognition (10 items) and behaviors (6 items). table 3. parallel analysis with and without item 10. parallel analysis based on minimum rank factor analysis factor with all-30 items without item 10 real-data % of variance mean of random % of variance 95 percentile of random % of variance real-data % of variance mean of random % of variance 95 percentile of random % of variance 1 26.3854* 69.362 74.966 25.2298* 7.1391 7.6882 2 16.9582* 64.800 69.601 16.4008* 6.6773 7.1497 3 9.7375* 61.474 65.788 9.2254* 6.3391 6.7502 4 6.6179* 58.574 62.246 5.7940 6.0279 6.3777 5 48.204 55.969 59.386 4.6558 5.7579 6.0850 6 40.024 53.474 56.637 3.9223 5.4936 5.8099 7 30.881 51.249 54.152 3.4597 5.2535 5.5521 8 30.179 48.883 51.499 3.2321 4.9963 5.2848 table 4. oblimin rotated loading matrix for models 1-3. model 1 model 2 model 3 item f1 f-2 f3 f-4 f1 f2 f-3 f4 f-1 f2 f-3 p1     0.567       0.587     0.456   p2 0.439 0.383   0.542   p3     0.737       0.776     0.522   p4 0.766 0.819   0.474 0.353 p5     0.594       0.543     0.411   p6 0.583 0.583   0.372   p7     0.443       0.563     0.543   p8 0.465 0.383   0.324   p9     0.544       0.450     0.330 0.314 p10 0.438       p11     0.401       0.353     0.362   p12 0.755 0.742     0.749 p13 0.935             0.951     0.893 p14 0.894 0.888     0.879 p15   0.577       0.576     0.603     p16 0.679 0.680 0.700     continue 7 campos et al. model 2 a second model was executed after eliminating item 10. a significant bartlett test of sphericity was obtained (p< 0.001) and a kmo of 0.85483 and an overall 0.8761 cronbach’s alpha. the ml and pa analysis advised a three-dimension model (table 3b). the oblimin rotation for the four-dimension model (table 4b) showed a good internal consistency (factor 1= 0.8918; factor 2= 0.9038; factor 3= 0.8332; factor 4= 0.8315). in table 5, indices of the fitted model can be observed. the same structure as model 1 was observed. model 3 as the ml and pa analysis advised a three-dimension structure, we executed the oblimin rotation for the three-dimension model without item 10 (table 3c), which showed good internal consistency (factor 1= 0.8918; factor 2= 0.9038; factor 3= 0.8506). however, this model presented a different structure, as the dimension negative affections was blended with the dimension behaviors. discussion the four-factor structure without item 10 in this efa presented an adequate cronbach’s alpha, suggesting acceptable internal consistency. moreover, the rmsea, tli-nnfi, rmsr, p17   0.760       0.755     0.782     p18 0.658 0.668 0.661     p19   0.798       0.790     0.799     p20 0.766 0.755 0.751     p21   0.622       0.639     0.590     p22 0.506 0.517 0.472     p23   0.748       0.749     0.732     p24 0.653 0.654 0.617     p25       0.869 0.862         0.760   p26 0.834 0.837   0.735   p27       0.632 0.615         0.621   p28 0.680 0.702   0.647   p29       0.681 0.677         0.704   p30       0.346 0.329         0.304   continuation table 5. comparison of indices of the adjusted models. model 1 model 2 model 3 expected values rmsea 0.076 0.075 0.095 ≤0.08 tli-nnfi 0.911 0.916 0.868 ≥0.85-0.90 rmsr 0.0501 0.0473 0.0656 ≤0.0568 gfi 0.971 0.975 0.920 ≥0.95 cfi 0.935 0.939 0.895 ≥0.90 root mean square error of approximation (rmsea), comparative fit index (cfi), incremental fit index (ifi), goodness of fit index (gfi), tucker-lewis fit index (tli-nnf), and root mean square of residuals (rmsr). 8 campos et al. gfi and cfi indices suggested a good fit of the model. also, it showed the best fitted model and was consistent with the literature19-22. all items loaded in a factor with the same four-factor structure, suggesting similarities with the colombian version of the mas19. when examining the pattern of item loadings, though all 30 items loaded with an exceeding absolute value of 0.3 into a factor, item 10 was removed from the scale because of cultural differences. although the word “depresión” is a direct translation of “depression”, in chile, it only has the strong connotation of a diagnosed mental illness, unlike in other countries which it also has the connotation of emotions such as “sadness” or “sorrow”. in the adaptation process, that item was identified as likely problematic, a situation which was confirmed in the efa, which finally suggested its exclusion from this adapted version. in this sense, depression was not considered a consistent part of the attitudes toward deaf persons in this sample. criterion validation was not possible because there were no validated instruments in chile regarding attitudes towards pwd. moreover, as this instrument was already in spanish, a confirmatory analysis (cfa) might have been suggested, but as the stimulus “person in a wheelchair” was changed to “deaf person”, an efa was mandatory in other to pursue further analysis. several limitations should be considered when interpreting the results of this study. first, the sample consisted of individuals receiving undergraduate dental education, as we were interested in determining the effect of an elective course24 on attitudes towards deaf persons. as it has been determined that higher levels of education are associated with more positive attitudes, future studies should also be conducted with different samples and with lower or higher education levels. secondly, the mas is an explicit instrument, so it is possible that social desirability or other factors could have influenced how participants responded. this is contrasted with what was previously reported, where the results of the implicit association test (iat) did not correlate generally with the results of explicit scales25,26. therefore, future studies should focus on designing iats toward people with disabilities in chile. third, the sample size did not allow us to divide the sample in two in order to run an efa with one subsample and a cfa with the other, so it is recommended to consider sample sizes of 500 participants and over. finally, although the tli-nnfi, rmsr, gfi and cfi indices suggested a good fit of the model for the four-factor solution, the rmsea suggested an adequate – but not good – fit. among the strengths of this study is that this spanish modified version of the mas may serve as a useful tool in identifying attitudes towards deaf persons and, from there, generate strategies to address them. different entities such as the world health organization have stated the urgent need for policies that promote the inclusion of pwd1-2. however, to achieve the latter in chile, validated instruments are needed in order to determine these current attitudes. secondly, no student refused to participate, which strengthens our results. another strength is the diverse background of the expert panel, supporting an adequate adaptation process. despite these limitations, it is important to remember that the validation process is permanent, and there is no study that can assure that any instrument has full validity, as each validation study adds evidence on different aspects of the validity for specific uses and particular populations for an individual instrument. 9 campos et al. in conclusion, the spanish modified version of the mas towards deaf persons has a four-factor structure, which in consistent with previous version of the mas. ethics approval this study was approved by the research and bioethics committee of the universidad de concepción school of dentistry (c.i.y.b. n°053/17) and was conducted in full accordance with the world medical association declaration of helsinki. consent to participate each student was informed of the study aim and their voluntary participation was obtained through signed informed consent. conflict of interest none acknowledgements none references 1. world health organization. disability and health: fact sheet nº 352. geneva: who; 2015. 2. world health organization. the world bank. world report on disability. malta: who; 2011. 3. ministry of social development. second national study of disability. chile: ministry of social development; 2015 4. stone dl, colella a. a model of factors affecting the treatment of disabled individuals in organizations. acad manag rev. 1996 apr;21(2):352-401. 5. eagly ah, chaiken s. the advantages of an inclusive definition of attitude. soc cognition. 2007;25(5):582-602. doi: 10.1521/soco.2007.25.5.582. 6. patka m, keys cb, henry db, mcdonald ke. attitudes of pakistani community members and staff toward people with intellectual disability. am j intellect dev disabil. 2013 jan;118(1):32-43. doi: 10.1352/1944-7558-118.1.32. 7. meadow-orlans k, erting c. deaf people in society. in: hindley p, kitson n, editors. mental health and deafness. london: whurr publishers; 2000. p.3-24. 8. ralston e, zazove p, gorenflo dw. physicians’ attitudes and beliefs about deaf patients. j am board fam pract. 1996;9(3):167-73. 9. goodman b, clemow r. nursing and collaborative practice: a guide to interprofessional learning and working. 2nd ed. exeter uk: learning matters; 2010. 10. eagly ah, chaiken s. the psychology of attitudes. orlando: harcourt brace jovanovich college publishers; 1993. 11. maio g, haddock g. the psychology of attitude and attitude change. london: sage publications; 2010. 12. power mj, green am, whoqol-dis group. the attitudes to disability scale (ads): development and psychometric properties. j intellect disabil res. 2010 sep;54(9):860-74. doi: 10.1111/j.1365-2788.2010.01317.x. 10 campos et al. 13. yuker he, block jr, young jh. the measurement of attitudes toward disabled persons. albertson, ny: human resources center; 1996. 14. cooper a, rose j, mason o. measuring the attitudes of human service professionals toward deafness. am ann deaf. 2004;148(5):385-9. doi: 10.1353/aad.2004.0001. 15. belmar n, quappe i, luengo l, campos v. exploratory factor analysis of the chilean deafness attitude scale. int j med surg sci. 2018;5(2):80-8. doi: 10.32457/ijmss.2018.020. 16. campos v, cartes-velásquez r, luengo l. chilean health professionals’ attitudes towards deafness: a cross-sectional study. pesq bras odontopediatria clín integr. 2020:20: e0020. doi: 10.1590/pboci.2020.087. 17. morin d, crocker g, beaulieu-bergeron r, caron j. validation of the attitudes toward intellectual disability: attid questionnaire. j intellect disabil res. 2013 mar;57(3):268-78. doi: 10.1111/j.1365-2788.2012.01559.x. 18. findler l, vilchinsky n, werner s. the multidimensional attitudes scale toward persons with disabilities (mas) construction and validation. rehab couns bull. 2007;50(3):166-76. doi: 10.1177/00343552070500030401. 19. stevens lf, getachew ma, perrin pb, rivera d, olivera plaza sl, arango-lasprilla jc. factor analysis of the spanish multidimensional attitudes scale toward persons with disabilities. rehabil psychol. 2013 nov;58(4):396-404. doi: 10.1037/a0034064.. 20. radlińska i, starkowska a, kożybska m, flaga-gieruszyńska k, karakiewicz, b. the multidimensional attitudes scale towards persons with disabilities (mas)–a polish adaptation (mas-pl). ann agric environ med. 2020;27(4):613-20. doi: 10.26444/aaem/114531. 21. dachez j, ndobo a, ameline a. french validation of the multidimensional attitude scale toward persons with disabilities (mas): the case of attitudes toward autism and their moderating factors. j autism dev disord. 2015 aug;45(8):2508-18. doi: 10.1007/s10803-015-2417-6. 22. lu m, pang f, luo j. chinese validation of the multidimensional attitude scale toward persons with disabilities (mas): attitudes toward autism spectrum disorders. j autism dev disord. 2020 oct;50(10):3777-89. doi: 10.1007/s10803-020-04435-1. 23. beaton de, bombardier c, guillemin f, ferraz mb. guidelines for the process of cross-cultural adaptation of self-report measures. spine (phila pa 1976). 2000 dec 15;25(24):3186-91. doi: 10.1097/00007632-200012150-00014. 24. campos v, cartes-velásquez r. developing competencies for the dental care of people with sensory disabilities: a pilot inclusive approach. cumhuriyet dent j. 2020;23(2):107-15. doi: 10.7126/cumudj.706518. 25. wilson mc, scior k. attitudes towards individuals with disabilities as measured by the implicit association test: a literature review. res dev disabil. 2014 feb;35(2):294-321. doi: 10.1016/j.ridd.2013.11.003. 26. hein s, grumm m, fingerle m. is contact with people with disabilities a guarantee for positive implicit and explicit attitudes? eur j spec needs educ. 2011;26(4):509-22. doi: 10.1080/08856257.2011.597192. 1http://dx.doi.org/10.20396/bjos.v21i00.8665580 volume 21 2022 e225580 original article 1 meridional faculty imed, passo fundo, rs, brazil. corresponding author: lilian rigo meridional faculty/imed, passo fundo, rio grande do sul, brazil senador pinheiro street, 304 passo fundo (rs) – brazil 99070-220 (+55 54) 99927-0441 e-mail: lilian.rigo@imed.edu.br editor: dr altair a. del bel cury received: may 10, 2021 accepted: august 28, 2021 factors associated with decision-making for replacing the temporary coronal restoration after endodontic treatment bárbara scarton fornari1, caroline solda1 , lara dotto1 , lilian rigo1 aim: this study aimed to evaluate the decision-making by patients to replace temporary restorations with permanent restorations after endodontic treatment and to verify the associated factors and evaluate the quality/integrity of the temporary restorative material within one month. methods: this is a cross-sectional study using non-probabilistic sampling which analyzed patients after one month of endodontic treatment. the self-administered questionnaire contained sociodemographic, treatment decision-making and endodontic treatment questions. the restoration present in the mouth was evaluated in the clinical oral examination. the poisson regression test was used to verify the prevalence ratio. results: the prevalence failure to perform permanent restorations was 61.1% of patients, and 42.7% reported not having adhered. the reasons are lack of time and not knowing the importance of replacing the restoration with a definitive one. the glass ionomer temporary restorative frequency was higher among those who chose not to replace the temporary restoration with a permanent one (pr=5.19; 95%ci 2.10-12.33). in addition, there was an association between the quality of the restorative material and the type of material, and the best clinical quality of the restoration was statistically associated with glass ionomer and composite resin. conclusions: the findings show the importance of guidance by the dental surgeon in helping patients decide to replace their temporary restoration. keywords: definitive dental restoration. temporary restoration. endodontics. https://orcid.org/0000-0003-4254-931x https://orcid.org/0000-0003-1535-4736 http://orcid.org/0000-0003-3725-3047 2 fornari et al. introduction the careful performance of the chemical-mechanical preparation, filling and sealing the root canals are among the stages of endodontic treatment. if there are failures in one of these stages, the treatment case may fail1. it is also important to point out that the immediate coronary and quality sealing directly influences the longevity of the endodontic treatment and survival of these elements2-4. the final restoration can be direct or indirect, depending on the amount of coronal structure and the type of cavity that the element shows, promoting a shield at the mouth of the root canal to prevent bacterial microleakage and enhance and protect the tooth structure5. provisional restorations are usually made with materials that meet the marginal integrity requirements and establish masticatory function for a short time until the final restoration can be performed. some studies highlight that one of the causes which can lead to root canal therapy failure is the fluid pathway from the oral cavity to the tooth through the temporary restorative material called coronary microleakage6. these microleakages can promote recontamination of the root canal system and a new periapical pathology requiring further intervention. therefore, endodontically treated teeth should be restored with definitive materials as soon as possible to avoid this type of failure. if a final coronal restoration is not possible, selecting a good quality temporary restorative material is a crucial factor2,6,7. temporary restorations may be necessary when the cavity is extensive and there is the loss of dental structure with the indication of indirect restorations8. thus, if it is not possible to perform a definitive restoration immediately after the endodontic treatment, it is essential to select a temporary material with more excellent color stability and resistance to different liquid pigments to optimize the aesthetics of the restorations9. therefore, the objectives of provisional restorations are to meet requirements such as marginal integrity and to establish masticatory function for a certain period until the final restoration is performed10. then, the endodontic treatment is only considered finished once this final restoration is performed. patients must be educated about the need to change the provisional restorative material for a permanent restoration and the possible consequences if the procedure is neglected11. thus, this research aimed to evaluate the adherence of patients to replace the temporary restorations after endodontic treatment for permanent restorations and the associated factors, as well as to assess the quality/integrity of the provisional restorative material within one month. materials and methods the study was submitted to the research ethics committee of the faculty and approved on april 10, 2019 (3.257.716, caae 11117319.0.0000.5319). all participants filled out the consent form to participate in the research, according to resolution number 466/12. 3 fornari et al. study design and sample this study implemented a cross-sectional design. the study was sampled by convenience, with all patients who returned for dental care one month after completing endodontic treatment on permanent teeth in a postgraduate center in the period of four months (december 2019 to march 2020), regardless of age. the study population was 239 patients. only 38 patients did not return for the post-endodontic revision, with a loss of 16%. data collection data collection was carried out during the post-endodontic review consultation after one month. an oral clinical examination evaluated the endodontically treated tooth and the self-application of a questionnaire between december 2019 and march 2020. the instrument used was a self-application questionnaire on sociodemographic questions such as gender, age, marital status, education level, family income, occupation, and city of residence. treatment decision-making if the restoration was final, with no reason to make the final restoration, type of permanent restorative material and place of performing the final restoration were also collected. the questions related to endodontic treatment were taken from the patients’ medical records and included endodontically-treated tooth, endodontic filling date and type of provisional material used. the oral clinical examination evaluated the patients’ adherence to the restoration and the temporary restoration quality in the mouth. variables the outcome variable of this study was “non-adherence to permanent restoration after endodontic treatment” after one month. the variable was constructed and evaluated as follows: ‘yes’ represents the performance of the final restoration (= 0), and ‘no’ represents the failure to perform the final restoration during the allotted time period (= 1). exposure variables were: 1. sociodemographic: gender (male / female); age group (14-29 / 30-45 / 46-71 years); marital status (single, widowed, divorced / married, common-law marriage); level of education (elementary and high school / higher education and postgraduate); family income (up to 1 minimum monthly salary / 2 to 10 minimum monthly salaries); occupation (employee / unemployed, retired); city of residence (passo fundo / other municipalities in the interior of the state). 2. restoration decision-making: a reason for not getting the final restoration (i did not know it was important to do it so soon / i did not have time / i did not have money / i do not have access to the service); type of temporary restorative material (composite resin and glass ionomer/cotosol®); place of the final restoration (public service / private service). 3. provisional restoration integrity, infiltration, fracture and loss the quality of the provisional restoration was defined according to the criteria of the world dental federation / fdi12, however, observing the item that refers to functional properties, and only evaluating the topic material fracture and retention (5 evaluation items): 4 fornari et al. 1. clinically excellent / very good no fractures or cracks; 2. clinically good small fracture lines; 3. clinically sufficient / satisfactory two or more or extensive fracture line and/or splinter (not affecting marginal integrity); 4. clinically unsatisfactory splinter fractures that cause damage to marginal integrity, volume fracture with or without a partial loss (less than half of the restoration). or fracture with partial loss of material (less than half of the restoration); 5. weak satisfactory loss of restoration (partial or complete) or multiple fractures. for statistical analysis purposes, items 1 and 2 were defined as clinically satisfactory restorations and items 3, 4 and 5 as clinically unsatisfactory and unsatisfactory restorations. examiner training and calibration a pilot test was conducted on ten patients to test the methodology used and the possible difficulties encountered by the participants and the researcher. the questionnaires were analyzed after conducting the data collection for the pilot test, and the instrument’s questions were observed, which is considered adequate for this study. a specialist in endodontics performed all clinical examinations and established uniform standards and determined acceptable levels of internal examiner consciousness. next, 8 hours of training was performed with intact restorations of images and failures: infiltrated with wear and fractures, according to the criteria of the “functional properties” of the fdi12. the kappa concordance test (p<0.05) was subsequently performed to test the intra-examiner agreement. the kappa results’ agreement in the clinical examination in verifying the quality of the restoration was 85%, which is considered a good to excellent agreement. analysis of results the data obtained were organized in excel form and exported to the ibm spss® statistical software program (statistical package for the social sciences, version 20.0, armonk, new york). descriptive analyzes of all variables were performed to present their relative and absolute frequencies. pearson’s chi-squared test was used in the bivariate analyzes. crude and adjusted poisson regression with robust variance was used in the multivariable analysis to obtain the prevalence ratios (pr) and respective 95% confidence intervals (95% ci). for the confusion adjustment, all exploratory variables that entered the model had a p-value <0.20, but only those with a p-value <0.05 remained in the adjusted analysis. results of the patients who took part in the study, 47.7% are female and 52.35 are male, with a mean age of 40 years (sd ± 15.34), a minimum age of 15 years, and a maximum age of 70 years. sociodemographic characteristics about marital status, income, education, occupation and city of residence are shown in table 1. the majority of endodontically-treated teeth were upper posterior (50.2%), followed by lower posterior teeth (28.9%), and finally upper and lower anterior teeth (20.9%). the temporary materials used were glass ionomer cement (61.9%), followed by composite resin (20.5%) and provisional restorative / coltosol® (17.6%). 5 fornari et al. table 1. distribution of the variable of all patients returned one month after endodontic treatment, brazil, 2020 (n = 239). variables n % gender female 114 47,7 male 125 52,3 age group 15-29 years 64 26,8 30-45 years 85 35,6 46-70 years 90 37,7 marital status single / widowed / divorced 104 43,5 married / common-law marriage 135 56,5 education level elementary / high school 161 67,4 higher education / postgraduate 78 32,6 family income up to 2 minimum wages 101 42,3 3 to 10 minimum wages 138 57,7 occupation employee / retired 202 84,5 unemployed 37 15,5 city passo fundo 99 41,4 others municipalities (interior) 140 58,6 endodontically treated teeth upper and lower anterior teeth 50 20,9 upper posterior teeth 120 50,2 lower posterior teeth 69 28,9 temporary restoration material composite resin 49 20,5 glass ionomer cement 148 61,9 temporary restorative (coltosol®) 42 17,6 final restoration no 146 61,1 yes 93 38,9 place where permanent restoration was performed (n = 93) public service 72 77,4 private service 21 22,6 restoration material (n=93) composite resin 93 100,0 6 fornari et al. of the 239 patients, 61.1% did not make the final restoration after the endodontic treatment. of the 38.9% who carried out the exchange of final restoration, 77.4% did in public service, and the restorative material was composite for 100%. table 2 shows only the patients (61.1%) who did not adhere to the definitive restoration. of these, most reported a lack of time (26.4%), followed by not knowing the importance of exchanging provisional for permanent material (16.3%). furthermore, when evaluating the clinical quality of the temporary restoration in these patients who did not make a final restoration, it is observed that clinically satisfactory restorations represented 26.4%. in comparison, the unsatisfactory restorations were 61.1%. table 3 shows the analysis of the prevalence of non-adherence to permanent restoration after one month of endodontic treatment. the prevalence of maintenance of provisional restorations for those who did not adhere to permanent restoration was 58.2% in the youngest, 81.2% in the posterior teeth, and 67.1% in the individuals who had their teeth restored with glass ionomer cement and 24.7% whose provisional restorative material was coltosol®. table 2. distribution of the frequencies of the variables of patients who did not adhere to the definitive restoration after endodontic treatment, brazil, 2020 (n = 146). variables n % reason for non-realization i didn’t know it was necessary to do 39 16,3 there was no time 63 26,4 had no money 3 1,3 did not have access to the service 41 17,2 quality of provisional restoration satisfactory 63 26,4 unsatisfactory 83 61,1 table 3. crude and adjusted prevalence (%) and prevalence ratio (pr) of non-adherence to definitive restoration after endodontic treatment, brazil, 2020 (n = 239). prevalence p-value* crude pr (ic95%) p-value** adjusted rp (ic95%) p-value** gender female 45,9 male 54,1 0,285 age 15-50 years 58,2 1,00 1,00 51-70 years 41,8 0,065 1,33 (0,86;2,07) 0,199 1,31 (0,84;2,03) 0,228 city passo fundo 39,7 others municipalities 60,3 0,297 continue 7 fornari et al. all variables associated with the crude analysis (age group, groups of teeth and restored material at p<0.20) entered the multivariate model to perform the poisson regression. after adjusting for confusion, the variables age group and groups of teeth left the model (p>0.05), with only the type of restorative material remaining. the probability of the provisional glass ionomer restorative material was higher among those who decided not to exchange the restoration for the final one (pr=5.19; 95%ci 2.10-12.33). a bivariate analysis was performed to verify the association between the type of provisional material (grouping the glass ionomer and the composite resin together; and coltosol® separately) and not completing the permanent restoration with the variable quality of the temporary restorative material. after the analysis, a statistically significant association was observed between the quality of the material and the type of restorative material variables. the best quality of the restoration was statistically associated with glass ionomer and composite resin (p<0.001) (table 4). continuation marital status single/widowed/ separated 41,8 0,293 married/common-law marriage 58,2 eduaction elementary/high school 67,1 higher education/ postgraduate 32,9 0,518 family income up to 1 minimum wage 43,2 two or more minimum wages 56,8 0,415 occupation employee/retired 84,2 unemployed 15,8 0,519 tooth upper and lower anterior teeth 18,5 1,00 1,00 upper and lower posterior teeth 81,5 0,160 1,24 (0,77;1,98) 0,375 1,13 (0,71;1,82) 0,589 temporary material composite resin 8,2 1,00 1,00 glass ionomer cement 67,1 <0,001 5,28 (2,23;2,52) <0,001 5,19 (2,10;12,33) <0,001 coltosol® 24,7 2,36 (1,01;5,51) 0,046 2,37 (0,01;5,54) 0,056 * pearson’s chi-square test (p <0.20)** teste de wald (p<0,05) pr prevalence ratio; 95% ci 95% confidence interval. it is adjusted for the variables: age group, groups of teeth and temporary material. 8 fornari et al. discussion adhesion of the temporary restoration exchange after endodontic treatment by patients was assessed in the present study. their sociodemographic characteristics, the type of provisional restorative material, and in the case of non-adhesion of the exchange, the integrity of these restorations was evaluated after one month. most patients were male, employed or retired and had the temporary glass ionomer cement restoration. it was observed that more than half of patients did not undergo the final restoration after the endodontic treatment. among the justifications, the majority reported a lack of time, followed by not knowing the importance of exchanging provisional material for the permanent one. still, of those who carried out the exchange of the final restoration, the vast majority performed it in some public service, and the restorative material in all of them was composite resin. according to sadaf4 (2020), it is possible to increase the survival rate of an endodontically-treated tooth with a well-performed coronary restoration. in a retrospective study evaluating private clinical data in germany, approximately 86% of the 795 endodontically-treated tooth restorations with an average follow-up of 4.5 years were considered successful, and the annual failure rate was minimal13. authors agree that the survival of endodontically-treated teeth is associated with permanent coronal restorations14. factors such as the restorative material used, cusp coverage and direct or indirect procedure can also affect the performance of endodontic restorations over time. the materials most used as temporary restorers in the present study were glass ionomer cement and composite resin7. soares et al.7 (2018) recommend using a cement 1-2 mm glass ionomer to cover root canal filling to reduce stress inside the pulp chamber and in the furcation area of the posterior teeth. they also recommend using some bulk filler composite resins to minimize deflection of the cusp and the stress concentration in weakened regions. table 4. bivariate analysis between the temporary material, the reason for not performing the definitive restoration and the clinical quality of the provisional restoration of patients who did not adhere to the final restoration after endodontic treatment, brazil, 2020 (n = 136). variables clinical quality of the restoration *psatisfactory unsatisfactory n % n % temporary materials <0,001 glass ionomer / composite resin 88 56,0 54 65,1 temporary material (coltosol®) 7 11,1 29 34,9 reason for not performing the final restoration 0,285 lack of money and access 38 63,3 45 54,2 lack of time 25 39,7 38 45,8 * pearson’s chi-square test (p <0.05) statistically significant 9 fornari et al. in this study, it was observed that two-thirds had pigmented, infiltrated, fractured provisional restorations and/or there was no such restoration of the patients who did not adhere. thus, the quality of the temporary restoration was found to be associated with the type of material. those with the worst quality were those who had the coltosol® provisional material related to those who had temporary restoration with ionomer cement of glass and composite resin. endodontically-treated teeth lose substantial structure due to previous restorations, dental caries and the preparation of access for endodontic treatment. thus, the restoration of these teeth is complex and their long-term prognosis is directly related to the quality of the final restoration15. an excellent temporary restorative material must prevent the root canal system from being contaminated by saliva, fluids and microorganisms16. the composite resin and glass ionomer cement adhere to the tooth structure, preventing the infiltration of oral fluids at the cement-tooth interface17. in a retrospective study evaluating 220 endodontically-treated permanent molars, the authors observed that composite restorations had a longer average survival time than those constructed with amalgam. furthermore, the amount of remaining tooth structure was the most significant factor for the longevity of the restorations18. however, stenhagen et al.19 (2020) assessed the choice of coronary restorations for endodontically-treated teeth, examined the survival of restorations and the coronary restoration on the success of endodontic treatment. however, no significant correlations were found between the type of coronary restoration and the quality of endodontic treatment. in the present study, there was a greater probability with the type of provisional material after logistic regression analysis of the adhesion of the restorative material, influencing the failure to perform the permanent restoration. glass ionomer cement is similar to the tooth in terms of color, texture, adaptation, and durability, which may have influenced patients not to change it. the glass ionomer properties are biocompatibility, physical-chemical adhesion to enamel, dentin and cement and having a similar thermal expansion coefficient to the natural dental structure20-22. although the glass ionomer cement was the most efficient between the moment of root canal filling and the evaluation consultation, one study investigated the association between the type of coronary restoration and the survival of endodontically treated teeth. the results of the study showed that the survival of endodontically-treated teeth was significantly longer when restored with molten restorations, amalgam restorations or composite restorations than teeth restored with provisional materials14. in another retrospective study investigating the influence of endodontic retreatment in the choice of definitive restoration, extensive restorations involving the insertion of pins and indirect crowns’ manufacture had a higher retreatment rate23. solubility, thermal expansion, porosity and contraction are significant variables in the clinical performance of provisional materials24,25; however, there are other factors capable of altering the sealing of these restorations, including: improper procedures and techniques; inadequate adaptation of the material to the cavity by carelessness or haste; maintenance of impurities between the cavity and the temporary restoration; cavity depth; and the number of dentinal tubules on the tooth surface26. thus, endodontic treatment must be completed with an adequate coronary seal; in some 10 fornari et al. cases, it is necessary to use provisional restorative materials due to time limitations or more extensive rehabilitation treatments. materials should be replaced with better adherence to permanent restorations as soon as possible. it is up to the professional to explain the importance and potential implications, such as the doubtful prognosis of the treatment performed. one of the limitations of this study was the absence of the return of some patients for consultation after one month of endodontic treatment. this fact led to sample loss in the study, which may seem that these patients did not consider it essential for the consultation, and perhaps the replacement of the restoration was also neglected, which could result in higher non-adherence prevalence. another limitation was concerning the study’s design being cross-sectional and not establishing a cause-effect relationship because it does not longitudinally follow the endodontic prognosis of patients inferring accurate long-term findings. the findings of this study highlight the importance of the clinician in helping and instructing patients in making decisions regarding the fundamental need to replace the provisional restoration with the permanent one after filling the root canal. the lack of adequate and precise information generates doubts. in the absence of visible flaws in the provisional restoration, the decision to substitute the temporary restorations for the permanent one is often neglected. however, even with minor imperfections in the temporary restorations which are invisible to patients, such as pigmentation and slight fracture, there may be recontamination of the root canal system, leading to unsuccessful treatment. although the present study is one of the few carried out with this methodology, other studies with methodological control and longer follow-up are necessary. more specific and practical materials and protocols should be created in the final coronary seal of endodontic treatment, considering its importance and lack of awareness of patients to replace the temporary material for the final restoration. in conclusion, this study observed a low prevalence of adherence to perform the permanent restoration after one month of the endodontic treatment. two-thirds of the patients did not do it, presenting the provisional restorations with infiltrations, pigmented, fractured material and/or with an absence of the material. it was also observed that the worst quality of the temporary restorations was directly related to the type of temporary material after endodontics, suggesting a poor long-term prognosis. place or institution where the work was developed, city and country: meridional faculty/imed, passo fundo, rio grande do sul, brazil conflict of interest: the authors declare no conflict of interest author’s participation: fornari bs worked in structuring the article, designed the method, preparation of database and analysis of results. solda c worked in project design, in data collection and entering the database. dotto l worked in discussion of the methodology, in the statistical analysis. rigo l worked in review of the english language and the final wording of article. 11 fornari et al. references 1. siqueira jf jr. a etiology of root canal treatment failure: why well-treated teeth can fail. int endod j. 2001 jan;34(1):1-10. doi: 10.1046/j.1365-2591.2001.00396.x. 2. ng yl, mann v, gulabivala k.  tooth survival following non-surgical root canal treatment: a systematic review of the literature. int endod j. 2010 mar;43(3):171-89. doi: 10.1111/j.1365-2591.2009.01671.x. 3. gillen bm, looney sw, gu ls, loushine ba, weller rn, loushine rj, et al. impact of the quality of coronal restoration versus the quality of root canal fillings on success of root canal treatment: a systematic review and meta-analysis. j endod. 2011 jul;37(7):895-902. doi: 10.1016/j.joen.2011.04.002. 4. sadaf d. survival rates of endodontically treated teeth after placement of definitive coronal restoration: 8-year retrospective study. ther clin risk manag. 2020 feb 21;16:125-31. doi: 10.2147/tcrm.s223233. 5. shu x, mai q q, blatz m, price r, wang x d, zhao k. direct and indirect restorations for endodontically treated teeth: a systematic review and meta-analysis, iaad 2017 consensus conference paper. j adhes dent. 2018;20(3):183-94. doi: 10.3290/j.jad.a40762. 6. deepak s, nivedhitha ms. comparison of coronal microleakage of three temporary restorative material using dye penetration methods. j adv pharm educ res. 2017;7(3):232-5. 7. soares cj, rodrigues pm, silva alf, santos-filho pcf, veríssimo c, kim hc, et al. how biomechanics can affect the endodontic treated teeth and their restorative procedures? braz oral res. 2018 oct;32(suppl 1):e76. doi: 10.1590/1807-3107bor-2018.vol32.0076. 8. jensen al, abbott pv, castro salgado j. interim and temporary restoration of teeth during endodontic treatment. aust dent j. 2007 mar;52(1 suppl):s83-99. doi: 10.1111/j.1834-7819.2007.tb00528.x. 9. mickeviciute e, ivanauskiene e, noreikiene v. in vitro color and roughness stability of different temporary restorative materials. stomatologija. 2016;18(2):66-72. 10. guler au, yilmaz f, kulunk t, guler e, kurt s. effects of different drinks on stainability of resin composite provisional restorative materials. j prosthet dent. 2005; 94(2):118-24. doi: 10.1016/j.prosdent.2005.05.004. 11. han gs, shim ys, choi yr, jang so. viscosity, micro-leakage, water solubility and absorption in a resin-based temporary filling material. ind j sci technol. 2015; 8(25):1-5. doi: 10.17485/ijst/2015/v8i25/80262. 12. hickel r, peschke a, tays m, major i, bayne s, peters m, et al. fdi world dental federation: clinical criteria for the evaluation of direct and indirect restorations update and clinical examples. clin oral invest. 2010 aug;14(4):349-66. doi: 10.1007/s00784-010-0432-8. 13. laske m, opdam nj, bronkhorst em, braspenning jc, huysmans mc. longevity of direct restorations in dutch dental practices. descriptive study out of a practice-based research network. j dent. 2016 mar;46:12-7. doi: 10.1016/j.jdent.2016.01.002. 14. lynch cd, burke fm, ní ríordáin r, hannigan a. the influence of coronal restoration type on the survival of endodontically treated teeth. eur j prosthodont restor dent. 2004 dec;12(4):171-6. 15. morgano sm, rodrigues ah, sabrosa ce. restoration of endodontically treated teeth. dent clin north am. 2004 apr;48(2):vi, 397-416. doi: 10.1016/j.cden.2003.12.011. 16. balkaya h, topçuoğlu hs, demirbuga s. the effect of different cavity designs and temporary filling materials on the fracture resistance of upper premolars. j endod. 2019 may;45(5):628-33. doi: 10.1016/j.joen.2019.01.010. 12 fornari et al. 17. kameyama a, saito a, haruyama a, komada t, sugiyama s, takahashi t, et al. marginal leakage of endodontic temporary restorative materials around access cavities prepared with pre-endodontic composite build-up: an in vitro study. materials (basel). 2020 apr;13(7):1700. doi: 10.3390/ma13071700.  18. nagasiri r, chitmongkolsuk s. long-term survival of endodontically treated molars without crown coverage: a retrospective cohort study. j prosthet dent. 2005 feb;93(2):164-70. doi: 10.1016/j.prosdent.2004.11.001. 19. stenhagen s, skeie h, bårdsen a, laegreid t. influence of the coronal restoration on the outcome of endodontically treated teeth. acta odontol scand. 2020;78(2):81-6. doi: 10.1080/00016357.2019.1640390. 20. seiler kb. an evaluation of glass ionomer-based restorative materials as temporary restorations in endodontics. gen dent. 2006;54(1):33‐6. 21. sivakumar js, suresh kumar bn, shyamala pv. role of provisional restorations in endodontic therapy. j pharm bioallied sci. 2013;5(suppl 1): s120‐4. doi: 10.4103/0975-7406.113311. 22. dowling ah, fleming gj. are encapsulated anterior glass-ionomer restoratives better than their hand-mixed equivalents? j dent. 2009 feb;37(2):133-40. doi: 10.1016/j.jdent.2008.10.006. 23. agrafioti a, giannakoulas dg, kournetas n, grigoriou s, kontakiotis eg. different patterns of restoration provision between initial endodontic treatment and retreatment: a retrospective clinical study. int j prosthodont. 2017 jul/aug;30(4):354-6. doi: 10.11607/ijp.5121. 24. ciftçi a, vardarli da, sönmez is. coronal microleakage of four endodontic temporary restorative materials: an in vitro study. oral surg oral med oral pathol oral radiol endod. 2009;108(4):e67-70. doi: 10.1016/j.tripleo.2009.05.015. 25. pieper cm, zanchi ch, rodrigues-junior sa, moraes rr, pontes ls, bueno m. sealing ability, water sorption, solubility and toothbrushing abrasion resistance of temporary filling materials. int endod j. 2009;42(10):893-9. doi: 10.1111/j.1365-2591.2009.01590.x. 26. zmener o, banegas g, pameijer ch. coronal microleakage of three temporary restorative materials: an in vitro study. j endod. 2004;30(8):582-4. doi: 10.1097/01.don.0000121610.63000.f2. 1http://dx.doi.org/10.20396/bjos.v19i0.8658336 volume 19 2020 e201674 original article 1 department of operative dentistry, endodontics and dental materials, bauru school of dentistry, university of sao paulo, bauru, sp, brazil 2 department of comprehensive care at case western reserve university, school of dental medicine, cleveland, oh, usa corresponding author: adilson yoshio furuse https://orcid.org/0000-0003-4705-6354 email: furuse@usp.br departamento de dentística, endodontia e materiais odontológicos faculdade de odontologia de bauru universidade de são paulo alameda dr. octávio pinheiro brisola, 9-75 cep 17012-901 telefone: (14) 3235-8000 bauru sp received: may 31, 2019 accepted: january 22, 2020 color evaluation of white spot lesions treated with resin infiltration after water or grape juice storage adilson yoshio furuse1,*, constantino fernandes neto1, genine moreira de freitas guimarães1, bianca rodrigues terrabuio1, fabio antonio piola rizzante2, linda wang1 aim: to evaluate the color stability of bovine enamel with artificial white spot lesions treated with resin infiltration (icon) or remineralization with fluoride using two storage methods. methods: sixty incisors were submitted to artificial white spot lesion induced by demineralization-remineralization (de-re) cycling. initial color was evaluated with cie-lab to measure δeab. demineralized teeth were divided according to the treatment of the white spot lesion (n = 20): 1) remineralization with 2% neutral fluoride gel for 4min (control); 2) icon application following manufacturer’s recommendations; and 3) icon with decreased drying time after the application of ethanol. after 24h, color was evaluated and samples were subdivided (n = 10) according to storage: 1) distilled water for 1 month; 2) grape juice for 10min daily. after storage, color was evaluated. l*, a* and b* data were analyzed by one-way anova and δeab data by two-way anova followed by tukey’s hsd (α = 0.05). results: l* was affected by juice storage, and decreased when icon was applied with decreased drying time after the ethanol application. the same behavior occurred with a* (increase with reduced drying time), while b* was not affected. for δeab significant differences were observed between groups (p = 0.0219) and storage methods (p = 0.0007). there was no interaction effect (p = 0.1118). remineralization with fluoride presented the lowest color changes after storage in water. conclusion: treatment of artificial carious lesions with resin infiltration presented greater color changes than fluoride remineralization after storage in both solutions in vitro. keywords: dental caries. dental enamel. esthetics. https://orcid.org/0000-0003-4705-6354 2 furuse et al. introduction dental caries is a common multifactorial disease that depends on host susceptibility, microorganism contamination, sugar rich diet and time for progression1. enamel carious lesions are initially characterized by a pseudo intact surface layer and loss of minerals within the lesion body2. its first clinical sign is the presence of opaque white spot lesions, which consist of non-cavitated subsurface lesions that can be treated conservatively using preventive approaches3. among these approaches, the application of fluoride in the form of solution, gel or varnish are the most common in dental clinics, since it is cost accessible and easy to use4. in addition, the introduction of resinous materials to seal pits and grooves and to infiltrate white spot lesions brought the advantage of being an immediate treatment. however, studies with infiltrant in anterior teeth have questionable esthetic in masking white spot lesions in the long-term5-7. the appearance of a white spot occurs due to the imbalance in the processes of demineralization and remineralization that precedes the presence of early caries in enamel8. the increased porosity of the tissue allows air penetration within the enamel structure, resulting in altered optical properties of the dental surface that occur due to the difference in the refractive index between hydroxyapatite and air, which are 1.62 and 1.00, respectively8. these changes cause the appearance of opaque white spots, which can be reversed through remineralization procedures. because of this reversible characteristic, any white spot lesion should preferably be detected and treated, thus avoiding further loss of tooth structure and possible need of restoration9. the method for treatment of incipient caries with the use of infiltrating agents was introduced in the past decade. the product, known commercially as icon (dmg, hamburg, germany), can be used on proximal or smooth areas and is a highly fluid resin, which improves its penetration and results in an increased microhardness of the lesion surface10. besides, icon promotes masking of white spot lesions based on changes in light scattering previously described11. icon infiltrant is presented in a kit consisting of three separate steps: 1) icon etch (15% hydrochloric acid), to remove the mineralized surface layer; 2) icon dry (99% ethanol), to penetrate in the demineralized enamel and dentin, to remove water and to improve the penetration of resin monomers, and 3) icon infiltrant, a light-curing resin, composed of triethylene glycol dimethacrylate (tegdma). through a non-invasive procedure, the resin penetrates the subsurface lesions without further damaging the tissues and prevents infiltration and diffusion of cariogenic acids inside the lesion, arresting caries progression12.the low viscosity of resin monomer and the high penetration coefficient are favorable characteristics of this material13. clinical studies concluded that the use of infiltrating agents to control caries disease by non-operative approaches were more effective in reducing the progression of proximal caries than using invasive methods10,14. although resin infiltration of white spot lesions has been showing promising results10,15. the application of this material should be properly indicated, since the infiltrant acts differently in primary and permanent teeth due toto differences in the degree of miner3 furuse et al. alization of each dentition16. besides, that effect on white spots depends on the depth of penetration of the resin infiltrant17. previous studies have reported limitations for the infiltrant to penetrate in lesions affecting dentin, attributing it to the presence of water in this tissue which restricts the penetration of resin monomers10. in addition, depending on the sorption and solubility of the resinous matrix, the long-term exposure to dyes and acidic solutions may degrade resin monomers through processes of swelling, plasticization, softening, oxidation, and hydrolysis, affecting its color stability18. based on these previous assumptions, the infiltrant application technique should be strictly performed according to the manufacturer’s recommendation and further studies are necessary to determine the long-term benefits of the infiltrant application19,20. thereby, a hypothesis to be studied is whether possible variations and/or errors in the infiltrating technique could compromise the success of the final treatment. in addition, the esthetic aspects related to the use of icon should also be further investigated, evaluating its effectiveness of resin infiltrants to mask white spots lesions in anterior teeth. for this reason, it is important to consider the esthetic aspects after the application of infiltrants, since changes in color should be expected, considering the material aging, in which it is exposed to staining agents found in patient’s diet. these extrinsic color changes can be found in foods and beverages, such as soft drinks, fruit juices and coffee. due to the solubility and water absorption rate, the extrinsic coloring susceptibility of this resinous material may be linked to the resin matrix used13. due to these factors, analyzing patient’s habits, age and resin properties are of great relevance in order to avoid future esthetic problems. furthermore, variations in the application technique related to inappropriate drying of the tooth may also play an important role in the final color. thus, it is important to evaluate whether variations in the application technique and regarding exposure to staining solutions would influence the color stability of teeth with artificial white-spot lesions treated with icon. therefore, the purpose of this in vitro study was to evaluate the color stability of bovine enamel with artificial white spot lesions, treated with infiltrating resin (icon) or remineralization with neutral fluoride using different application techniques of icon and two forms of storage (water or juice). materials and methods sixty sound, free of cracks or malformations, bovine teeth had their color evaluated (initial sound) using a cie-lab based spectrophotometer (vita easyshade 3d master, vita zahnfabrik, bädsackingen, germany). the color readings were standardized by performing the analysis in the same room with controlled illumination by the same operator. the tip of the device was held perpendicular to the enamel surface and three readings were performed for each specimen. subsequently, artificial white spot lesion was induced by demineralization-remineralization (de-re) cycling at ph 4.7 and 37°c for 7 days21. the bovine tooth samples were immersed in demineralizing solution during 6h for 5 days and after immersed in remineralizing solution during 18h in the last 2 days of cycling. the demineralizing and remineralizing solutions used in this process are composed respectively of: 2.0  mm ca(no3)2.4h20, 2.0mm nah2po4.2h2o, 0.077mm acetate buffer, 0.02 ppm f (demineralizing solu4 furuse et al. tion); and 1.5mm ca(no3)2.4h2o, 0.9mm nah2po4.2h2o, 150mm kcl, 0.1mol/l tris buffer, 0.03ppm f (remineralizing solution). after the induction of artificial white spot lesion, a second color measurement was performed (initial white spot). for the sake of confirming the white spot lesion formation, the color change caused de-re cycling (δeab) was calculated using the cie-lab color system from the individual values of l*, a* and b*, according to the following equation: δeab = [(δl*) 2 + (δa*)2 + (δb*)2]1/2 δl *, δa * and δb * represent the differences between the readings of the color parameters before and after ph cycling. all teeth had δeab considered visible to the human eye being δeab > 3 (mean value of 4.85) 22. groups were divided according to the treatment of white spot lesions (n = 20 per group): 1) remineralization with neutral fluoride gel 2% (flugel, dfl indústria e comércio sa, rio de janeiro, rj, brazil) for 4 min (control); 2) icon (dmg do brasil, são paulo, sp, brazil) application according to the manufacturer’s recommendations; 3) icon application with decreased drying time after the application of ethanol (icon dry). for determination of the sample size, α and β errors of 5% and 20% were respectively set and a standard deviation of 1.5 obtained in the pilot study was used. for group 1, the surface was cleaned with fine pumice and rubber cups, followed by application of thee neutral fluoride for 4 min. excesses were removed with a microbrush. for groups 2 and 3, the surface of the specimens was also cleaned, followed by acid etch with 15% hydrochloric acid (icon-etch) for 2 min, washing with water and drying with air jets for 30s. ethanol (icon-dry) was applied for 30s followed by drying with air for 30s (group 2) and for 5s (group 3). the infiltrator (icon-infiltrant) was applied to the surface twice, the first time for 3 min and the second time for 1 min according manufacturer’s recommendations followed by photoactivation (valo cordless, ultradent products inc., south jordan, ut, usa) for 40s both application steps. the led device was used in standard mode at 1000 mw/cm2. a digital flat-response power-meter (new port model 2936-r; irvine, ca, usa) was used to measure the power in w with the tip of the light source placed in close contact with the device. the irradiance (in mw/cm2) was obtained by dividing the power (in mw) by the area of the led tip23. after photoactivation a third color reading was performed (icon0). the specimens were stored in artificial saliva24, composed of: 1.5mm/l ca(no3)2. 4h2o, 0.9mm/l nah2po4.2h2o, 150mm/l kcl, 0.1mol/l tris buffering, 0.03 ppm f for 24h to rehydrate the surface of the tooth and the infiltrant matrix and another color measurement was performed (icon24h). each group (n=20) was subdivided into 2 subgroups (n = 10 per subgroup) according to the storage form: 1) distilled water for one month; 2) grape juice (frupic, ind. and palazzos ltda. trade, jaboticabal, sp, brazil) for 10 minutes daily, followed by storage in distilled water for 23:50h for 5 days. the color stability (δe) was calculated using the cie-lab color system to obtain δeab from the individual values of l*, a* and b*, according to the following equation: 5 furuse et al. δeab = [(δl*) 2 + (δa*)2 + (δb*)2]1/2 (1) δl *, δa * and δb * represent the differences between the readings of the color parameters. δeab represents the difference between the measurements obtained after artificial white spot lesion induction and in sound enamel. δej/w stands for the difference between measurements taken after water or juice storage and values assessed after 24h on artificial saliva. the data was evaluated for normality using kolmogorov-smirnov test. l*, a* and b* data were evaluated with one-way anova and tukey’s hsd. δeab data were analyzed by two-way anova considering treatments considering treatments and storage methods as independent variables. tukey’s hsd was applied for multiple comparison. a global level of significance of 5% was adopted (α = 0.05). data was analyzed using the software statistica 13.3 (statsoft, dell software, round rock, tx, usa). results l*, a*, and b* mean values and standard deviations are presented in figures 1 to 3, respectively. l* was mainly affected by the juice storage, and decreased when icon was applied with decreased drying time after the application of ethanol. the same behavior occurred with a* (increase with reduced drying time), while b* was not affected. analysis of variance of δeab is presented in table 1. means and standard deviations for δeab data are presented in figure 4 and table 2. significant differences were observed between the groups (p = 0.0217) and between storage methods (p = 0.0007). for measurements made after storage in water/juice, δeab values were lower for samples treated with fluoride and icon with recommended time, both stored in water. the highest δeab values was found for samples treated with icon with recommended time stored in juice. 100 90 abcde abcde de cde e e bc b cde bc bcd a ab a initial sound 80 70 60 50 40 30 20 10 0 after ph cycling after treatment icon recommended time – after treatment icon reduced time – after treatment fluoride – after treatment 24h after treatment ju ic e w at er ju ic e w at er ju ic e w at er ic o n r ec om m en de d tim e ic o n re du ce d tim e fl uo rid e ic o n r ec om m en de d tim e ic o n re du ce d tim e fl uo rid e figure 1. graphic representation of l* mean values and standard deviations. different letters indicate statistical significant difference (p < 0.05). 6 furuse et al. all δeab means, which were taken after white spot lesion induction, presented values greater than 1. there was no interaction effect between groups and storage methods (p = 0.1119). treatments with fluoride have shown lower δeab values after storage methods when compared to both treatments with icon. no statistically 8 7 ab ab b a ab ab ab ab ab ab ab c b c initial sound 6 5 4 3 2 1 0 -1 -2 after ph cycling after treatment icon recommended time – after treatment icon reduced time – after treatment fluoride – after treatment 24h after treatment ju ic e w at er ju ic e w at er ju ic e w at er ic o n r ec om m en de d tim e ic o n re du ce d tim e fl uo rid e ic o n r ec om m en de d tim e ic o n re du ce d tim e fl uo rid e figure 2. graphic representation of a* mean values and standard deviations. different letters indicate statistical significant difference (p < 0.05). 40 35 ab ab b a ab ab ab ab ab ab a a ab ab initial sound 30 25 20 15 10 5 0 after ph cycling after treatment icon recommended time – after treatment icon reduced time – after treatment fluoride – after treatment 24h after treatment ju ic e w at er ju ic e w at er ju ic e w at er ic o n r ec om m en de d tim e ic o n re du ce d tim e fl uo rid e ic o n r ec om m en de d tim e ic o n re du ce d tim e fl uo rid e figure 3. graphic representation of b* mean values and standard deviations. different letters indicate statistical significant difference (p < 0.05). 7 furuse et al. significant difference was observed between icon applications with reduced drying time. storage in grape juice resulted in higher δeab mean values compared with storage in water. table 1. results from 2-way anova of δeab means. variables of interest degree of freedom sum of squares mean of squares f p intercept 1 2232.342 2232.342 197.6146 0 group 2 92.87969 46.43985 4.111016 0.021784* storage 1 145.864 145.864 12.91239 0.000706* group*storage 2 49.89429 24.94714 2.208408 0.119704 error 54 610.0078 11.29644 * statistical significant difference for p<0.05. table 2. mean ∆eab and standard deviation for treatment groups and storage methods. group storage δeab fluoride water 2.15 (1.24)a,a juice 6.53 (2.09)a,b icon recommended time water 4.83 (2.33)b,a juice 9.25 (3.38)b,b icon reduced time water 6.64 (4.73)b,a juice 7.18 (4.75)b,b uppercase letters indicate difference between groups of treatment and lowercase letters indicate difference between storage methods (p < 0.05). 14 12 a,a a,b b,a b,a b,b b,b 10 8 6 4 2 icon – reduced time juicewater icon – recommended timefluoride juicewaterjuicewater ∆ ea b figure 4. graphic representation of ∆eab. uppercase letters indicate difference between groups of treatment (fluoride, icon – recommended time and icon – reduced time) and lowercase letters indicate difference between storage methods (water and juice). 8 furuse et al. discussion resin infiltration is currently indicated to mask defects in enamel staining, such as white spot lesions. in this case, icon can prevent the progression of non-cavitated enamels caries5,12,25. this material improves the esthetic aspects of the lesion due to monomer infiltration through porosities of white spot lesions, decreasing their opacity5. the present study was designed to evaluate the color stability of bovine teeth with artificial white spot lesions treated with remineralization (flugel, dfl indústria e comércio sa) or resin infiltration (icon, dmg) using two different application techniques. the cie-lab represents a uniform color space with three distinct axes: l*, a* and b*. l* is the measure of brightness, and the closer to zero, the less brightness the object has and the closer to 100, the brighter. the negative value of a* indicates a measure of green color and, when positive, indicates a measure of red color. the positive b* is a measure of the yellow color and, when negative, of the color blue. when a* and b* are close to zero, the color is neutral6. the cie-lab was used in this study because it is able to evaluate the lightness-darkness, red/green and blue/yellow color measurements. previous studies have demonstrated that such parameters are enough to evaluate white spot lesions, since these lesions are more opaque and whiter than healthy enamel. although some studies use ciede2000 system (δe00) to assess color measurements, the cie-lab system is still a reliable system used and has been used in recent studies to evaluate color stability of white spot lesions7,26.based on this cie-lab system, it is possible to calculate the δeab and compare it with a visual perception already reported in the literature (color variation is imperceptible when <1, clinically acceptable when ≤3.3 and unacceptable when above 3.3)22. in the present study, color coordinates were obtained at several time intervals as described in the materials and methods section. however, since the aim was to address color changes through δl*, δa*, and δb* calculation after two protocols of icon application as compared to fluoride remineralization, only l*, a*, and b* values collected 24 h after storage in artificial saliva were considered for δeab calculation. it should be noted that, immediately after the application of icon and fluoride, samples were dehydrated, leading to altered measurements and for this reason these values were not applied to the δeab equation. changes on color coordinates l*, a*, and b* were presented in figures 1 to 3, which revealed significant differences in mean values for l* and a* when icon was applied with decreased drying time after the application of ethanol. a values were also higher for samples treated with icon reduced time after storage in juice. l*, a*, and b* values provide a clue to possible color changes, δeab values are more appropriate to indicate significant color changes visible to the human eye. thereby, the present study was effective in inducing the formation of artificial white spot lesions, which can be verified by the value of δeab of 4.85, which is greater than 3 for samples of all experimental groups and assess color differences between different treatments of white spot lesions. the first hypothesis of this study was that a simple error in the application protocol of icon, such as a marked decrease in the time of evaporation of ethanol after acid conditioning, could influence its color stability. this hypothesis, however, was not confirmed, since no statistically significant difference was observed between icon 9 furuse et al. experimental groups. as the application of ethanol has the purpose of dehydrating the surface and increase the penetration power of the infiltrate within the lesion due to the removal of water molecules from the enamel portions, an error in this step could decrease the subsequent penetration of the infiltrating agent or, eventually, disrupt its polymerization14. however, the decrease in ethanol evaporation time after the acid conditioning step did not influence the color stability of the infiltrator. this result can be possibly attributed to better penetration capabilities of tegdma-based resins10, because it has a lower viscosity. a previous study accomplished by gray and shellis9 (2002) show that the efficacy of infiltration penetration depth is related to the increase of etching time. however, increasing the etching time can lead to wear of the enamel surface27. it should be noticed that not only the change in manufacturer’s protocols could influence the curing efficiency of resin-based materials, but also variables related to the operator have a major impact in the final outcomes28. another hypothesis evaluated is that storage in grape juice would cause significant color change. the present study demonstrated that icon is susceptible to staining when exposed to a natural agent such as grape juice. despite adequate polymerization and polishing, resin-based materials are susceptible to pigmentation caused for common food substances and can produce significant discoloration of these materials13,29,30. this hypothesis was confirmed, since all δew/j values in which icon was applied were higher than 3.3 units, agreeing with the work of borges et al.13 (2014) which explains the clinically unacceptable color change after exposure to staining solutions. in general, all groups and subgroups had δeab greater than 3.3 units, except when fluoride was applied followed by storage in water. this means that storage in water caused a color change in resin-based materials that can be considered clinically significant and noticeable, whilst storage in grape juice induced color alterations for all experimental groups. these results are in agreement with other studies which demonstrated significant changes in color after coloring processes related to storage in pigmented solutions6,26,29,31. these changes can be explained by the composition of the icon, since it has tegdma on this matrix. tegdma monomer has high sorption of water and has a hydrophilic behavior in comparison with other monomers, which can either facilitate its depth of penetration in the white spot lesion and increase its susceptibility to degradation in water14,32,33. as a result it can be assumed that the infiltrant can easily absorb the dyes present in beverages and food, affecting its color stability34. additionally, the sole absorption of water in the resinous materials matrix could be enough to cause color changes, which are illustrated in figure 1. trying to avoid this effect, some authors suggested that the polishing of the specimens could minimize the staining effect13,14. however, the polishing alternative may result in unnecessary enamel wear caused by abrasion34. an alternative would be to perform a bleaching procedure, which does not promote the removal of enamel34. resin infiltration was shown to reliably mask artificial caries lesions in vitro, following the trend of a previous studies7,35. within the limitation of this in vitro experiment, the results obtained from the present study could be of clinical relevance. a decrease in the ethanol evaporation time after an acid conditioning stage did not influence the color stability of the infiltrator. however, l* and a* were affected, without influenc10 furuse et al. ing δe. additionally, this study did not evaluate the infiltrator penetration depth. further studies could be conducted evaluating the color changes occurring in the resin matrix maturation of icon itself, without the influence of environmental factors, such as saliva, food dyes, water, dental structure or ions from oral cavity and the effect of infiltrator penetration depth on color stability. both materials tested showed significant color changes after exposure to staining dyes. it is assumed that while the icon may help the initial aesthetic problem associated with white spot lesions, the resin may suffer from spotting over the time, especially when the patient ingests foods and beverages containing dyes. for this reason, the use of fluoride remineralization seems to be a good alternative, since it is clinically easy to perform and provides better long-term color stability in comparison to resin infiltration. icon on the other hand, is known to provide satisfactory esthetic masking of the white spot lesion in an immediate one session application, requiring further polishing in future sessions to maintain its effectiveness. therefore, such factors should be considered by the clinicians when choosing the treatment of white spot carious lesions. within the limitations of an in vitro study, the use of fluoride is preferable for treating white spot lesions rather than the application of resinous infiltrant because it’s ability to prevent staining. furthermore, the decrease in the ethanol evaporation time did not influence the color stability of the infiltrator, allowing the reduction of this clinical step time. more in vivo studies are necessary to validate the potential of icon resin infiltration technique in providing color stability for white spot lesion treatment, since other clinical factors may be related. acknowledgements this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior – brasil (capes) – finance code 001 and conselho nacional de desenvolvimento científico e tecnológico/ pibic: 145207/2014-5. references 1. keyes ph. the infectious and transmissible nature of experimental dental caries. findings and implications. arch oral biol. 1960 mar;1:304-20. 2. askar h, lausch j, dörfer ce, meyer-lueckel h, paris s. penetration of micro-filled infiltrant resins into artificial caries lesions. j dent. 2015 jul;43(7):832-8. doi: 10.1016/j.jdent.2015.03.002. 3. buzalaf mar, pessan jp, honorio hm, ten cate jm. mechanisms of action of fluoride for caries control. monogr oral sci. 2011;22:97-114. doi: 10.1159/000325151. 4. palacio r, shen j, vale l, vernazza cr. assessing the cost‐effectiveness of a fluoride varnish programme in chile: the use of a decision analytic model in dentistry. community dent oral epidemiol. 2019 jun;47(3):217-24. doi: 10.1111/cdoe.12447. 5. abbas ba, marzouk es, zaher ar. treatment of various degrees of white spot lesions using resin infiltration—in vitro study. prog orthod. 2018 aug 6;19(1):27. doi: 10.1186/s40510-018-0223-3. 6. cohen-carneiro f, pascareli am, christino mr, vale hf, pontes dg. color stability of carious incipient lesions located in enamel and treated with resin infiltration or remineralization. int j paediatr dent. 2014 jul;24(4):277-85. doi: 10.1111/ipd.12071. 11 furuse et al. 7. knösel m, eckstein a, helms h-j. long-term follow-up of camouflage effects following resin infiltration of post orthodontic white-spot lesions in vivo. angle orthod. 2019 jan;89(1):33-39. doi: 10.2319/052118-383.1. 8. kidd e, fejerskov o. what constitutes dental caries? histopathology of carious enamel and dentin related to the action of cariogenic biofilms. j j dent res. 2004;83 spec no c:c35-8. 9. gray g, shellis p. infiltration of resin into white spot caries-like lesions of enamel: an in vitro study. eur j prosthodont restor dent. 2002 mar;10(1):27-32. 10. paris s, schwendicke f, seddig s, müller w-d, dörfer c, meyer-lueckel h. micro-hardness and mineral loss of enamel lesions after infiltration with various resins: influence of infiltrant composition and application frequency in vitro. j dent. 2013 jun;41(6):543-8. doi: 10.1016/j.jdent.2013.03.006. 11. manoharan v, arun kumar s, arumugam sb, anand v, krishnamoorthy s, methippara jj. is resin infiltration a microinvasive approach to white lesions of calcified tooth structures?: a systemic review. int j clin pediatr dent. 2019 jan-feb;12(1):53-58. doi: 10.5005/jp-journals-10005-1579. 12. paris s, hopfenmuller w, meyer-lueckel h. resin infiltration of caries lesions: an efficacy randomized trial. j dent res. 2010 aug;89(8):823-6. doi: 10.1177/0022034510369289. 13. borges a, caneppele t, luz m, pucci c, torres c. color stability of resin used for caries infiltration after exposure to different staining solutions. oper dent. 2014 jul-aug;39(4):433-40. doi: 10.2341/13-150-l. 14. paris s, schwendicke f, keltsch j, dorfer c, meyer-lueckel h. masking of white spot lesions by resin infiltration in vitro. j dent. 2013 nov;41 suppl 5:e28-34. doi: 10.1016/j.jdent.2013.04.003. 15. belli r, rahiotis c, schubert ew, baratieri ln, petschelt a, lohbauer u. wear and morphology of infiltrated white spot lesions. j dent. 2011 may;39(5):376-85. doi: 10.1016/j.jdent.2011.02.009. 16. wilson p, beynon a. mineralization differences between human deciduous and permanent enamel measured by quantitative microradiography. arch oral biol. 1989;34(2):85-8. 17. yoo hk, kim sh, kim si, shin ys, shin sj, park jw. seven-year follow-up of resin infiltration treatment on noncavitated proximal caries. oper dent. 2019 jan/feb;44(1):8-12. doi: 10.2341/17-323-l. 18. silva tmd, sales a, pucci cr, borges ab, torres crg. the combined effect of food-simulating solutions, brushing and staining on color stability of composite resins. acta biomater odontol scand. 2017 jan 16;3(1):1-7. doi: 10.1080/23337931.2016.1276838. 19. domejean s, ducamp r, leger s, holmgren c. resin infiltration of non-cavitated caries lesions: a systematic review. med princ pract. 2015;24(3):216-21. doi: 10.1159/000371709. 20. swamy df, barretto es, mallikarjun sb, dessai ssr. in vitro evaluation of resin infiltrant penetration into white spot lesions of deciduous molars. j clin diagn res. 2017 sep;11(9):zc71-4. doi: 10.7860/jcdr/2017/28146.10599. 21. vieira ae, delbem acb, sassaki kt, rodrigues e, cury ja, cunha rf. fluoride dose response in ph-cycling models using bovine enamel. caries res. 2005 nov-dec;39(6):514-20. 22. seghi rr, johnston wm, o’brien wj. performance assessment of colorimetric devices on dental porcelains. j dent res. 1989 dec;68(12):1755-9. 23. andreatta lm, furuse ay, prakki a, bombonatti jf, mondelli rf. pulp chamber heating: an in vitro study evaluating different light sources and resin composite layers. braz dent j. 2016 oct-dec;27(6):675-80. doi: 10.1590/0103-6440201600328. 24. francisconi l, honório hm, rios d, magalhães a, machado mda, buzalaf mar. effect of erosive ph cycling on different restorative materials and on enamel restored with these materials. oper dent. 2008 mar-apr;33(2):203-8. doi: 10.2341/07-77. 12 furuse et al. 25. horuztepe sa, baseren m. effect of resin infiltration on the color and microhardness of bleached white-spot lesions in bovine enamel (an in vitro study). j esthet restor dent. 2017 sep;29(5):378-85. doi: 10.1111/jerd.12308. 26. ceci m, rattalino d, viola m, beltrami r, chiesa m, colombo m, et al. resin infiltrant for non-cavitated caries lesions: evaluation of color stability. j clin exp dent. 2017 feb 1;9(2):e231-e237. doi: 10.4317/jced.53110. 27. arnold wh, haddad b, schaper k, hagemann k, lippold c, danesh g. enamel surface alterations after repeated conditioning with hcl. head face med. 2015 sep 25;11:32. doi: 10.1186/s13005-015-0089-2. 28. soares cj, bragança gfd, pereira rads, rodrigues mdp, braga ssl, oliveira lrs, et al. irradiance and radiant exposures delivered by led light-curing units used by a left and right-handed operator. braz dent j. 2018 may-jun;29(3):282-9. doi: 10.1590/0103-6440201802127. 29. rey n, benbachir n, bortolotto t, krejci i. evaluation of the staining potential of a caries infiltrant in comparison to other products. d dent mater j. 2014;33(1):86-91. 30. silva lo, signori c, peixoto ac, cenci ms, faria esal. color restoration and stability in two treatments for white spot lesions. int j esthet dent. 2018;13(3):394-403. 31. villalta p, lu h, okte z, garcia-godoy f, powers jm. effects of staining and bleaching on color change of dental composite resins. j prosthet dent. 2006 feb;95(2):137-42. 32. fonseca as, labruna moreira ad, de albuquerque pp, de menezes lr, pfeifer cs, schneider lf. effect of monomer type on the cc degree of conversion, water sorption and solubility, and color stability of model dental composites. dent mater. 2017 apr;33(4):394-401. doi: 10.1016/j.dental.2017.01.010. 33. sideridou id, karabela mm, bikiaris dn. aging studies of light cured dimethacrylate-based dental resins and a resin composite in water or ethanol/water. dent mater. 2007 sep;23(9):1142-9. 34. araújo g, naufel f, alonso r, lima d, puppin-rontani r. influence of staining solution and bleaching on color stability of resin used for caries infiltration. oper dent. 2015 nov-dec;40(6):e250-6. doi: 10.2341/14-290-l. 35. theodory tg, kolker jl, vargas ma, maia rr, dawson dv. masking and penetration ability of various sealants and icon in artificial initial caries lesions in vitro. j adhes dent. 2019;21(3):265-72. doi: 10.3290/j.jad.a42520. 1http://dx.doi.org/10.20396/bjos.v19i0.8660537 volume 19 2020 e200537 original article 1 faculty of dentistry, federal university of bahia (ufba), salvador, ba, brazil. 2 bahiana school of medicine and public health (ebmsp), salvador, ba, brazil. 3 faculty of dentistry, feira de santana state university (uefs), feira de santana, ba, brazil. corresponding author: emilena maria castor xisto lima av. araújo pinho, 72, canela. salvador-ba, brazil. cep 40301-155 email: emilenalima@gmail.com received: july 20, 2020 accepted: october 29, 2020 analysis of the marginal adaptation of different crowns fabricated with computer-aided technology using an intraoral digital scanner roniel kappler1, michelle villa oliveira2, ingrid de oliveira bandeira2, thayara coelho metzker2, adriana oliveira carvalho2,3, emilena maria castor xisto lima1,2,* aim: the aim of this study was to evaluate the marginal adaptation of ceramic and composite resin crowns fabricated with computer-aided design and computer-aided manufacturing (cad/cam) technology using an intraoral digital scanner. methods: a human mandibular right second molar was prepared for a ceramic crown. the impressions were made using intraoral scanning device and crowns were milled. ten crowns were fabricated for each group (n=10): gf feldspathic ceramic (cerec blocs, sirona), gl lithium disilicate ceramic (ips e.max cad, ivoclar), gg composite resin (grandio blocs, voco) and gb composite resin (brava block, fgm). the marginal gap was measured for each specimen at 4 points under magnification with a stereomicroscope. all data were statistically analyzed using one-way anova followed by the tukey’s test (α=.05). results: the lowest marginal discrepancy value was observed in gb (60.95 ± 13.64 μm), which was statistically different from the gl (84.22 ± 20.86 μm). however, there was no statistically significant difference between these groups when compared with the other groups, gf (73.26 ± 8.19 μm) and gg (68.42 ± 11.31 μm). conclusion: it can be concluded that the composite resin presented the lowest variance compared to the lithium disilicate glass ceramic, although the marginal gap of all materials tested was within the acceptable clinical limit (120 μm). keywords: computer-aided design. crowns. dental marginal adaptation. ceramics. composite resins. https://orcid.org/0000-0001-8233-7392 2 kappler et al. introduction in recent years, newer technologies have been developed in dentistry with the purpose of improving the outcome of indirect restorations1. as the name suggests, the computer-aided design and manufacturing (cad/cam) system is an innovative technology, wherein planning and fabrication of prostheses are performed using a computer2. with this technology, it is possible to create a virtual model of the prosthetic preparation, occlusal relationship of the arches, and plan the restoration. after virtual planning, fabrication of the restoration is carried out without intermediate manufacturing steps, thereby decreasing the cost, time, and risk of contamination during the interim restoration phase3. there are two main types of dental cad/cam scanners namely intraoral and extraoral scanners. intraoral scanners are used chairside to scan the dental arches of patients; while extraoral scanners are used in the dental laboratory to scan casts4. the extraoral technique may lead to errors during the final impression stage and master cast production. intraoral scanners aim to eliminate dimensional changes of the impression materials and expansion of the dental stone5,6; however, certain factors in the oral environment, such as saliva, sulcular fluid, patient movement, or limited space may interfere in obtaining the digital model7,8. in order to be considered an acceptable alternative to conventional impression methods, intraoral scanning devices should yield crowns with similar or better clinical success3,9. for the success of prosthetic crowns, good marginal adaptation is essential9-11. marginal gap can be defined as the distance from edge of the finish line of the prepared tooth to cervical margin of the restoration12. presence of marginal gaps contributes to exposure of cement to the oral environment, thereby raising the possibility of dissolution, biofilm accumulation, secondary caries, pulp and periodontal inflammation10,13-15. reference values for a clinically acceptable marginal discrepancy have been described in the literature as less than 120 µm16, and the recommended threshold for cad/cam crowns is between 50 and 100 µm17-20. several factors may influence marginal adaptation, including design of the preparation, location of the margin, impression and waxing techniques21, accuracy of the milling system, size of the milling bur, thickness of the cementation space and restorative material22,23. as chairside cad/cam technology is gaining a foothold in dentistry, several restorative materials such as ceramics and composite resins are being increasingly developed and marketed24. ceramics are highly aesthetic, with optical characteristics of translucency and opalescence superior to resinous materials. moreover, ceramics have high fracture resistance and low material wear25,26; however, may have a potential abrasive effect on opposing dentition27. resin composites consist of a polymeric matrix reinforced by fillers that could be inorganic (ceramics, glass-ceramics, or glasses), organic, or composite28,29. according to awada et al.24 (2015) polymer-based materials appear capable of producing acceptable margins with more conservative preparations, possibly due to relatively high flexural strength combined with low flexural modulus. polymer-based materials appear to exhibit smoother milled margins compared to ceramic materi3 kappler et al. als24. in addition, resinous composites are easy to fabricate, repairable intraorally, and allow for less visible intra-oral repair of minor defects induced by function29. there are controversial data in the literature regarding marginal adaptation of crowns fabricated with resin composites and lithium disilicate ceramics. a study by de paula silveira et al.2 (2017) showed no difference among these materials. tabata et al.1 (2020) found that the composite resin presented significantly lower values of marginal discrepancy (56 ± 27 mm) than ceramic (71 ± 35 mm), however, el ghoul et al.30 (2020) reported that ceramic-based groups showed smaller gaps than resin-based groups. further studies are required on marginal adaptation of cad/cam restorative materials considering the lack of sufficient data. thus, the purpose of this in vitro study was to evaluate the marginal adaptation of ceramic and composite resin crowns fabricated with cad/cam technology using intraoral digital scanner. the null hypothesis was that marginal adaptation values of crowns are not influenced by the type of material. material and methods the research ethics committee of the faculty of dentistry of the federal university of bahia approved this study (number 3,082,332). one caries-free human mandibular molar was selected, cleaned by scaling, and stored in 0.01% thymol to prevent bacterial proliferation. the tooth was stored in a metal box with a damp sponge to prevent it from drying and becoming brittle, throughout the study. the human mandibular right second molar was mounted with its adjacent teeth on a typodont and prepared to receive an all-ceramic crown with chamfer finish line. the tooth preparation was as follows: 2 mm reduction of the occlusal surface, convergence angle of approximately 6 degrees, 1.0 to 1.5 mm axial reduction, and location of the finish line was above the cementoenamel junction. diamond tips were used for the tooth preparation adapted on a multiplier contra-angle t3-line handpiece (sirona dental systems gmbh, bensheim, germany) in the following sequence: diamond tip fg 3216 (kg sorensen, cotia, brazil) for delimitation of the buccal, lingual and occlusal orientation grooves and union of the grooves, diamond tip fg 3203 (kg sorensen, cotia, brazil) was used to make contact point rupture, fg 3216 (kg sorensen, cotia, brazil) was used to create a chamfer finish line and diamond tip fg 4138 was used for finishing (kg sorensen, cotia, brazil). the composition and information regarding manufacturer of the tested materials are listed in table 1. table 1. type, composition, and manufacturer of the four tested materials material composition manufacturer cerec blocs feldspathic ceramic – gf sirona (bad säckingen – bensheim – germany) ips e.max cad lithium dissilicate glass ceramic – gl ivoclar vivadent (schaan – liechtenstein). grandio blocs resin composite (hybrid nano ceramic) – gg voco (cuxhaven – germany) brava block resin composite (glass ceramic composite) – gb fgm (joinville – santa catarina – brazil) 4 kappler et al. ten digital impressions of the prepared tooth were made for each group using the cerec omnicam intraoral scanner (sirona dental systems gmbh, bensheim, germany). the appropriate software cerec inlab sw 4.5 (sirona dental systems gmbh, bensheim, germany) was used to design the crowns on the virtual model. the operator determined design parameters were as follows: radial and occlusal spacer = 80 µm, resistance of proximal contacts = -25 µm, resistance of occlusal contacts = -25 mm, dynamic contact force = -25 µm, minimum thickness (radial) = 700 µm, minimum thickness (occlusal) = 900 µm, and margin thickness = 80 µm. all restorations were designed to have similar occlusal anatomy and the same occlusogingival height. after each crown was designed, the information was exported to the milling unit cerec inlab mcxl ((sirona dental systems gmbh, bensheim, germany). ten crowns were fabricated for each group (n = 10): gf feldspathic ceramic, gl lithium disilicate ceramic, gg composite resin (grandio blocs) and gb composite resin (brava block). following the manufacturers’ instructions, specimens in group gl were subjected to the crystallization process (programat cs2; ivoclar vivadent, schaan, liechtenstein), while specimens in gf, gg and gb groups did not need any crystallization firing. analysis of marginal discrepancy the crowns were adapted to the prepared dental unit (mandibular right second molar) with the aid of a “c” clamp and maintained in a standardized position during the analysis in a stereomicroscope lupe31 (optima mdce-5ª 2.0, hiperquímica, santo andré, brazil) (figure 1). photographs were obtained at 45x magnification from the buccal, lingual, mesial, and distal surfaces, and images were transferred to the coreldraw x7 program. marginal discrepancy was determined by measuring the space (marginal opening) between margin of the crowns and finish line of the human mandibular right second molar. for each crown, the measurements were made at four vertical reference lines previously marked at the midpoint of the dental unit finish line (figure 2) at four locations to represent the buccal, lingual, mesial, and distal surfaces of tooth32. the measurements were made thrice along the long axis of the tooth at each of the four reference points. the arithmetic mean of twelve readings (three on each face) was calculated for each specimen. all procedures were performed by one calibrated operator. figure 1. crown adapted to the prepared dental unit and maintained in a standardized position during the analysis in a stereomicroscope lupe. 5 kappler et al. figure 2. crown adapted to the prepared dental unit (see vertical reference lines) with the aid of a “c” clamp. statistical analysis the normality and variance homogeneity assumptions were verified using the shapiro-wilk and levene tests, neither of which violated this assumption. the amounts of marginal discrepancy were compared between the four materials with the one-way analysis of variance (anova) followed by the tukey’s test for multiple comparisons (a=.05). the analyses were performed using the statistical program, spss statistics v19.0 (ibm corp chicago, united states). results means and standard deviations of marginal adaptation are described in table 2. table 2. means and standard deviation (sd) of marginal discrepancy (values in micrometer μm) within each of four groups tested group material marginal discrepancy mean ±sd (mm) gf feldspathic ceramic 73.26 ± 18.19 ab gl lithium disilicate ceramic 84.22 ± 20.86 a . gg resin composite – grandio blocs 68.42 ± 11.31 ab gb resin composite – brava block 60.95 ± 13.64 b 1-way anova test and post-hoc tukey test (p <0.05). averages followed by distinct letters represent significant differences. the lowest marginal discrepancy value was observed in gb (60.95 ± 13.64 μm) which was statistically different from the gl (84.22 ± 20.86 μm), that showed the highest value of marginal discrepancy. however, there was no statistically significant difference 6 kappler et al. between these groups when compared with the other groups gf (73.26 ± 8.19 μm) and gg (68.42 ± 11.31 μm). all groups showed marginal discrepancies within the clinically acceptable value. discussion the null hypothesis that marginal adaptation values of crowns are not influenced by the type of material was rejected because a significant difference was observed between the lithium disilicate ceramic and composite resin (brava block). however, it was verified that there was no statistically significant difference when these materials were individually compared with feldspathic ceramic and composite resin (grandio blocs). a gap between 50 and 100 μm has been considered acceptable for adequate marginal adaptation of cad/cam restorations17-20. in this study, the mean value of marginal discrepancy of the four groups (gf, gl, gg, and gb) was in the range of 60.95 ± 13,64 μm to 84.22 ±20,86 μm; therefore, were clinically acceptable. group gl displayed the largest gaps while group gb displayed the smallest gaps. some studies2,24 have reported that resin materials demonstrated better machinability and adaptation. according to awada et al.24 (2015) these materials tend to be less brittle and more flexible probably due to the resin component. tabata et al.1 (2020) evaluated the marginal adaptation of crowns fabricated with two materials (ceramic and composite resin) and two internal spacings using the cad/cam system. they reported statistically significant difference between materials for marginal adaptation with spacing of 80 μm. this result is consistent with that of the present study, wherein the same internal spacing measure was used and a difference was observed between the lithium disilicate ceramic and composite resin (brava block), although there was no difference between the former and composite resin (grandio blocs). el ghoul et al.30 (2020) compared the marginal adaptation of lithium disilicate ceramic crown (ips e.max cad) and resin composite endocrown (cerasmat) fabricated using the cad/cam system and observed that there was a statistically significant difference between the tested groups. however, the composite resin crown showed higher marginal discrepancy values (143.0 ± 21.7 μm) than lithium disilicate ceramics (104.8 ± 14.1 μm). in the present study, resin composites showed smaller marginal discrepancy than the tested ceramics, with a statistically significant difference between the lithium disilicate ceramic and resin composite (brava block). de paula silveira et al.2 (2017) evaluated the marginal adaptation of lithium disilicate (ips e.max cad) and composite resin (lava ultimate) total crowns fabricated by cad/cam technology using intraoral digital scanner and reported no statistically significant difference between the materials. these data partially corroborate with the present study, wherein there was no statistically significant difference between lithium disilicate ceramics and the composite resin (grandio blocs). however, statistically significant difference was observed when compared to the other composite resin (brava block). 7 kappler et al. in the present study, photographs were taken at 45x magnification using stereoscopic magnifying glass, while in the studies by tabata et al.1 (2020) and de paula silveira et al.2 (2017) the method used was microtomography. variations may also be related to the different resin composite materials tested, namely lava ultimate, cerasmat, brava block, and grandio blocs. the manufacturing method, scanning, and milling system accuracy could also influence the observations. when comparing the materials used in the present study with those in other studies, the resin composite grandio blocs had 86% inorganic filler particles in a polymer matrix, but the particle size was not reported33 whereas brava block had 80% inorganic filler encased in a resin matrix with particle size ranging from of 40 nanometers (nm) to 5 μm34. in the other studies, the resin composite used (lava ultimate) had 80% inorganic fillers by weight with individual particles in the size range of 4 to 20 nm35. the variation in particle size may be related to the differences between the materials and results obtained. few other studies have compared the marginal adaptation of feldspathic ceramic crowns with other restorative materials. das neves et al.36 (2014) evaluated the marginal adaptation of feldspathic ceramic total crowns manufactured by the cad/cam system and found that the marginal discrepancy was 62.6 ± 65.2 μm. this was within close range of the values observed in the present study, wherein marginal adaptation of feldspathic ceramic was 73.26 ± 18.19 μm. the in vitro nature of this study could be considered a limitation, the results of which may differ from a clinical study, where the scanning and processing would be less precise due to constraints such as presence of saliva and limited access of the scanner in the oral cavity. within the limitations of this study, it can be concluded that the composite resin brava block presented the lowest variance when compared with the lithium disilicate glass ceramic, although the marginal gap of all materials tested was within the acceptable clinical limit (120 μm). acknowledgments the authors thank the biochemistry 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hunter aj, hunter ar. gingival margins for crowns: a review and discussion. part ii: discrepancies and configurations. j prosthet dent. 1990 dec;64(6):636-42. doi: 10.1016/0022-3913(90)90286-l. 14. sailer i, fehér a, filser f, gauckler lj, lüthych, hämmerle ch. five-year clinical results of zirconia frameworks for posterior fixed partial denture. int j prosthodont. 2007 jul-aug;20(4):383-8. 15. jei jb, mohan j. comparative evaluation of marginal accuracy of a cast fixed partial denture compared to soldered fixed partial denture made of two different base metal alloys and casting techniques: an in vitro study. j indian prosthodont soc. 2014 mar;14(1):104-9. doi: 10.1007/s13191-013-0286-2. 16. mclean jw, von fraunhofer já. the estimation of cement film thickness by an in vivo technique. br dent j. 1971 aug;131(3):107-11. doi: 10.1038/sj.bdj.4802708. 17. akbar jh, petrie cs, walker mp, williams k, eick jd. marginal adaptation of cerec 3 cad/cam composite crowns using two different finish line preparation designs. j prosthodont. 2006 may-jun;15(3):155-63. doi: 10.1111/j.1532-849x.2006.00095.x. 18. att w, komine f, gerds t, strub jr. marginal adaptation of three different zirconium dioxide three-unit fixed dental prostheses. j prosthet dent. 2009 apr;101(4):239-47. doi: 10.1016/s0022-3913(09)60047-0. 19. euán r, figueras-álvarez o, cabratosa-termes j, brufau-de barberà m, gomes-azevedo s. comparison of the marginal adaptation of zirconium dioxide crowns in preparations with two different finish lines. j prosthodont. 2012 jun;21(4):291-5. doi: 10.1111/j.1532-849x.2011.00831.x. https://www.ncbi.nlm.nih.gov/pubmed/?term=l%c3%bcthy h%5bauthor%5d&cauthor=true&cauthor_uid=17695869 https://www.ncbi.nlm.nih.gov/pubmed/?term=h%c3%a4mmerle ch%5bauthor%5d&cauthor=true&cauthor_uid=17695869 9 kappler et al. 20. euán r, figueras-álvarez o, cabratosa-termes j, oliver-parra r. marginal adaptation of zirconium dioxide copings: influence of the cad/cam system and the finish line design. j prosthet dent. 2014 aug;112(2):155-62. doi: 10.1016/j.prosdent.2013.10.012. 21. shamseddine l, mortada r, rifai k, chidiac jj. marginal and internal fit of pressed ceramic crowns made from conventional and computer-aided design/computer-aided manufacturing wax patterns: an in vitro comparison. j prosthet dent. 2016 aug;116(2):242-8. doi: 10.1016/j.prosdent.2015.12.005. 22. boeddinghaus m, breloer e.s, rehmann p, wöstmann b. accuracy of singletooth restorations based on intraoral digital and conventional impressions in patients. clin oral investig. 2015 nov;19(8):2027-34. doi: 10.1007/s00784-015-1430-7. 23. renne w, wolf b, kessler r, mcpherson k, mennito as. evaluation of the marginal fit of cad/cam crowns fabricated using two different chairside cad/cam systems on preparations of varying quality. j esthet restor dent. 2015;27(4):194-202. doi: 10.1111/jerd.12148. 24. awada a, nathanson d. mechanical properties of resin-ceramic cad/cam restorative materials. j prosthet dent. 2015 oct;114(4):587-93. doi: 10.1016/j.prosdent.2015.04.016. 25. kois de, isvilanonda v, chaiyabutr y, kois jc. evaluation of fracture resistance and failure risks of posterior partial coverage restorations. j esthet restor dent. 2013 apr;25(2):110-22. doi: 10.1111/jerd.12018. 26. zahran m, el-mowafy o, tam l, watson pa, finer y. fracture strength and fatigue resistance of all-ceramic molar crowns manufactured with cad/cam technology. j prosthodont. 2008 jul;17(5):370-7. doi: 10.1111/j.1532-849x.2008.00305.x. 27. sripetchdanond j, leevailoj c. wear of human enamel opposing monolithic zirconia, glass ceramic, and composite resin: an in vitro study. j prosthet dent. 2014 nov;112(5):1141-50. doi: 10.1016/j.prosdent.2014.05.006. 28. ferracane jl. resin composite-state of the art. dent mater. 2011 jan;27(1):29-38. doi: 10.1016/j.dental.2010.10.020. 29. ruse nd, sadoun mj. resin-composite blocks for dental cad/cam applications. j dent res. 2014 dec;93(12):1232-4. doi: 10.1177/0022034514553976. 30. el ghoul wa, özcan m, ounsi h, tohme h, salameh z. effect of different cad-cam materials on the marginal and internal adaptation of endocrown restorations: an in vitro study. j prosthet dent. 2020 jan;123(1):128-34. doi: 10.1016/j.prosdent.2018.10.024. 31. cerqueira cl, kappler r, cavalcanti aan, lima emcx. marginal adaptation of provisional crowns made of acrylic and bisacrylic resins using different impression materials. braz j oral sci. 2019;18:e191603. doi: 10.20396/bjos.v18i0.8657257. 32. nejatidanesh f, lofti hr, savabi o. marginal accuracy of interim restorations fabricated from four interim autopolymerizing resins. j prosthet dent. 2006 may;95(5):364-7. doi: 10.1016/j.prosdent.2006.02.030. 33. voco gmbh. grandio blocks. cuxhaven, germany; 2018 jan [cited 2019 oct 11]. available from: https://www.voco.dental/br/portaldata/1/resources/products/instructions-for-use/e1/grandioblocs_ifu_e1.pdf. 34. fgm dental group. brava bloc amann.joinvile, sc: fgm; c2020 [cited 2019 oct 11]. available from: https://www.fgm.ind.br/site/wp-content/uploads/2018/02/perfil_bravablock.pdf. 35. 3m. lava ultimate. in: catálogo técnico de produtos 3m oral care [cited 2019 oct 11]. p.28. available from: http://dentalprimecwb.com.br/wp-content/uploads/2017/09/atualizadocat%c3%a1logo-t%c3%a9cnico-de-produtos-3m.pdf. 36. das neves fd, carneiro tapn, do prado cj, prudente ms, zancopé k, davi lr, et al. micrometric precision of prosthetic dental crowns obtained by optical scanning and computer-aided designing/computer-aided manufacturing system. j biomed opt. 2014 aug;19(8):088003. doi: 10.1117/1.jbo.19.8.088003. https://doi.org/10.20396/bjos.v18i0.8657257 https://www.voco.dental/br/portaldata/1/resources/products/instructions-for-use/e1/grandio-blocs_ifu_e1.pdf https://www.voco.dental/br/portaldata/1/resources/products/instructions-for-use/e1/grandio-blocs_ifu_e1.pdf 1 volume 21 2022 e226698 original research braz j oral sci. 2022;21: e226698http://dx.doi.org/10.20396/bjos.v21i00.8666698 1 department of pediatric dentistry, dental school of ribeirão preto, university of são paulo, ribeirão preto, são paulo, brazil. corresponding author: clara marina pereira cavalcanti silva rua cavalheiro torquato rizzi, 2025 – apto. 75, jardim são luiz, ribeirão preto – sp, 14020-300 clarac@usp.br +5582993611190 editor: altair a. del bel cury received: august 17, 2021 accepted: april 02, 2022 the psychological impact of social distancing related to the covid-19 pandemic on undergraduate and graduate students in brazil angélica aparecida de oliveira1 , lea assed bezerra da silva1 , paulo nelson-filho1 , carolina maschietto puccinelli1 , clara marina pereira cavalcanti silva1,* , raquel assed bezerra segato1 aim: to evaluate the psychological impact of covid-19 on undergraduate and graduate students of the dental school of ribeirão preto, university of são paulo. methods: three questionnaires were used: sociodemographic, who abbreviated quality of life inventory, and general anxiety disorder-7. data were analyzed using graph pad prism 7a (α = 5%). mann-whitney or kruskal-wallis and dunn post-tests were used for statistical comparisons. the spearman test was used as the correlation test. results: 257 students responded to the online form that assessed their quality of life and anxiety level. on a scale from 1 to 100, with 100 being the best quality of life, the average obtained was 64.71 (± 13.36). in addition, 87.6% of the students rated their quality of life as good or very good. 74.7% reported good or very good health. the anxiety analysis resulted in an average of 10.04 (± 4.5), indicating moderate anxiety levels. there was an inversely proportional correlation between age and degree of anxiety (p = 0.008, r = -0.1628) and self-perceived learning and student commitment (r = 0.69). conclusion: despite the good quality of life and the students’ good self-perception of health, they showed a moderate degree of anxiety during the social distancing caused by covid-19, also demonstrating a decrease in interest and commitment during distance education. keywords: coronavirus infections. covid-19. patient health questionnaire. anxiety. students, dental. https://orcid.org/0000-0002-0447-8939 https://orcid.org/0000-0001-7118-6859 https://orcid.org/0000-0001-8802-6480 https://orcid.org/0000-0003-4876-6892 https://orcid.org/0000-0001-5099-1074 https://orcid.org/0000-0002-0230-1347 2 oliveira et al. braz j oral sci. 2022;21: e226698 introduction covid-19, caused by the infection of the sars-cov-2 virus, has become a pandemic1 because of its rapid spread and high mortality and morbidity rate worldwide2. as a result, there have been several social and educational changes since the beginning of the pandemic, employing preventive measures of social distancing, the use of face masks, and regular handwashing with soap or disinfection with 70% alcohol3. the sudden change in students’ daily lives in social isolation, combined with distance learning on a scale never experienced before, has impacted their quality of life, negatively affecting their feelings, eating habits, sleep, among others4,5. in addition, several studies describe that the pandemic had a tremendous emotional impact, causing an increase in psychological problems, such as anxiety, stress, and depression6,7, both in the general public8 and in university students in china9 and greece10. about 24.9% of the university students in china experienced anxiety due to the covid-19 outbreak. the protective factors against anxiety found were living in an urban area, living with parents, and having a stable family income. conversely, having a family member or acquaintance infected with covid-19 was a risk factor. likewise, academic delays, different effects on daily life, and economic instability acted as stressors9. university students in greece showed increased anxiety, depression, and suicidal thoughts. in addition, they also reported worsening of sleep quality, sex life, and quality of life10. both studies concluded that the mental health of university students was significantly affected by the covid-19 pandemic and emphasized the need for specific psychological care for this population group9,10. dental professionals are at the highest risk of spreading covid-19 due to the generation of aerosols, contact with blood and saliva, and the proximity to the patient during consultations11-13. therefore, there was a change in the recommendations for dental care, with restrictions to only perform urgent and emergency care14. in addition, care at the schools of dentistry was suspended in some countries, which harmed the ability of these students, and new biosafety protocols were proposed to minimize the risk of infection, with the addition of new personal protective equipment (ppe)15,16. all these changes and concerns related to dentistry students have affected their present and future lives. however, there is no study on the mental health situation of university students in dentistry in brazil. thus, the objective of this work is to evaluate the psychological impact of the covid-19 pandemic on undergraduate and graduate students in dentistry at the ribeirão school of dentistry of the university of são paulo (forp/usp), using validated questionnaires to assess the quality of life and the degree of anxiety. material and methods this cross-sectional study followed the strobe recommendations (strengthening the report on observational studies in epidemiology) through the validated portuguese version published in 2010. the data were collected remotely in brazil, between july 10, 2020 and august 4, 2020, during the covid-19 pandemic period. 3 oliveira et al. braz j oral sci. 2022;21: e226698 initially, the research project was submitted to the research ethics committee of the school of dentistry of ribeirão preto, university of são paulo, approved under nº 4.143.131. in a non-probabilistic approach, a convenience sample was formed by stricto sensu undergraduate and graduate students from school of dentistry of ribeirão preto. the criteria for inclusion in the study include being a student regularly enrolled at the school of dentistry and agreeing to participate in the study by signing the informed consent form (cif). lato sensu graduate students and students from different colleges of the same university were not included in the study, even if they carried out their research or elective courses at the school of dentistry. no other criteria were added. it was determined that the main variables of interest were quality of life and anxiety. personal information was based on the students’ self-perception, and no additional method was used to test these variables. all of this information was collected by using three study instruments: a sociodemographic questionnaire developed by the authors with specific information about the students during quarantine, whoqol-bref (world health organization abbreviated quality of life) to assess their quality of life, and gad-7 (general anxiety disorder 7) to evaluate the signs and symptoms of anxiety. 1. sociodemographic questionnaire (annex a) addressed the topics of age, gender, marital status, and specific information about students during the quarantine to contextualize the data obtained in the other questionnaires. 2. who abbreviated quality of life (whoqol-bref) (annex b) validated in portuguese by fleck et al. (2000)17, containing 26 questions, two of which are general quality of life questions, and the remaining 24 represent each of the 24 facets that make up the original whoqol-100 instrument. the data were transformed into a scale of 0-100, with 100 being the best quality of life18.   3. general anxiety disorder – 7 (gad-7) (anexo c) an instrument for assessing, diagnosing, and monitoring anxiety, validated by kroenke et al. (2010)19, according to the criteria of the diagnostic and statistical manual of mental disorders (dsm-iv). this questionnaire contains seven questions about how the individual has been feeling in the past two weeks, with four possible answers: 0 (not once), 1 (several days), 2 (more than half the days), and 3 (almost every day), with a final score ranging from 0 to 21. a positive indicator of signs and symptoms of anxiety disorders is considered to be a value equal to or greater than 10, with values from 1 to 5 indicating mild anxiety, 6 to 10 moderate, and 11 to 15 severe. the questionnaires were sent to students by the google forms platform, whereas the invitation to participate and explain the work via email, by the undergraduate and graduate sector of forp usp. in addition, they were posted on instagram and whatsapp to reach a more significant number of students at the school of dentistry of ribeirão preto. the data were analyzed using the graph pad prism 7a statistical program (graph pad software in., san diego, california, usa), using the appropriate tests for each evaluation. the level of significance adopted was 5%. the mann-whitney or kruskal-wallis 4 oliveira et al. braz j oral sci. 2022;21: e226698 and dunn post-test were used for statistical comparisons. the spearman test was used as the correlation test. results a total of 257 undergraduate and graduate students answered the online questionnaires; 65% were undergraduate students, and 35% were graduate students (50% master’s and 50% doctoral levels). the majority of the sample was composed of women (76.3%). the age group with the most significant number of participants was 20 to 25 years old, with 53.7%, and only 5.1% declared to be over 36 years old. further sociodemographic and specific information about students during the quarantine period is described in table 1. table 1. results of the online form related to sociodemographic data and some specific information. age gender level of education marital status spirituality <20 (12.1%) 20-25 (53.7%) 26-30 (22.6%) 31-35 (6.6%) > 36 (5.1%) female (76.3%) male (23.7%) graduate (65%) masters (17.5%) doctorate (17.5%) single (70%) relationship (19.8%) stable union (1.6%) married (7%) divorced (0.8%) widowed (0.8%) yes (81.3%) no (18.7%) scholarship moved from your city to study at forp during the pandemic you works to complement income income decrease yes (48.2%) no (51.8%) yes (78.6%) no (21.4%) stayed in ribeirão preto (26.8%) returned to your parents’ home (63.4%) other (9.7%) yes (18.7%) no (81.3%) yes (58.8%) no (41.2%) attended any online course offered during the pandemic how do you classify your learning level during the pandemic how do you classify your performance in courses during the pandemic compared to presential pedagogical activities, your productivity in remote (distance) activities was: have a quiet and calm place to attend online classes and study yes (89.5%) no (10.5%) excellent (3.9%) good (45.9%) average (34.2%) bad (10.5%) terrible (5.4%) excellent (10.5%) good (45.9%) average (33.1%) bad (8.9%) terrible (1.6%) much better (5.4%) better (12.5%) neither better nor worse (42%) yes (82.9%) no (17.1%) have good internet access have children the need to care for your child (ren) influenced your studies in any aspect wanted to drop the course during the pandemic you or someone in your family tested positive for covid yes (96.9%) no (3.1%) yes (3.9%) no (96.1%) yes (70%) no (3%) yes (31.5%) no (68.5%) yes (16.7%) no (83.3%) the average score for quality of life was 64.71 (± 13.36), indicating good quality of life. the results are described in figure 1. 5 oliveira et al. braz j oral sci. 2022;21: e226698 0% 21-30 41-50 61-70 81-90 1.13% 3.40% 9.81% 24.53% 28.68% 20% 11.32% 1.13% percentage of total people q ua lit y of li fe s co re 30%25%20%15%10%5% figure 1. graphical representation of the results from the evaluation of quality of life. of the total participants, 87.6% rated their quality of life as good or very good. 74.7% of the respondents also reported having good or very good health. the students’ degree of anxiety analysis resulted in an average of 10.04 (± 4.5), indicating a moderate degree of anxiety. figure 2 shows the results from this analysis. 6.61% 43.97% 28.79% 20.62% 0% absent light moderate severe a nx ie ty s co re percentage of total people 50%40%30%20%10% figure 2. graphical representation of the results after analyzing the students’ level of anxiety. when age and degree of anxiety were analyzed (figure 3), an inversely proportional correlation was observed, with p=0.008 and r=-0.1628, demonstrating that the older the student, the lower the degree of anxiety. 6 oliveira et al. braz j oral sci. 2022;21: e226698 a ge absent light moderate severe anxiety < 35 years 31–35 years 26–30 years 20–25 years < 20 years 0 figure 3. graphical representation of results after the correlation between anxiety and age. the students’ performance showed a positive correlation with their self-perception of learning (r=0.69), meaning that the greater the students’ commitment, the more they perceived how much they had learned (figure 4). s el fpe rc ep tio n of le ar ni ng very bad bad neither good nor bad good effort very good good neither good nor bad bad very bad 0 very good r = 0.6957 p ≤ 0.0001 0 figure 4. graphical representation of the results from the correlation between the efforts and self-perception of learning. students whose income was reduced during the pandemic had a statistically greater degree of anxiety, as depicted in figure 5. 7 oliveira et al. braz j oral sci. 2022;21: e226698 * severe moderate light absent a nx ie ty yes no income decrease figure 5. results from the correlation between anxiety and income. (*) represents statistical difference discussion this survey revealed that 93.39% of those students were experiencing some degree of anxiety during the period of confinement caused by the covid-19 pandemic. this result is consistent with what was reported by agius et al. (2021)20, iosif et al. (2021).21, hakami et al. (2021)22, and cayo-rojas et al. (2021)23; in their studies at universities in malta, bucharest, saudi arabia, and peru, respectively; they concluded that most students developed anxiety. other studies also reported that the high percentage of anxious university students might be related to several factors, such as changes in teaching methodology that went from in-person to entirely online due to the pandemic24, in the feeling of uncertainty about their academic and professional careers7,24 and the potential negative impact on their academic progress25. furthermore, because dental courses rely heavily on practical training and manual skills throughout preclinical and clinical studies, recent studies have shown that the leading cause of anxiety in dental students was the loss of dexterity, insofar as various clinical procedures require manual and fine motor skills20,21. in a group of university students in spain, odriozola-gonzáles et al. (2020)26 observed that the participants had moderate to extremely severe anxiety, depression, and stress scores and noticed that students seem to have suffered a significant psychological impact during the first weeks of the quarantine resulting from covid-19. likewise, in our study, we observed that students had moderate anxiety. student anxiety may be associated with the gradual increase in distance between people resulting from social isolation. anxiety disorders are more likely to occur and worsen without interpersonal communication26. some factors can have prevented severe anxiety in this study, as most students reported they had spirituality, returned to their parents’ home, nobody tested positive for covid, and had good internet access, facilitating social interaction and decreasing boredom9,27. most students were women, aged between 20-25, aspects that have already been associated with risk for mental illness28. despite this, our study did not find signifi8 oliveira et al. braz j oral sci. 2022;21: e226698 cant differences between male and female students regarding the quality of life or anxiety, corroborating caio-rojas et al. (2020)29. these findings indicate that male and female students have experienced similar tensions and negative emotions due to the pandemic. regarding the decrease in family income during the period of social distancing, this was a factor that significantly increased the anxiety experienced by students during the covid-19 pandemic, also found in other studies9,30. this could be explained by adversities in dealing with job losses, difficulties in paying bills in addition to the challenges of the pandemic itself. therefore, not only this factor, but others already mentioned, may have been considered by the students as contributing to their degree of anxiety, which explains the wide distribution of anxiety data in this analysis. there was an inversely proportional correlation between age and degree of anxiety. according to other studies, younger students also suffered greater psychological impacts23,31, which could be explained by factors including perceptions about the future and how media is consumed, as evidenced by another study32. however, this may be different for older students because they have less social mobility and probably have more life experience and, therefore, another perspective on their future. the present study showed that the greater the students’ commitment, the more they perceived learning during the social distance. however, it is essential to highlight that mental health disorders, such as anxiety, negatively influence students’ commitment and learning, decreasing motivation and concentration. according to projections regarding covid-19 cases in progress at the time of this study and their impacts, university students need immediate attention and psychological support32. as suggested by a recent study33 based on the italian experience of the pandemic, it is essential to assess the population’s stress levels and psychosocial adjustment to plan the necessary support mechanisms, especially during the recovery phase and similar events in the future. after applying questionnaires to undergraduate and graduate students during the social distancing caused by the covid-19 pandemic, it can be concluded that, despite the good quality of life and good self-perceived health of the students, they had a moderate degree of anxiety during social distancing, also demonstrating a decrease in interest and commitment during distance education. thus, to better understand the current situation and how it will impact the post-pandemic period, it is necessary to investigate how the pandemic has caused anxiety and its consequences. to ensure the mental health of their students due to social isolation, the authorities of various universities should develop plans and strategies34,35. they can prevent anxiety levels from rising by identifying them early and taking action29. the main limitation of the present study is that it was carried out on a particular population. therefore, the results cannot be convincingly extrapolated to the general population. data availability datasets related to this article will be available upon request to the corresponding author. 9 oliveira et al. braz j oral sci. 2022;21: e226698 conflict of interest none author contribution conceptualization: angélica aparecida de oliveira, léa assed bezerra da silva e raquel assed bezerra segato. methodology: angélica aparecida de oliveira, carolina maschietto puccinelli, formal analysis: carolina maschietto puccinelli. investigation: angélica aparecida de oliveira. resources: angélica aparecida de oliveira, léa assed bezerra da silva e raquel assed bezerra segato. data curation: angélica aparecida de oliveira, raquel assed bezerra segato. writing—original draft preparation: angélica aparecida de oliveira, carolina maschietto puccinelli. writing—review and editing: paulo nelson filho, clara marina pereira cavalcanti silva. visualization: angélica aparecida de oliveira, clara marina pereira cavalcanti silva. supervision: léa assed bezerra da silva e raquel assed bezerra segato. project administration: raquel assed bezerra segato. funding acquisition: none. all authors have read and agreed to the published version of the manuscript. references 1. pan american health organization. [who says covid-19 is now characterized as a pandemic]. paho; 2020 mar 11 [cited 2020 jul 6]. available from: https://www.paho.org/pt/news/11-3-2020who-characterizes-covid-19-pandemic. portuguese. 2. world health organization. weekly epidemiological update 12 january 2021. who; 2021 jan 12 [cited 2021 jand 30]. available from: https://www.who.int/publications/m/item/weeklyepidemiological-update---12-january-2021. 3. davenne e, giot jb, huynen p. coronavirus et covid-19 : le point sur une pandémie galopante [coronavirus and covid-19 : focus on a galopping pandemic]. rev med liege. 2020 apr;75(4):218-25. french. 4. silva pgb, de oliveira cal, borges mmf, moreira dm, alencar pnb, avelar rl, et al. distance learning during social seclusion by covid-19: improving the quality of life of undergraduate dentistry students. eur j dent educ. 2021 feb;25(1):124-34. doi: 10.1111/eje.12583. 5. elsalem l, al-azzam n, jum’ah aa, obeidat n, sindiani am, kheirallah ka. stress and behavioral changes with remote e-exams during the covid-19 pandemic: a cross-sectional study among undergraduates of medical sciences. ann med surg (lond). 2020 dec;60:271-9. doi: 10.1016/j.amsu.2020.10.058. 6. duan l, zhu g. psychological interventions for people affected by the covid-19 epidemic. lancet psychiatry. 2020 apr;7(4):300-2. doi: 10.1016/s2215-0366(20)30073-0. 7. wang c, pan r, wan x, tan y, xu l, ho cs et al. immediate psychological responses and associated factors during the initial stage of the 2019 coronavirus disease (covid-19) epidemic among the general population in china. int j environ res public health. 2020 mar;17(5):1729. doi: 10.3390/ijerph17051729. 8. chen q, liang m, li y, guo j, fei d, wang l et al. mental health care for medical staff in china during the covid-19 outbreak. lancet psychiatry. 2020 apr;7(4):e15-e16. doi: 10.1016/s2215-0366(20)30078-x. 10 oliveira et al. braz j oral sci. 2022;21: e226698 9. cao w, fang z, hou g, han m, xu x, dong j et al. the psychological impact of the covid-19 epidemic on college students in china. psychiatry res. 2020 may;287:112934. doi: 10.1016/j.psychres.2020.112934. 10. kaparounaki ck, patsali me, mousa dv, papadopoulou evk, papadopoulou kkk, fountoulakis kn. university students’ mental health amidst the covid-19 quarantine in greece. psychiatry res. 2020 aug;290:113111. doi: 10.1016/j.psychres.2020.113111. 11. harrel sk, molinari j. aerosols and splatter in dentistry: a brief review of the literature and infection control implications. j am dent assoc. 2004 apr;135(4):429-37. doi: 10.14219/jada.archive.2004.0207. 12. ather a, patel b, ruparel nb, diogenes a, hargreaves km. coronavirus disease 19 (covid-19): implications for clinical dental care. j endod. 2020 may;46(5):584-95. doi: 10.1016/j.joen.2020.03.008. 13. becker k, brunello g, gurzawska-comis k, becker j, sivolella s, schwarz f, et al. dental care during covid-19 pandemic: survey of experts’ opinion. clin oral implants res. 2020 dec;31(12):1253-60. doi: 10.1111/clr.13676. 14. gurzawska-comis k, becker k, brunello g, gurzawska a, schwarz f. recommendations for dental care during covid-19 pandemic. j clin med. 2020 jun;9(6):1833. doi: 10.3390/jcm9061833. 15. pan y, liu h, chu c, li x, liu s, lu s. transmission routes of sars-cov-2 and protective measures in dental clinics during the covid-19 pandemic. am j dent. 2020 jun;33(3):129-34. 16. ren yf, rasubala l, malmstrom h, eliav e. dental care and oral health under the clouds of covid-19. jdr clin trans res. 2020 jul;5(3):202-10. doi: 10.1177/2380084420924385. 17. fleck mp, louzada s, xavier m, chachamovich e, vieira g, santos l, et al. [application of the portuguese version of the abbreviated instrument of quality life whoqol-bref]. rev saude publica. 2000 apr;34(2):178-83. portuguese. doi: 10.1590/s0034-89102000000200012. 18. malibary h, zagzoog mm, banjari ma, bamashmous ro, omer ar. quality of life (qol) among medical students in saudi arabia: a study using the whoqol-bref instrument. 2019 sep;19(1):344. doi: 10.1186/s12909-019-1775-8. 19. kroenke k, spitzer rl, williams jb, löwe b. the patient health questionnaire somatic, anxiety, and depressive symptom scales: a systematic review. gen hosp psychiatry. 2010 jul-aug;32(4):345-59. doi: 10.1016/j.genhosppsych.2010.03.006. 20. agius am, gatt g, vento zahra e, busuttil a, gainza-cirauqui ml, cortes arg, et al. self-reported dental student stressors and experiences during the covid-19 pandemic. j dent educ. 2021 feb;85(2):208-15. doi: 10.1002/jdd.12409. 21. iosif l, ţâncu amc, didilescu ac, imre m, gălbinașu bm, ilinca r. self-perceived impact of covid-19 pandemic by dental students in bucharest. int j environ res public health. 2021 may;18(10):5249. doi: 10.3390/ijerph18105249. 22. hakami z, khanagar sb, vishwanathaiah s, hakami a, bokhari am, jabali ah, et al. psychological impact of the coronavirus disease 2019 (covid-19) pandemic on dental students: a nationwide study. j dent educ. 2021 apr;85(4):494-503. doi: 10.1002/jdd.12470. 23. cayo-rojas cf, castro-mena mj, agramonte-rosell rc, aliaga-mariñas as, ladera-castañeda mi, cervantes-ganoza la, et al. impact of covid-19 mandatory social isolation on the development of anxiety in peruvian dentistry students: a logistic regression analysis. j int soc prev community dent. 2021 apr;11(2):222-9. doi: 10.4103/jispcd.jispcd_52_21. 24. cornine a. reducing nursing student anxiety in the clinical setting: an integrative review. nurs educ perspect. 2020 jul/aug;41(4):229-34. doi: 10.1097/01.nep.0000000000000633. 25. pillay n, ramlall s, burns jk. spirituality, depression and quality of life in medical students in kwazulu-natal. s afr j psychiatr. 2016 mar;22(1):731. doi: 10.4102/sajpsychiatry.v22i1.731.  11 oliveira et al. braz j oral sci. 2022;21: e226698 26. odriozola-gonzález p, planchuelo-gómez á, irurtia mj, de luis-garcía r. psychological effects of the covid-19 outbreak and lockdown among students and workers of a spanish university. psychiatry res. 2020 aug;290:113108. doi: 10.1016/j.psychres.2020.113108. 27. kmietowicz z. rules on isolation rooms for suspected covid-19 cases in gp surgeries to be relaxed. bmj. 2020 feb;368:m707. doi: 10.1136/bmj.m707. 28. wang c, zhao h. the impact of covid-19 on anxiety in chinese university students. front psychol. 2020 may;11:1168. doi: 10.3389/fpsyg.2020.01168. 29. cayo-rojas cf, agramonte-rosell rc. challenges of virtual education in dentistry in times of covid-19 pandemic. rev cubana estomatol. 2020;57(3):e3341. 30. fancourt d, steptoe a, bu f. trajectories of anxiety and depressive symptoms during enforced isolation due to covid-19 in england: a longitudinal observational study. lancet psychiatry. 2021 feb;8(2):141-9. doi: 10.1016/s2215-0366(20)30482-x. 31. al-shayea ei. perceived depression, anxiety and stress among saudi postgraduate orthodontic students: a multi-institutional survey. pak oral dental j. 2014 jul;34(2):296-303. 32. holmes ea, o’connor rc, perry vh, tracey i, wessely s, arseneault l et al. multidisciplinary research priorities for the covid-19 pandemic: a call for action for mental health science. lancet psychiatry. 2020 jun;7(6):547-50. doi: 10.1016/s2215-0366(20)30168-1. 33. de girolamo l, peretti gm, maffulli n, brini at. covid-19-the real role of nsaids in italy. j orthop surg res. 2020 may;15(1):165. doi: 10.1186/s13018-020-01682-x. 34. sahu p. closure of universities due to coronavirus disease 2019 (covid-19): impact on education and mental health of students and academic staff. cureus. 2020 apr;12(4):e7541. doi: 10.7759/cureus.7541. 35. ozamiz-etxebarria n, dosil-santamaria m, picaza-gorrochategui m, idoiaga-mondragon n. stress, anxiety, and depression levels in the initial stage of the covid-19 outbreak in a population sample in the northern spain. cad saude publica. 2020 apr;36(4):e00054020. english. doi: 10.1590/0102-311x00054020. 1 volume 22 2023 e230408 original article braz j oral sci. 2023;22:e230408http://dx.doi.org/10.20396/bjos.v22i00.8670408 1 department of clinical dentistry, area of integrated clinic, school of dentistry, state university of piauí (uespi), parnaíba, pi, brazil. corresponding author: ana de lourdes sá de lira university of piauí, school of dentistry rua senador joaquim pires 2076 ininga. fone (86) 999595004 cep: 64049-590 teresina-pi-brasil email: anadelourdessl@hotmail.com editor: dr. altair a. del bel cury received: july 18, 2022 accepted: february 28, 2023 prevalence and factors associated with gummy smile in adolescents: a cross-sectional analysis millena lopes de brito1 , marcelo lucio sousa silva junior1 , breno wesley leal carvalho1 , elen maria carvalho da silva1 , ana de lourdes sá de lira1* aim: to evaluate the prevalence and factors associated with gummy smiles in adolescents aged between 15 and 18 years attending high school. methods: the cross-sectional and quantitative study was carried out during the covid-19 pandemic, with 160 adolescents, from two public (a1) and two private (a2) schools chosen by lottery, divided into two groups: g1 (with gummy smile) and g2 (no gummy smile). a clinical examination was carried out on the adolescents, investigating the presence or absence of a gummy smile (gs), by analyzing the variables (interlabial distance at rest, upper incisor exposure at rest, smile arc, measurement from the subnasal to the upper lip philtrum, upper lip length, upper lip thickness, hypermobility and lower/middle third ratio). means and dispersion were obtained, and the chi-square association test was applied, to compare the results between a1 and a2 and between g1 and g2. results: the prevalence of gs was 33.8% (=54). it was found that no statistically significant associations were found (p > 0.05), regarding the type of school and gender with the presence of gs in adolescents. there was no statistically significant difference (χ² = 1.82; p = 0.07) between the groups and the age of adolescents. there was a significant association between the studied variables and gs (p < 0.05). conclusion: the prevalence was high with a predominance of females. there were no statistically significant associations regarding the type of school and gender, but there was a significant association between gummy smile and lip dimensions. keywords: esthetics, dental. smiling. orthodontics. adolescent. https://orcid.org/0000-0001-6859-7219 https://orcid.org/0000-0002-0961-1700 https://orcid.org/0000-0001-5115-3929 https://orcid.org/0000-0003-3012-3178 https://orcid.org/0000-0002-9299-1416 2 brito et al. braz j oral sci. 2023;22:e230408 introduction an agreeable smile is considered a symbol of beauty and well-being, and depends not only on correct dental and skeletal configuration, but also on the structure and function of the lip muscles, as well as the amount of gingival exposure1,2. in a spontaneous smile, the smile arc is formed by the incisal edges of the canines and maxillary incisors and the lower lip. ideally, the incisal edges are parallel to, and slightly apart from or lightly touching, the lower lip. this is only possible if the lower lip develops a natural curvature, with the corners of the mouth facing upward, and the incisal edges follow this curvature3-5. a smile with excessive exposure of the gingiva, known as gummy smile (gs), is characterized by gingival exposure of more than 3 millimeters and may negatively influence the aesthetics of the smile. this affects about 10% of the population6, with a higher prevalence among women than men because of greater muscle flaccidity6,7. other authors have highlighted that both sex and age are influencing factors, suggesting that women have higher smiles than men and that dentogingival exposure decreases with age5-7. regarding etiological factors, bone, muscular, or dentogingival factors, or a combination of several factors, may play a role in gs. concerning bone, excess vertical maxillary growth or excess vertical growth of the alveolar bone may be observed. with regard to muscular factors, there may be a short upper lip or lip hypermobility. regarding dentogingival factors, the maxillary incisors may project excessively toward the buccal area, with a short clinical crown, which may be associated with gingival hyperplasia8. the smile is classified as high when there is the total exposure of the clinical crowns of the maxillary anterior teeth and excessive exposure of gingival tissue. a medium smile exposes most (75%) or all (100%) of the clinical crowns of the anterior maxillary teeth, but only the interdental or interproximal papillae are visible. a low smile shows less than 75% of the clinical crowns of these teeth, without showing gingival tissue9,10. a certain amount of exposed gingiva is aesthetically acceptable, being considered an important factor of joviality in interpersonal relationships. however, when more than 3 mm of gingiva is exposed, the smile becomes unsightly, inciting many patients to seek dental treatment11-15. although gs can be found in individuals of any age, adolescents are one of the most psychologically affected groups as aesthetic standards have a direct influence on their self-esteem and on the way they relate and live in society16-18. the null hypothesis is that there is a low prevalence of gs in adolescents, with no association with being ashamed to smile and with total ignorance of the possible associated factors. knowledge of the distribution and factors associated with gs in adolescents is of considerable importance for the design of treatment strategies and public policies aimed at improving oral health. thus, it is justifiable to investigate the prevalence of gs among adolescents, taking into account that smile aesthetics may interfere with self-esteem. after diagnosis and identification of associated factors, treatment possibilities can be suggested early. 3 brito et al. braz j oral sci. 2023;22:e230408 the objective of this research was to evaluate the prevalence and factors associated with gs in adolescents between 15 and 18 years of age in high school. materials and methods ethical aspects after ethical approval of the research ethics committee of the state university of piauí (cep/uespi) had been obtained, with number: 3.289.714, a cross-sectional and quantitative study was carried out from november 2020 to july 2021. study population the sample calculation was based on the target population—the number of people between 15 and 18 years of age enrolled in public and private schools in the city of parnaíba in the state of piauí, brazil—which totaled 6209 students in 2020 according to a survey carried out by the brazilian institute of geography and statistics (ibge)18. the required sample size was 362 participants. this minimum number of participants was considered sufficient for the proposed analyses, with a sampling error of 5% and 95% confidence level, indicating that the probability of the error made by the research did not exceed 5%19. two researchers were provided with a letter of consent from the directors of two public (a1) and two private (a2) schools chosen by lot in the city of parnaíba and that authorized the research. the schools were adopting the hybrid education system because of the current conditions of the covid-19 pandemic. students were chosen by lottery according to their schoolbook number. eligibility criteria although, according to the world health organization, adolescents are between 12 and 18 years of age, the inclusion criteria were those between 15 and 18 years of age who were attending high school in public and private schools, an age group that would present greater emotional maturity to analyze their own smile; having no harmful habits; with facial pattern i (sagittal and vertical balance of the face in the frontal and lateral views); and who were not undergoing orthodontic treatment. adolescents below the chosen age group and those unable to understand and answer the questionnaires, such as those with cognitive impairment syndromes or hearing and/or visual impairment, were excluded from the study. calibration to standardize the diagnosis of gs, clinical training was carried out for examiners at the clinic school of dentistry (csd) as calibration. questionnaires were applied and 20 adolescents who did not participate in the study were examined to determine intraand inter-examiner agreement, with kappa values of 0.83 and 0.82, respectively. these examinations were carried out twice, with an interval of 2 weeks. pilot study before data collection, a pilot study was carried out with 30 adolescents from municipal and private schools who were not part of the study sample, to evaluate the meth4 brito et al. braz j oral sci. 2023;22:e230408 ods and verify if there was a need to make changes to the methodology initially proposed. no changes were required. data collection data collection was undertaken during the covid-19 pandemic, when schools were adopting the hybrid teaching system. the research was carried out three times a week in both class shifts, with a small number of participants (2–5 adolescents). two researchers, wearing appropriate personal protective equipment (ppe), carried out the investigations. the questionnaire consisted of patient identification and related questions, and the epidemiological profile of the participant; type of school they belonged to; gender; age; and whether they were satisfied with their smile. participants who were not satisfied with their smile were asked what made it unpleasant and the reason for not having treatment, using the questionnaire developed by mokhtar et al.8 (figure 1). simple and objective language was employed to explain to the adolescents how the research would be carried out. at a subsequent time, in the school environment, participant smiles were examined under artificial light by the researchers, using a flat mouth mirror and a millimeter stainless steel ruler, previously sterilized in an autoclave and for individual use. the participants were seated in a school chair, with the head in the natural position. this position is standardized and easy to reproduce; the head is positioned vertically, with the patient looking at a distant point at eye level, which implies a horizontal visual axis9. in the clinical examination, the presence or absence of gs was investigated, and the variables (interlabial distance at rest, exposure of the maxillary incisors at rest, smile arc, philtrum measurement from the base of the nose to the upper lip, upper lip length, upper lip thickness, hypermobility, and lower/middle third ratio) were analyzed, based on a study previously carried out by seixas et al.5. the results were recorded in the clinical file (figure 2). students who presented gs participated in group 1 (g1), and those who did not were in group 2 (g2), in both a1 and a2. figure 1. epidemiological questionnaire applied to research participants. 5 brito et al. braz j oral sci. 2023;22:e230408 figure 2. clinical form for smile analysis statistical analysis spss statistical software (version 25) was used to perform descriptive statistics, and then statistically analyzed. means and dispersion were obtained and the chi-square association test was applied, with a statistical significance level of 5%, to compare the results between a1 and a2 and between g1 and g2. results as the teaching adopted by the schools was hybrid, it was not possible to obtain the predicted sample. from november to december 2020, a sample of 40 students was obtained, 20 students per month. from january to july 2021, 140 students participated, 5 students per week, but 15 male students and 5 female students were excluded because they only answered the questionnaire. in total, the participation rate in the research was only 160 adolescents, including 90 multiracial, 55 white, and 15 black people. the prevalence of gs was 33.8%, representing 54 adolescents. the distribution of the sample in relation to gender and school is shown in figure 3. chi-square statistical calculations revealed no statistically significant associations (p > 0.05) of the type of school and gender with the presence of gs. in addition, the t test was performed to compare the mean age between participants with and without gs, and there was no statistically significant difference (χ² = 1.82; p = 0.07) between the age of adolescents in the groups with and without gs. the mean age was 15.7 (± 1.02), and the mean age for g1 was 15.5 (± 0.96) and for g2 was 15.8 (± 1.04). 6 brito et al. braz j oral sci. 2023;22:e230408 90 80 70 60 50 40 30 20 10 0 24 30 14 40 44 62 26 80 public private male female with gummy smile 24 50 14 40 no gummy smile 44 62 26 80 figure 3. prevalence of gummy smile according to gender and school of adolescents (15-18 years old) all adolescents in g1 (n = 54) were satisfied with their smile, were not ashamed to smile, and did not seek orthodontic treatment out of disinterest. only g2 students (n = 106) who had dental crowding (n = 39) and diastema between the incisors (n = 28) were embarrassed to smile and did not seek orthodontic treatment for financial reasons. table 1 shows the variables and their association with the presence or absence of gs. there was an association between the presence of gs and all the variables studied (p < 0.05). adolescents who presented with an interlabial gap at rest of >5 mm (n = 28), maxillary incisor exposure of >4.5 mm (n = 28), or a flat smile arc (n = 16) had gs. table 1. distribution of variables associated with gummy smile in adolescents (15 to 18 years old). parnaíba-pi. 2021. variables (with gummy smile) (no gummy smile) total χ² p value interlabial distance at rest 1-5mm (normal) 26 106 132 χ² = 66.62 p = 0.001>5mm 28 0 28 upper incisor exposure at rest <1mm 0 0 0 χ² = 60.94 p = 0.001 1 – 4.5 (normal) 26 106 132 > 4.5mm 28 0 28 smile arch parallel to the curve of the lip ------------------------χ² = 34.89 p = 0.001 lower(normal) 38 106 144 plan 16 0 16 reverse 0 0 0 continue 7 brito et al. braz j oral sci. 2023;22:e230408 continuation measurement from the subnasal to the upper philtrum 12 mm (normal) 26 106 132 χ² = 66.62 p = 0.001<12mm 28 0 28 upper lip length >18mm (normal) 26 106 132 χ² = 66.62 p = 0.001≤ 18mm (short) 28 0 28 upper lip thickness ½ of the lower lip (normal) 44 106 150 χ² = 20.94 p = 0.001< ½ do lower lip 10 0 10 hypermobility yes 50 106 156 χ² = 8.05 p = 0.001no 4 0 4 lower/ middle third ratio lower ≤ medium (normal) 24 106 130 χ² = 39.81 p = 0.001lower >medium 30 0 30 in adolescents with gs, the philtrum length from the base of the nose to the upper lip was <12 mm and the upper lip was short, with a length of ≤18 mm (n = 28); upper lip thickness was less than half that of the lower lip (n = 10); lip hypermobility was present and the lower third of the face was larger than the middle third (most of g1) (p = 0.01). discussion data collected with the participation of 160 adolescents revealed a prevalence of gs in adolescents of 33.8%. this is higher than in another study7 that found a prevalence of 10% in the adult population. this may be due to greater hyperactivity of the upper lip elevator muscles and the nose wing in young people than in adults, especially in female individuals, as well as a short upper lip in most of the young population, which favors greater exposure of gingival tissue during smiling5,12,13,19,20. in the present study, there was a difference in terms of gender, with female participants having a higher prevalence of gs, corroborating the findings of other researchers that women have greater gingival exposure during smiling, with a significant influence on aesthetic perception21. it is worth mentioning that all subjects with gs were satisfied with their smile. despite having gs, they were not ashamed to smile and therefore did not feel the need to go in search of dental treatment. these findings corroborate those of other authors who found that laypersons do not know how to assess how much gingival exposure is considered acceptable when smiling22,23. the majority of participants in g2 presented with dental crowding and diastema between the incisors, were ashamed to smile, and for financial reasons had not yet sought dental care. it was found that the adolescents considered dentoskeletal malocclusion to be more aesthetically relevant than an excessive amount of gingival exposure when smiling, probably because they considered this to be normal physiology in their age group, unlike other deviations from normal occlusion. a similar fact was observed by other authors who found that for most adolescents, malocclusion did not interfere with the level of self-esteem, although more than 90% of the par8 brito et al. braz j oral sci. 2023;22:e230408 ticipants mentioned that they wanted to undergo orthodontic treatment to improve their appearance18. in this study, possible etiological factors in gs were disproportions of the lips and upper lip hypermobility, as observed in other studies8,13,16. if a patient with gs has an upper lip measurement that does not correspond to prescribed norms, the compromised smile aesthetics can be attributed, at least in part, to insufficient lip length. however, if face height, gingival levels, labial length, and central incisor length are all within acceptable limits in a patient with gs, the etiology of the smile is likely due to an overactive upper lip from excessive contraction of the upper lip elevator muscles9,16,17,24,25. skeletal discrepancy due to excess vertical growth, as a possible etiological factor in gs, was not investigated because this measurement requires cephalometric analysis. this can be considered a limitation of this study. this research was clinically relevant as it proved that the aesthetic issue of gs is not a worrying factor among adolescents. for them, the smile is unattractive if malocclusion is present. in this study, there were more female than male participants. this can be considered a limitation of this study. another relevant factor limiting this study was that the sample number was less than the minimum value indicated by the sample calculation, with a different number of participants in the groups. it is suggested that future studies be carried out with a greater number of brazilian participants, addressing both the prevalence and possible etiological factors. in addition, variations in gs highlight the need to establish data from various geographic regions to examine the effect of genetics and environment on tooth development. in conclusion the prevalence of gs was high in adolescents, with a predominance in female participants. there were no associations regarding the type of school and gender, but there was a significant association between gs and lip dimensions. acknowledgment this study was not financed. conflict of interest none. data availability datasets related to this article will be available upon request to the corresponding author. author contribution milena lopes de brito: design, literature review; marcelo lucio s. silva junior: materials, writer; breno wesley leal carvalho: data colletion and/or processing, critical review; elen maria carvalho da silva: interpretation and presentation of results; 9 brito et al. braz j oral sci. 2023;22:e230408 ana de lourdes sá de lira: conception, critical reviewing. all authors actively participated in the manuscript’s findings and revised and approved the final version of the manuscript. references 1. mahardawi b, chaisamut t, wongsirichat n. gum smile: a review of etiology, manifestations, and treatment. siriraj med j. 2019 mar;71(2):168-74. doi: 10.33192/smj.2019.26. 2. pausch nc, katsoulis d. gender-specific evaluation of variation of maxillary exposure when smiling. j craniomaxillofac surg. 2017 jun;45(6):913-20. doi: 10.1016/j.jcms.2017.03.002. 3. mercado-garcía j, rosso p, gonzalvez-garcía m, colina j, fernández jm. gummy smile: mercado-rosso classification system and dynamic restructuring with hyaluronic acid. aesth plast surg. 2021 oct;45(5):2338-49. doi: 10.1007/s00266-021-02169-8. 4. camara ca. aesthetics in orthodontics: six horizontal smile lines. dental press j. orthod. 2010 jan/feb;15(1):118-31. 5. seixas mr, costa-pinto ra, araújo tmd. checklist of aesthetic features to consider in diagnosing and treating excessive gingival display (gummy smile). dental press j orthod. 2011;16(2):131-57. doi: 10.1590/s2176-94512011000200016. 6. pavone af, marjan ghassemian bds, verardi s. gummy smile and short tooth syndrome-part 1: etiopathogenesis, classification, and diagnostic guidelines. compend contin educ dent. 2016 feb;37(2):102-7; quiz 108-10. 7. bynum j. treatment of a “gummy smile”: understanding etiology is key to success. compend contin educ dent. 2016 feb;37(2):114–22. 8. mokhtar ha, abuljadayel lw, al-ali r, yousef m. the perception of smile attractiveness among saudi population. clin cosmet investig dent. 2015 jan 20;7:17-23. doi: 10.2147/ccide.s74764. 9. dym h, pierre r 2nd. diagnosis and treatment approaches to a “gummy smile”. dent clin north am. 2020 apr;64(2):341-9. doi: 10.1016/j.cden.2019.12.003. 10. lemos i, avitos r, noronha r, urquiza m. gummy smile: diagnosis and treatment options. int j oral maxillofac surg. 2019;48(1):263-9. doi: 10.1016/j.ijom.2019.03.803. 11. bolas-colvee b, tarazona b, paredes-gallardo v, arias-de luxan s. relationship between perception of smile esthetics and orthodontic treatment in spanish patients. plos one. 2018 aug;13(8):e0201102. doi: 10.1371/journal.pone.0201102. 12. monaco a, streni o, marci mc, marzo g, gatto r, giannoni m. gummy smile: clinical parameters useful for diagnosis and therapeutical approach. j clin pediatr dent. 2004 fall;29(1):19-25. doi: 10.17796/jcpd.29.1.y01l3r4m06q3k2x0. 13. izraelewicz-djebali e, chabre c. gummy smile: orthodontic or surgical treatment? j dentofacial anom orthod. 2015;18(1):102. doi: 10.1051/odfen/2014036. 14. diaspro a, cavallini m, piersini p, sito g. gummy smile treatment: proposal for a novel corrective technique and a review of the literature. aesthet surg j. 2018 nov;38(12):1330-8. doi: 10.1093/asj/sjy174. 15. park j h. gummy smile: its etiology and treatment options. ajo-do clin comp. 2022 jan;2(2):123-4. doi: 10.1016/j.xaor.2022.01.006. 16. levi ylas, cota lvs, maia lp. digital smile design for gummy smile correction. indian j dent res. 2019 sep-oct;30(5):803-806. doi: 10.4103/ijdr.ijdr_132_18. 10 brito et al. braz j oral sci. 2023;22:e230408 17. gatto rcj, garbin a, corrente j, garbin c. self-esteem level of brazilian teenagers victims of bullying and its relationship with the need for orthodontic treatment. rev gauch odontol. 2017 jan-mar;65(1):30-6. doi: 10.1590/1981-863720170001000053304. 18. brazilian institute of geography and statistics. [an overview of health in brazil: access and use of services, health conditions and risk factors and health protection 2008]. rio de janeiro: ibge; 2010 [cited 2022 may 5]. available from: https://biblioteca.ibge.gov.br/bibliotecacatalogo?id=244356&view=detalhes. portuguese. 19. fontelles mj, simões mg, almeida jc, fontelles rgs. [research methodology: guidelines for calculating the sample size]. rev para med. 2010;24(2):57-64. portuguese. 20. pinho t, bellot-arcís c, montiel-company jm, neves m. esthetic assessment of the effect of gingival exposure in the smile of patients with unilateral and bilateral maxillary incisor agenesis. j prosthodont. 2015 jul;24(5):366-72. doi: 10.1111/jopr.12216. 21. durigon m, alessi bp, neves m, trentin ms. perception of dentists, dental students, and patients on dentogengival aesthectics. rev odont unesp. 2018 mar;47(2):92-7. doi: 10.1590/1807-2577.08917. 22. cotrin er, vasconcelos av jr, haddad ac, reis sa. perception of adults’ smile esthetics among orthodontists, clinicians and laypeople. dental press j orthod. 2015 jan-feb;20(1):40-4. doi: 10.1590/2176-945/20.1.040-044.oar. 23. polo m. gummy smile treatment: a 40year journey. ajo-do clim comp. 2022 apr;2(1):25-35. doi: 10.1016/j.xaor.2022.01.007. 24. antoniazzi rp, fischer ls, balbinot cea, antoniazzi sp, skupien ja. impact of excessive gingival display on oral health-related quality of life in a southern brazilian young population. j clin periodontol. 2017 oct;44:996-1002. doi: 10.1111/jcep.12753. 25. souza sml, araújo idt, abrantes os, borges bcd, assunção iv. [harmonization of the smile with gingivoplasty and composite resin: case report]. rev cienc plural, 2019;5(3):143-52. portuguese. 1 volume 21 2022 e225263 original article braz j oral sci. 2022;21:e225263http://dx.doi.org/10.20396/bjos.v21i00.8665263 1 department of restorative dentistry, dental school, shahid beheshti university of medical sciences, tehran, iran. 2 islamic azad university, dental branch, department of restorative dentistry, tehran, iran. 3 department of restorative dentistry, faculty of dentistry, kashan university of medical sciences, kashan, iran. 4 dental clinic of amir alam hospital, tehran university of medical science, tehran, iran. corresponding author: sahebeh haghi email: sahebeh.h.63@gmail.com editor: altair a. del bel cury received: april 11, 2021 accepted: october 24, 2021 comparative of flexural strength, hardness, and fluoride release of two bioactive restorative materials with rmgi and composite resin shahin kasraei1 , sahebeh haghi2,* , azin farzad3 , mona malek4 , sogol nejadkarimi1 aim: this study was fulfilled to evaluate the flexural strength, micro-hardness, and release of two fluoride ions of bioactive restorative materials (cention n and activa bioactive), a resin modified glass ionomer (fuji ii lc), and a resin composite (filtek z250). methods: forty samples from four restorative materials (activa bioactive, fuji ii lc, cention n, and filtek z250) were provided according to the current standards of iso 4049/2000 guide lines. subsequently, the samples were stored for 24 hours and 6 months in artificial saliva, and successively, flexural strength and micro-hardness of the samples were measured. for each studied groups the ph was decreased from 6.8 to 4 in storage solution. the rate of changes in fluoride ion release was measured after three different storage periods of 24 hours, 48 hours, and 6 months in distilled water, according to the previous studies’ method. two-way anova, one-way anova, tukey hsd pair wise comparisons, and independent t-tests were used to analyze data (α= 0.05). results: the highest flexural strength and surface micro-hardness after 24 hours and also after 6 month were observed for cention n(p<0.001).flexural strength of all samples stored for 6 months was significantly lower than the samples stored for 24 hours(p<0.001). the accumulative amount of the released fluoride ion in rmgi, after six-month storage period in distilled water was considerably higher (p<0.001) than 24 hours and 48 hours storage. the amount of fluoride ion release with increasing acidity of the environment (from ph 6.8 to 4) in fuji ii lc glass ionomer was higher than the bioactive materials (p<0.05). conclusion: the flexural strength of rmgi was increased after storage against the activa bioactive,cention n and z250 composite. storage of restorative materials in artificial saliva leads to a significant reduction in micro hardness. the behavior and amount of released fluoride ions in these restorative materials, which are stored in an acidic environment, were dependent on the type of restorative material. keywords: materials testing. physical phenomena. dental materials. saliva, artificial. https://orcid.org/0000-0003-0167-4704 https://orcid.org/0000-0002-2061-403x https://orcid.org/0000-0002-7503-4454 https://orcid.org/0000-0002-9926-2363 https://orcid.org/0000-0001-5226-2502 2 kasraei et al. braz j oral sci. 2022;21:e225263 introduction nowadays, the application of resin-based restorative materials is getting increased in dentistry due to esthetic issues, ease of application, and the capability of chemically bonding to dental structures1. different types of direct esthetic restorative materials are available to dentists, including composites and glass ionomer cements2. formulation improvement, leading to the increase in durability of these restorative materials, and made them as the preferable materials for dentists3. despite the existence of these benefits, there are some problems related to usage of these materials. marginal integrity, polymerization shrinkage, secondary caries, and post-operative hypersensitivity are recognized as the problems in usage of these substances in the practical work4. glass-ionomer cements are one of the most useful dental materials used in restorative dentistry due to their properties such as the ability of fluoride release, the intrinsic adhesion to the dental structures, the coefficient of thermal expansion similar to the dental structures and their biocompatibility5. despite these benefits, glass ionomers have some limitations such as high wear, solubility, poor mechanical properties, and low strength against occlusal forces6,7. advancement in the science of dental materials leaded to the introduction of bioactive restorative materials in recent years8. these materials present a combination of benefits for using glass ionomers, resin modified glass ionomers and composites. bioactive products actively participate in ion-exchange cycles and help to maintain dental structures and oral health9. these materials react to the changes in oral cavity environment to produce useful changes in salivary, dental, and restorative properties. this issue is introduced as “smart” behavior in this type of restorative materials10. most of the bioactive materials, in addition to their optical and chemical polymerization capabilities, contain polyacid components and glass particles, which affect the reaction of acid-based hardening and therefore include three step hardening mechanisms11. activa bioactive restorative material was introduced in 2013 by pulpdent company. it was reported that, this restorative material resemblance to composite resin, is durable and resistant to abrasion wear, and also can stimulate remineralization and apatite formation9. this process can lead to a greater adaptation and marginal seal at the edge of restorations and can ultimately reduce microleakage and secondary caries12,13. activa lacks bis-phenol a, bis-gma, and bpa; therefore, biocompatibility of the material is higher than ordinary composites14. it was reported that, this restorative material reacts to continuous changes in ph in oral environment to help the reinforcement and recharge of ionic properties of saliva, tooth, and the substance itself11,15,16. cention n is restorative substance belongs to a new group of materials known as alkazite. there is a resin based tooth-colored material, which has a self-curing set3 kasraei et al. braz j oral sci. 2022;21:e225263 ting mechanism with selective light curing capability. it is used as bulk-fill to repair teeth too17. fundamentally, alkazites are able to release acid neutralizing ions due to their alkaline fillers; and hence, cention n is able to release calcium, fluoride, and hydroxide ions in to the oral environment18. it was reported that, some mechanical and chemical properties of bioactive restorative materials were lower, compared to restorative composites19,20. it was stated that, the release of ions from bioactive materials could lead to the establishment of micro cracking and reduction in mechanical properties21. there are few studies that have evaluated and compared the physical and mechanical properties of these materials with the two groups of accepted restorative materials in usual services in dental clinic (composites and glass ionomer cements). on the other hand, in these few studies, the properties of materials in medium or long term storage in the similar conditions of the oral cavity environment have not been studied19. the aim of this study was to compare the flexural strength, microhardness, and release of fluoride ion in two bioactive materials and resin modified glass ionomer cement with conventional composite after a six-month storage period in aqueous and acidic environments. the null hypothesis is the flexural strength, micro-hardness and release of fluoride ions of cention n and activa bioactive have no different with resin modified glass ionomer (fuji ii lc) and a resin composite (filtek z250). materials and methods this laboratory-experimental study evaluated four direct restorative materials as follows: filtek z 250 (3m espe, st paul, mn, usa), fuji ii lc (gc corporation, tokyo, japan), activa bioactive (pulpdent corporation, watertown, ma, usa), cention n (ivoclar vivadent, liechtenstein, switzerland) to measure the properties of flexural strength, microhardness, and release of fluoride ions (table 1). table 1. investigated materials in the study materials manufacturer composition details activa bioactive pulpdent, corporation, watertown, ma, usa mix of diuretane and other methacrylates with the modified poly acrylic acid (44.6 %), reactive glass filler (21.8 wt.%), inorganic filler (56 wt.%),patented rubberized resin(embrace), water. cention n ivoclar – vivadent, liechtenstein, switzerland powder: inorganic fillers (ba-al-ca-ba-f silicate glass, ca-f-silicate glass and customized fillers. liquid: urethane dimethacrylate, triclodecan-dimethanol dimethacrylate, poly ethyleneglycol dimethacrylate. filtek z250 3m espe, st paul, mn, usa bisgma, udma, bisema with small amount of tegdma, filler 60%vol silanized zirconia/silica particle. fuji ii lc (capsulated) gc corporation, tokyo, japan powder: alumino-fluorosilicate glass, liquid: 35%hema, 25% distilled water, 24%polyacrylic acid, 6%tartaric acid and 0.10% camphorquinone, bis-gma and traces of tegdma. depending on the type of test to determine the characteristics, suitable samples were prepared from four restorative materials. 4 kasraei et al. braz j oral sci. 2022;21:e225263 assessment of flexural strength for each one of the restorative materials, 10 bar-shaped samples were prepared using metal molds with dimensions of 2*2*25 mm, in terms of the iso 4049/2000 guidelines. each restorative material was prepared in terms of the manufacturer’s instructions and placed in metal molds. one transparent celluloid strip was placed on the samples’ surface, and light-cured (drs light at, good doctors co., ltd, incheon, korea) with an intensity of 1200 mw/cm2 for 20 seconds over a glass slide. the intensity of the device was assessed by a radiometer (demetron l.e.d. radiometer, sds/kerr) and curing was performed based on the length of samples, as overlapping of the cured areas at 4 regions for each one of the samples. after removing the samples from metal molds, they were randomly divided into four groups. so, each group included two subgroups involved five samples (n=5) to assess their flexural strength at 24 hours and 6 months (in total, eight subgroups) (chart 1). artificial saliva with ph=7 was prepared, and the samples were stored in artificial saliva. the composition of artificial saliva included the followings: 7.0 mmol/l cacl2, 2.0 mmol/l mgcl2, 6h2o, 4 mmol/l kh2po4, 30 mmol/l kcl, and 20 mmol/l hepes buffer, and ph=722. the samples in first group, after being removed from the artificial saliva and rinsed with water, were dried by a paper towel and placed in a mechanical jig (universal testing machine, zwick/ roell z020, gmbh co, germany) to test the three-point bending . the speed of applied force was 0.5 mm/min, and the span in between supports was 20 mm. flexural strength valueswere calculated according to the following formula in terms of mega pascal’s: ∂=3 fi / 2 b d2 flexural strength values of the second group of samples were assessed after six-month storage in artificial saliva according to the above-mentioned method. data were analyzed by two-way anova and one-way anova for subgroups of 48-hour and 24-hour. tukey hsd pairwise comparisons was performed (α= 0.05). flexural strength activa biocative n = 10 cention n n = 10 24h storage n = 5 6m storage n = 5 24h storage n = 5 6m storage n = 5 24h storage n = 5 6m storage n = 5 24h storage n = 5 6m storage n = 5 rmgi n = 10 z250 n = 10 chart 1. how to distribute samples for assessment of flexural strength. 5 kasraei et al. braz j oral sci. 2022;21:e225263 assessment of surface micro-hardness ten disk-shaped samples with a diameter of 6 mm and a thickness of 2 mm were prepared for each material (totally 40 samples) by using metal molds. after placing the materials in the molds, celluloid strip was placed on the bottom and surface of samples, and the samples were cured beyond the glass slide for 20 seconds. the bottom surface of the samples were marked with sharp tip of scalpel, and the superficial surface was polished with 800, 1000, 1200, 1500, 2000, 2500 and 3000 grit sandpaper with reciprocating motions along with water stream, and then were washed by distilled water. the samples were randomly divided into two subgroups to assess 24-hr and 6-month vickers hardness (8 subgroups) and there were5 samples in each sub-group (n=5).artificial saliva with ph=7 was prepared and samples were placed in artificial saliva. surface micro-hardness was assessed using vickers hardness test by diamond indenter with apex angle of 136°. after 24-hour each of the samples of subgroups 1 to 4 dried and the surface hardness was determined by a surface hardness tester system (zhvµ, zwick / roell, zwick, gmbh, germany) in three areas with a distance of at least 300 microns with a force of 100g and a standstill of 15 seconds. the mean values of three regions were recorded as superficial micro-hardness of each sample in kg/mm2. in the second group, surface micro-hardness was assessed after a six-month period of storage in saliva for the subgroups 5-8 in terms of the above-mentioned method. data were analyzed by two-way anova and one-way anova for 48-hour and 24-hour subgroups and tukey hsd pairwise comparison was performed (α = 0.05). assessment of fluoride ion released before and after increasing, the acidity of the sample storage environment was performed as follows: eighteen disk-shaped samples were prepared using metal molds with a diameter of 6 mm and a thickness of 2 mm from each one of the study materials (fig1.). after the placement of materials in the molds, celluloid strip was placed on the bottom and surface of the samples, and after placing a glass slab on the molds, the samples were figure 1. preparation of disk shaped samples. 6 kasraei et al. braz j oral sci. 2022;21:e225263 cured beyond glass slide for 20 seconds. both surfaces of samples were polished using sandpaper of 600,800, and 1000 grit with the water stream, and were washed by distilled water. sample distribution method is specified in chart-2. three of the six study samples (cen 24 ph=6.8) in group 1 were separately placed for 24 hours in a plastic screw-top container containing 5 ml of distilled water (37°c) with a ph of 6.8. the value of ph was measured directly for each solution by ph-meter system (metrohm 744, metrohm ltd, herisa, switzerland). then one ml of solution of each plastic container was picked using micropipette and was diluted in 9 ml of distilled water. two ml of fluoride reagent solution (cat 21060-69) was added to solution and then was properly shaken for 20-30 seconds to achieve a homogeny solution. the prepared solution was placed in the spectrophotometer (dr-5000, hach co, loveland, usa) and the amount of the related fluoride was recorded in mg/l. the other three samples(cen 24 ph=4) in group 1 were individually placed for 24 hours in plastic packs containing 5 ml distilled water at 37°c with ph=6.8. afterward, using 50 ml/mol lactic acid, the ph of solutions reached 4 and the samples were kept for 1 hour23. one ml of solution was picked from the each plastic container and was diluted in 9 ml distilled water. the amount of fluoride ions was recorded in mg/l in terms of the above-described method. therefore, changes in the amount of fluoride ion released from the samples after one hour of reaction with acidic environment and ph reduction from 6.8 to 4 were calculated in one hour with repeated measures at time points of 0, 10, 20, 30, 40, 50, and 60 minutes. in order to make the obtained results similar to clinical situation and determining the effect of sample free surface area on amount of releasing ion from the materials, the data were recorded using the following equation in μg/cm2 24. ion release= m2 μg × volume of solution/area of sample (surface area (cm2) = 2πr (r+h) in group 2 and 3 the measurement was performed similar to group 1, except the storage was done in distilled water for 48 hours and 6 months. chart 2. sample distribution for determining fluoride ion release. 18 samples of each material 6 samples 24h storage ph=6.8 3 samples fluoride ions ph=6.8 3 samples fluoride ions ph=4 3 samples fluoride ions ph=6.8 3 samples fluoride ions ph=4 3 samples fluoride ions ph=4 3 samples fluoride ions ph=6.8 6 samples 6m storage ph=6.8 6 samples 48h storage ph=6.8 7 kasraei et al. braz j oral sci. 2022;21:e225263 this method was also used for three other materials and the changes for ion released from the samples were calculated before and after being placed in the acidic environment at three time points of 24-hr, 48-hr and six months storage. statistical analysis two-way anova, one-way anova, tukey hsd pairwise comparisons, and independent t-tests were used to analyze data of ion release among different materials and subgroups (α= 0.05). results two-way anova showed that the type of restorative material and storage time in artificial saliva had a significant effect on the amount of flexural strength value of restorative materials (respectively, p=0.001 and p=0.001). in addition, the interaction between storage time and type of restorative material factors was significant (p = 0.006). therefore, for 48-hour and 24-hour subgroups analysis used to compare the type of restorative materials with one-way anova and tukey’s post hoc tests. table 2 presents the statistical indices of flexural strength for 4 study materials at two timepoints of 48-hour and 24-hour measurement. the lowest flexural strength was for rmgi (fuji ii lc) and the highest one was for cention n at two time points of 24-hr and 6-month storage. table 2. mean flexural strength values of study materials at two storage timepoints in artificial saliva. material 24 hours *p-value 6 months *p-value activa bioactive 111.82±1.28 a 0.0001 90.11±0.94 a 0.0001 cention n 130.41±3.11 b 101.61±1.53 b rmgi 26.71±2.12 c 40.55±1.43 c z250 100.68±1.83 d 70.58±2.61 d * one way-anova, values with different capital letters in each column show a significant difference according to tukey tests (p <0.05) comparison of the materials with tukey hsd tests showed a significant difference in the amount of flexural strength between all the studied materials and at both storage timepoints of 24 hours and 6 months (p <0.005). due to the results, except for fuji ii lc that showed an increase in flexural strength after 6-month storage in artificial saliva (p=0.001), the flexural strength in other groups after 6 month was lower than 24-hr storage. the micro-hardness tests showed that the minimum surface hardness was observed for fuji ii lc and the maximum value was for cention n by passing after 24 hours and six months storage in artificial saliva (p=0.001 and p=0.001, respectively) 8 kasraei et al. braz j oral sci. 2022;21:e225263 two way anova showed that the type of restorative material and storage time in artificial saliva had a significant effect on surface microhardness value of restorative materials (respectively, p=0.001 and p=0.001). in addition, the interaction between storage time and type of restorative material factors was significant (p = 0.001). therefore, one-way anova and tukey post hoc tests were used to compare the effect of restorative materials in 24-hour and 6month subgroups (table 3). independent t-tests show that except for cention n, which had no statistical significant difference at two timepoint of storage in artificial saliva (p=0.083), the surface hardness of other materials, after 6 month was considerably less compared to 24-h storage time (p<0.01). in addition, it was identified that, after 24 hours, there was no significant difference in mean value of micro-hardness between two materials of activa bioactive and z250 (p = 0.284), and also between activa bioactive, cention n and z250 (p = 0.748), while comparison among other groups showed statistically significant difference (p<0.001). there was a significant difference among all groups during 6 months for surface micro-hardness (p<0.001). table 3. mean micro-hardness (kg/mm2) values of the study materials at two timepoints of storage in artificial saliva material 24 hours *p-value 6 months *p-value activa bioactive 58.73±1.03 a 0.0001 40.84±2.38 d 0.0001 cention n 62.19±1.95 cb 59.03±2.97 cb rmgi 40.91±2.52 d 36.78±1.27 e z250 60.97±1.83 ab 54.96±1.16 f * one way anova, similar lower cases letters show no difference, which was achieved by post hoc tukey hsd tests(p>0.05) two way analysis of variance showed that the main effect of two factors, type of restorative material and storage time in artificial saliva, had a significant effect on the release of fluoride ions from restorative materials (p = 0.001 and p = 0.001, respectively). also, the interaction between storage time and type of restorative factors was significant (p = 0.000). in other words, the release rate of fluoride ions for different restorative materials at different times of 24, 48 hours and 6 months of storage in artificial saliva were different significantly. therefore, one-way anova and tukey post hoc tests were used to compare the effect of restorative materials on ion release in subgroups of 48-hour, 24-hour and 6-month after storage in artificial saliva. results of fluoride ion release in table 4 and chart 3 show that, storage at three time-points of 24-hr, 48-hr, and 6-month after changes in ph of storage environment from 6.8 to 4, resulted in the significant differences in all the study materials of fuji ii lc, cention n, activa bioactive (p=0.001, p=0.001, and p=0.001, respectively). at three timepoints of storage in distilled water at 24-hr, 48-hr, and 6-month, and with a decline in ph of the storage medium from 6.8 to 4, the highest rate of changes in fluoride ions release was for fuji ii lc(p<0.001), which was followed by cention n and activa bioactive, respectively (p<0.05). 9 kasraei et al. braz j oral sci. 2022;21:e225263 also, the highest rate of fluoride ions release with a decline in ph of the environment for two materials of activa bioactive and fuji ii lc was at 6-month storage and the least rate was at 48-hr (p=0.001 and p=0.009, respectively). the rate of changes in fluoride ions release at 24 hours was between these two timepoints (p=0.001). in cention n restorative material, the highest rate of change in fluoride ions release was observed at 6-months, and the storage times at 24 and 48 h had no statistically significant difference (p=0.747). table 4. mean change values of fluoride ion release (µg/cm2) of the study materials after reduce ph of storage solution from 6.8 to 4 at three time points. storage material 24 hours p-value* 48 hours p-value* 6 months p-value* activa bioactive 5.57±0.23 0.0001 3.07±0.46 0.0001 38.82±1.03 0.0001 cention n 12.72±0.20 a 13±0.64 a 42.88±0.43 rmgi 24.89±0.05 20.1±0.25 168.32±1.05 z250 0 0 0 * one way-anova ,only the same lower cases letters show no difference, which was achieved using poh tukey hsd tests(p>0.05) discussion being aware of the mechanical and physical efficacies of various materials in dentistry can provide better treatment for the patients13. continuous progresses in the science of materials lead to introducing bioactive restorative materials, which it was claimed that, they have better properties such as apatite construction, stimulating time activa bioactive cention n rmgi z250 es tim at ed m ar gi na l m ea ns o f f lu or id e io n re le as e (µ g/ cm 2 ) chart 3. comparison of the mean values change in fluoride ion release (μg/cm2) from the stored restorative materials following ph reduction from 6.8 to 4 at different time-points. 10 kasraei et al. braz j oral sci. 2022;21:e225263 remineralization, ions release, and control of environment acidity compared to other usual restorative materials12,13. however, there are few studies conducted on the effect of time on the properties of bioactive materials. the current study compares some mechanical and physical properties of two tooth-colored bioactive restorative materials, with resin composite, and resin modified glass ionomer cement after 24 hours and 6 months storage in artificial saliva. the results of study showed that, cention n has the highest flexural strength at both timepoints of 24-hr and 6-month storage in artificial saliva, followed by activa bioactive, z250, and glass ionomer fuji ii lc, respectively. a higher flexural strength of cention n could be attributed to the composition of monomer used (udma). the findings obtained after 24-hr storage confirmed previous studies, which reported that, glass ionomer flexural strength is lower than resin composite and activa bioactive11,19,25. due to the higher amount of resin matrix lead to increase water sorption and softening the samples, it is also obvious that flexural strength of all groups have decreased after 6-month storage in artificial saliva except fuji ii lc. long-term durability of restorative materials depends on their mechanical and physical properties, and flexural strength is known as the best scale of dental material resistance and a good index of durability of materials in clinical applications26. it was reported that, minimum flexural strength of 80 mpa required for performing the acceptable clinical application in restorative dentistry27. dental composites and restorative materials containing resin are prone to hydrolytic degradation mediated by the effect of water on matrix and filler interface, and also their matrix softening and weakening resulted by water absorption by resin component28-30. this issue can be considered as an explanation for reduction in flexural strength of composite z250, cention n, and activa bioactive after 6-month storage in artificial saliva. it was reported that, ion release from filler particles can result in separating of matrix and filler, and also by micro-cracks formation in interface of filler-matrix interface21. this issue can lead to reduction in flexural strength of two bioactive materials after 6-month storage in artificial saliva. by the way, reduction in flexural strength of z250 composite was significantly higher in this study compared to activa bioactive. although there are two mechanisms for reducing flexural strength in the activa bioactive over time, one is the softening and weakening of the resin base and the other is the release of ions. on the other hand, continuous acid-base reaction after initial setting of glass ionomer and water absorption, can increase the flexural strength over time. this progress could be related to their dual cure setting reaction. the polymerization of resin starts with light curing but acid base reaction progress slowly until further maturation occurs maximum strength over time31. thereby, this might lead to a decline in flexural strength in activa bioactive after storage in artificial saliva, which was lower than z250 composite. increase in flexural strength of fuji ii lc in the current study was in line with previous studies 11 kasraei et al. braz j oral sci. 2022;21:e225263 showing that, 6-monthsstorage of rmgi leads to an increase in flexural strength31. this improvement in strength is due to continuation in setting reaction, which in addition to polymerization of resin presented in ionomer glass, acid-base reaction also continues until achieving the highest rate of material’s strength. delayed substitution of calcium ions by aluminum ions leads to an increase in crosslink and improvement of flexural strength of glass ionomer materials over time. further investigations are needed to compare these materials in clinical conditions due to temperature and acidity changes, and the presence of enzymes and salivary proteins in oral environment. surface hardness is one of the most important mechanical properties of restorative materials, which provides important information on abrasion and setting characteristics of materials32. surface hardness is affected by various factors such as material matrix, the amount and size of filler particles, and the way of filler distribution. in this study, surface micro-hardness of the studied materials was measured using vickers test, which is commonly used for dentistry materials. after 24-hr storage in artificial saliva, minimum surface hardness was observed for fuji ii lc, and no statistically significant difference was observed among restorative materials of z250, cention n, and activa bioactive. difference in hardness of composite compared to rmgi confirmed in previous studies33. inconsistent with the current studies, garoushiin et al.25 2018 reported that, the value of surface hardness in fuji ii lc after 24-hr storage in dry environment was higher than activa bioactive. although this study is inconsistent with the current study regarding force, conditions of storage, duration, and surface polishing of material. resin-modified glass ionomer storage in aqueous environment that is inconsistent with dry environments can lead to softening the it’s superficial layers, and finally to reducing surface micro-hardness by absorption of water and releasing some ions such as strontium, calcium, phosphate and fluoride from glass ionomer matrix25. according to studies by valanezhad et al.34, physical characteristics of materials are increased along with an increase in bioactive glass particles. however, the results of this study show that, except for cention n in other study restorative materials, 6-month storage leads to a significant decline in superficial hardness. in agreement with this study, most previous studies also reported that, long-term storage of restorative materials in aqueous environment can reduce the surface micro hardness11,17,33,35.water absorption in resin matrix leads to an increase in volume and its softening, and finally, to a decline in the micro-hardness9,25. in resin modified glass ionomer fujiii lc, despite observing an increase in flexural strength during 6 months storage in artificial saliva, the value of surface hardness was significantly decreased. this reduction may be due to the presence of resin components like hema as hydrophilic resin component in rmgi, which can increase water absorption at surface layers and leads to approximate decrease of 50% in vickers hardness36. it has been claimed that the release of certain ions, such as fluoride, can play an important role in reducing the incidence of secondary caries, which is the most important cause of failure in tooth-colored restorations37,38. so, in this study, we assessed 12 kasraei et al. braz j oral sci. 2022;21:e225263 ion release in aqueous environment and after acidifying the environment. about 50 years ago, stephan reported that, demineralization of dental structures could be occurred in long-term by exposure to acidic environment with ph lower than critical limit (ph=5.5), which the highest effect of this process could be observed in the first hour of acidity decline23. in the current study, the changes in ph and the release of calcium, phosphate, and fluoride ions were individually assessed after decline in environment acidity from 6.8 to 4 in one hour with repeated measures at time points of 0, 10, 20, 30, 40, 50, and 60 minutes. this method of assessment was also done after 24 hours, 48 hours, and 6 months of storage in distilled water. ion resin of bio-mineral material of activa includes acidic groups of phosphate, which improves the reciprocal effect between glass resin and fillers existed in it, and causes releasing of fluoride ion39,40 as well as high amounts of phosphate ions39. along with the initiation of ionization process, which depends on water uptake, hydrogen ions were separated from phosphate groups, and were replaced by calcium in dental structure. it was reported that, this ionic interaction links resin matrix to minerals in tooth, and forms a strong complex of resinhydroxy apatite, and can also be effective on causing marginal edge flooding41 and a decline in microleakage17,42,43. fluoride release from restorative materials was affected by various factors. rmgi cement (fuji ii lc) due to aqueous base and hema monomer and the presence of porosity in its structure, has more water uptake and subsequently more ion release in aqueous environments20,25. in this study, similar to most of the previous studies, fluoride ion release was higher at all the timepoints compared to two materials of activa bioactive and cention n17,20,35,44. cention n might be the leading cause of producing porosity and bubble during the combination of components of powder and liquid and having alkaline fillers (calcium fluorosilicate), which leads to more ion release in comparison to activa bioactive, as is a paste material with resin base17,35. the results show that, fluoride release was higher in materials fuji ii lc and cention n compared to activa bioactive. the reason of this issue might be related to producing bubble and porosity at mixing time of cention n or fuji ii lc in the form of powder and liquid in comparison to activa bioactive as a paste material. these porosities can lead to more release of ions in these restorative materials45. inconsistent with the current study, naghi et al.44 in 2018 showed that, the amount of fluoride ion release in material of rmgi (fuji ii lc) and activa bioactive at time points of storage after 1, 2, 7, 14, 21, and 28 days in distilled water were not significantly different44. in their study, the experimental method was different, and the effect of ph decline in storage medium was not investigated on amount of fluoride ion release. it was stated that, monthly fluoride release at 200 to 300 μg/cm2 is required to prevent demineralization of enamel46. in the current study, the rate of fluoride release from materials, specifically activa bioactive and cention n during 6 months storage, was lower than this value; therefore, it is possible that, it does not clinically affect the prevention of demineralization of enamel. 13 kasraei et al. braz j oral sci. 2022;21:e225263 although, the presence of fluoride ion in remineralization process of teeth is not effective as alone, and calcium, phosphate, and ohions had important role. therefore, it is necessary to conduct further studies on the effect of these materials, regardless of the type of ions, in reducing decay. most of the previous studies stated the rate of ions release in unit of volume25. however, it should be noted that reporting of ions release rate per surface unit can provide more accuracy for clinical assessments45. accordingly, in the current study, the results of ions release were reported as surface ratio (μg/cm2). it was claimed that, some of the bioactive tooth colored restorative materials such as rmgi, activa bioactive, and cention n have the capability of ion release with ph change of environment. these materials through more release of ions in exposure with acidic conditions lead to a significant increase in environmental ph and subsequently prevention of demineralization process17,47. the results showed significant increase in ph value after placement at acidic condition. however, it does not seem that, increase acidity of environment (increase in ph between 0.5-0.6) was not enough to be an obstacle for enamel demineralization process. if slump of the ph was remained under critical limit, demineralization process of tooth structure goes on to occur clinically, despite reduction in its speed48. since ph changes can be very important in limited environment of microbial plaque, clinical research is definitely required in this context. due to the novelty of these materials and the lack of sufficient research, further studies are needed to determine whether the release of ions leads to the deposition of minerals in the gap between the teeth and restorations, and in this case, can reduce marginal microleakage and clinical recurrent caries. since the association among release of ions such as calcium, fluoride, phosphate, and ohwith decline in dental caries was not investigated in this study, by considering the obtained results on bioactive restorative materials, clinical assessments of them in oral environment are required. in conclusion, by considering limitations of this study the mechanical properties of activa bioactive and cention n as new bioactive materials are comparable to composite resins and rmgi, but their storage in an acidic environment can alter ion diffusion behavior and reduce their mechanical properties. due to the fluoride ion release 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dent mater. 2010 nov;26(11):e227-35. doi: 10.1016/j.dental.2010.07.004. 1http://dx.doi.org/10.20396/bjos.v20i00.8661076 volume 20 2021 e211076 original article 1 associate professor, department of orthodontic and pediatric dentistry, college of dentistry, qassim university, qassim, saudi arabia. 2 associate professor, department of prosthetic dental sciences, college of dentistry, qassim university, qassim, saudi arabia. 3 college of dentistry, qassim university, qassim, saudi arabia. corresponding author: sanaa n. al-haj ali department of orthodontic and pediatric dentistry college of dentistry, qassim university qassim, saudi arabia po box: 6700 postal code: 51452 e-mail: dr.sanaa.alhajali@qudent.org telephone number: 00966504603339 fax number: 009660163801762 author contributions: r.f, a.a, i.a– research concept and design; a.a, i.a – collection and/ or assembly of data; s. al. h, r.f – data analysis and interpretation; all authors– writing the article; s. al. h – critical revision of the article; all authors– final approval of article. editor: dr altair a. del bel cury received: august 28, 2020 accepted: january 28, 2021 comparison of color difference and reliability of two intraoral spectrophotometers sanaa n. al-haj ali1,* , ra’fat i. farah2 , abdullah aldhafeeri3 , ibrahim alduraibi3 aim: to evaluate the intra and inter-device reliability of two intraoral spectrophotometers in measuring the commission internationale de l’éclairage (cie) l*a*b* color coordinates and to compare the color difference (δe) between both devices. methods: the central region of the labial surface of the maxillary central incisor of 31 participants was measured twice by each of the devices (vita easyshade and degudent shadepilot) by one examiner. cie l*a*b* color coordinates were obtained for all teeth and δe was measured and compared. intraclass correlation coefficient (icc) and mann-whitney u test were used to analyze the data (p<0.05). results: inter-device reliability iccs in measuring cie l*a*b* color coordinates ranged between 0.08-0.49 with significant difference between devices only concerning the b coordinate (p<0.05). while intra device reliability iccs ranged between 0.86-0.89 for vita easyshade and 0.81-0.86 for degudent shadepilot. the mean δe for cie l*a*b* color coordinates of vita easyshade was 3.61 (±1.93) compared to 3.60 (± 1.45) for degudent shadepilot with insignificant difference between both devices (p>0.05). conclusions: high intra device reliability in measuring cie l*a*b* color coordinates was achieved particularly of vita easyshade, and both devices had clinically acceptable color difference (δe <3.7) however, inter device reliability was low to moderate. consequently, the same spectrophotometer should be used throughout the steps of performing any toothcolored restoration. keywords: data accuracy. collor. spectrophotometry. https://orcid.org/0000-0001-5210-5128 https://orcid.org/0000-0003-3542-2775 https://orcid.org/0000-0001-9387-1603 https://orcid.org/0000-0002-1459-3244 2 al-haj ali et al. introduction correct tooth shade selection is a vital step that significantly affects the esthetic result of toothcolored restorations, particularly in the anterior region1. tooth shade selection can be done either visually or by instrumental methods which include spectrophotometers, digital color analyzers, or colorimeters1,2-4. visual shade selection using a dental shade guide which consists of tabs of various shades, despite being popular, is subjective and influenced by several factors which include color deficiency, gender, experience, and eye fatigue, consequently, inconsistencies in shade selection can result which will eventually affect the ability to select the optimum tooth shade5. spectrophotometers are among the most accurate, useful, and flexible instruments for tooth shade selection6-8. chen et al.8 (2012) reported in a systematic review that instrumental color measurements using a spectrophotometer provide the most precise and accurate shade-matching outcomes. spectrophotometers convert tooth shade into color coordinates as they are operated through the commission internationale de l’éclairage (cie) l*a*b* color coordinates system (l lightness, a chromaticity along the red–green axis, and b chromaticity along the yellow–blue axis), which allow for an objective shade determination as compared to the visual method6. consequently, they are capable of estimating color by measuring the intensity of reflected light in all visible wavelengths9. several studies have compared visual shade selection with instrumental shade selection using spectrophotometers and favored spectrophotometers for tooth shade selection8,10-12. among spectrophotometers investigated, vita easyshade advance (vita zahnfabrik gmbh, bad säckingen, germany) and shadepilot (degudent shadepilot gmbh, hanau, germany) were repeatedly assessed in terms of reliability. reliability is important as it provides a means of measuring the predictability of the device. a device that produces reliable measurements would likely perform more predictably than a device that is inconsistent13. moderate to high reliability of vita easy shade and degudent shadepilot was observed in the studies which assessed these devices9,13-18. however, different l*a*b* coordinates were reported to be given by each device17. the objective of this in vivo study was (1) to evaluate the intra-device reliability (agreement of measurements across the same device) and inter-device reliability (agreement of measurements between devices) in measuring cie l*a*b* color coordinates under clinical conditions of two intraoral spectrophotometers (vita easyshade and degudent shadepilot), and (2) to compare the color difference (δe) measured from the cie l*a*b* color coordinates between the two devices. the null hypotheses of the current study were that (1) significant agreement exists between the two clinical spectrophotometers, with high intra and inter device reliability and (2) no significant difference in color difference would be present between the two devices. material and methods subjects and ethical approval a total of 31 male patients participated in this in vivo study. ethical approval was obtained from the ethical committee of the college of dentistry/ qassim university 3 al-haj ali et al. before the study was conducted. a written informed consent was also obtained from every participant after a full explanation of the study protocol. the inclusion criteria for the study were: • participants in the age range between 20 and 45 years. • participants who had intact maxillary right central incisor; free from dental caries, restorations, previous endodontic treatment, and previous bleaching treatment or use of whitening toothpaste. • participants must have signed informed written consent. study measures the tooth color of the upper right central incisor of each of the 31 participants was measured at the dental clinics of the college of dentistry/qassim university. shade was recorded for the selected tooth region in each participant by one operator at the central region of its labial surface using vita easyshade and degudent shadepilot spectrophotometers in two different occasions separated by a one-hour interval. vita easyshade and degudent shadepilot were selected in this study as they were reported to be among the most reliable spectrophotometers in clinical settings17,19. also, they are both capable of giving different measurements according to the selected tooth region (incisal, central, or cervical). during the measurements, the participants were asked to keep their tongue in a relaxed position away from the selected tooth, lean their head against the headrest of the dental chair, and keep their mouth slightly opened to prevent moving that could affect the measurements. the devices were used and calibrated according to the manufacturer’s instructions. before measuring the tooth central region color of each participant with the vita easyshade, the central incisor was cleaned with a rubber cup and polishing paste for 15 seconds then it was thoroughly washed with water. a disposable infection control shield (vita zahnfabrik gmbh, bad säckingen, germany) was used over the face of the probe tip, then the device was calibrated by placing the instrument probe in the calibration block holder such that the probe tip is flush with and perpendicular to the calibration block and depresses the calibration block. following successful calibration, the central region of the labial surface of the central incisor was measured by placing the instrument probe perpendicular and flush to the tooth surface. tooth colors expressed in cie l*a*b* coordinates were obtained and recorded after each measurement. according to manufacturer instructions, measurements with vita easyshade were repeated until 2 identical, consecutive measurements of the central region of the tooth were achieved. on the other hand, measurement with degudent shadepilot was preceded by disinfecting its mouthpiece, then the device was calibrated. for measurement, the device was put in the triple-zone measurement mode then the device mouthpiece was put at a right angle to the labial surface of the tooth. correct positioning was confirmed with the appearance of green horizontal lines. the results were analyzed from the device and the cie l*a*b* color coordinates of the central region of the teeth were selected and obtained. 4 al-haj ali et al. statistical analysis data were statistically analyzed using the spss computer software (statistical package for the social sciences version 22, chicago, il, usa). descriptive statistics for the cie l*a*b* color coordinates obtained from each device were computed, and δe was calculated using the following equation and compared between both devices: δe= ½[(δl)2+ (δa)2+ (δb)2] it has been shown that color difference (δe) between 2.0 and 3.7 was visually detectable under clinical conditions, but it resulted in an acceptable color difference20 therefore clinically relevant data in the current study were obtained by counting the number of cases of δe <2.0 and δe<3.7, respectively. because the color differences for each device (δe values) were not normally distributed, a nonparametric mann-whitney u test was used to compare the color difference between both devices. intra and inter-device reliability of the cie l*a*b* color coordinates were verified by calculating the intraclass correlation coefficient (icc). icc measurements > 0.75 were employed for reliable measurements. probability values of p <0.05 were considered statistically significant. results the mean cie l*a*b* color coordinates (n=31) obtained after measurement of the teeth with vita easyshade and degudent shadepilot are summarized in table 1. the mean color difference (δe) for cie l*a*b* coordinates of each device as well as the medians are summarized in table 2. the mean color difference (δe) for cie l*a*b* color coordinates of vita easyshade was 3.61 (±1.93) while it was 3.60 (± 1.45) for degudent shadepilot. mann-whitney u test showed that the mean δe for cie l*a*b* color coordinates between both types of spectrophotometers were significantly indifferent (p=0.794). table 1. mean cie l*a*b* color coordinates (standard deviations) obtained in 31 participants measured with the vita easyshade and degudent shadepilot l* a* b* vita easyshade 77.54 (7.83) -0.177 (1.13) 16.37 (5.35) degudent shadepilot 68.67 (12.54) 4.26 (2.80) 18.80 (6.0) table 2. means (standard deviations) and medians of the calculated e values with both devices with e cut-off values of 2.0 and 3.7 δe (vita easyshade) δe (degudent shadepilot) p value mean (sd) 3.61 (1.93) 3.60 (1.45) 0.794 median 3.20 3.30 δe < 2 19.4 9.7 δe<3.7 54.8 61.3 using mann-whitney u test 5 al-haj ali et al. according to icc, low inter-device reliability with regards to both l and a color coordinates (icc: 0.08 and 0.22 respectively) was achieved, while moderate inter-device reliability was noted with regards to the b coordinate (icc: 0.49) with statistically significant difference (p=0.02) (table 3). on the other hand, high intra device reliability of the cie l*a*b*color coordinates of both devices was achieved with slightly higher intra device reliability of vita easyshade when compared to degudent shadepilot (icc: 0.86-0.89 for vita easy shade compared to 0.81-0.86 for degudent shadepilot (table 4)). table 3. inter-device reliability of color coordinates (l*, a* and b*) cie l*a*b*color coordinate icc p-value l 0.22 0.17 a 0.08 0.23 b 0.49 0.02* *p < 0.05 means statistically significant agreement. table 4. intra device reliability of the color coordinates vita easyshade degudent shadepilot cie l*a*b*color attribute icc icc l 0.88* 0.82* a 0.86* 0.86* b 0.89* 0.81* *icc > 0.75 indicates reliable measurements discussion in this in vivo study, the cie l*a*b* color coordinates of the participants’ teeth were measured in two different occasions separated by one hour at the central region of the labial surface of the teeth using two commercially available clinical spectrophotometers and both intra device and inter-device reliability of the color coordinates were compared using icc which is a well-known statistical test for reliability analysis21. in addition, the color difference (δe) was calculated in this study for each device and compared. having such a study in a patient treatment setting instead of a laboratory setting is an advantage as it should represent actual clinical conditions that we face every day more closely7. one should consider; however, the complexity involved in clinical instrumental tooth shade selection using a spectrophotometer as compared to in vitro settings as the patient’s movement, presence of fogging, the angle and position of the probe, different inclinations, and lightning conditions all can influence the measurements taken and cause misreadings7,11,19. in the current study, the central region of the labial surface of the teeth was chosen as it has been observed that this region lacks the presence of influencing variables which can produce bias in measurements such as the reddish color of adjacent gingiva, the 6 al-haj ali et al. greater curvature of the tooth surface in the cervical region, or higher translucency in the incisal region9. according to the results of the current study, the first null hypothesis is partially rejected as the reported inter-device reliability of the color coordinates was low with regards to both l and a coordinates (icc: 0.08-0.22) and moderate with regards to the b coordinate (icc: 0.49), which indicates that the color coordinates (l*a*b) given by the two devices varied widely and cannot be exchanged particularly with regards to both l and a coordinates. having different color coordinates by different spectrophotometers including the ones assessed in the current study was also reported in previous studies9,14,17,20. on the other hand, the portion of the null hypothesis which concerns intra device reliability can be accepted as the reported intra device reliability of color coordinates was higher than the inter-device reliability in the current study (icc value >0.86 for vita easyshade and >0.81 for degudent shadepilot) with slightly higher intra device reliability of vita easyshade when compared to degudent shadepilot. these findings are consistent with the findings of previous studies9,22. the resultant low inter-device reliability can be justified by the fact that the color coordinates in cie l*a*b* color space depend not only on the spectral reflectance curve of an object but also on the light source and the geometrical orientation of the measuring device7,11,19. in the absence of a positioning device for both spectrophotometers, it is difficult to ascertain a hundred percent that minor movement while measuring did not occur despite that manufacturers’ instructions were strictly followed in both devices as holding the devices steady intraorally is more difficult when compared to having a positioning device13. on the other hand, the slightly lower intra device reliability of the surface measurement device degudent shadepilot when compared to the contact type vita easyshade can be justified by the fact that the measurements of surface measurement devices are more affected by ambient lighting conditions than those of contact-type devices18. when the mean color difference (δe) of each device is compared to the other in the current study, the result was almost the same with both devices (3.61 with vita easyshade vs 3.60 for degudent shadepilot) and consequently no significant difference could be found which leads us to accept the second null hypothesis; however, the fact that 45.5% of the cases with vita easy shade and 38.75% with degudent shadepilot led to an unacceptable color measurement (δe > 3.7) can be interpreted as an unsatisfactory outcome. nevertheless, when that is compared to visual shade selection, where shade matches in only 26.6% of the cases, this can still be considered acceptable20. few limitations of the current study should be noted; the first limitation would be that this study compared both devices without having a reference for comparison and comparison with visual shade selection was not also considered. consequently, the design of the current study does not allow us to know the true color of the participant’s teeth. the second limitation would be that this study focused on measuring the central region of the tooth to minimize the bias although in a clinical scenario a reliable spectrophotometer should give reliable measurements for all tooth regions which are prone to be restored. therefore, future clinical studies should focus on comparing these devices with a reference system and preferably involve a larger number of par7 al-haj ali et al. ticipants and several teeth and teeth regions. before conducting these studies, it is recommended that manufacturers train dentists, and even dental technicians, on how to use these devices in ways that can lessen misreadings clinically and improve comparability of readings between different devices. in conclusion, low to moderate inter-device reliability of vita easy shade and degudent shadepilot was achieved in measuring cie l*a*b* color coordinates with a significant difference concerning the b coordinate, while high intra device reliability was achieved particularly of vita easyshade. in addition, clinically acceptable mean color difference (δe< 3.7) was achieved by both spectrophotometers. therefore, in a clinical setting and throughout the steps of performing any tooth-colored restoration, which may involve a dental technician, one should consider using the same spectrophotometer; either vita easyshade or degudent shadepilot for tooth shade selection, without using them interchangeably until manufacturers provide more standardization for the shade selection process. conflicts of interest the authors declare that they have no conflicts of interest. funding statement this study received no external funding. references 1. bahannan sa. shade matching quality among dental students using visual and instrumental methods. j dent. 2014 jan;42(1):48-52. doi: 10.1016/j.jdent.2013.11.001. 2. yılmaz b, irmak ö, yaman bc. outcomes of visual tooth shade selection performed by operators with different experience. j esthet restor dent. 2019 sep;31(5):500-7. doi: 10.1111/jerd.12507. 3. farah ri, elwi h. spectrophotometric evaluation of color changes of bleach-shade resin-based composites after staining and bleaching. j contemp dent pract. 2014 sep 1;15(5):587-94. doi: 10.5005/jp-journals-10024-1584. 4. farah ri. agreement between digital image analysis and clinical spectrophotometer in ciel*c*h° coordinate differences and total color difference (δe) measurements of dental ceramic shade tabs. int j esthet dent. 2016;11(2):234-45. 5. haddad hj, jakstat ha, ametzl g, borbely j, vichi a, dumfahrt h, et al. does gender and experience influence shade matching quality. j dent. 2009;37 suppl 1:e40-4. doi: 10.1016/j.jdent.2009.05.012. 6. paul sj, peter a, rodoni l, pietrobon n. conventional visual vs. spectrophotometric shade taking for porcelain-fused-to-metal crowns: a clinical comparison. int j periodontics restorative dent. 2004 jun;24(3):222-31. 7. witkowski s, yajima nd, wolkewitz m, strub jr. reliability of shade selection using an intraoral spectrophotometer. clin oral investig. 2012 jun;16(3):945-9. doi: 10.1007/s00784-011-0590-3.  8. chen h, huang j, dong x, qian j, he j, qu x, et al. a systematic review of visual and instrumental measurements for tooth shade matching. quintessence int. 2012 sep;43(8):649-59. 9. lehmann km, devigus a, igiel c, wentaschek s, azar ms, scheller h. repeatability of colormeasuring devices. eur j esthet dent. 2011;6(4):428‐35. 8 al-haj ali et al. 10. kalantari mh, ghoraishian sa, mohaghegh m. evaluation of accuracy of shade selection using two spectrophotometer systems: vita easyshade and degudent shadepilot. eur j dent. 2017 apr-jun;11(2):196-200. doi: 10.4103/ejd.ejd_195_16. 11. igiel c, lehmann km, ghinea r, weyhrauch m, hangx y, scheller h, et al. reliability of visual and instrumental color matching. j esthet restor dent. 2017 sep;29(5):303-8. doi: 10.1111/jerd.12321. 12. liberato wf, barreto ic, costa pp, de almeida cc, pimentel w, tiossi r. a comparison between visual, intraoral scanner, and spectrophotometer shade matching: a clinical study. j prosthet dent. 2019 feb;121(2):271-5. doi: 10.1016/j.prosdent.2018.05.004. 13. kim-pusateri s, brewer jd, davis el, wee ag. reliability and accuracy of four dental shadematching devices. j prosthet dent. 2009 mar;101(3):193-9. doi: 10.1016/s0022-3913(09)60028-7. 14. lehmann km, igiel c, schmidtmann i, scheller h. four color-measuring devices compared with a spectrophotometric reference system. j dent. 2010;38 suppl 2:e65-70. doi: 10.1016/j.jdent.2010.07.006. 15. gehrke p, riekeberg u, fackler o, dhom g. comparison of in vivo visual, spectrophotometric and colorimetric shade determination of teeth and implant-supported crowns. int j comput dent. 2009;12(3):247-63. 16. browning wd, chan dc, blalock js, brackett mg. a comparison of human raters and an intra-oral spectrophotometer. oper dent. 2009;34(3):337-43. doi: 10.2341/08-106. 17. yuan k, sun x, wang f, wang h, chen jh. in vitro and in vivo evaluations of three computer-aided shade matching instruments. oper dent. 2012;37(3):219-27. doi: 10.2341/11-230-c. 18. sarafianou a, kamposiora p, papavasiliou g, goula h. matching repeatability and interdevice agreement of 2 intraoral spectrophotometers. j prosthet dent. 2012 mar;107(3):178-85. doi: 10.1016/s0022-3913(12)60053-5. 19. dozić a, kleverlaan cj, el-zohairy a, feilzer aj, khashayar g. performance of five commercially available tooth color-measuring devices. j prosthodont. 2007;16(2):93-100. doi: 10.1111/j.1532-849x.2007.00163.x. 20. khashayar g, dozic a, kleverlaan cj, feilzer aj. data comparison between two dental spectrophotometers. oper dent. 2012;37(1):12-20. doi: 10.2341/11-161-c. 21. sabour s, moezizadeh m, dastjerdi ev. reliability of shade selection using an intraoral spectrophotometer: common mistakes in reliability analysis. clin oral investig. 2013 apr;17(3):1025. doi: 10.1007/s00784-013-0930-6.  22. lagouvardos pe, fougia ag, diamantopoulou sa, polyzois gl. repeatability and interdevice reliability of two portable color selection devices in matching and measuring tooth color. j prosthet dent. 2009 jan;101(1):40-5. 1http://dx.doi.org/10.20396/bjos.v20i00.8661606 volume 20 2021 e211606 original article 1 department of health sciences and children´s dentistry, piracicaba dental school, university of campinas, são paulo, brazil. av. limeira, 901 p.o. box 52. piracicaba, sp, 13414-903, brazil. 2 faculty of dentistry of uberlândia, federal university of uberlândia, brazil. av. pará, 1720, block 4l, annex b, room 34 campus umuarama, uberlândia, mg, 38400-902. 3 department of preventive medicine, federal university of são paulo, brazil. r. sena madureira, 1500 vila clementino, são paulo, sp, 04021-001. *corresponding author: inara pereira da cunha e-mail: inara-pereira@hotmail.com department of community dentistry, piracicaba dental school, state university of campinas, são paulo, brazil. av. limeira, 901 p.o. box 52. piracicaba, sp, 13414-903, brazil. received: october 13, 2020 accepted: january14, 2021 caries experience in children under 5 years old in the xingu indigenous park in brazil alana cristina guisilini1 , jaqueline vilela bulgareli2 , luciane miranda guerra1 , antonio carlos pereira1 , inara pereira da cunha1,* , pablo natanael lemos3 , rosana de fátima possobon1 , karine laura cortellazzi1 aim: the present study sought to investigate dental caries experience and its association with sociodemographic, postnatal and breastfeeding variables in children in the agerange from 6 to 71  months of age, in the xingu indigenous park, mato grosso, brazil. methods: this was an analytical cross-sectional study that used secondary data pertaining to 402 indigenous children of the low, middle and eastern xingu regions, who participated in the oral health epidemiological survey in 2013. the dependent variable was dental caries, dichotomized by the median (dmf-t≤1 and dmf-t>1). the  data of independent variables were obtained by means of instruments of the local health information system of the xingu indigenous special sanitary district (dsei). raw analyses were performed to test the association of the independent variables with the dependent variable. the  variables were tested in the multiple logistic regression model. results: the mean value of the dmf-t index was 2.60 and the prevalence of affected children was 51%. in the multiple analysis, only children older than 36 months (or: 6.64; ci95%: 4.11 to 10.73) and those that were breastfed for a longer period of time (or: 1.88; ci95%: 1.16 to 3.02) showed significant association with the dmf-t>1 index. conclusion: childhood dental caries among indigenous children was associated with age and breastfeeding prolonged for over 26  months, therefore, pointing out the need to offer dental follow-up care at earlier ages. keywords: indians, south american. dental caries. breast feeding. oral health. mailto:inara-pereira@hotmail.com https://orcid.org/0000-0001-7525-824x https://orcid.org/0000-0001-7810-0595 https://orcid.org/0000-0002-7542-7717 https://orcid.org/0000-0003-1703-8171 https://orcid.org/0000-0002-5330-6869 https://orcid.org/0000-0003-0585-3187 https://orcid.org/0000-0001-6179-3030 https://orcid.org/0000-0001-9584-9477 2 guisilini et al. introduction in brazil, there are approximately 820 thousand indigenous children, who represent almost a third of the total number of brazilian children1, and they are the ones who suffer most with health problems, when compared with non-indigenous children2. as far as oral health is concerned, an unequal distribution of caries disease among children is suspected. the data of the last national oral health survey, known as “sb brasil 2010,” revealed that 5-year-old indigenous children have double the number of cases of dental caries when compared with non-indigenous children in the same age group3. this warning has instigated the need for investigations into the   area, and it would be interesting to find out about the trajectory of the disease and its early onset, which occurs before children reach the age of 71 months of life4. early childhood caries causes pain, difficulties with eating, speaking, sleeping, and socializing. it may compromise children’s development and growth and increase the probability of developing disease in their permanent dentition5. this disease develops as a result of the interaction of multiple individual, social and environmental components6, such as sex, age, dietary habits, social conditions, and access to oral hygiene instructions7-9. moreover, the causal relations between the time of breastfeeding and caries disease, especially among younger children, must be emphasized10-12. reports in the literature have indicated that the concentration of lactose in human milk has the capacity to reduce the ph of dental plaque, thereby contributing to the establishment of caries, particularly when plaque is not removed from the tooth surface by means of correct brushing13. indigenous women of some brazilian ethnicities are known to have the habit of offering breastfeeding on free demand and for an indefinite period of time14, and this is a behavior that must be checked in the indigenous communities, in view of the occurrence of oral health problems, such as dental caries15. the prevalence of dental caries has remained high among the people of the xingu indigenous park (pix) over the last few years16, and the oral health conditions of indigenous children under the age of 5 years, together with the factors associated with them, have hardly been investigated. therefore, the aim of the present study was to find out about dental caries experience among children of pix, under the age of 5 years, and test the hypothesis that caries would be associated with the sociodemographic conditions, post natal and breastfeeding variables. this information could contribute to subsidize strategies for the prevention and control of caries disease among the indigenous children. material and methods ethical aspects the project was summited to and approved by the research ethics committee (cep) of the piracicaba dental school of the state university of campinas (fop/unicamp), report number 135/2014, and by the national commission of ethics in research (conep), report number 1.018.666/2015. 3 guisilini et al. study population the xingu indigenous park (pix) was created by a federal government act in 1961, and is regulated by decree no. 51.084. localized in the northeast of the state of mato grosso, brazil, it covers a total area of 2,825,470 hectares17. according to local health information system (slis) records dated 2013, relative the xingu indigenous special sanitary district (dsei xingu), the pix is inhabited by 16 indigenous ethnicities, totaling 5,859 inhabitants, distributed among 78 villages that have no fluoridated water. the pix is divided into 4 regions that coincide with the following 4 basic poles of health reference: “polo base leonardo villas bôas”, in the high xingu region, “polo base diauarum”, in the low xingu, “polo base pavuru”, in the middle xingu and “polo base wawi”, in the east xingu regions. structured with primary health units, these basic poles are served by the activities operated by multidisciplinary indigenous health teams [“equipes multidisciplinares de saúde indígena (emsi)”] with the first reference being the indigenous health agents [“agentes indígenas de saúde (ais)” and sanitary indigenous health agents [“agentes sanitários indígenas de saúde (aisan)] who work in the villages17. the major portion of the pix population consists of youngsters, of whom 38.2% under the age of 15 years; and approximately 5% are persons over the age of 60 years. as regards proportion between sexes/ethnicity in the territory, a higher number of women (51%) is observed, and predominance of the kaiabi ethnicity, of which 43% of the local population is composed18. eligibility criteria the survey involved 44 villages in the low, middle and east xingu regions, between the months of april 2012 and april 2013. in the present study, the dmf-t data considered were those of all the children between the ages of 6 and 72 months, who participated in the epidemiological survey. of the total of 431 existent children under five years of age, 29 (7.7%) did not participate in the inquiry because they refused or were absent during the period when the exams were performed. calibration of examiners the examiners were calibrated in accordance with the standards adopted in epidemiological surveys, which consisted of the following steps: a) training in the theoretical framework of the variables used; b)acceptance of the criteria adopted for defining each observation of the exam and its respective codes; c) application of the criteria in real situations; that is, the calibration itself; and e) calculation of intraand inter-examiner errors, based on the coefficient. the pondered kappa coefficient for each examiner considered the minimum acceptable limit to be the value 0.65. the kappa inter-examiner value obtained was 0.90. the data were noted on paper forms by 3 indigenous oral health agents, who acted as note-takers. study variables and instruments in the survey, clinical oral exams were performed to identify decayed, missing and/or filled primary teeth, in order to calculate the dmf-t index. the sociodemographic, post-natal and breastfeeding data were obtained from the local health information system (slis) 4 guisilini et al. and dsei xingu, instruments of records dated 2013. the sociodemographic (basic pole, sex, age, village population size and presence or absence of indigenous health agent or indigenous sanitation agent) were obtained from the demographic census of the villages of dsei xingu17. the post-natal data (type of delivery and birth weight) were obtain from the birth spreadsheet records, and data about time of breastfeeding were localized on the daily follow-up map of children under the age of five years. this instrument is used by the indigenous health agents and nurses for filling out the breastfeeding data that are inserted into the local health information system of dsei xingu. statistical analysis the sample size of 402 participants provided a test power of 90% (β = 0.10) with a significance level of 5% (α = 0.05) for the effect size found in the study (odds ratio of 2.0 and 30% of response in the unexposed group). the time of breastfeeding was calculated by measuring the time interval from the child’s birth up to the time when the inquiry was conducted. therefore, if the child were 24 months old on the date of the exam and was still breastfeeding, this was the total period of breastfeeding recorded. therefore, the time of exclusive breastfeeding was not considered, instead, the time of breastfeeding either associated with or without complementary feeding was considered. the data collected were tabulated in a microsoft excel® 2010. the variable dental caries was described by stratifying the data into age groups and presented as absolute and relative frequency distributions, mean dmf-t values, standard deviations, and dental caries prevalence. the dental caries index (dmf-t) was considered the dependent variable, dichotomized by the median of dmft (value=1) into: dmf-t≤1 and dmf-t>1).the  independent variables, child’s age and time of breastfeeding were also dichotomized by the median into ≤36 months and >36 months and ≤26 months and >26 months, respectively. the other variables were grouped in the following manner: presence of indigenous (ais) or sanitary (aisan) health agent in the village: yes or no; basic pole: diauarum, pavuru or wawi; child’s sex: female or male; type of delivery: vaginal or cesarean; birth weight: <2500g or ≥2500g; village population size: small, for villages with population smaller than or equal to 50 inhabitants, medium for villages with population between 50 and 250 inhabitants or large, for villages with population larger than 250 inhabitants. association of the independent variables with the dependent variable was tested by raw analysis. the variables with p<0.20 in the simple analysis were tested in the multiple logistic regression model by using the stepwise procedure. the odds ratio (or) and respective confidence intervals of 95% (ic) were estimated for the variables that remained in the final model, at the level of significance of 5%. all statistical tests were performed with sas program 9.3 (institute inc., cary, nc, usa). a residual analysis was performed and applied the hosmer and lemeshow goodness-of-fit test that showed a good fit of the model (p=0.6880). results of the total number of 402 indigenous children studied, aged between 6 and 71 months, 208 (51.70%) were of the female sex. over half of the children had had previous caries 5 guisilini et al. experience, with a mean dmf-t index of 2.60; with children in the group between 60 and 71 months old showing the highest dmf-t index (5.81). the highest caries prevalence was found in children from 48 to 59 months old, with 87.87% of these children being affected by the disease (table 1). table 1. distribution, mean value of dmf-t index, standard deviation and prevalence of dental caries among indigenous children, according to age-group. age group months n (%) mean dmf-t (sd) caries prevalence (%) 6 17 73 (18.15) 0.10 (0.54) 5.47 18 36 128 (31.84) 1.43 (2.32) 36.71 37 47 77 (19.15) 3.18 (3.89) 59.74 48 59 66 (16.41) 4.36 (3.35) 87.87 60 71 58 (14.42) 5.81 (4.23) 86.20 total 402 (100) 2.60 (3.60) 50.99 note: n=number of children examined; dmf-t=caries index for primary teeth; sd=standard deviation table 2 shows that there were a higher number of participants in the low region of xingu indigenous park (54.98%), with predominance of the female sex (51.74%), and children delivered by vaginal birth (92.54%). the majority of the children had a birth weight equal to or higher than 2500 g (88.81%), and a time of breast feeding shorter than or equal to 26 months (51.00%). the majorities of the villages were of medium size (53.48%), and had the presence of an ais/aisan (97.51%). in the multiple logistic regression, the children over the age of 36  months had 6.64 times more chance of having a caries index higher than 1 than those of an age lower than or equal to 36  months. furthermore, children who were breastfeed for longer than 26 months (or=1.88) had more chance of having a dmf-t index higher than 1 (table 2). discussion the growing interest in childhood dental caries has been emphasized in the latest national researches that have investigated the indigenous populations19-21. among the children under 71 months old, in the present research over half of them were found to have experienced caries, which showed association with age and time of breastfeeding. in indigenous children, the association of dental caries experience with the increase in their age is plausible since a previous study found that among the indigenous women of kaiowa ethnicity in mato grosso do sul, the mean dmf-t tripled with the increase in children’s age, especially between 12 and 24 months of life15. while among the indigenous baniwa of alto rio negro, the mean dmf-t index more than doubled between the ages of 36 and 48  months19; and among the indigenous kotiria in alto rio negro, the mean dmf-t index was 4.72 among children from 1 to 5 years of age20. when all the ethnicities studied in the xingu indigenous park were considered, an increase was also observed in the mean dmf-t index in the age group from 0 to 36 months and from 48 to 60 months old21. 6 guisilini et al. ta bl e 2. r aw a nd a dj us te d an al ys es f or a ss oc ia tio n be tw ee n th e dm ft in de x an d so ci od em og ra ph ic , p os t na ta l a nd b re as tf ee di ng , a nd m ul tip le lo gi st ic a na ly si s fo r dm ft in de x va ria bl es l ow , m id dl e an d ea st x in gu , 2 01 3 t ot al dm ft≤ 1 dm ft> 1 v ar ia bl e c at eg or y n ( % ) n ( % ) n ( % ) c ru de o r ic ( 95 % ) pva lu e o r a dj us te d ic ( 95 % ) pva lu e b as ic p ol ea (p ix r eg io n) d ia ua ru m (l ow ) 22 1 (5 4. 98 ). 12 0 (5 4. 30 ) 10 1 (4 5. 70 ) 1. 18 0. 75 1. 86 0. 53 35 p av ur u (m id dl e) 11 8 (2 9. 35 ). 69 (5 8. 47 ) 49 (4 1. 53 ). r ef w aw i ( ea st ) 63 (1 5. 67 ). 35 (5 5. 56 ). 28 (4 4. 4) . 1. 13 0. 61 -2 .0 9 0. 82 54 se x fe m al e 20 8 (5 1. 74 ). 11 7 (5 6. 25 ) 91 (4 3. 75 ) r ef m al e 19 4 (4 8. 26 ). 10 7 (5 5. 15 ) 87 (4 4. 85 ) 1. 04 0. 70 -1 .5 5 0. 90 41 a ge <3 6  m on th s 20 1 (5 0. 00 ). 16 0 (7 9. 60 ) 41 (2 0. 80 ) r ef r ef >3 6  m on th s 20 1 (5 0. 00 ). 64 (3 1. 84 ) 13 7 (6 8. 16 ) 8. 35 5. 31 13 .1 5 <0 .0 00 1 6. 64 4. 11 10 .7 3 <0 .0 00 1 ty pe o f d el iv er y v ag in al 37 2 (9 2. 54 ). 20 5 (5 5. 11 ) 16 7 (4 4. 89 ) 1. 63 0. 68 3. 90 0. 37 18 c es ar ea n 24 (5 .9 7) . 16 (6 6. 67 ) 8 (3 3. 33 ) r ef b ir th w ei gh t <2 50 0g 31 (7 .7 1) . 17 (5 4. 84 ) 14 (4 5. 16 ) 1. 05 0. 50 2. 19 0. 95 12 >2 50 0g 35 7 (8 8. 81 ). 20 0 (5 6. 02 ) 15 7 (4 3. 98 ) r ef ti m e of b re as tf ee di ng <2 6  m on th s 20 5 (5 1. 00 ). 14 4 (7 0. 24 ) 61 (2 9. 76 ) r ef r ef >2 6  m on th s 18 8 (4 6. 77 ). 77 (4 0. 96 ) 11 1 (5 9. 04 ) 3. 40 2. 24 5. 16 <0 .0 00 1 1. 88 1. 16 3. 02 0. 00 75 p re se nc e of a is / a is a n c ye s 39 2 (9 7. 51 ). 21 9 (5 5. 89 ) 17 3 (4 4. 13 ) r ef n o 10 (2 .4 9) . 5 (5 0. 00 ) 5 (5 0. 00 ) 1. 26 0. 36 4. 44 0. 96 29 v ill ag e po pu la tio n si ze d sm al l 92 (2 2. 89 ). 61 (6 6. 30 ) 31 (3 3. 70 ) r ef m ea n 21 5 (5 3. 48 ). 11 0 (5 1. 16 ) 10 5 (4 8. 84 ) 1. 88 1. 13 3. 12 0. 02 03 la rg e 95 (2 3. 63 ). 53 (5 5. 79 ) 42 (4 4. 21 ) 1. 56 0. 86 2. 82 0. 18 56 n ot e: a b as ic p ol es : s tr uc tu re d te rr ito rie s w ith p rim ar y he al th u ni ts , i n w hi ch m ul tid is ci pl in ar y in di ge no us h ea lth t ea m s (e m si ) w or k, w ith th e fir st re fe re nc e be in g th e in di ge no us h ea lth a ge nt s w ho w or k in th e vi lla ge s. e ac h ba si c po le c ov er s a se t o f v ill ag es 22 ; b t im e of b re as tf ee di ng 25 c a is (i nd ig en ou s h ea lth a ge nt ) a nd a is a n (i nd ig en ou s sa ni ta tio n a ge nt ); d v ill ag e p op ul at io n si ze : s m al l ≤ 5 0 in ha bi ta nt s; m ed iu m > 50 a nd ≤ 2 50 in ha bi ta nt s; l ar ge > 25 0 in ha bi ta nt s. o r =o dd s r at io ; c =c on fid en ce in te rv al ; d m ft> 1 is th e le ve l o f r ef er en ce o f th e in de pe nd en t v ar ia bl e. 7 guisilini et al. the justification for the association between these two variables is based on the cumulative nature of the caries index over the course of years22 and on the relations between the development of caries lesions and chronology of tooth eruption. this is because the tooth surfaces are affected as a result of the time during which they remain exposed to the risk factors present in the oral cavity23. this is a natural fact that has been observed, however, the factors that cause concern are the magnitude of differences in the prevalence rates and levels of severity of the disease among the age groups studied. when the epidemiological findings were compared with the results of the last national oral health survey in brazil, the mean dmf-t of children from 60 to 71 months of age in this study was observed to be higher than the national mean value of non-indigenous children (2.43 teeth with caries experience)24. similarly, the prevalence of children with caries was also higher than the brazilian mean (53.4%) for the age group from 60 to 71 months, demonstrating the possibility of inequalities existent between indigenous and non-indigenous populations, and revealing the need to implement strategies of preventive actions directed towards this segment of the population. indeed, progressive reduction in dental caries had been expected in the low, middle and east xingu regions, as a result of a series of public and social actions in the region, especially the increase in financial investments, from the time the xingu indigenous special sanitary district (dsei) was created in 1999. this strategy, together with the training and presence of the indigenous health agents (ais), and indigenous sanitation agents (aisan) has improved and extended the structure of the health services25. in spite of the importance of these professionals in the villages, no association of this variable with the reduction in prevalence  of childhood caries experience was found in the present study. the competence of  these professionals is not confined exclusively to oral health actions, as they need to provide general guidance about health care during breastfeeding and healthy eating habits of small children. moreover, public health surveillance forms part of public health practice and involves actions of health promotion developed by these professionals25. therefore, we suggest that reflections about educational methods in oral health should be shared with the technical professionals that work in the villages. the high dental caries indexes in both pix and other indigenous populations are influenced by various determinants such as living conditions7-9. the sociodemographic conditions were investigated from this aspect relative to the outcome. the variable village population size was found to be associated with the dmf-t index exceeding 1, however, it did not remain in the adjusted multiple logistic analysis model. village population size is a factor that depends on the sociocultural characteristics of each ethnicity, since some ethnic groups form large communities, and others live in small family nuclei, exhibiting extensive ethnic diversity (16 ethnicities). therefore, association between village population size and dental caries could be related to the different factors, e.g., economic, sociocultural, localization, infrastructure, type of access and use of health services, and the different ethnicities of residents in the park. alves filho  et  al.24 (2013) and arantes and frazão 8 guisilini et al. (2016)26  have previously demonstrated the association of these variables with the occurrence of dental caries in other indigenous populations. the authors point out that that the world health organization recommends the practice of breastfeeding up to the age of two years; moreover, it should be complemented with other foods as from six month of age, as this is fundamental for healthy development18. in the present study, almost half of the children (46.7%) of the villages in the low, middle and east xingu regions were breastfed for extended periods of over 26 months. this clearly benefitted both mothers and children, as has been described in the literature12. nevertheless, although this practice apparently was advantageous to indigenous community, the children breastfed for a longer period of time, and older children were observed to be those most affected by caries experience. each ethnic group has its own peculiarities that influence the practice of breastfeeding14,19. among the indigenous of xakriabá ethnicity, interior of the state of minas gerais, the median time of breastfeeding was from six to twelve months, with high prevalence of early weaning, in which 49% of the children no longer received mother’s milk before they completed one year of life. the short duration of breastfeeding was explained by the introduction of water and teas, according to guidance provided by the village elders and contact with non-indigenous people14. whereas in the xingu indigenous park, indigenous children culturally tended to be breastfed from the first year through to the third year of life, irrespective of the introduction of solid foods27,28. the relationship of association between breastfeeding for longer than 24  months and being affected by dental caries has previously been shown in the literature. chaffee et al.10 (2014) and tham et al.11 (2015) affirmed that dental caries was associated with the time of breastfeeding when the child was offered the breast frequently during the day and night. this led to reduced saliva production and facilitated milk stagnating in the mouth, so that occurrence of the two factors with absence of oral hygiene led to caries. there is a hypothesis that mother’s milk has factors that provide protection against dental caries, such as the presence of casein protein and imunoglobulin a (iga), which inhibit the growth and adhesion of cariogenic bacteria on tooth surfaces, particularly streptococcus mutans. in breastfed children, another complementary mechanism of initial protection against caries disease is the healthy oral microbiome established by exposure to mother’s milk and her skin29. neves et al. (2016)30 evaluated the acidogenicity of human milk in children with and without caries lesions and revealed that breastfeeding did not cause a reduction in the ph of dental biofilm, irrespective of the presence or absence of caries lesions. therefore, breastfeeding in early childhood may well protect against dental caries, however, the time of breastfeeding and behavioral components (such as cleaning the teeth and supporting structures) may significantly contribute to the increase in dental caries in children breastfed after 12  months of age11. furthermore, the risk of dental caries increases, depending on the carbohydrate content, acidity and frequency of consumption of these types of foods. when associated with poor oral hygiene, these foods become essential factors for development of the disease, making them factors that could be confounded with the time of breastfeeding11. therefore, it was perceived 9 guisilini et al. that breastfeeding of itself was not the sole factor contributing to caries disease progression, but that other factors, including its practice in conjunction with a cariogenic diet, were involved. the above-mentioned conditions are exacerbated by the absence of behaviors relative to oral health care, especially as the children get older, new primary teeth erupt, and they are exposed to certain groups of foods, possibly leading to changes in the microbiome and increasing the risk of developing dental caries. the present study had some important limitations related to the methodological approach, with respect to controlling potential confounding factors, such as cariogenic diet and oral hygiene that were not studied, and could have had a negative impact on the children’s oral  health. another limitation was the cross-sectional study design that did not allow causal inferences to be made, therefore, only associations between the independent variables and the dependent variable dental caries (dmf-t index) could be demonstrated. however, the study was conducted with the intention of identifying caries experience in indigenous children under the age of five years, and possible factors related to this outcome, in order to produce new knowledge, based on strategies with actions to improve the health of this segment of the population the findings on dental caries in indigenous children of pix demonstrated the need for actions promoting oral health, preventing oral diseases and providing dental care for children in the age group studied, within the oral health program of dsei xingu. as a coping strategy, we suggest that the oral health teams become more involved in periodic actions of prenatal care and puericulture. continual guidance and encouragement of exclusive breastfeeding up to 6 months, and complementary feeding without restrictions of time or frequency, up to at least 2 years of age, as recommended by the world health organization, must be provided, considering the benefits of breastfeeding to both mother and child11,12. moreover, oral hygiene instructions and healthy dietary practices must be reinforced, considering the sociocultural and local characteristics of the community. lastly, we suggest that educational actions implemented should involve members of the family, such as caregivers (sibling, uncles and aunts, grandparents) and that members of the community (midwives, shamans, teachers) should participate in demonstrating the traditional care practices. since the villages have no fluoridated water, we suggest periodical distribution of oral hygiene products, extension of preventive actions of topical fluoride application, including children under 5 years of age. the ias/aisan in the villages could also develop periodical actions of guidance on mother and child self care, which must be guaranteed by dsei xingu, with a view to improving the oral health conditions of this group. this study revealed that dental caries experience in indigenous children in the pix, between the ages of 48 and 71  months, and of those older than 36  months was associated with breastfeeding for a period of longer than one year. therefore, implementing preventive measures and oral health care for indigenous children under five years of age, and providing the mothers with guidance about oral care to be performed during the breastfeeding period are indispensable actions for diminishing the incidence of caries in this segment of the population. 10 guisilini et al. references 1. ministry of planning, budget and management of brazil. brazilian institute of geography and statistics ibge. 2010 [population census: general characteristics of indigenous people results of the universe]. rio de janeiro: ibge; 2012 [cited 2019 jun 27]. available from: http://www.ipea.gov.br/redeipea/images/pdfs/base_de_informacoess_por_setor_censitario_universo_ censo_2010.pdf. portuguese. 2. united nations children’s fund (unicef). [30 years of the convention on the rights of the child: advances and challenges for girls and boys in brazil]. são paulo: unicef; 2019 [cited 2020 jan 15]. available from: https://www.unicef.org/brazil/relatorios/30-anos-da-convencao-sobre-os-direitos-da-crianca. portuguese. 3. miranda kco, souza tac, leal sc. caries prevalence among brazilian indigenous population of urban areas based on the 2010 national oral health survey. cienc saude colet. 2018 apr;23(4):1313-22. doi: 10.1590/1413-81232018234.18082016. 4. drury tf, horowitz am, ismail ai, msertens mp, rozier rg, selwitz rh. diagnosing and reporting early childhood caries for research purposes. j public health dent. 1999;59(3):192-7. doi: 10.1111/ j.1752-7325.1999.tb03268.x. 5. anil s, anand ps. early childhood caries: prevalence, risk factors, and prevention. front pediatr. 2017 jul;5:157. doi: 10.3389/fped.2017.00157. 6. peltzer k, mongkolchati a. severe early childhood caries and social determinants in three-yearold children from northern thailand: a birth cohort study. bmc oral health. 2015 sep;15:108. doi: 10.1186/s12903-015-0093-8. 7. arantes r, santos rv, frazão p. between-group differences in dental caries in xavante indians from central brazil. rev bras epidemiol. 2010;13(2):223-36. portuguese. doi: 10.1590/s1415790x2010000200005. 8. sampaio fc, freitas chsm, cabral mbf, machado atab. dental caries and treatment needs among indigenous people of the potiguara indian reservation in brazil. rev panam salud publica. 2010 apr;27(4):246-51. doi: 10.1590/s1020-49892010000400002. 9. alves filho p, santos rv, vettore mv. [factors associated with dental caries and periodontal disease in indigenous people of latin america: systematic review]. rev panam salud publica. 2014;35(1):6777. portuguese. 10. chaffee bw, feldens ca, vítolo mr. association of long-duration breastfeeding and dental caries estimated with marginal structural models. ann epidemiol. 2014 jun;24(6):448-54. doi: 10.1016/j. annepidem.2014.01.013. 11. tham r, bowatte g, dharmage sc, tan dj, lau mxz, dai x, et al. breastfeeding and the risk of dental caries: a systematic review and meta-analysis. acta paediatr. 2015 dec;104(467):62-84. doi: 10.1111/apa.13118. 12. victora cg, barros ajd, frança gva, bahl r, rollins nc, horton s, et al. breastfeeding in the 21st century: epidemiology, mechanisms, and lifelong effect. lancet. 2016 jan;387(10017):475-90. doi: 10.1016/s0140-6736(15)01024-7. 13. peres kg, nascimento gg, peres ma, mittinty mn, demarco ff, santos is, et al. impact of prolonged breastfeeding on dental caries: a population-based birth cohort study. pediatrics. 2017 jul;140(1):e20162943. doi: 10.1542/peds.2016-2943. 14. sírio mao, freitas sn, figueiredo am, gouvêa gdr, pena jl, machado-coelho gll. [duration of breastfeeding among the indigenous xakriabá people living in the minas gerais state, southeast brazil]. rev nutr. 2015;28(3):241-52. portuguese. doi: 10.1590/1415-52732015000300002. http://www.ipea.gov.br/redeipea/images/pdfs/base_de_informacoess_por_setor_censitario_universo_censo_2010.pdf http://www.ipea.gov.br/redeipea/images/pdfs/base_de_informacoess_por_setor_censitario_universo_censo_2010.pdf https://www.unicef.org/brazil/relatorios/30-anos-da-convencao-sobre-os-direitos-da-crianca 11 guisilini et al. 15. parizotto spcol. [prevalence of dental caries in primary dentition of children from the kaiowá guarani indigenous community of mato grosso do sul and association with risk factors] [thesis]. são paulo: faculty of dentistry, university of são paulo; 2004. 16. lemos pn, rodrigues da, frazão p, coelho cc, campos jns, narvai pc. dental caries in peoples of xingu indigenous park, brazil, 2007 and 2013. epidemiol serv saude. 2018 feb;27(1):e20171725. doi: 10.5123/s1679-49742018000100005. 17. dsei xingu. [demographic census and forms of the local health information system of the xingu indigenous sanitary district]. canarana, mato grosso; 2013. portuguese. 18. lemos pn, rodrigues da, frazão p, hirooka lb, guisilini ac, narvai pc. [oral health care in the xingu indigenous park, brazil, from 2004 to 2013: an analysis based on evaluation indicators]. cad saude publica 2018;34(4), e00079317. portuguese. doi: 10.1590/0102-311x00079317. 19. carneiro mcg, santos rv, garnelo l, rebelo mab, coimbra jr cea. [dental caries and need for dental care among the baniwa indians, rio negro, amazonas]. cienc saude colet. 2008;13(6):198592. portuguese. doi: 10.1590/s1413-81232008000600034. 20. côrtes g. [dental caries and associated factors in indigenous kotiria do alto rio uaupés, am, brazil] [dissertation]. manaus: federal university of amazonas; 2013. portuguese. 21. pacagnella, rc. [epidemiological profile of oral health of the population of the xingu indigenous park, between 2001 and 2006] [dissertation]. ribeirão preto: ribeirão preto school of medicine, university of são paulo; 2007. 22. ministry of health of brazil. health care secretariat. health surveillance secretariat. [sb brazil 2010: national research on oral health: main results]. brasília: ministry of health of brazil; 2012 [cited 2020 sep 23]. available from: http://bvsms.saude.gov.br/bvs/publicacoes/pesquisa_nacional_saude_bucal.pdf. portuguese. 23. barros sg, alves ac, pugliese ls, reis sr. [contribution to the study of dental caries in 0-30-monthold infants]. pesqui odontol bras. 2001 jul-sep;15(3):215-22. portuguese. doi: 10.1590/s151774912001000300007. 24. alves filho p, santos rv, vettore mv. social and environmental inequities in dental caries among indigenous population in brazil: evidence from 2000 to 2007. rev bras epidemiol. 2013;16(3):692704. doi: 10.1590/s1415-790x2013000300013. 25. ministry of health of brazil. secretariat for management of work and education in health. department of management of education in health. [qualification program for indigenous health agents (ais) and indigenous sanitation agents (aisan)]. brasília: ministry of health; 2016. portuguese. 26. arantes r; frazão p. income as a protective factor for dental caries among indigenous people from central brazil. j health care poor underserved. 2016;27(1a):81-9. doi: 10.1353/hpu.2016.0043. 27. mattos a, morais mb, rodrigues da, baruzzi rg. nutritional status and dietary habits of indian children from alto xingu (central brazil) according to age. j am coll nutr. 1999 feb;18(1):88-94. doi: 10.1080/07315724.1999.10718832. 28. fagundes-neto u, baruzzi rg, wehba j, silvestrini ws, morais mb, cainelli m. observations of the alto xingu indians (central brazil) with special reference to nutritional evaluation in children. am j clin nutr. 1981 oct;34(10):2229-35. doi: 10.1093/ajcn/34.10.2229. 29. holgerson pl, vestman nr, claesson r, öhman c, domellöf m, tanner acr et al. oral microbial profile discriminates breastfed from formula-fed infants. j pediatr gastroenterol nutr. 2013 feb;56(2):127-36. doi: 10.1097/mpg.0b013e31826f2bc6. 30. neves pa, ribeiro cc, tenuta lm, leitão tj, monteiro-neto v, nunes am, et al. breastfeeding, dental biofilm acidogenicity, and early childhood caries. caries res. 2016;50(3):319-24. doi: 10.1159/000445910. http://www.ncbi.nlm.nih.gov/pubmed/?term=neves%20pa%5bauthor%5d&cauthor=true&cauthor_uid=27226212 http://www.ncbi.nlm.nih.gov/pubmed/?term=ribeiro%20cc%5bauthor%5d&cauthor=true&cauthor_uid=27226212 http://www.ncbi.nlm.nih.gov/pubmed/?term=tenuta%20lm%5bauthor%5d&cauthor=true&cauthor_uid=27226212 http://www.ncbi.nlm.nih.gov/pubmed/?term=leitão%20tj%5bauthor%5d&cauthor=true&cauthor_uid=27226212 http://www.ncbi.nlm.nih.gov/pubmed/?term=monteiro-neto%20v%5bauthor%5d&cauthor=true&cauthor_uid=27226212 http://www.ncbi.nlm.nih.gov/pubmed/?term=nunes%20am%5bauthor%5d&cauthor=true&cauthor_uid=27226212 http://www.ncbi.nlm.nih.gov/pubmed/?term=nunes%20am%5bauthor%5d&cauthor=true&cauthor_uid=27226212 http://www.ncbi.nlm.nih.gov/pubmed/?term=cury%20ja%5bauthor%5d&cauthor=true&cauthor_uid=27226212 1 volume 21 2022 e226427 original research braz j oral sci. 2022;21:e226427http://dx.doi.org/10.20396/bjos.v21i00.8666427 1 department of odontology, federal university of bahia, salvador, bahia, brazil. 2 department of odontology, federal university of sergipe, aracaju, sergipe, brazil. 3 department of morphology, federal university of sergipe, sergipe, brazil. 4 department of oral surgery and pathology, dentistry faculty of federal university of minas gerais, minas gerais, brazil. corresponding author: liciane dos santos menezes address: department of odontology, federal university of bahia, avenida reitor miguel calmon s/n vale do canela, 4º andar (sala 404), salvador, bahia, brazil, 40110-902 e-mail: licianesmenezes@gmail.com phone number: (+55 79) 998985676 editor: altair a. del bel cury received: july 21, 2021 accepted: february 25, 2022 mutagenicity in oral cells of individuals exposed to radiofrequency generated by different smartphones liciane dos santos menezes1,* , itana silva santos2 , marcos antônio lima dos santos2 , andrea ferreira soares3 , sílvia ferreira de sousa4 , wilton mitsunari takeshita2 aim: this study aimed to investigate whether non-ionizing radiation emitted by smartphones is likely to cause genotoxic effects on oral epithelial cells. methods: thirty adults were distributed into two groups according to the mobile phone brand used, namely samsung (samsung, seoul, south korea) and apple (apple, california, usa). the material was collected with gentle swabbing of the right and left buccal mucosa using a cervical brush, then the micronucleus test was performed. results: the mann-whitney test with a 5% significance level did not reveal statistically significant differences in micronuclei frequency between the exposed and non-exposed sides (p=0.251). the different brands do not seem to cause risks of inducing genetic damage because there were no statistically significant differences between them (p=0.47). conclusion: therefore, our results suggest no correlations of micronuclei frequency in the exposed buccal cells of mobile phone users at the exposure standard levels observed. keywords: radio waves. micronucleus tests. mutagenicity tests. https://orcid.org/0000-0002-4971-2354 https://orcid.org/0000-0003-3194-5894 https://orcid.org/0000-0002-7589-4809 https://orcid.org/0000-0002-1442-6462 https://orcid.org/0000-0001-7820-4749 https://orcid.org/0000-0001-5682-1498 2 menezes et al. braz j oral sci. 2022;21:e226427 introduction the advent of globalization and the importance of communication networks in this context has increased the use of technologies such as smartphones exponentially all over the world. more than two-thirds of the world population, meaning over five billion people, are mobile telephony subscribers. this technology is based on the exchange of signals between smartphones and base stations through electromagnetic waves known as radiofrequency electromagnetic fields (rf-emf)1,2. international organizations are responsible for establishing guidelines on limits of exposure to radiofrequency. according to the international commission on non-ionizing radiation protection3 (2009), the acceptable limit, which is used in several countries, is a maximum of 300 ghz. the rate at which rf-emf is absorbed by the human body is called specific absorption rate (sar), which is a standardized unit that measures the impact of radiofrequency electromagnetic waves on the human body and is expressed as watt/kg. the maximum legal sar level limited to any mobile phone is 1.6 watt/kg2. therefore, it is important to investigate the effects of exposure to these radiofrequency values. radiofrequency exposure limits are necessary because the human body absorbs a part of electromagnetic waves, implying serious biological risks. biological consequences such as a higher risk of neurological and auditory diseases have been reported in the literature but the results are contradictory4,5. the effects of these radiations are classified into thermal and non-thermal6. the first case happens because non-ionizing radiation can release a sufficient amount of energy to warm up the biological tissue, and serious damage may occur when exceeding the limit levels6,7. according to christ and kuster8 (2005), several factors influence the amount of radiation absorbed by the head of users, namely the power required to transmit and receive the signal from the radio base station (tower), the model of the antenna, phone design, and the positioning relative to the head. the energy emitted by rf-emf is not sufficiently capable of causing direct dna damage but may interfere with the genome through indirect mechanisms such as the production of reactive oxygen species, chromatin disorganization, and impairment of dna repair9. given the carcinogenic potential of rf-emf in human cells, classified as a group 2b agent10, the study of potential mutagenic alterations in the oral epithelium of individuals exposed to this radiation is relevant. de oliveira et al.4 (2017) reported that dna damage can trigger important cellular changes such as senescence, death, or malfunction. these genotoxic changes can be diagnosed with methods such as the micronucleus (mn) test, which allows observing chromatin fragments from chromosomal breakage due to clastogenic or aneugenic events also classified as genotoxicity biomarkers11. the mn test, performed through exfoliative cytology of the oral epithelium, is a useful and minimally invasive diagnostic tool for assessing genetic damage in humans12. according to ros-llor et al.13 (2012), the test is fast and practical, considering that oral mucosa cells are easier to collect than others, such as blood cells. 3 menezes et al. braz j oral sci. 2022;21:e226427 considering the growing number of smartphone users, the risk of genetic damages may contribute to implementing measures to help eradicate long-standing misconceptions about the radiation emitted by these devices. thus, the present study aimed to evaluate, by microscopic observation using the mn test, whether non-ionizing radiation emitted by mobile phones of different brands caused mutagenic effects on oral mucosa cells. materials and methods study population thirty adults (12 men and 18 women) were selected for the study. the sample size calculation was based on the studies by daroit et al.5 (2015), souza et al.9 (2014), and yadav and sharma14 (2008), in which the mean and standard deviation values of 3.75 and 3.791, respectively, were used for the variable of “total micronucleus”. considering a study with 90% power and α = 0.05, the acceptable sample size was at least 13 participants in each group. volunteers who reported not using a mobile phone or having no preference of side (right and left) when using the device were excluded. individuals who reported using non-traditional mechanisms to answer calls such as headsets, hands-free devices, and bluetooth were not included in the survey. other exclusion criteria were the presence of diseases such as diabetes and anemia; use of medications; reports of facial trauma; pregnancy; chronic use of alcohol or drugs; smoking; use of orthodontic appliances; exposure to oral x-rays one month before the study; use of mouthwashes, and use of tooth-desensitizing or bleaching agents 21 days before the study. volunteers with the following characteristics were included in the study: male or female individuals aged between 20 and 30 years and individuals with good general and oral health without changes in the oral mucosa. all participants signed the informed consent, and the study was approved by the human research ethics committee (caae: 53233716.5.0000.5546), following the declaration of helsinki. the participants included responded to a questionnaire on sociodemographic data, past medical history, family history, habits (e.g., alcohol and tobacco consumption), diet, and history of exposure to rf-emf (time using smartphones, the number of minutes a day using smartphones, and side of the face preferred when using the device). the groups were divided according to the questionnaire answers and two mobile phone brands were compared: samsung (head sar value = 0.52 w/kg; body sar value= 0.99w/kg) (samsung, seoul, south korea) and apple (head sar value = 1.2w/kg; body sar value= 1.13w/kg) (apple, california, usa). collection of material after a mouth rinse with water, cells were collected by gentle swabbing of the right and left buccal mucosa with a cytobrush cervical brush (adlin, jaraguá do sul/sc-brazil). the cells were transferred to a vial containing a fixative solution 4 menezes et al. braz j oral sci. 2022;21:e226427 (sra medical, balneário camboriú/sc-brazil). then, they were homogenized in a vortex shaker at speed four for 30 seconds (ni 1059 novainstruments equipamentos para laboratórios ltda., piracicaba/sp-brazil), centrifuged for 10 minutes at 1000 rpm, 130 × g (baby i 206 – fanem, guarulhos/sp-brazil), and finally placed on glass slides and allowed to dry at room temperature for about one hour. the cells were fixed on the glass slides with 80% ethanol for 48 hours before staining. after drying, the slides were stained with hematoxylin-eosin (he), an acid-basic stain that produces a contrast between the cytoplasm and the nucleus. first, the samples were exposed to hematoxylin, a basic dye that binds to substances containing acid groups. then, the samples were exposed to eosin, a weak acid colorant that stains basic structures. considering this characteristic, he has a high affinity with nuclear cells presenting great blue and pink colorations15. analysis of slides an oral pathologist with over 10 years of experience performed a blind evaluation. calibration was performed with the joint analysis of five slides, totaling approximately 6,000 cells. the intraclass correlation coefficient (icc) value was 0.79, indicating excellent agreement. an olympus cx31 transmitted light microscope model (são paulo/sp-brazil) was used for slide analyses. the slides were analyzed from left to right and top to bottom with a 40× objective. then, an immersion objective was used for micronucleus analysis. micronuclei were searched in 2,000-cell nuclei per cytological smear15,16, and an additional 2,000 cells were analyzed when the frequency of micronuclei was higher than 2%. the micronuclei were identified according to the criteria by sarto et al.17 (1987) for measuring dna damage/genotoxicity. data analysis the results of the microscopic analysis of the cell counts of the oral mucosa exposed to radiofrequency radiation were tabulated in microsoft excel, version 2010 for windows 64-bit (microsoft corporation, redmond, wa, usa). the shapiro-wilk test was used to verify the normality of distribution. as a non-gaussian distribution was found, the mann-whitney test was used. a t-test was used to compare the mobile phone brands, as gaussian distribution was observed in this case. the statistical tests were performed using the r software with the rcmdr package, version 3.2.1 for windows 64-bit (the r foundation, vienna, austria). a 5% significance level was set for all statistical analyses. results cells of the right and left buccal mucosa of 30 individuals (15 users of apple and 15 users of samsung mobile phones) were evaluated, resulting in 60 samples. table 1 shows the most important characteristics of the study population. there were male (40%) and female (60%) participants aged between 20 and 30 years. the total period of exposure in this study was predominantly in the range of over 10 years (73%). 5 menezes et al. braz j oral sci. 2022;21:e226427 table 1. characteristics of the study population. patients: n 30 age: mean 23.93 sex: n (%) male 12 (40) female 18 (60) time of exposure to mobile phones: n (%) < 5 years 0 (0) 5 – 10 years 8 (27) > 10 years 22 (73) mobile phone use (h/week): n (%) 0 0 0 2 26 (87) 2 4 4 (13) hand used to answer calls: n (%) right 28 (93) left 2 (7) according to the mann-whitney test, the micronuclei count was not statistically different between exposed and non-exposed sides (p = 0.251) (figure 1). differences between brands were not statistically significant (table 2). 20 18 16 14 12 10 8 6 4 2 0 p = 0.251 exposed buccal mucosa non-exposed buccal mucosa m ic ro nu cl ea te d ce lls /1 00 0 ce lls figure 1. median micronuclei count (maximum and minimum) regardless of brand (mann-whitney test). 6 menezes et al. braz j oral sci. 2022;21:e226427 table 2. micronuclei count according to mobile phone brand (t-test). micronuclei mean sd p-value apple 2.70 ±1.45 0.47 samsung 3.23 ±2.46 sd: standard deviation. discussion in the global communication era, mobile phones are often used and some assumptions regarding their side effects are questioned. the present study aimed to evaluate, with the mn assay, whether the radiation emitted by mobile phones can cause mutagenic effects on oral epithelium cells. the results presented in this study suggest that ionizing radiation associated with mobile phones does not induce the formation of micronuclei in buccal cells at the exposure levels observed. these results agree with some studies3,4,9,17 that demonstrated that using smartphones does not cause genotoxicity, considering that exposed and non-exposed sides did not show statistically significant differences. however, the literature on this topic is controversial, as other studies with exfoliated cells5,14,18 showed a significantly higher number of micronuclei, indicating that mobile phones may cause genotoxicity in contrast with the results presented in this study. daroit et al5. (2014) showed a slight increase in the number of micronucleated cells in the oral mucosa of individuals who used their phones more than 60 minutes per week over eight years. banerjee18 et al. (2016) investigated micronuclei count in mobile phone users, comparing 150 “low-frequency users” (less than 3 h/ week using cell phones) with 150 “high-frequency users” (more than 10 h/week). considering the “high-frequency users” group, a comparative evaluation of both sides of the buccal mucosa was performed, which showed a statistically significant higher frequency of micronuclei in exfoliated buccal cells of the exposed side than those of the contralateral side. yadav and sharma14 (2008) found twice as many micronuclei in mobile phone users than in non-users and reported an increased frequency of micronuclei related to the total time of exposure. however, they used orcein, a non-dna-specific stain that may stain dna containing micronuclei and other artifacts not associated with genomic instability, which could imply false-positive micronuclei count. the present study used hematoxylin and eosin (he), an acid-basic stain that produces a contrast between the cytoplasm and the nucleus and may mark mn frequency considerably19. the samples are first exposed to hematoxylin, a basic dye that binds to any substance containing acid groups, such as the phosphate groups in dna structure, and to nuclear proteins with a negative charge. then, samples can be exposed to eosin, a weak acid colorant that stains basic structures. the basophil structures such as nuclei are stained in blue with hematoxylin, while eosin stains acidophil structures such as collagen fibers in pink. some complications may occur during colorant precipitation, which could facilitate a false-positive result. however, 7 menezes et al. braz j oral sci. 2022;21:e226427 if there is sufficient precaution during slide preparation and staining, this is a reliable method for mn detection15. the mn assay is often used in the oral mucosa due to rapid renewal, and the collection of oral cells involves minimal invasion and high representation of the epithelial tissue4,12. the genetic analysis with exfoliated epithelial cells of the oral mucosa provides several advantages because it is the primary target of exposure and the minimally invasive technique allows monitoring populations exposed to genotoxic agents and the association of lifestyles with the epithelial damages detected12. accordingly, the micronucleus assay with exfoliated cells was chosen because it is well established as a reliable assessment test. there were no statistically significant differences between the brands compared. each cell phone model has its specific absorption rate (sar), which is the amount of energy absorbed per unit mass of tissue during a given time interval, determined by the icnirp3 (2009). the acceptable rate used in brazil is two watts per kilogram of body weight5. the sar for each device used in the study was lower than the recommendations of the responsible institution. other features are directly linked to the increase in mn count. as increasing age (> 40 years) and cigarette consumption (> 40/day) exert a highly significant influence on micronucleus frequency15,20, the participants of the present study were carefully selected to exclude biases. reducing the age difference of patients was attempted, establishing an age range of 20 to 30 years, and smoking patients were excluded. regarding the count of the number of cells, it must be scored in order to obtain statistically results needs to be addressed. tolbert et al.21 (1992), recommended scoring at least 1000 cells per plate, which represents a great method for determining the frequency of all the various types of cells. most recent studies, have scored between 1000 and 3000 cells, which are in accordance to the methodology adopted in our study4,9,16,19,22. a few studies3,4,17 have analyzed the potential correlation of micronuclei frequency with demographic data (sex, age, and place of birth), social origin, and environmental factors (occupation, duration and recent work changes, proximity of homes to helipads or airports, alcohol and tobacco consumption, diet, vitamin supplementation, family history of cancer, chronic medication, and risk factors). however, none showed statistically significant results, which agrees with most studies using the mn test for the oral mucosa. several investigations involving the use of mobile phones are limited due to the challenge to establish a control group4 because the vast majority of the population uses mobile phones, making it nearly impossible to find a sufficient number of individuals who do not use cell phones regularly. due to this difficulty, the present study used the side of the face that was not preferred when answering calls as the control group. the side and the duration of mobile phone use are subject to errors associated with self-reporting methods because underestimations and overestimations are common. although bias is a tangible obstacle to epidemiological research, self-reporting is often the only alternative available to evaluate certain variables. considering the increase in the number of mobile phone users and the dilemma regarding their 8 menezes et al. braz j oral sci. 2022;21:e226427 biological consequences, the present study is important and further research is still required to better elucidate such effects. in conclusion, this study suggests that the mobile phone brands investigated do not have genotoxic potential when comparing mn frequency between the exposed buccal mucosa side and the non-exposed side. data availability datasets related to this article will be available upon request to the corresponding author. conflict of interests none. author contribution contributor 1 liciane contributor 2 itana contributor 3 marcos contributor 4 andrea contributor 5 silvia contributor 6 wilton conception of the work √ √ √ √ design of the work √ √ √ √ data acquisition √ √ √ √ data analysis √ √ √ √ √ √ data interpretation √ √ √ √ √ √ manuscript preparation/ work draft √ √ √ √ √ √ manuscript review/ work review √ √ √ √ √ √ final approval of the version to be published √ √ √ √ √ √ references 1. smith-roe sl, wyde me, stout md, winters jw, hobbs ca, shepard kg, et al. evaluation of the genotoxicity of cell phone radiofrequency radiation in male and female rats and mice following subchronic exposure. environ mol mutagen. 2020;61(2):276-90. doi: 10.1002/em.22343. 2. revanth mp, aparna s, madankumar pd. effects of mobile phone radiation on buccal mucosal cells: a systematic review. electromagn biol med. 2020;39(4):273-81. doi: 10.1080/15368378.2020.1793168. 3. international commission on non-ionizing radiation protection (icnirp). icnirp statement on the “guidelines for limiting exposure to time-varying electric, magnetic, and electromagnetic fields (up to 300 ghz)”. health phys. 2009 sep;97(3):257-8. doi: 10.1097/hp.0b013e3181aff9db. 4. de oliveira fm, carmona am, ladeira c. is mobile phone radiation genotoxic? an analysis of micronucleus frequency in exfoliated buccal cells. mutat res genet toxicol environ 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genotoxic effects of mobile phone radiation using buccal micronucleus assay: a comparative evaluation. j clin diagn res. 2016 mar;10(3):zc82-5. doi: 10.7860/jcdr/2016/17592.7505. 19. metgud r, neelesh bt. effect of staining procedures on the results of micronucleus assay in the exfoliated buccal mucosal cells of smokers and nonsmokers: a pilot study. j cancer res ther. 2018;14(2):372-6. doi: 10.4103/0973-1482.157351. 20. bonassi s, coskun e, ceppi m, lando c, bolognesi c, burgaz s, et al. the human micronucleus project on exfoliated buccal cells (humn(xl)): the role of life-style, host factors, occupational exposures, health status, and assay protocol. mutat res. 2011 nov-dec;728(3):88-97. doi: 10.1016/j.mrrev.2011.06.005. 10 menezes et al. braz j oral sci. 2022;21:e226427 21. tolbert pe, shy cm, allen jw. micronuclei and other nuclear anomalies in buccal smears: methods development. mutat res. 1992 feb;271(1):69-77. doi: 10.1016/0165-1161(92)90033-i. 22. thomas p, holland n, bolognesi c, kirsch-volders m, bonassi s, zeiger e, et al. buccal micronucleus cytome assay. nat protoc. 2009;4(6):825-37. doi: 10.1038/nprot.2009.53. 1http://dx.doi.org/10.20396/bjos.v20i00.8663587 volume 20 2021 e213587 original article 1 department of orthodontics, college of stomatology, the first affiliated stomatological hospital, xi’an jiaotong university, xi’an 710004, pr china 2 iranian association of periodontology, tehran, iran 3 department of endodontics, school of dentistry, shahid beheshti university of medical sciences, tehran, iran 4 school of dentistry, kermanshah university of medical sciences, kermanshah, iran corresponding author: ali amiri department of orthodontics, college of stomatology, the first affiliated stomatological hospital, xi’an jiaotong university, xi’an 710004, pr china tell: +86-2982655450 email: draliamiri2020@gmail.com received: december 12, 2020 accepted: december 29, 2020 evaluation of the potential for transmission of coronavirus disease via saliva: a systematic review and meta-analysis ali amiri1,* , pantea moradinejad2 , navid nasrabadi3 , marzieh ghasemi vojoodi4 aim: one of the main factors that play a pivotal role in the transmission of covid-19 from human to human is saliva; according to the subject’s importance, the present study aimed to evaluate the potential of transmission via the saliva of coronavirus disease. methods: pubmed, isi, embase, scopus, medicine have been used until september 2020 to search for articles. therefore, endnote x9 used to manage electronic resources. a 95% confidence interval (ci) effect size, fixed effect model, inverse-variance methods have been calculated. the positive rate of sars-cov2 assessed with meta analysis. to deal with potential heterogeneity, random effects were used, and i2 showed heterogeneity. i2 values above 50% signified moderate-to-high heterogeneity. the meta-analysis has been evaluated with stata/mp v.16 (the fastest version of stata) statistical software. results: according to the study’s purpose, in the initial search with keywords, 19 articles were found, the full text of 3 studies was reviewed, and finally, three studies were selected. the positive rate of sars-cov2 was 86% (86%; 95% ci 67 %-100%). conclusion: saliva can be a non-invasive specimen type for diagnosis of covid-19. dentists should be aware that saliva plays a major role in the transmission of covid-19 from human to human, and failure to follow prevention protocols can contaminate them. keywords: covid-19. dental health services. saliva. https://orcid.org/0000-0001-9416-808x https://orcid.org/0000-0002-6857-6482 https://orcid.org/0000-0002-6365-6619 https://orcid.org/0000-0003-0966-2759 2 amiri et al. introduction in the current context of the world, since the advent of covid-19 in december 2019 in wuhan, china1, the disease has become a major global health concern. on february 11, 2020, the world health organization (who) has declared a new title for the 2019-ncov: corona virus disease (covid-19) epidemic infection2. according to the who, the global mortality rate over time was estimated at 5.7% using a 14-day estimate3. early studies reported transmitted from animals to humans, but studies have illustrated human-to-human transmission of the covid-19 through droplets or direct contact4,5. so far, the 2019-ncov has affected more than 43,150,456 reported cases, according to a new report by the center for systems science and engineering (csse) at johns hopkins university (jhu) (october 26, 2020). in the sixth edition of the covid-19 treatment regimen (trial implementation)6, it was reported that the possible routes of transmission of covid-19 are direct contact and transmission through respiratory particles. in a closed environment, aerosols may be transferred, and more people are at risk for aerosol transmission7. covid-19 transmission is expected to be seen during contact during dental clinical procedures with droplets and aerosols; aerosols can pose potential risks to the dentist and reciprocally to the patient. dentists need to evaluate effective strategies to prevent covid-19 infection during the aerosol production process. one of the main factors that play a pivotal role in the transmission of covid-19 from human to human is saliva. according to the subject’s importance, the present study aimed to evaluate the effect size of 2019-ncov and the potential of transmission via the saliva of coronavirus disease. materials and methods pubmed, isi, embase, scopus, medicine have been used until september 2020 to search for articles. therefore, endnote x9 is used to manage electronic resources. pubmed search was conducted using mesh terms: (“covid-19 vaccine” [supplementary concept] or “covid-19 diagnostic testing” [supplementary concept] or “covid-19” [supplementary concept] or “severe acute respiratory syndrome coronavirus 2” [supplementary concept]) and (“saliva”[mesh] or “saliva-interacting cell wall protein, streptococcus” [supplementary concept] or “saliva natura” [supplementary concept] )) and “dental health services”[mesh]. the present study answers the following question: what is the probability of transmitting the coronavirus disease through saliva? inclusion criteria included were randomized controlled trials studies, controlled clinical trials, in vitro studies, case studies, case reports, and prospective and retrospective cohort studies, only studies have reported the transmission of covid-19 through saliva and aliva specimens were collected at 0–14 days after hospitalization. review studies were excluded from the present article. 3 amiri et al. data extraction methods data extracted from the studies were included sample size, study, study design. newcastle-ottawa scale (nos) used to assessed quality of the cohort studies, the scale scores range from 0 (lowest grade) to 6 (highest grade). effect size with 95% confidence interval (ci), fixed effect model, inverse-variance methods were calculated. to deal with potential heterogeneity, random effects were used, and i2 showed heterogeneity. i2 values above 50% signified moderate-to-high heterogeneity. the meta-analysis has been evaluated with stata/mp v.16 (the fastest version of stata) statistical software. results according to the study’s purpose, in the initial search with keywords, 19 articles were found. in the first step of selecting studies, 14 studies were selected to review the abstracts. then, studies that did not meet the inclusion criteria were excluded from the study. in the second step, the full text of the three studies was reviewed. finally, three studies were selected (figure1). studies identified (n=19) studies after copies expelled (n=14) studies screened (n=14) studies excluded (n=11) 11 reviews studies full content article surveyed for eligibility (n=3) full content article excluded (n=3) the included studies (n=3) id en tif ic at io n sc re en in g el ig ib ili ty in cl ud ed figure 1. study attrition characteristics the sample size total was 74 patients with 62 years mean of age between 37-75 years. saliva specimens in to et al. (2020)8, meng et al.9 (2020) and williams et al. (2020)10 4 amiri et al. were 2 , 4 and 6 days, respectively. overall, 11/12, 20/23, and 33/39 patients detected in saliva in to et al. (2020)8, meng et al.9 (2020) and williams et al. (2020)10, respectively. the positive rate of sars-cov2 the effect size of studies included in the present systematic review and meta-analysis was 86% (86%; 95% ci 69 %-100%). this result has shown saliva’s potential to be a non-invasive type of specimen for 2019-ncov diagnosis and viral load monitoring. table 1. details of selected studies according to inclusion criteria study. years study disegn place sample size mean/range of age (years) patient specimens saliva specimens* sars-cov-2 detected in saliva to et al., 20208 cohort public health laboratory services branch in hong kong 12 female:5 male: 7 62.5/37 to 75 2 ml saliva / cough sterile two days 0-7 days 11/12 meng et al., 20209 cohort hospital of stomatology, wuhan university 23 female:10 male: 13 62 / 37–75 -----four days 0-13 days 20/23 williams et al., 202010 cohort royal melbourne hospital 39 nr nr six days 0-14 days 33/39 * saliva specimens were collected at a median of days after hospitalization figure 2. forest plot showed an effect size of 2019-ncov was detected in the initial saliva specimens. discussion the most common symptoms are: fever, tiredness, cough, aches and pains, sore throat, conjunctivitis, diarrhea, loss of taste or smell, headache, skin rash, or finger or toe discoloration are less common symptoms. and most importantly, there are severe symptoms require special attention, which includes: shortness of breath 5 amiri et al. or difficulty breathing, chest pain or pressure, loss of speech or movement11,12. although coronavirus infection is mild, severe acute respiratory syndrome coronavirus (sars-cov) leads to high mortality rates13. it should be noted that some patients are carriers and have only mild symptoms (carriers). it takes an average of 5-6 days for a person to get the virus to show symptoms, but it can take up to 14 days. some of the characteristics of the virus are still unknown due to the novelty of the virus. covid-19 has recently been reported to be detected in infected patients’ saliva, so its spread through saliva is possible. casaroto et al.13, 2020, evaluate the effect of cooling water temperatures on changes in pulp chamber temperatures and showed when using a high-speed handpiece, too much heat is generated, which should use a water coolant. water coolant can create aerosols, and when combined with saliva in the oral cavity, bio-aerosols are formed. bio-aerosols originate from various sources and may be hazardous to healthcare workers and patients due to their potential pathogenic nature. zemouri et al. 14, 2017, in a review reported that 38 types of microorganisms found in the dental clinic air, and for both patients and healthcare workers, all aerosols may be hazardous. several studies show that the transmission of covid-19 occurs mostly through oral droplets15-18. studies in the field of oral dentistry provide an opportunity to determine if a diagnosis of non-invasive saliva for covid-19 could assist detect such viruses and reduce their spread. in china, researchers can develop unique pcr tests focusing on covid-19 diagnosis by examining viral genome sequence data from international genbank databases12,19. to date, the routes of transmission of covid-19 have not been fully elucidated; however, human-to-human transmission has been confirmed. throat samples and blood tests can be used to diagnose the virus20-22. potential routes of transmission of covid-19 include cough, sneezing, and aerosols produced during the clinical process and even talking. as a result, it can be said that the source of the droplets can be the throat or pharyngeal cavity, which are generally associated with saliva23. small droplets can remain suspended in the air, and larger droplets can help transmit the virus. it has also been reported that transmission by contaminated blood may occur. dentists may carelessly or unknowingly care for carrier patients21,24. studies have shown that about 29% of patients with covid-19 are health care workers25. because the airborne particles and aerosols produced during the dental process are inhaled, covid-19 transmission occurs rapidly in dentistry, especially in dentists who directly contact the patient26. as a result, dentists should be aware of prevention methods and adopt preventive strategies. according to the mentioned transmission routes, covid-19 can spread to dental offices. as a result, hands should be washed regularly, all equipment and surfaces should be disinfected regularly, and personal protective equipment and preferably disposable items should be used. aerosol droplets are produced when coughing, laughing, talking, or sneezing. aerosol droplets are droplets smaller than five μm diameter and larger droplets larger than 5 μm diameter. small droplets settle faster than larger droplets and stay in the air longer, while large droplets fall to the ground quickly and can be transferred to another person in less time27. otter et al. 28, 2013 evaluate the evidence that contaminated surfaces contribute to the transmission of pathogens 6 amiri et al. in hospitals; the result showed contaminated surfaces contribute to transmission. baghizadeh fini. 29, 2020, examine the routes of respiratory virus transmission among humans; the result showed respiratory viruses, droplets, or aerosols are transmitted via contact. one way to diagnose covid-19 is to use salivary diagnosis platforms, which may detect saliva infection in some virus strains for up to 29 days30,31. in this type of test, because the person has close contact with infected patients to collect the sample, there may be a transmission risk. the covid-19 virus is present in saliva in three different pathways: 1. lower or upper respiratory tract presence 2. enter with fluids21 3. minor or major salivary gland infection21 in an animal study, it was shown that salivary gland cells could be the main source of covid-1922. further studies on saliva and its effect on the transmission of the virus should be performed to confirm these findings. to suggest more effective follow-up methods, especially in dentists who perform aerosol production methods themselves. the current systematic review and meta-analysis have aimed to evaluate the potential of transmission via the saliva of coronavirus disease. present meta-analysis showed overall 86% patients (86%; 95% ci 67 %-100%) had sars-cov-2 detected in saliva. personal protective equipment and hand cleanliness practices, personal protective equipment (ppe), preprocedural mouth rinse, single-use (disposable), periapical radiography. cone-beam computed tomography (cbct) and periapical (pa), rubber dam, sodium hypochlorite for root canal irrigation, disinfect inanimate surfaces, ultrasonic scaling instruments and, airborne infection isolation. the advantages of using saliva samples in the diagnosis of 2019-ncov can be the following: 1. saliva samples can be easily prepared by the patient without any invasive methods. 2. the use of saliva samples can reduce the risk of nosocomial transmission 2019-ncov. 3. saliva samples can be collected in outpatient or community clinics. 4. in an environment where large numbers of people need screening, saliva is a non-invasive sample. 5. using saliva samples reduces the waiting time to collect samples, so results will be available faster. this method is especially important in crowded hospitals where the number of available staff is limited. the results of the present systematic review and meta-analysis study show that saliva can be a non-invasive type of specimen used to diagnose covid-19. saliva can be easily obtained from the patient, saliva use also reduces the risk of transmission, and 7 amiri et al. saliva samples can be useful for patients who cannot be pcred. salivary transmission is also possible and should be observed in dental clinics so that patients are not exposed to saliva. finally, all dentists should be aware that saliva plays a major role in the transmission of covid-19 from human to human, and failure to follow prevention protocols can contaminate them. references 1. wei ff, moradkhani a, hemmati hezaveh h, miraboutalebi sa, salehi l. evaluating the treatment with favipiravir in patients infected by covid-19: a systematic review and meta-analysis. int j sci res dent med sci. 2020;2(3):87-91. doi: 10.30485/ijsrdms.2020.241494.1079. 2. alhazzani w, møller mh, arabi ym, loeb m, gong mn, fan e, et al. surviving sepsis campaign: guidelines on the management of critically ill adults with coronavirus disease 2019 (covid-19). intensive care med. 2020 may;46(5):854-87. https://doi.org/10.1007/s00134-020-06022-5. 3. gaye b, fanidi a, jouven x. denominator matters in estimating covid-19 mortality rates. eur heart j. 2020 oct 1;41(37):3500. doi: 10.1093/eurheartj/ehaa282. 4. aponte mendez m, rivera marval ek, talebzade toranji m, amini f, casaroto ar. dental care for patients during the covid-19 outbreak: a literature review. int j sci res dent med sci. 2020;2(2):42-5. doi:10.30485/ijsrdms.2020.232096.1058. 5. borges do nascimento ij, cacic n, abdulazeem hm, von groote tc, jayarajah u, weerasekara i, et al. novel coronavirus infection (covid-19) in humans: a scoping review and meta-analysis. j clin med. 2020 mar 30;9(4):941. doi: 10.3390/jcm9040941. 6. china nhc. 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[expert consensus for bronchoscopy during the epidemic of 2019 novel coronavirus infection (trial version)]. zhonghua jie he he hu xi za zhi. 2020 mar 12;43(3):199-202. doi: 10.3760/cma.j.issn.1001-0939.2020.03.012. chinese. 27. world helath organization. infection prevention and control of epidemic-and pandemic-prone acute respiratory infections in health care. 2014 apr 7 [cited 2020 nov 20]. available from: https://www.who.int/publications/i/item/infection-prevention-and-control-of-epidemic-andpandemic-prone-acute-respiratory-infections-in-health-care. 28. otter ja, yezli s, salkeld ja, french gl. evidence that contaminated surfaces contribute to the transmission of hospital pathogens and an overview of strategies to address contaminated surfaces in hospital settings. am j infect control. 2013 may;41(5 suppl):s6-11. doi: 10.1016/j.ajic.2012.12.004. 29. baghizadeh fini, m. transmission routes of sars-cov-2 in dentistry: a literature review. int j sci res dent med sci. 2020;2(4):135-7. doi: 10.30485/ijsrdms.2020.252402.1088. 30. zuanazzi d, arts ej, jorge pk, mulyar y, gibson r, xiao y, et al. postnatal identification of zika virus peptides from saliva. j dent res. 2017 sep;96(10):1078-1084. doi: 10.1177/0022034517723325. 31. barzon l, pacenti m, berto a, sinigaglia a, franchin e, lavezzo e, et al. isolation of infectious zika virus from saliva and prolonged viral rna shedding in a traveller returning from the dominican republic to italy, january 2016. euro surveill. 2016;21(10):30159. doi: 10.2807/1560-7917.es.2016.21.10.30159. 1 volume 22 2023 e237338 original article braz j oral sci. 2023;22:e237338http://dx.doi.org/10.20396/bjos.v22i00.8667338 1 graduate program in dentistry, department of restorative dentistry, federal university of pelotas, pelotas, rs, brazil. orcid 2 graduate program in dentistry, são leopoldo mandic college, são paulo, sp, brazil. corresponding author: catarinacumerlato@hotmail.com federal university of pelotas, graduate program in dentistry rua gonçalves chaves 457. 96015-560, pelotas-rs, brazil tel./fax: +55-53-3222.6690 editor: dr. altair a. del bel cury received: october 21, 2021 accepted: may 5, 2022 fracture strength and failure load of cad/cam fabricated endocrowns performed with different designs fernanda angeloni de souza1 , catarina borges da fonseca cumerlato1,* , pedro paulo feltrin2 , ricardo tatsuo inoue2 , raquel virginia zanetti2 aim: endocrown restorations are commonly used to rehabilitate endodontically treated posterior teeth and their use is wellfounded in these cases. however, to date, there is little scientific evidence of their behavior in anterior teeth. the aim of this in vitro study was to evaluate the compressive strength of upper central incisors teeth, restored with glass-ceramic total crowns by the conventional anatomical core technique, and compare them to teeth restored with endocrowns with and without the presence of ferrule. methods: thirty teeth were randomly distributed into three groups: ge2 endocrown group with 2 mm ferrule, ge0 endocrown group without a ferrule, and gc conventional crown with intraradicular post group. crowns were cemented and teeth submitted to the 45o compression test until the fracture happened. fractured specimens were analyzed to determine the fracture pattern. descriptive analysis of the variables was performed and one-way analysis of variance was utilized to analyze the data for significant differences at p < 0.05. results: the results of the control group (284.5 ± 201.05n) showed the highest fracture resistance value, followed by the 2mm group (274.54 ± 199.43n) and by the 0mm group (263.81 ± 80.05n). there was no statistically significant difference between all the groups (p = 0.964). conclusions: the absence of a cervical enamel necklace favored a debonding of the pieces and endodontically treated anterior teeth could be restored with endocrown, which could be considered a conservative and viable treatment option. keywords: endodontics. computer-aided design. crowns. flexural strength. https://orcid.org/0000-0001-5591-2507 https://orcid.org/0000-0001-5943-6958 https://orcid.org/0000-0001-8570-3773 https://orcid.org/0000-0001-7960-3995 https://orcid.org/0000-0003-3823-856x 2 souza et al. braz j oral sci. 2023;22:e237338 introduction restoration of endodontically treated teeth (ett) is often a clinical challenge because the ett are frequently fragile and more susceptible to fracture than vital teeth due to loss of structural integrity, as a result of caries, trauma, previous restorations, and endodontic treatment1-3. with the development of adhesion methods, endocrowns can be used to restore supragingival structure of posterior demaged teeth. the advantages of using these restorations include fewer dental structures preparation compared with post and cores, as well as reduced intervention in the root canals4,5. this is an important advantage since it is know that the higher the ferrule of tooth, the higher the fracture resistance6. moreover, compared to traditional methods they need less time to be made and fewer interfaces between each part of the restorations and the teeth. five-year clinical observations reveal that 87.1% of endocrowns in posterior teeth functioned well, without fracture or debonding7,8. considering all of these critical factors, endocrowns restorations are commonly used for restoring endodontically treated teeth9-11. besides that, an in vitro study has investigated the fracture resistance of restorations from endodontically treated anterior teeth and promising results for endocrowns were found12. however, there is limited evidence about the endocrown’s behaviour with and without ferrule component, compared to conventional crown technique. in addition, it is not possible to find, in the available literature, accurate information on the use of endocrowns for the rehabilitation of incisors. in this sense, this study aimed to evaluate the fracture strength of teeth restored with glass-ceramic total crowns by the conventional anatomical core technique and compare them to teeth restored with endocrowns with and without the presence of ferrule. material and methods ethical issues all stages of this study were approved by the human research ethics committee of the school of dentistry and dental research, são leopoldo mandic (process number 2.604.244/18). specimen preparation thirty maxillary central incisors with complete root formation were collected from the tooth bank of the school of dentistry at the health science center/federal university of são paulo. teeth were examined under ×4 magnification and cleaned to remove all tissues and debris. then, specimens were randomly distributed into three groups (10 specimens for each): endocrown group with 2mm ferrule (ge2), endocrown group without ferrule (ge0) and conventional crown with intraradicular post group (gc) (figure 1). 3 souza et al. braz j oral sci. 2023;22:e237338 gc ge2 ge0 fiber post remaining of gutta percha remaining of gutta percha height of crown 10mm height of crown 2mm composite crown composite endorown figure 1. image of the section type according to the amount of wear. gc control group; ge2 – endocrown with 2 mm ferrule group; ge0 – endocrown with 0 mm ferrule group. all teeth received conventional endodontic treatment, where root canals were prepared up to no. 60 k file using manual instrumentation (dentsply sirona, bensheim, germany) and irrigation with 2% chlorhexidine. each canal was then obturated using lateral condensation of the gutta-percha (dentsply sirona, bensheim, germany), and sealed with sealing cement (ah26, dentsply sirona, bensheim, germany). the anatomic crowns were reduced according to the predetermined height for the experimental groups. the cement enamel junction (cej) served as the circumferential reference for the linear measurement of the remaining coronal heights for each specimen. endocrown preparation and conventional crown preparation preparations for endocrowns were performed using a standardization device adapted from an optical microscope. entrances and undercuts were made 2mm from the cervical margin, limited by the canal anatomy, and then, protected using an adhesive system (ambar universal, fgm, joinville, brazil) and a flowable resin (opallis flow, fgm, joinville, brazil). roots from group gc received glass fiber post #1 (white post, fgm, joinvile, sc, brazil) with was anatomized with composite resin (vitra aps, fgm, joinville, brazil) and cemented using an adhesive system (ambar universal; fgm, joinville, brazil) and dual cure resin cement (allcem core – fgm, joinville, brazil). to standardize the core height a transparent addition silicone mold (transil, ivoclar-vivadent ag, schaan, switzerland), was filled and with the mold sitting on the tooth was light activated for 60 seconds. it resulted in final margins with width of 1.7 mm. small adjustments were also performed in cases in which the core exceeded the height of 7 mm. laboratory phase specimens were scanned using a desktop scanner (ceramil mind, amann girrbach, koblach, austria), and the virtual planning of the standardized crowns was started using the cad system from the software total ceramil mind. and then, crowns were 4 souza et al. braz j oral sci. 2023;22:e237338 milled on a 3d printer (amann girrbach dna motion 2, koblach, austria) using blocks of glass-ceramic resin (brava block, fgm, joinville, brazil). marginal adaptation was checked with the aid of a 4x magnifying glass (eyemag smart, zeiss, jena, germany) and samples that showed mismatch were excluded and a new crown milled. cimentation aluminum oxide blasting (coejet, 3m espe, minnesota, usa) was carried out with a pressure of about 3 bar in the internal surface of the crown until it became matte, and then was rinsed for 180 seconds. subsequently, the adhesive system was applied, waiting for 20 seconds, and a light jet of air was applied for 5 seconds. the remaining coronary structure was cleaned with pumice and water prophylaxis and washed for 30 seconds to remove debris. acid conditioning was performed on the enamel for 30 seconds and on the dentin for 15 seconds, and then ilhe structure was rinsed and blot-dried (ultra-etch indispense 35% ultradent). afterwards an adhesive system was applied actively for 20 seconds, and dried for 5 seconds. the cementation of the glass-ceramic pieces was carried out with dual cure resin cement (allcem core, fgm, joinville, brazil). after reconstruction, the teeth were fixed in cylinders of a self-curing resin having a height of 30mm and 22mm in diameter and taken to the universal testing machine, model dl 2000 (emic, são josé dos pinhais, brazil). fracture strength test to perform the fracture strength test, each specimen in a fixation device was placed obliquely on the base of a universal testing machine (emic, são josé dos pinhais, brazil). a compressive load was applied at a 135-degrees angle to the long axis of the tooth, on the internal and central face of the lingual cuspid of all ceramic crowns5. this was done by means of a metal rod 6 mm in diameter at a speed of 1.0 mm/min until failures occurred, represented by fracturing and/or debonding of the tooth and/or crown. the amount of force required to cause failure was recorded for each specimen in newton (n)5 (figure 2). 135.4° 135° 45° 45° figure 2. schematic drawing of test specimens subjected to load at 45° in a universal testing machine. pressure from the rod tip was applied at a crosshead speed of 1 mm/min (3 mm below the incisal edge) on the palatal surface of the crown. 5 souza et al. braz j oral sci. 2023;22:e237338 data analysis statistical analysis was conducted with spss 23.0 (spss inc., chicago, usa). descriptive analysis was performed, presenting the maximum, minimum, means and standard deviations of the variables. one-way analysis of variance (anova) was utilized to analyze the data for significant differences at p < 0.05. results fracture resistance table 1 shows the descriptive analysis of the samples from each group tested. the results of the control group (284.5 ± 201.05n) showed the highest fracture resistance value, followed by the 2mm group (274.54 ± 199.43n) and by the 0mm group (263.81 ± 80.05n). the analysis of variance (anova) showed no statistically significant difference among the tested groups (p = 0.964). table 1. descriptive analysis of the sample including the maximum, minimum, mean values of load failure with standard deviation for each tested group (newton). group n max. min. mean ± sd p value 0.964 ge0 10 763.77 93.2 263.81±80.05 ge2 10 374.07 80.05 274.54±199.43 gc 10 664.89 131.02 284.50±201.05 failure mode types of fractures were classified according to the position of the failure and the damage to the prosthetic crown and the dental remnant. the classification was developed by us based on the observation of flaws presented (figure 3). type i type ii type iii figure 3. failure pattern of the specimens according to the type of fracture (icatastrophic fracture of the crown and/or remnant (below cej); iifracture of the crown with remnant above or at cej; iiidebonding of the glass-ceramic crown). 6 souza et al. braz j oral sci. 2023;22:e237338 the delimited groups were: i catastrophic fracture of the crown and/or remnant (below cej); ii fracture of the crown with remnant above or at cej; iii debonding of the glass-ceramic crown. in relation to the failure pattern, endocrowns with no ferrule (group 0mm) obtained the highest rates of debonding without fracture of the part and/or dental remnant in 50% of the total sample (type iii), followed by catastrophic fracture of the dental remnant in 30% (type i). and only 20% of the sample obtained a favorable fracture (type ii). the endocrown group with 2mm of ferrule showed a catastrophic failure pattern (type i) in 60% of the sample followed by 30% of the crown fracture with the dental remnant at gingival level (type ii). control group showed a higher fracture rate (50%) of the glass-ceramic piece without compromising the post or the dental remnant (type ii), followed by a fracture pattern involving the post and/or root remnant (type i 40%). discussion the decision to restore a non-vital tooth with loss of coronary structure can be complex so aspects such as planning, selection of the restorative system and adequate cavity preparation must be carefully considered13. the classic alternative to rehabilitate endodontically treated teeth is through the use of intraradicular posts as retainers of total crowns4,14. however, with the placement of post and cores, there is a risk of root perforation and thinning of the canal walls due to excessive preparation15. the advantages of endocrown restorations include less preparation of dental structures compared to posts and cores, as well as reduced intervention in the root canals. compared to traditional methods, they need less laboratory and clinical time for the treatment to be completed16-18. in addition, the masticatory stresses and forces received to the tooth are better dissipated when endocrows are placed19. nevertheless, some studies show that the full crown is still more reliable than endocrown4,20. from the results of this study, we could observe that the group with no ferrule effect showed greater resistance to compression. however, when it was compared to the group with 2mm of ferrule, the group with no ferrule exhibited a higher rate of debonding. this may be attributed to the fact that the surface available for the adhesive joint is reduced, the cementation is only in dentin substrate and there is no ferrule for a better distribution of force, corroborating with another studies2-5,7,10,11,18. on the other hand, the group with 2mm of ferrule, showed lower rates of fracture resistance, and the worst results when assessing the fracture pattern of the pieces, since 60% of the samples had catastrophic fractures (type i) corroborating with the findings from recent studies8,21. in contrast, some studies observed that the presence of ferrule increased fracture resistance2,6. extrapolating to the clinical 7 souza et al. braz j oral sci. 2023;22:e237338 environment, this type of fracture are critically and normally irreparable leading to tooth extraction. the control group has shown a higher fracture rate (50%) of the glass-ceramic piece without compromising the fiber post or the remnant (type ii), agreeing with several authors4,18-23. in relation to failure mode, it has been reported in literature a maximum incisal forces of almost always below 200n for restorations in anterior teeth24,25. our research demonstrated the need of a greater force for fracturing the central incisors than the normal values of oblique loads described in the literature, corroborating with some recent studies10,19,22,23. moreover, it is worth mentioning that this study applied only a static dynamic load, being a limitation that should be considered. in conclusion, endodontically treated anterior teeth with a ferrule effect of at least 2 mm can be restored with endocrown, as well as using a glass fiber post with an adhesive crown/endocrown. no statistically significant difference was found in fracture resistance and failure mode in upper central incisors cemented with glass-ceramic resin comparing total crown with glass fiber post and endocrown with 0mm and 2mm of a template. in this sense, crowns and endocrowns fabricated from machinable glass-ceramic resin blocks are a viable alternative to the restoration of anterior endodontically treated teeth. acknowledgments the authors declare no potential conflicts of interest. this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior brasil (capes) finance code 001. author contribution conceptualization: zanetti rv. data curation: de souza fa and cumerlato cbf. formal analysis: de souza fa and zanetti rv. investigation: de souza fa. methodology: feltrin pp, inoue rt. project administration: zanetti rv. resources: souza fa and zanetti rv. software: de souza fa. supervision: zanetti rv. validation: de souza fa. visualization: cumerlato cbf, feltrin pp, inoue rt. writing original draft: de souza fa and cumerlato cbf. writing review & editing: zanetti rv, feltrin pp, inoue rt. all authors actively participated in the discussion of the manuscript’s findings, revised, and approved the final version of the manuscript. references 1. skupien ja, opdam nj, winnen r, bronkhorst em, kreulen cm, pereira-cenci t, et al. survival of restored endodontically treated teeth in relation to periodontal status. braz dent j. 2016 jan-feb;27(1):37-40. doi: 10.1590/0103-6440201600495.  2. santos pantaleón d, morrow br, cagna dr, pameijer ch, garcia-godoy f. influence of remaining coronal tooth structure on fracture resistance and failure mode of restored endodontically treated maxillary incisors. j prosthet dent. 2018 mar;119(3):390-6. doi: 10.1016/j.prosdent.2017.05.007. 8 souza et al. braz j oral sci. 2023;22:e237338 3. gulec l, ulusoy n. effect of endocrown restorations with different cad/cam materials: 3d finite element and weibull analyses. biomed res int. 2017;2017:5638683. doi: 10.1155/2017/5638683. 4. dejak b, mlotkowski a. strength comparison of anterior teeth restored with ceramic endocrowns vs custom-made post and core. j prosthet dent. 2018 apr;62(2):171-6. doi: 10.1016/j.jpor.2017.08.005. 5. kanat-ertürk b, saridağ s, köseler e, helvacioğlu-yiğit d, avcu e, yildiran-avcu y. fracture strengths of endocrown restorations fabricated with different preparation depths and cad/cam materials. dent mater j. 2018 mar;37(2):256-65. doi: 10.4012/dmj.2017-035. 6. pereira jr, de ornelas f, conti pcr, do valle al. effect of a crown ferrule on the fracture resistance of endodontically treated teeth restored with prefabricated posts. j prosthet dent. 2006 jan;95(1):50-4. doi: 10.1016/j.prosdent.2005.10.019. 7. bindl a, richter b, mörmann wh. survival of ceramic computer-aided design/manufacturing crowns bonded to preparations with reduced macroretention geometry. int j prosthodont. 2005 may-jun;18(3):219-24. doi: 10.1016/j.prosdent.2005.09.002. 8. salameh z, sorrentino r, ounsi hf, sadig w, atiyeh f, ferrari m. the effect of different fullcoverage crown systems on fracture resistance and failure pattern of endodontically treated maxillary incisors restored with and without glass fiber posts. j endod. 2008 jul;34(7):842-6. doi: 10.1016/j.joen.2008.03.025. 9. zarone f, sorrentino r, apicella d, valentino b, ferrari m, aversa r, et al. evaluation of the biomechanical behavior of maxillary central incisors restored by means of endocrowns compared to a natural tooth: a 3d static linear finite elements analysis. dent mat. 2006 nov;22(11):1035-44. doi: 10.1016/j.dental.2005.11.034. 10. el-badawy aa, el aziz mh, omar ea. failure load of maxillary central incisor restored with cad/cam endocrown using different designs. int j dent sci res. 2019;7(1):5-9. doi: 10.12691/ijdsr-7-1-2. 11. el-damanhoury hm, haj-ali rn, platt ja, fracture resistance and microleakage of endocrowns utilizing three cad-cam blocks. oper dent. 2015 mar-apr;40(2):201-10. doi: 10.2341/13-143-l. 12. ramírez-sebastià a, bortolotto t, cattani-lorente m, giner l, roig m, krejci i. adhesive restoration of anterior endodontically treated teeth: influence of post length on fracture strength. clin oral invest. 2014;18(2):545-54. doi: 10.1007/s00784-013-0978-3. 13. ploumaki a, bilkhair a, tuna t, stampf s, strub jr. sucess rates of prosthetic restorations on endodontically treated teeth: a systematic review after 6 years. j oral rehabil. 2013 aug;40(8):618-30. doi: 10.1111/joor.12058. 14. mannocci f, bertelli e, sherriff m, watson tf, pitt ford tr. three-year comparison of survival of endodontically teeth restores with full case coverage or with direct composite restoration. j prosthet dent. 2002 sep;88(3):297-301. doi: 10.1067/mpr.2002.128492. 15. clavijo vgr, souza nc de, kabbach w, calixto lr, andrade mf de, susin ah. [endocrown restorations: an approach for non-vital posterior teeth]. clin inter j braz dent. 2007;3(3):246-52. portuguese. 16. shin y, park s, park jw, kim km, park yb, roh bd. evaluation of the marginal and internal discrepancies of cad-cam endocrowns with different cavity depths: an in vitro study. j prosthet dent. 2017 jan;117(1):109-15. doi: 10.1016/j.prosdent.2016.03.025. 17. helal ma, wang z. biomechanical assessment of restored mandibular molar by endocrown in comparison to a glass fiber post-retained conventional crown: 3d finite element analysis. j prosthodont. 2019 dec;28(9):988-96. doi: 10.1111/jopr.12690. 18. bankoglu gungor m, turhan bal b, yilmaz h, aydin c, karakoca nemli s. fracture strength of cad/cam fabricated lithum disilicate and resin nano ceramic restorations used for endodocntically treated teeth. dent mat j. 2017 mar;36(2):135-41. doi: 10.4012/dmj.2016-017. 9 souza et al. braz j oral sci. 2023;22:e237338 19. chang cy, kuo js, lin ys, chang yh. fracture resistance and failure modes of cerec endo-crowns and conventional post and core-supported cerec crowns. j dental sci. 2009 sep;4(3):110-7. doi: 10.1016/s1991-7902(09)60016-7. 20. silva-sousa ac, moris icm, simões barbosa af, corrêa silva-sousa yt, sousa-neto md, ferreira pires cr, et al. effect of restorative treatment with endocrown and ferrule on the mechanical behavior of anterior endodontically treated teeth: an in vitro analysis. j mech behav biomed mat. 2020 dec;112:104019. doi: 10.1016/j.jmbbm.2020.104019. 21. clausson c, schroeder cc, goloni pv, farias far, passos l, zanetti rv. fracture resistance of cad/cam lithium disilicate of endodontically treated mandibular damaged molars based on different preparation designs. int j biomater. 2019 may;2019:2475297. doi: 10.1155/2019/2475297. 22. casagrande dda. [analysis of the compressive strength at 45° of full lithium disilicate crowns cemented in endodontically treated maxillary central incisors with 2mm and 4mm reconstruction (buildup) with composite resin] [dissertation]. campinas: são leopoldo mandic college; 2019. portuguese. 23. gresnigt mmm, özcan m, van den houten mla, schipper l, cune ms. fracture strength, failure type and weibull characteristics of lithium disilicate and multiphase resin composite endocrowns under axial and lateral forces. dent mater. 2016 may;32(5):607-14. doi: 10.1016/j.dental.2016.01.004. 24. qing h, zhu z, chao y, zhang w. in vitro evaluation of the fracture resistance of anterior endodontically treated teeth restored with glass fiber and zircon posts. j prosthet dent. 2007 feb;97(2):93-8. doi: 10.1016/j.prosdent.2006.12.008. 25. tan pl, aquilino sa, gratton dg, stanford cm, tan sc, johnson wt, et al. in vitro fracture resistance of endodontically treated central incisors with varying ferrule heights and configurations. j prosthet dent. 2005 apr;93(4):331-6. doi: 10.1016/j.prosdent.2005.01.013. 1 volume 21 2022 e228274 original article braz j oral sci. 2022;21:e228274http://dx.doi.org/10.20396/bjos.v21i00.8668274 1 department of pediatric dentistry, school of dentistry, ahvaz jundishapur university of medical sciences, ahvaz, iran. 2 department of orthodontics, school of dentistry, ahvaz jundishapur university of medical sciences, ahvaz, iran. 3 infectious and tropical diseases research center, health research institute, ahvaz jundishapur university of medical sciences, ahvaz, iran. corresponding author: samaneh khanehmasjedi address: infectious and tropical diseases research center, health research institute, ahvaz jundishapur university of medical sciences, ahvaz, iran phone number: 00989169225100 e-mail: masjedi.samaneh@yahoo.com; khanehmasjedi.s@ajums.ac.ir editor: dr. altair a. del bel cury received: january 31, 2022 accepted: april 13, 2022 knowledge, attitudes, and practices regarding the oral health of children: a cross-sectional study among iranian parents leila basir1 , mashallah khanehmasjedi2 , samaneh khanehmasjedi3,* parents are responsible for their children’s health care, and their oral health-related knowledge, attitude, and habits can affect their children’s oral health. aim: the objective of this study was to evaluate parents’ knowledge, attitudes, and practices regarding their children’s oral health. methods: in this study, a sample of 398 parents of 4to 6-year-old children completed a self-designed questionnaire. the parents’ oral health-related knowledge, attitudes, and practices were assessed. children’s oral health was evaluated using decayed, missing, and filled tooth index (dmft). data were analyzed using the spss version 23.0 with a p < 0.05 as statistically significant. categorical data were reported as frequency (%), and continuous data were reported as mean ± sd. moreover, spearman’s correlation, multiple regression, mann-whitney test, kruskal wallis test, and kolmogorov-smirnov test were used. results: most of the parents had a satisfactory level of knowledge and positive attitudes regarding their children’s oral health. the knowledge and attitude scores were higher among parents with higher education (p<0.001), and the knowledge score was higher among mothers (p=0.004). also, the attitude score was correlated with the number of decayed, missed, and filled teeth of children (p=0.01, p=0.04, and p=0.007, respectively). however, there was no significant relationship between dmft and the parents’ knowledge, attitudes, and practices using multiple regression. the mean dmft of children was 6.86 ± 3.56, and most of the parents had poor oral health-related practices. conclusion: the parents’ level of knowledge and attitudes were satisfactory, but they had poor oral health practices. moreover, there was no significant relationship between children’s oral health and their parents’ level of knowledge, attitudes, and practices. educating programs and strategies are needed to enhance parents’ oral health-related attitudes and knowledge and, more importantly, change their oral health practices. keywords: knowledge. habits. health behavior. dental caries. tooth, deciduous. https://www.scopus.com/authid/detail.uri?authorid=57190230828 https://orcid.org/0000-0002-1684-1774 https://www.scopus.com/authid/detail.uri?authorid=55753667000 https://orcid.org/0000-0001-9537-5339 https://orcid.org/0000-0001-7736-7773 https://orcid.org/0000-0001-7736-7773 2 basir et al. braz j oral sci. 2022;21:e228274 introduction oral health as an essential part of overall health1 has been the center of attention of researchers for many years. however, compared to general health, little significance is given to it2. in young children, dental caries is an essential facet of oral health3, related to many risk factors such as poor oral hygiene and a highly cariogenic diet4. regardless of numerous strategies and interventions to promote oral health and prevent dental caries, evidence shows that the prevalence of dental caries has increased among children in middle eastern countries5. according to a recent meta-analysis, the mean dmft of children in iran was 3.866. parents play a crucial part in maintaining good oral health in preschool children. due to manual incompetency, preschoolers cannot clean their teeth, and due to mental immaturity, they are ignorant of the importance of preserving their teeth7. moreover, children under six spend most of their time with their parents, and through a period that is called “primary socialization,” they acquire their parents’ everyday routines (including health behaviors)8.9. it has been reported that parents’ oral health-related knowledge, attitudes, and practices can affect their children’s oral health3,10,11. therefore, parents must have favorable oral health behaviors and satisfactory knowledge and attitude to inculcate necessary oral health habits in their children12. since the prevalence of dental caries in children has increased over the last 15 years in iran5, it is important to assess parents’ knowledge and attitudes to discover which aspects need improvement to enhance children’s oral health11. therefore, in this study, we intended to evaluate parents’ knowledge, attitudes, and practices regarding their 4to 6-year-old children’s oral health. our null hypothesis was that iranian parents had low-to-moderate levels of knowledge, attitudes, and practices. methods this study was conducted under the strobe guidelines13. ethical approval the ethic committee of the ahvaz jundishapur university of medical sciences approved this study (ir.ajums.rec.1397.559). additionally, after explaining the purpose of the study, written informed consent was obtained from the parents. study sample and design this was an analytical, cross-sectional study conducted on an initial sample of 414 subjects (using cochran formula, α=0.05, β=0.2, pilot sampling variance=0.52, and minimum effect size=0.1) selected using the convenience sampling method. the inclusion criteria were parents of 4to 6-year-old healthy children presenting to the department of pediatric dentistry, school of dentistry, the ahvaz jundishapur university of medical sciences. the exclusion criteria were the presence of any disease 3 basir et al. braz j oral sci. 2022;21:e228274 and the usage of orthodontic appliances. the sample was recruited in the fall of 2019, from october 9th to december 15th. data collection parents’ knowledge, attitudes, and practices regarding their children’s oral health were assessed using a self-designed questionnaire based on previous studies14. before the study, ten pediatric dentists and health education specialists validated the questionnaire, and based on their review, modifications were made. the test-retest method was used to evaluate the reliability of the questionnaire. for this purpose, the questions were given to the parents (not included in the main study) in two terms with a two-week interval. cronbach’s α (0.81) confirmed the questionnaire’s reliability15,16. the questionnaire consisted of four sections: 1) demographic data of parents and their children (age and gender of the accompanying parent, their level of education, their occupation, their source of acquiring oral health-related information, and age and gender of the child), 2) knowledge section (10 questions), 3) attitude section (ten statements), and 4) oral health-related practices (three statements). the knowledge section included ten multiple-choice questions regarding the etiology of dental caries, the importance of primary teeth, fluoride, oral health, oral health-related practices, and the time of the permanent teeth eruption. each correct answer scored a point. the maximum score for this section was 10 points. it was further categorized as unsatisfactory, less than 6 points (the median), and satisfactory, 6 points and above. the attitude section was a five-point likert scale that included ten statements from “strongly agree” to “strongly disagree” regarding primary teeth eruption and caries, oral health-related practices, and nutrition of children under six. the response of each statement was given a value from 1 to 5, with the response anchors having 1 or 5 points. the maximum score of the attitude section was 50, and the minimum was 10 points. this score was also categorized as negative, less than 36 points (the median), and positive, 36 points and above. the practices section included four binary-option questions on the frequency of children’s tooth brushing, flossing, dental visits, and whether they ever had a fluoride varnish. the questions were rated using “yes,” “no,” and “don’t know” on a modified likert scale. approximately 10 min was required to fill out the questionnaire. a calibrated pediatric dentist examined the children’s oral health status to evaluate the children’s oral health status. according to the who standard diagnostic criteria, the dmft index was obtained by calculating the number of decayed, missing, and filled primary teeth17. data analysis data were analyzed using the spss statistical software (version 23.0, ibm corporation, armonk, ny, usa) with a p < 0.05 as statistically significant. categorical data were reported as frequency (%), and continuous data were reported as mean ± sd. kolmogorov-smirnov statistic tested the normal distribution of variables. spearman’s correlation coefficient was used to discover possible correlations between variables. mann-whitney u (two-category variables) and kruskal wallis (more than 4 basir et al. braz j oral sci. 2022;21:e228274 two-category variables) evaluated the mean differences in knowledge, attitude, and dmft scores between different groups. multiple regression was used to indicate whether there was a relationship between dmft and parents’ knowledge, attitudes, and practices. results questionnaires with unanswered questions were excluded to secure authentic results, and the final sample size included 398 completed questionnaires (the response rate was 96.13%). in total, 207 mothers (52.0%) and 191 fathers (48.0%) participated in the study. of the children, 200 (50.3%) were girls and 198 (49.7%) were boys, and their mean age was 5.16 ± 0.77. of the mothers, 83 (40%) were unemployed. other demographic data are presented in table 1. the mean knowledge score of the parents was 5.81 ± 1.62 (females: 6.009 ± 1.59, males: 5.602 ± 1.63), and 57% of them had a satisfactory level of knowledge. the mothers had a significantly higher knowledge score (mann-whitney u; p = 0.004). there was a statistically significant difference between the mean knowledge scores table 1. frequency distribution of the demographic data of the participating parents demographic data n (%) age (years) 20-29 65 (16.3) 30-39 259 (65.1) 40-49 74 (18.6) occupation employed 197 (49.5) self-employed 83 (20.9) laborer 29 (7.3) unemployed 89 (22.4) education higher education 254 (63.8) high school diploma or less 144 (36.2) source of oral health-related information* dentist 222 (55.7) magazines 1 (0.2) personal experience 40 (10.0) tv 50 (12.5) family & friends 57 (14.3) internet 84 (21.1) * 46 participants chose more than one source of oral health-related knowledge 5 basir et al. braz j oral sci. 2022;21:e228274 of parents from different age groups. it was higher among parents aged 30-39 years (kruskal wallis; p= 0.007). the mean knowledge score was also higher among parents with higher education (mann-whitney u; p< 0.001) and correlated with the mean attitude score (spearman’s correlation; p< 0.001). however, there was no significant relationship between dmft and the parents’ knowledge, attitudes, and practices using multiple regression (p>0.05) and (table 2). table 3 shows the knowledge questions and the frequency of the parents’ true or false answers. the mean attitude score of the parents was 35.60 ± 4.97 (females: 35.97 ± 4.86, males: 35.21 ± 5.07), and 54.3% of them had positive attitudes regarding their children’s oral health. this score was significantly higher among parents with higher education (mann-whitney u; p< 0.001). in addition, it was correlated with the number of decayed, missing, and filled teeth of the children (spearman’s correlation; p= 0.01, p= 0.04, and p= 0.007, respectively). table 4 shows the questionnaire’s statements regarding the parents’ attitudes and the frequency of their answers. table 5 shows spearman’s correlation between the parents’ knowledge and attitude scores and the children’s dmft score, and the parents’ practices regarding their children’s oral health are presented in table 6. table 2. multiple regression between the dmft index and the parents’ knowledge, attitudes, and practices variable coefficient (β) p-value constant 8.230 <0.001 knowledge 0.002 0.984 attitude -0.037 0.331 practice -0.061 0.743 table 3. frequency distribution of the parents’ knowledge regarding their children’s oral health questions true (%) false (%) 1what causes dental caries? 193 (48.5) 205 (51.5) 2what is the importance of primary teeth? 253 (63.9) 145 (36.4) 3which one is more effective in the incidence of dental caries? 369 (92.7) 29 (7.3) 4which one is more effective in preventing dental caries? 178 (44.7) 220 (55.3) 5what is the color of healthy gums? 348 (87.4) 50 (12.6) 6what is the best instrument to clean the interdental surfaces? 314 (78.9) 84 (21.1) 7from what age can we use fluoride-containing toothpaste for children? 68 (17.1) 330 (82.9) 8from what age can we use fluoride-containing mouthwashes for children? 186 (46.7) 212 (53.3) 9how many times a day should children brush their teeth? 119 (29.9) 279 (70.1) 10at what age does the first permanent tooth erupt? 286 (71.9) 112 (28.1) 6 basir et al. braz j oral sci. 2022;21:e228274 table 4. frequency distribution of the parents’ attitudes regarding their children’s oral health statement strongly disagree (%) disagree (%) neutral (%) agree (%) strongly agree (%) 1going to the dentist for children under 6 is a waste of time. 214 (53.8) 118 (29.6) 26 (6.5) 16 (4.1) 24 (6.0) 2gargling with salt water is a good alternative for children who do not brush their teeth. 41 (10.3) 85 (21.4) 107 (26.9) 132 (33.1) 33 (8.3) 3experience has shown that infants who use breast milk are less likely to develop caries. 33 (8.3) 71 (17.8) 85 (21.4) 92 (23.1) 117 (29.4) 4mouthwashes have no protective effect on primary teeth. 53 (13.3) 122 (30.7) 154 (38.7) 47 (11.8) 22 (5.5) 5it is not necessary to visit a dentist for check-ups for a child who is brushing. 99 (24.9) 205 (51.5) 41 (10.3) 34 (8.5) 19 (4.8) 6the cause of early dental caries in children is neglecting tooth brushing and using floss. 7 (1.8) 48 (12.0) 31 (7.8) 148 (37.2) 164 (41.2) 7all permanent teeth erupt as a substitute for primary teeth. 20 (5.1) 36 (9.0) 79 (19.8) 164 (41.2) 99 (24.9) 8only in case of pain should children be referred to a dentist. 111 (27.9) 207 (25.1) 17 (4.3) 41 (10.2) 22 (5.5) 9if the child has a decayed tooth, i prefer for the tooth to be extracted. 144 (36.2) 174 (43.7) 42 (10.6) 24 (6.0) 4 (3.5) 10proper nutrition in children is very effective in maintaining healthy teeth. 2 (0.5) 4 (1.0) 10 (2.5) 136 (34.2) 246 (61.8) table 5. spearman’s correlation between the parents’ knowledge and attitude scores and children’s dmft score variable knowledge score attitude score dmft d m f knowledge score 1 0.315** -0.309 -0.074 -0.032 0.094 attitude score 1 -0.079 -0.121* -0.100* 0.134** dmft 1 0.836** 0.172** 0.085 d 1 -0.175** -0.325** m 1 0.205** f 1 * p < 0.05 ** p < 0.01 table 6. frequency distribution of the parents’ practices regarding their children’s oral health questions yes (%) no (%) don’t know (%) do you brush your child’s teeth twice a day? 98 (24.6) 288 (72.4) 12 (3) do you floss your child’s teeth daily? 56 (14.1) 326 (81.9) 16 (4) do you take your child to dental check-ups every six m.? 84 (21.1) 288 (72.4) 26 (6.5) has your child ever had a fluoride varnish? 109 (27.4) 255 (64.1) 34 (8.5) 7 basir et al. braz j oral sci. 2022;21:e228274 the mean dmft of the children was calculated 6.86 ± 3.56 (d = 6.01 ± 3.69, m = 0.38 ± 0.97, f = 0.46 ± 1.26). as demonstrated in table 7, the dmft mean was higher among boys (p = 0.80). also, the number of filled teeth increased with the child’s age (kruskal wallis; p = 0.009). only 3.5% (n = 14) of the children were caries-free. discussion through this study, which intended to evaluate parents’ knowledge, attitudes, and practices regarding their children’s oral health, we discovered that the majority of parents of 4to 6-year-old children had a satisfactory level of knowledge and positive attitudes but poor practices in this regard. in the present study, more than half of the parents had a satisfactory level of knowledge. the knowledge score was higher among the mothers, as stated by previous studies7,18. a conflicting study by mehdipour et al. reported that 51.1% of the iranian parents had poor knowledge about the care of primary teeth13. this difference can be due to the level of education of the participants in that study, of which only 38.2% had higher education. in our study, more than 60% of the parents were university educated. similar to our results, it is generally accepted that individuals with higher levels of education have a higher oral health knowledge and a better understanding of their overall health8,17 (appendix 1). regarding attitude in the present study, most of the parents had positive attitudes about their children’s oral health. in a study conducted by dhull et al., the overall attitude of indian mothers regarding the oral health care of their children was poor, which may be a result of their low education19. consistent with our result, it has been reported that the attitude of individuals is related to their education level1,12. moreover, similar to the study by mehdipour et al.14, in our study, parents with a higher knowledge score had a higher attitude score. additionally, children whose parents had a higher attitude score had better oral health. that is, they had fewer decayed and missed teeth and more filled teeth. this is mainly important because children with less caries experience have a higher oral health-related quality of life. furthermore, untreated caries affects children’s oral and general health20 (appendix 2). in the present study, 29.9% of the parents knew that children should brush their teeth twice a day, and about a quarter of the children did so. complementary results table 7. descriptive statistics of the dmft index among girls and boys of the study sample variable mean + sd p* girls boys dmft 6.82 ± 3.46 6.91 ± 3.67 0.80 d 5.96 ± 3.59 6.07 ± 3.79 0.96 m 0.32 ± 0.90 0.45 ± 1.04 0.13 f 0.54 ± 1.43 0.38 ± 1.06 0.30 * mann-whitney u 8 basir et al. braz j oral sci. 2022;21:e228274 have been found in a study in saudi arabia3. contrary to a study by kameli et al.21, our result showed that about three-fourths of the parents knew flossing was the best way to clean the interdental surfaces, but less than 15% of the children used dental floss daily. since parents in the mentioned study were mostly housewife mothers, it can be reasoned that they had more free time to spend caring for their children. however, more than half of the mothers in our study were either employed or self-employed. we found that only about 27% of the children have had a fluoride varnish application. as reported by a study, less than 10% of the children in trinidad have had fluoride varnish applied to their teeth9. moreover, only about 15% of the parents in this study believed that children should visit dentists only in case of pain. nevertheless, only about 20% took their children for a dental visit every six months. similarly, ramakrishnan et al. stated that 18% of indian parents took their children for regular dental check-ups. at the same time, the majority of them preferred taking their children to the dentist only if they were in pain10. given the evidence, even though most of the parents had a satisfactory level of knowledge and positive attitudes toward their children’s oral health, most of them could not translate this knowledge and attitudes into good oral practices to maintain their children’s oral health. as a result, dental caries had a 96.5% prevalence in children in this study. this neglect toward children’s oral health can be the result of daily workload, expenses of dental care, fear of dental treatments, and past painful experiences 8,22. for interested readers, a detailed description of each question and statement of the questionnaire is presented in the appendix. this was a cross-sectional study, and all the limitations of this type of study should be considered. also, regarding oral health practices, since our data were collected through a questionnaire, parents may have given socially desired answers rather than describing their real habits. in conclusion, the parents’ level of knowledge and attitudes were satisfactory, but they had poor oral health practices. moreover, we found no significant relationship between the children’s oral health and their parents’ level of knowledge, attitudes, and practices. our findings give an insight into parents’ knowledge, attitudes, and practices and can be of great importance to policymakers to develop strategies that can improve oral health-related behaviors of the population. future research can focus not only on education programs and strategies needed to improve parents’ attitudes and increase their knowledge but also on changing their oral health practices. acknowledgment the ahvaz jundishapur university of medical sciences financially supported this study. the authors thank dr. saki for her assistance with statistical analysis. author contribution study concept and design: l.b. and m.kh.; acquisition of data: l.b.; analysis and interpretation of data: l.b. and m.kh.; drafting of the manuscript: s.kh.; critical revision of 9 basir et al. braz j oral sci. 2022;21:e228274 the manuscript for important intellectual content: l.b. and m.kh.; statistical analysis: s.kh. all authors actively participated in the discussion of the manuscript’s findings, and have revised and approved the final version of the manuscript. conflicts of interest the authors declare no conflicts of interest. however, they state that they have a familial connection; that is, s.kh. is l.b. and m.kh.’s daughter. references 1. mahmoud n, kowash m, hussein i, hassan a, al halabi m. oral health knowledge, attitude, and practices of sharjah mothers of preschool children, united arab emirates. j int soc prev community dent. 2017;7(6):308-14. doi: 10.4103/jispcd.jispcd_310_17. 2. vanobbergen j, lesaffre e, garcia-zattera m, jara a, martens l, declerck d. caries patterns in primary dentition in 3-, 5-and 7-year-old children: spatial correlation and preventive consequences. caries res. 2007;41(1):16-25. doi: 10.1159/000096101. 3. salama f, alwohaibi a, alabdullatif a, alnasser a, hafiz z. knowledge, behaviours and beliefs of parents regarding the oral health of their children. eur j paediatr dent. 2020;21(2):103-9. doi: 10.23804/ejpd.2020.21.02.03. 4. harris r, nicoll ad, adair pm, pine cm. risk factors for dental caries in young children: a systematic review of the literature. community dent health. 2004 mar;21(1 suppl):71-85. 5. elamin a, garemo m, mulder a. determinants of dental caries in children in the middle east and north africa region: a systematic review based on literature published from 2000 to 2019. bmc oral health. 2021;21(1):1-30. doi: 10.1186/s12903-021-01482-7. 6. soltani mr. dental caries status and its related factors in iran: a meta-analysis. j dent (shiraz). 2020 sep;21(3):158-76. doi: 10.30476/dentjods.2020.82596.1024. 7. suma sogi hp, hugar sm, nalawade tm, sinha a, hugar s, mallikarjuna rm. knowledge, attitude, and practices of oral health care in prevention of early childhood caries among parents of children in belagavi city: a questionnaire study. j family med prim care. 2016;5(2):286-90. doi: 10.4103/2249-4863.192332. 8. kotha sb, alabdulaali ra, dahy wt, alkhaibari yr, albaraki asm, alghanim af. the influence of oral health knowledge on parental practices among the saudi parents of children aged 2–6 years in riyadh city, saudi arabia. j int soc prev community dent. 2018;8(6):565-71. doi: 10.4103/jispcd.jispcd_341_18. 9. naidu rs, nunn jh. oral health knowledge, attitudes and behaviour of parents and caregivers of preschool children: implications for oral health promotion. oral health prev dent. 2020 apr;18(1):245-52. doi: 10.3290/j.ohpd.a43357. 10. ramakrishnan m, banu s, ningthoujam s, samuel va. evaluation of knowledge and attitude of parents about the importance of maintaining primary dentition-a cross-sectional study. j family med prim care. 2019 feb;8(2):414-8. doi: 10.4103/jfmpc.jfmpc_371_18. 11. setty jv, srinivasan i. knowledge and awareness of primary teeth and their importance among parents in bengaluru city, india. int j clin pediatr dent. 2016;9(1):56-61. doi: 10.5005/jp-journals-10005-1334. 12. gurunathan d, moses j, arunachalam sk. knowledge, attitude, and practice of mothers regarding oral hygiene of primary school children in chennai, tamil nadu, india. int j clin pediatr dent. 2018;11(4):338-43. doi: 10.5005/jp-journals-10005-1535. 10 basir et al. braz j oral sci. 2022;21:e228274 13. von elm e, altman dg, egger m, pocock sj, gøtzsche pc, vandenbroucke jp, et al. the strengthening the reporting of observational studies in epidemiology (strobe) statement: guidelines for reporting observational studies. j clin epidemiol. 2008 apr;61(4):344-9. doi: 10.1016/j.jclinepi.2007.11.008. 14. mehdipour a, montazeri hedeshi r, asayesh h, karimi a, omidi kopayee r, asgari h. [evaluation of knowledge, attitudes and performance of the parents of preschool and primary school children referred to health centers of qom city about the importance of preserving primary teeth and its related factors, iran]. qom uni med sci j. 2016 [cited 2022 feb 2];10(6):94-105. persian. available form: https://journal.muq.ac.ir/article-1-1098-en.pdf. 15. alshehri a, nasim v. infant oral health care knowledge and awareness among parents in abha city of aseer region, saudi arabia. saudi j dent res. 2015;6(2):98-101. doi: 10.1016/j.sjdr.2015.01.001. 16. mohamed ys. assessment of the knowledge and awareness among egyptian parents in relation to oral health status of their children. egypt dent j. 2020;66(2):737-46. doi: 10.21608/edj.2020.25196.1058. 17. chen l, hong j, xiong d, zhang l, li y, huang s, hua f. are parents’ education levels associated with either their oral health knowledge or their children’s oral health behaviors? a survey of 8446 families in wuhan. bmc oral health. 2020 jul;20(1):203. doi: 10.1186/s12903-020-01186-4. 18. nagarajappa r, kakatkar g, sharda aj, asawa k, ramesh g, sandesh n. infant oral health: knowledge, attitude and practices of parents in udaipur, india. dent res j (isfahan). 2013 sep;10(5):659-65. 19. dhull ks, dutta b, devraj im, samir p. knowledge, attitude, and practice of mothers towards infant oral healthcare. int j clin pediatr dent. 2018;11(5):435-9. doi: 10.5005/jp-journals-10005-1553. 20. duangthip d, gao ss, chen kj, lo ecm, chu ch. oral health-related quality of life and caries experience of hong kong preschool children. int dent j. 2020;70(2):100-7. doi: 10.1111/idj.12526. 21. kameli s, mehdipour a, montazeri hedeshi r, nourelahi m. [evaluation of parental knowledge, attitudes and practices in preschool children on importance of primary teeth and some related factors among subjects attending semnan university of medical sciences dental clinic]. koomesh. 2017 [cited 2022 feb 2];19(1):191-8. persian. available from: http://eprints.semums.ac.ir/1083/1/ koomesh-v19n1p191-en.pdf. 22. sanguida a, vinothini v, prathima gs, santhadevy a, premlal k, kavitha m. age and reasons for first dental visit and knowledge and attitude of parents toward dental procedures for puducherry children aged 0–9 years. j pharm bioallied sci. 2019 may;11(suppl 2):s413-9. doi: 10.4103/jpbs.jpbs_54_19. 1 volume 22 2023 e237798 original article braz j oral sci. 2023;22:e237798http://dx.doi.org/10.20396/bjos.v22i00.8667798 1 nitte (deemed to be university), ab shetty memorial institute of dental sciences (absmids), department of oral medicine and radiology, mangalore, india. corresponding author: dr renita lorina castelino nitte (deemed to be university), ab shetty memorial institute of dental sciences (absmids), department of oral medicine and radiology, mangalore, india. contact info-9880143370 email: renita.castelino@yahoo.com editor: dr. altair a. del bel cury received: december 6, 2021 accepted: may 6, 2022 prevalence of soft tissue calcifications in the maxillofacial region – a radiographic study deepthi darwin1 , renita lorina castelino1,* , gogineni subhas babu , mohamed faizal asan1 aim: to evaluate the prevalence of soft tissue calcifications in orofacial region and their panoramic radiographic characteristics using digital panoramic radiographs among patients reporting to a tertiary dental hospital. methods: 1,578 digital panoramic radiographs were retrieved from the archives and scrutinized for the presence of calcifications. soft tissue calcifications were recorded according to age, gender, site (left or right). data were analysed using chi-square and fisher’s exact test using spss software and a p < 0.05 was considered statistically significant. results: among the total number of radiographs, calcified carotid artery (34.3%), calcified stylohyoid ligament (21%), tonsillolith (10.3%), phlebolith (17.6%), antrolith (6.3%), sialolith (5.9%), rhinolith (2.5%) and calcified lymph nodes (1.9%) were identified. the most commonly observed calcifications were calcification of carotid artery and stylohyoid ligament and the least commonly observed calcifications were rhinolith and calcified lymph node. a statistically significant association of the presence of calcifications of carotid artery and stylohyoid ligament on the left and right side was observed in females and tonsillolith on the right side in males (p-value < 0.05). considering the gender and age group, the occurrence of antrolith among males and rhinolith among females of young-adult population, tonsillolith among the males, calcified carotid artery and stylohyoid ligament among the females of middle-aged population was found to be significant. conclusion: soft tissue calcifications are often encountered in dental panoramic radiographs. our study revealed that the soft tissue calcifications in orofacial region were more common in women and were found to be increased above 40 years of age. keywords: radiography, panoramic. calcinosis. plaque, atherosclerotic. eagle syndrome. diagnostic imaging. https://orcid.org/0000-0003-2440-1012 https://orcid.org/0000-0002-8696-549x https://orcid.org/0000-0001-9383-7886 https://orcid.org/0000-0001-9747-1914 2 darwin et al. braz j oral sci. 2023;22:e237798 introduction calcium is a micronutrient that is required for a variety of physiological functions such as cellular activities, neuronal activity, tooth and bone formation, etc.1. deposition of calcium salts in the tissues can be manifested in various physiological and pathological conditions2. calcium salts, primarily calcium phosphate gets accumulated in the soft tissues in an unstructured manner resulting in pathological calcifications which is also referred to as heterotopic calcification3. pathologic calcifications can be either dystrophic which occurs in degenerating and necrotic tissues or metastatic which occurs due to the precipitation of excess calcium and phosphate in the normal tissues4. occasionally, such calcifications may occur bilaterally and symmetrically secondary to the skeletal deposits from a malignancy5,6. extra-skeletal calcifications such as calciphylaxis, calcifications within the brain such as primary familial brain calcification, calcifications of tumour, arthritic bone spurs, gall and kidney stones are some of the typical sites of soft tissue calcifications6,7. soft tissue calcifications in the orofacial region are commonly detected as incidental findings during routine radiographic examinations with orthopantomograms (opgs)4,8. evaluation of such calcification should be carried out in a systematic manner considering the anatomical location, distribution, shape, size and number of calcifications to arrive at an appropriate diagnosis9. digital panoramic imaging is a routinely employed modality for diagnosing pathologies of the jawbones. it is considered as an initial imaging modality that allows appropriate discernibility of the structures of the maxillofacial region10. there are various pieces of evidence available in the literature that delineates the detection of soft tissue calcifications in opg, but the prevalence of such calcinosis differs widely among various studies and the population studied5. the calcifications that can be encountered in panoramic radiographs include rhinolith, antrolith, tonsillolith, phlebolith, sialolith, carotid artery calcifications, calcified lymph nodes and stylohyoid ligament11. with the increased utilization of digital panoramic imaging in routine dental practices, understanding of the soft tissue calcifications and their characteristics is necessary for formulating a diagnosis and establishing an appropriate referral strategy12,13. hence, considering the wide span of pathologies and the paucity of research, we undertook this retrospective study with the aim of evaluating the prevalence of soft tissue calcifications using digital panoramic radiographs among patients reporting to a tertiary dental hospital. materials and methods selection of patients the present retrospective study was carried out after the approval of the institutional ethical committee (ethics/absmids/149/2021). panoramic radiographs of individuals who had visited the department of maxillofacial radiology from january 2020 to december 2020 were retrieved from the archives and screened based on our inclusion criteria. the panoramic radiographs of all age groups and gender with 3 darwin et al. braz j oral sci. 2023;22:e237798 good diagnostic quality were included in the study. we obtained a total of 1693 radiographs which were scrutinized for the presence of soft tissue calcifications. however, 115 radiographs were excluded due to the presence of radiographic artifacts, facial deforming pathologies, obscure images with superimposition of structures and lack of diagnostic quality.  image acquisition the radiographic images were procured with an orthopantomogram unit, planmeca promax s2-2d (helsinki, finland, 2008) using planmeca romexis software (version 2.4.2) and viewed on a full-screen monitor. the panoramic radiographs were taken by a trained radiographer with the exposure parameters of 64-70 kv; 7-14 ma and an average exposure time of 16 seconds. all the images were evaluated by two independent oral and maxillofacial radiologists with a minimum of ten years of clinical experience. the evaluation of the radiographs was done based on parameters such as age, gender, site and type of calcification. image analysis soft tissue calcifications were classified according to their number, location, shape, distribution (side of involvement) and appearance. for the ease of identification of soft tissue calcifications, the panoramic radiographs were divided into eight equal quadrants by sketching one horizontal and three vertical lines following the method described by rajkumar et al.1. a horizontal line was drawn across the occlusal plane. vertical lines were drawn across the midline passing through the nasal septum and along the anterior aspect of the mandibular ramus on either side and numbered from 1-8 as shown in figure-1. the soft tissue calcifications were documented based on their anatomical location and the quadrant in which they were present. table-1 enumerates the calcifications in the maxillofacial region based on their characteristic radiographic appearance as found in our study. the description of the characteristic radiographic features based on the location of the soft tissue calcifications is cited from the previous literature1,5,9,14-16. figure 1. division of opg divided into 8 boxes arbitrarily. 1 5 6 7 2 3 4 8 l 4 darwin et al. braz j oral sci. 2023;22:e237798 statistical analysis statistical analysis was performed with spss statistics program (ibm, armonk, ny, usa) version 22 for windows. numerical data were represented as mean and standard deviation values. the categorical data were represented as frequencies and percentages and analysed with the help of fisher’s exact test and chi-squared tests. a p-value of 0.05 was considered to assess the level of significance.  results the demographic data of the study subjects are enlisted in table-2. our study included a total of 1578 panoramic radiographs which comprised of 644 males and 934 females, and the age of subjects ranged from 7 to 82 years with a mean age of 45.60 ± 15.9 years. out of 1578 radiographs, only 204 (12.9%) had visible soft tissue calcifications of which 78 (38.2%) were males and 126 (61.8%) were females. table 1. characteristics of calcifications located in the maxillofacial region s.no type of calcification number radiographic appearance location 1 antrolith 2nd or 3rd quadrant • commonly appears as a solitary radiopacity which is well-defined, round to ovoid, irregular or smooth in outline. • seen in the antrum of maxillary sinus. 2 rhinolith medial aspect of 2nd or 3rd quadrant • these are heterogenous or homogenous radiopacities with a laminar appearance. • seen in the nasal fossa. 3 phlebolith 5th or 8th quadrant • seen as small, multiple circular or oval layers of concentric radiolucent and radiopaque rings. • often, these calcified bodies have the characteristic radiopaque “bulls eye” or “target” appearance. common sites are: • posterior region of body of mandible. • internal aspect of inferior alveolar canal. 4 tonsillolith 1st or 4th quadrant • appears as unilateral ill-defined radiopacities in clusters. • seen superimposed over the mid-portion of ramus along the oro-pharyngeal air space. 5 sialolith 1st or 4th quadrant • single or multiple, irregularly shaped calcified structures with a smooth outline. • submandibular gland sialolith (83-94%) – along the ramus or angle of the mandible. • parotid gland sialolith (4-10%) – along the upper-third of mandibular ramus (within parotid space). 6 calcified lymph nodes 5th, 8th or lateral aspect of 6th or 7th quadrant • calcified structures with “cauliflower-like” lobulated appearance and an irregular periphery. • commonly located at the inferior border of mandible near the angle. 7 atherosclerosis (carotid artery calcifications) 5th or 8th quadrant • irregular, heterogenous vertico-linear radiopacities with a characteristic “pipe-line” or “tram-track” appearance. • seen at the level of intervertebral space between c3 and c4. 8 calcified stylohyoid ligament from 1st to 6th quadrant or 4th to 7th quadrant • ossified radiopaque ligaments which can be slender, segmented, tapering and often longer than 30 mm. • extends from mastoid region to the hyoid bone, crossing the posterior-inferior aspect of mandibular ramus. 5 darwin et al. braz j oral sci. 2023;22:e237798 the study samples were categorized based on the age group classification given by world health organization as subjects, between 3 to 39 years as young adults; subjects between 40 to 59 years as middle age and subjects between 60 to 99 years as old age. the majority of soft tissues calcifications observed in our study were more common in the middle-aged (44.1%) population followed by the young adult population (40.7%). among the 204 calcifications, calcifications of carotid artery (34.3%) were the most predominantly observed calcification followed by calcified stylohyoid ligament (21%), phlebolith (17.6%), tonsillolith (10.2%), antrolith (6.4%) and sialolith (5.9%). the presence of calcification in the nasal cavity (2.4%) and lymph nodes (1.9%) were the least encountered calcifications (figures-2,3,4 and 5). subjects identified with soft tissue calcifications comprised of 33% and 35.7% of carotid artery calcifications, 19.8% and 22.4% of calcified stylohyoid complex, 17.9% and 16% of phleboliths, 11.3% and 8.5% of tonsilloliths, 7.5% and 5.1% of antroliths, 6.6% and 5.1% of sialoliths, 2.8% and 2% of rhinoliths, 0.9% and 3% of calcified lymph nodes on left and right sides of the radiographs respectively. analysis of soft tissue calcifications based on various age groups and gender: in the present study, the occurrence of antrolith, rhinolith, tonsillolith, calcifications of the carotid artery and stylohyoid ligament (p > 0.05) was significant. we found a table 2. demographic data of the study sample. total no. of panoramic radiographs collected and screened 1693 no. of panoramic radiographs excluded 115 no. of panoramic radiographs included 1578 characteristics of the included radiographs gender n % males 644 40.8 females 934 59.2 age group n % 03-39 (young adults) 498 31.5 40-59 (middle aged) 516 32.7 60-99 (old aged) 564 35.7 characteristics of the radiographs with calcifications no. of panoramic radiographs with soft tissue calcifications 204 gender n % males 78 38.2 females 126 61.8 age group n % 03-39 (young adults) 83 40.7 40-59 (middle aged) 90 44.1 60-99 (old aged) 31 15.1 6 darwin et al. braz j oral sci. 2023;22:e237798 figure 2. (a) the arrow on the panoramic radiograph showing a radiopaque mass in the right maxillary sinus suggestive of antrolith. (b) the arrows on the panoramic radiograph showing well-defined elongated radiopacity at the level of third cervical vertebrae suggestive of carotid artery calcification. l l a b figure 3. (a) the arrows on the panoramic radiograph showing well-defined circular radiopacities in the internal aspect of inferior alveolar canal bilaterally suggestive of phleboliths. (b) the arrow on the panoramic radiograph showing a radiopacity near the angle of mandible on right side suggestive of sialolith. l l a b 7 darwin et al. braz j oral sci. 2023;22:e237798 figure 4. (a) the arrow on the panoramic radiograph showing a well-defined radiopacity with a “cauliflower appearance” near the mandibular angle suggestive of calcified lymph node. (b) the arrow on the panoramic radiograph showing a well-defined radiopacity close to the nasal fossa on left side suggestive of rhinolith (the contrast of the radiograph is altered for better visualisation of the calcification). l l a b figure 5. (a) the arrows on the panoramic radiograph depicting calcified stylohyoid ligament bilaterally. (b) the arrow on the panoramic radiograph showing multiple radiopaque masses superimposed on the right mid-ramus region suggestive of tonsilloliths (the contrast of the radiograph is altered for better visualisation of the calcification). l l a b 8 darwin et al. braz j oral sci. 2023;22:e237798 statistically significant association of the occurrence of antrolith (p=0.018) among the males and rhinolith (p=0.025) among the females and it was predominantly seen in the young-adult population. we also observed a significant prevalence in the occurrence of tonsillolith (p=0.007) among the males, carotid artery calcifications (p=0.001) and calcified styloid ligament (p=0.022) among the females and was predominantly seen among the middle-aged population respectively (table-3). analysis of soft tissue calcifications based on site and gender: our results revealed that most of the calcifications occurred on the left side and the prevalence of calcifications on the left and right sides comprised 51.9% and 48% respectively. there was a statistically significant association of occurrence of tonsillolith in the right side of males (p=0.011). in addition, the occurrence of carotid artery calcification in the left (p=0.011); right (p=0.029) sides and calcified stylohyoid ligament in the left (p=0.041); right (p=0.009) sides in females were found to be statistically significant (table 4). however, there was no significant difference in the occurrence of antrolith, rhinolith, phlebolith, sialolith, calcified lymph nodes among males and females (p-value > 0.05). table 3. distribution of various soft tissue calcifications based on age group and gender type of calcification age p-value03-39 (young adults) 40-59 (middle aged) 60-99 (old aged) antrolith m 6 (100%) 0 0 0.018 f 4 (57.1%) 3 (42.6%) 0 rhinolith m 0 1 (20%) 0 0.025 f 4 (80%) 0 0 phlebolith m 11 (50%) 5 (22.7%) 6 (27.3%) 0.111 f 4 (28.6%) 8 (57.1%) 2 (14.3%) tonsillolith m 3 (17.7%) 10 (58.8%) 4 (23.5%) 0.007 f 4 (100%) 0 0 sialolith m 3 (33.3%) 4 (44.4%) 2 (22.2%) 0.513 f 2 (66.7%) 1 (33.3%) 0 calcified lymph node m 1 (100%) 0 0 0.248 f 1 (33.3%) 0 2 (66.6%) carotid artery calcifications m 12 (80%) 3 (20%) 0 0.001 f 8 (14.5%) 35 (63.6%) 12 (21.8%) calcified styloid ligament m 4 (57.1%) 1 (14.3%) 2 (28.6%) 0.022 f 16 (44.4%) 19 (52.8%) 1 (2.8%) 9 darwin et al. braz j oral sci. 2023;22:e237798 discussion panoramic radiography (opg) is an imaging modality that can aid in simultaneous preliminary radiographic examination of both maxillary and mandibular jaws. despite being a two-dimensional radiograph, opg is one of the frequent radiographic investigations employed in maxillofacial radiology due to its low cost and reduced patient radiation exposure17. thereby, given its popularity, dental clinicians should be familiar with the typical patterns and characteristic radiographic features of various soft-tissue calcifications that are encountered in the maxillofacial region. these calcifications are often asymptomatic and detected as incidental findings4. according to the recommendations of the american academy of oral and maxillofacial radiology, the use of a three-dimensional imaging modality like cbct, which has the advantage of providing high-quality diagnostic images, cannot be justified in the initial evaluation of soft tissue calcifications unless the patient is symptomatic18,19. in our study, we observed soft tissue calcifications in 12.9% of the total radiographs, which is comparatively higher than the reports of previous studies which revealed a prevalence rate varying between 2.6% and 19.7%3,5,10,20. this difference in the prevalence can be attributed to several factors such as racial, age, geographical and ethnic variations. apart from this, the possibility to alter the image density and contrast in the radiographic images can also attribute to the variations as reported by monsour et al.20. we found that the majority of calcifications within the soft tissues were more common in middle-aged (44.1%) population followed by young adults (40.7%). these table 4. frequency of various soft tissue calcifications based on gender type of calcification gender p-value male female antrolith l 4 (30.8%) 4 (30.8%) 0.717 r 2 (15.4%) 3 (23%) 0.401 rhinolith l 1 (20%) 2 (40%) 0.668 r 0 2 (40%) 0.167 phlebolith l 12(33.3%) 7 (19.4%) 0.806 r 10 (27.8%) 7 (19.4%) 1.0 tonsillolith l 8 (38%) 4 (19%) 0.762 r 9 (42.9%) 0 0.011 sialolith l 5 (41.7%) 2 (16.7%) 0.701 r 4 (33.3%) 1 (8.3%) 0.649 calcified lymph node l 0 1 (25%) 0.410 r 1 (25%) 2 (50%) 0.668 carotid artery calcifications l 7 (10%) 28 (40%) 0.011 r 8 (11.4%) 27 (38.6%) 0.029 calcified styloid ligament l 4 (9.3%) 17 (39.5%) 0.041 r 3 (6.9%) 19 (44.2%) 0.009 10 darwin et al. braz j oral sci. 2023;22:e237798 findings were consistent with the results of icoz et al. and ribeiro et al. who also suggested that the prevalence of calcifications increased above 40 years4,10. the process of calcinosis begins at an early age and progresses as the patient ages thus becoming radiographically identifiable. the most commonly observed soft tissue calcification in the present study was calcification of the carotid artery constituting 4.4% of all included radiographs. the results were statistically significant and were consistent with the findings of bayer et al., saati et al. and garay et al. who reported a higher prevalence of calcifications of carotid artery in the middle-aged female population3,17,21. women were more typically affected in the post-menopausal period (>50 years) owing to the diminished oestrogen levels. the role of oestrogen in the metabolism of lipoproteins is well established in the literature, and it is known to inhibit the formation of atheromatous plaques4,22. the link between the risk of ischemic stroke and the occurrence of carotid artery calcifications has been a source of debate. significant narrowing of an arterial lumen with a lucent defect is indicated by a massive calcification. according to the literature, the calcium levels in cervical carotid arteries serves as an independent marker for the detection of ischemic symptoms23. literature evidences have accounted stroke to be the second-leading cause of death globally (11.6%). risk of stroke in the presence of atherosclerosis of the carotid artery is 8%. hence, active observation of the asymptomatic patients with the aid of panoramic radiographs can potentially reduce the occurrence of any life-threatening consequences such as stroke and myocardial infarction5,10,17,24. out of the total radiographs evaluated, our study reported an overall prevalence of 2.7% of calcified stylohyoid ligament. previous studies have reported varied prevalence rates ranging from 7.9% to 38.57%5,14,25. we observed the occurrence of calcified stylohyoid process to be significantly higher in women and predominantly on the right side compared to the left. we also observed an increasing trend in the calcification of the stylohyoid ligament in the middle-aged population which was statistically significant. our findings are in accordance with the study by guimarães et al. and oztas et al., which reported patients above 40 years of age to have higher prevalence of calcification of the stylohyoid complex26,27. this could be attributed to the anatomical variations, variability in the muscle stress owing to occlusal interferences and racial differences of the population studied. the stylohyoid ligament was considered to be calcified if the length of the stylohyoid complex exceeded more than 30mm extending from the inferior border of the external acoustic meatus10. till date, there is an absence of a consensus regarding the standard size of this medially angulated ligament14,17. opg is a preferred choice for the evaluation and visualization of the calcified stylohyoid complex which is one of the major etiological factors for eagle’s syndrome10. this syndrome which was initially described by wett eagle in 1937 is characterized by persistent dull pain in the facial and oropharyngeal region. the affected individuals may present with dysphagia and subjective sensation of a foreign body in the throat accompanied by unexplained headache and increased salivation28. in our study, tonsilloliths constituted 1.3% of the total included radiographs. our findings can be matched with the reports by garay et al. and ribeiro et al. who reported 11 darwin et al. braz j oral sci. 2023;22:e237798 prevalence rates of 1.4% and 0.9% respectively3,10. occurrence of tonsilloliths in the right side were significant in males which is consistent with the findings reported by saati et al.17. in addition, we observed a significant association of tonsillolith with age, as majority of these calcifications were seen in individuals above the age of 40. garay et al.3 in their study have also reported similar findings. often, these small concretions are asymptomatic and are detected incidentally on panoramic radiographs29. however, the presence of these calcifications in a relatively larger size may cause difficulty in swallowing, reflex otalgia, odynophagia, halitosis and may predispose to peritonsillar abscess due to superinfection30. the prevalence of antrolith, observed in our study was 0.8% and most of them were found on the left side. rhinoliths were observed in 0.3% of the total radiographs studied and was more frequent on the left side. in our study, the occurrence of antroliths was more in the young-adult males and rhinoliths in the young-adult females. these results were found to be significant and was similar to the findings of ribeiro et al.10. in most individuals, calcifications in the antrum or nasal cavity may not cause any clinical symptoms. occasionally, such calcifications can cause facial pain, epistaxis, epiphora, perforation of the mucosa16,31. of the total radiographs studied, 36 (2.3%) of the radiopacities represented the characteristic features of phleboliths, which are the calcifications of the venous system. similar findings were reported by saati et al. who observed a prevalence rate of 0.29% of phleboliths17. in the present study, 0.7% of sialoliths and 0.2% of calcified lymph nodes were observed on the radiographs. according to the findings of ayranci et al.32, calcification of the salivary glands is known to affect one in every 10,000 to one in every 30,000 individuals. these calcifications may obstruct the ductal flow causing acute pain that intensifies with meals. the understanding of various calcifications occurring in the maxillofacial region is necessary to aid in their diagnosis and to predict their possible consequences, which could be beneficial for patients. the present study has certain inherent limitations of cross-sectional studies like the inability to acquire the entire medical history of the patient. there exists a lack of consensus in the standardization of criteria for analysing soft tissue calcifications in the panoramic radiographs. further, better-designed multi-centric prospective studies using three-dimensional radiographic modalities focusing on long-term follow-up are necessary to define the typical radiographic appearances of specific calcifications and to describe their clinical effects on symptomatic and asymptomatic patients. such future researches would not only provide more relevant information to an individual’s medical care but also permits an accurate referral so as to prevent any potential morbidities. in conclusion, the present study emphasizes the radiographic appearances of various soft tissue calcifications occurring in the maxillofacial region, so as to aid in a better diagnosis. we found the soft tissue calcifications to be more predominant in women with increased occurrences over 40 years of age. optimal knowledge of the normal anatomy of the maxillofacial skeleton is inevitable to arrive at a precise radiographic diagnosis of such calcifications. 12 darwin et al. braz j oral sci. 2023;22:e237798 acknowledgements no acknowledgements. conflicts of interest none. data availability datasets related to this article will be available upon request to the corresponding author. author contribution deepthi darwin: methodology, investigation, writing – original draft preparation, review and editing. renita lorina castelino: conceptualization, methodology, formal analysis, validation. gogineni subhas babu: conceptualization, resources, validation. mohamed faizal asan: writing – review and supervision, investigation, visualization. all authors actively participated in the discussion of the manuscript’s finding, revised and approved the final version of the manuscript. references 1. rajkumar m, siva b, sudharshan r, srinivas h, vishalini a, sivakami p. prevalence of soft tissue calcification in orthopantamograph. asian j dent sci. 2021;4(1):20-8. 2. nasseh i, sokhn s, noujeim m, aoun g. considerations in detecting soft tissue calcifications on panoramic radiography. j int oral health. 2016;8(6):742-6. doi: 10.2047/jioh-08-06-20. 3. garay i, netto hd, olate s. soft tissue calcified in mandibular angle area observed by means of panoramic radiography. int j clin exp med. 2014 jan;7(1):51-6. 4. icoz d, akgunlu f. prevalence of detected soft tissue calcifications on digital panoramic radiographs. srm j res dent sci. 2019;10(1):21-5. doi: 10.4103/srmjrds.srmjrds_60_18. 5. vengalath j, puttabuddi jh, rajkumar b, shivakumar gc. prevalence of soft tissue calcifications on digital panoramic radiographs: a retrospective study. j indian acad oral med radiol. 2014;26(4):385-9. doi: 10.4103/0972-1363.155676. 6. tseung j. book review: robbins and cotran pathologic basis of disease. 7th ed. pathology. 2005;37(2):190. doi: 10.1080/00313020500059191. 7. romano n, silvestri g, castaldi a. the ‘abc’ of neck calcifications: a practical guide. sn compr clin med. 2021 sep;3(2):1-10. doi: 10.1007/s42399-021-01061-5. 8. adhami f, ahmed a, omami g, mathew r. soft-tissue 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soft tissue calcifications of the head and neck region on maxillofacial cone beam computed tomography [master’s thesis]. university of louisville; 2011. doi: 10.18297/etd/1545. 13. schroder agd, de araujo cm, guariza-filho o, flores-mir c, de luca canto g, porporatti al. diagnostic accuracy of panoramic radiography in the detection of calcified carotid artery atheroma: a meta-analysis. clin oral investig. 2019 may;23(5):2021-40. doi: 10.1007/s00784-019-02880-6. 14. safabakhsh m, johari m, bijani a, haghanifar s. prevalence of soft tissue calcification in panoramic radiographs in northern of iran. j babol univ medical sci. 2018;20(6):41-5. doi: 10.18869/acadpub.jbums.20.6.41. 15. aoun g, nasseh i, diab ha, bacho r. palatine tonsilloliths: a retrospective study on 500 digital panoramic radiographs. j contemp dent pract. 2018 oct;19(10):1284-7. 16. white sc, pharoah mj. oral radiology: principles and interpretation. saint louis: mosby/elsevier; 2009. p.526-40. 17. saati s, foroozandeh m, alafchi b. radiographic characteristics of soft tissue calcification on digital panoramic images. pesq bras odontoped clin integr. 2020;20:e5053. doi: 10.1590/pboci.2020.068. 18. khojastepour l, haghnegahdar a, sayar h. prevalence of soft tissue calcifications in cbct images of mandibular region. j dent (shiraz). 2017 jun;18(2):88-94. 19. kumar m, shanavas m, sidappa a, kiran m. cone beam computed tomography know its secrets. j int oral health. 2015 feb;7(2):64-8. 20. monsour pa, romaniuk k, hutchings rd. soft tissue calcifications in the differential diagnosis of opacities superimposed over the mandible by dental panoramic radiography. aust dent j. 1991 apr;36(2):94-101. doi: 10.1111/j.1834-7819.1991.tb01336.x. 21. bayer s, helfgen eh, bös c, kraus d, enkling n, mues s. prevalence of findings compatible with carotid artery calcifications on dental panoramic radiographs. clin oral investig. 2011 aug;15(4):563-9. doi: 10.1007/s00784-010-0418-6. 22. sisman y, ertas et, gokce c, menku a, ulker m, akgunlu f. the prevalence of carotid artery calcification on the panoramic radiographs in cappadocia regionpopulation. eur j dent. 2007 jul;1(3):132-8. 23. yoon sj, yoon w, kim os, lee js, kang bc. diagnostic accuracy of panoramic radiography in the detection of calcified carotid artery. dentomaxillofac radiol. 2008 feb;37(2):104-8. doi: 10.1259/dmfr/86909790. 24. çakur b, yıldırım e, demirtaş ö. [an investigation of relationship between tonsillolith and carotid artery calcification on panoramic radiography]. atatürk üniv. diş hek. fak. derg. 2014;24(1):1-5. turkish. 25. de andrade km, rodrigues ca, watanabe pc, mazzetto mo. styloid process elongation and calcification in subjects with tmd: clinical and radiographic aspects. braz dent j. 2012;23(4):443-50. doi: 10.1590/s0103-64402012000400023. 26. guimarães ac, pozza dh, guimarães as. prevalence of morphological and structural changes in the stylohyoid chain. j clin exp dent. 2020 nov;12(11):e1027-32. doi: 10.4317/jced.57186. 14 darwin et al. braz j oral sci. 2023;22:e237798 27. öztaş b, orhan k. investigation of the incidence of stylohyoid ligament calcifications with panoramic radiographs. j investig clin dent. 2012 feb;3(1):30-5. doi: 10.1111/j.2041-1626.2011.00081.x. 28. yavuz gy, keskinrüzgar a. clinical and radiological evaluation of elongated styloid process in patients with temporomandibular joint disorder. cumhur dent j. 2019;22(1): 37-41. doi: 10.7126/cumudj.498907. 29. babu b b, tejasvi m l a, avinash ck, b c. tonsillolith: a panoramic radiograph presentation. j clin diagn res. 2013 oct;7(10):2378-9. doi: 10.7860/jcdr/2013/5613.3530. 30. ghabanchi j, haghnegahdar a, khojastehpour l, ebrahimi a. frequency of tonsilloliths in panoramic views of a selected population in southern iran. j dent (shiraz). 2015 jun;16(2):75-80. 31. aoun g, nasseh i. maxillary antroliths: a digital panoramic-based study. cureus. 2020 jan 17;12(1):e6686. doi: 10.7759/cureus.6686. 32. ayranci f, omezli mm, torul d, sunar c, koc l. sialolith of the submandibular gland: a case report. mbsjhs. 2020;6(2):407-11. doi: 10.19127/mbsjohs.817042. 1http://dx.doi.org/10.20396/bjos.v21i00.8665686 volume 21 2022 e225686 original article braz j oral sci. 2022;21:e225686 1 private practice, porto alegre, rs, brazil. 2 school of dentistry, lutheran university of brazil (ulbra), canoas, rs, brazil. 3 department of conservative dentistry, federal university of rio grande do sul (ufrgs), porto alegre, rs, brazil. *corresponding author: theodoro weissheimer department of conservative dentistry, federal university of rio grande do sul rua ramiro barcelos, 2492 90035-003, porto alegre rio grande do sul (rs) brazil. telephone: +55 51 996924725 e-mail: theodoro.theo@hotmail.com editor: altair a. del bel cury received for publication: may 19, 2021 accepted: october 24, 2021 assessment of quality of life in total edentulous patients rehabilitated with implants and fixed prosthesis alexandre da silveira gerzson1 , brenda ledur lauxen2 , theodoro weissheimer3,* , elizangela paludo2 , luís artur zenni lopes2 aim: tooth loss is very prevalent in brazil, reflecting high demand for dental services, especially those related to oral rehabilitation. this study aimed to assess the quality of life in total edentulous patients rehabilitated with implants and fixed prosthesis. methods: thirty-two patients were evaluated before and after rehabilitation with dental implants and fixed prosthesis using the ohip-14 questionnaire and the visual analogue scale (vas) after 6 months follow-up. results: ohip-14 revealed a significant improvement after treatment in all seven parameters and in the global score (p < 0.001). vas presented positive results related to patient satisfaction regarding oral rehabilitation, except for the hygiene of the fixed dentures. conclusion: at the end of this study, ohip-14 scores decreased by 50% in most of the questions raised, and vas presented positive results, except for hygiene of the fixed dentures, presenting an improvement in the quality of life of total edentulous patients after rehabilitation with implants and fixed prosthesis. keywords: dental implants. dental prosthesis. oral health. quality of life. https://orcid.org/0000-0001-7571-2289 https://orcid.org/0000-0002-8789-9981 https://orcid.org/0000-0001-6810-1877 https://orcid.org/0000-0001-5465-7225 https://orcid.org/0000-0003-4504-8625 2 gerzson et al. braz j oral sci. 2022;21:e225686 introduction in brazil, there is a high prevalence of edentulousness1. edentulism is an important public health issue, presenting a strong negative impact on the patients’ quality of life, expressed by the loss of functional abilities, as mastication and phonation, as well as nutritional, aesthetic and psychological losses, with direct influence in the reduction of self-esteem and social integration2-5. it is known that teeth are key factors to facial harmony and to the smile, which are determinant factors in the patients’ quality of life1,6. fully edentulous patients show a preference towards implant-supported fixed rehabilitations, because they provide greater masticatory effectiveness and comfort, require less repair and lower maintenance, besides favoring the psychological aspect, since it remains still in the mouth3-5. the scientific and technical advances in dentistry, especially in the field of oral rehabilitation, have been seeking to restore occlusal stability and, consequently, promoting facial harmony with the aid of implants, which can delay the physiological resorption of the bone, increasing the removable prostheses retention and seeking to improve the patients’ quality of life7. . it is important to verify if the rehabilitation of edentulous patients with implants and fixed prosthetics can present some impact on its quality of life, in order to determine the best treatment modality and factors that can affect this outcome. therefore, this study aimed to access the quality of life of total edentulous patients before and after rehabilitation with implants and fixed prosthesis. the hypothesis of the study was that the rehabilitation of total edentulous patients with implants and fixed prosthesis improves the quality of life. material and methods the sample size calculation determined a required sample of 32 patients for a 95% confidence level, 80% power and 50% estimated reduction in ohip scores, using as base reference a mean (standard deviation) ohip score of 13.5 (13.0) before intervention8. after the approval of the research ethics committee of the lutheran university of brazil (caae 49943415.0.0000.5349), informed consent was obtained from the participants to the performance of this prospective study. all procedures performed in this study involving human participants were in accordance with ethical standards of the institutional and/or national research committee, as well as with the 1964 helsinki declaration and its later amendments or comparable ethical standards. patients treated at the implant dentistry specialization course were included using the following criteria: a) fully edentulous patients or those with indication of extraction, treated with dental implants and full fixed rehabilitation in the maxilla and / or mandible; b) both sexes; c) patients who agreed to participate by signing an informed consent form; d) treatment completed within at least six months of follow-up. 3 gerzson et al. braz j oral sci. 2022;21:e225686 the ohip questionnaire 14 was used as reference, due to its great reliability in international research, giving more credibility to the research to evaluate the impact of oral rehabilitation on patients’ quality of life.. this questionnaire uses seven categories (functional limitation, physical pain, psychological discomfort, physical limitation, psychological limitation, social disability and incapacity) with two objective questions each, with responses ranging from 1 to 5. responses were summarized in a 6-point likert scale. for each of the categories, the average of the two questions addressed was calculated, and the total average of the seven categories before and after the rehabilitation treatment was calculated. the visual analogue scale (vas), which ranges from 0 to 10 (0: completely dissatisfied and 10: completely satisfied), was used to evaluate the patients’ general satisfaction, comfort and stability, aesthetics, ease of cleaning, ability to speak, self-esteem and functionality after rehabilitation with dental implants. the patients answered the ohip-14 questionnaire before and after 6 months of the treatment with dental implants and fixed prosthesis, and the vas only at 6 months follow-up. vas ratings were transferred to a ruler tabulated in millimeters and centimeters (10cm) in order to obtain a numerical value referring to the satisfaction corresponding to each patient. a descriptive analysis of the results was performed to analyze the results of the vas, which also has full digit data, ranging from zero to 10, and that evaluated the degree of satisfaction of patients treated with dental implants and fixed prosthesis, in relation to the seven previously mentioned parameters. data obtained were tabulated and statistically analyzed (spss 21.0 software, ibm corp, armonk, ny). the non-parametric wilcoxon test was applied, with a significance level of 5%. results thirty-two patients were interviewed using the ohip-14 questionnaire before and after implant placement, and the vas exclusively related to implant rehabilitation. analyzing the ohip-14 questionnaires before and after treatment, there was a significant difference in the results presented before and after the treatment, and it was verified that dental implants and fixed prosthodontic rehabilitation had a positive impact in all seven parameters and in the global score evaluated by the ohip-14 questionnaire (p < 0.001), as presented in table 1. table 1. results (mean ± standard deviation) of the parameters before and after treatment with dental implants obtained through the ohip-14 questionnaire. parameters before after p-value functional limitation 3.89 ± 0.95 2.11 ± 1.48 p < 0.001 physical pain 4.38 ± 0.88 1.38 ± 0.77 p < 0.001 psychological discomfort 4.48 ± 0.94 1.42 ± 0.87 p < 0.001 physical limitation 4.25 ± 0.74 1.06 ± 0.31 p < 0.001 psychological limitation 4.59 ± 0.71 1.09 ± 0.43 p < 0.001 social disability 4.05 ± 0.85 1.06 ± 0.35 p < 0.001 incapacity 4.11 ± 1.04 1.09 ± 0.55 p < 0.001 global score 4.25 ± 0.91 1.32 ± 0.85 p < 0.001 4 gerzson et al. braz j oral sci. 2022;21:e225686 as for the vas score, in terms of general satisfaction with implants, comfort and stability, aesthetics, ability to speak, self-esteem and functionality, patients were totally satisfied. all 32 patients answered 10 for these questions. regarding the ease of cleaning (7.8 ± 2.72), 12 of the 32 patients gave results below 7 and eight of them below 5. discussion oral rehabilitation with dental implants has resulted in great patient satisfaction, although there are still few studies that address the patient’s opinion and aspirations regarding this approach9. rehabilitation is of paramount importance when it comes to improving the quality of life, by reestablishing the edentulous patient’s health, physical, psychological and social well-being. based on the presented results, the study hypothesis can be confirmed. the ohip questionnaire was created by slade & spencer as a tool to evaluate the impact of the oral condition on the quality of life of the individuals and population. initially, it comprised 49 questions (ohip-49) and was later reduced to 14 questions (ohip-14), which was considered effective to determine the same associations with clinical and socio-demographic factors that were observed using ohip-49. the reduced questionnaire accurately assesses the efficacy and success of the treatment through different prosthetic parameters10,11. this study followed the methodology of another study, which also used two ohip14 questionnaires to identify the patient’s perception regarding restorative treatment with implants, where the final mean values closer to zero indicate a better quality of life of the individuals after oral rehabilitation with implants and fixed prosthesis12. a study performed with a sample of 22 patients fully edentulous and maxillae rehabilitated with zygomatic implants and fixed prosthesis, used the ohip-14 questionnaire before and after treatment and found that the end-time rehabilitation had a positive impact in most categories (p < 0.05)13, which is in accordance with the results of this study. it is important to emphasize that the results of the present study on the ohip scores was, in most aspects, a reduction of 50% of the baseline values, indicating a great improvement related to the patients’ quality of life. another study evaluated 58 patients rehabilitated with implant-supported prostheses and a mean follow-up of 13.7 years. patients also answered the ohip14 questionnaire and it was noted that they were satisfied with the quality of the rehabilitation, with older patients showing greater satisfaction when compared to younger patients14. in the present study, it was verified an average ohip score of 13.5 before intervention. at the end of the study, it was managed to reach an average score of 4, representing a score reduction greater than 50%, confirming a very significant improvement in quality of life. a survey of patients that used conventional prostheses and replaced them with implant-supported prostheses evaluated the effects of treatment, and concluded that there was improvement in neuromuscular adaptation, bone tissue preservation and improvement of masticatory function15. 5 gerzson et al. braz j oral sci. 2022;21:e225686 as for the parameters analyzed in the vas scale (comfort and stability, aesthetics, ease of cleaning, ability to speak, self-esteem and functionality after rehabilitation with dental implants), it was verified an 100% satisfaction in four questions (comfort and stability, aesthetics, ability to speak, self-esteem and functionality), and nearly 100% in one question (ease of cleaning), demonstrating a high degree of satisfaction of the patients involved in this research. the issue that addressed the ease of cleaning was the last degree of contentment after treatment, where patients reported that oral hygiene became more complex in comparison to the dentures previously used, which is easily understandable, since the hygiene of a fixed implant based prosthesis require more training and more dedication time, besides the use of specific hygiene instruments such as dental floss guides, interdental brushes, among others. it is necessary to emphasize that this study is limited on the evaluation of patients reports after a short follow-up period (6 months), an that not necessarily reflects the patient long-term satisfaction. however, a previous study that evaluated patients satisfaction and oral-health related quality of life after ten years of implant placement presented results similar to those of this study16, suggesting that such parameters are mantained over time. still, further studies evaluating the patient’s quality of life after a shortand long-term follow-up periods are necessary. in conclusion, based on the findings of this study, it is possible to conclude that the rehabilitation of total edentulous patients with implants and fixed prosthesis positively influenced on their quality of life. acknowledgments the authors declare no conflict of interest. data availability datasets related to this article will be available upon request to the corresponding author. author contribution alexandre da silveira gerzson: conceptualization, data curation, formal analysis, investigation, methodology, project administration, resources, supervision, validation, visualization, writing original draft. brenda ledur lauxen: data curation, investigation, methodology, project administration, resources, supervision, validation, visualization, writing original draft. theodoro weissheimer: data curation, formal analysis, writing – review & editing. elizangela paludo: data curation, formal analysis, writing – review & editing. luís artur zenni lopes: data curation, formal analysis, writing – review & editing. all authors have actively participated in the discussion of the manuscript’s findings and have revised and approved the final version of the manuscript. 6 gerzson et al. braz j oral sci. 2022;21:e225686 references 1. peršić s, čelebić a. influence of different prosthodontic rehabilitation options on oral health-related quality of life, orofacial esthetics and chewing function based on patient-reported outcomes. qual life res. 2015 apr;24(4):919-26. doi: 10.1007/s11136-014-0817-2. 2. musacchio e, perissinotto e, binotto p, sartori l, silva-netto f, zambon s, et al. tooth loss in the elderly and its association with nutritional status, socio-economic and lifestyle factors. acta odontol scand. 2007 apr;65(2):78-86. doi: 10.1080/00016350601058069. 3. beloni wb, vale hf, takahashi jmfk. 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[assessment of quality of life in total edentulous patients rehabilitated with zygomatic implants.] rev implantnews. 2010;7(3):183-90. portuguese. 14. kuoppala r, näpänkangas r, raustia a. quality of life of patients treated with implant-supported mandibular overdentures evaluated with the oral health impact profile (ohip-14): a survey of 58 patients. j oral maxillofac res. 2013 jul 1;4(2):e4. doi: 10.5037/jomr.2013.4204. 15. heckmann sm, heussinger s, linke jj, graef f, pröschel p. improvement and long-term stability of neuromuscular adaptation in implant-supported overdentures. clin oral implants res. 2009 nov;20(11):1200-5. doi: 10.1111/j.1600-0501.2009.01722.x. 16. wang y, bäumer d, ozga a-k, körner g, bäumer a. patient satisfaction and oral health-related quality of life 10 years after implant placement. bmc oral health. 2021 jan;21(1):30. doi:10.1186/s12903020-01381-3 1http://dx.doi.org/10.20396/bjos.v20i00.8663867 volume 20 2021 e213867 original article 1. student fellow, federal university rio grande do sul, porto alegre, rio grande do sul, brazil. 2. student fellow, post-graduate program in dentistry, federal university rio grande do sul, porto alegre, rio grande do sul, brazil. 3. post-doctoral student fellow, post-graduate program in bioscience and biotechnology applied to pharmacy, department of clinical analysis, são paulo state university, araraquara, brazil. 4. professor of oral microbiology and biochemistry and cariology, department of preventive and community dentistry, federal university of rio grande do sul, porto alegre, rio grande do sul brazil. 5. master degree student, postgraduate program in dentistry, federal university rio grande do sul, porto alegre, rio grande do sul brazil. 6. professor of periodontology, department of semiology and clinic, federal university of pelotas, pelotas, rio grande do sul, brazil. *corresponding author: francisco wilker mustafa gomes muniz. federal university of pelotas. rua gonçalves chaves, 457, pelotas/rs, brazil, zip code: 96015-560. e-mail: wilkermustafa@ gmail.com. telephone: +55 53 9 91253611. received: january 7, 2021 accepted: january 28, 2021 editor: dr altair a. del bel cury antimicrobial potential of essential oils mouthrinses with and without alcohol: a randomized clinical trial matheus grunevald1, romulo cantarelli2, harry juan rivera oballe2, thais de cássia negrini3, rodrigo alex arthur4  , eduardo liberato da silva5, tiago luís herpich1, francisco wilker mustafa gomes muniz6,* aim: this study aimed to compare the microbiological potential and gustatory perception of essential oils (eo) mouthrinses containing and not containing alcohol. methods: twenty healthy adult volunteers rinsed with 10ml of the following test solutions: eo with alcohol, eo without alcohol, or a control solution (saline solution with mint essence). a washout period of at least seven days was adopted after a single-use protocol of the respective solution. all participants used all three tested substances. antimicrobial potential was assessed by counting salivary total viable bacteria both before and after each rinse. gustatory perception was evaluated using the visual analogue scale (vas). multiple comparisons were performed with the wilcoxon test, using bonferroni correction. results: both eo solutions presented a higher antimicrobial potential in comparison to the control solution (p<0.017). however, no significant difference in antimicrobial potential was observed between eo containing or not containing alcohol (p=0.218). vas of eo with alcohol (median: 2.7) was similar to control solution (median: 1.6) (p=0.287). a better gustatory perception was observed of the eo without alcohol (median 7.6) when compared to the control solution (p<0.0001). when eo groups were compared, eo without alcohol also demonstrated a significantly better gustatory perception (p=0.001). conclusion: mouthrinse containing eo without alcohol presented a better taste perception when compared to the eo with alcohol, but no difference was observed in the antimicrobial potential of both eo solutions after a single rinse protocol. keywords: oils volatile. bacteria. mouthwashes. alcohols. taste perception. https://orcid.org/0000-0001-8107-413x https://orcid.org/0000-0002-3945-1752 2 grunevald et al. introduction the control of supragingival biofilm is essential for preventing the development of several oral diseases, including gingivitis, periodontitis, and caries. to accomplish this, the most common strategy is the mechanical removal of biofilm with toothbrushes1. however, the sole use of toothbrushes may not be sufficient to maintain healthy conditions in all individuals. patients with motor or cognitive problems, lack of motivation, those undergoing post-surgical phases, and those with orthodontic devices may require the use of antimicrobial-containing mouthrinses in order to achieve effective biofilm control2,3. several antimicrobial substances are available on the general market, among which essential oils (eo) present the most favorable results for gingivitis and dental plaque control in the long term. the literature constantly demonstrates better results using eo mouthrinse as an adjunct to the mechanical control of biofilm when compared to other oral hygiene regimens4,5. one study showed that eo presented 36.1% and 24.1% higher antiplaque and antigingivitis effects, respectively, when compared to a placebo solution5. alcohol is present in the composition of several mouthrinses as a vehicle solution, despite the fact that it does not demonstrate important effects on gingivitis or plaque control6. moreover, the flavor of mouthrinses is critical, as flavor may interfere with patients’ adherence to treatment, especially when prescribed for long-term use7. alcohol is likely responsible for the notoriously strong flavors of such solutions, which may be unpleasant for most individuals. as a result, over the last few years the industry has developed mouthrinses without alcohol. recently, a randomized clinical trial demonstrated no significant difference in the antiplaque and antigingivitis effects of eo with or without alcohol8. however, at the moment, no published study has evaluated the impact of alcohol on the gustatory perception of mouthrinses containing eo. therefore, the aim of the present study is to compare the microbiological potential and gustatory perception of eo mouthrinses containing and not containing alcohol. materials and methods ethical aspects and study design this is a crossover, randomized, double-blind, clinical trial that followed the consolidated standards of reporting trials (consort) statement. the study was approved by the ethical committee of the associação dos funcionários do estado do rio grande do sul (protocol #1.020.949). all volunteers signed an informed consent form prior to the beginning of the study. sample selection twenty participants were included in this study (17 women and three men). this  study was conducted between april and june of 2015, at the dental faculty of the federal university of rio grande do sul. all participants answered a questionnaire and were 3 grunevald et al. clinically examined to verify the following eligibility criteria: at least 18 years of age, nonsmokers, with at least 24 natural teeth. the study criteria excluded individuals with a presence or history of periodontitis, active dental caries, pregnant or lactating women, users of removable partial dentures, those with fixed dental prostheses or orthodontic appliances, both alcohol abstainers and alcoholics, and those who had used antibiotics within three months prior to the start of the study. interventions and gustatory perception assessment all participants used all three tested substances, with a washout period of at least seven days between tests. the solutions were as follows: • eo+ group: essential oils in an alcoholic solution (listerine®, johnson & johnson, são paulo, brazil). ingredients: aqua, sorbitol, alcohol, poloxamer 407, benzoic acid, sodium saccharin, eucalyptol, aroma (d-limonene), thymol, methyl salicylate, sodium benzoate, menthol and ci 42053. • eogroup: essential oils in an aqueous solution (listerine zero®, johnson & johnson, são paulo, brazil). ingredients: aqua, sorbitol, propylene glycol, sodium lauryl sulfate, poloxamer 407, eucalyptol, benzoic acid, aroma (d-limonene), thymol, methyl salicylate, sodium benzoate, menthol and ci 42053. • control group: saline solution with mint essence. all participants were instructed not to drink, eat or perform any control of biofilm, either chemical or mechanical, within one hour of the experimental procedure. each  participant rinsed for one minute using 10  ml of the predetermined solution. in order to assure blindness, all mouthrinses were stored in opaque bottles and coded accordingly by an external researcher not involved in any other study process. the order in which participants used the mouthrinses was randomly determined by a researcher not involved in data collection (fwmgm), using a randomizing website (randomization.com). the sequence was kept in an opaque envelope until the end of all experimental procedures to assure allocation concealment. during this period, only the researchers responsible for the randomization had contact with these envelopes. the participants were not aware of which solutions they used at any experimental period. immediately after the rinse was performed, participants’ gustatory perceptions were evaluated using a visual analogue scale (vas). the vas was composed of a straight line of 10cm. markers on the left indicated the most unpleasant taste, while markers on the right indicated the most pleasant taste. the participants were instructed to mark at any point on the line based on this scale. using a ruler, the distance between the beginning of the line and the participant’s mark was measured by one researcher who was blind to the group allocation (rc). microbiological analysis in order to assess the antimicrobial potential of the test solutions, stimulated saliva was collected from all participants both before and after the use of each solution. 4 grunevald et al. all participants were asked to chew a piece of unflavored and inert gum to stimulate salivary flow. saliva produced during the first minute was spat out and discarded. participants chewed the gum for an additional five minutes, with the saliva produced during this period of time collected in proper sterile and coded bottles. all  preand post-rinse codified saliva samples were stored on ice and processed within two hours of their collection. preand post-rinse saliva samples were kept on ice. saliva samples were serially diluted in sterile 0.89% nacl solution, and aliquots of 25 µl of each dilution were plated using the drop technique on the surface of brain heart infusion agar (bhi) supplemented with 5% sheep blood. plates were incubated aerobically at 37°c for 48 hours9. saliva was collected and analyzed only once. colony-forming units (cfu) were counted on each drop by one blinded researcher (rc) under a stereomicroscope, and were expressed as cfu/ml of saliva according to the formula described below: cfu/ml saliva = (cfu x 1000/25) x 10f (1) f = dilution factor from serially diluted samples. sample size calculation the primary outcome of the present study was the microbiological potential. therefore, the sample size estimation was based on data from a previously published study10. it considered a mean (±standard deviation) of aerobic bacteria levels in the essential oil and placebo groups of 11.35±13.11 and 56.41±38.72, respectively. when it was considered a power of 90%, an alpha of 5%, and a total of 16 individuals were necessary. an attrition rate of 20% was expected, totaling 20 participants. statistical analysis for each individual, the percentage of reduction in salivary total bacterial viability was calculated considering preand post-rinse saliva samples. the differences in the antimicrobial potential and gustatory perception were analyzed using the friedman test, as a non-normal distribution was detected in both outcomes. we analyzed data distribution using the shapiro-wilk test. as a p-value <0.001 was detected in the shapiro-wilk test, multiple pair-wise comparisons were performed using the wilcoxon test. a bonferroni correction was established, and the new p-value for statistical significance was <0.017. results twenty-four individuals were recruited for the present study, among whom four were excluded because they did not fit the inclusion criteria. reasons for exclusion are reported in figure 1. among the included individuals, the response rates were 100% for all follow-up periods. furthermore, no adverse events were reported throughout the study. 5 grunevald et al. 24 subjects screened 20 subjects randomized (sequence of mouthwash used was randomized) 20 subjects in the essential oil with alcohol group 20 subjects in the essential oil without alcohol group 20 subjects in the control group 20 subjects analyzed 20 subjects analyzed 20 subjects analyzed 4 excluded diagnosis of periodontitis = 1 taken antibiotics 3 months prior the study = 3 figure 1. flowchart of the participants in the study. during microbiological analysis, no differences were found regarding counts of salivary viable bacteria among groups by the pre-rinsing analysis (p=0.387). table 1 shows the mean percentage reduction of the total viable bacteria of all three groups. by comparing the percentage of reduction in counts of viable bacteria before and after each rinse, a significant reduction was found (p<0.05), with 60.33±29.33, 62.61±39.25 and 4.16±156.55 in the eo with alcohol, eo without alcohol and control groups, respectively. the antimicrobial potential of both eo mouthrinses was significantly higher than that of the control group (p=0.007 for eo with alcohol and p=0.005 for eo without alcohol). however, when both eo groups were compared, no significant difference was demonstrated (p=0.218). table 1. mean±standard deviation values for each time point and groups of the colony forming-units of total aerobes. group mean±sd p-value before rinsing after rinsing mean percent reduction within groups between groups (percentage reduction) eo + 1.87x108±1.62x108 0.71x108±1.06x108 60.33±29.33 <0.001# 0.218ω 0.007µ 0.005α eo 3.73x108±12.5x108 0.52x108±1.31x108 62.61±39.25 <0.001# control 6.46x108±11.66x108 5.29x108±12.99x108 4.16±156.55 0.029# p-value between groups 0.387* 0.074* 0.017* legend: sd: standard deviation; *friedman test; #wilcoxon test for the comparison within groups; ω wilcoxon test for the comparison between eo+ and eogroups; µwilcoxon test for the comparison between control and eo+ groups; α wilcoxon test for the comparison between control and eogroups. the gustatory perceptions of all tested solutions are reported in table 2. these results demonstrated a statistically significant difference in gustatory perception among groups. the control group reported the worst flavor (median 1.6), the eogroup reported the most pleasant gustatory perception (median 7.6), and the eo+ group pre6 grunevald et al. sented an intermediate perception (median 2.7). when groups eo+ and control were compared, no statistically significant difference was detected (p=0.287). however, in the comparison between eoand control groups, a statistically significance difference was detected, showing a better gustatory preference for the eomouthrinse (p<0.001). regarding the comparison between eo groups, a significantly better gustatory preference was observed for the eomouthrinse (p=0.001). table 2. gustatory perception after a single rinse with essential oils with alcohol, without alcohol, and control substance. control eo+ eop-value median (min./max.) 1.6 (0.0 – 8.4) 2.7 (0.1 – 10.0) 7.6 (0.5 – 9.9) <0.001* α 0.001 ω 0.301#mean±sd 2.81±2.72 3.55±2.95 6.90±2.25 legend: eo+: essential oil with alcohol; eo-: essential oil without alcohol; *friedman test; #wilcoxon test for the comparison between control and eo+ groups; α wilcoxon test for the comparison between control and eo groups; ω wilcoxon test for the comparison between eo+ and eogroups. discussion the present study aimed to compare the microbiological potential and gustatory perceptions of eo mouthrinses containing and not containing alcohol. overall, it was observed that both solutions presented a significantly higher antimicrobial potential when compared to a negative control solution. however, both eo solutions presented similar antimicrobial potential. regarding the gustatory potential, participants reported the eo without alcohol as having the most pleasant taste. the chemical control of supragingival biofilm may be performed with mouthrinses. among these, chlorhexidine is considered the gold standard substance, as it demonstrates a good antiplaque effect and long substantivity; however, several adverse events may be detected after long periods of use11-13. other mouthrinses also demonstrate antiplaque and antigingivitis effects with fewer reported adverse events, and these substances may be used for longer periods14. however, a strong, unpleasant flavor and a burning sensation have been reported by the patients who use these mouthrinses. among other available mouthrinses, eo may be the most important one for patient outcomes5. in low concentrations, eo may inactivate bacterial enzymes, interfering with growth velocity and biofilm maturation. additionally, its utilization may be an encouraging factor to enhance patient adherence to this method of supragingival biofilm control15. traditionally, mouthrinses containing eo also contain alcohol in their composition. the alcohol is used to dilute or solubilize the oils, and it is also used to extend the product’s expire date. one study has shown that alcohol may present some antimicrobial effects16, but a systematic review showed that an alcoholic vehicle demonstrated very limited effects in terms of antiplaque and antigingivitis efficacy6. in addition, short-17 and long-term8 clinical trials have demonstrated no statically significant difference in the antiplaque and/or antigingivitis efficacy of eo with or without alcohol. these results are in accordance with the present study, which demonstrated no significant difference in the antimicrobial potential of the two eo solutions. however, fur7 grunevald et al. ther long-term clinical trials are necessary to determine the clinical efficacy of both eo mouthrinses. the mechanical process of chewing an inert piece of gum detaches microorganisms from mucosal and tooth sites. using this method, microbial diversity found on stimulated saliva is representative of other oral niches. moreover, stimulated saliva has the advantage of carrying a greater microbial diversity than unstimulated saliva18. considering that one of the outcomes of this study was to assess the antimicrobial effects of eo-containing mouthrinses, it was decided that this study would use stimulated saliva to evaluate this effect on a broad load and diversity of microorganisms (which could not be found by using unstimulated saliva). the presence of alcohol can also negatively affect the gustatory perception of some individuals due to the strong flavor4, although a previously published study demonstrated that alcohol was not capable of interfering in the taste perception of chlorhexidine solutions9. the literature reports that the flavor of mouthrinses is an important factor in their usage, and it is part of the criteria used by patients when choosing a mouthrinse7. it must be highlighted that several mouthrinses are sold on the general market. a prescription may or may not be necessary to buy these products, depending on the laws of different countries. additionally, as many of these products are designed for continuous usage, a pleasant flavor is a pivotal factor for the ongoing use of the mouthrinse and adherence to the recommended treatment. a vas was used in order to measure participants’ gustatory perceptions. this is a valid method for quantifying the taste19,20. in the present study, participants performed the evaluation immediately after rinsing, without having access to their previous evaluations, which did not allow comparisons. it was found that the most pleasant gustatory perception was reported for the eo not containing alcohol. therefore, these results must be taken into consideration when prescribing an eo mouthrinse, as individuals with alcohol restrictions and those with higher sensitivity to alcohol should not receive an eo mouthrinse with alcohol. the present study used commercially available solutions with essential oils. additionally, a saline solution with mint essence was used. this was a double-blind and cross-over study, which reduced the chance of any interference from knowledge of which substance was tested. furthermore, the washout period of at least seven days allowed a decrease in any potential residual effects during the study. the order of the tested solutions was determined randomly, avoiding any adaptation among the individuals throughout the study. in the present study, twenty individuals were included. although this number may seem small, previous studies regarding the chemical control of biofilm have used similarly sized and valid samples21,22. regarding  the study participants, it is important to highlight that no dropouts occurred and no adverse events were reported. some limitations to the present study must be acknowledged, such as the fact that each rinsing was performed only once. additionally, the included individuals were young and some of them were students of the school of dentistry of the federal university of rio grande do sul. in this sense, these characteristics may have interfered with the study results, decreasing the external validity of the present study23. therefore, further clinical trials involving only non-health professionals are warranted. 8 grunevald et al. in addition, the present study included a higher number of female participants, and a lower internal validity might be expected for male individuals. in conclusion, no significant difference was observed in the antimicrobial potential of eo with and without alcohol. however, it was concluded that the mouthrinse containing eo without alcohol was widely evaluated as having a more pleasant taste when compared to the flavor of an eo mouthrinse with alcohol. acknowledgment this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior brasil (capes) finance code 001. all other funding was self-supported by the authors. references 1. van der weijden fa, slot de. efficacy of homecare regimens for mechanical plaque removal in managing gingivitis a meta review. j clin periodontol. 2015 apr;42 suppl 16:s77-91. doi: 10.1111/ jcpe.12359. 2. haas an, pannuti cm, andrade ak, escobar ec, almeida er, costa fo, et al. mouthwashes for the control of supragingival biofilm and gingivitis in orthodontic patients: evidence-based recommendations for clinicians. braz oral res. 2014 jul 11;28(spe):1-8. doi: 10.1590/18073107bor-2014.vol28.0021. 3. de andrade meyer ac, de mello tera t, da rocha jc, jardini ma. clinical and microbiological evaluation of the use of toothpaste containing 1% chlorhexidine and the influence of motivation on oral hygiene in patients with motor deficiency. spec care dentist. 2010;30(4):140-5. doi: 10.1111/j.1754-4505.2010.00140.x. 4. van leeuwen mp, slot de, van der weijden ga. essential oils compared to chlorhexidine with respect to plaque and parameters of gingival inflammation: a systematic review. j periodontol. 2011 feb;82(2):174-94. doi: 10.1902/jop.2010.100266. 5. haas an, wagner tp, muniz fwmg, fiorini t, cavagni j, celeste rk. essential oils-containing mouthwashes for gingivitis and plaque: meta-analyses and meta-regression. j dent. 2016 dec;55:715. doi: 10.1016/j.jdent.2016.09.001. 6. van leeuwen mp, slot de, van der weijden ga. the effect of an essential-oils mouthrinse as compared to a vehicle solution on plaque and gingival inflammation: a systematic review and metaanalysis. int j dent hyg. 2014 aug;12(3):160-7. doi: 10.1111/idh.12069. 7. cantarelli r, ribas me, daudt farl, rosing ck. [use profile of mouthwash used by patients at the ufrgs school of dentistry]. perionews. 2011;5:361-7. portuguese. 8. lynch mc, cortelli sc, mcguire ja, zhang j, ricci-nittel d, mordas cj, et al. the effects of essential oil mouthrinses with or without alcohol on plaque and gingivitis: a randomized controlled clinical study. bmc oral health. 2018 jan 10;18(1):6. doi: 10.1186/s12903-017-0454-6. 9. cantarelli r, negrini tc, muniz fw, oballe hj, arthur ra, rösing ck. antimicrobial potential and gustatory perception of chlorhexidine gluconate mouthwashes with or without alcohol after a single rinse a randomized controlled crossover clinical trial. int j dent hyg. 2017 nov;15(4):280-6. doi: 10.1111/idh.12255. 10. cortelli jr, cogo k, aquino dr, cortelli sc, ricci-nittel d, zhang p, et al. validation of the antibacteremic efficacy of an essential oil rinse in a brazilian population: a cross-over study. braz oral res. 2012 sep-oct;26(5):478-84. doi: 10.1590/s1806-83242012005000021. 9 grunevald et al. 11. erriu m, pili fm, tuveri e, pigliacampo d, scano a, montaldo c, et al. oil essential mouthwashes antibacterial activity against aggregatibacter actinomycetemcomitans: a comparison between antibiofilm and antiplanktonic effects. int j dent. 2013;2013:164267. doi: 10.1155/2013/16426. 12. ajay rao ht, bhat ss, hegde s, jhamb v. efficacy of garlic extract and chlorhexidine mouthwash in reduction of oral salivary microorganisms, an in vitro study. anc sci life. 2014;34(2):85-8. doi: 10.4103/0257-7941.153465. 13. ros-llor i, lopez-jornet p. cytogenetic analysis of oral mucosa cells, induced by chlorhexidine, essential oils in ethanolic solution and triclosan mouthwashes. environ res. 2014 jul;132:140-5. doi: 10.1016/j.envres.2014.03.032. 14. gunsolley jc. a meta-analysis of six-month studies of antiplaque and antigingivitis agents. j am dent assoc. 2006 dec;137(12):1649-57. doi: 10.14219/jada.archive.2006.0110. 15. stoeken je, paraskevas s, van der weijden ga. the long-term effect of a mouthrinse containing essential oils on dental plaque and gingivitis: a systematic review. j periodontol. 2007 jul;78(7):1218-28. doi: 10.1902/jop.2007.060269. 16. sissons ch, wong l, cutress tw. inhibition by ethanol of the growth of biofilm and dispersed microcosm dental plaques. arch oral biol. 1996 jan;41(1):27-34. doi: 10.1016/0003-9969(95)00103-4. 17. marchetti e, tecco s, caterini e, casalena f, quinzi v, mattei a , et al. alcohol-free essential oils containing mouthrinse efficacy on three-day supragingival plaque regrowth: a randomized crossover clinical trial. trials. 2017 mar 31;18(1):154. doi: 10.1186/s13063-017-1901-z. 18. simón-soro a, tomás i, cabrera-rubio r, catalan md, nyvad b, mira a. microbial geography of the oral cavity. j dent res. 2013 jul;92(7):616-21. doi: 10.1177/0022034513488119. 19. miller md, ferris dg. measurement of subjective phenomena in primary care research: the visual analogue scale. fam pract res j. 1993;13(1):15-24. 20. kimberlin cl, winterstein ag. validity and reliability of measurement instruments used in research. am j health syst pharm. 2008 dec;65(23):2276-84. doi: 10.2146/ajhp070364. 21. sennhenn-kirchner s, wolff n, klaue s, mergeryan h, borg-von zepelin m. decontamination efficacy of antiseptic agents on in vivo grown biofilms on rough titanium surfaces. quintessence int. 2009;40(10):e80-8. 22. marchetti e, casalena f, capestro a, et al. efficacy of two mouthwashes on 3-day supragingival plaque regrowth: a randomized crossover clinical trial. int j dent hyg. 2017 feb;15(1):73-80. doi: 10.1111/idh.12185. 23. kim kj, komabayashi t, moon se, goo km, okada m, kawamura m. oral health attitudes/behavior and gingival self-care level of korean dental hygiene students. j oral sci. 2001 mar;43(1):49-53. doi: 10.2334/josnusd.43.49. 1 volume 22 2023 e239183 original article braz j oral sci. 2023;22:e239183http://dx.doi.org/10.20396/bjos.v22i00.8669183 1 department of oral and maxillofacial surgery, faculty of dentistry, damascus university, damascus, syria. 2 department of prosthodontics, faculty of dentistry, syrian private university, damascus, syria. corresponding author: rami shurbaji mozayek department of prosthodontics, faculty of dentistry, syrian private university. damascus, syria. email: ramishm88@gmail.com editor: valentim a. r. barão received: may 06, 2022 accepted: sep 22, 2022 success and survival rates of immediate anatomic zirconia implants: a prospective clinical and radiographic evaluation mohammed yamen al-shorbaji al-moziek1 , issam alkhouri1 , rami shurbaji mozayek2* modern immediate titanium implants have two major drawbacks which are the black metal appearance that might be seen through the mucosa and the gap between implant and extraction socket. immediate anatomical zirconia implants were introduced to match the shape of the extracted root and fill the socket without gaps while still providing better metal-free appearance. aim: this study aims to investigate success and survival rates of immediate anatomical zirconia implants. methods: this prospective interventional study was held between 2017 and 2020 in the faculty of dental medicine, damascus university, syria. the sample consisted of 27 immediate anatomical zirconia implants in 21 patients from both genders. implants were designed and manufactured starting from cbct image and prior to extraction. specialized software applications were used to modify implant design. implants went through different processing procedures to make them ready for insertion immediately after tooth extraction. restorations were made after a minimum period of 3 months, clinical and radiographic follow ups were performed after 10 13.5 months from restoring the implants in order to evaluate their success/ survival. repeated measures anova was used to assess marginal bone loss, t test for probing depth assessment. results: immediate anatomical zirconia implants showed success in (n=17) 63% of total cases, satisfactory survival (n=3) 11.1%, compromised survival (n=2) 7.4% and they failed in (n=5) 18.5%. conclusions: immediate anatomical zirconia implants had low success/survival rates when compared to conventional immediate implants. therefore, they cannot be considered as a predictable alternative in their current form. keywords: dental implants. tooth extraction. tooth root. https://orcid.org/0000-0001-9669-2643 https://orcid.org/0000-0003-1957-6651 https://orcid.org/0000-0002-8399-4025 2 al-moziek et al. braz j oral sci. 2023;22:e239183 introduction dental implants are the most favorable choice for replacing missing teeth since they achieve high success rate (90-100%)1. however, it was preferred to wait 6-9 months after tooth extraction to have a complete healing before the insertion of dental implants which was known as late implant placement, this period is becoming shorter with more advancement in dental materials and surface treatment methods and nowadays implant insertion can be done after 2-3 months of the extraction, that was called early implant placement2,3. recent studies showed that dental implants can be inserted in the same day of extraction in carefully selected cases (immediate implant placement)4. after clinical and radiographic follow-up, immediate implantation showed similar results and success rate compared to late and early implantation2,3. on the other hand, screw shaped immediate implants have the disadvantage of mismatching the alveolar socket which leads to gap formation that needs to be filled with bone graft to prevent epithelial and connective tissues growth toward this gap space especially when the distance between alveolar crest and implant neck is more than 2mm5. in 1969, hodosh was the first to try solving this mismatching problem in immediate implants by using custom made implants that matches the extracted root, this technique reduced bone and soft tissue trauma. but since pmma (poly methyl methacrylate) was used to make the implants osseointegration could not be achieved but rather a soft-tissue capsule was formed resulting in implant failure6. in 1992, titanium was used instead of pmma for making implants in the same previous technique and osseointegration was achieved in 88%7. in 2001 zirconia rootshaped implants were introduced and 100% primary stability in the first month was obtained but due to high failure rate in 12months follow-up these implants were not recommended for clinical use until more modifications were made and clinical evidence for stability and osseointegration was confirmed8,9. pirker and kocher added proximal macro-retentions for the root-shaped zirconia immediate implants. this addition increased the survival rate to 92% in 12 months follow-up period and achieved excellent aesthetic and functional aspects with minimal bone resorption and gingival recession4. literature has so few studies regarding zirconia root-shaped immediate implants and most of them are just case reports6-8. more studies are needed to confirm this technique as an alternative treatment plan. thus, this study aims to: investigating success and survival rates of immediate anatomical zirconia implants materials and methods a prospective interventional study was conducted between september 2017 and july 2020 at the department of oral and maxillofacial surgery, faculty of dental medicine, damascus university, syria. this study was approved by the ethics committee of damascus university (scientific research council decision no.940 date 30/1/2017) and an informed consent was signed by every participant. 3 al-moziek et al. braz j oral sci. 2023;22:e239183 27 immediate anatomical zirconia implants were inserted for 21 patients with indication for one or more dental extraction (3 patients received 2 implants each, one patient received 4 implants and 17 patients received on implant each). 5 implants were placed in the anterior region of the maxilla, 3 implants were placed in the anterior region of the while 12 implants were placed in the premolars region of the maxilla, 7 implants were placed in the premolars region of the mandible; sample size was calculated using g-power software with significance level 0.05 and effect size 0.76310. 17 implants were inserted in the maxillary arch and 10 implants in the mandible. these patients fulfilled the following eligibility criteria: eligibility criteria patients with age over 18 years with clear indication for extraction such as (unrestorable tooth, deep root caries, and longitudinal fracture), having normal position of the tooth that needs to be extracted with natural opposing dentition and no traumatic occlusion were included. the integrity of the surrounding alveolar bone was checked with no acute infection in the surgical site and good oral hygiene was included. no systemic diseases or conditions preventing surgical procedures such as diabetes, pregnancy and chemotherapy were present. alcoholic, heavy smokers (more than 20 cigarettes per day) and teeth with irregular root shape were excluded. after thorough clinical examination, cbct was obtained to evaluate tooth dimensions then designing a 3d model of the anatomical implant by using several software programs: mimics® (materialise’s interactive medical image control system) (materialise n.v., leuven, belgium), 3-matic® v13.0 (materialise n.v., leuven, belgium), autodesk meshmixer v3.5. 0.5mm macro retentions were added on proximal surfaces, bucco-lingual dimension was reduced by 0.1-0.2 mm whereas the coronal part was modified as a prepared abutment with shoulder finishing line (fig. 1). 4 al-moziek et al. braz j oral sci. 2023;22:e239183 figure 1. designing the 3d model of the anatomical implant. 5 al-moziek et al. braz j oral sci. 2023;22:e239183 with the help of a cad-cam system, zirconia (y-tzb) implants were manufactured according to the designed models. implant root surface was sandblasted with 50µ aluminum oxide powder for 0.5 second under pressure of 5 bar11. then implant was put in a furnace in 1500ºc for 8 hours to complete zirconia sintering followed with 99% ethanol bath in ultrasonic cleaning device for 10 minutes and another 10 minutes with distilled water. after that implant root was submerged in 70% hydrofluoric acid solution for 24 hours in room temperature to have surface micro-roughness12, then it was returned to the ultrasonic ethanol and distilled water baths for 10 minutes each to completely clean the implant surface from any residual contaminants. the implant was packaged and sterilized with gamma radiation (2.5 rad)13. surgical stage oral cavity was disinfected with chlorhexidine (chx) mouthwash, then atraumatic extraction with suitable elevators and forceps was performed. immediate anatomical zirconia implant was inserted with finger pressure or by gentle taps on a surgical mallet when needed. primary stability was evaluated with palpation and percussion, and radiographic evaluation with cbct was done immediately after surgery. prosthetic stage was initiated after at least 3 months and zirconia restoration was cemented (fig. 2). follow up clinical and radiographic follow-ups were made according to the following time table: pre-surgery stage, immediately after surgery (t0), prosthetic stage (t1): 3 4.5 months after t0, follow up stage (t2): 10 13.5 months after t1. radiographical settings were field of view (fov) 5x5 cm, voxel size 0.3mm, 85kv, 15ma and exposure time 9 seconds; radiographical follow-ups included the assessment of vertical marginal bone loss around implant in t1 and t2 (fig. 3). 6 al-moziek et al. braz j oral sci. 2023;22:e239183 a b c d e f figure 2. clinical stages for placing immediate anatomical zirconia implant. (a) before tooth extraction (b) immediately after extraction (c) natural extracted tooth and correspondent immediate anatomical zirconia implant (d) inserting the immediate anatomical zirconia implant into the socket. (e) implant inside alveolar socket (f) after definitive restoration cementation. clinical follow-ups included the evaluation of the following implant success/survival was evaluated according to the international conference of oral implantologists held in italy in 2007 (table 1)14, probing depth (fig. 4) was measured in 4 sides and the average was calculated for every stage then the differences between averages were assessed, percussion test in t1 and t2 (as positive percussion is the unique crystal sound indicating rigid fixation or osseointegration)15,16, clinical mobility was assessed by palpation with blunt end instrument in t1 and t2, pain on pressure in t1 and t2, implant success and survival in t1 and t2. statistical analysis repeated measures anova was used to assess marginal bone loss, t test for probing depth assessment. p-value <0.05 was considered statistically significant. statistical analysis was carried out by spss v.25 software. 7 al-moziek et al. braz j oral sci. 2023;22:e239183 a b c d e f figure 3. vertical marginal bone loss around immediate anatomical zirconia implant (a,b) bone measurement in t0 (c,d) bone measurement in t1 (e,f) bone measurement in t2. table 1. implant quality scale i. success (optimum health) a) no pain or tenderness upon function b) 0 mobility c) less than 2 mm radiographic bone loss from initial surgery d) no exudates history ii. satisfactory survival a) no pain on function b) 0 mobility c) 2–4 mm radiographic bone loss d) no exudates history iii. compromised survival a) may have sensitivity on function b) no mobility c) radiographic bone loss more than 4 mm (less than 1/2 of implant body) d) probing depth more than 7 mm e) may have exudates history iv. failure (clinical or absolute failure) any of following: a) pain on function b) mobility c) radiographic bone loss more than 1/2 length of implant d) uncontrolled exudate e) no longer in mouth 8 al-moziek et al. braz j oral sci. 2023;22:e239183 figure 4. probing depth. results sample consisted of 27 immediate anatomical zirconia implants inserted for 21 patients [(n=7) 33.3% males and (n=14) 66.7% females] aged between (21-55 years), 17 implants were inserted in the maxilla and 10 in the mandible, and their total length ranged between 13.1 20mm. at t1, (n=23) 85.2% implants survived and (n=4) 14.8% of the implants failed which were excluded from further statistical study. the implants were considered successful in (n=21) 77.8% of cases, satisfactory survival in (n=2) 7.4% and compromised survival in (n=0) 0%. at t2, (n=22) 95.65% implants survived and (n=1) 4.35% of the implants failed. the implants were considered successful in (n=17) 73.91% of cases, satisfactory survival in (n=3) 13.04% (3 implants showed vertical bone resorption more than 2 mm) and compromised survival in (n=2) 8.7% (2 implants showed vertical bone resorption more than 4mm with bleeding index of 2,3). in 3 of the failed cases the patients stated that they were having hard food when they first felt mobility in their implants whereas for the remaining 2 implants the patients stated that they started to feel an increasing mobility till the failure occurred. follow-up results of implants success/failure results are shown in (table/fig. 2). 9 al-moziek et al. braz j oral sci. 2023;22:e239183 table 2. count and percentage for implants success/ failure count percentage osseointegration 22 81.5% success 17 63.0% satisfactory survival 3 11.1% compromised survival 2 7.4% failure 5 18.5% before restoration 4 14.8% after restoration 1 3.7% total 27 100% the average vertical bone loss was 0.70±0.61 mm between t0 t1, 0.68±0.58 mm between t1 t2 and in total 1.38±1.19 mm between t0 t2, the results were statistically significant for the difference in vertical bone averages in studied time groups (p<0.05) (table 3). table 3. repeated-measures anova for average vertical bone loss side mean std. deviation minimum maximum 95% confidence interval for mean p value lower bound upper bound t0 – t1 mesial 0.68 0.61 0.20 2.40 0.41 0.95 distal 0.72 0.66 0.10 2.50 0.43 1.01 buccal 0.75 0.64 0.00 2.50 0.47 1.03 lingual 0.65 0.60 0.00 2.50 0.38 0.92 mean 0.70 0.61 0.08 2.45 0.43 0.97 0.000284 t1 – t2 mesial 0.72 0.62 0.10 2.30 0.45 1.00 distal 0.70 0.63 0.20 2.40 0.43 0.98 buccal 0.67 0.59 0.10 2.20 0.41 0.93 lingual 0.62 0.54 0.10 2.00 0.38 0.86 mean 0.68 0.58 0.18 2.20 0.42 0.94 0.000056 t0 – t2 mesial 1.40 1.20 0.50 4.60 0.87 1.94 distal 1.42 1.24 0.40 4.90 0.87 1.97 buccal 1.42 1.15 0.10 4.60 0.91 1.93 lingual 1.27 1.09 0.10 4.50 0.79 1.75 mean 1.38 1.15 0.30 4.65 0.87 1.89 0.000035 repeated-measures anova test: p < 0.0005 average probing depth was 2.19±1.10 mm in t1 and 2.55±1.22 mm in t2 with statistical significance (p<0.05) (table 4). 10 al-moziek et al. braz j oral sci. 2023;22:e239183 table 4. t test for probing depth difference between t1 and t2 t value p value mean difference std. error difference 95% confidence interval of the difference lower upper -4.247 0.0001 -0.46 0.31 -0.87 -0.30 percussion test results were positive in (n=23) 85.2% of cases and negative in (n=4) 14.8% in t1. in t2 the four failed implants were not included in statistical calculations so the total number of studied implants in t2 was 23 implants, (n=22) 95.65% of the remaining surviving implants were positive and (n=1) 4.3% were negative. mobility was recorded in (n=4) 14.8% of implants in t1 and in (n=1) 4.3% of the surviving implants in t2. pain on pressure was observed in (n=4) 14.8% of implants in t1 and (n=1) 4.3% of the surviving implants in t2. follow up results are shown in (table 5). table 5. follow-ups results t1 t2 average vertical bone loss 0.70±0.61 mm 0.68±0.58 mm average probing depth 2.19±1.10 mm 2.55±1.22 mm positive percussion test (n= 23) 85.2% (n=22) 95.7% mobility (n= 4) 14.8% (n= 1) 4.3% pain on pressure (n= 4) 14.8% (n= 1) 4.3% discussion using immediate anatomical implants eliminate the gap formation between implant and alveolar socket so there will be no need for using bone grafts4. besides, using zirconia implants enhances aesthetic aspects especially in the anterior region and reduces plaque accumulation with less inflammation in the surrounding soft tissue. in addition, zirconia possesses high biocompatibility and mechanical properties which suits dental implants4,17-18. the method described in this study for immediate anatomical zirconia implants introduced the advantage of having anatomical implants prior to extraction so they can be applied to the fresh socket in the same appointment unlike what was used in previous studies for immediate zirconia anatomical implants where laser scanning was done to the extracted tooth and then the implant was manufactured, this procedure usually takes 4-7 days and will increase the chance of failure due to fibrous tissue formation around implant instead of osseointegration4,8,19,20. proximal protrusions were added to the implant design to play the role of macro-retentions which provided more primary stability by engaging to the bone of the proximal spaces, also bucco-lingual dimension was reduced by 0.1-0.2 mm to protect the thin 11 al-moziek et al. braz j oral sci. 2023;22:e239183 buccal plate from fracturing while inserting the implant and to prevent bone resorption due to implant pressure on buccal bone4. in this study, the immediate anatomical zirconia implant success, survival and failure rates after one year follow-up were evaluated in accordance with the classification that describes implant condition from the consensus conference of the international congress of oral implantologists held in pisa, italy, 200714. percussion test is one of the simplest tests that can be done to evaluate osseointegration of dental implants. however, this test is considered subjective and depends mainly on the practitioner’s expertise and cannot be solely relied on, thus this study also used pain on pressure and clinical implant mobility as indices to determine osseointegration in addition to percussion test15,16. 4 out of 27 of the immediate anatomical zirconia implants (14.8%) showed no resonant (crystal) sound on percussion and some pain when applying finger pressure on their abutments in addition to having clinical mobility in the pre-prosthetic stage between t0 -t1, these implants were considered failure. one more of the remaining implants showed no resonant (crystal) sound, pain on mastication and clinical mobility at follow-up in t2 and was also considered failure; this increased the total failed implants to 18.5%. the negative percussion test result, presence of pain and clinical mobility were present altogether in all failing cases and absent when osseointegration is observed, this is consistent with what pirker and kocher stated4. pain on percussion was found only in the cases of failed implants and it was associated with clinical mobility and dull percussion sound, this was also consistent with what pirker and kocher stated4. probing depth mean increased from t1 to t2 by 0.36mm with statistical significance, that was consistent with what pirker and kocher stated about soft tissue response in their study of immediate anatomical zirconia implants (soft tissue retraction ranged from 0–1.5 mm (0.5±0.7mm)4. in the current study, survival rate in t2 was 81.5%. in other studies regarding immediate screw-shaped titanium implants survival rate ranged between 94.6 – 96.9% which was higher than the result for immediate anatomical zirconia implants discussed in this article21-23. the survival rate was 100 % for anatomical implants made with direct laser metal sintering technique (dlms)10, 94.4% for anatomical hybrid implants (replicate system/ ndi) with titanium root and zirconia abutment24. thus zirconia anatomical implants as they were described in this study are still less predictable than other techniques and materials in immediate implantation. the total failure rate was 18.5%, 14.8% were before prosthetic stage and 3.7% after. failing in early stage was also stated in other immediate implantation studies4,24,25 [table/figure 10] and it can be explained with: not achieving enough primary stability to withstand the immediate load on implants, trauma caused by having hard food before osseointegration, failure to achieve osseointegration because of zirconia implants surface treatment. all failed implants were removed and sockets were curetted and washed with normal saline, no inflammatory signs or other changes were seen in the sockets. this could be due to zirconia high biocompatibility. 12 al-moziek et al. braz j oral sci. 2023;22:e239183 the limitations of this study were: bone density around implants could not be assessed in cbct images due to metal artifact around zirconia. primary stability could not be measured using resonance frequency analysis because this technique uses a transducer that connects firmly to implants or abutments and that transducer is not available for custom made implants. in conclusion, immediate anatomical zirconia implants as they were described in this study showed low survival and cannot be considered a predictable treatment plan. also the increase of early stage failure in immediate anatomical zirconia implants can be explained with not achieving adequate primary stability for the implant. this type of implants has strict indications and application criteria and still needs more research. data availability datasets related to this article will be available upon request to the corresponding author. conflict of interests none. author contribution study conception and design: mohammed yamen al-shorbaji al-moziek, issam alkhouri, rami shurbaji mozayek. data collection: mohammed yamen al-shorbaji al-moziek. analysis and interpretation of results: mohammed yamen al-shorbaji al-moziek, issam alkhouri, rami shurbaji mozayek. draft manuscript preparation: mohammed yamen al-shorbaji al-moziek, rami shurbaji mozayek. all authors actively participated in the discussion of the manuscript’s findings, and have revised and approved the final version of the manuscript. references 1. telleman g, meijer hj, raghoebar gm. long-term evaluation of hollow screw and hollow cylinder dental implants: clinical and radiographic results after 10 years. j periodontol. 2006 feb;77(2):203-10. doi: 10.1902/jop.2006.040346. 2. chen st, buser d. clinical and esthetic outcomes of implants placed in postextraction sites. int j oral maxillofac implants. 2009;24 suppl:186-217. 3. schropp l, wenzel a, spin-neto r, stavropoulos a. fate of the buccal bone at implants placed early, delayed, or late after tooth extraction analyzed by cone beam ct: 10-year results from a randomized, controlled, clinical study. clin oral implants res. 2015 may;26(5):492-500. doi: 10.1111/clr.12424. 4. pirker w, kocher a. immediate, non-submerged, root-analogue zirconia implants placed into single-rooted extraction sockets: 2-year follow-up of a clinical study. int j 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in humans: a comparison of 2 different rough surfaces. int j oral maxillofac implants. 2007 may-jun;22(3):430-5. 1http://dx.doi.org/10.20396/bjos.v20i00.8660624 volume 20 2021 e210624 original article 1 department of forensic dentistry, piracicaba dental school, university of campinas, são paulo, brazil. 2 forensic expert, police department of federal district, brazil. 3 national museum, federal university of rio de janeiro, rio de janeiro, brazil. 4 forensic expert, police department of minas gerais, brazil. 5 department of forensic dentistry, university of são paulo, brazil. 6 department of forensic dentistry, federal university of paraíba, brazil. corresponding author: rachel tinoco email: rachelrtinoco@gmail.com received: july 25, 2020 accepted: january 22, 2021 sex dimorphism according to the nasozygomatic triangle fábio delwing1,2 , rachel lima ribeiro tinoco3,* , geraldo elias miranda1,4,5 , laíse nascimento correia lima1,6 , luiz francesquini júnior1 , eduardo daruge júnior1 sex is one of the first features to be diagnosed in human identification, composing, with age, ancestry and stature, the so called “big four”. aim: the present study aimed to metrically analyze the sexual dimorphism in skulls of known age and sex from rio grande do sul – brazil. methods: this was a crosssectional study of metrical analysis, which used a sample comprised of 209 human skulls (106 male and 103 female) older than 22 years old at the time of death, undamaged and without signs of trauma or abnormalities. the point nasion and the most superior points on the zygomaticotemporal sutures from each side were connected forming a triangle. this area was calculated using heron’s formula, and the results were submitted for statistical analysis. results: all measurements showed significant values for sexual dimorphism. through the area of the triangle, it was possible to determine sex with an accuracy of 83.97% for males and 83.50% for females. conclusion: this simple method requires only a caliper, and still can be reliable for forensic human identification. it must be diffused and tested on other samples, and can be used as a good and inexpensive tool for experts in day-to-day practice. keywords: forensic anthropology. sex determination by skeleton. sex characteristics. skull. mailto:rachelrtinoco@gmail.com https://orcid.org/0000-0002-9003-2350 https://orcid.org/0000-0003-3043-0661 http://orcid.org/0000-0003-1240-3256 https://orcid.org/0000-0002-1773-847x https://orcid.org/0000-0002-6344-3488 https://orcid.org/0000-0001-6565-3203 2 delwing et al. introduction historically, human identification is one of the biggest challenges faced by forensic science. the existence of sexual dimorphism in human skeletons and its importance in investigative methods has long been recognized. krogman and íscan1 asserted that sex assessment was possible, with levels of reliability of 100% when the entire skeleton is present, 92% using the skull alone, and 98% when combining pelvis and skull. together with pelvic bones, the skull remains among the most dimorphic segments of the skeleton, although this determination has its reliability totally established only after puberty2,3. morphological analysis, being an even faster process, brings a high degree of subjectivity, decreasing its reliability. for this reason, metric techniques, being intrinsically more objective, can offer a better data achievement, with less variability between experts1,4,5. craniofacial structures have the advantage of being composed largely of hard tissue with a higher resistance to decomposition6, allowing their analysis even after mass disasters, or other forms of violence. krogman and iscan1 and meindl et al.7 have stressed that anatomical variations are population-dependent, and any method for human identification should be tested and validated in the target population, prior to being used. patil and mody8 claim that large and robust skulls tend to be male, and delicate skulls tend to be female. to kranioti et al. 9, the males are statistically larger in all their dimensions in relation to females. several authors concluded in their research, among different craniometric points analyzed, that the bizygomatic distance exhibited a high degree of sexual dimorphism9-11. the purpose of this study was to evaluate the accuracy of a new sexing method using the area of the triangle formed from the measurements of three craniometrics points in the upper face of skulls. materials and methods this research was conducted in the city of porto alegre, rio grande do sul brazil, after being approved by the ethics committee of the faculty of dentistry of piracicaba, unicamp, são paulo – brazil, approval number 138/2010. the sample consisted of 209 skulls (106 males and 103 females) selected by convenience during six months of data collection, according to the routine exhumations of the cemetery. individuals from 22 years of age or older at time of death were included, to ensure full development of the skull and end of facial growth. exclusion criteria were any kind of trauma, visible anomalies, or post-mortem damage, like bone breaking or cracking during exumation, that could interfere with the measurements taken. the possibility of edentulism was not an exclusion criterion, since the presence or absence of teeth does not influence the used measures. sex verification was ensured by the burial records and codified to allow a blind analysis. 3 delwing et al. the metric analysis of the skulls was performed by a single examiner previously calibrated after measuring 50 skulls, with a digital caliper (mitutoyo, são paulo – brazil), and registered in an excel sheet. the data consisted of the measurements between nasion (point n) and the most superior point of the zygomatic-temporal suture on both sides – which was called, for this study, zygomatic-temporal point (point zt). table 1 shows the measurement abbreviations and definitions, and figure 1 shows their representations on the skull. table 1. abbreviation and definition of the measures taken measure definition n – zt.r from nasion to the most superior point of the zygomatic-temporal suture on the right side n – zt.l from nasion to the most superior point of the zygomatic-temporal suture on the left side zt.r – zt.l between the most superior point of the zygomatic-temporal suture from right to left sides area a b figure 1. measure taken from nasion to point zt.l (a); and graphic representation of the triangle formed by the three points (b). after the three measurements had been taken, the triangle formed by their connection (figure 1) had its area calculated by heron´s formula12. this is used in plane geometry to determine the area of a triangle when only the length of the sides a, b and c are known, as shown below: triangle area = s (s a)(s b)(s c), in which s = a + b + c2 the area of the proposed nasozygomatic triangle can only be calculated by this formula, since the triangle has its base backwards from its apex, making it an angled figure relative to the anatomic coronal plane. the measurements taken were subjected to student t-test (p <0.001) for assessment of sexual dimorphism. in order to test for intra-examiner error, the skulls of 20% of the sample were randomly selected, including both sexes, and were analyzed twice, within a two week interval. the two sets of values for these individuals were compared by means of a paired students´t-test, showing non statistically significant difference (p>0.05). 4 delwing et al. results for the three measurements taken from the sample (209 skulls), and the triangle area calculated by their connection, sexual dimorphism was statistically significant (p<0.001) in all values, as shown in table 2. on analyzing the ratio between the male and female average of the measurements and triangle area, the male average was always higher than the female, thereby showing relevant sexual dimorphism of this anatomic triangle, as shown in table 3. table 2. student t-test with average male and female and their minimum and maximum limits male female mean (sd) min max mean (sd) min max p n – zt.r (mm) 76.67 (4.207) 75.866 77.487 71.546 (2.896) 70.988 72.104 <0.001* n – zt.l (mm) 76.857 (4.395) 76.011 77.704 71.606 (2.861) 71.054 72.157 <0.001* zt.r – zt.l (mm) 117.998 (5.368) 116.964 119.031 111.117 (4.344) 110.280 111.954 <0.001* area (mm2) 2,896.006 (338.470) 2,830.821 2,961.191 2,503.607 (200.644) 2,464.952 2,542.262 <0.001* * all significant at p<0.001 table 3. quotient between male and female average quotient % interpretation n – zt.r 1.072 7.20 average male 7.2% higher than female n – zt.l 1.073 7.30 average male 7.3% higher than female zt.r – zt.l 1.062 6.20 average male 6.2% higher than female triangle area 1.157 15.7 average male 15.7% higher than female figure 2 shows a diagram with the range of the triangle area for males and females. to obtain it, the average area of the triangle for each sex and their respective standard deviation was used. from that, it can be said that if the triangle area value is lower than 2,558mm2, the skull is female; if the value is greater than 2,703mm2, it is male. however, if the value is between 2,558mm2 and 2,703mm2, the method does not contribute to sex diagnosis, since this range was found to be doubtful. skulls with nasozygomatic triangle area greater than 3,234mm2 were considered hypermales, and those with area below 2,303mm2 were considered hyperfemales, as can be seen in figure 2. female (14m, 47f) ambiguous (15m, 22f) 2,303 2,558 2,703 3,234 male (71m, 17f) figure 2. sex diagnosis according to the nasozigomatic triangle area (in mm2) 5 delwing et al. for skulls with this triangle area higher than 2,703mm2, or lower than 2,558mm2, this sexing method has a reliability of 83.97% for males and 83.50% for females, respectively, as shown in table 4. intra-observer error showed non-significant values between the two groups of measures (p=0.773 for n – zt.r; p=0.100 for n – zt.l; and p=0.266 for zt.r – zt.l). table 4. sex determination accuracy and area values for male and female area (mm2) sex accuracy > 2,703 male 83.97% <2,558 female 83.50% discussion forensic anthropologists are continually challenged by the human identification issue, and develop new methods or improve the accuracy of existing ones, applied on various parts of the skeleton so that the method can be admissible in court10,13,14. it is known that anthropological research is more susceptible to errors when purely morphological criteria are considered, due to phenotypic variations, pathological signs and even according to the observer, making the analysis undesirably subjective. thus, a quantitative method, for its objectivity and reproducibility by any researcher and expert, can join to the group of procedures for human identification, with accuracy and reliability5,8. in a forensic anthropological analysis of a skeleton, with identification as its primary aim, sex is one of the first and most important piece of information to be obtained, being part of the so called “big four”, together with age, stature, and ancestry15-17. human bones have low sexual dimorphism, if compared with other primates18; still, the most dimorphic parts of the skeleton is the pelvis, followed by the skull. therefore, whenever the pelvis is not available, sexing methods must be based on cranial anatomy1,5. the percentage of correct answers regarding sex diagnose based exclusively on skull features range from 70.56% to 92%7,19. results obtained in this study, using the area of the proposed nasozygomatic triangle (in mm2) reached a reliability of 83.97% for males and 83.50% for females. before puberty, sexual characteristics are not very pronounced; it is only after this period, under hormonal influence, environment and muscles, that the human skeleton begins to show sexual dimorphism. due to this, in this study, as inclusion criteria, only skulls of individuals aged from 22 years old and up at time of death were analyzed. mainly due to the variation between groups of different ancestries, the methods of identification in forensic anthropology must be regionalized and validated for specific populations. this is the reason why previous studies should test and find the reliability of a given method in their respective target population, so that it can be used for human identification5,7. craniometric traits show a level of regional differentiation comparable to genetic markers, with high levels of variation within populations as 6 delwing et al. well as a correlation between phenotypic expression and geographic distance, which allows high levels of classification reliability when comparing skulls from different parts of the world20. the brazilian population has a high degree of biological variation. this is due to the hybridization of the amerindians as first settlers with european and sub-saharan groups, after colonization occurred in the 16th century, which was studied by ross et al.21, and stressed by urbanová et al.22. this showed higher misclassification of sex and ancestry for the afro-brazilian sample, according to software tools. it also must be noted that genetic marker studies23,24, showed a regional variation according to the mtdna lineages, with high european influence in the southern region, where this study was performed. the use of craniometric methodology published in tables and indexes from studies of foreign authors or other regions of brazil must be cautious, and craniometric variations for any method should be validated, for forensic purposes. large and robust skulls tend to be male, and delicate skulls tend to be female8. as known from anthropological studies, males skulls are statistically higher in all their dimensions in relation to females9. this study also observed the preponderance of all measures in males. the average area of the nasozygomatic triangle in males was 15.7% higher than females. among the measurements, bizygomatic width was classified as the most dimorphic measure for sex diagnosis9,10. after this, measurements of the upper portion of the facial skull have also been shown as sexually dimorphic11. this study used craniometric measurements of the upper face in search of this dimorphism reported in literature, and applied a new method to examine an old issue in forensic anthropology: sexual diagnosing by use of the skull. the use of techniques such as computerized tomography25, 3d graphics26, and morphometric geometry27 can be very helpful, but if a simple method that requests only a caliper and can still be reliable for forensic human identification, it must be diffused and tested on other samples. the accuracy of the cutoff points showed that the method can be used as a good and inexpensive tool for experts in day-to-day practice, dealing with unidentified individuals. however, the predictive values (sensitivity, specificity, positive predictive value, negative predictive value, and likelihood ratio) were not calculated, and can be investigated by future researchers. in skulls that show results between 2,558mm2 and 2,703mm2 the method does not contribute to sexual diagnosis, which was considered as a limitation, as well as skulls younger than 22 years old, not included in the sample. future projects should also consider the possibility of adapting the method for children and young adults. to conclude, the proposed method uses points which are easily identifiable and of rapid measurement, with which experts can diagnose sex when only the skull is available. if the value found of the nasozygomatic triangle area is within the doubtful range, nothing can be said about sex by the proposed technique; however, if this value is not between 2,558mm2 and 2,703mm2, sex information can be achieved with a reliability of 83.97% for males and 83.50% for females. 7 delwing et al. references 1. krogman w, iscan my. the human skeleton in forensic medicine. 2nd ed. springfield: charles c thomas; 1986. 2. christensen am, passalacqua nv, bartelink ej. sex estimation. in: christensen am, passalacqua nv, bartelink ej. forensic anthropology: current methods and practice. cambridge: academic press; 2019. p.243-70. doi: 10.1016/b978-0-12-815734-3.00008-7. 3. klales ar, editor. sex estimation of the human skeleton: history, methods, and emerging techniques. cambridge: academic press; 2020. 4. walrath de, turner p, bruzek j. reliability test of the visual assessment of cranial traits for sex determination. am j phys anthropol. 2004;125(2):132-7. doi: 10.1002/ajpa.10373. 5. i̇şcan my. forensic anthropology of sex and body size. forensic sci int. 2005;147(2-3):107-12. doi.org/10.1016/j.forsciint.2004.09.069 6. latham ke, baterlink ej, finnegan m. new perspectives in forensic human skeletal identification. cambridge: academic press; 2017. 7. meindl rs, lovejoy co, mensforth rp, don carlos l. accuracy and direction of error in the sexing of the skeleton: implications for paleodemography. am j phys anthropol. 1985;68(1):79-85. doi.org/10.1002/ajpa.1330680108. 8. patil kr, mody rn. determination of sex by discriminant function analysis and stature by regression analysis: a lateral cephalometric study. forensic sci int. 2005;147(2-3):175-80. doi.org/10.1016/j.forsciint.2004.09.071. 9. kranioti ef, işcan my, michalodimitrakis m. craniometric analysis of the modern cretan population. forensic sci int. 2008;180(2-3):110.e1-5. doi.org/10.1016/ j.forsciint.2008.06.018. 10. steyn m, işcan my. sexual dimorphism in the crania and mandibles of south african whites. forensic sci int. 1998;98(1-2):9-16. doi.org/10.1016/s0379-0738(98)00120-0. 11. naikmasur vg, shrivastava r, mutalik s. determination of sex in south indians and immigrant tibetans from cephalometric analysis and discriminant functions. forensic sci int. 2010;197(1-3):122.e1-6. doi.org/10.1016/j.forsciint.2009.12.052. 12. pappas t. heron’s theorem. in: pappas t. the joy of mathematics. san carlos: wide world publishing/tetra; 1989. p.62. 13. rogers tl. determining the sex of human remains through cranial morphology. j forensic sci. 2005;50(3):493-500. 14. williams ba, rogers t. evaluating the accuracy and precision of cranial morphological traits for sex determination. j forensic sci. 2006;51(4):729-35. doi: 10.1111/j.1556-4029.2006.00177.x. 15. gill gw. craniofacial criteria in the skeletal attribution of race. in: reichs kj, editor. forensic osteology: advances in the identification of human remains. 2nd ed. springfield: charles c thomas; 1998. p.293-315. 16. white td, black mt, folkens pa. human osteology. san diego. academic press; 2011. 17. byers sn. introduction to forensic anthropology: a textbook. boston: allyn and bacon; 2002. 18. stanford c, allen js, antón sc. biological anthropology: the natural history of humankind. london: pearson education; 2016. 19. đurić m, rakočević z, đonić d. the reliability of sex determination of skeletons from forensic context in the balkans. forensic sci int. 2005;147(2-3):159-64. doi: 10.1016/j.forsciint.2004.09.111. 20. relethford jh. race and global patterns of phenotypic variation. am j phys anthropol. 2009;139(1):16-22. doi: 10.1002/ajpa.20900. 8 delwing et al. 21. ross ah, ubelaker dh, falsetti ab. craniometric variation in the americas. hum biol. 2002;74(6):807-18. doi: 10.1353/hub.2003.0010. 22. urbanová p, ross ah, jurda m, nogueira mi. testing the reliability of software tools in sex and ancestry estimation in a multi-ancestral brazilian sample. leg med tokyo. 2014;16(5):264-73. doi: 10.1016/j.legalmed.2014.06.002. 23. pena sdj. [retrato molecular do brasil]. cienc hoje. 2000;27(159):16-25. portuguese. 24. alves-silva j, da silva santos m, guimarães pem, ferreira ac, bandelt hj, pena sd, et al. the ancestry of brazilian mtdna lineages. am j hum genet. 2000;67(2):444-61. doi: 10.1086/303004. 25. gulhan o, harrison k, kiris a. a new computer-tomography-based method of sex estimation: development of turkish population-specific standards. forensic sci int. 2015;255:2-8. doi: 10.1016/j.forsciint.2015.07.015. 26. mediavilla er, pérez bp, gonzález el, sánchez jas, fernández ed, sáez as. determining sex with the clavicle in a contemporary spanish reference collection: a study on 3d images. forensic sci int. 2016;261:163.e1-163.e10. doi: 10.1016/j.forsciint.2016.01.029. 27. cavaignac e, savall f, faruch m, reina n, chiron p, telmon n. geometric morphometric analysis reveals sexual dimorphism in the distal femur. forensic sci int. 2016;259:246.e1-246.e5. doi: 10.1016/j.forsciint.2015.12.010. microsoft word annex 6 2 21 .docx 1http://dx.doi.org/10.20396/bjos.v20i00.8661717 volume 20 2021 e211717 original article 1 orthodontics program. unicieo university, bogotá, colombia. corresponding author: dr. judith patricia barrera chaparro, assistant professor, orthodontic department, unicieo university. cra. 5 #118-10, bogotá, colombia. e-mail: jp.barrera@unicieo.edu.co phone: (+57) 3002123863 received: october 27, 2020 accepted: january 13, 2021 modified short version of the oral health impact profile for patients undergoing orthodontic treatment judith patricia barrera-chaparro1,* , sonia patricia plaza-ruíz1 , tania camacho-usaquén1 , jairo andrés pasuy-caicedo1 , ada katherine villamizar-rivera1 aim: to derive and validate a short version of the oral health impact profile (ohip) in spanish to measure oral health quality of life (ohrqol) for subjects wearing fixed orthodontic appliances. methods: cross-sectional study (data for sensitivity to change analysis were collected longitudinally). the data of 400 subjects (27.34 years, sd 11.66 years, 231 women, and 169 men) were used to develop a short-form instrument, and the data of 126 other subjects (25.95 years, sd 12.39 years, 62 women, and 64 men) were used for its validation. the original ohips were translated into spanish using an iterative forward-backward sequence. after face and content validity were evaluated by an expert committee, an exploratory factorial analysis (efa) was used to derive the spanish short-form instrument (ohip-s14 ortho). to validate the ohip-s14 ortho, validity (content validity assessed by efa, construct validity assessed by confirmatory factor analysis (cfa), discriminative validity assessed by the kruskal-wallis test, and reliability (internal consistency assessed by cronbach’s α test-retest, and inter-observer reliability assessed by correlation coefficients) were evaluated. sensitivity to change and usefulness of the scale were also evaluated. results: the ohip-s14 ortho included only six of the items in slade´s original ohip-14 short-form. a two-factor structure with adequate discriminative validity was found. high internal consistency (α=0.912), excellent inter-observer (lin’s correlation=0.97±0.011; rho= 0.97), test-retest agreement (lin’s correlation=0.80±0.059) and adequate sensitivity to change were also found. conclusions: the ohip-s14 ortho is a valid and reliable instrument to measure ohrqol in spanish-speaking patients with fixed orthodontic appliances. keywords: quality of life. reproducibility of results. orthodontic appliances. https://orcid.org/0000-0002-6308-7241 https://orcid.org/0000-0002-4577-3096 https://orcid.org/0000-0002-7334-6606 https://orcid.org/0000-0002-3854-9205 https://orcid.org/0000-0002-9234-610x 2 barrera-chaparro et al. introduction oral health-related quality of life (ohrqol) is multidimensional and impacts the functional, psychological, and social aspects of daily life1. individuals seek orthodontic treatment mainly because they are dissatisfied with their appearance, dental malposition, deformity of the teeth, or spaces between them2; therefore, ohrqol after orthodontic treatment tends to improve3. however, wearing fixed orthodontic appliances can cause pain and difficulty with eating, speaking, or smiling, and the ohrqol seems to deteriorate during orthodontic treatment4,5. the most widely used instrument to measure ohrqol is the oral health impact profile (ohip), which was proposed by slade and spencer6. the long-form of this instrument has 49 items (ohip-49), the short form has 14 items (ohip-14), and the instrument covers seven dimensions (i.e., functional limitation, physical pain, psychological discomfort, physical disability, psychological disability, social disability, and handicap). a long questionnaire may not be feasible to administer in clinical settings because it has a high cost of administration and data provision, takes a long time to complete and score, and causes a burden on the respondent, which may lead to the exclusion of a substantial proportion of respondents or problems arising from the need to impute missing data7. because of respondents’ different cultural backgrounds, short forms of the ohip-49 have been derived and validated in different languages and countries8-13, and most of them were validated in an adult population with oral health or oral rehabilitation needs. although the world health organization (who)14 does not consider malocclusion an illness and most patients continue to have good oral health during orthodontic treatment, wearing fixed appliances may affect aspects of a patient’s quality of life in different ways when compared to patients who have caries, tooth loss, or periodontal disease. pain is a near pervasive unpleasant experience encountered during orthodontic treatment. numerous authors15-18 have linked orthodontic treatment to the experience of pain, finding that orthodontic patients are most likely to experience increased levels of pain for one to three days following the placement of their appliance and subsequent adjustment visits. to date, to the best of our knowledge, a short form of ohip has not been developed for patients with orthodontic treatment. for this reason, the present research study aims to derive and validate a short form of the ohip-49 (ohip-s14 ortho) in spanish to measure ohrqol in patients wearing orthodontic appliances. materials and methods this was a cross-sectional study. however, the data for sensitivity to change analysis was collected longitudinally. the study included individuals who were wearing fixed orthodontic appliances provided by unicieo university in bogotá, colombia, between january 2016 and november 2017. this study was approved by the unicieo university ethics committee. all participants and parents/guardians of minors signed the informed consent form. the research was conducted in full accordance with the world medical association declaration of helsinki. 3 barrera-chaparro et al. from 1,151 eligible subjects during the study period, 400 were chosen by non-probabilistic sampling (for convenience) for the ohip short version derivation. additionally, another 126 subjects were chosen for the ohip-s14 ortho validation. the sample size was estimated using the criterion recommended by streiner19, which recommends that 5 to 10 subjects per variable be included in the sample. for the reliability assessment, the calculation of the sample size for the intraclass correlation coefficient (icc) was considered to achieve an icc equal to or greater than 0.6, a confidence interval (ci) of 95% (α = 0.05) and 90% power was calculated, resulting in a sample size of 25 subjects. a total of 126 subjects were included in the internal consistency analysis. randomly (simple random sampling) selected were 40 for the test-retest reliability analysis, 30 for the inter-observer agreement analysis, 25 for the sensitivity to change analysis, and 50 for the analysis of the instrument’s usefulness. the inclusion criteria involved patients who had active orthodontic treatment with fixed orthodontic appliances, who were ≥12 years old, and whose native language is spanish. patients with a physical or mental disability that hindered administration of the survey and patients with other oral health conditions (e.g., cavities, muscle pain, periodontal pain) were excluded. the ohrqol was measured by the ohip-49 scale6. the questionnaire was self-administered by the participants on paper, except for the assessments related to the sensitivity to change (the scale was administered at three time points: t0=immediately before bracket placement, t1=24 to 48 hours after bracket placement, and t2=2 weeks after bracket placement), interobserver agreement (two observers administered the instrument in 5-minute intervals) and test-retest reliability (application of the instrument repeatedly, at two time points separated by 24 to 48 hours), which were completed through telephone interviews by three researchers previously trained in the implementation of the instrument (kappa: 0.88). the time intervals were chosen because the changes in the short time of the status of patients wearing fixed orthodontic appliances, due to the activation of the devices, the type of arch, etc. demographic variables (i.e., sex; age) were recorded at the time that the questionnaire was applied. two clinical variables were retrieved from clinical records: the time having worn the fixed appliances at survey’s administration, in days, and time since the last orthodontic adjustment visit. discriminative validity was evaluated by a visual analogue scale for pain (vas pain). scores were assigned, with 0 corresponding to the absence of pain and 10 corresponding to the highest intensity of pain. the scores were categorized as mild (0 to 2), moderate (3 to 7), or intense (8 to 10). the time required to complete the questionnaire for both the long-form and the short-form was also recorded. as recommended by who, a systematic approach to translation and adaptation was conducted20. it requires five steps: forward-translation, expert panel discussion, backward translation, a pre-test, a cognitive briefing, and a consensus on the final version. the english version of the ohip-496 was translated into spanish by four bilingual professionals (three colombian and one british professional) using an iterative forward-backward sequence. four orthodontists and four orthodontic patients assessed the face validity (i.e., does the test “look like” a measure of the construct of interest?) and content 4 barrera-chaparro et al. validity (i.e., does the test contain items from the desired “content domain”?). a subsequent preliminary fit test was performed with 10 orthodontic patients. statistical analysis the statistical analysis was performed with stata14 software (version 14.0; statacorp, college station, tex). the statistical analysis process was conducted in consecutive steps as follows: 1. ohip-s14 ortho derivation: to derive a subgroup of 14 questions, an exploratory factor analysis (efa) was used (assumptions previously verified: sphericity by using bartlett’s sphericity test (p<0.05) and sampling adequacy by using the kaiser-meyer-olkin (kmo) >0.80). the factors to be extracted were determined by the percentage and variance explained (minimum of 80%). the factor loads for each question were estimated to identify those that exceeded 0.4, which was the cutoff point for moderate to high loads. then, the items that presented the highest factor loads were chosen, and no more than two items of each conceptual dimension were included. to determine whether the removal of the individual items affected the internal consistency of the derived ohip short version, an analysis using cronbach’s α coefficient was carried out. 2. ohip-s14 ortho validation 2.1 validity of the ohip-s14 ortho: content validity was evaluated by exploratory factor analysis (efa). construct validity was evaluated by confirmatory factor analysis (cfa) using the maximum likelihood method. the fit of a model was considered adequate when the ratio of the chi-squared value to the degrees of freedom (χ2/df) <2.0, root mean square error of approximation (rmsea) <0.10, comparative adjustment index (cfi) >0.90, tucker-lewis index (tli) >0.9 and low values of the akaike information criterion (aic), browne-cudeck criterion and the bayes information criterion (bic) were obtained21. discriminative validity was evaluated by comparing the ohip-s14 ortho with a vas pain categorized scale (mild, moderate, severe) by the kruskal wallis test. 2.2 reliability of the ohip-s14 ortho: internal consistency was evaluated by the cronbach’s α coefficient. intra-observer reliability (test-retest) was evaluated by lin’s concordance correlation coefficient. the inter-observer reliability was evaluated by the spearman correlation coefficient and lin’s concordance correlation coefficient. 2.3 sensitivity to change: this was evaluated by comparing the measurements at t0, t1, and t2 by the wilcoxon signed-rank test for paired samples. our approach to avoiding missing data was to maximize the data collection by explaining to participants the importance of their responses and motivating them to fill out the surveys. however, in the cases where missing data happened, a listwise deletion method was used. 5 barrera-chaparro et al. results the descriptive statistics of the samples are shown in table 1. the sample to derivate the ohip-s14 ortho was composed of 400 subjects (27.34 years, sd 11.66, 231 women, and 169 men). another sample of 126 subjects was used for its validation (25.95 years, sd 12.39, 62 women, and 64 men). the mean time spent wearing fixed appliances was 420.76 (sd = 331.46) days for the short version derivation sample and 146.167 (sd = 262.43) days for the ohip-s14 ortho validation sample. the experts’ panel removed three of the ohip-49 questions from the questionnaire; two referred to edentulous patients (q17 and q18), and one (q3: a tooth that does not look good) was redundant and confusing for orthodontic patients. this is because patients seeking orthodontic treatment perceive that crowded teeth do not look good. a modification in the questions related to time was also made; the text was modified to 1 month to match the orthodontic appointment interval. table 1. demographic and clinical characteristics of the subjects sample for short version derivation (n=400) sample for ohip-s14 ortho validation (n=126) categorical variables n % n % sex male 169 42.25 64 50.79 female 231 57.75 62 49.21 age categorized <18 years old 71 17.75 33 26.19 ≥ 18 years old 329 82.25 93 73.81 quantitative variables mean (sd) median (min-max) mean (sd) median (min-max) age total sample 27.375 (11.633) 24 (12-66) 26.055 (12.440) 22 (12-66) <18 years old 15.154 (1.348) 15 (12-17) 14.654 (1.486) 14.71 (12-17) ≥ 18 years old 30.012 (11.177) 26 (18-66) 30.101 (12.097) 26 (18-66) time wearing fixed appliances (days) 420.76 (331.46) 364 (1-1932) 146.167 (262.43) 80.5 (1-1334) time since the last orthodontic adjustment visit (days) 28.81 (14.03) 28 (7-84) 32.18 (25.09) 28 (0-112) ohip-49 total score 26.53 (19.591) 23 (0-108) <18 years old 21.452 (14.974) 19 (0-64) ≥ 18 years old 27.626 (20.304) 23 (0-108) ohip-s14 ortho total score 10.849 (9.138) 9 (0-45) <18 years old 8.333 (7.056) 6 (0-31) ≥ 18 years old 11.742 (9.647) 10 (0-45) vas pain total score (scale 10mm) 3.40 (2.43) 3 (0-10) 6 barrera-chaparro et al. 1. ohip-s14 ortho derivation: efa was performed after it was verified that the data met the assumptions (bartlett’s sphericity test (x2 =9808.468, p<0.0001), kmo =0.911). four factors were extracted by the percentage and variance explained (minimum of 80%); the first had an eigenvalue of 13.06, which accounted for 50.71% of the variance, and the four factors accounted for 80.34% of the variance (table 2). table 2. exploratory factor analysis ohip-49 and ohip-s14 ortho. the factors were extracted by the percentage and variance explained (minimum of 80%). ohip-49 ohip-s14 ortho factor eigenvalue % of variability % of cumulative variability factor eigenvalue % of variability % of cumulative variability 1 13.06 50.71 50.71 1 6.50 78.9 78.9 2 3.6 13.98 64.69 2 1.02 12.4 91.23 3 2.46 9.55 74.24 4 1.57 6.1 80.34 table 3 shows the non-response item frequency, item prevalence (% of responses corresponding to occasionally, fairly often, or very often), item severity (item mean), and the ohip-s14 ortho development procedure with the highest factorial load questions. efa correlation loads greater than 0.4 were represented almost entirely by factor 1, except for seven questions (q2, q11, q12, q25, q44, q45, q48). the 14 items selected according to their factor loads were q1, q4, q16, q15, q23, q22, q32, q31, q34, q35, q41, q42, q47, and q46. internal consistency (cronbach’s α coefficient) for the ohip-49 was in total of α= 0.93 (annex 1). the comparison of the obtained questions of the ohip-s14 ortho with those of the ohip-14 derived by slade22 are shown in table 4. only six items from slade’s original ohip-14 version were included in the ohip-s14 ortho. ohip s14ortho agreed in six questions with the original ohip-14 short form. table 3. prevalence, mean, exploratory factor analysis of ohip long-form questions item and conceptual dimension no response % % prevalence mean general factor load functional limitation q1 difficulty chewing 0 46.25 1.28 0.53* q2 difficulty pronouncing words 0 17 0.6 0.4 q3 tooth that does not look good . . . . q4 affected appearance 0.25 20 0.657 0.58* q5 bad breath 0 28 0. 787 0.4 q6 worsened taste 0.25 7.25 0.276 0.47 q7 stuck food 0.75 7.25 2.065 0.48 q8 digestion worsened 0 5.75 0.255 0.45 continue 7 barrera-chaparro et al. physical pain q9 pain in a wound mouth 0 53 1.497 0.51 q10 discomfort in the jaw 0.50 21 0.693 0.50 q11 headaches 0 9 0. 32 0.38 q12 sensitive teeth 0.25 41 1.22 0.36 q13 pain in your teeth 0.25 53 1.464 0.45 q14 pain in your gums 0.25 24 0.819 0.47 q15 eating discomfort 0.25 58 1.569 0.59* q16 painful sites in the mouth 0 45.5 1.28 0.62* q17 unfitted dentures . . . . q18 uncomfortable dentures . . . . psychological discomfort q19 worried 0 15 0.522 0.45 q20 self-conscious 0 12 0.42 0.65 q21 unhappy 0 7.25 0.23 0.60 q22 appearance of brackets 0 14.25 0.485 0.67* q23 tense 0 13.75 0.455 0.66* physical disability q24 speak badly 0 15.75 0.545 0.49 q25 people do not understand my words 0 14.75 0.482 0.37 q26 less flavor in food 0 8.25 0.307 0.51 q27 unable to brush your teeth 0 35.5 1.04 0.48 q28 avoid eating 0.25 61.25 1.697 0.53 q29 unsatisfactory diet 0 17.5 0.547 0.54 q30 unable to eat 0 17 0.597 0.61 q31 avoid smiling 0.50 19.75 0.623 0.67* q32 discontinuing meals 0 21.75 0.685 0.66* psychological disability q33 interrupting sleep 0 6.25 0.26 0.56 q34 upset 0 17.25 0.55 0.72* q35 difficulty to relax 0.75 2.5 0.325 0.70* q36 depressed 0 3.75 0.147 0.62 q37 affected attention 0 2.25 0.157 0.54 q38 ashamed 0.25 11 0.383 0.69 social disability q39 avoid leaving 0.50 2.75 0.131 0.58 q40 less tolerant with others 0.25 3.25 0.15 0.52 q41 interacting with others 0.25 3.75 0.15 0.61* q42 irritable with others 0.25 3.5 0.168 0.59* q43 difficulty of working 0.25 2.25 0.113 0.50 handicap q44 general affected health 0.25 1.25 0.078 0.31 q45 financial loss 0.25 5.75 0.215 0.23 q46 enjoy company 0.25 2.75 0.095 0.49* q47 unsatisfactory life 0.25 2.5 0.12 0.54* q48 unable to operate 0.25 0 0.03 0.34 q49 unable to work 0.25 1.5 0.083 0.43 * highest factorial load questions (selected two for each dimension). continuation 8 barrera-chaparro et al. table 4. comparison of ohip-14 original slade questions and ohip-s14 ortho questions ohip -14 (original slade) ohips14 ortho question functional limitation 2 have you had trouble pronouncing any words because of problems with your teeth, mouth or dentures? 1 ¿en el último mes ha tenido dificultad para masticar algún tipo de comida debido a sus brackets? in the last month have you had difficulty chewing any type of food because of your brackets? 6 have you felt that your sense of taste has worsened because of problems with your teeth, mouth or dentures? 4 ¿en el último mes ha tenido la sensación de que su apariencia se ve afectada debido a sus brackets? in the last month have you had the feeling that your appearance is affected due to your brackets? physical pain 9 have you had painful aching in your mouth? 16 ¿en el último mes ha tenido sitios dolorosos en su boca debido a sus brackets? in the last month have you had painful sites in your mouth because of your brackets? 15 have you found it uncomfortable to eat any foods because of your teeth, mouth or dentures? 15 ¿en el último mes ha sentido incomodidad al comer algunos alimentos debido a sus brackets? in the last month have you felt discomfort when eating some foods due to your brackets? psychological discomfort 20 have you been self-conscious because of your teeth, mouth or dentures? 22 ¿en el último mes se ha sentido incomodo debido a la apariencia de sus brackets? in the last month, have you felt uncomfortable due to the appearance of your brackets? 23 have you felt tense because of problems with your teeth, mouth or dentures? 23 ¿en el último mes se ha sentido tenso debido a sus brackets? in the last month, have you felt tense because of your brackets? physical disability 29 has your diet been unsatisfactory because of problems with your teeth, mouth or dentures? 31 ¿en el último mes ha evitado sonreír debido a sus brackets? in the last month have you avoided smiling because of your brackets? 32 have you had to interrupt meals because of problems with your teeth, mouth or dentures? 32 ¿en el último mes ha tenido que interrumpir sus comidas debido a sus brackets? in the last month you had to interrupt your meals because of your brackets? psychological disability 35 have you found it difficult to relax because of problems with your teeth, mouth or dentures? 35 ¿en el último mes ha encontrado dificultad para relajarse debido a sus brackets? in the last month have you found difficulty relaxing due to your brackets? 38 have you been a bit embarrassed because of problems with your teeth, mouth or dentures? 34 ¿en el último mes se ha sentido molesto debido a sus brackets? in the last month, have you felt upset due to your brackets? social disability social 42 have you been a bit irritable with other people because of problems with your teeth, mouth or dentures? 42 ¿en el último mes ha estado un poco irritable con otras personas debido a sus brackets? have you been a bit irritable with other people because of problems with your teeth, mouth or dentures? 43 have you had difficulty doing your usual jobs because of problems with your teeth, mouth or dentures? 41 ¿en el último mes ha tenido dificultad para interactuar con otras personas debido a sus brackets? in the last month have you had difficulty interacting with other people because of your brackets? handicap 47 have you felt that life in general was less satisfying because of problems with your teeth, mouth or dentures? 47 ¿en el último mes ha tenido la sensación que su vida en general ha sido menos satisfactoria debido a sus brackets? in the last month you have had the feeling that your life in general has been less satisfactory due to your brackets? 48 have you been unable to function because of problems with your teeth, mouth or dentures? 46 ¿en el último mes ha sido incapaz de disfrutar de la compañía de otras personas debido a sus brackets? in the last month have you been unable to enjoy the company of other people because of your brackets? 9 barrera-chaparro et al. 2. ohip-s14 ortho validation 2.1 validity of the ohip-s14 ortho: content validity was evaluated by an efa, and the assumptions were previously verified (bartlett sphericity test (x2 =981.003, p<0.0001), kmo=0.863). two factors were extracted, which accounted for 91.23% of the variance (table 2). construct validity assessed by the cfa model showed two latent dimensions (functional and social) from the ohips14 ortho. three bi-dimensional models were evaluated by the chi-squared value (χ2/df), aic and tli. the third model, with associations (q4, q46, q1, q15, q34, q22, q23) and covariances between most of the items, was deemed best, as it had strong item loadings and a strong model fit (aic=3711.92; tli=0.972, χ2/df=1.29, cfi=0.981, rmsa=0.049; srmr=0.037) (table 5 and annex 2). table 5. confirmatory factor analysis (cfa) ohip-s14 ortho chi2 x2/df rmsa aic bic cfi tli srmr ave func ave social model 1 223.33 2.93 0.125 3895.58 3946.85 0.84 0.813 0.075 0.51 0.517 model 2 186.84 2.70 0.117 3803.09 3944.1 0.87 0.835 0.054 0.531 0.533 model 3 77.67 1.29 0.049 3711.92 3878.31 0.981 0.972 0.037 0.507 0.513 rmsea (mean square error of approximation); aic akaike bic information criterion (bayesian information criterion); cfi (comparative adjustment index); tli (tucker-lewis index); srmr (root square of the average of the sum of the squares of the residues); ave (average variance extracted); func (functional). the mean scores of ohip-s14 ortho that showed statistically significant differences by sex were q15, q16, and q42, with higher values in females. the questions with the highest negative impact on the ohrqol were q15 and q16 (figure 1). 2.0 1.5 1.0 0.5 0 m ea n q1 q4 q22 q23 q31 q32 q34 q35 q41 q46 q47q15* q16* q42* male mean female variables figure 1. mean scores of the ohip-s14 ortho questions by sex. 10 barrera-chaparro et al. discriminative validity showed significantly (p<0.05) higher ohip-s14 ortho scores in the intense pain perception group (19.56) than in the moderate (13.45) and mild (6.1) pain perception groups. 2.2 reliability of the ohip-s14 ortho: internal consistency (cronbach’s α coefficient) for the ohip-s14 ortho was in total of α= 0.91 (annex 1). the intra-observer reliability assessment (test-retest) showed excellent correlation (lin’s correlation=0.80±0.059; 95% ci:0.68;0.91). the inter-observer agreement analysis showed a high correlation (lin’s correlation=0.97±0.011; 95% ci:0.68;0.91 and spearman correlation coefficient=0.97). 2.3 sensitivity to change: the ohip-s14 ortho scores recorded at different time points were t0=2.64, sd 6.59, t1=14, sd 10.27, and t2=11.92, sd 7.76, showing adequate sensitivity to change. there were significant differences (p=0.001) between the ohip-s14 ortho scores at t0-t1 and t0-t2, but there were no differences between t1 and t2 (p=0.0937) (annex 3). additionally, there was a 6.13-minute reduction in the scale administration time of the ohip-s14 ortho compared with that of the long form of the ohip. discussion ohip-s14 ortho development according to our results, a version of ohip-14 was extracted for subjects with fixed orthodontic appliances. the ohip-s14 ortho had eight items that were different from those in the original short-form ohip developed by slade22. two were from the functional limitation dimension (q1: difficulty chewing, q4: “appearance affected”), and six were from each of the other six dimensions (q16: “sore spots”, q22: “appearance”, q31: “avoid smiling”, q34: “upset”, q41: “trouble getting on with others”, and q46: “unable to enjoy people’s company”). compared with the ohip-s14 ortho, a different version in spanish developed by castrejón-perez and borges-yañez9 had seven different items, and another version in spanish developed by león et al.13 did not have any matching items. likewise, other short forms of the ohip derived by other authors8,23-26 differ from the original shortform ohip derived by slade22. the differences might be explained by the cultural distinctiveness of the populations studied, the short-form development methodologies, or the specific impact that fixed orthodontic appliances have on the subject. the ohip’s versions mentioned were about populations with dental needs other than patients wearing fixed orthodontic appliances. fixed orthodontic appliances affect functional and physical dimensions, making it difficult to consume certain hard and sticky foods, which can cause pain or damage to the appliance. they can also affect appearance, which can generate a social impact on the daily life of patients, preventing them from smiling, and participating in social activities27,28. 11 barrera-chaparro et al. one important aspect to be considered is the different statistical methods used to derive the different ohip short forms. in our study, we used the statistical methods suggested by slade and spencer6 that were also applied by león et al.13, (reliability analysis, principal component factor analysis, and least squares regression analysis), whereas other studies used a statistically significant association about the clinical variables9, the item frequency method8, or efa29. the sample’s age range is another important factor to consider. most of the studies were conducted with subjects older than 60 years9,13,22 who had oral rehabilitation needs, while the sample in our research mainly included patients over 11 years old, as adolescents and young adults mostly represent the population undergoing orthodontic treatment. ohip specifically evaluates problems with the mouth, teeth, or dentures, as proposed by slade and spencer6. however, as shown by the results of this study, the scale can be adapted by an appropriate method to another target population, such as patients undergoing orthodontic treatment. likewise, the scale can be adapted to assess the impact on ohrqol in the last month, which, in patients with orthodontic treatment, accounts for the most recent impact of orthodontic treatment, whereas, with the original ohip-496,22,30, it was assessed over the previous year. the two items from each dimension from the original ohip were maintained in the ohip 14 s-ortho. also, the high cronbach’s alpha (0.912) indicates that the scale measures the same construct. therefore, we suggest that this number of items satisfactorily evaluates the ohrqol construct for orthodontic patients31. ohip-s14 ortho validation this study demonstrated appropriate validity of the ohip-s14 ortho scale across two main dimensions (functional limitation and social disability) and the associations and covariances between all the items. santos et al.32 compared one-dimensional and tri-dimensional structures of the ohip-14 and reported that the scale measures one single construct. john et al.33 compared the psychometric performance of three models: a unidimensional model, a four-factor model, and a bifactor model, showing that the model with the best fit was the four-factor model. however, the other two models also showed a good fit, suggesting that one ohip summary score is sufficient to characterize ohrqol. the ohip-s14 ortho showed excellent discriminative validity compared with the visual analog scale for pain (vas pain). other authors used clinical variables such as periodontal status, caries, or missing teeth to evaluate the discriminative validity8-11,13,22 because these variables affect ohrqol. moreover, in orthodontic patients, oral health must be at an optimum level to initiate tooth movement. pain and discomfort occur as part of orthodontic mechanotherapy, but it is an individual and subjective response dependent on factors such as age, sex, individual pain thresholds, the magnitude of the force applied, the current emotional state and stress, cultural differences, and previous pain experiences27. thus, this research allows clinicians to improve communication with patients so that they can plan treatment and use the ohip-s14 ortho in daily practice. 12 barrera-chaparro et al. according to the reliability results, the ohip-s14 ortho indicated to have very good internal consistency (cronbach’s α coefficient = 0.912), a good level of test-retest agreement (lin’s=0.80; icc=0.894), and inter-observer agreement (lin’s=0.97; spearman=0.97). similar results were reported in other studies9,13,33. the scale also demonstrated good sensitivity to change in the initial stages of orthodontic treatment, showing that the ohip-s14 ortho has a good capacity to respond to changes in the ohrqol that occur during orthodontic treatment, but more extensive testing of the measure’s responsiveness to change needs to be carried out to confirm this statement. although in previous studies8-10,12,13,22 sensitivity to change has not been evaluated, it is important to measure the impact of orthodontic treatment on ohrqol over time. mansor et al.5, found that ohrqol became highly deteriorated within 24 hours after the placement of fixed orthodontic appliances. streiner19 and johal et al.28 determined that the initial stages of fixed appliance treatment result in a negative impact on the quality of life and pain experience but that pain and discomfort intensity significantly decrease three days after the bracket’s placement and over the following three months. in our study, q15 (eating discomfort) and q16 (painful sites in the mouth) had a higher negative impact on the ohrqol, suggesting that pain and discomfort are the main impact during orthodontic treatment. to assess the impact on ohrqol in a wide range of time wearing fixed orthodontic appliances since discomfort is experienced in the first 24 hours after brackets placement and subsequent adjustment visits5,19,28. however, this assessment could be a limitation due to the inaccuracy which is derived from this aspect, so future research with homogeneous ranges of time of wearing fixed orthodontic appliances is recommended. within the limitations of the current study, the data did not show ceiling effects, as the maximum score of 56 of the ohip-14 scale was not reported by any of the participants. meanwhile, there were floor effects (scores of 0), which might suggest that the questionnaire is not picking up all the potential impacts of a fixed appliance, and it might be helpful to implement further qualitative work to determine this. kettle et al.34 identified from young people a multi-dimensional social process of managing everyday life with an orthodontic appliance. this study only included subjects wearing buccal fixed appliances; therefore, the impact of other removable appliances, retainers, or lingual brackets was not measured with the ohip-s14 ortho, and the results are not generalizable to all kinds of orthodontic treatment. although the original ohip-14 version was derived from adult patients, in orthodontics, it has been widely used with patients under 18 years of age5,28 as it was with our study; however, it would be more appropriate to use the ohip-14 with individuals’ questionnaires developed for young people as is the child oral health impact profile (cohip)35. in our results, both ohip-49 and ohip-s14 ortho total scores were higher in adult patients (≥ 18 years old) than in younger patients, suggesting a different impact in the ohrqol according to age. further research considering important variables that were not examined in this study, such as socioeconomic status and psychological parameters (self-esteem, depression, and stress), must be done. regarding the usefulness of the ohip-14 scale, in our study, the time to completion of the long-form ohip was 8.93 minutes, while for the ohip-s14 ortho, it was 2.8 13 barrera-chaparro et al. minutes. this shows that there was a significant decrease in the time to completion of the questionnaire. currently, people’s time and their compliance with surveys are very important; therefore, long-form questionnaires must be avoided. however, other authors have suggested that reducing the number of questions in the instrument can affect their psychometric properties and that an excessive simplification of the scale can lead to negative interpretations7. conversely, other studies3,31 have suggested that a reduction in the number of questions does not affect the responsiveness of the instrument, but it does affect its validity and reliability. another factor to take into account is the response scale used in this study. although it is easily quantifiable and understood, it might not measure the true attitudes of respondents, which could underestimates the effects of impacts of high concern to individuals, as the impact of the malocclusion is largely in the emotional and social well-being subscales34.on the other hand, our study analysis approach was by classical test theory (ctt) instead of the item response theory (irt), based on the notion that ctt does not invoke a complex theoretical model to relate an examinee’s ability to succeed on a particular item and that is easier to apply in many testing situations. however, readers must be aware of the weakness of ctt in terms of its circular dependency on item/person statistics. as the conclusions of the present study, the ohip-s14 ortho is a valid and reliable instrument to measure ohrqol in spanish-speaking patients with fixed orthodontic appliances, and the construct validity of the ohip-s14 ortho showed a two-dimensional structure with associations and covariances between all the items. ethics statement: individuals who were wearing fixed orthodontic appliances were provided by the unicieo university in bogotá, colombia. this study was approved by the unicieo university ethics committee. conflicts of interest: none. references 1. sischo l, broder hl. oral health-related quality of life: what, why, how, and future implications. j dent res. 2011 nov;90(11):1264-70. doi: 10.1177/0022034511399918. 2. bernabé e, sheiham a, tsakos g, messias c. the impact of orthodontic treatment on the quality of life in adolescents: a case-control study. eur j orthod. 2008 oct;30(5):515-20. doi: 10.1093/ejo/cjn026. 3. andiappan m, gao w, bernabé e, kandala n-b, donaldson an. malocclusion, orthodontic treatment, and the oral health impact profile (ohip-14): systematic review and meta-analysis. angle orthod. 2015 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oral health impact profile on responsiveness, validity and reliability in edentulous populations. community dent oral epidemiol. 2008 feb;36(1):12-20. doi: 10.1111/j.1600-0528.2006.00364.x. 32. santos c, branca h, nadanovsky p, balbinot j, keller r, neves f. the oral health impact profile-14: a unidimensional scale? cad saude publica. 2013 apr;29(4):749-57. doi: 10.1590/s0102-311x2013000800012. 33. john mt, feuerstahler l, waller n, baba k, larsson p, čelebić a, et al. confirmatory factor analysis of the oral health impact profile. j oral rehabil. 2014 sep;41(9):644-52. doi: 10.1111/joor.12191. 34. kettle je, hyde ac, frawley t, granger c, longstaff sj, benson pe. managing orthodontic appliances in everyday life: a qualitative study of young people’s experiences with removable functional appliances, fixed appliances and retainers. j orthod. 2020 mar;47(1):47-54. doi: 10.1177/1465312519899671. 35. broder hl, mcgrath c, cisneros gj. questionnaire development: face validity and item impact testing of the child oral health impact profile. community dent oral epidemiol. 2007 aug;35 suppl 1:8-19. doi: 10.1111/j.1600-0528.2007.00401.x. annex 1. reliability: cronbach’s alpha values for ohip-49 and ohip-s14 ortho ohip-49 questio n item cronbach´s α question item cronbach´s α q1 difficulty chewing 0.93 q27 unable to brush your teeth 0.93 q2 difficulty pronouncing words 0.93 q28 avoid eating 0.93 q4 affected appearance 0.93 q29 unsatisfactory diet 0.93 q5 bad breath 0.93 q30 unable to eat 0.93 q6 worsened taste 0.93 q31 avoid smiling 0.93 q7 stuck food 0.93 q32 interrupting meals 0.93 q8 worsened digestion 0.93 q33 interrupting sleep 0.93 q9 pain due to wounded mouth 0.93 q34 annoyed 0.93 q10 discomfort in the jaw 0.93 q35 difficulty to relax 0.93 q11 headaches 0.93 q36 depressed 0.93 q12 sensitive teeth 0.93 q37 affected attention 0.93 q13 pain in their teeth 0.93 q38 ashamed 0.93 q14 pain in your gums 0.93 q39 avoid leaving 0.93 q15 discomfort when eating 0.93 q40 less tolerant with others 0.93 q16 painful sites in the mouth 0.93 q41 interact with others 0.93 q19 worried 0.93 q42 irritable with others 0.93 q20 self-conscious 0.93 q43 difficulty working 0.93 q21 unhappy 0.93 q44 general health affected 0.93 q22 appearance of the brackets 0.93 q45 financial loss 0.93 q23 tense 0.93 q46 enjoy company 0.93 q24 speaking badly 0.93 q47 unsatisfactory life 0.93 q25 people do not understand their words 0.93 q48 unable to function 0.93 q26 less flavor in food 0.93 q49 unable to work 0.93 total 0.93 ohip-s14 ortho q1 difficulty chewing 0.91 q32 interrupting meals 0.91 q4 affected appearance 0.90 q34 annoyed 0.90 q15 discomfort when eating 0.90 q35 difficulty to relax 0.91 q16 painful sites in the mouth 0.90 q41 interact with others 0.91 q22 appearance of the brackets 0.91 q42 irritable with others 0.91 q23 tense 0.91 q46 enjoy company 0.91 q31 avoid smiling 0.91 q47 unsatisfactory life 0.91 total 0.91 annex 2. association between ohip-49, ohips14 ortho and demographic and clinical characteristics categorical variables ohip-49 mean (ds) ohips14 ortho mean (ds) sex male 24.89 (18.84) 7.53 (6.84) female 27.72 (20.07) 9.16 (8.0) p value 0.120 0.059 occupation unemployed 25.92 (27.09) 7.65 (9.26) independent worker 29.92 (27.09) 9.78 (7.89) employee 27.21 (19.71) 8.70 (7.97) depend on family resources 24.04 (17.42) 7.45 (6.39) p value 0.132 0.077 education level primary school 28.67 (20.45) 9.17 (6.71) middle school 23.39 (15.10) 6.35 (5.72) high school 22.73 (19.10) 7.37 (7.52) post-secondary education 26.71 (12.10) 8.14 (3.63) technical education 25.85 (18.08) 8.02 (6.65) university education 30.69 (22.92) 10.34 (9.06) specialization course/master 27.35 (16.59) 8.65 (6.55) doctoral degree 12 (4.24) 4.5 (0.719) p value 0.190 0.128 orthodontic technique standard 25.62 (17.62) 7.81 (6.54) self-ligating 27.54 (20.55) 9.40 (8.55) mbt 26.60 (20.65) 8.33 (7.70) p value 0.889 0.558 age 0.099 0.125 p value 0.048 0.013 last control time -0.092 -0.062 p 0.067 0.216 treatment time -0.004 -0.062 p 0.937 0.216 chi2 test for categorical variables and independent t-test for quantitative variables annex 3. fca model 1. fca model 2. fca model 3. annex 4. sensitivity to change. mean (sd). t0 t1 t2 t0t1 t0t1 t1-t2 mean 2.64 (6.59) 14 (10.27) 11.92 (7.76) 0.001a 0.001a 0.0937a t0 (before treatment); t1 (24 and 48 hours); t2 (15 days). signed wilcoxon rank test of paired samples. a p <0. 05. 1http://dx.doi.org/10.20396/bjos.v21i00.8665272 volume 21 2022 e225272 original article 1 senior assistant professor, department of pediatric dentistry, faculty of dental medicine, medical university of plovdiv, bulgaria. 2 professor, department of pediatric dentistry, faculty of dental medicine, medical university of plovdiv, bulgaria. corresponding author: maria shindova, dds, msc, phd senior assistant professor department of pediatric dentistry faculty of dental medicine medical university plovdiv 3 hristo botev bvd 4000 plovdiv, bulgaria mobile: + 359 898 390 935 email: mariya.shindova@gmail.com mariya.shindova@mu-plovdiv.bg editor: altair a. del bel cury received: april 12, 2021 accepted: august 24, 2021 attitudes of dental students towards paediatric dental behaviour guidance maria shindova1,* , ani belcheva2 aim: to compare the pre-clinical and clinical students` perceptions about the non-pharmacological behaviour management techniques in paediatric dentistry and to investigate the influence of the dental curriculum on the students` knowledge regarding this issue. methods: a total of 283 students from the iv-and x-semester completed a questionnaire, consisted of 12 statements, describing the nonpharmacological behaviour management techniques for the treatment of paediatric dental patients. the acceptability rate was evaluated with a likert scale ranging from 1 to 5. results: the students from all courses demonstrated high acceptance for reinforcement and desensitization techniques and low for the negative reinforcement and physical restraint. the comparison between the perceptions of the pre-clinical and clinical students demonstrated a statistically significant increase in the acceptance of the physical restraint, along with nonverbal communication, modelling and parental presence/absence (p<0.05). conclusion: the results provide information about the students’ knowledge and skills in behaviour management techniques together with some insights about how the educational process can modify the students` perceptions and views in dealing with paediatric dental patients. keywords: students. behavior. methods. pediatric dentistry. mailto:mariya.shindova@gmail.com mailto:mariya.shindova@mu-plovdiv.bg https://orcid.org/0000-0003-2996-3700 https://orcid.org/0000-0002-9625-8684 2 shindova et al. introduction one of the most challenging aspects in the dental practice is working with uncooperative patients and their behaviour management. dentists are expected to be aware of the wide variety of behaviour management techniques (bmts) and to use the most suitable one according to the individual clinical situation in order to provide adequate clinical care. in addition to this, dental practitioners should be tolerant and flexible in incorporating behaviour management strategies1. the american academy of pediatric dentistry (aapd) has issued a set of guidelines on behaviour guidance for paediatric dental patients with recommendations for focusing and implementing them during the entire period of dental education2. it identifies both basic and advanced bmts, as well as the indications for their use2. as to the advanced bmts such as positive stabilization, hand over mouth, use of papoose boards, sedation and general anesthesia, it is recommended for use only for those dentists who have completed postdoctoral training2,3. while for the basic bmts, students receive theoretical and clinical training during their dentistry training at the university. dental education in paediatric dentistry should provide the opportunity for students to learn, observe, put into practice the different behaviour management strategies and techniques. al-jobair et al. considered that the content of the educational curriculum and the degree of the training in bmts have an impact on the students` perceptions and practice of such techniques4. there have been some empirical studies evaluating the acceptability of the bmts during dental treatment. they can be divided into three approaches. the first one corresponds to the parent`s views and аttitudes with a child currently receiving dental treatment5-7. the second approach has compared how children faced these strategies8,9. and the third one has examined the views of individuals who have not had direct experience of the treatment10. dental practitioners` and students` views are associated not only with the dental experience but also with the educational level during the learning process10. to a large extent, dentist`s perceptions about bmts for the treatment of dental patients are based on the information obtained during their dental education and on the experience derived from the contact with patients11. at the beginning of the course of paediatric dentistry, students have no or limited knowledge of bmts, thus their views could be compared with those of parents and the general public12. these perceptions are important and play a significant role in determining the acceptance by the public and their implementation12. nevertheless, as they receive didactics classes about bmts and start to provide treatment for children, their view may be changed by the influence of the educational process11. thus, the purpose of the present study was to compare the fourth(pre-clinical) and tenth(clinical) semester students` perceptions about the non-pharmacological bmts in paediatric dentistry and to investigate the influence of the dental curriculum on the students` knowledge regarding this issue. material and methods the study consisted of an anonymous, self-completed e-mailed survey. potential subjects received an email describing the study and inviting their participation. a total of 460 students (250 students from the fourth semester (iind course) and 210 3 shindova et al. from the tenth semester (vth course) were invited to participate in the study. the mail included a brief cover letter explaining the purpose of the survey. the study was conducted in january 2021 (within three weeks) and consisted of 12 statements, describing the non-pharmacological bmts for the treatment of paediatric dental patients. to obtain students` acceptability scores, they had to mark on a 5-point likert scale from 1 to 5 (figure 1). ethical principles the study is conducted in accordance with the conditions and principles of the declaration of helsinki, the existing eu clinical trial directive (ec) no. 2001/20/ec, the recommendations of the ethical committee at the medical university of plovdiv, bulgaria and the international ethical and scientific quality standard for designing, recording and reporting trials that involve the participation of human subjects good clinical practices (gcp). the study was approved by the committee for scientific research ethics, medical university plovdiv, bulgaria (reference number p-1371/30.04.2018, protocol of approval no. 2/01.04.2021) before circulating the questionnaire. 0 0.9 total disagreement 1.0 -1.9 disagreement 2.0 -2.9 neutral 3.0 -3.9 agreement 4.0 -5.0 total agreement figure1.rating of acceptability to determine students` perceptions statistical analysis the obtained data were tabulated, processed and analysed using a spss (statistical package for social science software) version 21.0 (ibm, usa). independent t-test was used to evaluate the statistically significant differences between the fourth-semester means and those obtained at the tenth-semester. the level of significance was set at p<0.05. results out of the 460 surveys that were e-mailed, 283 subjects (61.52% response rate) were included in the statistical analysis for this study. the sample size was n1 = 131 (for the fourth-semester) and n 2=152 (for the tenth-semester). table 1 shows the means and standard deviations of the acceptability scores as well as the statistical differences between the students` rates from the two investigated courses. the most acceptable bmts among the students from the two semesters were nonverbal communication, stop signals, positive reinforcement (pr) and tell-show-do (tsd). there were no changes in the levels of acceptability of these bmts when the perceptions of the students from the two courses were compared (p>0.05), except for nonverbal communication (p=0.011). considering the desensitization techniques only the acceptability of modelling increased significantly, changing from `agreement` to `total agreement` (p<0.01). http://ec.europa.eu/health/human-use/clinical-trials/directive/index_en.htm 4 shindova et al. the analysis of the views of all participants of our study showed that the least acceptable techniques were the aversive ones – negative reinforcement (nr) and restraint. both showed a statistically significant increase in the scores of the group of clinical respondents (p<0.001 and p=0.006). however, voice control (vc) as an aversive technique was defined as `acceptable` among the two investigated groups of students, no statistically significant difference was observed in the comparison (p>0.05). statistically significant differences in the acceptability rates concerning other bmts were also found. the perceptions of latent inhibition were `acceptable` for the pre-clinical group and `completely acceptable` for the clinical group of respondents (p=0.003). as to the parent involvement in the dental treatment, the present results demonstrated that the parental presence/absence technique became more acceptable throughout the semesters. table 1. means and standard deviations of the acceptability scores and comparisons between students` rates from the pre-clinical and clinical students bmt means±sd comparison fourth semester tenth semester p nonverbal communication 4.64±.72 4.81±0.42 0.011* tell-show-do (tsd) 4.44±0.67 4.50±0.74 0.533 voice control (vc) 3.32±1.24 3.52±1.09 0.174 positive reinforcement (pr) 4.51±0.70 4.34±0.93 0.063 negative reinforcement (nr) 1.89±1.13 2.56±1.26 0.000* distraction 4.13±0.85 4.15±0.87 0.836 stop signals 4.55±0.81 4.47±0.76 0.397 modelling 3.72±0.94 4.01±0.93 0.008* desensitization 3.82±0.95 3.97±1.01 0.197 parental presence/absence 3.20±1.19 3.65±1.10 0.001* latent inhibition 3.90±1.05 4.21±0.82 0.003* restraint 1.97±1.29 2.38±1.30 0.006* *statistically significant difference, p<0.05 discussion the results of the present study showed that some of the students` perceptions about several techniques changed significantly throughout the under-graduation program. in line with other similar studies, most students demonstrated an acceptance of methods categorized as reinforcement and desensitisation techniques such as pr, tsd, distraction, communication regardless of their course in the university3,4,11-13. a significant increase in the acceptability of nonverbal communication was observed in the views of the vth-course students. the technique is simple, easy to be remembered and applied, as well as communicative techniques are included as a first option in the behaviour guidance strategies of the dental curriculum. another desensitizing bmt also demonstrated a significant positive change in the present study – modelling. the 5 shindova et al. effectiveness of using it in the management of a child`s dental behaviour during the clinical training classes by students could be a possible reason for our results4. in contrast to other basic techniques, not many students accepted aversive methods. the analysis of the current results demonstrated a significantly higher acceptability mean score amongst clinical year students for the investigated aversive bmts, except for vc, in comparison with their pre-clinical counterparts. similar results were noticed by sotto et al.12, al-jaboir et al.4, ali et al.13. in many countries, the use of physical restraint on dental patients is guided by protocols and guidelines, which highlights the need for careful consideration of patients’ physical, physiological, psychological and medical conditions14. as to vc, students from the fourth and the tenth semesters reported comparable perceptions for its use in the treatment of paediatric dental patients. the present findings appeared to contrast to the results found by york et al.3 and ali et al.13 who indicated a significant positive change throughout the semesters in the university. students initially disagreed with nr but their perceptions shifted significantly towards `neutral` at the end of the course. this could be explained by achieving better patient cooperation during dental treatment using this bmt. following the current trend in the dental profession for a loss of the aversive techniques and an increase in pharmacological management, future exploration of the perceptions about the pharmacological bmts will be interesting. although latent inhibition is time-consuming, requires attention to detail, more effort and special equipment in the dental office, this bmt became more acceptable throughout the semesters and interestingly more students reported using this technique with their patients3. in recent years, dental education and clinical practice implemented psychological principles and techniques into dental training. york et al. emphasized that cognitive-behavioural techniques, such as latent inhibition, have become standard of patient care and were included in many dental curricula3. in the present study, significantly higher acceptability scores were given by clinical students at the end of the dental course of education for parental presence/absence. on one hand, they found that the involvement of the parent in the management and control of dental behaviour is useful during the treatment of patients in the clinical training classes. on the other hand, students followed the present clear trend for parents preferring to stay with their children during dental treatment15. al-jobair et al.4 also found that students` perceptions shifted significantly towards acceptance at the end of the educational process4,11. in contrast, the results of a study from 2020 demonstrated that significantly fewer year 5 students, in comparison with students in years 1 to 4, and amongst those in clinical years, accepted the presence of parents in the clinic during the procedure13. the dental curriculum includes clinical training classes where students receive instructions for the use of different bmts. moreover, they have the opportunity to put them into practice and develop patient management skills4. the present findings indicated that dental education has adapted to the changing needs of patients and parents12. for future research, it is recommended the study be conducted on the same cohort of students to investigate the impact of education on their acceptance of various bmts as well as the factors that influence such acceptance. 6 shindova et al. in conclusion, there were significant differences in students’ acceptance of modelling and the aversive techniques—across academic years, between pre-clinical and clinical groups of respondents. students’ acceptance of the various techniques provides useful information for the faculty of dental medicine about the students’ knowledge and skills in bmts, as well as it is an indication about how and to what extent this educational material can be modified in dental programmes. data availability datasets related to this article will be available upon request to the corresponding author. conflicts of interests: none the investigators have no conflicts of interest to declare. they agree with the study and there is no financial interest to report.  acknowledgements the authors would like to show their gratitude to all students for participating in the survey. funding this research will receive no specific grant from any funding agency in the public, commercial or not-for-profit sectors. reference 1. feigal rj. guiding and managing the child dental patienт. j dent educ. 2001 dec;65(12):1369-77. 2. american academy of pediatric dentistry. behavior guidance for the pediatric dental patient. the reference manual of pediatric dentistry. chicago, ill.: american academy of pediatric dentistry; 2020:292-310. 3. york km, mlinac me, deibler mw, creed ta, ganem i. pediatric behavior management techniques: a survey of predoctoral dental students. j dent educ. 2007 apr;71(4):532-9. 4. al-jobair am, al-mutairi ma. saudi dental students’ perceptions of pediatric behavior guidance techniques. 2015 sep 10;15:120. doi: 10.1186/s12909-015-0382-6. 5. luis de león j, guinot jimeno f, bellet dalmau lj. acceptance by spanish parents of behaviour-management techniques used in paediatric dentistry. eur arch paediatr dent. 2010 aug;11(4):175-8. doi: 10.1007/bf03262739. 6. elango i, baweja dk,shivaprakash pk. parental acceptance of pediatric behaviour management techniques: a comparative study. j ind soc pedod prev dent.2012;30:195-200. 7. peretz b, kharouba j, blumer s. pattern of parental acceptance of management techniques used in pediatric dentistry. j clin pediatr dent. 2013 fall;38(1):27-30. 8. davies eb, buchanan h. an exploratory study investigating children’s perceptions of dental behavioural management techniques. int j paediatr dent. 2013 jul;23(4):297-309. doi: 10.1111/ipd.12007. 9. kantaputra pn, chiewcharnvalijkit k, wairatpanich k, malikaew p, aramrattana a. children’s attitudes toward behavior management techniques used by dentists. j dent child (chic). 2007;74(1):4–9. 10. newton jt, sturmey p. students’ perceptions of the acceptability of behaviour management techniques. eur j dent educ. 2003 aug;7(3):97-102. https://www.jisppd.com/searchresult.asp?search=&author=pk+shivaprakash&journal=y&but_search=search&entries=10&pg=1&s=0 7 shindova et al. 11. oliveira r, angelo ac, brito db, de medeiros rc, forte fd, sousa sa. student’s perceptions about pediatric dental behavior guidance techniques throughout a five-year dental curriculum. pesq bras odontopediatr clin integrada. 2015 jun;15(1):143-52. doi: 10.4034/pboci.2015.151.16. 12. sotto jj, azari af, riley j, bimstein e. first-year students’ perceptions about pediatric dental behavior guidance techniques: the effect of education. j dent educ. 2008;72(9):1029-41. 13. ali nm, husin in, ahmad ms, hamzah sh. perceptions of behavioural guidance techniques for paediatric patients amongst students in a malaysian dental school. eur j dent educ. 2021 feb;25(1):18-27. doi: 10.1111/eje.12573. 14. stirling c, west m, editors. guidelines for ‘clinical holding’ skills for dental services for people unable to comply with routine oral health care. british society for disability and oral health; 2009. 15. vishwakarma ap, bondarde pa, patil sb, dodamani as, vishwakarma py, mujawar sa. effectiveness of two different behavioral modification techniques among 5-7-year-old children: a randomized controlled trial. j indian soc pedod prev dent. 2017 apr-jun;35(2):143-9. doi: 10.4103/jisppd.jisppd_257_16. https://www.researchgate.net/profile/rosa-oliveira?_sg%5b0%5d=n3z6rpnlzrynrmeky8agis5hxww9ed25g_dqc-6t95et2z67hsq-dh5ecbcs_bs0aoaxmhc.gzodzvljnuy9onzwovnkmj04euwmhykj5uga-mnqzy0hxe_tuskr56ywaiunlsztekzfgjn55byrm3rv4avahg&_sg%5b1%5d=7atij2aye6h7fzd5jqzmxaknza8nxwkfj5r3lzaadyozuh1xchpsum3sbpjjsmcawzb1sbi.cniwaxd_rug_kzbp2yoylkndj31ig26qh6v_otochkocc7vet3ibgj25s3ymytdznp5fawzsnkofciw2mmwnea 1http://dx.doi.org/10.20396/bjos.v19i0.8658518 volume 19 2020 e208518 original article 1 graduate program of the federal university of santa maria, santa maria, rs, brazil 2 department of restorative dentistry, federal university of santa maria, santa maria, rs, brazil 3 clinical practice, federal university of santa maria, santa maria, rs, brazil corresponding author: letícia brandão durand department of restorative dentistry, federal university of santa maria, santa maria, rs, brazil email: leticia_durand@yahoo.com received: february 27, 2020 accepted: july 30, 2020 marginal staining of ultra-thin ceramic veneers renata ragagnin zago1 , luciana abitante swarowsky1 , gabriela simões teixeira1 , marcela marquezan3 , alexandre henrique susin2 , letícia brandão durand2,* the pigmentation of the resin cement at the tooth/ceramic interface compromises the esthetic and longevity of ultra-thin ceramic veneers. aim: the aim of this study was to evaluate marginal staining of ultra-thin ceramic veneers cemented to intact enamel (non-prepared) and prepared enamel. methods: thirty-two (32) permanent central incisors were selected and randomly divided into two groups: intact enamel (ie) and prepared enamel (pe). the ceramic veneers of pe group were bonded to the prepared enamel and the ceramic veneers of ie group were cemented directly onto the intact enamel, with no preparation. both preparation and cementation were standardized and performed by a single operator. each group was subdivided into two subgroups (n = 8) with different immersion media coffee and water. after an immersion period of 10 days, stereomicroscope images were made at 20x magnification of the mesial, distal, cervical and incisal surface of each specimen. three blinded, trained and calibrated examiners evaluated the images of the resin cement interface of each surface. the data were subjected to kruskalwallis and mannwhitney statistical analysis. immersion media and enamel preparation influenced the marginal staining of the tooth/ceramic interface. results: when immersed in coffee, prepared interfaces presented greater marginal staining than unprepared interfaces. when immersed in water, there was no statistically significant difference between the groups. conclusion: the cementation of ultra-thin ceramic veneers onto intact enamel is associated with less marginal staining and, consequently, improved esthetics. keywords: coffee. dental enamel. dental veneers. esthetics. staining and labeling. mailto:leticia_durand@yahoo.com https://orcid.org/0000-0002-0621-948x https://orcid.org/0000-0002-7001-3339 https://orcid.org/0000-0003-2167-2204 https://orcid.org/0000-0002-7908-6083 https://orcid.org/0000-0002-7083-6028 https://orcid.org/0000-0002-4853-4015 2 zago et al. introduction ultra-thin ceramic veneers have become one of the main treatment options for esthetic improvement of discolored, fractured, worn, malformed and misaligned anterior teeth1,2. the conservation of dental structures, high success rate and excellent esthetic outcomes contributed to expand the indications and increase the popularity of this procedure3,4. despite the brittleness of the ceramic materials, resistance is improved due to the strong and stable bond produced when adhesive luting agents are used for cementation4,5. thus, success is highly dependent on the quality of the adhesion on restricted enamel preparations, adequate ceramic surface treatments, and the use of proper luting agents6. luting agents provide a link between tooth and restoration. various types of resin cements are indicated for the cementation of ceramic veneers, such as auto-, light-or dual-cured7. light cured resin cements are preferable because of the superior color stability due to the absence of tertiary amines. in addition, the presence of aliphatic amines reduces the oxidation susceptibility7,8 and the activation by light allows longer working time, as well, as optimization of the technique9. extensive enamel preparations with accidental dentine exposure are associated with reduced bond strength, increased microleakage10 and marginal defects11. even when these circumstances do not result in absolute failure, they may complicate clinical outcomes1,11. contrarily, long-term survival rates are improved with conservative enamel preparations10. moreover, when ceramic veneers are supported exclusively by enamel, the load-bearing capacity is increased, due to the similar modulus of elasticity between the structures12. the trend toward the indication of conservative treatments, based on the principles of adhesion and minimally invasive dentistry, is widely encouraged. prepless veneers are consolidated as an esthetic elective treatment. conservative preparations are associated with increased esthetics and longevity13, nevertheless, the importance of the preservation of dental structures is unquestionable. the esthetic outcomes are important concerns and the high expectations may be compromised by marginal staining of the tooth/ceramic interface11. the above-listed arguments motivated this study, that aims to evaluate the marginal staining at the tooth/ceramic interface of ultra-thin veneers placed on intact and prepared enamel. the null hypothesis was that the enamel preparation would not affect the marginal staining of the tooth/ceramic interface of ultra-thin veneers. materials and methods ethics the research ethics committee of the participating institution (certificate number – 7100, caee number 00564612.4.0000.5346) approved this study. thirty-two non-carious human mandibular incisors with a cervico-incisal length of 9.5mm and a mesio-distal width of 5.5mm (±1 mm) were selected from a tooth bank. 3 zago et al. the teeth were kept in 0.1% of thymol solution for two weeks for disinfection and then, stored in distilled water (37°c), until specimen preparation. specimen preparation the teeth were randomly assigned to two groups according to the different preparation alternatives: 1. intact enamel with no preparation (ie) and 2. prepared enamel (pe). half of the specimens of each group were immersed in water (n=8) and the remaining half were immersed in a coffee solution (n=8). the root of each tooth was embedded in acrylic resin blocks, 2 mm below the cement-enamel junction to facilitate specimen preparation. enamel preparation dental enamel preparation was standardized and prepared by a trained operator. initially, a silicon based impression was taken from the tooth crown (virtual, ivoclar vivadent, schaan, liechtenstein). afterwards, the impression was sectioned and used as a reference guide for enamel reduction. a 0.5 mm-enamel reduction was performed with a diamond bur #2135 (kg sorensen, cotia, sp, brazil) (figure 1a), using the window preparation technique. the cervical margin was placed 0.5mm above the cement-enamel junction with a chamfer finishing line. in order to produce a smooth and well-finished surface, fine and extra fine grit diamond burs # 2135 (kg sorensen, cotia, sp, brazil) were used. impression the impressions were taken by a thimble shaped tray (figure 1b) through the single-step technique, which provides the simultaneous polymerization of the heavy and light-body impression material (virtual, ivoclar vivadent, schaan, liechtenstein). figure 1. a) enamel reduction using a silicon based impression as a reference guide. b) impressions of specimens were taken using a thimble and through the single-step technique a b 4 zago et al. ceramic veneers ceramic veneers were produced from a lithium disilicate glass ceramic (ips e.max press, ivoclar vivadent, schaan, liechtenstein), by one experienced dental technician, using the heat-pressed technique. the veneers produced for non-prepared enamel specimens were 0.3mm-thick, and for the prepared enamel specimens (pe) 0.5mm-thick. bonding procedures and cementation the ceramic veneers were cemented to the tooth surfaces with light-curing resin cement (variolink veneer, ivoclar vivadent, schaan, liechtenstein), according to the manufacturer’s instructions. each specimen was light-cured for 150s, with a led unit (emmiter c, schuster eq. odont ltd, santa maria, rs, brazil), with 800mw/cm2 of irradiance. the sequence of adhesive procedures is summarized in figure 2 and the materials used in the study are described in figure 3. after complete polymerization, the margins were polished with a silicon carbide finishing kit (kg sorensen, cotia, sp, brazil), and washed in an ultrasonic bath (l100, schuster eq odon. ltd, santa maria, rs, brazil) for 2 cycles of 480s to remove residual particles from the resin cement or finishing points. staining half of the ie and pe specimens were stored in water at 37°c, and the remaining specimens of each group were immersed in coffee. the coffee solution was prepared in a standardized manner, using 25g of instant coffee powder (nestlé, caçapava, sp, brazil) and 250 ml of water, for 10 days at 37° c. the immersion solutions were replaced every 24h. evaluation marginal staining was assessed by observing digital images, similar to other studies reported in the literature5,14. standardized images of the mesial, distal, incisal and cervical margins of each restoration were used for evaluation. the images were registered with a stereomicroscope (carl zeiss, göttingen, germany), using the axiovision program at 20x magnification. the examiners underwent a three-day training period, in which they received instructions on the evaluation procedure and became familiar with the scores. in addition, the examiners observed and rated 20 selected images of mesial, distal, cervical and incisal margins from specimens used in a previous pilot study. after seven days, the examiners evaluated the same images without receiving any initial instructions, and kappa intraand interexaminers agreement was tested. kappa values for intra-examiner agreement ranged from 0.73 to 0.93, and inter-examiners agreement ranged from 0.77 to 1.0. afterwards, the staining of the marginal finishing line was assessed for the 128 images according to alfa, bravo, charlie and delta scores (figure 4). each image was displayed on a full hd monitor in a random sequential order. the viewing distance was 50 cm and no time limit was set for each evaluation. 5 zago et al. statistical analysis the scores were tabulated and analyzed with spss software (version 18.0; spss, inc., chicago, il). the data were analyzed with the kruskal-wallis test, considering that it was not normally distributed. subsequently, a mann-whitney u-test was used for pairwise comparisons among groups at a 0.05 level of significance (p≤ 0.05). tooth surface (sequence) ceramic laminate veneers (sequence) step action step action 1 cleaning with pumice 1 10% hydrofluoric acid etching of inner side of the ceramic veneer for 20s 2 acid etching for 30s with 37% phosphoric acid gel 2 rinsing with water spray for 60s at 5 cm of distance 3 rinsing with water for 60s at 5cm of distance 3 air drying for 10s at 5cm of distance 4 air drying for 5s at 5cm of distance 4 silane coupling agent application 60s 5 bonding agent application and air thinning for 10s at 5cm of distance 5 bonding agent application and air thinning for 10s at 5cm of distance 6 light-curing for 10s 6 light-curing for 10s 7 cementation with light curing resin cement 7 application of light-cure resin cement on the inner side of the ceramic laminate veneer 8 positioning of the ceramic laminate veneer on the tooth 9 light-curing for 30s 10 removal of excess resin cement 11 glycerin gel application in all interfaces 12 light-curing of each interface for 30s figure 2. sequence of adhesive procedures on tooth surfaces and ceramic veneers product type chemical composition manufacturer condac porcelana ceramic etching gel low viscosity gel with 10% hydrofluoric acid, water, thickening agent and colorants fgm, joinvile, sc, brazil condac 37 acid conditioner low viscosity gel with phosphoric acid (37 wt.% in water), thickening agent and color pigments variolink veneer micro-filled, light-curing luting cement urethane dimethacrylate, decamethylene dimethacrylate, inorganic fillers, ytterbium trifluoride, initiators, stabilizers, pigments and catalysts ivoclar vivadent; schaan, liechtenstein monobond s silane coupling agent 1% 3-methacryloxypropyltrimethoxy-silane, ethanol-/ water-based solution tetric n-bond light-curing, single-component bonding agent phosphoric acid acrylate, hema, bis-gma, urethane dimethacrylate, ethanol, film-forming agent, catalysts and stabilizers virtual addition reaction silicone impression material vinyl polysiloxane, methyl hydrogen siloxane, an organoplatinic complex, silica and food dyes ips e.max press pressable lithiumdisilicate glass ceramic lithium disilicate, quartz, lithium dioxide, phosphorous oxide, alumina, potassium oxide figure 3. manufacturers and chemical compositions of the materials used in the study 6 zago et al. results a significant difference was observed between the immersion conditions. specimens stored in water presented less marginal staining, in comparison with specimens immersed in coffee (p=0.000). scores showing more intense pigmentation as score criteria image alfa absence of marginal pigmentation of the resin cement interface. bravo presence of slight, discontinuous, grayish marginal pigmentation of resin cement interface, with a predominance of regions with no staining. charlie resin cement interface completely pigmented in yellow or marginal pigmentation in yellow and brown colors, with a predominance of yellow. delta resin cement interface completely pigmented in brown, or marginal pigmentation in yellow and brown colors, with a predominance of brown. figure 4. criteria used for image evaluation and representative images of each score 7 zago et al. charlie and delta were observed only in specimens immersed in coffee mostly pe specimens whereas alfa and beta scores were noticed for both groups in water immersion. the score charlie presented tendency to be more equally distributed in all interfaces for ie and pe, while score delta, was more expressively present on pe in the mesial and distal interfaces. the absolute score distribution for each surface (cervical, mesial, distal and incisal) of pe and ie groups, immersed in water and coffee, is displayed in table 1. a comparison of the marginal staining scores of ie and pe specimens immersed in water and coffee solutions is presented in table 2. no statistical significance was found between ie and pe in water storage (p=0.45). however, pe presented significantly more marginal staining in the coffee solution than ie (p=0.01). table 3 compares all surfaces marginal staining of ie and pe in the coffee immersion. the mesial surface presented significantly more marginal staining in pe group (p=0.02). table 1. surface score distribution for prepared and intact enamel restored with ceramic veneers, immersed in water and coffee solution solution surface score prepared enamel (pe) intact enamel (ie) m d c i m d c i water alfa 0 0 0 3 2 0 1 2 bravo 8 8 8 5 6 8 7 6 charlie 0 0 0 0 0 0 0 0 delta 0 0 0 0 0 0 0 0 coffee alfa 0 0 0 0 0 0 0 0 bravo 0 0 0 0 1 0 0 0 charlie 4 6 8 7 7 8 8 7 delta 4 2 0 1 0 0 0 1 *abbreviations: m mesial, d distal; c cervical and i incisal. table 2. comparison between pe and ie in water and coffee solution mann-whitney test solution median rank values “p” value prepared enamel (pe) intact enamel (ie) water 33.50 31.50 0.45 coffee 35.89 29.11 0.01 table 3. score comparison between ie and pe specimens on each surface (mesial, distal, cervical and incisal) immersed in coffee solution mann-whitney test (n=8) solution surface ie pe “p” value coffee mesial 86.00 50.00 0.020 distal 76.00 60.00 0.44 cervical 68.00 68.00 1.0 incisal 68.00 68.00 1.0 8 zago et al. discussion the present study demonstrated that both immersion solutions and enamel preparation influenced the marginal staining of the tooth/restoration interface. marginal staining of the prepared group was significantly greater than the intact group, when immersed in coffee. based on these results, the null hypothesis was rejected. extrinsic discoloration is associated with high intake of dietary colorants15, whereas intrinsic discoloration is influenced by physicochemical reactions of resin matrix monomers, size and content of inorganic particles, as well as the characteristics of the luting agents16. therefore, esthetic failures are not due to the color change of the ceramic but as a result of the surface degradation from underlining cement and extrinsic color pigmentation17. coffee was chosen as an immersion solution, because of the high staining potential and because it is one of the most consumed beverages worldwide15. a period of 10 days of immersion corresponds to approximately one year of regular coffee consumption18. after the tenth day, the marginal finish line was visually perceptible on the prepared group. projecting these findings to clinical practice, it may be assumed that marginal staining could be a clinical issue among coffee consumers after 1 year. further studies with long-term clinical follow-up periods are required to confirm this assumption. in the present study, the mesial surface showed significantly greater marginal staining of prepared specimens immersed in coffee. such unexpected finding has no plausible explanation. the cervical area commonly presents increased staining and microleakage due to the lack of enamel in this region19. conversely, jha et al.20 (2013) found no difference in marginal adaptation between cervical, mesial, distal and incisal margins, in veneers fabricated by both the heat pressing and the refractory die techniques. restoration of lower incisors with ceramic veneers is considered a challenging task due to the limited dimensions and small amount of enamel available for bonding21. however, a retrospective study, showed similar success rates on both mandibular and maxillary incisors after 36 months22. the preparation design and enamel margins were carefully examined prior to cementation, however, the presence of thin enamel in the cervical area of the lower incisors often creates undetected dentine exposure, which may explain the increased marginal staining of the prepared specimens19. controversial results regarding the bond strength of ground and unground enamel can be observed. some studies found no difference between prepared and intact enamel, whereas others consider that unprepared enamel yields lower bond strength values23,24. in the present study, the quality of the adhesion was not evaluated, however, the least amount and intensity of staining observed on the intact surfaces suggests that the adhesion was not affected by the lack of preparation. research involving this issue should be addressed to elucidate this hypothesis. lithium disilicate reinforced ceramic was used in the fabrication of the veneers because of the excellent esthetic characteristics, biocompatibility and adhesive properties of these ceramics25. in addition, the high strength in small thickness enables the indication of minimally invasive procedures12. in this study, 0.3 mm and 0.5 mm-thick high translucency bl 3 shade ingots were used. the ceramic veneers were cemented 9 zago et al. with resin cement shade lv 1, the color shade and translucency of the cement may have affected the perception of marginal staining of specimens immersed in coffee. resin cements with opaque and more saturated shades, as well as ceramic veneers with reduced thickness, may affect the overall color change in translucent 0.5 mm-thick veneers15,26. water may induce aging of resin composites and subsequent staining27. it was possible to state that the specimens stored in water did not exhibit intense staining. the majority of the pigmentation scores were associated with no staining (alfa score) or slight discontinued grayish staining with predominance of no staining (bravo score), probably not compromising the esthetic results. the composition of the light-cured cement used in this study could explain this slight pigmentation, since tegdma may be associated with water intake and staining8, 18. artificial aging and thermocycling were not conducted. if applied, they would probably have contributed to more intense staining in either groups. apart from that, the microscopic images could have overestimated the findings. this experimental study confirms that the enamel preparation was associated with greater marginal staining, thus suggesting that maintaining unprepared intact enamel contributes to long-term esthetic outcomes. moreover, marginal staining may be influenced by different factors that should be studied individually. ceramic veneers placed on unprepared enamel are still a recent conservative trend, and long-term clinical trials are needed to evaluate if conservation of tooth structures will meet expectations and lead to high success rates. in conclusion, marginal staining was affected by the enamel preparation. cementation of ultra-thin ceramic veneers to intact enamel provides a dental restoration interfaces with less marginal staining. clinical relevance ultra-thin veneers with no enamel 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doi: 10.1016/j.dental.2002.11.001. 1http://dx.doi.org/10.20396/bjos.v20i00.8661223 volume 20 2021 e211223 original article 1 academic of the speech therapy course – federal university of health sciences of porto alegre ufcspa, porto alegre, brazil 2 department of public health, federal university of health sciences of porto alegre – ufcspa, porto alegre, brazil 3 department of speech-language pathology, federal university of health sciences of porto alegre – ufcspa, porto alegre, brazil 4 cristo redentor hospital – conceição hospital group, porto alegre, brazil corresponding author: esther da cunha rodrigues. 245, sarmento leite. porto alegre, rio grande do sul. brasil. zip code: 90050-170 e-mail: esthercunha.rs@gmail.com editor: dr altair a. del bel cury received: september 15, 2020 accepted: february 10, 2021 diagnostic properties of sensitivity changes in patients with maxillofacial fractures: a systematic review esther cunha rodrigues1,* , eliana márcia da ros wendland2 , deisi cristina gollo marques vidor3 , karoline weber dos santos4 aim: verify the accuracy of objective assessments compared to subjective tests in detecting changes in somatosensory perception in individuals affected by maxillofacial trauma. methods: the review (prospero n ° crd42019125546) used the databases: medline, cochrane, embase, lilacs and other bibliographic resources. prospective and retrospective studies that used objective and subjective methods of assessing facial sensitivity in maxillofacial fractures were included. there was no restriction on language or publication date. risk of bias was assessed using the quadas-2. data extraction and analysis were performed using a form developed for the study. results: 21 studies were included. the clinical objective examination mainly includes assessments of: tactile sensitivity (95.24%) and nociceptive sensitivity (57.14%).the subjective assessment was based on the patient’s report, spontaneously (61.90%), guided by structured questionnaires (33.33%) and/or using scales (9.52%) to measure the degree of impairment. in risk of bias assessment, zhuh�revhuyhg�qr�dghtxdwh�lqwhusuhwdwlrq�dqg�fodvvlͤfdwlrq�ri� changes in subjective sensitivity, subject to inappropriate analysis of the data. in addition, the studies bring several instruments without standardization for assessing sensory modalities. conclusion: the objective assessment is a complement to the subjective assessment, using the touch assessment as the main sdudphwhu�lq�wkh�surͤoh�ri�wkh�idfldo�shulskhudo�lqwhjulw\��dvvrfldwhg� or not with nociceptive assessment. lack of consensus on the lqglfdwlrq�ri�vshflͤf�lqvwuxphqwv�iru�whvwlqj�lv�d�olplwlqj�idfwru�� thus, based on the studies, is proposed a minimum battery of sensitivity assessment to obtain an overview of the patient’s peripheral nervous situation. keywords: facial injuries. zygomatic fractures. jaw fracture. somatosensory disorders. sensation disorders. systematic reviews as topic. https://orcid.org/0000-0001-5745-0094 https://orcid.org/0000-0001-6356-6413 https://orcid.org/0000-0003-4805-6145 https://orcid.org/0000-0003-0524-5878 2 rodrigues et al. introduction trauma involving the skull and face are among the leading causes of morbidity and mortality, especially in the young population1. it is estimated that, globally, there are 7.5 million new cases of facial fractures with 1.8 million people living with their comorbidities2��:lwk�rffxuuhqfh�lq�pruh�vljqlͤfdqw�qxpehuv�lq�pdoh�lqglylgxdov�� lqmxulhv�duh�wkh�uhvxow�ri�wudiͤf�dfflghqwv��idoov��sk\vlfdo�ylrohqfh��dvvdxowv��rffxsdtional, and sports accidents1,3. in general, besides bone fractures, individuals have other injuries that can limit their functional capacity3. among these traumas, maxillofacial traumas, as well as their repair procedures, cause bone dislocations that can result in lesions in the peripheral nerve, which are responsible for facial sensation and perception. thus, compression, sectioning, or stretching of the branches of the trigeminal nerve (v1, v2, and v3) and the nerves of the cervical plexus (c1 and c2)4 may result in somatosensory changes that impact functionality, quality of life, and well-being of individuals. it may impair the functions of chewing, breathing, swallowing, sucking, and speaking5. the diagnosis of these changes is based on clinical and instrumental assessment, which considers the patient’s report, the use of subjective questionnaires and quantitative neurosensory tests5,6. 6xemhfwlyh�yhulͤfdwlrq�edvhg�rq�wkh�v\pswrp�uhihuuhg�e\�wkh�sdwlhqw�lv�wkh�jrog� standard to determine the diagnosis, as it considers aspects of somatosensory perception more comprehensively. in it, individuals are submitted to a qualitative assessment of changes in sensory perception5,6. it considers parameters such as the presence or absence of change and the description of the change sensation5. the objective assessment of sensory changes, represented by quantitative tests, is based into parameters that assess the patient’s perception according to the different somatosensory modalities explored. it determines the profile of the detection of thermal, painful, touch, and proprioceptive stimuli, using instruments to identify perception and quantitative measurement of perceptual thresholds6. the objective assessment of facial sensitivity uses different techniques. they can be classified according to the type of fiber being stimulated. it may be dvvrfldwhg�zlwk�$ƌ�ilehuv��p\holqdwhg��wkurxjk�whvwv�lqyroylqj�wrxfk�vhqvdwlrq�� ru�p\holqdwhg�$�ǝ�ilehuv�dqg�&�ilehuv��qrw�p\holqdwhg��e\�yhuli\lqj�wkhupdo�dqg� nociceptive sensation)6. thus, the present study aims to conduct a systematic review of the literature to verify the accuracy of objective tests compared to subjective tests of facial sensitivity in detecting changes in somatosensory perception in individuals affected by maxillofacial trauma. materials and methods this review was conducted based on the guidelines proposed by the cochrane handbook for systematic reviews of diagnostic test accuracy reported follow3 rodrigues et al. ing the prisma7 recommendations and registered in prospero under number crd42019125546. studies that used objective and subjective methods of assessing facial sensitivity in patients with maxillofacial trauma were included. criteria for including studies in this review types of studies studies that used objective and subjective methods to assess facial sensitivity to detect peripheral somatosensory changes resulting from maxillofacial trauma were included. prospective and retrospective studies were considered, provided they had both exams. participants studies with an assessment of patients with sensory changes in the peripheral nervous system originating from trauma or postoperative traumatic maxillofacial injuries. participants underwent at least one of the modalities of objective assessment and at least one modality of subjective sensitivity assessment. index test changes in facial sensitivity must have been assessed objectively using quantitative tests or scales. reference standard all patients must have been subjected to a subjective assessment of changes in sensory perception considering the following parameters: presence or absence of change or description of the sensation. target conditions changes in the peripheral sensory perception of the face. 6hdufk�phwkrgv�iru�vwxg\�lghqwlͤfdwlrq� the search was carried out in the medline, cochrane, embase, scopus, and lilacs databases of articles published until march 2019, using the following terms and their correlates: “facial fractures,” “zygomatic-orbital fracture,” “mandibular fractures,” and “somatosensory disorders.” the search strategy for each database is available in appendix 1. the search was complemented by the manual review of rwkhu�eleolrjudsklf�uhvrxufhv�lq�wkh�khdowk�ͤhog��vxfk�dv�*rrjoh�6fkrodu��2shq*uh\�� proquest, dissertations, theses, and reference lists. there was no restriction on language or publication date. the authors of the selected studies were contacted to uhtxhvw�plvvlqj�ru�lqvxiͤflhqw�gdwd� 4 rodrigues et al. data collection and analysis selection of studies the studies were initially analyzed by title and abstracts by two independent evaluators (kws and ecr), including studies that met the eligibility criteria. a third evaluator (dcgmv) judged doubts regarding the inclusion to obtain consensus. those eligible in this stage were read in full for a final decision on their inclusion. those selected were registered on a form regarding inclusion or exclusion in the study at each step of the selection process, as well as the respective reasons. data extraction and management 7kh�gdwd�iurp�wkh�lqfoxghg�vwxglhv�zhuh�h[wudfwhg�lqwr�d�irup�ghyhorshg�vshflͤfdoo\� for this analysis. first, data on the characteristics of the studies and their population were tabulated. data were also extracted regarding the objective and subjective assessment methods used, as well as a description of the facial sensitivity assessment techniques performed. assessment of methodological quality the studies were assessed regarding quality using the quality assessment tool for diagnostic accuracy studies (quadas-2)8 by two independent evaluators (kws and ecr) and, in case of disagreement, a third evaluator (dcgmv) was consulted. divided into four domains (patient selection, index test, reference standard, and flow and timing) the quadas-2 tool analyzes the methodological quality of the included studies, judging the risk of bias and applicability8. results study selection out of the 7782 titles and abstracts analyzed from the search strategies, 135 met the eligibility criteria for reading the full manuscript. the authors of four articles9-12 were contacted for more information on the methodology used in their studies, but they were excluded due to a lack of responses. thus, for quality analysis, 21 studies13-33 were included. the prisma flow diagram (figure 1) provides, according to the different phases of the systematic review, the registration of the identified, included and excluded studies, and the reasons for the exclusions. 5 rodrigues et al. study characteristics the characteristics of the studies included are described in table 1. the studies were predominantly observational (85.71%), with a sample composed of individuals aged between 11 and 83 years, mostly males. individuals from 11 years old were included when they presented the same type of intervention used in adults34. despite the literdwxuh�flwlqj�vshflͤf�idfldo�vhqvlwlylw\�whvwv�wkdw�vkrz�gliihuhqw�dffruglqj�wr�wkh�lqglvidual’s age and sex4, the studies included in this review do not show differences in results in the assessments regarding the age and sex variables. among the causes of trauma, from the most recurring to the least common are: wudiͤf�dfflghqwv��sk\vlfdo�ylrohqfh��dvvdxowv��idoov��vsruwv�dfflghqwv��ͤuhdup�lqmxulhv�� work accidents, and domestic accidents. the most frequent type of fracture was the middle third of the face (52.38%), involving the regions of the zygoma, maxilla, and orbit; mandibular fractures (38.10%), including the regions of the body, angle, branch, v\psk\vlv�� sdudv\psk\vlv�� khdg�� dqg� frqg\odu� surfhvv�� dqg� ͤqdoo\�� elpd[loodu\� ���������&rqfhuqlqj�vxujlfdo�lqwhuyhqwlrqv��prvw�ri�wkhp�kdg�d�uljlg�lqwhuqdo�ͤ[dwlrq� with open reduction (76.19%), with intra-oral (37.5%), extra-oral (37.5%), or combined ������lqflvlrqv��6rph�vwxglhv���������phqwlrq�wkh�xvh�ri�pd[loorpdqglexodu�ͤ[dwlrq� to stabilize the fracture, and others also bring conservative treatment (14.28%) as an option for trauma management. records identified through database search (n = 5733) additional records identified through other sources (n = 2111) id en tifi ca tio n sc re en in g el ig ib ili ty in cl ud ed records after duplicates removed (n = 7782) records screened based on title and abstract (n = 7782) records excluded (n = 7647) did not fulfill screening/ inclusion criteria full-text articles assessed for eligibility (n = 135) studies included in qualitative synthesis (n = 21) full-text articles excluded, with reasons (n = 114) 91 studies without index test, reference test or both; 4 studies not fulfill methodology set out in inclusion criteria; 15 studies the population is not adequate to the elegibility criteria; 4 unavailable studies. figure 1.�46-71%�ƽs[�hmekveq� 6 rodrigues et al. ta bl e 1. c ha ra ct er is tic s of th e st ud ie s in cl ud ed a ut ho r, y ea r c ou nt ry s tu dy d es ig n a ge n (f /m ) c au se o f t ra um a lo ca ti on o f f ra ct ur e m ax ilo fa ci al s ur ge ry s ur gi ca l i nc is io n a nc hl ia , 20 18 13 in di a 3 ri �e vq �g pmr mg ep � tr ia l a ve ra ge 3 0 ye ar s 20 (6 /1 4) 8v eƾ g� eg gm hi rx w� �� �� � �� -r xi vt iv ws re p�z ms pi rg i� �� �� � m an di bl e fr ac tu re s: b od y fr ac tu re s (p as si ng xl vs yk l� xl i� q ir xe p�j sv eq mr e o r if in tr ao ra l v es tib ul ar in ci si on n eo vi us , 20 17 14 7[ ih ir r et ro sp ec tiv e c oh or t n d 81 (1 6/ 65 ) 8v eƾ g� eg gm hi rx w� �� � % ww ey pxw ��� � *e pp� mr ��� � 3 xl iv w� �� � -w sp ex ih �^ ]k sq ex mg �jv eg xy vi � �� � -w sp ex ih �f ps [ �s yx �jv eg xy vi ��� � >] ks q ex mg �jv eg xy vi �g sq fm ri h� [ mxl �e �f ps [ �s yx �jv eg xy vi �e rh � �� & mpe xi ve p�s v�q yp xmt pi �jv eg xy vi � �� �� o r a nd / or o r if 0s [ iv �i ]i pmh �mr gm wm sr o ko ch i, 20 15 15 ja pa n r et ro sp ec tiv e c oh or t �� �� �r �� �� � 10 (4 /6 ) *e pp� mr ��� � 7t sv xw �e gg mh ir xw ��� � � 8v eƾ g� eg gm hi rx w� �� � 9 rm pe xi ve p�^ ]k sq ex mg sq e\ mpp ev ]� gs q tp i\ �jv eg xy vi o r if 0e xi ve p�i ]i fv s[ ��p s[ iv � ey el id s ub ci lia ry a nd in tr ao ra l a pp ro ac h sc ot t, 20 14 16 u k r et ro sp ec tiv e c oh or t n d 15 0 n d & sh ]� �e rk pi �e rh �ve q yw ��t ew wm rk � fi x[ ii r� xl i� q er hm fy pe v�e rh � q ir xe p�j sv eq ir o r if in tr ao ra l a pp ro ac h m ay rin k, 20 12 17 b ra zi l p ro sp ec tiv e c oh or t a ve ra ge �� �� �� (r an ge 1 5 to 6 8) 19 (4 /1 5) 8v eƾ g� eg gm hi rx w� �� �� �� *e pp� �� �� � -r xi vt iv ws re p�z ms pi rg i� �� �� �� 7] q tl ]w mw ��f sh ]� �e rk pi ��v eq yw � an d co nd yl ar p ro ce ss , i so la te d sv �g sq fm ri h o r if )\ xv e� fy gg ep ��x ve rw ��e rh � in tr ab uc ca l ( in b od y an d co nd yl e fr ac tu re s mr �xl i� we q i� te xmi rx b ag he ri, 20 09 18 u sa r et ro sp ec tiv e c oh or t a ve ra ge �� �� ��v er ki � 11 to 6 1) 42 (1 7/ 2 5) n d >] ks q ex mg sq e\ mpp ev ]� gs q tp i\ jv eg xy vi ��t ev ew ]q tl ]w mw ��e rk pi �� ve q yw �e rh �q er hm fp i� fs h] n d n d sa ka vi ci us , 20 08 19 li th ua ni a r et ro sp ec tiv e c oh or t m ea n 32 ,1 7 (r an ge 1 5 to 7 8) 47 8 (8 6/ 39 2) % ww ey pxw ��� �� �� � 6 se h� xv eƾ g� eg gm hi rx w� �� �� �� � 7t sv xw �e gg mh ir xw ��� �� �� � 3 xl iv w� �� �� �� 9 rm pe xi ve p�^ ]k sq ex mg sq e\ mpp ev ]� gs q tp i\ �jv eg xy vi w c lo se d re du ct io n or o r if if hm wt pe gi q ir x c lo se d re du ct io n or ps [ iv �i ]i pmh �w yf gm pme v] � ap pr oa ch b ar ry , 20 07 20 ir el an d r et ro sp ec tiv e c oh or t 1 ie r� �� �� � (r an ge 1 6 to 4 2) 50 (2 /4 8) in te rp er so na l v io pi rg i� �� � 7t sv xmr k� mr ny v] ��� � *e ppw ��� � 1 sx sv �z il mg pi �e gg mh ir xw ��� % rk pi �e rh �ve q yw ��� �� [ iv i� ew ws gm ex ih �[ mxl �e r� mq te gx ih �s v� iv yt xi h� xl mvh �q sp ev ��� xi ix l� mr �xl i� lin e of fr ac tu re ) 3 6 -* �[ mxl �1 1 *� if oc cl us al hm wt pe gi q ir x )\ xi rh ih �xl mvh �q sp ev � in ci si on c on tin ue 7 rodrigues et al. c on tin ua tio n iiz uk a, 19 91 21 fi nl an d p ro sp ec tiv e c oh or t 1 ie r� �� �� � (r an ge 1 6 to 8 3) 13 3 (2 5/ 10 8) n d & sh ]� �e rk pi �e rh �ve q yw ��t ew wm rk � fi x[ ii r� xl i� q er hm fy pe v�g er ep o r if )\ xv es ve p� �w yf q er hm fy pe v�s v� vi xv sq er hm fy pe v �e rh � in tr ao ra l a pp ro ac h fo ga ça , 20 04 22 b ra zi l r et ro sp ec tiv e c ro ss -s ec tio na l n d 25 n d 9 rm pe xi ve p�^ ]k sq e� jv eg xy vi w o r if n d fa ya zi , 20 13 23 + iv q er ] p ro sp ec tiv e ' sl sv xɸ �� r� �� � (r an ge 10 to 6 5 ye ar s) 49 (5 /4 4) 1 sx sv �z il mg pi �e gg mh ir x�� �� �� � 7t sv x�i zi rx w� �� �� � -r xi vt iv ws re p�z ms pi rg i� �� �� � � *e ppm rk ��� �� 4 ev ew ]q tl ]w mw ��w ]q tl ]w mw �� co nd yl ar p ro ce ss a nd h ea d, fs h] ��e rk pi ��v eq yw ��g sv sr sm h� tv sg iw w� jv eg xy vi wɸ m m f er ic h ar ch b ar s si dd iq ui , 20 07 24 u k 6 er hs q m^ ih � gs rx vs ppi h� xv me pɸ r an ge 1 7 to 5 7 85 (1 0/ 75 ) n d % rk pi �e rh �ve q yw ��g sq q mr yx ih � jv eg xy vi w� [ iv i� i\ gp yh ih o r if % x�x li �i \x iv re p�s fp mu yi � rid ge o r t ra ns bu cc al 1 g+ mq tw i] �� 20 00 25 u k c ro ss -s ec tio na l r an ge 1 1 to 8 0 45 (4 5m ) % ww ey px� �� �� �� �* ep pw ��� �� �� � 8v eƾ g� eg gm hi rx w� �� �� �� sp or ts eg gm hi rx w� �� �� � >] ks q ex mg �g sq tp i\ g ill ie s el ev at io n, 4 sw [ mpp s� ls so �� e� gs q fm re xms r� of b ot h g ill ie s er h� 4 sw [ mpp s� sv � co ns er va tiv e 8i q ts ve p�� + mpp mi w � ap pr oa ch v rie ns , 19 98 26 n et he rla nd s p ro sp ec tiv e c oh or t �� �� r� �� �� � (r an ge 1 4 to 7 7) 65 n d >] ks q ex mg sq e\ mpp ev ]� gs q tp i\ fr ac tu re a nd o rb it �j vs rx s^ ]k sq ex mg �w yx yv i� sv � sv fm xe p�f ps [ �s yx � o r if 8i q ts ve p�� + mpp mi w � ap pr oa ch k ip pe r, 20 16 27 b ra zi l c ro ss -s ec tio na l st ud y a bo ve 1 8 ye ar s 14 n d >] ks q e� er h� sx li v�j ve gx yv iw n d n d fo ga ça , 20 08 28 b ra zi l c ro ss -s ec tio na l st ud y n d 25 (9 /1 6) n d >] ks q e� er h� sv fm x o r if n d / iw ev [ er m�� 19 89 29 c an ad a r et ro sp ec tiv e c oh or t m ea n 37 (r an ge 1 8 to 5 7) 20 (4 /1 6) n d 4 er je gm ep �jv eg xy vi w� �q e\ mpp e� � sv fm x�� re ws ix lq sm h� �^ ]k sq e� er h� q er hm fp i o r if ' sv sr ep �ƽ et w� � su bc ili ar y, u pp er a nd ps [ iv �f yg ge p�w yp gy wɸ a bd el -k ad er , 20 11 30 eg yp t 3 ri �e vq �g pmr mg ep � xv me pɸ m ea n 31 (r an ge 2 0 to 4 2) 12 (2 /1 0) 8v eƾ g� eg gm hi rx w� �� �� �� -r xi vt iv ws re p�z ms pi rg i� �� �� �� >] ks q ex mg sq e\ mpp ev ]� gs q tp i\ � fr ac tu re o r if su bc ili ar y, bu cc og in gi va l a nd pe xi ve p�i ]i fv s[ � ap pr oa ch es c on tin ue 8 rodrigues et al. c on tin ua tio n le on ha rd t, 20 05 31 + iv q er ] p ro sp ec tiv e c oh or t m ea n 31 (r an ge d 15 to 7 0) 30 n d % rk pi �e rh �ve q yw o r if in tr ao ra l m ar zo la , 20 06 32 b ra zi l p ro sp ec tiv e c oh or t r an ge 1 1 to 5 1 10 0 (1 9/ 81 ) -r xi vt iv ws re p�z ms pi rg i� �� � � 8v eƾ g� eg gm hi rx w� �� � ( sq iw xmg �e gg mh ir xw ��� � 7t sv xw �e gg mh ir xw ��� ; sv om rk �e gg mh ir xw ��� 3 xl iv w� �� � � >] ks q ex mg sq e\ mpp ev ]� gs q tp i\ � fr ac tu re o r if o r co ns er va tiv e n d % lq ih �� 20 10 33 in di a p ro sp ec tiv e c oh or t *v sq �� �� ye ar s 13 3 (3 /1 30 ) n d >] ks q e n d n d 0i ki rh ��2 ( ��r sx �h iw gv mf ih ��3 6 -* ��3 ti r� 6 ih yg xms r� -r xi vr ep �* m\ ex ms r� �1 1 *� �1 e\ mpp sq er hm fy pe v�* m\ ex ms r 9 rodrigues et al. risk of bias figure 2 gathers the results of the quality analysis, which is described below. patient selection thirteen (61.90%) studies13-17,19,21,25-31 had a high risk of bias and 8 (38,10%) studies17,18,20,22-24,32,33 had a low risk due to comparing individuals with changes to healthy individuals in case-control designs. as for applicability, all studies were convlghuhg�dghtxdwh��vlqfh�wkh�sdwlhqw�surͤoh�zhuh�lq�dffrugdqfh�zlwk�wkh�holjlelolw\�fulteria listed for this review. index test as for the risk of bias concerning the objective sensitivity test, 20 (95.24%) studies13-21,23-33 had high risk, and 1 (4.76%) study22 had low risk. the high rate of bias was due to the lack of blinding to subjective assessment by the examiner to perform the objective tests. the non-independent assessment may have distorted the execution or interpretation of the objective test. also, there was no adequate description of the interpretation of the tests, without description of diagnostic thresholds. as for applicability, all studies were considered adequate, as they contemplate the review proposal. reference standard 3 (14.29%) studies22,29,33 had high risk and 18 (85.71%) studies13-21,23-27,29-32 low risk. it was considered a low-risk criterion when the reference test was conducted according to the patient’s report, without adaptation of the terms by the researchers. as for applicability, 19 (90.48%) studies13-21,23-32 showed good applicability, and 2 (9.52%) studies22,33 showed flaws in their applicability. in one of the studies, there was an interpretation of the perceptual responses by the authors33, which may distort the data obtained. in another, the assessment procedures were not adequately described iru�dffxudwh�fodvvlͤfdwlrq22. figure 2.�1ixlshspskmgep�izepyexmsr�eggsvhmrk�xs�59%(%7���sj�xli�mrgpyhih�wxyhmiw� patient selection index test reference standard flow and timing 0% 25% 50% 75% 100% risk of bias applicability concerns high unclear low 0% 25% 50% 75% 100% 10 rodrigues et al. flow and timing $oo�vwxglhv�zhuh�fodvvlͤhg�dv�kdylqj�d�orz�ulvn�ri�eldv��dv�lw�zdv�frqvlghuhg�wkdw�wkh� application interval between the reference test and the index test is not a variable that can interfere with the test results. all patients in the studies were submitted to the index and reference tests and included in the data analysis. assessments the characteristics of the facial sensitivity assessment are described in table 2. the assessment moments involved periods of the preoperative period (4.76%), postoperative period (47.62%), and both (42.86%), and some had followed up to complete nervous recovery (4.76 %). the postoperative follow-up time varied, being shuiruphg�lq�wkh�shulrg�ehwzhhq�wkh�ͤuvw�zhhn�����������wkh�ͤuvw�prqwk����������� the second month (14.28%), the third month (42.86%), the sixth month (42.86%), dqg�wkh�ͤuvw�\hdu�����������6rph�vwxglhv14-16,29 extend the follow-up to more than one year (19.05%) after surgery. facial sensitivity assessments were performed to check the activity of the following nerve portions: infraorbital (61.90%), lower alveolar (38.10%), supraorbital (4.76%), lingual (4.76%), and buccal (4.76%). thus, classifying the assessments from the main branch of the trigeminal nerve, it is observed: 61.90% ophthalmic branch, 42.86% mandibular branch, and 4.76% maxillary branch. as for the assessed facial region, the assessment of the ophthalmic branch was performed on the upper lip (53.85%), cheeks (38.46%), nasal and paranasal region (46.15%), eyelids (23.08%), gingiva (7.69%), and forehead (7.69%). the activity of the maxillary branch was observed in the region of the cheeks (100%) and the assessments of the mandibular branch in the lower lip (80%), chin (40%), labial commissures (10%), and lower border of the mandible (10%). :khq� dvvhvvlqj� $ƌ� w\sh� ͤehuv�� wrxfk� vhqvdwlrq� ���������� wkh� iroorzlqj� phwkods were used: light touch/static light touch (59.09%), two-point discrimination (45.45%) moving (20%) or static (60%), mechanical detection threshold (18.18%), direction sensation (13.63%), moving-touch discrimination (9.09%), stimulus localization (4.54%), vibratory sensation (4.54%), and trigeminal somatosensory evoked potential (4.54%). $v�iru�$ǝ�dqg�&�ͤehuv��wkhupdo�vhqvdwlrq����������sdlqixo���������ru�erwk����������� the following measurements were used: painful stimuli/pinprick (75%), pain detection threshold (25%), thermal sensation (25%), and thermal discrimination (8.33%). 11 rodrigues et al. ta bl e 2. �* eg me p�w ir wm fm pmx ]� ew wi ww q ir x a ut ho r an d ye ar pu bl is he d m om en ts o f ev al ua ti on m om en ts o f ev al ua ti on – fo llo w u p n er ve as se ss ed ev al ua ti on r eg io n $ ƌ� ͤe hu v� �w rx fk � $ ǝ� ͤe hu v� dq g� & � ͤe hu v� �w hp sh ud wx uh � an d pa in ) s ub je ct iv e ev al ua ti on p re op . p os op . a nc hl ia , 20 18 13 n y 1s t, 4t h an d 12 th [ ii ow �e jx iv �w yv ki v] ia n ( v 3) 0s [ iv �pm t 0m kl x�x sy gl ��x [ s� ts mr x� hm wg vmq mr ex ms r p in p ric k q ue st io nn ai re n eo vi us , 20 17 14 n y �� ɓ �� �� �] ie vw io n ( v 1) a la r b as e an d up pe r l ip 0m kl x�x sy gl ��q ig le rm ge p� de te ct io n th re sh ol d n e sc al e (0 -1 00 ) o ko ch i, 20 15 15 y y -q q ih me xi p] ��� �] ie v� an d 5 ye ar io n ( v 1) ey el id 0m kl x�x sy gl ��q ig le rm ge p� de te ct io n th re sh ol d c ur re nt p er ce pt io n th re sh ol d p at ie nt ’s re po rt sc ot t, 20 14 16 n y 1 mr mq yq �s j�� �� q sr xl w� �q e\ mq yq � rs x�w ti gm ƽi h ia n ( v 3) 0s [ iv �pm t li gh t t ou ch n e q ue st io nn ai re an d sc al e (0 -1 0) m ay rin k, 20 12 17 y y �� [ ii o� �� �� ��� � q sr xl w� er h� �] ie v ia n ( v 3) 0e fm ep �g sq q mw wy vi ��g lm r� er h� ho riz on ta lly b y la bi al in fe rio r fs vh iv ��q ir xs pe fm ep �js ph iv �e rh � ps [ iv �f sv hi v�s j�q er hm fp i st at ic li gh t t ou ch , b ru sh di re ct io na l s tr ok e 8l iv q ep � hm wg vmq mr ex ms r� er h� pi n p ric k q ue st io nn ai re b ag he ri, 20 09 18 y y % jx iv �xv ey q e� � tv i� st ��� �[ ii o� �� �� �� �� �q sr xl w� er h� 1 ye ar ia n ( v 3) , i o n (v 1) , l n ( v 3) an d b n ( v 3) n d 7x ex mg �pm kl x�x sy gl ��q sz mr k� fv yw l� wx vs oi w� �w xmq yp yw �ps ge pm^ ex ms r� � wx ex mg �� �t sm rx �h mw gv mq mr ex ms r 4 em rj yp �w xmq yp m p at ie nt ’s re po rt sa ka vi ci us , 20 08 19 y y �� [ ii ow ��� ��� ��� �e rh � �� �q sr xl w io n ( v 1) rs wi ��g li io ��p s[ iv �i ]i pmh �� up pe r l ip , g in gi va l a nd te et h n e p ai n de te ct io n th re sh ol d p at ie nt ’s re po rt an d cl in ic al w] q tx sq w� b ar ry , 2 00 72 0 y y n d ia n ( v 3) n d �� ts mr x�h mw gv mq mr ex ms r� �h mvi gx ms r� se ns at io n 8l iv q ep �w ir we xms r q ue st io nn ai re iiz uk a, 19 91 21 y y �� �� [ ii ow �e rh �� �� �� q sr xl w� sr �e zi ve ki ia n ( v 3) ' lm r� er h� ps [ iv �pm t 0m kl x�x sy gl ��[ mxl �g sx xs r� [ ss p p in pr ic k (s ha rp /b lu nt hm jj iv ir xme xms r� [ mxl � a sh ar p de nt al p ro be ) p at ie nt ’s re po rt fo ga ça , 20 04 22 n y n e io n ( v 1) >] ks q ex mg sx iq ts ve p�v ik ms r� � pa ra na sa l r eg io n, a nd u pp er lip 7x ex mg �e rh �q sz mr k� xs yg l� hm wg vmq mr ex ms r� �w xe xmg �x[ s� ts mr x� hm wg vmq mr ex ms r n e p at ie nt ’s re po rt c on tin ue 12 rodrigues et al. c on tin ua tio n le on ha rd t, 20 05 31 + iv q er ] p ro sp ec tiv e c oh or t m ea n 31 (r an ge d 15 to 7 0) 30 n d % rk pi �e rh �ve q yw o r if in tr ao ra l m ar zo la , 20 06 32 b ra zi l p ro sp ec tiv e c oh or t r an ge 1 1 to 5 1 10 0 (1 9/ 81 ) -r xi vt iv ws re p�z ms pi rg i� �� � � 8v eƾ g� eg gm hi rx w� �� � ( sq iw xmg �e gg mh ir xw ��� � 7t sv xw �e gg mh ir xw ��� ; sv om rk �e gg mh ir xw ��� 3 xl iv w� �� � � >] ks q ex mg sq e\ mpp ev ]� gs q tp i\ � fr ac tu re o r if o r c on se rv at iv e n d % lq ih �� 20 10 33 in di a p ro sp ec tiv e c oh or t *v sq �� �� ]i ev w 13 3 (3 /1 30 ) n d >] ks q e n d n d v rie ns , 19 98 26 n y % zi ve ki �� �� �q ��7 ( � �� �� �ve rk i� �� �� � q sr xl w io n ( v 1) c he ek , o n ha irbe ar in g sk in o f up pe r l ip 7x ex mg �xs yg l� �w xe xmg �x[ s� ts mr x� hm wg vmq mr ex ms r c ol d se ns at io n an d pi np ric k p at ie nt ’s re po rt k ip pe r, 20 16 27 y n n e io n ( v 1) c he ek a nd u pp er li p st at ic to uc h n e qu es tio nn ai re fo ga ça , 20 08 28 n y n e io n ( v 1) >] ks q ex mg �vi km sr ��t ev er ew ep � re gi on , u pp er li p 7x ex mg �x[ s� ts mr x� hm wg vmq mr ex ms r� gy xe ri sy w� pr es su re th re sh ol d (s ta tic p oi nt ), cu ta ne ou s pr es su re th re sh ol d �h ]r eq mg �t sm rx �� gy xe ri sy w� pr es su re th re sh ol d (s ta tic x[ s� ts mr x ��g yx er is yw �t vi ww yv i� xl vi wl sp h� �h ]r eq mg �x[ s� ts mr x n e p at ie nt ’s re po rt / iw ev [ er m�� 19 89 29 n y �] ��q ie r� xi wx mr k� xmq i� �� ] su pr ao rb ita l ne rv e (v 1) , io n ( v 1) , i a n (v 3) fo re he ad , c he ek , c hi n an d th e zi vq mpm sr �s j�x li �ps [ iv �pm t 1 sz mr k� er h� wx ex mg �x[ s� ts mr x� hm wg vmq mr ex ms r� �z mf ve xs v] �� cu ta ne ou s pr es su re th re sh ol ds n e p at ie nt ’s re po rt an d vi br at or y pe rc ep tio n a bd el -k ad er , 20 11 30 y y �� �e rh �� �� [ ii ow io n ( v 1) 0s [ iv �i ]i pmh ��p ex iv ep �w om r� sj � th e no se , u pp er li p 8v mk iq mr ep �w sq ex sw ir ws v] � ev ok ed p ot en tia l ( ts ep ) n e q es tio nn ai re c on tin ue 13 rodrigues et al. c on tin ua tio n le on ha rd t, 20 05 31 + iv q er ] p ro sp ec tiv e c oh or t m ea n 31 (r an ge d 15 to 7 0) 30 n d % rk pi �e rh �ve q yw o r if in tr ao ra l m ar zo la , 20 06 32 b ra zi l p ro sp ec tiv e c oh or t r an ge 1 1 to 5 1 10 0 (1 9/ 81 ) -r xi vt iv ws re p�z ms pi rg i� �� � � 8v eƾ g� eg gm hi rx w� �� � ( sq iw xmg �e gg mh ir xw ��� � 7t sv xw �e gg mh ir xw ��� ; sv om rk �e gg mh ir xw ��� 3 xl iv w� �� � � >] ks q ex mg sq e\ mpp ev ]� gs q tp i\ � fr ac tu re o r if o r c on se rv at iv e n d % lq ih �� 20 10 33 in di a p ro sp ec tiv e c oh or t *v sq �� �� ]i ev w 13 3 (3 /1 30 ) n d >] ks q e n d n d le ge nd : n e: n ot e va lu at ed ; n d : n ot d es cr ib ed ; i a n : i nf er io r a lv eo la r n er ve ; i o n : i nf ra or bi ta l n er ve ; l n : l in gu al n er ve ; b n : b uc ca l n er ve ; 14 rodrigues et al. procedures and measurements as for subjective assessment, it is always performed before the objective clinical examination, from touching the affected region, using materials, or the gloved hand. the subjective assessment was carried out based on the patient’s report, spontaneously (61.90%) or guided by structured questionnaires (33.33%), or using scales created for the respective studies (9.52%). when assessments based on the reports are used, they could take place from unstructured conversations between the researcher and the patient or contain questions with yes or no answers. the questions were related to changes in sensitivity, numbness, burning and tingling sensation, thermal sensitivity, pain, functional changes (mainly during feeding, such as bites on the lips and escape of food from the oral cavity) and interference in the individual’s daily life and quality of life. some studies guide the comparison of sensory differences on the injured side with a region of the face with uninvolved innervation or a sensitive region of another part of the body. the use of scales sugjhvwv�wkdw�wkh�sdwlhqw�fodvvlͤhv�wkh�fkdqjh�lq�fdwhjrulhv��7kh�prvw�frpprq�duh� represented visually by numbers, where zero corresponds to the absence of sensory complaints, and ten/hundred corresponds to severe sensory changes. for the subjective assessment to be reliable, the patient’s report must be considered. for this, the evaluator must investigate the sensory complaint, asking the patient to explain and describe the altered sensation. as for the objective assessment, studies advise that patients should be examined in a quiet room, with their eyes closed and in a comfortable position, preferably with a headrest. for each type of assessment, procedures are cited for carrying out the different measurements proposed. the studies bring the following measurements and procedures/techniques for assessing touch and nociceptive sensation: • light touch/static light touch (61.90%) assessment of detection of light touch vwlpxoxv��vorzo\�dgdswlqj�qhuyh�ͤehuv���2swl+dlu�yrq�)uh\�ͤodphqwv�(marstock nerve test, marburg, germany)14, 6hpphv�:hlqvwhlq� prqrͤodphqw� (esthesiometer)15,17,28,28, 0.7-mm-gauge needle (bd precision glidetm)17, pressure-speciͤhg�6hqvru\�'hylfh�(pssd)22,28 and cotton roll25; • mechanical detection threshold (19.04%) a gradual measurement of the detection of light touch stimulus, of ascending and descending character to determine wkh�wkuhvkrog��vorzo\�dgdswlqj�qhuyh�ͤehuv���2swl+dlu�yrq�)uh\�ͤodphqwv (marstock nerve test, marburg, germany)14 and 6hpphv�:hlqvwhlq� prqrͤodphqw (esthesiometer)15,28,29; • direction sensation (14.28%) assessment of the detection of the direction of movement, differentiation of movements up, down, right or left (rapidly-adapting qhuyh�ͤehuv���0.7-mm-gauge needle (bd precision glidetm)17 and dental cotton swab25; • two-point discrimination static or moving (47.62%) assessment of the miqlpxp� glvwdqfh� ehwzhhq� wzr� vwdwlf� srlqwv� �vorzo\�dgdswlqj� qhuyh� ͤehuv�� ru� prylqj��udslgo\�dgdswlqj�qhuyh�ͤehuv��wkdw�wkh�sdwlhqw�fdq�glvfulplqdwh�� presvxuh�6shflͤhg�6hqvru\�'hylfh (pssd)22,28, mackinnon-dellon disk-crimínator® (north coast medical, inc.) or aesthesiometer 2 point26,28,29; 15 rodrigues et al. • vibratory sensation (4.76%) assessment of the detection of vibration and determination of the threshold of the disappearance of the stimulus (rapidly-adapting qhuyh�ͤehuv���vibrometer and 256-cps tuning fork29; • thermal discrimination (9.52%) detection of temperature differences and determination of cold or hot stimuli. cotton-tipped applicator saturated with a spray freeze of -50°c temperature17, and ethyl chloride vapor was sprayed onto a spherical dental cotton bud (cold sensation) (diameter: 5 mm)26; • painful stimuli/pinprick (33.33%) assessment of painful stimulus detection: qhhgoh�khog�wkh�ehwzhhq�wkxpe�dqg�lqgh[�ͤqjhu17 and 27-gauge needle25; • pain detection threshold (14.29%) a gradual measurement of the detection of painful stimuli, of an ascending and descending character to determine the threshold (aid in the determination of hypoalgesia): neurometer cpt (neurotron inc)15and non-invasive electrocutaneous stimulation19; • sensory assessment/ sensory changes: • $vvhvvphqw�ri�vhqvrulqhxudo�ghͤflwv�ri�wkh�lqihulru�dqg�phqwdo�doyhrodu�qhuyhv�� thermography25 (4.76%); • assessment of nerve function latency and amplitude: trigeminal somatosensory evoked potential30 (4.76%). • details on how to conduct facial sensitivity assessment procedures described in the articles are listed in appendix 2. it was not possible to carry out a meta-analysis because the studies did not have vxiͤflhqw�txdqwlwdwlyh�gdwd�dqg�vkrzhg�kljk�txdolwdwlyh�khwhurjhqhlw\�lq�wkh�dvshfwv� of nomenclature, procedures, and equipment used in the sensitivity objective assessment procedures. discussion in this study, we found a varied number of procedures used to assess each sensory modality. considering the high incidence of traumatic events that cover the facial region1-3,14,16,17,25 and the occurrence of sensitivity changes resulting from these episodes9,10,13-33, it is necessary to have tests that assess these changes accurately. bearing in mind that the subjective procedures were considered as reference tests in wklv�uhylhz��lw�zdv�lghqwlͤhg�wkdw�wklv�dvvhvvphqw�rffxuv�lq�d�yhu\�gliihuhqw�zd\��xvlqj� questionnaires with questions aimed at guiding the patient’s report and/or scales to phdvxuh�wkh�ghjuhh�ri�uhsruwhg�lpsdluphqw��,q�erwk�dvvhvvphqw�prgdolwlhv��gliͤfxowlhv�uhodwhg�wr�wkh�lqwhusuhwdwlrq�dqg�fodvvlͤfdwlrq�ri�wkh�fkdqjhv�phqwlrqhg�e\�wkh� patient are found, and the results are subject to inappropriate analyzes, distortion of the report, and inadequate diagnoses of the change. also, there is qualitative heterogeneity in the scales used by the authors, who create scales for the punctual assessment using variations of the visual analog scale14,16. based on this, what is effective in most studies is the realization of a questionnaire with structured questions13,17,20,27,30,33 and the consideration of the patient’s report as a marker of change15,18,19,21-26,28,29,31,32 to guide the use of objective tests. 16 rodrigues et al. the objective assessment of facial sensitivity must be seen as a complement to the subjective assessment, and it must involve a large number of procedures that fdq�eh�olvwhg�dffruglqj�wr�wkh�w\sh�ri�qhuyh�ͤehu�whvwhg��wrxfk��dqg�qrflfhswlyh�vhqsitivity (pain and temperature). most of the articles used the touch assessment as wkh�pdlq�sdudphwhu�lq�wkh�surͤoh�ri�wkh�shulskhudo�lqqhuydwlrq�lqwhjulw\�ri�wkh�idfh�� being13,15,16,20,21,25,26,30,32 or not14,17,18,22-24,27-30,33 associated with nociceptive assessment. about touch assessment, the method used in most studies is the detection of light touch stimulus, usually associated with the mechanical detection threshold, with wkh�xvh�ri�prqrͤodphqwv�zlwk�irufh�ydoxhv�douhdg\�vwdqgdugl]hg�iru�phdvxulqj� cutaneous sensitivity thresholds. the method allows a gradual assessment of impairment and nervous recovery over time, in cases where there is a follow-up after the intervention14,15,17,27,28,30. the nociceptive assessment, on the other hand, yhulͤhv�wkh�qhuyh�ͤ ehuv�lqyroyhg�lq�wkh�vhqvdwlrq�ri�sdlq�dqg�whpshudwxuh��7kh�vwxglhv�suhvhqw�juhdwhu�yhulͤfdwlrqv�ri�wkh�sdlqixo�vhqvdwlrq13,15,17-19,21,25,26,31,32, eventually being accompanied by the sensation of temperature17,26,31. regarding these modalities, when researching the sensation of pain, studies use the prick test13,17,18,21,25,26,32, and when researching the sensation of temperature, they determine if the patient differentiates cold and hot stimuli17,26. a limiting factor of these assessments refers wr�wkh�odfn�ri�frqvhqvxv�rq�wkh�lqglfdwlrq�ri�vshflͤf�lqvwuxphqwv�wr�fduu\�rxw�wkh� tests, using heterogeneous equipment, which results in several protocols. thus, based on the studies, a minimum battery of facial sensitivity assessment is proposed with the modalities and procedures that should be performed so that the applicator has a complete overview of the patient’s peripheral nervous situation and the regions affected. assessments should be carried out, if possible, preoperatively and postoperatively (in cases of surgical intervention)15,17-21,25,30,31 because it is known that the changes may be the result of trauma or type of surgical treatment used21. in wkh�srvwrshudwlyh�shulrg��lw�lv�vxjjhvwhg�wkdw�uh�dvvhvvphqwv�eh�pdgh�lq�wkh�ͤuvw� week13,15-19,23,24,26,29-33��lq�wkh�ͤuvw month13,17-19,23,31-33, in the third month13,17-19,23,24,30,31,33, in the sixth month17-19,23,26,31-33��dqg�lq�wkh�ͤuvw�\hdu15-19,23,29,33 after surgery or trauma. it is recommended to start with the subjective assessment, which is important to identify the patient’s complaint and to delimit what results are expected from the objective tests later. at this stage, it is suggested questions to guide the patient’s report (chart 1). chart 1. questions to guide the patient’s report questions to guide the patient’s report: 1. do you notice changes in the sensitivity of the face? 2. do changes in sensitivity involve numbness, burning, tingling, pain or sensitivity to cold? can you explain with your words how the sensation is? 3. are your functionality and quality of life impaired? in what situations? (situations can be exemplified for the patient, such as: food runs through the mouth, drooling, biting of the lips.) 4. comparing with the unaffected side (or with some other region of the face, in cases of bilateral fracture), do you feel differences in sensitivity? 17 rodrigues et al. $iwhu�hvwdeolvklqj�wkh�idfh�vhqvlwlylw\�surͤoh�edvhg�rq�wkh�sdwlhqw̵v�uhsruw��surjuhvv�vkrxog�eh�pdgh�zlwk�wkh�remhfwlyh�dvvhvvphqw��,w�lv�qhfhvvdu\�wr�frqͤup�wkh� patient’s report, since changes, even if slight, may be present despite the patient not reporting complaints. in the objective assessment, it is necessary to perform procedures of the touch and nociceptive modalities, to stimulate different receptors dqg�qhuyh�ͤehuv��7kh�wrxfk�prgdolw\�yhulͤhv�wkh�lqwhjulw\�ri�wkh�phfkdqlfdo�idfldo� uhfhswruv�wkdw�lqyroyh�$ƌ�ͤehuv��shuiruplqj�wkh�vwlpxodwlrq�ri�wkh�0hunho�glvf�dqg� wkh�5xiͤql�frusxvfoh��uhvsrqvleoh�iru�ghwhfwlqj�udslgo\�dqg�vorzo\�dgdswlqj�wrxfk� stimuli; and meissner corpuscles and kdlu�iroolfoh�ͤehu, which are involved in the transduction of nerve signals. the nociceptive modality (perception of pain and whpshudwxuh�� lv�qrw�phgldwhg�e\�wkh�uhfhswruv�ri�wkh�frusxvfohv�vr�wkdw�wkh�$ǝ� dqg�&�ͤehuv�duh�lqyroyhg�lq�wkh�wudqvplvvlrq�ri�wkhvh�vhqvru\�prgdolwlhv4. thus, in the case of touch stimulation, it is recommended, due to the frequency of use in the articles included and the ease of application, the light touch/static light touch test and, consequently, the mechanical detection threshold, which can also be periruphg�dffruglqj�wr�wkh�lqvwuxphqw�xvhg�iru�fkhfnlqj��li�prqrͤodphqwv�duh�xvhg��� these tests will allow the stimulation of corpuscular receptors and stimulation of $ƌ�ͤehuv4. for nociceptive stimulation, the use of the prick test or thermal stimulation is recommended. however, it is emphasized that for proper stimulation and central transmission of painful stimuli, cutaneous thresholds must be between 23g and 51g, and if thermal stimulation is used, temperatures below 0°c or above 47°c4. the tests are carried out with the patient with eyes closed, informing the applicator from which point the stimulation is perceived. in conclusion, the instruments for investigating facial sensitivity used in the clinic in cases of maxillofacial trauma involve, for subjective assessment: the patient’s report guided by structured questions; and for objective assessment: predominantly the evaluation of touch and nociceptive sensitivity, the latter also comprising thermal evaluation. from this, it is proposed a standardization to investigate changes in idfldo�vhqvlwlylw\��%hvlghv��wkh�vwxg\�ri�wkh�surͤoh�ri�wkhvh�fkdqjhv�frqwulexwhv�wr�wkh� improvement of surgical techniques and to a safe return about the long-term results of the patient’s sensory situation14. limitations it was not possible to carry out a meta-analysis of this systematic review because wkh�lqfoxghg�vwxglhv�glg�qrw�kdyh�vxiͤflhqw�txdqwlwdwlyh�gdwd�iru�uhjlvwudwlrq�lq�frqtingency tables. also, they showed high qualitative heterogeneity in the aspects of nomenclature, procedures, and equipment used in the sensitivity objective assessment procedures. for this, more studies should investigate the validity of the tests used in practice, to favor the use of effective diagnostic procedures, since the accuracy analysis of the tests was not possible due to the low availability of data in the studies. conflicts of interest statement no conflicts of interest exist. 18 rodrigues et al. references 1. bogusiak k, arkuszewski p. characteristics and epidemiology of zygomaticomaxillary complex fractures. j craniofac surg. 2010 jul;21(4):1018-23. doi: 10.1097/scs.0b013e3181e62e47. 2. lalloo r, lucchesi lr, bisignano c, castle cd, dingels zv, fox jt, et al. epidemiology of facial fractures: incidence, prevalence and years lived with disability estimates from the global burden of disease 2017 study. inj prev. 2020 oct;26(supp 1):i27-i35. doi: 10.1136/injuryprev-2019-043297. 3. allareddy v, allareddy v, nalliah rp. epidemiology of facial fracture injuries. j oral and maxillofac 6xuj�������2fw����������������grl����������m�mrpv�������������b 4. siemionow m, gharb bb, rampazzo a. the face as a sensory organ. plast reconstr surg. �����)he����������������grl����������356��e���h����ihg�ig�b 5. devine m, hirani m, durham j, nixdorf dr, renton t. identifying criteria for diagnosis of post-traumatic pain and altered sensation of the maxillary and mandibular branches of the trigeminal nerve: a systematic review. oral surg oral med oral pathol oral radiol. �����-xq����������������grl����������m�rrrr�������������b 6. 6dlg�<hnwd�6��6phhwv�5��(vwhyhv�2olyhlud�0��6whlq�-0��5lhgljhu�'��/dpshuw�)��9hulͤfdwlrq�ri�qhuyh� integrity after surgical intervention using quantitative sensory testing. j oral maxillofac surg. 2012 feb;70(2):263-71. doi: 10.1016/j.joms.2011.03.065. 7. mcinnes mdf, moher d, thombs bd, mcgrath ta, bossuyt pm, clifford t, et al. preferred reporting items for a systematic review and meta-analysis of diagnostic test accuracy studies: the prisma-dta statement. jama. 2018 jan 23;319(4):388-396. doi: 10.1001/jama.2017.19163. erratum in: jama. 2019 nov 26;322(20):2026. 8. whiting pf, rutjes aw, westwood me, mallett s, deeks jj, reitsma jb, et al. quadas-2: a revised tool for the quality assessment of diagnostic accuracy studies. ann intern med. 2011 oct;155(8):529-36. doi: 10.7326/0003-4819-155-8-201110180-00009. 9. faria jcm, freitas rs, busnardo ff, munhoz am, fogaça wc, alonso n, et al. 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[sensitivity assessment objective versus objective after fracture of zygoma]. rev odontol unesp. 2016;45(5):265-70. 3ruwxjxhvh��grl��������������������������b 28. fogaça wc, alonso n, naufal rr, ferreira mc. [late evaluation of facial sensibility post orbitozygomatic complex fractures]. rev soc bras cir craniomaxilofac. 2008;11(4):119-25. portuguese. 29. kesarwani a, antonyshyn o, mackinnon se, gruss js, novak c, kelly l. facial sensibility testing in the normal and posttraumatic population. ann plast surg. 1989 may;22(5):416-25. doi: 10.1097/00000637-198905000-00006. 30. abd el-kader aa, azmy rm, mahmoud ga, allam km, hassanien hr. infraorbital nerve assessment diwhu�ͤ[dwlrq�ri�]\jrpdwlfrpd[loodu\�frpsoh[�iudfwxuhv��(j\swldq�-�1hxuro�3v\fkldw�1hxurvxuj�� 2011;48(4):399-403. 31. leonhardt h, meinecke d, gerlach kl. quantitative evaluation of thermosensitivity in patients with mandibular fractures. mund kiefer gesichtschir. 2005 sep;9(5):312-6. doi: 10.1007/s10006-005-0631-7. 32. marzola c, pastori cm, toledo jlf, zorzetto dlg, sant’anna aj. [paresthesias of the infraorbital nerve in fractures of the middle third of the face]. rev eletron odontol acad tiradentes odontol. 2006;6(1):153-73. portuguese. 20 rodrigues et al. 33. $kphg�66��$ivkdq�%��+dvkpl�*6��+dvkpl�6+��1hxurvhqvru\�ghͤflw�lq�fdvhv�ri�]\jrpdwlf�frpsoh[� fractures. curr neurobiol. 2010;1(1):51-4. 34. boyette jr. facial fractures in children. otolaryngol clin north am. 2014 oct;47(5):747-61. doi: 10.1016/j.otc.2014.06.008. appendix 1 pubmed search search strategy hits #1 facial fractures maxillofacial injuries[mesh terms]) or maxillofacial fractures) or maxillofacial trauma) or maxillofacial injury) or maxillary fractur*) or maxillary fractures[mesh terms]) or facial injuries[mesh terms]) or facial injury) or facial fractur* 59.092 #2 maxilla fractures zygomatic fractures[mesh terms]) or zygomatic bone fracture) or zygomatic fracture) or zygomatico-orbital fracture) or zygoma fractures) or zygomatic complex fractures) or blow-out fractures) or orbital fractures[mesh terms]) or orbital fractur*) or orbitozygomatic complex) or orbitozygomatic fractur* 6.641 #3 jaw fractures jaw fracture[mesh terms]) or jaw fractur*) or mandibular fractures[mesh terms]) or mandibular injuries[mesh terms]) or mandibular injur*) or mandib* fractur*) or mandibular condyle fracture) or angle fracture) or multiple mandibular fractures) or mandibular trauma) or condyle fractures 102.18 7 #4 patient #1 or #2 or #3 144.95 3 #5 tests and outcomes sensation disorders[mesh terms]) or somatosensory disorders[mesh terms]) or somatosensory function) or cutaneous sensibility disturbances) or allodynia) or dysesthesia) or paradoxical heat sensation) or facial sensibility testing) or cutaneous sensibility) or facial sensibility) or sensib*) or cutaneous sensory function) or sensory profile) or somatosensory nervous system 247.23 0 #6 search #4 and #5 4.412 google search fraturas maxilomandibulares or fraturas mandibulares or fraturas maxilares or fraturas orbitárias or fraturas zigomáticas = 2.030 lilacs, opengrey and proquest search fraturas maxilomandibulares or fraturas mandibulares or fraturas maxilares or fraturas orbitárias or fraturas zigomáticas = 0 cochrane search strategy hits #1 facial fractures maxillofacial injuries [mesh terms] or maxillofacial fractures or maxillofacial trauma or maxillofacial injury or maxillary fractur*[mesh terms] or facial injur*[mesh terms] or facial fractur* 1288 #2 maxilla fractures zygo* fractur*[mesh terms] or zygomatic bone fracture or zygomatico-orbital fracture or zygomatic complex fractures or blow-out fractures or orbital fractur*[mesh terms] or orbitozygomatic fractur* 148 #3 jaw fractures jaw fractur*[mesh terms] or mandibular injur* or mandib* fractur[mesh terms] or mandibular condyle fracture or angle fracture or multiple mandibular fractures or mandibular trauma 1022 #4 patient #1 or #2 or #3 2105 #5 tests and outcomes sensation disorders[mesh terms] or somatosensory disorders[mesh terms] or cutaneous sensibility disturbances or allodynia or dysesthesia or paradoxical heat sensation or facial sensibility* or cutaneous sensibility or sensib* or cutaneous sensory function or sensory profile or somatosensory nervous system 5478 #6 search #4 and #5 224 embase search search strategy hits #1 facial fractures 'maxillofacial injury'/exp or maxillofacial) and fractures or 'maxilla fracture'/exp or 'face injury'/exp or 'face fracture'/exp 68.385 #2 maxilla fractures 'zygoma arch fracture'/exp or 'orbit fracture'/exp 5.745 #3 jaw fractures 'jaw fracture'/exp or 'mandible fracture'/exp 10.000 #4 patient #1 or #2 or #3 68.531 #5 tests and outcomes 'sensory dysfunction'/exp or 'somatosensory disorder'/exp or 'allodynia'/exp or 'dysesthesia'/exp 573.36 2 #6 result #4 and #5 8.764 #7 age #6 and ([adult]/lim or [aged]/lim or [middle aged]/lim or [very elderly]/lim or [young adult]/lim) and [embase]/lim not ([embase]/lim and [medline]/lim) 1.097 appendix 2. facial sensibility procedures instrument modality method optihair von frey filaments (marstock nerve test, marburg, germany)10 static light touch and mechanical detection threshold examinations were started on the uninjured side on unilateral fractures. the filaments were applied perpendicular to the face in a descending order of magnitude to assess the threshold at which sensation disappeared. procedure was repeated four times. semmes-weinstein monofilament (estesiometer) 11, 13, 23, 24, 25 static light touch and mechanical detection threshold monofilament is placed perpendicular to the skin and pressed until the filament begins to deform. the monofilament gently touched the skin and patients were asked about sensibility. each measurement was repeated 3/4 times and filament was applied for 1.5 seconds, held for 1.5 seconds, and released for 1.5 seconds. if the patients were able to feel the monofilament minor caliper, testing ceased. if they were unable, the test followed until the patient could feel the monofilament touching the skin. neurometer cpt (neurotron inc) 11 current perception threshold (identify and evaluate nerve fiber damage sense of pressure, temperature and pain) fixing an electrode to the test site using the attached tape. two different current intensities were passed from the electrode to the patient, who was then asked to say which current the patient believed was stronger. minimum perceivable current was measured when an electrical stimulation at 2,000, 250, or 5 hz was applied. the measurements were taken 3 times at each frequency. 0.7-mm-gauge needle (bd precision glidetm) 13 static light touch (slt) and brush directional stroke (bds) slt: the needle gently touched the skin, and the point that the patient feel sensibility was noted. bds: it was applied in a 1-cm stroke in each point. the examiner decided if move it from right to left or from left to right in each interval, and the patient was asked about the direction of the movement. a needle held the between thumb and index finger 13 pinprick discrimination the intensity was applied sufficiently so the patient would feel pain or to draw as small drop of blood at the puncture side. cotton-tipped applicator saturated with a spray freeze of −50 c temperature 13 thermal discrimination the patients were asked about cold or normal/not cold feeling immediately upon application of each respective applicator. noninvasive electrocutaneous stimulation 15 pain detection threshold (pdt) is performed applying noninvasive electrocutaneous stimulation of the dry skin in the region by active 2 mm diame e elec de a d a i e elec de fi ed b a ie ha d h mb a d f efi ge . pdt a a e ed using ascending method of limits. the stimulating current was gradually increased by fixed rate until the subject indicated first pain sensation. three pdts were evaluated. pressure-specified sensory device (pssd) 18, 25 one-point static discrimination, two-point static discrimination and moving-touch discrimination the small blue pssd is hand held by the person doing the testing, and the two small metal probes are touched gently to the skin area being tested. the cutaneous pressure thresholds for one-point static and moving-touch discrimination were recorded in grams per square millimeter, and the pressures required for two-point static and moving-touch discrimination were recorded as the pressure for a given interprong distance (in millimeters). cotton roll 22 light touch sensation not described 27 gauge needle 22 pain test not described dental cotton swab 22 directional test not described thermography 22 sensory changes each subject was asked not to eat, drink, or smoke for an hour before the examination. all cosmetics were washed off and the skin surface allowed to dry in the air. hair was held off the face with hair grips. no sources of radiation were allowed in this environment and sunlight was excluded. all air convection sources were minimized and only two operators were allowed into the room while the examination was in progress. once stabilized, baseline measurements were recorded for the frontal and left and right profiles of each patient in a sitting position at a focal distance of one metre perpendicular to the region of interest. mackinnon-dellon diskcrimínator® (north coast medicai, inc.) or aesthesiometer 2 point 23, 25, 26 static two-point discrimination and moving two-point discrimination series with either ascending or descending increments with a successively longer or shorter pin distance in the device, during which the subject reported on a present or absent sensation of two separate points of stimulation. a test series was terminated after a response reversal, i.e. when a particular type of response (positive/negative) on a stimulus increment was followed by two responses of the opposite type on successive increments. each of the tests consisted of four alternating series. the initial two point testing distance was 24 mm, proceeding in stages down to 2mm. the stimulus was randomly alternated between one and two points. if the patient correctly perceived the changes, the distance was decreased. this testing pattern was continued until the patient answered incorrectly, at which time the observer returned to the next higher distance. in the two-point limit, two of three correct answers were required for this distance to be chosen as the end-point. ethyl chloride vapour was sprayed onto a spherical dental cotton bud (diameter: 5 mm)23 cold sensation after ice crystals had been formed, the bud was placed on the test site for at most 1 s. the drop in temperature varied within a range from 22 to 24~ at the interface between cotton bud and skin. vibrometer 26 vibratory threshold using a fixed-frequency (120hz) variable amplitude instrument. the vibrating portion of the instrument was applied to the test area, and the voltage was gradually increased until the patient was first able to perceive vibration. the threshold is converted into microns (amplitude) of displacement. trigeminal somatosensory evoked potential (tsep) 27 sensory assessment the recording electrode was placed contralateral to the side of stimulation 2cm posterior to c3 and c4 at the coronal suture. a reference electrode was placed at mid frontal site and the array was earthed by ground electrode placed around neck. the electrical stimulator provided stimuli at a rate of 2 sec and each stimulus lasted for 0.1 sec. the stimulus intensity was adjusted by gradual increasing up to the level where minimal lower eye lid twitch could be observed. in order to achieve pure sensory stimulation with maximum activation of the nerve fibers and minimum electrical artifact, the infra-orbital nerve (ion) stimulation was performed at the ion foramen using the stimulator electrode of tsep. tsep was at least repeated twice to confirm the reproducibility and reliability of the response; 1 volume 22 2023 e239389 original article braz j oral sci. 2023;22:e239389http://dx.doi.org/10.20396/bjos.v22i00.8669389 1 school of health sciences, positivo university, brazil. 2 graduate program in dentistry, federal fluminense university, brazil. 3 department of restorative dentistry, school of dentistry, oregon health & science university, usa. corresponding author: amanda mahammad mushashe school of health sciences, universidade positivo rua professor pedro viriato parigot de souza 5300, campo comprido, 81280-330 curitiba – pr-brazil. e-mail: amandamushashe@ hotmail.com phone: (+55 41 3317-3094) fax: (+55 41 3317-3082) editor: dr. altair a. del bel cury received: may 24, 2022 accepted: january 18, 2023 effect of s. mutans biofilm on the hybrid ceramic-resin cement bond strength assessed by different methods amanda mahammad mushashe1,* , sarah aquino de almeida2 , jack libório ferracane3 , justin merritt3 , carla castiglia gonzaga1 , gisele maria correr1 aim: the purpose of this study was to investigate the biofilm effect on the hybrid ceramic-resin cement bond strength (bs) by comparing two methods. methods: teeth were distributed into groups (n=5), according to the resin cement (maxcem elite-(mc) or nx3 nexus-(nx)) and degradation method (24h or 7 days in distilled water; 7 or 30 days incubated with biofilm and 30 days in sterile media). treated surfaces of vita enamic blocks (5x6x7mm) were luted to treated or no treated dentin surfaces and light-cured. after 24h, beams were obtained (1x1x10mm) and stored accordingly. the flexural bond strength (fbs) was assessed by four-point bending test. additional beams were obtained from new teeth (n=5), stored for 24h or 7 days in distilled water, and submitted to a microtensile bond strength (µtbs) assay. failure modes were determined by scanning electron microscopy (100x). the flexure strength of the cements (n=10) was assessed by a four-point bending test. data were analyzed by 1 and 2-ways anova, and tukey’s test (α=0.05). results: there was no significant difference between the degradation methods for the fbs groups. for the µtbs, the significant difference was as follows: nx 7days > nx 24h > mc 7days = mc 24h. failure mode was mainly adhesive and mixed, but with an increase of cohesive within cement and pre-failures for the mc groups assessed by µtbs. nx had better performance than mc, regardless of the method. conclusions: the biofilm had no effect on the materials bs and fbs test was a useful method to evaluate bs of materials with poor performance. keywords: biofilms. resin cements. dental bonding. tensile strength. https://orcid.org/0000-0002-7710-0857 https://orcid.org/0000-0002-5396-7499 https://orcid.org/0000-0002-6511-5488 https://orcid.org/0000-0002-3464-0490 https://orcid.org/0000-0001-6374-1605 https://orcid.org/0000-0002-5032-0948 2 mushashe et al. braz j oral sci. 2023;22:e239389 introduction success of indirect restorations depends on a combination of several factors, such as aesthetics, occlusal balance and long-term bond stability between substrate, adhesive layer and restorative material1. in the challenging oral environment, dental materials are subjected to biodegradation, which is caused by the deleterious effect of oral biofilm on their structure and properties. bacterial acids can promote an increase in surface roughness, matrix and interfacial softening, decrease in surface hardness and chemical degradation of the hybrid layer, directly affecting the bond strength of indirect restorations and promoting the loss of cervical sealing2-5. to date, few authors4 have evaluated the effect of actual biofilm growth on the bond strength of restorative materials to dentin. vita enamic (vita zahnfabrik, bs, germany) is a hybrid cad-cam material, composed by feldsphatic ceramic (75 wt%) and a dimethacrylate polymer network (25wt%)6-9. cad-cam materials are preferably adhesively cemented in order to promote better bond stability, and conventional resin cements with dental adhesives are typically used. however, in an attempt to diminish the technique sensitivity of the process, self-adhesive luting agents can be used to eliminate the need for treating the surface of the teeth with an adhesive before applying the cement10,11. while the resin cements and ceramics have different resistance to biodegradation, the bond strength of hybrid materials to conventional and self-adhesive resin cements when subjected to the action of a growing biofilm is yet to be determined. bond strength can be assessed by different methods, with microtensile bond strength (μtbs) being the most popular test used in the literature3,12,13. if performed correctly, it produces a uniform interfacial stress distribution, resulting in reliable outcomes13. despite its popularity, it is a time-consuming and highly technique-sensitive assay12. the mounting of specimens to the proper jig can lead to premature stress, resulting in many pre-test failures and data with high standard deviation, especially for materials with low bond strength values10,13. flexure bond strength assessed by a four-point bending test (fbs) has been shown to be a promising method to evaluate the bonding performance of materials13-15. four-point bending geometry concentrates the maximum tensile stress on the convex surface (bottom), removing the stress concentration at the surface of the interface, which is claimed to be more clinically relevant than the direct tension test13-15. also, the easy placement of samples on the four-point bending device leads to a less technique sensitive assay. however, there is still a lack of evidence regarding the reliability of such results, raising the importance of studies comparing fbs to other well-stablished bonding methods, such as the microtensile test. therefore, the aims of this study were to evaluate the effect of a growing s. mutans biofilm on the bond strength of a hybrid cad-cam material to two different luting agents and to compare the bonding performance by two assays: flexure and microtensile bond strength. the hypotheses of this research were: (1) the biofilm will negatively affect the bond strength, and (2) both bond strength methods will provide similar outcomes. 3 mushashe et al. braz j oral sci. 2023;22:e239389 methods and materials specimen preparation fifty caries-free human third molars were stored in an aqueous solution of 0.5% chloramine-t for 7 days and stored in distilled water until use. before extraction, patients had been informed about the use of the teeth for research purposes, and verbal consent had been obtained. deep dentin was exposed by removing the occlusal enamel with a low speed, water-cooled diamond saw (isomet 1000 precision saw, buehler, il, usa). dentin surfaces were abraded on #600 silicon carbide paper for 30s to create a standardized smear layer, and then ultrasonically cleaned in water for 5 min. teeth were then randomly distributed into ten groups (n=5), according to the luting agent (maxcem elite or nx3 nexus, kerr, ca, usa) and the storage condition (table 1). table 1. group distribution (n=5). storage condition/ resin cement nx3 nexus (nx) maxcem elite (mc) 24hrs in distilled water nxc mcc 7 days in distilled water nx7w mc7w 7 days with biofilm nx7b mc7b 30 days in sterile media nx30m mc30m 30 days with biofilm nx30b nx30b dentin surfaces were treated according to the resin cement group. for the self-adhesive cement (mc), no further surface preparation was performed. for the conventional luting agent (nx), surfaces were etched for 15s with a 37.5% phosphoric acid gel (gel etchant, kerr, ca, usa) and then rinsed abundantly with water. after removing excess moisture with an absorbent paper, leaving a glistening surface, an etch-andrinse adhesive system (optibond s, kerr, ca, usa) was actively applied by means of a microbrush for 15 s, gently air-dried for 3 s at a standardized distance of 5 cm and light-cured for 20s, using a curing unit (elipar s10, 3m espe, mn, usa), with an output of at 900 mw/cm2, monitored with a radiometer (sds kerr model 100, optilux radiometer, kerr, ca, usa). the light-curing unit tip (9.8mm of diameter) was at standardized distance of 5 mm from the dentin surface. rectangle-shaped hybrid ceramic (vita enamic, vita zahnfabrik, bs, germany) specimens (5 x 6 x 7 mm) were prepared cut from standard blocks with a diamond saw. the bonding surface of each specimen was then etched with a 5% hydrofluoric acid (vita ceramics etch, vita zahnfabrik, bs, germany) for 60 s and rinsed ultrasonically with distilled water for 5 min. the surfaces were then air-dried, and one layer of a silane primer (relyx ceramic primer, 3m espe, mn, usa) was applied for 20 s and then allowed to dry for 30 s. each luting agent was prepared by aid of auto-mixing dispenser provided by the manufacturer and applied on the treated hybrid ceramic surface by the same mixing tips. 4 mushashe et al. braz j oral sci. 2023;22:e239389 the rectangular specimen was then placed on the dentin surface with a constant pressure of 100g to standardize the thickness of the cement layer (~130 µm). after gently removing the excess cement with a microbrush, the complex was light-cured for 10 s at 900 mw/cm2 from two opposite sides at a 90º angle with the edge of the light guide resting on the dentin surface. the specimens were then stored in distilled water at 37ºc for 24hrs. the specimens were then cut into beams (1 x 1 x 10 mm) with the bonded interface in the middle using a low-speed, water-cooled diamond saw at 300 rpm. the cross-sectional area of each stick was measured for subsequent calculation of the bond strength. degradation methods the bonded sticks originating from the same teeth were then assigned to each group, according to the degradation method: 1 or 7 days in distilled water at 37ºc; 30 days storage in sterile todd-hewitt (th) media (thermo fisher scientific, ma, usa), changed each 4 days, and 7 or 30 days co-incubated with an inoculate of idh-reng luciferase reporter strain streptococcus mutans grown as a biofilm16. for the samples tested with living biofilm, an overnight culture of s. mutans was added in fresh sterile th media, and the optical density was set at 0.8 at 600 nm (od600). this particular strain of s. mutans is genetically modified to result in a bioluminescent phenotype, able to provide quantitate data regarding cell viability under light emission conditions16. after the beams were sterilized by storage in 70% ethanol for 5 min and rinsed with autoclaved water, they were placed in a sterile 24-well plate along with 0.5 ml of the bacterial suspension. to encourage biofilm formation, 1% of a 40% sucrose solution was also added. the specimens were then incubated at 37ºc in 5% co2 for 7 or 30 days. bacterial growth medium was refreshed every day without disturbing the formed biofilm. after the incubation period, a luciferase assay was performed to assess the viability of the bacteria in the biofilm, essentially as previously described16. briefly, samples were moved carefully with the aid of sterile tweezers and placed into a luminescence 24-well plate and incubated with fresh media for 1 hr at 37ºc in 5% co2. after, light emission from growing bacteria cells was measured by adding 5 µl of a substrate solution (1 mm d-luciferin in 0.1 m sodium citrate buffer [ph 5.0]) to each well. the plate was immediately placed in an optical plate reader (glomax discover multimode microplate reader, promega, madison, wi, usa) and light emission recorded, representing the quantity of viable s. mutans cells. four-point bending assay to determine the flexural bond strength (fbs) after the various degradation methods, beams were washed in tap water for 5 min, carefully dried and subjected to a fourpoint bending test. the 10 mm beams were fixed between the four supports with the bonded interface centered within the inner rollers and loaded until fracture using a universal testing machine (q-test, mts, eden prairie, wi, usa) at 1 mm/min crosshead speed17,18 (figure 1). 5 mushashe et al. braz j oral sci. 2023;22:e239389 figure1. example of a beam accordingly positioned between the supports for the flexural bond strength assay. the fbs (mpa) was calculated using the following equation: 9 x f x l 8 x w x t2 fbs = where f (n) was the load at fracture, l the support span (8.48mm), w and t the specimen width and thickness, respectively. microtensile bond strength the enamel crown of an additional twenty caries-free human third molars was removed to expose dentin by cutting with the diamond saw. these teeth were randomly distributed into four groups (n=3), according to the storage period (24 hrs or 7 days at 37ºc) and luting agent (maxcem elite and nx3 nexus). specimen preparation was performed in the same way as previously described for the fbs. there were many pretest failures for maxcem specimens (more than 50% of the sticks for the 24 h specimens and about 40% for the 7 days specimens), but essentially no pre-test failures for nexus. though more teeth were prepared, only teeth in which at least three sticks could be tested were included in the analysis, leaving n=3 for all four groups19. after the respective storage periods, each ceramic-dentin stick was removed from the solution and gently dried. they were attached to a microtensile testing device (odeme dental research, luzerna, sc, brazil) using cyanoacrylate adhesive and subjected to a tensile force in the universal testing machine at 1 mm/min cross-head speed. failure mode bond test samples were mounted on metallic stubs, coated with 60% gold:palladium in a sputter coater (anatech,hayward, ca, usa) and observed under a scanning elec6 mushashe et al. braz j oral sci. 2023;22:e239389 tron microscope (sem) (quanta 200 sem, fei company, or, usa), at magnification x100, in order determine the failure modes. flexural strength of the cements the flexural strength of the luting agents was assessed using the four-point bending test. to prepare these specimens, 1 x 1 x 10 mm polyvinylsiloxane molds were filled with each cement (n=10), sandwiched between glass slides and light-cured at 900 mw/cm2 for 10s on each side (2 exposures of 5 s each to cover the entire surface). after polishing the samples to remove any excess, they were stored in distilled water at 37ºc for 24h. the specimens were gently dried and mounted in the four-point bending device to measure the flexural strength, using the same conditions previously described for the fbs test. statistical analysis the data from the fbs test and the µtbs were analyzed by 2-way anova, followed by tukey’s multiple comparison test (α = 0.05). regression analysis was used to correlate the results from both tests. comparison of the flexure strength of the two cements was test done with a student’s t-test (α = 0.05). results flexural bond strength mean and standard deviation (sd) values for the fbs are presented in figure 2. m p a 70 60 50 40 30 20 10 0 b b b b b a a a a a mc c mc 7w mc 30m mc 30bmc 7b nx c nx 7w nx 30m nx 30bnx 7b flexural bond strength figure 2. flexural bond strength (mpa) of the different groups after the degradation methods (mean ± sd). bars with dissimilar letters indicate values that are significantly different from each other (p <0.05). analysis of variance showed a significant difference among the cements, with the fbs of nx being higher for all conditions than mc (p<0.001). because there was no significant difference between the degradation methods and no interaction effect., 7 mushashe et al. braz j oral sci. 2023;22:e239389 individual one-way anovas were run for the two cements. no significant differences between the aging conditions was shown for either cement. the failure modes were classified as adhesive, cohesive within cement, cohesive within dentin and mixed (figure 3). the failure modes of the different groups are presented in figure 4. figure 3. examples of the failure modes assessed by sem (x100): (a) adhesive; (b) cohesive within cement; (c) cohesive within dentin and (d) mixed. mc c mc 7w mc 30m mc 30b mc 7b nx c nx 7w nx 30m nx 30b nx 7b chart area 0% 20% 40% 60% 80% 100% mixed cohesive on dentin cohesive on cement adhesive pre failure fbs failure mode figure 4. failure mode assessed by sem (x100) after four-point bending assay. 8 mushashe et al. braz j oral sci. 2023;22:e239389 for all the groups, there was a predominance of mixed and adhesive failures. pre-test failures only occurred for the mc cement stored in water for 24 h and 7 days and co-incubated with bacteria for 7 days. the viability of biofilm of the samples co-incubated with s.mutans were assessed by a luciferase assay. the biofilm was considered viable, without significant difference between the groups (p > 0.05). microtensile bond strength mean and standard deviation (sd) values for the microtensile bond strength (mpa) of the different groups are presented in figure 5. m p a 50 45 40 35 30 25 20 15 10 5 0 µtbs mc 24h mc 7dnx 24h nx 7d d b c a figure 5. microtensile bond strength (mpa) of the different groups after the degradation methods (mean ± sd). bars with dissimilar letters indicate values that are significantly different from each other (p <0.05) regardless of the storage period, nx presented higher values than mc. also, the values at 7 days were greater than those at 24 hours for both cements. the failure mode of the different groups is presented in figure 6. 9 mushashe et al. braz j oral sci. 2023;22:e239389 mc 24h mc 7days nx 24h nx 7days 0% 20% 40% 60% 80% 100% mixed cohesive on dentin cohesive on cement adhesive pre failure µtbs failure mode figure 6. failure mode assessed by sem (x100) after µtbs assay. for the nx groups, there was a predominance of mixed and adhesive failures. for the mc groups, more cohesive within cement and pre-test failures were observed. flexural strength of the cements mean and standard deviation (sd) values for flexural strength of both cements (mpa) after 24 h in distilled water at 37ºc are presented in figure 7. m p a 90 80 70 60 50 40 30 20 10 0 flexural strength mc nx figure 7. flexure strength of cements (mpa) after 24 h in water (mean ± sd; n=9). the fs for nx was significantly higher than for mc (p <0.001). comparison between bond strength methods a regression plot between the fbs and µtbs results for both cements at 24 h and 7 days in distilled water at 37ºc is represented in figure 8. 10 mushashe et al. braz j oral sci. 2023;22:e239389 60 50 40 30 20 10 0 fb (m p a) mtbs (mpa) mc 24h mc 7d nx 24h comparison of fb and mtbs for mc and nx cements nx 7d y = 0.9308x + 10.316 r2 = 0.6479 0 5 10 15 20 25 30 35 40 45 figure 8. correlation between fbs and µtbs assays (r2= 0.664). for the 1-week period, both test methods gave similar values. at 24h, fbs values were higher (~50%) than µtbs values for both cements. discussion the bond strength stability of the restorative material-cement-dentin interface is a key factor in the success of indirect restorations. the aim of this study was to investigate the effect of different degradation methods on the hybrid ceramic-resin cement flexure bond strength, and to compare the results from the flexure bond strength to the more common microtensile bond strength test for the two cements. viable oral biofilms produce significant concentration of acids, mainly propionic, acetic and lactic3,20. the hydroxyl and carboxyl functional groups of these acids can establish a high level of hydrogen bonds with the polar sites of the methacrylate monomers present in the adhesive and cement, increasing the acid uptake by the polymeric phase of the hybrid layer. synergistically, these entrapped acid molecules can reduce the local ph, favoring the hydrolysis of ester groups and leading to the degradation of the hybrid layer that results in a reduction in the interfacial bond strength2,3,21,22. amaral et al.2 (2015) showed evidence for decreased bond strength values for resin composites bonded to bovine teeth after storage in lactic and propionic acids. similar results were found by reis et al.3 (2015), showing approximately a 33% decrease of the resin composite-human dentin bond strength after storage in acetic acid for 1 week and propionic acid for 1 month. in contrast, the present study showed no significant difference between the degradation methods when samples were assessed by a four-point bending test (fig. 2), rejecting the first hypothesis. actual biofilms may produce cariogenic acids at a slow rate and these may have accumulated at a lower concentration than those utilized in the studies that tested the direct effect 11 mushashe et al. braz j oral sci. 2023;22:e239389 of the acids on the bonded interface, thus explaining the different outcomes. within the limitations of in vitro studies, incubation of restorative materials with cariogenic bacteria may be considered more clinically relevant in comparison with chemical degradation alone (e.g. storage in cariogenic solution), since more variables, such as bacterial metabolism and biofilm structure, are simulated. another hypothesis for the lack of difference between the degradation methods, especially for those stored for 30 days, may be related to the time frame. one of the major causes for decrease of bond strength is the degradation produced by water sorption3,13,14. in an aqueous environment, the plasticization of the resin matrix23 and the hydrolysis of the unprotected collagen fibrils by host-derived proteases6,9 can promote the collapse of the hybrid layer. this hydrolytic degradation, however, is time-dependent. several studies showed that hydrolytic bonding degradation occurs only after 6 months of water storage1,3,11,24,25. therefore, the period of time chosen for this study may have not been sufficient to produce significant degradation of the adhesive interface. for the µtbs, samples stored for 7 days had higher bond strength results than those tested after 24h (fig. 4). both luting agents used in this study were dual-cured, achieving an adequate degree of cure by the synergistic effect of light exposure and a redox initiator for the free radical formation. while the immediate photo-activation will guarantee an initial mechanical stability, enhanced properties will be obtained after the chemical curing reaction occurs26. although authors claimed that most of the curing occurs within 24 h27, a residual setting may still occur after this period, explaining the better bonding performance after 7 days. also, in the short-term, the presence of an aqueous environment can increase the bond strength by forming hydrogen bonds between the polar components in the resin with water13,28. regardless of the bond strength testing method, the nx cement presented better bonding performance as compared with the mc (figs. 2 and 4). others have shown that self-adhesive luting agents have lower bond strengths to dentin than conventional resin cements that are used in conjunction with a dentin adhesive10,11,29-31. characteristics such as low etching potential of the functional acid monomers of the self-adhesive cements and their high viscosity promote only partial or no modification of the smear layer, resulting in a weaker hybrid layer in comparison with conventional resin cements when used with their associated primer and etchants24,32,33. maxcem also presented poorer mechanical properties than nexus, as shown by the comparison of their flexural strengths (fig.4). the lower values for mc are consistent with the literature. fuirichi et al.34 (2016) showed that mc, when compared with other self-adhesive and conventional resin cements, had presented the lowest flexural strength. although the mechanical properties of self-adhesive cements are material-dependent, it has been shown that some self-adhesive luting agents tend to present poorer mechanical behavior due to specific factors: incompatibility between the acidic functional monomers and the others resinous components31, reduced degree of conversion34,35, resin matrix hydrophilicity and unprotected surfaces on filler particles36, these latter may be responsible for a higher susceptibility to wear. the poorer mechanical properties of mc can be also observed in this study in the failure mode analysis after the µtbs test (fig.5). for both storage periods, mc presented a higher 12 mushashe et al. braz j oral sci. 2023;22:e239389 percentage of ‘cohesive within the cement’ failure, indicating that the weaker cement failed before the interface failed. a direct comparison between µtbs and fbs is somewhat dubious considering the different types of forces and dynamics acting in each test. during the assay, µtbs specimens are subjected exclusively to tensile forces distributed over a well-defined bonding area. in contrast, during the four-point bending test, a mixture of tensile (bottom) and compression (top) forces are produced in the area within the supports spans13-15. another difference that may complicate the comparison concerns sample preparation. for most fbs studies, beams of each of the substrates are produced separately and are then bonded to each other with the cement materials, which likely incorporates more variables and less sample standardization13-15. in the present study, specimen preparation was performed identically for both test methods, thus eliminating variables such as irregular beam cutting and luting procedures, making the comparison between methods potentially more accurate. the major difference between both tests pertains to the sample mounting before the actual test. for the µtbs, the beams must be glued to a jig prior to the test, which, besides being time-consuming, may lead to premature stress on the beams13. this is especially critical for materials with poor bonding performance, such as aged specimens and the self-adhesive cement tested in this study. in contrast, fbs samples must only be aligned horizontally within the supports, reducing any excessive manipulation of the beams13-15, as shown in the analysis of the failure mode assessed after each experiment (figs. 3 and 5). the µtbs specimens presented more pre-test failures than the fbs specimens, many of which occurred while mounting the specimen to the testing jig. this was especially critical for the mc 24h group. this corroborates the results presented by this material on the assays performed, demonstrating that its poorer mechanical and adhesion properties can influence its performance during the microtensile bond strength test. for the samples tested after 7 days, the number of pre-test failures decreased, which correlated with the increase of the bond strength that likely occurred due to the completion of cure and maturation of the bond. the sensitivity of the µtbs method can also be observed on the correlation plot between the assays (fig.7). when tested at 24 hours, specimens in the fbs test produced higher values than those from the µtbs test. however, at 7 days, the two methods gave essentially identical results. as mentioned, at 24h, the polymerization of the cements may not have been completed, resulting in specimens that were more sensitive to the application of manipulation stresses, e.g. to possible shear forces induced during µtbs assembling. nevertheless, it was possible to observe that both assays presented a similar trend between the different materials and storage periods, validating the second hypothesis. considering a restoration loaded/cemented interface in tension, the microtensile bond strength provides a closer representation of what is occurring at the adhesive interface than the four-point bending13. additionally, few data are available regarding this test in comparison with the abundant evidence related to µtbs, the latter being considered the gold-standard test method. on the other hand, the ease of performing 13 mushashe et al. braz j oral sci. 2023;22:e239389 the fbs, the lower sensitivity of the fbs technique, and the ability to determine the different trends between the materials, as shown by this study, makes the fbs a useful method to determine bond strengths of dental interfaces. regarding the effect of the biofilm on the bond strength of the interface of the materials tested, the limitations of this study were related to the limited verosimilarity conditions that an in vitro design promotes. further studies, including in situ analysis should be performed, in order to assess the alternations of ph, bacterial flora, temperature, salivary flow, etc, that occur on the oral cavity. furthermore, additional analyses including a wider range of resin cements and hybrid/ceramic materials may provide more consistent information regarding the comparison of different bond strength methods. in conclusion, within the limitations of this study, it can be concluded that biofilm exposure did not affect the hybrid ceramic-resin cement flexural bond strength. both bond strength methods provided similar outcomes, stating that nx presented higher bond strength than mc for both storage periods. therefore, fbs was a useful method to compare different materials, especially for those with low mechanical properties which are more sensitive to pre-test manipulation. acknowledgments this work was supported by the brazilian federal agency for support and evaluation of graduate education – capes (pdse 88881.134979/2016-01). data availability datasets related to this article will be available upon request to the corresponding author. conflicts of interest none. author contribution amanda mahammad mushashe: conception, design, experiment performance analysis and interpretation of data, paper writing. sarah aquino de almeida: conception, design, experiment performance analysis and interpretation of data. jack libório ferracane: conception, design, literature review, analysis and interpretation of data. justin merritt: design, analysis, and interpretation of data. carla castiglia gonzaga: literature review and critical review of the manuscript. gisele maria correr: literature review and critical review of the manuscript. 14 mushashe et al. braz j oral sci. 2023;22:e239389 all authors actively participated in the manuscript’s findings and revised and approved the final version of the manuscript. references 1. de oliveira lino lf, machado cm, de paula vg, vidotti ha, coelho pg, benalcázar jalkh eb et al. effect of aging and testing method on bond strength of cad/cam fiber-reinforced composite to dentin. dent mater. 2018;34(11):1690-701.doi: 10.1016/j.dental.2018.08.302 2. amaral cd, correa ds, miragaya lm, silva em. influence of organic acids from the oral biofilm on the bond strength of self-etch adhesives to dentin. braz dent j. 2015;26(5):497-502. doi: 10.1590/0103-6440201300260. 3. reis a, martins gc, de paula ea, sanchez ad, loguercio ad. alternative aging solutions to accelerate resin-dentin bond degradation. j adhes dent. 2015 aug;17(4):321-8. doi: 10.3290/j.jad.a34591. 4. follak ac, miotti ll, 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bond strength of self-adhesive resin cements to dentin. j prosthet dent. 2011;105(4):227-35. doi: 10.1016/s0022-3913(11)60037-1. 34. fuirichi t, takamizawa t, tsujimoto a, miyasaki m, barkmeier ww, latta ma. mechanical properties and sliding-impact wear resistance of self-adhesive resin cements. oper dent.2016;41(3):e83-92. doi: 10.2341/15-033-l. 35. almeida cm, meereis ctw, leal fb, ogliari ao, piva e, ogliari fa. evaluation of long-term bond strength and selected properties of self-adhesive resin cements. braz oral res. 2018;32:e15. doi: 10.1590/1807-3107bor-2018.vol32.0015. 36. ferracane jl, stansbury jw, burke fj. self-adhesive resin cements chemistry, properties and clinical considerations. j oral rehabil. 2011;38(4):295-314. doi: 10.1111/j.1365-2842.2010.02148.x. 1 volume 21 2022 e226709 original research braz j oral sci. 2022;21:e226709http://dx.doi.org/10.20396/bjos.v21i00.8666709 1 department of stomatology, health sciences center, federal university of santa maria, santa maria, rs, brazil. 2 department of oral and maxillofacial rehabilitation, university of talca, talca, chile. 3 paulista university (unip), campinas, sp, brazil. 4 department of gerontology, university of campinas, campinas, sp, brazil. 5 department of health sciences and pediatric dentistry, piracicaba dental school, university of campinas, piracicaba, sp, brazil. corresponding author: maria da luz rosário de sousa, department of health sciences and pediatric dentistry, piracicaba dental school, state university of campinas, av. limeira, 901 areião, piracicaba sp, 13414-018, +55 19 2106-5209. e-mail: luzsousa@fop.unicamp.br editor: altair a. del bel cury received: august 18, 2021 accepted: january 20, 2022 functional dentition and associated factors: the evaluation of three indicators luísa helena do nascimento tôrres1 , maría jesús arenas-márquez2 , débora dias da silva3, roberta barros de held4, talita bonato de almeida5 , anita liberalesso neri4 , maria da luz rosário de sousa5,* studying the different indicators of functional dentition classification can contribute to the understanding of the associated factors, and thus help in the definition of strategies associated with oral health care. this approach has been little explored in the literature, especially when considering the older age group. aim: the aim of this study is to evaluate the factors associated with three distinct functional dentition classification. methods: crosssectional exploratory study using secondary data from the frailty in older brazilians (fibra) project of 876 older adults living in campinas, brazil. the indicators of dental function assessed was number of natural teeth present, occluding pairs of teeth and the eichner index, which were verified by trained dentists, following the world health organization criteria for epidemiological studies in oral health. the explanatory variable assessed was the selfperception of oral health-related quality of life measured by the geriatric oral health assessment index (gohai) and its dimensions. it was also collected sociodemographic information such as age, gender, race/ethnicity, schooling, family income, smoking behavior and frailty status. the association was verified through poisson regressions for number of teeth and pairs of teeth in occlusion and multinomial regression for the eichner index, adjusted by sociodemographic and health variables. results: lower prevalence of participants with less than 21 teeth who negatively perceived gohai´s pain and discomfort dimension and higher prevalence of having less teeth among the ones that negatively perceived gohai´s physical and functional dimensions. no association was found between the perception of quality of life and occlusion pairs of teeth and the eichner index. conclusion: two out of three indicators assessed were associated with quality of life. therefore, it is important to select sensitive indicators to be able to identify and better comprehend this relationship. keywords: aged. dentition. quality of life. https://orcid.org/0000-0003-0740-2785 https://orcid.org/0000-0002-5017-4303 https://orcid.org/0000-0002-0533-7126 https://orcid.org/0000-0002-6833-7668 https://orcid.org/0000-0002-0346-5060 2 tôrres et al. braz j oral sci. 2022;21:e226709 introduction oral health tends to decline with aging. cumulative and progressive changes throughout life can result in tooth loss, which impairs the functionality of the dentition1. the reduction in the number of teeth can lead to a loss of masticatory efficiency, affecting nutrition2, communication, self-esteem, general well-being3, and even being associated with a higher risk of morbidity and mortality4. few studies evaluate the relation between functional condition of dentition and quality of life5, and there are even less about older populations. this is the age group with more sequelae in the dentition1. the multidimensional impairment of tooth loss can affect and be affected by quality of life, and thus evaluating the different types of classification can contribute to the understanding of associated factors and especially the quality of life evaluated globally and through different dimensions, such as pain/ discomfort, physical and psychological aspects. this relationship is frequently studied with a focus on the number of teeth1, but this impact may vary depending on the degree of the dentition functionality, as in the case of the eichner indicator that is still barely used in the literature. as seen in a recent systematic review6, which verified the association between oral health factors associated with oral health-related quality of life (ohqol) in people aged 65 or more. in addition, the study found that the literature shows a consensus about the positive association between the number of natural teeth and occluding pairs of teeth with ohqol. one of the factors that hinder this relationship is the lack of consensus on the functional dentition definition5. the world health organization (who), fdi world dental federation (fdi) and international association for dental research (iadr) have jointly established that to have a functional dentition, a person must retain at least 21 natural teeth7. this definition is widely used in research8; however, it does not consider the quantity and location of occlusal contacts. the evaluation of occlusal contacts seems to be more descriptive and discriminatory in determining the functional condition of the dentition9,10. however, studies in the literature are heterogeneous for study designs, populations, assessments of the measurements, and outcome tools6. this makes the evidence not conclusive and insufficient to determine the extent to which the functional condition of the dentition affects the quality of life of older adults. considering the aforementioned, the functional condition of the dentition was verified using three clinical indicators, aiming to evaluate the factors associated with each type of functional dentition classification focusing on the association with oral health-related quality of life. material and methods study design and participants the data employed in the present cross-sectional study were taken from the “fragilidade em idosos brasileiros” – fibra (frailty in older brazilians) study, conducted in 2008-2009. the fibra survey was a population-based, multicenter study designed to investigate conditions of frailty regarding health, sociodemographic, psychosocial, 3 tôrres et al. braz j oral sci. 2022;21:e226709 and functionality variables in older people of the community. methodological details have been previously published11. the minimum sample size was estimated at 601 elderly people living in the community (campinas – sp). for this calculation, the formula and parameters were used as described: n={z2 x [p x q / e2]} (formula for calculating sample size, without correction for finite populations); z=1.96 (95% significance level); p=q=0.50 (maximum confidence values to estimate prevalence in sample studies); e=4% (sampling error margin). a total of 90 census sectors were drawn, of which 88 sectors were part of the sample of 900 elderly people, totaling an average of 10.2 elderly people per sector. the sample was probabilistic by conglomerates, with the urban census sectors as the sampling unit. the number of census tracts was defined by dividing the number of existing urban census tracts by the desired number of elderly people. the recruiters received the map of each census sector and visited all the households to recruit as well as to identify those in which there were one or more elderly residents and interviewed those who met the inclusion criteria. in addition, they scheduled sessions for the next week with the elderly who agreed to participate. this sample, representative of the older population of campinas, brazil, included participants aged 65 or over who had complete data for the dependent variables “functionality of dentition”, as well as for the independent variables: “quality of life” (explanatory variable), and “sociodemographic/health conditions” (control variables). all procedures were approved by the ethics committee of the school of medical sciences of the university of campinas (process nº 208/2007). functionality of dentition three clinical indicators were evaluated, through dental examinations performed by three trained dentists, with a gold-standard examiner with experience on data collection following the who criteria for epidemiological studies in oral health12. individuals using dentures were asked to remove them. the variable occluding pairs of teeth was constructed based on the natural teeth present. measurements: • number of natural teeth present: categorized into up to 20 teeth (impaired) and 21 or more teeth (functional), according to the global goals for oral health 2020, proposed by who, fdi and iadr7. • occluding pairs of teeth: categorized according to the average number of occlusive teeth estimated in this research: up to 2 pairs and 3 or more pairs. • eichner index: posterior occlusal contacts were classified into four support regions (two molars and two premolars), and three categories were determined: “a” occlusal contacts in four posterior regions; “b” contacts in up to three posterior regions or only in the anterior area; and “c” without occlusal contacts13. oral health-related quality of life (ohrqol) we used the geriatric oral health assessment index (gohai) validated in brazil14, designed to assess the perception of oral health problems that impact quality of life in older adults15. the instrument consists of 12 questions, whose answers “always”, “sometimes”, and “never” were weighted on a scale of 1 to 3 points in ascending 4 tôrres et al. braz j oral sci. 2022;21:e226709 order, from the more negative condition to the more positive one, according to the context of the question. the higher the sum of the scores, more positive the evaluation is, therefore, lower perception of the impact of oral health on quality of life. the questionnaire was analyzed globally and according to dimensions of oral health problems. two categories were dichotomized based on a previous study16: positive perception that corresponded to a high score (in the global index greater than 34 points, and in the dimensions: physical/functional greater than 10 points, psychosocial/psychological 15 points, and pain/discomfort 9 points), and negative perception, corresponding to a moderate/low score (lower than the scores already indicated as high for the global index and for each dimension). sociodemographic/behavior/health conditions sociodemographic data were collected: age, gender, race/color dichotomized according to the sample distribution in “caucasians” and “non-caucasians” (category that included those who declared themselves as “black”, “mulato/caboclo/ pardo”, “indigenous” or “yellow/oriental”), literacy, schooling dichotomized in “up to three years of study” and “four or more years of study”, if retired, family income according to the minimum wage (mw) in 2008 equivalent to r$ 415.00/us $ 231, being dichotomized into “up to two mw” and “three or more mw”, and possession of residence (owner or not). data on self-reported smoking of older adults were also collected. frailty according to the phenotype of fried et al.17 (2001), whose evaluation details were previously published15. regarding oral health, access to dental services (“insurance/private” or “public”) and self-assessed oral health dichotomized as “positive” (when the older person assessed it as “excellent” or “good”) and “negative” (when the evaluation was “regular” or “bad”). statistical analysis associations between each dentition functionality indicator and independent variables were verified using the chi-square and fisher’s exact tests. multivariate models were constructed with the variables that showed an association of p <0.25 in the bivariate analyzes, performing poisson regressions for number of teeth (model 1) and pairs of teeth in occlusion (model 2); and multinomial regression for the eichner index (model 3). in the models, variables with a statistical significance of p <0.05 were presented, showing prevalence and odds ratios with 95% confidence intervals. it was used the backward stepwise method in this exploratory study on which the variables considered in the analysis were based on the p value and the epidemiological relevance on the association. results research participants of the 900 participants in the fibra study, 876 had complete data to be included in the analysis; their characteristics are described in table 1. the mean age of the vol5 tôrres et al. braz j oral sci. 2022;21:e226709 unteers was 72.78 (± 5.8) years, with a predominance of women (69.3%), caucasians (70.9%), four years or more of schooling (58.7%), and a family income above two minimum wages (71.8%). most of them assessed positively both their oral health (72.4%) and their quality of life in relation to oral health (69.8% global gohai). gohai ranged from 12 to 36 points. functional dentition condition only 14.1% of the older adults had 21 or more teeth, 25.3% had three or more occluding pairs of teeth and 71.3% had no occlusal contact (eichner c index), with a low prevalence of functional dentition (table 1). table 1. characteristics of the participants according to the studied variables (n= 876). variables n (%) gender man 276 (30.7) woman 624 (69.3) race/color caucasian 636 (70.9) non-caucasian 261 (29.1) literate no 196 (21.9) yes 699 (78.1) schooling 4 or more years 371 (41.3) up to 3 years 528 (58.7) retired no 261 (29.2) yes 634 (70.8) family income* up to 2 mw 221 (28.2) 3 or more mw 562 (71.8) home-owneship no 162 (18.0) yes 738 (82.0) smoking no 612 (88.8) yes 77 (11.2) frailty not frail 359 (39.9) pre-frail 469 (52.1) continue 6 tôrres et al. braz j oral sci. 2022;21:e226709 continuation frail 72 (8) use of dental service insurance/private 440 (67.7) public 210 (32.3) oral health self-assessment negative 186 (27.6) positive 487 (72.4) gohai global index negative (12 to 33 points) 265 (30.2) positive (34 to 36 points) 612 (69.8) gohai – physical / functional dimension negative (4 to 9 points) 108 (12.3) positive (10 to 12 points) 769 (87.7) gohai – psychological /psychosocial dimension negative (5 to 14 points) 320 (36.5) positive (15 points) 557 (63.5) gohai – pain/ discomfort dimension negative (3 to 8 points) 236 (26.9) positive (9 points) 641 (73.1) number of teeth up to 20 teeth 753 (85.9) 21 or more teeth 124 (14.1) paired teeth in occlusion up to 2 pairs 655 (74.7) 3 or more pairs 222 (25.3) eichner index a (occlusal contact in 4 posterior regions) 68 (7.7) b (occlusal contact in up to 3 posterior regions/ anterior only) 184 (21) c (without occlusal contact) 626 (71.3) gohai, geriatric oral health assessment index (positive perception: high score; negative perception: moderate/low score). *mw, minimum wage (in 2008 2mw = r$ 830.00; on average us$ 462). perception of problems associated with functional condition of dentition in figure 1, the oral health problems that impact on quality of life (gohai) associated with the studied clinical conditions stood out. regardless of the functional condition of the dentition, most older adults estimated that they had no problems that limited their diet (type and quantity), or speech, dissatisfaction with the smile, or discomfort eating in front of other people. the number of older adults with impaired dentition that indicated problems in chewing was higher. 7 tôrres et al. braz j oral sci. 2022;21:e226709 figure 1. frequency of response of older people to the gohai questions according to the functional condition of the dentition. the bar at the end of the figure symbolizes the range of colors that each category could acquire according to the number of older people who answered within it. it ranged from 0 individuals (white) increasing in intensity to black (876 responses), which is the maximum number of participants in this study. the categories of the eichner index mean: a, occlusal contact in 4 posterior regions; b, occlusal contact in up to 3 posterior regions/anterior only; and c, without occlusal contact. *p <0.05 for chi-square and fisher’s exact tests. perception of quality of life with different degrees of functionality in dentition regarding the presence of teeth and pairs of teeth in occlusion, table 2 showed the profile of older adults impaired dentition: women, non-caucasian, with low education and low family income, who negatively perceived their quality of life due to problems in the physical/functional dimension. however, older adults with functional dentition negatively perceived the pain/discomfort dimension. as for the eichner index, table 3 presented profiles of older people according to the number and location of occlusal contacts. individuals with occlusal contact in all posterior regions (eichner a) had higher education and higher family income. this characteristic is shared with those with occlusal contact in up to three posterior regions or only anterior (eichner b). in addition, the latter group assessed their oral health negatively. no association was found between the perception of quality of life and these profiles. 8 tôrres et al. braz j oral sci. 2022;21:e226709 table 2. poisson regression models with variables associated with the number of teeth (model no. 1) and pairs of teeth in occlusion (model no. 2). variables model no 1 model no 2 number of teeth* (up to 20 teeth) paired teeth in occlusion** (up to 2 pairs of teeth in occlusion) crude pr (95% ci) p adjusted pr (95% ci) p crude pr (95% ci) p adjusted pr (95% ci) p schooling up to 3 years (ref. 4 or more years) 1.17 (1.11-1.23) < 0.001 1.12 (1.07-1.18) < 0.001 1.40 (1.26-1.55) < 0.0001 1.28 (1.19-1.39) < 0.0001 family income up to 2 mw (ref. 3 or more mw) 1.12 (1.06-1.18) < 0.001 1.07 (1.02-1.12) 0.004 1.29 (1.17-1.43) < 0.0001 1.15 (1.07-1.24) < 0.0001 gender man (ref. female) 0.91 (0.85-0.97) 0.008 0.91 (0.85-0.97) 0.01 0.83 (0.73-0.95) 0.001 0.87 (0.79-0.96) 0.02 race/color non-caucasian (ref. caucasian) 0.87 (0.83-0.91) < 0.001 1.10 (1.05-1.16) < 0.001 gohai physical/functional dimension negative perception (ref. positive) 1.12 (1.07-1.18) < 0.001 1.12 (1.05-1.20) < 0.001 gohai pain/discomfort dimension negative perception (ref. positive) 0.91 (0.85-0.98) 0.014 0.91 (0.84-0.98) 0.01 *reference category: 21 or more teeth ** reference category: 3 or more pairs pr, prevalence ratio; ci, confidence interval; sm, minimum wage. table 3. multinomial regression with variables associated with the eichner index * (model no. 3). variables a (occlusion in 4 posterior regions) b (occlusion in up to 3 posterior regions/anterior only) crude or (95% ci) p adjusted or (95% ci) p crude or (95% ci) p adjusted or (95% ci) p schooling up to 3 years (ref. 4 years or more) 0.17 (0.09-0.35) < 0.001 0.27 (0.12-0.60) 0.001 0.35 (0.24-0.51) < 0.001 0.35 (0.21-0.56) < 0.001 family income up to 2 mw (ref. 3 or more mw 0.13 (0.04-0.38) < 0.001 0.18 (0.05-0.61) 0.005 0.39 (0.25-0.61) <0.001 0.44 (0.24-0.78) 0.005 gender man (ref. female) 1.20 (0.70-2.06) 0.51 1.32 (0.70-2.49) 0.38 2.01 (1.43-2.83) < 0.001 1.97 (1.28-3.03) 0.002 oral health self-assessment negative (ref. positive) 0.64 (0.32-1.26) 0.20 0.69 (0.33-1.44) 0.32 1.56 (1.05-2.32) 0.027 1.57 (1.01-2.48) 0.04 *reference category: c (without occlusion) or, odds ratio; ci, confidence interval; mw, minimum wage. 9 tôrres et al. braz j oral sci. 2022;21:e226709 discussion this research contributes to broaden the understanding of factors that affect oral health on older adults, with a still little explored approach in the literature. the associations found reveal two realities: older adults who perceive physical and functional problems have impaired dentition; and, surprisingly, those who do have functional dentition perceive problems related to pain and discomfort, probably because of the presence of unhealthy teeth. these two conditions (physical/functional and pain/discomfort) seem to have a negative impact on quality of life. in general, the study shows low prevalence of functional dentition. similar information was found in the literature18,19. although there has been a worldwide trend in the last few decades to preserve more teeth in aging20, this reality will probably occur in brazil by 205021. the causes are multifactorial; on the one hand, the current generation of older adults has belatedly benefited from the preventive public policies implemented in recent years21, added to the limited use of dental services, and the legacy of a care model in which mutilating practices prevailed22. even with the low prevalence of functional dentition, most older people have a positive perception of their oral health and overall quality of life, showing a difference between self-perception and real condition, also seen in other studies16,22. this finding can be explained because older adults are more resilient related to oral health10 and they adapt to oral conditions, devaluing the impact of diseases because they assume that it is an inevitable consequence of aging22. for this reason, evaluating quality of life from a global point of view could mask the identification of specific functions that harm older adults. and therefore, each dimension in particular was analyzed, finding a negative impact on physical/functional and pain/discomfort dimensions when older adult has less than 20 teeth. the physical/functional dimension of gohai assess several factors14, including chewing ability, which was affected in most older people without functional dentition. this finding seems to be expected and confirmed in previous studies23,24. many studies prove that older people with impaired dentition are more likely to have masticatory problems8,23 and to perceive them as negative for their quality of life10,24 emphasizing the need to maintain a functional dentition. precarious condition of the remaining teeth can explain the pain and discomfort that affect older people with functional dentition, as already observed in a similar study25 endorsed by world statistics that indicate an increase in oral diseases and need for treatment not attended due to reduction of tooth loss20. therefore, this is probably the cause of negative perception in this area. the profiles also reveal that they come from different segments of society. women, non-caucasians, with low educational level, and whose family group lives in poor economic conditions characterize the profile of older adults with impaired dentition. as the oral condition improves, the profile is associated with higher education and income. this social gradient was also observed for tooth loss21. consequently, the functional condition of dentition could be considered an indicator of social inequality. 10 tôrres et al. braz j oral sci. 2022;21:e226709 the low socioeconomic level is related to the lack of use of dental services21,26, limited information on prevention habits26,27, and difficulty in recognizing a health need28. despite the efforts to reduce social inequalities, they still persist among older adults28 and have an impact on the observed oral condition. gender seems to have a different effect according to the outcome evaluated. being men was associated with a lower prevalence of having less teeth and less occluding pairs of teeth but a higher prevalence of having occlusal contact in up to three posterior regions or only in anterior region (eichner b). women tend to use more regularly the oral health services and consequently are more susceptible to have their teeth extracted as a result of overtreatment29. as a differential of other studies, the functional condition of dentition was assessed considering three clinical indicators8, and as for quality of life it was used two indicators, one subjective (a self-perception single question) and another objective (the gohai instrument and its dimensions). the number of teeth was the indicator of dentition functionality that identified more associated factors, including quality of life, even though more precise measures such as occluding pairs of teeth and eichner index were adopted8. on the other hand, the eichner index characterized profiles in older adults, which to our understanding, have not yet been described in brazil using this instrument, showing a differential of this study. hence the importance of this study in expanding the understanding of associated factors according to the indicator used. as a limitation, we recognize the lack of oral health variables to check the condition of the remaining teeth and the use of prosthesis, that could confirm the hypothesis of discussion about the association of functional dentition and the perception of pain/discomfort. as it is an exploratory cross-sectional study, it advances in recognizing the variability of associated factors according to the selected indicator. finally, we emphasize that despite the worldwide trend to preserve more teeth throughout life, older brazilians still do not retain a number of teeth or adequate occlusal contacts to have a functional dentition and they perceive the negative impact of this condition. this reality reveals the need for preventive and therapeutic measures to maintain a healthy and functional dentition throughout life. two out of the three indicators assessed in this study were associated with quality of life. therefore, it is important to select sensitive indicators to be able to identify and better comprehend the relationship between a functional dentition and quality of life, especially in this age group that has great tooth loss. acknowledgments the authors thank espaço da escrita – pró-reitoria de pesquisa – unicamp – for the language services provided. funding sources: national council for scientific and technological development (cnpq) – 555082/2006-7 and são paulo research foundation (fapesp) no. 2008/03919-7. 11 tôrres et al. braz j oral sci. 2022;21:e226709 data avaliability the datasets related to this article belong to the fibra research group and will be available upon request from the author responsible for the project. data availability datasets related to this article will be available upon request to the corresponding author. conflict of interest none author contribution conceptualization: dds, mlrs. methodology: rbh, lhnt, dds, aln, mlrs. formal analysis: lhnt, dds, mlrs. investigation: rbh, dds. resources: aln. data curation: dds. writing—original draft preparation: rbh, lhnt, dds. writing—review and editing: rbh, lhnt, mjam, tba, dds, aln, mlrs. supervision: dds, aln, mlrs. project administration: dds, aln, mlrs. funding acquisition: dds, aln. all authors have read and agreed to the published version of the manuscript. references 1. müller f, shimazaki y, kahabuka f, schimmel m. oral health for an ageing population: the importance of a natural dentition in older adults. int dent j. 2017 sep;67 suppl 2:7-13. doi: 10.1111/idj.12329. 2. gil-montoya ja, mello al, barrios r, gonzalez-moles ma, bravo m. oral health in the elderly patient and its impact on general well-being: a nonsystematic review. clin interv aging. 2015 feb;10:461-7. doi: 10.2147/cia.s54630. 3. bidinotto ab, santos cm, torres lh, sousa md, hugo fn, hilgert jb. change in quality of life and its association with oral health and other factors in community-dwelling elderly adults-a prospective cohort study. j am geriatr soc. 2016 dec;64(12):2533-8. doi: 10.1111/jgs.14482. 4. lamster ib. geriatric periodontology: how the need to care for the aging population can influence the future of the dental profession. periodontol 2000. 2016 oct;72(1):7-12. doi: 10.1111/prd.12157. 5. chalub l, ferreira rc, vargas amd. influence of functional dentition on satisfaction with oral health and impacts on daily performance among brazilian adults: a 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10.1590/s0034-89102009000100003. 1 volume 22 2023 e230467 original article braz j oral sci. 2023;22:e230467http://dx.doi.org/10.20396/bjos.v22i00.8670467 1 restorative dentistry department, school of dentistry, university of são paulo (fousp), são paulo, sp, brazil. corresponding author: maria angela pita sobral address: department of restorative dentistry, school of dentistry, university of são paulo av. prof. lineu prestes, 2227 cidade universitária, são paulo sp, 05508-000. e-mail: mapsobra@usp.br tel: +55 1126488014 editor: altair a. del bel cury received: jul 22, 2022 accepted: sep 22, 2022 patient care in the restorative clinic of a public dental school after covid-19 lockdown kennedy santana de macedo1 , alana cristina machado1 , maria angela pita sobral1* aim: this study, with the aim of checking some of the changes in patients’ daily habits and their reasons for needing restorative treatment was conducted at a public university immediately on return to attendance after the covid-19 pandemic lockdown. methods: this survey consisted of interviews held by applying 2 questionnaires to students and patients. a single researcher collected data in all the restorative dentistry clinics at the university after the return to face-to-face activities in the period between 02/02/2021 and 07/30/2021. data obtained by means of the questionnaires were submitted to descriptive analysis. results: ninety (90) participants answered the patient questionnaire. when considering possible associations between being in social isolation or not, significant values were found for changed type of diet (p=0.0011), frequency of eating (p=0.011) and toothbrushing (p=0.034). data about 417 restorations were collected and among the reasons for restorative intervention, 33.03% were restoration replacement, 31.87% treatment of primary caries and 24.40% of dental wear/non-carious lesions. conclusion: patients who isolated themselves during the pandemic tended to change their eating habits and frequency of oral hygiene. toothache was the main reason for seeking dental treatment and the replacement of pre-existing restorations was the main reason for restorative treatments. keywords: covid-19. coronavirus. dental clinics. operative dentistry. universities. https://orcid.org/0000-0002-7853-7743 https://orcid.org/0000-0003-1772-0090 https://orcid.org/0000-0003-0976-3031 2 macedo et al. braz j oral sci. 2023;22:e230467 introduction on march 11, 2020, the world health organization (who) declared a pandemic status, making the outbreak of the new coronavirus (covid-19) a major public health challenge1-6. due to its high rate of transmissibility and in order to reduce the number of people infected with the virus, preventive measures were implemented around the world, the main ones being lockdown or social distancing3-7. considering the pandemic scenario, the authors speculated about what the conduct of patients would be in relation to oral health in this critical period of time. perhaps they would be more careful because of the fear of visiting a dental office during the pandemic and this would, therefore, lead to patients developing fewer or no oral pathologies. or an opposite situation might be found, in which patients might neglect their oral health because they were at home without social contact, or their smile would be covered by a face mask whenever they needed to be in public places in person, consequently they would be more likely to develop oral pathologies. previous studies have investigated the reasons for placement and replacement of direct restorations8-14 and have shown that primary caries was the main reason for performing restorations. wilson et. al. 8 (1997) revealed that over half of direct restorative practice consisted of replacing existing restorations. replacements of pre-existing restorations occur mainly due to secondary caries, and to a lesser extent, due to restorative material failure, such as, for example, marginal degradation, fractures and loss of anatomical shape8-14. moreover, in a study conducted in brazilian’s private clinics, braga et al.12 (2007) reported that they found similar results, and the second ranked reason for performing restorations was non-carious lesions, and the patients’ desire for the use of more aesthetic material, such as composite resin, was the main reason for replacement of amalgam restorations12. there are few studies in the literature that have proposed to evaluate the profile of patients who receive care at dental university clinics, specifically where restorations placed with direct materials are concerned. the results were similar to those found in studies conducted in private clinics. primary caries lesions were the main reasons for performing restorations and the main reason for replacing pre-existing restorations was the presence of secondary caries9,13. in view of the possibility of changing the patient’s behavioral profile, the advances in public oral health policies and considering the context of the covid-19 pandemic, the aim of this study was to check some of the changes in daily habits of patients who sought dental treatment at the dentistry school of the university of são paulo, in addition to verifying the reasons that caused the need for restorative treatment, immediately on return to face-to-face activities after the covid-19 pandemic lockdown. 3 macedo et al. braz j oral sci. 2023;22:e230467 material and methods experimental design and ethical aspects this clinical, observational, analytical, cross-sectional, single-center study was conducted at the school of dentistry of the university of são paulo after approval by the local research ethics committee (process number 4.520.017/caae 40941820.9.0000.0075). this survey consisted of interviews with application of two questionnaires: 1. to students enrolled in the clinical discipline of restorative dentistry and, 2. to patients who were treated by these students. a single researcher collected the data at all the clinics available at the university after the return to face-to-face activities in the period between 02/02/2021 and 07/30/2021. sample and survey to qualify students for answering the questionnaire, they had to meet the following inclusion criteria: they had to be enrolled in the restorative dentistry 2 discipline; have performed restoration(s) in their patients and had to accept participation in the research. for patients, the inclusion criteria were being over 18 years old and accepting to participate in the research. after signing the term of free and informed consent, 78 pairs of students and 90 volunteer patients who met the inclusion criteria and accepted to participate in the research answered the questionnaires. the student questionnaire, based on previous studies12, was applied in all clinics on the conclusion of the restorative procedure, and it took approximately 2 minutes to answer. the patient questionnaire was applied only once during the entire study, in the waiting room while the patient was waiting to be called. on average, patients took approximately 8 minutes to answer all the questions. all patients and students that met the inclusion criteria answered the questionnaire. statistical analyses the data obtained by means of the questionnaires were tabulated in spreadsheets and submitted to descriptive statistical analysis that allowed the researchers to discuss the impact of the pandemic on the patients’ oral health-related behaviors and whether this impact had any influence on the service profile of the restorative dentistry clinic. to compare some of the data collected in the two questionnaires obtained, contingency tables were constructed to enable the relationship between two sets of variables to be understood. the chi-square test (p<0.05) was applied to assess statistical significance and fisher’s exact test was applied to some data (p<0.05). results patients’ age varied between 18 and 72, with a mean age of 43 years; 52% were male and 48% female. among the participants 14% were students, 11% retired, 10% unemployed and 65% reported that they had a profession. 4 macedo et al. braz j oral sci. 2023;22:e230467 as regards the reasons for seeking dental care at the university, 123 reasons were reported by the participants, considering that among the 90 participants, some had more than one reason. the main reason for seeking care was pain (22.8%); waiting for a long time for treatment (15.4%); aesthetics (14.6%); tooth/restoration fracture (13.8%); presence of cavity (11.4%); indication of another discipline (11.4%) and return consultation (10.6%) (figure 1). return consultation 10.6% pain 22.8% indication of another discipline 11.4% presence of any cavity 11.4% teeth/restoration fracture 13.8% aesthetics 14.6% had been waiting for a long time for treatment 15.4% figure 1. distribution (%) of factors that led to patients seeking dental treatment at the dental clinic of the school of dentistry of the university of são paulo. distribution of patient responses related to covid-19, eating, oral care, and socioeconomic factors during the pandemic are shown in table 1. table 1. distribution of patients’ answers related to covid-19, eating, oral care, and socioeconomic factors during the pandemic. n % did you have covid-19? yes 10 11.11% no 80 88.89% did any close family member have covid-19? yes 42 46.67% no 48 53.33% did you practice social isolation? yes 66 73.33% no 18 20.00% just a few days (less than 30 days) 6 6.67% continue 5 macedo et al. braz j oral sci. 2023;22:e230467 continuation did your pattern of eating behavior change during the pandemic? yes 55 61.11% no 35 38.89% did your frequency of eating change during the pandemic? increased 47 52.22% decreased 13 14.44% continued the same 30 33.33% did your frequency of toothbrushing change during the pandemic? increased 19 21.11% decreased 16 17.78% continued the same 55 61.11% did you have toothache during the pandemic? yes 37 41.11% no 53 58.89% did any tooth or restoration fracture during the pandemic? yes 44 48.89% no 46 51.11% did you have to seek dental emergency care during the pandemic? yes 33 36.67% no 57 63.33% did you face financial difficulty? yes 55 61.11% no 35 38.89% have you avoided going back for/ or seeking dental treatment? yes 24 26.67% no 66 73.33% were you worried about your dental aesthetic appearance? yes 62 68.89% no 28 31.11% for statistical purposes, we compared patients that did practice isolation with those that did not. in the group “no = did not practice isolation”, we included patients who had not practiced isolation and patients who did so, but within a period of less than 30 days (table 2.). 6 macedo et al. braz j oral sci. 2023;22:e230467 table 2. frequency of the outcomes evaluated considering social isolation. outcome evaluated social isolation yes (n = 66) no (n = 24) p-value changed type of eating n = 47 (71.21%) n = 8 (33.33%) 0.0011 frequency of eating n = 49 (74.24%) n = 11 (45.83%) 0.011 frequency of toothbrushing n = 30 (45.45%) n = 5 (20.83%) 0.034 toothache n = 27 (40.91%) n = 10 (41.67%) 0.95 tooth or restoration fracture n = 33 (50.00%) n =11 (45.83%) 0.73 when applying statistics to these data, patients in social isolation changed their type of eating pattern to a significantly greater extent than patients who did not remain in social isolation (p=0.0011), as well as the frequency of eating (p=0.011) and frequency of tooth brushing (p=0.034). there was no significant difference for toothache (p=0.95) and tooth or restoration fracture (p=0.73) between those who were in social isolation and those who were not. from the student questionnaire were collected data about 417 restorative procedures; that is, 417 teeth were submitted to intervention at the direct dentistry clinic during the period of data collection. relative to these 417 teeth a total of 433 reasons were given to justify the restorative procedure. it is worth mentioning that of these 433 reasons, 143 were replacements, 101 were composite resin replacements with 147 reasons that justified the intervention; 21 amalgam replacements that had 23 reasons for the intervention and 21 replacements of temporary restorations by definitive types, as shown in table 3. the table also shows the distribution (%) of the following topics: teeth that underwent the procedures, cavities (black’s classification), and restorative material used. table 3. distribution of students’ answers related to the restorative procedure they performed n % tooth group incisor 107 25.66% canine 41 9.83% premolar 119 28.54% molar 150 35.97% black’s cavity classification i 115 27.58% ii 106 25.42% iii 54 12.95% iv 35 8.39% v 93 22.30% continue 7 macedo et al. braz j oral sci. 2023;22:e230467 continuation restorative material used composite resin 375 89.93% amalgam 4 0.96% glass ionomer cement 34 8.15% others 4 0.96% reasons for restorative intervention 433 restoration replacement 143 33.03% primary caries 138 31.87% dental wear/non-carious lesion 110 25.40% dental fracture 28 6.47% others 14 3.23% replacements 143 composite resin 101 70.63% amalgam 21 14.69% temporary restorations for definitive ones 21 14.69% reasons for composite resin replacement 147 secondary caries 55 37.41% material wear 21 14.29% inappropriate anatomical shape 20 13.61% fracture of the restoration 19 12.93% discoloration of the restoration 13 8.84% displacement of restoration 7 4.76% marginal discoloration 6 4.08% pain/sensitivity 6 4.08% reasons for amalgam replacement 23 fracture of the restoration 15 65.22% secondary caries 6 26.09% displacement of the restoration 1 4.35% inappropriate anatomical shape 1 4.35% discussion it is well known that due to covid 19 pandemic many daily habits and social conditions were altered and one of these was the recommendation of practicing social isolation. this study raised important information regarding the impacts of this condition on restorative dentistry procedures to help the scientific community and clinical dentists to recognize its possible after effects and to provide guidance to avoid an increase in oral diseases. quarantine or social isolation is an unfamiliar and unpleasant experience that involves separation from friends and family, and a change in usual everyday routines15. the 8 macedo et al. braz j oral sci. 2023;22:e230467 majority of patients in this survey, 73.33%, were concerned about the social isolation proposed as a health measure to contain the virus. they complied with this recommendation and reported that they did not have covid-19, although over 50% of the patients knew a close family member who had the disease. patients who complied with social isolation reported more changes in daily habits such as eating pattern, frequency of eating and tooth brushing, when compared with those who were not isolated. with regard to change in eating habits, our results were in agreement with those of a systematic review16 that suggested that people exposed to the preventive measures of restricting physical contact during the covid-19 pandemic may have experienced changes in food intake from several aspects such as increase in consumption of both healthy and unhealthy foods, restrictive eating behaviors, uncontrollable eating, behavior of eating outside of the home, and/or binging food from outside into the home16. whereas relative to brushing frequency, previous studies have shown that the subjects were brushing their teeth fewer times per day due to the use of masks, and people were less concerned about oral hygiene17. as regards toothache, social isolation did not seem to be an impact factor, since both patients who were isolated and those who were not isolated had toothache in the same proportion, with no statistical differences. however, toothache was the most prevalent condition for patients seeking dental care, as has also been reported by previous studies in which the most frequently mentioned reason for seeking dental treatment during the covid 19 pandemic period was toothache17,18. among the participants, only 37% reported having sought emergency dental care during the pandemic. it can be speculated that this number was not higher because patients were afraid of exposing themselves to the dental environment or leaving home during the period of social isolation19. this number could also have been attributed to the financial difficulty faced by the population during the pandemic19,20, which prevented them from seeking care by private professionals, since the free service at the university was suspended for a long period during the pandemic. financial difficulty was confirmed by over half of the patients in the sample collected. 61% of the patients reported having faced financial difficulties, which also impacted the access to health services20. the university clinic serves a more socioeconomically vulnerable population, which may explain the main reasons for seeking care, such as pain and dental or restoration fractures. something intriguing that emerged from the survey was the fact that 69% of participants claimed that they were concerned about dental aesthetics during the pandemic. theoretically, people would end up caring less about this factor since they were isolated and when in public they would be wearing a facial mask, however, even in the pandemic scenario, aesthetics was a concern. this result corroborated the findings of previous studies that showed that even with the use of face masks, aesthetic factors such as tooth color followed by tooth alignment were the main complaints of the subjects, as had occurred before the covid 19 pandemic period17,21,22. the results found in this survey are of great concern because an unbalanced diet can represent a cariogenic or acidic diet. when this is associated with deficient tooth9 macedo et al. braz j oral sci. 2023;22:e230467 brushing and reduced opportunity to seek dental care, the dietary factor may increase the risk for oral diseases such as dental caries, erosive tooth wear, gingivitis and periodontal infection and could perhaps change the profile of the restorative dentistry procedures performed in the dental clinic. considering the factors that determined the reason for the restorations, the main factor would appear to be the replacement of restorations, which has become an increasing part of the day-to-day work at the dental clinic. this topic was also reported in a recent literature review that included studies with similar methodology about the reasons for placement and replacement of direct restorations. the research was last updated in 201714 and demonstrated an increase in the percentage of restoration replacements since 1981 up to the present time. as regards the replacement of composite resin restorations, the main reasons for replacement were the recurrence of caries (37.41%); material wear (14.29%); inadequate anatomical shape (13.61%); restoration fracture (12.93%); restoration discoloration (8.84%); displacement of the restoration (4.76%); marginal discoloration (4.08%) and pain/sensitivity (4.08%). in other studies, including systematic reviews, the literature has also pointed out that the main reason for replacing composite resin restorations was the recurrence of caries followed by material fracture and aesthetics23,24. in our results, there were no statistical differences between patients who were in social isolation and those who were not, relative to the factor tooth/restoration fracture (p=0.73), since 44 patients (48.89%) reported dental/restoration fractures. although the literature has shown that the high rate of anxiety due to the covid-19 pandemic led to the increase of bruxism25-27, an oral condition that may have dental and restorations fractures28 as consequences. in this study, only 54 restorations were related with fractures: 28 teeth, 19 composite resin restorations and 15 amalgam restorations. considering the change in eating pattern, frequency of eating and toothbrushing, there was statistical difference for those who were isolated and those who were not, and these factors can represent a clinical impact on carious lesions. this fact could also explain why the main reasons for placement and replacement of restorations were primary or secondary caries, and not fractures. as far as the restorative material was concerned, composite resin was the most frequently used for restorations (89.93%), followed by glass ionomer cement (8.15%), amalgam (0.96%) and others (0.96%). composite resins with better mechanical characteristics have been developed, consequently they are materials with good clinical performance. the adhesive and aesthetic characteristics combined with the best mechanical behavior of the composite resins developed in recent years, have made them the direct restorative material of choice24,29. in view of the results observed, some difficulties faced during the research period could be pointed out, such as the fact that the university clinics in general only attended a reduced number of patients and many of the requested and scheduled appointments made by patients could not take place. at the above-mentioned clinics, consultations for clinical care were restricted to a single patient per stu10 macedo et al. braz j oral sci. 2023;22:e230467 dent pair per day. the time of clinical care duration was reduced. many patients were rotated through 2 or 3 different classes, in other words, the same patient was being treated by several different students. there was no prior triage due to the pandemic and several patients who arrived for treatment by the discipline were referred to another pair of students or class. these facts were the reality in dental clinics all over the world since dentistry was profoundly impaired by the pandemic scenario and the consultation model that was used could not be sustained until there was improvement in covid-19 pandemic situation, which probably resulted in impact on patients’ oral health. in addition, it could explain some important limitations of this study such as the impossibility of conducting a pilot study, the low number of attendances permitted, and low number of responses obtained in the patients’ questionnaires (90 responses) for the period proposed for the survey. in conclusion, patients who isolated themselves during the pandemic tended to change their eating habits and the frequency of performing oral hygiene and eating meals. dental pain was the main reason for seeking dental treatment, in patients whether they were isolated or not, while fear and economic difficulties led to postponed treatment despite concerns about aesthetics. the replacement of pre-existing restorations was the main reason for the restorative treatment, with composite resin being the restorative material most frequently used. acknowledgments this work was supported by the são paulo research foundation (fapesp) brazil. under grant nº 2020/13341-4. data availability datasets related to this article will be available upon request to the corresponding author. conflict of interest none. author contribution kennedy santana de macedo: conceptualization, methodology, survey application, data collection, writing original draft and funding acquisition. alana cristina machado: conceptualization, methodology, writing review and editing. maria angela pita sobral: project administration, conceptualization, methodology, data curation, formal analysis, writing review and editing. all authors actively participated in the manuscript’s findings, revised, and approved the final version of the manuscript. 11 macedo et al. braz j oral sci. 2023;22:e230467 references 1. guo j, xie h, liang m, wu h. covid-19: a novel coronavirus and a novel challenge for oral healthcare. clin oral investig. 2020 jun;24(6):2137-8. doi: 10.1007/s00784-020-03291-8. 2. dziedzic, a, wojtyczka, r. the impact of coronavirus infectious disease 19 (covid‐19) on oral health. oral 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10.1016/j.jdent.2020.103387. 21. dudea d, lasserre jf, alb c, culic b, pop ciutrila is, colosi h. patients’ perspective on dental aesthetics in a south-eastern european community. j dent. 2012 jul;40 suppl 1:e72-81. doi: 10.1016/j.jdent.2012.01.016. 22. lawson j, warren jj, levy sm, broffitt b, bishara se. relative esthetic importance of orthodontic and color abnormalities. angle orthod. 2008 sep;78(5):889-94. doi: 10.2319/080207-361.1. 23. demarco ff, corrêa mb, cenci ms, moraes rr, opdam nj. longevity of posterior composite restorations: not only a matter of materials. dent mater. 2012 jan;28(1):87-101. doi: 10.1016/j.dental.2011.09.003. 24. demarco ff, collares k, correa mb, cenci ms, moraes rr, opdam nj. should my composite restorations last forever? why are they failing? braz oral res. 2017 aug 28;31(suppl 1):e56. doi: 10.1590/1807-3107bor-2017.vol31.0056. 25. cerqueira trdc, batista sg, de mello eb, dossantos mf, tuñas itc. impact of the covid-19 pandemic on stress, sleep, and oral health in university students. front pain res (lausanne). 2021 oct 26;2:744264. doi: 10.3389/fpain.2021.744264. 26. emodi-perlman a, eli i, smardz j, uziel n, wieckiewicz g, gilon e, et al. temporomandibular disorders and bruxism outbreak as a possible factor of orofacial pain worsening during the covid-19 pandemic-concomitant research in two countries. j clin med. 2020 oct;9(10):3250. doi: 10.3390/jcm9103250. 27. carrillo-diaz m, ortega-martínez ar, romero-maroto m, gonzález-olmo mj. lockdown impact on lifestyle and its association with oral parafunctional habits and bruxism in a spanish adolescent population. int j paediatr dent. 2022 mar;32(2):185-93. doi: 10.1111/ipd.12843. 28. beddis h, pemberton m, davies s. sleep bruxism: an overview for clinicians. br dent j. 2018 sep;225(6):497-501. doi: 10.1038/sj.bdj.2018.757. 29. rasines alcaraz mg, veitz-keenan a, sahrmann p, schmidlin pr, davis d, iheozor-ejiofor z. direct composite resin fillings versus amalgam fillings for permanent or adult posterior teeth. cochrane database syst rev. 2014 mar 31;(3):cd005620. doi: 10.1002/14651858.cd005620.pub2. update in: cochrane database syst rev. 2021 aug 13;8:cd005620. 1http://dx.doi.org/10.20396/bjos.v21i00.8664809 volume 21 2022 e224809 original article ¹ school of health sciences, department of dentistry, positivo university, curitiba, paraná, brazil. ² latin american institute of dental research and education, ilapeo, curitiba, paraná, brazil. 3 department of stomatology, federal university of paraná, curitiba, paraná, brazil. corresponding author: rafaela scariot e-mail: rafaela_scariot@yahoo.com.br 632 prefeito lothario meissner avenue, curitiba, pr, brazil 80210-170 telephone/fax number: +55 41 33604020 editor: dr altair a. del bel cury received: march 2, 2021 accepted: june 14, 2021 microbial evaluation of implant surface – a clinical study comparing submerged, non-submerged and immediately loaded implants fabio furquim¹ , tatiana miranda deliberador² , maria cecília lopes giacomel1 , suyany gabriely weiss¹ , joão césar zielak1 , rafaela scariot3,* aim: the aim of the present study was to evaluate the microbial contamination in internal and external walls of cone morse implant walls. methods: eleven patients with edentulous mandibular posterior area were selected to received dental implants, divided into groups: submerged (s), non-submerged (ns), and immediately loaded (il). microbiological evaluations (microorganisms’ number, aerobic and anaerobic colony forming units (cfu) number and microorganisms’ qualification) were divided into internal and external collection of the implant walls, at different stages: t0 (surgical procedure), t2 (suture removal), t4 (reopening s group), t6 (suture removal s group), and t8 (abutment placement in s and ns). all data were submitted to statistical analyses, with confidence level of 0.05. results: there was difference in number of microorganisms observed over time within the same group (p < 0.05). a difference was observed in cfu when evaluated within the same group over time (p < 0.05), except for the il group. in internal collection, a predominance of non-formation of microorganisms was observed at t0 in all groups, while formation of gram-positive diplococci and gram-positive bacilli was observed at t8 (p>0.05). in external collection, an increase in number of microorganisms was observed at t0. conclusion: there was no difference in microbial contamination among the evaluated groups. the microorganism’s colonization changed over time. keywords: dental implants. surgery, oral. actinobacteria. https://orcid.org/0000-0001-5148-2706 https://orcid.org/0000-0003-4076-4905 https://orcid.org/0000-0003-3659-0797 https://orcid.org/0000-0002-5778-3144 https://orcid.org/0000-0003-3393-3491 https://orcid.org/0000-0002-4911-6413 2 furquim et al. introduction dental implants are currently a safe and predictable reality for patients that wish to rehabilitate their oral health1. there are two surgical protocols for dental implants insertion: one-stage surgery or non-submerged implants; two-stage surgery or submerged implants. in the first technique, the implant is placed so that the soft tissue flap remains around the coronal portion of the implant body or healing abutment. in the second surgical technique, the implant is placed at or below the alveolar crest level and the soft tissue is closed around the implant. then, after a suitable period, a second procedure is performed to expose the implant platform and install a healing or a final abutment2. during the initial phase of osseointegration, most implant systems recommend that the implant remain submerged3-5. there are several reasons for the use of submerged implants, including minimizing the risk of infection, reducing vertical bone level loss and reducing the risk of excessive early loading6. a radiographically randomized clinical trial conducted by giacomel et al., in 20177, compared changes occurred in marginal bone level between immediately loaded implants (il) delayed loaded non-submerged implants (ns) and delayed loaded submerged implants (s). the different protocols used for implant placement and loading did not show statistically significant differences in bone level changes around the implants. since the implant is composed of an endosseous part that connects it to the prosthetic part, even if these structures are closely attached, a microgap of up to 50μ may exist between them8. within this context, it is essential that the connection between abutment and implant is as effective as possible in order to prevent or reduce the risk of bacterial colonization, since the greater the proximity between the microgap and the alveolar bone, the greater the expected bone loss9. although there is lack of in vivo studies comparing bacteria in submerged, non-submerged and immediately loaded implants, outcomes from a clinical study suggested that all the external hexagon, double internal hexagon, internal hexagon with external collar and conical connection contaminated after 5 years of functional loading10. progressive colonization by periodontopathogenic bacteria in the internal cavities of two-piece implants has been previously described11. besides, submerged implants with screw-retained structures are considered to be more susceptible to pathogenic microflora than non-submerged implants12. therefore, the aim of the present study was to evaluate quantitatively the microbial contamination in internal and external walls of cone morse implant walls in submerged (s), non-submerged (ns) and immediately loaded (il) implants at different stages. material and methods ethical approval was obtained from the ethics research committee of the university, under protocol number 69244216.5.0000.0093. study population the inclusion criteria were adult patients with at least three missing teeth that should be mandibular premolars and/or mandibular molars. patients who entered the study 3 furquim et al. received oral hygiene instructions. in addition, all patients in the study were submitted to prophylaxis every three months. thus, we were able to submit the same patient to submerged, non-submerged and immediately loaded implants, under the same oral and systemic conditions, decreasing bias. patients were excluded from the study if any of the following criteria was present: (1) smoking, pregnancy or use of bisphosphonates (2) uncontrolled systemic disorders; (3) parafunction; (4) poor oral hygiene; (5) the presence of active periodontal disease or caries; (6) a need for bone reconstruction before implant insertion. patients were consecutively selected according to the inclusion and exclusion criteria, and to the period planned for patient recruitment. sample size calculation was not performed but was based on a previous study of our group7 that evaluated the same three different approaches: submerged, non-submerged and immediately loaded implants in marginal bone level changes. anamnesis and clinical examination in the first evaluation, a complete anamnesis and physical/clinical examination, definition of the treatment plan and oral hygiene instructions were performed. an occlusal evaluation to determine the prosthetic space available was also carried out. in this case, the occlusal height and the possibility of rehabilitation after implant placement were verified. the patients were maintained, until the end of the research, in an adequate plaque control regimen. all treatment steps were performed and registered by the same calibrated examiner with experience in implantology (ff). in addition, all patients had a mandible tomography performed on the i-cat classic (imaging sciences international, hatfield, pennsylvania, usa). with this examination, the size of the implant to be installed in the region was defined. surgical procedures all patients received three dental implants divided into three groups according to the installation protocol. the submerged group (s) implants received a delayed loading and remained submerged until the reopening phase and subsequent prosthetic rehabilitation. the non-submerged group (ns) received delayed loading with non-submerged implants until the prosthetic rehabilitation phase. the immediately loaded group (il) implants received immediate loading without abutment removal for prosthetic rehabilitation. surgery began for all implant groups with a mid-crestal incision in the edentulous area. the same milling sequence was used for the confection of the bone window and implant placement and consisted of initiating drilling with a 2.0 mm-diameter spear drill, followed by a 2.0 mm-diameter helical drill and by a milling cutter specific for 3.5 mm-diameter sw morse implants (sin – implant system, são paulo, brazil). all implants were placed 1 mm below the bone crest level. after implant insertion, the final torque was evaluated. patients were advised regarding postoperative care and mechanical control of bacterial plaque. to control postoperative pain, patients were instructed to use ibuprofen 600 mg every eight hours for three to five days. amoxicillin 500 mg was also prescribed every eight hours for seven days for patients not allergic to penicillin. sutures were removed seven days after implant placement. 4 furquim et al. collection and microbiological analysis microbial collection was performed in two ways: internal and external. the internal collection was held in the internal part of the surrounding walls of the implant and the external collection in the external part of the implant walls, always at a point located in the buccal surface. the internal collection analysis was conducted in the following stages: t0 internal collection in s group, ns group and il group on the day of surgery. in the il group, a single internal collection was performed since the abutment was not removed after placement (figure 1a); t4 after a period of 90 days, a new collection was executed in the s group, on the day of reopening and insertion of the healing screw; t8 after the removal of the healing screw and before the insertion of the final abutment, a new internal collection was carried out in s group and ns group. the final abutment was then placed, and a provisional acrylic resin restoration was cemented. a b figure 1. (a): internal collection at t0, ns group. (b): external collection at t0, located on the periimplant groove, ns group. the external collection analysis was conducted in the following stages: t0 for il group and ns group on the day of implant insertion, in which material was collected in the abutment transmucosal area in the il group and in the transmucosal area of the healing screw in the ns group. at this stage, there was no collection in s group implants, as they remained submerged (figure 1b); t2 material collection in the abutment transmucosal area in the il group and in the transmucosal area of the healing screw in the ns group, on the day of suture removal; t4 on the day of reopening in the s group, the material was collected in the transmucosal area of the healing screw; t6 material collection around the transmucosal area in the s group, on the day of suture removal; t8 the material was collected around the transmucosal area in ns group and s group after placement of the final abutment. evaluation of colony forming units (cfu) qualitative evaluations were performed by inoculating the material in petri dishes with blood agar culture. bacterial proliferation was evaluated after a period of 24 hours at a temperature of approximately 37°c. for the cfu quantification, the criterion 5 furquim et al. used was “no growth”, “countable growth” and “uncountable growth” as expressed: no growth = < 1 cfu; countable growth 1 a 250 cfu and uncountable growth if it is not possible to count the number of colonies on the plate, as they are too numerous to count (> 250 cfu). spectrophotometric analysis of optical density to perform the analysis of optical density of all groups, the material was collected in t0, t2, t4, t6 and t8 and read immediately. in addition, a new reading occurred after a period of 24 hours (t1, t3, t5, t7 and t9) in which the collection was incubated at a temperature of approximately 37°c. gram staining the gram staining process started by covering the entire surface of the plate on which the smear was performed with crystal violet (purple dye), for two minutes, draining the excess. the plate was again covered with lugol solution (mordant) for one minute. once it was rinsed with distilled water, acetone-alcohol solution was dripped over it for about fifteen seconds, followed by a new wash. again, the plate was covered with carbol fuchsin, followed by rinsing and drying. once the plate was completely dry, a drop of entellan was applied on the side where the smear layer was performed and covered with a coverslip, waiting for the entellan to dry completely. after a period of 24 hours, the plate was analyzed using a microscope. after staining, the plates were classified according to the presence of gram-negative bacilli, gram-positive bacilli, gram-negative diplococci, gram-positive diplococci, gram-negative cocci, gram-positive cocci and funghi. the amount of growth was quantified as no growth (-), little growth (+), average growth (++) and substantial growth (+++) which is considered in values in percentage. no growth (-) corresponds to 0%, little growth (+) corresponds to 1 – 33.33%, average growth (++) corresponds to 33.34 to 66.66% and substantial growth (+++) corresponds to 66.67 to 100%. statistical analysis the data were tabulated and submitted to analysis using spss software for windows 24.0 (spss inc., chicago, illinois, usa). the data were submitted to normality test. after the test, the numerical variables were considered non-parametric, being cataloged by the median, minimum and maximum. the cfus were evaluated as an ordinal variable. statistically significant differences were considered when p < 0.05. results of the eleven patients evaluated, the majority of the sample was female (63.6%) and caucasian (90.9%). mean age was 49.91 years (±10.23). table 1 presents the results of spectrophotometric analysis of optical density for all groups over time, both in internal and external collections. it is possible to observe that there was a statistically significant difference in the number of microorganisms when the same group was assessed over time (p < 0.05). in t1, t3, t5, t7, and t9 there was greater bacterial growth than in other phases since the other stage correspond to the analysis performed after 24 hours of culture of the collection. 6 furquim et al. ta bl e 1. s pe ct ro ph ot om et ric a na ly si s of o pt ic al d en si ty e va lu at in g th e su bm er ge d (s ), no nsu bm er ge d (n s) a nd im m ed ia te ly lo ad ed (i l) g ro up s at th e di ff er en t s ta ge s c ol le ct io n g ro up s t0 t1 t2 t3 t4 t5 t6 t7 t8 t9 pva lu e (s ta ge ) m ed ia n (m in -m ax ) m ed ia n (m in -m ax ) m ed ia n (m in -m ax ) m ed ia n (m in -m ax ) m ed ia n (m in -m ax ) m ed ia n (m in -m ax ) m ed ia n (m in -m ax ) m ed ia n (m in -m ax ) m ed ia n (m in -m ax ) m ed ia n (m in -m ax ) in te rn al s 0. 03 b (0 .0 1– 0. 32 ) 1. 15 a (0 .5 3– 1. 59 ) 0. 72 ab (0 .1 2– 0. 82 ) 1. 54 a (0 .8 1– 2. 11 ) 0. 00 b (0 .0 3– 0. 09 ) 1. 28 a (1 .0 6– 1. 72 ) < 0. 00 1 n s 0. 03 6a b (0 .0 1– 0. 23 ) 0. 89 c (0 .1 3– 1. 51 ) 0. 46 a (0 .0 1– 0. 20 ) 1. 22 b (0 .8 2– 1. 44 ) < 0. 00 1 il 0. 04 a (0 .0 1– 0. 64 ) 1. 24 b (0 .1 7– 1. 58 ) 0. 00 5 ex te rn al s 0. 44 a (0 .1 0– 0. 59 ) 1. 08 a (0 .3 6– 2. 21 ) 0. 04 b (0 .0 1– 0. 10 ) 1. 28 a (0 .9 1– 1. 55 ) 0. 03 bc (0 .0 1– 0. 05 ) 1. 28 a (1 .0 6– 1. 72 ) < 0. 00 1 n s 0. 15 a (0 .0 9– 0. 34 ) 1. 54 ab (0 .9 7– 1. 71 ) 0. 25 a (0 .0 90. 36 ) 1. 83 b (1 .3 12. 25 ) 0. 03 c (0 .0 2– 0. 15 ) 1. 35 b (1 .0 2– 1. 70 ) < 0. 00 1 il 0. 22 a (0 .1 3– 0. 70 ) 1. 74 b (1 .1 5– 1. 86 ) 0. 22 a (0 .1 4– 0. 72 ) 1. 95 b (1 .0 52. 26 ) < 0. 00 1 n ot e: f rie dm an n te st fo r t hr ee o r m or e ca te go rie s, w ilc ox on te st fo r t w o ca te go rie s; d iff er en t l et te rs m ea n st at is tic al s ig ni fic an ce a t p < 0 .0 5 7 furquim et al. table 2 shows the comparison among groups of the results obtained in the spectrophotometric analysis of optical density at each stage. there was no statistically significant difference between groups (p > 0.05). the comparison among the different groups of implants (s, ns, and il) regarding the number of aerobic and anaerobic cfu formed at t0, t2, and t8 did not present a statistically significant difference (table 3). the intragroup comparison showed a statistically significant difference in the number of aerobic and anaerobic cfu formed over time in s group and ns group in the internal collection and in ns group in the external collection (p < 0.05) (table 4). table 2. spectrophotometric analysis of optical density comparing the studied groups at each stage collection stages submerged group non-submerged group immediately loaded group p-value median (min-max) median (min-max) median (min-max) median (min-max) internal t0 0.04 (0.01–0.32) 0.04 (0.01–0.30) 0.07 (0.01–0.64) 0.896 t1 1.15 (0.30–1.59) 1.17 (0.13–1.56) 1.25 (0.17–1.58) 0.929 t8 0.05 (0.02–0.09) 0.05 (0.01–0.20) 1 t9 1.34 (1.06–1.95) 1.19 (0.82–1.56) 0.104 external t0 0.15 (0.02–0.36) 0.22 (0.10–0.70) 0.06 t1 1.53 (0.99–1.71) 1.62 (0.66–1.86) 0.695 t2 0.18 (0.09–0.36) 0.22 (0.14–0.76) 0.292 t3 1.83 (1.31–2.25) 1.73 (1.05–2.26) 0.794 t8 0.03 (0.01–0.05) 0.03 (0.02–0.33) 0.179 t9 1.42 (1.06–1.72) 1.35 (1.02–1.70) 0.479 note: kruskall-wallis test for three or more categories, mann-whitney test for 2 categories; significance level of 0.05. the external collection was not performed in s group at t2 and t3 since implants remained submerged in these stages. table 3. comparison among the submerged, non-submerged and immediately loaded groups regarding the aerobic and anaerobic cfu count. t0 p value t2 p value t8 p value internal aerobic (s, ns, il) 0.423 1 anaerobic (s, ns, il) 0.378 1 external aerobic (s, ns, il) 0.365 0.748 0.730 anaerobic (s, ns, il) 0.562 0.748 1 note: kruskall-wallis test for three or more categories, mann-whitney test for 2 categories; significance level of 0.05. the internal collection could not be compared between groups at t2 since implants in s group remained submerged in this stage. 8 furquim et al. ta bl e 4. q ua nt ifi ca tio n of a er ob ic a nd a na er ob ic c ol on y fo rm in g un its (c fu ) w ith in th e su bm er ge d (s ), no nsu bm er ge d (n s) a nd im m ed ia te ly lo ad ed (i l) g ro up s ov er ti m e c ol le ct io n g ro up s s ta ge s a er ob ic c fu (n ) s ta ge s a na er ob ic c fu (n ) n o g ro w th (n ) c ou nt ab le (n ) u nc ou nt ab le (n ) pva lu e n o g ro w th (n ) c ou nt ab le (n ) u nc ou nt ab le (n ) pva lu e in te rn al s t0 a 5 6 0 >0 .0 01 t0 a 8 3 0 0. 02 t4 b 3 6 t4 bc 2 3 4 t8 bc 9 t8 b 9 n s t0 7 4 0 0. 03 t0 9 2 0 0. 02 t8 9 t8 9 il t0 8 3 0 t0 6 5 0 ex te rn al s t4 1 2 6 0. 05 t4 9 1 t6 9 t6 9 t8 9 t8 9 n s t0 a 5 2 4 0. 02 t0 a 5 3 3 0. 02 t2 ab 4 7 t2 ab 9 t8 b 9 t8 b 9 il t0 3 3 5 0. 10 t0 2 5 4 0. 18 t2 2 9 t2 4 7 n ot e: k ru sk al l-w al lis te st fo r t hr ee o r m or e ca te go rie s, m an nw hi tn ey te st fo r 2 c at eg or ie s; s ig ni fic an ce le ve l o f 0 .0 5. d iff er en t l et te rs m ea n st at is tic al s ig ni fic an ce a t p < 0 .0 5 9 furquim et al. table 5 compares the number of aerobic and anaerobic cfu formed within groups at the different stages, according to the type of microorganism identified by gram staining. table 5. classification of the different types of microorganisms present within groups at the different stages collection groups microorganisms t0 t2 t4 t6 t8 internal submerged non-formation aerobic +++ x x anaerobic +++ x + x g+ diplococci aerobic +++ x ++ x +++ anaerobic ++ x ++ x +++ g+ bacilli aerobic x +++ x +++ anaerobic x +++ x + non-submerged non-formation aerobic +++ x x x anaerobic +++ x x x g+ diplococci aerobic +++ x x x +++ anaerobic + x x x +++ gdiplococci aerobic x x x anaerobic x x x g+ bacilli aerobic x x x ++ anaerobic x x x + immediately loaded non-formation aerobic +++ x x x x anaerobic +++ x x x x diplococo g+ aerobic ++ x x x x anaerobic ++ x x x x funghi aerobic + x x x x anaerobic + x x x x external submerged non-formation aerobic x + anaerobic x g+ diplococci aerobic x ++ +++ +++ anaerobic x +++ +++ +++ gdiplococci aerobic x + anaerobic x + g+ bacilli aerobic x +++ + ++ anaerobic x +++ + + gbacilli aerobic x + + anaerobic x g+ cocci aerobic x + anaerobic x + gcocci aerobic x + anaerobic x + funghi aerobic x ++ +++ anaerobic x + continue 10 furquim et al. discussion the concern of implant clinicians regarding the best choice of implant for a specific area is a constant point of discussion. one of the main topics that guide the decision is based on the choice of whether to use submerged, non-submerged or immediately loaded implants. therefore, the main objective of the present study was to investigate if there is a difference among the studied groups regarding bacterial contamination externally or internally to dental implants. the microbiota in the oral environment determines to a large extent the composition of the flora developing around implants13. it has been hypothesized that submerged implants may present a lower risk of infection, less vertical bone loss and lower risk of implant overloading3-5,14. furthermore, it has been hypothesized that immediately loaded implants would present lower internal microbial contamination when compared to other implants. as for the assessment of the same implant over time, the present study found a significant difference in microbial contamination both by spectrophotometric analysis continuation external non-submerged non-formation aerobic +++ x x + anaerobic +++ x x + g+ diplococci aerobic +++ +++ x x +++ anaerobic +++ +++ x x +++ gdiplococci aerobic + x x anaerobic + x x g+ bacilli aerobic + ++ x x +++ anaerobic x x + g+ cocci aerobic + x x anaerobic x x gcocci aerobic + x x anaerobic x x funghi aerobic + x x anaerobic x x + immediately loaded non-formation aerobic ++ x x x anaerobic + x x x g+ diplococci aerobic +++ +++ x x x anaerobic +++ +++ x x x gdiplococci aerobic + x x x anaerobic x x x g+ bacilli aerobic +++ x x x anaerobic ++ x x x funghi aerobic x x x anaerobic + x x x x – collection was not performed; no growth; + cfu little growth; ++ cfu average growth; +++ cfu substantial growth. 11 furquim et al. and by cfu count. one fact that draws attention is that, in some specific stages it was demonstrated greater bacterial contamination than in others. gaps and hollow spaces within the implant system, for example the gap between implant and abutment in the two-part implant system, may provide a bacterial reservoir causing or maintaining inflammation. the bacterial spectrum involved is similar to that found in periodontitis15. in general, in our study there was more microorganism growth in the external collections than in the internal collections, except for the group of submerged implants (s group), in which values remained similar. this may be explained by the existence of a microgap between the abutment and the implant, which is an area where there is a higher concentration of microorganisms. thus, the greater the gap between the implant and the abutment, the greater the microbial contamination in the area, leading to greater bone loss8-9,16-18. in the present study, no difference in microbial contamination was found among the different groups of implants, either by spectrophotometric analysis or by cfu count, contradicting the initial hypothesis in which it was thought that immediately loaded implants would be advantageous since they would present lower microbial contamination. there are no studies in the literature comparing the three types of implants evaluated up until the writing of this study. however, some interesting studies have been previously carried out assessing microbial contamination, mainly comparing different abutment designs. in a study conducted by de moraes rego et al.16 significantly higher counts of a. gerencseriae and s. constellatus were found in implants placed at the supracrestal level compared to the ones placed at the bone level. no relation was found between the installation level of dental implants and peri-implant bone remodeling. peruzetto et al.19, in 2016 evaluated the bacterial seal at the implant-abutment interface using two morse taper implant models, by in vitro microbiological analysis. the authors concluded that both tapered components failed to provide adequate sealing to bacterial leakage, although the indexed type components showed a superior seal compared with non-indexed components. another important issue of the study refers to the cfu quantification for each of the groups in each one of the stages. it was possible to observe that there was greater microbial contamination within the same group of implants over time, and that there were more cfu externally than internally. it is known that contamination of internal implant and suprastructure components has shown considerable great biodiversity, indicating bacterial leakage along the implant-abutment interface, abutment-prosthesis interface, and restorative margins. cosyn et al.17 compared microbiologically the peri-implant sulcus to these internal components on implants with no clinical signs of peri-implantitis and in function for many years. the authors concluded intra-coronal compartments of screw-retained fixed restorations were heavily contaminated. the restorative margin may have been the principal pathway for bacterial leakage. contamination of abutment screws most likely occurred from the peri-implant sulcus via the implant-abutment interface and abutment-prosthesis interface. in order to qualify the microorganisms present, gram staining was used. thereby making it possible to observe the presence of gram-positive and gram-negative 12 furquim et al. microorganisms. normal microbiota of healthy implants includes gram-positive rods and cocci. peri-implantitis is caused by pathogens, especially gram-negative bacteria like veillonella sp. and spirochetes, including treponemadenticola20,21. in this study, in the internal collection of the implant wall, it was observed a predominance of non-formation of microorganisms at t0 in all groups, with formation of gram-positive diplococci and gram-positive bacilli at t8. on the other hand, in the external collection, an increase in the number of microorganisms was observed at t0, when compared to the internal collection. fungi were also predominant in the external collection. one limitation of the study is that no characterization of the evaluated bacteria was carried out. with our results it is possible to know the morphology of the bacterium and if it positive or negative. we cannot say, however, whether the quantification of the bacteria indicates a greater predisposition to peri-implantitis and/or bone loss. moreover, it is important to consider that the bacteria found could be part of the normal flora and the hygiene of each patient could influence the results during the collection of biological material. we recommend a more detailed analysis in future studies. another important point is that at t4 (reopening s group), the external collection was not performed in the ns group since the healing process of the tissue around the healing screw prevented the insertion of the paper cone in that region. also, it is important to highlight the choice for the use of antibiotics in the postoperative period. we chose to use this type of medication in order to reduce the possibility of infectious processes related to the procedure. however, it should be emphasized that the primary objective of the study is to compare the three groups and all of them were subject to the same effects of the antibiotic in the seven-day period, which we believe does not make the results unfeasible. there was also no control over the systemic drugs used by patients during the collection of biological material. however, it is worth mentioning that patients who take systemic medications continued the intake during all evaluations, therefore, we understand that this does not interfere with the results, as the comparisons are performed on the same individual. based on the present results, it can be stated that regardless the insertion of submerged or non-submerged implants, no difference is expected in their microbial contamination. thus, considering the reduction of surgical stages, the insertion of non-submerged implants, may present as an advantage if the basic principles of osseointegration are respected, such as good primary stability and sufficient bone quantity4. finally, it is possible to infer that there is no difference concerning the amount of microbial contamination among the studied groups, only for microorganism’s colonization over time. acknowledgements there are no conflicts of interest in this study. 13 furquim et al. references 1. palaska i, tsaousoglou p, vouros i, konstantinidis a, menexes g. influence of placement depth and abutment connection pattern on bone remodeling 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department of oral and maxillofacial surgery, school of dentistry, university of são paulo, brazil 2 department of oral and maxillofacial surgery, hospital m. dr. arthur r. de saboya, são paulo, brazil *correponding author: joão gualberto c. luz department of oral and maxillofacial surgery, school of dentistry, university of são paulo – usp. av. prof. lineu prestes, 2227 cidade universitária, 05508–900, são paulo – sp, brazil. phone: 5511 3091-7887, fax: 5511 3091-7879. e-mail: jgcluz@usp.br received: november 02, 2020 accepted: january 11, 2021 retrospective study of sports-related maxillofacial fractures in a brazilian trauma care center julia souza ribeiro-dos-santos1 , vinícius paes de assis santos2 , joão gualberto cerqueira luz1,2 * aim: maxillofacial fractures occur frequently in the general population, and sports-related fractures represent some of these cases. however, few studies have been carried out in brazilian populations aimed at sports-related maxillofacial fractures. this study assessed the demographic and fracture characteristics of patients with sports-related maxillofacial fractures who were seen at a brazilian trauma care center. methods: medical records of patients with a history of sports-related maxillofacial fractures treated between january 2018 and december 2019 were retrospectively evaluated. personal data, fracture characteristics, sport type, treatment performed and need for hospitalization were collected. the data were subjected to statistical analyses with likelihood ratio test using the statistical package for social sciences (spss) 25.0 (p ≤ 0.050). results: forty cases (4.96% of the total) of facial fractures were included. the mean age was 24.9 (± 9.8) years, with a predominance of males (92.5%). the use of protective equipment was rare. the most frequently involved sport modality was soccer (47.5%), followed by cycling (27.5%). the most frequent fracture location was nose (45%), followed by mandible (25%) and zygomatic complex (17.5%). soccer was responsible for most nose fractures (61.1%), while cycling caused the majority of mandibular fractures (60%). conservative treatment predominated (60%). there was only a significant difference between fracture location and the need for hospitalization (p = 0.021). conclusion: patients with sportsrelated maxillofacial fractures were typically young adult males, the injury was more often located in the nose and mandible and related to soccer or cycling. the use of protective equipment must be reinforced. keywords: athletic injuries. fractures, bone. mandibular fractures. zygomatic fractures. nasal bone. https://orcid.org/0000-0002-9908-7876 https://orcid.org/0000-0002-2352-6481 https://orcid.org/0000-0002-7686-7829 2 ribeiro-dos-santos et al. introduction practicing sports is a frequent cause of facial injury. a wide range of facial injuries occur during sports, and their severity varies greatly1. in addition, sports-related maxillofacial injuries represent a significant proportion of the workload in a maxillofacial unit2. and the majority of patients are amateur athletes3. sports account for 3% to 29% of facial injuries and 10% to 42% of facial fractures4. it is also important to note that participation in sporting activities has grown worldwide, and the number of cases of sports-related injuries has also increased5,6. at the london olympics in 2012, 11% of athletes suffered at least one traumatic injury, confirming the high prevalence of trauma in sports7. indeed, football, soccer, hockey, rugby, baseball, and handball are frequently associated with facial bone fractures2-4,8. in addition, the need to educate all players regarding the use of personal protective equipment and adherence to the rules of sports has been proposed to reduce the frequency of impacts during matches2,8. on the other hand, sport cycling is the most common noncontact sport causing maxillofacial trauma, with the injuries resulting from slipping6. additionally, participation in mixed martial arts (mma) striking-predominant disciplines, such as boxing, karate, and muay thai, may result in high rates of head and facial injuries, especially during training9. soccer is the most popular sport in brazil, and soccer-related maxillofacial trauma is reported to be quite frequent10. additionally, a common sport worldwide, soccer generates a vast number of maxillofacial injuries, predominantly fractures, resulting in esthetic or functional problems2,5,11. although less common than orthopedic injuries in soccer players, maxillofacial trauma may occur. indeed, knowledge of this situation is important to first responders, nurses, and professionals who have initial contact with patients10. on the other hand, the maxillofacial fractures most commonly related to sports are nasal, mandibular and zygomatic fractures4,12. additionally, sports-related dentoalveolar trauma has been most frequently described among basketball and handball players13,14. there are many studies in the literature from abroad reporting the importance of sports in the occurrence of maxillofacial fractures. however, there are still few studies in this regard in brazilian populations, and some of these works refer to dentoalveolar trauma during sports practice. thus, this study aimed to assess the demographic and fracture characteristics of sports-related maxillofacial fractures in patients seen at a brazilian trauma care center. materials and methods a retrospective study was conducted with information collected from the medical charts of patients treated at the oral and maxillofacial surgery clinic of a trauma hospital that provides coverage for the southern area of são paulo city, são paulo state, brazil. patients with sports-related maxillofacial fractures seen between january 2018 and december 2019 were included. this study received approval from the human research ethics committee of the school of dentistry, university of são paulo, brazil (protocol # 14086819.7.0000.0075). 3 ribeiro-dos-santos et al. age, sex, medical history, history of drug abuse, use of mouthguards, and characteristics of the fracture were obtained from the medical charts of patients. next, the type of sport involved was noted. taekwondo, muay thai and jiu jitsu were included in the  martial arts category. fractures were distributed according to their location. next,  the fractures were classified according to zygomatic complex, as described by knight and north; the maxila, as described by le fort; nasal by nasal bone and septal fracture; orbital by blow-out, nasoorbitoethmoid and fronto-orbital fractures; frontal sinus by anterior table and upper orbit; and the mandible by location15-17. dentoalveolar trauma included cases of dental fracture and injuries such as concussion, subluxation, lateral dislocation, extrusion and avulsion. conventional radiographic views were verified and to improve the description of the fractures, computed tomography features and intraoperative findings were used. patients with incomplete medical records were excluded. the performed treatment and the need for hospitalization were noted. surgical treatment included the reduction of nasal fractures with forceps and surgical access and fixation with plates and screws for the other locations, all under general anesthesia. all  patients were treated in a municipal public system. dentoalveolar trauma cases that required reduction and fixation procedures were also included in the surgical treatment category. conservative treatment was used for nasal fractures without deviation, fractures of the zygomatic complex with little or no deviation and favorable maxillomandibular fractures that received intermaxillary fixation. finally, regarding complications, it was verified whether a second surgery was necessary and the reason for it. the data obtained were tabulated and subjected to statistical analyses. the likelihood ratio test was used to assess possible differences among age groups for the variables “type of sport” and “fracture location”, among types of sports for the variables “fracture location” and “treatment performed”, and among fracture location for the variables “treatment performed” and “need for hospitalization”. the statistical package for social sciences (spss) version 25.0 (ibm software group, chicago, usa) was used for the analysis. the level of significance adopted was p ≤ 0.050 for all statistical analyses. results during the period comprised by this study, 806 cases of maxillofacial fractures were assessed, and 40 cases (4.96%) of sports-related fractures were included. the  remaining cases in the sample were excluded because they were due to other causes of fracture. the mean age was 24.9 (± 9.8) years, with a predominance of males, who accounted for 37 cases (92.5%). the most affected age group was 12-20 years old with 18 cases (45%), followed by 21-30 years old with 11 cases (27.5%), 31-40  years  old with 7 cases (17.5%) and 41 years or older with 4 cases (10%). the most frequent sport involved was soccer with 19 cases (47.5%), followed by cycling with 11 cases (27.5%), martial arts with 2 cases (5%), and unspecified sport with 8 cases (20%), according to the findings on the medical charts. all patients were amateur sportsmen. no relevant data were found regarding medical history or drug abuse. data establishing the use or absence 4 ribeiro-dos-santos et al. of mouthguards were scarce. the location of the fracture in descending order of prevalence was the nose with 18 cases (45%), the mandible with 10 cases (25%) including three cases of bilateral fractures, the zygomatic complex with 7 cases (17.5%), dentoalveolar trauma with 3 cases (7.5%), frontal sinus with 1 case (2.5%) and orbit with 1 case (2.5%). a detailed description of the fracture characteristics is shown in table 1. table 1. demographic and fracture characteristics of patients with sports-related maxillofacial fractures (n= 40). age (mean ±sd) 24.9 (9.8) gender [n (%)] male 37 (92.5) female 3 (7.5) location of fractures [n (%)] mandible (n= 13)* parasymphyseal 1 (7.7) body 4 (30.8) angle 2 (15.4) condyle 6 (46.1) zygomatic complex (n= 7)** group i 2 (28.5) group ii 1 (14.3) group iii 2 (28.5) group iv 1 (14.3) group v (-) group vi 1 (14.3) nose (n= 18) nasal bone 13 (72.2) septal fracture 5 (27.8) frontal sinus (n= 1) anterior table fracture 1 (100) orbit (n= 1) blow-out 1 (100) dentoalveolar trauma (n= 3) lateral luxation 1 (33.3) avulsion 1 (33.3) extrusion 1 (33.3) sd: standard deviation. *the number of mandibular fractures exceeds that of patients as three cases were bilateral fractures. **according to knight & north. the nose was the predominant location by age group for both the 12-20-year-old group (55.6%) and the 41-year-old and above group (50%). zygomatic complex fractures occurred more frequently in the 21-30-year-old group (45.5%), while mandible fractures were more common in the 31-40-year-old group (42.9%). the distribution of patients by type of sport and fracture location according to age group is depicted in table 2. with the application of the likelihood ratio test in relation to the age group, there was no significant difference for the type of sport (p = 0.774) or for the location of the fracture (p = 0.112) (table 2). 5 ribeiro-dos-santos et al. table 2. age-specific distribution of patients by type of sport and fracture location according to the likelihood ratio test significance. variable category age group p value12-20 21-30 31-40 ≥ 41 n % n % n % n % type of sport martial arts 1 5.6 0 0.0 1 14.3 0 0.0 0.774 cycling 5 27.8 3 27.3 1 14.3 2 50.0 unspecified sport 4 22.2 3 27.3 1 14.3 0 0.0 soccer 8 44.4 5 45.5 4 57.1 2 50.0 fracture location zygomatic complex 0 0.0 5 45.5 1 14.3 1 25.0 0.112 mandible 4 22.2 2 18.2 3 42.9 1 25.0 nose 10 55.6 4 36.4 2 28.6 2 50.0 orbit 0 0.0 0 0.0 1 14.3 0 0.0 frontal sinus 1 5.6 0 0.0 0 0.0 0 0.0 dento-alveolar trauma 3 16.7 0 0.0 0 0.0 0 0.0 with regard to the type of sport and the location of the fractures, soccer was responsible for the majority of nasal fractures, with 11 cases (61.1%), followed by zygomatic complex fractures, with 3 cases (42.8%), orbit and frontal sinus fractures, with 1 case each, and dentoalveolar trauma, with 1 case. cycling was responsible for the majority of fractures of the mandible, with 6 cases (60%), followed by zygomatic complex fractures, with 3 cases (42.8%), and nasal fracture and dentoalveolar trauma accounted for 1 case each. martial arts were responsible for 1 case of nasal fracture (5.5%) and 1 case of mandible fracture (10%). regarding the treatment performed, there was a predominance of conservative treatment (24 cases 60%) upon surgical treatment (16 cases 40%). soccer and cycling required a higher percentage of conservative treatment (63.2% and 54.5%, respectively). the distribution of patients by fracture location and type of treatment performed according to the type of sport is shown in table 3. with the application of the likelihood ratio test in relation to the type of sport, there was no significant difference in the location of the fracture (p = 0.332) or the type of treatment performed (p = 0.957) (table 3). table 3. distribution of patients by fracture location and treatment performed according to the type of sport and likelihood ratio test significance. variable category type of sport p valuemartial arts cycling soccer unspecified sport n % n % n % n % fracture location zygomatic complex 0 0.0 3 27.3 3 15.8 1 12.5 0.332 mandible 1 50.0 6 54.5 2 10.5 1 12.5 nose 1 50.0 1 9.1 11 57.9 5 62.5 orbit 0 0.0 0 0.0 1 5.3 0 0.0 frontal sinus 0 0.0 0 0.0 1 5.3 0 0.0 dento-alveolar trauma 0 0.0 1 9.1 1 5.3 1 12.5 treatment performed surgical 1 50.0 5 45.5 7 36.8 3 37.5 0.957 conservative 1 50.0 6 54.5 12 63.2 5 62.5 6 ribeiro-dos-santos et al. regarding the location of the fractures, the treatment of nasal fractures was predominantly conservative, with 10 cases (55.6%). the same occurred with mandible fractures in 7 cases (70%), zygomatic complex fractures in 5 cases (71.4%) and orbital fractures (100%). surgical treatment was more prevalent in dentoalveolar trauma, with 2 cases (66.7%), as follows: 1 case of lateral luxation and 1 case of extrusion that were treated by dental repositioning and fixation with application of a resin splint involving intact neighboring teeth, and in frontal sinus fracture (100%). no cases of second surgery were observed. most fractures did not require hospitalization: nasal fractures represented 18 cases (100%); mandibular 7 cases (70%); zygomatic complex 5 cases (71.4%); orbital (100%); and dentoalveolar trauma with 3 cases (100%). the distribution of patients by type of treatment performed and need for hospitalization according to the location of the fractures is detailed in table 4. with the application of the likelihood ratio test in relation to the location of the fractures, there was no significant difference for the type of treatment performed (p = 0.454), but a significant difference was found for the need for hospitalization (p = 0.021) (table 4). table 4. distribution of patients by treatment performed and the need for hospitalization according to fracture location and likelihood ratio test significance. variable category fraclure location p value zygomatic complex mandíble nose orbit frontal sinus dentoalveolar trauma n % n % n % n % n % n % treatment performed surgical 2 28.6 3 30.0 8 44.4 0 0.0 1 100.0 2 66.7 0.454 conservative 5 71.4 7 70.0 10 55.6 1 100.0 0 0.0 1 33.3 need for hospitalization no 5 71.4 7 70.0 18 100.0 1 100.0 0 0.0 3 100.0 0.021 yes 2 28.6 3 30.0 0 0.0 0 0.0 1 100.0 0 0.0 discussion in this study, an analysis of the characteristics of sports-related maxillofacial fractures was performed, aiming at the profile of the affected patients and the anatomical singularities. in addition, the particularities of the treatment, such as the need for surgical treatment and hospitalization, were evaluated. thus, it was possible to determine that there were characteristics specific to personal data, with most patients being male, younger aged, amateur sportsmen, participants in specific types of sports, prone to fractures at specific locations, and treated with conservative treatment with an infrequent need of hospitalization. sports accidents accounted for approximately 5% of the causes of maxillofacial fractures in this study. studies carried out in european countries demonstrated a higher prevalence of sports-related maxillofacial fractures, with percentages ranging from 11.4 to 31%15,18-20. a brazilian study reported that 6.6% of facial fractures resulted from sports accidents21. on the other hand, two other studies with university sportsmen or professional athletes have described even higher frequencies of orofacial trauma associated with sports activities13,22. the occurrence of orofacial trauma in brazilian basketball players has been described as 50% of athletes13. 7 ribeiro-dos-santos et al. male sex was more frequently affected in this study. this statement is corroborated by other studies in which the percentage ranged from 70.3% to 100% of cases8,11,23. a male to female ratio of 12.3:1 was observed; other studies established proportions ranging from 6.6:1 to 9:13,8,18. when considering various etiologies, the male to female ratio of maxillofacial trauma ranged from 2.1:1 to 4.7:119-21,24,25. one possibility for this result would be that in our study, the most frequent sport involved was soccer and that in brazil and europe, soccer is an essentially male sport, with a series of soccer-related maxillofacial fractures with almost all or nearly all patients being male8,10,11. the mean age of the patients in this study was at the midpoint of the third decade, with the most affected age group being 12-20 years old. this suggests a lower mean age for sports-related trauma cases, corroborated by many studies in which the mean age of patients ranged from 18.3 to 25.0 years8,23. however, in maxillofacial trauma, when considering various etiologies the mean age increases, ranging from 25.5 to 40.7 years15,20,21,25. the most prevalent fracture locations found in this study were nasal, the mandible and the zygomatic complex. in the literature, the same locations are the three most prevalente; however, they have different distributions. in such studies, there was a predominance of mandible fractures24,25, while zygomatic complex fractures had a higher prevalence in others19,20. only one study determined the predominance of nasal fractures above the other two21. in studies that exclusively assessed sports-related trauma, the proportions also differed. in these other studies, there was a predominance of mandible fractures3,18,26, fractures of the zygomatic complex2,8,11 or nasal fractures27. in the present study, in contrast to other studies, there were few cases of dentoalveolar trauma13,14,28. in this study, the sport modalities more frequently involved in maxillofacial fractures were soccer, followed by cycling and martial arts. in studies in other countries, in both europe (italy, greece and ireland), and in the united states, we found a profile similar to the current study that was conducted in brazil, with a higher frequency of injury in soccer but with a greater participation in sports-related cases of facial trauma2-4,8,18. soccer has presented a high number of incidents against the head and face during professional practice in brazil, representing a real risk to athletes29. according to our findings, soccer practice resulted in the highest prevalence of nasal fractures, followed by fractures of the zygomatic complex, similar to other studies27. comparing the results of this study with other research directed at the location of maxillofacial trauma in this sport, conflicting results were found, with the predominance of the zygomatic complex followed by mandibular or nasal fractures8,11,18. in general, ball sports, such as soccer and rugby, have contributed to higher rates of zygomatic complex or mandible fractures2,3. a brazilian study on soccer-related facial trauma determined that the most common fracture sites were nasal bones, the zygomatic complex and the mandible10. direct contact between players generally causes soccer-related maxillofacial fractures, such as head-elbow or head-head impacts, which take place mainly when the ball is played with the forehead8. unlike soccer, cycling had a different fracture profile in this study. it was responsible for most of the mandibular fractures, followed by fractures of the zygomatic com8 ribeiro-dos-santos et al. plex and, with the same participation, nasal and dentoalveolar trauma. the literature on cycling-related maxillofacial trauma demonstrated that the mandible is the most frequent bone involved in these accidents, which corroborates our findings26. sports cycling is reported as an important cause associated with maxillofacial trauma6. with regard to the treatment performed, in this study, there was a predominance of conservative treatment. similar results were observed in a study of general facial trauma, in which nonsurgical procedures were performed at a greater proportion in comparison with patients who underwent surgery24. in studies that comprised only sports-related fractures, on the other hand, the proportion of surgical treatment was higher2,5,11,18. this fact might reflect the philosophy of the service, thus resulting in a greater or smaller number of cases treated surgically. no cases of second surgery were observed, probably because of following the precepts of the surgical technique, as well as no relevant data on comorbidities or drug abuse among patients. a few patients needing treatment of maxillofacial fractures required hospitalization, while the majority were treated on an outpatient basis. this finding mainly reflects the decision-making process to perform surgical treatment of nasal fractures under general anesthesia but on an outpatient basis. in most studies on sports-related facial trauma, cases of fracture treated surgically were admitted as inpatients5,18, thus generating additional costs for the health system30. considering the increasing incidence of sports-related injuries, oral and maxillofacial surgeons have to be more concerned with maxillofacial trauma during sports practice6. in terms of ball sports, stricter regulations are needed to reduce the percentage of impacts during matches, rather than relying on the use of protective equipment8,18. changes in rules and safety standards have been suggested for the prevention of such injuries5. considering the current reality, the use of protective equipment may safeguard athletes when returning to play after facial injuries have occurred1,23. one of the limitations of this study could be the sample size. although 40 cases of sports-related maxillofacial fractures were detected, the sample size was not calculated. this could be the reason that there was no significant differences in relation to the age group for type of sport or for location of the fracture. another possible limitation would be the number of cases of unspecified sports, with 8 cases (20%), reducing the characterization of sports. based on the data collected in this study, it was concluded that the typical patient with sports-related maxillofacial fracture is a young male adult and that the fractures are located more often in the nose and mandible and related to soccer or cycling. conservative, out-of-hospital treatment prevailed over surgical treatment and inpatient care. considering the limited use of mouthguards, campaigns are necessary for the prevention of sports-related maxillofacial fractures. statement of ethics: this study was approved by the research ethics committee of the school of dentistry, university of são paulo, brazil. disclosure statement: the authors declare that there are no 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ff. video analysis of craniofacial soccer incidents: a prospective study. j sci med sport. 2012 jan;15(1):14-8. doi: 10.1016/j. jsams.2011.07.011. 30. saperi bs, ramli r, ahmed z, nur am, ibrahim mi, rashdi mf, et al. cost analysis of facial injury treatment in two university hospitals in malaysia: a prospective study. clinicoecon outcomes res. 2017 feb 7;9:107-13. doi: 10.2147/ceor.s119910. 1http://dx.doi.org/10.20396/bjos.v19i0.8661088 volume 19 2020 e201088 original article 1 graduate program in dentistry, school of dentistry, university of vale do taquari (univates); lajeado, rs, brazil. 2 graduate program in dentistry, school of dentistry, federal university of pelotas, pelotas, rs, brazil. corresponding author: luiz alexandre chisini dds, ms, phd professor (school of dentistry – university of vale do taquari) university of vale do taquari, school of dentistry, graduate program of dentistry address: 171, avelino talini st. lajeado rs brazil 95914-014 phone: (55) 53981121141e-mail: alexandrechisini@gmail.com received: august 29, 2020 accepted: december 8, 2020 is obesity associated with tooth loss due to caries? a cross-sectional study luiz alexandre chisini1,2* , ana beatriz de lima queiroz2 , filippe vareira de lima2 , lucas jardim da silva2 , mariana gonzalez cademartori2 , francine dos santos costa1 , flávio fernando demarco2 , marcos brito corrêa2 aim: to investigate the association between obesity, overweight, and tooth loss due to caries among university students of (federal university of pelotas) in southern brazil. methods: a cross-sectional study with all first-year students who regularly enrolled in the first semester of 2016 who were invited to respond to a self-administered questionnaire contain socioeconomic and demographic; psychosocial; oral health; behavioral questions. the body mass index (bmi) was calculated through the self-reported data of weight and height. the main outcome of the present study was determined by the person’s that answer having had at least one tooth extracted due to caries. a poisson regression using a backward stepwise procedure was performed. two models were tested: i) including socioeconomic and behavioral variables; ii) without behavioral variables. results: from 3,237 eligible students, 2,089 (64.5%) participated in the present study. almost 23% of students presented overweight and 8.4% obesity, whereas 362 individuals (17.5%) reported having had at least one tooth extracted due to caries. regarding the final model adjusted by behavioral variables, it was observed that obese university students presented a 32.0% higher prevalence of tooth loss (pr=0.32,ci95%[1.17–1.49]). however, overweight was not associated with tooth loss in the present sample. when the model was not associated with behavioral variables, overweight was associated with tooth loss (pr=1.44; ci95%[1.15–1.81]), just as obesity (pr=2.13; ci95%[1.63 – 2.78]). conclusions: obesity and overweight were associated with tooth loss due to caries in the present sample of university students. keywords: epidemiology. tooth loss. dental caries. obesity. https://orcid.org/0000-0002-3695-0361 https://orcid.org/0000-0001-5773-2745 https://orcid.org/0000-0002-0548-0109 https://orcid.org/0000-0002-0127-7832 https://orcid.org/0000-0002-2433-8298 https://orcid.org/0000-0001-9558-937x https://orcid.org/0000-0003-2276-491x https://orcid.org/0000-0002-1797-3541 2 chisini et al. introduction obesity is a health disturb featured by fat surplusage due to unhealthy eating habits, sedentary lifestyle, genetic, social, and behavioral aspects, which affects people’s quality of life in many ways1. this disorder is frequently associated with comorbidities such as hypertension, osteoarthritis, certain types of cancer and diabetes, and caries2-4. according to the world health organization (who), obesity is increasing worldwide, affecting both developed and developing countries1,5. from an economic perspective, obesity is also an impairment owing to great direct medical expenditure and drugs’ costs1,5. the diet of individuals is a determinant in obesity occurrence, mainly due to the high levels of lipids, carbohydrates and sugars intake6. similarly, studies have presented solid evidence of the role of sugar consumption on dental caries experience7,8 which is a common cause of obesity4,6, and can culminate in tooth loss. in this way, systematic reviews have reported contradictory results between the association of obesity and dental caries, mainly based on children and adolescents samples4,6. mainly, studies that investigate the association between caries and obesity were performed with children9-13. a recent study showed an elevated prevalence of obesity among university students14. few studies report how obesity correlates with caries and tooth loss in the undergraduate population, which is relevant considering the high prevalence of overweighed students14. oppositely to studies that report the relation between periodontal disease and overweight, which already have concise results in literature15, the possible association between tooth loss due to caries and obesity is not well established yet9,10,16,17. thus, the purpose of this study was to investigate if there is an association between obesity and tooth loss due to caries among university students in southern brazil. the present study hypothesizes that obesity and overweight are associated with tooth loss. materials and methods the present study was reported following the recommendation of strengthening the reporting of observational studies in epidemiology (strobe) for cross-sectional studies. full details concerning the methods of the present study’s baseline have been published previously18,19. ethical issues the institution (ufpel) and research ethics committee of the school of medicine / ufpel approved this study under protocol 49449415.2.0000.5317. setting and study design a cross-sectional analytical study was carried out with students from federal university of pelotas (ufpel). the sample consisted of students that joined the university in the first semester of 2016, excluding distance learning courses (626 students).  the average new enrollments of ufpel by semester is 3,000 students. thus, we applied a self-reported questionnaire with first-year students of ufpel. previously, a pilot questionnaire was tested in other students (n=100 from the second semester) from five courses selected randomly. the official questionnaire was improved after the pilot and the meantime spent to answer was 20 minutes. 3 chisini et al. participants (eligible and non-eligible participants) all students regularly enrolled in the first semester of 2016 at ufpel composed the eligible sample. first-year students were searched in their respective academic units with the authorization of academic directors and professors. the questionnaires were applied before or after lessons, respecting professors’ preference. students that were not regularly enrolled in the course and students from distance learning courses were excluded from the sample. data collection the fieldwork team was composed of undergraduate and graduate students under the supervision of professors of ufpel dental school. the participants were part of the oral health epidemiology study group (epibucal). the questionnaire was constructed in the epibucal meetings. the final questionnaire was self-administered designed, presented 74 multiple-choice questions which were divided into 5 wide blocks: 1) socioeconomic and demographic; 2) psychosocial; 3) oral health; 4) behavioral of oral health; 5) drugs use. outcome the outcome of the present study was the self-report of tooth loss due to caries. the individual answered if he or she had at least one dental extraction due to dental caries, in the question: “have you ever had a tooth extracted because it was affected by caries?” yes or no were the possible answers. exposure the body mass index (bmi) was calculated using the self-report of current weight (in kilograms) and height (in meters). these variables were assessed as continuous and were posteriorly converted to categorical, as recommended by the world health organization criteria20. the bmi is defined as the weight divided by the square of the height (kg/m2). the established cut-off points20 were the following: a) eutrophic (bmi <25); b) overweight (bmi between 25 and 30); and c) obesity (bmi>30.0). confounding/mediator variables two models were used to analyze the data. the first was considering the socioeconomic variables and the second model was controlled by socioeconomic and behavioral variables (stress and symptoms of depression). regarding the socioeconomic variables, systematic reviews have reported that age, income, skin color, and sex are important confounders in the association of obesity and dental caries4,6. theoretically, stress and depression symptoms could be considered confounders and mediators of the relationship between bmi and tooth loss because they present a reciprocal link21. in other words, stress and depression can be considered as confounders because are associated with bmi21 and with dental caries12 and can be considered mediators because a decrease of bmi can decrease the risk of depression22,23 and depression could influence tooth loss24 (figure 1). therefore, we excluded behavioral variables from the second analysis considering this possibility. 4 chisini et al. sociodemographic variables were collected in the first block of the questionnaire. the sex of the participants was asked, as the age of individuals, which was categorized in ≤ 18, 19 to 34, and 35 or more. skin color variable was collected according to the brazilian institute of geography and statistics (ibge) as white, light-black, dark-black, yellow, american indian. they were categorized in white and non-white (light-black, dark-black, yellow, american indian). family income was collected categorically in brazilian reals (brl): (a) up to 500; b) 501 up to 1,000; c) 1,001 up to 2,500; d) 2,501 up to 5,000; e) 5,001 up to 10,000; and f) more than 10,001) and stratified in three categories: a) ≤ 1,000; b) 1,001 to 5,000 and c) ≥ 5,001 brazilian reals.   also, we have performed control in the second model by behavioral variables: symptoms of depression and stress. symptoms of depression were assessed with patient health questionnaire-2 (phq-2), an abbreviated version of the patient health questionnaire depression (phq-9) used for depression screening. the phq-2 is comprised of two questions about the frequency of depressive mood over the past two weeks. the phq-2 score ranges from 0 to 6 points and a cutoff of 3 was adopted. the stress level was measured with a modified version of the perceived stress scale (pss). the ten questions referred to the past month and present five answer options on a likert scale (from score 1 to 5). higher scores corresponded to an elevated level of stress and the variable was categorized in quartiles. statistical methods statistical analysis was performed with the software stata 16.0 (stata corporation, college station, tx, usa). relative and absolute frequencies and the respective confidence interval of 95% (ci95%) of each variable of interest were estimated. the use of poisson regression models for binary outcomes has been proposed and extensively figure 1. theoretical model for analysis of association of overweight and obesity with tooth loss due to caries. block 1 socieconomic variables block 2 behavioral varibles sex skin color age family income depression symptoms stress block 3 body mass index (overweight and obsity) outcome tooth loss 5 chisini et al. applied in the last decade because it provides the estimation of prevalence or risk ratios instead of the odds ratio provided in the logistic model. we have chosen the multivariate poisson model because the odds ratio can overestimate prevalence or risk ratio, complicating results interpretations. also, poisson regression with robust variance provides correct estimates for the analysis of binary outcomes and the prevalence or risk ratio is easier to interpret than the odds ratio. moreover, independent variables were included in the multivariate model according to a theoretical model described in figure 1. variables with p values ≤ 0.250 in the crude analyses were included in the adjusted model fitting. a backward stepwise procedure was used to include or exclude explanatory variables in the model fitting. variables included in the final adjusted model should present a p-value of ≤ 0.250. for the final model, variables were considered significant if they had a p-value of ≤ 0.05 after adjustments. prevalence ratios (pr) were obtained for tooth loss and effect measure was estimated with 95% confidence intervals (ci 95%). results a total of 3,237 students enrolled in the university were considered eligible for the study. from these, 2,118 (65.4%) students were found and invited to participate in the present study, since 34.6% of students did not attend classes for three consecutive weeks and were considered as losses. further, 2,089 (64.5%) individuals agreed to participate in this study, assigning the consent term. only 29 students (1.4%) did not agree to participate in the study. the majority of the sample (54.0%) was between 19 and 34 years old and 71.4% of students had a family income ranging from 1,001.00 to 5,000.00 brl (table 1). regarding the behavioral variables, we observed that the most stressed students (22.9%) presented scores of the perceived stress scale ranging from 22 to 40, and around 16.3% of students presented depressive symptoms. besides, almost 23% of students were overweighed and 8.4% were obese. also, 362 individuals (17.5%) reported having had at least one tooth extracted due to caries (table 1). table 1. description of the general characteristics of the sample (n=2,089). brazil, 2016 variable/category n % sex male 993 47.7 female 1,087 52.3 age (years) ≤ 18 777 37.4 19 to 34 1,125 54.0 ≥ 35 178 8.6 skin color white 1,530 74.8 non-white 515 25.2 continue 6 chisini et al. in table 2, the crude and adjusted prevalence ratio for the association of body mass index and tooth loss due to caries is displayed. in the final model adjusted by behavioral variables, obese university students presented a 32.0% higher prevalence of tooth loss (pr = 0.32, ci95% [1.17 – 1.49]). however, overweight was not associated with tooth loss in this model (pr = 1.01; ci95% [0.91 – 1.13]).  when the model was not associated by behavioral variables, overweight was associated with tooth loss (pr = 1.44; ci95% [1.15 – 1.81]) just as obesity (pr = 2.13; ci95% [1.63 – 2.78]). family income (brl) ≤ 1000 280 16.3 1001 to 5000 1,057 61.6 ≥ 5001 380 22.13 depression symptoms no 1,743 83.7 yes 339 16.3 stress (quartile) 1nd (pss scores 0 11) 550 26.3 2nd (pss scores 12 – 16) 571 27.3 3rd (pss scores 17 – 21) 489 23.4 4rd (pss scores 22 – 40) 479 22.9 body mass index eutrophic 1,387 68.8 overweight 460 22.8 obesity 169 8.4 tooth loss due to caries no 1,710 82.5 yes 362 17.5 continuation table 2. crude (c) and adjusted (a) prevalence ratio (pr) for association of body mass index and tooth loss control by independent variables. pelotas, rs, brazil. poisson regression (n=1902). brazil, 2016. variable/category prc (ci95%) p-value pra1(ci95%) p-value pra2(ci95%) p-value sex (ref=male) 0.702 0.231 female 0.98 (0.900 – 1.07) 1.13 (0.92 – 1.38) skin color (ref=white) 0.009 0.009 0.181 non-white 1.13 (1.03 – 1.24) 1.13 (1.03 – 1.24) 0.85 (0.68 – 1.07) age (years) (ref=16 to 17) 0.972 18 to 24 1.05 (0.93 – 1.19) 25 to 34 1.01 (0.85 – 1.21) 35 or more 1.02 (0.84 – 1.22) family income (ref= ≤ 1000) 0.069 <0.001 1001 to 5000 0.98 (0.87 – 1.11) 1.93 (1.35 – 2.7) ≥ 5001 0.88 (0.75 – 1.02) 2.85 (1.98 – 2.70) continue 7 chisini et al. discussion tooth loss and obesity are significant health issues, affecting an elevated number of individuals worldwide15. the previous studies that evaluate the relationship between obesity and tooth loss have only considered the periodontal disease as a causal factor for tooth loss, disregarding losses due to caries15,25. in the present study, it was hypothesized that tooth loss due to caries can be influenced by obesity or overweight. in the examined sample of university students, obese individuals presented a higher prevalence of tooth loss due to caries and overweight individuals only showed this association when the model was not adjusted by behavioral variables, possibly due to a mediator effect of depressive symptoms. the main explanation of the connection between tooth loss due to caries and obesity is the highly cariogenic diet of obese individuals mainly in industrialized countries8. beyond a lipid-rich diet, obese individuals frequently have a sugar and carbohydrate-rich diet26, which is a prominent factor for the development and proliferation of caries8. also, some studies have demonstrated that a decrease of salivary flow is observed in individuals with a body mass index higher than 2527. thus, a decrease of salivary flow can be observed in obese individuals, potentially increasing the progression of caries and conducting to tooth loss. this occurs because of the low-grade and generalized inflammation caused by obesity, which influences immune mediators such as proinflammatory cytokines28. the cytokines and the hypothalamic–pituitary–adrenal axis can modulate the central nervous system function and decrease the salivary flow27. the relation between caries and obesity is not well established, considering the conflicting results, maybe because bmi cannot distinguish fat mass, muscle, and bone mass9,10,16,17. however, this is a simple and accessible tool for obesity and overweight measurement, employed in most epidemiological studies. when the etiological factors of the disease are not treated, a progression of caries is expected29, which can lead to tooth loss. systematic reviews have presented conflicting results regarding the presence of a positive association between obesity and dental caries6,30 and the studies have not investigated the relationship between obesity or overweight and tooth loss due to cardepression symptoms (ref=no) 0.001 yes 1.18 (1.07 – 1.31) stress (quartile) (ref=1st pss scores 0 – 11) <0.001 <0.001 2nd (pss scores 12 – 16) 1.19 (1.05 – 1.35) 1.19 (1.05 – 1.35) 3rd (pss scores 17 – 21) 1.27 (1.12 – 1.44) 1.26 (1.11 – 1.44) 4rd (pss scores 22 – 40) 1.30 (1.15 – 1.48) 1.29 (1.13 – 1.46) body mass index (ref= eutrophic) 0.001 0.001 <0.001 overweight 1.01 (0.91 – 1.13) 1.01 (0.91 – 1.13) 1.44 (1.15 – 1.81) obesity 1.32 (1.16 – 1.49) 1.32 (1.17 – 1.49) 2.13 (1.63 – 2.78) a1 included behavioral variables in the model; a2 behaviorall variables were not included in the model continuation 8 chisini et al. ies. generally, the studies have investigated the association of bmi with dmft-index and not considered only the tooth loss due to caries27,31-34. a study carried out in chile considered only the decayed teeth, not those restored or extracted due to caries, and have not observed association with obesity35. on the other hand, our study included only tooth loss and did not include decayed teeth. therefore, we included in our outcome the most severe caries experience and its factor could be responsible for differences observed among the studies. corroborating, silva et al.6 related that both methods of diagnosis (to bmi and caries) probably influenced the results observed in the review: i.e., some studies used more sensible to diagnosis caries as interproximal radiography36,37; and all studies included in the systematic review6 used bmi to measure obesity/overweight, but using different cut-offs. these findings indicate that the way in which obesity criteria are measured can be an important factor in the study’s conclusions. similarly, different adjustments in analytical models could be another important factor observed in the results of studies6. in the present study, we tested two analytical models. in the first, we considered the possibility of behavioral variables (stress and depression) having confounders of the association between obesity/overweight and tooth loss, and observed that only obesity was associated, possible due to a mediation effect. on the other hand, when the analysis was not adjusted by symptoms of depression, both obesity and overweight were associated with tooth loss. considering that the present study is cross-sectional and this is not the best design to investigate mediation variables, we did not explore possible mediation effects. therefore, further studies with longitudinal design could be addressed to investigate this gap and explore the causal mechanism. although several variable adjustments were performed for the present analysis, we did not conduct diet intake control, being this one of the main frail points of the present study. in addition, the use of self-reported measures could be over or underestimated, as this is a subjective measurement. however, university students are barely investigated in the literature, consisting of an interesting sample for studies although it presents different features compared to the general population. thus, extrapolation of the results to the general population must be avoided. moreover, the self-reported oral health measurement used in the present study can vary in data accuracy compared to objectively measured data38. however, this approach is a valid tool widely used in epidemiologic studies, which promotes ease and quickness, aiming to reduce the time of examination and lowering the costs39,40. beyond that, it is important to consider a probable underestimation of bmi, mainly in overweight and obese participants41,42. a significant number of eligible students did not participate in the study, although field teamwork returned for three consecutive weeks in classrooms. thus, the losses were mainly caused by the impossibility of the location of individuals in their respective classrooms. however, similar questionnaire-based studies also showed this difficulty43. regardless, the present study evaluated a wide sample of individuals and presented control by socioeconomic and behavioral factors, being the first to evaluate psychosocial variables with the present outcome. most of the studies found in the literature evaluate the association of obesity and tooth loss due to periodontal disease25. nevertheless, the relationship between obesity and tooth loss due to caries is poorly investigated and was positively associated with the present study. 9 chisini et al. therefore, obesity and overweight were associated with tooth loss due to caries in the present sample of university students. overweight lost the association when the analysis was adjusted by behavioral variables. these findings suggest underlining causal mechanisms that can be explored in further studies, preferably carried out with a longitudinal design. moreover, further studies could explore the bidirectional effects of the association of obesity and tooth loss. compliance with ethical standards conflict of interest: the authors declare that there is no conflict of interest. ethical approval: all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 helsinki declaration and its subsequent amendments, or comparable ethical standards. informed consent: informed consent was obtained from all individuals participating in the study or their legal responsible (<18 years). references 1. collaboration ncdrf. worldwide trends in body-mass index, underweight, overweight, and obesity from 1975 to 2016: a pooled analysis of 2416 population-based measurement studies in 128.9 million children, adolescents, and adults. lancet. 2017 dec 16;390(10113):2627-42. doi: 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2015;26(6):566-71. doi: 10.1590/0103-6440201300409. 1 volume 21 2022 e226469 original article braz j oral sci. 2022;21:e226469http://dx.doi.org/10.20396/bjos.v21i00.8666469 1 federal university of pelotas, pelotas, rs, brazil. 2 catholic university of pelotas, pelotas, rs, brazil. 3 university of vale do taquari, lajeado, rs, brazil. corresponding author: catarina borges da fonseca cumerlato, dds, msc; graduate program in dentistry, federal university of pelotas rua gonçalves chaves 457, 96015-560, pelotas-rs, brazil tel./fax: +55-53-3222.6690 e-mail: catarinacumerlato@hotmail.com editor: dr. altair a. del bel cury received: july 22, 2021 accepted: june 10, 2022 analysis of dentistry content on instagram® and the use of social networks by brazilian dental students catarina borges da fonseca cumerlato1,* , rodrigo nunes rotta2 , luiz alexandre chisini3 , luísa jardim corrêa de oliveira2 , marcos britto correa1 aim: the aim of our study was to analyze the content related to dentistry published on instagram® and to investigate the students’ profiles related to the use of social networks in a sample from a dentistry school. methods: this study was carried out in two stages. first, research was conducted to analyze content published on instagram® related to dentistry. the second part investigated the profiles of students’ from a dental school in relation to the use of social networks through a questionnaire. a descriptive analysis was performed and associations between the variables were tested using fisher’s exact test. results: we found a diversified and large amount of content related to dentistry published on instagram®, and publications that involved advertising were more prevalent in our study. there are also many publications that are violating the code of dental ethics. in addition, in the second part of our study, we observed an enormous use of social networks by dental students (98.4%). conclusions: as a result, we believe that it is important to update professionals about the ethical infractions related to social networks which could be approached during graduate courses in universities. finally, the analyzed social network seems to be an interesting alternative for disseminating health information in order to dialogue with greater proximity to the large number of users who participate in it. keywords: social media. dentistry. epidemiology. ethics. https://orcid.org/0000-0001-5943-6958 https://orcid.org/0000-0003-0401-1543 https://orcid.org/0000-0002-3695-0361 https://orcid.org/0000-0001-7738-4338 https://orcid.org/0000-0002-1797-3541 2 cumerlato et al. braz j oral sci. 2022;21:e226469 introduction virtual social networks are social structures consisting of a set of individuals who regularly interact online, facilitating the relations of people and/or organizations that share the same interests, goals, experiences or values1. the most popular social network today is facebook®, which has 1.73 billion active users in the world on average per day2. there are several other networks available on the internet such as instagram®, which in recent years has shown a strong growth rate, currently resulting in more than 1 billion registered users3. the use of social networks in health has increased exponentially4, challenging health professionals to achieve the best use of this new communication mode, considering all ethical standards in its use. social networks have been widely used as a vehicle for communication by dental professionals, for example, to advertise in order to attract the labor market which is increasingly competitive5. brazilian law prevents the use of any communication vehicle, including the internet, to advertise dental services (i.e. dental surgeons may use social networks as an advertisement vehicle) respecting the ethical restrictions that are available in the brazilian federal code of ethical dentistry (ceo)6. the number of articles that reviewed the use of social networks in medicine have grown over the last few years7. it is also possible to notice a lack of commitment and professionalism on the parts of professionals and students on social networks in the dental field. a study which evaluated facebook® profiles of dental and dental hygiene students found that 5.8% of the analyzed profiles contained non-professional content such as sexism, racism, privacy violations, and lack of respect for the teacher or faculty8. in addition, there is a daily increase in the publication of content on social networks by dental professionals and students, often violating the profession’s code of ethics, which, for example, does not allow disseminating before-and-after images or selfies of dental surgeons accompanied by patients or not, without their previous authorization9. on the other hand, another study published in 2011 showed that social networks have been used to communicate experiences of dental pain, including the actions taken in an attempt to cease pain and descriptions of the impacts caused in the individual’s daily routine, being an interesting instrument for behavior analysis and disseminating health information10. social networks can also be used in a beneficial way both for professionals who can publish health content and information, and for the general population to facilitate interaction and communication between the professional and the patient. however, there is a limited number of studies in the literature which have scientifically analyzed the content related to dentistry on the instagram® social network. therefore, it is important to perform an analysis of the relation between dentistry and social networks so that they are best used without infringing on important ethical aspects involved in the profession. in view of the above, the aim of this study was to analyze the contents related to dentistry published on instagram®, to evaluate the compliance with ethical and legal 3 cumerlato et al. braz j oral sci. 2022;21:e226469 aspects, and to investigate the students’ profiles related to the use of social networks in a sample from a brazilian dentistry school. material and methods this study was carried out in two stages. firstly, research was conducted to analyze content published on instagram® related to dentistry. the second part investigated the profiles of students’ from a dental school in relation to the use of social networks. part 1: analyzing content published on instagram® it is necessary to use an instagram® profile to perform the search for the most followed profiles and identify the most used hashtags. thus, we created a totally empty profile on the network without connection to any other users to perform the research in order to reduce confirmation bias. research was carried out in two assessment periods. the first was performed in 2016 and the second assessment was performed in 2020. we searched the 20 users who publish content about dentistry and who have the largest number of followers on the instagram® network at the time of the search, totaling 40 profiles. we used the keyword “odonto” (abbreviation of odontology in portuguese) to search these profiles, and after obtaining the results of the search, we entered all the returned profiles to verify the amount of followers that each user had at the date of the search. after identifying the 40 users with the largest number of followers, the initial pages of these profiles were collected through print screens for later analysis of the published content type and classification of the profile type of those users. likewise, we conducted the search for the most frequently used hashtags by instagram® users related to dentistry using the keyword “odonto”. from the hashtags found, the five which had the largest number of publications in both assessment periods were selected, totaling 10 hashtags. the 100 most recent posts of each hashtag were selected through print screens, and were registered for further descriptive analysis of their contents. after the images were recorded, we collected the data of interest and organized it in a database created in excel 2013, so the hashtag posts and the profiles of the users could be classified into categories according to their content. disagreements were discussed until a consensus between two researchers was reached. a third author made the final decision in case of non-consensus. first, we classified the 40 selected profiles into three categories according to the type of user to understand whether the profile belonged to a person, company or dental clinic. this identification of the user type was done through the user name, profile image and through the biography present on the profile. after evaluating the postings of these 40 profiles individually, we classified them in relation to the type of publication they generally post as follows: clinical case publication (before and after), advertising, oral health information, entertainment, and tips. we considered the images and subtitles of the posts to perform this evaluation and classification. as instagram® does not provide the characteristics of the users, information such 4 cumerlato et al. braz j oral sci. 2022;21:e226469 as gender and age were recorded according to the appearance of the people in the photographs (when possible). these characteristics were collected for both the 40 profiles and the selected hashtag posts. the registered publications from the hashtags were also classified into categories related to the type of publication (e.g., if they were clinical case postings, oral health information, tips, entertainment, advertising, photo with patient, selfie, patient photo or collective activity photo). regarding user type, we collected if who did the posting was a person, company or clinic/dental clinic. this information was obtained by analyzing the images and the content of the subtitles of the publications. in addition, when explicitly stated (by image/caption), we classified the publications according to the dental specialization covered on the post. finally, we analyzed the presence of non-professional content on publications such as racism, sexism, privacy violations involving recognizable photographic images of patients, negative patient descriptions, and negative comments about the profession by professionals. in practice, social networking sites report that content being published on the platforms is being made public, and that in those where there are privacy options (such as twitter and instagram®, for example), the only content that can be searched is that characterized as public11,12. in our study, all publications reviewed were free from privacy, meaning they were publicly available for access by any instagram® user. part 2: investigating the students’ profiles all of the students from the faculty of dentistry from federal university of pelotas/ brazil were invited to participate in the study. the students should sign the consent form and then answer the questionnaire to participate, which was applied in the classroom with the prior authorization of the course lecturers. the questionnaire had 18 questions regarding the use of social networks and the students’ knowledge about the brazilian code of dental ethics. the questionnaire application took place from february to july 2018. after the data collection, the questionnaires were double typed independently into excel spreadsheets. data analysis statistical analysis was performed using the stata 12.0 software program (statacorp lp, college station, united states). a descriptive analysis was performed presenting the relative and absolute frequencies of the variables of interest. associations between the age and publication type variables were tested using fisher’s exact test. ethical aspects both phases of this study were approved by the research ethics committee of the school of dentistry of federal university of pelotas under protocol number #1,793,486. all study participants signed the informed consent form. 5 cumerlato et al. braz j oral sci. 2022;21:e226469 results part 1 data collection was carried out on august 13, 2016 and on october 17, 2020. out of the 20 users surveyed on instagram®, the five that had the highest number of followers up to the date of the first search were: @odontologiadicas (181k), @odontoespacio (51.1k), @odonto.sopensoemdente (42.3k), @sigaodontofantasy (38.2k), and @odontologiapreventiva (33.4k). in relation to the five hashtags analyzed, the one that presented the largest number of publications was #odontologia (863,932), followed by #odonto (501,600), #odontolove (166,485), #odontopediatria (150,019), and #odontoporamor (124,786). in the second assessment, the five users that had the highest number of followers were: @odontologiadicas (243k), @odontologiavfh (104k), @odontoemdicas (89k), @odontofada (75.8k) and @draarielycaramori (64.9k). furthermore, the hashtags that presented the largest number of publications in this second assessment were: #odontologia (7.6m), #odonto (3m), #odontopediatria (1.4m), #odontologiaestetica (1.3m) and #odontoporamor (1m). table 1 presents the descriptive analysis of the collected variables related to the 40 most followed users selected. considering the type of user, 8 (40%) were classified as a company, 7 (35%) as personal and 5 (25%) as a dental clinic in 2016. in contrast, we can observe an increase in personal profiles (65%) in 2020 in comparison to 2016 (35%). we table 1. descriptive analysis of the variables collected from profiles with the largest number of followers related to the term “odonto” on the instagram® social network (40 profiles). variable/category 2016 2020 p* n % 95%ci n % 95%ci user type 0.024 personal 7 35.0 15.4-59.2 13 65.0 40.8-84.6 clinic 5 25.0 8.7-49.1 6 30.0 11.9-54.3 company 8 40.0 19.1-63.9 1 5.0 0.1-24.9 sex 0.500 male 2 10.0 1.2-31.7 female 6 30.0 11.9-54.3 8 40.0 19.1-63.9 age 0.258 young 3 15.0 3.2-37.9 adult 5 25.0 8.7-49.1 6 30.0 11.9-54.3 publication type 0.002 advertising and marketing 11 55.0 31.5-76.9 12 60.0 36.1-80.9 health information 13 65.0 40.8-84.6 13 65.0 40.8-84.6 clinical case 12 60.0 36.1-80.9 5 25.0 8.7-49.1 entertainment 11 55.0 31.5-76.9 8 40.0 19.1-63.9 tips 11 55.0 31.5-76.9 14 70.0 45.7-88.1 others 2 10.0 1.2-31.7 *fisher’s exact test. 6 cumerlato et al. braz j oral sci. 2022;21:e226469 can also visualize a changing in the publication pattern and type in table 1, where 2020 had a decrease in clinical case posts and an increase in posting tips about dentistry. the descriptive analysis of the variables collected from publications associated with the 10 most used hashtags related to the term “odonto” on the instagram® social network is presented in table 2. most of the publications regarding the user type were published in personal profiles in both assessment periods. the predominant gender in 2016 was female with 68.3% and the adult age group with 81.0%. in addition, in 2020 we could observe an increase in adults in social media (95.9%). advertising posts (31.8%) were the most published in 2016, followed by selfie posts (18.6%). otherwise, we observed a reduction in selfie posts (6.6%) and a significant increase of marketing publications (52.7%) in 2020. table 2. descriptive analysis of the variables collected from publications associated with the 10 most used hashtags related to the term “odonto” on the instagram® social network (1,000 publications). variable/category 2016 2020 p* n % 95%ci n % 95%ci user type 0.000 personal 253 50.6 46.1-55.1 237 47.6 43.1-52.1 clinic 89 17.8 14.5-21.4 139 27.9 24.1-32.2 company 158 31.6 27.5-35.9 122 24.5 20.9-28.6 sex 0.363 male 80 31.8 27.7-36.1 60 27.7 23.9-31.9 female 172 68.3 64.1-72.5 157 72.3 68.3-76.3 age 0.000 young 48 19.0 15.7-22.7 9 4.1 2.6-6.3 adult 204 81.0 77.3-84.3 208 95.9 93.9-97.5 publication type 0.000 selfie 93 18.6 15.3-22.3 32 6.6 4.6-9.1 advertising and marketing 159 31.8 27.7-36.1 257 52.7 48.3-57.2 photo with patient 37 7.4 5.3-10.1 26 5.3 3.6-7.8 patient’s photo 40 8.0 5.8-10.7 9 1.8 0.8-3.4 health information 66 13.2 10.4-16.5 88 18.0 14.7-21.7 clinical case 25 5.0 3.3-7.3 54 11.1 8.6-14.3 entertainment 74 14.8 11.8-18.2 21 4.3 2.8-6.6 collective activity photo 6 1.2 0.4-2.6 1 0.2 0.005-1.1 * fisher’s exact test and chi-squared test. table 3 presents the distribution of the posts according to publication type and dental specializations. the specialization that presented the greatest number of publications of both photos with patients (24.2%) and photos of patients (18.1%) was pediatric dentistry. advertising publications were more prevalent in the specialization of esthetic dentistry (47.7%), as well as publications of clinical cases (23.6%). 7 cumerlato et al. braz j oral sci. 2022;21:e226469 table 3. distribution of selected posts from the instagram® social network according to publication type and dental specializations. specialization, n (%) p* surgery esthetic orthodontics pediatric dentistry periodontics others publication type 0.000 selfie 2 (3.1) 6 (3.0) 5 (11.9) 7 (4.7) 1 (3.7) 0 (0.0) advertising and marketing 26 (40.0) 95 (47.7) 17 (40.5) 33 (22.2) 2 (7.4) 7 (31.8) photo with a patient 3 (4.6) 11 (5.5) 3 (7.1) 36 (24.2) 0 (0.0) 0 (0.0) patient’s photo 3 (4.6) 9 (4.5) 1 (2.4) 27 (18.1) 1 (3.7) 1 (4.6) health information 18 (27.7) 26 (13.1) 4 (9.5) 33 (22.2) 18 (66.7) 6 (27.3) clinical case 5 (7.7) 47 (23.6) 9 (21.4) 5 (3.4) 5 (18.5) 4 (18.2) entertainment 8 (12.3) 5 (2.5) 3 (7.1) 8 (5.4) 0 (0.0) 4 (18.2) * chi-squared test. figure 1 shows 50 hashtags which have appeared most frequently on the subtitles of posts with #odonto publications. the five hashtags that were presented in the largest number of posts after #odonto were: #odontologia (dentistry in portuguese), #dentistry, #dentist, and #odontoporamor (dentistry for love). it should also be noted that other hashtags related to aesthetic procedures such as: #odontologiaestetica (esthetic dentistry), #botox, and #dentesbrancos (white teeth) were also well represented. figure 1. most used hashtags with #odonto. pelotas, rs/brazil, 2016. 8 cumerlato et al. braz j oral sci. 2022;21:e226469 part 2 from a total of 459 eligible students, a total of 380 (82,8%) individuals participated in the study. from these, 371 (98.4%) reported using social networks and only 6 (1.6%) answered they do not use it, as we can visualize in table 4. the most used social network by the students is facebook® (95.5%), followed by instagram® (88.2%). we can also see in table 4 that most students (89.5%) reported that they search content about dentistry on social networks. the type of content most searched by the students was clinical cases (71.8%), followed by professional tips (70.5%), information about oral health (56.3%), and publicity/entertainment (45.8%). in addition, 34.8% of individuals reported having published some content related to dentistry on the networks, with oral health information (16.1%) and photo with patient (14.5%) being the most published content. regarding the code of dental ethics, 136 students (35.9%) reported not being aware of the code aspects related to dental posts on social networks. table 4. descriptive analysis from the collected variables related to student profiles of the faculty of dentistry from federal university of pelotas/brazil. variables n % gender male 125 32.9 female 255 67.1 use of social network yes 371 98.4 no 6 1.6 social network used facebook 362 95.5 instagram 335 88.2 twitter 65 17.1 snapchat 96 25.3 others 39 10.7 search for content about dentistry yes 339 89.5 no 40 10.5 dentistry-related content type clinical cases/before and after type 273 71.8 professional tips 268 70.5 information about oral health 214 56.3 publicity 53 14.0 entertainment 121 31.8 others 13 3.5 continue 9 cumerlato et al. braz j oral sci. 2022;21:e226469 continuation have published some content related to dentistry on the networks yes 131 34.8 no 245 65.2 type of publication clinical cases/before and after type 35 9.2 tips 30 7.9 information about oral health 61 16.1 publicity 7 1.8 entertainment 31 8.2 photo with patient 55 14.5 patient’s photo 6 1.6 others 13 2.7 aware of the code aspects related to dental posts on social networks yes 243 64.1 no 136 35.9 discussion the present study is one of the first to analyze content related to dentistry on instagram®. it was possible to observe that there was a great amount of content related to dentistry published on this network. as can be seen by the increase in number of followers and posts from 2016 to 2020, the use of social networks by health professionals has been considerably increasing over the years. this increasing use can be attributed to a number of factors, such as the popularization of these networks and the increase in competitiveness in the labor market. brazil currently concentrates around 10% of dentists in the world, with an unequal distribution of professionals with concentration in the major urban centers and in the more economically developed regions5. this fact generates an increase in competition among dental professionals in the country, which makes dentist surgeons seek to give greater visibility to their work through advertising on social networks in order to attract patients5. in fact, the publications which involved advertising were more prevalent in our study. from the total of publications, advertising posts increased from 31.8% in 2016 to 52.7% in 2020. moreover, we could observe that the number of adults in instagram® social media had increased. it is important to highlight that social networks have emerged as an inexpensive, accessible and rapidly-diffusing opportunity for users to carry out this marketing. thus, it is important emphasize that there is no legislation in brazil that prohibits the use of social networks by dental professionals. however, in order to advertise their services, it is necessary for the dental surgeon to respect the ethical constraints of the federal code of dental ethics (ceo)6. publications containing patient pictures and/or photos of the dentist with the patients were more present in posts related 10 cumerlato et al. braz j oral sci. 2022;21:e226469 to pediatric dentistry. according to the brazilian ceo, it is an ethical infraction to refer to identifiable clinical cases, patient display, images or any other element that identifies the patient, using expressions of self-promotion, sensationalism, and commercialization of the profession, without the patient’s or legal guardian’s authorization (article 14, iii)9. this becomes more critical considering the significant amount of images of children published by pediatric dentists. the brazilian statute of the child and adolescent (eca) is clear in guaranteeing children’s right to respect, which includes preserving their image and identity13. thus, even with the presence of parents at the time of the photo, publishing images of children in a dental clinic can at least be considered inadequate. a recent study evaluated 123 images of instagram® postings by dental surgeons, clinics or dental academics, and found that most posts were before-and-after images for advertising purposes14. our findings revealed that before and after clinical case reports were in greater quantity in esthetic dentistry posts, which were also characterized by a greater number of posts for the purpose of advertising. allied to this fact, purely esthetic procedures which have been gaining popularity recently such as bichectomy and the application of botulinum toxin have been widely mentioned as hashtags alongside #odontologia (odontology in portuguese), being even more mentioned than terms related to health. the main oral health public problems are caries and periodontal disease, which have an unequal distribution, affecting the lower socioeconomic level of the population15. this population group is the one who generally less use and access dental services, and when they do, they mostly use public services16. considering that ads on social networks are carried out by companies and dentists working in the private service which focus on patients with higher incomes and therefore have a lower burden of oral diseases, it is not surprising that only aesthetic procedures gain prominence in the posts and that the content related to dentistry on the networks is increasingly focused on the market and not on health promotion. it is also worth noting that brazil is one of the countries which performs the greatest number of aesthetic medical procedures in the world, which also confirms the high value given to esthetics by the brazilian population17. in addition, in the second part of our study we observed a huge use of social networks by dental students (98.4%). from the total of students, 34.8% reported having published some content related to dentistry on the networks. furthermore, more than one third of students reported not being aware of the ethical code aspects related to dental posts on social networks. in this sense, educational institutions should guide students from the beginning of their graduate course on how to correctly use social networks, avoiding future problems in their professional lives, such as those detected in the analyzes of the present work. a study conducted in 2014 on twitter® demonstrated that online social networks can become a powerful complement to traditional sources of health information; in addition, the study showed that the users of the research use the twitter® social network for the purpose of expressing feelings, being able to be positive and/or negative18. in agreement, another study published in 2020 regarding instagram® demonstrated that social networks are used to communicate experiences of dental pain, including 11 cumerlato et al. braz j oral sci. 2022;21:e226469 actions taken to stop pain and descriptions of the impacts on their daily routines, and can be an interesting tool for behavioral analysis and disseminating health information19. given these findings, we can see that social networks can be very beneficial and useful for both professionals and patients. for example, in the case of professionals they can be used to disseminate health information for patients, and thus guide their followers how to face some kind of difficulty regarding dental pain or any other dental problem. from the users’ perspective, the use of social media for health purposes can provide access to information on healthcare, as well as for them to express their daily states. finally, social networks could even be used by government and health professionals with a focus on health surveillance. health surveillance actions, which have the principle to observe and analyze the health situation of the population, articulate themselves in a set of actions aimed at controlling determinants, risks and damage to health20, and could also be put into practice on the social networks, where one could partially analyze the health situation and behaviors of the population, and in turn promote virtual actions aiming to disseminate health information as well as control determinants. furthermore, disseminating health information could be performed by researchers to communicate relevant findings of studies and by professors in order to assess students in another way of communication in a more modern and direct language, as people nowadays are connected to their smartphones and have wide access to the internet. in this sense, once any content can be published on the networks, the publication of health information by professors would also help to ensure that higher quality content was made available to users in order to increase knowledge of the population about oral health based on the best evidence21. it is also important to highlight some limitations of the present study. first, we did not investigate all content linked with dentistry published on instagram®. we have investigated a sample of this content and most of the analyzed posts were from brazil; therefore, the results must be interpreted carefully. in addition, the second part of the study was carried out in a public dental school in southern brazil, so the findings can only be compared with dental schools with a similar profile. on the other hand, this study shed light on a contemporary topic, such as the use of social media in dentistry. the presence of dentistry in social networks is a reality, with an increasing trend over the next years, following society’s pattern. studies about the use and content related to oral health on these networks are of great interest to understand how dental professionals are behaving and what we can expect in the future. in conclusion, there is a very diversified and large amount of content related to dentistry on the instagram® social network, and there are also many publications which are violating the code of dental ethics (ceo). thus, a concern arises with respect to professionals who are violating the ceo, since a part of the analyzed publications were not professionals in the exercise of teaching, but posts aiming at self-promotion, sensationalism, and commercialization of the profession. in addition, we believe that it is important to update professionals about the ethical infractions present in the ceo related to social networks/the internet, which could be approached during the graduate course in universities so that future dentists do not commit the same infractions. finally, the analyzed social network seems to be an interesting alternative 12 cumerlato et al. braz j oral sci. 2022;21:e226469 for disseminating health services and information in order to dialogue with greater proximity to the large number of users who participate in it. acknowledgments this study was in part financed by the coordenação de aperfeiçoamento de pessoal de nível superior – brasil (capes) – finance code 001. disclosure statement the authors declare no conflict of interest. author 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edmonds a, sanabria e. medical borderlands: engineering the body with plastic surgery and hormonal therapies in brazil. anthropol med. 2014;21(2):202-16. doi: 10.1080/13648470.2014.918933. 18. henzell mr, knight am, morgaine kc, antoun js, farella m. a qualitative analysis of orthodontic-related posts on twitter. angle orthod. 2014 mar;84(2):203-7. doi: 10.2319/051013-355.1. 19. da fonseca cumerlato cb, rotta rn, de oliveira ljc, corrêa mb. #dentalpain: what do the brazilian instagram® users want to mean. braz j oral sci 2020; 19:e208591. doi: 10.20396/bjos.v19i0.8658591. 20. ministry of health of brazil. national health surveillance guidelines. brasília: ministry of health; 2010. 21. almaiman s, bahkali s, alabdulatif n, bahkali a, al-surimi k, househ m. promoting oral health using social media plataforms: seeking arabic online oral health related information (ohri). stud health technol inform 2016; 226:283-6. https://www.instagram.com/press/ 1 volume 21 2022 e226252 original article braz j oral sci. 2022;21:e226252http://dx.doi.org/10.20396/bjos.v21i00.8666252 1 department of dentistry, state university of maringá, maringá, pr, brazil. 2 global research and innovation network – grinn, curitiba, pr, brazil. 3 mcgill university, faculty of dentistry, mcgill division of oral, health and society, montreal, quebec, canadá. corresponding author: mitsue fujimaki department of dentistry, state university of maringá. av. mandacaru 1.550, 87080000 – maringá, pr, brazil. phone: (44) 991116464. e-mail: mfujimaki@uem.br editor: altair a. del bel cury received: july 2, 2021 accepted: december 12, 2021 oral healthcare management practices in brazil: systematic review and metasummary tânia harumi uchida1 , uhana seifert guimarães suga1 , clarissa garcia rodrigues2 , josely emiko umeda1 , mark tambe keboa3 , raquel sano suga terada1 , mitsue fujimaki1,* universal health coverage is a global target included in the united nations sustainable development goals agenda for 2030. healthcare in brazil has universal coverage through the unified health system (sus), which guarantees health as basic right to the brazilian population. considering the principles of sus, public oral healthcare management is a huge challenge. aim: to identify good management practices for quality care adopted by local public oral healthcare managers and teams around brazil. methods: this study was registered with prospero (crd42017051639). five databases (pubmed, embase, web of science, scopus and lilacs) as well as the reference lists and citations of the included publications were searched according to prisma guidelines. results: a total of 30,895 references were initially found, which were evaluated according to the defined eligibility criteria. twenty qualitative studies, eight surveys and two mixed-model studies were selected. the practices (codes) were organized into three main groups (families), and the frequency of the effect size (fes) of each code was calculated. among the 20 codes identified, the most relevant ones were: diagnosis and health planning (fes=80%) and family health strategy (fes=66,7). the intensity of the effect size of each study was also calculated to demonstrate the individual contribution of each study to the conclusions. conclusion: the evidence emerging from this review showed that healthcare diagnosis, planning, and performance based on the family health strategy principles were the most relevant practices adopted by public oral healthcare managers in brazil. the widespread adoption of these practices could lead to improved oral healthcare provision and management in brazil. keywords: public health. dentistry. policy making. health policy. practice management. https://orcid.org/0000-0001-8170-1092 https://orcid.org/0000-0003-3150-0123 https://orcid.org/0000-0002-1821-5697 https://orcid.org/0000-0003-1106-4344 https://orcid.org/0000-0001-9754-7819 https://orcid.org/0000-0003-1344-9870 https://orcid.org/0000-0002-7824-3868 2 uchida et al. braz j oral sci. 2022;21:e226252 introduction health is a valuable resource for sustainable human development. it contributes to national social equality, justice and peace, and increased quality of life. the importance of health in global development is exemplified in the sustainable development goals (sdgs) agenda for 2030 proposed by the united nations1. specifically, the third sdg seeks to “ensure a healthy life and promote well-being for all, at all ages”. the public health system in brazil is aligned with this broad sdg, and has been described as a reference model for neighbouring countries2. for over 30 years, public healthcare has been enshrined in the brazilian constitution as an inalienable right of all members of the population. brazil operates a unified health system (sus sistema único de saúde, in portuguese) that was created on the core principles of equity, integrality, and universality of healthcare provision. under sus, every person in the country is entitled to free healthcare, and are invited to take part in the formulation, evaluation and control of health policies3. because oral health is inherent to a healthy life, the brazilian national oral health policy (bnohp política nacional de saúde bucal, in portuguese), a program also known as “smiling brazil”, was created and incorporated into sus. this policy has steadily been implemented by stakeholders at various levels, such as consumer protection agencies, public health professionals, and oral health professional4. over the last decade, newer links have been forged between the bnohp and non-dental actors within sus such as health workers, managers, and the community. to further integrate oral healthcare within the universal system provided by sus, practice transformation, and the introduction of new concepts, contents, and forms of organization are required with the overarching intent of improving the oral health of the population5. however, managing sus presents various levels of challenges. the complexity of a universal system in association with the fragmentation of health policies and programs, lack of management qualification and social control, and a hierarchical, regionalized network structure are some of the issues that encumber health actions and services6. under such circumstances, unqualified management can become a critical leadership bottleneck, impairing the implementation of health policies7. furthermore, the role of managers in the public sector is dependent on regulations that sometimes limit their autonomy. indeed, difficulties experienced by managers in promoting healthcare integration at all levels of the public service have created barriers to full access to proper healthcare8. hence, the combination of inadequate management qualification and the organizational complexities of a health system that is intended to be universal can compromise the very foundational principles of sus7. sus management has become a major public health issue in brazil3, and more effective and efficient public management is required to facilitate the implementation of oral healthcare actions in line with the principles and guidelines of the national healthcare system. yet, insufficient attention has been given to the role of managers and the qualification they require as a way to achieve sus objectives. 3 uchida et al. braz j oral sci. 2022;21:e226252 a possible approach to the problem is to highlight health management models employed in different parts of the country, which have the potential to face the challenges and change the predominant traditional practices that are not in accordance with the bnohp9. therefore, the aim of this systematic review and metasummary was to identify good management practices for quality care adopted by local public oral healthcare managers and teams around brazil. materials and methods protocol and registration this systematic review was conducted in accordance with the preferred reporting items for systematic review and meta-analyses (prisma) statement10 and was registered with the international register of prospective systematic reviews (prospero) under the registration number crd42017051639. literature search the research question that guided this systematic review, according picos, was: “what practices have been adopted by local managers and teams within the public health service in brazil aimed at improving oral healthcare management?”. to answer this question, a search was performed in the following electronic databases: pubmed, embase, web of science, scopus and lilacs. a search was conducted until september 2021. for the search in the databases, no terms related to the type of study were used, since the term “qualitative research” was introduced only in 1988 in the embase database and in 2003 as a mesh term in pubmed. search strategy it was used the picos strategy, following terms that were used in the final search strategy: patient (p) “policymaker”, “policy making”, “public health”, intervention (i) “dentistry”. “mesh terms” (pubmed), “entry terms” (embase) and “decs” (lilacs) were also used to “construct” a highly sensitive search strategy. some initial keywords were selected. different strategies were tested in the databases, and key words were added or rejected according to the results obtained. terms related to study type were not used because the term “qualitative research” was only introduced in embase in 1988, and as a mesh term in pubmed in 200311. eligibility criteria the inclusion criteria were as follows: qualitative studies, surveys, or mixed-model (qualitative-quantitative) articles that indicated the practices adopted by local public health managers to improve oral healthcare management in the brazilian public sector. no limits were imposed on the date, language, or type of study. moreover, no study was excluded a priori for reasons of quality. according to supplementary guidance for inclusion of qualitative research in cochrane systematic reviews of interventions12, this is a strategy that allows that potentially valuable themes remain included. 4 uchida et al. braz j oral sci. 2022;21:e226252 study selection, quality assessment, and data extraction followed a similar procedure. two reviewers (thu and usgs) initially performed the task independently, and then met with a third reviewer (mf) for consultation and consensus. study selection all titles and abstracts of the articles retrieved were independently assessed by two reviewers (thu and usgs). these reviewers held weekly meetings for 18 weeks in the presence of a third reviewer (mf) with experience in public management, qualitative research and systematic reviews. abstracts that did not provide sufficient information in relation to the eligibility criteria were maintained for full text evaluation. afterwards, manual searches were performed in the references of the included articles, and citations were analyzed using google scholar. the authors of the included studies were contacted by e-mail for the identification of possible additional studies. study quality assessment qualitative studies were evaluated according to quality items adapted from the critical appraisal skills program (casp)13; surveys were assessed based on quality items adapted from bennett et al.14 (2010), while mixed-model studies were analyzed according to o’cathain et al.15 (2008). quality items were assessed and classificated as being present (yes) or absent (no). studies which presented a prevalence of “yes” (>60% of the evaluated items) in the quality evaluation were considered as presenting low risk of bias. studies with 40% 60% of “yes” were considered moderate risk of bias. and studies with a prevalence of “no” (<40% of the items) were classified as presenting high risk of bias. data extraction the following general data were collected from the studies: authors, year of publication, and geographic region of the first author. additionally, the following specific characteristics were also retrieved: study objective, type of study, place of research, intervention, number of participants in the sample, inclusion and exclusion criteria, participant characteristics, data collection methods, data analysis, main results, and conclusions. data analysis data analysis was conducted through a metasummary of the retrieved data. this is a quantitatively oriented aggregation approach for the synthesis of both qualitative studies and surveys. the methodology involves extracting, grouping and formatting the results to allow the calculation of the frequency of the effect size (fes) of each practice, and the intensity of the effect size (ies) of each study16. after extracting the results of the included studies, and grouping the relevant findings, major topics (concise but comprehensive representations) termed “families” were created referring to the practices adopted by oral healthcare managers for https://translate.google.com/translate?hl=pt-br&prev=_t&sl=pt-br&tl=en&u=http://media.wix.com/ugd/dded87_29c5b002d99342f788c6ac670e49f274.pdf https://translate.google.com/translate?hl=pt-br&prev=_t&sl=pt-br&tl=en&u=http://media.wix.com/ugd/dded87_29c5b002d99342f788c6ac670e49f274.pdf https://translate.google.com/translate?hl=pt-br&prev=_t&sl=pt-br&tl=en&u=http://media.wix.com/ugd/dded87_29c5b002d99342f788c6ac670e49f274.pdf https://translate.google.com/translate?hl=pt-br&prev=_t&sl=pt-br&tl=en&u=http://media.wix.com/ugd/dded87_29c5b002d99342f788c6ac670e49f274.pdf 5 uchida et al. braz j oral sci. 2022;21:e226252 quality care. in each family, individual practices, termed “codes”, were grouped based on similarity. for the coding step, the software atlas.ti 8.0 – qualitative data analysis (atlas.ti® scientific software development, berlin, germany) was used. fes was calculated in order to evaluate the magnitude of the extracted results. it consisted on verifying the number of times a particular code emerged from all included articles. to do so, the number of studies that presented an individual code was divided by the total number of studies included, and the result was presented as a percentage. the ies of each study was calculated by checking the number of times codes emerged in each of the included articles. the calculation was performed to indicate which codes with fes > 25% contributed to answering the research question. in order to do this, the number codes contained in one study was divided by the total number of codes in all the studies. with this calculation, the articles were considered “stronger” or “weaker” based on their contribution to answering the research question. thus, the number of codes with fes > 25% in one particular study was divided by the number of codes with fes > 25% across all studies11. this information assisted in interpreting the data in the metasummary, determining the individual contribution of studies to the conclusions of this systematic review17. results study selection figure 1 show the flowchart of the study selection process. the initial search in the electronic databases yielded 30,895 references. after the removal of duplicates (3,485 references); title and abstract evaluations (27,385 references), 25 articles were considered potentially eligible. full texts were retrieved and analyzed by applying the eligibility criteria. after the analysis of the references of these articles, quotes in google scholar, and studies indicated by the authors of the selected texts, 35 new articles were included for further eligibility evaluation. of the 60 articles selected, 23 were excluded for the following reasons: 3 studies were non-scientific research; 19 articles did not present any practices to improve oral healthcare management; 2 articles were not on dentistry; and in 6 studies, the subject interviewed could not be clearly identified. in the end of the evaluations, 30 articles were included in the systematic review and metasummary: 20 qualitative studies, 8 surveys and 2 mixed model studies. study characteristics table 1 presents information on the included studies (number of participants, setting, context of the study). the total number of participants was 1,010, among whom 498 were dentists and 512 were managers. the geographical distribution of the studies was as follows: amazonas (1 study); bahia (5 studies); ceará (2 studies); minas gerais (2 studies); paraíba (2 studies); paraná (4 studies); pernambuco (1 study); rio grande do norte (2 studies); santa catarina (6 studies); são paulo (4 studies). only 1 study was multicentric, involving the states of paraná and são paulo. 6 uchida et al. braz j oral sci. 2022;21:e226252 most of participants were enrolled in the family health strategy (fhs estratégia saúde da família, in portuguese), and most health managers held municipal or state positions (table 1). id en ti fi ca ti on s cr ee ni ng el ig ib ili ty in cl ud ed total of 30.895 records pubmed: 10.394 records embase: 9.809 records web of science: 3.129 records scopus: 7.117 records lilacs: 446 records 3.485 duplicates removed 24.410 records after duplicetes removed 25 full-text articles for eligibility assessment 27.385 articles excluded based on review of titles and/ or abstract 60 full-text articles were assessed for eligibility 35 new records included for evaluation of eligibility after evaluation of citations, references and papers indicated by the authors of the complete texts 20 qualitative studies included 8 surveys included exclusions: 03 studies were not scientific research; 19 records did not present practices for qualified management in dentistry; 02 records were not about dentistry; in 06 studies it was unclear who the research subject interviewed was. 2 mixed studies included figure 1. flowchart of the study selection process. 7 uchida et al. braz j oral sci. 2022;21:e226252 table 1. study characteristics and risk of bias (n = 30). study number of participants setting context of the study dentists managers aguilera et al.18 (2013) 0 17 paraná municipal health office aquilante and aciole19 (2015) 38 11 são paulo regional department of health of são paulo araújo and dimenstein20 (2006) 21 0 rio grande do norte dentists from the family health strategy (fhs) cavalcanti et al.21 (2012) 17 0 paraíba basic health unit (bhu) chaves and da silva22 (2007) 9 0 bahia dentists in the primary healthcare systems correa et al.23 (2010) 6 2 amazonas dentists inserted in the fhs, municipal health department and oral health coordinator at fhs fernandes et al.24 (2015) 11 0 santa catarina dentists inserted in the fhs lippert et al.25 (2020) 14 0 paraná dentists from the basic health units (bhu) and dental specialties centers mello et al.26 (2014) 0 10 santa catarina sus managers moimaz et al.27 (2008) 0 3 são paulo regional department of health nascimento et al.28 (2009) 58 0 são paulo and paraná dentists worked in the esf padilha et al.29 (2005) 74* paraíba dentists worked in the fhs and as managers pimentel et l.30 (2010) 3 12 pernam-buco fhs health district vi rodrigues et al.31 (2011) 31* bahia fhs rossi and chaves32 (2015) 8 5 bahia oral health management team sá et al.33 (2015) 23 1 santa catarina fhs santos et al.34 (2007) 4 0 bahia fhs santos and assis35 (2006) 11* bahia fhs silva junior et al.36 (2020) 0 9 ceará state health managers vieira et al.37 (2013) 8 0 são paulo public sector baldani et al.38 (2005) 105 0 paraná oral health team (oht) at the fhs colussi and calvo39 (2011) 0 207 santa catarina municipal health managers godoi et al.40 (2013) 0 1 santa catarina municipal health managers godoi et al.41 (2014) 0 12 santa catarina municipal health managers lessa and vettore42 (2010) 0 3 ceará municipal health office lourenço et al.43 (2009) 278** 166 minas gerais oht at the fhs mattos et al.5 (2014) 43 14 minas gerais oht at the fhs souza and roncalli44 (2007) 25 19 rio grande do norte oht at the fhs aquilante and aciole45 (2015) 38 11 são paulo regional department of health of são paulo moretti et al.46 (2010) 67 9 paraná oht total 498 512 * subjects excluded from the total sum, since it was not clear in the methodology how many were dentists and how many were managers. ** subjects excluded from the total sum, because it was not clear in the methodology how many were dentist. 8 uchida et al. braz j oral sci. 2022;21:e226252 quality assessment the overall risk of bias of the selected studies is presented in table 1. of the 20 qualitative articles, 20 (100%) presented high risk of bias (table 2). among the 8 surveys included in this systematic review, 2 (25%) had low risk of bias, 4 (50%) had moderate risk of bias and 2 (25%) high risk of bias (table 2). the 2 mixed-model studies (100%) presented high risk of bias (table 2). frequency of the effect size (fes) twenty practices (codes) were identified after analysis and coding of the 30 included articles. similar codes were grouped into three families: “oral healthcare structure”, “oral healthcare provision”, and “staff management” (table 3). codes belonging to the family “oral healthcare structure” with highest fes values, showed that the main practices adopted by managers to achieve qualified management were: care diagnosis and planning (80%), healthcare networks (63,3%), infrastructure and materials, and information systems and evaluation (30%). as for the family “oral healthcare provision”, the following codes stood out: family health strategy (66,7%), expanded clinical service (56,7%), and intersectoriality (46,7%). codes within the family “staff management” with the highest fes were: interprofessional teamwork (40%), continuing education (26,7%), creativity, initiative, motivation and innovation (10%), and university-health service integration (6,7%). intensity of the effect size (ies) the ies was calculated to verify the individual contribution of each study to the conclusions of this systematic review. all the qualitative studies, surveys and mixed-model studies contributed significantly to the practices for the qualification of oral healthcare management (table 4). the study that presented the highest ies was vieira et al. 2013 with 55%, followed by aquilante and aciole 2015 with 50%, baldani et al. 2005 with 45%, and lourenço et al. 2009 with 40%. among the other 24 selected studies, 8 had scores between 5% and 15%, and 24 studies presented scores between 25% and 35%. ten codes: care diagnosis and planning, family health strategy, healthcare networks, expanded clinical service, intersectoriality, interprofessional teamwork, ongoing health education, infrastructure and materials, information systems and evaluation and continuing education presented fes > 25%, which resulted in ies > 25% in all included studies. 9 uchida et al. braz j oral sci. 2022;21:e226252 ta bl e 2. q ua lit y as se ss m en t o f i nc lu de d st ud ie s. q ua lit at iv e st ud ie s ac co rd in g c a s p (n =2 0) in cl ud ed a rt ic le s q ua lit y va ri ab le s as se ss m en t* risk of bias assessment clarity of purpose qualitative methodology appropriateness justification for the qualitative methodology participant recruitment strategy data collection relationship between researcher and participants ethical issues data analysis clarity of results study relevance a gu ile ra e t a l.1 8 ( 20 13 ) h ig h a qu ila nt e an d a ci ol e1 9 ( 20 15 ) h ig h a ra új o an d d im en st ei n2 0 ( 20 06 ) h ig h c av al ca nt i e t a l.2 1 ( 20 12 ) h ig h c ha ve s an d da s ilv a2 2 ( 20 07 ) h ig h c or re a et a l.2 3 ( 20 10 ) h ig h fe rn an de s et a l.2 4 ( 20 15 ) h ig h li pp er t e t a l. 25 (2 02 0) h ig h m el lo e t a l.2 6 ( 20 14 ) h ig h m oi m az e t a l.2 7 ( 20 08 ) h ig h n as ci m en to e t a l.2 8 ( 20 09 ) h ig h c on tin ue 10 uchida et al. braz j oral sci. 2022;21:e226252 c on tin ua tio n p ad ilh a et a l.2 9 ( 20 05 ) h ig h p im en te l e t a l.3 0 ( 20 10 ) h ig h r od rig ue s et a l.3 1 ( 20 11 ) h ig h r os si a nd c ha ve s3 2 ( 20 15 ) h ig h sá e t a l.3 3 ( 20 15 ) h ig h sa nt os e t a l.3 4 ( 20 07 ) h ig h sa nt os a nd a ss is 35 (2 00 6) h ig h si lv a et a l.3 6 ( 20 20 ) h ig h v ie ira e t a l.3 7 ( 20 13 ) h ig h s ur ve ys a cc or di ng b en ne tt (n =8 ) q ua lit y va ria bl es a ss es sm en t* in cl ud ed a rt ic le lo ur en ço e t a l.4 3 (2 00 9) b al da ni e t a l.3 8 (2 00 5) m at to s et a l.5 (2 01 4) g od oi e t a l.4 1 (2 01 4) s ou za a nd r on ca lli 44 (2 00 7) le ss a an d v et to re 42 (2 01 0) c ol us si a nd c al vo 39 (2 01 1) g od oi e t a l.4 0 (2 01 3) ju st ifi ca tio n of th e re se ar ch qu es tio n ex pl ic it re se ar ch q ue st io n c la rit y of p ur po se c on tin ue 11 uchida et al. braz j oral sci. 2022;21:e226252 c on tin ua tio n d es cr ip tio n of m et ho ds u se d fo r d at a an al ys is m et ho d of a dm in is te rin g th e qu es tio nn ai re lo ca tio n an d da te m et ho ds s uffi ci en tly de sc rib ed fo r r ep lic at io n ev id en ce o f r el ia bi lit y ev id en ce o f v al id ity u se o f e nc od in g sa m pl e si ze c al cu la tio n r ep re se nt at iv en es s of th e sa m pl e sa m pl e se le ct io n m et ho d d es cr ip tio n of th e sa m pl e po pu la tio n d es cr ip tio n of th e se ar ch to ol d es cr ip tio n of to ol de ve lo pm en t p re -t es t i ns tr um en t r el ia bi lit y an d va lid ity in st ru m en t c on tin ue 12 uchida et al. braz j oral sci. 2022;21:e226252 c on tin ua tio n c on se nt et hi ca l a pp ro va l ev id en ce o f e th ic al tr ea tm en t o f r es ea rc h pa rt ic ip an ts r is k of b ia s as se ss m en t lo w lo w m od er at e m od er at e m od er at e m od er at e h ig h h ig h m ix ed -m od el s tu di es a cc or di ng o ’c at ha in (n =2 ) in cl ud ed a rt ic le s q ua lit y va ri ab le s as se ss m en t* r is k of b ia s as se ss m en t a ss es sm en t o f st ud y su cc es s a ss es sm en t o f st ud y de si gn a ss es sm en t of th e qu an ti ta ti ve co m po ne nt a ss es sm en t of th e qu al it at iv e co m po ne nt a ss es sm en t o f st ud y in te gr at io n a ss es sm en t o f st ud y in fe re nc es a qu ila nt e an d a ci ol e4 5 ( 20 15 ) h ig h m or et ti et a l.4 6 ( 20 10 ) h ig h * a da pt ed fr om c oc hr an e’ s c ol la bo ra tio n: y es n o 13 uchida et al. braz j oral sci. 2022;21:e226252 ta bl e 3. f am ili es (o ra l h ea lth ca re s tr uc tu re , o ra l h ea lth ca re p ro vi si on , a nd s ta ff m an ag em en t) a nd c od es w ith th ei r r es pe ct iv e fr eq ue nc y of th e ef fe ct s iz e (f es ). s tr u c tu r a l m a n a g em en t c a r e m a n a g em en t m a n a g em en t o f th e w o r k c od es / r ef er en ce s ef fe ct s iz e fr eq ue nc y (% ) c od es / r ef er en ce s ef fe ct s iz e fr eq ue nc y (% ) c od es / r ef er en ce s ef fe ct s iz e fr eq ue nc y (% ) h ea lth ca re n et w or ks a gu ile ra e t a l.1 8 ( 20 13 ), a qu ila nt e an d a ci ol e4 5 ( 20 15 ), c av al ca nt i e t a l.2 1 ( 20 12 ), c ha ve s an d da s ilv a2 2 ( 20 07 ), c ol us si a nd c al vo 39 (2 01 1) , f er na nd es e t a l.2 4 ( 20 15 ), li pp er t e t a l.2 5 ( 20 20 ), g od oi e t a l.4 0, 41 (2 01 3, 2 01 4) , l es sa a nd v et to re 42 (2 01 0) , lo ur en ço e t a l.4 3 ( 20 09 ), m at to s et a l.5 (2 01 4) , m el lo e t a l.2 6 ( 20 14 ), p ad ilh a et a l.2 9 (2 00 5) , p im en te l e t a l.3 0 ( 20 10 ), r os si a nd c ha ve s3 2 ( 20 15 ), si lv a et a l.3 6 ( 20 20 ), so uz a an d r on ca lli 44 (2 00 7) , v ie ira e t a l.3 7 ( 20 13 ) 63 ,3 u se r s at is fa ct io n c av al ca nt i e t a l.2 1 ( 20 12 ) 3, 3 a ut on om y of m an ag em en t v ie ira e t a l.3 7 ( 20 13 ) 3, 3 c ar e d ia gn os is a nd p la nn in g a gu ile ra e t a l.1 8 ( 20 13 ), a qu ila nt e an d a ci ol e1 9, 45 (2 01 5) , b al da ni e t a l.3 8 ( 20 05 ), c av al ca nt i e t a l.2 1 ( 20 12 ), c or re a et a l.2 3 ( 20 10 ), fe rn an de s et a l.2 4 ( 20 15 ), g od oi e t a l.4 0, 41 (2 01 3, 2 01 4) , l es sa a nd v et to re 42 (2 01 0) , lo ur en ço e t a l.4 3 ( 20 09 ), m at to s et a l.5 (2 01 4) , m el lo e t a l.2 6 ( 20 14 ), m oi m az e t a l.2 7 ( 20 08 ), m or et ti et a l.4 6 ( 20 10 ), n as ci m en to e t a l.2 8 (2 00 9) , p ad ilh a et a l.2 9 ( 20 05 ), r os si an d c ha ve s3 2 ( 20 15 ), sá e t a l.3 3 ( 20 15 ), sa nt os e t a l.3 4 ( 20 07 ), sa nt os a nd a ss is 35 (2 00 6) , s ilv a ju ni or e t a l.3 6 ( 20 20 ), so uz a an d r on ca lli 44 (2 00 7) , v ie ira e t a l.3 7 ( 20 13 ) 80 ex pa nd ed c lin ic al s er vi ce a qu ila nt e an d a ci ol e1 9, 45 (2 01 5) , b al da ni e t a l. (2 00 5) 38 , c ha ve s an d da s ilv a2 2 ( 20 07 ), c ol us si an d c al vo 39 (2 01 1) , c or re a et a l. 23 (2 01 0) , fe rn an de s et a l.2 4 ( 20 15 ), g od oi e t a l.4 1 ( 20 14 ), m at to s et a l.5 ,2 6 ( 20 14 ), m or et ti et a l.4 6 ( 20 10 ), n as ci m en to e t a l.2 8 ( 20 09 ), p im en te l e t a l.3 0 (2 01 0) , s an to s et a l.3 4 ( 20 07 ), sa nt os a nd a ss is 35 (2 00 6) , s ou za a nd r on ca lli 44 (2 00 7) , v ie ira e t a l.3 7 (2 01 3) 56 ,7 in di vi du al p er fo rm an ce c ha ve s an d da s ilv a2 2 ( 20 07 ) 3, 3 b ud ge t a nd f un di ng lo ur en ço e t a l.4 3 ( 20 09 ), m oi m az e t a l.2 7 (2 00 8) , r os si a nd c ha ve s3 2 ( 20 15 ), v ie ira e t a l.3 7 ( 20 13 ) 13 ,3 s oc ia l c on tr ol a qu ila nt e an d a ci ol e4 5 ( 20 15 ), b al da ni e t a l.3 8 (2 00 5) , c av al ca nt i e t a l.2 1 ( 20 12 ), c ol us si an d c al vo 39 (2 01 1) , m oi m az e t a l.2 7 ( 20 08 ), n as ci m en to e t a l.2 8 ( 20 09 ), sa nt os e t a l.3 4 ( 20 07 ) 23 ,3 c re at iv ity , i ni tia tiv e, m ot iv at io n an d in no va tio n a qu ila nt e an d a ci ol e1 9 ( 20 15 ), c ha ve s an d da si lv a2 2 ( 20 07 ), c or re a et a l.2 3 ( 20 10 ) 10 c on tin ue 14 uchida et al. braz j oral sci. 2022;21:e226252 c on tin ua tio n in fr as tr uc tu re a nd m at er ia ls a gu ile ra e t a l.1 8 ( 20 13 ), b al da ni e t a l.3 8 ( 20 05 ), c or re a et a l.2 3 ( 20 10 ), lo ur en ço e t a l.4 3 ( 20 09 ), m el lo e t a l.2 6 (2 01 4) , s ou za a nd r on ca lli 44 (2 00 7) , r os si an d c ha ve s3 2 ( 20 15 ), sa nt os e t a l.3 4 ( 20 07 ), v ie ira e t a l.3 7 ( 20 13 ) 30 o ng oi ng h ea lth e du ca tio n a qu ila nt e an d a ci ol e1 9, 45 (2 01 5) , b al da ni e t a l.3 8 ( 20 05 ), c av al ca nt i e t a l.2 1 ( 20 12 ), fe rn an de s et a l.2 4 ( 20 15 ), g od oi e t a l.4 0, 41 (2 01 3, 20 14 ), lo ur en ço e t a l.4 3 ( 20 09 ), m or et ti et a l.4 6 (2 01 0) , n as ci m en to e t a l.2 8 ( 20 09 ), sa nt os a nd a ss is 35 (2 00 6) , s ou za a nd r on ca lli 44 (2 00 7) , v ie ira e t a l.3 7 ( 20 13 ) 43 ,3 c on tin ui ng e du ca tio n a gu ile ra e t a l.1 8 ( 20 13 ), a qu ila nt e an d a ci ol e4 5 ( 20 15 ), c ha ve s an d da s ilv a2 2 ( 20 07 ), lo ur en ço e t a l.4 3 ( 20 09 ), n as ci m en to e t a l.2 8 (2 00 9) , r od rig ue s et a l.3 1 ( 20 11 ), sa nt os a nd a ss is 35 (2 00 6) , v ie ira e t a l.3 7 ( 20 13 ) 26 ,7 in fo rm at io n sy st em s an d ev al ua tio n a qu ila nt e an d a ci ol e4 5 (2 01 5) , b al da ni e t a l.3 8 ( 20 05 ), c av al ca nt i e t a l.2 1 ( 20 12 ), c ol us si a nd c al vo 39 (2 01 1) , g od oi e t a l40 ,4 1 ( 20 13 , 2 01 4) , m oi m az e t a l.2 7 ( 20 08 ), p im en te l e t a l.3 0 (2 01 0) , s ou za a nd r on ca lli 44 (2 00 7) 30 fa m ily h ea lth s tr at eg y a gu ile ra e t a l.1 8 ( 20 13 ), a qu ila nt e an d a ci ol e1 9, 45 (2 01 5) , a ra új o an d d im en st ei n2 0 ( 20 06 ), b al da ni e t a l.3 8 ( 20 05 ), c av al ca nt i e t a l.2 1 ( 20 12 ), c ha ve s an d da s ilv a2 2 (2 00 7) , f er na nd es e t a l.2 4 ( 20 15 ), g od oi e t a l40 ,4 1 ( 20 13 , 20 14 ), le ss a an d ve tt or e4 2 ( 20 10 ), lo ur en ço e t a l.4 3 (2 00 9) , m at to s et a l.5 (2 01 4) , m or et ti et a l.4 6 ( 20 10 ), n as ci m en to e t a l.2 8 ( 20 09 ), p ad ilh a et a l.2 9 ( 20 05 ), p im en te l e t a l.3 0 ( 20 10 ), sa nt os a nd a ss is 35 (2 00 6) , so uz a an d ro nc al li4 4 ( 20 07 ), v ie ira e t a l.3 7 ( 20 13 ) 66 ,7 a ux ili ar y te am (o ra l h ea lth t ec hn ic ia n an d o ra l h ea lth a ss is ta nt ) b al da ni e t a l.3 8 ( 20 05 ), c or re a et a l.2 3 ( 20 10 ), fe rn an de s et a l.2 4 ( 20 15 ), 10 in te rs ec to ria lit y a qu ila nt e an d a ci ol e1 9, 45 (2 01 5) , b al da ni e t a l.3 8 (2 00 5) , c ha ve s an d da s ilv a2 2 ( 20 07 ), c ol us si a nd c al vo 39 (2 01 1) , g od oi e t a l.4 1 ( 20 14 ), m at to s et a l.5 (2 01 4) , m el lo e t a l.2 6 ( 20 14 ), m or et ti et a l.4 6 ( 20 10 ), p ad ilh a et a l.2 9 ( 20 05 ), p im en te l e t a l.3 0 ( 20 10 ), sa nt os a nd a ss is 35 (2 00 6) , s ou za a nd r on ca lli 44 (2 00 7) , v ie ira e t a l.3 7 ( 20 13 ) 46 ,7 q ua lifi ed m an ag er a qu ila nt e an d a ci ol e4 5 ( 20 15 ), fe rn an de s et a l.2 4 (2 01 5) , r os si a nd c ha ve s3 2 ( 20 15 ) 10 u ni ve rs ity -h ea lth s er vi ce in te gr at io n g od oi e t a l.4 0, 41 (2 01 3, 2 01 4) , m el lo e t a l.2 6 ( 20 14 ) 6, 7 in te rp er so na l r el at io ns hi p v ie ira e t a l.3 7 ( 20 13 ) 3, 3 in te rp ro fe ss io na l t ea m w or k a qu ila nt e an d a ci ol e1 9, 45 (2 01 5) , b al da ni e t a l.3 8 (2 00 5) , c av al ca nt i e t a l.2 1 ( 20 12 ), c ha ve s an d da s ilv a2 2 ( 20 07 ), c ol us si a nd c al vo 39 (2 01 1) , fe rn an de s et a l.2 4 ( 20 15 ), lo ur en ço e t a l.4 3 ( 20 09 ), m el lo e t a l.2 6 ( 20 14 ), m or et ti et a l.4 6 ( 20 10 ), n as ci m en to e t a l.2 8 ( 20 09 ), p ad ilh a et a l.2 9 ( 20 05 ), p im en te l e t a l.3 0 ( 20 10 ) 40 15 uchida et al. braz j oral sci. 2022;21:e226252 discussion a total of 20 specific practices (codes) were identified in the metasummary of the retrieved data and allocated into three families: “oral healthcare structure”, “oral healthcare provision” and “staff management”. adequate structure, including not only the physical structure itself, but also the knowledge about health system organization, is a basic requirement to address table 4. intensity of the effect size (ies) for all codes and codes with fes > 25%. article ies % all codes (n = 20) ies % codes > 25% (n= 10) aguilera et al.18 (2013) 15 30 aquilante and aciole19 (2015) 35 60 aquilante and aciole45 (2015) 50 90 araújo and dimenstein20 (2006) 10 20 baldani et al.38 (2005) 45 80 cavalcanti et al.21 (2012) 30 40 chaves and da silva22 (2007) 35 50 colussi and calvo39 (2011) 15 30 correa et al.23 (2010) 25 30 fernandes et al.24 (2015) 30 50 godoi et al.40 (2013) 30 40 godoi et al.41 (2014) 35 60 lessa and vettore42 (2010) 15 30 lippert et al.25 (2020) 5 20 lourenço et al.43 (2009) 40 70 mattos et al.5 (2014) 25 50 mello et al.26 (2014) 15 20 moimaz et al.27 (2008) 15 10 moretti et al46. (2010) 25 50 nascimento et al.28 (2009) 35 60 padilha et al.29 (2005) 35 70 pimentel, moura and acioli30 (2010) 30 60 rodrigues et al.31 (2011) 5 10 rossi and chaves32 (2015) 25 30 sá et al.33 (2015) 5 10 santos et al.34 (2007) 25 30 santos and assis35 (2006) 25 40 silva et al.36 (2020) 10 10 souza and roncalli44 (2007) 25 50 vieira et al.37 (2013) 55 80 16 uchida et al. braz j oral sci. 2022;21:e226252 the demands of universal health coverage. apart from oral health clinical care, the involvement of the community, other health professionals, and other sectors of society are of great value to amplify habit changing and health promotion47. additionally, healthcare staff motivation, interprofessional integration and continuing qualification are also important characteristics to improve working processes through individual contribution, and to strengthen interpersonal relationships. although all the 20 specific practices were not applied at the same time in the same place, it seems that their widespread implementation could place oral healthcare managers/teams onto a more progressive path to promote a healthier population in long-term practice. in all the included studies, samples were composed by dentists and/or oral health managers. the majority of the participants were oral health managers, who directly contributed to the implementation of oral healthcare practices within sus. dentists emerged as important protagonists, either working at basic health units (bhus), leading oral health teams (ohts), or occupying management positions. however, the evidence also shows that good management is not only dependant on qualified managers, but also on the efficient performance of oht members. among the 20 codes identified in the metasummary, three main practices emerged as being the most relevant: care diagnosis and planning (fes = 82%), family health strategy (fes = 71%), and interprofessional teamwork (fes = 46%). in the family “oral healthcare structure”, the code care diagnosis and planning emerged from 23 of 28 studies included in the metasummary, clearly indicating that situational diagnosis based on the epidemiological status of care provision along action planning are essential for a quality service45. this finding is in agreement with the bnohp guidelines, which indicate that epidemiology and information about the geographic area covered by the ohts should be used to subsidize action planning30. planning has been considered the instrument to consolidate the foundational principles of sus (universality, integrality and equity), and promote health improvements32. in several studies, the authors registered the need for managers to structure and organize oral healthcare provision based on action planning to increase access and ensure the continuity of treatment19,27,32,33,42-46. moreover, proposed actions need to be permanently evaluated to ensure that improvements in the healthcare system and in the general health of the population are implemented step by step. the practices conducted by managers in municipalities with no water fluoridation and high prevalence of dental caries is a good example. oht members should be guided on the need to perform fluoridated mouthwashes or distribute sachets with fluoride to the local population23,30,41,45. therefore, oht professionals should be responsible for planning, organizing, developing and evaluating actions according to the requirements of their local community, seeking articulation with the most varied social actors involved in health promotion48. in the family “oral healthcare provision”, the code family health strategy emerged as the most important practice. most of the studies analyzed proposed that ohts should be more closely integrated into the fhs through group activities, regular home visits, and seeing the patient in a more holistic sense22,29,43,46,49. the fhs has been designed to renew the rationale of care, which must go beyond interventions directed to the cure of the individual34. the fhs philosophy involves the reorgani17 uchida et al. braz j oral sci. 2022;21:e226252 zation of care practices, by replacing the traditional model oriented to the treatment of diseases, to focusing on how families live and their immediate needs49. the fhs is responsible for monitoring a defined number of families, located in a defined geographical area, with focus on health promotion, prevention, recovery, and rehabilitation of more frequent diseases50. the fhs endeavours to redirect the work flow through the interaction of multiprofessional teams, aiming at implementing the most resolutive and integral practices within the perspective of health surveillance. hence, primary care organization based on fhs principles has been deemed as essential to the development of the service. managers have reported on the importance the fhs and community health agent programs, in addition to specific programs for women’s and children’s health, control of systemic diseases such as diabetes and hypertension, and oral health programs24,30,42. in the family “staff management”, the code interprofessional teamwork highlights the importance of teamwork for the improvement of the fhs, emphasizing the integrality aspect of healthcare provision3. thus, ohts is the way to break away from more conventional models, by incorporating the expanded concept of health and sharing the burden of oral healthcare provision among different professional30. ohts should not only assist in dealing with health issues, but also motivate the population to be engaged in selfcare. moreover, ohts are required for the collective construction of health actions. when difficulties arise, these can be the subject of discussion before they are eventually overcome. thus, the presence of ohts allows for the exchange of information and search of more adequate therapeutic plans for the user21,29,45. the evidence arising from this systematic review shows that the integration of the oht members within the fhs has been occurring through the development of activities designed to draw stakeholders together and integrate health actions in an interprofessional manner19,29,43. for instance, the inclusion of dentists in vaccination campaigns, ludic-educational activities, supervised brushing, and children’s diet evaluation30. the evidence emerging from the three families of codes indicate that care diagnosis, health planning, oht/fhs integration, and interprofessional teamwork were the most relevant adopted practices. as a result, oral healthcare managers tend to perform well when: 1. they know the legislation, and sus and bnohp guidelines; 2. their oht members participate in ongoing health education; 3. they stimulate intersectionality within their local communities; and 4. they put into effect their leadership role. thus, qualified oral healthcare managers provide support and guidance, foster cooperation while implementing government health policies, involve all healthcare stakeholders collectively, and are in close contact with the community. reliable situational diagnosis, establishment of coherent goals, and optimization of physical and financial resources are fundamental requirements for reorganizing and strengthening basic oral healthcare. action planning, appropriate to the needs and priorities of the population in question through the fhs, can allow the provision of higher quality care and more comprehensive and resolute attention to sus users. concerning the relevance of individual studies to the outcome of this review, four studies stood out with the higest ies, two qualitative studies37,45 and two surveys38,43. qualitative studies showed codes that surveys and mixed-model studies did not, rein18 uchida et al. braz j oral sci. 2022;21:e226252 forcing the importance of the qualitative methodology as a powerful tool for in-depth research in dentistry. while all the 20 codes emerged from qualitative studies, 5 of them (user satisfaction, management autonomy, individual performance, qualified management and interpersonal relationship) appeared exclusively in qualitative studies. the advantage of qualitative studies resides in its design, which may permit a deeper insight into the perceptions, feelings and opinions that are sometimes difficult to be captured by surveys11. nonetheless, surveys can also make an important contribution when they are adequately designed. the quality of the included studies was evaluated by assessing the risk of bias, which considers the characteristics of individual studies that contributed to the outcome51. overall, most studies presented low risk of bias. important quality limitations were observed in the majority of the selected studies. for instance, many qualitative studies did not mention the type of relationship between researchers and participants; did not present an adequate sample description; did not disclose the criteria used to select research subjects or the way data were analyzed; some results lacked clarity; and the relevance of the study and ethical issues were also absent. therefore, future qualitative studies in the area should make use of the confidence in the evidence from reviews of qualitative research (cerqual) and the consolidated criteria for reporting qualitative research (coreq). cerqual provides a clear method for assessing confidence in the synthesis of qualitative findings52. coreq is an instrument that defines verification criteria to help researchers to report important aspects related to research teams, methods, context, findings, analysis and interpretations53. although most of the surveys (62%) included in this systematic review was identified as having low risk of bias, none of the selected studies presented any type of questionnaire validation. the use of a validated instrument would have significantly contributed to increasing the quality of the evidence, since the validation process shows the reliability and veracity of the questionnaire applied to research subjects. the two mixed-model studies also showed a high risk of bias. none of the items evaluated by the instrument were found in the included studies, with weaknesses in both the quantitative and qualitative evaluation. in relation to the quantitative component, not enough information on the methodological outline could be found. on the other hand, in the qualitative component, there was no information on items related to sampling, methodology and the presence an experienced researcher. in relation to the metasummary, an important limiting factor concerns the absence of a quality assessment instrument to integrate qualitative studies, surveys and mixed-model studies. in conclusion, the evidence emerging from this systematic review and metasummary demonstrate that oral healthcare diagnosis, planning, and basic care based on the fhs principles were the most relevant practices adopted by public oral healthcare managers in brazil to provide quality care. although most studies included in this systematic review presented a high risk of bias, the emerging evidence makes a significant contribution to the improvement of oral healthcare management within sus in brazil. other countries with universal health systems, as well as those seeking to follow the united nations sdgs, may also benefit from the present findings. 19 uchida et al. braz j oral sci. 2022;21:e226252 acknowledgments the authors would like to thank the coordination for the improvement of higher education personnel (capes) for the scholarships granted to the graduate student participating in the study and the national council of technological and scientific development (cnpq) for the research funding grant no. 401514/2013-7. author contribution tânia harumi uchida: data curation, investigation, methodology, project administration, validation, original draft, writing, review and editing. uhana seifert guimarães suga: data curation, investigation, methodology. clarissa garcia rodrigues: methodology. 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2015;1(2):56-70. portuguese. http://cqrmg.cochrane.org/supplemental-handbook-guidance http://cqrmg.cochrane.org/supplemental-handbook-guidance file:///c:\fop-5 mar 2015\bjos\revisoes\2022\ 21 uchida et al. braz j oral sci. 2022;21:e226252 25. lippert ao, mendonça ff, carvalho bg, caldarelli pg. the dental specialties center as a point of attention in the oral health care network in a region of paraná. braz j oral sci. 2020;19:e209984. doi: 10.20396/bjos.v19i0.8659984. 26. mello alsf, de andrade sr, moysés sj, erdmann al. oral health care in the health network and the regionalization process. cien saude colet. 2014;19(1):205-14. doi: 10.1590/1413-81232014191.1748. 27. moimaz sas, garbin cas, garbin aji, ferreira nf, gonçalves pe. challenges and difficulties of financing oral health: a qualitative analysis. rev adm publica. 2008;42(6):1121-35. portuguese. doi: 10.1590/s0034-76122008000600005. 28. nascimento ac, moysés st, bisinelli jc, moysés sj. oral health in the family health 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2015;39(spe):196-206. portuguese. doi: 10.5935/0103-1104.2015s005186. 33. sá cr, kuhnen m, santos if, arruda mp, toassi rfc. oral health planning in primary health care: from theory to practice. rev aps. 2015;18(1):92-101. portuguese. 34. santos am, assis mma, rodrigues aaao, nascimento maa, jorge m. conflicting situations in the reception of oral health teams from the family health program in alagoinhas, bahia, brazil. cad saude publica. 2007;23(1):75-85. portuguese. doi: 10.1590/s0102-311x2007000100009. 35. santos am dos, assis mma. from fragmentation to integrality: constructing and reconstructing the practice of buccal health in the alagoinhas (ba) family health program. cien saude colet. 2006;11(1):53-61. portuguese. doi: 10.1590/s1413-81232006000100012. 36. silva junior cl, almeida pf, martins filho mt, lima ld. implementation of dental specialty centers in the context of health regionalization in the state of ceará, brazil. physis. 2020;30(4):1-22. portuguese. doi: 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[inclusion of oral health services in the family health program in the state of paraná, brazil]. cad saude publica. 2005 jul-aug;21(4):1026-35. portuguese. doi: 10.1590/s0102-311x2005000400005. 39. colussi cf, calvo mcm. an evaluation model for oral health in primary care. cad saude publica. 2011 sep;27(9):1731-45. portuguese. doi: 10.1590/s0102-311x2011000900007. 40. godoi h, de mello alsf, caetano jc, zanardi e. oral health care network: limitations and challenges in a large municipality of santa catarina, brazil. saude transform soc. 2013;4(4):69-77. portuguese. 22 uchida et al. braz j oral sci. 2022;21:e226252 41. godoi h, mello alsf de, caetano jc. an oral health care network organized by large municipalities in santa catarina state, brazil. cad saude publica. 2014 feb;30(2):318-32. portuguese. doi: 10.1590/0102-311x00084513. 42. lessa cfm, vettore mv. primary health care management in oral health in fortaleza, ceará, between 1999 and 2006 saude soc. 2010;19(3):547-56. portuguese. doi: 10.1590/s0104-12902010000300007. 43. lourenço ec, silva acb, meneghin mc, pereira ac. the insertion of oral health services in the family health program at minas gerais state, brazil. cien saude colet. 2009 oct;14 suppl 1:1367-77. portuguese. doi: 10.1590/s1413-81232009000800009. 44. souza tms de, roncalli ag. oral health in the brazilian family health program: a health care model evaluation. cad saude publica. 2007 nov;23(11):2727-39. portuguese. doi: 10.1590/s0102-311x2007001100020. 45. aquilante ag, aciole gg. building a “smiling brazil”? [a “smiling brazil”? implementation of the brazilian national oral health policy in a health region in the state of são paulo. cad saude publica. 2015 jan;31(1):82-96. portuguese. doi: 10.1590/0102-311x00193313. 46. moretti ac, teixeira ff, suss fmb, lawden jac, lima lsm, bueno re, et al. intersectoriality in health promotion actions carried out by the oral health team of curitiba, paraná state. cien saude colet. 2010 jun;15 suppl 1:1827-34. portuguese. doi: 10.1590/s1413-81232010000700095. 47. peduzzi m, agreli hf. teamwork and collaborative practice in primary health care. interface. 2018;22(supl.2):1525-34. doi: 10.1590/1807-57622017.0827. 48. melo lmll, moimaz sas, garbin cas, garbin aji, saliba na. oral health assessment from the perspective of the municipal manager. rgo. 2016;64(4):402-10. doi: 10.1590/1981-863720160003000063096. 49. assis mma, alves js sa. human resources policies and the (re)signification of workers’ practices in the unified health system (sus): notes for the debate. rev baiana saude publica. 2008;32(suppl 1):7-15. portuguese. doi: 10.22278/2318-2660.2008.v32.n0.a1453. [referência não citada no texto] 50. moraes dos santos ml, zafalon ej, bomfim ra, kodjaoglanian vl, mendonça de moraes sh, do nascimento ddg, et al. impact of distance education on primary health care indicators in central brazil: an ecological study with time trend analysis. plos one. 2019 mar 26;14(3):e0214485. doi: 10.1371/journal.pone.0214485. 51. neves rg, flores tr, duro sms, nunes bp, tomasi e. time trend of family health strategy coverage in brazil, its regions and federative units, 2006-2016. epidemiol serv saude 2018;27(3):e2017170. doi: 10.5123/s1679-49742018000300008. 52. galvão tf, pereira mg. rating the quality of evidence of systematic reviews. epidemiol serv saude. 2015;24(1):173-5. portuguese. doi: 10.5123/s1679-49742015000100019. 53. lewin s, glenton c, munthe-kaas h, carlsen b, colvins cj, gülmezoglu m, et al. using qualitative evidence in decision making for health and social interventions: an approach to assess confidence in findings from qualitative evidence syntheses (grade-cerqual). plos med 12(10): e1001895. doi: 10.1371/journal.pmed.1001895. 54. tong a, sainsbury p, craig j. consolidated criteria for reporting qualitative research (coreq): a 32-item checklist for interviews and focus groups. int j qual health care. 2007 dec;19(6):349-57. doi: 10.1093/intqhc/mzm042. http://dx.doi.org/10.1590/1981-863720160003000063096 23 uchida et al. braz j oral sci. 2022;21:e226252 s up po rt in g in fo rm at io n a pp en di x 1. s ea rc h st ra te gy u se d in p ub m ed . g ro up k ey w or ds s ea rc h st ra te gy (m es h an d en tr y te rm s) p = p at ie nt de nt is t, de nt is ts / ge ne ra l d en ta l pr ac tit io ne r / ge ne ra l d en ta l pr ac tit io ne rs po lic ym ak er , po lic ym ak er s / po lic y m ak er , po lic y m ak er s (“ d en tis t” [a ll fi el ds ] o r “ d en tis ts ”[a ll fi el ds ] o r “ g en er al d en ta l p ra ct iti on er ”[a ll fi el ds ] o r “ g en er al d en ta l p ra ct iti on er s” [a ll fi el ds ] o r “p ol ic ym ak er s” [a ll fi el ds ] o r “p ol ic ym ak er ”[a ll fi el ds ] o r “p ol ic y m ak er s” [a ll fi el ds ] o r “p ol ic y m ak er ”[a ll fi el ds ] o r “ he al th ca re m an ag er ”[a ll fi el ds ] o r “ he al th ca re m an ag er s” [a ll fi el ds ] o r “ he al th p er so nn el ”[a ll fi el ds ] o r “a dm in is tr at iv e pe rs on ne l”[ a ll fi el ds ] o r “p er so nn el , a dm in is tr at iv e” [a ll fi el ds ] o r “a dm in is tr at or s” [a ll fi el ds ] o r “a dm in is tr at or ”[a ll fi el ds ] o r “ he al th fa ci lit y ad m in is tr at or s” [a ll fi el ds ] o r “ he al th fa ci lit y ad m in is tr at or s” [a ll fi el ds ] o r (“ he al th fa ci lit y ad m in is tr at or s” [m es h t er m s] o r (“ he al th ”[a ll fi el ds ] a n d “f ac ili ty ”[a ll fi el ds ] a n d “a dm in is tr at or s” [a ll fi el ds ]) o r “ he al th fa ci lit y ad m in is tr at or s” [a ll fi el ds ] o r (“ ad m in is tr at or ”[a ll fi el ds ] a n d “h ea lth ”[a ll fi el ds ] a n d “ fa ci lit y” [a ll fi el ds ])) o r (“ he al th fa ci lit y ad m in is tr at or s” [m es h t er m s] o r (“ he al th ”[a ll fi el ds ] a n d “ fa ci lit y” [a ll fi el ds ] a n d “a dm in is tr at or s” [a ll fi el ds ]) o r “ he al th fa ci lit y ad m in is tr at or s” [a ll fi el ds ] o r (“ ad m in is tr at or s” [a ll fi el ds ] a n d “ he al th ”[a ll fi el ds ] a n d “ fa ci lit y” [a ll fi el ds ])) o r (“ he al th fa ci lit y ad m in is tr at or s” [m es h t er m s] o r (“ he al th ”[a ll fi el ds ] a n d “ fa ci lit y” [a ll fi el ds ] a n d “a dm in is tr at or s” [a ll fi el ds ]) o r “ he al th fa ci lit y ad m in is tr at or s” [a ll fi el ds ] o r (“ fa ci lit y” [a ll fi el ds ] a n d “a dm in is tr at or ”[a ll fi el ds ] a n d “ he al th ”[a ll fi el ds ])) o r (“ he al th fa ci lit y ad m in is tr at or s” [m es h t er m s] o r (“ he al th ”[a ll fi el ds ] a n d “ fa ci lit y” [a ll fi el ds ] a n d “a dm in is tr at or s” [a ll fi el ds ]) o r “ he al th fa ci lit y ad m in is tr at or s” [a ll fi el ds ] o r (“ fa ci lit y” [a ll fi el ds ] a n d “a dm in is tr at or s” [a ll fi el ds ] a n d “h ea lth ”[a ll fi el ds ])) o r “ he al th fa ci lit y ad m in is tr at or s” [a ll fi el ds ] o r “d ec is io n m ak er s” [a ll fi el ds ] o r “d ec is io n m ak er ”[a ll fi el ds ] o r “m an ag er ”[a ll fi el ds ] o r “m an ag er s” [a ll fi el ds ] o r “d ec is io nm ak er s” [a ll fi el ds ] o r “d ec is io n m ak er ”[a ll fi el ds ] o r “ le ad d en tis ts ”[a ll fi el ds ] o r (( “le ad ”[m es h t er m s] o r “ le ad ”[a ll fi el ds ]) a n d (“ de nt is ts ”[m es h t er m s] o r “d en tis ts ”[a ll fi el ds ] o r “d en tis t” [a ll fi el ds ])) o r “ le ad er s” [a ll fi el ds ] o r “ le ad er ”[a ll fi el ds ] o r “d en ta l l ea de rs ”[a ll fi el ds ] o r “d en ta l l ea de r” [a ll fi el ds ] o r “d en ta l p ub lic h ea lth co ns ul ta nt s” [a ll fi el ds ] o r “a ct or s” [a ll fi el ds ]) (“ po lic y m ak in g” [a ll fi el ds ] o r “ he al th p ol ic ym ak in g” [a ll fi el ds ] o r “d en ta l p ra ct ic e m an ag em en t” [a ll fi el ds ] o r (( “o rg an iz at io n an d ad m in is tr at io n” [m es h t er m s] o r (“ or ga ni za tio n” [a ll fi el ds ] a n d “a dm in is tr at io n” [a ll fi el ds ]) o r “o rg an iz at io n an d ad m in is tr at io n” [a ll fi el ds ] o r “m an ag em en t” [a ll fi el ds ] o r “d is ea se m an ag em en t” [m es h t er m s] o r (“ di se as e” [a ll fi el ds ] a n d “m an ag em en t” [a ll fi el ds ]) o r “d is ea se m an ag em en t” [a ll fi el ds ]) a n d (“ d en t p ra ct ”[j ou rn al ] o r (“ de nt al ”[a ll fi el ds ] a n d “p ra ct ic e” [a ll fi el ds ]) o r “d en ta l p ra ct ic e” [a ll fi el ds ] o r “ d en t p ra ct (e w el l)” [j ou rn al ] o r (“ de nt al ”[a ll fi el ds ] a n d “p ra ct ic e” [a ll fi el ds ]) o r “d en ta l p ra ct ic e” [a ll fi el ds ] o r “a us t d en t p ra ct ”[j ou rn al ] o r (“ de nt al ”[a ll fi el ds ] a n d “p ra ct ic e” [a ll fi el ds ]) o r “d en ta l p ra ct ic e” [a ll fi el ds ])) o r (“ pr ac tic e m an ag em en t” [m es h te rm s] o r (“ pr ac tic e” [a ll fi el ds ] a n d “m an ag em en t” [a ll fi el ds ]) o r “p ra ct ic e m an ag em en t” [a ll fi el ds ] o r (“ m an ag em en t” [a ll fi el ds ] a n d “p ra ct ic e” [a ll fi el ds ])) o r (“ pr ac tic e m an ag em en t” [m es h t er m s] o r (“ pr ac tic e” [a ll fi el ds ] a n d “m an ag em en t” [a ll fi el ds ]) o r “p ra ct ic e m an ag em en t” [a ll fi el ds ] o r (“ m an ag em en ts ”[a ll fi el ds ] a n d “p ra ct ic e” [a ll fi el ds ])) o r “p ra ct ic e m an ag em en t” [a ll fi el ds ] o r “p ra ct ic e m an ag em en t s er vi ce s” [a ll fi el ds ] o r (“ pr ac tic e m an ag em en t” [m es h t er m s] o r (“ pr ac tic e” [a ll fi el ds ] a n d “m an ag em en t” [a ll fi el ds ]) o r “p ra ct ic e m an ag em en t” [a ll fi el ds ] o r (“ m an ag em en t” [a ll fi el ds ] a n d “s er vi ce ”[a ll fi el ds ] a n d “p ra ct ic e” [a ll fi el ds ])) o r “p ra ct ic e m an ag em en t s er vi ce ”[a ll fi el ds ] o r c on tin ue 24 uchida et al. braz j oral sci. 2022;21:e226252 c on tin ua tio n p = p at ie nt de nt is t, de nt is ts / ge ne ra l d en ta l pr ac tit io ne r / ge ne ra l d en ta l pr ac tit io ne rs po lic ym ak er , po lic ym ak er s / po lic y m ak er , po lic y m ak er s (“ pr ac tic e m an ag em en t” [m es h t er m s] o r (“ pr ac tic e” [a ll fi el ds ] a n d “m an ag em en t” [a ll fi el ds ]) o r “p ra ct ic e m an ag em en t” [a ll fi el ds ] o r (“ se rv ic e” [a ll fi el ds ] a n d “p ra ct ic e” [a ll fi el ds ] a n d “m an ag em en t” [a ll fi el ds ])) o r (“ pr ac tic e m an ag em en t, de nt al ”[m es h t er m s] o r (“ pr ac tic e” [a ll fi el ds ] a n d “m an ag em en t” [a ll fi el ds ] a n d “d en ta l”[ a ll fi el ds ]) o r “d en ta l p ra ct ic e m an ag em en t” [a ll fi el ds ] o r (“ de nt al ”[a ll fi el ds ] a n d “p ra ct ic e” [a ll fi el ds ] a n d “m an ag em en t” [a ll fi el ds ] a n d “s er vi ce s” [a ll fi el ds ])) o r (“ pr ac tic e m an ag em en t, de nt al ”[m es h te rm s] o r (“ pr ac tic e” [a ll fi el ds ] a n d “m an ag em en t” [a ll fi el ds ] a n d “d en ta l”[ a ll fi el ds ]) o r “d en ta l p ra ct ic e m an ag em en t” [a ll fi el ds ] o r (“ pr ac tic e” [a ll fi el ds ] a n d “m an ag em en t” [a ll fi el ds ] a n d “s er vi ce s” [a ll fi el ds ] a n d “d en ta l”[ a ll fi el ds ])) o r “m an ag ed c ar e” [a ll fi el ds ] o r “p ub lic h ea lth m an ag em en t” [a ll fi el ds ] o r “ he al th m an ag em en t” [a ll fi el ds ] o r “ he al th p ol ic ym ak in g” [a ll fi el ds ] o r “d ec is io n m ak in g” [a ll fi el ds ] o r “ he al th c ar e m an ag em en t” [a ll fi el ds ] o r “p ub lic p ol ic ie s” [a ll fi el ds ] o r “p ub lic p ol ic y” [a ll fi el ds ] o r “ he al th p ol ic ie s” [a ll fi el ds ] o r “ he al th p ol ic y” [a ll fi el ds ] o r “p ol ic ie s, h ea lth ”[a ll fi el ds ] o r “p ol ic y, h ea lth ”[a ll fi el ds ] o r “n at io na l h ea lth p ol ic y” [a ll fi el ds ] o r (“ he al th po lic y” [m es h t er m s] o r (“ he al th ”[a ll fi el ds ] a n d “p ol ic y” [a ll fi el ds ]) o r “ he al th p ol ic y” [a ll fi el ds ] o r (“ he al th ”[a ll fi el ds ] a n d “p ol ic ie s” [a ll fi el ds ] a n d “n at io na l”[ a ll fi el ds ])) o r (“ he al th p ol ic y” [m es h t er m s] o r (“ he al th ”[a ll fi el ds ] a n d “p ol ic y” [a ll fi el ds ]) o r “ he al th p ol ic y” [a ll fi el ds ] o r (“ he al th ”[a ll fi el ds ] a n d “p ol ic y” [a ll fi el ds ] a n d “n at io na l”[ a ll fi el ds ])) o r “n at io na l h ea lth p ol ic ie s” [a ll fi el ds ] o r (“ he al th po lic y” [m es h t er m s] o r (“ he al th ”[a ll fi el ds ] a n d “p ol ic y” [a ll fi el ds ]) o r “ he al th p ol ic y” [a ll fi el ds ] o r (“ po lic ie s” [a ll fi el ds ] a n d “n at io na l”[ a ll fi el ds ] a n d “ he al th ”[a ll fi el ds ])) o r (p ol cy [a ll fi el ds ] a n d (“ fe de ra l g ov er nm en t” [m es h t er m s] o r (“ fe de ra l”[ a ll fi el ds ] a n d “g ov er nm en t” [a ll fi el ds ]) o r “ fe de ra l g ov er nm en t” [a ll fi el ds ] o r “n at io na l”[ a ll fi el ds ]) a n d (“ he al th ”[m es h t er m s] o r “ he al th ”[a ll fi el ds ])) o r “g ov er na nc e” [a ll fi el ds ] o r “c lin ic al g ov er na nc e” [a ll fi el ds ] o r “m an ag em en t” [a ll fi el ds ] o r “d ec is io nm ak in g” [a ll fi el ds ] o r “m an ag in g re so ur ce s” [a ll fi el ds ] o r “ le ad er sh ip ”[a ll fi el ds ]) i = in te rv en tio n de nt is tr y / or al he al th / d en ta l ca re / d en ta l he al th s er vi ce (“ de nt is tr y” [a ll fi el ds ] o r “o ra l h ea lth ”[a ll fi el ds ] o r “ he al th , o ra l”[ a ll fi el ds ] o r “d en ta l c ar e” [a ll fi el ds ] o r “c ar e, d en ta l”[ a ll fi el ds ] o r “d en ta l he al th s er vi ce s” [a ll fi el ds ] o r (“ de nt al h ea lth s er vi ce s” [m es h t er m s] o r (“ de nt al ”[a ll fi el ds ] a n d “ he al th ”[a ll fi el ds ] a n d “s er vi ce s” [a ll fi el ds ]) o r “d en ta l h ea lth s er vi ce s” [a ll fi el ds ] o r (“ se rv ic es ”[a ll fi el ds ] a n d “d en ta l”[ a ll fi el ds ] a n d “ he al th ”[a ll fi el ds ])) o r (“ de nt al he al th s er vi ce s” [m es h t er m s] o r (“ de nt al ”[a ll fi el ds ] a n d “ he al th ”[a ll fi el ds ] a n d “s er vi ce s” [a ll fi el ds ]) o r “d en ta l h ea lth s er vi ce s” [a ll fi el ds ] o r (“ he al th ”[a ll fi el ds ] a n d “s er vi ce s” [a ll fi el ds ] a n d “d en ta l”[ a ll fi el ds ])) o r “d en ta l h ea lth s er vi ce ”[a ll fi el ds ] o r (“ de nt al h ea lth se rv ic es ”[m es h t er m s] o r (“ de nt al ”[a ll fi el ds ] a n d “ he al th ”[a ll fi el ds ] a n d “s er vi ce s” [a ll fi el ds ]) o r “d en ta l h ea lth s er vi ce s” [a ll fi el ds ] o r (“ he al th ”[a ll fi el ds ] a n d “s er vi ce ”[a ll fi el ds ] a n d “d en ta l”[ a ll fi el ds ])) o r (“ de nt al h ea lth s er vi ce s” [m es h t er m s] o r (“ de nt al ”[a ll fi el ds ] a n d “h ea lth ”[a ll fi el ds ] a n d “s er vi ce s” [a ll fi el ds ]) o r “d en ta l h ea lth s er vi ce s” [a ll fi el ds ] o r (“ se rv ic e” [a ll fi el ds ] a n d “d en ta l”[ a ll fi el ds ] a n d “h ea lth ”[a ll fi el ds ])) o r “p ub lic d en ta l s er vi ce ”[a ll fi el ds ]) 25 uchida et al. braz j oral sci. 2022;21:e226252 p r is m a 2 00 9 c he ck lis t s ec ti on /t op ic # c he ck lis t i te m r ep or te d on pa ge # ti tl e ti tle 1 id en tif y th e re po rt a s a sy st em at ic re vi ew , m et aan al ys is , o r b ot h. ti tle a b s tr a c t st ru ct ur ed s um m ar y 2 p ro vi de a s tr uc tu re d su m m ar y in cl ud in g, a s ap pl ic ab le : b ac kg ro un d; o bj ec tiv es ; d at a so ur ce s; s tu dy e lig ib ili ty c rit er ia , p ar tic ip an ts , a nd in te rv en tio ns ; s tu dy a pp ra is al a nd s yn th es is m et ho ds ; r es ul ts ; l im ita tio ns ; c on cl us io ns a nd im pl ic at io ns o f k ey fi nd in gs ; s ys te m at ic re vi ew re gi st ra tio n nu m be r. a bs tr ac t in tr o d u c ti o n r at io na le 3 d es cr ib e th e ra tio na le fo r t he re vi ew in th e co nt ex t o f w ha t i s al re ad y kn ow n. in tr od uc tio n o bj ec tiv es 4 p ro vi de a n ex pl ic it st at em en t o f q ue st io ns b ei ng a dd re ss ed w ith re fe re nc e to p ar tic ip an ts , i nt er ve nt io ns , c om pa ris on s, o ut co m es , a nd st ud y de si gn (p ic o s) . in tr od uc tio n m et h o d s p ro to co l a nd re gi st ra tio n 5 in di ca te if a re vi ew p ro to co l e xi st s, if a nd w he re it c an b e ac ce ss ed (e .g ., w eb a dd re ss ), an d, if a va ila bl e, p ro vi de re gi st ra tio n in fo rm at io n in cl ud in g re gi st ra tio n nu m be r. m et ho ds el ig ib ili ty c rit er ia 6 sp ec ify s tu dy c ha ra ct er is tic s (e .g ., p ic o s, le ng th o f f ol lo w -u p) a nd re po rt c ha ra ct er is tic s (e .g ., ye ar s co ns id er ed , l an gu ag e, p ub lic at io n st at us ) u se d as c rit er ia fo r e lig ib ili ty , g iv in g ra tio na le . m et ho ds in fo rm at io n so ur ce s 7 d es cr ib e al l i nf or m at io n so ur ce s (e .g ., da ta ba se s w ith d at es o f c ov er ag e, c on ta ct w ith s tu dy a ut ho rs to id en tif y ad di tio na l s tu di es ) i n th e se ar ch a nd d at e la st s ea rc he d. m et ho ds se ar ch 8 p re se nt fu ll el ec tr on ic s ea rc h st ra te gy fo r a t l ea st o ne d at ab as e, in cl ud in g an y lim its u se d, s uc h th at it c ou ld b e re pe at ed . a pp en di x st ud y se le ct io n 9 st at e th e pr oc es s fo r s el ec tin g st ud ie s (i. e. , s cr ee ni ng , e lig ib ili ty , i nc lu de d in s ys te m at ic re vi ew , a nd , i f a pp lic ab le , i nc lu de d in th e m et aan al ys is ). m et ho ds d at a co lle ct io n pr oc es s 10 d es cr ib e m et ho d of d at a ex tr ac tio n fr om re po rt s (e .g ., pi lo te d fo rm s, in de pe nd en tly , i n du pl ic at e) a nd a ny p ro ce ss es fo r o bt ai ni ng a nd co nfi rm in g da ta fr om in ve st ig at or s. m et ho ds d at a ite m s 11 li st a nd d efi ne a ll va ria bl es fo r w hi ch d at a w er e so ug ht (e .g ., p ic o s, fu nd in g so ur ce s) a nd a ny a ss um pt io ns a nd s im pl ifi ca tio ns m ad e. m et ho ds r is k of b ia s in in di vi du al s tu di es 12 d es cr ib e m et ho ds u se d fo r a ss es si ng ri sk o f b ia s of in di vi du al s tu di es (i nc lu di ng s pe ci fic at io n of w he th er th is w as d on e at th e st ud y or ou tc om e le ve l), a nd h ow th is in fo rm at io n is to b e us ed in a ny d at a sy nt he si s. m et ho ds su m m ar y m ea su re s 13 st at e th e pr in ci pa l s um m ar y m ea su re s (e .g ., ris k ra tio , d iff er en ce in m ea ns ). m et ho ds sy nt he si s of re su lts 14 d es cr ib e th e m et ho ds o f h an dl in g da ta a nd c om bi ni ng re su lts o f s tu di es , i f d on e, in cl ud in g m ea su re s of c on si st en cy (e .g ., i2 ) fo r e ac h m et aan al ys is . m et ho ds 1 volume 22 2023 e237397 original article braz j oral sci. 2023;22:e237397http://dx.doi.org/10.20396/bjos.v22i00.8667397 1 federal university of santa maria, school of dentistry, santa maria, rs, brazil. 2 federal university of santa maria, school of dentistry, department of stomatology, santa maria, rs, brazil. 3 federal university of santa maria, school of dentistry, department of restorative dentistry, santa maria, rs, brazil. corresponding author: mariana marquezan av. roraima, 1000. prédio 26f. cidade universitária. camobi. 97105-900. santa maria, rio grande do sul, brazil. mariana.marquezan@ufsm.br editor: dr. altair a. del bel cury received: october 27, 2021 accepted: october 9, 2022 temporomandibular disorders and associated comorbidities among brazilian dental students during covid-19 francyéllen teixeira da silva1 , jessica klöckner knorst1 , lucas machado maracci1 , vilmar antônio ferrazzo2 , gabriela salatino liedke2 , tatiana bernardon silva3 , mariana marquezan2* aim: this study aimed to assess the prevalence of temporomandibular disorders (tmd) and psychosocial comorbidities in undergraduate dental students in a southern brazilian university, during the covid-19 pandemic. also, it aimed to verify the association between psychosocial factors and tmd. methods: fonseca anamnestic index, depression anxiety stress scale (dass-21), a socioeconomic questionnaire, and questions about academic performance and social distancing were applied. poisson regression analysis was used to assess the association of predictive variables with tmd. results: the prevalence of tmd was found to be 82.4%, and more than half of the students had some degree of stress, anxiety, and depression. students who had symptoms of stress (rr 1.11; 95% ci 1.04-1.19), anxiety (rr 1.19; 95% ci 1.12-1.27) and reported academic performance worsening (rr 1.12; 95% ci 1.07-1.19) had higher tmd scores. conclusion: the findings suggest that tmd was highly prevalent among dental students at a federal university in southern brazil during the pandemic, being associated with high levels of stress, anxiety, poor academic performance, and greater social distancing. keywords: covid-19. temporomandibular joint disorders. depression. anxiety. https://orcid.org/0000-0002-6717-5062 https://orcid.org/0000-0001-7792-8032 https://orcid.org/0000-0003-4668-8130 https://orcid.org/0000-0002-2792-9034 https://orcid.org/0000-0002-0967-9617 https://orcid.org/0000-0001-7280-5068 https://orcid.org/0000-0001-6078-5194 2 silva et al. braz j oral sci. 2023;22:e237397 introduction temporomandibular disorders (tmd) encompass a group of multifactorial conditions that cause pain and/or loss of function of the masticatory muscles, temporomandibular joint (tmj), and associated structures1. scientific evidence indicate a relationship between tmd and psychosocial aspects, such as stress, anxiety and depression2,3, being the last both highly associated with painful tmd3. undergraduate students have high stress levels and feel greater distress than the general population, which leads to a high prevalence of mental health problems and greater risk for psychopathological problems4. intense pace of life, increased hours of study, and geographical distance from family and friends might provoke feelings such as disappointment, irritability, concern, and impatience5. in addition, several studies have shown that stress levels among dental students are significantly higher when compared to students from other undergraduate programs6. this may be related to a complex curriculum that involves theoretical learning, clinical practice, and patient care7, being a possible reason why most dental students report symptoms that are already identified as risk factors for tmd and pain conditions8. on march 11, 2020, the world health organization (who) declared the covid-19 pandemic, caused by the sars-cov-2 virus9. due to its high contagion, in the absence of mass vaccination the most effective means of controlling virus propagation was practicing social distancing10. therefore, factors that generate stress may be increased during the covid-19 pandemic. in this sense, the suspension of in-person activities, such as classes, for an indefinite period, as well as the excess of negative information may trigger important psychological disorders11. in addition, the adoption of a remote educational model can lead to several access difficulties that can also be considered stressful and generate anxiety in this period12. the stress present in a daily routine during a pandemic might cause damage to health, that is, it can contribute to the development of tmd, depression, anxiety, and stress. thus, this study aimed to verify the prevalence of these pathologies in undergraduate dental students at federal university of santa maria (ufsm), as well as to analyze the association between tmd and the presence of comorbidities, academic performance, and social distancing, during the covid-19 pandemic. the conceptual hypotheses were: 1) the prevalence of tmd is high among dental students at ufsm; 2) the highest occurrence of tmd is associated with high levels of stress, anxiety, and depression; 3) the highest occurrence of tmd is associated with worse academic performance and greater social distancing. materials and methods this is a cross-sectional observational study carried out on dental students in the city of santa maria, southern brazil. federal university of santa maria is a higher education institution located in santa maria, in which 25,951 students are enrolled. it has a teaching staff composed of 2,020 teachers and offers 266 undergraduate programs. in march 2020, in-person classes were suspended due to the beginning of the covid-19 pandemic in brazil and were replaced by remote education until this 3 silva et al. braz j oral sci. 2023;22:e237397 date. all 343 students enrolled at the dentistry undergraduate program were invited to participate in this study. the volunteer participants filled out a digital questionnaire on google forms platform. data collection was carried out in the period between august 11 and november 20, 2020, when the questionnaire was available on google forms. students were invited to participate in the survey via social networks, email, and whatsapp messaging application. the sample size was calculated considering the following parameters: 5% sampling error, 95% confidence interval (ci), finite population of students enrolled in the ufsm dentistry undergraduate program (estimated 350 students), and the expected prevalence of tmd (50%). adding 20% for possible losses, the minimum sample size required was 221 individuals. prevalence value of 50% was used when the prevalence of the phenomenon in question was unknown in some context, for example. the value of 50% gave the biggest possible size of the necessary sample13. the questionnaire was composed of the fonseca anamnestic index (fai)14; depression, anxiety and stress scale (dass-21)15; questions regarding academic performance and social distancing during the covid-19 pandemic; and a demographic and socioeconomic questionnaire. the fai14 was used to assess the presence of tmd. this index consists of 10 questions: 1) “do you have difficulty opening your mouth wide?”; 2) “do you have difficulty moving your jaw to the sides?”; 3) “do you feel fatigue or muscle pain when you chew?”; 4) “do you have frequent headaches?”; 5) “do you have neck pain or a stiff neck?”; 6) “do you have ear aches or pain in that area (temporomandibular joint)?”; 7) “have you ever noticed any noise in your temporomandibular joint while chewing or opening your mouth?”; 8) “do you have any habits such as clenching or grinding your teeth?”; 9) “do you feel that your teeth do not come together well?”; 10) “do you consider yourself a tense (nervous) person?”. each question has three possible answers: “yes”, “no”, or “sometimes”, which are assigned values of “10”, “0”, and “5”, respectively. the total sum of these values classifies the severity of tmd: “absence of tmd” (0-15), “mild tmd” (20-40), “moderate tmd” (45-65), and “severe tmd” (70-100). this index is one of the few instruments available in the portuguese language that is able to assess tmd severity. despite being very sensitive to identifying tmd patients, it has low specificity in identifying non-tmd individuals, being recommended for screening of patients16. in the present study, the total scores of the tmd questionnaire were used. thus, the higher the questionnaire score, the greater the severity of tmd. stress, anxiety, and depression levels were assessed using the reduced version of the depression, anxiety and stress scale (dass-21), validated for the brazilian population15,17. it consists of three subscales of seven items each, which aim to assess depression, anxiety, and stress experienced by the individual in the past seven days. the answers to each question are reported on a likert scale, ranging from “strongly disagree” (0) to “strongly agree” (3). the overall scores for the three components are calculated by the sum of the scores of the 7 relevant items multiplied by two. the scoring ranges correspond to the levels of symptoms, ranging from “normal” to “extremely severe”15. in the analysis, the presence of stress, anxiety and depression 4 silva et al. braz j oral sci. 2023;22:e237397 was dichotomized into “absent” (normal) or “present” (scores considered medium, moderate, severe, or extremely severe). regarding academic performance, a question about self-perceived performance was used: “during the pandemic, do you consider that your academic performance: 1) has improved, 2) has remained the same, 3) has worsened”. in the analysis, the variable was dichotomized into “remained the same/improved” (scores 1 and 2) and “has worsened” (score 3). the attitude towards social distancing during the pandemic was also assessed: “regarding the social distancing that is being guided by health authorities, that is, staying at home and avoiding contact with other people, how much do you think you are managing to do?”, with the following possible answers: 1) practically isolated; 2) isolated enough; 3) more or less isolated; 4) little isolated; 5) very little isolated18. for the analysis, the variable was categorized as very isolated (scores 1 and 2) and more or less/little/very little isolated (scores 3, 4 and 5). the demographic and socioeconomic variables assessed were gender (female and male), age, skin color (white and non-white), income, and family structure (living alone or with other people). the age was collected in years and later dichotomized according to the median into ≤21 years or >21 years. the monthly income was collected in real (brazilian currency; 1.00 us dollar corresponds to 5.13 reais) and later dichotomized by the median of ≤5000 or >5000 reais per month. the period (semester) in which the student was enrolled in was also considered for the analysis. data analysis was performed using stata 14.0 program (statacorp. 2014. stata statistical software: release 14.0. college station, tx: statacorp lp). a descriptive analysis of the demographic, behavioral, and clinical characteristics of the sample was carried out. unadjusted and adjusted poisson regression analysis was used to assess the association between the predictor variables and the total tmd scores. the predictor variables that presented a p-value <0.20 in the unadjusted analysis were considered in the adjusted model. the results are presented as a rate ratio (rr) and a 95% confidence interval (95% ci). poisson regression analysis was used in our study for modeling count data, as recommended by previous literature for this approach19,20. all procedures were in accordance with the ethical standards. the study protocol was approved by the institutional ethics committee of federal university of santa maria (date of approval: august 2020; approval number: 34721820.0.0000.5346). all participants signed an informed consent form. results from 343 students enrolled in the dentistry undergraduate program at ufsm in august 2020, 222 participated in this study (response rate of 64.72%). the sample included students from all semesters, with an average of 22 years of age (standard deviation [sd] 2.9). among the assessed individuals, 74.3% were female and 82.4% were white. regarding the socioeconomic level, 53.3% belonged to families whose monthly income was up to 5000 reais. the suggested prevalence of tmd was found to be 82.4%, and the mean score was 37.1 (sd 18.8). most students had some degree of stress, anxiety, and depression (57.5%, 54.2%, and 58.6%, respectively) (table 1). 5 silva et al. braz j oral sci. 2023;22:e237397 table 1. distribution of demographic, socioeconomic, and clinical characteristics of students in the dentistry undergraduate program at ufsm (n = 222). variables n* % demographic and socioeconomic variables gender female 165 74.3 male 57 25.7 age ≤ 21 years 116 52.3 > 21 years 106 47.7 skin color white 183 82.4 non-white 39 17.6 monthly income (in reais)a ≤ 5000 116 52.3 > 5000 106 47.7 dwelling situation living alone 25 11.3 living with other people 197 88.7 semester of dental course 1st – 5th 111 50.0 6th – 10th 111 50.0 behavioral and psychosocial characteristics social distancing during the covid-19 pandemic very isolated 141 63.5 more or less/little/very little isolated 81 36.5 how do you consider your academic performance before the covid-19 pandemic? same/better 55 24.8 worse 167 75.2 stress absent 95 42.8 present 197 57.2 anxiety absent 101 45.5 present 121 54.5 depression absent 92 41.4 present 130 58.6 continue 6 silva et al. braz j oral sci. 2023;22:e237397 continuation tmd classification absent 39 17.6 mild 92 41.4 moderate 78 35.1 severe 13 5.9 tmd score (mean [sd]) 37.1 (18.8) *values less than 222 are due to missing data; a1 usd corresponds to 5.60 reais. sd, standard deviation; tmd, temporomandibular disorder. table 2 shows the unadjusted and adjusted association between the predictor variables and the total tmd scores during the covid-19 pandemic. in the unadjusted analysis, sex, age, social distancing, academic performance during the pandemic, stress, anxiety, and depression were associated with the total tmd scores (p <0.05). in the adjusted analysis, only depression lost association with tmd. male individuals had tmd scores 18% lower (rr 0.82; 95% ci 0.77-0.86) than females. in addition, individuals over the age of 21 presented about 10% higher degrees of tmd (rr 1.10; ci 95% 1.05-1.15) than their counterparts. considering behavioral characteristics, students who performed low social distancing presented tmd scores 16% lower than individuals that performed high isolation (rr 0.84; 95% ci 0.80-0.88). dental students who had a worse academic performance showed higher tmd scores (rr 1.12; 95% ci 1.07-1.19). considering the psychosocial characteristics, the presence of stress (rr 1.11; 95% ci 1.04-1.19) and anxiety (rr 1.19; 95% ci 1.12-1.27) symptoms were responsible for 11% and 19% higher scores of tmd among students during the pandemic, respectively. table 2. unadjusted and adjusted association between the predictor variables and the total tmd scores, determined by poisson regression. variables unadjusted rr (ci 95%) p-value adjusted rr (ci 95%) p-value demographic and socioeconomic variables gender <0.01 <0.01 female 1.00 1.00 male 0.76 (0.72-0.80) 0.82 (0.77-0.86) age <0.01 <0.01 ≤ 21 years 1,00 1.00 > 21 years 1.06 (1.02-1.11) 1.10 (1.05-1.15) skin color 0.787 white 1,00 non-white 1.02 (0.85-1.22) continue 7 silva et al. braz j oral sci. 2023;22:e237397 continuation monthly income (in reais) 0.325 ≤ 5000 1.00 > 5000 1.06 (0.93-1.22) dwelling situation 0.394 living alone 1.00 living with other people 1.09 (0.88-1.36) semester of dental course 0.901 1st – 5th 1.00 6th – 10th 1.00 (0.88-1.15) behavioral and psychosocial characteristics social distancing <0.05 <0.01 very isolated 1.00 1.00 more or less/little/very little isolated 0.83 (0.72-0.96) 0.84 (0.80-0.88) academic performance <0.01 <0.01 same/better 1.00 1.00 worse 1.15 (1.09-1.21) 1.12 (1.07-1.19) stress <0.01 <0.01 absent 1.00 1.00 present 1.34 (1.28-1.40) 1.11 (1.04-1.19) anxiety <0.01 <0.01 absent 1.00 1.00 present 1.35 (1.29-1.41) 1.19 (1.12-1.27) depression <0.01 0.098 absent 1.00 1.00 present 1.28 (1.23-1.34) 1.05 (0.99-1.12) *tmd, temporomandibular disorder; rr = rate ratio; ci = confidence interval; a1 usd corresponds to 5.60 reais. discussion during a pandemic, many studies on the biological risk of the disease or on the pathogen are developed, but it is also necessary to pay attention to psychosocial aspects. these generally tend to be neglected and can cause several disorders21,22, such as stress, anxiety, and depression, which can lead to the development of tmd11. in addition, patients with tmd have a higher level of anxiety and depression than the general population23. thus, the findings of this study partially suggest the confirmation of its conceptual hypothesis, because in the unadjusted analysis tmd was associated with stress, anxiety, depression, and low academic performance, whereas in the adjusted analysis, depression lost association with tmd. the findings suggest that the prevalence of tmd in undergraduate dental students at ufsm during the covid-19 pandemic was high (82.4%), higher than previous studies with undergraduate students that also used the fai to assess prevalence 8 silva et al. braz j oral sci. 2023;22:e237397 of tmd, ranging between 53.2% and 66.3%24-26. in a study carried out in saudi arabia that also used the fai, the prevalence of tmd in dental students before the covid-19 outbreak was 49.9% and a positive association was reported between the level of perceived stress and anxiety and tone of both masseters27. during the pandemic, a cross-sectional study28 found the prevalence of tmd to be 77.5%, corroborating our findings. on the other hand, another recent study29 found the prevalence of symptoms of tmd to be only 27.4%, which can be partially explained by the low percentage of individuals that were performing greater social isolation in the sample (21.2%). based on the findings of this study, this high prevalence of tmd might be an effect of stressors caused by the pandemic, which intensify the symptoms of bruxism and tmd11,30, as well as the difficulty of access and consequent learning during remote education31. in addition, students who reported worse academic performance had higher tmd scores, which suggest the confirmation of the hypothesis raised. moreover, it was observed that individuals who performed greater social distancing had higher levels of stress and, consequently, probable higher tmd scores. during the pandemic, social distancing is necessary to mitigate contamination32, although it is associated with negative psychological effects, such as post-traumatic stress, confusion, and anger, mainly due to prolonged isolation, frustration, boredom, and fear of infection33. social isolation also leads to a decrease in the practice of physical activity; those who exercised during the pandemic had 13% lower levels of stress34. therefore, the stress levels during the pandemic influence the development of tmd (or its worsening in those individuals who already had the disorder)28 and might also lead to greater depressive and painful symptoms35. some of the main side effects caused by the covid-19 outbreak are high anxiety and depression levels, due to great exposure to negative news, fear of contagion, unemployment, and the loss of loved ones36.more than half of the study sample self-reported stress, anxiety, and depression. two metanalyses found divergent prevalences of anxiety37 and depression38 on dental students during the post-pandemic period, ranging from 26-45% and 26-49%, respectively. regarding brazilian dental students, a cross-sectional study found that 31.3%, 29.6%, and 24.2% of students had mild, moderate, and severe anxiety symptoms, respectively, totalizing 85.1% of the study sample39. before the covid-19 outbreak, the prevalence of anxiety and depression ranged from 24% to 50.5%27,40 on dental students, similar to the findings after the pandemic, possibly explained by the extensive curriculum of this undergraduate program, involving theoretical learning, clinical practice, and patient care7. considering the gender of the students, females presented higher tmd scores. studies also point out that the occurrence of tmd is twice as common among women41, and that psychological overload during the pandemic is greater in female individuals42. also, individuals who were over 21 years of age presented higher tmd scores. this age group is closer to graduation, with additional stressors such as concerns about the future and the labor market42. the literature considers that the peak of development of tmd occurs between 20 and 40 years of age43, which corroborates the findings of the present study. 9 silva et al. braz j oral sci. 2023;22:e237397 this is the first study to assess the impact of the covid-19 pandemic on the prevalence of tmd among undergraduate dental students in the south of brazil. in addition, this study used validated instruments for the diagnosis of tmd and comorbidities. the depression, anxiety and stress scale questionnaire is a method of easy reproducibility and data collection without the examiner’s influence. another positive aspect is its representative sample of undergraduate dental students at ufsm, which allowed the analysis and extrapolation of findings. some limitations of this study must be recognized. the fact that it was a cross-sectional observational study did not allow any causal relations. therefore, future studies are suggested to assess causality, following participants until a change in the pandemic scenario occurs. besides, the study used a digital questionnaire, thus no clinical examinations were performed and therefore the origin of the tmd (myogenous or arthrogenous) could not be assessed. lastly, even though more complete instruments for the diagnosis of tmd are available (e. g., the diagnostic criteria for temporomandibular disorders dc/tmd)44, the fai is a very sensitive index in identifying tmd patients (despite its low specificity in identifying patients who do not have tmd), recommended for patient screening. furthermore, this instrument has already been used in previous studies that evaluated tmd45-47. sociodemographic variables should also be considered when interpreting the results. even though all students were invited to participate, some biased answers toward those who were concerned with their oral health or who wanted to participate in research projects might be seen. participants’ age is another relevant issue. since this study evaluated undergraduate students, other studies might observe a different impact of the pandemic in older subjects. in addition, questions about academic performance and social distancing were created for this study, and their results reflect the students’ self-report. however, the findings are extremely relevant to the academic environment. this study can be taken as a warning to other educational institutions, in view of its findings. mental health care should be emphasized by the university, based on activities that encourage self-care and dialogue strategies with students. this is essential because, as demonstrated in this study, students are stressed, anxious, and with a high prevalence of tmd. as a result of these problems, the performance and development of daily activities are affected. in conclusion, the findings of this study suggest a high prevalence of tmd (82.4%) among dental students at ufsm during the covid-19 pandemic. tmd was associated with high levels of stress, anxiety, poor academic performance, and greater social distancing. furthermore, women and individuals over the age of 21 were more likely to have higher tmd scores. acknowledgements the authors wish to thank the undergraduate students who helped with data collection. conflicts of interest the authors report no conflict of interest. 10 silva et al. braz j oral sci. 2023;22:e237397 funding none. data availability the authors declare that data is unavailable to access. author contribution francyéllen teixeira da silva: conception of the work, original draft of the work, final approval of the version to be published, agreement to be accountable for all aspects of the work; jessica klöckner knorst: analysis of data for the work, drafting the work, final approval of the version to be published, agreement to be accountable for all aspects of the work; lucas machado maracci: interpretation of data, drafting the work, final approval of the version to be published, agreement to be accountable for all aspects of the work; vilmar antônio ferrazzo: interpretation of data, critical review, final approval of the version to be published, agreement to be accountable for all aspects of the work; gabriela salatino liedke: design of the work, critical review, final approval of the version to be published, agreement to be accountable for all aspects of the work; tatiana bernardon silva: acquisition of data, critical review, final approval of the version to be published, agreement to be accountable for all aspects of the work mariana marquezan: design of the work, critical review, final approval of the version to be published, agreement to be accountable for all aspects of the work. all authors have made substantial contributions to the conception or design of the work, actively participated in the manuscript’s findings, and revised and approved the final version. references 1. de leeuw r, klasser gd. orofacial pain: guidelines for assessment, diagnosis, and management. 6th ed. chicago: quintessence publishing; 2018. 2. manfredini d, lombardo l, siciliani g. temporomandibular disorders and dental occlusion. a systematic review of 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3 dental surgeon from the state university of feira de santana uefs, bahia, brazil. 4 dental surgeon from metropolitan union for education and culture – unime, lauro de freitas, bahia, brazil. 5 master and specialist in oral and maxillofacial surgery and traumatology from the state university of pernambuco, phd in stomatology from the university of paraíba, professor at the state university of feira de santana (ba) and unime college of agrarian health sciences (ba). * corresponding author: sheinaz farias hassam rua juca marques, 401 campo formoso, bahia, brazil 44790-000 email: sheinazhassam@hotmail.com received for publication: october 30, 2020 accepted: january 7, 2021 prevalence of tumours of the maxillomandibular complex diagnosed in a reference center in brazil alessandra laís pinho valente pires1 , izana santos borges nascimento2, ana letícia marques de souza assis³, sheinaz farias hassam4,* , jener gonçalves de farias5 tumors of the maxillomandibular complex are a heterogeneous group of lesions with a wide spectrum of clinical and histopathological characteristics. aim: to evaluate the prevalence of odontogenic and non-odontogenic tumors associated with maxillary bones in a reference center for oral lesions. methods: a cross-sectional study based on the medical records of a reference center for oral lesions at the state university of feira de santana, from 2006 to 2018. the data was initially analyzed in a descriptive manner. for bivariate analysis, pearson’s chi-square test was applied. the level of significance was set at 5%, where p≤ 0.05 is considered significant. results: the prevalence of tumors was 2.27%. the average age of the individuals was 22.2 (± 15.1) years, the majority being up to 39 years (79.59%) and female (69.40%). a statistically significant difference was observed in relation to age (p = 0.00), as well as regarding the location of tumors in the anterior or posterior region (p = 0.02). odontogenic tumors were benign, with odontoma being most frequent (46.90%), followed by ameloblastoma (16.30%). as  for the non-odontogenic, neurofibroma (4.10%) and osteoma (4.10%) were the most common across the benign, while osteosarcoma accounted for 6.10% of cases. conclusion: odontogenic tumors were the most frequent in women, with age up to 39 years, odontoma being most common in the posterior region of the mandible. among nonodontogenic tumors, central neurofibroma and osteoma were the most common. osteosarcoma was more frequent in men over 40 years old and in the mandible region. keywords: diagnosis. prevalence. pathology, oral. crosssectional studies. epidemiology. mailto:sheinazhassam@hotmail.com https://orcid.org/0000-0002-6848-8992 https://orcid.org/0000-0002-8567-6207 2 pires et al. introduction the maxillomandibular complex is subject to the development of several conditions1. intraosseous lesions of the jaws constitute a heterogeneous group of lesions that present a wide spectrum of clinical and histopathological characteristics, ranging from cysts, tumors and bone-associated lesions2. the first internationally accepted classification for maxillofacial lesions was published by the world health organization (who) in 1971, which has been modified over the years (1992 and 2005), in an attempt to better define its diagnostic criteria3. the most recent who edition, in 2017, introduced some changes in the 2005 classification, and, in addition to the return of odontogenic cysts, two new entities were included: sclerosing odontogenic carcinoma and primordial odontogenic tumor4. this  new classification focuses on those that are biologically benign or malignant, signaling a simplification of the previous version5. odontogenic tumors form a complex group of lesions that range from hamartomatous or non-neoplastic proliferations to malignant neoplasms with metastatic capacity6, originating from the remnants of the tooth-forming apparatus7. regarding non-odontogenic tumors, their classification is not yet well established, whether this would be according to the original tissue or according to its topography. according to the who, these tumors have a predilection for the mandibular bone, but some of the lesions, such as chondroma, chondrosarcoma and osteosarcoma, although occurring in the mandible, do not essentially show a greater predilection for this site8,9. in brazil, epidemiological studies show that, among all diagnosed oral lesions, 1.3 to 4.8% are odontogenic tumours10, presenting results similar to other latin american countries, such as chile and mexico11. regarding non-odontogenic tumors, a study conducted in queensland, australia, showed that benign non-odontogenic lesions were 6.8 times more likely to appear than malignant non-odontogenic lesions12. national epidemiological studies related to bone lesions are scarce13. these studies have an important relevance for the knowledge of the population profile, as well as the injuries that can occur in the stomatognathic system, helping in the early diagnosis and treatment. thus, this study aimed to describe the prevalence of odontogenic and non-odontogenic tumors of the maxillomandibular complex in a reference center in brazil. materials and methods design, area of study and characterization of the sample this is a retrospective cross-sectional study, based on secondary data from medical records and conclusive anatomopathological reports of individuals diagnosed at a reference center for oral lesions (crlb) in the department of health sciences, state university of feira de santana (uefs). this research was registered and approved by research ethics committee of the institution where it was carried out (protocol number: 015/2008, caae 0015.0.059.000-08). cases of odontogenic and non-odontogenic tumors were selected between 2006 to 2018 and classified according to the current who classification (2017)5. the criteria 3 pires et al. for exclusion were: medical records that had only descriptive reports; reports with the same registration number; and different diagnoses for the same patient. data collection and selected variables this collection was performed by a single examiner trained to complete the collection worksheet. data regarding gender, age, anatomical site (maxilla versus mandible; posterior versus anterior) and histopathological type were obtained from patients’ records, which also contained the informed consent form duly signed by the patient or guardian. data analyses the data was initially analyzed descriptively. for the bivariate analysis, the normality of the data was verified with the kolmogorov-smirnov test. then, pearson’s chi-square test was applied for categorical variables. the level of significance used was 5%, where p≤ 0.05 was considered significant. analysis was carried out using the statistical package for the social sciences software, version 17.0. (spss inc., chicago, il, usa). results during the study period, all 2,156 histopathological reports of oral lesions, diagnosed at crlb, were evaluated. of these, 290 were diagnosed with some type of intraosseous lesion, and 49 were conclusive for tumors of the maxillofacial complex, representing a prevalence of 2.27%. the mean age of the individuals was 22.2 (± 15.1) years. the majority, 39 (79.59%), were in the group of up to 39 years and were female (69.40%). statistically significant differences were observed between tumors of the maxillomandibular complex and other intraosseous lesions in relation to age (p = 0.00), as well as regarding the anatomical site (anterior versus posterior) (p = 0.02) (table 1). table 1. bivariate analysis of tumors of the maxillomandibular complex and other intraosseous lesions, crlb, uefs, 2006-2018. variables tumors other intraosseous lesions n (%) n (%) p age range up to 39 years 39 (79.59%) 123 (51.10%) 0.00* from 40 years 10 (20.41%) 118 (48.90%) gender female 34 (69.40%) 164 (68.00%) 0.85 male 15 (30.60%) 77 (32.00%) anatomical site (mandible vs maxilla) mandible 27 (55.10%) 140 (58.10%) 0.70 maxilla 22 (44.90%) 101 (41.90%) anatomical site (posterior vs anterior) anterior 27 (55.10%) 90 (37.30%) 0.02* posterior 22 (44.90%) 151 (62.70%) * p<0.05 4 pires et al. benign odontogenic tumors (39) were the most common. odontoma was the most frequent 23 (46.90%), followed by ameloblastoma 08 (16.30%). cementoblastoma 3 (6.10%) was the third most common. no malignant odontogenic tumor was diagnosed. as for non-odontogenic tumors (10), central neurofibroma 02 (4.10%) and osteoma 02 (4.10%) were the most common, while osteosarcoma was the malignant tumor present in 3 (6.10%) of the cases (table 2). table 2. tumors of the maxillomandibular complex according to frequency and percentage, crlb, uefs, 2006-2018. tumors of the maxillomandibular complex n % odontogenic tumors odontoma 23 46.90 ameloblastoma 08 16.30 cementoblastoma 03 6.10 myxoma 02 4.10 aot* 02 4.10 ceot** 01 2.05 non-odontogenic tumors benign central neurofibroma 02 4.10 osteoma 02 4.10 fibroblastoma 01 2.05 hemangiopericitoma 01 2.05 malignant osteossarcoma 03 6.10 small round cell sarcoma 01 2.05 total 49 100 * adenomatoid odontogenic tumor; ** calcifying epithelial odontogenic tumor table 3 shows the distribution of odontogenic and non-odontogenic tumors by age, gender and location. table 3. distribution of odontogenic and non-odontogenic tumors according to age, gender and location, crlb, uefs, 2006-2018. tumors age (years) gender location 1-39 ≥ 40 female male maxilla mandible odontogenic odontoma 21 02 16 08 08 08 ameloblastoma unicystic 05 00 05 00 00 05 ameloblastoma 02 01 01 02 00 03 cementoblastoma 02 01 03 00 01 02 mixoma 02 00 02 00 00 02 aot* 01 01 01 01 01 01 ceot** 01 01 01 01 01 01 continue 5 pires et al. tumors age (years) gender location 1-39 ≥ 40 female male maxilla mandible non-odontogenic benign centra neurofibroma 01 01 01 01 00 02 osteoma 00 02 01 01 02 00 fibroblastoma 01 00 01 00 01 00 hemangiopericitoma 01 00 01 00 00 01 malignant osteossarcoma 01 02 01 02 01 02 small round cell sarcoma 01 00 00 01 00 01 * adenomatoid odontogenic tumor; ** calcifying epithelial odontogenic tumor discussion in this study, intraosseous lesions of the jaws showed a prevalence of 13.45%, based on the 13-year analysis. a low prevalence (25%) was reported by jaafari-ashkavandi and akbari2 (2017) after 22-year of collecting data, which differs from the findings of ali14 (2011), whose percentage was 31% in 5-year follow-up. most tumors were of odontogenic origin, corroborating with parkins  et  al.15 (2007). however, a study conducted in ghana found a greater number of non-odontogenic tumors. this can be justified by the fact that non-aggressive odontogenic lesions, such as odontomas, have not been diagnosed, since only symptomatic patients with facial edema were included in the sample16. odontogenic tumors corresponded to 1.80% of all cases, a result that is similar to other studies17,18 and these lesions mainly occurred in female patients. there are studies in the literature reporting similar occurrence between males and females19; however, some studies have reported a higher prevalence in males20,21, while others also have shown females to be more affected22, corroborating with our result. there is no plausible explanation proven for these differences. souto et al.23 (2014) described that a higher prevalence in women can be explained through the fact that women are more likely to seek healthcare, making these lesions more detectable and raising the number of cases in the female gender. the age group with the most cases was the fourth decade of life, as noted by kebede, et al.21 (2017). in contrast, pereira, et al.24 (2010) described a higher frequency in individuals over 50 years old. the variation may be related to the different samples and populations analyzed. malignant odontogenic tumors were not diagnosed in this study. they are extremely rare lesions, with reported incidences of 1.1%25 and 1.18%26. in silvera et al.27 (2021) study, the malignant lesions were more common in males and in the mandible, affecting individuals of 55 ± 21.6 years. overall, odontomas were the most prevalent of the group, followed by unicystic ameloblastoma, similar to the data from previous publications19. however, alsheddi et al.28 continuation 6 pires et al. (2015) found that odontogenic keratocysts were the most common, followed by ameloblastomas, which can be justified by the difference in the who classification, as they used the third edition (2005), which classifies keratocysts as tumors, whereas we used the fourth, which classifies them as a cyst. in some studies, myxoma was the third most common odontogenic tumour27,29. in our study, adenomatoid odontogenic tumor (aot) and myxoma had a similar frequency (4.10% each) and were in the fourth position. calcifying epithelial odontogenic tumor (ceot) comprised a lower occurrence (2.05%). ali14 (2011) observed that ceot appears as the least frequent among odontogenic tumors. the majority of the studies on odontogenic tumors demonstrated a strong predilection for the mandible, especially the posterior region20,30. jaw-specific genetic mechanisms that regulate the evolution and development of upper and lower dentitions appear to differ and this may provide a partial explanation to the difference in the incidence of odontogenic tumors in the mandible versus maxilla31. however,  açikgoz, et al.32 (2012) and kambalimath,  et  al.33 (2014), suggested involvement in the maxilla. regarding  odontoma, the highest prevalence was in the maxilla, being in line with other authors19,28. it is worth mentioning that among the group of non-odontogenic tumors, neurofibroma and osteoma were the most common among the benign, diverging from the findings of rodrigues,  et  al.34 (2010) and johnson,  et  al.12 (2013), in which the central giant cell granuloma was the most frequent. osteosarcoma was the most frequent among the malignant. these are rare, aggressive, with a high mortality rate35. jaafari-askavandi and akbari2 (2017) pointed out that this lesion corresponded to 28.1% of the neoplasms in their findings. due to this being a descriptive study, the variables analyzed do not allow for inferring causality, requiring further longitudinal studies. however, the results contribute to a better understanding of the clinical-epidemiological profile of individuals and in the development of strategies and actions destined towards diagnosis and treatment of such lesions. the present study was carried out in a reference center, in which oral pathologists issued a histopathological diagnosis, enabling the evaluation of lesions that affect the jaws. new epidemiological studies on tumors of the maxillomandibular complex should be carried out in brazil, in order to obtain greater knowledge surrounding their behavior, thus improving their diagnosis and treatment. in conclusion, the prevalence of tumors of the maxillomandibular complex was 2.27%. odontogenic tumors were the most frequent in women, aged up to 39 years, odontoma being the most common in the posterior region of the mandible. among  non-odontogenic tumors, central neurofibroma and osteoma were the most common. osteosarcoma was more frequent in men over 40 years old and in the mandible region. references 1. zanda mj, poleti ml, fernandes tmf, sathler r, sant’ana e, consolaro a. 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[maxillofacial lesions: a survey of 762 cases from the federal university of sergipe, brazil]. rev odontol unesp. 2014;43(3):185-90. doi: 10.1590/ rou.2014.029. portuguese. 24. pereira jv, figueirêdo du, souza ea, holmes tsv, gomes dqc, cavalcanti al. [prevalence of odontogenic cysts and tumors in patients treated at the paraíba health assistance foundation: a retrospective study]. arq odontol. 2010;46(2):75-81. portuguese. 25. mosqueda-taylor a, ledesma-montes c, caballero-sandoval s, portilla-robertson j, ruíz-godoy rivera lm, meneses-garcía a. odontogenic tumors in mexico: a collaborative retrospective study of 349 cases. oral surg oral med oral pathol oral radiol endod. 1997 dec;84(6):672-5. doi: 10.1016/ s1079-2104(97)90371-1. 26. fernandes am, duarte ec, pimenta fj, souza ln, santos vr, mesquita ra, et al. odontogenic tumors: a study of 340 cases in a brazilian population. j oral pathol med. 2005 nov;34(10):583-7. doi: 10.1111/j.1600-0714.2005.00357.x. 27. silveira fm, macedo ccs, borges cmv, mauramo m, vasconcelos acu, soares ab, et al. odontogenic tumors: an 11-year international multicenter study. oral dis. 2021 mar;27(2):320-4. doi: 10.1111/odi.13550.  28. alsheddi ma, alsenani ma, aldosarib aw. odontogenic tumors: analysis of 188 cases from saudi arabia. ann saudi med. 2015;35(2):146-50. doi: 10.5144/0256-4947.2015.146. 29. pontes cgc, trindade neto ai, ribeiro ilh, sarmento va, santos jn, azevedo ra. [epidemiology of odontogenic cysts and tumors treated under general anesthesia in a philanthropic hospital in salvador, bahia]. rev cir traumatol buco-maxilo-fac. 2012 jan-mar;12(1):93-100. portuguese. 30. mascitti m, togni l, troiano g, caponio vca, sabatucci a, balercia a, et al. odontogenic tumours: a 25-year epidemiological study in the marche region of italy. eur arch otorhinolaryngol. 2020 feb;277(2):527-38. doi: 10.1007/s00405-019-05683-3. 31. ferguson ca, tucker as, sharpe pt. temporospatial cell interactions regulating mandibular and maxillary arch patterning. development. 2000 jan;127(2):403-12. 32. açikgöz a, uzun-bulut e, özden bk, gunduz k. prevalence and distribution of odontogenic and nonodontogenic cysts in a turkish population. med oral patol oral cir bucal. 2012 jan;17(1):e108-15. doi: 10.4317/medoral.17088. 33. kambalimath dh, kambalimath hv, agrawal sm, singh m, jain n, anurag b, et al. prevalence and distribution of odontogenic cyst in indian population: a 10 year retrospective study. j maxillofac oral surg. 2014 mar;13(1):10-5. doi: 10.1007/s12663-012-0450-y. https://www.ncbi.nlm.nih.gov/pmc/articles/pmc6074140/ 9 pires et al. 34. rodrigues tlc, rodrigues fg, carodos ab, gandelmann iha, cavalcante maa. [benign tumors of the jaws: a 10-year retrospective analysis]. rev cir traumatol buco-maxilo-fac. 2010;10(2):91-6. portuguese. 35. elkordy ma, elbaradie ts, elsebai hi, khairalla sm, amin aae. osteosarcoma of the jaw: challenges in the diagnosis and treatment. j egypt natl canc inst. 2018 mar;30(1):7-11. doi: 10.1016/j. jnci.2018.02.001. erratum in: j egypt natl canc inst. 2018 sep;30(3):123. 1 volume 22 2023 e238902 original article braz j oral sci. 2023;22:e238902http://dx.doi.org/10.20396/bjos.v22i00.8668902 1 kaher’s kle vk institute of dental sciences, jnmc campus, belagavi 590010, india. 2 department of oral medicine and radiology, kle vk institute of dental sciences, belagavi 590010, india. corresponding author: dr. sulem ansari 3rd-year post-graduate department of oral medicine and radiology kaher’s kle vk institute of dental sciences, jnmc campus belagavi-590010, india email: ansarisulem@gmail.com phone no: 9773723359 editor: altair a. del bel cury received: apr 02, 2022 accepted: oct 07, 2022 a hospital-based observational study on clinical and radiographic findings in covid-19 associated rhinomaxillary mucormycosis: revealing the pandora box sulem ansari1* , shivayogi charantimath2 , vasanti lagali jirge2 , vaishali keluskar2 mucormycosis is a rare, rapidly spreading, fulminant, opportunistic infection that is caused by a group of filamentous molds. during the second wave of covid-19 india reported most of the cases of mucormycosis which is termed as covid-19-associated mucormycosis (cam). aim: the purpose of this study is to describe and understand the clinical and radiographic findings related to covid-19 associated rhinomaxillary mucormycosis. methods: in this observational study 76 individuals with proven rhinomaxillary mucormycosis were included. the demographic profile, predisposing factors, anatomic structures involved, oral manifestations, radiographic findings management, and 90-day mortality were recorded and analyzed. results: among 76 individuals with covid-19-associated rhinomaxillary mucormycosis diabetes mellitus was present in 93.42% of cases. almost all patients received corticosteroids during covid-19 treatment. the maxilla was most commonly involved in around 98.6% of cases. interestingly 1 case involving the mandible was noted and the maxillary sinus was the most commonly involved. mortality occurred in 1.31% (n=1) of cases. conclusion: diabetes was the most common predisposing factor. administration of corticosteroids was evident. a considerable number of patients developed diabetes during the treatment of covid-19. early signs and oral manifestations of rhinomaxillary mucormycosis play a pivotal role in the early diagnosis and prompt treatment to reduce mortality and morbidity in covid-19 associatedrhinomaxillary mucormycosis patients. keywords: mucormycosis. covid-19. https://orcid.org/0000-0003-3727-5772 https://orcid.org/0000-0002-9721-3939 https://orcid.org/0000-0003-1752-1948 https://orcid.org/0000-0001-6956-6035 2 ansari et al. braz j oral sci. 2023;22:e238902 introduction although the primary pathology of covid-19 is pneumonia and respiratory failure, secondary infections are common and attribute significantly to morbidity and mortality1. fungal infections are least common than bacterial infections, nevertheless are usually more invasive and fatal2. mucormycosis is a fungal infection caused by fungi known as mucorales. it is highly aggressive with a tendency for widespread infection. this infection occurs by inhalation of spores. the fungal spores adhere to the respiratory epithelium and transform into hyphae causing angioinvasion, leading to endothelial injury, thrombosis, and necrosis. the fungus can then spread to various organs rapidly to cause disseminated mucormycosis. if the diagnosis and management are delayed, the prognosis is poor1. several factors increase the risk of invasive fungal infections and mucormycosis in patients with covid-19. the most important predisposing factor is hyperglycemia which may decrease the ability of the body to fight infections3. prolonged use of a high dose of corticosteroids and drugs like tocilizumab used in moderate and severe disease may also increase predisposition for fungal infections4. severe covid-19 disease is associated with the cytokine storm, and it is associated with producing insulin resistance and hyperglycemia and the use of steroids aggravates it, as corticosteroids have hyperglycemic action thus providing the milieu for seeding by mucor. this cytokine storm triggers a hyperinflammatory and hypercoagulatory response, which disrupts endothelial cell integrity thus causing organ damage such as lung injury and pancreatic injury5. the sars-cov-2 confers pancreatic islet injury and acute diabetes onset by binding to the ace2 receptor, one more assumed factor for increased risk of mucormycosis in covid-19 is intracellular iron overload signified by increased ferritin levels, which leads to the formation of reactive oxygen species6. widespread endothelial injury in patients with severe disease can upregulate endothelial receptor glucose-regulated protein (grp 78), which is responsible for increased adhesion and penetration of mucorales to the endothelium7. the treatment modalities available for the treatment of mucormycosis are aggressive debridement of infected hard and soft tissue and parenteral antifungal therapy such as liposomal amphotericin b, lipid amphotericin b, posaconazole, and itraconazole. functional endoscopic sinus surgery (fess) is an endoscopic surgical debridement of the paranasal sinuses used in the cases of rhinomaxillary mucormycosis8. the commonest reported form of mucormycosis in literature is rhinomaxillary mucormycosis9. there are other recently published reports as well as case series that reported similar findings10,11. this study aims to describe and understand the clinical and radiographic findings related to covid-19-associated rhinomaxillary mucormycosis. this study aimed to describe the predisposing factors, gender predilection, age, clinical signs and symptoms, oral manifestations, anatomical structures involved, treatment received and mortality related to covid-19associated rhinomaxillary mucormycosis in patients who reported to the dept of oral medicine and radiology with dental complaints. 3 ansari et al. braz j oral sci. 2023;22:e238902 materials and methods this observational hospital-based study was conducted in a dental hospital-based setting. the study sample consisted of confirmed cases of rhinomaxillary mucormycosis with a previous history of covid-19 who reported during the 2nd wave of covid-19 from march 2021 to december 2021 in india. confirmation of diagnosis was based on clinical and radiological features in the paranasal sinus view, computed tomography (pns ct), and demonstration of mucor on potassium hydroxide (koh) staining or histopathological examination using periodic acid-schiff stain (pas) (figure 1) and grocott’s methenamine silver stain (gms) (figure 2). patients with fungal culture positive for mucorales were included in the study. figure 1. histopathological section showing non-septate fungal hyphae on periodic acid-schiff stain figure 2. histopathological section showing septate fungal hyphae on grocott’s methenamine silver stain (gms) 4 ansari et al. braz j oral sci. 2023;22:e238902 a total of 76 patients were included in the study. patients who had rhinomaxillary mucormycosis with negative history of covid-19 were excluded from this study. clinical data was retrieved from the clinical records. these included demographic data like age and gender. details of covid-19 infection were noted which included duration of hospitalization and intensive care unit (icu) stay, the need of a ventilator, and use of corticosteroids and other medicines. history of co-morbidities like diabetes, coronary heart disease, kidney disease, cancer, organ transplant, chronic lung diseases, etc. was recorded. finally, the clinical details of mucormycosis were extracted including the oral manifestations, anatomical structure involved, sinus involvement, treatment provided, and outcome. all data were entered in a central google spreadsheet and the patient information was de-identified. the data was checked by two investigators. results a total of 76 patients with covid-19-associated “rhinomaxillary mucormycosis” were included in this study. the mean age of occurrence was 52 years with 72 males (94.73%) and 4 females (5.26 %) given in table-1. diabetes mellitus (dm) type-ii was found in 93.42% (n=71) of the patients. the previous history of dm type-ii was noted in 46.47 % (n=33) of patients, and 53.52% (n=38) developed dm type-ii during covid-19 treatment. the list of co-morbidities is given in table 1. history of hospitalization was present in 71 patients (93.42%). intensive care (icu) was needed in 22 patients (28.94%). all patients had received systemic corticosteroids. supplemental oxygen was required in 72.36 % (n=55) of cases. various antifungal drugs used such as liposomal amphotericin b were received by 34% (44.73%), although due to acute shortage of the drug 37 (48.68%) patients received lipid amphotericin b parenterally during hospitalization, non-hospitalized individuals received oral isavuconazole (2.63%) and posaconazole (3.94%) [table 1] table 1. age and gender correlation, predisposing factors and covid-19 treatment related parameters in covid-19 associated rhinomaxillary mucormycosis parameters no. of patients (n) range age 76 32-75 years mean age52 years sex 76 males-94.73% (n=72), females-5.26 % (n=4) m: f ratio – 16:1 predisposing factors no. of patients (n) percentage (%) 1) diabetes mellitus type-ii 71 93.42% a) previous history of diabetes 33 46.47 % b) developed diabetes type-ii during covid 19 treatment 38 53.52% 2) renal disease 2 2.63 % 3) myocardial infarction 1 1.31 % parameters no. of patients (n) percentage (%) hospitalization 71 93.42% continue 5 ansari et al. braz j oral sci. 2023;22:e238902 continuation stay in intensive care unit (icu) 22 28.94% oxygen support 40 56.33% systemic corticosteroids 76 100% oral 5 6.58 % iv 71 93.42 % antifungal agents i) liposomal amphotericin b (iv) 34 44.73% ii) lipid amphotericin b (iv) 37 48.68% iii) isavuconzole (oral) 2 2.63% iv) posaconazole (oral) 3 3.94% the maxilla was the most commonly involved anatomic structure in 98.6% (n=75) of the cases, although 1 case of mucormycosis involving the mandible was noted. the right maxilla 64% (n=48) was more commonly involved than the left maxilla 36% (n=27). the most common oral manifestation was a periodontal abscess (figure 3) with mobility seen in 50% (n=38) of patients, followed by necrotic bone (figure 4) with pus discharge in 42.1% (n=32) of patients [table 2]. in radiographic findings, right maxillary sinus 36% (n=27) was more commonly involved than the left side 26.6% (n=20), although in advanced cases a bilateral sinus involvement was observed in 37.3% (n=28). other paranasal sinuses were involved in clinically advanced disease. orbit involvement and other structures of the skull were involved in severe cases. none of the patients showed evidence of brain involvement. [table 3]. figure 3. necrotic bone involving the right maxilla in a patient with rhinomaxillary mucormycosis 6 ansari et al. braz j oral sci. 2023;22:e238902 figure 4. periodontal abscess i.r.t left maxillary premolar in a patient with rhinomaxillary mucormycosis table 2. oral manifestations and anatomic structures involvement in covid-19 associated rhinomaxillary mucormycosis parameters no. of individuals (n) percentage (%) maxilla involvement 75 98.6% mandible involvement 1 1.31% oral manifestations i) necrotic bone with pus discharge 32 42.1% ii) single/ multiple periodontal abscess with mobility 38 50% iii) extra-oral swelling with necrotic bone 6 7.89 % table 3. radiographic findings of rhinomaxillary mucormycosis in covid-19 associated rhinomaxillary mucormycosis parameters no. of individuals (n) percentage (%) paranasal sinus involvement 75 98.6% i) maxillary sinus 75 98.6% a) right 27 36% b) left 20 26.6% c) bilateral involvement 28 37.3% ii) ethmoid sinus 18 24% a) right 8 44.4% b) left 7 38.8 c) bilateral involvement 3 16.6% iii) orbital involvement a) medial wall 2 2.6% b) lateral wall 2 2.6% c) intra-orbital extension 3 3.9% continue 7 ansari et al. braz j oral sci. 2023;22:e238902 continuation other structures pterygopalatine fossa 3 3.9% infra-temporal fossa 2 2.6% sphenopalatine foramen 1 1.3% ethmoid air cells 2 2.6% nasal cavity 20 26.3% sphenoid bone 2 2.6% zygoma 1 1.3% functional endoscopic sinus surgery (fess) under general anesthesia (ga) was done in 96.68 % (n = 75) of patients. orbital decompression was done in 3.94 % (n = 3) of patients. depending upon the bone involvement various surgical procedures were performed as given in table-4. mortality occurred in 1.31% (n=1) of the patient, it was due to a delay in the treatment due to underlying cardiac disease. [table 4] table 4. treatment provided and mortality associated with in covid-19 associated rhinomaxillary mucormycosis parameters no. of individuals (n) percentage (%) treatment provided 76 100% i) fess 75 96.6% ii) micro-debridement of orbit 4 5.26 % iii) orbital decompression 3 3.94 % iv) partial maxillectomy 28 39.47 % v) alveolectomy 46 60.52 % vi) subtotal maxillectomy 1 1.31% vii) debridement 1 1.31% mortality associated 1 1.31% discussion mucormycosis was one of the comorbidities associated with covid 19. in this observational study majority of patients had diabetes. covid-19-associated rhinomaxillary mucormycosis was more prevalent in males (94.73%) than females (5.26%) and m: f ratio was 16:1. the age range was between 32-75 years with a mean age of 52 years. the majority of patients had diabetes mellitus in this observational study; a study conducted by gupta et al 2021 reported the presence of diabetes in the majority of patients12. prominently, the majority of the reported mucormycosis cases during the covid-19 pandemic were from india. a systematic review of 101 cases of cam 8 ansari et al. braz j oral sci. 2023;22:e238902 globally published in july 2021 revealed diabetes in 80% of patients and steroid use in 76% of patients13. a retrospective countrywide study revealed that diabetes mellitus is predominant in 79% and steroid use in 87% of patients with rhino-orbito-cerebral mucormycosis associated with covid-1911. in an updated systematic review comprised of 233 patients from india and 42 from the rest of the world, diabetes was identified as the most common primary risk factor for cam in india than in other countries. a study from egypt in 21 patients with cam also found a high prevalence of diabetes (90%)14. in our study, all patients received systemic corticosteroids during the treatment of covid-19. the interesting fact here is although the prevalence of diabetes was similar in most studies, the use of corticosteroids is less in some studies (85% in a recent systemic review)15. another notable finding was that, 53.52% developed diabetes mellitus during the covid-19 treatment, new onset of diabetes was reported in association with covid-1916. the nasal mucociliary clearance is the primary distinctive defense mechanism of the paranasal sinus against various antigens. this mechanism guards the upper respiratory system against numerous inhaled particles and microorganisms. however, it becomes one of the critical factors for the growth of fungus together with the inflammation of the upper airway, in patients requiring prolonged hospital stay with supplemental oxygen therapy17, mild thrombocytopenia has been detected in 58–95% of patients with sars-cov-2 infection. platelets possibly hamper the growth of fungi are (i) directly by adhering to mucorales hyphae to form a thrombus. (ii) indirectly, platelets secrete pro-inflammatory and anti-inflammatory cytokines such as tgf-β and thrombocidins which may act against mucorales18. corticosteroids used for suppression of inflammatory mediators and cytokines in severe covid-19 cause suppression of lymphocytes and also cause hyperglycemia by promoting gluconeogenesis and inhibiting glycolysis thus there is an increased incidence of mucormycosis in patients with a history of corticosteroid use19. the maxilla (98.6%) was the most commonly involved jaw bone in our study. interestingly 1 case involving the mandible was seen, although in bacterial osteomyelitis, the mandible is most common than the maxilla20. the most common oral manifestations were multiple periodontal abscesses with mobility (50%) and necrotic bone with pus discharge (42.1%) followed by extra-oral swelling with necrotic bone (7.89 %). bacterial osteomyelitis is associated with the symptoms of localized intense pain, fever, tenderness, etc.21 although in the cases of covid-19-associated rhinomaxillary mucormycosis no such symptoms were reported by the patients. on radiographic examination, the maxillary sinus was the most commonly involved. bilateral maxillary sinus involvement (37.3%) was most commonly seen followed by right maxillary sinus involvement (36%). ethmoidal sinus was the second most involved paranasal sinus. orbital involvement was found in advanced cases of rhinomaxillary mucormycosis. other vital structures such as infratemporal fossa, pterygopalatine fossa, sphenopalatine foramen, ethmoid air cells, sphenoid bone, and zygoma were seen in clinically advanced cases. this is the first study reporting oral manifestations and radiographic findings of covid-19associated rhinomaxillary mucormycosis. functional endoscopic sinus surgery under 9 ansari et al. braz j oral sci. 2023;22:e238902 general anesthesia (fess) was employed in the majority of patients reported with sinus involvement. depending upon the area of bone involvement resection of jaw bone was done. due to resection of the bone, there was loss of a masticatory function. the mortality rate observed was 1.31 %, possibly due to early diagnosis and aggressive treatment. another interesting fact is that the fatality rate of cases reported from india (36.5%) is less than the globally reported cases (61.9%) this might be due to the predominance of rhinomaxillary mucormycosis type of mucormycosis18. the aggressive nature of mucor species and mucormycosis warrants attention. dentists should be able to identify the early signs of mucormycosis so that early treatment is instituted thereby reducing morbidity and mortality. delay in the diagnosis of rhinomaxillary mucormycosis has led to many patients becoming severely disfigured19,22. the increase in mucormycosis cases in the indian context appears to be due to diabetes (high prevalence genetically). rampant use of corticosteroids in covid-19 was reported in many studies. corticosteorids lead to an increase in blood glucose and consequently increase the chances of developing opportunistic fungal infection. covid-19 leads to reduced immune functions due to cytokine storm, lymphopenia, and endothelial damage. the combined damaging effects of corticosteroid therapy and sars-cov 2 virus seem to have led to a high prevalence of rhinomaxillary mucormycosis23. all efforts should be made to maintain optimal hyperglycaemia and only judicious evidence-based use of corticosteroids in patients with covid-19 is recommended to reduce the burden of fatal mucormycosis23. oral manifestations of mucormycosis in covid-19 patients are frequently seen in the palate and may include varying degrees of mucosal discoloration, swelling, ulcerations, and superficial necrotic areas involving the palate (figure 1), tooth mobility, multiple periodontal abscess, bone necrosis and exposure with dark eschar formation24. hence, palatal ulcerations could be the primary presenting symptom, leading the patient to the dentist, who can be the first clinician to recognize an infection leading to the diagnosis of rhinomaxillary mucormycosis25. therefore, a non-specific palatal ulcer can be considered as the presenting sign of mucormycosis, and a dental practitioner must be alert to initial signs and symptoms of this disease, specifically when evaluating the high-risk patients. early diagnosis of mucormycosis is critical, as treatment should start as soon as possible in an attempt to decrease mortality26. the role of dentists is critical because mucormycosis primarily occurs around rhinomaxillary or rhinocerebral areas involving facial tissues, palate, alveolar bone, and mandibular bone. therefore, dental professionals should be alert of symptoms of mucormycosis. in addition to palatal lesions, atypical symptoms such as sinus pain, facial pain, unanticipated odontalgia of otherwise sound teeth, or patient deterioration after dental therapeutic interventions should alert clinicians to seek confirmation of the diagnosis and promptly start optimal treatment27. limitations we could not assess the prevalence of covid-19 associated rhinomaxillary mucormycosis as this is a single-center study conducted in a dental opd in india. 10 ansari et al. braz j oral sci. 2023;22:e238902 in conclusion, this single-center observational study from india found a higher prevalence of diabetes mellitus and steroid administration in covid-19-associated rhinomaxillary mucormycosis. maxillary sinus was most commonly involved in the paranasal sinus. many patients presented with aggressive periodontal disease. in the era of the covid -19 pandemic, any patient who presents with aggressive periodontal disease and palatal ulcerations should be investigated for mucormycosis. conflict of interest authors declare no conflict of interest. this study is self-funded. ethics obtained ethical clearance from the institutional ethical committee. ethical clearance no: 1382 source of funding self-funded. author contribution 1) dr. sulem ansari 1* study design, collection of data, interpretation of ct scans, statistical analysis 2) dr. shivayogi charantimath 2 – interpretation of ct scans 3) dr. vasanti jirge 3 – study design, editing of the manuscript 4) dr. vaishali keluskar 4 – final editing of the manuscript all authors have revised and approved the final version of the manuscript. data availability datasets related to this article will be available upon request to the corresponding author. references 1. palanisamy n, vihari n, meena ds, kumar d, midha n, tak v, et al. clinical profile of bloodstream infections in covid-19 patients: a retrospective cohort study. bmc infect dis. 2021 sep;21(1):933. doi: 10.1186/s12879-021-06647-x. 2. zia m, goli m. predisposing factors of important invasive fungal coinfections in covid-19 patients: a review article. j int med res. 2021 sep;49(9):3000605211043413. doi: 10.1177/03000605211043413. 3. unnikrishnan r, misra a. infections and diabetes: risks and mitigation with reference to india. diabetes metab syndr. 2020 nov-dec;14(6):1889-94. doi: 10.1016/j.dsx.2020.09.022. 4. garg d, muthu v, sehgal is, ramachandran r, kaur h, bhalla a, et al. coronavirus disease (covid-19) associated mucormycosis (cam): case report and systematic review of literature. mycopathologia. 2021 may;186(2):289-98. doi: 10.1007/s11046-021-00528-2. 11 ansari et al. braz j oral sci. 2023;22:e238902 5. oh h, ghosh s. nf-κb: roles and regulation in different cd4(+) t-cell subsets. immunol rev. 2013 mar;252(1):41-51. doi: 10.1111/imr.12033. 6. jose a, singh s, roychoudhury a, kholakiya y, arya s, roychoudhury s. current understanding in the pathophysiology of sars-cov-2-associated rhino-orbito-cerebral mucormycosis: a comprehensive review. j maxillofac oral surg. 2021 sep;20(3):373-80. doi: 10.1007/s12663-021-01604-2. 7. alqarihi a, gebremariam t, gu y, swidergall m, alkhazraji s, soliman ss, et al. grp78 and integrins play different roles in host cell invasion during mucormycosis. mbio. 2020 jun;11(3):e01087-20. doi: 10.1128/mbio.01087-20. 8. rai s, yadav s, kumar d, kumar v, rattan v. management of rhinomaxillary mucormycosis with posaconazole in immunocompetent patients. j oral biol craniofac res. 2016 nov;6(suppl 1):s5-s8. doi: 10.1016/j.jobcr.2016.10.005. 9. dilek a, ozaras r, ozkaya s, sunbul m, sen ei, leblebicioglu h. covid-19-associated mucormycosis: case report and systematic review. travel med infect dis. 2021 nov-dec;44:102148. doi: 10.1016/j.tmaid.2021.102148. 10. patel a, agarwal r, rudramurthy sm, shevkani m, xess i, sharma r, et al. multicenter epidemiologic study of coronavirus disease–associated mucormycosis, india. emerg infect dis. 2021 sep;27(9):2349-59. doi: 10.3201/eid2709.210934. 11. sen m, honavar sg, bansal r, sengupta s, rao r, kim u, et al. epidemiology, clinical profile, management, and outcome of covid-19-associated rhino-orbital-cerebral mucormycosis in 2826 patients in india–collaborative opai-ijo study on mucormycosis in covid-19 (cosmic), report 1. indian j ophthalmol. 2021 jul;69(7):1670-92. doi: 10.4103/ijo.ijo_1565_21. 12. patel a, kaur h, xess i, michael js, savio j, rudramurthy s, et al. a multicentre observational study on the epidemiology, risk factors, management and outcomes of mucormycosis in india. clin microbiol infect. 2020 jul;26(7):944.e9-944.e15. doi: 10.1016/j.cmi.2019.11.021. 13. singh ak, singh r, joshi sr, misra a. mucormycosis in covid-19: a systematic review of cases reported worldwide and in india. diabetes metab syndr. 2021 jul-aug;15(4):102146. doi: 10.1016/j.dsx.2021.05.019. 14. alfishawy m, elbendary a, younes a, negm a, hassan ws, osman sh, et al. diabetes mellitus and coronavirus disease (covid-19) associated mucormycosis (cam): a wake-up call from egypt. diabetes metab syndr. 2021 sep-oct;15(5):102195. doi: 10.1016/j.dsx.2021.102195. 15. pal r, singh b, bhadada sk, banerjee m, bhogal rs, hage n, et al. covid‐19‐associated mucormycosis: an updated systematic review of literature. mycoses. 2021 dec;64(12):1452-9. doi: 10.1111/myc.13338. 16. qadir mm, bhondeley m, beatty w, gaupp dd, doyle-meyers la, fischer t, et al. sars-cov-2 infection of the pancreas promotes thrombofibrosis and is associated with new-onset diabetes. jci insight. 2021 aug;6(16):e151551. doi: 10.1172/jci.insight.151551. 17. jose a, singh s, roychoudhury a, kholakiya y, arya s, roychoudhury s. current understanding in the pathophysiology of sars-cov-2-associated rhino-orbito-cerebral mucormycosis: a comprehensive review. j maxillofac oral surg. 2021 sep;20(3):373-80. doi: 10.1007/s12663-021-01604-2. 18. muthu v, rudramurthy sm, chakrabarti a, agarwal r. epidemiology and pathophysiology of covid-19-associated mucormycosis: india versus the rest of the world. mycopathologia. 2021 dec;186(6):739-54. doi: 10.1007/s11046-021-00584-8. 19. weprin be, hall wa, goodman j, adams gl. long-term survival in rhinocerebral mucormycosis: case report. j neurosurg. 1998 mar;88(3):570-5. doi: 10.3171/jns.1998.88.3.0570. 20. sosale a, sosale b, kesavadev j, chawla m, reddy s, saboo b, et al. steroid use during covid-19 infection and hyperglycemia–what a physician should know. diabetes metab syndr. 2021 jul-aug;15(4):102167. doi: 10.1016/j.dsx.2021.06.004. 12 ansari et al. braz j oral sci. 2023;22:e238902 21. selvamani m, donoghue m, bharani s, madhushankari gs. mucormycosis causing maxillary osteomyelitis. j nat sci biol med. 2015 jul-dec;6(2):456-9. doi: 10.4103/0976-9668.160039. 22. gudmundsson t, torkov p and thygesen th. diagnosis and treatment of osteomyelitis of the jaw – a systematic review of the literature. j dent oral disord. 2017 jun;3(4):1066. doi: 10.26420/jdentoraldisord.2017.1066. 23. singh ak, singh r, joshi sr, misra a. mucormycosis in covid-19: a systematic review of cases reported worldwide and in india. diabetes metab syndr. 2021 jul-aug;15(4):102146. doi: 10.1016/j.dsx.2021.05.019. 24. brandão tb, gueiros la, melo ts, prado-ribeiro ac, nesrallah ac, prado gv, et al. oral lesions in patients with sars-cov-2 infection: could the oral cavity be a target organ? oral surg oral med oral pathol oral radiol. 2021 feb;131(2):e45-e51. doi: 10.1016/j.oooo.2020.07.014. 25. amorim dos santos j, normando ag, carvalho da silva rl, acevedo ac, de luca canto g, sugaya n, et al. oral manifestations in patients with covid-19: a living systematic review. j dent res. 2021 feb;100(2):141-54. doi: 10.1177/0022034520957289. 26. sanath ak, nayak mt, sunitha jd, malik sd, aithal s. mucormycosis occurring in an immunocompetent patient: a case report and review of literature. cesk patol. 2020 winter;56(4):223-6. 27. bains mk, hosseini-ardehali m. palatal perforations: past and present. two case reports and a literature review. br dent j. 2005 sep;199(5):267-9. doi: 10.1038/sj.bdj.4812650. 1 volume 22 2023 e238329 original article braz j oral sci. 2023;22:e238329http://dx.doi.org/10.20396/bjos.v22i00.8668329 1 department conservative dentistry, federal university of rio grande do sul, school of dentistry, porto alegre, rio grande do sul, brazil. 2 department of production and control of medicines, federal university of rio grande do sul, school of pharmacy, porto alegre, rio grande do sul, brazil. corresponding author: patricia weidlich ramiro barcelos, 2492, santa cecília, porto alegre, rio grande do sul, brazil. telephone: +55 51 99288 7959, e-mail: patricia.weidlich@ufrgs.br editor: dr. altair a. del bel cury received: february 6, 2022 accepted: october 19, 2022 fixed and on-demand regimens of acetaminophen in periodontal surgery: randomized clinical trial carla cioato piardi1 , cristiane galli vaz1 , maria beatriz cardoso ferreira1 , diogo pilger2 , marilene issa fernandes1 , patrícia weidlich1,* aim: to evaluate the clinical efficacy of an acetaminophen analgesic by comparing its prescription in fixed versus ondemand schedules after periodontal surgery. the hypothesis of the study was that the fixed regimen would be more effective than the on-demand regimen for postoperative analgesics following periodontal surgery. methods: an open randomized clinical trial was conducted. the 68 patients who needed total flap surgery to restore supracrestal tissue attachment or surgical treatment of periodontitis were randomized”. visual analogue scale was used to assess pain. the fixed group (n = 34) received 500 mg of acetaminophen every 4 hours for 2 days. the on-demand group (n = 34) was instructed to use the acetaminophen “as needed,” at intervals of no less than 4 hours between doses. ibuprofen was the rescue medication for both groups. pain scores and medication use were recorded 2, 6, 12, 24 and 48 hours after the surgical procedure. the study was registered at the brazilian registry of clinical trials under rbr-7wv259. results: the two groups did not differ in relation to the frequency or the intensity of pain in a 48-hour period (n=20 in the fixed group, and n=22 in the on-demand group), or even in the intention-to-treat (n=34 in each group). individuals who experienced moderate to severe pain used rescue medication more frequently in both groups. no adverse events were reported. conclusion: both regimens were effective in controlling postoperative pain after periodontal surgery. keywords: pain, postoperative. randomized controlled trials as topic. periodontal diseases. acetaminophen. https://orcid.org/0000-0001-6040-8153 https://orcid.org/0000-0001-6219-5630 https://orcid.org/0000-0002-6814-6773 https://orcid.org/0000-0002-8171-2688 https://orcid.org/0000-0003-2358-3758 https://orcid.org/0000-0003-2013-8229 2 piardi et al. braz j oral sci. 2023;22:e238329 introduction surgical procedures are routine in a periodontal clinic1, and the most commonly performed surgeries involve total flaps2, either for surgical treatment of periodontitis, or for the reestablishment of supracrestal attached tissues. the intensity of postoperative pain in periodontal surgery varies greatly, and the prevalence of patients who do not need postoperative analgesic medication is not consistent in the literature. in a cross-sectional study that included different types of periodontal surgery, 32% of the patients did not use postoperative analgesics3. in another study, 20,6% of the patients undergoing periodontal surgery for different indications reported absence of postoperative pain4. in yet another study involving periodontal surgery to treat periodontitis, only 8.6% of the patients reported no postoperative pain5. different factors may interfere with the intensity of pain during the postoperative period, including anxiety6-8 and such surgical factors as surgery duration and osteotomy9. the disparity in the occurrence of postoperative pain raises doubts about the indication and prescription of analgesic drugs. the vast clinical efficacy of analgesic protocols can be seen in the literature, specifically in the cases that compare different medications used either alone or in combination to treat dental pain. a variety of studies can be found that test non-opioid analgesics for postoperative pain control10-12. among these, acetaminophen is one of the most commonly used agents available on the market. its widespread use can be attributed to its efficacy in relieving dental pain, its low incidence of adverse events12, and particularly its distinction as a first-choice medicine for pregnant women and the elderly, or for situations when non-steroidal anti-inflammatory drugs are contraindicated13. acetaminophen is commonly prescribed in fixed or on-demand schedules in dental clinical practice. in the latter case, it is important for the doctor to inform the maximum daily dose and the minimum interval between doses, since the patients are in charge of their own pain management. the on-demand schedule has potential risks that include the use of doses beyond those recommended as the daily maximum limit. on the other hand, a fixed regimen poses the risk of a potential increase in adverse effects due to continued, higher total drug consumption. this is because the pain level at the moment of administration is not under consideration, unlike on-demand use14. regarding the management of postoperative pain after periodontal total flap surgery, the literature shows variable and conflicting data that may hinder establishing an effective and safe postoperative analgesic regimen. thus, the aim of the present study was to compare the efficacy of acetaminophen analgesics, when prescribed in a fixed or an on-demand regimen, in patients undergoing periodontal total flap surgery. the hypothesis of the present study was that the fixed regimen would be more effective than the on-demand regimen for postoperative analgesics following periodontal surgery. 3 piardi et al. braz j oral sci. 2023;22:e238329 materials and methods study design and sample an open label randomized controlled trial was conducted to test the analgesic efficacy of acetaminophen in two different prescription schedules, according to the consort statement. although it was an open study, participants were unaware of the working hypothesis. the participants were recruited from the periodontology residence clinic at the federal university of rio grande do sul (ufrgs), in porto alegre, brazil. data collection took place between may 2016 and july 2017. adult patients who needed either total flap surgery to restablish supracrestal tissue attachment or surgical treatment of periodontitis were included. patients using systemic analgesic, anxiolytic, anti-infective or anti-inflammatory agents, muscle relaxants or antidepressants were excluded, as well as those who had a previous condition of chronic or acute pain, or those who found it difficult to understand the instructions given by the research team. this study was performed according to the declaration of helsinki. the research ethics committee of the federal university of rio grande do sul approved this study (caae 38637714.2.0000.5347). the registration at the brazilian registry of clinical trials can be found at http://www.ensaiosclinicos.gov.br/rg/rbr-7wv259/. data collection after reading and signing the informed consent form, the participants answered questions about gender, age, years of schooling, and smoking. the visual analogue scale (vas)15,16 was applied to assess the basal level of pain before the beginning of the dental procedure. the following scales were applied to assess the degree of anxiety: (a) the corah dental anxiety scale (cdas), proposed by corah17 and validated in brazil by hu et al.18, and (b) the reduced version of the state-trait anxiety inventory (stai), proposed by kaipper et al.19 and validated in brazil by biaggio et al.20. the cdas comprises four questions concerning how patients feel about dental treatments. the cdas scores range from 4 to 20, with greater scores indicating higher levels of anxiety18. the stai scores range from 13 to 52 (state section) and 13 to 48 (trait section), with greater scores indicating higher levels of anxiety. two previously trained researchers conducted the interview. the surgical treatments were performed by dentists from the periodontology residence clinic of the school of dentistry, ufrgs. surgical procedures the surgeries were performed using full thickness flaps as follows: the initial incision was performed parallel to the long axis of the tooth and placed approximately 1 mm from the buccal/palatal gingival margin, or intracrevicular when esthetic considerations were important. buccal and palatal full‐thickness flaps were carefully elevated. subsequently, intracrevicular incisions were made around the teeth to the alveolar crest and the third and last incision was made in a horizontal direction and in a position close to the surface of the alveolar bone crest separates the soft tissue collar of the root surfaces from the bone. the granulation tissues were removed by 4 piardi et al. braz j oral sci. 2023;22:e238329 means of manual curettes. ostoeotomy was performed to reestablish supracrestal tissues dimensions or radicular debridement took place in cases of surgical treatment of periodontitis. local anesthesia was administered with both infiltrative and regional techniques. the duration of the surgeries was recorded. chlorhexidine digluconate solution (0.12%) was prescribed for topical use every 12 hours for 14 days. allocation after the surgical procedures were completed, the participants were randomly assigned to one of the two groups for postoperative pain control: (1) fixed-time or (2) on-demand analgesic regimens. simple randomization was performed using a computer-generated table. allocation was concealed using numbered opaque envelopes, which contained the code generated by the random number table, set up by an individual not involved in the study. in the fixed-time regimen group, the patients were instructed take 500 mg of acetaminophen every 4 hours for 48 hours, as prescribed. administration was to start 2 hours after surgery ended. the patients received a written form to record the time periods when they used the medicine. the proposed total dose of acetaminophen in this group was 3 g/day. in the on-demand regimen group, the patients were instructed to use 500 mg of acetaminophen when they felt pain, with an interval of no less than 4 hours between two doses, during a period of 48 hours, as prescribed. they received a written form to record the time periods when they used the medicine. the total daily dose of acetaminophen in this group could differ, up to a maximum dose of 3 g/day. ibuprofen 600 mg was prescribed as a rescue analgesic for both groups. the patients were instructed to use it when pain persisted after 1 hour following administration of acetaminophen, with an interval of no less than 6 hours between two doses. the total daily dose of rescue analgesic could differ, up to a maximum dose of 2.4 g/day. each participant received 16 acetaminophen tablets (500 mg each) in a non-electronic container that could be opened manually, identified by a blue label, and 6 ibuprofen tablets (600 mg each), identified by a yellow label. pain assessment after the surgical procedure, the participants were given instructions on how to fill out a postoperative pain control form (or a “pain diary”). the pain control form was composed of the vas21, and included a section to record analgesic consumption (yes/no), and time of use. for detailed information about the pain scale, see schirmer et al.8. the patients were instructed to fill out the pain control form 2, 6, 12, 24 and 48 hours after surgery. to improve adherence, the patients received a phone call every 24 hours reminding them to fill out the postoperative pain control form. in the return visit for postoperative evaluation and suture removal, the participants were instructed to bring back the pain diary and remaining medication, to determine adherence. the number of tablets left was counted and recorded. patients were also questioned about the occurrence of drug-related adverse events. 5 piardi et al. braz j oral sci. 2023;22:e238329 adherence to treatment adherence to the randomized scheme in both regimens was based on the participants’ notes in their pain diary, and the number of remaining tablets. adherence in the fixed regimen was assumed when the participants (a) used the acetaminophen as indicated in the prescription, and (b) respected the period of 1 hour following its administration before taking the rescue medication. forgotten doses, observed from the records in the pain diary, were considered non-adherence to treatment. the presence of a high number of tablets in the acetaminophen container suggested non-use, and a lower number of tablets than estimated suggested excessive use, and was also considered as non-adherence. adherence in the on-demand regimen was affirmed when the individual used the acetaminophen respecting intervals of no less than 4 hours between doses, and 1 hour after its administration before taking the rescue medicine, according to the pain diary. the presence of fewer acetaminophen tablets than estimated suggested excessive use, and constituted non-adherence. sample calculation the sample calculation considered 70% prevalence of analgesic use in the fixed scheme group, and 35% in the on-demand group3,5. a significance level of 5%, and a beta error probability of 20% were assumed. based on these data, a sample size of 31 individuals per group was estimated. considering a 10% attrition rate in both groups, 34 subjects were randomized to each group, totaling 68 participants. data analysis the data were expressed as either absolute or relative frequencies, mean and standard deviation, or median and interquartile range. the continuous variable comparisons between groups were performed using the student t-test for independent samples, or the mann-whitney u test. the data of categorical variables were expressed as absolute frequencies and percentages. comparisons were made using the chi-square test. the value of the 75th percentile was considered the reference point to analyze the anxiety levels. participants within the 75th percentile and with higher scores were categorized as “high anxiety,” and those with lower scores, as “without high anxiety.” this procedure was used for the “cdas level variables,” and the “state” (32) and “trace” levels (30) of the state-trait anxiety inventory (stai) (11). for example, subjects with “high anxiety” presented values equal to or higher than the 75th percentile for each variable. vas data were categorized as mild (vas 1-39), moderate (vas 40-69), or severe (vas ≥70) pain, according to collins et al.15 and al-hamdan5. participants were dichotomized into those with mild pain and those with moderate to severe pain within 6 hours after surgery. a period of 6 hours was selected because it is used routinely in the literature for acute postoperative pain analysis, inasmuch as it is the period of highest pain intensity for the surgical procedure in quesion22-24. the results were shown in intention-to-treat and protocol analyses. in the intentionto-treat analysis, all 68 subjects in the sample were considered. the analysis by protocol included only participants categorized as adherent to the scheme proposed by 6 piardi et al. braz j oral sci. 2023;22:e238329 the study. statistical software spss® for windows, version 18.0, was used for data analysis. the individual was considered the unit of analysis, and the significance level was established at 5%. results regarding the study eligibility criteria, 97 individuals indicated to undergo periodontal surgery were evaluated (figure 1). of these, 29 were excluded either because they did not meet the criteria (n=22), or because they did not return to perform the procedure (n = 7). the remaining 68 participants who agreed to participate were included. characteristics of the sample and the surgical procedures are described in table 1. there was no difference between the fixed and on-demand groups in regard to the variables analyzed. adherence to the proposed schedule was observed in 61.76% of the total sample, with no statistically significant difference between the two groups (58.8% in the fixed regimen group versus 64.7% in the on-demand regimen group, p = 0.54; chi-square test). figure 1. flow diagram. enrollment allocation follow-up analysis assessed for eligibility (n = 97) randomized (n = 68) excluded (n = 29) • not meeting inclusion criteria (n = 29) • declined to participate (n = 0) • other reasons (n = 0) allocated in the on-demand scheme group (n = 34) • received allocated intervention (n = 34) • did not receive allocated intervention (n = 0) allocated in the fixed scheme group (n = 34) • received allocated intervention (n = 34) • did not receive allocated intervention (n = 0) interntion-to-treat analysis (n = 34) analysis by protocol (n = 20) • excluded from analysis (n = 0) interntion-to-treat analysis (n = 34) analysis by protocol (n = 22) • excluded from analysis (n = 0) lost to follow-up (n = 0) discontinued intervention (n = 0) lost to follow-up (n = 0) discontinued intervention (n = 0) 7 piardi et al. braz j oral sci. 2023;22:e238329 table 1. demographic and descriptive data of groups who received prescription of acetaminophen in fixed or on-demand schemes for analgesics after periodontal procedures. variable fixed (n = 34) on-demand (n = 34) p age (± sd) 41.5 (± 16.15) 41.2 (± 14.31) 0.93 a sex – n (%) 0.60 amale 11 (32.4) 11 (32.4) female 23 (67.6) 23 (67.6) schooling – mean (years) (± sd) 10.5 (± 3.22) 11 (± 2.76) 0.49 a smoking habit – n (%) non-smoker 27 (79.4) 24 (70.6) 0.60 asmoker 4 (11.8) 7 (20.6) ex-smoker 3 (8.8) 3 (8.8) reason for surgery – n (%) reestablish supracrestal tissue attachment 29 (85.3) 31 (91.2) 0.71 a surgical treatment of periodontitis 5 (14.7) 3 (8.8) local anesthetic solution – n (%) lidocaine 18 (52.9) 23 (67.6) 0.71 a mepivacaine 11 (32.4) 8 (23.5) prilocaine 2 (5.9) 2 (5.9) not registered 3 (8.8) 1 (2.9) number of local anesthetic tubes used – n (± sd) 2.72 (± 0.86) 2.49 (± 0.81) 0.26 a removal of bone tissue – n (%) no 11 (32.4) 8 (23.5) 0.59 a yes 23 (67.6) 26 (76.5) duration of surgery (h & min) (mean ± sd) 1:31 (± 0:40) 1:23 (± 0:25) 0.37 a furcation injury – n (%) yes 4 (11.8) 2 (5.9) 0.67 a no 30 (88.2) 32 (94.1) anxiety-trace (idate) total scores median (p25p75) 26 (24.5-32.2) 27.5 (23-31.2) 0.48 b anxiety-state (idate) total scores median (p25p75) 28 (24-31) 26 (24-30) 0.53 b cdas total scores – median (p25p75) 8 (5-11.2) 8 (5-10.5) 0.29 b a chi-square test for categorical variables, and student t test for independent samples for continuous variables. b mann-whitney test. idate: trait-state anxiety inventory. cdas: corah dental anxiety scale. p25 e p75 (p25p 75): interquartile range the frequency of the postoperative pain levels at 2, 6, 12, 24 and 48 hours for participants who received an analgesic prescription in a fixed or on-demand schedule is shown in table 2. when the frequency of the pain levels was compared with each of the schemes in the different periods, no statistically significant difference was 8 piardi et al. braz j oral sci. 2023;22:e238329 observed (p>0.05 for all the time periods, using the friedman test). the pain scores of patients who received a prescription for fixed or on-demand analgesics is shown as supplementary material (supplementary table 1). the frequency of participants using rescue medication at each time interval is shown in the supplementary table 2. there was no difference between the groups in any of the periods evaluated in the protocol analysis or the intention-to-treat analysis. table 2. distribution frequency of pain levels assessed at 2, 6, 12, 24 and 48 hours after periodontal procedures for the groups that received a prescription of acetaminophen in fixed or on-demand regimens, in an intention-to-treat analysis (n = 68). the vas scores are categorized as follows: mild pain scores = from 1 to 39; moderate pain scores = from 40 to 69; severe pain scores = ≥ 70.  pain levels analgesic scheme 2 hours 6 hours 12 hours 24 hours 48 hours mild fixed 85.3% 82.4% 91.2% 97.1% 97.1% on-demand 85.3% 73.5% 79.4% 85.3% 85.3% moderate fixed 11.8% 14.7% 2.9% 2.9% 2.9% on-demand 5.9% 11.8% 14.7% 8.8% 8.8% severe fixed 2.9% 2.9% 5.9% 0% 0% on-demand 8.8% 14.7% 5.9% 5.9% 5.9% p a 0.92 0.30 0.20 0.85 0.85 a friedman test. in table 3, the sample was categorized into two groups according to the reported pain intensity over a period of 6 hours – participants with mild pain or those with moderate to severe pain. there were no significant differences between the two groups regarding sociodemographic variables related to the surgical procedure or anxiety. patients that experienced moderate to intense pain used rescue medication more frequently than those feeling mild to moderate pain. there were no reports of adverse events related to consumption of the prescribed drugs in either group. table 3. distribution of individuals who presented mild pain or moderate to severe pain 6 h after periodontal procedures, according the sociodemographic and surgical characteristics, anxiety scores and prescription of acetaminophen in fixed or on-demand schemes. variable mild pain n=53 moderate to severe pain n=15 p age (years) (mean±sd) a 40.2 (±15.2) 45.0 (±15.37) 0.28 sex – n (%) c female 35 (66) 11 (73.3) 0.59 male 18 (34) 4 (26.7) continue 9 piardi et al. braz j oral sci. 2023;22:e238329 continuation smoking habit – n (%) c smoking 9 (17.0) 2 (13.3) 0.76non-smoking 40 (75.5) 11 (73.3) ex-smoker 4 (7.5) 2 (13.3) duration of the surgery (h & min) (mean±sd) a 01:26 (±00:35) 01:29 (±00:26) 0.73 osteotomy c yes 39 (73.6) 10 (66.7) 0.59 no 14 (26.4) 05 (33.3) anxiety-trace (idate)d median (p25p 75) b 27 (24-30) 26 (24-31) 0.43 anxiety-state (idate)d median (p25p 75) b 29 (24-32) 28 (23-31) 0.86 cdasd median (p25p75) b 8 (5-11) 9 (5-12) 0.32 acetaminophen administration scheme n (%)c fixed 28 (52.8) 6 (40.0) 0.38 on-demand 25 (47.2) 9 (60.0) adherence to the scheme prescribed (n %)c yes 36 (67.9) 9 (60.0) 0.49 no 17 (32.1) 6 (49.0) use of acetaminophen in the proposed period n (%)e yes 48 (90.6) 14 (93.3) 0.60 no 5 (9.4) 1 (6.7) use of rescue medication n (%)e yes 8 (15.1) 9 (60) 0.001 no 45 (84.9) 6 (40) dose of ibuprofen used in the proposed period (mg) median (p25p75) b 0 (0/0) 600 (0/600) 0.001 a student t test for independent samples b mann-whitney u test c chi-square test d total scores e exact fisher test discussion the present clinical trial compared the analgesic efficacy of two acetaminophen prescription schedules—fixed-dose or on-demand—in the postoperative period of total flap periodontal surgery. both prescribed regimens were found to be effective in reducing pain levels, and rescue medication was used more frequently by patients who experienced moderate to intense pain. few studies have compared fixed to on-demand schedules – the latter is known as the pro-rata regimen, or “as needed” in more popular vernacular. only two studies were found for the postoperative period of periodontal surgery. one compared the preop10 piardi et al. braz j oral sci. 2023;22:e238329 erative use of etodolac in a fixed regimen with acetaminophen and hydrocodone in a pro-rata regimen, and found no difference between the pain scores for the two regimens25. the other study compared the preoperative use of ibuprofen associated to a fixed prescription of the same drug in the postoperative period with the preoperative use of placebo associated to ibuprofen in an “as needed” regimen in the postoperative period of periodontal surgery26. there was no difference in the postoperative pain levels, thus corroborating the results of the present study. however, the design of these studies is not adequate enough to allow an effective comparison to be made between the prescription drug regimens. the reason there is no difference between the two analgesic regimens tested could be attributed to the fact that postoperative pain after periodontal surgery is predominantly mild and moderate in intensity3; this would make the efficacy of acetaminophen suffice even when used sporadically22. in the present clinical trial, there was a predominance of mild pain throughout the 48 hours of observation. pain peaks, expressed as the highest median scores on the vas scale, were observed at the 2nd and 6th postoperative hours, in both the fixed and on-demand groups. in fact, the literature shows that pain is more intense in the first 6 to 12 hours after periodontal surgery. one clinical trial involving surgery and reestablishment of supracrestal attached tissues observed a reduction in pain scores after the 6th and the 8th postoperative hour23. another study with surgical treatment of periodontitis showed higher levels of pain in 6 hours, and a reduction in pain levels between 24 and 72 hours24. it is of fundamental importance to evaluate adverse events in drug studies, with the aim of drawing a safety profile of the studied drug12. in this study, there were no reports of adverse events associated with acetaminophen or ibuprofen. however, the sample size calculation was not based on this outcome; therefore, there could be a beta error. in two systematic reviews, the adverse effects attributed to acetaminophen (nausea, vomiting, and drowsiness) were classified as mild and transient, similar to those described by the placebo group12,22. several additional factors have been associated with reports of higher levels of postoperative pain, including longer surgical procedures and bone tissue removal3,6,9. these variables were analyzed in our study, and no significant association with pain intensity was observed. non-adherence to the proposed scheme was not related to pain intensity either. our findings corroborate those of another study, which also used acetaminophen to control acute pain, and observed no association between adherence to fixed or on-demand protocols and pain levels27. smoking is another condition that may also be associated with higher levels of postoperative pain. in a study on factors associated with pain and analgesic consumption, smokers were 47% more likely to report pain after non-surgical scaling and root planing than nonsmokers8. however, in the present study, no association was found between smoking and higher levels of postoperative pain, in line with the findings of beaudette6 involving the occurrence of pain following soft tissue grafting or implant surgery. it should be pointed out that only 20 to 30% of the participants in this study were smokers, which may not be enough to show a statistical difference. 11 piardi et al. braz j oral sci. 2023;22:e238329 anxiety is also a factor that can influence pain levels28-32, but the state and trait anxiety, and the dental anxiety scores in the present study did not show any significant differences between patients who reported mild pain or moderate to severe pain. it can be postulated that periodontal patients who undergo surgical procedures as part of their entire periodontal treatment are already familiar with the periodontist and dental staff, hence representing a anxiety-reducing factor. at the same time, procedures involving periodontal surgery most commonly receive mild postoperative pain scores, associated with less anxiety and fear. the present study has limitations. all the efforts made in data collection focused on increasing adherence to a patient-randomized scheme. however, in addition to cases of non-adherence (35.3%), there were also cases where the information on medication use and pain control recorded on a specific form by patients may not have reflected exactly what occurred in the postoperative period. it is also important to consider that a small group of patients in both groups experienced pain 48 hours after the procedure. a longer follow-up period should be considered for these patients. considering that the anesthetic used was not standardized, the pain levels reported may have been influenced by the duration of the different anesthetics. in addition, the surgeries were performed by different professionals and although they were all in the periodontics residency course, they could be at different stages of training and with different clinical and surgical skills. in conclusion, the present study demonstrated that the use of acetaminophen, in a fixed-dose or on-demand regimen is effective in postoperative pain control after periodontal total flap surgery. since both regimens were effective, other parameters, such as patient safety and convenience, should be considered before prescribing either one or the other. funding sources this study did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. conflict of interest none. data availability datasets related to this article are available upon request to the corresponding author. author contribution c. piardi originated and designed the study, acquired data, analyzed and interpreted data, drafted the manuscript and critically revised it; c. g. vaz acquired and interpreted data and critically revised the manuscript; m. b. c. ferreira originated and designed the study, analyzed and interpreted data, and revised the manuscript for important intellectual content; d. pilger analyzed and interpreted data, and revised the manuscript for important intellectual content m. i. fernandes originated and designed 12 piardi et al. braz j oral sci. 2023;22:e238329 the study, analyzed and interpreted data, and revised the manuscript for important intellectual content the manuscript; p. weidlich originated and designed the study, analyzed and interpreted data, and critically reviewed the manuscript for important intellectual content. all authors approved the final version of the document to be published. all authors agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the manuscript were appropriately investigated and resolved. supplementary table 1. visual analog scale (vas) scores for pain evaluated 2, 6, 12, 24 and 48 hours after periodontal procedures, in the groups that received acetamoniphen prescription in fixed or on-demand schedules, based on intention-to-treat and per protocol analysis. data are expressed as median and interquartile range (p25p 75). vas 2 h vas 6 h vas 12 h vas 24 h vas 48 h intention-totreat analysis (n=68) fixed (n=34) 10.5 (1.5–30.7)a 10.0 (7.5–30.0)a 3.0 (0–18.5)ac 2.0 (0–8.2)bc 1.0 (0–4.0)b on-demand (n=34) 8.5 (0.75–27.7)ac 14.0 (1.0–2.5)a 5.5 (0.75–21)ac 2.0 (0–11.2)bc 2.0 (0–10.7)bc per protocol analysis (n=42) fixed (n=20) 12.0 (0.5–36.2)a 10.0 (0.25-2.0)a 2.0 (0–36.0)ab 2.0 (0–10.0)b 1.0 (0–4.0)b on-demand (n=22) 6.0 (0–29.75)a 11.0 (1.0–9.5)a 5.5 (0–26.75)a 2.0 (0–10.5)a 2.0 (0–7.75)a different letters represent intragroup differences, friedman and dunn’s test. supplementary table 2. frequency of individuals who used rescue medication at 6, 12, 24 and 48 hours after periodontal procedures, in the groups that received acetamoniphen prescription in fixed or on-demand schemes, based on intention-to-treat and by per protocol analysis. analysis period frequency of use of rescue medication intention-to-treat analysis (n=68) per protocol analysis (n=42) fixed group n=34 on-demand group n=34 p a fixed group n=20 on-demand group n=22 p a 6 h 14.7% 35.3% 0.17 0% 18.2% 0.13 12 h 8.8% 29.4% 0.85 0% 13.6% 0.23 24 h 5.9% 17.6% 0.31 0% 4.5% 0.33 48 h 11.7% 20.6% 0.17 5% 9.0% 0.62 a chi-square test, comparing fixed and on-demand groups using or not using rescue medication, in each period of time. references 1. graziani f, karapetra d, mardas n, leow n, donos n. surgical treatment of the residual periodontal pocket. periodontol 2000. 2018 feb;76(1):150-63. doi: 10.1111/prd.12156. 2. 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implants. int j oral maxillofac implants. 2013 mar-apr;28(2):531-5. doi: 10.11607/jomi.2713. 1http://dx.doi.org/10.20396/bjos.v20i00.8663961 volume 20 2021 e213961 original article 1 department of pediatric dentistry, school of dentistry of ribeirão preto, university of são paulo, ribeirão preto, são paulo, brazil. 2 department of pharmacology, faculty of medicine of ribeirão preto, university of são paulo, ribeirão preto, são paulo, brazil. 3 department of restorative dentistry, school of dentistry of ribeirão preto, university of são paulo, ribeirão preto, são paulo, brazil. 4 department of restorative dentistry, piracicaba dental school, university of campinas, piracicaba, são paulo, brazil. corresponding author: maria gerusa brito aragão address: department of pediatric dentistry, school of dentistry pf ribeirão preto, university of são paulo. avenida do café, s/n, cep 14040-904 email: mariagerusa@usp.br phone: + 55 88 996744119 preprint information: https://doi.org/10.1101/2020.08.2 4.20179614 research data repository: https://doi.org/10.7910/dvn/cma editor: dr altair a. del bel cury received: january 13, 2021 accepted: april 5, 2021 information sources of brazilian undergraduate dental students during the covid-19 outbreak: a cross-sectional study maria gerusa brito aragão1,* , francisco isaac fernandes gomes2 , letícia pinho maia paixão-de-melo3 , camila siqueira silva coelho4 , silmara aparecida milori corona3 aim: this cross-sectional study aimed to investigate where brazilian dental students seek information about covid-19 by a self-administered web-based questionnaire. methods: a social network campaign on instagram was raised to approach the target population. the dental students responded to a multiple-response question asking where or with whom they get information about covid-19. the possible answers were government official websites or health and education institutions websites, tv programs, professors, social media, scientific articles, health professionals, and family members. the data were analyzed by descriptive statistics, and the frequency distributions of responses were evaluated by gender, age, type of institution, and year of enrollment. results: a total of 833 valid responses were received. the main source of information used by the dental students were government official websites or health and education institutions websites, which were reported by 739 (88.7%) participants. in the sequence, 477 (57.3%) participants chose health professionals while 468 (56.2%) chose scientific articles as information sources. the use of social media was reported by 451 (54.1%) students, while tv programs were information sources used by 332 (39.9%) students. the least used information sources were professors, reported by 317 (38.1%) students, and family members, chosen only by 65 (7.8%) participants. conclusion: brazilian dental students rely on multiple information sources to stay informed about covid-19, mainly focusing their information-seeking behavior on governmental and health professional’s websites. keywords: covid-19. students, dental. information seeking behavior. social media. schools, dental. https://orcid.org/0000-0002-3334-1800 https://orcid.org/0000-0002-8843-2945 https://orcid.org/0000-0002-7661-4506 https://orcid.org/0000-0002-4315-3854 https://orcid.org/0000-0002-1733-3472 2 aragão et al. introduction covid-19 is a virus-mediated disease caused by a member of the coronavirus family sars-cov-2 originated in wuhan, hubei, china in december 20191,2. a few months after the first cases, the disease became pandemic, affecting more than 100 million people and causing over 2.5 million deaths (march 8th of 2021), according to the world health organization (who) covid-19 dashboard3. in latin american countries, brazil has risen to the spotlight of leading nations with the highest number of cases and deaths episodes due to covid-19, experiencing health system collapse and ranking number 3 globally. since the first diagnosed case in brazil, there have been more than 10 million cases confirmed along with staggering 264,000 numbers of death episodes by 8th march 20213. as the cases of covid-19 increased globally, and the pathophysiology of the disease began to be constantly studied, an avalanche of information on this issue started to be shared. by 21 august 2020, the keyword “covid-19” yielded over 42,000 and 60,000 indexed articles on pubmed and google scholar, respectively, let alone over 6,5 billion results were acquired on google search engine only in 2020. even more impressive is the number of posts on social media platforms, which spread rapidly and easily4,5. this social media content has a high potential to carry on misleading information, hindering public health policies. ultimately, it can create a global epidemic of misinformation6-9. recent reports revealed that individuals who get their news from social media are more likely to have misperceptions about covid-19, whereas those who consume more traditional news media have fewer misperceptions and are more likely to follow public health recommendations like social/physical distancing10. along with these concerning findings, the information-seeking patterns can also modulate attitudes and behaviors towards this crisis11. once the exposure to online health information has been associated with health-related behaviors in different populations and contexts12-15, understanding the information-seeking behavior of dental students and their infosphere can be a necessary step toward building efficient educational planning in the context of covid-19. these students are in direct contact with patients16,17 and can funnel information to their niches. in this sense, educational institutions have such a role in fostering and providing the academic community with scientifically-oriented and official information to battle the current wave of infodemic, so that dental students can be better prepared to tackle any related issue16. in brazil, 350 tertiary institutions are responsible for the formal dental education of up to 125,585 students across the country18. during the current university recess and social/physical distancing, the e-learning regimen has taken place while hands-on experience has been discontinued for a while, profoundly affecting up to the totality of these students. however, considering social inequalities regarding information access19,20, the overload of information regarding covid-199, and the infosphere as a behavioral modulator15, identifying the information sources of dental students about covid-19 is paramount to fight off misperceptions and misinformation among this population. moreover, knowing from where and with whom 3 aragão et al. the dental students get informed about the pandemic will facilitate the schools to reach their attention and enforce educational policies toward this end. thus, this study aimed to identify the source of information brazilian dental students use regarding covid-19. materials and methods ethical aspects this research protocol was approved by the research and ethics committee of the school of dentistry of ribeirão preto at the university of são paulo (caae: 33608320.5.0000.5419). the research was performed following the helsinki declaration. all participants signed a written consent informing that they accepted to participate in the investigation. the study consists of a cross-sectional survey directed to a sample of undergraduate dental students. all data used for this manuscript are available in portuguese at https://doi.org/10.7910/dvn/cmad57 . survey content this cross-sectional study is part of a broader investigation and the details on how the questionnaire was developed and administered have been published elsewhere21. in summary, a self-administered questionnaire about the awareness and knowledge of brazilian dental students about covid-19 and its impact on the undergraduate dental practice was hosted online (google forms). the questionnaire contained 20 mandatory close-ended items, divided into four sections: 1) demographic and academic profile (6 questions); 2) general knowledge about covid-19 (4 questions); 3) knowledge about the preventive measures to avoid covid-19 spread on the undergraduate dental practice (2 questions); 4) perceptions about the covid-19 impacts on the undergraduate dental courses (8 questions). the current work covers only the part of the 4th section in which the students responded where or with whom they usually get information about covid-19. the possible answers were government official websites or health and education institutions websites, tv programs, professors, social media, scientific articles, other health professionals, and family members. this was asked as a multiple-response question. thus, each participant could choose more than one option as their information source. recruitment and data collection according to data from the last brazilian tertiary education census18, there are 125,585 undergraduate students enrolled in dentistry courses in brazil, considering public and private institutions. all these students were eligible to participate in the research. recruitment was conducted through an instagram® (facebook, menlo park, ca) social networking campaign, which started on july 4 and lasted until july 14. data analysis the data collected were extracted from google forms and converted to excel (microsoft, usa) sheets. the frequency distribution for the source of information about covid-19 was analyzed by gender, age, and type of institution (public and private). https://doi.org/10.7910/dvn/cmad57 4 aragão et al. it was considered valid the response obtained from a questionnaire answered by a participant who signed yes to the question: “do you agree to participate in this research?”, which was provided after the presentation of the consentient form. moreover, the participants had to fill in the entire questionnaire for their answers to be considered valid. results government official websites or health and education institutions websites were the answer more frequently chosen, reported by 739 (88.7%) participants. interestingly, 477 (57.3%) students also referred to other health professionals as those with whom they seek information about the disease. a very small proportion of dental students, 65 (7.8%) in total, obtained their information from family members. more than half of the students, accounting for 468 (56.2%) respondents, used scientific articles as a source of information about covid-19, while the use of social media was reported by 461(54.1%). such a proportion overcame the one for tv programs and professors, accounting for 332 (39.9%) and 337 (38.1%) responses, respectively. we observed that 88.9% of men and 88% of women sought information twice as much in official websites than in tv programs (40.3% for male participants and 38% for female participants) or professors, who were chosen only by 37% of male respondents and 42.2% of female participants (table 1). scientific articles were more used as information sources by men than social media (59% and 50%, respectively), which was not observed for female respondents (table 1). table 2 shows that official websites were more frequently used as sources of information by those who study dentistry in private institutions (91%), while the acquisition of information from scientific articles was more frequently used by students from public dental schools (60%). moreover, comparable proportions of dental students from the public (54.2%) and private (54.1%) institutions used social media as an information source (table 2). table 1. dental students’ source of information about covid-19 by gender possible answers female male n % n % government official websites or health and education institutions websites 593 88.9 146 88.0 other health professionals 374 56.1 103 62.0 scientific articles 369 55.3 99 59.6 social media 368 55.2 83 50.0 tv programs 269 40.3 63 38.0 professors 247 37.0 70 42.2 family members 53 7.9 12 7.2 total (multiple-response question) 667   166   table 3 displays that the use of official websites increased with age, being it absolute in those older than 39 years old. on the other hand, the use of social media decreased 5 aragão et al. with age, dropping from 56.6% in the group of students younger than 25 years old to 20% in the group older than 39 years old. the use of scientific articles by students aging 25 to 32 years old was more frequent (68.6%) than by those aging between 18 to 25 years old (54.4%). likewise, the use of scientific articles doubled in the group of dental students older than 39 years (87%) in comparison to the ones aging 25 to 32 years old (43.5%). tv programs were a source of information less frequently used by students older than 32 years old, and those older than 39 years old were less likely to obtain information with their family members. table 2. dental students’ source of information about covid-19 by type of institution possible answers public private n % n % government official websites or health and education institutions websites 347 86.3 392 91.0 scientific articles 241 60.0 227 52.7 other health professionals 225 56.0 252 58.5 social media 218 54.2 233 54.1 tv programs 175 43.5 157 36.4 professors 166 41.3 151 35.0 family members 28 7.0 37 8.6 total (multiple-response question) 402   431   table 4 shows that the use of official websites increased as the year of undergraduate enrollment increased, reaching 91.9% in the group of students from the 5th year of dental school. on the other hand, the frequency of students who had professors as information sources decreased as the time of enrollment increased from the third to the fifth year of dental school. moreover, while 52.1% of last-year dental students used social media to stay informed about covid-19, only 30.8% of them saw their professors as someone with whom they could obtain information. table 3. dental students’ source of information about covid-19 by age possible answers 18 ≤ 25 25 ≤ 32 32 ≤ 39 ≥39 n % n % n % n % government official websites or health and education institutions websites 627 88.4 75 87.2 22 95.7 15 100 other health professionals 409 57.7 46 53.5 12 52.2 10 67 social media 401 56.6 38 44.2 9 39.1 3 20 scientific articles 386 54.4 59 68.6 10 43.5 13 87 tv programs 286 40.3 37 43.0 6 26.1 3 20 professors 267 37.7 33 38.4 11 47.8 6 40 family members 58 8.2 5 5.8 2 8.7 0 0 total (multipleresponse question) 709 86 23 15 6 aragão et al. table 4. dental students’ source of information about covid-19 by year of enrolment possible answers first-year second-year third-year fourth-year fifth-year n % n % n % n % n % government official websites or health and education institutions websites 89 83.2 118 88.7 153 87.4 185 89.4 194 91.9 other health professionals 60 56.1 74 55.6 98 56.0 117 56.5 128 60.7 scientific articles 56 52.3 82 61.7 97 55.4 112 54.1 121 57.3 social media 54 50.5 70 52.6 105 60.0 112 54.1 110 52.1 professors 45 42.1 60 45.1 73 41.7 74 35.7 65 30.8 tv programs 42 39.3 52 39.1 69 39.4 81 39.1 88 41.7 family members 13 12.1 9 6.8 14 8.0 13 6.3 16 7.6 total (multiple response question) 107 133 175 207 211 discussion the outbreak of covid-19 has challenged individuals, communities, and educational institutions given the need for social distancing and due to the sanitary measures imposed by the pandemic2,21. health care students and professionals have been strongly affected given the imminent risk of infection spread associated with their process of learning and working22-25. as dentists are at the top of professionals at risk22,26, so are dental students, who have been facing a hard time trying to complete their education during these uncertain times16,27-30. as it has been demonstrated, the course of infection control can be shaped by how governments enact timely policies and disseminate information11. thus, here we investigated where brazilian dental students usually seek information about covid-19. these data might aid dental schools in choosing the best platforms to display educative campaigns. as far as we know, this is the first study to investigate information-seeking behavior of brazilian dental students about covid-19. our study might be subjected to sample selection bias as we used social media to disseminate the questionnaire, allowing the participation of any dental student who had an instagram account. in this regard, we believe that given the sanitary measures imposed by the pandemics, the use of social media to disseminate web-based surveys is an alternative to aid in recruiting difficult to reach populations. besides that, the participants’ responses might also present the social desirability bias, which consists of choosing the options that are more socially accepted as right. to avoid such an effect, we asked where or with whom the students usually seek information about covid-19, making it clear in the way the question was written that there was not a right nor a wrong answer. moreover, we allowed the participants to choose more than one option as the information sources more frequently used by them. by doing that, we allowed them to express their true behavior. regarding our results, we observed that regardless of gender, age, type of institution, and year of enrollment, dental students had government official websites or health and education institution’s websites as their main source of informa7 aragão et al. tion about covid-19. thus, we stress the importance of government websites to provide information with transparency. moreover, as dental schools in brazil offer dental undergraduate courses to over 125,000 students18, knowing that they are familiarized with getting informed on official websites might reveal the students’ precaution in getting information through reliable online platforms. in this context, data of the national portals of the 193 united nations member states showed that by 8 april 2020, around 86 percent of nations (167 countries) had included information and guidance about covid-19 in their portals31. however, it has been shown that a more advanced strategy is having a dedicated portal or section about covid-1911. thus, we suggest that dental schools could display a covid-19 page on their school’s website, where they could provide information about preventive measures and on the statistics about the outbreak, focusing on the local situation of the city and campus17. such information helps people make informed decisions about their daily routines and build public trust11. contrary to what we expected, social media did not figure among the top source of information used by dental students. such findings are also contrary to what other investigations with university students have been showing32. in this regard, we decided to use instagram to recruit the participants because the use of social media in brazil increased significantly during the social distancing period33. thus, we saw instagram as a tool to spread our call for participants. instagram as a recruitment tool was a choice also based on the need to reach a large population (dental students) during a time of movement restrictions and heavy sanitary measures. as we recruited the respondents via instagram, we expected that such a tool would be more frequently reported by the students as information sources. in this sense, even not being at the top of the most used information source, more than half of the respondents used social media to get informed about the pandemics, mainly the younger ones. these findings are an alert to dental school directors to the importance of social media in disseminating information to the students, especially during the pandemic. in this scenario, it is concerning the fast and uncontrolled spread of news, which might be associated with misinformation6,7,9,11,13-15,34. moreover, social media users are more likely to believe false information. for instance10, manipulation of information with doubtful intent might be amplified through social networks, spreading farther and faster like a virus, the so-called infodemics11,34. thus, an effort should be made by dental schools to keep their social media active and updated to provide their students with trustable information, fighting fake news17. interestingly, we also observed that the respondents considered other health professionals as someone with whom they obtain information. such data represents the acknowledgment of the critical role of healthcare workers during the pandemic, as it has also been stressed by health care authorities35. surprisingly, professors were one of the least chosen sources of information of the dental students, which might be associated with the social distancing imposed by the pandemics. in this aspect, it is known that covid-19 has strongly impacted teaching and learning in dental schools, which had to move online, challenging the interaction among students and professors17,36,37. corroborating with which has been shown to other uni8 aragão et al. versity students, family members were at the bottom of the list when evaluating with whom the students get informed about covid-1932. moreover, as it has been shown elsewhere, university students seem to seek information in scientific publications38, as we observed in our sample of dental students. in this regard, according to the nature index, there have been published 67,753 scientific publications about covid-1939. as the rise in publications represents the massive effort of scientists to overcome the pandemic crisis, it also signifies that it is becoming difficult to follow all daily updates, mainly for undergraduate students, who are at the beginning of their academic life. thus, we reinforce the role of dental schools in funneling the available information, providing the students with reliable sources, and leading them to a safer return to hands-on activities. contrary to other studies with university students, the most used information sources of the brazilian dental students who participated in our research where official government and educational websites, followed by other health professionals and scientific articles. however, the use of social media was also reported by a high proportion of the respondents. therefore, we might conclude that the brazilian dental students rely on multiple information sources to stay informed about covid-19, mainly focusing their information-seeking behavior on governmental and health professional’s websites. moreover, as a final remark, we emphasize that knowing where the dental students seek information about covid-19 might facilitate dental school directors to approach such public continuously, providing them with trustable information on different platforms. once it has been shown that when individuals face risks, they seek information to reduce uncertainty, dental schools in countries such as brazil, where the epidemic is rocketing in cases and deaths, should implement strategies to keep their students updated. such precaution would provide students with the knowledge, guiding them to proper attitudes. acknowledgment we especially acknowledge all students who participated in this study, as well as those who engaged in our divulgation via social media. the first and second authors of this manuscript hold fapesp ph.d. scholarships (2020/02658-7 and 2019/14285-3, respectively). the third author holds a cnpq scientific initiation scholarship (project 2020-1004) conflicts of interest the authors declare no conflicts of interest. references 1. gralinski le, menachery vd. return of the coronavirus: 2019-ncov. viruses. 2020 jan 24;12(2):135. doi: 10.3390/v12020135. 2. wang c, horby pw, hayden fg, gao gf. a novel coronavirus outbreak of global health concern. lancet. 2020 feb ;395(10223):470-3. doi: 10.1016/s0140-6736(20)30185-9. 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among university students in jordan: a cross-sectional study. front public health. 2020 may;8:254. doi: 10.3389/fpubh.2020.00254. 1http://dx.doi.org/10.20396/bjos.v21i00.8665757 volume 21 2022 e225757 original article braz j oral sci. 2022;21:e225757 1 department of dentistry, state university of ponta grossa, ponta grossa, paraná, brazil. 2 inga university center uninga, maringá, brazil. 3 departament of prosthodontics and periodontology, piracicaba dental school, university of campinas, piracicaba, são paulo, brazil. corresponding author: alfonso sánchez-ayala department of dentistry, state university of ponta grossa avenida general carlos cavalcanti n° 4748, uvaranas, 84030-900, ponta grossa, paraná, brazil. e-mail: snzcd@uepg.br, phone: +55-42-3220-3104, fax: +55-42-3220-3102 editor: altair a. del bel cury received for publication: may 25, 2021 accepted: october 26, 2021 effect of frozen storage on preservation of a silicone-based test food material garcia dutra de castro1 , alfonso sánchez-ayala1* , giancarlo de la torre canales2 , olívia maria costa figueredo3 , mariana barbosa câmara-souza3 , camilla fraga do amaral3 , renata cunha matheus rodrigues garcia3 aim: this study aimed to evaluate the effect of frozen storage on the physical properties of a silicone-based test food material, highly used to evaluate the masticatory performance in research settings. methods: a total of 1,666 silicone cubes of optosil comfort® with 5.6-mm edges were shaped and stored at -18°c. the cubes were subsequently tested for flexural strength (maximum force, displacement, stress, and strain) before breaking (n = 136), changes in weight and size (n = 170), and masticatory performance (n = 1360) at eight timepoints: immediately after cube preparation (baseline, no freezing), and 1, 2, 3 and 4 weeks, and 2, 4 and 6 months after frozen storage. the cubes were thawed 8 h before each assessment. results: the maximum force, stress, maximum displacement, and deformation values for the cubes were not affected by freezing (p > 0.05). at all of the time points, the cubes exhibited similar weight (p = 0.366) and size (identical values). the masticatory performance for the cubes also showed no differences from baseline through 6 months (p = 0.061). conclusion: freezing optosil comfort® silicone cubes did not alter the physical and mechanical properties of the material, being suitable to optimize the assessment of masticatory parameters for research purposes. keywords: mastication. flexural strength. freezing. silicone elastomers. mailto:snzcd@uepg.br https://orcid.org/0000-0003-0237-0171 https://orcid.org/0000-0003-3426-0997 https://orcid.org/0000-0002-0921-342x https://orcid.org/0000-0001-9829-8607 https://orcid.org/0000-0002-9961-121x https://orcid.org/0000-0001-6542-8284 https://orcid.org/0000-0001-8486-3388 2 de castro et al. braz j oral sci. 2022;21:e225757 introduction a proper masticatory function is fundamental to individuals’ nutrition, being related to long-term health and quality of life. thus, studies have been developed to quantify this aspect in different population groups and to test the impact of oral rehabilitation, by using an objective masticatory performance test1. this test determines the comminuted median particle size (x50) of a test food after a certain number of masticatory cycles2. several artificial and natural materials have been used to measure the crushing and grinding capacity of teeth2. however, the traditional, reliable and validated option for evaluating masticatory performance in dentate employs a portion with 17 cubes of optosil® silicone (optosil® 1980, optosil p plus®, and optosil-comfort® versions) each with an edge length of 5.6 mm, and the total amount of ~3.4 g/3 cm3 (heraeus kulzer gmbh & co. kg, hanau, germany)3. optosil® 1980 version has traditionally been described as presenting a texture quality similar to apples, raw carrots, peanuts, chocolate, or coconut4. the current version, optosil-comfort® (optosil-c®), shows higher fracture force (18% n), degree of deformation (21% mm), and fracture work (42% n.mm) than the classic version1. these cubes have been used to evaluate the masticatory function of both dentate subjects and fixed prostheses wearers3. the weight of 17 cubes corresponds to ~7% of the mean weight of a freely chosen mouthful by a dentate subject. they also represent ~30% of a test food maximum weight which a subject can store in their mouth5. the average time needed to form optosil-c® cubes in a metallic mold and trim them is approximately 30 min. to achieve complete polymerization of the material, the cubes are then incubated in an oven at 65° for 16 h6, and an additional 24 h is needed to disinfect the cubes7. when optosil-c® cubes are used in masticatory performance tests, sieving, fractioning, and weighing of comminuted particles are usually completed within 45 min. thus, the processing of optosil-c® cubes involves a time-consuming and systematic laboratory sequence3. moreover, to guarantee dimensional stability, it is recommended that optosil® test food is used or stored within 7 days after polymerization8. to date, only one study has been reported for long-term preservation of optosil® materials, where optosil® 1980 version and its rarely-used modified versions, optoweak (by the use of another catalyst) and optosoft (by heating the silicone base), were stored in a freezer at -20 °c and did not show alterations in their properties after 26 weeks1. the physical characteristics of a test food determine the probability of the teeth grind the particle, the size reduction when a selected particle breaks, and the distribution of all food particles in mouth compartments during mastication9. therefore, the type and quality of materials used to measure the masticatory function of individuals are critical for reliable results. given the time-consuming process which is needed to prepare optosil-c® cubes for clinical research, storing these materials without affecting their comminution attributes would be of great value. thus, this study aimed to analyze the flexural strength, weight, size, and masticatory performance of optosil-c® cubes after their storage at -18 °c for up to 6 months. 3 de castro et al. braz j oral sci. 2022;21:e225757 materials and methods study design a total of 1,666 specimens of optosilc® cubes, each with an edge length of 5.6 mm, were prepared for this cross-sectional study. optosilc® comfort silicone elastomer and activator universal plus (heraeus kulzer gmbh & co. kg, hanau, germany) were manipulated according to the manufacturer’s recommendations3. optosilc® cubes were shaped and initially polymerized into steel matrices by a single calibrated examiner. to achieve complete polymerization, all of the specimens were incubated in an electrical stove (sp-400, splabor, presidente prudente, sp, brazil) at 60°c for 16 h4. after confection, the optosilc® cubes were randomly divided into experimental groups to undergo a flexural strength test (n = 136), weight and size paired comparisons (n = 170), and a clinical assessment of masticatory performance (n = 1360). specimens in each experimental group were subsequently divided into eight groups to undergo testing at specific time points: immediately after confection (baseline), and then 1-week, 2-weeks, 3-weeks, 4-weeks, 2-months, 4-months, and 6-months after freezing of the cubes at -18°c in a fe 22 freezer (electrolux, pinhais, pr, brazil). the freezer was maintained at an average temperature and humidity of -18°c and 63%8, respectively, according to a digital hygro-thermometer (cotronic technology ltd, bao’an, shenzhen, china) with precisions of ±1°c and ± 5%, respectively. during storage, specimens were kept inside closed plastic containers. before each evaluation, the cubes were removed from the fridge and thawed for 8 h at room temperature. the optosilc® cubes used for weight and size comparisons were frozen again after each evaluation. the sample size was determined by using the medcalc® (version 18.2.1, medcalc software, ostend, belgium). a type i error α=0.05 (significance) and a power of 0.8 were considered. in a pilot study, five optosilc® cubes were evaluated in two flexural strength tests which were performed with an interval of one week between the tests. the difference of means (4.02) and standard deviation for each test (3.20 and 3.49) were used in a comparison of means test. it was calculated that a minimum of 12 observations per group was needed. thus, a sample size of 17 optosilc® cubes for each of the eight timepoints was determined (n = 136 cubes). for masticatory performance, five dentate volunteers were evaluated twice with an interval of one week between the evaluations. the mean difference (0.21) and standard deviation of differences (0.14) from these data were used in a paired samples t-test. thus, a sample size of 10 portions of test food (17 optosilc® cubes each) for each timepoint was needed for a total of 1,360 cubes. this sample size was also considered for weight and size paired evaluations (10x17 = 170 cubes). flexural strength a three-point flexural test was applied to 17 optosilc® cubes at each of the eight timepoints evaluated (n = 136). the test was performed to calculate the maximum force (n), maximum displacement (mm), maximum stress (n/mm2), and maximum strain (%) before breaking the material. the assay was performed with a universal testing machine (ag-i, shimadzu, tokyo, japan) with a 10 kn-load cell capacity. the speed of 4 de castro et al. braz j oral sci. 2022;21:e225757 the probe was 30 mm/min. the cubes were placed on two parallel supporting cylindrical pins separated by 4.2 mm. a loading force was applied to the middle of cubes by using a cylindrical loading pin10. to guarantee uniform loading of the specimen and prevent friction between the specimen and the supporting pins, supporting and loading pins were mounted to achieve free rotation around the axis parallel to the pin axis, or around the axis parallel to the specimen axis (figure 1). figure 1. a: optosilc® cube placed on the universal testing machine; b-e: sequence of flexion and breakage; f: recovery of the cube after the breakage. weight and size at each of the eight timepoints evaluated, 10 portions of 17 optosilc® cubes were weighed on a 0.001-g analytical balance (mark 2060, bel engineering, monza, italy). the cubes were also sized with a sieving method by using a mesh of 5.6 mm in a shaker (bertel indústria metalúrgica ltd., caieiras, sp, brazil) for 20 min3. thus, the weight and size of the cubes were evaluated by paired comparisons at each timepoint. masticatory performance inclusion criteria for participation in the clinical phase of this study were: (1) complete healthy natural dentition, (2) mesofacial biotype, (3) convex anteroposterior profile, (4) normodivegent vertical pattern, and (5) natural normocclusion, showing angle class i molar and canine relationships, 3 mm overjet and overbite, slight dental crowding with a midline deviation < 1 mm, and mutually protected articulation. the volunteer must have also been free of any systemic disease which could interfere with motricity, and not be under any drug which would alter saliva secretion9. thus, a 37-year-old male volunteer met these criteria and was included in the study. before enrolment, the 5 de castro et al. braz j oral sci. 2022;21:e225757 research was approved by the local ethics committee (caae: 15107313.8.0000.0105), and the single volunteer read and signed a consent form, which was following the helsinki declaration and its later amendments. the masticatory performance test was performed by asking the volunteer to chew ten portions of 17 optosilc® cubes (~3.4 g / 3 cm3) in a habitual manner for 20 chewing cycles, counted by the examiner. this procedure was repeated at each of the eight timepoints evaluated, with a 5-min interval between the mastication of each additional portion of cubes3,6,11. to recover all of the material, the volunteer expelled the particles onto a paper filter placed on a beaker and rinsed the oral cavity with 200 ml water. after the water was drained through the filter, it was dried in an oven at 80°c for 25 min. the particles were then sieved through a stack of nine sieves with √2-progression mesh sizes (8.0–0.5 mm) in a shaker (bertel indústria metalúrgica ltd., caieiras, sp, brazil). after 20 min, the particles retained in each sieve were weighed on a 0.001-g analytical balance (mark 2060, bel engineering, monza, italy). masticatory performance was calculated according to rosin-rammler equation: qw – (x)=1–2– (x/x50 )b, with qw – representing the cumulative weight percentage of the particles smaller than x (certain sieve aperture), x50 representing the aperture of a theoretical sieve through which 50% of the weight can pass, and b representing a broadness of size particle distribution. then, it was verified if comminution was altered after each freezing timepoint3,6. statistical analysis data were examined with prism (version 7; graphpad software, inc., ca, usa). all inferences were based on two-tailed tests performed with a significance level of 95% and power of 80%. parametric assumptions were discarded according to the d’agostino-pearson normality test, bartlett’s and brown-forsythe tests for homogeneity of variances, and mauchly’s test of sphericity. the results for maximum displacement and strain were compared by using one-way analysis of variance (anova) on ranks and dunn’s multiple comparisons tests, respectively. maximum force and stress data were compared by employing one-way anova and tukey post hoc tests. meanwhile, friedman and dunn’s multiple comparisons tests were applied to comparisons of weight and masticatory performance paired values. when non-significant p-values were close to the level of significance (p = 0.05), the observed power was calculated by using the spss statistics® software (v. 25, ibm corporation, amonk, ny, usa) with a 95% confidence level. results flexural strength data for the optosilc® cubes are presented in table 1. outcomes related to resistance, as maximum force and stress achieved until breaking, presented no significant differences over time (p = 0.071 and p = 0.069, respectively). regarding cube deformation during flexion, displacement and strain values were statistically significant for anova on ranks test (p = 0.030 and p = 0.030, respectively), but the posthoc analyses did not identify significant differences among time points (p > 0.05). 6 de castro et al. braz j oral sci. 2022;21:e225757 table 1. mean (sd) from the flexural strength test results (n = 17 optosil® cubes). follow-up variables * force (n)† displacement (mm)‡ stress (n/mm2)† strain (%)‡ baseline 63.30 ± 6.38 a 3.32 ± 0.45 a 2.27 ± 0.23 a 63.24 ± 8.62 a one week 63.06 ± 8.96 a 3.18 ± 0.52 a 2.26 ± 0.32 a 60.63 ± 9.99 a two weeks 58.97 ± 4.97 a 3.36 ± 0.24 a 2.12 ± 0.18 a 64.02 ± 4.49 a three weeks 58.83 ± 5.89 a 3.47 ± 0.32 a 2.11 ± 0.21 a 66.14 ± 6.01 a four weeks 61.19 ± 4.66 a 3.49 ± 0.22 a 2.20 ± 0.17 a 66.42 ± 4.11 a two months 63.09 ± 4.50 a 3.55 ± 0.24 a 2.26 ± 0.16 a 67.67 ± 4.58 a four months 58.91 ± 5.70 a 3.43 ± 0.32 a 2.11 ± 0.20 a 65.28 ± 6.19 a six months 61.10 ± 4.00 a 3.51 ± 0.22 a 2.19 ± 0.14 a 66.80 ± 4.20 a †one-way anova and tukey’s post hoc tests (α = 0.05); ‡one-way anova on ranks and dunn’s multiple comparisons tests (α = 0.05); * power observed (1-β error probability): force = 0.741, displacement = 0.804, stress = 0.744, strain = 0.802; different letters indicate statistically significant differences among time points. the weights of the optosilc® cubes (range: 3.388–3.391 g) remained unchanged (p = 0.366) between the baseline and all time points. furthermore, the cubes preserved their original dimensions and were retained in a 5.6-mm sieve. there were no differences among the x50 mean values obtained at the eight timepoints examined (p = 0.061). similarly, no differences in the broadness of particle size distribution were observed (p = 0.054) (table 2). table 2. mean (sd) from the masticatory performance results (n = 10 portions 17 optosil® cubes). follow-up time variables * x50 (mm) b baseline 3.10 ± 0.14 a 3.38 ± 0.25 a one week 3.11 ± 0.13 a 3.20 ± 0.29 a two weeks 3.15 ± 0.22 a 2.98 ± 0.32 a three weeks 3.12 ± 0.25 a 2.97 ± 0.44 a four weeks 2.97 ± 0.33 a 3.07 ± 0.50 a two months 3.06 ± 0.13 a 3.29 ± 0.24 a four months 3.03 ± 0.09 a 3.11 ± 0.35 a six months 2.86 ± 0.16 a 3.09 ± 0.34 a friedman and dunn’s multiple comparisons tests (α = 0.05); * power observed (1-β error probability): x50 = 0.801, e b = 0.652; different letters indicate statistically significant differences among time points. discussion the results of this study demonstrate the stability of optosilc® cubes after being stored at -18°c for up to six months. this long-term behavior may be explained by the chemical and physical characteristics of silicone material. polydimethylsiloxanes are organosilicon polymers that are composed of a si–o– linked backbone, repeating units of –[(ch3)2si–o–]n, and silanol end groups 12. as a result, silicone exhibits ther7 de castro et al. braz j oral sci. 2022;21:e225757 mal stability, minimal temperature effects, and low-temperature performance13. this molecular configuration also provides excellent release features and surface activity, antifriction and lubricity, good damping behavior, shear stability, hydrophobic and physiological inertness, and weak intermolecular forces12,13. it is hypothesized that continuous degradation of optosilc® is inhibited or at least retarded by storage at -18°c, and partly by complementary polymerization which occurs over 16 h at 60°c during the preparation of optosilc® cubes. however, there is no scientific proof to support such hypotheses. only one research indirectly detected a slight influence of freezing at -18°c on elastomer stability through impressions of a single steel gauge block to cast stone dies after 24h14. our laboratory findings from a 3-pint flexural strength test simulating dental occlusion also show that similar maximum force and maximum stress were needed to break the optosilc® cubes at each timepoint. the low-temperature performance of silicone is due to its highly stable chemical structure. optosilc® is composed of a polymeric matrix of polydimethylsiloxane and a 24.74% volumetric fraction of inorganic particles measuring 11.66 μm [e.g., zn (6.39%), mg (15.30%), si (72.89%), and na (5.42%)]12. moreover, the catalyst paste contains alkylsilicate and a tinbased activator (stannous octoate). a condensation reaction is driven by crosslinking (van der waals forces) between the hydroxyl groups (from silanol ends) and the alkyl, which produces alcohol as a byproduct12,15. silicone is then polymerized by irreversible formation of a three-dimensional network which prevents the silicone chains from sliding over each other, while still maintaining the flexibility of the material15. the u.s. food and drug administration recommends -18°c as an ideal temperature for food preservation (https://www.fda.gov/consumers/consumer-updates). the thermal behavior of optosilc® cubes allows them to be stored at this temperature without alteration of their properties. moreover, lower temperatures slow down the natural degradation of the polymer by decreasing the activation energy for these reactions and also slowing the propagation of microorganisms. however, no uniform reduction in reaction rate has been observed as the temperature is lowered, although there is a certain extent of adherence to van’t-hoff’s rule (the velocity of a chemical reaction increases two-fold or more for each 10°c increase in temperature)13. the latter is generally true when a temperature approximates that at which a reaction normally occurs. the combination of very high siloxane chain flexibility and very few methyl/methyl interchain interactions produces polydimethylsiloxanes which have extremely low glass (-123.15°c) and low melting (-45.15 – -41.15°c and -37.15°c) transitions16,17. the freezing point of polydimethylsiloxanes may also play a relevant role in defining the low-temperature use limit of this material because its mechanical properties undergo changes that are similar to a harder rubber within just a few degrees of temperature change. the freezing temperature for a material is highly dependent on the rate of cooling of that material. for example, a rapid cooling rate of 10°c per minute can result in a freezing temperature between -70°c and -80°c. in contrast, a slower cooling rate of 1°c per minute can result in a freezing temperature between -60°c and -65°c18. the freezing temperature for the optosil® https://www.fda.gov/consumers/consumer-updates 8 de castro et al. braz j oral sci. 2022;21:e225757 material in the present study was far from optosil®’s critical temperature. besides, the hydrophobic nature of optosil® (even at -18°c)19, may have prevented it from absorbing or adsorbing water. regarding the weight and size of the optosilc® cubes tested, no differences were observed among all time points. these results are consistent with the long-term behavior of silicone materials. when silicone is stored at room temperature, its dimensional stability depends directly on its properties of elastic recovery, polymerization shrinkage, and evaporation of volatile components from the material15,20. all condensation silicones exhibit a slight volume reduction due to cross-linking, bond rearrangements in their polymer chains, and alcohol evaporation15. moreover, as alcohol is produced, silicone material is distorted as it is released12. the mechanical properties of silicone have been improved with low polydispersity, long molecular chains between crosslinking points, and a faultless network with fewer dangling ends20,21. after 48 h at room temperature, polymer degradation is accentuated, thereby resulting in increased shear and greater young’s moduli and dynamic viscosity22. degradation of polydimethylsiloxanes has been found to depend on the physical magnitude to be evaluated, failure time, temperature (e.g., at the lowest temperature, at room temperature, or highest temperature), apparent activation energy, and gas constancy. when polydimethylsiloxanes undergo artificial aging (thermal), the polymer network and the chemical structure of the backbone will be altered significantly. the degradation course mainly involves depolymerization and chain scission reactions which lead to cleavage of the main chain and the production of dangling ends (cyclic oligomeric siloxanes, higher oligomeric siloxane residues, and a smaller proportion of other components)20,23. overall, degradation can lead to “backbiting” of hydroxyl-terminated polydimethylsiloxanes and intramolecular depolymerization of end blocking polymers20. meanwhile, clinical evaluations of masticatory performance support the hypothesis that optosilc® cubes can be frozen since no significant differences were found. this result is supported by the mechanical properties of this material, which did not alter over time. the single volunteer in the present study acted as a “chewing device”9. considering that the masticatory process involves muscle activity to generate mandibular movements and exert bite forces for food comminution by teeth1, standardizing the masticatory performance test by one volunteer reduces possible bias related to muscle strength. although the variables flexural strength and broadness of particle size distribution (b) showed a significance level near 0.05, the power of the tests presented appropriate values (1-β). in general, an 80% power or higher is considered statistically powerful. in the present study, the power achieved was around 75%, which could be considered enough to avoid a type ii error. it is important to emphasize that the present study combined in vitro and in vivo assessment of mastication. the loading geometry for optosilc® cubes can be explained by a crack in the surface, which may start adjacent to a cusp tip as the particle is indented, or more remotely from cusps through bending of the cubes 9 de castro et al. braz j oral sci. 2022;21:e225757 against a threeor greater-point cuspal support9. thus, an eventual cusp-fossa or cusp-embrasure occlusion can be simulated by the in vitro flexural strength test and the masticatory performance test allowed an in vivo evaluation, evidencing the reproducibility of the present results. studies have demonstrated that the test food portion may change its presentation by decreasing the number and size of cubes (two half cubes of 9.6x9.6x4.8mm)24, to avoid a functional high-test load (number of chewing cycles and bite strength) and increase the comminution degree25. however, cubes with a 5.6 mm edge are still the most used method and the gold standard in masticatory research in dentistry1,26. it is worthy to highlight that, despite our attempt to standardize all steps of this study, it would have been relevant to have control groups without freezing to investigate cubes’ degradation over time. thus, it could be considered a limitation of this study and considered in future researches. in conclusion, the storage of optosilc® cubes at -18°c did not modify its physical and mechanical properties. thus, freezing this silicone-based test food material may reduce time-consuming laboratory processes during clinical research. acknowledgments we acknowledge the coordenação de aperfeiçoamento de pessoal de nível superior brasil (capes) — finance code 001 for graduate program support and the scholarships granted; and the são paulo research foundation fapesp, brazil for the post-doctoral scholarship to gdltc (2017/21674-0) and the phd scholarship granted to mbcs (2017/23429-3). data availability datasets related to this article will be available upon request to the corresponding author. conflict of interest none. please add, before references: author contributions this research is part of the undergraduate coursework of garcia dutra de castro, who made most of the experimental methodology, obtained the data, and contributed with paper drafting. olívia maria costa de figueredo, mariana barbosa câmara de souza and camilla fraga do amaral significantly contributed to execution of the methodology and paper draft. giancarlo de la torre canales contributed with interpretation of the results, manuscript writing, and revision. alfonso sánchez-ayala and renata cunha matheus rodrigues garcia conceived the idea for the study and study design. these authors were also responsible for the main data interpretation and manuscript writing. all authors have actively participated in the discussion of the manuscript’s findings and have revised and approved the final version of the manuscript. 10 de castro et al. braz j oral sci. 2022;21:e225757 references 1. van der 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10.1016/j.polymdegradstab.2009.04.030. 24. van der glas hw, liu t, zhang y, wang x, chen j. optimizing a determination of chewing efficiency using a solid test food. j texture stud. 2020 feb;51(1):169-84. doi: 10.1111/jtxs.12477. 25. liu t, wang x, chen j, van der glas hw. determining chewing efficiency using a solid test food and considering all phases of mastication. arch oral biol. 2018 jul;91:63-77. doi: 10.1016/j.archoralbio.2018.04.002. 26. elgestad stjernfeldt p, sjögren p, wårdh i, boström am. systematic review of measurement properties of methods for objectively assessing masticatory performance. clin exp dent res. 2019 jan 31;5(1):76-104. doi: 10.1002/cre2.154. 1 volume 22 2023 e237812 original article braz j oral sci. 2023;22:e237812http://dx.doi.org/10.20396/bjos.v22i00.8667812 1 graduate program teaching in health, federal university of rio grande do sul, porto alegre, rs, brazil. 2 department of dentistry and oral health, section for periodontology, aarhus university, aarhus, denmark. 3 department of public health, graduate program teaching in health, federal university of rio grande do sul, porto alegre, rs, brazil. 4 department of dentistry of state university of ponta grossa. ponta grossa, pr, brazil. 5 department of public health, federal university of santa catarina, florianópolis, sc, brazil. 6 professor and head of graduate program teaching in health, federal university of rio grande do sul, porto alegre, rs, brazil. corresponding author: fernando valentim bitencourt fvbitencourt@dent.au.dk (+45 25770902) department of dentistry and oral health, section for periodontology aarhus university vennelyst boulevard 9, building 1610, office 2.76 8000 aarhus c, denmark editor: altair a. del bel cury received: december 07, 2021 accepted: july 11, 2022 how brazilian oral health care workers face covid-19: surveillance, biosafety, and education strategies luciana zambillo palma1 , fernando valentim bitencourt2,* , gabriel ricardo velho1 , fabiana schneider pires3 , márcia helena baldani4 , claudia flemming colussi5 , cristine maria warmling6 aim: to investigate surveillance, biosafety, and education strategies of brazilian oral health care workers (ohcws) during the first wave of the covid-19 outbreak. methods: this was a cross-sectional study covering ohcws from a single multicenter research centre. a self-administered and validated online questionnaire was used for data collection, including the following variables: sociodemographic, medical history, biosafety, professional experience, surveillance, and education. results: the sample consisted of 644 ohcws (82.5% dentists, 13.2% dental assistants and 4.3% technicians), most without comorbidities (84.8%), from the public (51.7%) and private (48.3%) health systems, in 140 cities of a southern state. the most prominent measures of surveillance were waiting room distancing and visual alerts, symptom assessment, and availability of guidelines on covid-19. regarding biosafety measures, the lowest adherence was related to intraoral radiographs (2.7±1.4; 95%ci: 2.6–2.9), use of dental dams (2.1±1.4; 95%ci: 2.0–2.2), and availability of high-power suction systems (2.5±1.7; 95%ci: 2.3–2.6). among ohcws, 52.6% received guidance on measures to take during dental care in the workplace. continuing education was mainly through documents from non-governmental health authorities (77.4%). conclusion: surveillance and biosafety measures were adopted, but activities that reduce the spread of aerosols had less adherence. these findings underscore the importance of considering dental practices, and surveillance and education strategies to formulate policies and relevant support to address health system challenges during the covid-19 pandemic. a coordinated action of permanent education by policymakers is necessary. keywords: sars-cov-2. covid-19. health workforce. education, dental. public health dentistry. https://orcid.org/0000-0003-1187-0784 https://orcid.org/0000-0002-7310-2767 https://orcid.org/0000-0002-4155-6801 https://orcid.org/0000-0001-6545-524x https://orcid.org/0000-0003-1310-6771 https://orcid.org/0000-0002-3395-9125 https://orcid.org/0000-0003-2259-4199 2 palma et al. braz j oral sci. 2023;22:e237812 introduction with the worldwide spread of the covid-19 pandemic caused by the sars-cov-2 virus, the policies adopted by brazil to contain the disease have had no positive effect. the mismanagement of the public health system, the delay in vaccine acquisition, and the lack of tests, combined with political instability, worsened the country’s handling of covid-19. brazil was an epicenter of the spread of covid-19, considered the third most-affected country globally, with excessive cases and deaths1. in this challenging scenario for health systems, oral health care workers (ohcws) in particular have been part of the frontline struggle against covid-19. dental care requires proximity to the patient, and it demands procedures that generate aerosols containing saliva, oral fluids, and blood. with the increase of covid-19 cases combined with the high risk of transmission of sars-cov-2 and the intensification of healthcare work, especially in dental emergencies, strict biosecurity and infection control measures were recommended2,3. given the need to maintain dental care, brazil adopted restrictive measures and actions to prevent the spread of the virus. the ministry of health (ms) and the federal council of dentistry (cfo) published guidelines with specific recommendations for clinical dental management to be followed by ohcws. among them, dental care was restricted to urgencies and emergencies with subsequent release of elective procedures. covid-19 symptom investigation during anamnesis, education in the waiting room, appropriate use of personal protective equipment (ppe), and reduction of aerosols to prevent the spread of the virus were emphasized4,5. the bleak context for brazilian public health and particularly dental care, aggravated by the absence of a national emergency plan, prompted collaboration and research networks to face the challenges of covid-19. this investigation is part of a broader multicenter study, carried out in three states in the southern region of brazil between four universities, the cfo, and the brazilian dental education association (abeno). thus, the study aimed to evaluate the surveillance, biosafety, and education strategies of brazilian ohcws in the context of the first wave of the covid-19 pandemic. materials and methods study design and population this study was descriptive and cross-sectional design using data on surveillance and biosafety measures and access to education activities among ohcws (dentists, dental assistants, and technicians) from the public and private sectors. the study population corresponds to one of the states obtained from the multicenter research encompassing three states of the south of brazil.   this investigation was conducted between august 2020 and october 2020. ethical approval for data collection in the state was obtained from the research ethics committee, the federal university of rio grande do sul, porto alegre, brazil (caae 3 palma et al. braz j oral sci. 2023;22:e237812 no. 31720920.5.2002.5530). all participants provided written informed consent. the study followed the strengthening the reporting of observational studies in epidemiology (strobe) guidelines6. selection of participants and eligibility criteria participants were identified through the cfo registration of professionals at june 2020 in the state of rio grande do sul. this record contained a total of 30,588 ohcws. they were invited to participate by email to the address provided by the cfo. to maximize participation, before recruiting participants through email, instagram social networking campaigns targeting ohwcs were held to promote this research. informed consent for participation in the study was incorporated. after the first invitation, the response to the form was monitored. two more invitations were made within 15 days. data collection a structured, self-administered, unidentified, and validated questionnaire was applied via email using a google forms® (appendix 1). briefly, the instrument was submitted to eight experts with experience from the public health, biosafety, and education for evaluation to verify its performance and reliability. after modifications requested by the experts, the questionnaire was evaluated through a pilot study with 52 ohcws in the three professional categories. this step aimed to ascertain the degree of understanding concerning the questions that were developed and the reproducibility of the instrument. the agreements obtained in the test retest ranged from 84% to 100%. the time to complete the instrument was approximately 20 minutes. it was composed of three thematic axes: (1) sociodemographic characteristics; (2) surveillance and biosafety measures; and (3) professional experience, management, education, work, and staff. the questionnaire had 47 closed questions with answer options on a 5-point likert scale (1: never, 2: rarely, 3: sometimes, 4: almost always, 5: always).  data analysis the data compilation, organization, and codification were performed using microsoft excel tm (microsoft corp., seattle, usa). data were subsequently analyzed for inconsistencies and incomplete data. missing data were excluded from the study. the statistical analysis was performed using ibm spss version 20.0 (ibm corp, armonk, usa). absolute and percentage frequencies were measured for categorical variables and means (± standard deviations) for likert scale scores. proportions and confidence intervals for the study population were estimated.  results the study included 644 ohcws in 140 cities of the state of rio grande do sul. the sample was composed of 82.5% dentists, 13.2% dental assistants and 4.3% technicians, being 73.8% women. the length of professional experience was well-distributed 4 palma et al. braz j oral sci. 2023;22:e237812 in the sample, with the highest rates in the groups over 20 years (29.5%) and between 6 and 10 years (22.4%). concerning the professional category of the participating dentists, 47.5% were specialists, 19.1% of whom were in public health. of the participants, 51.7% worked in the public sector, 46.5% worked in primary health care, and private clinics represented 36.6%. a total of 68.5% reported an absence from work in the first wave of the covid-19 outbreak. regarding health aspects, 84.8% of the participants reported no risk factors or conditions. testing for covid-19 was not performed by 43.0% (table 1). table 1. sociodemographic, education, work, and health characteristics of the sample of oral health care workers from the rio grande do sul, brazil, august-october, 2020. variables n % ci 95% gender female 475 73.8 70.3 – 77.1 male 169 26.2 22.9 – 29.7 age (years) 18-24 24 3.7 2.4 – 5.4 25-39 307 47.7 43.8 – 51.5 40-59 277 43.0 39.2 – 46.9 >60 36 5.6 4.0 – 7.5 occupation dentists 531 82.5 79.4 – 85.3 dental assistant 85 13.2 10.7 – 16.0 technicians 28 4.3 3.0 – 6.1 conclusion of professional training (years) up to 5 131 20.3 17.4 – 23.6 6-10 144 22.4 19.3 – 25.7 11-15 95 14.8 12.2 – 17.6 16-20 84 13.0 10.6 – 15.8 >20 190 29.5 26.1 – 33.1 higher graduate level* specialization/residency 306 47.5 43.7 – 51.4 master 87 13.5 11.0 – 16.3 phd 57 8.9 6.8 – 11.2 none 194 30.1 26.7 – 33.7 postgraduate areas# public health 123 19.1 16.8 – 20.5 clinical specialties# 327 69.9 67.2 – 72.5 none 194 11.0 9.8 – 14.2 continue 5 palma et al. braz j oral sci. 2023;22:e237812 continuation workplace sus¶ – primary health care (phc) 300 46.5 44.3 – 48.4 sus¶ dental specialty centers (dsc) 32 5.0 3.9 – 7.4 sus¶ – urgency care 1 0.2 0.1 – 0.4 private clinic 236 36.6 32.8 – 40.5 dental teaching clinic 43 6.7 3.8 – 8.1 corporate entities health system 11 1.7 0.7 – 2.6 security forces (army, police, etc.) 4 0.6 0.3 – 0.8 hospital 6 1.0 0.6 – 1.7 management 4 0.6 0.3 – 0.8 other 7 1.1 0.8 – 1.5 risk factors for severe forms of covid-19 only age over 60 years old 25 3.8 3.0 – 4.5 health condition only 62 9.7 6.8 – 11.2 age over 60 and health condition 11 1.7 1.4 – 2.1 none 546 84.8 80.6 – 87.1 absence from work during the pandemic yes 441 68.5 65.4 – 71.4 no 203 31.5 28.0 – 35.2 testing for covid-19 no 277 43.0 39.2 – 46.9 yes rt pcr 153 23.8 20.6 – 27.1 rapid test 173 26.9 23.5 – 30.4 serological test 41 6.4 4.7 – 8.4 * only dentists included. # most cited areas of dentistry: orthodontics, implantology, dental prosthesis, endodontics, periodontics, pediatric dentistry and dentistry. ¶ brazilian national health system. the covid-19 preventive practices with the highest average responses were the availability of guidelines (3.9±1.2) and visual alerts in the office (3.9±1.4), investigation of possible respiratory symptoms (4.1±1.3), and adoption of distancing in the waiting room (4.2±1.1). however, lower averages were registered for questions relating to innovative practices in dental care: working directly in covid-19 fast-tracking procedures (2.2±1.4) or the use of tools for telemonitoring of patients (2.4±1.6; table 2). 6 palma et al. braz j oral sci. 2023;22:e237812 table 2. sample distribution regarding the adoption of surveillance, planning and risk management measures to control the dissemination of covid-19 in health services. oral health care workers from the rio grande do sul, brazil, august-october, 2020. organization of health services (surveillance, planning and management) always (score 5) often (score 4) sometimes (score 3) ever (score 2) never (score 1) do not know mean (dp) ci 95% n (%) n (%) n (%) n (%) n (%) n (%) suspended elective procedures and care restricted to urgency/emergency 213 (33.1) 198 (30.7) 124 (19.3) 64 (9.9) 42 (6.5) 3 (0.5) 3.7 (1.2) 3.6 – 3.8 participation in decision-making about changes in work during the pandemic 238 (37.0) 69 (10.7) 96 (14.9) 59 (9.2) 178 (27.6) 4 (0.6) 3.1 (1.6) 3.0 – 3.3 reduced workload or professional turnover to minimize the risk of contamination 165 (25.6) 74 (11.5) 100 (15.5) 63 (9.8) 236 (36.6) 6 (0.9) 2.7 (1.6) 2.6 – 2.9 worked directly in covid-19 reception/ sorting/fast track procedures 89 (13.8) 53 (8.2) 89 (13.8) 84 (13.0) 325 (50.5) 4 (0.6) 2.2 (1.4) 2.0 – 2.3 investigation of respiratory infection symptoms in appointment scheduling 394 (61.2) 98 (15.2) 60 (9.3) 39 (6.1) 32 (5.0) 21 (3.3) 4.1 (1.3) 4.0 – 4.2 patients with symptoms of respiratory tract infection immediately isolated 384 (59.6) 84 (13.0) 40 (6.2) 29 (4.5) 65 (10.1) 42 (6.5) 3.8 (1.6) 3.7 – 4.0 waiting room respecting the minimum distance of 01 meter between people 386 (59.9) 125 (19.4) 72 (11.2) 34 (5.3) 16 (2.5) 11 (1.7) 4.2 (1.1) 4.1 – 4.3 availability of visual alerts in the health service 346 (53.7) 110 (17.1) 61 (9.5) 47 (7.3) 63 (9.8) 17 (2.6) 3.9 (1.4) 3.7 – 4.0 urgency based on pre-established clinical protocols 318 (49.4) 141 (21.9) 73 (11.3) 29 (4.5) 58 (9.0) 25 (3.9) 3.8 (1.4) 3.7 – 3.9 orientation of patients about covid-19 303 (47.0) 135 (21.0) 125 (19.4) 42 (6.5) 30 (4.7) 9 (1.4) 3.9 (1.2) 3.8 – 4.0 use of digital tools for teleorientation or telemonitoring 121 (18.8) 62 (9.6) 102 (15.8) 81 (12.6) 255 (39.6) 23 (3.6) 2.4 (1.6) 2.3 – 2.5 interaction with other health professionals 221 (34.3) 153 (23.8) 157 (24.4) 67 (10.4) 44 (6.8) 2 (0.3) 3.6 (1.2) 3.5 – 3.7 regarding the adoption of biosafety measures by ohcws, the highest averages were related to routine care with ppe and decontamination of environments: disinfection of the face shield (4.7±0.9), proper removal of personal barrier protection (3.9±1.3), reuse of n95/pff2 masks following appropriate criteria (3.8±1.5), and disinfection of environments (3.8±1.4). lower averages were identified in practices to minimize the generation of aerosols and oral secretions: avoiding intraoral radio7 palma et al. braz j oral sci. 2023;22:e237812 graphs (2.7±1.4), use of dental dams in high-speed care (2.1±1.4) and availability of high-power suction systems (2.5±1.7; table 3). table 3. sample distribution regarding the adoption of biosafety measures in health services. oral health care workers from the rio grande do sul, brazil, august-october, 2020. work biosafety always (score 5) often (score 4) sometimes (score 3) ever (score 2) never (score 1) do not know mean (dp) ci 95% n (%) n (%) n (%) n (%) n (%) n (%) disinfection of the environment by a trained professional with appropriate ppe 322 (50.0) 118 (18.3) 69 (10.7) 45 (7.0) 82 (12.7) 8 (1.2) 3.8 (1.4) 3.7 – 3.9 disinfection of suction hoses 272 (42.2) 86 (13.4) 89 (13.8) 56 (8.7) 105 (16.3) 36 (5.6) 3.4 (1.7) 3.2 – 3.5 use of sterile micromotors at every dental appointment 269 (41.8) 62 (9.6) 67 (10.4) 80 (12.4) 152 (23.6) 14 (2.2) 3.2 (1.7) 3.1 – 3.4 intraoral radiographic examinations were avoided 75 (11.6) 159 (24.7) 160 (24.8) 75 (11.6) 156 (24.2) 19 (3.0) 2.7 (1.4) 2.6 – 2.9 performing fourhanded dental procedures 165 (25.6) 114 (17.7) 110 (17.1) 103 (16.0) 137 (21.3) 15 (2.3) 3.0 (1.5) 2.9 – 3.1 use of the dental dam in high rotation services 68 (10.6) 77 (12.0) 98 (15.2) 84 (13.0) 278 (43.2) 39 (6.1) 2.1 (1.4) 2.0 – 2.2 procedures that generate aerosols were avoided 135 (21.0) 176 (27.3) 135 (21.0) 86 (13.4) 98 (15.2) 14 (2.2) 3.1 (1.4) 3.0 – 3.3 use of suction system (vacuum pump) 176 (27.3) 51 (7.9) 53 (8.2) 31 (4.8) 309 (48.0) 24 (3.7) 2.5 (1.7) 2.3 – 2.6 proper removal of personal barrier protection 317 (49.2) 167 (25.9) 61 (9.5) 34 (5.3) 50 (7.8) 15 (2.3) 3.9 (1.3) 3.8 – 4.0 n95/pff2 mask reuse with proper criteria 357 (55,4) 101 (15,7) 66 (10,2) 24 (3,7) 70 (10,9) 26 (4,0) 3,8 (1,5) 3.7 – 4.0 disinfection of face shield 569 (88,4) 31 (4,8) 12 (1,9) 13 (2,0) 8 (1,2) 11 (1,7) 4,7 (0,9) 4.6 – 4.7 table 4 shows how the participants accessed technical standards and recommendations on dental care during the covid-19 pandemic. of the ohcws, 77.4% searched for documents without identifying the agency responsible for the information accessed. the responses related to accessing official recommendations showed similar scores: 58.8% accessed cro recommendations, and 58.0% accessed the technical note no. 04/2020 anvisa. 8 palma et al. braz j oral sci. 2023;22:e237812 table 4. aspects related to access to technical standards and recommendations on dental care during covid-19 pandemic. oral health care workers from the rio grande do sul, brazil, august-october, 2020. variables total dentists dental assistants technicians n (%) n (%) n (%) n (%) access to technical standards and recommendations technical note gvims/ggtes/anvisa nº 04/2020 374 (58.0) 324 (50.3) 40 (6.2) 10 (1.5) recommendations booklet of the federal council of dentistry (cfo) 377 (58.5) 334 (51.8) 33 (5.1) 10 (1.5) recommendations booklet of the regional council of dentistry (cro) from own state 361 (56.0) 303 (47.0) 44 (6.8) 14 (2.1) recommendations booklet of the regional council of dentistry (cro) from other state 92 (14.2) 84 (13.0) 6 (0.9) 2 (0.3) recommendations from the municipal/ state secretariat 341 (52.9) 270 (41.9) 51 (7.9) 20 (3.1) none 33 (5.1) 29 (4.5) 4 (0.6) 0 (0.0) other documents * 499 (77.4) 405 (62.8) 70 (10.8) 24 (3.7) * any source of information without identification of the agency responsible for the information accessed. the results related to continuing education show that 52.6% of the participants received guidance on measures to be taken during dental care in the workplace. however, 22.2% reported not having applied the acquired information, with no changes in dental practices. clarity and security to work correctly in the pandemic were positive, with 41.3% of participants partially agreeing and 39.3% fully agreeing. however, 33.4% felt anxious or worried about working properly during the pandemic (table 5). table 5. sample distribution regarding training/education during covid-19 pandemic. oral health professionals from the rio grande do sul, brazil, august-october, 2020. training on covid-19 strongly agree (score 5) agree (score 4) undecided (score 3) disagree (score 2) strongly disagree (score 1) do not know mean (dp) ci 95% n (%) n (%) n (%) n (%) n (%) n (%) i consider that i received guidance at my workplace regarding measures to be taken during the covid-19 pandemic 339 (52.6) 177 (27.5) 43 (6.7) 39 (6.1) 42 (6.5) 4 (0.6) 4.1 (1.2) 4.0 – 4.2 i was able to apply the knowledge acquired in training/education about covid-19 to modify my practice 255 (39.6) 180 (28.0) 35 (5.4) 17 (2.6) 14 (2.2) 143 (22.2) 4.6 (1.1) 4.5 – 4.6 continue 9 palma et al. braz j oral sci. 2023;22:e237812 continuation i feel sufficiently enlightened and secure to work properly in dental practice during the covid-19 pandemic 253 (39.3) 266 (41.3) 42 (6.5) 51 (7.9) 27 (4.2) 5 (0.8) 4.0 (1.1) 3.9 – 4.1 i feel anxious and concerned to work properly in dental practice during the covid-19 pandemic 215 (33.4) 208 (32.3) 57 (8.9) 72 (11.2) 88 (13.7) 3 (0.5) 2.3 (1.4) 2.2 – 2.4 discussion this study emphasizes the surveillance, biosafety and education strategies by ohcws during the first wave of the covid-19 outbreak in the south of brazil. although the pandemic’s effects on dentistry in brazil have been discussed7-9, scarce information exists regarding actions to respond to the challenges facing ohwcs. the pandemic has amplified the need for instituting biosafety processes and actions and professional updating in the area. the context of the high risk of contagion faced by health professionals is one of the vulnerabilities of health systems. in addition to human risks, the decrease in front-line workers can compromise the potential response of health services. our findings demonstrate the adherence of ohcws to covid-19 procedures, guidelines, and surveillance, especially for activities close to the dental office such as screening and fast-tracking a rapid-flow tool for triage and care of covid-19 cases. the results are consistent with studies indicating that dentists know about methods to investigate patients10 with suspected covid-19 and inform the population about widespread disease issues11,12. adherence to fast-tracking of ohcws working in the sus was low. this performance may have influenced oral health policies at the time of the pandemic, which induced a financing model based on the productivity of specific indicators for dentistry, that do not include activities such as fast-tracking13. the restrictions imposed by the pandemic impacted the offer of dental treatments, and dentistry mediated by remote technologies emerged as a possibility, but with controversies, especially in the regulation of this professional practice. therefore, during the pandemic, a resolution has regulated the types of use of teledentistry: teleorientation (guidance by digital means or telephone) and telemonitoring (verification of health issues and clinical developments), prohibiting its use for consultation, diagnosis, prescription, or preparation of a treatment plan14. in the present study, ohcws demonstrated moderation in the use of digital tools in daily dental work. obstacles to the use of teledentistry are related to the conservatism of managers, clinical acceptance (willingness by professionals to use telehealth tools)15, the perception of its benefits by professionals, and demanding technological and personnel resources16. with teledentistry, the workflow and the 10 palma et al. braz j oral sci. 2023;22:e237812 participation of the patient can be streamlined in more personalized and accessible care7,17. in the resumption of activities during the pandemic, teledentistry was used to face the reduction of preventive procedures, allowing the monitoring of groups in health surveillance18. the highest scores in the biosafety themes were those related to the care of professionals’ ppe. specifically, cleaning and disinfection of the face shield were reported as always performed by 88.4% of the participants, and the appropriate reuse of n95/pff2 masks was always performed by 55.4%. additionally, 50.0% reported always cleaning the environments (table 3). sars-cov-2 can be found in the saliva of covid-19 patients in the pre-symptomatic period, which demands the correct use of ppe to avoid exposure to contaminated aerosols19,20. the survival of this virus on surfaces for many days can be considered one of the reasons for the care reported by ohcws in the frequent cleaning of dental environments. these locations can be vehicles for indirect contact between patients and professionals2. proper removal of personal barrier protection for ohcws is essential. the operator’s body and arms, visors, glasses, and masks can become highly contaminated19. in this study, adequate removal was indicated as always performed by 49.2% of the participants. considering that one of the main ways of contamination of health professionals is during the removal of ppe, all steps must be strictly followed. health services must carry out training with teams to achieve mastery in these skills21. this study showed lower adherence to the recommendations associated with controlling the generation and spread of aerosols and oral secretions. of the participants, 10.6% stated that they always used dental dams. additionally, 27.3% reported adherence with high-power suction systems, and 11.6% always avoided intraoral radiographic examinations (table 3). in the context of the covid-19 pandemic, biosafety needs have made dental practice more costly. in the sus, the place of employment of half of the study participants (51.7%) and many brazilian dentists (around 58,000), adapting to guidelines and norms has implied a high investment economy22,23. the pandemic represented an unprecedented situation, a disease with high morbidity and mortality caused by an etiological agent that can be airborne, which caused fear and high demand for technical information24. an important finding of the study regarding access to technical standards and recommendations for dental care during the pandemic was that 77.4% of participants reported accessing publications that did not identify the agency responsible, regardless of whether they also accessed materials from reputable agencies (table 04). much access to publications via the media, the internet, or direct communication has been observed in other studies12,25,26. this reality, which presents difficulties in clinical practice is based on the best evidence during such times20,27. searching without scientific criteria can lead to false information and corroborate inappropriate conduct in dealing with the pandemic26. as for continuing education, 52.6% of the participants stated that they had received guidance on the measures to be adopted in their workplaces, but 22.2% did not know how to answer this question (table 5). given the panorama of social iso11 palma et al. braz j oral sci. 2023;22:e237812 lation imposed by the pandemic, the privileged education strategies were elaborated online, lacking a foundation in the problematization of realities28. digital tools (applications, online courses) allow for fast and constant updating. it is noteworthy that this type of pedagogical tool is an essential resource in the face of social isolation. however, it disfavors human interaction that facilitates learning and sociability28,29. thus, coordinated actions based on a national education program for health professionals should be proposed with a broad scope, contributing to safety in work processes26. in this study, 33.4% strongly agreed and 32.3% agreed that they felt anxious or worried about working during the pandemic (table 5). fear and anxiety are natural in pandemics, especially with an increase in infected individuals and mortality rates11. the highest scores for anxiety, depression, and stress were related to increased risk factors for contracting the disease30. the highest indices of fear and anxiety were associated with low searching for knowledge7,31, not following biosafety rules11,25 and receiving updates by social media26. some strengths and weaknesses should be highlighted. the study was carried out in the context of the first wave of covid-19. therefore, the generalizability of the results must be extrapolated with caution. considering the country’s regional inequalities, epidemiological differences, and subnational government response to the covid-19 pandemic, the results may not fully reflect the brazilian reality since the responses varied widely in terms of the type, timing, and rigor of policy implementation in each state32. nevertheless, our study was intended to be discussed at the moment of the first wave, as the pandemic impacts unfold around us daily. the results reveal for researchers and policymakers the evidence needed for planning and evaluating surveillance and biosafety measures in the context of the brazilian political severe crisis. our findings revealed that ohcws adopted surveillance measures in dental environments, such as providing covid-19 guidelines and visual alerts in the office, investigating possible respiratory symptoms, and adopting distancing in the waiting room. biosafety measures to reduce the generation or propagation of aerosols, including avoiding intraoral radiographs, using dental dams in high-speed care, and availability of high-power suction systems, had less adherence because they conflict with team management. however, disinfection of face shields, proper removal of personal barrier protection, and reuse of n95/pff2 masks following appropriate criteria had greater compliance. the substantial access to information on dental care during the covid-19 pandemic reflected awareness of the high risk of work exposure. most access to technical standards and recommendations was through non-governmental health authorities. coordinated and purposeful action by policymakers for permanent education of the entire workforce is necessary. conflict of interest none declared. 12 palma et al. braz j oral sci. 2023;22:e237812 funding and acknowledgements none declared. ethical approval ethical approval for data collection was obtained from the research ethics committee, the federal university of rio grande do sul, porto alegre, brazil (caae no. 31720920.5.2002.5530) in accordance with the helsinki declaration of 1975 on experiments involving human subjects. data availability datasets related to this article will be available upon request to the corresponding author. author contribution all authors substantially contributed to the analysis and interpretation of the data; significantly contributed to the critical review of the content; and participated in the approval of the final version of this manuscript. lzp, grv, fsp, mhb, cfc and cmw contributed to the project elaboration and conception of the present study. references 1. boschiero mn, capasso palamim cv, ortega mm, mauch rm, lima marson fa. one year of coronavirus disease 2019 (covid-19) in brazil: a political and social overview. ann glob heal. 2021 may;87(1):44. doi: 10.5334/aogh.3182. 2. cirillo n. covid-19 outbreak: succinct advice for dentists and oral healthcare professionals. clinical oral investigations. 2020 jul;24(7):2529-35. doi: 10.1007/s00784-020-03323-3. 3. dave m, seoudi n, coulthard p. urgent dental care for patients during the covid-19 pandemic. lancet. 2020 apr;395(10232):1257. doi: 10.1016/s0140-6736(20)30806-0. 4. national health surveillance agency of brazil. echnical note gvims/ggtes/anvisa nº 04/2020. [guidelines for health services: measures of prevention and control that must be adopted during case assistance suspected or confirmed infection by new coronavirus (sars-cov-2) – updated on 02/25/2021]. rio de janeiro: anvisa, feb 2021 [cited 2021 jun 6]. available from: https://www. gov.br/anvisa/pt-br/centraisdeconteudo/publicacoes/servicosdesaude/notas-tecnicas/2020/notatecnica-gvims_ggtes_anvisa-04_2020-25-02-para-o-site.pdf. portuguese. 5. brazilian association of intensive care medicine/federal council of dentistry. [amib/cfo recommendations for coping with covid-19 in dentistry.2nd update]. brasília-df: amib/cfo; 2020. [cited 2021 sep 26]. available from: https://www.amib.org.br/fileadmin/user_upload/amib/2020/ junho/22/220620recomendacoes_amib_-_cfo_para_atendimento_odontologico_covid-19.pdf. portuguese. 6. vandenbroucke jp, von elm e, altman dg, gøtzsche pc, mulrow cd, pocock sj, et al. strengthening the reporting of observational studies in epidemiology (strobe): explanation and elaboration. plos medicine. 2007 oct;4(10):e297. doi: 10.1371/journal.pmed.0040297. 7. moraes rr, correa bm, queiroz ab, daneris â, lopes pj, pereira-cenci t, et al. covid-19 challenges to dentistry in the new pandemic epicenter: brazil. 2020 nov;15(11):e0242251. doi: 10.1371/journal.pone.0242251. 13 palma et al. braz j oral sci. 2023;22:e237812 8. novaes tf, jordão mc, bonacina cf, veronezi ao, de araujo car, olegário ic, et al. covid-19 pandemic impact on dentists in latin america’s epicenter: são-paulo, brazil. plos one. 2021 aug;16(8):e0256092. doi: 10.1371/journal.pone.0256092. 9. candeiro gtm, gavini g, vivan rr, carvalho fdmb, duarte ham, feijão pc, et al. knowledge about coronavirus disease 19 (covid-19) and its professional repercussions among brazilian endodontists. braz oral res. 2020 sep 4;34:e117. doi: 10.1590/1807-3107bor-2020.vol34.0117. 10. hesaraki m, akbarizadeh m, ahmadidarrehsima s, moghadam mp, izadpanah f. knowledge, attitude, practice and clinical recommendations of health care workers towards covid-19: a systematic review. rev environ health. 2020 nov 23;36(3):345-57. doi: 10.1515/reveh-2020-0099. 11. ahmed ma, jouhar r, ahmed n, adnan s, aftab m, zafar ms, et al. fear and practice modifications among dentists to combat novel coronavirus disease (covid-19). int j environ res public health. 2020 apr;17(8):2821. doi: 10.3390/ijerph17082821. 12. kamate sk, sharma s, thakar s, srivastava d, sengupta k, hadi aj, et al. assessing knowledge, attitudes and practices of dental practitioners regarding the covid-19 pandemic: a multinational study. dent med probl. 2020 jan-mar;57(1):11-7. doi: 10.17219/dmp/119743. 13. azevedo e silva g, giovanella l, camargo jr rk. brazil’s national health care system at risk for losing its universal character. am j public health. 2020 jun;110(6):811-2. doi: 10.2105/ajph.2020.305649. 14. brazilian federal council of dentistry. [resolution cfo-226, of june 4, 2020. provides for the practice of distance dentistry, mediated by technologies, and other measures]. brasília: cfo; 2020. [cited 2021 oct 15]. available from: https://sistemas.cfo.org.br/visualizar/atos/ resolu%c3%87%c3%83o/sec/2020/226. portuguese. 15. smith ac, thomas e, snoswell cl, haydon h, mehrotra a, clemensen j, et al. telehealth for global emergencies: implications for coronavirus disease 2019 (covid-19). j telemed telecare. 2020 jun;26(5):309-13. doi: 10.1177/1357633x20916567. 16. wosik j, fudim m, cameron b, gellad fz, cho a, phinney d, et al. telehealth transformation: covid-19 and the rise of virtual care. j am med inform assoc. 2020 jun;27(6):957-62. doi: 10.1093/jamia/ocaa067. 17. gasparoni a, michael k. covid-19 and dental emergencies: reflections on teledentistry. brazilian dental science. 2020; 23(2 supp 2):1-4. doi: 10.14295/bds.2020.v23i2.2270. 18. chisini al, costa f dos s, sartori lrm, corrêa mb, otávio pereira d’avila po, et al. covid-19 pandemic impact on brazil’s public dental system. braz oral res. 2021 jul;35:e082. doi: 10.1590/1807-3107bor-2021.vol35.0082. 19. innes n, johnson ig, al-yaseen w, harris r, jones r, kc s, et al. a systematic review of droplet and aerosol generation in dentistry. j dent. 2021 feb;105:103556. doi: 10.1016/j.jdent.2020.103556. 20. peng x, xu x, li y, cheng l, zhou x, ren b. transmission routes of 2019-ncov and controls in dental practice. int j oral sci. 2020 mar;12(1):9. doi: 10.1038/s41368-020-0075-9. 21. sinjari b, rexhepi i, santilli m, d´addazio g, chiacchiaretta p, di carlo p, et al. the impact of covid-19 related lockdown on dental practice in central italy-outcomes of a survey. int j environ res public health. 2020 aug;17(16):5780. doi: 10.3390/ijerph17165780. 22. cavalcanti yw, da silva ro, ferreira lf, lucena ehg, de souza amlb, cavalcante df, et al. economic impact of new biosafety recommendations for dental clinical practice during covid-19 pandemic. pesq bras odontoped clin integr. 2020.20(sup 1):1-9. doi:10.1590/pboci.2020.143. 23. müller a, melzow sf, göstemeyer g, paris s, schwendicke f. implementation of covid-19 infection control measures by german dentists: a qualitative study to identify enablers and barriers int j environ res public health. 2021 may;18(11):5710. doi: 10.3390/ijerph18115710. https://ajph.aphapublications.org/doi/abs/10.2105/ajph.2020.305649 https://ajph.aphapublications.org/doi/abs/10.2105/ajph.2020.305649 https://doi.org/10.14295/bds.2020.v23i2.2270 14 palma et al. braz j oral sci. 2023;22:e237812 24. consolo u, bellini p, bencivenni d, iani c, checchi v. epidemiological aspects and psychological reactions to covid-19 of dental practitioners in the northern italy districts of modena and reggio emilia. int j environ res public health. 2020 may;17(10):3459. doi: 10.3390/ijerph17103459. 25. al-amad sh, hussein a. anxiety among dental professionals and its association with their dependency on social media for health information: insights from the covid-19 pandemic. bmc psychol. 2021 jan;9(1):9. doi: 10.1186/s40359-020-00509-y. 26. costa sm, lacerda gt, villafort rn, silveira rl, amaral mbf. what do we know about covid-19?: maxillofacial surgeons survey. j craniofac surg. 2020 sep;31(6):e661-3. doi: 10.1097/scs.0000000000006658. 27. sezgin gp, şirinoğlu çapan b. assessment of dentists’ awareness and knowledge levels on the novel coronavirus (covid-19). braz oral res. 2020;34:e112. doi: 10.1590/1807-3107bor-2020.vol34.0112. 28. warmling cm, moysés sj. essay on visibilities and blindness in the formative world of dentistry: part 2 internships and online (remote) education. rev abeno. 2021;21(1):1599. doi: 10.30979/revabeno.v21i1.1599. 29. ghai, s. are dental schools adequately preparing dental students to face outbreaks of infectious diseases such as covid‐19? j dent educ. 2020 jun;84(6):631-3. doi: 10.1002/jdd.12174. epub 2020 may 11. 30. alencar cm, silva am, jural la, magno mb, campos ea, silva cm, et l. factors associated with depression, anxiety and stress among dentists during the covid-19 pandemic. braz oral res. 2021 aug;35:e084. doi: 10.1590/1807-3107bor-2021.vol35.0084. 31. duruk g, gumuşboğa zş, colak c. investigation of turkish dentists’ clinical attitudes and behaviors towards the covid-19 pandemic: a survey study. braz oral res. 2020;34:e054. doi: 10.1590/1807-3107bor-2020.vol34.0054. 32. touchton m, knaul fm, arreola-ornelas h, porteny t, sánchez m, médez o, et al. a partisan pandemic: state government public health policies to combat covid-19 in brazil. bmj global health. 2021 jun;6(6):e005223. doi: 10.1136/bmjgh-2021-005223. https://doi.org/10.30979/revabeno.v21i1.1599 15 palma et al. braz j oral sci. 2023;22:e237812 appendix 1. research data collection instrument. questions categories sociodemographic and health profile gender male female other age years occupation dentists dental assistants technicians conclusion of professional training years higher graduation level specialization/residency master phd none postgraduate area specialty list city where you work municipality workplace sus – primary health care (phc) sus dental specialty centers (dsc) sus – urgency care private clinic dental teaching clinic corporate entities health system security forces (i.e., army, police) hospital management other risk group for covid-19 only age over 60 years old health condition only age over 60 and health condition none absence from work during the pandemic yes no testing for covid-19 no yes, rt pcr yes, rapid test yes, serological test continue 16 palma et al. braz j oral sci. 2023;22:e237812 continuation adoption of surveillance, planning and risk management measures to control the dissemination of covid-19 in health services answer options: 0 – do not know; 1 – never; 2 – ever; 3 – sometimes; 4 – often; 5 always suspended elective procedures and care restricted to urgency/emergency participation in decision-making about changes in work during the pandemic reduced workload or professional turnover to minimize the risk of contamination worked directly in covid-19 reception/sorting/fast track procedures investigation of respiratory infection symptoms in appointment scheduling patients with symptoms of respiratory tract infection immediately isolated waiting room respecting the minimum distance of 01 meter between people availability of visual alerts in the health service urgency based on pre-established clinical protocols orientation of patients about covid-19 use of digital tools for teleorientation or telemonitoring interaction with other health professionals adoption of biosafety measures in health services answer options: 0 – do not know; 1 – never; 2 – ever; 3 – sometimes; 4 – often; 5 always disinfection of the environment by a trained professional with appropriate ppe disinfection of suction hoses use of sterile micromotors at every dental appointment intraoral radiographic examinations were avoided performing four-handed dental procedures use of the dental dam in high rotation services procedures that generate aerosols were avoided use of suction system (vacuum pump) proper removal of personal barrier protection n95/pff2 mask reuse with proper criteria disinfection of face shield permanent health education access to technical standards and recommendations on dental care access to standards and recommendations on dental care during the covid-19 pandemic technical note gvims/ggtes/anvisa nº 04/2020 recommendations booklet of the federal council of dentistry (cfo) recommendations booklet of the regional council of dentistry (cro) from own state recommendations booklet of the regional council of dentistry (cro) from other state recommendations from the municipal / state secretariat none other documents continue 17 palma et al. braz j oral sci. 2023;22:e237812 continuation training/education during covid-19 pandemic answer options: 0 – do not know; 1 – strongly disagree; 2 – disagree; 3 – undecided; 4 – agree; 5 – strongly agree i consider that i received guidance at my workplace regarding measures to be taken during the covid-19 pandemic i was able to apply the knowledge acquired in training/education about covid-19 to modify my practice i feel sufficiently enlightened and secure to work properly in dental practice during the covid-19 pandemic i feel anxious and concerned to work properly in dental practice during the covid-19 pandemic 1 volume 21 2022 e225337 original article braz j oral sci. 2022;21:e225337http://dx.doi.org/10.20396/bjos.v21i00.8665337 1 santa casa of belo horizonte college, belo horizonte, mg, brazil. 2 newton paiva university center, belo horizonte, mg, brazil. 3 federal university of minas gerais ufmg, belo horizonte, mg, brazil. corresponding author: janice sepúlveda santa casa de misericórdia – belo horizonte, mg, brazil email: janicesepulveda@gmail.com editor: altair a. del bel cury received: april 19, 2021 accepted: october 27, 2021 a validated instrument to assess dentists’ knowledge about diabetes: the dental -diabetes questionnaire margarete aparecida gonçalves melo guimarães1 , amanda augusto de oliveira prates1 , vladimir reimar augusto de souza noronha2 , adriana pagano3 , aleida nazareth soares1 , janice sepúlveda reis1,* aim: to elaborate and validate an instrument for brazilian portuguese speakers, to assess dentists’ knowledge about care of patients with diabetes mellitus (dental-diabetes). methods: methodological study comprising four stages: a) elaboration of instrument; b) content validation (computing content validity index cvi) based on expert committee assessment; c) pre-test with 30 dentists, followed by assessment of suggestions by expert committee; d) psychometric validation through instrument application in a sample of 127 dentists by means of the web tool e-surv. cronbach’s alpha and intraclass correlation coefficients were used to evaluate, respectively, internal consistency and reproducibility. results: the final version of the instrument consists of 22 questions (7 on sociodemographic data and 15 querying dentists’ knowledge) and those submitted for validation attained a cvi of 0.95 [95% ci 0.916-0,981], showing satisfactory internal consistency, with 0.794 cronbach’s alpha [95% ci 0.741-0.842] and an intraclass correlation coefficient of 0.799 [95% ci: 0.746-0.846] between the test and retest scores. conclusions: dental-diabetes is a comprehensive instrument, culturally adequate and validated to assess dentists’ knowledge about care of patients with diabetes. keywords: diabetes mellitus. dentists. knowledge. validation studies as topic. https://orcid.org/0000-0003-2464-5789 https://orcid.org/0000-0001-7719-9773 https://orcid.org/0000-0002-2809-0859 https://orcid.org/0000-0002-3150-3503 http://orcid.org/0000-0002-2671-3661 http://orcid.org/0000-0002-2465-862x 2 guimarães et al. braz j oral sci. 2022;21:e225337 introduction diabetes mellitus (dm), a chronic disease, is a public health problem that affects a large number of individuals from all social backgrounds. in 2019, the international diabetes federation (international diabetes federation, idf) estimated that diabetes affected 9.3% of the world population, with an estimated prevalence of more than 10.9% people in 20451. if uncontrolled, systemic complications of dm can include heart attack, kidney disease, limb loss, blindness, and peripheral nerve damage2. due to the complexity of diabetes, how well the disease is controlled is an important issue in dental treatment planning. patients with dm manifest a high prevalence of oral problems such as periodontal disease, tooth loss, xerostomia, caries, burning mouth disorder, taste and salivary gland dysfunction, delayed wound healing, lichen planus, geographic tongue, and candidiasis3. being a common disease in dental practice, dentists are expected to be part of a multidisciplinary team, collaborating especially with endocrinologists. they are expected to base their care on strategies to provide effective management of dm and its oral consequences, identifying oral disease impacting glycemic control, which, in turn, can impact oral health4. they are also expected to be aware of the pathophysiology of dm, its oral manifestations, signs and symptoms, how to react in case of an emergency, risks involved, systemic repercussions of the use of medications and anesthetics, which can all add up to provide better care for patients5-7. assessing dentists’ conduct regarding dm is crucial to understand their knowledge, contributing to establishing targets for their training in public and private services and teaching institutions and defining guidelines for educational content and contributing to better care for patients with dm. given the importance of dentists’ role and the lack of tools to assess their knowledge about dm, an instrument was felt to be needed to gather information about dentists’ knowledge and promote their education on related topics. a specific instrument for this purpose is not currently available. the aim of the study was to develop and validate an instrument for the assessment of dentists’ knowledge about diabetes (dental-diabetes). materials and methods this is a methodological and exploratory study carried out from january 2017 to august 2018 in the city of belo horizonte, in the state of minas gerais, brazil. the project was approved by the ethics and research committee involving human beings (caae number 65656117.6.1001.5138) at santa casa of belo horizonte hospital. agreement to participate in the study was obtained by using a free informed consent form signed by participants when accessing an electronic questionnaire by means of the webtool e-surv. an expert committee made up of five dentists, a nurse, an endocrinologist, a linguist and a statistician took part in elaboration of the instrument and assessed all stages until its final version (figure 1). 3 guimarães et al. braz j oral sci. 2022;21:e225337 stage 1 instrument’s development prior to elaboration of the instrument, three authors (m.a.g.m.g., a.a.o.p., j.s.r.) conducted a literature review in pubmed (u.s. national library of medicine), lilacs (latin american and caribbean literature in health sciences databases), and scielo (scientific electronic library online) databases to obtain state-of-the-art information about diabetes, national and international recommendations on dental treatment of people with diabetes and questionnaires used to assess professionals’ knowledge about a particular disease. the descriptors used for the queries were ‘diabetes mellitus’, ‘dentists’, ‘knowledge’, and ‘validation studies’. national and international publications yielded by the database queries were screened2,5-13. based on the gathered insights we decided to elaborate an instrument in the form of a questionnaire. in establishing a general conceptual structure dentistry-endocrinology interface, our instrument was developed in two parts: the first section focusing on dentists’ sociofigure 1. stages in the instrument’s elaboration. v1: first version of the instrument; v2: second version of the instrument; v3: third version of the instrument *05 dentists; 01 nurse; 01 endocrinologist; 01 linguist; 01 statistician **22 dentists; 05 linguists; 06 endocrinologists • judge committee assessment** • expert committee assessment* • content validity index (cvi) instrument development content validation pre-test validation • literature review • construct definition • elaboration of items • expert committee assessment* • face-to-face tests • expert committee assessment* • test • retest • analysis of internal consistency and temporal stability v1 v2 v3 instrument validated 4 guimarães et al. braz j oral sci. 2022;21:e225337 demographic profile (7 questions); and the second section aimed at assessing dentists’ knowledge about key aspects of dm and related care expected to be performed as part of their work (16 questions) (version 1-v1). stage 2 content validation for content validation, a web address to access a web assessment form was sent by e-mail to 22 dentists, six endocrinologists and five linguists (judges’ committee), who evaluated each item of the instrument’s first version (v1). criteria for participation in the committee were either to be a professional dentist with or without clinical practice implicating diabetes (dentist profile); or have taken part in questionnaire elaboration or translation in the healthcare area (linguist profile); or to have clinical practice in diabetes (endocrinologist profile). judges were selected based on their curriculum vitae. the judges’ committee assessed clarity and relevance of each item in v1 and rated them with the following options: one star standing for need for full reformulation; two stars, partial reformulation (substantial revision needed); three stars, need for partial reformulation, with minor editing to enhance text style; and four stars in case of no need for reformulation. a comment box was also provided for the experts’ remarks and suggestions. once the evaluation was completed, the content validity index (cvi – the level of agreement of experts on adequacy of the items) was computed: number of scores 3 and 4 divided by total number of scores by all committee members. cvi indicates the degree to which a scale has an adequate sample of items to represent a construct of interest that is, whether a domain of content for the construct is adequately represented by the items. results higher than or equal to 0.78 are considered acceptable14. upon computing cvi (higher than 0.78) and implementing the committee’s suggestions to improve, v2 was obtained. v2 is substantially similar to v1, except for minor editing and spelling correction. stage 3 – pre-test the author (m.a.g.m.g.) carried out the pre-test through face-to-face interviews with 20 dentists15,16 in 10 meetings following participants’ schedules 2 group meetings with 5 dentists, 2 pair meetings and 6 individual meetings. first, the whole instrument was read by each participant individually; secondly, items were discussed to ensure whether they were clear, accurate, relevant and adequately arranged. participant’s feedback was then assessed by the expert committee, who considered all relevant comments and redrafted those items that obtained less than 80% agreement14. version 3 (v3) was thus obtained and tested on a newly selected group of 10 dentists (in 8 individual meetings and 2 in pair meetings). stage 4 validation a web address to access v3 in digital format on the e-surv platform was sent via e-mail to 127 dentists selected by convenience from both public and private services and universities. the sample size was adequate considering a level of significance equal to 5%, test power equal to 80%, standard deviations equal to the test and retest 5 guimarães et al. braz j oral sci. 2022;21:e225337 scores and a correlation coefficient equal to 0.30 (minimum value detected in the consistency assessment). a minimum sample size requirement was 85 professionals. retest was performed with those 127 dentists with a minimum interval of 7 days and a maximum of 21 days between the tests (average 16 days)17. statistical analysis absolute and relative frequencies were used to describe the sample characteristics and the proportion of correct answers to the instrument items. internal consistency and reproducibility were verified to analyze the reliability of the construct. cronbach’s alpha (ca) was used to assess the internal consistency of the instrument. internal consistency is an assessment of whether items intended to measure the same construct produce similar scores. a high degree of internal consistency indicates that items meant to assess the same construct yield similar scores. there are a variety of internal consistency measures. usually, they involve determining how highly these items are correlated and how well they predict each other. cronbach’s alpha is a commonly used measure. the instrument’s reproducibility was evaluated through test-retest (temporal stability), computing the intraclass correlation coefficient (icc). the kappa index was added for reproducibility and refers to the percentage of concordant responses in the test and retest, defined as the ratio between the number of individuals who selected the same answer (regardless of being correct or incorrect) at both test and retest and the total number of individuals16,18. floor and ceiling effects were measured by the number of respondents receiving the minimum and maximum scores, respectively. the significance level adopted for the statistical tests was 5%. for data analysis, spss version 20.0 was used. results instrument development and content validation the development spanned 6 months. v1 of the instrument consisted of 23 questions. after reviewing v1 following the experts’ suggestions, a second version (v2) was obtained, with 23 questions. the instrument achieved a good score by the committee regarding clarity and relevance, with a total cvi of 0.95 [95% ci 0.916-0,981]. pre-test in face-to-face tests, which lasted 3 months, 30 dentists participated. 67% were female; 47% had a diploma course and were working in different areas such as surgery, dentistry, endodontics, periodontics and others; 47% had more than 20 years’ experience; 100% reported having provided dental care to patients with diabetes; 67% declared not having had any training to treat patients with diabetes; however, 63% reported feeling empowered to provide care for diabetes patients. (table 1). https://en.wikipedia.org/wiki/construct_(philosophy) https://en.wikipedia.org/wiki/construct_(philosophy) 6 guimarães et al. braz j oral sci. 2022;21:e225337 as an outcome of the first meeting, with suggestions by 20 dentists, 2 questions were merged in order to adapt terms and increase understanding, a total of 22 items remaining in the questionnaire, yielding a third version (v3). v3 was tested with ten other dentists, no need for further redrafting having been requested. v3 was hence considered adequate to be submitted to psychometric validation. the 15 questions on knowledge about diabetes were then submitted for validation (table 2). table 1. sociodemographic data of participants in the adaptation and validation stages. variables   pre-test (n=30) validation (n=127) n (%) n (%) sex female 20 (67) 89 (70.1) male 10 (33) 37 (29.1) i’d rather not say 1 (0.8) education first degree 09 (30) 45 (35.4) diploma course 14 (47) 68 (53.5) master’s degree 02 (07) 8 (6.3) doctor’s degree 04 (13) 6 (4.7) post-doctoral degree 01 (03) 0 main area of professional expertise general clinic 11 (37) 52 (40.9) surgery 2 (7) 5 (3.9) dentistry 1 (3) 6 (4.7) endodontics 2 (7) 17 (13.4) pediatric dentistry 1 (3) 6 (4.7) orthodontics 0 21 (16.5) periodontics 1 (3) 11 (8.7) lecturing 6 (20) 0 research 1 (3) 0 others 5 (17) 9 (7.2) first degree obtained less than 1 year ago 03 (10) 9 (7.1) 1 to 5 years ago 06 (20) 19 (15) 5 to 10 years ago 0 11 (8.7) 10 to 20 years ago 07 (23) 21 (16.5) more than 20 years ago 14 (47) 67 (52.8) have you ever provided dental treatment to any patient with diabetes? yes 30 (100) 120 (94.5) no 0 7 (5.5) have you ever taken any training or course about diabetes? yes 10 (33) 10 (7.9) no 20 (67) 117 (92.1) do you feel empowered to provide care for diabetes patients? yes 19 (63) 73 (57.5) no 11 (37) 54 (42.5) 7 guimarães et al. braz j oral sci. 2022;21:e225337 validation this stage lasted about 28 days and 127 dentists answered the final version of the instrument (test and retest) (table 1). 70% were female, 53.5 % had a diploma course, 40.9% had expertise in general clinical practice., and 52.8% had obtained their first degree 20 years ago. 94.5% had already provided dental care to people with diabetes. the total ca alpha value was 0.794 (95% confidence interval 0.7410.842). floor effects (percent with minimum score) were 0%, and ceiling effects (percent with maximum score) 3,2%. an icc value of 0.799 (95% ci: 0.746-0.846) was obtained. the kappa coefficient, which assesses the degree of agreement, varied between 0.5-1.0 (mean: 0.80). when the alpha absence index was calculated, there was a slight impact on reducing ac and no questions needed to be removed (table 3). table 2. items reviewed along the process of elaboration and adaptation. v1 v2 v3 initial number of questions (part 1/ part 2) 23 (7/16) 23 (7/16) 22 (7/15) number of questions requiring redrafting or exclusion (part 1/ part 2) 21 (7/14) 9 (0/9) 0 suggestions deemed necessary by the expert committee 17 5 na questions excluded due to agreement below 80% 0 1 na final number of questions (part 1/ part 2) 23 (7/16) 22 (7/15) 22 (7/15) v= version; part 1: socio-demographic assessment; part 2: knowledge assessment; na: not applicable. table 3. correlation between test and retest, answers agreement percentage and cronbach’s alpha coefficient for the dentaldiabetes instrument. item* kappa index percentual agreement test-retest cronbach’s alpha if item is removed 95% ci for alpha q1 0.629 76.27 0.813 0.763 – 0.857 q2 0.368 61.34 0.787 0.729 – 0.837 q3 0.658 79.17 0.797 0.743 – 0.845 q4 0.594 88.50 0.798 0.745 – 0.846 q5a 0.174 69.75 0.796 0.741 – 0.844 q5b 0.22 85.12 0.796 0.741 – 0.844 q5c 0.158 85.12 0.798 0.744 – 0.845 q5d 0.501 97.58 0.797 0.743 – 0.845 q5e 0.195 72.48 0.8 0.747 – 0.847 q6 0.474 83.76 0.804 0.751 – 0.850 q7a 0.335 91.13 0.794 0.794 – 0.842 q7b 0.601 97.64 0.795 0.741 – 0.843 q7c 0.315 76.72 0.792 0.736 – 0.840 continue 8 guimarães et al. braz j oral sci. 2022;21:e225337 the mean final score during test was 19.40, with a standard deviation of 4.49. at the time of the test, the percentages of minimum and maximum correct answers were 8.7% (question 2) and 95.3% (question 5d), respectively. all participants spent between 22 and 16 minutes on testing and retesting. the final version of the instrument is available in supplementary material. discussion the treatment of patients with diabetes requires knowledgeable professionals, dentists being fundamental member in a multidisciplinary team; therefore, dentists are expected to be updated regarding diabetes and its implications for daily care, with a greater knowledge about the onset, duration and control of the disease, resulting in a more effective and satisfactory approach19. a good interaction between the dentist and the multidisciplinary team is essential for a safer dental treatment, with lower chances of complications for the patient20. given the importance of the dental approach for the patient with dm, elaborating and validating an instrument to evaluate the dentists’ knowledge about dm was considered important to identify possible flaws in the knowledge of diabetes of these professionals that could impact treatment decisions and the objectives of the patient. in brazil, dentists are not yet part of teams in diabetes centers as is the case in other countries, despite the clear need for these professionals to share the knowledge and duties of a multidisciplinary team. continue q7d 0.368 77.87 0.799 0.746 – 0.846 q8 0.605 87.70 0.778 0.718 – 0.830 q9 0.434 69.72 0.784 0.727 – 0.835 q10 0.418 80.36 0.786 0.729 – 0.837 q11 0.352 70.09 0.8 0.747 – 0.847 q12a 0.478 79.65 0.8 0.746 – 0.847 q12b 0.191 89.47 0.799 0.745 – 0.846 q12c 0.239 85.84 0.796 0.741 – 0.844 q13 0.675 89.34 0.798 0.744 – 0.845 q14a 0.534 86.09 0.794 0.739 – 0.842 q14b 0.391 80 0.793 0.738 – 0.842 q14c 0.263 69.57 0.795 0.740 – 0.843 q14d 0.3 7.27 0.796 0.741 – 0.844 q15a 0.563 77.27 0.785 0.728 – 0.836 q15b 0.464 76.85 0.787 0.730 – 0.837 q15c 0.549 76.32 0.781 0.722 – 0.832 q15d 0.464 84.35 0.781 0.723 – 0.833 q15e 0.544 73.50 0.784 0.727 – 0.835 * instrument in supplementary file. 9 guimarães et al. braz j oral sci. 2022;21:e225337 the collaborative work by the expert committee pooling expertise in diabetes, dentistry and language issues made it possible to elaborate a comprehensive instrument, solving problems encountered during the process of drafting and adapting concepts and terms to the language used by the target subjects13,16,18,21-23. interaction between healthcare professionals and applied linguists is a fundamental piece in the elaboration and cultural adaptation of new instruments. assessment by the expert committee through the web tool e-surv is a reliable and efficient methodology24, allowing for remote application and quick data extraction, avoiding potential errors in transcriptions and gathering of results25. the instrument successfully passed the committee’s examination in terms of clarity and relevance, with an excellent cvi (0.95). it should be noted that the maximum value for cvi is equal to 1, the results achieved being close to the maximum score18 , well above the cvi cutoff point of 0.80 for new instruments18,26. in the pre-test, the face-to-face meetings with a sample of dentists proved successful, favoring adjustments in the instrument, and ensuring the prospective understanding of the items by the target audience27,28. in carefully developed instruments, two or three face-to-face tests can be satisfactory, which was the case in our study, with two rounds being required21. for validation (test-retest), a ca index of 0.794 was obtained, which indicates good internal consistency29-32. the time span for retest met the recommendations in the literature: a 7 to 21 day interval (a mean of 16 days). there are controversies regarding interval between test and retest, a desirable interval being not too short for participants to recall their answers in the test and too long for the study to be impacted33-35. there are recommendations of an interval of one to two weeks between test and retest; however, no fixed amount of time is prescribed, the main concern being the need to account for whatever interval span chosen36. the time interval in our study adhered to the above recommendations, variation being due to participants’ agendas. our ca, icc (0,799) and kappa index (mean 0.80) indicate that our instrument showed adequate stability, reproducibility and confidence18. icc being satisfactory, we computed kappa to corroborate it. items with a low kappa (5a, 5c e 5d, 12b e 12c) revealed topics that were less familiar to dentists. ca absence index was carried out. removing items (1, 5e, 6, 11 e 12a) yielded alpha scores higher than those for the whole set of items. therefore, no questions were excluded, due to the small difference that would result in the final ca37 and the possibility of leaving out important information13. in addition, the value was above 0.799 for all items and thus considered satisfactory. when we analyzed the performance of dentists in the test, the questions that had the lowest percentage of correct answers were question 2 (time period considered in the glycated hemoglobin test to assess mean blood glucose levels); 5 (hypoglycemia and signs of mood change and/or irritability); and 14c (use of sedatives). this percentage of incorrect answers was somehow predictable, since those are the most common questions asked to endocrinologists by dentists before dental procedures in daily care. in this respect, it is worthy of note that both in the pre-test and the validation stages, the majority of participants had over twenty years’ experience and 10 guimarães et al. braz j oral sci. 2022;21:e225337 had provided dental care to patients with diabetes; nevertheless, most had had no training whatsoever in diabetes care. still, despite the lack of training, most reported feeling confident to treat patients with diabetes. this finding reveals a major problem in diabetes education and clinical practice regarding a highly prevalent condition as is diabetes and showcases the need for questionnaires such as the one we have elaborated and validated in our study as an instrument to assess the level of knowledge about diabetes by dentists and propose educational initiatives to contribute to better dental treatment for people with diabetes. no studies were found describing the development and validation of instruments to assess dentists’ knowledge about diabetes, which did not allow for our results to be compared. our instrument comprises items implicating knowledge compatible with themes indicated as priorities for dentists’ care of patients with diabetes: diagnostic criteria, symptoms, urgency and emergency, dental risk, conduct in care, clinical signs in the oral cavity and use of anesthetics and medications. in conclusion, our study yielded an instrument that proved useful, reliable and stable for use by dentists. the instrument is useful to evaluate dentists’ knowledge and promote professionals’ training, with potential impact to enhance treatment for people with diabetes. author contribution magmg and aaop participated in all steps of the study and was a major contributor in writing the manuscript. asp and vrasn took part in pre-test. ans analyzed and interpreted data and results. jsr and ans provided guidance to magmg and aaop. all authors read and approved the final manuscript and actively actively participated in the discussion of the manuscript’s findings. disclosure no potential conflict of interest is relevant to this article. references 1. saeedi p, petersohn i, salpea p, malanda b, karuranga s, unwin n, et al. global and regional diabetes prevalence estimates for 2019 and projections for 2030 and 2045: results from the international diabetes federation diabetes atlas, 9th edition. diabetes res clin pract. 2019 nov;157:107843. doi: 10.1016/j.diabres.2019.107843. 2. brazilian diabetes society. 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terwee cb, bot sd, de boer mr, van der windt da, knol dl, dekker j et al. quality criteria were proposed for measurement properties of health status questionnaires. j clin epidemiol. 2007 jan;60(1):34-42. doi: 10.1016/j.jclinepi.2006.03.012. 37. valim md, marziale mh, hayashida m, rocha fl, santos jl. validity and reliability of the questionnaire for compliance with standard precaution. rev saude publica. 2015;49:87. doi: 10.1590/s0034-8910.2015049005975. 1http://dx.doi.org/10.20396/bjos.v20i00.8661236 volume 20 2021 e211236 original article 1 faculty of dentistry, universitas gadjah mada, indonesia. corresponding author: ryna dwi yanuaryska email: ryanuaryska@ugm.ac.id received: september 15, 2020 accepted: january 21, 2021 alteration of mucosa cell maturation pattern after exposure to different radiographic imaging methods ryna dwi yanuaryska1,* , sabrina ceasy anggraeni1, adhara harvita puspitasari1, rurie ratna shantiningsih1 , munakhir mudjosemedi1 , rellyca sola gracea1 aim: dental imaging has been widely used for diagnosis in dentistry. however, dental x-ray may induce cytotoxicity leading to apoptosis in oral mucosa cells. the present study aimed to observe the maturation pattern of buccal and gingival cells after exposure to x-ray radiation from analog/digital panoramic scanning and cone beam computed tomography (cbct). methods: the research samples were 40 subjects who fulfilled the inclusion and exclusion criteria. the subjects were divided into the exposed (patients who received analog/digital panoramic radiography or cbct) and controlled (patients who had no radiography examinations) groups, with 10 subjects in each group. exfoliative cytology smears were obtained from buccal mucosa and gingiva before exposure (or on day 0 for the control group) and 10 days later. the cells were stained with the papanicolaou method. then, the superficial, intermediate, and parabasal cells were counted in each glass slide. results: no significant differences (p > 0.05) were observed among all cell types between day 0 and 10 in the control group. meanwhile, after exposure to three kinds of radiography examinations, the frequency of intermediate cells in buccal mucosa and gingiva increased (p < 0.05), but that of superficial cells decreased (p < 0.05) significantly. no significant difference was found in the parabasal cells (p > 0.05). the frequency differences between intermediate and superficial cells showed no significant difference between the buccal mucosa and gingiva. conclusion: analog/digital panoramic radiography and cbct exposure can induce cytotoxicity by altering the maturation pattern of buccal mucosa cells and gingiva, so it is strongly recommended to only perform these procedures if necessary and avoid repeated exposure to the same patient. keywords: papanicolaou test. mouth mucosa. cone-beam computed tomography. gingiva. radiography, panoramic. https://orcid.org/0000-0001-6195-245x https://orcid.org/0000-0001-5885-9545 https://orcid.org/0000-0003-0091-6839 https://orcid.org/0000-0002-3940-0891 2 yanuaryska et al. introduction the use of ionizing radiation in imaging science in dentistry has been rapidly increasing. the essential diagnostic method in dental practice is radiography, but x-ray provokes genotoxic and cytotoxic outcomes in cells. the genotoxic effects of x-rays are still debatable, whereas its cytotoxic effects are more proven. x-ray exposure through panoramic scanning initiates a cytotoxic consequence by increasing cell apoptosis1,2. the patients exposed to a series of radiographs had a significant increase in cellular death marked by pyknosis, karyolysis, and karyorrhexis in buccal mucosa smears. cone beam computed tomography (cbct) is a widely used oral-maxillofacial imaging modality which provides accurate 3d imaging of hard tissue structure3. radiation exposure from cbct also acts as a cytotoxicant to oral mucosa cells due to its high radiation dose. the patients submitted to cbct show higher cell death than conventional radiographs group4. cytotoxicants act by disrupting the molecules involved in cell growth and cell death5. the oral epithelium has been widely used to analyze x-ray effects because it is directly exposed to radiation, and the tissue is easily collected by scraping the mucosa. the effects of x-ray exposure can be studied using exfoliative cytology because the method is simple, low-cost, and non-invasive6. papanicolaou staining is a cytology method that analyzes the proportion of exfoliated cells as the maturation pattern. thus, this method was applied in this study. the oral epithelium is continuously renewed; new cells migrate from the parabasal layer to the outer tissue layer, causing epithelial maturation7. normally, oral mucosa smears consist of cells from the superficial, intermediate, and rare cases, the parabasal layer. normal maturation pattern indicates the predominance of intermediate cells accompanied by superficial cells and rarely parabasal cells. lesions in oral mucosal cells such as leucoplakia and oral squamous cell carcinoma have been associated with the maturation pattern changes indicated by a greater number of intermediate or parabasal cells8. studies have evaluated the influence of extrinsic factors, such as tobacco and alcohol consumption, on the process of oral epithelium maturation, given that they have been suggested as risk factors for oral cancer development8-10. dental x-rays stimulate cytotoxic consequences in oral mucosa cells, leading to cellular death, and may be considered as a nongenotoxic mechanism of carcinogenesis1,2. however, whether x-ray radiation exposure affects epithelial maturation’s normal process in the oral cavity remains uncertain. thus, this study aimed to evaluate the outcomes of x-ray exposure from analog/digital panoramic radiography and cbct on the maturation patterns of buccal and gingival cells. material and methods study design and sample this cross-sectional observational study evaluated patients aged 25 years or older and who had analog or digital panoramic radiography or cbct examinations requested 3 yanuaryska et al. by a dentist independent of this study at dental hospital, universitas gadjah mada, indonesia and were considered eligible for the study. the subjects of this study fulfilled the following inclusion criteria: (1) non-cigarette smoker and/nor alcohol consumer; (2) no exposure to radiographic imaging for the last 3 weeks; (3) no visible lesions in the oral mucosa; (4) not using orthodontic and prosthodontic appliances. ethical clearance had been granted by the research ethics commission of the faculty of dentistry, universitas gadjah mada, indonesia. the informed consent for each patient was obtained. the sample consisted of ten subjects for each radiological exam selected by purposive sampling design based on the previous study conducted by shantiningsih and diba11 (2018). the control group included an equal number of subjects who had no radiography examination and were observed under the same research protocol. analog panoramic radiographs were obtained using a yoshida panoura deluxe system (the yoshida dental mfg. co., ltd., tokyo, japan) with the following exposure parameters: 90 kvp, 8–10 ma, and 20 s. a digital system using a pax-i (vatech, gyeonggi-do, republic of korea) was operated with the following exposure parameters: 72 kvp, 10 ma, and 16 s. the cbct images were obtained using a volux 3d dental ct system (genoray, gyeonggi province, republic of korea) with parameter settings of 85 kvp, 6 ma, and 16.6 s. sample collection and cytopathological analysis smears were collected immediately from buccal mucosa and gingiva cells before x-ray exposure and 10 days later. before smear collection, the patients were instructed to rinse their mouths with water for 30 s. after rinsing, a cytobrush was used to collect smears over the buccal mucosa and gingiva. both buccal and gingival mucosa samples were taken twice, before the x-ray exposure on the left region and after the procedure on the right region. the smears were spread onto a glass slide and fixed in 96% alcohol. the slides were stained by the papanicolaou method. the stained slides were analyzed under a light microscope (ys100, nikon, japan) and optilab viewer 2.1. all slides were analyzed by an experienced and blinded observer. cells were classified as superficial, intermediate, and parabasal according to the criteria described by montgomery6. the cells were counted until 100 visible, and non-overlapping cells were obtained at 400x magnification on each slide horizontally, from left to right (figure 1). the results are expressed in percentages. similar analyzes have been established in previous published studies8,10,12. statistical data analysis all data were presented as mean ± standard deviation (s.d.). statistical analysis was performed using ibm® spss® statistics 25 version software (ibm corporation, armonk, new york). the normality criterion was evaluated using the shapiro–wilk test. based on the shapiro-wilk test results, the normally distributed data were further analyzed using a paired  t-test, otherwise using wilcoxon signed-rank test to examine the difference in the number of superficial, intermediate, and parabasal cells, before and after x-ray exposure. a paired  t-test was also used to measure 4 yanuaryska et al. the differences between buccal mucosa and gingiva of the oral cavity in the study group. the statistical differences were significant if  p < 0.05. the intraclass correlation coefficient (icc) was used to assess the reproducibility of measurements. intra-rater reliability statistics for cell numbers were calculated after the same examiner re-evaluated a randomly selected subset of 8 slides after 2 weeks. based on the 95% confidence interval of the icc estimate, the values were interpreted as follows: poor for less than 0.5, moderate for 0.5–0.75, good for 0.75–0.9, and excellent reliability for values greater than 0.913. results intra-rater reliability measurement was excellent for parabasal cells (icc = 1) and good for intermediate and superficial cells (icc = 0.8). the cell number was normally distributed by shapiro–wilk test, except for parabasal cells. tables 1 and 2 show the mean frequency differences in percentages of parabasal, intermediate, and superficial cells of buccal mucosa and gingiva, respectively, before and after obtaining analog/digital panoramic or cbct radiographs and in the control group. a small number of parabasal cells were detected after radiation exposure, but the number was not significant (p > 0.05) by wilcoxon signed-rank test. the paired t-test showed a significant increase in intermediate cells (p < 0.05), while superficial cells were significantly decreased (p < 0.05) after radiation exposure using the three types of radiography methods. no significant differences were observed among all cell types in the control group (p > 0.05). parabasal cell intermediate cell superficial cell figure 1. papanicolaou staining showing three types of oral mucosa cells. magnification: 400×. 5 yanuaryska et al. table 3 shows the frequency differences between the intermediate and superficial cells in buccal mucosa and gingiva after x-ray exposure under the three types of radiographic methods. no significant differences were observed between the buccal mucosa and gingiva in any radiography methods (p > 0.05). table 1. comparison of mean frequencies (in percentages) of parabasal, intermediate, and superficial cells before and after exposure to each radiography imaging in the buccal mucosa. variable parabasal p*a intermediate p*b superficial p*b control before 0 ref. 67.80 + 3.88 ref. 32.20 + 3.88 ref. after 0 1 69.8 + 5.31 0.254 30.20 + 5.31 0.254 analog panoramic before 0 ref. 69.50 + 4.42 ref. 30.50 + 4.42 ref. after 0.083 + 0.29 0.32 75.17 + 6.89 0.036 24.83 + 6.89 0.036 digital panoramic before 0 ref. 69.70 + 6.74 ref. 30.50 + 8.26 ref. after 0.33 + 0.65 0.10 81.20 + 6.37 0.003 17.83 + 7.41 0.000 cbct before 0 ref. 67.80 + 3.88 ref. 32.89 + 3.41 ref. after 0.5 + 0.71 0.059 77.6 + 9.80 0.013 22.67 + 9.51 0.018 a wilcoxon signed-rank test b paired t-test *comparison of the reference group (ref.); differences were considered to be statistically significant when p < 0.05. table 2. comparison of mean frequencies (in percentages) of parabasal, intermediate, and superficial cells before and after exposure to each radiography imaging in the gingiva. variable parabasal p*a intermediate p*b superficial p*b control before 0 ref. 58.10 + 7.49 ref. 41.9 + 7.49 ref. after 0 1 63.00 + 9.59 0.186 37.00 + 9.59 0.186 analog panoramic before 0 ref. 61.08 + 6.71 ref. 38.92 + 6.71 ref. after 0.083 + 0.29 0.32 67.75 + 5.24 0.039 31.42 + 5.55 0.017 digital panoramic before 0 ref. 60.60 + 8.06 ref. 41.83 + 9.28 ref. after 0.33 + 0.65 0.10 79.40 + 6.42 0.000 20.83 + 7.48 0.000 cbct before 0 ref. 58.1 + 7.49 ref. 43.33 + 6.32 ref. after 0.5 + 0.71 0.059 73.70 + 7.36 0.001 26.11 + 9.02 0.001 a wilcoxon signed-rank test b paired t-test *comparison of the reference group (ref.); differences were considered to be statistically significant when p < 0.05. 6 yanuaryska et al. table 3. comparison of frequency differences of intermediate and superficial cells in buccal mucosa and gingiva before and after exposure to each radiography imaging between buccal mucosa and gingiva cell types buccal gingival p-value*a analog panoramic intermediate 5.67 6.67 0.773 superficial −5.67 −7.50 0.577 digital panoramic intermediate 11.50 18.80 0.069 superficial −12.75 −21.00 0.085 cbct intermediate 9.80 15.60 0.252 superficial −11.30 −14.10 0.584 a paired t-test *differences were considered to be statistically significant when p < 0.05. discussion the cytotoxic effects of panoramic and cbct lead to cell death3,4,14,15. cellular death is believed to be a nongenotoxic mechanism induced by carcinogenesis1,2. genotoxic and cytotoxic x-ray effects on exfoliated buccal mucosal and gingival cells were detected when a series of x-ray dental imaging exams, including full mouth radiograph, panoramic, lateral cephalometric radiographs, posteroanterior cephalometric radiographs, and cbcts, were performed14-16. further, analog panoramic radiographs showed a significant increase in post-exposure micronuclei relative to digital panoramic radiographs12. exposure to carcinogens also affects the maturation pattern of the oral mucosa, as detected by papanicolaou10. this study aimed to evaluate whether x-ray radiation from dental imaging affects buccal mucosa and gingiva epithelium maturation patterns. this research is the first study to assess the maturation patterns after dental imaging by cytopathology. oral epithelium maturation involves several widely studied confounding factors, such as mouth rinse utilization, oral lesion, the use of orthodontic and prosthodontic appliances, smoking, and alcohol usage8,10,17-19. in this study, the patient with those confounding factors was excluded. the first cell count represented any consequences of cytotoxic agents before radiation exposure. therefore, any differences emerging between before and after radiation exposure can be attributed to radiation. this study showed that normal mucosa smears contained no parabasal cell, and no significant change was noted in the intermediate and superficial cell numbers for 10 days. burzlaff et al. have confirmed that cells in the superficial and intermediate layers and, in rare cases, the parabasal layer are normally observed in oral mucosal smears8. normal maturation pattern shows the balanced proportions of cells categorized as superficial, intermediate, and parabasal in exfoliated cells. the alteration in oral epithelial cell composition is related to the abnormal composition of cell 7 yanuaryska et al. types. cytopathological studies to assess the maturation pattern of oral mucosal cells of patients exposed to tobacco and alcohol found fewer superficial cells and an increased number of intermediate cells8. these findings are in line with our results that more intermediate cells were detected after radiation exposure, implying the alteration of maturation pattern. in the present study, the number of intermediate cells increased (p < 0.05), whereas that of superficial cells decreased (p < 0.05) after exposure to analog/digital panoramic or cbct. this finding may correlate with the cytotoxicity effect of dental imaging on oral mucosa cells. x-ray exposure during panoramic dental radiography triggers a cytotoxic effect by increasing apoptosis1. the number of karyolytic, karyorrhexic, and pyknotic cells showed a significant increase after panoramic radiography and cbct scan3,14. these types of cells represent the cell death process, which enables the elimination of defected cells, resulting in a massive discharge of superficial cells followed by increased mitosis on the parabasal layer as replacement20. the homeostatic mechanism of the parabasal layer that produces new cells replaces the loose cells; the more severe the lesion, the more parabasal cells are found8,21. moreover, parabasal cells were detected after x-ray radiation, although the statistic calculation was not significant (p > 0.05) compared with that before radiation. we consider that the cytobrush swab may not reach the deepest layers of the oral mucosal epithelium. cytohistological techniques showed a limited sensitivity of between 79 and 97%22. buccal mucosa and gingiva showed no difference in the number of cells before and after analog/digital panoramic radiography and cbct (p > 0.05). yang et al. verified that the number of nuclear changes in both sites, including those in karyorrhexis, condensed chromatin, pyknosis, and karyolysis, showed no significant increase before and after cbct scan14. however, kesidi et al. compared the differences in micronuclei and other nuclear alterations in buccal mucosa and gingiva after exposure by conventional full mouth radiograph procedure, which revealed a substantial difference with the mean difference being high in the buccal mucosa compared to the gingiva14. this condition may be explained by the differences in radiography, indicating variations in the dosage. differences in radiation dose and repeated exposure may increase the radiation effect on the body, as studies have proven that cellular death increases with radiation dose23. there may be some possible limitations in this study. the first limitation is the reliability of cell frequency measurement within an observer. to avoid this limitation, we assessed intra-rater reliability that reflects the variation of data measurement performed by the observer. intra-rater measurement showed good to excellent reliability in this study. the second limitation concerns the absence of anucleated superficial cell measurement; hence the keratinization index cannot be determined. the keratinization index may be established in future research to provide additional information as a marker to detect early cell changes in oral cancer. the results from the present study suggest that routine x-ray dental imaging exams, such as analog/digital panoramic and cbct, can induce changes in the maturation pattern of buccal mucosa and gingiva epithelium. 8 yanuaryska et al. acknowledgement the authors would like to thank the faculty of dentistry, universitas gadjah mada for ryna dwi yanuaryska (grant number 4305/un1/fkg1/set.kg1/pt/2019) for providing the financial support to execute this study. references 1. cerqueira emm, gomes-filho is, trindade s, lopes ma, passos js, machado-santelli gm. genetic damage in exfoliated cells from oral mucosa of individuals exposed to x-rays during panoramic dental radiographies. mutat res. 2004;562(11):111-7. doi: 10.1016/j.mrgentox.2004.05.008. 2. angelieri f, de oliveira gr, sannomiya ek, ribeiro da. dna damage and cellular death in oral mucosa cells of children who have undergone panoramic dental radiography. pediatr radiol. 2007;37(6):561-5. doi: 10.1007/s00247-007-0478-1. 3. suomalainen a, esmaeili e p, robinson s. dentomaxillofacial imaging with panoramic views and cone beam ct. insights into imaging. 2015;6(1):1-16. doi: 10.1007/s13244-014-0379-4. 4. lorenzoni dc, fracalossi acc, carlin v, ribeiro da, sant’anna ef. mutagenicity and cytotoxicity in patients submitted to ionizing radiation. angle orthod. 2013 jan;83(1):104-9. doi: 10.2319/013112-88.1. 5. mally a, chipman jk. non-genotoxic carcinogens: early effects on gap junctions, cell proliferation and apoptosis in the rat. toxicology. 2002 dec;180(3):233-48. doi: 10.1016/s0300-483x(02)00393-1. 6. montgomery pw. a study of exfoliative cytology of normal human oral mucosa. j dent res. 1951 feb;30(1):12-8. doi: 10.1177/00220345510300010501. 7. holland n, bolognesi c, kirsch-volders m, bonassi s, zeiger e, knasmueller s, et al. the micronucleus assay in human buccal cells as a tool for biomonitoring dna damage: the humn project perspective on current status and knowledge gaps. mutat res. 2008;659(1-2):93-108. doi: 10.1016/j.mrrev.2008.03.007. 8. burzlaff jb, bohrer pl, paiva rl, visioli f, sant’ana filho m, da silva vd, et al. exposure to alcohol or tobacco affects the pattern of maturation in oral mucosal cells: a cytohistological study. cytopathology. 2007 dec;18(6):367-75. doi: 10.1111/j.1365-2303.2007.00473.x. 9. abdelaziz ms, osman te. detection of cytomorphological changes in oral mucosa among alcoholics and cigarette smokers. oman med j. 2011 sep;26(5):349-52. doi: 10.5001/omj.2011.85. 10. baumgart cds, daroit nb, maraschin bj, haas an, visioli f, rados pv. influence of factors in the oral mucosa maturation pattern: a cross-sectional study applying multivariate analyses. braz j oral sci 2016;15:27-34. doi: 10.20396/bjos.v15i1.8647094. 11. shantiningsih rr, diba sf. biological changes after dental panoramic exposure: conventional versus digital. dent j (majalah kedokteran gigi) 2018;51(1):25-8. doi: 10.20473/j.djmkg.v51.i1.p25-28. 12. sandhu m, mohan v, kumar js. evaluation of genotoxic effect of x-rays on oral mucosa during panoramic radiography. j indian acad oral med radiol. 2015;27(1):25-8. doi: 10.4103/0972-1363.167070. 13. koo tk, li my. a guideline of selecting and reporting intraclass correlation coefficients for reliability research. j chiropr med. 2016 jun;15(2):155-63. doi: 10.1016/j.jcm.2016.02.012. 14. kesidi s, maloth kn, reddy kk, geetha p. genotoxic and cytotoxic biomonitoring in patients exposed to full mouth radiographs – a radiological and cytological study. j oral maxillofac radiol. 2017;5(1):1-6. doi: 10.4103/jomr.jomr_47_16. https://doi.org/10.1016/j.mrgentox.2004.05.008 https://doi.org/10.1007/s00247-007-0478-1 https://doi.org/10.20396/bjos.v15i1.8647094 https://doi.org/10.4103/jomr.jomr_47_16 9 yanuaryska et al. 15. yang p, hao s, gong x, li g. cytogenetic biomonitoring in individuals exposed to cone beam ct: comparison among exfoliated buccal mucosa cells, cells of tongue and epithelial gingival cells. dentomaxillofac radiol. 2017;46(5):20160413. doi: 10.1259/dmfr.20160413. 16. li g, yang p, hao s, hu w, liang c, zou bs, et al. buccal mucosa cell damage in individuals following dental x-ray examinations. sci rep. 2018;6:8(1):2509. doi: 10.1038/s41598-018-20964-3. 17. ribeiro da, angelieri f. cytogenetic biomonitoring of oral mucosa cells from adults exposed to dental x-rays. radiat med. 2008;26(6):325-30. doi: 10.1007/s11604-008-0232-0. 18. tandelilin rtc, jonarta al, widita e. maturation index assessment of sodium tripolyphosphate and tetra potassium pyrophosphate based calculus dissolution mouthrinse (periogen®) in moderate gingivitis patients: a histopathological study. jdhodt 2017;6:166-70. doi: 10.15406/jdhodt.2017.06.00218. 19. arruda ep, trevilatto pc, camargo es, woyceichoski ie, machado ma, vieira i, et al. preclinical alterations of oral epithelial cells in contact with orthodontic appliances. biomed pap med fac univ palacky olomouc czech repub. 2011;155(3):299-303. doi: 10.5507/bp.2011.043. 20. squier ca, kremer mj. biology of oral mucosa and esophagus. j natl cancer inst monogr. 2001;(29):7-15. doi: 10.1093/oxfordjournals.jncimonographs.a003443. 21. thomas p, holland n, bolognesi c, kirsch-volders m, bonassi s, zeiger e, et al. buccal micronucleus cytome assay. nat protoc. 2009;4(6):825-37. doi: 10.1038/nprot.2009.53. 22. jagannathan n, ramani p, premkumar p, natesan a, sherlin hj. epithelial maturation pattern of dysplastic epithelium and normal oral epithelium exposed to tobacco and alcohol: a scanning electron microscopic study. ultrastruct pathol. 2013;37(3):171-5. doi: 10.3109/01913123.2013.766292. 23. he jl, chen wl, jin lf, jin hy. comparative evaluation of the in vitro micronucleus test and the comet assay for the detection of genotoxic effects of x-ray radiation. mutat res. 2000;469(2):223-31. doi: 10.1016/s1383-5718(00)00077-2. https://doi.org/10.1259/dmfr.20160413 https://doi.org/10.1007/s11604-008-0232-0 https://doi.org/10.15406/jdhodt.2017.06.00218 https://doi.org/10.5507/bp.2011.043 https://doi.org/10.1093/oxfordjournals.jncimonographs.a003443 https://doi.org/10.1038/nprot.2009.53 https://doi.org/10.3109/01913123.2013.766292 https://doi.org/10.1016/s1383-5718(00)00077-2 1 volume 22 2023 e230106 original article braz j oral sci. 2023;22:e230106http://dx.doi.org/10.20396/bjos.v22i00.8670106 1 department of health, piracicaba city hall, piracicaba, são paulo, brazil. 2 department of health sciences and pediatric dentistry, piracicaba dental school, university of campinas, piracicaba, são paulo, brazil. 3 state university of southwest bahia, bahia, brazil. 4 australian research centre for population oral health, the university of adelaide, adelaide, sa, australia. corresponding author: prof. dr. fábio luiz mialhe avenida limeira 901, areão piracicaba, sp, brasil zip 13414-903 e-mail: mialhe@unicamp.br editor: altair a. del bel cury received: jun 14, 2022 accepted: oct 10, 2022 factors associated with oral health literacy among users of primary health care: a cross-sectional study fernanda maria rovai bado1 , alcir josé de oliveira júnior2 , manoelito ferreira silva junior3 , gustavo hermes soares4 , karine laura cortellazzi2 , fábio luiz mialhe2* aim: to analyze associations between sociodemographic factors, self-perception, self-care practices in health with oral health literacy (ohl) levels among users of primary health care (phc). methods: a cross-sectional and analytical study was performed in piracicaba (são paulo), brazil, in 2018, with a convenience sample of users of phc that were aged over 18 years. data were collected with a questionnaire by a trained dentist in two family health units. the outcome variable was ohl, measured by the ohla-b instrument, which was dichotomized by median into low (≤8 point) and high (>8 point). the independent variables were sociodemographic conditions (age, sex, self-declared skin color, and education), self-perceived oral health and self-care practices in health (tooth brushing frequency, smoking habits, reason for the last visit to the dentist, and source of health information search). unadjusted and adjusted analyses were performed between ohl and independent variables for multiple logistic regression model (p≤0.05). results: the sample consisted of 450 adults. a total of 54.7% had a low ohl. after adjusting for sex and age, ohl was associated with skin color (or=0.57; 95%ci=0.37-085), educational level (or=4.92; 95%ci=3.16-7.79), health information from health professionals (or=2.40; 95%ci=1.42-4.16) and internet (or=2.88; 95%ci=1.59-5.32), toothbrushing >1 time a day (or=3.23; 95%ci=1.27-9.92) and smokers (or=0.42; 95%ci=0.23-0.73). after adjusting for sex, age and income, ohl was associated with (or=0.63; 95%ci=0.41-096), educational level (or=4.06; 95%ci=2.57-6.51) and smokers (or=0.48; 95%ci=0.26-0.84). conclusions: low ohl was associated with socioeconomic factors, source of information and smoking. this fact highlights the importance of health professionals to promote ohl. keywords: health literacy. oral health. primary health care. https://orcid.org/0000-0002-7974-5456 https://orcid.org/0000-0001-9117-6295 https://orcid.org/0000-0001-8837-5912 https://orcid.org/0000-0001-6122-4399 https://orcid.org/0000-0001-9584-9477 https://orcid.org/0000-0001-6465-0959 2 bado et al. braz j oral sci. 2023;22:e230106 introduction primary health care (phc) ensures the well-being of the population by focusing on its needs1. in the oral health field, phc is responsible for prevent and controlling the main oral diseases. in brazil, oral diseases represent one of the three main reasons why people seek health treatments, and this fact indicate the need for coordinated actions between society and health services2. however, for health services to be able to solve problems and monitor them, it is necessary efficient communication processes between users and providers, since it is a critical component in health care3. in this context, it is essential to reflect on how people understand and use the information provided by oral health teams to manage their self-care4. individuals access health information from several sources, which have different degrees of usefulness and accuracy5. all of these issues are dealt with by the field of health literacy (hl), which is defined as the cognitive and social skills that determine motivation and the ability of individuals to gain access to, understand and use information in order to promote and maintain good health6. although there are multiple definitions, it is of mutual agreement that hl contemplates more than reading pamphlets, scheduling appointments, understanding medicine labels or performing actions prescribed by health professionals7-9. high levels of hl enable better decision-making about health self-care, as well as the use of services in an optimized form, including healthier lifestyles and successful management of the social determinants of health8. limited levels of ls are associated with individuals with low education and worse socioeconomic conditions, which together cause worse health outcomes8,9. likewise, oral health literacy (ohl) is a variable associated with the maintaining and promotion of good oral health10-12. studies have shown associations of this construct with the modality and frequency of dental visits by the population and diverse outcomes in oral health10-12. therefore, ohl should be considered an important factor by oral health teams in treatment planning and management strategies for the population. however, little is known about the factors that are associated with ohl in users of health services in the brazil despite the several studies already carried out in the country10-18. this study aimed to investigate the associations between sociodemographic factors, self-perception and self-care practices with ohl among adult users of phc. materials and methods this cross-sectional study was conducted with adult users of family health units (fhu) in a medium-sized city located at the state of são paulo, brazil, in 2018. it was approved by the research ethics committee of the piracicaba dental school (protocol 140/2014), and all subjects signed the free and informed consent form. 3 bado et al. braz j oral sci. 2023;22:e230106 the sample size of 450 participants provided a test power of 90% (β=0.10) with a significance level of 5% (α=0.05) for the effect size found in the study (odds ratio of 2.0 and 50% response in the unexposed group) based on dependent variable ohl. a sample of 535 individuals were invited. thus, the non-response rate was 15.9%. as inclusion criteria for the research, individuals should aged between 18 and 80 years old, self-reported ability to read and speak brazilian portuguese and absence of diagnosis of dementia and visual or hearing impairment. data were collected by a trained dentist (fmrb) in two fhu with dental teams. these units were selected because they were the places where the researcher worked as a dentist. the convenience sample was composed by volunteers who were looking for dental care in the fhu and were contact to participate in the research when waiting for dental or general consultation. questionnaires were applied in the form of an interview. ohl was the outcome variable of this study, and it was measured using the ohla-b instrument, the version of the oral health literacy assessment – spanish (ohla-s) validated to brazilian portuguese13. ohla-b is an instrument for measuring ohl through the pronunciation and comprehension of 15 words in the dental vocabulary. for the evaluation of the ohl score, the pronunciation and comprehension of the words were considered, adding a point for each item when both were correct. if one of the tests were incorrect, the score for this item would be 0. considering the total of 15 items, the score could vary from 0 to 15 points. the higher the score, the higher the ohl levels. ohla-b scores were dichotomized by median of the sample into low (≤ 8 point) and high (>8 point). the independent variables were sociodemographic factors, self-care practices in health and self-perceived oral health. the sociodemographic factors were dichotomized as follows: age (≤36 years old or >36 years old), sex (woman or man), self-declared skin color (white/yellow or brown/black), and education level (low: ≤8 years or high: >8 high years of study). the variables related to self-care practices were dichotomized as: toothbrushing frequency (≤1 time a day or > 1 time a day), smoking (yes or no), reason for the last visit to the dentist (pain/caries or others) and the main source of health information search (tv, radio, books, others/internet/health professionals). in addition, the variable self-perceived oral health was evaluated with a single question “how do you evaluate your oral health?” and was dichotomized as (excellent, very good and good or regular and poor). all statistical tests were performed using the sas 9.4 program (sas institute inc., cary, nc, usa. release 9.4, 2010)19. descriptive analyses of the data were performed, and the associations between each independent variable and the dependent variable ohl were analyzed were using regression analyses. logistic regressions were performed on ohl controlled by age and sex (model 1) and age, sex and socioeconomic status (model 2). the level of significance adopted was 5%. 4 bado et al. braz j oral sci. 2023;22:e230106 results the sample consisted of 450 participants and all the questionnaires were filled completely. most participants were ≤36 years old, declared being white/yellow, having more than 8 years of study, good /very good/ excellent self-perceived oral health, brushing their teeth more than once a day, not smoking, going to the last dentist appointment due to pain/caries, and consulting a health professional as a main source of health information. a total of 54.7% was classified as presenting low ohl taking into account the cut-off point used for the olha instrument. after adjusting for sex and age, ohl was associated with skin color (or=0.57; 95%ci=0.37-085), educational level (or=4.92; 95%ci=3.16-7.79), health information from health professionals (or=2.40; 95%ci=1.42-4.16) and internet (or=2.88; 95%ci=1.59-5.32), toothbrushing >1 time a day (or=3.23; 95%ci=1.27-9.92) and smokers (or=0.42; 95%ci=0.23-0.73). after adjusting for sex, age and income, ohl was associated with skin color (or=0.63; 95%ci=0.41-096), educational level (or=4.06; 95%ci=2.57-6.51) and smokers (or=0.48; 95%ci=0.26-0.84) (table 1) table 1. adjusted analysis between ohl with sociodemographic factors and self-care practices in health, piracicaba, sp, 2018. characteristic crude or 95% ci p-value adjusted or* 95%ci p-value adjusted or┼ 95% ci p-value socioeconomic white / yellow 1 1 1 brown / black 0.86 0.38-0.86 0.007 0.57 0.37-0.85 0.006 0.63 0.41-0.96 0.032 low educational level 1 1 high educational level 4.50 2.98-6.88 <0.001 4.92 3.16-7.79 <0.001 4.06 2.57-6.51 <0.001 self-perception of health poor/regular self-perceived oral health 1 1 1 good/very good/excellent self-perceived oral health 0.81 0.54-1.22 0.314 0.82 0.54-1.23 0.349 0.75 0.49-1.14 0.174 source of health information tv, radio, books, others 1 1 health professional 2.45 1.45-4.24 <0.001 2.40 1.42-4.16 0.001 2.18 1.27-3.83 internet 3.03 1.70-5.53 <0.001 2.88 1.59-5.32 <0.001 2.18 1.18-4.10 0.052 continue 5 bado et al. braz j oral sci. 2023;22:e230106 continuation oral health behaviours tooth brushing ≤ 1 time a day 1 1 1 tooth brushing >1 time a day 3.33 1.3110.19 0.018 3.23 1.27-9.92 0.022 2.75 1.06-8.57 non-smoker 1 1 1 smoker 0.41 0.23-0.72 0.002 0.42 0.23-0.73 0.002 0.48 0.26-0.84 0.013 reason for the last visit to the dentist others 1 1 1 pain/caries 1.30 0.89-1.91 0.175 1.27 0.86-1.87 0.224 1.04 0.70-1.57 0.830 *adjusted for age and sex ┼adjusted for age, sex and income discussion this study showed that low levels of ohl among adult users of phc were associated with sociodemographic factors, source of health information and smoking habits. our results corroborate findings in the literature using other ohl instruments in other countries20-26. brown and black individuals had a lower level of ohl, indicating the presence of ethnic inequity, corroborating other studies that verified this issue among non-whites9,20-23, even considering a great variability in the studies design, in the composition of the samples, and in the instruments used. all of these studies suggest that patients that declared being brown or black may have greater difficulties in understanding health information, which shows the need to direct health improvement programs to these populations. the association between low education and low ohl is well described in the literature20,22-25. therefore, the longer the years of study, the better the processes of understanding health information, reverberating in better levels of health and oral health literacy26,27. our results corroborate this association in the context of primary health care in brazil, a fact that brings new information for the planning of health teams working at this level of care and reinforce the importance of public health policies to focus on socially disadvantaged people in order to promote their acess to health services and resolution capacity28. among oral health behaviors, it was observed associations between smoking behaviour with low ohl, corroborating the fact that risky health choices can be associated with inadequate ohl. to our knowledge, very few studies have evaluated this variable as a predictor of ohl levels23,29,30, although others studies found associations with general health literacy31-33, a fact that should be investigated in future studies in larger populations. considering that the use of cigarettes influences the activity of periodontal disease34, it is assumed that low levels of ohl may be predictors of a worse periodontal condition, therefore, health professionals need to pay more attention in patients with low ohl levels. 6 bado et al. braz j oral sci. 2023;22:e230106 in relation to the influence of source of information of health on ohl, it was observed that health professionals and internet presented considerable importance in the verified associations when model were adjusted for sex and age. however, they did not remain statistically significant with the outcome when adjusted for sex, age and income. despite not remaining in the model 2 (p>0.05), the use of internet as the main source of health information presented a value very close to statistical significance (p=0.052). some studies have shown that the internet and health professionals were the most sought-after sources of information on oral health24. these results suggest that individuals with high ohl are more proactive on the search for health information but these hypotheses should be tested in future studies. the variable brushing teeth more than once a day remained associated with ohl levels in the model adjusted for age and sex, but lost its significance in the income-adjusted model. studies have shown that those who have better levels of health literacy also have better brushing habits12,35 this is probably a bidirectional relationship, as those who take better care of their oral health also seek more knowledge on the subject. the above findings reinforce the influence of social determinants of health on ohl levels. therefore, it highlights the importance of health professionals and services in providing accurate and easy-to-understand information to users in order to reduce health inequities. some of the limitations of this study include the use of an ohl instrument that has a limited capacity to assess wider aspects of ohl, as it assesses just functional, but not communicative and critical ohl36-37 . in addition, the variables included in the analysis, the measurement methods of behaviors variables and the self-reporting nature of data related to oral health outcomes are limitations of this study that should be overcome in future studies. lastly, the results found in the selected sample may not reflect the reality of all adults in the city. in conclusion, functional ohl, measured through the olha-b scores, was associated with skin color, educational level and smoking habits after adjusted by age, sex and income. because ohl is associated with better health outcomes, actions to increase their levels based on individuals’ characteristics and their context should be considered by health professionals working on phc with intersectorial partnerships. acknowledgments the brazilian national council for scientific and technological development (cnpq). data availability datasets related to this article will be available upon request to the corresponding author. conflict of interest none. 7 bado et al. braz j oral sci. 2023;22:e230106 author contribution fmrb, flm and klc: conception of the work, interpretation of data, drafting the work; critical review; ghs: analysis and interpretation of data; mfsj and ajoj: drafting the work. all authors approved the final version to be published. all authors approved the final submitted version. references 1. world health organization & united nations children’s fund (unicef). a vision for primary health care in the 21st century: towards universal health coverage and the sustainable development goals. world health organization; 2018 [cited 2021 mar 10]. avaible from: https://apps.who.int/iris/ handle/10665/328065. 2. antunes jl, toporcov tn, bastos jl, frazão p, narvai pc, peres ma. oral health in the agenda of priorities in public health. rev saude publica. 2016 sep; 50:57. doi: 10.1590/s1518-8787.2016050007093. 3. gutierrez n, kindratt tb, pagels p, foster b, gimpel ne. health literacy, health information seeking behaviors and 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strategies into the 21st century. health promot int. 2020 sep; 15(3):259-67. doi: 10.1093/heapro/15.3.259. 37. dickson-swift v, kenny a, farmer j, gussy m, larkins s. measuring oral health literacy: a scoping review of existing tools. bmc oral health. 2014 dec; 14:148. doi: 10.1186/1472-6831-14-148. 1http://dx.doi.org/10.20396/bjos.v20i00.8661632 volume 20 2021 e211632 original article 1 national university federico villarreal, lima, peru corresponding author: percy alfonso delgado rojas national university federico villarreal, lima, peru pdelgadorojas20@gmail.com editor: dr altair a. del bel cury received: october 14, 2020 accepted: may 12, 2021 relationship between extrinsic factors and non-carious cervical lesions in patients of the national hospital “hipólito unánue” percy alfonso delgado rojas1* non-carious cervical lesions cause destructive dental disorders that actively contribute to the progressive loss of dental structure and the immediate need for dental treatment, due to their multiple symptoms and factors that produce them. aim: the purpose of the study was to determine the relationship between extrinsic factors and non-carious cervical lesions in patients of the national hospital hipólito unánue. methods: the research was of a descriptive correlative type. the sample consisted of male and female patients between 18 and 65 years old, who attended the carielogy service of this hospital. for data collection, 2 questionnaires were used to estimate the values of the extrinsic factors that allowed us to obtain the necessary information on the variables to be studied. spearman’s rho was applied to determine the relationship between the variable’s study. results: according to spearman’s rho of 0.622, compared to p-0.000 <0.01. between the variables studied; extrinsic factors and noncarious cervical lesions there is a moderate and significant positive correlation. conclusion: through this section it was possible to demonstrate the existing relationship between extrinsic variable factors and non-carious cervical lesions, therefore it was concluded that there is a moderate and significant positive correlation in the sample comprised by the patients of the hospital in mention. keywords: tooth abrasion. tooth erosion. tooth wear. dentin. dental stress analysis. https://orcid.org/0000-0001-6084-6129 2 rojas et al. introduction oral diseases represent a serious health problem of high frequency with which pain, functional impairment and discomfort are produced, so immediate attention is necessary, as well as its effective and rapid diagnosis to achieve an adequate treatment1. indeed, today’s lifestyle follows a regular common pattern in most individuals, in such a way that factors, such as the high rate of stress, eating habits and social demands to which humans are constantly subjected have increased the degree and types of non-carious cervical lesions2. in this order of ideas, it is possible to say that non-carious cervical lesions are the loss of tooth structure, located at the amelocementall boundary. it comes in various forms that include sensitivity, in some cases, the dental pulp become compromised3. nevertheless, it should be taken into account that on many occasions these ones are not noticed by health professionals, and even less by patients, which is why it is important to have a correct diagnosis and treatment of these lesions. unlike cavity-affected dentin, non-carious cervical lesions (nccls) present a high degree of sclerosis, with partial or complete obliteration of the dentin tubules, preventing the formation of hybrid layer4. in addition, epidemiologically, it is indicated that lesions caused by imperfections and reduction of dental tissue on the incisal and occlusal surfaces, creates a greater sensation of hypersensitivity. this situation occurs if they originate on the cervical surface, loss of esthetics and gingival retraction, as do non-carious dental lesions, whose origin is multifactorial and with different clinical characteristics1,5. however, gonzález garcía et al.1 argue that lesions in the neck of the teeth are seen and most of the time go unnoticed because of chronic symptomatology, except for exceptions. in fact, chronic loss of tooth structure in the neck of the teeth characterize  this kind lesions  without having a bacterial etiology, but considering that more than one factor causes it. they can be present in different forms, mainly on the vestibular or oral side of the teeth, with or without dentin sensitivity6. indeed, patients with non-carious cervical lesions disguise their esthetic discomfort by avoiding the visibility of these lesions due to enamel loss and gingival regression. although, these lesions have not aroused much interest in researchers or clinicians, they are now common in clinical practice due to the large increase that has made it one of the frequent stomatological conditions7. in this sense, several studies indicate a highly variable prevalence and propose a positive correlation with demographic factors, age and the use of toothpastes with abrasive components, at least in one of the varieties8. regarding the etiology of nccls, toothbrushing was historically considered and identified as the main cause, defining it as toothpaste abrasion1. several factors influence the rate of abrasion, including the toothbrushing technique, brushing force, brushing frequency, bristle stiffness and toothpaste abrasiveness9. due to the aforementioned fact and because of the increasing number of patients retaining natural teeth at older ages.  the widespread availability of preventive oral care information, the large number of oral hygiene products in home, has increased 3 rojas et al. considerable interest toward a better understanding of the functions of toothbrush, toothpaste and toothbrushing behaviors in dental use10. in addition, the consumption of alcohol was also correlated with nccls, which is explained due to the erosive potential of some of these beverages11.  a useful concept concerning nccls is the abfraction concept, which is defined as lateral forces that create stress in the cervical areas of the teeth and disrupt the enamel microstructure12. in this study, the non-carious cervical lesions that will be considered are erosion, abrasion, abfraction and stress corrosion. it is important to keep in mind that the initial signs that occur in these types of lesions are not taken as important and are associated with other types of more common or morbid pathologies in the oral cavity.  some of them, such as cariogenic disease or gingivitis are deemed a common condition, without realizing that it is much more worrisome, as it can occur indiscriminately in people regardless of sex, but these pathologies increase over the years. therefore, this research considers important aspects of oral health at the national level, those that have not been frequently addressed by previous researchers5. in this line of thought, this research verified the relationship between extrinsic factors and non-carious cervical lesions in patients of the hipólito unánue national hospital. in addition, the following research hypothesis was devised: there is a link between extrinsic factors and non-carious cervical lesions in patients of the national hospital hipólito unánue. the types of non-carious cervical lesions can be found individually in a patient or comprehensively in multiple combinations in the same patient. due to the etiological factors that can produce them, the cervical third of the dental pieces and the sensitivity caused by these lesions are part of the daily consultation for the dentist. this is due to the difficulties in performing basic and necessary actions such as eating, chewing or laughing, all this product of the loss of dental enamel7. in other circumstances, these types of lesions can also be associated with professional practices that have failed. throughout history, many authors have defined, based on their evidence, the multifactorial and triggering aspects of non-carious cervical lesions, as well as providing alternative solutions or measures for early diagnosis and treatment with the aim of improving the quality of life of people and avoiding esthetic and functional disability. in this study, the classification established by cuniberti and rossi2 of non-carious cervical lesions is considered in four types: abrasion, erosion, abfraction and stress corrosion, most of them located at the level of the amelocementary junction border (ajb). the first one  abrasion according to cuniberti and rossi2,  is the pathological wear of the tooth structure caused by abnormal mechanical processes from foreign objects or substances introduced into the mouth. this process occurs when contacting teeth generate the loss of hard tissues by using mechanisms such as polishing, rubbing or scraping. these sorts of injuries are noticed all through dental structures such as lacquer, dentin, and even cementum. furthermore, they happen because of the poor toothbrushing method, kinds of toothpaste with high rough substances, and related to the use of mouthwashes12. it is important to highlight the role played by harmful habits in the appearance of abrasion.  such habits  as holding nails or other work instruments between the teeth and lips,  as occurs with shoemakers and construction workers. in 4 rojas et al. other cases, certain musical instruments such as the harmonica and factors associated with work such as environmental dust in patients who work with abrasive substances such as carborundum dust3. the application of incorrect and inadequate periodontal, prosthetic and orthodontic treatment should also be considered in its etiology. the second one erosion, considered also by cuniberti and rossi3, as the loss of the surface structure of the teeth by chemical action in the continuous presence of demineralizing agents, especially acids. this type of lesion does not involve the presence of bacteria. in addition, ortuño et al.6 state that this kind of lesion due to the continuous presence of demineralizing agents, especially acids of non-bacterial origin, act as catalysts by preparing the dental tissue and accelerating the action of mechanical factors such as incorrect brushing or poorly adapted prostheses, thus favoring faster wear. this type of lesion is characterized by being multifactorial since they include chemical and mechanical factors, and the acids produce demineralization of the enamel in early stages. in the third place, another lesion that can be presented is  abfraction, wedgeshaped.  this lesion is  distinguished by being rapidly advancing and destructive, which in advanced cases can lead to tooth fracture5. its etiology involves the lateral or eccentric component in the lingual-vestible direction of the occlusal forces. that case results into the concentration of forces in the amelocemental junction border and thus determining the bending of the tooth, which will oppose this force with another, but in the opposite direction, of equal resistance.  that  situation will cause tension that manifests itself as fatigue in the cervical third causing the aforementioned bending. likewise, cuniberti and rossi2 defined it as compression syndrome because the lesion is evidence of a set of signs (loss of tooth structure in the form of a wedge, and fracture and repeated detachment of restorations) and symptoms (dentin hypersensitivity, when the lesion is active). the fourth one according to rodríguez chala7, is stress corrosion. this lesion is defined as the synergistic interaction of mechanical forces and corrosive reactions, with climatic, environmental or electrochemical factors, which degenerate or destroy a material. for ruiz candina8. it is the physicochemical degradation resulting from the biodynamics occurring in the oral cavity. this lesion is referred to as flexural stress in an acidic environment and is the reason why the loss of enamel is approximately 10 times greater than in an environment that is not exposed to these substances. materials and methods the present investigation is descriptive and correlational, since the variables were described and the degree of between them was measured13. in this sense, variables, extrinsic factors such as polishing, rubbing or scraping to mention some and also non-carious cervical lesions were measured, and then the relationship between them was determined. with regard to the population used for the research, it consisted of patients over 18 years of age who attended the dental surgery service of the dental department of the national hospital hipólito unánue between the months of january and march 2018, which according to the statistical information source consulted would be limited with an average of 252 monthly patient care. the population was a sample of 100 persons between 18 and 65 years of age was randomly drawn. 5 rojas et al. the formula used was as follows: n = z2 (p * q) z2 (p * q) ne 2 + where: n: sample size z: desired confidence level p: proportion of the population to the desired characteristic (success) q: proportion of population without desired characteristic (failure) e: level of error willing to commit n: population size the data were obtained through the application of two instruments; the first was a questionnaire own designed by the author, for the estimation of values in relation to extrinsic factors evaluated, structured by 31 items, under the dichotomous scale (yes/ no); which allowed determining the levels of presence of the dimensions of the variable: factors associated with erosion, abrasion, abfraction and stress of the patients studied, detailing these elements in table 1. it is important to point out that the questionnaire was validated and its reliability was determined, obtaining a cronbach’s alpha coefficient equal to 0.83. subsequently, the patients who constituted the units of analysis in this research were surveyed through the use of a questionnaire to collect information on extrinsic factors. table 1. operational definition of the variable extrinsic factors dimensions indicators items levels associated with the abrasion presence / no presence from 9 to 16 low: 0 7 regular: 8 16 intermediate: 17-24 high: 25-31 associated with the abfraction presence/ no presence from 16 to 23 associated with the corrosion by stress presence/ no presence from 24 to 31 the second was an observation guide, of own design and applied to measure the estimation of values in relation to medically assessed non-carious cervical lesions, structured by 13 items, under the dichotomous scale; which allowed determining the levels of presence of the dimensions of the referred variable: erosion, abrasion, abfraction and stress corrosion of the studied patients, attending the clinical evaluation of the specialists; these elements are detailed in table 2. it is worth mentioning that the guide was validated and its reliability was determined, obtaining a cronbach’s alpha coefficient equal to 0.80. in addition, the observation guide was calibrated according to the criteria used by specialists. this was made in order to conduct the respective clinical evaluation of non-carious cervical lesions, and by these criteria, the levels of these lesions were determined. 6 rojas et al. table 2. operational definition of variable non-carious cervical lesions. dimensions indicators items levels erosion presence /no presence from 1 to 3 low: 0 3 regular: 4 -7 intermediate: 8 10 high: 11-13 abrasion presence/no presence from 4 to 7 abfraction presence/non-presence from 8 to 10 corrosion by stress presence/no presence from 11 to 13 for the analysis of the data obtained, a descriptive scheme based on percentages was used and to measure the variables, scales were used to determine the levels of presence according to the scores obtained in the instruments applied. in addition, spearman’s rho coefficient was applied to establish the relationship between the two variables studied. the software used was the static package for social sciences spss version no 22, the results are presented in tables, under the use of the scales established for the analysis. ethical aspects all persons who participated in the research were notified of the purpose of the research and were assured of strict confidentiality, anonymity and that the results would be for the exclusive use of the study. thus, after agreeing to participate, they signed an informed consent form. moreover, this research study was submitted to and approved by the institutional research ethics committee of the hospital nacional hipólito unánue, under the registration code rcei-54. results as figure 1 shows, 80% of the patients were diagnosed with dental erosion, while 71% of the patients were diagnosed with dental abfraction, and additionally 57% presented cases of abrasion and stress corrosion; these rates correspond to the population of 100 evaluated patients who attended the karyography section of the hospital nacional hipólito unánue. 10 20 30 40 50 60 70 80 90 erosion abrasion abfraction stress corrosion 0 number of cases by type of cervical lesion non-carious figure 1. number of cases by type of non-carious cervical lesion. 7 rojas et al. the first dimension of the 4 cervical lesions evaluated was erosion. figure 2 shows that there is a tendency from an intermediate to a regular level in terms of extrinsic factors and this type of lesion, in this regard 30% of the patients evaluated presented an intermediate level of extrinsic factors, showing intermediate values associated with dental erosion; on the other hand, 22% of the patients presented a regular level of extrinsic factors compared to regular levels of presence of erosion, while 12% presented a low level of extrinsic factors compared to low levels of presence of dental erosion and additionally 10% of the patients presented high levels of extrinsic factors compared to high levels of presence of dental erosion, the results being evident in relation to the population studied, which were the patients of the national hospital hipólito unánue. low regular intermediate high 0 extrinsic factors and erosion 5 10 15 20 25 30 35 figure 2. percentage levels of extrinsic factors and erosion. the second dimension, of the 4 cervical lesions evaluated, was abrasion, determining that the tendency would be to an intermediate and regular level. in that sense, it has been shown that 27% of the patients have intermediate levels of extrinsic factors showing intermediate levels of dental abrasion; 24% had regular levels of extrinsic factors versus regular presence of dental abrasion, 14% high levels of extrinsic factors versus high levels of dental abrasion, and only 12% showed low levels of extrinsic factors, showing low presence of dental abrasion as shown in figure 3. 0 5 10 15 20 25 30 r ec ou nt low regular intermediate high low regular intermediate high extrinsic factors and abrasion figure 3. percentage of extrinsic factors and abrasion levels. 8 rojas et al. the third dimension, of the four cervical lesions evaluated, was dental abfraction. figure 4 shows that there is a tendency from high to intermediate levels in terms of extrinsic factors, in this sense, 22% of the patients showed intermediate levels of extrinsic factors, showing intermediate levels of the presence of dental abfraction; likewise, 14% of the patients presented high levels of extrinsic factors versus a high presence of dental abfraction. meanwhile, 12% showed regular levels of extrinsic factors versus regular levels of dental abfraction, and only 13% of the patients showed low levels of extrinsic factors, showing a low presence of dental abfraction in the patients of the national hospital hipólito unánue. r ec ou nt low regular intermediate high low regular intermediate high extrinsic factors and abfraction 0 5 10 15 20 25 figure 4. percentage of extrinsic factors and abfraction. the fourth and last dimension, of the 4 cervical lesions evaluated, was stress corrosion, showing the existence of an intermediate to low-level trend in extrinsic factors; 25% of the patients evaluated had intermediate levels of extrinsic factors in the presence of intermediate onset stress corrosion; 15% had a low level of extrinsic factors in the presence of low stress corrosion; 10% showed a high level of extrinsic factors expressed in high levels of stress corrosion; and finally, 10% of patients had regular levels of extrinsic factors when they had regular levels of cervical carious lesions, as it is shown in figure 5. 0 5 10 15 20 25 30 r ec ou nt low regular intermediate high low regular intermediate high extrinsic factor levels and stress corrosion figure 5. percentage of extrinsic factor levels and stress corrosion. 9 rojas et al. hypothesis testing from the results obtained, shown in table 3, the statistics are presented in terms of the degree of correlation between the variables determined by spearman’s rho which is equal to 0.622 indicating that there is a positive, moderate and significant relationship between the variables compared to the degree of statistical significance p < 0.01, a sufficient condition to reject the null hypothesis (ho) and accept the alternative hypothesis (ha): there is a relationship between extrinsic factors and cervical injuries in patients of the national hospital hipólito unánue. table 3. correlation between extrinsic factors and non-carious cervical lesions rho of sperman extrinsic factors non-carious cervical lesions factors extrinsic correlation coefficient next. (bilateral) n 1000 0.622** 0.000 n non-carious cervical lesions correlation coefficient next. (bilateral) n 0.622** 0.000 1000 discussion according to the results obtained, it was possible to determine that there is a positive relationship between extrinsic factors and non-carious cervical lesions, which coincides with the statement of several authors that these factors have a great influence on the development of this type of lesions14. among these extrinsic factors, some foods are mentioned as in contact with acid vapors by inhalation15 and even by activities such as swimming16. regarding abrasion, it is evident that the relationship between this non-carious cervical lesion, in terms of extrinsic factors would be between intermediate and regular, which coincides with a study in which this same alteration was evaluated in young people and adults17,18. alternatively, it was determined that the analysis of the relationship of extrinsic factors with respect to stress corrosion was medium-low, which coincides with the work that shows that although stress corrosion can cause damage, its incidence remains at intermediate levels with respect to external factors such as external acids19. however, as the dental cover significantly decreases the stress in the area will increase20, especially due to toothbrush misuse, which according to several studies is observed as the main external factor responsible for the appearance of non-carious cervical lesions due to stress21,22.   when comparing the results obtained using other studies, the derivations of the research conducted by busleimán23 are confirmed.  in that research he concludes that the most frequent lesions found in the cervical third of the dental elements were non-cariogenic cervical lesions. also, he stated that the evaluation of the intermediate factors indicated that the lesions were associated with a low and moderate cariogenic risk, coinciding with the levels reported in this research. however, in the research conducted by abarca pineda24, he found that functional habits are the main etiological factors in the appearance of abrasion. furthermore, in addition, he also found a significant relationship between acidic diets 47.8% and the 10 rojas et al. existence of non-carious cervical lesions (erosion), which indicates that the consumption of acidic foods predisposes to the appearance of such damage, corroborating these results with this study. in the same line of research, the work of castillo guerra25 is reaffirmed, who concluded that there is a relationship between the presence of non-carious lesions and a healthy lifestyle. therefore, a direct correlation was found between variable lifestyle habits and variable non-carious lesions. likewise, in the research performed by viera jácome26. the data obtained demonstrate the lack of knowledge of the population about the factors associated with this type of lesion. these results indicate the need for a treatment plan to control hypersensitivity. it is important to point out that the results obtained by romero27 also confirm the results of this investigation, since it was concluded that a high presence of abfraction lesions and the factors influences their appearance. the level of abfraction was high in patients who suffered emotional stress. this study also reports stress as a determining factor of the referred injuries. in the research conducted by cerna bacerra28, he reports that there is a high prevalence of non-carious cervical lesions in this study group, the most significant being dental wear, coinciding with the derivations of the research conducted. in the research performed by guzmán zavaleta29, among the main results a prevalence of abfractions of 24% was found, although a greater prevalence of abfractions was observed in patients with higher levels of stress, no statistically significant relationship was found between abfractions and stress levels, these results differ from those of this study, since it was verified that there is a positive relationship between extrinsic factors and non-carious cervical lesions. alternatively, the research performed by segura escudero30 found similar results to this study: among the types of non-carious cervical lesions present in the population, the most frequent were abrasion with a percentage of 60%, followed by abfraction with 48% and then erosion with 15%. it also showed that there is a relationship between the frequency of abrasion and the time patients take to start brushing. likewise, the research performed by reyes et al.31 reported that 83.3% had dental caries, there was dental wear due to attrition in 43.3% and wear due to abfraction in 31.7%, concluding that stress has a direct and significant relationship with the presence of non-carious lesions in the population studied; the aspect that is confirmed by similar results in this study. and in the work carried out by latorre lópez32. the results showed that 40.7% of the population studied non-carious lesions. these results reveal some similarities with the results of our investigation., which indicates the existence of a moderate and significant positive correlation between the extrinsic factors of the variable and non-carious cervical lesions in patients at the national hospital hipólito unánue. regarding the positive relationship shown between extrinsic factors and dental abrasion, abfraction and stress corrosion, it is recommended that the factors that have a direct impact on the production of these pathologies be consistently and continuously discouraged through educational and demonstration sessions given daily in the waiting rooms by patients and/or students who collaborate daily in the care services. 11 rojas et al. there were not significant limitations on this study due to the access of the information, however, like all research, this one required time and dedication for its pertinent development. in conclusion, there is a moderate and significant positive correlation between the variables extrinsic factors and non-carious cervical lesions of the patients of the hipólito unánue national hospital. on the other hand, this study will serve as a basis for the development of future research on this topic, which could be aimed at investigating the risk factors and the correct treatment plan for non-carious cervical lesions. as a scientific contribution of great relevance to future research and projects related to this topic. conflict of interest the author states that he has no conflict of interest. references 1. gonzález garcía x, cardentey garcía j, martínez pérez mb. [non-carious cervical injuries in adolescents in a health care area]. rev cienc med pinar rio. 2020;24(2): e4324. spanish. 2. cuniberti n, rossi gh. [non-carious cervical lesions]. rev ateneo argent. odontol. 2017;57(2):35-8. spanish. 3. cuniberti n, rossi g. [a different view on non-carious cervical lesions why cervical wedge lesions are not caused by acid erosion]. rev oper dent biomater. 2017; 6(2): 1-10. spanish. 4. loguercio ad, luque-martinez iv, fuentes s, reis a, muñoz ma. effect of dentin roughness on the adhesive performance in non-carious cervical lesions: a double-blind randomized clinical trial. j dent. 2018 feb;69:60-9. doi: 10.1016/j.jdent.2017.09.011. 5. cerón xa, narváez rf, madroñero ae, chávez ls, tobar as. [prevalence of non-carious lesions causing hypersensitivity in patients at the pasto dental clinic]. rev colomb invest odontol. 2016;7(19);25-33. spanish. doi: 10.25063/21457735.234. 6. ortuño d, mellado b, prado s, vargas jp, rada g. [restorations of non-carious cervical lesions: a review protocol systematic for clinical practice]. ars med. 2018;43(2):33-41. spanish. doi: 10.11565/arsmed.v43i2.1073. 7. rodríguez chala h, hernández pampim y, gonzález fernández c. [non carious cervical lesions in patient of the area of health “electric”,arroyo naranjo municipality, 2015]. rev cubana estomatol. 2016;53(4):188-97. spanish. 8. ruiz candina hj, herrera batista aj, gamboa sousa j. [non-carious dental lesions in patients treated at the siboney stomatology clinic]. rev cuba invest biomed. 2018;37(2):46-53. spanish. 9. sawlani k, lawson nc, burgess jo, lemons je, kinderknecht ke, givan da, et al. factors influencing the progression of noncarious cervical lesions: a 5-year prospective clinical evaluation. j prosthet dent. 2016 may;115(5):571-7. doi: 10.1016/j.prosdent.2015.10.021. 10. turssi cp, kelly ab, hara at. toothbrush bristle configuration and brushing load: effect on the development of simulated non-carious cervical lesions. j dent. 2019 jul;86:75-80. doi: 10.1016/j.jdent.2019.05.026. 11. yoshizaki kt, francisconi-dos-rios lf, sobral ma, aranha ac, mendes fm, scaramucci t. clinical features and factors associated with non-carious cervical lesions and dentin hypersensitivity. j oral rehabil. 2017 feb;44(2):112-8. doi: 10.1111/joor.12469. 12 rojas et al. 12. heasman pa, holliday r, bryant a, preshaw pm. evidence for the occurrence of gingival recession and non-carious cervical lesions as a consequence of traumatic toothbrushing. j clin periodontol. 2015 apr;42 suppl 16:s237-55. doi: 10.1111/jcpe.12330. 13. hernández r, fernández c, baptista p. [research methodology]. méxico: grawhill; 2018. spanish. 14. coupal i, sołtysiak a. dental erosion in archaeological human remains: a critical review of literature and proposal of a differential diagnosis protocol. arch oral biol. 2017 dec;84:50-7. doi: 10.1016/j.archoralbio.2017.09.011. 15. nascimento mm, dilbone da, pereira pn, duarte wr, geraldeli s, delgado aj. abfraction lesions: etiology, diagnosis, and treatment options. clin cosmet investig dent. 2016 may;8:79. doi: 10.2147/ccide.s63465. 16. paryag a, rafeek r. dental erosion and medical conditions: an overview of aetiology, diagnosis and management. west indian med j. 2014 sep;63(5):499-502. doi: 10.7727/wimj.2013.140. 17. jaeggi t, lussi a. prevalence, incidence and distribution of erosion. monogr oral sci. 2014;25:55-73. doi: 10.1159/000360973. 18. teixeira dnr, zeola lf, machado ac, gomes rr, souza pg, mendes dc, et al. relationship between noncarious cervical lesions, cervical dentin hypersensitivity, gingival recession, and associated risk factors: a cross-sectional study. j dent. 2018 sep;76:93-7. doi: 10.1016/j.jdent.2018.06.017. 19. sugita i, nakashima s, ikeda a, burrow mf, nikaido t, kubo s, et al. a pilot study to assess the morphology and progression of non-carious cervical lesions. j dent. 2017 feb;57:51-6. doi: 10.1016/j.jdent.2016.12.004. 20. leal nms, silva jl, benigno mim, bemerguy ea, meira jbc, ballester ry. how mechanical stresses modulate enamel demineralization in non-carious cervical lesions? j mech behav biomed mater. 2017 feb;66:50-7. doi: 10.1016/j.jmbbm.2016.11.003. 21. cerezo-román j, anderson b. deconstructing non-carious cervical lesions on teeth in forensic contexts. in: schmidt cw, watson jt, editors. dental wear in evolutionary and biocultural contexts. london: elsevier; 2020. p.123-42. doi: 10.1016/b978-0-12-815599-8.00006-x. 22. matarrita ae, sotela truque p. [case report: treatment of abfraction due to bruxism]. rev electron fac odontol ulacit. 2014;7(2):16-32. spanish. 23. busleiman fj. [evaluation of factors associated with lesions of the cervical third of the dental elements and their relationship with the periodontium] [thesis]. córdoba: universidad nacional de córdoba; 2015. spanish. 24. abarca pineda jp. [etiological factors involved in the appearance of non-carious injuries in the university students aged 18-30 national of loja of the modality of studies presential period may-july 2014] [thesis]. ecuador: universidad nacional de loja; 2014. spanish. 25. castillo guerra d. [incidence of non-carious lesions and its relationship with habits that are part of a lifestyle considered healthy in 300 people in the cumbayá valley] [thesis]. ecuador: universidad san francisco de quito; 2011. spanish. 26. viera jácome dg. [diagnostic study on the incidence of hypersensitivity in patients with non-carious cervical lesions at the san miguelito parish health center in santiago de píllaro county] [thesis]. píllaro, ecuador: universidad autónoma regional de los andes; 2011. spanish. 27. romero f. abfractions: [abfractions: non-carious cervical in wedge, its relationship with stress]. acta odontol venez. 2012; 50(2):1-7. spanish. 28. cerna becerra al. [prevalence of tooth enamel alterations in older adults in the “hogar san josé” nursing home in trujillo, 2016] [thesis]. trujilio, perú: universidad privada antenor orrego; 2016. spanish. 29. guzmán zavaleta ve. [prevalence of abfractions and its relationship with stress in adult patients served at the moche dental clinic, 2013] [thesis]. trujilio, perú: universidad nacional de trujillo; 2013. spanish. 13 rojas et al. 30. segura escudero js. [frequency of non-carious cervical lesions in adult patients between 18 and 60 years of age who attend the clinic of the faculty of dentistry of the unmsm] [thesis]. perú: universidad nacional mayor de san marcos; 2013. spanish. 31. reyes j, paz soldán r, palian r, apaza e, garcía v. [characteristics of dental wear and occupational stress in technical nursing staff in a psychiatric hospital]. rev psiq salud mental “hermilio valdizán”. 2011;12(1):51-62. spanish. 32. latorre lópez ea. [prevalence of non-carious dental lesions in the community of santa rosa annex 22 of jicamarca san juan de lurigancho in 2009] [thesis]. lima, perú: universidad inca garcilaso de la vega. facultad de estomatología; 2011. spanish. 1 volume 22 2023 e236508 original article braz j oral sci. 2023;22:e236508http://dx.doi.org/10.20396/bjos.v22i00.8666508 1 department of dentistry, federal university of juiz de fora (ufjf), governador valadares campus. corresponding author: jean soares miranda, phd department of dentistry, federal university of juiz de fora (ufjf) governador valadares campus rua são paulo, 745, governador valadares, mg, 35010-180 tel.: +55(32)988239151 e-mail: jean.miranda@ufjf.br editor: dr. altair a. del bel cury received: july 7, 2021 accepted: july 13, 2022 the effectiveness of edta 17% as a cleaning solution for the fiber post space after filling with cements luciana arruda mendes de paula1, lohara campos de abreu reis1, jean soares miranda1,* , francielle silvestre verner1 , rafael binato junqueira1 , rodrigo furtado de carvalho1 aim: to evaluate the resistance of the union between a glass fiber post and radicular dentine after cleaning the root with 17% edta and filling with different endodontic cements. methods: forty uniradicular bovine incisors were removed to obtain root lengths of 18 mm. endodontic treatment was performed on all roots using different filling cements (zinc oxide and eugenol-based, oze; cement based on epoxy resin, ah) and cleaning solutions (saline, sa or edta), which made it possible to obtain four groups: ozesa, ozeedta, ahsa and ahedta. subsequently, 12 mm of filling material was removed from the roots, and they were prepared to receive fiber posts luted with resin cement. to execute the mechanical cycles (2x106 cycles, 90 n, 4 hz), coronal reconstruction was performed with a silicon matrix. the roots were then sliced (2-mm thick) to perform the push-out test. the results were analyzed using analysis of variance (one factor and two factors) and tukey’s test (α=0,05). results: bond strength (mpa) was significantly higher for ozeedta (9,18) and ahedta (8,70) than for ozesa (6,06) ahsa (8,7). ozeedta also presented the highest values in the cervical region (15,18) but was significantly lower in the apical region (2,99). however, ahedta had a homogeneous bond strength in all thirds. conclusion: regardless of the endodontic cement used, edta was used as an irrigating solution, culminating in a higher bond strength between the glass fiber post and dentin. keywords: tooth, nonvital. endodontics. zinc oxide-eugenol cement. edetic acid. https://orcid.org/0000-0001-5379-0155 https://orcid.org/0000-0001-5770-316x https://orcid.org/0000-0002-0732-2753 https://orcid.org/0000-0002-8271-8571 2 paula et al. braz j oral sci. 2023;22:e236508 introduction several clinical and in vitro studies have already highlighted the advantages of fiber glass posts over fused metallic cores and pre-fabricated metallic posts1-3. when adhesively cemented, these posts may show biomechanical properties similar to the dental structure, an elastic module, which favors the formation of a monoblock structure of the cement with the posts and root canal walls, providing a junction with high retention, equal stress distribution, low microleakage, and high root fracture resistance2,4-6. moreover, they allow for a more conservative treatment of the dental structure, do not undergo corrosion, and present satisfactory esthetics7. however, this adhesion between glass fiber posts and root dentin may be compromised by the presence of remnants of the filling material, which is not totally removed during clearance8-9. one of the most common filling material is the zinc oxide and eugenol-based cements (oze)10; however, the presence of eugenol in its composition prevents a complete polymerization of the resin cement, reducing the bond strength with the post10-12. therefore, cements with other compositions, such as calcium hydroxide, tungsten hydroxide, or ah (ah), have been developed. among them, ah plus (ah; dentsply maillefer, ballaigues, switzerland) is one of the best endodontic sealers to fill the root canal before fiber post cementation4,13. this epoxy resin-based sealer has physicochemical properties that reduce viscosity and improve flow, adhesion, and capacity for filling empty space without compromising adhesion of the post4. after the removal of the gutta-percha and cement from the canal, a variety of solutions can be used in the chemical cleaning of the space before post luting. sodium hypochlorite (naocl) and ethylenediaminetetraacetic acid (edta) are commonly used to remove dentinary remnants and smear layers from dentinal walls14,15, which may increase the penetration of the adhesive in dentin and, consequently, increase the bond strength of the resin cement16-18. however, the application of edta for an exacerbated time can result in a dental demineralizing effect, widening the dentinal tubules, softening dentin, and denaturing the collagen fibers14,19. nonetheless, while much is known about the influence of cleaning the post and the mode of cement application to optimize bond strength9, little is known about the cleaning protocols prior to resinous cementation of the post. therefore, it is important to investigate protocols that seek to effectively remove the remaining filling materials from the canals to ensure adequate retention of the glass fiber post to the dental root20-22. therefore, the objective of this study was to evaluate the influence of 17% edta as a cleaning solution on the bond strength between a glass fiber post and a root filled with different endodontic cements subjected to mechanical aging. the null hypothesis was that the type of sealer (1) and use of 17% edta (2) did not influence the bond strength. methods and materials tooth preparation forty uniradicular bovine incisors were randomly selected. the teeth were decoronated to their longitudinal axis using a slow-speed, water-cooled diamond disc; thus, 3 paula et al. braz j oral sci. 2023;22:e236508 the root length was standardized at 18 ± 0.2 mm. the canal diameter was measured with a digital caliper and must have equal or smaller dimensions to the post used in the study (ø= 2.0 mm, white post dc #2, fgm, joinvile, sc, brazil). a single operator, specialist in endodontics and who experienced all of the techniques used in this study, performed the root canal preparation, obturation, and post cementation procedures. the bovine roots were endodontically treated using gates glidden drills and hand files (dentsply/maillefer, petrópolis, brazil). a 2.5% naocl solution was used to irrigate the canals (10 ml in total), followed by drying using absorbent paper points (dentsply sirona, rio de janeiro, brazil). randomization of the teeth in the different groups was performed using the “random allocator” software. the oze (endofill, dentsply maillefer, petrópolis, rj, brazil) (n=20) and ah plus (dentsply/maillefer, petrópolis, brazil) (n =20) sealers used to fill the root canals were prepared according to the manufacturers’ recommendations, and the gutta-percha cone (dentsply/maillefer, petrópolis, brazil) was covered with a layer of sealer and inserted into the canal (with gentle brushing movements against the root canal walls). subsequently, the gutta-percha cone was slowly positioned. twenty teeth were filled with gutta-percha cones (dentsply/maillefer; petrópolis, brazil). the lateral condensation technique was used for thermofilling, with the foramen considered as the apical obturation limit. the entrance of the canal was closed with a ketactm cem (3m, espe, st paul, mn, usa) conventional restorative glass ionomer to avoid contamination. the roots were radiographed to evaluate the quality of the endodontic treatment and stored in distilled water at 37 °c for 7 days. cleaning protocols and fiber post luting the teeth were mounted individually in plastic cylinders (tigre, rio claro, sp, brazil), and the roots were embedded in resin up to 3 mm below the coronary portion. the method used to reproduce the periodontal ligament was embedded in the root with a polyether impression material (impregum f, 3m-espe, seefeld, germany). the periodontal ligament was simulated by covering the root, including up to 3 mm of the coronary portion of the specimens with an elastomer (impregum soft, 3m-espe, seefeld, germany), with a thickness of approximately 0.3 mm. they were then included in pipes with chemically activated acrylic resin (dencrilay, dencril, caieiras, sp, brazil), according to the methodology described by junqueira et al.23. the filling material was initially removed from the root with heated paiva condensers (golgran, são paulo, brazil) supplemented with a drill corresponding to the white post dc #2 glass fiber post (fgm, joinville, sc, brazil) in the first 12 mm. a total of 6 mm of the filling material remained in the apical third. after the preparation, the roots were irrigated according to their respective groups: saline solution (ozesa and ahsa) or 17% edta (ozeedta and ahedta). for both groups, the dentinal walls were cleaned with 10 ml of the specific solution using a syringe and an irrigation needle (ultradent products, indaiatuba, sp, brazil) for 1 min and irrigated with 5 ml distilled water. the canal was then aspirated with microcannulas (capillary tips, ultradent, são paulo, brazil) and dried using # 80 absorbent paper points (dentsply sirona, rio de janeiro, brazil). 4 paula et al. braz j oral sci. 2023;22:e236508 all glass fiber posts (white post dc #2, fgm produtos odontológicos, joinvile, sc, brazil) were treated by conditioning with phosphoric acid 37% (15 s), washing with water (60 s), and air drying (15 s). then, a silane agent (silano, ângelus brazil) was applied using a brush, covering its entire surface (60 s), and laid on a glass plate for a period of 4 min and then air-dried (15 s). before adhesive cementation, the fiber posts were disinfected with alcohol 70° gl and then a silane layer (prosil fgm, joinville sc, brazil) was applied on the post’s surface using a micro-brush for 1 min and then dried by blowing compressed air for 20 s. after post preparation, the post space was etched with 37% phosphoric acid (fgm; joinivile, sc, brazil) for 15 s, rinsed with water for 60 s, and dried with #80 absorbent paper points. subsequently, an adhesive (ambar, fgm, joinivile, sc, brazil) was applied to the radicular canal using a microbrush (cavibrush longo, fgm) for 10 s, and the excess was removed with absorbent paper tips. finally, the adhesive resin cement system (allcem, fgm, joinivile, sc, brazil) was manipulated and placed in the post space with a lentulo #40 tip. the endodontic post was also covered with a sealer and seated to full depth using finger pressure for 60 s. light curing was performed for 40 s using halogen light at 1200 mw/cm2 (radii cal; sdi, melbourne, australia). for coronary reconstruction, the cervical dentin was etched for 15 s with 37% phosphoric acid (fgm; joinivile, sc, brazil), rinsed with water for 60 s, and dried with absorbent paper. subsequently, a layer of dentin adhesive (ambar, fgm) was applied to the dentin using a microbrush (cavibrush, fgm, joinivile, sc, brazil) with a light air jet used to evaporate the solvent and homogenize the thickness of the adhesive. the cure was performed for 30 s (radii cal; sdi, melbourne, australia). then, a standardized plastic matrix, obtained from a human central incisor with a 10 mm crown, was filled with micro-hybrid composite resin (opallis, fgm, joinville, santa catarina, brazil), positioned on the post coronary surface, and light-curing was performed for 20 s on each surface of the tooth (bergoli et al. 2014). the teeth were immersed in distilled water and placed at 100% relative humidity and 37 °c for 24 h. mechanical cycling the teeth were placed at 45º in a mechanical cycling machine (er 11000; erios, são paulo, brazil). a 90 n load was applied with a piston (ø = 1.6 mm), 2 mm below the incisal edge on the palatal face of the specimen, for 1.2 x 106 cycles, at a frequency of 4 hz in a humid environment. the piston (ø = 1.6 mm) was loaded 2 mm below the incisal border on the palatal face of each tooth. removal by extrusion (push-out) each root post space was cut into four 1.5-mm-thick slices using a cutting machine (isomet 100 precision saw, buehler ltd., lake bluff, il, usa) at a speed of 325 rpm and weight of 75 g under constant water irrigation. two 1.0 ± 0.1-mm-thick slices were obtained from each third of the post. the cervical side of each test specimen was placed in contact with a support coupled to the base of a universal test machine (emic 2000, são josé dos pinhais, paraná, brazil). loading was per5 paula et al. braz j oral sci. 2023;22:e236508 formed at a crosshead speed of 0.5 mm/min on the surface of the post, without reaching the cement and/or dentin, using a cylindrical tip with a 0.8-mm diameter until the post was completely dislodged from the root slice. data were recorded in newtons (n). the bond strength (bs) (mpa) was calculated using the formula bs=n/π(r + r)√h2 + (r − r), where “π” is the constant 3.14, “r” is the measure of the radius of the resin cement/post junction in its coronal portion, “r” is the measure of the radius of the resin cement/post junction in its apical portion, and “h” is the height/thickness of the slice. the obtained values were subjected to descriptive (mean and standard deviation) and inferential statistical analysis, using parametric anova (one factor and two factors) and tukey test (α = 0.05). failure analysis the failure mode was determined using a stereomicroscope at 50x magnification (vanox, olympus, tokyo, japan) and classified as (1) adhesive between resinous cement and root dentin; (2) adhesive between the post and resinous cement; (3) mixed between post, resinous cement, and root dentin; (4) cohesive in dentin; (5) cohesive in post; and (6) cohesive in cement. results when calculating the total bond strength, there was no statistical difference between the ozesa and ahsa groups, demonstrating no influence of the sealing material. however, these values were significantly lower than those of ozeedta and ahedta, which demonstrates the influence of the irrigation solution on these results (table 1). table 1. mean and standard deviations (mpa) of the total teeth bond strength according to the filling cement and irrigation solution used for cleaning the post space. lower case letters indicate differences pointed by the anova 1-factor and tukey test. groups average (±dp) p value groups ozesa ozeedta ahsa ahedta ozesa 6,06 (±0,80)a ozesa 0,006 0,817 0,022 ozeedta 9,18 (±0,76)b ozeedta 0,010 0,666 ahsa 6,32 (±0,78)a ahsa 0,037 ahedta 8,70 (±0,81)b ahedta table 2 compares the bond strengths of the root third of each sample. zs and rs also did not show statistically significant differences between the thirds. in the ze groups, it was observed that the cervical third presented union values that were significantly higher than those of the others. 6 paula et al. braz j oral sci. 2023;22:e236508 table 2. mean and standard deviations (mpa) of bond strength of each root third. upper case letters indicate difference on the same line. lower case letters indicate difference in the same column pointed by anova 2-factors and tukey test (p<0,05). groups root thirds cervical middle apical ozesa 7,60 (±1,40)aa 5,52 (±1,31)aa 5,06 (±1,46)aab ozeedta 15,18 (±1,31)ab 9,36 (±1,27)bb 2,99 (±1,35)ca ahsa 7,58 (±1,31)aa 5,63 (±1,52)aab 5,76 (±1,24)aab ahedta 10,91 (±1,35)aa 7,27 (±1,58)aab 7,92 (±1,24)ab the frequencies of the failure modes are listed in table 3. there was a predominance of adhesive between the resinous cement and root dentin, and mixed between the post, resinous cement, and root dentin. table 3. frequency of failure mode between groups (%). groups failure mode 1 2 3 4 5 6 ozesa 60% 6.6% 18.4% 1.7% 13.3% ozeedta 63.3% 5.1% 16.7% 3.3% 11.6% ahsa 61.7% 6.6% 16.7% 1.7% 13.3% ahedta 63.3% 8.3% 13.5% 3.3% 11.6% (1) adhesive between resinous cement and root dentin; (2) adhesive between the post and resinous cement; (3) mixed between post, resinous cement and root dentin; (4) cohesive in dentin; (5) cohesive in post and (6) cohesive in cement. discussion based on the results of this study, it was possible to answer our questions. the first null hypothesis, that the type of sealer would not influence the results of bond strength, was rejected because although the results of the totality of the dental root showed no differences between the oze and ah groups, the results of the third cervical and apical regions showed differences when edta was used to irrigate the canals. when analyzing the thirds, another interesting fact to note is that only the ozeedta group showed a difference in bond strength between all its thirds, with the apical one showing statistically lower bond strength results between the post and dentin, which is also commonly observed in other studies4,9, because the dentin tissue morphology in the cervical region shows a greater number of dentinal tubules and wider diameters than the others4. thus, the cervical third is the most adequate for the adhesion of different types of adhesive sealers4. however, this is not always the case15. the ahedta group, for example, presented homogeneity between its thirds, a fact that was also observed in previous studies15,24. this group also presented 7 paula et al. braz j oral sci. 2023;22:e236508 the highest bond strength in the apical third, which was statistically superior to the ozeedta group. normally, most studies show a better bond strength of the posts when the ah sealer is used at the expense of oze4,13,25, because zinc oxide eugenol-based sealer penetrates into the dentinal tubules26, inhibits the polymerization of resin cement, significantly reducing bond strength, and is avoided in these cases4,9,13,26-28. conversely, the epoxy resin, considered the gold standard13, does not interfere with the free radicals that initiate the polymerization of the composite resin. the high bs between resin cement and endodontic epoxy resin sealer is due to the affinity between their components4. thus, remnants of the epoxy resin sealer on the dentinal walls in prosthetic preparation may improve the adhesion of resin cement4. however, it is interesting to observe that in the other thirds, ozeedta had the highest bond strength values in the cervical and middle thirds, but this result was statistically similar to that of ahedta. the cervical ozeedta results may be associated with easy access and application of adhesive materials and greater efficiency of the cement cure in this region, which is next to the light source10,27. another fact that justified the higher values of bond strength found in cervical ozeedta may be due to the short time between root canal filling and post placement10,13. greater penetration of eugenol into the interior of the dentinal tubules may occur if the post luting was made after some days, which probably would cause a drop in the bond strength results10,13,26. therefore, it is suggested that an oze-based sealer would be better in the cervical third only; however, as it is not possible to use different sealers for each third of the root, an ah sealer may be more preferrable. the second null hypothesis was also rejected because regardless of the analysis of the entire dental root or each third, the groups with irrigation performed with edta, except ozeedta in the apical third, obtained better results. edta is also the most commonly used chelating solution to remove this smear layer created by post space preparation so that a hybrid layer is achieved to increase the retention when resin cement is used14,15. it has a calcium ion chelating capacity and is highly efficient in removing this smear layer15 because edta reacts with calcium ions in the dentin and forms soluble calcium chelates14. the results found highlight the importance of cleaning the fiber post space in the cementation protocol. the push-out test is a valid method to measure the adhesion of fiber posts to the root canal walls. it is considered more reliable than the conventional shear test and the micro-tensile test for evaluation of the bond strength of posts and allows the measurement of such values among different root regions1. it can be observed, when using this test, that there was a predominance of adhesive failure between resinous cement and root dentin, which represents the interface of interest to the study and can also be related to other studies4,13,15. some limitations can be attributed to this study; however, the use of the bovine tooth is not among them, because previous studies have shown that the type of tooth (bovine or human) does not influence the bond between the post and radicular dentin9,29. however, although this methodology has proposed a mechanical fatigue test, it would be interesting to obtain clinical outcomes of the longevity of these treatments, which should be conducted in the future. 8 paula et al. braz j oral sci. 2023;22:e236508 given this, it may be suggested that the use of ah and subsequent use of edta for irrigation of dentinal tubules may be the most suitable protocol for the treatment of root canals prior to the adhesive cementation of the fiberglass posts; even in the cervical third, the use of oze + edta has obtained better results, and the maintenance and predictability of post adhesion in the other thirds proved to be safer when using ah. in conclusion, edta (17%) was an effective cleaning solution for the post space. regardless of the endodontic cement, its use led to an increase in the bond strength between the glass fiber post and the dentinary structure. conflicts of interest the authors deny any conflicts of interest. author contribuitions luciana arruda mendes de paula: acquisition of data, drafting the article. lohara campos de abreu reis: acquisition of data, drafting the article. jean soares miranda: acquisition of data, drafting the article. francielle silvestre verner: formal analyses, revising it critically for important intellectual content. rafael binato junqueira: conceptualization, revising it critically for important intellectual content, formal analyses. rodrigo furtado de carvalho: conceptualization, drafting the article, revising it critically for important intellectual content. all authors actively participated in the discussion of the manuscript’s findings, revised, and approved the final version of the manuscript. references 1. ferreira r, prado m, soares aj, zaia aa, souza-filho fj. influence of using clinical microscope as auxiliary to perform mechanical cleaning of post space: a bond strength analysis. j endod. 2015 aug;41(8):1311-6. doi: 10.1016/j.joen.2015.05.003. 2. monticelli f, grandini s, goracci c, ferrari m. clinical behavior of translucent-fiber posts: a 2year prospective study. int j prosthodont. 2003 nov-dec;16(6): 593-6. 3. makade cs, meshram gk, warhadpande m, patil pg. a comparative evaluation of fracture resistance of endodonticaly treated teeth restored with different post core systems – an in vitro study. j adv prosthodont. 2011 jun;3(2):90-5. doi: 10.4047/jap.2011.3.2.90. 4. soares imv, crozeta bm, pereira rd, silva rg, da cruz-filho am. influence of endodontic sealers with different chemical compositions on bond strength of the resin cement/glass fiber post junction to root dentin. clin oral investig. 2020 oct;24(10):3417-3423. doi: 10.1007/s00784-020-03212-9. 5. ayub, kv; ebeling, lc; zavanelli, ac; mazaro, jvq. [tensile strength evaluation of prefabricated posts: literature review]. rev odontol araçatuba. 2009 jul-dec;30(2):50-6. portuguese. 9 paula et al. braz j oral sci. 2023;22:e236508 6. malferrari s, monaco c, scotti r. clinical evaluation of teeth restored with quartz fiber reinforced epoxy resin posts. int j prosthodont. 2003 jan-feb;16(1):39-44. 7. guldener ka, lanzrein cl, siegrist guldener be, lang np, ramseier ca, salvi ge. long-term clinical outcomes of endodontically treated teeth restored with or without fiber post-retained single-unit restorations. j endod. 2017 feb;43(2):188-93. doi: 10.1016/j.joen.2016.10.008. 8. perdigão j, gomes g, augusto v. the effect of dowel space on the bond strengths of fiber posts. j prosthodont. 2007 may-jun;16(3):154-64. doi: 10.1111/j.1532-849x.2006.00166.x. 9. skupien ja, sarkis-onofre r, cenci ms, moraes rr, pereira-cenci t. a systematic review of factors associated with the retention of glass fiber posts. braz oral res. 2015;29:s180683242015000100401. doi: 10.1590/1807-3107bor-2015.vol29.0074. 10. hagge ms, wong rd, lindemuth js. retention, of posts luted with phosphate monomerbased composite cement in canals obturated using a eugenol sealer. am j dent. 2002 dec;15(6):378-82. 11. tjan ahl, nemetz h. effect of eugenol containing endodontic sealer on retention of prefabricated posts luted with an adhesive composite resin cement. quintessence int. 1992 dec;23(12):839-44. 12. hagge ms, wong rdm, lindemuth js. effect of three root canal sealers on the retentive strength of endodontic post luted with a resin cement. int endod j. 2002 apr;35(4):372-8. doi: 10.1046/j.0143-2885.2001.00493.x. 13. vilas-boas da, grazziotin-soares r, ardenghi dm, bauer j, de souza po, de miranda candeiro gt, et al. effect of different endodontic sealers and time of cementation on push-out bond strength of fiber posts. clin oral investig. 2018 apr;22(3):1403-9. doi: 10.1007/s00784-017-2230-z. 14. miranda js, marques ea, landa fv, leite app, leite fpp. [effect of three final irrigation protocols on the smear layer removal from the middle third of endodontically treated teeth: a qualitative analysis]. dental press endod. 2017 may-aug;7(2):72-7. doi: 10.14436/2358-2545.7.2.072-077.oar. 15. vangala a, hegde v, sathe s, dixit m, jain p. effect of irrigating solutions used for postspace treatment on the push-out bond strength of glass fiber posts. j conserv dent. 2016 jan-feb;19(1):82-6. doi: 10.4103/0972-0707.173206. 16. gu xh, mao cy, liang c, wang hm, kern m. does endodontic post space irrigation affect smear layer removal and bonding effectiveness? eur j oral sci. 2009 oct;117(5):597-603. doi: 10.1111/j.1600-0722.2009.00661.x. 17. mao h, chen y, yip kh, smales rj. effect of three radicular dentine treatments and two luting cements on the regional bond strength of quartz fibre posts. clin oral investig. 2011 dec;15(6):869-78. doi: 10.1007/s00784-010-0453-3. 18. kul e, yeter ky, aladag li, ayrancı lb. effect of different post space irrigation procedures on the bond strength of a fiber post attached with a self-adhesive resin cement. j prosthet dent. 2016 may;115(5):601-5. doi: 10.1016/j.prosdent.2015.10.010. 19. calt s, serper a. time-dependent effects of edta on dentin structures. j endod. 2002 jan;28(1):17-9. doi: 10.1097/00004770-200201000-00004. 20. coniglio i, magni e, goracci c, radovic i, carvalho ca, grandini s, et al. post space cleaning using a new nickel titanium endodontic drill combined with different cleaning regimens. j endod. 2008 jan;34(1):83-6. doi: 10.1016/j.joen.2007.10.019. 21. zhang l, huang l, xiong y, fang m, chen j-h, ferrari m. effect of post-space treatment on retention of fiber posts in different root regions using two self-etching systems. eur j oral sci. 2008 jun;116(3):280-6. doi: 10.1111/j.1600-0722.2008.00536.x. 22. bitter k, eirich w, neumann k, weiger r, krastl g. effect of cleaning method, luting agent and preparation procedure on the retention of fibre posts. int endod j. 2012 dec;45(12):1116-26. doi: 10.1111/j.1365-2591.2012.02081.x. about:blank about:blank about:blank about:blank about:blank about:blank about:blank about:blank about:blank about:blank about:blank about:blank about:blank 10 paula et al. braz j oral sci. 2023;22:e236508 23. junqueira rb, de carvalho rf, marinho cc, valera mc, carvalho cat. influence of glass fiber post length and remaining dentine thickness on the fracture resistance of root filled teeth. int endod j. 2017 jun;50(6):569-577. doi: 10.1111/iej.12653. 24. freitas tl, vitti rp, miranda me, brandt wc. effect of glass fiber post adaptation on push-out bond strength to root dentin. braz dent j. 2019 jul 22;30(4):350-5. doi: 10.1590/0103-6440201902491. 25. dibaji f, mohammadi e, farid f, mohammadian f, sarraf p, kharrazifard mj. the effect of bc sealer, ah-plus and dorifill on push-out bond strength of fiber post. iran endod j. 2017 fall;12(4):443-8. doi: 10.22037/iej.v12i4.15863. 26. rosa ra, barreto ms, moraes ra, broch j, bier cas, so mvr, et al. influence of endodontic sealer composition and time of fiber pos t cementat ion on sealer adhesiveness to bovine root dentin. braz dent j. 2013;24(3):241-6. doi: 10.1590/0103-6440201302154. 27. baldissera p, zicari f, valandro lf, scotti r. effect of root canal treatments on quartz fiber posts bonding to root dentin. j endod. 2006 oct;32(10):985-8. doi: 10.1016/j.joen.2006.04.013. 28. altmann asp; leitune, vcb; collares fm. influence of eugenol-based sealers on push-out bond strength of fiber post luted with resin cement: systematic review and meta-analysis. j endod. 2015 sep;41(9):1418-23. doi: 10.1016/j.joen.2015.05.014. 29. carvalho mff, leijôto-lannes acn, rodrigues mcn, nogueira lc, ferraz nkl, moreira an, yamauti m, et al. viability of bovine teeth as a substrate in bond strength tests: a systematic review and meta-analysis. j adhes dent. 2018;20(6):471-9. doi: 10.3290/j.jad.a41636. 1 volume 22 2023 e230883 original article braz j oral sci. 2023;22:e230883http://dx.doi.org/10.20396/bjos.v22i00.8670883 1 faculty of dentistry, federal university of santa maria, santa maria, rs, brazil. 2 eastman dental center, university of rochester, ny, usa. 3 indiana university school of dentistry, indianapolis, in, usa. 4 piracicaba dental school, university of campinas, piracicaba, sp, brazil. corresponding author: prof. jaime a cury piracicaba dental school, unicamp cep 13414-903, piracicaba, sp, brazil e-mail: jcury@unicamp.br editor: dr. altair a. del bel cury received: august 31, 2022 accepted: september 09, 2022 effect of phenylmethylsulfonyl fluoride, a protease inhibitor, on enamel surface remineralization paulo edelvar corrêa peres1, jean fu2 (in memorian), domenick t zero3 , jaime aparecido cury4,* phenylmethylsulfonyl fluoride (pmsf) is a protease inhibitor widely used in research, but fluoride is released during its action and this knowledge has been neglected in dental research. aim: to evaluate if fluoride released by salivary protease action on pmsf affects enamel remineralization and fluoride uptake. methods: groups of 10 enamel slabs, with caries-like lesions and known surface hardness (sh), were subjected to one of the following treatment groups: stimulated human saliva (shs), negative control; shs containing 1.0 μg f/ml (naf), positive control; and shs containing 10, 50 or 100 µm pmsf. the slabs were subjected to a ph-cycling regimen consisting of 22 h/day in each treatment solution and 2 h/day in a demineralizing solution. after 12 days, sh was again measured to calculate the percentage of surface hardness recovery (%shr), followed by enamel fluoride uptake determination. the time-related fluoride release from 100.0 µm pmsf by shs action was also determined. data were analyzed by anova followed by newman-keuls test. results: the release of fluoride from pmsf by shs was rapid, reaching a maximum value after 10 min. fluoride released from pmsf was more effective in enhancing %shr and increasing fluoride uptake in enamel compared with shs alone (p < 0.05); furthermore, it was equivalent to the positive control (p > 0.05). conclusion: in conclusion, fluoride released by saliva from pmsf is available to react with enamel and needs to be taken into account in research using this protease inhibitor. keywords: protease inhibitor. dental enamel. fluorides. tooth remineralization. https://orcid.org/0000-0001-7499-2282 https://orcid.org/0000-0003-1046-5605 2 peres et al. braz j oral sci. 2023;22:e230883 introduction even before the salivaomics era in dentistry1, protease inhibitors had already been used to preserve the structure of proteins to be analyzed. particularly, for human salivary proteome analysis, the subject is relevant because the proteins in saliva collected suffer rapid degradation2, requiring the use of a cocktail of protease inhibitors during storage to stabilize the structure of the proteins3. phenylmethylsulfonylfluoride (pmsf) has been used in research as a protease inhibitor to avoid the breakdown of proteins. it inhibits serine-proteases by a covalent linkage to the active site of the enzyme and, during the reaction, the serine hydroxyl links to sulfonyl group and fluoride is released into the media4. pmsf is an efficient inhibitor of salivary proteases5 and has been used in dental research for a long time, e.g.: (1) to avoid proteolysis during saliva collection6; (2) in studies about adsorption of salivary proteins to enamel7; (3) in studies on salivary gland function8; (4) determination of gtf activity9; (5) inhibition of bacteria coaggregation in saliva10, and (6) in isolation of salivary proteins11 and proteomics analysis12. in these studies, pmsf has been used from 100 µm to 0.5 m. if it was totally hydrolyzed, the final f concentration into the batch media would range from 1.9 to 9,500 μg f/ml (ppm f). this high f concentration released from pmsf could have an indirect effect depending on the research in question as fluoride at a concentration of only 1.0 μg f/ml can interfere with protein adsorption to and desorption from hydroxyapatite13. likewise, the enzymes enolase and f1foatpase of streptococci species are inhibited by f at 20 to 45 μg /ml14 and 10 ppm f prevents the enrichment of s. mutans in biofilms15. furthermore, sub-ppm fluoride concentrations are sufficient to enhance enamel remineralization16. although fluoride released from pmsf by salivary action can have an indirect effect in research and even producing an artifact, it could be a new approach in development of products for caries prevention. therefore, the aims of this research were (1) to evaluate if fluoride is released by saliva from pmsf, and (2) to investigate its effect on fluoride uptake and remineralization of dental enamel. materials and methods experimental design fifty bovine enamel blocks with caries-like lesions and of known surface hardness (sh) were randomly distributed into five groups of 10 each and allocated to one of the following treatments groups: (i) simulated human saliva (shs) as negative control; (ii) experimental groups containing 10, 50, and 100 mmol of pmsf/l of shs; and (iii) shs containing 1.0 μg f/ml (naf) as a positive control. the blocks were placed for 22 h in the treatment solutions and 2 h in a demineralising solution, simulating a ph-cycling remineralizing regimen. after 12 days the enamel blocks were recovered, microhardness was again measured to calculate enamel sh recovery (%shr), and enamel fluoride uptake was also determined. 3 peres et al. braz j oral sci. 2023;22:e230883 fluoride released from 100 μm pmsf by saliva action was assessed according to the time of incubation at 37 oc. the experiment was repeated 6 times and the increase of fluoride concentration in shs was determined. this study was conducted according to resolution no. 196 from national health council, health ministry, brasília, df, brazil. enamel blocks preparation and lesion creation one hundred and nine enamel slabs (4 x 4 x 2 mm) of sound bovine incisors were prepared17 and their baseline surface hardness (sh) measured using a 50 g load with a shymadzu tester. ninety-nine slabs, presenting indentations length from 40 to 46 mm, were selected for lesion creation. the slabs were painted with an acid-resistant varnish, except for a circular central area of 3.14 mm2, in which caries-like lesions were induced18. the demineralising solution contained 0.05 m lactic acid, 0.2% carbopol c907, and was 50% saturated with respect to hydroxyapatite at a ph of 5.0. each specimen was placed in 6.3 ml of this solution for 16 h at 37 oc. the enamel sh was again determined and 50 slabs with indentations length from 110 to 150 mm were selected for the present study. treatments and ph-cycling remineralizing regimen shs was collected twice/day over ice in the morning and afternoon from seven (7) healthy adult volunteers by parafilm chewing and was then pooled. sodium azide was added to the pooled saliva as a preservative (final concentration 0.02%). the pools were split into five fractions to prepare the treatment solutions. one fraction was separated to be used as a negative control treatment, and to three other, pmsf (sigma) 10 μm (dissolved in isopropanol) was added to obtain final concentrations of pmsf at 10, 50 and 100 μm. a fluoride solution of 100 μg/ml (orion) was added to the 5th fraction to obtain a final concentration of 1.0 μg f/ml (positive control treatment). fluoride in these treatment solutions was determined daily with an ion-specific electrode, before the ph-cycling regimen. each enamel block was immersed individually in the shs treatment solutions prepared each morning from 9:30 to12:30 h and from 14:30 to 17:00 h. between these periods, the enamel blocks were immersed individually in the demineralising solution, which composition was identical to that described for the formation of early artificial caries. from 17:00 h until the next day, the enamel blocks were individually immersed in the shs treatment solution prepared with shs collected in the afternoon. each slab was immersed in 4.0 ml of shs solutions and in 12.0 ml of the demineralizing solution. the enamel blocks stayed in all solutions at 37 oc, and after each soaking they were washed with deionized water. the shs solutions were changed twice a day and the demineralizing solution after the 6th day of cycling. this ph-cycling model used is similar to that used by white18 to evaluate the ability of fluoride dentifrice to remineralize enamel. surface hardness analysis (sh) after ph-cycling, the sh of the treated enamel blocks was measured again. five indentations spaced 100 µm from each other, from the baseline and from those made 4 peres et al. braz j oral sci. 2023;22:e230883 after the artificial caries development were made. a micro-hardness tester (shimadzu hmv 2000) with a knoop diamond indenter was used with a 50-g load for 15 seconds. the mean values of all five measurements at the three different times (baseline, after lesion creation and after ph cycling) were used to calculate the percentage surface microhardness recovery (%shr) using the equation: %shr = (hardness after ph cycling hardness after caries production) x 100 baseline hardness values hardness after caries production sh was evaluated because there is a good correlation (0.94) between remineralization of early carious lesions measured by this technique and by microradiography18. after surface microhardness analysis, all slabs were prepared for fluoride enamel analysis. analysis of fluoride concentration in enamel five layers of enamel were sequentially removed from each dental slab under agitation in 0.5 ml of 0.5 m hydrochloric acid for 30, 30, 30, 60 and 60 s. an equal volume of tisab ii ph 5.0, modified with 20 g naoh/l, was added to the acid extracts containing the dissolved enamel layer19. fluoride was determined using an ion specific electrode (orion 96-09) and an ion analyzer orion e 940. the thickness of the enamel layer removed was calculated from the inorganic phosphorus concentration, determined by the fiske and subarrow method20. phosphorus content of 17.4% and enamel density of 2.92 were assumed in order to calculate the amount of enamel removed and to estimate the depth of each enamel layer. fluoride release from pmsf by saliva shs was pre-incubated at 37 oc for 5 min. a volume of 0.51 ml of pmsf 5 μm, dissolved in isopropanol, was added to 25 ml of shs. aliquots of 1 ml of this saliva solution containing pmsf 100 µm were distributed in 18 assays tubes. after 5, 10, 20, 40, 60 and 120 min at 37 oc, three tubes were removed and 1 ml of tisab (acetate buffer 1.0 m, ph 5.0, containing 1.0 m nacl and 0.4% 1,2-cyclohexanediaminotetraacetic) was added to them. this procedure was repeated for six days. fluoride released, and that in the shs, was determined using an ion-selective electrode orion 96-09 and an ion analyzer orion ea-940. statistical analysis the results were analyzed by analysis of variance (anova) followed by newman-keuls test, with exception of comparison between fluoride concentration found in the solutions fresh and after the ph-cycling, which was evaluated by paired t test. for all statistical analysis, bioestat 2.0 software21 [ayres et al., 2000] was used and the significance limit was set at 5%. results the anova showed statistically significant effects for fluoride release into saliva over time of incubation with pmsf (p < 0.0001), fluoride in the treatment solutions 5 peres et al. braz j oral sci. 2023;22:e230883 used in ph-cycling, enamel fluoride uptake (p < 0.0001), and % of sh recovery (p<0.0001) after ph-cycling. the effect of saliva on pmfs is shown in figure 1. fluoride release was very rapid and reached a plateau within 20 min. fluoride concentration found at all times was statistically higher than that at time zero (p < 0.05), but after 10 min of incubation the concentrations were not statistically different (p > 0.05). 0 0.5 1.0 1.5 2.0 0 µg f /m l time (min) 120110100908070605040302010 figure 1. means (n=6) of fluoride concentration (μg f/ml) in saliva over time of incubation with pmsf 100 μm. bars denote se; the statistical significance is described in results section. table 1 shows that all treatments were more effective in increasing %shr than the negative control (p<0.05). the %shr of enamel blocks treated with shs containing pmsf 50 and 100 μm was statistically higher than that treated with pmsf 10 μm (p < 0.05), but the difference between them was not significant (p> 0.05). all treatments with pmsf were equivalent to the positive control treatment (p> 0.05). table 1. means (±se) of enamel surface hardness recovery (%shr, and fluoride concentration (μg f/ml) in the treatment solutions before and after the ph-cycling, according to the treatment groups. treatments groups %shr (n=10) μg f/ml (n=24) before after shs (negative control) 9.5 ± 1.8 a a 0.076 ± 0.006 a b 0.061±0.003a shs+pmsf 10 µm 32.9 ± 2.0 b a 0.216±0.007 b b 0.1308±0.010 a shs+pmsf 50 µm 47.9 ± 2.7 c a 0.852±0.016 c b 0.697±0.035 b shs+pmsf 100 µm 48.6 ± 3.1 c a 1.888±0.032d b 1.484±0.051 c shs+1.0 μg f/ml(positive control) 43.2 ± 5.9 b,c a 1.081±0.011 e b 0.866±0.047 d means followed by different letters are statistically significant (p<0.05); lower case letters represent significant differences among treatments and capital letters represent significant differences between before and after ph-cycling for each treatment. 6 peres et al. braz j oral sci. 2023;22:e230883 all treatment solutions containing pmsf presented greater (p < 0.05) fluoride concentrations than the negative control (table 1). in addition, fluoride concentration in all treatment solutions decreased (before vs. after) significantly after the ph-cycling (p < 0.05). also, all treatment solutions containing pmsf presented either before or after ph-cycling greater (p < 0.05) fluoride concentrations than the negative control (shs). before cycling, all treatment groups differed statistically in terms of fluoride concentration in the solutions (p < 0.05), but after the ph-cycling the difference between shs containing 10 μm of pmsf and shs (negative control) was no longer statistically significant (p > 0.05). figure 2 shows the enamel fluoride uptake data. all treatments were statistically more effective (p < 0.05) in incorporating fluoride into enamel than the negative control (shs), up to approximately 30 mm from the dental surface (first three layers of enamel removed). the treatments with shs containing pmsf 50 and 100 μm did not differ from the positive control treatment (1.0 μg f/ml) and between each other (p > 0.05). pmsf 10 μm formed lower fluoride concentration in enamel than the positive containing 1.0 μg f/ml (p < 0.05). 0 10 20 30 40 50 60 70 80 15000 10000 5000 0 m g f/ kg depth (µm) shs (negative control) shs + pmsf 10 µm shs + pmsf 50 µm shs + pmsf 100 µm shs + 1.0 µg f/ml (positive control) figure 2. means (n=10) of fluoride concentration (μg f/kg) in enamel according to the treatment groups and the distance from dental surface (µm). bars denote se; difference statistically significant (p < 0.05) between treatments are described in the text. discussion the present findings showed that fluoride released from pmsf by saliva action has the same ability to enhance the remineralizing properties of human saliva as fluoride from the positive control (naf) treatment. the release of fluoride by whole saliva is very fast (figure 1) but the origin of the proteases is unknow, because they usually originate from oral mucosa tissue, salivary glands, or oral microorganisms22. the amount (mol) of fluoride released (figure 1 and table 1) is according to stoichiometry of the equation in figure 3: 7 peres et al. braz j oral sci. 2023;22:e230883 figure 3. stoichiometric reaction between serine-enzyme and pmsf. the hydroxyl residue of ser amino acid of the active site of enzyme links to sulfonyl group of pmsf and due to an electronic balance, free ion fluoride (f-) is released to the media. the reaction is equimolecular, 1 mol of pmsf produces 1 mol of f-. thus, the concentration of fluoride found after 10 min of incubation of pmsf 100 µm with shs (figure 1) was very close 100 µm of f (1.9 µg f/ml = 1.9 ppm f). in addition, the fluoride concentrations found in the treatment solutions of groups of shs containing pmsf 10, 50 and 100 µm (table 1) is according to the stoichiometry of the above chemical equation. the results of enamel surface remineralization (table 1) are supported by the fluoride concentrations present in the treatment solutions used. a positive correlation of 0.79 was found (data not shown) between %shr and the concentrations of fluoride (μg f/ml) in the treatments used before the ph-cycling. after the ph-cycling, the fluoride concentrations in all groups decreased significantly (table 1). this can be explained by the enamel fluoride uptake data (figure 2), whereas the surface of enamel was remineralized with fluorapatite-like minerals. indeed, a positive correlation of 0.97 was found (data not shown) between the mean of fluoride concentrations in the three first layers of enamel (~30 mm depth) and the %shr (table 1 and figure 2). therefore, the robust findings of the present study show that during its action as protease inhibitor, pmsf releases fluoride into the batch media. considering that pmsf has been used in research at concentration up to 0.5 m, it might result in up to 9,500 ppm f in the treatment solution. this unexpected very high fluoride concentration is not only a concern for research conducted in dentistry, because protease inhibitors are used for other research and therapeutic applications. thus, the findings are an alert because artefacts in research may occur. on the other side, although fluoride released from pmsf by salivary action can have indirect effect on research in progress, it could be a new approach in development of new products for caries prevention. in fact, fluoride has been used for a long time chemically bound to phosphate as monofluorophosphate (fpo3 2-). in the past, it was believed that fpo3 2per se was the active moiety against caries. nowadays it is accepted that its anticaries activity is due to the fluoride ion released by hydrolysis catalyzed by enzymes found in saliva and dental biofilm23. however, fpo3 2is hydrolyzed by salivary enzymes at a very slow rate24, what could explain the relatively lower anticaries effect of dentifrice containing fpo3 2in comparison with naf-based ones25. on the other hand, fpo3 2is indispensable as source of fluoride in a formulation containing ca-based abrasives26. thus, a molecule containing bound fluoride that was rapidly hydrolyzed by salivary action could have a better anticaries effect. on the other hand, the present in vitro study presents some limitations. first, it was only evaluated the effect of fluoride released on surface enamel remineralization. 8 peres et al. braz j oral sci. 2023;22:e230883 the effects on caries lesions remineralization and mainly the effect on reduction of enamel demineralization were not evaluated. also, the effect on the enzymes inactivated by pms was not evaluated in terms of reversibility. in conclusion, fluoride released by saliva from pmsf is active to react with enamel and possibly may have other effects in research using this protease inhibitor. acknowledgements the 1st author was supported with a scholarship from coordenação de aperfeiçoamento de pessoal de nível superior brazil (capes) (finance code 001) during his master of science course in piracicaba dental school. the 4th author was supported with a scholarship from conselho nacional de desenvolvimento científico e tecnológico (cnpq) (finance proc, 450685/95) during his laboratory training about “analytical methodologies in cariology” done in eastman dental center, rochester university, rochester, usa. the 4th author is grateful to dr domenick zero and jean fu (in memorian) for the scientific support had in rochester, and to pedro luiz rosalen family for the hospitality. data availability all data are available in unicamp repository. conflict of interest the authors have no conflicts of interest to declare. author contributions conceived and designed the experiment: jac. performed the experiment: pecp. analyzed the data: jac, jf and dtz. wrote the paper: jac. reviewed the paper: pecp and dtz. declaration of originality, interests, and financing: this ms is original, and it was not supported by any manufacturer of oral hygiene products references 1. papale f, santonocito s, polizzi a, giudice al, capodiferro s, favia g, et al. the new era of salivaomics in dentistry: frontiers and facts in the early diagnosis and prevention of oral diseases and cancer. metabolites. 2022 jul;12(7):638. doi: 10.3390/metabo12070638. 2. siqueira wl, dawes c. the salivary proteome: challenges and perspectives. proteomics 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j dent res. 1997 mar;76(3):734-43. doi: 10.1177/00220345970760030501. 12. bassim cw, ambatipudi ks, mays jw, edwards da, swatkoski s, fassil h, et al. quantitative salivary proteomic differences in oral chronic graft-versus-host disease. j clin immunol. 2012 dec;32(6):1390-9. doi: 10.1007/s10875-012-9738-4. 13. rölla g, melsen b. desorption of protein and bacteria from hydroxyapatite by fluoride and monofluorophosphate. caries res. 1975;9(1):66-73. doi: 10.1159/000260144. 14. hamilton ir. biochemical effects of fluoride on oral bacteria. j dent res. 1990 feb;69 spec no:660-7; discussion 682-3. doi: 10.1177/00220345900690s128. 15. bradshaw dj, marsh pd, hodgson rj, visser jm. effects of glucose and fluoride on competition and metabolism within in vitro dental bacterial communities and biofilms. caries res. 2002 mar-apr;36(2):81-6. doi: 10.1159/000057864. 16. cury ja, tenuta lm. enamel remineralization: controlling the caries disease or treating early caries lesions? braz oral res. 2009;23 suppl 1:23-30. doi: 10.1590/s1806-83242009000500005. 17. zero dt. in situ caries models. adv dent res. 1995 nov;9(3):214-30; discussion 231-4. doi: 10.1177/08959374950090030501. 18. white dj. reactivity of fluoride dentifrices with artificial caries. i. effects on early lesions: f uptake, surface hardening and remineralization. caries res. 1987;21(2):126-40. doi: 10.1159/000261013. 19. koo rh, cury ja. soluble calcium/smfp dentifrice: effect on enamel fluoride uptake and remineralization. am j dent. 1998 aug;11(4):173-6. 20. fiske cm, subarrow y. the colorimetric determination of phosphorus. j biol chem. 1925 dec;66(2):375-400. 21. ayres m, ayres m jr, ayres dl, santos as: bioestat 2.0: statistical applications in biological sciences and medicine. belém: sociedade civil mamiraua; 2000. portuguese. 10 peres et al. braz j oral sci. 2023;22:e230883 22. feng y, li q, chen j, yi p, xu x, fan y, et al. salivary protease spectrum biomarkers of oral cancer. int j oral sci. 2019 jan;11(1):7. doi: 10.1038/s41368-018-0032-z. 23. shellis rp, duckworth rm. studies on the cariostatic mechanisms of fluoride. int dent j. 1994 jun;44(3 suppl 1):263-73. 24. pearce eif, jenkins gn: the decomposition of monofluorophosphate by enzymes in whole human saliva. arch oral biol 1977;22(6):405-7. doi: 10.1016/0003-9969(77)90064-4. 25. bowen wh. relative efficacy of sodium fluoride and sodium monofluorophosphate as anti-caries agents in dentifrices: proceedings of a conference sponsored by unilever research, held at the royal society of medicine, london, 5 november 1994. london: royal society of medicine press; 1995. 66p. 26. tenuta lma, cury ja. laboratory and human studies to estimate anticaries efficacy of fluoride toothpastes. monogr oral sci. 2013;23:108-24. doi: 10.1159/000350479. 1http://dx.doi.org/10.20396/bjos.v21i00.8666262 volume 21 2022 e226262 original article 1 department of dentistry, universidade federal do rio grande do norte (ufrn), natal, rio grande do norte, brazil. 2 department of restorative dentistry – school of dentistry – facult of pharmacy, dentistry and nurse, universidade federal do ceará (ufc), fortaleza, ceará, brazil. corresponding author: isauremi vieira de assunção 1787 sen. salgado filho avenue, natal rn, 59056-000, brazil; mobile.: +55-84-99935-4263; phone/fax: +55-84-3215-4100; email: isauremi@gmail.com editor: dr altair a. del bel cury received: july 3, 2021 accepted: november 16, 2021 dual-cured adhesive system improves adhesive properties of dentin cavities restored with a bulk-fill resin composite ana margarida dos santos melo1 , anne kaline claudino ribeiro1 , diana araújo cunha2 , nara sousa rodrigues2 , vicente de paulo aragão saboia2 , boniek castillo dutra borges1 , isauremi vieira de assunção1,* aim: to evaluate the impact of a dual-cured adhesive system on the in situ degree of conversion (dc), bond strength (bs) and failure mode (fm) of adhesive interfaces in dentin cavities restored with a bulk-fill resin composite. methods: 4-mm-deep dentin cavities with a 3.1 c-factor were created in 68 bovine incisors (n = 17 per group). the lightcured (scotchbond™ universal) or the dual-cured (adper™ scotchbond™ multi-purpose plus) adhesive system was applied to the cavities, which were then restored with a bulkfill resin composite (filtek™ bulk fill). in situ dc analysis was performed by means of micro raman spectroscopy at the top and bottom interfaces. push-out bs was measured in a universal testing machine after 24-h or 6-month water storage. fm was determined with a stereomicroscope. data of in situ dc and bs were analyzed by two-way analysis of variance (anova) and tukey test (p<0.05), while the fm was analyzed descriptively. results: the groups that received the dual-cured adhesive system showed statistically higher in situ dc and bs than those that received the light-cured adhesive system. cohesive failure mode was the most frequent in all conditions. conclusion: in situ dc and bs were influenced by the curing strategies of the adhesive systems with better performance of the dual-cured material. keywords: aging. composite resins. polymerization. dentinbonding agents. physical properties. mailto:isauremi@gmail.com https://orcid.org/0000-0001-7234-226x https://orcid.org/0000-0001-7839-1476 https://orcid.org/0000-0002-8718-9947 https://orcid.org/0000-0003-4922-2686 https://orcid.org/0000-0002-1143-6390 https://orcid.org/0000-0003-4313-5776 https://orcid.org/0000-0003-3171-6514 2 melo et al. introduction the proper cure of the adhesive system is one of the factors needed to achieve the required mechanical properties1 and longevity2,3 of dental restorations. inappropriate monomer conversion and the consequent residual monomers in the hybrid layer increase the local permeability and water sorption2-4, weakening the hybrid layer structure, and impairing the adhesive capacity4. therefore, a higher degree of conversion of the adhesive system, would provide longer-lasting adhesive integrity to dental restorations. the dual-curing strategy can promote the material self-cure when a proper light activation might be compromised. therefore, its use is traditionally recommended in indirect restorations or fiber post bonding in the root canal5. the benzoyl peroxide and tertiary amine in the catalyst allow the cure reaction without light energy6, which can increase the degree of conversion and, consequently, promote long-lasting bonds. areas of the cavity preparation for direct resin composite restorations that are too far from the curing light tip may receive insufficient energy, negatively affecting the degree of conversion and bond stability7. to overcome this inconvenience, the use of dual-cured adhesive systems has been suggested in 2-mm-deep cavities resulting in greater dentin bonding durability8, probably due to their higher degree of conversion in the hybrid layer and faster cure. however, when such tooth preparations occur in posterior teeth, the tip of the curing light can be set even further than 2 mm in certain dentin sites, negatively affecting interfacial properties in such areas. thus, regular viscosity bulk-fill resin composites, which allow light-curing single increments of up to 4 to 5 mm thickness9, could be used to fill deep preparations with a single resin composite increment, saving chair time. however, it is not well known if using a dual-cured adhesive system in 4-mm-depth would benefit the adhesive conversion and dentin bonding durability. thereby, the aim of this study was to evaluate the in situ degree of conversion (dc) of light-cured and dual-cured adhesive systems and their impact on bond strength (bs) and failure modes (fm) in 4-mm-deep dentin cavities restored with a regular viscosity bulk-fill resin composite. the null hypothesis tested was that different curing strategies of adhesive systems and storage times would not affect the physical properties of the adhesive interface. materials and methods experimental design and specimen preparation this in vitro study involved the following response variables: in situ dc, bs and fm. for in situ dc (n=17), the factors studied were two adhesive systems with different curing strategies (light-cured: scotchbond™ universal, 3m espe, st. paul, usa; and dual-cured: adper™ scotchbond™ multi-purpose plus, 3m espe, st. paul, usa), and the regions of the specimen (top and bottom). for bs and fm (n=17), the factors studied were the two adhesive systems with different curing strategies and the time of water storage (24 h and 6 months aging). materials used in this study are described in table 1. 3 melo et al. table 1. materials used in this study. material chemical composition (wt%) lot number filtek™ bulk fill (3m espe, st. paul, mn, usa) silane treated ceramic (60 – 70) aromatic urethane dimethacrylate (10 – 20) diurethane dimethacrylate (udma) (1 – 10) ytterbium fluoride (ybf3) (1 – 10) silane treated silica (1 – 10) 1,12-dodecane dimethacrylate (dddma) (<10) silane treated zirconia (<5) water (<5) 1717800606 scotchbond™ universal (3m espe, st. paul, mn, usa) 2-hydroxyethyl methacrylate (15 – 25) bisphenol a diglycidyl ether dimethacrylate (bisgma) (15 – 25) 2-propenoic acid, 2-methyl-, reaction products with 1,10-decanediol and phosphorous oxide (p2o5) (10 – 20) ethanol (10 – 15) water (10 – 15) 2-propenoic acid, 2-methyl-, 3-(trimethoxysilyl)propyl ester, reaction products with vitreous sílica (7 – 13) copolymer of acrylic and itaconic acid (1 – 5) camphorquinone (<2) dimethylaminobenzoat(-4) (<2) (dimethylamino)ethyl methacrylate (<1) 1718500452 adper™ scotchbond™ multi-purpose plus (3m espe, st. paul, mn, usa) primer: water (40 – 50) 2-hydroxyethyl methacrylate (hema) (35 – 45) copolymer of acrylic and itaconic acids (10 – 20) 1723700298 activator: ethyl alcohol (>95) sodium benzenesulfinate (<5) 1728600477 catalyst: bisphenol a diglycidyl ether dimethacrylate (bisgma) (60 – 70) 2-hydroxyethyl methacrylate (hema) (30 – 40) benzoyl peroxide (<2.5) triphenylantimony (<0.5) triphenylphosphine (<0.5) hydroquinone (<0.05) 1727600198 source: safety data sheet (sds) figure 1 shows a schematic representation of the specimens’ preparation and analyses performed. sixty-eight bovine incisors without enamel cracks or structural defects were selected for dental preparations according to a previously described method10,11. teeth were decontaminated in a water solution of thymol (0.1%) at 4ºc for a week, and then the roots were removed at the cementoenamel junction (cej) with a diamond saw using a precision cutting machine (isomet 1000; buehler, lake forest, il, usa) under irrigation. such surface will be the future top of the sample. a parallel cut was made 4 mm from the cej, surface that will be the bottom of the sample, resulting in a 4-mm-high specimen. the top and bottom surfaces of the specimens were sanded with 400 and 600 grits sandpapers (labopol-21, struers, copenhagen, denmark). the central void of the specimen was prepared with maxicut burs (komet inc, lemgo, germany) mounted in a handpiece under air-water cooling, resulting in standardized conical cavities (4.8 x 2.8 x 4 mm) with a 3.1 c-factor. 4 melo et al. a b c d e f 4,8 mm light-cured strategy (n = 17) dural-cured strategy (n = 17) top bottom region 24 hours 6 months storage time adhesive failure cohesive failures mixed failure failure mode stereomicroscope bond strength utm in situ degree of conversion micro-raman spectroscopy 2,8 mm 4 mm 4 mm figure 1. specimen preparation and groups of curing strategies (a). regions of specimens (b). storage times (c). analysis of the in situ degree of conversion through the micro-raman spectroscopy (d). analysis of the bond strength in the universal testing machine (utm) (e). analysis of failure mode through scanning electron microscopy (sem) (f). curing strategies were as follow: light-cured: no acid etching was done. the adhesive was applied and rubbed for 20 s, followed by gentle air drying for approximately 5 s to evaporate the solvent, and light cured for 10 s (coltolux led, coltène/whaledent, altstätten, switzerland 1200 mw/cm²). dual-cured: the surface was etched with 37% phosphoric acid for 15 s and rinsed for 30 s. dentin was maintained moist after water excess removal with absorbent paper. the activator was applied and air-dried gently for 5 s. then, the primer was applied and air dried gently for 5 s, leaving the surface with a shiny aspect. a coat of the catalyst was applied and light-cured for 10 s 5 melo et al. (coltolux led 1200 mw/cm²). the adhesive system was applied according to the manufacturers’ instructions. the bond component was not applied because it is part of the adper scotchbond multi-purpose plus catalyst formulation. after adhesive procedures, the teeth were set on a glass slab and filled with a single increment of filtek™ bulk fill (3m espe, st. paul, mn, usa) resin composite. a glass slide was pressed onto the specimen in order to standardize the smooth surface and the distance of 1,0 mm between the light source and the resin composite during photoactivation. the resin composite was cured for 20 s (coltolux led 1200 mw/cm²). all the adhesive, restorative and curing procedures were performed by a single operator. samples were polished with 400, 500, and 800 grit sandpapers and cleaned in an ultrasonic bath for 20 min. half of samples were stored in distilled water at 37 ºc for 24 hours. another thirty four samples were stored at the same conditions for aging in distilled water changed weekly for 6 months. “in situ” degree of conversion (dc) the degree of conversion (dc) of each material tested in this study was evaluated through micro-raman spectroscopy (xplora micro-raman, horiba, paris, france). spectroscope calibration was done using a silicon sample. raman spectra were collected using in the range between 1590 and 1670 cm-1 using the 638 nm laser emission wavelength with 10 s acquisition time and 3 accumulations. the diameter of the laser beam used over the specimen was 1 µm and the analysis was performed with a 100x magnification lens (olympus uk, london, uk). the spectrum was obtained in the middle of hybrid layer. six random spots of each sample were analyzed (three at the top and three at the bottom). a noncured amount of each adhesive system was used as reference. processing was performed with the opus spectroscopy software version 6.5 (bruker optik gmbh, ettlingen, baden-wurttemberg, germany). the % dc was calculated using the monomers to polymer double bonds ratio in the adhesive, according the formula (i) below, in which “r” is the ratio between aliphatic and aromatic bond peaks at 1635 and 1605 cm-1 in the adhesive layer, as performed in a previous study12. dc (%) = 100 × (1 − [r cured/r uncured]) (i) bond strength (bs) evaluation bs was evaluated by the push-out method in a universal testing machine (emic dl 2000, são josé dos pinhais, pr, brazil) as previously reported10,11. an acrylic device with a central hole was adapted on the machine base where specimens were placed with its larger surface facing down. the smaller surface was pushed by a cylindrical plunger (1-mm diameter) with a compressive force (0.5 mm/min) until failure. data were obtained in n and transformed in mpa using the following equation (ii), in which “n” is the bond strength in newton, “r” is the larger surface radius, and “r” is the smaller surface radius and “h” is the sample height10,11. mpa = n/π (r + r) [(h2 + (r r)2] (ii) 6 melo et al. failure mode (fm) analysis after bs test, the fractured specimens were examined on the surface of the dentin and on surface of resin composite using a stereomicroscope (stereo zoom, bausch & lomb, new york, ny, usa) at 30x magnification. the failure modes were categorized into cohesive (dentin or resin composite failure), adhesive (dentin/resin composite interface failure), or mixed (cohesive failure in resin composite and adhesive in interface), as previously described13. statistical analysis data normality and homoscedasticity were analyzed through, respectively, d’agostino & pearson test (p>0.05) and bartlett’s test in graphpad prism 7 software (san diego, ca, usa). in situ dc and bs data were analyzed using two-way anova and tukey’s post hoc tests (p<0.05) in graphpad prism 7 software. failure modes patterns were descriptively analyzed. results “in situ” degree of conversion significant differences in dc were found between curing strategies (p<0.01). multiple comparisons are shown in table 2. top and bottom interfaces showed similar in situ dc, while the dual-cured adhesive system showed higher dc than light-cured adhesive system in both regions. table 2. means (standard deviations) of in situ dc (%) according to the curing strategy of the adhesive system and specimen region. region curing strategy light-cured dual-cured top 53.4 (8.6) ba 62.6 (6.9) aa bottom 54.4 (8.9) ba 62.2 (10.1) aa distinct uppercase letters indicate statistically significant differences between curing strategies for the same region (p<0.05). distinct lowercase letters indicate statistically significant differences between regions for the curing strategy (p<0.05). bond strength significant differences were found between curing strategies (p<0.05), storage times (p<0.01), and the interaction between the two factors was significant (p<0.01). multiple comparisons are shown in table 3. dual-cured adhesive system provided higher bs then light-cured adhesive system in both storage times. for light-cured material, 24-h and 6-month storage times provided similar bs. specimens bonded with dualcured adhesive system stored for 6 months provided lower bs than those stored for 24 h, even though they showed higher bs than the light-cured specimens. 7 melo et al. table 3. means (standard deviations) of bs (mpa) according to the curing strategy of the adhesive system and storage time in water. storage time curing strategy light-cured dual-cured 24 h 7.7 (1.4) ba 12.2 (1.5) aa 6 months 8.0 (0.7) ba 9.7 (0.7) ab distinct uppercase letters indicate statistically significant differences between curing strategies for the same storage time (p<0.05). distinct lowercase letters indicate statistically significant differences between storage times for the curing strategy (p<0.05). failure mode most failures were of cohesive mode regardless of the adhesive and storage time. the light-cured adhesive system showed more adhesive failures than the dual-cured (table 4). table 4. number of adhesive, cohesive, and mixed failures according to curing strategy of adhesive systems and storage times. curing strategy light-cured dual-cured storage time adhesive cohesive mixed adhesive cohesive mixed 24 h 6 8 3 13 4 6 months 3 13 1 2 9 6 total 9 21 4 2 22 10 discussion/conclusion the null hypothesis tested – that different curing strategies of adhesive systems and storage times do not affect physical properties of adhesive interfaces – was rejected, as specimens in which dual-cured adhesive system was used had significant differences in the analyzed variables. an universal adhesive system was chosen as the light-cured material since it represents the newest eighth-generation adhesives that can be applied according to the dentist’s preference, with or without dentin etching (etch-and-rinse or self-etch application)14. previous studies showed that scotchbond™ universal adhesive presents similar dentin bond strength15 and bond stability16 for etch-and-rinse and self-etch techniques, or higher bond strength for self-etch technique17, which was used in this investigation. although in situ dc was similar for top and bottom regions with both curing strategies, the dual-cured adhesive system provided higher dc and bs than the light-cured. the dual-cured adhesive used in this study requires the application of an activator and a catalyst before and after the primer, respectively. the activator contains components derived from sulfinate salts, such as sodium benzenesulfinate, that react with acidic monomers of the primer to produce phenyl or benzenesulfonyl free radicals and initiate polymerization18. the catalyst contains benzoyl peroxide that can chemically acti8 melo et al. vate the polymerization reaction without light energy by reacting with tertiary amine to produce free radicals6. these characteristics can contribute to increase the degree of conversion of the adhesive system in order to promote long-lasting bonds, which could justify the better results obtained with the dual-cured adhesive system to in situ dc and bs. in addition, higher dc values were reported for multi-step adhesives compared to simplified adhesive systems due to the relatively hydrophobic and un-solvated bond layer from the primer of multi-step adhesive systems3,19. on the other hand, the higher percentage of hydrophilic monomers and water presence in simplified adhesives (as scotchbond™ universal) impair the curing reaction20, decreasing dc. although differences regarding bond strength were found among groups, the low occurrence of adhesive failures may reflect an adequate interaction between materials and dentin21. the decreased bs values after 6-month aging for dual-cured strategy is possibly associated with the degradation of collagen fibers exposed to acid etching that were not completely covered by the primer, enabling the enzymatic action on matrix metalloproteinases (mmps) and cysteine cathepsins22. on the other hand, as the light-cured strategy had a universal adhesive system applied in self-etching mode, it is likely that fewer collagen fibrils were left uncovered after adhesive application23, favoring bond stability. although presenting lower bs values after water storage, the dual-cured adhesive system provided higher bs means than the light-cured material, which is an important finding. this might have been due to the better mechanical strength of multi-step adhesive systems, which is in part related to their higher dc. further studies should be conducted to evaluate bonding stability of dual-cured adhesives after longer aging times. the results of this laboratorial investigation indicated that the use of a dual-cured adhesive system may result in higher dc within the hybrid layer and better bonding performance, increasing the longevity of tooth restorations. the choice for a three-step dual-cured adhesive system was made since three-step adhesive systems are the gold standard24. however, there are chemical activators that can transform some universal adhesives into dual-cured adhesives. therefore, the stability of dentin bond strength after 6-month water storage has been previously reported for scotchbond™ universal with the self-etching technique16. thus, further investigations should be performed to compare which dual-cured material would provide better performance, such as the outcomes of dual-cured in cavities filled by the incremental technique and longer aging times. concerning the findings of this study, the dual-cured adhesive system influenced the bond strength and degree of conversion in 4-mm deep dentin cavities filled with a bulk-fill resin composite. although the dual-cured adhesive bond strength had reduced after 6 months, it was higher compared to the light-cured independently of the storage time. the degree of conversion was greater in both regions for the dualcured adhesive system. these results suggest a positive effect on bond strength, and better performance of the dual-cured adhesive system, which might contribute to the success and longevity of resin composite restoration in deep cavities filled with a bulk-fill resin composite. 9 melo et al. data availability datasets related to this article will be available upon request to the corresponding author. references 1. ferracane jl, greener eh. the effect of resin formulation on the degree of conversion and mechanical properties of dental restorative resins. j biomed mater res. 1986 jan;20(1):121-31. doi:10.1002/jbm.820200111. 2. breschi l, mazzoni a, ruggeri a, cadenaro m, di lenarda r, de stefano dorigo e. dental adhesion review: aging and stability of the bonded interface. dent mater. 2008 jan;24(1):90-101. doi: 10.1016/j.dental.2007.02.009. 3. cadenaro m, antoniolli f, sauro s, tay fr, di lenarda r, prati c, et al. degree of conversion and permeability of dental adhesives. eur j oral sci. 2005 dec;113(6):525-30.  doi: 10.1111/j.1600-0722.2005.00251.x. 4. tay fr, pashley dh, suh bi, carvalho rm, itthagarun a. single-step adhesives are permeable membranes. j dent. 2002 sep-nov;30(7-8):371-82. doi: 10.1016/s0300-5712(02)00064-7. 5. faria-e-silva al, casselli ds, lima gs, ogliari fa, piva e, martins lr. kinetics of conversion of two dual-cured adhesive systems. j endod. 2008 sep;34(9):1115-8. doi: 10.1016/j.joen.2008.06.016. 6. arrais ca, rueggeberg fa, waller jl, de goes mf, giannini m. effect of curing mode on the polymerization characteristics of dual-cured resin cement systems. j dent. 2008 jun;36(6):418-26. doi: 10.1016/j.jdent.2008.02.014.  7. shirai k, de munck j, yoshida y, inoue s, lambrechts p, suzuki k, et al. effect of cavity configuration and aging on the bonding effectiveness of six adhesives to dentin. dent mater. 2005 feb;21(2):110-24. doi: 10.1016/j.dental.2004.01.003. 8. borges bc, vilela ar, da silva-junior ca, souza-junior ej, sinhoreti ma, pinheiro fh, et al. dual-cured etch-and-rinse adhesive systems increase the bond durability of direct coronal dentin restorations. oper dent. 2013 sep-oct;38(5):512-8. doi: 10.2341/12-246-l.  9. campodonico ce, tantbirojn d, olin ps, versluis a. cuspal deflection and depth of cure in resin-based composite restorations filled by using bulk, incremental and transtooth-illumination techniques. j am dent assoc. 2011 oct;142(10):1176-82. doi: 10.14219/jada.archive.2011.0087. 10. sousa-lima rx, silva l, chaves l, geraldeli s, alonso r, borges b. extensive assessment of the physical, mechanical and adhesion behavior of a low-viscosity bulk fill composite and a traditional resin composite in tooth cavities. oper dent. 2017 sep/oct;42(5):e159-66. doi: 10.2341/16-299-l.  11. chaves lvf, oliveira sn, özcan m, acchar w, caldas mrgr, assunção iv, et al. interfacial properties and bottom/top hardness ratio produced by bulk fill composites in dentin cavities. braz dent j. 2019 oct 7;30(5):476-83. doi: 10.1590/0103-6440201902741. 12. rodrigues ns, souza lc, feitosa vp, loguercio ad, d’arcangelo c, sauro s, et al. effect of different conditioning/deproteinization protocols on the bond strength and degree of conversion of self-adhesive resin cements applied to dentin. int j adhes adhes. 2018 mar;81:98-104. doi: 10.1016/j.ijadhadh.2017.03.013. 13. sousa-lima rx, melo ams, guimarães lkc, souza roa, caldas mrgr, assunção iv, et al. mechanical properties of low and regular viscosity bulk fill composites in a 3d dentin cavity model. j adhes sci tech. 2020 sep 10; 35(3):325-35. doi: 10.1080/01694243.2020.1802134. 14. van meerbeek b, yoshihara k, van landuyt k, yoshida y, peumans m. from buonocore’s pioneering acid-etch technique to self-adhering restoratives. a status perspective of rapidly advancing dental adhesive technology. j adhes dent. 2020 22(1):7-34. doi: 10.3290/j.jad.a43994. https://doi.org/10.1002/jbm.820200111 https://doi.org/10.1080/01694243.2020.1802134 10 melo et al. 15. chen c, niu ln, xie h, zhang zy, zhou lq, jiao k, et al. bonding of universal adhesives to dentine old wine in new bottles? j dent. 2015 may;43(5):525-36. doi: 10.1016/j.jdent.2015.03.004. 16. muñoz ma, luque-martinez i, malaquias p, hass v, reis a, campanha nh, et al. in vitro longevity of bonding properties of universal adhesives to dentin. oper dent. 2015 may-jun;40(3):282-92. doi: 10.2341/14-055-l.  17. valizadeh s, moradi a, mirazei m, amiri h, kharazifard mj. microshear bond strength of different adhesive systems to dentin. front dent. 2019 jul-aug;16(4):265-71. doi: 10.18502/fid.v16i4.2085. 18. ikemura k, endo t. effect on adhesion of new polymerization initiator systems comprising 5-monosubstituted barbituric acids, aromatic sulphonate amides, and tert-butyl peroxymaleic acid in dental adhesive resin. j appl polym sci. 1999 apr;72(13):1655-68. doi: 10.1002/(sici)1097-4628(19990624)72:13<1655::aid-app2>3.0.co;2-0. 19. breschi l, cadenaro m, antoniolli f, sauro s, biasotto m, prati c, et al. polymerization kinetics of dental adhesives cured with led: correlation between extent of conversion and permeability. dent mater. 2007 sep;23(9):1066-72. doi: 10.1016/j.dental.2006.06.040.  20. navarra co, cadenaro m, armstrong sr, jessop j, antoniolli f, sergo v, et al. degree of conversion of filtek silorane adhesive system and clearfil se bond within the hybrid and adhesive layer: an in situ raman analysis. dent mater. 2009 sep;25(9):1178-85. doi: 10.1016/j.dental.2009.05.009. 21. flury s, peutzfeldt a, lussi a. influence of increment thickness on microhardness and dentin bond strength of bulk fill resin composites. dent mater. 2014 oct;30(10):1104-12. doi: 10.1016/j.dental.2014.07.001. 22. peumans m, wouters l, de munck j, van meerbeek b, van landuyt k. nine-year clinical performance of a hema-free one-step self-etch adhesive in noncarious cervical lesions. j adhes dent. 2018 20(3):195-203. doi: 10.3290/j.jad.a40630.  23. van meerbeek b, yoshihara k, yoshida y, mine a, de munck j, van landuyt kl. state of the art of self-etch adhesives. dent mater. 2011 jan;27(1):17-28. doi: 10.1016/j.dental.2010.10.023. 24. scotti n, cavalli g, gagliani m, breschi l. new adhesives and bonding techniques. why and when? int j esthet dent. 2017 12(4):524-35. https://doi.org/10.1002/(sici)1097-4628(19990624)72:13%3c1655::aid-app2%3e3.0.co;2-0 1http://dx.doi.org/10.20396/bjos.v20i00.8664873 volume 20 2021 e214873 original article ¹ department of prosthodontics and periodontology, university of campinas piracicaba dental school, piracicaba, sp, brazil. 2 department of periodontology and oral implantology, dental research division, univeritas – university of guarulhos, guarulhos, sp, brazil. 3 department of prosthodontics and periodontology, university of são paulo bauru dental school, bauru, sp, brazil. 4 department of prosthodontics, faculty of technology and sciences (uniftc), salvador, ba, brazil. corresponding author: dr. raissa micaella marcello machado department of prosthodontics and periodontology university of campinas piracicaba dental school limeira avenue, 901 – piracicaba, sp, brazil. e-mail address: raissammm@ gmail.com editor: dr altair a. del bel cury received: march 5, 2021 accepted: march 29, 2021 marginal misfit of heat-pressed milled wax-pattern and cad/cam crowns and its effect on stress distribution in implant-supported rehabilitations michele costa de oliveira ribeiro¹ , raissa micaella marcello-machado1,* , dimorvan bordin2 , edmara t. p. bergamo3 , rafael soares gomes4 aim: to compare the marginal fit of lithium disilicate cad/cam crowns and heat-pressed crowns fabricated using milled wax patterns, and evaluate its effect on stress distribution in implantsupported rehabilitation. methods: a cad model of a mandibular first molar was designed, and 16 lithium disilicate crowns (8/group) were obtained. the crown-prosthetic abutment set was evaluated in a scanning electron microscopy. the mean misfit for each group was recorded and evaluated using student’s t-test. for in silico analysis, a virtual cement thickness was designed for the two misfit values found previously, and the cad model was assembled on an implant-abutment set. a load of 100 n was applied at 30° on the central fossa, and the equivalent stress was calculated for the crown, titanium components, bone, and resin cement layer. results: the cad/cam group presented a significantly (p=0.0068) higher misfit (64.99±18.73 µm) than the heat-pressed group (37.64±15.66 µm). in silico results showed that the heat-pressed group presented a decrease in stress concentration of 61% in the crown and 21% in the cement. in addition, a decrease of 14.5% and an increase of 7.8% in the stress for the prosthetic abutment and implant, respectively, was recorded. for the cortical and cancellous bone, a slight increase in stress occurred with an increase in the cement layer thickness of 5.9% and 5.7%, respectively. conclusion: the milling of wax patterns for subsequent inclusion and obtaining heat-pressed crowns is an option to obtain restorations with an excellent marginal fit and better stress distribution throughout the implant-abutment set. keywords: dental materials. dental marginal adaptation. dental prosthesis, implant-supported. microscopy, electron, scanning. finite element analysis. https://orcid.org/0000-0001-7679-0502 https://orcid.org/0000-0001-7661-703x https://orcid.org/0000-0002-8466-9558 https://orcid.org/0000-0002-5006-2184 https://orcid.org/0000-0002-7989-0098 2 ribeiro et al. introduction the marginal misfit of dental restorations has been associated with clinical failures. it is commonly related to microleakage, caries, margin staining, debonding, and restoration fracture1-3. in addition, the misfit between the crown and implant-abutment set can lead to biofilm and food accumulation, which could result in peri-implant complications4. some studies have reported that marginal misfit can influence the stress distribution around restorations, where a thick cement layer increases the stress in itself and is harmful to the longevity of the restoration1,2. a 120 µm misfit was considered as a minimum clinically acceptable value in the past, and the current studies still consider this value as a reference even with the higher accuracy of the current techniques and devices3,5,6. technology devices such as computer-aided design/computer-aided manufacturing (cad/cam) systems have been successfully used to improve restorative procedures in the dental field. this technology offers faster and more practical procedures to obtain ceramic restorations compared to the conventional manual method3,7 because it allows a chairside digital workflow without the need for physical models8. a clinical study9 assessing implant-supported single crowns in the posterior region showed that the use of the cad/cam technique produced crowns with excellent adaptation in relation to interproximal and occlusal contacts, without the need for adjustments. another option for fabricating dental restorations is the heat-press technique (hpt)3,7,10,11, where a tooth is waxed-up, invested in refractory material, and heated in an oven3,7,12. the space created by wax elimination is filled with a ceramic ingot that is heat-pressed to obtain the restoration12,13. the waxing-up procedure can be handmade (conventional method), or computer-aided designed and milled in wax blocks10-12. milling restoration directly from ceramic blocks decreases one step compared to milling those in wax blocks, which needs to be invested and heat-pressed. however, some studies report that the latter procedure is related to the production of a better fit than the former7,10,14,15. furthermore, when several restorations are made, the milling process directly from single ceramic blocks could be slow to obtain a large number of restorations because of its hardness15. in contrast, milling from a wax block is faster, and the investment of the restorations for pressing can be made with several restorations at the same time16. cad/cam restorations have the advantage of good accuracy and a computer-controlled process that can provide well-defined and fitted margins17. in practice, the milled edges of thin crowns on hard materials can produce defects in their margins which worsens their fit and produce stresses in that region, which could lead to restoration of failure14,18. a possible solution would be a combination of cad/cam and hpt. from a digital design, a crown can be milled in a wax block10,12. since wax presents a soft surface with low hardness, it is an easy material to be milled and consequently to produce high margin accuracy restorations18,19. this wax crown can be invested to create a ceramic restoration by hpt afterwards16,20. 3 ribeiro et al. different commercial presentations of the same material are available sometimes21. one of these materials is lithium disilicate, a glass-ceramic material that has been well studied; however, it is still controversial whether the material provides better edge stability and marginal fit7,22. currently, this material is available in blocks for cad/cam or ingots for hpt to furnish all market demand3,7. although many studies have compared the marginal fit of lithium disilicate cad/cam crowns to those made by hpt, the wax patterns of the hpt are often produced manually by dental technician7,12. as all manual labor, reproducibility is a factor that can compromise the comparison between such techniques7. however, this problem can be solved by a controlled milling process23. additionally, the stress distribution in lithium disilicate implant-supported single crowns manufactured by the two techniques remains unclear, and its influence on the implant components and bone is still unclear. the objective of the present study was to compare the marginal fit of lithium disilicate cad/cam crowns and heat-pressed crowns fabricated using milled wax patterns and evaluate its effect on stress distribution in implant-supported rehabilitation. material and methods in vitro analysis using a cad software (ceramill mind; amann girrbach, koblach, vorarlberg, austria) a mandibular first molar (height, 10.6 mm; buccal-lingual width, 10.8 mm; mesio-distal width, 11.4 mm) was designed over a universal prosthetic abutment (4.5 diameter, 6 mm height, 2.5 mm collar height). the relief adopted followed the standard of the software used, which is 0.05 mm. from this cad, sixteen crowns were milled, eight from lithium disilicate blocks (ips e.max cad; ivoclar), and eight from a wax block (odontofix; ribeirão preto, são paulo, brazil). the crowns were milled under irrigation using a 5-axis milling unit (ceramill motion 2 5x; amann girrbach, koblach, vorarlberg, austria) using a new bur for each group. for the heat-pressed group, the wax-up was invested with a phosphate-bonded universal investment (ips pressvest premium; ivoclar vivadent) and after heat pressing with a lithium disilicate ingot (ips e.max press; ivoclar vivadent) in a furnace (programat p310, ivoclar vivadent) according to the manufacturer’s instructions. the crowns were sputter-coated with gold for evaluation using a scanning electron microscope (sem) (jsm-5600lv, jeol, boston, massachusetts, usa)24. the crown was fixed with carbon adhesive tape from the occlusal surface to the base of the prosthetic abutment and positioned perpendicular to the stub. to avoid bias, the crowns were evaluated exactly in the way they were manufactured, without any kind of adjustment. the measurement was standardized on the center of the buccal, lingual, mesial, and distal faces with a zoom of 550x24,25. four measures were made in each face with a distance of approximately 50 µm between them, and a mean of misfit was obtained for each crown (figure 1). 4 ribeiro et al. normal data distribution was confirmed by the shapiro-wilk test and homogeneity by levene’s test. the mean misfit between the cad/cam and heat-pressed groups was evaluated by student’s t-test. statistical analysis was performed using the sas system release 9.3 (sas institute inc., cary, nc, usa), and a significance level of 5% (α=0.05) was adopted. in silico analysis the same mandibular first molar cad model used for milling the crowns was exported to solidworks software (solidworks 2013; dassault systèmes solidworks corp). the crown was assembled in a universal prosthetic abutment (4.5 mm width × 2.5 mm collar height × 6 mm height), which was screwed in a 4 mm width x 11 mm height morse taper implant (intraoss, itaquaquecetuba, são paulo, brazil). both universal prosthetic abutment and implant cads were supplied by the manufacturer (intraoss). the implant was inserted into a jaw segment with cortical and cancellous bones. a virtual cement thickness was designed for the two values found previously in the marginal fit evaluation to form the two experimental models (figure 2). the two models were exported to the ansys workbench software for mathematical analysis (ansys workbench 15.0; canonsburg, pa, usa). a 0.6 mm tetrahedral mesh was generated after 5% convergence analysis. the elastic modulus and poisson’s ratio of each material were used in the simulations (table 1). a load of 100 n was applied at 30° to the central fossa. the maximum principal stress (σmax) was calculated for the prosthetic crown, von mises stress (σvm) for titanium comfigure 1. measurement of the gap existing between the crown and the prosthetic abutment. 15 kv x550 20 µm 46.5 µm 47.6 µm46.9 µm 48.4 µm table 1. material properties used in finite element models. material elastic modulus (gpa) (e) poisson’s ratio (δ) lithium disilicate26 95 0.20 resin cement27 18.3 0.33 titanium28 110 0.35 cortical bone28 13.6 0.26 cancellous bone28 1.36 0.31 5 ribeiro et al. ponents (implant and prosthetic abutment), and maximum shear stress (τmax) for bone (cancellous and cortical) and resin cement layer26,28. the results were evaluated qualitatively by the stress distribution and quantitatively by the peak stress (mpa) generated in each model. all models were assumed to be homogeneous, isotropic, and linearly elastic. results the mean misfit for the heat-press group was 37.64 ± 15.66 µm, statistically different (p = 0.0068) from the cad/cam group, which presented a mean of 64.99 ± 18.73 µm. these values were used to simulate the cement thickness in the finite element analysis (fea) (figure 2). the fea results (table 2) revealed an important influence of the cement thickness on the stress distribution in the two studied models. the most substantial difference occurred in the crown and cement layer, where the model restored with the lowest cement thickness (heat-press group) presented a decrease of 61% in the σmax of the crown and 21% in the τmax of the cement, both compared to the cad/cam group, restored with the highest cement thickness layer (figure 3). figure 2. occlusal and sectional schematic view showing the crown dimensions and cement thickness used in the cad/cam (a) and heat-pressed milled wax-pattern (b) groups. 11.4 mm 10.6 mm 65 µm 38 µm a b 10.8 mm table 2. peak stress (mpa) and difference between groups after load. component cad/cam heat-press % stress crown (σmax) 132 51 *61% cement layer (τmax) 21.2 16.7 *21% prosthetic abutment (σvm) 302 258 *14.5% implant (σvm) 152 165 #7.8% cortical bone (τmax) 29.9 31.8 #5.9% cancellous bone (τmax) 11.4 12.1 #5.7% (*) stress decrease. (#) stress increase. 6 ribeiro et al. the 38-µm cemented thickness model presented a decrease of 14.5% and an increase of 7.8% in the σvm for the prosthetic abutment and implant, respectively, compared to the 65-µm cemented thickness model (figure 4). for the cortical and cancellous bone, a slight increase in τmax occurred with a decrease in the cement layer thickness of 5.9% and 5.7%, respectively (figure 5). a c b d 132 max 120 107 95.3 83.2 71 58.9 46.7 34.6 22.4 10.2 -1.92 -14.1 26.2 -38.4 min 21.2 max 19.7 18.2 16.7 15.3 13.8 12.3 10.8 9.34 7.86 6.39 4.91 3.43 1.95 -0.47 min 132 max 121 110 99 88 76.9 65.9 21,2 19,6 51 max 16,7 max 32.9 21.9 10.9 -0.125 -11.1 -22.1 min 14,8 13,3 11,7 10,1 8,5 6,91 5,32 3,74 2,15 0,562 min figure 3. stress distribution in the crown (σmax) and cement layer (τmax). cervical view of the crown restored with a 65 µm (a) and 38 µm (b) cement layer showing the stress peak on the inner face. isometric view of the cement layer with 65 µm (c) showing the stress peak on the occlusal face, and 38 µm (d) with the stress peak on the axial face. 7 ribeiro et al. figure 4. stress distribution in the prosthetic abutment and implant (σvm). vestibular view of the prosthetic abutment of the model restored with a 65 µm (a) and 38 µm (b) cement layer showing the stress peak on the prosthetic abutment collar. isometric view of the implant of the model restored with a 65 µm (c) and 38 µm (d) cement layer showing the stress peak on the corresponding abutment collar level. a c b d 302 max 280 259 237 216 194 173 151 130 108 86.7 65.2 43.7 22.2 0.666 min 165 152 max 130 118 107 94.8 83 71.2 59.4 47.7 35.9 24.1 12.3 0.541 min 165 max 154 142 130 118 107 94.8 83 71.2 59.4 47.6 35.8 24 12.2 0.452 min 302 280 -22.1 min 237 216 194 173 151 130 108 86,6 65,1 43,6 22 0,488 min 8 ribeiro et al. discussion the concerns related to the study of restoration marginal fit have been addressed for many years29. whenever a new material or technique arises, some studies resort to this methodology18. the concern about poorly fitting restorations is justifiable. several studies have shown that a poor fit can cause many problems in the restoration such as cement dissolution, microleakage, and lower fracture strength7,18,23,30. clinically acceptable values of 120 µm were established many years ago, regardless of the material and technique that are likely capable of generating better adjustment values than those reported in the past as acceptable5,23. thus, this study evaluated, through in vitro and in silico analysis, the marginal fit and stress distribution of implant-supported rehabilitations restored with lithium disilicate crowns manufactured by cad/cam and the heat-pressed technique. regardless of the technique used for crown manufacture, the present study found values lower than 120 µm for both groups. this finding is supported by most studies related to the marginal fit of this material7,13,18,31. however, the result of a better fit figure 5. stress distribution in the cortical and cancellous bone (τmax). exterior view of the cortical bone of the model restored with a 65 µm (a) and 38 µm (b) cement layer showing τmax in the cervical inferior area at the buccal portion. exterior view of the cancellous bone of the model restored with a 65 µm (c) and 38 µm (d) cement layer showing τmax at buccal region. a c b d 29.9 max 27.3 25 22.7 20.4 18.2 15.9 13.6 11.4 9.09 6.82 4.55 2.28 0.0112 min 11.4 max 10.3 9.43 8.58 7.72 6.86 5.15 4.3 3.44 2.58 1.73 0.872 0.0165 min 12.1 max 11.2 10.4 9.49 8.63 7.77 6.91 6.04 5.18 4.32 3.46 2.6 1.74 0.878 0.0166 min 31.8 max 29.9 27.2 25 22.7 20.4 18.2 15.9 13.6 11.4 9.08 6.82 4.55 2.28 0.0117 min 31.8 12 9 ribeiro et al. to the heat-pressed group in this study is controversial12. some others consider that the cad/cam process, owing to its high accuracy, produces the best values for the marginal fit of the restorations12,13,30. however, these studies do not consider chipping that may occur at the margin of the thin restorations during the milling process, which could lead to higher misfit values18,19. one of the most accepted theories for the best fit of the heat-pressed group is precisely the fact that it was made based on a milled wax pattern, which combined the high accuracy of the cad/cam system with the easy milling from wax, causing less occurrence of cervical defects on them12,18,19. usually, the inaccuracies of the restoration fit occur in techniques where the manual skill of the technician is indispensable, as in the conventional lost-wax method, to fabricate porcelain fused to metal crowns12. although marginal fit problems are minimized with cad/cam restorations, when compared to manual techniques, the final fit quality of restoration will further depend on the type of material milled18,19. the ease of how a material is milled depends directly on its hardness, which together with fracture toughness will be responsible for the final restorations edge quality19. the greater the hardness and the lower the material fracture toughness, the greater will be the difficulty of milling and achieving a good quality margin18,19. the difference between the two cement layers, although statistically significant, could not be clinically relevant because such a small difference found could not present different behaviors in the clinical environment. however, fea seems to show a relevant influence of the cement layer on the stress behavior through rehabilitation, mainly for the crown and the cement itself. this stress distribution difference, over time, could lead to different fatigue behaviors with different failure load32. it is possible that the lower cement thickness in the heat-pressed group, as it presented the lowest stress value, would take longer to fail, which could decrease the chance of failure due to crown debonding when compared to the cad/cam group. it can also be seen that when a thicker cement layer is used, the stress peak in the crown is 2.5 times higher. this suggests that thinner cement layers favor the stress distribution throughout the crown ad cement layer and at the same time do not compromise in a relevant way the adjacent structures, such as the prosthetic abutment, implant, and bone, as the heat-pressed group showed only slightly higher values of stress for that component. moreover, it is better for rehabilitation that the highest stress concentration is in the titanium components; ceramic restorations, due to their brittleness index, are more vulnerable to chipping33 than prosthetic abutments and implants that are ductile and therefore withstand a certain level of plastic deformation before failure34. hence, the higher stress in the ceramic crown could increase the possibility of crown chipping/fracture over time1,35 and increase the risk of infiltration and solubility of the cement layer. although the heat-pressed group showed better results in both evaluations, this study had some limitations. this includes the absence of a mechanical test that allows the identification of the failure modes of the rehabilitation tested in the fea, as it is numerical theoretical analysis. in addition, the lack of evaluation of the axial and occlusal discrepancies, since it is not possible to visualize the interior of the crown-prosthetic abutment set using the sem, as the assessment restricted only to the margin of the 10 ribeiro et al. restoration. hence, further in vitro studies in this regard are needed to validate the results of the fea, and to assess the internal misfit of the crowns. despite these limitations, it is worth remembering that although one technique has excelled the other, even the worst result can be considered as a good performance, being approximately half of what is considered clinically acceptable5. therefore, it is up to each dentist and prosthetic technician to consider which procedure would work better in the workflow of their office or laboratory15. in conclusion, both methods achieved marginal misfit values within the clinically acceptable limits. the milling of wax patterns for subsequent inclusion and obtaining heat-pressed crowns is an option to obtain restorations with an excellent marginal fit and better stress distribution throughout the rehabilitation. conflicts of interest the authors state no conflicts of interest. funding this study was supported by the são paulo research foundation (fapesp) (grant nº. 2014/23358-0), national council for scientific and technological development (cnpq) (grant nº. 308141/2006-7), and coordination for the improvement of higher education personnel brazil (capes) – (grant nº. 001) acknowledgements the authors are grateful to intraoss for its support with the implant system cads used in this study. references 1. rojpaibool t, leevailoj c. fracture resistance of lithium disilicate ceramics bonded to enamel or dentin using different resin cement types and film thicknesses. j prosthodont. 2017 feb;26(2):141-9. doi: 10.1111/jopr.12372. 2. tuntiprawon m, wilson pr. the effect of cement thickness on the fracture strength of all-ceramic crowns. aust dent j. 1995 feb;40(1):17-21. doi: 10.1111/j.1834-7819.1995.tb05607.x. 3. dolev e, bitterman y, meirowitz a. comparison of marginal fit between cad-cam and hotpress lithium disilicate crowns. j prosthet dent. 2019 jan;121(1):124-8. doi: 10.1016/j. prosdent.2018.03.035. 4. vargas sp, neves acc, vitti r, amaral m, henrique mn, silva-concílio lr. influence of different ceramic systems on marginal misfit. eur j prosthodont restor dent. 2017 sep;25(3):127-30. doi: 10.1922/ejprd_01702vargas04. 5. mclean jw, von fraunhofer ja. the estimation of cement film thickness by an in vivo technique. br dent j. 1971 aug;131(3):107-11. doi: 10.1038/sj.bdj.4802708. 6. daou ee, baba nz. evaluation of marginal and internal fit of presintered co-cr and zirconia three-unit fixed dental prosthesis compared to cast co-cr. j prosthodont. 2020 dec;29(9):792-9. doi: 10.1111/jopr.13183. 11 ribeiro et al. 7. azar b, eckert s, kunkela j, ingr t, mounajjed r. the marginal fit of lithium disilicate crowns: press vs. cad/cam. braz oral res. 2018;32:e001. doi: 10.1590/1807-3107/2018.vol32.0001. 8. joda t, zarone f, ferrari m. the complete digital workflow in fixed prosthodontics : a systematic review. bmc oral health. 2017 sep;17(1):124. doi: 10.1186/s12903-017-0415-0. 9. joda t, brägger u. time-efficiency analysis of the treatment with monolithic implant crowns in a digital workflow: a randomized controlled trial. clin oral implants res. 2016 nov;27(11):1401-6. doi: 10.1111/clr.12753. 10. homsy f, bottin m, özcan m, majzoub z. fit accuracy of pressed and milled lithium disilicate inlays fabricated from conventional impressions or a laboratory-based digital workflow. eur j prosthodont restor dent. 2019 feb;27(1):18-25. doi: 10.1922/ejprd_01828homsy08. 11. schestatsky r, zucuni cp, dapieve ks, burgo tal, spazzin ao, bacchi a, et al. microstructure, topography, surface roughness, fractal dimension, internal and marginal adaptation of pressed and milled lithium-disilicate monolithic restorations. j prosthodont res. 2020 jan;64(1):12-9. doi: 10.1016/j.jpor.2019.05.004. 12. shamseddine l, mortada r, rifai k, chidiac jj. marginal and internal fit of pressed ceramic crowns made from conventional and computer-aided design and computer-aided manufacturing wax patterns: an in vitro comparison. j prosthet dent. 2016 aug;116(2):242-8. doi: 10.1016/j.prosdent.2015.12.005. 13. sadid-zadeh r, li r, miller lm, simon m. effect of fabrication technique on the marginal discrepancy and resistance of lithium disilicate crowns: an in vitro study. j prosthodont. 2019 dec;28(9):1005-10. doi: 10.1111/jopr.13014. 14. reich s, gozdowski s, trentzsch l, frankenberger r, lohbauer u. marginal fit of heat-pressed vs. cad/cam processed all-ceramic onlays using a milling unit prototype. oper dent. 2008;33(6):644-50. doi: 10.2341/07-162. 15. zeltner m, sailer i, mühlemann s, özcan m, hämmerle chf, benic gi. randomized controlled within-subject evaluation of digital and conventional workflows for the fabrication of lithium disilicate single crowns. part iii: marginal and internal fit. j prosthet dent. 2017 mar;117(3):354-62. doi: 10.1016/j.prosdent.2016.04.028. 16. santos mjmc, costa md, rubo jh, pegoraro lf, santos gc. current all-ceramic systems in dentistry: a review. compend contin educ dent. 2015 jan;36(1):31-7; quiz 38, 40. 17. steinmassl o, dumfahrt h, grunert i, steinmassl p-a. cad/cam produces dentures with improved fit. clin oral investig. 2018 nov;22(8):2829-35. doi: 10.1007/s00784-018-2369-2. 18. gomes rs, souza cmc de, bergamo etp, bordin d, del bel cury aa. misfit and fracture load of implant-supported monolithic crowns in zirconia-reinforced lithium silicate. j appl oral sci. 2017;25(3):282-9. doi: 10.1590/1678-7757-2016-0233. 19. tsitrou ea, northeast se, van noort r. brittleness index of machinable dental materials and its relation to the marginal chipping factor. j dent. 2007 dec;35(12):897-902. doi: 10.1016/j.jdent.2007.07.002. 20. sailer i, benic gi, fehmer v, hämmerle chf, mühlemann s. randomized controlled within-subject evaluation of digital and conventional workflows for the fabrication of lithium disilicate single crowns. part ii: cad-cam versus conventional laboratory procedures. j prosthet dent. 2017 jul;118(1):43-8. doi: 10.1016/j.prosdent.2016.09.031.. 21. guess pc, schultheis s, bonfante ea, coelho pg, ferencz jl, silva nrfa. all-ceramic systems: laboratory and clinical performance. dent clin north am. 2011 apr;55(2):333-52, ix. doi: 10.1016/j.cden.2011.01.005. 22. kim j-h, jeong j-h, lee j-h, cho h-w. fit of lithium disilicate crowns fabricated from conventional and digital impressions assessed with micro-ct. j prosthet dent. 2016 oct;116(4):551-7. doi: 10.1016/j.prosdent.2016.03.028. 12 ribeiro et al. 23. boitelle p, mawussi b, tapie l, fromentin o. a systematic review of cad/cam fit restoration evaluations. j oral rehabil. 2014 nov;41(11):853-74. doi: 10.1111/joor.12205. 24. castillo-oyagüe r, lynch cd, turrión as, lópez-lozano jf, torres-lagares d, suárez-garcía m-j. misfit and microleakage of implant-supported crown copings obtained by laser sintering and casting techniques, luted with glass-ionomer, resin cements and acrylic/urethane-based agents. j dent. 2013 jan;41(1):90-6. doi: 10.1016/j.jdent.2012.09.014. 25. barbosa jr sa, bacchi a, barão var, silva-sousa ytc, bruniera jf, caldas ra, et al. implant volume loss, misfit, screw loosening, and stress in custom titanium and zirconia abutments. braz dent j. 2020 sep;31(4):374-9. doi: 10.1590/0103-6440202003643. 26. schmitter m, schweiger m, mueller d, rues s. effect on in vitro fracture resistance of the technique used to attach lithium disilicate ceramic veneer to zirconia frameworks. dent mater. 2014 feb;30(2):122-30. doi: 10.1016/j.dental.2013.10.008. 27. lü l-w, meng g-w, liu z-h. finite element analysis of multi-piece post-crown restoration using different types of adhesives. int j oral sci. 2013 sep;5(3):162-6. doi: 10.1038/ijos.2013.50. 28. cruz m, wassall t, toledo em, da silva barra lp, cruz s. finite element stress analysis of dental prostheses supported by straight and angled implants. int j oral maxillofac implants. 2009 may-jun;24(3):391-403. 29. holmes jr, bayne sc, holland ga, sulik wd. considerations in measurement of marginal fit. j prosthet dent. 1989 oct;62(4):405-8. doi: 10.1016/0022-3913(89)90170-4. 30. mostafa nz, ruse nd, ford nl, carvalho rm, wyatt ccl. marginal fit of lithium disilicate crowns fabricated using conventional and digital methodology: a three-dimensional analysis. j prosthodont. 2018 feb;27(2):145-52. doi: 10.1111/jopr.12656. 31. toniollo mb, macedo ap, silveira rodrigues rc, ribeiro rf, de mattos mg. a three-dimensional finite element analysis of the stress distribution generated by splinted and nonsplinted prostheses in the rehabilitation of various bony ridges with regular or short morse taper implants. int j oral maxillofac implants. 2017;32(2):372-6. doi: 10.11607/jomi.4696. 32. bonfante ea, coelho pg. a critical perspective on mechanical testing of implants and prostheses. 2016 mar;28(1):18-27. doi: 10.1177/0022034515624445. 33. flask jd, thompson ga, singh m, berzins dw. edge chipping of translucent zirconia. j prosthet dent. 2021 feb 11;s0022-3913(20)30801-5. doi: 10.1016/j.prosdent.2020.12.009. 34. yamaguchi h, takahashi m, sasaki k, takada y. mechanical properties and microstructures of cast dental ti-fe alloys. dent mater j. 2021 jan;40(1):61-7. doi: 10.4012/dmj.2019-254. 35. rezende cee, borges afs, gonzaga cc, duan y, rubo jh, griggs ja. effect of cement space on stress distribution in y-tzp based crowns. dent mater. 2017 feb;33(2):144-51. doi: 10.1016/j.dental.2016.11.006. 1 volume 22 2023 e237434 original article braz j oral sci. 2023;22:e237434http://dx.doi.org/10.20396/bjos.v22i00.8667434 1 state university of piauí (uespi), school of dentistry, department of clinical dentistry, area of integrated clinic, parnaíba, pi, brazil. corresponding author: ana de lourdes sá de lira state university of piauí (uespi), school of dentistry rua senador joaquim pires 2076 ininga. fone (86) 999595004 cep: 64049-590 teresina-pi-brasil email: anadelourdessl@hotmail.com editor: dr. altair a. del bel cury received: october 31, 2021 accepted: july 1, 2022 prevalence and influence of dental development anomalies in smile esthetics: a cross-sectional analysis marcelo lucio sousa silva junior1, millena lopes de brito1, breno wesley leal carvalho1 , elen maria carvalho da silva1 , ana de lourdes sá de lira1,* aim: to determine the prevalence of dental development anomalies and type of influence on the smile of adolescent students. method: this was a cross-sectional and analytical study carried out in two public (a1) and two private (a2) schools chosen by lot in the city of parnaíba-piauí. the sample calculation was based on the target population: number of people enrolled in public and private schools between 15 and 19 years, in the city of parnaíba-pi, which totaled 6209 students in 2020, according to a survey carried out by the brazilian institute of geography and statistics – ibge. a questionnaire on epidemiological data and aesthetic self-perception of the smile was applied to 160 adolescents between 15 and 19 years old, from august 2020 to july 2021. the clinical examination was carried out under natural light, to check for the presence of anomaly(s) in the dental development. students who presented only one pathology would be called group 1 (g1), those who presented two would be called group 2 (g2) and those who presented 3 or more would be called group 3 (g3). on the other hand, adolescents in whom no anomaly was evidenced would participate in the control group (cg), both in a1 and a2. results: it was observed that 37.5% of the sample had only a type of dental anomaly, corresponding to 60 individuals. the most prevalent were enamel hypoplasia, fusion, transposition, agenesis, ectopic eruption, microdent and dens-in-dent. it was possible to verify a higher prevalence in the maxilla, private schools (76.6%) and females (86.6%). in 45% of adolescents with dental anomalies, embarrassment was observed when smiling. conclusion: the prevalence was relatively high, highlighting the enamel hypoplasia, influencing the smile esthetics of a reasonable number of adolescents, whether for acquaintances, strangers or even for photographs. keywords: tooth abnormalities. prevalence. adolescents. esthetics, dental. https://orcid.org/0000-0001-5115-3929 https://orcid.org/0000-0003-3012-3178 https://orcid.org/0000-0002-9299-1416 2 junior et al. braz j oral sci. 2023;22:e237434 introduction dental developmental anomaly (dda) is any disorder or deviation of an anatomical characteristic or structure, relative to normality, whose etiology may be congenital, genetic in the periods of prenatal and postnatal development, or due to environmental factors, during training and cell differentiation. they can be characterized by disorders in the following aspects: shape, size, number, position, and eruption1-4. in a recent study with panoramic radiographs of brazilian children, a prevalence of dda of 27.5% was observed, with hypodontia being the most common (7.3%)3. compared to other common oral cavity diseases and disorders, such as tooth decay and periodontal diseases, they are less common, however, treatment and control are often associated with difficulty and complexity5,6. a radiographic examination should be performed to confirm the diagnosis when dda is suspected after the clinical examination. early diagnosis in primary, mixed or early permanent dentition is recommended, suggesting greater simplicity in the treatment plan, with a reduction in complications. thus, controlling the eruption and developing the dentition is an integral part of achieving occlusal, functional and esthetic harmony7-12. although dda can be found in any age group, adolescents are one of the groups most affected psychologically by the effects of these anomalies, because the aesthetic standards exert a direct influence on their self-esteem and social behavior13-17. the early diagnosis of a given dental anomaly, such as agenesis or the presence of a conoid tooth, can alert the clinician to the possibility of developing other associated anomalies in the same patient or in other family members, allowing for early diagnosis and timely dental intervention12-14. in this context, as it is a public health problem, dental aesthetics acts by improving social interaction, improving the patient’s self-esteem and self-confidence, reinforcing the importance of dental treatment in adolescents, both in the aesthetic-functional and psychosocial scope18. the null hypothesis of this research was that the prevalence of dda among adolescents is low, but when present, an association of anomalies can be found in the same individual, with the predominant etiological factor being genetic. based on this context, it became justifiable to investigate the presence of dda, the self-perception and the impact generated by it, to encourage the incorporation of treatment in the public service, and thus make dental care more accessible to less socioeconomically favored. the aim of this research was to determine the prevalence of dental development anomalies and type of influence on the smile of adolescent students. material and methods the research was carried out after the ethical opinion of approval of the research ethics committee of the state university of piauí cep/uespi, with caae 3 junior et al. braz j oral sci. 2023;22:e237434 number: 26139419.0.0000.5209. this was a cross-sectional and analytical study carried out in from august 2020 to july 2021. the sample calculation was based on the target population: number of people enrolled in public and private schools between 15 and 19 years, in the city of parnaíba-pi, which totaled 6209 students in 2020, according to a survey carried out by the brazilian institute of geography and statistics ibge19. in this way, from the sample size formula, a number of 396 was obtained, with approximation to 400. according to the sample calculation, the minimum number of 396 participants for this research would be enough, taking into account the proposed analyses, a sampling error of 5%, in addition to a 95% confidence level, according to the guidelines addressed by fonteles et al.20. due to the research being carried out during the covid-19 pandemic period, the minimum estimated population value was not obtained. the inclusion criteria adopted were: adolescent students between 15 and 19 years old, who were studying in public and private schools and accepted to participate in the research, with permission from their parents (underage). the exclusion criteria were all students unable to understand and answer the questionnaires, such as those with cognitive impairment, syndromes or hearing and visual impairment, those who did not wish to participate in the research or those whose parents not authorized. before the pilot study, in order to standardize the diagnosis of dda, clinical and radiological training was carried out to calibrate two examiners at the clinic school of dentistry (csd), based on the study previously carried out by other authors16. twenty adolescents who did not participate in the study were examined to determine intraand inter-examiner agreement. kappa values were 0.84 for inter-examiner agreement (between the two examiners), 0.85 and 0.87 for intra-examiner agreement, and 0.86 and 0.85 for inter-examiner agreement between each examiner and the gold standard. for this, the individuals were examined twice, with an interval of two weeks. the same was done with regard to the interpretation of panoramic radiography. before data collection, a pilot study was carried out with 30 adolescents from municipal schools that did not participate in the sample, to evaluate the methods and check whether there would be a need to make changes in the initially proposed methodology. there was no need to reformulate the method. two public (a1) and two private (a2) schools were chosen by drawing lots, so that the number of students participating in the sample was evenly distributed. the schools were adopting the hybrid education system due to the current moment of the covid-19 pandemic. students were also chosen by lottery according to their schoolbook number. a questionnaire was applied to each research participant about epidemiological data and the aesthetic self-perception of the smile, based on a previously validated study with brazilians21 (figure 1). the clinical examination was carried out under natural light, in a school environment, by two examiners using previously calibrated personal protective equipment (ppe’s), with the aid of a wooden spatula, mouth 4 junior et al. braz j oral sci. 2023;22:e237434 mirror and dental probe. students who presented only one pathology would be called group 1 (g1), those who presented two would be called group 2 (g2) and those who presented 3 or more would be called group 3 (g3). on the other hand, adolescents in whom a dda was not evidenced participated in the control group (cg), both in a1 and a2. 1. gender: female ( ) male ( ) 2. age of adolescent: 15 years ( ) 16 years ( ) 17 years ( ) 18 years ( ) 19 years ( ) 3. race: white ( ) black ( ) brown ( ) 4. school: public ( ) private ( ) 5. are you embarrassed to smile in photographs? yes ( ) no ( ) 6. are you ashamed to smile at acquaintances? yes ( ) no ( ) 7. are you ashamed to smile at strangers? yes ( ) no ( ) 8. if yes, why not have sought dental treatment? ( ) lack of interest ( ) financial issues ( ) lack of information ( ) fear of treatment ( ) family characteristic figure 1. epidemiological questionnaire and self-perception of smile after the initial clinical examination to verify the presence of the pathology, the students were examined again for classification of the dda (number, shape, position or eruption disorder) in csd, one week after, if present in the upper, lower arch or in both arches, if on the right side or left, or on both sides. then, panoramic radiography was taken to confirm the diagnosis of dda. spss statistical software (version 25) was used to perform descriptive statistics, with percentages and frequencies, association analyzes using the chi-square and mean comparisons using the t test, all with the significance level measured by p value >0.05. the chi-square test was applied because, in the data collection, only two groups were found: students who had a single dental anomaly (g1) and those who did not (gc). results due to the period of covid-19, the schools adopted the hybrid or strictly remote mode of classes, making it difficult to obtain an estimated sample, and the fact that 12 male students after answering the questionnaire did not wish to be examined, there was only participation of 160 adolescents. of these, 37.5% had anomalies, corresponding to 60 individuals, and these had only one type of anomaly, corresponding to g1, being 46.6% brown, 41.6% white and 11.8% black. in figure 2, it is possible to verify the frequency distribution according to gender and type of school. 5 junior et al. braz j oral sci. 2023;22:e237434 80 70 60 50 40 30 20 10 0 public private male female 14 46 8 52 14 8 30 with anomalies public private male female no anomalies 54 46 30 68 46 52 54 46 68 figure 2. prevalence of anomalies regarding gender and type of school of adolescents (15-19 years old) it was observed that 37.5% of the sample had only a type of dental anomaly, corresponding to 60 individuals. the most prevalent were enamel hypoplasia, fusion, transposition, agenesis, ectopic eruption, microdent and dens-in-dent. it was possible to verify a higher prevalence in the maxilla, private schools (76.6%) and females (86.6%). in addition, the t test was performed to compare the mean age between the sample with and without anomalies, and it was possible to verify that there was no statistically significant difference (t = 0.24; p = 0.81) between the groups with and without anomalies in adolescents. finally, in general, tables 1 and 2 show the variables and their association with the presence or absence of anomalies in the arch and side, respectively. based on table 1, using the chi-square test, it was possible to observe that it is associated with the presence of anomalies in the arch in 83.3% of adolescents in the upper,15% in the lower and in no adolescent in both arches. specifically, no anomalies of the supernumerary type, conoid tooth, gemination, retained, infraocclusion and supraocclusion were found. table 1. distribution of variables associated with arch anomalies in adolescents (15 to 19 years old). parnaíba, 2021. cg χ² p valoranomalies (n = 100) upper arch (n = 51) lower arch (n = 9) agenesis no 100 43 7 χ² = 18.76 p = 0.001yes 0 8 2 continue 6 junior et al. braz j oral sci. 2023;22:e237434 continuation supernumerary no 100 51 9 --yes 0 0 0 microdontics no 101 47 9 χ² = 9.03 p = 0.001yes 0 4 0 macrodontia no 101 48 9 χ² = 4.56 p = 0.09yes 0 3 0 dens-in-tooth no 101 48 9 χ² = 4.56 p = 0.09yes 0 3 0 conoid tooth no 101 51 9 ---yes 0 0 0 fusion no 101 40 9 χ² = 23.47 p = 0.001yes 0 11 0 twinning no 101 51 9 --yes 0 0 0 transposition no 101 40 9 χ² = 23.47 p = 0001yes 0 11 0 ectopic eruption no 101 46 6 χ² = 19.58 p = 0.001yes 0 5 3 withheld no 101 51 9 --yes 0 0 0 impacted no 101 51 5 χ² = 68.83 p = 0.001yes 0 0 4 infraocclusion no 101 51 9 --yes 0 0 0 superocclusion no 101 51 9 --yes 0 0 0 enamel hypoplasia no 101 40 9 χ² = 23.47 p = 0.001yes 0 11 0 foot note: cg: control group; *p < 0.05; (--)chi-square test not applicable however, anomalies with a statistically significant distribution (p < 0.05) were found from the agenesis-type chi-square test in the upper arch of 8 and the lower arch in 2 adolescents. in the upper arch, 4 adolescents had microdontia, 10 had fusion, 10 had transposition, and finally, 10 had enamel hypoplasia. as for the ectopic eruption, there were 4 adolescents with this type of anomaly in the upper arch and 3 in the lower arch. in this same arch, 4 anomalies of the impaction type of third molars were also found. 7 junior et al. braz j oral sci. 2023;22:e237434 table 2. distribution of variables associated with dental development anomalies on the dental arch side in adolescents (15 to 19 years old). parnaíba, 2021. dental arch side χ² p valoranomalies none (n = 101) right (n = 23) left (n = 4) both (n = 32) agenesis no 101 21 4 25 χ² = 18.60 p = 0.001yes 0 2 0 7 supernumerary no 101 23 4 32 --yes 0 0 0 0 microdontics no 101 23 4 28 χ² = 16.41 p = 0.001yes 0 0 0 4 macrodontia no 101 23 4 30 χ² = 8.10 p = 0.001yes 0 0 0 2 dens-in-tooth no 101 21 4 32 χ² = 12.06 p = 0.01yes 0 2 0 0 conoid tooth no 101 23 4 32 ---yes 0 0 0 0 fusion no 101 13 4 32 χ² = 63.,57 p = 0.001yes 0 10 0 0 twinning no 101 23 4 32 --yes 0 0 0 0 transposition no 101 25 2 32 χ² = 53.89 p = 0.001yes 0 8 2 0 ectopic eruption no 101 22 2 27 χ² = 29.99 p = 0.001yes 0 1 2 5 withheld não 101 23 4 32 --yes 0 0 0 0 impacted no 101 23 4 28 χ² = 16.41 p = 0.001yes 0 0 0 4 infraocclusion no 101 23 4 32 --yes 0 0 0 0 superocclusion no 101 23 4 32 --yes 0 0 0 0 enamel hypoplasia no 101 23 4 22 χ² = 42.67 p = 0.001yes 0 0 0 10 foot note: *p < 0.05 such anomalies were found with a statistically significant distribution from the chi-square: agenesis in 2 adolescents on the right side and in 7 on both sides, microdontia in 4 adolescents on both sides and macrodontia in both sides of 2 adolescents. dens-in-dental anomaly was found only in 2 adolescents on the right side and fusion was also found only on the right side in 10 adolescents. 8 junior et al. braz j oral sci. 2023;22:e237434 as for the type of transposition anomaly, this was found in 8 adolescents on the right side and in 2 on the left side. ectopic eruption appeared in 1 adolescent on the right side, in 2 on the left side, and in 5 on both sides. on both sides, impacted tooth position anomaly was found in 4 adolescents on both sides and enamel hypoplasia in 10 adolescents. discussion it was possible to observe the prevalence rate of dental anomalies of 37.5%, corroborating the values found by other authors9,10,22 which presented approximate values to those of this research, 39.2%, 31.3%, 39.31%. but diverging from the value found by carneiro et al.23 (2021), of 22.7%. this probably explains the fact that there are divergent values of prevalence, due to the studies being carried out in different populations, under the influence of specific genetic and environmental factors. the presence of these anomalies suggests that they are related to genetic, hereditary and environmental factors, with the exception of enamel hypoplasia, which may be exclusively associated with environmental factors that interfere with odontogenesis. some authors2,12,13 have verified this statement by stating that there is a genetic interrelationship in the development of some dda, with different degrees of severity. in the present study, there was a difference between genders, with females having a higher prevalence, possibly due to the fact that all students accepted to be examined in schools, unlike some males who objected. this fact was also observed by some authors4,9 when they found that females seek dental treatment more frequently than males. however, in the studies carried out by braga et al.24 (2020) the prevalence was higher in males. as for location, it was observed in this study that dda was more prevalent in the maxilla than in the mandible, with the most common being: enamel hypoplasia, fusion, transposition, agenesis, ectopic eruption, microdent, dens-in-dent, corroborating the findings by carneiro et al.23 (2021) and diverging from the research by martins neto et al.4 (2019) who observed a greater presence of number anomalies in the mandible. the null hypothesis was rejected because the prevalence of dda in this study sample was high and no teenager had more than one type of anomaly. in the current study, anomalies in number, position, shape and eruption disorders were observed, with enamel agenesis, fusion, transposition and hypoplasia being the most prevalent. however, some authors22,24-26 found that the number anomaly (agenesis) was more prevalent, emphasizing that the genetic mutation is the most relevant etiological factor. in this research, the teeth most affected by agenesis were the maxillary central incisors, maxillary lateral incisors, and third molars. tooth transposition observed, as in another study27, had a high prevalence, affecting mainly the canine and first premolar on the right side. however, the number of fusions in this study was as high as the transposition, mainly affecting the central and lateral incisors on the right side, in contrast to other studies28,29, with microdontia and conoid tooth more prevalent. 9 junior et al. braz j oral sci. 2023;22:e237434 regarding eruption disorders, the prevalence of ectopic eruption was high, as observed by lagana et al.28 (2017), with a decreasing number of upper canines and lower second premolars. enamel hypoplasia, as in the research by ramos et al.30 (2019) affected the upper central incisors on both sides in adolescents. the presence of dda can also be highlighted which, although not having a high prevalence, reached a part of the sample, such as microdontia and dens-in-dent in upper lateral incisors, as in other studies4,31. macrodontia was observed in the maxillary central incisors, as well as in the studies by yassin29 (2016). impacted third molars were also observed by other authors3,22 probably because they are the last teeth to erupt in the oral cavity. it is noteworthy that regardless of the etiological factors, knowledge of the prevalence of dda serves as a guide for dentists to pay attention to the early diagnosis to prevent malocclusions, delay in tooth eruption or deviations from its trajectory. it was possible to observe that 45% of adolescents who had anomalies are afraid to smile in some situation during social life, whether for acquaintances, strangers or even for photographs, probably due to the aesthetic value of the smile. such findings corroborate those found by other authors17,18 whose presence of these anomalies generates both situations of bullying and self-criticism, however, diverging from the findings of other authors32 who found no association between the practice of bullying and the presence or absence of malocclusion. it is believed that with globalization, a greater number of patients are aware of their dental conditions, due to the ease of access to information on smile esthetics on social networks. this reinforces the need for early diagnosis, intervention and treatment of such anomalies. in this study, the sample of females was larger than males, with the finding that dental anomalies directly affect aesthetics, with females being more concerned with appearance. this can be considered a limitation of this study. another relevant factor limiting this study was that the sample number was lower than the minimum value indicated by the sample calculation, with a different number of participants in the groups. it is suggested that other studies be carried out with a greater number of brazilian participants, addressing both the prevalence and possible etiological factors, since variations in dental anomalies highlight the need to establish data from various geographic regions to examine the effect of genetics and environment on tooth development. in conclusion, the prevalence was relatively high, highlighting the enamel hypoplasia, influencing the smile esthetics of a reasonable number of adolescents, whether for acquaintances, strangers or even for photographs. conflict of interests all our affiliations, corporate or institutional, and all sources of financial support to this research are properly acknowledged, except when mentioned in a separate letter. we certify that do not have any commercial or associate interest that represents a conflict of interest in connection with the submitted manuscript. 10 junior et al. braz j oral sci. 2023;22:e237434 author contribution all authors actively participated in  the manuscript’s findings and have revised and approved the final version of the manuscript. references 1. torres pf, simplício ahm, luz arca, lima mdm, moura lfad, moura ms. [dental anomalies of number in orthodontic patients]. rev odontol unesp.2015;44(5):280-4. portuguese. doi:10.1590/1807-2577.0066 portuguese. 2. bandaru bk, thankappan p, nandan srk, amudala r, annem sk, santosh abr. the prevalence of developmental anomalies among school children in southern district of andhra pradesh, india. j oral maxillofac pathol. 2019 jan-apr;23(1):160. doi: 10.4103/jomfp.jomfp_119_18. 3. reis jc, fraga maa, de azevedo-vaz sl, miotto mhmb, pereira tcr, camisasca dr. prevalence of dental anomalies and bone alterations in children’s panoramic radiographies. rgo. 2020;68:e20200058. doi: 10.1590/1981-863720200005820180073. 4. martins neto rs, alves ifs, machado al, barbosa neto la, alencar aa, esses dfs. 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[prevalence of malocclusion in schoolchildren aged 10 to 15 years in the city of santo antônio de pádua-rj]. j orofac invest. 2019;6(2):47-58. portuguese. 16. luke am, kassem rk, dehghani sn, mathew s, shetty k, ali ik, et al. prevalence of dental developmental anomalies in patients attending a faculty of dentistry in ajman, united arab emirates. pesq bras odontop clin integr. 2017;17(1):1-5. doi: 10.4034/pboci.2017.171.38. 17. gatto rcj, garbin aji, corrente je, garbin cas. self-esteem level of brazilian teenagers victims of bullying and its relation with the need of orthodontic treatment. rgo. 2017;65(1):30-6. doi:10.1590/1981-863720170001000053304. 18. boffi jc, franzin lcds. [bullying and practice of dentistry]. rev uningá rev. 2017; 29(2):38-41. portuguese. 19. brazilian institute of geography and statistics. [an overview of health in brazil: access and use of services, health conditions and risk factors and health protection]. rio de janeiro: ibge; 2010. 20. fontelles mj, simões mg, almeida jc, fontelles rgs. [research methodology: guidelines for calculating the sample size]. rev para med. 2010;24(2):57-64. 21. pithon mm, santos cr, santos nl, lima soas, coqueiro rs, santos r l. impact of malocclusion on affective / romantic relationships among young adults. angle orthod. 2016;86(4):638-43. doi: 10.2319/030915-146.1. 22. da silva bx, rossi sb, ribeiro ra, sendyk wr, roman-torres cvg, de melo quintela m. prevalence of anomalies of shape and number in orthodontic patients: observational study. res soc develop. 2021;10(9):e3910917504–e3910917504. portuguese. doi: 10.33448/rsd-v10i9.17504. 23. carneiro gkm, rodrigues mc, araújo waf, cremonese ppf. [radiographic analysis of dental anomalies in children from 4 to 12 years of age]. arch health invest. 2021;10(2):282-6. portuguese. doi: 10.21270/archi.v10i2.5282. 24. braga hf, lima lmnb, sá lma, nascimento rsg, firmino bs, sousa ala, et al. the prevalence of dental anomalies in patients 4 to 12 years of age: a radiographical study. focus oral res. 2020;2(3):140-8. 25. fauzi nh, ardini yd, zainuddin z, lestari w. a review on non-syndromic tooth agenesis associated with pax9 mutations. jpn dent sci rev. 2018 feb;54(1):30-6. doi: 10.1016/j.jdsr.2017.08.001. 26. paravizo ca, pinto ms, caetano rm, netto ag, cassab mtf. [prevalence of dental agenesis in patients submitted to orthodontic treatment: a radiographic study]. rev uningá. 2021;58:euj3643–euj3643. portuguese. doi: 10.46311/2318-0579. 27. alassiry a. prevalence and distribution of selected dental anomalies in najran city of saudi arabia. egypt dent j. 2020;66(3):1471-82. doi: 10.21608/edj.2020.31399.1133. 28. laganà g, venza n, borzabadi-farahani a, fabi f, danesi c, cozza p. dental anomalies: prevalence and associations between them in a large sample of non-orthodontic subjects, a cross-sectional study. bmc oral health. 2017;17(1):1-7. doi: 10.1186/s12903-017-0352-y. 29. yassin sm. prevalence and distribution of selected dental anomalies among saudi children in abha, saudi arabia. j clin exp dent. 2016 dec;8(5):e485-90. doi: 10.4317/jced.52870. 30. ramos lp, suárez vor, rodríguez sg, soler db. [structural anomalies of the enamel and aesthetic affection in schools children of 6-17 years of cojímar]. rev elect medimay. 2019;26(1):4-13. spanish. 31. magalhaes gp, paz ec, silva ytcs, carvalho leite cm, falcão cam, et al. diagnosis of anomalies in dental panoramic x-ray]. robrac. 2019;28(87):244-7. portuguese. doi: 10.36065/robrac.v28i87.1315. 32. alves do, barbosa fr, colares v, santos cfbf, menezes va, godoy f. maloclusão e bullying em adolescentes escolares. res soc devel. 2020;9(10):1-18. portuguese. doi: 10.33448/rsd-v9i10.8403. 1 volume 22 2023 e236839 original article braz j oral sci. 2023;22:e236839http://dx.doi.org/10.20396/bjos.v22i00.8666839 1 department of restorative dentistry, ribeirão preto school of dentistry, university of são paulo, ribeirão preto, sp, brazil. 2 department of chemistry, center of nanotechnology and tissue engineering, photobiology and photomedicine research group, faculty of philosophy, sciences and letters of ribeirão preto, university of são paulo, ribeirão preto, sp, brazil. corresponding author: thiago vinicius cortez school of dentistry of ribeirão preto university of são paulo avenida do café, s/n, ribeirão preto, sp, brazil, 14040-904 phone: +55-16-33154075 e-mail: thiago.cortez@usp.br editor: dr. altair a. del bel cury received: august 30, 2021 accepted: september 23, 2022 longitudinal analyses of composite resin restoration on erosive lesions: effect of dentin treatment with a chitosan nanoformulation containing green tea renato gonçalves dos reis1 , antônio cláudio tedesco2 , fabiana almeida curylofo-zotti1 , thiago vinicius cortez1* , hiago salge borges2 , aline evangelista souza-gabriel1 , silmara aparecida milori corona1 aim: to evaluate the influence of the biomodification of erosive lesions with a chitosan nanoformulation containing green tea (nanocsq) on the clinical performance of a composite resin. methods: the study was performed in a split-mouth, randomized and double-blinded model with 20 patients with 40 erosive lesions. the patient’s teeth were randomized into two groups (n=20) according to the surface treatment: 1) without biomodification (control), and 2) biomodification with nanocsq solution (experimental). the lesions were restored with adhesive (tetric n-bond, ivoclar) and composite resin (ips empress direct, ivoclar). the restorations were polished and 7 days (baseline), 6 months, and 12 months later were evaluated according to the united states public health service (usphs) modified criteria, using clinical exam and photographics. data were analyzed by friedman’s and wilcoxon signed-rank tests. results: no significant differences were found between the control and experimental groups (p=0.423), and also among the follow-up periods (baseline, six months, and 12 months) (p=0.50). regarding the retention criteria, 90% of the restoration had an alpha score in the control group. only 10% of the restorations without biomodification (control) had a score charlie at the 12-month follow-up. none of the patients reported post-operatory sensitivity. conclusion: the nanocsq solution did not negatively affect the performance of the composite resin restorations after 12 months. keywords: clinical trials as topic. tea. chitosan. tooth erosion. nanoparticles. https://orcid.org/0000-0002-7689-4421 https://orcid.org/0000-0003-4198-9321 https://orcid.org/0000-0002-0482-6956 https://orcid.org/0000-0002-1490-1884 https://orcid.org/0000-0002-6927-6051 https://orcid.org/0000-0002-9280-2945 https://orcid.org/0000-0002-1733-3472 2 reis et al. braz j oral sci. 2023;22:e236839 introduction tooth erosion starts with the softening of the surface by acidic materials. it can be caused by both intrinsic (acid gastric regurgitation) and/or extrinsic (acidic drinks and food) acids, and is modified by changes in salivary flow and constituents. the dentine is more susceptible to erosion than enamel because the crystals in dentine are much smaller than those of enamel and the carbonate content of dentine is greater than in enamel1. the enamel prisms of the tooth with erosion are dissolved, which creates a surface with fissures that resembles a honeycomb. the progression of the lesion affects the peri and inter-tubular dentin, enlarging the dentin tubules1. erosive dentin lesions are heterogeneous, highly permeabilized, have high crystallinity, and partially denatured collagen. altogether, these structural alterations negatively impact the longevity of restorative treatments performed on eroded dental tissues2,3. the organic matrix of the eroded dentin hinders the adhesive penetration due to its high content of water and fibers and because might be already collapsed, which affects the hybridization and increases the adhesive interface degradation4. in this context, the use of protective substances, such as chitosan, might improve the mechanical bond resistance of collagen fibrils, which are the scaffold to the adhesive interface5,6. chitosan is a hydrophile biopolymer of chitin composed of reactive groups, a linear polycation of high density and charge, and hydrogen bonds5,6. such hydrogen bonds are arranged in parallel, ensuring high resistance7. chitosan is biocompatible and non-toxic to human cells, promotes bio-adhesion, biodegradability, and presents antimicrobial activity8-12. the use of chitosan straightens13,14 and stabilizes15,16 the dentin collagen, increasing the number of cross-links among the collagen’s fibers. chitosan neutralizes the matrix metalloproteinases, creating collagen arrangements with better mechanical properties17. the chitosan bonds are formed by the union of molecules through covalent linkages5,18, a process used to stabilize biological tissues19. camellia sinensis (c. sinensis) as chitosan, another biomodifier has been investigated in dentistry, is a rich polyphenol, extract containing epicatechin (ec), epigallocatechin (egc), and epigallocatechin-3-gallate (egcg)20. c. sinensis extract had an inhibitory effect over enzymes that degrade dentin collagen21. the catechins, mainly egcg, can induce cross-linking of fibers, impeding the access of the collagenases to the active sites22. in vitro tests showed that the egcg is effective in preserving the bond resistance of the resin-dentin for 6 months23. due to the multiple aromatic rings in the structure of egc, this molecule has higher antioxidative activity than non-phenolic or mono-phenolic compounds24. egc also has antimicrobial activity, affecting acid production, and the metabolisms of streptococcus mutans enzymes such as glycosyltransferases25. egc also inhibits the synthesis of extracellular polysaccharides (eps), reducing microbial cellular adhesion and biofilm formation26. 3 reis et al. braz j oral sci. 2023;22:e236839 there is a lack of studies on the biomodification of the eroded dentin surface. this study evaluated the influence of the biomodification of erosive lesions with a chitosan nanoformulation containing green tea (nanocsq) on the clinical performance of a composite resin. the hypotheses tested were that there is no correlation between the longevity of restorations and the application of the experimental solution considering retention, marginal discoloration, marginal adaptation of the resin restorations, secondary caries, and post-operatory sensitivity. material and methods experimental design the sample was composed of 20 patients (n=20) aged between 20 to 50 years old who had non-carious erosive lesions of medium to a deep depth at the buccal, palatal/lingual, or incisal/occlusal faces. the study was performed in a split-mouth, randomized, and double-blinded model. the teeth of each participant were received one of the dentin treatments: 1) control (without biomodification); 2) nanocsq biomodification with a nanoformulation of chitosan containing green tea extract (c. sinensis). the response variables were: 1) longitudinal evaluation of the restorative treatment through a clinical examination using the modified usphs criteria at 7 days, 6-months, and 12-months; 2) photographic evaluation of the restorative treatment at the same time points of the clinical follow-up. all consort guidelines were followed (figure 1). ethical and legal aspects and sample size calculation this study was approved by our local ethics committee (caae: 23972619.8.0000.5419) and registered on the brazilian platform of clinical trials registration (rebec) (utn: 1111-1245-1517). a consent form was signed by each participant. the website www.sealedenvelope.com was used to calculate the sample size of this study. the parameters were set to α=5%, 90% of power, 98% of success to the experimental and control groups, and an equivalence limit of 15%. the tests pointed to required 19 participants. the calculation values were carried out considering the possibility of dropouts during the experiment and based on a previous clinical study27. selection and preparation of the participants male and female patients aged from 20 to 50 years old underwent a clinical examination performed during the activities of the restorative dentistry clinics. patients received professional teeth cleaning with pumice-water slurry with rotating rubber cup and brushes at low-speed handpiece. the clinical examination was performed on dried surface under adequate light exposure. the participants received diet and dental hygiene instructions, and information about dental erosion. the inclusion criteria were the presence of at least two non-carious erosive lesions of medium to deep depth at the buccal/lingual/palatal or incisal/occlusal faces of two restored teeth. all teeth must have a positive response to thermic stimuli, which was performed using endo-frost (roeko, langenau, germany). about:blank 4 reis et al. braz j oral sci. 2023;22:e236839 100 patients evaluated 30 patients excluded 70 patients in need restorations 50 excluded patients 20 selected patients inclusion criteria • age between 20 and 50 years old; • at least two non-carious erosive lesions; • homolog teeths; • vital tooths initial (7 days) patients: 20 tooths: 40 6 months patients: 20 tooths: 40 12 months patients: 20 tooths: 40 drop out patients: 0 drop out patients: 0 drop out: there were not alfa and charlie does represents the retention criteria control (n = 20) alfa (n = 20) charlie (n = 0) nanocsq (n = 20) alfa (n = 20) charlie (n = 0) control (n = 20) alfa (n = 20) charlie (n = 0) nanocsq (n = 20) alfa (n = 20) charlie (n = 0) control (n = 20) alfa (n = 18) charlie (n = 2) nanocsq (n = 20) alfa (n = 20) charlie (n = 0) figure 1. consort flow chart. all patients who had spontaneous pain, sensitivity, fistulae, or edema were excluded from this study. the teeth of the participants that were not selected for this investigation but needed intervention were adequately treated. the anamnesis regarding the participant’s general health and odontogram charting of the patients were filled. preparation of the chitosan nanoformulation containing green tea extract (nanocsq) low-molecular-weight chitosan nanoparticles (75-85% of deacetylation) (sigma-aldrich, saint louis, mo, eua) were dissolved in 0.33% (vol/vol) glacial acetic acid for a stock solution of 2 mg/ml. the ph was adjusted to 5 using 0.1 n sodium hydroxide. under mild stirring, tpp solution (1 mg/ml) was slowly added, drop by drop, to chitosan solution. the proportion of chitosan to tpp was 5:1. 5 reis et al. braz j oral sci. 2023;22:e236839 a 0.3% extract of c. sinensis (green tea extract 400mg, now supplements, usa) was submitted to ultrasound bath for 10 min and centrifuged at 10,000 rpm for 15 min. the supernatants were pooled. under constant stirring, supernatants were added to chitosan solution, drop by drop continuously and slowly. after 30 min of stirring, the tpp was added to the solution, following the same method of our previous in vitro study28. clinical procedures the randomization of the participants was done using a sheet of random number generation available at http://randomnumbergenerator.intemodino.com/pt/. the participants were codified with numbers to organize the order of the treatments. participants’ teeth were randomly separated according to the treatment to be applied on dentin: control or nanocsq, using the coin tossing method, patients and operator were blinded as to the type of treatment. the two teeth of each patient were treated at the same dental visit by a single operator. before the treatment, initial photographs were taken (canon eos rebel t2i 18.0 megapixels, cannon, japan). the color selection of the composite resin (ips empress direct, ivoclar vivadent, liechtenstein, germany) was performed using the vita 3d color scale (wilcos do brazil indústria e comércio ltda, petrópolis, rj, brazil). all teeth were treated under rubber dam (madeitex, são josé dos campos, sp, brazil) and dental clamps were chosen according to the dental anatomy (duflex, sswhite, rio de janeiro, rj, brazil). all teeth received selective acid conditioning with 35% phosphoric acid for 30 seconds applied only on the enamel. after the acid conditioning, the cavity was washed with water for 1 minute to ensure that acid was removed from the surface. then, the excess water was removed with a suction cannula and the surface was dried with cotton. in the experimental group nanocsq. solution actively applied with a brush (kgbrush, kg sorensen, cotia, sp, brazil) for 1 minute. then the surface was dried with absorbent paper29. a layer of the adhesive system (tetric n-bond, ivoclar vivadent, liechtenstein, germany) was actively applied for 20 seconds using a micro brush (kgbrush, kg sorensen, cotia, sp, brazil). then, the adhesive was light-cured for 10 seconds with radii-cal led light curing device (1200 mw/cm2) (sdi, bayswater, australia) previously measured with a radiometer. this procedure was performed in the all-treated teeth following the manufacturer’s recommendations. to restore erosive dental lesions, the ips resin (ivoclar vivadent) was used in the incremental filling technique. each increment up to 2 mm in depth, was cured for 20 seconds (radii-cal led curing, sdi), restoring dental anatomy. the adjacent teeth were protected with polyester straps. once finished, the rubber dam was removed and occlusal adjustments were done with carbon paper (angelus, londrina, pr, brazil). the dental premature conabout:blank 6 reis et al. braz j oral sci. 2023;22:e236839 tacts and occlusal interferences were removed using diamond-finishing burs (kg sorensen, cotia, sp, brazil). diamond-finishing burs (kg sorensen, cotia, sp, brazil) and sof-lex discs (3m espe, st paul, mn, usa) were used for finishing the restorations. the patients returned 7 days later for a final polishing using impregnated abrasive burs and discs (enhance, dentsply industria e comercio ltda, petropolis, rj, brazil). clinical and photographic assessment of dental restorations the restored teeth were assessed by a clinical and photographic examination seven days after the clinical procedures (after polishing baseline), 6 and 12 months. the clinical examination was performed by three examiners, following the cvar&ryge modified usphs criteria30, which include the analyses of retention, marginal discoloration, secondary caries, marginal adaptation, and postoperative sensitivity (table 1), the different raters were previously calibrated. table 1. modified usphs criteria used to the clinical evaluation of the restorative treatments. category score criteria retention alpha loss of restorative material not detected charlie loss of restorative material detected marginal discoloration alpha marginal discoloration not detected bravo minor marginal discoloration without axial penetration charlie axial discoloration with axial penetration secondary caries alpha secondary caries not detected charlie secondary caries detected marginal adaptation alpha the restoration adapts closely to the tooth and there are no visible margins bravo there are visible yet clinically acceptable margins charlie there is no marginal adaptation. clinical failure. post-operatory sensitivity alpha no post-operatory sensitivity detected charlie post-operatory sensitivity detected intraoral photographs of restored teeth were taken with a digital camera (canon eos rebel t2i 18.0 megapixels, canon, japan), intraoral mirror, and circular flash, standardized as best as possible. the photographs evaluation was blindly and individually carried out by three examiners by visualization of the photographs 7 reis et al. braz j oral sci. 2023;22:e236839 in a laptop screen under the same environmental and light conditions, including the analyses of color, marginal pigmentation, and anatomical shape of the dental restorations30 (table 2). table 2. modified usphs criteria used to the photographic evaluation of the restorative treatments. category score criteria color alpha the color of the restoration corresponds to the tooth structure in terms of color and translucency bravo there are minor alterations in the color, hue, and translucency between the restoration and the tooth charlie there is a clear alteration in the color and translucency of the restoration marginal pigmentation alpha there is no pigmentation on the margin between the restoration and the tooth bravo there is minor pigmentation between the restoration and the tooth charlie there is pigmentation between the restoration and the tooth anatomy alpha there is continuity with the tooth anatomy bravo there is no continuity with the tooth anatomy charlie there is loss of restorative material exposing dentin or the restoration inner layers data statistical package for the social sciences (spss v 25.0 chicago, il, usa) software was used for data analyses at a significance level of 5%. data analyses were based on inferential and descriptive statistics. descriptive statistics described the frequency and distribution of usphs modified scores, including the percentage of dental restorations with failures. the analyses of inferential statistics used friedman’s non-parametric test and wilcoxon signed-rank test for the different time points (baseline, 6 and 12 months) and interactions between treatment and period of analysis. cohen’s kappa test was used to compare inter-examiner and intra-examiner reliability. results patients’ age ranged between 20 to 50 years old (mean age ~ 30 years old) for both genders. regarding the restored teeth, 12 anterior and 28 posterior teeth were restored, totaling 40 teeth. the percentage of surface erosion found was 60% buccal/lingual/palatal and 40% incisal/occlusal. 8 reis et al. braz j oral sci. 2023;22:e236839 table 3. data obtained upon clinical examination based on the modified usphs criteria. treatment time point retention discoloration secondary caries marginal adaptation post-operative sensitivity control (without biomodifying agent) baseline a b c a b c a b c a b c a b c (7 days) n = 20 (%) 20 100 20 100 20 100 20 100 20 100 (6 months) n = 20 (%) 20 100 20 100 20 100 20 100 20 100 (1 year) n = 20 (%) 18 90 2 10 20 100 20 100 20 100 20 100 biomodification with nanocsq baseline a b c a b c a b c a b c a b c (7 days) n = 20 (%) 20 100 20 100 20 100 20 100 20 100 (6 months) n = 20 (%) 20 100 20 100 20 100 20 100 20 100 (1 year) n = 20 (%) 20 100 20 100 20 100 20 100 20 100 clinical examination the intra-examiner kappa index equaled 1.0 for the same examiner (a compared to a, b compared to b, c compared to c). the inter-examiner index (a and b and c) equaled 0.98. as for the retention criteria, an alpha score of 100% was achieved after 6 and 12 months after dental restoration with nanocsq (n=20). in the control group (without nanocsq pre-treatment), the alpha score was found in 100% (n=20) of the restorations 6 months later. after 12 months, the alpha score was found in 90% of the restorations (n=18), and 10% (n=2) had the charlie score. only two restorations from the control group obtained charlie score for the criteria retention upon 12-month follow-up and were replaced by new ones. the statistical analyses regarding the criterion “retention” did not show a significant difference between groups (green tea-containing chitosan nanoformulation versus no treatment) (p=0.432). also, no significant difference between time points (baseline, 6 and 12 months) (p=0.500) and the interaction treatment between time points (p=0.126). regarding the remaining criteria analyzed in this study (marginal discoloration, secondary caries, marginal adaptation, and post-operatory sensitivity), no changes were observed for the scores at the different time points for both groups (control and experimental gruop), so that all restorations maintained the alpha score. photographic evaluation within 12 months, 100% of the dental restorations with nanocsq received alpha scores for all the photographic criteria analyzed: restoration color, marginal pigmentation, and anatomical shape. in the control group, (without biomodification), 90% of 9 reis et al. braz j oral sci. 2023;22:e236839 the restorations received an alpha score of 90% and 10% of them received a charlie score for the criteria anatomic shape (figure 2 and table 4). l a b c d e f g h i j k figure 2. a. initial clinical aspect of the teeth 44 and 45; b. baseline (7 days); c. 6-month follow-up of; d. 12-month follow-up; e. initial clinical aspect of the teeth 34 and 35; f. baseline (7 days) g. 6-month follow-up; h. 12-month follow-up; i. initial clinical aspect of the teeth; j. baseline (7 days); k. 6-month follow-up; l. 12-month follow-up. table 4. data obtained from photographic analyses based on the modified usphs criteria. treatment time point color of restoration marginal pigmentation anatomic shape control (without biomodifying agent) baseline a b c a b c a b c (7 days) n = 20 (%) 20 100 20 100 20 100 (6 months) n = 20 (%) 20 100 20 100 20 100 (1 year) n = 20 (%) 20 100 20 100 18 90 2 biomodification with nanocsq baseline a b c a b c a b c (7 days) n = 20 (%) 20 100 20 100 20 100 (6 months) n = 20 (%) 20 100 20 100 20 100 (1 year) n = 20 (%) 20 100 20 100 20 100 discussion dental erosion is a complex process characterized by mineral dissolution that exposes the organic matrix to bacterial and enzymatic degradation, such as metalloproteinases (mmps)31,32. 10 reis et al. braz j oral sci. 2023;22:e236839 biomodifying substances such as chitosan and green tea have potential against erosive dentin lesions6,33. chitosan has been used to improve the mechanical resistance of collagen, thereby, increasing its resistance to the degradation of its fibrils, which are used as support to the establishment of an adhesive interface obtained during restorative procedures with dental composites5,6. benefits were demonstrated through the use of chitosan on the dentin such as the increase of surface resistance of the dentin16 and the mechanical resistance of dental restorations, contributing to less hydrolytic degradation of collagen fibrils by collagenases14. chitosan also promotes collagen preservation and efficacy in preventing and treating dentin erosion34,35, factors that can contribute to the clinical longevity of dental restorations in erosive lesions. the protective effects of chitosan on the collagen fibrils34,35 and the increase of the bond resistance of adhesive restorations when green tea was used36,37. could have contributed to the longevity of the restorations. the possible explanation for absence of statistical difference for control group is the short period of clinical evaluation. the literature is lacking in studies on the efficacy of green tea in chitosan nanoformulations to improve adhesive restorations, especially in eroded substrate. among the mmps inhibitors, green tea, a natural inhibitor of mmps, as well as its active components had therapeutic potential along with egcg, a polyphenol found in the green tea. egcg when applied on the dentin has satisfactory results, for example, protective effect against dentin erosion33 and improvement in the adhesive resistance when used combined with resin composites37. in our study, clinical and photographic evaluations of the baseline-, 6-, and 12-month time points showed no significant difference for both clinical (retention, marginal discoloration, secondary caries, marginal adaptation, and post-operative sensitivity) and photographic criteria (restoration color, marginal pigmentation, and anatomic shape). the main limitations of this study were the “n” of samples that meet the inclusion criteria although adequate was relatively low, and the heterogeneity of erosive lesions. a possible explanation for the outcomes is the restorative materials’ quality, such as the tetric n-bond adhesive (ivoclar), ips empress direct composite (ivoclar), as well as the careful clinical protocol. the selective enamel etching can be another factor that collaborated with the final results in this study once, the retention rates observed here were satisfactory38,39. the use of selective enamel etching increases the bond resistance of the enamel40, which might have contributed to the low loss index of restorations we observed. as for the polymerization of the composite resin, the light-curing unit used had its power previously measured (1200 mw/cm2) and was fully charged. the increments were standardized in 2 mm and the light source was really close to the dental surface. literature demonstrates that the constant use of the light-curing for more than 25 times without previous loading reduces the polymerization intensity41. in this study, only two restorations were performed per each appointment, to avoid possible overload or radiant power loss. two dental restorations from the control group obtained charlie score for the criteria retention upon 12 months and needed to be replaced by new ones. one of them 11 reis et al. braz j oral sci. 2023;22:e236839 was done in the maxillary central incisor (palatal face) and the other was done in the vestibular face of a mandibular pre-molar. during 12 months of follow-up, no failure regarding retention was observed in the experimental group, the one that underwent biomodification of the dental surface, we can thus accept the two hypotheses raised initially. in vitro33,37 and situ42 studies highlight that green tea has a promising protective effect against dentin erosion33, as well as it improves the adhesive resistance in restorative procedures that involve the dentin36,37, and increased the microhardness of dentin28. in corroboration with the present study, souza et al.27(2021) noted that the application of 2.5% chitosan nanoformulation on eroded dentin did not increase failures of resin restorations after 1 year and it can be used as a pre-treatment solution. vailati et al.3 (2013) found that restorations of erosive lesions had marginal integrity and absence of infiltration 6 years after the clinical procedure. wilder et al.2 (2009) observed that the retention rates after 12 years were approximately 93% in the group that underwent selective enamel etching and 84% in the group subjected to non-selective etching, with a retention rate of 89%, the restorations in both groups had classification “alpha” of 88% or above in all the categories of clinical evaluation, except for marginal coloration. the outcomes of our study showed that overall, the use of a green tea-containing chitosan nanoformulation did not affect the performance of dental composite restorations after 12 months. due to the promising results found in the literature concerning the use of these modifying agents on the eroded dentin, more studies need to be conducted to indicate this protocol on clinical practice. acknowledgments the authors acknowledge the national council for scientific and technological development for the funding concession (#130194/2019-0). f.a.c.z. and s.a.m would like to thank the são paulo research foundation (fapesp) for the scholarships [#2017/11582-1 and #2019/04807-2] awarded. a.c.t thank (cnpq) for the grants support #404416/2021-7 and #441673/2020-1. author contribution study conception and design: antônio cláudio tedesco, hiago salge borges, fabiana almeida curylofo-zotti, silmara aparecida milori corona; data collection: renato gonçalves dos reis, thiago vinicius cortez; analysis and interpretation of results: renato gonçalves dos reis, fabiana almeida curylofo-zotti , aline evangelista souza-gabriel, silmara aparecida milori corona. author; draft manuscript preparation: renato gonçalves dos reis, thiago vinicius cortez, aline evangelista souza-gabriel. all authors actively participated in the discussion of the manuscript’s findings, and have revised and approved the final version of the manuscript. 12 reis et al. braz j oral sci. 2023;22:e236839 references 1. warreth a, abuhijleh e, almaghribi ma, mahwal g, ashawish a. tooth surface loss: a review of the literature. saudi dent j. 2020 feb;32(2):53-60. doi: 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hannas a, attin t, buzalaf ma. chlorhexidine and green tea extract reduce dentin erosion and abrasion in situ. j dent. 2009 dec;37(12):994-8. doi: 10.1016/j.jdent.2009.08.007. 1 volume 21 2022 e225368 short communication braz j oral sci. 2022;21:e225368http://dx.doi.org/10.20396/bjos.v21i00.8665368 1 federal university of juiz de fora, governador valadares mg brazil 2 university of vale do taquari, lajeado rs – brazil 3 federal university of pelotas, pelotas rs brazil corresponding author: luiz alexandre chisini institute of health sciences, department of dentistry, federal university of juiz de fora, 1167 moacir paleta st., governador valadares mg zip:35020-360, brazil e-mail: alexandrechisini@gmail.com editor: dr. altair a. del bel cury received: april 20, 2021 accepted: february 12, 2021 flipped classroom and the use of role-play in the covid-19 pandemic: challenges and learning luiz alexandre chisini1,* , marcus cristian muniz conde2 , alessandro menna alves2 , francine dos santos costa3 aim: in brazil, covid-19 remains one of the epicenters of the pandemic, thus, presential educational activities are suspended. the study aimed to describe the approach of flipped classroom performed to provide social distancing and to maintain teaching activities during covid-19-pandemic. methods: flipped classroom was chosen to be associated with the role-play technique as a central methodology. to bring students closer to activities performed in the non-pandemic period, some actions and assessments were also role-played by professor simulating patients in virtual meetings. results: although the changes in the format of the lessons, our approaches stimulated the students maintained the high frequency of students in the activities (almost 100%) providing continuity activities. conclusions: this report can reinforce that meaningful learning can be taught by using a virtual/remote approach. however, the potential positive results observed cannot be used as an excuse to maintain remote teaching activities with the objective of cost-cutting by the institutions. keywords: covid-19. coronavirus infections. students, dental. curriculum. https://orcid.org/0000-0002-3695-0361 https://orcid.org/0000-0003-2662-3305 https://orcid.org/0000-0001-6702-2739 https://orcid.org/0000-0001-9558-937x 2 chisini et al. braz j oral sci. 2022;21:e225368 introduction coronavirus disease 2019 (covid-19) starts in wuhan – china in december of 2019 and rapidly affects most of the countries worldwide1,2. recent data indicate that above 363 million individuals are infected and 5.6 million were dead due to covid-193. the first case of covid-19 was confirmed in brazil in february 2020 with significant growth in cases in the following months; therefore, brazil became (and remains) one of the epicenters of the pandemic along with the united states of america1,4-7. the pandemic led to changes in dental education, especially in dentistry8,9, considering the significant number of practical activities and the wide range of skills needing to be developed. in this way, following global recommendations, the theoretical classroom teaching activities have been suspended since then10,11. this scenario has required the development of innovative learning approaches to minimize losses in the teaching and learning process as well as to continue the training of dental students in a new environment12,13. thus, the flipped classroom has been proposed as an active teaching strategy used in different academic environments14. the general guiding principle of the flipped classroom is that the traditional teaching process takes place outside of the classroom through videos performed by professor, which is watched and studied before the classroom activity. so, the activities and projects related to upper-level cognitive field steps are carried out during classroom time. therefore, the study aimed to describe the approach of flipped classroom associated to role-play technique performed in our school to provide social distancing and maintain active learning as close as possible to the pre-covid-19 period. material and methods third-year dental students of the university of vale do taquari (located in the south region of brazil) starts to study principles of occlusion being the activities mostly theoretical permeated by hands-on classes. it is important to highlight that the curriculum is not divided into disciplines, but is organized into integrated modules that aim to develop skills and competencies guided by the use of active teaching and learning methodologies. in this way, professors should organize activities aiming to integrate the content covered by different areas. thus, when classroom activities were suspended due to covid-19, the curriculum was reorganized to promote students to meaningful learning and some hands-on activities. the professor:students ratio (1 professor to 10 students) has been maintained in the period. activities that were not possible to be virtualized will be performed after the return of normal activities. flipped classroom was chosen as a central methodology to carry out teaching activities. the theory of meaningful learning proposes that information is more easily learned when it makes connections with other previously known knowledge, helping to understand new knowledge based on prior knowledge15. thus, the making of the videos by the professor who knows the students is fundamental, 3 chisini et al. braz j oral sci. 2022;21:e225368 since the new knowledge and the examples presented will always be anchored in content that the students already know of. so, flipped classroom consists of the inversion of actions taking place in the classroom and outside it, the study materials must be made available in advance for students to access and understand the proposed contents16. it was created a virtual classroom (in google classroom; https://classroom.google.com) where study materials and videos recorded by professors were uploaded and activities were made available. thus, students must attend classes previously recorded and take notes. the virtual meetings were carried out synchronously by google meet (https://meet.google.com); initially, the students presented their questions regarding the video content. whenever possible, students were encouraged to address their colleagues’ questions and thus, teachers complemented their answers. after this initial moment, students were frequently separated into smaller groups, containing up to 5 individuals, where they carried out virtually group activities. these activities ranged from directed studies, concept maps, case studies, depending on the instructional objectives. some hands-on activities could be maintained. for example, an important skill to be developed is the anamnesis interview and clinical exams. in this way, in small groups, one of the students received a clinical case, which should be role-played. the student should represent, for colleagues, the patient described in the case reporting symptoms and characteristics related to temporomandibular dysfunctions while the other students should carry out the anamnesis interview and reach a consensus regarding the diagnosis. during all the activities, the teacher supervised the various virtual rooms. in general, assessments performed in the pre-covid-19 period have been based on objective structured clinical examination (osce)17,18 with real patients. to bring students closer to activities that were carried out in the pre-pandemic period, assessments were also performed by the professor’s role-play. thus, professor role-played a patient condition, describing the clinical symptoms. then, the student asked questions regarding the patient and tmd. the professor observed the path by which the student investigated and gave the diagnosis of tmd following the osce. results although the changes in the format of the lessons, the present approaches stimulated the students maintained the high frequency in the activities (almost 100%) with a high level of academic performance (mean of 8.4, standard deviation ± 0.5) considering all evaluations, providing continuity of theoretical and some possible hand-on activities. one of the main problems faced by us concerns the connection’s instability of students residing in the. in this way, the flipped classroom proved to be an interesting tool because such students have been able to watch the videos which allowed students to master the content that would be discussed in class. this was one of the main advantages pointed out by the students. video records show that all students watched at least once (mean of 2 times; standard deviation of ± 0.7) while some viewed multiple times. however, we have no record of the average time spent watching activities. it was conducted in all in-person virtual classrooms, frequently with small group settings. 4 chisini et al. braz j oral sci. 2022;21:e225368 discussion presents results can reinforce that significant knowledge can be taught using a virtual/remote approach and could have a promising future perspective of pandemic prolongation. indeed, students were assessed according to three domains: cognitive, affective, and psychomotor19. in each of these domains, there is a hierarchical organization, classified based on the pedagogical learning objectives in increasing levels of complexity and specificity19. the first of them is the cognitive domain, which is mainly linked to the skills of different levels of knowledge acquisition and use. the affective domain concerns feelings and emotions, including how we face difficulties and the strategies developed to overcome them. the psychomotor domain, in turn, is linked to the student’s ability to perform psychomotor activities. to assess the cognitive domain, the different works performed (i.e., conceptual map or case studies) over the period were used, where students presented high performances (>8.0). the affective assessment was carried out considering the criteria of responsibility, decision-making, leadership and communication developed throughout the module. in this evaluation, the students’ perceptions were contrasted with the professors’ perceptions until a consensus was reached. finally, psychomotor assessments were performed using the use of role-play. considering all domains, the class average was 8.4 and no student had a score lower than 6.0. although we use the flipped classroom combined with a role-play approach to minimize the pedagogical damage due to the covid-19 pandemic, it needs to emphasize that will not be feasible to overcome the inherent limitations of remote/virtual approach, such as lack of patient contact and face-to-face professor-student, which stimulate a wide number of emotional skills essential for the professional development of future dentists. in this way, the potential positive results observed cannot be used as a loan for the maintenance of non-face-to-face activities with the objective of cost-cutting by the institutions. in conclusion, although the changes in the format of the lessons, our approaches stimulated the students maintained the high frequency in the activities providing continuity activities. flipped classroom and the use of role-play can be a pedagogical strategy to be used during the covid-19 pandemic. disclosure statement the authors have no conflicts of interest to declare. funding no funding was obtained for this article. competing interests the authors have no conflicts of interest to declare. 5 chisini et al. braz j oral sci. 2022;21:e225368 author contribution lac contributed to conception, design, literature review and wrote the manuscript. mcmc, ama and fsc contributed to conception, design, literature review and critically revised the manuscript. all authors revised and approved the final version of the manuscript. references 1. who. world health organization. who coronavirus disease (covid-19) dashboard. [real time] [cited 2020 sep 7]. available from: https://covid19.who.int. 2. chisini la, costa fds, salvi lc, demarco ff. endodontic treatments in the brazilian public health system: influence of covid-19 pandemic. health pol technol. 2021 jun;10(2):100514. doi: 10.1016/j.hlpt.2021.100514. 3. who. world health organization. coronavirus disease (covid-19) situation report – 195. 2020 aug 2 [cited 2020 aug 3]. available from: https://www.who.int/docs/default-source/coronaviruse/ situation-reports/20200802-covid-19-sitrep-195.pdf?sfvrsn=5e5da0c5_2. 4. chisini l, sartori l, costa f, salvi l, demarco f. covid-19 pandemic impact on prosthodontics treatments in the brazilian public health system. oral dise. 2022;28(suppl. 1):994-6. 5. chisini la, costa fds, demarco gt, da silveira er, demarco ff. covid-19 pandemic impact on paediatric dentistry treatments in the brazilian public health system. int j paediatr dent. 2021 jan;31(1):31-4. doi: 10.1111/ipd.12741. 6. chisini la, castilhos ed, costa fds, d’avila op. impact of the covid-19 pandemic on prenatal, diabetes and medical appointments in the brazilian national health system. rev bras epidemiol. 2021 may 28;24:e210013. doi: 10.1590/1980-549720210013. 7. chisini la, costa fds, sartori lrm, corrêa mb, d’avila op, demarco ff. covid-19 pandemic impact on brazil’s public dental system. braz oral res. 2021 jul 16;35:e082. doi: 10.1590/1807-3107bor-2021.vol35.0082. 8. aquilanti l, gallegati s, temperini v, ferrante l, skrami e, procaccini m, et al. italian response to coronavirus pandemic in dental care access: the decade study. int j environ res public health. 2020 sep 24;17(19):6977. doi: 10.3390/ijerph17196977. 9. liu x, zhou j, chen l, yang y, tan j. impact of covid-19 epidemic on live online dental continuing education. eur j dent educ. 2020 nov;24(4):786-9. doi: 10.1111/eje.12569. 10. sukumar s, dracopoulos sa, martin fe. dental education in the time of sars-cov-2. eur j dent educ. 2021 may;25(2):325-31. doi: 10.1111/eje.12608. 11. gurgel bcv, borges sb, borges rea, calderon pds. covid-19: perspectives for the management of dental care and education. j appl oral sci. 2020 sep 28;28:e20200358. doi: 10.1590/1678-7757-2020-0358. 12. parsegian k, ayilavarapu s, gardner al, angelov n. predoctoral periodontal education and covid-19: challenges, actions, and learned lessons. j dent educ. 2020 oct 4:10.1002/jdd.12451. doi: 10.1002/jdd.12451. 13. quinn b, field j, gorter r, akota i, manzanares mc, paganelli c, et al. covid-19: the immediate response of european academic dental institutions and future implications for dental education. eur j dent educ. 2020 nov;24(4):811-4. doi: 10.1111/eje.12542. 6 chisini et al. braz j oral sci. 2022;21:e225368 14. halasa s, abusalim n, rayyan m, constantino re, nassar o, amre h, set al. comparing student achievement in traditional learning with a combination of blended and flipped learning. nurs open. 2020 mar 31;7(4):1129-38. doi: 10.1002/nop2.492. 15. ausubel dp. the acquisition and retention of knowledge: a cognitive view. kluwer academic publishers; 2000. 16. vanka a, vanka s, wali o. flipped classroom in dental education: a scoping review. eur j dent educ. 2020 may;24(2):213-26. doi: 10.1111/eje.12487. 17. zayyan m. objective structured clinical examination: the assessment of choice. oman med j. 2011 jul;26(4):219-22. doi: 10.5001/omj.2011.55. 18. donn j, scott ja, binnie v, bell a. a pilot of a virtual objective structured clinical examination in dental education. a response to covid-19. eur j dent educ. 2021 aug;25(3):488-94. doi: 10.1111/eje.12624. 19. ferraz apcm, belhot rv. bloom’s taxonomy and its adequacy to define instructional objective in order to obtain excellence in teaching. gest prod. 2010;17(2):421-31. doi: 10.1590/s0104-530x2010000200015 1 volume 22 2023 e239042 original article braz j oral sci. 2023;22:e239042http://dx.doi.org/10.20396/bjos.v22i00.8669042 1 department of prosthodontics, dental education unit, jss dental college and hospital, jss academy of higher education and research, mysuru, karnataka, india. 2 jss dental college and hospital, jss academy of higher education and research, mysuru, karnataka, india. 3 department of biochemistry, j.j.m.medical college, davangere, karnataka, india. corresponding author: dr. sunila bukanakere sangappa mds pg dip hpe, faimer fellow 2014 associate professor, department of prosthodontics convener, dental education unit jss dental college and hospital jss academy of higher education and research email: drsunilasangappa@gmail.com mob:9591613824 editor: altair a. del bel cury received: apr 15, 2022 accepted: jul 13, 2022 correlation of fasting blood, salivary glucose and malondialdehyde in subjects with & without type 2 diabetes sunila bukanakere sangappa1* , tamal das2 , basavaraj patil preethi3 oxidative stress is identified as the common pathogenic factor that leads to insulin resistance in diabetics. malondialdehyde is a product of lipid peroxidation. aim: the aim of this study was to determine the variation in the salivary malondialdehyde (mda) among subjects with and without t2dm in comparison to the fasting blood and salivary glucose. methods: this study involved  29 healthy participants as controls (group i) and 29 participants with type 2 diabetes mellitus as cases (group ii). salivary glucose was analysed by glucose oxidase end-point assay. thiobarbituric acid (tba) assay method was considered for estimation of mda in fasting saliva. data was statistically analysed using spss20. parametric test was performed to analyse the data. results: the correlation calculated between fbg with fsg level was found to be highly significant. a positive correlation between mda levels with fbg was found. the relationship between fbg and fsg (r = 0.7815, p < 0.05), fbg and mda (r =0.3678, p < 0.05) and fsg and mda (r = 0.2869, p < 0.05) were found to be positively significant. conclusion: saliva as a unique body fluid can serve as a medium for biochemical analysis only in standard settings and with multiple measures to be used as a diagnostic tool in par with the gold standard serum. salivary mda levels can be considered as one of the oxidative stress markers in type 2 diabetic condition. keywords: diabetes mellitus. glucose oxidase. malondialdehyde. oxidative stress. biomarker. https://orcid.org/0000-0002-8926-4971 https://orcid.org/0000-0001-8328-0826 2 sangappa et al. braz j oral sci. 2023;22:e239042 introduction type 2 diabetes mellitus is a multifactorial disease with its prevalence increasing at an alarming rate in modern day1. hyperglycaemia, the characteristic feature of diabetes increases the risk for many serious health complications leading to deteriorated quality of life and expectancy2. elevated levels of free radicals in the plasma and saliva are observed because of the biochemical alterations of glucose and lipid peroxidation3. diabetes control and complications trial (dcct) has emphasised the need of maintaining glycaemic control in order to delay or reduce the complications of diabetes4. although insulin resistance is the real root cause of type 2 diabetes it is evidenced that the role of oxidative stress is pivotal in contributing to the devastating effects of the acute and delayed systemic complications. oxidative stress is identified as the common pathogenic factor that leads to insulin resistance with impaired function of pancreatic beta cells ultimately resulting in type 2 dm (t2dm)5. evidence points at the overwhelming concentrations of reactive oxygen species (ros) particularly superoxide anion generated through mitochondrial oxidative metabolism among diabetics6. hyperglycaemia generating reactive oxygen species (ros) is suggested as the “dangerous metabolic route in diabetes” that results in tissue damage by a variety of mechanisms7. exposure to a relatively high concentration of ros and/or a decrease in antioxidant defence system against ros leads to oxidative stress8. estimation of malondialdehyde (mda), a product of lipid peroxidation in plasma is documented as a primary biomarker of the level of oxidative stress in clinical situations9,10. the percentage of glycated haemoglobin (hba1c) in blood gives an indication of the glycaemic level of an individual. although practice of hba1c test as a routine diagnostic test for detection of diabetes has several limitations, its role as an important marker for risk of microvascular complication in diabetics is well established in the literature11. saliva as a diagnostic tool for the assessment of oxidative stress is widely considered12. routine use of invasive procedures causes a great deal of mental trauma, discomfort, and anxiety especially in paediatric and old aged patients. step up in the prevalence of type 2 diabetes mellitus (t2dm) necessitates an efficient non-invasive screening strategy13. therefore, this study aims at correlating the levels of fasting blood and salivary glucose, salivary malondialdehyde (mda), levels in subjects with and without t2dm to append the role of salivary mda in determining the oxidative stress in t2dm. materials and methods study subjects selection this study was designed and performed following the helsinki declaration of 1975 (revised in the year 2000) after obtaining approval from the institutional review 3 sangappa et al. braz j oral sci. 2023;22:e239042 board (irb) of jagadguru sri shivarathreeswara dental college and hospital, jss aher, mysore, karnataka, india (iec research protocol no. 34/2019). study subjects were recruited from the outpatient department of jss hospital following attainment of informed consent. based on our pilot study sample size of 58 was determined with 29 for each amongst the diabetic and control group, assuming even group sizes to achieve 80% power and a significance level of 5% for detecting a true difference means between the test and reference group of 2.9 i.e., 5.22 – 2.23 units. this comparative cross-sectional study had subjects with age group ranging between 30–60 years. subjects with blood glucose levels in the fasting state (fbg) of 126 mg/dl or higher and ppbs (post prandial blood sugar) of 200 mg/dl or higher and hba1c level more than 6.5% were considered under group ii: cases. apparently healthy subjects with no history of type 2 diabetes mellitus who visited the hospital for routine check-up, not on any systemic medication, whose fasting blood glucose (fbg) less than 100 mg/dl and ppbs less than 140 mg/dl and hba1c level 5.9 % or less were categorised under group i: controls. the exclusion criteria for both groups included subjects with chronic systemic illness, infection, on vitamin supplements, pregnant woman, uncooperative patients, mentally compromised, completely edentulous patient. clinical evaluation participants selected for this study were subjected to oral examination. demographics along with details of physical dependency, oral habits, history of exposure to medication, daily medication intake were recorded in the study proforma. a unique barcode was assigned for each of the subject record for identification and confidentiality. age and gender of the subjects recruited were matched. collection of saliva sample clinical evaluation was performed followed by saliva collection. subjects were provided with prior instruction and guided to rinse their mouth with 10 ml of tap water for 20 second and expectorate. unstimulated whole saliva was collected using modification of method reported by navazesh14 in the literature using 5ml prelabelled sterile sample collection tubes. subjects were instructed not to perform any kind of oral hygiene measures minimum 1 hour prior to sample collection such as flossing, brushing, using mouthwash etc. saliva samples collected from the hospital were immediately stored on ice and transferred to the lab where it was stored at −80°c until analysis. plasma glucose and hba1c estimation: instructions were given to subjects for overnight fasting (minimum 8 hours) and to visit hospital for fasting blood glucose estimation in the morning. disposable syringe was used to withdraw 2 ml of peripheral venous blood from the antecubital vein and collected in a sterile tube. samples collected were immediately transported to the biochemistry laboratory for analysis the same day. 4 sangappa et al. braz j oral sci. 2023;22:e239042 salivary glucose estimation: unstimulated whole saliva sample was subjected to centrifugation at 8000 rpm for twenty minutes. when the clear supernatants appeared, it was immediately processed for fasting salivary glucose estimation with glucose oxidase end-point assay15 and hitachi 902 automatic analyser for the estimation. oxidative stress estimation: thiobarbituric acid (tba) assay method as reported by baliga et al.16 was used for estimation of mda in fasting saliva. saliva samples were diluted with distilled water (10 times). each sample (10µl) was mixed with 1ml tba reagent. the mixture was heated in a boiling water bath at 950c for 60minutes. the test tubes were cooled at room temperature and absorbance was measured at 532nm using uv visible spectrophotometer. statistical analysis data collected was transferred to a spreadsheet application for statistical analysis using spss20 software. parametric test was performed to analyse the data. independent t test was performed to measure the variability between test and control groups and correlations among blood glucose, hba1c level, fasting salivary glucose and fasting salivary mda was analysed with karl pearson’s correlation coefficient. the statistical significance was set at 5% (p < 0.05). results subjects catering to the inclusion and exclusion criteria were recruited to the study. study population consisted of a cohort of 23 male (41.66%) and 35 females (58.34%) with a mean age of 50.79±6.69 for group i (control) and 42.55±10.62 for group ii (cases). a descriptive analysis of fbg levels among controls (≤100 mg/dl) and type ii diabetic status (>126mg/dl) was then calculated in comparison with salivary levels of mda (µmol/l), hba1c (%), and fsg (mg/dl) as depicted in table 1. normality of fasting blood glucose level (mg/dl), fasting salivary glucose (mg/dl) and mda and hba1c were calculated using kolmogorov smirnov test. the scores of fasting blood and salivary glucose level (mg/dl) and mda, hba1c, followed a normal distribution as demonstrated table 2 and therefore parametric tests were applied. table 3 demonstrates the result of the independent t test. the results showed that mean fasting blood glucose, hba1c, salivary mda and glucose levels significantly higher amongst cases when subjected to comparison against the healthy controls. fbg levels among controls were 92.24±9.32 mg/dl and among cases were 157.21±39.30 mg/dl. fsg levels among controls were 2.89±2.60 mg/dl and among cases were 9.27±6.74 mg/dl. the levels of mda among controls were 0.41±0.26 μmol/l and among cases were 1.16±1.21 μmol/l. hba1c levels among the controls were 4.75±0.59 % and among cases were 6.79±1.34 %. these differences were found to be statistically significant. 5 sangappa et al. braz j oral sci. 2023;22:e239042 table 1. comparison of t2dm and control groups with fbg, fsg and mda and hba1c, test group % control group % total % chi-square p-value fbg <100mg/dl 0 0.00 25 86.21 25 43.10 49.1110 0.0001* 100-126 mg/dl 5 17.24 4 13.79 9 15.52 127-153 mg/dl 13 44.83 0 0.00 13 22.41 154-180 mg/dl 4 13.79 0 0.00 4 6.90 >180 mg/dl 7 24.14 0 0.00 7 12.07 fsg 0.305.00 mg/dl 12 41.38 25 86.21 37 63.79 18.7100 0.0001* 5.019.71 mg/dl 3 10.34 4 13.79 7 12.07 9.7214.42 mg/dl 3 10.34 0 0.00 3 5.17 > 14.42 mg/dl 11 37.93 0 0.00 11 18.97 mda 0.100.60 μmol/l 10 34.48 20 68.97 30 51.72 15.5830 0.0010* 0.611.11 μmol/l 7 24.14 9 31.03 16 27.59 1.121.62 μmol/l 7 24.14 0 0.00 7 12.07 > 1.62 μmol/l 5 17.24 0 0.00 5 8.62 hba1c <5.9 mg/dl 0 0.00 29 100.00 29 50.00 35.4440 0.0001* 6-7.9 mg/dl 23 79.31 0 0.00 23 39.65 >8.1 mg/dl 6 20.69 0 0.00 6 10.35 total 29 100.00 29 100.00 58 100.00 table 2. normality of fasting blood glucose level (mg/dl), fasting salivary glucose and mda and hba1c, by kolmogorov smirnov test. parameters test group control group z-value p-value z-value p-value fasting blood glucose level (mg/dl) 1.0320 0.2370 1.2910 0.0710 fasting salivary glucose 1.2210 0.1010 1.0340 0.2350 fasting salivary mda 1.1590 0.1360 1.1600 0.0890 hba1c 0.3890 0.9980 0.7510 0.6250 note: the scores of fasting blood glucose level (mg/dl), fasting salivary glucose, fasting salivary mda and hba1c follow a normal distribution. therefore, the parametric tests were applied table 3. comparison of t2dm and control groups with mean of fbg, fsg, mda, hba1c by independent t test variables groups min max mean sd se t-value p-value fasting blood glucose level (mg/dl) control 74.00 107.00 92.24 9.32 1.73 8.6610 0.0001* test 126.00 268.00 157.21 39.30 7.30 total 74.00 268.00 124.72 43.30 5.69 continue 6 sangappa et al. braz j oral sci. 2023;22:e239042 continuation fasting salivary glucose control 0.22 9.45 2.89 2.60 0.48 4.7580 0.0001* test 0.31 18.20 9.27 6.74 1.25 total 0.22 18.20 6.08 6.00 0.79 fasting salivary mda control 0.13 0.89 0.41 0.26 0.05 3.2340 0.0020* test 0.13 6.41 1.16 1.21 0.22 total 0.13 6.41 0.79 0.94 0.12 hba1c (mg/dl) test 4.25 10.24 6.79 1.34 0.25 7.4870 0.0001* control 3.56 5.96 4.75 0.59 0.11 total 3.56 10.24 5.77 1.45 0.19 *p<0.05 table 4 depicts the degree and direction of the relationship between fasting blood glucose level (mg/dl), hba1c (mg/dl), fasting salivary glucose and fasting salivary mda (μmol/l), in test and control groups calculated by karl pearson’s correlation coefficient. the strong positive correlation (r = 0.7815, p < 0.05) was found between fbg with fsg level and a moderate positive correlation between mda levels with fbg (r =0.3678, p < 0.05) and fsg and mda (r = 0.2869, p < 0.05) inferring that the value of salivary mda and fsg and fbg were positively correlated to each other and the rise of glycaemic level in blood is reflected in saliva and with increase in fbg there is a moderately significant rise in mda level in saliva. figure 1 represents incorporation of these pattern of data on a scattered plot diagram. it showed that even though the points are observed to be somewhat scattered a positive relationship is indicated in a wider band. table 4. correlations among fasting blood glucose (mg/dl), fasting salivary glucose (mg/dl), mda (μmol/l), hba1c (%), in test and control groups by karl pearson’s correlation coefficient groups variables fbg fsg mda hba1c test group fasting blood glucose level (mg/dl) r value -p value -fasting salivary glucose (mg/dl) r value 0.7815 -p value 0.0001* -fasting salivary mda (μmol/l) r value 0.3678 0.2869 -p value 0.0500* 0.1310 -hba1c (%) r value 0.7785 0.7426 0.0695 -p value 0.0001* 0.0001* 0.7200 -7 sangappa et al. braz j oral sci. 2023;22:e239042 control group fasting blood glucose level (mg/dl) r value -p value -fasting salivary glucose (mg/dl) r value 0.4298 -p value 0.0200* -fasting salivary mda (μmol/l) r value 0.3744 0.1549 -p value 0.0450* 0.4220 -hba1c (%) r value 0.6060 -0.0013 0.3026 -p value 0.0001* 0.9950 0.1110 -*p<0.05 discussion type ii diabetes mellitus is a chronic disease associated with impairment of multiple metabolic functions leading to secondary pathophysiological consequences in multiple organ systems eventually resulting in microand macrovascular complications. generation of oxidative stress plays a pivotal role in the pathogenesis of both type 1 and type 2 diabetes which is well established in the literature5. diabetic hyperglycaemia is accompanied with a rise in generation of free radicals in all tissues from auto-oxidation of glucose molecules and protein glycosylation leads to damage of enzymes, intricate cellular functionality and also increased insulin resistance17. lipid peroxidation is a biochemical process that leads to formation of malondialdehyde figure 1. correlation of fbg, fsg, and fasting salivary mda and hba1c in test group fasting salvary glucose fasting salvary mca fasting blood glucose level (mg/dl) hba1g (mg/dl) 8 sangappa et al. braz j oral sci. 2023;22:e239042 (mda) which is considered as a critical biomarker of oxidative stress. a need arises to find out a quick easy and non-invasive method to diagnose this silently growing pandemic. in this regard, this study was planned and constructed to ascertain and compare levels of malondialdehyde (mda) and glucose in fasting saliva with fasting plasma glucose18. american diabetes association recommends the fasting plasma glucose test (fpg) to be the preferred method for diagnosing diabetes owing to its ease of performance, convenience, and cost effectiveness when compared to other tests. fasting (for at least 8 hours before the test) and 2-hour blood glucose concentrations correlate closely with β-cell function of the pancreas, the impairment of which has been identified as the principal factor responsible for the pathogenesis of type 2 diabetes19. the american diabetes association also recommends glycated haemoglobin (hba1c) level as an alternative method of diagnosing diabetes. it is considered an important indicator of long-term glycaemic control as it reflects the cumulative glycaemic status of previous two to three months20. it has been documented that in diabetics a significant correlation also exists between blood glucose and salivary glucose and hba1c levels21. in our study subjects with type 2 diabetes mellitus who have been diagnosed for minimal of two years with fasting blood glucose (fbg) higher than 126 mg/dl and ppbs of 200 mg/dl or higher and hba1c level more than 6.5% were involved to eliminate other causes of increased blood glucose. it was noticeable that 44.83% of subjects in the t2dm groups had their fbg values in the range of 127-153 mg/dl. saliva is a unique kind of bodily fluid, and its innate potential as diagnostic media is being explored and developed. it is a well-known fact that saliva is an ultrafiltrate of blood produced in various salivary glands. study has shown that glucose value in saliva can vary depending on the fasting duration of the subjects and data showed glucose levels can also vary depending on the time of day of blood sample is withdrawn, subject’s physical activity or intake of alcohol22. in this study unstimulated 12hour fasting saliva was collected from the recruited subjects. literature has evidenced that type2 diabetics with and without complication were susceptible to oxidative stress and elevated blood glucose level had a significantly high positive correlation with serum mda level23,24. further ahead it was observed that salivary mda level increases in patient with type 2 diabetes10,25. studies have also concluded that salivary mda appears to be an indicator and also reflects the value of serum mda concentration which in turn reflects precisely the severity of the oxidative stress12,21. the paucity of studies on the extent of association between fbg and salivary mda levels in type 2 diabetics directed us to undertake this as one of the objectives. our findings confirmed the evidence of significant and positive correlation between fasting salivary mda levels with fbg with pearson’s correlation coefficient of 0.3678 among diabetics thus concluding that estimation of salivary mda levels in type 2 diabetic condition can be used as an oxidative stress marker and further as an adjuvant diagnostic aid with extended research. glucose is one of the many components of blood that is transferrable to salvia in proportion to their blood concentration. as per biochemical investigations it has 9 sangappa et al. braz j oral sci. 2023;22:e239042 been documented in the literature the normal value of salivary glucose in a healthy non diabetic is less than 2 mg/dl15. studies have documented the association with salivary glucose levels and blood glucose levels suggesting that salivary glucose level can be used as a noninvasive tool for monitoring glycaemic level in dm in a dental setup15,26. systematic review and meta-analysis of observational studies on effect of salivary glucose in type 2 dm has concluded that saliva can be a biomarker especially when it is used for screening of type 2 dm in large scale18. our study findings were in consensus with existing literature to have been able to establish a positive correlation between fasting blood glucose and fasting salivary glucose and salivary mda levels among type 2 diabetics21,23,24. this study also found a significant correlation that exists between fbg and fsg with hba1c levels which adds onto diagnostic potential of saliva for t2dm and requires further exploration. as the study estimates were one time measures it can be concluded that multiple measures are required for conclusive results to indicate consideration of saliva as a diagnostic parameter in diabetic condition among different population groups. literature has highlighted the application of salivary diagnostic tests in clinical sciences27. therefore it is valuable to integrate salivary diagnostics into clinical practice by advancing dentistry into primary health care28. with emerging need for chair side diagnostics to overcome the inconvenience of invasive procedures to the patients the authors would recommend routine estimation of salivary mda to evaluate oxidative stress following further research. in conclusion, the use of saliva as a “diagnostic tool” offers the advantage over serum as the collection process of saliva is not invasive and it’s cost effective. with the limitations of this study, it can be concluded that saliva can indeed be used as a medium for biochemical analysis only in standard settings and with multiple measures to consider saliva as a diagnostic tool in par with the gold standard serum. salivary mda levels can be considered as one of the oxidative stress markers in type 2 diabetic condition. this further strengthens the inferences in the literature. acknowledgements the authors of this study would like to thank indian council medical research (icmr) for providing studentship and encouragement to take up short studies. the authors would also like to thank dr rohit jain for helping in arrangement of sample collection. funding source i wish to quote that this project submitted to your esteemed journal is an icmr short term studentship (ref :2029-07738) which is awarded fellowship with faculty guide dr.sunila sangappa and student mr.tamal das. data availability datasets related to this article will be available upon request to the corresponding author. 10 sangappa et al. braz j oral sci. 2023;22:e239042 author contribution dr. sunila bukanakere sangappa has mentored this study, involved in conception, design, analysis and/or interpretation of data. dr preethi.b.p is involved in analysis, interpretation of data and review of manuscript. all the authors of 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10.4103/jomfp.jomfp_222_15. 16. baliga s, chaudhary m, bhat s, bhansali p, agrawal a, gundawar s. estimation of malondialdehyde levels in serum and saliva of children affected with sickle cell anemia. j indian soc pedod prev dent. 2018 jan-mar;36(1):43-7. doi: 10.4103/jisppd.jisppd_87_17. 17. tiongco re, bituin a, arceo e, rivera n, singian e. salivary glucose as a non-invasive biomarker of type 2 diabetes mellitus. j clin exp dent. 2018 sep;10(9):e902-e907. doi: 10.4317/jced.55009. 18. mascarenhas p, fatela b, barahona i. effect of diabetes mellitus type 2 on salivary glucose--a systematic review and meta-analysis of observational studies. plos one. 2014 jul;9(7):e101706. doi: 10.1371/journal.pone.0101706. 19. abdul-ghani ma, defronzo ra. plasma glucose concentration and prediction of future risk of type 2 diabetes. diabetes care. 2009 nov;32 suppl 2(suppl 2):s194-8. doi: 10.2337/dc09-s309. 20. ghazanfari z, haghdoost aa, alizadeh sm, atapour j, zolala f. a comparison of hba1c and fasting blood sugar tests in general population. int j prev med. 2010 summer;1(3):187-94. 21. gupta s, sandhu sv, bansal h, sharma d. comparison of salivary and serum glucose levels in diabetic patients. j diabetes sci technol. 2015 jan;9(1):91-6. doi: 10.1177/1932296814552673. 22. moebus s, göres l, lösch c, jöckel kh. impact of time since last caloric intake on blood glucose levels. eur j epidemiol. 2011 sep;26(9):719-28. doi: 10.1007/s10654-011-9608-z. 23. mahreen r, mohsin m, nasreen z, siraj m, ishaq m. significantly increased levels of serum malonaldehyde in type 2 diabetics with myocardial infarction. int j diabetes dev ctries. 2010 jan;30(1):49-51. doi: 10.4103/0973-3930.60006. 24. bhutia y, ghosh a, sherpa ml, pal r, mohanta pk. serum malondialdehyde level: surrogate stress marker in the sikkimese diabetics. j nat sci biol med. 2011 jan;2(1):107-12. doi: 10.4103/0976-9668.82309. 25. smriti k, pai km, ravindranath v, pentapati kc. role of salivary malondialdehyde in assessment of oxidative stress among diabetics. j oral biol craniofac res. 2016 jan-apr;6(1):41-4. doi: 10.1016/j.jobcr.2015.12.004. 26. kadashetti v, baad r, malik n, shivakumar km, vibhute n, belgaumi u, et al. glucose level estimation in diabetes mellitus by saliva: a bloodless revolution. rom j intern med. 2015 jul-sep;53(3):248-52. doi: 10.1515/rjim-2015-0032. 27. javaid ma, ahmed as, durand r, tran sd. saliva as a diagnostic tool for oral and systemic diseases. j oral biol craniofac res. 2016;6(1):66-75. doi: 10.1016/j.jobcr.2015.08.006. 28. wong dt. salivaomics. j am dent assoc. 2012 oct;143(10 suppl):19s-24s. doi: 10.14219/jada.archive.2012.0339. 1http://dx.doi.org/10.20396/bjos.v20i00.8661883 volume 20 2021 e211883 original article 1 federal university of pernambuco, recife, pernambuco, brazil. *corresponding author: renata cimões email: renata.cimoes@globo.com received: november 02, 2020 accepted: january 12, 2021 family functioning and dental trauma, malocclusion and anthropometry in adolescents adelaine maria de sousa1 , thais carine lisboa silva1 , bruna de carvalho vaigel1 , roberto carlos mourão pinho1 , renata cimões1,* aim: the aim of the study was to investigate perceived family cohesion and adaptability and its association with trauma, malocclusion and anthropometry in school adolescents. methods: cross-sectional study with a representative sample of 921 adolescents from 13 to 19 years old of both sexes, enrolled in state public schools of a northeastern brazilian municipality. a questionnaire with sociodemographic questions, the faces iii scale was applied and a clinical oral examination (dental trauma and malocclusion) and anthropometric (bmi by age) were performed. for statistical analysis, was evaluated by the chi-square test. the variables that presented significance in the bivariate analysis of up to 25% were taken to the multivariate analysis (multinomial logistic regression), variables that presented significance in bivariate analysis of up to 25% were taken to multivariate analysis and all conclusions were drawn considering the significance level of 5%. results: as a result, it was identified that displaced families were associated with low maternal education, agglutinated families associated with the absence of caries. rigid families were associated with marked overjet and caries. the prevalence of dental trauma (37.5%) was considered high. conclusion: it was concluded that family cohesion and adaptability were associated with oral health and socioeconomic factors. keywords: oral health. family. adolescent. epidemiology. holistic health. https://orcid.org/0000-0003-3964-8122 https://orcid.org/0000-0001-9878-6280 https://orcid.org/0000-0002-2756-0767 http://orcid.org/0000-0002-2831-2722 https://orcid.org/0000-0003-3673-8739 2 sousa et al. introduction the family plays an important role in the care of its members. this care is characterized by meeting the physical and psychological needs of family members1. thus, it is valuable to understand the relationship between family members and the degree of union. this makes the functioning of the family an important unit of study and action2. this understanding is essential to optimize relationships and improve health conditions and quality of life for family members3. dental trauma is not the consequence of an illness, but of several factors that occur throughout life4. therefore, investigating factors other than biological ones is of paramount importance, since it is they that influence the lifestyle, the health habits that are taken to the future. and adolescence represents a fundamental moment for health promotion, since it does not always enjoy more of the care and attention one has in childhood5. and behavior can be important in the occurrence of dental trauma in adolescents, because aggressive behaviors are risk factors4. a systematic review reveals that dental trauma is one of the most prevalent aggravations in the world and affects people of various ages and social conditions, and that severe overjet is one of the risk factors, traumatic dental injuries have important physical, psychological and economic consequences, with etiological factors varying between countries and age groups6, along with socio-demographic, behavioral and overweight factors, the importance of social, economic and structural factors on overweight children’s eating patterns and sedentary life style must be considered and this prevention strategy would reduce all types of injuries, diabetes, arteriosclerosis and hypertension7,8. therefore, this study aimed to investigate the perceived family cohesion and adaptability and its association with dental trauma and other factors such as overjet, anthropometrics and demographics in school adolescents, or that there is no relationship between the variables or phenomena measured. materials and methods this study was approved by the research ethics committee of ufpe, under the report number 1,903,021 and in compliance with the ethical requirements and legal criteria of the research a cross-sectional, analytical and school-based study was conducted in state schools in the municipality of camaragibe, state of pernambuco, brazil, on adolescents regularly enrolled in high school. camaragibe had 21 state public schools, of which 15 offered high school. of the 15, 11 accepted to participate. the municipality has an estimated population of 157,8289 inhabitants and a state high school network with a total of 4,78410 adolescents enrolled. to carry out the sample calculation, the prevalence of 50% was used, a proportion that maximizes the sample size, as it generates the greatest variance (p = 0.50) (44). this means assuming the worst case scenario, that is, for any prevalence the sample is sufficient and significant, with 95% confidence with an error of 5%. the minimum value for a representative sample was 786 adolescents. to this minimum number was added 20% to 3 sousa et al. compensate for losses, leaving a total of 921 adolescents. in each of the eleven schools, the terms of informed consent were given to the students of the randomly selected classrooms (in each school, three classes were randomly selected, one from each high school grade) to obtain the authorization of those responsible. those over 18 years of age signed another informed consent form and minors also signed the informed consent form. from the list made available by the pernambuco information system, contacts were made for all schools to participate and eleven accepted to participate in the study, offering the lists of the rooms of all high school classes in the morning and afternoon shifts. in each of these schools, a draw was made for three classrooms, one class from each grade of high school, in order to make the sample more heterogeneous and representative. all students in the selected class participated, were excluded, in addition to the exclusion criteria, those who refused to participate and / or without authorization from the responsible. adolescents who did not present the guardian’s authorization were excluded. adolescents between the ages of 13 and 19, of both sexes were included. users of orthodontic appliances and adolescents with disabilities that made it impossible to complete the questionnaire or perform the clinical examination were excluded. the adolescents received explanations on how to correctly fill out the forms. the exams were performed in a school environment by two examiners assisted by note takers. millimeter probes and odontoscopes were used for the examination of dental trauma and overjet. for anthropometric evaluation, a digital scale and a stadiometer were used. the researchers were calibrated and this process began with a theoretical stage in which the criteria and indexes were presented; followed by clinical exams performed by two examiners and by a more experienced researcher, here considered the gold standard, in twenty adolescents, assisted by note-takers. the calibration process was performed only once the result of the kappa degree of agreement was above 0.81 for all indexes seen (trauma and overjet). the reasons for dropping out on the part of the adolescents was not wanting to take the clinical exam, even though they answered the questionnaire, especially when checking their weight and height. the variable dependent on the study was family cohesion and adaptability, collected from the faces iii11 scale, validated in brazil by falceto and bozzetti12. for family cohesion, adolescents were divided into three groups. values below and above the standard deviation corresponded to families with low (disconnected) and high family cohesion (agglutinated), respectively; and values between standard deviations corresponded to families with mean family cohesion (those separated or connected)3. for family adaptability, there were also three groups low (rigid family), moderate (structured or flexible family), and high adaptability (chaotic family), also based on mean and standard deviation. a questionnaire on socio-demographic data aspects was also applied. in the clinical chart, data concerning the identification of the adolescent, the clinical exams, as well as weight and height for anthropometric calculation were recorded. dental trauma was seen through the codes and criteria proposed by garcía-godoy13 and, for the association test, categorized by absence or presence. the overjet was measured in millimeters and divided into normal ones, those with less than 5mm, and those with 5mm or more were considered accentuated6. 4 sousa et al. regarding anthropometric data, weight and height were collected. for the classification, the bmi for the age was used, which is the who’s and recommended by the ministry of health14. for this study we divided into two groups, adolescents without overweight (underweight and eutrophic) and those with overweight (overweight, obesity and severe obesity). the results are presented through the description of absolute and relative frequencies (absolute distribution, percentage and standard deviation of variables). statistical significance was evaluated by the chi-square test. the variables that presented significance in the bivariate analysis of up to 25% were taken to the multivariate analysis (multinomial logistic regression). the bank was made in epi info software, with double typing to avoid errors and the analyses were performed in spss version 21 and the significance level of 5% was adopted. results of the 921 adolescents, 790 composed the final sample. the majority (93.7%) were between 15 and 19 years old, with an average age of 16.15 years. of these, 400 (50.6%) were female. the most frequent family income was up to 2  minimum wages with 50.5% of the adolescents and in 59.4% of the adolescents the mother had more than 9 years of study, that is, they had at least incomplete high school. the prevalence of dental trauma was 37.5% and chart 1 shows the distribution of dental trauma according to the tooth and type of fracture. chart 1. distribution of dental trauma according to the tooth and type of fracture dental traumatism teeth dt12 dt11 dt21 dt22 dt42 dt41 dt31 dt32 n(%) n(%) n(%) n(%) n(%) n(%) n(%) n(%) 0 no fracture 767 (97.1) 672 (85.1) 632 (80.1) 767 (97.2) 781 (98.9) 782 (99.0) 782 (99.0) 782 (99.1) 1enamel crack 13 (1.6) 61 (7.7) 66 (8.4) 13 (1.6) 5 (0.6) 5 (0.6) 2 (0.3) 1 (0.1) 2 enamel fracture 8 (1.0) 48 (5.8) 73 (9.3) 9 (1.1) 4 (0.5) 3 (0.4) 6 (0.8) 5 (0.6) 3 enamel and dentin fracture 2 (0.3) 11 (1.4) 18 (2.3) (0.0) (0.0) (0.0) (0.0) 1 (0.1) total 790 (100.0) 790(100.0) 789(100.0) 789(100.0) 790(100.0) 790(100.0) 790(100.0) 789(100.0) legend :dt 12 – dental trauma tooth 12; dt11dental trauma tooth 11; dt21dental trauma tooth 21; dt22 dental trauma tooth 22; dt42dental trauma tooth 42; dt41dental trauma tooth 41; dt31dental trauma tooth 31; dt32dental trauma tooth 32 . the average for family cohesion was 30.43 with a standard deviation of 5.33 and the average for adaptability was 22.83 with a standard deviation of 3.12, so families were divided where: 29.4% had low cohesion, 50.6% moderate cohesion, 20% high cohesion, 25.3% low adaptability, 44.4% moderate adaptability and 30.3% high adaptability. table 1shows the bivariate analysis in relation to oral, anthropometric and socio-demographic clinical conditions and family cohesion. only trauma was associated with high cohesion (p=0.031). 5 sousa et al. table 1. bivariate analysis of family cohesion according to clinical conditions and socio-demographic factors. variables low n(%) family cohesion p-value (low x medium) p-value (high x medium) medium high total n(%) n(%) n(%) trauma no 144 (62.1) 262 (65.5) 88 (55.7) 494 (62.5) 0.386 0.031* yes 88 (37.9) 138 (34.5) 70 (44.3) 296 (37.5) total 232 (100.0) 400 (100.0) 158 (100.0) 790 (100.0) bmi no overweight 162 (69.8) 302 (75.5) 119 (75.3) 583 (73.8) 0.120 0.964 overweight 70 (30.2) 98 (24.5) 39 (24.7) 207 (26.2) total 232 (100.0) 400 (100.0) 158 (100.0) 790 (100.0)     maxillary overjet normal 208 (89.7) 352 (88.0) 139 (88.0) 699 (88.5) 0.528 0.993 enlarged 24 (10.3) 48 (12.0) 19 (12.0) 91 (11.5) total 232 (100.0) 400 (100.0) 158 (100.0) 790 (100.0) family income up to 2 mw 123 (72.4) 197 (65.2) 79 (62.2) 399 (66.6) 0.112 0.550 above 2 mw 47 (27.6) 105 (34.8) 48 (37.8) 200 (33.4) total 170 (100.0) 302 (100.0) 127 (100.0) 599 (100.0)   how many people live up to 2 people 156 (68.1) 272 (69.6) 102 (65.4) 530 (68.3) 0.708 0.342 3 or more 73 (31.9) 119 (30.4) 54 (34.6) 246 (31.7) total 229 (100.0) 391 (100.0) 156 (100.0) 776 (100.0) 1pearson’s chi-square test; *statistically significant (p<0.05) in table 2 we have the bivariate analysis between oral, anthropometric and sociodemographic clinical conditions and family adaptability, where income of up to two minimum wages (p=0.030) and accentuated overjet (p=0.002) were associated with low adaptability. table 2. bivariate analysis of family adaptability according to clinical conditions and sociodemographic factors. variables low n(%) family adaptability p-value1 (low x medium) p-value1 (high x medium) medium high total n(%) n(%) n(%) trauma no 126 (63.0) 221 (63.0) 147 (61.5) 494 (62.5) 0.993 0.720 yes 74 (37.0) 130 (37.0) 92 (38.5) 296 (37.5) total 200 (100.0) 351 (100.0) 239 (100.0) 790 (100.0) bmi no overweight 145 (72.5) 253 (72.1) 185 (77.4) 583 (73.8) 0.916 0.146 overweight 55 (27.5) 98 (27.9) 54 (22.6) 207 (26.2) total 200 (100.0) 351 (100.0) 239 (100.0) 790 (100.0)     maxillary overjet normal 165 (82.5) 321 (91.5) 213 (89.1) 699 (88.5) 0.002* 0.343 enlarged 35 (17.5) 30 (8.5) 26 (10.9) 91 (11.5) total 200 (100.0) 351 (100.0) 239 (100.0) 790 (100.0) family income up to 2 mw 112 (73.2) 162 (62.8) 125 (66.5) 399 (66.6) 0.030* 0.421 above 2 mw 41 (26.8) 96 (37.2) 63 (33.5) 200 (33.4) total 153 (100.0) 258 (100.0) 188 (100.0) 599 (100.0)     how many people live up to 2 people 145 (73.6) 236 (68.4) 149 (63.7) 530 (68.3) 0.203 0.237 3 or more 52 (26.4) 109 (31.6) 85 (36.3) 246 (31.7) total 197 (100.0) 345 (100.0) 234 (100.0) 776 (100.0) 1pearson’s chi-square test;, *statistically significant (p<0.05) 6 sousa et al. after bivariate analysis, using chi-square test, all variables with significance less than 0.25 were included in the multinominal logistic regression model. for cohesion, no variables were associated. table 3 reveals that in relation to family adaptability, the multivariate analysis showed that the maxillary overjet (p=0.010) was associated with low adaptability, showing that adolescents with severe overjet are 1.766 times more likely to have low adaptability in relation to average family adaptability. table 3. multivinomial logistic regression for family adaptability   variables coef. d.e. c2 value of p or1 ci 95% minimum maximum low constant -1.064 0.336 10.035 0.002 accentuated overjet 0.569 0.239 5.646 0.018* 1.766 1,105 2,823 high constant -1.061 0.343 9.580 0.002 accentuated overjet 0.213 0.236 0.815 0.367 1.238 0,779 1,966 likelihood ratio test (p-value) goodness-of-fit test (p-value) r2 of nagelkerke 15,213 0.019 pearson 0.143 deviance 0.107 0.017 legend: c2 – chi-square; 1-or – odds ratio; ci – confidence interval; coefcoefficient of the variable; d.e. default error; r2 – coefficient of determination. discussion there was an association between family cohesion and adaptability with sociodemographic factors and clinical conditions. this result was similar to ferreira  et  al.3 where it showed that family cohesion perceived by the adolescent was associated with behavioral variables, oral health and socioeconomic factors, another important point was that and increased overjet was associated with adaptability. the prevalence of dental trauma (37.5%) was considered high when compared with other studies15-17, therefore, early interventions are necessary, in order to reduce the impact of malolusions, and especially overjet on the quality of life and self-esteem of adolescents. in the present study, a result that, although it did not remain significant in the multivariate analysis, should not be overlooked, is the low income associated with low adaptability. in adolescence, it is important to emphasize that the decision to consult the service is usually made by the parents and not by the youngster himself; furthermore, the socioeconomic condition is one of the most important social determinants in the use of dental services5,18. and, therefore, it is important to emphasize that the population studied was of students from public schools, thus being able to be considered homogeneous, economically low; where approximately half has income of up to two minimum wages. more than half of the adolescents perceived moderate family cohesion and, regarding adaptability, the highest percentage perceived was from families with moderate adaptability. there was an association between severe overjet and low adaptability. silva and katz19, suggest that a family with low adaptability has a low adherence to 7 sousa et al. proposed treatments, thus justifying the association between having a more pronounced overjet and having a family with low adaptability. low family adaptability was associated to the accentuated overjet families like this have insufficient adaptive power and, for this reason, would have problems of adherence to treatment protocols19,20. this may call attention to the need for new approaches to prevention and health promotion, focusing on family functioning, besides what is already known in the health-disease process. overjet is one of the most prevalent types of malocclusion and interferes negatively on the psychological well-being and social interaction of adolescents18, besides being a risk factor for dental trauma. however, it is a risk factor that can be altered by the use of orthodontic appliances and can reduce the risk of dental trauma6. maintaining  the oral health of adolescents must also involve education in the oral health of parents and guardians, so the achievement of results in modifying maintenance attitudes or interventions in the oral health of adolescents must involve and reach parents and guardians. there was no association between bmi and family functioning. a similar result to another study with adolescents in which there was no difference between overweight and eutrophic adolescents with the family relationship, showing that unhealthy family functioning can be common in this phase of life, regardless of nutritional status21. in brazil, part of the basic care in the public health system is family-centered. this  reveals that it should not be limited to clinical issues, but also include family factors in the actions of prevention and health education, promotion and recovery22. since it is a cross-sectional study, it does not allow a causal relationship, however, this type of design is widely used in health research. and this research is one of the few already carried out involving adolescents’ oral health with family functioning3,19. and this is important, since it can create guidelines for monitoring the oral health of adolescents at school, as well as family relationships that can contribute to success or failure in both oral health and school performance. another result that also deserves attention, despite not remaining significant in the multivariate analysis, is the association between trauma and high cohesion. according to barber and buehler23, it is possible that families with high cohesion may be more permissive, not establishing rules against, for example, aggressive behavior, which could explain a greater risk for trauma. nevertheless, new studies are suggested, with other designs and populations, in order to clarify the involvement of dental trauma and factors associated with it with the family functioning in adolescents. in conclusion, families with low adaptability, the rigid ones, were associated to the accentuated overjet and there was no association between family cohesion and adaptability with anthropometric data, and the prevalence of dental trauma was considered high and increased overjet was associated with adaptability. 8 sousa et al. references 1. serapioni m. 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[validation of diagnostic scales of family functioning for use in primary health care services]. pan am j public health 2000 apr;7(4):255-63. portuguese. doi: 10.1590/ s1020-49892000000400007. 13. garcía-godoy fm. prevalence and distribution of traumatic injuries to the permanent teeth of dominican children from private schools. community dent oral epidemiol. 1984 apr;12(2):136-9. doi: 10.1111/j.1600-0528.1984.tb01426.x. 14. ministry of health of brazil, department of health care, department of primary care. [guidelines for collection and analysis of anthropometric data in health services: technical standard system of food and nutrition surveillance – sisvan]. brasília: ministry of health of brazil; 2011 [cited 2020 oct 2]. available from: http://bvsms.saude.gov.br/bvs/publicacoes/orientacoes_coleta_analise_dados_antropometricos.pdf. portuguese 15. ministry of health of brazil. health care secretariat. health surveillance secretariat. [sb brazil 2010: national research on oral health: main results]. brasília: ministry of health of brazil; 2012 [cited 2020 oct 2]. available from: http://bvsms.saude.gov.br/bvs/publicacoes/pesquisa_nacional_saude_ bucal.pdf. portuguese. 9 sousa et al. 16. vieira em, cangussu mct, vianna mip, cabral mbb, roque rn, anjos es. prevalência, gravidade e fatores associados ao traumatismo dentário em escolares de 12 e 15-19 anos de idade em salvador, bahia. feira de santana: rev. saúde col. uefs; 2017;7(1):51-7. 17. bendo cb, paiva sm, torres cs, oliveira ac, goursand d, pordeus ia, et al. association between treated/untreated traumatic dental injuries and impact on quality of life of brazilian schoolchildren. health qual life outcomes. 2010 oct 4;8:114. doi: 10.1186/1477-7525-8-114. 18. martins lp, bittencourt jm, bendo cb, vale mp, paiva sm. malocclusion and social vulnerability: a representative study with adolescents from belo horizonte, brazil. cienc saude colet. 2019 feb;24(2):393-400. doi: 10.1590/1413-81232018242.33082016. 19. silva jfo, katz crt. [caries experience, treatment needs, and family functioning in brazilian preschool children: a casecontrol study]. arq odontol. 2015;51(2):76-87. portuguese. doi: 10.7308/ aodontol/2015.51.2.03. 20. olson dh. circumplex model of marital and family systems. j fam ther. 2000;22(2):144-67. 21. almeida ccjn, mora po, oliveira va, joão ca, joão cr, riccio ac, et al. [variables associated with family breakdown in healthy and obese/ overweigh adolescents]. rev paul pediatr. 2014 mar;32(1):70-7. portuguese. doi: 10.1590/s0103-05822014000100012. 22. department of primary care secretariat of health policies of brazil. [family health program. technical institutional reports]. rev saude publica. 2000;34(3):316-9. portuguese. doi: 10.1590/ s0034-89102000000300018. 23. barber bk, buehler c. family cohesion and enmeshment: different constructs, different effects. j marriage fam. 1996 may;58(2):433-41. doi: 10.2307/353507. 1 volume 21 2022 e227878 original article braz j oral sci. 2022;21:e227878http://dx.doi.org/10.20396/bjos.v21i00.8667878 1 department of restorative dentistry, faculty of dentistry, university of north parana – unopar, londrina-pr. 2 department of restorative dentistry, faculty of dentistry, paranaense university– unipar, londrina-pr. corresponding author: dr. murilo baena lopes, university north of parana unopar, faculty of dentistry, rua marselha, 183, jd. piza, londrina-pr, 86041-140, brazil, tel +554333717820, baenalopes@gmail.com editor: dr. altair a. del bel cury received: december 12, 2022 accepted: april 2, 2022 assessing the bleaching effect of an experimental stabilized chlorine dioxide agent used for internal bleaching purposes bruno shindi hirata1 , sandrine bittencourt berger1 , ricardo danil guiraldo1 , klissia romero felizardo2 , nádia buzignani pires ramos1 , murilo baena lopes1* aim: assessing the intracoronal bleaching effectiveness of an experimental chlorine dioxide product, based on the walking bleach technique. methods: extracted bovine incisors were artificially stained with bovine blood and filled with zinc phosphate cement at cementoenamel junction level. teeth were divided into 3 groups (n=10): (sp) sodium perborate added with distilled water, (cd) chlorine dioxide and (c) control – dry cotton inserted into the pulp chamber. bleaching agents were used at 0, 7 and 14 days. vita easyshadetm (∆eab) was used to analyze tooth color at the 7th, 14th and 21st days, based on the cie2000 system. data were analyzed through anova and tukey’s test. results: there were no statistically significant differences in δb, δe, δe00 and δwid between cd and the control group. these parameters have shown significant differences between cd and sp, which differed from the control. however, they did not show significant differences either in the control group or in cd at the 7th, 14th and 21st days. values recorded for sp at the 7th day differed from those recorded at the 14th and 21st days. δa has shown differences within the same group at the 7th, 14th and 21st days. there was no difference between groups, when they were compared at the same day (7th and 14th). the control group differed from sp at the 21st day. cd did not differ from the other two groups. δl did not show differences between groups and times. conclusion: stabilized chlorine dioxide (0.07%, at ph 3.5) should not be used as intracoronal bleaching agent along with the walking bleach technique. keywords: tooth bleaching. borates. chlorine compounds. dental materials. https://orcid.org/0000-0001-7043-8858 https://orcid.org/0000-0002-7915-3207 https://orcid.org/0000-0002-1625-3120 https://orcid.org/0000-0002-6296-4185 https://orcid.org/0000-0001-5845-8995 https://orcid.org/0000-0001-6198-7695 2 hirata et al. braz j oral sci. 2022;21:e227878 1. introduction tooth discoloration can result from intrinsic or extrinsic factors. procedures such as polishing tooth surfaces, microabrasion, direct or indirect restoration and dental bleaching techniques have been developed to treat this cosmetic impairment. dental bleaching treatments provide fast and expressive results without wearing the dental structure away1. consequently, they have become remarkably popular2. internal bleaching is the technique most widely used in non-vital teeth due to its quite reasonable cost, safety and pleasing aesthetic results3. root-filled teeth whitening was firstly performed with hydrogen peroxide or sodium perborate, since the late nineteenth century4. spasser5 (1961) has introduced a technique, according to which, sodium perborate and distillated water solution is injected into the pulp chamber for a few days; subsequently, this procedure is repeated until the enamel reaches the desired color. this technique is known as walking bleach. a simultaneous internal and external bleaching technique based on peroxide carbamide was described in the literature in 19976. the adverse effect known as external cervical root resorption can result from certain internal bleaching techniques7. this issue appears to be directly linked to hydrogen peroxide diffusion in the periradicular area. thus, hydrogen peroxide should not be used as treatment agent, either in association with sodium perborate, or not8,9. reports on external bleaching carried out with chlorine dioxide precursors were published, mainly in the uk, in the first decade of the current century. this technique applies sodium chloride, in association with anhydrous citric acid, to whiten tooth surface; its effects result from chlorine dioxide release on tooth surface10. several studies have shown that this technique can harm the dental tissue due to the solution’s low ph; thus, its application should be discouraged10-14. however, chlorine dioxide has oxidative antimicrobial activity and it may remain used as alternative to other internal bleaching agents15. nevertheless, the literature lacks studies focused on investigating its use in a lesser acidic stabilized formula. therefore, the aim of the current study was to assess the effects of stabilized chlorine dioxide used as internal bleaching agent in comparison to those of sodium perborate and the control group. 2. materials and methods sixty (60) permanent bovine mandibular incisors with intact crowns were used in the current study. all organic and inorganic debris were removed from extracted teeth with the aid of scalers; dental elements were stored in chloramine-t solution, at 37°c/ph 7, for one week, for disinfection purposes. teeth were selected and only the ones presenting a2 color, in compliance with the vittapan classical scale, were used. root canals were sectioned right below the amelocemental junction, with the aid of diamond disk attached to a handpiece, whereas crowns were opened with spherical diamond burs (kg sorensen, barueri, sp, brazil, model 1018hl). tooth pulp was removed with the aid of scalers and the first third of the root canal was expanded with spherical diamond burs (kg sorensen model 1016hl). 3 hirata et al. braz j oral sci. 2022;21:e227878 all teeth were stained by using a variation of the technique described by freccia and peters16 (1982). crowns were centrifuged together with fresh bovine blood, at high speed (5,000 rpm), three times a day. the aforementioned blood was replaced on a daily basis; the pigmentation procedure was repeated for 15 days. then, dental elements were rinsed with running water for 2 minutes, and stored in saline solution. thirty (30) stained crowns were selected and covered with 1-mm thick protective base (znpo4, ss white, juiz de fora, brazil), which was fixed 1 mm below the buccal cementoenamel junction. each crown was randomly assigned to one of the three groups. the sp group was treated with sodium perborate (odontofarma, londrina, brazil) and distilled water solution at 2:1 mg/ml. the bleaching agent was applied to the tooth until it filled the buccal surface inside the pulp chamber; then, a provisional restoration (coltosol, coltrane, france) was used to seal the cavity. the bleaching agent was applied at treatment days 0, 7 and 14. specimens were rinsed with distilled water and gently dried with triple syringe, before each bleaching exchange. the cd group was treated with 0.07% chlorine dioxide (from the tescaclor product, which has 5% chlorine dioxide), in association with carbopol, and it generated a product with ph 3.5. the gel was applied to the tooth until it filled the buccal surface inside the pulp cavity; then, a provisional restoration (coltosol, coltrane, france) was used to seal the cavity. the bleaching agent was applied at treatment days 0, 7 and 14. specimens were rinsed with distilled water and gently dried with triple syringe, before each bleaching exchange. dry cotton was inserted into the pulp chamber of teeth in the control group at 0, 7 and 14 days after the procedures described above. all dental elements were stored in gauze (soaked in water), at 37°c and relative humidity of 100%. easyshade intraoral spectrophotometer (vita, zahnfabrik h. rauter gmbh & co. kg, bad sackingen, germany) was used for tooth color assessment under controlled conditions (artificial light and temperature of 16°c). a mold, whose opening had the same diameter as the tip of the spectrophotometer, was prepared with hot glue to ensure that the same point was assessed on each specimen. color change (t1 and t2) was herein obtained through ciede2000 formula, which uses coordinates h (hue) and c (chroma): δe00 = [(δl 0 /klsl)2 + (δc 0 / kcsc)2 + (δh 0 /khsh)2 + rt * (δc 0 /kcsc) * (δh 0 /khsh)]1/2. (23) in addition, the perceptibility threshold was set at δe00 ≤ 0.8, whereas the clinical acceptability threshold was set at δe00 ≤ 1.8.17 data were also obtained through a formula recommended by cielab, according to which, coordinates l (indicating brightness), a and b (red-green and yellow-blue, respectively) interact as follows: δeab = [(δl) 2 + (δa) 2 + (δb) 2] 0.5. color variations δa, δb and δl, as well as the whiteness index δwid, were calculated 18. data were subjected to kolmogorov-smirnov test, which has shown that they were within the normality range; then, they were subjected to analysis of variance (anova) and to tukey’s test, at 5% significance level. 3. results there were no statistically significant differences in color measurement (δb, δe and δe00) and whiteness index (δwid) between cd and the control group. there were sig4 hirata et al. braz j oral sci. 2022;21:e227878 nificant differences in these parameters between the cd and sp groups, which also differed from the control group (p<0.05). moreover, no significant differences were observed either in the control group or in the cd group at the 7th, 14th and 21st days. however, values recorded for the sp group at the 7th day differed from those recorded at the 14th and 21st days. data are shown in table 1. δa did not show differences inside the same group at the 7th, 14th and 21st days (table 1). there was also no difference between groups, when they were compared at the same day (7th and 14th). the control group differed from sp in the analysis conducted at the 21st day. cd did not differ from any of the two other groups. the analysis applied to δl (table 1) did not show differences between groups and times. 4. discussion sodium perborate efficiency, either in association with distilled water or with hydrogen peroxide, has been known for years4,5,19-23. in addition, it is widely used for internal bleaching due to its reasonable cost and reliability. sodium perborate was herein associated with distilled water at 2:1 g/ml, rather than with hydrogen peroxide, which may be linked to external root resorption3,8. however, sodium perborate, either in its mono-, trior tetrahydrate form, releases hydrogen peroxide. h2o2 release can generate different radicals and reactions in the body. besides being related to external root resorption, free radicals can act on lipids, proteins and nucleic acids8. thus, the discovery of a bleaching agent that does not release hydrogen peroxide would be a remarkable breakthrough. chlorine dioxide has been randomly used in beauty salons in the uk10,11. however, precursor elements such as sodium chlorite and citric acid are used to obtain chlorine dioxide, since they cause a chemical reaction that releases it right away10-12. however, table 1. effect of bleaching agents on the color change of materials subjected to bleaching treatment groups period (days) δa* δb* δl* δe δ00 δwid control 7 -0.15±1.14 a 0.72±5.82 a 1.22±3.02 a 5.6±3.4 c 2.87±1.84 c 0.18±8.16 c 14 -0.33±1.43 ab 0.62±5.96 a 1.72±3.20 a 5.6±3.8 c 3.14±1.93 c 0.96±8.14 c 21 -0.30±1.46 ab 1.12±6.37 a 2.32±2.80 a 6.4±3.6 c 3.40±1.77 c 0.65±8.48 c sodium perborate 7 -1.66±1.32 abc -12.34±8.92 b 3.41±2.77 a 14.5±6.3 b 7.98±3.36 b 19.17±10.36 b 14 -2.02±0.77 bc -25.44±2.82 c 4.01±2.33 a 26.1±2.8 a 14.12±1.30 a 34.73±3.90 a 21 -2.22±1.09 c -28.84±3.40 c 6.01±2.99 a 29.7±3.8 a 16.04±1.71 a 39.95±5.37 a chlorine dioxide 7 -0.03±1.55 a -0.09±3.28 a 1.38±4.52 a 5.3±2.3 c 3.4±1.28 c 0.87±8.22 c 14 -0.42±1.31 abc 1.21±3.75 a 2.28±5.11 a 6.2±2.3 c 3.92±1.44 c 0.81±7.45 c 21 -0.56±1.43 abc -0.59±5.17 a 3.28±4.24 a 6.9±2.6 c 4.02±1.41 c 3.63±8.17 c different letters indicate statistically significant difference based on tukey’s test, at 5% significance level. 5 hirata et al. braz j oral sci. 2022;21:e227878 this reaction has severe impact on hard dental tissue, since it leads to increased dentinal sensitivity and enables faster future staining11. studies have shown that topographic changes take place when dental tissues get in contact with solutions at ph 2, or lower10. this factor leads to enamel tissue deterioration, tooth surface roughening, minerals’ erosion and to changes at atomic level, including calcium leaching12. several studies have shown that chlorine dioxide works as bleaching agent in discolored teeth10-13 and composites24. chlorine dioxide attacks stains, be them organic or inorganic, at subatomic level, such as clo2 + e= clo212. however, the chlorine dioxide used in these studies derived from anhydrous citric acid and sodium chloride. because of the acidic nature of this association, apart from its other deleterious effects, the aforementioned studies have emphasized that sodium chloride should be recommended to be used in tooth bleaching procedures. chlorine dioxide ph level was selected due to its proximity to the agents10,12, as well as to its comparability to that of hydrogen peroxide bleaching solutions, which range from 2.113 to 3.720. the adopted chlorine dioxide concentration was based on ablal et al.13 (2013), although the aforementioned authors used an agent produced by mixing sodium chloride with anhydrous citric acid, rather than a stabilized agent. the bleaching observed in their study was associated with teeth dehydration caused by the temperature resulting from light activation; this factor could explain the immediate bleaching effect observed in their study13. thus, prolonged exposures did not improve bleaching effect14. if one takes into consideration the non-difference in luminosity (δl) among all assessed groups, the significant difference in color measurement (δb, δe and δe00) and whiteness index (δwid) recorded for sp, in comparison to the cd and control groups, can be associated with the potential of sp bleaching to remove stains and, consequently, to decrease δa and δb. this factor provided the analyzed specimens with stronger greenish and bluish color, and it has changed the perceptibility threshold. although the perceptibility threshold observed for the control and cd groups was slightly higher than 1.8 (δ00), which clinically indicated discoloration, it happened due to elimination of easily-removed extrinsic pigments from bovine teeth. it is possible saying that immersing the specimens into water, similar to the control group, did not have effective bleaching effect like the one observed for chlorine dioxide. the citric acid and sodium chlorite combination enabled fast chlorine dioxide release, in its gaseous form, which acted almost immediately on nearby surfaces11. however, the chlorine dioxide-based agent was not capable of bleaching teeth discolored with bovine blood in the current study. the negative result observed for the chlorine dioxide-based experiment was likely associated with the use of stabilized chlorine dioxide liquid, rather than with the active destabilized form deriving from the association between citric acid and sodium chloride, which, however, is unsafe for such a use10,11. the use of bovine dental elements in the current study was justified by common human teeth standardization difficulties associated with both dentin thickness and the incidence of reactionary dentin. bovine incisors are similar to human teeth; thus, they have been used as substitutes in assays focused on assess the effectiveness of dental products23. other studies have used cattle incisors in mandibular tooth whiten6 hirata et al. braz j oral sci. 2022;21:e227878 ing tests, which included both external25 and internal bleaching26,27. according to these tests, specimens were pigmented with bovine blood in order to simulate discoloration types often found in clinical practice16. the herein used teeth were standardized in a2 and randomly distributed among the analyzed groups, after staining protocol application. although teeth with slight difference in color were used in the current study, the visually different ones were removed from it. this factor can be considered a limitation of the present study, since spectrophotometry-based analysis was not performed at that time. although shade evaluation could have been performed by calibrated observers, based on the vita scale, in controlled environment20, the use of the spectrophotometer provides lower margin of error, enables more accurate results and presents higher reproducibility28. chlorine dioxide is categorized as primary oxidizing agent. however, its stabilized form was not capable of bleaching bovine incisors in the current study, unlike sodium perborate, which is also a well-known oxidizing product. using chlorine dioxide at other concentrations or ph levels, or adding certain adjuvant agents to it, may help achieving the desired effect. in conclusion, keeping in mind the limitations of the current study, it was possible concluding that stabilized chlorine dioxide (0.07% at ph 3.5) cannot perform as internal bleaching agent. data availability datasets linked to the current article will be available upon request to the corresponding author. disclosure statement the authors declare no conflict of interest. author contribution conceptualization: lopes mb, berger sb. data curation: felizardo kr. formal analysis: lopes mb, berger sb funding acquisition: lopes mb investigation: hirata bs, felizardo kr methodology: hirata bs, guiraldo rd project administration: lopes mb resources: ramos nbp, guiraldo rd. supervision: lopes mb validation: guiraldo rd visualization: berger sb writing original draft: lopes mb, guiraldo rd, ramos nbp, hirata bs writing review &editing: felizardo kr, berger sb, lopes mb all authors actively participated in the discussion of the manuscript’s findings and have revised and approved the final version of the manuscript. 7 hirata et al. braz j oral sci. 2022;21:e227878 references 1. baia jcp, oliveira rp, ribeiro mes, lima rr, loretto sc, silva esjmh. influence of prolonged dental bleaching on the adhesive bond strength to enamel surfaces. int j dent. 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dentistry department, school of dentistry, tehran university of medical sciences, north karegar street, tehran, iran. postal code: 1439955991 tel: + 98-21-88015801 e-mail: kermanshahhamid@yahoo. com received: january 15, 2021 accepted: april 8, 2021 editor: dr altair a. del bel cury comparison of microleakage of an alkasite restorative material, a composite resin and a resin-modified glass ionomer fariba motevasselian1 , hamid kermanshah1* , ebrahim rasoulkhani2, mutlu özcan3 aim: to compare the microleakage of cention n, a subgroup of composite resins with a resin-modified glass ionomer (rmgi) and a composite resin. methods: class v cavities were prepared on the buccal and lingual surfaces of 46 extracted human molars. the teeth were randomly assigned to four groups. group a: tetric n-bond etch-and-rinse adhesive and tetric n-ceram nanohybrid composite resin, group b: cention n without adhesive, group c: cention n with adhesive, and group d: fuji ii lc rmgi. the teeth were thermocycled between 5°-55°c (×10,000). the teeth were coated with two layers of nail vanish except for 1 mm around the restoration margins, and immersed in 2% methylene blue (37°c, 24 h) before buccolingual sectioning to evaluate dye penetration under a stereomicroscope (×20). the data were analyzed by the kruskal-wallis and wilcoxon tests (α=0.05). results: type of material and restoration margin had significant effects on the microleakage (p<0.05). dentin margins showed a higher leakage score in all groups. cention n and rmgi groups showed significant differences at the enamel margin (p=0.025, p=0.011), and for the latter group the scores were higher. no significant difference was found at the dentin margins between the materials except between cention n with adhesive and rmgi (p=0.031). conclusion: microleakage was evident in all three restorative materials. cention n groups showed similar microleakage scores to the composite resin and displayed lower microleakage scores compared with rmgi. keywords: cention n, composite resins. dental leakage. glass ionomer cements. https://orcid.org/0000-0002-3856-5096 https://orcid.org/0000-0002-4208-3276 https://orcid.org/0000-0002-9623-6098 2 motevasselian et al. introduction class v cervical carious lesions remain a major oral health problem in the elderly and those at high risk of caries1. a wide variety of restorative materials have been suggested for restoration of these lesions; among which, composite resins and resin-modified glass ionomer (rmgi) cements are most commonly used1. rmgi has been recommended for restoration of class v lesions because it has the combined benefits of chemical adhesion to the tooth substrate, fluoride release potential, and caries-preventive effect2. achieving adequate marginal integrity at the dentin margins of class v restorations extending beyond the cementoenamel junction (cej) remains a challenge1,3. secondary carious lesions develop in absence of adequate marginal seal of restorations3. several factors can affect the marginal adaptation of adhesive restorative materials such as polymerization shrinkage and contraction stress4,5, and the difference in the linear coefficient of thermal expansion (lcte) of restorative material and that of tooth structure6. all these factors can lead to gap formation and marginal microleakage4-6. during the past two decades, researchers have extensively focused on developing dental restorative materials with improved physical and bioactive properties to minimize interfacial gap and secondary caries. recently, a bulk-fill resin-based powder-liquid composite containing alkaline fillers (alkasite) was introduced to the market by ivoclar vivadent (schaan, liechtenstein). it is a bioactive restorative material, and the manufacturer claims that it has low polymerization shrinkage. also, the manufacturer claims that it releases large amounts of fluoride and calcium ions at low ph and deposits minerals in the form of calcium phosphate and calcium fluoride layers. furthermore, the hydroxide ions released from cention n have been claimed to have a protective buffering capacity to neutralize cariogenic acids. this material can be used with or without an adhesive7-10. preclinical screenings and in vitro studies simulating oral conditions are useful for estimation and predication of the performance of restorative materials. various in vitro methods are used for evaluation of marginal quality of restorations such as penetration test in class ii and v cavities and assessment of marginal interface under a light microscope or a scanning electron microscope. dye penetration test is still the most commonly used method for evaluation of the sealability of restorative materials. various types of dyes can be used for this purpose. no specific dye tracer has been recommended by the iso standard for this test. the most commonly used dyes for this test include basic fuchsine, methylene blue, and silver nitrate. thermocycling (tc) and/or mechanical loading have been recommended for microleakage tests to better simulate the clinical conditions11. the available studies about the microleakage mostly compared the conventional gics rather than the rmgis with cention n12-14. cention n is a resin-based (udma-based) material containing fillers. rmgis contain resin and their composition is more similar to alkasite restorative materials than conventional gics7,9,10. in addition, the frequency of thermal cycles in previous studies on this topic was 500 or less while 500 cycles are the minimum cycles recommended by iso 1140515. 3 motevasselian et al. the aim of this study was to evaluate the effect of three restorative materials namely an alkasite restorative material (cention n®; ivoclar vivadent, schaan, liechtenstein) with and without an adhesive (tetric® n-bond vivadent, schaan, liechtenstein), a nanohybrid composite resin (tetric-n® ceram, ivoclar vivadent, schaan, liechtenstein), and a rmgi (gc fuji ii lc®, gc corporation., tokyo, japan) on the marginal integrity of class v restorations submitted to 10,000 thermal cycles using the dye penetration test. the null hypotheses were that the location of restoration margin (in the enamel or dentin) or type of restorative material would have no significant effect on the marginal microleakage of restorations. materials and methods the commercial materials used in this study and their composition are presented in table 1. the materials were used according to the manufacturers’ instructions. the study was approved by the ethics committee in research of school of dentistry of tehran university of medical sciences (ir.tums.dentistry. rec.1398.055). specimen preparation forty-six sound, non-carious, unrestored human third molars, extracted for periodontal reasons or as part of orthodontic treatment, were collected after informed consent was obtained from the patients. the minimum sample size for each study group was calculated to be 23, based on a previous study12 contable 1. the materials used and their classification, manufacturer, and composition6,8,29,30 material (manufacturer) liquid powder batch number cention® n (ivoclar vivadent, schaan, liechtenstein) udma, dcp, aromatic aliphatic-udma peg-400 dma ca-f-silicate glass, ba-al silicate glass, ca-ba-al fluorosilicate glass, ytf3, isofiller (78.4 wt% ) w07418 gc fuji ii lc® (gc corp., tokyo, japan) polyacrylic acid, hema, 2,2,4 tmhedc, tegdma fluoro-alumino-silicate glass 1704011 tetric® n-ceram (ivoclar vivadent, schaan, liechtenstein) bis-gma, bis-ema udma ba glass; ybf3; mixed oxide; prepolymer (80%wt%) w84901 tetric® n-bond (ivoclar vivadent, schaan, liechtenstein) bis-gma, udma, hema, phosphonic acid acrylate, ethanol, nanofiller, catalysts and stabilizer, nanofiller w83533 udma: urethane dimethacrylate; dcp: tricyclodecan-dimethanol dimethacrtylate; an aromatic aliphatic-udma: tetramethyl-xylylen diurethane dimethacrylate; peg-400 dma: polyethylene glycol 400 dimethacrylate; ca-f-silicate glass: calcium fluorosilicate glass; ba-al silicate glass: barium aluminum silicate glass; ca-ba-al-f glass: calcium barium aluminum fluorosilicate glass; ytf3: ytterbium trifluoride; isofiller: copolymer, hema: hydroxyethyl methacrylate; 2,2,4 tmhedc: trimethyl hexamethylene dicarbonate; tegdma: triethylene glycol dimethacrylate; bis-gma: bisphenol a diglycidylether methacrylate; bis-ema: ethoxylated bisphenol-a dimethacrylate; ba glass: barium glass. 4 motevasselian et al. sidering α=0.05, β= 0.2 and pooled standard deviation of 2.1 using spss 11 (spss  inc., chicago, il). the teeth were debrided of residual plaque, calculus and residual soft tissue, and stored in a solution of distilled water and 0.5% chloramine t at 4°c until usage. all teeth had been extracted within the past 3 months. the teeth were visually inspected under a stereomicroscopic at ×10 magnification (leica, leica ez4d, mel sobel microscopes, italy), and sound teeth without fracture lines and cracks were selected for this study. class v cavities (with 3 mm occlusogingival width, 3 mm mesiodistal width, and 1.5 mm depth) were prepared in the buccal and lingual surfaces of each tooth using a cylindrical diamond bur (≠ 838-012-fg; hager & meisinger gmbh, neuss, germany) and a high-speed handpiece under copious water irrigation. the occlusal margin of the cavities was located 2 mm coronal to the cej, while the gingival margin was located 1 mm apical to the cej. the diamond bur was replaced after five preparations. non-retentive cavities with divergent walls were prepared as such. all the internal line-angles were rounded. the dimensions of all cavities were measured using a periodontal probe. restorative procedure before restoration, the prepared cavities were gently cleaned with a slurry of pumice paste and water using a prophy cup, and thoroughly rinsed with tap water. the prepared teeth were randomly divided into four groups (n=23) according to the type of material used, as follows: group a: (composite resin): 37% phosphoric acid gel (ivoclar vivadent, schaan, liechtenstein) was applied on the enamel and subsequently on the dentin margins for 15 s. afterwards, the etchant was thoroughly rinsed off with water spray for 15 s, and the excess water was removed with a small cotton pellet to avoid excessive drying. tetric-n bond nanofilled single-component adhesive (ivoclar vivadent, schaan, liechtenstein) was applied in one thick layer and rubbed on the enamel and dentin surfaces with a micro-applicator brush for 10 s. excess adhesive in the line angles and the solvent were removed by gentle air stream for 10 s. the adhesive was light-cured for 10 s using a light-emitting diode (led) curing unit with a light intensity of 1200 mw/cm2 (bluephase; ivoclar vivadent, schaan, liechtenstein). light output was measured using a radiometer (bluephase meter ii, ivoclar vivadent ag, schaan, lichtenstein). a2 shade of tetric-n ceram nanohybrid composite resin (ivoclar vivadent, schaan, liechtenstein) was used to restore the cavities in two layers with oblique incremental application technique. the first oblique layer was applied and extended from the gingival floor to the axial wall. the second increment was applied to fill the remainder of the cavity. each layer was polymerized with led curing unit (bluephase, ivoclar vivadent, schaan, liechtenstein) for 20 s. group b: (cention n without adhesive) this product is only available in a2 shade. the prepared cavities were gently dried with air stream. one spoon of powder and one drop of liquid were dispended on a 5 motevasselian et al. mixing pad according to the manufacturer’s instructions. the powder was gradually added to the liquid and thoroughly mixed for 60 s until a homogenous mass with a slight shine was obtained to wet the tooth substrate. the restorative material was immediately applied and condensed in the cavity with a spatula in one increment. excess material was carefully removed, and the restoration was cured for 20 s using a led curing unit. group c: (cention n with adhesive) the same steps were followed for adhesive application in this group as in group a. cention n was then mixed and delivered into the cavity with the same sequence as in group b. group d: (rmgi) a2 vita shade of rmgi was chosen. the cavities were conditioned with 10% polyacrylic acid (dentin conditioner, gc corporation, tokyo, japan) applied with a micro-applicator brush for 20 s, and were then thoroughly rinsed with water spray for 20 s and blot-dried with cotton pellets to avoid desiccation. one level scoop of powder and two drops of liquid were placed on a mixing pad according to the manufacturer’s instructions. the powder was divided by half and mixed with the liquid within 25 s until a homogenous mass was achieved and applied and packed in bulk into the cavities with a spatula as long as the surface of the  mixed  cement was shiny. afterwards, it was polymerized for 20 s using a led curing unit. all restored cavities were stored in distilled water at 37°c for 24 h, and were then finished and polished with graded series of sof-lex discs (3m espe, dental products st paul, mn, usa). the same operator prepared all the specimens. microleakage test the specimens were thermocycled for 10,000 cycles between 5˚ c and 55˚c with a dwell time of 30 s and a transfer time of 10 s. following tc, the teeth were dried, and the root apex of each tooth was sealed with sticky wax. the entire tooth surface including the crown and root structures were covered with two layers of nail varnish, except for a 1 mm band around the restoration margins. all the specimens were then immersed in freshly prepared 2% methylene blue solution for 24 h at 37°c. the teeth were then rinsed with running water. afterwards, the specimens were mounted in auto-polymerizing acrylic resin (acropars, marlic medical co., tehran, iran) and longitudinally sectioned in half at the center of the restoration in buccolingual direction with a low-speed diamond saw under water coolant. the sectioned teeth were evaluated under a stereomicroscope (leica, leica ez4d, mel sobel microscopes, italy) at ×20 magnification. the extent of dye penetration at the restorative material-tooth interface was scored from zero to three along the occlusal and cervical walls13(figure. 1). the dye penetration scores were determined by one single operator who was blinded to the type of restorative material used for each group. 6 motevasselian et al. score 0: no dye penetration score 1: dye penetration extending to 1/3 of the occlusal or cervical wall score 2: dye penetration extending to two-thirds of the occlusal or cervical wall score 3: dye penetration extending to the axial wall and beyond statistical analysis the statistical analysis of microleakage data was performed using spss version 25 (spss inc., chicago, il, usa). the kruskal-wallis test was applied for multiple comparisons followed by the dunn test. the occlusal and gingival microleakage scores were compared using the wilcoxon signed rank test. level of significance was set at 0.05 for the main analysis, and dunn adjusted p-values were used for multiple comparisons. results table 2 presents the microleakage scores (number and percentage) at the enamel and dentin margins of the four groups. the microleakage scores at the enamel margin were significantly lower than the corresponding values at the dentin margins in all groups (p<0.001). figure 1. schematic view of the cavity prepared in the buccal and lingual walls of a molar tooth. the maximum degree of dye penetration was recorded according to the followiing scoring system :0, no dye penetration; 1, dye penetration to 1/3 of the cavity wall; 2, dye penetration up to 2/3 of the cavity wall, 3, dye penetration extending to the axial wall and beyond. cej: cementoenamel junction; dej: dentinoenamel junction gingival wall gingival wall enamel dentin occlusal wall occlusal wall occlusal wall pulpal tissue p ulpal tissue cej dej 0 1 2 3 0 1 2 3 gingival wall cej 7 motevasselian et al. the kruskal-wallis test indicated significant differences between the restorative materials for the occlusal and gingival margin microleakage scores (p=0.041 for the enamel margins, and p=0.020 for dentin margins). table 2 shows pairwise comparisons of the differences in the microleakage scores of the study groups at 0.05 level of significance. the only significant differences at the enamel margins were found between cention n with/without adhesive and rmgi groups (p=0.011 and p= 0.025, respectively). however, no significant difference was found between the other groups (p≥0.121). there was no statistically  significant difference in microleakage between the groups at dentin margins (p≥0.076), except for cention n with adhesive and rmgi (p=0.031). discussion the purpose of this in vitro study was to compare the microleakage of different types of restorative materials for restoration of class v cavities in the cervical region of the teeth using the dye penetration test. furthermore, microleakage scores of cention n cavities restored with or without adhesive were compared. the recommended adhesives by the manufacturer of cention n product are either universal bonding agents such as tetric n-bond universal or etch and rinse adhesive systems such as tetric n-bond8. in the current study the latter surface treatment was selected to compare the microleakage scores since the phosphoric acid agent removes the smear layer13. based on the results of the present study, the score of microleakage was greater at the dentin margin than the enamel margin in all groups regardless of the type of restorative material used. thus, the first null hypothesis of the study was rejected. with regard to the type of restorative material, the results of the present study showed significant differences between cention n and rmgi after immersion in 2% methylene blue for 24 h. therefore, the second null hypothesis regarding insignificant effect of type of restorative material on microleakage score was also rejected. the tooth samples were exposed to 10,000 thermal cycles corresponding to 1-year of clinical service in the oral cavity, as claimed by gale and darvell. tc simulates the thermal alterations in the oral cavity that lead to stress build-up at the interface and table 2. microleakage (number and percentage) of the study groups at the enamel and dentin margins and pairwise comparison material enamel number/percentage dentin number/percentage 0 1 2 3 0 1 2 3 a tetric n-ceram 14(61%) 8(34%) 1(4%) 0 ab 1(4.34%) 3(13%) 2(8.6%) 17(73.9%) cd b cention n without adhesive 18(78%) 5(22%) 0 0 b 1(4.34%) 1(4.34%) 4(17.39%) 17(73.9%) cd c cention n with adhesive 19(83%) 4(17%) 0 0 b 3(13%) 3(13%) 8(34.8%) 9(39%) c d rmgi* 12(25%) 6(26%) 3(13%) 2(8.6%) a 0 2(8.6%) 3(13%) 18(78%) d p value=0.041 p=0.020 similar letters show that the distribution of the leakage scores are not significantly different. *rmgi: resin-modified glass ionomer 8 motevasselian et al. can adversely affect marginal integrity of the restoration, causing microleakage. tc was performed between 5-55 ˚c according to iso1140514,15. the results of the present study regarding lower microleakage at the enamel margins were similar to the findings of a previous study16. bonding of adhesive restorative materials to dental substrate depends on either micromechanical interlocking and hybridization due to the penetration of bonding resin into the microscopic porosities on the surface of enamel and dentin, or chemical interactions with the inorganic content of dental substrate. both mechanisms depend on the amount of surface free energy of dental substrate, which is directly proportional to the mineral content of the tooth structure and inversely correlated with the percentage of organic content. enamel has a more homogenous structure than dentin due to higher mineral content and lower water and organic content. therefore, one primary requisite for better marginal seal is already provided17,18. the comparison between rmgi and cention n showed that fuji ii lc group revealed significantly higher leakage scores at the enamel margins compared with cention n groups with/without adhesive. dentin margins in class v cavities restored with fuji ii lc showed significantly higher leakage scores than cention n group with adhesive. there are several factors that may explain these findings such as viscosity, polymerization rate, monomer conversion and linear coefficient of thermal expansion (lcte). there is an inverse relationship between viscosity of a resin-based material and its rate of polymerization19. it has been demonstrated that powder/liquid (p/l) ratio of a resin-based cement affects its viscosity. higher p/l ratio leads to higher viscosity20. viscosity also depends on the filler loading, size (and hence the surface area), and shape of fillers as well as the heterogeneity of particle sizes and monomer types in the mixture. it was demonstrated that decreasing the filler size increased the viscosity of experimental composites19. the recommended p/l ratio for cention n is 4.6:1, which is higher than that of fuji ii lc (3.2:1)20. the average particle size of cention n is between 0.1 μm and 35 μm8, a wide size distribution; while, that of fuji ii lc is 5.9 μm21. considering the abovementioned explanation, cention n appears to have higher viscosity. high viscosity decreases the mobility of free radicals, leading to a reduction in polymerization rate, which has a great impact on shrinkage stress relief and interfacial gap reduction4,5,19. therefore, the authors assume that lower microleakage scores in cention n groups compared with the rmgi group might be explained by its probably lower rate of polymerization. furthermore, it has been stated that lower particle content can cause higher volumetric shrinkage4. therefore, it might be assumed that fuji ii lc with lower p/l ratio can undergo higher volumetric shrinkage and contraction stress, that can cause interfacial debonding and higher leakage scores. also, a direct correlation exists between the degree of polymerization and volumetric shrinkage4,5. it also has been demonstrated that monomers with lower molecular weight and viscosity and higher mobility have higher degree of monomer conversion22. udma and hema are the base monomers of cention n and fuji ii lc, respectively6,8,23. udma has higher molecular weight and viscosity than hema22. as a result, higher degree of conversion is expected in fuji ii lc. panpisut and toneluck found the same results20. this is another factor which might explain higher leakage scores in fuji ii lc group. 9 motevasselian et al. lcte is another parameter influencing the volumetric change of restorative materials in the oral cavity. if the difference between the lcte of the tooth substrate and that of a restorative material is high, marginal seal at the tooth substrate-restoration interface might be breached6. pinto-sinai et al. explained that lcte of resin-based restorative materials is influenced by the amount of filler. rmgi cements do not contain fillers6. cention n has 78.4% filler content by weight in the final mass8,23, and a higher p/l ratio than fiji ii lc20, which might decrease its lcte. it has been shown that fuji ii lc has a high lcte during heating and cooling cycles (15ºc-50ºc) i.e. 25.4 and 30 ppm, respectively. the lcte of dentin and enamel is 11 and 17 ppm, respectively in this temperature range6. the lcet of molar teeth in the cervical region has been reported to be around 5 ppm24. in the current study, the difference in expansion and contraction at the tooth and rmgi interface might have caused marginal deterioration and higher leakage score in the rmgi group, compared with cn groups. rmgi and composite resin displayed no difference in leakage score in the current study. the bonding of composite resin to tooth structure is mediated by micromechanical adhesion following etching of dental substrate and penetration of bonding resin17. the bond strength of composite resin to tooth structure is higher than that of rmgi18. an interfacial gap is expected if the adhesion of the restorative material to the tooth structure does not compensate for the shrinkage stress induced during setting and polymerization4,5. similar leakage scores of these two materials can be explained by several properties of the materials such as their modulus of elasticity, hygroscopic expansion, and the application technique of the material. rmgi has lower modulus of elasticity than composite resin25, which can relieve the induced polymerization shrinkage stress. a material with low modulus of elasticity has higher capacity for plastic flow and stress relaxation during polymerization4,5. studies have shown that both composite resins and rmgi cements absorb water26,27. in the humid oral environment, polymerization shrinkage may be partly relieved by hygroscopic expansion following water sorption. therefore, marginal gaps caused by polymerization shrinkage can be decreased by hygroscopic expansion26,27. water uptake by a restorative material is a diffusion-controlled process through the resin matrix. the diffusion coefficient of water sorption is controlled by hydrophilicity/hydrophobicity of the resin matrix and filler level/resin content ratio27,28. panpisut and toneluck showed that the studied nanofilled composite resin had lower water sorption than fuji ii lc, and they attributed this finding to the hydrophilic nature of resin matrix (hema) and polyacrylic salt network in the studied rmgi cement20. hema accounts for nearly 25%-50% of the liquid content of fuji ii lc6; while, more hydrophobic and rigid monomers such as bis-gma and udma comprise the polymer network of tetric n-ceram29. the filler content of the composite is 80-81% by weight29; while, fuji ii lc has no filler content6. furthermore, there is a relationship between the cement maturity and water balance. acid-base reactions in light-cure rmgi are slower than photo-initiated polymerization25. thus, a longer time for water uptake and maturation is required, and the maturation occurs over a prolonged period of time. however, lower post-curing is expected to occur in composite resin, and the majority of monomer to polymer conversion reactions possibly occur upon the initial light irradiation. this means that composite resin is close to maturity. all these factors probably cause 10 motevasselian et al. higher water sorption in rmgi than resin composite25. several studies found that water exposure of rmgi cements and consequent hygroscopic expansion were beneficial for reversing tensile or pulling stresses into compressive stress to minimize gaps in teeth restored with resin-based materials26,27. furthermore, the composite resin was placed in two oblique increments to avoid contact with the opposing occlusal and gingival margins at the same time. the rational was that this technique decreases the overall polymerization shrinkage and consequently the polymerization stress4,5. incremental application is also helpful to reduce the probability of bond failure along the gingival margins located in dentin. dentin provides a weaker bond compared with enamel17, and polymerization shrinkage in one increment may cause debonding at the weaker interface. composite resin and cention n groups with/without adhesive also showed comparable leakage scores. this finding could be attributed to degree of conversion (dc), polymerization shrinkage and contraction stress of these materials. degree of polymerization and polymerization stress are related to interfacial gap formation4,5. dc is influenced by the filler/resin ratio and resin content. the former factor for the composite resin and cention n is relatively the same (table 1). however, they do not contain the same resin content (table 1). tetric n-ceram is a bis-gma-based composite resin29; while, cention n is a udma-based polymer7,8. bis-gma monomer has higher molecular weight and viscosity but lower dc than udma monomer22. panpisut and toneluck20 found a higher monomer conversion in cention n compared with a bis-gma nanofilled composite resin. therefore, cention n is expected to have higher microleakage score. however, there are other factors that might compensate for the higher dc, and consequent polymerization shrinkage and contraction stress. ilie23 showed that cention n polymerization initiates instantly following light irradiation in 2 mm material thickness and reaches a plateau in 1 h; whereas, composite resin polymerization continues for more than 24 h. this polymerization behavior might explain the similar leakage scores in these two groups. it was interesting that cavities restored by cention n with or without adhesive did not reveal significant differences in leakage scores. it shows that marginal adaptation of cavities restored with this material is unrelated to micromechanical retention provided by the adhesive resin. according to the manufacturer and several authors, it is a self-adhering bulk fill restorative material that obviates the need for a separate adhesive7-9. the improved adaptation of the material to tooth margins and smear layer might be related to its hydrophilic character and its ability to wet the tooth surface which is attributed to its resin composition containing a hydrophilic dimethacrylate (polyethylene glycol dimethacrylate)8,10 (table 1). in summary, cention n with and without adhesive showed comparable leakage scores to the composite resin, and the values were lower than those in fuji ii lc. tetric n-ceram nanohybrid composite resin, which was incrementally applied on the tooth substrate showed similar leakage scores in comparison with cention n groups and rmgi. comparison of the abovementioned commercial materials is not simple. there are many factors related to different formulations of resin polymers that complicate a precise comparison such as the initiator type and concentration, and filler dispersion 11 motevasselian et al. that can affect the physicochemical properties. in addition, thermomechanical loading can better simulate the harsh oral environment and its adverse effect on the longevity of restorations. therefore, future studies are required on other properties of materials such as their dc, polymerization shrinkage, shrinkage stress, volumetric shrinkage, elastic modulus, and water sorption after thermomechanical cycling of specimens to provide more detailed information and explain the variations in data. conclusions in vitro microleakage of cention n was comparable to tetric n-ceram at enamel and dentin margins. however, cention n showed significantly lower leakage scores than fuji ii lc. references: 1. wierichs r, kramer e, meyer-lückel h. risk factors for failure of class v restorations of carious cervical lesions in general dental practices. j dent. 2018;77:87-92. 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evaluation of marginal adaptation and microleakage of three glass ionomer-based class v restorations: in vitro study. eur j dent. 2019;13(4):599-606. doi: 10.1055/s-0039-3401435. 17. sofan e, sofan a, palaia g, tenore g, romeo u, migliau g. classification review of dental adhesive systems: from the iv generation to the universal type. ann stomatol. (roma)2017; 3;8(1):1-17. doi: 10.11138/ads/2017.8.1.001. 18. nicholson jw. adhesion of glass-ionomer cements to teeth: a review. int j adhes adhes. 2016;69:33-8. doi: 10.1016/j.ijadhadh.2016.03.012. 19. habib e, wang r, zhu x. correlation of resin viscosity and monomer conversion to filler particle size in dental composites. dent mater. 2018;34(10):1501-8. doi: 10.1016/j.dental.2018.06.008. 20. panpisut p, toneluck a. monomer conversion, dimensional stability, biaxial flexural strength, and fluoride release of resin-based restorative material containing alkaline fillers. dent mater j. 2020; 39(4):608-15. doi: 10.4012/dmj.2019-020. 21. bala o, arisu hd, yikilgan i, arslan s, gullu a. evaluation of surface roughness and hardness of different glass ionomer cements. eur j dent. 2012;6(1):79-86. 22. pratap b, gupta rk, bhardwaj b, nag m. resin based restorative dental materials: characteristics and future perspectives. jpn den sci rev. 2019;55(1):126-38. doi: 10.1016/j.jdsr.2019.09.004. 23. 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https://doi.org/10.1016/j.jdsr.2019.09.004 https://doi.org/10.1016/j.jdent.2015.10.001 https://doi.org/10.1016/j.dental.2013.05.001 1http://dx.doi.org/10.20396/bjos.v20i00.8663641 volume 20 2021 e213641 original article 1 professor, department of oral rehabilitation, school of dentistry, university of cartagena, cartagena de indias, colombia. 2 undergraduate student, school of dentistry, university of cartagena, cartagena de indias, colombia. * corresponding author at: department of oral rehabilitation, school of dentistry, university of cartagena, cartagena de indias, colombia. phone: (+57) 6698172, ext: 118. e-mail address: cmadridt@unicartagena.edu.co. received: december 15, 2020 accepted: march 9, 2021 editor: dr. altair a. del bel cury bond strength of selfadhesive flowable resin composites to tooth structure: a systematic review cristhian camilo madrid troconis1,* , samantha molina pérez2 aim: to review the current literature regarding the bond strength of self-adhesive flowable resin composites (safrcs) to tooth structure, comparing the outcomes with conventional flowable resin composites (cfrcs). methods: pubmed/ medline, ebscohost and scopus databases were screened (last update on november 2020) using related medical subject headings (mesh) and free terms. we included in vitro studies published in english language assessing the bond strength of safrcs and cfrcs to enamel and/or dentin from primary and/or permanent teeth. results: in total, 23 articles were included. unlike cfrcs, safrcs such as vertise® flow and fusio™ liquid dentin exhibited statistically lower bond strength to enamel and dentin from permanent teeth. there  were limited studies comparing the enamel bond strength of cfrcs and safrcs (prior phosphoric acid etching and/or adhesive system use). also, we found few studies that evaluated the bonding effectiveness of constic® and other safrcs to primary teeth. conclusions: current safrcs showed low bond strength to permanent teeth, which impedes to recommend them as a reliable alternative to cfrcs. the bonding performance of constic® on both hard dental tissues should be evaluated on future studies. also, more evidence assessing the bond strength of safrcs to primary teeth and etched enamel is needed. keywords: composite resins. dental bonding. systematic review as topic. mailto:cmadridt@unicartagena.edu.co https://orcid.org/0000-0003-4058-1447 https://orcid.org/0000-0002-6776-7391 2 troconis et al. introduction the simplification of dental techniques represents one of the main goals and tendencies in current restorative dentistry. interestingly, clinical studies have shown that dental restorations performed with simplified dental materials such as universal adhesive systems, self-adhesive resin cements and bulk-fill resin composites have an acceptable performance1-4. recently, another simplified dental materials known as self-adhesive flowable resin composites (safrcs) were introduced into the market. safrcs are indicated for pit and fissure sealants, base/ liner and restorative material in small cavities5-7, the same clinical indications than conventional flowable resin composites (cfrcs). according to manufacturer’s instructions, safrcs could be used without previous phosphoric acid etching and adhesive systems, especially for dentin bonding procedures5-7. this was possible by acidic functional monomers such as glycerol phosphate dimethacrylate (gpdm), 10-methacryloyloxi-decyl-dihydrogen-phosphate (10-mdp) and 4-methacryloxyethyl trimellitic acid (4-meta) incorporated into vertise® flow, constic® and fusio™ liquid dentin, respectively. these functional monomers establish a chemical interaction with inorganic phase of hard dental tissues which theoretically would guarantee acceptable bond strength. in some cases, only previous phosphoric acid etching on uncut enamel surface is recommended to increase the bond strength of safrcs5, but findings from some in vitro studies using this approach are controversial8-9. safrcs could represent a good alternative to perform dental restorative/preventive procedures because they would reduce clinical time, operative errors and post-operative sensitivity5-7. nonetheless, the number of clinical trials assessing the performance of safrcs restorations or pit and fissure sealants are extremely limited and controversial10-12 to contraindicate or recommend these novel dental materials. however, there are fairly available in vitro studies which evaluate microleakage, nanoleakage, solubility, water sorption and bond strength of safrcs13-16. this latter is one of the most important and critical features on self-adhesive materials due to it reflects the physico-chemical interaction with hard dental tissues, which could partially predict common clinical problems such as microleakage and retention loss. until now, no consensus on the bonding effectiveness of safrcs has been established to determine if these novel dental materials could be used as a reliable alternative to conventional flowable resin composites (cfrcs). therefore, a compilation of in vitro studies on this issue is urgently needed to indicate whether current safrcs should be used on future research or more technological developments are required. the  aim of this study was to review the current literature regarding the bond strength of self-adhesive flowable resin composites to tooth structure, comparing the results with conventional flowable resin composites. materials and methods the present systematic review was conducted following all parameters described in prisma guidelines (preferred reported items for systematic reviews and meta-anal3 troconis et al. ysis)17. the research question was: do safrcs exhibit comparable enamel and dentin bond strength to cfrcs? selection criteria we included studies that used human enamel and/or dentin from primary and/or permanent teeth, independently if dental substrates were cut, grounded and/or laser ablated (patient). the studies had to evaluate safrcs (intervention) such as vertise® flow, fusio™ liquid dentin and/or constic® used with or without previous phosphoric acid etching and/or adhesive system. also, cfrcs (control/comparison) used as pit and fissure sealant and/or restorative material bonded by etch-and-rinse adhesive systems (eras), self-etch adhesive systems (seas) or universal adhesive systems (uas). all included studies had to compare the bond strength between safrcs and cfrcs to enamel and/or dentin (outcome). reports not published in english language, literature reviews, clinical studies, case reports/case series, book chapters, congress abstracts, editor letters and studies which exclusively evaluated the bond strength of experimental safrcs were excluded from the analysis of the current systematic review. search strategy and study selection different systematic searches were conducted by two trained and independent reviewers (c.m.t and s.m.p) until november 2020. we screened pubmed/medline, ebscohost and scopus, using search strategies as follows; pubmed/medline, ((((((((self-adhesive flowable composite resin) or (self adhesive flowable resin composites)) or (self-adhering flowable resin composite)) or (self-adhering flowable composite resin) or (vertise flow)) or (fusio liquid dentin)) or (constic)) and (bond strength); ebscohost, self-adhesive flowable composite resins or self-adhesive flowable resin composite or self-adhering flowable composite resin or self-adhering flowable resin composite or vertise flow or fusio liquid dentin or constic and bond strength; scopus, self-adhesive and flowable and resin and composite or self-adhering and resin and composite or self-adhesive and composite and resin or self-adhering and flowable and composite and resin or vertise and flow or fusio and liquid and dentin or constic and bond and strength. article titles were exported to microsoft excel® 2016 (microsoft corporation, redmond, washington, usa) to eliminate repeated hits in the same database and between them. later, remaining titles and abstracts were screened in detail by two reviewers (c.m.t and s.m.p), excluding those that seem not to meet inclusion criteria. when abstracts presented limited information to be classified or seemed to meet all inclusion criteria, articles were downloaded for full-text reading. the titles were codified into 6 categories according to selection criteria, as follows: c1 (articles not published in english language), c2 (clinical studies/case reports/ case series), c3 (articles which did not compare the bond strength of safrcs with cfrcs), c4 (studies that exclusively evaluated the bond strength of experimental safrcs), c5 (others types of papers such as literature reviews, book chapters, congress abstracts and editor letters) and c6 (included studies). finally, reference lists from selected studies were screened in detail to find possible articles which could meet inclusion  criteria. 4 troconis et al. data extraction data extraction was performed by two trained reviewers (c.m.t and s.m.p), using a standardized form containing information such as first author name, publication year, sample size (n), type of teeth, tested materials, type of materials, dental substrate (enamel, dentin or both), bonding test, aging technique, sample dimensions/load speed, failure mode analysis and predominant failure mode in safrcs. if relevant methodological information was missed from a study, we contacted the correspondence author via e-mail. if no answer was received after 2 weeks, we sent other mail, requesting the same methodological information. finally, if no response was obtained four weeks following the first attempt, the article was included in the systematic review with not reported data (nr). data analysis after methodological data extraction, meta-analysis was considered inappropriate due to great methodological divergences among included studies, especially in terms of bonding tests, load speed, adhesive systems and cfrcs. nevertheless, means and standard deviations of bond strength values of safrcs and cfrcs groups from individual studies, were extracted and tabulated, indicating statistically significant differences (p≤0.05) among groups. risk of bias assessment risk of bias assessment was conducted in duplicate by two trained reviewers (c.m.t and s.m.p) and both analyses were later contrasted to find possible inconsistencies. to assess evidence quality, we employed an adapted instrument previously used in other systematic reviews about dental adhesion18-19. this instrument contains the following domains or items: randomization, sample size calculation, teeth free of caries, sample with similar dimensions, failure mode evaluation, manufacturer instructions, single operator and operator blinded. each item was checked in individual studies, judging as “yes” when reported in the methodology, but if not, the specific domain received “no”. the number of positive responses obtained in each included study were counted to determine the overall risk of bias, as follows: high risk of bias (yes:1 to 3), medium risk (yes: 4 or 5) and low risk of bias (yes: 6 to 8). results search and selection figure 1 summarizes the selection process, according to prisma guidelines. overall, electronic searches on three databases yielded 196 articles. after excluding repeated hits, screening and full-text reading, 20 articles remained. complementary searches resulted in 3 new papers that met inclusion criteria. finally, 23 articles were included in the qualitative analysis of the current systematic review. 5 troconis et al. id en tifi ca tio n p u b m ed (n =8 7) eb s c o h o s t (n =8 5) s c o p u s (n =2 4) s el ec tio n el ig ib ili ty in cl ud ed articles identified through database search (n=196) articles removed by duplicate (n=15) articles reviewed (n=181) full-text articles evaluated for eligibility (n=20) full-text articles included in qualitative analysis (n=23) articles identified through another strategy (n=3) excluded articles (n=161) reports not published in english literature reviews, clinical cases, book chapters articles that did not compare the bonding performance of safrcs with cfrcs bond strength of experimental safrcs others types of papers figure 1. selection process, according to prisma guidelines. study characteristics table 1 presents the main methodological aspects from included studies. in total, 23 in vitro studies met inclusion criteria (published between 2012 and 2019)8,9,16,20-39 and most of them (n=18) used permanent teeth (ranging from 30 to 160) for the bonding tests. the bond strength of safrcs to primary teeth was evaluated in six studies, published between 2013 and 201916,22,26,30,34,38. vertise® flow (kerr corp, orange, ca, usa) (n=22) followed by fusio™ liquid dentin (pentron clinical, orange, ca, usa) (n=5) were the most tested safrcs, while constic® was evaluated only in three studies29,35,37. the bond strength of safrcs was mainly assessed by shear bonding test (n=20)8,16,2027,29-33,35-39 and tensile bonding test (n=3)9,28,34, using dentin (n=13), enamel (n=3) or both tissues (n=7) as substrates. most studies tested the immediate bond strength of safrcs to enamel and dentin. only six studies employed thermocycling as an aging method and the number of cycles varied from 500 to 5000 (temperature from 5ºc to 55ºc)26,29,33,35,36,38. failure mode analysis was evaluated in 20 of 23 studies, using stereomicroscope/optical microscope, digital microscope and/or scanning electron microscopy (sem), showing a predominant adhesive failure pattern in safrcs groups8,9,20-24,27-39. 6 troconis et al. ta bl e 1. m ai n m et ho do lo gi ca l d at a fr om in cl ud ed s tu di es . a ut ho r (y ea r) s am pl e si ze ty pe o f te et h te st ed m at er ia ls ty pe o f m at er ia ls d en ta l su bs tr at e b on di ng te st a ge in g/ te ch ni qu e s am pl e di m en si on / lo ad s pe ed fa ilu re m od e an al ys is p re do m in an t fa ilu re m od e in s a fr c ju lo sk i j (2 01 2) 8 en am el (n =5 0) d en tin (n =5 0) p er m an en t m ol ar s pa + o pt ib on d™ f l + p re m is e ™ fl ow ab le o pt ib on d™ x tr + p re m is e™ fl ow ab le pa + o pt ib on d™ x tr + p re m is e™ fl ow ab le v er tis e fl ow ® pa + v er tis e fl ow ® er a s + c fr c 2s -s ea s+ c fr c 2s -s ea s+ c fr c sa fr c sa fr c en am el an d de nt in sb s n ot / n a 3m m in di am et er / 0. 5m m / m in st er eo m ic ro sc op e a f w aj do w ic z (2 01 2) 20 n r th ird m ol ar s v er tis e fl ow ® fu si o™ l iq ui d d en tin pa + o pt ib on d™ f l + v er tis e fl ow ® pa + o pt ib on d™ f l + fu si o™ l iq ui d d en tin pa + o pt ib on d™ f l + r ev ol ut io n™ sa fr c sa fr c er a s+ sa fr c er a s+ sa fr c er a s+ c fr c en am el sb s n ot / n a 2. 4m m in di am et er / 1m m /m in st er eo m ic ro sc op e a f in f us io ™ li qu id d en tin v ic hi a (2 01 3) 21 en am el (n =6 0) d en tin (n =6 0) p er m an en t m ol ar s ea sy b on d® + f ilt ek ™ s up re m e x t fl ow x en o® v + x f lo w ® g -b on d™ + g ra di a® d ire ct l of lo a dh es e o ne ® + t et ric e vo fl ow ® ib on d® + v en us f lo w ® v er tis e fl ow ® 1s -s ea s+ c fr c 1s -s ea s+ c fr c 1s -s ea s+ c fr c 1s -s ea s+ c fr c 1s -s ea s+ c fr c sa fr c en am el an d de nt in sb s n ot / n a 3m m in di am et er / 0. 5m m / m in o pt ic al m ic ro sc op e a f p ac ifi ci e (2 01 3) 22 50 p rim ar y m ol ar s o pt ib on d™ a llin -o ne +p re m is e ™ f lo w pa + o pt ib on d™ f l+ p re m is e ™ f lo w p ol ya +f uj i i i® p ol ya +f uj i i x ® v er tis e fl ow ® 1s -s ea s+ c fr c er a s+ c fr c g la ss io no m er g la ss io no m er sa fr c d en tin sb s n ot / n a 3m m in di am et er / 1m m /m in st er eo m ic ro sc op e a f ya zi ci a r (2 01 3) 23 80 p er m an en t m ol ar s pa +o pt ib on d™ s ol o p lu s+ p re m is e™ f lo w v er tis e fl ow ® er a s+ c fr c sa fr c d en tin sb s n ot / n a 2. 38 m m in di am et er / 1m m /m in o pt ic al m ic ro sc op e a f m ar gv el as hv ili m , ( 20 13 )2 4 30 p er m an en t m ol ar s pa + v er tis e fl ow ® pa + g ua rd ia n se al ® a dp er ™ p ro m pt l -p op + c lin pr o™ s ea la nt sa fr c c fr c 1s -s ea s+ c fr c en am el sb s n ot / n a 3m m in di am et er / 0. 5m m /m in o pt ic al m ic ro sc op e se m a f b ek ta s o o (2 01 3) 25 30 th ird m ol ar s o pt ib on d™ a llin -o ne + r ev ol ut io n™ f or m ul a2 v er tis e fl ow ® o pt ib on d™ a llin -o ne + v er tis e fl ow ® 1s -s ea s+ c fr c sa fr c 1s -s ea s+ sa fr c d en tin µ sb s n ot / n a 0. 7m m in di am et er / 1m m /m in n a n a c on tin ue ... https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow http://www.ncbi.nlm.nih.gov/pubmed/?term=vichi%25252525252525252525252520a%2525252525252525252525255bauthor%2525252525252525252525255d&cauthor=true&cauthor_uid=23086332 https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.ncbi.nlm.nih.gov/pubmed/?term=pacifici%2525252525252525252520e%25252525252525252525255bauthor%25252525252525252525255d&cauthor=true&cauthor_uid=24683779 https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow http://www.ncbi.nlm.nih.gov/pubmed/?term=yazici%25252525252525252525252520ar%2525252525252525252525255bauthor%2525252525252525252525255d&cauthor=true&cauthor_uid=22821150 https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow 7 troconis et al. p oi te vi n a (2 01 3) 9 en am el (n =4 0) d en tin (n =5 5) th ird m ol ar s fu si o™ l iq ui d d en tin v er tis e fl ow ® pa + v er tis e fl ow ® a dh es e o ne ® + t et ric e vo fl ow ® a dp er ™ p ro m pt l -p op + fi lte k™ s up re m e x t fl ow ab le ib on d® + v en us f lo w ® x en o® v + x fl ow ® pa +o pt ib on d™ f l + p re m is e ™ fl ow ab le sa fr c sa fr c sa fr c 1s -s ea s+ c fr c 1s -s ea s+ c fr c 1s -s ea s+ c fr c 1s -s ea s+ c fr c e r a s+ c fr c en am el an d de nt in µt b s n ot / n a 1m m in di am et er / 1m m /m in st er eo m ic ro sc op e f eg -s em a f tu lo gl u n (2 01 4) 26 60 30 p rim ar y m ol ar s 30 pe rm an en t m ol ar s v er tis e fl ow ® o pt ib on d™ a llin -o ne + f ilt ek ™ u lti m at e fl ow ab le o pt ib on d™ a llin -o ne + v er tis e fl ow ® sa fr c 1s -s ea s+ c fr c 1s -s ea s+ sa fr c d en tin sb s tc 5 00 cy cl es (b et w ee n 5° c a nd 55 °c fo r 10 s) 2 m m in di am et er / 1m m /m in n a n a r us so d (2 01 4) 27 72 p er m an en t m ol ar s pa +o pt ib on d™ f l + p re m is e™ f lo w ab le o pt ib on d™ x tr + p re m is e ™ f lo w ab le v er tis e fl ow ® sm ar t c em 2® r el yx ™ u ni ce m 2 sp ee dc em ® m ax c em e lit e™ r el yx ™ u ni ce m k et ac ™ c on di tio ne r r efi ll + k et ac ™ f il p lu s a pl ic ap er a s+ c fr c 2s -s ea s+ c fr c sa fr c sa r c sa r c sa r c sa r c sa r c sa g ic d en tin µs b s n ot / n a 0. 95 1. 45 m m in di am et er / 1m m /m in se m a f yu an h (2 01 5) 28 40 th ird m ol ar s a dp er ™ e as y o ne + f ilt ek ™ z 35 0 fl ow ab le c le ar fil ™ s e b on d+ f ilt ek ™ z 35 0 fl ow ab le pa +p rim e  &  b on d n t® + fi lte k™ z 35 0 fl ow ab le d ya d™ f lo w δ 1s -s ea s+ c fr c 2s -s ea s+ c fr c er a s+ c fr c sa fr c d en tin µt b s n ot / n a 1m m in di am et er / 0. 5m m / m in st er eo m ic ro sc op e se m a f sc hu ld t (2 01 5) 29 90 th ird m ol ar s c on st ic ® pa + c on st ic ® pa + h el io se al f ® sa fr c sa fr c c fr c en am el sb s tc 5 00 0 cy cl es (b et w ee n 5° c a nd 55 °c ) 2. 38 in di am et er / 1m m /m in st er eo m ic ro sc op e a f c on tin ue ... ta bl e 1. c on tin ua tio n. http://www.ncbi.nlm.nih.gov/pubmed/?term=poitevin%25252525252525252525252520a%2525252525252525252525255bauthor%2525252525252525252525255d&cauthor=true&cauthor_uid=23107191 https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow http://www.ncbi.nlm.nih.gov/pubmed/?term=tuloglu%25252525252525252525252520n%2525252525252525252525255bauthor%2525252525252525252525255d&cauthor=true&cauthor_uid=24127036 https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow 8 troconis et al. sa ch de va p (2 01 6) 16 60 p rim ar y te et h d ya d™ f lo w δ fu si o™ l iq ui d d en tin a dh es iv e (n r ) + g -a en ia l u ni ve rs al f lo ® sa fr c sa fr c n r +c fr c d en tin sb s n ot / n a 2. 5m m in di am et er / 0. 5m m / m in n a n a m em ar po ur m (2 01 6) 30 en am el (n =6 0) d en tin (n =6 0) p rim ar y ca ni ne s o pt ib on d™ a llin -o ne + p re m is e™ f lo w ab le v er tis e fl ow ® o pt ib on d™ a llin -o ne + v er tis e fl ow ® 1s -s ea +c fr c sa fr c 1s -s ea +s a fr c en am el an d de nt in sb s n ot / n a 3m m in di am et er / 1m m /m in d ig ita l m ic ro sc op e se m a f an d m f. a lm az m , (2 01 6) 31 48 p er m an en t m ol ar s v er tis e fl ow ® c le ar fil ™ s e b on d+ c le ar fil ™ m aj es ty f lo w a llb on d se ® + a el ite ™ f lo a dp er ™ e as y o ne + fi lte k™ u lti m at e fl ow sa fr c 2s -s ea s+ c fr c 1s -s ea s+ c fr c 1s -s ea s+ c fr c d en tin sb s n ot / n a 3 m m in di am et er / n r ilu m in at ed m ic ro sc op e a f m os le m i (2 01 6) 32 40 th ird m ol ar s si c + e r,c r:y sg g la se r + s in gl eb on d® + c fr c (n r ) si c + er ,c r:y sg g la se r + d ya d™ f lo w δ si c + s in gl eb on d® + c fr c (n r ) si c + d ya d™ f lo w δ er a s+ c fr c sa fr c er a s+ c fr c sa fr c d en tin µs b s n ot / n a 0. 7m m in di am et er / 0. 5m m /m in st er eo m ic ro sc op e a f in sa fr c s w ith ou t la se r. b um ru ng ru an (2 01 6) 33 60 th ird m ol ar s v er tis e fl ow ® pa +o pt ib on d™ f l+ p re m is e™ f lo w ab le o pt ib on d™ a llin -o ne + p re m is e™ f lo w ab le sa fr c er a s+ c fr c 1s -s ea +c fr c d en tin µs b s b et w ee n 5c a nd 55 c fo r 50 00 cy cl es 0. 8 in di am et er / 1m m /m in st er eo m ic ro sc op e a f d ur m uş la r s (2 01 7) 34 60 p rim ar y m ol ar s v er tis e fl ow ® g -a en ia l b on d® + g -a en ia l u ni ve rs al f lo ® pa + te tr ic ® n -b on d+ t et ric ® n -f lo w sa fr c 1s -s ea s+ c fr c er a s+ c fr c d en tin µt b s n ot / n a 3m m in di am et er / 1m m /m in se m a f p et er so n j (2 01 7) 35 en am el (n =6 4) d en tin (n =6 4) p er m an en t m ol ar s c on st ic ® fu si o™ l iq ui d d en tin v er tis e fl ow ® pa +o pt ib on d™ f l + v en us d ia m on d fl ow ® sa fr c sa fr c sa fr c er a s+ c fr c en am el an d de nt in sb s tc (5 00 0 cy cl es (b et w ee n 5° c a nd 55 °c ) 3m m in di am et er / 1m m /m in st er eo m ic ro sc op e a f b ru ec kn er c (2 01 7) 36 en am el (n =8 0) d en tin (n =8 0) p er m an en t m ol ar s v er tis e fl ow ® fu si o™ l iq ui d d en tin a dp er ™ p ro m pt l -p op + f ilt ek ™ s up re m e x t flo w ab le sa fr c sa fr c 1s -s ea s+ c fr c en am el an d de nt in sb s tc (1 50 0 cy cl es (b et w ee n 5° c a nd 55 °c ) 3m m in di am et er / 0. 75 ± 0. 25  m m / m in se m a f c on tin ue ... ta bl e 1. c on tin ua tio n. https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow 9 troconis et al. r an ga pp a a (2 01 8) 37 64 p er m an en t m ol ar s c on st ic ® d ya d™ f lo w δ pa + te tr ic ® n -b on d+ t et ric ® n -f lo w sa fr c sa fr c er a s+ c fr c d en tin sb s n ot / n a 3m m in di am et er / 1m m /m in se m a f p oo rz an dp ou sh (2 01 9) 38 48 p rim ar y ca ni ne s an d fir st m ol ar s pa + o pt ib on d™ f l+ p re m is e™ f lo w ab le v er tis e fl ow ® er a s+ c fr c sa fr c d en tin sb s 10 00 cy cl es be tw ee n 555 °c 3m m in di am et er / 1m m /m in st er eo m ic ro sc op e a f a bd el ra ou f (2 01 9) 39 en am el (n =2 4) d en tin (n =1 2) p er m an en t m ol ar s d ya d™ f lo w δ pa + u ni ve rs al s in gl e b on d® + fi lte k™ z 35 0x t sa fr c u a s+ c fr c en am el an d de nt in sb s n ot / n a 3m m in di am et er / 0. 5m m / m in d ig ita l m ic ro sc op e a f n a : n ot a pp lie d; n r : n ot re po rt ed p ol ya : p ol ya cr ili c a ci d; p a : p ho sp ho ric a ci d; s ic : s ili co n ca rb id e sa nd pa pe r; sb s: s he ar b on d st re ng th ; µ sb s: m ic ro -s he ar b on d st re ng th ; µ tb s: m ic ro -t en si le b on d st re ng th ; s em : s ca nn in g el ec tr on m ic ro sc op y; f eg -s em : fi el dem is si on g un s ca nn in g el ec tr on m ic ro sc op y; 1 sse a s: o ne -s te p se lfet ch -a dh es iv e sy st em ; 2 sse a s: tw ost ep s se lfet ch a dh es iv e sy st em ; e r a s: e tc h an d rin se a dh es iv e; u a s: u ni ve rs al a dh es iv e sy st em ; s a r c : s el fad he si ve re si n ce m en t; sa g ic : s el fad he si ve g la ss io no m er c em en t; tc : t he rm oc yc lin g; a f: a dh es iv e fa ilu re ; c f: c oh es iv e fa ilu re ; m f: m ix ed fa ilu re . δ v er tis e® f lo w is m ar ke te d as d ya d™ f lo w in s om e co un tr ie s. o pt ib on d™ f l (k er r, o ra ng e, c a , u sa ), p re m is e™ f lo w ab le (k er r, o ra ng e, c a , u sa ), o pt ib on d™ x tr (k er r, o ra ng e, c a , u sa ), v er tis e fl ow ® (k er r, o ra ng e, c a , u sa ), fu si o™ l iq ui d d en tin (p en tr on c lin ic al , o ra ng e, u sa ), r ev ol ut io n™ (k er r, o ra ng e, c a , u sa ), ea sy b on d (3 m e sp e, s t. p au l, m n , u sa ), fi lte k™ s up re m e x t fl ow (3 m e sp e, s t. p au l, m n , u sa ), x en o® v (d en ts pl y, d et re y, k os ta nz , g er m an y) , x f lo w ® (d en ts pl y, d et re y, k os ta nz , g er m an y) , g -b on d™ (g c , t ok yo , j ap an ), g ra di a® d ire ct l of lo (g c , t ok yo , j ap an ), a dh se o ne ® (i vo cl ar v iv ad en t, sc ha an , l ie ch te ns te in ), te tr ic ® e vo f lo w (i vo cl ar v iv ad en t, sc ha an , l ie ch te ns te in ), r ev ol ut io n™ f or m ul a2 (k er r, o ra ng e, c a , u sa ), ib on d® (h er ae us k ul ze r, h an au , g er m an y) , o pt ib on d™ a ll in o ne (k er r, o ra ng e, c a , u sa ), fu ji ii® (g c , t ok yo , j ap an ), fu ji® ix (g c , t ok yo , j ap an ), o pt ib on d™ s ol o p lu s (k er r, o ra ng e, c a , u sa ), g ua rd ia n se al ® (k er r, o ra ng e, c a , u sa ), a dp er ™ p ro m pt l -p op (3 m e sp e, s t. p au l, m n , u sa ), c lin pr o™ s ea la nt (3 m e sp e, s t. p au l, m n u sa ), fi lte k™ u lti m at e fl ow ab le (3 m e sp e, s t. p au l, m n , u sa ), a dp er ™ e as y o ne (3 m e sp e, s t. p au l, m n , u sa ), fi lte k™ z 35 0 fl ow ab le (3 m e sp e, s t. p au l, m n , u sa ), sm ar t c em 2® (d en ts pl y, y or k, p a , u sa ), r el yx ™ u ni ce m 2 (3 m e sp e, g er m an y) , sp ee dc em ® (i vo cl ar v iv ad en t, sc ha an , l ie ch te ns te in ), m ax c em e lit e™ (k er r, o ra ng e, c a , u sa ), r el yx ™ u ni ce m (3 m e sp e, g er m an y) , k et ac ™ c on di tio ne r r efi ll( 3m e sp e, g er m an y) , k et ac ™ f il p lu s a pl ic ap (3 m e sp e, g er m an y) , c le ar fil ™ s e b on d (k ur ar ay , o ka ya m a, j ap an ), p rim e  &  b on d n t® (d en ts pl y, y or k, p a , u sa ), c on st ic ® (d m g , h am bu rg , g er m an y) , h el io se al f ® (i vo cl ar v iv ad en t, sc ha an , l ie ch te ns te in ), g -a en ia l™ u ni ve rs al f lo (g c , t ok yo , j ap an ), c le ar fil ™ m aj es ty f lo w (k ur ar ay , o ka ya m a, j ap an ), a ll b on d se ® (b is co in c, sc ha um bu rg , i l, u sa ), a el ite ™ f lo (b is co in c, s ch au m bu rg , i l, u sa ), si ng le b on d® (3 m e sp e, s t. p au l, m n , u sa ), g -a en ia l™ b on d (g c , t ok yo , j ap an ), te tr ic ® n -b on d (i vo cl ar v iv ad en t, sc ha an , l ie ch te ns te in ), te tr ic ® n -f lo w (i vo cl ar v iv ad en t, sc ha an , l ie ch te ns te in ), v en us d ia m on d fl ow ® (h er ae us k ul ze r, h an au , g er m an y) ; u ni ve rs al s in gl e b on d® (3 m es p e, g er m an y) . ta bl e 1. c on tin ua tio n. https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow 10 troconis et al. risk of bias assessment table 2 summarizes the risk of bias of the included studies. only one of the studies reported sample size was calculated, but none of the studies reported if operators were blinded. most included studies (n=21) did not report in the methodology section whether experiments were conducted by a single operator. conversely, aspects such as randomization, teeth free of caries, samples with similar dimensions and manufacturer instructions were reported. overall, 20 studies scored medium risk of bias, two studies had low risk and other one scored high risk. synthesis of results enamel bond strength of safrcs and cfrcs table 3 presents means, standard deviations and statistically significant differences on enamel bond strength between safrcs and cfrcs. mean enamel bond strength of safrcs (without prior phosphoric acid etching and/or adhesive system use) in permanent teeth showed the following variations: vertise® flow (from 2.035 to 15.3  mpa9), fusio™ liquid dentin (from 3.035 to 13.0 mpa9) and constic® (from 3.929 to 4.5 mpa35). overall, mean bond strength of safrcs to previously etched enamel varied from 9.878 to 23.1 mpa9. conversely, the mean enamel bond strength of cfrcs associated with different types of adhesive systems ranged between 5.021 and 28.0 mpa9 in permanent teeth. the only study that used primary teeth to evaluate the bond strength of a safrc (vertise® flow) reported mean values of 9.29 mpa and 14.84 mpa for sic and laser treated surfaces, respectively30. most studies showed significant lower enamel bond strength values on safrcs compared to cfrcs. dentin bond strength of safrcs and cfrcs table 4 presents means, standard deviations and significant differences on dentin bond strength between safrcs and cfrcs. mean bond strength values of safrcs used without prior phosphoric acid etching and/or adhesive system use in permanent teeth showed the following variations: vertise® flow (from 1.035 to 32.66 mpa28), fusio™ liquid dentin (from 2.835 to 17.7 mpa9) and constic® (from 0.835 and 12.2  mpa37). overall, the bond strength of safrcs used on previously etched dentin or associated to an adhesive system ranged between 5.488 and 35.08 mpa25. two studies employed thermocycling33,36 and the results revealed that bond strength of fusio™ liquid dentin decreased from 4.4 mpa to 1.6 mpa36 following thermocycling while the values for vertise® flow diminished from 3.0 to 1.0 mpa36 and from 22.1 to 21.1 mpa33. six studies evaluated the bonding performance of safrcs to primary teeth16,22,26,30,34,38 without prior acid etching or adhesive system use. the mean dentin bond strength values were: vertise® flow (from 2.334 to 12.17 mpa30) and fusio™ liquid dentin (14.15 mpa16). two studies26,30 evaluated the dentin bond strength of vertise® flow associated to optibond™ all-in-one adhesive system and mean bond strength ranged between 8.726 and 16.89 mpa30. on the other hand, mean bond strength of cfrcs associated to different adhesive systems varied from 14.8738 to 21.11 mpa16. overall, most studies reported statistically significant lower dentin bond strength on safrcs compared to cfrcs groups. 11 troconis et al. ta bl e 2. r is k of b ia s as se ss m en t i n in cl ud ed s tu di es . a ut ho r r an do m iz at io n s am pl e si ze ca lc ul at io n te et h fr ee o f ca ri es s am pl e w ith s im ila r di m en si on s fa ilu re m od e ev al ua tio n m an uf ac tu re r’s in st ru ct io ns s in gl e op er at or o pe ra to r bl in de d r is k of b ia s ju lo sk i e t a l8 ye s n o ye s ye s ye s ye s n o n o m ed iu m w aj do w ic z et  a l20 n o n o ye s ye s ye s ye s n o n o m ed iu m v ic hi e t a l21 ye s n o ye s ye s ye s ye s ye s n o lo w p ac ifi ci e t a l22 ye s n o ye s ye s ye s ye s n o n o m ed iu m ya zi ci e t a l23 ye s n o ye s ye s ye s ye s n o n o m ed iu m m ar gv el as hv ili e t a l24 ye s n o ye s n o ye s ye s ye s n o m ed iu m b ek ta s et  a l25 ye s n o ye s ye s n o ye s n o n o m ed iu m p oi te vi n et  a l9 ye s n o ye s ye s ye s ye s n o n o m ed iu m tu lo gl u et  a l26 ye s n o ye s n o n o ye s n o n o h ig h r us so e t a l27 ye s n o ye s ye s ye s n o n o n o m ed iu m yu an e t a l28 ye s n o ye s ye s ye s ye s n o n o m ed iu m sc hu ld t e t a l29 ye s n o ye s ye s ye s ye s n o n o m ed iu m sa ch de va e t a l16 ye s n o ye s ye s n o ye s n o n o m ed iu m m em ar po ur e t a l30 ye s n o ye s ye s ye s ye s n o n o m ed iu m a lm az e t a l31 ye s n o ye s ye s ye s ye s n o n o m ed iu m m os le m i e t a l32 ye s n o ye s ye s ye s ye s n o n o m ed iu m b um ru ng ru an e t a l33 ye s n o ye s ye s ye s ye s n o n o m ed iu m d ur m uş la r e t a l34 ye s n o ye s ye s ye s ye s n o n o m ed iu m p et er so n et  a l35 ye s n o ye s ye s ye s ye s n o n o m ed iu m b ru ec kn er e t a l36 ye s n o ye s ye s ye s ye s n o n o m ed iu m r an ga pp a et  a l37 ye s n o ye s ye s ye s ye s n o n o m ed iu m p oo rz an dp ou sh e t a l38 ye s ye s ye s ye s ye s ye s n o n o lo w a bd el ra ou f e t a l39 ye s n o ye s ye s ye s ye s n o n o m ed iu m 12 troconis et al. table 3. means, standard deviations and statistically significant differences on enamel bond strength between safrcs and cfrcs. permanent teeth author (year) materials enamel bond strength in mpa mean ± standard deviation significant difference juloski (2012)8 pa + optibond™ fl + premise ™ flowable pa + vertise flow® optibond™ xtr + premise™ flowable. pa + optibond™ xtr + premise™ flowable vertise flow® 16.83±2.93 9.87±4.24 8.59±4.39 7.04±3.63 6.61±2.41 a b b b b wajdowicz (2012)20 pa + optibond™ fl + vertise flow® pa + optibond™ fl + fusio™ liquid dentin pa + optibond™ fl + revolution™ fusio™ liquid dentin vertise flow® 10.2±nr 8.5±nr 8.3±nr 3.6±nr 3.5±nr a a a b b vichi a (2013)21 easybond + filtek™ supreme xt flow xeno® v + x flow® g-bond™ + gradia® direct loflo adhese one + tetric evo flow® ibond® + venus flow® vertise flow® 12.1±5.0 10.4±4.0 7.7±1.9 6.0±4.0 5.0±1.8 2.6±2.6 a ab abc bcd cd d margvelashvili (2013)24 pa + vertise flow® adper™ prompt l-pop + clinpro™ sealant pa + guardian seal 17.9±2.9 12.9±6.0 11.7±4.6 a ab b poitevin (2013)9 adper™ prompt l-pop + filtek™ supreme xt flowable pa + vertise flow® fusio™ liquid dentin vertise flow® bur-cut:28.0±9.8/sicground:25.5±8.2 bur-cut:23.1±7.1/sicground:22.6±7.6 bur-cut:13.0±4.3/sicground:10.8±5.8 bur-cut:11.0±4.2/sicground:15.3±6.0 a/a a/a b/b b/b schuldt (2015)29 pa + helioseal f® pa + constic® constic® 19.1±6.2 / tc:15.6±4.4 17.1±5.1 / tc:13.0±3.8 4.3±1.6 / tc:3.9±1.4 a/a a/ab c/c peterson (2017)35 pa+optibond™ fl + venus diamond flow® constic® fusio™ liquid dentin vertise flow® 13.0±5.1 4.5±nr 3.0±nr 2.0±nr a b b b brueckner (2017)36 adper™ prompt l-pop + filtek™ supreme xt flowable experimental flowable vertise flow® fusio™ liquid dentin 9.8±3.6 /tc: 8.3±3.7 4.4±3.0 / tc: 0.7±0.4 4.0±2.1 / tc: 0.4±0.4 3.5±2.3 / tc: 0.5±0.1 a/a b/b b/bc c/c abdelraouf (2019)39 pa+ universal single bond®+ filtek™ z350xt dyad™ flowδ uncut: 24.6±6.2/ cut: 12.7±4.5 uncut: 3.5±1.6/ cut: 4.5±2.7 a/b c/c primary teeth memarpour (2016)30 optibond™ all-in-one+vertise flow® optibond™ all-in-one+premise™ flowable vertise flow® sic:15.05±2.12 / er:yag laser:16.16±3.16 sic:13.06±2.36 / er:yag laser:13.90±2.76 sic:9.29±1.56 / er:yag laser:14.84±1.32 a/a a/a b/a different capital letters mean statistically significant difference (p≤0.05) among study groups, reported on individual studies. sic: silicon carbide sandpaper; polya: polyacrilic acid; pa: phosphoric acid; tc: thermocycling; nr: not reported; er:yag laser: erbium:yttrium aluminum garnet laser. δvertise® flow is marketede as dyad™ flow in some countries. https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow 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https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow 13 troconis et al. table 4. means, standard deviations and statistically significant differences on dentin bond strength between safrcs and cfrcs. permanent teeth author (year) materials dentin bond strength in mpa mean ± standard deviation significant difference juloski (2012)8 optibond™ xtr + premise™ flowable. pa + optibond™ xtr + premise™ flowable pa + optibond™ fl + premise ™ flowable pa + vertise flow® vertise flow® 10.60±5.0 9.60±4.91 8.15±3.88 5.48±4.94 2.94±2.79 a a ab bc c vichi a (2013)21 easybond + filtek™ supreme xt flow adhese one + tetric evo flow xeno® v + x flow g-bond™ + gradia® direct loflo ibond® + venus flow® vertise flow® 12.2±3.6 11.3±5.7 10.7±4.7 6.9±3.2 5.8±1.2 3.4±1.6 a a a ab ab b yazici (2013)23 pa+ optibond™ solo plus + premise™ flow vertise flow® sic:14.64±6.75 / er:yag laser:16.81±6.76 sic:7.92±2.91 / er:yag laser:12.61±3.49 a/a b/a bektas (2013)25 optibond™ all-in-one/vertise flow® optibond™ all-in-one/ revolution™ formula2 vertise flow® 35.08±7.0 29.33±5.19 23.70±5.28 a b c poitevin (2013)9 pa+optibond™ fl + premise™ flowable xeno® v + x-flow® adper™ prompt l-pop + filtek™ supreme xt flowable ibond® + venus flow® pa + vertise flow® fusio™ liquid dentin adhese one® + tetric evoflow® vertise flow® bur-cut:44.8±13.6/sic-ground:nr bur-cut:29.4±11.7/sic-ground:nr bur-cut:25.4±10.0/sicground:34.9±13.4 bur-cut:23.9±10.3/sic-ground:nr bur-cut:18.7±11.0/sic-ground:nr bur-cut:17.7±8.6/sicground:17.19.5 bur-cut:7.9±5.3/sic-ground:nr bur-cut:1.8±2.7 /sic-ground:5.36.7 a/nr a/nr a/a b/nr b/nr b/b c/nr c/c tuloglu (2014)26 optibond™ all-in-one + filtek™ ultimate flowable optibond™ all-in-one + vertise flow® vertise flow® 35.7±2.9 25.6±3.0 19.3±2.3 a b c russo (2014)27 optibond™ xtr + premise™ flowable pa+ optibond™ fl + premise™ flowable smart cem2® relyx™ unicem 2 speedcem® maxcem elite™ vertise flow® relyx™ unicem ketac™ fil plus aplicap 25.3±13.0 20.8±7.8 11.6±6.9 11.3±7.3 10.7±5.5 9.6±5.3 7.1±4.0 6.3±3.2 5.8±3.0 a a b bc bcd bcde cde de e yuan h (2015)28 pa+prime & bond nt®+ filtek™ z350 flowable clearfil™ se bond+ filtek™ z350 flowable adper™ easy one+ filtek™ z350 flowable dyad™ flowδ 37.96±7.15 35.63±5.23 34.90±8.33 32.66±8.20 a b b c almaz (2016)31 clearfil™ se bond+clearfil™ majesty flow adper™ easy one +filtek™ ultimate flow all-bond se® +aelite™ flo vertise flow® 14.70±2.47 12.90±2.40 8.29±2.66 2.94±1.95 a b c d continue... https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow 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https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow 14 troconis et al. moslemi (2016)32 sic + er,cr:ysgg laser+pa+single-bond®+ cfrc (nr) sic + pa+single bond® + cfrc (nr) sic+ er,cr:ysgg laser + dyad™ flowδ sic + dyad™ flowδ 20.62±0.125 19.72±0.01 16.42±0.01 12.85±0.01 a a a b bumrungruan (2016)33 pa+optibond™ fl+ premise™ flowable optibond™ all-in-one+ premise™ flowable vertise flow® 32.2±8.94 / tc: 31.8±6.80 24.4±6.21 / tc: 23.9±7.14 22.1±6.13 / tc: 21.1±5.39 a/a b/b b/b peterson (2017)35 pa+optibond™ fl + venus diamond flow® fusio™ liquid dentin vertise flow® constic® 11.2±6.3 2.8±nr 1.0±nr 0.8±nr a b b b brueckner (2017)36 adper™ prompt l-pop + filtek™ supreme xt flowable fusio™ liquid dentin vertise flow® self-adhesive experimental flowable 11.6±3.5 / tc:5.4±3.7 4.4±1.3 / tc:1.6±2.1 3.0±2.6 / tc:1.0±1.6 2.4±4.1 / tc:0.7±0.0 a/a b/b b/b b/b rangappa a (2018)37 pa+ tetric® n-bond+ tetric® n-flow dyad™ flowδ constic® carbide bur:23.0±3.1 /diamond bur:18.2±2.6 carbide bur:14.6±2.1 /diamond bur:11.9±1.7 carbide bur:12.2±3.1 /diamond bur:10.2±2.7 a/a b/b c/c abdelraouf (2019)39 pa+ universal single bond®+ filtek™ z350-xt dyad™ flowδ 6.7±1.7 4.3±1.6 a b primary teeth pacifici (2013)22 optibond™ all-in-one + premise™ flowable pa + optibond™ fl + premise™ flowable polya + fuji ix® polya + fuji ii® vertise flow® 16.59±1.77 16.02±3.15 6.04±3.76 5.91±4.80 4.31±2.66 a a b b b tuloglu (2014)26 optibond™ all-in-one + filtek™ ultimate flowable optibond™ all-in-one + vertise flow® vertise flow® 15.6±2.6 8.7±1.7 4.1±2.3 a b c sachdeva (2016)16 adhesive system (nr) +g-aenial universal flo® fusio™ liquid dentin dyad™ flowδ 21.11±1.168 14.15±1.168 12.03±1.168 a b b memarpour (2016)30 optibond™ all-in-one+premise™ flowable optibond™ all-in-one+vertise flow® vertise flow® sic:17.41±1.20 / er:yag laser:17.65±1.25 sic:16.89±1.05 / er:yag laser:13.93±0.97 sic:12.17±1.31 / er:yag laser:12.09±1.26 a/a a/b b/c durmuşlar s (2017)34 g-aenial bond® + g-aenial universal flo® pa+ tetric® n-bond+ tetric® n-flow vertise flow® 15.5±10.06 13.0±6.99 2.3±2.93 a a b poorzandpoush (2019)38 pa+ optibond™+ premise™ flowable vertise flow® 14.87±3.42 6.60±1.97 a b different capital letters mean statistically significant difference (p≤0.05) among study groups, reported on individual studies. sic: silicon carbide sandpaper; polya: polyacrilic acid; pa: phosphoric acid; tc: thermocycling; nr: not reported; er:yag laser: erbium:yttrium aluminum garnet laser. δ vertise® flow is marketed as dyad™ flow in some countries. table 4. continuation. https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow 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https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow https://www.dentalbauer.de/praxis/fuellungen/composite-lichthaertend/112766/els-extra-low-shrinkage-flow 15 troconis et al. discussion according to our knowledge, this is the first systematic review that critically approaches the bonding performance of safrcs on permanent and primary teeth, comparing the outcomes with cfrcs associated to different adhesive systems. there were considerable variations in enamel bond strength values among included studies, probably due to methodological divergences such as type of teeth, specimen preparation technique, bonding test, enamel treatment, bonding area and load speed which made it impossible to conduct a meta-analysis. the results of this systematic review revealed low enamel bond strength of safrcs (table 3), which was in agreement with previous studies testing the same safrcs40,41 or self-etching sealants exhibiting similar chemical composition42,43. these findings may be explained due to enamel is a very complex and mineralized dental structure44, which requires a surface treatment prior to composite resin restorations or resin-based sealant placement. phosphoric acid etching is the most used strategy to promote micro morphological alterations on enamel surface, leading to an effective resin interlocking and enhanced bond strength45,46. some included studies revealed that safrcs applied under etched enamel exhibited higher bond strength values compared to safrcs used in self-etch mode9,29. however, only two studies aimed to compare the findings between resin-based sealants and safrcs24,29, highlighting the need for future research using this approach to confirm if safrcs applied on etched enamel could show the same bonding performance than resin-based sealants. functional monomers such as gpdm and 4-meta incorporated into vertise® flow and fusio™ liquid dentin, respectively, are highly acidic but do not promote the same enamel demineralization pattern that phosphoric acid etching47. therefore, the bonding effectiveness of these functional monomers relies largely on the chemical interaction with dental hap, which is lower and less stable compared to that promoted by 10-mdp monomer48. these facts may explain why vertise® flow and fusio™ liquid dentin used without prior phosphoric acid etching performed significantly worse than cfrcs8,9,20,21,30,36 other strategies to improve the bonding effectiveness of adhesive restorations involve lasers such as erbium:yttrium aluminum garnet laser (er:yag) or neodymium-doped yttrium aluminum garnet (nd:yag)49,50. this was confirmed in one included study30 using er:yag laser (120 mj, 10 hz, 1.20 w) at 1 mm of distance from primary enamel. the  results demonstrated that the bond strength of vertise® flow increased up to 38% following laser irradiation compared to sic treatment, being comparable to bond strength values in control group (optibond™ all-in-one and premise™ flowable). the authors argued that the ablation effect promoted by er:yag laser on enamel surface resulted in a more irregular and microretentive morphological pattern, increasing surface area for micromechanical interlocking of flowable resin composites30, as reported in other micromorphological studies51,52. despite er:yag laser treatment increased the enamel bond strength of tested safrc, high cost and learning curve to manipulate the device makes it an unfeasible option compared to phosphoric acid etching. regarding dentin bond strength of safrcs, considerable mean variations were also found among included studies, probably due to the same reasons explained for enamel bonding tests. safrcs exhibited statistically lower dentin bond strength in contrast to cfrcs (table 4)8,9,16,21-23,25-28,30-32,34-39 as well as predominant adhesive fail16 troconis et al. ures8,9,22,23,28,30,31,33,34-39. this indicates a deficient and non-stable chemical interaction between functional monomers incorporated into safrcs and dentin microstructure. these hypotheses were also demonstrated by a chemical study48 as well as on transmission electron microscopy (tem)53 and scanning electron microscopy (sem) studies assessing vertise® flow54. this self-adhesive flowable resin composite followed by fusio™ liquid dentin were the most evaluated materials, especially in primary and permanent dentin, showing similar bond strength values16,35,36. in contrast to gpdm and 4-meta monomers, 10-mdp monomer promotes a superficial demineralization of dentin collagen fibers and enables a stable ionic interaction between phosphate group and remaining calcium ions of hap55, leading to satisfactory dentin bonding performance as demonstrated in other dental materials56,57. nonetheless, two included articles35,37 tested constic®, a 10-mdp containing safrcs which revealed deficient dentin bond strength values, being comparable35 or lower37 than other safrcs that do not incorporate this phosphate monomer. this raises the suggestion that 10-mdp monomer by itself did not guarantee acceptable bonding performance of this safrc. there are other material-dependent factors such as water content, purity and functional monomer concentration which may negatively impact the bond strength of self-adhesive dental materials58,59. self-adhesive resin cements60 and safrcs are flowable materials that present similar chemical composition. self-adhesive resin cements also incorporate silanized inorganic fillers, methacrylate monomers and an activator-initiator system. in addition, self-adhesive resin cements contain functional monomers such as 10-mdp, 4-meta, dipentaerythritol penta-acrylate monophosphate (penta-p) or others60. one  study27 included in this systematic review additionally compared the bond strength of vertise® flow and some self-adhesive resin cements, showing similar bonding performance to dentin. however, it is not possible to indicate safrcs as alternatives for metallic crowns, posts, inlays, onlays, or ceramic crowns cementation because dualcured luting materials are desired for these clinical applications60. safrcs are not even recommended as light-cured resin cements because film thickness is not suitable for that purpose and limited color availability5. besides bond strength of safrcs to hard dental tissues, other relevant aspects such as color stability61, water sorption, solubility13, nanoleakage14, microleakage16,62, polymerization stress, gap formation63 need further research. main strengths of this systematic review were extensive searches on different databases, strict selection criteria, risk of bias assessment and data extraction. conversely, one limitation was that most included studies evaluated the immediate bond strength of safrcs to hard dental tissues. this is not clinically relevant due to mechanical loading, chemical and hydrolytic degradation of laboratorial samples are important issues to predict the possible mechanical performance of adhesive restorations. in addition, the findings should be carefully interpreted due to most included evidence showing medium risk of bias (table 2) which appears to be usual in systematic reviews of in vitro studies on dental adhesion18,19. the lack of methodological homogeneity was another limitation that made it impossible to conduct a meta-analysis. based on the results of the current systematic review, it is possible to affirm that chemical changes on safrcs as well as additional studies are required to consider 17 troconis et al. these dental materials as a possible alternative in restorative and preventive dentistry. for a while, the use of phosphoric acid on enamel is essential on resin-based sealants placement. also, the acid etching, especially in enamel and adhesive systems in both hard dental tissues remains as mandatory steps for successful restorative treatments involving resin composites8,9,20-23,26,27,28,31,33,36,37. self-adhesive flowable resin composites, such as vertise® flow and fusio™ liquid dentin used in self-etch mode exhibited lower bond strength to enamel and dentin from permanent teeth, compared to conventional flowable resin composites. the  bonding performance of constic® on both hard dental tissues should be evaluated on future studies. the evidence is still limited to support that self-adhesive flowable resin composites applied under etched enamel exhibit comparable bond strength to resin-based sealants. the number of studies assessing the bond strength of self-adhesive flowable resin composites to primary teeth 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10.18502/fid.v16i4.2082. 63. nakano el, de souza a, boaro l, catalani lh, braga rr, gonçalves f. polymerization stress and gap formation of self-adhesive, bulk-fill and flowable composite resins. oper dent. 2020 nov;45(6):e308-16. doi: 10.2341/19-166-l. 1 volume 22 2023 e238354 original article braz j oral sci. 2023;22:e238354http://dx.doi.org/10.20396/bjos.v22i00.8668354 1 department of dental materials and prosthodontics, são paulo state university (unesp), school of dentistry, araraquara, são paulo, brazil. 2 department of health sciences, implantology post graduation course, dental school, university center of araraquara (uniara), araraquara, são paulo, brazil corresponding author: lucas portela oliveira department of dental materials and prosthodontics, são paulo state university (unesp), school of dentistry, araraquara, são paulo, brazil. 1680 humaitá street, zip code 14801–903 phone: +55(16) 33016424; fax: +55(16) 33016406 email: lp.oliveira@unesp.br editor: altair a. del bel cury received: february 08, 2022 accepted: april 10, 2022 implant digital impression accuracy using extraoral scanners: a three-dimensional analysis grazielle franco gomes1 , mónica estefanía tinajero aroni1 , lucas portela oliveira1* , joão neudenir arioli filho1 , carolina mollo binda2 , francisco de assis mollo júnior1 aim: to analyze the accuracy of extraoral systems (ceramill map400+, autoscan-ds200+, and e2) in full implantprosthetic rehabilitation three-dimensionally. methods: a metallic edentulous maxilla with four implants was digitalized by a contact scanner (mdx-40 roland, control) and used as a control image to compare with other images generated by three laboratory scanners (10 samples per group). letters identified all the four components: a and d angled 45º, and b and c parallel. the biocad software exported the images (.stl) to compare and verify deviations of the analogs on the x, y, and z axes. the nonparametric kruskal-wallis test and the two-way anova on ranks with a post hoc tukey test analyzed the data with 5% significance. results: no statistical differences were observed in the accuracy between the extraoral scanners (p=0.0806). however, when analyzing only the components, component d was more accurate when scanned with ceramill map400+ compared with autoscan ds200+ (p<0.001) and with e2 (p=0.002). conclusions: all extraoral systems assessed showed digitalization accuracy but with more deviations in angled implants. the ceramill map400+ scanner showed the best results for the digital impression of a complete arch. keywords: dental impression technique. dental implants. dental prosthesis. dental prosthesis, implant-supported. https://orcid.org/0000-0003-0474-7059 https://orcid.org/0000-0002-8346-5561 https://orcid.org/0000-0002-7136-3488 https://orcid.org/0000-0003-3582-9233 https://orcid.org/0000-0002-3675-4360 https://orcid.org/0000-0003-0742-2145 2 gomes et al. braz j oral sci. 2023;22:e238354 introduction the use of conventional complete dentures is one of the most common options for treatment in cases of complete edentulism1. nevertheless, low retention and stability in patients with considerable bone resorption resulted in a greater demand for implants2. therefore, the all-on-four concept is an option in cases of anatomical limitations and severe bone resorption. this protocol uses four implants, two parallel and two 45º angled, in the anterior and posterior region, respectively—this aim to reduce the cantilever length and improve the transmission of strength3. the implant impression technique has the objective of transferring intraoral positions of implants. an accurate impression is vital to obtain a passive fit: a clinical condition in prosthetic rehabilitation which avoids static load on the prosthetic system or alveolar bone4-6. however, incompatibility may cause mechanical and biological failures, such as poor adjustment, fracture of screws or components, and loss of osseointegration7,8. the literature mentions several impression techniques, such as using stable impression material, splinted or non-splinted, or even using only implants or with abutments9-11. however, the contraction of impression materials and clinical and laboratory processes, such as improper leakage time and the plaster type used, can influence the accuracy of the final impression9,12,13. in addition, impression on implants, distance, and angulation may negatively affect the final passivity14. due to these problems caused by conventional impressions, cad/cam (computer-aided-design/manufacturing) systems were introduced to eliminate impression materials and some laboratory processes15. cad/cam systems comprise three stages: data acquisition, prosthesis design, and manufacturing processes16,17. besides, two scan modalities are available: extraoral and intraoral18,19. intraoral digitalization is performed directly in the patient’s mouth. the advantages include eliminating impression material, patient comfort, and a faster treatment18,20-22. however, studies show that buccal humidity, patient’s head movement, and restrictions in the scanner movement can limit the use of this technique23-25. however, there are two systems concerning extraoral scanners: (1) one allows the digitalization of a cast created from the conventional impression; (2) another digitalizes the impression. unfortunately, both modalities may have errors resulting from the impression, manufacture of the dental cast, or even failures in digitalization26. during the process, the scanning is performed with light sources, such as light rays, laser, infrared light, led, or structured light16. for example, laser scanners use a pattern of one-dimensional lines, whereas structured light scanners project a two-dimensional light to obtain three-dimensional data of the scanned object27. in addition, scanners with blue led technology have a shorter wavelength, resulting in better accuracy17. thus, although digitalization is a simple process, the operating mechanism of scanners is complex and may influence its final accuracy, characterized by the combination of trueness and precision28. trueness is the scanner’s ability to digitalize an object 3 gomes et al. braz j oral sci. 2023;22:e238354 with its real dimensions. precision is the scanner’s ability to create repeatable images using different measurements of the same object12,29. other factors that can influence the extraoral scanner precision include device hardware, software algorithms, digitalization technology, and the shape and size of the master model17; however, there is literature lacking about the accuracy of extraoral scanners in angled implants associated with the all-on-four technique. due to the importance of obtaining an accurate final impression, this study assessed, and three-dimensionally compared, the accuracy of different extraoral scanners (ceramill map400+, autoscan-ds200+, and e2) in parallel and angled implants. our null hypothesis states that different laboratory scanners do not present differences in accuracy. materials and methods sample size estimation the sample size was calculated using a software program (gpower; heinrich-heine-universität düsseldorf). in this study the parameters for analysis of variance (anova) were used, which effect size f = 3.60, α = 5%, power = 80%, number of groups= 3 (extraoral scannings). the sample size was calculated to be 6. considering a loss of 30%, the final sample of this study consisted of 10 scanning for each extraoral system analyzed. obtaining the master impression initially, an edentulous maxilla cast model was used to obtain a metallic model (figure 1a) through the lost-wax casting technique. next, a precision lathe performed four 4.1-mm-diameter perforations in this metallic model and installed external hexagon implants with a regular platform (conexão, sao paulo, brazil). then, two parallel perforations were done in the premaxilla region to install 13-mm-long implants; two other perforations angled 45º were conducted in the canine fossa’s posterior area, installing 15-mm-long implants. the implants were named a, b, c, and d (figure 1a) to facilitate analysis. a b figure 1. (a) scheme of the metallic master cast. (b) a metallic model with scan bodies in position. 4 gomes et al. braz j oral sci. 2023;22:e238354 abutments with a 3-mm collar were installed on anterior implants (micro unit, conexão), and 30º angled abutments (micro unit) with a 3-mm collar were installed on 45º angled posterior implants, which compensated for implant angulation (a 15º final angulation). all abutments were applied a 20 n.cm torque, as recommended by the manufacturer. digital impression due to high accuracy, the metallic master cast was initially digitalized with an industrial contact scanner (mdx-40, roland, centro de tecnologia da informação cti, campinas, sp, brazil) due to high accuracy30. the distance between the contact tip and the model surface was calibrated to 0.2 mm, resulting in a high-precision digital model, which was then exported as an stl file to be used as a control image and compared with the other scanners. subsequently, we used three laboratory scanners, including two structured light (ceramill map400+, amann girrbach charlotte usa; autoscan ds200+, shining 3d, zhejiang china) and a multilinear blue led light (e2, 3shape copenhagen, denmark) (table 1), to digitalize the metallic master model and generate the stl images. in addition, scan bodies were installed on the abutments of the master cast (scan-connect micro unit, conexão) (figure 1b), which allowed the components to shift position. table 1. experimental groups system scanner technology manufacturer country mdx-40 control contact scanner ronald são paulo, brazil ceramill map400+ scanner 1 structured light lab scanner amann girrbach charlotte, usa autoscan-ds200+ scanner 2 structured light lab scanner shining 3d zhejiang, china e2 scanner 3 multilinear blue led light 3shape copenhagen, denmark scanners used (laboratory scanner and contact scanner) and their features. a thin, uniform layer of titanium dioxide powder (d70, metal chek, uberaba, brazil; skd-s2 spotcheck, magnaflux, glenview, usa) was used on the surface of the master model to be digitalized by all three scanners to generate an opaque surface and avoid the reflection of light on the metallic model, thus preventing interferences on the final accuracy of the digital model. subsequently, each scanner performed 10 scans following the manufacturer’s instructions, and stl images were obtained (n=30). after obtaining the digital models, the digitalization system replaced the scan bodies present in the digital images for mini pillars available at the digital library, generating the images to be analyzed; we then used interest areas (pyramid and components) for a subsequent 3d analysis. the professionals trained in each system used conducted the digitalization processes: nb for ceramill map400+, aps for autoscan ds200+, and np for e2. 5 gomes et al. braz j oral sci. 2023;22:e238354 determining the distances between the pyramids and the components all models were digitalized in stl files, including one control image (contact scanner) and 30 experimental (extraoral scanners), and then these files were imported to a bio-cad program (computer assisted design; rhino3d, rhinoceros, usa) to determine measures to be later compared (figure 2). initially, each image was imported to select the reference points and build references between the pyramid (creating schemes to represent the pyramidal geometry and obtain the pyramid’s edges and apex) and the components to measure distances (figure 3a). contact scanner mdx-40 (control group) reference model 31 digital images in stl bio-cad software reference model reference model reference model n = 1 n = 10 n = 10 n = 10 2 3 1 ceramill map400+ extraoral scanner e2 autoscan ds200+ figure 2. flowchart of the steps performed. 6 gomes et al. braz j oral sci. 2023;22:e238354 figure 3. (a) selection of reference points of the components. (b) measurements of the distances between the origin and the center of the analogs. (c) x, y, and z axes to determine the measurements of all three axes. after obtaining the reference points in the experimental images (extraoral scanners), we imported the control image to the bio-cad program, repeating the previous steps described to create the reference points to analyze the images. the pyramid’s apex was used as the origin of the coordinate systems of models to calculate the distance between the origin and the centers of analogs (figure 3b), generating the measurements necessary for verifying the deviations. these measurements were performed in the axes of the pyramid (x, y, and z), the x-axis being the vertical deviation, the y-axis being the anteroposterior deviation, and the z-axis being the lateral deviation (figure 3c). as a result, we obtained three measurements for each component. the process was conducted with all 30 images generated by the laboratory scanners and compared with the control image generated by the contact scanner. 7 gomes et al. braz j oral sci. 2023;22:e238354 a b 40 30 20 10 0 a a a a a a b autoscan ds200+ ceramill map400+ e2 150 100 50 0 d ev ia tio n (r an k) d ev ia tio n (µ m ) e2 au to sc an d s2 00 + co m po ne nt a co m po ne nt b co m po ne nt c co m po ne nt d ce ra m ill m ap 40 0+ figure 4. (a)deviations of scanners related to manufacturers compared with the master model. (b) components a, b, c, and d, when compared with the master model, about manufacturers of extraoral scanners. the components were analyzed individually and with no multiple comparisons between a, b, c, and d. same letters represent no statistical difference (a=0.05). statistical analysis this study has one dependent variable (accuracy) and two independent (extraoral scanning and components). however, before performing a statistical test, the data were treated: the master model deviation values were subtracted from all images, and the value of each sample was acquired. next, two variables were analyzed: scanners and components. when analyzing scanners, an average of the values (from the four components, considering all axis) was used to obtain a mean of each model. besides, a mean of the components for each model was performed to analyze the components. a normality test (shapiro-wilk) analyzed the measurements, and the nonparametric kruskal-wallis test was applied to analyze the scanners. the average values of components a, b, c, and d were determined by a two-way anova on ranks and a post hoc tukey test. all the tests with a 5% significance level. graphpad prism6 software (san diego, ca, usa) was used to perform the statistical tests. results considering the scanners variable, this study did not find any difference (p=0.0806). however, when analyzing by component (a, b, c, and d) and the different scanners technologies (figure 4a), there is an interaction (p<0.001) between component (p=0.001) and scanner (p=0.262). 8 gomes et al. braz j oral sci. 2023;22:e238354 this interaction is related to scanners accuracy in each component, as observed in component d, despite greater deviations, was more accurate for the ceramill map400+ model when compared with autoscan ds200+ (p<0.001) and e2 (p=0.002) (figure 4b). however, all the other components (a, b, and c) presented no statistical differences, independent of the scanners. discussion our null hypothesis was partially accepted, as we did not find statistically significant differences in accuracy among the laboratory scanners; however, we found such differences between the components. component d was the only one to present a statistical difference in digitalization accuracy, as the ceramill map400+ scanner had a better performance than autoscan ds200+ and e2. probably the difference found in the last quadrant to be scanned, precisely the component d, occurred due to an increase in the area to be digitalized. vecsei et al.31 found that the digitalization accuracy of laboratory scanners was influenced by the length of the arch included in the impression the longer the arch to be scanned, the lower the accuracy of the digital impression32,33. several images are merged when digitalizing a more extensive area, leading to progressive distortion and more significant errors17. thus, the digital impression of a complete-arch is less accurate due to the overlapping of partial scans of quadrants12. our results showed greater deviations in all extraoral systems, in components a and d: ceramill map400+ (93.7 mm / 32.4 mm), autoscan-ds200+ (113.1 mm/ 144.11 mm), and e2 (64.3 mm / 97.8 mm), respectively. these errors may be related to the interaction between the angulation of implants and the distance between the scan bodies, as both implants are positioned in the reference model extremities. these extremities might distort the last components in a complete scan. concerning the distance between scan bodies, only four implants in a completely edentulous arch result in a greater distance between the pillars. additionally, distal angulation of posterior implants may increase the final interimplant distance. referring to angulation, pan et al.34, using an experimental block that simulates the all-on-four concept, found that laboratory scanners had a significant distortion in tilted sites. in addition, sizeable interimplant distance magnified the errors induced by the 45° implants. pan et al.34 explained this finding based on light scattering and rotation. in a 3d structured light scan, light patterns are projected on the target surface and captured by cameras. therefore, minimal light obstruction from projectors to cameras is fundamental for such a difference in accuracy. thus, the undercut areas of angulated implants might be avoided because the cameras did not receive sufficient signals due to shadows, affecting scanning accuracy34. studies assessing implant angulation on digital models of intraoral scanners showed that ≤ 15º angulation does not affect scanning accuracy9,35. furthermore, regarding the distance between scan bodies, studies showed that the accuracy of laboratory scanners was not affected by interimplant distances31,33. nevertheless, according to vandeweghe et al.32, if the distance between scan bodies increases, scanning processes would become more complex, which would decrease scanning accuracy. 9 gomes et al. braz j oral sci. 2023;22:e238354 scan bodies b and c positioned parallel to each other in the anterior region showed minor deviations in scanning accuracy, probably due to the morphology of the anterior arch, which presents a linear scan path. concerning scanners, ceramill map400 showed the best results for the digital impression of a complete-arch, considering even the extremities quadrant with minor deviations. furthermore, we did not find differences between the structured light and blue led technologies. emir and ayyıldız17 analyzed the accuracy of eight different extraoral scanners and their respective technologies. the authors concluded that the blue light scanners had more accurate results than white light ones17. structured light scanners project a bi-dimensional pattern and have good scanning velocity; however, they lack repeatability and may present errors in narrow and deep areas. on the other hand, led light scanners have better scanning repeatability and fewer errors due to short wavelengths17. in this study, the scanners or the product software technology might have reduced this repeatability error in structured light scanners. despite our results, some limitations must be considered. because this is an in vitro study whose methodology was standardized, in everyday clinical practice, several variables may influence accuracy in the cad/cam method, such as the stage of the impression, material used, and scanning procedures31, as well as the device hardware, software algorithms, and scanning technology. even the shape and size of a model may significantly impact the accuracy of an extraoral scanner17. some scanners use powder during digitalization, and its thickness may contribute to differences between scanners in the final accuracy of digital impression16,23,36. although there are advances in the launch of laboratory scanners on the market, few studies have approached the accuracy of extraoral scanners in complete-arch implant rehabilitation. scientific literature is scarce, and results are divergent, meaning there is no agreement on the best extraoral systems. in conclusion, all extraoral systems showed accuracy in digitalization. however, the angulated components may result in insufficient scanning accuracy. the ceramill map400+ scanner showed the best results for the digital impression of a complete-arch, which suggests that the autoscan ds200+ and e2 scanners should be used for single or partial prostheses. acknowledgment the work was supported by são paulo research foundation – fapesp (grazielle franco gomes was supported by fapesp grant #2019/22509-9) and capes (coordination for the improvement of higher education personnel finance code 001). data availability datasets related to this article will be available upon request from the corresponding author. conflicts of interest none. 10 gomes et al. braz j oral sci. 2023;22:e238354 author contribution all authors declare that they actively participated in the discussion of the results, reviewed, and approved the final version for submission. grazielle franco gomes: substantial contributions to the conception of the work; the acquisition, analysis, interpretation of data and final approval of the version to be published. mónica estefanía tinajero aroni: drafting the work and revised it critically for important intellectual content and and final approval of the version to be published. lucas portela oliveira: substantial contributions to the conception of the work; the acquisition of data, analysis and final approval of the version to be published. joão neudenir arioli filho: interpretation of data and final approval of the version to be published. carolina mollo binda: interpretation of data and final approval of the version to be published. francisco de assis mollo júnior: substantial contributions to the conception of the work, acquisition, analysis, interpretation of data and final approval of the version to be published. references 1. carlsson ge, omar r. the future of complete dentures in oral 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for cad/cam systems an in vitro study. j prosthodont res. 2017;61(2):177-84. doi: 10.1016/j.jpor.2016.07.001. 32. vandeweghe s, vervack v, dierens m, de bruyn h. accuracy of digital impressions of multiple dental implants: an in vitro study. clin oral implants res. 2017;28(6):648-53. doi: 10.1111/clr.12853. 33. pan y, tam jmy, tsoi jkh, lam wyh, pow ehn. reproducibility of laboratory scanning of multiple implants in complete edentulous arch: effect of scan bodies. j dent. 2020;96:103329. doi: 10.1016/j.jdent.2020.103329. 34. pan y, tam jm, tsoi jk, lam wy, huang r, chen z, et al. evaluation of laboratory scanner accuracy by a novel calibration block for complete-arch implant rehabilitation. j dent. 2020;102:103476. doi: 10.1016/j.jdent.2020.103476. 35. giménez b, özcan m, martínez-rus f, pradíes g. accuracy of a digital impression system based on parallel confocal laser technology for implants with consideration of operator experience and implant angulation and depth. int j oral maxillofac implants. 2014;29(4):853-62. doi: 10.11607/jomi.3343. 36. runkel c, güth jf, erdelt k, keul c. digital impressions in dentistry-accuracy of impression digitalisation by desktop scanners. clin oral investig. 2020;24(3):1249-57. doi: 10.1007/s00784-019-02995-w. 1http://dx.doi.org/10.20396/bjos.v20i00.8661322 volume 20 2021 e211322 original article 1 graduate program in dentistry, school of dentistry, university of vale do taquari (univates), lajeado, brazil. 2 graduate program in dentistry, school of dentistry, federal university of pelotas, pelotas, brazil. 3 postgraduate program in dentistry, dental school, university of passo fundo, brazil. corresponding author: luiz alexandre chisini university of vale do taquari avelino talini st. 171 lajeado, rio grande do sul, 95914-014, brazil tel: + 55 53 981121141 e-mail: alexandrechisini@gmail.com received: september 21, 2020 accepted: december 10, 2020 are stress and symptoms of depression associated with halitosis? a cross-sectional study luiz alexandre chisini1,2,* , mariana gonzalez cademartori2 , kauê collares3 , francine dos santos costa2 , marina sousa azevedo2, flávio fernando demarco2 , marcos britto correa2 mood disorders such as stress and depression can promote alterations of several hormones aim: the present study aimed to evaluate if symptoms of depression and stress are associated with halitosis. the hypothesis is that halitosis is associated with stress and depression symptoms. methods: all university students’ entrants in the federal university of pelotas (ufpel) were invited to participate. self-reported halitosis was measured using a visual analog scale. students were also asked about the presence of halitosis perceived by close individuals. the stress level was measured using a modified version of the perceived stress scale and symptoms of depression were screened by patient health questionnaire-2. poisson regression models were performed. results: of a total of 2,058 students who participated in the study, only 16% of them have reported not having any degree of halitosis and 17.6% were informed of closed individuals having halitosis. after adjustments, stress and depression symptoms remained associated with halitosis selfreported. individuals from the higher stressed quartile presented higher scores of halitosis (rr 1.37 ci95% [1.24–1.53]). students who presented depressive symptoms showed higher scores of self-perceived halitosis (rr 1.20 ci95% [1.10–1.32]). when the association of stress and halitosis reported by close individuals was tested, symptoms of depression lost the association in the adjusted model, while individuals from the highest quartile from stress remained associated (pr 1.36 ci95% [1.02–1.81]). conclusion: halitosis was associated with stress even after control for oral health and socioeconomic variables. keywords: halitosis. depression. stress. psychological. https://orcid.org/0000-0002-3695-0361 https://orcid.org/0000-0002-2433-8298 https://orcid.org/0000-0002-7276-1074 http://orcid.org/0000-0001-9558-937x http://orcid.org/0000-0003-2276-491x http://orcid.org/0000-0002-1797-3541 2 chisini et al. introduction genuine halitosis is an oral malodor caused mainly due to volatile sulfur compounds produced by microorganisms present in the mouth affecting about 22% to 50% of individuals worldwide1. a recent systematic review investigating the global prevalence observed that above 32% of individuals are affected by bad breath1. the etiology of malodor can be originated from systemic or local factors1. in most cases, inadequate oral hygiene practices are strongly associated with self-reported bad breath2. in this way, poor oral hygiene causes oral health problems as carious lesions and periodontal disease, which in turn have also been attributed as the main reasons for malodor. moreover, unhealthy habits such as alcohol use, smoking, and an unbalanced diet can be predisposing factors to its development2,3. on the other hand, systemic diseases including gastrointestinal tract disorders, diabetes, renal and hepatic insufficiencies even as chronic sinusitis are some of the systemic factors correlated to self-report of halitosis2. the literature has also shown that psychological factors can play an important role in the prevalence of bad breath4. two different explanations have been addressed for this association. the use of medication for these disorders could alter the flow and composition of saliva leading to increased bacterial colonization and degradation of proteins present in the mouth, resulting in an elevation of the volatile sulfur-containing responsible for bad breath3,4. besides, individuals with mood disorders, such as depressive, stressed, and anxious people could present a decrease in self-care5. these individuals are less motivated to maintain oral health, culminating in poor hygiene. halitosis can be measured through the objective (i.e. organoleptic test, volatile sulfur compounds -vsc) and subjective (self-report) parameters6. objective tests require trained and calibrated examiner even as specific equipment making these measurements more expensive and difficult to conduct in population-based studies6. in this way, self-reported assessment can reduce the cost as well as the time to the examination being widely used in epidemiological studies4,7. self-reported halitosis cannot always be correlated with objective measurements (organoleptic test and vsc). in some cases, only the patient perceives the malodor, being this condition classified as pseudo-halitosis, which represents a minor part of the total diagnosed halitosis8. report of halitosis perceived by other persons, close to the individuals, could be an interesting measurement to complement the self-report measurement. although different forms of measurement and classification of halitosis are used, self-perception is perhaps the most relevant measure, since it can reflect how an individual feel about his bad breath, which strongly affects not only the social life of the individual but also interpersonal contact, which could compromise the relationships, mainly when perceived by other individuals, impacting the quality of life9. systemic disorders such as diabetes and renal/hepatic problems are less presented in young adults, so the development of halitosis in this population is more related to local and psychological factors3. in this way, individuals in academic activities are more vulnerable to develop mood disorders10. few studies have evaluated factors associated with halitosis in young individuals, in representative samples. thus, 3 chisini et al. the present study aimed to evaluate if symptoms of depression and stress are associated with self-reported halitosis and halitosis perceived by close individuals in university students in the south of brazil. the study hypothesis was that stress and depressive symptoms are associated with both measurements of halitosis (self-related and perceived by other individuals) even after adjustments by socioeconomic and oral health factors. materials and methods the present study was reported as recommended by the strobe statement (strengthening the reporting of observational studies in epidemiology) for cross-sectional studies. compete methodology is available elsewhere11-13. setting and study design pelotas is a city in southern brazil with approximately 400,000 inhabitants, considered one of the main educational poles of the state of rio grande do sul. the city has five higher education institutions being only one public: federal university of pelotas (ufpel). currently, this university has five campi dispersed in the city. approximately 13,000 undergraduate students are enrolled in this institution, where about 3,000 students enter each selection process that occurs once a year. in the first semester of 2016, a cross-sectional study was carried-out being applied to a self-reported questionnaire with entrants students of ufpel. sample size and power of the study considering the estimated number of entrants in the first half of 2015 (3000 students) and a prevalence of 50% (unknown) for the variables of interest, the margin of error of the study would be1.8 percentage points within a 95% confidence interval. for analysis of association, this sample size is sufficient to detect a prevalence ratio of 1.4, considering a prevalence of exposure of 50%, prevalence of the outcome in exposed individuals of 5%, power of 80%, and α = 5%. participants all regular entrants in the first semester of 2016 in ufpel were considered eligible for this study. the students were located by a list sent by their respective academic units. the questionnaire was applied in before lessons with the authorization of lecturers of each course. students who were not able to complete the questionnaire and those who are studying subjects without regular enrollment with the institution were excluded. students of courses out of pelotas and from distance learning courses were also excluded from the sample. data collection the fieldwork team was comprised of undergraduate and postgraduate students from ufpel dental school. all team experienced a previous theoretical training of 4 hours with a presentation of the research instruments, logistics of the study with discussion and clarification of possible doubts. in order to test the applicability of the questionnaires, a pilot study was conducted with 100 university students (n = 100) of 4 chisini et al. the second semester (not eligible for the study). five courses were randomly selected and the pilot test was performed. the questionnaire was adjusted to facilitate participants’ understanding, and the average time of 20 minutes for completing the instrument was estimated. thus, in academic units, students received an explanation of the nature of the research, and students who agreed to participate signed a free and informed consent form. the questionnaire was self-administered with multiple-choice questions. this questionnaire was divided into 5 large blocks: block asocioeconomic, demographic, and social support data; block bpsychosocial variables; block cself-perceived / subjective measures of oral health; block dbehavioral variables of oral health; block e use of alcohol, tobacco, and other substances. outcomes self-reported halitosis was measured using a visual analog scale7 accompanied by the following question: “in the scale of zero to 10, being zero no odor and 10 extremely malodor, mark how do you rate your breath?”4,7. responses from 0 to 10 were obtained and the variable was analyzed in discrete form. the students were also asked about the presence of bad breath referred by a close person through the following question: “has any family member/friend/boyfriend ever reported that you have bad breath?” with the possible answers “yes” and “no”. independent variables demographic characteristics including sex and age (18 to 24; 25 to 34; and 35 or more) were collected. family income was collected categorically in brazilian reals brl: (a) up to 500.00; b) 5,001.00 up to 1,000.00; c) 1,001.00 up to 2,500.00; d) 2,501.00 up to 5,000.00; e) 5,001.00 up to 10,000.00; and f) more than 10,001.00) and categorized in three categories: a) ≤ 1,000.00; b) 1,001.00 to 5,000.00 and c) ≥ 5,001.00. oral health status was investigated using self-report measures. gingival bleeding was estimated by the question: “do your gums bleed when brushing your teeth?” (no; sometimes; and always). experience of dental caries was verified using the question: “do you actually have or have you ever had any tooth affected by tooth decay?” (yes or no). the use of alcohol and tobacco was collected in a separate questionnaire where only one numeric code linked the information of this questionnaire with the other information. in addition, the questionnaires were collected separately in an opaque box to increase the impersonality and consequently improve the reliability of the data collection. thus, the students were asked about smoking habits and consumption of alcoholic beverages, as well as their frequency. those who reported that “drink daily or almost daily” were considered as consumers of alcoholic beverages and those who reported “smoking at least weekly” were considered as smokers. stress’ level was measured using a modified version of the perceived stress scale (pss) validated for the portuguese language. this questionnaire is comprised of ten questions related to the past month, which reflect events and situations, four questions positives, and six negatives. each question presents five options in a likert scale, ranging from 1 = never; 2 = almost never; 3 = sometimes; 4 = less common; and 5 = very often. scores of the positive questions are reverse-scored and negative scores are normally scored, thus all scales range from 0 to 40 points. a higher score 5 chisini et al. indicates greater stress. thus, the variable was categorized in quartiles14. depression symptoms were investigated using patient health questionnaire-2 (phq-2), an abbreviated version of patient health questionnaire-9 (phq-9) for depression screening at the population level. the phq-2 represents the first two questions of phq-9: “over the last 2 weeks, how often have you been bothered by any of the following problems? 1) “little interest or pleasure in doing things” and 2) “feeling down, depressed, or hopeless” with possible responses “not at all”, “several days”, “more than half the days”, and “nearly every day”. the responses were scored respectively 0 to 3. the two questions can range from 0 to 6. individuals that presented a score of 3 or higher were classified with symptoms of depression. statistical methods data were double entered in a spreadsheet of the software excel 2013 (microsoft corporation). statistical analyses were performed using stata 12.0 software (stata corporation, college station, tx, usa). the relative and absolute frequencies of the variables of interest were estimated. multivariate poisson regression models with robust variance were performed to analyze the associations between halitosis and stress and symptoms of depression. self-reported halitosis was analyzed as a discrete outcome and halitosis perceived by other individuals was evaluated in a dichotomous way. variables with p values of ≤0.250 in the crude analyses were included in the model fitting. a backward stepwise procedure was used to include or exclude explanatory variables in the model fitting. variables included in the final adjusted model should present a p-value ≤ of 0.250. for the final model, the variables were considered significant if they had a p-value of ≤ 0.05 after adjustments. prevalence ratios were obtained for halitosis referred by close persons and rate ratios were obtained for self-reported halitosis. both effect measures were estimated with 95% confidence intervals. due to the high correlation between stress and symptom of depression (correlation coefficients = 0.422), the results for depression were not adjusted by stress. ethical issues the institution (ufpel) and the research ethics committee of the school of medicine / ufpel approved this study under protocol 49449415.2.0000.5317. results of total eligible students identified (n=3,237), 63.6% (n= 2,058) signed the consent term and participated of the study, being 52.3% female (ic 95% 50.0 – 54.4). refusals represented only 1.4% of the total sample. around 66% of the sample were between 18 to 24 years old, with a family income ranging from 1,001.00 to 5,000.00 brl (71.4%) (table 1). besides, about 16% of students presented symptoms of depression. scores mean of stress was 16.3 (sd ± 6.8), of which 31.1% present more than 20 points in the score. concerning oral health variables, almost 50% have reported no presence of gingival bleeding and nearly 33% have reported never presented dental caries. only 16% of subjects have reported not having some degree of halitosis, while 61.3% reported a score between one and three vas (figure 1). furthermore, 363 students (17.6%) were informed by closed individuals that they had halitosis. 6 chisini et al. figure 1. self-reported halitosis in analog visual scale (n=2054) se lfre po rt ed h al ito si s (% ) analogue visual scale 25 20 15 10 5 0 0 2 4 6 108 table 1. description of the general characteristics of the sample. variable/category total sample n (%) (ci 95%) sex 2080 female 1,087 (52.2) (50.0 – 54.4) male 993 (47.7) (45.6 – 49.9) age (years) 2080 16 to 17 312 (15.0) (13.5 – 16.6) 18 to 24 1,375 (66.1) (64.0 – 68.1) 25 to 34 215 (10.3) (09.6 – 11.7) 35 or more 178 (8.6) (07.4 – 09.8) family income 1717 ≤ 1000 280 (16.3) (14.6 – 18.1) 1001 to 5000 1,057 (61.6) (59.2 – 63.9) ≥ 5001 380 (22.1) (20.2 – 24.2) gingival bleeding 2,073 no 1,020 (49.2) (47.0 – 51.4) some times 955 (46.1) (43.9 – 48.2) always 98 (4.7) (03.9 – 05.7) dental caries experienced 2,077 no 684 (32.9) (30.09 – 35.0) yes 1,393 (67.1) (65.00 – 69.1) smoking 2,025 no 1,812 (89.5) (88.1 – 90.8) yes 213 (10.5) (09.2 – 11.9) alcohol 1,929 no 1,845 (95.7) (94.6 – 96.5) yes 84 (4.3) (03.5 – 05.4) depression symptoms 2,082 no 1,743 (83.7) (82.06 – 85.3) yes 339 (16.3) (14.72 – 17.9) stress (quartile) 2,089 1st (pss scores 0 – 11) 550 (26.3) (24.5 – 28.3) 2dn (pss scores 12 – 16) 571 (27.4) (25.4 – 29.3) 3rd (pss scores 17 – 21) 489 (23.4) (24.6 – 25.3) 4rd (pss scores 22 – 40) 479 (22.9) (21.1 – 24.8) perceived stress scale (pss) 7 chisini et al. table 2 shows the association between stress/symptoms of depression and self-perceived halitosis. in the crude model, individuals whom self-perceived halitosis showed to be associated with stress (p < 0.001). individuals in the higher quartile of stress showed a rate ratio (rr) of halitosis 38% greater than individuals from the less stress quartile (rr 1.38 ci95% [1.25 – 1.52]). individuals with depression symptoms too showed association with self-perceived halitosis (rr 1.23 ci9% [1.13 – 1.34]). after adjustments by sex, age, family income, and gingival bleeding (control variables that continued associated with self-perceived halitosis in the final model), both stresses even as depression symptoms remained associated with self-reported halitosis. individuals from higher stressed quartile presented higher scores of halitosis (rr 1.37 ci95% [1.24 – 1.53]) as well as individuals form intermediates stressed quartiles 3rd (rr 1.22 ci95% [1.09 – 1.35]) and 2nd (rr 1.14 ci95% [1.02 – 1.27]). similarly, students who presented depressive symptoms showed higher scores of self-perceived halitosis (rr 1.20 ci95% [1.10 – 1.32]) in the final model. table 2. crude (c) and adjusted (a) rate ratio (rr) of independent variables for halitosis self-perception in university students. pelotas, rs, brazil. poisson regression (n=1668). variable/category rrc (ci95%) p-value rra(ci95%) p-value sex(ref=female) 0.244 0.029 male 1.04 (0.97 – 1.11) 1.09 (1.01 – 1.17) age (yrs)(ref=16 to 18) 0.005 0.002 18 to 24 1.07 (0.98 – 1.17) 1.02 (0.92 – 1.11) 25 to 34 1.09 (0.95 – 1.24) 1.06 (0.92 – 1.22) 35 or more 1.23 (1.07 – 1.40) 1.25 (1.08 – 1.44) family income(ref= ≤ 1000) 0.001 0.018 1001 to 5000 0.90 (0.81 – 0.99) 0.92 (0.83 – 1.01) ≥ 5001 0.81 (0.71 – 0.91) 0.86 (0.76 – 0.97) gingival bleeding(ref=no) < 0.001 < 0.001 some times 1.21 (1.13 – 1.31) 1.19 (1.10 – 1.29) always 1.54 (1.34 – 1.76) 1.51 (1.30 – 1.74) dental caries experienced(ref=no) 0.714 yes 1.01 (0.94 – 1.09) smoking(ref=no) 0.744 yes 1.02 (0.91 – 1.14) alcohol(ref=no) 0.431 yes 0.93 (0.79 – 1.11) depression symptoms(ref=no) * < 0.001 < 0.001 yes 1.23 (1.13 – 1.34) 1.20 (1.10 – 1.32) stress (quartile)(ref= 1st pss scores 0 – 11) < 0.001 < 0.001 2nd (pss scores 12 – 16) 1.14 (1.03 – 1.26) 1.14 (1.02 – 1.27) 3rd (pss scores 17 – 21) 1.22 (1.10 – 1.34) 1.22 (1.09 – 1.35) 4rd (pss scores 22 – 40) 1.38 (1.25 – 1.52) 1.37 (1.24 – 1.53) -2 loglikelihood empty model = 8,158.3 final model = 6,562.2 perceived stress scale (pss); * depression was not adjusted by stress; the results displayed in final model are adjusted by stress 8 chisini et al. when the association of stress and halitosis reported by close individuals was tested (table 3), stress (p = 0.012) and symptoms of depression (p = 0.039) were associated with outcome in the bivariate analysis. individuals that presented the greatest scores of stress have shown a prevalence of almost 50% higher than the individual with less stress. similarly, students with symptoms of depression presented a prevalence of 27% higher than those individuals without depressive symptoms. on the other hand, after adjustments (by sex, family income, gingival bleeding, and dental caries), symptoms of depression lost the association, while individuals from the highest quartile from stress remained associated with halitosis reported by close individuals (pr 1.36 ci95% [1.02 – 1.81]). table 3. crude (c) and adjusted (a) prevalence ratio (pr) of independent variables for halitosis reported by the closed person in university students. pelotas, brazil. poisson regression. (n=1686). variable/category prc (ci95%) p-value pra (ci95%) p-value sex(ref=female) 0.039 0.008 male 1.22 (1.01 – 1.47) 1.31 (1.08 – 1.61) age (yrs)(ref=16 to 18) 0.335 18 to 24 0.93 (0.71 – 1.21) 25 to 34 0.84 (0.57 – 1.25) 35 or more 1.27 (0.89 – 1.82) family income(ref= ≤ 1000) 0.008 0.034 1001 to 5000 0.82 (0.64 – 1.06) 0.85 (0.66 – 1.09) ≥ 5001 0.65 (0.47 – 0.90) 0.70 (0.51 – 0.98) gingival bleeding(ref=no) < 0.001 < 0.001 some times 1.82 (1.48 – 2.23) 1.69 (1.35 – 2.11) always 3.09 (2.26 – 4.20) 2.87 (2.05 – 4.02) dental caries experienced(ref=no) 0.044 0.037 yes 1.24 (1.01 – 1.53) 1.27 (1.01 – 1.59) smoking(ref=no) 0.956 yes 1.01 (0.74 – 1.37) alcohol(ref=no) 0.890 yes 0.97 (0.59 – 1. 57) depression symptoms(ref=no)* 0.039 0.335 yes 1.27 (1.01 – 1.60) 0.335 stress (quartile)(ref= 1st pss scores 0 – 11) 0.012 0.061 2nd (pss scores 12 – 16) 1.23 (0.94 – 1.61) 1.10 (0.82 – 1.46) 3rd (pss scores 17 – 21) 1.10 (0.82 – 1.46) 1.03 (0.76 – 1.40) 4rd (pss scores 22 – 40) 1.48 (1.14 – 1.93) 1.36 (1.02 – 1.81) -2 loglikelihood empty model = 1,988.8 final model = 1,610.9 perceived stress scale (pss); * depression was not adjusted by stress; the results displayed in final model are adjusted by stress 9 chisini et al. discussion the present study has evaluated the occurrence of halitosis from two points of view; self-reported and perceived by close individuals. from the total students investigated, 84% have reported some degree of halitosis and near 18% reported that they were informed they had halitosis by close individuals. even after controlling by socioeconomic and gingival bleeding – the main local factor related to halitosis – more stressed individuals remained associated using both measures showing to be an important factor related to halitosis. on the other hand, students who showed depressive symptoms remained associated only with self-reported halitosis losing the association in the adjusted models from halitosis perceived by close individuals. genuine halitosis has been classified as the halitosis confirmed for organoleptic tests being objectively perceived while the pseudo-halitosis or halitophobia can occur even in cases when the bad breath is not detected by this test and for other individuals15. although the organoleptic test has been considered the “gold standard” on halitosis evaluation, the use of a self-reported questionnaire has been used in the literature4,7. a recent study of meta-analysis comparing the prevalence obtained with clinical and self-reported measurements observed that the method adopted to evaluate halitosis did not influence the heterogeneity among studies1. to decrease this possible bias, we have asked students also if some close person had ever reported him/her about the presence of bad breath. this question was used to evaluate if the presence of halitosis perceived by other persons could also be associated with psychosocial variables and, thus, compare with results of self-perceived halitosis (pseudo-halitosis), promoting more robust evidence. in this way, when self-perceived halitosis was investigated, a strong association with stress was observed. even after adjustments, individuals with some degree of stress were related to higher scores of self-perceived halitosis. this can be explained due to the high correlation between self-reported measurements and pseudo-halitosis15 since that patient with some mood disorder could present a self-perception or self-judgment worse than other people due to psychological alterations that can change their perception of health5. on the other hand, in halitosis perceived by close individuals, the association was only observed in the students from the highest quartile of stress. these results corroborate with a study that has shown a strong relationship between halitosis and the levels of cortisol present in the saliva, mainly in pseudo-halitosis15. moreover, another hypothesis is that it may due to the inclusion of only cases of genuine halitosis (perceived by other individuals) in the second halitosis question, which may show that only high levels of stress can act influencing genuine halitosis while the self-perception could be more easily influenced, although to confirm this hypothesis it is necessary an objective evaluation. despite stressed individuals presents an association with halitosis, we cannot establish a causal relationship between halitosis and stress, due to the design of the present study. individuals with an elevated level of stress could present the tendency to answer more easily bad outcomes (bad breath) than individuals with a low level of stress. also, stress can act leading to other conditions that cause halitosis (such as gastritis and reduced salivary flow). besides, the presence of halitosis could conduct individuals to present with higher stress, since that bad breath impacts on quality of life9. 10 chisini et al. stress and depression are comorbidities and normally can coexist as conditions related to mood disorders presenting elevate correlation16. similarly to the observed in the literature, we found a significant coefficient of correlation among these variables and, thus, we performed individual regression models to these variables. an explanation of this correlation is the act of mood disorders promotes the alteration of several hormones, stimulating biochemical modifications17,18, i.g., the increase of levels of cortisol promoted by stress, that could significantly have altered the serotonin uptake, hence, influencing the depressive symptoms17,18. this biochemical mechanism is attributed to the induction by the cortisol that promotes an increase in the expression of the gene that encodes the serotonin transponder, resulting in a drastic decrease of serotonin involved in brain synapses18. however, not all individuals exposed to stress displaying depression and some genetic factors could influence this relationship19. a longitudinal birth cohort tested the influence of stress in the polymorphism of gene 5-htt displaying results that support this theory19. this gene presents a short allele “s” in the region 5-httlpr (prevalence near 50% of caucasian individuals with one “s” allele) that promotes less efficiency in the gene transcription, which could influence the response of serotonin to stressful events. a direct association of this gene and depression was not observed, but when the stressful events that occurred in the life course were considered in the analysis, the positive interaction of the investigated alleles and depression was observed19. these results highlight the associations of stress and depression and the possible mechanisms of correlation of these diseases. in the present study, while stress remained associated in both halitosis questions, this did not occur with depression, which lost the association in the adjusted model in the question of halitosis perceived by close individuals. young individuals presented a less prevalence of depression compared to stress20 as well as stress prevailed over signs of depression or act as a trigger for depression21. thus, being a young population (mean of 23 years), it seems that stress was the variable that presented a key role related to halitosis. besides, in the present study, we use an instrument for screening symptoms of depression and did not perform a clinical diagnostic. however, the use of phq-2 in screening depression symptoms is a useful and time-saving validated instrument. the evaluation of the occurrence of halitosis by two points of view promotes greater robustness to the outcome and helps to explain with more detail the effect of stress and depression in halitosis. therefore, we observed that stress was the main psychological factor that remained associated in both halitosis measurements at the final model in the studied population. it is important to evidence that in both measurements of halitosis the socioeconomic and oral health variables were similarly associated, showing a consistent measurement among the outcome variables. moreover, the combined use of both questions can provide major robustness of the results. universities’ students are a low investigated population, which presents a vulnerability to develop mood disorders10. our results showed an important part of the sample with symptoms of depression and with elevated levels of stress, corroborating with previous studies, an indication of the necessity of intervention programs to reduce these disorders16. the use of cognitive and behavioral approaches was used to decrease bruxism associated with stress22 and can be recommended to decrease the stress 11 chisini et al. levels in this population and a more precise investigation of depressive symptoms is necessary16. besides, the investigation of the association between halitosis and stress/symptoms of depression in this population is very important, since that halitosis can impact the oral health-related quality of life9. however, some limitations have to be highlighted. due to the sample of university students, individuals with low schooling were not included in the present study, which may have influenced our results by underestimating the prevalence. therefore, extrapolation data should be performed with high caution. the present findings can be extrapolated to a population with high educations levels and with elevated socioeconomic status. the losses were mainly caused by the impossibility of the location of individuals in their respective classrooms. however, similar questionnaire-based studies also showed this difficulty23. also, the oral health variables were investigated by self-report measurements. although no oral health clinical examination has been performed, the self-reported oral health condition is a valid tool used. besides, we observed that self-reported gingival bleeding (presents in the gingivitis and periodontitis) was a variable strongly associated in final models with both halitosis measurements. although the most precise measure is the organoleptic test, self-referred measures are fundamental in epidemiological studies because they are simple, low-cost measures, besides being able to reduce the time of examination4,7. in conclusion, both halitosis measures (self-reported and reported by close individuals) were associated with stress even after control for oral health and socioeconomic variables. in contrast, symptoms of depression were associated only with self-reported halitosis. declaration of interest statement: the authors report no conflict of interest. references 1. silva mf, leite frm, ferreira lb, pola nm, scannapieco fa, demarco ff, et al. estimated prevalence of halitosis: a systematic review and meta-regression analysis. clin oral investig. 2018 jan;22(1):47-55. doi: 10.1007/s00784-017-2164-5. 2. al-ansari jm, boodai h, al-sumait n, al-khabbaz ak, al-shammari kf, salako n. factors associated with self-reported halitosis in kuwaiti patients. j dent. 2006 aug;34(7):444-9. doi: 10.1016/j.jdent.2005.10.002. 3. kim sy, sim s, kim sg, park b, choi hg. prevalence and associated factors of subjective halitosis in korean adolescents. plos one. 2015 oct;10(10):e0140214. doi: 10.1371/journal.pone.0140214. 4. lee e, lee yh, kim w, kho hs. self-reported prevalence and severity of xerostomia and its related conditions in individuals attending hospital for general health examinations. int j oral maxillofac surg. 2014 apr;43(4):498-505. doi: 10.1016/j.ijom.2013.10.011. 5. takiguchi t, yoshihara a, takano n, miyazaki h. oral health and depression in older japanese people. gerodontology. 2016 dec;33(4):439-46. doi: 10.1111/ger.12177. 6. yaegaki k, coil jm. genuine halitosis, pseudo-halitosis, and halitophobia: classification, diagnosis, and treatment. compend contin educ dent. 2000 oct;21(10a):880-6, 888-9; quiz 890. 7. rosenberg m, kozlovsky a, gelernter i, cherniak o, gabbay j, baht r, et al. self-estimation of oral malodor. j dent res. 1995 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pires a, corrêa m, demarco f. desire of university students for esthetic treatment and tooth bleaching: a cross-sectional study. braz j of oral sci. 2019;18:e191648. doi: 10.20396/bjos.v18i0.8657267. 13. chisini l, queiroz a, lima f, silva l, cademartori m, costa f, et al. is obesity associated with tooth loss due to caries? a cross-sectional study. braz j of oral sci. 2020;19:e201088. doi: 10.20396/bjos.v19i0.8661088. 14. white rs, jiang j, hall cb, katz mj, zimmerman me, sliwinski m, et al. higher perceived stress scale scores are associated with higher pain intensity and pain interference levels in older adults. j am geriatr soc. 2014 dec;62(12):2350-6. doi: 10.1111/jgs.13135. 15. fukui m, hinode d, yokoyama m, yoshioka m, kataoka k, ito ho. levels of salivary stress markers in patients with anxiety about halitosis. arch oral biol. 2010 nov;55(11):842-7. doi: 10.1016/j.archoralbio.2010.07.014. 16. beiter r, nash r, mccrady m, rhoades d, linscomb m, clarahan m, et al. the prevalence and correlates of depression, anxiety, and stress in a sample of college students. j affect disord. 2015 mar;173:90-6. doi: 10.1016/j.jad.2014.10.054. 17. tafet ge, toister-achituv m, shinitzky m. enhancement of serotonin uptake by cortisol: a possible link between stress and depression. cogn affect behav neurosci. 2001 mar;1(1):96-104. doi: 10.3758/cabn.1.1.96. 18. tafet ge, idoyaga-vargas vp, abulafia dp, calandria jm, roffman ss, chiovetta a, et al. correlation between cortisol level and serotonin uptake in patients with chronic stress and depression. cogn affect behav neurosci. 2001 dec;1(4):388-93. doi: 10.3758/cabn.1.4.388. 19. caspi a, sugden k, moffitt te, taylor a, craig iw, harrington h, et al. influence of life stress on depression: moderation by a polymorphism in the 5-htt gene. science. 2003 jul 18;301(5631):386-9. doi: 10.1126/science.1083968. 20. ben loubir d, serhier z, diouny s, battas o, agoub m, bennani othmani m. prevalence of stress in casablanca medical students: a cross-sectional study. pan afr med j. 2014 oct;19:149. doi: 10.11604/pamj.2014.19.149.4010. 21. monroe sm, slavich gm, gotlib ih. life stress and family history for depression: the moderating role of past depressive episodes. j psychiatr res. 2014 feb;49:90-5. doi: 10.1016/j. jpsychires.2013.11.005. 22. chisini la, san martin as, cademartori mg, boscato n, correa mb, goettems ml. interventions to reduce bruxism in children and adolescents: a systematic scoping review and critical reflection. eur j pediatr. 2020 feb;179(2):177-89. doi: 10.1007/s00431-019-03549-8. 23. chisini la, conde mc, correa mb, dantas rv, silva af, pappen fg, et al. vital pulp therapies in clinical practice: findings from a survey with dentist in southern brazil. braz dent j. 2015;26(6):566-71. doi: 10.1590/0103-6440201300409. 1 volume 22 2023 e230961 original article braz j oral sci. 2023;22:e230961http://dx.doi.org/10.20396/bjos.v22i00.8670961 1 post graduate program in oral and maxillofacial surgery, piracicaba dental school, university of campinas (unicamp), piracicaba, sp, brazil. 2 post graduate program in human movement sciences, methodist university of piracicaba (unimep), piracicaba, sp, brazil. 3 graduate program in physical therapy, state university of northern paraná (uenp), jacarezinho, pr, brazil. 4 post graduate program in oral and dental biology, piracicaba dental school, university of campinas (unicamp), piracicaba, sp, brazil. corresponding author: elisa bizetti pelai post graduate program in oral and maxillofacial surgery, piracicaba dental school, state university of campinas – unicamp – av. limeira, 901 areião, piracicaba sp, 13414-903. e-mail: elisabpelai@gmail.com editor: dr. altair a. del bel cury received: september 8, 2022 accepted: may 2, 2023 normalization of the electromyographic signals of masticatory muscles during non-habitual chewing activity elisa bizetti pelai1* , ester moreira de castro-carletti2 , fabiana foltran-mescollotto2 , paulo fernandes pires3 , fausto berzin4 , marcio de moraes1 , delaine rodrigues-bigaton1 there is no consensus on the most appropriate method for normalizing electromyography (emg) signals from masticatory muscles during isotonic activity. aim: to analyze the best method for data processing of the emg signal of the masticatory muscles during isotonic activity (non-habitual chewing), comparing raw data and different types of normalization. methods: this is a cross-sectional study. women aged between 18 and 45 years were selected. anthropometric data were collected (age, height, body mass index – bmi, masticatory preference) as well as emg signal (root mean square – rms) data for the anterior temporal and masseter bilaterally, and for the suprahyoid muscles, during isotonic (non-habitual chewing) and isometric tasks. emg data were processed offline using matlab® software. the normalization of the emg signal was carried out using the 2nd masticatory cycle, chosen at random, of the 20 cycles collected, the maximum rms value, and the maximum voluntary contraction (mvc). to analyze the best method of data processing for the isotonic data, the coefficient of variation (cv) was calculated. descriptive data analysis was adopted, using the mean and standard deviation. anova with repeated measures was used to detect significant differences between the methods of normalization. statistical significance was set at 5% (α<0.05). results: the final sample of this research was composed of 86 women. the volunteers presented an average age of 27.83±7.71 years and a mean bmi of 22.85±1.91 kg/m2. regarding masticatory preference, 73.25% reported the right side, and 26.75% the left side. considering the comparison between the methods, the https://orcid.org/0000-0003-0826-9744 https://orcid.org/0000-0002-0688-165x https://orcid.org/0000-0002-6145-7448 https://orcid.org/0000-0003-3191-4772 https://orcid.org/0000-0002-9179-1893 https://orcid.org/0000-0002-5229-5723 https://orcid.org/0000-0002-3423-5575 2 pelai et al. braz j oral sci. 2023;22:e230961 introduction with the increase in the use of surface electromyography (emg) in clinical and academic practice, it is extremely important to develop protocols to ensure the correct interpretation of data, such as the normalization of the emg signal1. adherence to standardized methods allows the acquisition of reliable and valid data that, in turn, facilitates the interpretation and correct comparisons between the results obtained2. the current literature reports the need to standardize the emg signal normalization procedure3. emg normalization is a mathematical procedure, whereby the absolute emg data is divided by a reference value4. anatomical and physiological factors can significantly influence the amplitude of the electromyographic signal, leading to high variation and heterogeneity in the signal for comparison. to truly compare the emg signal between individuals, muscles, and/or days, normalization is necessary5-7. a recent systematic review found that only 50% of studies with electromyography (that evaluated the isotonic condition) of the masticatory muscles described the method used to normalize the data, and there is no standardization8. it is worth mentioning that in the research carried out by the authors, studies were found that evaluated normalization for isometric conditions, however, no studies were found that evaluated normalization for isotonic conditions taking into account the masseter, temporal, and suprahyoid muscles. there are many ways that the literature shows to normalize the emg data. the maximum isometric contraction (mvc) is a common method of normalization, however, individuals with control-limiting conditions or muscle weakness are unable to efficiently participate in this method9. the maximum rms and dynamic tasks (ie. second cycle) are also usual to normalize this data. but there is no consensus in the literature regarding this topic in the masticatory muscles during isotonic activity. the current study is justified due to the heterogeneity of the studies that use emg for masticatory muscles and, in addition, to analyze the different forms of normalization and propose the format with the least data variability. the importance of using normalization of the emg signal that reduces signal variability while preserving its characteristics is known7, therefore, it is important to define the best way to process the data. within the literature search carried out by the authors, this is the first study to compare raw data and methods of normalization during isotonic activity. therefore, the current study aimed to analyze the best method of data %cv measure of the 2nd cycle showed the lowest variation coefficient during biting for all the muscles from the raw data, rms max, and mvc (p=0.001, p=0.003, and p=0.001 respectively). conclusion: in conclusion, for non-habitual chewing activity, the results of this study recommend data processing using normalization with the second cycle during chewing. keywords: electromyography. masticatory muscle. mastication. muscles. 3 pelai et al. braz j oral sci. 2023;22:e230961 processing of the emg signal of the masticatory muscles during isotonic activity (non-habitual chewing) comparing raw data and different types of normalization. methods study design this is a cross-sectional study, approved by the research ethics committee of the local university, protocol no. 25/2015. subjects a sample size calculation was performed, based on a pilot study, composed of 20 volunteers. the outcome used was surface emg, the main outcome of the present study. considering the evaluated muscles, the mean normalized rms value of the anterior temporal muscle during the biting phase was considered because it has a lower standard deviation value (86.25±7.42%). according to the variables, an effect size of 0.34 was found. after setting power of 95% and a 5% alpha, an n of 82 volunteers was determined. the sample size calculation was performed using gpower® software, version 3.1.9.2. the volunteers were recruited in a surgery sector of a school of dentistry in a city in the interior of são paulo state, brazil. the volunteers were recruited through an advertisement at the university between january and december of 2017. all volunteers signed a statement of informed consent. inclusion and exclusion criteria healthy women aged between 18 and 45 years, with a body mass index (bmi)<25 kg/ m² were selected for the study. volunteers who were toothless, wear dental prothesis, and/or present osteoarthrosis of the temporomandibular joint were excluded. the sample consisted only of women because this study intended to perform analysis with electromyography, so the inclusion of both sexes in the same comparison is not indicated. materials electromyography the emg 830c signal acquisition module (emg system do brasil, são josé dos campos, brazil) was used for reading the semg signals, with an impedance of >10 mω, analog/digital converter, 16-bit resolution, sampling frequency of 2000 hz, and fourth-order butterworth filter (high-pass filter set at 20 hz and low-pass filter set at 1000 hz). five differential surface electrodes were used, (self-adhesive ag/agcl electrodes had a conductive gel), with a fixed inter-electrode distance of 10 mm, gain of 20×, common rejection mode greater than 130 db, input impedance of 10 gω, and signal/noise ratio of less than 3 μv rms. 4 pelai et al. braz j oral sci. 2023;22:e230961 the skin was cleaned with 70% alcohol before the placement of the electrodes, positioned following the criteria proposed by cram10. in addition. a reference electrode (30 × 40 mm) consisting of a metal plate was positioned on the manubrium of the sternum. on the anterior temporalis muscles, the electrodes were positioned vertically, 3cm along the zygomatic arch, just lateral to the eyebrow. on the masseter muscles, the electrodes were positioned parallel to the muscular fibers, between the cheekbone and the corner of the jaw, with the upper pole of the electrode at the intersection between the tragus-labial commissure and the evocation–gonion lines. on the suprahyoid muscles, one electrode was fixed on the midline under the chin, running in the anterior-to-posterior direction, over the muscle mass felt in the submandibular region10,11 (figure 1). a b figure 1. positioning of the different bipolar electrodes on the masseter, anterior temporal, and suprahyoid muscles, and electrode monopolar reference: a) anterior view and b) profile. procedures anthropometric data were collected (age, height, body mass). to collect the emg signal, all volunteers were asked to sit on a chair with their feet flat on the floor, hands on the lower limbs (knee and hip 90°), feet and chair on a rubber mat, and head parallel to the ground, concerning the frankfurt plane. for the data collection, isotonic and isometric tasks were performed as described below (figure 2): non-habitual chewing a sheet of parafilm m® (american national can tm, chicago, il, usa) was used, folded three times in length and then in the middle in width. the volunteers were 5 pelai et al. braz j oral sci. 2023;22:e230961 required to position the parafilm m® on the occlusal surfaces of the first and second upper and lower molars bilaterally during collection to protect the teeth11,12. thus, the volunteers were familiarized with the task. two chewing repetitions were requested for 20 seconds each. the chewing was required to be carried out according to the rhythm of an ma-30 digital metronome korg brand (new market, usa), set at 60 beats per minute. for each repetition of chewing, the volunteers performed 20 full mouth biting/opening cycles, one cycle per second according to the pre-determined rhythm of the metronome. the volunteers were previously trained to perform the task. one cycle was considered as one bite and open. isometric procedure the volunteers were asked to perform a bilateral molar bite on a dynamometer, with the maximum voluntary contraction (mvc). they were asked to bite with maximum strength, even if they felt pain in the temporomandibular joint. the mvc was collected for 5 seconds, and this evaluation was performed twice. 15 mm a b figure 2. a) sheet of parafilm m®; b) bite dynamometer. data processing emg data were processed offline using matlab® software 8.5.0.1976.13 (r2015a, mathworks inc., natick, massachusetts, usa). for the processing of the signal, the cycles of activity were used. one phase of clenching the teeth (a contraction of the mandibular elevating muscles, denominated the biting phase) and mouth opening (a contraction of the depressor muscles of the mandible, denominated the mouth opening phase) was defined as the masticatory cycle. six central masticatory cycles of each collection in the emg signal were considered to avoid interferences that could have occurred at the beginning and end of the collection, as well as to guarantee the standardization of the signal analyzed. in this way, the average of the sum of all the rms values of the six cycles performed was used. it is important to note that all muscles started and ended the contraction of the biting 6 pelai et al. braz j oral sci. 2023;22:e230961 phase simultaneously, and there were no changes in the action potential of the motor units in the agonist muscles of the biting phase. the six central masticatory cycles of each collection were manually selected through matlab® software. three cuts were performed for each of the six cycles selected in the analysis for each attempt by the volunteers. the first cut was performed at the beginning of the biting phase, the second cut at the end of the biting phase (which also represented the beginning of the mouth opening phase), and the third cut at the end of the mouth opening phase (which in turn represented the beginning of the next cycle biting phase). for the statistical analysis, the mean of the six cycles of each repetition was calculated, to obtain the average of the electrical activity of each muscle (anterior temporal and masseter bilaterally and suprahyoid) in the biting phase and the average of each muscle in the mouth opening phase. for the emg signal processing and maximum bite force, a 4th order digital butterworth filter was applied to the emg signal, with zero phase delay (high pass of 10 hz, low pass of 400 hz). the first and second emg signals were always eliminated to avoid interferences that occurred at the beginning and end of each collection. the emg indices were processed in the amplitude domain to determine the root mean square (rms) values, through the evaluation of the magnitude of the electrical activity of the masticatory muscles during the mvc. from this process the result was the raw data. subsequently, three different methods were performed to normalize the electromyographic signal, as follows: the second masticatory cycle of the 20 cycles collected, which was chosen at random; the maximum rms value; and the mvc. the normalization is usually calculated by dividing the raw electromyographic data by a reference value, in this case, the normalization of the emg signal during the biting and mouth opening phases of the muscles using each normalization value (second masticatory cycle, maximum rms, and medium rms) was performed using the following formulas4: x 100 mean rms of the six cycles in the bite phase normalization value ; average rms of the six cycles in the mouth phase normalization value x 100 statistical analysis to analyze the best normalization method for the isotonic data the coefficient of variation (cv) was calculated. the cv is a relative measure of variability that indicates the size of a standard deviation from its mean. it is a standardized, unitless measure that enables comparison of the variability between disparate groups and characteristics13. the cv was calculated for the raw signal, and for the three types of normalization (i.e., second masticatory cycle, maximum rms, and mvc), to verify which normalization method presented the smallest variation. the cv measures the relationship between the standard deviation and the mean. thus, lower values indicate a more homogeneous data set. the values are expressed in percentages. a cv is considered low (indicating a reasonably homogeneous data7 pelai et al. braz j oral sci. 2023;22:e230961 set) when it is less than or equal to 25%. the formula used to calculate the coefficient of variation was described by reed et al. (2017), as follows: cv = x̄ • 100 σ the descriptive data are expressed in mean and standard deviation. to compare the means of each four methods of data processing and detect significant differences with a fewer type i errors, analysis of variance (anova) with repeated measures was used. statistical significance was set at 5% (α<0.05). data analysis was performed using spss software, version 13.0. results the final sample of this research was composed of 86 women, according to the flowchart shown in figure 3. volunteers recruited for the research n = 127 eligible volunteers n = 86 raw data not eligible n = 41 normalized by the 2nd cycle normalized by the rms max normalized by the mvc male (n = 9) bmi > 25 kg/m (n = 6) age > 40 years old (n = 9) age < 18 years old (n = 3) refused to participate in this study (n = 14) figure 3. flowchart of the recruitment of volunteers. the volunteers presented an average age of 27.83±7.71 years and a mean bmi of 22.85±1.91 kg/m2. regarding masticatory preference, 73.25% (63 volunteers) reported preferring the right side, and 26.75% (23 volunteers) preferred the left side. table 1 presents the mean, standard deviation, and coefficient of variation (cv) values for raw data and data normalized by the second cycle, maximum rms, and mvc. this value shows what is expected in the literature. in the mouth opening phase, the suprahyoid muscles are in contraction therefore, their muscle activity is greater than other muscles (temporalis and masseter) which are at rest. in the biting phase is the opposite, the suprahyoid muscles are at rest and the temporalis and masseter are in contraction because they are mandibular elevators, so, they have a higher muscle activity. 8 pelai et al. braz j oral sci. 2023;22:e230961 the cv values are expressed in percentages for the masticatory muscles (temporalis and masseter bilaterally), for the mouth opening and biting phases. a cv with values lower than 25 % is considered low which means that it indicates a reasonably homogenous dataset. therefore, normalizing by 2nd cycle value would be ideal table 1. mean, standard deviation (sd), and %cv values for raw data, data normalized by the second cycle, maximum rms, and mvc, for the phases of mouth opening and biting (n=86). muscle raw data 2nd cycle rms max mvc mean±sd %cv mean±sd %cv mean±sd %cv mean±sd %cv mouth opening lt 7.24±4.12 56.91 94.77±31.30 33.03 2.05±1.49 72.68 6.28±4.83 76.91 rt 8.25±5.42 65.70 94.60±34.22 36.17 1.95±1.28 65.64 6.99±6.93 99.14 lm 9.13±6.96 76.23 96.97±33.37 34.41 1.77±1.31 74.01 5.64±5.46 96.81 rm 8.54±5.24 61.36 97.98±40.01 40.83 1.68±1.31 77.98 5.25±5.63 107.24 sh 28.22±34.62 122.68 86.52±18.46 21.34* 23.49±10.70 45.55 84.25±60.22 71.48 biting lt 143.09±61.51 42.99 90.37±10.47 11.59* 35.84±6.47 18.05* 114.84±57.55 50.11 rt 166.86±77.23 46.28 90.83±11.13 12.25* 36.53±6.04 16.53* 125.99±75.57 59.98 lm 207.99±114.33 54.97 88.34±12.88 14.58* 34.73±6.02 17.33* 114.89±83.29 72.50 rm 207.63±89.23 42.98 88.52±13.53 15.28* 34.55±6.15 17.80* 105.96±59.77 56.41 sh 26.96±29.87 110.79 86.09±20.59 23.92* 20.77±8.45 40.68 85.93±81.69 95.07 rms=root mean square; mvc=maximum voluntary contraction; %cv=% coefficient of variation; lt=left temporalis; lm=left masseter; rt=right temporalis; rm=right masseter; sh=suprahyoid; *cv low (indicating a reasonably homogeneous dataset). regarding the comparison between the methods, the measure %cv of the 2nd cycle showed the lowest variation coefficient during the biting phase for all the muscles from the raw data, rms max, and mvc (p=0.001, p=0.003, and p=0.001 respectively). discussion the current study aimed to analyze the best method of data processing of the emg signal of the masticatory muscles during isotonic activity (non-habitual chewing) comparing raw data and different types of normalization. normalization by the second masticatory cycle showed the lowest variation coefficient during biting for all the muscles, followed by the rms maximum for the mandibular lift muscles (anterior temporalis and master bilateral). however, during mouth opening, values lower than 25% were found only for the second cycle for the suprahyoideos, which is the agonist of the task. emg studies with dynamic activities such as walking and cycling have been widely studied in the literature and provide a lot of information about normalization14-17. the result of the present study corroborates the study of albertus-kajee et al.14, which indicates that the use of static isometric methods is not appropriate for the normalization of emg signals in dynamic tasks. 9 pelai et al. braz j oral sci. 2023;22:e230961 an isometric maximal voluntary contraction (mvc) is mostly used for emg normalization, a procedure described in the scientific literature to compare muscle activity among different muscles and subjects. however, the use of mvc presents certain limitations17. in the second cycle, muscle activation is more stable, so variability is lower. normalization by mvc demonstrates very different contractions in terms of action and pattern of mvc biomechanics and motor control. the normalization procedure of the electromyographic signal, when performed from dynamic contractions, can be influenced by extrinsic factors of data collection, such as electrode displacements during movements. however, in addition to the current study, others that aim to verify the reproducibility of measurements of normalization procedures from the values in dynamic and isometric contractions indicated the use of normalization by the mean and peak of the emg signal during dynamic activity, due to the lowest coefficients of variation found18. regarding contributions to clinical practice and research, for clinicians, publications that include isotonic evaluations are an advantage, since this enables understanding of how muscles behave in functional tasks and not only during isometric tasks. considering research, this study demonstrates standardized methods to process and normalize the data, which also facilitates future comparisons among studies and provides more reliable results. as strengths of the study, this is a pioneer study, since in the search carried out by the authors, no studies were found that address normalization during mastication of the temporal, masseter, and suprahyoid masticatory muscles. in addition, assessment of the suprahyoid muscle is rare. the present study is also relevant because it evaluates a more functional activity than an isometric task. the method used to analyze the cycle is also a differential of the study due to its complexity. as a limitation, it can be pointed out that the collection was not carried out more than once, to test the reliability of the measurements. it is suggested that future studies carry out analyses to verify which normalization has a lower cv of the masticatory muscles at rest and in isometry conditions. in conclusion, for non-habitual chewing activity, the results of this study recommend data processing using normalization with the second cycle during chewing. conflicts of interest the authors have no personal or financial conflicts of interest related to the present work. acknowledgments the authors would like to thank all the subjects who participated in the study. this work was funded by grants from the comissão de aperfeiçoamento de pessoal de nível superior (capes), brasília, df, brazil. https://www.sciencedirect.com/topics/medicine-and-dentistry/electromyography 10 pelai et al. braz j oral sci. 2023;22:e230961 author contribution elisa bizetti pelai: conceptualization, methodology, data curation, data analysis, writingreviewing and editing; ester moreira de castro carletti: conceptualization, methodology, data curation, data analysis, writing; fabiana foltran mescollotto: conceptualization, writingreviewing and editing; paulo fernandes pires: conceptualization, methodology, data curation, data analysis, writing; fausto berzin: conceptualization, methodology; marcio de moraes: conceptualization, methodology, writingreviewing and editing delaine rodrigues bigaton: conceptualization, methodology, writingreviewing and editing. all authors actively participated in the manuscript’s findings, and have revised and approved the final version of the manuscript. references 1. duarte-kroll c, bérzin f, alves mc. [clinical evaluation of masticatory muscle activity during habitual mastication a study on the normalization of electromyographic data]. rev odontol unesp. 2010;39(3):157-62. portuguese. 2. cabral eea, fregonezi gaf, melo l, basoudan n, mathur s, reid wd. surface electromyography (semg) of extradiaphragm respiratory muscles in healthy subjects: a systematic review. j electromyogr kinesiol. 2018 oct;42:123-35. doi: 10.1016/j.jelekin.2018.07.004. 3. schwartz c, tubez f, wang fc, croisier jl, brüls o, denoël v, forthomme b. normalizing shoulder emg: an optimal set of maximum isometric voluntary contraction tests considering reproducibility. j electromyogr kinesiol. 2017 dec;37:1-8. doi: 10.1016/j.jelekin.2017.08.005. 4. soderberg gl, knutson lm. a guide for use and interpretation of kinesiologic electromyographic data. phys ther. 2000 may;80(5):485-98. 5. burden a. how should we normalize electromyograms obtained from healthy participants? what we have learned from over 25 years of research. j electromyogr kinesiol. 2010 dec;20(6):1023-35. doi: 10.1016/j.jelekin.2010.07.004. 6. lehman gj, mcgill sm. the importance of normalization in the interpretation of surface electromyography: a proof of principle. j manipulative physiol ther. 1999 sep;22(7):444-6. doi: 10.1016/s0161-4754(99)70032-1. 7. tabard-fougère a, rose-dulcina k, pittet v, dayer r, vuillerme n, armand s. emg normalization method based on grade 3 of manual muscle testing: withinand between-day reliability of normalization tasks and application to gait analysis. gait posture. 2018 feb;60:6-12. doi: 10.1016/j.gaitpost.2017.10.026. 8. pelai eb, foltran-mescollotto f, de castro-carletti em, de moraes m, rodriguesbigaton d. comparison of the pattern of activation of the masticatory muscles among individuals with and without tmd: a systematic review. cranio. 2023 mar;41(2):102-111. doi: 10.1080/08869634.2020.1831836. 9. jones t, shinohara m. a submaximal normalization of emg signals in trunk muscle groups. 2022 may [2022 aug 15. available from: https://repository.gatech.edu/server/api/core/bitstreams/ eec034f5-0b4f-4a74-9588-2d82d4cac7a0/content. 10. cram jr. electrode placement. in: cram’s introduction to surface electromyography. boston: jones & bartlett publishers; 2005. chapter 14, p.237-389. 11 pelai et al. braz j oral sci. 2023;22:e230961 11. berni kc, dibai-filho av, rodrigues-bigaton d. accuracy of the fonseca anamnestic index in the identification of myogenous temporomandibular disorder in female community cases. j bodyw mov ther. 2015 jul;19(3):404-9. doi: 10.1016/j.jbmt.2014.08.001. 12. pitta nc, nitsch gs, machado mb, de oliveira as. activation time analysis and electromyographic fatigue in patients with temporomandibular disorders during clenching. j electromyogr kinesiol. 2015 aug;25(4):653-7. doi: 10.1016/j.jelekin.2015.04.010. 13. brown ce. coefficient of variation. in: brown ce. applied multivariate statistics in geohydrology and related sciences. springer; 1998. p. 155-7. doi: 10.1007/978-3-642-80328-4_13. 14. reed p, romano m, re f, roaro a, osborne la, viganò c, rt al. differential physiological changes following internet exposure in higher and lower problematic internet users. plos one. 2017 may;12(5):e0178480. doi: 10.1371/journal.pone.0178480. 15. albertus-kajee y, tucker r, derman w, lamberts rp, lambert mi. alternative methods of normalising emg during running. j electromyogr kinesiol. 2011 aug;21(4):579-86. doi: 10.1016/j.jelekin.2011.03.009. 16. chuang td, acker sm. comparing functional dynamic normalization methods to maximal voluntary isometric contractions for lower limb emg from walking, cycling and running. j electromyogr kinesiol. 2019 feb;44:86-93. doi: 10.1016/j.jelekin.2018.11.014. 17. fernández-peña e, lucertini f, ditroilo m. a maximal isokinetic pedalling exercise for emg normalization in cycling. j electromyogr kinesiol. 2009 jun;19(3):e162-70. doi: 10.1016/j.jelekin.2007.11.013. 18. fraga chw, candotti ct. [estudo comparativo sobre diferentes métodos de normalização do sinal eletromiográfico aplicados ao ciclismo]. rev bras biomec. 2008;9:124-9. portuguese. 1 volume 22 2023 e230130 original article braz j oral sci. 2023;22:e230130http://dx.doi.org/10.20396/bjos.v22i00.8670130 1 division of prosthodontics, são leopoldo mandic dental institute, campinas, sp, brazil. 2 division of cariology and restorative dentistry, são leopoldo mandic dental institute, campinas, sp, brazil. corresponding author: cecilia pedroso turssi instituto e centro de pesquisas odontológicas são leopoldo mandic rua josé rocha junqueira, 13 cep 13045-755 campinas, sp, brazil telephone: +55-19-3211-3600; email: cecilia.turssi@slmandic.edu.br editor: altair a. del bel cury received: jun 15, 2022 accepted: aug 31, 2022 interplay between resin cements and surface-treated poly-ether-ether-ketone (peek): effect of aging aline mometi joly1 , guilherme da gama ramos1 , cecilia pedroso turssi2* aim: this study assessed the effect of thermal aging on the interfacial strength of resin cements to surface-treated peek. methods: ninety-six peek blocks were allocated into 4 groups (n=24), according to following surface treatments: sb sandblasting with aluminum oxide; sa acid etched with 98% sulfuric acid; ca – coupling agent (visio.link, bredent) and co control group (untreated). surface roughness (ra) was measured and one cylinder (1-mm diameter and height) of rely-x ultimate ult (3m/espe) and another one of panavia v5 pan (kuraray) were constructed on the treated or untreated peek surfaces. half of the samples of each group were thermal aged (1,000 cycles). samples were tested at a crosshead speed of 1 mm/min in shear mode (µsbs). ra and µsbs data were compared using oneand three-way anova, respectively, and tukey’s tests. results: sa and sb samples had the roughest surfaces, while ca the smoother (p<0.001). thermal aging reduced µsbs regardless the surface treatment and resin cement used. there was interaction between surface treatment and resin cement (p <0.001), with ult showing higher µsbs values than pan. sa provided higher µsbs than sb for both resin cements, while for ca µsbs was higher (pan) or lower than sb (ult). conclusion: aging inadvertently reduces interfacial strength between peek and the resin cements. if ult is the resin cement of choice, reliable interfacial strength is reached after any peek surface treatment. however, if pan is going to be used only sa and ca are recommended as peek treatment. keywords: polymers. resin cements. shear strength. aging. https://orcid.org/0000-0003-4393-6397 https://orcid.org/0000-0001-7342-083x https://orcid.org/0000-0002-0078-9895 2 joly et al. braz j oral sci. 2023;22:e230130 introduction poly-ether-ether-ketone (peek) is a thermoplastic polymer with attractive properties such as low allergenic potential, non-metallic color, high polishing, wear resistance, lightness and reduced biofilm formation make it as an alternative to prosthetic and restorative materials1,2. in dentistry, the clinical applications of peek include framework for fixed and removable prostheses, crowns, abutments, dental implants, occlusal guards, orthodontic wires, and posts2-4. despite its versatility, peek has low free energy and inert hydrophobic surface which pose challenges to bonding procedures to dental materials5-7. in order to increase surface energy and provide functional groups for improved bond strength with resin materials, as a previous step to bonding, peek surface has been subjected to physical or chemical treatments, including sulfuric acid etching, sandblasting, silica coating, coupling agent, laser and plasma4,8-11. however, the bonding result depends not only on the peek surface treatment, but also on the adhesive or resin cement itself and on the interplay between surface-treated peek surface and adhesive/resin cement7,11. these two later aspects are especially important if one considers the myriad of available adhesives and resin cements and their compositions, which can affect bonding to peek. one example are resin cements containing 10-methacryloxydecyl dihydrogen phosphate (10-mdp). although such component contributes to the overall polymerization process of some resin cements, such as in panavia v5, there are speculations that 10-mdp negatively affect bonding to peek due to its phosphate group, which does not react with peek12. the understanding of the interaction of surface-treated peek-resin cement is even more important if one considers that such materials face biochemical and physicomechanical degradation processes in the oral cavity. factors including saliva, acidic conditions, temperature oscillations, and masticatory stresses may hinder the properties of resin cements over time. aging by simulating oral conditions, such as thermocycling, has been used to anticipate the impact of degradation processes13. however, to the best authors’ knowledge, to date, the effect of thermal aging has been investigated between surface-treated peek and resin cement has only been investigated plasma-treated peek14, which is less tangible to the clinicians. as for the combination surface-treated peek/adhesive/composite system, chances are that the repetitive temperature changes could strain the interface between surface-treated peek and resin cement, and affect the bonding stability, which would have the influence of the composition of the resin cement. based on the aforementioned rationales, this study aimed to assess the effect of thermal aging on the interfacial strength between surface-treated peek and resin cements. we tested the null hypothesis that there would be no effect of surface treatment of peek, resin cement and thermal aging, neither alone nor interacting, on micro-shear bond strength (µsbs) between peek-resin cement. 3 joly et al. braz j oral sci. 2023;22:e230130 material and methods experimental design this study had two parts. in part one, the samples were 24 peek blocks whose surface was subjected to four different surface treatments as follows: 98% sulfuric acid etching (sa); sandblasting (sb); pentaerythritol triacrylate (petia)-containing coupling agent (ca, visio.link, bredent, germany) and untreated control surface (co). the dependent variable was surface roughness. in part two of this study samples of part one were bonded to two dual-cure resin cements (relyx ultimate – ult and panavia v5 pan, table 1) and unaged or aged using thermocycling. the dependent variable was µsbs. table 1. description of the resin cements. characteristics relyx ultimate (ult) panavia v5 (pan) monomers base paste: methacrylate monomers catalyst paste: methacrylate monomers paste a: bis-gma, tegdma, hydrophobic aromatic dimethacrylate, hydrophilic aliphatic dimethacrylate paste b: bis-gma, hydrophobic aromatic dimethacrylate, hydrophilic aliphatic dimethacrylate inorganic fillers 43% by volume silanized filler particles, alkaline filler particles size: 13 µm 38% by volume silanized barium glass particles, silanized fluoralminosilicate glass particles, colloidal silica, silanized aluminum oxide particles size: 0.01-12 µm initiators sodium p-toluenesulfonate, sodium persulfate, terc.butil 3,5,5-trimethylperoxyhexanoate dl-camphorquinone shade a1 clear manufacturer, batch # 3m/espe; 4471448 kuraray; 000001 bis-gma: bisphenol a-glycidyl methacrylate; tegdma: triethylene glycol dimethacrylate. based on a pilot study, in which the effect size was 0.183, a total of 21 samples per group would be required to detect significant difference, at 5% significance level and 80% of power. three samples were added in each group in order to compensate for eventual sample loss due to premature failure during thermocycling. each group had therefore 24 samples. part one – sample preparation, surface treatment, surface roughness evaluation and afm imaging using a milling system (cnc discovery d600, indústrias romi sa, brazil), 96 peek blocks (mgm plásticos de engenharia, brazil) were machined to 10x10x5.5 mm. peek blocks were then randomly allocated into four groups (n = 24) to receive one of the following surface treatments: 4 joly et al. braz j oral sci. 2023;22:e230130 sa: 200 µl of 98% sulfuric acid etching (ecibra/cetus, brazil) for 60 s11, followed by immersion in distilled water for 15 s to stop the chemical reaction and rinsing with distilled water for 15 s; sb: sandblasting with aluminum oxide particles11 (average particle size: 125 µm) for 20 s under 3 bar (pressure), at an angle of 45 degrees and 10 mm-distance between the surface and the nozzle (sandblaster basic master and cobra, renfert, germany), and rinsing with distilled water for 15 s; ca: application of petia-containing coupling agent11 (visio.link, bredent, germany), using a microbrush® applicator and light-curing for 90 s (valo, ultradent products, usa); co: control (untreated surface). after the surface treatments, peek blocks were measured for average surface roughness (ra) using a profilometer (mitutoyo sj210, mitutoyo sul americana ltda, brazil). the cut-off was set at 0.25 mm and total transverse length was 1.25 mm. measurements were made in three different directions (0, 45 and 90o) of the sample. representative images of surface-treated-samples were obtained under atomic force microscopy (dimension® icon afm system with scanasyst®, bruker nano surfaces division, usa), operating in intermittent mode, with a scanning area of 2x2 µm. part two – fabrication and bonding of resin cement cylinders, thermal aging, µsbs testing and failure mode examination directly on the surface of each sample, two translucent tygon tubes with an internal diameter of 1.0 mm15 and a height of 1.0 mm were used as matrices. one trained operator using magnifying loupes (galilean hd 3.3, examvision, denmark) positioned the matrices on the peek surface. the resin cements ult e pan were mixed according manufacturers’ direction. each matrix carefully received one of each resin cement. a mylar strip was positioned over the filled tube and gently pressed. the resin cements were light-cured through the mylar strip, according to the recommendations of each manufacturer: 20 s for ult and 10 s for pan, with the valo curing light (ultradent products, usa) at standard power (1000 mw/cm2). matrices were then carefully removed using a sharp blade to expose the resin cement cylinders. each cylinder was examined using magnifying loupes to identify possible defects (bubbles and flow of resin cement beyond the limits of the bonding area). all the samples, formed by the peek surface and one cylinder of each resin cement, were stored in distilled water at 37ºc for 24 h and randomly allocated to be either thermal aged or remain unaged. the samples thermal aged underwent 1,000 hydrothermal cycles in water between 5ºc and 55ºc, with 30 s dwell time (mct, elquip, brazil). the samples were mounted into a jig attached to a universal testing machine (dl 200, emic, brazil). a 0.2-mm diameter orthodontic wire was looped around the base of the resin cement cylinder as close as possible to the peek-cylinder interface and a shear force was applied to cylinder (figure 1) at a crosshead speed of 1 mm/min until failure occurred16. the µsbs values was calculated in megapas5 joly et al. braz j oral sci. 2023;22:e230130 cals (mpa) by dividing the load at failure point (newtons) by the surface area of the peek-resin cement bonding. fractured µsbs samples were then examined for their failure modes with a stereomicroscopic loupe (ek3st, eikonal equip, brazil) at 10x magnification and classified into: adhesive failure (between peek and resin cement), cohesive failure in peek, cohesive failure in the resin cement and mixed failure (figure 1). f p rc assembly f – force p – peek rc – resin cement adhesive failure between p and rc mixed failure between p and rc cohesive failure within rc cohesive failure within p figure 1. schematic drawing of the sample tested for micro-shear bond strength (on the left) and the four different possible failure modes. statistical analysis due to the lack of normality, data were square-root transformed. one-way analysis of variance compared surface roughness data (part one), while the effect of surface treatment, resin cement, thermocycling and their interactions (part two) were tested using three-way analysis of variance. all multiple comparisons were performed with tukey’s test. the calculations were run on spss (spss inc., usa), at a significance level of 5%. results surface pre-treatments significantly affected roughness (p < 0.001), with both sb and sa groups significantly rougher than co, whereas ca presented the smoothest surface (table 1). figure 2 shows afm images and revealed that co samples (figure 2d) had a primary texture featuring some grooves caused by the extrusion process after casting, whereas samples that received ca (figure 2c) expressed a flat surface with a micellar aspect. the samples of sb group (figure 2b), on the other hand, exhibited an irregular surface, with few and sparse pits, while those etched by sa (figure 2a) had the surface changed to a spongy pattern with marked and wider depressions. 6 joly et al. braz j oral sci. 2023;22:e230130 table 1. means and standard deviations of surface roughness (ra, µm) of peek after different physical or chemical treatments. surface treatment surface roughness sa 1.412 (0.546) c sb 1.528 (0.140) c ca 0.127 (0.073) a co 0.465 (0.107) b sa (98% sulfuric acid etching); sb (sandblasting with aluminum oxide particles);ca (petia-based coating agent visio.link). groups followed by dissimilar capital letters differ from each other. a b c d figure 2. afm images of peek etched with 98% sulfuric acid (a), sandblasted (b), subjected to coupling agent (c) and untreated (control, d). table 2 presents µsbs data which demonstrated no significant interaction among surface treatment, resin cement and thermal aging (p = 0.575), but a significant interaction was noticed between surface treatment and resin cement (p < 0.001). this interaction was explored using tukeys’ test and showed that compared to sb, sa provided higher µsbs to both pan and ult resin cements. however, while for pan no 7 joly et al. braz j oral sci. 2023;22:e230130 difference existed between the µsbs when peek surface received sa or ca, for ult, ca resulted in lower µsbs values. regardless of the surface pretreatment performed, ult resulted in higher values of µsbs to peek (table 3). as no other significant interaction was detected (surface treatment x thermal aging: p = 0.182; resin cement x thermal aging: p =0.458), then it was checked the effect of the main variable, which was shown to be statistically significant. specifically, regardless of the surface treatment and resin cement used, thermal aging significantly reduced µsbs between resin cements and peek surface by 15.6%, [thermal unaged: 18.46 mpa (11.85 mpa); aged: 15.57 mpa (12.64 mpa)]. table 2. means and standard deviations (mpa) of bond strength between resin cements and surface-treated peek, unaged and thermal aged. surface treatment panavia v5 (pan) rely-x ultimate (ult) unaged thermal aged unaged thermal aged sa 16.05 (11.91) 11.69 (10.33) 30.44 (8.83) 33.22 (11.62) sb 3.43 (2.76) 5.28 (10.07) 27.52 (7.97) 23.23 (5.77) ca 14.44 (7.91) 7.18 (4.04) 18.89 (6.00) 12.51 (3.43) sb (sandblasting with aluminum oxide particles); sa (98% sulfuric acid etching); ca (petia-based coating agent visio.link). according to three-way analysis of variance: interaction among surface treatment x resin cement x thermal cycling (p = 0.575); interaction between surface treatment x resin cement: p < 0.001; interaction between surface treatment x thermal cycling: p = 0.182; interaction resin cement x thermal cycling: p = 0.458; main variable thermal cycling: p = 0.013). table 3. bond strength means and standard deviations (mpa) between resin cements and peek subjected to different surface treatments, regardless whether thermal aged. surface treatment panavia v5 (pan) rely-x ultimate (ult) sa 13.96 aa (11.16) 31.83 ab (10.19) sb 4.35 ba (7.28) 25.38 bb (7.15) ca 10.81 aa (7.17) 15.70 cb (5.79) sa (98% sulfuric acid etching); sb (sandblasting with aluminum oxide particles); ca (petia-based coating agent visio.link). means followed by different capital letters indicate difference among surface treatments within each column. means followed by different lower-case letters indicate difference between resin cements within each row. adhesive failure was predominant in all groups. mixed failures occurred in samples bonded with ult but not with pan. in samples that received ca pre-treatment, those thermal aged had adhesive failures only, while 8.33% of unaged samples had mixed failures. the same proportion of mixed failures was seem in the sa pre-treated group that was unaged. still in unaged samples, 16.6% of sb group samples had mixed failures. when thermal aged, sa and sb groups mixed failures occurred in 50,0% of 33.3% of the samples. cohesive failure within peek occurred in a single sample (8.33%) pertaining to sa group (unaged). 8 joly et al. braz j oral sci. 2023;22:e230130 discussion the findings of this study demand rejection of the null hypotheses as thermal aging and the interplay between surface treatment of peek and resin cement significantly affected µsbs values. the reasons why thermal aging reduced the µsbs values are twofold: a) causing water sorption and hydrolytic degradation at bonding interfaces and, b) causing thermal stress due to differences in the coefficient of thermal expansion and condutivity between peek and resin cement17,18. water sorption can plasticize, break hydrogen bonds within the resin matrix, cause polymer swelling and ultimately hinder the properties of resin cements17. water sorption can also cause hydrolytic degradation of the resin matrix, the filler/matrix interface, or the filler. in effect, there are reports showing that both ult and pan present water sorption. ult contains phosphoric acid modified methacrylate monomers, which have the capability to bind water at hydroxyl groups18. in addition, ult has alkaline fillers, which bind water by starting an acid-base reaction18. pan, on the other hand, presents water sorption because it contains hydrophilic aliphatic dimethacrylate, but as there are no phosphate/hydroxyl groups or alkaline fillers, water sorption has been shown to be reduced18. as a result, for both resin cements (ult e pan) thermocycling increases water sorption and solubility18. still with respect to the explanations why thermocycling reduced µsbs values in the current study, cyclic temperature changes can generate expansion and contraction stresses, leading to microcracks within the resin cement18. such events can cause microcracks and thereby increase water sortion and solubility of resin cements18. however, stress can concurrently occur at the peek-resin cement interface, as the coefficient of thermal expansion of pure peek has been described to be half of resin cements such as ult19,20. one can argue that a higher number of thermal cycles could better represent the long-term aging, especially because 10,000 cycles have been described to correspond to approximately one year of clinical service21 and higher numbers of thermal cycles have been described in peek experiments5. however, it is worth mentioning that in these publications the samples were prepared for shear bond testing not for µsbs, as used in the current paper5. preliminary experiments of our group showed that 10,000 thermal cycles caused debonding of 92% of the samples during thermocycling. even during 5,000 thermal cycles an extensive proportion of samples prematurely failed (67%). the explanation for debonding may be probably found in the aggravated action of temperature oscillations in the peek-resin cement interface, because of a lower bonding area in µsbs testing in comparison to the shear bond method. thus, in order to have minimal premature failure and make it feasible to mearure µsbs values, we run 1,000 cycles. interesting to notice is that previous literature data in which the authors thermocycled ult 10,000x the bond strength of this resin cement was reduced in 14.7%22, an amount equivalent to that observed in our study (15.6%) using 1,000 thermal cycles. this similar reduction despite the different number of thermal cycles may be ascribed to the fact that in the cited paper the bonding area was increased and samples were tested in tensile rather than microtensile mode. 9 joly et al. braz j oral sci. 2023;22:e230130 besides the effect of thermal aging, surface treatment also played a role on µsbs values. regardless of the resin cement, sa provided higher µsbs than sb. figures 1a and 1b substantiate this finding showing, respectively, marked versus sparse pits on the peek surface. the effect of sa stems from the cleavage of benzene rings by attacking peek carbonyl and ether groups and the introduction of sulfonic acid groups in the peek polymer chains23,24. a micromorphological change is generated, but probably in a range not significantly different from sb in terms of ra values, in accordance with a previous study25. however, other papers have indicated that sb promotes smoother26 or rougher surface than sa27-29. such differences may be attributed to variation in the size of aluminum oxide particles, the pressure and duration of blasting30,31. in effect, in the present study, the pressure used during blasting was higher than that used in some previous studies7,32. the pressure of 3 bar was chosen in an attempt to achieve greater bond strength, since it has been reported that peek bond strength is enhanced by increasing blasting pressure28,33. however, bonding to sandblasted or any pretreated surface proved to be dependent on the resin cement used, as pan systematically provided lower µsbs than ult. this result substantiates the speculation that 10-mdp present in pan can negatively affect bonding to peek is correct. in this regard, however, it is relevant to verify whether the µsbs values reached the 10 mpa threshold, considered as a clinically acceptable value in a number of published papers as cited elsewhere10. our data showed that in only one combination of surface treatment (sb) and resin cement (pan) the µsbs was below the 10 mpa threshold. despite the proximity between the average µsbs and the 10-mpa threshold, the combination between ca as a pretreatment for peek and pan as the resin cement is electable. ca (figure 2c) created a surface with micellar aspect promoted by the chemical interaction between peek and methylmethacrylate (mma) and petia12 that constitutes the coupling agent (visio.link). however, the efficiency of such interaction has been significantly higher following air abrasion and sulfuric acid etching11. it is noteworthy noting that in a previous study that tested peek bonded to titanium bases showed that the weaker interface was between the peek and a resin cement34. this finding validates the importance of the present paper in further explores the interfacial strength between peek and different resin cements, especially under aging. however, one should bear in mind that in continuation to this study, it would be valuable to test whether or not the bonding capacity of resin cements to peek and its longevity would hold when resin cements are sandwiched between peek and dental substrates (or composite resins). this set up would be feasible through micro-tensile testing. if possible obtaining micro-tensile samples using resin cements sandwiched between peek and other substrates, the results would allow gaining additional insights into the predictability of the interfacial strength under clinical circumstances involving peek usage. based on the current findings, thermal aging reduced the interfacial strength between peek and resin cements, but if ult is the resin cement of choice, reliable interfacial strength is reached after any peek surface treatment. however, if pan is going to be used only sa and ca are recommended as peek treatment. 10 joly et al. braz j oral sci. 2023;22:e230130 data availability datasets related to this article will be available upon request to the corresponding author. conflict of interest none. authors contribution joly am: collected data, drafted the manuscript; ramos gg: interpreted the data, revised the manuscript; turssi cp: conceived and designed the study, data analysis, revised the manuscript. all the authors actively participated in discussing the manuscript’s findings and have revised and approved the final version of the manuscript. references 1. blanch-martínez n, arias-herrera s, martínez-gonzález a. behavior of polyether-ether-ketone (peek) in prostheses on dental implants. a review. j clin exp dent. 2021;13:e520-6. doi: 10.4317/jced.58102. 2. papathanasiou i, kamposiora p, papavasiliou g, ferrari m. the use of peek in digital prosthodontics: a narrative review. bmc oral health. 2020 aug;20(1):217. doi: 10.1186/s12903-020-01202-7. 3. nahar r, mishra sk, chowdhary r. evaluation of stress distribution in an endodontically treated tooth restored with four different post systems and two different crownsa finite element analysis. j oral biol craniofac res. 2020 oct-dec;10(4):719-26. doi: 10.1016/j.jobcr.2020.10.004. 4. qin l, yao s, zhao j, zhou c, oates tw, weir md, et al. review on development and dental applications of polyetheretherketone-based biomaterials and restorations. materials (basel). 2021 jan;14(2):408. doi: 10.3390/ma14020408. 5. gama lt, duque tm, özcan m, philippi ag, mezzomo lam, gonçalves tmsv. adhesion to high-performance polymers applied in dentistry: a systematic review. dent mater. 2020 apr;36(4):e93-e108. doi: 10.1016/j.dental.2020.01.002. 6. bathala l, majeti v, rachuri n, singh n, gedela s. the role of polyether ether ketone (peek) in dentistry: a review. j med life. 2019 jan-mar;12(1):5-9. doi: 10.25122/jml-2019-0003. 7. tsuka h, morita k, kato k, kawano h, abekura h, tsuga k. evaluation of shear bond strength between peek and resin-based luting material. j oral biosci. 2017;59(4):231-6. doi: 10.3290/j.jad.b2288283. 8. rocha rf, anami lc, campos tm, melo rm, souza ro, bottino ma. bonding of the polymer polyetheretherketone (peek) to human dentin: effect of surface treatments. braz dent j. 2016 octdec;27(6):693-9. doi: 10.1590/0103-6440201600796. 9. parkar u, dugal r, madanshetty p, devadiga t, khan as, godil a. assessment of different surface treatments and shear bond characteristics of poly-ether-ether-ketone: an in vitro sem analysis. j indian prosthodont soc. 2021 oct-dec;21(4):412-9. doi: 10.4103/jips.jips_199_21. 10. escobar m, souza jcm, barra gmo, fredel mc, özcan m, henriques b. on the synergistic effect of sulfonic functionalization and acidic adhesive conditioning to enhance the adhesion of peek to resinmatrix composites. dent mater. 2021 apr;37(4):741-54. doi: 10.1016/j.dental.2021.01.017. 11 joly et al. braz j oral sci. 2023;22:e230130 11. soares machado p, cadore rodrigues ac, chaves et, susin ah, valandro lf, pereira gkr, et al. surface treatments and adhesives used to increase the bond strength between polyetheretherketone and resin-based dental materials: a scoping review. j adhes dent. 2022 may;24(1):233-45. doi: 10.3290/j.jad.b2288283. 12. keul c, liebermann a, schmidlin pr, roos m, sener b, stawarczyk b. influence of peek surface modification on surface properties and bond strength to veneering resin composites. j adhes dent. 2014 aug;16(4):383-92. doi: 10.3290/j.jad.a32570. 13. morresi al, d’amario m, capogreco m, gatto r, marzo g, d’arcangelo c, et al. thermal cycling for restorative materials: does a standardized protocol exist in laboratory testing? a literature review. j mech behav biomed mater. 2014 jan;29:295-308. doi: 10.1016/j.jmbbm.2013.09.013. 14. stawarczyk b, bähr n, beuer f, wimmer t, eichberger m, gernet w, et al. influence of plasma pretreatment on shear bond strength of self-adhesive resin cements to polyetheretherketone. clin oral investig. 2014 jan;18(1):163-70. doi: 10.1007/s00784-013-0966-7. 15. armstrong s, geraldeli s, maia r, raposo lh, soares cj, yamagawa j. adhesion to tooth structure: a critical review of “micro” bond strength test methods. dent mater. 2010 feb;26(2):e50-62. doi: 10.1016/j.dental.2009.11.155. 16. muñoz ma, baggio r, mendes ybem, gomes gm, luque-martinez i, loguercio, ad, et al. the effect of loading method and cross-head speed on resin dentin microshear bond strength. int j adhes adhes. 2014 apr;50:136-41. doi: 10.1016/j.ijadhadh.2014.01.024. 17. walker mp, spencer p, david eick j. mechanical property characterization of resin cement after aqueous aging with and without cyclic loading. dent mater. 2003 nov;19(7):645-52. doi: 10.1016/s0109-5641(03)00008-3. 18. müller ja, rohr n, fischer j. evaluation of iso 4049: water sorption and water solubility of resin cements. eur j oral sci. 2017 apr;125(2):141-50. doi: 10.1111/eos.12339. 19. goyal rk, tiwari an, mulik up, negi ys. thermal expansion behaviour of high performance peek matrix composites. j phys d: appl phys. 2008 mar;41(8):085403. doi: 10.1088/0022-3727/41/8/085403. 20. cakan u, saygılı g. comparison of thermal stress on various restorative post and core materials generated by oral temperature changes using three dimensional finite element analysis. clin dent res. 2015;39(1):27-35. 21. gale ms, darvell bw. thermal cycling procedures for laboratory testing of dental restorations. j dent. 1999 feb;27(2):89-99. doi: 10.1016/s0300-5712(98)00037-2. 22. correr-sobrinho l, costa ar, fugolin app, sundfeld neto d, ferracane jl, pfeifer cs. effect of experimental resin cements containing thio-urethane oligomers on the durability of ceramic-composite bonded interfaces. biomater investig dent. 2019 nov 27;6(1):81-9. doi: 10.1080/26415275.2019.1693274. 23. stawarczyk b, jordan p, schmidlin pr, roos m, eichberger m, gernet w, et al. peek surface treatment effects on tensile bond strength to veneering resins. j prosthet dent. 2014 nov;112(5):1278-88. doi: 10.1016/j.prosdent.2014.05.014. 24. yee rsl, zhang k, ladewig bp. the effects of sulfonated poly (ether ether ketone) ion exchange preparation conditions on membrane properties. membranes. 2013 aug;3(3):182-95. doi: 10.3390/membranes3030182. 25. silthampitag p, chaijareenont p, tattakorn k, banjongprasert c, takahashi h, arksornnukit m. effect of surface pretreatments on resin composite bonding to peek. dent mater j. 2016;35(4):668-74. doi: 10.4012/dmj.2015-349. 12 joly et al. braz j oral sci. 2023;22:e230130 26. rosentritt m, preis v, behr m, sereno n, kolbeck c. shear bond strength between veneering composite and peek after different surface modifications. clin oral investig. 2015 apr;19(3):739-44. doi: 10.1007/s00784-014-1294-2. 27. çulhaoğlu ak, özkır se, şahin v, yılmaz b, kılıçarslan ma. effect of various treatment modalities on surface characteristics and shear bond strengths of polyetheretherketone-based core materials. j prosthodont. 2020 feb;29(2):136-41. doi: 10.1111/jopr.12702. 28. stawarczyk b, beuer f, wimmer t, jahn d, sener b, roos m, et al. polyetheretherketone a suitable material for fixed dental prostheses? j biomed mater res part b appl biomater. 2013 oct;101(7):1209-16. doi: 10.1002/jbm.b.32932. 29. adem n, bal b, kazazoğlu e. comparative study of chemical and mechanical surface treatment effects on the shear bond strength of polyether-ether-ketone to veneering resin. int j prosthodont. 2022 march/april;35(2):201-7. doi: 10.11607/ijp.6938. 30. ourahmoune r, salvia m, mathia tg, mesrati n. surface morphology and wettability of sandblasted peek and its composites. scanning. 2014 jan-feb;36(1):64-75. doi: 10.1002/sca.21089. 31. elawadly t, radi wia, el khadem a, osman rb. can peek be an implant material? evaluation of surface topography and wettability of filled versus unfilled peek with different surface roughness. j oral implantol. 2017 dec;43(6):456-61. doi: 10.1563/aaid-joi-d-17-00144. 32. tsuka h, morita k, kato k, kimura h, abekura h, hirata i, et al. effect of laser groove treatment on shear bond strength of resin-based luting agent to polyetheretherketone (peek). j prosthodont res. 2019 jan;63(1):52-7. doi: 10.1016/j.jpor.2018.08.001. 33. stawarczyk b, taufall s, roos m, schmidlin pr, lümkemann n. bonding of composite resins to peek: the influence of adhesive systems and air-abrasion parameters. clin oral investig. 2018 mar;22(2):763-71. doi: 10.1007/s00784-017-2151-x. 34. yilmaz b, gouveia d, schimmel m, lu we, özcan m, abou-ayash s. effect of adhesive system, resin cement, heat-pressing technique, and thermomechanical aging on the adhesion between titanium base and a high-performance polymer. j prosthet dent. 2022 may 2;s0022-3913(22)00207-4. doi: 10.1016/j.prosdent.2022.03.026. 1 volume 21 2022 e225946 original article braz j oral sci. 2022;21:e225946http://dx.doi.org/10.20396/bjos.v21i00.8665946 1 periodontics, bauru school of dentistry, university of são paulo. corresponding author: matheus völz cardoso bauru school of dentistry alameda octávio pinheiro brisolla 9-75 17012-901 bauru-sp phone: 55 14 32358278 fax: 55 14 32234679 cell phone 55 14 98112-2293 matheusvolz@alumni.usp.br editor: dr. altair a. del bel cury received: june 6, 2022 accepted: january 17, 2022 reproducibility and comparison between methods for gingival color evaluation: a validation study matheus völz cardoso1,* , giovanna vicenzoti1, adriana campos passanezi sant’ana1 , mariana schutzer ragghianti zangrando1 , carla andreotti damante1 aim: this study aims to evaluate and validate the sensibility and the level of agreement between different gingival color measures obtained by a spectrophotometer (spectro) and a photography (photo) method. methods: among 40 patients, the color was measured 2 mm apical to the gingival margin by cie l*, a*, b* system using a reflectance spectrophotometer and the photography’s plus software. the level of agreement between three different measures (m1, m2, m3) in parameters l*, a*, b*, and ∆e (color variation) was evaluated by random and systematic errors, as well as the limits and coefficient of concordance. a comparison between the methods was performed by the bland-altman test and the sensibility level was evaluated accordingly to the ∆e: 3.7 thresholds with p<0.05 as the level of significance for these comparisons. results: the spectro method has not presented the systematic error (p>0.05) and had reproducibly and agreement level in three variable measures l* (r: 0.6), a* (r: 0.3), and b* (r: 0.5) as to the photo method l* (r: 0.6), a* (r: 0.5), and b* (r: 0.5), which presented systematic error in l* values (p<0.05). the means of ∆e between measurements were: 6.5 spectro and 5.9 photo. there was no good level of sensitivity ∆e> 3.7 and agreement between the methods, mainly for the a* values. on the other hand, for the l* and in for the most comparisons of b* values, the level of agreement was higher. conclusion: both methods could quantify the gingival color from the coordinates l *, a *, and b *, which has shown greater reliability between the measurements acquired by the spectro method. keywords: color. gingiva. photography, dental. https://orcid.org/0000-0002-7609-8003 https://orcid.org/0000-0001-5640-9292 https://orcid.org/0000-0003-0286-7575 https://orcid.org/0000-0002-6782-8596 2 cardoso et al. braz j oral sci. 2022;21:e225946 introduction color is a complex phenomenon and an interesting field of study in several areas of science. complexity is derived from the physical, physiological, geometric, and sensorial color presentation characteristics, and this conditions bring complications for the evaluation and categorization of this phenomenon1. color has a wide utility in dentistry. color perception and selection are a daily maneuver in dental office attendance. several factors can modify the dental2 and gingival color perception, such as visual organ fatigue, changing backgrounds, or differences in light incidence. for the understanding of the subjectivity of color perception in science, the commission internationale de l’eclairage (cie) has established different tridimensional color spaces. these measurement systems aim to upgrade global communication and to exclude the major subjective factor of color analyses. the l* a* b* color space had three fundamental components, illumination: l*, variations between red/green: a*, and yellow/ blue: b*. the cielab color space had values with major correlation compared to human perception3. through tridimensional color spaces, it is possible to calculate and compare objects, specimens, and periods4, using the color variation (∆e). due to scientific advances, the color of dental enamel can be evaluated5,6 with a higher level of evidence compared to gingiva color analysis4. the primary methods of color acquisition were derived from colorimeters and spectrophotometers devices. under specific adjustments, digital cameras showed potential as an alternative method to color evaluation7. when dental enamel is evaluated, digital cameras associated with software were accurate tools to evaluate l* and b*, but not for the analysis of the a* axis8,9. studies to the gingival color evaluation have tried to establish gingival color shade guides3,10-12, as exhibited by dental enamel6. however, in order to classify the gingival color, the main variations are race/ethnicity and gender13,14. besides that, other not-yet evaluated factor, are the possible interference of different gingival phenotypes15 and had an importance in the validation of gingival color methods. despite that, the gingival color was studied with these tools after the gingival graft procedures16, tissue changes derived by different colors and materials of dental implant abutments17, differences between natural and artificial gingival colors18, also reduction in gingival inflammation (redness) after periodontal treatment against gingivitis19. photographs were used to compare the ∆e threshold color values among different evaluators. this study has reported that professionals and patients had a distinct sense of color perception and different ∆e thresholds20. comparisons between color analysis methods for dental enamel have shown that photography and colorimeters have obtained ∆e values below of recognized by the human eye21,22. they have also presented reliable sensitivity, due to the error of the analysis of the measurement for both methods, which were below of 2 units in the central incisors21. in clinical conditions, the accurate gingival color evaluation is achievable to compare results in randomized clinical trials (rcts) before and after periodontal plastic surgery procedures (gingival grafts and aesthetic crown lengthening), in implant dentistry, as described before16,17 to compare different 3 cardoso et al. braz j oral sci. 2022;21:e225946 implant abutment materials, the gingiva luminescence, and to explain for a patient the acquired soft tissue color match. in the future, it will be possible the patient conducts a self-evaluation of the gingival inflammation levels by photographs. these methods can reduce the subjective analyses currently executed in rcts, highly dependent on patients and on professional individual criteria examination. in addition, objective color parameters could be used as a complementary result, not a substitute, in gingival aesthetic evaluation executed by the patient. this would facilitate the gingival color comparison between different populations, ethnicities, and cultural realities of the participants of the study. despite that, the comparison amongst methods for gingival color evaluation and the measurements errors were poorly evaluated and validated in literature. when one method is established, the level of reproducibility is important to the validation of the color acquisition protocols tested. this study has aimed to evaluate the sensibility (agreement, reproducibility, and assertive grades) for three different measurements (m1, m2, and m3) for each method of gingival color evaluation by spectrophotometer (spectro) and photography (photo), and to compare both methods. the null hypothesis (1) is that the level of agreement between each measure (m1, m2, m3)/ intra-method shows equivalent results, and the second hypothesis (2) is that both methods present similar values in the l*, a*, b*, and ∆e outcomes. material and methods forty patients were invited to participate in this observational study that evaluated the gingival color in the same period by two the spectro and photo methods. the local human ethics committee from bauru school of dentistry approved the study protocol (nº 2.505.538 /caae: 79080117.4.0000.5417). the inclusion criteria were: 1) patients with teeth without signals of disease activity (periodontitis or gingivitis), 2) full month bleeding and plaque index ≤ 20%; 3) probing depth < 3 mm on teeth; 4) no restored and/or endodontically treated teeth; 5) aligned teeth with arch and adjacent teeth; 6) health and intact alveolar mucosa (without irritation signals, burn injuries or other lesions); 7) nonsmoker patients; 8) at least twice a day brush frequency. the exclusion criteria were: 1) smokers; 2) systemic diseases that affect the cicatricial course or blood dyscrasias; 3) using medications such as anticonvulsant, antihypertensive, also cyclosporine; 4) pregnant woman; 5) patients with notable cutaneous alteration due to excessive tanning and/or manifestations of skin diseases; 6) patients already submitted to a periodontal surgical procedure on the included site; 7) teeth (crown or root) with color alteration due to endodontic lesions; 8) remarkable alterations in alveolar or keratinized mucosa (pigmentations; trauma; amalgam tattoo, and melanic pigmentation). the reflectance spectrophotometer11,12,17 (direct color acquisition method) and the photography plus software (indirect color acquisition method)16,19,20 were used methods for the gingival color evaluation. the measurements were performed in triplicate by the same evaluator (gv). the reproducibility of methods was evaluated in each patient by three different measurements (m1, m2, m3) at the same site both for the spectro and the photo methods. 4 cardoso et al. braz j oral sci. 2022;21:e225946 the measures in the spectro group were performed by a reflectance spectrophotometer for dental color analysis easy shade (vita). the selected area was in the center of a buccal site, 2 mm apical to the gingival margin. the equipment was protected by a plastic film and was calibrated as determined by the manufacturer. the range of the spectrum was set between 400 to 700 nm, and it was programmed to generate values of l*, a*, and b* axes. in the photo group, a photograph was taken in the same area evaluated by the spectrophotometer. a standardized photography protocol was established based on previous studies9,19,23. the digital camera (t6 model, canon do brasil indústria e comércio) with a magnification ratio of 1:1 was selected in the 100 mm macro lens (canon do brasil indústria e comércio). it was also used a macro ring flash (canon do brasil indústria e comércio). the camera focus was adjusted manually. the equipment configuration was standardized at iso 100 and diaphragm aperture of 32, using 4500 kelvin as color temperature. the same operator (mvc) was responsible for obtaining all the images, ensuring the acquisition process. the color was measured in the center of a buccal site 2 mm apical to gingival margin in each tooth, and the size of program cursor was adjusted to dropper>color sorter tool in the software (adobe photoshop cs6®)19. all the measures were performed by the same operator (gv) on the same screen computer (sony vaio®) with standardized screen settings24. the software was configured to generate values of l*, a*, and b* axes. in the tested methods, the patient was positioned in a comfortable chair, with the head positioned in the headrest of a dental chair. it was used lip retractors on both sides of the mouth (maquira, maringa, paranábrazil) to promote access of to the anterior upper teeth, which was included in study. the external conditions of color measures were set under natural clinical room light always during the day in a period of 10:00 a.m. to 4:00 p.m. for each method, the ∆e was calculated by the equation 1, which was based on previous methodologies16,20,25. equation 1: ∆e = (∆l)2 + (∆a)2 + (∆b)2 sample size it was used a previous study26, which evaluated the measurement error, in order to calculate the sample size of this study. it was determined that the minimum number of pairs of comparisons that needed to be executed was of 25-30 pairs27 to evaluate the random error. 20 pairs of comparisons were the minimum number to evaluate the dahlberg error method28. the sample size was estimated in forty patients and sites, and the null hypotheses assumed a comparison power >80% (β: 0.83) determined by software gpower 3.1. statistical analyses the data distribution was tested by the shapiro-wilk test (n<50). for the reproducibility analyses, l*, a*, and b* values were compared using the three different measurements of mean and median to each method separately, using the wilcoxon or the 5 cardoso et al. braz j oral sci. 2022;21:e225946 t-tests and the pearson or spearman correlation tests29,30. in order to calculate the dahlberg coefficient (measurement error)31, the concordance limits, and the standard error of the method sensitivity, it was evaluated the dental enamel studies, considering the value of ∆e <3.721,22, which is under the threshold of the human eye, considered as adequate sensitivity. to evaluate the second null hypothesis, for the comparison between the spectro versus the photo method, on which the values of l*, a*, b*, and ∆e (direct-indirect) were equal to zero5, were considered to be the perfect concordance. the bland-altman analysis was performed for the comparison between methods in each measurement, besides the 2d graphic comparisons presentation with the confidence interval32,33. all analyses adopted the significance level of p<0.05 and the data was analyzed in the statistical software (ibm spss statistics)34 and rproject35. results from the patients (n: 40) enrolled in this study (n: 10 men and n: 30 women), the average age of the group was of 39.4 years (maximum 57 and minimum 20 years). all the participants evaluated themselves as of the white race-ethnicity. the gingival sites evaluated were located on the upper right central incisor (n: 18/45%) and on the upper left central incisor (n: 22/55%). the values obtained in the three different measures for any method were presented in table 1. these values were calculated using the ∆e equation for each measure and method (table 1). overall, the ∆e values were similar for each method, with ∆e: 6.5 for the spectro and 5.9 for the photo. comparing these three measurements, all of them have surpassed the reference value (∆e: 3.7), which is already considered perceptible by the human eye. the level of reproduction between the measures was evaluated in three comparison levels (m1 versus m2, m1 versus m3, and m2 versus m3) (table 2). mainly, in the photo method, the values of the l* axis in two comparisons surpassed the significance value (p<0.05). table 1. values of l*a*b*and ∆e between measures (m1, m2, and m3) for spectro and photo methods and (n: 40). spectro m1_ l a b m2 _l a b m3 _l a b ∆e m1 vs m2 ∆e m1 vs m3 ∆e m2 vs m3 mean ± sd (max;min) 57.3±8.6 (77.2; 40.3) 18.2±10.3 (58; 6.2) 15.2±11.0 (61; 4.5) 58.4±7.7 (74.7; 37.5) 17.8±5.8 (38; 11.3) 14.3±6.2 (39; 9.6) 59.4±9.2 (72.4; 35) 17.3±6.6 (37.3;10.2) 15.0±9.3 (58.8; 9.1) 6.7 ± 4.5 (23; 0) 7.2 ±5.2 (19.8; 0) 5.6 ± 4.3 (15.9; 0) median, 25%,75% 57.1 17 12.7 57.1 16.7 12.7 60.6 16.4 12.8 5.5; 5.8; 5.3; 51.8 13.8 11.4 53.4 14.2 11.1 57.2 12.9 11.6 4; 5.3; 2; 63.5 20.1 13.8 63.8 19.3 14.2 65.8 19.0 14.8 8.6 10.4 8 photo mean ± sd (max;min) 59.3±6.1 (77; 47) 33.1±5.5 (47; 21) 18.2±3.3 (28; 11) 59.8±6.2 (75; 46) 33.6±5.3 (46; 20) 18.8±3.3 (26; 11) 57.4±5.5 (70; 47) 35.4±5.0 (44; 26) 19.6±3.6 (31; 11) 3.9 ± 3.8 (15.4; 0) 6.7 ± 4.5 (22; 0) 7.1 ± 4.0 (17.2; 0) median, 25%,75% 59.5 33 19 59.0 34 18 57 35 20 3; 5.9; 6.3; 56 30 17 56.3 31 16 53.7 31 17 0; 3.5; 4.6; 62.5 36.5 20 63.8 37.7 20 61 38 21 6.2 8.4 8.9 max.: maximum; min.: minimum 6 cardoso et al. braz j oral sci. 2022;21:e225946 the obtained concordance limits for the spectro were l*: ≈1, a*: 0.4-0.8, and b*: 0.2-0.5. as for the concordance coefficients (closest values for maximum agreement), they have exhibited intervals of l*: 25, a*: 15, and b*: 12 units. for the photo method, obtained the concordance limits were l*: 0.1-1.8, a*: 0.2-0.9, and b*: 0.3, and the concordance coefficients were l*: 15.3, a*: 16 e b*: 13 units. predominantly in both methods, the values have shown reduced concordance limits and standard error (next to 1 and <1, respectively). the random error was 3-4 units to l* and a* axes, and 2 to b* axis, with 3 units (1.8 to -1.2) of variations between means. the similarity between measures is satisfactory (icc: intra-class correlation coefficient: 0.44 spectro/0.57 photo) in both comparisons. table 2. reproducibility between measures. (n: 40) concordance limits concordance coefficient* dahlberg casual error mean of differences standard deviation standard error paired t test ⸸ pearson(r)⸸ coefficient correlation icc m1 vs m2 l spectro -1.158 -11.5 – 9.19 3.77 -1.15 5.28 1.31 0.19 0.80 0.79 a -0.86 -10.4 – 8.75 3.47 -0.86 4.91 1.05 0.29 0.31 0.30 b -0.20 -6.99 – 6.58 2.42 -0. 20 3.46 0.36 0.89# 0. 5# 0.34 l photo -0.13 -6.99 – 6.71 2.44 -0. 13 3.49 0.23 0.81 0.83 0.83 a -0.25 -6.22 – 5.72 2.13 0. 25 3.04 0.49 0.62 0.83 0.83 b 0.02 -5.68 – 5.73 2.03 0.02 2.91 0.05 0.98# 0.5# 0.64 m1 vs m3 l spectro -1.25 -14.2 – 11.7 4.70 -1.25 6.62 1.13 0.26 0.69 0.67 a -0.42 -8.83 – 7.98 3.0 -0.42 4.28 0.59 0.59 0.55 0.47 b 0.32 -7.48 – 8.14 2.79 0.32 3.98 0.49 0.59# 0.5# 0.03 l photo 1.72 -7.05 – 10.4 3.35 1.72 4.47 2.30 <0. 05 0.72 0.69 a -0.91 -11.3 – 9.4 3.75 -0.91 5.30 1.03 0.30 0.56 0.56 b 0.38 -7.05 – 7.83 2.66 0.38 3.79 0.61 0.69# 0.5# 0.49 m2 vs m3 l spectro -0.09 -11.7 – 11.5 4.13 -0.09 5.92 0.09 0.92 0.71 0.71 a -0.44 -5.23 – 6.12 2.04 0.44 2.89 0.91 0.36 0.68 0.67 b 0.53 -4.72 – 5.79 1.91 0.53 2.68 1.19 0.33# 0.5# 0.44 l photo 1.72 -7.05 – 10.4 3.35 1.72 4.47 2.30 <0. 05 0.72 0.69 a -0.91 -11.3 – 9.4 3.75 -0.91 5.30 1.03 0.30 0.56 0.56 b 0.38 -7.05 – 7.83 2.66 0.38 3.79 0.61 0.69# 0.5# 0.49 * 95% confidence interval; ⸸p values; # b axis values show nonparametric distribution (shapiro-wilk test. p<0.05): wilcoxon test and spearman correlation was executed; icc: intraclass correlation coefficient. 7 cardoso et al. braz j oral sci. 2022;21:e225946 the results for the comparisons between the measurements to each method were presented in figures 1 and 2 (fig. 1 spectrophotometer, fig. 2 photography). the values of the spectro method showed major proximity to zero (value of maximal concordance between measures) and had shortest confidence intervals (ellipses), except for the l* axis. in the photo method, a major level of differences was observed with values that move away from zero. l m1 vs m2 80 60 40 20 0 -30 -20 -10 0 10 20 30 delta l: -1.15 ± 5.28 l a m1 vs m2 80 60 40 20 0 -30 -20 -10 0 10 20 30 delta a: -0.86 ± 4.95 a b m1 vs m2 80 60 40 20 0 -30 -20 -10 0 10 20 30 delta b: -0.20 ± 3.46 b l m1 vs m3 80 60 40 20 0 -30 -20 -10 0 10 20 30 delta l: -1.25 ± 6.62 l a m1 vs m3 80 60 40 20 0 -30 -20 -10 0 10 20 30 delta a: -0.42 ± 4.28 a b m1 vs m3 80 60 40 20 0 -30 -20 -10 0 10 20 30 delta b: 0.32 ± 3.98 b l m2 vs m3 80 60 40 20 0 -30 -20 -10 0 10 20 30 delta l: -0.09 ± 5.92 l a m2 vs m3 80 60 40 20 0 -30 -20 -10 0 10 20 30 delta a: 0.44 ± 2.89 a b m2 vs m3 80 60 40 20 0 -30 -20 -10 0 10 20 30 delta b: 0.53 ± 2.68 b figure 1. comparison between measurements (to each measure blue or red) in the spectro method (ellipses represent the 95% confidence interval) (r project). 8 cardoso et al. braz j oral sci. 2022;21:e225946 figure 2. comparison between measurements (to each measure blue or red) in the photo method (ellipses represent the 95% confidence interval) (r project). l m1 vs m2 80 60 40 20 0 -30 -20 -10 0 10 20 30 delta l: -0.13 ± 3.49 l a m1 vs m2 80 60 40 20 0 -30 -20 -10 0 10 20 30 delta a: -0.25 ± 3.04 a b m1 vs m2 80 60 40 20 0 -30 -20 -10 0 10 20 30 delta b: 0.02 ± 2.91 b l m1 vs m3 80 60 40 20 0 -30 -20 -10 0 10 20 30 delta l: -1.72 ± 4.47 l a m1 vs m3 80 60 40 20 0 -30 -20 -10 0 10 20 30 delta a: -0.91 ± 5.30 a b m1 vs m3 80 60 40 20 0 -30 -20 -10 0 10 20 30 delta b: 0.38 ± 3.79 b l m2 vs m3 80 60 40 20 0 -30 -20 -10 0 10 20 30 delta l: -1.86 ± 4.75 l a m2 vs m3 80 60 40 20 0 -30 -20 -10 0 10 20 30 delta a: -0.66 ± 5.00 a b m2 vs m3 80 60 40 20 0 -30 -20 -10 0 10 20 30 delta b: 0.36 ± 4.00 b 9 cardoso et al. braz j oral sci. 2022;21:e225946 comparison between methods the secondary hypothesis of the study is the comparison between methods. in table 3 it was shown the ∆s values for each method. the general mean was ∆: 20.5, higher than the expected value for the concordant methods. in the space color axes, the concordance between methods has presented that a* values do not acquire similarity in all measures (table 4). the l* values had good concordance and only in one comparison, the b* value did not have a concordance (table 4). moreover, a* and b* values presented central and adequate distribution in most comparisons. one measure (m3) has shown a proportion bias (allocation trend outside the reference values), as was shown in table 4. the comparison between methods can be viewed in the scatter diagram (figure 3). it was evaluated the concordance coefficient, the value of the outlier, and the point intersections, as the greater the intersection of blue and red points, the greater the similarity between methods. the major intersection was presented by l* values, followed by b*. table 4. bland-altman concordance analysis between methods. spectro vs photo n: 40 pearson (r) correlation p-value (paired t test/ one sample) mean of differences (sd) icc bland-altman concordance (inferior; superior limits) proportion bias (linear simple regression) p-value for means m1 l 0.12 0.21 -2.13 (10.0) 0.11 -21.9 – 17.6 0.31 a 0.11 <0. 05* -16.3 (6.72) 0.02 -29.5 – -3.19 0.39 b > 0.050# 1.00# -6.11 (4.54) 0.02 -15.0 – 2.78 0.85 m2 l -0.04 0.50 -1.19 (9.92) -0.04 -20.5 – 18.3 0.14 a 0.17 <0. 05* -15.7 (5.75) 0.02 -27.0 – -4.47 0.38 b > 0.050# <0. 05#* -5.88 (3.95) 0.08 -13.6 – 1.86 0.12 m3 l -0.31 0.65 0.83 (11.0) -0.29 -20.8 – 22.5 0.94 a 0.11 <0. 05* -16.8 (6.5) 0.01 -29.7 – -3.95 <0. 05§ b 0. 05# > 0.050# -6.05 (5.2) -0.25 -16.4 – 4.28 <0. 05§ *methods are not concordant; # b* axis values show nonparametric distribution (shapiro-wilk test. p<0. 05): executed wilcoxon test and spearman correlation; icc: intraclass correlation coefficient. icc < 0.4 poor; 0.4 <= icc < 0.75 satisfactory; icc >= 0.75 excellent (fleiss, j.l. the design and analysis of clinical experiments. new york: wiley, 1986). linear regression significance of mean: if value was smaller than 0.05 there is a proportion bias§; (the reference levels had the tendency to concentrate above or below the averages / central reference), it means that the method tends to error only for high or low values. table 3. ∆e values for the comparison between methods photo and spectro. ∆e between methods (n: 40) m1 m2 m3 mean ± sd (max.; min.) 20.4 ± 7.4 (33.2; 6.0) 19.8 ± 5.8 (34.1; 8.3) 21.38 ± 7.3 (35.1; 9.2) median, 25%,75% 21.7; 14. 7; 26.5 19.7; 15.8; 24.1 20.8; 15. 9; 27.9 max.: maximum; min.: minimum 10 cardoso et al. braz j oral sci. 2022;21:e225946 focusing on the shape of the ellipses, the greater deformation of the ellipse, the greater the correlation between the x and y coordinate values. perfect circles indicated perfect independence and normality between measurement errors. it was possible to observe that the agreement index between methods was poor (figure 3). however, in the intra method comparison, the measure distribution, confidence intervals, and the number of values of the outliers can be suitable for the not yet validated methods for gingival color evaluation. also, when comparing ∆l*, ∆a*, figure 3. scatter diagram, the spectro method are represented by blue color and photo method are represented by red color, (ellipses represent the 95% confidence interval) (r project). clearest darkest reddest greenest yellowest bluest l spectro vs photo l spectro vs photo l spectro vs photo 80 60 40 20 0 -30 -20 -10 0 10 20 30 delta l m1: -2.13 ± 10.8 l -30 -20 -10 0 10 20 30 delta l m2: -1.19 ± 9.92 delta l m3: 0.8 ± 11.05 -30 -20 -10 0 10 20 30 50 40 20 0 -30 -20 -10 0 10 20 30 delta a m1: -16.37 ± 6.72 a -30 -20 -10 0 10 20 30 delta a m2: -15.75 ± 5.72 -30 -20 -10 0 10 20 30 delta a m3: -16.86 ± 6.58 40 20 0 -30 -20 -10 0 10 delta b m1: -6.11 ± 4.54 b -30 -20 -10 0 10 delta b m2: -5.88 ± 3.95 -30 -20 -10 0 10 delta b m3: -6.05 ± 5.27 100 -40 -40 -40 a spectro vs photo a spectro vs photo a spectro vs photo 30 10 b spectro vs photo b spectro vs photo b spectro vs photo 30 10 11 cardoso et al. braz j oral sci. 2022;21:e225946 and ∆b* between methods (figure 3), it was observed that the biggest variation was in the a* values, approximately 16 units of difference compared to l* that had 2 and b* 5 units of difference. discussion the level of agreement between the three measures of gingival color was evaluated and has exhibited better reproducibility and agreement grades in l*, a*, and b* axis, with adequate proportion and within the confidence intervals. for the ∆e values of 6.5 (spectro) and 5.9 (photo), the results assumed values above the threshold of the human eye (3.7) and those are already known for dental enamel, approximately ∆e: 3.3 (spectrophotometer) and ∆e: 2.9 (photography)21,22. despite that, the quantification of gingival color by the tested methods has obtained an agreement level between the measures, and represents the main result of the study, mainly for l*, a*, and b*, when evaluated separately. it is emphasized that the methods were developed using conventional devices in the dental office (reflectance spectrophotometer to select enamel color and digital camera with software). the clinical relevance of this study was the research with common tools used in dentistry, the spectrophotometer (easyshade), and intra-oral photography to measure gingival color. one of the most used features recently in dentistry, photography can contribute to the auto evaluation of the state of health/disease of the patient, and as the gingival color can be measured through them and daily monitored by dentists after a periodontal plastic procedure to evaluate the cicatricial tissue course, inflammation levels, and aesthetic. the devices used on the tested methods are alternatives36, when compared to studies that use colorimeters10 or spectrophotometers12,37 , specific for gingival scanning and entail additional costs. this explains how the ∆e values and the sensibility of both methods have exceeded the threshold of the human eye. on the other hand, the quantification of gingival color was possible, having a great potential for future use in research. in addition, usual and common tools in the dental office were also used and have presented with an acceptable agreement level between the measures. spectrophotometers has already been used in dental rehabilitation and dental bleaching to evaluate dental color. the present study has shown a possible use of this tool in periodontology and implant dentistry, for the evaluation of gingival color, having acceptable agreement and concordance rates. in the photo method, a digital camera or cell phone camera is able to acquire the images, but in order to reproduce the results observed in the study, it is necessary to have access to a payable software, in order to execute the examinations of l* a* b* values. despite that, mobile apps with color scanning functions are free and available on different digital platforms. even though these tools have not been yet validated for gingival color measurement, they may be tested and therefore, expand the universalization of the method. in the first hypothesis (intra method comparison), in the systematic error (evaluate the method accuracy measured by presence or absence of bias) and in the random error (accuracy between measurements)38,39, the bias or systematic error was evaluated by continuous values. in the wilcoxon or on the ttests, it was revealed that in 12 cardoso et al. braz j oral sci. 2022;21:e225946 the photo group there was bias between the l* axis values (p<0,05), differently to the spectro group, which the axis needs to be adjusted to maintain similarly in the photo acquisition protocol, which interferes in the reading the other outcomes a * and b *. the difficulty of controlling the luminosity can explain the observed difference in the l* axis, beyond the level of sensibility of software to capture minimal different values of l*. a random error is not predictable and it uses the estimate through the agreement coefficients (bland-altman). measures differences were not observed in this concept, since all the limits of agreement were <1.7, and the agreement coefficients (value close to the maximum agreement) presented similar intervals between the measures (maximum of 25 and minimum of 12 units). the b* axis represented the minor interval of agreement coefficients and the lowest limit of agreement, thus obtaining the major approximation to the perfect concordance among measures. the a* axis presented intermediate agreements intervals and the l * axis presented the largest agreement intervals between the measures and the highest agreement limits, values that deviate from the perfect agreement (perfect agreement order: b*>a*>l*). regarding what was observed in the systematic error, the spectro method had major reliability in different measures. for a method to be reliable, the systematic and random errors must be known and contained in the statistical limit of difference. using the concordance limits has the benefit of not requiring data with parametric distribution and fewer comparisons27. to measure the “strength” of reliability, pearson or spearman’s correlation coefficient was used and the similarity was considered satisfactory between measurements (icc mean: 0.44 spectro/0.57 photo). however, this analysis had limitations, since only the values were used to measure the agreement between methods32. the real interpretation of this concept is that the differences between the measurements were not large enough to be detected in the sample size. thus, the model that best express all the information about the comparisons is based on two-dimensional scatter plots with confidence intervals33. the second study hypothesis analyzes the sensibility between the spectro and the photo methods. for the a* values, the results have not shown a good agreement among methods. however, for the l* and in most of b* values, a reasonable agreement was observed. for the dental enamel evaluation, the a* values have varied beyond expectations when compared in the same methods and the values of l* and b* have shown an excellent level of agreement8. the results favor the photography method (plus software) as an alternative, compared to the spectrophotometer, for the reliable acquisition of the variables l* and b*. the agreement level of any measurement method needs information. in periodontology, the probing depth exam helps in the clinical identification of periodontal parameters (sulcus or pocket probing depth, clinical attachment level, beyond bleeding upon probing, and inflammation signals). thus, agreement and sensitivity of the method of measurement combined with the instruments help the operator in identifying the disease/health outcomes. in comparison, the level of error obtained between probing exams is above 1 mm for systematic error and between 0.3 to 0.7 mm as a random error for establishing the attachment clinical level. among evaluators with both manual and electronic probes, the intra-class correlation coef13 cardoso et al. braz j oral sci. 2022;21:e225946 ficient is from 0.41 to 0.90 (reasonable to excellent)8. even a conventional instrument shows changes in its measurements depending on the operators. determining all types of instruments variation is essential to establish fair regimes and the most adequate research protocols. the use of photographs is not recent in dentistry40, not even in the study of tooth enamel color6. new tools were included to facilitate the outcomes from the collection of disorders and diseases41. intraoral scanners are the most current technology for dental impressions and on acquiring oral characteristics. depending on the file format generated, colors are also present in this analysis. nevertheless, it is not yet possible to use the polychromatic scanned files for color evaluation with quality in analysis42,43. despite that, with advances and improvements in technology, it is not difficult to imagine that color will be another factor better incorporated into these tools. when this alternative is available in a quality, the validation for color analysis will also be necessary, even for the comparison between methods and their sensitivity. thus, the next steps for understanding and validating methods used in this study (and their execution format) are the comparison between different operators of the software and the photographs. mainly due to the photo method that had more variables and still needs to be continually tested for its effective potential and for being the cheapest gingival color analysis tool21. even though the used spectrophotometer had a lower number of variables also needs calibrations focused on colors of the natural gingiva, with comparison to directories related to the race, age, and sex/gender of patients13,14. with these elements, the accuracy of the methods would be effectively tested, and the quality of the results better debated. this study had limitations, such as the photograph protocol and adjustments beyond the patient head position7, that could interfere in the measurement21,22. the spectro method needed a better calibration system, aiming to measure gingival color. another limitation was in the point of both analyses of the methods, which was executed 2 mm the gingival margin. this point was defined without guides and the periodontal phenotype was not evaluated as a possible interference. also, comparison with specific colorimeters10 or spectrophotometers12,37, able to evaluate the gingival color, was not executed. nevertheless, this study protocol could compare three different measures, using for each method a feasible comparison pair and an adjusted and complementary statistical analysis system described by previous studies26,27,29-33,38,39. in conclusion, both methods could quantify the gingival color from the coordinates l *, a *, and b *. the evaluation of the intra method has shown slight variations between the measurements and greater reliability for the spectro method. the comparison between methods showed little agreement between them, mainly for a* values. acknowledgments we are grateful to professor dr. adilson yoshio furuse (furuse@usp.br) from the dentistry discipline of the department of dentistry, endodontics and dental materials at the bauru school of dentistry -the university of são paulo, to loan borrowing the spectrophotometer used throughout our study. also, dr. shin-jae lee (nonext. shinjae@gmail.com) and dr. richard donatelli (rdonatelli@dental.ufl.edu) for the prompt help and availability of the r language scripts. 14 cardoso et al. braz j oral sci. 2022;21:e225946 data availability datasets related to this article will be available upon request to the corresponding author funding this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior brasil (capes) finance code 001 (process number 88882.182735/2018-1), and national council for scientific and technological development – cnpq/pibic (n° 653-process number119839/2019-2). disclosure statement the authors declare that there is no conflict of interest. author contribution m.v.c. and c.a.d. designed the study model, the computational framework, and analyzed the data. m.v.c. and g.v. carried out the implementation. m.s.r.z. performed the calculations. m.v.c. and a.c.p.s. wrote the manuscript with input from all authors. m.v.c. and g.v. conceived the study and were in charge of overall direction and planning. all authors actively participated in the discussion of the manuscript’s findings and have revised and approved the final version of the manuscript. references 1. bach junior j. 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shade guides using an intraoral digital scanner. j prosthodont. 2018;27(7):670-5. doi: 10.1111/jopr.12559. http://www.r-project.org/ 1 volume 21 2022 e227095 original research braz j oral sci. 2022;21:e227095http://dx.doi.org/10.20396/bjos.v21i00.8667095 1 graduate program in dentistry, school of dentistry, federal university of pelotas, pelotas, rs, brazil. 2 graduate program in epidemiology, federal university of pelotas, pelotas, brazil. corresponding author: mariana gonzalez cademartori. graduate program in dentistry, school of dentistry, federal university of pelotas, pelotas, rs, brazil. email: marianacademartori@gmail.com phone number: (053) 981351584 editor: altair a. del bel cury received: september 24, 2021 accepted: may 29, 2022 number of erupted teeth at the age of 12 and 24 months: a maternal report validation study ethieli rodrigues da silveira1 , mariana gonzalez cademartori1,* , francine dos santos costa1 , andréia hartwig1 , fernando celso barros1 , andréa dâmaso bertoldi2 , marina sousa azevedo1 , flávio fernando demarco1 aim: to verify the validity of maternal reports on the number of deciduous teeth erupted in their children. methods: this cross-sectional study was performed with children enrolled in a birth cohort at the age of 12 months in the first stage and 24 months in the second stage. at both stages, children were clinically examined, and mothers reported the number of teeth of their children. comparison between groups was performed using the mann-whitney non-paired wilcoxon test. level of agreement between two methods were estimated by the observed agreement, weighted kappa and intraclass correlation coefficients. results: a total of 125 children were examined in the first stage, with mean number of reported teeth of 6.2. in the second stage, 149 children were examined, with mean number of reported teeth of 15.9. high level of agreement, kappa values and intraclass correlation coefficients were observed for both arches in both periods (p<0.001). conclusions: maternal report on the number of teeth erupted in children was reliable and valid. thus, it seems to be a useful instrument for collecting data in population-based epidemiological studies targeting young children. keywords: pediatric dentistry. epidemiology. tooth eruption. tooth, deciduous. https://orcid.org/0000-0002-3444-9282 https://orcid.org/0000-0002-2433-8298 https://orcid.org/0000-0001-9558-937x https://orcid.org/0000-0002-3597-9445 https://orcid.org/0000-0001-5973-1746 https://orcid.org/0000-0002-4680-3197 https://orcid.org/0000-0002-7519-6808 https://orcid.org/0000-0003-2276-491x 2 silveira et al. braz j oral sci. 2022;21:e227095 introduction follow up and evaluation of children’s development stages is crucial to obtain health data, allowing the early identification of any disorder and the establishment of an appropriate care plan1. the eruption of primary teeth usually begins 6–8 months after birth and it is an important marker of the child’s development process, associated with overall growth and metabolic functions2,3. the eruption process can be altered due to several genetic and environmental issues, such as maternal habits, maternal childbearing age, gestational period, child’s head circumference, birth length and weight, eating habits, ethnicity and socioeconomic factors2. the process of deciduous teeth emergence has already been associated with many aspects of children growth, including motor development, where infants developing faster in the first 6 months of life have also earlier onset of tooth emergence3. in addition, primary teeth eruption should be regarded as a potential predictor for nutritional status. significant delay may occur in tooth eruption when a malnutrition episode occurs in the first year of life4. on the other hand, children with accelerated weight gain in the first three months of life have earlier eruption of the first tooth2. a longitudinal follow-up evaluated the anthropometric indices of teenagers at the age of 17 years and found that the early eruption of primary teeth can be an important predictor of obesity in adolescence5. in addition, when tooth eruption does not follow a regular pattern, it may negatively influence children’s chewing pattern, mouth growth6 and can also be a predictor for dental caries7. clinical oral examination has been considered the best method to determine the oral health status. nevertheless, clinical examinations present many difficulties concerning operational aspects, such as high cost, need for trained personnel, time constraints and exam protocol accuracy8,9. in large epidemiological population-based studies, these difficulties may preclude the data collection process. thus, using self-reported oral health measurements has become a common practice in large-scale oral health epidemiological studies, as it is a simple, fast and low cost method10. previous studies have used the number of erupted deciduous teeth reported by the mother as an instrument to monitor the tooth eruption process11,12. however, despite the recognized advantages of using the self-reported number of teeth, there is lack of information in literature on its validity and reproducibility when compared to clinical examination. since primary teeth eruption may be an important marker of child development, there is a need for a validated instrument suitable for use in population-based epidemiological studies. we hypothesized that mothers can correctly recognize and report the number of teeth of their children. thus, the aim of this study was to test the validity of maternal reports regarding the number of deciduous teeth erupted at the age of 12 and 24 months compared to clinical examination. material and methods this cross-sectional study was reported in accordance with the strobe guidelines and involved a sample of participants of the 2015 birth cohort of pelotas13. pelotas 3 silveira et al. braz j oral sci. 2022;21:e227095 is a city in southern brazil recognized worldwide for birth cohort studies carried out in its population. unlike previous studies, the 2015 birth cohort started monitoring its participants during the gestational period with mothers of potential participants living in the urban area of pelotas. the cohort study has performed prenatal, perinatal, three-, twelveand twenty-four-month follow-ups of the children so far. as inclusion criteria, child should be enrolled in the 2015 birth cohort of pelotas and have already participated in 12-month or 24month cohort follow-ups. exclusion criterion considered mothers who were not able to answer the question or perform the tooth count. three dentists, graduate students, with experience in epidemiological surveys were selected for the fieldwork. to ensure reliability, examiners participated on a calibration process prior to oral examinations, evaluating children at the same age group of those who were not included in the final sample. the lowest intra-class correlation coefficient (icc) was 0.82. sample size calculation was carried out using the tool proposed by arifin14. sample size of 55 and 253 children was required for icc and kappa estimation, respectively. the sample size calculation for icc considered 0.7 as the lowest acceptable icc, 0.80 as expected icc, significance level of α = 0.05 for one-tailed test and power of 80%. the measurement was taken on one occasion, two times (two replicates) and the expected dropout rate was 10%. the sample size calculation for kappa statistics used the follow parameters: 0.6 as minimum acceptable kappa, 0.95 as expected icc, 96% as the proportion of any erupted teeth at 12 months (p=0.96) (data not shown), significance level of α=0.05, statistical power of 80% and expected dropout rate of 10%. the validity of maternal reports of deciduous teeth erupted at 12 and 24 months was evaluated in two distinct samples of children enrolled in the 2015 birth cohort of pelotas. about 10% of the 1,384 mother-child dyads who had been monitored in the birth cohort between april and july/2016 and met the inclusion criteria were randomly selected and invited to participate. the sample was selected with the aim of verifying the quality of information collected in the general cohort study (with the reapplication of general questionnaires) and as a source for the validation study. interviews with mothers and clinical examination of children were performed. mothers were asked about the number of deciduous teeth in the superior and inferior arch through the following questions: 1) how many teeth does your child have in the maxillary part of the mouth? and” 2) how many teeth does your child have in the mandibular part of the mouth?”. mothers were instructed to consider as erupted even elements with only a small visible part. standardized figures of deciduous teeth with different eruption degrees were used to illustrate and guide mothers (figure 1a and figure 1b). mothers who were not aware about the number of teeth in their children were encouraged to give an approximate answer. subsequently, children were clinically evaluated using the knee-to-knee technique with the participation of mothers. examiners used disposable gloves and mask, natural lighting, and gauze when necessary. all biosafety precepts were in accordance with the original world health organization recommendation. the number of teeth was counted separately for each arch. to validate the number of deciduous teeth erupted at 24 months, all mother-child dyads that had been monitored between october and november/2017 and met the 4 silveira et al. braz j oral sci. 2022;21:e227095 inclusion criteria were invited to participate. interviews with mothers and clinical examination of children were performed at the dental clinic of the epidemiological research centre during the 24-month follow-up period. mothers were asked about the number of deciduous teeth in the superior and inferior arch using the same questions used in the 12-month evaluation, and oral examination was conducted using the same previously described methodology. sociodemographic information was obtained from the interview performed at the 12 and 24-month follow-ups. maternal age was categorized into less than 30 years and 30 years or more, maternal schooling categorized until 8 years of study and 9 years or more, skin color (self-referred) classified in white, or non-white and family income divided into tertiles. statistical analysis absolute and relative frequencies were described for each variable. nonparametric distribution was identified using the shapiro-wilk test. comparison between groups was performed using the mann-whitney non-paired wilcoxon test (non-parametric test). the number of teeth in the superior and inferior arch reported by mothers was compared with the number of teeth observed in dental examinations. the level of agreement between the two methods was estimated by the observed agreement (oa) and the weighted kappa statistics. kappa-value equal to one means that the methods are in complete agreement. intraclass correlation coefficients were also estimated. all analyses were performed using the stata 14.0 software (statacorp. college st, tx, usa). ethical aspects this study was approved the human research ethics committee of the federal university of pelotas under protocol number 717.271. interviews with mothers and clinical examination of children were performed after signing the informed consent form. orientations regarding child oral health care were given after examination. in cases of the need for dental treatment or dental follow-up, the child was referred to the school of dentistry (federal university of pelotas) or to the public health system. results a total of 125 children aged 11-14 months were evaluated for the validation of maternal reports about number of deciduous teeth erupted at 12 months and 149 children aged 23-25 months participated of the validation of maternal reports at 24 months. the mean age of children was 12.2 months and 24.3 months, respectively (data not shown). table 1 shows the samples according to demographic and socioeconomic characteristics. in both samples, more than 60% of mothers were younger than 30 years, presented nine or more years of formal education and self-declared as white skin color. 5 silveira et al. braz j oral sci. 2022;21:e227095 table 1. distribution of total number of teeth according to demographic and socioeconomic characteristics considering clinical examination. 12-months follow up (125 children) and 24-months follow up (n=149 children). pelotas, brazil, 2019. variables clinical examined number of teeth 12-months follow up 24 months follow up n p-value* n p-value* demographic and socioeconomic characteristics maternal age 0.19 0.90 <30 years 61 76 ≥30 years 46 75 maternal schooling 0.82 0.49 0-8 years 42 49 ≥9 years 83 102 household income 0.89 0.73 1st tertile (lower) 41 51 2nd and 3rd tertiles 80 100 skin color 0.10 0.12 white 79 106 non-white 29 45 nonparametric distribution was identified using the shapiro-wilk test. comparison between groups was performed using the mann-whitney non-paired wilcoxon test (non-parametric test). *p value <0.05. the minimum number of erupted teeth was zero and the maximum number was twelve at the 12-month follow up. in the 24-month follow up, the number of teeth ranged from seven to twelve (data not shown). table 2 presents descriptive analysis of self-reported and dental clinical examination of the number of teeth in the total sample and stratified by sex. in the 12-month follow up, the mean number of present teeth was 6.2, being 3.3 the mean of maxillary teeth and 2.9 the mean of mandibular teeth. in the 24-month follow up, children presented mean number of 15.9 erupted teeth. the same mean number (7.9) for maxillary and mandibular teeth was observed. no differences were observed regarding number of teeth among boys and girls at 12 and 24-month follow-ups (table 2). table 3 describes the observed agreement, kappa statistics and intraclass correlation coefficient for the number of maxillary and mandibular teeth reported by mothers compared with clinical examination for both samples. high level of agreement, kappa values and intraclass coefficients were observed for both arches. table 4 presents the validity of the number of teeth according to sociodemographic characteristics. the findings revealed that sociodemographic variables did not influence the agreement between self-reporting and clinical examination. 6 silveira et al. braz j oral sci. 2022;21:e227095 table 2. distribution of the number of teeth identified in clinical examination according to children’s sex. pelotas, brazil, 2019. variables total sample children’s sex mean (sd) female mean (sd) male mean (sd) p-value* 12 months of age (n=125 children) number of present teeth 6.2 (2.2) 6.6 (3.1) 5.9 (2.5) 0.11 number of present maxillary teeth 3.3 (1.5) 3.5 (1.7) 3.0 (1.6) 0.04 number of present mandibular teeth 2.9 (1.2) 3.1 (1.6) 2.8 (1.9) 0.40 24 months of age (n=149 children) number of present teeth 15.9 (1.6) 15.7 (2.1) 16.0 (1.9) 0.31 number of present maxillary teeth 7.9 (0.7) 7.78 (1.1) 8.05 (0.8) 0.07 number of present mandibular teeth 7.9 (0.9) 7.94 (1.1) 7.96 (1.2) 0.80 *p value <0.05 table 3. observed agreement, kappa index and intraclass correlation coefficient of number of teeth reported by mothers compared to clinical examination. 12-month (125 children) and 24-month (149 children) followup. pelotas, brazil, 2019. variables mother report mean (sd) clinical examination mean (sd) oa (%) wk icc p-value* 12 months of age (n=125 children) number of present maxillary teeth 3.27 (1.63) 3.26 (1.64) 98.7 0.947 0.973 <0.001 number of present mandibular teeth 2.90 (1.38) 2.96 (1.37) 98.5 0.927 0.949 <0.001 24 months of age (n=149 children) number of present maxillary teeth 7.97 (1.15) 7.92 (0.98) 95.9 0.659 0.749 <0.001 number of present mandibular teeth 8.01 (1.33) 7.95 (1.18) 95.4 0.738 0.812 <0.001 oa = observed agreement. icc = intraclass correlation coefficient. wk = weighted kappa. sd = standard deviation. *p value <0.05. table 4. observed agreement, kappa index and intraclass correlation coefficient of clinical examination and self-reported number of teeth according to sociodemographic information. 12-months (125 children) and 24 months (149 children) follow-up. pelotas, brazil, 2019. 12 months follow up 24 months follow up variables oa (%) wk icc p-value* variables oa (%) wk icc p-value* number of maxillary teeth number of maxillary teeth maternal age maternal age <30 years 99.0 0.964 0.985 <0.001 <30 years 96.1 0.681 0.826 <0.001 ≥30 years 97.8 0.917 0.959 <0.001 ≥30 years 93.1 0.634 0.192 <0.001 maternal schooling maternal schooling 0-8 years 98.9 0.962 0.987 <0.001 0-8 years 92.5 0.614 0.618 <0.001 ≥9 years 98.4 0.938 0.965 <0.001 ≥9 years 96.1 0.677 0.818 <0.001 continue 7 silveira et al. braz j oral sci. 2022;21:e227095 continuation household income household income 1st tertile (lower) 98.3 0.929 0.972 <0.001 1st tertile (lower) 92.5 0.585 0.607 <0.001 2nd and 3rd tertiles 98.9 0.962 0.982 <0.001 2nd and 3rd tertiles 96.2 0.698 0.852 <0.001 skin color skin color white 98.9 0.959 0.983 <0.001 white 95.8 0.674 0.814 <0.001 non-white 97.9 0.935 0.961 <0.001 non-white 93.2 0.600 0.656 <0.001 number of mandibular teeth number of mandibular teeth maternal age maternal age <30 years 98.8 0.952 0.974 <0.001 <30 years 95.6 0.773 0.892 <0.001 ≥30 years 96.3 0.842 0.855 <0.001 ≥30 years 93.2 0.694 0.719 <0.001 maternal schooling maternal schooling 0-8 years 97.8 0.911 0.937 <0.001 0-8 years 89.4 0.595 0.716 <0.001 ≥9 years 98.6 0.943 0.967 <0.001 ≥9 years 96.8 0.805 0.863 <0.001 household income household income 1st tertile (lower) 97.1 0.881 0.933 <0.001 1st tertile (lower) 94.7 0.774 0.829 <0.001 2nd and 3rd tertiles 98.7 0.944 0.954 <0.001 2nd and 3rd tertiles 95.4 0.714 0.789 <0.001 skin color skin color white 97.9 0.922 0.945 <0.001 white 96.2 0.786 0.874 <0.001 non-white 97.1 0.896 0.900 <0.001 non-white 88.4 0.608 0.550 <0.001 oa = observed agreement. icc = intraclass correlation coefficient. wk = weighted kappa. *p value <0.05. discussion this study investigated the agreement level between number of teeth reported by mothers and number of teeth observed in clinical examination in both samples (mothers and children) enrolled in the 12 and 24-month follow-ups of the 2015 birth cohort study of pelotas. our findings revealed high agreement level between maternal report and clinical examination regarding number of teeth in both periods, showing that the maternal report is a reliable and valid instrument for this purpose. to the best of our knowledge, this is the first study to demonstrate that the use of maternal report to collect data on the number of deciduous erupted teeth in children is a valid tool. there is substantial variability among individuals in the tooth eruption time15-17, however, there is no consensus about the eruption time being different in terms of sex. some studies have indicated that tooth eruption occurs more quickly in boys17, compared to girls, other studies have shown the opposite relationship18. in this study, no difference in the tooth eruption time was observed between sexes. other child and maternal factors could explain the difference in tooth eruption. findings of previous studies have shown that lower mean number of teeth was observed in early preterm children, shorter children at birth and at 12 months of age. also, higher number of teeth was observed in children whose mothers had excessive weight gain during 8 silveira et al. braz j oral sci. 2022;21:e227095 pregnancy, those who smoked during pregnancy, children with greater weight at birth and at 12 months of age. bastos et al.19 also identified relationship between dental eruption and nutritional status of children, with children shorter than 49 cm having, on average, less pairs of erupted teeth at 6 months of age. twelve-month-old children presenting height-for-age deficit at 6 months of age and females also presented less pairs of erupted teeth in this sample. recent researches about the use of oral-health self-report have pointed out that great variation on agreement levels may occur across socioeconomic strata, which could affect the validity of self-reported measures20,21. regarding self-reported information on dental caries in adolescents, for instance, this method presents high specificity and sensitivity in relation to clinical examination, but higher schooling levels led to even higher sensitivity and positive predictive values. socioeconomic level also influences maternal report on the presence of dental plaque in their children, which demonstrated not to be a valid method22. however, our study evidenced that counting the number of erupted teeth in young children reported by mothers is a reliable method, despite sociodemographic conditions like maternal schooling or family income. it could be hypothesized that this influence was not found because the question about the number of teeth does not have a correct or desirable answer, opposed to results obtained when questioning about the presence of caries or dental plaque. the number of erupted teeth in the first two years of life may also be related to anthropometric measures in childhood2,5 and adolescence. it has been suggested that early deciduous teeth eruption may be an indicator of obesity at the age of 17 years5. likewise, association between pattern of weight gain and eruption of the deciduous dentition has been observed. according to un lam and colleagues2, the higher the weight gain in the first three months of life, the earlier the eruption of the first deciduous teeth2. therefore, the eruption process may be an important indicator of future health changes. regarding younger children, distress behavior can be observed when facing health procedures23, due to their difficulties to understand the process and control emotions24. the success of procedures in pediatric dentistry depends mainly on child behavior and uncooperative temperament may predict the failure of dental care25. this process may be a barrier to the development of epidemiological surveys targeting children. thus, in pediatric dentistry, finding a suitable tool to evaluate deciduous teeth, which that does not rely only upon children’s collaboration during the execution of procedures and possible to be used in epidemiological surveys is of fundamental importance. regarding oral health, this measure is also significant since early eruption of deciduous teeth may be considered a risk factor for dental caries in the future7. knowledge about the process of deciduous teeth eruption is fundamental to guide health actions for the promotion and prevention of oral diseases in children. in brazil, the last national oral health survey showed that the lowest rate of dental caries reduction was observed among 5-years-old children. in addition, it showed that 80% of primary teeth affected by caries remained untreated26. dental caries in the deciduous dentition affects approximately 9% of the world population27, being ranked among the most prevalent chronic diseases worldwide. it is of relevance for clinicians and 9 silveira et al. braz j oral sci. 2022;21:e227095 policy makers, since dental caries in deciduous teeth is a risk factor of dental caries in permanent teeth28. in oral health surveys, data collection through clinical examination has already been considered as the only valid source of information28; however, it has disadvantages such as the need for large number of people involved, professionals with higher level of academic training (dentists), with high costs and requiring larger structures8. maternal reporting has already been used in research involving children, such as motor development evaluation29, and pubertal development30, and the present study demonstrated the feasibility of using maternal report to count the number of erupted teeth in children, which is a simple, quick, and low-cost method. despite the promising results observed in this study, they should be analyzed with caution. children participating in this sample were very young; therefore, presenting low number of teeth. in addition, at this stage of life, children are heavily dependent on maternal care, so mothers are probably more attentive and more participatory than usual. it could be concluded that the maternal report on the number of teeth erupted in young children is a reliable and valid instrument compared to clinical examination performed by dentists. thus, it seems to be a useful instrument for collecting deciduous teeth data in population-based epidemiological studies. acknowledgements this article is based on data from the study “pelotas birth cohort, 2015” conducted by postgraduate program in epidemiology at universidade federal de pelotas, with the collaboration of the brazilian public health association (abrasco). the 2015 pelotas (brazil) birth cohort is funded by the wellcome trust (095582). funding for specific follow-up visits was also received from the conselho nacional de desenvolvimento científico e tecnológico (cnpq) and fundação de amparo a pesquisa do estado do rio grande do sul (fapergs). this study was also financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior brasil (capes) finance code 001. the authors declare that are not conflict of interests. data availability datasets related to this article cannot be shared at this moment because they are part of an ongoing research. conflict of interest none author contributions e.r.s., m.g.c., f.f.d., a.h.d. and m.s.a. conceived the idea; e.r.s., f.s.c. and a.h. collected the data; m.g.c. and e.r.s. analysed the data; and all authors wrote and reviewed the manuscript. 10 silveira et al. braz j oral sci. 2022;21:e227095 references 1. sabatés al, mendes lcdo. 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[a cross-sectional survey on the patterns of primary teeth eruption in 2 581 children]. zhonghua er ke za zhi. 2017 jan 2;55(1):37-41. chinese. doi: 10.3760/cma.j.issn.0578-1310.2017.01.007. 11 silveira et al. braz j oral sci. 2022;21:e227095 17. oziegbe eo, adekoya-sofowora c, esan ta, owotade fj. eruption chronology of primary teeth in nigerian children. j clin pediatr dent. 2008 summer;32(4):341-5. doi: 10.17796/ jcpd.32.4.9571r10781044217. 18. burgueño torres l, mourelle martínez mr, de nova garcía jm. a study on the chronology and sequence of eruption of primary teeth in spanish children. eur j paediatr dent. 2015 dec;16(4):301-4. 19. bastos jl, peres ma, peres kg, barros aj. infant growth, development and tooth emergence patterns: a longitudinal study from birth to 6 years of age. arch oral biol. 2007 jun;52(6):598-606. doi: 10.1016/j.archoralbio.2006.12.001. 20. genco rj, falkner kl, grossi s, dunford r, trevisan m. validity of self-reported measures for surveillance of periodontal disease in two western new york population-based studies. j periodontol. 2007 jul;78(7 suppl):1439-54. doi: 10.1902/jop.2007.060435. 21. nico ls, andrade ss, malta dc, pucca júnior ga, peres ma. self-reported oral health in the brazilian adult population: results of the 2013 national health survey. cien saude colet. 2016 feb;21(2):389-98. english, portuguese. doi: 10.1590/1413-81232015212.25942015. 22. cascaes am, peres kg, peres ma, demarco ff, santos i, matijasevich a, et al. validity of 5-year-old children’s oral hygiene pattern referred by mothers. rev saude publica. 2011 aug;45(4):668-75. english, portuguese. doi: 10.1590/s0034-89102011005000033. 23. miranda-remijo d, orsini mr, corrêa-faria p, costa lr. mother-child interactions and young child behavior during procedural conscious sedation. bmc pediatr. 2016 dec;16(1):201. doi: 10.1186/s12887-016-0743-2. 24. klingberg g, broberg ag. dental fear/anxiety and dental behaviour management problems in children and adolescents: a review of prevalence and concomitant psychological factors. int j paediatr dent. 2007 nov;17(6):391-406. doi: 10.1111/j.1365-263x.2007.00872.x. 25. aminabadi na, pourkazemi m, babapour j, oskouei sg. the impact of maternal emotional intelligence and parenting style on child anxiety and behavior in the dental setting. med oral patol oral cir bucal. 2012 nov;17(6):e1089-95. doi: 10.4317/medoral.17839. 26. roncalli ag. [sb brasil project 2010 national oral health survey reveals significant reduction in dental caries in the country]. cad saude publica. 2011;27(1):4-5. portuguese. 27. marcenes w, kassebaum nj, bernabé e, flaxman a, naghavi m, lopez a, murray cj. global burden of oral conditions in 1990-2010: a systematic analysis. j dent res. 2013 jul;92(7):592-7. doi: 10.1177/0022034513490168. 28. grund k, goddon i, schüler im, lehmann t, heinrich-weltzien r. clinical consequences of untreated dental caries in german 5and 8-year-olds. bmc oral health. 2015 nov;15(1):140. doi: 10.1186/s12903-015-0121-8. 29. jamieson lm, thomson wm, mcgee r. an assessment of the validity and reliability of dental selfreport items used in a national child nutrition survey. community dent oral epidemiol. 2004 feb;32(1):49-54. doi: 10.1111/j.1600-0528.2004.00126.x. 30. simcock g, kildea s, elgbeili g, laplante dp, stapleton h, cobham v., et al. age-related changes in the effects of stress in pregnancy on infant motor development by maternal report: the queensland flood study. dev psychobiol. 2016 jul;58(5):640-59. doi: 10.1002/dev.21407. 1http://dx.doi.org/10.20396/bjos.v21i00.8664977 volume 21 2022 e224977 original article 1 departement of periodontogy, university of padjadjaran, indonésia. corresponding author: puspa puspita lasminingrum adress: bandung, west java, indonesia e-mail adress: puspa16002@mail. unpad.ac.id editor: dr altair a. del bel cury received: march 15, 2021 accepted: june 30, 2021 the assessment of periimplant soft tissue condition with morse taper abutment connection: a rapid review puspa puspita lasminingrum1,* , aldilla miranda1 , dyah nindita carolina1 , prajna metta1 aim: this study aims to evaluate the clinical assessment results of periimplant soft tissue with morse taper (internal abutment connection). methods: the study was conducted using a rapid review by searching the articles from pubmed ncbi and cochrane by using keywords. all articles were selected by the year, duplication, title, abstract, full-text, and finally, all selected articles were processed for final review. following clinical parameters were included; periimplant probing pocket depth (ppd), plaque score (ps), modified plaque index (mpi), mucosal thickness (mth), gingival height (gh), periimplant mucosal zenith, pink esthetic score (pes), bleeding on probing (bop), sulcus bleeding index (sbi), and modified gingival index (mgi). results: 9 selected articles were obtained from the initial literature searching count of 70 articles. the overall samples included 326 morse taper implants. based on the evaluation, 3 out of 4 articles reported pocket depth < 4 mm, no bleeding was reported in 2 out of 4 articles. 4 out of 4 articles reported low plaque accumulation, low soft tissue recession was reported in 3 out of 3 articles, and 4 out of 4 articles reported acceptable pes values. conclusion: the evaluations indicate that the morse taper (internal abutment connection) has favorable assessment results based on various clinical parameters. keywords: dental implant-abutment design. soft tissue injuries. dental abutments. dental implants. gingiva. mouth mucosa. https://orcid.org/0000-0002-8771-3649 https://orcid.org/0000-0003-1474-0053 https://orcid.org/0000-0003-0208-1353 https://orcid.org/0000-0001-8508-1919 2 lasminingrum et al. introduction dental implants have been widely used for the replacement of missing teeth1. the survival rate of dental implant treatment for five years reached 96.3%2. however, this treatment still caused several complications such as; technical complications or screw loosening 8.8%; soft tissue complications 7.1%; marginal bone loss 5.2%; and aesthetic complications 7.1%2. the implant system can be differentiated based on the material used in dental implants, surface topography, morphology, and geometry of the abutment connection3. the geometry of the abutment connection influences the incidence of screw loosening and may play a crucial role of the bacterial composition in the neck area of the implant4. the formation of microgap due to bacterial leakage on the abutment connection causes microleakage to occur5. microleakage allows penetration and accumulation of bacteria through the microgap which leads the periimplant soft tissue inflammation5. therefore, this can affect the stability of marginal bone, periimplant soft tissue, and aesthetics condition5. basically, the geometry of the abutment connection is divided into external connection and internal connection1. external connection was first implemented in the branemark implant system and has a hexagonal structure3,6. internal connection has several variants, including internal hexagon and morse taper7. morse taper abutment connection that was placed in the anterior maxilla had the lowest global annual failure rate (0.2%)8. this design can reduce microgap and micromovement formation due to its ability to resist leakage9. therefore, better load distribution on the abutment connection obtained and minimal marginal bone loss observed with morse taper9. the stability of periimplant soft tissue is one of the key aspects of successful dental implant treatment because it provides sealing ability and stability of the marginal bone, prevents penetration of oral microorganisms, and enables good aesthetic results10. a considerable amount of literature from ncbi pubmed and cochrane databases regarding morse taper internal abutment connection have been published. however, no previous study has been conducted using a rapid review method for evaluating the periimplant soft tissue. based on the explanation that has been presented, this paper aims to systematically evaluate the clinical assesment results of periimplant soft tissue using morse taper (internal abutment connection) with the rapid review method. material and methods this rapid review was performed according to the prisma (preferred reporting items for systematic reviews and meta-analyses) statement (figure 1)11,12. selected articles were english language articles between 2009-2019 that discussed morse taper and the clinical assesment result of periimplant soft tissue with a minimum follow-up period of 12 months. non full -text article and narrative review were excluded. 3 lasminingrum et al. an electronic search through pubmed ncbi and cochrane was conducted using the boolean operators method with keywords “(((((morse taper[title/abstract]) or conical interface[title/abstract]) or conical connection[title/abstract])) and (((periimplant[title/abstract]) or soft tissue[title/abstract])) and clinical[title/abstract]”. all articles were selected by the year, duplication, title, abstract, full-text, and finally, all articles were obtained and analyzed using the thematic analysis method. following data were assessed: periimplant probing pocket depth (ppd), bleeding on probing (bop), sulcus bleeding index (sbi), modified gingival index (mgi), modified plaque index (mpi), plaque score (ps), mucosal thickness (mth), gingival height (gh), periimplant mucosal zenith, and pink esthetic score (pes). levels of evidence were identified using the strength of recommendation taxonomy (sort). results through the literature searching, 70 articles were identified, selected by the year, duplication, title, and abstract. finally, 9 full-text articles matched the inclusion criteria and were processed for the final review (figure 1)13-21. five articles were randomized controlled trials (sort level of evidence-1)13,15,16,19,20. three articles were prospective cohort studies (sort level of evidence-2)14,17,18. one case series article (sort level of evidence-3)18. the overall samples included 326 morse taper connection implants (mt); 151 non-morse taper internal connection implants (nmti); and 69 non-morse taper external connection implants (nmte)13-21. table 2 presents the results of clinical periimplant soft tissue assessment parameters obtained from selected articles. figure 1. flowchart of the study selection process id en fic ic at io n sc re en in g el ig ib ili ty in cl ud ed records identified through databasa searching (n = 70) records selected for the year of publication (n = 69) records after duplicates removed (n = 61) records screened for title and abstract (n = 31) full-text articles assessed for eligibility (n = 9) articles included in the review (n = 9) record excluded by title and abstract (n = 30) articles excluded for full-text analysis (n = 22) 4 lasminingrum et al. ta bl e 1. c lin ic al p er iim pl an t s of t t is su e a ss es sm en t p ar am et er s fr om s el ec te d a rt ic le s a ut ho r (y ea r) s tu dy de si gn fo llo w -u p pe ri od n um be r of s am pl es / c on ne ct io n ty pe c lin ic al p ar am et er s r es ul ts r em ar ks c oo pe r, et a l.1 3 (2 01 9) r an do m iz ed co nt ro lle d tr ia l 3 ye ar s 79 im pl an ts : ex pe rim en ta l • m t: 4 5 (o ss eo sp ee d) c on tr ol • n m ti : 3 4 (n ob el sp ee dy ) 1. p p d 2. p er iim pl an t m uc os al z en ith re ce ss io n 1. p p d ≥ 4 m m b uc ca l a sp ec t: • m t 7% • n m ti 1 8% p p d ≥ 4 m m li ng ua l a sp ec t: • m t 2% • n m ti 2 4% 2. p er iim pl an t m uc os al z en ith re ce ss io n ≤ 5 m m : • m t 80 % • n m ti 6 1% 1. p p d ≥ 4 m m b uc ca l a nd li ng ua l as pe ct th e le as t o cc ur re d in m t 2. p er iim pl an t m uc os al z en ith re ce ss io n ≤ 5 m m m os tly oc cu rr ed in m t p es so a, e t a l.1 5 (2 01 7) r an do m iz ed co nt ro lle d tr ia l 1 ye ar 48 im pl an ts : ex pe rim en ta l • m t: 2 4 (u n i-t it e) c on tr ol • n m te : 2 4 (u n i-t it e) 1. p p d 2. m th 3. m g i 1. a ve ra ge p p d : • m t 1. 36 ± 0 .7 m m • m te 1 .5 7 ± 0. 9 m m 2. a ve ra ge m th : • m t 2. 27 ± 0 .8 5 m m • n m te 2 .1 6 ± 0. 94 m m 3. m g i • v er y lo w in b ot h ty pe s 1. l ow er p oc ke t d ep th a nd m uc os al ap pe ar an ce w er e fo un d w ith m t 2. n o si gn ifi ca nt d iff er en ce fo r po ck et d ep th a nd m uc os al th ic kn es s in b ot h ty pe s 3. v er y lo w b le ed in g in de x fo un d in bo th ty pe s b ar w ac z, e t a l.3 6 (2 01 6) r an do m iz ed co nt ro lle d tr ia l 3 ye ar s 97 im pl an ts : ex pe rim en ta l • m t: 4 8 (o ss eo sp ee d) c on tr ol • n m ti : 4 9 (n ob el sp ee dy ) 1. p es 1. a ve ra ge p es : • m t 10 .4 • n m ti 1 0. 0 1. b ot h ty pe s sh ow ed fa vo ra bl e p es v al ue s w ith n o si gn ifi ca nt di ff er en ce m cg ui re , e t a l.1 9 (2 01 5) r an do m iz ed co nt ro lle d tr ia l 1 ye ar 8 im pl an ts : ex pe rim en ta l • m t: 4 (o ss eo sp ee d) c on tr ol • n m ti : 4 (n ob el sp ee dy ) 1. p es 1. a ve ra ge p es v al ue s: • m t 11 .3 5 • n m ti 1 1. 55 1. b ot h ty pe s sh ow ed fa vo ra bl e p es v al ue s w ith n o si gn ifi ca nt di ff er en ce c on tin ue 5 lasminingrum et al. p oz zi e t a l.2 0 (2 01 4) r an do m iz ed co nt ro lle d tr ia l 3 ye ar s 68 im pl an ts : ex pe rim en ta l • m t: 3 4 (n ob el a ct iv e) c on tr ol • n m te : 3 4 (n ob el sp ee dy ) 1. s b i 2. p s 1. s b i f ou nd in 1 n m t 2. p s fo un d in 1 m t an d 1 n m te (y ea r 1 ) 1. n o si gn ifi ca nt d iff er en ce in th e as se ss m en t o f s b i a nd p s 2. n o bl ee di ng in a ll so ft ti ss ue su rf ac es w ith m t 3. p la qu e ac cu m ul at io n w as fo un d in 1 im pl an ts w ith m t an d 1 im pl an t w ith n m te a ft er 1 y ea r in fu nc tio n h ey de ck e, e t a l.1 4 (2 01 9) p ro sp ec tiv e c oh or t 3 ye ar s m t: 9 9 (n ob el r ep la ce c on ic al c on ne ct io n) 1. b o p 2. m p i 3. p es 1. n o b o p in 8 0. 7% s of t t is su e su rf ac es 2. n o pl aq ue w as o bs er ve d on 6 5. 9% im pl an t si te s fr om m p i a ss es sm en t 3. a ve ra ge p es : 9 .8 7 ± 2. 19 1. n o bl ee di ng w as fo un d in 8 0. 7% pe rii m pl an t s of t t is su e su rf ac es 2. n o pl aq ue a cc um ul at io n w as fo un d in 6 5. 9% im pl an t s ite s 3. a cc ep ta bl e p es re su lt c os yn , e t a l.1 8 (2 01 6) p ro sp ec tiv e c as e se rie s 5 ye ar s m t: 1 7 (n ob el a ct iv e) 1. p p d 2. b o p 3. p s 4. p es 1. a ve ra ge p p d : 3 .1 m m 2. a ve ra ge p s: 1 5% , 3. a ve ra ge b o p : 3 2% 4. a ve ra ge p es : • 12 .1 5 (1 y ea r) • 11 .1 8 (5 y ea r) 1. f av or ab le p p d a nd p s re su lts 2. b le ed in g w as fo un d in b o p as se ss m en t 3. s ig ni fic an t p es v al ue re du ct io n (0. 97 ) w ith in 5 y ea rs fo llo w -u p g ul te ki n, e t a l.2 1 (2 01 3) p ro sp ec tiv e c oh or t 15 m on th s 93 im pl an ts : ex pe rim en ta l • m t: 4 3 (n ob el a ct iv e) c on tr ol • n m ti : 5 3 (n ob el r ep la ce ta pe re d g ro ov y) 1. p p d 2. m p i 1. a ve ra ge p p d v al ue s: • m t 2. 6 ± 0. 46 m m • n m ti 3 .3 3 ± 0. 51 m m 2. a ve ra ge m p i v al ue s: • m t 0. 64 ± 0 .2 8 • n m ti 0 .6 1 ± 0. 36 1. s ig ni fic an t d iff er en ce o f t he av er ag es o f p oc ke t d ep th w as fo un d, th e lo w es t p oc ke t d ep th ob se rv ed in m t 2. n o si gn ifi ca nt p la qu e in de x va lu es d iff er en ce . a cc ep ta bl e re su lts w er e re po rt ed in b ot h ty pe s k am in ak a, e t a l.1 7 (2 01 5) p ro sp ec tiv e c oh or t 1 ye ar 22 im pl an ts : ex pe rim en ta l • m t: 1 2 (n ob el a ct iv e) c on tr ol • n m te : 1 1 (n ob el sp ee dy g ro ov y) • n m ti : 1 1 (n ob el r ep la ce ) 1. g h re du ct io n (t he m ea su re m en t fr om th e im pl an t pl at fo rm to th e m ar gi na l s of t tis su e le ve l ) us in g c b c t 1. δ g h v al ue s: • m t 0. 06 ± 0 .1 0 m m • n m te 0 .3 9 ± 0. 19 m m • n m ti 0 .2 8 ± 0. 30 m m 1. s ig ni fic an t d iff er en ce s of g h re du ct io n va lu es w er e ob se rv ed , lo w es t r ed uc tio n w as fo un d in m t c on tin ua tio n 6 lasminingrum et al. favorable ppd results with morse taper were reported in 3 out of 4 articles (75%). cooper et al.13 (2019) found ppd ≥ 4 mm the least occurred with morse taper. three studies reported the ppd averages of 1.36 ± 0.7 mm, 2.6 ± 0.46 mm, and 3.1 mm15,18,21. no bleeding presence on most of the periimplant soft tissue surfaces was reported in 2 out of 4 articles (50%). pessoa et al.15 (2017) stated low mgi score in the evaluated region with morse taper. heydecke et al.14 (2019) observed no bleeding occurred in 80.7% of surfaces and another study by pozzi et al.20 (2014) reported no bleeding found on all surfaces. on the contrary, as stated by cosyn et al.18 (2016) bleeding was found in 32% of periimplant soft tissue surfaces. low plaque accumulation was reported in 4 out of 4 articles (100%). pozzi et al.20 (2014) found a slight plaque accumulation in one morse taper implant after one year in function. cosyn et al.18 (2016) stated that the ps was on average of 15% after 5 years follow-up period. heyecke et al.14 (2015) found no plaque accumulation in 66.9% implants. a study by gultekin et al.21 (2013) stated the mean mpi value was 0.64 ± 0.28 after 12 months follow-up period. low soft tissue recession was reported in 3 out of 3 articles (100%). kaminaka et al.17 (2015) stated that among other designs, morse taper showed the lowest gingival height reduction, which was 0.06 ± 0.10 mm. cooper et al.13 (2019) reported periimplant mucosal zenith reduction of less than 5 mm found in 80% of implants with morse taper. pessoa et al.15 (2017) reported the average mth value of 2.27 ± 0.85 mm after implant placement. acceptable pes values were reported in 4 out of 4 articles (100%). two studies that assessed pes in implants with morse taper internal connection and non-morse taper internal connection, stated that there was no significant difference and both types of abutment connection had equally good pes values13,16. other authors reported pes value with the averages of 9.87 ± 2.19 and 11.1814,18. discussion the examinations of ppd and bop have been considered as the assessment to define a successful dental implant treatment22-24. takei and carranza4 (2019) stated that pocket depth around 3 mm without any presence of bleeding on all surfaces could be identified as a healthy periimplant soft tissue condition. in this review, several studies showed the average of ppd ≤ 3.1 mm15,18,21 low percentages of bop and sbi were also found14,20. this result might be explained by the concept of morse taper connection, which is a particular kind of internal abutment connection with a matching conical or taper shape and an equal angle (5-16° of conicity) between the wall of abutment and the implant3. this creates an intimate contact and a significant amount of mechanical friction locking. hence, this design can stabilize under static load, eliminates microgap below dynamic load, and prevents micromovement14. recent study reported that no type of abutment connection could 100% prevent microleakage25. however, the microgap formed in morse taper (2-3 μm) was smaller than in external connection (10 μm), which pointed out that morse taper had a better bacterial seal compared to the external connection6,26-30. bacterial contamination through microgap causes an inflamma7 lasminingrum et al. tory reaction in the periimplant soft tissue and triggers an osteoclastic process that may precipitate on marginal bone resorption around the implant5,15,31. gingival recession due to bone resorption caused by the absence of supracrestal connective tissue as established in periodontal tissue leads the periimplant soft tissue to thoroughly rely on the supporting marginal bone4. this view is supported by the recent systematic review by caricasulo et al.9 (2018) who stated that the least marginal bone loss was found in the internal connection, especially in the morse taper. favourable periimplant soft tissue assessment results in this review are likely to be related to the optimal support from the marginal bone due to low bone resorption13,15,17,20. consequently, a healthy periimplant soft tissue condition and a satisfactory aesthetic result will be obtained15. this is consistent with that of kaminaka et al.17 (2015) who concluded that morse taper was more effective in preserving the stability of the periimplant hard and soft tissue. another factor that might influence this condition is the presence of platform switch, which is a concept of the placement of narrower diameter abutment. therefore, if the bacterial contamination on the microgap persists; a certain distance between the microgap and the neck of the implant that attached to the marginal bone will be maintained15. hence, minimal marginal bone resorption obtained and adequate biological width will be established15. this accords with another study by macedo et al.26 (2016), that reported low marginal bone resorption and wide biological width observed in apical and lateral directions with morse taper and platform switch abutment. in this article, all types of abutment connections reported acceptable pes results13,14,16,18. however, only some appeared to be in healthy periimplant soft tissue conditions and some of the results showed the presence of bleeding on probing and pocket depth ≥ 4 mm which may lead to periimplant mucositis and periimplantitis20,21. according to some studies, there were several factors other than the variation of the implant abutment connection that may influence the pes results such as, the presence of implant thread, the surface roughness of the implant neck, tissue biotype, and the experience of the clinicians15,16. dani et al.32 (2018) found that higher soft tissue recession was observed with inexperienced clinicians after 3 years following implant placement. time may also play as a contributing factor to this outcome. cosyn et al.18 (2016) stated that recession more than 1 mm was found in 3 out of 17 implants after 5 years following implant placement, which caused a significant change in pes results. this finding seems to be consistent with a study by pozzi et al.20 (2014) which stated that statistically significant results may be obtained by the longer research period. high plaque accumulation within the study period may increase the risk of periimplant disease33. likewise, dhir34 (2013) and prado et al.25 (2016) stated that bacterial contamination might exist in the implant abutment connection causing fluid leakage into the microgap and reduce the mechanical friction between surfaces. therefore, this could affect the periimplant soft and hard tissue condition25,34. better sealing capability from morse taper can reduce microgap and prevent any leakage6. this view is supported by low plaque accumulation results around the periimplant soft tissue surfaces with morse taper in this review14,18,35. in contrast to earlier findings, recent study have failed to demonstrate significant changes of plaque index results between morse taper (0.64 ± 0.28) and internal connection (0.61 ± 0.36)21. mishra et al.6 (2017) stated that 8 lasminingrum et al. implant abutment connection plays an important role in preventing bacterial leakage. however, infiltration of inflammatory substances may occur regardless of the amount of plaque accumulation6. a possible factor that may increase the formation of biofilms other than the type of connection is the implant surface roughness ≥ 0.2 µm34. a limitation of this study is that only a few articles have assessed the role of implant abutment connection towards periimplant soft tissue condition in 5 years follow-up period with comprehensive assessment and randomized control trial method. another source of uncertainty is the varied specifications of morse taper that came from several implant systems. therefore, it is difficult to determine which factors have a significant impact on the assessment results. future researches on the current topic are therefore suggested. in conclusion, the results of this evaluation indicate that the morse taper (internal abutment connection) has favorable assessment results based on various clinical parameters such as; ppd; ps; mpi; mth; gh; periimplant mucosal zenith; and pes. the low percentage of bleeding around periimplant soft tissue surfaces were reported from the assessment of bop, sbi, and mgi examinations. acknowledgment the authors appreciate the financial support for this review provided by lpdp, ministry of finance, republic of indonesia. references 1. goiato mc, pellizzer ep, da silva evf, bonatto l da r, dos santos dm. is the internal connection more efficient than external connection in mechanical, biological, and esthetical point of views? a systematic review. oral maxillofac surg. 2015 sep;19(3):229-42. doi: 10.1007/s10006-015-0494-5. 2. jung re, zembic a, pjetursson be, zwahlen m, thoma ds. systematic review of the survival rate and the incidence of biological, technical, and aesthetic complications of single crowns on implants reported in longitudinal studies with a mean follow-up of 5 years. clin oral implants res. 2012 oct;23 suppl 6:2-21. doi: 10.1111/j.1600-0501.2012.02547.x. 3. ceruso fm, barnaba p, mazzoleni s, ottria l, gargari m, zuccon a, et al. implant-abutment connections on single crowns: 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10.1111/clr.12640. 1http://dx.doi.org/10.20396/bjos.v21i00.8666288 volume 21 2022 e226288 original article 1 department of public health dentistry, faculty of dentistry, universitas jember, jember, indonesia. 2 department of biostatistics, faculty of public health, universitas jember, jember, indonesia. 3 department of pediatric dentistry, faculty of dentistry, universitas jember, jember, indonesia. corresponding author: elyda akhya afida misrohmasari faculty of dentistry, universitas jember, jl kalimantan 37, kampus tegal boto, jember, jawa timur, indonesia tel/fax: +62331 333536 e-mail: elyda.fkg@unej.ac.id editor: altair a. del bel cury received: july 6, 2021 accepted: january 11, 2022 toothache experiences: findings from 21 years longitudinal survey elyda akhya afida misrohmasari1,* , dimas wicaksono2 , hestieyonini hadnyanawati1 , berlian prihatiningrum3 aim: to describe the pattern of toothache experience in a cohort of children aged 2-5 over 21 years and to find the relationship between previous toothache experience and later reports of toothache to get finding on the most critical period of toothache as a problem in life. methods: this is a secondary data analysis from indonesia family life survey (ifls). a total of 1,927 children from ifls-1 with complete data were included as baseline participants. they were followed up four times within 21 years (age 6-9y, age 9-12y, age 16-19y, age 2326y). toothache was based on the question of self-reported toothache experience during the last four weeks. after 21 years, a total of 1,098 individuals could be traced and completed every cohort of the survey. toothache experiences were reported for frequencies in every cohort and accumulative experiences over 21 years. logistic regression tests were performed to analyze the association of previous toothache experience and later toothache experience. results: almost 40% of the respondents reported toothache at least once in their life. the age of 6-9 years is the period when a high percentage of children had teeth-related pain. the experience of toothache at this period was significantly related to every period of age in life. conclusions:  the period of early mixed dentition is important. oral health status in this period is associated with future oral health. a comprehensive dental health prevention program targeting this population is essential to increase the quality of life. keywords: toothache. longitudinal studies. oral health. https://orcid.org/0000-0002-1791-113x https://orcid.org/0000-0002-8148-7433 https://orcid.org/0000-0002-8974-7201 https://orcid.org/0000-0002-0744-6123 2 misrohmasari et al. braz j oral sci. 2022;21:e226288 introduction a toothache is a public health problem because of its high prevalence and impacts among all age groups1-3. it is often mentioned as the most frequent type of orofacial pain4 and one of the major consequences of dental and oral health problems among children5,6. toothache was also found to be experienced by 21.8% of adolescents7. more than one in ten people ≥65  years were also reported had a recent toothache experience. moreover, toothache may have detrimental effects on individuals and society. many studies showed that dental pain reduces sleep duration, increases school/work absences, and affects nutrition, which can negatively impact the quality of life8-10. because of its significant effects on the life of people and society, the global goals for oral health in the year 2020 includes reduction of the episodes of oral pain in the population as one of the targets on oral health programs11. information that toothache is the most mentioning problem in life is abundant, but understanding the importance of how the sequences is still questioning. understanding the sequences of toothache experience in a lifetime is crucial to prevent toothache in an appropriate period of life. the individual life-course approach to diseases is the critical period of development, which presumes that having experience of certain conditions in early life can have long term impacts on later health outcomes12. we may understand that people in certain periods of life experience a higher rate of toothache in their life. however, there is limited study on toothache using a life course approach. most of the studies in dental pain are cross-sectional1,13-15. few studies have focused on changes in toothache experiences at two or more points in time12,16-18. some studies examined the trend of toothache over the different periods of life and found socioeconomic conditions were related to toothache experience. previous studies on toothache using longitudinal data in brazil highlighted the relation of toothache to maternal education inequality remained stable from childhood and adolescence2. the prevalence of toothache among brazilian adolescents increased and was found linked to race and region18. another study in brazil showed that the economically disadvantaged group had a higher prevalence of toothache in primary and mixed dentition17. furthermore, a study in australia found that lower-income family was related to more frequent toothache across the 14-years follow up12. australian adults in the lowest socioeconomic group were found to have an increased prevalence of toothache from 1994-201316. considering the rare availability of recent studies on dental pain using a life course approach. this study aimed to address these problems by analyzing a 21 years longitudinal study in indonesia. the main advantage of this longitudinal study was the ability to estimate the trend in toothache over 21 years. within this study period, children had primary dentition to permanent dentitions and became more independent and mature in adulthood. to our knowledge, there are no studies on toothache in which the same individuals have been followed up over a period as long as 21 years. most studies examined dental pain using longitudinal data limited from childhood to adolescence4-6,18. in particular, this study bridging the understanding of toothache 3 misrohmasari et al. braz j oral sci. 2022;21:e226288 experience in childhood, adolescence, and adulthood periods. therefore, the aims were to describe the prevalance of toothache experience in a cohort of children aged 2-5 over 21 years to find the most critical period when toothache became the most serious problem and find the relationship between previous experience and later reports of toothache. materials and methods study design this study is a secondary data analysis from the indonesia family life survey (ifls). the ifls is an ongoing longitudinal household and community survey conducted by rand corporation in collaboration with many indonesian institutions. the survey is a broad range survey that includes topics on health. the first survey started in 1993, with four subsequent rounds of data collection in 1997, 2000, 2007, and 2014. the survey dataset was obtained from the rand indonesia family life survey website19. setting and participants the first ifls in 1993 covered a sample of around 7,200 households across 13 provinces in indonesia20. this ifls-1 encompassed approximately 83% of the indonesian population. this survey collected data on individual household and community levels using multistage stratified sampling. this survey’s full details, including sampling methods and related protocols, can be found in the overview and field report20,21. the baseline data of this present study is the data of ifls-1 in 1993 for 2-5 years old children as they were at the early stage of primary teeth. the flowchart for the sample collection carried out in this study can be seen in figure 1. a total of 1,927 children from ifls-1 with complete data on toothache experience and sociodemographic characteristics were included as baseline participants. this study excluded samples of missing data in all variable interests from statistical analysis. they were followed up four times within 21 years. the ifls interviewer revisited the household in every follow-up period and conducted direct interviews with respondents from previous surveys. target respondents who moved from the original residence will be tracked if only they moved in any one of the 13 ifls provinces. the participants were at the age of 6-9 years when ifls-2 was held in 1997 and 9-12 years old when ifls-3 was held in 2000. the ifls-4 was conducted in 2007 as the participants reached adolescence at 16-19 years old. the recent survey in 2014 was ifls-5 when the participants were in early adulthood (age 23-26 years). in 2014, 1,098 individuals (57% of baseline participants) could be traced and completed questionnaires on toothache and demographic characteristics in every follow up period. 4 misrohmasari et al. braz j oral sci. 2022;21:e226288 toothache toothache as a variable interest in this study was assessed based on a question of toothache experience in the last four weeks with yes-no answers. the interviews were conducted in indonesian and the local language. at the 1st, 2nd, and 3rd follow-up, interviewers collected the data of toothache from parents or guardians, by the question “has (child’s name) ever suffered from a toothache during the past 4 weeks?”. the toothache question can be found in the ifls’s book 5, section maa (acute morbidity), which asked questions on the health status and symptoms of children under 15 years old. at the 4th and 5th follow-up, toothache data was collected from a direct interview of the respondents by the question, “did you ever experience a toothache in the last 4 weeks?”. for adults 15 years or older, the toothache question was presented in ifls’s book 3b, section ma (acute morbidity) that asked questions about adult health symptoms. sociodemographic characteristics in this study, sociodemographic characteristics of respondents were obtained from ifls-1 data, which were included child sex, maternal education, number of persons in the house, and area of living. sex was categorized into male and female. the maternal ifls-1 in 1993 age 2-5 y (n = 1927) ifls-2 in 1997 age 6-9 y (n = 1,786) ifls-3 in 2000 age 9-12 y (n = 1,740) ifls 5 in 2014 age 23-26 y (n = 1,098) ifls-4 in 2007 age 16-19 y (n = 1,434) dropouts n = 141 (7.3%) dropouts n = 46 (2.6%) dropouts n = 306 (17.6%) dropouts n = 336 (23.4%) figure 1. ifls flowchart in this study 5 misrohmasari et al. braz j oral sci. 2022;21:e226288 education variable was simplified into three categories based on the highest education level attended by the participants’ mother from baseline data: primary or less, high school, and higher education level. in this study, the number of persons living in the house was categorized into three levels: 3 people or less, 4-5 people, and more than 5 people. the area of living categorization was urban and rural that were based upon 1993 susenas (national economic and social survey) listings obtained from regional bps (statistic indonesia) office20. ifls 1-5 conducted training for field coordinators, supervisors, interviewers, and editors to establish inter-rater reliability21. the training was to ensure that people who gathered and entered the data in the field understood the same method for the details and technical knowledge of conducting the survey. this present study examined carefully how the data was collected and coded to minimize potential bias. ethics this study is a secondary data analysis of a publicly available dataset. procedures of the surveys were reviewed and approved by the institutional review boards (irbs) in the united states (at rand) and in indonesia at the university of gadjah mada (ugm). all respondents were given written informed consent before data collection. data analysis descriptive statistics of sociodemographic characteristics were retrieved from the participants at age 2-5 (ifls-1). toothache experiences were reported for frequencies in every cohort and accumulative experience over 21 years. binary logistic regression tests were performed to analyze the association of previous toothache experiences and later toothache experiences. the level of significance was at p<0.05. the analysis was performed using spss 25. results there were 1,927 children age 2-5 at baseline in 1993. in the follow-up periods, the number of participants decreased because they either died or moved to other provinces outside the survey areas. the participants were 1,786 in 1997, 1,740 in 2000, 1,434 in 2007, and 1,098 participants in 201completing all follow-ups. for every cohort, sociodemographic characteristics of participants were similar, except for sex. in 1993, the number of male participants was higher, but in 2014 more females (52.1%) were reinterviewed. more than 65% of the participants in this study had low education mothers. in this study, more than 50% of participants lived in rural areas with a family of 4-5 people. detailed information on the sociodemographic characteristics of respondents can be found in table 1. the toothache experience varied over time, which was reported among 155 children 2-5 years at baseline, 251 children in 1997, 208 children in 2000, 138 children in 2007, and 131 children in 2014 (table 1). 6 misrohmasari et al. braz j oral sci. 2022;21:e226288 table 1. distribution of sociodemographic characteristics and toothache experience among participants in every cohort (ifls1-5) variables ifls-1 in 1993 (age 2-5y) (n=1,927) ifls-2 in 1997 (age 6-9y) (n=1,786) ifls-3 in 2000 (age 9-12y) (n=1,740) ifls-4 in 2007 (age 16-19y (n=1,434) ifls-5 in 2014 (age 23-26y) (n=1,098) sex male 1,017 (52.8%) 935 (52.4%) 908 (52.2%) 736 (51.3%) 526 (47.9%) female 910 (47.2%) 851 (47.6%) 832 (47.8%) 698 (48.7%) 572 (52.1%) maternal education ≤ primary 1,270 (65.9%) 1199 (67.1%) 1,174 (67.5%) 949 (66.2%) 745 (67.9%) high schools 547 (28.4%) 487 (27.3%) 469 (27.0%) 405 (28.2%) 288 (26.2%) higher edu 110 ( 5.7%) 100 ( 5.6%) 97 (5.6%) 80 (5.6%) 65 (5.9%) persons per household ≤ 3 324 (16.8%) 300 (16.8%) 288 (16.6%) 249 (17.4%) 197 (17.9%) 4-5 993 (51.5%) 993 (51.8%) 908 (52.2%) 744 (51.9%) 567 (51.6%) ≥ 6 610 (31.7%) 560 (31.4%) 544 (31.3%) 441 (30.8%) 334 (30.4%) area urban 854 (44.3%) 750 (42.0%) 725 (41.7%) 608 (42.4%) 440 (40.1%) rural 1,073 (55.7%) 1,036 (58.0%) 1,015 (58.3%) 826 (57.6%) 658 (59.9%) toothache yes 155 (8.0%) 251(14.1%) 208 (12.0%) 136(9.5%) 131(11.9%) no 1,772 (92.0%) 1,535(85.9%) 1,532 (88.0%) 1,298(90.5%) 967(88.1%) figure 2 illustrates the trend of toothache prevalence in a lifetime. in an early dentition period at age 2-5 years, toothache was reported by 8% of the population. when they reached the age of 6-9 years, 14% of children had teeth-related pain, which is the highest percentage over the surveys. at the age of 9-12 years, the proportion of participants who reported toothache decreased to 12%. over the 21 years survey period, the prevalence of self-reported toothache was at the lowest level when they figure 2. toothache experiences in every period of life of the cohort participants 8% 14.10% 12% 9.50% 11.90% age 2-5y age 6-9y age 9-12y age 16-19y age 23-26y prevalence of toothache experince 7 misrohmasari et al. braz j oral sci. 2022;21:e226288 reached the age of 16-19 years (9.5%). however, at the period of early adulthood at 23-26 years old, the percentage of participants reporting toothache was higher than the previous period at 11.9%. the 21 years accumulative reported toothache experience was presented in table 2. almost 40% of the respondents reported toothache at least once in their life. table 2. accumulative frequencies of toothache experiences over 21 years (n=1,098) n (%) none 662 (60.3%) once 308 (28.1%) twice 95 (8.7%) 3 times 27 (2.5%) 4 times 6 (0.5%) the result from binary logistic regression tests, as shown in table 3, found that the age of 6-9 years consistently had a significant association with toothache experience in every model. participants who experienced a toothache at age 6-9 were more likely to experience a toothache in every period of age later in life. experience toothache at age 2-5 was more likely to experience a toothache at age 6-9 (p=0.036; or=1.60). toothache experience at age 9-12 was significantly related only to toothache experience at age 6-9 (p <0.000; or=2.20). at age 16-19, toothache experince was related to toothache experience at age 6-9 (p<0.000; or=2.53) and age 9-12 (p=0.001; or=2.17). the experience of toothache at early adulthood (age 23-26 years) was significantly associated with toothache experience at the age of 6-9 years (p=0.042; or=1.58) and 16-19 years (p<0.000; or=2.86). table 3. regression model of the association between previous toothache experience and later toothache experience toothache model 1 age 6-9y (n=1786) model 2 age 9-12y (n=1740) model 3 age 16-19y (n=1434) model 4 age 23-26y (n=1098) sig or 95% ci sig or 95% ci sig or 95% ci sig or 95%ci age 2-5y 0.036 1.60 1.03-2.47 0.387 1.32 0.70-2.48 0.431 1.28 0.70-2.34 0.885 0.95 0.47-1.91 age 6-9y <0.000 2.20 1.42-3.41 <0.000 2.53 1.68-3.81 0.042 1.58 1.02-2.45 age 9-12y 0.001 2.17 1.39-3.40 0.924 0.97 0.56-1.70 age 16-19y <0.000 2.86 1.72-4.76 p < 0.05; binary logistic regression or=odds ratio; ci=confidence interval discussion this longitudinal study followed a group of children age 2-5 years in ifls 1993 over 21 years. the aims were to find the prevalence of toothache in every age period and seek the association between previous toothache experience and later life. the 8 misrohmasari et al. braz j oral sci. 2022;21:e226288 toothache experience was based on the self-reported toothache during the last four weeks. this study found that the highest percentage of toothache was reported at age 6-9 years. the toothache experience in this period was also related to the toothache experience in subsequent periods of life. the prevalence of toothache at the age of 2-5 years is 8%, the lowest compared to the later periods of life. this result is lower than the prevalence of toothache in preschool children in brazil at age 3-4 years with 11.8%22 and of preschool children age 2-4 years in australia (15%)23. all primary teeth erupt into the mouth by two years of age. children learn to brush their teeth under supervision in this period of age24. most of the toothache in children found related to caries6. caries development into a symptom needs a period of time5. therefore, most children reported no toothache due to the early tooth wear in this age group. it is essential that children’s oral health was assessed from a very young age, and oral health behavior was formulated to prevent negative impacts on later life. when children reached the age of 6-9 years in 1997, the toothache was reported by 14.1% of the participants in this study. this prevalence is the highest over 21 years of study. the toothache experience in this age period is significantly associated with every period of age in later life, from adolescence to early adulthood. age of 6–7 years, children go through a critical transition phase of both psychological and biological development25. general anxiety, physical injury, and fears such as separation from parents are present during this period of life25. this age period is also an early transitional period of the mixed dentition, with exfoliation of primary teeth and eruption of permanent teeth. tooth mobility in this transition phase can be a factor in reporting toothache25. this age is also the period when children become more independent and develop more mature motoric skills5. the prevalence of toothache in 6-9 years children in this study is similar to a study in the united states (14%)26 and slightly lower than toothache experience of children age seven years in sweden with about 15%27. this prevalence is lower than 49.9% reported from 6-12 years old school children in mexico14. however, a study in canada reported a lower percentage of toothache at around 5% of children28. the difference in toothache prevalence can be related to many factors, including the development context of different locations and countries, variations of the disease level between populations, health system management and actions on population oral health problems, and also methodologies applied in every study. this study found that in the late phase of mixed dentition, children age 9-12, reported less frequent toothache experience than the previous age period (12%). this result was lower than a study in south brazil for children 8-12 years that reported toothache experience for 17.3% of the population29 and a study for children age 9-12 years in australia (27.4%)12. this number is also lower than a study in taiwan where more than 30% of children experience dental pain during the past year15. in this period of life, children begin to have the ability to judge their behavior and emotion, including the awareness of their physical appearance and other’s thought30. in the adolescent period (age 16-19 years), 9.5% of the participants in this study reported toothache experience in the last four weeks. this result is slightly lower 9 misrohmasari et al. braz j oral sci. 2022;21:e226288 than around 10% of canadian adolescents who reported toothache28. a study of adolescence age 15-19 in brazil found that 28.3% of participants experience toothache during the last six months31. adolescence is an intermediate period between childhood and adulthood, characterized by intense psychological and physical changes32. in this transitional phase, individuals may involve in particular behaviors that can compromise their health status4. the experience of toothache in this period is significantly related to the toothache experience in childhood. self-reported toothache at the age of 23-26 years was 11.9%. this number is lower than a study in brazil among 20-29 years adult (17.2%)33 and a young adult population in canada 14%28. the experience of toothache in this period was significantly associated with toothache experience at early mixed dentition and adolescence. a similar result was also found in which followed individuals from late childhood into early adulthood. the dmft and cpi values at age 18 years were associated with dmft and cpitn scores at younger ages34. over 21 years of study, almost 40% of the respondents had ever experienced a toothache. this study reported that having toothache experience in early life was an important determinant for future toothache experience in this life course study5. the high prevalence of toothache in the early mixed dentition period and its significant association with future toothache experiences suggest the need to create comprehensive dental health prevention programs targeting this population to reduce the impact of pain on their quality of life. the strengths of this study were the participants’ follow-up over 21 years and the high number of participants involved. this study could be a reason to describe the trend in toothache experience over time. a longitudinal study design had a reliable estimate of the change in toothache experience over the 21 years of the study. the life course models are useful to understand the long-term effects of exposures during childhood, adolescence, young adulthood, and later adult life on chronic disease35. some limitations are found in this study. first is the loss of follow-up at the survey. a considerable number of participants in this study cohort were lost at the ifls-4 and ifls-5 when they reached adolescence and adulthood. second, due to secondary data analysis of this study, data available limited in terms of information related to oral health, limiting the authors to draw a more comprehensive analysis. future studies may consider more comprehensive variables, including socioeconomic factors and other oral health behaviors, such as dietary habits, tooth brushing frequency, to explore the mechanisms of the statistical association found in this study. data availability datasets related to this article can be found at  https://www.rand.org/well-being/ social-and-behavioral-policy/data/fls/ifls/access.html. authors’ contribution elyda misrohmasari: design, interpretation, drafting, revising dimas wicaksono: acquisition, analisis, revising 10 misrohmasari et al. braz j oral sci. 2022;21:e226288 hestieyonini hadnyanawati: design, interpretation, revising berlian prihatiningrum: interpretation, revising all of the authors listed have been read and approved to submit the manuscript to this journal. all authors agree to be accountable for all aspects of the work. references 1. muirhead ve, quayyum z, markey d, weston-price s, kimber a, rouse w, et al. children’s toothache is becoming everybody’s business: where do parents go when their children have oral pain in london, england? a cross-sectional analysis. bmj open. 2018 feb 28;8(2):e020771. doi: 10.1136/bmjopen-2017-020771. 2. freire mcm, jordão lmr, peres ma, abreu mhng. six-year trends in dental pain and maternal education 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dokki, giza, p.o. 12622, egypt. phone: +2 0111 282 6250 fax: +2 02 33370931 email: omniaelhiny@yahoo.com editor: altair a. del bel cury received: november 02, 2020 accepted: august 20, 2021 evaluation of an open access generic 3d software for orthodontic diagnosis and treatment planning hany salah eldin eid1, omnia a. elhiny2* aim: to evaluate the accuracy and the validity of orthodontic diagnostic measurements, as well as virtual tooth transformations using a generic open access 3d software compared to orthoanalyzer (3shape) software; which was previously tested and proven for accuracy. methods: 40 maxillary and mandibular single arch study models were duplicated and scanned using 3shape laser scanner. the files were imported into the generic and orthoanalyzer software programs; where linear measurements were taken twice to investigate the accuracy of the program. to test the accuracy of the program format, they were printed, rescanned and imported into orthanalyzer. finally, to investigate the accuracy of editing capabilities, linear and angular transformation procedures were performed, superimposed and printed to be rescanned and imported to orthoanalyzer for comparison. results: there was no statistically significant difference between the two groups using the two software programs regarding the accuracy of the linear measurements (p>0.05). there was no statistically significant difference between the different formats among all the measurements, (p>0.05). the editing capabilities also showed no statistically significant difference (p>0.05). conclusion: the generic 3d software (meshmixer) was valid and accurate in cast measurements and linear and angular editing procedures. it can be used for orthodontic diagnosis and treatment planning without added costs. keywords: software. imaging, three-dimensional. dental models. casts, surgical. https://orcid.org/0000-0002-8435-1021 2 eid e elhiny introduction orthodontists are currently facing the challenge of continuous technological advancements in computer sciences. digitization in orthodontics has transformed from digital records and images into digital study casts, digital analysis, digital virtual tooth movements and fabrication of aligners and appliances1,2. in addition, the availability of 3d orthodontic software programs has facilitated the study of maxillary, mandibular, intra-arch and inter-arch relations, as well as performing virtual setups and treatment plans1,3,4. study models are crucial for orthodontic diagnosis and treatment planning. the recent use of digital casts has allowed for a wider scale of communication between orthodontists and easier marketing to attract patients without the need for a physical storage space5. in early 1999, the first digital system was introduced and nowadays many systems are commercially available6. this presence of different technologies calls for a proper standardization and the investigation of the accuracy of digital measurements compared to those on plaster study models. many systems were evaluated; such as digimodel,4 o3dm7, orthocad1 and 3shape orthoanalyzer2,5,6, and were found to be as reliable and accurate as plaster models; which means that digital models can be considered as the new gold standard5,8-10. there are increasing office expenses and training that have to be met in order to gain maximum benefits using such a fast-developing technology. hence, the presence of a free software; that is easy to operate and accessible for download on the web, appears to be an appealing alternative to many. the question lies here in whether this software program is valid and accurate or not. the aim of this study was to evaluate the accuracy and the validity of orthodontic diagnostic measurements, as well as virtual tooth transformations using a generic open access 3d software compared to orthoanalyzer (3shape) software; which was previously tested and proven for accuracy. material and methods forty single arch sets of maxillary and mandibular study models were included in the study. the sample size calculation showed that this number provided a statistical power of 80%, α = 0.05 and β = 0.20. the study models were duplicated from those of patients attending a private orthodontic practice. the selection criteria were intact full set of permanent dentition from permanent first molar to permanent first molar upper and lower. no previous orthodontic treatment, no gingival inflammation or recession, no dental attrition or erosion, and no crowding or mild to moderate crowding (not more than 4 mm) and no voids or blebs in the stone or digital models. the models were duplicated using jeltrate fast-set alginate (sirona dentsply) in stock impression trays and poured into type iv dental stone (gc fujirock ep polar white, 3 eid e elhiny gc europe n.v, belgium). digital impressions were recorded using laser scanner (3shape d500, 3shape a/s, copenhagen, denmark). the specifications and accuracy of the laser scanner have been validated in previous studies11. after scanning all the sample by the laser scanner, default stl files (standard triangle language) were created and imported one by one on microsoft windows 7 desktop computer to both software programs; generic open access software (autodesk meshmixer 3.5 for windows, usa) and orthoanalyzer (version 1.5; 3shape, copenhagen, denmark). once imported, the formats of scanned models were changed automatically to its corresponding format specific to the software. the investigation was then conducted in three steps. firstly, linear measurements were made twice on the scanned images to investigate the accuracy of the generic software. the first measurements were done using the meshmixer; those measurements were given the symbol gm; figure 1a. the second measurements were done using orthoanalyzer, and those measurements were given the symbol sm1; figure 1b. the orthoanalyzer measurements were considered as control since their accuracy and validity were established in previous studies1,2,6,7,12,13. all the measurements were performed by the same investigator. the linear measurements taken were: a = intercanine width: the distance between the cusp tips. b = intermolar width: the distance between the mesiopalatal cusp tips of maxillary first permanent molars. the distance between the mesiobuccal cusp tips of mandibular first permanent molars. c = arch depth: the perpendicular distance from the incisal edge at the midline to the mesiobuccal cusp tip of mandibular molars and the mesiopalatal cusp tip for maxillary molars. figure 1. a meshmixer measurements gm. b orthoanalyzer measurements sm1. a b 4 eid e elhiny secondly, the accuracy of the generic software format was tested where the scanned models with the meshmixer format (generic software format) were exported to a digital printer (formlabs,  somerville, ma, usa). they were retransformed into physical models which were rescanned and a second stl file was created, imported and reanalyzed using the orthoanalyzer (sm2). after that, sm1 and sm2 measurements were analyzed to compare the stability of the initial model measurements (sm1) to those taken after the models’ format was changed from stl to meshmixer format and then again to the second stl and orthoanalyzer format (sm2). thirdly, the accuracy of the editing capabilities of the generic software was tested using copies of all the original models. the copies were saved on both software programs and were subjected to two editing transformation procedures on two anterior teeth; one movement per tooth. using the generic software, the first movement was an unspecified labial tipping movement not exceeding the labiolingual thickness of that particular tooth. the second movement was a 5-degree labial rotation around the mesial axis of the tooth. the edited model copies were then superimposed over the original models of the same case. the amount of labial linear transformation for the first tooth (gt1) and the linear transformation of the distal contact point of the second tooth, as a result of tooth rotation around its mesial axis (gt2), were measured; figure 2a. both movements were exported and printed in the superimposed form. this printed form was rescanned and imported to the orthoanalyzer software figure 2. a meshmixer measurement of the amount of labial linear transformation of the first tooth and linear transformation of distal contact point of the second tooth; gt1 and gt2. b the same measurements as in a, but using orthoanalyzer; st1 and st2. a b 5 eid e elhiny where the same measurements were performed (st1 & st2); figure 2b. measurements from both software programs were then compared. the mean and standard deviation values were calculated for each group in each test. data were explored for normality using kolmogorov-smirnov and shapiro-wilk tests and showed parametric (normal) distribution. paired sample t-test was used to compare between two groups in related samples. the significance level was set at p ≤ 0.05. statistical analysis was performed with ibm® spss® statistics version 20 for windows. results i. measuring the accuracy of generic software to perform measurements on scanned models gm (gm vs sm1): the accuracy of generic software to perform measurements is represented in figure 3. the means and standard deviations of gm vs sm1 are shown in table 1. 1. inter-molar width: there was no statistically significant difference between (generic gm) and (specialized sm1) groups where (p=0.635). figure 3. bar chart representing gm vs sm1. 0 5 10 15 20 25 30 35 40 45 50 inter-molar width inter-canine width arch depth gm vs sm1 generic gm specialized sm1 table 1. mean and standard deviation (sd) values of gm vs sm1 variables gm vs sm1 generic gm specialized sm1 p-value mean sd mean sd inter-molar width 38.60 4.31 38.74 4.47 0.635ns inter-canine width 31.82 4.20 31.96 4.32 0.263ns arch depth 32.16 6.97 31.52 6.44 0.137ns ns; non-significant (p>0.05) 6 eid e elhiny the highest mean value was found in (specialized sm1) group, while the lowest mean value was found in (generic gm) group. 2. inter-canine width: there was no statistically significant difference between (generic gm) and (specialized sm1) groups where (p=0.263). the highest mean value was found in (specialized sm1) group, while the lowest mean value was found in (generic gm) group. 3. arch depth: there was no statistically significant difference between (generic gm) and (specialized sm1) groups where (p=0.137). the highest mean value was found in (specialized sm1) group, while the lowest mean value was found in (generic gm) group. i. measuring the accuracy of original cast analysis measurements after changing the scanned models’ format to generic software format sm (sm1 vs sm2): the accuracy of the original cast analysis measurements is represented in figure 4. the means and standard deviations of sm1 vs sm2 are shown in table 2. figure 4. bar chart representing sm1 vs sm2. 0 5 10 15 20 25 30 35 40 45 50 inter-molar width inter-canine width arch depth sm1 vs sm2 specialized sm1 specialized sm2 table 2. mean and standard deviation (sd) values of sm1 vs sm2 variables sm1 vs sm2 specialized sm1 specialized sm2 p-value mean sd mean sd inter-molar width 39.02 4.43 38.74 4.47 0.066ns inter-canine width 31.46 4.38 31.96 4.32 0.403ns arch depth 30.22 4.02 30.26 3.93 0.477ns ns; non-significant (p>0.05) 7 eid e elhiny 1. inter-molar width: there was no statistically significant difference between (specialized sm1) and (specialized sm2) groups where (p=0.066). the highest mean value was found in (specialized sm1) group, while the lowest mean value was found in (specialized sm2) group. 2. inter-canine width: there was no statistically significant difference between (specialized sm1) and (specialized sm2) groups where (p=0.403). the highest mean value was found in (specialized sm2) group, while the lowest mean value was found in (specialized sm1) group. 3. arch depth: there was no statistically significant difference between (specialized sm1) and (specialized sm2) groups where (p=0.477). the highest mean value was found in (specialized sm2) group, while the lowest mean value was found in (specialized sm1) group. i. measuring the accuracy of generic software to perform editing transformation on scanned casts t (gt 1&2 vs st 1&2): the accuracy of generic software to perform editing transformation is represented in figure 5. the means and standard deviations of gt 1&2 vs st1&2 are shown in table 3. table 3. mean and standard deviation (sd) values of gt 1&2 vs st 1&2 variables gt1 st1 gt2 st2 mean sd mean sd mean sd mean sd gt 1&2 vs st 1&2 1.56 0.31 1.32 0.20 1.26 0.23 1.08 0.08 p-value 0.118ns 0.137ns ns; non-significant (p>0.05) figure 5. bar chart representing gt 1&2 vs st 1&2. 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 gt1 and st1 gt2 and st2 gt and st gt st 8 eid e elhiny 1. there was no statistically significant difference between (gt1) and (st1) groups where (p=0.118). the highest mean value was found in (gt1) group, while the lowest mean value was found in (st1) group. 2. there was no statistically significant difference between (gt2) and (st2) groups where (p=0.137). the highest mean value was found in (gt2) group, while the lowest mean value was found in (st2) group. discussion it is an undeniable fact that we currently live in an electronic era where the utilization of virtual models has become increasingly important in the orthodontic practice2,14-17. consequently, the availability of a well-established software has become crucial. this has been manifested by the abundance of a generous market; providing a rather noticeable number of well-established software programs, investigated for validity and accuracy, and capable of executing multifunctional tasks. however, most of those software programs are considered rather expensive, considerably complicated and require extensive training to master their potentials in full4,18-20, which restrains their use to a limited fraction of the realm of orthodontic practices. on the other hand, there is another domain of open access software programs available; not necessarily originally designed for dental work, and haven’t been investigated for their potentials. the availability of such software programs provides a golden opportunity for enhancing the performance of the orthodontic clinic, have they been proven to be as effective as the market provided programs without the burden of the extensive finances. therefore, the present study compared the accuracy of a generic open access 3d software (meshmixer) to that of orthoanalizer (version 1.5; 3shape, copenhagen, denmark). the investigation was conducted in three steps, where linear cast measurements’ accuracy was compared between the two software programs; as the first step. there was no significant difference between the meshmixer (gm) and 3shape (sm1) measurements regarding the inter-molar and inter-canine widths as well as the arch depth which were needed on the diagnostic level of comparison. the dental cast measurements taken by the 3shape orthoanalizer software are known from previous studies to be as accurate and valid as the measurements taken on plaster or stone dental casts1,2,6,11-13, which concludes that the meshmixer is an accurate and valid software as a diagnostic measurement tool. the investigated meshmixer software was not designed specifically for orthodontic use and it is well known that the accuracy of orthodontic measurements is crucial to the diagnosis and treatment planning. this necessitated the investigation of the accuracy of the meshmixer file format to determine the stability and reproducibility of its measurements12. when the dental casts are scanned, they are normally stored as stl files and then imported to the analyzing software where they are converted into its suitable 9 eid e elhiny format10,11,21. the previously measured casts in the meshmixer format (sm1) were exported and printed, then rescanned and imported as stl files into the 3shape orthoanalizer software. those files were then converted into the 3shape format and the casts were then reanalyzed (sm2). when sm1 was compared to sm2, there was no significant difference which indicated that there was an accurate smooth conversion from the stl format to the meshmixer format and comparable to the conversion provided by 3shape orthoanalizer. there is a worldwide increased demand for simulated treatment plans and customized orthodontic appliances for the sake of providing a more efficient and calculated treatment4. therefore, the virtual software used must be able to face this challenge by providing accurate angular and linear editing capabilities. this transformation potential is considered the corner stone in moving forward towards digital treatments. in this study, there was no statistically significant difference between the meshmixer and the 3shape in either of the linear or angular editing movements performed; which indicated that the meshmixer was valid and accurate in performing the different editing movements. most of the previous studies addressed the accuracy of the virtual measurements and model analysis, using software programs available in the market, and showed their comparability to the measurements on plaster models. nonetheless, they seldom explored the software potential to accurately select and transform, digitally, a unit or multiple units within the dental model for correcting teeth positions. further, they didn’t investigate the accuracy of the file format for assessing the stability and reproducibility of the measurements of the used programs. this could be clearly seen in the studies conducted by bukhari et al.6 where they investigated the accuracy of measurements of 3shape ortho analyzer and camardella et al.4 who compared the measurements of 3 shape and digimodel orthoproof. sousa et al.1 also examined the accuracy of geometric studio 5 software and westerlund et al.5 compared 4 market software programs; cadent, ortholab, orthoproof and 3 shape. this drops the light on the importance of this investigation and its value in the future of digitization in orthodontics. the constant advances in the digital world have reduced the obstacles in diagnosis and treatment planning. the ability to plan treatment, design appliances and make treatment simulations and store every step for reference has escalated the need for such digital technology in every orthodontic practice. the main disadvantage of such technology discussed in the literature was the high costs and extensive training that come with it4,18-20. the meshmixer software has proven to be a valid, accurate and reproducible measurement and editing tool; with the advantage of being an open access software on the internet that is easy to comprehend; such a finding will allow the delivery of a more efficient and predictable treatment for a wider base of patients. on the other hand, this study was burdened by the limitation of the need to increase the vectors of the mechanical virtual movements performed on the units transformed; which should serve as an eye opener to software providers for making future improvements in their programs to address a wider base of users. 10 eid e elhiny digital transformation has become the feature of today and not the future and the existence of such digital potentials, via open access programs, in one’s hand represents a leap forward towards a new era of digital orthodontics. it also puts an additional responsibility on every operator to learn such new techniques as quickly as possible since their learning and tutoring has become, now, a must and not an additional luxury. in conclusion, the generic software (meshmixer) was accurate and valid in cast measurements, as well as linear and angular editing procedures. it provided stable and reproducible measurements. the generic software (meshmixer) can be used in orthodontic diagnosis and treatment planning without any added costs for the orthodontist. references 1. sousa mvs, vasconcelos ec, janson g, garib d, pinzan a. accuracy and reproducibility of 3-dimensional digital model measurements. am j orthod dentofacial orthop. 2012 aug;142(2):269-73. doi: 10.1016/j.ajodo.2011.12.028. 2. moreira dd, gribel bf, torres gdr, vasconcelos kf, de freitas dq, ambrosano gmb. reliability of measurements on virtual models obtained from scanning of impressions and conventional plaster models. braz j oral sci. 2014 oct;13(4):297-302. doi: 10.1590/1677-3225v13n4a11. 3. hajeer my, millett dt, ayoub af, siebert jp. applications of 3d imaging in orthodontics: part i. j orthod. 2004 mar;31(1):62-70. doi: 10.1179/146531204225011346. 4. camardella lt, rothier ekc, vilella ov, ongkosuwito em, breuning kh. virtual setup: application in orthodontic practice. j orofac orthop. 2016 nov;77(6):409-19. 5. westerlund a, tancredi w, ransjo m, bresin a, psonis s, torgersson o: digital casts in orthodontics. a comparison of 4 software systems. am j orthod dentofacial orthop 2015;147:509-16. doi: 10.1007/s00056-016-0048-y. 6. bukhari saa, reddy ka, reddy mr, shah sh. evaluation of virtual models (3shape ortho system) in assessing accuracy and duration of model analyses based on the severity of crowding. saudi j dent res 2017;8(1-2):11-8. doi: 10.1016/j.sjdr.2016.05.004. 7. mac kriel ea. accuracy of orthodontic digital study models [thesis]. faculty of dentistry, university of the western cape; 2012. 8. fleming ps, marinho v, johal a. orthodontic measurements on digital study models compared with plaster models: a systematic review. orthod craniofac res. 2011 feb;14(1):1-16. doi: 10.1111/j.1601-6343.2010.01503.x. 9. luu ns, nikolcheva lg, retrouvey jm, flores-mir c, el-bialy t, carey jp, et al. linear measurements using virtual study models. angle orthod. 2012 nov;82(6):1098-106. doi: 10.2319/110311-681.1. 10. rossini g, parrini s, castroflorio t, deregibus a, debernardi cl. diagnostic accuracy and measurement sensitivity of digital models for orthodontic purposes: a systematic review. am j orthod dentofacial orthop. 2016 feb;149(2):161-70. doi: 10.1016/j.ajodo.2015.06.029. 11. michelinakis g, apostolakis d, tsagarakis a, kourakis g, pavlakis e. a comparison of accuracy of 3 intraoral scanners: a single-blinded in vitro study. j prosthet dent. 2020 nov;124(5):581-8. doi: 10.1016/j.prosdent.2019.10.023. 12. camardella lt, breuning h, vilella ov. are there differences between comparison methods used to evaluate the accuracy and reliability of digital models? dental press j orthod. 2017 feb;22(1):65-74. doi: 10.1590/2177-6709.22.1.065-074.oar. 11 eid e elhiny 13. camardella lt, ongkosuwito em, penning ew, kuijpersjagtman am, vilella ov, breuning kh. accuracy and reliability of measurements performed using two different software programs on digital models generated using laser and computed tomography plaster model scanners. korean j orthod. 2020 jan;50(1):13-25. doi: 10.4041/kjod.2020.50.1.13. 14. whetten jl, williamson pc, heo g, varnhagen c, major pw. variations in orthodontic treatment planning decisions of class ii patients between virtual 3-dimensional models and traditional plaster study models. am j orthod dentofacial orthop. 2006 oct;130(4):485-91. doi: 10.1016/j.ajodo.2005.02.022. 15. okunami tr, kusnoto b, begole e, evans ca, sadowsky c, fadavi s. assessing the american board of orthodontics objective grading system: digital vs plaster dental casts. am j orthod dentofacial orthop. 2007 jan;131(1):51-6. doi: 10.1016/j.ajodo.2005.04.042. 16. akyalcin s, dyer dj, english jd, sar c. comparison of 3-dimensional dental models from different sources: diagnostic accuracy and surface registration analysis. am j orthod dentofacial orthop 2013;144: 831-7. 17. wiranto mg, engelbrecht wp, nolthenius het, meer wj, ren y. validity, reliability, and reproducibility of linear measurements on digital models obtained from intraoral and cone-beam computed tomography scans of alginate impressions. am j orthod dentofacial orthop. 2013 dec;144(6):831-7. doi: 10.1016/j.ajodo.2013.08.014. 18. leifert mf, leifert mm, efstratiadis ss, cangialosi tj. comparasion of space analysis evaluations with digital models and plaster dental casts. am j orthod dentofacial orthop. 2009 jul;136(1):16. e1-4; discussion 16. doi: 10.1016/j.ajodo.2008.11.019. 19. santoro m, galkin s, teredesai m, nicolay of, cangialosi tj. comparison of measurements made on digital and plaster models. am j orthod dentofacial orthop. 2003 jul;124(1):101-5. doi: 10.1016/s0889-5406(03)00152-5. 20. brusco n, andreetto m, lucchese l, carmignato s, cortelazzo gm. metrological validation for 3d modeling of dental plaster casts. med eng phys. 2007 nov;29(9):954-66. doi: 10.1016/j.medengphy.2006.10.011. 21. chiu a, chen yw, hayashi j, sadr a. accuracy of cad/cam digital impressions with different intraoral scanner parameters. sensors (basel). 2020 feb 20;20(4):1157. doi: 10.3390/s20041157. 1 volume 22 2023 e239056 critical review braz j oral sci. 2023;22:e239056http://dx.doi.org/10.20396/bjos.v22i00.8669056 1 federal university of santa maria (ufsm), post-graduate program in oral sciences, santa maria, rs, brazil. 2 hodos-rs dentistry, graduate school, porto alegre, rs, brazil. corresponding author: dds, mscid. doctorate degree student helder callegaro velho. federal university of santa maria, mscid-phd post-graduate program in oral science, prosthodontics unit, 1000 roraima av, t street, building 26f, ufsm campus, 97105-900, brazil. e-mail: heldercvelho@hotmail.com editor: altair a. del bel cury received: apr 19, 2022 accepted: jul 11, 2022 is the adhesive or mechanical behavior of glass ceramics influenced by the adhesive layer application after etching and silanization? a literature review helder callegaro velho1* , pablo soares machado1 , lucas saldanha da rosa1 , catina prochnow2 , jatyr pisani-proença2 aim: this review investigated the effect of applying an adhesive after surface treatment of glass-ceramics on the bonding, mechanical or clinical behavior. methods: studies comparing the adhesive, mechanical or clinical behavior of glass-ceramics, with or without adhesive application after surface treatment, were included. searches were performed in pubmed, scopus, and web of sciences databases (january 2022), resulting in 15 included studies. results: regarding the evaluated outcomes, 13 studies assessed bond strength, 2 studies assessed biaxial flexural strength and 1 study assessed fatigue failure load, while no study evaluating clinical outcomes was included. it was possible to observe that the adhesive application after ceramic surface treatment was unfavorable or did not influence the evaluated outcomes. conclusion: most of the evidence available in the literature shows that the adhesive application after surface treatment does not improve the adhesive and mechanical behavior of glass-ceramics. keywords: dental adhesives. ceramics. https://orcid.org/0000-0002-5996-3755 https://orcid.org/0000-0002-7702-0013 https://orcid.org/0000-0002-2159-3787 https://orcid.org/0000-0003-3649-8296 https://orcid.org/0000-0002-6202-728x 2 velho et al. braz j oral sci. 2023;22:e239056 introduction dental ceramics can currently be classified into three categories according to their composition: glass-matrix ceramics (glass-ceramics): non-metallic inorganic ceramic materials containing glass phase; polycrystalline ceramics: non-metallic inorganic ceramic materials without glass phase; and resin-matrix ceramics: polymeric matrix containing inorganic compounds, which may include glass-ceramics1. glass-ceramics have been widely used for indirect restorations since they combine excellent physical and chemical properties and present excellent esthetics2,3. in addition to choosing the glass-ceramic according to the clinical indication, another fundamental factor for the longevity of ceramic restorations is the use of an adequate protocol of adhesive luting4. the conventional protocol for glass-ceramics includes etching the ceramic surface with hydrofluoric acid (hf), which selectively attacks the glassy phase and exposes the silicon dioxide (sio2), causing morphological changes that contribute to micromechanical retention of the resin cement to the material5-7; and the application of the silane coupling agent, which chemically reacts with the exposed silicon dioxide and promotes a chemical bond between the ceramic and the resin cement8,9. for adequate restoration behavior, it is essential that the surface irregularities resulting from the etching of hf are completely filled in by the resin cement, since unfilled spaces at the adhesive interface can negatively influence the performance of ceramic restorations10. in this sense, studies have suggested applying an adhesive layer on the ceramic surface before applying resin cement11-14. this adhesive layer could improve the wettability of the ceramic surface, as its viscosity is lower than that of the resin cement, which would facilitate the filling of irregularities. however, there are still conflicting statements in the literature about the use of an adhesive after ceramic surface treatment, which makes it difficult to define an ideal technique. although nogueira et al15 (2021) showed that the application of an adhesive layer on glass-ceramics after surface treatment does not improve the bond strength values, an updated synthesis of the literature addressing other outcomes becomes relevant. thus, the aim of the present review was to investigate the effect of applying an adhesive after surface treatment of glass-ceramics on the adhesive, mechanical or clinical behavior. materials and methods focused question does the application of an adhesive after surface treatment improve the adhesive, mechanical or clinical behavior of glass-ceramics? picos this literature review adopted the population, intervention, comparison, and outcomes process (i.e. the “picos” process), as follows: 3 velho et al. braz j oral sci. 2023;22:e239056 population: glass-ceramics. intervention: adhesive layer application. comparison: non-adhesive layer application. outcomes: adhesive, mechanical and clinical behavior. study design: in vitro and clinical studies. eligibility criteria inclusion criteria studies in dentistry which considered the adhesive, mechanical or clinical behavior of all glass-ceramics cemented using adhesive strategies were selected (i.e. ceramics used as intra radicular posts, or at implant abutment or pillar contexts were not considered). studies comparing the adhesive, mechanical or clinical behavior of glass-ceramics, with or without adhesive application after surface treatment, regardless of the glass-ceramic used (e.g., feldspathic, leucite, lithium disilicate, lithium silicate, among others), the processing method for ceramic manufacturing (layering, pressing, or cad/cam techniques, among others), bond strength methodology (shear, micro-shear, tensile, micro-tensile, among others), mechanical property measured (strength, hardness, toughness, among others), regardless of the testing method (monotonic, fatigue, among others) and clinical outcome were included. all existing in vitro or clinical studies on such themes were included regarding the adopted study design. exclusion criteria studies which did not adopt ceramic surface pretreatment including hf etching and application of silane coupling agent were excluded. search the pubmed, web of science and scopus databases were consulted, without date restriction (last executed on january 10, 2022). the search strategy (table 1) was based on the mesh terms and the specific free-text terms of pubmed, which were then adapted, if necessary, for the other databases. table 1. search strategy. pubmed (“ceramics” [mesh] or ceramic [tiab] or “dental porcelain” [mesh] or porcelain [tiab] or glass ceramic [tiab] or feldspathic [tiab] or lithium disilicate [tiab] or lithium silicate [tiab] or leucite [tiab]) and (adhesives [mesh] or “tissue adhesives” [mesh] or “dentin-bonding agents” [mesh] or dentin bonding [tiab] or bonding agent [tiab] or dental adhesive system [tiab] or luting strategies [tiab]) and (adhesion [tiab] or bond strength [tiab] or “survival rate”[mesh] or clinical survival [tiab] or clinical performance [tiab] or mechanical behavior [tiab] or mechanical properties [tiab] or “fatigue” [mesh] or fatigue [tiab] or load bearing or fracture strength [tiab] or failure load [tiab] or resistance [tiab] or compression [tiab] or retention [tiab] or tensile [tiab]) continue 4 velho et al. braz j oral sci. 2023;22:e239056 continuation web of science ts=(ceramic or porcelain or glass ceramic or feldspathic or lithium disilicate or lithium silicate or leucite) and ts=(adhesives or dentin bonding or bonding agent or dental adhesive system or luting strategies) and ts=(adhesion or bond strength or survival rate or clinical survival or clinical performance or mechanical behavior or mechanical properties or fatigue or load bearing or fracture strength or failure load or resistance or compression or retention or tensile) and su= (dentistry) not ts=(review) scopus title-abs-key (“ceramic” or “porcelain” or “glass ceramic” or “feldspathic” or “lithium disilicate” or “lithium silicate” or “leucite”) and title-abs-key (“adhesives” or “dentin bonding” or “bonding agent” or “dental adhesive system” or “luting strategies”) and title-abs-key (“adhesion” or “bond strength” or “survival rate” or “clinical survival” or “clinical performance” or “mechanical behavior” or “mechanical properties” or “fatigue” or “load bearing” or “fracture strength” or “failure load” or “resistance” or “compression” or “retention” or “tensile”) and not (“review”) and ( limit-to ( subjarea,”dent” ) ) screening screening was performed using a reference manager (endnote x9, thomson reuters, new york, ny) by two independent researchers (h.c.v. and p.s.m.). first, titles and abstracts were analyzed for relevance and the presence of the eligibility criteria and then classified as included, excluded or uncertain. the full text of the studies included in the first phase was analyzed again in a second moment regarding the eligibility criteria by the same two reviewers mentioned above (acting independently). discrepancies in the review of titles/abstracts and full text were resolved by discussion. data collection the following data were collected in a spreadsheet (microsoft excel, redmond, wa): year of publication, country of origin, type of vitreous ceramic, adhesive system, cementing agent, aging protocol, evaluated outcome / type of test, predominant failure type and main result in relation to the use of adhesive (favorable to the outcome, no difference or unfavorable). data analysis data were summarized in tables and figures in order to describe the main characteristics of the included studies. results a total of 3,133 studies were initially identified. then, a total of 40 studies were considered eligible for full-text evaluation after removing duplicates and evaluating titles and abstracts, of which 15 were included for qualitative analysis (figure 1). 5 velho et al. braz j oral sci. 2023;22:e239056 id en ti fi ca ti on s cr ee ni ng in cl ud ed records identified from: databases (n = 3133) (pubmed – n =534; scopus – n = 549; web of science – n = 2035) records removed before screening: duplicate records removed (n = 1301) records screened (n = 1832) records excluded (n = 1792) reports assessed for eligibility (n = 40) reports excluded: absence of the main comparison (hydrofluoric acid + silane x hydrofluoric acid + silane + adhesive) (n = 25) studies included in review (n = 15) figure 1. study selection diagram. table 2 presents a qualitative synthesis of the articles included in the review. the articles included were published between 2003 and 2021, with most of them published from 2015 onwards and by brazilian authors. a total of 13 commercial adhesive brands were evaluated. all studies that met the criteria were in vitro studies, without clinical studies entering the final review. it was possible to observe that the adhesive application after ceramic surface treatment was unfavorable or at least did not influence the evaluated outcomes regarding the adhesive and mechanical behavior of glass-ceramics, except for particular groups in non-aged regimes12,16,17. table 2. descriptive synthesis of the included studies. author (year) country type of glass-ceramic adhesive system resin cement aging outcome evaluated/ type of test predominant failure type results* el zohairy et al.12 (2003) netherlands feldspathic syntac optibond solo plus scotchbond tetric flow nexus 2 relyx arc 24h bond strength/ microtensile adhesive + for optibond = for syntac and scotchbond el zohairy et al.18 (2004) netherlands feldspathic syntac optibond solo plus visio bond tetric flow nexus 2 1 day, 7 days and 28 days. bond strength/ microtensile adhesive ou = peumans et al.19 (2007) japan leucite heliobond variolink ii 24h bond strength/ microtensile adhesive = meng et al.20 (2008) japan leucite heliobond variolink ii 24h or 10,000 cycles of tc. bond strength/ microshear mixed continue 6 velho et al. braz j oral sci. 2023;22:e239056 continuation passos et al.21 (2008) brazil feldspathic scotchbond variolink ii immediate or 12,000 cycles of tc+ 50 days of storage bond strength/ microtensile mixed lise et al.22 (2015) brazil lithium disilicate excite f dsc variolink ii multilink automix relyx unicem 2 24h bond strength/ microshear adhesive = elsayed et al.23 (2017) germany lithium disilicate scotchbond universal; optibond xrt; all bond universal; prime e bond nt variolink esthetic dc relyx ultimate nx 3 calibra esthetic 3 days, 30 days and 7500 cycles of tc or 150 days 37,500 cycles of tc bond strength/ tensile cohesive = murillogómez et al.13 (2017) brazil lithium disilicate single bond plus; scotchbond universal relyx ultimate 24h or 6 months bond strength/ microshear cohesive = ataol and ergun24 (2018) turkey lithium disilicate zirconiareinforced lithium silicate clearfil universal bond clearfil majesty es-2 24h or 5,000 cycles of tc bond strength/ shear adhesive romaninijunior et al.16 (2018) brazil lithium disilicate xp bond; scotchbond universal surefil sdr flow 24h and 12 months bond strength/ microshear adhesive + in 24h or = in 12 months barbon et al.17 (2019) brazil feldspathic adper single bond 2 relyx veneer and 3 experimental resin cements immediate bond strength/ microtensile mixed = or + depending on resin cement. flexural strength/ biaxial fracture = or depending on resin cement. chen et al.25 (2019) china lithium disilicate single bond plus; all bond universal relyx veneer clearfil as luting relyx unicem 24h or 20,000 cycles of tc and 120 days of storage bond strength/ shear mixed + murillogómez et al.26 (2019) brazil lithium disilicate adper single bond plus; single bond universal relyx ultimate 24h flexural strength/ biaxial fracture = tribst et al.27 (2019) brazil lithium disilicate single bond universal; multilink n primer a and b multilink n 24h up to a maximum of 7 days fatigue failure load/ staircase test radial crack = südbeck et al.28 (2021) germany leucite or lithium disilicate scotchbond universal variolink esthetic dc relyx ultimate 24h or 6 months bond strength/ microtensile mixed * + the use of adhesive was favorable to the outcome; the use of adhesive was unfavorable to the outcome; = the use of adhesive was not altered to the outcome. tc= thermocycling. 7 velho et al. braz j oral sci. 2023;22:e239056 discussion the longevity of the adhesion of resin materials to glass-ceramics is associated with a correct treatment of the ceramic surface29. conventional surface treatment involving hf etching and silanization is well established for glass-ceramics30,31. however, modifications have been suggested, such as the application of an adhesive after ceramic surface treatment15. in addition, based on the data of this review, this additional step does not seem to improve the adhesive and mechanical behavior of glass-ceramics, since the results in most studies were similar or worse than conventional treatment. only four studies showed favorable results from the adhesive application for the bond strength outcome12,16,17,25. however, the results of el zohairy et al.12 (2003) were only favorable for the optibond adhesive, while the results for the syntac and scotchbond adhesives were similar to the control, with the authors justifying this fact due to the greater filler content in the optibond adhesive. the results found by romanini-junior et al.16 (2018) were in favor of adhesive layer application only when tested after 24h, which was not maintained after 12 months of storage, since the hydrophilic characteristic of the adhesives used favors hydrolytic degradation over time. for barbon et al.17 (2019), the adhesive layer application favors the bond strength values when associated with experimental resin cements of higher viscosity, as they facilitate filling in irregularities by hf etching on the ceramic surface. a common characteristic among the studies in which the adhesive layer application was unfavorable to the outcome20,21,28 is the hydrophilicity of the adhesives used. the adhesives are present in hydrophobic or hydrophilic form, with the latter being characterized by its affinity for water. water absorption is influenced by the material’s affinity for water and by the amount of hydroxyl groups (oh) in the resin matrix , which form hydrogen bonds with water, favoring water absorption and consequently worsening adhesion over time32. in this sense, applying an adhesive with hydrophilic properties on the ceramic surface can make the adhesive interface more susceptible to hydrolytic degradation over time. it is important to highlight that restorative materials are exposed to the presence of moisture, chewing loads, changes in temperature and ph in the oral environment33. these factors tend to degrade the adhesive interface over time. in this sense, it is important that this degradation is simulated in in vitro studies through the storage and/or thermocycling of the specimens34. some kind of aging protocol was used in most of the included studies in the present review, demonstrating the authors’ concern in this regard. however, especially in studies that showed no influence of the adhesive application after ceramic surface treatment, the specimens were not subjected to aging protocols, and consequently the results may have been overestimated. therefore, they must be interpreted with caution. in addition, when it comes to adhesion tests, it is known that micro tests are the most reliable since they tend to include a smaller number of defects in the substrate or at the bond interface35. most of the included articles adopted microshear or microtensile tests, demonstrating the authors’ concern with this point. in observing the overall findings, the adhesion test methodology did not influence the results’ 8 velho et al. braz j oral sci. 2023;22:e239056 trends. another important point in relation to adhesion studies is the presence of a careful analysis regarding the types of failure found (adhesive, mixed or cohesive) and their relationship with the findings34. in this context, all included studies presented such analyzes. mechanical outcomes were only evaluated by 3 studies17,26,36. flexural strength data were obtained from the biaxial tests using ceramic discs resin-cement coated17,26. the data regarding fatigue failure load data come from simplified restorations (ceramic discs cemented on a supporting substrate) subjected to cyclic fatigue36. in both studies the adhesive application did not improve the mechanical outcomes, yet more studies employing these methodologies are encouraged due to the scarce available evidence. universal adhesives (uas) were the most used adhesives in the studies (table 2). uas were launched with the purpose of simplifying the adhesive technique, and can be used on dental substrates with or without acid etching, in addition to promoting adhesion to different substrates due to the presence of methacryloyloxydecyl dihydrogen phosphate (mdp) monomers and silane incorporation in their composition37-40. in silane-containing uas, manufacturers suggest that the adhesive can replace silane application after hf etching on glass-ceramics16. however, studies show that the amount of silane present in the uas would not be enough to replace the application of a silane layer9,16,41. one of the inclusion criteria required in the present review was that there were comparison groups (hf + silane) x (hf + silane + adhesive); thus, studies which only applied uas were not included. in this sense, application of silane-containing uas would add an additional layer of silane, but there was no improvement in the bond strength values16,24. adhesive technique is an extremely sensitive procedure and subject to operator experience and skill42,43. therefore, the inclusion of additional steps such as the adhesive application after ceramic surface treatment can make the procedure even more complex and subject to operator errors. in addition, in view of most of the available evidence demonstrating that application of an adhesive layer was unfavorable or without influence on the evaluated outcomes, this may be a dispensable step. the aim of the present review was to perform a qualitative synthesis of the studies available in the literature, but a quantitative synthesis and risk of bias analysis of the studies were not performed. in addition, all included studies were laboratory studies, since only this design is able to evaluate adhesive outcomes in an isolated form. clinical studies may evaluate the survival rate of dental restorations with a higher level of evidence; however, such an outcome may be influenced at the same time by cyclic loads, wear and/or parafunction habits, which may generate cracks and fractures. therefore, extrapolating the results of in vitro studies to the clinical practice should be done with caution. another important point is how the application of an adhesive could influence the adaptation of indirect ceramic restorations, however the lack of evidence on the subject makes the discussion difficult. the absence of clinical studies on the subject until this time impairs being able to indicate the application of an adhesive after surface treatment of glass-ceramics. in this sense, the conduction of clinical studies and studies of mechanical properties within the theme is suggested. 9 velho et al. braz j oral sci. 2023;22:e239056 in conclusion, most of the evidence available in the literature demonstrates that the adhesive application after surface treatment does not improve the adhesive or mechanical behavior of glass-ceramics. however, the literature still lacks clinical studies on the subject. conflict of interest none data availability datasets related to this article will be available upon request from the corresponding author. author contribution helder callegaro velho: conceptualization, data curation, formal analysis, methodology, writing – original draft; pablo soares machado: conceptualization, data curation, formal analysis, methodology, writing – review & editing; lucas saldanha da rosa: formal analysis, methodology, writing – review & editing; catina prochnow: formal analysis, methodology, writing – review & editing; jatyr pisani proenca: conceptualization, supervision, formal analysis, data curation, writing – review & editing; all authors actively participated in the manuscript’s findings and have revised and approved the final version of the manuscript. references 1. gracis s, thompson vp, ferencz jl, silva nr, bonfante ea. a new classification system for all-ceramic and ceramic-like restorative materials. int j 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breschi l, paolino d, et al. influence of operator experience on non-carious cervical lesion restorations: clinical evaluation with different adhesive systems. am j dent. 2016 feb;29(1):33-8. 1http://dx.doi.org/10.20396/bjos.v20i00.8663690 volume 20 2021 e213690 original article 1 department of endodontics, endodontics specialization career, school of dentistry, university of buenos aires (uba), buenos aires, argentina. 2 department of histology and embryology, school of dentistry, university of buenos aires (uba), buenos aires, argentina 3 institute of cell biology and neurosciences “prof. e. de robertis “(ibcn) – school of medicine, university of buenos aires (uba) – national research council (conicet), buenos aires, argentina. corresponding author: mariela c. canzobre marcelo t. de alvear 2142 (1-a), buenos aires, argentina +5491151076672 mariela.canzobre@odontologia.uba.ar editor: dr altair a. del bel cury received: december 19, 2020 accepted: may 25, 2021 regenerative endodontics model in immature infected rat molars using two step protocol liliana sierra1 , ángeles castrillo1 , elizabeth ritacco1, haydeé miño cornell1, mariela c canzobre2,3,* aim: to develop a model for regenerative endodontics using newly-weaned wistar rats immature molars with pulp necrosis to histologically describe the evolution of apical tissues following treatment with a bi-antibiotic paste, induced bloodclot formation and mta. methods: ten 25-day-old female wistar rats were divided into an initial control group (ci) and two experimental groups in which pulp necrosis was experimentally induced on the left mandibular first molar by exposing the pulp chamber and leaving it open to the oral environment. one of the experimental groups was left untreated (e1) while the other was submitted to a protocol of regenerative endodontics 10 days thereafter (e2). fifteen days after placement of a bi-antibiotic paste, bleeding was induced into the root canal space and mta was placed upon. animals were euthanized 30 days later. right mandibular first molars served as an 80-day-old final control group (cf). each hemimandible was histologically processed to analyse parameters associated with root development. statistical analysis was carried by means of anova; p values below 0.05 were considered statistically significant. results: baseline (i.e. 25-days old) mean root length and apical diameter of the distal root canal were 1.84±0.25 and 0.38±0.02mm respectively. following the regenerative endodontic protocol, cells lining the walls of the root canal and significant increase to both length (2.37±0.22mm) and diameter (0.32±0.03 mm) were observed. conclusions: newly-weaned wistar rats serve as a suitable model to evaluate regenerative endodontic protocols. however, further research is needed in order to disclose the nature of the cells and/or cell mediators involved. keywords: regenerative endodontics. dental pulp necrosis. root canal therapy. anti-bacterial agents. https://orcid.org/0000-0002-0313-3031 https://orcid.org/0000-0002-2969-630x https://orcid.org/0000-0002-9460-204x 2 sierra et al. introduction the american association of endodontists has defined regenerative endodontic therapy as “biologically based procedures designed to replace damaged structures, including dentine and root structures, as well as cells of the pulp-dentine complex”1. continued root development, tooth survival and function retention have been long pursued; from the very first studies carried on humans and dogs by ostby2 in 1961, when a new apical tissue was observed after the formation of an intracanal blood clot, to the more recent advances in tissue engineering. considering the core principles of tissue engineering, a triad that includes an appropriate source of stem/progenitor cells, growth factors, and scaffolds – regenerating functioning pulp tissue would be the ideal therapeutic approach when treating immature teeth with open apexes and a diagnosis of pulp necrosis3,4. immature open apexes are richly vascularised and some cells of the apical papilla and hertwig’s epithelial root sheath (hers) might be able to survive infection5. consequently, if noxious stimuli are removed, these cells become able to proliferate in proximity to the periapical tissues leading to the regeneration of radicular and periradicular tissues such as alveolar bone, periodontal ligament, cementum and dentine-pulp complex thereby allowing the root to mature increasing both in length and thickness6. a number of clinical studies have reported positive pulpal testing results after complete maturation of the root7. notwithstanding, the nature of the tissues regenerated within the root canal space is still unknown and under investigation. considering human samples are difficult to harvest, animal models might contribute to shedding some light on this matter and identifying cell populations as well as the intrinsic and extrinsic factors that might participate in the process of apexogenesis/maturogenesis6,8,9. a paradigm shift in endodontics currently presents us with the alternative to perform a protocol based in endodontic regenerative procedures (reps) when treating an immature permanent tooth with an open apex and diagnosis of pulp necrosis: to induce, by generation an intracanal blood clot, the formation of new tissues that can assist continued root maturation. when unsuccessful, apexification protocols always remain an option10. developing a standardised protocol using laboratory animals and materials such as bi-antibiotic paste and mineral trioxide aggregate (mta) that permits the study of the histologic characteristics of the newly formed tissues within the canal and in the periapical region remains a challenge11-13. the aim of the present study was to develop a model for regenerative endodontics using newly-weaned wistar rat immature molars with pulp necrosis by means of a bi-antibiotic paste followed by evoked bleeding and formation of a blood clot within the root canal and its contact with a biomaterial. moreover, we sought to assess the progress of root maturation in length and diameter of the root canal following treatment and describe the histology of the apical and periapical tissues materials and methods this study was approved by “ethics committee of the school of dentistry of the university of buenos aires (cd n° 012/2016 cicual)” 3 sierra et al. ten 25-day-old female wistar rats were used. animals were housed in cages in groups of five animals per cage maximum within the animal facility of the department of histology and embryology of the school of dentistry of the university of buenos aires under standard controlled conditions (temperature 18-22°c, relative humidity 40-60% and 12:12 light/dark cycles approximately 100 lux -) and were allowed ad libitum access to food (standard chow, cooperación, buenos aires, argentina) and water. both, animals and environmental conditions, were monitored daily to guarantee they remained stable throughout the entire study. newly-weaned rats (i.e. 25-day-old) animals were randomly allocated to one of the following groups: control group (ci; n=3): animals weighing about 60 g average were used as an initial control group to study the histological characteristics of the mandibular first molar of the rat molar at the age of 25 days. experimental group 1 (e1; n=3): pulp necrosis was experimentally induced on the left mandibular first molar as explained below and received no further treatment. experimental group 2 (e2; n=4): following experimentally-induced pulp necrosis, a protocol of regenerative endodontics was performed. experimental induction of pulp necrosis: animals in the experimental groups were anaesthetised with a combination of 50mg/kg of ketamine (ketamid; holliday-scott s.a., beccar, buenos aires, argentina) and 10 mg/kg of xylazine (kensol; könig s.a., avellaneda, buenos aires, argentina) administered i.p. once anaesthetised, each animal was laid in a dorsal position on a stretcher immobilising its head by the upper incisors, which are placed through a metal wire attached to the headrest. mouth opening, tongue separation and mandibular first molar isolation were guaranteed by an aluminium clamp as proposed by a protocol presented recently14. in order to achieve complete necrosis of the dental pulp, the pulp chamber was accessed by drilling through its roof using a size 1/4 round dental bur (ss white, lakewood, new jersey) mounted on an electric-engine driven micromotor and left open to the oral environment for 10 days. regenerative endodontic therapy protocol: left mandibular first molars of animals allocated to the e2 group were further treated following the protocol previously described by thibodeau and trope15. ten days after pulp exposure, the pulp chamber was cleaned using a dentine excavator and a 2.5% sodium hypochlorite solution; the distal canal was catheterised using a #08 k-file (dentsply sirona, ballaigues, switzerland) calibrated at a working length of 3 mm and slowly irrigated with a 2.5% sodium hypochlorite (clorox, argentina) solution followed by saline; no further mechanical preparation was performed. the root canal was then dried using sterile paper points #25 (meta biomed; cheongju, north chungcheong, south korea) and filled with a bi-antibiotic paste containing equal amounts of 200 mg ciprofloxacin and 500 mg metronidazole (2mix) mixed with propylene glycol15. the access cavity was sealed with glass-ionomer cement (ketac molar easymix, 3m espe, neuss, germany). fifteen days after intervention, the pulp chamber of the left mandibular first molar was accessed again and cleaned using a dentine excavator. the bi-antibiotic paste was 4 sierra et al. gently flushed out of the canal with 2.5% sodium hypoclorite (clorox, argentina). after final irrigation with 17% edta (tedequim, argentina) for 5 minutes, the canal was dried using sterile paper points (meta biomed; cheongju, north chungcheong, south korea). a #15 k-file (dentsply sirona, ballaigues, switzerland) was inserted through the distal canal beyond the foramen to induce bleeding from the periapical tissues16 and formation of a blood clot inside the root canal. mta was then placed upon the distal root canal entrance orifice and compacted using a wet cotton pellet to assist its setting reaction; the access cavity was finally sealed with glass-ionomer cement (ketac molar easymix, 3m espe, neuss, germany). coronal seals were daily monitored for wear derived from rodents’ diet so as to prevent their loss and root canal reinfection. animals were euthanized by i.p. injection of a lethal dose of pentobarbital sodium (euthanyle, brouwer s.a., buenos aires, argentina) 30 days after placement of mta (e2). experimental group e1 which received no further treatment was matched in age by the time of euthanasia, set to take place at approximately the same time of the day in order to minimise the effect of changes owed to circadian rhythm. all right mandibular first molars were kept as an 80 day-old control group (cf) (fig. 1). in order to perform bright-field microscopy evaluation, right and left hemimandibles were dissected and fixed in buffered formalin (ph 7.3-7.5) for 48 hs. following decalcification in edta 10% ph 7 for 5 weeks, hemimandibles were dehydrated and embedded in paraffin (paraplast, sigma-aldrich) and serial mesio-distally oriented sections of the mandibular first molars of approximately 7 µm were obtained using a minot microtome. sections were stained with hematoxylin and eosin (h-e) and digitalised microphotographs were then taken with a light microscope (nikon eclipse ni, nikon instruments inc, melville, ny, usa) and analysed using imagepro plus4.5.0.29 software to measure (fig. 2) the length and apical diameter of the distal root canal as described in previous studies14. in order to do this, one line was drawn passing through the mesial and distal cement enamel junctions (anatomic limit between the crown and root surface) and another one tangent to the apex of the mesial and distal roots. with these as references, two lines were drawn: a perpendicular one running through the centre of the distal canal (a: root canal length) and a parallel one at the figure 1. the experimental protocol and the procedures carried upon both experimental and control groups are shown in a. photographs depict the isolation of the mandibular first molar with its pulp exposed (b) and the intact mandibular first molar that served as control (c). female wistar rats experimental groups necrosis (e1) treatment (e2) 25 days 35 days 50 days 80 days biantibiotic paste blood clot eutanasiaeutanasia exposed tooth pulp eutanasia eutanasia initial (ci) final (cf) control groups ba c 5 sierra et al. level of the apical third of the root from inner dentine surface located on the distal side to the one on the mesial side (b: apical diameter of the distal root canal). furthermore, we measured the size of the periapical area as enclosed by the line tangent to the apex of the distal root and the surface of the alveolar bone proper as well as the osteoclastic cells within this area considering all cells close to the bone surface and containing two or more nuclei (fig. 2). statistical analysis graph pad prism 5.0 was employed. all the results were expressed as mean and standard deviation (sd), and statistically analysed using anova followed by tukey’s posthoc test setting the significance level at 5%. results histomorphometric analysis of root development as observed on the h-e stained histological sections, molars of the 25-day-old control group (ci) exhibited thin root canal walls as well as wide root canals, pulp with a normal histological pattern and wide open apexes where hers could still be observed (fig 2a). mean values for root canal length and apical diameter of the canal of the distal root of the mandibular first molar are shown in table 1; statistically significant differences were found between ci and e2 (p<0.05) and between ci and cf (p<0.001). with regards to the periapical area, this group showed a normal width without signs of inflammation. a high number of osteoclasts was observed, as expected for a developing alveolus which is being remodelled constantly in order to accommodate a maturing root. figure 2. microphotographs showing he stained histological sections of the mandibular first molar corresponding to the initial (a; i.e. 25-day-old) and final (b; i.e. 80-day-old) control groups. dashed lines served as landmarks passing through the cement enamel junction and the apex of both roots. lines a and b were drawn to measure the length of the distal root and the apical diameter of its root canal, respectively. the asterisk indicates the location of the periapical area. note that, a presents a wide root canal as well as very thin walls and hers can still be devised. a b 6 sierra et al. group e1 (80 days old) revealed that pulps exposed to the oral environment and left untreated until euthanasia were entirely necrotic; these molars exhibited no sign of hers and very thin root canal walls. a greater width of the periapical periodontal ligament space was observed (ci vs e1 p<0.001) at expense of the presence of an inflammatory infiltrate and resorption of the surrounding alveolar bone by osteoclasts (e1 vs cf p<0.001). however, no odontoclasts were found (fig. 3) which translated into a lack of statistically significant differences when comparing root length and apical diameter of the root canal to the ci group (table 1). group e2 (80 days old), in which the distal root of the left mandibular first molar was treated by means of a regenerative protocol, revealed an increase in root length and a decrease in the width of the root canal and its apical diameter as well as slightly thicker root canal walls (e2 vs e1 p<0.05 and e2 vs cf p<0.001) (table 1). the width of the periapical periodontal ligament space was significantly greater than that of the control groups (ci vs e2 p<0.05; cf vs e2 p<0.05) but a reduction in the inflammatory infiltrate, size of the periapical area and the number of osteoclasts were found when compared with e1 (no significant difference). cells lining the dentinal surface of the root canal and osteoblasts on the alveolar bone surface could be observed (fig. 4). table 1. mean values and standard deviations of the distal root length and canal diameter, size of the periapical area and osteoclastic activity. wistar rat mandibular first molar distal root ci: control (25 days old) e1: pulp necrosis e2: reps cf: control (80 days old) length (mm) 1.84 ± 0.25ab 1.65 ± 0.16cd 2.37 ± 0.22ace 3.20 ± 0.13bde apical canal diameter (mm) 0.38 ± 0.02ab 0.39 ± 0.02cd 0.32 ± 0.03ace 0.08 ± 0.01bde periapical area (mm2)  0.19 ± 0.02ab 1.23 ± 0.34a 0.88 ± 0.46bc 0.14 ± 0.05c osteoclasts/mm 1.79 ± 0.55 a 1.72 ± 0.45 b 1.19 ± 0.23 0.36 ± 0.42 ab values sharing the same letter within a row show a statistical difference between groups (anova p<0.05) figure 3. microphotographs showing he stained histological sections of the experimental distal root of the mandibular first molar (e1) with pulp necrosis and immature apex (a). necrotic tissue can be seen inside the root canal; neither odontoblasts lining the dentine surface or hers can be observed (b). actively resorbing osteoclasts and howship lacunae can be seen on the alveolar bone surface (arrow in c). bars represent 100µm. a b c 7 sierra et al. in spite of the improved root development, the length of the distal root in the group that received the regenerative endodontic protocol did not get to match the degree of maturation of the right mandibular first molar (cf) used as an 80 day-old control given the latter measured 3.2±0.12mm, presented thick dentinal walls and a completely developed apex narrowed by the continuous deposition of cellular cementum (hypercementosis). in this group, lateral canals were also evident probably as a consequence of the rapid deposition of cellular cementum in relation to blood vessels. the periapical area exhibited normal width and histological characteristics and an alveolar wall with little cell activity (fig 2b and table 1). discussion stem cell research brought along new therapeutic approaches to treat immature permanent teeth with a diagnosis of pulp necrosis. reps constitute a promising alternative but the results derived from different protocols, whether they be currently approved or under investigation, require experimental studies to guarantee more predictable patient outcomes in the future. several biological aspects related to tissue regeneration and repair are still missing likely due to the ethical and casuistic limitations associated with human trials. for this reason, animals such as dogs, cats, monkeys, rabbits, ferrets and rats are used by different groups in the search for an experimental model in vivo that would prove to be suitable and reproducible in order to study an analyse regenerative processes12. the animal models of reps currently available in the literature11-13,15,17 do not adhere to the three stages as presented in clinical protocols. for example, scarparo et al.17 also used necrotic immature teeth but, unlike our model, they only performed root canal disinfection with no further evoked bleeding and blood clot formation. previous reports using animal models have more commonly used a triple antibiotic paste to achieve disinfection of the root canal11,13,17; we, however, opted for a bi-antibiotic paste figure 4. microphotographs showing he stained histological sections of the experimental distal root of the left mandibular first molar (e2) that received regenerative endodontic therapy (a). blood cells and mta particles can be seen inside the root canal (b). although there is no evidence of hers, some cells can be seen lining the root canal walls (arrow in c). active osteoblasts and a thin layer of osteoid can be seen on the alveolar bone surface (arrow in d). bars represent 100µm. a b c d 8 sierra et al. thus ruling minocycline out, as suggested by thibodeau and trope15 in 2007, thereby implementing a protocol that is presently considered more appropriate in a clinical setting as minocycline is associated with dental discoloration. we used wistar rats as they can be easily housed, anaesthetised and handled, and enable histological sections to be obtained relatively fast, at a lower cost of acquisition and maintenance with less concerned breeding management and ethical considerations when compared to dogs11, ferrets12 or sheep13. moreover, their pulpal, periapical and healing response is comparable to that of humans17,18. given we used rats of a very young age in our experimental model, we waited for them to be weaned at the age of 21 days. even though mouth opening at this age is still limited, the mandibular first molars are indeed fully erupted and present short roots and wide open apexes. based on recent observations14,18 that pointed out that the development of the mandibular first molar is still incomplete by the age of 35 days old, younger rats were chosen for this model – i.e. four days post-weaning (25 days old (ci)) – so as to guarantee roots were still immature but access was sufficient for rotary instruments to expose dental pulp and cause its necrosis by leaving it open to the oral environment. unfortunately, working with small animals in vivo does not permit to take intraoperative intraoral radiographs that would allow to take measurements and follow up the evolution and response of hard tissues to treatment thus making it necessary to await dissection of the hemimandibles and obtention of post-mortem radiographs and histological sections in order to be able to analyse results. following complete necrosis of the dental pulp, root development was halted in the group that received no further treatment (e1); the result was a wide root canal with thin walls and no further lengthening of the root which occurred as a consequence of the death of odontoblasts and cells of hers, respectively. in the experimental group that received treatment (e2), a regenerative endodontic therapy model that adhered to the principles applied to treating immature permanent human teeth was followed. for this purpose, newly-weaned rats with experimentally-induced necrosis were used and the regenerative procedure was carried in two steps (disinfection and blood-clot formation). it is worth mentioning that procedures related to the regenerative protocol, including initial debridement of the root canal, placement of the bi-antibiotic paste and coronal seal could be carried out without major inconveniences even in spite of the reduced dimensions with which we were set to work. likewise, the distal root was accessed again two weeks after to induce bleeding and formation of the intracanal blood clot and to place mta upon the root canal orifice. the biggest challenge was perhaps encountered when manipulating mta given the presence of the blood clot, the shallowness of the pulp chamber and the consistency of the material making both ability and practice necessary to achieve a coronal seal that remained in place until the moment of euthanasia. for a number of authors5-7, the results following regenerative endodontics can be explained by the mobilisation of undifferentiated stem cells from the apical region which in combination with growth factors supplied by the bleeding are fundamental to trigger tissue regeneration. in other words, the granulation tissue presents in the apical region as a response to pulp necrosis acts as a potential niche and reservoir for regenerative processes. 9 sierra et al. in the present model, complete development of the root was not achieved, but we were still able to observe an increase in the length of the root and the thickness of its walls therefore suggesting that the cells that were observed lining the walls of the root canal could be responsible for the synthesis and mineralisation of mineralised radicular tissues. moreover, after performing regenerative endodontic procedures, histological characteristics of the periapical tissues were analysed observing a decrease in the periodontal ligament width with reduced inflammatory infiltrate and fewer osteoclasts per mm of bone surface. even osteoblasts were identified on the surface of the surrounding alveolar bone. nonetheless, there is still much to be unravelled in regenerative endodontics, whether it is developing new substances for root canal disinfection that are safer than antibiotics or irrigants that might attract certain cell populations or using scaffolds for more biomimetic approaches19. in conclusion, in our newly-weaned wistar rat model we demonstrated that after experimental induction of pulp necrosis and root development arrest, an increase in length of the distal root and decrease in the apical diameter of its root canal were achieved by means of reps. therefore, the present model of pulp necrosis, canal disinfection using a bi-antibiotic paste and formation of an intracanal blood clot, is suitable and reproducible in order to study an analyse regenerative processes. it would be interesting to continue our work with immunohistochemical determinations to determine the kind of cells involved in the process and the tissues formed within the root canal. ethics the ethics committee of the school of dentistry of the university of buenos aires approval with the reference number (cd n° 012/2016 cicual). acknowledgments this work was supported by project ubacyt: 20720150200012ba. the authors are grateful to carlos bárcenas for his expert assistance in animal care and in handling animals during procedures, laboratory technicians mariela lacave and paula rocha for processing the samples and obtaining the histological sections, and sharon r. oyhanart for kindly reviewing the manuscript. conflicts of interest the authors declare no conflict of interest. references 1. american association of endodontists. glossary of endodontic terms. 10th ed. chicago: aae; 2019. 2. ostby bn. the role of the blood clot in endodontic therapy. an experimental histologic study. acta odontol scand. 1961 dec;19:324-53. 3. hargreaves km, diogenes a, teixeira fb. treatment options: biological basis of regenerative endodontic procedures. j endod. 2013 mar;39(3 suppl):s30-43. doi: 10.1016/j.joen.2012.11.025. 10 sierra et al. 4. diogenes a, ruparel nb, shiloah y, hargreaves km. regenerative endodontics: a way forward. j am dent assoc. 2016 may;147(5):372-80. doi: 10.1016/j.adaj.2016.01.009. 5. huang gt, sonoyama w, liu y, liu h, wang s, shi s. the hidden treasure in apical papilla: the potential role in pulp/dentin regeneration and bioroot engineering. j endod. 2008 jun;34(6):645-51. doi: 10.1016/j.joen.2008.03.001. 6. huang gt. apexification: the beginning of its end. int endod j. 2009 oct;42(10):855-66. doi: 10.1111/j.1365-2591.2009.01577.x. 7. nosrat a, kolahdouzan a, hosseini f, mehrizi ea, verma p, torabinejad m. histologic outcomes of uninfected human immature teeth treated with regenerative endodontics: 2 case reports. j endod. 2015 oct;41(10):1725-9. doi: 10.1016/j.joen.2015.05.004.  8. huang gt, garcia-godoy f. missing concepts in de novo pulp regeneration. j dent res. 2014 aug;93(8):717-24. doi: 10.1177/0022034514537829. 9. alexander a, torabinejad m, vahdati sa, nosrat a, verma p, grandhi a, et al. regenerative endodontic treatment in immature noninfected ferret teeth using blood clot or synoss putty as scaffolds. j endod. 2020 feb;46(2):209-15. doi: 10.1016/j.joen.2019.10.029. 10. pulyodan mk, paramel mohan s, valsan d, divakar n, moyin s, thayyil s. regenerative endodontics: a paradigm shift in clinical endodontics. j pharm bioallied sci. 2020 aug;12(suppl 1):s20-6. doi: 10.4103/jpbs.jpbs_112_20. 11. stambolsky c, rodríguez-benítez s, gutiérrez-pérez jl, torres-lagares d, martín-gonzález j, segura-egea jj. histologic characterization of regenerated tissues after pulp revascularization of immature dog teeth with apical periodontitis using tri-antibiotic paste and platelet-rich plasma. arch oral biol. 2016 nov;71:122-8. doi: 10.1016/j.archoralbio.2016.07.007. 12. torabinejad m, corr r, buhrley m, wright k, shabahang s. an animal model to study regenerative endodontics. j endod. 2011 feb;37(2):197-202. doi: 10.1016/j.joen.2010.10.011. 13. altaii m, cathro p, broberg m, richards l. endodontic regeneration and tooth revitalization in immature infected sheep teeth. int endod j. 2017 may;50(5):480-91. doi: 10.1111/iej.12645. 14. oyhanart sr, canzobre mc. methodological considerations for a model of endodontic treatment in wistar rats. acta odontol latinoam. 2020 dec;33(3):153-64. 15. thibodeau b, trope m. pulp revascularization of a necrotic infected immature permanent tooth: case report and review of the literature. pediatr dent. 2007 jan-feb;29(1):47-50. 16. hameed mh, gul m, ghafoor r, badar sb. management of immature necrotic permanent teeth with regenerative endodontic procedures a review of literature. j pak med assoc. 2019 oct;69(10):1514-20. 17. scarparo rk, dondoni l, böttcher de, grecca fs, rockenbach mi, batista el jr. response to intracanal medication in immature teeth with pulp necrosis: an experimental model in rat molars. j endod. 2011 aug;37(8):1069-73. doi: 10.1016/j.joen.2011.05.014. 18. yoneda n, noiri y, matsui s, kuremoto k, maezono h, ishimoto t, et al. development of a root canal treatment model in the rat. sci rep. 2017 jun;7(1):3315. doi: 10.1038/s41598-017-03628-6. 19. kaushik sn, kim b, walma am, choi sc, wu h, mao jj, et al. biomimetic microenvironments for regenerative endodontics. biomater res. 2016 jun;20:14. doi: 10.1186/s40824-016-0061-7. 1 volume 21 2022 e228356 original article braz j oral sci. 2022;21:e228356http://dx.doi.org/10.20396/bjos.v21i00.8668356 1 post graduate program in human movement sciences, methodist university of piracicaba unimep piracicaba (sp), brazil. 2 post graduate program of dental clinic, concentration area oral and maxillofacial surgery, piracicaba dental school, university of campinas – unicamp – piracicaba (sp), brazil. corresponding author: adriana pertille graduate program in human movement sciences, methodist university of piracicaba e-mail address: pertille.adri@gmail. com editor: dr. altair a. del bel cury received: february 8, 2022 accepted: april 2, 2022 strength of scapular elevation in women with tmd and asymptomatic women a cross-sectional study lúcio ferreira dos santos1, fabiana foltran-mescollotto1 , ester moreira de castro-carletti1 , elisa bizetti pelai2 , marcio de moraes2 , delaine rodrigues-bigaton2 , adriana pertille1 temporomandibular disorder (tmd) is recognized for its high prevalence, presenting characteristic signs and symptoms. cervical spine pain is present in 70% of diagnosed tmd cases. aim: to verify if women with tmd present changes in isometric muscle strength in the scapula elevation. methods: this is an observational, cross-sectional study. thirty-five women, aged 22.89±2.04 years, were divided into the tmd group (tmdg), diagnosed with tmd according to the dc/tmd, and control group (cg), with asymptomatic individuals. the volunteers accessed a online link by the smartphone in order to answer questions on personal data, the fonseca anamnestic index (fai), neck disability index (ndi), and masticatory preference. in all participants, evaluation of the force of the scapula elevation muscles was performed, using a load cell model mm-100 (kratos® sp, brazil). data were analyzed descriptively using the maximum, mean, and standard deviation and a two-way ancova test was applied for all variables. a significance level of 5% was considered. results: there were no statistically significant differences between the tmdg and cg for the maximal and mean muscle strength of scapular elevation. there were statistically significant differences in fai (p <0.001*) between the cg and the tmdg. conclusion: based on the results, it was not possible to confirm the hypothesis that women diagnosed with tmd present lower isometric strength during scapular elevation (right/left). keywords: temporomandibular joint. isometric contraction. muscle strength. https://orcid.org/0000-0002-6145-7448 https://orcid.org/0000-0002-0688-165x https://orcid.org/0000-0003-0826-9744 https://orcid.org/0000-0002-5229-5723 https://orcid.org/0000-0002-3423-5575 https://orcid.org/0000-0002-6979-342x 2 santos et al. braz j oral sci. 2022;21:e228356 introduction temporomandibular disorder (tmd) is recognized by the american academy of orofacial pain (aaop) as a group of musculoskeletal and neuromuscular conditions involving the temporomandibular joint (tmj), masticatory muscles1, and all other associated craniocervical structures2,3. the clinical signs and symptoms present in individuals with tmd can be listed as muscle and/or joint pain, tmj sounds or noises (in cases of disc displacement and/or degenerative dysfunction), restrictions, limitations, and deviations during mouth opening and/or closing2. moreover, pain in the cervical region is present in 70% of cases of tmd4-6. the prevalence of tmd ranged from 21.1% to 73.3% with the occurrence of painful tmd signs/symptoms varying from 3.4% to 65.7%, and non-painful signs/symptoms between 3.1% and 40.8%7. according to silveira et al. (2015)5, the presence of signs and symptoms in the cervical region of patients diagnosed with tmd, as well as the presence of painful points in the cervical region, are very common. the presence of signs and symptoms in the cervical region was confirmed on palpation, with 23 to 67% of patients with tmd presenting muscle tenderness (presence of tender points) in the sternocleidomastoid and upper trapezius muscles5. muscle tension in the cervical and mandibular regions is associated with high levels of disability, according to the neck disability index (ndi)8. the cervical spine and tmj present a neuro-anatomical and functional relationship5,6. in addition, alterations in the cervical posture can be related to patients with tmd and vice versa. high rates are reported for the coexistence of tmd and cervical spine abnormalities. according to armijo-olivo et al.9, the prevalence of neck pain in their sample of subjects with tmd was high; approximately 88% of subjects with mixed tmd had self-reported neck pain. several studies demonstrate correlations and associations between tmd and signs and symptoms of cervical dysfunctions and despite increasing evidence, this is still explained by the intimate anatomical connections10,11, neurophysiological mechanisms that connect these two regions12-14, and pain15. the work of truong quang dang et al.16, investigated the relationship between dental occlusion and arm strength; in particular, jaw imbalance could cause a loss or decrease in upper limb strength. during functioning, mechanical loads on the scapula are transferred to the shoulder and cervical spine, through the double orientation of the musculature (upper trapezius and scapular elevation)17-19, and this may be altered in patients with cervical dysfunction20, in addition to being frequently cited for involvement in myofascial neck pain21. based on the literature cited above, the current study hypothesized that women with tmd would present decreased isometric strength generation of scapular elevation in the orthostatic position when compared with asymptomatic women. measurements of the function and strength of scapular elevation were not found in the existing literature, especially when related to patients with tmd. this informa3 santos et al. braz j oral sci. 2022;21:e228356 tion may help to elucidate the functionality of this structure in patients with tmd, using an inexpensive, simple, and objective evaluation method. the possibility of correlations between muscle strength in scapula elevation and subjects with tmd could bring elements that are easy to access for the clinical evaluation of these subjects in the future. thus, the current study aimed to verify if there is a difference in developing the strength of scapular elevation between women with tmd and asymptomatic women. methods study design this is an observational, cross-sectional study. the study was approved by the research ethics committee of the university (caae 65444417.1.0000.5507) and led by laret (therapeutic resources laboratory) from unimep (methodist university of piracicaba). sample size a sample size calculation was performed, based on a pilot study comparing the groups, composed of 6 volunteers in each group. the measure used was the movement of scapular elevation strength. the mean and standard deviation values of the control group (cg) and the temporomandibular disorder group (tmdg) were, respectively, 23.23±6.38 kgf and 43.98±6.91 kgf. according to the variables, for an effect size of 0.32, power of 80%, and 20% alpha, following the analysis of the results, 15 volunteers were required per group. the sample size calculation was performed using gpower® software, version 3.1.9.2. inclusion criteria for inclusion in the tmdg, volunteers were required to be female university students, aged between 18 and 40 years, with a body mass index (bmi) ≤25kg/m², and a diagnosis of tmd according to the diagnostic criteria for temporomandibular disorders (dc/tmd)22. the diagnoses accepted in this study were myalgia, local myalgia, myofascial pain with spreading, myofascial pain with referral, disc displacement with reduction, disc displacement with reduction and with intermittent locking, disc displacement without reduction and with a limited opening, disc displacement without reduction and limited opening, degenerative joint disease, and arthralgia. for inclusion in the cg, the subjects were required to be female university students aged between 18 and 40 years, with a bmi<25kg/m², and no diagnosis of tmd according to the dc/tmd. exclusion criteria in the tmd group, volunteers that did not present a tmd diagnosis or presented a diagnosis of degenerative joint diseases in the tmj were excluded. for both groups (tmd and control) the following exclusion criteria were considered; volunteers that were under physiotherapeutic or pharmacological treatment (analgesic, 4 santos et al. braz j oral sci. 2022;21:e228356 anti-inflammatory, and muscle relaxant), presented tooth loss, using a full or partial dental prosthesis, history of trauma in the face and/or tmj, and history of dislocation and/or subluxation of the tmj. assessment tools diagnostic criteria of temporomandibular disorder (dc/tmd) the dc/tmd is a validated biaxial questionnaire for myogenic tmd diagnosis23. axis i of the dc/tmd includes a physical evaluation and considers recurrent factors in the patient’s daily life, while axis ii considers the previous history, including the beginning and perpetuating factors of the dysfunction22-24. the evaluator was trained and calibrated to use this tool. fonseca anamnestic index (fai) the fai is a scale developed in portuguese that assesses the severity of the myogenic temporomandibular disorder. the short-form fonseca anamnestic index (sfai) demonstrates a high level of diagnostic accuracy and may be used as a new version of the index for the diagnosis of myogenous tmd25. the fai also presents multidimensionality, with dimension one (primary) consisting of five reliable and well fitting items for the composition of its structure26. the high degree of diagnostic accuracy demonstrates that the fai can be employed for the identification of myogenous tmd in women27. this instrument consists of ten self-answered questions, which include information on difficulty opening the mouth and moving the jaw sideways; tiredness and/or muscle pain when chewing; frequent headaches; neck pain and/or a stiff neck; ear or joint pain in the face; noises in the joint when opening the mouth or chewing; if the teeth are well articulated; and whether a person is tense/nervous and has the habit of teeth clenching or grinding. the scale presents three possible answers: “yes” with a score of 10, “sometimes” with a score of 5, and “no” with a score of 0. neck disability index (ndi) the ndi measures the extent to which neck pain affects activities of daily living such as personal care, lifting, reading, headaches, concentration, work, driving, and sleeping. the ndi is a relatively short validated, reliable, and responsive questionnaire that is easy to administer8,28. the 10 items produce a score from zero to 50. the level of neck disability for the ndi was determined as follows: 0–4 points: ‘no disability’, 5–14 points: ‘mild disability’, 15–24 points: ‘moderate disability’, 25–34 points: ‘severe disability’, and >35 points: ‘complete disability’. the total score on the questionnaire was used for statistical purposes. maximal voluntary isometric contraction (mvc) to evaluate the muscle strength of the right and left scapular elevations, a model mm-100 (kratos ®, são paulo, sp, brazil) load cell was used. the volunteers remained standing, on an eight-centimeter-high platform, to better accommodate the load cell. one end of the load cell was fixed to the ground by a metal hook and chain, and the 5 santos et al. braz j oral sci. 2022;21:e228356 other end was held by the volunteer to pull; the load cell did not allow movement, only isometric contraction. all subjects were asked to perform a unilateral shoulder elevation (shoulder blade) to keep the shoulder blade muscles in voluntary isometric contraction. the volunteers were instructed not to perform elbow flexion and/or trunk inclination, and to avoid possible postural compensations. a therapist observed the data collection to prevent any compensations (figure 1). two interspersed measurements of ten seconds were performed for each side, that is, two attempts for the right and two for the left. a two-minute interval was given between each attempt to avoid interference of muscle fatigue in the results. the choice of the right or left side was randomized. procedures at the beginning of the evaluation, all volunteers signed the consent form agreeing to participate of the study. the volunteers were instructed to answer an online questionnaire regarding personal and anthropometric data (body mass, height, age, sex), dominant hand, side preference for chewing, the fai, and ndi. next, the volunteers underwent the dc/tmd evaluation, and were divided into the control and tmd groups. finally, the volunteers performed the mvc of the scapular elevation. data from the mvc were obtained through emg 830c signal acquisition module (emg system do brasil, são josé dos campos, brazil) software. data on the mean and maximum values of each attempt made using the load cell were extracted from this software and tabulated for later analysis. figure 1. muscle strength evaluation model of scapular elevation. a): anterior view; b): posterior view. a b 6 santos et al. braz j oral sci. 2022;21:e228356 statistical analysis data were initially analyzed descriptively (means and standard deviations). data normality was assessed by the shapiro-wilk test, which indicated normal data distribution. to verify if there was a difference in the maximum muscle strength of right and left scapular elevations, and to compare the values of the ndi and fai between the tmdg and cg, the t-test for independent samples was performed. to compare the mvc of right and left scapular elevations between groups, and sides of masticatory preference, a two-way ancova was used. the analysis was adjusted for age. data analysis was performed using spss software, version 13.0. it was considered statistically significant when the p-value was less than 5%. results figure 2 presents a flow chart of the recruitment and assignment to the groups. table 1 presents the characterization of the sample, highlighting the mean age of the volunteers of 22.89 ± 2.04 years, and chewing and manual preference on the right side. table 1. sample characterization presented as mean and standard deviation for age, weight, height, and presented as the frequency of occurrence for chewing and manual preference (right/left) for the total sample, tmdg, and cg (n=35). n = 35 tmdg = 17 cg = 18 age (years) 22.8±2.0 22.5±3.6 23.0±1.9 weight (kg) 61.6±7.2 61.8±11.5 62.5±6.5 height (cm) 160.9±10.3 163.7±6.3 166.7±4.9 chewing preference (right/left) 30/5 13/4 17/1 manual preference (right/left) 31/4 13/4 18/0 n: total sample; tmdg: temporomandibular disorder group; gc: control group. figure 2. volunteer recruitment flowchart. individuals recruited for research (n = 38) not elegible (n = 3) elegible volunteers (n = 35) tmdg (n = 17) cg (n = 18) 7 santos et al. braz j oral sci. 2022;21:e228356 table 2 presents the classification according to the dc/tmd for all volunteers. the maximum strength values and means found for right and left scapular elevations for each of the groups are presented in table 3. no statistically significant differences were found between the tmdg and cg for the mvc of the right and left scapula elevations. there was a statistically significant difference (p=0.001*) in fai scores between the tmdg (71.76±11.71) and cg (36.94±22.89). however, there was no difference between groups in relation to the ndi. table 4 shows the analysis of mean and standard deviation of maximal strength of scapular elevations, right and left sides, and masticatory preference between the tmdg and cg, using a two-way ancova test. a t-test for independent groups was applied. all outcomes were grouped and none of the differences found were statistically significant. table 2. diagnosis of volunteers according to the dc/tmd, presented as frequency of occurrence (n=35). diagnosis control group 18 myalgia 16 myofascial pain 16 disc displacement with reduction, with intermittent locking (r/l) 9 (7/6) disc displacement without reduction, with limited opening (r/l) 1(1/1) disc displacement without reduction, without limited opening (r/l) 1 (1/1) arthralgia 14 (12/11) dc/tmd (diagnostic criteria for temporomandibular disorders)r/l= right/left. table 3. maximum and mean of strength measures for the variables (m.s.s.l.), total ndi and fai scores for the tmdg and cg (n=35). tmdg (mean±sd) cg (mean±sd) p value m.s.s.l. right (kgf) maximum 33.1±10.9 27.3±7.2 0.059 m.s.s.l. right (kgf) mean 28.7±9.7 23.0±6.7 0.053 m.s.s.l. left (kgf) maximum 33.4±10.9 27.9±7,3 0.090 m.s.s.l. left (kgf) mean 29.0±9.5 23.6±6.8 0.059 ndi 8.5±3.8 5.7±3.3 0.270 fai 71.7±11.7 36.9±22.8 0.001* sd: standard deviation; m.s.s.l.: maximal strength of the scapular elevations; tmdg: temporomandibular disorder group; cg: control group; ndi: neck disability index; fai: fonseca anamnestic index. *statistically significant difference between groups (p<0.05). a t-test was applied. 8 santos et al. braz j oral sci. 2022;21:e228356 discussion the present results did not confirm the hypothesis that women with tmd present decreased isometric strength generation of scapular elevation in the orthostatic position when compared with asymptomatic women. no statistically significant differences were found between groups. statistically significant differences were only found in fai (p <0.001*) between the cg and the tmdg. in the current study, the fai presents significantly higher values in the tmd group, reaching a severe index, and the cg, unexpectedly showed a level that was between mild and moderate. no statistically significant differences were found between groups for the mean and maximum mvc test, respectively. indeed, when the mvc values were adjusted by age and compared considering the masticatory preference, no statically significant differences were found between mean and maximum in the mvc test. however, even without significance, the tmdg showed greater (28.7±9.71right; 29.07±9.56 left) development of isometric strength (mean) than the cg (23.03±6.79 right; 23.60±6.88 left). for the ndi, the groups were not statistically significantly difference and showed a mild disability index. the result presented by the tmdg in the measurement of strength may be related to the low values found in the ndi. the level of cervical disability was mild. thus, we could suggest that the cervical musculature (upper trapezius and scapular elevation)17,29, even with dysfunction, can still generate table 4. maximum and mean of strength measures (kilogram force) for the variables (m.s.s.l.) in the comparison between masticatory preference, and tmg and cg (n=35). muscle group masticatory preference mean±sd p value m.s.s.l. right mean tmdg right 30.8±8.1 0.46 left 21.8±12.3 control right 23.4±7.0 left 21.6±0.0 m.s.s.l. right max tmdg right 35.1±9.2 0.54 left 26.6±14.9 control right 27.4±7.4 left 25.3±0.0 m.s.s.l. left mean tmdg right 30.9±9.1 0.34 left 22.8±9.3 control right 23.9±7.1 left 24.7±0.0 m.s.s.l. left max tmdg right 35.0±9.8 0.52 left 28.2±14.2 control right 28.3±7.6 left 28.1±0.0 m.s.s.l.: maximal strength of the scapular elevations; max: maximum; tmdg: temporomandibular disorder group; cg: control group; sd: standard deviation. an ancova test was applied. *statistically significant difference between variables (p<0.05). 9 santos et al. braz j oral sci. 2022;21:e228356 isometric strength normally. in addition, the fai results showed severe levels in the tmdg and mild to moderate levels in the cg, yet despite this significant difference between the groups, the levels of isometric strength were similar. some studies in the literature which also investigated the mvc of scapular elevation, evaluated women workers with self-reported neck/shoulder pain. the findings showed that the group with pain generated significantly lower force during maximal scapula elevation compared to healthy controls30-32. it is proposed that pain may impair the activation of the painful muscles. however, the same findings were not confirmed by the present study. this could occur due to the different population evaluated in this study, as well as the similar values in the ndi for both groups, showing that the control and tmd groups presented mild neck disability and, therefore, both groups presented pain, which could interfere in the strength measures. another study33 that compared an asymptomatic group with a group presenting myofascial trigger points (mtp) and measuring the mvc of the scapular elevation, for both sides at the same time without trunk motion for 5 seconds, did not find a statistically significant difference between the groups. these findings agree with those in the present study since no difference was found between the groups. in addition, the authors reported that when scapular elevation was restricted, shoulder abductor strength was significantly lower in the mtp group than in the non-mtp group, suggesting that overuse of the upper trapezius can cause mtp by compensatory movements in shoulder abduction33. however, the present study did not evaluate the presence of myofascial trigger points or scapular restriction, therefore, this cannot be confirmed by our findings. the current study also did not find significant differences when grouping and relating the masticatory preference with the mean and maximum forces of the control and tmd groups. the tmdg showed higher values of mean and maximum force, with the highest values predominating on the right side (p=0.46 and 0.54), but without statistical significance. these high values on the right side could be explained by the right hand and right chewing side dominance. in addition, bech et al.34 (2017) found that individuals with neck/shoulder pain compared with healthy subjects presented lower values in the mvc of scapula elevation, even after the data were adjusted for sex, which would confirm less capacity to generate mvc in muscles with pain or disability. however, when comparing the methodologies and resources used for the assessment with the current study, many differences stand out, such as self-assessment of pain and measurement of strength, in addition, of course, to the fact that the symptomatic group did not perform the tmd assessment34. it is well known that individuals with tmd present higher ndi scores than control groups14. according to the current literature, the prevalence of self-reported cervical pain ranges from 75 to 87.8% in individuals with tmd, and on average they present an ndi score 7.9 points greater than asymptomatic individuals3. this was partially found in the present study (tmdg: 8.59±3.82, cg: 5.78±3.37), however, although the tmd presented higher values than the control, no significant difference was found between the groups, and the scores of both groups can be classified as mild disability. although this fact may influence cervical dysfunction, the focus of the study was the difference in strength between tmdg and cg individuals. 10 santos et al. braz j oral sci. 2022;21:e228356 the fai has been used as a complementary tool in the evaluation of tmd, due to its simplicity and the fact that it does not require specific pre-training for use in research, clinical practice, and in characterizing the severity of tmd. according to the study by berni et al.27 (2015) in the fai accuracy assessment, a cut-off point was found, which determines the absence or presence of tmd, with a score between 0 and 45 and 50 to 100, respectively. this fact was demonstrated by the results of the fai in the present study (tmdg: 71.76±11.71, cg: 36.94±22.89), with a statistically significant difference. the contributions of this study are related to daily clinical practice and the belief that regions of dysfunction or muscles with local pain have less capacity to generate isometric strength. this fact reaffirms the importance of individualized assessment, independent of the diagnosis, to treat not only the pathology but also its causes and compensations. this study proved to be innovative in the method of evaluation used and the methodological care taken, considering the muscle group evaluation, and the use of valid and reliable tools. in addition, a sample calculation was performed. the limitations include the small sample size, age range of the participants, absence of analysis of antagonist muscle strength, which could serve as a parameter for comparison. other criteria for evaluating the cervical spine, such as range of motion, could provide further evidence of the relationship between the structures evaluated, especially regarding the functionality of both structures. both groups presented a mild disability score on the ndi, which could interfere in the findings of the present study. finally, it is known that young adult women can easily use adaptation and compensation mechanisms in presence of mild tmd, which can interfere with the results. it is suggested that future studies using emg evaluation be performed, as this tool could provide clarifications about the specific behavior of each muscle (scapula elevator, upper trapezius) and its antagonists (cervical flexors and sternocleidomastoid). it is also suggested that future studies evaluate a larger sample and men volunteers. in conclusion, it was not possible to confirm the hypothesis that women diagnosed with tmd present lower isometric strength (mean and maximum) during scapular elevation (right /left). the results of the other comparisons and analyses between the groups also did not show statistically significant results, except for the results of the fai. acknowledgments the authors would like to thank all the subjects who participated in the study. this study was financed in part by the coordination of superior level staff improvement brazil (capes) finance code 001. data availability datasets related to this article will be available upon request to the corresponding author. 11 santos et al. braz j oral sci. 2022;21:e228356 lúcio ferreira dos santos: data curation, data analysis, writing; fabiana foltran mescollotto: conceptualization, methodology, data curation, data analysis, writing; ester moreira de castro carletti: methodology, writingreviewing and editing; elisa bizetti pelai: methodology, writingreviewing and editing; marcio de moraes: writing reviewing and editing; delaine rodrigues bigaton: conceptualization, methodology, writingreviewing and editing; adriana pertille: writingreviewing and 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trapezius pain sensitivity is not associated with increased tissue hardness. j pain. 2010 may;11(5):491-9. doi: 10.1016/j.jpain.2009.09.017. 33. kim ha, hwang uj, jung sh, ahn sh, kim jh, kwon oy. comparison of shoulder strength in males with and without myofascial trigger points in the upper trapezius. clin biomech (bristol, avon). 2017 nov;49:134-8. doi: 10.1016/j.clinbiomech.2017.09.001. 34. bech kt, larsen cm, sjøgaard g, holtermann a, taylor jl, søgaard k. voluntary activation of the trapezius muscle in cases with neck/shoulder pain compared to healthy controls. j electromyogr kinesiol. 2017 oct;36:56-64. doi: 10.1016/j.jelekin.2017.07.006. 1 volume 22 2023 e237471 original article braz j oral sci. 2023;22:e237471http://dx.doi.org/10.20396/bjos.v22i00.8667471 1 post-graduate program in dentistry, federal university of pelotas, pelotas, rs, brazil. 2 faculty of dentistry, federal university of pelotas, rs, brazil. corresponding author: marcos britto correa 457, gonçalves chaves st. room 506, pelotas rs brazil zip code 96015-560 pelotas tel: +55 53 98115-5031 e-mail: marcos.britto@ufpel.edu.br editor: dr. altair a. del bel cury received: april 11, 2021 accepted: february 12, 2022 covid-19 pandemic and impact on dental education: perception of brazilian dental students clarissa de aguiar dias1 , sarah arangurem karam1 , catarina borges da fonseca cumerlato1 , letícia regina morello sartori1 , matheus dos santos fernandez2 , nathália ribeiro jorge da silva1 , camilla hubner bielavski2 , alexandre emidio ribeiro silva1 , eduardo dickie de castilhos1 , marcos britto correa1,* aim: the aim of the present study is to assess the perception of undergraduate students on the impact of the covid-19 pandemic on brazilian dental education. methods: this crosssectional study was conducted using an online questionnaire hosted in google forms platform and publicized on instagram® and facebook®. the questionnaire was available between july 8-27, 2020. absolute and relative frequencies were obtained for variables of interest using pearson’s chi-squared and considering 95% confidence intervals. prevalence of learning resource variables according to self-reported skin color, educational institution, and brazilian region were presented using equiplots. results: a total of 1,050 undergraduate dental students answered the questionnaire. most students reported being in full-distance learning mode. among the undergraduate students, 65.4% reported perceiving a very high impact in dental education, and 16.6% of students reported not being able to follow distance learning. in addition, 14% reported not having a personal laptop or desktop to study with, with this condition being more prevalent among non-white than white individuals. conclusion: we conclude that brazilian dental students perceived a high impact of covid-19 on dental education, as well as one-sixth of the students reported not having adequate resources to continue with distance learning. it is important that different policies are developed at the institutional and governmental levels to reduce the impact of the pandemic on dental education. keywords: education, dental. students, dental. dentistry. covid-19. pandemics. mailto:marcos.britto@ufpel.edu.br https://orcid.org/0000-0002-6556-0282 https://orcid.org/0000-0002-3921-0182 https://orcid.org/0000-0001-5943-6958 https://orcid.org/0000-0002-1082-0534 https://orcid.org/0000-0001-7781-3083 https://orcid.org/0000-0002-2691-3970 https://orcid.org/0000-0002-2928-0582 https://orcid.org/0000-0001-6402-0789 https://orcid.org/0000-0002-7072-6558 https://orcid.org/0000-0002-1797-3541 2 dias et al. braz j oral sci. 2023;22:e237471 introduction coronavirus disease 2019 (covid-19) emerged in december 2019 in wuhan, china, as pneumonia cases of an unknown cause1. after drawing the attention of international authorities and the world health organization (who), there were an increasing number of cases in different countries. then in march 2020, the who determined that the world was experiencing a pandemic2. more than 10 million cases and 244,737 deaths by covid-19 were recorded in brazil between february 2020 (first case reported of covid-19) and february 20213. this scenario led the country to occupy the third place in the ranking of countries with the most confirmed cases of covid-19, being considered one of the epicenters of the pandemic3. many countries in the world, including brazil, experienced episodes of exponential growth in the number of cases, hospitalizations and overload of health systems3 between march and december 2020 due to the high infection rate, enhanced by transmission via oral and nasal secretion mainly in direct contact4,5, and the absence of a vaccine and/or specific treatment against the sars-cov-2 virus for most of world’s population. thus, different measures of individual and collective protection against contagion were instituted, such as social distancing, hygiene of hands, objects and surfaces and the use of masks in order to mitigate the infection spreading in different populations1,4,6. in this context, one of the fields most susceptible to exposure to the virus is dentistry, increasing the risk of contagion for professionals and patients5,7. most dental procedures require proximity to the patient, in addition to the fact that aerosols and droplets are present during various dental procedures5,8. moreover, brazil is one of the countries with the largest number of professionals in the dental field, around 350,0009, and the covid-19 pandemic introduced important impacts for public and private dentists in the country10. there were 412 public and private dental schools in brazil until 2019, totaling approximately 100,000 students9. in addition to the impacts reported by professionals, dental education has been facing several other challenges in this period11. an undergraduate dental clinic requires professors to supervise students and the model followed in most of institutions is a dental clinic with simultaneous assistance12. moreover, the need for provisional closure of clinical activities in educational institutions tends to interfere in dental training, mainly due to the need for practical activities12. therefore, the possibility of distance learning resources can partially supply the pedagogical need. however, internet access is not yet a reality for all brazilian students13, which can be a barrier to e-learning, in addition to depending on the type of interactive learning of teachers and also on the relationship of students with technology14. to the best of our knowledge, no study has assessed the impact of the covid-19 pandemic in the brazilian dental education. thus, the aim of the present study is to assess the impact of the covid-19 pandemic on brazilian dental education through the perception of undergraduate students. methods this study was reported following surge (the survey reposting guideline)15 and the strobe (strengthening the reporting of observational studies in epidemiology) statement16 for cross-sectional studies. 3 dias et al. braz j oral sci. 2023;22:e237471 study design this study protocol was approved by research ethics committee of the faculty of medicine federal university of pelotas (#4121205/2020). this cross-sectional study was conducted using an online questionnaire developed and pre-tested with questions that aimed to evaluate the impact of the covid-19 pandemic on brazilian dental education17. questionnaire development and pre-testing the self-administered questionnaire used in this study was developed based on previous findings in the literature considering adversities faced by dental professionals and students related to access to technology and previous experience of distance learning10,12. after ethical approval, the questionnaire was hosted online on the google forms platform and submitted to a pre-test carried out with 12 graduate dental students in order to assess the clarity, writing, organization and internal consistency of questions. students were instructed to answer the questionnaire, record the time to complete, and then fill in a clarity scale for each question from 1 (not clear) to 5 (very clear)10. after the evaluation, 6 questions with a rating ≤3 were discussed among the researchers and were edited later. the questions together obtained an average clarity score of 4.78 (0.49). in addition, the mean response time to complete the questionnaire was 12 (2.2) minutes. the present study did not aim to validate the questionnaire used in this study. questionnaire content the questionnaire should be accessed from the link in the invitation to participate in the study. the questionnaire was introduced on the first page, where the participant was informed about the study objective, the average response time and contact of the researchers involved. the participants had to read the informed consent term and agree to participate in the study by selection of an affirmative answer “yes” in order to access the following pages and questionnaire sections. the final questionnaire was composed of 41 mandatory and closed-ended questions in five sections. the sections covered the following themes: student characteristics (n=10); dental education during the covid-19 pandemic (n=17); knowledge about covid-19 (n=4); biosafety and dental care (n=7); alcohol consumption and psychological symptoms (n=3). the options “i’d rather not say” and “does not apply” were available on all questions. setting, participant recruitment and survey administration participants were recruited through online resources without assuming a convenience sample17. the final questionnaire hosted on the google forms platform was available on the instagram® social network (facebook, menlo park, ca) together with images and informative texts about the study content, eligible participants, average response time and ethical approval. the link which hosted the questionnaire was made available in instagram in the @ensino.odonto_covid19 bio, a professional profile specifically created for the study. in addition, the researchers involved acted directly in sharing content of the research profile and content and inviting professors and students at dental schools around the country to share the research link with their colleagues through feed and stories posts in their personal profiles on instagram®, twitter® and 4 dias et al. braz j oral sci. 2023;22:e237471 facebook®. responses were collected between july 8-27, 2020 and the project’s profile had a maximum of 1,389 followers17. in addition to the social network approach, e-mails with information and a link to access the survey were sent to 250 coordinating departments of brazilian dental schools across the country17. of these, 121 institutions responded to the e-mail (48.4% response rate)17. participants and sample size participants were undergraduate dental students in public or private brazilian dental schools. considering ~100.000 undergraduate students in public and private dental schools in brazil9, we estimated that 500 responses would be necessary to obtain a 95% confidence interval, admitting 30% losses and unknown prevalence (50%). variables considering the students’ characteristics section, the variables included gender (female or male), self-reported skin color (white vs non-white brown, black, yellow and indigenous), residence area (rural or urban). age was collected in years and categorized into ranges of 17-19 years, 20-25 years, 26-31 years, 32 years or more. additionally, the variable brazilian regions was obtained from the categorization of brazilian states following the classification of the brazilian institute of geography and statistics ibge (north, northeast, midwest, southeast and south)18. variables associated with the context of dental education during the covid-19 pandemic collected were the type of institution (public, private or other), undergraduate stage (semester) categorized as initial stage (up to the fourth semester), intermediate stage (fifth to seventh semester) and final stage (eighth to tenth semester), and the learning methodology used in the dental school during the pandemic (“full distance learning”, “partial distance learning”, “no activities”). the students self-reported impact on dental education was collected through the question “how do you consider the impact of covid-19 pandemic on dental education?”, with answers ranging in a five-point scale from 0 “no impact” to 4 “very high impact”. additionally, data were collected regarding the students’ access to infrastructure to follow the classes including “adequate resources for distance learning”, “internet access”, “smartphone”, “laptop”, “desktop”, all dichotomized as “yes” or “no” questions. moreover, when a computer was available, the number of persons sharing the computer was collected (“none”, “one”, “two”, “three or more”). data analysis the data were imported from the google forms platform in spreadsheet format to microsoft excel software. all analysis was performed in the stata 15.0 software program (statacorp. collegestation, tx, usa). absolute and relative frequencies were obtained for variables of interest using pearson’s chi-squared test with 5% significance level and 95% confidence intervals. the options “i’d rather not say” and “does not apply” were treated as missing data. in addition, prevalence of learning resource variables according to self-reported skin color (white and non-white), educational institution (public and private) and brazilian region (south, southeast, midwest, northeast, north) were presented using equiplots (http://www.equidade.org/equiplot). 5 dias et al. braz j oral sci. 2023;22:e237471 results a total of 1,050 undergraduate dental students answered the questionnaire online and were considered part of the final sample. among the participants, most were female (70.5%), aged 20 to 25 years (64.2%) and with self-declared skin color as white (66.2%). a majority of the students reported living in urban area (95.5%) and in the south region of brazil, followed by the northeast and southeast regions (table 1). table 1. descriptive analysis of the sample according to student characteristics (n = 1,050). student characteristics (n) n (%) ci 95% gender (1 048) female 739 (70.5) 67.7-73.2 male 309 (29.5) 26.8-32.3 age (1 050) 17 – 19 years 150 (14.3) 12.3-16.5 20 – 25 years 674 (64.2) 61.2-67.0 26 – 31 years 162 (15.4) 13.4-17.7 32 years or more 64 (6.1) 4.8-7.7 skin color (1 046) white 682 (65.2) 62.3-68.0 brown 243 (23.2) 20.8-25.9 black 106 (10.1) 8.4-12.1 yellow 12 (1.2) 0.6-2.0 indigenous 3 (0.3) 0.1-0.9 residence area (1 044) rural 47 (4.5) 3.4-5.9 urban 997 (95.5) 94.1-96.6 brazilian region (1 048) north 51 (4.9) 3.7-6.4 northeast 247 (23.5) 21.1-26.2 midwest 68 (6.5) 5.1-8.2 southeast 222 (21.2) 18.8-23.8 south 460 (43.9) 40.9-46.9 table 2 shows a similar distribution of students between public and private institutions and that the majority of students were at the intermediate stage of their graduate course (41.5%). additionally, considering the learning methodology during the pandemic, most students reported being in full distance learning mode (48.9%). among the undergraduate students, 65.4% reported having perceived a very high impact on their dental education. in table 3 it is possible to observe that 16.6% of students reported not being able to follow distance learning, 19.7% of students reported not having a laptop, and 84.7% not having a desktop. in addition, only 1.3% have no access to internet and 0.4% declared no access to a smartphone. most of those who owned a computer (37.5%) reported not sharing with another person. 6 dias et al. braz j oral sci. 2023;22:e237471 table 2. descriptive analysis of the sample according to dental education characteristics in the pandemic (n = 1,050). dental education and pandemic (n) n (%) ci 95% institution (1 050) public 536 (51.0) 48.0-54.1 private 506 (48.2) 45.2-51.2 other 8 (0.8) 0.4-1.5 undergraduate stage (semester) (1 050) initial stage (1st 4th semester) 333 (31.7) 29.0-34.6 intermediate stage (5th 7th semester) 436 (41.5) 38.6-44.5 final stage (8th 10th semester) 281 (26.8) 24.2-29.5 teaching methodology during the pandemic (1 041) full distance learning 509 (48.9) 45.9-51.9 partial distance learning 255 (24.5) 22.0-27.2 without activities 277 (26.6) 24.0-29.4 impact of the pandemic on dental education (1 041) no impact 7 (0.7) 0.3-1.4 low impact 10 (1.0) 0.5-1.8 intermediate impact 95 (9.1) 7.5-11.0 high impact 248 (23.8) 21.3-26.5 very high impact 681 (65.4) 62.5-68.3 table 3. descriptive analysis of the sample according to students’ resources for distance learning (n = 1,050). students’ resources for distance learning (n) n (%) ci 95% absence of any resources for distance learning (1 050) no 876 (83.4) 81.0-85.6 yes 174 (16.6) 14.4-18.9 internet access (1 050) no 14 (1.3) 0.8-2.2 yes 1036 (98.7) 97.8-99.2 have a smartphone (1 050) no 4 (0.4) 0.1-1.0 yes 1046 (99.6) 99.0-99.9 have a laptop (1 050) no 207 (19.7) 17.4-22.2 yes 843 (80.3) 77.8-82.6 have a desktop (1 050) no 889 (84.7) 82.4-86.7 yes 161 (15.3) 13.3-17.6 people sharing the computer (958) none 359 (37.5) 34.5-40.6 one 175 (18.3) 15.9-20.8 two 254 (26.5) 23.8-29.4 three or more 170 (17.7) 15.4-20.3 7 dias et al. braz j oral sci. 2023;22:e237471 data on two variables about adequate resources for distance learning and computers (laptop or desktop) are presented by brazilian region, educational institution, and self-declared skin color in figures 1 and 2. among students, 14% reported not having a personal laptop or desktop to study, being more prevalent among non-whites than whites (18.1% vs 11.9%, respectively, p<0.001) (figure 2). in addition, it is possible to observe a gap in the prevalence of absence of personal computer to study between regions, varying from 10.5% and 12.1% in northeast and southeast to 19.1% and 21.6% in midwest and north regions, respectively. an absence of resources for distance learning was more frequently reported by participants classified as non-white (19.0%), studying in private institutions (18.0%) and living in midwest brazilian region (19.0%); however, no statistical significance was observed (p > 0.05) (figure 1). brazilian region white/private/south non white/public/southeast midwest northeast north educational institution skin color 0 10 20 30 40 50 60 70 80 prevalence (%) abscence of adequate students’ resources for distance learning by brazilian region, educational institution and skin color figure 1. equiplot of prevalence about abscence of adequate student resources for distance learning by brazilian region, eucational institution and skin color (n = 1,050). brazilian region white/private/south non white/public/southeast midwest northeast north educational institution skin color 0 10 20 30 40 50 60 70 80 prevalence (%) abscence of personal notebook/desktop to study by brazilian region, educational institution and skin color figure 2. equiplot of prevalence about abscence of personal laptop/desktop to study by brazilian region, educational institution and skin color (n = 1,050). 8 dias et al. braz j oral sci. 2023;22:e237471 discussion the results obtained by this study are a source of concern, mainly because dental students reported a high impact from the covid-19 pandemic. additionally, our findings shed some light on the fact that a significant number of students present important limitations to follow distance learning, with inequalities in distribution. about one sixth of students reported not having adequate resources for distance learning, and an important part reported being in full or partial learning distance mode. among dental students, 19.7% reported not having a laptop, and more than 80% not having a desktop; of those who have these resources, more than 60% share with at least one person. additionally, not having a laptop or desktop to study was significantly more prevalent among students who self-declared non-white, which comprises racial and ethnic minorities in brazil, such as blacks, browns and indigenous. although some dental schools had already adopted the system of e-learning before the pandemic19, most of essential courses had to adhere to the remote emergency education (ree) modality to meet the pedagogical needs required after the covid-19 pandemic and social distancing measures. medical schools in brazil similarly also suspended face-to-face theoretical and undergraduate activity practices13. in the present study, most students were only studying by distance learning methods, totally (48.9%) or partially (24.5%), as well as most institutions worldwide12,20-23. the ree may have contributed to the fact that majority of dental students reported a high impact of the covid-19 pandemic on their education. still, many institutions completely suspended their activities at times during 2020, and the impact for these students is assumed to be even greater. as dentistry is a very practical course, the absence of clinics and laboratories directly affect student performance and clinical skills development12,20,21. this is one of the greatest challenges for dental education because although some face-to-face classroom lectures were replaced by e-learning, distance methods cannot replace clinical practice12,24. the great impact on activities reported by students in this study may also be due to feelings arising from students such as insecurity, fear, anguish, and stress, as well as a difficulty in virtual learning by some undergraduate students25. a study carried out through an online questionnaire applied in april 2020 at a private dental school in the northeast region in brazil observed one of the most affected domains of dental student quality of life was the psychological domain26. other studies carried out in different regions and countries, such as north america12,27, indonesia20 jordan21 and new zealand22 using online surveys had similar results, with undergraduate dental students reporting fear from covid-19, anxiety and depressive symptoms due to social distancing situations, family economic issues and return to face-to-face activities. the context of the covid-19 pandemic in a middle-income country, routine changes promoted by social distancing, socioeconomic and family environment factors may intensify students’ stress17,24. the covid-19 pandemic is disproportionately affecting the most vulnerable strata in different societies, especially in lowand middle-income countries, which already have important inequalities in living conditions, health and education, such as brazil28. as found in the present study, although most also have the necessary resources, a 9 dias et al. braz j oral sci. 2023;22:e237471 significant portion of students (16.6%) still do not have resources for distance learning classes. according to data from the national continuous household survey, in 2018, almost 46 million of the included brazilian households do not have an internet connection29. internet access for learning is not yet a reality for all students13. it is therefore necessary to consider students who do not have sufficient resources for distance learning classes and who are in a situation of social vulnerability28. differences in the access to a personal computer was also found considering brazilian regions, where it was observed that students from the north and midwest regions presented less access to a desktop or laptop to study. it is also worth mentioning that, although it is possible to watch classes by a mobile device, the presence of a computer is absolutely necessary to perform e-learning activities. north and midwest regions are less economically developed in brazil compared with the southeast30. thus, differences between regions can be explained by socioeconomic differences, calling attention to the need for digital inclusion actions mostly directed at these vulnerable regions. emergency e-learning maintains inequalities and provides a window into the colonial process of social exclusion of the ethnicity and racial minority population in brazil31,32. a statistically significant difference was found in the present study, showing inequality in the access to a personal computer between whites and blacks/browns. in addition, it is observed that most respondents in this study are white, even though the majority of brazilians self-declared black or brown. racial inequalities in different outcomes are well described in the literature and occurs as a result of structural racism32. structural racism is an oppressive manifestation through interconnected systems, with racial discrimination occurring from the space of the individual, employment, residential location, education, health care, justice, reflecting on the history, culture and institutions present in our society33. this historical racial discrimination generates a marginalized population, with worse housing conditions, lower income and fewer opportunities for professional advancement, and in turn makes it impossible or delays access of blacks/browns to more modern technologies in relation to whites33. consequently, whites are more likely to finish high school than blacks/browns and thus they are also more likely to enter and complete an undergraduate course, as there is racial disparity in access, training and resources available in different schools, corroborating the majority of university students interviewed who declared themselves whites34. in view of the reduction of these inequalities, brazilian universities went through a huge expansion at the end of 2000s and at the beginning of the last decade. as part of this expansion, racial quotas were implemented in public institutions, allowing black/brown people an increase in possibilities to access all careers, including those with more prestige in society, where dentistry can be included35. federal universities increased policies directed to offer home, transport and food for this group to guarantee the permanence of students in situations of vulnerability35. at this moment, it is also necessary that digital inclusion actions should be promoted, allowing all students to have adequate conditions to follow on-line courses. a survey which studied digital inclusion in the poorest sectors of the population found that while the percentage of white people with a computer exceeds the average (9.0%), and that of mixed race is equal to it, the black population has a level equivalent to half the average36. brazilian 10 dias et al. braz j oral sci. 2023;22:e237471 data showed that only 55% of black people used computers at least once in their lives, demonstrating the differences in access to technology in the country29. this study presents some limitations that should be discussed. although the sampling method is not representative, the dental community is very present on social networks, where more than 1000 students answered the questionnaire, alleviating this limitation. in addition, with limitation, we can point out that the sample was not from the region of the country where most dentistry courses are concentrated, which may have impacted the representativeness and generalization of our results for the country. by the online application method, attendance by the participant is prevented when the participant does not understand a question, there is exclusion of people who do not usually use social networks or do not have access to the internet, in addition to not knowing the circumstances in which the questionnaire was answered. thus, the results regarding access to devices for e-learning could be overestimated since students with difficulty to access the internet had less chance to answer the questionnaire. as a strength, the use of the social networking tool is a good option in times of social isolation where contact between people has been avoided, as well as being important to emphasize the educational inequalities that occur due to the pandemic. moreover, knowledge of the dental education context in the covid-19 era is necessary in order to understand the limitations and losses caused by the period and to outline strategies to qualify and reduce the impacts, mainly in a middle-income country. the covid-19 pandemic caused many challenges related to education to arise, both for educational institutions and for students and teachers. however, distance learning resources can meet the orientation of social distancing and at the same time preserve the connection and support to academics13. however, it is necessary that the distance learning discipline has some criteria to be attended, such as organization, planning, technological availability to meet the needs of the students, demands and training of teachers. furthermore, the need to maintain healthy students, teachers and staff with the presence of individual protection equipment, knowledge about the disease and adequacy of work spaces and routines in a context of theoretical and practical activities is highlighted23. public policies aimed at democratizing higher distance education should not only take into account the increase in the number of courses and vacancies, but also variables that impact the access, permanence and conclusion of the course of these students, such as individual characteristics and regional inequalities in the country35. providing classes without these adjustments puts the teaching proposal in a safe and correct way at risk in the current pandemic scenario of covid-19. in this study, we concluded that brazilian dental students reported a high impact of covid-19 on their dental education, and one sixth of the students reported not having adequate resources to continue with distance learning. in addition, self-declared nonwhite students reported more than whites not having a laptop or computer, which is an important obstacle in being able to follow class activities. the social inequalities that already exist among students are accentuated in a pandemic moment, and thus it is important that different policies are developed at institutional and governmental levels to reduce the impact of the pandemic on dental education, providing assistance to students to guarantee internet access and availability of electronic devices. 11 dias et al. braz j oral sci. 2023;22:e237471 acknowledgment the authors gratefully acknowledge all brazilian undergraduate dental students who participated in this study and professors and other collaborators who helped disseminate the research on their social networks and institutions. this study was partly supported by capes/brazil (coordenação de aperfeiçoamento de pessoal de nível superior/brasil) – finance code 001. declaration of conflicting interests the authors report that there are no direct or indirect conflicts of interest involving associations with commercial entities that supported the work reported in the submitted manuscript, associations with commercial entities that might have an interest in the submitted manuscript, financial associations involving family members and other relevant non-financial associations. data availability datasets related to this article will be available upon request to the corresponding author. author contribution dias ca contributed to conception, design and drafted the manuscript. sartori lrm drafted the manuscript. karam sa and cumerlato cbf analysis and interpretation of the data. fernandez ms, silva nrj and bielavski ch participated in the elaboration of the questionnaire and data collection. silva aer, castilhos ed and correa mb contributed to critically revised the manuscript. all authors gave final approval and agreed to be accountable for all aspects of the work. references 1. zhu n, zhang d, wang w, li x, yang b, song j, et al. a novel coronavirus from patients with pneumonia in china, 2019. n engl j med. 2020 feb;382(8):727-33. doi: 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[digital divide: conceptual issues, empirical evidence and public policy]. novos est cebrap. 2005;(72):101-17. portuguese. doi: 10.1590/s0101-33002005000200006. 1 volume 22 2023 e231303 original article braz j oral sci. 2023;22:e231303http://dx.doi.org/10.20396/bjos.v22i00.8671303 1 department of restorative dentistry, faculty of dentistry, çanakkale onsekiz mart university, çanakkale, turkey. 2 department of endodontics, faculty of dentistry, çanakkale onsekiz mart university, çanakkale, turkey. 3 department of dentomaxillofacial radiology, faculty of dentistry, ankara university, ankara, turkey. 4 department of dentomaxillofacial radiology, faculty of dentistry, eskişehir osmangazi university, eskişehir, turkey. 5 department of pharmaceutical microbiology, faculty of pharmacy, düzce university, düzce, turkey. 6 department of endodontics, faculty of dentistry, bahçeşehir university, i̇stanbul, turkey. corresponding author: tuğba misilli department of restorative dentistry, faculty of dentistry, çanakkale onsekiz mart university, 17100, çanakkale, turkey. phone: +90 505 9392100 e-mail: dt.tugbay@outlook.com editor: dr. altair a. del bel cury received: october 23, 2022 accepted: january 17, 2023 investigation of the effect of indirect pulp capping materials on dentin mineral density tuğba misilli1* , gülşah uslu2 , kaan orhan3 , i̇brahim şevki bayrakdar4 , demet erdönmez5 , taha özyürek6 aim: to evaluate the potential of inducing mineral density changes of indirect pulp capping materials applied to demineralized dentin. methods: a total of 50 cavities were prepared, 5 in each tooth, in extracted ten molars without caries, impacted or semi-embedded. the cavities were scanned by microcomputed tomography (µ-ct) after creating artificial caries by microcosm method (pre-treatment). each cavity was subjected to one of 5 different experimental conditions: control (dental wax), conventional glass ionomer cement (fuji ix gp extra), resin-modified calcium silicate (theracal lc), resin-modified calcium hydroxide (ultra-blend plus), mta (mm-mta) and the samples were kept under intrapulpal pressure using simulated body fluid for 45 days. then, the second µ-ct scan was performed (post-treatment), and the change in dentin mineral density was calculated. afterward, elemental mapping was performed on the dentinal surfaces adjacent to the pulp capping agents of 5 randomly selected samples using energy dispersive x-ray spectroscopy (eds) apparatus attached to a scanning electron microscope (sem). the ca/p ratio by weight was calculated. friedman test and wilcoxon signed ranks test were used to analyze the data. results: there was a significant increase in mineral density values of demineralized dentin after treatment for all material groups (p<0.05). resin-modified calcium silicate had similar efficacy to mta and conventional glass ionomer cement, but was superior to resin-modified calcium hydroxide in increasing the mineral density values of demineralized dentin. conclusions: demineralized dentin tissue that is still repairable can be effectively preserved using materials with remineralization capability. keywords: calcium hydroxide. calcium compounds. silicates. spectrometry, x-ray emission. glass ionomer cement. x-ray microtomography. https://orcid.org/0000-0003-0019-4872 https://orcid.org/0000-0003-3176-1251 https://orcid.org/0000-0001-6768-0176 https://orcid.org/0000-0001-5036-9867 https://orcid.org/0000-0002-5331-2206 https://orcid.org/0000-0003-3299-3361 2 misilli et al. braz j oral sci. 2023;22:e231303 introduction the main theme of contemporary dentistry is the development of minimally invasive biological-based treatments aimed at preserving pulp vitality. a vital pulp is of great importance in keeping the tooth in the mouth, as it provides nutrition and defense of the tooth and acts as a biosensor detecting pathogenic stimuli1. treatment of caries lesions includes removal of infected dentin and coating of the affected dentin tissue with a bioactive material with remineralization capacity to preserve pulp vitality2,3. the repair concept of demineralized dentin aims to promote the renucleation of mineral crystals into the hydroxyapatite structure4. bioactive materials used for this purpose consist of calcium silicate, calcium hydroxide or hydroxyapatite-based materials, and glass ionomer cement. calcium hydroxide is a material with a long history of clinical success, considered the “gold standard” among pulp capping agents5. in recent years, calcium silicate-containing materials such as mineral trioxide aggregates (mta) have attracted considerable attention. however, calcium hydroxide and calcium silicate show poor physical properties, such as high solubility and gradual resorption. by adding resin monomers to these materials, physical properties such as light polymerization and low solubility in water have been improved, so the use of resin-modified versions in clinical routine has become widespread6. however, this situation raises the concern that the moisture diffusion required for the ion release, which stimulates dentin formation, may be prevented, especially in calcium silicate-based materials3. in investigating the bioactive potential of these materials, non-destructive laboratory techniques that examine the mineral changes in dentin come to the fore. many recent studies have proven that microcomputed tomography (µ-ct) can be successfully used to evaluate the mineralization dynamics of enamel and dentin tissues7,8. however, as a common result of previous studies evaluating the remineralization capacity of dentin, it can be said that the changes in mineral density vary due to the use of different experimental designs, such as the ph cycle or biofilm model, in simulating the caries formation process2,9. on the other hand, while µ-ct analysis does not provide information about the mineral content, energy dispersive x-ray spectroscopy (eds) can be used to determine the percentages and distributions of elements on the adjacent dentin surface to which the material is applied3. therefore, this study aimed to evaluate the effect of pulp capping agents with different contents, such as resin-modified calcium hydroxide/calcium silicate, mta, and conventional glass ionomer cement on dentin tissue demineralized with an artificial caries model using human bacterial inoculum by µ-ct and eds analysis. the first null hypothesis of the study was that, regardless of the mechanism of action of bioactive materials, when applied to demineralized dentin tissue, there would be no difference in mineral density between preand post-treatment. the second null hypothesis was that there would be no difference between the bioactive materials in terms of mineral density increase. materials and methods sample preparation for mineral density analysis (µ-ct imaging), sample size calculation was performed based on a previous study by pires et al.9 (2018), with α = 0.01, power (1-β) = 0.95, effect 3 misilli et al. braz j oral sci. 2023;22:e231303 size = 1.719, using g*power software (version 3.1, heinrich-heine-universität düsseldorf, düsseldorf, germany). the resulting total sample size required was n = 10. in the present study, ten non-carious, fully or partially erupted third molars, extracted for clinical reasons, belonging to individuals over the age of 18 were used under the approval of çanakkale onsekiz mart university clinical research ethics committee (2011-kaek-27/2019-e.1900029346). written informed consent was obtained from all individual participants included in the study. all procedures performed in research involving human biological material were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. the teeth were kept in a 0.1% thymol solution at 4°c for a period of 3 months, until they were used in the experimental phase. a flat dentin surface was obtained by removing the occlusal enamel of the samples, the apical two-thirds of the root was removed to reveal the pulp canal space. a total of 50 cavities were obtained by preparing five cavities with a depth of at least half the bur diameter (± 1mm) on the occlusal dentin surface of each sample using a diamond round bur. biofilm formation with a microcosm model samples were fixed on polystyrene tissue-culture plates with wax all around to ensure that the formed biofilm only came into contact with the occlusal surface. plates were sterilized under uv light for 40 minutes before microbial inoculum. the artificial caries model used in the study was developed by pires et al.9 (2018) and was carried out in the same way. for this, after obtaining written informed consent from 3 volunteers who met the inclusion criteria, 1 ml of saliva sample was collected, mixed with 1 ml of synthetically prepared sterile saliva in a sterile tube and homogenized by vortex. a total of 9 x 107 cfu ml-1 microorganisms were counted in this suspension. in this microorganism community, 2.3 x 104 cfu ml-1 of streptococcus mutans, 3.7 x 105 cfu ml-1 of lactobacilus ssp. and 2.9 x 102 cfu ml-1 of candida ssp. were determined in selective culture medium (mitis salivarius bacitracin, candida chromagar) by conventional analysis method. 20 µl of homogeneous culture medium was seeded into each cavity on the tooth surface and incubated in a brain-heart infusion (bhi) containing 10% glucose. it was incubated at 37°c for 7 days under microaerophilic conditions for biofilm formation to occur. bhi and glucose were replenished every 24 hours (1000 µl per cavity). all procedures were performed in a laminar air flow chamber in an aseptic environment. biofilm-forming tooth samples were washed three times with phosphate-buffered saline solution after the medium was removed. µ-ct examination after the biofilm growth was obtained on the tooth surfaces, the culture medium was removed, and the samples were scanned with µ-ct after clearing the biofilm by sonication, and initial images were obtained. a high-resolution, desktop µ-ct system (bruker skyscan 1275, kontich, belgium) was used to scan the samples. the scanning parameters were: 100 kvp, 100 ma, 0.5 mm al/cu filter, 10.2 µm pixel size, rotation in 0.5 steps. air calibration of the detector was performed before each scan to minimize the rate of ring artifact. each sample was rotated 360° in an integration 4 misilli et al. braz j oral sci. 2023;22:e231303 time of 5 minutes. average scan time was around 2 hours. other settings included beam hardening correction and entry of optimum contrast limits according to manufacturer’s instructions based on pre-scanning and reconstructing teeth. each tooth was scanned twice before and after the application of the experimental procedure, ensuring standardization with the same scanning parameters. experimental procedure with bioactive cements for indirect pulp capping, the most commonly used current materials in the clinical routine of today’s dentistry were selected. in all tooth samples, capping materials were placed in the prepared cavities according to the manufacturer’s instructions. the central cavity (no. 1) was filled with dental wax as the control group. the properties of the materials are shown in table 1. then, the samples were kept in simulated body fluid (sbf) with a ph of 7.4, where the intrapulpal pressure was simulated for 45 days at room temperature, in a 100% humidity environment. table 1. pulp-capping materials used in the study cavity no. material type commercial name compositions manufacturer c1 dental wax (control) cavex set up regular paraffin waxes, micro-crystalline paraffin waxes, pigment cavex c2 glass ionomer cement (gic) fuji ix gp extra polyacrylic acid, fluoro-aluminosilicate glass, distilled water gc c3 resin-modified calcium silicate (rmcs) theracal lc portland cement (calcium silicates), fumed silica, bis-gma, polyglycol dimethacrylate bisco c4 resin-modified calcium hydroxide (rmch) ultra-blend plus calcium hydroxide, tricalcium phosphate, diurethane, dimethacrylate, tegdma ultradent c5 mineral trioxide aggregate (mta) mm mta tricalcium silicate, dicalcium silicate, tricalcium aluminate, bismuth oxide, calcium sulphate dehydrate, magnesium oxide micromega mineral density analysis the modified algorithm described by feldkamp et al.10 (1989) was used to obtain axial, two-dimensional (2d), 1000×1000 pixel images for the visualization and quantitative measurements of the samples10. ring artifact correction and smoothing for reconstruction parameters were fixed to zero and beam artifact correction was set to 40%. contrast limits were applied following skyscan’s instructions. using the nrecon software (version 1.6.10.4, skyscan, kontich, belgium), the images acquired by the scanner were reconstructed to show 2d slices. reconstructed images were further processed with skyscan ctvox (version 3.3.0, skyscan, kontich, belgium) for visualization. skyscan ctan software (version 1.17.7.2, skyscan, kontich, belgium) provides an integrated calibration of datasets for these two density scales: hounsfield unit (hu) and mineral density (md). for this purpose, conical md phantom (rods) with two different mineral concentrations of 0.25 and 0.75 g cm-3 were used for appropri5 misilli et al. braz j oral sci. 2023;22:e231303 ate calibration phantom scans and measurements. thus, to aid calibrations, samples placed and scanned with md phantom sticks were placed in an identical tube for the calibration scan, allowing the density value to be calibrated independently for each scan. after scanning, the grayscale values were converted to mineral density values with a linear calibration curve based on the grayscale values obtained from two different mineral concentration conical phantoms of 0.25 and 0.75 g cm-3. pixel size, rotation step, frame average, voltage, filter, etc. parameters were kept constant for all scans. a global thresholding was applied to separate dentin from other structures using ctan software. to analyze dentin mineral density in 3d volumes, the original grayscale images were processed with a gaussian low-pass filter for noise reduction and an automatic segmentation threshold was used. a thresholding (binary) process was used, which required processing of the gray level range to obtain the image of only black/white pixels. next, a region of interest was selected for each slice individually to analyze the mineral density (fig. 1). figure 1. processed images of preand post-treatment longitudinal sections of a representative sample. calculations of the difference in mineral loss (δz: ghap cm-3) of each sample were made by calculating the md values as an average loss of the entire demineralized area and the interior of the demineralized area (fig. 2). mineral loss from the sample was defined after subtracting the demineralized md values from the baseline phantom rod md values to correct for misalignment. also, a mineral density threshold (1.2 g cm-3) for dentin caries was used as the cut-off point for carious and intact dentin, as previously described11. the remineralization potential was measured as the percent change in mineral density values of demineralized dentin before and after the experimental procedures. 6 misilli et al. braz j oral sci. 2023;22:e231303 a b c d figure 2. preand post-treatment 2d representation of dentin demineralization areas of a representative sample. elemental composition analysis materials in the cavities of 5 randomly selected samples were removed under a dental microscope (zumax oms2360, surgical microscope, suzhou, china) with the help of a slow speed micromotor and excavator, and cleaned of cement residues in an ultrasonic bath (sonorex, bandelin, germany) for 10 minutes. then, the samples were dehydrated in ethyl alcohol with 70% concentration for 30 minutes and at 95% concentration for 1 hour. then, the elemental distribution was mapped using energy dispersive x-ray spectroscopy (x-max 80, oxford instruments, uk) on the images obtained by scanning electron microscopy (sem) (jsm-7001f, jeol, tokyo, japan) from the dentin surfaces adjacent to the capping materials of each cavity, including the entire cavity area. also, calcium and phosphorous contents (percentages by weight) were converted into ca/p ratio for each material group. statistical analysis statistical package program (spss 19, ibm-spss inc, chicago, il) was used for data analysis. shapiro-wilk test was used to determine whether the data was normally distributed. since the data were obtained from dependent samples, the friedman test followed by the wilcoxon signed ranks test was used to analyze the differences between groups. wilcoxon signed ranks test was used to analyze preand post-treatment differences of the same sample. the results were considered significant for p<0.05. results the values of mineral density in intact and demineralized dentin and the percentage of decrease in mineral density in the cavity floor before treatment are shown in table 2. no statistically significant difference was found within the intact and demineralized dentin tissues in terms of mineral density (p>0.05). also, there was no significant difference between the groups in terms of the percentage of decrease in mineral density after demineralization (p>0.05). 7 misilli et al. braz j oral sci. 2023;22:e231303 table 2. mean (sd) mineral density values (g cm-3) of intact and demineralized dentin and decrease in mineral density (%) at the cavity floor cavity no. n intact dentine (mean ± sd) demineralized dentine (mean ± sd) decrease in mineral density (%) c1 (control) 10 2.22 ± 0.07a 0.17 ± 0.02a 88.82 ± 4.07a c2 10 2.29 ± 0.06a 0.18 ± 0.02a 91.82 ± 1.65a c3 10 2.28 ± 0.08a 0.18 ± 0.03a 89.56 ± 2.33a c4 10 2.29 ± 0.05a 0.18 ± 0.03a 88.87 ± 2.55a c5 10 2.32 ± 0.07a 0.19 ± 0.04a 89.33 ± 2.28a different superscripts indicate statistical differences between groups in the same column. p<0.05 table 3 shows the mean mineral density values for the entire volume of demineralized dentin for each group before and after treatment. a significant increase was observed in the mineral density values after treatment in all cement groups, except for the control group in which dental wax was applied. when the groups were compared among themselves, the group with the highest increase after treatment was rmcs, followed by the gic and mta groups. table 3. mean (sd) mineral density values (g cm-3) for the entire volume of demineralized dentin before and after treatment cavity no. material group n pre-treatment (mean ± sd) post-treatment (mean ± sd) effect size c1 control 10 0.26 ± 0.11a,a 0.26 ± 0.10a,a c2 gic 10 0.20 ± 0.06a,a 0.35 ± 0.06b,ab 2.5 c3 rmcs 10 0.16 ± 0.07a,a 0.42 ± 0.08b,b 3.44 c4 rmch 10 0.20 ± 0.05a,a 0.33 ± 0.02b,a 2.98 c5 mta 10 0.15 ± 0.06a,a 0.34 ± 0.03b,ab 3.66 different capital letters indicate preand post-treatment statistical differences in the same row. different lowercase letters indicate statistical differences between groups in the same column. p<0.05 eds mapping images of the dentin surfaces adjacent to the post-treatment material of a representative sample are shown in figures 3a-e, and the ca and p weight percentages and ca/p ratios of the groups are listed in table 4. the analysis revealed that the different capping materials made no statistically significant difference in the ca/p ratio, with a relatively high ratio in the mta group (p>0.05). table 4. mean (sd) of elemental levels (wt%) for eds analysis of post-treatment dentin surface (friedman’s test) groups elements (wt %) ca p ca/p control 12.28 ± 1.78 11.46 ± 0.81 1.07 ± 0.11 gic 10.04 ± 2.58 10.32 ± 0.48 0.97 ± 0.22 rmcs 11.03 ± 5.51 9.86 ± 2.52 1.06 ± 0.28 rmch 8.05 ± 2.25 8.19 ± 1.35 0.97 ± 0.13 mta 5.8 ± 1.96 3.67 ± 0.75 1.55 ± 0.28 p value 0.308 8 misilli et al. braz j oral sci. 2023;22:e231303 a b c d e figure 3. representative eds mapping images of the dentin surfaces adjacent to the test materials (a) control (b) gic (c) rmcs (d) rmch (e) mta. 9 misilli et al. braz j oral sci. 2023;22:e231303 discussion for endodontic and restorative materials, bioactivity is explained by the ability to induce specific and deliberate mineral loading to the dentin substrate. these materials are expected to release significant amounts of ions to enable specific biomineralization in the clinical setting4. this study evaluated the effectiveness of different bioactive materials in restoring the mineral structure of demineralized dentin tissue using the indirect pulp capping technique by µ-ct and eds analysis. regardless of the bioactive material used, the first null hypothesis was rejected because a significant increase in mineral density was observed in the demineralized dentin tissue after treatment compared with before. the second null hypothesis was rejected because there was a difference between the materials in terms of the change in mineral density. recently, µ-ct has been widely used as a three-dimensional analytical technique in studies of changes in mineral density of dental hard tissues2,7,8,12. the method allows three-dimensional visualization of the internal structure of an object, analyzing the entire mass and obtaining volumetric results. microradiography, which is used to evaluate the remineralization capacity of dental hard tissues, is a non-destructive method, unlike microhardness and traditional microscopic methods13. therefore, in this study, the µ-ct method was used to evaluate the samples before and after the experimental procedures. the µ-ct evaluation after demineralization showed that the mineral density values of the cavities were below the threshold value determined for dentin caries (1.2 g cm-3 of hydroxyapatite). this indicated that biofilm formation with the microcosm model could lead to mineral loss on the dentin surface, similar to natural caries lesions. in the artificial caries model called microcosm, it is possible to mimic an actual dental biofilm, which is a complex structure, more accurately in vitro, thanks to the biofilm model created by the saliva pool14. thus, in contrast to methods that only deal with the physicochemical aspects of demineralization and cannot simulate the primary factors of the natural process, such as collagen degradation, saliva, and biofilm, in vivo caries lesion progression can be more accurately mimicked15. using this model, it can be said that the decrease in the mineral density of the intact dentin tissue was very high in all the cavity samples in the present study. similar results were obtained in dentin demineralization with this model used in previous studies by pires et al.9,15 (2018). dentin is naturally moist, and induction of hydroxyapatite formation occurs as a result of the reaction of calcium ions released from the material with phosphates in the dentinal fluid5,16. for this reason, intrapulpal pressure was simulated in this study, and the materials were contacted with simulated body fluid, a metastable solution supersaturated with apatite and containing ions, mainly calcium and phosphate ions, in concentrations similar to human blood plasma. the components of the simulated body fluid were prepared as described by kokubo and takadama17 (2006). the purpose of simulating intrapulpal pressure was to reproduce what occurs in the oral environment when these materials come into contact with dentin, thus obtaining results closer to a clinical scenario. a constant positive pressure value of approximately 15 cm h2o reported in previous studies was used to simulate normal 10 misilli et al. braz j oral sci. 2023;22:e231303 pulp pressure in vital teeth18. the experimental setup was prepared as described by scheffel et al.19 (2014). in addition, cavities were prepared on the dentin surface of the same tooth for five different materials to be tested so that the dentin substrate structure had similar mineralization properties. all indirect pulp capping materials significantly increased the mineral density values of demineralized dentin after the experimental conditions. among the materials, the most significant increase was observed in the resin-modified calcium silicate group (theracal lc), while the least effective material group was resin-modified calcium hydroxide (ultrablend plus). besides the superior physical properties of mta/calcium silicate-based materials, such as better sealing ability and biocompatibility than calcium hydroxide, their mechanism of action is similar. calcium hydroxide is released as a by-product of the hydration reaction of mta/ calcium silicates with water6. in this study, resin-added versions were tested for both material types, which were developed to improve their weak physical properties and increase bond strength by facilitating immediate permanent restoration (thanks to their light-curing feature)5. a previous study reported that the presence of a resin matrix in resin-modified calcium silicate cement (theracal lc) restricts moisture diffusion into the material so that hydration cannot be completed20. it has been thought that this situation may alter calcium ion kinetics and result in a lower remineralization potential with a reduced ability to release calcium ions5. in contrast, in this study, the increase in mineral density in the resin-modified calcium silicate group was similar to that in the mta group, while it was higher than that in the resin-modified calcium hydroxide group. this can be explained by the differences in the resin content of the calcium hydroxide and calcium silicate material groups in which the resin-modified forms were investigated. the resin-modified calcium hydroxide (ultra-blend plus) used in the study contains a hydrophobic monomer/polymer matrix, while the resin-modified calcium silicate (theracal lc) contains a hydrophilic monomer/polymer matrix. the hydrophilic matrix preserves its ability to stimulate dentin formation by allowing higher calcium and hydroxide release21,22. another material group tested in the study is glass ionomer cement. when freshly mixed glass ionomer cement is placed on the wet dentin surface, an ion exchange interaction occurs between the material and the dentin substrate. the release of fluoride and calcium/strontium caused by the dissolution of the fluoro-aluminosilicate glass particles in these cements promotes the balance to be reversed in favor of apatite formation by ion supplementation in demineralized tissues, and the process is considered bioactive4,23. in the glass ionomer cement (fuji ix gp) tested in this study, strontium was added instead of calcium due to its radiopaque properties23. however, it has been reported that this change does not have any effect on the remineralization ability of the cement24. the findings of the present study showed that the tested glass ionomer cement caused a similar increase in mineral density as the resin-modified calcium silicate and mta groups. this is in line with the results of previous µ-ct studies exhibiting that glass ionomer cement has similar bioactive potential to calcium silicate-based materials2,9. 11 misilli et al. braz j oral sci. 2023;22:e231303 chemical analysis can be used to determine the composition and distribution of elements on the sample surface. with eds analysis, it is possible to detect the elemental composition and obtain semi-quantitative data from the sem images of the sample surface25. the weight-based ca/p ratio determines the amount of hydroxyapatite mineralization26. considering the ca/p ratios obtained as a result of the eds analysis of the dentin surfaces on which the capping material was placed in this study, it can be said that the tested materials did not induce any significant difference in the apatite structures. although the mta group had lower ca and p levels, it showed a higher ca/p ratio, which was not statistically significant. this result can be explained by the relative decrease in the proportions of the investigated minerals (ca and p) since different minerals are released depending on the material placed on the cavity. the higher ca/p ratio of the mta group in this study was in line with a previous study reporting that calcium silicate-based material compositions immersed in simulated body fluid for 6 weeks showed a higher ca/p ratio3. on the other hand, the lower ca/p ratio in the resin-modified calcium silicate group, another calcium silicate-based material group, may be due to the lower solubility of this material due to the shorter curing time resulting from its resin content. as a limitation of the current study, pure material properties were evaluated independently of stem cells and biochemical and hormonal stimuli that have proven to be effective in their activity in previous studies27,28. further investigation that also considers the activation mechanisms of these cells is needed to assess the role of hybrid, composite, or mineral molecules in inducing controlled cell differentiation. in conclusion, within the limitations of this study, it can be concluded that the resin-modified calcium silicate material has similar potential to mta and conventional glass ionomer cement, but higher potential than resin-modified calcium hydroxide cement to induce mineral density changes as an indirect pulp capping agent. acknowledgments the work was supported by çanakkale onsekiz mart university the scientific research coordination unit (project number [tsa-2019-2954]). data availability datasets related to this article will be available to the corresponding author upon request. author contribution all authors contributed to the study’s conception and design. material preparation, data collection, and analysis were performed by [tuğba misilli], [gülşah uslu], [kaan orhan], [demet erdönmez]. the first draft of the manuscript was written by [tuğba misilli], [demet erdönmez], [i̇brahim şevki bayrakdar] and all authors commented on previous versions of the manuscript. all authors read and approved the final manuscript. 12 misilli et al. braz j oral sci. 2023;22:e231303 references 1. zhang w, yelick pc. vital pulp therapy-current progress of dental pulp regeneration and revascularization. int j dent. 2010;2010:856087. doi: 10.1155/2010/856087. 2. neves ab, bergstrom tg, fonseca-gonçalves a, dos santos tmp, lopes rt, de almeida neves a. mineral density changes in bovine carious dentin after treatment with bioactive dental cements: a comparative micro-ct study. clin oral invest. 2019;23(4):1865-70. doi: 10.1007/s00784-018-2644-2. 3. sadoon ny, fathy sm, osman mf. effect of using biomimetic analogs on dentin remineralization with bioactive cements. braz dent j. 2020;31(1):44-51. doi: 10.1590/0103-6440202003083. 4. vallittu pk, boccaccini ar, hupa l, watts dc. bioactive dental materials-do they exist and what does bioactivity mean? dent mater. 2018;34(5):693-4. doi: 10.1016/j.dental.2018.03.001. 5. kunert m, lukomska-szymanska m. bio-inductive materials in direct and indirect pulp capping—a review article. materials. 2020;13(5):1204. doi: 10.3390/ma13051204. 6. chen l, suh bi. cytotoxicity and biocompatibility of resin-free and resin-modified direct pulp capping materials: a state-of-the-art review. dent mater j. 2017;36(1):1-7. doi: 10.4012/dmj.2016-107. 7. kucuk eb, malkoc s, demir a. microcomputed tomography evaluation of white spot lesion remineralization with various procedures. am j orthod dentofac orthop. 2016;150(3):483-90. doi: 10.1016/j.ajodo.2016.02.026. 8. gomes mn, rodrigues fp, silikas n, francci ce. micro-ct and fe-sem enamel analyses of calcium-based agent application after bleaching. clin oral invest. 2018;22(2):961-70. doi: 10.1007/s00784-017-2175-2. 9. pires pm, santos tp, fonseca‐gonçalves a, pithon mm, lopes rt, neves aa. mineral density in carious dentine after treatment with calcium silicates and polyacrylic acid based cements. int endod j. 2018;51(11):1292-300. doi: 10.1111/iej.12941. 10. feldkamp la, goldstein sa, parfitt am, jesion g, kleerekoper m. the direct examination of three‐dimensional bone architecture in vitro by computed tomography. j bone miner res. 1989;4:3-11. doi: 10.1002/jbmr.5650040103. 11. neves aa, coutinho e, vivan-cardoso m, jaecques s, van meerbeek b. micro-ct based quantitative evaluation of caries-excavation. dent mater. 2010;26(6):579-88. doi: 10.1016/j.dental.2010.01.012. 12. zan kw, nakamura k, hamba h, sadr a, nikaid t, tagami j. micro‐computed tomography assessment of root dentin around fluoride‐releasing restorations after demineralization/ remineralization. eur j oral sci. 2018;126(5):390-9. doi: 10.1111/eos.12558. 13. swain mv, xue j. state of the art of micro-ct applications in dental research. int j oral sci. 2009;1(4):177-88. doi: 10.4248/ijos09031. 14. santos dmsd, pires jg, braga as, salomão pma, magalhães ac. comparison between static and semi-dynamic models for microcosm biofilm formation on dentin. j appl oral sci. 2019;27:e20180163. doi: 10.1590/1678-7757-2018-0163. 15. pires pm, dos santos tp, fonseca-gonçalves a, pithon mm, lopes r, de almeida neves a. a dual energy micro-ct methodology for visualization and quantification of biofilm formation and dentin demineralization. arch oral biol. 2018;85:10-15. doi: 10.1016/j.archoralbio.2017.09.034. 16. darvell bw, wu rc. “mta”an hydraulic silicate cement: review update and setting reaction. dent mater. 2011;27:407-22. doi: 10.1016/j.dental.2011.02.001. 17. kokubo t, takadama h. how useful is sbf in predicting in vivo bone bioactivity? biomaterials. 2006;27(15):2907-15. doi: 10.1016/j.biomaterials.2006.01.017. https://doi.org/10.1155/2010/856087 https://doi.org/10.1016/j.dental.2018.03.001 https://doi.org/10.3390/ma13051204 https://doi.org/10.4012/dmj.2016-107 https://doi.org/10.1016/j.ajodo.2016.02.026 13 misilli et al. braz j oral sci. 2023;22:e231303 18. alamoudi nm, baik am, el-housseiny aa, haimed tsa, bakry as. influence of povidone-iodine on micro-tensile bonding strength to dentin under simulated pulpal pressure. bmc oral health. 2018;18(1):1-7. doi: 10.1186/s12903-018-0645-9. 19. scheffel dl, estrela rp, pires pm, mariusso mr, costa ca, hebling j. effect of time between adhesive application and photoactivation on adhesion and collagen exposure. am j dent. 2014;27(6):330-4. 20. camilleri j, laurent p, about i. hydration of biodentine, theracal lc, and a prototype tricalcium silicate–based dentin replacement material after pulp capping in entire tooth cultures. j endod. 2014;40:1846-54. doi: 10.1016/j.joen.2014.06.018. 21. chen l, shen h, suh bi. bioactive dental restorative materials: a review. am j dent. 2013;26:219-27. 22. gandolfi mg, siboni f, prati c. chemical-physical properties of theracal, a novel light-curable mta-like material for pulp capping. int endod j. 2012;45:571-9. doi: 10.1111/j.1365-2591.2012.02013.x. 23. watson tf, atmeh ar, sajini s, cook rj, festy f. present and future of glass-ionomers and calcium-silicate cements as bioactive materials in dentistry: biophotonics-based interfacial analyses in health and disease. dent mater. 2014;30(1):50-61. doi: 10.1016/j.dental.2013.08.202. 24. ngo hc, mount g, mc intyre j, tuisuva j, von doussa rj. chemical exchange between glass-ionomer restorations and residual carious dentine in permanent molars: an in vivo study. j dent. 2006;34(8):608-13. doi: 10.1016/j.jdent.2005.12.012. 25. coceska e, gjorgievska e, coleman nj, gabric d, slipper ij, stevanovic m, et al. enamel alteration following tooth bleaching and remineralization. j microsc. 2016;262(3):232-44. doi: 10.1111/jmi.12357. 26. velo mmdac, farha alh, da silva santos ps, shiota a, sansavino sz, souza at, et al. radiotherapy alters the composition, structural and mechanical properties of root dentin in vitro. clin oral invest. 2018;22(8):2871-8. doi: 10.1007/s00784-018-2373-6. 27. bhandi s, alkahtani a, reda r, mashyakhy m, boreak n, maganur pc, et al. parathyroid hormone secretion and receptor expression determine the age-related degree of osteogenic differentiation in dental pulp stem cells. j pers med. 2021;11(5):349. doi: 10.3390/jpm11050349. 28. bhandi s, alkahtani a, mashyakhy m, abumelha as, albar nhm, renugalakshmi a, et al. effect of ascorbic acid on differentiation, secretome and stemness of stem cells from human exfoliated deciduous tooth (sheds). j pers med. 2021;11(7):589. doi: 10.3390/jpm11070589. https://doi.org/10.1016/j.jdent.2005.12.012 https://doi.org/10.1007/s00784-018-2373-6 https://doi.org/10.3390/jpm11050349 1 volume 22 2023 e230171 original article braz j oral sci. 2023;22:e230171http://dx.doi.org/10.20396/bjos.v22i00.8670171 1 department of science, college of education, university of technology and applied sciences, rustaq, sultanate of oman. 2 faculty of dentistry, the asoulaldeen university college, baghdad, iraq. 3 faculty of dentistry, university of babylon, babel, iraq. 4 school of medicine, international medical university, kuala lumpur, malaysia. 5 apellis pharmaceuticals, waltham, ma, usa. 6 department of restorative dentistry, faculty of dentistry, universiti malaya, kuala lumpur, malaysia. corresponding author: mazen m jamil university of technology and applied sciences, rustaq college of education, science department, biology unit. 329 sultanate of oman. mazen.alobaidi@utas.edu.om editor: dr. altair a. del bel cury received: june 19, 2022 accepted: september 23, 2022 antibiotic prescription in the management of endodontic infections amongst iraqi final-year undergraduate dental students mazen m jamil al-obaidi1,2* , elaf ahmed hadi2 , zeyad nazar al-talib3 , aqil m daher4 , mohammed al-adhamy5 , hany mohamed aly ahmed6 aim: this study aimed to examine the prescription of antibiotics for endodontic infections among undergraduate dental students. methods: two government iraqi dental schools [(the university of baghdad (uob) (n=99) and university of babylon (ub) (n=70)], and one private dental school [osouldeen university college (ouc) (n=103)] were included in this survey study. a paper-based questionnaire composed of seven questions was distributed to students, and collected. a chi-square test was used for data analysis, and the level of significance was set at 0.05 (p=0.05). results: a statistically significant difference (p<0.05) was identified between students’ answers in the three dental schools regarding antibiotic selection for endodontic infections in which patients had no known allergies (p=0.001). in comparison to other dental schools, a statistically significantly higher proportion of respondents from uob (32%) favored azithromycin 500mg for treating patients with penicillin hypersensitivity (p=0.003). a high percentage of participants (62.1%) selected antibiotic prescription in cases with necrotic pulp and symptomatic apical periodontitis (with swelling and moderate/severe preoperative symptoms). however, there were no significant differences between the 3 dental schools (p>0.05). conclusion: in conclusion, a significantly greater percentage of ub chose amoxicillin for the treatment of endodontic infection in patients with no medical allergies. azithromycin 500mg was selected by uob as the preferred option in patients who were sensitive to penicillin. our findings support the need for the implementation of strategies to raise awareness of good antibiotic prescribing practices among dentists in iraq. keywords: anti-bacterial agents. students, dental. endodontics. iraq. https://orcid.org/0000-0001-5950-4498 https://orcid.org/0000-0002-1284-5516 https://orcid.org/0000-0002-4310-3304 https://orcid.org/0000-0002-3504-4340 https://orcid.org/0000-0003-2021-747x https://orcid.org/0000-0003-0776-9288 2 al-obaidi et al. braz j oral sci. 2023;22:e230171 introduction antimicrobial resistance (ar) is one of the most significant threats to global public health1. approximately, 400,000 infections and 25000 deaths were recorded as a result of persistent multidrug-resistant bacteria in europe2. consequently, by 2050, it is suspected that 10 million people will be killed annually if no action is taken against drug-resistant infections2. gram-positive and gram-negative facultative anaerobes are the main reasons for endodontic infections3. systemic antibiotics are usually prescribed for patients with endodontic infections as a prevention measure against the development of apical abscesses and to inhibit subsequent progression and persistence of infections4. this will lead to the increasing prescription of broad‐spectrum antibiotics even in cases where antibiotics are not indicated, such as symptomatic irreversible pulpitis, necrotic pulps, and localized acute abscesses4. however, literature shows that immunocompromised patients with congenital immuno-deficiencies are encouraged to take antibiotics as a compensation for their compromised immune systems that cannot withstand the spread of microbial infections4. the ar of microorganisms may develop due to three main factors, including improper dosage, prolonged antibiotic treatment, and unnecessary antibiotic prescription5. according to the guidelines of the american association of endodontics (aae), systemic indication of antibiotic prescription is only appropriate when there is a spread of infection characterized by fever, swelling, cellulitis, and lymphadenopathy6. unfortunately, most general dental practitioners (gdps) have shallow knowledge of prescription patterns to treat endodontic infections7. the majority of gdps prescribe about 10% of all common antibiotics for endodontic infection treatment8, which is considered very prevalent nowadays9. in recent literature, it has been noticed that some inappropriate prescriptions are undertaken by gdps due to a lack of knowledge, social factors or traditional beliefs10. therefore, undergraduate as well as postgraduate dental students should be equipped with sufficient knowledge to follow the prescription guidelines for systemic antibiotics in endodontic infections and frequent survey studies should be undertaken to evaluate current practices and ways to improve in instances of inappropriate prescription profiles. up to date, there is no congruous data for antibiotic prescription in endodontic infections amongst iraqi-final year undergraduate dental students. this study aimed to determine the knowledge of final-year undergraduate dental students in three iraqi dental schools in terms of prescribing antibiotics for patients with endodontic infections. materials and methods ethical approval the study was approved by the medical ethics committee, school of dentistry, university of baghdad (reference: ref247mec). 3 al-obaidi et al. braz j oral sci. 2023;22:e230171 preparation of the questionnaire a one-page paper-based questionnaire that was adopted from previous studies11,12 has been distributed. the questionnaire is composed of 7 questions (figure 1). three questions were related to the demographic information of the participants, such as age, gender, and dental school name. two questions were related to the type of antibiotic used in the treatment of endodontic infections in an adult patient without and with medical allergy. one question was related to the duration of an antibiotic prescription. another last question was related to the antibiotic indication for the following clinical situations: 1. irreversible pulpitis; moderate to severe preoperative symptoms (case 1). 2. irreversible pulpitis with symptomatic apical periodontitis; moderate to severe preoperative symptoms (case 2). 3. necrotic pulp with asymptomatic apical periodontitis; no swelling, no/mild preoperative symptoms (case 3). 4. necrotic pulp with symptomatic apical periodontitis; no swelling; moderate/severe preoperative symptoms (case 4). 5. necrotic pulp with asymptomatic apical periodontitis; sinus tract present; no/mild preoperative symptoms (case 5). 6. necrotic pulp with symptomatic apical periodontitis; swelling present; moderate/ severe preoperative symptoms (case 6). when the questionnaire was posted on the website, it was stated that this questionnaire is utilized for research. the dental schools involved in the survey the survey was undertaken at two government dental schools [university of baghdad (uob), university of babylon (ub)], and one private dental school [osouldeen university college (ouc)] from january 2019 to june 2019. a total of 446 students were invited to participate in the survey. based on sample size calculation (https://www.surveymonkey.com/mp/sample-size-calculator/), a total of 211 participants are needed to participate in the survey (95% confidence interval and 5% margin of error). two hundred and seventy-two participants contributed to this study (uob =99, ub =70, and ouc =103). the response rate to the survey was 61% from all three dental schools. distribution and collection of the survey final year undergraduate dental students from those three iraqi dental schools were invited to fill out a one-page paper-based questionnaire that was adopted from previous studies (figure 1)11,12. the questionnaire was distributed by a representative from each dental school. students were allowed to decline participation in the survey, and those that agreed were assured anonymity. 4 al-obaidi et al. braz j oral sci. 2023;22:e230171 age: gender: male female university: 1. which is the first type of antibiotic used in the treatment of endodontic infections in an adult patient without medical allergy? (please encircle your answer) • amoxicillin 250 mg 500 mg • amoxicillin + clavulanic acid (augmentin®) 625 mg 825 mg 1000 mg • clindamycin 150 mg 300 mg • metronidazole (flagyl®) 250 mg 500 mg • azithromycin (zithromax®) 250 mg 500 mg 1000 mg • cephalexin (keflix®) 250 mg 500 mg • others: 2. if the patient has a sensitivity to pencilline, which type of the antibiotic used in the treatment of endodontic infections • clindamycin 150 mg 300 mg • azithromycin 250 mg 500 mg 1000 mg • erythromycin 500 mg • metronidazole 250 mg 500 mg • others: 3. determine the duration of antibiotic prescription: _______ days 4. the antibiotics are indicated in which of the following situations? (indicate your choice by √) • irreversible pulpitis; moderate to severe preoperative symptoms. (case 1) • irreversible pulpitis with symptomatic apical periodontitis; moderate to severe preoperative symptoms. (case 2) • necrotic pulp with asymptomatic apical periodontitis; no swelling, no/mild preoperative symptoms. (case 3) • necrotic pulp with symptomatic apical periodontitis; no swelling, moderate/severe preoperative symptoms. (case 4) • necrotic pulp with asymptomatic apical periodontitis; sinus tract present, no/mild preoperative symptoms. (case 5) • necrotic pulp with symptomatic apical periodontitis; swelling present, moderate/severe preoperative symptoms. (case 6) figure 1. questionnaire used for the survey in this study statistical analysis spss version 24 was used to analyse the data. categorical variables were described in terms of frequency and percentage. the difference in proportions explained by selected explanatory variables was tested with the chi-square test. the level of significance was set at 0.05 (p=0.05). questionnaire internal consistency was assessed with cronbach’s alpha, which was between 0.7 and 0.81. 5 al-obaidi et al. braz j oral sci. 2023;22:e230171 results socio-demographic data of the study samples table 1 shows the gender distribution among the three dental schools. two hundred and seventy-two participants were involved in this study (uob =99 out of 186, ub =70 out of 116, and ouc =103 out of 143) with a response rate of 61%. about two thirds of the participants were female, while one-third were male with a mean age of 22 years old. the highest number of participants were from ouc (37.9%), followed by uob (36.4%), followed by ub (25.7%). table 1. socio-demographic data for the present study among 3 dental schools. variable n percent (%) gender male 97 35.7 female 175 64.3 dental schools oub 99 36.4 ub 70 25.7 ouc 103 37.9 analysis of endodontic infection treatment for patients with no medical allergies figure 2 shows the drug of choice that was selected by students to treat adult patients with endodontic infections with no known allergies. the majority of the students selected amoxicillin 500 mg (32.7%) and amoxicillin + clavulanic acid 625 mg (32.4%) as the first choice of endodontic infection treatment. amoxicillin 250 mg was listed as a second choice, in which was selected by (18.8%) of the participants. the other types of antibiotics included in the survey were selected by fewer participants, as shown same figure. table 2 illustrates the comparison between the genders and dental schools in terms of antibiotic selection for endodontic infections in an adult patient without medical allergy. table 2 showed that there was a significant difference (p value=0.001) between the responses of participants in the three dental schools regarding the choice of antibiotics for endodontic infections for patients without medical allergies, with ub (44.3%) coming in first, followed by the participants from ouc (40.8%), and finally the participants from uob (16.2 percent). 6 al-obaidi et al. braz j oral sci. 2023;22:e230171 clindamycin 150 mg cephalexin 500 mg cephalexin 250 mg azithromycin 500 mg azithromycin 250 mg metronidazole 500 mg clindamycin 300 mg amoxicillin+clavulanic acid 1000 mg amoxicillin+clavulanic acid 825 mg amoxicillin+clavulanic acid 625 mg amoxicillin 500 mg amoxicillin 250 mg 0 10 20 30 40 % figure 2. descriptive analysis of antibiotics used in the treatment of endodontic infections in adult patients without medical allergy. table 2. comparisons between the genders and dental schools in terms of antibiotic selection for endodontic infections in adult patients without medical allergy. question 1 p-valueanswer 1 answer 2 n % n % gender male 73 75.3 24 24.7 0.037 female 110 62.9 65 37.1 dental schools uob 83 83.8 16 16.2 0.001*ub 39 55.7 31 44.3 ouc 61 59.2 42 40.8 answer 1:different type of antibiotics. answer 2:amoxicilline 500mg. * p value for comparison of row percentage. endodontic infection treatment analysis for penicillin sensitive patients figure 3 shows the selection of the most appropriate penicillin replacement for patients that are allergic to penicillin. the majority of participants chose azithromycin 500 mg (33.1%), while clindamycin 300 mg (22.8%) and erythromycin 500 mg (21.7%) were listed as second choice. according to table 3, several antibiotic classes were recommended by students to patients who had penicillin allergies, where uob had a significant higher percentage (32.3%) who selected azithromycin 500mg compared with the other 2 dental schools, as shown in table 3. 7 al-obaidi et al. braz j oral sci. 2023;22:e230171 others clindamycin 150 mg cephalexin 500 mg cephalexin 250 mg erythromycin 500 mg azithromycin 1000 mg azithromycin 500 mg azithromycin 250 mg metronidazole 500 mg metronidazole 250 mg clindamycin 300 mg clindamycin 150 mg 0 10 20 30 40 % figure 3. descriptive analysis of antibiotic used in the treatment of endodontic infections in adult patients allergic to penicillin. table 3. comparison between the genders and dental schools in terms of antibiotic selection for endodontic infections treatment in adult patients allergic to penicillin. question 2 p-valueanswer 1 answer 2 n % n % gender male 74 76.3 23 23.7 0.072 female 136 77.7 39 22.3 dental schools uob 67 67.7 32 32.3 0.003*ub 63 90 7 10 ouc 80 77.7 23 22.3 answer 1: different type of antibiotic. answer 2:azithromycin 500mg. * p value for comparison of row percentage the suitable duration of antibiotic prescription upon endodontic infection figure 4 shows the appropriate duration of antibiotic prescription selected for endodontic infection. the majority of the participants selected 7 days as the first choice (36.4%), while 25.4% and 23.9% selected 5 and 3 days, respectively. lesser percentages of participants selected other durations for antibiotic usage, as shown in the figure. there was a significant difference between the participants of the three dental schools (p =0.001), where the participants of ub (97.1%) and ouc (92.2%) were significantly higher in terms of selecting 7 days compared to the participants of uob (79.8%), as shown in (table 4). 8 al-obaidi et al. braz j oral sci. 2023;22:e230171 15 14 12 10 7 6 5 4 3 2 1 0 10 20 d ay s 30 40 % figure 4. descriptive analysis of the duration of antibiotic prescription for endodontic infections. table 4. comparison between the genders and dental schools in terms of the duration of antibiotic prescription for endodontic infections. question 3 p-valueanswer 1 answer 2 n % n % gender male 13 13.4 84 86.6 0.352 female 17 9.7 158 90.3 dental schools uob 20 20.2 79 79.8 0.001*ub 2 2.9 68 97.1 ouc 8 7.8 95 92.2 answer 1:different duration for treatment. answer 2:3 to 7 days treatment. * p value for comparison of row percentage. an antibiotic prescription profile in different clinical situations table 5 shows the antibiotic prescription for the six clinical scenarios. in the first clinical case, 28.3% of the participants indicated the use of antibiotics. a high percentage of participants (62.1%) selected antibiotic prescriptions in case 6 in comparison with other clinical cases. table 5. descriptive analysis of the indicated antibiotics in six different clinical situations. question n % q4_1 not indicated 195 71.7 indicated 77 28.3 continue 9 al-obaidi et al. braz j oral sci. 2023;22:e230171 continuation q4_2 not indicated 129 47.4 indicated 143 52.6 q4_3 not indicated 226 83.1 indicated 46 16.9 q4_4 not indicated 179 65.8 indicated 93 34.2 q4_5 not indicated 191 70.2 indicated 81 29.8 q4_6 not indicated 103 37.9 indicated 169 62.1 discussion previous studies conducted in different countries have shown a lack of information and inappropriate antibiotic prescribing forms amongst dentists for endodontic infections7,13. studies have demonstrated that it is essential to modify the antibiotic-prescribing practices of dentists to effectively control endodontic infections14-16. the information of dental students on the use of systemic antibiotics in pulp and periodontal infections should be improved by developing new strategies. therefore, it is essential to identify the level of knowledge possessed by dental students on this particular subject. this is the first report examining iraqi dental students’ awareness of antibiotic usage on endodontic infections. results revealed that the majority of iraqi final-year dental students opted for the appropriate antibiotic for the treatment of endodontic infections, although there were still students that unsuitably specified antibiotics for clinical situations that do not really require a prescription of systemic antibiotics. a total of 272 iraqi final-year undergraduate dental students (97 male and 175 female) from 3 iraqi dental schools located in baghdad and babylon cities participated in this survey, which is comparable to other similar surveys17,18. our results showed that 18.8%, 32.7%, and 32.4% of respondents chose amoxicillin 250 mg, amoxicillin 500 mg, and amoxicillin+clavulanic acid 625 mg, respectively. ub and ouc were significantly higher compared with the participants of uob in terms of amoxicillin prescription. this can be explained by the fact that antibiotic prescriptions at uob are undertaken at the endodontic department where postgraduate dental students receiving the cases have shallow knowledge of the current guidelines for antibiotic prescription in endodontic infections, whereas finalyear undergraduate dental students at ouc and ub are directly supervised by their respective lecturer’s specialist in the field of endodontics. it is worth noting that the dental curriculum at iraqi dental schools shows considerable variations in terms of the teaching profile and materials provided including textbooks and scientific publications in addition to guidelines and protocols followed for endodontic diagnosis and treatment procedures. 10 al-obaidi et al. braz j oral sci. 2023;22:e230171 according to the spanish endodontic society, the first-choice antibiotic for non-allergic patients is amoxicillin (44%) or a combination with clavulanate (42%)19. gpds practising in europe designated amoxicillin as the first antibiotic option for the treatment of endodontic infections7. guzmán-álvarez et al.20 demonstrated that 78.9% of fourth-year undergraduate dental students have chosen amoxicillin as the first-choice drug for the management of odontogenic infections in one dental school in mexico. in contrast, lesser percentages of amoxicillin prescriptions were noticed among finalyear dental students in other regions21. one study showed that the second antibiotic of choice for non-allergic patients to penicillin (30.9%) is a combination of amoxicillin and clavulanic acid22. thus, the first option for endodontic infections is the combination of amoxicillin + clavulanic acid, as an β-lactamase inhibitor23. β-lactam allergic patients were prescribed azithromycin 1000 mg and erythromycin 500 as the first antibiotics of choice24, which is similar to our results, where final year dental students selected the same medication (uob16 %, ub7% and ouc 23%). endodontic specialists usually prefer clindamycin 300 mg as the firstchoice antibiotic for patients who are allergic to penicillin21. erythromycin and clindamycin are the two universally most commonly chosen antibiotics for the management of pulp and periapical infections7. streptococcus viridans and fusobacterium streptococci are commonly associated with odontogenic infections that cannot be treated by erythromycin25. furthermore, erythromycin has a relatively high occurrence of gastrointestinal adverse effects (5-30%)26. this result corresponds with (ese 2018) protocols that mention patients who are allergic to beta-lactam antibiotics may use clindamycin, clarithromycin, or azithromycin as an alternative treatment27. the final year dental students suggested 3-7 days, with 7 days being the most common (89.0%), as a typical duration for the antibiotic therapy, with substantial variances between the dental schools (p >0.05). according to aae, amoxicillin 500 mg (3 times/ day) is the desirable dose for adult patients, where 3 to 7 days are the recommended duration of antibiotic prescription6. clindamycin (300 mg every 6 hours) is the most commonly chosen alternative for patients with a penicillin allergy. one study found that 99% of italian students preferred 3–7 days as the duration for endodontic infection treatment2. it seems that there is confusion amongst dental students about the interval of antibiotic usage. one report has shown that endodontic infections have a fast commencement and short period of time, determining in 3 to 7 days, or less if the cause is cured or removed5. in addition, toxicity and/or allergy, and the risk of developing resistant microorganisms might be reduced with the short duration of therapy of antibiotics. the development of resistant microbial species might occur because of prolonged antibiotic usage or due to an insufficient dosage of antibiotics with narrow coverage of all microbial species22. in this study, the number of students proposing antibiotics for the 6 periapical and pulpal clinical cases confirms that the topic of antibiotic usage in endodontics should be included in the curriculum of dental schools. our results demonstrated that a relatively high percentage of students would prescribe antibiotics in case 1 (28.3%) or case 2 (52.6%). several reports4,28 demonstrated that these two clinical scenarios can be treated without antibiotics as long as patients do not show symp11 al-obaidi et al. braz j oral sci. 2023;22:e230171 toms of systemic involvement. in terms of antibiotic prescription, a low percentage for this clinical situation was found in lithuania29 and belgium30, whilst this percentage was higher in studies carried out in iran (80.6%) and india (71.6%)31,32. as a general rule, pain reduction, percussion pain or the number of analgesic medications taken by patients with untreated irreversible pulpitis does not require antibiotic administration33. results showed that 16.9% of students indicated the use of antibiotics in patients with case 3. a similar percentage (14%) has been reported in one study21, while 31% of spanish oral surgeons used to prescribe antibiotics in similar clinical situations10. previous studies4,28 reported that healthy individuals with this clinical condition do not require antibiotics; root canal treatment is sufficient to resolve the problem. our results also showed that 34.2% of students would recommend antibiotics to a patient with case 4. however, previous studies have described that this clinical situation requires only root canal treatment and in some cases, painkillers4,28. it is obvious that about one third of undergraduate iraqi dental students had an inappropriate conception of antibiotic utilization in endodontic infection, which is relatively higher than survey studies10,19. the presence of asymptomatic apical periodontal diseases associated with the presence of a sinus tract is the 5th clinical case that is ideally indicated for root canal treatment without the use of antibiotics7. about one-third of dental students preferred antibiotic prescriptions in this circumstance, which is considered a high percentage. it seems that students require more attention to differentiate between acute and chronic infections of the periapical tissues. it is well-known that systemic involvement such as fever, malaise, as well as cellulitis and lymphadenitis require antibiotic prescriptions because such cases cannot be solely controlled by the immune system, and if left, may turn into a life-threatening situation34. the 6th case is pulp necrosis, symptomatic apical periodontal disease, swelling, and moderate/severe symptoms. based on this survey, 62.1% of dental students prescribed antibiotics for this case. undoubtedly, the existence of systemic involvement makes it necessary to use antibiotics in addition to endodontic treatment, incision, and drainage7. results of this study showed that a large proportion of final-year undergraduate dental students are not aware of the scientific basis for prescribing antibiotics in endodontic infections. thus, iraqi dental schools are recommended to revise the guidelines for antibiotic prescriptions in the endodontic curriculum to provide students with more information about antibiotics and their proper use in endodontic infections. worldwide, the contents and quality of medical and dental education systems provide limited focus on the principles of antimicrobial stewardship and resistance in terms of knowledge, attitude, and behaviour to medical or dental students35. there is a lack of studies, particularly in the middle east, evaluating the efficacy of an educational syllabus on antibiotic prescribing for dental students36. it is essential to equip dental students with the necessary educational tools for analysing endodontic infections and ways to manage them with the aid of problem-based as well as case-based learning modules18,37. for instance, a 12 al-obaidi et al. braz j oral sci. 2023;22:e230171 program-based module on antibiotic policy dealing with the history of infectious diseases and antibiotic guidelines should be followed, as it has been structured by one of the netherlands universities35. thus, iraqi dental students need to be further educated in controlling the irrational outlooks and demands of patients as they leave dental school and go into a world where less-than-responsible prescribing is the norm. in conclusion, among patients without medical allergies, a significantly higher proportion of ub respondents chose amoxicillin for the treatment of endodontic infection. uob decided that azithromycin 500 mg would be the best treatment for people who have penicillin sensitivity. furthermore, in the first clinical case, 28.3% of the participants indicated the use of antibiotics. therefore, results of the present study confirm the necessity of putting policies in place to increase iraqi dentists’ understanding of appropriate antibiotic prescription techniques. conflict of interest the authors declare no conflict of interest author contribution mmj al-obaidi: concepts, design, literature search, data acquisition, manuscript preparation, manuscript editing, funding ea hadi: concepts, design, literature search, data acquisition, manuscript preparation zn al-talib: design, literature search, data acquisition, manuscript preparation am daher: data analysis, statistical analysis m al-adhami: data acquisition, data analysis, statistical analysis hma ahmed: review and manuscript editing all authors declare that they were actively involved in revising and approving the manuscript’s final form. acknowledgments we acknowledge all dental colleges written in the articles for their permission allowing us to obtain the answers for the dental students. references 1. world health organization. the evolving threat of antimicrobial resistance: options for action. who published; 2014 [cited 2022 mar 10]. available from: https://apps.who.int/iris/handle/10665/44812. 2. salvadori m, audino e, venturi g, garo ml, salgarello s. antibiotic prescribing for endodontic infections: a survey of dental students in italy. int endod j. 2019 sep;52(9):1388-96. doi: 10.1111/iej.13126. 3. siqueira jf, rôças in, silva mg. prevalence and clonal analysis of porphyromonas gingivalis in primary endodontic infections. j endod. 2008 nov;34(11):1332-6. doi: 10.1016/j.joen.2008.08.021. 13 al-obaidi et al. braz j oral sci. 2023;22:e230171 4. segura-egea jj, gould k, şen bh, jonasson p, cotti e, mazzoni a, et al. antibiotics in endodontics: a review. int endod j. 2017 dec;50(12):1169-84. doi: 10.1111/iej.12741. 5. epstein jb, chong s, le d. a survey of antibiotic use in dentistry. j am dent assoc. 2000 nov;131(11):1600-9. doi: 10.14219/jada.archive.2000.0090. 6. aae position statement: aae guidance on the use of systemic antibiotics in endodontics. j endod. 2017 sep;43(9):1409-13. doi: 10.1016/j.joen.2017.08.015. 7. segura-egea jj, martín-gonzález j, jiménez-sánchez m del c, crespo-gallardo i, saúco-márquez jj, velasco-ortega e. worldwide pattern of antibiotic prescription in endodontic infections. int dent j. 2017 aug;67(4):197-205. doi: 10.1111/idj.12287. 8. ajantha gs, hegde v. antibacterial drug resistance and its impact on dentistry. n y state dent j. 2012 jun-jul;78(4):38-41. 9. dutta a, smith-jack f, saunders wp. prevalence of periradicular periodontitis in a scottish subpopulation found on cbct images. int endod j. 2014 sep;47(9):854-63. doi: 10.1111/iej.12228. 10. segura-egea jj, velasco-ortega e, torres-lagares d, velasco-ponferrada mc, monsalve-guil l, llamas-carreras jm. pattern of antibiotic prescription in the management of endodontic infections amongst spanish oral surgeons. int endod j. 2010 apr;43(4):342-50. doi: 10.1111/j.1365-2591.2010.01691.x. 11. whitten bh, gardiner dl, jeansonne bg, lemon rr. current trends in endodontic treatment: report of a national survey. j am dent assoc. 1996 sep;127(9):1333-41. doi: 10.14219/jada.archive.1996.0444. 12. yingling nm, byrne be, hartwell gr. antibiotic use by members of the american association of endodontists in the year 2000: report of a national survey. j endod. 2002 may;28(5):396-404. doi: 10.1097/00004770-200205000-00012. 13. perić m, perković i, romić m, simeon p, matijević j, mehičić gp, et al. the pattern of antibiotic prescribing by dental practitioners in zagreb, croatia. cent eur j public health. 2015 jun;23(2):107-13. doi: 10.21101/cejph.a3981. 14. bansal r, jain a, goyal m, singh t, sood h, malviya hs. antibiotic abuse during endodontic treatment: acontributing factor to antibiotic resistance. j fam med prim care. 2019 nov;8(11):3518-24. doi: 10.4103/jfmpc.jfmpc_768_19. 15. licata f, di gennaro g, cautela v, nobile cga, bianco a. endodontic infections and the extent of antibiotic overprescription among italian dental practitioners. antimicrob agents chemother. 2021 sep;65(10):e0091421. doi: 10.1128/aac.00914-21. 16. b. abraham s, abdulla n, himratul-aznita wh, awad m, samaranayake lp, ahmed hma. antibiotic prescribing practices of dentists for endodontic infections; a cross-sectional study. plos one. 2020 dec;15(12):e0244585. doi: 10.1371/journal.pone.0244585. 17. scaioli g, gualano mr, gili r, masucci s, bert f, siliquini r. antibiotic use: a cross-sectional survey assessing the knowledge, attitudes and practices amongst students of a school of medicine in italy. plos one. 2015 apr;10(4):e0122476. doi: 10.1371/journal.pone.0122476. 18. jain a, gupta d, singh d, garg y, saxena a, chaudhary h, et al. knowledge regarding prescription of drugs among dental students: a descriptive study. j basic clin pharm. 2015 dec;7(1):12-6. doi: 10.4103/0976-0105.170584. 19. rodriguez-núñez a, cisneros-cabello r, velasco-ortega e, llamas-carreras jm, tórres-lagares d, segura-egea jj. antibiotic use by members of the spanish endodontic society. j endod. 2009 sep;35(9):1198-203. doi: 10.1016/j.joen.2009.05.031. 20. guzmán-álvarez r, medeiros m, reyes lagunes li, campos-sepúlveda ae. knowledge of drug prescription in dentistry students. drug healthc patient saf. 2012;4:55-9. doi: 10.2147/dhps.s30984. 14 al-obaidi et al. braz j oral sci. 2023;22:e230171 21. martín-jiménez m, martín-biedma b, lópez-lópez j, alonso-ezpeleta o, velasco-ortega e, jiménez-sánchez mc, et al. dental students’ knowledge regarding the indications for antibiotics in the management of endodontic infections. int endod j. 2018 jan;51(1):118-27. doi: 10.1111/iej.12778. 22. bolfoni mr, pappen fg, pereira-cenci t, jacinto rc. antibiotic prescription for endodontic infections: a survey of brazilian endodontists. int endod j. 2018 feb;51(2):148-56. doi: 10.1111/iej.12823. 23. stein ge, schooley s, tyrrell kl, citron dm, goldstein ejc. human serum activity of telithromycin, azithromycin and amoxicillin/clavulanate against common aerobic and anaerobic respiratory pathogens. int j antimicrob agents. 2007 jan;29(1):39-43. doi: 10.1016/j.ijantimicag.2006.08.041. 24. baumgartner jc, xia t. antibiotic susceptibility of bacteria associated with endodontic abscesses. j endod. 2003 jan;29(1):44-7. doi: 10.1097/00004770-200301000-00012. 25. kuriyama t, williams dw, yanagisawa m, iwahara k, shimizu c, nakagawa k, et al. antimicrobial susceptibility of 800 anaerobic isolates from patients with dentoalveolar infection to 13 oral antibiotics. oral microbiol immunol. 2007 aug;22(4):285-8. doi: 10.1111/j.1399-302x.2007.00365.x. 26. australian medicines handbook 2017. australian medicines handbook; 2017. 27. segura-egea jj, gould k, şen bh, jonasson p, cotti e, mazzoni a, et al. european society of endodontology position statement: the use of antibiotics in endodontics. int endod j. published online 2018 jan;51(1):20-25. doi: 10.1111/iej.12781. 28. agnihotry a, fedorowicz z, van zuuren ej, farman ag, al-langawi jh. antibiotic use for irreversible pulpitis. cochrane database syst rev. 2016 feb;2:cd004969. doi: 10.1002/14651858.cd004969.pub4. 29. skučaitė n, pečiulienė v, manelienė r, mačiulskienė v. antibiotic prescription for the treatment of endodontic pathology: a survey among lithuanian dentists. medicina. 2010;46(12):806. doi: 10.3390/medicina46120113. 30. mainjot a, d’hoore w, vanheusden a, van nieuwenhuysen jp. antibiotic prescribing in dental practice in belgium. int endod j. 2009 dec;42(12):1112-7. doi: 10.1111/j.1365-2591.2009.01642.x. 31. pallasch tj. how to use antibiotics effectively. j calif dent assoc. 1993 feb;21(2):46-50. 32. nabavizadeh mr, sahebi s, nadian i. antibiotic prescription for endodontic treatment: general dentist knowledge + practice in shiraz. iran endod j. 2011 spring;6(2):54-9. doi: 10.22037/iej.v6i2.2091. 33. nagle d, reader a, beck m, weaver j. effect of systemic penicillin on pain in untreated irreversible pulpitis. oral surg oral med oral pathol oral radiol endod. 2000 nov;90(5):636-40. doi: 10.1067/moe.2000.109777. 34. montagner f, jacinto rc, correa signoretti fg, scheffer de mattos v, grecca fs, gomes bp. betalactamic resistance profiles in porphyromonas, prevotella, and parvimonas species isolated from acute endodontic infections. j endod. 2014 mar;40(3):339-44. doi: 10.1016/j.joen.2013.10.037. 35. pulcini c, gyssens ic. how to educate prescribers in antimicrobial stewardship practices. virulence. 2013 feb;4(2):192-202. doi: 10.4161/viru.23706. 36. lee cr, lee jh, kang lw, jeong bc, lee sh. educational effectiveness, target, and content for prudent antibiotic use. biomed res int. 2015;2015:214021. doi: 10.1155/2015/214021. 37. stevens nt, bruen c, boland f, pawlikowska t, fitzpatrick f, humphreys h. is online case-based learning effective in helping undergraduate medical students choose the appropriate antibiotics to treat important infections? 2019 dec;1(3):dlz081. doi: 10.1093/jacamr/dlz081. 1 volume 21 2022 e226585 systematic review braz j oral sci. 2022;21:e226585http://dx.doi.org/10.20396/bjos.v21i00.8666585 1 dentistry faculty, arthur sá earp neto university, petrópolis, rio de janeiro, brazil. 2 department of periodontology, federal university of pelotas, pelotas, rio grande do sul, brazil. 3 graduate program in dentistry, federal university of pelotas, pelotas, rio grande do sul, brazil. corresponding author: francisco wilker mustafa gomes muniz. rua gonçalves chaves, 457 – pelotas, rio grande do sul, brazil. zip code: 96015-560. telephone: +5553991253611. e-mail: wilkermustafa@gmail.com editor: altair a. del bel cury received: august 6, 2021 accepted: january 25, 2022 a systematic review assessing occurrence of medication-related osteonecrosis of the jaw following dental procedures shimelly monteiro de castro lara1, francisco wilker mustafa gomes muniz2,* , ana beatriz caetano gerônimo1, cinthia studzinski dos santos3 , thayanne brasil barbosa calcia1 aim: this study aimed to systematically review existing literature regarding the association between dental procedures—such as tooth extractions and periodontal therapy—and occurrence of medication-related osteonecrosis of the jaw (mronj) in individuals using bone-modifying drugs. methods: search strategies were performed in pubmed, scopus, web of science and cochrane library for a timeframe ending in december 2021. study selection, data extraction and risk of bias were analyzed independently by two researchers. three meta-analyses were performed, estimating the crude risk ratio (rr), the adjusted odds ratio (or) and the adjusted hazard ratio (hr) for the association between tooth extraction and mronj. results: of the 1,654 studies initially retrieved, 17 were ultimately included. the majority of patients with mronj in these studies were female, with a mean age of 64 years. zoledronic acid was the most commonly used drug among patients with mronj, and cancer was the most frequent underlying health condition. regarding the performed meta-analyses, crude and adjusted analyses demonstrated that tooth extraction increased the risk for mronj by 4.28 (95% confidence interval [95%ci]: 1.73–10.58), the or for mronj by 26.94 (95%ci: 4.17–174.17), and the hr for mronj by 9.96 (95%ci: 4.04–24.55). conclusion: it was concluded that performing dental procedures, especially tooth extraction, in patients using bone-modifying drugs increased the risk of mronj occurrence and, therefore, should be avoided. further studies, using adjusted data, are warranted. keywords: bisphosphonate-associated osteonecrosis of the jaw. bone density conservation agents. diphosphonates. osteonecrosis. surgery, oral. https://orcid.org/0000-0002-3945-1752 https://orcid.org/0000-0002-2022-6221 https://orcid.org/0000-0001-7641-2915 2 lara et al. braz j oral sci. 2022;21:e226585 introduction dental treatment currently presents new challenges among professionals due to the increasing prevalence of patients with cancer and other comorbidities, conditions which are frequently treated with bone-modifying drugs such as bisphosphonates and antiresorptive agents1. bisphosphonates are well-tolerated and extensively used as treatment for bone-related diseases, reducing risk of vertebral fractures and bone loss due to steroid-based treatment2. however, in the early 2000’s, an important adverse effect related to these drugs was reported. several patients exhibited necrotic bone in the jaw, often refractory towards surgical debridement, a clinical feature later denominated as bisphosphonate-related osteonecrosis of the jaw (bronj)3-5. in ensuing decades, this condition has also been connected to other drugs such as antiresorptive and angiogenesis-inhibiting agents, including (respectively) denosumab and bevacizumab, despite their different mechanisms of action6,7. recently, the newer terminology of medication-related osteonecrosis of the jaw (mronj) was adopted in order to include these drugs8. the occurrence of mronj seems to be highly variable, running from very rare to common (0.01–1%) depending on multiple factors both drug-related and independent9. clinically, this condition presents as an avascular exposed bone or bone that can be probed through an intraoral or extraoral fistula on the maxilla or mandible8,10. furthermore, an mronj diagnosis is based on current or previous exposure to antiresorptive or antiangiogenic agents with no history of radiotherapy or metastatic jaw disease8. mronj is most often observed in older women, since this group of patients is more likely to be treated with antiresorptives due to conditions such as breast cancer or osteoporosis11. this disease can present different degrees of complexity, and a classification according to staging systems has been proposed; at present, every patient under antiresorptive therapy is considered at risk10. in this sense, mronj presentation can vary from an asymptomatic exposed bone with no further clinical complication to a lesion with extensive bone involvement, extraoral communication and infection10. thus, it is an important oral complication, one for which treatment can be difficult and often ineffective12. its etiology seems to be related to drug dosage, administration route, therapy duration and comorbidities (e.g., diabetes or a smoking habit)13. antiresorptives and/or antiangiogenics usually demand higher doses when prescribed in cancer treatment, which has also been associated with an increased risk8. additionally, use of corticosteroids and/or immunosuppressants may also be related to mronj14. however, there is no consensus about how these factors can influence mronj occurrence12. crucially, oral surgical procedures are frequently identified as mronj precipitating events; therefore, elective procedures are often unadvised8. as a result, mronj occurrence has become a challenge for dental professionals in recent years due to the absence of predictive factors providing security for dental intervention10. however, mronj can also occur without a history of oral surgical treatment15. thus, this study aimed to systematically review current literature regarding the association 3 lara et al. braz j oral sci. 2022;21:e226585 between dental procedures and mronj in patients with current or historical use of bone-modifying agents. material and methods pico question a systematic review was performed according to preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines16. the present study focused on answering the following clinical question: “do dental extractions and periodontal surgeries increase the risk of mronj in patients in current therapy or with a history of antiresorptive and/or antiangiogenic use?” therefore, the pico question was structured as follows: population: antiresorptive and/or antiangiogenic users or those with a use history; intervention: extractions and periodontal therapy; comparison: absence of dental interventions; outcome: occurrence of mronj. search strategy electronic search strategies were performed in order to include studies published up to december 2021. the search strategies were applied in the medline-pubmed, web of science, scopus, and cochrane library databases, and are presented in table 1. grey literature was also searched using the new york academy of medicine (nyam) gray literature report and google scholar databases. an adaptation of the search strategies described in table 1 was performed on both databases. in google scholar, the first 300 studies were searched, as recommended by the literature17. in order to be included in this study, studies from the search results had to involve clinical trials (randomized or not), case-controls, case series (minimum of ten patients) and prospective or retrospective cohorts. furthermore, studies had to involve current or former users of bone-modifying agents (antiresorptive and/or antiangiogenic drugs). the study group had to be composed of individuals submitted to dental extraction and/or periodontal surgical procedures. the control group had to include those who had not undergone the previously repored dental procedures. literature reviews, in vitro studies, animal studies and case reports were excluded. study selection references obtained through the described search strategy were organized, and duplicates were removed using the endnoteweb® program (clarivate analytics). study selection was carried out independently by two reviewers (abgc and smcl) in consideration of the inclusion criteria. in case of disagreement, a consensus was reached through discussion. subsequently, full texts were independently evaluated for final selection. three reviewers (abgc, smcl and tbbc) were included in this step and a fourth reviewer (fwmgm) was consulted to resolve possible disagreements. additionally, a manual search was performed on the references list of selected studies. 4 lara et al. braz j oral sci. 2022;21:e226585 table 1. search strategies performed and number of studies detected in all dabatases. database search strategy number of studies retrieved medline-pubmed #1 – surgery, oral[mesh terms] or oral surgery[title/abstract] or dental care[mesh terms] or oral surgical procedures[mesh terms] or oral surgical procedures[title/abstract] or tooth extraction[mesh terms] or tooth extraction[title/abstract] or dental extraction[title/abstract] or dental extractions[title/abstract] or teeth extraction[title/abstract] or teeth removal[title/abstract] or tooth extraction[title/abstract] or crown lengthening[mesh terms] or crown lengthening[title/abstract] or gingivectomy[mesh terms] or gingivoplasty[mesh terms] or dental scaling[mesh terms] or root planing[title/abstract] or open flap debridement[title/abstract] or periodontal surgery[title/abstract] or guided tissue regeneration[mesh terms] or root coverage[title/abstract] or gingival graft[title/abstract] or periodontal osseous surgery[title/abstract] or periodontal treatment[title/abstract] #2 – diphosphonates[mesh terms] or bisphosphonate[title/abstract] or bisphosphonates[title/abstract] or alendronate[mesh terms] or alendronate sodium[title/abstract] or risedronic acid[mesh terms] or risedronate[title/abstract] or pamidronate[mesh terms] or amidronate[title/abstract] or ibandronate[title/abstract] or ibandronic acid[mesh terms] or zoledronate[title/abstract] or zoledronic acid[title/ abstract] or clodronic acid[mesh terms] or clodronate[title/abstract] or etidronic acid[mesh terms] or denosumab[mesh terms] or antiresorptive drugs[title/abstract] or bevacizumab[mesh terms] or sorafenib[mesh terms] or sunitinib[mesh terms] or pazopanib[title/ abstract] or axitinib[mesh terms] #3 – osteonecrosis[mesh terms] or osteonecrosis[title/abstract] or bone necrosis[title/abstract] or aronj[title/abstract] or bronj[title/abstract] or onj[title/abstract] or mronj[title/abstract] or “osteonecrosis of the jaw”[title/abstract] or “bisphosphonate-related osteonecrosis of the jaw”[title/abstract] or “bisphosphonate-related onj”[title/abstract] or “bisphosphonate-associated osteonecrosis of the jaw”[title/abstract] or “medication-related osteonecrosis of the jaw” [title/abstract] #4 #1 and #2 and #3 918 web of science #1 – ts=(surgery, oral or “oral surgery” or “dental care” or “oral surgical procedures” or “tooth extraction” or “dental extraction” or “dental extractions” or “teeth extraction” or “teeth removal” or “tooth extraction” or “crown lengthening” or gingivectomy or gingivoplasty or “dental scaling” or “root planing” or “open flap debridement” or “periodontal surgery” or “guided tissue regeneration” or “root coverage” or “gingival graft” or “periodontal osseous surgery” or “periodontal treatment”) #2 – ts=(diphosphonates or bisphosphonate or bisphosphonates or alendronate or alendronate sodium or “risedronic acid” or risedronate or pamidronate or amidronate or ibandronate or “ibandronic acid” or zoledronate or “zoledronic acid” or “clodronic acid” or clodronate or “etidronic acid” or denosumab or “antiresorptive drugs” or bevacizumab or sorafenib or sunitinib or pazopanib or axitinib) #3 – ts=(osteonecrosis or “bone necrosis” or aronj or bronj or onj or mronj or “osteonecrosis of the jaw” or “bisphosphonate-related osteonecrosis of the jaw” or “bisphosphonate-related onj” or “bisphosphonate-associated osteonecrosis of the jaw” or “medication-related osteonecrosis of the jaw”) #4 #1 and #2 and #3 899 continue 5 lara et al. braz j oral sci. 2022;21:e226585 data extraction data extraction was performed in a spreadsheet specifically developed for this study. two researchers (smcl and tbbc) were involved in this process, with a third continuation scopus (( title-abs-key (surgery, oral) or title-abs-key (“oral surgery”) or title-abs-key (“dental care”) or title-abs-key (“oral surgical procedures”) or title-abs-key (“tooth extraction”) or title-abs-key (“dental extraction”) or title-abs-key (“dental extractions”) or title-abskey (“teeth extraction”) or title-abs-key (“teeth removal”) or titleabs-key (“tooth extraction”) or title-abs-key (“crown lengthening”) or title-abs-key (gingivectomy) or title-abs-key (gingivoplasty) or title-abs-key (“dental scaling”) or title-abs-key (“root planing”) or title-abs-key (“open flap debridement”) or title-abs-key (“periodontal surgery”) or title-abs-key (“guided tissue regeneration”) or title-abs-key (“root coverage”) or title-abs-key (“gingival graft”) or title-abs-key (“periodontal osseous surgery”) or title-abs-key (“periodontal treatment”))) and ((title-abs-key (diphosphonates) or title-abs-key (bisphosphonate) or title-abs-key (bisphosphonates) or title-abs-key (alendronate) or title-abs-key (alendronate sodium) or title-abs-key (“risedronic acid”) or title-abs-key (risedronate) or title-abs-key (pamidronate) or title-abs-key (amidronate) or title-abs-key (ibandronate) or title-abs-key (“ibandronic acid”) or title-abs-key (zoledronate) or title-abs-key (“zoledronic acid”) or title-abs-key (“clodronic acid”) or title-abs-key (clodronate) or title-abs-key (“etidronic acid”) or title-abs-key (denosumab) or title-abs-key (“antiresorptive drugs”) or title-abs-key (bevacizumab) or title-abs-key (sorafenib) or title-abs-key (sunitinib) or titleabs-key (pazopanib) or title-abs-key (axitinib))) and ((title-abs-key (osteonecrosis) or title-abs-key ( “bone necrosis”) or title-abskey (aronj) or title-abs-key (bronj) or title-abs-key (onj) or title-abs-key (mronj) or title-abs-key (“osteonecrosis of the jaw”) or title-abs-key (“bisphosphonate-related osteonecrosis of the jaw”) or title-abs-key (“bisphosphonate-related onj”) or title-abs-key (“bisphosphonate-associated osteonecrosis of the jaw”) or title-abs-key (“medication-related osteonecrosis of the jaw”))) 1535 cochrane library #1 surgery, oral or “oral surgery” or “dental care” or “oral surgical procedures” or “tooth extraction” or “dental extraction” or “dental extractions” or “teeth extraction” or “teeth removal” or “tooth extraction” or “crown lengthening” or gingivectomy or gingivoplasty or “dental scaling” or “root planing” or “open flap debridement” or “periodontal surgery” or “guided tissue regeneration” or “root coverage” or “gingival graft” or “periodontal osseous surgery” or “periodontal treatment” #2 – diphosphonates or bisphosphonate or bisphosphonates or alendronate or alendronate sodium or “risedronic acid” or risedronate or pamidronate or amidronate or ibandronate or “ibandronic acid” or zoledronate or “zoledronic acid” or “clodronic acid” or clodronate or “etidronic acid” or denosumab or “antiresorptive drugs” or bevacizumab or sorafenib or sunitinib or pazopanib or axitinib #3 – osteonecrosis or “bone necrosis” or aronj or bronj or onj or mronj or “osteonecrosis of the jaw” or “bisphosphonaterelated osteonecrosis of the jaw” or “bisphosphonate-related onj” or “bisphosphonate-associated osteonecrosis of the jaw” or “medicationrelated osteonecrosis of the jaw” #4 #1 and #2 and #3 73 nyam gray literature report oral surgery and osteonecrosis and (bisphosphonate or denosumab or bevacizumab) 0 google scholar oral surgery and osteonecrosis and (bisphosphonate or denosumab or bevacizumab) 300 6 lara et al. braz j oral sci. 2022;21:e226585 researcher involved in case of discrepancy (fwmgm). the following parameters were collected: author, publication year, country, study design, patient gender, mean (standard deviation [sd]) age, number of patients with and without mronj, mronj diagnosis criteria, number of patients submitted to dental procedures in both test and control groups, dental prosthesis users, smokers, use of bone-modifying drugs and its clinical indication and other medications reported. in cases of insufficient information, authors were contacted by email to obtain additional information. risk of bias assessment both retrospective and prospective cohorts as well as case-control studies were included in this review. therefore, the newcastle-ottawa quality assessment tool was used to analyze the risk of bias in this study. this tool consists of eight criteria and classifies the involved research according to a score ranging from zero to nine stars. on this scale, a study can be awarded a maximum of one star for each item within the selection and outcome categories. in the comparability category, it is possible to award the study with two stars. the first star is received by studies that performed control for the most important factor. the second is received by studies that carried out controls on other additional factors. the risk of bias analysis was performed independently by two reviewers (css and tbbc), and discrepancies were solved by a third researcher (fwmgm). data synthesis and statistical analysis three different meta-analyses were conducted during this study. meta-analyses were performed if at least two studies provided enough information regarding the occurrence of mronj following dental surgical procedures. no study clearly reported the occurrence of mronj after periodontal surgical procedures. thus, three meta-analyses were performed for tooth extraction procedures regardless of the study follow-up time. firstly, mronj risk involving dental extraction was assessed using risk ratio (rr). additionally, two other meta-analyses were performed using the pooled log of odds ratio (or) and the pooled log of hazard ratio (hr); this was done only for studies that provided multivariate estimates for the occurrence of mronj following dental extraction, controlling for at least two important confounding variables. heterogeneity was assessed using the q test and quantified by i2. for both analyses, a random model was used within the review manager software (version 5.3). results search results firstly, a total of 3,725 studies were obtained through our search strategy. four additional studies were included as the result of a manual search on the final references list of included articles. after duplicates removal, 1,654 studies were screened; of those, 85 were assessed for eligibility. seventeen studies fulfilled the outlined inclusion criteria and were included in the qualitative synthesis. therefore, 13 retrospective cohorts18-30, one prospective cohort31 and three case-control studies32-34 were included in the present study, as shown in figure 1. 7 lara et al. braz j oral sci. 2022;21:e226585 risk of bias assessment the risk of bias assessment conducted on the cohort studies is summarized in figure 2a. among the different cohort studies, eight20-23,25,27-29 presented low risk of bias in all analyzed criteria. four cohort articles presented a high risk of bias regarding comparability between groups, given the presence of possible confounders18,19,24,31. additionally, four studies failed to demonstrate that the outcome was not present at the beginning of the study19,26,30,31. figure 2b presents the risk of bias assessment for the case-control studies. only one case-control study presented a low risk of bias in all analyzed criteria34. moreover, among case-control studies, risk of bias in relation to non-response rate between groups was detected in two studies, which were thus classified as presenting a high risk of bias32,33. main results the main characteristics of the included studies with patients subject to bone-modifying therapies are summarized in table 2. the number of patients enrolled had great variability among studies, with a range from 39 to 164,926 individuals. overfigure 1. prisma flowchart of study selection process. legend: rr: risk ratio; aor: adjusted odds ratio; ahr: adjusted hazard ratio. id en ti fi ca ti on s cr ee ni ng el ig ib ili ty in cl ud ed records identified through database searching (n = 3725) records after duplicates removed (n = 1654) records screened (n = 1654) full-text articles assessed for eligibility (n = 85) studies included in qualitative synthesis (n = 17) studies included in quantitative synthesis (meta-analysis) (n = 11 – rr; n = 3 – aor; n = 4 – ahr) records excluded (n = 1569) additional records identified through other sources (n = 4) full-text articles excluded, with reasons data extraction not possible (n = 2) lack of group without intervention (n = 25) only included patients with mronj (n = 41) 8 lara et al. braz j oral sci. 2022;21:e226585 figure 2. risk of bias analysis of cohort (a) and case-control (b) studies, according to newcastle-ottawa scale. 9 lara et al. braz j oral sci. 2022;21:e226585 ta bl e 2. m ai n ch ar ac te ris tic s of p at ie nt s us in g bo ne -m od ify in g dr ug s en ro lle d in th e st ud ie s. a ut ho r/ y ea r c ou nt ry s tu dy d es ig n m r o n j/ a ll pa ti en ts in ci de nc e (% ) m ea n ag e d ru gre la te d fa ct or s p at ie nt -r el at ed fa ct or s m ai n re su lt s b on em od ify in g dr ug c lin ic al in di ca ti on ; c ur re nt m ed ic at io ns o ra l s ur gi ca l p ro ce du re s; d en ta l p ro st he si s; o ra l in fe ct io ns d ia be te s; s m ok er s m r o n j n on -m r o n j m r o n j n on -m r o n j m r o n j n on -m r o n j m r o n j n on -m r o n j b on ac in a (2 01 1) c an ad a; p ro sp ec tiv e co ho rt . 7/ 65 (1 0. 7% ) n r zo le dr on at e (7 ) zo le dr on at e (5 5) zo le dr on at e an d su ni tin ib (3 ) c an ce r ( 7) ; c or tic oi ds (0 ) c an ce r ( 7) ; c or tic oi ds (7 ) ex tr ac tio n (1 ); n r ; n r ex tr ac tio n (3 ); n r ; n r n r ; 0 n r ; 1 3 a m on g th e ev al ua te d pa tie nt s, th er e w as a co rr el at io n be tw ee n th e nu m be r o f z ol ed ro na te in fu si on s an d m r o n j. in a dd iti on , a ll th es e pa tie nt s pr es en te d pr ev io us e xp os ur e to bi sp ho sp ho na te s. c al vo -v ill as (2 01 6) sp ai n; r et ro sp ec tiv e co ho rt . 7/ 64 (1 0. 9% ) 75 y o zo le dr on at e (3 ) p am id ro na te a nd zo le dr on at e (4 ) zo le dr on at e (5 7) p am id ro na te an d zo le dr on at e (4 3) m ul tip le m ye lo m a (7 ); c or tic oi ds (7 ) th al id om id e (3 ) m ul tip le m ye lo m a (5 7) ; c or tic oi ds (3 5) th al id om id e (1 4) ex tr ac tio n (5 ); n r ; o ra l in fe ct io n (6 ) ex tr ac tio n (1 ); n r ; o ra l in fe ct io n (4 4) n r ; 2 n r ; 1 8 o cc ur re nc e of m r o n j w as d ire ct ly re la te d to d ur at io n of b is ph os ph on at e th er ap y an d pr es en ce of lo ca l f ac to rs , a s or al in fe ct io ns . c ar m ag no la (2 00 8) it al y; r et ro sp ec tiv e co ho rt . 20 /3 9 (5 1. 7% ) 65 y o zo le dr on at e (1 9) p am id ro na te a nd zo le dr on at e (1 ) zo le dr on at e (1 8) p am id ro na te (1 ) c an ce r ( 14 ) m ul tip le m ye lo m a (6 ); n r c an ce r ( 12 ) m ul tip le m ye lo m a (6 ) ly m ph om a (1 ); n r ex tr ac tio n (1 0) ; d en tu re s (7 ); n r ex tr ac tio n (5 ); n r ; n r n r ; n r n r ; n r th e au th or s di d no t fi nd an a ss oc ia tio n be tw ee n de nt al e xt ra ct io ns a nd m r o n j. in cr ea se d do se s of z ol ed ro na te w er e re la te d to m r o n j. g un al di (2 01 5) tu rk ey ; c as ec on tr ol 24 /4 4 (5 4. 4% ) 60 ,5 y o zo le dr on at e (2 3) zo le dr on at e + b ev ac iz um ab (1 ) zo le dr on at e (1 9) zo le dr on at e + b ev ac iz um ab (1 ) c an ce r ( 14 ) m ul tip le m ye lo m a (8 ) o th er (2 ); c or tic oi ds (7 ) th al id om id e (5 ) c an ce r ( 9) m ul tip le m ye lo m a (7 ) o th er (4 ); c or tic oi ds (7 ) th al id om id e (4 ) ex tr ac tio n (1 3) ; n r ; n r ex tr ac tio n (4 ); n r ; n r 1; 4 1; 4 ti m e of e xp os ur e to zo le dr on at e an d to ot h ex tr ac tio n w er e fa ct or s re la te d to m r o n j. c on tin ue 10 lara et al. braz j oral sci. 2022;21:e226585 c on tin ua tio n h of f ( 20 08 ) u sa r et ro sp ec tiv e co ho rt . 29 /3 96 5 (0 .7 9% ) 62 yo p am id ro na te (7 ) zo le dr on at e (9 ) p am id ro na te + zo le dr on at e (1 3) p am id ro na te (2 28 1) zo le dr on at e (1 17 1) p am id ro na te + zo le dr on at e (5 13 ) c an ce r ( 16 ) m ul tip le m ye lo m a (1 3) ; c or tic oi ds (2 3) c an ce r ( 23 92 ) m ul tip le m ye lo m a (5 35 ) o st eo po ro si s (2 71 ) p ag et ’s d is ea se (1 1) ; c or tic oi ds (3 27 2) ex tr ac tio n (1 6) ; d en tu re s (3 ); o ra l in fe ct io n (1 4) ex tr ac tio n (1 36 ); n r ; n r n r ; n r n r ; n r m ea n tim e of tr ea tm en t an d de nt al e xt ra ct io ns w er e re la te d to oc cu rr en ce o f m r o n j. u se o f z ol ed ro na te in cr ea se s ris k of os te on ec ro si s. ik es ue (2 02 1) ja pa n r et ro sp ec tiv e co ho rt 34 /3 74 (9 .0 9% ) 70 y o d en os um ab (2 7) zo le dr on at e (7 ) d en os um ab (1 88 ) zo le dr on at e (1 52 ) c an ce r ( 28 ) m ul tip le m ye lo m a (4 ) o th er s (2 ) o ra l bi sp ho sp ho na te (1 ) a nt ia ng io ge ni c ag en ts (1 1) c or tic oi d (2 2) c an ce r ( 24 6) m ul tip le m ye lo m a (5 2) o th er s (4 0) o ra l bi sp ho sp ho na te (1 2) a nt ia ng io ge ni c ag en t ( 62 ) c or tic oi d (1 81 ) ex tr ac tio n (1 0) ; n r ; n r ex tr ac tio n (1 0) ; n r ; n r 3; n r 67 ; n r in ci de nc e of m r o n j w as g re at er in th e de no su m ab g ro up th an in z ol ed ro na te u se rs . m r o n j oc cu rr en ce w as re la te d to a ge > 65 y ea rs a nd to ot h ex tr ac tio n be fo re o r af te r s ta rt in g th er ap y. ik es ue (2 02 1) ja pa n r et ro sp ec tiv e co ho rt 58 /7 99 (7 .3 % ) n r d en os um ab (3 9) zo le dr on at e (1 9) d en os um ab (3 67 ) zo le dr on at e (3 74 ) n r n r n r n r n r ; n r ; n r n r ; n r ; n r n r ; n r n r ;n r r is k of d ev el op in g m r o n j w as h ig he r in d en os um ab u se rs . to ot h ex tr ac tio n af te r st ar tin g th er ap y w as a si gn ifi ca nt ri sk fa ct or to de ve lo p m r o n j. ik es ue (2 02 1) ja pa n r et ro sp ec tiv e co ho rt 65 /7 95 (8 .2 % ) n r d en os um ab a ft er zo le dr on at e (7 ) zo le dr on at e (1 9) d en os um ab (3 9) d en os um ab af te r zo le dr on at e (3 6) zo le dr on at e (3 31 ) d en os um ab (3 63 ) n r n r n r n r n r ; n r ; n r n r ;n r ;n r n r ;n r n r ; n r m r o n j oc cu rr en ce w as as so ci at ed w ith to ot h ex tr ac tio n, c on co m ita nt us e of a nt ia ng io ge ni cs , de no su m ab th er ap y an d sw itc hi ng z ol ed ro na te to de no su m ab . c on tin ue 11 lara et al. braz j oral sci. 2022;21:e226585 c on tin ua tio n ja du (2 00 7) c an ad a r et ro sp ec tiv e co ho rt 21 /6 55 (3 .2 % ) n r p am id ro na te (2 1) p am id ro na te (6 34 ) m ul tip le m ye lo m a (2 1) n r m ul tip le m ye lo m a (6 34 ) n r n r ; n r ; n r n r ;n r ;n r n r ;n r n r ; n r d en ta l e xt ra ct io ns w er e as so ci at ed w ith m ro n j on se t. o th er fa ct or s w er e du ra tio n of p am id ro na te th er ap y, ad va nc ed a ge , an d co nc om ita nt u se of s om e dr ug s, s uc h as co rt ic oi ds . k im (2 02 1) so ut h k or ea r et ro sp ec tiv e co ho rt 16 6/ 16 4, 92 6 (0 ,1 % ) n r b is ph os ph on at e no t s pe ci fie d (1 66 ) b is ph os ph on at e no t s pe ci fie d (1 64 ,7 60 ) o st eo po ro si s (1 66 ) n r o st eo po ro si s (1 64 ,7 60 ) n r n r ; n r ; n r n r ;n r ;n r n r ;n r n r ; n r d en ta l e xt ra ct io n, gi ng iv iti s an d pe rio do nt iti s w er e st ro ng ly a ss oc ia te d w ith m r o n j on se t. k yr gi di s (2 00 8) g re ec e c as ec on tr ol 20 /6 0 (n a ) 59 .5 zo le dr on at e (2 0) zo le dr on at e (4 0) c an ce r ( 20 ); n r c an ce r ( 40 ); n r ex tr ac tio n (1 0) ; 8 ; n r ex tr ac tio n (3 ); 7; n r n r ; 1 6 n r ; 2 3 o dd s of m r o n j oc cu rr en ce in cr ea se d to w ar ds u se o f d en tu re s an d de nt al e xt ra ct io n. m cg ow an (2 01 9) a us tr al ia c as eco nt ro l 44 /1 59 (n a ) 67 .8 a le nd ro na te (1 0) zo le dr on at e (1 7) p am id ro na te (5 ) r is ed ro na te (5 ) d en os um ab (7 ) a le nd ro na te (2 8) zo le dr on at e (3 8) p am id ro na te (1 4) r is ed ro na te (1 5) d en os um ab (1 8) o st eo po ro si s (1 6) m ul tip le m ye lo m a (1 3) c an ce r ( 12 ) r he um at oi d ar th rit is (3 ) o st eo po ro si s (4 9) m ul tip le m ye lo m a (3 1) c an ce r ( 26 ) r he um at oi d ar th rit is (7 ) ex tr ac tio n (2 8) p er io do nt al tr ea tm en t ( 3) p er io do nt al ac ut e in fe ct io n (4 ) d en ta l pr os th es is (2 4) p er io do nt al tr ea tm en t ( 3) p er io do nt al ac ut e in fe ct io n (3 ) d en ta l pr os th es is (6 0) 13 ; 1 1 19 ; 1 4 n on -s ur gi ca l t he ra py an d to ot h ex tr ac tio n w er e as so ci at ed w ith m r o n j oc cu rr en ce . m at su i ( 20 15 ) ja pa n r et ro sp ec tiv e co ho rt 23 /1 06 (2 1. 6% ) 74 .8 zo le dr on at e (7 ) m in od ro na te (3 ) r is ed ro na te (2 ) a le nd ro na te (5 ) d en os um ab (5 ) u nk no w n (1 ) r is ed ro na te (3 4) a le nd ro na te (2 6) m in od ro na te (1 4) u nk no w n (7 ) et id ro na te (1 ) o st eo po ro si s (1 0) c an ce r ( 11 ) o th er s (1 ) u nk no w n (1 ) o st eo po ro si s (7 2) c an ce r ( 4) o th er s (5 ) u nk no w n (2 ) to ot h ex tra ct io n (9 ) d en ta l pr os th es is (2 ) p er io do nt al di se as e (3 ) to ot h ex tr ac tio n (7 9) c ys te ct om y (3 ) b io ps y (1 ) n r ; n r n r ; n r to ot h ex tr ac tio n w as th e m ai n ca us e re la te d to m r o n j on se t. m os t p at ie nt s w ith m r o n j ha d a hi st or y of zo le dr on at e us e. c on tin ue 12 lara et al. braz j oral sci. 2022;21:e226585 c on tin ua tio n so ut om e (2 02 1) ja pa n r et ro sp ec tiv e co ho rt 33 ja w s/ 35 9 ja w s n a 65 .9 b is ph os ph on at e no t s pe ci fie d (1 2 ja w s) d en os um ab (2 0 ja w s) b is ph os ph on at e no t s pe ci fie d (1 49 ja w s) d en os um ab (1 69 ja w s) b is ph os ph on at e to d en os um ab c an ce r ( 33 ja w s) c or tic os te ro id (3 ja w s) c an ce r ( 32 6 ja w s) c or tic os te ro id (3 8 ja w s) to ot h ex tr ac tio n (5 ja w s) o ra l in fe ct io n (1 3 ja w s) to ot h ex tr ac tio n (2 2 ja w s) o ra l i nf ec tio n (1 7) ja w s 4; 3 34 ; 5 4 p re se nc e of lo ca l sy m pt om s of in fe ct io ns or in fe ct ed te et h w er e in de pe nd en t r is k fa ct or s to m r o n j oc cu rr en ce , su ch a s du ra tio n of an tir es or pt iv e th er ap y. d en ta l e xt ra ct io n w as no t a ri sk fa ct or to m r o n j on se t. u ed a (2 02 1) ja pa n r et ro sp ec tiv e co ho rt 43 /7 45 n a n r n r n r c an ce r ( 43 ) c an ce r ( 70 2) to ot h ex tr ac tio n (6 ) to ot h ex tr ac tio n (1 3) n r ;n r n r ;n r to ot h ex tr ac tio ns w er e as so ci at ed w ith m r o n j oc cu rr en ce . r ad io gr ap hi c fin di ng s as o st eo sc le ro tic a re as at th e fir st e xa m w er e re la te d w ith fu tu re m r o n j oc cu rr en ce . u ed a (2 02 1) ja pa n r et ro sp ec tiv e co ho rt 42 /3 98 (1 0. 6% ) 70 .5 b is ph os ph on at e no t s pe ci fie d (1 1) d en os um ab (2 6) b is ph os ph on at e no t s pe ci fie d pl us de no su m ab (5 ) b is ph os ph on at e no t s pe ci fie d (1 36 ) d en os um ab (1 79 ) bi sp ho sp ho na te no t s pe ci fie d pl us de no su m ab (4 1) c an ce r ( 42 ) m ul tip le m ye lo m a (3 ) c or tic oi d (1 ) c an ce r ( 31 0) m ul tip le m ye lo m a (4 6) c or tic oi d (1 3) to ot h ex tr ac tio n (5 ) to ot h ex tr ac tio n (7 ) 4; 14 51 ; 6 8 a lv eo la r bo ne lo ss in vo lv in g m or e th an ha lf t he r oo t on pa no ra m ic r ad io gr ap hs an d to ru s m an di bu la ri s ca rr y a hi gh r is k of m r o n j d ev el op m en t. to ot h ex tr ac ti on w as no t as so ci at ed w it h m r o n j on se t. v ah ts ev an os (2 00 9) g re ec e r et ro sp ec tiv e co ho rt 80 /1 62 1 (4 .9 3% ) 63 .6 b is ph os ph on at e no t s pe ci fie d (8 0) b is ph os ph on at e no t s pe ci fie d (1 54 1) c an ce r ( 34 ) m ul tip le m ye lo m a (4 6) c an ce r ( 10 48 ) m ul tip le m ye lo m a (4 93 ) to ot h ex tr ac tio n (4 6) d en tu re s (2 4) to ot h ex tr ac tio n (6 9) d en tu re s (1 99 ) n r ;4 2 n r ;8 26 to ot h ex tr ac tio ns a nd us e of d en tu re s w er e ris k fa ct or s fo r m r o n j de ve lo pm en t. m r o n j – m ed ic at io nre la te d os te on ec ro si s of th e ja w ; n r = n ot rp or te d; y o ; y ea rs o ld ; n a = n ot a pp lic ab le 13 lara et al. braz j oral sci. 2022;21:e226585 all, 175,004 patients were included in the seventeen studies, with mean age ≥ 60 years. a higher percentage of female patients was reported among the majority of included studies. male patients were more prevalent in only five studies21-23,28,29, while three studies did not provide data regarding sex24,31,32. aaoms classification diagnostic criteria35 was adopted in ten studies21-24,26-31. among the included studies, a total of 683 patients with mronj were enrolled. this condition had a slightly higher diagnosis rate among female patients (n = 129/256), when considering studies that provided this information. among individuals with mronj, 159 patients had a history of tooth extraction, representing 23.2% of the total. use of dental prosthesis was reported in 60 patients, after an analysis of 196 individuals (30.6%). however, it is important to note that this variable was not reported in most of the studies. smoking was also assessed as a possible risk factor for mronj; this factor was reported in two studies. among 966 individuals who reported smoking habits, 89 patients developed mronj18,28,30-34. the main reason for clinical indication of the use of antiresorptive and antiangiogenic drugs was cancer (209 patients, 30.6%). among the drugs used, zoledronate was the most cited (143 patients, 20.9%). when considering corticosteroid use and occurrence of mronj, studies reported prevalence varying from 2.3%28 to 29%33, 64.7%21, 79%20 and up to 100%18 of patients, among five studies that provided this information (table 2). it is important to highlight that, aside from the provided data, one study was not included in the quantitative synthesis, as only the number of jaws (not patients) was available27. regarding patients without mronj, the total number of patients varied among studies, with a total sample of 174,132. there was once again a higher prevalence of female individuals involved, but the main underlying disease reported was osteoporosis (165,152 patients, 94.8%), mostly due to one expressive cohort study27. for patients without mronj, pamidronate was the most frequently reported drug used; however, several studies did not specify the bisphosphonate agent. the use of dental prostheses was reported for 259 patients. additionally, smoking was observed in 0.5% of patients, for a total of 1,010 individuals (table 2). meta-analyses only dental extraction procedures were reported in all selected studies. for this reason, this was the only dental procedure available to include in meta-analyses. crude analysis revealed an increase in the relative risk of occurrence of mronj following tooth extraction (rr = 4.28; 95%ci: 1.73–10.58), as presented in figure 3. for this analysis, eleven studies were included18-21,26,28,30-34. it is important to highlight the high heterogeneity among these studies (i2 = 95%, p<0.001). furthermore, two additional meta-analyses were performed in consideration only of the studies that reported adjusted analysis for the association between dental extraction and mronj. three studies assessed the adjusted or20,32,34, and four estimated the adjusted hr20,21,25,28. as shown in figures 4 and 5, similar results were demonstrated, as dental extraction significantly increased the or for the occurrence of mronj (or = 26.94; 95%ci: 4.17–174.17%) and the hr for the occurrence of mronj (hr = 9.96; 95%ci: 4.04–24.55). moreover, high heterogeneity was demonstrated in both analyses (respectively: i2 = 84%, p=0.002; and i2 = 80%, p=0.002). 14 lara et al. braz j oral sci. 2022;21:e226585 discussion mronj is a recently described complication, first reported in 2003 through a series of cases in patients who used bisphosphonates3. since then, several reports have associated avascular bone necrosis with oral and intravenous bisphosphonates therapy, in addition to antiresorptives and antiangiogenics8,36. due to its multifactorial nature and complex management, many authors have tried to associate risk factors with mronj development in order to avoid its occurrence. the present study identified that the occurrence of mronj is generally higher among women, which is in accordance with the literature37. osteoporosis and breast cancer, the therapeutic indications of which are related to the use of antiresorptives, bisphosphonates and antiangiogenics, are more common among female patients, which may explain this result8. in addition to these factors, literature reports that women seek more often dental care; this could result in a higher diagnosis rate for several conditions, including mronj38. figure 3. forest plot for the crude association between medication-related osteonecrosis of the jaw (mronj) and tooth extraction. figure 4. forest plot for adjusted odds ratio analysis between medication-related osteonecrosis of the jaw (mronj) and tooth extraction. figure 5. forest plot for adjusted hazard ratio analysis between medication-related osteonecrosis (mronj) of the jaw and tooth extraction. 15 lara et al. braz j oral sci. 2022;21:e226585 the mean age of patients diagnosed with mronj was greater than 60 years in the included studies, which is in accordance with the literature39,40. among these patients, there is a higher incidence of chronic diseases—a common reason for prescribing bone-modifying drugs—and few studies failed to detect this relationship30. trigger factors for mronj remain subject to critical investigation. marx3 reported its occurrence more often following dental procedures in the mandible. among the studies included here, tooth extraction, periodontal disease or trauma induced by poorly adapted prostheses have been involved in the onset and even exacerbation of the condition20,31. bone exostosis, trauma induced by intubation and poor dental implant placement have also been discussed20. dental extractions can be justified as a risk factor for the development of mronj, as they may induce bone exposure in the oral cavity with a consequent reduction in blood support and bone metabolism and greater osteoclast apoptosis19. the present study confirms the proposed association between tooth extraction and mronj onset, including in the adjusted analysis. in fact, mronj occurs frequently at the procedure site, although it can also occur spontaneously (i.e., without an identifiable precipitating clinical event)41. according to carmagnola et al.19, patients with mronj reported extractions twice as frequently as those without mronj. however, analysis also revealed a high heterogeneity, which can be attributed to different experimental designs and varied sampling. moreover, due to the lack of randomized controlled trials, this research included only cohort and case-control studies; as a result, it is difficult to provide solid evidence regarding this research topic, which explains the fact that several recommendations for the management of mronj are based on expert consensus10. hence, this must be taken into consideration when interpreting the results of the present study. other local factors may also be involved in the occurrence of mronj. teeth with dental extraction indication are often associated with an infectious process, and the presence of inflammation or infection would be a predisposing factor for the appearance of lesions caused by avascular bone necrosis41. in this regard, one study found that mronj occurrence may also be related to preexisting inflammatory dental disease; in such a scenario, dental extraction is fundamental in order to prevent mronj onset27. furthermore, the use of dental prostheses, which is common among older adults, is considered another potential local risk factor for mronj. unretained dental prosthesis may cause low-grade chronic trauma, usually resulting in oral lesions due to rupture of the protective barrier which allow the entry of highly contaminated oral microbiota into the bone29,32. in addition, systemic factors can also influence the occurrence of mronj. one critical example is diabetes, a disease that can predispose a patient to the occurrence of this dental complication40. in the present study, two included studies suggested that type ii diabetes and smoking represent important risk factors for the occurrence of mronj31,33. moreover, exposure to smoke predisposes a patient to cancer, making the need for administration of antiresorptive and antiangiogenic drugs more likely and thus intrinsically increasing the possibility of mronj, complicating the identification of a cause-and-effect relationship33. conversely, two previous studies did not state an association between smoking and mronj18,30. 16 lara et al. braz j oral sci. 2022;21:e226585 current use of other medications, such as corticosteroids, has also been listed as a risk factor in the literature24. one included study18 reported that all mronj patients were also using corticosteroids, while another reported this percentage as 2.3%28. therefore, based on compiled data, there is no way to draw a definitive conclusion on this topic. bisphosphonate or antiresorptive type, form of administration (oral or iv), number of infusions and the time of exposure are also factors considered in relation to the development of mronj10. among the included studies, a higher percentage of patients with mronj undergoing cancer treatment was observed. however, due to lack of information regarding time of therapy and dosage, it was not possible to further analyze these characteristics in the present study. that said, the majority of studies found a correlation between cumulative doses and mronj. the included studies ultimately reveal that there is not yet enough evidence to ensure safe invasive dental procedures in such patients, even after the interruption of the therapeutic protocol. therefore, communication between health care professionals is essential to provide preventive treatment prior to drug administration. routine and preventive appointments for early identification of infectious areas are fundamental for preventing mronj and reducing the significant impact of this complication. it must be noted that the present study was not previously registered in any database of protocols for systematic reviews. unfortunately, a posteriori registrations are not permitted by these databases. this must therefore be understood as a major limitation of the present study. in summary, this systematic review showed that dental extractions increased the risk of mronj occurrence 4.28 times. risk factors such as smoking, diabetes and the use of corticosteroids are possible variables related to this condition, which is more frequent in women over 60 years of age. our work adds vital evidence in order to provide better care for these patients. preventive oral hygiene measures are the best options available for patients using bone-modifying drugs, preferably before the start of medical and dental procedures. acknowledgments this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior brasil (capes) finance code 001. all other funding was self-supported by the authors. the authors report no conflict of interest. data availability datasets related to this article will be available upon request to the corresponding author. conflict of interests none. author contribution shimelly monteiro de castro lara has contributed with study selection, data extraction and writing; ana beatriz caetano gerônimo was enrolled in study selection and data 17 lara et al. braz j oral sci. 2022;21:e226585 extraction; francisco wilker mustafa gomes muniz has contributed with conceptualization, methodological advice, designing search strategies, writing and final revision; cinthia studzinski santos was enrolled in bias assessment and writing; thayanne brasil barbosa calcia has contributed with conceptualization, bias assessment, writing and final revision. list of all captions aaoms: american academy of oral and maxillofacial surgery bronj: bisphosphonate related osteonecrosis of jaw ci: confidence interval mronj: medication related osteonecrosis of the jaw or: odds ratio prisma: preferred reporting items for systematic reviews and meta-analyses rr: risk ratio references 1. mishra mb, mishra s, mishra r. dental care in the patients with bisphosphonates therapy. int j dent clin 2011;3(1):60-4. 2. allen cs, yeung jhs, vandermeer b, homik j. bisphosphonates for steroid-induced osteoporosis. cochrane database syst rev. 2016 oct;10(10):cd001347. doi: 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with advanced cancer treated with bisphosphonate therapy. oncologist. 2008 aug;13(8):911-20. doi: 10.1634/theoncologist.2008-0091. 41. saad f, brown je, poznak cv, ibrahim t, stemmer sm, stopeck at, et al. incidence, risk factors, and outcomes of osteonecrosis of the jaw: integrated analysis from three blinded active-controlled phase iii trials in cancer patients with bone metastases. ann oncol. 2012 may;23(5):1341-7. doi: 10.1093/annonc/mdr435. 1http://dx.doi.org/10.20396/bjos.v21i00.8666321 volume 21 2022 e226321 original article 1 department of prosthodontics and periodontology, piracicaba dental school, university of campinas, piracicaba, são paulo, brazil. corresponding author: renata cunha matheus rodrigues garcia department of prosthodontics and periodontology piracicaba dental school, university of campinas avenida limeira, nº 901. piracicaba, são paulo, brazil 13414-903 phone: + 55 19 2106-5240/ fax: + 55 19 2106-5211 e-mail: regarcia@fop.unicamp.br editor: dr altair a. del bel cury received: july 8, 2021 accepted: september 6, 2021 academic perspectives and psychosocial aspects of final-year dental students during covid-19 pandemic mariana barbosa câmara-souza1 , fabiana han na kim1 , olívia maria costa de figueredo1 , renata cunha matheus rodrigues garcia1 aim: this cross-sectional observational study aimed to evaluate the influence of the universities lockdown measures on academic perspectives and psychosocial aspects of brazilian finalyear dental students. methods: 268 undergraduate students regularly enrolled in a dentistry course at public universities were asked about anxiety, depression, stress sensitivity, and their academic perspectives by using an online survey. the hospital anxiety and depression scale (hads) was used to measure anxiety and depression, while the perceived stress scale (pss) evaluated stress levels. the academic perspective was evaluated by five affirmatives regarding possible difficulties that will be faced when school reopens and after graduation. the possible association between fear of having covid-19 with psychosocial outcomes and covid-19 association with academic perspectives were analyzed by anova and chisquare tests, respectively, considering a significance level of 5%. results: considering possible associations between the fear of having covid-19 and psychosocial aspects, significant values were found for anxiety (p = 0.018) and stress sensitivity (p = 0.002). regarding students’ academic perspectives, covid-19 had significant impact on less opportunity to perform procedures (p = 0.023), additional expenses with personal protective equipment (p = 0.007), and concerns of consulting elderly people (p = 0.012). conclusion: the covid-19 pandemic led to increased psychological impairments and enlarged concerns with learning and biosecurity, which might impact academic perspectives. thus, being aware of these apprehensions, university professors and staff can improve the clinical training of final-year dental students in an empathetic way. keywords: anxiety. depression. pandemics. students, dental. https://orcid.org/0000-0002-9961-121x https://orcid.org/0000-0002-8470-9687 https://orcid.org/0000-0001-9829-8607 https://orcid.org/0000-0001-8486-3388 2 câmara-souza et al. introduction covid-19 was first identified in december 2019 in the city of wuhan, china1, and declared a pandemic on march 11, 2020, by the world health organization (who)2. among the guidelines established for the control and prevention of covid-19, social distancing was the most important and widely spread, leading to lockdown in several countries. the lockdown changed the daily routine, highlighting challenges as working from home and education via internet, as well as led to the loss of social connections by the closure of parks and commercial centers, which modified lifestyle. thus, the lockdown protocol has provoked the feeling of loneliness, frustration, anger, uncertainty, and powerlessness3,4, which may impact the mental health. researchers investigating the immediate psychological response during the initial stage of the covid-19 outbreak in china showed that 53.8% of the respondents had moderate to severe psychological impact5, and it could be increased as the pandemic scenario continues. this perception can be further aggravated when university students are considered. even before covid-19, it was estimated that 15 to 25% of university students have some mental disorder6, being those aged from 18 to 20 the most vulnerable to depression and anxiety7. the covid-19 pandemic led to the suspension of in-person academic activities, and most of them were transferred to online platforms. remote education has been a temporary strategy to enable classes, exams, and group discussions, maintaining teaching-related activities. nevertheless, it cannot replace in-person lessons, which are essential for the development of skills and attitudes for integral education8. teaching dentistry encountered significant challenges because it requires hands-on training and patient-care experiences to fully learn dental procedures. therefore, especially for pre-clinical and clinical students, online education has several limitations9, which may lead to higher levels of stress considering the uncertainties10. thus, dental students are at high risk of developing mental health problems caused by concerns about the redesigned classes, inability to fulfill clinical requirements for graduation and its consequences on their future profession, additional spending on personal protective equipment (ppe), and safety concerns, as the fear of possible cross-contamination between students, patients, and family members11. moreover, considering the negative impact of the pandemic on the economy, it is not known whether patients will be able to continue dental treatments when the university reopens. it is also expected to lower the number of clinical appointments to minimize possible contact among people in the waiting room and to ensure proper disinfection of the office, dental chair, and materials10, which will consequently reduce the number of hours of patient-care practice. the final-year students’ concerns may still be associated with the fear of a potential economic recession and lower job offer, besides the occupational risks post-pandemic. the occupational safety and health administration (osha.gov) has classified the dentist’s profession at “very high risk” to potential exposure to 3 câmara-souza et al. coronavirus through aerosol-generating procedures11,12. thus, it is expected higher costs to implement additional precautions in infection control, which may discourage professionals from starting the career10. therefore, this study aimed to evaluate the influence of the covid-19 pandemic on academic perspectives and psychosocial aspects of brazilian final-year dental students. material and methods experimental design this cross-sectional observational study was previously approved by the local ethics committee (caae: 38129220.7.0000.5418) and included undergraduate students regularly enrolled in the last year of dentistry course at public universities of são paulo, brazil. this survey evaluated the effects of the covid-19 pandemic on the academic perspectives of dental students, as well as on anxiety, depression, and stress levels. the research was conducted by using an online form, via google forms (google; mountain view, ca, usa). sample and e-survey students enrolled in the final year of public dental schools in são paulo were considered for this research. são paulo is the most populous state in brazil, with 46.3 million inhabitants, which represents 21.9% of the brazilian population. it has seven public dental schools, affiliated with three universities: the university of são paulo, the university of campinas, and the são paulo state university. thus, the sample size calculation considered the 597 students registered on them. for an 80% power, design effect of 1.0 and 95% confidence level, 233 students should participate in this study. the first page of the online questionnaire presented the informed consent form, which explained the research aims, reported potential risks and highlighted the benefits. participation was voluntary and not paid; thus, those who accepted the invitation, digitally signed the informed consent form before proceeding to the structured questionnaires. the average time to fill out the entire questionnaire was approximately 15 minutes. invitation to participate was sent by email and whatsapp®, and the form could be filled out via smartphone, tablet, computer, or any compatible electronic device. data collection was conducted from september 28 to october 10, 2020. initial data collection comprised socioeconomic data, such as sex, monthly income, house sharing, alcohol consumption, smoking habits, and use of medications. moreover, participants should answer in which dental school was enrolled, and if they already had or were afraid of having covid-19. anxiety, depression and stress levels the validated portuguese version13 of the hospital anxiety and depression scale (hads) was used to measure anxiety and depression. hads is a 14-item questionnaire, seven related to anxiety and seven exploring depression. the hads questions 4 câmara-souza et al. address fears, insecurities, joy, slowness, restlessness, among others, and answers are related to the frequency in which the respondent experienced the situations. the score for each item can vary from 0 to 3, with a maximum score of 21 points. thus, scores from 0 to 7 indicated no anxiety/depression, 8 to 10 mild anxiety/depression, from 11 to 14 moderate anxiety/depression, and scores from 15 to 21 would denote severe anxiety/depression13. the portuguese version of the perceived stress scale (pss)14 was used to measure stress levels. the pss was constructed to evaluate how unpredictable and uncontrollable life events were in the last 30 days, from the individual viewpoint. it is a 10-item questionnaire, answered on a likert scale, ranging from never (0) to always (4) for questions in a negative tone, while for questions with a positive tone this punctuation was inverse14. the pss may be scored from 0 to 40, and higher values mean higher stress levels. academic perspectives the academic perspectives of dental students dealing with the school closure due to the covid-19 pandemic were evaluated by five affirmatives regarding possible difficulties that will be faced when school reopens and after graduation. the sentences were constructed based on published literature regarding dental education during and post-covid-19 era9-11. volunteers should indicate whether they agreed, disagreed, or ‘do not have an opinion’ (neither agree nor disagree), on each of the statements below: 1. “due to the covid-19 pandemic, i will have fewer opportunities to perform dental procedures when the clinical practice return”; 2. “due to the covid-19 pandemic, i will have less chance to learn techniques and i will be less prepared as a dentist”; 3. “due to the covid-19 pandemic, it will be necessary a higher clinical time to sanitize the environment and change the ppe, which will hinder clinical care”; 4. “i am worried about the additional expenses with ppe, mandatory for the clinical care”; and 5. “when the clinics restart, it will be very worrisome to consult elderly patients, considered a risk group for covid-19”. statistical analysis the data collected on the digital platform were exported and tabulated. initially, descriptive statistics were performed to identify frequencies and distributions of the outcomes. then, analysis of variance was used to test the possible association between fear of having covid-19 with psychosocial outcomes, while likelihood ratio chi-square was used to verify its association with academic perspectives. the study hypotheses were tested considering a significance level of 5% and minimum power of 80%. the sas software version 9.3 (sas institute, inc., cary, nc, usa) was used for all analyses. 5 câmara-souza et al. results considering the total amount of final-year students from public universities in são paulo, the response rate was 52.8% (n = 315). however, exclusion due to the use of antidepressants (n = 47) yielded a final rate of 44.9% (n = 268). participants’ age varied between 19 and 55 years, mean age of 23.7 ± 2.8 years (median = 23 years). volunteers’ distribution according to each dental school is shown in table 1. most participants were female (82.5%), with monthly income between 4 and 10 minimum wages (brazilian currency), representing the middle and upper-middle class. while during regular academic activities only 29.1% used to live with parents, during lockdown this amount increased to 85.5% (see table 2). from the respondents, only 9 students reported that already had covid-19, but the fear of having covid-19 was confirmed by 82.1%. table 2. socioeconomic characteristics of participants. characteristic n % gender female 221 82.5% male 47 17.5% monthly income (brazilian minimum wage – r$) ≤ 4 70 26.12% > 4 and ≤ 10 134 50.00% > 10 58 21.64% not reported 6 2.24% housing before covid-19 outbreak alone 53 19.78% with family 78 29.10% with friends 126 47.01% with a partner 11 4.10% continue table 1. distribution of volunteers according to the dental school. participants (n) response rate (%) usp – são paulo school of dentistry 58 (13 m; 45 f) 43.6 usp – bauru school of dentistry 17 (5 m; 12 f) 37.8 usp – school of dentistry of ribeirão preto 22 (5 m; 17 f) 26.5 unicamp piracicaba dental school 51 (8 m; 43 f) 69.9 unesp school of dentistry of araçatuba 48 (10 m; 38 f) 49.0 unesp araraquara school of dentistry 32 (2 m; 30 f) 41.0 unesp – school of dentistry of são josé dos campos 40 (4 m; 36 f) 46.0 usp, university of são paulo; unicamp, university of campinas; unesp, state university of são paulo; m, male; f, female. 6 câmara-souza et al. considering the psychological outcomes, the frequency of participants classified as having no, mild, moderate, or severe anxiety and depression are presented in figure 1. as for the academic perspectives after the covid-19 pandemic, most of the volunteers are in accordance that they will have difficulties when returning clinical activities, as shown in table 3. (a) anxiety (b) depression 13.8% 32.8% 22.4% 31% 4.5% 57.5%22% 16% absent mild moderate severe figure 1. distribution of anxiety (a) and depression (b) symptoms among the volunteers. continuation housing during lockdown alone 9 3.36% with family 229 85.45% with friends 12 4.48% with a partner 18 6.72% table 3. possible worries associated with clinical care after covid-19 pandemic, and the percentage of agreement of final-year dental students. worries about academic perspectives agree disagree no opinion w1due to the covid-19 pandemic, i will have fewer opportunities to perform dental procedures when the clinical practice return. 89.2% 3.7% 7.1% w2due to the covid-19 pandemic, i will have less chance to learn techniques and i will be less prepared as a dentist. 72.8% 13.1% 14.2% w3due to the covid-19 pandemic, it will be necessary a higher clinical time to sanitize the environment and change the ppe, which will hinder clinical care. 86.2% 4.9% 9.0% w4i am worried about the additional expenses with ppe, mandatory for the clinical care 82.1% 9.0% 9.0% w5when the clinics restart, it will be very worrisome to consult elderly patients, considered a risk group for covid-19. 85.4% 4.5% 10.1% 7 câmara-souza et al. possible associations between fear of having covid-19 and mean values of anxiety, depression, and stress are reported in table 4. moreover, it was also associated with the affirmatives representing academic perspectives for school reopening. discussion this internet-based research assessed final-year dental students from brazilian public universities in lockdown, regarding academic perspectives and psychosocial outcomes, as well as their association with students’ fear of having covid-19. it was found moderate levels of anxiety and stress, while depression symptoms were absent in more than half of the participants. furthermore, the great majority of participants agreed that they will have technical difficulties in clinical care and spend significantly more time and money on biosecurity protocols. brazil is now considered the epidemic center of the covid-19 pandemic, due to the steadily increasing caseloads15. while several countries had overcome covid-19 first wave and are now suffering the so-called second wave, brazil has only experienced a downward trend in hospitalizations between august and november 2020 but has never completely controlled the pandemic. the richest and most populous state of the country, são paulo, is responsible for approximately 20% of all cases and deaths, despite the efforts to implement lockdown measures as soon as the who declared covid-19 as a pandemic. thus, public dental schools have been closed since march 13, 2020, and remained without clinical practice until january 2021. the three universities evaluated are highly developed and the only brazilian ones included in the top 50 qs world university ranking. however, the pandemic challenges imposed on dental teaching seem similar to several dental schools around the country and worldwide. table 4. mean (standard deviation) or frequencies of the evaluated outcomes considering the fear of having covid-19. afraid of having covid-19 (n = 220) not afraid of having covid-19 (n = 48) p-value psychosocial assessment҂ anxiety 10.00 (4.03) 8.35 (5.52) 0.018 depression 7.01 (3.99) 6.35 (4.64) 0.318 stress 23.87 (5.51) 20.90 (7.21) 0.002 academic perspectives ¥ w1 90.91% 81.25% 0.023 w2 75.45% 60.42% 0.107 w3 87.27% 81.25% 0.457 w4 85.45% 66.67% 0.007 w5 88.58% 70.83% 0.012 w1, w2, w3, w4, w5, affirmatives about academic perspectives that can be found in table 3. ҂ data represented by mean (standard deviation); ¥ data represents the relative frequency of agreement for each affirmative. 8 câmara-souza et al. the sample was mainly represented by female subjects. although it could be considered a limitation, when evaluating students enrolled in dental schools, the response rates for each sex are similar. it can be supposed that this trend occurs in the entire country, since the number of female dental students are expressively higher than males, with approximately 83,500 females enrolled in 201716. as for monthly income, our results are comparable with those obtained in a previous study conducted in brazilian northeast17, and volunteers could be deemed as middle-to-upper middle class. considering the anxiety levels, 53.4% of volunteers had mild to moderate anxiety, and only 13.8% showed severe anxiety. these results are in accordance with several published studies, in which participants had mostly mild to moderate anxiety related to the covid-19 pandemic18-23. the anxiety of undergraduate students during the pandemic is especially related to uncertainties about future employments, worries for familiar and own safety, loss of social connections, media speculation and ‘culture of fear’, prolonged home lockdown, and loss of familiar income18-23. all these factors are also possibly related to the moderate stress levels found in the present study. khan et al. (2020)22 found event higher values, reporting that more than 69% of college students were suffering from mental stress. conversely, less than half of the volunteers (42.5%) had mild to severe depressive symptoms. a previous study, conducted between february and april 2020, found that 35.5% of participants reported mild to moderate depression24. prolonged self-isolation may modify neurotransmitters’ function, reducing serotonin levels. it is relevant to observe that this survey was assessed in october 2020, seven months after school closure, which represented a sample after long periods of lockdown and uncertainties regarding graduation. despite 42.5% of depressive symptoms are a considerably high percentage, another study found that 82.4% of university students from bangladesh presented some symptoms of depression25. the authors report that bangladesh students have a history of high levels of depression and anxiety due to the lack of family and social support, and concerns about the professional future. as for the present study, although students were worried about their future, during lockdown they had moved in with their parents, which could be psychological support26. for the affirmative that the covid-19 pandemic would result in fewer opportunities to perform dental procedures when the clinical practice return, 89.2% agreed with the statement. considering a lower possibility of learning techniques and being less prepared for the job market, 72.8% of the participants demonstrated this concern. van doren et al. (2020)9 corroborate our findings, stating that the lack of practice and clinical experience will occur due to the limitations of pre-clinical and clinical teaching. it is known that dentistry is a course that requires hands-on training, and despite online teaching is vital in continuing didactic learning during the covid-19 pandemic, it does not replace in-person clinical experiences. regarding biosafety, concerns about longer clinical time for environmental sanitization and ppe replacement were confirmed by 86.2% of the respondents, while worries due to additional costs with ppe needed for clinical care were expressed by 82.1% of the 9 câmara-souza et al. participants. to the best of our knowledge, no previous studies have addressed those issues. due to the covid-19 pandemic, students and professionals have to redouble infection control practices, demanding more protective materials to be used (face shield, n95 mask, cloak), and, consequently, increasing expenses with these materials that have already gone through an astonish inflation. these extra expenses may discourage or even hinder some of these students from public universities to continue their studies. therefore, institutions should empower students in need, aiming to do not compromise their performance, and, thereby, increase students’ stress levels. finally, considering that elderly patients are at increased risk of severe illness from the virus that causes covid-19, 85.1% of the students reported being very worried about consulting this population. the literature reports that aerosol production, violation of cross-infection protocols, and inadequate prevention for infection control in dental clinics may increase the possibility of transmitting the disease27-29. dental students have reported being afraid not only of becoming infected but also of contaminating people around them. procedures, such as endodontics and surgery, are the ones with higher-reported concerns, possibly due to the increased aerosol production28. although this research has obtained important answers on psychosocial aspects and academic perspectives of university students, some limitations should be considered. data collection was conducted only in october 2020, during the seventh month of lockdown. different results could have been obtained if we had evaluated the students in the early stages of pandemic onset, or even by january 2021, ten months after dental school closure. also, it would be interesting to assess these students when universities reopen to confront the academic perspectives reported here with the reality faced by them. a possible shortcoming of this study was volunteers’ selection, which was limited only to public universities of são paulo. it would be relevant to compare this population with students from all over brazil, and also consider those from private universities. therefore, further assessments and comparisons are highly encouraged, since they may assist professors and universities to deal with dental students during and after the pandemic. in conclusion, considering final-year dental students, the covid-19 pandemic led to increased anxiety, depression, and stress levels, alongside enlarged concerns with learning and biosecurity, that might impact academic perspectives. acknowledgments this work was supported by the são paulo research foundation (fapesp) brazil under grant 2017/23429-3; and by the coordenação de aperfeiçoamento de pessoal de nível superior brasil (capes), finance code 001. data availability datasets related to this article will be available upon request to the corresponding author. conflict of interest none. 10 câmara-souza et al. references 1. zhu n, zhang d, wang w, li x, yang b, song j, et al. a novel coronavirus from patients with pneumonia in china, 2019. n engl j med. 2020 fev;382(8):727-33. doi: 10.1056/nejmoa2001017. 2. sohrabi c, alsafi z, o’neill n, khan m, kerwan a, al-jabir a, et al. world health organization declares global emergency: a review of the 2019 novel coronavirus (covid-19). int j surg. 2020;76:71-6. doi: 10.1016/10.1016/j.ijsu.2020.02.034. 3. ioannidis jpa. coronavirus disease 2019: the harms of exaggerated information and non-evidence-based measures. eur j clin invest. 2020 apr;50(4):e13222. doi: 10.1111/eci.13222. 4. xiang yt, yang y, li w, zhang l, zhang q, cheung t, et al. timely mental health care for the 2019 novel coronavirus outbreak is urgently needed. lancet psychiatry. 2020 mar;7(3):228-9. doi: 10.1016/s2215-0366(20)30046-8. 5. wang c, pan r, wan x, tan y, xu l, ho cs, et al. immediate psychological responses and associated factors during the initial stage of the 2019 coronavirus disease (covid-19) epidemic among the general population in china. int j environ res public health. 2020 mar;17(5):1729. doi: 10.3390/ijerph17051729. 6. cavestro jm, rocha fl. 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10.1007/s11469-019-00144-8. 27. loch c, kuan ibj, elsalem l, schwass d, brunton pa, jum’ah a. covid-19 and dental clinical practice: students and clinical staff perceptions of health risks and educational impact. j dent educ. 2021 jan;85(1):44-52. doi: 10.1002/jdd.12402. 28. ataş o, talo yildirim t. evaluation of knowledge, attitudes, and clinical education of dental students about covid-19 pandemic. peerj. 2020 jul;8:e9575. doi: 10.7717/peerj.9575. 29. agius am, gatt g, vento zahra e, busuttil a, gainza-cirauqui ml, cortes arg, et al. self-reported dental student stressors and experiences during the covid-19 pandemic. j dent educ. 2021 feb;85(2):208-15. doi:10.1002/jdd.12409. https://doi:10.1007/s11469-019-00144-8 https://doi:10.1002/jdd.12409 1 volume 21 2022 e226337 original article braz j oral sci. 2022;21:e226337http://dx.doi.org/10.20396/bjos.v21i00.8666337 1 department of oral medicine, school of dentistry, tehran university of medical sciences, tehran, iran. 2 assistant professor, razi vaccine and serum research institute, agricultural research, education and extension organization (areeo), karaj, iran. 3 associate professor, department of oral medicine, imam khomeini hospital, tehran university of medical science, tehran, iran. corresponding author: soheila manifar, associate professor, department of oral medicine, imam khomeini hospital, tehran university of medical science, tehran, iran. tel: +98-2188351150, +98-912-033-0580 e-mail: mariakoopaie@gmail.com address: north kargar st,tehran, iran po. code: 14399-55991 p.o.box:14395-433. editor: dr. altair a. del bel cury received: july 10, 2021 accepted: april 2, 2022 lineage and phylogenetic analysis of hpv-16, -18 in saliva of hnscc patients maryam koopaie1 , mohamad amin nematollahi1 , maryam dadar2 , soheila manifar3,* aim: head and neck squamous cell carcinoma (hnscc) is a global health problem whose incidence varies by geographic region and race according to risk factors. human papillomavirus (hpv) infection is a significant risk factor for hnscc. hpv-16 and hpv-18 are two forms of hpv that are carcinogenic. hnsccs that are hpv positive have a better prognosis rather than hpv negative. the purpose of this research was to characterize hpv-16, -18 variations in the saliva of hnscc patients by examining the genetic diversity of hpv-16, -18 utilizing the full e6, e7, and l1 genes. methods: the case-control research included 15 patients with hnscc and 15 healthy volunteers. unstimulated entire saliva samples were obtained from the case and control groups by spitting method. genomic dna was isolated from all saliva samples. a pcr reaction was used to determine the presence of hpv in saliva. hpv-positive samples were genotyped and data were analyzed. we conducted a variant study on the hpv-16, -18 e6, and e7 genes. results: three patients with hnscc were hpvpositive for two hpv genotypes out of 30 people diagnosed with hpv-dna. hpv-16 and -18 were the most common genotypes. the hpv-16, -18 e6, and e7 genes were sequenced and compared to the hpv-16, -18 (e6, e7) prototype sequence. in all, hpv-16 lineages a1 and hpv-18 lineages a3 were discovered. conclusion: regarding the variation of hpv found in iranian hnscc patients, the need for further studies in hpv genotyping was seen. sequencing hpv genes in hnscc may help answer questions about hpv genotyping in the iranian population. hpv genotype analysis aids in the development of vaccinations against hnscc, halting disease progression and preventing hpv-associated hnscc. keywords: squamous cell carcinoma of head and neck. saliva. alphapapillomavirus. phylogeny. https://orcid.org/0000-0002-9999-1443 https://orcid.org/0000-0003-0605-781x https://orcid.org/0000-0001-5831-801x https://orcid.org/0000-0002-5898-3893 2 koopaie et al. braz j oral sci. 2022;21:e226337 introduction squamous cell carcinoma (scc) accounts for 90% of oral cavity cancers, and about 400000 new instances of head and neck squamous cell carcinoma (hnscc) are diagnosed each year1. men are 1.5 times more likely than women to develop hnscc2. the prevalence of hnscc varies by geographic region and race due to risk factor differences3. the incidence of hnscc is higher in india, pakistan, and bangladesh in the southern part of asia1,4. it may appear on the mucosa as a red, white, or red-white prominence. it may also be portrayed as a severe wound with noticeable edges. it is often linked to lymph node swelling5. hnscc is a complex illness with a variety of intrinsic and extrinsic risk factors that contribute to its development. tobacco and alcohol usage, infections, and dietary variables are examples of extrinsic risk factors. iron deficiency anemia and alterations in cell cycle control genes are among the inherent risk factors for hnscc6,7. in contrast to other viruses, human papillomavirus (hpv) has a significant role in hnscc pathogenesis among extrinsic risk factors8. hpv is a dna virus that attacks the skin and mucous membranes. there are about 30 different types of hpv viruses that have been linked to benign and malignant epithelial lesions9. furthermore, high-risk hpv strains such as hpv-16 and hpv-18 are thought to be etiologic factors for hnscc10,11. the presence of hpv-16 in exfoliated oral epithelial cells may raise the risk of hnscc and oropharyngeal cancer by 4 and 14 times, respectively12. according to reports, hpv16 is the most common kind in nearly all geographical areas of iran, followed by hpv-1813,14. hpv-16, hpv-18, hpv-11, and hpv-52 as the most frequently identified hpv types in iranian patients with hnscc12,15. although the data on hpv types in iran is well-documented, there is little information on hpv variations in hnscc. the current research sought to examine basic data of hpv-16 and hpv-18 variants in iranian patients with hnscc by examining the genetic variability of hpv-16 and hpv-18 utilizing the whole e6, e7, and l genes. the findings of our study might provide justification for future research into hpv genetic epidemiology, pathogenicity, and evolution. material and method ethical statement this study was approved by the tehran university of medical sciences ethical committee (ethical code ir.tums.dentistry.rec.1398.036). after describing the study objectives, written informed consent was obtained from all case and control group subjects. study population this case-control study was performed in imam khomeini hospital, tehran university of medical sciences (tehran, iran). 15 hnscc patients and 15 healthy volunteers as the control group enrolled in the study. hnscc of all patients was confirmed with the histopathologic examination, and they had not undergone surgery, chemotherapy, and radiotherapy. the study participants were not affected by any 3 koopaie et al. braz j oral sci. 2022;21:e226337 other malignancy or systemic disease. in the intraoral examination, there was no active dental and periodontal infection. the control group was matched with the case group for age, sex, tobacco, and alcohol consumption. unstimulated whole saliva samples were collected from the case and control group using the spitting method with a minimum of 5 ml saliva. all participants in the case and control group were asked not to eat, drink or brush their teeth, smoke at least one hour prior to the trial, and not consume any alcoholic beverages 24 hours prior to collection1. the authors confirm that all methods were performed in accordance with the relevant guidelines and regulations. evaluation of the presence of hpv with my09 / 11 primer genomic dna was extracted from saliva samples of the case (hnscc patients) and control group using the high pure viral nucleic acid kit (roche diagnostics gmbh, roche applied science, mannheim, germany) as directed by the manufacturer. the dna concentration was measured using the nd-1000 nanodrop at 260/280 nm. furthermore, the dna integrity was tested using a 1.5 percent agarose gel and kept at 20 °c for future study. the presence of hpv in the saliva was determined using the my09 / my11 oligonucleotide primers 5’-cgt ccm arr gga wac tga tc-3’ and 5’-cgt cag ggw cat aay aat gg-3’, which amplify a region of about 450 pb in the genome of most hvp types16. a 25 l reaction mixture containing 10 pmol of each primer, 50 m of each dntp, 200 ng of dna template, 2 u of taq dna polymerase, 20 mm tris-hcl, and 3 mm mgcl2 was used for the pcr process. the pcr was started with a 5 minute denaturation at 95 °c, followed by 35 cycles of denaturation for 30 seconds at 95 °c, annealing for 30 seconds at 55 °c, and extension for 30 seconds at 72 °c, with a final extension at 72 °c for 7 minutes. a negative pcr control, consisting of a reaction mixture devoid of template dna, was added to each pair of pcr reactions. electrophoresis on a 1 percent agarose gel was used to examine the pcr results. variant analysis of hpv-16 e6 and e7 genes pcr was used to examine the hpv-16 e6 and e7 genes, with an amplicon size of 762 bp targeted (table 1). a 50 l reaction mixture containing 10 pmol of each primer, 50 m of each dntp, 200 ng of dna template, 2 u of taq dna polymerase, 20 mm tris-hcl, and 3 mm mgcl2 was used for the pcr process. an initial denaturation of 5 minutes at 95 °c was followed by 40 cycles of denaturation at 95 °c for 30 seconds, annealing at 55 °c for 50 seconds, extension at 72 °c for 50 seconds, and final elongation at 72 °c for 10 minutes. a negative control, consisting of a reaction mixture devoid of template dna, was added to each pair of pcr reactions. electrophoresis on a 1 percent agarose gel was used to examine the pcr results. variant analysis of hpv-16 l1 gene hpv-16 l1 gene was analyzed by pcr (table 1), targeting an amplicon size of 7932 bp. 4 koopaie et al. braz j oral sci. 2022;21:e226337 table 1. primers for pcr analyzing of hpv-16 e6, e7 and l1 and hpv-18 e6, e7 and l1 primer sequences genes 5’-cga aac cgg tta gta taa aag cag ac-3’ hpv-16 e6 5’-tag att atg gtt tct gag aac a-3’ hpv-16 e7 5’-atg tgc ctg tat aca cgg gtc-3’ hpv-16 l1, forward 5’-tta ctt cct ggc acg tac acg c hpv-16 l1, reverse 5’-atg gcg cgc ttt gag gat cc -3’ hpv-18 e6 5’tta ctg ctg gga tgc aca cc -3’ hpv-18 e7 5’atg tgc ctg tat aca cgg gtc -3’ hpv-18 l1, forward 5’tta ctt cct ggc acg tac acg c -3’ hpv-18 l1, reverse pcr reaction method is similar to that described for hpv-16 e6 and e7 genes, and the schematic of hpv-16 e6 primers design is depicted in figure 1. figure 1. schematic primers design for pcr analyzing of hpv-16 e6. variant analysis of hpv-18 e6 and e7 genes the hpv-18 e6 and e7 genes were amplified using polymerase chain reaction (pcr) with an amplicon size of 835 base pairs as the target (table 2). a 50-l reaction mixture containing 10 pmol of each primer, 50 m of each dntp, 200 ng of dna template, 2 u of taq dna polymerase, 20 mm tris-hcl, and 3 mm mgcl2 was used for the pcr process. the primers were desaturated for 5 minutes at 95 °c, followed by 40 cycles of denaturation at 95 °c for 30 seconds, annealing at 55 °c for 50 seconds, extension at 72 °c for 50 seconds, and final elongation at 72 °c for 10 minutes. each set of pcr reactions contained a negative control consisting of a reaction mixture devoid of template dna. electrophoresis on a 1% agarose gel was used to verify the pcr results. 5 koopaie et al. braz j oral sci. 2022;21:e226337 variant analysis of hpv-18 l1 gene the hpv-18 l1 gene was examined using a pcr assay with an amplicon size of 7857 bp (table 1). the pcr reaction technique is identical to that described previously for the hpv-16 e6 and e7 genes, and a schematic primer design is shown in figure 2 for reference. figure 2. schematic primers design for pcr analyzing of hpv-18 l1, r. abi prism 377 sequencer genetic analyzer equipment (applied biosystems foster city, canada) was used to sequence all of the pcr products from the e6 and e7 genes to evaluate the hpv-16 and hpv-18 variants. the sequences were run according to the big-dye terminator protocol (applied biosystems) (geneali, seoul, south korea). it was determined that the reference sequences were unique to each lineage and sublineage by aligning them to all previously known lineages and sublineages of a1-4, b1-4, c2-4, and d1-316. the maximum likelihood approach was employed to build the phylogenetic tree, which was done using mega software version 6. in order to analyze the hpv-18 lineages and sublineages, all hpv-18 sequences were analyzed using the mega software version, which was used to depict the phylogenetic tree according to the maximum likelihood method with the reliability of bootstrap on 1000 replicates using the maximum likelihood method with bootstrap reliability on 1000 replicates. the a1-5, c, and b1-3 reference sequences for hpv-18 were also obtained from the genbank database, as were the lineage and sublineage-specific reference sequences for hpv-18. 6 koopaie et al. braz j oral sci. 2022;21:e226337 results this research comprised 30 people diagnosed with hpv-dna over a year (from march 2020 to march 2021). ten patients were male and five were female in the patient group. the control group consisted of 9 males and 6 females. the mean age and standard deviation (sd) of patient and control group were 53.84±13.61 and 45.23±12.80, respectively. four patients in the hnscc group had tongue scc, six had oropharyngeal scc, three had soft palate scc, and two had laryngeal scc. figure 3. hpv-16 and hpv-18 (e6, e7) were sequenced in genomes isolated from saliva of subjects. when the sequences recovered in this research were compared to the sequences of samples in the hpv-18 e6, e7, and l1 gene banks, it was discovered that the viral strain isolated from the patient’s saliva belonged to lineage a3 (figure 4-d, e, f). overall, lineages a1 of hpv-16 (figure a, b, c) and a3 of hpv-18 variations (figure 4-d, e, f) were discovered (figure 4), as shown by the red outlined data. 7 koopaie et al. braz j oral sci. 2022;21:e226337 a b c d e f fi gu re 4 . t he p hy lo ge ne tic tr ee in c om pa ris on w ith th e se qu en ce o f s am pl es in th e h p v -1 6 e6 , e 7 an d l1 a nd h p v -1 8 e6 , e 7 an d l1 g en e ba nk s ho w ed th at th e vi ru s st ra in is ol at ed fr om th e pa tie nt ’s s al iv a be lo ng s to ty pe 1 6; li ne ag es a 1 (a , b a nd c ) a nd ty pe 1 8; li ne ag es a 3 (d , e a nd f ). 8 koopaie et al. braz j oral sci. 2022;21:e226337 discussion hnscc is a group of heterogenic cancers with multiple risk factors. hpv positivity may be an important contributing factor in the histologic grade and prognosis of cases with hnscc2. risk factors of hpv transmission include multiple sex partners, tobacco consumption, and hiv infection. the roots of hpv transmission include sexual or nonsexual contact with the affected person, transmission through saliva contaminated with the viruses, breastfeeding, transmission during labor3. in the elderly, the most important predisposing factors for hnscc are alcohol and tobacco use, but in younger people, the role of alcohol, and tobacco in hnscc is lower and the number of young people with hpv are more than older ones. hence, the hpv virus may be one of the major risk factors for hnscc in the young population4. the annual incidence of oropharyngeal scc was increased, while scc incidence in the other sites of oral cavity was decreased. these differences may be attributed to the role of hpv in hnscc pathogenesis5. the role of hpv virus in anogenital cancers is confirmed. furthermore, hpv has a role in 20 – 40 % of hnscc6. pcr test to detect the expression of e6 and e7 oncogenes in hpv virus is the best diagnostic test for detection of hpv in hnscc7. the carcinogenic’s mechanism of hnscc is attributed to e6 and e7 oncogenes. hpv-e7 protein disrupts the prb-mediated cell-cycle control and causes a reduction in cyclin d1 synthesis and the over-expression of the cyclin-dependent kinase 4/6 inhibitor p16ink4a. hpv e6 protein promotes the destruction of p53 tumor suppressor gene8. hpv positive hnscc patients have a better prognosis than hpv negative patients, and their response to treatment is better9. some studies suggested that hpv-18 is the dominant type of hpv in iranian hnscc patients, but these reports are controversial and this controversy attribute to the geographic, cultural, and habit difference10,11. wood et al.13 showed that the prevalence of oral hpv varied significantly in different geographical areas. seifi et al.11 introduced hpv-18 as the dominant type of virus in east azerbaijan province of iran. delavarian et al.14 found no correlation between oral scc and hpv infection. these results were in line with sibers et al.12 and sisk et al.15 but in contrast with kreimer et al.17. a systematic review study by jalilvand et al. stated that hpv was detected in 44.4% of patients with head and neck cancers16. the most common hpv types were hpv-16 and -18. there was a strong association between hpv-16 and oropharyngeal cancer. these studies reflect the controversial reports about hpv prevalence and its subtypes in iran. this controversy recently confirmed by a systematic review study18. in conclusion, the hpv vaccine is available for the prevention of cervical cancer. gardasil is a type of hpv vaccine which induces immunity against hpv-6, -11, -16, -18. clinical trials on the effectiveness of this vaccine showed over 98% efficacy in the prevention of cervical, anal, vulvar, and vaginal cancers19. genomic analysis of hpv in the saliva of hnscc patients in the present study is a starting point for designing a vaccine to prevent hpv-associated hnscc. disclosure statement no potential conflict of interest was reported by the authors. 9 koopaie et al. braz j oral sci. 2022;21:e226337 acknowledgments the authors would like to acknowledge the help and support by imam khomeini hospital, tehran university of medical sciences and school of dentistry, tehran university of medical sciences for their constant assistance with the research. data availability datasets related to this article will be available upon request to the corresponding author. authors contribution mk and md conceived the study idea and led data collection. mk, man, and md created the study protocol and wrote the original draft. man and sm contributed in collecting the saliva sample. md, mk and man contributed to data analysis / interpretation and preparation of the manuscript. mk, man and sm led the writing review & editing. mk and md interpreted the results. all authors read and approved the final manuscript. references 1. jasim h, carlsson a, hedenberg-magnusson b, ghafouri b, ernberg m. saliva as a medium to detect and measure biomarkers related to pain. sci rep. 2018 feb 19;8(1):3220. doi: 10.1038/s41598-018-21131-4. 2. wang s, zhuang x, gao c, qiao t. expression of p16, p53, and tlr9 in hpv-associated head and neck squamous cell carcinoma: clinicopathological correlations and potential prognostic significance. onco targets ther. 2021 feb 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